Pathogen Identification And Agrobacterium Tumefaciens-mediated Transformation Of Corynespora Cassiicola From Muskmelon

Melon（Cucumis melo L.）is one of the most economically important cucurbit crops in China.Dozens of diseases infect melon,of which the fungal diseases are the most common.There are still new emerging outbreak because of the species number of fungi.Morphological characteristics,multiple-gene sequencing,and pathogenicity tests were used to identify a new outbreak disease of melon.In order to evaluate fungicides on the pathogen,indoor toxicity of fungicides for C.cassiicola were determined in laboratory.At the same time,Agrobacterium tumefaciens-mediated transformation（ATMT）technique has been used to create the mutant library of C.cassiicola.The main progresses were as follows:1、Corynespora cassiicola was identified as a pathogen infecting muskmelon（Cucumis melo L.）in China.A leaf spot disease was observed on muskmelon in October,2015,in Wuming,Guangxi province,China.The disease exhibited symptoms suggestive of target spot,caused by Corynespora cassiicola.Morphological characteristics,multiple-gene sequencing,and pathogenicity tests were used to identify the causal organism.The fungal isolate produced a colony with brownish or greenish concentric growth rings and abundant aerial mycelia.The conidia were solitary,hyaline,cylindrical,oval or obclavate,straight or curve,0-13-septate,non-constricted at the septa and 3.1-6.4×14-138.9μm.The isolate was sequenced using four genomic loci,from ribosomal DNA internal transcribed spacer sequences[ITS1,5.8S rRNA,ITS2（ITS1,2）,ITS4 and ITS5（ITS4,5）],and the actin（ACT）,β-tubulin（TUB2）genes.The purified Guangxi isolate infected all tested genotypes of muskmelon,watermelon（Citrullus lanatus）,and cucumber（Cucumis sativus L.）,in seedlings and detached leaves,and was consistently reisolated from diseased leaves,satisfying Koch’s postulates.Our results identified the pathogen as C.cassiicola.Glucose and soluble starch were the suitable carbon sources for mycelial growth;KNO₃,NaNO₃ and peptone were the suitable nitrogen sources for mycelial growth;the optimal temperature was 25-29°C,with an optimal pH range of 6-9.Optimal carbon sources for conidial germination were sucrose and glucose,with an optimal pH range of 5-8.2、Toxicity of 22 fungicides against C.cassiicola causing of melon leaf spot was evaluated.Melon target spot（C.cassiicola）is an emerging disease as a potential risk for melon production.In order to evaluate fungicides on the pathogen,indoor toxicity of 22fungicides for C.cassiicola Cc-SY strain and Cc-12 strain of melon were determined in laboratory.The results indicated that EC₅₀0 value of 50%Sporgeon WP was the most effective for both Cc-SY strain and Cc-12 strain.30%difenoconazole SC,40%flusilazole EC and 80%thiram WG also had significant inhibition activity on Cc-12 strain mycelium growth,with the EC₅₀0 values less than 5μg/mL,but the three fungicides were less effective on Cc-SY strain.25%azoxystrobin SC,75%chlorothalonil WP and 90%carbendazim WG were least effective on Cc-SY strain and Cc-12 strain.3、The whole genome sequence of C.cassiicola.The genome of Cc-12 was sequenced,44 Mb genome was assembled.Through a phylogenetic analysis with model species of fungi,the evolutionary divergence time of C.cassiicola was estimated to be 82.5 Mya.The carbohydrate-active enzyme genes in C.cassiicola was estimated and 351 glycoside hydrolases were identified.4、Transformation of C.cassiicola by A.tumefaciens was developed.Understanding the molecular mechanism underlying the pathogenicity of C.cassiicola is essential to design new strategies to control the disease.Thus,Agrobacterium tumefaciens-mediated transformation（ATMT）might be useful to obtain transformants of C.cassiicola for further investigating the genes determining its pathogenicity.In the present work,we established and optimized an ATMT protocol using A.tumefaciens strain AGL-1 carrying the vector pATMT1 for C.cassiicola.Efficiency of ATMT was 102-148 transformants per 10⁶ conidia and successive subculturing of transformants on non-selective and selective media demonstrated that the integrated transfer（T）-DNA could be stably inherited in C.cassiicola transformants.The integration of the hygromycin B phosphotransferase（hph）gene into C.cassiicola was validated by PCR and Southern blot analyses,which revealed that nearly 90%of the transformants contained single-copy T-DNA.Additionally,transformants with altered phenotypes were characterized.Three of these transformants completely lost pathogenicity（CT0001,CT0022 and CT0013）and other one showed strongly impaired pathogenicity（CT0005）relative to the wild-type strain.With the aid of TAIL-PCR technique and Cc-12 genome database,the flanking sequences of the 4 single T-DNA inserted and low virulent mutants were obtained.These results pave the way to identify genes relevant to pathogenicity of C.cassiicola.