The Presence of Tobacco Mosaic Virus in the Compost Extract of Cigar Tobacco Debris

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1 HAYATI Journl of Biosciences, September 28, p Vol. 1, No. 3 ISSN: The Presence of Tobcco Mosic Virus in the Compost Extrct of Cigr Tobcco Debris WIWIEK SRI WAHYUNI, MUHAMMAD HANAPI, IGNASIUS HARTANA Deprtement of Entomology nd Plnt Pthology, Fculty Agriculture, Jember University Jln Klimntn 37, Jember 68121, Indonesi Received Mrch 3, 28/Accepted August 12, 28 Tobcco mosic virus (TMV) is resistnce to high temperture nd ble to survive over 1 yers on dried leves, nd plnt debris is considered s source of inoculums of TMV in the field. In order to inctivte TMV, TMVinfected cigr tobcco debris ws composted t strting temperture of ºC for two to three dys; however, TMV ws still infective in the extrct compost. If hlf lef cigr tobcco 'H877' ws inoculted with compost extrct, the symptoms ppered s necrotic locl lesion (NLL) nd did not develop systemic lesions. The dilution end point of TMV in extrct compost ws 1 3. The number of lesion ws higher in the glsshouse with verge dylight temperture of 32 ºC thn in the field with verge dylight temperture of 293 ºC. The number NLL ws lower nd NLL size seemed to be smller on the first nd second inoculted leves with extrct thn tht of on the first nd second inoculted leves with TMV inoculums. There ws dely of time bout 816 hours fter of NLL from extrct compost inoculums to pper thn those of from TMV inoculums. These could be hppened becuse of minerl nutrients of compost nd lso the temperture of mintining tobcco plnt which inhibited the infections, nd of therml composting process which destroyed some TMV prticles, prticulrly degrded it s cot protein. Key words: TMV, extrct wter compost, cigr tobcco debris INTRODUCTION Cigr tobcco is mjor commercil plnt grows in Jember, Indonesi. Mostly, either Tobcco mosic virus (TMV) or Cucumber mosic virus (CMV) is still dominnt infected on the cigr tobcco frms. TMV hs mny strins, i.e. strin U1, U2, B (ben), C (cowpe), S (soyben), P (pes), nd vulgre, ll those strins could induce vry symptoms on tobcco. Strins U1, S, B, nd C induced necrotic locl lesion on Nicotin glutinos, N. tbcum Burley 21, Xnthi nc., nd Smsun (McDniel et l. 199). Some cultivrs resistnce to TMV hve been produced, i.e. H877 nd H894. They crry the N gene derived from Nicotin glutinos, the resistnce gene to TMV which induce necrotic locl lesion (Hrtn 1981). In H877, TMV in the glsshouse could develop systemic lesions or systemic necrotic burned, lso fter top pruning developed systemic necrosis on the stem, if the plnts were exposed to the temperture of 364 o C (Whyuni et l. 1999). TMV is considered s the most het resistnt plnt pthogen, stble in vitro, nd ble to survive over 1 yers in dried leves nd cigrettes or cigrs (Lucs 197). Therefore, the infected dried leves, cigr or cigrettes cn be the source of inoculums of TMV to helthy plnt. In the field, the most common source of virus inoculums is the debris of infected plnts which serve s reservoirs for virus trnsmission to helthy plnts, nd the improper disposl of these plnt Corresponding uthor. Phone: , Fx: , Emil: wswhyuni@fpert.unej.c.id residues cn contribute to recycling of virus s pthogens (Conwy 1996). In this pper, we wnted to observe whether processing compost of tobcco plnt debris would inctivte TMV. Formerly, this extrct ws suggested cn be used s pesticide becuse it contins nicotine. However, its nicotine concentrtion ws low bout.2.4%; so this extrct could only be used for pursuing wy smll insects nd this is not considered s pesticide (Whyuni 28, unpublished dt). MATERIALS AND METHODS Compost nd Compost Extrct Preprtion. TMVinfected 'H386' cigr tobcco debris ws collected from tobcco government frms t Jember. The debris ws cut into smll pieces, plced in the plstic continer nd dded with Effective Microorgnism 4 (EM4) nd rice brn, then incubted for 46 weeks. The strting temperture of composting process ws o C nd during the composting process, the debris ws stirred severl times to mintin semierobic conditions. About g CCO 3 ws dded to 1 kg degrded debris to increse the ph during decomposition process, nd the excess of decomposed debris solution ws llowed to drin off. The mtured compost ws extrcted with wter (1:2) nd the slurry ws strined through muslin cloth. Inoculte TMV from the Extrct Compost to Tobcco 'H877'. The hypersensitive cigr tobcco 'H877' to TMV ws used s model to observe whether TMV ws still survive in the compost extrct. TMV inoculums ws collected from cigr tobcco 'H382' t Jember, nd used s the positive control. About 1 g dried leves of TMVinfected tobcco ws ground

2 Vol. 1, 28 TMV IN THE COMPOST EXTRACT OF TOBACCO DEBRIS 119 with 1 ml of.1m phosphte buffer ph 7., then diluted to 1 nd inoculted on hlf lef of tobcco 'H877'. Compost extrct ws seril diluted t 1 1 to 1 7 with wter nd inoculted on hlf lef, ech tretment ws replicted six times. The development of symptoms nd the number of locl lesions were observed three times dy, on 7..m., 12. p.m., nd 17. p.m. for 9 dys fter. Distribute Locl Lesion. To ensure tht the necrotic locl lesion (NLL) of TMV my develop systemiclly on the subsequence leves of hypersensitive tobcco, the experiment ws done by inoculting lef with 1 ml extrct t dilution of 1 2, then fter 7 dys lter the subsequence lef ws inoculted with 1 ml extrct t dilution of 1 2. For the positive control, the first ws done with 1 ml TMV inoculums t dilution of 1 nd the second ws done fter 7 dys lter with the sme TMV dilution. For the negtive control, the first ws done with 1 ml H 2 O, nd second ws done fter 7 dys lter with the extrct t dilution of 1 2, s shown on Figure 1. This experiment ws repeted twice nd the lter ws locted in two different conditions of mintining of plnts which might effect to the development of locl lesions. The first condition ws under glsshouse, nd the second condition ws in the field. The development locl lesions were observed on 1 to 1 dys fter the first of lef with either compost extrct t 1 2 dilution or H 2 O. RESULTS Compost from TMVInfected Tobcco Debris. During the composting process, the strting temperture in the plstic continer incresed up to bout o C for the first of twothree dys, nd fter tht decresed bout 4 to 4 o C until plnt debris well composted. The mesurement of temperture might be ws not ccurte becuse done only during the dylight, but it should be lso done during the night where the temperture outside the continer ws cooler. Plnt debris composted well for two months processing, then extrcted with wter (1:3 w/v) nd this ws designted s compost extrct. Number Locl Lesions Produced by Inocultion Lef with Compost Extrct in Seril Dilution. Inocultion of compost extrct in seril dilution on tobcco 'H877' lef produced smll NLL. It ws stted tht the number of smll necrotic lesion expressed the virus mount in compost extrct. The number of locl lesion ws ffected by the temperture in the experiment loction nd the dilution of extrct (Figure 2). In the glsshouse with the verge dylight temperture of 32 o C, the first symptoms ws observed on 12 hours fter (h..i.), while in the field with the verge temperture of 293 o C, the first symptoms ws observed on 1169 h..i. The first NLL produced from TMV inoculums were observed t 44 h..i. (Figure 2). Sometimes during with compost the 3 rd lef Dy 7 fter the first Second with compost the th lef fter 7 dys lter with compost the 3 rd lef with H 2 O on the 3 rd lef Second with compost the th lef fter 7 dys lter with H 2 O on the 3 rd lef Figure 1. Inocultion of compost extrct t dilution of 1 2 on the subsequence leves of cigr tobcco 'H877'.. inoculted lef with compost extrct nd second inoculted lef with compost extrct, b. inoculted lef with H 2 O nd second inoculted lef with compost extrct. b Number of locl lesions Positive control Hours fter b Number of locl lesions Positive control Hours fter Figure 2. The number of locl lesions produced by inoculted hlf tobcco lef with compost extrct in seril dilutions, on 9 dys fter the first.. The glsshouse experiment with verge of dylight temperture 32 o C. b. The field experiment with verge of dylight temperture 293 o C. All extrct tretments were replicted six times. The positive control ws with TMV inoculums t 1 dilution

3 12 WAHYUNI ET AL. HAYATI J Biosci 11.3.m.1.3 p.m. the temperture in the glsshouse could increse to 3 o C. TMV in the compost extrct ws still infective t 1 3 dilution, lthough compost ws stored for 4 months since compost ws extrcted. Thus, the dilution end point (DEP) of TMV in the compost extrct of tobcco debris ws 1 3, in the field experiment ws 2 NLL nd in the glsshouse ws NLL (Figure 2). Distribute Locl Lesion. After 9 dys in the glsshouse, the number of NLL ws lower on the first inoculted lef with H 2 O nd the second inoculted lef with compost extrct thn those of on the first inoculted lef with H 2 O nd the second inoculted lef with TMV inoculums. The number of NLL ws lso lower on the first nd second inoculted leves with extrct thn those of on the first nd second inoculted leves with TMV (Tble 1). At the sme time of observtion, the distribution of NLL on the second inoculted lef ws less thn on the first inoculted lef with either compost extrct or TMV inoculums. The size of NLL ws smller in the second inoculted lef thn in the first inoculted lef. The NLL of TMV inoculums on 67 h..i. ws shown s individul NLL but on 7 h..i. some individuls NLL were joint together to form lrger necrotic lesion (Figure 3). However, this phenomenon did not shown with the compost extrct, until 14 h..i. the necrotic lesions were still observed s individul NLL (Figure 3 & 4); with the mximum individul NLL dimeter of..6 cm 2 nd the minimum NLL dimeter ws less thn.2 cm 2 (Tble 1). In the field experiment, there ws reduction in number of NLL followed with the smller NLL size on the second inoculted lef. The NLL dimeter on the first inoculted lef ws more thn.4 cm 2 nd on the second inoculted lef with compost extrct ws less thn.2 cm 2. The number NLL ws lower on the second inoculted lef with compost extrct, followed with the smller NLL size thn tht of on the first inoculted lef, t the sme time observtion (Tble 2). b c d b c Figure 3. Development of TMV lesion on hlf lef inoculted with 1 ml TMV inoculums t dilution of 1 under the glsshouse conditions.. Symptoms on 14 h..i, b. on169 h..i, nd c. helthy lef. Figure 4. Comprison the number nd size of NLL from. compost extrct of tobcco debris nd b. TMV inoculums, in the glsshouse. The number of NLL on the second inoculted lef ( nd b) ws lower thn those of on the first inoculted lef ( nd c), nd NLL size on b nd d ws smller thn those of on nd c. Tble 1. Number NLL nd NLL size on the leves inoculted with either compost extrct or TMV inoculums in the glsshouse NLL on the first inoculted lef NLL on the second inoculted lef Inocultion Number NLL NLL size Number NLL NLL size (1) extrct (2) extrct (1) H2O (2) extrct (1) TMV (2) TMV (1) H2O (2) TMV A = 2, B =, C = 4 A = 16, B = 1, C = B = 2, C = 2 B = 7, C = 2 B = 1, C = 7 A = 2, B = 24, C = 11 (1) The first ws done on the lef number 3 from the bse nd (2) the second ws on the lef number from the bse, nd this ws done on 7 dys fter first. The first symptoms for TMV inoculums were observed t 44 h..i. nd for the compost extrct t 12 h..i. The extrct volume ws 1 ml nd diluted t 1 2. Experiment ws done in the glss house with the verge dylight temperture of 32 o C. The dimeter of NLL A >.4 cm 2 ; B =.2.39 cm 2 ; C <.2 cm 2. Dt ws the verge of six replictions nd ws observed t the sme time.

4 Vol. 1, 28 TMV IN THE COMPOST EXTRACT OF TOBACCO DEBRIS 121 Tble 2. Number NLL nd NLL size on the leves inoculted with either compost extrct of tobcco debris or TMV inoculums in the field Inocultion NLL on the first inoculted lef NLL on the second inoculted lef Number NLL NLL size Number NLL NLL size (1) extrct (2) extrct (1) H2O (2) extrct (1) TMV (2) TMV (1) H2O (2) TMV 27 A = 1, B = 2, C = 2 A = 6, B = 1, C = C B = 2, C = 1 C A = 3, B = 4, C = 2 (1) The first ws done on the lef number 3 from the bse, nd (2) the second ws on the lef number from the bse, nd this ws done on 7 dys lter. The first symptoms for TMV inoculums were observed t 6 h..i. nd for the compost extrct 1 h..i. The extrct volume ws 1 ml nd diluted t 1 2. The experiment ws done in the field with the dylight temperture 293 o C. The dimeter of NLL A >.4 cm 2 ; B=.2.39 cm 2 ; C <.2 cm 2. Dt ws the verge of six repliction nd ws observed t the sme time. DISCUSSION Although tobcco plnt debris ws composted with EM4, the compost extrcts still contined TMV. This ws shown by NLL produced on cigr tobcco 'H877' which crries the N gene. This compost ws processed t bout 4 o C for two to three dys, nd the compost extrct t dilution of 1 3 still contined TMV, it ws shown by 2 NLL in the field experiment nd NLL in the glsshouse fter 217 hours. However, Ghly et l. (26) found tht compost of tomto debris infected with TMV which process in biorector with temperture of 636 o C for four dys could inctivted TMV. A therml process is cpble of destroying TMV s other plnt pthogens. They lso found tht under the sme conditions, of tht compost of TMV infected tomto which processed with temperture 636 o C reduced of,, 2, 12, 3 NLL on tobcco, nd of,,, 2, 6, 1 NLL on tomto fter 24, 48, 64, 96, 12, nd 144 hrs of thermophilic tretments, respectively, while the untreted TMV produced 1 NLL on tobcco, nd 22 NLL on tomto. It seemed there ws different host rection on those tretments. Inocultion hlf lef with either compost extrct or TMV inoculums showed tht NLL did not distribute systemiclly to djcent hlf lef, under dylight temperture of 3 o C in the field. But under glsshouse conditions, NLL from TMV inoculums seemed to develop s systemic lesions. As Hull (22) mentioned tht hypersensitive rection (HR) of TMV which resulted in NLL occur t temperture bove 28 o C. Becuse of the temperture in this experiment ws 32 o C, NLL could develop to systemic necrotic lesions. In the other experiment, Whyuni et l. (1999) found when the temperture in the glsshouse ws bove 34 o C, the NLL of TMV on tobcco 'H877' ws firstly distributed loclly on the inoculted lef, nd then this necrotic developed well, very fst, nd distributed systemiclly to big necrotic lesions nd cused some leves dried. This ws lso determined by White nd Antoniw (1991), if tobcco contins the N gene ws mintined on bove 34 o C, TMV firstly produced locl lesion then developed to systemic lesions nd sometimes to mosic systemic. Temperture ws lso effect the number nd size of NLL. In the field experiment, the number nd size of NLL ws smller thn in the glsshouse. Besides it ffected the number of NLL, the different temperture lso effected the incubtion period of TMV on tobcco 'H877' in producing NLL. In the glsshouse with the verge temperture of 32 o C, the first NLL produced from compost 12 h..i. nd from TMV inoculums on 44 h..i., while in the field with the verge dylight temperture 293 o C, the fist NLL from compost extrct produced on 169 h..i. It seemed there ws dely of time (816 h..i.) for TMV from compost extrct to pper s NLL compred to TMV from TMV inoculums. Tht dely of time could be cused by the minerlnutrition contined in the extrct tht influenced on virl infection nd it inhibited during the incubtion period (Ghly et l. 26), nd on the hypersensitive tobcco, symptoms of TMV ws inhibited by the temperture more thn 28 o C (Hull 22). In 1 ml of 1 2 dilution of compost extrct produced bout to 11 NLL per lef, while in 1 ml TMV inoculums which diluted to 1 produced 3 to 4 NLL. This ws indicted tht lthough tobcco debris ws composted nd the compost extrct ws stored for over 3 months, TMV ws still present in the extrct which diluted to 1 3. As Lucs (197) mentioned the DEP of TMV in lef sp ws bout 1 7, however, the DEP of compost extrct ws reltively high, 1 3, nd this difference could be cused by the het during the composting process which inctivted some TMV prticles. The production of NLL indictes tht the resistnce tobcco H877 to TMV is systemic cquired resistnce (SAR). This SAR showed by the reduction in the number nd size NLL (Hull 22), nd this could be cused by slicylic cid which ctivte the gene PR1 protein to induce hypersensitive rection to TMV (Linthorst 1991; Whyuni et l. 1999). TMV is considered s the most resistnt plnt pthogens to high temperture (Hull 22). By observing the low NLL produced, some TMV prticles might be destroyed by the temperture of composting process of o C for bout twothree dys, prticulrly degrded its cot protein. REFERENCES Conwy KE An overview of the influence of sustinble griculturl system on plnt diseses. Crop Protection 1: Ghly AE, Alkoik F, Snow A, Singh R. 26. Effective thermophilic composting of crop residues for inctivtion of tobcco mosic virus. Am J Biochem Biotech 2: Hrtn I Control of mosic disese in Besuki cigr tobcco through resistnce. Ind Agric Res Dev 3:3238. Hull R. 22. Mtthews Plnt Virology. Sn DiegoLondon: Acd Pr. Linthorst HJM Pthogenesis relted proteins of plnts. Crit Rev Plnt Sci 1:1231.

5 122 WAHYUNI ET AL. HAYATI J Biosci Lucs GB Disese of Tobcco. 3 rd Ed. RleighNorth Crolin: Biologicl Consult Assoc. McDniel LL, Mrtos ML, Goodmn JE, Tolin SA Prtil chrcteristic of soyben strin of tobcco mosic virus. Plnt Dis 79: Whyuni WS, Trisusilowti EB, Sulistynto D Totl protein content nd pthogenesis reltedproteins in lef intercellulr fluid of cigr tobcco cultivr H877 infected with tobcco mosic virus (TMV). J Perlind Tn Ind :117. White RF, Antoniw JF Virus induced resistnce responses in plnts. Crit Rev Plnt Sci 9: