海南柑橘黄龙病发生分布调查及病原种类鉴定

本研究在海南全省范围进行了柑橘黄龙病的发生分布调查和病原种类鉴定,共采集了14个市县62处柑橘种植点的不同柑橘品种样品1227份。用柑橘黄龙病菌亚洲种和非洲种rpIA-rpIJ基因片段的通用检测引物A2/J5和柑橘黄龙病菌美洲种16S rRNA基因片段的特异检测引物GB1/GB3,对样品进行PCR扩增,发现海南柑橘黄龙病的病原为柑橘黄龙病菌亚洲种,检测样品中阳性率为79.01%,未发现柑橘黄龙病菌非洲种和美洲种。结果发现,柑橘黄龙病已分布在海南的琼中、澄迈、儋州、琼海等8个市县。同时选取海南澄迈、儋州、琼海等4个市县有代表性的甜橙、柚子、柠檬感染黄龙病的样品,扩增样品的rpIA-rpIJ基因A2/J5片段,进行序列比对和系统进化分析。序列比对结果发现,海南各地不同品种中的柑橘黄龙病菌rpIA-rpIJ基因A2/J5片段序列一致性为99.87%,序列十分保守。系统进化分析表明,海南各地不同品种的柑橘黄龙病菌都与黄龙病菌亚洲种聚集为同一进化分枝,与黄龙病菌非洲种和美洲种分属不同的进化分枝。本研究明确了海南柑橘黄龙病的发生分布情况和病原种类,对柑橘黄龙病的防控具有重要的理论意义。

Investigation and distribution of the Citrus Huanglongbing and identification of Huanglongbing liberobacter species in Hainan

In this study,the occurrence and distribution of Citrus Huanglongbing disease and the identification of its pathogenic species were investigated from 14 cities,62 orchard in Hainan Province,and 1227 samples were collected.Using the universal detection primers A2/J5 of rpIA-rpIJ gene fragment of Candidatus Liberibacter asiaticus and Candidatus Liberibacter africanus and the specific detection primers GB1/GB3 of 16S rRNA gene fragment of Candidatus Liberibacter americanus,PCR amplification was carried out on the samples.It was found that the pathogen of Huanglongbing disease in Hainan was the Candidatus Liberibacter asiaticus,and the positive rate was 79.01%.No Candidatus Liberibacter africanus and Candidatus Liberibacter americanus were found.The results show that Citrus Huanglongbing has been occurred and distributed in 8 cities and counties in Hainan,such as Qiongzhong,Chengmai,Danzhou,Qionghai,etc.At the same time,the representative samples of orange,pomelo and lemon infected with Huanglongbing disease were selected from Chengmai,Danzhou,Qionghai,and so on of Hainan Province.The rpIA-rpIJ gene A2/J5 fragment of the samples was amplified for sequence comparison and phylogenetic analysis.The results of sequence alignment showed that the sequence consistency was 99.87%,which was very conservative.Phylogenetic analysis showed that different varieties of citrus in Hainan gathered in the same evolutionary branch with the Candidatus Liberibacter asiaticus,and they belonged to different evolutionary branches with Candidatus Liberibacter africanus and Candidatus Liberibacter americanus.In this study,we identified the occurrence and distribution of Citrus Huanglongbing and the species of pathogens,which is of great theoretical significance for the prevention and control of Citrus Huanglongbing.