Epigenetic priming and in vitro mutagenesis in sugarcane (Saccharum Spp. Hybrids) for resistance to fusarium species and Eldana saccharina (Lepidoptera: Pyralidae).

Masters Degree. University of KwaZulu-Natal, Durban.

In the South African sugar industry, there have been substantial economic losses of R1 billion/annum caused by the indigenous pyralid borer, Eldana saccharina (Lepidoptera: Pyralidae). To develop control measures for E. saccharina in sugarcane, it is important to understand the interactions between the stalk borer and Fusarium spp. In previous studies, in vitro assays have shown that Fusarium strains may be antagonistic (e.g., F. sacchari PNG40) or beneficial (e.g., F. pseudonyamai SC17). F. pseudonyamai SC17 is a potential endophytic indicator of E. saccharina infestation, as the association between borer infestation and infection by the fungus causes Fusarium stalk rot in sugarcane. Studies have reported that the presence of endophytic fungi may have several benefits to the host plant, e.g., the production of phytohormones such as indole-3-acetic acid (IAA), which promotes plant development. The study aimed to: 1) choose a suitable resistance priming agent between hexanoic acid (Hx) and cis-jasmone (CJ); 2) determine an appropriate culture filtrate (CF) concentration for in vitro screening of calli and plantlets for tolerance to F. pseudonygamai; 3) develop a protocol (epigenetic priming and mutagenesis) to generate mutants: primed only (0.6 mM Hx), a combination of priming and mutagenic agents (100 μM 5-AzaC + 16 mM EMS-induced); 4) screen for indole-3-acetic acid production by F. pseudonygamai; and 5) characterise in vitro selected mutants for E. saccharina and F. pseudonygamai resistance by comparing the levels of resistance between unprimed, primed, and primed + mutagenic plantlets through ex vitro screening. When cis-jasmone (CJ) and hexanoic acid (Hx) were investigated for their effect on priming for pathogen resistance, 0.6 mM Hx was selected as the optimum priming agent concentration for both the callus and plantlet regeneration stages. At the highest CF concentration (100 ppm) at the embryo germination stage, the number of plantlets was greatly reduced to 58 and 98 plantlets/0.1 g of callus, for cultivars 88H0019 and N41 respectively, compared to more than 600 plantlets/0.1 g of callus in the no CF control. Unexpectedly, in the plantlet regeneration stage all the tested CF concentrations had a significant positive effect on the percentage of plantlets that re-rooted compared with the control. Both cultivars showed a 95 - 100 % rooting ability of the plantlets, which was significantly higher than the percentage of plantlets that rooted in the embryo germination media (EGM1) containing no CF (60 - 70 %) (p < 0.001). Likewise, all the concentrations of the CF had a positive effect on the root length of plantlets, with 1500 ppm CF resulting in the highest root length of 31.5 mm ± 4.3 for 88H0019 and 34.05 mm ± 3.9 for N41. Hence, F. pseudonygamai SC17 could not be used as an in vitro selection agent in a root re-growth assay. Due to the enhanced effect of F. pseudonygamai SC17 CF on root growth, the fungal isolate’s potential to produce indole acetic-3-acid (IAA) was assessed. F. pseudonygamai produced the highest IAA concentration (743.1 nM) in the presence of L-tryptophan than in the treatment without L-tryptophan (457.2 nM). This suggests that the observed enhanced root growth may be due in part to the production of auxin (IAA) in the F. pseudonygamai SC17 CF. Acclimatised in vitro plantlets (8-9 months old) were inoculated only with F. pseudonygamai SC17 or dual inoculated: firstly, with F. pseudonygamai SC17, then 1-2 2nd instar E. saccharina larvae that were placed into the leaf whorls 2 weeks later. To confirm tolerance of the putative mutants, fungal isolations were performed on the stem sections above the inoculation lesion from symptomatic and asymptomatic plants. The results revealed that the putative mutant plants that were primed with Hx only and treated with a combination of mutagens (EMS and 5-AzaC) and priming agent exhibited a significant decrease in lesion severity as compared with controls. For both treatments, a mild lesion severity rating was recorded for plants inoculated with only SC17 for cultivars N41 and 88H0019. For the plants that were dual inoculated there was a significant difference in the lesion severity ratings between the two treatments (p < 0.001). The lesion severity rating was moderate for cultivar 88H0019 (primed with Hx) and mild for cultivar N41 (primed with Hx). Plants from the combined treatment for both cultivars resulted in a mild lesion severity rating. This protocol could be valuable in generating commercially important cultivars that are tolerant and resistant to F. pseudonygamai SC17 and possibly other sugarcane pathogens. Planting resistant cultivars is recommended as an economical and the best method for controlling diseases and pests. This approach used in this study will have the least impact on the environment and increase yields without the need for expensive chemical applications and labour.