| The production of Kiwifruits in China,has been rapidly developed since the 21st century,and China has become the country with the largest kiwifruits production and the bigest planting area in the world.Shaanxi Province is currently the major kiwifruit production base in China.In which,Zhouzhi and Meixian Prefectures,have the largest planting area,accounting for more than 70%of the whole province.With the expansion of cultivation area,frequent occurrences of diseases have increasingly affected the quality and yield of kiwifruits.Phytophthora de Bary,belonging to Oomycota,is a very harmful plant pathogen that can cause strong damage to agricultural production,landscape plants and ecological environment.According to reports,three Phytophthora species include Phytophthora cactorum,Phytophthora cinnamomi and Phytophthora lateralis can cause the kiwifruits root rot disease.This study investigated 35 kiwifruit orchards in all in Meixian and Zhouzhi Prefectures of Shaanxi Province.Twenty strains of P.cactorum and 3 strains of P.cinnamomi were isolated.No P.lateralis was isolated.At the same time,a quantitative real-time molecular detection technology was established for these three pathogens,and their distributions in kiwifruits orchard were analyzed.A total of 166 rhizospheric soil samples were collected from the kiwifruit trees with disease histories in April,June and October of 2017,and in May,October of 2018 in Zhouzhi and Meixian Prefecture,respectively.After baiting,isolation,and cultivation,the isolates were identified as P.cactorum and P.cinnamomi by the morphological observations and genetic identifications.The Ypt1(Ras-related Protein Gene)sequences of 50 Phytophthora species and 3 Pythium species were aligned to develop species-specific primers and TaqMan probes for three Phytophthora species include P.cactorum,P.cinnamomi and P.lateralis,respectively.And 52 isolates(including 44 Phytophthora,3 Pythium and 3 other soil borne pathogens)were used to verify the specificity of primers and TaqMan probes.The results showed that the designed molecular markers had good specificity.In addition,the multiplex quantitative real-time PCR detection system had been optimized,and the detection sensitivity of the Ypt1 gene for these three pathogens reached 100 fg.Finally,an absolute quantitative detection method was established based on the DNA gradient concentration of the pathogens and the Ct value of the Ypt1 gene,and the quantity and distribution characteristics of these three Phytophthora in the main producing areas of kiwifruits in Shaanxi Province were investigated.By measuring 90 soil samples from Zhouzhi,P.cactorum were found in 24 soils,with the concentration of Ypt1 gene ranging from 15 to 89 pg(g soil)-1.P.cinnamomi were found in 6 soils,with the concentration of Ypt1 gene ranging from 15 to 43 pg(g soil)-1.P.lateralis were found in 6 soils,with the concentration of Ypt1 gene ranging from 25 to 56 pg(g soil)-1.By measuring 76 soil samples from Meixian,P.cactorum were found in 14 soils,with the concentration of Ypt1 gene ranging from 10 to 67 pg(g soil)-1.P.cinnamomi were found in 3 soils,with the concentration of Ypt1 gene ranging from 17 to 151 pg(g soil)-1.P.lateralis were found in 7 soils,with the concentration of Yptl gene ranging from 9 to 59 pg(g soil)-1.Overall,P.cactorum was found to be the most widely distributed,while P.cinnamomi and P.lateralis were relatively less frequent.By analyzing the correlation between three Phytophthora species with soil environmental factors,it was found that soil pH and soil temperature had the greatest influence on the quantity and distribution of these Phytophthora species.Kiwifruits production is one of the important economic industries in Shaanxi Province.However,affected by human behaviors and environmental factors,Kiwifruit disease has become the main factor restricting its yield and quality.This study will be very helpful in the early diagnosis,monitoring and prevention of Phytophthora disease for kiwifruits cultivation. |
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