Insecticidal secondary metabolites from entomogenous fungi: Entomophthora virulenta

JOURNAL

OF INVERTEBRATE

insecticidal

PATHOLOGY

Secondary

32, 319-324(1978)

Metabolites

from Entomogenous

Fungi

En tomoph thora virulen ta NORMAN~LAYDON Agricultural

Research

Council,

Unit of Invertebrate Falmer, Brighton,

Chemistry and Physiology, BNI 9QJ, England

University

of Susser

Received August 18, 1977 Two strains of the insect pathogenic fungus Enfomophfhora virulenta have been examined for their pathogenicity to adult blowflies, Calliphora erythrocephala, for their ability to grow on defined media, and for the production of biologically active secondary metabolites. The fungus proved highly pathogenic to blowflies and hyphal bodies isolated from diseased insects were successfully cultivated on egg yolk medium and subsequently grown in shake culture on a glucoseasparagine medium. Both strains were found to produce a mixture of 4,4’-azoxybenzene dicarboxylic acid and 4,4’-hydroxymethyl azoxybenzene carboxylic acid. The hydroxy acid proved toxic by intrahemocoelic injection into adult blowflies and accounted for all the extractable insecticidal activity. KEY WORDS: Entomophfhora virulenta; insecticidal secondary metabolites; fungal toxins; natural aryl azoxy compounds..

INTRODUCTION

Species of Entomophthora causing disease in insect populations are well documented and many studies relate to the morphology and pathogenesis of these organisms. The role of mycotoxin production in the disease process has attracted minimal attention and the reports available indicate without elaboration that toxins may be produced by certain species. In particular, Yendol et al. (1968) demonstrated toxin production by a strain of E. virulenta. Reconstituted, freeze-dried filtrates from shake cultures of this fungus were injected into adult face flies, Musca autumnalis and found to produce a 52% mortality after 48 hr. Similar injections into the larvae of the greater wax moth, Galleria mellonella, failed to elicit a toxic effect. Prasertphon and Tanada (1969) confirmed this latter finding and further demonstrated the lack of toxicity shown by E. virulenta filtrates to a range of lepidopterans. To date, no further publications relating to toxin production by E. virulenta have appeared despite the encouraging results by

Yendol and his associates. The present study examines two strains of E. virulenta for their capacity to infect an insect, the blowfly, Calliphora erythrocephala, and to subsequently produce in vitro a material demonstrably toxic to that insect. The isolation and identification of a toxic metabolite and its analog is reported in summary only. Full chemical details will be published elsewhere (Claydon and Grove, 1978).

319

MATERIALS

AND METHODS

Fungi. Two strains of the fungus E. virulenta, previously isolated from aphids and kept on egg yolk medium (MullerKiigler, 1959) at the Rothamsted Experimental Station, Harpenden, Hertfordshire, were kindly donated by Neil Wilding. The strains were numbered Cth. 1 and Cth. 2 in Dr. Wilding’s collection and 11 and 12 in our collection. Pathogenicity tests. Both strains were grown on egg yolk medium in Petri dishes and 10 adult blowflies, Calliphora erythrocephala (three replicates), were allowed to crawl over the mycelial surfaces for 30 min. 0022-201 l/78/0323-0319$01.00/0 Copyright All rights

0 1978 by Academic Ress. Inc. of reproduction in any form reserved.

320

NORMANCLAYDON

The flies were removed to cylindrical glass dishes inverted onto a fine nylon gauze affixed across the neck of a glass jar containing 20 ml of water. Sucrose and water were provided and the dishes were placed in an incubator at 23°C. Mortality was recorded daily and compared with a control. Dead flies were removed from the dishes and examined for fungal development within the hemocoel. Cultivarion offingus. Hemolymph from infected blowflies was removed by micropipet through a small abdominal incision and examined microscopically. Twenty microliters of the hemolymph was placed on coagulated egg yolk slopes and incubated at 23°C for 8 days. Five-millimeter disks were cut from the rapidly growing cultures and affixed centrally onto the inside surfaces of Petri dish lids. The lids were placed onto their complementary bases containing egg yolk medium and incubated for 5 hr. The lids were all replaced with new sterile lids and the plates were kept for a further 8 days. Dry weight determinations were made for growth in liquid media. An aqueous basal solution of L-asparagine, 2 g/liter, was prepared, and glucose was added at the rates of 0.5, 1, 3, 5, 10 g/liter. Each solution was dispensed into 250-ml Erlenmeyer flasks in four replicates of 100 ml. After sterilization at 121°C for 10 min, the cooled flasks were inoculated with a l-ml spore/mycelial suspension and placed on a rotary shaker at 120 rpm for 18 days at 23°C. The mycelial suspension from each flask was collected and dried in vacua and the average dry weight for the four flasks in each series was recorded. The inoculum was prepared by washing three egg yolk slopes of the respective strains with a glucose, 20 g/liter, Lasparagine, 2 g/liter solution. The washings were made up to 500 ml with the same solution and dispensed as IOO-ml aliquots into each of five 250-ml Erlenmeyer flasks. The flasks were kept on a rotary shaker, as described above, for 10 days. The flask contents were bulked and the spore/mycelial

suspensions were separated by filtration and resuspended in 50 ml of sterile distilled water. Aseptic procedures were employed throughout. Fermentations. Both strains were grown in parallel in shake culture on a medium containing 20 g of glucose, 4 g of Lasparagine, and 2 liters of distilled water. The medium, pH 4.9, was dispensed as loo-ml aliquots into 250-ml Erlenmeyer flasks, inoculated with a l-ml mycelial suspension as described above, and placed on a rotary shaker at 120 t-pm. The fermentation was kept for 18 days at 23°C. Control flasks of uninoculated medium were kept under the same conditions. Analysis ofculturejuid. Periodic analyses were performed by removing lo-ml aliquots of the culture fluid under sterile conditions and after filtration by measuring bothp H and optical rotation for the aqueous solutions. Five-milliliter aliquots of these solutions were acidified top H 2.0 with 2 N HCl and extracted (2 x 2 ml) with ethyl acetate. After drying over anhydrous sodium sulfate, the ethyl acetate was removed under vacuum and the residues were taken up in 10 ml of methanol and examined for uv absorbtion. Isolation of secondary metabolites. After 18 days, the fermentations were harvested and the mycelium was removed by filtration. The respective culture filtrates were extracted (2 x l/3 vol) at natural pH with chloroform followed by a further extraction at pH 2.0 with ethyl acetate. The solvents in each case were removed under vacuum. Similar extractions were made on the uninoculated glucose-asparagine medium. Tests for insecticidal activity. Twomilligram quantities of the extracts were dissolved in 0.05 ml of dimethyl sulfoxide and added to 0.05 ml of a fly Ringer solution consisting of 3.3 ml of 0.45 M NaCl, 1 ml of 0.4 M KCI, and 0.3 ml of 0.3 M MgCl. These extract solutions were tested for toxicity against adult blowflies. One-microliter quantities were injected into the thoracic hemocoel of 4-day postemergence flies in three replicates of 10 (Claydon et al., 1977).

SECONDARY

METABOLITES

Mortality was recorded daily and compared with control flies injected with a dimethyl sulfoxide: Ringer solution (1: 1). Aqueous solutions of azoxybenzene dicarboxylic acid and hydroxymethyl azoxybenzene carboxylic acid were prepared as follows: Ten milligrams of the diacid was dissolved in 3 ml of 1 M KOH solution and acidified topH 9 with HCl, bringing the total volume to 3.6 ml. Five milligrams of the hydroxy acid was dissolved in 1.5 ml of 1 M KOH solution and acidified to pH 7.5 with HCl, bringing the total volume to 2.0 ml. One, two, and three microliters were injected into blowflies as before in four replicates of 10, and mortality was compared with control solution injections of 1 M KOH: HCl at pH 9 and 7.5, respectively. RESULTS

The fungus proved highly pathogenic to blowflies (Table 1). At 72 hr, a 30% mortality was recorded with a high proportion of the surviving insects clearly in a moribund state. Total mortality was achieved by 96 hr and examination of the dead flies did not reveal any extensive fungal development. Hyphal bodies were evident in the hemolymph but in small numbers. Cadavers retained for 48 hr at 23°C and 100% RH did not develop any of the classical external symptoms of entomophthoraceous disease. Growth in the glucose-asparagine media was not dense and appeared as a fine suspension of hyphal strands. Figure 1 shows the TABLE PATHOCENICITY

1

OF E. virulenta etythrocephala”

FROM Entomophrhora

321

virulenta

60

0

A0 .A--------

----______

*’

E

0

.A’

/

,*‘. /’

=m

40

P

4” ;

s E

I: 30 I

9’ ,’

20 o-o

.5 1

Strain

12

3

5

10

Glucose

Concentration

gm

FIG. 1. Graph showing the average dry weights attained in 18 days by two strains of E. virulenta. The fungus was grown in shake culture on a glucose-rasparagine medium with the asparagine concentration fixed at 2 @liter throughout.

average dry weights obtained for each strain on the various solutions. The solution chosen for the production fermentation was initially faint yellow but had progressively darkened to a deep yellow coloration by the time of harvest. Ultraviolet absorbing materials, A,,, 275 nm, appeared late in the fermentation. The pH rose during the first 6 days and remained at a steady value of 7.6 until harvesting. The optical rotation measurement remained constant throughout (Table 2). For both strains (11 and 12) the neutral extracts were reddish brown gums 14 and TABLE

2

COURSE OF A TYPICAL FERMENTATION ON GLUCOSE-ASPARAGINE MEDIUM

Strain

24

48

72

96

11 12

0 0

6.6 10

26.6 30

96.6 100

OF E. virulenta (STRAIN 11)

Days 0

PH a Scores marked as percentage mortality with time. Values quoted are corrected against controls by Abbotts’ formula (Busvine, 1971).

I I

TO Calliphora

Hours

~

,.=

50

Optical rotation Amax

4.9 2.591 -

6

9

15

18

7.3 7.6 7.6 7.6 2.584 2.547 2.552 2.560 275 275

322

NORMAN

CLAYDON

9 mg, with no specific uv absorption between 200 and 390 nm, or any toxicity to blowflies at the concentrations tested. The acid extracts were orange yellow powders, 122 and 96 mg, solutions of which produced 33 and 37% mortality when injected into blowflies at 20 pg/pl (Table 3). Mass spectra of the acid extracts indicated mixtures of at least two components and, by various physicochemical techniques (Claydon and Grove, 1978), the compounds azoxybenzene dicarboxylic acid (I) and hydroxymethyl azoxybenzene carboxylic acid (II) were isolated and identified. Extracts of the uninoculated medium were completely free of these compounds. Toxicity testing of these compounds was hindered by their poor solubility in carrier solvent systems. For the diacid a concentration of 2.7 pg/j.~l atpH 9.0 was the maximum achieved and 34 injections of this solution into blowflies did not produce any mortalities. The hydroxy-acid had a greater solubility and 3-4 injections of a 2.5 pg/pl solution atpH 7.5 produced a 77% mortality at 72 hr (Table 3). DISCUSSION

The ability high mortality

of E. virulenta to produce a in blowflies without extensive TABLE

TOXICITY

OF CRUDE ACIDS

EXTRACTS

TO Calliphora

3 AND

AZOXYBENZENOID

erythrocephahP Mortality

Volume injected WI)

Solution Crude extract (neutral) Crude extract (neutral) Crude extract (acid) Crude extract (acid)

I1 12 11 12

Diacid Diacid Diacid Hydroxy Hydmxy Hydroxy o Values 1971).

acid acid acid corrected

against

at 72 hr (%)

Contrcd

observed

COIrected

1 1

IO 10

10 7

0 0

I I

10 10

40 43

33 37

I 2 3

0 2.5 22.5

2.5 12.5 17.5

0 10 0

I 2 3

0 0 0

12.5 22.5 77.5

12.5 22,s 77.5

controls

by Abbotts‘

formula

(Busvine,

(1)

4.4’

azoxybenzene

(11)

4.4’

hydroxymethyl

dicarboxylic

azoxybenzene

carboxylic

(III)

azobenzene

(IV)

azoxybenzene SCHEME

acid.

acid

1

colonization of the host is suggestive of mycotoxin production. This is in agreement with the findings of earlier workers (Yendol and Paschke, 1965) in relation to laboratory pathogenicity tests in which Eastern subteranean termites, Reticulotermes flavipes, were infected with the fungus Conidiobolus coronatus. It was seen that upon the death of a high proportion of the infected termites the fungus had not penetrated deeply into the hemocoel and its growth was extremely restricted. These observations conflict with those of other investigators (Speare and Colley, 1912; Sawyer, 1933; Hutchinson, 1962) who found the entomophthoraninfected host to succumb only after the fungus had become well established in the body cavity. In a subsequent study of myco-

SECONDARY

METABOLITES

toxin production from these fungi (Yendol et al., 1%8), it was found that culture filtrate preparations of C. coronatus fermentations were toxic by injection to wax moth larvae, Galleria mellonella. The effect of similar preparations from E. virulenta on adult face flies has been described above. No attempts seem to have been made to identify the toxins involved but in a later study (Prasertphon and Tanada, 1969) it was claimed that the C. coronatus toxin had properties characteristic of a protein. In the present work, examination of the acid extracts shows that E. virulenta has the capacity to produce substituted azoxybenzenoid compounds. Under the conditions chosen in this study, the compounds 4,4’azoxybenzene dicarboxylic acid and 4,4’hydroxymethyl azoxybenzene carboxylic acid were produced. The former compound has been described on numerous occasions as a synthetic product but the latter has never been described. These compounds are the only low molecular weight metabolites to be reported from entomophthoraceous fungi and this is the first account of the isolation of an aromatic azoxy compound from a natural source. The production of a toxic azoxybenzenoid compound by an insect pathogen is of particular interest since attention has previously been focused on the use of such compounds in insect control. Azobenzene (III) and some of its substitution products were first reported to be effective as insecticides against mosquitoes (Fink and Vivian, 1936). Activity was also demonstrated against the European corn borer, Pyruusta nubilalis (Questel et al., 1949), and formulations with azobenzene as the active ingredient were found particularly effective against a range of economically important insect pests (Haring, 1946). Sharp (1948), using adult cockroaches, Periplaneta americana, and housefly larvae, Musca domestica, demonstrated the action of azobenzene as an insecticide. Fly larvae fed with azobenzene-contaminated food assumed a yellow coloration and became progressively less active until death ensued.

FROM Entomophthora

virulenta

323

Cockroaches with sealed mouth parts died when dusted with the compound indicating cuticular absorption. Dissection of the treated insects revealed that the fat body, malpighian tubules, parts of the alimentary canal, and to some extent the hemolymph had taken on a yellow coloration. Sharp contended that azobenzene was changed in vivo to a water-soluble derivative which became stored in the fat body when production exceeded excretion. Death seemingly occurred when saturation of the tissues had been reached. Azoxybenzene (IV), too, has been shown to possess a potent activity as both an acaritide and as an insecticide (Eaton and Davies, 1948, 1950; Travis et al., 1949; Bailey and Carlisle, 1956). In a study by Eaton and Davies, the relationship between molecular structure and pesticidal activity was examined. It was found that substitution in the aromatic rings tended to produce lower activity than for the unsubstituted parent compound. An exception to this was 4chloro substitution where activity was enhanced. None of the compounds tested however contained a carboxyl or hydroxymethyl side chain. The production of azoxybenzenoid compounds by E. virulenta in vivo has not been demonstrated and it is purely speculative that such compounds are produced within the insect host. Studies are currently being undertaken to establish whether the compounds isolated in vitro are produced during the disease process. REFERENCES BAILEY, L., AND CARLISLE, E. 1956. Tests with acaricides onAcarapis woodi. Bee World, 37,85-94. BUSVINE, J. R. 1971. “A Critical Review of the Techniques for Testing Insecticides.” Commonwealth Agriculture Bureaux. CLAYDON, N., AND GROVE, J. F. 1978. Metabolic products ofEntomophthora virulenta. J. Chem. Sot. (Perkin Trans. I.). 171-173. CLAYDON, N., GROVE, J. F., AND POPLE, M. 1977. Insecticidal secondary metabolic products from the entomogenous fungus Fusarium solani. J. Invertebr. Pathol., 30, 216-223.

324

NORMAN

EATON, J. K., AND DAVIES, R. G. 1948. Toxicity of azo compounds and other substances to the fruit tree red spider mite. Nature (London), 161,644-645. EATON, J. K., AND DAVIES, R. G. 1950. Toxicity of certain synthetic organic compounds to the fruit tree spider mite. Ann. Appl. Biol., 37, 471-489. FINK, D. E., AND VIVIAN, D. L. 1936. Toxicity of certain azo compounds to mosquito larvae. .Z.Econ. Entomol., 29, 804-805. HARING, R. 1946. Azobenzene as an acaricide and insecticide. .I. Econ. Entomol., 39, 78-80. HUTCHINSON, J. A. 1962. Studies on a new Entomophthora species attacking Calyptrate flies. Mycologiu, 54,258-271. MUELLER-K~GLER, E. 1959. Zur isolierung und Kultur insektenpathogener entomophthoraceen. Entomophaga, 4,261-274. PRASERTPHON, S., AND TANADA, Y. 1969. Mycotoxins of entomophthoraceous fungi. Hilgardia, 39, 4, 581-600. QUESTEL, D. D., CONNIN, R. V., AND GERTLER, S. I. 1949. “The Toxic or Repellent Action to the European Corn Borer of Corn Plants Grown in Soil

CLAYDON Treated with Various Compounds.” U. S. Department of Agriculture, Bureau of Entomology and Plant Quarantine, E-785. SAWYER, W. H. 1933. The development ofEntomophthora sphaerosperma upon Rhopobata vacciniana. Ann. Bot. (London), 47, 799-809. SHARP, S. S. 1948. Toxicity of azobenzene and certain related compounds to insects. Iowa State Coil. J. Sci., 23,78-79. SPEARE, A. T., AND COLLEY, R. H. 1912. “The Artificial Use of the Brown Tail Fungus in Massachusetts,” Wright and Potter, Boston. TRAVIS, B. V., MORTON, F. A., JONES, H. A., AND ROBINSON, J. H. 1949. The more effective mosquito repellents tested at the Orlando Fla., laboratory, 1942-47. J. Econ. Entomol. 42, 686-694. YENDOL, W. G., MILLER, E. M., AND BEHNKE, C. N. 1968. Toxic substances from entomophthoraceous fungi. J. Znvertebr. Pathol., 10, 313-319. YENDOL, W. G., AND PASCHKE, J. D. 1965. Pathology of an Entomophthora infection in the Eastern subterranean termite Reticulotermes Jlavipes. J. Znvertebr. Pathol. 7, 414-422.