Identification Of Causing Agents For Tartary Buckwheat Leaf Spot And Transcriptomics Analysis On Leaves Infected By Alternaria Alternata

Tartary buckwheat disease is an important limiting factor for the yield and quality of Tartary buckwheat.In the two consecutive years of disease investigations on the Tartary buckwheat breeding base in Xiangxiang City,Hunan Province in 2019 and 2020,two leaf spot diseases with different symptoms were found,both of which occurred throughout the life cycle of Tartary buckwheat after emergence,and cause the leaves to fall off.The symptoms of the first type of leaf spot disease showed several small round or nearly round spots on the upper leaf surface at the early stage of onset,light yellow to light brown.After a few days,the lesions expanded with reddish brown edges.The symptoms of the second type of leaf spot disease showed several round or oval spots in the initial stage,chlorosis,water-stained,and easy to develop on the tip and edge of the leaf,and then expand and merge into large round or irregular spots,pale to gray brown.The different symptoms suggest that the pathogens that cause the two leaf spot diseases are different.In this paper,the field incidence,disease index and resistance differences of Tartary buckwheat leaf spot disease were investigated and analyzed.The pure culture of the pathogen was obtained by the tissue separation method,and the pathogen was determined by Koch’s postulates.The RNA-Seq technique was used to analyze the differentially expressed genes at the transcriptome level in Tartary buckwheat leaves after infection by one of the leaf spot pathogens — Alternaria altrenata.The identification of differentially expressed genes related to disease resistance will lay a theoretical foundation for developing effective control methods,clarifying the molecular mechanism of disease resistance of Tartary buckwheat,and screening excellent genetic resources for the selection of resistant varieties.The results are summarized as follows:1.Morphological observation,pathological inoculation test and molecular biology analysis showed that the first leaf spot disease was caused by Nigrospora osmanthi,and the second leaf spot disease was caused by Alternaria alternata.To our knowledge,it is the first report that Nigrospora osmanthi and Alternaria alternata cause Tartary buckwheat leaf spot diseases in China and worldwide.To our knowledge,this is the first report of N.osmanthi causing leaf spot on F.tataricum in China and worldwide.2.Field investigations showed that the average incidence of N.osmanthi leaf spot and A.alternata leaf spot were about 51.7% and 65.3%,and the average disease index was about40.5 and 49.1,respectively.A.alternata leaf spot had a higher incidence and degree of damage.Pinku 1 and Yunqiao 2 had the strongest resistance to N.osmanthi leaf spot,while Guimiku 1 and Yunqiao 1 had the weakest resistance.Similarly,Pingku 1 had the strongest resistance to A.alternata leaf spot,and Yunqiao 1 and Guimiku 1 had the weakest resistance.3.Based on RNA sequencing technology,transcriptome analysis was carried out on the samples of Tartary buckwheat leaves at 0 h,12 h,and 36 h after infection of A.alternata,a more harmful pathogen of leaf spot.Each sample obtained over 6.44 Gbof high-quality bases,and the percentage of Q30 bases in each group of samples was above 93.94%.Transcription factor family analysis and R gene annotation analysis were performed on all Unigenes,and1786 and 2061 annotation records were obtained,respectively.The GO annotation showed that the number of differential genes involved in cellular processes is the largest,followed by cellular components and molecular functions.KEGG pathway analysis showed that the enrichment of differential genes was mainly in the plant-pathogen interaction,plant hormone signal transduction and MAPK signal transduction pathways.The results suggest that the plant hormone pathway may play an important function during the defense responses of Tartary buckwheat to A.alternata Tartary buckwheat.4.Ten differentially expressed genes of JA-,ET-and SA-dependent defense signal transduction pathways and 4 Pathogenesis-related protein genes were chosen to verify the validness of the RNA-seq using real-time fluorescence quantitative PCR under the treatment of A.altrenata.The results showed that the expression trend of these genes under A.altrenata infection was consistent with transcriptome sequencing,which proved the reliability of transcriptome data.