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Biocontrol Science and Technology (1996) 6, 35±50

Effect of Bait Substrate and Formulation on Infection of Grasshopper Nymphs by Beauveria bassiana G. DOUGLAS INGLIS,1,2 DAN L. JOHNSON 1

Downloaded By: [University of Arizona] At: 21:59 15 November 2010

1

AND

MARK S. GOETTEL 1

Agriculture and Agri-Food Canada Research Centre, PO Box 3000, Lethbridge, Alberta, Canada T1J 4B1; 2 Centre for Pest Management, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6 (Received for publication 10 May 1995; revised manuscript accepted 15 October 1995)

The effects of tw o form ulation s (oil and water ) and tw o bait substrat es (lettuc e and bran) on infectio n of grassho pper nymphs (M elanoplus sanguin ipes) by Beauveri a bassian a were investi gated. More nymphs died of mycosis afte r they had ingeste d substrat es inoculat ed with conidi a in oil than in water , but ther e was no differen ce betwee n the lettuc e and bran substrat es. Of the conidi a recovere d in frass, most (95% ) were recover ed within 24 h of ingestio n of the lettuce and bran substrat es by nymphs. Signi®cantly m ore conidi a average d over time were recover ed in frass from the water than from the oil form ulation . A highe r incidenc e (33±82% ) and m ore rapid onset of mycosis was observe d in nymphs that were surface- sterilize d before ingestio n, com pare d with those surface- sterilize d after ingestio n of lettuc e and bran substrat es inoculat ed with B. bassiana in both formulations . A sim ilar tren d was observe d in nym phs receivin g the steriliza tion treatm ent before , rathe r than after, ingestio n of whea t leave s spraye d with conidi a in oil or water. Numerou s conidi a were observe d on the heads, thoraxe s and abdo m ens of nymphs that ingeste d treate d lettuc e or bran . M odes t numbers of conidi a were also recover ed from the surface s of nym phs, but no differen ces were observe d betwee n formulation s or substrat es. However , when nym phs ingeste d lettuce disk s treate d with ¯uoresce nt dye in either oil or water, m ore dye was observe d on nymphs with the oil formulation . This laborato ry stud y demonstrat es that grassho pper nymphs are highly suscepti ble to infectio n by conidi a formulate d on bait substrat es, and that the ef®cacy of the bait relie s on the exten t to which nymphs beco me surface- conta minate d durin g ingestio n. Keyw ord s: Beauveri a bassian a, conidia , grassho pper , M elanopl us sanguini pes, nym ph, bait formulation , oil, infectio n

INTR OD UCT ION Grasshop pers are econom ically importan t pests in arid agro-sy stem s. D urin g populati on outbreaks in N orth Am erica, contro l m easure s rely alm ost exclusi vely on the use of chem ical insectici des. Recogniti on of the deleteri ous effects of pesticid es has prom pted the develop ment of alternati ve, less obtrusiv e m anage ment strategi es, such as the use of m icrobia l contro l agents . The ento m opathog enic fungus , Beauveri a bassian a (B alsa mo) V uille m in, has shown consider able potentia l for the m anage ment of insects (Feng et al., 1994), and its pathoge nicity agains t 0958±3157/96/010035±16 $7.50

1996 Canadian Government

G . D . ING LIS E T A L .

36


grassho ppers in a ®eld environ m ent has been demonstrate d (Johnso n & Goettel, 1993). H ow ever, the ef®cacy of B. bassian a in ®eld environ m ents has been inconsi stent, and im prove d form ulatio n strategie s that enhanc e the persisten ce and targetin g of B. bassiana agains t grassho ppers is desirabl e. B ait formulation s reduc e the quantit y of insectici des require d to contro l grassho ppers by improved targetin g (M ukerji et al., 1981 ; Ew en & M ukerji, 1987 ; Johnso n & Henry, 1987). Formulatio n of ento mopathog ens as baits is receivin g increasi ng attentio n, and bran has been used successf ully as a substrat e for Nosema locusta e agains t grasshop pers in ®eld environ m ents (Henry, 1971 ; H enry et al., 1973 ; Onsage r et al., 1980 ; Johnson , 1989). The use of Steinern em a feltiae (Capiner a & Hibbard , 1987 ) and ento mopoxvir us (EPV ) (M cGuire et al., 1991) in baits agains t grassho ppers has also been reported , but the ef®cacy of these pathoge ns in ®eld environ m ents is unkno wn. The form ulation of entom opathog enic fung i as baits agains t grassho ppers has not been extensi vely studied , and if baits are to be develop ed as a bioratio nal formulatio n for B. bassian a, an understa ndin g of the m echanis m of infectio n of nymphs ingestin g baits is of param oun t im portanc e. Therefor e, the objectiv es of this stud y w ere: (1) to determ ine the ef®cacy of B . bassian a in tw o formulation s (oil and w ater) and on tw o bait substrat es (lettuc e and bran ) agains t grassho pper nymphs; (2) to elucidat e the m echanis m (s) by which B. bassian a infects grasshop per nymphs that have com e in contac t with the bait substrat es.

M ATER IALS AN D M ETH OD S Preparatio n of Inoculum Dry conidi a of B. bassian a (GH A isolate ) were supplie d by M ycotec h C orp. (B utte , M T, USA ). Conidia w ere suspend ed in distille d water and germinatio n percentages w ere determ ined on potato dextros e agar (PDA ; D ifco L aborator ies, Detroit, M I, U SA) am ended with 0.005 % benlat e (DuPont, W ilmington , DE , U SA), 0.04 % penicill iun G and 0.1% strepto m ycin . A fter 24 h at 25 6 1 C, conidi a were ®xed with lactophe nol and germ ination rate s w ere determ ined by examinatio n of a m inim um of 500 conidi a from each of tw o replicat e cultures . Conidia were consider ed viable if germ tubes were greate r than 3 m m in length . Im m ediatel y prior to use, conidi a w ere suspend ed in eithe r sterile distille d water or oil; unless indicate d otherwise, the oil used was sun¯ow er oil (Saf¯o, Culina r Food s Inc., Toronto , ON , Canada) . To facilitat e suspens ion, conidi a were m ixed w ith a m icropest le. C oncentra tion s of conidi a were estim ated using a hem ocyto m eter and the numbers of viable conidia /m l w ere calculat ed as the percenta ge germ ination 3 the concent ratio n of conidia , as determ ined from the hem ocyto meter counts . Excep t where indicate d otherwise, conidia l concent ration s were adjuste d to 2 3 10 8 viable conidia /m l and 0.5- ml aliquot s (10 5 conidia ) were pipette d on to the bait substrat es.

Grasshoppers Nym phs of a non-dia pause strain of M elanoplus sanguin ipes (F.) were reared on a diet of bran and w heat leaves at an am bien t tem peratur e of 20±25 C ; a vertica l heat gradien t was produce d in the cage (40 3 40 3 30 cm) by a 25-W incande scen t light-bul b. Third instar nymphs were individu ally collecte d in sterile 20-m l glass vials stoppere d with a sterile polyuret hane foam plug and starve d for 12 h before inoculat ion. Durin g the experi menta l period , nym phs were maintained on a diet of wheat leaves in a controlled environ ment cham ber under a 16 h/8 h light:da rk photope riod and a 25/20 C day/nigh t temperatur e regim e. The relativ e hum idity (RH) w ithin the chamber varie d from 20 to 55% durin g the experi m ents.

B AIT FOR M U LATIO N O F B . BASSIA NA

37


Effect of Substrate and D ose Aliquots of conidia l inoculu m in w ater or oil containi ng 10 3, 10 4 or 10 5 conidi a were pipette d on to lettuce disks (5 m m in diam eter) or w heat bran ¯akes (approxi m ately 3 3 3 mm ). For contro l treatments, water or oil alone was placed on the bait. The baits were m aintaine d in the dark for 15±30 m in before presenta tion to nymphs in the vials. To preven t loss of the oil carrie r and/or stickin g of the bait substrat e to the wall of the vial, the inoculat ed bait was pierce d w ith a pin and then suspend ed from the foam plug approxi m ately 2 cm into the vial. Nym phs in vials were placed at 25 C unde r arti®cial ligh t provide d by incande scen t and ¯uoresce nt bulbs. A lthoug h the majority of the nym phs consum ed the bait w ithin 30 m in, nym phs were kept in the vials for 12 h befor e transfe r to individu al 240-m l clea r plastic containe rs. Nymphs that m olte d durin g the 12-h period or did not consu me the entir e bait were rem oved from the experi ment. During the subsequ ent 14 days, unconsu med diet and frass were rem oved once daily and dead nym phs were rem oved twice daily . Cadaver s w ere place d on moistene d ®lter papers at 25 C for 3±5 days in the dark, and those that produce d hyphal growth charact eristic of B. bassian a were recorde d as infected . N ym phs that survive d the 14-day period of the experi ment w ere killed by freezing , and place d on m oistene d ®lter pape r and incubat ed as above. The total num ber of nym phs/treatment com binatio n ranged from 39 to 46.

Effect of Steriliz ation and Freezing on Conidial Viability Conidia l suspens ions (100 m l) in oil or w ater were applie d with an airbrus h (Artek, Rockford , IL, USA ) at 103 kPa to nym phs in a 10.8-c m diam eter by 35.5-c m high Plexiglas s cylinder . Nym phs were killed by freezing , thawed, weighed and placed on their ventra l surfac e before spra y applicat ion. Droplet size, densit y and distribut ion pattern s w ere monitore d using w ater - and oil-sensi tive papers . To quantif y the depositi on of conidia , thre e sterile roun d glass coverslips (13 m m in diameter) were placed equidist ant at the bottom of the spray cylinder . Following the applicat ion of conidia , the coversli ps w ere aseptica lly placed in 5 m l of 0.01 M -sodiu m phospha te buffe r (pH 7.0) amende d with 0.05 % Tw een 80 (buffer ±Tw een) in 20-m l vials with approximately 0.5 g of sterile sand and w ashe d at am bien t temperatur e for 2 h on a rotary shaker at 300 rpm . After shaking , the w ash solutio n was diluted two to thre e tim es in a 10-fol d dilutio n series and 100 - m l aliquot s for each dilutio n w ere sprea d on to sem i-selecti ve oatm eal±dodin e agar (consisti ng of 17.5 g of oatm eal agar, 2.5 g of agar, 0.45 g of cyprex , 2.5 mg of crysta l violet, 0.2 g of penicilli n G and 0.5 g of strepto m ycin in 500 m l of deioniz ed water; Chase et al., 1986). Culture s w ere incubated at 25 C for 6±7 days and the num bers of colony- form ing units (CFU) were enumerate d at the dilutio n yieldin g 30±300 B. bassiana CFU /dish. The m ean num ber of CFU cm 2 2 of coversli p area w as then calculat ed. Follow ing inoculat ion, nymphs were m aintaine d in the dark at am bien t tem peratur e for 15±20 m in. Subseque nt treatm ents were: (1) nym phs submerged in 1.0% sodiu m hypoch lorite with 0.05 % Tw een 80 for 2 m in and then rinse d twice in sterile w ater ; (2) nym phs subm erge d in 1.0% sodiu m hypochl orite w ithou t Tween 80 for 2 min and then rinse d twice in sterile w ater ; (3) nym phs frozen at 2 20 C for 6 h; (4) nym phs m aintaine d at ambien t tem peratur e in the dark . Five nym phs for each treatm ent w ere individu ally macerate d in buffer ±Tween and CFU were enumerate d on oatmeal±dodin e agar. D ata w ere calculated as the num ber of C FU/standard ized nym ph (adjuste d for differen tial bod y w eight). Reduction s in conidia l num ber/replicat e (x i) w ere determ ined relativ e to the mean value of the contro l treatm ent (X c ) and were calculat ed as: o(((C FU/nym ph X c 2 C FU/nym ph x i)/CFU/nymph X c ) 3 100)/n


38

If the num ber of conidi a recover ed for x i w as large r than for X c , the percenta ge increas e in num ber was calculat ed as: o((( CFU/nymph X c 2 CFU/nym ph x i)/CFU /nym ph x i) 3 100)/n


The experi m ent was repeate d once. Effect of Surface Steriliza tion of G rass hopper on M ortalit y To m easure the relativ e importanc e of externa l versu s per os infection , the m ortalit y of nym phs surface- sterilize d before or afte r feedin g on lettuc e or bran substrat es treate d with B. bassian a conidi a in oil or w ater was m easured . N ymphs w ere surface- sterilize d at 10 C by submersio n in 1.0% sodiu m hypochl orite for 2 min follo wed by tw o rinses in sterile distille d w ater . Nym phs were surface- sterilize d eithe r befor e place ment in the glas s vial w ith the bait substrat e or after they had ingeste d the bait in the vial. N ymphs w ere then transferr ed to 21 3 28 3 12 cm Plexiglas s cages ®tted w ith a perforat ed m etal ¯oor to m inim ize contac t with frass. There were no tem peratur e differen ces between the am bient and the cage environ m ent in the controll ed environ m ent cham ber during the dark period , but the temperatur e within the cages was 2.5±3.1 C higher than ambien t durin g the ligh t period . Althoug h R H value s within the cages w ere sim ilar to ambien t for most of the day and ranged from 24 to 34% , they rose 4±8% higher than am bien t levels for a 2±3 h perio d afte r place ment of the wheat leave s in cages. Cadaver s were collecte d twice daily and were incubate d on moistene d ®lter paper to detec t interna l B . bassiana . The total num ber of nymphs/treatm ent combinatio n range d from 48 to 69. C onidia suspend ed in an emulsi®able paraf®nic-base d oil (M ycotec h Corp. #9209 ) or w ater (1.8 3 10 8 conidia m l 2 1 w ere applie d to w heat leave s (100 m l) with the Artek airbrush . At the time of applicat ion, the num bers of conidi a cm 2 2 on rando mly selecte d leaf segments /replicat e were quanti ®ed usin g a wash/dilution- sprea d plate techniq ue (Inglis et al., 1993). Nym phs were place d in plastic cage s (21 3 6.5 3 8.5 cm ) and one-hal f of the nym phs w ere surface- sterilize d as describe d previous ly. Sterilize d and non-ster ilized nym phs were allo wed to feed ad libitum on 1.5 g (fresh weight) of the treate d wheat within 5 min of conidia l applicat ion. A fter 6 h, unconsu m ed wheat was removed , air dried and weighed and the rem ainin g nymphs were surface- sterilize d. Nym phs w ere then placed in the controll ed environ ment cham ber for 16 days , and dead nymphs were rem oved daily and checke d for interna l B. bassiana . The total num ber of nym phs/treatment com binatio n ranged from 42 to 49. Passag e of C onidia T hrough the A limentary C anal Nym phs were fed lettuce or bran bait inoculat ed with conidi a in oil or water and maintaine d in the controll ed environ m ent cham ber in 240-m l clear plastic contain ers as describe d previou sly. Control treatments consiste d of lettuce disk s or bran ¯akes inoculat ed with either water or oil. Frass w as collected from individu al nym phs at 24-h interval s for 5 days, w eighe d and stored at 5 C for a maximum of 48 h. Frass from nym phs which had molted in the previou s 24 h w as exclude d, and the m ean quantit y of frass was calculat ed only for the non-moltin g nym phs in each replicate . Frass w as macerate d in 1 m l of buffer ±Tw een, diluted and sprea d on oatm eal±dodin e agar to recove r conidia . Nym phs w hich survive d for 5 days were killed by freezin g at 2 20 C for approxi m ately 6 h, surface- sterilize d in 70% ethano l for 1 m in and rinse d tw ice in sterile deionize d water. T he alimentary cana l was then removed aseptical ly. It w as then m acerate d and the num ber of CFU in the m acerat e enum erated on oatm eal±dodin e agar. To determ ine if conidi a passed in frass could act as a sourc e of inoculu m, fras s collecte d from 13± 20 B. bassian a-treate d and -untreated nym phs/replicat e w as place d daily in plastic cages containi ng 12±20 nym phs not previou sly expose d to B. bassian a. All nymphs were m aintaine d in the controll ed environ m ent chamber for 14 days, and dead nymphs w ere removed daily and checke d for interna l B. bassiana . Introduc ed frass w as not w eighed , but the am oun t of frass produce d by nym phs inoculat ed with conidi a decrease d noticeab ly after 3±4 days , due to the effects of mycosis . The total num ber of nymphs expose d to frass range d from 45 to 48 nym phs/treatment.


39


Deposition of C onidia and Dye on N ym phs Nym phs w ere fed bran or lettuc e inoculat ed w ith conidi a suspend ed in w ater or oil. Contro l treatments consiste d of nym phs allo wed to ingest lettuc e treate d w ith just w ater or oil. W ithin 1 h of ingestion of the substrat es, nym phs were killed by freezing . A fter freezing , head s and the last abdo m inal segm ent w ere subm erge d in sterile molten paraf®n to preven t possibl e conta minatio n by conidi a from fras s or regurgit ation . Nym phs w ere then placed individu ally in 5 m l of buffer ±Tween with sterile sand and washe d for 2 h on a rotary shaker . CFUs w ere enumerate d on oatm eal±dodin e agar; 12±15 nym phs/bait form ulatio n treatm ent were used . Ten addition al nym phs fed bran or lettuc e inoculat ed w ith conidi a in water or oil w ere kille d by freezing , ®xed in 2% glutaral dehyde , dehydra ted in ethanol , critical- poin t drie d in liquid C O 2 sputter- coated w ith gold and exam ined w ith a H itachi S-570 scannin g electro n microsco pe (Nissei Sangy o Canad a Inc., Rexdale , Ont., Canada) . Tw enty- ®ve nym phs were also fed bran ¯akes or lettuc e disk s treated with 0.1% (w/w ) ¯uorescen t dye (D ay-G lo Color Corp., Cleveland , OH , USA ) in w ater or oil. Follo w ing ingestion, nymphs w ere im mediately kille d by freezing , thawed and photogr aphe d unde r long-wave UV light. Statistica l Analyses All computation s w ere perfor med usin g the G LM , REG and LIFEREG procedu res of SAS (SA S Institut e Inc., 1988) . Nym phs that died and subsequ ently produce d hypha l growth of B. bassian a follo w ing incubati on on m oistene d ®lter pape r were conside red to have died from m ycosis ; those not produci ng hypha l grow th w ere recorde d as `othe r mortality ’. W ith the exceptio n of the in¯uenc e of steriliza tion procedu re and depositi on of conidi a and dye experi ments w hich were repeated , all experi m ents were replicat ed in tim e (m inim um of thre e replicate s) and analyze d as rando mized com plete block designs . The in¯uence of steriliza tion and freezin g on conidia l viabilit y and the effect of sterilizat ion and form ulation on ingestio n of wheat leave s were analyze d usin g one-w ay analysi s of varianc e (ANO VA ). In conjunc tion with a signi®cant F-test (P < 0.05), T ukey ’s studenti zed rang e test ( a 5 0.05 ) was used to separat e treatm ent m eans . W hen diseas e progres s or passag e of conidi a over tim e was include d in the model, the experi m ents were analyze d as split-pl ots in tim e (Gom ez & G om ez, 1984). A Box correction w as used as a conserv ativ e test for time (t) and time±treatment interacti ons (M illike n & Johnson , 1984); the Box correcti on reduce s the degree s of freedo m for tim e (t), the time±treatment interacti ons and the residua l erro r (tim e) by t 2 1. In all instance s, residuals were plotted agains t predicte d values , and, w here necessa ry, the appropr iate transfor mation s w ere used to norm aliz e the variance . The conidia l populati on data w ere alw ays log 10transfor m ed, and log value s for the means and standar d errors of the m ean (SE) are presente d in parenth eses througho ut the text. In severa l instance s, an arcsin e transfor m atio n w as applie d to the incidenc e of m ycosis data , but untransf ormed means and SE in parenth eses are present ed. For each treatment±dose combinatio n (in¯uenc e of substrat e and dose experi ment), the m ycosis data w ere ®tted to a W eibull function , and m edia n letha l tim es w ith uppe r and low er 95% con®dence lim its (CL) were estim ated . RESU LTS Effect of Substrate and D ose The grassho pper m ortalit y from mycosis increase d w ith conidia l dose , and this effec t w as greate r for nymphs consum ing baits inoculat ed w ith conidi a suspend ed in oil than those treate d with conidi a in water (F 5 11.0 ; degree s of freedo m (df) 5 2, 21; P 5 0.0006 ) (Figure 1). Disease develop m ent was m ore rapid am ong nym phs fed bait substrat es inoculat ed w ith conidi a in oil on lettuce (F 5 18.2; df 5 1, 4; P , 0.01 ) and bran (F 5 28.5, df 5 1, 4; P , 0.01). For the oil form ulation , LT 50 values at the 10 5 dose w ere 4.9 (95% C L 5 4.6±5.2) and 6.0 (95% CL 5 5.3±6.7) days for the lettuce and bran substrat es respecti vely. For the w ater formulation , LT 50 values at the sam e dose were 9.8 (95% C L 5 8.2±11.8) and 11.0 (95% CL 5 9.2±13.2 ) days on lettuce and bran respecti vely . In contras t to the conspic uous differen ces between formulations , there was no differen ce between substrat es (F 5 1.8;



40

FIGUR E 1.

Cumulative mortality due to mycosis (% ) in grasshopper (M . sanguinipes) nymphs that fed on lettuce and bran substrates inoculated with B. bassiana conidia in oil or water.

df 5 1, 21; P 5 0.20 ) in the inciden ce of total mycosis . Also the rate of diseas e develop m ent did not differ between substrat es for eithe r the oil (F 5 5.45; df 5 1, 4; P . 0.05 ) or water (F 5 1.23; df 5 1, 4; P . 0.05 ) formulation . M ortalit y not attribut ed to B. bassian a range d from 2.4 (2.4 ) to


TABLE 1.

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Effect of freezing or exposure to sodium hypochlorite on recovery of viable B. bassiana conidia from the surface of M . sanguinipes nymphs sprayed with conidia in oil or water Oil

Treatment Untreated (post-spray) Frozen ( 2 20 C for 6 h) NaOC l (1% ) NaOC l 1 Tween (0.05% )

log1 0 CFU/nym ph 3.92 3.91 2.05 1.91

b

(0.06)a (0.05)a (0.25)b (0.28)b

a

W ater Reduction (% ) 8.5 10.3 97.6 97.4

(10.8) (9.5) (0.6) (0.9)

log 10 CFU/nymph 3.96 4.13 1.75 1.45

b

(0.13)a (0.05)a (0.33)b (0.18)b

a

Reduction (%) 10.5

2 8.8 98.2 99.6

(14.5) (9.4) (0.7) (0.1)

a


Conidia (log 1 0 CFU/nymph) recovered from nymphs (standardized by weight) sprayed with conidia in oil or water. Values in parentheses following means represent standard errors of the means. b Means not followed by the same letter within each formulation±substrate group are signi®cantly different ( a 5 0.05) according to Tukey’s studentized range test. The experiment was conducted twice.

20.6 % (10.7), and w as sim ilar between formulation s (F 5 0.11; df 5 1, 29; P 5 0.74), substrat es (F 5 0.03; df 5 1, 29; P 5 0.87 ) and doses (F 5 2.2; df 5 3, 29; P 5 0.10).

Effect of Steriliz ation and Freezing on Conidial Viability Surface-s terilizati on of nym phs by subm ersio n in 1% sodiu m hypochl orite for 2 m in reduce d ( a 5 0.05) the number of viable conidi a on nym phs by more than 97% (Table 1). N evertheless, consider able num bers of conidi a (46±240 CFU/nymph) were recover able from treated nym phs. Am endment of sodiu m hypochl orite with Tw een 80 (0.05 % ) did not signi®cantly increas e ( a 5 0.05) the ef®cacy of steriliza tion . Freezin g nym phs ( 2 20 C for 6 h) had no effec t ( a 5 0.05) on conidia l surviva l (Table 1). W hether conidi a were applie d to nymphs in oil or w ater also had no effect (F 5 0.86; df 5 1, 63; P 5 0.36).

Effect of Surface Steriliza tion of G rass hoppers on M ortalit y The incidenc e of total m ycosis (14 days) in nymphs that were surface- sterilize d before ingestin g lettuce or bran substrat es infeste d w ith conidi a in oil or w ater w as 33±82% greate r (F 5 47.0; df 5 1, 21; P < 0.0001 ) than that observe d in nym phs sterilize d afte r ingestin g the substrat es (Figure 2). For all formulation ±substrat e combination s except oil on lettuce (F 5 4.2, df 5 1, 6; P . 0.05), the rate of m ycosis develop m ent w as slow er (F 5 8.0 to 21.2; df 5 1, 6; P , 0.05) for the post-ster ilizatio n treatm ent. W ith both steriliza tion treatm ents, the cumulativ e incidenc e of mycosis (F 5 54.6; df 5 1, 21; P < 0.0001 ) was highe r am ong nym phs ingestin g conidi a applie d in oil on lettuc e than in nym phs ingestin g conidi a in oil on bran or water applie d to either substrat e; a w eak interacti on (F 5 5.8; df 5 1, 21; P 5 0.025) w as observe d betw een form ulatio n and substrat e. Total mortality due to mycosis was higher (F 5 27.1; df 5 1, 21; P < 0.0001) in nym phs that ingeste d conidi a on lettuc e than bran for both the oil and water form ulations . M ortalit y not attribut ed to B. bassiana ranged from 18.4 (11.2 ) to 33.5 % (11.8), and w as not in¯uenced by steriliza tion treatm ent (F 5 0.06; df 5 1, 21; P 5 0.80), form ulatio n (F 5 2.1; df 5 1, 21; P 5 0.17 ) or substrat e (F 5 0.01; df 5 1, 21; P 5 0.93). To obtain a distribu tion of conidi a on foliag e that would be m ore represen tative of a ®eld test, conidi a in oil or w ater w ere applie d to w heat leave s with an airbrush . Conspicu ously less free oil w as eviden t on the wheat leave s than on the lettuc e substrat e of the precedi ng experi ment. Both oil and water droplet s appeare d to be unifor m in distribu tion and size on water- and oil-sensi tive papers , and similar num bers of conidi a applie d in oil (4.72 , standar d error (SE) 5 0.03 9 log CFU cm 2 2) and w ater (4.58 , SE 5 0.034 log CFU cm 2 2 were recovere d from wheat leaves . For the oil form ulation , 44.4 % (5.0 ) m ycosis w as observe d over 16 days in nym phs sterilize d prio r to ingestio n of inoculat ed wheat as compared w ith 10.0 % (5.8) mycosis in



42

FIGUR E 2.

Cumulative mortality due to mycosis (%) in grasshopper (M . sanguinipes) nymphs surface-sterilized 5 before (pre-) and after (post-) ingestion of lettuce and bran substrates inoculated with 10 B. bassiana conidia in oil or water.

those sterilize d after ingestio n (Figure 3). For the water form ulation , the incidenc e of m ycosis was 34.2% (11.1) and 13.2 % (9.4) nym phs sterilize d before and after ingestio n of inoculat ed wheat respecti vely . The sterilizat ion treatm ent (F 5 10.9; df 5 1, 6; P 5 0.016 ) but not form u-



FIGUR E 3.

43

Cumulative mortality due to mycosis (%) in grasshopper (M . sanguinipes) nymphs surface-sterilized before (pre-) and after (post-) ingestion of wheat leaves sprayed with B. bassiana conidia in oil or water.

lation (F 5 0.17; df 5 1, 6; P 5 0.69 ) nor the interacti on between form ulatio n and treatment (F 5 0.63; df 5 1, 6; P 5 0.46 ) in¯uence d the am oun t of m ycosis observe d afte r 16 days . For the oil (F 5 15.9; df 5 1, 4; P , 0.05 ) but not the water (F 5 1.7; df 5 1, 4; P . 0.05 ) formulation , the develop m ent of mycosis was m ore rapid in nymphs sterilize d before than after ingestio n of conidia . The incidenc e of non -Beauveria mortalit y was relativel y high , rangin g from 20.9 (12.1) to 53.7% (6.7) , but w as not in¯uenced by form ulation (F 5 0.07; df 5 2, 10; F 5 0.94) or sterilizat ion treatment (F 5 0.13; df 5 1, 10; P 5 0.72). To determ ine if the oil form ulation on wheat leave s or treatment of nym phs with sodiu m hypochl orite affecte d consu m ption , uninges ted leaf m aterial w as collecte d after 6 h. W eights of unconsu m ed leaves from cages w ith nymphs sterilize d before feedin g on treate d foliag e were 92.8 (7.1), 131.4 (10.0 ) and 56.8 (19.2 ) m g for the water, oil and contro l (untreat ed) treatm ents respecti vely . For nymphs sterilize d after feedin g on treate d foliage , the weights of uningest ed foliag e w ere 91.3 (3.4), 111.1 (11.9 ) and 61.8 (8.0 ) for the water, oil and contro l treatm ents respecti vely . The tim ing of the sterilizat ion treatm ent (befor e or afte r food present ation ) had no effect (F 5 0.32; df 5 1, 10; P 5 0.58) on the am oun t of foliage consum ed. However, form ulatio n in¯uenced the amount of leaves ingeste d (F 5 13.1; df 5 2, 10; P 5 0.0016 ) indepen dent of the sterilizat ion treatm ent (F 5 0.59; df 5 2, 10; P 5 0.57). Less ( a 5 0.05 ) foliag e treate d with conidi a in oil was consu med by grassho ppers relativ e to untreate d leaves , w hereas sim ilar am ounts ( a 5 0.05 ) of water and untreate d leave s were consu m ed. Passag e of C onidia T hrough the A limentary C anal M ost of the conidi a recover ed from the frass were recover ed w ithin 24 h of ingestio n, but small num bers of conidi a w ere recover ed for up to 5 days afte r ingestio n (Figure 4). O nly 1.3±2.5% and 7.7±11.7 % of the conidi a inoculat ed on to baits in oil or water respecti vely were recover ed


44


in frass . For both form ulations , the num ber of conidi a recover ed in fras s decrease d over tim e (F 5 83.7; df 5 1, 10; P , 0.01). There was no interacti on between form ulatio n and tim e (F 5 2.5; df 5 1, 10; P . 0.05) but average d over tim e, few er conidi a (F 5 9.3; df 5 1, 7; P 5 0.019) w ere recover ed from the frass of nym phs fed baits treate d with the oil formulation . The substrat e had no effec t (F 5 0.47; df 5 1, 7; P 5 0.52) on conidia l passage . After 5 days , conidi a w ere recovere d from the alimentary canals of only 7/47 nym phs (14.9 %). There w as no in¯uenc e of formulatio n (F 5 4.5; df 5 1, 10; P 5 0.060 ) or substrat e (F 5 0.46; df 5 1, 10; P 5 0.51 ) on the presenc e of conidi a in guts. M ean num bers of conidi a recover ed from alim entar y canals range d from 0.13 (0.13 ) to 0.79 (0.16 ) log C FU/nym ph. M ost nym phs m olte d durin g the 5-da y incubati on period , causin g consider able variabil ity in frass product ion despite the rem oval of individu als that molted during each 24-h collecti on period . No differen ces (F 5 1.1± 2.0; df 5 1, 5; P . 0.05 ) w ere observe d in the amoun t of frass produce d by nym phs between the contro l and B. bassian a treatments over time w ithin each of the formulation ±substrat e com bination s. N o m ycosis w as observe d in test nymphs expose d to fras s from the nymphs fed conidi a in oil on lettuce , even thoug h the incidenc e of mycosis in the nymphs from which the frass w as collecte d w as 93.8% (3.5 ) afte r 10 days . T he densit y of conidi a in this frass was measure d in only one of the thre e replicat es and only in frass produce d durin g the ®rst 24-h perio d after inoculat ion; the conidia l populations average d 2.41 (0.23 ) log C FU mg 2 1 of frass. No conidi a were recover ed from the frass of contro l nymphs. The incidenc e of othe r mortality w as sim ilar (F 5 0.27, df 5 3, 6; P 5 0.84 ) between treatm ents and ranged from 8.6 (1.7 ) to 13.9 % (4.9).

Deposition of C onidia and Dye Relativel y small num bers of conidi a w ere recover ed from the surface s of nym phs ingestin g lettuce or bran inoculat ed w ith conidi a in either oil or w ater. C onidia l populati ons range d from 1.7 (0.30 ) to 2.1 (0.26) log C FU/nymph. There was no effec t due to formulatio n (F 5 0.03; df 5 1, 34; P 5 0.87), substrate (F 5 0.06; df 5 1, 34; P 5 0.80 ) or the interacti on between them (F 5 1.6; df 5 1, 34; P 5 0.22). Conidia were unevenl y distribut ed on the integu ment, and were observe d on the thorax , abdo men and head . In m ost instance s, conidi a were clum ped (Figure 5). M ost of the nym phs examined had regurgit ated, and conidi a observe d near their m outhpart s were frequent ly observe d to be regurgit ate-coat ed (Figure 5(a)). W hen nym phs were fed bait treate d with ¯uoresce nt dye in eithe r oil or water, consider ably more dye w as observe d on nym phs ingestin g baits treated with oil (Figure 6). M ost of the dye w as observe d on the head (includi ng antenna e and m outhpart s), legs (in particula r the forelegs ) and thorax (Figure 6(a)).

DISC USSIO N Grasshop per nym phs ingestin g bait substrat es (lettuc e or w heat bran ) inoculat ed with conidi a in oil or water are suscepti ble to infectio n unde r laborat ory conditio ns. The form ulation of conidi a in oil was consiste ntly m ore ef®caciou s than in w ater, but the superior ity of the lettuc e substrat e relativ e to bran was less evident . Althoug h B. bassiana formulate d as baits has been used agains t a variety of non-acri did insec t pests (Feng et al., 1994), bait form ulation s of ento m opathog enic fung i for the contro l of grassho ppers have been little studied , despit e the com m on use of baits for the applicati on of insectic ides and N. locustae . M oore and Erlandso n (1988) reporte d that B. bassiana (10 5 conidia ) applie d in w ater to lettuce disk s cause d signi®cant m ortalit y in nym phs, and the incidenc e of diseas e that they observe d (53% ) w as sim ilar to that observe d in the presen t study . Johnso n et al. (1988 ) also found that the applicati on of Verticilli um lecanii in bran cause d signi®cant mortalit y in cage d grassho ppers . A lthoug h B. bassiana conidi a form ulated as a bran bait cause d signi®cant reductio ns of ®eld populati ons of grassho ppers (Johnso n & Goettel, 1993), it w as speculat ed that infectio n occurre d from conidi a liberate d from the bran substrat e durin g applicat ion. Further m ore, Johnso n and Goette l (1993) observe d that m ycosis w as low in nym phs



45

FIGUR E 4.

21

Recovery of ingested B. bassiana conidia in frass (log 10 CFU mg fresh weight) from grasshopper 5 (M . sanguinipes) nymphs that were fed lettuce and bran substrates inoculated with 10 B. bassiana conidia in oil or water or with oil or water alone (control).

ingestin g the bran in the laborato ry, raising a questio n as to the ef®cacy of B. bassian a applied as a bran bait. Despite anecdot al account s of success ful use of bait form ulation s of entom opatho genic fungi, very little infor mation is availabl e on the m ode of action of thes e fung i in/on baits .


46

FIGUR E 5.


Scanning electron micrographs of B. bassiana conidia on grasshopper (M . sanguinipes) nymphs that ingested lettuce treated with conidia in oil (bars 5 5 mm): (a) regurgitate-coated conidia on head (labrum); (b) conidia on abdominal tergum.

An underst andin g of the m echanis m(s) of actio n is necessa ry if ento mopathog enic fung i are to be effective ly deploye d in bait formulations . This stud y indicate s that grasshop per nym phs beco m e surface- contam inated with conidi a durin g ingestio n of the bait substrat e, and that infectio n then occur s throug h the externa l integu m ent. Numerou s conidi a w ere observe d on the surface s of nym phs that ingeste d baits, and conidi a were also recovere d from nymphs that had been surface- w ashe d ( , 130 conidia /nym ph on average ), even though the head s and posterio r abdo m inal segments had been coate d with paraf®n. Further m ore, when nym phs were fed baits treated with ¯uoresce nt dye, substant ial quantiti es of dye w ere observe d on the nym phs, mostly on bod y parts involve d in the detectio n and handlin g of food (mouthpar ts, antenna e and forelegs ) or on the parts of the body that w ould most likely com e into contac t with the bait substrat e (hea d sclerite s and the ventra l surfac e of the



FIGUR E 6.

47

Deposition of dye (dots) on M . sanguinipes nymphs (bar 5 1 mm). Nymphs were fed lettuce disks treated with ¯uorescent dye in oil (a) and water (b), and representative drawings were traced from color photographs taken under UV light.

thorax ) (Figure 6). Infectio n of insects by B. bassiana occurs m ost frequen tly throug h the externa l integu m ent (Ferron , 1978). Per os infectio n of insects by ento m opathog enic fung i has also been dem onstrate d using both histopat hologica l and indirec t m ethod s (D illon & C harnley , 1991), and V een (1966 ) observe d hyphae in the maxillar y palp s and heads of locust nym phs sterilize d afte r they ingeste d M . anisopli ae conidi a on leaves , thereb y suggesti ng an infectio n site in the mouth region . In this study , a surface- steriliza tion techniq ue was employe d in an attem pt to show quantita tively the importanc e of externa l versu s per os infectio n in grassho pper nym phs feedin g on baits . Conidia of B. bassiana are rapidly kille d by low concent ration s of sodiu m hypochl orite (Ignoff o & Dutky, 1963), and sodium hypochl orite was con®rm ed to kill the majority ( . 97% ) of conidi a on grassho pper nymphs. In nymphs sterilize d after the ingestio n of substrat es inoculat ed with B . bassiana , 33±82% less m ycosis w as observe d than in nym phs sterilize d before ingestio n of the baits. This suggest s that infectio n occurs largely throug h the externa l integu m ent. H ow ever, some m ycosis also occurre d in grasshop per nymphs sterilize d afte r ingestio n, and althoug h infectio n may have occurre d per os, addition al possibil ities includ e infectio n follow ing the surfac e conta minatio n of nym phs with B. bassiana conidi a excrete d in feces , as describe d previous ly for Colorad o potato beetle s (Allee et al., 1990) and/or externa l infectio n due to the incom plete sterilizat ion of nym phs. Inocula tion of nymphs from conidi a in



48

frass seem s unlikel y in grasshop per nym phs, as frass pellets are encapsu late d by peritrop hic mem bran e (D illon & Charnley , 1991). Further , w e observe d no m ycosis in nymphs expose d to frass that contain ed conidia . A threshol d of inoculu m is require d to caus e diseas e in insects , and althoug h the quantities of viable conidia presen t on nym phs afte r steriliza tion w ould not normally be suf®cient to cause mycosis , it is possibl e the subm ersio n of nym phs in sodiu m hypochl orite predispo sed them to infection . H istologi cal studies will be necessa ry to ascertai n if per os infectio n by B. bassian a occurs in acridids . The alimentary canal of insects is consider ed to be a relativel y inhospit able environ ment for fung i (Dillon & Charnley , 1991). How ever, many microorg anis ms can surviv e passag e throug h the alim entary canal of acridids , includin g bacteri a (M ead et al,, 1988 ; C hapc o & Kelln , 1994) and ®lam entou s fungi (Dillon & Charnley , 1991). It was observe d that som e ingeste d B. bassian a conidi a passed throug h the alim entary cana l of M . sanguini pes nym phs and that viable conidi a could be recover ed from frass. Acridids have a rapid alim entary transit time of 2±6 h (Ueckert & Hansen , 1972; Dillon & Charnley , 1986 ; M ead et al., 1988). The m ajority of the B. bassian a conidi a that passe d throug h the alimentary cana l of grassho pper nym phs did so within 24 h of ingestio n, as has been reporte d for othe r funga l propagu les (H asan , 1982; H uang & H arper, 1985 ; Dillon & C harnley , 1986). However, very small proporti ons of the conidi a placed on the bait substrat es w ere subsequ ently recover ed in frass (1.3 ±11.7 %). Loss of conidia l viabilit y m ay have occurre d durin g transit throug h the alim entary canal. In addition , the m aceratio n spread- plate techniqu e measure s CFU and not necessar ily individu al conidia , and therefor e populations may have been underest im ated due to the clum ping of conidia . For successf ul infectio n via the gut, propagu les of a fungu s must rem ain in the alim entary cana l for a suf®cient tim e to perm it germ ination and penetrat ion, as has been observe d for M . anisopli ae conidi a in the hindgu t of the desert locust (D illon & C harnley , 1986) . H ow ever, in M . sanguini pes nym phs, the rapid excretio n of conidi a in frass alon g with the low incidenc e of conidia l retentio n in alim entary canals (viable conidi a w ere foun d in the guts of only 15% of the nymphs 5 days afte r bait ingestio n) is consiste nt w ith the apparen t im portanc e of an externa l m echanis m of infectio n in grassho ppers ingestin g bait form ulation s of B. bassian a. W hen oil was used as a carrier for B. bassian a, more m ycosis was observe d in nym phs than when w ater w as used , regardle ss of the bait substrat e ingested . This observat ion is in agree ment with the ®nding s from a preliminary stud y (G oettel & Johnson , 1992). Possible advanta ges of the use of oil, rather than w ater, as a carrie r of funga l propagu les include : (1) (2) (3) (4) (5) (6)

infectio n at lower hum iditie s (Batem an et al., 1993); stimulate d germinatio n (W inde r & Van Dyke, 1990); longer duratio n of viabilit y (Prior et al., 1988); decrease d sensitivi ty to high temperatur e in storag e (M cC latchie et al., 1994); decrease d sensitivi ty to U V radiatio n (M oore et al., 1993; Inglis et al., 1995); enhance d attach m ent to the hydroph obic surface s of insec t integu ment.

In addition , the form ulatio n of B. bassiana in oil m ay improv e the adhesio n of the conidi a to the bait, and thereb y reduce the dislodge m ent of inoculu m from the bait substrat e durin g applicat ion (Johnso n & G oettel, 1993). D espite the superior ity of form ulatio n in oil relativ e to water in the presen t study , little differen ce was observe d in m ycosis when conidi a in oil or w ater were applie d to w heat leave s with an airbrus h. Inglis et al. (1993 ) also observe d no differen ce in infectio n of nym phs fed wheatgra ss or alfalfa leave s spraye d w ith B. bassian a conidi a in oil or w ater in the ®eld, and that the oil was rapidly absorbe d into leaf tissues follow ing applicati on at ultra-lo w volu mes (ULV ). In this study , in contras t to U LV applicati on, a consider able volu m e of the oil pipette d onto the lettuce , and to a lesse r degree on to the bran substrat e, rem ained unabsor bed; in the surface- sterilizat ion experim ent, absorpti on of oil into bran m ay explai n the decreas ed ef®cacy observe d relativ e to the lettuc e substrat e. Conspicu ously m ore ¯uoresce nt dye was also observe d on nym phs ingestin g bait substrat es treate d with dye in oil than in w ater, suggesting the ef®cacy of the oil relativ e to the water formulatio n w as due to the increase d coverag e of nym phs with the oil carrier .


49

This stud y dem onstrate s that grassho pper nym phs are highly suscepti ble to infectio n by B. bassiana form ulated on bait substrates, and that the ef®cacy of bait formulatio n appear s to be depende nt on the degree to which nymphs beco me surface- contam inate d durin g handlin g and ingestio n of the bait. Therefor e, future develop m ent of bait formulation s should focus on baits that prom ote tactile handlin g by the grasshop pers , thereb y enhanci ng transfe r of conidi a to the integu m ent surface .

AC KNO W LED GEM E NTS


The author s than k the follo wing people at the Agricultu re and A gri-Food Canad a (A AFC) Lethbridg e R esearc h C entre (LRC ): R on Feniuk for his technica l assistan ce, Eva Pavlik for providin g grasshop per eggs, K en Sinclair for photogr aphin g the nym phs and T oby Entz for his statistic al advice . The author s also w ish to than k Dr M artin Erlandso n (AA FC, Saskatoon ) and Dr K evin Floate , Dr Bob Byers and M s H elen M cM enamin (AA FC, Lethbridg e) for criticall y revie wing the m anuscript. Researc h was funde d in part by a gran t from Farming for the Future , Alberta Agricultu ral Researc h Institute . This m anuscri pt is LRC contribu tion #387950 4.

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