| Located on the northwest border of China,Xinjiang has sufficient light,large temperature difference between day and night,and no pollution of soil.It is the main producing area of high-quality jujube in China.Xinjiang red jujube mainly grows in areas such as Kashgar,Aksu,Hotan,Bazhou,and the Production and Construction Corps.Most of the jujube seedlings of susceptible varieties show typical arbuscular symptoms in the second year after one grafting,and a few occur in the third year.In 2012,reports of jujube witches’ broom disease outbreaks in Aksu and Kashgar were published.In the same year,the risk analysis results showed that jujube witches’ broom disease is a highly dangerous forestry pest in Xinjiang.Currently,as no vector for the pathogen has been test in Xinjiang,infected seedlings or grafts remain the main route for the spread of the disease.In 2021,a diseased tree with typical symptoms of jujube witches ’ broom was found on the campus of Tarim University in Alaer City,Xinjiang.The tree was34 years old and the variety was Zanhuang jujube.No grafting was carried out after planting,and there was no possibility of disease transmission by scion.In addition,only Tetranychus truncatus occurred on the tree through annual observation.This study aimed to analyze the geographical distribution of phylogenetic strains of jujube witches’ broom phytoplasma based on the 16 S r RNA,Sec A,and Sec Y sequences of the existing strains at home and abroad.Subsequently,the study focused on typical symptoms of jujube witches ’broom found in Tarim University were identified and traced by molecular biology methods,and the Real-time LAMP detection method of jujube witches ’broom was established.The main research results are as follows:(1)This study conducted sequence polymorphism analysis,phylogenetic tree analysis,and homology analysis of 82 jujube witches’ broom phytoplasma strains based on their 16 S r RNA,15 Sec A,and 16 Sec Y sequences from both domestic and international sources.The results showed that the genetic distance of the16 S r RNA gene among the 82 strains from different geographic locations was small,and they could be roughly divided into four groups: I-Indian strains,II-Shandong strains,III-Hebei strains,and IV-Shaanxi and Henan strains.The Indian strains had the farthest phylogenetic distance and obvious geographical isolation from other strains,while the Shaanxi and Henan strains had a closer relationship with the Hebei strains,followed by the Shandong strains.Compared with the 16 S r RNA gene,the genetic distance of the Sec A and Sec Y genes from isolates from different geographic sources was larger,and they could be clearly divided into two clusters: cluster I-Indian strains and cluster II-Shandong strains.These findings indicate that there is a certain geographical correlation between the phytoplasma isolates of jujube witches’ broom.This study can provide some reference for further research on the interaction and adaptability between the host(plants,insects)and the ecological environment of the jujube witches’ broom phytoplasma,as well as the origin tracing of its related strains.This research results can provide reference for related research on the traceability of jujube witches ’ broom phytoplasma Aral isolate.(2)PCR amplification of the total DNA of Arar jujube mad plants with generic or semi-generic specific primers of 16 S r RNA,23 S r RNA,16-23 S r RNA,Sec A,Imp,rp,and Sec Y was performed to construct a systematic evolutionary tree to determine the taxonomic status of Arar jujube mad disease using Blast comparison and homologous comparison.In addition,the physicochemical properties and structures of Sec A,Imp,rp,and Sec Y were analyzed and predicted based on the sequencing results of Sec A,Imp,rp S3,rpl22,rpl15,and Sec Y.Sequencing revealed that we obtained 16 S r RNA genes with 1334 bp length,2680 bp 23S r RNA genes,380 bp 16-23 Sr RNA genes,838 bp Sec A genes,457 bp Imp genes,1123 bp rp genes and 1341 bp Sec Y genes.It was confirmed that the susceptible date plant was caused by phycoplasma infection.The obtained gene sequence was highly homologous to the related sequence of Candidatus Phytoplasma ziziphi(JWB)in the 16 S r V-B subgroup and named as JWB-Alar.Based on the homology comparison of the nucleotide sequences of the above specific genes and the constructed phylogenetic tree,JWB-Alar was determined to belong to the 16 S r V-B subgroup,rp V-C subgroup and Sec Y V-C subgroup.The JWB-Alar 16 S r RNA gene,16-23 S r RNA and Sec Y gene sequence had the highest similarity with the phytoplasma of jujube witches ’ broom from Baoding,Hebei and Shandong,reaching 100%.The JWB-Alar23 S r RNA gene sequence had the highest similarity with the phytoplasma from Shandong,reaching99.92%.The JWB-Alar Sec A gene sequence had the highest similarity with the phytoplasma from Shandong,and the homology was more than 99.76%.The sequence of Imp gene and rp gene of JWB-Alar had the highest similarity with the phytoplasma of jujube witches ’ broom from Baoding,Hebei Province,reaching 100%.The seedlings of jujube witches ’ broom were introduced from Hebei Province to Alar 35 years ago,and the source of JWB-Alar was traced to Hebei Province.Protein analysis and prediction revealed that Sec A,Imp,rp S3,rpl22,rpl15,and Sec Y were all unstable proteins and phosphorylated by kinases.The second and third levels were dominated by irregular curls and alpha helices,with the exception of Sec A for hydrophilic acid,Sec Y for hydrophobic alkaline,The remaining four proteins belong to hydrophilic basic proteins,Imp and Sec Y for trans-membrane proteins,and four others for non-membrane proteins.This research results lays a foundation for screening which protein for serological detection.(3)For rapid qualitative and quantitative detection of jujube phytoplasma,Based on the existing LAMP detection,Real-time LAMP was developed.The reaction system components and temperature were optimized,and the specificity,repeatability,and field applicability of the method were evaluated.The optimal reaction conditions for the developed Real-time LAMP method for jujube witches’ broom phytoplasma were as follows: ACLR-F3/ACLR-B3(10 μmol/L)0.5 μL,ACLR-FIP/ACLR-BIP(20μmol/L)1.0 μL,Bst II DNA polymerase 1.5 μL,5×LAMP reaction mix 5 μL,TS LAMP μL Green(20×)0.45 μL,Nuclease-free water was added to make up the volume to 25 μL,followed by incubation at 61°C for 60 minutes.The method specifically amplified JWB(16S r V-B subgroup)but not SCWB(16S r XXX group),with a detection limit as low as 3.1×102 copies/μL,which is 100 times more sensitive than direct PCR.In addition.The intra-assay repeatability was high.Application of the method to field samples with visible symptoms jujube stock,jujube hanging,jujube leaf and root(4 samples)of a jujube tree with obvious symptoms in the field and 11 samples(44 samples)of the corresponding parts of the surrounding jujube garden were 75%and 4.55%,respectively,while the positive detection rates of direct PCR were 50%and 0%,respectively.The established Real-time LAMP could detect the recessive diseased plants in the field.The Real-time LAMP established in this study can provide a reference for the early diagnosis and prevention of jujube witches’ broom disease isolate in Alar. |
