Huixian SHI Yimin GU Xingsheng LI Xiping SUN Yangdong GUO Xiaowei YUAN
Abstract ［Objectives］In 2019， virus diseases occurred widely on zucchini planted in Shandong Province. The disease symptom was different from previous reports. This study aimed to identify the pathogen causing the zucchini virus disease.
［Methods］ Ten diseased zucchini leaves were collected in the field and used as materials for PCR and sequencing.
［Results］ PCR detection and sequencing showed that the nucleotide sequence of the amplified fragment had the highest identity with the squash isolate of squash leaf curl China virus （SLCCNV） （MW389919.1） in Guangdong Province. Primers were further designed for amplifying the full-length SLCCNV. The full-length DNA-A was 2 730 bp （OM692270.1）， and the full-length DNA-B was 2 711 bp （OM692269.1）. Through sequence alignment， it was found that the DNA-A sequence shared identity of 89.65%-99.42% with registered SLCCNV， and the identity with the SLCCNV-GDHY pumpkin isolate （MW389919.1） in Guangdong was the highest， at 99.42%. The DNA-B sequence was identical with registered SLCCNV in the range of 81.82%-97.29%， and the identity with the SLCCNV-GDHY pumpkin isolate （MW389918.1） in Guangdong， was the highest， at 97.29%. Therefore， it was speculated that SLCCNV is the pathogen of zucchini virus disease. Since the virus was first found on zucchini in Shandong， it was named SLCCNV-SD.
［Conclusions］ This study provides materials for the research on the spread of SLCCNV in China and the analysis of population genetic characteristics， as well as a reference for the prevention and control of the virus in zucchini.
Key words Zucchini; Viral disease; Squash leaf curl China virus
Chinese squash leaf curl China virus （SLCCNV） is an important virus that harms cucurbit crops worldwide. It belongs to the genus Begomovirus of the Geminiviridae family， and has a two-component genome， including DNA-A and DNA-B genomes ［1］. The virus is transmitted by Bemisia tabaci in a persistent， circulatory manner and cannot be transmitted by mechanical friction or seeds ［2］. Infected Cucurbitaceae crops show dwarf plants with shriveled leaves， and downward curled leaf edges， causing serious economic losses to Cucurbitaceae crops ［3］.
The research on SLCCNV at home and abroad mainly focuses on its genome sequence， composition of the encoded protein and the discovery of new hosts. In 2003， the SLCCNV-［Pumpkin：
Coim］ isolate was first discovered in India to cause yellowing and shrinking of pumpkin leaves ［4］. In the same year， it was first confirmed that SLCCNV caused yellowing of squash leaves and dwarf plants through genome-wide analysis in Vietnam ［5］. In 2011， it was first confirmed that SLCCNV caused zucchini mosaic and leaf shrinkage in the Varanasi region of India ［6］.
In China， Begomovirus can infect squash， tobacco and tomato ［7-9］. In 1994， China first reported the infection of squash by a geminivirus. According to the analysis of its coat protein cp gene sequence， the virus may be SLCCNV ［10］， but SLCCNV infection of zucchini has not been reported in China. In the autumn of 2019， a large-scale virus disease outbreak occurred in zucchini cultivated in facilities in Qingzhou City， Shandong Province. The symptoms in the field were dwarf plants， curled leaves， and heavy losses. The diseased zucchini planting greenhouses suffered serious losses， and even a total crop failure in severe cases. PCR detection showed that it was not caused by cucumber mosaic virus （CMV）， watermelon mosaic virus （WMV） and zucchini yellow mosaic virus （ZYMV）. In the spring of 2020， the zucchini in Qingzhou City， Shandong Province also showed similar symptoms of virus disease. The successive occurrence of disease has attracted our attention， and we suspected that it was caused by a new virus infection. Because the observed symptoms in the field were similar to those of SLCCNV infecting squash， 10 diseased leaf samples were collected and amplified with specific primers， and the PCR products were subjected to sequence analysis and alignment to identify the pathogen causing the zucchini virus disease.
Materials and Methods
Materials
Plant materials：
In the experimental greenhouse in Qingzhou City， Shandong Province， dwarfed zucchini plants with shriveled leaves and downwardly curled leaf edges were found. Ten new leaves of zucchini with the symptoms were randomly collected， and healthy new leaves of zucchini were used as negative control.
Reagents：
Reagents for DNA extraction were purchased from Tiangen Biotech （Beijing） Co.， Ltd.
Primer synthesis and gene sequencing were performed in Sangon Biotech （Shanghai） Co.， Ltd.
Methods
Virus detection of field zucchini samples
The total DNA of leaves was extracted by the CTAB method， and the specific steps were carried out according to the instructions of Tiangen kit （TIANGEN DP350-02）.
The SLCCNV gene sequences were found from GenBank， and primers SLCCNV-729AF/SLCCNV-729AR and SLCCNV-808BF/SLCCNV-808BR were designed for PCR （Table 1）. The PCR system was 20 μl in volume， containing 10×Easy Taq Buffer （Mg2+） 2 μl， High Pure dNTPs 1.6 μl， Easy Taq DNA Polymerase （5 U/μl） 0.2 μl， DNA 1 μl， 10 μmol/L upstream and downstream primers 0.4 μl each， and double-distilled water to 20 μl. The PCR amplification conditions were 95 ℃ for 3 min， 34 cycles of 95 ℃ for 30 s， 55 ℃ for 30 s and 72 ℃ for 1 min， and 72 ℃ for 5 min. The PCR amplification product was detected by 1.0% agarose gel electrophoresis， and then the gel was cut and recovered and sent to Sangon Biotech （Shanghai） Co.， Ltd. for sequencing. In order to further confirm the virus， according to the obtained gene sequence， primers SLCCNV-AF/SLCCNV-AR and SLCCNV-BF/SLCCNV-BR （Table 1） were designed according to the genome characteristics of SLCCNV to amplify full-length DNA-A and DNA-B， respectively.
Phylogenetic tree analysis
According to the full-length gene sequence of SLCCNV obtained by sequencing， BLAST was used for similarity search in GenBank database， and Clustalx was used for multiple sequence alignment. Phylogenetic trees were constructed by the neighbor-joining method （NJ） of MEGA6.0， with the bootstrap of 1 000 ［11］.
Pathogenicity assay
Bemisia tabaci were taken from the leaves of zucchini with virus disease and put into 800-mesh tuck nets， and the nets containing B. tabaci were placed on healthy zucchini seedlings for virus inoculation. Each healthy zucchini seedling was placed with 20 individuals that probably carried the virus. After 3 d， insecticide thiamethoxam was used to spray the B. tabaci， and the tuck nets were removed. The incidence of zucchini seedlings was counted after 2 weeks of normal management， and multiple mixed B. tabaci individuals and inoculated zucchini plants were detected by PCR. Three replicates were set up in this experiment， and a total of 30 zucchini seedlings were inoculated.
Results and Analysis
Field symptom observation
At different growth stages of zucchini in the field， infected zucchini seedlings and adult plants showed yellowing of young leaves， and severe curling of the leaf edge downwards， and dwarf plants appeared simultaneously （Fig. 1）.
PCR test results of suspected squash leaf curl China virus infection
Ten parts of symptomatic zucchini leaves collected in the field were amplified using primers SLCCNV-729AF/SLCCNV-729AR and SLCCNV-808BF/SLCCNV-808BR， respectively. The results showed that target bands were amplified at 729 and 808 bp， respectively， while healthy samples did not show the target bands （Fig. 2）. The sequencing of the PCR products showed that the DNA-A fragment sequence shared identity of 90.27%-99.56% with registered SLCCNV， and the identity with the SLCCNV-GDHY pumpkin isolate （MW389919.1） in Guangdong， China was the highest， at 99.56 %. The DNA-B fragment sequence was identical with registered SLCCNV in the range of 81.04%-97.28%， and the identity with the SLCCNV-GDHY pumpkin isolate （MW389918.1） in Guangdong， China was the highest， at 97.28%.
In order to further obtain the full-length sequence of SLCCNV （zucchini isolate）， we designed primers SLCCNV-AF/SLCCNV-AR and SLCCNV-BF/SLCCNV-BR to amplify the full-length sequences of DNA-A and DNA-B， respectively. After PCR and sequencing， the full-length gene sequences DNA-A 2 730 bp and DNA-B 2 711 bp were obtained， and uploaded under the GenBank registration numbers of OM692270.1 and OM692269.1， respectively. Since the virus was first discovered in zucchini in Shandong Province， it was named SLCCNV-SD. In order to clarify the nucleotide differences between the SLCCNV-SD genome sequence and other isolates， we selected the nucleotide sequences of some SLCCNV isolates from NCBI for comparative analysis. The results showed that the nucleotide identity of SLCCNV-SD DNA-A and DNA-B with other isolates was 89.65%-99.42% and 81.82%-97.29%， respectively， and the nucleotide sequences of SLCCNV-SD DNA-A and DNA-B shared the most identity with SLCCNV GDHY pumpkin isolates （MW389917.1 and MW389918.1）， at 99.42% and 97.29%， respectively.
Phylogenetic tree analysis
In the GenBank database， the obtained DNA-A and DNA-B sequences were analyzed for kinship with other reported isolates. The SLCCNV-SD DNA-A and DNA-B sequences were phylogenetically analyzed with the DNA-A and DNA-B sequences of 20 SLCCNV isolates with the closest relationship. The results showed that SLCCNV-SD was clustered with SLCCNV-GDHY pumpkin isolate from Guangdong Province together （Fig. 3）.
SLCCNV pathogenicity verification
In order to verify the pathogenicity of SLCCNV to zucchini， B. tabaci that probably carried the virus were collected from field diseased zucchini plants and inoculated into 30 healthy zucchini seedlings. After 2 weeks， 29 of them were observed to have obvious symptoms （Fig. 4）. In order to further confirm that the diseased plants were infected by SLCCNV， the leaves of naturally diseased zucchini， leaves of artificially inoculated zucchini and B. tabaci that transmitted the virus were collected for PCR detection. The results showed that the virus was detected on all of the leaves of naturally diseased zucchini， leaves of artificially inoculated zucchini and B. tabaci that transmitted the virus （Fig. 5）. Therefore， we speculated that B. tabaci can carry SLCCNV to infect zucchini seedlings and cause viral disease.
Huixian SHI et al. Isolation and Identification of Squash Leaf Curl China Virus in Zucchini
Conclusions and Discussion
In this study， the detection of SLCCNV was first reported in diseased zucchini samples from Shandong Province. The SLCCNV-SD DNA-A sequence was more than 90% similar to 59 SLCCNV isolates from India， Bangladesh， Vietnam， Australia， Thailand， the Philippines and seven different provinces （cities） in China. Its similarity with SLCCNV-GDHY pumpkin isolate from Guangdong， China was the highest， at 99.42%. The SLCCNV DNA-B sequence
was more than 81% similar to 35 SLCCNV isolates from seven countries of Vietnam， Thailand， India， the Philippines， Indonesia， Bangladesh， Pakistan and five different provinces （cities） in China. Its similarity with SLCCNV-GDHY pumpkin isolate from Guangdong， China was the highest， at 97.29%.
The results of phylogenetic tree analysis showed that the SLCCNV-SD zucchini isolate might be transmitted from other Cucurbitaceae crops， and it was further speculated that the virus infecting zucchini plants in Shandong Province might be caused by the circulation of plant materials and the activity of B. tabaci. The increase in the host range of SLCCNV and the enhancement of its adaptability suggest that SLCCNV may occur on a larger scale， endangering the healthy production of cucurbit crops. In addition， B. tabaci has become one of the disastrous mediators of virus transmission in vegetable crops. Coupled with the extensive use of facility greenhouses， a suitable environment has been provided for the annual growth of B. tabaci. With the migration and activity of B. tabaci， the infection range of SLCCNV will gradually increase. In this study， SLCCNV was detected in zucchini， which provides materials for the research on the spread of SLCCNV in China， analysis of population genetic characteristics， and virus mutation and evolution， as well as a reference for the prevention and control of the virus in zucchini. Next， we can continue to study the molecular characteristics and pathogenic mechanism of the virus in depth， and analyze the incidence of the disease， so as to provide theoretical support for the prevention and control of the large-scale occurrence of the disease.
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