Identification, Molecular Detection And Population Diversity Of Colletotrichum In Anthurium Andraeanum

Both morphology and the molecular biology were used for identification ofColletotrichum in Anthurium andraeanum, rapid and accurate methods for specificdetection of which were also studied. Best PCR reactions of RAPD and ISSR forColletotrichum destructivum were set to study the population diversity of five geographicalpopulation of C. destructivum and one geographic population of Colletotrichumgloeosporioides.To collect and characterize Colletotrichum from A. andraeanum: Among theColletotrichum strains, which were isolated from A. andraeanum in Guozhou, Nanjing,Yangzhou, there were two different morphologies exhibited. Two swains of each group ofisolates were chose for identification with the morphological characteristics, pathogencity,host range and the sequence of ribosomal DNA-ITS. The results indicated that these strainsbelong to two species, C. gloeosporioides and C. destructivum, the latter of which was firstreported as a pathogen in A. andraeanum and was dominant. Both species could infectsolely, crossly and latently.To detect Colletotrichum pathogen from A. andraeanum: Based on differences ininternal transcribed space (ITS) sequences of Colletotrichum genus, two pairs ofspecie-specific primers were synthesized. Two pairs of primers amplified a single 329 bpand 486bp products from C. gloeosporioides and C. destructivum respectively, while otherrelative strains within different species had no corresponding band. The detectionsensitivity of primers E1/E2 and F1/ITS4 were both 10 pg genomic DNA. Using ITS1/ITS4as the first round primes and E1/E2 or F1/ITS4 as the second round primes respectively,the detection sensitivity both increased 10000-fold to 10fg. The detection sensitivity for thesoil pathogens was 200-conidia/g soil. The PCR-based method developed here could stablyand quickly detect the pathogen from water samples, diseased plant and could enablediagnosis and monitor of the pathogens caused these plant diseases and guide the plantdisease management.To set the best RAPD and ISSR-PCR reaction of C. destructivum in A. andraeanum: The effect of different concentrations of Mg²⁺,dNTP,Taq DNA polymerase and primer onthe PCR reaction were analyzed and the optimized RAPD and ISSR-PCR systems in C.destructivum were established. The RAPD-PCR reaction was performed in a 25μl solutioncontaining 2.5mmol/L of Mg²⁺, 200μmol/L of dNTP, 1U Taq DNA polymerase and1μmol/L of RAPD primer, and the ISSR-PCR reaction was performed in a 25μl solutioncontaining 2mmol/L of Mg²⁺, 150mmol/L of dNTP, 0.5U Taq DNA polymerase and1.25μmol/L of primer.To carry out RAPD and ISSR-based fingerprinting analysis of C. destructivum fromA. andraeanum: The genetic relationships of five geographic populations of C.destructivum from Guazhou, Nanjing and one geographic population of C. gloeosporioideswere determined using RAPD and ISSR markers. Eleven RAPD primers of total 105random primers amplified 155 bands (alleles), of which 96.13 percent bands werepolymorphic. Ten primers of total 52 ISSR primers amplified 101 bands (alleles), of which98.02 percent bands were polymorphic. Genetic similarity analysis based on RAPD andISSR fingerprints showed that the C. gloeosporioides population of Guazhou, Huadu hadremarkably higher genotypic diversity and genotypic variation than others, while thesecond genotypic diversity population was C. destructivum from Guazhou, Huadu. TheHuadu and Fanyu populations of C. destructivum were more similar to each other than toother populations, and the Huadu population of C. gloeosporioides had the farthest geneticdistance.