Isolate-specific conidiation in Trichoderma in response to different nitrogen sources
Introduction
Trichoderma spp. are ubiquitous ascomycetous fungi found commonly in agricultural, grassland, forest, saline, and desert soils, and are particularly prevalent in the humic layer of hardwood forests where they represent up to 3 % of all fungal propagules (Papavizas, 1985, Klein and Eveleigh, 1998). Many species of Trichoderma are considered superior saprophytes able to enzymatically attack and metabolise a wide range of substrates, which has led to their exploitation in industry and agriculture. Their antagonistic abilities towards pathogenic fungi have been harnessed globally as a means of biocontrol and consequently Trichoderma spp. represent one third of all fungal species used in biocontrol of plant pathogens (Chernin & Chet 2002). In general, commercial preparations of Trichoderma spp. for biological control consist of bulk produced conidia, whereas good biocontrol activity in the environment relies upon the fungus remaining vegetative and, thus, antagonistically active. Therefore, efficient and effective use of Trichoderma biological control agents (BCAs) involves achieving a balance between ample cost-effective conidiation during production and vigorous vegetative growth during usage.
Conidiation in Trichoderma can be stimulated in vitro through the manipulation of nutrients, exposure to light and by injury to the mycelium (Betina and Farkaš, 1998, Casas-Flores et al., 2004). Though light induction is not wholly exploited in commercial preparations of conidia, it does provide a means for conducting controlled, precise experiments on conidiation. In constant light, conidiation occurs continuously across the colony. In contrast, under alternating light/dark conditions, concentric rings of conidial formation can be seen and in response to a single light exposure a single ring of profuse conidiation can be observed at the colony margin (Gutter, 1957, Betina and Farkaš, 1998, Casas-Flores et al., 2004). Fungal conidiation in response to light is known to be genetically regulated. The effective wavelength range for photoconidiation in Trichoderma spp. lies within the blue/UVA spectrum and, in T. atroviride, the proteins BLR-1 and BLR-2 (Blue-Light Regulators) have been demonstrated to be the key regulators of this response (Casas-Flores et al. 2004). BLR-1 and BLR-2 are orthologues of the WC (White Collar) proteins, which control all known blue-light responses in Neurospora crassa (Rodriguez-Ortiz et al. 2009).
Commercial production of conidia typically relies on manipulation of nutrients and substrates to promote conidiation, which has led to much research into the optimal growth conditions for in vitro conidiation in many species of Trichoderma. Together, these studies suggested that the carbon and nitrogen status and the C:N ratio, in addition to the ambient pH, are the main environmental factors influencing conidiation in Trichoderma (Brian and Hemming, 1950, Aube and Gagnon, 1969, Lewis and Papavizas, 1983, Jackson et al., 1991, Bastos, 2001, Kredics et al., 2004, Gao et al., 2007). However, conidiation is not simply and directly dependent on environmental stimuli. Photoinduction, for example, is dependent on cell competency (Gutter, 1957, Gressel and Galun, 1967, Galun, 1971). Fungal development is further mediated and regulated by intercolony communication through volatile signalling molecules (Nemcovic et al. 2008). Environmental factors such as light and nutrients can also interact in more complicated ways to modify the conidiation response. For example, while conidiation in T. atroviride has been suggested to be primarily carbon-source dependent, the source of carbon cross-regulates photoconidiation by BLR-1/BLR-2 (Friedl et al. 2008). The extent of light-induced conidiation has also been suggested to be dependent upon the nitrogen status of the medium (Ellison et al. 1981). Light exposure and the manipulation of carbon or nitrogen might be factors that could be exploited for controlled, precise induction of conidiation.
In the presence of preferred carbon or nitrogen sources, organisms repress expression of genes required for the utilisation of secondary sources and this is referred to as catabolite repression. Under carbon or nitrogen deprivation, or when primary sources are low and secondary sources are high, derepression occurs. Further, in T. atroviride sudden carbon deprivation has been shown to induce a ring of conidia at the colony perimeter and this was dependent on expression of blr-1 and blr-2, which clearly demonstrated a link between carbon sensing and the light-induction pathway (Casas-Flores et al. 2006). Sudden nitrogen deprivation in T. atroviride resulted in a disk-like conidial response and this was independent of the BLR pathway, however studies in N. crassa have demonstrated cross-regulation of the WC-1/WC-2 photoconidiation pathway by nitrogen starvation. Nitrogen deprivation induced expression of blue-light inducible genes in N. crassa and this was independent of wc-1/wc-2 (Sokolovsky et al. 1992). These studies showed a clear interaction between catabolite repression and aspects of light induction and conidiation.
Nitrogen catabolism has also been implicated in other light-associated responses in filamentous fungi. Production of the toxin cercosporin, by the phytopathogenic fungus Cercospora nicotianae, is primarily induced by light exposure and is also affected by environmental conditions including carbon and nitrogen sources and pH (Dekkers et al., 2007, You et al., 2008). Similarly, carotenoid production by the filamentous fungi Gibberella fujikuroi is induced by blue light and is affected by environmental conditions including nitrogen supply (Garbayo et al., 2003, Rodriguez-Ortiz et al., 2009).
While we have some understanding of the chemical and physical factors that influence conidiation in some Trichoderma species, the majority of conidiation studies have focused on T. viride and more recently T. atroviride. This work seeks to expand our understanding of conidiation by investigating both light- and injury-induced conidiation in T. atroviride and four additional species of Trichoderma (T. hamatum, T. asperellum, T. virens, and T. pleuroticola), and by investigating the influence of primary and secondary nitrogen sources on the induction of conidiation.
Section snippets
Isolates
Four Trichoderma biocontrol isolates (T. hamatum LU592, T. atroviride LU298, T. asperellum LU697, and T. virens LU540) and one isolate from the mushroom pathogen species T. pleuroticola (LU675) were obtained from the Lincoln University culture collection for use in this study. All isolates were derived from New Zealand soil or plant material and had been previously identified on the basis of morphology and ITS/tef1 sequence analysis (Bio-Protection Research Centre, Lincoln University, New
Effect of alternating light/dark conditions on conidiation on PDA
Under 12 h light/12 h dark alternating cycles, all isolates produced concentric rings of conidiation and conidial maturation proceeded from the centre outwards (Fig 1). Each ring represented the colony front on consecutive days and the number of rings varied between isolates due to differences in growth rate.
Effect of a single blue-light exposure on conidiation on PDA
Response to a single blue-light exposure varied significantly between the species. Trichoderma asperellum and T. virens produced a clearly defined ring of pigmented conidia similar to that
Discussion
Research into photoconidiation in Trichoderma has largely been limited to the species T. viride and T. atroviride. These species have dominated Trichoderma conidiation research and served as both the morphological and molecular models. In this study, photoconidiation was induced and compared between four biocontrol isolates and one mushroom pathogen in five separate experiments. The isolates represented different species of Trichoderma, which have been identified on the basis of ITS and tef1
Acknowledgments
This research was funded by a grant from the New Zealand Tertiary Education Commission.
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Nutrient amendments affect Trichoderma atroviride conidium production, germination and bioactivity
2016, Biological ControlCitation Excerpt :Excessive nutrient availability will also cause nutrient catabolite repression. For example, Steyaert et al. (2010a) demonstrated that nutrient-rich media containing C and N repressed C/N catabolite genes in Trichoderma spp., while under N or C starvation, conidiation was induced by carbon/nitrogen catabolite de-repression under gene regulation. In the present study, the amount of sugar in the media varied from 10 g/l (4.2 g/l C) to 40 g/l (16.8 g/l C), this last concentration being excessive for T. atroviride LU132.
Rhythmic conidiation in the blue-light fungus Trichoderma pleuroticola
2010, Fungal BiologyCitation Excerpt :Some ras-1 mutations have been shown to cause an increase in ROS (reactive oxygen species) and indeed (2007) demonstrated that an increase in the cellular levels of ROS in the wild type resulted in clear circadian banding with periods similar to the ras-1bd mutant. The effects of CO2 on photoconidiation in Trichoderma spp. are not known, however an absence of oxygen does inhibit development of conidia in response to light in Trichoderma viride (Gutter 1957; Gressel et al. 1975) and it has been observed that sealing of the plates significantly reduced conidiation in Trichoderma atroviride LU298 and Trichoderma hamatum LU592 (Steyaert et al. 2010a,b). Thus it is possible that use of the inverted tube assay and/or promotion of cellular ROS may enable calculation of the free-running period of the T. pleuroticola conidiation rhythm.
Ambient pH intrinsically influences Trichoderma conidiation and colony morphology
2010, Fungal BiologyCitation Excerpt :Thus it is proposed that photoconidiation is dependent on a low intracellular pH in T. atroviride. Conidiation in response to injury also required a low pH on buffered media, whereas injury-induced conidiation has been observed on standard PDA which is unbuffered and at the higher pH value of 5.6–5.8 and was more intense at the perimeter (Steyaert et al. 2010). If conidiation in response to injury is also dependent on low intracellular pH, as postulated for photoconidiation, then injury-induced conidiation at pH values above pH 4.0 on unbuffered PDA is likely due intracellular acidification.