Pathogen Identification Of A New Disease On Pepper And Comparative Study Of Colletotrichum Acutatum

Colletotrichum spp. causes anthracnose diseases on many crops. It has a wide host range,including vegetable plants, ornamental plants, fruit plants, medicinal plants, and weeds. Anthracnose on pepper was reported happened on leaf and mature fruit (red pepper) normally.In2009,a special anthracnose was happened on pre-matured fruit (green pepper).To find out the pathogen and the differences between the pathogen with other strains of the same species, related researches were conducted.1. Pathogen IdentificationSymptoms Characteristics Diseased pepper fruit submitted to our lab has similar symptoms to anthracnose with concentric pink dot arranged peripherally on the lesion.But there are two obvious differences from the normal pepper anthracnose. First,the disease was happened on green chili pepper;Second,the leaf did not been infected.Pathogen Morphology Typical anthracnose acervuli was observed under microscope.Fungal conidia were15.8×4.1μm, fusiform or oval with one end acute, single,colorless celled with several oil globules. No setae were found on the acervuli.Conidia characteristics were similar to C.acutatum.No sexual stage was seen.Cultivate Characters Eight isolates (HNZJ001-HNZJ008) showed no difference in colony feature.The isolate HNZJ001formed white colonies on PDA at the early stage,then pink,finally dark with obvious grey black concentric rings. Colony diameter was63.2mm-67.4mm under28℃after seven days cultivation. The comparison C.gloeosporioides strain,LSQ1formed grey colony on PDA,and it grew faster than HNZJ001.Its diameter reached to64.0mm after five days. Cultivate characters reflected that HNZJ001was different from C.gloeosporioides.Carbendazim Sensibility Fungistasis test in vitro showed that HNZJ001was not sensitive to carbendazim while C. gloeosporioides strain LSQ1was sensitive to it.It convinced the foreign report that C.acutatum was not sensitive to carbendazim.Molecular Identification PCR amplification of HNZJ001was carried out by utilizing universal rDNA-ITS primer pair ITS4/ITS5. Sequencing of PCR products of HNZJ001(GenBank Accession No. GU059863) showed100%identity to Glomerella acutata (GenBank Accession No. EU008863) and C.acutatum (GenBank Accession No. AF207794) after a BLAST search. In view of sexual stage’s disappearance,the pathogen was tentatively identified as C.acutatum (sexual stage:G. acutata) combined with asexual stage characteristic. This was the domestic report.2. Pathogenic and Biological Characteristics Comparative Study of Chinese C.acutatumPathogenic Comparative Study2011,HNAH003(GenBank Accession No.JN697579) was isolated from Mangnolia officinalis in An’hua country,Hunan province. Then its rDNA-ITS sequence was amplified and sequenced. Accession number of HNAH003was JN697579after submitted to GeneBank. Another ten C.acutatum strains from other plants were collected from the domestic.All the twelve C.acutatum strains and one C.gloeosporioides isolates were inoculated to pepper, apple,mango,lemon and strawberry in vitro respectively.The results showed that Hunan province isolates HNZJ001and HNAH003were more aggressive than the other isolates.And all C.acutatum isolates were much more pathogenic than the C.gloeosporioides isolates.Characteristics Comparative Study Spore morphology,cultivation characters and temperature,illumination conditions etc. to hapha growth and sporulation of each strain were complex. Isolates from different hosts and different isolates from the same host were both different in biological characteristics.But in whole, the differences were obvious between C.acutatum and C.gloeosporioides.3. rDNA-ITS Phylogenetic AnalysisrDNA-ITS sequences of C.acutatum,including four domestic C.acutatum from other plant hosts were downloaded from NCBI GeneBank. rDNA-ITS sequences of another six C.acutatum isolates were amplified and sequenced. Phylogenetic tree was conducted together with the two Hunan C. acutatum strains and four C. gloeosporioides strains.The results indicated that we can distinguish fungi species quite good based on ITS area and abundant diversities existed among C.acutatum species.Innovation Points:(1) The first time reported pre-matured pepper anthracnose in China and identified the pathogen as C.acutatum;(2)The first time compared pathogenic and biological Characteristics of Chinese C.acutatum strains.Proved that virulence of Hunan isolates were stronger than the other provinces’and virulence of C.gloeosporioides were stronger than C.acutatum;(3)Sequenced the C.acutatum isolates of Hunan chili pepper and Mangnolia officinalis and the other six C.acutatum.Constructed rDNA-ITS system evolutionary tree and surpported rDNA-ITS region as the fungus species of bar code.