Studies On PCR Detection Of Citrus Variegated Chlorosis And On The Transmissible Rate And The Distribution In Spring Leaves Of Huanglongbing

Citrus Variegated Chlorosis (CVC) is an economically important citrus disease caused by Xyllela fastidiosa, distributed in Brazil, Argentina and Paraguay. CVC has been causing serious damage to citrus industry in Brazil since it was found in 1980's and also a potential threat to the citrus industry of USA.CVC has not been found in China, along with the importation of overseas citrus materials, there exists potential risk that CVC could be spred into China, so strict quarantine based on the effective detection technique is an effective method to prevent spreading of CVC. PCR is one of the rapid and accurate methods to detect CVC. In this study, we have established 5 conventional PCR and 1 nested-PCR based on 5 pairs of primers by optimizing the PCR conditions, compared the sentivity and specificity of the 6 PCR products and compared the nucleotide sequences of the PCR products with CVC standard sequence in NCBI. The main results are as follows:1. The specific bands of the 5 conventional PCR products sequences were 137 bp, 500 bp, 603 bp, 745 bp and 700bp, and their minimum detection concentration of the template were 1ng, 10pg, 0.1ng, 0.1ng, respectively; the specific band of the nested-PCR product was 700bp and the detection sensitivity was 1pg. All of the 6 PCR were not able to detect HLB and Citrus canker.2. Comparison results showed that the nucleotide sequences of PCR products shared 98% homology with standard sequences of CVC in the NCBI database, so all of the 6 PCR products were the CVC target bands.3. Total DNA of thirty-five suspective CVC samples from 4 farms in Brzail were extracted by the mocro and rapid total nucleic acid extraction; and all of the 35 extracts were used for detection of CVC by conventional PCR based on 272-1-int /272-2-int and by nested-PCR. The two PCR methods showed the same detection result.Huanglongbing (HLB) is a destructive citrus disease caused by Liberibacter. In this study, we did preliminary experiments on the transmissible rate by two kinds of inoculation (bark-inocualtion and bud-inoculation) and on the distribution in spring leaves of HLB in order to choose the right inoculation method for conserving HLB and definitude the right parts of leaves for PCR in spring. The main results are as follows:1. From August to December in 2006 and April in 2007, leaf samples collected monthly from citrus trees by two different methods of inoculation (bark-inocualtion and bud-inoculation) were used for the detection of HLB by conventional PCR. The result showed that the transmissible rate by bud-inoculation was obviously higher than that by bark-inoculation. The transmissible rates by bud-inoculation and bark-inoculation were 50% and 15.7%, respectively.2. From April to July in 2006, spring leaf samples collected every half month from sweet orange were used for the detection of the distrbution of HLB in citrus spring shoots by conventional PCR. The result showed that HLB wasn't detectable in spring young leaves in April. As the matrue of the leaves, HLB was detectable in spring leaves in May, and after two months when the leaves matured, almost all of the leaves were positive in HLB.