Biological Control 61 (2012) Contents lists available at SciVerse ScienceDirect. Biological Control

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1 Biologicl Control 61 (2012) Contents lists vilble t SciVerse ScienceDirect Biologicl Control journl homepge: Appliction of modified tmosphere pckging (gs flushing nd ctive pckging) for extending the shelf life of Beuveri bssin conidi t high tempertures Mrcos Fri,, Joseph H. Hotchkiss b,1, Stephen P. Wright c EMBRAPA Recursos Genéticos e Biotecnologi, Brsili, Brzil b Deprtment of Food Science, Cornell University, Ithc, NY, USA c Robert W. Holley Center for Agriculture nd Helth, USDA-ARS, Ithc, NY, USA highlights " Shelf life of Beuveri bssin conidi is extended in tmospheres with CO 2,N 2,H 2 or He. " Vibilities P80% were recorded fter 6 months t 40 C in pckges with O 2 /moisture scvengers. " Wter ctivity under O 2 -free storge should be lower thn previously determined in open storge. " An equilibrtion period t moderte temperture is necessry when initil w of conidi is high. grphicl bstrct rticle info bstrct Article history: Received 9 September 2011 Accepted 16 December 2011 Avilble online 24 December 2011 Keywords: Mycoinsecticides Storge Shelf life Modified tmosphere pckging Active pckging Beuveri bssin Shelf life determintions under non-refrigerted conditions, especilly high temperture regimes chrcteristic of tropicl/subtropicl regions, deserve more ttention. In this study, we investigted effects of modified tmosphere pckging (MAP) on longevity of conidi of Beuveri bssin (Bb) strin GHA. Similr rtes of conidil survivl were observed fter storge for 60 dys t 50 C in tmospheres of pure CO 2, N 2,H 2, or He (49 51% vibility), but few conidi (62%) survived storge in O 2 -rich tmospheres. Vibility of conidi stored in n tmosphere of 20% CO 2 /80% N 2 decresed to <80% within 180 dys t 40 C nd within 30 dys t 50 C but remined high (87%) fter 16-month storge period t 25 C. O 2 concentrtions in the storge continers rnged from 0.3% t the strt to s high s 12.4% t the end of experiments (due to continer lekge). When ctive pckging (hermeticlly seled pckges with O 2 /moisture scvengers) ws employed, shelf lives were substntilly improved. Vibilities P80% were consistently recorded fter 6 months t 40 C or 2 months t 50 C when dul O 2 /moisture bsorber or combintion of schets (dul O 2 bsorber/co 2 genertor + desiccnt) were used. Wter ctivities ( w ) supporting gretest survivl were , suggesting tht optiml w for long-term storge under nerobic conditions is lower thn determined in previous studies of storge in the presence of O 2. Additionlly, we hve shown tht ctively pckged conidi with higher thn desirble initil w should be llowed n equilibrtion period t moderte temperture before exposure to high storge tempertures. Active pckging of dried conidi ( w ) preserved 71% vibility for 16 months t 40 C nd 63 65% for Corresponding uthor. Fx: E-mil ddress: fri@cenrgen.embrp.br (M. Fri). 1 Current ddress: School of Pckging, Michign Stte University, Est Lnsing, MI, USA Ó 2011 Elsevier Inc. Open ccess under the Elsevier OA license. doi: /j.biocontrol

2 M. Fri et l. / Biologicl Control 61 (2012) months t 50 C. To our knowledge, these re the longest survivl times yet reported for Bb conidi under high-temperture conditions. Ó 2011 Elsevier Inc. Open ccess under the Elsevier OA license. 1. Introduction Improved shelf life of fungl propgules under non-refrigerted storge conditions is of prmount importnce, since mycopesticides my be exposed to tempertures in the high 30 s to 50 C for extended periods during trnsport, wrehousing, or on-frm storge, prticulrly in the tropics or subtropics (Stthers et l., 1993; Alves et l., 1996; Hong et l., 1997; Burges, 1998; Jin et l., 1999; Roberts nd Leger, 2004). Shelf life my vry ccording to species (Hong et l., 1997), isolte (Aregger, 1992; Hong et l., 2001), nd propgule type (Elzein et l., 2004; Cliquet nd Zeeshn, 2008). It is obvious tht fctors such s temperture (Doust nd Roberts, 1983; Sndhu et l., 1993; Moore et l., 1996; Hong et l., 1999), moisture (Sndhu et l., 1993; Connick et l., 1996; Hong et l., 2001), nd pckging system (Clerk nd Mdelin, 1965; Jin et l., 1999; Abelln et l., 2000) ply vitl role during storge. Studies deling with shelf life of entomopthogenic fungi hve focused primrily on cold or moderte mbient tempertures (Wlstd et l., 1970; Aregger, 1992; Silv, 2006). Also, most studies hve involved open storge, in which ir exchnge tkes plce between the externl nd internl tmosphere of pckges (nonhermetic continers). Vrible results hve been reported in open storge studies of Beuveri bssin (Bb) conidi t high mbient tempertures (P30 C). Mrques nd Alves (1996) reported tht vibility of conidi with 15.5% moisture content (MC) stored t 30 C ws gretly reduced within 30 dys, while conidi formulted in sunflower oil retined 80% vibility for 90 d but only c. 30% vibility for 120 d. Time for initil vibility to drop to 80% ws less thn one week t 37 C for powder with unspecified moisture content (Jin et l., 1999). Significnt reduction in vibility of formulted eril conidi with unspecified moisture content occurred 2 3 months fter storge t 30 C (Kss, 2003). When Bb conidi were stored in glss jrs in which equilibrium reltive humidities (ERHs) were controlled with sturted slt solutions to levels between 0% nd 98%, highest vibilities were observed t lower tempertures nd ERHs, nd fter one yer t 40 nd 30 C vibilities were completely lost or shrply reduced, respectively, for ll tested ERHs (Sndhu et l., 1993). Hermetic storge of formulted nd unformulted Bb conidi with MC in the 11 19% rnge resulted in loss of P52% vibility fter 30 d t 35 C(Silv, 2006). On the other hnd, hermetic storge of Bb conidi with % MC led to estimted times for initil vibility (96%) to drop to 80% of s long s 80 nd 17 d t 40 nd 50 C, respectively (Hong et l., 2001). To our knowledge, neither O 2 -free hermetic pckging nor ctive pckging (AP), commercilly used by the food industry since 1976 (Robertson, 2006), hs been tested for storge of Bb. According to Robertson (2006), AP is defined s pckging in which subsidiry constituents hve been delibertely included in or on the pckging mteril or in the pckge hedspce to enhnce the performnce of the pckge system. The objectives of this work were to elucidte the effects of vrious gseous tmospheres on shelf life of Bb conidi nd ssess the potentil of n nerobic pckging strtegy for extension of Bb conidi longevity under high- temperture regimes. 2. Mteril nd methods 2.1. Conidil powders Unformulted eril conidi (technicl powders) of Bb strin GHA were cquired from Lverlm Interntionl Corp. (Butte, MT, USA) nd stored t 20 C until use. Unless indicted otherwise, ech of the replicte smples ssigned to tretments in ll tests comprised 0.6 g of conidil powder, n mount suitble for wter ctivity mesurements Germintion counts Two protocols were used for ssessing pre- nd post-tretment vibility of conidil powders. In the first (slow-rehydrtion protocol), smll mount of conidil powder ( mg) ws picked up with sptul, trnsferred to glss Petri dish nd incubted for 24 h in 24-cm internl dimeter glss desicctor with wter-sturted tmosphere. The conidi were then trnsferred to screw-cp glss vils contining glss beds nd 7 ml of 0.05% Lutensol Ò (Ethoxylted Tridecyl Alcohol, BASF Corportion, Florhm Prk, NJ, USA), possessing hydrophilic lipophilic blnce number of 10. Suspensions were gitted for 10 min on wrist ction shker (Burrel Scientific, Pittsburgh, PA, USA), nd 10-ll liquots were inoculted onto cm blocks (1 droplet/ block) of yest extrct gr-bsed solid medium (Meikle et l., 2003). The gr blocks (on glss slides) were plced in Petri dishes, nd the dishes were seled with Prfilm nd incubted t 25 C in drkness. Germintion ws ssessed 24 h post-inocultion (p.i.). In the second germintion protocol (fst-rehydrtion protocol), vibility ws determined s described bove except tht the dry conidi were directly suspended in the wter/surfctnt solution without prior exposure to humid tmosphere. As it ws previously shown tht dry Bb conidi with high vibility (high qulity) re not susceptible to imbibitionl dmge t moderte tempertures (Fri et l., 2009), solutions used for prepring suspensions (wter/surfctnt) were equilibrted t room temperture. Conidi were considered to hve germinted when germ tube of ny size ws visible t 400X mgnifiction with phse-contrst illumintion. A minimum totl of 200 conidi were exmined in severl microscope fields for ech replicte suspension of ech experimentl tretment Gs flushing Flushing with different gses Conidil smples (0.6 g) were dded to 3.4 cm dim. 1.1 cm plstic smple cups (code ; Novsin, Pfäffikon, Switzerlnd) nd kept inside irtight 125-mL glss jrs (Bll Ò, Jrden Corp., Muncie, IN, USA) seled with metllic lids fitted with rubber sept. Ech glss jr ws flushed for 40 min t 40 ml/min flow rte with pure crbon dioxide, nitrogen, helium or hydrogen, s well s 100% or 21% oxygen, blnced with N 2 (Airgs Est, Inc., Slem, NH, USA). In jrs not flushed with O 2, the concentrtion of this gs following this procedure ws checked to mke sure mbient ir hd been successfully removed from continers. Smples of gs (500 ll) were extrcted from ech jr with gs-tight syringe (model 1750, Hmilton Compny, Reno, NV, USA) nd injected into gs chromtogrph (Vrin Aerogrph, Wlnut Creek, CA, USA) equipped with therml conductivity detector. Pek heights were compred ginst commercil stndrd contining 6.96% O 2 nd 4.91% CO 2, blnced with N 2. Ech gs exposure tretment ws replicted three or four times. In order to minimize ir lekge (O 2 ingress) into storge continers, the 125-mL glss jrs were kept inside lrger irtight Bll Ò jr (0.95-L) contining the sme mixture of gses. Using this setup, glss jrs were incubted t 50 C for 60 d. Following this storge, the O 2 concentrtion in ech jr ws re-ssessed s n indiction of lekge. Conidil wter

3 80 M. Fri et l. / Biologicl Control 61 (2012) ctivity ( w ) ws mesured t 25 C with n w meter (LbMster w, Novsin, Pfäffikon, Switzerlnd), nd germintion counts were determined s previously described. The experiment ws repeted on different dte, without the 21% O 2 tretment. Experimentl tempertures for this nd ll subsequent experiments were ±1 C, s determined from continuous monitoring with two digitl dt loggers per incubtor (Hobo Ò, Onset Computer Corp., Bourne, MA, USA) Flushing with 20% CO 2 nd storge t different tempertures Using the sme set up described in the previous section, jrs with conidil smples were flushed with 20% CO 2 (+80% N 2 ). After ech storge period t 40 C (45, 91, 180 nd 240 d), four replicte jrs were destructively smpled for mesurement of the finl O 2 concentrtion nd w. The experiment ws repeted, beginning on different dte. Following the sme protocol, seprte experiments were conducted investigting effects of storge t 25 C (ssessments t 46, 120, 180, 365, nd 480 d) nd 50 C (ssessments t 15, 30, 47, 61, 75, nd 90 d). In ll cses, vibility ws lso ssessed t 0 d (just prior to storge) Active pckging of conidi Comprison between luminized films In order to select n luminized plstic film for the ctive-pckging experiments, conidil smples were held over MgCl 2 6H 2 O for 2 d t 25 C, resulting in men (±stndrd error) w of ± Conidi were then trnsferred to pouches (10 12 cm) fbricted from two different luminized films: one of unknown composition provided by mycopesticide compny (overll thickness 114 lm) nd the other three-lyered lminte composed of 12 lm polyethylene terephthlte, 8 lm luminum foil, nd n 85 lm crystllized polypropylene het-sel lyer (mnufcturer unknown). One dul-ction O 2 /H 2 O-bsorbing schet (RP-3A, Mitsubishi Gs Chemicl Co., Jpn) ws enclosed loose in ech pouch before het seling nd incubtion t 40 C for 5 months. Ech tretment ws replicted four times. After 5 months t 40 C, finl conidil w s nd vibilities for smples stored with the respective films were virtully identicl (90.9 ± 0.1 vs ± 2.0), nd both mterils were employed in the subsequent tests (one mteril used for ll tretments within test). Hereinfter, pouches composed of these lmintes will be referred to s foil pouches. We opted not to mesure finl (poststorge-tretment) concentrtions of O 2 in the luminized pouches, which would hve required instlltion of rubber sept, becuse we did not wnt to risk compromising continer integrity. Het-seled luminized pouches (high-brrier foil pouches) re specificlly designed to function s hermetic storge continers Comprison between gs flushing nd ctive pckging (AP) gents Bb conidi were dried over NOH in 125-ml glss jrs for 1 d t 25 C, resulting in ± w. A number of rndomly selected smples were then trnsferred to glss jrs nd flushed with N 2.To minimize dusting during the flushing procedure, conidi in glss jrs were kept inside smll, uncovered plstic smple cups. O 2 lekge ws reduced by using the previously-described double-continer system. Ech of the remining 0.6-g smples ws rndomly ssigned to one of three AP tretments comprising foil pouches (8 8.5 cm) with either one RP-3A O 2 /H 2 O-bsorbing schet, one O 2 -bsorbing film (code M-0034, lot 19208A, mm; CSP Technologies, Auburn, AL, USA) plus one H 2 O-bsorbing film (CSP Technologies, code M-0026, lot 02208A, mm), or one O 2 /H 2 O-bsorbing film (CSP Technologies, code M-0033, lot 10808A, mm). As control, conidi were kept in polyethylene pouches (8 8.5 cm, wll thickness 30 lm; code P ; Empc Agroindustril de Plásticos Ltd, Brsili, Brzil) with one RP-3A O 2 /H 2 O-bsorbing schet. Following preprtion, ll continers with conidi were preincubted t 25 C for 5 d nd then trnsferred to 50 C. O 2 concentrtions in glss jrs flushed with N 2 were checked immeditely prior to high-temperture incubtion. For ll tretments, three replicte continers were destructively smpled to ssess conidil w nd vibility immeditely before the trnsfer to 50 C. Conidi were incubted t 50 C for either 56 or 129 d. After storge, conidil w ws mesured nd germintion counts performed. For ech tretment nd ssessment dte, four independently prepred replicte pouches were destructively evluted Comprison mong AP schets Bb conidil smples were stored in 125-mL glss jrs over the desiccnt clcium sulphte (eight-mesh indicting drierite, W.A. Hmmond Drierite Co., Xeni, OH, USA) for 2 d t 25 C. Conidil w just before pckging ws ± Alterntively, conidi were stored over sturted NCl solution for 2 d t 25 C, which resulted in ± w prior to pckging. Smples were then trnsferred to foil pouches (10 12 cm) contining one of the following AP schets: RP-3A O 2 /H 2 O bsorber, Ageless Ò ZPT 1000 O 2 bsorber (Mitsubishi Gs Chemicl Co., Jpn), OxyFree 504A O 2 /CO 2 bsorber (Tinhu Tech, Chin), OxyFree 504E O 2 bsorber/co 2 genertor (Tinhu Tech, Chin), or drierite H 2 O bsorber (56.7 g). As control, foil pouches without ny AP schet were employed. Pouches were incubted t 50 C without preincubtion equilibrtion period, nd conidil w ws quntified nd germintion counts were performed fter 45 d. Ech tretment (schet type vs. initil w ) ws replicted four times Combintion of AP schets for shelf-life extension Conidil smples were dried over drierite for 2 d t 25 C (resulting in ± w ) nd then trnsferred to cm foil pouches with different schets: one RP-5A O 2 /H 2 O bsorber (sme s RP-3A but indicted for lrger pckges), one 504E O 2 bsorber/ CO 2 genertor, or one 504E plus one schet of drierite (56.7 g). Ech tretment ws replicted three times, nd conidil w nd germintion ssessments were performed 148 nd 180 d post-storge t 40 C Effect of pre-incubtion regime on shelf-life Due to lower thn expected vibilities fter 6-month period t 40 C in the previous experiment, we decided to test the importnce of llowing conidil powders with high initil w to equilibrte to lower O 2 /moisture levels within the pckge before exposure to high temperture regimes. Bb smples were kept over drierite or NCl for 2 d t 25 C, resulting in ± nd ± w s, respectively. Then, conidi were trnsferred to foil pouches ech with one RP-3A O 2 /H 2 O bsorber schet nd pre-incubted for n dditionl 5 dys t 25 C (equilibrtion period) before storge t trget tempertures (25, 40, nd 50 C). Alterntively, smples were kept over drierite or NCl for 7 d t 25 C, trnsferred to foil pouches with RP-3A schets nd immeditely stored t trget tempertures without 5-d pre-incubtion period t 25 C. Ech tretment ws replicted four times nd ssessment of conidil w nd germintions were crried out fter 60 d t 50 C, nd 180 d t either 25 or 40 C Effects of w on long-term conidil survivl t 40 nd 50 C under nerobic conditions Smples of conidi (0.6 g) were initilly dried over drierite in 125-mL glss jrs for 3 dys t 25 C nd then trnsferred to foil pouches. Conidil w ws then djusted to different levels vi the following tretments: (1) ddition of schet 504E (O 2 scvenger/ CO 2 nd H 2 O genertor), (2) ddition of schet RP-3 K (O 2 scven-

4 M. Fri et l. / Biologicl Control 61 (2012) ger/h 2 O genertor), (3) ddition of schet RP-3A (O 2 /H 2 O scvenger), nd (4) hydrtion of conidi for 6.5 h over 30 ml distilled wter (in glss jr) prior to ddition of schet RP-3A. For ech tretment, three independently prepred replicte pouches were destructively evluted fter 7 dys t 25 C (equilibrtion period) in order to determine vibilities of conidi prior to high-temperture storge, nd four pouches were destructively evluted in ssessments following long-term storge for 3 months t 50 C or 16 months t 40 C. The experiment ws repeted on different dte, the only difference being tht drierite (10 g) ws lso enclosed in ech pouch with 504E schet (tretment 1) A Lower w (0.099±0.0248) 2.5. Sttisticl nlyses In experiments conducted to determine the effect of different gses on storge of Bb conidi, jrs flushed with gses other thn O 2 in which considerble lekge took plce (finl O 2 concentrtion >3.5%) were discrded. With the exception of the tests with helium (see Section 3), no more thn one or two of the eight replictes of ech tretment were discrded. In gs flushing experiments with 20% CO 2 /80% N 2 t different tempertures, ll glss jrs were considered in sttisticl nlyses independently of O 2 ingress, s finl conidil vibilities cross replictes within tretments were similr, regrdless of finl O 2 concentrtion. Dt in the form of percentges were rcsine squre root trnsformed nd exmined using one-wy nlysis of vrince. Mens were compred by the Tukey Krmer HSD test or t-test nd considered to be sttisticlly different t the 5% significnce level. Dt nlyses were performed using the JMP sttisticl pckge (SAS Institute Inc, Cry, NC, USA). Germintion (%) B N 2 CO 2 H 2 He O 2 (21%) O 2 (100%) Higher w (0.119±0.0021) Fst rehydrtion Slow rehydrtion b b b c 3. Results Actul tempertures in closed, equilibrted incubtors set t 25, 40, nd 50 C were ±0.5, ±1, nd ±1 C, respectively (in the 40 nd 50 C tretments, tempertures momentrily dropped 2 3 C nd 4 6 C, respectively, when incubtor were opened). Germintion percentges determined fter fst versus slow rehydrtion re reported in most cses, but we regrd estimtes from the fst-rehydrtion protocol s being better indictors of conidil qulity (Fri et l., 2010). Thus, unless otherwise stted, the use of the terms vibility nd germintion refer to germintion percentges mesured following fst rehydrtion. In ll experiments of the present study, the estimted initil vibilities determined by the fst- nd slow-rehydrtion protocols were P96.1% nd P96.8%, respectively Flushing with different gses Finl w for conidi in the first series of ssys (with lower w ) did not vry with gs tretment (P = 0.41, F [5,13] = 1.1); overll men w ws ± Significnt germintion differences were recorded fter 60 d t 50 C (P < , F [5,13] = 122.0), nd while exposure to N 2,CO 2,H 2, nd He produced equivlent vibilities in the 49 51% rnge, very low germintion rtes nd no survivors were recorded for 21% nd 100% O 2, respectively (Fig. 1A). Finl O 2 concentrtions did not differ mong jrs flushed with gses other thn O 2 (P = 0.29, F [3,9] = 1.5), rnging from 0.3% to 1.1%. In the second series of ssys (with higher w ), excessive O 2 lekge occurred inexplicbly in ll continers flushed with helium (finl O 2 >3.5%), nd the dt were discrded. As in the first ssy, the tretments produced no significnt differences in finl conidil w (P = 0.29, F [3,11] = 1.4); w verged ± cross tretments. Flushing with 100% O 2 resulted gin in no survivors (21% O 2 ws not tested). Storge with ll other gses resulted in low, equivlent vibility (rnge of 10 13%) (Fig. 1B). 0 N 2 CO 2 H 2 O 2 (100%) Gs Fig. 1. Effect of different gses on percent vibilities (men ± SE) fter fst vs. slowrehydrtion protocols for Beuveri bssin conidi fter storge t 50 C for 60 dys. In both experiments, vibilities for the 100% O 2 tretment were 0%. Within ech germintion protocol, brs (±SE) with the sme letter re not significntly different (Tukey HSD, = 0.05). These germintion rtes were mrkedly lower thn the rnge of 49 51% observed in the ssys with lower w. Finl O 2 concentrtions ( %) did not differ mong jrs flushed with gses other thn O 2 (P = 0.73, F [2,8] = 0.3) Flushing with 20% CO 2 nd storge t different tempertures In the 25 C storge experiment, significnt drop in vibility ws observed (P = , F [5,18] = 8.8), but the decrese ws smll nd grdul, from 96% to 90% within 365 d nd to 87% within 480 d (Fig. 2A). Conidil w incresed from t dy 46 to t the end of the experiment (P < , F [4,15] = 36.3), nd O 2 concentrtions incresed from n verge of % (P < , F [4,15] = 38.0). In the experiment t 40 C (Fig. 2B), 2-fctor ANOVA (min effect of ssy nd storge time) followed by the Tukey HSD test reveled sttisticlly significnt loss of vibility during the first 3 months of storge, but the decrese ws only 6 percentge points (from 93% to 87%). This ws followed by rpid decline to just 4% vibility within 240 d (overll ANOVA P < , F [4,34] = 361.7). During the intervl between 45 nd 240 dys, men O 2 concentrtion incresed from 1.2% to 6.6% (P = , F [3,28] = 9.4) nd w incresed from to (P < , F [3,27] = 35.4). At 50 C, initil vibility dropped quickly, from 96% to 81% within the first 15 d to 10% fter just 90 d of storge (P < , F [6,21 = 129.1) (Fig. 2C). O 2 concentrtion incresed from n verge b b

5 82 M. Fri et l. / Biologicl Control 61 (2012) A Germintion (%) Slow rehydrtion Fst rehydrtion Slow rehydrtion Fst rehydrtion B C 25 ºC 40 ºC 50 ºC Slow rehydrtion Fst rehydrtion Dys post-storge Fig. 2. Vibilities fter fst- vs. slow-rehydrtion protocols of Beuveri bssin conidi flushed with 20% CO 2 (+80% N 2 ) fter storge t either 25 (A), 40 (B), or 50 C (C). of 0.8% t 15 d to 3.2% t 90 d (P = , F [5,18] = 4.5), wheres conidil w did not chnge significntly during this period (from t 15 d to t 90 d; P = , F [5,18] = 1.2) Comprison between gs flushing nd AP gents Use of foil pouches, ech with one O 2 /H 2 O-bsorbing RP-3A schet, significntly lowered conidil w to following 5- d equilibrtion period t 25 C (P = 0.016, F [4,10] = 5.2) (Tble 1). Considerble differences in vibilities were observed mong tretments 56 d (P < , F [4,10] = 5.2) nd 129 d fter storge t 50 C (P < , F [4,15] = 427.9). The combintion of one O 2 - nd one H 2 O-bsorbing film provided stisfctory vibilities up to 56 d post-storge, but results fter 129 d were not s good s the use of RP-3A. O 2 concentrtion in glss jrs flushed with N 2 ws 0.2% immeditely prior to incubtion t 50 C. Gs flushing Tble 1 Wter ctivities ( w ) nd vibilities of Beuveri bssin conidi stored t 50 C in gs-flushed jrs or foil pouches with bsorbing schets/films. Continer/tretment 2 Dy 0 1 Dy 56 Dy 129 Percent germintion fter Percent germintion fter Percent germintion fter Slow rehydrtion Finl w Fst rehydrtion Slow rehydrtion Finl w Fst rehydrtion Slow rehydrtion Initil w Fst rehydrtion Glss jr flushed with N ± b 95.0 ± ± ± ± 1.8 d 59.7 ± 2.4 b ± ± 0.1 cd 1.3 ± 0.3 d Foil pouch + RP-3A O 2 /H 2 O-bsorbing schet ± b 96.5 ± ± ± c 89.4 ± ± ± b 54.9 ± ± 1.7 Foil pouch + M-0033 O 2 /H 2 O-bsorbing film ± b 96.0 ± ± ± c 33.8 ± 2.2 b 70.4 ± 2.0 b ± b 1.0 ± 0.5 c 12.9 ± 1.8 c Foil pouch + M-0034 O 2 -bsorbing film + M-0026 H 2 O ± b 97.3 ± ± ± c 86.0 ± ± ± b 24.8 ± 0.8 b 72.5 ± 1.6 b bsorbing film Polyethylene pouch + RP-3A O 2 /H 2 O-bsorbing schet ± ± ± ± b 16.0 ± 0.9 c 33.8 ± 3.9 c ± b 0.0 d 0.0 e Dt recorded immeditely prior to high-temperture storge, following n initil 5-dy pre-incubtion (equilibrtion) period t 25 C. Within columns, mens (±stndrd errors) followed by the sme letter re not significntly different (Tukey HSD, = 0.05). 1 2

6 M. Fri et l. / Biologicl Control 61 (2012) Tble 2 Wter ctivities ( w ) nd vibilities of Beuveri bssin conidi stored for 45 dys t 50 C in foil pouches with different bsorbing nd/or generting schets. AP schet Low initil w (0.019) High initil w (0.738) Percent germintion fter Percent germintion fter 1 Finl w Fst rehydrtion Slow rehydrtion 1 Finl w Fst rehydrtion Slow rehydrtion Ageless O 2 bsorber; H 2 O genertor ± c 0.0 c ± c 0.0 c Drierite H 2 O bsorber ± de 5.2 ± 0.7 b 9.0 ± 1.0 b ± e 7.3 ± 1.3 b 10.8 ± 0.9 b RP-3A O 2 /H 2 O bsorber ± e 79.0 ± ± ± e 72.8 ± ± A O 2 /CO 2 bsorber; H 2 O genertor ± c 0.0 c 0.0 c ± c 0.0 c 0.0 c 504E O 2 bsorber; CO 2 /H 2 O genertor ± b 0.0 c 0.0 c ± b 0.0 c 0.0 c No schet (control) ± d 3.8 ± 0.4 b 8.3 ± 0.9 b ± d 0.0 c 0.0 c 1 Within ech column, mens (±stndrd errors) followed by the sme letter re not significntly different (Tukey HSD, = 0.05). Tble 3 Effects of n O 2 bsorber/co 2 genertor, with or without desiccnt schet, on wter ctivities ( w ) nd vibilities of Beuveri bssin conidi stored in foil pouches t 40 C for either 5 or 6 months. Schet type Stored 148 dys Stored 178 dys Percent germintion fter Percent germintion fter 1 w, 2 Fst rehydrtion Slow rehydrtion 1 w, 2 Fst rehydrtion Slow rehydrtion 504E O 2 bsorber; CO 2 /H 2 O genertor ± b 0.0% b ± b 0.0 b 504E + Drierite H 2 O bsorber ± b 81.0 ± ± ± b 79.3 ± ± 0.3 RP-5A O 2 /H2O bsorber) ± b 83.5 ± ± ± b 81.8 ± ± Initil w ws ± , nd conidi were not pre-incubted (equilibrted) t moderte temperture before exposure to 40 C. 2 Within ech column, mens (±stndrd errors) followed by the sme letter re not significntly different (Tukey HSD, = 0.05). or use of polyethylene pouch (+RP-3A) resulted in poor vibilities compred to ctive pckging with dul O 2 /H 2 O bsorber Comprison mong AP schets Use of the vrious AP schets resulted in highly significnt differences in conidil vibility both for conidi with low (P < , F [5,12] = ) nd high initil w (P < , F [5,12] = 522.4) (Tble 2). As expected, considering the bsorption qulities of the different schets, the finl w of conidi in tretments with either low (P < , F [5,12] = 69,902) or high initil w (P < , F [5,12] = 84,526) were lso mrkedly different. Use of schets tht relesed moisture during storge (Ageless, 504A, nd 504E), or tht bsorbed moisture but not O 2 (drierite), resulted in lower vibility compred to use of dul-ction O 2 /H 2 O bsorber (RP-3A) Combintion of AP schets for shelf-life extension The AP schet tht bsorbs O 2 but releses moisture (504E) ws efficient when tested in conjunction with desiccnt (drierite), but the use of schet 504E lone resulted in totl loss of vibility (Tble 3). The combo pproch ws s good s the use of dul O 2 / H 2 O bsorber (RP-5A), both t 148 d (P < , F [2,6] = 309.0) nd 178 d post-storge t 40 C (P < , F [2,6] = 2035) Effect of pre-incubtion regime on shelf-life Finl conidil w did not vry mong tretments within ech of the storge temperture regimes (Tble 4). Vibilities fter 180 d t 25 C were high (91 94%) for ll tretments, except high initil w / no pre-incubtion, which reduced vibility to 88%; however, there were few significnt differences (P = , F [3,8] = 5.8 nd see Tble 4). After 180 d t 40 C, vibility ws 87 89% for ll tretments nd significntly lower (75%) in the high initil w /no pre-incubtion tretment (P = , F [3,8] = 8.7). Finlly, fter 60 d t 50 C the sme trend ws gin observed, with vibilities from ll tretments in the rnge 83 86% except for the high initil w /no preincubtion tretment, which significntly reduced vibility to 60% (P < , F [3,8] = 37.8) Effect of w on long-term vibility of conidi stored nerobiclly t 40 nd 50 C Anerobic pckging of conidi with different w ws chieved, nd initil vibilities (fter fst rehydrtion) cross tretments were not different following 7-dy equilibrtion period t 25 C (Tble 5), either in experiment 1 (P = 0.560, F [3,8] = 0.7) or experiment 2 (P = 0.248, F [3,8] = 1.7). Initil w significntly ffected conidil survivl t both 40 nd 50 C. In experiment 1, percent germintion fter 3 months t 50 C rnged from 0 or ner zero (conidil w P 0.214) to 66% ( w = 0.026) (P < , F [3,12] = 625.3). The most severely ffected conidi (those exhibiting 60.1% vibility) were drker color thn less-ffected conidi. Conidi dried to very low w of retined 71% vibility fter storge for 16 months t 40 C. A doubling of initil w to 0.05 resulted in significntly lower vibility (53%). It ws observed lso tht spore powders with n initil w of exhibited slight cking (clumping) when smpled with the sptul, wheres smples lbeled w retined their originl loose consistencies. In experiment 2, vibilities fter 3 months t 50 C rnged from 0( w = 0.214) to 65% ( w = 0.025) (P < , F [3,12] = 514.1). After 16 months t 40 C, vibility of conidi initilly dried to very low w s of either = or ws 71% in both cses (P = , F [1,5] = 0.3). 4. Discussion Shelf lives observed in the present study re longer thn previously reported for Bb. Modified tmospheres following flushing with gses other thn O 2 (CO 2,N 2,H 2, nd He) resulted in comprble vibilities following 2-month storge t 50 C. When n tmosphere with 20% CO 2 (+80% N 2 ) ws tested in jrs, times for conidil vibility to drop to 80% (fst-rehydrtion protocol) were >91 nd >15 d t 40 nd 50 C, respectively. These times re similr

7 Tble 4 Effects of initil w nd pre-incubtion t moderte temperture (25 C) on germintion of Beuveri bssin conidi stored in foil pouches with n O 2 /H 2 O-bsorbing schet. Condition Dy 180 t 25 C Dy 180 t 40 C Dy 60 t 50 C Percent germintion fter Percent germintion fter Percent germintion fter Finl 1 w, 2 Fst rehydrtion Slow rehydrtion Finl 1 w, 2 Fst rehydrtion Slow rehydrtion Finl 1 w, 2 Fst rehydrtion Slow rehydrtion Low initil w /pre-incubtion ± ± 0.4 b 95.2 ± ± ± ± ± ± ± 0.8 b Low initil w /no pre-incubtion ± ± ± ± ± ± ± ± ± 1.9 b High initil w /pre-incubtion ± ± 1.3 b 93.7 ± ± ± ± ± ± ± 1.0 High initil w /no pre-incubtion ± ± 0.4 b 91.0 ± ± ± 2.2 b 93.8 ± ± ± 3.0 b 94.7 ± 1.0 b 1 Within ech column, mens (±stndrd errors) followed by the sme letter re not significntly different (Tukey HSD, = 0.05). 2 Low nd high initil w s were ± nd ± , respectively. Tble 5 Long-term effects of wter ctivity ( w ) on vibility of Beuveri bssin strin GHA conidil powders stored t 40 nd 50 C under nerobic conditions. Dy 0 2 Stored 3 months t 50 C Stored 16 months t 40 C Tretment 1 Initil w Percent germintion 3 Finl w Percent germintion Finl w Percent germintion Experiment 1 4 Desicction + RP-3A (O 2 /H 2 O bsorber) ± ± ± ± ± ± 1.51 Hydrtion + RP-3A ± ± ± ± 2.98 b ± ± 5.02 b Desicction + RP-3K (O 2 bsorber; H 2 O genertor) ± ± ± ± 0.13 c nd 5 Desicction + 504E (O 2 bsorber; CO 2 /H 2 O genertor) ± ± ± c nd Experiment 2 4 Desicction + RP-3A ± ± ± ± ± ± 0.92 Desicction + 504E + drierite ± ± ± ± ± ± 1.16 Hydrtion + RP-3A ± ± ± ± 2.25 nd Desicction + RP-3K ± ± ± b nd 84 M. Fri et l. / Biologicl Control 61 (2012) Initil w of conidi ws djusted either by desicction over drierite for 3 d or hydrtion over wter for 6.5 h prior to pckging in foil pouches with indicted schet. 2 Dt recorded immeditely prior to high-temperture storge, following n initil 7-dy equilibrtion period t 25 C. 3 Germintion recorded 24 h fter fst rehydrtion. 4 Within ech experiment nd column, mens (±stndrd errors) followed by the sme letter re not significntly different (Tukey HSD, = 0.05). 5 Not determined.

8 M. Fri et l. / Biologicl Control 61 (2012) to estimtes clculble from dt published by Hong et l. (2001), in which Bb conidi dried to c. 5% moisture content nd stored in ir in hermeticlly seled continers retined 80% vibility for 80 nd 17 d t 40 nd 50 C, respectively. These were previously the longest shelf lives recorded for this fungl species t these high tempertures. However, when optiml ctive pckging (with schets tht bsorb both O 2 nd moisture in hermetic pckges) ws employed for isolte GHA, vibilities consistently in the 80 90% rnge were recorded following 6 months t 40 C or 2 months t 50 C. Even fter 16 months t 40 C, ctively pckged conidi displying w (in equilibrium with 3.2% ERH t 25 C) exhibited germintion >70% ( sorption isotherm relting w to moisture content of Bb strin GHA conidi is presented in Fri et l. (2009)). Shelf life of the mycoherbicide Sclerotini minor long with its substrte ws lso substntilly incresed when mbient ir ws replced by CO 2 nd/or N 2 (Teshler et l., 2007). Likewise, shelf lives of ir-dried mycelil lginte pellets of the nemtophgous fungi Pecilomyces lilcinus nd Pochoni chlmydospor were improved under vcuum, CO 2,orN 2 -rich environments when compred to ir tmosphere (Dun et l., 2008). According to these uthors, replcement of O 2 by other gses slows metbolism nd limits oxidtive rections, llowing for shelf life extension of fungi. We recently demonstrted tht tmosphere in which ir ws replced by CO 2 /N 2 incresed longevity of Bb conidi (Fri et l., 2010). All other ttempts to extend the shelf life of this fungus hve been crried out in ir, even though the beneficil effects of O 2 exclusion (or incresed CO 2 concentrtion) during short-term storge of fungus identified s Metrhizium nisoplie ws demonstrted decdes go by Clerk nd Mdelin (1965). Indeed, these uthors suggested the doption of modified tmosphere pckging s strtegy for prolonging conidil longevity, so the limited progress mde since then is surprising. On the other hnd, much of our knowledge of optiml storge conditions for microbil biocontrol gents is developed nd held in secret or ptented by commercil enterprises. Most notbly, ptent by Jin et l. (1999) climed tht Metrhizium conidi dried to very low w s with drierite nd stored in O 2 -free tmospheres creted by use of the Ageless schets inside moisture- nd gs-impermeble foil pouches showed 74% vibility fter 2 months t 37 C, wheres 0% vibility ws reported in foil pouches either without n O 2 bsorber or with ERH s high s 40 or 100%. Preservtion of dry myceli of Btko sp. nd Furi sp. for 3 months t 23 C by use of Ageless nd silic gel lso proved successful (Leite et l., 2002), but we re not wre of dditionl studies on modified tmosphere pckging of entomopthogenic fungi. The present study focused lmost exclusively on conidil survivl under high-temperture storge conditions; however, the excellent shelf life exhibited by Bb conidi stored t the moderte temperture of 25 C in glss jrs flushed with CO 2 /N 2 (Fig. 2A) is lso noteworthy. We unfortuntely did not include norml tmosphere (ir) control in the experiment, but 87% vibility fter 16 months is nevertheless one of the longest recorded shelf lives of this fungus in the bsence of refrigertion. In 25 C storge studies reported by Jronski (1997), wettble powder formultion of Bb strin GHA retined 89% vibility fter nerly 9 months, nd technicl powder remined 70% vible fter 1 yer. Gs flushing ws shown to be less efficient thn high brrier foils (+AP gents) for shelf life extension of Bb conidi in the present study due to higher thn desirble w following the flushing procedure, persistent problems with ir lekge, nd likely lso due to the incpcity of gs flushing protocols to remove ll O 2. Teshler et l. (2007) reported residul O 2 concentrtion of 0.26% following gs flushing of foil bgs. On the other hnd, in our study w remined t constnt low levels following hermetic pckging with foil nd use of n efficient O 2 /moisture bsorber. In nhydrobiotic orgnisms, isolted enzymtic rections leding to production of free rdicls nd free-rdicl medited non-enzymtic rections my tke plce. For exmple, phospholipid degrdtion rections my occur, with detrimentl ccumultion of their byproducts (ftty cids) in membrnes (McKersie et l., 1988). However, ging under O 2 -free nd extremely dry tmospheric conditions is considerbly slower thn ging under non-hermetic conditions. Shelf life predictbility for mycopesticides is difficult due to vribility with respect to ctive ingredients (composition nd qulity of propgules my vry from btch to btch) nd uncontrolled reltive humidity nd temperture regimes during trnsporttion nd storge. Improved predictbility cn be chieved by use of controlled conidil w nd gs composition, which cn be ccomplished by pproprite AP pckging systems s previously discussed. In non-hermetic pckging, vilbility of ir to conidi is fr greter (Hong et l., 2005), nd since O 2 penetrtion through currently used storge continers is not stndrdized, considerble vrition in terms of O 2 concentrtion nd conidil w nd consequent shelf life is not surprising. As shown in this work, doption of plstic polymers with high permebility to O 2 nd moisture re totlly undesirble for pckging of mycopesticides, even if combined with n efficient AP schet. Most of the AP schets used in the food industry tht we tested were not effective for extending Bb conidi longevity, either becuse conidil w incresed to undesirble levels or O 2 ws not removed. A dul O 2 /moisture bsorbing schet ws more efficient thn schets with only one ttribute. Although CO 2 is known to possess fungisttic ctivity ginst some growing fungi (Tbk nd Cooke, 1968; Abelln et l., 2000), no deleterious effect hs been observed on stored entomopthogenic conidi, which cretes the possibility for use of ctive pckging through dul O 2 bsorbing/co 2 -emitting schets. This ws the reson for inclusion of gs flushing experiments with c. 20% CO 2 in this nd previous work (Fri et l., 2010). Unfortuntely, moisture relesed when the O 2 -bsorbing/co 2 -emitting schet ws used lone resulted in incresed conidil w nd, therefore, reduced longevity. However, ssocition of this kind of schet with desiccnt schet proved successful, nd the development of efficient three-wy O 2 bsorbing/co 2 generting/moisture bsorbing schets would be welcomed, especilly if considerbly less expensive thn the commercil O 2 /moisture bsorbing schets currently vilble. Active pckging is not new in insect pthology, nd combintion of O 2 bsorbing schets nd desiccnts (Jin et l., 1999; Leite et l., 2002), nd doption of dul O 2 /moisture bsorbing schets by some compnies hve been observed by the uthors. Although ctive pckging is n ttrctive wy of replcing gs flushing to control tmosphere composition in commercil pckges, we hve noticed mistken suppositions concerning the overll O 2 - nd moisture-bsorbing cpbilities of AP schets. The Ageless Ò system uses n O 2 -scvenging technology bsed on iron powder oxidtion, nd 1 g of this inorgnic substnce rects with 300 cc of O 2, reducing its concentrtion to less thn 0.03% (Flodberg, 1997; Vermeiren et l., 1999). Unlike Ageless, tht requires moisture to ctivte its O 2 -bsorbing mechnism, the Revolutionry Preservtion (RP) system lso bsorbs moisture nd corrosive gses, nd is comprised of 5 15% unsturted orgnic compounds; 10 45% clcium oxide, 10 50% mordenite, 5 15% ctivted crbon, nd 10 30% polyethylene (Dy, 2005). RP-3A nd RP-5A schets used in this study re cpble of bsorbing both the moisture nd oxygen potentilly present in 300 nd 500 ml of ir, respectively. Due to the limited moisture bsorbing cpcity of AP schets, voidnce of lrge mounts of high moisture mteril (such s colonized substrtes) into pckges nd pre-drying of mycoinsecticides prior to pckging re strongly recommended in order to extend shelf-life. Field dosges of fungl conidi employed for control of pests such s highly susceptible locusts cn be s low s 25 g per hectre

9 86 M. Fri et l. / Biologicl Control 61 (2012) (Kooymn, 2007). In experiments with commercil volume of pure conidi (30 g), lowering the initil w from (equivlent to 8.2% MC) to (equivlent to 3.6% MC) ws chieved when one RP-5A schet ws enclosed per pouch (dt not shown). For lrge volumes of conidi in commercil pckges, strtegies such s the use of more potent O 2 /moisture bsorbers (higher RP numbers) or combintion of O 2 bsorbers with potent desiccnts could work stisfctorily, lthough selection of the best pckging strtegy depends on vrious prmeters such s composition nd size of the pckge, volume nd initil w of the mycoinsecticide, trget w,o 2 - nd moisture-bsorbing cpcities of the schets, nd the rtes of O 2 nd moisture bsorption by AP schets. The ltter is required for definition of the optiml equilibrtion period t mild tempertures before storge under high temperture regimes. Minimum desirble shelf lives for biologicl insecticides re 3 6 months under mbient storge for products supplied by contrct for ppliction t specific time, nd 18 months otherwise (Couch nd Ignoffo, 1981). Shelf life (which we define s the time during which P80% vibility is retined under certin temperture) of c. one yer hs been recorded for the reltively thermotolernt Metrhizium cridum with 6.2% MC (but not 7.0% MC) t C stored under vcuum (Hong et l., 1999), nd prffinic oil formultions of Bb t 25 C(Wright et l., 2001). Longevities observed in our study t 25 nd 40 C would comply with Couch & Ignoffo s recommendtions. Gs flushing nd storge of Bb conidi with MC = 4.4% led to high germintion rtes (87%) fter 16 months t 25 C, nd it is very likely tht 80% vibility would hve been retined for t lest 18 months. We lso chieved shelf life of t lest 6 months t 40 C when conidi were ctively pckged with MC = %, which is sufficient time for deliveries under contrct in regions with effective men tempertures of pproximtely 40 C. Even longer shelf lives could be expected in more commonly encountered tropicl climtes where conditions re less extreme. Tempertures s high s or higher thn 50 C cn be reched in some regions (Hong et l., 1997) or during trnsporttion (Ostrem nd Godshll, 1979), nd we chieved 2-month shelf life t this extreme temperture. Our principl objective in testing t such high constnt temperture, however, ws to ccelerte the initil experiments (reduce response times to expedite identifiction of importnt vribles for subsequent reserch). This strtegy ws successful in testing the effects of different gses, compring different pckging strtegies (gs flushing vs. ctive pckging), nd selecting AP schets with gretest potentil to extend the shelf life of Bb conidi, s subsequently demonstrted in experiments crried out t 40 C. Definitions of shelf life such s vibility fter storge (Dun et l., 2008) re incomplete, nd inclusion of time-relted component nd storge conditions re necessry. So, shelf life should be defined s the period of time tht mycopesticide cn be stored under specified conditions without considerble loss of ttributes tht would compromise its effectiveness. This is dpted from definitions used by food technologists (Mn nd Jones, 2000; Robertson, 2006). Vibility is the most common ttribute used by insect pthologists to ssess conidi qulity, nd ccording to Jenkins nd Grzywcz (2000) it should be greter thn 85%, lthough in this work we trgeted 80%. Therefore, time for initil vibility to drop to 80% t given temperture (s well s RH for non-hermetic pckges), could be ssigned s the shelf life for stndrdized mycoinsecticides under those conditions. As guideline to regultors nd mnufcturers, n open dting system tht clerly displys the pckging dte nd the estimted shelf lives under specific conditions, would be highly desirble. The specific conditions should refer to the ones tht users must follow nd/or the ones tht re likely to be dopted by them before product ppliction. For instnce, disply on commercil pckges of time for initil vibility to drop to 80% t both 4 C (refrigertion) nd, for exmple, 35 C (for products commercilized in regions or sesons with effective men tempertures round this vlue) would be very useful. In ddition to the storge fctors previously mentioned (see Introduction), pre-storge fctors such s the initil qulity of fungl propgules, which in turn is influenced by culturing conditions (Agosin et l., 1997; Frey nd Mgn, 2001; Trocco et l., 2005), drying nd hrvesting processes (Sndovl-Corondo et l., 2001; Btemn, 2004; Jckson nd Pyne, 2007), nd formultion (Sndovl-Corondo et l., 2001; Btt, 2003; Friesen et l., 2006) hve profound impcts on longevity. We hve shown tht in-pckge drying of conidi under moderte conditions before exposure to high temperture regimes is required to llow undesirbly high initil w s to rech desirble levels nd, therefore, void premture deth or conidil debilittion. Post-storge fctors, such s germintion protocol, lthough not directly relted to shelf life, cn lso gretly ffect vibility estimtes. Results from this nd previous studies hve indicted, for exmple, tht estimtes of vibility obtined for conidil preprtions removed from dry storge vry mrkedly depending on the speed of rehydrtion nd length of the incubtion period (Moore et l., 1997; Fri et l., 2009, 2010). Shelf lives presented in this study were estimted bsed on fst-rehydrtion protocol (without previous exposure of conidi to slow-rehydrtion regime inside wet chmber for 24 h). Estimtes of time for initil vibility to drop to 80% would be much greter (in some cses doubled) if estimtes from the slow-rehydrtion protocol were ccepted s the stndrd. However, the biologicl/microbil control significnce of this improvement is questionble, nd we hve shown tht vibility determintions following the more gentle process of slow rehydrtion re inflted by germintion of debilitted conidi, including those tht hve lost the cpcity for rpid germintion or re hypersensitive to imbibitionl dmge (Fri et l., 2010). In the forementioned study (Fri et l., 2010), we ultimtely recommended incubtion for generlly 624 h following fst rehydrtion in protocol imed t providing the most conservtive/rigorous ssessment of mycoinsecticide qulity. With respect to Bb strin GHA, we suggested n incubtion time of h. The lck of ccord with the current study, in which we hve used 24 h incubtion time, is explined by the fct tht the experiments reported herein were initited prior to the studies tht led us to propose the bove-cited recommendtions. It is likely tht if we hd performed our ssessments fter 18 rther thn 24 h, we would hve recorded somewht lower estimtes of germintion, trnslting to shorter shelf lives. These observtions underscore gret need for empiricl studies of diverse pestpthogen systems to determine which ssessment methods provide the most meningful estimtes of vibility (i.e., which methods produce vibility estimtes tht re the best indictors of pthogen virulence nd biopesticide qulity). Wter ctivity, term initilly proposed by microbiologists (Troller, 1980), is defined s the rtio of the wter vpor pressure of mteril to the vpor pressure of pure wter t the sme temperture (Robertson, 2006). It is strightforwrd mesure of wter vilble for chemicl nd biologicl rections nd, therefore, meningful prmeter in studies with dehydrted microorgnisms. A w s of pre-dehydrted conidi kept in hermetic foil pouches with drierite or O 2 /moisture bsorbers were consistently in the rnge ( % ERH). This smll vrition ws observed between redings performed in wintertime (colder nd drier ir in the lbortory) nd sesons with higher T/RH. The importnce of drying eril Bb conidi for extended shelf life hs been demonstrted previously (Clerk nd Mdelin, 1965; Feng et l., 1994; Shimizu nd Mitni, 2000). In hermetic storge studies in which ir ws not removed from pckges, Hong et l. (2001) reported tht longevity of two Bb isoltes ws not significntly incresed when storge moisture content ws reduced below

10 M. Fri et l. / Biologicl Control 61 (2012) %, corresponding to moisture contents in equilibrium with bout 11 14% ERH t 20 C (see Fri et l., 2009 for sorption isotherms tht llow conversion of w mesurements to MC). In our study, best conidil w s were consistently ssocited with drierite, which dried the conidi to considerbly <5% MC (see lso Fri et l., 2010). It is possible tht under nerobic conditions (<0.03% O 2 ) optiml wter ctivities for storge re lower thn under erobic tmospheres. A gret number of mycoinsecticides re commercilized worldwide (Fri nd Wright, 2007) nd improvements in their longevity under non-refrigerted conditions re urgently needed for greter mrket cceptnce. Mycoinsecticides cpble of displying predictble nd stisfctory germintion counts following longdistnce trnsporttion nd relistic uncontrolled storge should be gol for product developers. We hve shown tht MAP, especilly ctive pckging, could be brodly dopted in order to extend shelf life of Bb nd possibly other fungl gents. Acknowledgments We thnk Jrrod Lelnd (Novozymes Biologicls, Inc., USA) for providing smples of one of the luminized pouches. We re thnkful to Billy Abrms from CSP Technologies (Auburn, USA) for smples of O 2 nd/or moisture bsorbing films. Mention of proprietry product does not constitute n endorsement or recommendtion by the UDSA, Michign Stte University, or EMBRAPA. References Abelln, M., Rmos, A.J., Snchis, V., Nielsen, P.V., Effect of modified tmosphere pckging nd wter ctivity on growth of Eurotium mstelodmi, E. chevlieri nd E. herbriorum on sponge cke nlogue. Journl of Applied Microbiology 88, Agosin, E., Volpe, D., Munoz, G., Sn-Mrtin, R., Crwford, A., Effect of culture conditions on spore shelf life of the biocontrol gent Trichoderm hrzinum. 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