Fungal endophyte diversity in soybean.

AIM: To determine the identity and diversity of endophytes in soybean plants using culture-dependent (CD) and culture-independent (CI) methods. METHODS AND RESULTS: Stem samples were collected from three field-grown soybean cultivars grown to a reproductive stage in Minnesota, USA. Samples were surface disinfested, and CD and CI methods were used to assess the endophytes. For the CD method, fungi were isolated and grouped based on colony morphology, and the rDNA ITS region was sequenced to identify the cultures. The most frequently isolated genera were Cladosporium (36%), Alternaria (13%), Diaporthe (9%) and Epicoccum (9%). For the CI method, DNA was extracted from the stems, and the ITS region was amplified, cloned and sequenced for identification. The most prevalent genus detected using CI method was Cladosporium (85%). CONCLUSIONS: Soybean contains a diverse array of endophytic fungi that were identified in this study. The CD method detected greater endophyte diversity (H' = 2·12) than the CI method (H' = 0·66). SIGNIFICANCE AND IMPACT OF THE STUDY: The results improve our understanding of the identity and diversity of endophytic fungi that likely have different kinds of interactions with soybean plants. The results suggest that CD and CI methods should be used to study endophytes in soybean and perhaps other annual crop plants.

Characterizing Reaction of Soybean to Phialophora gregata Using Pathogen Population Density and DNA Quantity in Stems.

Evaluation of soybean germplasm for resistance to brown stem rot (BSR) is typically based on symptom severity. However, this approach may not reflect the level of colonization of soybean by the casual agent, Phialophora gregata. A potentially more accurate method to characterize resistance to BSR is to estimate pathogen quantity. The primary goal of this study was to evaluate soybean accessions for resistance to BSR based on the quantity of pathogen in stems. Plants were collected from experiments in field and controlled environments, and CFU and pathogen DNA quantity were determined using dilution plating techniques and real-time quantitative PCR (qPCR), respectively. In the field, the BSR-susceptible cultivars Corsoy 79 and Century 84 expressed greater than 73% foliar and stem symptom severity and had the highest pathogen population density, with a range from log10 4.3 to 4.7 CFU per gram of stem tissue. The resistant cultivar Bell expressed less than 10% foliar symptom severity, but had a pathogen population density that was not statistically different from the susceptible accessions. CFU measured in Dwight and L84-5873 were consistently lower than CFU in susceptible accessions and several resistant accessions. The amount of pathogen DNA differed among accessions in controlled environments. For example, Corsoy 79 and Century 84 had the highest pathogen DNA quantity, ranging from log10 6.19 to 6.65 copies, whereas the resistant cultivars Bell, Dwight, and L84-5873 had significantly lower DNA quantities, ranging from log10 2.04 to 2.91 copies. PI 437833 and IA2008R expressed low symptom severity but contained high DNA quantities. Pella 86, a highly symptomatic cultivar, had fewer CFU and lower DNA quantity in comparison to two other highly symptomatic cultivars and some cultivars with low symptom severity. These results suggest that some accessions express resistance to both pathogen colonization and symptom development, while others are resistant to symptom development but not to pathogen colonization. Results also indicate that resistant and susceptible accessions can be distinguished based on DNA quantity in controlled environments. In the field, differences between the pathogen population in resistant and susceptible cultivars were less distinct, possibly due to when plants were assayed.

Detection and Quantification of Phialophora gregata in Soybean and Soil Samples with a Quantitative, Real-Time PCR Assay.

Brown stem rot of soybean, caused by the soilborne fungus Phialophora gregata, is a common and widespread disease of soybean (Glycine max) in the midwestern United States. This pathogen is challenging to study due to a long latent period and slow growth. A TaqMan probe-based quantitative, real-time polymerase chain reaction (qPCR) assay was developed for sensitive and specific detection and quantification of genotypes A and B of P. gregata in plant and soil samples. It is sensitive with detection limits of 50 fg of pure genomic DNA, 100 copies of the target DNA sequence, and approximately 400 conidia. The qPCR assay is approximately 1,000 times more sensitive in detecting DNA and conidia of P. gregata, and is more rapid and less sensitive to PCR inhibitors from soybean stems than a standard PCR (sPCR) assay. Using this single-step qPCR assay, low levels of infection were detected in soybean stems at least 1 to 2 weeks prior to symptom development and before P. gregata was detected with sPCR. This assay also was used to detect the pathogen in field-grown plants and in naturally infested field soils. This new qPCR assay is a powerful tool for rapid, specific, and sensitive detection, diagnosis, and quantification of P. gregata in plants and soil, and for advancing studies of the ecology of P. gregata and its interactions with host plants.

Population Dynamics of Phialophora gregata in Stem Residue of a Resistant and a Susceptible Soybean Cultivar.

Previous studies on the saprophytic survival of Phialophora gregata were conducted with soybean residue derived from a susceptible cultivar and did not address genotypes of P. gregata. This current study monitored the saprophytic population density of P. gregata in stem residue derived from a susceptible and a resistant soybean cultivar placed in the field. A second phase of the study followed the frequencies of genotypes A and B of P. gregata in stem residue derived from a susceptible cultivar. The population density of P. gregata declined 10-fold in stem residue from the initiation of sampling to the end of this 16-month study, regardless of cultivar or whether residue was positioned on the soil surface or buried. The population density of P. gregata was greater in buried residue of the resistant cultivar compared with the susceptible cultivar after 12 to 14 months, but equalized after 16 months. The population density of P. gregata was similar in residue derived from the susceptible and resistant cultivars if positioned on the soil surface. Genotype B was detected more frequently than genotype A of P. gregata at each sampling date regardless of residue placement.