Laboratory Evaluation of Beauveria bassiana as a Pathogen of the Larval Stage of the Large Elm Bark Beetle, Scolytus scolytus

JOURNAL OF INVERTEBRATE PATHOLOGY

29, 361-366 (1977)

Laboratory Evaluation of Beauveria bassiana as a Pathogen of the Larval Stage of the Large Elm Bark Beetle, Scolytus scolytus G. BARSON 1 Natural Environment Research Council, Unit of Invertebrate Virology, 5 South Parks Road, Oxford OXI 3RB, England Received May 21, 1976 8eauveria bassiana was occasionally isolated in culture from the body surfaces of apparently healthy adults of Scolytus multistriatus collected from the field in one locality in Worcestershire, Great Britain. The mortality of S. scolytus larvae exposed to a series of spore dilutions, from 4.8 x 107 to 4.8 x 104/ml, decreased with decreasing amounts of inoculum. At spore concentrations of 4.8 x 104/ml, the mortality of the treated larvae was significantly greater than that of the uninoculated controls. The LDs0 after 5 days of exposure at 23°C and 100% RH was calculated as 1.0 x 106 spores/ml. At 100% RH, all larvae treated with 2.8 x 107 spores/ml were killed over a temperature range of 5-30°C (with 5°C intervals); 98% were killed at 25°C. The mortality rate was slowest at 5°C (LTs0 = 45 days) and quickest at 25°C (LTs0 = 6 days).

INTRODUCTION Beauveria bassiana was first recognized as a disease-causing organism by Agostino Bassi in 1835 and has been used as a biological control agent for about 85 years. It is a nonspecific entomogenous fungus with a broad geographical range (Pascalet, 1939; Charles, 1941; Tanada, 1953; Leatherdale, 1970; Kenneth et al., 1971). It has not previously been recorded in Britain as a pathogen of elm bark beetles, but, in the United States, Charles (1941), Mook and Wolfenbarger (1943), and Doane (1959) frequently recorded the fungus infecting larvae and adults in field populations of the small elm bark beetle, Scolytus multistriatus, one of the vectors of Dutch elm disease in Britain and the United States. B. bassiana has also been recorded in Poland (Balazy, 1965), infecting both S. multistriatus and S. scolytus, the other vector of Dutch elm disease in Britain. During a survey for potential insect 1 Present address: Pest Infestation Control Laboratory, Ministry of Agriculture, Fisheries and Food, Insecticides and Storage Department, London Road, Slough, Berkshire, England.

pathogens in field populations of S. multistriatus and S. scolytus, conducted in the south and west of England from 1972 to 1974 (Barson, 1976), no sign of infection of larvae ( > second instar) or adults by B. bassianna was found, but the fungus was isolated in culture from the body surface of several apparently healthy S. multistriatus adults from Madresfield, Worcestershire, during the summer of 1973. The laboratory investigations reported here were to determine (1) the pathogenicity of the Madresfield strain ofB. bassiana for healthy final instar larvae ofS. scolytus, and (2) the rate of infection at various temperatures, at 100% RH.

MATERIALS AND METHODS The techniques used for laboratory assays were modifications of those of Pesson et al. (1955) and Doane (1959) and are given in detail by Barson (1976). Pathogenicity. B. bassiana was grown on Oxoid nutrient agar, p H 7.4, in the dark at 23°C. Spores of B. bassiana washed under sterile conditions from cultures were suspended in 25 ml of sterile water to 361

Copyright © 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.

ISSN 0022-2011

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standard inoculation technique described before. Sixty larvae were incubated on inoculated (2.8 x 107 spores/ml) media at each temperature (10/dish), and forty uninoculated larvae were incubated at each temperature as controls. The dishes were incubated in the dark, and assessments of mortality were made daily.

3

RESULTS

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o

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'Z I I

LDso

I

4.8X10 4

1.Ox

106

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4.8X I0 6

4.8xi0 5

4.8X10 7

Log. Dose

FIG. 1. Mortality of Scolytus scolytus larvae after exposure to a logarithmic series of spore dilutions of Beauveria bassiana for 5 days. LDs0 = 1.0 × 106 spores/ml.

which was added one drop of Tween 20 to act as a wetting agent for the dry spores. Petri dishes (9 cm in diameter), each containing a sterilized mixture of 10g of pulverized elm bark and 15 ml of sterile water, were used in the tests. There were four treatments, in a logarithmic series of spore dilutions from 4.8 × 107 to 4.8 × 104/ml. Forty final instar larvae were used at each spore dilution (10/dish). Four milliliters of spore inoculum were evenly applied to the surface of each treatment dish, and four control dishes were treated with 4 ml of sterile water mixed with wetting agent. Ten final instar larvae of S. scolytus were then placed in circular depressions in each dish. The dishes were incubated in the dark at 23°C, at approximately 100% RH. Assessments of mortality were made daily. Spores were taken from cultures about 6 weeks old. Temperature assay. The rate of infection of overwintering final instar larvae of S. scolytus by B. bassiana was tested at 5°, l0 °, 15°, 20°, 25°, and 30°C using the

The mortality rate of S. scolytus final instar larvae decreased with decreasing amounts of spore inoculum (Table 1A, Fig. 1). Levels of statistical significance between mortalities at each spore dilution after 5 days of exposure are given in Table lB. The LDs0 after 5 days of exposure was calculated to be 1.0 × l0 s spores/ml (Fig. 1). After 7 days of exposure at 4.8 x 107 to 4.8 x 105 spores/ml (Table 1A), all the larvae had died; these mortalities were significantly greater than control mortality (P < 0.001). All larvae at the lowest spore concentration of 4.8 x l& were killed after 7

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5

10

15

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25

30

21.6 Temperature (°C)

FIG. 2. LT~ [(days)i] to kill Scolytus scolytus larvae exposed to spore concentrations (2.8 x 10Vml) of Beauveria bassiana at different temperatures. The optimum temperature required for B. bassiana to kill its host was interpolated at 21.6°C.

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B. B A S S I A N A AS A P A T H O G E N

TABLE 1 (m) MORTALITY (%) OF SCOLYTUS SCOLYTUS FINAL INSTAR LARVAE EXPOSED TO A LOGARITHMIC SERIES OF SPORE CONCENTRATIONS OF BEAUVERIA BASSIANA D a y s after incubation Spore concentration

3

4

5

7

11

4.8 x 107 4.8 x 106 4.8 x 104 4.8 x 104 Control

2.5 7.5 2.5 5.0 2.5

27.5 32.5 15.0 7.5 2.5

85.0 67.5 52.5 15.0 2.5

100 100 100 67.5 5.0

100 35.0

(B) STATISTICAL SIGNIFICANCE BETWEEN THE LARVAL MORTALITIES AT EACH SPORE CONCENTRATION AFTER 5 DAYS OF EXPOSURE TO BEAUVERIA BASSIANA a

P values Spore concentration

4.8 x 10r

4.8 x 106

4.8 x 105

4.8 x 104

4.8 x 10r 4.8 x 106 4.8 x 104 4.8 x 104 Control

NS P < 0.01 P < 0.001 P < 0.001

-NS P < 0.001 P < 0.001

-P < 0.01 P < 0.001

NS

Control

a Calculated from 2 x 2 Xz c o n t i n g e n c y tables.

11 days; control mortality at this time was significantly lower than treatment mortality (P < 0.001).

Temperature Assay B. bassiana killed 100% of the larvae in all but the 25°C treatment (Table 2).

At 15°C, all larvae were killed by the twelfth day, but the optimum temperature for 50% kill interpolated from Figure 2 was 21.6°C, although the LTg0 was shortest at 25°C. The mortality rate was slowest at 5°C, all the larvae being killed in 56 days. At this temperature, no larvae were killed during the first 38 days of exposure,

TABLE 2 INTERVALS AFTER INOCULATION WITH BEAUVERIA BASSIANA (2.8 X 107 SPOREs/ml) AT SIX TEMPERATURES WHEN MORTALITY OF SCOLYTUS SCOLYTUS FINAL INSTAR LARVAE REACHED 50, 90, 95, AND 100%

M e a n n u m b e r of days to Temperature (°C)

50% Mortality

90% Mortality

5 10 15 20 25 30

45.10 14.00 7.50 7.00 6.13 8.44

52.90 18.15 8.50 8.50 8.00 23.50

95% Mortality

D a y s to 100% mortality

Control a

54.00 18.80 8.90 9.00 10.00 25.40

56 21 12 13 -29

5.0 0.0 2.5 10.0 5.(P 85.0

a Percentage of mortality w h e n treated = 100%. b Control mortality was 5% w h e n mortality of treated larvae had reached 95%.

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but, once the first larvae had been killed, it only required a further 18 days for larval mortality to reach 100%. At 30°C, all the larvae were killed after 29 days, but there was also a high control mortality though this was significantly lower than treatment mortality (P <0.01). Disease symptoms in both treatment and uninoculated controls at this temperature suggest death by bacteriosis; only a few treated larvae displayed symptoms of Beauveria infection. A random sample of 10 larvae from each treatment and from the uninoculated controls was tested for B. bassiana infection after each test. B. bassiana was isolated in culture from all treated larvae except those exposed at 30°C, where the fungus was isolated twice. B. bassiana was not isolated from any control larvae. Levels of mortality between treatments and controls in the pathogenicity tests were compared using X2 contingency tables. The probit line (Fig. 1) expresses the mortality of S. scolytus related to the log of the dosage of B. bassiana spores. The slope of this line is b =0.667 and the SE is b = 0.1091. In Figure 2, the regression line describing the death rate [LTs0, expressed as (Days)t] related to different temperatures is expressed as a quadratic relationship, where (days)½ to 50% kill = 9 . 1 2 - 0.625 (°C) + 0.0143 (°C) 2. This equation accounted for 93% of the total variation in the root number of days.

infected larvae became opaque creamywhite in color, unlike their usual translucent appearance. About 24 hr after death at 23°C and 100% RH, their color changed to pale pink, and the hemocoel was partially filled with fungal mycelium, causing the larvae to become turgid. After a further 48 hr, aerial mycelium appeared and gradually covered the whole body surface. Sporulation on the surface of the dead larvae occurred from the sixth day after death.

DISCUSSION

B. bassiana was highly pathogenic to final instar larvae of S. scolytus in laboratory assays, but was not isolated in culture from diseased larvae found in field populations sampled from 1972 to 1974. The larval mortality in these samples (estimated from third to fifth instar) attributed to pathogens appeared to contribute very little to the total population mortality (Barson, 1976). Beaver (1966) demonstrated that natural regulation of S. scolytus populations is due mainly to parasites and subcortical predators. Although it appears that entomogenous fungi contribute very little to the population mortality of S. scolytus in the field, the microclimate, particularly during the summer months inside the bark of recently dead trees, would seem to be an ideal habitat for the germination and development of the fungus (Barson, 1976). However, varying conditions of temperature and humidity can determine the success or Disease Symptoms failure of a pathogenic fungus. Doane There is much experimental evidence, (1959) has shown that mortality of S. multiparticularly that presented by Palllot (1930) striatus larvae in a natural epizootic caused and Schaerffenberg (1957), which shows by B. bassiana reached, on the average, that B. bassiana, like many other ento- 97% in trees in the shade, whereas only mogenous fungi, kills its insect host by the 4% were killed in trees in the open. action of hyphae that germinate from Direct sunlight on the bark of trees in the spores, penetrate the exocuticle, and subse- open reduced phloem moisture and inquently invade and destroy the internal creased bark temperature and thus, pertissues. Larvae ofS. scolytus were probably haps, minimized the action of Beauveria. infected the same way. At the time of death, Some workers (Hart and MacLeod, 1955;

B. B A S S I A N A AS A PATHOGEN

Walstad et al., 1970) have shown that very few spores of B. bassiana germinate at humidities below 94% RH. Dunn and Mechalas (1963) claim that high mortality of Lygus hesperus occurred at RH as low as 40-50%, although at high humidities B. bassiana kills its host more rapidly. In my experiments with B. bassiana and S. scolytus larvae, the humidity was maintained at 100%, as the moisture content in elm bark phloem recently colonized by S. scolytus is also high. Mortality rates of S. scolytus larvae infected by B. bassiana are in part a function of spore inoculum concentrations (F4ig. 1). More than 108 spores/ml of B bassiana gave a rapid kill of S. scolytus larvae in 7 days, whereas, at the lower spore concentration of 4.8 × 104/ml, all treated larvae were killed after 11 days of exposure. In a preliminary pathogenicity test with the same strain of B. bassiana (6.5 x 107/ml), all 40 treated larvae were killed after only 5 days of exposure (23°C, 100% RH). Doane (1959) obtained 90% mortality of S. multistriatus larvae after only 5 days of exposure to B. bassiana spores at 22°C 90% RH, but the spore concentration used was not given. The calculated LDs0 value of 1.0 × 108 spores/ml is probably too high as the test method used does not take into account the spore viability or the number of spores which comes into contact with the test larvae. Further investigations into the relationships between spore inoculum concentrations and larval mortality are needed. The temperature limits for germination of B. bassiana vary from 0° to 40°C (Schaerffenberg, 1957). The limits for growth generally fall between 5° and 35°C, with the optimum between 20° and 30°C (Roberts and Yendol, 1971). G6sswald (1938) has recorded infections of laboratory insects with B. bassiana at 0°C. At temperatures above 30°C, growth and infection are delayed (Headlee and McColloch, 1913; Pascalet, 1939; Hart and MacLeod, 1955). Such wide variations in tolerance of temper-

365

ature may explain the broad climatic range of the fungus. Strains of B. bassiana from warm climates grow well at high temperatures, whereas strains from temperate climates are better adapted to low temperatures (Yevlakhova and Shvetsova, 1965). The S. multistriatus strain of B. bassiana that infected S. scolytus larvae in the laboratory assays was perhaps typical of a temperate strain causing high mortalities at low temperatures (5°C). The interpolated optimum of 21.6°C was at the lowest end of the optimum range reported by Roberts and Yendol (1971), whereas, at 30°C, the effects of the fungus on the larvae were much slower. At 30°C, B. bassiana was unable to germinate and grow rapidly on S. scolytus to produce a quick kill. During the first 12 days at this temperature, mortality reached 75%, after which the rate of mortality decreased considerably (Table 2). Infection by B. bassiana may not have been the main cause of mortality at 30°C, as many uninoculated larvae also died of unidentified bacterial infection. From the 30°C treatment, B. bassiana was isolated in culture only twice from a random sample of 10 treated larvae. Both the host and the fungus may have been adversely affected by the high temperature (B. bassiana grew very slowly on nutrient agar at 30°C), and the fungus by competition from saprophytic fungi, mainly Penicillium spp., which grew rapidly at 30°C. Waldstad et ai. (1970) have shown that B. bassiana will grow on soil and leaf litter only when it is sterilized; even then, growth is negligible. They conclude that B. bassiana is inhibited by other microorganisms. These conclusions may help to explain the diminished mortality rate at 30°C. At temperatures lower than 20°C, the saprophytes grew more slowly, or not at all at 5°C, and appeared not to interfere with the development of Beauveria on its host. The various methods of entry of entomogenous fungi into the beetle gallery systems have been discussed by Barson (1976). No infection by B. bassiana was

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found in any of the field populations of DOANE,C. C. 1959. Beauveria bassiana as a pathogen of Scolytus multistriatus. Ann. Entomol. Soc. Amer. , larvae and adults of S. scolytus and S. $2, 109-111. multistriatus that were sampled. The beau- DUNN, P. H., AND MECHALAS, B. J. 1963. The veria strains used in the assay were potential of Beauveria bassiana (Balsamo) Vuillerisolated in culture from apparently healthy min as a microbial insecticide. J. Insect Pathol., 5, 451-459. S. multistriatus adults. The numbers of spores carried by these adults may have GOSSWALD, K. 1938. Ober den insektent6tenden Pilz Beauveria bassiana (Bals.) Vuill. Bisher been sublethal, thus ensuring that the fungus Bekanntes and eigene Versuche. Arb. Biol. Reichreaches the maternal galleries. Spores of sanst., 22, 399-452. B. bassiana will germinate and grow on HART, M. P., AND MACLEOD, D. M. 1955. An apparatus for determining the effects of temperature sterile elm bark, but it is not known if and humidity on germination of fungous spores. spores from this strain will grow on bark (Canad. J. Bot., 33, 289-292. contaminated with other fungi and bacteria. HEADLEE, T. J., AND McCOLLOCH, J. W. 1913. Owing to the difficulties of disseminating The chinch bug (Blissus leucopterus Say.). Bull. the disease inside the bark, it appears Kansas State Agri. Coll., 191, 287-353. that natural control by this fungus is KENNETH, R., WALLIS, G., OLMERT, Y., AI~D HALPERIN, J. 1971. A list of entomogenous unlikely to be important (Barson, 1976). fungi of Israel. Isr. J. Agr. Res., 21, 63-66. It would also seem unlikely that artificial LEATHERDALE, D. 1970. The arthropod hosts of application of B. bassiana spores to entomogenous fungi in Britain. Entomophaga, trees suitable for beetle breeding would 15, 419-435. significantly reduce beetle populations and MOOK, P. V., AND WOLFENBARGER, D. O. 1943. Distribution of Beauveria bassiana on elm insects thus affect the rate of spread of Dutch elm in the United States. Phytopathology, 33, 76-77. disease in Britain (Barson, unpub.).

ACKNOWLEDGMENTS This work was carried out during a contract appointment with the Natural Environment Research Council. I would like to thank Mr. P. F. Entwistle for his helpful criticism of the manuscript. Mrs. M. Smith kindly supplied the B. bassiana isolates.

REFERENCES BALAZV, S. 1965. Grzyby owadob6jcze z rzedu Hyphomyctes na szkodliwych owadach lesnych w Polsche. Summary: Entomophathogenous fungi from the order Hyphomyctes damaging forest insects in Poland. Roczn. Wy~. Szk. Rdn. Pozn6n, 27, 21-30. BARSON, G. 1976. Laboratory studies on the fungus Verticillium lecanii, a larval pathogen of the large elm bark beetle (Scolytus scolytus). Ann. Appl. Biol., 83, 207-214. BEAVER, R. A. 1966. The development and expression of population tables for the bark beetle Scolytus scolytus (F). J. Anim. Ecol., 35. 27-41. CrtARLES, V. K. 1941. A preliminary check-list of the entomogenous fungi of North America. U.S. Dept. Agr. Bur. Entornol., Plant Quarant., Insect Pest Survey Bull, 21, Suppl. to No. 9, 707-785.

PAILLOT, A. 1930. "Trait( des Maladies Due Ver soie." G. Doin, Paris. PASCALET, P. 1939. La lutte biologique contre Stephanoderes hampei on Scolyte due caf6ier au Cameroun. Rev. Bot. Appl. Agr. Trop., 19, 753-764. PESSON, P., TOUMONOFF, C., AND HARARAS,C. 1955. l~tude des epizooties bact6riennes observ6es dans les 61evages d'insectes xylophages. Ann. I~piphyt., 6, 315-328. ROBERTS, D. W., AND YENDOL, W. G. 1971. Use of fungi for microbial control of insects. In "Microbial Control of Insects and Mites" (H. D. Burges and N. W. Hussey, eds.). Academic Press, London and New York. SCHAERFFENBERG,B. 1957. Infektions- und Entwicklungsverlauf des insektent6tenden Pilzes Beauveria bassiana (Vuill.) Link. Z. Angew. Entomol., 41, 395-402. TANADA, Y. 1953. Applied insect pathology in Hawaii. Hawaii Farm Sci., 2, 7-8. WALSTAD,J. O., ANDERSON,R. F., AND STAMBAUGH, W. J. 1970. Effects of environmental conditions on two species of muscardine fungi (Beauveria bassiana and Metarrhiziurn anisopliae). J. lnvertebr. Pathol., 16, 221-226. YEVLAKHOVA, A. A., AND SHVETSOVA, O. I. 1965. Basic aspects of research on the microbiological control of insect pests. (with English translation). Entornol. Rev. (USSR) 44, 423-426.