Characteristics relating to the pathogenicity of Metarhizium anisopliae toward Nilaparvata lugens

IOURNALOF

INVERTEBRATE

PATHOLOGY

53, 25-31 (1989)

Characteristics Relating to the Pathogenicity of Mefarhizium anisopliae toward Nilaparvata lugens K.D.

ZSAMUELS,'

J.B.

HEALE,AND

M. LLEWELLYN

Department of Biology, King’s College London, Kensington, London W8 7AH, England Received January 27, 1988; accepted May 9, 1988 Laboratory bioassays were conducted to assess the pathogenicity of 24 wild-type isolates of Metarhizium anisopliae toward Nilaparvata lugens. LT,, values ranged from 5 days (highly pathogenic) to >14 days (nonpathogenic). In vitro germination, growth rate, spore production, extracellular enzyme production, and spore characteristics were assessed in relation to pathogenicity. Rapid germination and growth, and small spore volume, were correlated with high pathogenicity. Highly pathogenic isolates produced no detectable extracellular amylase and detectable amounts of extracellular chitinase, lipase, and protease activity. Evidence for the stable diploid nature of var. majus isolates is presented. 8 1989 AC&~~C Press, Inc. KEY WORDS: Metarhizium anisopliae; Nilaparvata lugens; extracellular enzymes, amylase. chitinase, lipase, protease; sporulation; trait expression; pathogenicity: ploidy.

INTRODUCTION

traits involved in, or responsible for, conferring pathogenicity and other desirable characteristics (necessary if a fungal strain is to be successfully used as a biological control agent), is an essential prerequisite in any strain improvement program. The natural variation in pathogenicity and other characteristics, exhibited by isolates of the same fungus, is one starting point (Heale. 1982; Jackson et al., 1985). Further improvement in such Hyphomycete fungi may be induced by mutagenesis, recombination via the parasexual cycle (Drummond, 1986; Jackson and Heale, 1987; Samuels, 1986), or manipulation of single genes where possible/feasible (Heale, 1988). In this paper, we examine the wide variation in a number of characteristics exhibited by 24 wild-type isolates of M. anisopliae and assess their significance and correlation to pathogenicity.

Planthoppers are now considered among the most serious pests of rice (Wilson and Claridge, 1985). Of these Niluparvata lugens (Homoptera: Delphacidae) is the most important. Five biotypes of N. lugens are known (Pathak and Khush, 1977) with ability to overcome rice cultivar resistance (Khush et al., 1977), and resistance to insecticides is now widespread (Chung et al., 1982). It is therefore important to develop all possible options available for the control of this pest. A number of Hyphomycete fungi are known to cause considerable natural control of planthoppers. Metarhizium anisopliae is a natural pathogen of N. lugens and attention has been drawn to its use as a potential biological control agent (Gillespie, 1984). There is a clear need to improve many existing strains of fungi currently being considered for use as mycoinsecticides. The majority of the present wild-type isolates offer little potential as candidates for commercialization. The identification of

MATERIALS

AND METHODS

Isolates. Attempts were made to passage 38 wild-type isolates of M. anisopliae, obtained from a wide variety of hosts and geographical locations, through N. lugens. Eighteen isolates were successfully passaged and these, together with six M. an-

’ Present address: Department of Entomology, The University of Adelaide, Waite Agricultural Research Institute, Glen Osmond, South Australia 5064. 25

0022-201 l/89 $1.50 Copyright AU rights

0 1989 by Academic Press. Inc. of reuroduction in anv form rewrwd

26

SAMUELS,

HEALE,

AND LLEWELLYN

isopliae var. majus isolates from Oryctes rhinoceros, were single-spored and se-

lected for further investigation (Table 1). Media. Stock cultures were maintained on Czapek-Dox complete media (CM) (Typas and Heale, 1976) with 2% agar at 4°C. Stock cultures were inoculated onto fresh Sabouraud’s dextrose agar media (SDA), consisting of 1% mycological peptone (Oxoid), 4% D-glucose, and 1.5% agar, as required. Water agar (WA) consisted of 1.5% purified agar (Oxoid). Incubation was at 28°C in the dark. Spore characteristics. Spore length measurements were recorded using a microscope fitted with an eye-piece screwmicrometer. Spore volume measurements

were recorded on a Coulter counter (Typas and Heale, 1977). Spore nucleus diameter measurements were recorded as for spore length, following staining in 100 kg/ml of acridine orange (Clutterbuck and Roper, 1966) and observed under ultraviolet light. All methods involved the use of spores harvested from 7-day-old “spread plates.” Spore germination. Spores harvested from 7-day-old SDA spread plate cultures were used to prepare WA and SDA spread plates, using 0.1 ml of a 1 x lo* spores/ml suspension in 0.05% Tween 80, incubated at 28°C. Plates were assessed to determine the T,, germination value. Radial growth rate. Five-millimeter plugs from 5-day-old colonies were incu-

TABLE VARIETY,

ORIGINAL

ISOLATE

Isolate number 410 413 415 416 414 401 412 403 402 411 431 420 417 427 433 404 501

anisopiiae anisopliae anisopliae anisopliae anisopliae anisopliae anisopliae anisopliae anisopliae anisopliae anisopliae anisopliae anisopliae anisopliae anisopliae anisopliae anisopliae

502 503 504 505 So6 507 508

majus majus majus majus majus majus

ORIGINAL anisopliae

Original isolate number”

Var.

(QWb

NUMBER, Metarhizium

Intermediate’

-

ARSEF 45.5 ARSEF 485 ARSEF 487 ARSEF 488 ARSEF 486 GCRI 35-79 ARSEF 457 GCRI 83-82 GCRI 82-82 ARSEF 456 ARSEF 588 ARSEF 551 ARSEF 489 ARSEF 576 ARSEF 683 CM1 98-374 GCRI 100-82 GCRI 134-82 GCRI 135-82 GCRI 137-83 GCRI 148-83 GCRI 170-83 GCRI 171-83 GCRI 172-83

1 AND GEOGRAPHIC LOCATION OF ISOLATES

HOST,

Original host Nilaparvata N. lugens N. lugens N. lugens N. lugens

lugens

Vine weevil N. lugens Spittlebug Zigzag leafhopper N. lugens Soil Spittlebug N. lugens N. lugens

Cane grub Pericoptus truncatus Melolontha melolontha M. melolontha Oryctes rhinoceros 0. rhinoceros 0. rhinoceros 0. rhinoceros 0. rhinoceros 0. rhinoceros

Geographic location Philippines Philippines Philippines Philippines Philippines England Philippines Brazil Unknown Philippines Columbia Brazil Philippines Philippines P.R. China New Zealand Unknown Unknown Unknown New Zealanc Unknown Philippines Philippines Philippines

’ ARSEF, Collection of entomopathogenic fungi, U.S. Department of Agriculture, Agricultural Researcl Service Plant Protection Research Unit, Boyce Thompson Institute, Ithaca, New York. CMI, Commonwealtl Mycological Institute, Kew, Surrey, United Kingdom. GCRI, Institute for Horticultural Research, Littlehamp ton, West Sussex, United Kingdom. b QEC, King’s College London, Kensington, London, United Kingdom. c Isolate 502 was intermediate between var. anisopliae and var. majus isolates (Tulloch, 1976) on the basis o spore length (P < 0.001) and spore volume (P < 0.001).

PATHOGENICITY

OF M. anisopliae

bated inverted on SDA plates at 20”, 23”, 25”, and 28”C, for 10 days. Sporulation. SDA spread plates were set up as for spore germination, but using a 1 x 10’ spores/ml suspension, incubated at 28°C. Spores were harvested following agitation in 10 ml of 0.05% Tween 80 daily for 7 days. Spore concentration was determined with a hemocytometer. Extracellular enzyme production. Extracellular production of amylase and lipase (Hankin and Anagnostakis, 1975), chitinase (Gabriel, 1968), and protease (Bucher, 1960) were visualized using agar plate clearing techniques, or as a visible precipitate (lipase). Enzyme activity was expressed as the colony diameter/halo diameter ratio (Rosato et al., 1981) after incubation at 28°C for 4 days (lipase), 6 days (amylase and protease), or 9 days (chitinase). After 9 days, chitinase plates were placed at 4°C for 7 days to enhance the clearing (Hall, 1977). Substrates and modifications were as follows: 0.2% soluble starch (amylase), 2% Tween 20 and 0.5% yeast extract (Oxoid) (lipase), 0.2% colloidal chitin prepared from crab shell (Campbell and Williams, 1951) (chitinase), and 2.4% nonfat milk powder (protease). Insect bioassays. Pathogenicity of all wild-type isolates toward adult male N. lugens was tested by spraying with a standard volume of a 1 X lo6 spores/ml suspension. This concentration was found to distinguish between highly, moderately, and poorly pathogenic isolates (Samuels, 1986). Inoculated N. lugens were placed within individual leaf clip-cages, allowing access to both surfaces of a supported rice leaf on intact plants. Insects were observed daily to determine the LTS, mortality. Spore concentration was determined with a hemocytometer. N. lugens stock cultures and bioassays were maintained on Taichung Native 1 rice in a regime of 16 hr of light at 28°C and 8 hr of dark at 24°C. RESULTS

The 24 wild-type isolates bioassayed against N. lugens gave LTsa mortality val-

TO N. lugens

‘7

ues (Table 2) ranging between 5 and > 14 days. Isolates with a LTS, >14 days were considered to be nonpathogenic. Control insects, inoculated with 0.05% Tween 80 alone, exhibited a LT,, > 14 days. Spore lengths (Fig. 1) varied between 6.7 and 14.2 pm, with three distinct groups: (I) 17 isolates, 6.7-8.2 pm (95% c.i. 6.52-8.69, pathogenic toward N. lugens); (2) 1 isolate, 10.3 km (95% c.i. 10.03-10.57, poorly pathogenic); and (3) 6 isolates, 12.5-14.2 km (95% c.i. 12.07-14.70, nonpathogenic). Nucleus diameter and spore volume readings also gave the same pattern of three groups (Fig. 1). On WA the T,, germination value varied between 11 and >24 hr at 28”C, while on SDA it decreased to between 7.25 and >24 hr (Fig. 1). Most isolates of var. anisopliue achieved maximum germination by 18-24 hr on SDA, whereas few isolates did so on WA. All var. mujus isolates failed to germinate by 24 hr on WA and germination on SDA was poor when compared to var. ar!isopliae isolates. In general, radial growth rate increased between 20” and 28”C, although three isolates exhibited a significant decrease between 25” and 28°C (isolates 401 and 404, P < 0.01, and 501, P < 0.001). Five isolates exhibited a significantly increased radial growth rate at 28°C compared with that at 25°C (P < 0.001) and these were the most pathogenic isolates tested. In addition, var. anisopliae isolates generally exhibited faster radial growth than var. mujus isolates at all temperatures investigated. Most isolates originally from N. lugens exhibited poor total spore production and the production of sterile over-growing mycelium preventing easy spore detachment. Extracellular amylase production (Table 2) varied between 1 (no detectable halo) and 0.54 (high production). The five most pathogenic isolates (Table 2) had an amylase rating of 1. However, all var. mujus isolates also had an amylase rating of 1 and were nonpathogenic (LTw > 14 days). Extracellular chitinase production (Table 2)

28

SAMUEL&

HEALE,

AND LLEWELLYN

TABLE BIOASSAY

Isolate number

(QEC) 410 413 415 416 414 401 412 403 402 411 431 420 417 427 433 404 501 502 503 504 505 506 507 508

RESULTS

2

AGAINST Nilaparvata lugens AND EXTRACELLULAR ENZYME WILD-TYPE ISOLATES OF Metarhizium anisopliae

LT,, days (SE) 5 6 6.5 7 7 8 9 9 9.5 10.5 10.5 10.5 11 11 11 12 12 13 >14 >14 >14 >14 >14 >14

(0.3) (0.4) (0.4) (0) (0.4) (0.8) (0.4) (0.8) (0.2) (0.4) (0.4) (0.6) (0.6) (0) (0.6) (1.2) (0.8) (0.4) (-) (-) (-) (-) (-) (-)

Extracellular Amylase 1 1 0.59 0.91 0.78 0.74 1 0.85 0.93 1 0.54 0.77 1 1 1

PRODUCTION

BY

24

enzyme

Chitinase

Lipase

0.92 0.94 0.95 0.93 0.95 0.89 0.94 0.93 0.95 0.93 0.93 0.94 0.94 0.95 0.91 0.93 0.95 0.95 0.95 0.94 0.93 0.95 0.96 0.96

0.77” 0.68 0.72 0.64 0.71 0.77” 0.70 0.97 0.73” 0.67” 0.86 0.67 0.57” 0.81 0.81’ 0.68 0.65 0.61 0.61 0.70 0.52 0.62 0.58

0.79” 0.77 0.70 0.68 0.75 0.6Sb 0.78” 0.98 0.71 0.72 0.77” 0.75” 0.76” 0.65 0.90 0.69 0.77 0.66 0.68 0.66 0.64 0.66 0.62

Note. Isolates are ranked according to LT,, values. LTSo values are the means for three replicates of 12 insects to the nearest half day, following treatment with 1 x lo6 spores/ml. Data for enzymes are the means for four replicates, expressed as colony diameter/halo diameter (Rosato et al., 1981). SE’s are all CO.01 unless indicated: == 002 ., b’O03., ‘=005. .

was detected in all isolates, but varied little. Extracellular lipase production (Table 2) varied between 1 and 0.52. One isolate (403) failed to produce a detectable halo and was moderately pathogenic (LT,, 9 days). Extracellular protease production (Table 2) varied between 1 and 0.54. One isolate (420) failed to produce a detectable halo and exhibited low pathogenicity (LT,, 10.5 days). Spearman’s Rank analyses resulted in significant correlations between the following traits and pathogenicity (LT,,): small spore volume (P = O.Ol), rapid T,, germination on both WA (P = 0.01) and SDA (P = 0.02), and rapid radial growth at 28°C (P = 0.04) (Fig. 1).

DISCUSSION All var. mujus isolates were nonpathogenie, probably due in part to the low viability of newly harvested spores (0% on WA and 21-52% on SDA, by 24 hr) and slow germination even on supplemented media, suggesting a requirement for specific nutrients for the initiation of germina. tion. This is supported by the finding thal var. mujus isolates exhibited an increase ir percentage germination on addition of col, loidal chitin to the culture media (Samuels 1986). The presence of undetermined nutri ents in or on the cuticle may explain the specificity toward Oryctes rhinoceros ex hibited by most var. mujus isolates.

PATHOGENICITY

14

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FIG. 1. Scatter diagrams of pathogenicity toward Nilaparvaru lugens (LT,,) and individual characteristics of 24 isolates of Metarhizium anisopliae. (A) Spore length, (B) nucleus diameter, (C) spore volume, (D) T,, germination value on WA and (E) SDA, and (F) radial growth rate at 28°C.

Germination occurred more rapidly on SDA than on WA. However, the germination and growth requirements of many M. anisopliue isolates do not appear to be complex; WA was sufficient for a high percentage germination and hyphal growth in a number of isolates (Samuels, 1986). Mutants of M. anisopliae exhibiting rapid germination were found to be hypervirulent toward Culex pipiens, when compared to the wild types (Al-Aidroos and Roberts, 1978). Hall (1984) also found that rapid germination (T,, < 9 hr) was a characteristic of Verticillium lecanii isolates pathogenic toward Macrosiphoniella

sanborni. M. anisopliae

may penetrate the insect cuticle only after appressorial formation (Zacharuk, 1973), a process that may extend the time required from spore attachment to entry into the hemocoel. As germination only occurs during periods of high humidity (>92.5%) (Walstad et al., 1970), which generally occurs only at night, and as spores may be

TO N. lugens

29

dislodged from the insect by moulting, preening, or other activity, rapid germination is an extremely important trait. Cuticular invasion is likely to involve both enzymatic and physical activities. Although no direct correlation was observed between enzyme production and pathogenicity, chitinase, lipase, and protease activity together with a lack of detectable amylase activity were features of all the highly pathogenic isolates. These isolates all originated from N. lugens. The inference is that strain adaptation to the original host is more important than nonspecific enzyme production. Total spore production by N. lugens isolates of M. anisopliae was particularly low and spore release was prevented due to the production of over-growing sterile mycelium. All N. lugens isolates exhibited this morphology, possibly explaining why M. anisopliae rarely causes epizootics in the field. Sterile over-growing mycelium is also implicated in the inability of some strains of V. lecanii to spread efficiently among aphids (Hall, 1984). M. anisopliue isolates with a spore volume in excess of 20 pm3 were either nonpathogenic or only poorly pathogenic toward N. lugens. Spore volume, by Coulter counter analysis, has been shown to be correlated with ploidy levels of Verticillium spp. (Typas and Heale, 1977). The possibility that var. mu&s isolates are naturally occurring diploids has been suggested (Tinline and Noviello, 1971) and observations during the course of this investigation support this view. Acridine orange staining revealed that the spore nucleus diameter measurements of the smallest spore group (with volumes 11.5-21.4 Fm3) were, on average, 1.4 times smaller than the average for the largest group (with volumes 53.257.8 km3). This represents slightly more than a doubling in nucleus volume. DAPI fluorescent staining (Hamada and Fujita, 1983) gave similar results (unpubl. data). The 2% survival level following ultraviolet irradiation, most commonly used in the iso-

30

SAMUELS,

HEALE,

lation of mutants, occurred between 60 and 80 set for var. anisopliae isolates (254 nm at 20 cm) while var. mujus isolates required over 120 sec. Isolate 506 required in excess of 140 sec. Isolate 502 exhibited a survival curve similar to var. anisopliae isolates. In a haploid cell, any auxotrophic mutation will be evident in the phenotype. In a diploid, any inactivated gene on one chromosome will invariably be complemented by an unaffected gene on its homolog. Only simultaneous mutations in the same locus of both homologs, or a single mutation that produces a dominant effect, will be expressed in diploids. Auxotrophic mutants were readily isolated from all var. anisopliae isolates investigated, following ultraviolet irradiation. However, no auxotrophic mutants of var. mujus isolates were isolated and there are no reports of them in the literature. Thus the increased resistance to ultraviolet light and the failure to isolate auxotrophic mutants, together with spore and nucleus volume measurements, provides evidence for the stable diploid nature of var. mujus isolates. The results presented here indicate that there are a number of characteristics which contribute significantly to the expression of pathogenicity in M. anisopliue isolates toward N. lugens, although the present wildtype isolates appear to offer little hope as potential control agents, particularly due to the poor epizootic potential of the most pathogenic isolates. However, the variation between isolates is such that a number of desirable traits are present, albeit individually, or may be isolated or enhanced following mutagenesis. Strain improvement may select strains of M. anisopliue suitable for use as control agents against N. lugens. Attempts to select pathogenic recombinants with enhanced epizootic potential, by employing parasexual genetics, have been carried out successfully and will be reported later. ACKNOWLEDGMENTS The authors thank the Science and Engineering Re-

AND

LLEWELLYN

search Council for the Quota Award which supported this investigation. We gratefully acknowledge the United States Department of Agriculture and the Institute for Horticultural Research, Littlehampton, for provision of fungal cultures; The University of London Botanical Gardens for their help in providing rice plants; and University College, CardiIf, for the original N. lugens stock. We also acknowledge the advice and encouragement of Dr. S. Lisansky and Dr. R. Quinlan.

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PATHOGENICITY

OF

HEALE, J. B. 1988. The potential impact of fungal genetics and molecular biology on biological control, with particular reference to entomopathogens. In “Fungi in Biological Control Systems” (M. N. Burge, Ed.), Manchester Univ. Press, in press. JACKSON, C. W., AND HEALE, J. B. 1987. Parasexual crosses by hyphal anastomosis and protoplast fusion in the entomopathogen Verticillium lecanii. .I. Gen. Microbial., 133, 3537-3547. JACKSON, C. W., HEALE, J. B., AND HALL, R. A. 1985. Traits associated with virulence to the aphid Macrosiphonielfa sanborni in eighteen isolates of Verticillium lecanii. Ann. Appl. Biol., 106, 39-48. KHUSH, G. S., PATHAK, M. D., AND SIDHU, G. S. 1977. Breeding for and genetics of resistance. In “The Brown Planthopper: Symposium of the International Rice Research Institute.” Los Banos. PATHAK, M. D., AND KHUSH, G. S. 1977. Studies on varietal resistance to the brown planthopper at IRRI. In “The Brown Planthopper: Symposium of the International Rice Research Institute.” Los Banos. ROSATO, Y. B., MESSIAS, C. L., AND AZEVEDO. J. L. 1981. Production of extracellular enzymes by isolates of Metarhizium anisopliae. J. Invertebr. Pathol., 38, l-3. SAMUELS, K. D. Z. 1986. “Genetical Studies and Strain Selection in Metarhizium anisopliae

M.

anisopliae

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TO N. lugens

(Metschnikoff) Sorokin for the Control of Nilaprtrvata lugens (Stal), the Brown Planthopper of Rice ” Ph.D. thesis, University of London. TINLINE, R. D., AND NOVIELLO, C. 1971. Heterokaryosis in the entomogenous fungus Metarrhizium anisopliae. Mycologia, 63, 701-712. TULLOCH, M. 1976. The genus Metarhizium. Trans. Brit. Mycol. Sot., 66, 407-411. TYPAS, M. A.. AND HEALE, J. B. 1976. Heterokaryosis and the role of cytoplasmic inheritance in dark resting structure formation in Verticillium spp. Mol. Gen. Genet., 146, 17-26. TYPAS, M. A., AND HEALE, J. B. 1977. Analysis of ploidy levels in strains of Verticillium using a coulter counter. J. Gen. Microbial., 101, 177-180. WALSTAD, J. D., ANDERSON, R. F., AND STAMBAUGH, W. J. 1970. Effects of environmental conditions on two species of muscardine fungus (Beauveria bassiana and Metarrhizium anisopliae). J. II!vertebr. Pathol., 16, 221-226. WILSON, M. R., AND CLARIDGE, M. F. 1985. The leafhopper and planthopper faunas of rice fields. In “The Leafhoppers and Planthoppers” (L. R. Nault and J. G. Rodriguez, Eds.). Wiley, New York. ZACHARUK, R. Y. 1973. Electron-microscope studies of the histopathology of fungal infection by Metarrhizium Amer.,

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