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Genetic Diversity in Colletotrichum gloeosporioides from Stylosanthes spp. at Centers of Origin and Utilization.

ABSTRACT Using molecular markers, this work compares the genetic diversity in Colletotrichum gloeosporioides infecting species of the tropical forage legume Stylosanthes at the center of origin in Brazil and Colombia with that of Australia, China, and India, where Stylosanthes spp. have been introduced for commercial use. There was extensive diversity in the pathogen population from Brazil, Colombia, China, and India. The Australian pathogen population was least diverse probably due to its geographical isolation and effective quarantine. The extensive diversity in China and India means that threats from exotic pathogen races to Stylosanthes pastures can potentially come from countries outside the South American center of origin. In Brazil and India, both with native Stylosanthes populations, a high level of genetic differentiation in the pathogen population was associated with sites where native or naturalized host population was widely distributed. There was limited genetic diversity at germplasm evaluation sites, with a large proportion of isolates having identical haplotypes. This contrasts recent pathogenicity results for 78 of the Brazilian isolates that show hot spots of complex races are more common around research stations where host germplasm are tested, but few are found at sites containing wild host populations. For a pathogen in which the same races arise convergently from different genetic backgrounds, this study highlights the importance of using both virulence and selectively neutral markers to understand pathogen population structure.

Genetic diversity in South American Colletotrichum gloeosporioides isolates from Stylosanthes guianensis, a tropical forage legume

The degree of genetic diversity of 127 Colletotrichum gloeosporioides isolates from Stylosanthes guianensis genotypes in South America was measured at the molecular level by random amplified polymorphic DNA (RAPD) with nine arbitrary primers of 10 bases, and by restriction fragment length polymorphism (RFLP) with a non-LTR (long terminal repeats) retrotransposon DNA sequence. The RAPD products revealed scorable polymorphism among the isolates, and a total of 80 band positions were scored. Sixty-three of the 127 isolates were clustered into 13 distinct lineages usually correlating with geographic origin. Where isolates from various regions were clustered together, most had identical host genotype origin. The pathogen population sampled from Carimagua, Colombia, a long-time Stylosanthes breeding and selection site, with a savanna ecosystem, was highly diverse. A set of 12 S. guianensis genotype differentials was used to characterize pathogenic variability of 104 isolates and their virulence patterns were grouped into 57 pathotypes. However, when they were tested on four Australian differentials, they grouped into 11 pathotypes. As shown in previous studies, no strict correlations existed between genetic diversity measured by RAPD or RFLP, and pathotype defined by pathogenicity pattern on the differentials. Southern blot analysis of the 127 isolates revealed 23 hybridizing fragments, resulting in 41 fingerprint patterns among the 127 isolates. Relationships between RFLP and RAPD variables were examined using Spearmans Rank Correlation Coefficient, which showed that the two measures of genotypic variation are in agreement.

Acremonium implicatum, a Seed-Transmitted Endophytic Fungus in Brachiaria Grasses

The pan-tropical grass genus Brachiaria comprises about 100 species, several of which are forages of economic importance, particularly in tropical America. Acremonium implicatum is a fungus that forms an endophytic association with at least some of these economically important grasses. To ascertain whether A. implicatum can be seed transmitted in Brachiaria species, we vegetatively propagated, under greenhouse conditions, 20 tillers from an endophyte-infected mother plant obtained from each of 14 Brachiaria hybrids and species. Ten tillers of each genotype were treated with the fungicide tebuconazole to eliminate the endophyte, and the other 10 were left untreated. Seeds were then harvested individually from all 20 of these genetically identical plants, germinated, and the seedlings grown. A previously developed polymerase chain reaction-based method used a pair of endophyte-specific primers to amplify a diagnostic 500-bp DNA fragment. The seedlings generated from seeds harvested from endophyte-infected plants also tested positive, whereas those from seeds of endophyte-free plants showed no amplification products. This is the first report of A. implicatum being transmitted through seeds of Brachiaria grasses.

Virulence spectrum of South American isolates of Colletotrichum gloeosporioides on selected Stylosanthes guianensis genotypes

Anthracnose, caused by Colletotrichum gloeosporioides, is the most important and widespread disease of Stylosanthes, a diverse tropical and subtropical forage legume naturally distributed in Central and South America. Although South American isolates of C. gloeosporioides are known to be variable in pathogenicity, no suitable differential host lines have been identified to fully characterize them. This study takes advantage of new S. guianensis inbred lines, cultivars, and accessions to describe the pathogenicity of South American isolates of C. gloeosporioides and to select potentially useful differentials. Seedlings of 23 S. guianensis genotypes were inoculated with 45 isolates from various regions of South America. Virulence patterns were used to select 12 differentials and determine 23 pathotypes in C. gloeosporioides. In contrast, the same isolates were grouped into nine pathotypes with the four Australian differentials currently in use.

Agrobacterium-mediated transformation of the endophytic fungus Acremonium implicatum associated with Brachiaria grasses

Acremonium implicatum is a seed-transmitted endophytic fungus that forms symbiotic associations with the economically significant tropical forage grasses, Brachiaria species. To take advantage of the endophytes plant protective properties, we developed an efficient Agrobacterium-mediated transformation system for Acremonium implicatum, using green fluorescent protein (GFP) expression and vector pSK1019 (trpC promoter) or pCAMBIA1300 (CaMV35S promoter). We found that transformation efficiency doubled for both mycelial and conidial transformation as the co-cultivation period for Agrobacterium tumefaciens and Acremonium implicatum was increased from 48 to 72h. Significantly, optimal results were obtained for either mycelial or conidial transformation with Agrobacterium tumefaciens strain AGL-1 and vector pSK1019 under the control of the trpC promoter. However, mycelial transformation consistently generated a significantly higher number of transformants than did conidial transformation. The mitotic stability of the transferred DNA was confirmed by growing ten transformants in liquid and agar media for six generations. In all cases, resistance to the selection pressure (hygromycin B) was maintained. Fluorescence emission was retained by the transformants and also expressed in Brachiaria tissues from plants inoculated with GFP-transformed A. implicatum. This technology will help in the transfer and expression of agronomically important genes in host plants.

Distribution and relationship of chromosome-specific dispensable DNA sequences in diverse isolates of Colletotrichum gloeosporioides

DNA hybridization probes specific to a dispensable 1·2 Mb chromosome identified in some isolates of biotype B of Colletotrichum gloeosporioides pathogenic on the legume Stylosanthes guianensis were used to assess the distribution and chromosomal location of homologous sequences in a diverse collection of other Colletotrichum isolates. Homologous sequences were not detected in isolates representing C. lindemuthianum, C. musae, C. crassipes, C. trifolii, C. dematium and C. truncatum but were in some C. gloeosporioides isolates. Thus these sequences were not conserved in the genus and deleted in some C. gloeosporioides strains. Partly homologous DNA sequences were detected in genetically distinct biotypes of C. gloeosporioides pathogenic on hosts other than Stylosanthes , which suggests that these DNA sequences may have evolved within the species C. gloeosporioides prior to host specialization. Highly homologous chromosomes were identified in some isolates of the fungus from Stylosanthes from outside Australia including one isolate from the Philippines which appeared to have a 1·2 Mb chromosome almost identical to that of Australian biotype B strains. The DNA probes revealed a range of sizes of homologous chromosomes in other strains of C. gloeosporioides but in general the chromosome-specific DNA probes were linked, suggesting a common origin with subsequent gross chromosomal rearrangement or exchange. The possibility that the chromosome-specific markers may be useful diagnostic probes for biotype B isolates virulent on S. guianensis cv. Graham was tested using 10 new isolates with this virulence phenotype and three of these isolates lacked both the chromosome and the DNA sequence homology. This suggests that the 1·2 Mb mini-chromosome is not associated with race-specificity. The significance of these results for the origin and function of dispensable chromosomes in C. gloeosporioides is discussed.