| Decline disease is one of the most devastating viral diseases of citrus, has resulted in thousands of cirus trees died around the citrus producing areas of world. Citrus tristeza virus(CTV)mainly transmitted by grafting and aphids, its genome contains 12 open reading frame(ORF), ORF6, which encodes the coat protein(CP), covers about 95% of the virion genome and is associated with pathogenicity and assembly process. There is obvious strain differentiation among CTV strains, it can be divided into yellow seedings( YS), stem pitting( SP), Quick decline( QD)according to different pathogenic performance in the host and also can be classified into seven genotypes based on its genome characteristics. This study mainly focus on investigating the infection rates of CTV in Gannan citrus producing areas, analysing phylogenetic and genetic sequence information; Use two types of genotype primer to identify genotype, and some samples were cloned and sequenced to analyse the isolates genetic diversity and genotypic characteristics of Gannan citrus producing areas. Main results are as follows:1. 351 samples of Newhall navel orange were collected from 15 counties, cities and districts of Ganzhou in Jiangxi province. The presence of CTV detection was performed by RT-PCR. Results showed that 175 samples were infected with CTV, the infecting rate of single site ranged from39.3% ~ 69.3%, and the total rate was up to 49.85%. The results showed that the widespread occurrence of CTV in Gannan orchards.2. 78 positive samples were selected to analyse recombination and base content of CP gene.Then calculated the nucleotide genetic distance, sequence homology and building phylogenetic tree of CP gene with 10 different pathogenic foreign CTV isolates. The results showed that JK4 exist recombination between 35~490 nt region, the recombination event was detected by five kinds of algorithms, P values between 3.746×10-4~1.811×10-2; The average GC content of samples was52.8%, AT(U) content of samples was 47.2%, the average base content of samples GC>AT; the CP gene nucleotide genetic distance of 78 samples ranged 0.000~0.072, and with 10 foreign CTV isolates ranged 0.005~0.100. The nucleotide and amino acid homology of CP gene were94.7%~100% and 92.3%~100%, with 10 foreign CTV isolates were 76.5%~84.1% and73.6%~80.3%; in phylogenetic tree, multiple samples collected from the same area clusted into different branches, some of them clusted with stem pitting isolate and recession of foreign isolate clustere and high homology of sequence between them. The results showed that the CP gene of CTV isolates in Gannan citrus producing areas were highly conserved; a trifling variation exsit in these sequnces; isolates among the various regions of high homology but no correlation with geographical origin; and low homology between foreign CTV isolates, The existence of mixed infection isolates in the area..3. Use primers designed by Hilf and Roy to identify the genotype of 135 samples respectively.Roy’s primer identification results showed that: 43 samples were single genotype, contained VT, T3,B165 genotypes and all of them were composed by servere isolate; 41 samples were mixed infection by two identififiable viral, of these, 5 sample contained servere and mild isolates, 36 samples contained servere isolates, of which 29 samples genotype were VT + T3 and the remaining number of samples contained VT isolate. 8 samples were mixed infection by three identififiable viral, four of them were VT+T3+T36 combination, rest of them contained servere and mild isolate;8 were mixed infectiom by four identififiable viral, two samples were VT+T3+T36+B165combination, 6 samples were VT+T3+T30+T36 combination; 35 of the samples could not be assigned to a know genotype. Hilf’s primer identification results showed that: 44 samples were single genotype and all of them composed of servere isolate, While VT was the most genotypes, the T3 genotype was least; 35 samples were mixed infection by two identififiable viral, 25 of them genotype were VT+T36 and VT+T3 combination and rest of them mixed servere isolates; 8samples were mixed infection by three identififiable viral, 4 of them were VT+T3+T36combination, 3 of them were VT+T30+T3 combination; only one sample contained four isolates;47 of the samples could not be assigned to a know genotype. In total, more than 30 percent of samples contain one isolate, multiple samples contained mixed infections of 2 to 5 of the know genotypes, the results of that use two types of primers to identified the genotype were inconsistent,and some of them could not be assigned to a know genotype.4. Select nine isolates that the results of genotype identification similarities and differences by two type of genotype primers, according to amplified fragment by Hilf’s primers to cloned and sequenced to analyzed their genetic information. The results show: in 5’ and k17 phylogenetic tree,the samples clustered into a group with reference isolate that identified by two types of primers showed contained the isolate, sequence homology between them were high and the genetic distance closer; some samples contained some kind isolates but did not clustered into one group with this isolates, the sequence homology between them were lower and the genetic distance farther; the genotype composition of samples were complex and might contained molecules variant or unknown genotype. |
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