WU Jia-yu,ZHANG Yu,ZHOU Xue-ping,QlAN Ya-juan

1 Department of Applied Biological Science,College of Agriculture and Biotechnology,Zhejiang University,Hangzhou 310058,P.R.China

2 State Key Laboratory of Rice Biology,Institute of Biotechnology,College of Agriculture and Biotechnology,Zhejiang University,Hangzhou 310058,P.R.China

3 State Key Laboratory for Biology of Plant Diseases and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing 100193,P.R.China

Abstract Vegetative propagation of seed potato often allows passaging of viruses to seed tubers,resulting in significant yield losses and reduction of potato tuber quality.Thus,virus detection approach is crucial for effective virus management programs and the production of virus-free seed potatoes.Among the reported potato-infecting viruses,potato virus A (PVA) is considered as one of the most important viruses in potato-growing regions worldwide.This study prepared four hybridoma lines secreting PVA-specific monoclonal antibodies (MAbs) (2D4,8E11,14A6 and 16H10) using purified PVA virions as an immunogen.Western blotting results indicated that all the four MAbs reacted strongly and specifically with the putative capsid protein of PVA.Using these four MAbs,this study developed antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA),Dot-ELISA and Tissue print-ELISA for detection of PVA infection in potato plants.The results indicated that PVA can be detected in crude tissue extracts from infected potato plants diluted up to 1:327 680 (w/v,g mL-1) by ACP-ELISA or up to 1:10 240 by Dot-ELISA.The Tissue print-ELISA is the quickest and easiest approach among the three serological assays,and is more suitable for onsite large-scale potato screening programs.Further analyses of field-collected potato samples showed that the sensitivities and specificities of the three serological approaches were similar to those of RT-PCR in PVA detection and confirmed that PVA is currently widespread in Yunnan and Zhejiang provinces of China.Hence,the results strongly suggest that these highly sensitive serological approaches based on PVA-specific MAbs are useful and powerful for PVA-free seed potato production programs and PVA field surveys.

Keywords:potato virus A,monoclonal antibody,serological approach,antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA),Dot-ELISA,Tissue print-ELISA

1.lntroduction

Potato (Solanum tuberosumL.) is currently the fourth largest food crop after rice,wheat,and maize.Potato is also an important vegetable crop worldwide and a globally traded commodity (Heet al.2012;Zhanget al.2020).However,virus infection in potato seed tubers causes significant potato yield losses and reduction of tuber quality.Currently,potato virus A (PVA),potato virus Y (PVY),potato virus S (PVS),potato virus X (PVX),potato leafroll virus (PLRV) and potato virus M (PVM) are known to be the most widely spread viruses in potato fields worldwide (Salazar 1996;Singhet al.1996;Singh 1999;Palmaet al.2013;Hameedet al.2014;Yardimciet al.2015;Zhanget al.2020).PVA,a member of the genusPotyvirusin the familyPotyviridae,has been reported in many potato-growing countries,causing about 40% potato yield reduction each year (Singh and Singh1998;Hameedet al.2014).

PVA virions are flexuous filaments of 680-750 nm long and 11-13 nm wide,which contain a single-stranded,polyadenylated,positive-sense RNA genome.PVA is transmitted through aphid vectors in the fields in a nonpersistent manner (Foxet al.2017).It can also spread over a long distance through trade using infected seed tubers(Singh and Singh 1998).In potato field,the PVA-infected potato plants often develop mild mosaic symptom in leaves.When potato plants are co-infected with PVA and PVX,the plants often show severe disease symptoms in leaves known as“potato crinkle”(Singh and Singh 1998).When potato plants are co-infected with PVA and PVY,the plants often show severe leaf mosaic and stunting symptoms (Singh and Singh 1998).In most cases,the symptoms in PVA-infected plants are difficult to be distinguished from those in the plants infected by PVY,PVS or PVX.

It is well known that virus-free seed potato is essential for high-yield and high-quality potato productions.Thus,the establishment of fast,simple,sensitive,low-cost and high-throughput detection approaches for potato-infecting viruses is critical for the production of virus-free potato seed tubers and virus controls.Currently,the approaches for PVA detection include reverse transcription PCR (RTPCR) (Singh and Singh 1998;Xu and Niee 2006;Crosslin and Hamlin 2011;Zhanget al.2017),real-time RT-PCR(Xu and Niee 2006;Agindotanet al.2007;Chenget al.2013),polyclonal antibody (PAb)-based double antibody sandwich enzyme-linked immunosorbent assay (DASELISA) (Maat and De Bokx 1978;Vettenet al.1983;Singhet al.1996;Hameedet al.2014;Yardimciet al.2015)and Dot-ELISA (Singhet al.1996;Palmaet al.2013).The PCR-based virus detection is highly sensitive and specific.However,this method requires total RNA isolation from infected plant tissues and dedicated equipment and reagents.Furthermore,PCR-based method can not be used to process the large number of samples demanded by a typical virus-free seed potato production program,especially for the onsite virus detection programs.Serological approaches are rapid,simple,reliable,costeffective and high-throughput,well-suited for plant virus detections (Zhanget al.2018).In a large-scale virus screening program,some samples may have high levels of viruses that can be readily detected using various serological assays,while some others may contain very low levels of viruses that can be misdiagnosed as virusfree samples.Many studies have demonstrated that the misdiagnosis of virus infection in plant tissues based on serological assays can be avoided through highly sensitive and specific antibodies.To establish reliable and sensitive serological approaches for monitoring PVA infection in potato plants,this study firstly produced four highly specific and sensitive monoclonal antibodies (MAbs) using purified PVA virions as the immunogen.Then these four MAbs were used to establish three fast,highly sensitive and reliable PVA detection approaches,such as antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA),Dot-ELISA and Tissue print-ELISA.Finally,this study demonstrated that these three serological approaches could be used to accurately detect PVA infection in field-collected potato plants and tuber samples,and it also confirmed that PVA is currently widespread in Yunnan and Zhejiang provinces,China.Hence,these results strongly suggest that these four highly sensitive MAbs and serological approaches are useful and powerful for PVA-free seed potato production programs and PVA field surveys.

2.Materials and methods

2.1.Sources of viruses and collection of potato samples

Potato plants showing virus-like symptoms were randomly collected from a potato field in Yunnan Province,China.After the RT-PCR analyses using a set of PVA-specific primers,a PVA-infected potato plant was identified and maintained in the laboratory.PLRV-,PVS-,PVM-,PVY-and PVX-infected potato samples and watermelon mosaic virus(WMV)-infected watermelon samples were kindly supplied by Dr.Ding Ming (Institute of Biotechnology and Germplasm Resources,Yunnan Provincial Academy of Agricultural Sciences,Kunming,China).Prior to further assays,PVA was propagated in potato test-tube plantlets,cultivar Hezuo 88 and PVA virions were purified from infected leaf tissues as described previously (Singh and McDonald 1981).The purified PVA virions were then used to produce PVA-specific MAbs.To confirm the validity of the serological approaches,22 potato samples randomly collected from potato fields in 2019 in Yunnan and Zhejiang provinces of China were tested for the presence of PVA.

2.2.Preparation of MAbs

Purified PVA virions were injected into four 8-wk-old BALB/c female mice as described (Songet al.2017).Preparation of hybridomas was done as described previously (Zhanget al.2018).Briefly,spleen cells from immunized mice were fused with murine myeloma cells Sp2/0 using 50%polyethylene glycol (PEG,MW 1 500,Sigma-Aldrich,St.Louis,MO,USA) solution.The fused cells were resuspended and cultured in an RPMI-1640 medium supplemented with 100 µmol L-1hypoxanthine,0.4 µmol L-1aminopterin,16 µmol L-1thymidine,and 15% fetal calf serum in wells of 96-well cell culture plates.At 10 days post cell fusion,cell culture supernatant was collected from each well and screened for the presence of PVA-specific antibody through an indirect ELISA using microtiter plates coated with purified PVA virions.Hybridomas secreting anti-PVA antibodies were cloned at least three times using the limiting dilution method (Songet al.2017) to obtain monoclonal hybridoma line.The resulting hybridoma lines were individually injected into syngeneic BALB/c mice to produce ascitic fluids containing MAbs.The titer of MAb in ascitic fluid was determined through an indirect-ELISA using microtiter plates coated with purified PVA virions (Guoet al.2020).The isotype of MAb was identifiedviaa DAS-ELISA using a Mouse MAb Isotyping Kit (Sigma-Aldrich,St.Louis,MO,USA).Specificity and sensitivity of individual MAb were determined by Western blotting assays and ACP-ELISA as described (Liuet al.2017;Yuet al.2018).

2.3.Detection of PVA using ACP-ELlSA

Working dilutions of individual PVA MAbs and an alkaline phosphatase (AP)-conjugated goat anti-mouse IgG were determinedviaa phalanx test as described (Shanget al.2011;Wuet al.2013).The ACP-ELISA was performed as described by Zhanget al.(2020).Briefly,an ELISA plate was coated with plant crude tissue extracts from potato plants and incubated at 4°C overnight.The plate was blocked for 30 min in 0.01 mol L-1phosphate buffered saline (PBS) containing 3% skimmed milk powder followed by a 1-h incubation in a diluted anti-PVA MAb solution.Then,the plate was incubated for another 1 h in a diluted AP-conjugated goat anti-mouse IgG second antibody(Sigma-Aldrich).Each step above was followed by four washes with PBS containing 0.05% Tween-20.Finally,the plate was incubated for 30 min in ρ-nitrophenyl phosphate substrate solution,and the optical density at 405 nm (OD405)of each well was read with a microplate reader (BIO-RAD,Hercules,CA,USA).

2.4.Dot-ELlSA and Tissue print-ELlSA

Dot-ELISA was carried out as described previously (Wuet al.2014;Guoet al.2020) with specific modifications.Briefly,approximately 100 mg potato leaf tissues or tuber tissues were ground in 3-5 mL PBS.The resulting homogenate was centrifuged at 5 000×g for 3 min and the supernatant was used as plant crude extract for PVA test.Leaf or tuber crude exracts from a PVA-infected and a non-infected potato plant were used as the positive and negative control,respectively,during assays.Crude extracts from different samples (3 µL per sample) were blotted onto nitrocellulose membranes with an Eppendorf pipettor.After dried at 37°C for 5 min,the membranes were blocked for 30 min in PBS containing 5% skimmed milk,and then incubated for 1 h in one of the four anti-PVA MAb solutions.The membranes were then incubated for 1 h in an AP-conjugated goat anti-mouse IgG second antibody (Sigma-Aldrich) followed by an incubation for 15-20 min in the nitro-blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate substrate solution.After each incubation,the membranes were washed with PBST for four times.Under optimized antibody dilutions,specificities of Dot-ELISAs were analyzed using crude extracts from the PVA-infected potato leaf and tuber tissues and the PVY-,PVS-,PVX-,PVM-or PLRV-infected potato leaf and tuber samples,with those from non-infected potato leaf and tuber samples as the negative controls.For sensitivity analysis assays,the crude extract from a PVA-infected or a noninfected potato plant was diluted from 1:20 to 1:10 240 in a 0.01 mol L-1PBS solution (w/v,g mL-1) prior to Dot-ELISA assays.

Tissue print-ELISA was done as described (Chenet al.2017).Briefly,stems of potato plants or tubers were harvested from the PVA-,PVS-,PVA-,PVX-,PVY-or PLRVinfected plants.A transverse cut was made to each stem or tuber using a razor blade,and the fresh-cut surface was immediately printed onto the membranes.The membranes were then probed for PVA infection using different MAb solutions as described above for Dot-ELISA.

2.5.Reverse transcription PCR (RT-PCR) and sequence analysis

Primers used for RT-PCR were designed based on the conserved region in the PVACPgenes available in the GenBank database.Sequences of the primers are 5´-ATTTAGGTACTGCTGGGACT-3´ (forward,nucleotide position 8632-8651) and 5´-TCAGGTTGCGTTGAAGAC-3´(reverse,nucleotide position 9099-9082).Total RNA was extracted from potato leaf tissues using a TRIzol reagent as instructed (Invitrogen,Carlsbad,CA,USA).PVACPcDNA was synthesized using the reverse prime and the AMV reverse transcriptase (TaKaRa Biotechnology,Dalian,China) according to the manufacturer’s instructions.PCR amplifications were performed in a volume of 25-µL that contained 12.5 µL LOD OneTMPCR Master Mix (Toyobo Biotechnology,Shanghai,China),1 µL of each primer(10 µmol L-1),1 µL cDNA,and 9.5 µL ddH2O.The PCR protocol was as follow:at 98°C for 2 min (initial denaturation),30 cycles at 98°C for 10 s (denaturation),at 54°C for 5 s(annealing),at 68°C for 5 s (extension),and at 68°C for 10 min (final extension).The resulting PCR products were cloned,sequenced and then aligned with the known PVACPgene sequences in the GenBank database.

3.Results

3.1.Virus purification

PVA virions were purified from PVA-infected potato leaf tissues through differential centrifugation.Numerous flexuous filamentous viral particles of 680-750 nm long and 11-13 nm wide,similar to viruses in the genusPotyvirus,were found in the purified virion sample under an electron microscope (Fig.1).

Fig.1 An electron micrograph showing purified potato virus A(PVA) virions. The purified PVA virions were negatively stained with 1% phosphotungstic acid,pH 7.5,prior to examination.Bar=0.2 µm.

3.2.Preparation and characterization of MAbs

After four cell fusion experiments,this study has screened about 500 hybridoma wells containing antibodies against PVA,and finally obtained four hybridoma cell lines (i.e.,2D4,8E11,14A6 and 16H10) secreting supersensitive and highly specific anti-PVA MAbs through sensitivity and specificity analyses of antibodies and cell cloning.It was also found that the vast majority of the screened antibodies were less sensitive and/or less specific for PVA detection.These four hybridoma cell lines were individually and intraperitoneally injected into BALB/c mice to produce ascites.Isoforms and subclasses of the four MAbs were identified as the IgGl,κ light chain (Table 1).Titers of the four MAbs in ascites were all above 10-9by an indirect-ELISA using the purified PVA virions as the antigen (Table 1).Yields of IgG in ascites of 2D4,8E11,14A6 and 16H10 cell lines were 25.92,10.14,13.51 and 8.02 mg mL-1,respectively (Table 1).

Table 1 Properties of the four potato virus A (PVA) monoclonal antibodies

Western blotting assays demonstrated that all the four MAbs reacted strongly and specifically with a～30 kDa protein in the PVA-infected potato leaf sample (Fig.2-A).No protein signal was detected in extracts from the PVY-or PVX-infected or non-infected potato plant tissues.According to the molecular weight of the protein recognized by MAbs,this study suggests that the detected protein band in gels is the putative capsid protein (CP) of PVA (Fig.2-A).

3.3.ACP-ELlSA for PVA detection

Results of the three independent phalanx assays showed that PVA could be readily and reliably detected in crude extracts from PVA-infected potato plants using 1:20 000(v/v) diluted MAbs as the primary antibody and 1:10 000(v/v) diluted AP-conjugated goat anti-mouse IgG as the secondary antibody.Specificity analyses of ACP-ELISA using the above optimal working concentrations of the primary and second antibody revealed that this assay gave a strong positive reaction with crude extracts from the PVA-infected potato plants,and negative reactions with crude extracts from the PVY-,PVS-,PVX-,PVM-,PLRV-,or WMV-infected and non-infected plants (Fig.2-B).ACPELISA assays using serially diluted crude extracts from a PVA-infected and a PVA-free potato plant tissue showed that the highest PVA detection endpoints of the assays were up to 1:327 680 dilution (w/v,g mL-1) (Fig.2-C),indicating that ACP-ELISAs using these four MAbs were highly sensitive for PVA detection.

Fig.2 Specificity and sensitivity analyses of the four monoclonal antibodies (MAbs) (2D4,8E11,14A6 and 16H10) based on Western blotting and Antigen-coated plate (ACP)-ELISAs.A,specificity analyses of MAbs through Western blotting assays.B,specificity analyses of MAbs through ACP-ELISA.The OD405 absorbance values were measured at 30 min after the addition of the substrate at room temperature.The results are presented as the mean value±SD from three independent assays.C,sensitivity analyses of MAbs through ACP-ELISA.CK- was a non-infected potato plant,used as a negative control.The OD405 absorbance values were measured and the results are presented as described in B above.

3.4.Dot-ELlSA and Tissue print-ELlSA for PVA Detection

To establish Dot-ELISA and Tissue print-ELISA for PVA detection,this study firstly investigated the optimal working dilutions of the four MAbs and the AP-conjugated goat anti-mouse IgG second antibody through phalanx assays.Results revealed that the optimal working dilutions of the four MAbs were at 1:20 000 (v/v) and the optimal working dilution of AP-conjugated goat anti-mouse IgG was at 1:8 000.Under optimized antibody dilutions,specificity analysis based on Dot-ELISAs indicated that these assays gave a strong positive reaction with the crude extract from a PVA-infected potato plant,and negative reactions with crude extracts from the PVY-,PVS-,PVX-,PVM-or PLRV-infected potato plants and non-infected potato plants (Fig.3-A and B).Sensitivity analysis results of these assays showed that using MAbs 2D4,8E11,14A6 and 16H10,PVA could be detected in the infected plants’ crude extract diluted up to 1:10 240,1:5 120,1:5 120 and 1:5 120 (w/v,g mL-1),respectively (Fig.3-C).Furthermore,results of Tissue print-ELISA showed that these four MAbs can be used to detect PVA infection in potato stems and tubers (Fig.4-A and B).When stems and tubers of the PVY-,PVS-,PVX-,PVM-or PLRV-infected or non-infected potato plants were used in the assays,no positive reaction was detected (Fig.4-A and B),indicating the specificity of Tissue print-ELISAs for PVA detection.

Fig.3 Specificity and sensitivity analyses of the four monoclonal antibodies (MAbs) based on Dot-ELISAs method.A,specificity analyses of the Dot-ELISAs for potato virus A (PVA) detection in potato plants.For this assay,two dots (upper and lower)representing a crude leaf extract from a PVA-,PVY-,PVS-,PVX-,PVM-or PLRV-infected potato plant or from a non-infected potato plant were made.Purple color dots are PVA-positive and green dots are PVA-negative.B,specificity analyses of the Dot-ELISAs for PVA detection in potato tubers.For this assay,two dots (upper and lower) representing a crude extract from PVA-,PVS-,PVM-,PVX-,PVY-or PLRV-infected potato tubers or from non-infected potato tubers were made.C,sensitivity analyses of Dot-ELISAs for PVA detection in potato plants.

Fig.4 Detection of potato virus A (PVA) infection in potato stems and tubers using Tissue print-ELISAs. A,detection of PVA infection in potato stems using Tissue print-ELISAs.Prints made with non-infected potato stems were used as negative controls.Purple color prints indicate positive reactions and light green color prints indicate negative reactions.B,detection of PVA infection in potato tubers using Tissue print-ELISAs.Tubers were collected from PVA-,PVY-,PVS-,PVX-,PVM-or PLRV-infected potato plants.Prints made with non-infected potato tubers were used as negative controls.Purple color prints indicate positive reactions and colorless prints indicate negative reactions.

3.5.Detection of PVA infection in field-collected potato samples

To confirm that the above three serological approaches are reliable for the detection of PVA infection in field-collected potato samples,this study randomly sampled 22 potato plants showing virus-like symptoms from potato fields in Yunnan and Zhejiang provinces in 2019.These samples were tested for PVA infection using ACP-ELISA,Dot-ELISA and Tissue print-ELISA.Among these samples,three were found to be PVA-positive by all the three approaches(Fig.5-A,B and C).To validate this result,this study further analyzed these 22 samples through RT-PCR using PVACPgene specific primers (Fig.5-D).RT-PCR results showed that a～500-bp PCR product was obtained from the three PVA-positive samples identified through the three serological approaches.Further DNA sequencing and sequence alignment confirmed that these three PCR products mapped with the PVACPgene and shared 88-98%sequence similarity with the reported PVACPgenes.

Fig.5 Detection of potato virus A (PVA) infection in 22 field-collected potato plant samples by Tissue print-ELISA (A),Dot-ELISA(B),antigen-coated plate (ACP)-ELISA (C) and RT-PCR (D). Samples labeled as a1-6,b1-6,c1-6,d1-4 are 22 field-collected potato plants.Samples labeled as d5 and d6 were collected from a PVA-infected and a non-infected potato plant,used as a positive and a negative control.The sample labeled as d2 were confirmed to be co-infected by PVA and PVY through ACP-ELISA and RT-PCR.Lane M is a 1-kb DNA ladder.

4.Discussion

Potatoes are vegetatively propagated.Without the proper certification,vegetatively propagated seed potatoes often contain one or more viruses,resulting in significant yield losses and reduction of tuber quality (Yardimciet al.2015;Zhanget al.2020).To overcome this important limiting factor,potato growers have developed various strategies,especially high-throughput viral detection approaches,to ensure productions and applications of virus-free seed potatoes through different postharvest inspections (Crosslinet al.2007).

Among the current approaches for plant virus detection,serological assays have the advantages of simple,rapid,cost-effective and high-throughput detection,which have been used extensively in plant virus detection (Wuet al.2011,2014;Liuet al.2016),including the detection of potatoinfecting viruses (Songet al.2017;Zhanget al.2020).Among these reported serological approaches,ELISA is particularly useful for this purpose (Clark and Adams 1977;Karasevet al.2010;Zhanget al.2020).However,it is well known that the reliability of any given serological assay depends largely on the sensitivity and specificity of the viral detection antibody.Consequently,serological approaches using less sensitive and/or less specific detection antibodies often give non-conclusive test results (Huttinga 1996;Singh and Singh 1996).

To date,some serological approaches for PVA detection have been developed.Gugerliet al.(1979) reported that a PVA-specific antiserum,which was produced by immunizing rabbits with purified PVA virions,was used to detect PVA infection in potato plants through ELISA.Although detection of PVA infection in potato sprouts using ELISA is accurate,detection of PVA infection in leaves of greenhouse-grown potato plants is less reliable (Maat and De Bokx 1978).Singhet al.(1992) reported a DAS-ELISA for PVA detection and showed that this assay could be used to monitor PVA in 1:640 (w/v,g mL-1) diluted crude extracts collected from infected potato leaves.Noticeably,Boonekampet al.(1991) produced five PVA-specific MAbs and used these MAbs to characterize epitopes on PVA capsids.In that report,however,the authors did not indicate the sensitivity and specificity of the MAbs or use these MAbs to detect PVA infection in potato leaves or tubers.Singh and Singh(1998) reported that the results of ELISA used to detect PVA infection in dormant potato tubers were often inaccurate due mainly to the low level of virus accumulation in this tissue.In a separate report,Vettenet al.(1983) recommended to detect virus infection in potato tubers by ELISA and indicated that it is necessary to break the dormancy of tubers prior to the assay.Although PVA is one of the most prevalent potato-infecting viruses,its accumulation level in infected potato leaves and tubers is often lower than that in other potyviruses (Singh and Singh 1998).Moreover,PVA concentration in potato tubers is much lower than that in potato leaves (Singh and Singh 1998).Hence,Vetten and colleagues considered that serological detection of PVA in potato tubers was difficult and unreliable (Vettenet al.1983).To overcome the limitations and drawbacks of the current serological methods applied for PVA detection,supersensitive serological approaches are urgently required.

This study has screened about 500 hybridoma wells containing antibodies against PVA and finally obtained four highly sensitive and specific anti-PVA MAbs to develop three highly specific and sensitive serological assays,ACP-ELISA,Dot-ELISA and Tissue print-ELISA assays,for PVA detection in potato leaves and tubers.The results revealed that ACPELISA and Dot-ELISA can be used to detect PVA infection in infected potato plants’ crude extracts diluted up to 1:327 680 and 1:10 240 (w/v,g mL-1),respectively.To the best of our knowledge,these two assays are the most sensitive and specific serological approaches for PVA detection in potato.

Tissue print-ELISA is a simple,fast and practical approach,which can be used to detect different potato viruses (Zhanget al.2020).Tissue print-ELISA does not require sample extraction that is needed by Dot-ELISA,ACP-ELISA and DAS-ELISA.Additionally,the resulting tissue prints from field samples can be stored for several weeks prior to laboratory tests.Thus,Tissue print-ELISA is particularly useful for potato growers in the developing countries when they need to investigate virus infection in the fields during potato-growing seasons.Compared the three serological assays described above,the Tissue print-ELISA is considered to be the quickest,easiest and most suitable approach for onsite large-scale surveys.

Using field-collected potato samples,this study finds that PVA is commonly present in Yunnan and Zhejiang provinces of China.It is noteworthy that these results indicate that the sensitivity and specificity of these three serological approaches are similar to those of RT-PCR using PVACPgene specific primers,suggesting that these three serological approaches have the great potential to conduct large-scale PVA screening and contribute to the production of PVA-free seed potatoes.However,it remains to be determined whether the three serological approaches developed in the present study can be used to detect different PVA strains in field-collected potato samples from other potato-growing regions of China or different potatogrowing countries.In view of the fact that PVA can infect aphid as well as plant host to threaten potato production,it is also important to carry out further experiments to elucidate whether the serological approaches developed in this study are sensitive to PVA detection in PVA-infected aphid body.

5.Conclusion

In the present study,four supersensitive and highly specific anti-PVA MAbs were produced individually.Using these MAbs,ACP-ELISAs,Dot-ELISAs and Tissue print-ELISAs were developed for quick,sensitive,specific,accurate and large-scale detection of PVA infection in potato.Further field survey result suggests that PVA is now common in Yunnan and Zhejiang provinces of China.It is believed that these three serological approaches can be used to conduct routine PVA screenings and PVA epidemiological studies,and facilitate PVA-free seed potatoes production and PVA control program development.

Acknowledgements

We are grateful to Dr.Ding Xinshun (Noble Research Institute (retired),Ardmore,USA) for his help during preparation of this manuscript.This work was supported by the National Key Research and Development Program of China (2017YFD0201604) and the National Natural Science Foundation of China (31571976).

Declaration of competing interest

The authors declare that they have no conflict of interest.