Cross-protection of cotton against Verticillium wilt by Verticillium nigrescens

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1 Emirtes Journl of Food nd Agriculture (9): doi: /ejf REGULAR ARTICLE Cross-protection of cotton ginst Verticillium wilt y Verticillium nigrescens Ionnis Vgels*, Stefnos Leontopoulos Deprtment of Agronomy Technology, Lortory of Plnt Protection, Technologicl Eductionl Institute of Thessly, 41110, Lriss, Greece ABSTRACT Two species of plnt pthogenic fungi cusing Verticillium wilt of cotton, Verticillium dlhie Kle. nd V. nigrescens Pethyr., proved ggressive nd wekly pthogen of cotton, respectively, nd were used in this study. Cotton cultivrs cvs 4S nd Stoneville 453, susceptile to Verticillium wilt disese, were treted with oth orgnisms in order to determine possile cross protection effects of V. nigrescens ginst V. dhlie. Results showed tht the cotton plnts which were inoculted on the sme dy s V. dhlie prior to inocultion y V. nigrescens were showing ggressive wilt symptoms of Verticillium wilt of cotton. However, the plnts inoculted on the sme dy s V. nigrescens prior to inocultion y V. dhlie resulted low to mild wilt symptoms. Control of Verticillium wilt of cotton ws otined when V. nigrescens ws inoculted four dys prior to inocultion y V. dlhie. Those promising results where further explined y dt otined from the trnspirtion rtes of the mss flow of sp in cotton stems nd demonstrted tht conidi of V. nigrescens could protect the cotton plnts from virulent conidi of V. dhlie. Keywords: Biocontrol; Vsculr diseses; Defense mechnisms induced y fungi INTRODUCTION Verticillium wilt cused y Verticillium dlhie Kle., is well-known serious disese of mny economicl crops, especilly of cotton where it cn reduce cotton yield gretly under certin environmentl conditions (Pegg, 1984; Bht nd Suro, 1999). The fungus is widespred in most cotton cultivted res nd is one of the gretest threts to cotton production worldwide (Erdogn nd Benlioglum, 2010) s well s in cotton res in Greece where this disese is one of the min constrints to cotton production especilly in the region of Thessly (Tjmos et l., 2000). Most cotton plnts re symptomtic t initil infection (Shen, 1992; Xio et l., 1998; M, 2007), thus, cotton yield losses cn e reduced effectively when cotton Verticillium infection is predicted erly nd ccurtely, nd prophylctic mesures re properly tken (Jing nd Hung, 2010). However, ccording to Pplomts et l., (1992) when infection occurs t lter stges infected plnts usully exhiit symptoms of mrginl necrosis in leves, discolortion of the stem vsculr undles, decrese in photosynthesis nd increse in respirtion, resulting in significnt reduction of the plnt iomss nd significnt loss in yield. The study of the symptoms on the infected plnts depends minly on the mnul investigtion nd smpling in fields which is time nd effort consuming nd difficult to prctice for lrge res. Thus, correltion nlysis of lef spectrl reflectnce nd disese severity of cotton Verticillium hs shown tht there re numer of sensitive nds tht spred in severl spectrums (Ning et l., 2013). In generl, different methods of controlling Verticillium wilt of cotton plnt like het tretment, fumigtion with rodspectrum iocides nd eco-friendly friendly lterntive methods like rhizocteri (Weller, 1998; Frvel nd Lrkin, 2000; Lnd et l., 2004) mycoprsites (Grunden et l., 2001; Reknovic et l., 2007; Zheng et l., 2011) nd endophytes (Yng et l., 2013) hve een well-reported. Furthermore, ccording to Xue et l., (2013) shown tht the ppliction of powdered iocontrol gent contining living ctinomycetes could e useful for iocontrol of the plnt pthogen in griculturl production. The isoltes *Corresponding uthor: Ionnis Vgels, Deprtment of Agronomy Technology, Lortory of Plnt Protection, Technologicl Eductionl Institute of Thessly, 41110, Lriss, Greece. E-mil: s_leontopoulos@yhoo.com Received: 11 April 2015; Revised: 22 June 2015; Accepted: 26 June 2015; Pulished Online: 26 June 2015 Emir. J. Food Agric Vol 27 Issue

2 Vgels nd Leontopoulos: Cross-protection of cotton ginst Verticillium wilt successfully colonized the cotton rhizosphere in wilt-sick field study when the powdered iocontrol gent ws pplied either s seed coting or s soil inocultion. Moreover, plnting resistnt vrieties is possile strtegy for protection of cotton from V. dhlie, ut few uplnd cotton cultivrs showed resistnce towrds Verticillium wilt (Bolek et l., 2005; Gore et l., 2009; Go et l., 2011). Furthermore, orgnic mendments in pplied soil lso reduced disese severity in oth inoculted pots nd nturlly infested cotton field plots. According to Hung et l., (2006) the most effective control ws chieved with cr shell (chitin), soyen stlk nd lflf. The im of this study ws to to determine possile cross protection effects of V. nigrescens ginst V. dhlie in susceptile cotton cultivrs in the region of Thessly, Greece. MATERIALS AND METHODS In the present study the severe pthogen (V. dlhie) ws isolted, in the lortory of plnt protection of Technologicl Eductionl Institute of Thessly (T.E.I.), severl times from cotton plnts showing symptoms of Verticillium wilt t severl loctions in centrl Greece. In order to determine protection ginst virulence isolte of V. dlhie, n isolte of V. nigrescens, comprtively weker vsculr pthogen, recovered from cotton plnts showing wek wilt symptoms, ws tested. Following tht we tested cross protection effects in cotton ginst disese induced y virulent isolte of V. dlhie with the weker vsculr pthogen V. nigrescens four dys prior to inocultion with chllenger strin sed on the ide presented (Melouk nd Horner, 1975). Moreover, tht cross protection effects were tested in process iochemicl mechnisms, ntifungl mechnisms, such s phytolexins of cotton plnts in response to inoculums of the ggressive pthogen (V. dlhie) s represented in previous reserch work (Zki et l., 1972). Both pthogens were isolted from cotton vsculr tissues nd grown on PDA pltes which were incuted in drkness t 22 o C for 20 dys. Pthogenicity tests were determined y immersing cotton plnt roots (shoot tip cutting) for 45 min in Verticilliums solution, which contined conidi/ml. This high spore suspension cn ffect plnt tissues nd cuse Verticillium disese symptoms (Pegg nd Brdy, 2002). Treted plnts were trnsplnted in pots nd kept in glsshouse with o C for 45 dys. Two cotton (Gossypium hirsutum L.) cultivrs, 4S nd Stoneville 453, susceptile to Verticillium wilt were used. The experiment ws designed with seven tretments nd 21 replictions, s follows: ) Immersing the root cut ends into V. dhlie (Vd), in conidi suspension ( conidi/ml), for 45 min (Melouk nd Horner, 1975). ) Immersing the root cut ends into V. nigrescsens (Vn), in conidi suspension ( conidi/ml), for 45 min. c) Immersing the root cut ends into V. nigrescens, in conidi suspension ( conidi/ml), for 45 min nd susequently, on the sme dy, inocultion with V. dhlie (Vn + Vd), y dding spore suspension (5x10 5 spores/ml) in the pot (10 ml/pot). d) Immersing the root cut ends into V. nigrescens, in conidi suspension (5x10 5 conidi/ml), for 45 min nd fter 4 dys inocultion with V. dhlie (Vn + 4Vd), y dding spore suspension (5x10 5 conidi/ml) in the pot (10 ml/pot). e) Immersing the root cut ends into V. dhlie, in conidi suspension (5x10 5 conidi/ml), for 45 min nd susequently, on the sme dy, inocultion with V. nigrescens (Vd + Vn), y dding spore suspension (5x10 5 conidi/ml) in the pot (10 ml/pot). f) Immersing the root cut ends into V. dhlie, in conidi suspension ( conidi/ml), for 45 min nd fter 4 dys inocultion with V. nigrescens (Vn 4Vd), y dding spore suspension (5x10 5 conidi/ml) in the pot (10 ml/pot). g) Immersing the root cut ends into sterile wter (Control plnts). Numer of lef discolortion (folir dmge y 0-5 rting scle where 0: no folir symptoms; 3: 25% of plnts showing evidence of lef yellow; 5: severe folir dmge), stem vsculr stining (drker in color y 1-4 rting scle where 1: light vsculr stining to 4: vsculr stining drker in color), stem height, dimeter, fresh weight nd dry weight of inoculted plnts were ssessed t the end of the experiment, fter 45 dys of incution. All dt were nlysed y nlysis of vrince (ANOVA) nd presented with grphs using Microsoft Excel. Xylem sp flow investigtion in cotton plnts infected with or without Verticillium species ws determined s follows. The study ws crried out fter 40 dys of inocultion in eight periods (30 to 240 min, Fig. 4), using two rndom plnts for ech period. The proes of phytometric system LPS-03 Phytomonitor (PhyTech Ltd) ws estlished into the stem up to height of 20 cm from soil surfce to mesure sp flow density. RESULTS AND DISCUSSION The results showed tht cotton plnts inoculted with only V. nigrescens were symptomless, otined significnt high 688 Emir. J. Food Agric Vol 27 Issue

Vgels nd Leontopoulos: Cross-protection of cotton ginst Verticillium wilt vlues for stem height, dimeter, fresh nd dry weight nd significnt less vlues for numer of lef discolortion nd stem vsculr

dlhie, mild wilt symptoms occurred in ll cotton tested plnts, greeing with Korolev nd Ktn, (1999). Plnts which were inoculted on the sme dy s V. nigrescens efore the inocultion with V.

3 Vgels nd Leontopoulos: Cross-protection of cotton ginst Verticillium wilt vlues for stem height, dimeter, fresh nd dry weight nd significnt less vlues for numer of lef discolortion nd stem vsculr stining compred to those cotton plnts inoculted with only V. dlhie (Figs. 1-3). When inocultion with V. nigrescens precedent V. dlhie, mild wilt symptoms occurred in ll cotton tested plnts, greeing with Korolev nd Ktn, (1999). Plnts which were inoculted on the sme dy s V. nigrescens efore the inocultion with V. dhlie were sttisticlly different from those inoculted on the sme dy s V. dhlie efore the inocultion with V. nigrescens, in numer of lef discolortion, stem vsculr stining, dimeter, fresh nd dry weight (Figs. 1-3). When V. nigrescens ws inoculted four dys fter V. dlhie, significnt wilt symptoms were oserved s mesurements such s stem dimeter, stem height, folir dmge, stem discolourtion, stem dry weight nd stem fresh weight were done (Figs. 1-3). Moreover, the results of Figs. 1-3 lso showed tht when V. dlhie ws inoculted four dys fter V. nigrescens, significnt less wilt symptoms ws oserved, nd we cn sy tht V. nigrescens induced crop protection effect ginst V. dlhie when introduced first in cotton plnt rhizosphere. As determined y mesurement the trnspirtion rtes of mss flow of sp in the cotton stem (Figs. 4), the rte of mss flow of sp incresed significntly in plnts inoculted with only V. nigrescens nd in plnt when V. dlhie ws inoculted four dys fter V. nigrescens. This is in greement with previous reserch work, where cross protection effects were oserved in peppermint when inocultion with V. nigrescens preceded inocultion with V. dhlie y two dys (Melouk nd Horner, 1975). CONCLUSION In the present investigtion results show tht it is possile to reduce disese induced y virulent isolte of V. dlhie, when inoculted first with wek pthogen, V. nigrescens. It is elieved tht cross-protection of cotton to severe isolte of V. dlhie ws possile due to ccumultion into xylem of ntifungl secondry metolites in response to Fig 1. Cotton stems dimeter () nd height (), in inoculted plnts with V. nigrescens nd V.dhlie. Fig 2. Cotton folir dmge () nd stem discolortions (), in inoculted plnts with V. nigrescens nd V.dhlie. Emir. J. Food Agric Vol 27 Issue

Vgels nd Leontopoulos: Cross-protection of cotton ginst Verticillium wilt Fig 4. Mss flow of sp in the cotton Stems inoculted with V. nigrescens nd V.dhlie. Fig 3.

4 Vgels nd Leontopoulos: Cross-protection of cotton ginst Verticillium wilt Fig 4. Mss flow of sp in the cotton Stems inoculted with V. nigrescens nd V.dhlie. Fig 3. Cotton stems fresh weight () nd dry weight (), in inoculted plnts with V. nigrescens nd V.dhlie. inocultion with V. nigrescens, (Zki et l., 1972; Schnthorst nd Mthre, 1996). Finlly, we cn concluded tht the crossprotection phenomenon reported in the present study is form of iologicl control occurred in nture nd my hve ppliction in control of wilt diseses in plnt (Price nd Sckston, 1989; Huerts-Gonzlez et l., 1999; Erdogn et l., 2013). Author contriutions The work is product of the intellectul environment of oth uthors which hve contriuted in vrious degrees to the nlyticl methods used, to the reserch concept, nd to the experiment design. Dr. I. Vgels designed the study, developed the methodology, performed the nlysis while Dr. S. Leontopoulos collected the dt, nd mde the isoltions. Both uthors contriuted in writing the mnuscript. REFERENCES Bht, R. G. nd K.V. Suro Host rnge specificity in Verticillium dhlie. Phytopthology. 89: Bolek, Y., K. M. El-Zik nd A. E. Pepper Mpping of Verticillium wilt resistnce genes in cotton. Plnt Sci. 168: Erdogn, O. nd K. Benlioglu Biologicl control of Verticillium wilt on cotton y the use of fluorescent Pseudomons spp. under field conditions. Biol. Cont. 53: Erdogn, O., S. Nemli, T. Oncu nd B. Tnyolc Genetic vrition mong pthotypes of Verticillium dhlie Kle. from cotton in western Turkey reveled y AFLP. Cn. J. Plnt Pth. 35: Frvel, D. R. nd R. P. Lrkin Effect of sulethl stresses on microscleroti of Verticillium dhlie. In: Tjmos, E. C., R. C. Rowe., J. B. Hele nd D. R. Frvel., (Eds.). Advnces in Verticillium Reserch nd Disese Mngement. Americn Phytopthologicl Society (APS) Press, St. Pul, Minessot., Pp Go, X., T. Wheeler., Z. Li., C.M. Kenerley., P. He. nd L. Shn Silencing GhNDR1 nd GhMKK2 compromised cotton resistnce to Verticillium wilt. Plnt J. 66: Gore, E. M., O. K. Cner., N. Altın., M. H. Aydın., O. Erdogn., F. Filizer nd A. Buyukdogerlioglu Evlution of cotton cultivrs for resistnce to pthotypes of Verticillium dhlie. Crop Prot. 28: Grunden, E., W. Chen nd J. L. Crne Fungi colonizing microscleroti of Verticillium dhlie in urn environments. Fungl Divers. 8: Hung, J., H. Li nd H. Yun Effect of orgnic mendments on Verticillium wilt of cotton. Crop Prot. 25: Huerts-Gonzlez, M. D., M. C. Ruiz-Roldn., A. Di Pietro nd 690 Emir. J. Food Agric Vol 27 Issue

5 Vgels nd Leontopoulos: Cross-protection of cotton ginst Verticillium wilt M. I. G. Roncero Cross protection provides evidence for rce-specific virulence fctors in Fusrium oxysporum. Phys. Mol. Plnt Pth. 54: Jing, X., W. J. Hung nd Y. Ju Remote sensing monitoring severity level of cotton Verticillium wilt sed on prtil lest squres regressive nlysis. Trns. CSAE. 26: Jin, N., W. Hung., Y. Ren., J. Luo., Y. Wu., Y. Jing nd D. Wng Hyperspectrl identifiction of cotton Verticillium disese severity. Optik. 124: Lnd, B. B., J. A. Nvs-Cortés nd R. M. Jiménez-Díz Influence of temperture on plnt: Rhizocteri interctions relted to iocontrol potentil for suppression of Fusrium wilt of chickpe. Plnt Pthol. 53: M, C Studying on Verticillium wilt nd Fusrium wilt of cotton. In: Jin, G. L., J. R. Yng., J. Feng nd J. X. He. (Eds.). Breeding of Cotton Resistnt Vrieties. Vol. 1. Chinese Agriculturl Press, Beijing, Pp Melouk, H. A. nd C. A. Horner Cross protection in mints y Verticillium nigrescens ginst V. dhlie. Phytopthology. 65: Pplomts, E. J., D. M. Bssett., J. C. Broome nd J. E. Devy Incidence of Verticillium wilt nd yield losses of cotton cultivrs (Gossypium hirsutum) sed on soil inoculum density of Verticillium dhlie. Phytopthology. 82: Pegg, G. F The impct of Verticillium diseses in griculture. Phytopthol. Mediterr. 23: Pegg, G. F. nd B. L. Brdy Verticillium Wilts. CABI Pulishing, Oxon, UK. Price, D. nd W. E. Sckston Cross protection mong strins of Verticillium dhlie on sunflower. Vsculr wilt diseses of plnts. NATO ASI Series. 28: Reknovic, E., S. Milijsevic., B. Todorovic nd I. Potocnik Possiilities of iocontrol nd chemicl control of Verticillium wilt in pepper. Phytoprsitic. 35: Shen, Q. Y Cotton disese-sic reserch nd protection. In: Deng, Y. S., Q. J. Li nd Q. Y nd Chen, Q. Y. (Eds.). Identifiction of the Resistnce of Cotton Vrieties. Vol. 1. Science Press, Beijing, Pp Schnthorst, W. C. nd D. E. Mthre Cross protection in cotton with strins of Verticillium lo-trum. Phytopthology. 56: Tjmos, E. C., R.C. Rowe., J. B. Hele nd D. R. Frvel Advnces in Verticillium Reserch nd Disese Mngement. APS Press. The Americn Phytopthologicl Society, St. Pul, MN, p Weller, D. M Biologicl control of soilorne plnt pthogens in the rhizosphere with cteri. Ann. Rev. Phytopthol. 26: Xio, C. L., K. V. Suro., K. F. Schulch nd S. T. Koike Effects of crop rottion nd irrigtion on Verticillium dhlie microscleroti in soil nd wilt in culiflower. Phytopthology. 88: Xue, L., Q. Xue, Q. Chen, C. Lin, G. Shen nd J. Zho Isoltion nd evlution of rhizosphere ctinomycetes with potentil ppliction for iocontrol of Verticillium wilt of cotton. Crop Prot. 43: Yng, P., Z. Sun., S. Liu., H. Lu., Y. Zhou nd M. Sun Comining ntgonistic endophytic cteri in different growth stges of cotton for control of Verticillium wilt. Crop Prot. 47: Zki, A. I., N. T. Keen nd D. C. Erwin Impliction of vergosin nd hemigossypol in the resistnce of cotton to Verticillium lotrum. Phytopthology. 62: Zheng, Y., Q. Y. Xue., L. L. Xu., Q. Xu., S. Lu., C. Gu nd J. H. Guo A screening strtegy of fungl iocontrol gents towrds Verticillium wilt of cotton. Biol. Cont. 56: Emir. J. Food Agric Vol 27 Issue

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