柑橘—黄龙病病菌-Wolbachia-柑橘木虱互作关系的研究

发布时间：2018-07-24 11:02

【摘要】：柑橘木虱Diaphorina citri Kuwayama是取食植物韧皮部汁液的刺吸式口器昆虫。柑橘木虱是柑橘黄龙病病菌Candidatus Liberibacter asiaticus唯一的自然传播媒介。柑橘黄龙病是柑橘毁灭性病害之一,给世界柑橘产业造成了巨大的损失。本文以柑橘木虱为研究对象,利用常规PCR、实时荧光定量PCR(q RT-PCR)、荧光原位杂交(FISH)等技术,从分子生态学的角度来研究柑橘-黄龙病病菌-Wolbachia-柑橘木虱互作关系。本研究通过寄主植物生理状态对柑橘木虱生物学特性的影响,系统研究了健康与感病砂糖橘对柑橘木虱适合度的影响;从氨基酸利用的角度分析了黄龙病病菌影响柑橘木虱适合度的营养机制;基于昆虫内共生菌Wolbachia和黄龙病病菌感染与位置分布的相关性,分析了Wolbachia影响柑橘木虱传播黄龙病病菌的分子机制,从而为利用昆虫内共生菌研究柑橘木虱和黄龙病的防控措施提供理论基础。研究结果如下:1.柑橘木虱的形态特征通过对柑橘木虱卵和各龄期若虫形态特征的观察,归纳了各相邻龄期之间的形态差异。柑橘木虱各龄期若虫长宽差距较大:二龄比一龄长0.145 mm,宽0.166 mm;三龄与二龄间差距分别为0.193 mm和0.182 mm;四龄与三龄差距分别为0.351 mm和0.279 mm;五龄与四龄间差距分别为0.572 mm和0.358 mm。在田间一般可先根据虫体大小对各龄期进行初步判定,不确定龄期的再在显微镜下进一步的确认。2.寄主植物对柑橘木虱生活史的影响柑橘木虱卵、一龄若虫以及整个若虫的发育历期受寄主植物的影响较为明显。在柚子寄主植物上若虫存活率最高,在黄皮上最低,且两者差异显著。寄主植物影响柑橘木虱成虫寿命,在柚子上的寿命显著长于黄皮上的寿命。黄皮上的木虱单雌产卵量显著低于其他4种寄主。柑橘木虱在九里香上的内禀增长率最高、酸橘上最低;而净增值率在砂糖橘上最高、黄皮上最低。结果表明在本实验的5中寄主植物中,除黄龙病隐症寄主九里香之外,显症寄主中砂糖橘是柑橘木虱的最适寄主。3.黄龙病病菌对柑橘木虱适合度的影响实验发现,带菌柑橘木虱或者在感病砂糖橘上取食的柑橘木虱世代发育历期均缩短,如卵、若虫以及卵到成虫的整个发育历期,均显著小于健康柑橘木虱在健康砂糖橘上的发育历期。但对于柑橘木虱的存活率来讲其变化趋势正好和发育历期相反,带菌柑橘木虱在感病砂糖橘上的存活率最低,健康柑橘木虱在健康砂糖橘上最高,健康砂糖橘上柑橘木虱的存活率显著高于感病砂糖橘上的存活率。感病砂糖橘上柑橘木虱F1代的单雌平均产卵量显著高于健康砂糖橘。本研究结果表明黄龙病病菌有利于柑橘木虱在砂糖橘寄主上的种群增长。4.黄龙病病菌对寄主植物及柑橘木虱氨基酸组分的影响当砂糖橘感染黄龙病病菌后,韧皮部汁液中各游离氨基酸的组分以及比例均发生了改变。表明黄龙病病菌对柑橘木虱的有利影响可能与砂糖橘寄主韧皮部汁液营养成分改变有关。柑橘木虱取食感病砂糖橘时分泌的蜜露中游离氨基酸天冬酰胺和脯氨酸下降明显,同时这两种氨基酸在感病砂糖橘韧皮部汁液中又明显增加,说明黄龙病病菌增加了柑橘木虱对天冬酰胺和脯氨酸的吸收,这可能利于柑橘木虱若虫发育。同时黄龙病病菌增加了柑橘木虱体内必需氨基酸的含量,必需氨基酸的增加可能有利于柑橘木虱的生长发育。带菌柑橘木虱若虫和健康柑橘木虱若虫在九里香上分泌的蜜露中游离氨基酸的组分差异很小,表明黄龙病病菌对柑橘木虱若虫氨基酸营养吸收的影响不大。5.次生共生菌Wolbachia在柑橘木虱体内分布及感染动态广州柑橘木虱种群体内7种共生菌的感染率存在差异,Carsonella、Profftella和Wolbachia感染率均是100%;Arsenophonus感染率为51.7%;Rickettsia、Cardinium和Hamiltonella三种共生菌的感染率很低。Wolbachia在柑橘木虱卵、各龄期若虫及雌雄成虫体内的感染率存在差异。Wolbachia的含量随着木虱龄期的增加而增多,且雄虫体内Wolbachia的含量高于雌虫。Wolbachia在卵中的分布随着卵的发育而不断变化,从卵柄到卵中部再到远离卵柄一端,最后在老熟卵体内呈现散布型分布。Wolbachia在若虫体内主要集中在含菌体内,而在雌雄成虫体内是局限在虫体腹部。6.黄龙病病菌在柑橘木虱体内分布及感染动态初羽化的柑橘木虱成虫接到感病柑橘树上,分别在15d、30d、45d以及60d收集不同取食时间的柑橘木虱进行检测。结果表明柑橘木虱体内黄龙病病菌含量在取食15d时,黄龙病病菌含量达到最高,此后,随着取食时间的进一步延长而减少。而Wolbachia的含量变化趋势则是随着取食时间的延长而增加。利用常规PCR可以在3-5龄若虫以及成虫体内检测到黄龙病病菌,而q RT-PCR除了3-5龄若虫外,还可以在二龄若虫体内检测到黄龙病病菌,但仍然不能在卵和一龄若虫体内检测到。卵和各龄期若虫中黄龙病病菌的含量是随着龄期的变大而不断增多。对于柑橘木虱的成虫,在产卵盛期的木虱体内黄龙病病菌的含量最高,显著高于产卵前期以及产卵后期。荧光原位杂交技术在柑橘木虱卵、1-3龄若虫体内不能检测到黄龙病病菌,而在四、五龄若虫体内可以检测到黄龙病病菌,病菌主要分布在若虫的U型含菌体内,个别虫体在若虫胸部也有分布。黄龙病病菌在柑橘木虱若虫中的分布位置与Wolbachia在若虫的位置相似,表明了在四、五龄若虫体内黄龙病病菌和Wolbachia可在含菌体内共生。黄龙病病菌在雌雄成虫体内均是散布型分布,且这种分布形式利于黄龙病病菌的水平传播。概括而言,通过砂糖橘-黄龙病病菌-柑橘木虱互作关系研究发现,黄龙病病菌有利于柑橘木虱种群的增长;黄龙病病菌的感染增加了柑橘木虱对氨基酸的吸收与利用;另外,柑橘木虱体内Wolbachia的感染动态与黄龙病病菌的感染动态均是随着龄期的变大而增加,且两者在柑橘木虱若虫体内均分布于含菌体内,二者共生关系可能与柑橘木虱若虫阶段获菌率高有关,推测Wolbachia参与砂糖橘-黄龙病-柑橘木虱三者间的互作来影响柑橘木虱的获菌率,具体分子机制尚不明确,需要进一步研究。
[Abstract]:Citrus Diaphorina citri Kuwayama is a spiny mouthed insect that takes the juice of the phloem of plants. Citrus lice is the only natural medium for Candidatus Liberibacter asiaticus of Citrus Yellow Dragon's disease pathogen. Citrus Yellow Dragon disease is one of the devastating diseases of citrus, which has caused great loss to the world citrus industry. By using conventional PCR, real-time fluorescence quantitative PCR (Q RT-PCR), fluorescence in situ hybridization (FISH) and other techniques, the interaction relationship between Citrus Yellow Dragon disease pathogen -Wolbachia- citrus plant lice was studied from the angle of molecular ecology. The study on the effects of the physiological state of host plant on the biological characteristics of citrus plant lice was studied. The effect of the disease on the fitness of citrus citrus and the effects of the amino acid utilization on the nutrition mechanism of the citrus lice were analyzed from the angle of amino acid utilization. Based on the correlation between the infection of insect endonicine Wolbachia and the infection of Yellow Dragon's disease and the location distribution, the molecular machine for the effect of Wolbachia to spread the Yellow Dragon disease pathogen of citrus fruit louse was analyzed. In order to provide a theoretical basis for the study of the control measures for citrus and yellow dragon disease by endophytic bacteria, the results are as follows: 1. the morphological characteristics of the citrus lice were observed through the observation of the morphological characteristics of the egg and the age nymphs of the citrus, and the difference between the adjacent ages was summed up. The length and width gap of the nymphs of the citrus plant lice at all ages The two years of age were 0.145 mm longer than the first age, 0.166 mm in width, 0.193 mm and 0.182 mm for two years, respectively, and 0.351 mm and 0.279 mm, respectively. The gap between four and three years was 0.351 mm and 0.279 mm respectively. The effects of.2. host plants on the life history of citrus plant lice were further confirmed by microscope. The growth of the first instar nymphs and the whole nymphs were significantly affected by the host plants. The highest survival rate of the nymphs on the pomelo host plants was the lowest on the yellow skin, and the difference between the two was significant. The host plant affected the adult life of citrus citrus. Life expectancy on pomelo was significantly longer than that on yellow skin. The number of egg laying of single female on yellow skin was significantly lower than that of the other 4 hosts. The intrinsic increase rate of citrus citrus on the citrus was the highest and the lowest in the acid orange. The net increment rate was the highest on the tangerine and the lowest in the yellow peel. The result indicated that in the 5 host plant of this experiment, the Yellow Dragon disease was found. The effect of sugar orange on the host of the symptomatic host is the best host of the citrus plant lice, the most suitable host of the citrus.3., the effect of the pathogen on the citrus lice. However, the survival rate of citrus citrus on Citrus sugar orange was the lowest, the healthy citrus fruit was the highest in healthy citrus, and the survival rate of citrus citrus on healthy citrus was significant. The average oviposition of the F1 generation of citrus citrus on the sugar orange was significantly higher than that of the healthy sugar orange. The results showed that the pathogen of Yellow Dragon disease was beneficial to the population growth of Citrus louse in the sugar orange host. The effect of.4. Yellow Dragon disease on the amino acid components of the host plant and citrus citrus The composition and proportion of free amino acids in phloem juice were changed after orange infection with yellow dragon disease. The results showed that the beneficial effect of Yellow Dragon disease on citrus fruit juice may be related to the change of nutrient content in the phloem juice of citrus fruit. The decrease of amines and proline was obvious, and the two kinds of amino acids increased obviously in the phloem juice of the citrus sugar orange. It indicated that the pathogen of Yellow Dragon's disease increased the absorption of citrus to asparagine and proline, which may be beneficial to the development of the nymph of citrus melice. The increase of amino acids may be beneficial to the growth and development of citrus plant lice. The differences in free amino acids of free amino acids in the nymphs of citrus citrus and healthy citrus lice on the nectar of the nymphs are small, indicating that the effect of the Yellow Dragon disease pathogen on the amino acid nutrient absorption of the nymphs of citrus citrus is not.5. Wolbachia in Citrus The infection rates of 7 species of symbiotic bacteria in the species of Guangzhou citrus plant species were different, the infection rates of Carsonella, Profftella and Wolbachia were 100%, the infection rate of Arsenophonus was 51.7%, and the infection rates of Rickettsia, Cardinium and Hamiltonella were low in.Wolbachia in the citrus lice eggs, all the nymphs of each age, and The infection rate of male and female adults increased with the increase of the age of the lice, and the content of Wolbachia in the male was higher than that of the female.Wolbachia in the egg, which changed with the egg development, from the egg handle to the middle of the egg and then to the end of the egg crank. Finally, the distributed distribution of.Wo was presented in the old mature eggs. Lbachia is mainly concentrated in the body of the nymphs, while in the male and female adults, the distribution of.6. Yellow Dragon disease pathogen in the abdomen of the insect body and the infection dynamic initial emergence of the adult citrus citrus adults are received on the citrus trees, and the citrus tree lice are collected at different feeding times in 15d, 30d, 45d and 60d respectively. The results showed that the content of Yellow Dragon disease pathogen in the citrus plant was highest when feeding 15d, and then decreased with the further prolongation of feeding time. The trend of Wolbachia content was increased with the prolongation of feeding time. The use of conventional PCR could detect Yellow Dragon disease in 3-5 years old nymphs and adults. In addition to 3-5 instar nymphs, Q RT-PCR can also detect the pathogen of Yellow Dragon disease in two instar nymphs, but still can not be detected in the egg and the one age nymph. The content of the Yellow Dragon disease in the eggs and the nymphs of each age is increasing with the age. The content of the pathogenic bacteria was the highest, which was significantly higher than the preoviposition and anaphase. Fluorescence in situ hybridization was found in the citrus lice eggs, and the pathogen of Yellow Dragon's disease could not be detected in the 1-3 instar nymphs, but the pathogen of Yellow Dragon disease could be detected in the four and five nymph. The pathogen was mainly distributed in the U type of the worm, and the individual body was also in the nymph breasts. Distribution of the pathogen of Yellow Dragon's disease in the nymphs of Citrus lice is similar to that of Wolbachia in the nymph. It shows that the pathogen of Yellow Dragon disease and Wolbachia in the four and five nymphs can be symbiotic in the body containing bacteria. The pathogen of Yellow Dragon's disease is distributed in the body of the adult and male and female adults, and this distribution form is beneficial to the horizontal transmission of the pathogen of Yellow Dragon disease. In summary, it was found that the pathogen of Yellow Dragon disease was beneficial to the population growth of citrus citrus by the study of the interaction between Citrus Yellow Dragon disease pathogen and citrus plant lice, and the infection of Yellow Dragon disease bacteria increased the absorption and utilization of Citrus lice to amino acids. In addition, the dynamics of Wolbachia infection in citrus citrus and the infection dynamics of Yellow Dragon disease pathogens were both dynamic. As the age increased, and both of the two were distributed in the body of the nymphs in the citrus plant, the two symbiotic relationship may be related to the high bacteria rate in the nymphs of citrus citrus. It is speculated that Wolbachia participates in the interaction between the three kinds of sugar orange - Yellow Dragon disease - Citrus plant lice to influence the rate of the citrus fungus. The specific molecular mechanism is not clear. Further research is needed.
【学位授予单位】：华南农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S436.66

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