Distribution Of Huanglongbing In Citrus Orchards And Analysis Of Pathogen And Associated Bacterium

Citrus Huanglongbing(HLB)is one of the most devastating diseases of citrus.Up to date,there’s no effective cure methods available.Comprehensive integrated control of Huanglongbing in citrus production area is of vital importance.To know the infection status of Huanglongbing bacteria in citrus orchard in epidemic areas,and the characteristics of infected citrus orchards are important basis for comprehensive integrated control of citrus Huanglongbing.In order to understand the distribution of Huanglongbing pathogen(Ca.L.asiaticus,CLas)in orchards,and the reason why the old citrus orchard infected by Huanglongbing bacteria for many years but still kept stable production.We carried out current research.In the present study,newly-built orchards in Anyuan County,Ganzhou,Jiangxi province,newly-built(5 years)Orchard of Tianma Orchard in Yongchun County,Fujian Province,and 60-year Old Menghu Orchard in Fujian province were chosen for analysis.Nested PCR was used to detect the presence of Ca.L.asiaticus in citrus plants collected from the above orchards.Main results obtained were summarized as follows:1.Investigation of pathogen distribution in diseased orchards: a nested PCR method was used to detect CLas in 6 orchards in same place with same management mode in Anyuan County,Ganzhou,Jiangxi Province.The results showed that the CLas was widely distributed among these orchards,and the disease rate was different,ranging from 27% to 64.5%.The titer of CLas in infected plants was generally low,and more than 70% of infected plants’ were of disease level1(only 1 to 2 samples were HLB positive out of 8 samples).In the same orchard,the ratio of CLas in different positions(edge,center)of the orchard was obviously different,and the distribution of CLas in different directions(East,South,West and North)was distinct.2.Analysis of the efficiency of the new planting model on the prevention and control of Huanglongbing in epidemic area.HLB investigation in newly-built orchard of Tianma was compared with Anyuan County Orchard,and Menghu Orchard.According to the survey results in April to May in 2016,HLB disease rate of in Tianma Orchard was 8.33%,which was much lower than 40% of Anyuan County Orchard,and 71% of Menghu Orchard.After HLB-infected plants were removed,a second round of HLB survey was conducted in Tianma Orchard in April2017.The results showed that the rate of infected plants was zero.It indicated that the integrated control strategies against HLB bacteria in Tianma Orchard achieved significant success.3.Illustration of the reason of old orchard with continuous production even infected by CLas.The results showed that the disease rate of in this orchard was over 70% in three investigations.The high throughput sequencing of 16 s rDNA was carried out on the phloem samples of infected and healthy plants separately.The results showed that CLas was extremely high in OTUs cluster.There were 62 and 60 unique OTUs were found in healthy and CLas-infected samples respectively.The bacteria represented by OTUs in diseased plants contained a large number of bacteria resistant to different stresses.OTUs endemic to healthy plants contained many biodegradable or drug-resistant bacteria.T-A cloning was used to verify these OTUs,13 of the 24 gene sequences obtained from infected plants were consistent with those obtained by high throughput sequencing,which proved that the results of high throughput sequencing were reliable.4.Analysis of prophages in CLas isolates in Ganzhou,Jiangxi Province and in Menghu Orchards,Fujian Province.Hol in phage SC1 and Taa in prophage SC2 were selected for comparison and analysis.The results showed that there was no significant difference in the sequence of Hol and Taa gene in the isolates from two different origin.And showed 100%similarity with corresponding genes deposited in GenBank(Accession number: CP004005.1,CP019958.1,CP010804.1,KX879602.1).Our results confirmed that both prophage of SC1 and SC2 were found in Fujian and Jiangxi HLB samples.Whether prophages affect the population diversity of CLas in both proviences,and whether there are other prophages existed need to be further studied.