柑橘—黃龍病病菌-Wolbachia-柑橘木虱互作關(guān)系的研究
發(fā)布時(shí)間：2018-07-24 11:02
【摘要】：柑橘木虱Diaphorina citri Kuwayama是取食植物韌皮部汁液的刺吸式口器昆蟲。柑橘木虱是柑橘黃龍病病菌Candidatus Liberibacter asiaticus唯一的自然傳播媒介。柑橘黃龍病是柑橘毀滅性病害之一,給世界柑橘產(chǎn)業(yè)造成了巨大的損失。本文以柑橘木虱為研究對象,利用常規(guī)PCR、實(shí)時(shí)熒光定量PCR(q RT-PCR)、熒光原位雜交(FISH)等技術(shù),從分子生態(tài)學(xué)的角度來研究柑橘-黃龍病病菌-Wolbachia-柑橘木虱互作關(guān)系。本研究通過寄主植物生理狀態(tài)對柑橘木虱生物學(xué)特性的影響,系統(tǒng)研究了健康與感病砂糖橘對柑橘木虱適合度的影響;從氨基酸利用的角度分析了黃龍病病菌影響柑橘木虱適合度的營養(yǎng)機(jī)制;基于昆蟲內(nèi)共生菌Wolbachia和黃龍病病菌感染與位置分布的相關(guān)性,分析了Wolbachia影響柑橘木虱傳播黃龍病病菌的分子機(jī)制,從而為利用昆蟲內(nèi)共生菌研究柑橘木虱和黃龍病的防控措施提供理論基礎(chǔ)。研究結(jié)果如下:1.柑橘木虱的形態(tài)特征通過對柑橘木虱卵和各齡期若蟲形態(tài)特征的觀察,歸納了各相鄰齡期之間的形態(tài)差異。柑橘木虱各齡期若蟲長寬差距較大:二齡比一齡長0.145 mm,寬0.166 mm;三齡與二齡間差距分別為0.193 mm和0.182 mm;四齡與三齡差距分別為0.351 mm和0.279 mm;五齡與四齡間差距分別為0.572 mm和0.358 mm。在田間一般可先根據(jù)蟲體大小對各齡期進(jìn)行初步判定,不確定齡期的再在顯微鏡下進(jìn)一步的確認(rèn)。2.寄主植物對柑橘木虱生活史的影響柑橘木虱卵、一齡若蟲以及整個(gè)若蟲的發(fā)育歷期受寄主植物的影響較為明顯。在柚子寄主植物上若蟲存活率最高,在黃皮上最低,且兩者差異顯著。寄主植物影響柑橘木虱成蟲壽命,在柚子上的壽命顯著長于黃皮上的壽命。黃皮上的木虱單雌產(chǎn)卵量顯著低于其他4種寄主。柑橘木虱在九里香上的內(nèi)稟增長率最高、酸橘上最低;而凈增值率在砂糖橘上最高、黃皮上最低。結(jié)果表明在本實(shí)驗(yàn)的5中寄主植物中,除黃龍病隱癥寄主九里香之外,顯癥寄主中砂糖橘是柑橘木虱的最適寄主。3.黃龍病病菌對柑橘木虱適合度的影響實(shí)驗(yàn)發(fā)現(xiàn),帶菌柑橘木虱或者在感病砂糖橘上取食的柑橘木虱世代發(fā)育歷期均縮短,如卵、若蟲以及卵到成蟲的整個(gè)發(fā)育歷期,均顯著小于健康柑橘木虱在健康砂糖橘上的發(fā)育歷期。但對于柑橘木虱的存活率來講其變化趨勢正好和發(fā)育歷期相反,帶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顯著高于感病砂糖橘上的存活率。感病砂糖橘上柑橘木虱F1代的單雌平均產(chǎn)卵量顯著高于健康砂糖橘。本研究結(jié)果表明黃龍病病菌有利于柑橘木虱在砂糖橘寄主上的種群增長。4.黃龍病病菌對寄主植物及柑橘木虱氨基酸組分的影響當(dāng)砂糖橘感染黃龍病病菌后,韌皮部汁液中各游離氨基酸的組分以及比例均發(fā)生了改變。表明黃龍病病菌對柑橘木虱的有利影響可能與砂糖橘寄主韌皮部汁液營養(yǎng)成分改變有關(guān)。柑橘木虱取食感病砂糖橘時(shí)分泌的蜜露中游離氨基酸天冬酰胺和脯氨酸下降明顯,同時(shí)這兩種氨基酸在感病砂糖橘韌皮部汁液中又明顯增加,說明黃龍病病菌增加了柑橘木虱對天冬酰胺和脯氨酸的吸收,這可能利于柑橘木虱若蟲發(fā)育。同時(shí)黃龍病病菌增加了柑橘木虱體內(nèi)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長發(fā)育。帶菌柑橘木虱若蟲和健康柑橘木虱若蟲在九里香上分泌的蜜露中游離氨基酸的組分差異很小,表明黃龍病病菌對柑橘木虱若蟲氨基酸營養(yǎng)吸收的影響不大。5.次生共生菌Wolbachia在柑橘木虱體內(nèi)分布及感染動態(tài)廣州柑橘木虱種群體內(nèi)7種共生菌的感染率存在差異,Carsonella、Profftella和Wolbachia感染率均是100%;Arsenophonus感染率為51.7%;Rickettsia、Cardinium和Hamiltonella三種共生菌的感染率很低。Wolbachia在柑橘木虱卵、各齡期若蟲及雌雄成蟲體內(nèi)的感染率存在差異。Wolbachia的含量隨著木虱齡期的增加而增多,且雄蟲體內(nèi)Wolbachia的含量高于雌蟲。Wolbachia在卵中的分布隨著卵的發(fā)育而不斷變化,從卵柄到卵中部再到遠(yuǎn)離卵柄一端,最后在老熟卵體內(nèi)呈現(xiàn)散布型分布。Wolbachia在若蟲體內(nèi)主要集中在含菌體內(nèi),而在雌雄成蟲體內(nèi)是局限在蟲體腹部。6.黃龍病病菌在柑橘木虱體內(nèi)分布及感染動態(tài)初羽化的柑橘木虱成蟲接到感病柑橘樹上,分別在15d、30d、45d以及60d收集不同取食時(shí)間的柑橘木虱進(jìn)行檢測。結(jié)果表明柑橘木虱體內(nèi)黃龍病病菌含量在取食15d時(shí),黃龍病病菌含量達(dá)到最高,此后,隨著取食時(shí)間的進(jìn)一步延長而減少。而Wolbachia的含量變化趨勢則是隨著取食時(shí)間的延長而增加。利用常規(guī)PCR可以在3-5齡若蟲以及成蟲體內(nèi)檢測到黃龍病病菌,而q RT-PCR除了3-5齡若蟲外,還可以在二齡若蟲體內(nèi)檢測到黃龍病病菌,但仍然不能在卵和一齡若蟲體內(nèi)檢測到。卵和各齡期若蟲中黃龍病病菌的含量是隨著齡期的變大而不斷增多。對于柑橘木虱的成蟲,在產(chǎn)卵盛期的木虱體內(nèi)黃龍病病菌的含量最高,顯著高于產(chǎn)卵前期以及產(chǎn)卵后期。熒光原位雜交技術(shù)在柑橘木虱卵、1-3齡若蟲體內(nèi)不能檢測到黃龍病病菌,而在四、五齡若蟲體內(nèi)可以檢測到黃龍病病菌,病菌主要分布在若蟲的U型含菌體內(nèi),個(gè)別蟲體在若蟲胸部也有分布。黃龍病病菌在柑橘木虱若蟲中的分布位置與Wolbachia在若蟲的位置相似,表明了在四、五齡若蟲體內(nèi)黃龍病病菌和Wolbachia可在含菌體內(nèi)共生。黃龍病病菌在雌雄成蟲體內(nèi)均是散布型分布,且這種分布形式利于黃龍病病菌的水平傳播。概括而言,通過砂糖橘-黃龍病病菌-柑橘木虱互作關(guān)系研究發(fā)現(xiàn),黃龍病病菌有利于柑橘木虱種群的增長;黃龍病病菌的感染增加了柑橘木虱對氨基酸的吸收與利用;另外,柑橘木虱體內(nèi)Wolbachia的感染動態(tài)與黃龍病病菌的感染動態(tài)均是隨著齡期的變大而增加,且兩者在柑橘木虱若蟲體內(nèi)均分布于含菌體內(nèi),二者共生關(guān)系可能與柑橘木虱若蟲階段獲菌率高有關(guān),推測Wolbachia參與砂糖橘-黃龍病-柑橘木虱三者間的互作來影響柑橘木虱的獲菌率,具體分子機(jī)制尚不明確,需要進(jìn)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.
【學(xué)位授予單位】：華南農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S436.66
本文編號：2141165
[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.
【學(xué)位授予單位】：華南農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S436.66
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
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