Sorghum ergot affects seed production and grain usage in stock feed due to concerns of animal toxicity. Worldwide, three species of Claviceps are known to cause ergot of sorghum with different epidemiological, animal toxicity, and management implications. Effective management strategies for the control of sorghum ergot require a thorough knowledge of the biology, epidemiology and genetics of the pathogen and of the host-pathogen interaction. Sorghum ergot was discovered in Australia in 1996 and was ascribed to Claviceps africana, based solely on the morphology of conidia. At that time there was a suggestion that other Claviceps spp. might also be present. The major aims of this study were to identify the Claviceps spp. causing sorghum ergot in Australia, and to establish the morphological, genetic and pathogenic diversity in the Australian sorghum ergot pathogen population.
Pathogenicity, symptom development, spore morphology, sequencing of the internal transcribed spacer, ITS1l region and randomly amplified DNA fingerprinting (RAF), were used to confirm that ergot of sorghum in Australia is caused by C. africana. Over 200 isolates were collected from various sorghum-cropping regions. All isolates were pathogenic on a grain sorghum A-line. The morphology of sphacelia, microconidia, macroconidia and secondary conidia of a subset of 44 Australian isolates studied matched the published descriptions of C. africana. DNA sequence of the ITS 1 region of 2 selected Australian isolates was identical to that of C. africana. Based on RAF analysis of 110 Australian and overseas isolates of Claviceps spp., C. africana isolates could be clearly distinguished from the other sorghum ergot pathogens C. sorghi and C. sorghicola; and C. pusilla and a Claviceps sp. isolated from Panicum maximum (PM).
Considerable phenotypic variability for cultural characteristics, growth rate and spore morphology was detected among 44 Australian isolates. Numerical classification showed that isolates could be grouped into 5 clusters based on mycelium density, colony elevation and colour. Variability was also detected in the in vitro production of the alkaloids dihydroergosine (DHES), dihydroelymoclavine (DHEL) and festuclavine (FEST) in the mycelium of 49 Australian and overseas C. africana isolates. Although most C. africana isolates produced only trace amounts of alkaloids, up to 8 mg/kg were detected in the mycelium of some Australian isolates, which is approximately eight times above the industry limit of DHES considered safe for livestock.
RAF analysis was used to establish the genotypic diversity of 106 Australian and overseas C. africana isolates. Isolates formed two distinct clusters. Cluster 1 contained 72 Australian isolates and 21 overseas isolates, while the 13 isolates in cluster 2 (all from Australia) were more diverse than those in cluster 1. This high level of genotypic diversity of C. africana isolates in Australia is unexpected given that ergot was only reported in Australia in 1996. Similarity to isolates from India point to possible introductions of the pathogen from Asia.
Gene diversity was investigated using the introns of the glyceraldehyde-3- phosphate dehydrogenase (gpd) gene and the gene for Cu/Zn-superoxide dismutase (sod). Only the gpd intron allowed a clear differentiation of six Claviceps spp. (C. africana, C. purpurea, C pusilla, C sorghicola, C. sorghi and Claviceps sp. from PM). It showed a higher sequence divergence compared to the sod gene and appears more suitable for phylogenetic studies of the genus Claviceps. There was almost no intra-specific gene diversity detected among 23 C. africana isolates from Australia, India, America and Africa for the gpd gene intron and among 8 isolates for the sod gene intron. There was no correlation between genetic and phenotypic groupings of C. africana isolates.
Potential sources of genetic variability in Australian isolates of C. africana were investigated. Isolates were paired to determine mycelial compatibility and could be grouped into a number of putative mycelial compatibility groups suggesting that there is a potential for asexual recombination. Mixed inoculations with pairs of compatible but phenotypically different isolates showed that both isolates successfully infected and colonized the ovary. Mixed inoculations resulted in the development of new colony phenotypes but no genotypic changes were found using RAF analysis.
Southern hybridisation with heterologous probes of the Pot2 transposon from Pyricularia grisea and the impala transposon from Fusarium oxysporum did not detect any homologies in the C. africana genome. PCR primers for the conserved region of transposases of the Fot1 fransposon family found a nucleotide sequence in the C. africana isolate SE86S, approximately 50% similar, with an inferred amino acid sequence up to 35% similar to other transposases of the Fot1 family. Further studies are required to confirm the identity of this putative transposable element.
A host range study determined that 15 native and cultivated Sorghum spp. from Australia are susceptible to C. africana infection. Additionally, C. africana also infected four other grass species, Bothriochloea pertusa, Dichantium aristatum, Pennisetum glaucum and P. occidentale. Further assessment is needed to determine their potential role in the survival of C. africana, as sources of primary inoculum and if these potential collateral hosts may serve as sources of resistance. The specialization in the host - pathogen interaction was studied using 16 C. africana isolates inoculated onto four sorghum A-lines and a further subset of 8 isolates on to 9 A-lines. Components of pathogen aggressiveness were assessed by measuring incubation and latent periods, the time to the onset of secondary conidiation, ergot severity, and honeydew and spore production. All isolates infected all A-lines and most isolates showed similar levels of aggressiveness. A-lines showed small but consistent differences in their susceptibility to C. africana infection. A296 and AT x 623 were most susceptible, while AOK11, AKS4 and AQL41 were least susceptible. Differential rankings of sorghum lines using the different components of aggressiveness point to more than one underlying resistance mechanism.
A fluorescent staining technique using chitin specific fluorescein isothiocyanate-conjugated wheat germ agglutin and aniline blue was modified to follow the process of colonization of sorghum ovaries by C. africana. Conidia of C. africana germinated within 24 hrs after inoculation (a.i.); by 79 hrs a.i., the pathogen had established itself in the sorghum ovary and colonization of the host tissue proceeded rapidly; by 120 hrs a.i. at least half of the ovary had been completely converted into sphacelial tissue and at 8 days a.i. the conversion of host tissue was virtually completed. Histochemical changes in host and fungal cell walls during penetration and invasion of the ovary were detected. The technique was applied to study pathogen aggressiveness of five C. africana isolates and three of the A-lrnes used in the pathogenic diversity study (A296, AKS4, A3IS8525). The rate of ovary colonization supported the findings of the pathogenicity study and confirmed the small but significant differences in A-lines after inoculation with C. africana isolates. These findings present a significant step towards an improved understanding of the host - pathogen biology. Potential quantitative resistance identified in the A-lines, when improved by breeding may effectively reduce or delay infection and the development of epidemics and potentially reduce the number of fungicide applications or the R-line: A-line ratios required in hybrid seed production.