亚洲柑橘木虱传播黄龙病菌的特性与机制研究进展

黄龙病Huanglongbing(HLB)是世界性的重大柑橘病害,在中国主要由亚洲柑橘木虱Diaphorina citri Kuwayama传播。该害虫的传病特性与机制一直是相关研究热点之一。本文综述了近些年该领域的研究成果,包括亚洲柑橘木虱传播黄龙病的方式、传病过程与机制,以及影响传病效率的因素等方面,为该木虱及黄龙病的防控与深入研究提供参考。     

亚洲柑橘木虱的起源、分布和扩散能力研究进展

亚洲柑橘木虱Diaphorina citri Kuwayama是柑橘的毁灭性病害—黄龙病(huanglongbing,HLB)的传播媒介,是柑橘生产中最重要的害虫。本文综述了亚洲柑橘木虱的起源、分布和扩散情况,揭示中国亚洲柑橘木虱由南向北的扩散规律,为有效防治木虱、阻止黄龙病进一步蔓延传播、保障我国柑橘产业的健康发展提供依据。     

龙葵上柑橘木虱存活观察与黄龙病检测

柑橘木虱Diaphorina citri是毁灭性病害黄龙病的媒介,其寄主范围比较严格,仅为芸香科内的一些植物。研究发现,在自然状态下,柑橘木虱成虫可在柑橘园中常见茄科Solanaceae杂草-龙葵Solanum nigrum上停留。对比试验显示,木虱成虫在龙葵上的存活期最长可达45 d,而在假臭草Eupatorium catarium、含水海绵和无水海绵上分别为24 d、9 d和2 d;通过实时荧光PCR检测发现部分龙葵叶片中含有黄龙病病原菌。这些非寄主植物可能有助于柑橘木虱躲避不良环境或长距离迁移扩散,成为柑橘木虱和黄龙病的潜在库源。     

越南地区柑橘黄龙病发生及防治

黄龙病是全球广泛发生的毁灭性病害,柑橘木虱为其主要媒介昆虫。越南最早于上个世纪六十年代有相关报道。通过一些国际合作项目,越南对柑橘木虱和黄龙病进行了一系列研究并积极采取措施有效防控:包括消除病株、筛选抗病虫品种、改变种植密度和时间、化学防治与生物防治、作物间种、喷施矿物油乳剂、施用有机肥、使用无病苗和无病接穗并在运输过程中封闭保护等。其中控制黑臭蚁Dolichoderus thoracicus增殖黄猄蚁Oecophylla smaragdina、交错式间种番石榴以控制柑橘木虱和黄龙病值得中国借鉴和参考。     

基于COI基因的华南地区柑橘木虱系统发育研究

柑橘木虱Diaphorina citri是重要的柑橘害虫,其传播的黄龙病对华南地区柑橘产业造成严重危害。为了解华南地区柑橘木虱不同地理种群间的遗传多样性和进化关系,测定该地区5个地理种群线粒体COI基因900 bp序列,对不同地理种群柑橘木虱遗传距离和系统发育进行分析。结果表明,5个地理种群柑橘木虱间的遗传距离小(0.0%~0.2%)。其中,广州种群和湛江种群的柑橘木虱之间的遗传距离,以及南宁种群、桂林种群和厦门种群的柑橘木虱之间的遗传距离均为0,序列间的相似度极高。基于COI基因系统发育分析显示,位于中国的5个柑橘木虱地理种群之间分化程度较低,与国外其他地区21个种群相比,各种群间的同源性与地理距离无显著相关性。     

利用虫生真菌生物防治柑橘木虱的研究进展

柑橘木虱是柑橘黄龙病的重要传播虫媒。目前对柑橘黄龙病的防治尚缺有效的药剂和抗病品种,加强对柑橘木虱的防治,对控制柑橘黄龙病的蔓延具有重要意义。目前防治柑橘木虱多采用化学防治,杀虫剂的频繁使用造成了农药残留、环境污染、生物多样性被破坏和害虫产生抗药性等诸多问题,生物防治以其高效、低毒、低残留、不易产生抗药性等优点逐渐受到重视。昆虫病原真菌能侵入昆虫寄主体内,导致昆虫发病死亡,具有良好的病害流行潜力及生产应用便利性,利用昆虫病原真菌防治柑橘木虱具有广阔的发展空间。本文总结了用于柑橘木虱生物防治的虫生真菌种类,重点介绍了国内外利用球孢白僵菌、玫烟色棒束孢、淡紫紫孢菌、宛氏拟青霉、蜡蚧菌等虫生真菌在防治柑橘木虱中的应用,并对虫生真菌防治柑橘木虱的发展前景进行了展望,以期为柑橘黄龙病的防控提供借鉴。     

亚洲柑橘木虱成虫和5龄若虫在感染黄龙病的柑橘上的取食行为及获菌效率比较

【目的】亚洲柑橘木虱Diaphorina citri是柑橘的毁灭性病害——黄龙病亚洲种‘Candidatus Liberibacter asiaticus’(‘C las’)的主要传播媒介。本研究的目的是明确木虱成虫和5龄若虫的取食行为、获菌效率是否有差异,以及寄主感染黄龙病是否对5龄若虫取食产生影响。【方法】利用直流型刺吸电位仪(DC-EPG Giga-4)记录柑橘木虱成虫和5龄若虫在携带黄龙病的酸橘Citrus reticulata cv.Sunki嫩梢上10 h的取食行为,用qPCR单头检测其获得黄龙病病原菌的效率,并比较5龄若虫在感病和健康植株嫩梢上的取食行为。【结果】柑橘木虱成虫与5龄若虫在感染黄龙病的酸橘上的取食行为有显著差异。5龄若虫比成虫更快地开始在韧皮部和木质部进行吸食,口针在韧皮部的总过程以及吸食时间显著长于成虫。此外,5龄若虫和成虫在EPG测定(同时饲菌)10 h后获菌率分别为37.5%和20.0%,若虫明显高于成虫。寄主植物感染黄龙病对5龄若虫的取食行为有一定的影响,表现在感病植株上的刺探次数、唾液分泌次数和韧皮部吸食次数都显著少于健康植株,而两者分泌唾液和韧皮部吸食时间没有显著差异。另外,在感病植株上首次韧皮部取食出现时间较在健康植株上要早。【结论】在同等时间下,柑橘木虱5龄若虫比成虫在感染黄龙病的酸橘上的取食能力更强、取食量更大、获菌率更高,原因可能是若虫需要更多的营养物质供其生长发育所致。寄主感染黄龙病对木虱5龄若虫的取食有利,使其更快地开始取食,而且更改取食位点次数变少,推测可能与黄龙病菌破坏了植物的防御有关。     

柑橘木虱抗药性研究进展

柑橘木虱Diaphorina citri Kuwayama主要危害柑橘等芸香科植物,传播柑橘黄龙病病菌。柑橘树感染黄龙病后,叶片黄化,果实早熟脱落,畸形,最终死亡,给柑橘产业带来了极大的损失。柑橘木虱对各种常见的杀虫剂已产生了不同程度的抗药性。本文从柑橘木虱的抗性测定方法、抗药性现状、交互抗性、抗药性机理和抗性治理等方面进行了综述,为今后柑橘木虱的防治提供一定的参考。     

柑橘木虱抗药性研究进展

柑橘木虱Diaphorina citri Kuwayama主要危害柑橘等芸香科植物,传播柑橘黄龙病病菌。柑橘树感染黄龙病后,叶片黄化,果实早熟脱落,畸形,最终死亡,给柑橘产业带来了极大的损失。柑橘木虱对各种常见的杀虫剂已产生了不同程度的抗药性。本文从柑橘木虱的抗性测定方法、抗药性现状、交互抗性、抗药性机理和抗性治理等方面进行了综述,为今后柑橘木虱的防治提供一定的参考。     

Infection and distribution of Candidatus Liberibacter asiaticus in citrus plants and psyllid vectors at the cellular level

Huanglongbing (HLB) is currently considered the most destructive disease of citrus worldwide. In the major citrus-growing areas in Asia and the US, the major causal agent of HLB is the bacterial pathogen Candidatus Liberibacter asiaticus (CLas). CLas is vectored by the Asian citrus psyllid, Diaphorina citri, in a persistent propagative manner. CLas cannot be cultured in vitro because of its unclear growth factors, leading to uncertainty in the infection mechanism of CLas at the cellular level in citrus and in D. citri. To characterize the detailed infection of CLas in the host and vector, the incidence of HLB was first investigated in citrus-growing fields in Fujian Province, China. It was found that the positive association of the level of CLas infection in the leaves correlated with the symptoms. Then antibodies against peptides of the outer membrane protein (OMP) of CLas were prepared and tested. The antibodies OMP-225, OMP-333 and OMP724 showed specificity to citrus plants in western blot analyses, whereas the antibodies OMP-47 and OMP-225 displayed specificity to the D. citri vector. The application of OMP-225 in the immunofluorescence assay indicated that CLas was located in and distributed throughout the phloem sieve cells of the leaf midribs and axile placenta of the fruit. CLas also infected the epithelial cells and visceral muscles of the alimentary canal of D. citri. The application of OMP-333 in immunoelectron microscopy indicated the round or oval CLas in the sieve cells of leaf midribs and axile placenta of fruit as well as in the epithelial cells and reticular tissue of D. citri alimentary canal. These results provide a reliable means for HLB detection, and enlighten a strategy via neutralizing OMP to control HLB. These findings also provide insight for the further investigation on CLas infection and pathogenesis, as well as CLas–vector interaction.     

基于纳米孔测序技术的柑橘黄龙病检测方法建立

柑橘黄龙病(Huanglongbing)是柑橘生产上最具毁灭性的病害,及时快速地进行早期检测和诊断是防控黄龙病的关键措施之一.本文利用掌上纳米孔测序仪MinION对携带黄龙病菌Candidatus Liberibacter asiaticus的DNA样品进行测序,并利用Blast、GraphMap、minimap2以及两种bwa的比对方法将测序结果比对到黄龙病菌基因组上,比对结果均匀的比对到黄龙病菌基因组上,并未发现严重的偏倚现象,验证了其测序结果的可靠性.本技术可弥补因柑橘木虱Diaphorina citri(Kuwayama)虫体过小或损坏难以进行光学识别的不足,并可同时检测虫体是否携带有黄龙病菌,对有黄龙病发生风险但尚未有黄龙病实际发生的柑橘种植区提供实时实地的监控与预警.     

Diversity and characterization of Candidatus Liberibacter asiaticus strains causing huanglongbing disease in Iran,based on two prophage loci

Huanglongbing (HLB), also known as citrus greening, is a destructive disease of citrus; it is considered a newly emerging disease which has spread to the Middle East and North Africa (MENA). In Iran, the disease was first found in 2009. In this study, two hypervariable prophage and phage‐related loci, bacteriophage repressor protein C1 (CLIBASIA_ 01645 locus) and prophage terminase gene (CLIBASIA_05610 locus), were used to determine the diversity and characterization of Candidatus Liberibacter asiaticus ( CLas) strains associated with HLB samples. Analyses of the CLIBASIA_01645 locus, characteristic of variable tandem repeat numbers (VTRNs), revealed the homogeneity of Iranian CLas isolates: However, this result showed two distinct genotypes (TRN < 10 and TRN > 10) of CLas in Iran. This is the first report documenting the presence of two differentially distributed genotypes of CLas in Iran. Sequence analysis of prophage terminase revealed the presence of two putative prophages (prophage I and prophage II) in the genome of CLas isolates of Iran. Frequency analysis of these two prophages by specific loci revealed the association between prophages populations, the development HLB symptoms and CLas genotypes and their interactions with another obligate symbiontic, HLB phytoplasma.     

Plant response and huanglongbing disease development against heat treatments on ‘Siam Purworejo’ (Citrus nobilis (Lour)) and ‘Nambangan’ (C. maxima (Burm.) Merr.) under field condition

Huanglongbing is known as a destructive disease in citrus production. Investigation on plant response and development of huanglongbing disease against heat treatment on ‘Siam Purworejo’ (Citrus nobilis (Lour)) and ‘Nambangan’ (C. maxima (Burm.) Merr.) was done. High-temperature treatment was applied by covering plants with an enclosed screen. The treatment plant has three types of CLas pathogen infection status. The result showed that heat treatments could significantly increase new flushes. It was found that intensity of HLB infected plant decreased. The treatments effect on the proline level was dependent on cultivar, leaves age and HLB-infection status. Chlorophyll content of HLB infected plants increased due to high temperature. High-temperature increased Fe content in the artificially infected plant, whereas Zn contents increased in the leaves of infected plants by vector. ‘Nambangan’ had higher Fe content than ‘Siam Purworejo’ on uncovered plants. CLas bacteria still persisted in infected plant based on real-time PCR analysis.     

Metabolic Interplay between the Asian Citrus Psyllid and Its Profftella Symbiont: An Achilles’ Heel of the Citrus Greening Insect Vector

‘Candidatus Liberibacter asiaticus’ (CLas), the bacterial pathogen associated with citrus greening disease, is transmitted by Diaphorina citri, the Asian citrus psyllid. Interactions among D. citri and its microbial endosymbionts, including ‘Candidatus Profftella armatura’, are likely to impact transmission of CLas. We used quantitative mass spectrometry to compare the proteomes of CLas(+) and CLas(-) populations of D. citri, and found that proteins involved in polyketide biosynthesis by the endosymbiont Profftella were up-regulated in CLas(+) insects. Mass spectrometry analysis of the Profftella polyketide diaphorin in D. citri metabolite extracts revealed the presence of a novel diaphorin-related polyketide and the ratio of these two polyketides was changed in CLas(+) insects. Insect proteins differentially expressed between CLas(+) and CLas(-) D. citri included defense and immunity proteins, proteins involved in energy storage and utilization, and proteins involved in endocytosis, cellular adhesion, and cytoskeletal remodeling which are associated with microbial invasion of host cells. Insight into the metabolic interdependence between the insect vector, its endosymbionts, and the citrus greening pathogen reveals novel opportunities for control of this disease, which is currently having a devastating impact on citrus production worldwide.     

Detection of <Emphasis Type="Italic">Diaphorina citri</Emphasis> Kuwayama (Hemiptera: Liviidae) in Kenya and potential implication for the spread of Huanglongbing disease in East Africa

The Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae) is a damaging pest of citrus globally and has recently been detected in Tanzania. Although direct damage by the pest is seldom of economic importance, the insect is more notorious for its ability to vector the fastidious phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas), the putative causal bacterium of Huanglongbing or Asian citrus greening disease. For many years, Trioza erytreae (Del Guercio) (Hemiptera: Triozidae) was known to be the main vector of the African citrus greening disease caused by Candidatus Liberibacter africanus (CLaf), but the recent arrival of D. citri on the continent adds to the dynamics of infection and spread of both diseases on mainland Africa. Following the recent report of the presence of D. citri in Tanzania, an additional delimiting survey was carried out in the region, focusing on Kenya, mainland Tanzania and Zanzibar to detect the presence and ascertain the extent of spread of D. citri. We employed molecular tools based on the use of DNA barcoding to confirm the identity of D. citri. In addition to D. citri, the occurrence of T. erytreae in the same sampling locations is also reported. Adults and nymphs of either D. citri or T. erytreae were collected from citrus at many of the surveyed sites ranging from 19 to 668 m above sea level (masl) in Tanzania, 20–1666 masl in Kenya, and 42–48 masl in Zanzibar. Diaphorina citri was sympatric with T. erytreae at the mid to higher elevations of 1375–1666 masl and no T. erytreae or its open-gall symptoms were detected below 523 masl. Sequences obtained were queried via BLAST and all linked to D. citri of different accession numbers already available on GenBank. This is the first report of the presence of D. citri in Kenya and Zanzibar. The potential implication of the detection and spread of the two pathogens, CLaf and CLas to the citrus industry in East Africa and movement of suitable host plants is discussed.     

Predominance of Single Prophage Carrying a CRISPR/cas System in “Candidatus Liberibacter asiaticus” Strains in Southern China

“Candidatus Liberibacter asiaticus” (CLas) is an uncultureable α-proteobacterium associated with citrus Huanglongbing (HLB, yellow shoot disease), a highly destructive disease affecting citrus production worldwide. HLB was observed in Guangdong Province of China over a hundred years ago and remains endemic there. Little is known about CLas biology due to its uncultureable nature. This study began with the genome sequence analysis of CLas Strain A4 from Guangdong in the prophage region. Within the two currently known prophage types, Type 1 (SC1-like) and Type 2 (SC2-like), A4 genome contained only a Type 2 prophage, CGdP2, namely. An analysis on CLas strains collected in Guangdong showed that Type 2 prophage dominated the bacterial population (82.6%, 71/86). An extended survey covering five provinces in southern China also revealed the predominance of single prophage (Type 1 or Type 2) in the CLas population (90.4%, 169/187). CLas strains with two and no prophage types accounted for 7.2% and 2.8%, respectively. In silico analyses on CGdP2 identified a CRISPR (clustered regularly interspaced short palindromic repeats)/cas (CRISPR-associated protein genes) system, consisting of four 22 bp repeats, three 23 bp spacers and 9 predicted cas. Similar CRISPR/cas systems were detected in all 10 published CLas prophages as well as 13 CLas field strains in southern China. Both Type 1 and Type 2 prophages shared almost identical sequences in spacer 1 and 3 but not spacer 2. Considering that the function of a CRISPR/cas system was to destroy invading DNA, it was hypothesized that a pre-established CLas prophage could use its CRISPR/cas system guided by spacer 1 and/or 3 to defeat the invasion of the other phage/prophage. This hypothesis explained the predominance of single prophage type in the CLas population in southern China. This is the first report of CRISPR/cas system in the “Ca. Liberibacter” genera.     

柑橘木虱两地理种群的内共生菌群落组成及传菌能力

柑橘黄龙病（Huanglongbing, HLB）是经柑橘木虱Diaphorina citri传播的最主要柑橘病害之一, 危害严重时能对柑橘产业造成毁灭性的破坏。为了鉴定福建和海南2个地理种群柑橘木虱的内共生菌群落组成, 本研究对16S rRNA部分保守序列进行PCR扩增, 并利用特异性引物对不同内共生菌进行了感染率检测; 另外, 还通过人工接虫的方法, 探索柑橘木虱成虫在带黄龙病菌蕉柑Citrus reticulata cv. Tankan上的获菌能力, 以及带菌柑橘木虱成虫对黄岩蜜橘C. reticulata cv. Subcompressa的传菌能力。研究发现, 这2个地理种群的柑橘木虱含有相同的内共生菌组成, 包括α-Proteobacteria, Wolbachia spp., γ-Proteobacteria, mycetocyte symbionts, β-Proteobacteria, Oxalobacter和β-Proteobacteria, Herbaspirillum, 而且这2个地理种群柑橘木虱的4种内共生菌的携带率均在95%以上。柑橘木虱成虫在带菌蕉柑上饲菌28 d后, 带菌率可达到82%, 而带菌柑橘木虱成虫在黄岩蜜橘上传菌75 d后, 可导致橘树整体带菌。本研究为柑橘木虱的进一步研究和防虫治病途径提供了一些理论依据。