SHORT COMMUNICATION BIOCONTROL OF FUSARIUM WILT OF SPINACH BY THE PLANT GROWTH PROMOTING FUNGUS FUSARIUM EQUISETI GF183

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1 Journl of Plnt Pthology (2010), 92 (1), Edizioni ETS Pis, SHORT COMMUNICATION BIOCONTROL OF FUSARIUM WILT OF SPINACH BY THE PLANT GROWTH PROMOTING FUNGUS FUSARIUM EQUISETI GF183 H. Horinouchi 1, A. Muslim 2 nd M. Hykumchi 3 1 Gifu Prefecturl Agriculturl Technology Center, 729 Mtmru, Gifu , Jpn 2 Deprtment of Plnt Pests nd Diseses, Fculty of Agriculture, Sriwijy University, Indrly Cmpus, Plembng 30662, Indonesi 3 Lbortory of Plnt Pthology, Fculty of Applied Biologicl Sciences, Gifu University, 1-1 Yngido, Gifu , Jpn SUMMARY The plnt growth promoting fungus Fusrium equiseti GF183 effectively controlled Fusrium wilt of spinch cused by Fusrium oxysporum f. sp. spincie in trnsplnting systems using pper pots. Reduction in disese severity rnged from 43.5 to 91.8%. Double ppliction of F. equiseti GF183 incresed the protective effects. The number of colony-forming units of F. oxysporum f. sp. spincie per grm fresh weight of roots ws significntly reduced (P<0.05) in plnts treted with F. equiseti. Root extrcts from both F. equiseti-treted plnts nd F. equiseti nd pthogen-treted plnts significntly inhibited new production of budding-cells of F. oxysporum f. sp. spincie. Key words: Fusrium oxysporum f. sp. spincie, PGPF Fusrium equiseti, biocontrol, spinch. Fusrium wilt of spinch (Spinci olerce L.) (FWS) cused by Fusrium oxysporum Schlechtend: Fr. f. sp. spincie (Sherb) W.C. Snyder & H.N. Hnsen (FOS) is serious constrint to spinch production worldwide (Correl et l., 1994; Lrsson nd Gerhrdson, 1992). It cuses dmping-off, wilting, root rot nd discolortion of the vsculr system of seedlings nd mture plnts. Although the use of resistnt cultivrs is considered n effective mens to control Fusrium wilt (Beckmn, 1987), FWS-resistnt cultivrs hve been reported to hve only some resistnce to the disese (O Brien nd Winter, 1977). Although soil fumigtion with chloropicrin or methyl bromide hs been used to control the disese in Jpn, its use hs cused severe environmentl problems. Thus, lterntive control methods must be mde vilble s soon s possible. The use of biocontrol gents to control Fusrium wilt hs been reported for mny crops including tomto, cucumber, melon, strwberry, bnn nd crntion. There re lso some reports on the use of biocontrol Corresponding uthor: M. Hykumchi Fx: E-mil: hykumc@gifu-u.c.jp ginst FWS, including non-pthogenic F. oxysporum (Ktsube nd Aksk, 1997), hypovirulent binuclete Rhizoctoni (Muslim et l., 2003) nd Enterobcter cloce (Tsud et l., 2001). A pper pot trnsplnting system is nother method used for controlling soil-borne diseses such s FWS (Ktsube nd Aksk, 1997) nd common potto scb (Nito et l., 1998). Ktsube nd Aksk (1997) reported tht high suppression effect ginst FWS ws obtined by trnsplnting spinch seedlings grown in pper pots contining non-pthogenic F. oxysporum. The sprophyte plnt growth promoting fungus (PGPF) Fusrium equiseti, obtined from turfgrss rhizospheres, hs been reported to enhnce plnt growth significntly nd suppress severl soil-borne diseses cused by Pythium spp., Rhizoctoni solni, Sclerotium rolfsii, Geumnnomyces grminis vr. tritici nd Cochliobolus stivus (Hykumchi, 1994; Hykumchi nd Kubot, 2004). We hve previously shown tht F. equiseti cn lso control Fusrium crown nd root rot of tomto cused by F. oxysporum f. sp. rdicis-lycopersici in both hydroponic rock-wool system nd soil system (Horinouchi et l., 2007, 2008). From the results of our preliminry experiments, we found tht mong severl bcteri nd fungi used s biocontrol gents, the isolte of PGPF F. equiseti effectively controls FWS. The present study ws conducted to investigte the effect of F. equiseti in controlling FWS. We used F. equiseti isolte GF183. The FOS isolte, S1HI-4, obtined from the Iwte Prefectul Agriculturl Experiment Sttion, ws used s pthogen. Spinch cv. Snrito (Skt Seed, Jpn), populr cultivr susceptible to FWS, ws used throughout the experiment. All seeds were surfce-disinfected with 1% sodium hypochlorite solution for 5 min nd rinsed three times in sterile distilled wter prior to sowing. GF183 nd S1HI-4 were seprtely cultured on potto dextrose gr (PDA) in 9-cm Petri dishes for 5 dys in the drk t 25 o C. Five mycelil disks (5 mm in dimeter), tken from the edges of 5-dy-old cultures, were trnsferred into 100 ml of potto dextrose broth (PDB) in 300 ml Erlenmeyer flsks nd incubted for 7 dys t 25 o C on rotry shker (NR-150, Titec Co., Jpn) t 120 rpm. To obtin budding-cells of Fusrium isoltes,

2 250 Biocontrol of Fusrium wilt of spinch Journl of Plnt Pthology (2010), 92 (1), the fungl cultures were filtered through three lyers of sterile guze. Fungl suspensions were then diluted to 10 7 budding-cells per ml with sterile distilled wter nd ech ws used s n inoculum source. In ll experiments, pper pot set ( cm) contining 162 pots (5.0 cm depth 1.5 cm dimeter per pot), mnufctured by Nippon Beet Sugr Co., Jpn, ws filled with potting soil Str Bed (Zen-Noh, Tokyo, Jpn). The potting soil contins cly, pet, zeolite, nd composted plnt mteril. The nitrogen, phosphorus nd potssium contents of the potting soil were 200:1500:200 mg per liter, nd the ph rnged from 6.0 to 7.0. A 10 ml suspension of GF183 ws poured into ech pot, nd one spinch seed ws sown. The seedlings were grown for 14 dys in greenhouse. The spinch seedlings grown in pper pots were trnsplnted pproximtely 15 cm prt into concrete blocks ( cm) filled with pthogen-infested soil (FOS concentrtion ws 1.0x10 4 CFU g -1 soil) in polytunnels. Seedlings not treted with GF183 nd chllenged or not chllenged with FOS were set up s controls. Experiments 4 nd 5 were conducted to compre the effectiveness of three pplictions of GF183 ginst FWS. The three pplictions were: GF183 single ppliction t seeding, single ppliction one dy before trnsplnting, nd GF183 double ppliction t both seeding nd one dy before trnsplnting. In experiment 5, n dditionl test ws conducted for comprison, in which the spinch seeds were sown directly in pthogen-infested soil. Experiments 1, 2, 3, 4 nd 5 were conducted for 22, 24, 27, 22 nd 26 dys fter trnsplnting to pthogen-infested soil between My 18 nd June 9, 1999, June 4 nd June 28, 2000, June 28 nd July 28, 2003, My 26 nd June 18, 2004 nd June 6 nd July 5, 2006, respectively. Disese severity bsed on the folir symptoms of wilting ws ssyed t the end of the experiments using scle of 0 to 4: 0 = helthy; 1 = yellowing; 2 = slight wilting; 3 = severe wilting; nd 4 = ded plnt, ws clculted with the following formul: (4A+3B+2C+D)/4N 100 in which A = number of plnts on scle 4; B: = number of plnts on scle 3; C = number of plnts on scle 2; D = number of plnts on scle 1; N = totl number of plnts. The experiments were rrnged in rndomized complete block design of four replictions, with nine plnts per repliction. First, nlysis of vrince ws performed for the tretment mens obtined for disese severity. When the F test results were significnt, these mens were compred using Fisher s protected lest significnt difference test t P<0.05. Dt on disese severity were nlyzed fter trnsformtion to rc sine x. Spinch plnts treted with GF183 hd less disese thn pthogen-control plnts in experiments 1, 2, nd 3 (Tble 1). The protective effects bsed on disese severity were 91.8%, 51.5% nd 79.9%, respectively, t the end of experiments 1, 2, nd 3. In experiment 4, the GF183 single ppliction t seeding nd GF183 double ppliction t both seeding nd one dy before trnsplnting significntly (P<0.05) reduced disese severity by 52.5% nd 58.1%, respectively, compred to the pthogen control (Tble 2). The GF183 single ppliction one dy before trnsplnting ws not effective. In experiment 5, the protective effect of the GF183 double ppliction ws higher thn tht of the GF183 single ppliction t seeding, though the difference ws not significnt. The GF183 double ppliction significntly (P<0.05) reduced disese severity by 87.3% compred to the pthogen control. The protective effect of the GF183 double ppliction ws further enhnced, reching 94.8% when compred to direct sowing. In this study, GF183 effectively controlled FWS, bsed on the results of five different experiments. Protective effects of GF183 were % nd % by single ppliction t seeding nd double ppliction t seeding nd one dy before trnsplnting, respectively. These effects were lmost the sme s with non-pthogenic F. oxysporum (Ktsube nd Aksk, 1997), lthough the tretment methods were slightly different. In the ltter cse, non-pthogenic F. oxysporum ws tested singly t 1 or 5 dys fter sowing (protective Tble 1. Effects of Fusrium equiseti GF183 on disese severity of Fusrium wilt of spinch cused by F. oxysporum f. sp. spincie under polytunnel conditions. Tretment Fusrium equiseti (GF183) + Pthogen Pthogen only Disese severity Exp. 1 Exp. 2 Exp b 22.9 b 6.3 b 41.7 c 47.2 c 31.3 c Vlues with the sme letter in ech column re not significntly different (P<0.05) ccording to Fisher s protected lest significnt difference test.

3 Journl of Plnt Pthology (2010), 92 (1), Horinouchi et l. 251 Tble 2. Effects of single nd double ppliction of Fusrium equiseti GF183 on disese severity of Fusrium wilt of spinch cused by F. oxysporum f. sp. spincie under polytunnel conditions. Tretment GF183 single ppliction t seeding + Pthogen Disese severity Exp. 4 Exp b 20.4 bc GF183 single ppliction one dy before trnsplnting + Pthogen 71.3 bc ND GF183 double ppliction t both seeding nd one dy before trnsplnting + Pthogen Pthogen only 36.7 b 4.6 b 87.5 c 36.1 c Direct sowing + Pthogen ND 89.8 d ND: Not done. Vlues with the sme letter in ech column re not significntly different (P<0.05) ccording to Fisher s protected lest significnt difference test. effects %), or 5 dys before trnsplnting (87.1%). A different strin, PGPF GF191 of F. equiseti, could lso effectively decrese Fusrium crown nd root rot of tomto both in rock wool nd soil systems (Horinouchi et l., 2007; 2008). The protective effects were % nd 53-62% in rock wool nd soil systems, respectively. GF191 could lso decrese Fusrium wilt of tomto (unpublished dt). Like them, F. equiseti could be potentil cndidte s biocontrol gent. GF183 ws frequently re-isolted from spinch roots (dt not shown), suggesting tht the high bility of GF183 to colonize spinch roots might be relted to the mechnism of disese suppression. Reports indicte tht PGPF isoltes, including F. equiseti, colonize the epiderml nd outer corticl cell lyer of roots (Hykumchi, 1994; Shivnn et l., 1996). The continuous presence of PGPF isoltes on/in roots my trigger the plnts to produce defence responses (Meer et l., 1995). Recently, Mciá-Vicente et l. (2009) demonstrted endophytic coloniztion by F. equiseti nd indicted tht root coloniztion by the biocontrol gent is n importnt fctor for the control of root pthogens. Although single ppliction of GF183 one dy before trnsplnting to pthogen-infested soil did not significntly reduce disese severity, single ppliction of GF183 t seeding nd double ppliction t both seeding nd one dy before trnsplnting to pthogeninfested soil served to control FWS (Tble 2). These results lso suggest tht high degree of coloniztion of GF183 is necessry for disese suppression. FOS popultions in roots were estimted t the end of experiment 4. The roots in the pper pots were collected from ll plnts in ech disese ctegory within ech tretment, were pooled, wshed in tp wter nd homogenized in sterile distilled wter (1:10 w/v) using blender (Ace Homogenizer, Model AM, Nihonseiki Kish, Jpn) t 8,000 rpm for 5 min. The homogente ws filtered through two lyers of guze, diluted 10- to 1000-fold nd plted on Komd s selective medium (Komd, 1975) with six pltes per dilution. The experiment included four replictes. The number ws recorded of CFU of FOS per grm of fresh weight of the roots in ech scle within ech tretment. Discolortion of the vsculr tissue ws ssessed fter trnsversely cutting the bsl prt of the tproot of ech plnt on the following scle: 0 = no vsculr discolortion; 1 = < 33%; 2 = > 34 to 67%; nd 3 = > 67 to 100% discolortion. The verge FOS popultions in ech repliction within the tretment were clculted using the formul: (P 0 A+P 1 B+P 2 C+P 3 D)/N 100, where P 0, 1, 2, nd 3 = popultions of the pthogen in scles 0, 1, 2 nd 3, respectively; A = fresh weight of roots on scle 0; B = fresh weight on scle 1; C = fresh weight on scle 2; D = fresh weight on scle 3; nd N = totl weight of roots. Sttisticl nlysis ws performed s previously described. The FOS popultion densities in roots treted with the GF183 single ppliction t seeding nd double ppliction t seeding nd one dy before trnsplnting

4 252 Biocontrol of Fusrium wilt of spinch Journl of Plnt Pthology (2010), 92 (1), Tble 3. Effects of Fusrium equiseti GF183 tretments on popultion density of Fusrium oxysporum f. sp. spincie in spinch roots in experiment 4. Tretment GF183 single ppliction t seeding + Pthogen GF183 double ppliction + Pthogen Pthogen only FOS popultions in different discolortion scores (x10 2 CFU g -1 fresh weight) b 7.3 b b 95.3 b b Averge popultion of FOS (x10 2 CFU g -1 fresh weight) 98.9 b 96.5 b c 0.0 : No smple. Vlues with the sme letter in ech column re not significntly different (P<0.05) ccording to Fisher s protected lest significnt difference test. were significntly (P<0.05) reduced by 75.7% nd 81.8%, respectively, t scle 3 of vsculr discolortion, compred to the pthogen control (Tble 3). Furthermore, the verge FOS popultions in roots treted with the GF183 single nd double pplictions were significntly (P<0.05) reduced by 85.3% nd 85.6%, respectively, reltive to the pthogen control. Pre-inocultion of spinch seedlings with GF183 not only suppressed the disese but lso reduced pthogen popultions in the roots. This result is similr to tht of Nelson et l. (1992), who showed tht pre-inocultion of tomto nd cucumber with non-pthogenic Fusri reduced the multipliction of F. oxysporum f. sp. lycopersici nd F. oxysporum f. sp. cucumerinum. In the present study, pthogen popultions in the roots treted with GF183 were significntly lower (reduced by bout 85%) thn those of the pthogen control. These results suggest tht, lthough FOS cn enter spinch roots treted with F. equiseti, it cnnot multiply there. This further suggests tht, in this system, the observed disese reduction is ttributble to indirect ntgonism medited through the host plnt in response to root coloniztion by F. equiseti, nd tht induced resistnce my be one of the mechnisms of FWS biocontrol. The effect of root extrcts on the prolifertion of FOS ws studied further using spinch plnts treted or not with GF183 t seeding, chllenged with FOS, nd showing discolortion scle of 3. Roots were collected t the end of experiment 4. Spinch plnts treted with GF183 nd unchllenged with FOS were lso used. Roots were exmined 30 dys fter seeding. Homogenized root filtrtes were centrifuged t 3,000 rpm for 10 min, superntnts were collected nd filtered through 0.45 µm Millipore ( Millex-HV, Millipore USA). The effect of these extrcts on pthogen prolifertion ws evluted by counting newly formed budding-cells in the root extrcts. Nine milliliters of root extrct nd 1 ml of FOS budding-cell suspension (1.0x10 6 buddingcells per ml) were mixed in 100 ml Erlenmeyer flsk which ws then incubted t 25 C on reciprocl shker t 120 rpm. The numbers of budding-cells formed were determined with hemocytometer. Ech Petri dish nd Erlenmeyer flsk constituted one replicte, nd ech experiment ws conducted four times. Sttisticl nlysis ws performed s previously described. Root extrcts from spinch plnts treted with GF183 nd chllenged with pthogen inhibited significntly (P<0.05) the production of new budding-cells of FOS compred with extrcts of untreted nd unchllenged plnts 3, 5 nd 7 dys fter inocultion (Fig. 1A), nd the rtes of budding-cell formtion were reduced by 67.9%, 48.8% nd 53.8%, respectively. Root extrcts from spinch plnts untreted with GF183 nd chllenged with pthogen lso significntly (P<0.05) inhibited FOS prolifertion compred with extrcts of untreted nd unchllenged plnts, nd the rtes of budding-cell formtion were reduced by 33.3%, 32.9% nd 31.3%, respectively. Furthermore, root extrcts from spinch plnts treted with GF183 nd unchllenged with pthogen lso significntly (P<0.05) inhibited the production of new FOS budding-cells compred with extrcts of untreted nd unchllenged plnts (Fig. 1B), nd the rtes of budding-cell formtion were reduced by 19.7%, 24.1% nd 30.9%, respectively. In the present study, we found n inhibitory effect of spinch root extrcts from pthogen-treted plnts on pthogen prolifertion (inhibitory effect ws % for 3-7 dys) but the effect ws stronger ( %) in F. equiseti-pthogen-treted plnts (Fig. 1A). An inhibitory effect ws lso given by root extrcts from F. equiseti-treted plnts ( %) (Fig. 1B), but it ws not s strong s tht from F. equiseti-pthogentreted plnts. These results suggest tht F. equiseti might induce physiologicl chnges in the composition of plnt extrcts nd enhnce defence mechnisms tht re further triggered when the plnt senses potentil pthogen in phenomenon known s potentition or conditioning (Kuss et l., 1993; Kessmnn et l., 1994).

5 Journl of Plnt Pthology (2010), 92 (1), Horinouchi et l. 253 A B Density of budding-cells (x10 4 /ml) Density of budding-cells (x10 4 /ml) Fig. 1. Suppression of production of new budding-cells of Fusrium oxysporum f. sp. spincie in extrcts of root of spinch (A) treted with F. equiseti-gf183 nd pthogen, (B) treted with F. equiseti-gf183 nd unchllenged with pthogen. Vlues with the sme letter in ech column re not significntly different (P<0.05) ccording to Fisher s protected lest significnt difference test. An sterisk denotes significnt difference ccording to Student s t-test t P<0.05. Brs indicte stndrd error of mens. Although F. equiseti is implicted in few diseses, such s rotting of cucurbit fruits in contct with soil (Burgess et l., 1988), it is considered n unimportnt or opportunistic pthogen. This fungus, however, hs been chrcterized s nturl root endophyte ble to colonize plnt roots nd endowed with properties tht could mke it promising cndidte for the biologicl control of root pthogens nd nemtodes (Mciá-Vicente et l., 2008; Nito et l., 2001). We hve now shown tht F. equiseti GF183 is ble to control FWS, which, to our knowledge, is the first report of such n ctivity. Further reserch is nevertheless wrrnted to determine the complete mechnisms of disese suppression involved in the F. equiseti system, nd to develop methods for its prcticl use in biocontrol. REFERENCES GF183 + Pthogen Pthogen GF183 Beckmn C.H., The Nture of Wilt Disese of Plnts. APS Press, St. Pul, MN, USA. c b * * * Time (dys) b c b Burgess L.W., Liddell C.M., Summerell B.A., Lbortory Mnul for Fusrium Reserch. 2nd Ed. The nture of Wilt Disese of Plnts. Fusrium Reserch Lbortory Deprtment of Plnt Pthology nd Agriculturl Entomology, University of Sydney, Austrli. Correll J.C., Morelock T.E., Blck M.C., Koike S.T., Brndenberger L.P., Dinello F.J., Economiclly importnt diseses of spinch. Plnt Disese 78: Horinouchi H., Muslim A., Suzuki T., Hykumchi M., Fusrium equiseti GF191 s n effective biocontrol gent ginst Fusrium crown nd root rot of tomto in rock wool systems. Crop Protection 26: Horinouchi H., Ktsuym N., Tguchi Y., Hykumchi M., of Fusrium crown nd root rot of tomto in soil system by combintion of plnt growth-promoting fungus, Fusrium equiseti, nd biodegrdble pots. Crop Protection 27: Hykumchi M., Plnt growth-promoting fungi from turfgrss rhizosphere with potentil for disese suppression. Soil Microorgnisms 44: Hykumchi M., Kubot M., Fungi s plnt growth promoter nd disese suppressor. In: Aror, D.K. (ed). Mycology Series. Vol. 21. Fungl Biotechnology in Agriculturl, Food, nd Environmentl Applictions, pp Mrcel Dekker, New York, NY, USA. Ktsube K., Aksk Y., of Fusrium wilt of spinch by trnsplnting seedlings pretreted with nonpthogenic Fusrium oxysporum. Annls of the Phytopthologicl Society of Jpn 63: Kuss H., Frnke R., Kruse K., Conrth U., Jeblick W., Grimmig B., Mtern U., Conditioning of prsley (Petroselinum crispum L.) suspension cells increses elicitor-induced incorportion of cell wll phenolics. Plnt Physiology 102: Kessmnn H., Stub T., Hofmnn C., Metzke T., Herzog J., Wrd E., Uknes S., Ryls J., Induction of systemic cquired disese resistnce in plnts by chemicls. Annul Review of Phytopthology 32: Komd H., Development of selective medium for quntittive isoltion of Fusrium oxysporum from nturl soil. Review of Plnt Protection Reserch (Tokyo) 8: Lrsson M., Gerhrdson B., Disese progression nd yield losses from root diseses cused by soilborne pthogens of spinch. Phytopthology 82: Mciá-Vicente J.G., Jnsson H-B., Tlbot N.J., Lopez-Llorc L.V., Rel-time PCR quntifiction nd live-cell imging of endophytic coloniztion of brley (Hordeum vulgre) roots by Fusrium equiseti nd Pochoni chlmydospori. New Phytologist 182: Meer M.S., Shivnn M.B., Kgeym K., Hykumchi M., Persistence of induced systemic resistnce in cucumber in reltion to root coloniztion by plnt growth promoting fungl isoltes. Crop Protection 14: Muslim A., Horinouchi H., Hykumchi M., Suppression of Fusrium wilt of spinch with hypovirulent binuclete Rhizoctoni. Journl of Generl Plnt Pthology 69: Nito S., Kto M., Sto A., The probbility of escping potto tubers from scb disese by pper-pot trnsplnting method. Annls of the Phytopthologicl Society of Jpn 64: 580.

6 254 Biocontrol of Fusrium wilt of spinch Journl of Plnt Pthology (2010), 92 (1), Nelson H., Ouchi S., Shirishi T., Oku H., Induced resistnce to fusrium wilt of tomto nd cucumber: Symptoms nd pthogen prolifertion. Annls of the Phytopthologicl Society of Jpn 58: Nito J.K., Meyer S.L.F., Schmidt W.F., Fettinger J.C., Chitwood D.J., Nemtod-ntgonistic trichothecenes from Fusrium equiseti. Journl of Chemicl Ecology 27: O Brien M.J., Winter H.F., Evlution of spinch ccessions nd cultivrs for resistnce to Fusrium wilt. I. Greenhouse-bench method. Journl of the Americn Society for Horticulturl Science 102: Shivnn M.B., Meer M.S., Hykumchi M., Role of root coloniztion bility of plnt growth promoting fungi in the suppression of tke-ll nd common root rot of whet. Crop Protection 15: Tsud K., Kosk Y., Tsuge S., Kubo Y., Horino O., Evlution of the endophyte Enterobctor cloce SM10 isolte from spinch roots for biologicl control ginst Fusrium wilt of spinch. Journl of Generl Plnt Pthology 67: Received Mrch 19, 2009 Accepted July 29, 2009