广西柑橘黄龙病发生情况及黄龙病菌的遗传多样性分析

Present status of citrus Huanglongbin and genetic diversity of Candidatus Liberibacter asiaticus in Guangxi

【目的】了解广西柑橘主产区黄龙病(Huanglongbing,HLB)危害情况及黄龙病菌(Candidatus Liberibacter asiaticus, CLas)的遗传多样性,为黄龙病的田间诊断和科学防控提供参考。【方法】对广西柑橘主产区8个地级市有代表性的柑橘园进行2个年度(2020和2021年)的跟踪调查并随机采集1048份柑橘叶片样品,利用qPCR技术对采集的样品进行CLas检测,并对阳性样品CLas菌株的基因组高变区的扩增产物进行测序,分析CLas菌株的遗传多样性。【结果】与2020年相比,除采自百色的柑橘样品CLas检出率2个年度保持不变(14.29%)外,2021年采自玉林、桂林、河池、梧州、南宁、贺州和来宾的柑橘样品CLas检出率均呈上升趋势,其中2021年采自梧州的柑橘样品CLas检出率最高(52.94%)。不同种类柑橘的CLas检出率差异明显,2021年甜橙类、柚类、杂交柑类和金柑类的样品CLas检出率均较2020年升高,2个年度的宽皮柑橘类CLas检出率相同(14.29%)。不同症状类型的柑橘样品CLas检出率也存在较大差异,其中2021年采集的缺素黄化型样品CLas检出率最高(61.64%),其次是均匀黄化型(24.00%)和褪绿斑驳型(20.00%),无症状样品的CLas检出率亦高达11.54%,均较2020年的检出率增加。通过对CLas基因组高变区的PCR产物进行测序,发现CLas菌株存在丰富的遗传多样性,37株CLas菌株可聚为7个类群,与寄主柑橘的种类有一定关系,但其与症状类型及地理来源无明显的相关性。【结论】不同年份、地理来源、柑橘种类和症状类型的CLas检出率差异较大,且发病率有逐年上升趋势。田间缺素黄化型植株确诊为感染黄龙病的概率最大,其次是均匀黄化型和褪绿斑驳型,无症状植株也有很大概率感染黄龙病。CLas菌株存在丰富的遗传多样性,与寄主柑橘的种类有一定关系,但与症状类型及地理来源无明显的相关性。

【Objective】To investigate the hazards of Huanglongbing(HLB)and the genetic diversity of Candidatus Liberibacter asiaticus(CLas)in citrus producing areas of Guangxi,so as to provide reference for field diagnosis and scientific prevention and control of Huanglongbing.【Method】A two-year(2020 and 2021)follow-up survey was carried out in representative citrus orchards in 8 cities in the main citrus producing areas of Guangxi,and 1048 citrus leaf samples were randomly collected.The collected samples were detected for CLas by qPCR technology,and the amplified products of the hypervariable regions of the genome of CLas strains in HLB positive samples were sequenced to analyze the genetic diversity of the CLas strains.【Result】Compared with the citrus samples collected in 2020,the detection rate of CLas in citrus samples collected from Yulin City,Guilin City,Hechi City,Wuzhou City,Nanning City,Hezhou City and Laibin City showed an upward trend in 2021,among which the detection rate of CLas in the citrus samples collected from Wuzhou City was the highest(52.94%),while the detection rate of CLas in the citrus samples from Baise City remained unchanged(14.29%)in the 2 years.There were great differences in the detection rate of CLas among different citrus varieties.The detection rates of CLas in sweet orange,pomelo,hetero citrus and kumquat samples in 2021 was higher than those in 2020,and the detection rate of CLas in mandarin orange in the two years remained unchanged(14.29%).There were also great differences in the detection rate of CLas in citrus samples with different symptom types.Among them,the detection rate of CLas in chlorosis deficient samples collected in 2021 was the highest(61.64%),followed by uniform chlorosis(24.00%)and chlorosis mottled(20.00%),and the detection rate of CLas in asymptomatic samples was also as high as 11.54%,all of them were higher than those in 2020.By sequencing the PCR products of the hypervariable regions of the CLas genome,it was found that CLas strains had rich genetic diversity.The 37 CLas strains could be clustered into 7 groups,which were related to the species of the host citrus,but they had no obvious correlation with symptom types and geographical origin.【Conclusion】The detection rate of CLas varies greatly in different years,geographical sources,citrus species and symptom types,and the incidence rate has an increasing trend year by year.The plants with element deficiency chlorosis in the field are most likely to be diagnosed with HLB,followed by uniform yellowing and chlorotic mottle.Asymptomatic plants also have a high probability of being infected with CLas.CLas strains have rich genetic diversity.It is related to the host citrus varieties at certain degrees,but there is no obvious correlation with the symptom types and geographical origin.