A characteristic feature of species of Trichoderma is the production of concentric rings of conidia in response to alternating light-dark conditions. In response to a single burst of light, a single ring of conidia forms at what was the colony perimeter. On the basis of these observations, competency to photoconidiate has been proposed to be due to the age and metabolic rate of the hyphal cell. In this study, conidiation was investigated in five biocontrol isolates (T. hamatum, T. atroviride, T. asperellum, T. virens and T. harzianum) using both a morphological and molecular approach. All five isolates produced concentric conidial rings under alternating light-dark conditions on potato-dextrose agar (PDA), however, in response to a 15 min burst of blue light, only T. asperellum and T. virens produced a clearly, defined conidial ring which correlated with the colony margin at the time of light exposure. Both T. harzianum and T. hamatum photoconidiated in a disk-like fashion and T. atroviride produced a broken ring with a partially filled in appearance. On the basis of these results, it was postulated that competency to photoconidiate is a factor of the metabolic state of the hyphal cell rather than chronological age or metabolic rate. The influence of the source of nitrogen on photoconidiation was assessed on pH-buffered (pH 5.4) minimal medium (MM) amended with glutamine, urea or KNO₃. In the presence of glutamine or urea, T. asperellum and T. harzianum conidiated in a disk, whereas, when KNO₃ was the sole nitrogen source, a ring of conidia was produced. Further, in the presence of increasing amounts of glutamine, the clearly defined photoconidial ring produced on PDA by T. asperellum became disk-like. These results clearly demonstrated that primary nitrogen promotes photoconidiation in these isolates and strongly suggests that competency of a hyphal cell to conidiate in response to light is dependent on the nitrogen catabolite repression state of the cell. The experiments were repeated for all five isolates on unbuffered MM. Differences were apparent between the buffered and unbuffered experiments for T. atroviride. No photoconidiation was observed in T. atroviride on buffered medium whereas on unbuffered medium, rings of conidia were produced on both primary and secondary nitrogen. These results show that photoconidiation in T. atroviride is influenced by the buffering capacity of the medium. Conidiation in response to light by T. hamatum and T. virens was absent in all nitrogen experiments, regardless of the nitrogen source and buffering capacity, whereas both isolates conidiated in response to light on PDA. These results imply that either both sources of nitrogen are required for photoconidiation, or a factor essential for conidiation in these two isolates was absent in the minimal medium. Mycelial injury was also investigated in five biocontrol isolates of Trichoderma. On PDA, all isolates except T. hamatum conidiated in response to injury. On nitrogen amended MM, conidiation in response to injury was again observed in all isolates except for T. hamatum. In T. atroviride, injury-induced conidiation was observed on all medium combinations except the pH-buffered MM amended with glutamine or urea and T. virens conidiated in response to injury on primary nitrogen only, regardless of the buffering capacity. These results have revealed conidiation in response to injury to be differentially regulated between isolates/species of Trichoderma. On unbuffered MM amended with glutamine or urea, conidiation in response to injury occurred at the colony perimeter only in T. atroviride. It was hypothesised that the restriction of conidiation to the perimeter may be due to changes in the pH of the agar. The experiment was repeated and the pH values of the agar under the growing colony measured at the time of light induction (48 h) or injury (72 h). The areas under the hyphal fronts were acidified to below the starting value of the medium (pH 5.4) and the centres of the plates were alkalinised. The region of acidification at the time of stimuli correlated with the production of conidia, which implicates a role for crossregulation of conidiation by the ambient pH. The influence of the ambient pH on injury-induced conidiation was investigated in T. hamatum and T. atroviride on MM amended with glutamine and PDA, pH-buffered from pH 2.8 to 5.6. Thickening of the hyphae around the injury site was observed at the lowest pH values on MM in both T. atroviride and T. hamatum, however no conidia were produced, whereas both Trichoderma species conidiated on pH-buffered PDA in a strictly low pH-dependent fashion. This is the first observation of injury-induced conidiation in T. hamatum. The influence of the ambient pH on photoconidiation was assessed in T. hamatum, T. atroviride and T. harzianum using both buffered and unbuffered PDA from pH 2.8 to 5.2. On buffered PDA, no conidiation in response to light was observed above pH 3.2 in T. hamatum, above 4.0 in T. atroviride and above 4.4 in T. harzianum, whereas on unbuffered PDA it occurred at all pH values tested. It was postulated that conidiation at pH values above 4.4 on unbuffered PDA was due to acidification of the agar. The pH values of the agar under the growing colony were measured at the time of light exposure and in contrast to the MM with glutamine experiments, alkalisation of the agar had occurred in both T. atroviride and T. hamatum. No change in medium pH was recorded under the growing T. harzianum colony. These results indicate that low pH-dependence of photoconidiation is directly related to the buffering capacity of the medium. Recent studies have linked regulation of conidiation in T. harzianum to Pac1, the PacC orthologue. In fungi, PacC regulates gene expression in response to the ambient pH. In these studies pH-dependent photoconidiation occurred only on buffered PDA and on unbuffered PDA conidiation occurred at significantly higher ambient pH levels. It is proposed that the influence of ambient pH on conidiation in the isolates used in this study is not due to direct Pac1 regulation. The T. harzianum isolate used in this study produced profuse amounts of the yellow anthraquinone pachybasin. Production of this secondary metabolite was strictly pH-dependent, irrespective of the buffering capacity of the medium. Studies in T. harzianum have linked Pac1 regulation to production of an antifungal α-pyrone. pH-dependence on both buffered and unbuffered media strongly suggests that pachybasin production may also be under the control of Pac1. Photoconidiation studies on broth-soaked filter paper, revealed rhythmic conidiation in the pachybasin producing T. harzianum isolate. Diffuse rings of conidia were produced in dark-grown cultures and, in cultures exposed to light for 15 min at 48 h, the rings were clearly defined. These results show that conidiation is under the control of an endogenous rhythm in T. harzianum and represent the first report of circadian conidiation in a wild-type Trichoderma. A Free-Running Rhythm (FRR) assay was used to investigate rhythmic gene expression in T. atroviride IMI206040 and a mutant derivative, in which the wc-2 orthologue, blr-2, was disrupted. Over a 3 d period, expression of gpd, which encodes the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, oscillated with a period of about 48 h. In the Δblr-2 mutant, the gpd rhythm was absent. These results revealed that in T. atroviride, gpd expression is under the control of an endogenous clock and that clock-regulated expression of gpd is associated with a functional BLR complex. Using degenerate primers, a portion of frq, which encodes the N. crassa clock oscillator FREQUENCY, was isolated from T. atroviride and used to probe the FRR assay northern blots. No frq expression was detected at any time point, which suggests that the circadian clock in Trichoderma does not involve FREQUENCY. In a concurrent study, orthologues of rco-1 (rcoT) were isolated and sequenced from T. atroviride and T. hamatum using a combination of degenerate, inverse and specific PCR. RcoT is an orthologue of the yeast global co-repressor Tup1 and in the filamentous fungi, RcoT orthologues have been demonstrated to negatively regulate conidiation. Genomic analysis of all available rcoT orthologues revealed the conservation of erg3, a major ergosterol biosynthesis gene, upstream from rcoT in ascomycetous filamentous fungi, but not in the ascomycetous yeast or in the basidiomycetes. These studies have significantly contributed to our understanding of the regulatory factors controlling conidiation in Trichoderma and have multiple implications for Trichoderma biocontrol; most notable the promotion of conidiation by primary nitrogen and low pH. Incubation conditions can be altered to suit the nitrogen and pH preferences of a biocontrol strain in order to promote cost effective conidial production, however this is not easily achieved in the soil, where the biocontrol strain must perform in a highly buffered environment optimised for plant growth. Successful use of Trichoderma biocontrol strains may involve the screening and targeting of strains to the appropriate pH conditions or the selection of new strains on the basis of capacity to perform under a given range of conditions.
| Identifer | oai:union.ndltd.org:ADTP/183331 |
| Date | January 2007 |
| Creators | Steyaert, Johanna M. |
| Publisher | Lincoln University |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://purl.org/net/lulib/thesisrights |
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