Role of Bacillus thuringiensis var. thuringiensis on the larval survivability and egg hatching of Meloidogyne spp., the causative agent of root knot disease

IOLTRS.\L

OF

Kole

~NV~l<‘rt:titt.\‘rh;

of Bacillus

Survivability

I’ATHOLOGY

377.-378

20,

(1972)

thuringiensis and

Egg

Causative

var. Hatching

Agent

377 0 1973 by Academic Press, Inc. of reproduction in any form reserved.

on

the

of Meloidogyne

of Root

In rcwnt scars an ever increasing volume of literature, has bwn accumulated on the wntrol of various pests of crops 1vit.h the USI’ r)t’ Bacillus thurinqiet~sis. However, thaw is no published report concerning t,hc uw of this insect pat.hogen against plant parasitic nematodes. This paper embodies thr inwstigations 011 the rffccts of Bacillus tlmr’irqiek5 var. thuri7lgierks on the larval survivnbilit’y and egg hatching of dIeloitlogyne spp., the causative agent of root knot disease of vegetable crops. The product,ion of toxins and spores of Bacillus thuri~giensis vt\r. thuringiensis in the laboratory was attained by growing t’he organism on the medium reported by F. P. DaMrld, H. J. Somerville, and S. C. Rittcknbcrg (,I. Bacterial., 96, 713-720, 196s). The whc~lc culture obtained after 72 hr of incubation on a rotar? shaker was centrifugrd at 3,000 rpm to separate the heatstable cxotoxin and spore crystal complex. Thea purified exotoxin was obtained by the procedure of Y. T. Kim and H. T. Huang (J. IrlleMw. I%ll/Ol., 51, 100-108, 1971). I~resh, vigorous second-stage larvae of Meloirloyyne spp. were obtained bg a modifknt.ion of method folked by A. 31. Fallis (Can. Pub. Health J., 34, 44, 194s). The lnrvw thus obtained were sterilized a?cr)rding to t,hr method of E. C. Dough&J and H. C. Calhon (9wt. Rec., 100, 395, 1948). To identify the nematicidal fraction, the cffwts of spore-crystal complex and crude, aut,oclavcd exotoxin were compared with those of whole culture at different dilutions. In addition, the effects of the crude, autoclavcd esotoxin were also compared with those of the purified exotoxin. The dilutions \v(‘r(’ made with sterile distilled water. Copyright All rights

thuringiensis Knot

Larval

spp.,

the

Disease

Into 2 ml of the test fraction of the dcsired concentration in the cavity of sterilized cavity block, 50 sterile, second-stage larvae wwe introduced by a capillary tube. The cavity block was covered with a glass plate and incubated at 30°C. It was wplicntcd thriw. The percent mortality in all thr treatments nas obwrved after 1 day, 2 days, and 7 days of t~rc~atment~(Tabk 1 ). TABLIS EFFICCT

r F VMILUS

Bacillus

ihuringiensis

SURVIVAL

OF

v.\tt. SEC!CtRD

Meloidogyne

1 g (/: y 2 3

4 .‘J 6 7 8

1

CULTURIL

(>cJMPI)NI’:NTS

thuringiensis 8T.iGtC

L:WVAt:

OF

SPP.

~

T<> Mortality 3 ad 4 -__

Purified exotoxin Purified exotoxin Whole culture Whole culture Autoclaved exotoxin (crude) Autoclaved exotoxin (crude) Spore-crystal complex Control

OF

ON THE

10 100 Undiluted 10 Undiluted 10 ITndiluted

2 -1

R d tr __

1LOO 100 100 4 9.3 26.8 9.5 100 100 0 3 20 94 100

100

0

-4

1 18

0 00

0

0 0

i I li Percentage mortality was calculated by following Abbot,t’s formula: (r<, mortality = (S - I’)/S X 100. S = 74 skival in control; I- = W, survival in treatment. In a similar manner t,he effects of test fractions were also studied, using the percentage of eggs of equal size hatching as the criterion. Six replications were made for every concentration of each fraction. The percentage of eggs hakhing was recorded aft.er 7 days.

378

NOTES TABLE

2

EFFECT OF Hac~il1u.s thuringieruis V.W. giensis EXOT~XIN ON EGG H.ITCHING ikfeloifiog,yne sm. -

thrtrinOP'

7

Treatment

5; hatch

52 cn 1 2

3 -

-

Exotoxin (undiluted) Exotoxin diluted (10 times) Control

The hatched larvae were counted and removed at’ regular intervals. At the end of 7 days all the hat’ched larvae \vere removed by use of a glass capillary, and the egg masses wrre teased with needles under a stereoscopic microscope. Two to 3 drops of Clorox was added in order to release the free, unhatched eggs. In every replication the volume was made up to 100 ml, and t’he total number of unhatched eggs n-as count’ed. From the total number of eggs, unhatched and hatched, the percentage hatch was calculated (Table 2). From t,he results it is evident that only t,he heat-stable exotoxin is highly nematicida1 whereas the sport-crystal complex did not show any effect. The effectiveness of exot,oxin was further confirmed with purified exotoxin, which showed 100 % mortality within 24 hr, ruling out t’hc possibility of other bacterial metabolites.

From the above experiment it is evident that the cxotoxin which is produced during growth and sporulation of B. thuringiensis var. thwin~ier~~is has a high nematicidnl activity under the laboratory conditions. B. thwirlgiensis was abscnb in soils with high root knot nematode disease infestation and was observed in soils with low infestation of the disease under natural conditions was reported by S. 8. S. V. Prasad and I<. V. B. R. Tilak (I&. J. fllz’crobiol. 11, 5940, 1972). l’urthcr investigations on the effectiveness of B. thurirryiensis VU. thwi~nqierlsis on the rootknot’ disease incidence under natural conditions are in progress. Sincere thanks are due to Indian Council of Agricultural Besearch for awarding a Junior Fellowship to the senior aut,hor. The assistance and facilities extended by I>r. Maharaj Hingh, Addit,ional Director, Experiment Station; Drs. Y. I>. Nene, S. M. Sicora, and Mr. K. Hitaramaih are also gratefully acknowledged. This report represents Research Publication Journal Series So. 405 from the Experiment Stntion of this university. S.

SATYA

I<.

CT.

VARA

SAI

Ii. V. B. R.

PRAHAD'

TILAK

C:OLLAKOTA

Department of Microbioloyy, College of Basic Sciemes and Huma?lities G. B. Parlt I;tli~ersity of Agriculture and Techtrology Panttlaqar (Naitli Tal), C:.P., Idia Receiljed

Xeptembes

14, 1971

i Present address : Jlepartment of and Pharmacology, Indian Institute Bangalore-12, India.

Microbiology of Science,