Effective molecular detection of Diaphorina citri Kuwayama (Hemiptera: Liviidae) in bulk insect samples from sticky traps

Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is the principal vector of citrus greening (huanglongbing) disease. Invasion of new areas by the vector increases the risk of further spread of the disease and has economic impacts on the global citrus industry. Effective implementation of vector surveys is essential to contain disease outbreaks. This is especially true in countries such as Japan, where most of the major citrus‐producing areas are free from citrus greening. Recently, vector surveys have been routinely conducted to maintain ‘disease‐free’ and ‘disease‐ and vector‐free’ areas in Japan, and improvement of methods that can detect D. citri in native insect populations is imperative. Here, we developed a method of using conventional and real‐time PCR to detect D. citri among bulk insects captured in sticky traps without the need for preliminary differentiation steps based on morphology. DNA fragments of D. citri were specifically detected by both conventional and real‐time PCR in a mixture of a 10^?3 dilution (ca. 0.008–0.009 ng/μl) of D. citriDNA and 100 ng/μl of bulk insect DNA, indicating that small body parts such as pieces of leg or parts of wings of D. citri were detectable in the bulk insect samples. No misleading amplification of fragments from the other psyllid species and citrus pests we used occurred under our PCR conditions. Our results suggest that the technique is applicable to extensive surveys of D. citri in early warning programmes.     

柑桔木虱及其防治

柑砧术虱是柑桔新梢期重要害虫,刺吸为害。福建福州在柑桔上一年发生8代,在月桔、九里香上一年约发生10代左右,世代重叠,以成虫在柑桔叶背群集越冬,无完全滞育。4—5月气温22．3℃世代历期42．5天．6-7月27．2℃为24．4天．8月28．1℃为23．2天。10-12月19.6℃为52．9天。成虫寿命长,约历1个月半,越冬代长达半年。田间种群消长与柑桔芽梢抽发期相对应．一年中虫口数量出现3个高峰期,第一峰期3-4月为柑桔春梢主要抽发期,第二峰期5-6月为夏梢主要抽发期,第三峰期8～9月为秋梢主要抽发期,其中以秋梢期虫口数量最大．秋芽受害最重,次为春梢期。本还对柑桔术虱天敌进行调查,并对柑桔术虱卵、若虫和成虫进行药剂防治试验。     

Long PCR Is a Sensitive Method for Detecting Liberobacter asiaticum in Parasitoids Undergoing Risk Assessment in Quarantine

The accidental importation of plant pathogens in or on the bodies of parasitoids imported as natural enemies has been raised as a potential risk of classical biological control projects involving insects that serve as vectors of plant diseases. During quarantine evaluation of two parasitoids, Tamarixia radiata Waterston and Diaphorencyrtus aligarhensis (Shafee, Alam and Agarwal), imported for classical biological control of Asian citrus psylla Diaphorina citri (Kuwayama) in Florida, we were asked to determine whether these parasitoids were free of the causal agent of Asian greening disease, the bacterium Liberobacter asiaticum. Preliminary tests with allele-specific polymerase chain reaction (Standard PCR) suggested that the assays were prone to false negatives. Another PCR protocol, Long PCR, previously was shown to be more reliable than Standard PCR for screening of insects for another bacterium (Wolbachia). The sensitivity of Long and Standard PCR protocols was compared by use of plasmid DNA containing two DNA fragments from the greening disease agent or plasmid mixed with DNA extracted from host plants, psyllids, or parasitoids. Results indicated that inhibitors of the PCR were present in both plant and insect DNA, making the Standard PCR relatively insensitive and allowing high levels of false negatives. Long PCR, which incorporates a second DNA polymerase with proof-reading activity, yielded consistent results and was orders of magnitude more sensitive than the Standard PCR. As few as 100 copies of plasmid mixed with either plant or insect DNA consistently could be detected. Long PCR assays conducted on pooled and individual T. radiata and D. aligarhensis, their psyllid hosts, or their host plants over a period of 6 months failed to produce any positives, indicating that release of these two parasitoids should elicit little concern that greening bacteria would be introduced accidentally into Florida through this classical biological control program.     

Automated Needle‐free Injection Method for Delivery of Bacterial Suspensions into Citrus Leaf Tissues

A prototype needle‐free device was evaluated for delivery of Xanthomonas citri subsp. citri bacteria into the leaves of cultivars susceptible and resistant to citrus canker. The device delivered a precisely controlled volume of bacterial suspension through infiltration of stomata by injection with pressurized gas. The device produced a uniform inoculation of bacteria into the leaves as measured by the volume of infiltration and diameter of the infiltrated area. No damage to the leaves was observed after inoculation with the automated device, even though a higher number of canker lesions developed compared to a hand‐held needleless syringe injection method. The level of practice needed for operation of the automated device was minimal compared to considerable skill required to perform the hand‐held injection. Results from inoculations with the automated device are in accord with the results with the hand‐held syringe method that demonstrated kumquats are highly resistant to citrus canker while rough lemon and ‘Hamlin’ sweet orange are susceptible.     

桔全爪螨与柑桔相互作用的研究

研究了不同柑桔品种对桔全爪螨Ｐａｎｏｎｙｃｈｕｓｃｉｔｒｉ（ＭｃＧ）种群的影响,桔全爪螨危害对柑桔生理的影响以及柑桔叶片受受后对桔全爪螨种群的反馈作用,结果表明：供试的１０个柑桔品种对桔全爪螨种群生长发育,繁殖时明显的影响,品种间存在感螨性差异。     

释放巴氏钝绥螨对沙田柚上橘全爪螨的防治效果

2007～2008年开展释放巴氏钝绥螨Amblyseius barkeri(Hughes)防治沙田柚上橘全爪螨Panonychus citri McGregor试验示范。结果表明:巴氏钝绥螨能在江西省吉安市沙田柚果园大量繁殖,每株成年沙田柚树上释放3～4盒巴氏钝绥螨,每盒含巴氏钝绥螨600头以上,可有效控制橘全爪螨的危害,减少施药次数和农药用量,节约防治成本,改善橘园生态环境和果品品质。     

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。     

园艺矿物油乳剂对亚洲柑橘木虱成虫刺吸行为的影响

目的】亚洲柑橘木虱Diaphorina citri是传播柑橘最重要病害黄龙病(huanglongbing, Candidatus Liberibacter asiaticus)的媒介昆虫,树冠喷施园艺矿物油(horticultural mineral oil, HMO)可以减少其在柑橘上的取食和产卵。本研究旨在探索植物组织内残留的园艺矿物油如何影响亚洲柑橘木虱成虫的取食行为。【方法】采用直流型刺吸电位仪(DC-EPG Giga-8)记录亚洲柑橘木虱成虫12 h内在喷施不同浓度(0.25%, 0.5%, 1%和2%, v/v)HMO(nC24)乳剂和清水(对照)时柑橘嫩叶上的刺探和取食行为并转换为波形信号,分析比较各处理的波形参数。【结果】不同浓度HMO乳剂处理显著提高了亚洲柑橘木虱成虫在柑橘叶片上的C波(路径波)持续时间百分比,显著减少了E2波(韧皮部吸食汁液)持续时间百分比,而对D(口针第一次接触韧皮部组织), E1(韧皮部出现唾液分泌) 和G(木质部吸食汁液) 波持续时间百分比没有显著影响。HMO处理组C波的每成虫波形累积时间(WDI)显著长于对照组,而各浓度处理之间没有显著差异,但D波WDI明显短于对照组。随着HMO浓度的增加, E1, E2和G波WDI显著下降,2% HMO处理组E1, E2和G波WDI明显低于其他浓度处理组。HMO乳剂处理明显减少了刺探次数;在HMO处理中,每成虫刺探次数和每成虫各波形次数均显著少于对照组,其中以1%和2% HMO乳剂处理最少。HMO乳剂处理组np波 (非刺探波)、D波、E2波的每成虫单次波形持续时间(WDE)均短于对照组,其中2% HMO乳剂处理后,D, E1和E2波WDE显著短于其他浓度处理和对照。同时,2% HMO乳剂处理推迟了从口器接触叶片到第1次开始刺探的时间和从口器接触叶片到第1次刺探到韧皮部的时间。【结论】结果表明,2% HMO乳剂显著减少亚洲柑橘木虱成虫在柑橘叶片上的取食次数和有效取食时间,同时还缩短了其在韧皮部分泌唾液和吸食汁液时间,因此可推荐矿物油用于柑橘黄龙病和亚洲柑橘木虱的综合防治体系。同时初步推断矿物油的作用机制可能是其进入植物气孔阻止植物挥发性物质的释放,抑制或掩盖柑橘叶片表面的挥发性物质从而减少了亚洲柑橘木虱在寄主上取食。     

Ecological effects of multi‐species,ant–hemipteran mutualisms in citrus

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