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As part of the development of alternative and environmentally friendly control against phytopathogenic fungi, Burkholderia cepacia could be a useful species notably via the generation of hydrolytic enzymes like chitinases, which can act as a biological control agent. Here, a Burkholderia contaminans S614 strain exhibiting chitinase activity was isolated from a soil in southern Tunisia. Then, response surface methodology (RSM) with a central composite design (CCD) was used to assess the impact of five factors (colloidal chitin, magnesium sulfate, dipotassium phosphate, yeast extract, and ammonium sulfate) on chitinase activity. B. contaminans strain 614 growing in the optimized medium showed up to a 3-fold higher chitinase activity. This enzyme was identified as beta-N-acetylhexosaminidase (90.1 kDa) based on its peptide sequences, which showed high similarity to those of Burkholderia lata strain 383. Furthermore, this chitinase significantly inhibited the growth of two phytopathogenic fungi: Botrytis cinerea M5 and Phoma medicaginis Ph8. Interestingly, a crude enzyme from strain S614 was effective in reducing P. medicaginis damage on detached leaves of Medicago truncatula. Overall, our data provide strong arguments for the agricultural and biotechnological potential of strain S614 in the context of developing biocontrol approaches.

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Alfalfa spring black stem and leaf spot disease (ASBS) is a cosmopolitan soil-borne and seed-borne disease caused by Phoma medicaginis, which adversely affects the yield, and nutritive value and can stimulate production of phyto-oestrogenic compounds at levels that may adversely affect ovulation rates in animals. This review summarizes the host range, damage, and symptoms of this disease, and general features of the infection cycle, epidemic occurrence, and disease management. ASBS has been reported from over 40 countries, and often causes severe yield loss. Under greenhouse conditions, reported yield loss was 31-82% for roots, 32-80% for leaves, 21% for stems and 26-28% for seedlings. In field conditions, the forage yield loss is up to 56%, indicating that a single-cut yield of 5302 kg/ha would be reduced to 2347 kg/ha. P. medicaginis can infect up to 50 species of plants, including the genera Medicago, Trifolium, Melilotus, and Vicia. ASBS is more severe during warm spring conditions before the first harvest than in hot summer and cooler winter conditions, and can infect alfalfa roots, stems, leaves, flowers, pods, and seeds, with leaf spot and/or black stem being the most typical symptoms. The primary infection is caused by the overwintering spores and mycelia in the soil, and on seeds and the cortex of dead and dry stems. The use of resistant cultivars is the most economical and effective strategy for the control of ASBS. Although biological control has been studied in the glasshouse and is promising, chemical control is the main control method in agriculture.

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Salinity and Phoma medicaginis infection represent significant challenges for alfalfa cultivation in South Africa, Europe, Australia, and, particularly, Tunisia. These constraints have a severe impact on both yield and quality. The primary aim of this study was to establish the genetic basis of traits associated with biomass and growth of 129 Medicago sativa genotypes through genome-wide association studies (GWAS) under combined salt and P. medicaginis infection stresses. The results of the analysis of variance (ANOVA) indicated that the variation in these traits could be primarily attributed to genotype effects. Among the test genotypes, the length of the main stem, the number of ramifications, the number of chlorotic leaves, and the aerial fresh weight exhibited the most significant variation. The broad-sense heritability (H²) was relatively high for most of the assessed traits, primarily due to genetic factors. Cluster analysis, applied to morpho-physiological traits under the combined stresses, revealed three major groups of accessions. Subsequently, a GWAS analysis was conducted to validate significant associations between 54,866 SNP-filtered single-nucleotide polymorphisms (SNPs) and seven traits. The study identified 27 SNPs that were significantly associated with the following traits: number of healthy leaves (two SNPs), number of chlorotic leaves (five SNPs), number of infected necrotic leaves (three SNPs), aerial fresh weight (six SNPs), aerial dry weight (nine SNPs), number of ramifications (one SNP), and length of the main stem (one SNP). Some of these markers are related to the ionic transporters, cell membrane rigidity (related to salinity tolerance), and the NBS_LRR gene family (associated with disease resistance). These findings underscore the potential for selecting alfalfa genotypes with tolerance to the combined constraints of salinity and P. medicaginis infection.

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Plant disease occurs simultaneously with insect attack. Arbuscular mycorrhizal fungi (AMF) modify plant biotic stress response. Arbuscular mycorrhizal fungi and pathogens may modify plant volatile organic compound (VOC) production and insect behavior. Nevertheless, such effects are rarely studied, particularly for mesocosms where component organisms interact with each other. Plant-mediated effects of leaf pathogen (Phoma medicaginis) infection on aphid (Acyrthosiphon pisum) infestation, and role of AMF (Rhizophagus intraradices) in modifying these interactions were elucidated in a glasshouse experiment. We evaluated alfalfa disease occurrence, photosynthesis, phytohormones, trypsin inhibitor (TI) and total phenol response to pathogen and aphid attack, with or without AMF, and aphid behavior towards VOCs from AMF inoculated and non-mycorrhizal alfalfa, with or without pathogen infection. AM fungus enhanced alfalfa resistance to pathogen and aphid infestation. Plant biomass, root : shoot ratio, net photosynthetic rate, transpiration rate, stomatal conductance, salicylic acid, and TI were significantly increased in AM-inoculated alfalfa. Arbuscular mycorrhizal fungi and pathogen significantly changed alfalfa VOCs. Aphids preferred VOCs of AM-inoculated and nonpathogen-infected to nonmycorrhizal and pathogen-infected alfalfa. We propose that AMF alter plant response to multiple biotic stresses in ways both beneficial and harmful to the plant host, providing a basis for strategies to manage pathogens and herbivore pests.

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Spring black stem and leaf spot, caused by Phoma medicaginis, is an issue in annual Medicago species. Therefore, in this study, we analyzed the response to P. medicaginis infection in a collection of 46 lines of three annual Medicago species (M. truncatula, M. ciliaris, and M. polymorpha) showing different geographic distribution in Tunisia. The reaction in the host to the disease is explained by the effects based on plant species, lines nested within species, treatment, the interaction of species × treatment, and the interaction of lines nested within species × treatment. Medicago ciliaris was the least affected for aerial growth under infection. Furthermore, the largest variation within species was found for M. truncatula under both conditions. Principal component analysis and hierarchical classification showed that M. ciliaris lines formed a separate group under control treatment and P. medicaginis infection and they are the most vigorous in growth. These results indicate that M. ciliaris is the least susceptible in response to P. medicaginis infection among the three Medicago species investigated here, which can be used as a good candidate in crop rotation to reduce disease pressure in the field and as a source of P. medicaginis resistance for the improvement of forage legumes.

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Plant growth-promoting rhizobacteria (PGPR) such as the root colonizers Bacillus spp. may be ideal alternatives to chemical crop treatments. This work sought to extend the application of the broadly active PGPR UD1022 to Medicago sativa (alfalfa). Alfalfa is susceptible to many phytopathogens resulting in losses of crop yield and nutrient value. UD1022 was cocultured with four alfalfa pathogen strains to test antagonism. We found UD1022 to be directly antagonistic toward Collectotrichum trifolii, Ascochyta medicaginicola (formerly Phoma medicaginis), and Phytophthora medicaginis, and not toward Fusarium oxysporum f. sp. medicaginis. Using mutant UD1022 strains lacking genes in the nonribosomal peptide (NRP) and biofilm pathways, we tested antagonism against A. medicaginicola StC 306-5 and P. medicaginis A2A1. The NRP surfactin may have a role in the antagonism toward the ascomycete StC 306-5. Antagonism toward A2A1 may be influenced by B. subtilis biofilm pathway components. The B. subtilis central regulator of both surfactin and biofilm pathways Spo0A was required for the antagonism of both phytopathogens. The results of this study indicate that the PGPR UD1022 would be a good candidate for further investigations into its antagonistic activities against C. trifolii, A. medicaginicola, and P. medicaginis in plant and field studies.

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Introduction: Arbuscular mycorrhizal (AM) fungi are important for the resistance of plants to insect infestation and diseases. However, the effect of AM fungal colonization of plants response to pathogen infection activated by pea aphid infestation is unknown. Pea aphid (Acyrthosiphon pisum) and the fungal pathogen Phoma medicaginis severely limit alfalfa production worldwide. Methods: This study established an alfalfa (Medicago sativa)-AM fungus (Rhizophagus intraradices)-pea aphid-P. medicaginis experimental system to clarify the effects of an AM fungus on the host plant response to insect infestation and subsequent fungal pathogen infection. Results: Pea aphid increased the disease incidence of P. medicaginis by 24.94%. The AM fungus decreased the disease index by 22.37% and enhanced alfalfa growth by increasing the uptake of total nitrogen and total phosphorus. The aphid induced polyphenol oxidase activity of alfalfa, and the AM fungus enhanced plant-defense enzyme activity against aphid infestation and subsequent P. medicaginis infection. In addition, the AM fungus increased the contents of jasmonic acid and abscisic acid in plants exposed to aphid infestation or pathogen infection. Abscisic acid and genes associated with the gene ontology term "hormone binding" were upregulated in aphid-infested or pathogen-infected alfalfa. Discussion: The results demonstrate that an AM fungus enhances plant defense and signaling components induced by aphid infestation, which may contribute to improved defense against subsequent pathogen infection.

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Phoma black stem and leaf spot disease of annual Medicago spp., caused by Phoma medicaginis, not only can devastate forage and seed yield but can reduce herbage quality by inducing production of phytoestrogens (particularly coumestrol and 4'-O-methylcoumestrol), which can also reduce the ovulation rates of animals grazing infected forage. We determined the consequent phytoestrogen levels on three different annual Medicago species/cultivars (Medicago truncatula cultivar Cyprus, Medicago polymorpha var. brevispina cultivar Serena, and Medicago murex cultivar Zodiac) after inoculation with 35 isolates of P. medicaginis. Across the isolate × cultivar combinations, leaf disease incidence, petiole/stem disease incidence, leaf disease severity, petiole disease severity, and leaf yellowing severity ranged up to 100, 89.4, 100, 58.1, and 61.2%, respectively. Cultivars Cyprus and Serena were the most susceptible and cultivar Zodiac was the most resistant to P. medicaginis. Isolates WAC3653, WAC3658, and WAC4252 produced the most severe disease. Levels of phytoestrogens in stems ranged from 25 to 1,995 mg/kg for coumestrol and from 0 to 418 mg/kg for 4'-O-methylcoumestrol. There was a significant positive relationship of disease incidence and severity parameters with both coumestrol and 4'-O-methylcoumestrol contents, as noted across individual cultivars and across the three cultivars overall, where r = 0.39 and 0.37 for coumestrol and 4'-O-methylcoumestrol, respectively (P < 0.05). Although cultivar Serena was most susceptible to P. medicaginis and produced the highest levels of phytoestrogens in the presence of P. medicaginis, cultivar Zodiac contained the highest levels of phytoestrogens in comparison with other cultivars in the absence of P. medicaginis. There was a 15-fold increase in coumestrol in cultivar Serena but only a 7-fold increase in cultivar Zodiac from infection of P. medicaginis. The study highlights that the intrinsic ability of a particular cultivar to produce phytoestrogens in the absence of the pathogen, and its comparative ability to produce phytoestrogens in the presence of the P. medicaginis, are both important and highly relevant to developing new annual Medicago spp. cultivars that offer improved disease resistance and better animal reproductive outcomes.

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Didymella pinodella is the major pathogen of the pea root rot complex in Europe. This wide host range pathogen often asymptomatically colonizes its hosts, making the control strategies challenging. We developed a real-time PCR assay for the detection and quantification of D. pinodella based on the TEF-1 alpha gene sequence alignments. The assay was tested for specificity on a 54-isolate panel representing 35 fungal species and further validated in symptomatic and asymptomatic pea and wheat roots from greenhouse tests. The assay was highly consistent across separate qPCR reactions and had a quantification/detection limit of 3.1 pg of target DNA per reaction in plant tissue. Cross-reactions were observed with DNA extracts of five Didymella species. The risk of cross contamination, however, is low as the non-targets have not been associated with pea previously and they were amplified with at least 1000-fold lower sensitivity. Greenhouse inoculation tests revealed a high correlation between the pathogen DNA quantities in pea roots and pea root rot severity and biomass reduction. The assay also detected D. pinodella in asymptomatic wheat roots, which, despite the absence of visible root rot symptoms, caused wheat biomass reduction. This study provides new insights into the complex life style of D. pinodella and can assist in better understanding the pathogen survival and spread in the environment.

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AIMS: One of the major limitations to the production of alfalfa (Medicago sativa) is the fungus Phoma medicaginis, which infects alfalfa and causes leaf spots. This study aims to understand alfalfa's response to P. medicaginis infection, the colonization of arbuscular mycorrhizal fungus (AMF) and the effect of AMF on plant-pathogen interactions. METHODS AND RESULTS: Transcriptome analysis (RNA-seq) was used to identify differentially expressed genes (DEGs) in alfalfa infected by P. medicaginis and colonized by AMF Rhizophagus intraradices. AMF ameliorated the effects of P. medicaginis infection on alfalfa by reducing leaf spot incidence and disease index by 39·48 and 56·18% respectively. Inoculation with pathogen and AMF induced the activity of defence pathways, including peroxidase (POD), polyphenol oxidase activities and jasmonic acid (JA), salicylic acid concentration. Plants showed differential expression of P. medicaginis resistance-related genes, including genes belonging to pathogenesis-related (PR) proteins, chitinase activity, flavonoid biosynthesis, phenylpropanoid biosynthesis, glutathione metabolism, phenylalanine metabolism and photosynthesis. Inoculation with AMF led to changes in the expression of genes involved in PR proteins, chitinase activity, phenylalanine metabolism and photosynthesis. CONCLUSION: The physiological and transcriptional changes caused by P. medicaginis infection in non-mycorrhizal and mycorrhizal alfalfa provides crucial information for understanding AMF's association with pathogenic systems. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that AMF alleviated alfalfa leaf spots demonstrating that AMF can serve as a biocontrol strategy for alfalfa disease management.

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Phoma medicaginis (syn. Ascochyta medicaginicola Qchen & L. Cai) causes spring black stem and leaf spot of alfalfa and the model legume Medicago truncatula. Phoma medicaginis produces uninucleate conidia in melanized pycnidia and is genetically tractable through Agrobacterium tumefaciens-mediated transformation (ATMT), which can result in insertional mutants. One T-DNA-tagged mutant, P1A17 produced conidia in non-melanized (hyaline) pycnidia. Pycnidial melanization recovered if the mutant was supplemented with melanin precursors or allowed to age. DNA sequences flanking the insertion did not predict any disrupted open reading frames (ORF) unless a Coccidioides prediction algorithm was used. Pmhyp gene was expressed in the wild type, but not the mutant, and has not been annotated in any genomes, to date. Expression of two conserved genes flanking the T-DNA disrupted Pmhyp was unchanged from the wild type. Knockout of Pmhyp strain displayed same cultural phenotype (non-melanized pycnidia). Complementation of Pmhyp strains with wild type PmHYP partially recovered pycnidial melanization. Both knockout and complementation transformants were confirmed using RT-PCR and southern blot analysis. Taken together, PmHYP appears to be a novel regulator of pycnidium specific melanization.

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Phoma medicaginis (syn. Ascochyta medicaginicola Qchen & L. Cai) causes spring black stem and leaf spot, an important disease of alfalfa and annual medics. P. medicaginis forms uninucleate conidia in melanized pycnidia and is genetically tractable using Agrobacterium mediated transformation (ATMT), resulting in random integration of T-DNA that occasionally generates pycnidial mutants. The T-DNA tagged mutant, P265 displayed smaller pycnidia and more aerial hyphae than the wild type. A single T-DNA disrupted a putative noncanonical poly(A) RNA polymerase gene, Pmncpap1, which in yeast interacts with ribonucleotide reductase (RNR). As in yeast mutants, P265 showed sensitivity to hydroxyurea (HU), a RNR inhibitor. To characterize the role of Pmncpap1, targeted ΔPmncpap1 mutants were created using a hygromycin selectable marker flanked by 1 Kbp regions of Pmncpap1. ΔPmncpap1 mutants possessed similar morphological features to those of P265. The plasmid for rescue of PmncPAP1, pCAM-Nat1 (nourseothricin selection) was constructed and used to introduce full-length PmncPAP1 into mutants. Rescued P265 showed partial recovery of wild type and the original T-DNA was lost due to homologous integration. To our knowledge, this is the first ncPAP to be examined in a filamentous fungus.

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