The effect of certain insecticides on the entomogenous fungi Beauveria bassiana and Metarrhizium anisopliae

JOURNAL

OF

INVERTEBRATE

The

Effect

Fungi

PATHOLOGY

of

9,

Certain

Beauveria

3!%-d@3

1967)

(

Insecticides

bassiana

and

on

the

Entomogenous

Metarrhkium

anisopliae

N. V. RAMARAJE URS, 1 H. C. GOVINDU, AND K. S. SHIVASHANKARA SHASTRY Division

of Pknt

Pathology,

Research

AgTicdtuTal

Accepted

September

Institute,

Hebbal,

Bangalore,

India

20, 1966

Testing the effect of Dimecron, Folidol, DDT, Malathion, Endrin, and BHC, at 0.04%, 0.05%, 0.06%, O.l%, and 0.5% on the development of the fungi Beauvetia bassiuna and Metarrhizium anisopliae compared to controls indicated that BHC 50% WP was the most toxic. It inhibited the growth of both fungi in all concentrations; Dimecron 100% was the most innocuous. The other insecticides had varying effects on the two fungi. Endrin, DDT, Malathion, and Folidol were inhibitory in the order mentioned. Different concentrations of insecticides had different effects on the growth and sporulations of the fungi, and the two fungi also reacted differently to the same insecticide.

INTRODUCTION

Beauveria bassiana and Metarrhizium anisopliae are two of the more important entomogenous fungi which offer promise in microbial control of certain economic crop pests. A revival of interest in the use of these and other similar microorganisms individually or in combination with sublethal doses of an insecticide is to some extent an outcome of the current insecticide resistance and nonselectivity problems in pest control (Laird, 1962). One of the most promising aspects of microbial control of insects is its integration with other pest control measures, particularly the chemical method. In 1956 Steinhaus pointed to the compatibility of pathogens and many insecticides as one of the advantages of microbial methods of control. Franz ( 1961) has given an excellent review on the results obtained by Telenga, Dyadechko, and Zakharchenko, who have utilized these fungi in ’ Present address: Department of Botany, versity of Iowa, Iowa City, Iowa.

Uni-

398

combination with sublethal doses of insecti. tides against certain insect pests. Franz ( 1961) and later Hall ( 1963)) have discussed the principles underlying the integration of microbial pathogens with insecticides and refer to the specific adjuvants admitting the maximum effectiveness of the pathogen. They also mention the possibility of the two materials assisting each other, the pathogen making the pest sick enough to become less resistant to the chemical and the chemical in turn weakening the pest sufficiently to make it more susceptible for the infection by the pathogen. However, as pointed out by Hall ( 1963), “Very little information is available on the effect of agricultural chemicals on the new microbial insecticides and any speculation without the support of thorough laboratory and field studies at best would be guess work.” An attempt has been made in this laboratory to study the effect of normally recommended and high concentrations of certain common insecticides on the development of B. bassiana and M. anisopliae;

EFFECT

the findings this paper.

of this study

VETHODS

;~ND

OF

INSECTICIDES

are presented

in

MATERIALS

The cultures of B. bassiana and M. anisopliae, originally isolated from the silkworm (Ramaraje Urs et al., 1965) and root grubs ( Ramaraje Urs et al., 1964), respectively, were employed for these studies. The following six insecticides were tested: 1. BHC 50% WP (1,2,3,4,5,6-hexachlorocyclohexane) . 2. DDT 50% WP (l,l,l-trichloro-2,2-di( P-chlorophenyl) ethane ) . 3. Dimecron (2-chloro-2-diethyl-carbomoyl-1-methyvinyldimethyl phosphate). 4. Endrin 20% ( 1,2,3,4,10-hexachloro-6, 7-epoxy-1,4,4a,5,6,7,8,8a-octohydro-exo-1,4exo-5,B-dimethanonapthalene) . 5. Folidol E 605, 46.7% (O,O-diethylop-nitrophenyl phosphorothioate ). 6. Malathion (O,O-dimethyl-S-1,2-diphosphorodithio( ethoxycarbonyl ) ethyl ate ). Solutions of Malathion, Folidol, Endrin, and Dimecron at concentrations of 0.04%, 0.05% ) 0.06%, O.l%, and 0.5%, and of BHC and DDT at 0.04%, 0.05%, 0.06%, and 0.1% were prepared in separate flasks by adding calculated quantities of each of the insecticides to 200 ml of M, medium (Horsfall, 1956). M2 medium was prepared as suggested by Ramachandran and Sukapure ( 1963). The medium was adjusted to pH 7 and autoclaved for 20 minutes at 15 lb pressure before incorporation of the insecticides. Inoculum was prepared by making a heavy spore suspension of the fungi in 5 ml of sterile distilled water from a I2-day-old culture. Preliminary studies were conducted first with liquid medium and then on solid medium. In the latter 2% agar was used. Ten milliliters of liquid medium incorporated with an insecticide was inoculated with a loopful of spore suspension of B. bassiana

ON

FUNGI

399

and M. ankopliae separately, and they were incubated at room temperature for 10 days. Four replications were set up for each concentration. The observations were recorded from the 3rd day after inoculation. The dcvelopment of the fungus was assayed by the total dry weight of the fungus in all the four replications. The response of the fungus to the insecticide was recorded on the basis of mortality of the fungus. Similar studies were undertaken with solid (agar) medium; a 20-ml quantity of the medium with the insecticide incorporated in it was poured, while liquid, into plates, and a loopful of the inoculum of the two fungi under study was placed scparately at the center. The growth of the fungi on solid medium was measured along the diameter. The mean value of the four replications was recorded. Controls were maintained for each ~OIIcentration in the solid and liquid media. RESULTS

The observations of experiments to dctermine the inhibitory concentrations of the six insecticides-BHC, Endrin, Malathion, Folidol, DDT, and Dimecron-on the growth of the two fungi, B. bassianu and M. anisopliae, are recorded separately under “liquid medium” and “solid medium.” Liquid

Mediunz

In the case of BHC the growth of 13. bassiana and M. anisopliae was completely inhibited at all concentrations (see Table 1). In the case of Endrin, B. bassianu did not develop in any of the concentrations. while scanty vegetative growth of A4. anisopliae was noticed only after the 8th day at concentrations of 0.04%) 0.05%) and 0.06%. However, there was complete inhibition at higher concentrations. Malathion completely inhibited growth of the two fungi at 0.5% concentration, while

0.1350 0.92959

0.0339 0.2417 -

0.04

OF DIFFERENT

Malathion BHC WP DDT Endrin Folidol Dimecron Control (no insecticide)

Concentrations:

EFFECT

0. ‘3196 0.0474 0.3038

0.1558 -

0.05

INSECTICIDES

TABLE

1

0.06

bassiana

0.1335 O.Sl%O 0.2264

0.0500 0.1094 -

Beam&a

0.006 0.0804 0.2634

0.1

0.106% 0.2476 0.0780 0.1305 0.2815

0.0736 0.1874 0.0358 0.0410 0. P890

0.0630 0.0118

0.05

0.04

0.0475 0.2274 0.0350 0.1330 0.9960 0.2851

0.06

anisopliae

AND Metarrhizium

Meiarrhizium

bassiana

0.5

OF VARYING CONCENTRATION ON THE GROWTH OF Beauveria IN LIQUID MEDIUM EXPRESSED IN MG DRY WEIGHT

0.0348 0.091% 0.1406 0. !24!26

0.01

anisopliae

0.0804 0. a500

-

-

0.5

1 9 g 5 $ P

5 u

“8

2

2

s “J

E 1

EFFECT

OF

INSECTICIDES

it permitted only vegetative growth at 0.04%, 0.05%, 0.06%, and 0.1%. Folidol favored growth of B. bassiana in all concentrations, but its sporulation was retarded for the first 4 days under 0.04% to 0.06% and for the first 6 days at 0.1%. However, sporulation was noticed at 0.04% to 0.06% on the 5th day and after, and on the 7th day at 0.1%. At the highest concentration (0.5% ) there was no sporulation. Similarly, RI. anisopliae sporulated after 4 days at the lower concentrations (0.04% to 0.06%), and only vegetative growth took place at higher concentrations (0.1% and 0.5%). DDT inhibited the growth of I?. bussiunu at 0.1% only and permitted growth in all other concentrations. But the sporulation was retarded for the first 6 days at 0.04%, 0.05%. and 0.06% and was noticed on the 7th day at 0.04% and 0.05%, while there was no sporulation at 0.06%. M. unisopliue grew normally in all concentrations. Sporulation commenced on the 7th day at 0.04% and 0.05%. No sporulation was noticed at 0.06% and 0.1%. Dimecron was the most innocuous and favored growth of both fungi at all concentrations. However, inoculum from all such media where sporulation only was inhibited, sporulated when transferred to insecticide-free media. Solid hlediunr BHC inhibited growth in all the concentrations 2).

of the two fungi tried (see Table

B. bussiana and M. unisopliue grew only at 0.04% Endrin, and were completely inhibited at higher concentrations, viz. 0.05% to 0.5%. Malathion inhibited growth of the two fungi at the highest concentration only. Crowth was normal at all other concentrations.

ON

FUNGI

401

In the case of Folidol, M. unisopliue grew only at 0.04% but was completely inhibited at all higher concentrations, while B. budana grew unchecked in all concentrations. DDT permitted growth of B. hassianu and Al. unisopliue in all the concentrations tested. Dimecron was the most innocuous of the insecticides tried. It favored growth of both fungi in all the concentrations.

DISCUSSION

The six insecticides tested had varying effects on the two fungi B. bassianu and M. anisopliae. Dimecron was the most innocuous, favoring maximum growth at all concentrations, while BHC 50% WP was the most toxic and completely inhibited growth. The inhibitory effect of the other four insecticides-Endrin, Malathion, Folidol, and DDT-on the growth of the two fungi declined in the order listed. Similarly, Hall and Dunn (1959) in their studies on Entomophthoru in combination with several insecticides, showed that the most innocuous of the materials tested were Parathion and DDT, while Malathion was fungicidal in its action. Telenga ( 1960) reported that BHC had synergistic effects on B. bussiunu and ,ZZ. anisopliae when used with low doses of insecticides, while in the present studies BHC was considerably toxic to both the fungi tested. This variation may be due to the method and concentration in which BHC was combined by Telenga with the particular fungus used for such tests, both of which are different from those studied at present. Different concentrations of an insecticide have varying effects on the fungi. Endrin inhibited the growth of M. anisopliue at high concentrations (0.05% to 0.5% ) while at 0.04% concentration it did not inhibit (in solid media). Malathion inhibited the

OF DIFFERENT

a Expressed

in cm radial

Malathion BHC WP DDT Endrin Folidol Dimecron Control (no insecticide)

Concenirations:

EFFECT

growth

1.5 2.?2 0.5 2.1 4.5

0.04

2.1 3.6

2.0 a.9

Rrauwria

OF VAHYING

0.05

of colony.

INSECTICIDES

1.9 3.0

1.6 -

1.6 2.3 3.4 3.5

1.5

0.1

SOLID

TABLE

1.5

0.06

bavviana

0N

CONCEXTRATIOKS

2 GHOWTH

1.3 a.6

-

-

0.5

MEDIUM@

ON THE

1.7 -

1.6 2.8

3

2.8 3.5

1.5

0.06

anisopliae

ill rfnr~hizium

1.4

0.05

AND

Metarrhizium

bassiana

1.4 1.6 1.3 2.5

0.04

OF Beaurrria

2.7

1.5 1.7 -

0.1

m~isopliae

1.7

-. -

0.5

EFFECT

OF

INSECTICIDES

growth of both fungi at 0.5% concentration only. Dirimanov and Angelova ( 1963) recorded similar observations. According to them, development of B. bussiana was greatly inhibited by 12% BHC dust; 0.1% Parathion reduced its growth very much, and 0.2% DDT and 0.5% Parathion permitted its normal growth. The effect of a particular insecticide is not the same on both fungi. DDT completely inhibited the growth of B. bassiana under 0.1% concentration but not of M. ~Msopliae in the same concentration (liquid media). Similarly, Hall and Dunn ( 1959) showed that different species of Entomophthoru reacted differently when combined with the same insecticide. The reactions of fungi have varied with the same medium in combination with the same insecticide when tried with or without agar. In the case of DDT in liquid medium, there was complete inhibition of B. bassiana at 0.1%) whereas in solid medium normal growth occurred. With Endrin of 0.04% concentration there was normal growth of B. bassiana in solid medium, whereas it was inhibited in liquid media at the same concentration. Parry and Wood ( 1958), Raghava Reddy and Appa Rao ( 1964 ) have made similar observations in their studies of adaptation of Botrytis cineriu and Sclerotium rolfsi in the presence of certain fungicides. However, in the case of Folidol the position was reversed. Al. unisopliae was completely inhibited in 0.05% to 0.5% concentrations in solid media, while there was normal growth in the same concentrations in liquid media. The sporulation of the two fungi studied was inhibited in media combined with certain concentrations of the insecticides. However, they sporulated normally when a loopful of inoculum was transferred to insecticide-free media. This indicates that addition of certain concentrations of insecticides to the medium brings about inhibition in sporulation.

ON

FUNGI

403

ACKNOWLEDGMENTS

The senior author is deeply indebted Martin A. Rosinski, Associate Professor University advice in

of Iowa, reviewing

for his criticism the manuscript.

to Dr. of Botany, and valuable

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M.,

AND

KA~~AHAJE

Crw.

i%.

V.,


II.

C:..

.~uIp

M. J. 1964. L,aborxtory studirs on Meturrhi~izln~ anisopliue mtl its host range’ in Mysore State. In press. RA~IARAJE CT+ N. \‘.. CovmDu, II. C., ANIJ SHIVASHANKAHA SEIASTRY, K. S. 1965. A preliminary study of white nmscardine fungus on cabbage semilooper from Mysorc. (:lirr. Sci., 34, 382-383. STEINHALE, E. A. 19.56. Potentialities for mierot~ial control of insects. Agri. Food Chcm.. 4. 676-680. TELENGA, 6. A. 1960. Die Anwendang der hlusskardinenpilze im Verein mit Insektiziden fiir cler Schgdlingsinsekten. die BekPmpfung Trans. 1st Intern. Conf. Insect Pathol. Riol. Control, Prahu 19.58, 155-168. THIRUhIALACHAR,