Identification and distributive investigation of Fig mosaic viruses in Xinjiang

Abstract: 【Objective】Fig mosaic disease is one of the main diseases that damage figs. It is widespread in Xinjiang, and produces many types of complex symptoms, which seriously threatens the sustainable development of the fig industry in Xinjiang, but the types of the pathogen are not clear yet. The patho- gen identification and distributive investigation of fig mosaic disease in Xinjiang will provide a basis for further clarifying the potential harm of different fig viruses and guiding the disease prevention and control.【Methods】We conducted disease investigations and recorded the damages in 21 orchards and roadsides in 8 regions of Xinjiang’s Kashgar, Atushi, Korla, Aksu, Hotan, Turpan, Yili and Urumqi, and took pictures of the disease symptoms in the field. 135 samples of different types with ring spots, chlo- rotic mottles, malformation, and asymptomatic leaves were collected. Through high- throughput sequencing, the virus was preliminarily identified in samples of susceptible figs, and the sequencing re- sults were verified by PCR or RT-PCR. Using plant DNA and RNA extraction kit (Tiangen) to extract total RNA and DNA from 135 leaf samples with different symptoms and asymptomatic mosaic disease collected from 8 regions in Xinjiang, and electrophoresis with 1.0% agarose gel was used to detect the integrity of RNA and DNA and determine the concentration before storing in a -80 ℃ refrigerator. Fig total RNA was used as a template to synthesize cDNA using a reverse transcription kit (TaKaRa), and the specific operation was carried out according to the instructions. DNA or cDNA was used as a tem- plate to detect 13 types of viruses and viroides in 135 samples with different symptoms using PCR or RT-PCR. PCR reaction system was as follows: 2×Taq PCR Master Mix 12.5 μL, cDNA 2 μL and 10 μmol·L-1. Each downstream primer was 1 μL, ddH2O was made up to 25 μL, and reaction program was the following: pre-denaturation at 94 °C for 3 min; denaturation at 94 °C for 30 s, annealing at 52-61 °Cfor 30-45 s, extension at 72 ℃ for 30 s, total 30-35 cycles; and extension at 72 °C for 5 min. The 6 μL product of PCR was detected by 1.2% agarose gel electrophoresis, and the target fragment was recov- ered by the gel extraction kit, and the target fragment was connected to the vector. The obtained positive clone was sent to the company for sequencing, and the result of BLAST comparison was performed.【Results】Symptoms such as leaf shrinkage, ring spots, chlorotic mottled, leaf vein emergence, bright veins and fruit spots were observed in 21 orchards and 13 sites out of the orchards, and the incidence was high. In one orchard, the disease was mild, the symptoms were not obvious, and no fruit spots were observed. The figs on 7 orchards and the street roadside were asymptomatic. The mosaic disease in Kashgar, Atushi and Korla orchards was more serious in 8 regions, the incidence rate in orchards with lighter diseases was generally 50%-80%, and the incidence rate in severe orchards reached 100%. There were no obvious disease symptoms in other areas. Results from high-throughput sequencing anal- ysis showed that there were 5 types of fig viruses in susceptible samples, fig mosaic virus (FMV), fig badnavirus 1, (FBV-1), fig badnavirus 2, (FBV-2), fig leaf mottle-associated virus 4 (FLMaV-4), andfig fleck-associated virus (FFkaV). In the transcriptome sequencing, 94.6% of the sequence of FMV was obtained, and the sequence similarity was between 93.6% and 98.8%, 95.1% of the sequence of FBV-1 was also obtained, and the sequence similarity was between 99.8% and 100%. PCR and RT-PCR tests found that the above 5 viruses were present in the samples, and they had high sequence similarity with the registered virus sequences on the GenBank. This result further clarified the accuracy of the high-throughput sequencing. PCR and RT-PCR detection results showed that six viruses were detected from 135 samples, and the detection rates were FMV (100%), FBV-1 (100%), FBV-2 (99.3%), FLMaV- 4 (74.8%), FFkaV (31.9%), and Fig leaf mottle-associated virus 1 (FLMaV-1) (19.3%). Multiple infec- tions were common in Xinjiang figs, with 127 samples infected with more than 4 viruses, accounting for 94%. The detection rates of compound infections of 2, 3, 4 and 5 viruses were 0.7%, 5.2%, 62.2% and 31.9%, respectively. There was no significant difference in the incidence of FMV, FBV-1 and FBV- 2 in different regions. FMV, FBV-1 and FBV-2 can be detected in the symptoms of ring spots, chlorotic mottles, malformation and asymptomatic samples. These three viruses may be the main viruses infect- ing Xinjiang figs.【Conclusion】Fig mosaic disease was more serious in Kashgar, Atushi and Korla of Xinjiang and produced a variety of complex symptoms. The spots on the fruit would not disappear, which affected the appearance and taste of the fruit. Mosaic symptoms in other areas were relatively slight or asymptomatic. There were 6 types of viruses that infected Xinjiang figs. The types of viruses detected in this study were basically the same as previous studies, but the detection rate increased signif- icantly. Among them, FMV, FBV-1 and FBV-2 had the highest detection rate.