| Citrus tristeza disease caused by Citrus tristeza virus(CTV)is of economical importance,which has destroyed over 100 million citrus trees grafted on sour orange worldwide until now.Due to the perennial and evergreen natures of citrus,and the common occurrence of citrus brown aphids(Aphis citricidus,CTV vector)in field,as well as farmers’ preference to top-graft new varieties/cultivars with uncertain viral infection,moreover,viral genetic mutation and recombination are somehow encountered,therefore,CTV isolates collected from field trees usually are mixtures of various strains,with complex differentiation and genetic diversity.According to the syndrome of citrus induced by CTV,three viral types are described as seeding yellow(SY),stem pitting(SP),and quick decline(QD).In recent studies,to quickly identify different viral variants,CTV were divided into multiple genotypes such as T36,VT,T3,T30,RB,S1,HA,and T68 based on sequence differences of the whole genome.Among them,T36 and VT genotypes were mostly associated with QD tristeza,the T3,VT,RB and T68 genotypes mostly associated with SP tristeza,while S1 and T30 genotypes usually do not cause obvious symptoms.With the development of bioinformatics and techniques,transcriptome sequencing has been used to obtain the complete genome sequence of plant viruses,and it can provide a more systematic and in-depth analysis than traditional techniques.Ganzhou,Jiangxi province,is one of the most famous navel sweet orange(NSw O)producing area in the world.Although CTV was found to be prevalent throughout the citrus orchards in Ganzhou,no serious damage was caused.More recently,severe stem pitting tristeza has been breaking out on some newly planted NSw Os in Ganzhou,causing stunted growth,tree decline,and the decrease in yield and fruit quality,with huge economic losses.However,the cause in detail of this pandemic remains unclear.In this study,based on field investigations,the CTV genotypes of NSw O samples collected from Ganzhou were initially identified using specific genotype primers.In order to understand the structure and change of CTV populations in Ganzhou,then PCR products of p33 from 20 distinct CTV isolates from Ganzhou were selected for deep sequencing to investigate the relative proportion of each genotype in each sample,so as to reveal the possible relationship between the genotype composition and the symptoms.In addition,we have obtained the complete genome sequences of four severe and three mild CTV isolates from Ganzhou,and bioinformatics analysis has been approached to identify their molecular characteristics,which may provide new genomic information on the origin and evolution of severe SP-associated strains.Finally,to explore the possible association of severe SP isolates derivered from Ganzhou of Jiangxi and Zigui of Hubei,respectively,the isolates from the two regions were analyzed through bioinformatics method.The results of this study are as follows:1.The occurrence of CTV is common for the 10 investigated districts/counties of Ganzhou,since 199 NSw O samples were collected there,among which 112 were found to be infected with CTV.The incidence rate for each district/county and average for the whole area was within 22.2-86.0% and 56.3%,respectively.Eight genotypes,including T36,VT,T3,T68,T30,RB,HA16-5,and S1,were found in 112 CTV positive samples collected during 2019 to 2020,of which T36 was predominant with an infection rate of 99.1%,followed by genotypes T3 and VT with an infection rate of95.5% and 94.6%,respectively.With regards to the early CTV samples derived from Ganzhou and preserved by our research group,only five genotypes(T36,VT,T3,T68,S1)could be detected,among which T3 and VT genotypes were common in all samples.Comparing with the samples collected from 2004 to 2007,the detection rates of six genotypes except VT and T3 in the samples collected from 2019 to 2020 increased,even with much significant increase for T36 and RB.In this study,all Ganzhou CTV samples were infected with multiple genotypes and the genotype complexity of CTV samples collected from 2019 to 2020 was higher than that of samples collected from 2004 to 2007.Among the samples collected from 2019 to 2020,the mixed infection of six genotypes was the most common structure(ca 31.3%),followed by the structure of four genotypes(ca 26.8%).Comparing among the Newhall NSw O samples collected from 2004 to 2007,the most common structure of CTV genotypes include VT+T3+T68 and VT+T3,both with occurrences of 33.3%,followed by VT+T3+S1 and T36+VT+T3,with occurrences of 22.2% and 11.1%,respectively.All samples collected from 2004 to 2007 did not show any apparent SP symptoms.In contrast,among 112 CTV-infected Newhall NSw O samples collected from 2019 to 2020,58 showed non to mild SP symptoms(scale 0-1),10 moderate symptoms(scale 2),and 44 severe symptoms(scale 3).2.The genomes of seven Ganzhou isolates,including the four severe(2019-6-DY,2019-7-DY,2019-30-2,2019-30-3),and the three mild(2019-20-XF,2019-33-DY,CY-77),were fully sequenced.The results of genomic sequence analysis showed that,the nucleotide sequence similarity among these isolates was 80.72-99.74%,while the similarity with 84 CTV isolates from all over the world ranged from 79.20% to 99.63%.In addition,the four severe isolates(2019-30-2,2019-30-3,2019-6-DY,2019-7-DY)shared the maximal sequence identities with NZRB-M17(FJ525435,99.03%),FL202-VT(KC517493,99.30%),HU-PSTS(KU720382,99.63%),and NZRB-M17(FJ525435,99.01%),respectively.The three mild isolates(2019-20-XF,2019-33-DY,CY-77)shared the maximal sequence identities with B301(JF957196,98.77%,causing mild vein-clearing on Mexican lime),NZRB-M17(FJ525435,98.99%),and Maxi(KU883266,99.31%),respectively.According to phylogenetic tree analysis,the 91 CTV isolates were classified into 12 genotypes,namely,T36,T30,VT,T68,HA16-5,S1,T3,M1,L1,SY568,A18 and RB.Among which CTV isolates 2019-30-2,2019-7-DY,2019-20-XF and 2019-33-DY belong to genotype RB,2019-30-3 and CY-77 belong to genotype T3,and 2019-6-DY belongs to genotype VT.The recombination analyses showed that recombination events of 2,6,3,5,3,5 and 4 times were found within isolates 2019-6-DY,2019-7-DY,2019-20-XF,2019-30-2,2019-30-3,2019-33-DY and CY-77,respectively.Based on the previously reported analysis for the association of CTV genotypes and the symptoms,and combined with the multiple recombination events within CTV isolates found in this study,it is speculated that recombination among multiple genotypes may be one of the major causes of the severe SP tristeza in citrus groves all over Ganzhou.3.The results of deep sequencing of PCR products of the p33 gene showed that the deep sequencing identified more genotypes than PCR with the specific primers for the same sample,except for genotype T36.In addition,when the relative proportion of genotype VT or RB within CTV populations exceeded 90%,or the relative proportion of genotype HA16-5 exceeded 30%,CTV populations would cause severe SP tristeza;when the relative proportion of genotype T30 or S1 exceeded 1%,CTV populations did not cause obvious SP tristeza.4.Because the NSw O scions recently replanted in Ganzhou were mainly from Zigui,Hubei province,39 citrus samples from Zigui were tested,of which 13 were found to be infected with CTV,with genotypes T36,VT,T3,T68,T30,RB,HA16-5,and S1 detected,among which genotypes T36,VT,T3 and T30 could be detected in all samples,followed by genotypes S1 and RB,both with an infection rate of 61.5%.It was found that CTV samples collected from Zigui and Ganzhou were similar in genotype composition.Contig4247,contig436,and contig629 obtained from Zi G-1through the deep sequencing shared the maximal sequence identities with the corresponding regions of CTV isolates 2019-30-2(99.29%),CN-RB-L13(97.13%),and T3-KB(93.45%),respectively.The results of recombination analysis showed that contig4247 as the major parent,participated in the recombination event of Ganzhou isolate 2019-20-XF at 227-2445 nt.In addition,the phylogenetic trees constructed via p23,cp and p33 genes showed that CTV Zi G-1 from Zi Gui was grouped with CTV isolates 2019-6,2019-10,2019-10,and 2019-18 from Ganzhou,of which 2019-6,and2019-10 were associated with the severe SP tristeza.The above results suggest that there is a close relationship,and may exist possible genetic exchanges of CTV isolates between Zigui and Ganzhou. |
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