Isolation Of Streptomyces Species From Potato Scab Lesions And Establishment Of Detection System

Potato?Solanum tuberosum L.?is the world's fourth largest staple crop after rice,wheat and corn,which is also an important crop to increase farmers'income and guarantee the food security in China.Common scab caused by Streptomyces spp is an important epidermal disease in northern potato regions of China.However,potato common scab also occurred frequently in some southern potato regions in recent years,which seriously affects the appearance quality of the potato tubers.In this study,potato tubers with scab symptoms were collected from Guangdong and Hubei provinces,the causal agents were isolated and identified.Furthermore,related detection methods were developed,which laid a foundation of the prevention and control of potato common scab.The main results are as follows:1.102 tubers with scab symptoms were collected in five areas of Guangdong and Hubei provinces,and their symptom types and the diversity of spore chain morphology were observed and analyzed.A single colony named XY-12 was isolated from scab tuber collected at Xiangyang of Hubei province;2.By phenotypic and microscopic observation,and 16s rDNA sequencing,isolate XY-12 was preliminary identified as Streptomyces bottropensis,further inoculation confirmed that XY-12 at concentration of 1.68×10³ CFU/mL or more could induce scab symptoms on tubers of potato cv.Zhongshu 5;3.Based on 66 txtA gene sequences from 5 Streptomyces species,Streptomyces scabies,S.acidiscabies,S.bottropensis,S.stelliscabiei,S.europeascabiei,which is known causing common scab in potato,a specific PCR primer pair CS-67s/379a was designed.The annealing temperature,concentration of primer and template were optimized.The field samples assay showed that the PCR method could specially detect common scab as less as 10^-6 ng DNA.4.5 pairs of RPA primer were designed based on txtA gene of Streptomyces species,they all performed well for detection of DNA of Streptomyces bottropensis,one pair of primers named CS-RPA126s/395a was selected for further analysis.PCR and RPA assays using CS-RPA126s/395a showed that RPA could detect 10^-4 ng DNA of Streptomyces bottropensis,which was 10 times less than the sensitivity of PCR.The RPA technology could realize isothermal amplification without using any complex instruments,and the results from RPA assay had a high degree of consistency with that of PCR test using primers CS-67s/379a.