Occurrence of Ascospores and White Mold Caused by Sclerotinia sclerotiorum in Dry Bean Fields in Alberta, Canada.

White mold caused by the fungal pathogen Sclerotinia sclerotiorum (Lib.) de Bary is one of the most important biological constraints to dry bean (Phaseolus vulgaris L.) production in Canada. Disease forecasting is one tool that could help growers manage the disease while reducing fungicide use. However, predicting white mold epidemics has remained difficult due to their sporadic occurrence. In this study, over the course of four growing seasons (2018 to 2021), we surveyed dry bean fields in Alberta and collected daily in-field weather data and daily in-field ascospore counts. White mold levels were variable and generally high in all years, confirming that the disease is ubiquitous and a constant threat to dry bean production. Ascospores were present throughout the growing season, and mean ascospore levels varied by field, month, and year. Models based on in-field weather and ascospore levels were not highly predictive of final disease incidence in a field, suggesting that environment and pathogen presence were not limiting factors to disease development. Rather, significant effects of market class on disease were found, with pinto beans, on average, having the highest disease incidence (33%) followed by great northern (15%), black (10%), red (6%), and yellow (5%). When incidence of these market classes was modeled separately, different environmental variables were important in each model; however, average wind speed was a significant variable in all models. Taken together, these findings suggest that white mold management in dry bean should focus on fungicide use, plant genetics, irrigation management, and other agronomic factors.

Inoculum dose-disease response relationships for the pea root rot pathogen, Aphanomyces euteiches, are dependent on soil type and other pathogens.

The oomycete pathogen, Aphanomyces euteiches, was implicated for the first time in pea and lentil root rot in Saskatchewan and Alberta in 2012 and 2013. Subsequent surveys from 2014 to 2017 revealed that Aphanomyces root rot (ARR) was widespread across the Canadian prairies. The absence of effective chemical, biological, and cultural controls and lack of genetic resistance leave only one management option: avoidance. The objectives of this study were to relate oospore levels in autoclaved and non-autoclaved soils to ARR severity across soil types from the vast prairie landscape and to determine the relationship of measured DNA quantity of A. euteiches using droplet digital PCR or quantitative PCR to the initial oospore inoculum dose in soils. These objectives support a future end goal of creating a rapid assessment method capable of categorizing root rot risk in field soil samples to aid producers with pulse crop field selection decisions. The ARR severity to oospore dose relationship was statistically significantly affected by the soil type and location from which soils were collected and did not show a linear relationship. For most soil types, ARR did not develop at oospore levels below 100/g soil, but severity rose above this level, confirming a threshold level of 100 oospores/g soil for disease development. For most soil types, ARR severity was significantly higher in non-autoclaved compared to autoclaved treatments, demonstrating the role that other pathogens play in increasing disease severity. There was a significant linear relationship between DNA concentrations measured in soil and oospore inoculum concentration, although the strength of the relationship was better for some soil types, and in some soil types, DNA measurement results underestimated the number of oospores. This research is important for developing a root rot risk assessment system for the Canadian prairies based on soil inoculum quantification, following field validation of soil quantification and relationship to root rot disease severity.

Specific Detection and Quantification of Major Fusarium spp. Associated with Cereal and Pulse Crops.

Plant pathogenic Fusarium spp. are widespread and cause important diseases on a wide host range, including economically important cereal and pulse crops. A number of molecular methods have been used to detect, identify, and quantify a long list of plant pathogenic Fusarium spp. In general, these methods are much faster, highly specific, more sensitive, and more accurate than culture-based methods and can be performed and interpreted by personnel with no specialized taxonomical expertise. The accurate isolation and identification of these pathogens is required to effectively manage diseases caused by pathogenic Fusarium spp. In this chapter, we present detailed molecular methods for detection, quantification, and differentiation between many of the Fusarium spp. associated with cereal and pulse crops.

Predictive modeling of Pseudomonas syringae virulence on bean using gradient boosted decision trees.

Pseudomonas syringae is a genetically diverse bacterial species complex responsible for numerous agronomically important crop diseases. Individual P. syringae isolates are assigned pathovar designations based on their host of isolation and the associated disease symptoms, and these pathovar designations are often assumed to reflect host specificity although this assumption has rarely been rigorously tested. Here we developed a rapid seed infection assay to measure the virulence of 121 diverse P. syringae isolates on common bean (Phaseolus vulgaris). This collection includes P. syringae phylogroup 2 (PG2) bean isolates (pathovar syringae) that cause bacterial spot disease and P. syringae phylogroup 3 (PG3) bean isolates (pathovar phaseolicola) that cause the more serious halo blight disease. We found that bean isolates in general were significantly more virulent on bean than non-bean isolates and observed no significant virulence difference between the PG2 and PG3 bean isolates. However, when we compared virulence within PGs we found that PG3 bean isolates were significantly more virulent than PG3 non-bean isolates, while there was no significant difference in virulence between PG2 bean and non-bean isolates. These results indicate that PG3 strains have a higher level of host specificity than PG2 strains. We then used gradient boosting machine learning to predict each strain's virulence on bean based on whole genome k-mers, type III secreted effector k-mers, and the presence/absence of type III effectors and phytotoxins. Our model performed best using whole genome data and was able to predict virulence with high accuracy (mean absolute error = 0.05). Finally, we functionally validated the model by predicting virulence for 16 strains and found that 15 (94%) had virulence levels within the bounds of estimated predictions. This study strengthens the hypothesis that P. syringae PG2 strains have evolved a different lifestyle than other P. syringae strains as reflected in their lower level of host specificity. It also acts as a proof-of-principle to demonstrate the power of machine learning for predicting host specific adaptation.

First Report of Root and Hypocotyl Rot of Dry Bean Caused by Aphanomyces euteiches in Alberta, Canada.

Dry bean (Phaseolus vulgaris L.) is a high-value crop grown under irrigation on 45,000 ha in southern Alberta, Canada. In 2019, one field of red beans showed premature yellowing and stunting of shoots in mid-July near Bow Island, AB. Roots had brown lesions along the length of hypocotyl and tap root, and decay and sloughing of the root cortex of lateral roots (Fig. 1). Roots were washed for 10 min under running tap water, and portions of the lesions were excised for DNA extractions using the Plant DNeasy kit (Qiagen) and multiplex PCR assays for an array of pathogens, which included Aphanomyces euteiches (Chatterton et al. 2019). Lesion pieces were also surface sterilized and plated onto PDA or cornmeal agar amended with metalaxyl, benomyl and vancomycin (CMBV) and visualized microscopically for oospores. The roots were PCR positive for A. euteiches, Rhizoctonia solani, and Pythium ultimum. Isolations yielded R. solani, and Fusarium spp. on PDA, but cultures on CMBV were over-grown with Rhizopus. Oospores measuring 24.7 +/- 1.08 m, consistent with the expected size of A. euteiches oospores of 18 - 25 m (Papavizas and Ayers, 1974), were visible in some of the root pieces. To obtain an A. euteiches isolate, soil was collected from three areas of the red bean field and used in a bait assay. Tests were performed using pea (cv. CDC Meadow) and dry bean (cv. Redbond) seeds treated with metalaxyl. Five seeds per pot were planted into field soils in 10-cm pots with 4 replicate pots/field. Soils were watered as needed until the 2nd node stage and then kept at saturation for 14 days, at which time roots were washed. Roots from the dry bean, but not the pea, plants showed severe root rot, including honey-brown discolouration of the lateral roots and extending to the hypocotyl, degradation of the root cortex, and presence of oospores. Diseased root pieces were plated onto CMBV without surface sterilization. Cultures with fast growing, white, aerial mycelia characteristic of A. euteiches on CMBV were recovered from dry bean roots from the three soil samples, considered as three isolates, and were transferred to PDATo confirm identity, total DNA was extracted from 7-day old cultures of the three isolates growing on PDA using the Qiagen DNeasy Plant Kit. The ribosomal DNA internal transcribed spacer (ITS) region was amplified using the primer pair ITS1 and ITS4, and sequenced (White et al. 1990). The sequences, deposited in GenBank with accession numbers OM976770, OM976771, and OM976772, were 100% identical to the ITS rDNA sequence of several isolates of A. euteiches (e.g. KM486066.1; 669/669 bp; 670/670 bp; 670/670 bp) using a BLASTn query and 99.7% identical to the CBS15773 voucher specimen (HQ643116.1, 661/663 bp; 662/664 bp; 662/664 bp). To confirm pathogenicity of these three isolates, cultures were used to produce zoospores for inoculating 14-day old red bean and pea seedlings (Chatterton et al. 2015). Control plants received minimal salt media solution and did not develop symptoms. Dry bean, but not pea, seedlings displayed root rot symptoms (Fig. 2). A. euteiches was re-isolated from dry bean seedlings by plating onto CMBV, and identity confirmed as described above. Although a previous survey detected A. euteiches in a small number of dry bean fields in southern Alberta (Chatterton et al. 2019), tests to confirm Koch's postulates were not performed. Aphanomyces root rot can be a devastating pathogen of pulse crops. Precautionary measures should be taken to prevent spread of this pathogen in the small dry bean growing area of southern Alberta by encouraging producers to use rotation intervals, monitoring movement of the pathogen, and evaluating cultivars for resistance to this pathogen.

Characterization of Aphanomyces euteiches Pathotypes Infecting Peas in Western Canada.

Aphanomyces root rot, caused by the soilborne oomycete Aphanomyces euteiches Drechs., has developed into a serious disease in the pea- and lentil-producing areas of the Great Plains of North America. Based on six pea differentials previously used to differentiate 11 pathotypes in France, pathotypes were identified among field isolates from Saskatchewan (14) and Alberta (18). Four isolates from the U.S.A. and standard isolates for pathotypes I and III designated in the French study were also included. Each isolate was tested twice in replicated experiments by inoculating French pea differentials 'Baccara', 'Capella', MN 313, 902131, 552, and PI 80693, along with the Canadian susceptible pea cultivar 'CDC Meadow' and partially resistant USDA line PI 660736 under controlled conditions. Pea plants grown in vermiculite were inoculated 10 days after seeding by pipetting 5 ml of a suspension containing 1 × 103 zoospores ml-1 to the base of each plant. Root discoloration was scored 10 days postinoculation using a 0 to 5 scale. Testing revealed that 38 of the isolates, including standard pathotype I isolate RB84, belonged to pathotype I; four isolates including standard pathotype III isolate Ae109 were pathotype III; and U.S.A. isolate Ae16-01 was a pathotype II isolate. An alfalfa isolate from Quebec was avirulent on all pea genotypes. These findings indicate that pathotype I is predominant on the Canadian prairies.

Enniatin Production Influences Fusarium avenaceum Virulence on Potato Tubers, but not on Durum Wheat or Peas.

Fusarium avenaceum is a generalist pathogen responsible for diseases in numerous crop species. The fungus produces a series of mycotoxins including the cyclohexadepsipeptide enniatins. Mycotoxins can be pathogenicity and virulence factors in various plant-pathogen interactions, and enniatins have been shown to influence aggressiveness on potato tubers. To determine the role of these mycotoxins in other F. avenaceum-host interactions, enniatin synthase 1 (ESYN1) disruption and overexpression mutants were generated and their ability to infect wheat and peas investigated. As a preliminary study, the transformants were screened for their ability to cause potato tuber necrosis and, consistent with a previous report, enniatin production increased necrotic lesion size on the tubers. By contrast, when the same mutants were assessed in their ability to cause disease in pea roots or durum wheat spikes, no changes in disease symptoms or virulence were observed. While it is known that, at least in the case of wheat, exogenously applied enniatins can cause tissue necrosis, this group of mycotoxins does not appear to be a key factor on its own in disease development on peas or durum wheat.

Potential effects of a high CO2 future on leguminous species.

Legumes provide an important source of food and feed due to their high protein levels and many health benefits, and also impart environmental and agronomic advantages as a consequence of their ability to fix nitrogen through their symbiotic relationship with rhizobia. As a result of our growing population, the demand for products derived from legumes will likely expand considerably in coming years. Since there is little scope for increasing production area, improving the productivity of such crops in the face of climate change will be essential. While a growing number of studies have assessed the effects of climate change on legume yield, there is a paucity of information regarding the direct impact of elevated CO2 concentration (e[CO2]) itself, which is a main driver of climate change and has a substantial physiological effect on plants. In this review, we discuss current knowledge regarding the influence of e[CO2] on the photosynthetic process, as well as biomass production, seed yield, quality, and stress tolerance in legumes, and examine how these responses differ from those observed in non-nodulating plants. Although these relationships are proving to be extremely complex, mounting evidence suggests that under limiting conditions, overall declines in many of these parameters could ensue. While further research will be required to unravel precise mechanisms underlying e[CO2] responses of legumes, it is clear that integrating such knowledge into legume breeding programs will be indispensable for achieving yield gains by harnessing the potential positive effects, and minimizing the detrimental impacts, of CO2 in the future.

Chemical Activation of the Ethylene Signaling Pathway Promotes Fusarium graminearum Resistance in Detached Wheat Heads.

Plant signaling hormones such as ethylene have been shown to affect the host response to various pathogens. Often, the resistance responses to necrotrophic fungi are mediated through synergistic interactions of ethylene (ET) with the jasmonate signaling pathway. On the other hand, ET is also an inducer of senescence and cell death, which could be beneficial for some invading necrotrophic pathogens. Fusarium graminearum, a causative agent in Fusarium head blight of wheat, is a hemibiotrophic pathogen, meaning it has both biotrophic and necrotrophic phases during the course of infection. However, the role of ET signaling in the host response to Fusarium spp. is unclear; some studies indicate that ET mediates resistance, while others have shown that it is associated with susceptibility. These discrepancies could be related to various aspects of different experimental designs, and suggest that the role of ET signaling in the host response to FHB is potentially dependent on interactions with some undetermined factors. To investigate whether wheat genotype can influence the ET-mediated response to FHB, the effect of chemical treatments affecting the ET pathway was studied in six wheat genotypes in detached-head assays. ET-inhibitor treatments broke down resistance to both initial infection and disease spread in three resistant wheat genotypes, whereas ET-enhancer treatments resulted in reduced susceptibility in three susceptible genotypes. The results presented here show that the ET signaling can mediate FHB resistance to F. graminearum in different wheat backgrounds.

Lack of efficacy of transgenic pea (Pisum sativum L.) stably expressing antifungal genes against Fusarium spp. in three years of confined field trials.

Fusarium root rot is a major pea disease in Canada and only partial tolerance exists in germplasm. Transgenic technologies may hold promise but the economic benefits of genetically modified (GM) pea will need to surpass the regulatory costs, time and labor involved in bringing a GM crop to market. European pea (Pisum sativum L.) cultivars expressing four antifungal genes, 1-3 ß glucanase (G), endochitinase (C) (belonging to PR proteins family), polygalacturonase inhibiting proteins (PGIPs) (P) and stilbene synthase (V) have been transformed for disease tolerance and showed disease tolerance under laboratory conditions. Transgenic lines with four antifungal genes inserted either individually or stacked through crossing were tested for their efficacy against Fusarium root rot (Fusarium avenaceum) in confined trials over three years (2013 to 2015) in comparison with two parental German lines and three Canadian lines. Superior emergence, higher fresh weight or lower disease ratings above and below ground, of transgenic lines in presence of disease inoculum were not observed consistently in the three years of field experiments when compared to the parental and Canadian lines in the presence of disease inoculum. No indication of an advantage of stacked genes over single genes was observed. Most transgenic lines had lower relative gene expression in the roots than in the leaves in greenhouse trials suggesting a possible explanation for poor tolerance to Fusarium root rot. Field trials are necessary to verify the agronomic performance and ecological relevance of the promising effects detected under laboratory conditions.

Interactions of Root-Feeding Insects with Fungal and Oomycete Plant Pathogens.

Soilborne fungal and oomycete pathogens are the causal agents of several important plant diseases. Infection frequently co-occurs with herbivory by root-feeding insects, facilitating tripartite interactions that modify plant performance and mortality. In an agricultural context, interactions between pathogens, herbivores, and plants can have important consequences for yield protection. However, belowground interactions are inherently difficult to observe and are often overlooked. Here, we review the impact of direct and indirect interactions between root-associated insects, fungi, and oomycetes on the development of plant disease. We explore the relationship between insect feeding injury and pathogen infection, as well as the role of insects as vectors of fungal and oomycete pathogens. Synergistic interactions between insects and phytopathogens may be important in weed suppression, and we highlight several promising candidates for biocontrol. Bridging the gap between entomological and pathological research is a critical step in understanding how interactions between insects and microorganisms modify the community structure of the rhizosphere, and how this impacts plant functioning. Furthermore, the identification of belowground interactions is required to develop effective pest monitoring and management strategies.

Identifying and Managing Root Rot of Pulses on the Northern Great Plains.

Pulse crops (annual grain legumes such as field pea, lentil, dry bean, and chickpea) have become an important component of the cropping system in the northern Great Plains of North America over the last three decades. In many areas, the intensity of damping-off, seedling blight, root rot, and premature ripening of pulse crops is increasing, resulting in reduction in stand establishment and yield. This review provides a brief description of the important pathogens that make up the root rot complex and summarizes root rot management on pulses in the region. Initially, several specific Fusarium spp., a range of Pythium spp., and Rhizoctonia solani were identified as important components of the root rot disease complex. Molecular approaches have recently been used to identify the importance of Aphanomyces euteiches on pulses, and to demonstrate that year-to-year changes in precipitation and temperature have an important effect on pathogen prevalence. Progress has been made on management of root rot, but more IPM tools are required to provide effective disease management. Seed-treatment fungicides can reduce damping-off and seedling blight for many of the pathogens in this disease complex, but complex cocktails of active ingredients are required to protect seedlings from the pathogen complex present in most commercial fields. Partial resistance against many of the pathogens in the complex has been identified, but is not yet available in commercial cultivars. Cultural practices, especially diversified cropping rotations and early, shallow seeding, have been shown to have an important role in root rot management. Biocontrol agents may also have potential over the long term. Improved methods being developed to identify and quantify the pathogen inoculum in individual fields may help producers avoid high-risk fields and select IPM packages that enhance yield stability.

Chitinase and beta-1,3-glucanase enzyme production by the mycoparasite Clonostachys rosea f. catenulata against fungal plant pathogens.

Clonostachys rosea f. catenulata (syn. Gliocladium catenulatum) is an effective fungal biological agent against Fusarium root and stem rot and Pythium damping-off diseases on cucumber plants. Both chitinase and beta-1,3-glucanase enzymes were produced when C. rosea was grown on a synthetic medium containing chitin or laminarin as a sole carbon source, respectively. Chitinase production was also induced by Fusarium cell walls, while beta-1,3-glucanase activity was induced by both Fusarium and Pythium cell walls, as well as by growth on homogenized cucumber roots and on low-carbon media. Mycelial growth of Fusarium and Pythium, when exposed to C. rosea culture filtrates that contain glucanase activity, was significantly reduced compared with the controls, and cell walls of both pathogens were degraded. On excised cucumber roots, hyphae of C. rosea formed appressorium-like structures and coiled around hyphae of Pythium. In culture, C. rosea caused localized degradation of Fusarium hyphae. Cucumber root tissues colonized by C. rosea showed higher levels of beta-1,3-glucanase activity at 7 days post-application compared with untreated controls. To determine if this activity was derived from C. rosea, glucanase isoforms were separated on activity gels. Fungal culture filtrates and root extracts contained the same predominant 20 kDa isoform. Reverse-transcription polymerase chain reaction (RT-PCR) using primers designed to amplify a beta-1,3-glucanase gene in C. rosea confirmed glucanase expression on roots. These results show that C. rosea produces beta-1,3-glucanase in situ, which can degrade hyphae of Fusarium and Pythium and contribute to biological control efficacy.