Ent. exp. & appl. 14 (1971): 457--466. North-Holland Publishing Co., Amsterdam

M O D I F I C A T I O N O F T H E D E V E L O P M E N T O F B E A U V E R I A

T E N E L L A M Y C O S I S I N M E L O L O N T H A M E L O L O N T H A L A R V A E ,

B Y M E A N S O F R E D U C E D D O S E S O F O R G A N O P H O S P H O R U S

I N S E C T I C I D E S

BY

P. F E R R O N Institut National de la Recherche Agronomique (I.N.R.A.), Station de Recherches de Lutte

biologique et de Biocoenotique, La Mini~re 78, Versailles, France

Infection experiments have been carried out in the laboratory by mixing increasing quantities of spores of Beauveria tenella and reduced doses of parathion or trichloronate (organophosphorus insecticides) with the rearing medium o{ white grubs (MeloIontha melo- lontha) to verify whether it is possible to favour the development of the muscardine disease. The results are similar to those which were obtained previously with reduced doses of BHC : with low dosage of spores (5 X 104 per gram of peat) which does not produce the mycosis after 4 months at 20°C, the addition of small quantities of parathion or trichloronate gives a real increase of muscardine; with an inoculum of 1 )< 106 spores per gram, the mortality by the fungus is approximately the same with or without addition of insecticide, but the development of the disease is more rapid in the case of a simultaneous contamination by the spores and one of the two insecticides. These results give the possibility of carrying out field experiments with low quantities of a non long lasting insecticide so as to favour the development of the muscardine disease in a natural population of white grubs.

Previous experiments (Ferron, 1967) have shown that development of Beauveria tenella (Delacroix) Siemaszko mycosis (Fungi imperfecti, Moniliales) in larvae of the common cockchafer, Melolontha meIolontha L. (Coleoptera, Scarabaeidae), is rather slow and is related mainly to the number of spores infecting the rearing

substrate. For example, a temperature of 20 ° for at least 2 months is required to obtain a 5 0 % mortality rate due to mycosis for these larvae in wet peat infected with 106 fungus spores/g dry weight. The possibility of increasing the mortality due to mycosis has been tested earlier by weakening the insect either by the milky disease due to Bacillus popilliae strain melolontha (Ferron et al., 1969) or by reduced doses of chemical insecticides (Ferron, 1970a). The latter result was achieved by using BHC, an organochlorine insecticide. Because current regulations are limiting the use of this type of insecticide more and more severely, similar

experiments with various organophosphorus compounds have now been performed

under laboratory conditions.

Experimental conditions The white grubs used in these experiments were in the 3rd instar; they originat-

458 P. FEggOU

ed from the Belfort area (East of France), where they were collected after plowing and subsequently kept in wet peat at 6 ° . Sanitary checks made during collection and storage showed that this population was practically without apparent disease, especially free of white muscardine, the most frequently occurring infection.

The Beauveria tenella strain used (B.t. No. 6; Ferron, 1967) was multiplied at 23 ° as conidiospores, in Roux bottles on a solid medium, the composition of which had been worked out by the Mycology Service of the Institut Pasteur in Paris (KH2PO 4 0.36 g, NaH2PO4 1.42 g, MgSO4"7H20 0.60 g, KC1 1 g, Berthelot mineral solution 10 drops, NH4NO3 0.7 g, glucose 10 g, agar 15 g, distilled water 1000 ml).

The organophosphorus insecticides were selected from a range of products that had been investigated for their laboratory effects on white grubs by the I.N.R.A. Plant Protection Research Station in Versailles and for their field effects on these insects by the I.N.R.A. Zoological Research Station in Colmar (Blaisinger et al., 1970). These insecticides were commercial formulations of parathion and triehloronate.

The basis of the experiment was the simultaneous contamination of the larval rearing substrate with reduced doses of one of the insecticides and with increasing quantities of Beauveria spores. This result was achieved by mixing these materials, suspended in water, into the peat by means of a blendor. In accordance with the technique of rearing white grubs recommended by Hurpin (1964), the larvae were placed individually in 250 cm3 polystyrene boxes which were two-thirds filled with wet peat; they were fed with a carrot slice that was renewed every week at the time of the pathological checks. The experiments were carried out at a constant temperature of 20 ° for 4 months; after this time the surviving larvae pupated. For each variant of an experiment 25 larvae were selected randomly from the insects stored in a cold chamber.

Pathological diagnosis was based on post-mortem symptoms in order to dis- criminate clearly between cases of septicemia due to poisoning by the insecticides and of mycosis due to B. tenella. Only those dead larvae which were mummified by the fungus were attributed to the cryptogamic infection. It is possible that a systematic error by default was thus made, since Pristavko (1966) in similar experiments with B. bassiana and reduced doses of DDT on larvae of Leptinotarsa decemlineata Say found some cases of mixed infection developing into the septicemia type. With practical microbial control in view it is of most interest to obtain the greatest possible number of insects affected by mycosis, in order to multiply the foci of secondary infection in the biotopes treated; therefore we made our diagnosis according to two criteria only, either septicemia or mycosis.

Before performing experiments in which the fungus spores and the two organo- phosphorus insecticides were associated, we had to define the doses of these materials that would induce only a low mortality, and to check the effect of these reduced quantities upon the germination and growth of the fungus.

MYCOSIS AND INSECTICIDE TREATMENT IN MELOLONTHA 459

Definition of the reduced doses of insecticides According to laboratory and field experiments reported by Blaisinger et al.,

(1970), parathion and trichloronate are the two most efficient organophosphorus insecticides for the chemical control of white grubs. In pastures 5 kg of active ingredient of parathion and 5 to 10 kg of trichloronate/ha are required to reach a larval mortality of 70--85%, treatment being made in a granulated form during the spring that follows the year of flight.

In order to determine the quantities of these insecticides that would weaken the white grubs sufficiently without inducing significant mortality, a series of pre- liminary tests was carried out by treating the rearing peat with decreasing quan- tifies of both materials. In order to compare these doses with the one used in field applications, it has been assumed that the insecticide spread on the ground is distributed through a depth of 10 cm. Thus, treatment with 10 kg/ha of active agent is equivalent to 10 rag/1 of soil.

Since the batch of 25 white grubs used in each variant of the experiment corresponds to a total volume of 4500 ml of substrate, every rearing box having a capacity of 250 cm3 and being only two-thirds full, the dose of 10 kg/ha active ingredient corresponds to 46 mg/25 boxes. With an insecticide preparation having 5~/0 active ingredient, the quantity of material that corresponds to the dose used in practice is therefore 920 mg. By the same reasoning, treatment with 5 kg/ha active ingredient is equivalent to mixing 460 mg of the preparation, having 5% active ingredient, with 4.5 1 of peat.

After some preliminary experiments, a dose scale ranging from 40--300 mg of the commercial preparation of parathion and from 120--300 mg of the trichloro- hate formulation (both insecticides containing 5°/o of active ingredient) was selected and mixed with 800 g of dry peat, the weight required to make a batch of 25 rearing boxes. For 4 months the sanitary condition of the larvae was checked each week, with results presented in Table I, where the types of mortality, either by septicemia (S) or by mycosis (M), have been distinguished.

In order to induce only a low rate of mortality from insecticide we used the following doses each in 800 g of peat:

parathion: 40, 80 and 120 mg of preparation (5%); trichloronate: 120, 150 and 180 mg of preparation (50/o).

As had occurred in the experiments with BHC, it became evident in one of the variants with parathion (80 rag) that by means of the effect of the insecticide alone in a medium not artificially infected with Beauveria, it is possible to induce a significant development of muscardine (48% after 4 months of rearing; see Table I). Since we have no reason to suspect an accidental infection of this batch, we assume once again that some larvae collected in the field were naturally infected, so that mycosis may exist in a latent form in the insect, and that it may develop only in consequence of some promoting factor. Laboratory experiments are now being performed in order to investigate this phenomenon.

460 v. FERRON

TABLE I

Cumulative mortality in % of the larvae o] M. melolontha reared during 4 months at 20 ° in peat contaminated by reduced doses of parathion or trichloronate (25 third-instar larvae /or

every variant). S : septicemia, M : mycosis.

reduced doses of insecticides in mg for 80'0 g of peat

Control

Parathion mg

Trichloronate mg

days after infection

30 60 90 120 S M S M S M S M

0 0 4 0 4 0 4 0

40 0 0 0 4 4 4 8 4 80 8 0 24 8 36 48 40 48

120 12 0 72 0 84 0 92 4 150 48 0 92 0 96 0 100 0 180 88 0 100 0 . . . . 240 84 0 92 8 . . . . 300 84 8 92 8 . . . .

120 8 0 12 0 24 8 28 8 150 16 0 20 0 32 0 56 0 180 8 0 20 0 52 0 68 4 240 56 0 92 4 96 4 - - - - 300 72 0 100 0 . . . .

Effect of reduced doses of insecticide upon germination and growth of the fungus

Since the rearing substrate had been infected with an aqueous suspension of

spores of B. tenella that conta ined a known dose of insecticide, and the patho-

logical symptoms were studied for several months, it was essential to ascertain

whether the chemicals had an unfavourable effect on the fungus. The germination

rate of conidiospores and mycelial growth were selected as criteria of this possible

effect. Germinat ion was checked at 20, 24 and 28 hours after initiation of culture at

23 ° in a nutrient liquid medium (malt extract) shaken constantly. The chosen dose

of insecticide was transferred into the autoclaved medium as an aqueous suspension of constant volume, in order to avoid any change of the produc t during sterilization

and finally 0.05% of chloramphenicol was added. The medium was then incubated

with a suspension of conidiospores in sterile water in order to obtain a final

concentrat ion of about 1 X 106 spores/ml. I t is thus possible to read the percent-

age of germination directly on culture samplings, without dilution. Counting was carried out on slides under the light microscope. Fo r each check, three drops of medium f rom three successive samplings were placed separately on the slide,

air-dried, and fixed in alcohol before bi-acid Man staining. Fo r every dose of insecticide three Er lenmeyer flasks were prepared; that is to say there were nine

samples, and a counting on approximately 1000--1200 spores (Table II). Mycelial growth was measured on mycelial discs of a constant initial diameter

that were inoculated into a solid nutrient medium to which increasing quantities

MYCOSIS AND INSECTICIDE TREATMENT IN MELOLONTHA 461

TABLE II

Percentages of germination of conidiospores (B. tenella) after 20, 24 and 28 hours at 23 ° according to the quantities of parathion or trichloronate mixed with the nutrient medium.

Quantities of commercial preparations of insecticides in 120 ml of nutrient medium

Time of control 0 0,5 mg 2,5 mg 5 mg 10 mg

Parathion, after 20 hours 85 78 80 70 32 after 24 hours 96 96 96 93 76 after 28 hours . . . . 96

Trichloronate, after 20 hours 71 68 69 63 42 after 24 hours 95 93 92 92 82 after 28 hours . . . . 96

N.B. After 28 hours, it was impossible to count the germinated spores in the cultures, where the germination was already higher than 90% after 24 hours, because the mycelial development was too great.

of insecticide had been added. The discs were stamped out from cultures incubat- ed uniformly in Petri-dishes for 4 days, and were removed into cells of the same diameter made in the solid medium of another Petri-dish that had not been incubated but that contained insecticide. The discs thus removed were incubated for 6 days at 23 ° and their diameter then measured. For each insecticide dose, four Petri-dishes were thus prepared; therefore, the values shown in Table III are the average of 16 measurements.

Calculations of the doses of insecticides to be incorporated into 120 ml of liquid medium in order to study germination in 300 ml Erlenmeyer flasks, and into 20 ml of solid medium in order to test mycelial growth in Petri-dishes, were made by correlating a peat volume with a volume of nutrient medium. Thus, the doses selected (0.5, 2,5, 5, 10, and 50 mg for 120 ml of medium) correspond to the values 19, 95, 190, 380, 950, and 1900 mg in the range used to infect the peat.

From the results shown in Tables II and III it is clear that parathion and trichloronate retard the germination and growth of B. tenella only when the doses are very high, obviously higher than those used in association with the Beauveria spores in infecting the peat. Therefore it appears that the results of our

TABLE I l I

Variation of mycelial development according to the quantities o£ parathion or trichloronate mixed with the nutrient medium (This variation is measured by the diameter of myceIial

discs inoculated into the agar medium containing the insecticide)

Insecticides Quantities of commercial preparations of insecticides in 12Oral of nutrient medium (mg)

0 0.5 2.5 5 I0 25 50 Parathion 26 24 22 23 22 20 15 Trichloronate 26 25 26 26 25 22 20

462 P. FERRON

experiments are not likely to have been distorted by a possible effect of the insecticide on the fungus.

]oint effect o[ B. tenella and reduced doses of parathion or trichIoronate on the

larvae of Melolontha melolontha

The infection experiments consisted in rearing third-instar larvae in wet peat infected simultaneously with increasing doses of B. tenella conidiospores and of parathion or trichloronate. The amounts of insecticide used were, respectively, 40, 80 and 120 mg 5 % parathion and 120, 150 and 180 mg 5 % trichloronate for 800 g of dry peat. Additionally, this rearing medium was infected with 5 X 104, 1 )~ 105, 5 X 105 and 1 × 106 conidiospores/g.

The results achieved after 4 months of rearing are presented in Figs 1 and 2, which compare the development of the mycosis in batches treated only with the

fungal spores and in those treated with the association, Beauveria + insecticides. Table IV shows complementary data on the mortality in the control batches and the cases of septicemia or muscardine in the treated batches.

TABLE IV

Cumulative mortality (in %) by mycosis (M) or septicemia (S) of the larvae reared at 20 ° in peat simultaneously contaminated by conidiospores oJ B. tenella and reduced doses oJ

parathion or trichloronate

Spores of B. tenella Insecticides in per g of peat Parathion

0 40 80 120

Control S 4 4 36 68 M 8 4 48 28

5 × 104 spores S 12 4 12 28 M 0 48 84 72

l × 10 ~ spores S 4 0 28 24 M 20 32 48 68

5 × 10 ~ spores S 12 12 28 28 M 32 60 76 64

l × 106 spores S 4 4 8 44 M 76 72 84 52

mg for 800 g o~ peat Tfichloronate

0 120 150 180

8 24 56 68 0 8 0 4

8 20 20 24 4 64 56 72

0 0 24 40 28 76 64 60

4 4 8 32 52 76 84 64

4 24 20 16 64 76 72 84

In all instances studied there was a higher percentage of mycosis in the insects infected by the two materials than in those infected by Beauveria alone. This was particularly apparent with low doses of spores (5 )< 104 conidiospores/g peat) when, during the 4 months of the experiment, only one case of muscardine was noted among the controls infected by the fungus alone whereas, depending on the size of the dose of parathion or trichloronate added to the same amount of spores, 48 to 8 4 % of the larvae were affected by the disease when insecticide was also present (Figs l a and 2a).

MYCOSIS AND INSECTICIDE TREATMENT IN MELOLONTHA 463

Mortal i fy by mycosis %

'[00.

80,

60.

40,

20

BEAUVERIA 5.104/g a

400.

80 .

o j , 3

o / / . , I 60 .

/o . - -~ . / - - - . .o 2 o./ / , /

/ oj:,~./-! ~o.

. . ~ / .I • c;B. ~ 1 ¢ " ' 1 - - - - I I I ° * ' " ' ° 1 . . . . . I

d 2 5 4

BEAUVERIA -1.10S/g b

/ / /.,.:--::~

.......... .%B .-'""

/ o / :/// , . . . . . . .

o~ a

o. / 7 , , , ,

- - --- ,~-/ / - ; ("" I I i I I i 1 2 3 4

400 BEAUVERIA 5.105/g C

8C .o . . . . 0 I " ' ' ° 2 0 . 1 ' ,__ _,1

/ o ~ " / / o/:--o / o o 03

60 / ~ . , I ~ - - /I/ /

l id.." / . . . . . ..... . - .

40 / / / / / .,'""" c ~

I /.o / ./"

/ i / / / / / 20 /*"'

/ / / . / I 2 3 4

I°° I ~° I

I 50

4O

20

BEAUVERIA I JOB/g d

o ~ O ~ O o ~ o ~

~.1, -'~ . o , - - , o ~ ,.o 2

/.j.'~'-'°' ........ / / , / . , °" c.B.

/// l / I/,'*''['" /

7 • oo"~

, ~ z

LT 50~=38 days

Months

L T50~.=84 days

Fig. I. Cumulative mortality (in %) by mycosis of the larvae reared during 4 mouths at 20 ° in peat contaminated by different quantities of spores of B. tenella and reduced doses of trichlorouate. (i = 120mg, 2 = 150mg, 3 = 180mg of 5% trichlorouate for 800g of peat.

C.B. = cheek with Beauveria without insecticide).

464 P . F E R R O N

Morfclfity by mycosis %

400 BEAUVERIA 5 . t 0 4 / 0

/ o . - - . o2 80 /

o 3 o o--/- . . . . .

60. / . / ° / / , • ~.,. o - - - - 0 1

• . o ~ / 40. / / o • / / ! ,/.o

'-°t / / o / / / / - I ~ ! .L . I ] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,c. B

1 2 3 4

100

80

60.

40.

20.

BEAUVERIA t.t05/0 b

o / O / . . . . . 3

./o . . . . o--.-o . . . . 0 2

/

/ ' . / / o ~ _oi

l / 1 `.° / /o .to / / o / ,, . . . . . . . . GB

. . . / / / 7 ..... "'"

7/.-;"? ..... "" I I ~ ~ " I f I I I

I 2 3 4

100

80

60

40,

20.

BEAUVERIA 5.10S/g C

. .o 2

. o . . . . o ' ' 1 "

o.,~ ~ " / o . - ~ - - o - - o _ _ 0 3

o / o 1 o - - - o / . .o. t "

o' /

o .o /

/ / i // ......... c. a

I 2 3 4

1 0 0

8 0

60.

40.

20.

BEAUVERIA 1.10S/g d

~o . . . . o . . . . o , - - . o ~'

o , / • . . . . . ,c.B

# o ~L'o __ - oi / .I.-"

/ y 7 , / / /

/ o . . ~ / ~ / _ _ _ . . . . 3

f / //

_ I " : ( , , , , ,

• I 2 3 i i

4 months

Fig. 2. Cumulative mortali ty (in %) by mycosis of the larvae reared during 4 months at 20 ° in peat contaminated by different quantities of spores of B. tenella and reduced doses of

parathion. (1 = 40mg, 2 = 80mg, 3 = 120mg of 5% parathion for 800 g of peat. C.B. = check with Beauveria without insecticide).

MYCOSIS AND INSECTICIDE TREATMENT IN MELOLONTHA 465

With a pathogenic inoculum that by itself induces a high mortality (for example, 1 X 106 spores/g peat: Figs ld and 2d), the cumulative results do not of them- selves display clearly the advantage of the association Beauveria + insecticides; but if the development of the disease is investigated it can be established that addition of the insecticide accelerates the infective process (LT500/0 = 84 days with Beauveria alone, but = 38 days with Beauveria + trichloronate). When blastospores, which are less resistant to natural conditions than conidiospores, are employed, it might be preferred to use simultaneously a reduced dose of insec- ticide, in order to infect the insects more quickly.

It is of interest to emphasize that, whatever the amount of spores infecting the rearing medium, approximately the same mortality due to mycosis is achieved after 4 months of rearing (60 to 80o/0 of the larvae in the experiment), this being especially evident in the series of experiments with trichloronate, Therefore it should be possible, theoretically, if experimental conditions in the field were exactly the same as in the laboratory, to reduce by a factor of 20 the amount of inoculum needed to spray a unit area. This should produce a saving of the bio- logical preparation, and thus permit experimentation on agricultural areas.

CONCLUSION

In these experiments we have confirmed that mortality of white grubs killed by B. tenella muscardine may be promoted by weakening host resistance with reduced doses of insecticides. The first results of this kind were obtained by inducing muscardine development in larvae previously infected by the milky disease agent, the bacterium Bacillus popilliae (Ferron et al., 1969); then similar facts were observed when both the fungus and reduced doses of BHC were used (Ferron, 1970a); and they have now been checked by substituting organophosphorus compounds like parathion or trichloronate for the chlorinated hydrocarbons.

We are thus provided with a technique that can be used readily under natural conditions, avoiding the risks of soil pollution, since these organophosphorus insecticides, which are only slightly persistent, may be used at low dosages. Besides, in the Soviet Union this same procedure has been applied in agricultural practice for several years (Telenga, 1964), particularly for controlling such pests as Leptinotarsa decemlineata, Ostrinia nubilalis (Hiibn.) and Carpocapsa pomo- nella L., using B. bassiana conidiospores associated with reduced doses of DDT (Ferron, 1970b). Application of this method to underground insects may now be considered, by promoting epizootic diseases via the multiplication of secondary foci of infection.

466 P. FERRON

I ~ $ U M I ~

MODIFICATIONS DU D~VELOPPEMENT DE LA MYCOSE .'~ BEAUVERIA TENELLA DES LARVES DE M E L O L O N T H A M E L O L O N T H A AU MOYEN DE DOSES

R~DUITES D'INSECTICIDES ORGANO-PHOSPHOR~S

Des experiences d'infectioa des larvcs de M. melolontha par le champignon entomopatho- gSne Beaaveria teneIla, cn pr6sence de doses r6duites d'insccticides organo-phosphor~s (para- thion on trichloronate), ont 6t6 r6alis6es au laboratoire pour v6rifier s'il est ainsi possible de favoriser le d6veloppement de la mycose. Les rdsultats confirment ceux obtenus ant6rieurc- ment avec des doses r6duites de H C H : avec de faibles quantit6s de spores (5 × 104 conidio- spores par gramme de tourbe utilis~e comme milieu d'61evage des vcrs blancs), qui ~ elles seules ne provoquent pas l'apparition de la maladie apr~s 4 mois d'observatio~ ~ 20 °, l'addition de faibles doses de parathion ou de trichloronate assure une r~elle augmentation des cas de muscardine ; avec un inoculum de 1 × 106 conidinspores par gramme, la mortalit6 par mycose est approximativement la m~me avec ou sans addition d'insecticide, mais le d6veloppement de l'infecti(m est nettement plus rapide lorsque la tourbe est contamin6e simultan6ment par le champignon et l'tm des deux insecticides.

R E F E R E N C E S

BLAISINGER, P., VARLET, G., BOURDIN, J. & ROBERT, P. CH. (1970). Essais insecticides contre les vers blames, larves du Hanneton eommun MeloIontha melolontha L., dons les prairies. Vll~me Congr. Int. Protect. Plantes, Paris, 156--157.

FERRON, P. (1967). t~tude en laboratoire des conditions 6cologiques favorisant le d6veloppe- ment de la mycose h Beauveria tenella du ver blanc. Entomophaga 12 : 257--293.

- - (1970a) Augmentation de la sensibilit6 des larves de MeIolontha melolontha L. (Col6o- pt~re Scarabaeidae) ~ Beauveria tenella (Delacr.) Siemaszko an moyen de quantit6s r6duites de HCH. 1V~me Colloque Inter. Pathol. Insectes, College Park, (sous presse).

- - (1970b). Orientations des recherches effectu6es en U.R.S.S. stir les champignons ento- mopathog~nes. Ann. Zool. ~col. anita. N ° hors-s6rie 3 : 117--134.

FERRON, P., HURPIN, B. & ROBERT, P. (1969). Sensibilisation des larves de Melolontha melo- lontha L. ~t la mycose ~t Beauveria tenella (Delacr.) Siemaszko par une infection pr6alable

Bacillus popilliae. Entomophaga: 14: 429--437. HUm'IN, B. (1964). I~levage des vers blancs ou larves de MeloIontha meloIontha L. Rev. Path.

vdg. Ent. agric. France, 43: 153--177. PRISTAVKO, V.P. (1966). Processus pathologiques cons6cutifs ~t Faction de Beauveria bassiana

(Bals.) Vuillemin associ6 ~t de foibles doses de DDT chez Leptinotarsa decemlineata Say. Entomophaga 11 : 311--324.

TELENGA, N . A . (1964). Le probl~me de l'utilisation des microorganismes entomopathog~nes en combinaison avec les insecticides. Colloque Int. Pathol. Insectes, Lutte microbiol., Paris 1962, Entomophaga, M6m. Hors S6r. (2): 531--544.