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Alcohol oxidases (AOXs) are ecologically important enzymes that facilitate a number of plant–fungal interactions. Within Ascomycota they are primarily associated with methylotrophy, as a peroxisomal AOX catalysing the conversion of methanol to formaldehyde in methylotrophic yeast. In this study we demonstrate that AOX orthologues are phylogenetically conserved proteins that are common in the genomes of nonmethylotrophic, plant‐associating fungi. Additionally, AOX orthologues are highly expressed during infection in a range of diverse pathosystems. To study the role of AOX in plant colonization, AOX knockout mutants were generated in the broad host range pathogen Sclerotinia sclerotiorum. Disease assays in soybean showed that these mutants had a significant virulence defect as evidenced by markedly reduced stem lesions and mortality rates. Chemical genomics suggested that SsAOX may function as an aromatic AOX, and growth assays demonstrated that ΔSsAOX is incapable of properly utilizing plant extract as a nutrient source. Profiling of known aromatic alcohols pointed towards the monolignol coniferyl alcohol (CA) as a possible substrate for SsAOX. As CA and other monolignols are ubiquitous among land plants, the presence of highly conserved AOX orthologues throughout Ascomycota implies that this is a broadly conserved protein used by ascomycete fungi during plant colonization. [ABSTRACT FROM AUTHOR]

Autophagy is a genetically regulated, eukaryotic cellular degradation system that sequestrates cytoplasmic materials in specialised vesicles, termed autophagosomes, for delivery and breakdown in the lysosome or vacuole. In plants, autophagy plays essential roles in development (e.g., senescence) and responses to abiotic (e.g., nutrient starvation, drought and oxidative stress) and biotic stresses (e.g., hypersensitive response). Initially, autophagy was considered a non-selective bulk degradation mechanism that provides energy and building blocks for homeostatic balance during stress. Recent studies, however, reveal that autophagy may be more subtle and selectively target ubiquitylated protein aggregates, protein complexes and even organelles for degradation to regulate vital cellular processes even during favourable conditions. The selective nature of autophagy lends itself to potential manipulation and exploitation as part of designer protein turnover machinery for the development of stress-tolerant and disease-resistant crops, crops with increased yield potential and agricultural efficiency and reduced post-harvest losses. Here, we discuss our current understanding of autophagy and speculate its potential manipulation for improved agricultural performance. [ABSTRACT FROM AUTHOR]

A lack of complete resistance in the current germplasm complicates the management of Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum in soybean. In this study, we used bean pod mottle virus (BPMV) as a vehicle to down-regulate expression of a key enzyme in the production of an important virulence factor in S. sclerotiorum , oxalic acid (OA). Specifically, we targeted a gene encoding oxaloacetate acetylhydrolase (Ssoah1), because Ssoah1 deletion mutants are OA deficient and non-pathogenic on soybean. We first established that S. sclerotiorum can uptake environmental RNAs by monitoring the translocation of Cy3-labeled double-stranded and small interfering RNA (ds/siRNAs) into fungal hyphae using fluorescent confocal microscopy. This translocation led to a significant decrease in Ssoah1 transcript levels in vitro. Inoculation of soybean plants with BPMV vectors targeting Ssoah1 (pBPMV-OA) also led to decreased expression of Ssoah1. Importantly, pBPMV-OA inoculated plants showed enhanced resistance to S. sclerotiorum compared to empty-vector control plants. Our combined results provide evidence supporting the use of HIGS and exogenous applications of ds/siRNAs targeting virulence factors such as OA as viable strategies for the control of SSR in soybean and as discovery tools that can be used to identify previously unknown virulence factors. [ABSTRACT FROM AUTHOR]

Plant diseases caused by necrotrophic fungal pathogens result in large economic losses in field crop production worldwide. Effectors are important players of plant-pathogen interaction and deployed by pathogens to facilitate plant colonization and nutrient acquisition. Compared to biotrophic and hemibiotrophic fungal pathogens, effector biology is poorly understood for necrotrophic fungal pathogens. Recent bioinformatics advances have accelerated the prediction and discovery of effectors from necrotrophic fungi, and their functional context is currently being clarified. In this review we examine effectors utilized by necrotrophic fungi and hemibiotrophic fungi in the latter stages of disease development, including plant cell death manipulation. We define "effectors" as secreted proteins and other molecules that affect plant physiology in ways that contribute to disease establishment and progression. Studying and understanding the mechanisms of necrotrophic effectors is critical for identifying avenues of genetic intervention that could lead to improved resistance to these pathogens in plants. [ABSTRACT FROM AUTHOR]

While fungal biotrophs are dependent on successfully suppressing/subverting host defenses during their interaction with live cells, necrotrophs, due to their lifestyle are often confronted with a suite of toxic metabolites. These include an assortment of plant defense compounds (PDCs) which can demonstrate broad antifungal activity. These PDCs can be either constitutively present in plant tissue or induced in response to infection, but are nevertheless an important obstacle which needs to be overcome for successful pathogenesis. Fungal necrotrophs have developed a number of strategies to achieve this goal, from the direct detoxification of these compounds through enzymatic catalysis and modification, to the active transport of various PDCs to achieve toxin sequestration and efflux. Studies have shown across multiple pathogens that the efficient detoxification of host PDCs is both critical for successful infection and often a determinant factor in pathogen host range. Here, we provide a broad and comparative overview of the various mechanisms for PDC detoxification which have been identified in both fungal necrotrophs and fungal pathogens which depend on detoxification during a necrotrophic phase of infection. Furthermore, the effect that these mechanisms have on fungal host range, metabolism, and disease control will be discussed. [ABSTRACT FROM AUTHOR]

Clarireediajacksonii causes dollar spot disease of cool‐season turfgrasses in the United States and produces the phytotoxin oxalic acid. The role of oxalic acid in host–pathogen interactions of C. jacksonii is unknown and there are multiple challenges to studying these interactions in natural turfgrass hosts. Consequently, identification of model plants to study C. jacksonii–host interactions and the role of oxalic acid in pathogenesis is necessary. Controlled environment inoculation assays were used to evaluate pathogenesis of C. jacksonii in various model plants and investigate the role of oxalic acid in symptom development. Observations at microscopic and macroscopic levels demonstrated that infection progressed similarly in all monocots tested (creeping bentgrass, wheat, barley, rice, Brachypodiumdistachyon) but not in the dicot Arabidopsisthaliana. Plant oxalic acid content increased from near zero to around 0.2–0.4 mM following inoculation with C. jacksonii in creeping bentgrass, barley, and wheat. Conversely, oxalic acid content remained near zero in A. thaliana and was not well correlated with inoculation in rice and B. distachyon, both of which had higher endogenous oxalic acid levels than other monocots. Time‐course oxalic acid quantification experiments with creeping bentgrass and B. distachyon further supported a link between symptom development and in planta oxalic acid content and identified 48 hr postinoculation as a critical time‐point for investigating the role of oxalic acid in C. jacksonii pathogenesis. These studies demonstrate that various monocots can serve as tractable model systems for studying C. jacksonii–host interactions and that increases in oxalic acid content are associated with C. jacksonii symptom development. [ABSTRACT FROM AUTHOR]

Dollar spot is caused by the fungus Clarireedia jacksonii and is the most common disease of golf course turfgrass in temperate climates. Oxalic acid (OA) is an important pathogenicity factor in other fungal plant pathogens, such as the dicot pathogen Sclerotinia sclerotiorum , but its role in C. jacksonii pathogenicity on monocot hosts remains unclear. Herein, we assess fungal growth, OA concentration, and pH change in potato dextrose broth (PDB) following incubation of C. jacksonii. In addition, OA production by C. jacksonii and S. sclerotiorum was compared in PDB amended with creeping bentgrass or common plant cell wall components (cellulose, lignin, pectin, or xylan). Our results show that OA production is highly dependent on the environmental pH, with twice as much OA produced at pH 7 than pH 4 and a corresponding decrease in PDB pH from 7 to 5 following 96 h of C. jacksonii incubation. In contrast, no OA was produced or changes in pH observed when C. jacksonii was incubated in PDB at a pH of 4. Interestingly, C. jacksonii increased OA production in response to PDB amended with creeping bentgrass tissue and the cell wall component xylan, a major component of grass cell walls. S. sclerotiorum produced large amounts of OA relative to C. jacksonii regardless of treatment, and no treatment increased OA production by this fungus, though pectin suppressed S. sclerotiorum 's OA production. These results suggest that OA production by C. jacksonii is reliant on host specific components within the infection court, as well as the ambient pH of the foliar environment during its pathogenic development. [ABSTRACT FROM AUTHOR]

Soybean (Glycine max L.) is a major crop grown in the United States but is susceptible to many diseases that cause significant yield losses each year. Consistent threats exist across both northern and southern production regions and include the soybean cyst nematode, charcoal rot, and seedling diseases. In contrast, significant soybean diseases like Phytophthora stem and root rot, sudden death syndrome, and Sclerotinia stem rot (white mold) are intermittent threats that can be heavily influenced by environmental factors. Additional threats to soybean production that have emerged in recent years as more common problems in soybean production include root-knot and reniform nematodes, frogeye leaf spot, and Diaporthe diseases. Disease in any crop will only occur when the three components of the disease triangle are present: a susceptible host, a virulent pathogen, and a conducive environment. If an environment is becoming more conducive for a particular disease, it is important that farmers and practitioners are prepared to manage the problem. The information in this review was compiled to help assist agriculturalists in being proactive in managing new soybean diseases that may be emerging in new areas. To do this, we provide: 1) an overview of the impact and disease cycle for major soybean diseases currently causing significant yield losses in the United States, 2) a comprehensive review of the current management strategies for each soybean disease, and 3) insights into the epidemiology of each pathogen, including the likelihood of outbreaks and expansion to additional geographic regions based on current trends in climate change. [ABSTRACT FROM AUTHOR]

Summary: Sclerotinia sclerotiorum, a predominately necrotrophic fungal pathogen with a broad host range, causes a significant yield‐limiting disease of soybean called Sclerotinia stem rot. Resistance mechanisms against this pathogen in soybean are poorly understood, thus hindering the commercial deployment of resistant varieties. We used a multiomic approach utilizing RNA‐sequencing, gas chromatography–mass spectrometry‐based metabolomics and chemical genomics in yeast to decipher the molecular mechanisms governing resistance to S. sclerotiorum in soybean. Transcripts and metabolites of two soybean recombinant inbred lines, one resistant and one susceptible to S. sclerotiorum were analysed in a time course experiment. The combined results show that resistance to S. sclerotiorum in soybean is associated in part with an early accumulation of JA‐Ile ((+)‐7‐iso‐jasmonoyl‐L‐isoleucine), a bioactive jasmonate, increased ability to scavenge reactive oxygen species, and importantly, a reprogramming of the phenylpropanoid pathway leading to increased antifungal activities. Indeed, we noted that phenylpropanoid pathway intermediates, such as 4‐hydroxybenzoate, cinnamic acid, ferulic acid and caffeic acid, were highly accumulated in the resistant line. In vitro assays show that these metabolites and total stem extracts from the resistant line clearly affect S. sclerotiorum growth and development. Using chemical genomics in yeast, we further show that this antifungal activity targets ergosterol biosynthesis in the fungus, by disrupting enzymes involved in lipid and sterol biosynthesis. Overall, our results are consistent with a model where resistance to S. sclerotiorum in soybean coincides with an early recognition of the pathogen, leading to the modulation of the redox capacity of the host and the production of antifungal metabolites. [ABSTRACT FROM AUTHOR]

Abstract: Despite the importance of proper cell death regulation across broad evolutionary distances, an understanding of the molecular machinery underpinning this fundamental process in plants remains largely elusive. This is despite its critical importance to development, homeostasis, and proper responses to stress. The identification of endogenous plant regulators of cell death has been hindered by the fact that many core regulators of cell death in animals are absent in plant genomes. Remarkably, numerous studies have shown that the ectopic expression of animal prosurvival genes in plants can suppress cell death imposed by many stresses. In this study, we capitalize on the ectopic expression of one of these animal prosurvival genes, an inhibitor of apoptosis from Spodoptera frugiperda (SfIAP), to identify novel cell death regulators in plants. A yeast two‐hybrid assay was conducted using SfIAP as bait to screen a tomato cDNA library. This screen identified several transcription factors of the SQUAMOSA promoter‐binding protein (SBP) family as potential SfIAP binding partners. We confirmed this interaction in vivo for our top two interactors, SlySBP8b and SlySBP12a, using coimmunoprecipitation. Interestingly, overexpression of SlySBP8b and SlySBP12a induced cell death in Nicotiana benthamiana leaves. Overexpression of these two transcription factors also induced the accumulation of reactive oxygen species and enhanced the growth of the necrotrophic pathogen Alternaria alternata. Fluorescence microscopy confirmed the nuclear localization of both SlySBP8b and SlySBP12a, while SlySBP12a was also localized to the ER membrane. These results suggest a prodeath role for SlySBP8b and SlySBP12a and implicate ER membrane tethering as a means of regulating SlySBP12a activity. [ABSTRACT FROM AUTHOR]

The Bcl-2 associated athanogene (Bag) family is a multifunctional group of proteins distinguished by a conserved region known as the Bag domain (BD). Herein, we discuss the discovery and characterization of a Bag protein in the model genetic fungus Aspergillus nidulans, we designated BagA. BagA shares striking similarities in 3D structure, domain organization, amino acid properties, and Hsp70 binding surfaces to animal and plant Bags. While Hsp70 binding is a common feature of Bag proteins, our experimental evidence shows that BagA does not cooperate with A. nidulans Hsp70s, suggesting this association may not be a universal feature of Bag proteins. Gene expression of bagA was strongly induced during sexual development suggesting a role in developmental processes. Accordingly, the deletion of bagA (ΔbagA) negatively impacted sexual development, while its overexpression resulted in constitutive induction of sexual fruiting bodies and spores. Asexual and sexual development was linked to secondary metabolism in A. nidulans. Our data show that the deletion of bagA also provoked an altered secondary metabolite (SM) profile in both sexual and vegetative growth phases. Indeed, LC-MS analysis showed a significant enrichment of SMs in ΔbagA, including novel metabolites not produced by wild type strain. Enrichment of SMs in ΔbagA strain is particularly intriguing and suggest that altering cellular homeostasis can be used as a provocative strategy to activate cryptic metabolites and uncover novel bioactive compounds. Overall, our results indicate that Bag proteins in filamentous fungi share developmental regulatory roles with their animal and plant counterparts. We also show a potentially unique role for BagA in modulating secondary metabolism in A. nidulans. To our knowledge, this study provides a first insight into Bag function in filamentous fungi. [ABSTRACT FROM AUTHOR]

Summary: The plant membrane‐localized NADPH oxidases, also known as respiratory burst oxidase homologues (RBOHs), play crucial roles in various cellular activities, including plant disease responses, and are a major source of reactive oxygen species (ROS). Sclerotinia sclerotiorum is a cosmopolitan fungal pathogen that causes Sclerotinia stem rot (SSR) in soybean. Via a key virulence factor, oxalic acid, it induces programmed cell death (PCD) in the host plant, a process that is reliant on ROS generation. In this study, using protein sequence similarity searches, we identified 17 soybean RBOHs (GmRBOHs) and studied their contribution to SSR disease development, drought tolerance and nodulation. We clustered the soybean RBOH genes into six groups of orthologues based on phylogenetic analysis with their Arabidopsis counterparts. Transcript analysis of all 17 GmRBOHs revealed that, of the six identified groups, group VI (GmRBOH‐VI) was specifically and drastically induced following S. sclerotiorum challenge. Virus‐induced gene silencing (VIGS) of GmRBOH‐VI using Bean pod mottle virus (BPMV) resulted in enhanced resistance to S. sclerotiorum and markedly reduced ROS levels during disease development. Coincidently, GmRBOH‐VI‐silenced plants were also found to be drought tolerant, but showed a reduced capacity to form nodules. Our results indicate that the pathogenic development of S. sclerotiorum in soybean requires the active participation of specific host RBOHs, to induce ROS and cell death, thus leading to the establishment of disease. [ABSTRACT FROM AUTHOR]

Sclerotinia sclerotiorum, the causal agent of Sclerotinia stem rot, is a devastating fungal pathogen of soybean that can cause significant yield losses to growers when environmental conditions are favorable for the disease. The development of resistant varieties has proven difficult. However, poor resistance in commercial cultivars can be improved through additional breeding efforts and understanding the genetic basis of resistance. The objective of this project was to develop soybean germplasm lines that have a high level of Sclerotinia stem rot resistance to be used directly as cultivars or in breeding programs as a source of improved Sclerotinia stem rot resistance. Sclerotinia stem rot-resistant soybean germplasm was developed by crossing two sources of resistance, W04-1002 and AxN-1-55, with lines exhibiting resistance to Heterodera glycines and Cadophora gregata in addition to favorable agronomic traits. Following greenhouse evaluations of 1,076 inbred lines derived from these crosses, 31 lines were evaluated for resistance in field tests during the 2014 field season. Subsequently, 11 Sclerotinia stem rot resistant breeding lines were moved forward for field evaluation in 2015, and seven elite breeding lines were selected and evaluated in the 2016 field season. To better understand resistance mechanisms, a marker analysis was conducted to identify quantitative trait loci linked to resistance. Thirteen markers associated with Sclerotinia stem rot resistance were identified on chromosomes 15, 16, 17, 18, and 19. Our markers confirm previously reported chromosomal regions associated with Sclerotinia stem rot resistance as well as a novel region of chromosome 16. The seven elite germplasm lines were also re-evaluated within a greenhouse setting using a cut petiole technique with multiple S. sclerotiorum isolates to test the durability of physiological resistance of the lines in a controlled environment. This work presents a novel and comprehensive classical breeding method for selecting lines with physiological resistance to Sclerotinia stem rot and a range of agronomic traits. In these studies, we identify four germplasm lines; 91-38, 51-23, SSR51-70, and 52-82B exhibiting a high level of Sclerotinia stem rot resistance combined with desirable agronomic traits, including high protein and oil contents. The germplasm identified in this study will serve as a valuable source of physiological resistance to Sclerotinia stem rot that could be improved through further breeding to generate high-yielding commercial soybean cultivars. [ABSTRACT FROM AUTHOR]

Like all eukaryotic organisms, plants possess an innate program for controlled cellular demise termed programmed cell death (PCD). Despite the functional conservation of PCD across broad evolutionary distances, an understanding of the molecular machinery underpinning this fundamental program in plants remains largely elusive. As in mammalian PCD, the regulation of plant PCD is critical to development, homeostasis, and proper responses to stress. Evidence is emerging that autophagy is key to the regulation of PCD in plants and that it can dictate the outcomes of PCD execution under various scenarios. Here, we provide a broad and comparative overview of PCD processes in plants, with an emphasis on stress-induced PCD. We also discuss the implications of the paradox that is functional conservation of apoptotic hallmarks in plants in the absence of core mammalian apoptosis regulators, what that means, and whether an equivalent form of death occurs in plants. [ABSTRACT FROM AUTHOR]

Programmed cell death is characterized by a cascade of tightly controlled events that culminate in the orchestrated death of the cell. In multicellular organisms autophagy and apoptosis are recognized as two principal means by which these genetically determined cell deaths occur. During plant-microbe interactions cell death programs can mediate both resistant and susceptible events. Via oxalic acid (OA), the necrotrophic phytopathogen Sclerotinia sclerotiorum hijacks host pathways and induces cell death in host plant tissue resulting in hallmark apoptotic features in a time and dose dependent manner. OA-deficient mutants are non-pathogenic and trigger a restricted cell death phenotype in the host that unexpectedly exhibits markers associated with the plant hypersensitive response including callose deposition and a pronounced oxidative burst, suggesting the plant can recognize and in this case respond, defensively. The details of this plant directed restrictive cell death associated with OA deficient mutants is the focus of this work. Using a combination of electron and fluorescence microscopy, chemical effectors and reverse genetics, we show that this restricted cell death is autophagic. Inhibition of autophagy rescued the non-pathogenic mutant phenotype. These findings indicate that autophagy is a defense response in this necrotrophic fungus/plant interaction and suggest a novel function associated with OA; namely, the suppression of autophagy. These data suggest that not all cell deaths are equivalent, and though programmed cell death occurs in both situations, the outcome is predicated on who is in control of the cell death machinery. Based on our data, we suggest that it is not cell death per se that dictates the outcome of certain plant-microbe interactions, but the manner by which cell death occurs that is crucial. [ABSTRACT FROM AUTHOR]

Sclerotinia sclerotiorum is a necrotrophic ascomycete fungus with an extremely broad host range. This pathogen produces the non-specific phytotoxin and key pathogenicity factor, oxalic acid (OA). Our recent work indicated that this fungus and more specifically OA, can induce apoptotic-like programmed cell death (PCD) in plant hosts, this induction of PCD and disease requires generation of reactive oxygen species (ROS) in the host, a process triggered by fungal secreted OA. Conversely, during the initial stages of infection, OA also dampens the plant oxidative burst, an early host response generally associated with plant defense. This scenario presents a challenge regarding the mechanistic details of OA function; as OA both suppresses and induces host ROS during the compatible interaction. In the present study we generated transgenic plants expressing a redox-regulated GFP reporter. Results show that initially, Sclerotinia (via OA) generates a reducing environment in host cells that suppress host defense responses including the oxidative burst and callose deposition, akin to compatible biotrophic pathogens. Once infection is established however, this necrotroph induces the generation of plant ROS leading to PCD of host tissue, the result of which is of direct benefit to the pathogen. In contrast, a non-pathogenic OA-deficient mutant failed to alter host redox status. The mutant produced hypersensitive response-like features following host inoculation, including ROS induction, callose formation, restricted growth and cell death. These results indicate active recognition of the mutant and further point to suppression of defenses by the wild type necrotrophic fungus. Chemical reduction of host cells with dithiothreitol (DTT) or potassium oxalate (KOA) restored the ability of this mutant to cause disease. Thus, Sclerotinia uses a novel strategy involving regulation of host redox status to establish infection. These results address a long-standing issue involving the ability of OA to both inhibit and promote ROS to achieve pathogenic success. [ABSTRACT FROM AUTHOR]

Dollar spot is a destructive foliar disease of amenity turfgrass caused by Clarireedia spp. fungi, mainly C. jacksonii, on the Northern United States region's cool-season grass. Oxalic acid (OA) is an important pathogenicity factor in related fungal plant pathogens such as Sclerotinia sclerotiorum; however, the role of OA in the pathogenic development of C. jacksonii remains unclear due to its recalcitrance to genetic manipulation. To overcome these challenges, a CRISPR/Cas9-mediated homologous recombination approach was developed. Using this novel approach, the oxaloacetate acetylhydrolase (oah) gene that is required for the biosynthesis of OA was deleted from a C. jacksonii wild-type (WT) strain. Two independent knockout mutants, ΔCjoah-1 and ΔCjoah-2, were generated and inoculated on potted creeping bentgrass along with a WT isolate and a genome sequenced isolate LWC-10. After 12 days, bentgrass inoculated with the mutants ΔCjoah-1 and ΔCjoah-2 exhibited 59.41% lower dollar spot severity compared with the WT and LWC-10 isolates. OA production and environmental acidification were significantly reduced in both mutants when compared with the WT and LWC-10. Surprisingly, stromal formation was also severely undermined in the mutants in vitro, suggesting a critical developmental role of OA independent of plant infection. These results demonstrate that OA plays a significant role in C. jacksonii virulence and provide novel directions for future management of dollar spot. [ABSTRACT FROM AUTHOR]

Numerous studies have shown both the detrimental and beneficial effects of reactive oxygen species (ROS) in animals, plants, and fungi. These organisms utilize controlled generation of ROS for signaling, pathogenicity, and development. Here, we show that ROS are essential for the pathogenic development of Scierotinia sclerotiorum, an economically important fungal pathogen with a broad host range. Based on the organism's completed genome sequence, we identified two S. scierotiorurn NADPH oxidases (SsNoxt and SsNox2), which presumably are involved in ROS generation. RNA interference (RNAi) was used to examine the function of SsNoxl and SsNox2. Silencing of SsNoxl expression indicated a central role for this enzyme in both virulence and pathogenic (sclerotial) development, while inactivation of the SsNox2 gene resulted in limited scierotial development, but the organism remained fully pathogenic. Ssnox1 strains had reduced ROS levels, were unable to develop scierotia, and unexpectedly correlated with significantly reduced oxalate production. These results are in accordance with previous observations indicating that fungal NADPH oxidases are required for pathogenic development and are consistent with the importance of ROS regulation in the successful pathogenesis of S. scierotiorum. [ABSTRACT FROM AUTHOR]

Mycosphaerella graminicola causes Septoria tritici blotch (STB) in wheat (Triticum aestivum) and is considered one of the most devastating pathogens of that crop in the United States. Although the genetic structures of M. graminicola populations from different countries have been analyzed using various molecular markers, relatively little is known about M. graminicola populations from geographically distinct areas of the United States and, in particular, of those from spring versus winter wheat. These are exposed to great differences in environmental conditions, length and season of host-free periods, and resistance sources used in geographically separated wheat breeding programs. Thus, there is more likely to be genetic differentiation between populations from spring versus winter wheat than there is among those within each region. To test this hypothesis, 330 single-spore isolates of M. graminicola representing 11 populations (l from facultative winter wheat in California, 2 from spring wheat in North Dakota, and 8 from winter wheat in Indiana and Kansas) were analyzed for mating type frequency and for genetic variation at 17 microsatellite or simple-sequence repeat (SSR) loci. Analysis of clone-corrected data revealed an equal distribution of both mating types in the populations from Kansas, Indiana, and North Dakota. but a deviation from a 1:1 ratio in the California population. In total, 306 haplotypes were detected, almost all of which were unique in all 11 populations. High levels of gene diversity (H = 0.31 to 0.56) were observed within the 11 populations. Significant (P ≤ 0.05) gametic disequilibrium, as measured by the index of association (rBarD), was observed in California, one Indiana population (IN1), and three populations (KS1, KS2, and KS3) in Kansas that could not be explained by linkage. Corrected standardized fixation index (G''ST) values were 0.000 to 0.621 between the 11 populations and the majority of pairwise comparisons were statistically significant (P ≤ 0.001), suggesting some differentiation between populations. Analysis of molecular variance showed that there was a small but statistically significant level of genetic differentiation between populations from spring versus winter wheat. However, most of the total genetic variation (>98%) occurred within spring and winter wheat regions while <2% was due to genetic differentiation between these regions. Taken together, these results provide evidence that sexual recombination occurs frequently in the M. graminicola populations sampled and that most populations are genetically differentiated over the major spring- and winter-wheat-growing regions of the United States. [ABSTRACT FROM AUTHOR]

As complete host resistance in soybean has not been achieved, Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum continues to be of major economic concern for farmers. Thus, chemical control remains a prevalent disease management strategy. Pesticide evaluations were conducted in Illinois, Iowa, Michigan, Minnesota, New Jersey, and Wisconsin from 2009 to 2016, for a total of 25 site-years (n = 2,057 plot-level data points). These studies were used in network metaanalyses to evaluate the impact of 10 popular pesticide active ingredients, and seven common application timings on SSR control and yield benefit, compared with not treating with a pesticide. Boscalid and picoxystrobin frequently offered the best reductions in disease severity and best yield benefit (P < 0.0001). Pesticide applications (oneor two-spray programs) made during the bloom period provided significant reductions in disease severity index (D1X) (P < 0.0001) and led to significant yield benefits (P = 0.0009). Data from these studies were also used in nonlinear regression analyses to determine the effect of DIX on soybean yield. A three-parameter logistic model was found to best describe soybean yield loss (pseudo-R² = 0.309). In modem soybean cultivars, yield loss due to SSR does not occur until 20 to 25% DIX, and considerable yield loss (-697 kg ha-1 or -10 bu acre-1) is observed at 68% DIX. Further analyses identified several pesticides and programs that resulted in greater than 60% probability for return on investment under high disease levels. [ABSTRACT FROM AUTHOR]

In soybean, Sclerotinia sclerotiorum apothecia are the sources of primary inoculum (ascospores) critical for Sclerotinia stem rot (SSR) development. We recently developed logistic regression models to predict the presence of apothecia in irrigated and nonirrigated soybean fields. In 2017, small-plot trials were established to validate two weather-based models (one for irrigated fields and one for nonirrigated fields) to predict SSR development. Additionally, apothecial scouting and disease monitoring were conducted in 60 commercial fields in three states between 2016 and 2017 to evaluate model accuracy across the growing region. Site-specific air temperature, relative humidity, and wind speed data were obtained through the Integrated Pest Information Platform for Extension and Education (iPiPE) and Dark Sky weather networks. Across all locations, iPiPE-driven model predictions during the soybean flowering period (R1 to R4 growth stages) explained end-of-season disease observations with an accuracy of 81.8% using a probability action threshold of 35%. Dark Sky data, incorporating bias corrections for weather variables, explained end-of-season disease observations with 87.9% accuracy (in 2017 commercial locations in Wisconsin) using a 40% probability threshold. Overall, these validations indicate that these two weather-based apothecial models, using either weather data source, provide disease risk predictions that both reduce unnecessary chemical application and accurately advise applications at critical times. [ABSTRACT FROM AUTHOR]

Sclerotinia stem rot (SSR) epidemics in soybean, caused by Sclerotinia sclerotiorum, are currently responsible for annual yield reductions in the United States of up to 1 million metric tons. In-season disease management is largely dependent on chemical control but its efficiency and cost-effectiveness depends on both the chemistry used and the risk of apothecia formation, germination, and further dispersal of ascospores during susceptible soybean growth stages. Hence, accurate prediction of the S. sclerotiorum apothecial risk during the soybean flowering period could enable farmers to improve in-season SSR management. From 2014 to 2016. apothecial presence or absence was monitored in three irrigated (n = 1,505 plot-level observations) and six nonirrigated (n = 2,361 plot-level observations) field trials located in Iowa (n = 156), Michigan (n = 1,400), and Wisconsin (n - 2,3 10), for a total of 3,866 plot-level observations. Hourly air temperature, relative humidity, dew point, wind speed, leaf wetness, and rainfall were also monitored continuously, throughout the season, at each location using high-resolution gridded weather data. Logistic regression models were developed for irrigated and nonirrigated conditions using apothecial presence as a binary response variable. Agronomic variables (row width) and weather-related variables (defined as 30-day moving averages, prior to apothecial presence) were tested for their predictive ability. In irrigated soybean fields, apothecial presence was best explained by row width (r = -0.41, P < 0.0001), 30-day moving averages of daily maximum air temperature (r = 0.27, P < 0.0001), and daily maximum relative humidity (r = 0 .16, P < 0.05). In nonirrigated fields, apothecial presence was best explained by using moving averages of daily maximum air temperature (r = -0.30, P < 0.0001) and wind speed (r = -0.27, P < 0.0001). These models correctly predicted (overall accuracy of 67 to 70%) apothecial presence during the soybean flowering period for four independent datasets (n = 1,102 plot-level observations or 30 daily mean observations). [ABSTRACT FROM AUTHOR]

Dollar spot is a destructive foliar disease of amenity turfgrass caused by Clarireedia spp. fungi, mainly C. jacksonii , on the Northern United States region's cool-season grass. Oxalic acid (OA) is an important pathogenicity factor in related fungal plant pathogens such as Sclerotinia sclerotiorum ; however, the role of OA in the pathogenic development of C. jacksonii remains unclear due to its recalcitrance to genetic manipulation. To overcome these challenges, a CRISPR/Cas9-mediated homologous recombination approach was developed. Using this novel approach, the oxaloacetate acetylhydrolase ( oah ) gene that is required for the biosynthesis of OA was deleted from a C. jacksonii wild-type (WT) strain. Two independent knockout mutants, Δ Cjoah-1 and Δ Cjoah-2 , were generated and inoculated on potted creeping bentgrass along with a WT isolate and a genome sequenced isolate LWC-10. After 12 days, bentgrass inoculated with the mutants Δ Cjoah-1 and Δ Cjoah-2 exhibited 59.41% lower dollar spot severity compared with the WT and LWC-10 isolates. OA production and environmental acidification were significantly reduced in both mutants when compared with the WT and LWC-10. Surprisingly, stromal formation was also severely undermined in the mutants in vitro, suggesting a critical developmental role of OA independent of plant infection. These results demonstrate that OA plays a significant role in C. jacksonii virulence and provide novel directions for future management of dollar spot. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

Fungicide use is integral to reduce yield loss from Sclerotinia sclerotiorum on dry bean and soybean. Increasing fungicide use against this fungus may lead to resistance to the most common fungicides. Resistance has been reported in Brazil ( Glycine max ) and China ( Brassica napus subsp. napus ), however, few studies have investigated fungicide sensitivity of S. sclerotiorum in the United States. This work was conducted to determine if there was a difference in fungicide sensitivity of S. sclerotiorum isolates in the United States from: (i) dry bean versus soybean and (ii) fields with different frequencies of fungicide application. We further hypothesized that isolates with fungicide applications of a single active ingredient from tropical Brazil and subtropical Mexico were less sensitive than temperate U.S. isolates due to different management practices and climates. The EC 50(D) fungicide sensitivity of 512 S. sclerotiorum isolates from the United States (443), Brazil (36), and Mexico (33) was determined using a discriminatory concentration (DC) previously identified for tetraconazole (2.0 ppm; EC 50(D) range of 0.197 to 2.27 ppm), boscalid (0.2; 0.042 to 0.222), picoxystrobin (0.01; 0.006 to 0.027), and thiophanate-methyl, which had a qualitative DC of 10 ppm. Among the 10 least sensitive isolates to boscalid and picoxystrobin, 2 presented mutations known to confer resistance in the SdhB (qualitative) and SdhC (quantitative) genes; however, no strong resistance was found. This study established novel DCs that can be used for further resistance monitoring and baseline sensitivity of S . sclerotiorum to tetraconazole worldwide plus baseline sensitivity to boscalid in the United States., Competing Interests: The author(s) declare no conflict of interest.

Soybean production in the upper midwestern United States is affected by Sclerotinia stem rot (SSR) caused by the fungal pathogen Sclerotinia sclerotiorum . Genetic resistance is an important management strategy for this disease; however, assessing genetic resistance to S. sclerotiorum is challenging because a standardized method of examining resistance across genotypes is lacking. Using a panel of nine diverse S. sclerotiorum isolates, four soybean lines were assessed for reproducible responses to S. sclerotiorum infection. Significant differences in SSR severity were found across isolates ( P < 0.01) and soybean lines ( P < 0.01), including one susceptible, two moderately resistant, and one highly resistant line. These four validated lines were used to screen 11 other soybean genotypes to evaluate their resistance levels, and significant differences were found across genotypes ( P < 0.01). Among these 11 genotypes, five commercial and public cultivars displayed high resistance and were assessed during field studies across the upper midwestern United States growing region to determine their response to SSR and yield. These five cultivars resulted in low disease levels ( P < 0.01) in the field that were consistent with greenhouse experiment results. The yields were significantly different in fields with disease present ( P < 0.01) and disease absent ( P < 0.01), and the order of cultivar performance was consistent between environments where disease was present or absent, suggesting that resistance prevented yield loss to disease. This study suggests that the use of a soybean check panel can accurately assess SSR resistance in soybean germplasm and aid in breeding and commercial soybean development.

The plant membrane-localized NADPH oxidases, also known as respiratory burst oxidase homologues (RBOHs), play crucial roles in various cellular activities, including plant disease responses, and are a major source of reactive oxygen species (ROS). Sclerotinia sclerotiorum is a cosmopolitan fungal pathogen that causes Sclerotinia stem rot (SSR) in soybean. Via a key virulence factor, oxalic acid, it induces programmed cell death (PCD) in the host plant, a process that is reliant on ROS generation. In this study, using protein sequence similarity searches, we identified 17 soybean RBOHs (GmRBOHs) and studied their contribution to SSR disease development, drought tolerance and nodulation. We clustered the soybean RBOH genes into six groups of orthologues based on phylogenetic analysis with their Arabidopsis counterparts. Transcript analysis of all 17 GmRBOHs revealed that, of the six identified groups, group VI (GmRBOH-VI) was specifically and drastically induced following S. sclerotiorum challenge. Virus-induced gene silencing (VIGS) of GmRBOH-VI using Bean pod mottle virus (BPMV) resulted in enhanced resistance to S. sclerotiorum and markedly reduced ROS levels during disease development. Coincidently, GmRBOH-VI-silenced plants were also found to be drought tolerant, but showed a reduced capacity to form nodules. Our results indicate that the pathogenic development of S. sclerotiorum in soybean requires the active participation of specific host RBOHs, to induce ROS and cell death, thus leading to the establishment of disease., (© 2017 BSPP AND JOHN WILEY & SONS LTD.)

Genome-wide association (GWAS) and epistatic (GWES) studies along with expression studies in soybean [Glycine max (L.) Merr.] were leveraged to dissect the genetics of Sclerotinia stem rot (SSR) [caused by Sclerotinia sclerotiorum (Lib.) de Bary], a significant fungal disease causing yield and quality losses. A large association panel of 466 diverse plant introduction accessions were phenotyped in multiple field and controlled environments to: (1) discover sources of resistance, (2) identify SNPs associated with resistance, and (3) determine putative candidate genes to elucidate the mode of resistance. We report 58 significant main effect loci and 24 significant epistatic interactions associated with SSR resistance, with candidate genes involved in a wide range of processes including cell wall structure, hormone signaling, and sugar allocation related to plant immunity, revealing the complex nature of SSR resistance. Putative candidate genes [for example, PHYTOALEXIN DEFFICIENT 4 (PAD4), ETHYLENE-INSENSITIVE 3-LIKE 1 (EIL3), and ETHYLENE RESPONSE FACTOR 1 (ERF1)] clustered into salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) pathways suggest the involvement of a complex hormonal network typically activated by both necrotrophic (ET/JA) and biotrophic (SA) pathogens supporting that S. sclerotiorum is a hemibiotrophic plant pathogen.

The Bcl-2-associated athanogene (BAG) family is a multifunctional group of proteins involved in numerous cellular functions ranging from apoptosis to tumorigenesis. These proteins are evolutionarily conserved and encode a characteristic region known as the BAG domain. BAGs function as adapter proteins forming complexes with signaling molecules and molecular chaperones. In humans, a role for BAG proteins has been suggested in tumor growth, HIV infection, and neurodegenerative diseases; as a result, the BAGs are attractive targets for therapeutic interventions, and their expression in cells may serve as a predictive tool for disease development. The Arabidopsis genome contains seven homologs of BAG family proteins (Figure 1), including four with a domain organization similar to animal BAGs (BAG1-4). The remaining three members (BAG5-7) contain a predicted calmodulin-binding motif near the BAG domain, a feature unique to plant BAG proteins that possibly reflects divergent mechanisms associated with plant-specific functions. As reported for animal BAGs, plant BAGs also regulate several stress and developmental processes (Figure 2). The recent article by Li et al. focuses on the role of BAG6 in plant innate immunity. This study shows that BAG6 plays a key role in basal plant defense against fungal pathogens. Importantly, this work further shows that BAG6 is proteolytically activated to induce autophagic cell death and resistance in plants. This finding underscores the importance of proteases in the execution of plant cell death, yet little is known about proteases and their substrates in plants., Competing Interests: Conflict of interest: The authors declare no conflict of interest.

Numerous studies have shown both the detrimental and beneficial effects of reactive oxygen species (ROS) in animals, plants, and fungi. These organisms utilize controlled generation of ROS for signaling, pathogenicity, and development. Here, we show that ROS are essential for the pathogenic development of Sclerotinia sclerotiorum, an economically important fungal pathogen with a broad host range. Based on the organism's completed genome sequence, we identified two S. sclerotiorum NADPH oxidases (SsNox1 and SsNox2), which presumably are involved in ROS generation. RNA interference (RNAi) was used to examine the function of SsNox1 and SsNox2. Silencing of SsNox1 expression indicated a central role for this enzyme in both virulence and pathogenic (sclerotial) development, while inactivation of the SsNox2 gene resulted in limited sclerotial development, but the organism remained fully pathogenic. ΔSsnox1 strains had reduced ROS levels, were unable to develop sclerotia, and unexpectedly correlated with significantly reduced oxalate production. These results are in accordance with previous observations indicating that fungal NADPH oxidases are required for pathogenic development and are consistent with the importance of ROS regulation in the successful pathogenesis of S. sclerotiorum.

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, β-glucuronidase (GUS), with putative positive or negative regulators of cell death. In this system, N. benthamiana leaves are co-infiltrated with a 35S driven expression cassette containing the gene to be analyzed, and the GUS vector pCAMBIA 2301 using Agrobacterium strain LBA4404 as a vehicle. Because live cells are required for GUS expression to occur, loss of GUS activity is expected when this marker gene is co-expressed with positive regulators of cell death. Equally, increased GUS activity is observed when anti-apoptotic genes are used compared to the vector control. As shown below, we have successfully used this system in our lab to analyze both pro- and anti-death players. These include the plant anti-apoptotic Bcl-2 Associated athanoGene (BAG) family, as well as, known mammalian inducers of cell death, such as BAX. Additionally, we have used this system to analyze the death function of specific truncations within proteins, which could provide clues on the possible post-translational modification/activation of these proteins. Here, we present a rapid and sensitive plant based method, as an initial step in investigating the death function of specific genes., (Copyright © 2011 Journal of Visualized Experiments)