Browsing by Author "Mahlanza, Tendekai."

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In vitro generation of somaclonal variant plants of sugarcane (Saccharum spp. hybrids) for tolerance to toxins produced by Fusarium sacchari.
(2012) Mahlanza, Tendekai.; Watt, Maria Paula Mousaco Deoliveira.; Snyman, Sandra Jane.; Rutherford, Richard Stuart.
The fungus Fusarium sacchari (Butler) Gams causes stem rot in sugarcane especially in association with the stem borer Eldana saccharina Walker. Sugarcane plants tolerant to F. sacchari PNG40 were obtained by chemical mutagenesis and in vitro selection during somatic embryogenesis and plantlet regeneration on media containing F. sacchari culture filtrates (CF), followed by selection in the greenhouse. Somaclonal variants tolerant to F. sacchari PNG40 CF were established by treatment of calli with ethyl methanesulphonate (EMS) and various selection treatments. Investigations were conducted to test the effect of varying CF concentrations and the culture developmental stages (embryo maturation, embryo germination and plantlets) that were most effective in screening calli and plants. Incorporation of CF (0-100 ppm) in the media, at either embryo maturation or germination stages, resulted in significant callus necrosis, and consequent decreased plantlet yield. The highest callus necrosis of 95.55 ± 0.9 % and the lowest plant yield of 1.4 ± 0.45 plants/0.2 g were obtained after inclusion of 100 ppm CF in the germination medium compared with 61.5 ± 3.8 % and 43.8 ± 5.6 plants/0.2 g in the maturation medium, respectively. Exposure of whole plants with trimmed roots to 0-1500 ppm CF resulted in inhibition of root re-growth, with the 1500 ppm CF treatment having the greatest negative effect. Subsequent treatments involved immersing in vitro plantlets in varying concentrations of F. sacchari conidial suspensions. This resulted in 33.3 % and 100 % mortality with 103 and 105 conidia/ml treatments, respectively. Control and EMS-treated calli and potentially tolerant regenerated plants were selected using the established CF and inoculation treatments. Plants from EMS treatments displayed more varying root length. More plants with increased root growth, in the presence of CF, were produced from these treatments than from non-EMS treatments, indicating the ability of EMS to induce somaclonal variation. These putative tolerant plants were inoculated with PNG40 and those selected using CF in vitro were symptomless whilst the positive controls (plants unexposed to CF) were symptomatic. Re-isolation of Fusarium from the inoculated plants and identifying isolates as PNG40 using ISSR analysis confirmed tolerance of the asymptomatic plants and the fungus as the causal agent of the observed symptoms. This confirmed that tolerance to CF correlates to tolerance to F. sacchari PNG40. Future work includes testing stability of tolerance in the field and after sexual reproduction, and use of this protocol to produce plants that permit endophytic PNG40 colonisation towards biological control of E. saccharina.
The interaction between endophytic Fusarium species and Eldana saccharina (Lepidoptera) following in vitro mutagenesis for F. sacchari tolerance to control the borer in sugarcane.
(2015) Mahlanza, Tendekai.; Rutherford, Richard Stuart.; Snyman, Sandra Jane.
Eldana saccharina is a major pest in the South African sugar industry. Stalk damage by this borer and infection of bored tissue by opportunistic fungi result in loss of biomass and sucrose content, respectively. Amongst integrated management approaches, the best is employing E. saccharina-resistant genotypes. Resistance is attributed to physical stalk traits that impede boring and biochemical defences via nitrogen-based antiherbivory compounds. Further, in vitro assays have shown that Fusarium strains may be beneficial (e.g. F. pseudonygamai SC17) or antagonistic (e.g. F. sacchari PNG40) to the insect. The first objective of this study was, therefore, to establish the effect of sugarcane stalk traits and infection by Fusarium spp. on resistance to E. saccharina. In the first of two glasshouse trials, mature and immature stalk internodes of seven cultivars of known E. saccharina resistance ratings were inoculated with 2nd instar larvae via nodal wounds. Stalk rind hardness was greatest in both mature (42.2 units) and immature internodes (25 units) of the resistant cultivar N33. The softest of both mature and immature stalk regions were from the very susceptible N11 (32 units) and susceptible NCo376 (17.7 units), respectively. Percent fibre content in mature internodes was highest in the resistant N33 and N17 (12.8 - 14.2%) and lowest in the susceptible N11 and NC0376 (10.9 - 11.2%) cultivars. In all but one cultivar, % nitrogen content/dry mass was higher in immature internodes (0.65 - 1.2 %) than mature ones (0.36 - 0.91%) and lower in stalks of the resistant N41, N29 and N33 (0.36 - 0.75%) than in those of the susceptible NCo376 and N41 (0.48 - 1.27%) cultivars. Damage and mass gain by larvae retrieved from stalks were not entirely consistent with the cultivar resistance ratings, probably because the inoculation method by-passed the rind; N29 and N33 were unaffected by lack of rind protection. Hence, the tested stalk traits may contribute to E. saccharina resistance to varying extents in different sugarcane cultivars. In another trial, immature and mature stalks of NCo376 and N41 were inoculated with SC17 and PNG40 and then with E. saccharina larvae. The stalk area discoloured by Fusarium infection was smaller in the immature (6.1 - 7.1 cm²) than the mature (12.3 – 17.8 cm²) internodes. The smallest stalk length bored was in PNG40-infected NCo376 (3.3 cm) and N41 (1.7 cm) mature internodes, whilst NCo376 stalks colonised by SC17 (8.2 cm) were the most damaged. Hence, the proposal that Fusarium strains affect E. saccharina differently thereby impacting cultivar resistance/susceptibility to the borer, is supported. The in vivo activity of F. sacchari PNG40 against E. saccharina was also established, corroborating its potential as a biological control agent against the borer. As this application of PNG40 is impeded by the fungus being the causal agent of Fusarium stem rot in sugarcane, F. sacchari-tolerant plants were then produced via induced mutagenesis. Embryogenic calli of NCo376 and N41 were exposed to 32 mM ethyl methanesulphonate (EMS) for 4h. They were then placed on 100 ppm F. sacchari PNG40 culture filtrate (CF) at embryo maturation, germination or both stages, where 30.7 - 86.9% of the calli became necrotic and plantlet yield decreased by 59.2 - 99.2%. Roots of the regenerated plants were trimmed and placed on 1500 ppm CF. Plantlets with roots that regrew on CF medium beyond the 10 mm established threshold were deemed putatively tolerant (26.6 – 47.6% for EMS treatments, 5-24% for controls). These plants were acclimated and inoculated with PNG40 in the glasshouse. After 8 weeks, absence of symptoms, low lesion severity, re-isolation of PNG40 from the lesion and molecular identity of the isolates, confirmed some as PNG40 resistant. Re-isolation of PNG40 from undamaged tissue above the lesion, in plants with low lesion severity and no symptoms, confirmed endophytic colonisation and tolerance to the fungus in the mutants. Polymorphisms were detected in some mutants, using 24 RAPD primers. The use of the tolerant mutants in F. sacchari PNG40-mediated control of E. saccharina was then investigated. Stalks of five tolerant mutants and parents of each NCo376 and N41 cultivars were inoculated with PNG40 and with E. saccharina larvae, 3 weeks later. The length bored was less (1.0 - 4.7 cm) in stalks of PNG40 infected-mutants and parents than in the controls (3.9 - 9.0 cm). However, the % stalk discoloured area due to PNG40 infection was less in the mutants (10.6 - 22.0%) than in the parents (N41 - 28.9% and NCo376 - 30.2%). Re-isolation of PNG40 from undamaged tissue, within the inoculated internode and that above it, confirmed endophytic colonisation and fungal spread across internodes. Amongst stalks inoculated with PNG40, one mutant of NCo376 and two of N41 displayed limited boring (1 - 2 cm) and % discoloured area (10.6 - 15.1%), and the highest % of endophytically colonised stalk sections (50 - 75%) in the internodes immediately above those inoculated. There were no differences between the mutants and their respective parents in stalk rind harness, fibre and nitrogen contents. This work, therefore, resulted in the production of F. sacchari-tolerant mutants, demonstrated the toxicity of F. sacchari PNG40 against E. saccharina in vivo, and the ability of the PNG40-tolerant mutants to support endophytic colonisation by the fungus. Demonstration of these Fusarium - E. saccharina interactions in the mutants under field conditions will lead to the application of biological control of E. saccharina using PNG40, as part of integrated management approaches for the pest.