Studies on the genetic regulation of mycoparasitism in Trichoderma hamatum

Abstract

The ability of Trichoderma species to mycoparasitise other fungi as a source of carbon has led to their exploitation as biocontrol agents of soilborne fungal diseases, however widespread implementation has been hampered by inconsistent field performance. Usage recommendations based on field studies have provided a more consistent product, by optimising conditions for biocontrol. Genetic studies on genes implicated in mycoparasitism have revealed molecular targets for optimising biocontrol response, however much is unknown about the genetic regulation of mycoparasitism. The majority of reported Trichoderma biocontrol agents are T. harzianum or T. atroviride, however a T. hamatum isolate (6Sr4) with significant activity against Sclerotinia diseases of vegetable crops, has recently been developed for use as a commercial biocontrol agent of soilborne fungal diseases. In this study, three genes previously implicated in the mycoparasitic ability of T. atroviride were characterised from 6Sr4, and from two other T. Hamatum isolates of variable biocontrol potential to further current knowledge on the regulation of mycoparasitism, and identify molecular targets for research on optimisation of Trichoderma biocontrol activity in the field.

Using a combination of targeted, degenerate and inverse PCR, gene orthologues of chit42, prb1 and xbg1.3-110 were isolated, cloned and sequenced from all three T. Hamatum isolates and sequence analysis performed on the regulatory regions. Comparison between the different genes revealed regions of identity between chit42 and prb1, which supported the existence of a global inducer of mycoparasitism, suggested for T. atroviride. Identification of putative regulatory motifs in prb1 supported mutational work in T. atroviride prb1, which suggested an actively bound GATA site involved in nitrogen repression.

Northern blotting was performed to examine gene expression under alternate carbon sources and during mycoparasitism. In 6Sr4, chit42 and prb1 were strongly induced under high glycerol, whereas no induction was detected from xbg1.3-110. Conversely, no chit42 or prb1 induction was detected from a T. harzianum isolate (JD2) grown in the same study and only low induction of prb1 from T. atroviride has been reported. This suggested T. hamatum chit42 and prb1 to be under control of an additional pathway not active in T. harzianum and T. atroviride.

Both chit42 and prb1 were moderately induced during confrontation against Sclerotinia sclerotiorum, which supported a role in mycoparasitism. Xbg1.3-110 had been previously implicated in mycoparasitism by T. harzianum, however no northern analysis during mycoparasitism has not been reported. This study detected no xbg1.3-110 expression during confrontation and therefore questions its importance in mycoparasitism.

Genetic variation between the three isolates was assessed using UP-PCR, sequence alignments of chit42, prb1 and xbg1.3-110, and comparison of chit42 expression in response to elevated glycerol and during confrontation with S. sclerotiorum. 6Sr4 and 3Sr4-2 were more similar than either isolate with S1BYG, which did not correlate with observed biocontrol potential or colony morphology. Sequence analysis revealed multiple single nucleotide polymorphisms (SNPs) within the regulatory regions of chit42, prb1 and xbg1.3-110, some associated with putative regulatory sites. In S1BYG, chit42 expression was delayed relative to 6Sr4 and 3Sr4-2.

These studies identified multiple targets for future research. Most notable is the potential to incorporate glycerol into inoculant amendments to optimise chit42 and prb1 expression. It is likely field studies will be conducted to determine amendment effects on biocontrol activity.