Identification and control of Colletotrichum species associated with Eucalyptus seeds

Abstract

The South African forestry industry largely relies on seed for its replanting programs following clear-felling. The high cost of Eucalyptus seed and planting targets makes it imperative to start with pathogen-free seeds to avoid inconsistences of emergence and development of seedlings. Together with 34 other fungal species, two Colletotrichum species in the Colletotrichum gloeosporioides species complex were found to be naturally associated with 14 commercial Eucalyptus seed lots. Eucalyptus nitens seed lot had the highest incidence of fungi (92.4%), whereas the lowest incidence occurred on E. dorrigoensis seed lot (28.9%). Seed germination of seed lots inoculated with seed-borne fungi was less than 62% and as low as 25%. Multiloci phylogenetic analyses of ITS, β-tubulin, actin, and glyceraldehyde-3-phosphate dehydrogenase gene regions identified Colletotrichum isolates PPRI 24314 as C. fructicola Prihastuti. and PPRI 24315 as C. kahawae Waller and Bridge. Biochemical tests based on utilisation of ammonium tartrate confirmed the latter isolate as C. kahawae subsp. cigarro. Colletotrichum fructicola and C. kahawae subsp. cigarro were naturally associated with seed lots of E. dunnii (3.5 and 0.5%, respectively), E. nitens (2.6 and 1.2%, respectively) and E. macarthurii (0.8% for only C. fructicola). Greenhouse pathogenicity tests showed that both C. fructicola and C. kahawae subsp. cigarro caused the highest incidence of anthracnose leaf spot on E. nitens (65 and 55%, respectively), with a severity of 67 and 63%, respectively. Sowing seeds inoculated with C. fructicola demonstrated the pathogen to be vertically transmitted into E. camaldulensis, E. dunnii and E. nitens seedlings (25.5, 38.3 and 64.0%, respectively), whereas C. kahawae subsp. cigarro inoculated seeds transmitted the pathogen in 27.3, 30.5 and 56.3% of the seedlings, respectively. Since both pathogens were seed-borne and seed-transmitted, non-chemical seed treatments that included Bacillus and Trichoderma, hot-water, microwave radiation and hydrogen peroxide were evaluated for their efficacy at sanitising seed lots of E. nitens and E. viminalis artificially inoculated with either C. fructicola or C. kahawae subsp. cigarro. Soaking Eucalyptus spp. seed lots in 15% H2O2 for 10 min effectively reduced incidences of Colletotrichum spp. but negatively reduced seed germination. Instead, soaking Eucalyptus spp. seeds in 10% H2O2 for 5 min and 10 min were optimal treatment/time combinations with an acceptable reduction of Colletotrichum spp. and significantly high seed germination percentages. Hot water optimum treatment/time parameters were 50 °C for 30 min against C. kahawae subsp. cigarro, and 55 °C for 15 min against C. fructicola. Exposure of moist Eucalyptus spp. seeds to microwave radiation at powers levels of 1 400 w for 30 s significantly improved seed germination similar to that of the Celest® XL treatment. However, sowing seeds treated with H2O2, hot water and microwave radiation failed to control anthracnose leaf spot developing on seedlings grown under greenhouse conditions. Seeds treated with a Bacillus biocontrol agent consistently reduced incidences of Colletotrichum spp. on Eucalyptus spp. and effectively suppressed appearance of anthracnose leaf spot on seedlings grown under greenhouse conditions. Due to the high efficacy of Celest® XL and Bacillus in-vitro and in-vivo, they can be recommended as sanitisers of commercial Eucalyptus seed lots against anthracnose leaf spot.