Multiple Plant Pathogens Research Articles

The NRC0 gene cluster of sensor and helper NLR immune receptors is functionally conserved across asterid plants.

Nucleotide-binding domain and leucine-rich repeat-containing receptor (NLR) proteins can form complex receptor networks to confer innate immunity. An NLR-REQUIRED FOR CELL DEATH (NRC) is a phylogenetically related node that functions downstream of a massively expanded network of disease resistance proteins that protect against multiple plant pathogens. In this study, we used phylogenomic methods to reconstruct the macroevolution of the NRC family. One of the NRCs, termed NRC0, is the only family member shared across asterid plants, leading us to investigate its evolutionary history and genetic organization. In several asterid species, NRC0 is genetically clustered with other NLRs that are phylogenetically related to NRC-dependent disease resistance genes. This prompted us to hypothesize that the ancestral state of the NRC network is an NLR helper-sensor gene cluster that was present early during asterid evolution. We provide support for this hypothesis by demonstrating that NRC0 is essential for the hypersensitive cell death that is induced by its genetically linked sensor NLR partners in 4 divergent asterid species: tomato (Solanum lycopersicum), wild sweet potato (Ipomoea trifida), coffee (Coffea canephora), and carrot (Daucus carota). In addition, activation of a sensor NLR leads to higher-order complex formation of its genetically linked NRC0, similar to other NRCs. Our findings map out contrasting evolutionary dynamics in the macroevolution of the NRC network over the last 125 million years, from a functionally conserved NLR gene cluster to a massive genetically dispersed network.

Rouxiella badensis, a new bacterial pathogen of onion causing bulb rot.

Onion bulb rot can be caused by multiple plant pathogens including bacterial pathogens. During our routine survey of commercial onion farms in 2014, 2020, and 2021, seven putative Rouxiella spp. strains were isolated from symptomatic onion samples in Georgia, United States. Upon fulfilling Koch's postulates on onion, a genome analysis was conducted. Whole-genome indices (ANI and dDDH) showed that the strains belonged to Rouxiella badensis. Although the seven R. badensis strains were not pathogenic on onion foliage, the strains were able to cause bulb rot and could also produce necrotic lesions in a red onion scale assay. R. badensis populations increased significantly and to a level comparable to P. ananatis PNA 97-1R in a red onion scale infection assay. The core-genome analysis grouped all onion R. badensis strains from Georgia together, and distinct from R. badensis strains isolated from other sources and locations. Based on the genome analysis of strains (from the current study and available genomes in the repository), type I, III (Ssa-Esc and Inv-Mxi-Spa types), and V secretion systems are present in R. badensis genomes, while type II, IV, and VI secretion systems are absent. However, various secondary metabolite gene clusters were identified from R. badensis genomes, and a thiol/redox-associated enzyme gene cluster similar to the Pantoea alt cluster mediating thiosulfinate tolerance was also present in onion strains of R. badensis. This is the first report of R. badensis as a plant pathogen.

Paecilomyces variotii extract increases lifespan and protects against oxidative stress in Caenorhabditis elegans through SKN-1, but not DAF-16

A natural extract from Paecilomyces variotii ( P. variotii extract, PVE), an endophytic fungus, has been used widely to improve agricultural crop performance and control multiple plant pathogens. Most recent studies focused on its application as a plant growth promoter, while relatively few studies have been reported on the antioxidant potential in vivo and the underlying mechanism. The present study was designed to determine the antioxidant activities of PVE and its mechanisms using Caenorhabditis elegans . Results showed that, compared to the solvent control, PVE at 1.0, 10 and 100 ng/mL significantly extended the lifespan of C. elegans by 36.60%, 59.80% and 53.30%, respectively. PVE at 10 ng/mL consistently promoted nematodes growth, but all treatments did not influence nematode fecundity, locomotion behavior, and pharyngeal pumping. Furthermore, PVE at the three tested concentrations significantly reduced accumulation of reactive oxygen species (ROS), lipofuscin, lipid and malondialdehyde (MDA) content, meanwhile significantly promoted activities of superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) in the nematodes. Compared with the solvent control, PVE up-regulated gene expression of skn-1 , mev-1 , sod-3 , and daf-2 , but significantly down-regulated the expression of nhr-49 and daf-16 . Further evidence revealed that PVE at the three concentrations significantly promoted nuclear localization of SKN-1, but not affected that of DAF-16, indicating the complex roles of DAF-16 and SKN-1 in stress resistance and longevity regulation. Overall, our results demonstrated that SKN-1 played a critical role in increasing lifespan of C. elegans and protecting the nematodes from oxidative stress, independent of DAF-16.

Biocontrol potential of Trichoderma harzianum against Botrytis cinerea in tomato plants

Trichoderma harzianum (TM) is a soil-borne beneficial fungus that positively affects plant growth and defense. TM shows antagonism against multiple plant pathogens; however, the TM as a biocontrol agent against Botrytis cinerea (BC) and underlying mechanisms remain unclear. Herein we studied the effects of TM on BC by dual-culture assay and greenhouse bioassay in tomato plants. In vitro experiments showed that TM rapidly occupied most of the nutrient space and eventually covered BC on potato dextrose agar (PDA) medium. The TM inhibition rate against BC was 62.05% on the sixth day of culture. The inhibitory effect of TM spore suspension on gray mold inoculated with spore suspension and solid inoculants was 33.66% and 69.44%, respectively in tomato plants. Notably, TM decreased the leaf concentrations of BC-induced hydrogen peroxide and malondialdehyde but improved the redox state. TM inoculation caused biphasic peaks in the expression of secondary metabolism and defense-related genes and increased the concentrations of flavonoids, phenols and lignin in tomato leaves. Moreover, TM alleviated stomatal closure and enhanced the net photosynthesis rate and the activity of RuBisCO and FBPase compared with only BC inoculation. Our results suggest that TM spore suspension improves tomato resistance to gray mold disease by stimulating cellular redox, secondary metabolites and stomatal movement.

Expression of the ripAA Gene in the Soilborne Pseudomonas mosselii Can Promote the Control Efficacy against Tobacco Bacterial Wilt.

Simple SummaryTobacco bacterial wilt caused by Ralstonia solanacearum brings large economic losses every year. Currently, an increasing number of biocontrol agents have been widely used in the control of this disease, but they cannot replace chemical agents, mostly due to the poorer control effect. Therefore, in this study, the avirulence gene ripAA from Ralstonia solanacearum, which determines incompatible interactions with tobacco plants, was introduced into the biocontrol agent Pseudomonas mosselii to increase the efficacy against Ralstonia solanacearum. The newly engineered strain can improve the systemic resistance and elicit a primary immune response of plants. Our research not only provides a new strategy for the genetic modification of biocontrol agents, in which a number of avirulence genes from the pathogen or plant can be tested to be expressed in different biocontrol agents to antagonize this plant disease, but also helps the study of the interaction between the phytopathogenic avirulence gene and the host.The environmental bacterium Pseudomonas mosselii produces antagonistic secondary metabolites with inhibitory effects on multiple plant pathogens, including Ralstonia solanacearum, the causal agent of bacterial wilt. In this study, an engineered P. mosselii strain was generated to express R. solanacearum ripAA, which determines the incompatible interactions with tobacco plants. The ripAA gene, together with its native promoter, was integrated into the P. mosselii chromosome. The resulting strain showed no difference in antimicrobial activity against R. solanacearum. Promoter-LacZ fusion and RT-PCR experiments demonstrated that the ripAA gene was transcribed in culture media. Compared with that of the wild type, the engineered strain reduced the disease index by 9.1% for bacterial wilt on tobacco plants. A transcriptome analysis was performed to identify differentially expressed genes in tobacco plants, and the results revealed that ethylene- and jasmonate-dependent defense signaling pathways were induced. These data demonstrates that the engineered P. mosselii expressing ripAA can improve biological control against tobacco bacterial wilt by the activation of host defense responses.

QTL mapping and colocalization analysis reveal novel candidate genes for multiple disease resistance in maize

AbstractUnpredictable pathogen pressures resulting from changing climatic patterns have required plant breeders to breed crop cultivars with durable resistance to multiple plant pathogens. The genetic basis of multiple disease resistance (MDR) is an important component in building durable resistance in crop plants. Maize (Zea mays L.) inbred NY22613, developed at Cornell University, has shown resistance to northern leaf blight (NLB), gray leaf spot (GLS), common rust, and Stewart's wilt (SW). In order to unravel the genetic basis of MDR in NY22613, quantitative trait locus (QTL) mapping and colocalization studies were conducted. A BC3S3 biparental mapping population (resistant inbred NY22613 and susceptible recurrent inbred Oh7B) was used to map the QTL responsible for disease resistance. The analysis revealed that 16 QTL were associated with NLB resistance, 17 QTL with GLS resistance, and 16 QTL with SW resistance. No QTL were colocalized for all three diseases. Three QTL were shared for NLB and GLS in chromosomes 7, 9, and 10 and one QTL was shared for GLS and SW in chromosome 2. These QTL contain the following genes that have been previously associated with disease resistance: Zm00001d002005 (lg1, liguleless1), Zm00001d024007, Zm00001d024008, LOC103642379, and LOC103641009. These regions also contain the following novel genes, which have not been associated with any function: LOC541757, Zm00001d044974, Zm00001d044975, and LOC109941121. To select individuals with MDR, a novel selection method was developed that combines phenotypic data, QTL data, and high‐density marker information in a cluster analysis designated as the high‐density marker phenotype (HEMP) QTL selection strategy.

Complete Genome Sequence Resource for the Endophytic Burkholderia sp. Strain MS389 Isolated from a Healthy Soybean Growing Adjacent to Charcoal Rot Disease Patch.

Burkholderia sp. strain MS389, an endophytic bacterium, was isolated from a healthy soybean plant growing adjacent to a patch of plants affected by charcoal rot disease, caused by the fungal pathogen Macrophomina phaseolina. Preliminary studies demonstrated that strain MS389 possesses antimicrobial activities against multiple plant pathogens. Burkholderia sp. strain MS389 was found to have three circular chromosomes of 3,563,380 bp, 3,002,449 bp, and 1,180,421 bp in size, respectively. The 7,746,250-bp genome, with 66.73% G+C content, harbors 6,756 protein coding genes in the predicted 6,985 genes. In total, 18 rRNAs, 68 tRNAs, and four ncRNAs were identified and 139 pseudogenes were annotated as well. The findings of this study will provide valuable data to explore the antimicrobial mechanisms of the endophytic bacterial strain.

Synthesis and fungicidal activity of phenazine-1-carboxylic triazole derivatives

A total of 15 novel-substituted 3-(benzylsulfanyl)-1H-1,2,4-triazol-5-ylamine and 10 novel-substituted 3-benzylmercapto-1,2,4-triazol derivatives were synthesized based on the natural product phenazine-1-carboxylic acid (PCA). Their structures were confirmed by 1H-NMR, 13C-NMR, HRMS, and X-ray. Most substituted 3-benzylmercapto-1,2,4-triazol derivatives displayed very strong fungicidal activity against one or multiple plant pathogens in vitro and in vivo. Compounds 8b, 8h, and 8i showed a broad spectrum of fungicidal activity. Further field experiments indicated that compounds 8b, 8c, and 8h displayed better efficacy against rice blast (Pyricularia oryzae) than PCA. These data demonstrate that compounds 8b, 8c, and 8h are promising fungicidal candidates, deserving further studies.

Floral and Foliar Source Affect the Bee Nest Microbial Community.

Managed pollinators such as the alfalfa leafcutting bee, Megachile rotundata, are essential to the production of a wide variety of agricultural crops. These pollinators encounter a diverse array of microbes when foraging for food and nest-building materials on various plants. To test the hypothesis that food and nest-building source affects the composition of the bee-nest microbiome, we exposed M. rotundata adults to treatments that varied both floral and foliar source in a 2 × 2 factorial design. We used 16S rRNA gene and internal transcribed spacer (ITS) sequencing to capture the bacterial and fungal diversity of the bee nests. We found that nest microbial communities were significantly different between treatments, indicating that bee nests become inoculated with environmentally derived microbes. We did not find evidence of interactions between the fungi and bacteria within our samples. Furthermore, both the bacterial and fungal communities were quite diverse and contained numerous exact sequence variants (ESVs) of known plant and bee pathogens that differed based on treatment. Our research indicates that bees deposit plant-associated microbes into their nests, including multiple plant pathogens such as smut fungi and bacteria that cause blight and wilt. The presence of plant pathogens in larval pollen provisions highlights the potential for bee nests to act as disease reservoirs across seasons. We therefore suggest that future research should investigate the ability of bees to transmit pathogens from nest to host plant.

Detection and qPCR quantification of seven Fusarium species associated with the root rot complex in field pea

Multiple plant pathogens cause root rot on field pea, including Aphanomyces euteiches, Rhizoctonia solani and numerous Fusarium spp. In North Dakota, root rot pathogens have been a contributing factor in the decline in area planted to field pea over the past decade. Real-time quantitative PCR (qPCR) has proven valuable in quantifying a multitude of host–pathogen interactions. However, no qPCR assays are available to effectively quantify Fusarium spp. associated with root rot in field pea. The objective of this study was to develop multiplex qPCR assays to quantify seven Fusarium spp. commonly associated with field pea (F. acuminatum, F. avenaceum, F. culmorum, F. graminearum, F. redolens, F. solani and F. sporotrichioides). Primers and associated hydrolysis probes designed for each Fusarium sp. were combined into three multiplexed assays. Assay amplification efficiencies ranged from 93 to 108 and the regression coefficient for all assays was greater than 0.97 on serial dilutions of pure fungal DNA. All three multiplex assays amplified down to 40 pg µL−1 Fusarium DNA. Multiplex qPCR assays proved sensitive and specific based on evaluations of pea plants inoculated under greenhouse conditions and plants displaying symptoms of root rot collected from commercial pea fields. Annealing temperatures and reaction components were the same for all assays, providing interchangeability and versatility in each multiplexed assay. These assays will aid researchers in advancing the information available for the Fusarium–host interactions for many important crop systems.

Cyclic Dipeptides from Bacillus vallismortis BS07 Require Key Components of Plant Immunity to Induce Disease Resistance in Arabidopsis against Pseudomonas Infection.

Cyclic dipeptides (CDPs) are one of the simplest compounds produced by living organisms. Plant-growth promoting rhizobacteria (PGPRs) also produce CDPs that can induce disease resistance. Bacillus vallismortis strain BS07 producing various CDPs has been evaluated as a potential biocontrol agent against multiple plant pathogens in chili pepper. However, plant signal pathway triggered by CDPs has not been fully elucidated yet. Here we introduce four CDPs, cyclo(Gly-L-Pro) previously identified from Aspergillus sp., and cyclo(L-Ala-L-Ile), cyclo(L-Ala-L-Leu), and cyclo(LLeu-L-Pro) identified from B. vallismortis BS07, which induce disease resistance in Arabidopsis against Pseudomonas syringae infection. The CDPs do not directly inhibit fungal and oomycete growth in vitro. These CDPs require PHYTOALEXIN DEFICIENT4, SALICYLIC ACID INDUCTION DEFICIENT2, and NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 important for salicylic acid-dependent defense to induce resistance. On the other hand, regulators involved in jasmonate-dependent event, such as ETHYLENE RECEPTOR1, JASMONATE RESPONSE1, and JASMONATE INSENSITIVE1, are necessary to the CDP-induced resistance. Furthermore, treatment of these CDPs primes Arabidopsis plants to rapidly express PATHOGENESIS-RELATED PROTEIN4 at early infection phase. Taken together, we propose that these CDPs from PGPR strains accelerate activation of jasmonate-related signaling pathway during infection.

Bioprospecting the fungicides compatible Trichoderma asperellum isolate effective against multiple plant pathogens in vitro

Bioprospecting the fungicides compatible Trichoderma asperellum isolate effective against multiple plant pathogens in vitro

An accurate, specific, sensitive, high-throughput method based on a microsphere immunoassay for multiplex detection of three viruses and bacterial fruit blotch bacterium in cucurbits

To employ a microsphere immunoassay (MIA) to simultaneously detect multiple plant pathogens (potyviruses, Watermelon silver mottle virus, Melon yellow spot virus, and Acidovorax avenae subsp. citrulli) in actual plant samples, several factors need to be optimized and rigorously validated. Here, a simple extraction method using a single extraction buffer was successfully selected to detect the four pathogens in various cucurbit samples (cucumber, cantaloupe, melon, and watermelon). The extraction method and assay performance were validated with inoculated and field cucurbit samples. The MIA showed 98–99% relative accuracy, 97–100% relative specificity and 92–100% relative sensitivity when compared to commercial ELISA kits and reverse transcription PCR. In addition, the MIA was also able to accurately detect multiple-infected field samples. The results demonstrate that one common extraction method for all tested cucurbit samples could be applied to detect multiple pathogens; avoiding the need for multiple protocols to be employed. This multiplex method can therefore be instrumental for high-throughput screening of multiple plant pathogens with many advantages such as a shorter assay time (2.5h) with single assay format, a lower cost of detection ($5 vs $19.7 for 4 pathogens/sample) and less labor requirement. Its multiplex capacity can also be expanded to detect up to 50 different pathogens upon the availability of specific antibodies.

Pseudomonas syringae type III effector HopAF1 suppresses plant immunity by targeting methionine recycling to block ethylene induction

HopAF1 is a type III effector protein of unknown function encoded in the genomes of several strains of Pseudomonas syringae and other plant pathogens. Structural modeling predicted that HopAF1 is closely related to deamidase proteins. Deamidation is the irreversible substitution of an amide group with a carboxylate group. Several bacterial virulence factors are deamidases that manipulate the activity of specific host protein substrates. We identified Arabidopsis methylthioadenosine nucleosidase proteins MTN1 and MTN2 as putative targets of HopAF1 deamidation. MTNs are enzymes in the Yang cycle, which is essential for the high levels of ethylene biosynthesis in Arabidopsis We hypothesized that HopAF1 inhibits the host defense response by manipulating MTN activity and consequently ethylene levels. We determined that bacterially delivered HopAF1 inhibits ethylene biosynthesis induced by pathogen-associated molecular patterns and that Arabidopsis mtn1 mtn2 mutant plants phenocopy the effect of HopAF1. Furthermore, we identified two conserved asparagines in MTN1 and MTN2 from Arabidopsis that confer loss of function phenotypes when deamidated via site-specific mutation. These residues are potential targets of HopAF1 deamidation. HopAF1-mediated manipulation of Yang cycle MTN proteins is likely an evolutionarily conserved mechanism whereby HopAF1 orthologs from multiple plant pathogens contribute to disease in a large variety of plant hosts.

A simple, rapid and visual antibody array for the simultaneous detection of multiple plant pathogens

Most traditional methods for pathogen detection, such as the ELISA and PCR, can only detect a single target pathogen at any one time. Here, an antibody array assay system has been developed to detect multiple pathogens in plants at the same time. The system provides a rapid, visual and cheap strategy that combines the pore plate integrated antibody array with a classical double antibody sandwich method. After incubating the array with samples, reaction signals are amplified by specific detection antibodies linked with alkaline phosphatase. All the assays can be completed within 6 hours. Furthermore, the results can be directly examined by the naked eye or by scanning with a flat bet scanner for quantitative analysis. With almost the same sensitivity as the ELISA, the array can detect 10 pathogens in one well simultaneously. This array system is used to screen 24 seed samples (240 detection items in all) and shows a high consistency (240/240) with results from the ELISA, indicating that this antibody array assay system could be used as a rapid method to simultaneously analyse at least ten pathogens, not only for lab research but also for practical applications.

In vivo bioluminescence monitoring of defense gene expression in response to treatment with yeast cell wall extract

Previous studies indicated that Housaku Monogatari (HM), a plant activator made from yeast cell wall extract, is effective for control of various plant diseases. To investigate the effect of HM treatment on plant gene expression, we tested the expression of defense related gene induction by exploiting tobacco pathogenesis-related protein 1a (PR-1a) gene promoter- and the Arabidopsis plant defensin 1.2 (PDF1.2) gene promoter-luciferase fusions as reporter genes. Transgenic Arabidopsis plants harboring promoter-luciferase fusion genes were treated with HM and the promoter activity was monitored as changes in luciferase activity in planta. Results of bioluminescence monitoring assay indicated that the promoters were activated at different times after the treatment of test plants with HM. Maximum activation of the PR-1a promoter occurred 4 days, and of the PDF1.2 promoter 4 h, after treatment. These results suggest that HM might contain multiple microbe-associated molecular patterns (MAMPs) that activate systemic acquired resistance and induced systemic resistance signaling pathways at different times. This may explain the mechanisms involved in the induction of defense responses against multiple plant pathogens by HM treatment.

Quantitative multiplex detection of plant pathogens using a novel ligation probe-based system coupled with universal, high-throughput real-time PCR on OpenArrays.

BackgroundDiagnostics and disease-management strategies require technologies to enable the simultaneous detection and quantification of a wide range of pathogenic microorganisms. Most multiplex, quantitative detection methods available suffer from compromises between the level of multiplexing, throughput and accuracy of quantification. Here, we demonstrate the efficacy of a novel, high-throughput, ligation-based assay for simultaneous quantitative detection of multiple plant pathogens. The ligation probes, designated Plant Research International-lock probes (PRI-lock probes), are long oligonucleotides with target complementary regions at their 5' and 3' ends. Upon perfect target hybridization, the PRI-lock probes are circularized via enzymatic ligation, subsequently serving as template for individual, standardized amplification via unique probe-specific primers. Adaptation to OpenArrays™, which can accommodate up to 3072 33 nl PCR amplifications, allowed high-throughput real-time quantification. The assay combines the multiplex capabilities and specificity of ligation reactions with high-throughput real-time PCR in the OpenArray™, resulting in a flexible, quantitative multiplex diagnostic system.ResultsThe performance of the PRI-lock detection system was demonstrated using 13 probes targeting several significant plant pathogens at different taxonomic levels. All probes specifically detected their corresponding targets and provided perfect discrimination against non-target organisms with very similar ligation target sites. The nucleic acid targets could be reliably quantified over 5 orders of magnitude with a dynamic detection range of more than 104. Pathogen quantification was equally robust in single target versus mixed target assays.ConclusionThis novel assay enables very specific, high-throughput, quantitative detection of multiple pathogens over a wide range of target concentrations and should be easily adaptable for versatile diagnostic purposes.

Suppressiveness of 18 composts against 7 pathosystems: Variability in pathogen response

Compost is often reported as a substrate that is able to suppress soilborne plant pathogens, but suppression varies according to the type of compost and pathosystem. Reports often deal with a single pathogen while in reality crops are attacked by multiple plant pathogens. The goal of the present study was to evaluate the disease suppression ability of a wide range of composts for a range of plant pathogens. This study was conducted by a consortium of researchers from several European countries. Composts originated from different countries and source materials including green and yard waste, straw, bark, biowaste and municipal sewage. Suppressiveness of compost-amended (20% vol./vol.) peat-based potting soil was determined against Verticillium dahliae on eggplant, Rhizoctonia solani on cauliflower, Phytophthora nicotianae on tomato, Phytophthora cinnamomi on lupin and Cylindrocladium spathiphylli on Spathiphyllum sp., and of compost-amended loamy soil (20% vol./vol.) against R. solani on Pinus sylvestris and Fusarium oxysporum f. sp. lini on flax. From the 120 bioassays involving 18 composts and 7 pathosystems, significant disease suppression was found in 54% of the cases while only 3% of the cases showed significant disease enhancement. Pathogens were affected differently by the composts. In general, prediction of disease suppression was better when parameters derived from the compost mixes were used rather than those derived from the pure composts. Regression analyses of disease suppression of the individual pathogens with parameters of compost-amended peat-based mixes revealed the following groupings: (1) competition-sensitive: F. oxysporum and R. solani/cauliflower; (2) rhizosphere-affected: V. dahliae; (3) pH-related: P. nicotianae; and (4) specific/unknown: R. solani/pine, P. cinnamomi and C. spathiphylli. It was concluded that application of compost has in general a positive or no effect on disease suppression, and only rarely a disease stimulating effect.

Quantifying the relationship between disease severity and the amount of Aphanomyces euteiches detected in roots of alfalfa and pea with a real-time PCR assay

A real-time PCR assay was used to quantify the relationship in alfalfa and pea between disease severity and the amount of Aphanomyces euteiches detected in roots. The study included isolates of race 1 and race 2 of the alfalfa pathovar of A. euteiches and an isolate obtained from diseased pea. Spearman rank correlations between pathogen DNA content and disease severity index (DSI) ratings were positive ( ⩾̸ 0.57) and significant (P ⩽ 0.0007) for individual alfalfa plants, bulked alfalfa plant samples, and individual pea plants. In all experiments, significantly more pathogen was detected in susceptible populations than in resistant populations. The results clearly demonstrate that resistance to A. euteiches in both alfalfa and pea is characterized by a reduction in pathogen colonization relative to levels observed for susceptible reactions. The assay was very specific for A. euteiches, producing very linear assays with DNA extracted from pathogen isolates obtained from alfalfa, pea, and bean. Possible applications of the assay in conjunction with other real-time PCR assays specific to other legume pathogens are discussed in relation to simultaneous disease screening for multiple plant pathogens and the study of microbial population dynamics in mixed plant infections.