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
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.
University of Minnesota Ph.D. dissertation. April 2010. Major: Plant Pathology. Advisor: Dr. Dean Malvick. 1 computer file (PDF); x, 108 pages.
Impullitti, Ann E..
Colonization of soybean (Glycine max) by the pathogen Phialophora gregata colonization of soybean (Glycine max) by the pathogen Phialophora gregata and endophytic fungi..
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