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1 Biologicl Control 51 (2009) Contents lists vilble t ScienceDirect Biologicl Control journl homepge: Imbibitionl dmge in conidi of the entomopthogenic fungi Beuveri bssin, Metrhizium cridum, nd Metrhizium nisoplie Mrcos Fri,b, *, Ann E. Hjek, Stephen P. Wright c Cornell University, Deprtment of Entomology, Comstock Hll, Ithc, NY , USA b EMBRAPA Recursos Genéticos e Biotecnologi, Cix Postl 02372, Brsíli, DF , Brzil c USDA-ARS, Biologicl Integrted Pest Mngement Reserch Unit, Robert W. Holley Center for Agriculture nd Helth, Ithc, NY , USA rticle info bstrct Article history: Received 15 My 2009 Accepted 29 June 2009 Avilble online 2 July 2009 Keywords: Biologicl control Microbil control Mycoinsecticides Anhydrobiosis Rehydrtion Germintion Sorption isotherm When dried orgnisms re immersed in wter, rpid imbibition my cuse severe dmge to plsm membrnes; in unicellulr orgnisms, such dmge is usully lethl. This study investigted effects of wter ctivity (dryness) of orgnisms nd immersion temperture on imbibitionl dmge in three insect pthogenic fungi. Conidil powders of Beuveri bssin (Bb), Metrhizium nisoplie (M) nd Metrhizium cridum (Mc) were dried/hydrted to brod rnge of wter ctivities ( w ) ( ) prior to immersion in wter t C. Imbibitionl dmge in conidi of ech fungus occurred rpidly, with no differences in vibilities observed following immersion for 2 vs. 60 min. Dmge incresed with decresing wter ctivity of the conidi nd decresing temperture of the immersion wter. Dry ( w ) Metrhizium spp. conidi were highly susceptible to imbibitionl dmge, with vibility declining to 65% fter immersion t 0.5 C nd 663% following immersion t 15 C. Germintion of the driest M conidi ws reduced to 66% fter tretment t 25 C. In contrst, Bb ws highly tolernt to dmge, with significnt reductions in vibility (to levels s low s 43 65%) occurring only when dry conidi were immersed t 0.5 C. Dmge ws prevented when conidi were slowly rehydrted by humidifiction prior to immersion nd immersion temperture ws incresed to C; germintion of ll fungi ws P94% under these optiml conditions. However, immersion of the driest Bb, M, nd Mc powders in wrm wter (33 C) lso resulted in high vibilities (95%, 89%, nd 94%, respectively), nd slow-rehydrted conidi lso retined high vibility (87%, 92%, nd 83%, respectively) fter immersion in ice-cold wter (0.5 C). Formultion of conidi in pure (non-emulsifible) prffinic oil provided considerble protection from imbibitionl dmge. This study underscores need for estblishing stndrd protocols for prepring queous suspensions of sensitive fungi for both reserch nd commercil pplictions. Ó 2009 Elsevier Inc. All rights reserved. 1. Introduction Mny fungi disply the bility to survive profound dehydrtion (Burges, 1998; Crowe et l., 1992). In desiccted (nhydrobiotic) sttes, spores re ble to survive extreme environments chrcterized by dry het, freezing/thwing, nd cid conditions (Griffin, 1994). With respect to fungl entomopthogens employed s microbil biologicl control gents, enhnced thermotolernce imprted by desicction (Hedgecock et l., 1995; Moore et l., 1996; Chen et l., 2008; Connick et l., 1998) is prticulrly significnt s it cn substntilly increse the shelf-lives of fungus-bsed biopesticide products (Burges, 1998; Wright et l., 2001). When dried orgnisms re immersed in wter, rpid imbibition my cuse severe dmge to plsm membrnes (Crowe et l., * Corresponding uthor. Address: EMBRAPA Recursos Genéticos e Biotecnologi, Cix Postl 02372, Brsíli, DF , Brzil. Fx: E-mil ddress: fri@cenrgen.embrp.br (M. Fri). 1992; Hoekstr et l., 1999). In unicellulr orgnisms, such injury usully leds to cell deth (Nijsse et l., 2004). Numerous studies with vrious orgnisms, including seeds, pollens, nd yests, hve demonstrted tht imbibitionl dmge increses with decresing moisture content (dryness) of the orgnism subjected to rpid rehydrtion nd decresing immersion temperture. Imbibitionl dmge cn be prevented either by using wrm wter for rehydrtion or by slowly rehydrting the orgnism in humid tmosphere (vpor phse rehydrtion) prior to immersion in wter (see Crowe et l., 1992). Imbibitionl dmge nd methods for its prevention hve been studied in yests (vn Steveninki nd Ledeboer, 1974; Crowe et l., 1992), but we re wre of only one reference to imbibitionl dmge mong filmentous fungi, which cites Metrhizium cridum (Driver & Milner) Bischoff, Rehner, & Humber conidi dried to 4 5% moisture content (Moore et l., 1997) (M. cridum ws formerly identified s Metrhizium nisoplie vr. cridum; see Bischoff et l., 2009). Vibility of conidi of this fungus tht were slowly rehy /$ - see front mtter Ó 2009 Elsevier Inc. All rights reserved. doi: /j.biocontrol

2 M. Fri et l. / Biologicl Control 51 (2009) drted by exposure to wter-sturted tmosphere for 5 40 min ws >70%, wheres vibility of conidi tht were rpidly rehydrted by immersion in wter ws <25%. Rehydrtion of conidi to levels of norml metbolism without membrne dmge is of prmount importnce for successful pest control using dry formultions of fungl pthogens. In-depth studies of imbibitionl dmge s potentilly importnt fctor in the efficcy of mycopesticide products re lcking. The primry objective of this study ws to quntify the effects of wter ctivity nd temperture s key fctors determining severity of imbibitionl dmge in conidi of three commercilly importnt species of entomopthogenic fungi. Broder objectives included incresing wreness nd understnding of this phenomenon mong mycopesticide reserchers, producers, nd users nd ssessing prcticl mens of circumventing the problem. Towrd the ltter objective, n experiment ws conducted to determine if oil formultion hs potentil to protect conidi from imbibitionl dmge. 2. Mterils nd methods 2.1. Source of fungi Three fungi were tested: Beuveri bssin (Bls.) Vuill. (Bb) strin GHA originlly isolted from chysomelid in USA, M. nisoplie (Metschn.) Sorokın (M) isolte CB-10 (ARSEF 7981) from cercopid in Brzil, nd M. cridum (Mc) isolte IMI (AR- SEF 3341) from n cridid in Niger. Unformulted conidi of Bb were obtined from Lverlm Interntionl Corp. (Butte, MT, USA). Metrhizium isoltes were lb-produced using commonly employed biphsic process in which culture is initited in liquid nd completed on semi-solid substrte. In the initil phse, flsks contining 100 ml liquid medium (20 g glucose, 20 g yest extrct l 1 )(Jenkins nd Prior, 1993) were inoculted with conidi nd incubted on rotry shker (150 rpm) for 6 dys t C. In the second phse, brley mixed with distilled wter (dh 2 O) ws utoclved for 20 min in polyethylene bgs (100 g dry brley flkes + 60 ml dh 2 O per bg). Ech bg ws then inoculted with 70 ml of liquid culture from the initil production phse. Bgs were incubted t 25 C in drkness, nd eril conidi were hrvested fter 2 weeks by mnully shking the fungus-colonized substrte through series of two sieves, 20 nd 100 mesh. Conidi tht pssed through the 100-mesh sieve (150 lm pore size) were collected. The three stock technicl powders were stored t 20 C Vibility determintions For vibility determintions, 10 ll droplets of suspension comprising conidi suspended in wter with low concentrtion of surfctnt (see below) or pure prffinic oil were pipetted onto cm blocks (1 droplet/block) of yest extrct gr-bsed solid medium (YEA) contining 0.5 g yest extrct, 100 mg gentmicin, 0.1 g Tween 80, nd 16 g gr l 1 (Meikle et l., 2003), nd incubted on glss microscope slides in seled petri dishes in drkness t 25 C. After the desired incubtion time, coverslip ws pplied, nd the smple ws exmined t 400 mgnifiction with phse-contrst illumintion. Conidi were considered germinted if germ tube of ny length ws visible. A minimum totl of 200 conidi were exmined in severl microscope fields for ech replicte suspension of ech experimentl tretment. In ll experiments, incubtors providing experimentl tempertures were continuously monitored with digitl dt loggers (Hobo Ò, Onset Computer Corp., Bourne, MA, USA). Reported tempertures were ±0.5 C for 25 C nd ±1 C for ll other tempertures Initil tests to identify optiml procedures for ssessing germintion Three different concentrtions (0, 0.005, nd 0.1 g l 1 ) of benomyl (Bonide Chemicl Co., Yorkville, NY, USA) were tested in YEA. Benomyl slows hyphl growth following germ tube emergence, llowing germintion ssessments over prolonged periods (Milner et l., 1991). Conidil smples of Bb, M, or Mc (0.6 g/smple) were collected from the stock powders nd plced in 3.4 cm dim. 1.1 cm plstic smple cups (Novsin, Pfäffikon, Switzerlnd). Wter ctivity ( w ) ws mesured t 25 C with n electronic meter (LbMster- w, Novsin, Pfäffikon, Switzerlnd); w of the smples rnged from to From ech smple, three subsmples ( mg picked up on the tip of sptul) were tken. One of these subsmples ws mintined in its originl dry stte, while the other two were slow hydrted vi incubtion t 25 C in desicctor with wter-sturted tmosphere; one subsmple ws incubted for 40 min nd the other for 24 h. Following these tretments, ech of the three subsmples ws trnsferred to screw-cp glss vil (23 ml cpcity) contining pproximtely 1 g of 2 mm glss beds nd 7 ml of dh 2 O (temperture not recorded) with 0.05% of the surfctnt Lutensol Ò (Ethoxylted Tridecyl Alcohol, BASF Corportion, Florhm Prk, NJ, USA) possessing hydrophilic lipophilic blnce number of 10 (Jin et l., 1999). Resulting suspensions (contining conidi/ml) were gitted for 10 min with wrist ction shker set t 6.7 oscilltions/sec (Burrel Scientific, Pittsburgh, PA, USA), nd vibility ws determined using blocks of YEA with the bove-indicted rnge of benomyl concentrtions. Germintion ws recorded 18 h postinocultion (p.i.) for Bb nd 20 h p.i. for both M nd Mc. This experiment ws repeted with conidi dried to the w rnge using the desiccnt clcium sulfte (eight-mesh indicting drierite Ò, W.A. Hmmond Drierite Co., Xeni, OH, USA), nd germintion ws recorded 18, 21, nd 25 h p.i. for Bb, M nd Mc, respectively. In third experiment, the bove-described procedure ws repeted with the following ltertions. The 0.6-g smples of ech fungus were initilly dried t 25 C for 5 9 dys in hermeticlly seled 125 ml glss jrs (Bll Ò, Jrden Corp., Muncie, IN, USA) contining drierite. Distilled wter plus 0.05% Lutensol (herefter referred wter + Lutensol) used in the fst-rehydrtion tretments ws equilibrted t 25 C. Following the fst- vs. slow-rehydrtion tretments, conidi were inoculted onto blocks of YEA contining g l 1 benomyl (herefter referred to s YEA + benomyl), nd germintion counts were performed 24, 48, nd 72 h p.i Effect of fst rehydrtion time on vibility Conidil smples of ech fungl isolte were plced individully in 125-ml glss jrs contining drierite nd stored t 25 C for 10 dys. After drying, subsmples of conidi were fst rehydrted into wter + Lutensol previously equilibrted to 0.5 C in n ice bth (methods otherwise s described bove). Suspensions were mnully shken for 5 s nd then returned to the ice bth nd incubted for n dditionl 2 or 60 min. After tretment, ech suspension ws vortexed for 10 s t room temperture (23 24 C), nd smple ws plted on YEA + benomyl. Conidi were incubted for 48 h t 25 C nd germintion ws ssessed Effects of conidil wter ctivity nd imbibition temperture on imbibitionl dmge Conidil smples of the three fungl isoltes were plced in hermetic 125-ml glss jrs contining either dh 2 O, drierite, or sturted slt solutions bsed on sodium hydroxide (NOH), mgnesium chloride hexhydrte (MgCl 2 6H 2 0), or sodium nitrite (NNO 2 ) nd

3 348 M. Fri et l. / Biologicl Control 51 (2009) stored t 25 C for 3 dys in drkness. After storge, wter ctivity ws determined by rpidly trnsferring ech smple into the wter ctivity meter, previously clibrted with seven stndrds in the w rnge. Averge wter ctivities (±stndrd devition) of conidi held over drierite were ± , ± , nd ± for Bb, Mc nd M, respectively. Respective vlues generted by the other tretments were: ± , ± , nd ± for NOH; ± , ± , nd ± for MgCl 2 6H 2 0; ± , ± , nd ± for NNO 2 ;nd0.953± , ± , nd ± for dh 2 O. After w of the conidil smples ws determined, subsmples were collected nd deposited in vils contining wter + Lutensol tht hd been equilibrted in n ice bth (0.5 C) or in incubtors set t 15, 25, or 33 C. Vils contining conidi were vigorously shken for 5 s nd returned to the trget temperture for n dditionl 2 min. Germintion ws then ssessed s described in Section Assessing the protective potentil of oil An initil test exmined the effects of queous vs. oil immersion medi on imbibitionl dmge. M conidi were dried over sturted NOH solution t 25 C for 3 dys in the hermetic jrs, then immersed for 2 min in dh 2 0, dh Lutensol, or pure prffinic oil (without emulsifiers or other dditives; lot number ; provided by Lverlm Interntionl Corp.) t either 0.5 or 16 C. Dry conidi were lso dusted onto YEA + benomyl in 60-mm petri dishes equilibrted t either 1 or 16 C nd held t these tempertures for 2 min. As controls, conidil smples were either fst rehydrted by immersion in wrm wter (without Lutensol) t 34 C for 2 min or slow rehydrted for 24 h t 25 C in wter-sturted tmosphere before being immersed in wter equilibrted t 25 C. Following the imbibition tretments, smples of ech wteror oil-bsed suspensions were inoculted onto blocks of YEA + benomyl equilibrted t room temperture nd then incubted t 25 C for vibility determintions t 24 h p.i.; the 60-mm pltes inoculted (dusted) with dry conidi were lso incubted t 25 C for vibility ssessments. A complete list/description of the bove-described tretments is presented in Tble 5. A second experiment ws conducted to ssess the potentil for oil formultion to protect conidi from imbibitionl dmge. Dry M conidi were dded to prffinic oil equilibrted t room temperture, vortexed for 1 min, nd then plted onto benomylmended YEA equilibrted t 1 C s explined bove. In ddition, dry conidi were dusted directly onto the surfce of the medium in petri dishes t the sme temperture. As control, conidi were fst rehydrted by immersion in wrm wter + Lutensol t 34 C for 2 min nd then inoculted onto YEA + benomyl equilibrted t room temperture. Germintion counts were recorded t 24 nd 48 h p.i Sorption isotherms Sorption isotherms llow conversion of w mesurements to moisture content (MC), nd vice vers. Sturted slt solutions were prepred ccording to the procedure described by Vertucci nd Roos (1993). Smll cups (3.4-cm dim., fbricted from luminum foil) contining conidi were plced on plstic supports in hermeticlly seled 0.95 l wide-mouth glss jrs (Bll Ò, Jrden Corp., Muncie, IN, USA) contining drierite or different sturted slt solutions for period of 7 dys t 25 C. Zinc chloride (ZnCl 2 ), sodium hydroxide (NOH), lithium chloride (LiCl), mgnesium chloride hexhydrte (MgCl 2 6H 2 0), potssium nitrite (KNO 2 ), sodium nitrite (NNO 2 ), sodium chloride (NCl), potssium chloride (KCl), nd potssium sulfte (K 2 SO 4 ) were used to crete different equilibrium reltive humidities. After storge, wter ctivity ws determined by rpidly trnsferring ech smple cup into the clibrted wter ctivity meter. Conidil smples were then weighed nd plced in drying oven t 105 ± 2 C for 24 h. After drying, ech smple ws cooled for 45 min in seled jr with drierite. The dry smples were then reweighed nd wet-bsis moisture content (w.b. MC) ws clculted nd expressed s the percent weight loss due to desicction. In ech test, one 1.0-g smple of conidi ws exposed to ech slt, nd the entire procedure ws repeted on three different dtes Sttisticl nlyses For ll experiments (other thn sorption isotherms), four replicte smples of conidi were prepred (ll men vibilities nd stndrd errors reported in the results re bsed on n = 4). In most cses, subsmples were ssigned to the vrious tretments in rndomized complete block design. In the experiments tht ssessed the protective potentil of oil, subsmples were ssigned to the vrious tretments in completely rndomized design. In the second test with oil-formulted conidi, the 24 nd 48 h germintion counts were recorded from independent smples (not repeted mesures). Percent germintion dt were normlized by rcsine trnsformtion nd nlyzed by nlysis of vrince (ANOVA). Where indicted for nlyses cross fungi, vibility dt for ech fungus were djusted with respect to the mximum vibility recorded for tht fungus (ech dtum divided by the mximum). Mens were compred by pired t-test or Tukey Krmer HSD ( = 0.05). ANOVAs nd polynomil regression nlyses were performed using the JMP sttisticl softwre pckge (SAS Institute Inc., Cry, NC, USA). 3. Results 3.1. Identifiction of optiml procedures for ssessing germintion In the first test, w nd temperture were not stndrdized cross fungl species, nd therefore results from tests of ech fungus were nlyzed independently. For Bb nd M, there were no significnt benomyl rehydrtion tretment interctions (Bb: F 2,15 = 0.54, P = 0.59; M: F 2,15 = 0.40, P = 0.68). Amendment of solid medium with benomyl hd little or no effect on vibility of these two fungi (Tble 1). Slow rehydrtion hd significnt positive impct on Bb germintion, but the effect ws smll; germintion ws 91.8% following fst rehydrtion vs. 96.2% fter slow rehydrtion (F 1,15 = 59.1, P < ). In contrst, slow rehydrtion incresed vibility of M conidi by n verge of 34% (from 55.9% to 89.8%; F 1,15 = 947.8, P < ). A benomyl rehydrtion tretment interction ws detected in the cse of Mc (rehydrtion benomyl interction: F 4,24 = 16.0, P < ); however, the overll min effects of benomyl nd rehydrtion were still tested, s trends were unmbiguous (benomyl hd negtive effect nd slow rehydrtion hd positive effect on vibility). Addition of benomyl to the germintion medium reduced germintion by n verge of 26% or 40%, depending on concentrtion (F 2,24 = 181.3, P < , Tble 1). Slow rehydrtion incresed germintion by n verge of 20% or 49%, depending on the durtion of the slow rehydrtion period (F 2,24 = 253.1, P < ). As the rehydrtion period ws incresed, the conidi becme less susceptible to the benomyl effect. In the vibility checks of Mc in the first test, it ws evident tht mny non-germinted conidi were swollen nd pprently vible (dt not shown). It ws therefore decided to conduct second test in which germintion incubtion for this fungus ws incresed from 20 to 25 h. In ddition, conditions were stndrdized cross fungl species by drying the conidi to w nd control-

4 M. Fri et l. / Biologicl Control 51 (2009) Tble 1 Effect of benomyl concentrtions nd slow rehydrtion times on percent germintion of dry Beuveri bssin, Metrhizium cridum nd M. nisoplie conidi. Benomyl (g l 1 ) Men percent germintion (±stndrd error) fter slow rehydrtion for indicted times B. bssin (Bb) M. cridum (Mc) M. nisoplie (M) 0 40 min 24 h b Men 0 40 min 24 h Men 0 40 min 24 h b Men Test 1: wter ctivity ( w ) not stndrdized prior to tretment c ± ± ± ± ± ± ± ± ± ± ± ± ± 1.0 b 29.3 ± ± ± ± 7.5 b 54.6 ± ± ± ± ± ± 1.0 b 18.7 ± ± ± ± 7.8 c 57.0 ± ± ± 6.3 Men 91.8 ± 0.6 b 96.2 ± ± 6.0 c 57.4 ± 7.7 b 86.3 ± ± 0.8 b 89.8 ± 0.5 Test 2: w stndrdized t prior to tretment ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 3.6 c 54.0 ± ± ± ± ± ± ± ± ± ± ± ± 3.2 b 60.5 ± ± ± ± 4.1 Men 93.4 ± 0.4 b 93.9 ± 0.4 b 96.0 ± ± 4.6 b 80.5 ± ± ± 1.2 c 85.7 ± 0.8 b 91.6 ± 0.9 Conidi were slow rehydrted by holding in wter-sturted tmosphere (humid chmber) for the indicted time nd then suspended in wter with 0.05% Lutensol nd inoculted onto yest extrct gr for vibility ssessment; incubtion times t 25 C were: 18 h for Bb in both tests, 20 h for the Metrhizium species in test 1, nd 21 nd 25 h for M nd Mc, respectively, in test 2. Within ech test nd fungl species, mens followed by the sme letter re not significntly different (Tukey HSD or t-test, = 0.05). Germintion counts were not performed for Bb nd M in test 1 due to excessive mycelil growth in the tretment without benomyl. Initil wter ctivity levels: (Bb), (Mc), nd (M). b c ling imbibition temperture (set t 25 C). As result, mrkedly higher vibility percentges were recorded for Mc. Becuse tests 1 nd 2 were conducted independently, however, differences were not sttisticlly comprble. An overll nlysis of the djusted dt (see Section 2.8) from test 2 reveled significnt interctions mong fungl isolte nd both the rehydrtion nd benomyl tretments (fungus rehydrtion: F 4,78 = 28.6, P < ; fungus benomyl: F 4,78 = 32.4, P < ). Slow rehydrtion hd little effect on Bb compred to the Metrhizium isoltes, nd benomyl inhibited germintion of Mc, but not Bb or M (Tble 1). Due to these interctions, dt were nlyzed independently for ech fungl isolte. In the cses of Bb nd M, there were no benomyl rehydrtion tretment interctions (Bb: F 4,24 = 0.38, P = 0.82; M: F 4,24 = 1.03, P = 0.41). Apprently s result of the incresed incubtion time, the benomyl rehydrtion interction for Mc, which ws highly significnt in the first test, ws mrginlly insignificnt in this cse (F 4,24 = 2.71, P = 0.054). Slow rehydrtion significntly incresed vibility of Bb, but only by few percentge points, from 93.4% to 96.0% (F 2,24 = 13.7, P < ). There ws no significnt effect of benomyl on Bb germintion (Tble 1). In contrst, slow rehydrtion for 40 min or 24 h incresed vibility of M by 28% or 33%, respectively (F 2,24 = 237, P < ). As observed with Bb, benomyl hd no significnt effect on M germintion. Slow rehydrtion for 24 h hd n intermedite effect on Mc compred to Bb nd M, incresing germintion by 14 18%. Unlike Bb or M, Mc ws significntly susceptible to benomyl. The two benomyl concentrtions reduced vibility from 90% to men of 70% (F 2,24 = 92.6, P < ). Considering the pprent fungisttic effect of benomyl, third test ws conducted to ssess germintion over longer incubtion periods (24 72 h). An overll nlysis of the djusted dt showed significnt interctions mong fungl species nd both rehydrtion tretment nd incubtion time (fungus rehydrtion: F 2,58 = 27.3, P ; fungus incubtion time: F 2,58 = 4.72, P = ) (Tble 2). Most noticebly, conidi of Bb were less ffected by incresing incubtion or rehydrtion time thn M or Mc. Dt were consequently nlyzed independently for ech fungl isolte. Significnt incubtion time rehydrtion tretment interctions were observed for both Bb nd Mc (Bb: F 2,15 = 5.10, P = 0.021; Mc: F 2,15 = 8.82, P = 0.003). This interction ws not significnt for M (F 2,15 = 0.15, P = 0.86), likely due to the greter stndrd errors ssocited with the dt. The dt in ll cses indicted tht vibility of fst-rehydrted conidi incresed more over 72 h thn vibility of slow-rehydrted conidi (Tble 2). The min effect of incubtion time ws highly significnt for Mc (F 2,15 = 33.2, P ) nd mrginlly insignificnt for M (F 2,15 = 2.71, P = 0.10; one-tiled test). Incresing incubtion time from 24 to 72 h incresed germintion by verges of 13%, 7%, nd 1% for Mc, M nd Bb, respectively (Tble 2). At 72 h p.i., germintion counts for ll species becme more difficult due to clumping cused by extensive hyphl elongtion, nd high levels of debris from hyphl degrdtion (vesicles, etc.) in microscopic fields, lthough this ws less pronounced for Mc. The min effect of rehydrtion ws significnt for ll fungl isoltes, with slow rehydrtion incresing germintion, prticulrly for Metrhizium species (Tble 2). Slow rehydrtion incresed germintion by verges of 3%, 15%, nd 26% for Bb, Mc nd M, respectively (Bb: F 1,15 = 31.0, P ; Mc: F 1,15 = 124.5, P ; M: F 1,15 = 114.9, P ). Bsed on the results from the bove-described experiments, the subsequent studies investigting effects of rehydrtion time, imbibition temperture, nd wter ctivity on imbibitionl dmge used the g/l concentrtion of benomyl nd n incubtion time of 48 h for ll of the fungi.

5 350 M. Fri et l. / Biologicl Control 51 (2009) Tble 2 Effect of incubtion times on percent germintion of Beuveri bssin, Metrhizium cridum, nd M. nisoplie conidi following fst or slow rehydrtion. Incubtion time (h t 25 C) Percent germintion (±stndrd error) B. bssin M. cridum M. nisoplie Fst rehydrted b Slow Men c Fst rehydrted b rehydrted Slow rehydrted Men c Fst rehydrted Slow rehydrted ± ± ± ± ± ± 5.1 b 58.4 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 4.5 Men c 94.7 ± 0.5 b 97.5 ± ± 3.2 b 94.0 ± ± 2.0 b 90.0 ± 1.5 b Incubted on yest extrct gr with g l 1 benomyl. Fst-rehydrted conidi were immersed in wter with 0.05% Lutensol t 25 C without pre-rehydrtion, wheres slow-rehydrted conidi were held in wter-sturted tmosphere (humid chmber) t 25 C for 24 h before immersion. c Within ech fungl species, mens followed by the sme letter re not significntly different (Tukey HSD or t-test, = 0.05). Men c Tble 3 Effect of imbibition time on percent germintion of dry Beuveri bssin, Metrhizium cridum, nd M. nisoplie conidi following fst rehydrtion (immersion) t 0.5 C. Initil hydrtion stte of conidi Imbibition time t 0.5 C (min) Percent germintion ± stndrd error b B. bssin M. cridum M. nisoplie Control (pre-rehydrted) 97.4 ± ± ± 0.7 Dry ± ± ± 0.6 Dry ± ± ± 0.5 Control conidi were pre-rehydrted in wter-sturted tmosphere for 24 h t 25 C prior to fst rehydrtion in wter with 0.05% Lutensol t 25 C. Dry conidi were dried over drierite for 10 dys t 25 C prior to fst rehydrtion in wter + Lutensol equilibrted t 0.5 C. b Vibilities were determined by plting on yest extrct gr with g l 1 benomyl nd incubting for 48 h t 25 C. Mens within columns followed by the sme letter re not significntly different (t-test, = 0.05) Effect of fst rehydrtion time on vibility Incresing the fst-rehydrtion immersion time from 2 to 60 min did not ffect germintion (F 1,18 = , P = 0.99). Anlysis of the djusted dt showed tht the effects of fst rehydrtion vried gretly mong fungl species (F 2,18 = 94.2, P < ), with no fungus rehydrtion time interction (F 2,18 = 0.03, P = 0.97) (Tble 3). Following immersion of conidi in wter + Lutensol t 0.5 C, Bb retined >50% vibility, wheres Mc retined only 12 13%, nd vibility of M ws reduced to ner zero Effects of conidil wter ctivity nd immersion temperture on vibility Overll nlysis of the effect of conidil wter ctivities nd tempertures used for fst rehydrtion reveled highly significnt interctions mong fungl species nd these two fctors (fungus wter ctivity interction: F 8,180 = 38.4, P ; fungus temperture interction: F 6,180 = 2.37, P ). Drying hd greter negtive impct on vibility of M thn Mc or Bb (Tble 4). Across tempertures, germintion of Bb ws substntilly ffected only t 0.5 C, wheres Mc nd M vibilities were lso gretly reduced t 15 nd C, respectively. Becuse of these interctions, ech fungl species ws nlyzed independently. Within ech fungl species, significnt wter temperture vs. wter ctivity interction ws present (Bb: F 12,57 = 17.7, P ; M: F 12,57 = 34.8, P ; Mc: F 12,57 = 51.5, P ); however, min effects were cler in tht vibility of ll fungl species generlly incresed with incresing immersion temperture (Bb: F 3,57 = 274.0, P ; Mc: F 3,57 = 964.1, P ; M: F 3,57 = 472.6, P ) nd wter ctivity (Bb: F 4,57 = 34.6, P ; Mc: F 4,57 = 109.6, P ; M: F 4,57 = 161.3, P ) (Tble 4). Dry conidi were highly susceptible to imbibitionl dmge when fst rehydrted under chilled conditions. Germintion percentges for Bb, Mc, nd M conidi dried to w were 51.5%, 5.3%, nd 0.9%, respectively, following fst rehydrtion t 0.5 C, wheres the respective germintion percentges were 95.6%, 93.9%, nd 94.0% for ner-fully hydrted conidi imbibed t 33 C. With the exception of M, even the driest conidi were not susceptible to imbibitionl dmge when fst hydrted t 33 C, nd in the cse of M, dry conidi ( w ) fst rehydrted t 33 C exhibited only n verge 6.5% loss in vibility when compred to ner-fully hydrted conidi imbibed t the sme temperture. Similrly, vibilities of ner-fully hydrted conidi of Bb, Mc, nd M ( w P 0.954) were reduced only 9.0%, 10.9%, nd 2.5%, respectively, when fst rehydrted t 0.5 C vs. t 33 C. Bsed on sorption isotherms (Fig. 1), t 25 C Bb conidi with 0.335, 0.627, nd w hve w.b. MC equivlent to 8.4%, 15.1% nd 39.2%, respectively; Mc conidi with 0.321, 0.634, nd w hve MCs equivlent to 7.8%, 13.0%, nd 35.6%, respectively; M conidi with 0.333, 0.632, nd w hve MCs equivlent to 8.2%, 14.8%, nd 39.7%, respectively Assessing the protective potentil of oil s crrier As observed in the previous tests, vibility of M conidi ws high (in this cse P82%) when conidi were slow rehydrted prior to immersion nd when dry conidi were fst rehydrted in wrm wter (34 C). The technique used for ssessing vibility of conidi in queous suspension ws eqully effective for conidi formulted in pure prffinic oil. Conidi in the oil droplets settled onto the gr surfce nd germinted normlly. As the oil contined no emulsifier, there ws no formtion of oil micro-droplets, nd conidi were redily identifible. A2 4 fctoril ANOVA of the dt from the 0.5 nd 16 C tretments cross the three immersion medi (wter, wter + Lutensol, nd oil) reveled highly significnt immersion medium temperture interction (F 3,24 = 15.4, P < ), nd

6 M. Fri et l. / Biologicl Control 51 (2009) Tble 4 Effects of conidil wter ctivity nd imbibition temperture on percent germintion of three entomopthogenic fungi (Beuveri bssin, Metrhizium cridum, nd M. nisoplie). Fungus Wter temperture ( C) Percent germintion (men ± stndrd error) t indicted wter ctivity ( w ) Men B. bssin ± ± ± ± ± ± 4.4 b ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.4 Men 82.9 ± 4.7 c 81.5 ± 5.9 c 87.2 ± 3.6 b 93.8 ± ± 1.1 M. cridum Men ± ± ± ± ± ± 7.0 d ± ± ± ± ± ± 3.8 c ± ± ± ± ± ± 0.5 b ± ± ± ± ± ± 0.5 Men 62.3 ± 9.2 c 62.2 ± 9.5 c 63.9 ± 9.5 c 77.1 ± 7.3 b 90.1 ± 1.3 M. nisoplie Men ± ± ± ± ± ± 8.2 d ± ± ± ± ± ± 6.7 c ± ± ± ± ± ± 2.2 b ± ± ± ± ± ± 0.9 Men 44.3 ± 9.2 c 46.7 ± 8.8 c 50.8 ± 8.8 c 65.7 ± 8.7 b 92.3 ± 0.8 Conidi with different initil w were immersed in wter with 0.05% Lutensol equilibrted t different tempertures nd then incubted for 48 h on yest extrct gr with g l 1 benomyl. For ech fungl species, mens followed by the sme letter re not significntly different (Tukey HSD, = 0.05). one-wy ANOVA of the vrious tretments reveled highly significnt differences (F 9,30 = 198.3, P < ). Immersion of conidi in wter or wter + Lutensol t 0.5 or 16 C ws extremely detrimentl, reducing vibility to low levels ( %), wheres immersion in oil t low tempertures hd no negtive effect (vibility remined P83% nd did not differ significntly from the controls) (Tble 5). Vibility fter immersion in wter + Lutensol t 16 C ws lower thn vibility following immersion in wter without Lutensol (12% vs. 25%), but the difference ws not sttisticlly significnt. Inocultion of dry conidi onto cold gr substrte hd the sme negtive effect on vibility s immersion in cold wter. Dry M conidi formulted in prffinic oil retined 66% vibility fter inocultion onto gr equilibrted t 1 C, wheres only 0.1% of unformulted conidi survived this tretment (F 5,18 = 352.2, P , Tble 6). 4. Discussion The principl finding of strong inverse reltionships between imbibitionl dmge in conidi of filmentous fungi nd both immersion temperture nd wter ctivity is in ccord with the results of studies of imbibitionl dmge in seeds (Obendorf nd Hobbs, 1970; Nijsse et l., 2004), pollen (Hoekstr 1984; Hoekstr nd vn der Wl, 1988) nd yests (vn Steveninki nd Ledeboer, 1974). Studies conducted over the pst four decdes hve produced numerous hypotheses to explin imbibitionl dmge. One of the most prominent of these, referred to by Crowe et l. (1992) s the phse trnsition hypothesis, is bsed on observed chnges in membrne permebility during temperture-dependent phse trnsitions. The phospholipid bilyer comprising biologicl membrnes exists in either gel phse or liquid Moisture content (%, wb) B. bssin M. nisoplie M. cridum Wter ctivity ( w ) Fig. 1. Reltionships between conidil wter ctivity ( w ) nd moisture content [%, wet-bsis (w.b.)] determined t 25 C for Beuveri bssin, Metrhizium nisoplie, nd M. cridum for the rnge Curves were fitted by cubic polynomil regressions nd represent bsorption nd desorption isotherms bove nd below wter ctivity in the rnge B. bssin: y = w ( w 0.42) ( w 0.42) 3 (R 2 = ), M. nisoplie: y = w ( w 0.42) ( w 0.42) 3 (R 2 = ), nd M. cridum: y = w ( w 0.42) ( w 0.42) 3 (R 2 = ). Tble 5 Effects of immersion medium nd immersion temperture on vibility of Metrhizium nisoplie conidi. Tretments/initil hydrtion stte of conidi Immersion medium b Immersion temperture ( C) % vibility (men ± stndrd error) c Controls Pre-rehydrted Wter ± 1.9 Dry Wter ± 1.2 Tretments Dry Wter ± 3.8 b Dry Wter ± 0.1 c Dry Wter + Lutensol ± 2.6 b Dry Wter + Lutensol ± 0.1 c Dry Agr ± 6.4 b Dry Agr c Dry Prffinic oil ± 1.2 Dry Prffinic oil ± 0.9 Conidi were pre-rehydrted in wter-sturted tmosphere for 24 h or dried over sturted NOH solution for 3 dys (in both cses t 25 C) prior to testing. b Conidi were immersed in distilled wter (dh 2 O), dh 2 O with 0.05% Lutensol, or pure prffinic oil or were dusted onto yest extrct gr with g l 1 benomyl (YEA + benomyl) equilibrted t either 1 or 16 C. c Following the immersion tretments, conidi were incubted on YEA + benomyl t 25 C for 24 h for vibility determintions. Pltes dusted with dry conidi were lso incubted t 25 C for the sme period. Mens followed by the sme letter re not significntly different (Tukey HSD, = 0.05).

7 352 M. Fri et l. / Biologicl Control 51 (2009) Tble 6 Assessment of the protective effect of oil ginst imbibitionl dmge of Metrhizium nisoplie conidi. Incubtion time (h) Percent germintion (±stndrd error) Control b Oil c No crrier d ± 2.8 b 63.3 ± 3.4 c 0.0 d ± ± 4.2 bc 0.1 ± 0.1 d Following the imbibition tretments, conidi were incubted 24 or 48 h on yest extrct gr with g l 1 benomyl (YEA + benomyl) t 25 C for vibility determintions. Mens followed by the sme letter re not significntly different (Tukey HSD, = 0.05). b Control (dry) conidi were immersed in wrm (34 C) distilled wter (dh 2 O) with 0.05% Lutensol prior to plting on YEA + benomyl. c Conidi suspended in pure prffinic oil were inoculted onto YEA + benomyl equilibrted t 1 C. d Dry conidi were dusted onto YEA + benomyl equilibrted t 1 C. crystlline phse depending on the phse trnsition temperture (generlly designted s T m, the melting temperture); the two phses coexist trnsiently t T m. Dehydrtion gretly increses T m, generlly resulting in trnsition from the liquid crystlline phse to the gel phse. When dry membrne in gel phse is immersed in wter t temperture <T m, the membrne remins in gel phse until imbibition begins. Subsequent rehydrtion of the membrne reduces T m, nd reversion to the liquid crystlline phse is initited. During this phse trnsition, lekge of cell contents occurs, nd ccording to the phse trnsition hypothesis, lekge becomes severe nd lethl when the trnsition tkes plce in the presence of liquid wter (Crowe et l., 2002, 1992). More recently, Hoekstr et l. (1999) hypothesized tht membrnes in gel phse hve reduced elsticity, mking them susceptible to mechnicl dmge from the pressure of penetrting liquid wter. Regrdless of the exct mechnism, the most prominent effect of imbibitionl dmge is drmtic increse in cell permebility. The current hypotheses further suggest tht imbibitionl dmge is prevented when membrne phospholipids melt to the liquid crystlline phse prior to or t the onset of imbibition. The trnsition cn be induced either by slowly rehydrting membrnes vi exposure to wter vpor (lowering T m ) or by heting membrnes to temperture >T m (Crowe et l., 1992). In the ltter cse, heting of the membrne cn occur very rpidly. Thus, when dry membrne in gel phse is immersed in wter t >T m, trnsition to the liquid crystlline phse tkes plce before the liquid wter cn exert lethl effect (Hoekstr et l., 1999). Combintions of wter ctivity nd imbibition temperture tht resulted in very high germintion counts for conidi plunged into wter re presented herein. Mximl germintion counts for dry conidi of ll fungl species were observed t 33 C, but we did not determine if higher vibilities (especilly for dry M) could be chieved t higher imbibition tempertures. Incubtion for either 2 or 60 min t 0.5 C resulted in equivlent levels of dmge, indicting tht this phenomenon tkes plce in very short time. Over 90% of Typh ltifoli L. pollen with low initil moisture content showed irreversible loss of vibility when imbibed in ice-cold medium (Hoekstr, 1984). In the sme study, evidence tht imbibitionl stress occurs in just few seconds ws reported for pollen dried to 7% MC. When conidi were dried to w levels , substntil imbibitionl dmge (resulting in vibilities <65%) ws observed only t 0.5 C for Bb, wheres Mc conidi were lso susceptible to imbibitionl dmge t 15 C nd M ws susceptible t both 15 nd 25 C. These observtions strongly suggest tht Bb is less susceptible to imbibitionl dmge thn the Metrhizium species. This trit my hve contributed to the successful development of GHA nd other Bb strins s biocontrol gents. The results lso suggest tht Mc my be less susceptible to dmge thn M. It is noteworthy in this regrd tht Mglhães nd Boucis (2004) found no evidence of imbibitionl dmge when dried conidi of Brzilin isolte of Mc were immersed in queous Tween solution without previous slow rehydrtion, but the temperture of the solution ws not reported. It is importnt to underscore tht the results presented herein re bsed on single production btch of single strin of ech fungl species, nd it is cler tht rigorous conclusions regrding the reltive susceptibilities of these fungi to imbibitionl dmge will require more extensive ssys. Interestingly, t the moderte imbibition temperture of 25 C, germintion percentges for dry Mc were pproximtely 90%, which is considerbly higher thn the rte of 24.5% reported by Moore et l. (1997) for the sme isolte. The conflicting results could be relted to differences in the germintion medi, imbibition tempertures, or overll qulity of conidi used in the two studies. Moore et l. (1997) did not report the temperture of the wter + Tween solution used for fst rehydrtion; however, the conidi they subjected to this tretment hd low moisture content (4.5%, equivlent to n w of c. 0.11), which, ccording to our results, would hve mde them highly susceptible to imbibitionl dmge t tempertures 615 C. In ddition, conidil btches with low vibility, possibly indictive of stressed condition, might be more sensitive to imbibitionl dmge thn btches exhibiting high vibility. Vibility of the conidil powder used by Moore et l. (1997) ws 76 78% following slow rehydrtion for 40 min, wheres vibility of the powder used in our study ws >90% (on benomyl-free medium). We lso observed 22% increse in germintion of dry (non-slow rehydrted) Mc conidi fter incubtion for 25 vs. 20 h t 25 C (86% vs. 64%, Tble 1), nd in view of this, it is possible tht without slow rehydrtion, incubtion of this fungus for 24 h t 25 C (the protocol reported by Moore nd collegues) is only mrginlly dequte for ssessing germintion, with potentil for results to be significntly ffected by smll differences in incubtion tempertures or other experimentl prmeters. Studies of the effects of initil w on the speed of germintion of this nd other biocontrol fungi (both in the presence nd bsence of benomyl) re wrrnted. Conidi wettbility cn be correlted to presence nd stbility in the outer cell wll of hydrophobic rodlet lyer, present in Bb, M, nd Mc, but bsent or not well-orgnized in species tht produce hydrophilic conidi (Boucis nd Pendlnd, 1991). Possible roles of this hydrophobic lyer include protection ginst dehydrtion, dispersl in ir currents, nd ttchment to insect cuticle. Hydrophobicity seems to be t lest prtilly medited by components of rodlet lyers clled hydrophobins, group of low-moleculr weight proteins found in M (St. Leger, 1992). This extrcellulr polypeptide ws lso detected in Bb eril conidi but not in blstospores, which lck rodlet lyer (Holder nd Keyhni, 2005). Whether hydrophobicity is involved in differentil wter uptke by dry Bb nd M conidi nd consequently in vrible levels of imbibitionl dmge to the plsm membrne is not cler. Both species seem to be eqully hydrophobic, lthough size nd rrngement of rodlets on the cell wll surfce is quite distinct between these two species (Boucis et l., 1988). Differences in desicction tolernce between thick-wlled conidi nd thin-wlled blstospores of Mc lso hve been reported nd cell wll thickness suggested s possible explntion (Lelnd et l., 2005). The Mc conidil wll comprises double-lyered cell wll with totl thickness of 0.17 lm (Lelnd et l., 2005). Interestingly, the eril conidi of M, which we found more susceptible to imbibitionl dmge thn Mc, possess triple-lyered cell wll, yet totl wll thickness is only lm (Zchruck, 1970). The importnce of n outer brrier in voiding imbibitionl dmge ws demonstrted for lettuce seeds, which re tolernt to this kind of stress when intct, but highly sensitive if the endosperm

8 M. Fri et l. / Biologicl Control 51 (2009) lyer is removed (Nijsse et l., 2004). These uthors suggested tht membrne injury could be prevented by thick-wlled endosperm cting s brrier ginst rpid influx of liquid wter combined with network of intercellulr spces llowing diffusion of wter vpor with consequently slower nd more homogeneous rehydrtion. We hypothesize tht the cell wlls of conidi (especilly the cell wlls of Bb) ct similrly to slow the uptke of wter nd llow slow nd homogeneous membrne rehydrtion by wter vpor, preventing or lleviting membrne injury. During ultrstructurl studies with fungl conidi, Zchruck (1970) observed tht the cell wll ws brrier to rpid penetrtion of fixtives. Whether or not thinner cell wll is involved in the incresed susceptibility of M to imbibitionl injury compred to Mc conidi remins to be proven, but cell wlls likely ply mjor role in conferring vrying degrees of tolernce to this kind of stress. Tolernce to imbibitionl dmge ws reltively high for dry Bb nd Mc conidi t 25 C, but vibility of both Metrhizium isoltes dropped bruptly t 15 C. This is reson for concern for use of these fungi s pest control gents, s the temperture of wter from underground sources my be this low, even in tropicl res. Metrhizium spp. comprise more thn one-third of the mycoinsecticides nd mycocricides developed worldwide nd currently in the mrket, nd the ctive ingredients in mny of these products re dry conidi (in wettble powders or oil-bsed formultions) (Fri nd Wright, 2007). There re numerous dvntges to use of dry conidi in biopesticide products, including fcilittion of hrvesting nd formultion, reduced contmintion, nd reduction in shipping/storge volumes nd weights. Even more importntly, mximum storge stbility of conidi of commonly employed entomopthogenic fungi requires drying to low moisture content (4 5% ccording to Hedgecock et l. (1995) nd Hong et l. (2001)). Avoidnce of imbibitionl stress vi slow rehydrtion or suspension in wrm wter my be imprcticl for commercil pplictors, depending on opertionl scle or other conditions. Nevertheless, development of either producer- or user-friendly protocols to cope with this problem should be considered reserch priority. Our results suggest tht oil formultion might be beneficil, but much dditionl reserch is needed. Applictions of microbil biocontrol gents in pure oil, such s tht used in our study, do not involve mixing or other contct with wter unless the trget includes wet substrtes. More significnt would be demonstrtion of reduced imbibitionl dmge following queous suspension of conidi formulted in emulsifible oils. When emulsifible formultions re mixed in wter, conidi re encpsulted in very smll droplets of oil tht might hve only limited protective cpcity. In ddition to oils, however, there my be other formultion mterils with potentil, through encpsultion, to protect conidi or other fungl propgules from imbibitionl dmge. Benomyl is useful tool in ssessing vibility of entomopthogenic fungi. Slowing germ tube development enbles reserchers to incubte conidi for prolonged periods, providing greter flexibility in the set up nd reding of vibility ssys (Milner et l., 1991; Goettel nd Inglis, 1997). Nevertheless, it is importnt to recognize the cpcity of this mteril to inhibit germintion of t lest some fungi. In the presence of benomyl, we observed substntilly slower germintion of Mc compred to Bb or M. It should lso be noted tht conidi pretreted by exposure to wter-sturted environment (slow rehydrted) germinted more rpidly thn dry conidi. Our study underscores need for estblishing stndrd protocols for prepring queous suspensions of sensitive fungi for reserch nd commercil pplictions. Such informtion would be prticulrly importnt ddition to mycopesticide lbels. The gener Beuveri nd Metrhizium include diverse ssemblges of species nd strins with biopesticide development potentil, nd dditionl studies chrcterizing the rnge of susceptibilities of these fungi nd others to imbibitionl dmge re urgently needed. Acknowledgments We re grteful to Lverlm Interntionl Corp. for providing the prffinic oil, nd BASF Corportion for smples of the surfctnt used in this study. We re grteful to Stephen Rehner (USDA-ARS, Beltsville, MD, USA) nd Joseph Bischoff (USDA-APHIS, Beltsville, MD, USA) for genomic identifiction of the M isolte used in this work. We lso thnk Mrk Rmos (USDA-ARS, Ithc, NY, USA) for reviewing n erlier version of the mnuscript. References Bischoff, J.F., Rehner, S.A., Humber, R.A., A multilocus phylogeny of the Metrhizium nisoplie linege. Mycologi 101, Boucis, D.G., Pendlnd, J.C., Attchment of mycopthogens to cuticle: the initil event of mycoses in rthropod hosts. 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