Antiserum Production, Resistant Cultivar Screening And Cross-Protection Of Papaya Ringspot Virus-Watermelon Strain

Papaya ringspot virus(PRSV)is a member of the genus Potyvirus belonging to the family Potyviridae.According to the host range,PRSV can be divided into P and W strains.Among them,the W strain(PRSV-W)mainly infects cucurbitaceous crops and causes symptoms of leaf mosaic and fruit ring spots,which seriously affected the yield and quality.In recent years,the occurrence of PRSV-W has been increasing in Shandong,Henan and other provinces.In this study,the PRSV-W antiserum was prepared,the resistance of cucurbitaceous crops to PRSV was analyzed and the cross-protection efficiency of three attenuated mutants on wild-type PRSV was evaluated.The main results are shown as below:1.The PRSV-W antiserum with high titer and strong specificity was prepared using the prokaryotic expressed capsid protein.The PRSV-W capsid protein(CP)gene was ligated into the prokaryotic expression vector pEHISTEV to obtain a recombinant plasmid,which was transformed into E.coli Rosetta.After induction,a protein of about 37 kD was expressed,and the optimal IPTG final concentration was 0.2 mmol/L and the induction time was 4 h.The titer of the resultant antiserum was 1:8192 in ELISA.In Western blotting analysis,the antiserum showed specific positive reaction only with the zucchini plants infected with PRSV,but not with healthy zucchini plants or those infected with watermelon mosaic virus,or Nicotiana tabacum plants infected with potato virus Y.2.The resistance of 55 cucurbitaceous cultivars to PRSV-W was identified by indoor inoculation test.Of the 13 cucumber cultivars identified,the disease indices of Tangshan Qiumei,Tangshan Qiugua,Heat-resistant Wangzhong Wang and Liaoyan Cuiyu were 32.2,34.4,37.8 and 38.9,respectively,indicating that these cultivars had medium resistance;the other cultivars were susceptible or highly susceptible.The disease indices of Qiye Zaonanguan,Ruili Tianmiben,Japanese pumpkin and Yipin Tianhongli were between 31.1 and 39.4,indicating that these cultivars also had medium resistance.The disease indices of Changfeng Rousigua and Changfeng Changxiangsigua were 50.5 and 54.4,respectively,indicating that both cultivars were susceptible cultivars.The disease indices of 13 melon cultivars,11 watermelon cultivars,11 zucchini cultivars and 2 melon cultivars were higher than 60,indicating that all these cultivars were highly susceptible.3.The cross protection effiency of three attenuated mutants on wild-type PRSV was determined.With a protection interval of 5 days,none of the three attenuated mutants showed cross-protective effects.With an interval of 10 days,at 15 days after the challenge,the zucchini plants protected with the attenuated mutant N137 A had no obvious symptoms,and the protection efficiency was 100%.The virus accumulation level in the N137 A pre-inoculated plants was significantly reduced compared to that in plants inoculated with wild-type virus alone.The attenuated mutant N346 A delayed the appearance of PRSV-W symptom for 15 days,while attenuated mutant V417 A showed no cross-protective effect.With the interval of 15 days,the protective efficiencies of N137 A and N346 A were 100% and 26.7%,respectively,while V417 A still had no cross-protection effect.