Colonization of soybean (Glycine max) by the pathogen Phialophora gregata colonization of soybean (Glycine max) by the pathogen Phialophora gregata and endophytic fungi.

Abstract

Fungal pathogens, beneficial symbionts, and endophytes colonize plants and reduce, enhance, or have cryptic effects on plant productivity. My dissertation focused on colonization of soybean by the fungal pathogen Phialophora gregata and characterization of the endophytic fungal population within soybean. Soybean was used as a model plant because it is economically important, its colonization by P. gregata has well-defined latent and pathogenic phases, and endophytes within the stem were not characterized. The first goal of this research was to investigate infection and plant responses during latent and pathogenic phases of disease development. Susceptible and resistant cultivars were infected with type A of P. gregata that causes stem and leaf necrosis or type B that causes only stem necrosis. Latent infection occurs after plants are colonized by a pathogen and remain asymptomatic. The number of vessels, leaf area, stomatal conductance, and yield of photosystem II (PSII) of infected and noninfected plants were determined. During latent infection, differences in the number of vessels was observed between susceptible and resistant plants, P. gregata was rarely observed in stems, and leaf area of susceptible plants was reduced by infection with type A. During pathogenic infection by type A, the resistant cultivar had fewer than 10% of vessels colonized and 20 to 25% more vessels than uninfected plants, while more than 70% of vessels were colonized in the susceptible cultivar and 50% fewer vessels were present compared to uninfected plants. During pathogenic infection by type B, more than 10% of vessels were colonized and no differences in vessel numbers were observed compared to the uninfected resistant plants. Type A did not reduce the leaf area of the resistant cultivars, but the leaf area of the susceptible cultivar was reduced by 80%. Type B reduced the leaf area of susceptible and resistant plants by 30%. Stomatal conductance was reduced 80% by types A and B in susceptible plants and by 40% in resistant plants. No differences in yield of PSII iii were observed. Qualitative differences in colonization were observed during pathogenic infection using GFP and RFP-tagged isolates. Type B-RFP was observed in the primary xylem, while type A-GFP was observed outside of the primary xylem in the resistant cultivar. Whereas in the susceptible cultivar, PgAGFP was in the primary xylem and PgB-RFP was limited to the interfascicular region. In summary, latent infection reduced the photosynthetic area of infected plants, but did not significantly modify their vascular structure and may lead to reduction of photosynthetic efficiency and increased susceptibility to biotic and abiotic stress. During pathogenic infection, resistant cultivars produced more vessels, restricted or excluded P. gregata from the vascular system, and reduced stomatal conductance and photosynthetic area. These responses may compensate for reduced vessel function and allow water movement. The second goal was to determine the diversity of fungal endophytes in soybean stems. Stems from field-grown plants that were either treated or not treated with glyphosate were surface-disinfested, and fungal endophytes were assessed using culture-dependent (CD) and –independent (CI) methods. For the CD method, stem segments were dissected into an outer stem composed of the epidermal and vascular tissues and an inner stem composed of the pith tissues. Cultured fungi were grouped based on colony morphology and identified based on rDNA ITS sequences. For the CI method, DNA was extracted from stems and the ITS-region was amplified using fungal-specific primers, cloned, and sequenced for identification. More isolates were obtained from the outer than inner stems, and from the base of the stems compared to the apex. The most frequently isolated genera were Cladosporium (32%), Phomopsis/Diaporthe (15%), Alternaria (14%), Fusarium (11%), and Phoma (8%), The CD method detected more endophytic diversity (H’ = 2.35) than the CI method (H’ = 0.76). The most prevalent genus identified using the CI method was Cladosporium (83%). Soybean genotype influenced the diversity of endophytes more than glyphosate treatment. This research also suggests soybean harbors an endophytic fungal population much less diverse than plants in the tropics and in polycultures.