Pathogenicity And Genetic Diversity Of Hop Stunt Viroid Infecting Mulberry (Morus Alba L.) In China

Mulberry（Morus alba L.）is a woody plant with considerable economic and ecological benefits,but because it thrives in the natural environment all year,it is prone to disease invasion.Hop stunt viroid（HSVd）is a member of the genus Hostuviroid in the family Pospiviroidae,which has an extremely wide host range.Many key cash crops（such as hops,peaches,plums,citrus,etc.）are known to be infected with HSVd,causing obvious disease symptoms and severe economic losses.HSVd infection to mulberry has been reported abroad,but in China,the country with the largest mulberry fields,it is yet to be reported in any of the relevant journals or reports.In addition,there is a dearth of studies about the pathogenicity of HSVd on mulberry and the genetic diversity of HSVd communities infecting mulberry in China.In this study,the sequence of the most conserved region of HSVd was used to design universal primers,and RT-PCR was utilized to detect mulberry samples with the virus or virus-like symptoms in the mulberry field.We confirmed for the first time the presence of HSVd infection on mulberry plants in China.In this study,the pathogenicity of the HSVd mutation and population genetic variation were investigated.1.HSVd occurs in mulberry gardens in China.Mulberry leaf samples from 13 mulberry trees with the virus or virus-like symptoms were randomly collected from the Zhenjiang mulberry field.Double-stranded（ds）RNA was extracted as a template for reverse transcription-polymerase chain reaction（RT-PCR）and HSVd was detected with the universal primers.One of the 13 individual samples had a positive PCR product band which we confirmed from cloning and sequencing analysis to have a fragment size of 296 nt.BLAST analysis showed that this sequence has the highest similarity with the HSVd sequence in Gen Bank,from which we confirmed the presence of HSVd infection in the Chinese mulberry field.Specific primers designed from the obtained HSVd sequence were used to test 40mulberry samples in the mulberry field,of which 3 samples were HSVd positive,showing a7.5%incidence rate.Sequence similarity analysis showed that the sequence similarity of the four HSVd varieties identified from the Chinese mulberry plants was 99.7%-100%.The highest similarity is with the HSVd variant of the Turkish plum（96.68%）,while the similarities with HSVd varieties from Iranian,Italian and Lebanese mulberry plants are relatively lower（93.6%-95.6%）.Phylogenetic analysis showed that the four HSVd varieties identified from China formed a single evolutionary branch,which differed from the evolutionary sources of HSVd isolates from Iran and Italy.2.Pathogenicity of HSVd:The pathogenicity of HSVd（identified from Mulberry in China）,which cloning of HSVd c DNA infection was mediatied by Agrobacterium tumefaciens to mulberry and herbaceous plants cucumber and tomato showed that:the invasive clone successfully infected mulberry,cucumber and tomato.On the 32nd day after inoculation,the positive rate of HSVd was 17.64%,and the infected cucumber didn’t show any viroid-like symptoms.The positive rate of HSVd was 66.67%on the 40th day after inoculation,and some tomatoes showed dwarfing symptoms.However,one mulberry sample was detected to be positive on the 60th day after mulberry inoculation,and the HSVd positive rate reached the highest at 45.45%on the 78th day.The leaves of infected mulberry trees did not show any viroids-like symptoms,so it takes a longer time to observe whether they show dwarfing symptoms.3.The genetic diversity and population structure of HSVd identified in Chinese mulberry plants were studied by analyzing the sequences of 179 HSVd clones from 12isolates obtained from naturally infected mulberry plants（4 isolates with 60 clones）,Agrobacterium inoculated with HSVd-positive mulberry plants（4 isolates with 60 clones）and tomatoes（4 isolates with 59 clones）.Analysis of nucleotide sequence similarity and site differences of HSVd population showed that the similarity of the nucleotide sequences across all clones ranged between 97-100%.There are 111 variant sequences of HSVd in the179 isolates,and the mutation sites occurred mainly in the pathogenic and variable regions.The presence of multiple heterogeneous sequences with various sequences（mutation cloud）in one isolate,indicates that HSVd is a quasi-species.Genetic diversity and evolutionary analysis also showed that the nucleotide diversity（Pi）and haplotype diversity（Hd）of the HSVd population in inoculated tomatoes are the highest（0.00553 and 0.831,respectively）,and the HSVd population has the richest genetic diversity.The HSVd population in inoculated mulberry has the lowest Pi and Hd（0.00249 and 0.583,respectively）as well as the lowest genetic diversity.The Pi and Hd of the HSVd population in the naturally infected mulberry plants were 0.00413 and 0.727,respectively,and the genetic diversity of the HSVd population was among the top two.The mutation frequency of HSVd was found to be1.542×10^-2in inoculated tomatoes and 0.656×10^-2in inoculated mulberry,indicating that the evolution rate of HSVd in the unnatural host is faster than that in the natural host,which may be a result of HSVd adapting to different replication mechanisms.These findings provide a scientific basis for China to establish a special HSVd inspection and quarantine system,produce healthy and non-toxic commercial mulberry seedlings and block the spread of HSVd in mulberry fields across the country.This study will lay a foundation for further work to be done on the pathogenicity of HSVd and the molecular mechanism of HSVd pathogenesis,which has important value in the theoretical and practical application.