Kirk Broders

Evaluation of Fusarium graminearum Associated with Corn and Soybean Seed and Seedling Disease in Ohio

Fusarium graminearum is an important pathogen of cereal crops in Ohio causing primarily head blight in wheat and stalk and ear rot of corn. During the springs of 2004 and 2005, 112 isolates of F. graminearum were recovered from diseased corn and soybean seedlings from 30 locations in 13 Ohio counties. These isolates were evaluated in an in vitro pathogenicity assay on both corn and soybean seed, and 28 isolates were tested for sensitivity to the seed treatment fungicides azoxystrobin, trifloxystrobin, fludioxonil, and captan. All of the isolates were highly pathogenic on corn seed and moderately to highly pathogenic on soybean seed. Fludioxonil was the only fungicide that provided sufficient inhibition of mycelial growth; however, several fludioxonil-resistant mutants were identified during the sensitivity experiments. These results indicate that F. graminearum is an important pathogen of both corn and soybean seed and seedlings in Ohio, and that continued use of fludioxonil potentially may select for less sensitive isolates of F. graminearum.

Characterization of Pythium spp. Associated with Corn and Soybean Seed and Seedling Disease in Ohio

Cool, moist conditions in combination with minimum tillage, earlier planting, and recent shifts in commercial fungicide seed-treatment active ingredients have led to an increase in corn (Zea mays) and soybean (Glycine max) seedling establishment problems. This situation resulted in an investigation of Pythium spp. associated with seed and seedling diseases. Samples of diseased corn and soybean seedlings were collected from 42 production fields in Ohio. All isolates of Pythium recovered were identified to species using morphological and molecular techniques and evaluated in an in vitro pathogenicity assay on both corn and soybean seed, and a subset of the isolates was tested for sensitivity to fungicides currently used as seed treatments. Eleven species and two distinct morphological groups of Pythium were identified, of which six species were moderately to highly pathogenic on corn seed and nine species were highly pathogenic on soybean seed. There was significant variation (P < 0.05) in sensitivity to mefenoxam, azoxystrobin, trifloxystrobin, and captan both across and within species. Multiple species of Pythium had the capacity to reduce germination of both corn and soybean seed. Results indicated that mefenoxam, azoxystrobin, trifloxystrobin, or captan, when used individually, may not inhibit all pathogenic species of Pythium found in Ohio soils.

Population structure of Geosmithia morbida , the causal agent of thousand cankers disease of walnut trees in the United States

The ascomycete Geosmithia morbida and the walnut twig beetle Pityophthorus juglandis are associated with thousand cankers disease of Juglans (walnut) and Pterocarya (wingnut). The disease was first reported in the western United States (USA) on several Juglans species, but has been found more recently in the eastern USA in the native range of the highly susceptible Juglans nigra. We performed a comprehensive population genetic study of 209 G. morbida isolates collected from Juglans and Pterocarya from 17 geographic regions distributed across 12 U.S. states. The study was based on sequence typing of 27 single nucleotide polymorphisms from three genomic regions and genotyping with ten microsatellite primer pairs. Using multilocus sequence-typing data, 197 G. morbida isolates were placed into one of 57 haplotypes. In some instances, multiple haplotypes were recovered from isolates collected on the same tree. Twenty-four of the haplotypes (42%) were recovered from more than one isolate; the two most frequently occurring haplotypes (H02 and H03) represented 36% of all isolates. These two haplotypes were abundant in California, but were not recovered from Arizona or New Mexico. G. morbida population structure was best explained by four genetically distinct groups that clustered into three geographic regions. Most of the haplotypes isolated from the native range of J. major (Arizona and New Mexico) were found in those states only or present in distinct genetic clusters. There was no evidence of sexual reproduction or genetic recombination in any population. The scattered distribution of the genetic clusters indicated that G. morbida was likely disseminated to different regions at several times and from several sources. The large number of haplotypes observed and the genetic complexity of G. morbida indicate that it evolved in association with at least one Juglans spp. and the walnut twig beetle long before the first reports of the disease.

Infection of Soybean Seed by Fusarium graminearum and Effect of Seed Treatments on Disease Under Controlled Conditions

Fusarium graminearum causes seed decay and damping-off of soybean. This study evaluated the effect of inoculum density of F. graminearum, temperature, and fungicide seed treatments on disease development. To determine the optimum conditions for disease development, individual soybean seed was inoculated with 100 μl of a suspension of 2.5 × 102, 2.5 × 103, 2.5 × 104, or 2.5 × 105 macroconidia/ml in a rolled-towel assay at temperatures of 18, 22, and 25°C. Inoculum concentrations of 2.5 × 104 macroconidia/ml or higher were necessary for optimum disease development at all temperatures. The efficacy of captan, fludioxonil, mefenoxam + fludioxonil, azoxystrobin, trifloxystrobin, and pyraclostrobin as seed treatments was then evaluated with the same assay at 2.5 × 104 and 2.5 × 105 macroconidia/ml. Seed treated with captan at 61.9 g a.i. or fludioxonil at 2.5 or 5.0 g a.i. per 100 kg developed smaller lesions than other seed treatments and the nontreated control. Based on these results, there are limited choices in fungicide seed treatments for managing this seedling disease, and it is possible that shifts in seed treatment products may have played a role in the recent emergence of this soybean pathogen.

Association of Soil Chemical and Physical Properties with Pythium Species Diversity, Community Composition, and Disease Incidence

A high-throughput baiting and identification process identified more than 7,000 isolates of Pythium from 88 locations in Ohio. Isolates were identified using direct-colony polymerase chain reaction followed by single-strand conformational polymorphism, and communities were assembled using the Jaccard similarity coefficient and cluster analysis. Both univariate and multivariate statistics were used to evaluate differences in soil properties between communities, and canonical discriminant analysis (CDA) was used to assess the strength of the association of soil variables within communities from 83 of the locations. In all, 21 species of Pythium were identified but only 6 were recovered from >40% of the locations. Five communities were formed using the cluster analysis, and significant differences were observed in disease incidence, as well as soil pH, calcium, magnesium, and cation exchange capacity between communities. Stepwise multiple discriminant analysis and CDA identified pH, calcium, magnesium, and field capacity as contributing the most to the separation of the five Pythium communities. There was a strong association between abiotic soil components and the structure of Pythium communities, as well as diversity of Pythium spp. collected from agronomic production fields in Ohio.

A Vavilovian approach to discovering crop-associated microbes with potential to enhance plant immunity

Through active associations with a diverse community of largely non-pathogenic microbes, a plant may be thought of as possessing an “extended genotype,” an interactive cross-organismal genome with potential, exploitable implications for plant immunity. The successful enrichment of plant microbiomes with beneficial species has led to numerous commercial applications, and the hunt for new biocontrol organisms continues. Increasingly flexible and affordable sequencing technologies, supported by increasingly comprehensive taxonomic databases, make the characterization of non-model crop-associated microbiomes a widely accessible research method toward this end; and such studies are becoming more frequent. A summary of this emerging literature reveals, however, the need for a more systematic research lens in the face of what is already a metagenomics data deluge. Considering the processes and consequences of crop evolution and domestication, we assert that the judicious integration of in situ crop wild relatives into phytobiome research efforts presents a singularly powerful tool for separating signal from noise, thereby facilitating a more efficient means of identifying candidate plant-associated microbes with the potential for enhancing the immunity and fitness of crop species.

Priority of Lophophacidium over Canavirgella: taxonomic status of Lophophacidium dooksii and Canavirgella banfieldii, causal agents of a white pine needle disease

In 2009 unusual white pine needle discoloration was observed in eastern Canada and northeastern USA. While the symptoms were similar in most pine stands, the disease was diagnosed as Canavirgella banfieldii in several locations and Dooks needle blight caused by Lophophacidium dooksii in others. Because of the similarities in symptom development and morphological characters of the causal agents, it was suspected that C. banfieldii and L. dooksii are either the same or closely related species. To test this hypothesis we examined several collections representing C. banfieldii and L. dooksii, including the two type specimens. Phylogenetic analyses of nuc internal transcribed spacer rDNA sequences confirm the synonymy of C. banfieldii with the earlier described L. dooksii and provide the first evidence of the close evolutionary relationship of L. dooksii to other pine pathogens.

Pythium delawarii—a new species isolated from soybean in Ohio

A new species of Pythium isolated from soybean in Ohio is described. Pythium delawarii sp. nov. is characterized by globose internally proliferating sporangia, aplerotic oospores and diclinous antheridia that make broad lengthwise contact. Sporangia produce conspicuous papilla and germinate indirectly by producing zoospores via a vesicle and proliferate internally or the sporangia germinate directly with either one or more germ tubes. Pythium delawarii is pathogenic on soybean causing damping-off of seedlings. This oomycete can grow at 10–34 C with an optimum of 28 C. The sequence of the ITS1, 5.8S and ITS2 region of the rDNA did not match the sequence of any known Pythium species but was similar to P. citrinum, P. litorale and P. sterilum. P. delawarii can be distinguished from these three species based on the presence of aplerotic oospores and diclinous antheridia and the absence of hypogynous antheridia. Therefore biological, morphological and molecular data support the recognition of a new species.

Two new species of Pythium, P. schmitthenneri and P. selbyi pathogens of corn and soybean in Ohio

Two new species of Pythium, pathogens of corn and soybean in Ohio, are described. Pythium schmitthenneri sp. nov. and Pythium selbyi sp. nov. both have morphological and sequence characteristics that place them in clade E1 of the genus Pythium. Morphology and sequence analysis of the ITS1-5.8S-ITS2 regions of these species were different from previously described species. The ITS region of Pythium schmitthenneri was 99.9% similar to P. acrogynum and 99.8% similar to P. hypogynum. All three species are characterized by globose to limoniform sporangia and plerotic oospores. Pythium schmitthenneri has mostly diclinous antheridia, compared to the strictly hypogynous antheridia of P. acrogynum and P. hypogynum. The temperature for growth of P. schmitthenneri is below 4 C to 32 C, and optimum growth is 18–25 C compared to 31–34 C for P. hypogynum. The ITS region of P. selbyi was 97.1% similar to P. longandrum and 97.5% similar to P. longisporangium. All three species are characterized by globose sporangia, mostly plerotic oospores, with one to two oospores per oogonium, and hypogynous or monoclinous antheridia. The temperature for growth of P. selbyi is below 4 to 32 C, with an optimum 18–25 C. These new species were widely dispersed throughout the soybean- and corn-producing regions in Ohio, making their characterization critical for managing the Pythium complex that causes seedling and root-rot disease in Ohio soybean and corn fields.

Detection and Characterization of Xanthomonas vasicola pv. vasculorum (Cobb 1894) comb. nov.Causing Bacterial Leaf Streak of Corn in the United States

Bacterial leaf streak of corn (Zea mays) recently reached epidemic levels in three corn-growing states, and has been detected in another six states in the central United States. Xanthomonas vasicola was identified as the causal agent of this disease. A multilocus sequence alignment of six housekeeping genes and comparison of average nucleotide identity from draft genome sequence were used to confirm phylogenetic relationships and classification of this bacteria relative to other X. vasicola strains. X. vasicola isolates from Nebraska and South Africa were highly virulent on corn and sugarcane and less virulent on sorghum but caused water-soaking symptoms that are typical of X. vasicola infection on the leaves of all three hosts. Based on host range and phylogenetic comparison, we propose the taxonomic designation of this organism to X. vasicola pv. vasculorum ( Cobb 1894 ) comb. nov. Polymerase chain reaction-based diagnostic assays were developed that distinguish X. vasicola pv. vasculorum and X. vasicola pv. holcicola from each other and from other Xanthomonas spp.