Microbial control of coconut leaf beetle (Brontispa longissima) with green muscardine fungus, Metarhizium anisopliae var. anisopliae

JOURNAL

OF INVERTEBRATE

53, 307-314 (1989)

PATHOLOGY

Microbial Control of Coconut Leaf Beetle (Brontispa longissima) with Green Muscardine Fungus, Metarhizium anisopliae var. anisopliae s. D. LIU AND s. c. LIN Department

of Plant

Protection,

National

Pingtung Republic

Institute

of Agriculture,

Pingtung,

Taiwan

91207.

of China

AND

J. F. SHIAU Division

of Plant

Protection,

Department

of Agriculture and Forestry, Republic of China

Taiwan

Provincial

Government,

Received January 27, 1988; accepted August 1, 1988 A domestic strain of Metarhizium anz’sopliae var. anisopliae (MA-l) was isolated from infected coconut leaf beetles (CLB), Brontispa longissima. Mycelial growth of this green muscardine fungus was better in Sabouraud dextrose broth (SDB) or SDB supplemented with yeast extract than in Campbell’s and other media. In both liquid and solid phase, V-8 media effectively promoted sporulation of MA-l. The optimal temperatures and relative humidities for conidia germination were 24”-30°C and IOO-92.5% RH, respectively. In a pathogenicity test, the mortalities of larvae. pupae, and adults of CLB were 100% when they were inoculated with MA-l conidia suspension at a density of 2.15 x 10’ conidia!ml. Even at a density of 2.17 x lo3 conidialml, the mortality of larvae still reached 47% and pupae still reached 60%. Strain MA-l had a higher virulence on CLB than another isolate of M. anisopliae and Beauveria bassiana. Two field trials of microbial control of CLB were conducted in the Pingtung area (southern part of Taiwan) in 1986 and 1987. CLB could not be detected after three applications of MA-I formulated as a homogeneous biomass, in granules or in a conidial suspension. 0 1989 Academic PXSS. IX. KEY WORDS: entomopathogenic fungi; pathogenecity; biological control; coconut pest,

INTRODUCTION Coconut palm (Cocos nucifera) is one of the most economically important tropical trees. Over 600,000 are planted in southern Taiwan. Coconut leaf beetle (CLB), Brontispa longissima (Chrysomelidae), was first found to be a coconut pest in Taiwan in July 1975 and since then has spread widely and rapidly, causing severe damages and losses to coconut trees (Chen, 1976). A pupal parasitoid, Tetrastichus brontispae, was successfully introduced and liberated for biological control of CLB in Taiwan (Chiu et al., 1985). Use of microbial agents such as baculovirus and green muscardine fungus, Metarhizium anisopliae, for controlling coconut palm rhinoceros beetle and other pests of coconut palm has been observed (Ferron et al., 1975: Prior, 1985; Young,

1974), but no microbial control of CLB has yet been reported. This study was undertaken to investigate the infectivity of entomopathogenic fungi to CLB and to study methods for formulation of these agents for application in the field. MATERIALS

AND METHODS

Isolation and collection of entomogenous fungi. Diseased larvae and adults of CLB were collected from coconut palm trees in the Pingtung area. Potential pathogenic fungi were isolated by use Sabouraud-4% dextrose agar (SDA) (E. Merck, Cat. No. 5438, Federal Republic of Germany) medium. A fungus producing a green colony appeared most frequently and was cultured in slant and identified as Metarhizium anisopliae var. anisopliae 307 0022-2011189 $1.50 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reerved.

308

LIU,

LIN,

(MA-l) according to the system of Tulloch (1976). The conidia size averaged about 6.47 x 2.5 pm. Isolates of M. anisopliae (MA-2) and Beauveria bassiana were obtained from Dr. C. W. Kao, Specialist, Council of Agriculture. Preparation of test media and culture, harvesting techniques. There were six liquid media tested for mycelial growth of M. anisopliae, i.e., Sabouraud dextrose broth (SDB, 5 g peptone from meat, 5 g peptone from casein, 40 g D-( +)-glucose, 1 liter distilled water), SDB supplemented with 5 g yeast extract (SDYB), modified Sabouraud dextrose broth (MSDYB, 10 g neopeptone, 5 g yeast extract, 40 g D-(+)-glucose, 20 g agar, and 1 liter distilled water), V-8 medium (200 ml V-8 juice, 3 g CaCO,, distilled water made to 1 liter), Campbell’s medium with 20 g D-mannose (CMB) or with 20 g i-inositol (CIB) (Campbell et al., 1983). A 7-day-old mycelia disk (diameter 5 mm) of MA-l or MA-2 was added to a 250-ml Erlenmeyer flask containing 100 ml of test media and incubated at 28°C for 18 days. The mycelia mats were harvested by vacuum filtration of culture broth onto two layers of predried and weighed filter papers (Whatman No. 1). All mycelia were washed with 250 ml of distilled water, and papers and mycelia were then dried for 24 hr at 100°C and weighed. Each treatment had three replicates with 10 flasks in each replicate and mean separations were done by Duncan’s multiple range test. Optimum temperature and relative humidity for conidia germination. The conidia of MA-l and MA-2 were harvested from 21-day-old SDA culture plates by pouring sterilized water onto the surface of colonies and stirring with a microspatula. The spore suspension was counted on a hemocytometer and adjusted to a concentration of lo5 conidia/ml (0.2 ml of sterilized Tween 20 added to 100 ml of spore suspension). A drop of spore suspension was placed on sterilized slides in sealed Petrie dishes (RH 100%) and incubated at varying temperatures from 16” to 30°C. After 24- and 36-hr

AND

SHIAU

incubation periods, the conidia were examined under a light microscope for germination. The relative humidities of various saturated salt solutions (K,Cr,O,, &SO,, KNO,, BaCI,, KCl, NaCl, Mg(NO,),) ranged from 98.0 to 53.4% at 25°C and distilled water served as a RH 100% treatment. Two SDA disks (diameter 10 mm) were placed on slides and spore suspensions were applied to the disks and kept in sealed Petrie dishes filled with various saturated solutions. Conidia germination was examined after a 48-hr incubation. Pathogenicity of entomopathologenic fungi to CLB. CLB larvae were collected from infested coconut palm and reared artificially on detached young leaves in Petrie dishes (diameter 15 cm) covered with nylon net for offspring generation. The larvae, pupae, and adults were used for pathogencity tests. These were sterilized with 1% NaOCl for 2 min and washed with sterilized water. The larvae, pupae, or adults were dipped into conidial suspension at a concentration of 2.17 x 107, 2.17 X 105, or 2.17 x lo3 conidia/ml for 1 min, transferred to Petrie dishes, and fed aseptically for 8 days. The dead CLB were counted and then placed in moist Petrie dishes for observation of pathogen sporulation to determine whether the death of CLB was due to infection of pathogen. The mortality of insects was calculated by the percentage of infected dead CLB. Each treatment had three replicates and each replicate had 50 insects kept in five separate Petrie dishes. Histological study of infective insects. For electron microscopic observation, 10 infective and control larvae and adults were immobilized at 5°C. Small pieces of insect tissues were fixed in 5% glutaraldehyde in 0. 1 M phosphate buffer, pH 7.0, at 4°C for 2 hr. They were rinsed and posttixed in 1% osmium tetraoxide at 4°C for 2 hr. After tixation, the tissue pieces were dehydrated in 2,2-dimethoxypropane, treated with propylene oxide, and embedded in LX 112 epoxy resin. Ultrathin sections were cut with a glass knife fixed on Reichert-Jung’s Ul-

MICROBIAL

CONTROL

tracut E ultramicrotome. The thin sections were double-stained with m-any1 acetate and lead tartrate and examined under a JEM-200 CX electron microscope. Field trials of CLB control. Forty severely infested coconut palm trees near an aquaculture pond in Chao-Chou, Pingtung, were selected for biocontrol treatments. The homogeneous biomass of MA-1 was harvested from 14-day SDB culture and homogenized with a Waring blender, adjusted to a concentration of lo7 CFU/ml, and sprayed at l-month intervals from February 1986. The second trial was conducted in Lin-Ben, Pingtung, from March 1987. One hundred twenty trees were divided into three treatments, i.e., a sprayed conidia suspension (lo7 conidiaknl), a granule application, and a control. The granules were made from rice grain media in polypropylene bags cultured with MA-l for 10 days and conidia densities ranged from 2 to 6 x 10’ conidia/g. Each tree received 100-120 g of granules depending on the size of its crown. The insect population of each tree was calculated by counting the number of insects from half the young leaves and multiplying by 2. RESULTS Mycelial growth and sporulation of pathogen. The mycelial growth of MA-l

was greater in SDB or SDYB than in the other media. The dry weight of MA-l mycelia harvested from SDYB after 14 days of incubation at 28°C reached 1400 mg compared to 310 mg in that harvested from CIB. The mycelial growth of MA-2 was statistically less than that of MA-1 in each medium (Fig. 1). The sporulation of MA-l was enhanced in V-8 (7.6 x 10’ conidialg concentration compared with 2.4 x IO*, 7.3 x 108, 5.6 x lo’, 6.4 x 107, 4.8 x lo6 conidia/g in SDB, SDYB, MSDYB, CIB, and CMB, respectively). The sporulation of MA-2 in tested media also was similar. Effect of temperature and relative humidity on conidial germination. Only 10%

of the conidia of MA-l

germinated

at 20°C

309

OF B. longissima

SD6

V-6

Cl6

0

2

4 MYCELIA

6

6

16

DRY

WT

llo6mal

12

14

IS

FIG. 1. The effect of various media on mycelial growth of two strains (MA-l and MA-Z) of Metarhizium nnisopfiue var. anisopliae. The fungi were cultured on Sabouraud dextrose broth (SDB), SDB supplemented with yeast extract (SDYB), modified Sabouraud dextrose broth (MSDYB), V-8, Campbell’s media with o-mannose (CMB), or with i-inositol (CIB) at 28°C for 18 days. The horizontal columns of mycelia dry weight with diierent letters are significantly different according to Duncan’s multiple range test (P = 0.05).

after 24 hr of incubation; this increased to 72% at 24”C, but conidial germination of MA-2 was still below 10% at 16” to 30°C. After 36 hr of incubation, conidia germination of MA-l reached 70% at 20°C and 93% at 24°C. The conidia of MA-2 germinated much slower and were significantly less compared with those of MA-1 (Fig. 2). The relative humidity was an important factor greatly affecting conidial germination. The conidia germination of MA-1 was 100, 98, and 96% at RH 100, 98, and 97.5%, respectively. Decreased germination was ob-

MA-2

16

20 TEMPERATURE

24

28

30

,‘Cl

FIG. 2. The effect of temperature and incubation time on conidial germination of two strains (MA- I and MA-2) of Meiarhizium anisopliae var. anisopliac.

310

LIU, LIN, AND SHIAU TABLE

1

centrations decreased to lo3 conidia/ml but still induced 47% mortality of larvae and 60% mortality of pupae. Electron microscopy. Numerous cavities were found in fourth instar CLB larvae and caused disintegration of tissue (Fig. 3A). Fungal mycelia were found in varying amounts in tissues of adult CLB (Figs. 3B and C). Field experiments. In a small-scale trial the first year (1986), with three sprays of the biomass of MA-l on 20 severely infested coconut trees with an average of 150 larvae, 3.5 pupae, and 49 adults of CLB per tree, the insect population was significantly lower than that with 20 trees of the control (Fig. 4). In a second trial the microbial agents were applied through liquid spraying of conidia suspension and solid granule treatment to 120 coconut trees; the population density of CLB decreased significantly compared with control. In the granule treatment, the density of CLB was significantly lower than that in the spraying treatment (Table 3). The new leaves grew without symptoms after treatment, in contrast to the necrotic leaves of untreated trees.

THE EFFECT OF RELATIVE HUMIDITY ON SPORE GERMINATION OF Metarhizium anisopliae VAR. anisopliae

Saturated solution

Relative humidity (%)

MgW’,), NaCl KC1 BaCl, KNO, KS’, KG-D, Dist. H,O

53.4 75.5 85 90.2 92.5 97.5 98 100

Spore germination (%) 0’ 0’ 24.Sd 61.1’ 80.4’ 96” 98.1” 99.5”

Note. The percentages of spore germination with different letters are significantly different according to Duncan’s multiple range test (I’ = 0.05).

served when relative 90.2% (Table 1).

humidity

was below

Virulence of pathogens. Two isolates of M. anisopliae var. anisopliae, MA-1 and MA-2, and one isolate of B. bassiana were

cultured on SDA plates for 14 days and conidial suspensions were harvested and adjusted to varying concentrations to test for virulence. At a density of 2.17 x lo7 conidis/ml of MA- 1, there was 100% mortality of CLB larvae, pupae, and adults (Table 2). MA-2 and B. bassiana induced lower mortality to pupae and adults. The pathogenicity of MA-1 decreased when conidial con-

DISCUSSION The entomogenous fungi M. anisopliae and B. bassiana infect a wide variety of ag-

TABLE THE VIRULENCE

OF Two

STRAINS OF Metarhizium AGAINST COCONUT LEAF

2

atzisopliae VAR. anisopliae BEETLE, Brontispa longissima

AND Beauveria

bassiana

Mortality (%) Larvae Pathogen M. anisopliae

MA-l MA-2 B. bassiana

Pupae

Adults

10’

105

103

0

10’

10’

lo3

0

107

IO5

lo3

0

100” 97” 91”

97” 76’ 47’

47” 23’ 37’

0 0 0

100” 60b 35’

40” 29’ 20’

60” lSb 20’

0 0 0

100” 52’ 46b

17’ 13b 27”

6” 13” 10”

0 0 0

var. anisopliae

Note. The percentages of mortality in each vertical column with different letters are significantly different according to Duncan’s multiple range test (P = 0.05). The conidia of entomopathogenic fungi were harvested from Sabouraud dextrose agar plates incubated for 14 days and 28°C and adjusted conidia suspensions to three concentrations (2.17 X IO’, X lo’, x IO3 conidia/ml) with 0.1% (v/v) Tween 20. Each treatment had three replicates. Each replicate had 50 insects in five Petrie dishes fed with young coconut leaves. After 8 days, the infected and dead insects were counted and mortality of insects was determined.

MICROBIAL

CONTROL

OF B. longissima

FIG. 3. Transmission electron microscopy of ultrathin sections of coconut leaf beetle (Brontispa anisopliae var. anisopliae (MA-l). (A) Many empty longissima) larva infected with Metarhizium cavities were found in fourth instar larva tissue and disintegration was observed. (B and C) Fungal mycelia (FM) with cell walls (CW), mitochondria (M), and nuclei (N) were observed within adult tissue: muscle fibrils (MF) in cross section were found. The black bar in each figure measures 2 pm.

31

312

LIU, LIN, AND SHIAU

FIG. 3-Continued.

ricultural pests (Burgess and Hussey, 1971; Fen-on, 1978, 1981). To the author’s knowledge, this is the first study in which the biocontrol effect of these fungi on the coconut leaf beetle was assessed. The success of entomopathogenic fungi as microbial control agents is largely dependent upon

1966

FEB. 26 t)lp,q

MARCH 7 14 fSp,O”

APNIL 11 3 &I,.”

MAY 1

N JUNE 6

FIG. 4. The effect of Metarhizium anisopliae var. anisopliae (MA-l) on control of the coconut leaf beetle, Brontispa longissima. The biomass of MA-l at 10’ CFU/ml concentration with 0.1% (v/v) Tween 20 suspensions were sprayed on February 25, March 14, and April 11, 1986, respectively. The insect population of the spray treatment is shown as the solid lines and the control as the broken lines.

the use of highly virulent strains (Daoust and Roberts, 1982). Therefore, screening and selection of the pathogen to infect CLB were the focus of this study. MA-1 was most virulent against CLB (Table 2). The utilizaiton of nutrition may influence virulence and host range of entomopathogen (Campbell, 1978). As demonstrated in Figs. 1 and 2, MA-1 grew better than MA-2 and germinated over broader ranges of temperature and this appeared to be correlated with its greater virulence on CLB. But additional research with different agricultural pests as target host will be necessary to determine the host range of MA- 1. Epizootics are influenced by environmental conditions. Relative humidity appeared to be a limiting factor for infection of insect due to the low percentage of conidial germination below RH 90% (Table 1). However, the microenvironment associated with the infestation site of CLB in coconut palm leaves evidently provided sufftcient moisture during the process of infection in these tests for biocontrol. The ecological niche associated with CLB in heart leaves of the coconut

MICROBIAL

THE BIOCONTROL EFFECT OF Meturhizium Population densities before treatment per tree Treatment Spray Granule Control

Larvae

Pupae

Adults

170.0 130.0 62.7

12.1 11.5 2.7

36.3 40.3 23.8

CONTROL

TABLE 3 VAR. anisopliae

anisopliae Brontispa

311

OF B. longissimu

(MA-11 ON COCONUT LEAF BEETLE,

longissima

Population densities after treatment per tree __,-~~ Larvae Pupae Adults

~~~-. Larvae

0.9 0.0 9.1

-80.9' - 92.3” + 10.0’

32.4 10.6 69.0

15.1 10.1 30.9

Population change (5%) Pupae

Adult\

-92.Y - 100.0’ +334.4>

-58.4' .- 74.9,” + 30.0”

Note. Conidia suspension with Tween 20 (10’ conidia/ml) were sprayed in spraying treatment. Granule application was 100-120 g per tree MA-l formulated from rice grain medium. Each treatment was applied three times at l-month intervals. Each treatment had four replicates, each replicate had IO coconut trees. Population densities with different letters were significantly different according to Duncan’s multiple range test (P = 0.0s).

crown favors development of epizootic mycosis. In addition, heavy rainfall was present in the test area. Therefore, water, either liquid or vapor, is adequately provided by nature in this biocontrol system. Also, granule application had the same or even better effectiveness in biocontrol as spraying of conidial suspensions (Table 3). In the granule applications, a high density of conidia (approximately IO” conidia per tree) was applied compared with the spray treatments (approximately 10” conidia per tree). The abundant conidia produced from infected insects could serve as a secondary inocula dispersion accomplished by rain and wind. The high potential for inocula dispersal may play an important role in the propagation of the disease. It has already been established by many authors that there is a positive correlation between the number of infective spores and the mortality by mycosis (Ferron, 1978). Therefore, the application of this microbial control agent can be considered successful. Nevertheless, the influence of environmental conditions on the survival of the microbial control agent, the formulation, and the delivery system requires future study to establish a long-term application of this system. ACKNOWLEDGMENTS This research was supported by a grant from the Council of Agriculture, Executive Yuan, Republic of

China. We thank Dr. M. J. Chen for his assistance in electron microscopy and Dr. C. W. Kao for providing the isolates of Metarhizium anisopliue (MA-21 and Beauveria hassiana. The skillful technical assistance of Mr. M. C. Leu and S. T. Liu is greatly appreciated. The author is deeply indebted to Dr. R. Baker for his invaluable assistance in the preparation of the manuscript.

REFERENCES BURGESS, H. D., AND HUSSEY, N. W. 1971. “Microbial Control of Insects and Mites.” Academic Press, New York/London. CAMPBELL, R. K.. BARNES, G. L.. CARTWRIGHT, B. 0.. AND EIKENBARY, R. D. 1983. Growth and sporulation of Beauveria bassiana and Metarhizium anisopliae in a basal medium containing various carbohydrate source. J. Znvertebr. Pathol.. 41, 117121. CHEN, Z. C. 1976. The new pests of coconut palm in Taiwan. Sci. Agric.. 24, 481-485. CHIU, S. C.. LAI, P. Y.. CHEN, B. H.. CHEN, Z. C.. AND SHIAU. J. F. 1985. Introduction, propagation and liberation of a pupal parasitoid, Tetrastichus brontispae, for the control of the coconut leaf beetle in Taiwan. J. Agric. RPS. China, 34(2), 213-22’ DAOUST. R. A., AND ROBERTS, D. W. 1982. Virulence of natural and insect-passaged strains of Mrttrrhizium anisopliae to mosquito larvae. J. Invrrldw. Pathol.. 40, 107-117. FERRON. P. 1978. Biological control of insect pests hy entomogenous fungi. Annu. Rev. Entomoi.. 23, 40% 442. FERRON, P. 1981. Pest control by the fungi Beawericc and Metarhizium. In “Microbial Control of Pest and Plant Diseases, 1970-1980” (H. D. Burgess. Ed.). pp. 4655482. Academic Press, New York.

314 FERRON,

LIU, LIN, P., ROBERT,

P. H., AND DEOTTE,

A. 1975.

Susceptibility of Oryctes rhinoceros adults to Metarrhizium anisopliae. J. Invertebr. Pathol., 25, 313-319. PRIOR, C. 1985. The infectivity of Metarhizium anisopliae to two insect pests of coconuts. J. Znvertebr. Pathol., 45, 187-194. TEDDERS, W. L. 1981. In vitro inhibition of the ento-

AND SHIAU mopathogenic fungi Beauveria bassiana and Metarhizium anisopliae by six funngicides used in pecan culture. Environ. Entomol., 10, 346-349. TULLOCH, M. 1976. The genus Metarhizium. Trans. Brit. Mycol. Sot., 66, 407-411. YOUNG, E. C. 1974. The epizootiology of two pathogens of the coconut palm rhinoceros beetle. J. Invertebr. Pathol., 24, 82-92.