The soil-borne, rhizosphere-competent, filamentous fungus Trichoderma virens
is a well-known biocontrol agent able to control pathogenic fungi through the production
of antibiotics, the induction of systemic resistance in host plants, or by directly
parasitizing the competing fungus. Competition for iron is another means by which
Trichoderma can hinder competing microorganisms, and siderophores are a means by
which microorganisms obtain iron. In silico analysis of the T. virens genome suggested
that two genes putatively encoding extracellular siderophore-producing nonribosomal
peptide synthetases (NRPSs) were present.
In this study, a disruption was created in one of the genes, TvNPS6, to create a
mutant unable to produce the NRPS TvNps6 (DeltaTvnps6). Previously, a mutant
(DeltaTvsidD) had been generated with a disruption in the second gene (TvSIDD) encoding
an NRPS thought to be involved in siderophore biosynthesis. A double mutant
(DeltaDeltaTvsidDTvnps6) was generated by transformation of a DeltaTvsidD strain with a vector
targeting disruption of TvNPS6. This resulted in transformants disrupted within both the putative siderophore-producing NRPSs. Thus, three mutants were available for analysis
of the role of these genes in the ecology of T. virens. Transformants were confirmed by
PCR and Southern blotting analysis.
Phenotypic characterization of the mutants included both HPLC analysis of
siderophore production, growth on agar and in liquid media, conidiation, germination in
the presence of hydrogen peroxide, biocontrol against Pythium ultimum, in vitro
confrontation against Rhizoctonia solani and growth with iron chelators to determine the
contribution of reductive iron assimilation (RIA) compared to that of siderophores. The
HPLC analysis demonstrated that T. virens Gv 29-8 (wild-type) produced a single
siderophore peak when grown in an iron-depleted medium. This peak was not present in
the DeltaTvnps6 and DeltaDeltaTvsidDTvnps6 mutants but was apparent with the DeltaTvsidD
mutants. From the HPLC analysis, T. virens evidently produces a coprogen-type
siderophore. Few differences were observed in the other phenotypic tests, though
hydrogen peroxide showed some small inhibitory effects towards the DeltaTvnps6 mutants.
The addition of chelators, which inhibit RIA, exerted some negative effects on all strains
growing under iron-limited media, particularly the DeltaTvnps6 and DeltaDeltaTvsidDTvnps6
strains.
This study demonstrated that although T. virens has two genes putatively
encoding siderophore producing NRPSs, only the TvNPS6 gene was required for
extracellular siderophore production. The greater sensitivity of the mutants towards the
iron chelators suggests that unlike other other fungi studied, Trichoderma virens utilizes RIA, rather than siderophore production, as the primary means by which the fungus
obtains iron in an iron-limited environment.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/ETD-TAMU-2009-12-7410 |
| Date | 2009 December 1900 |
| Creators | Hurley, James Franklin |
| Contributors | Kenerley, Charles M. |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Thesis, text |
| Format | application/pdf |
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