Studies On The Transmission Mechanism Of Tomato Yellow Leaf Curl Virus Vectored By Different Biotypes Of Bemisia Tabaci

The sweet potato whitefly, Bemisia tabaci (Gennadius)(Hemiptera:Aleyrodidae), has been regarded as the one of the most destructive worldwide pest insects which can colonizes more than600species of host plants including vegetables, flowers and cotton. B. tabaci is a complex including at least32biotypes (also be called as cryptic species). B and Q biotypes are the most invasive and destructive. B. tabaci B biotype invaded China and caused great economic losses on the horticultural plants during the nineteen ninties. It was not until2005that B. tabaci Q biotype was firstly found in Yunnan, and displaced B. tabaci B biotype gradually in most areas in China during the recent years. It has been found that B. tabaci Q biotype replaced the original B. tabaci B biotype at least21provinces and cities in China at the end of2009. In the meantime, Tomato Yellow Leaf Curl Virus (TYLCV) is an important plant virus transmitted by B. tabaci in a circulative manner via the hemolymph, which have brought more destructive economical losses to the tomato production from south to north China, exploded along with the spread of B. tabaci Q biotype. However, the mechanism of the TYLCV transmission by B. tabaci and TYLCV disease explosion along with the replacement of B. tabaci B biotype by Q biotype are not clear now. Therefore, in this present study, the harmful whiteflies, B. tabaci B biotype and B. tabaci Q biotype are used as the experimental materials, the molecular markers were screened on the whiteflies including B. tabaci B biotype, B. tabaci Q biotype, and Trialeurodes vaporariorum through RAPD-PCR firstly TYLCV acquisition within different periods were compared between B. tabaci B and Q biotypes. Based on the sequence cloning of TYLCV transmission related protein, GroEL, the expression of B. tabaci B biotype and B. tabaci Q biotype were compared using the qRT-PCR technique. The RNAi technical system was established in B. tabaci and the function of GroEL gene were studied preliminarily. The results will be helpful for the demonstration for the mechanisms of TYLCV transmission in B. tabaci and also provide the theoretical guidance and scientific basis for the prevention and control of TYLCV disease vectored by B. tabci in the future. The main results are as follows.1RAPD markers for three whiteflies including Bemisia tabaci B and Q biotypes and Trialeurodes vaporariorum were studied. Two inter-specific random primers of OPA-17(5’-GACCGCTTGT-3’), OPE-03(5’-CCAGATGCAC-3’) were screened from120primers using the RAPD-PCR, which were regarded as the most suitable primers to discriminate B. tabaci B biotype, B. tabaci Q biotype and Trialeurodes vaporariorum after verification. Amplification spectrums were established for the three different whiteflies, and the spectrums for each whitefly were clear and stable for different individuals in each species. The field whiteflies collected from different places were also identified as expected according to the established spectrum.2SCAR markers of three whiteflies were obtained for Bemisia tabaci B. Q biotypes and Trialeurodes vaporariorum. Specific SCAR markers for each whitefly were obtained from the above RAPD amplifications. Specific PCR amplification primers for Bemisia tabaci B. Q biotypes and T. vaporariorum were successfully established, and the amplified fragments were507,387,412bp respectively. The corresponding bands of three pairs of primers could only be obtained from the corresponding specific whitefly, but not from other whitefly occurred in the same domain. The SCAR amplification was repeatable and stable. The amplification results using the SCAR marker for the field population collected from the different places in China showed the strong applicability for B. tabaci B and Q biotypes.3Using real-time qRT-PCR technique, the expression of TYLCV related gene, GroEL, in B. tabaci B biotype, Q biotype were studied. The results showed that the TYLCV acquisition of B. tabaci Q biotype is higher than B biotype within the tested five periods (6h-72h). Q biotype reached the steady level earlier than B biotype, after feeding on the TYLCV-infected tomato plants. The expressions of the GroEL gene were compared between B. tabaci B and Q biotypes and the results showed that the expression of GroEL in B. tabaci of virus-infected was higher than the non-infected B. tabci population in two biotypes. What’s more, the expressions in Q biotype exhibited1.55-fold and1.68-fold significantly higher than B biotype for the virus-infected and non-infected populations (P<0.05) respectively. The above results exhibted that the expression of GroEL was probably induced by the TYLCV infection, suggesting that there is close relation between them..4The expression of virus transmission related gene, GroEL, was studied using RNA interference (RNAi). Based on the sequence of GroEL gene, excreted by the symbiont named Hamiltonella in B. tabaci B biotype, the GroEL fragment was amplified using the gene-specific primers. The corresponding dsRNA was synthesized and was injected into the pupae of B. tabaci. The relative mRNA levels within different treatment time after injection of B. tabaci B biotype were quantified by real-time quantitative PCR.The results showed that different express levels in the whitefly adults changed with the different amounts of dsRNA and different treatment time. The GroEL expression decreased after dsRNA injection. When the dsRNA at concentration of3-4ng/μl was injected, the expression was found to be decreased significantly with60-70%when48h passed after injection. Then the expression recovered gradually and it reached the original level at96h after injection. The differences of TYLCV acquisition and transmission in B. tabci need further study.5Physiological characteristics of Bemisia tabaci infected TYLCV was studied. The activities of detoxification and protective enzymes of B and Q biotype were mesured after they were reared on the TYLCV infected tomato plants for72h and longer term. The whiteflies reared on the healthy tomato were regarded as the control. The results are as followed.(1) The activities of detoxification enzymes in B and Q biotype B. tabaci were found to be the similiar trend. Both the activities of CarE and GST of biotype B and Q were exposed on TYLCV-infected plants for72h was higher than those on healthy plants. The activity on the TYLCV-infected plants for the whole life cycle was the lowest. However, the activities of P450exposed to TYLCV-infected plants for long term> on TYLCV-infected plants72h> on healthy plants exhibited the same seen in B and Q biotypes.(2) For the protective enzyme including CAT, POD and SOD, all the activities of whiteflies on the TYLCV-infected plants forever showed the highest level than those on the TYLCV-infected plants for72h. The activities on the healthy plants were the lowest.The results suggest that the P450and protective enzymes increased probably due to the TYLCV infection and the activties increased higher with the infection lasting time.