List of flash talks & poster & winners
All accepted abstracts as flash talk or poster are avalaible here below. Click on the icon to view the abstract.
The winners of the poster prize can be viewed in the "Winning posters" tab.

Your poster must remain displayed during the entire display session.
Two displays sessions are planed:
- Display Session 1 from Monday 21 8h00 to Tuesday August 22, 2023 20h00
- Display Session 2 from Wednesday 23 8h00 to Thursday August 24, 2023 20h00.
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Posters viewing sessions will be scheduled as follows:
- on Monday and Tuesday from 18h00 to 20h00
- on Wednesday from 16h00 to 18h30
- on Thursday from 16h30 to 18h00.

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The fungal secretome comprises proteins that are involved in many aspects of fungal lifestyles, including adaptation to ecological niches. The aim of this study was to investigate the composition and function of predicted fungal secretomes in mycoparasitic and beneficial fungal-plant interactions of Clonostachys rosea, C. byssicola, C. chloroleuca, C. rhizophaga, C. solani and one unidentified Clonostachys species. The predicted secretomes of the analyzed species comprised around 8% of their proteomes. Mining of transcriptome data collected during previous studies showed that 18% of the genes encoding predicted secreted proteins were upregulated during the interactions with the mycohosts Fusarium graminearum and Helminthosporium solani. Functional annotation of the predicted secretomes revealed that the most represented protease family was subclass S8A, often involved in the response to nematodes and mycohosts, while the most numerous lipases and carbohydrate-active enzyme (CAZyme) groups appeared to be potentially involved in eliciting defense responses in the plants. Comparison with three Trichoderma spp., another genus known for its mycoparasitic activity, revealed profound differences in secretome composition, such as differences in the dominant CAZyme classes. This work sheds light on the role of the secretome in the interaction of Clonostachys spp. with plants and fungi, and highlights differences with Trichoderma spp. sharing similar lifestyles and ecological niches.
 

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Fire blight caused by Erwinia amylovora (Ea) represents a great threat to apple and pear production worldwide. For instance, the outbreak of fire blight occurred in Trentino caused a relevant reduction of crop yield in 2020. It is now widely accepted that apple flowers may harbor bacterial taxa that might hinder the ability of Ea to colonize apple flower. Based on this body of knowledge, we aimed at investigating the microbiota of apple flowers to select new potential biocontrol agents active against Ea. Flowers of Malus domestica cv. Golden Delicious from Trentino apple orchards were sampled at the ‘Baloon stage’ and surface sterilised to isolate only bacteria residing within the flowers. According to the 16S rRNA gene sequencing, the bacterial isolates mainly belonged to the Enterobacteriaceae, Pseudomonadaceae, and Microbacteriaceae families. One member of each bacterial family was selected and tested against Ea both on newly open apple flowers and on pear slices. Pantoea agglomerans AFF2001 and Curtobacterium flaccumfaciens AFF2009 effectively controlled Ea in both conditions. To characterize their mode of action, these bacterial strains were grown in a specific medium mimicking the apple stigma nutrient conditions and their cultural filtrates were tested to evaluate their impact on the growth and virulence of Ea. In the future, we will investigate the molecular mechanisms involved in the biocontrol activity of these bacterial strains.

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Potato scab, caused by Streptomyces spp., is a complex disease widespread in the main producing regions of the world. The control of this disease is very difficult and therefore a set of techniques is required for effective management. Biological control represents an alternative measurement and the bacteria Pseudomonas can be the solution. In the present study two strains of Pseudomonas sp. (IBSBF 3420 and IBSBF 3423), which are resistant to Fluazinam (fungicide widely used by potato growers), were tested with this chemical in vitro and in vivo against Streptomyces scabiei. In vitro assays, halos of inhibition with average of Ø 4.4 cm were observed in treatment with the two control agents (biological+chemical) while that those treatments with only one agent showed averages of  Ø 2.8 cm. In greenhouse assays, tubers treated with Pseudomonas sp. IBSBF 3420 and IBSBF 3423 resulted in the reduction of incidence (85 and 75%, respectively) and severity of disease (63.9 and 60.8%, respectively). Fluazinam treatment showed better performances when used together with Pseudomonas IBSBF 3420 (reduction of 79.6% incidence/64.1% severity) or Pseudomonas IBSBF 3423 (75.7% incidence/56.1% severity), while the treatment with only fungicide the reduction of incidence and severity were 68.5% and 50%, respectively. The results obtained herein showed that Pseudomonas strains can be used to reduce potato scab disease and the combination with Fluazinam does not affect the biological agent action.

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As for all viral pathogens, the establishment of turnip yellows virus (TuYV) cycle, belonging to the Polerovirus genus and newly to the Solemoviridae family, requires the expression of viral proteins whose function depends on their dynamic interactions with host factors. The investigation of these protein-protein interactions by yeast two-hybrid screen identified ALY proteins as potential interactants of two TuYV proteins. In Arabidopsis thaliana, four ALY family proteins (AtALY1 to 4) have been described. They participate redundantly in the nuclear export of cellular mRNAs mediated by the THO-TREX multiprotein complex.
Using co-immunoprecipitation experiments, we confirmed the interaction of all four AtALY proteins with the major capsid protein (CP) and also with the CP-RT fusion protein, which is notably involved in virus movement and in the plant aphid transmission.
Confocal microscopy analysis of agro-infiltrated Nicotiana benthamiana leaves confirmed the nuclear colocalization of the four AtALY:eGFP with the CP:RFP protein in viral context, while the CP-RT:tRFP protein expressed by the virus localized in perinuclear vesicles associated with viral infection.
Importantly, we have shown that the inhibition of expression of the four AtALY genes in a quadruple mutant of A. thaliana induced a significant increase in TuYV accumulation, proposing the AtALY as a potential target to elaborate a novel strategy to fight against TuYV infection.
 
 

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Stem-pitting (SP), an important malady of many fruit trees, is caused by Citrus tristeza virus (CTV) in citrus. CTV mutant with deletion of p13 displays very mild SP symptoms in Citrus macrophylla (Cmac). To reveal the role of this protein in SP development, we performed a combined study of transcriptome and microscopy analysis and characterization of phloem cell occlusions in Cmac infected with infectious clone of CTVΔp13 and CTV wild type (WT). CTVΔp13 induced non-visible SP symptoms and few defense response and development related genes, while the transcriptome responses of CTV WT infected trees, that caused moderate SP, was significantly affected. Transcriptomic and microscopic analyses of the stem-pitted area revealed that phloem regeneration is a characteristic manifestation of CTV-SP disease, while phloem regeneration was not observed in CTVΔp13 infected trees. CTVΔp13 induced less callose accumulation and lower PP2 gene expression comparing to CTV WT. To further define the role of p13 protein in SP, p13 transgenic Carrizo and Cmac were generated. In Cmac transgenic tree, we observed longitudinal stem-pit like symptoms and ropey-like appearance of the stem that mimics CTV-SP symptoms. Microscopic analysis of vasculature tissue of symptomatic stem indicated collapse of phloem cells. Collectively, p13 activates plant responses and induces phloem occlusion factors which disrupt phloem cells, resulting in regeneration of new cells that contribute to SP symptom development.

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The ubiquitin-proteasome system is a selective protein degradation pathway, which plays a vital role in the regulation of disease resistance in plants, such as the homeostasis of NLR proteins. However, most E3 ligases, the key subunits of ubiquitination enzymatic cascade, have not been functional characterized in rice, whose genome encodes large number E3 ligases. Here, we identified an E3 ligase OsFBX388, whose downregulation resulted in cell death, enhanced resistance against fungal and bacterial pathogens, and retardation of growth. OsFBX388 interacts with an Skp1 homolog OSK25 through its N-terminal F-box. Further, we showed that OsFBX388 targets an HMA domain-containing protein OsHIPP56 for ubiquitination and degradation via 26S proteasome. Disruption of OsFBX388 resulted in the overaccumulation of OsHIPP56. Overexpression of OsHIPP56 also resulted in enhanced disease resistance without yield penalty. In addition, both OsFBX388 and OsHIPP56 are required for pattern-triggered immunity. Furthermore, OsHIPP56 interacts with another HMA-domain containing protein RGA5, the sensor NLR protein of the NLR pair RGA4/RGA5. Co-expression of OsHIPP56 with RGA4/RGA5 induces cell death in both tobacco cells and rice protoplasts, suggesting OsHIPP56 functions as an innate mimic AVR protein. Together, our results provide a new mechanism in which the E3 ligase OsFBX388 balances the growth and immunity of rice by at least partially mediating the homeostasis of the AVR mimic OsHIPP56.

 

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Little is known about host susceptibility genes that facilitate the infection process of soil-borne, necrotrophic oomycete pathogen Pythium myriotylum Drechsler, which cause soft rot disease in spice crop ginger (Zingiber officinale Roscoe). From the differential accumulation of jasmonic acid (JA) and salicylic acid (SA) between the pathogen inoculated ginger and the highly resistant wild congener Z. zerumbet (L.) Smith, we presumed a susceptibility function for JA in Zingiber­-Pythium pathosystems. This presumption was vindicated when the exogenous application of Z. zerumbet with JA produced high susceptibility to P. myriotylum. Consistent with this, the application of SA prior to JA application in Z. zerumbet reverted the susceptibility. The susceptibility function of JA was confirmed following the real time expression analysis, phytohormone quantification and histopathological methods. With this background, in ginger, we silenced four key genes involved in the JA signalling pathway using virus induced gene silencing. The silenced plants showed tolerance to P. myriotylum. Histopathology of the silenced plant showed no pathogen ingress as compared to unsilenced inoculated and mock inoculated plants. The study depicted a key role for the relative concentration of SA/JA in deciding the host response to P. myriotylum and helped to identify four putative susceptibility genes for the CRISPR based genome editing in ginger for soft rot tolerance.

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Development of wheat resistant to Fusarium head blight (FHB), while improving yield and maintaining quality requirements is important in Ontario, Canada. FHB is a serious disease of wheat and deoxynivalenol (DON) is the most common mycotoxin produced by Fusarium graminearum (FG). All wheat commercially grown in Ontario is entered in the Performance Trials, and tested for agronomy traits and FHB resistance, in nurseries spray inoculated with FG isolates, at anthesis. FHB symptoms are recorded as incidence (percent of heads infected) and severity (percent of spikelets infected). FHB index is calculated as severity x incidence, divided by 100. DON level is estimated using ELISA method. Wheat cultivars and checks are grouped, based on FHB visual symptoms and DON level, using historical and the most recent data (www.gocereals.ca). The categories are: moderately resistant (MR), moderately susceptible (S), susceptible (S) and highly susceptible (HS). Some cultivars have different category for FHB index and DON level.  Phenotyping, genotyping and development of new cultivars, with increased level of FHB resistance and higher yield is in progress, in breeding program from University of Guelph, Ridgetown Campus. Correlation among morphological traits (plant height, heading date, awns presence), FHB related traits (severity, incidence, index, and DON level) and yield, will be presented. DON level of winter wheat populations, with different sources of FHB resistance, will be compared.

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Plant small RNAs (sRNAs) can trigger non-cell autonomous RNA interference (RNAi) in interacting eukaryotic pathogens or parasites possessing canonical RNAi factors. However, it is currently unknown whether a similar process could operate against a phytopathogic bacterium, which lacks a eukaryotic-like RNAi machinery. We recently demonstrated that Arabidopsis-encoded artificial sRNAs can trigger the sequence-specific silencing of a virulence factor from Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000). However, the sRNA species that are implicated in this phenomenon remain elusive. In the present study, we identified and characterized two populations of apoplastic sRNAs that orchestrate antibacterial gene silencing. The first one involves sRNAs that are associated with extracellular vesicles (EVs), and presumably incorporated in ribonucleoprotein complexes. Intriguingly, the second one involves sRNA duplexes that are in a free form, and thus referred to here as extracellular free small RNAs or efsRNAs. Here, I will present the experimental data supporting these findings. I will also discuss the relevance of these findings in the understanding of how plants regulate transcriptome, community composition and genome evolution of associated bacteria.
 

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Bacterial Leaf Blight of rice (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major threat for food security in many rice growing countries including Burkina Faso where the disease was reported first in the 1980’s. In line with the intensification of rice cultivation in West-Africa, BLB has been on the rise along the last 15 years. West-African strains of Xoo differ from their Asian counterparts as they (i) are genetically distant, (ii) belong to new races and, (iii) contain reduced repertoires of Transcription Activator Like (TAL) effector genes. In order to investigate the evolutionary dynamics of Xoo populations in Burkina Faso, 177 strains were collected from 2003 to 2018 in three regions where BLB is occurring. Multilocus VNTR Analysis (MLVA-14) targeting 10 polymorphic loci enabled to discriminate 24 haplotypes and showed that Xoo populations were structured according to their geographical localization and year of collection. Considering their major role in Xoo pathogenicity, we next surveyed the TAL effector repertoires of the 177 strains upon RFLP-based profiling. Surprisingly an important diversity was revealed with up to eight different RFLP patterns. Finally, comparing neutral vs. TAL effector gene diversity allowed to suggest scenarios underlying the evolutionary dynamics of Xoo populations in Burkina Faso, which could be helpful to guide the deployment of BLB resistant varieties in the country.

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Phytophthora infestans caused the Irish Potato Famine of 1845-1852. We performed targeted enrichment sequencing of dried, infected leaf samples from 29 herbarium specimens collected from 1845-1954. Our bait library was designed to enrich both pathogen effector genes and Solanum host R genes. We leveraged the targeted sequencing approach to generate high coverage of genes responsible for pathogen virulence and host resistance. Many modern well-characterized effector genes have historically been present in P. infestans. Both R genes and effectors showed signatures of selection. However, variant calling analysis revealed alternative alleles including avirulent forms compared to the reference genome for many effectors that likely impacted function. The effector Avr3b was the only well-described effector not present in Famine era samples but did appear in the mid-1900s. Interestingly, the resistance breaking allele of Avr1 was present during the famine before the Solanum R1 gene was deployed by plant breeders. Detailed ploidy analysis of 19 high coverage genomes showed that US-1 lineages appearing in the early 1940s were triploid, in contrast to the FAM-1 lineages from 1845 and thereafter that were diploid. This exploration of historic plant and pathogen genomes will shed light on the past host-pathogen evolutionary relationships of a globally important plant pathogen and could provide insight for future deployment of Solanum R genes.
 

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The field of ancient genomics has triggered considerable progress in the study of pathogens, including those affecting crops. Herbarium collections have been an important source of dated, identified and preserved DNA, whose use in comparative genomics and phylogeography may shed light into the emergence and evolutionary history of plant pathogens. I will present the reconstruction of 13 historical genomes of the bacterial crop pathogen Xanthomonas citri pv. citri (Xci) from infected citrus herbarium specimens using a shotgun-based deep sequencing strategy. After authentication of the historical genomes based on DNA damage patterns, we compared them to modern genomes to reconstruct their phylogenetic relationships, pathogeny-associated genes content and estimate several evolutionary parameters, using Bayesian tip-dating calibration and phylogeography inferences. Despite a challenging analysis of data, requiring adapted treatment before being compared to modern samples, our results reveal that Xci originated in Southern Asia ~11,500 years ago and diversified during the beginning of the 13th century, after Citrus diversification and before spreading to the rest of the world. This updated scenario links Xci specialization to Neolithic climatic change and the development of agriculture, and its diversification to the human-driven expansion of citriculture through the early East-West trade and later colonization. 

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Several plant viruses manipulate their hosts to modify the attractiveness of their insect vectors, in a way that enhances their own spread. Cucumber mosaic virus (CMV) emit volatile organic compounds (VOC) that attracts its main vector, Aphis gossypii. However, it is not known how aphids respond to plants double infected with additional viruses such as Cucurbit aphid-borne yellows virus (CABYV). Furthermore, plants often emit volatiles after aphid feeding that attract natural enemies to limit pest damage (“call for help”). However, few studies have been conducted on how aphid parasitoids react to VOC emitted by virus-infected plants. Thus, we conducted Y-tube olfactometer assays to understand how simple and double infection (CMV+CABYV) can modify volatile cues involved in aphid-attraction and in aphid-plant-parasitoid interactions. Our results confirmed that VOC emitted by CMV-infected melon attracted A. gossypii but when plants became infected with CMV+CABYV aphid attraction was suppressed. We also found that in the presence of aphids, Aphidius colemani was attracted to mock-inoculated plants but rejected VOC emitted by CABYV-infected plants. Thus, our work reports for the first time that a plant virus can counteract the “call for help” plant defense response involved in parasitoid attraction to its aphid host. This could ultimately induce lower rates of parasitism on CABYV-infected plants reducing the effectiveness of biological control of A. gossypii by A. colemani

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Plant pathogenic effector is secreted into the host and modulates the host immune system. Nuclear effectors translocated in the host nuclei and interact with proteins and DNA to regulate various defense mechanisms. Nuclear localization sequence (NLS) is the most well-known factor for nuclear transportation. However, the molecular mechanism of NLS-associated vehicles and the roles of NLS in transcriptional reprogramming still need to be understood. We previously reported that MoHTR1, a nuclear effector of the rice blast fungus, Magnaporthe oryzae, is translocated to rice nuclei but not to fungal nuclei. MoHTR1 was localized in the plant nucleus by interacting with rice importin alpha. We found one NLS (PGRSKKE) and further identified that RxKK residues were necessary for the nuclear localization of MoHTR1. MoHTR1 NLS altered the localization of cytoplasmic effectors of M. oryzae in the host. Furthermore, nuclear effector candidates which have RxKK sequence also localized in rice nuclei. SUMOylation, post-translational modification, was involved in the secretion and translocation of MoHTR1 to biotrophic interfacial complexes and host nuclei. In addition, MoHTR1 NLS was essential for the pathogenicity of M. oryzae by reprograming defense-related genes and host target gene candidates. Our findings will provide unprecedented details on the roles of plant-specific NLS on nuclear effector in pathogen-host interactions.
 

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Apple scab caused by the pathogenic fungi Venturia inaequalis is the most common disease in apple orchards. Resistance genes such as Rvi6 have been introduced in apple varieties to reduce the use of fungicides in the management strategies of this disease. However, in the 90’s, we observed a breakdown of Rvi6 resistance and the emergence of V. inaequalis virulent stains on trees carrying Rvi6. Using genomic analysis, we identified AvrRvi6, the first fungal avirulent gene responsible for a gene-for-gene interaction in the Apple-V. inaequalis pathosystem. AvrRvi6 codes for a small secreted protein with structure similar to the MAX effector family. On the plant side, Rvi6 is a receptor-like protein (RLP), similar to RXEG1 and predicted to localize at the plasma membrane. We observed that fungal virulent strains have mutations in the coding or regulatory sequences of AvrRvi6 which we hypothesize are responsible for the escape of Rvi6­ immunity. Using transient expression in Nicotiana benthamiana we were able to replicate the recognition of AvrRvi6 by Rvi6 resulting in a strong cell death phenotype. This N. benthamiana assay allowed us to test the AvrRvi6 alleles and identify essential residues for Rvi6 recognition. We also investigated the protein-protein interactions between AvrRvi6 and Rvi6 and the subcellular localization of this allele-specific immune recognition.

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Fusarium oxysporum (Fo) is a ubiquitous soilborne fungus that can cause Fusarium wilt (FW) in 100+ crops. Uncertainties in aspects of its epidemiology and a lack of global distribution data have historically challenged monitoring and containment efforts. Our NASA Interdisciplinary Sciences project seeks to address this need by integrating remote sensing, aerosol transport modeling, and comparative genomics to build a global disease surveillance system for FW incidence and Fo dispersal risk in aerosolized agricultural dust. As foundation, we released an interactive, global web map documenting 4500+ FW incidences reported in peer-reviewed literature. Here, we developed a global susceptibility assessment that integrates all three aspects of the disease triangle. We identified agricultural production zones conducive to FW, noting subsets capable of serving as dust sources, by overlapping the MODIS Deep Blue algorithm with a Landsat-based cropland product. We then restricted this assessment to only regions with reported Fo in the past 30 years. Conducive disease environment was modeled using multiple satellite-derived products with species distribution modeling. Results from this assessment along with aerosol transport modeling can inform how related incidence sites on opposite ends of dust events may be. This integrated approach to disease surveillance can provide key insights about drivers for current and future FW distribution and the spread of Fo on global dust currents.

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In Italy, the demand of fresh/processed pomegranates has recently grown due to the nutraceutical properties. Fruit can be cold stored no more than few months due to fruit rots caused by Botrytis, Alternaria, Coniella, and Penicillium fungal genera. The first three genera infect pomegranates during the blooming stage leading to latent infections, instead species belonging to Penicillium sensu lato are "wound" pathogens exploiting macro- and microinjuries occurring during harvest and postharvest handling. The scarcity of conventional and alternative fungicides for this minor crop has enhanced the need to find new solutions/strategies for disease control. In the blooming phase, 'Wonderful' plants were treated with low environmental impact compounds: a bio-stimulant made of red algae, a chitosan solution, and two formulations based on Aureobasidium pullulans and Bacillus amyloliquefaciens. Furthermore, harvested pomegranates with cracking were dipped in ozonated water or in neutral electrolyzed water (NEW) to evaluate the activity of these physical control means against “wound” pathogens. The research disclosed the effectiveness of B. amyloliquefaciens and of NEW in reducing postharvest rots due to latent and wound infections, respectively. In pomegranates, the integrated use of low environmental impact products and physical means can represent an ecofriendly alternative to fungicides, fitting into the concept of sustainable agriculture also in organic farming.

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Due to their specific indigenous microbial communities, soils vary in suppressiveness towards pathogens. To explore the effects of climate change and agricultural management on the ability of soil microbiota to suppress phytopathogenic fungi, we collected soils from conventional and organic farming (CF and OF, respectively) plots exposed to ambient (A) or future (F) climatic conditions at the "Global Change Experimental Facility" (Bad Lauchstädt, Germany). The preconditioned soils were cultivated with wheat at the greenhouse and tested for their suppressiveness towards Fusarium graminearum (Fg-1) and Gaeumannomyces tritici (Ggt). The soils inoculated with Fg-1 or Ggt presented a significantly lower microbial diversity (p<0.05) in the rhizosphere than the respective control soils. The preconditioning under ambient or future climatic conditions caused community differences in the conventional farming soil (CF-A vs. CF-F; p<0.001) for both inoculated treatments, while no such differences were observed for the organic farming soil. Overall, taxa affiliated with Actinobacteria and Proteobacteria were most abundant in all treatments and the most differentially abundant between CF-A and CF-F after inoculation with Fg-1. These taxa may contribute to disease suppressiveness and consequently support crop health. The study further revealed that soils preconditioned under different climatic conditions can help understand the impact of climate change on the functioning of soil microbiota.

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Food safety is intimately linked to plant health. It is threatened by pests whose impact varies greatly depending on the crop, agricultural practices, and regions of the world. In a global trade context, the transport of infested plant materials is an important factor in the spread of organisms to new territories, which can cause emerging diseases. It is critical to deploy strategies to limit this from happening. Identifying pests and their vectors is a first step towards risk analysis. International initiatives are bringing together research results on the role of seeds as vectors of pests. The International Seed Testing Association Reference Pest List (ISTA-RPL) focuses on seed-borne pathogenic organisms in about 50 plant species (field crops, legumes, fruit and forest trees, aromatic plants). The aim is to determine, based on scientific results, if these organisms can (or cannot) be transmitted vertically or transferred in the environment under natural conditions, making seeds a vector of dissemination. The ISTA-RPL currently inventories 333 pests, of which 146 are transmitted or transferred via seeds in 23 host species (v9.0; July 2022). It is a living tool, and an additional set of hosts is under investigation. This literature resource may be a valuable source for risk assessors and policymakers. It also opens avenues of R&D work by seed companies, academic laboratories or industry to develop diagnostics, detection methods, or treatments

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The ‘Stop the Rot’ project (https://alliumnet.com/stop-the-rot/), funded by the USDA National Institute of Food & Agriculture Specialty Crops Research Initiative, aims to help onion growers reduce losses to bacterial diseases through understanding interactions of the host, pathogens, and environment. Outcomes include improved diagnostic tools and enhanced bacterial disease management strategies. Surveys of >130 locations in 2020-2021 generated isolates of 116 bacterial genera from onion foliage and bulbs in seven regions of the USA, with distribution and pathogenicity varying across regions. Very few strains caused symptoms in onion bulb scale pathogenicity tests. Pantoea, Pseudomonas, Burkholderia, and Enterobacter were the most prevalent genera. Microbiome analyses revealed different complex bacterial communities in asymptomatic vs. symptomatic bulbs. Genomic analyses of Pantoea agglomerans strains showed some carry the HiVir gene cluster associated with virulence to onion. Copper tolerance genes found in ~50% of sequenced P. agglomerans isolates might explain the poor efficacy of copper bactericides in many trials. Trials of bactericides and irrigation, fertility, cultural, and post-harvest practices, evaluated across the USA, demonstrate that irrigation timing and method, and late-season cultural practices can be optimized to reduce losses to bacterial diseases. Economic evaluations of results ensure recommendations are practical, viable, and financially sustainable.
 

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Pepper (Capsicum annuum) is an important crop due to its massive consumption as a seasoning vegetable in Korea and many other countries. Anthracnose disease in chili pepper has caused serious damage to plant growth and reduced yield with apparent symptoms and signs on the fruits. In this study, we report a deep learning-enabled detection model for chili anthracnose among chili pepper disease based on a computer-vision algorithm. The model was developed based on a deep learning architecture based on a Convolutional Neural Network (CNN) that specializes in extracting features from image datasets. Large datasets of expert pre-screened pepper disease images were collected from ‘AI Hub’, a platform of AI infrastructure. We examined the effectiveness of image preprocessing and data augmentation to create a balanced dataset. The implemented model achieves higher than 90% classification accuracy compared with training and validation dataset. Our results showed that CNN could be the deployable method for digital disease detection. This meaningful success makes the model a useful disease detection tool, and this research could be further extended to develop a mobile application to help millions of farmers directly in the fields. Further results for the detection of chili pepper disease will be discussed.

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The cereal powdery mildews (Blumeria spp. of the family Erysiphaceae) are globally occurring fungal pathogens of grasses and cereals and pose a constant threat for agriculture. Blumeria species infect grasses and cereals in a host-specific manner. Ubiquitously distributed transposable elements make up >75% of the genomes of the cereal powdery mildews, which can be a source of genetic variation and genome instability. We study if and how Blumeria regulates and repurposes transposable elements to rapidly overcome host resistance. We found transcriptional activity of transposable elements in the barley powdery mildew pathogen B. hordei at specific stages of infection, particularly during early host cell penetration and haustoria establishment. Epigenetic profiling in conidia revealed increased 5mC methylated DNA levels in retrotransposons, while small RNA sequencing of isolated mycelia and haustoria indicated accumulation of phasiRNAs in >1,500 retrotransposon loci, suggesting dynamic control of transposon expression through epigenetic mechanisms and RNA interference. We further discovered long spliced antisense RNAs (antisense lncRNAs) at loci of transposon replication genes. These transposon antisense lncRNAs exhibit time point-dependent expression patterns as well as distinct co-expression patterns with transposons, indicative of both positive and negative regulation of transposons by antisense lncRNAs. 

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Under the National Clean Plant Network (NCPN) economic study, the sweetpotato clean centers started an experiment aiming to assess the value of clean seed in comparison to older generation seed. The goal of this study was to evaluate the performance and quality of foundation seed after it had been integrated into commercial sweetpotato operations. In NC, trials started in 2021 with Covington and Beauregard as evaluated varieties. G1 seed was used as a reference to compare the yield and virus incidence of growers’ generation 2 (G2), generation 3 (G3) and generation 4 (G4) seed roots. This experiment was repeated in 2022 with Averre and Bayou Belle added to the initial pool of varieties as well as older generations (G5 and G6). It is known that the accumulation and perpetuation of viruses in sweetpotato is a major constraint for production of seed and the commercial crop. The potyvirus complex is prevalent in North Carolina and comprises Sweet potato feathery mottle virus (SPFMV), Sweet potato virus G (SPVG), Sweet potato virus C (SPVC) and Sweet potato virus 2 (SPV2). In 2021, virus data suggested a low incidence of viruses (mainly SPFMV) on G1 material. Potyviruses (mainly SPVG, SPVC and SPFMV) started to be prevalent on G2 and G3 material. In the older generation evaluated (G4), all potyviruses (SPVG, SPVC, SPFMV and SPV2) were detected. In 2022, the same trend was observed as the prevalence of four potyviruses was associated to higher seed generations. 

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Investigating sporulation patterns is pivotal in plant disease epidemiology because it may shed light on relevant biological features of pathogens and on the environmental factors driving the release of infectious propagules. The ascomycetes Cryphonectria parasitica and Gnomoniopsis castaneae are major pathogens of chestnut (Castanea spp.) characterized by having both teleomorphic and anamorphic stages. In this study, we assessed and compared spatial and temporal spore deposition patterns of the two pathogens by using an aerobiological approach combining passive spore trapping, taxon-specific qPCR assays, and statistical modelling based on a novel index called Standardized Deposition Rate. Approximately 1300 samples collected at regular intervals over two years in three chestnut orchards in northern Italy were analyzed. Results showed that both species sporulate all year long, but while for C. parasitica peaks are seasonal and propagule loads are driven by the number of weekly rainfalls, spore deposition of G. castaneae increases with raising temperatures and wind gusts. Differences in the geospatial patterns of spore deposition between the two pathogens are discussed. Our results may contribute predicting the risk of infection of these two fungal plant pathogens.

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The QoIs, also known as strobilurins, represent a group of fungicides with arguably the greatest resistance risk. In 2023, the FRAC reported more than 50 species with a resistant status to QoIs. Resistance is mainly caused by the replacement of Glycine into Alanine (G143A) in the target cytochrome b protein. This substitution is conferred by a single nucleotide mutation in the cytochrome b gene. In fungi, a group I intron, situated directly after codon 143, prevents QoI resistance by blocking the G143A mutation. However, group I introns have the ability to move from a donor gene into an intronless acceptor gene. The mobility of group I introns has been speculated to be a compensation mechanism to restore the potential for QoI resistance mutation. On this basis, an extensive in silico analysis of whole mitochondrial fungal genomes was performed to characterize the distribution of the group I introns among CFR fungal species. Our results showed that one subtype of group I is associated with QoI resistance and that CFR fungi can be classified according to the presence/absence of this subtype in their genome, resulting in their capacity to acquire the G143A mutation, and thus become resistant. Accordingly, this classification allows to predict the potential of QoI resistance among fungi. At a time when the use of synthetic fungicides can lead to resistance and environmental problems, this predictive model could be a valuable tool in managing the use of QoIs for disease control.

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Immune receptors alarm the host plant about the presence of invading pathogens. Despite advances in understanding how receptor proteins are activated by non-self molecules, little is known about the regulation of their expression at the transcript level. Broad-spectrum disease resistance in Arabidopsis and crops can be conferred by mutations in the susceptibility genes DOWNY MILDEW RESISTANT 6 (DMR6) and its close paralog DMR6-LIKE OXIDOREDUCTASE 1 (DLO1). The DMR6 and DLO1 proteins are salicylic acid (SA) hydroxylases that act as negative regulators of immunity. RNAseq profiling of the transcriptomes of Arabidopsis DMR6/DLO1mutant and overexpression lines revealed a role of basal SA levels in the regulation of the expression of immune receptor genes. These DMR6/DLO1-affected and SA-dependent genes belong to specific groups of surface and intracellular immune receptors. We will present further experimental evidence for the importance of the expression control by SA through DMR6/DLO1 for early immune signaling. We propose that immunity in the dmr6 and dlo1 mutants involves enhanced basal expression of immune receptor genes that boosts the plant recognition potential.

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High temperatures are known to exacerbate rice panicle blight caused by Burkholderia glumae. Our long term goal is to develop rice with increased tolerance to combined heat and disease stresses. Our first steps have been to develop a robust experimental system to test how heat stress tolerance interacts with B. glumae infection. A set of rice genotypes with contrasting tolerance to heat was inoculated with B. glumae at anthesis stage. Genotype response to bacterial infection was measured by quantifying the proportion of empty spikelets as well as the total number of grains obtained from inoculated panicles. Preliminary results identified promising rice genotypes that can tolerate heat stress and the infection by B. glumae separately. These rice genotypes will be valuable for studying the molecular mechanisms responsible for tolerance and enable development of new rice varieties that withstand combined stresses under field conditions.

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Approximately 70% of fungicide applied on European wheat fields is primarily aimed towards the management of the fungal disease Septoria tritici blotch (STB), although the uptake of biological fungicides (biofungicides) in field-based cropping systems remains poor. The aim of this study was to integrate diagnostic tools into the fungicide decision process to improve disease prediction and refine biofungicide application. Another objective of the study was to investigate the mechanisms of a biofungicide in STB control.
A winter wheat field trial was conducted using traditional agronomy-based risk prediction and a novel method of disease forecasting- spore trapping and weather-based risk modelling- to influence the application rates of synthetic and biological treatments. The effects on STB control were assessed using visual disease assessments, while the data from molecular analyses (qPCR) of spores and symptomless leaves was used to retrospectively forecast disease severity in the crop.
In synthetic fungicide treatments, the diagnostics-based programme showed similar results to the agronomist-lead programme. However, the biofungicides did not effectively control STB, with significantly lower control in the diagnostics programme, prompting an investigation into the biological mechanisms for disease control, or lack thereof. This study will explore the effect of the biofungicide at various stages of STB ingress on the wheat phylloplane and STB control under greenhouse conditions.

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Effective management of soilborne pathogens is essential for commercial strawberry production in California. To minimize the impact of these pathogens a site-specific management program is under study that tracks pathogen load, disease incidence, and crop productivity for strawberry and vegetable crops grown in rotation to determine fumigant application rates within a field. Disease risk across the field is assessed by TaqMan soil quantification assays for three of the major lethal pathogens of strawberry (V. dahliae, M. phaseolina and F. oxysporum f. sp. fragariae) coupled with prior season disease incidence. Management decisions on the type of fumigant, a risk-based assessment of rates for different areas of a field, and application methods are based on this information. Plant vigor and disease incidence is monitored during the growing season on a field wide and individual plant basis by drone flights every 1-2 weeks and a tractor mounted sensor system. Two GPS-enabled systems are used to collect precision yield data with cumulative yield maps generated on a regular basis. Correlations between drone/tractor sensor data and yield are explored to evaluate efficacy of the fumigation treatment and develop yield prediction models. An economic analysis of precision pathogen management provides information to support grower decision making. Results from field trials indicate that variable rate fumigant application based on risk can effectively manage disease and maintain yield.

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The way in which we diagnose and respond to bacterial disease outbreaks has transformed over the past several decades. Serological and molecular methods were once the gold standard for diagnostics; however, these methods are labor-intensive and can make it difficult to identify and characterize pathogens. To combat these challenges, we used in-house, low-cost whole genome sequencing (WGS) to rapidly define and track an outbreak in real-time of Xanthomonas hortorum pv. pelargonii (Xhp) on US geraniums (Pelargonium x hortorum) that occurred in the spring and summer of 2022. We used short- and long-read sequencing to assemble 31 Xhp isolate genomes from the 2022 outbreak, and five Xhp genomes from previous decades. We compared the content and size of the core and plasmid genomes in order to investigate the evolution of virulence. The outbreak strains (Xhp2022) clustered in a new lineage and showed plasmid expansion when compared to older Xhp isolates. The new, larger plasmid acquired genes via horizontal gene transfer and cointegration of other plasmids, leading to expansion. These additional genes may have led to enhanced virulence and fitness, such as heavy metal resistance, and we speculate these factors led to the outbreak of 2022. WGS gave us insight within 2-3 weeks of receiving samples and allowed us to quickly characterize the outbreak, leading us to conclude that rapid sequencing can be used as a model for the surveillance and tracking of other pathogens.

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Emerging and re-emerging plant diseases pose an enormous threat to agricultural production and global food security. Early detection and identification of outbreaks using advanced high-throughput sequencing (HTS) technology and bioinformatics tools are playing increasingly important roles. At CFIA, the PolyChrome (PC) system and the Clasnip platform (www.clasnip.com) are developed for the early detection and identification of bacterial ring rot, zebra chip and soft rot of potato, as well as potato wart disease. The PolyChrome system, is comprisied of two command-line pipelines (PCC and PCD), an integrated state-of-the-art bioinformatics software and a high-quality genomic reference database. The analysis system allows for timely and accurate detection and identification of high-risk pathogens at the species/subspecies levels. The Clasnip platform is a web-based platform to quickly classify pathogens and their closely-relatives based on SNPs and/or whole-genome sequences. It was developed to allow users with minimum bioinformatics background to compare SNPs with curated, high-quality reference databases. Clasnip can accurately identify CLso haplotypes based on specific genetic signatures in seconds, and is also available for identifying bacterial ring rot pathogen from its close relatives of Clavibacter spp, differentiating different species of soft rot and blackleg bacteria, and different phylogroups of Potato Virus Y (PVY).

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The emergence of new and re-emergence of extant plant pathogens can lead to severe disease outbreaks in situations such as nurseries. Rapid responses to limit pathogen spread require quick and robust detection methods coupled to timely and appropriate management strategies. Whole-genome based diagnoses allow for fine-scale resolution and transmission-chain tracking, with potential to dramatically reduce the response time to an outbreak. However, the availability of infrastructure, tools, and expertise have delayed adoption in many diagnostic settings. To address this gap, we are developing an automated pipeline that processes raw whole genome sequencing reads, performs genome assembly and annotation, calls variants, generates a core genome phylogeny, and mines genomes for features of interest such as those implicated in virulence, antibiotic resistance, and management. We are using the Nextflow platform to develop this automated and reproducible bioinformatic pipeline. As a proof-of-concept, we applied the pipeline to analyze strains of the geranium pathogen Xanthomonas hortorum pv. pelargonii reportedly implicated in a common source outbreak that spread US wide within the nursery industry in 2022. This new bioinformatic pipeline is expected to support surveillance efforts and improve early detection as well as reduce response times to accelerate deployment of adequate management strategies against plant pathogens that afflict agriculturally important crops.

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Root-knot nematodes (Meloidogyne spp.) have evolved infection mechanisms that involve the secretion of effector proteins into host plants to suppress immune responses and facilitate parasitism in a wide range of land plants. Effector genes are, therefore, attractive targets for the genetic improvement of plant resistance to M. incognita. In this study, RNAi-mediated gene silencing of the Minc03328 and Minc16803 parasitism genes was used to generate transgenic Arabidopsis plants. We designed a T-DNA construct with the full-length regulatory region of the soybean E2 ubiquitin-conjugation promoter that modulates hairpin-type dsRNA expression in the nuclear genome of Arabidopsis thaliana. Data showed that transgenic Arabidopsis expressing the dsRNA-targeting Minc03328 and Minc16803 exhibited significantly increased resistance to nematode infection. Gall numbers and egg masses were reduced by up to 81% and 93%, respectively, in the dsRNA-Minc03328 transgenic lines, whereas the dsRNA-targeting Minc16803 showed 76% and 87% reduction in the same parameters. Interestingly, histopathological analyses of M. incognita-induced galls strongly suggest that both genes may play an important role during the early parasitism stages, encompassing amorphous giant cells with lower cytoplasmic content in the transgenic lines, besides a hallmarked effect on the nematode cuticle, reinforcing their potential as a promising specific target for application in modern crop protection development.
 

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Pathogen attacks elicit dynamic and widespread molecular responses in plants. While our understanding of plant responses has advanced considerably, little is known of the responses in the asymptomatic “green” regions (AGR) adjoining lesions. Here, we explore spatiotemporal transcriptome data and elemental maps to report the changes in the AGR of two wheat cultivars infected with a necrotrophic fungal pathogen, Pyrenophora tritici-repentis. We show, for the first time, that calcium oscillations are modified in the susceptible cultivar, resulting in possible “frozen” host defence signals at the mature disease stage, and silencing of the host’s recognition and defence mechanisms which would otherwise protect it from further attacks. In contrast, Ca accumulation and heightened defence response was observed in the moderately resistant cultivar. Other findings include the inability of the AGR of the susceptible wheat to recover post disease disruption and expression of eight predicted pathogen proteinaceous effectors. The targeted tissue sampling for gene expression analysis resulted in a unique dataset which creates capability to study plant-pathogen interactions at a higher resolution compared to classic bulk leaf tissue analysis. Collectively, our results highlight the benefits of spatially resolved molecular analysis in providing high-resolution spatiotemporal snapshots of host-pathogen interactions, paving the way for detangling complex disease interactions in crop plants.

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Frankliniella occidentalis (WFT) and Thrips tabaci (OT) are insect species that greatly impact horticultural crops through their transmission of tomato spotted wilt virus (TSWV) and iris yellow spot virus, members of the family Tospoviridae in the Bunyavirales. We have identified 64 viral segments in samples from 12 populations of OT and WFT from Italy, corresponding to 41 viruses. Fifteen were assigned to WFT, and 17 to OT, while 9 viruses could not be assigned to any species based on our stringent criteria for host association. All these viruses are putative representatives of new species, and some are the type members of new higher-ranking taxa. Repeated sampling in a subset of locations, and further virus characterization in a subset of four populations, reared in laboratory on a controlled diet for more generations, provided evidence of a locally persistent thrips core virome that characterizes each population.
Two WFT populations differentially infected by a virgavirus, a mononegavirus and a densovirus were tested for their efficiency in transmitting TSWV; some preliminary results indicate that densovirus infection inversely correlates with TSWV accumulation and transmission. Furthermore, the only virus that persisted over one year in an OT population is a member of the family Mitoviridae, among the first viruses of this family associated with insects: here we present different approaches to determine its host, tissue and subcellular localization unequivocally.
 

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Blackleg causes high economic losses for the seed potato industry worldwide. The disease is caused by bacteria belonging to the genera Pectobacterium or Dickeya. Recent developments in sequencing technology led to refine their taxonomy. Since 2016, the number of described species has increased from 12 to 33, highlighting their great genetic diversity. To date, few data are available about their specific behavior on potato host.
In order to compare the aggressiveness of 5 different Pectobacterium and 2 Dickeya species, we inoculated the pathogens on tubers just before plantation in trial fields. Each inoculum consisted in a mix of 5 strains belonging to a same species. Then, different parameters reflecting the aggressiveness and fitness of the inoculated strains were observed, as blackleg expression or vertical transmission in harvested tubers.
The results showed differences between species for all the parameters studied, highlighting different colonization strategies on potato host. Moreover, the qPCR analysis of blackleg symptoms obtained after inoculation with a mix of strains belonging to two different species can reveal a possible antagonistic relation between pectinolytic bacteria species association. Finally, a metabarcoding sequencing approach performed to monitor each inoculated strain revealed the predominance of one strain in each analyzed symptom, not always the same, highlighting a pioneer effect during the symptom development.
 

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Bacteria are ubiquitous and occupy a very wide range of ecological niches where oxygen can be rapidly limited. In this case, bacteria develop flexible metabolic respiration. Therefore, demonstrating the possibility of using carbon sources as alternative terminal electron acceptors (TEAs) and elucidating the underlying genetic pathways has great potential to help understand bacterial strategies to adapt and persist in the environment. This question is more relevant in the case of phytopathogenic bacteria which face many specific challenges to infect plants. It is the case for Dickeya, an emergent pathogen, found in various ecological niches, such as plant apoplast, and responsible for soft rot disease in a wide variety of plants. Understanding anaerobic respiratory pathways involved in Dickeya persistence and colonization will allow us to better understand its pathogenicity, and thus better counter it. In this study, we demonstrated that D. dadantii is able to use malate and asparagine as TEA. By constructing metabolic model of D. dadantii, we predicted pathways involved in anaerobic growth using asparagine and malate. Mutants affected in asparagine pathway were constructed and we evidenced the role of asparagine respiration in D. dadantii virulence. We also tested whether asparagine pathway is conserved among Dickeya and Pectobacterium genus.This study demonstrated that anaerobic respiration is an important trait involved in D. dadantii virulence.

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Macadamia is an Australian native tree that is grown for its edible kernel in tropical and subtropical regions worldwide. Young plants infected by fungi causing stem and leaf pathogens in the nursery may cause disease problems in commercial macadamia orchards. To investigate, we used a culture dependent approach to examine the structure and diversity of the fungal community in macadamia nursery plants at four different growth stages. Fungi isolated from roots, stems, and leaves of germinated seeds,1-3 month-old seedlings, 12-month-old non-grafted plants and 18-month-old grafted plant stages were characterised. Twenty-two fungal genera, mostly in the phylum Ascomycota were identified from the various macadamia tissues. The fungal community structure was significantly (P <0.05) influenced by the growth stages and plant organs. Grafted plants had the richest fungal composition and diversity (21 fungal genera). Neopestalotiopsis, Alternaria, Collectotrichum and Neofusicoccum populations were more frequent in leaf tissues than other organs. In contrast, Diaporthe, Lasiodiplodia and Pestalotiopsis populations were dominant in stem tissue. This study revealed that fungal community richness and diversity in macadamia plants are dependent on the growth stage. Ongoing investigations would determine the pathogenicity of these fungal species in mature macadamia plantations.
Keywords: Fungal diversity, Endophyte, Ascomycota, Tree nut.

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The Japanese beetle (Popillia japonica) is a polyphagous insect listed as priority pest by the EU phytosanitary legislation. The beetle was first detected in Continental Europe in 2014, in the Italian regions of Piedmont and Lombardy. Since then, it has quickly invaded a large portion of Northwestern Italy and Southern Switzerland, despite the eradication effort of regional phytosanitary services. Furthermore, several interceptions of living adults have occurred in distant locations as a result of unintended passive transport of the beetle. Indeed, it is well established in the literature that Popillia japonica is capable of being dispersed over large distances via passive human transportation (hitchhiking behavior). In this work we analyzed how the invaded areas of Northern Italy and Switzerland are connected to the rest of Europe via three main transportation networks: planes, trains and trucks. We built reachability maps from the invaded zone highlighting sites that are likely to act as stepping stones or entry points for the beetle, both close and further away from the currently infested zone. Combined with a suitability map, this allows to prioritize sites for early-detection surveillance based on how likely they are to be reached, as well as their potential for being a future outbreak of infestation. This is crucial, as experiences in North America proved that early detection followed by effective eradication protocols can prevent the establishment of the beetle.

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Microbial biological control agents (MBCAs) are used as an alternative to synthetic chemistries and the application of MBCAs implies that these confront microbial communities, including plant pathogenic fungi. The competitions for ecological niches are decided by the toxicity of secondary metabolites (SMs) produced by the microbes. The SM compounds produced and secreted by a microbe are taken up and are further modified by another, creating a SM cocktail, which might be highly toxic and putatively have an adverse effect on human health.
In order to test the hypothesis that confrontations between pathogenic fungi and MBCA transcriptionally de-regulate SM gene clusters (SMGCs) and SM synthesis, we established confrontation experiments with maize fungus Colletotrichum graminicola and the MBCA Bacillus amyloliquefaciens. Microscopy showed that large hyphal swellings are formed in the vicinity of B. amyloliquefaciens. To elucidate the response of SMGCs, transcriptome studies were performed, and we observed differentially regulated genes belonging to PKS and NRPS clusters. Of the 42 SM clusters in Colletotrichum, more than 30 clusters harbor genes that were differentially regulated in confrontations. The metabolites extracted from the fungus-bacterium interface were analyzed by LC-MS/MS to reveal the chemical interaction and these studies showed that confrontations between microbes induce the production of a large number of chemistries belonging to various classes.
 

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Trichoderma spp. are ubiquitous soil fungi occurring worldwide. Due to their mycoparasitic and endophytic properties, Trichoderma species are used in agriculture as biocontrol agents. However, in 2018, a massive occurrence of T. afroharzianum on maize cobs was observed for the first time in Germany. Since then, Trichoderma ear rot has been observed at several locations in Germany, France, and Italy, especially in dry and hot seasons. Symptoms of Trichoderma ear rot consist in massive production of green to gray-green conidia on infected cobs leading to significant reduction of cob weight and quality as well as reduced germination rate and malformed seedlings. In addition, several Trichoderma strains used in approved biological fungicides and soil additives were pathogenic and caused heavy cob infection. In inoculation trials in the greenhouse with barley, rye, sorghum and wheat, T. afroharzianum caused visual symptoms of infection, such as browning and discoloration on the ears of wheat and barley, leading to high colonization rates and reduction of grain weight. Climate chamber experiments confirmed that growth rate and disease severity of pathogenic T. afroharzianum isolates is increased above 25°C and show a broader temperature optimum, especially in the high temperature range.
 

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The use of biological control agents, allow a more ecological management for plant diseases, using population ecology.  This study evaluates the effect of applications of biological agents, in dormancy of Vitis vinifera, aiming to evaluate the effect of formulations of Trichoderma spp, (Mamull ®) and Bacillus spps (Nacillus pro), during the pruning period, on the control of wood diseases (Diplodia spp) incidence and parasitism of pycnidia’s, incidence of powdery mildew (Erysiphe necator), as well as incidence of gray rot (Botrytis cinerea) and parasitism in sclerotia. A field trial was established, with random blocks, with 5 replications by treatments, Control, pruning paste and chemical fungicides and winter biological, to foliage 24 hours post pruning and pruning debris. The results showed a significant effect (P< 0,05) of the treatments, both chemical (3.5%), and biological (0.5%) showed reduction damage to wood with respect to control (15.8%), only the biological showed a parasitism of pycnidia 73.4%, in Botrytis the control showed an 82.5% incidence, chemical 3.5% and the winter biological of 56.5% and 60% of sclerotia parasitism. In oidium, control 100% of incidence in clusters and 74% in leaves in veraison, while the chemical 5.2% in bunches, 1.2% in leaves, the biological reached 45% in clusters and 34% in leaves. This study shows the possibility to use biological control agents, in dormancy to reduce inoculum with significative effect in full season diseases.

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Coffee is one of the most important economic crops. Bacteria have been considered as the most abundant inhabitants of the phyllosphere while it is unknow for Coffea arabica Catimor7963, one of the main coffee cultivars in China. The objectives of this study were to reveal the status of the bacterial communities in the phyllosphere of Catimor7963 through sequencing by the Illumina HiSeq2500 of the 16S rDNA V4-V5 regions across 6 representative sampling sites located in the main cropping areas of China, including YN_HGGL, YN_ZFXH, YN_PE, YN_WS of Yunnan and HN_WN, HN_FS of Hainan. To the best of our knowledge, the current study revealed the status of the bacteria communities in the phyllosphere of Catimor7963 for the first time. The dominant family was Chloroplast norank followed with Mitochondria. PCoA based on Bray-Curtis dissimilarities displayed the differences of the phyllosphere bacteria communities were positively correlated with the geographical distances. RDA analysis of the alpha diversity index with geographical parameters demonstrated that the diversity and abundance of the phyllosphere bacteria communities in high altitude and high latitude were relatively higher than those in low altitude and low latitude. We aim to gain a better understanding of the biocontrol resources suitable for realizing ecological cultivation under different agroecological systems. Moreover, the ecological cultivation management should be adapted to the local conditions.

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Potato scab is widely distributed in major potato producing areas in the world, and can be transmitted through the infested soil and seed potatoes. Potato scab mainly harms the underground part of potatoes, and due to the properties of the soil are complicated, the effect of chemical pesticides on controlling potato scab is not good. This study developed a biocontrol agent (BCA) that can control potato scab. The naturally and artificially infested seed potatoes were used for preliminary field experiments. Both naturally and artificially infested seed potatoes were cut into pieces, coated them with wettable powder formulation of BCA (50-fold diluted with diatomaceous earth), planted them in the field, and then drenched them with suspension concentrate formulation of BCA (400-fold diluted with water). The results showed that drenching the naturally and artificially potatoes with BCA could reduce the diseased area of potato scab by 33.57 and 62.72% as compared with the control group, respectively. If the naturally and artificially potatoes coated with BCA alone, the diseased area of potato scab could be reduced by 7.99 and 47.3% as compared with the control group, respectively. In the future, if healthy seed potatoes can be used in conjunction with field sanitation to avoid discarding previously diseased potatoes in the field, the success rate of biological control of potato scab can be further increased.

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Most plant parasitic nematodes attack the underground parts of plants, and their life cycle can be completed in the soil. Because of the complex properties of soil, chemical pesticides are not effective in controlling plant parasitic nematodes. This study developed a biocontrol agent (BCA) that can control root-knot nematode (Meloidogyne graminicola) of green onion. The result of first preliminary field trial showed that treatment of green onion seedlings with 200-fold diluted BCA can reduce the nodulation rate by 65.5% as compared to the control group. The result of the second preliminary field trial showed that treatment of green onion seedlings with 200-fold and 400-fold diluted BCA can reduce the nodulation rate by 48.36% and 45.95% as compared to the control group, respectively. In addition, the effect of BCA on controlling Meloidogyne incognita of cucumber was evaluated in the greenhouse. Cucumber seedlings treated with 200 and 400-fold diluted BCA can reduce the nodulation rate by 65.2% and 59.4% as compared to the control group, respectively. At present, there is no BCA registered for managing root-knot nematode in Taiwan. This study develops a BCA that is easy to mass-produce, has a long shelf life, and has a good effect on the control of root-knot nematodes. It will provide an alternative method to control the nematode diseases for organic and conventional farming in Taiwan.

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Global climate change affects plant growth deficiency and yield loss with abiotic stresses such as drought, salinity, greenhouse gases and extreme temperature. In this regard, it is necessary to develop biostimulants to enhance tolerance to abiotic stress, uptake nutrients and promote plant growth. The aim of this study is utilizing of plant growth promoting rhizosphere microorganism as microbial biostimulants. Bacillus megaterium GEB3 and GEB13 were isolated from the rhizosphere of ginseng and identified by sequencing 16S rRNA. GEB3 and GEB13 has been showed plant growth related activity such as nitrogen fixation, siderophore secretion, and indole-3-acetic acid production. In addition, rice seeds were treated with GEB3 and GEB13 culture solutions, and seed germination rates were measured by culturing the seeds under drought conditions of 0 Mpa, -0.15 Mpa, and -0.49 Mpa. In drought conditions, the seed germination rate was higher in the GEB3 and GEB13 treatment groups than in the microbial-free control group. In greenhouse, GEB3 and GEB13 were treated to rice seedlings under drought conditions. As a result, the chlorophyll content was 34.1 SPAD for GEB3 treatment and 33.4 SPAD for GEB13 treatment after 4weeks, which was significantly increased compared to the control value of 21.6 SPAD without microbial treatment. These results indicate that B. megaterium GEB3 and GEB13 can be used as biostimulants that promote plant growth and abiotic stress resistance in crop cultivation.

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Indigenous Trichoderma strains  were isolated and characterized. Potential strains of Trichoderma spp. effective against six soil-borne fungal phytopathogens identified through  a series of studies. The mode of action of Trichoderma spp. was studied against the  targeted phytopathogens. PGP, enzyme release activity, the ability for siderophore production, P and Zn solubilization, tolerance to Al and Fe toxicity, and compatibility with soil microbiome studies showed a positive result for better plant health management. Through a continuous effort of 15 years in different agroecological conditions of NER of India, standardized bio-intensive strategies and popularized among the farming communities. More than 5000 farmers, FPOs, extension personnel, and tea garden managers were trained on technical aspects of the technology and its field use. Technology adopters gained more profit due to the result of higher yield with nutritious farm produce.  The farmers could fetch more income compared to the non-adopters and bankability increases. The success opens a hope that the technology may create a revolution among the farming communities of the nearby areas. Moreover, the low-cost technology has opened up a new vista for plant disease management and is likely to be a boon for seed industries that would like to provide protection to seeds as well as plants against a large number of seed, soil-borne, and foliar diseases.

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Southern blight caused by Sclerotium rolfsii is a major fungal and soil-borne disease damaging the economic crops worldwide. The management of southern blight relies primarily on the strategic application of synthetic fungicides. However, chemical fungicides are responsible for environmental pollution, health hazards, pest resurgence, development of resistance in pathogens, destruction of non-target species, and deterioration of natural habitats. Here, endophytic Bacillus spp. were evaluated the efficacy on control of southern blight. Results revealed that R8-25, R8-43, and PS6-2 strains showed best ability against S. rolfsii isolates with 59.1% to 71.1% mycelia inhibition rate. Moreover, these three strains could produce volatile organic compounds (VOCs) to inhibit mycelia growth and sclerotia germination. Characteristics analyses indicated that the three strains have abilities secrete amylase, cellulase, gelatinase, protease, iturin A, bacillaene and IAA. For the seed germination and plant-growth-promoting tests, the strains R8-25 and R8-43 have the best efficacy to induce seed germination and promote bell/chili pepper growth in greenhouse. In the control test, single strains or mixture of R8-25 and R8-43 could reduce the severity of southern blight in bell pepper based on seed coating combined with drenching method in greenhouse, especially two strains mixture. According to these results, the two strains have the potential to be agents on control of southern blight.

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During 2017-18 and 2019-20 surveys were conducted in 12 strawberry-producing districts of Pakistan. Out of 12, 08 districts of Punjab province, 03 of Khyber Pukhtunkhwa province and Islamabad for disease assessment and samples collection. Mean % disease prevalence and incidence ranged from 0-100% and 5-8% for dieback whereas, 0-100% and 0-16% for black root rot (BRR) respectively. Dieback symptoms appeared as wilting and dieback with collapsing of the entire plant. The BRR appeared as black discolorations on entire root and plant look stunted and collapsed. Purified cultures were identified on morpho-molecular basis. Total of 30 Lasiodiplodia theobromae (dieback) and 47 of Rhizoctonia solani (21), Macrophomina phaseolina (11) & Fusarium solani (15)  isolates of (BRR) were studied. Pathogens were by culturally & morphologically identified. For molecular studies, nucleotide sequencing of 20 highly pathogenic isolates (5 each of L. theobromae,  R. solani, M. phaseolina & F. solani were done by ITS, TEF1-α & EndoPG primers & phylogenetically analyzed. This is 1st detailed study of these diseases in Pakistan. For biological control 30 native bacterial isolates were processed of which 3 isolates viz. Bacillus subtilis (1) and Pseudomonas fluorescens (2) showed the highest antagonistic effectiveness (>70%) against the dieback and BRR pathogens during bioassays. The selected bacterial isolates were further tested in the greenhouse and also showed promising disease control of 21-57%

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The study was carried out to isolate and identify several fungi from Koya  soil.  (48) soil samples were collected from four different locations in Koya district. 6 fungal genera which include: P. spp., Asp. spp., Rh. spp., Aur. spp., Cl. spp. and Dip. spp. were isolated by used SDA, PDA, and MEA. Serial dilution  plate method was used for isolation of soil fungi.  the  results, reveal that the most predominant genera and highest number of colonies  were Pen. and Asp. sp..  the result showed that the  of occurrence of Asp. and Pen. were very high among the fungal isolates . Different species of Pen. obtained from soil samples especially from Shewashok soil. The P. coryl had significant antagonistic activity against five out of six (G-) and (G+) pathogenic ATCC . P. coryl. from Shewashok location was the best isolate for antibacterial production and it had more antagonistic activity against pathogenic bacteria than the other Pen.spp.  this isolate was used for antibacterial production. Ethanol solvent was used for the extraction of the antibacterial substances form fermented P. corylm.  The crude extract was dried in rotary evaporator , centrifuge at 35?C.  The antibacterial obtained was highly effective against Y. enterocolitica and S. aureus. (GC-MS) was used for analysis of the fungal extract obtained from P. coryl. The result had determined thirteen compounds from crude extract . Some of these compounds have antibacterial activity against some pathogenic microbes .

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Fusarium wilt of banana is becoming a serious challenge to the banana industry globally. Biological control is one of the most effective measures for this disease. In order to explore the biocontrol effects of a biological agent Serenade on banana plants, two different cultivars ‘Brazilian’ and ‘Yunjiao No.1’ were used in greenhouse pot experiments. Results showed that the plant height and pseudostem diameter of banana susceptible cultivar Brazilian increased by 11.68% and 11.94% respectively after Serenade application, while the plant height and pseudostem diameter of resistant cultivar Yunjiao No.1 increased by 14.87% and 12.51% respectively. The fresh weight of two cultivars increased by 20.66% and 36.68% respectively, these indicating that Serenade has positive effects on plant growth promotion. TR4 infection and Serenade application changed the bacterial community composition of two banana cultivars, and the fungal community composition of Yunjiao No.1 also changed significantly. Correlation analysis showed that the relative abundance of Bacillus and Pseudomonas in the rhizosphere of both cultivars increased significantly after Serenade application, which had significant positive correlation with plant height, psedustem girth, above ground fresh weight, leaf length and leaf width. Therefore, the outcome of this study suggests that Serenade could be used in banana field application for promoting plant growth and modification of soil microbial communities.

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?Coffee leaf rust (CLR) as one of the most devastated diseases of Coffea arabica, caused by the obligate fungal parasite Hemileia vastatrix (Hv). Mycoparasites frequently follow Hv in the field, interfering with the reproductive structures to stop further growth and dissemination of Hv, to offer a chance to potentially limit CLR. Here, we applied high throughput sequencing of the rDNA ITS1-ITS2 regions of coffee phyllosphere microbiota with 3 leaves symptoms (C, HV, PHV) across 6 sampling sites located in China coffee regions. Simplicillium was evaluated as the dominated mycoparasites of Hv in all the investigated populations through a series approach of metabarcoding analysis, revealing genetic diversity of associating with the geographic differences. At the 0.97 clustered threshold, 22 OTUs were 98-100% homologous to Simplicillium spp., of which, 11OTUs identified as S. lanosoniveum, 6 OTUs identified as S. subtropicum, others identified as S. lamellicola, S. obclavatum and Simplicillium sp. These sequences had 68 segregating sites and Pi of 0.05305. The MJ haplotype networks were built from a total of 17 haplotypes produced by 22 OTUs of Simplicillium spp. while divided into 2 groups, demonstrating great diversification with 0.996 Hd and 0.00023 VarHd and displaying genetic divergence with clear geographic patterns and leaf symptom selectivity. All in all, the current study is helpful for developing management biocontrol measures against CLR.

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Pantoea stewartii subsp. stewartii is the causative agent of "Jackfuit Bronzing," an emerging disease in jackfruit crops (Artocarpus heterophyllus L.). The disease was first discovered in Malaysia in 2017, affecting the J33 variety, Tekam Yellow. This disease degrades the quality of fresh jackfruit, leading to economic losses, and has remained a serious problem for the Malaysian jackfruit trade. In this study, 58 lactic acid bacteria (LAB) isolated from fruits and vegetables were screened and characterized for their antagonistic potential against P. stewartii subsp. stewartii. Fourteen cell-free supernatants (CFS) of the LAB isolates were found significantly inhibit the growth of P. stewartii subsp. stewartii in vitro (P<0.05). They were morphologically, biochemically, and genetically identified and based on the 16S rDNA sequencing analysis, the 6 LAB isolates showing the greatest antagonism are Lactiplantibacillus pentosus, Lactiplantibacillus argentoratensis, Leuconostoc holzapfelii, Weisella cibaria and Weisella paramesenteroides. CFS of the 6 potential LABs was extracted using ethyl acetate, diethyl ether, dichloromethane, and n-hexane solvents to examine their bioactive metabolites qualitatively and quantitatively. Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) identification of the purified bioactive metabolites from the extract that possessed the greatest effect against P. stewartii subsp. stewartii will be presented.

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Pepper mild mottle virus (PMMoV), a member of genus Tobamovirus, is the major viral pathogen of peppers (Capsicum annuum L.) in Taiwan.  Symptoms caused by PMMoV include various degrees of mottling and curling of leaves, distortion of fruit and dwarfing.  PMMoV is a rigid rod shaped virus that can be easily transmitted via mechanical inoculation when primary inoculum was present.  The primary inoculation of PMMoV is difficult to prevent because it often comes from contaminated cultivation media or seeds.  In our study, Bacillus velezensis was tested as a biocontrol agent for inducing systemic resistance of sweet pepper against PMMoV under nethouse conditions. Foliar application and soil irrigation of B. velezensis was performed once a week when the seedlings germinated.  Treatment and control sweet pepper plants were inoculated with PMMoV after 2 treatments. For challenge inoculation, diseased sweet pepper leaves were ground into 50 times (W/V) phosphate buffer, and the ground juice was mechanically inoculated on sweet pepper leaves.  The virus infection and virus concentration were detected by ELISA. All control plants were infected with PMMoV, and only 40% plants in the B. velezensis treatment were infected by the virus, and the virus concentration was lower than control plants.  In our results on sweet pepper, B. velezensis can be used as a biocontrol agent for induction of resistance against PMMoV or reduction of symptoms caused by this virus.

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Fire blight is a dangerous disease of fruit crops and causes enormous economic damage to fruit growing. The bacterium Erwinia amylovora, the causative agent of the disease, belongs to quarantine objects.
In order to reduce the spread of morbidity, screening of microorganisms isolated from garden cenosis of various regions of Kazakhstan was carried out against the causative agent of bacterial burn of fruit crops.
It was established that the MB-40 and 17M isolates showed the highest antagonistic activity against E. amylovora (the zones of pathogen growth inhibition were 48 mm and 30 mm, respectively).
It was determined that acetoin and 2,3-butanedione produced by B. amyloliquefaciens MB-40, as well as lactic and acetic acids produced by L. plantarum 17, are the components responsible for the inhibitory activity against E. amylovora.
It has been shown that double application of the culture broth of strains B. amyloleguefaciens MB-40 and L. plantarum 17M, containing inhibitory components, protects fruit trees from bacterial burn by 70%.
The study of the virulence properties of the B. amyloliguefaciens MB-40 and L. plantarum 17 M strains showed that they are not pathogenic for warm-blooded organisms and can be used as the basis of biological preparations against bacterial burn of fruit crops.

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Modern European agriculture increasingly relies on organic farming methods and ecological approaches, or conventional pesticides are partially replaced with biocontrol agents such Aureobasidium pullulans. Some strains of A. pullulans produce aureobasidin A, a cyclic antifungal peptide, catalyzed by an amino acid complex encoded by the aba1 gene. The aim of this field experiment, conducted in 2021 and 2022 in north-eastern Poland, was to select A. pullulans strains with the use of molecular markers linked to the aba1 gene, and to analyze the inhibitory effect of selected strains on trichothecenes concentrations in wheat grain. In 2021, all biological treatments reduced the severity of Fusarium head blight (FHB), and their effectiveness reached 62.6% (Ap CC2) and 57.1% (Ap 15). Wheat grain was contaminated with type-B trichothecenes: deoxynivalenol (DON), Fus-X, 3ADON, 15ADON, and nivalenol (NIV), and type-A trichothecenes: STO, HT-2 toxin, T-2 toxin tetraol, T-2 toxin triol, and diacetoxyscirpenol (DAS). Biological treatments involving a cell suspension of A. pullulans strain Ap 15 decreased the NIV content of grain from 11 mg/kg to 8 mg/kg in the first year of the study, and completely eliminated this mycotoxin from grain in the second year. When FHB severity was low, a A. pullulans treatments did not decrease DON concentrations in grain, although it reduced disease symptoms and the abundance of selected Fusarium species.
 

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ε-poly-L-lysine (ε-PL) produced by microorganisms has broad antimicrobial spectrum and good stability. miRNAs play an important role in various plant processes such as growth and development, defense stress and disease resistance. To investigate the antiviral mechanism of action of ε-PL in plants, we analyzed the expression profile of microRNA (miRNA) in tobacco mosaic virus (TMV)-infected Nicotiana tabacum after ε-PL treatment. The results showed that expression levels of 328 miRNAs were significantly altered by ε-PL. Some miRNAs were screened, and joint network analysis was performed on their target genes and gene-enriched GO/KEGG pathways. The results indicated that ε-PL regulates expression of miRNAs involved in pathways such as plant hormone signal transduction, host defense response and plant pathogen interaction, such as nta-miR6146, nta-miR1446 and nat-miR319a. Subsequently, TRV-VIGS gene silencing method combined with the short tandem targets mimic technology were used to functionally analyze these miRNAs and their target genes. The results of northern blot and RT-qPCR showed that the accumulation of TMV in N. benthamiana were increased after silencing miR1446 and miR6146. Meanwhile, the silencing of miR172, miR164, and miR319 reduced the content of TMV, indicating that these miRNAs may play diverse roles during ε-PL- mediated antiviral responses. Collectively, these results provide theoretical basis for further elucidating antiviral mechanisms of ε-PL.

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Soil-borne pathogens pose a significant threat to crops globally, leading to significant economic losses. These pathogens can survive without a host by producing resting structures that are resistant to environmental stress and pesticides. Pesticides are toxic and their accumulation in soil and plants is dangerous to the environment and humans. Hence, researchers are focused on developing sustainable solutions such as biocontrol agents (BCA). However, the complex interactions between BCAs and the environment pose a challenge in developing effective solutions. Biological soil crusts (BSCs) are the natural cover of many arid and semi-arid lands. The microorganisms inhibiting BSCs developed unique mechanisms to survive harsh environmental conditions such as desiccation, heat, and radiation. Green algae isolated from desert crusts show a remarkable ability to withstand harsh environmental conditions. Surprisingly, it is also one of the fastest-growing phototrophs known. Preliminary results show that co-culturing this alga significantly inhibits the growth as well as the production and viability of resting structure in various soil-borne pathogenic fungi, including Verticillium dahliae and Rhizoctonia solani. The combination of fast-growing (i), resilience (ii), and the observed antifungal activity (iii) make it a promising candidate for the development of a BCA against soil-borne pathogenic fungi, specifically for arid and semi-arid regions and in the face of climate change. 
 

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Lettuce is a popular vegetable crop worldwide, accounting for a production value of more than US billion in 2020. It is threatened by several bacterial pathogens, including Xanthomonas hortorum pv. vitians, Pseudomonas cichorii, and Pectobacterium carotovorum. These pathogens cause bacterial leaf spot, varnish spot, and bacterial soft rot, respectively. Control methods are limited and often ineffective. It is therefore important to develop and implement novel, effective, durable, and low environmental impact control methods, such as biocontrol. With this in mind, we screened a collection of 1,200 Pseudomonas strains for their ability to inhibit the growth of the three pathogens under study. In total, 35 effective antagonistic Pseudomonas strains where identified. Their genomes were fully sequenced and annotated, revealing their phylogenetic affiliation and potential genetic determinants involved in their antagonistic activity. These Pseudomonas strains belong to 27 different species distributed among the P. fluorescens and the P. putida phylogenomic groups. Only 15 of these species have been described to date. The presence of genes involved in microbial competition and antibiosis, including the biosynthesis of pyochelin, type VI secretion systems, tailocins, and hydrogen cyanide, correlated with their inhibition abilities. Some of these strains show promise for the development of efficient biocontrol products against lettuce bacterial diseases.

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Soil salinity is a prevalent environmental stress in agricultural production. Microbial inoculants could effectively help plants to alleviate salt stress. However, there is little knowledge of the Pseudomonas alcaliphila  Ej2 mechanisms aiding rice plants to reduce the adverse effects caused by salt stress. We performed integrated field and greenhouse experiments, microbial community profiling, and rice proteomic analysis to systematically investigate the Ej2 mechanism of action.  We found an increase in shoot/root lengths and fresh/dry weight in inoculated plants under salt stress. In the meantime, the strain Ej2 has important roles in controlling diseases and promoting rice growth. Furthermore, the alpha diversity of Ej2-inoculated plants was higher than the control plants, expect the Shannon index of the bacterial microbiome. The Ej2 inoculated samples clustered and separated from the control samples based on beta diversity analysis. Importantly, the enriched and specific OTUs after Ej2 inoculation at the genus level were Streptomyces, Pseudomonas, Flavobacterium, and Bacillus. Moreover, we observed that Ej2 inoculation influenced the rice proteomic profile, including metabolism, plant-pathogen interactions, and biosynthesis of unsaturated fatty acids. These results provide comprehensive evidence that Ej2 inoculation affects the rice endophytic microbiome and proteomic profiles under salt stress. 

 

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Potential of consortial application of bioagents against fungal, bacterial and nematodes can be utilized to fullest extent by selecting most potential strains and not by arbitrary use of consortia. Cumin (Cuminum cyminum L.) is an important seed spices crop in India is majorly infected by Fusarium oxysoprum f. sp. cumini (Fusarium wilt) and aphid (Myzus persicae (Sulzer)).The multi-location field trials were conducted at Zone III A semi-arid eastern plain zone (Jaipur- Jobner) and zone IVB southern humid zone (Banswara)to evaluate the efficacy biocontrol agents and insecticide against major pests of cumin (Variety: RZ19) at two different agroclimatic conditions for the years of 2017, 2018 and 2019 rabi seasons. The combined application of biocontrol agents for the management of wilt of cumin by soil application with T. harzianum Th3 and M. anisopliae Ma1 enriched FYM (1:20).  Seed treatment with Th3 and Ma1 @ 8g/kg seeds  also drenching at 30 and 60 days after sowing and three foliar spray with Flonicamid at 0.015 % clearly shows the maximum reduction of disease (62.16%) compared to control and also maximum yield of 570.94 kg/ha at Jaipur agroclimatic conditions. The maximum disease reduction of 60.22 per cent and increased yield up to 541.50 kg/ha was observed at Banswara agroclimatic conditions. Based on the bliss independence hypothesis, the synergy factor was >1 (1.01) which demonstrated the interaction was synergistic in both pests of cumin.
 

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Cucumis sativus L. is one of the most important vegetables worldwide. Cucumber anthracnose caused by Colletotrichum orbiculare can incite anthracnose symptoms on leaves, stems and fruits causing serious yield loss. Thrips palmi not only causes damage by sucking, but also transmits Tospovirus virus diseases. Here, we aimed to evaluate the potential of Bacillus subtilis for controlling cucumber anthracnose and thrip’s population, and to reveal potential mechanism in plant health management. B. subtilis WMA1 and B. subtilis 151B1 were isolated in native Taiwan. Both strains showed antagonistic activity to C. orbiculare COC3 by dual culture assay. Application of culture broth of either B. subtilis WMA1 or B. subtilis 151B1 exhibited preventive and curative efficacy against cucumber anthracnose. Culture filtrates of B. subtilis WMA1 and B. subtilis 151B1 inhibited conidial germination of C. orbiculare COC3 to 26.7 and 22%, respectively, compared to 70.7% of the SYB medium control. Additionally, both Bacillus strains promoted the growth of cucumber seedlings, inhibited the mycelium growth of C. orbiculare COC3, and reduced oviposition and modulated the development and reproduction of thrips, which may be due to the volatile compounds produced by B. subtilis WMA1 and B. subtilis 151B1. Our findings suggested that B. subtilis strains WMA1 and 151B1 exhibited potential for the control of cucumber anthracnose, and significantly reduced the fecundity and development rate of thrips.

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During 2006 to 2007, cucumber (Cucumis sativus L.) was infected by melon yellow spot virus (MYSV) with symptoms of mosaic, yellow and necrotic spots on the leaves, and transmited by melon thrips. So, the viral disease control mainly on preventing vector by insecticides. On this study, we develop a system to screen induced resistance microorganisms for cucumber virus disease control. Among 130 microorganisms, 23 exhibited good proteolytic activity with cleared more than >2.0 cm zone on milk agar plate. Bioassays of induce resistance to viral disease was conducted on indicator host quinoa (Chenopodium quinoa) and zucchini yellow mosaic virus (ZYMV). After spreading the lower five leaves with the culture filtrate of the above-mentioned microorganisms for 4 hours, the upper five leaves were inoculated with freshly prepared ZYMV inoculum. The local lesions were account at 7 days post inoculation (dpi). Results showed that Bacillus velezensis (B34) and Bacillus spp. (FYC14) is effective in inducing plant resistance. In vivo screening of endophytic, B. velezensis was selection for MYSV control in screenhouse cucumber. The results showed an approximately 47% reduction in disease incidence compared to the control after 4 weeks of planting and a 22% increase in total cucumber yield. In summary, B. velezensis is effective control with ability to cleave proteins, induce plant resistance, promote plant growth and endogenously. So far, the molecular mechanism is still under investigation.
 

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The present investigations were carried out to assess the interaction of rhizobacteria and target pathogen in soybean ecosystem at Molecular Genetics Lab,UAS,Dharwad to gain the insights into molecular basis of host-pathogen interaction during 2022. Defence related genes were analysed for their expression levels in response to pathogen (Sclerotium rolfsii Sacc.) and rhizobacterial isolates (AUUB209 Streptomyces enissocaesilis and AUDT626    (Streptomyces racemochromogenes) through quantitative real time polymerase chain reaction. A total of five defence related genes viz., Pathogenesis related protein 1 (PR 1), Pathogenesis related protein 2 (PR 2),Pathogenesis related protein 2 (PR 10), Polyphenol oxidase (PPO) and Chalcone synthase (CHS) were selected and analysed for their expression levels under different treatments where rhizobacteria were applied as seed treatment (10g/kg seeds) and drenching at 35-40 days after sowing. The results revealed that the highest expression levels of PR 1 (2.75 fold),PR 2 (7.88 fold), PR 10 (4.16 fold) and PPO (8.50 fold) observed in the treatment (Host + Pathogen + AUUB 209 + AUDT 626) and the highest (3.27 fold) CHS gene expression was recorded in the treatment (Host + AUDT 626). The positive check (Host + Pathogen + Trichoderma harzianum) recorded 2.42, 5.74, 4.15, 6.33 and 1.34 fold change of PR 1, PR 2, PR 10, PPO and CHS genes respectively.This is the first report on rhizobacteria and root rot pathogen interaction in soybean in India.

 
 

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The tomato is the most consumed fruit in France and in the world. Among the different diseases affecting tomatoes and potatoes, the late blight caused by the oomycete, Phytophthora infestans, is one of the most devastating pathogens. The oomycete causes significant yield losses, involving the repeated use of phytopharmaceutical products harmful to the environment and public health. In order to reduce the use of pesticides, the use of biocontrol agents is one of the possible alternatives for crop protection.  
In this context, my research project aims to characterize the mode of action of a biocontrol product (GA342) developed by the company Gaïago in order to improve its effectiveness against the late blight, using the pathosystem Phytophthora infestans (A36_A2 strain) - Micro-Tom, model cultivar for tomato. Preliminary results showed that 1) GA342 had a direct effect on the oomycete, decelerating or inhibiting totally the mycelial growth, in a dose dependent manner; 2) in planta, the treatment with GA342, prior to the infection, showed to reduced significantly the symptoms. The results of further studies will be presented on the direct effect of GA342 and more particularly on sporangia, zoospores and mycelia. In addition, targeted transcriptomic and biochemistry approaches in planta will be shown. 
The promising results related to the action mode of GA342 will allow us to optimize the efficiency of the product for field application.

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The negative impact of chemical inputs used in agriculture on the environment and human health calls for the development of more sustainable agricultural managements. Recent studies have highlighted that host-associated microbial diversity has an effect on plant resistance to biotic stresses. Biological processes related to this adaptation include microbiota-pest interactions (competition, antagonism, parasitism) and/or modulation of plant immunity. The objective of the presented work is to use microbiota-pest interactions to estimate Synthetic Microbial Communities (SynComs) composed by bacterial and fungal strains with protective effects on crops against a pathogen. The study focuses on two different pathosystems: Rhizoctonia solanii infecting Brassica napus and Fusarium graminearum infecting Triticum aestivum. During this congress, I propose to present the first results obtained during my PhD program. A microbial collection has been established and characterized to identify the microbiota of the two crops grew under contrasted agronomic and pedoclimatic conditions in France. The first SynComs could be formed quickly to be able to do in vitro tests on B. napus in the presence of R. solanii. These results could be presented at the congress in August.

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Phenolic substances are one of the important secondary metabolites of plants, which are of great significance in the resistance to the infection of pathogens and metabolic regulation. The early symptoms of poplar anthracnose are the oxidative allergic reactions of phenolic compounds, indicating that phenolic compounds play an important role in the occurrence and development of poplar anthracnose. Therefore, we determined the concentrations of phenolic compounds and their antifungal activities in three poplar species. The results showed that: (1) Three poplar species showed different resistance to poplar anthracnose: P. canadensis was resistant, P. tomentosa was susceptible, and P. beijingensis showed intermediate resistance; (2)This study selected 11 phenolic secondary metabolites with large difference and high content from three poplar species before and after inoculation. The results showed that these 11 substances showed different degrees of effects. Most of the substances showed the lowest and highest concentrations to inhibit the growth of pathogen, while the intermediate concentrations promoted the growth of pathogen; (3) The kinds and content of phenolic substances in P. tomentosa  are low, but P. tomentosa show resistance in the field. Therefore, we believe that phenolic substances do not play a major role in the interaction between P. tomentosa  and Colletotrichum gloeosporum, but other structures, such as waxy layers, are important in antifungal activities.

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Sweet acacia (Vachellia farnesiana) is a woody weed in Australia requiring Integrated Pest Management. Prospective biocontrol agents were sought among diseased plants from north and central Queensland. Symptoms were documented, samples collected, and stems were prepared for isolation using ½ strength Potato Dextrose Agar. Internal Transcribed Spacer sequencing and phylogenetic analysis revealed 53 fungal strains, several of which are undergoing formal description. Several isolates were also pathogenic on sweet acacia seedlings and were selected for use in a field study aimed to investigate the interaction between the fungi and a stress enhancer, glyphosate. The fungal inoculum was encapsulated and implanted into stems, while several were encapsulated with a sub-lethal dose of glyphosate. Untreated control and encapsulated glyphosate applications were included for comparison. All fungus-only and fungus+glyphosate treatments displayed significant stem lesions compared to control and glyphosate-only treatments. However, the canopy recovered and it is concluded that glyphosate addition did not increase fungal infection of sweet acacia within the year-long trial period. Investigation of these treatments is still ongoing to evaluate their long-term effect on sweet acacia. Further study on incorporating these isolates and investigating their pathogenicity as a potential biocontrol agent is also ongoing, particularly for the long-term management of sweet acacia.

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Grapevine trunk diseases (GTD) have become a major concern in viticulture. Since the ban in 2001 of sodium arsenate, the use of alternative methods, such as biocontrol, has become a major issue. Among the promising microorganisms, the oomycete Pythium oligandrum is known to improve the plants health by increasing their natural defenses and reducing diseases to up than 40%.
The BIOBESTicide project, funded by the European Commission, aims to industrialize the production of a biopesticide solution to fight GTD. Efficiency evaluation of the product formulated from P. oligandrum will be carried out, and the environmental impact of this solution will be assessed.
Thus, an experiment was carried out in greenhouse for assessing the impact of the biopesticide on microbial communities by a high-throughput sequencing approach. Vines were treated with a P. oligandrum formulation and were inoculated with a fungus involved in Esca disease: Phaeomoniella chlamydospora. A three-month follow-up was carried out with samples from wood and rhizosphere environment to allow the evaluation of potential changes on microbial communities, whether as part of grapevine trunk disease or after the action of P. oligandrum.
The results obtained revealed that the biopesticide acts efficacy against P. chlamydospora with a reduction in the size of induced necrosis. Moreover, it had few effect on the rhizosphere microbial communities, which may suggest that the biopesticide is environmentally safe.

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Fusarium Head Blight is a devasting disease in cereals caused mainly by Fusarium spp. (Fspp). Despite being primary inoculum sources, soil and crop residues have been less studied than grains. Knowledge of the diversity and dynamics of microbiota and Fspp populations in these compartments is relevant to elaborate efficient biocontrol strategies. Six min-till wheat fields were thus monitored for two years, with soil, maize residues, and wheat grains collected at four stages, before metabarcoding using 16S, ITS2, EF1α markers, and qPCR using Fspp-specific primers. F. graminearum (Fg) and F. avenaceum were dominant in both grains and residues. Despite similar Fspp loads in residues in both years, grains of 2021 were more severely infected than in 2022, most probably because of less conducive conditions (drier and hotter) at flowering for Fspp. Following metabarcoding, co-occurrence network analyses revealed significant negative correlations between Fg and Epicoccum nigrum as well as Sphingomonas sp.. In parallel, a collection of 1670 bacterial and fungal isolates from collected samples was built using two methods (culture on classical media or after confrontation with Fg using the double layer method). High throughput screening of their anti-Fg activities on wheat grain-based medium, followed by a taxonomic identification of positive isolates, is in progress and shows a high prevalence of Trichoderma spp. in soil, while Epicoccum spp. have also been found but to a lesser extent.

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Ralstonia solanacearum causes disease in more than 200 plant species including bacterial wilt of tomatoes and brown rot of potatoes. This bacterium is part of the EPPO A2 list of quarantine pathogens due to its soilborne and waterborne nature, worldwide distribution and lack of effective control measures. The use of bacteriophages as biocontrol agents is promising, as they exhibit high specificity to individual bacterial species, and do not affect eukaryotic cells. However, one aspect of the use of phage should not be underestimated: the quick ability of the bacteria to become resistant to the phage. This often leads to a trade-off, whereby in becoming resistant, bacteria lose virulence. This may be exploited to reduce the overall pathogenicity of Ralstonia populations, and can also help us to better understand plant-pathogen interactions. We have successfully identified phages which are effective at controlling R. solanacearum, and are now focused on understanding the transcriptome response of a susceptible tomato cultivar over time when inoculated with either a virulent ancestral strain, in-vitro evolved strains (non-virulent), or an ancestral strain plus phage treatment, which models co-evolution in the soil. The transcriptomic analysis reveals that there are clear differences in the immune response of the plant in each of the treatments. This work sheds light on the key genes activated in susceptible plants when phage resistance mutations occur in the bacteria.

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Fungicides are critical for modern grapevine (Vitis vinifera) production, but overuse has undeniable financial, environmental, and pathogen resistance consequences. This has led to a growing interest in biopesticides; however, it is yet unclear how best to integrate them into management programs. We conducted a metastudy of 7 years (2016-2022) of trial reports from the Cornell Pathology Vineyards (Chardonnay & Chancellor) in Geneva, NY to evaluate LifeGard® WG (Bacillus mycoides isolate J; BmJ) efficacy against grapevine downy mildew (Plasmopara viticola) and powdery mildew (Erysiphe necator). Disease incidence and severity was measured on leaves and clusters using the Horsfall-Barratt scale at harvest. We found that treatments containing LifeGard (e.g. alone, tank mix, or rotation), provided significantly greater protection compared to untreated control for both diseases. Generally, LifeGard provided better cluster control than foliar control. As expected, conventional materials provided more control than LifeGard when used alone, however, LifeGard provided exceptional control, beyond the commercial standards, when used in rotation. Overall, we find that integrating LifeGard into vineyard disease management can reduce conventional chemistry use while maintaining effective control. From these results, we speculate that increased biopesticide adoption can lengthen the useful life of highly effective yet resistance-prone fungicides by reducing resistance development pressure.

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Reducing the use of synthetic fungicides and implementing environmentally friendly alternatives are pivotal to achieving sustainable agriculture. However, the impact of these environmentally friendly fungicides on plant microbiomes has received limited attention. This study compared the effectiveness of two environmentally friendly fungicides (neutralized phosphorous acid (NPA) and sulfur) and one synthetic fungicide (tebuconazole) in controlling powdery mildew in cucumber. The differences in the phyllosphere microbiome are analyzed using high-throughput amplicon sequencing methods. The results showed that all three fungicides significantly reduced disease severity and the incidence of powdery mildew. However, while the α-diversity showed no significant differences of the phyllosphere microbial communities among treatments, tebuconazole had a significantly impact on the fungal community, as revealed by β-diversity analysis. The differential abundance analysis showed that tebuconazole altered the phyllosphere fungal composition by reducing the abundance of fungal OTUs, primarily from the Dothideomycetes and Sordariomycetes groups, which include potentially beneficial endophytic fungi. In contrast, NPA and sulfur had minimal effects on the phyllosphere fungal microbiome compared to the control. These findings indicated that the use of NPA and sulfur can effectively control powdery mildew while having fewer impacts on the phyllosphere fungal microbiome compared to tebuconazole.

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GroEL, which is a chaperone, plays a key role in maintaining protein homeostasis and, among other functions, serves to prevent protein misfolding and aggregation. In addition, the GroEL protein also has a significant effect on enhancing plant resistance and inhibiting plant diseases. However, the function of the GroEL protein in the inhibition of rice blast remains unknown. Field experiment results show that photosynthetic bacteria PSB-06 have a good control effect on M. oryzae. PSB-06 also can promote rice growth and enhance the stress resistance. A GroEL protein which was separated and purified from photosynthetic bacteria had a significant antagonistic effect on appressorial formation and pathogenicity of M. oryzae, meanwhile transcriptional analysis demonstrated that the GroEL protein could improve the expression of defense gene of rice. Our results show that the photosynthetic bacteria Rhodopseudomonas palustris significantly controls rice blast disease. Its action involves an extracellular GroEL protein, which inhibits appressoria formation, antagonizes the pathogenicity of Magnaporthe oryzae and promotes a host defense response. The research results provide evidence of the potential of this photosynthetic bacterium as biocontrol agent at least for rice blast control.

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Almond is the most extensive crop in California producing about 80% of the world production. Canker diseases caused by Botryosphaeriaceae, Ceratocystis destructans and Eutypa lata are the main limiting factor for almond production in California. Currently, management of canker diseases relies mainly on chemical treatments, which constitute a concern for environmental contamination in California. The efficacy of the biocontrol product Vintec (Trichoderma atroviride SC1) and benefits a spreader-sticker adjuvant to protect almond pruning wounds from infection by canker pathogens was evaluated using different spray application technologies [Solo 425 4-gallon backpack sprayer (Solo®); 25-gallon brushbuster spot sprayer Ag Spray Equipment and 100-gallon Pak-Blast air blast sprayer (Rears Manufacturing)]. Vintec treatments were compared to a water control and the chemical fungicide thiophanate-methyl. All treatments were applied on fresh pruning wounds 24 hours prior to inoculation with 100 µl of a spore suspension (1×104 spores/ml) of the fungi Botryosphaeria dothidea, Neofusicoccum parvum, E. lata and C. destructans. Results indicated that the efficacy of T. atroviride SC1 to protect pruning wounds was greater or equal to that of Thiophanate-methyl for E. lata and Botryosphaeriaceae fungi. Wound protection was improved when Vintec was amended with a spreader-sticker adjuvant and using the Solo 425 backpack sprayer and the brush spot sprayer.

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Fusarium wilt caused by Fusarium oxysporum is one of the most notorious diseases for cash crops. Using of microbial fungicides is one of the effective measures for controlling crop Fusarium wilt, and the genus of Bacillus is an important resource to develop microbial fungicides. Fusaric acid (FA) produced by F. oxysporum can inhibit the growth of Bacillus, thus affecting the control efficiency of microbial fungicides. Therefore, screening biocontrol Bacillus with FA-tolerant ability is hopeful to improve the biocontrol effect for Fusarium wilt. In this study, the method for screening biocontrol agents against crops Fusarium wilt was established based on the tolerant to FA and antagonism to F. oxysporum, three promising biocontrol bacteria, named as B31, F68 and 30833 were obtained to successfully control tomato, watermelon and cucumber Fusarium wilt. Strain B31, F68 and 30833 were identified as B. velezensis by phylogenetic analysis of 16S rDNA, gyrB, rpoB and rpoC genes sequences. Co-culture assays revealed that strain B31, F68 and 30833 enhanced the tolerance to F. oxysporum and its metabolites, when compared with B. velezensis strain FZB42. Further experiments confirmed that 10 µg/mL FA could completely inhibit the growth of FZB42, while strain B31, F68 and 30833 kept normal growth  at 20 µg/mL of FA and partial growth at 40 µg/mL of FA. Compared with strain FZB42, strain B31, F68 and 30833 significantly improved the tolerance to FA.

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Since 2017, our research team has built an extensive collection of microbes from different orchards and natural environments in Wisconsin and other states of the U.S. in order to identify natural metabolites produced from microbes for plant disease control. A novel metabolite named "RejuAgro" produced from Pseudomonas soli strain T3-07 was discovered. RejuAgro has been commercially formulated with a two-year shelf-life when stored at room temperature. RejuAgro shows high potency in suppressing multiple bacterial and fungal crop pathogens with large economic impact, including pathogens causing apple fire blight, citrus greening, and citrus canker. External field trials of RejuAgro have been performed to assess its inhibition efficacy on apple fire blight and citrus canker. RejuAgro was benchmarked against streptomycin, a commercial antibiotic that is considered the gold standard for controlling crop diseases such as fire blight in the U.S. A treatment of RejuAgro at 10-20 ppm can effectively control the fire blight and citrus canker in field trials.
There is no cure once a tree is infected with citrus greening. USDA predicted that citrus greening could destroy the entire U.S. citrus industry during our lifetime. RejuAgro suppresses Candidatus Liberibacter asiaticus, the cause of citrus greening by foliar spray. In addition, we observed a higher expression of pathogenesis-related defense genes PR1 and PR2 when RejuAgro was applied to the citrus.

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Kordyana brasiliensis, an obligate biotrophic fungal pathogen native to South America, was deliberately introduced to Australia as a classical biological control agent for the environmental weed Tradescantia fluminensis. A field-based monitoring study was established in New South Wales at 14 sites where K. brasiliensis was released across a broad latitudinal (~ 1000 km) and climatological gradient. The study’s objective was to evaluate the climatic conditions conducive to K. brasiliensis establishment and severe disease development as well as the ecological impacts of sustained disease on T. fluminensis. Sites were monitored 6, 18 and 24-months post release, assessing K. brasiliensis disease incidence (number of stems infected per plot) and severity (percentage of leaf area covered by lesions) and T. fluminensis abundance (cover and volume). Kordyana brasiliensis established at all release plots 6-months post release with an average disease incidence of ~ 80% at release plots. At 6, 18 and 24-months post release, disease severity was lower at southern, cooler, and drier sites compared to sites in the northern region with warmer, humid climates. At 18-months post release, K. brasiliensis high disease severity was strongly correlated with a significant negative decline in T. fluminensis abundance, with greatest reductions observed in the northern region.

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Soil-borne diseases are plant pathogens worldwide that cause disease in many economically important crops. Soil-borne pathogens are notoriously difficult to control. However, people using insecticides to control soil-borne diseases have developed pathogens that are resistant to the fungicides. Here, Bacillus spp. is used to control strawflowers stem rot caused by Sclerotinia sclerotiorum and cucumber damping-off caused by Pythium aphanidermatum. Seven strains of Bacillus spp. isolated from mushroom compost, it has the ability to promote plant growth. Among them, B13 and B36 inhibited the mycelial growth of S. sclerotiorum and P. aphanidermatum on PDA (potato dextrose agar) plates. In the greenhouse experiments, B13 and B36 bacterial cultures were irrigated on 5-week-old strawflowers and 14-day-old cucumber seedlings. After 7 dpi, plants inoculated with mycelium blocks of S. sclerotiorum which grew on PDA for 7 days, or inoculated with zoospores solution (10⁸ CFU/ml) of P. aphanidermatum. The disease incidence was recorded after 3 weeks and 10 days, respectively. Results showed that the incidence of plant irrigated of B36 were reduced 27% compared to control. The incidence of Pythium damping-off of cucumber seedlings was reduced from 92% of the control to 32% of Bacillus spp. B36 treatment. B36 was identified as B. velezensis and B13 was B. aryabhattai of 16S rDNA sequence. The strain B36 has the potential to control stem rot of strawflower and Pythium damping-off of cucumber.

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Phenazines are redox-active compounds exhibiting broad antibiotic activity. Lysobacter antibioticus OH13, a soil bacterium emerging as a potential biocontrol agent, produced phenazine 5,10-dioxides myxin with outstanding antimicrobial activity. Antibiotic-producing microbes always employ self-resistance mechanisms, mainly including efflux, target modification, sequestration, and enzymatic inactivation to escape self-toxicity. A monooxygenase encoding gene LaPhzX, located in myxin biosynthetic gene cluster, deletion of which caused the mutants more sensitive to myxin and prolonged existence of myxin. Meanwhile, myxin decreased significantly in the LaPhzX protein reaction, and heterologous expression of LaPhzX in Xanthomonas increased its resistance to myxin. So LaPhzX is a myxin detoxification enzyme for protecting L. antibioticus from suicide. In addition, we found a RND (resistance-nodulation-division) efflux pump encoding gene cluster lexABC in strain OH13, and their deficiency resulted in strains increased myxin susceptibility and reduction of myxin yield. Moreover, lexABC expression was induced by myxin and directly activated by a LysR type transcriptional regulator LexR. Myxin bound with LexR at valine (146) and lysine (195) residues. These results indicate a RND pump with regulation mediated self-protection strategy in L. antibioticus. The discovery of two self-resistance mechanisms against myxin in L. antibioticus is important for obtaining high-yield myxin strains.


 

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Pine pitch canker (PPC), caused by Fusarium circinatum, is an alarming forest disease that affects pine species differently. Pinus radiata is susceptible to PPC, while Pinus pinea is resistant. Previous work has shown that the Pinus pinea microbiome may be a source of plant growth promoting bacteria, which may also enhance resistance to PPC.
In this study, bacterial isolates were obtained from P. pinea and characterized. These were mixed into bacterial consortia (A1-A5 and B1-B5) presenting a progressively higher number of beneficial characteristics: IAA production, phosphate solubilization, siderophore production and ACC-deaminase. Pinus radiata seeds were soaked for 2 hours in solutions containing the bacterial consortia and sown in a peat:vermiculite (1:1, v/v) soil mixture. After 30 days of germination, seedlings’ height, biomass and biochemical parameters (i.e. pigments, sugars and starch (STA), free amino acids (FAA), phenolics, flavonoids and malondialdehyde (MDA)) were assessed.
Concerning germination, B3 and A4 consortia increased germination up to 20%. The A4 group displayed significantly more adventitious roots, and higher STA and FAA content. B1 and A5 showed higher MDA content.
All in all, this study points to a high potential of the selected bacterial consortia to affect the germination and primary metabolism of P. radiata. Further experiments with larger numbers of plants and subsequent inoculations are needed to fully assess the impact of these bacteria.

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Grapes have many diseases caused by plant pathogens that reduce the quality of grapes, such as white stain symptoms, and anthracnose, and have been controlled mainly through chemical control rather than biological control. In the case of beneficial microorganisms developed as biological control agents, due to their characteristics, much verification of grapefruit disease control has not been performed, and the control figures are insufficient. In this study, we selected the promising strain of Bacillus velezensis MWS28 which has induced systemic resistance and inhibitory effect on the plant pathogens such as White stain symptoms causing A. acutatum and T. roseum. Bacterial attachment increased as the concentration of alginate increased, and 0.3% alginate solution was most effective at gradient concentrations. As the attachment number increased, the biological control effect against grape white stain symptoms increased. At 3 weeks after MWS28 treatment, the grapefruits showed overweight, high sugar content, increased grapefruit size, and anthocyanin contents were significantly increased compared to the untreated control. These results showed that it can be used not only for grape disease control and yield increase during grape cultivation but also for eco-friendly pesticide-free cultivation.

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Mango and avocado are the main subtropical crops in southern Spain. These fruits are grown and processed in the same geographical area, and are transported to the rest of Europe. However, postharvest diseases during storage and transportation could damage the export market. In this work, symptoms of rot in mango fruits were detected, and analyzed in search of the causal agent. Parallely, asymptomatic avocado fruits were also analyzed. These two fruits share, on many occasions, farms, processing and even transport, and could act as cross inoculation source. The main fungal genera found, both in mango and avocado, were Alternaria sp. and Neofusicoccum sp. Of these two genera, only Neofusicoccum sp. was able to reproduce the symptoms of rot in mango similar to previously detected. Likewise, Neofusicoccum sp. isolates, but not Alternaria sp., could produce rot symptoms in avocado inoculated fruits.
To study the control of this disease through sustainable strategies, two microbial biological control agents were tested. Both are isolated antagonists against phytopathogenic fungi, and correspond to the bacterium Pseudomonas chlororaphis PCL1606. and Bacillus velezensis UMAF6639. The applications of both microorganisms on the fruit showed significant levels of protection, although only UMAF6639 showed greater persistence in the fruit during the preventive applications in the field.

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The decay of Ailanthus altissima due to Verticillium spp. (Fungi, Ascomycota) represents a relevant opportunity for the biocontrol of this invasive tree, which is not properly counteracted by traditional physical and chemical approaches. Verticillium wilt symptoms remember those of drought as they are caused by vessel occlusions, even if phytotoxins produced by the fungus are also involved in the pathogenic mechanism. Here, outcomes from an open air pot experiment aimed to investigate the physiochemical responses of Ailanthus trees stem inoculated with V. dahliae (VdGL16 strain, isolated from the same host in Tuscany) are reported. Inoculated plants showed foliar injuries starting from 2 weeks post inoculation (wpi), and a final severe defoliation. Already at 4 wpi, the infection induced a reduction in leaf water content (-14% compared with uninoculated plants), stomatal opening and net photosynthesis (-46 and -38%, respectively). Moreover, the disease altered the translocation of mineral elements and carbohydrates, that reached minimum values at 8 and 6 wpi, respectively (-84 and -14%). An accumulation of abscisic acid, proline and phenylalanine was also observed at 8 wpi (3-, 10- and 6-fold higher, respectively), suggesting a potential response mechanism. Despite this weak attempt to counteract the fungal colonization, plants were prematurely compromised and death inevitably occurred, confirming the great potential of using Verticillium to control Ailanthus invasion.

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The disease Septoria tritici blotch (STB), caused by Zymoseptoria tritici, is one of the most important and yield-reducing constraints in wheat production worldwide. Control relies heavily on fungicides, but there is an increasing desire to reduce fungicide use and the pathogen readily develops resistance against commonly used products. Biological control, using living microorganisms, is an upcoming trend within disease control, also for STB.
The mode of action of biocontrol agents is more complex than for traditional fungicides and therefore, the risk that they lose effect is considered much less than for chemical products. Potential biocontrol agents are often selected based on direct inhibitory effects in vitro and therefore, the mode of action is often not studied in detail. However, there is emerging evidence that one of the most important modes of action by biocontrol agents is induced resistance, which can only be discovered in assays using plants [1].
We use different fungi (e.g. endophytes and Clonostachys rosea) and bacteria to control STB and have found significant reductions of disease severity using spray applications of fungal biocontrol agents under controlled and field conditions [e.g. 2]. Whereas in vitro studies showed limited inhibition of the pathogen, microscopy and transcriptomics implicated induced resistance as important mechanisms.
 
1. Latz et al. (2018). Plant Ecology & Diversity. 11: 555-567.
2. Latz et al. (2020). Biological Control. 141: 104128.
 
 

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Wild Brassicaceae plant species synthetize defense compounds that can have an impact on the interactions between seed-borne fungi, with contrasting effects depending on the identity of the compounds and of the fungal species. Some fungi can metabolize some glucosinolates while other fungi such as Alternaria brassicicola, the seed-borne causal agent of the black leaf spot disease on many Brassica crops, are inhibited by camalexin and isothiocyanates. To gain insights into the role of the host plant on the interactions between A. brassicicola and the potential seed-borne fungal antagonists, characterization of fungi associated with the seeds of wild Brassicaceae described as resistant to A. brassicicola, was conducted. Their antagonistic effect on A. brassicicola was tested in the presence of the main defense compounds encountered in their host plants. The impact of defense compounds identified in non-host Brassicaceae species was also measured. Through a series of confrontations conducted on solid media and on liquid media (by using nephelometry) enriched or not with glucosinolates and camalexin, the main goal of the study is to gain a better understanding of the mechanisms involved in the interactions between the host plant defenses and seed-borne fungal communities towards future design of biological control of A. brassicicola.

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ABSTRACT
Tomato  is are regarded as one of the world’s most economical crops that is only second to potato. They are important horticultural crop with significant contribution to world’s food security as well as economic development from job creation. However, an invasive insect pest, Tuta absoluta, is a major threat to cultivation of tomato woldwide and is rapidly increasing its geographic presence. In a bid to develop an environmental-friendly control of this pest, next generation sequencing was used to unravel the microbiomes associated with the larvae of Tuta absoluta. DNA was extracted from larvae collected from different regions and sequenced using the illumina platform. Results of the sequencing showed that the dominant fungal phyla was Ascomycota, followed by Basidiomycota. Other fungal phyla present includes Mucoromycota, Glomeromycota, Mortiellomycota and Chrytridiomycota. Implications of these fungal occurrences in relation to biocontrol are discussed. There were also a large proportion of unidentified phyla, class, order, family, genus, and species.

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A study of the tomato endorhizosphere microbiome was performed in commercial greenhouse conditions. Results showed that keystone taxa were represented not only by bacteria with high relative abundance, such as Bacillus and Pseudomonas, but also by numerous and lesser-known genera. The systemic selection of cultivable bacteria enabled us to obtain representative isolates which, in vitro, showed diverse PGPR abilities and marked antagonistic activity. We selected ten bacterial isolates both from genera commonly used as bioinoculants, such as Bacillus and Pseudomonas, and also from unconventional genera like Arthrobacter, Paenarthrobacter, Acinetobacter, Glutamicibacter, and Enterobacter. Bacterial isolates were evaluated individually or in different combinations as consortia for their PGPR activity, induction of plant resistance and biocontrol potential against the soil-borne pathogen Fusarium oxysporum f. sp. radicis-lycopersici (Forl), causal agent of crown and root rot and the leaf pathogen Xanthomonas euvesicatoria pv. perforans (Xep), causal agent of bacterial spot. Most of the treatments significantly reduced the symptoms of both diseases although to a different extent. The efficacy against pathogens that infect different plant organs suggests a multifunctional potential that combines different modes of action. The genomes of the bacterial isolates were sequenced using Oxford Nanopore long read, and Illumina short read sequencing. Genome analysis is underway.

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Clover cyst nematode (CCN) is one of the important plant-parasitic nematodes worldwide, and mainly damage to Kimchi-cabbage production in Korea. Five chemical nematicides are available to control CCN in Kimchi-cabbage plant, but the nematicides are harmful to human and beneficial animals. Therefore, we need to develop the alternatives to control CCN. This study was performed to develop the bio-control agents (BCAs) using bacteria against CCN. Nematicidal activity of two bacteria (isolate BC1 and BC2) was assessed to CCN second-stage juveniles (J2s) using in vitro assay. As a result, culture solution of BC2 was highly toxicity to CCN J2s with 100% mortality. Culture filtrate of BC2 had also 74% of nematicidal activity to CCN J2s. To verify nematicidal activity of BC2 in vivo, pot experiment was conducted in a temperature-controlled room (25oC). The mean of fresh weight of Kimchi-cabbage in BC2 treatment were 1.4 and 2.2 times higher than that of the TSB (medium alone) and NemaO (nematode alone), respectively. The female reproduction on Kimchi-cabbage roots in BC2 treatment was inhibited by 77% (1st trial) and 73% (2nd trial) compared with control (NemaO), respectively. The BC2 treatments reduced the cyst size compared to NemaO treatment, but there was no significance. These results showed that the BC2 has nematicidal activity and potentials as BCAs against CCN.

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The microbiomes in rhizosphere, phyllosphere, and endosphere lived in symbiotic relationships with plants can influence plants to be holobiont by the phenotypic plasticity and extended phenotype. Last several decades, soil microbiota has taken notice as a source to identify specific species able to contorl resistance and tolerance to biotic and abiotic stresses. However, the mechanism of change about phenotype of plants by soil microbiota has yet to be fully understood. In this study, we aimed to identify soil microbiomes that could promote the growth and development of the tomato (cultivar Micro-Tom). Our results showed that the fruit maturity of the Micro-Tom grown with microbiomes from Gijang B soil was 22.8% higher on average compared to those produced in the mock-treated soil. Additionally, the growth of Micro-Tom treated with microbiomes from Gyeongju soil increased by 138% compared with those grown under mock-treated plants. To understand if the phenotypic changes of the Micro-Tom were due to epigenetic modifications by microbiome, we checked the transcript level of the representative genes related epigenetics in different tissues and conditions. Furthermore, we analyzed the similarities and differences among microbiomes used in this study. Our study will give new insight into how soil microbiome can regulate plant phenotype and suggest that soil microbiome has a role in shaping the epigenome of plants from an evolutionary perspective

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Bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) is the most destructive disease-causing great production loss to kiwifruit. Recent studies have shown that plant disease-resistant varieties can enrich beneficial microorganisms to inhibit the infection of pathogens. To investigate whether bacterial canker-resistant kiwifruit cultivars can enrich beneficial microorganisms, five common kiwifruit cultivars were identified for resistance by combining previous field surveys and laboratory resistance assays. Among them, the most resistant variety to Psa was A. deliciosa cv. Hayward, and the most susceptible was A. chinensis cv. HongYang. We researched the leaves, branches, root endospheric microbiome and rhizosphere soil microbiome of HongYang and Hayward by 16S rRNA gene amplicon sequencing in the field. The rhizosphere soil microbiome with the largest difference between the HongYang and Hayward, with more Flavobacterium in the resistant plants enriched. To identify whether Flavobacteria are involved in protecting plants against canker disease, we isolated and cultured 14 strains belonging to the high abundance of Flavobacteria OTU_1542, OTU_337, OTU_1542, OTU_6, OTU_193. It was proved that isolated Flavobacterium 55 and Flavobacterium B2 can reduce the occurrence of kiwifruit canker disease by inhibition zone methods and in the detached dormant experiments of leaves and branches. 

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Sustainable agriculture is crucial to ensure food security for the expected 9 billion people in 2050. However, plant stress has led farmers to rely on chemical fertilizers and pesticides. This is also the case with managing the devastating tomato (Solanum lycopersicum) disease caused by Fusarium oxysporum f. sp. lycopersici (FOL). To address this issue, it is important to find an efficient, cost-effective, and eco-friendly solution. Beneficial microbes, which enhance nutrient availability and have anti-microbial properties, can serve as green alternatives to agrochemicals. This study aims to identify rhizobacteria with multiple plant growth-promoting attributes and determine the key factors in stress mitigation. The bacterial strains were screened for various traits, such as solubilizing inorganic nutrients, tolerance to salt and drought, and production of indole acetic acid and siderophore. Strains were further assessed for in vitro inhibition on five economically significant phytopathogens, namely F. oxysporum f. sp. niveum, Magnaporthe oryzae, Colletotrichum gloeosporioides, and FOL. Thirteen potentially beneficial strains were obtained, and three of the best-performing strains were chosen for pot experiments in a greenhouse, which resulted in reducing disease severity of tomato infected by FOL. The use of these biocontrol agents is expected to regulate plant growth, defense-related genes, and chemical and physiological properties.

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The plant microbiota plays crucial roles in sustaining plant health and productivity. Advancing plant microbiome research and designing sustainable practices for agriculture requires in-depth assessments of microorganisms associated with different host plants; however, there is little information on functional aspects of many microorganisms of interest. Therefore, we enriched microorganisms from the phyllosphere of 110 rice genotypes and subjected them to shotgun metagenomic sequencing to reconstruct bacterial genomes from the obtained datasets. The approach yielded a total of 1.34 terabases of shotgun-sequenced metagenomic data. By separately recovering bacterial genomes from each of the 110 rice genotypes, we recovered 569 non-redundant metagenome-assembled genomes (MAGs) with a completeness higher than 50% and contaminations less than 10%. The MAGs were primarily assigned to Alphaproteobacteria, Gammaproteobacteria, and Bacteroidia. The presented data provides an extended basis for microbiome analyses of plant-associated microorganisms. It is complemented by detailed metadata to facilitate implementations in ecological studies, biotechnological mining approaches, and comparative assessments with genomes or MAGs from other studies.

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Mutualism is a dominant feature in a variety of host-bacteria interactions. Mutualistic bacteria confer plants with growth promotion and pathogen resistance, reciprocally, plants preferentially supply carbohydrates and a stable habitat to mutualistic bacteria to support their efficient colonization. However, whether these bilateral activities act independently or are interlinked via the shared molecular mechanism remains largely unexplored. In this study, the reactive intermediate deaminase A family protein Rhp-PSP secreted by phyllosphere bacterium Rhodopseudomonas palustris JSC-3b was required for both JSC-3b-generated plant health and efficient JSC-3b colonization. The growth of Nicotiana benthamiana seedlings was promoted as a result of Rhp-PSP-mediated plant auxin production. Constitutively, the expression of Rhp-PSP gene in N. benthamiana caused superior seedling growth, concomitantly, the altered leaf metabolite composition repressed the in-vitro and in-vivo proliferation of phytopathogens Xanthomonas oryzae and Pseudomonas solanacearum, but promoted that of JSC-3b. In addition, protein-protein interaction analysis revealed that Rhp-PSP interacted with plasma membrane-localized protein, which was responsible for the Rhp-PSP-mediated plant health and efficient colonization on leaves. Collectively, our study reveals a mode of mutualistic interaction between plants and bacteria, in which both partners benefit from the Rhp-PSP-mediated plant physiological alternations.

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Plant-associated microbiomes confer fitness advantages to the plant host, including growth promotion, nutrient uptake, stress tolerance and resistance to pathogens. While many studies have illustrated those key roles of the root microbiota, less is known about the role of the leaf microbiota and how it is maintained and assembled. Here, we applied shotgun metagenomic sequenced leaf microbiomes of 110 rice genotypes to characterize leaf microbiota grown in the field. We identified that Rhodopseudomonas palustris, Pseudomonas fluorescens and Ralstonia solanacearum are microbial hubs in the co-occurrence network of rice leaf microbiota. Furthermore, we found that amino acids and derivatives were the most possible metabolic exchanges in the leaf bacterial community. Using Genome-wide association studies (GWAS), We identified rice genetic loci connected with the abundance of Pseudomonadales, Burkholderiales, Xanthomonadales and Enterobacterales. Notably, those genes of rice genetic loci were enriched in metabolic pathway and biosynthesis of secondary metabolites pathway. Finally, we demonstrate that our results can be used to improve future studies of the microbe-microbe and microbe-host interaction.

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Calonectria leaf blight is a serious disease of Eucalyptus in India. In this study, the efficacy of five Trichoderma isolates was tested against two prominent Eucalyptus pathogens, Calonectria cerciana and Ca. pseudoreteaudii. All the Trichoderma isolates exhibited >80% mycelial growth inhibition of both the pathogens in inverted plate assays. Solvent extraction of Trichoderma liquid cultures with ethyl acetate followed by gas chromatography-mass spectrometry (GC-MS), revealed naphthalene, 2-methyl-5-formylfuran, ethenone, and other volatile organic compounds (VOCs). Additionally, GC-MS analysis also detected n-alkanes (n-C11 to n-C21), 1-tetradecene, quinoline, α-phellandrene, 1-propyldodecyl phenylacetate, phenol, 2,4-bis(1,1-dimethyl)-phosphite (3:1), benzaldehyde, 4-propyl, tetradecanoic acid, myristic acid, n-hexadecanoic acid, and 9(11)-dehydroergosterol tosylate. Calonectria fungal hyphae treated with Trichoderma VOCs subjected to electron microscopy revealed ultrastructural and morphological damage. To our knowledge, this is the first report implicating the ability of Trichoderma VOCs to suppress the growth of Calonectria fungi known to infect Eucalyptus in India. These results suggest that Trichoderma antifungals may be an eco-friendly alternative to chemical fungicides for the management of Calonectria leaf blight in Eucalyptus.
Keywords: VOCs, Trichoderma, Calonectria, biocontrol, GC-MS, electron microscopy, Eucalyptus, disease management
 

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The variation in Paenibacillus polymyxa E681, a plant growth-promoting rhizobacterium (PGPR), occurs spontaneously, changing B-type (wild-typeical) to F-type (variant). To better understand the difference between the two types, a high-throughput analysis was performed using Biolog Phenotype MicroArray. Compared to the B-type, the F-type showed significantly different growth rates on 17 out of 960 substrates. There was no significant difference in substrates, such as osmolytes, pH, and sodium salicylate. The spectrum of substrates available for F-type was relatively wider than for B-type. In inosine, D-melezitose, and cytidine as single nutrients, the growth of F-type was significantly higher than that of B-type, and interestingly, a few endospores that F-type could not form were observed. To verify the phenotypic microarray result, each type was cultured in a flask containing a minimal medium, including inosine as a single carbon source, and similar results to those of the phenotypic microarray were obtained. A known inosine-related metabolism in E681 is de novo IMP biosynthesis, and all related genes including pur operon (purEKBCSQLFMNHD) were overexpressed in F-type as a result of RNA-Seq in our previous studies. In conclusion, F-type generated by a naturally occurring variation biosynthesized and utilized inosine better than B-type. The association between among inosine biosynthesis, endospore formation, and phenotypic variation needs to be further investigationed.
 

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Plant disease control generally uses chemical pesticides. This method is not effective because it can cause environmental damage. To realize sustainable agriculture, effective control is needed and does not pollute the environment. One way that can be used is the use of antagonistic agents such as Bacillus sp. Encapsulation methods involve covering and protecting the microorganisms. The bioencapsulation formulation can protect Bacillus sp. from environmental stress such as chemical residues, unstable temperature , unsuitable pH, and sunlight. So that bioencapsulation can increase the effectiveness of Bacillus sp. as a biological control agent. The main topics discussed are bioencapsulation technology, bioencapsulation as a biopesticide formulation, materials, and manufacturing processes. This review presents a thorough analysis of the advantages and disadvantages of bioencapsulation technology. As well as closing with views on the prospects for bioencapsulation as a biopesticide formulation.

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This study aims to screen and identify potential biocontrol agents (BCAs) from Freshwater Bioresources Culture Collection (FBCC), Korea, against major phytopathogens under in vitro conditions. Of 856 strains, only nine strains exhibited antagonistic activity, from which only one representative isolate Brevibacillus halotolerans B-4359 has been selected based on in vitro antagonistic activity and enzyme production. Cell-free culture filtrate (CF) and volatile organic compounds (VOCs) of B-4359 have been shown to be effective against the mycelial growth C. acutatum. B-4359 showed an excellent biological control effect of anthracnose on red pepper fruits. Further, B-4359 has been found to show a growth promotion effect in red pepper seedlings. Based on in vitro results, B-4359 played a role to control anthracnose disease effectively in field conditions when compared to other treatments and a non-treated control. The genetic mechanism underpinning the biocontrol traits of B-4359 was characterized using the whole-genome sequence of B-4359, which was closely compared to related strains. In a whole-genome sequence, B-4359 consisted of a 5,761,776 bp length with a GC content of 41.0%, including 5,118 CDS, 117 tRNA, and 36 rRNA genes. The genomic analysis showed 23 putative biosynthetic secondary metabolite gene clusters. Therefore, our results provide a better understanding of the B-4359 strain as an effective biocontrol against red pepper anthracnose for sustainable agriculture.

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The improvement of crop management strategies is needed due to the demands of adaptation to climate change, the emergence of new and more aggressive phytopathogens and the deterioration of agricultural soil quality. Beneficial endophytes are non-pathogenic microbes that live within plant tissues and can provide sufficient protection of their hosts against biotic and abiotic stress. For potential use in agriculture, we isolated and characterized over 600 endophytic microbes from olive trees and crop wild relative halophytes. We investigated thoroughly 26 beneficial Bacillus isolates using a multi-disciplinary approach and under biotic and abiotic stress conditions. We sequenced full chromosomes and plasmids of selected 26 Bacillus isolates. Comparative genomics reveal high genetic/genomic dissimilarity, novel secondary metabolism gene clusters and the discovery of new species. Six isolates grow in-vitro in high salinity (>15%). 15 isolates inhibited the growth of important phytopathogens (eg, Ralstonia, Clavibacter, Fusarium, Botrytis, etc). Several isolates retain these characteristics in-planta. The inhibitions were intensified when testing eluents, obtained from Bacillus cultures using flash column chromatography. Our studies provide strong evidence that specific beneficial Bacillus endophytes demonstrate high metabolic and genetic diversity and are excellent candidates as Bioinoculants for the enhancement of growth and tolerance of crops under biotic and abiotic stress.

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One of the most efficient methods to control Heterobasidion root and butt rots is based on treatments of freshly cut coniferous stumps with biological or chemical products. Biological preparations based on the fungus Phlebiopsis gigantea are widely used in Europe as stump treatments against Heterobasidion spp., but these are more effective on pine stumps than on Norway spruce stumps. In the present study, we tested different Latvian isolates of Bjerkandera adusta and Sistotrema brinkmanii for their antagonistic potential in vitro against both H. annosum sensu stricto and H. parviporum, using native isolates of P. gigantea and Finnish Rotstop® as controls. The best isolates were chosen using several features: growth rate on agar, antagonistic ability against Heterobasidion spp. and oidia production. Some of the B. adusta and S. brinkmanii isolates performed similarly to P. gigantea isolates. For those isolates the growth rate was measured in wood of Pinus sylvestris, Picea abies, Larix decidua and L. x eurolepis.  
This study was funded by European Regional Development Fund  project Nr. 1.1.1.1/20/A/095 “Biological control of Heterobasidion root rot using Latvian fungal strains”.

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The club root of chinese cabbage can remain in the soi for a long period of 7 to 10 years, and after the dormant spores germinate, they break into the cabbage root hairs and multiply, forming root knots. Soil microbial community analysis and selection of useful microorganisms can be used as a basis for developing eco-friendly club root disease control technology from an agricultural point of view. Therefore, this study analyzed the microbial cluster difference between the outbreak and the undeveloped area of cabbage root knot disease for the development of the cabbage root knot disease eco-friendly control technology, and tested it to use as a basis for selecting eco-friendly biological control factors. The fungi soil microbiome examined the residual presence of club root pathogens in the soil to identify P. brassica in the site of the onset of club root disease, and not in the unendemic site. A comparison of the differences showed a 0.89-2.52% distribution difference in Trichoderma sp., Fusarium sp., and Purpureocillium sp.. The results of this study have provided a basis for the collection of root knot pathogen control microorganisms, especially, Trichoderma sp. is showed 47% club root disease inhibition effect in pot test. So it is believed that metagenomics data can be used as sufficient evidence when separating useful microorganisms for the development of eco-friendly club root disease control technology.

Key words : Chinese cabbage, clubroot, metagenomics, biocontrol
 

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Biocontrol agents are commonly used for disease management, however, biocontrol efficacy varies among plant genotypes, potentially because of genetic variation in plants for plant-biocontrol agent compatibility. This study aimed to explore the genetic variation in winter wheat for modulation of Clonostachys rosea-mediated biocontrol of septoria tritici blotch caused by Zymoseptoria tritici. In total, more than 200 wheat genotypes grown in the Scandinavian countries in the last 100 years were investigated under controlled greenhouse conditions. Foliar spray application of the pathogen and the biocontrol agent in two treatments, i.e. Z. tritici (Zt) alone and Z. tritici with C. rosea (ZtCr) was used to assess disease progress over time and biocontrol efficacy. There was significant phenotypic variation among plant genotypes for disease progress in Zt and ZtCr treatments. Moreover, individual plant genotypes differed significantly between Zt and ZtCr treatments, indicating the plant genotype-dependent variation in biocontrol efficacy. Genome-wide association mapping using a 20K single-nucleotide polymorphism (SNP) marker array identified four SNP markers associated with C. rosea biocontrol efficacy and one distinct SNP marker associated with disease resistance. This work will serve as a foundation to further characterize the genetic basis of plant-biocontrol agent interactions, facilitating opportunities for concurrent breeding for disease resistance and biocontrol efficacy.

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The soil-borne pathogen Ralstonia solanacearum causes bacterial wilt and thereby crop losses in the Solanaceae plants including tomato, potato, pepper, and eggplant. Although the susceptibility to the wilt disease primarily depends on the plant genotype, the microbial community in the rhizosphere also contributes to the severity. A flavobacterium TRM1, isolated from the wilt-resistant tomato cultivar Hawaii 7996, suppresses Ralstonia wilt in a susceptible tomato cultivar. The antagonistic activity of TRM1 against R. solanacearum was also observed from co-cultivation of the two bacteria in mCPG medium. To infer the wilt-suppressing mechanism, a large-scale transcriptional characterization was conducted. The transcriptional changes of TRM1 and R. solanacearum under the co-cultivation condition were compared to those in mono-cultivation. The transcriptome data of TRM1, R. solanacearum, and tomato were also collected in the plant rhizosphere. Genes for several secretion systems in R. solanacearum were inferred to be associated with virulence, while genes encoding some membrane-bound proteins in TRM1 appeared to be associated with virulence suppression. Integrating the results of these transcriptional data helped us systematically understand the wilt-suppressing mechanisms between the plant pathogen, the disease-suppressing microbe, and the host plant.

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Fireblight disease is caused by the bacterium Erwinia amylovora and has catastrophic consequences for apple and pear trees. The development of an innovative strategy against it is a priority challenge for fruit production worldwide. A potentially successful biocontrol tool could be the use of bacteriophages (or phages), the viruses of bacteria. To achieve this goal, a collection of 16 Erwinia phages newly isolated in the south of France were phenotypically and genotypically studied. The genomic analysis revealed the presence of 5 phage genera, including a new one, and 7 different phage species, including 4 new ones. Phage lifestyle analysis determined that all phages are virulent (only lytic cycle) and none can perform a lysogenic cycle, as advised for applied purposes. Their host range on a panel of 46 E. amylovora international strains and 4 closely related species was quantitatively assessed. Three phages have a broad host range (100% of strains), 12 phages have a medium host range (≥ 20 strains), and one has a narrow host range (13 strains). One phage can target two non-amylovora strains but the rest are restrained to the E. amylovora species, proving their specificity. The capacity of some candidate phages to inhibit bacterial growth in vitro was confirmed, and we are currently optimizing phage cocktails by adjusting phage types and ratios. Overall, this project seeks to prove the potential of phages as an efficient biocontrol tools against fireblight disease.

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During the vegetation season, natural stump colonization by Phlebiopsis gigantea plays a role in controlling Heterobasidion on coniferous stumps but this process may differ in cooler climate conditions. The aim of the study was i) to analyse P. gigantea spore deposition at low temperatures and gradients using Petri dishes and coniferous wood discs, ii) to compare P. gigantea and Heterobasidion spp. colonization in Pinus sylvestris discs and coniferous logs.
Results showed that viable spores of P. gigantea are released in November and December. When air temperature exceeds 0°C, one cm2 of P. gigantea hymenophore can discharge on average 330100 basidiospores per day. The number of P. gigantea spores decreases with increasing distance from fruitbodies. However, at a distance of 10 m, the number of spores can reach up to 120 000 per m2 per hour. P. gigantea better colonize pine wood compared to spruce. When the area occupied by P. gigantea exceeded 7% of the P. sylvestris disc surface area, Heterobasidion spore infection was not observed.
This research was funded by JSC Latvian State Forests project No. 5-5.9.1_007q_101_21_79, “Investigation of the impact of root rot and reducing risks caused by root rot”.

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Trichoderma is a versatile ascomycetes fungus with lucrative biocontrol potential and plant growth promoting activity. To understand their biocontrol abilities, an isolate of T. harzianum (Th) native to Meghalaya, India was evaluated in vitro against three major pathogens of maize viz., Rhizoctonia solani, Exserohilum turcicum and Sclerotium rolfsii with significant inhibitory effect. The bioactive metabolite of Th was isolated, characterized and evaluated against the targeted pathogens, and found inhibitory effect. A field experiment was conducted for two seasons against Banded leaf and sheath blight, Exserohilum leaf blight and Sclerotium wilt of maize with eight treatment combinations of bioactive metabolite of Th. The results revealed a decrease in per cent disease incidence and severity with enhanced plant growth parameters and yield attributing parameters. A positive effect on total soil organic carbon percentage and total microbial populations as compared to the control was also observed during the experimentation. The results are indicative of the antifungal activities and PGP abilities of the bioactive metabolite of Th. The findings of the experiments may be useful as an effective way of managing the targeted diseases of maize.

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King’s strawberry is one of large-fruited strawberries and is highly preferred by consumers due to their large fruits, but it is necessary to develop eco-friendly management technologies that can improve the disadvantages of soft fruits and weak powdery mildew. The purpose of this study was to evaluate the effects of phytopathogenic fungi on mycelial growth and quality of Strawberries when King’s strawberry was treated with microalgae and antifungal bacteria. 0.4% microalga, Chlorella fusca (CF) and antagonist bacteria, AFB2-2 were mixed and replaced with Botrytis cinerea (BC), Colletotrichum gloeosporioides (CG), Phytophthora capsici (PC) and Sclerotinia sclerotiorum (SS). It was found to inhibit all mycelial growth of five plant pathogenic fungi. Hardness of King’s strawberry with 0.2% and 0.4% CF and AFB2-2 increased by 22.7% and 9.1%, respectively, compared to untreated. In addition, the hardness of Seolhyang Strawberry mixed with 0.2% and 0.4% CF and AFB2-2 increased by 49.4% and 34.5%, respectively, compared to untreated. Through the above results, it is revealed that the combined treatment of chlorella (CF) and antagonist bacteria (AFB2-2) can be used as a biological management technology that can increase the hardness and sugar content of King’s and Seolhyang strawberry as well as inhibit the mycelial growth of plant pathogens.

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Plant-beneficial pseudomonads are promising candidates for the biological control of plant diseases and insect pests. Pseudomonas protegens bacteria are efficient root and insect colonizer with antifungal as well as insecticidal activities. This versatility in lifestyles makes them highly interesting to study. Although many traits enabling root colonization and insect pathogenicity are already known, it is not clearly understood how these bacteria are adapted to a life in insects. We performed an experimental evolution with P. protegens CHA0 based on serial infection cycles of larvae of the crop pest Plutella xylostella. Although some evolved populations displayed altered insect killing speed compared to the original strain, bacterial virulence did not substantially change during the experimental evolution, indicating that P. protegens CHA0 is already well adapted to this insect species. In vitro screens of the evolved populations showed changes in growth rate and antimicrobial activities whereas genotyping revealed mutations in genes which are connected to the bacterial membrane structure. The adaptational phenotype of the identified genetic variations needs yet to be determined. Our experimental evolution provides new knowledge on the adaption of plant-beneficial pseudomonads to insects which is also important for their application in biological pest control.

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Pepper (Capsicum annuum L.) is an economically and nutritionally important vegetable within the family Solanaceae. Biotic stresses impact the quality and productivity of the crop. Wilting caused by soil-borne Fusarium species are among the most challenging diseases of peppers to control. Integrated disease management (IDM) is a sustainable approach to control diseases. Biocontrol is an important component of IDM approaches to manage soil-borne pathogens. Plant microbiome analysis has allowed opportunities for identifying associated microbes with plant beneficial functions, including biocontrol. Samples of bulk soil, rhizosphere, roots, and stem of diseased pepper plants showing wilting symptoms and healthy pepper were collected from the experimental farm of the WorldVeg, Shanhua, Taiwan. Metagenomic sequencing and analysis of the 16S (V3-V4) and ITS (3-4) regions were compared among samples. Species composition showed an abundance of F. oxysporum, F. proliferatum, and F. solani associated with diseased peppers. The unique taxa directly associated with healthy peppers and distinct from the pepper-conserved microbial community were identified as potential biocontrol agents. Thus, analyzing plant-microbial communities can provide insights into key microbes for biocontrol against diseases and their interaction with host plants and pathogens.

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Atractylodes lancea is a perennial herb and an important medicinal plant with a long history of clinical application in China. With the massive market demand and the subsequent development of A. lancea farming, severe issues including root rot disease outbreaks resulted from the continuous cropping of perennial A. lancea. We performed extensive studies on the microbiota associated with A. lancea and the soil to seek solutions.
Via next-generation sequencing of the 16S and ITS amplicons of microbe communities in the root endosphere and rhizosphere of A. lancea samples of diverse origins. We identified a group of steadily co-existing microbes in the rhizome consisting of the bacterial genera Rhodococcus, Ralstonia, Burkholderia-Caballeronia-Paraburkholderia, Sphingomona, and Pseudomonas, and a fungal genus Ascomycota, which we believe comprised the specific core microbiome of this particular plant species. We managed to isolate 33 endophytic bacterial strains and 13 endophytic fungal strains from the rhizome of  A. lancea and performed inoculation experiments to study their resistance against root rot pathogens and their individual inducing effects on the medicinal compound accumulation and growth of A. lancea. We screened for endophytes with biocontrol potentials via dual-culture with two strains of Fusarium spp., the major pathogenic fungi of A. lancea root rot disease. The microbe strains we acquired showed promising application potential in future A. lancea farming. 
 

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Early blight and late blight are both economically significant potato diseases worldwide. Control of potato early blight and late blight are currently heavily reliant on chemical fungicides. In the last decade, the use of biological control agents (BCA’s) and plant resistant inducers (PRI’s) for the suppression of plant pathogens has increased rapidly and has become a viable alternative for chemical pesticides. The aim of the present study was to assess the efficacy of different BCA’s and PRI’s for the control of both potato diseases in an artificially inoculated greenhouse studies. The influence of timing and dosage of BCA’s and PRI’s on disease development was evaluated on one moderately resistant and one susceptible potato cultivar. Efficacy of disease suppression was expressed through the area under the disease progress curve (AUDPC). Efficacies of biological products were compared with chemical fungicide Revus Top (mandipropamide, difenoconazole). There were significant differences in efficacies of used biological products. Results indicated that timing of application and product as well their interactions had marked effect on development of both diseases in both used varieties. The best products provided significant disease reduction, but none of them had the efficacy in the same level as used chemical fungicide. The experiment was carried out within the framework of the ECOSOL project.

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Plant elicitors (PE) have the ability to activate pattern triggered immunity (PTI), via there recognition by pattern recognition receptors (PRRs). PRRs contain extracellular domains and bind to PE in a receptor-ligand manner. For instance, PRRs containing leucine-rich repeats (LRRs) domains, are known to bind to peptides as bacterial flagellin, whereas PRRs containing lysine motifs (LysMs) are implicated in recognition of N-acetylglucosamines compounds as fungal chitin. PE perception results in the induction of a series of events such as reactive oxygen species (ROS) production, phytohormone signalling and defence molecules accumulation.
 
Here, we evaluated how the combination of two elicitors, perceived by different PRRs, affect A. thaliana PTI activation. Using an untargeted metabolomic approach, we found that elicitors combination impacts a highest number of metabolites and deregulates specific metabolites pathway, when compared to the elicitors used alone. This highlights the interest to use a combination of elicitors in crop protection strategies. We then characterized a natural extract from Ulva algae from which we showed that plant eliciting activity was due to both ulvan polysaccharides and arabinogalactan-protein (AGP)-like compounds. We investigate defence response and metabolic changes induced by the Ulva extract treatment in A. thaliana and we studied how this type of complex extract is perceived by the plant.

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Nematodes have versatile lifestyles and provide a suitable lens to decipher the ecosystem conditions. Here, 18S rDNA metabarcoding was employed to study the effect on the nematode composition of arbuscular mycorrhizal fungi, bioeffector, and organic amendments in apple orchards, which were sampled during spring and autumn. Sampling time more than treatment had a significant effect on the nematode diversity and composition, and higher alpha-diversity indices were observed during spring as compared to autumn. Although treatments were able to reduce nematode richness and diversity, their effects varied. The composition of bacterivorous and herbivorous nematodes showed seasonal variations, and a higher number of bacterivorous- as compared to herbivorous- nematodes were seen during spring. The composition of nematode trophic guilds was driven by dominant families like Rhabditidae and Tylenchidae. Nematode-based indices like structure and enrichment indices revealed maturing and moderately disturbed soils for the two apple orchards; and maturity- and plant parasite- indices were generally low. This indicates potential soil nutrient enrichment in the two different orchards resulting in high primary productivity for the herbivorous nematodes. Our study provides insights into the effect of soil treatment on nematode, with implications for the development and modification of bioinoculants, as well as the potential to improve the soil ecosystem services.  
 

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Colletotrichum gloeosporioides can lead to huge economic losses during mango storage and transport, and dimethyl trisulfide (DMTS) was found in Streptomyces globisporus JK-1. The EC50 of DMTS to 66 representative strains of 13 mango Colletotrichum species in China was mainly from 0 to 20 µL/L, and the optimum treatment 80 µL/L for 6 h of DMTS to mango postharvest anthracnose could reach 66% control effect. A histological investigation demonstrated that DMTS exhibited strong inhibitory effects on the infection process of C. gloeosporioides in planta by inhibiting the germination of conidia and formation of appressoria, and contributing to deformation of appressoria prior to penetration. In vitro DMTS caused serious damage to the integrity of plasma membranes, which significantly reduced the survival rate of spores, and resulted in abnormal hyphal morphology. Moreover, DMTS caused deterioration of subcellular structures of conidia and mycelia, such as cell walls, plasma membranes, Golgi bodies, and mitochondria. In addition, to better understand the molecular antifungal mechanisms, the gene expression analysis showed DMTS significantly suppressed expression of ergosterol biosynthesis-related genes Cgerg6 and Cgerg11. The EC50 of ΔCgerg6 and ΔCgerg11 to DMTS was 3 folds and 1.9 folds of that of wild-type strain, respectively, and the wild-type phenotype was restored after the gene complements in situ, indicating that there was a close interaction between DMTS and the two genes.

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Intensification of agricultural management for yield maximization is associated with detrimental side effects on soil and plant health. We hypothesize that extensive management with integrated use of beneficial microorganisms (BM) promote plant stress resilience. The aim of this study was to examine whether the use of a consortium with BM affects the rhizosphere microbiome and plant health of maize depending on farming practices over two growing seasons. A long-term field experiment allowed the comparison of two tillage practices (mould-board plough vs. cultivator tillage) and two nitrogen (N) fertilization intensities (intensive vs. reduced extensive N-fertilization). In both years, successful root and soil colonization of the BM was detected in all treatments, associated with changes in the rhizosphere microbiome. A significant increase in biomass and nutrient content of the inoculated plants could only be detected in the growing season of 2020, which was characterized by severe spring drought. This was associated with increased expression of physiological stress indicators involved in drought stress defense, such as osmotic adjustment and detoxification of reactive oxygen species (ROS), and accordingly reduced leaf concentrations of ROS. This multidisciplinary study provides insights into the influence of BM applications on plant-microbe interactions and plant performance inclusive the relevance of abiotic stress factors under field conditions. 

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The Oomycete Phytophthora palmivora is the causal agent of Bud rot. This primary disease affects oil palm plantations in the Colombian North zone, which has reached high incidence and severity levels during the last five years. Endophytic microorganisms are of particular interest as biological control agents since they can colonize the internal tissue of plants without causing apparent damage, compete for nutrients and space within the vascular system, and act as inhibitors of pathogenic microorganisms. Fungi of the genus Trichoderma comprise a group of filamentous fungi widely used in the biocontrol of plant pathogens. This study was carried out in six plantations in northern Colombia, where root samples were taken from 18 oil palms (Elaeis guineensis), which were processed in selective media for Trichoderma. Subsequently, 30 endophytic isolates of Trichoderma spp. were morphologically identified, and their antagonistic capacity against P. palmivora was evaluated with dual culture and mycoparasitism assays. The potential of 15 Trichoderma isolates for the control of this pathogen with PICR values >70% in the dual culture test and 100% parasitism, where mycelium coiling, sporangia and chlamydospore parasitism were observed. This study highlights the potential of using endophytic Trichoderma as biological control agents in the integrated management program of Bud rot in oil palm plantations in Colombia.

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Forest trees frequently interact with a diverse range of microorganisms including ectomycorrhiza, bacteria, and fungal pathogens. Plant defense responses to individual pathogen have been widely studied, but very little is known on the effect of co-inoculation on host defenses. To study the impact of co-inoculation or tripartite interaction on plant growth and host defenses, Scots Pine (Pinus sylvestris) seedlings were inoculated with either Tricholoma sp or Streptomyces sp or both together with a root pathogen Heterobasidion annosum for three months. The inoculation with Streptomyces or Heterobasidion alone had negative effect on plant growth whereas co-inoculation of Tricholoma and Streptomyces sp in presence of the pathogen seems to promote plant growth (root length, number of lateral roots, seedling weight) of Scots pine over time. Based on the phenotypic examination, it was concluded that the ectomychorrhizal Tricholoma sp and Actinobacterial Streptomyces sp counteracts negative effect of H. annosum on plant growth. RNA-seq analysis of seedlings inoculated with Tricholoma sp, Streptomyces sp, infected with H. annosum will be analyzed for identification of differentially expressed genes (DEGs). The potential of pre-inoculation of seedlings to protect seedling roots before out-planting deserves to be further explored.

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The plant microbiome can modulate plants’ physiology and phenotype, such as development, growth, and responses to biotic and abiotic stresses. Previously, we demonstrated that a flavobacterial sp., isolated from the rhizosphere of a wilt-resistant tomato, suppresses the disease caused by Ralstonia solanacearum. Here, we defined bacterial species that are enriched in either resistant or susceptible tomatoes using 16S rRNA gene sequences and whole metagenome sequences. We established a collection of rhizosphere microbes, to constitute a synthetic microbial community for bacterial wilt resistance. We selected strains that belong to Flavobacteriaceae, Rhizobiaeceae, Xanthomonadaceae, Rhodobacteriaceae, and Cyclobacteriaceae as members of the synthetic community. An uncultured Sphigomonadaceae strain, which was abundant in the resistant tomato, was also successfully isolated by utilizing the metagenome-assembled genome information. The synthetic community and individual strains were subject to assays that assess their effects on bacterial wilt resistance and plant growth promotion. Our study on the rhizosphere microbiome for its involvement in disease resistance and plant health will shed light on understanding intricate relationships between plant, pathogen, and microbiome. Further, bacteria recovered in this work may serve as a useful resource to study microbe-host interactions and can be utilized as plant probiotics.

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The search for new approaches and solutions in the field of bacterial burn control is highly relevant. Microbial preparations based on microorganisms with antagonistic activity play an essential role. In this regard, the genera Bacillus, Pseudomonas, Lactobacillus and Saccharomyces, which are in the collection of the Scientific Production Center of Microbiology and Virology, have been screened and have inhibitory activity against Erwinia amylovora the causative agent of fire blight of fruit crops. It was established that in bacteria of the genus Bacillus only two strains suppressed the growth of the pathogen E. amylovora. B. amyloleguefaciens and Bacillus N2, the zone diameters of suppression were 30.0±0.6 mm and 14.0±1.0 mm, respectively. Among the bacteria of the genus Pseudomonas, no strains with inhibitory activity were found. Most strains of the genus Lactobacillus suppressed the growth of the pathogen. The maximum inhibition zone of E. amylovora was in strains L. paracasei 33-4 (39.6 ± 6.65 mm), L. plantarum M17 (35.6 ± 0.57 mm).
Among strains of the genus Saccharomyces, the largest growth inhibition zone (25.6 ± 2.08 mm) was found in S. cerevisiae (vini). It was found that the component composition of the culture fluid of B. amyloliquefaciens mainly contains acetoin and 2,3-butanedione. Strains of the genus Lactobacillus produce acetic acid and lactic acid to a greater extent. Strain S. cerevisiae forms phenylethyl alcohol, 1-butanol, 3-methyl.

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Mycoviruses are the only acellular agents that have been extensively investigated and utilized to combat crop fungal diseases. Recently, a novel class of viroid-like RNAs naturally infecting a filamentous fungus Botryosphaeria dothidea were isolated from apple, and tentatively named Botryosphaeria dothidea circular RNAs (BdcRNAs) 1 to 3, which have been characterized and termed as mycoviroids referring to viroid-like RNAs naturally infecting fungi besides its original definition. BdcRNAs 1 to 3 in size of 450 to 221 nt display no detectable nucleotide identity to known RNA sequences, but share different identity levels with each other, replicate autonomously in the nucleus via a rolling-circle mechanism following a symmetric pathway. BdcRNAs significantly affect the biological traits of B. dothidea by regulating the gene expression and metabolic pathways related to important cellular processes of the fungal host. More importantly, BdcRNAs 1 and 2 can significantly attenuate or even erase the fungal virulence, while enhance the growth (for BdcRNA1) and increase the tolerance to some osmotic stress of the host fungus. These features provide an important alternative candidate to serve as a biocontrol tool for attenuation of fungal diseases similar to some mycoviruses that cause hypovirulence.

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Below-ground pests are difficult to control because either no effective control methods exist or suitable insecticides are or will soon be banned due to their negative effects on the environment. We evaluated the potential of disease-suppressing Pseudomonas chlororaphis bacteria with entomopathogenic activity for insect control. Moreover, we combined the pseudomonads with entomopathogenic nematodes (Steinernema feltiae) and fungi (Metarhizium brunneum) with the aim to increase reliability and efficacy of biocontrol measures.
In a series of experiments ranging from the greenhouse to the field, P. chlororaphis emerged to be highly efficient in controlling the cabbage maggot Delia radicum, an important pest of Brassicacean crops. Furthermore, the triple consortium of P. chlororaphis with S. feltiae and M. brunneum increased the number of marketable radishes by 50% in a field trial. In several experiments, we observed increased pest control when combining the pseudomonads with the nematodes or the fungi. These synergistic effects were verified when applying the combinations against two further pests. The triple consortium was the most lethal and fastest killing treatment against Pieris brassicae and Diabrotica balteata larvae. In the early stages of the infection, all three agents established inside the larvae.
Our results show that entomopathogenic pseudomonads, nematodes and fungi are compatible and could potentially be used to control a variety of below-ground insect pests.

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In nature, bacterial pathogenicity is counteracted mainly by plant-derived antimicrobial defense molecules. In return, efflux pumps (EP) are part of the resistance mechanism employed by bacterial pathogens to promote their survival in a chemical hostile environment. We studied the effect of combinations of efflux pump inhibitors (EPIs) and plant-derived antimicrobial phenolic compounds on bacterial activity, using Pectobacteriun brasiliense 1692 (Pb 1692) as a model system. Specifically, we measured the minimal inhibitory concentration (MIC) of two phytochemicals, phloretin (Pht) and naringenin (Nar), and of one common antibiotic ciprofloxacin (Cip), either alone or in combinations with two known inhibitors of the AcrB EP of Escherichia coli, a close homolog of the AcrAB-TolC EP of Pb 1692. In addition, we also measured the expression of genes encoding for the EP, under similar conditions. Using the FICI equation, we observed synergism between the EPIs and the phytochemicals, but not between the EPIs and the antibiotic, suggesting that EP inhibition potentiated the antimicrobial activity of the plant-derived compounds, but not of Cip. Docking simulations were successfully used to rationalize these experimental results. Our findings suggest that AcrAB-TolC plays an important role in the survival and fitness of Pb1692 in the plant environment and that its inhibition is a viable strategy for controlling bacterial pathogenicity.  

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Mango (Mangifera indica L.) is a delectable fruit grown in less than 90 tropical and sub-tropical countries in the world. Mango anthracnose, caused by Colletotrichum gloeosporioides, is a highly destructive disease. Chemical control is most frequently practiced by the mango growers, which poses bad impacts on the environment and human health. Hence, alternate control strategies should be used for the management of disease and for increasing the potential yield of mango. The in vitro potential of neem, mint, and garlic for control of Colletotrichum gloeosporioides was investigated in the current study. The pathogen was isolated from diseased mango plant portions. Aqueous and methanolic extracts of neem, mint, and garlic were prepared and tested at different concentrations for their efficacy against fungal growth. All plant extracts significantly reduced the fungal growth in the poison food technique as compared to the control. The methanolic extract of neem was most effective in fungal growth suppression. Results of this study indicate that plant extracts can be used for the control of anthracnose disease in mango. It will be less expensive and safer, and it may be a viable alternative to synthetic fungicides. 

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In response to biotic and abiotic stresses, plants have evolved various defence strategies, including the recruitment of health-promoting root-associated microorganisms via a “cry for help” mechanism. This selected community of microorganisms is known to help minimize the damages caused by the stress, for example by modulating plant nutrition or immunity. However, the rules of microbiome assembly following foliar pathogen infection and the mechanisms that govern its assembly and function in the diseased host are still poorly understood. In particular, it is not known whether different pathogens can induce different root microbiome changes. Using Oryza sativa subsp. japonica cv. Nipponbare, we analysed the modifications of the root-associated microbiome after host exposure to five different foliar pathogens, including two bacteria (Xanthomonas oryzae and Xanthomonas oryzicola), two fungi (Pyricularia oryzae and Bipolaris oryzae) and one virus (Rice Yellow Mottle Virus). Rice was grown in a greenhouse on rice field-sampled soil, and inoculated with the respective pathogens. One week after inoculation, we collected the rhizosphere and analysed the diversity of the microbiome using 16S/18S/gyrB amplicon sequencing approaches. Whether the pathogen has an effect on the root microbiome and whether each pathogen affects it differently, with common signatures specific to each group of pathogens, will be presented in this poster.

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The resistance of plant pathogenic fungi to fungicides is becoming more and more serious. It is of great significance to develop new agricultural fungicides. Insects are widely distributed in a variety of ecological niches, and their ability to live in unique habitats is often to promote symbiosis with their microbes, which were sources of new antibiotic metabolites. For example, the new metabolite isochromophilone XV from the symbiont of Ectropis oblique significantly inhibited Colletotrichum graminicola with IC₅₀ value of 29.9 μg/mL. Insects are a group of organisms with the largest number of known species in the earth biosphere, and the special microorganisms symbiotic with insects are rich in diversity. However, compared with insect species, there is less research on insect symbionts and less research on their metabolites, so it is urgent to strengthen research.

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The fungal pathogen Magnaporthe oryzae causes one of the most important rice diseases: blast. It is found in rice-growing regions around the world and causes significant yield losses. Resistant cultivars and fungicides are the most widely used methods of control. However, the recent concept of the holobiont opens the way to promising sustainable alternatives for plant protection, based on plant-microbe interactions. In this context, we initiated a study of beneficial bacteria using two approaches: i) in vitro, to search for bacteria antagonistic to M. oryzae, through a bacterial/fungal confrontation test on two culture media, and ii) in planta, to search for bacteria with biocontrol activity, through the observation of blast symptoms on rice plants primed with the beneficial bacteria. The results allowed the selection of seven bacterial strains from the genera Azorhizobium, Bacillus, Burkholderia and Cupriavidus with the ability to reduce mycelial development by up to 57%, as well as four bacterial strains from the genera Azorhizobium, Bacillus and Burkholderia with the ability to reduce blast symptoms by up to 45%. Phylogenetic analysis based on 16S rDNA showed that the bacterial strains probably correspond to new species distinct from those already described. Two strains from the genera Bacillus and Burkholderia had both abilities. The bacterial genomes were sequenced, which should help us to further investigate the mechanisms involved.
 

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Composts have been shown to suppress soilborne pathogens in numerous greenhouse and field experiments. However, the effectiveness of disease suppression is highly variable between composts, and we currently lack reliable indicators to select composts for plant protection. We hypothesize that disease suppression is a complex interplay between abiotic and biotic compost properties. Investigating the microbial communities may help to develop tools for predicting suppressive properties and producing composts with strong biocontrol activity.
In the first part of the project, 17 composts were assessed for disease suppression in a cress–Globisporangium ultimum (syn. Pythium ultimum) system and assessed for their physico-chemical properties. Their microbial communities were analyzed using an Illumina metabarcoding approach, which identified bacterial taxa that are indicative for disease suppression. This data set has now been extended by 30 additional composts and a cucumber–G. ultimum and a cucumber–Rhizoctonia solani test system, which revealed differences in disease suppression between pathogens and plant species. The microbial communities are currently assessed by SMRT cell long-read sequencing with the goal to get a high taxonomic resolution to accurately relate the sequencing data with isolates obtained from the composts. Our comprehensive data set provides new insights into the contribution of different abiotic and biotic factors to disease-suppressive activity of composts.

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The autoaecious rust-fungus Puccinia punctiformis is an obligate biotroph pathogen of Cirsium arvense; a cosmopolitan weed and one of the most harmful noxious weeds in agricultural and forestry landscapes in North America and Europe. The pathogen completes its whole life cycle on C. arvense, occasionally causing a systemic infection characterized by the production of spore-bearing shoots that die before flowering or producing seeds, and a large reduction in above- and below-ground biomass. This high level of specificity and the severe damage caused by the systemic form of the rust disease makes P. punctiformis a promising biocontrol agent against C. arvense.
Although the pathogen has been found wherever its host is distributed and can persist in infected batches for years, it rarely reaches epidemic population levels. Since the viability of overwintering teliospores is crucial to forming basidiospores to establish systemic infection every new season, teliospore viability could be affected by field conditions of temperature and humidity through time. To test this hypothesis, teliospores collected in July-August 2022 were stored at -19°C, 6°C, or 23°C; and 5%, 22%, 62%, or 90% relative humidities (RH), to finally analyze their viability by measuring teliospore germination rate once each month later over 250 µl/L dodecyl-NSC in 1% agar. Germination results declined at 23°C under 90% and 62% RH suggesting that teliospore longevity decreases at high humidities and temperatures.

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The plant microbiome is an essential part of the host and is gradually recognized as playing a critical role in plant growth and health. But still, the formation and functions of plant microbiomes during pathogen invasion are not fully understood. We investigated how the soybean plant attracts helpful microbes to suppress soil-borne diseases. We found that the soil mycobiome determined whether the plants survived or succumbed to infection. Surviving plant microbiomes were linked to unique taxa, pathogen-suppressing fungi, and fungi that induce plant immunity. Our findings imply that soil mycobiome composition and function might be recruited under pathogen attack, influencing the consequences of plant-pathogen interactions.

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The use of pesticides for managing crop pests and plant diseases had shown several bottlenecks, stimulating research for developing alternative plant protection solutions. One of them relies on the use of biocontrol products to reduce pathogen populations and limit disease incidence. Plant Growth-Promoting Rhizobacteria (PGPR) could be considered as very good candidates.
In this work, we explore biocontrol potentials of a panel of PGPRs isolated from the wheat rhizosphere and belonging to several bacterial genera, against 2 major fungal wheat pathogens Fusarium graminearum and Zymoseptoria tritici. Characterization of bacterial volatiles and secreted secondary metabolites for their inhibition properties against mycelium and spore growth was made using in vitro antagonist confrontation tests on the two pathogens. It was followed by plant protection assays on wheat crown rot fusariosis under greenhouse conditions. This 2-step screening allowed us to identify strains with direct antagonist effects and others with indirect mechanisms related to plant defense stimulation.
Combining genome mining and metabolomics approaches, we were able to identify some key molecular determinants for the inhibition of fungal pathogens in our PGPR library. Metabolic profiling and consecutive bio-guided fractionation of secondary metabolites secreted by antagonist strains reveal that lipopeptides and dimethylpolysulfide volatile organic compounds are among the main antifungal active biomolecules.

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A diverse group of microorganisms inhabiting plant seeds can colonize the host during growth and development. Previous studies have demonstrated the capacity of fungal and bacterial species recovered from wheat seeds to manage plant disease. Trichoderma species are promising biocontrol agents. They are opportunistic, avirulent plant symbionts or endophytic fungi, and the molecular mechanisms employed by them to control fungal phytopathogens such as mycoparasitism and antibiosis have been previously described. Endophytic Trichoderma spp. have a broad host range with a remarkable ability to promote the host plant's performance. In the current study, a Trichoderma sp. was isolated from seeds of wheat cv. Tiregan through surface disinfection. Our infection assay under greenhouse conditions revealed that spray application of Trichoderma sp. could reduce Septoria tritici blotch (STB) symptoms caused by the Z. tritici IPO323 on inoculated plants. The percentage leaf area covered in lesions (PLACL) and percentage leaf area covered in pycnidia (PLACP) were reduced by 70% and 18%, respectively, in wheat plants treated with applied Trichoderma sp. compared with control plants. Therefore, this endophytic Trichoderma sp. isolated from wheat seed can reduce symptoms caused by Z. tritici IPO323 under glasshouse conditions.

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The massive use of chemical products not only affects the application effectiveness due to the risk of resistance, but also affects the environment. For this reason, the viticulture sector is facing a major challenge, which is an ecological transition. In recent years, the development of different vineyard management alternatives are trying to contribute to a sustainable viticulture model. Increasing biodiversity helps to reduce the use of pesticides and increase ecosystem services. The use of arbuscular mycorrhizal fungi improves the tolerance to abiotic stresses and protects the roots against pathogens. The main objective of this work was to analyze the effect on the biodiversity of vine rhizosphere under mycorrhizal products application. For a period of four years, the microbial rhizosphere biodiversity was manipulated in an experimental vineyard using seven different microbial products, with annual inoculations. Soil and root samples were taken annually for bacterial and fungal detection by the Illumina sequencing. For each treatment, the detected microorganisms and their respective number of reads was obtained. The microbial biodiversity was estimated by the indices: Shannon, Simpson, Pielou, species abundance and species richness. Preliminary results show that the products applied influence the rhizosphere biodiversity increasing the richness index and, in some cases, providing a promising induced resistance to downy mildew.

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Wireworms are very destructive in small grain production in the Pacific Northwest and difficult to control with insecticides. In a targeted survey to detect local populations of entomopathogenic nematodes (EPNs) in southern Idaho, USA, six populations of EPNs were isolated from the soils of cereal fields that were heavily infested with larval stage of click beetles (Coleoptera: Elateridae). Initial morphological characteristics placed the collected EPNs into the genus Steinernema (Travassos, 1927) which is a known biological control agent, with active nictation, cruiser locomotion, and infection dynamics that can be variable at intraspecific level. Additional molecular analysis was required as morphological characteristics will not delineate the isolated samples to species level. The sequence analysis of partial ribosomal RNA gene complexes including internal transcribed spacers (ITS1 and ITS2) and D2D3 expansions of 28S large subunit confirmed the occurrence of S. feltiae. Phylogenetic relationships inferred from maximum likelihood (ML) analysis of ITS-rRNA sequences distinguished the isolated EPNs to the Feltiae clade. Cytochrome c oxidase I (COI) of mitochondrial DNA resolved the phylogenetic relationships to within S. feltiae subclade using ML analysis of COI mtDNA sequences. This study is the first report and characterization of Steinernema feltiae in southeast Idaho. Additional studies on the infection dynamics and bacterial symbiosis for these isolates is required.

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Broomrapes (Orobanche and Phelipanche) are root parasitic plants that affect a wide range of economically important crops worldwide (rapeseed, sunflower, etc), thus posing tremendous threats as they can cause heavy yield losses. When broomrapes infest a field, farmers have to reduce their producing area using only non-infested plots. So far, crop protection against parasitic weeds is mostly based on the use of systemic chemical herbicides in combination with the use of crop genotypes that show tolerant behavior against broomrapes.
In conventional agriculture, large scale use of chemical plant protection products has led to the degradation of soil quality and has had a dramatic impact on natural flora and fauna. Living organisms able to protect plants against broomrapes, or any weed management strategies that are more environmentally friendly needs to be found.
We have identified and characterized several Pseudomonas rhizobacteria able to produce compound(s) that stop, under in vitro conditions, the germination of broomrape seeds, thus further affecting the growth of the parasitic plant under greenhouse conditions. One of this compound is a polyketide. It is efficient at concentrations below 25μM against P. ramosa and 50μM against O. cumana. Other Pseudomonas strains that do not produce this polyketide also shared an herbicide activity against these plant parasites, showing that a very wide diversity of novel secondary metabolites from Pseudomonas could be used as bioherbicides.

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Seed-bearing onion is an important crop in several area of production, and it can be affected by several fungal pathogens. Most of these infect the seed, which then become an efficient vehicle to disperse seedborne pathogens over long distances, resulting in potential severe crop losses. The need for high-quality seed and the increasingly stringent restrictions on the use of synthetic plant protection products imposed by the European Union fostered the search for alternative solutions to protect seedbearing vegetable crops from seedborne pathogens. Within the project “CleanSeed” promoted by PSR Marche, Central-Estern Italy, this work aimed to evaluate, on a company scale, the effectiveness of several innovative protection strategies based on the use of basic substances, biocontrol agents, plant extracts and low-risk active substances. Four strategies including chitosan, chito-oligosaccharides and oligo-galacturonides (COS-OGA), a mixture of terpenes, and Bacillus spp. were tested, and an assessment was carried out on the infections of the stem and flowers by Botrytis spp. Chitosan strategy reduced Botrytis spp. McKinney Index compared to untreated control on onion plant by 60%. The results obtained from this study open the way for new protection strategies based on the use of natural substances in the management of fungal diseases of seed-bearing vegetables.

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The aim of this study was to evaluate the essential oils of Pakistan`s local aromatic plants as biological control of chickpea blight caused by Ascochyta rabiei. Another objective was to evaluate the impact of selected essential oils on plant health and growth. Out of 30 plants essential oils only 10 showed the antifungal activities against A. rabiei. Cumin (Cuminum cyminum L.) essential oil produced the highest fungal mycelial growth and spore germination inhibition and was selected for the further study on planta. The minimal inhibitory concentration was revealed as 0.5 ml/L and the minimal fungicidal concentration was found 1 ml/L. Microscopic examination of C. cyminum EO on hyphal morphology revealed that the essential oil caused degenerations, less branching, loss of septations, vesicles formation, shriveling and lysis of hyphae. Cumin essential oil improved the plant growth especially when the seeds were treated with essential oil (≥0.5ml/L) after inducing the priming with water. In greenhouse, cumin essential oil protective spray on chickpea reduced the disease severity was significantly reduced (upto 70%). The biochemical changes and expression of resitance genes in planta due to the treatment by cumin essential is in progress. It will help to understand the possible role of C. cyminum essential oil in resistance mechanism in chickpea plants. The results of field experiments and These studies will hopefully be completed and presented in the conference.

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Because they are sessile organisms, plants have developed different strategies to fight pathogen infections. Although the plant immune system has been mainly described for plant-pathogen interactions, commonalities between beneficial microorganism and pathogen infections have been established. Among beneficial interactions, the nitrogen fixing symbiosis of alders with the actinobacteria Frankia has been well studied. Establishment of this symbiosis leads to the formation of root nodules. In the other well-known nitrogen-fixing symbiosis between legumes-rhizobia, it is recognized that the plant immune system is modulated in early stages of symbiosis. Much less data is available on Frankia-Alnus model and it is still unclear how plant immune system reacts when root nodules are well established and how it impacts plant ability to face pathogen infections. 
We hypothesised that the presence of Frankia in alder tissues activates the plant immune system and could play a role in the defence against Phytophthora alni.
Our objective is to understand whether the alder immune system is activated by the symbiosis with Frankia and whether this symbiosis can play a role in modulating the defences against the pathogen Phytophthora alni. For this purpose, we developed an experimental system of Alnus-Frankia interactions including the pathogen Phytophthora alni. We performed multi-omics analyses to compare plant defence reactions to face the pathogen when hosting or not its symbiont Frankia.

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Entomopathogenic fungi (EPF) offer potential biological solutions to manage plant sap-feeding insect pests, such as aphids. The plant-colonization capability of notable endophytic EPF strains ensures their survival and confers plant protection and growth functions. This work is aimed to discover novel EPF candidates with the potential for effective management of Barley Yellow Dwarf Virus (BYDV) and the aphids that vector the virus in barley crops.
For this study nineteen endophytic fungal candidates were utilized, which were isolated from different plant niches-seed, stem, leaf and root tissues of cereal plants. These endophytic fungi candidates were associated with ten different taxonomic genera. For the screening experiment, the adult aphids were exposed to endophytic fungal spores for three hours and then transferred to the plants. The results under controlled conditions have shown a promising reduction in the aphid population across different time points. There were up to two-fold reductions in the number of adult aphids and nymphs elicited by four different fungal candidates. Interestingly, >30% of the endophytic fungal candidates screened demonstrated the potential to suppress aphids and nymph numbers.
The key findings suggest that diverse representations of these endophytic taxa could potentially offer multi-choice effective biological control agents. Ongoing work is focused on establishing a mechanistic understanding of aphid inhibition by potential endophytes.

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Trichoderma genus has a good place in biological control market. Trichoderma spp are capable to recognize and attack on several soil borne plant pathogens but during current study, synergistic effects among four species were assessed against Fusarium wilt of tomato. Primarily, a comprehensive survey was conducted, and four dominating species of Trichoderma were identified and purified from different agro-ecological zones of Pakistan. These species were Trichoderma harzianum, T. viride, T. atroviride and T. virens. All these species were successfully cultivated in sets of two with different degree of success. In the present study an isolate of Fusarium oxysporum pv lycopersici was taken form First Fungal Culture Bank of Pakistan. After pathogenicity test, the pathogen was subjected to grow on PDA plates against all four Trichoderma species. In-vitro results showed that, T. harzianum and T. viride gave 92 and 84% reduction in colony growth over control whereas T. virens and atroviride gave 32 and 26% reduction respectively over control. Furthermore, sick plants were treated with all four strains of Trichoderma spp separately and in combinations of two with all possible blends. Statistical analyses revealed that, T. harzianum along with T. atroviride increased their antagonistic potential 3.6 fold where as T. atrovirude and T. viride gave 2.8 fold increase in protection value against Fusarium wilt of tomato with reference to single isolate application.       

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The large pine weevil (LPW) is a widespread forest pest in conifer clearfell restocking areas. Biological control agents are the best candidates to replace widely used synthetic chemicals with potential side effects on the environment and humans. Entomopathogenic nematodes (EPN) and fungi (EPF) are successful at controlling its immature stages and also present additive effects when applied together to tree stumps. We propose a new approach that also targets the adults that survived the control of immatures. Surviving emerging adults will be lured with volatiles like alpha-pinene to traps containing an EPF. A large-scale soil sampling campaign within various habitats has provided new locally sourced EPF to be used together with commercial EPN strains. High diversity of EPFs has been obtained from soil using a. The local strains of EPF are tested for pathogenicity on LPW to select the best entomopathogenic agent for adult traps and to study their additive/synergistic effects with commercial EPN. To test the efficacy of the approach in the field, mark-recapture experiments will be conducted, which will inform the development of an efficient chemical-free method to control H. abietis on forest clearfell areas.

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Antimicrobial volatile activity of rhizobacteria has been achieved by growing them on rich media, raising a question of whether the production and activity of antimicrobial volatiles are limited to a few reported media types. Effect of inoculum amount and inoculation method of antagonist on production and activity of antimicrobial volatiles has not been reported. Therefore, antimicrobial activity of Bacillus cabrialesii FA26 against soilborne fungi was investigated under different culture conditions. Results showed that FA26 when grown on NA showed higher suppression against Fusarium oxysporum, Rhizoctonia solani, and Phytophthora capsici by 39.83, 63.57, and 43%, respectively under double-dish chamber. However, FA26 exhibited 56.38% better suppression against Sclerotinia sclerotiorum when grown on LB. Effect of inoculum amount and inoculation method on the production and activity of antimicrobial volatiles of FA26 showed a clear dose-dependent potential and was highly correlated with the increase in the amount of inoculum using either of the inoculation methods; however, spreading mode of inoculation was better than that of the drops. Headspace SPME/GC-MS analysis revealed 26 volatiles’ production by FA26. Of these, 8 volatiles completely inhibited one of the four phytopathogens. The study suggests production and activity of rhizobacterial volatiles strongly depend on culture conditions. Furthermore, the results revealed a direct long-distance biocontrol mechanism of FA26.

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Rice (Oryzae sativa L) is suffering from several biotic and abiotic factors. Among biotic factors, brown leaf spot of rice (BLS) is potentially devastating disease of rice causing the severe yield losses up to 100%. The current study was designed to evaluate eco-friendly management strategy towards BLS to avoid environmental and human hazards. For this purpose, thirty botanicals were screened out under in vitro conditions and five most effective extracts were further demonstrated against the targeted pathogen with three different concentrations (10, 20 and 30%) by using poisoned food technique. The results revealed that Ginger and Eucalyptus showed the strong inhibitory effect against B. oryzae at 30% concentration followed by Mint, Turmeric and Dhatura respectively. The promising extracts (Ginger and Eucalyptus) under lab conditions were further evaluated in vivo against BLS disease by using 3 types of applications i.e. Preventive, Curative and after symptoms appearance. Findings showed that, preventive application was found most effective as compared to other applications, furthermore combination of ginger and eucalyptus showed significant reduction in disease incidence percent as compared to solo applications. The reduction in disease incidence percent in all application methods suggested that these extracts could be used as alternative of synthetic fungicides against BLS of rice. 
 

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Trichoderma are versatile beneficial fungi which can stimulate growth and plant resistance to biotic stress. Understanding the Trichoderma species for their diverse modes of action for management of Fusarium oxysporum, pv Lycopersici is a central goal of this research. In our ongoing studies most recently, we have tested the ability of T. harzianum to protect against salinity which adversely affects germination and growth of tomato seedlings. Trichoderma seed treatment improved plant tolerance. A model system for Trichoderma induced resistance to biotic stresses was provided by induction of a systemic response against fusarium wilt of tomato. Firstly 27 strains belonging to three species of Trichoderma were subjected to evaluate against F. oxysporum pv lycopersici through dual culture technique in-vitro.  Statistical analysis revealed that eight species showed better antagonism against tested pathogen and were selected for field studies. These eight species were cultivated on sorghum based solid state fermentation on large scale and applied in field on artificially inoculated tomato seedlings. The results of current study showed that, the isolates of T. harzianum isolated from soil sample taken from tomato field expressed promising results and gave 94.6% reduction in disease over control whereas other isolates gave protection value under 62.0%. Therefore, the isolate which showed better performance under way to be characterized and its molecular identification.

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Mycotoxins are one of the major threats to food and feed safety and quality worldwide. More specifically, aflatoxin AFB1 and its metabolite AFM1, have been classified by the International Agency for Research on Cancer (IARC) among the most carcinogenic compounds for humans. Due to the inability of chemical methods to control aflatoxin levels on maize and pistachios, the use of non-toxigenic strains of Aspergillus flavus has been characterized by numerous studies as the most effective control strategy. The purpose of the present study was to evaluate several non-toxigenic strains in terms of their ability to reduce aflatoxin production in situ, on artificially infected corn seeds with a highly toxigenic A. flavus strain from the collection of the Laboratory of Phytopathology, Agricultural University of Athens.Our experiments indicated the high effectiveness of these specific non-toxigenic isolates in inhibiting the biosynthesis of aflatoxins on pistachios, reaching reduction rates of aflatoxin levels between 80-90% both in laboratory experiments and in field experiments. In the context of biological management, the most effective of the non-toxigenic isolates in corn kernels were further applied to maize cultivation, under field conditions and their effectiveness was further confirmed at high percentages. Novel formulations of the application of the non-toxigenic strains in the field are also examined.  

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Present Study investigated the efficacy of PGPR as an alternative to chemical fungicides in controlling Alternaria blight, a severe fungal disease of tomato caused by Alternaria solani. At least 11 fungal isolates were recovered from symptomatic tomato plants and identified the most virulent one through virulence assay. A total of 17 bacterial strains belonging to two potential PGPR species; Bacillus and Pseudomonas were recovered from the rhizospheric soil of healthy tomato plants. Four rhizobacterial strains were selected based on their effectiveness in inhibiting the most virulent strain of A. solani in vitro. Two isolates belonging to Azotobacter and Rhizobium spp. with proven plant growth promoting traits provided by AARI Faisalabad were tested in vitro for their efficacy against the same virulent strain. Among all the bacterial isolates, Pseudomonas isolates showed the highest mycelial growth inhibition of A. solani. In repeated pot trials, all tested bacterial strains alone and in combination significantly improved seed germination and plant growth and provided substantial protection against early blight disease. The PGPR-pretreated tomato plants also exhibited increased chlorophyll content, total phenol, free proline, total protein, and the activities of peroxidase and polyphenol oxidase. All treatments showed increased levels of indole acetic acid, abscisic acid, salicylic acid, and jasmonic acid compared to the levels in infected plants used as control.

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Microbial biocontrol agents are promising tools to reduce the use of chemical pesticides in agriculture. Due to their characteristics of living organisms, their deployment is more complex than applying chemicals, and result in the variability of their efficacy, which can hinder their adoption. Taking this complexity into account would make their use more reliable.
Thus, it is necessary to develop decision support systems (DSS) based on biological properties of biocontrol agents, those of plant pathogens, and the characteristics of cropping systems. To develop such a DSS, a database has been set up to integrate information collected from scientific and technical literature and synthesize them into easily accessible datasheet.
However, an analysis of the database reveals that the available data are not sufficient, and many information is lacking in particular on the real conditions of use of biocontrol agents and few factors can be apply in commercial situation.
To solve this issue, it seems both necessary and promising to supplement available data with feedbacks from users of biocontrol agents directly from the field.  Therefore, an application is currently under development to collect these feedbacks on the use of biocontrol to enrich the database to allow better guideline when using biocontrol product.
 

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The world grape industry is seriously affected by grapevine trunk diseases (GTDs) and effective and sustainable management strategies are required. From 2019 to 2022, our laboratory evaluated biological and chemical fungicides as pruning wound protectants against GTD pathogens. In these trials, bacterial and fungal biocontrol agents (BCAs) obtained from healthy grapevine tissues were evaluated and compared to commercial chemical and biological fungicides. BCAs were prepared in liquid culture in the laboratory and applied on fresh pruning wounds of 10-years-old ‘Cabernet Franc’ vines. Commercial pruning wound protectants were applied at their label rate. After five days, treated pruning wounds were inoculated with spore suspensions (10,000 conidia) of Neofusicoccum parvum. After six months, pruning wounds were evaluated by performing isolations on potato dextrose agar. Results showed that fungal BCAs (Aureobasidium pullulans and Trichoderma spp.) exerted significant pruning wound protection when compared to synthetic chemicals and other biofungicides. In conclusion, fungal BCAs provided better pruning wound protection than bacterial BCAs in field conditions and constitute a suitable and sustainable management alternative for GTD management.
 

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Maize-associated microorganisms, established through the evolutionary mechanism of selection, change according to certain factors such as genotype, geographical location, and environmental variables. GEMMA project focuses on beneficial endophytic bacteria inhabiting the embryo of four traditional maize landraces (Nero Spinoso Valcamonica, Spinato Gandino, Rostrato Rosso Rovetta, Fiorine Clusone, preserved at CREA Bergamo Genebank) and an inbred line (B73). To highlight the effect of environmental selection and the influence of vertical microbiota inheritance, plants were grown for three years in four different locations (Landriano, Bergamo, Verderio, Carvico) under low-input farming systems. Since recent studies show how certain endophytes can fight pathogens, this study aims also to study the relationship between the isolated endophytes and the most common toxigenic maize fungal pathogen in Lombardy region: Fusarium verticillioides. In vitro and in vivo test antifungal assays show that, out of over 100 isolates, only 2 from Spinato Gandino significantly reduce fungal infection. Instead, field trial results from 2021 experimental fungal inoculations show that Spinato di Gandino is the most susceptible variety out of those analysed.
Pathogen resistance traits from landraces and their associated microbiome can be of interest for future organic maize production and contribute to more sustainable biotic stress management and higher yields, even in the scenario of climate change.
 

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Soil predators are of central importance in regulating the interaction between plants, soil, and microbiota via a top-down control of microbes, including plant pathogens, and increased nutrient cycling. Infections by plant pathogens trigger  defence, can alter the host metabolism and nutrient flow into the soil, leading to changes  which feed-back to the soil microbiome. However, the links between soilborne pathogens, soil predators and the soil microbiome are only starting to be explored. We used the clubroot pathogen Plasmodiophora brassicae - a major obstacle for the cultivation of Brassica worldwide with no effective control options -  to investigate disease induced changes of the soil microbiome and the role of soil predators in clubroot disease development. We aim to identify potentially disease suppressive and disease conducive predators and microbiome members, including bacteria, fungi and protist and other top-down controller. We combined soil physicochemical analyses  with long-amplicon sequencing to decipher underlying drivers of taxonomic and functional changes in the microbiome to clubroot infections in field and greenhouse experiments. Additionally, feeding behaviour of soil predators on clubroot spores was investgated. We present insights of the potential of soil predators to control soilborne diseases such as clubroot that might lead to new biocontrol applications for soilborne pathogens in the future. 

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Tar spot, caused by the obligate fungal pathogen Phyllachora maydis, is a foliar disease of corn that since 2015 has become a major concern in the USA. To test for interactions between other microbes and the tar spot pathogen, phyllosphere microbiomes were compared among corn inbreds with differential tar spot symptoms under natural infestation in the field. Leaf samples from sixteen inbred lines were assessed for tar spot symptoms, and bacterial and fungal microbiomes were characterized. Comparison of the phyllosphere microbiomes revealed distinct bacterial and fungal communities between resistant and susceptible lines. Bacterial and fungal species richness was significantly higher in resistant compared to susceptible inbred lines. In contrast, there were no clear differences in diversity when including evenness of bacterial communities between the resistant and susceptible lines. Diversity of fungal communities differed significantly, particularly between twelve of the fourteen susceptible versus resistant lines. Many of the bacterial and fungal species showed statistically significant correlations with P. maydis reads. Those that are positively associated could be mycoparasites that are more common with a more abundant food source. Species with significant negative correlations could be antagonistic with a potential for biocontrol. Further analyses of these distinct microbiota could lead to a better understanding of the potential role of foliar microbiomes on tar spot.

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In the last decades, the use of beneficial bacteria has become a promising strategy to combat plant diseases. Previous studies have shown that the Bacillus velezensis UMAF6639 strain had an excellent biocontrol capacity against fungal and bacterial diseases of cucurbits. In addition, it had been observed that it could also be effective against plant parasitic nematodes.
Currently, the application of chemical agents remains the most common method for managing and controlling these pathogens. However, due to the increasing concern about environmental and public health safety issues, many highly toxic chemical compounds have been restricted in their use. Therefore, there is an urgent need to develop more environmentally friendly ecological alternatives for controlling these pathogens.
Therefore, in this study, the identification, characterization, and mode of action description of a molecule produced by Bacillus velezensis UMAF6639, a cyclic dipeptide, which was demonstrated to have nematicidal and fungicidal activity, was carried out. The results indicated that the activity of this molecule was based on a common mechanism capable of altering the physical characteristics of the pathogens plasma membrane, which is key to the physiology and homeostasis of these organisms. This discovery is important because it provides a basis for the development of new biological control agents that are effective against plant diseases caused by pathogens other than fungi and bacteria.

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Crop inoculation with the eco-friendly strain of T. hamatum or their secondary metabolites could induce active biologicals against soilborne pathogens and reduce the use of pesiticides and fertilizers in the agricultural and environmental systems. Trichoderma species have garnered interest for decades due to their plant growth promoting and antimicrobial properties. Mycoparasitism by Trichoderma was first observed in the 1930s, followed more recently by the identification of small molecules such as peptiabols, gliotoxin and 6-pentyl pyrone. Sequencing of T. hamatum GD12 revealed that up to 38% of its genome is unique by comparison to other sequenced Trichoderma strains, and only 50% of the gene clusters identified by fungiSMASH have homology to clusters from other Trichoderma strains. These data combined imply that the genetic potential of T. hamatum hepA to produce novel specialised metabolites may be unprecedented within the Trichoderma genus. To access the biosynthetic potential of T. hamatum hepA, a mutant strain was constructed in which a gene encoding a heterochromatin protein – a global regulator of metabolism – was knocked out. This hepA mutant exhibits enhanced growth promotion in assays with lettuce, in addition to antifungal activity against Sclerotinia sclerotiorum. By a combination of comparative chromatography-mass spectrometry analyses and bioassays, we have begun to identify the cryptic metabolites responsible for the enhanced antimicrobial phenotype observed.

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Plants recruit soil microorganisms to provide various functions or protection against pathogens. Crop plants and their associated microbial communities are therefore increasingly studied together. However, the mechanisms that control the assembly of the root-associated microbiome remain difficult to disentangle, especially in rice ecosystems, which are poorly studied. Here, we compare the assembly of rice rootassociated microbiota sampled from 19 smallholder fields in the irrigated and rainfed lowlands of Burkina Faso. Using a 16S rRNA gene amplicon and ITS metabarcoding approach, we show that the rice production system is a major factor in the structure of the microbiome in addition to the expected structure by root compartments (root vs. rhizosphere) and geographic areas. In irrigated systems, we found greater diversity of rhizosphere prokaryotic communities and more complex co-occurrence networks, compared to rainfed lowlands, while fungal communities showed an opposite pattern. The main taxa were different between the two systems, and indicator species were identified: mostly within Bacillaceae in the rainfed lowlands, and within Burkholderiaceae and Moraxellaceae in the irrigated areas. Finally, a higher abundance in rainfed lowlands was found for mycorrhizal fungi. Our results highlight profound differences in the microbiome induced by contrasting rice production systems that should therefore be considered for microbial engineering applications.
 

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Almond is one the most important nut crops worldwide. The implementation of new management techniques such as high-density cultivation, prune intensification, drip irrigation and fertilization, mechanical harvest, use of more productive varieties, and the cultivation in agronomically and environmentally more favorable cropping areas, has increased the productivity in last decades. However, this new scenario, together with the current climate change situation, have increased almond diseases such as those caused by fungi of the Botryosphaeriaceae family. Symptoms include cankers on the trunk, extensive gummosis, internal tissue necrosis, and occasional death of the plant. Control methods are based on cultural practices, fungicide application and the search for resistant varieties. In this work, two collections of bacteria, rhizospheric and endophytic, have been obtained and characterized as potential biological control agents (BCA) of almond wood diseases. The in vitro and in planta antagonistic effect of 22 bacterial strains against Botryosphaeria dothidea, Neofusicoccum parvum, Diplodia seriata and Macrophomina phaseolina has been evaluated. Strains of Bacillus velezensis, Pseudomonas aeruginosa, B. mobilis and B. safensis could inhibit the in vitro growth and reduce the length of the lesions caused by these fungi in almond trees. The production of hydrolytic enzymes could be related to the mechanism of action of these potential BCAs

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The agriculture sector in Guyana as well as in the Caribbean region continues to face new challenges.
            Several advanced lines were evaluated and superior strains are under cultivation in Guyana. Among the various factors influencing the quality and quantity of rice produced, paddy bug Oebalus poecilus is known to be the principal insect pest of rice in Guyana
To evaluate potential microbial pesticides as biological control for paddy bug Oebalus poecilus in order to avoid ecological and toxicological hazards for chemical pesticides in Guyana.
The methods are used Screening, isolation and characterization of micro-organisms from soil, Culture medium for screening micro – organisms, Selection of bio pesticide (Microbial pesticide), Preparation of squash mounts from fungal cultures, In vitro- contact kill bioassay to control paddy bug using microbial insecticide, Mortality assessment of paddy bug under pot culture condition against microbial insecticide.
            Bio pesticide shown in control of paddy bug both in in vitro and pot culture condition.
 
 

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Olive knot disease caused by the Gram-negative bacterium Pseudomonas savastanoi pv. savastanoi is one of the most important diseases that affect olive trees (Olea europaea L.). The plant genotype has been recognized to be a key determinant of microbial community that is associated with plant health. We assessed the microbial community in phyllosphere and rhizosphere of olive varieties from the Olive Germplasm Collections. Varieties showed different susceptibility to olive knot disease. The trees showing the symptoms were compared with asymptomatic trees to elucidate the potential role of microbiome in protecting the host plants from the disease. DNA was extracted from root and leaf samples and whole metagenome shotgun sequencing of 16S rRNA gene was used to characterize microbiota in olive compartments. With regard to prokaryotic communities, both richness and diversity indices were compared. Bacterial communities were composed by dominant phyla Proteobacteria, Bacteroidota and Actinobacteriota in leaves and by Proteobacteria, Actinobacteriota and Chloroflexi in roots. The overall data suggest that genotype and pathogen infection may result in distinct microbial community structure. These results suggest that highly diverse microbiome may improve the plants ability to resist the effects of pathogens potentially contributing to plant health.
 
 

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Pathosystem drivers of change parameters have impacted directly and indirectly on the declining economic and nutritive value of groundnut (Peanut, Arachis hypogaea) in Kenya. PBMYV naturally infects Phasey bean (Macroptilium lathyroides) and has not been reported in Kenya. Transmission of PBMYV is by the groundnut-cowpea aphid (Aphis craccivora), whitefly (Bemisia tabaci) and grafting with infected scions. A survey in Bungoma, Busia, Kakamega and Siaya Counties during the short and long rains of 2020-2021, collected symptomatic leaves with mild yellowing, chlorotic streaking, chlorotic spots, mottling, bunching, puckering, curling downwards, stunting and those with evidence of aphid colonies. They were subjected to RT-PCR diagnostics and the positive samples were pooled then sequenced on the Illumina MiSeq platform for complete genome studies. Phylogenetic analysis was done using MEGA X software and Cucurbit aphid-borne yellows virus polerovirus (MZ508305.1) was used as a rooting outgroup. The PBMYV full genome sequences from Kenya (PBMYV_6, PBMYV_7) clustered together with other PBMYV and had closest sequence identity (91-95%) with PBMYV (KT963000.2, MT966033.1 and MT966038.1) from the GenBank. The PBMYV Kenyan strain is similarly diverse to the genetically distinct PBMYV Australian variants by descent. Further studies are needed to understand the contribution of climate change on the new disease distribution and molecular diversity of PBMYV in groundnut pathosystems.
 

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Increasing lines of evidence indicate that chloroplast-related genes are involved in plant–virus interactions. However, the involvement of photosynthesis-related genes in plant immunity is largely unexplored. RNA-Seq analysis showed that several chloroplast-related genes were strongly induced in response to infection with the avirulent strain of soybean mosaic virus (SMV), G5H, but were weakly induced in response to the virulent strain, G7H. For further analysis, we selected the PSaC gene from the photosystem I (PSI) and the ATP-synthase α-subunit (ATPsyn-α) gene whose encoded protein is part of the ATP-synthase complex. Overexpression of either gene within the G7H genome reduced virus levels in the susceptible cultivar Lee74 (rsv3-null). Both proteins are localized in the chloroplast envelope and the nucleus and cytoplasm. Because the chloroplast is the initial biosynthesis site of defense-related hormones, we determined whether hormone-related genes are involved in the ATPsyn-α- and PSaC-mediated defense. Interestingly, genes involved in the biosynthesis of several hormones were upregulated in plants infected with SMV-G7H expressing ATPsyn-α and PSaC. Both chimeras induced the expression of several antiviral RNA silencing genes, which indicates that such resistance may be partially achieved through the RNA silencing pathway. These findings highlight the role of photosynthesis-related genes in regulating resistance to viruses.

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The Potyvirus genus is one of the largest genera of plant RNA viruses responsible for serious diseases in crops worldwide. As potyviruses hijack the host secretory pathway and plasmodesmata (PD) for their transport, the goal of this study was to identify membrane and/or PD-proteins that interact with the 6K2 protein, a potyviral protein involved in replication and cell-to-cell movement of turnip mosaic virus (TuMV). Using Split-ubiquitin membrane Y2H assays we screened an Arabidopsis cDNA library for interactors of TuMV-6K2. We isolated AtHVA22a (Hordeum vulgare abscisic acid responsive gene 22) that belongs to a multigenic family of proteins homologous to DP1/Yop1 family proteins in yeast and interactors of reticulons. The role of HVA22 proteins in plants are not well-known, except the role in blast disease resistance in rice. Interestingly, proteomics analysis of PD fractions showed that AtHVA22a is highly enriched in Arabidopsis plasmodesmata proteome. We confirmed the interaction between 6K2 and AtHVA22a in yeast, as well as in planta by using bimolecular fluorescence complementation (BiFC) and showed that the interaction occurs at the level of the viral replication complexes (VRC) during TuMV infection. Finally, we showed that the propagation of TuMV in plants is increased when AtHVA22a is overexpressed but slowed down upon mutation of AtHVA22a by CRISPR-Cas9. Altogether, our results indicate that AtHVA22a plays an agonistic effect on TuMV propagation.

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Increasing evidence has suggested that NMD acts as a general virus restriction mechanism in eukaryotes. However, whether NMD factors, including SMG7 and UPF3, regulate virus infection is largely obscure. Here we show that overexpression of NbSMG7 and NbUPF3 attenuates cucumber green mottle mosaic virus (CGMMV) infection by targeting viral internal termination codon (iTC) and knock-down of NbSMG7 and NbUPF3 cooperatively facilitates virus infection. CGMMV infection upregulates the NbSMG7 transcription level but decreases its protein accumulation. Furthermore, NbSMG7, rather than NbUPF3, is subjected to autophagic degradation, which is executed by interacting with one of the autophagy-related proteins, NbATG8i. Mutation of the ATG8 interacting motif (AIM) in NbSMG7 (SMG7AIM1) abolishes its interaction with NbATG8i and comprises its autophagic degradation. Silencing of NbSMG7 and NbATG8i, or NbUPF3 and NbATG8i, compared to silencing of an individual one, leads to more virus accumulations, but overexpression of NbSMG7 and NbATG8i fails to achieve more potent virus inhibition compared to overexpressing one of them. However, overexpressing NbSMG7AIM1 and NbATG8i, or NbUPF3 and NbATG8i with CGMMV, exhibits more aggravated virus symptoms and more virus titers compared to their expression. These data show that both NMD and autophagy restrict virus infection, while NMD-mediated virus inhibition could be impaired by virus-induced autophagic degradation of NbSMG7.

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Cucumber green mottle mosaic virus (CGMMV)-induced watermelon blood flesh disease (WBFD) severely reduces the yield and edibleness of watermelon. We have found that application of exogenous boron can suppress CGMMV infection in watermelon fruit and alleviate WBFD symptoms. Here, the combined analysis results of proteomic and metabolomic showed that the main metabolic pathways of watermelon resistance to CGMMV infection by boron were concentrated in pyrimidine metabolism, sulfur metabolism, steroid biosynthesis, pyruvate metabolism, propanoate metabolism and photosynthesis. The most up-regulated differentially expressed genes (DEGs) were related to polyamine and auxin biosynthesis, abscisic acid catabolism, defense-related pathways, cell wall modification, and energy and secondary metabolism, while the down-regulated DEGs were mostly involved in ethylene biosynthesis, cell wall catabolism, and plasma membrane functions in transcriptome analysis. Additionally, in virus-induced gene silencing assays, silencing of SPDS, BG12, SBT, and TUBB1 expression in watermelon caused an inhibited CGMMV infection correlating with no WBFD symptoms. In contrast, silencing XTH23, PE/PEI7, GST, and ATPS1 expression promoted CGMMV accumulation, and UGDH, AST, 4CL4, RAP2-3, MYB6, WRKY12, H2A, and DnaJ11 are likely to participate in host antiviral resistance. Our results provide a novel molecular mechanism on the roles of boron in watermelon resistance to CGMMV-induced WBFD.

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Geminiviruses constitute the largest group of known plant viruses and cause devastating diseases and economic losses in many crops worldwide. Understanding plant antiviral defense against geminiviruses is critical for development of strategies for geminivirus control especially in the case of limited naturally occurring resistance genes. Here we identified NbWRKY1 as a positive regulator of plant defense against geminivirus infection. We found that NbWRKY1 was upregulated in response to tomato yellow leaf curl China virus/tomato yellow leaf curl China betasatellite (TYLCCNV/TYLCCNB) infection. Overexpression of NbWRKY1 attenuated TYLCCNV/TYLCCNB infection, whereas knockdown of NbWRKY1 enhanced plant susceptibility to TYLCCNV/TYLCCNB. We further revealed that NbWRKY1 bound to the promoter of the NbWHIRLY1 (NbWhy1) transcription factor and inhibited the transcription of NbWhy1. Consistently, NbWhy1 negatively regulates plant resistance against TYLCCNV/TYLCCNB. Furthermore, we demonstrated that NbWhy1 interfered with the antiviral RNAi defense and disrupted the interaction between NbCaM3 and NbCAMTA3. Moreover, the NbWRKY1-NbWhy1 also confers plant resistance toward tomato yellow leaf curl virus infection. Taken together, our findings suggest that NbWRKY1 positively regulates plant resistance to geminivirus infection by repressing NbWhy1. We propose that the NbWRKY1-NbWhy1 cascade could be further employed to control geminiviruses.

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Cucurbits are one of the highly cultivated and economically important crops worldwide but their production is often limited by plant viruses. In the recent years, yellowing diseases caused by viruses have been damaging to cucurbit crops in Korea. A survey of the incidence of yellowing viruses in melon, cucumber, and watermelon crops was conducted in Korea during 2022. Reverse-transcription polymerase chain reaction (RT-PCR) was performed with specific primers for cucurbit aphid-borne yellows virus (CABYV) and cucurbit chlorotic yellows virus (CCYV) in melon and cucumber and melon aphid-borne yellows virus (MABYV) in watermelon. The results showed that the infection rates of CABYV were 35.1% (n=259/738) for melon; 10.4% (n=78/747) for cucumber and the infection rates of CCYV were 9.2% (n=68/738) for melon; 11.2% (n=84/747) for cucumber. MABYV, which was first reported in 2021, was detected only in 6 greenhouses growing watermelon from 2 areas with an infection rate of 3.6% (n=20/562). Sequence analysis based on the complete genome sequences of CABYV, CCYV, and MABYV revealed 96~99% nucleotide identities, respectively, with previously reported sequences. Since the yellowing symptoms of the three viruses CABYV, CCYV, and MABYV are virtually identically and occur as mixed infections in cucurbits grown in Korea, we developed a multiplex RT-PCR method for rapid, sensitive, and simultaneous detection of the three viruses.

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Squash (Cucurbita moschata) is an important crop worldwide. However, cucurbit leaf curl disease (CuLCD), causing by whitefly-transmitted begomoviruses results significant yield losses. In Southeast Asia, three major bipartite begomoviruses, Tomato leaf curl New Delhi virus (ToLCNDV), Squash leaf curl China virus (SLCCNV) and Squash leaf curl Philippines virus (SLCuPV) are associated with CuLCD. Resistance squash cultivars are important for the disease control, but limited for resistant resources. So far, few squash lines were resistant to CuLCD. In Taiwan, SLCuPV is the major squash begomovirus. Two SLCuPV resistant lines, TOT8101A and Bi were screened by whitefly inoculation. The Bi line was also confirmed with SLCCNV resistance by agroinoculation of infectious virus. Furthermore, the resistance of TOT8101A is single recessive gene, and Bi is one dominance. Based on the F2 population generated by crossing of susceptible squash with Bi line, five resistance marker candidates were screened by sequence-related amplified polymorphism (SRAP). Consequently, one SRAP marker was verified in a F3 population. Following the sequence analysis of the resistance SRAP marker, it has 99.63% nucleotide identity with putative disease resistance protein at 3806932-3807467 nt of chromosome 6 of the squash whole genomic sequences. The co-dominant marker of the dominant resistance is under developing for marker-assessed selection in the squash breeding.

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Potato virus Y (PVY) is an economically important plant pathogen that reduces the productivity of a wide range of host plants. PVY causes symptoms such as stunted growth, severe chlorotic mosaic, and leaf curling affecting the lamina. To develop PVY-resistant cultivars, it would be essential to identify the plant-PVY interactome and decipher the biological significance of those molecular interactions. We performed a yeast two-hybrid (Y2H) screen of Nicotiana benthamiana cDNA library using PVY-encoded NIa-pro as the bait. The N. benthamiana Indole-3-acetic acid-amido synthetase (IAAS) was identified as an interactor of NIa-pro protein. The interaction was confirmed via targeted Y2H and bimolecular fluorescence complementation (BiFC) assays. We have shown the subcellular localization of both NIa-pro and IAAS protein in the nucleus and cytosol. IAAS converts free (active) IAA to the inactive, conjugated form, which plays a crucial regulatory role in auxin signaling. Transient silencing of IAAS in N. benthamiana plants interfered with the PVY-mediated symptom induction and virus accumulation. Conversely, overexpression of IAAS enhanced the symptoms induction and virus accumulation in the infected plants. In addition, the expression of several auxin-responsive genes was found to be downregulated during PVY infection. Our findings demonstrate that PVY NIa-pro protein potentially promotes disease development via modulating auxin homeostasis.

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Potato virus Y (PVY) is an important viral pathogen of potato. So far, the molecular plant-virus interactions underlying this pathogenicity are not fully understood. Gas chromatography coupled with mass spectroscopy (GC–MS) was used to study the changes in leaf metabolomes of PVY-resistant cv. Premier Russet (PR), and a susceptible cv. Russet Burbank (RB), following inoculation with three PVY strains, PVY-NTN, PVY N-Wi, and PVYO. Analysis of the resulting GC–MS spectra showed several common and strain-specific metabolites that are induced by PVY inoculation. In PR, the major overlap in differential accumulation was found between PVY N-Wi and PVYO. However, the 14 significant pathways occurred solely due to PVY N-Wi. In contrast, the main overlap in differential metabolite profiles and pathways in RB was between PVY-NTN and PVYO. Overall, limited overlap was observed between PVY-NTN and PVY N-Wi. As a result, PVY N-Wi induced necrosis may be mechanistically distinguishable from that of PVY-NTN. Furthermore, 10 common and seven cultivar-specific metabolites as potential indicators of PVY infection and susceptibility/resistance were identified. In RB, glucose-6-phosphate and fructose-6-phosphate were particularly affected by strain–time interaction. This highlights the relevance of the regulation of carbohydrate metabolism for defense against PVY. Some strain- and cultivar dependent metabolite changes were also observed. 

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Whether photosynthetic metabolites participate in host defenses against pathogens remains unclear. Here, we found the triose phosphate/phosphate translocator (TPT) located on the inner membrane of the chloroplast interacts with the movement protein (MP) of brassica yellows virus (BrYV) in vivo and in vitro, and its expression is significantly down-regulated by viral infection or transgenic expression of the MP. The loss-of-function mutants of tpt mutant shows increased accumulation of BrYV and conversely increased expression of TPT confers enhanced resistance. The antiviral activity of AtTPT requires its phosphate transport capacity, suggesting that it functions in fighting the virus via its transported substrate(s). To this end, we discovered that one of the TPT substrates, glyceraldehyde 3-phosphate (GAP), directly acts as an activator of the immune system to depress viral accumulation in leaves. At the mechanistic level, we revealed that exogenous application of GAP drastically triggers defense-related genes expression and prominently induces defense signaling pathways, such as MAPK. Both TPT and GAP demonstrate strong inhibitive activities against multiple types of plant pathogens. Collectively, we proposed that GAP exported by TPT to the cytosol triggers antimicrobial immunity and thus mediates broad-spectrum resistance to a variety of plant diseases by virtue of the chloroplast-to-nucleus/cytosol retrograde signaling.

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Plant viruses primarily consist of nucleic acids and proteins, and their degradation in host plants is considered as a defense mechanism against viruses. The RNA silencing mechanism, triggered by double-stranded RNAs derived from infected viruses, effectively degrade the viral genomic RNA. Meanwhile, growing evidences suggest that autophagy (AP), a major proteolytic system, plays a crucial role in antiviral defense in plants. Our earlier research demonstrated that RNA silencing and AP work in tandem against cucumber mosaic virus (CMV). To counteract antiviral RNA silencing, most plant viruses express RNA silencing suppressors; CMV has the 2b protein (2b). AP strengthens RNA silencing against CMV by targeting 2b through the calmodulin-like protein called rgs-CaM for degradation. In this study, we explored the interactions between CMV and AP by creating AP-defective Nicotiana benthamiana plants. Upon inoculating the CMV vectors that express foreign proteins by replacing 2b with the genes encoding these foreign proteins, we observed a great increase in CMV multiplication and accumulation of these foreign proteins. Our findings suggest that: 1) CMV-encoded protein levels may be affected by autophagy in addition to 2b, and 2) AP-defective N. benthamiana plants can serve as an excellent platform plant to produce useful proteins using the CMV vectors.