Preliminary Study On The Transmission Route Of Citrus Yellow Vein Clearing Virus

Yellow vein clearing disease(YVCD)caused by Citrus yellow vein clearing virus(CYVCV),an emerging viral disease,was first reported from Pakistan in 1988 in lemon(Citrus limon)and sour orange(C.aurantium).Since CYVCV was first detected in China in 2009,the virus was spreading rapidly and widely distributed in major citrus growing provinces of China.CYVCV can infect most citrus species,cultivars and hybrids,among which,lemon and sour orange were the most sensitive.Symptoms in affected lemon and sour orange consist of yellow vein clearing,leaf distortion and water soaking of the veins on the abaxial side of the leaf.YVCD usually does not cause tree death,but unthrifty growth and chronic yield reductions also cause high cumulative economic losses.CYVCV is transmitted through vegetative propagation of infected buds,scion or rootstocks,and by mechanical inoculations of sap extracts onto herbaceous indicator hosts and citrus.CYVCV can also be transmitted by Aphis craccivora and A.spiraecola from lemon to bean(Phaseolus vulgaris),and from bean to bean.However,until now no vector has been proven to transmit CYVCV from citrus to citrus,and it is not known whether there is other route of transmission existed.As a result,the prevention and control of citrus yellow vein disease has caused difficulties.In this study,the content of CYVCV in A.spiraecola,Toxoptera citricidus,Panonychus citri and Dialeurodes citri,and the transmissibility of CYVCV by these arthropod were investigate.Furthermore,the transmissibility of CYVCV by contaminative tools was also detected.This research fills in important gaps in knowledge of CYVCV transmission,which is critical for development of YVCD transmission and epidemiology.The main results are as follows,1.Quantification of CYVCV in arthropods.Adults of A.spiraecola,D.citri,P.citri and T.citricidus were collected in the field,and identified to species according to morphology using stereoscopy.These arthropods were reared on CYVCV-infected donor plants at 20-25 °C for 24 h,and then one hundred adults each of A.spiraecola,D.citri,P.citri and T.citricidus were mixed in sterile microfuge tube respectively.Total RNA extracts was obtained from each mixture with Trizol reagent,and used for quantitative PCR(q PCR)detection as previously described.These results suggested that CYVCV is acquired by different insect species.Moreover,a sensitive and reliable droplet digital PCR(dd PCR)assay for the detection of CYVCV was developed.The sensitivity of dd PCR was 100 times higher than the real-time RT-PCR.dd PCR can be used for early CYVCV diagnosis and detection of CYVCV in single arthropod.2.Transmission of CYVCV by A.spiraecola.Adults of A.spiraecola was placed on the young flushes of CYVCV-infected Pineapple sweet orange(C.sinensis)in perspex cage at 20-25 °C.After 48 h acquisition-access period,A.spiraecola were placed on young flushes of 15 virus-free-one-year old Pineapple sweet orange seedlings for 24 h using a small watercolor brush.Fifty aphids per receptor plant were used.The receptor plants were treated with the insecticide,and transferred to an insect proof glasshouse at 20 to 25 °C.Transmissions were repeated three times to insure consistency in transmission rate.Three months after inoculation,the young shoots of the sweet orange was analyzed by DTBIA(Direct tissue blot immunoassay)analysis.The result showed that the average transmission rate of CYVCV was 4.4 %.Six months post inoculation,the positive rate of CYVCV in receptor plants did not change.Three and six months after inoculation,RT-PCR assay got the same conclusions.After 48 h acquisition access period on CYVCV-infected plants,each 50 A.spiraecola were transferred to 30 virus-free Pineapple sweet orange receptor seedlings for 48 h.Transmission was repeated three times.Three months after inoculation,DTBIA and RT-PCR showed that the average transmission rate of CYVCV was 15.5 % and 17.8 %,respectively.Six months post inoculation,the transmission rate of CYVCV by A.spiraecola was 23.3 %.RT-PCR assay got the same conclusions.To our knowledge,this is the first experimental evidence that CYVCV is transmitted by A.spiraecola from citrus to citrus.3.Transmission of CYVCV by T.citricidus.Adults of T.citricidus was placed on the young flushes of CYVCV-infected Pineapple sweet orange in perspex cage at 20-25 °C.After 48 h acquisition access period,apterae adult aphids were placed on 29 one-year-old virus-free Pineapple sweet orange seedlings.Groups of 50,100 and 150 apterae adult aphids per receptor plant were given a 48 h inoculation access period,respectively.Three,six and twelve months after group T.citricidus inoculation,no CYVCV was detected in the receptor plants by DTBIA and RT-PCR.4.Transmission of CYVCV by P.citri.Adults of P.citri was placed on the young flushes of CYVCV-infected Dai Dai sour orange in perspex cage at 20-25 °C.After 48 h acquisition access period,adults of P.citri were transferred to 13 one-year-old virus-free Dai Dai sour orange seedlings using a small watercolor brush.Groups of 100,200 and 300 adults of P.citri per receptor plant were given a 24 h inoculation access period,respectively.Three,six and twelve months after group P.citri inoculation,no CYVCV was detected in the receptor plants by DTBIA and RT-PCR.5.Transmission of CYVCV by D.citri.Adults of D.citri was placed on the young flushes of CYVCV-infected Pineapple sweet orange in perspex cage at 20-25 °C.After a 48 h acquisition access period,approximately 1 000 adults of D.citri were collected with a mouth aspirator and caged with 23 one-year-old virus-free Daidai sour orange seedlings for 48 h.Three months after inoculation,CYVCV was detected in the receptor plants with an incidence of 31.9 % by DTBIA.Furthermore,the young flushes of CYVCV positive receptor plants showed characteristic symptoms including yellow vein clearing,leaf distortion and water soaking of veins on ventral side.Six months post inoculation,the average transmission was 39.1 %.Three and six months after inoculation,RT-PCR assay got the same conclusions.To our knowledge,we have confirmed for the first time the vector transmission of CYVCV by citrus whitefly,D.citri from citrus to citrus in laboratory conditions.6.Transmission of CYVCV by contaminated tools.The knife blade was twice sliced through the stem of the infected donor plant,and then sliced diagonally about 3mm into the stem of the recipient plant,50 cuts per receptor plant were made.Three months after inoculation,the young shoots of the sour orange was analyzed by DTBIA and RT-PCR analysis.The results showed that CYVCV was detected in 11.7 % and 16.5 %,respectively.All of the positive control plants were detected CYVCV at three-month post inoculation.Six months post inoculation,more sour orange receptor plants were tested positive by DTBIA and RT-PCR,and about 20.4 % and 23.3 % were CYVCV-positive.Three and six months post inoculation,CYVCV was detected in the rough lemon with an incidence of 20.0 % by DTBIA and RT-PCR.Therefore,contaminated cutting tools are probably one important route of CYVCV transmission in the field,and local spread.7.CYVCV has not been detected on Solanum nigrum which collocted from orchard.