新疆无花果花叶病病毒鉴定与发生调查

Identification and distributive investigation of Fig mosaic viruses in Xinjiang

【目的】无花果花叶病是危害无花果的主要病害之一,在新疆地区发生普遍,但导致该病害发生的病原种类尚不明确,因此进行了新疆无花果花叶病的病原鉴定与发生情况调查,为进一步明确不同无花果病毒的潜在危害以及病害防控提供依据。【方法】对新疆8个地区21个果园及路边的盆栽风景无花果进行病毒病的调查,通过高通量测序对感病无花果样品进行病毒初步鉴定,利用PCR或RT-PCR法,对采集自新疆8个地区的、不同花叶病症状及无症状的135份叶片样品进行病毒检测。【结果】高通量测序分析表明,感病样品中存在5种无花果病毒,分别为无花果花叶病毒(Fig mosaic virus,FMV)、无花果杆状病毒1(Fig badnavirus 1,FBV-1)、无花果杆状病毒2(Fig badnavirus 2,FBV-2)、无花果叶斑相关病毒4(Fig leaf mottle-associated virus 4,FLMaV-4)和无花果斑点相关病毒(Fig fleck-associated virus,FFkaV)。PCR及RT-PCR检测结果表明,135份无花果样品中存在6种无花果病毒,其检出率分别为FMV(100%)、FBV-1(100%)、FBV-2(99.3%)、FLMaV-4(74.8%)、FFkaV(31.9%)、无花果叶斑相关病毒1(Fig leaf mottle-associated virus 1,FLMaV-1)(19.3%)。新疆无花果病毒普遍存在复合侵染现象,感染4种以上病毒的样品有127份,占94%。2种、3种、4种和5种病毒复合侵染检出率分别为0.7%、5.2%、62.2%和31.9%。FMV、FBV-1、FBV-2在不同地区的发病率没有明显差异,并且症状为环斑、褪绿斑驳、褶皱及无症状样品中都能检测出FMV、FBV-1、FBV-2,这3种病毒可能是侵染新疆无花果的主要病毒。【结论】鉴定到侵染新疆无花果花叶病样的病毒性病原有6种,病毒检出率较前人报道的具有明显提升,并且普遍存在2种以上病毒复合侵染的现象,其中FMV、FBV-1、FBV-2检出率最高。

【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 pathogen 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,chlorotic mottles,malformation,and asymptomatic leaves were collected.Through high-throughput sequencing,the virus was preliminarily identified in samples of susceptible figs,and the sequencing results 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 template 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 12.5μL,cDNA 2μL and 10μmol·L^(-1).Each downstream primer was 1μL,ddH2O was made up to 25μL,and reaction program was the following:pre-denaturation at 94℃ for 3 min;denaturation at 94℃ for 30 s,annealing at 52-61℃ for 30-45 s,extension at 72℃ for 30 s,total 30-35 cycles;and extension at 72℃ for 5 min.The 6μL product of PCR was detected by 1.2% agarose gel electrophoresis,and the target fragment was recovered 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 analysis 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),and fig 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 infections 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 infecting 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 significantly.Among them,FMV,FBV-1 and FBV-2 had the highest detection rate.