|dc.description.abstract||Trichoderma cf. atroviride isolate LU132 is used commercially in New Zealand as a biological control agent (BCA) on various economically important plants to protect against plant pathogens. Its broad spectrum of antagonistic activity is linked to a wide range of biological parameters but we have limited knowledge of what specific attributes make this isolate particularly effective. While LU132 is a remarkably efficient BCA compared to T. cf. atroviride isolate LU140, LU132 was found to be highly similar to LU140 at the DNA sequence level. This unusual combination of phenotypic variability and high DNA sequence similarity between isolates begged the question of what precisely was the molecular basis of the superior biological control activity of LU132. The focus of this PhD study was the comparison of the two isolates on the microbiological and molecular level and linking the characteristics together.
The two isolates exhibited different growth rates, conidiation and metabolism. Superior pathogen control demonstrated by LU132 depended on its faster growth, which is a prerequisite for successful distribution and competition. Their whole genomes were sequenced and found to be almost identical. A comprehensive data analysis identified only two single nucleotide polymorphisms (SNPs) between LU132 and LU140. One of these two SNPs was non-synonymous, changing the amino acid sequence for the previously undescribed single copy gene small EDRK-rich factor (serf) in LU132 (compared to LU140 and the reference genome). Bioinformatics analysis of the serf promoter and the deduced SERF amino acid sequence indicated involvement of SERF in hyphal growth, cellular differentiation and photoreception. The SNP was predicted to change the protein’s tertiary structure in LU132 and to create and change binding motifs.
To study the function of serf and the impact of the SNP, three ∆serf mutants were generated for each isolate. The serf deletion had no impact on growth rates, conidiation or metabolism of the three LU140 mutants but the LU132 mutants exhibited slower growth and one of them a conidiation pattern and metabolic profile that resembled the LU140 WT more than the LU132 WT. However, the phenotypes of the LU132 mutants and the LU140 WT were not entirely identical, suggesting additional factors are involved.
Nucleic acid extract analysis revealed no evidence for the presence of extra-chromosomal elements, such as a virus or plasmid, in either LU132 or LU140. However, single-stranded RNA (ssRNA) bands were found in another T. cf. atroviride isolate that was used as positive control. This was the first evidence of ssRNA in Trichoderma.
The presence of the key epigenetic modification, cytosine methylation, was investigated via methylation sensitive AFLP, followed by capillary electrophoresis and MiSeq sequencing. LU132 and LU140 contained numerous differentially methylated regions in their genomes. Eight of these regions were located in or close to nine genes although none were in promoter regions. These modifications could potentially lead to altered gene regulation and alternative splicing and contribute to the phenotypic differences between LU132 and LU140. These results also provided the first evidence of cytosine methylation in Trichoderma.
The work contributed to a better understanding of phenotypic determinants and the underlying molecular causes for successful biocontrol in Trichoderma. Understanding the basis of biocontrol excellence will enable us to predict biocontrol potency, screen directly and more efficiently for BCAs and ultimately develop more successful biocontrol products.||en