Protoplast regeneration is an effective tool that can be used for strain improvement. This can lead to strains with improved characteristics more suited to the incorporation into an IPM programme. In this study, various strains of Trichoderma that showed bioactivity potential against Psa in kiwifruit orchards were used to produce protoplast regenerants that were tolerant to pesticides used in these orchards. The use of protoplast fusion (both intra-strain and inter-strain) is a common technique used to enhance biocontrol agents, however it has been have found that regenerating protoplasts on selective media enables selection for rare genetic variants at a rate that far exceeds that within the parental population. This highlights the potential for the usage of protoplast regeneration in place of traditional mutant forming methods such as UV or chemical mutagenesis.
Previously some researchers have described such progeny as arising from self-fusion (intra-strain). However, when only one parental strain is present, it is not possible to definitively state that the selected progenies were the result of any fusion event. Previous studies where protoplast technologies have been used to select or modify varying Trichoderma strains have not always offered proof of these changes at a molecular level. Prabavathy et al. (2006a) reported that regenerated protoplasts from a single T. reesei strain were mixed with the PEG solution (for self-fusion) and pairs of protoplasts were subsequently observed. They then stated the nuclei of the pairing protoplasts had fused together from having observed the two cells near to one another (Prabavathy et al., 2006a). Another study used intra- strain fusion to develop progeny from a single strain of T. harzianum to enhance the level of production of chitinase and the antagonistic potential against Rhizoctonia solani. Fifteen of these progeny were selected for further testing, and both the chitinase and antagonistic activity against pathogens had substantially increased (Prabavathy et al., 2006b). The authors postulated that this was the result of self-fusion, however no evidence of self-fusion was provided for this strain so the exact mechanism has not yet been identified. However, Sharma et al. (2016) showed that there were novel fragments between the progeny in the RAPD profiles of self-fused T. harzianum fusants. This highlights the importance of some form of molecular characterisation to prove that intra-strain fusion has occurred. After multiple protoplast fusion rounds, a stable fusant was unable to be produced. The putative fusion rate between the various regenerants varied between 0.0002% and 0.009% which is significantly lower than previously reported. For example, previous fusion rates with our research group on other Trichoderma strains were ~0.036% (unpublished data) and other studies have reported a fusion rate of up to 6.2% between T. harzianum and T. viride (Balasubramanian and Lalithakumari, 2008). Furthermore, any fusants produced lost their stability on the double amended media after three rounds of single spore selection. Previous studies producing protoplast fusants have encountered similar problems, in which some protoplast progeny form as the result of heterokaryosis or temporary
cytoplasmic fusion formation and lose their selection through successive cycles of mitotic division during mycelial growth (Hassan et al., 2011; Stasz et al., 1988; Stasz and Harman, 1990). Fungi are well known for having unstable genomes and even fungi undergoing purely asexual reproduction can produce offspring with different sized chromosomes over a few generations (Davière et al., 2001; Strom and Bushley, 2016). Genetic instability of intraspecific and interspecific hybrids formed through protoplast fusion is a commonly reported problem (Anné, 1982; Stasz and Harman, 1990; Wang et al., 2010).
Additionally stable heterokaryon formation can be rare due to the issues of heterokaryon or vegetative incompatibility, which is responsible for preventing the formation of these stable heterokaryons through expression of heterokaryon incompatibility genes (het) (Daskalov et al., 2017; Strom and Bushley, 2016). If two incompatible nuclei occupy the same cell, the expression of these het genes leads to the eventual death of the heterokaryotic cell. Hassan et al. (2011) also reported loss of double pesticide fungicide tolerance after successive subculturing in Trichoderma fusants. They determined the genetic stability of their double fungicide tolerant strains by their ability to maintain tolerance to both fungicides after subculturing three times in the absence of fungicide. Five out of the seven fusants they produced retained the double pesticide tolerance. This raises a notable difference in the production of fusants from other studies compared to this present one, that the parental strains used were either wild type fungi or mutants that were produced via UV-mutagenesis, not tolerant regenerated protoplasts. The unknown mechanism behind the phenotypic plasticity and remodelling that is observed from protoplast regeneration may be responsible for the incompatibility that was seen in the attempt to fuse these strains.
Due to the problems that were encountered with the low rate of fusion, it was postulated that there may be a potential interactive toxicity effect of the double amended pesticide media (between copper sulphate and the carbendazim of Chief®) on the strains tested. This was confirmed by the inability of parental strains to grow on combined media with sub-lethal amounts of the pesticides. The same result was obtained when pure carbendazim (rather than the commercial fungicide) was used. This is in contrast to a strain of T. longibrachiatum that was fused by Lalithakumari et al. (1996) which was found to be tolerant to 200 mmol/L copper sulphate and 10 µmol/L carbendazim. They used protoplast fusion to combine a T. harzianum strain that had a high biocontrol potential and the highly tolerant T. longibrachiatum that was mentioned above (Lalithakumari et al., 1996).
The results from this study raise some interesting questions with regards to the interactive effect of chemicals in nature, due to the fact that chemicals almost never occur in nature as a single solution, but as complex mixtures or part of a spray programme (Komárek et al., 2010; Kungolos et al., 2009). For kiwifruit alone, according to the 2018/2019 New Zealand NovaChem Agrichemical Manual
(https://www.novachem.co.nz/; last accessed 23.4.18) over 98 pesticide and herbicides are recommended for the protection of kiwifruit from pests (including fungi, bacteria and insects) and weeds. Numerous studies have shown that some interactions between pesticides influence both their behaviour in the soil and their toxicity (Kungolos et al., 2009; Schuster and Schroeder, 1990; Tsiridis et al., 2006; Zhang et al., 2003). Zhang et al. (2003) showed that the IC50 toxicity level (half minimal inhibitory concentration of a substance) of three agrochemicals that they tested (acetochlor, methamidophos and copper) under combinations were much lower that the individual IC50 values against overall indigenous microorganism populations in soil. Moreover, different toxicological tests on various bacterial species have shown that common environmental concentrations of copper based fungicides can pose a risk for other non-target organisms (Kungolos et al., 2009). Combining these chemicals may result in unexpected toxicity towards soil microbes, in particular beneficial ones. Further studies within our lab group are underway investigating the impact of intensive spraying regimes on the microbial communities within kiwifruit orchards, focusing particularly on Trichoderma populations. Soil was collected from seven sites around the Nelson and Bay of Plenty kiwifruit growing regions and the copper levels within these soils were tested (Christine Stark, pers. comm.). While at elevated levels (over 500 ppm) copper can become toxic most of the tested orchards had copper levels around 100 ppm. In contrast, the base level of selection for protoplasting (being also the cessation level of growth of the parents) was approximately 1000 ppm (4 mM). Although the level of copper toxicity may not be an issue for soil fungal communities currently, the regenerants that are copper tolerant could be used alongside copper sprays in an IPM programme within the same time frame when the levels are temporarily high during copper application unlike Kiwivax™ which cannot be used within three days of applying foliar coppers.
The production and isolation of protoplasts is a crucial step in strain enhancement of Trichoderma and other filamentous fungi that do not have an obvious sexual stage. The results presented here show that a significant increase in protoplast recovery is possible with the optimisation of each strain. Numerous studies have shown the importance of optimisation for the formation and regeneration of protoplasts, each with different recommendations. Tschen and Li (1994) highlighted the significance between species with differences in both the isolation of protoplasts and the regeneration frequency from two Trichoderma (T. koningii and T. harzianum) varying greatly. This most likely is dependent on the structural properties of the fungal cell wall as this can vary between species. They looked at eight different factors affecting protoplast formation: lytic enzymes, concentration of Novozym 234, culture age, concentration of mycelium, temperature used for lytic digestion, pH, osmotic stabiliser and incubation time in osmotic stabiliser and consequently gave a general recommendation that they suggested for all Trichoderma strains. This current study has found the isolation of protoplasts to be highly strain specific, with previous work within our group showing substantial differences in the
resistance of various strains to protoplasting techniques. Of four T. sp. “atroviride B” strains, the number of protoplasts that were produced varied from 5.1 x 107 protoplasts/mL to 8.19 x 109 protoplasts/mL and the methodology needed to be slightly modified for each strain. Strain-specificity has also been observed for T. viride and T. harzianum, with research having shown different optimisation methods varyingly greatly for the same species. For example, the release in protoplasts from different culture ages for the same species (between studies) varied. For T. viride, the optimal time for mycelial formation is between 14-18 h, and for T. harzianum that same figure varies from 16- 24 h (Balasubramanian and Lalithakumari, 2008; Mrinalini and Lalithakumari, 1998). Together these results highlight that significant optimisation of protoplast methodology for each strain is essential. Despite the frequent use of protoplast technology for strain enhancement in both the biotechnology industry and for biocontrol agents, very little is known of the underlying genetic consequences and the potential effects downstream. Further research is needed to understand the full extent of these changes. The phenotypic plasticity and remodelling that is induced in the production of protoplasts is beneficial for potential strain enhancement as seen in this study. One possible explanation for the protoplast-induced phenotypic plasticity is methylation as previously mentioned. The UP-PCR patterns were identical between both the parental strains and the respective progeny suggesting no major DNA rearrangements had occurred. Previous work within our group on other protoplast progeny using Methylation-Sensitive-AFLPs (MSAP) analysis detected remodelling of the methylation profile, suggesting that gene expression through methylation changes could be the cause of the enhanced phenotypic plasticity observed in the regenerant variants produced. Further chapters within this thesis attempt to characterise the influence of methylation changes in protoplast-induced phenotypic plasticity.