Novel Fusarium Wilt Resistance Genes Uncovered in the Wild Progenitors and Heirloom Cultivars of Strawberry

Fusarium wilt, a soilborne disease caused by Fusarium oxysporum f. sp. fragariae, poses a significant threat to strawberry (Fragaria × ananassa) production in many parts of the world. This pathogen causes wilting, collapse, and death in susceptible genotypes. We previously identified a dominant gene (FW1) on chromosome 2B that confers resistance to race 1 of the pathogen and hypothesized that gene-for-gene resistance to Fusarium wilt was widespread in strawberry. To explore this, a genetically diverse collection of heirloom and modern cultivars and wild octoploid ecotypes were screened for resistance to Fusarium wilt races 1 and 2. Here we show that resistance to both races is widespread and that resistance to race 1 is mediated by dominant genes (FW1, FW2, FW3, FW4, and FW5) on three non-homoeologous chromosomes (1A, 2B, and 6B). The resistance proteins encoded by these genes are not yet known; however, plausible candidates were identified that encode pattern recognition receptor or other proteins known to mediate gene-for-gene resistance in plants. High-throughput genotyping assays for SNPs in linkage disequilibrium with FW1-FW5 were developed to facilitate marker-assisted selection and accelerate the development of race 1 resistant cultivars. This study laid the foundation for identifying the genes encoded by FW1-FW5, in addition to exploring the genetics of resistance to race 2 and other races of the pathogen, as a precaution to averting a Fusarium wilt pandemic.

Identity and pathogenicity of fungi associated with root, crown and vascular symptoms related to winter squash yield decline

Winter squash (Cucurbita maxima cv. ‘Golden Delicious’) produced in Oregon’s Willamette Valley for edible seed production has experienced significant yield losses due to a soilborne disease. The symptoms associated with this disease problem include root rot, crown rot and vascular discoloration in the stems leading to a severe late season wilt and plant collapse. Through field surveys, Fusarium oxysporum, F. solani, F. culmorum-like fungi, Plectosphaerella cucumerina, and Setophoma terrestris were identified to be associated with diseased tissues, and each produced symptoms of root rot, crown rot or stem discoloration in preliminary pathogenicity trials. In this study, 219 isolates of these species were characterized by molecular identity analyses using BLAST of the ITS and EF1α genomic regions and by pathogenicity testing in outdoor, large-container trials. Molecular identity analyses confirmed the identity of isolates at 99 to 100% similarity to reference isolates in the database. In pathogenicity experiments, F. solani produced the most severe symptoms, followed by F. culmorum-like fungi, F. oxysporum, P. cucumerina, and S. terrestris. Some treatments of mixed species inoculum produced symptoms above what was expected from individual species. In particular, the mixture of F. culmorum-like fungi, F. oxysporum, and P. cucumerina and the mixture of F. culmorum-like fungi, F. solani, and S. terrestris had equally severe symptom ratings than that of F. solani by itself. Results indicate that this soilborne disease is primarily caused by Fusarium solani, but interactions among the complex of F. solani, F. culmorum-like fungi, F. oxysporum, and P. cucumerina, can exacerbate disease severity.

First report of root rot caused by Fusarium armeniacum on soybean in Korea

Fusarium wilt samples were collected in 2017 and 2019 from two soybean (Glycine max) fields, Yesan (36°73′N, 126°81′E) and Gimje (35°76′N, 126°80′E), in Korea. The disease incidence rate in each field was approximately 1%. For fungal isolation, root lesion fragments were surface-sterilized in 1% sodium hypochlorite for 2 min, rinsed thrice with sterile distilled water, and then incubated on water agar (WA) plates at 28 °C in an incubator for 5 days. Two isolates (YS37231 and GJ3050) were obtained using the hyphal tip method. Colonies of the isolates on potato dextrose agar (PDA) produced white aerial mycelia, which later turned pinkish yellow. The isolates on PDA formed abundant chlamydospores and macroconidia, but microconidia were absent. Macroconidia were 3–5 septate and prominently curved, measuring 12.4 to 41.2 × 3.3 to 4.3 µm (Leslie and Summerell, 2006). For the identification of the isolates, translation elongation factor 1 alpha (EF-1α) and RNA polymerase second largest subunit (RPB2) regions were amplified and sequenced using EF1, EF2, RPB2-5f2, and RPB2-7cr primers, respectively (O’Donnell et al. 2010). EF-1α sequences of YS37231 (MT445439) and GJ3050 (MT445440) showed 99.01 and 99.67% identity with F. armeniacum (FD_01843 and FD_01305; FUSARIUM-ID database), respectively. The RPB sequences of YS37231 (MT445442) and GJ3050 (MT445441) showed 100 and 98.48% identity with that of F. armeniacum (FD_01869; FUSARIUM-ID database), respectively. The sequences MT445439, MT445440, MT445441, and MT445442 were deposited in NCBI GenBank. The pathogenicity of the two isolates on the soybean cultivar Daewonkong was determined using two inoculation methods. In the first method, a pathogenicity assay was performed on seedlings using WA plates (Cruz Jimenez et al. 2018). Eight surface-sterilized seeds were transferred to WA plates, with or without actively growing cultures, for 3 days; and then incubated at 25 °C in a growth chamber (12 h photoperiod) for 7 days. After 7 days, brown lesions were observed on the roots in inoculated plates; however, no symptoms were observed in the control. -In the second method, 10-day old soybean seedlings were inoculated by cutting and soaking the roots in the spore suspension (1 × 106 conidia/mL) for 2 h. The inoculum was prepared by incubating isolates on PDA for 10 days, then adding sterile distilled water, scraping the growth medium, and filtering the suspension. The seedlings were then transplanted into 18 cm plastic pots (20 cm height) and grown under greenhouse conditions (26 °C ± 3 °C, 13 h photoperiod) for 2 weeks. After 7 days, the inoculated plants showed wilting symptoms, developed brown lesions in the roots, and eventually died within 2 weeks after inoculation. No such symptoms were observed in the control (inoculated with sterile distilled water). The isolates were re-isolated from the inoculated seedlings for confirmation. Although the fungus and associated mycotoxins have been reported in the rice produced in Korea (Hong et al. 2015), to the best of our knowledge, this is the first report of F. armeniacum causing Fusarium wilt on soybean in Korea. In the US, it was first reported by Ellis et al. (2012). Fusarium wilt is a soilborne disease of growing concern in soybean cultivation worldwide. Our findings will help increase awareness about the global spread of this disease.

Six de novo assemblies from pathogenic and non-pathogenic strains of Fusarium oxysporum f. sp. niveum

Fusarium wilt, caused by Fusarium oxysporum f. sp. niveum (Fon), is a soilborne disease which significantly limits yield in watermelon (Citrullus lanatus) and occasionally causes the loss of an entire year’s harvest. Reference-quality de novo genomic assemblies of pathogenic and non-pathogenic strains were generated using a combination of next-generation and third-generation sequencing technologies. Chromosomal-level genomes were produced with representatives from all Fon races facilitating comparative genomic analysis and the identification of chromosomal structural variation . Syntenic analysis between isolates allowed differentiation of the core and lineage-specific portions of their genomes. This research will support future efforts to refine the scientific understanding of the molecular and genetic factors underpinning the Fon host range, develop diagnostic assays for each of the four races, and decipher the evolutionary history of race 3.

Detection of soilborne disease utilizing sensor technologies: Lessons learned from studies on stem rot of peanut

Soilborne plant diseases are a major constraint to crop production worldwide. Effective and economical management of these diseases is dependent on the ability to accurately detect and diagnose their signs and/or symptoms prior to widespread development in a crop. Sensor-based technologies are promising tools for automated crop disease detection, but research is still needed to optimize and validate methods for the detection of specific plant diseases. The overarching goal of our research is to use the peanut-stem rot plant disease system to identify and evaluate sensor-based technologies that can be utilized for the detection of soilborne plant diseases. Here we summarize the current state of sensor-based technologies for plant disease detection and provide examples from our own research that illustrate the advantages and limitations of different sensor-based methods for detecting soilborne diseases. In addition, the potential to adapt different sensor-based technologies to practical use in the field is discussed.

Evaluation of Agricultural Byproducts and Cover Crops as Anaerobic Soil Disinfestation Carbon Sources for Managing a Soilborne Disease Complex in High Tunnel Tomatoes

Anaerobic soil disinfestation (ASD) is a viable option for disease management in tomato production and reduces damage due to a soilborne disease complex consisting of Pyrenochaeta lycopersici, Colletotrichum coccodes, Verticillium dahliae, and Meloidogyne spp. There are plentiful options for ASD carbon sources using byproducts of Midwestern United States agriculture or cover crops, yet these carbon sources have not been evaluated for use in Midwestern settings. Low (10.1 Mg/ha) and high (20.2 Mg/ha) rates of corn gluten meal, distillers dried grains, soybean meal, wheat bran, and dry sweet whey were evaluated as ASD carbon sources in growth chamber and greenhouse bioassays. Cover crops including buckwheat, cowpea, crimson clover, mustard, oilseed radish, sorghum-sudangrass, white clover, and winter rye were evaluated in similar bioassays with one amendment rate (20.2 Mg/ha). Reducing conditions developed in soils regardless of carbon source or rate. Use of high rates of corn gluten meal, distillers dried grains, soybean meal, and wheat bran led to the lowest levels of root rot severity compared to non-treated controls. The higher rate of any byproduct carbon source was always more effective than the lower rate in reducing root rot severity. Use of both rates of soybean meal or corn gluten meal and the high rate of distillers dried grains or dry sweet whey led to significant increases in dry root and shoot biomass compared to controls. For cover crops, ASD with crimson clover, sorghum-sudangrass, white clover, or winter rye amendments reduced root rot severity relative to the aerobic control, but not relative to the anaerobic control. Use of cover crops did not significantly impact plant biomass. A subset of three ASD carbon sources [distillers dried grains, soybean meal, and wheat middlings (midds), all 20.2 Mg/ha] were evaluated in five on-farm ASD trials in high tunnels. Soil temperatures were low during the application period, limiting treatment efficacy. Reducing conditions developed in all soils during ASD treatment, and a moderate but significant reduction in root rot severity was observed following ASD with the soybean meal or wheat midds compared to ASD with distillers dried grains. Tomato yield was not significantly affected by ASD treatment.

Plasmodiophora brassicae Inoculum Density and Spatial Patterns at the Field Level and Relation to Soil Characteristics

Clubroot, caused by Plasmodiophora brassicae, is an important soilborne disease of the Brassicaceae. Knowledge of the spatial dynamics of P. brassicae at the field level and the influence of soil properties on pathogen spatial patterns can improve understanding of clubroot epidemiology and management. To study the spatial patterns of P. brassicae inoculum density and their relationship to different soil properties, four clubroot-infested fields in central Alberta, Canada, were sampled in 2017 and 2019, and P. brassicae inoculum density, soil pH, and boron, calcium, and magnesium concentrations were quantified. Spatial autocorrelation of the inoculum density was estimated for each of the fields in both years with the Moran’s I and semi-variograms. A Bayesian hierarchical spatial approach was used to model the relationship between P. brassicae inoculum density and the soil parameters. Patchiness of the pathogen was detected, with most patches located at the field edges and adjacent to the entrance. Infested patches grew in size from 2017 to 2019, with an average increase in diameter of 221.3 m and with this growth determined by the maximum inoculum density and active dispersal methods such as movement by machinery and wind. Soil pH, boron, calcium, and magnesium concentrations were not found to have an important effect on the inoculum density of P. brassicae.

Mapping QTL Associated with Partial Resistance to Aphanomyces Root rot in Pea (Pisum Sativum L.) using a 13.2K SNP Array and SSR Markers

Aphanomyces root rot (ARR), caused by Aphanomyces euteiches Drechs., is a destructive soilborne disease of field pea (Pisum Sativum L.). No completely resistant pea germplasm is available, and current ARR management strategies rely on partial resistance and fungicidal seed treatments. In this study, an F8 recombinant inbred line (RIL) population of 135 individuals from the cross ‘Reward’ (susceptible) × ‘00-2067’ (tolerant) was evaluated for reaction to ARR under greenhouse conditions with the A. euteiches isolate Ae-MDCR1 and over 2 years in a field nursery in Morden, Manitoba. Root rot severity, foliar weight, plant vigor and height were used as estimates of tolerance to ARR. Genotyping was conducted with a 13.2K single-nucleotide polymorphism (SNP) array and 222 simple sequence repeat (SSR) markers. Statistical analyses of the phenotypic data indicated significant (P<0.001) genotypic effects and significant G×E interactions (P<0.05) in all experiments. After filtering, 3050 (23.1%) of the SNP and 30 (13.5%) of the SSR markers were retained for linkage analysis, which distributed 2999 (2978 SNP + 21 SSR) of the markers onto nine linkage groups representing the seven chromosomes of pea. Mapping of quantitative trait loci (QTL) identified 5 major-effect (R2 > 20%), 13 moderate-effect (10%<R2< 20%) effect and 10 minor-effect (R2 <10%) QTL. A genomic region on chromosome IV, delimited by the SNP markers PsCam037549_22628_1642 and PsCam026054_14999_2864, was identified as the most consistent region responsible for partial resistance to A. euteiches isolate Ae-MDCR1. Other genomic regions important for resistance were of the order chromosome III, II and VII.

Gliotoxin is an important secondary metabolite involved in suppression of Sclerotium rolfsii by Trichoderma virens T23

Sclerotium rolfsii causes destructive soilborne disease in numerous plant species, and biological control may be a promising and sustainable approach for suppressing this widespread pathogen. In this study, the antagonistic effect against S. rolfsii of ten Trichoderma strains was tested by the dual culture method, and a gliotoxin-producing strain, Trichoderma virens T23, was shown to be the most effective inhibiting growth of S. rolfsii in vitro by 70.2%. To clarify the antagonistic mechanism and gliotoxin biosynthesis regulation of T23, a gliotoxin-deficient mutant was constructed via Agrobacterium tumefaciens mediated gene knockout in vivo. As expected, disruption of the gene located in the putative gliotoxin biosynthesis gene cluster, gliI-T, resulted in gliotoxin deficiency and attenuation of the antagonistic effect against S. rolfsii, indicating that gliotoxin biosynthesis is regulated by gliI-T and that gliotoxin is an important antifungal metabolite of T23. Transmission electron microscopy revealed that gliotoxin treatment caused marked alterations of the hyphal cells of S. rolfsii depending on the drug concentration, whereby one of the prominent structural alterations was a reduction in the number and length of mitochondrial cristae. When exposed to 30 μg/mL gliotoxin for 12 h, striking plasmolysis and ultrastructural changes were induced in S. rolfsii. The results demonstrate that gliotoxin is an important secondary metabolite of T. virens T23 in its antagonism against S. rolfsii.