Abstract:
Agaricus bisporus is the most commonly cultivated mushroom in North America
and has a great economic value. Green mould is a serious disease of A. bisporus and
causes major reductions in mushroom crop production. The causative agent of green
mould disease in North America was identified as Trichoderma aggressivum f.
aggressivum. Variations in the disease resistance have been shown in the different
commercial mushroom strains.
The purpose of this study is to continue investigations of the interactions between
T. aggressivum and A. bisporus during the development of green mould disease. The
main focus of the research was to study the roles of cell wall degrading enzymes in green
mould disease resistance and pathogenesis.
First, we tried to isolate and sequence the N-acetylglucosaminidase from A.
bisporus to understand the defensive mechanism of mushroom against the disease.
However, the lack of genomic and proteomic information of A. bisporus limited our
efforts. Next, T. aggressivum cell wall degrading enzymes that are thought to attack
Agaricus and mediate the disease development were examined. The three cell wall
degrading enzymes genes, encoding endochitinase (ech42), glucanase (fJ-1,3 glucanase)
and protease (prb 1), were isolated and sequenced from T. aggressivum f. aggressivum.
The sequence data showed significant homology with the corresponding genes from
other fungi including Trichoderma species. The transcription levels of the three T.
aggressivum cell wall degrading enzymes were studied during the in vitro co-cultivation
with A. bisporus using R T -qPCR. The transcription levels of the three genes were
significantly upregulated compared to the solitary culture levels but were upregulated to a lesser extent in co-cultivation with a resistant strain of A. bisporus than with a sensitive
strain.
An Agrobacterium tumefaciens transformation system was developed for T.
aggressivum and was used to transform three silencing plasmids to construct three new
T. aggressivum phenotypes, each with a silenced cell wall degrading enzyme. The
silencing efficiency was determined by RT-qPCR during the individual in vitro cocultivation of each of the new phenotypes with A. bisporus. The results showed that the
expression of the three enzymes was significantly decreased during the in vitro cocultivation when compared with the wild type. The phenotypes were co-cultivated with
A. bisporus on compost with monitoring the green mould disease progression. The data
indicated that prbi and ech42 genes is more important in disease progression than the p-
1,3 glucanase gene. Finally, the present study emphasises the role of the three cell wall
degrading enzymes in green mould disease infection and may provide a promising tool
for disease management.
