Exogenous Melatonin For Virus Eradication From In Vitro-cultured Apple Shoots: Application And Mechanism

Viral diseases constitute a major constraint for the sustainable development of apple industry.Unlike other diseases induced by fungi and bacteria,viral diseases are difficult to control by use of any chemicals.Cultivation of virus-free plants is at present time widely used for effective control of apple viral diseases.Apple cholorotic leafspot virus(ACLSV),apple stem pitting virus(ASPV)and apple stem grooving virus(ASGV)are the three latent viruses that cause severe damage to the apple industry.ASPV did not infect meristematic issues and was easily eradicated,while ASGV can invade meristems and was difficulty eliminated from the infected plants.Availability of simple,highly efficient methods for virus eradication would facilitate production and cultivation of virus-free apple plants.Recently,melatonin has been proven to have multiple functions in regulating plant growth and development,and fortifying plants against abiotic stress(such as drought,salinity and extreme temperatures)and biotic stress(such as fungal and bacterial infections).However,studies on melatonin-mediated effects on virus resistance and virus eradication have been quite limited.Objective of the present study was therefore to:(1)investigate whether exogenous application of melatonin can improve resistance of plants to tobacco mosaic virus(TMV)and elucidate mechanisms involved in melatonin-mediated resistance of plants to TMV in the model plants;(2)test effects of exogenous application of melatonin on eradication of ASGV and ASPV from virus-infected in vitro apple shoots.Possible mechanisms as to why exogenous application of melatonin can efficiently eradicate ASGV and ASPV were revealed;(3)test if exogenous application of key substance of melatonin-mediated resistance to virus,salycilic acid(SA)and nitric oxide(NO),can improve ASGV and ASPV eradication from virus-infected in vitro apple shoots.Main results obtained in the present study were summarized as followings:(1)Nicotiana glutinosa,N.tabacum and Solanum lycopersicum were used to study melatonin-mediated resistance to TMV.Exogenous application of 100?M melatonin increased anti-virus infection activity to 37.4%in virus-infected N.glutinosa plants.The same treatment significantly reduced relative levels of virus RNA analyzed by RT-q PCR and virus titres measured by dot-ELISA,and increased the relative expression levels of the PR1 and PR5 genes analyzed by RT-q PCR in virus-infected S.lycopersicum.Melatonin treatment induced considerable accumulations of SA and NO,but did not significantly affect production of hydrogen peroxide(H₂O₂)in the virus-infected S.lycopersicum plants.Transgenic nah G N.tabacum was used to determine whether melatonin-induced TMV resistance was dependent on the SA pathway.The results showed that the relative RNA level of the TMV analyzed by RT-q PCR and virus titres analyzed by dot-ELISA were not significant reduced by the melatonin treatment in the nah G transgenic N.tabacum seedlings that had been treated twice with 100?M melatonin.The increased relative expression levels of PR1 and PR5 were considerably reduced when c PTIO,a NO scavenger,was included in the melatonin treatment.(2)Apple "Gala" single-infected with ASGV was used in this experiment.In vitro stock shoots were cultured for shoot proliferation on proliferation medium containing 0-20?M melatonin.Shoot tips(0.5 mm in size)containing 2 leaf primordia were excised from the shoots proliferated on melatonin-containing medium and cultured for shoot regeneration.Results showed that inclusion of 10-20?M melatonin in the proliferation medium significantly promoted shoot number and 10-15?M melatonin in the proliferation medium significantly increased shoot length.Melatonin treatment at 15?M resulted in the highest IAA level of the proliferating shoots,which was 1.5 times of the control.Shoot regrowth levels were significantly higher in shoot tips of the virus-infected shoots cultured for 4 weeks on the medium containing 15?M metalonin.Culture of shoot tips of the virus-infected in vitro shoots proliferated for 4 weeks on this medium resulted in 95%of shoots free of ASGV,while no virus-free shoots were obtained in those proliferated without melatonin.Exogenous application of 15?M melatonin significantly increased SA and NO level of the proliferating shoots.Analyses by RT-q PCR showed that ASGV concentration decreased by 60%in the virus-infected shoots proliferating on the medium containing 15?M melatonin for 4 weeks.Virus localization showed that exogenous application of melatonin enlarged the virus-free area in the virus-infected shoot tips.(3)Apple "Gala" double-infected with ASGV and ASPV was used in this experiment.The first three fully-opened leaves were taken from the virus-infected stock shoots.Leaf segments were trimmed to 0.8×0.6 cm,and four cuts at 1-mm intervals were made transversally to the midrib.Leaf segments were then cultured on the medium containing0-30?M melatonin for adventitious bud regeneration.After 3 weeks of bud regeneration,shoot tips(0.3-0.5 mm in size)containing 2-3 leaf primordia were excised from the adventitious buds and cultured for shoot regrowth.Exogenous application of 15?M and 30?M melatonin resulted in 100%organogenesis and produced 5.8 and 5.2 adventitious buds per leaf segment,which were significantly higher than 81.6%and 3.9 produced in the control.Melatonin treatment was also found to increase shoot regrowth and improve ASGV eradication.The highest virus eradication frequency was obtained when 15?M was used for induction of adventitious buds.Virus localization showed that exogenous application of melatonin enlarged the virus-free area in the adventitious buds regenerated from virus-infected leaf segments.These data provide explanations as to why exogenous application of melatonin can efficiently eradicate ASGV.(4)Apple "Gala" double-infected with ASGV and ASPV was used in this experiment.SA(0-100?M)and sodium nitroprusside(SNP,an NO donor,0-70?M)were added into shoot proliferation medium.Shoot tips(0.5 mm in size)with 2-3 leaf primordia were excised from the shoots proliferated on SA-and SNP-containing medium and cultured for shoot regeneration.The application of SA at 10?M,and SNP at 10-50?M to the shoot proliferation medium did not significantly affect shoot multiplication of in vitro shoots.Higher levels of SA(100?M)and SNP(70?M)inhibited the shoot growth,but improved ASGV and ASPV eradication.The results obtained from the present study demonstrated that melatonin treatments induced plant resistance to TMV.Exogenous applications of melatonin,NO and SNP promoted eradication of ASGV from the infected in vitro apple shoots,thus providing alternative methods for virus eradication.Therefore,the present study is of importance in theoretic and applied aspects.