Ergot caused by Claviceps africana is an important worldwide disease of sorghum attacking only the ovary of flowering spikelets. Conidia germinate on stigma, style or ovary wall, and hyphae grow into the ovaries replacing with a sphacelium. Five to ten days after infection clear 'honeydew' containing macroconidia and microconidia oozes from infected spikelets. In warm and dry weather the sphacelium develops into sclerotia, which remain inside the glumes. At low temperatures and high relative humidities macroconidia near the honeydew surface germinate producing secondary conidia. There is a lack of information on many critical aspects of biology and management of sorghum ergot. The research was aimed at to study some of the aspects of the biology and management of sorghum ergot disease.
Some of the factors which influence the germination of macroconidia and secondary conidia were studied in vitro. Macroconidia of C. africana germinated just as well on water agar as on potato dextrose agar at 20°C, and the addition of antibiotics did not affect germination. Water agar amended with sorghum flower extract stimulated higher macro- and secondary conidial germination, irrespective of temperature, compared to that on water agar. Although the optimum temperatures and the lower temperature for germination were similar for macroconidia and secondary conidia of C. africana (approximately 20°C and 8°C respectively), the germination of macroconidia on water agar ceased near 31 °C, but approximately 30% of secondary conidia germinated at 37°C after 48 hours of incubation. Germination of macroconidia and secondary conidia on water agar started after 4 hours at 20°C, with maximum germination occurring after 16 hours and 14 hour respectively. Germination of both macro- and secondary conidia was highest at water potentials ≥-5 bars at 20°C. Germination of secondary conidia ceased at -35 bars, whereas macroconidia germinated as low as -55 bars at 20°C. This is the first study on effect of temperature and water potential on germination of secondary conidia.
The factors influencing germination of conidia of C. africana on stigmas are poorly understood. The effects of various concentrations of macroconidia, and of the length of the stigma wetness period on infection of sorghum spikelets were studied in two experiments in growth cabinets at 20°C. The highest levels infection occurred when the conidial concentration was ≥106 conidia/ml. In the first trial, in which the length of stigma wetness ranged from 0 to 48 hours, maximum infection occurred after 6 hours of free moisture. In a second trial (stigma wetness period 0-6 hours), the greatest infection occurred when stigma wetness period was ≥4.5 hours. In both trials >10% infection occurred when the stigmas were dried immediately after inoculation. The relative humidity inside the growth cabinet was 60-80%, during the trials suggesting that infection may be possible in absence of free water on stigma.
The ability to survival of C. africana in honeydew on seed and in infected panicles was investigated. During my studies, it was observed that macroconidia of Claviceps africana survived in dried honeydew on soil for 13-14 weeks in a glasshouse at ambient temperatures, but for less than half that time on seed stored in a shadehouse over summer. Those on seeds stored at 4oC, however, survived for over a year (58-62 weeks). During summer, conidia on ergot-infected panicles buried in soil, or on the soil surface, survived for 7.5-12 weeks, while over winter the survival times were 4 weeks and 19-27 weeks respectively. Macroconidia on infected panicles held above the soil surface survived for >38 weeks (8 calendar months) over winter, suggesting that they may play a role in the perennation of C. africana in Australia.
Currently, ergot management practices in commercial sorghum crops are restricted to various agronomic practices such as planting time, tiller management, and harvester adjustment. Fungicides are economic only for high value seed production and breeders nurseries, and only one group [demethylation inhibitor (DMI)] has been shown to be effective when applied to flowering panicles. During my studies, I conducted trials on three aspects of management - the efficacy of (i) fimgicides/chemicals of other groups applied to flowering panicles, including timing of application, (ii) fungicides as seed dressings, and (iii) potential biocontrol agents against C. africana.
The efficacy of fungicides belonging in several groups for the control of ergot on flowering panicles was investigated in glasshouse and field trials. DMI fungicides (triadimenol and propiconazole) and benzothiadiazole (either alone or in combination with mancozeb) were the most effective. The timing of application of triadimenol and benzothiadiazole, and the number of application of triadimenol was found to affect ergot severity. In glasshouse trials triadimenol was most effective when first applied on the day of inoculation with C. africana, whereas benzothiadiazole was most effective when sprayed 4 days before inoculation. Applying triadimenol every 4 days was more effective than every 6, 8 or 12 days, with efficacy decreasing as the spray interval increased.
Seed lots contaminated with ergot honeydew are potential means of intra- and inter-country spread of sorghum ergot. Of the 11 fungicides assessed in the in vitro study, thiram and captan completely inhibited conidial germination of C. africana. Nine of the best fungicides from the in vitro test were selected for assessment as seed treatments using sorghum seed which had been artificially coated with ergot honeydew containing macroconidia. A quantitative test was developed to assess the efficacy of seed dressing fungicides on conidial germination. Thiram and captan were also the most effective, completely inhibiting macroconidial germination of one isolate and reduced germination of the other to <0.2%. Thiram applied to honeydew-coated sorghum seed completely inhibited macroconidium germination at a rate lower than that currently registered (2.4g a.i./kg) for use on sorghum seed in Australia. These results have important implications in the movement of sorghum seed from areas where C. africana is endemic to areas which are free from the pathogen.
Five fungi (two commercial products of Trichoderma spp, Penicillium citrinum (strain A and B) and Epicoccum nigrum) and six bacteria (Bacillus insolitus H60, B. subtilis T3, Burkholderia cepacia 526/1, Pseudomonas aeruginosa H68, and two unidentified isolates T2 and T4) were tested as potential biocontrol agents against C africana. The undiluted culture filtrates of all fungal biocontrol agents completely inhibited in vitro conidial germination of macroconidia of C. africana. P. citrinum A was the most effective fungus at the highest dilution (1:3). Only P. aeruginosa and B. cepacia completely inhibited macroconidial germination, with the former being more effective at high dilutions. In glasshouse tests, none of the bacterial biocontrol agents inhibited ergot infection, but all fungal biocontrol agents reduced the infection of sorghum panicles by C. africana, in some cases completely inhibiting infection. Another glasshouse trial showed that the best control was achieved when the biocontrol agents were applied 7 days before inoculation with conidia of C africana. In two preliminary field trials, none of the bacterial and fungal isolates significantly reduced infection by C. africana.
Current methods for the detection of C. africana in seed, such as seed inspection and washing tests are slow and unreliable. A standard protocol for detection of C. africana needs to be developed. Intron 3 of the β-tubulin gene was used to differentiate the species of Claviceps and other non-Claviceps fungi. Polymerase chain reactions (PCR) were conducted on four Claviceps and six non-Claviceps species using two primer pairs. Amplified bands were observed only on the isolates of C. africana with the BTAFR and BT5 primer pair, and on a Claviceps sorghicola isolate with BTSOR and BT5 primer pair. No amplified band was observed with the BTAFR and BT5 primer pair on any of the non-Claviceps species. An assay using this DNA-based technique could be further refined to quickly and accurately detect sorghum ergot pathogens in seeds.
This study has contributed significantly to our understanding of some important aspects of biology and management of sorghum ergot. The research in this thesis represents the first reports on some aspects of (i) the biology and ecology of secondary conidia, (ii) the effect of the stigma wetness period on infection by macroconidia, (iii) a quantitative technique for determining the efficacy of fungicidal seed dressing against C. africana, (iv) the efficacy of the systemic activated resistance chemical, benzothiadiazole, against C. africana, (v) the potential use of biocontrol agents against C. africana, and (vi) the possible use of a DNA-based technique to diagnose the pathogen. This study forms the foundation of further studies on many aspects of the biology of C africana , and will lead to a better understanding of the epidemiology and management of sorghum ergot.