| Stylo (Stylosanthes spp.) is important tropical legume forages, widely cultivated in tropical and subtropical regions of south China. The yield of stylo is restricted by anthracnose seriously. In stylo, the Colletotrichum gloeosporioides is reported as the main pathogenic fungi, whereas, the other species is barely reported in China. In this study,three stylo anthracnose pathogens were obtained, with the morphological classification and molecular characterization.In addition, the pathogenicity difference was compared with C. gloeosporioides. The main results were as follows:1) The morphological characteristics (compared to C. gloeosporioides) were as follows: colonies grew slower; the color of conidiomatas was lighter; conidia were fewer, the size of conidia was wider and shorter; no seta; appressoria was irregular in shape and strongly nodose. By conservative sequence of PCR amplification and sequencing and homology comparison, sequences of ITS, ACT,CHS and GAPDH of these isolates prepared from monoconidial cultures were obtained and BLAST analysis showed 99 to 100% compared to C. karstii. The isolates were identified as Colletotrichum karstii based on morphological and molecular analysis. This is the first reported in China for the pathogenic fungi of stylo anthracnose.2) Microscopic observation of C.karstii infecting stylo leaves: invasion stage was 3 to 36 hous post inoculation( hpi). conidia germination started at 3hpi. melanized appressoria and penetration pegs were also observed within 24 to 36 hpi.biotrophic stage was 36 hpi to 48 hpi. swollen primary hyphae was formed at 36phi which was the start of biotrophic phase, necrotrophic stage started at 60hpi.at this stage thin long secondary hyphae was differentiated from primary hyphae at 60hpi and invaded surrounding tissues at 72hpi. the stylo cells collapsed from 84 to 96 hpi, with brown tissue liquid flowing out.3) The pathogenicity of 3 isolates of C. karstii and 40 isolates of C. gloeosporioides was studied by inoculated detached leaves. All the isolates were classified into 4 groups, strong, medium strong, medium weak, weak pathogenicity strains. Srong groups contained 9 (20.9%) including C. karstii DZ-27,medium strong group contained 7 isolates (16.3%) including C.karstii DZ-30, medium weak group contained 10 isolates (23.3%), and weak group contained17 isolates (39.5%)including C.karstii DF-02.4) The results of strong and weak pathogenicity of C.karstii and C gloeosporioide inoculating stylo showed that: the disease index of stylo infected with strong pathpgenicity of Colletotrichum spp. were above 67, however, the disease index of stylo infected by weak ones were about 45. The physiological and bio-chemical variety of infected stylo indicated that: in the invasion stage, stylo enzyme activity of SOD, POD, CAT, APX, GPX, GR increased obviously infected by all the Colletotrichum spp. could leaded stylo and same trend in AsA and GSH. the strong pathogenicity of C.karstii caused high stylo response, while contrary to C gloeosporioide,the different response between C.karstii and C gloeosporioide interaction with stylo might due to the differerent mechanism.In summary, we first identified and reported C.karstii as a new pathogen of stylo anthracnose in China,and revealed the infection process of C.karstii. With the comprehensive evaluated the pathogenicity of C.karstii and C gloeosporioide, there existed a response difference of stylo inoculated of C.karstii and C gloeosporioide.Results further defined the Stylo anthracnose pathogen species in China, laying the theoretical basis for disease occurrence, prevention and treatment... |
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