PS801.69/2003 Pest Management Science

Esterase-mediated tolerance to a formulation of the organophosphate insecticide monocrotophos in the entomopathogenic fungus, Beauveria bassiana (Balsamo) Vuill—a promising biopesticide K Uma Devi,∗ N Nageswara Rao Reddy, D Sridevi, V Sridevi and C Murali Mohan•Q1 Department of Botany, Andhra University, Visakhapatnam, 530003 AP, India

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Abstract: The use of biopesticides compatible with chemical pesticides is recommended in pest management as an effective and ecologically sound strategy. The entomopathogenic fungus Beauveria bassiana (Balsamo) Vuill, which is used as a biopesticide, was found to exhibit and lose tolerance to two organophosphorus insecticides widely used in Indian fields. The pattern of response is similar to the esterase-mediated organophosphate tolerance in aphids achieved through esterase gene duplication. Therefore the role of esterase in the tolerance exhibited by isolates of B bassiana to the organophosphate, monocrotophos, was studied. Both the total soluble protein content and esterase activity were found to increase significantly in B bassiana cultures that were able to grow in the presence of monocrotophos. With the hitherto established linkage between esterase overproduction and organophosphate tolerance in insects and the observed similarity in response of the insects and B bassiana to the chemical, it is concluded that tolerance to monocrotophos in B bassiana may be due to a mechanism similar to that operating in insects. Habituation of the fungus to monocrotophos to initiate expression of esterase gene may help in achieving compatibility between the two.  2003 Society of Chemical Industry

tolerance to these chemicals; selecting an isolate tolerant to the chemical is required for combination treatment. A sample of 30 Beauveria bassiana isolates, well characterised for tolerance to abiotic stress, and whose DNA fingerprints and total protein profiles have been studied6,7 showed inconsistency in tolerance when screened for tolerance to monocrotophos [dimethyl (E)-2-(methylcarbamoyl)vinyl) phosphate], quinalphos [O,O-diethyl O-quinoxalin -2-yl phosphorothiate].6 Conidial germination, although delayed on occasions (1–8 h), was not inhibited in the presence of these chemicals.6 Inconsistency was observed in the growth bioassays with these chemicals. Understanding the mechanism of tolerance to organophosphorus insecticides in B bassiana is important for using it along with them in crop-pest management. The basis for tolerance was therefore investigated.

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1 INTRODUCTION The compatibility of the entomopathogenic fungal bioinsecticide, Beauveria bassiana (Balsamo) Vuill with agrochemicals applied for crop protection permits their simultaneous or sequential use, with the benefit of reducing the dosage of the toxic chemical and reaping the advantage of their synergistic effect.1,2 Several studies have been made on the compatibility of B bassiana with chemical pesticides.2 Monocrotophos, an organophosphate, and quinalphos, an organophosphorothiate, are two insecticides currently very widely used in Indian fields. Kaaya et al 3 reported normal growth and sporulation of B bassiana in a medium with organophosphate; a combined treatment with B bassiana and monocrotophos was found to be effective in insect pest management in tea crop.4 An earlier study5 reported an inhibitive effect of monocrotophos on B bassiana isolates. Thus, it appears that isolates of this fungus differ in their

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Keywords: Beauveria bassiana; biopesticide; esterase activity; habituation; monocrotophos tolerance; total protein content

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Pest Manag Sci 59:000–000 (online: 2003) DOI: 10.1002/ps.801

∗ Correspondence to: K Uma Devi, Department of Botany, Andhra University, Visakhapatnam, 530003 AP, India E-mail: [email protected] Contract/grant sponsor: Council for Scientific and Industrial Research (CSIR), India; contract/grant number: 38(098/97/EMR-II) Contract/grant sponsor: UGC, India (Received 1 April 2003; revised version received 19 June 2003; accepted 5 August 2003)

 2003 Society of Chemical Industry. Pest Manag Sci 1526–498X/2003/$30.00

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2.2 Fungal isolates Two isolates, one of which showed tolerance to the field recommended field (1.5 g litre−1 ) of monocrotophos on many occasions (three of six times bioassayed) and one only once in the six times tested, were chosen for the analysis (Table 1).

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Table 1. Total soluble protein content and esterase activity in isolates of the entomopathogenic fungus Beauveria bassiana exposed to an organophosphate insecticide, monocrotophos

BB4

ARSEF-1788a

BB2

Habituatedc

Normal

Normal

Normal

Helicoverpa armigera Warangal, India 3600 (±2.88)d 7400 (±4.04)f

Heliothis virescens Spain 2500 (±6.92)d —

Spodoptera litura Bangalore, India 500 (±0.577)d —

7933 (±7.5)d 11 416.5 (±2.3)f

7458 (±1.15)d —

11 729 (±3.46)d —

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ARSEF-1149a Normalb

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Helicoverpa armigera Cordoba, Spain 1800 (±1.54)d 3800 (±1.15)f

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Host insect Geographical origin Total soluble proteins (µg 100 mg−1 )g Esterase activity (Rappaport units 100mg−1 )g

108.3 (±2.88)d 2816.5 (±1.73)f

7400 (±1.15)d 7400 (±2.88)e 8800 (±5.77)f 12 541 (±11.54)d 41 000 (±17.32)e 42 400 (±11.54)f

a ARSEF isolates were obtained from USDA-ARS Collection of Entomopathogenic Fungal Cultures, Ithaca, New York; BB2 and BB4 were isolated from insects collected in local fields; they are yet to be accessioned. b Cultures established from conidia produced on a medium with no monocrotophos. c Cultures established from conidia produced in cultures grown in an medium with monocrotophos (1.5 g litre−1 ). d Control with no monocrotophos. e,f with 0.075% and with 0.15% of monocrotophos respectively. g 100 mg of culture filtrate. Differences of the values between d,f in normal cultures of isolates ARSEF 1149 and BB4 and d,e,f in habituated cultures of the isolate ARSEF 1149 and between the four isolates are significant (from ANOVA test, P < 0.0001).

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2.3 Culture of the fungus Beauveria bassiana isolates were cultured on Sabouraud dextrose (SD) agar slants maintained in an environmental chamber set at 25 (±2) ◦ C, 90% RH and 16:8 h light:dark regime. An aqueous conidial suspension of 106 conidia ml−1 containing 105 mg ml−1 Tween 80 was prepared from 14-dayold cultures. The viability of the conidia in these suspensions was tested as described previously17 and inocula with more than 95% conidial germination were used in the experiments. Cultures were initiated by inoculating 1 ml of the conidial suspension (with 106 conidia) in 100 ml of SD broth. Two cultures, one with monocrotophos (1.5 mg litre−1 , ie field recommended concentration) and another without the chemical were set up for each of the four isolates. The liquid cultures were maintained in an incubator shaker at 25 (±1) ◦ C and 100 rev min−1 for 10 days. The mycelium was harvested from 10-day-old cultures for analysis by filtering on a muslin cloth and washing twice with distilled water. In the case of the isolate ARSEF-1149 (which showed growth in the presence of monocrotophos), before harvesting the mycelium from the culture on the monocrotophos-containing medium, conidia on the surface of the culture were collected and suspended in water with 105 mg ml−1 Tween 80; volumes of this suspension equivalent to

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2 MATERIALS AND METHODS 2.1 Chemicals Monocrotophos was used as a 360 g litre−1 SL (Sudharshan Chemical Industries Ltd, Pune, India),

and quinalphos as a 250 g litre−1 EC (Tamil Nadu Agro Industries Corporation Ltd, Chennai, India).

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Decreased sensitivity to toxic compounds in organisms is achieved through various means: a change in the structure of the biological target leading to insensitivity to the toxic compound, modification in the cell membrane resulting in prevention of penetration/active excretion, isozymic alterations ensuing changes in specificity and enhanced metabolic detoxification through breakdown of the toxic compound into non-toxic substances (metabolic resistance).8,9 In insects, a positive correlation between resistance to organophosphate insecticides and total esterase activity has been demonstrated in several instances.9 – 13 Organophosphate tolerance in these cases was achieved through fast metabolic detoxification: hydrolysis by carboxylesterase10 or modification of acetylcholinesterase.13 In aphids, increased esterase production in response to organophosphate was traced to the duplication of the gene (E4) coding for an isozyme form of carboxylesterase with the number of gene copies duplicated, depending on the concentration of the chemical.12 In the absence of the insecticide, the resistant clone was found to revert to a susceptible one without any obvious rearrangement.11,12 The inconsistent expression of tolerance to the two organophosphorus chemicals observed in B bassiana isolates with frequent reversion from tolerance to sensitivity6 seemed a case parallel to the esterasemediated tolerance described in aphids.12 To test this presumption, the esterase activity was measured in B bassiana isolates exposed to monocrotophos. The isoesterase profiles of B bassiana have been studied earlier.14 – 16

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2.6 Statistical analysis The data of estimates of total soluble protein content and esterase activity was subjected to one-way analysis of variance (ANOVA) using STATISTICA ver 5.021 to assess the significance of the various effects. Pest Manag Sci 59:000–000 (online: 2003)

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3.2 Esterase activity Significant differences in esterase activity were observed among the four B bassiana isolates analysed •(F = 12 047.29, P < 0.001) (Table 1). The esterase activity in cultures of the isolates ARSEF 1149 and BB4 that grew in the presence of monocrotophos was found to be significantly higher than their corresponding controls [ARSEF 1149 •(F = 6471.47, P < 0.0001) and BB4 (F = 1957.22, P < 0.001)]. Among the four B bassiana isolates, esterase activity was lowest in ARSEF 1149 which increased ∼26 times when exposed to monocrotophos. In BB4, esterase activity was ∼70 times more than ARSEF 1149 and it increased 1.43 times on exposure to monocrotophos. The cultures of the isolate ARSEF 1149 that were established from conidia produced on monocrotophos-habituated culture, showed a further significant increase in esterase activity both in media without and with different concentrations of monocrotophos •(F = 3099.754, P < 0.0001).

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2.5 Esterase assays A colorimetric method has been used to detect enzymes expressed by conidia of entomopathogenic fungi.20 A similar approach was employed for estimating esterase activity. The kit supplied by Sigma Chemical Co (St Louis, MO) for estimation of acetylcholinesterase (catalogue number 420), which the manufacturers state estimates activity of all esterases, was used for assaying esterase activity in the fungal protein extracts. The protocol recommended by the manufacturers was followed with some modifications to suit the fungal system. The esterase activity was determined from a standard mixture consisting of aqueous sodium chloride (5.85 g litre−1 ; 0.2 ml), distilled water (3.0 ml), acetylcholine chloride solution (2.0 ml) and protein extract (0.2 ml, containing 10 µg of protein), with water as control. The reaction mixture was incubated at 32 ◦ C for 30 min in a water bath and absorbance was recorded at 400, 420 and 440 nm. m-Nitrophenol was used as the colour indicator. Acetic acid was used to draw a calibration curve. Absorbance of each sample recorded at each of the three wavelengths was plotted on the calibration curve. The corresponding esterase activity was noted and the mean of the values at the three wavelengths was taken as the esterase activity (Rappaport units) of the sample. Three assays were done from each extract. The experiment could not be repeated in time because the two isolates which showed tolerance in one experiment did not show growth in the presence of monocrotophos when the experiment was set up for the second time.

3.1 Total soluble proteins Significant differences were observed in the total soluble protein content among the four B bassiana isolates (F = 115, P < 0.0001) (Table 1). There was a sevenfold difference in total soluble protein content among the isolates. In the two isolates ARSEF 1149 and BB4, which showed growth in the presence of monocrotophos, the total protein content in the test was significantly higher than in the control [ARSEF 1149 •(F = 15 00 000, P < 0.0001) and BB4 (F = 103.62, P < 0.0001)]. Total soluble proteins in isolates ARSEF 1149 and BB4 more than doubled (2.15 and 2.12 times respectively) when exposed to monocrotophos. In the isolate ARSEF 1149 that was recultured after habituation in monocrotophos, the total protein content further increased •(F = 46 666.67, P < 0.0001).

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2.4 Extraction of total soluble proteins Protein sample was extracted from of the filtered culture (100 mg). It was ground to a fine powder in liquid nitrogen with a mortar and pestle and transferred to an Eppendorf tube and extraction buffer (Tris-HCl 10 mM, pH7 + EDTA 1 mM; 1 ml)18 was added to it. The mixture was centrifuged at 10 000 rev min−1 for 15 min at 4 ◦ C. The supernatant with the extracted proteins was decanted leaving the pellet. The quantity of total soluble proteins in the sample was determined by Lowry’s method using bovine crystallized albumin as standard and the Folin–Ciocaltaeu reagent.19

3 RESULTS The isolates ARSEF 1149 and BB4 showed growth in the presence of monocrotophos comparable to the controls. The other two isolates, ARSEF1788 and BB2, did not show growth in the presence of monocrotophos. The cultures set up from the inoculum of the monocrotophos-habituated isolate ARSEF 1149 showed growth in media with concentrations of 0.75 and 1.5 g litre−1 of monocrotophos but not in a concentration of 3 g litre−1 . When the cultures of the two isolates which showed tolerance to monocrotophos were set up a second time, from a subculture of the original culture, no growth was observed in the test.

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106 conidia were inoculated into 100 ml of SD media containing 0.75 g litre−1 , 1.5 g litre−1 and 3 g litre−1 monocrotophos, and incubated for 10 days under the conditions described above.

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4 DISCUSSION An increase in total soluble protein content was observed in B bassiana in the presence of monocrotophos, an organophosphate insecticide. Increase in 3

K Uma Devi et al

It is likely that the mechanism of tolerance to these two organophosphorus chemicals is similar. Whatever may be the mechanism of tolerance, habituating B bassiana to monocrotophos during its mass culture for application in the field is necessary to achieve compatibility with it.

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ACKNOWLEDGEMENTS KUD and VS are thankful to the Council for Scientific and Industrial Research (CSIR), of the government of India for financial assistance [grant number 38(098/97/EMR-II)]. NNRR and CMM are thankful to CSIR and UGC for their research fellowships. We thank Dr RA Humber, Curator, USDA-ARS Collection of Entomopathogenic Fungal Cultures, Plant Protection Research Unit, US Plant Soil and Nutrition laboratory, Tower Road, Ithaca, New York, for providing the ARSEF cultures.

REFERENCES

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1 Anderson TE, Hajek AE, Roberts DW, Preisler HK and Robertson JL, Colorado potato beetle (Coleoptera: Chrysomelidae)-effects of combinations of Beauveria bassiana with insecticides. J Econ Entomol 82:83–89 (1989). 2 Inglis GD, Goettel MS, Butt TM and Strasser H, Use of hyphomycetous fungi for managing insect pests, in Fungi As Biocontrol Agents, ed by Butt TM, Jackson C and Magan N, CAB International,• pp 23–69 (2001). 3 Kaaya GP, Mwangi EN and Ouna EA, Prospects for biological control of livestock ticks, Rhipicephalus appendiculatus and Amblyomma variegatum, using entomogenous fungi Beauveria bassiana and Metarhizium anisopliae. J Invertebr Pathol 67:15–20 (1996). 4 Sun JD, Wu GY, Lin A, Zeng MS, Wang QS and Xu DY, Investigations and demonstration of the integrated control the tea weevil by a mixture of pesticides and microbes. Tea Sci Technol Bull 3:32–34 (1993). 5 Aguda RM, Saxena RC, Litsinger JA and Roberts DW, Inhibitory effects of insecticides on entomogenous fungi Metarhizium anisopliae and Beauveria bassiana. IRR Newsl 9:16–17 (1984). 6 Murali Mohan C, Phenotypic and molecular characterization of Beauveria bassiana (Bals) Vuill isolates suitable for integrated pest management of cotton boll worm (Helicoverpa armigera Hub.) in semi-arid tropics, Doctoral thesis, Andhra University, Visakhapatnam, AP, India (2002). 7 Padmavathi J, A Study of morphology, host specificity and genetic structure through DNA fingerprinting of the insect pathogenic asexual fungal species Beauveria bassiana (Bals) Vuillemen—A potential biopesticide, Doctoral thesis, Andhra University, Visakhapatnam, AP, India (2002). 8 Brattsten LB, Resistance mechanisms to carbamate and organophosphate insecticides, in Managing Resistance to Agrochemicals: from Fundamental Research to Practical Strategies, ed by Green MB, Le Baron HM and Moberg WK, ACS Symposium Series 421, American Chemical Society, Washington, DC, pp 42–60 (1990). 9 Manikandan P and Ravisankar S, Carboxylesterase activity associated with organophosphate resistance in Helicoverpa armigera (Lepidoptera: Noctuidae) in Tamil Nadu. Curr Sci 75:186–187 (1998). 10 Devonshire AL, The properties of a carboxylesterase from the peach-potato aphid, Myzus persicae (Sulz.), and its role in conferring insecticide resistance. Biochem J 167:675–683 (1977).

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protein content on exposure to stress has been reported on several occasions.22 Esterase activity in B bassiana was found to increase on exposure to monocrotophos. The isolates which normally had higher esterase levels (equal to or greater than the elevated levels in tolerant isolates in the presence of monocrotophos) were not found to be tolerant to monocrotophos. In such isolates, though the esterase content is high, the isozyme form which confers tolerance may not have been present. In aphids, Devonshire10 reported that only the E4 isozyme form of esterase is involved in organophosphate tolerance. The isolates which showed normal growth in the presence of monocrotophos were found to have an elevated level of total esterase activity when grown in the presence of monocrotophos, compared with their controls (grown on normal medium with no monocrotophos). The increased esterase level may be due to the production of the esterase isozyme that metabolises organophosphates. Esterase is reported to hydrolyze the ester bonds in the toxic organophosphate (monocrotophos) and sequester it into a non-toxic form, thereby conferring tolerance.10,22 The pattern of response to monocrotophos in B bassiana is similar to the organophosphate tolerance reported in aphids, which was demonstrated to be achieved through duplication of E4 gene (coding for E4 type esterase isozyme).12 A time lag was shown by B bassiana isolates in establishing growth in the presence of monocrotophos.6 This further corroborates the role of gene-duplication-arbitrated expression of the esterase gene coding for a detoxifying isoenzyme, the time lag being due to the time taken for duplication of the gene before its expression. Alternatively, the expression of the gene may be modulated by extra genomic elements. We observed double-stranded RNA (ds RNA) in B bassiana isolates in genomic DNA preparations run before RNAse treatment. We did not make a detailed record of its occurrence, but isolates seemed to lose and attain them. These double-stranded RNAs earlier reported23 in B bassiana are considered viruses and it has been speculated that they modulate gene expression.24 All 30 screened B bassiana isolates showed tolerance in one or the other of the monocrotophos tolerance screening bioassays.6 Thus it appears that all isolates can tolerate this chemical. It is likely that B bassiana isolates show tolerance when they succeed in expressing the esterase gene that confers monocrotophos tolerance. The expression may depend on the readiness (affected by its physiological state) with which the isolate duplicated the esterase isozyme gene or, alternatively, may be due to the presence or loss of the ds RNA—the esterase gene expression being activated in its presence, leading to tolerance. The B bassiana isolates showed a similar response to the phosphorothioate (quinalphos) insecticide (data not shown) as to monocrotophos (organophosphate).

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18 Balesdent MH, Gall C, Robin P and Rouxel T, Intraspecific variation in soluble mycelial protein and esterase patterns of Leptosphaeria maculans French isolates. Mycol Res 96:677–684 (1992). 19 Lowry OH, Rosebrough NJ, Farr AL and Randall RJ, Protein measurement with Folin phenol reagent. J Biol Chem 193:265–275 (1951). 20 ElSayed GN, Ignoffo CM, Leathers TD and Gupta SC, Use of a colorimetric system to detect enzymes expressed by conidia of entomopathogenic fungi. Mycopathologia 118:29–36 (1992). 21 STATISTICA for Windows [Computer program manual], Tulsa, OK: StatSoft, Inc, 2325 East 13th Street, Tulsa, OK, 74104, USA (1995). 22 Hollomon DW and Butters JA, Molecular determinants for resistance to crop protection chemicals, in Molecular Biology in Crop Protection, ed by Marshall G and Walters D, Chapman and Hall, UK, pp 98–117 (1994). 23 Melzer MJ and Bidochka MJ, Diversity of double-stranded RNA viruses within populations of entomopathogenic fungi and potential implications for fungal growth and virulence. Mycologia 90:586–594 (1998). 24 Norman JA and Montgomery MM, RNA i and Cosuppression: double-stranded RNA as an agent of sequence-specific genetic silencing in animals and plants, in dsRNA Genetic Elements, Concepts and Applications in Agriculture, Forestry and Medicine, ed by Tavantzis SM, CRC Press, Boca Raton, FL, pp 1–55 (2002).

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11 Field LM and Devonshire AL, Insecticide resistance by gene amplification in Myzus persicae, in Resistance ’91: Achievements and Developments in Combating Pesticide Resistance, ed by Denholm I, Devonshire AL and Hollomon DW, Elsevier Science Publishers, London, pp 240–250 (1992). 12 Devonshire AL and Sawicki RM, Insecticide-resistant Myzus persicae as an example of evolution by gene duplication. Nature (London) 280:140–141 (1979). 13 Zhu KY and Gao J, Increased activity associated with reduced sensitivity of acetylcholinesterase in organophosphateresistant green bug, Shizaphis graminum (Homoptera: Aphididae). Pestic Sci 55:11–17 (1999). 14 Poprawski TJ, Riba G, Jones WA and Aioun A, Variation in isoesterase profiles of geographical populations of Beauveria bassiana (Deuteromycotina: Hyphomycetes) isolated from Sitona weevils (Coleoptera: Curculionidae). Environ Entomol 17:275–279 (1988). 15 Berretta MF, Lecuona RE, Zandomeni RO and Grau O, Genotyping isolates of the entomopathogenic fungus Beauveria bassiana by RAPD with fluorescent labels. J Invertebr Pathol 71:145–150 (1998). 16 Castrillo LA and Brooks WM, Differentiation of Beauveria bassiana isolates from the darkling beetle, Alphitobius diaperinus, using isozyme and RAPD analyses. J Invertebr Pathol 72:190–196 (1998). 17 Varela A and Morales E, Characterization of some Beauveria bassiana isolates and their virulence toward the coffee berry borer Hypothenemus hampei. J Invertebr Pathol 67:147–152 (1996).

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