Quantification and ecological study of ‘Candidatus Liberibacter asiaticus’ in citrus hosts,rootstocks and the Asian citrus psyllid

The use of proper management strategies for citrus huanglongbing (HLB), caused by ‘Candidatus Liberibacter asiaticus’ (Las) and transmitted by Asian citrus psyllid (ACP) (Diaphorina citri), is a priority issue. HLB control is based on healthy seedlings, tolerant rootstock cultivars and reduction of ACP populations. Here, dynamic populations of Las in different citrus hosts and each instar of ACP were studied, together with the seasonal growth and distribution of Las in different tissues, using conventional and TaqMan real‐time PCR. Different levels of susceptibility/tolerance to HLB were seen, resulting in different degrees of symptom severity and growth effects on hosts or rootstocks. Troyer citrange, Swingle citrumelo and wood apple were highly tolerant among 11 rootstock cultivars. Regarding distribution and seasonal analysis of Las, mature and old leaves contained high concentrations in cool temperatures in autumn and spring. Las was detected earlier through psyllid transmission than through graft inoculation, and the amounts of Las (AOL) varied in different hosts. Thus, different AOL (10⁴–10⁷ copy numbers μL^?1) and Las‐carrying percentages (LCP; 40–53.3%) were observed in each citrus cultivar and on psyllids, respectively. Furthermore, both AOL and LCP were lower in nymphs than in adult psyllids, whereas the LCP of psyllids were not affected by increasing the acquisition‐access time. The present study has significant implications for disease ecology. The combination of early detection, use of suitable rootstocks and constraint of psyllid populations could achieve better management of HLB disease.     

Identification of the Chinese Box Orange (Severinia Buxifolia) as an Alternative Host of the Bacterium Causing Citrus Huanglongbing

The Chinese box orange (Severinia buxifolia) was shown by graft-inoculation and psyllid-transmission tests to be an alternative host of the bacterium causing citrus Huanglongbing (HLB). A PCR-based assay for detection of the HLB bacterium (HLBB) was used to monitor HLBB. In graft-inoculation tests, the Chinese box orange (CBO) grafted with HLBB-infected scions of Luchen sweet orange (LSO) were positive for HLBB, 2–3 months after grafting. The back-grafting test demonstrated that HLBB-infected CBO scions could transmit HLBB back to LSO hosts via grafting. In psyllid-transmission tests, psyllids (insect vectors) transmitted HLBB to CBO plants, in which HLBB could be detected 3–4 months after inoculation. Acquisition-access tests of psyllids revealed that HLBB-free psyllids can acquire HLBB from diseased CBO hosts and can transmit HLBB back to the LSO plants. A field survey verified the presence of HLBB-infected CBO plants in the vicinity of citrus orchards. In this paper, CBO is shown to be a susceptible host plant in which HLBB can exist and replicate. It is also a donor plant from which HLBB can be transmitted to citrus hosts by grafting or by psyllid vectors.     

Detection of 'Candidatus Liberibacter asiaticus' in Diaphorina citri and its importance in the management of citrus huanglongbing in Florida

Citrus huanglongbing (HLB or citrus greening), is a highly destructive disease that has been spreading in both Florida and Brazil. Its psyllid vector, Diaphorina citri Kuwayama, has spread to Texas and Mexico, thus threatening the future of citrus production elsewhere in mainland North America. Even though sensitive diagnostic methods have been developed for detection of the causal organisms, Candidatus Liberibacter spp., the pathogen cannot be detected consistently in plants until symptoms develop, presumably because of low titer and uneven distribution of the causal bacteria in nonsymptomatic tissues. In the present study, TaqMan based real-time quantitative polymerase chain reaction methodology was developed for detection of 'Ca. L. asiaticus' in D. citri. Over 1,200 samples of psyllid adults and nymphs, collected from various locations in Florida, from visually healthy and HLB symptomatic trees at different times of the year were analyzed to monitor the incidence and spread of HLB. The results showed that spread of 'Ca. L. asiaticus' in an area may be detected one to several years before the development of HLB symptoms in plants. The study suggests that discount garden centers and retail nurseries may have played a significant role in the widespread distribution of psyllids and plants carrying HLB pathogens in Florida.     

柑橘黄龙病病原研究进展

本文对柑橘黄龙病(HLB)的发现、认识过程及病原菌的分类、检测鉴定技术进行了综述。HLB是一种十分严重的柑橘病害,主要通过木虱和嫁接进行传播,在不同的地区和国家具有不同的名字,已统一命名为HLB。对引起HLB的原因探索经历了一个漫长的过程,从水害、缺素、镰刀菌、病毒到类菌原体,最后确定为细菌。该病原属于α-亚纲,韧皮部杆菌属,分为亚洲种、非洲种和美洲种3个种,可以通过PCR技术进行检测鉴定。同时,对韧皮杆菌的人工培养以及与黄龙病相关植原体的最新研究进展进行了阐述。     

LncRNA在柑橘木虱与黄龙病病原菌互作中的差异表达分析及验证

为明确柑橘黄龙病的唯一自然传播媒介——柑橘木虱Diaphorina citri的长链非编码RNA(long non-coding RNA,lncRNA)是否参与调控黄龙病病原菌Candidatus Liberibacter asiaticus(CLas)的侵染及复制,采用生物信息学预测及PCR扩增方法进行lncRNA的预测、特征分析、验证及差异表达分析。结果显示,柑橘木虱的13个转录组RNA-Seq数据中共有10 192个lncRNA基因,对应15 747条lncRNA转录本;与蛋白质编码基因相比,柑橘木虱lncRNA具有更少的外显子数量和更短的转录本长度;随机选取的10条lncRNA基因中,有7条lncRNA基因在无菌柑橘木虱广州品系或赣州品系中有表达,其中1条lncRNA基因TCONS_00034665在无菌广州品系和无菌赣州品系中存在差异表达;带菌和无菌柑橘木虱成虫中预测获得2个差异表达的lncRNA基因TCONS_00096118和TCONS_00234564,实时荧光定量PCR验证发现TCONS_00234564与预测结果一致,在带菌柑橘木虱成虫中高表达。表明lncRNA参与了黄龙病病原菌与寄主柑橘木虱的互作。     

Predation capacity of phytoseiid mites (Mesostigmata: Phytoseiidae) from Brazil on eggs of Diaphorina citri (Hemiptera: Liviidae)

Phytoparasitica - The Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae) is a key vector of the causal agents of huanglongbing (HLB) in important citrus production regions of the...     

Effect of Candidatus Liberibacter asiaticus infection on susceptibility of Asian citrus psyllid, Diaphorina citri, to selected insecticides

BACKGROUND: In the present investigation, the effect of Candidatus Liberibacter asiaticus (Las), a bacterium considered to be responsible for causing huanglongbing (HLB) disease in citrus, on the physiology of its vector, the Asian citrus psyllid (ACP) Diaphorina citri Kuwayama, was determined. Specifically, the effects of Las infection on the susceptibility of ACP to selected insecticides were determined. Furthermore, total protein content and general esterase activity were quantified in Las‐infected and uninfected ACP to gain insight into the possible mechanism(s) responsible for altered susceptibility to insecticides owing to Las infection. RESULTS: LC₅₀ values were significantly lower in Las‐infected than in uninfected ACP adults for chlorpyrifos and spinetoram. Furthermore, there was a general trend towards lower LC₅₀ values for three other insecticides for Las‐infected ACP; however, the differences were not statistically significant. Total protein content (µg mL^?1) was significantly lower in Las‐infected (23.5 ± 1.3 in head + thorax; 27.7 ± 1.9 in abdomen) than in uninfected (29.7 ± 2.1 in head + thorax; 35.0 ± 2.3 in abdomen) ACP. Likewise, general esterase enzyme activity (nmol min^?1 mg^?1 protein) was significantly lower in Las‐infected (111.6 ± 4.5 in head + thorax; 109.5 ± 3.7 in abdomen) than in uninfected (135.9 ± 7.5 in head + thorax; 206.1 ± 23.7 in abdomen) ACP. CONCLUSION: Susceptibility of ACP to selected insecticides from five major chemistries was greater in Las‐infected than in uninfected ACP. The lower total protein content and reduced general esterase activity in Las‐infected than in uninfected ACP may partly explain the observed higher insecticide susceptibility of Las‐infected ACP. Copyright © 2010 Society of Chemical Industry     

The push–pull strategy for citrus psyllid control

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is the only natural vector of Candidatus Liberibacter asiaticus that causes citrus huanglongbing (HLB), a most destructive disease of citrus. Currently, no remedial therapy exists for the disease, and so effective control of ACP is very important in curbing the transmission of the disease. The push–pull strategy should be thoroughly explored as an approach to ACP management. This mini‐review summarises the current progress towards more effective repellent and attractant chemicals through investigating known repellent and attractive plants. Interactions between ACP and its host plants are also addressed, with emphasis on the possible involvement of the host biochemicals in attracting the insect. Potential ways to increase the effectiveness of the pull–push strategy are briefly discussed. It is expected that the pull–push strategy will be gradually developed following more extensive research. © 2014 Society of Chemical Industry     

Quantification of viable <Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter asiaticus in hosts using quantitative PCR with the aid of ethidium monoazide (EMA)

Citrus Huanglongbing (HLB) is a devastating disease of citrus known to be associated with a fastidious, phloem-limited Gram-negative, yet to be cultured bacterium in the genus Candidatus Liberibacter. In the present study we have developed a method to quantify viable Candidatus Liberibacter asiaticus (Las) with the aid of ethidium monoazide (EMA) which can differentiate live from dead cells. First, calibration curves were developed with the aid of quantitative real-time PCR (QPCR) by using a plasmid template consisting of a 703 bp DNA fragment of rplKAJL-rpoBC (β-operon) region. Standard equations were then developed to quantify Las genome equivalents in citrus, periwinkle, and Asian citrus psyllid, Diaphorina citri. To overcome the limitation of quantitative PCR in discriminating between live and dead bacterial cells, EMA was used to inhibit the amplification of DNA from the dead cells of Las in plant samples. By using the standard equations and EMA-QPCR methods developed in this study, we found that the proportion of viable cells in citrus and periwinkle ranged from 17–31% and 16–28%, respectively. It was determined that a minimum bacterial concentration is required for HLB symptom development by quantifying the population of Las in symptomatic and asymptomatic leaves. The EMA-QPCR methodology developed in the present study should provide an accurate assessment of viable HLB pathogen, providing a tool to investigate disease epidemiology and thus act as a crucial component for disease assessment and management. The authors P. Trivedi and U. S. Sagaram contributed equally to this work.     

13种常用农药对柑橘木虱田间种群防治效果室内评价

柑橘木虱Diaphorina citri Kuwayama是黄龙病的传播媒介, 快速、有效防治这种害虫是综合防控黄龙病的关键。目前, 其防治措施主要依赖化学农药。为了了解化学农药对柑橘木虱的田间防治效果, 本文以广东省博罗县柑橘木虱田间种群为测试对象, 以华南农业大学昆虫生态研究室饲养的种群为敏感对照, 室内测定了13种常用药剂推荐浓度对柑橘木虱的防治效果。结果表明, 24 h后77.5%敌敌畏EC和5%啶虫脒EC对田间种群的效果最好, 柑橘木虱死亡率分别为93.3%和90%, 48 h后两种药剂处理的死亡率已达100%; 48 h后, 350 g/L吡虫啉SC处理的死亡率也达93.3%; 72 h后, 20%丁硫克百威EC、30%噻虫嗪SC、12%高氯·毒死蜱EC、45%毒死蜱EC、2.5%高效氯氟氰菊酯EW处理的死亡率也达90%以上。两个种群相比, 所有药剂在相同时间对敏感种群的防治效果都高于田间种群。其中, 用20%甲氰菊酯EC、30%噻虫嗪SC、45%毒死蜱EC、25 g/L联苯菊酯EC、99%矿物油EC等5种药剂处理, 24 h后田间种群的校正死亡率显著低于敏感种群; 48 h后除上述药剂外, 4.5%高效氯氰菊酯ME处理的校正死亡率也显著低于敏感种群。72 h后, 4.5%高效氯氰菊酯ME、25 g/L联苯菊酯EC、99%矿物油EC处理的校正死亡率依然显著低于敏感种群。说明田间种群对以上药剂的敏感性有不同程度的下降。此外发现, 采用此测定方法22.4%螺虫乙酯SC对田间种群和敏感种群的防治效果均不理想。     

柑桔木虱(Diaphorina citri Kuwayama)与柑桔黄龙病流行关系的初步研究

 1973-1978年用从田间病树上采集的柑桔木虱成虫放饲在398株健康柑桔苗上,有32株发病。用病树上采集的柑桔木虱高龄若虫置健苗上所羽化的成虫放饲的56株,有5株发病。未接虫的110株没有发病。初步说明柑桔木虱成虫可以传病。     

应用Nested PCR技术检测柑桔木虱及其寄主九里香的柑桔黄龙病带菌率

应用PCR及Nested PCR技术检测柑桔木虱及其寄主九里香的结果表明:PCR只可检测最低2头带菌木虱,Nested PCR可检测到单个带菌木虱。100头带菌木虱中,单虫检出率为96％。检测田间重、中等、轻病的柑桔园内的木虱,其带菌率依次为87％、53％和21％。在病芦柑上饲菌不同天数的木虱均能检测出带菌,其饲菌时间最短为1d。城市九里香叶片及在其叶上取食的木虱单虫,均能用Nested PCR检测出病原。饲菌木虱接种九里香及芦柑健苗,在植株尚未表现症状时,常规PCR难检测出病原,但用Nested PCR则能检测到病原,说明九里香不仅是木虱的寄主,而且是黄龙病病原的隐症寄主。     

Antifeedant and sublethal effects of imidacloprid on Asian citrus psyllid,Diaphorina citri

BACKGROUND: Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, transmits the causal bacteria of the devastating citrus disease huanglongbing (HLB). Because of the variation in spatial and temporal uptake and systemic distribution of imidacloprid applied to citrus trees and its degradation over time in citrus trees, ACP adults and nymphs are exposed to concentrations that may not cause immediate mortality but rather sublethal effects. The objective of this laboratory study was to determine the effects of sublethal concentrations of imidacloprid on ACP life stages. RESULTS: Feeding by ACP adults and nymphs on plants treated daily with a sublethal concentration (0.1 µg mL^?1) of imidacloprid significantly decreased adult longevity (8 days), fecundity (33%) and fertility (6%), as well as nymph survival (12%) and developmental rate compared with untreated controls. The magnitude of these negative effects was directly related to exposure duration and concentration. Furthermore, ACP adults that fed on citrus leaves treated systemically with lethal and sublethal concentrations of imidacloprid excreted significantly less honeydew (7–94%) compared with controls in a concentration‐dependent manner suggesting antifeedant activity of imidacloprid. CONCLUSIONS: Sublethal concentrations of imidacloprid negatively affect development, reproduction, survival and longevity of ACP, which likely contributes to population reductions over time. Also, reduced feeding by ACP adults on plants treated with sublethal concentrations of imidacloprid may potentially decrease the capacity of ACP to successfully acquire and transmit the HLB causal pathogen. Copyright © 2009 Society of Chemical Industry     

Effects of pymetrozine, an antifeedant of Hemiptera, on Asian citrus psyllid, Diaphorina citri, feeding behavior, survival and transmission of Candidatus Liberibacter asiaticus

BACKGROUND: Vector‐borne plant disease management can be enhanced by deployment of antifeedants in addition to the use of broad‐spectrum neurotoxic insecticides. The effects of pymetrozine on Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, feeding behaviour, survival and transmission of Candidatus Liberibacter asiaticus (Las), the presumed causal pathogen of huanglongbing, were investigated. RESULTS: Pymetrozine applied at 52 and 104 µg mL^?1 to citrus plants [Swingle citrumelo (X Citroncirus webberi Ingram and Moore)] modified the feeding behavior of ACP and increased the amount of time spent performing non‐penetration behaviors while decreasing the time spent performing ingestion behaviors compared with the controls 1 day after treatment. However, the antifeedant effect of pymetrozine subsided 5 days after application. Pymetrozine reduced the survival of both adults and nymphs on treated plants compared with the control. However, it had a greater impact on survival of nymphs than on survival of adults. Pymetrozine applied at 52 and 104 µg mL^?1 on Las‐infected ‘Valencia’ sweet orange plants [Citrus sinensis L. (Osbeck)] reduced acquisition (12 and 21% respectively) and transmission (11 and 18% respectively) of Las by feeding ACP adults compared with the controls; however, these reductions were not statistically significant. CONCLUSIONS: Pymetrozine exhibited moderate antifeedant effects by modifying the feeding behavior of ACP adults with short residual activity. The impact of pymetrozine on survival of nymphs was greater than on adults at the higher concentrations tested. Pymetrozine also reduced the acquisition and transmission of Las by feeding ACP adults up to 21 and 18%, respectively, compared with untreated controls. Copyright © 2010 Society of Chemical Industry     

湖南省永州地区柑橘慢衰病与黄龙病病原鉴定及分布调查

为明确湖南省柑橘黄化症状与柑橘慢衰病和柑橘黄龙病的相关性,应用形态学和分子生物学方法对湖南省永州地区表现黄化症状的柑橘进行了两种病原的鉴定及分布调查。结果表明,造成该地区柑橘黄化现象的主要原因为柑橘半穿刺线虫和黄龙病菌对柑橘的侵染。所检样本中,柑橘慢衰病平均发生率为82.1%,土壤中的半穿刺线虫群体密度最高达到3 077条/100mL。永州地区柑橘黄龙病病菌属于韧皮部杆菌属类细菌亚洲种,平均检出率为64.3%。柑橘半穿刺线虫和黄龙病菌在柑橘园存在混合侵染现象,混合侵染率为53.6%。柑橘半穿刺线虫在永州地区柑橘产区分布广泛,是造成柑橘黄化症状的重要病因,重视并有效防控柑橘半穿刺线虫已迫在眉睫。     

柑桔木虱传递柑桔黄龙病的初步研究

柑桔木虱是柑桔黄龙病主要媒介昆虫。几年来的试验初步表明:无病植株上繁殖木虱成虫,在病树上吸毒20—30天以上就可传病。黄龙病病原在成虫体内约经20—30天循回期、带毒后木虱成虫,虫口密度大(20头/苗以上),吸食传毒1天即可发病;虫口密度小(1—5头/苗),吸食传毒7—14天也能传病。潜伏期一般2—8个月,个别株2年才发病,病梢于冬季回接,3—5个月后也表现出和虫传发病苗相同的斑驳状病症。     

中国不同地区亚洲韧皮杆菌遗传多样性分析

 柑桔黄龙病是最严重的柑桔病害之一,现已威胁世界柑桔产业。已被证实在中国南部该病害通过嫁接和木虱传播。因此黄龙病的流行病学研究就显得尤为重要。本研究中,基于外膜蛋白(omp)基因的PCR-RFLP的方法被用来研究中国南部各柑桔主产区的亚洲韧皮部杆菌的遗传多样性。研究采用不同的限制性内切酶消化各地区的亚洲韧皮部杆菌的omp基因产生了不同的RFLP指纹图谱,并对各地亚洲韧皮部杆菌的omp基因进行克隆测序然后构建系统发育树。结果显示:中国不同地理区域的亚洲韧皮部杆菌具有一定的遗传多样性,即便在某一特定的地区也有一定的差异。序列分析显示中国地区的亚洲韧皮部杆菌有很高的同源性。这些结果对揭示中国柑桔黄龙病的流行病学及制定科学有效的防治策略具有一定的指导意义。     

Effect of pyriproxyfen,a juvenile hormone mimic,on egg hatch,nymph development,adult emergence and reproduction of the Asian citrus psyllid,Diaphorina citri Kuwayama

BACKGROUND: The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is a vector of bacteria presumably responsible for huanglongbing (HLB) disease in citrus. In this laboratory study, an investigation was made of the activity of pyriproxyfen, a juvenile hormone mimic, on ACP eggs, nymphs and adults to evaluate its potential as a biorational insecticide for inclusion in an integrated pest management (IPM) program for ACP. RESULTS: Irrespective of egg age, timing or method of treatment, a significantly lower percentage of eggs (5–29%) hatched after exposure to 64 and 128 µg mL^?1 of pyriproxyfen. Only 0–36% of early instars (first, second and third) and 25–74% of late instars (fourth and fifth) survived to adults following exposure to 16, 32 and 64 µg mL^?1 of pyriproxyfen. However, 15–20% of adults that emerged following treatment as late instars exhibited morphological abnormalities. Furthermore, pyriproxyfen adversely affected reproduction (fecundity and fertility) of adults that emerged from treated fifth instars or that were treated topically with 0.04 µg as adults. CONCLUSIONS: Application of pyriproxyfen at 64 µg mL^?1 resulted in greater inhibition of egg hatch and suppression of adult emergence compared with lower rates. Pyriproxyfen also markedly reduced female fecundity and egg viability for adults that were exposed either as fifth instars or as newly emerged adults. The ovicidal, larvicidal and reproductive effects against ACP suggest that pyriproxyfen is suitable for integration into an IPM program for ACP. Copyright © 2009 Society of Chemical Industry     

Rapid and quantitative detection of citrus huanglongbing bacterium ‘Candidatus Liberibacter asiaticus’ by real-time fluorescent loop-mediated isothermal amplification assay in China

Huanglongbing (HLB) is a devastating citrus disease that is associated with bacteria of the species ‘Candidatus Liberibacter asiaticus (Las)’ in China. All currently popular cultivars are susceptible to HLB, and there are no effective approaches to control the disease once the plants have been infected. Therefore, rapid and easy diagnostic tools are needed for maintaining clean nursery stock and inoculum removal strategies to control HLB. A real-time fluorescence loop-mediated isothermal amplification (RealAmp) targeting16S rDNA was developed for the rapid and quantitative detection of Las in China. The detection sensitivity of the RealAmp assay was approximately 1 pg/μl template DNA (equal to 2.4 × 10⁵ target DNA copies/μl) and no cross-reaction was observed with other pathogens or virus-free seedlings. Besides the quantitative detection using SYTO-9 fluorescent dye, an improved closed-tube visual inspection technique using SYBR Green I staining was developed for rapid screening of samples and minimize the risk of cross-contamination. The RealAmp assay is an alternative quantitative method, which would be used as a routine detection service for nursery materials in China.     

Development of a loop-mediated isothermal amplification procedure as a sensitive and rapid method for detection of 'candidatus Liberibacter solanacearum' in potatoes and Psyllids

This study reports the development of a loop-mediated isothermal amplification procedure (LAMP) for polymerase chain reaction (PCR)-based detection of 'Candidatus Liberibacter solanacearum', the bacterial causal agent of potato zebra chip (ZC) disease. The 16S rDNA gene of 'Ca. Liberibacter solanacearum' was used to design a set of six primers for LAMP PCR detection of the bacterial pathogen in potato plants and the psyllid vector. The advantage of the LAMP method is that it does not require a thermocycler for amplification or agarose gel electrophoresis for resolution. Positive LAMP results can be visualized directly as a precipitate. The LAMP strategy reported here reliably detected 'Ca. Liberibacter solanacearum' and the closely related species 'Ca. Liberibacter asiaticus', the causative agent of huanglongbing disease of citrus, in plant DNA extracts. Although not as sensitive as quantitative real-time PCR, LAMP detection was equivalent to conventional PCR in tests of ZC-infected potato plants from the field. Thus, the LAMP method shows strong promise as a reliable, rapid, and cost-effective method of detecting 'Ca. Liberibacter' pathogens in psyllids and field-grown potato plants and tubers.