Genetic Structure of Rhizoctonia solani AG-2-2IIIB from Soybean in Illinois, Ohio, and Ontario.

Rhizoctonia solani AG-2-2IIIB is an important seedling pathogen of soybean in North America and other soybean-growing regions around the world. There is no information regarding the population genetics of field populations of R. solani associated with soybean seedling disease. More specifically, information regarding genetic diversity, the mode of reproduction, and the evolutionary factors that shape different R. solani populations separated in time and space are lacking. We exploited genotyping by sequencing as a tool to assess the genetic structure of R. solani AG-2-2IIIB populations from Illinois, Ohio, and Ontario and investigate the reproductive mode of this subgroup. Our results revealed differences in genotypic diversity among three populations, with the Ontario population having greatest diversity. An overrepresentation of multilocus genotypes (MLGs) and a rejection of the null hypothesis of random mating in all three populations suggested clonality within each population. However, phylogenetic analysis revealed long terminal multifurcating branches for most members of the Ontario population, suggesting a mixed reproductive mode for this population. Analysis of molecular variance revealed low levels of population differentiation, and sharing of similar MLGs among populations highlights the role of genotype flow as an evolutionary force shaping population structure of this subgroup.

Sensitivity of Rhizoctonia solani to Succinate Dehydrogenase Inhibitor and Demethylation Inhibitor Fungicides.

Soybean seedling diseases are caused by Rhizoctonia solani and can be managed with seed-applied fungicides that belong to different chemistry classes. To provide a benchmark for assessing a decline in sensitivities to these fungicide classes, R. solani isolates collected prior to 2001 were evaluated for their sensitivities to succinate dehydrogenase inhibitor (SDHI) (penflufen and sedaxane) and demethylation inhibitor (DMI) fungicides (ipconazole and prothioconazole). The effective concentration of each fungicide that reduced mycelial growth by 50% (EC50) was determined in vitro and compared with those of isolates recovered after 2011 from soybean plants with damping off and hypocotyl and root rot symptoms across different soybean-growing regions in the United States and Canada. All isolates, regardless of collection date, were extremely sensitive (EC50 < 1 µg/ml) to the SDHI fungicides but were either extremely sensitive or moderately sensitive (1 ≤ EC50 ≤ 10 µg/ml) to the DMI fungicides. For all four active ingredients, variation in sensitivities was observed within and among the different anastomosis groups composing both isolate groups. Isolates collected after 2011, which also had varying in vitro sensitivities, were further evaluated for in vivo sensitivity to the four fungicides in the greenhouse. In vitro fungicide sensitivity did not always coincide with fungicide efficacy in vivo because all isolates tested, regardless of in vitro sensitivity, were effectively controlled by the application of the seed treatment fungicides in the greenhouse. Overall, our results indicate no shift in sensitivity to the fungicide classes evaluated, although considerable variability in the sensitivities of the two groups of isolates examined was present. Based on this research, continued monitoring of fungicide sensitivities of R. solani populations should occur to determine whether sensitivities become further reduced in the future.

Identification and Characterization of Rhizoctonia Species Associated with Soybean Seedling Disease.

In an effort to identify the Rhizoctonia spp. associated with seedling diseases of soybean, Rhizoctonia isolates were recovered from soybean seedlings with damping off and root and hypocotyl rot symptoms from Arkansas, Illinois, Kansas, Michigan, Minnesota, and the Canadian province of Ontario between 2012 and 2014. Based on cultural morphology, polymerase chain reaction restriction fragment length polymorphism, and phylogenetic analysis of the internal transcribed spacer (ITS) region of the ribosomal RNA genes, 80 isolates were confirmed to be Rhizoctonia solani, 24 were binucleate Rhizoctonia spp., and 10 were R. zeae. Of the 80 R. solani isolates, one belonged to anastomosis group (AG) 2-1, 52 belonged to AG-2-2IIIB, five belonged to AG-3 PT, three belonged to AG-4 HGI, two belonged to AG-4 HGIII, nine belonged to AG-7, and eight belonged to AG-11. Bayesian inference of phylogeny using the ITS region revealed two clades of R. solani AG-7 that possibly correspond to different AG-7 subgroups. Phylogenetic analysis also provided evidence for genetic relatedness between certain binucleate Rhizoctonia and some R. solani isolates. On 'Williams 82' soybean, isolates of AG-2-2IIIB were the most aggressive, followed by isolates of AG-7, AG-4, and AG-11. On 'Jubilee', a sweet corn cultivar, AG-2-2IIIB and AG-4 isolates caused significant stunting and root damage, whereas the damage caused by the AG-11 isolates was mostly restricted to the mesocotyl. Isolates of R. zeae and the binucleate Rhizoctonia spp. were not pathogenic on soybean or corn. Our results indicate that soybean and corn are hosts to the predominant and aggressive AG of R. solani, implying that rotation between these two crops may not be an effective management practice.

Identification of Diverse Mycoviruses through Metatranscriptomics Characterization of the Viromes of Five Major Fungal Plant Pathogens.

UNLABELLED: Mycoviruses can have a marked effect on natural fungal communities and influence plant health and productivity. However, a comprehensive picture of mycoviral diversity is still lacking. To characterize the viromes of five widely dispersed plant-pathogenic fungi, Colletotrichum truncatum, Macrophomina phaseolina, Diaporthe longicolla, Rhizoctonia solani, and Sclerotinia sclerotiorum, a high-throughput sequencing-based metatranscriptomic approach was used to detect viral sequences. Total RNA and double-stranded RNA (dsRNA) from mycelia and RNA from samples enriched for virus particles were sequenced. Sequence data were assembled de novo, and contigs with predicted amino acid sequence similarities to viruses in the nonredundant protein database were selected. The analysis identified 72 partial or complete genome segments representing 66 previously undescribed mycoviruses. Using primers specific for each viral contig, at least one fungal isolate was identified that contained each virus. The novel mycoviruses showed affinity with 15 distinct lineages: Barnaviridae, Benyviridae, Chrysoviridae, Endornaviridae, Fusariviridae, Hypoviridae, Mononegavirales, Narnaviridae, Ophioviridae, Ourmiavirus, Partitiviridae, Tombusviridae, Totiviridae, Tymoviridae, and Virgaviridae More than half of the viral sequences were predicted to be members of the Mitovirus genus in the family Narnaviridae, which replicate within mitochondria. Five viral sequences showed strong affinity with three families (Benyviridae, Ophioviridae, and Virgaviridae) that previously contained no mycovirus species. The genomic information provides insight into the diversity and taxonomy of mycoviruses and coevolution of mycoviruses and their fungal hosts. IMPORTANCE: Plant-pathogenic fungi reduce crop yields, which affects food security worldwide. Plant host resistance is considered a sustainable disease management option but may often be incomplete or lacking for some crops to certain fungal pathogens or strains. In addition, the rising issues of fungicide resistance demand alternative strategies to reduce the negative impacts of fungal pathogens. Those fungus-infecting viruses (mycoviruses) that attenuate fungal virulence may be welcome additions for mitigation of plant diseases. By high-throughput sequencing of the RNAs from 275 isolates of five fungal plant pathogens, 66 previously undescribed mycoviruses were identified. In addition to identifying new potential biological control agents, these results expand the grand view of the diversity of mycoviruses and provide possible insights into the importance of intracellular and extracellular transmission in fungus-virus coevolution.