Study On Etiology Of Pepper Anthracnose In Sichuan Province

Pepper(Capsicum annuum)is an important vegetable and spice.Sichuan Province is one of the growing-provinces of pepper in China.The growing area is about 70 thousand hm2.Anthracnose is one of the destructive diseases of pepper.It causes extensive yield losses to both pre-and postharvest stages.There is no guidance to disease management,for few study about anthracnose on Pepper in Sichuan Province currently.173 infected fruit samples were collected from 28 regions of Sichuan,between 2012 and 2013.88 Colletotrichum isolates were selected after tissue separation and single-spore isolation.Morphology identification(including Colony characters,Growth rate,shape and size of Conidia,shape and size of Conidia appressoria and shape of Mycelial appressoria and phylogenetic analyses by GAPDH genes were used by all isolates.46 isolates were chosen to identify by three pairs of specific primer(The primer pairs were CgInt/ITS4,CaInt2/ITS4 and CcInt/ITS4 used for Colletotrichum gloeosporioides complex,C.acutatum complex and C.truncatum separately)and phylogenetic analyses based on multi-locus sequences,including the nuclear ribosomal internal transcribed spacer(ITS)region,?-tubulin(TUB2),actin(ACT),glyceraldehyde-3-phosphate dehydrogenase(GAPDH)and calmodulin(CAL).The combined datasets showed that eighty-eight strains were identified asColletotrichum gloeosporioides,C.simense,C.fructicola,C.truncatum,C.acutatum,C.brevisporum and Colletotrichum sp.suspected to be a new speices.Of them,C.gloeosporioides,C.simense and C.fructicola belonged to C.gloeosporioide complex,and C.scovillei belonged to C.acutatum complex.And the number of isolates belonged to C.truncatum was maximum.It comprising 32 isolates and occupying 36.4%of the total isolates was the predominant pathogenC.simense and C.scovillei was recorded for the first time as the cause of anthracnose on pepper in China.Pathogenicity test showed all species can infect Capsicum.All species are non-host-specific as isolates from each species can infect Pyrus pyrifolia.The results also suggested that these species associated with anthracnose in pepper may be the potential of other hosts for initial inoculum source.C.scovillei was the most virulent species to Capsicum spp.as the incidence of this disease achieve 100%on both species of Capsicum.116 bands contain 115 polymorphism bands were obtained by PCR amplification with the eight primers.The polymorphism proportion is 99.14%.88 isolates was analysed the genetic diversity by ISSR markers.We selected 8 primers giving clear bands and with good polymorphism from all screened 35 primers.The polymorphism proportion of seven primers achieved 100%,and only the polymorphism proportion of primer 841 was 93.33%.Clustering tree was built by UPGAM method.All isolates can be divided into seven groups at 0.56 genetic similarity coefficient.Group I comprised C.truncatum,Colletotrichum sp.and C.brevisporum.Group II comprised three species in C.gloeosporioides complex.Isolate in group III were C.scovillei.Three species in group I can be differentiate at 0.63 genetic similarity coefficient.At 0.7 genetic similarity coefficient,three species in group III can be divided.The results of clustering based on ISSR marker agreed with the results of phylogenetic analyses by GAPDH genes and multigene phylogeny,showed that ISSR molecular marker is useful to divided isolates at species level.88 isolates did not cluster in accordance with regions.Thus,the relationship between genetic diversity and regions was not significant.