基于3种原噬菌体基因的赣南地区黄龙病菌种群动态分析

柑橘黄龙病(citrus Huanglongbing,HLB)对全球柑橘产业造成了重大经济损失。为明确赣南地区柑橘黄龙病菌基因组上原噬菌体的分子特征和传播动态,本研究对采自赣南10个市县区的470份样品进行HLB检测,并对阳性样品中3种类型的原噬菌体（SC1、SC2和P-JXGC-3）上的SC1_gp050、SC2_gp040和PJXGC_gp08基因序列进行遗传多样性分析和谱系地理学分析。结果显示,共检出74份HLB阳性样品,包括7种原噬菌体组合类型:SC1、SC2、P-JXGC-3、SC1+SC2、SC1+P-JXGC-3、SC2+P-JXGC-3和SC1+SC2+P-JXGC-3,含SC2类型的原噬菌体检出41份,检出率为55.41%,说明赣南地区含SC2类型的原噬菌体菌株最为普遍。遗传多样性分析结果表明,赣南地区的3种原噬菌体的基因交流频繁,遗传分化现象不明显。选择压力分析结果显示,SC1和P-JXGC-3类型的原噬菌体均处于负向选择,SC2类型的原噬菌体处于正向选择。基于3种原噬菌体上的基因构建的中介网络图和单倍型分布表结果显示,3种原噬菌体均存在1个祖先单倍型,其他独有单倍型均是由该祖先单倍型经多次突变而来,其中PJXGC_gp08基因产生的独有单倍型最多。时空动态分析结果显示,SC1类型原噬菌体存在2条传播路径,分别为安远和全南地区间的相互传播(3相似文献   

Repellency of selected Psidium guajava cultivars to the Asian citrus psyllid,Diaphorina citri

Huanglongbing (HLB) is the most devastating disease of citrus worldwide. It is caused by bacteria of the genus ‘Candidatus Liberibacter’ and transmitted by two psyllid species, the Asian citrus psyllid (ACP) Diaphorina citri, and the African citrus psyllid Trioza erytreae. Considerable research has been conducted toward developing and implementing HLB and ACP management strategies. With respect to ACP control, of interest is that reports indicate guava, Psidium guajava, can be repellent to ACP. We conducted research to further evaluate repellency of guava to ACP. In one set of experiments, guava oil from five Brazilian guava cultivars (‘J3’, ‘Pedro Sato’, ‘Século XXI’, ‘Thailand’ and ‘Paluma’) was extracted from leaves (immature and mature) by hydro-distillation in a Clevenger-type apparatus and evaluated for psyllid repellency. In a second set of experiments, repellency of guava leaves to ACP was investigated using leaves (immature and mature) from two guava cultivars in Florida, ‘Pink’ and ‘Thai White’. In each set of experiments, repellency was evaluated by releasing ACP adults into a cage with two large vials, one containing a young flush shoot (= immature leaves) of Murraya exotica (a favored host plant of the psyllid, the flush of which is highly attractive to ACP) and one with M. exotica flush and the test material of interest (guava oil, immature guava leaf or mature guava leaf). The adults were free to move throughout the cage and into the vials, and the number of psyllids in each vial was counted after 24 h. The results showed that all guava materials tested had at least some repellency to ACP. Mature leaves tended to have a greater repellent effect than immature leaves. Each of the five oils exhibited repellency. A report in the literature suggested that sulfur compounds associated with guava may be responsible for ACP repellency. Interestingly, the five guava oil extracts we studied were repellent to ACP but none contained any sulfur compounds. Identification of the constituents responsible for repellency could lead to new ACP management tactics.     

柑橘黄龙病病原菌的鉴定及16S rDNA序列分析

采用Nested-PCR技术检测顺昌果园红心柚树中柑橘黄龙病病原的携带情况,通过黄龙病病原菌16S rDNA测序和构建系统发育树比较不同类型柑橘黄龙病原菌之间的进化关系,采用特异引物扩增16S rDNA片段鉴别不同黄龙病病原菌,结合限制性内切酶酶切确定该红心柚园黄龙病病原的类型,并分析不同类型柑橘黄龙病病原16S rDNA序列差异性.结果表明:所取的12株红心柚树叶片样品中有7株树的叶片中检测出黄龙病病原,黄龙病病原的阳性检出率为58.33％,其中4个为显性性状,3个为隐性性状.柑橘黄龙病病原菌16SrDNA进化分析及限制性内切酶XbaI酶切结果表明,顺昌柑橘黄龙病病原菌属于亚洲型.不同类型柑橘黄龙病病原菌16S rDNA的序列差异性分析表明,亚洲种与非洲种的差异性为2.75％,非洲种与美洲种的差异性为3.90％,亚洲种与美洲种的差异性为3.44％.     

中国柑橘黄龙病研究30年

综述了我国自1978年以来有关柑橘黄龙病的研究概况,包括柑橘黄龙病在我国的分布与危害、黄龙病寄主、病原研究、病害诊断与检测、传播途径与病害流行、防控技术等.     

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。     

常规和巢式PCR对柑橘黄龙病菌的检测灵敏度比较

根据柑橘黄龙病(citrus Huanglongbing,HLB)病菌16SrDNA、16S/23S核糖体基因间隔区(ribosomal intergenic region,RIR)、核糖体蛋白β操纵子(β-operon)和外膜蛋白(out membrane protein,OMP)基因序列设计了8对引物,通过常规和巢式PCR方法对各引物的检测灵敏度进行了比较。结果表明,不同引物的检测灵敏度不同。对于上述任何基因,巢式PCR的灵敏度均比常规PCR至少高103倍;对于同一种基因,扩增短片段的引物比扩增长片段的引物灵敏度高或相当;在扩增产物大小相同时,用以扩增核糖体蛋白β操纵子基因的引物较其他三种稍高。对不同症状柑橘样品的检测进一步验证了该结果。因此,对于柑橘黄龙病的检测,可优先考虑使用巢式PCR方法,若使用常规PCR,则宜选用具有高灵敏度的扩增小片段引物。     

Effect of chemical treatments on ‘Candidatus Liberibacter asiaticus’ infected pomelo (Citrus maxima)

Huanglongbing (HLB), also known as citrus greening, is one of the most destructive diseases affecting Rutaceae plants in many parts of the world. HLB is associated with three species of ‘Candidatus Liberibacter’ with ‘Ca. L. asiaticus’ (Las), being the most widely distributed bacterium in Thailand and Asia. The effectiveness of chemical treatment via trunk injection was evaluated in Las-infected pomelo (Citrus maxima) trees using quantitative real-time polymerase chain reaction. Our results demonstrated that a mixture of streptomycin (250 mg/L), ampicillin (2.5 g/L), penicillin G (2 g/L), and Bacicure^® (2 g/L) provided the highest efficiency in reducing and suppressing the Las-bacterium population indicated by the mean cycle threshold (Ct) values from 20.8 (≈7.2 × 10⁷ cells/g of plant tissue) to 36.6 (≈2140 cells/g of plant tissue) in greenhouse experiments, and from 19.4 (≈1.8 × 10⁸ cells/g of plant tissue) to 37.8 (≈969 cells/g of plant tissue) in the field experiments. In addition, our strategies remained therapeutically effective for one year. Finally, response of Las-infected pomelo to chemical treatment was elucidated using transmission electron microscopy.     

柑橘黄龙病植株内生菌PLFAs多态性研究

采用磷脂脂肪酸(PLFAs)生物标记法,分析柑橘黄龙病株不同部位及健康状态叶片内生菌群落结构。结果表明,从柑橘黄龙病植株叶片共检测到42种PLFAs,其中完全分布的生物标记有9种,不完全标记有33种。对PLFAs进行聚类分析表明,柑橘黄龙病株不同部位叶片内生菌PLFAs可以分为2大类群,类群I特点为脂肪酸生物标记均为不完全分布,类群Ⅱ的特点为脂肪酸生物标记在被检测的柑橘黄龙病植株的大部分叶片中均有分布。柑橘黄龙病株不同部位及健康状态叶片内生菌PLFAs组成及含量存在差异。总体来看,不同朝向中,东面叶片内生菌PLFAs含量最大;不同高度中,下部叶片内生菌PLFAs含量最大;不同健康状态中,带有黄龙病原叶片内生菌PLFAs含量比健康植株叶片更大。此外,不同朝向中,南面叶片真菌/细菌值最大,革兰氏阳性菌和革兰氏阴性菌的比值(G+/G)较大;不同高度叶片中,真菌/细菌值差异不明显,G+/G值差异明显,上部最大,下部次之,中部最小;不同健康状态中,健康叶片的真菌/细菌值高于黄龙病株叶片。对柑橘黄龙病株不同部位及健康状态叶片内生菌多样性研究表明,不同朝向和不同健康状态的叶片内生菌种群多样性指数差异显著,不同高度的叶片内生菌种群多样性指数差异不显著。主成分分析表明,主成分1和主成分2基本上能把柑橘黄龙病株不同朝向叶片内生菌种群区分开来。对柑橘黄龙病株不同部位及健康状态叶片进行聚类,结果表明带有黄龙病原的叶片聚为一类,不带黄龙病原的健康叶片分聚为两类,其中,不同朝向的叶片分聚在不同的亚类群中,表明柑橘黄龙病株内生菌PLFAs分布与叶片健康状态和叶片朝向均有紧密关系,与叶片健康状态关系更为密切。     

柑橘黄龙病灾变原因及避灾减灾主要措施

从柑橘黄龙病传播途径着手,分析黄岩橘区柑橘黄龙病灾变的原因,并针对检疫性有害生物的特点,提出了政府主导、经费保障和宣传培训,以及加强检疫监管、种植无病苗木、抓好三梢期木虱防治、切断毒源和健身栽培等减灾避灾控制措施,实现了柑橘优势产业的可持续生产。     

柑桔黄龙病综合防治的理论与实践

柑桔黄龙病是世界性的毁灭性病害，除原有的亚洲、非洲外，美国、巴西也已发现此病和其传播媒介－柑桔木虱。根据柑桔木虱是黄龙病唯一自然传播媒介和广西柑桔主产区无法实施隔离种植的特点以及柑桔黄龙病已成为广西柑桔发展最大障碍的实际和柑桔生产现状，提出在黄龙病区建立以柑桔黄龙病为第一重要病害，柑桔木虱为第一重要害虫的柑桔病虫害综合防治体系，实施以控制柑桔木虱为重点的柑桔黄龙病综合防治措施。3年的大面积田间验证试验结果表明，验证区累计发病率为0.72%，对照Ⅰ区达9.42%，对照Ⅱ区达7.14%。