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Distribution of Metarhizium anisopliae (Metsch.) Sorokin (Deuteromycotina:Hyphomycetes) in Tamil Nadu, India, its biocontrol potential on Dysdercuscingulatus (Fab.) (Hemiptera: Pyrrhocoridae)K. Sahayaraj a; J. Francis Borgio a

a Crop Protection Research Centre, Department of Advanced Zoology and Biotechnology, St. Xavier'sCollege (autonomous), Palayamkottai, Tamil Nadu, India

First Published:May2009

To cite this Article Sahayaraj, K. and Francis Borgio, J.(2009)'Distribution of Metarhizium anisopliae (Metsch.) Sorokin(Deuteromycotina: Hyphomycetes) in Tamil Nadu, India, its biocontrol potential on Dysdercus cingulatus (Fab.) (Hemiptera:Pyrrhocoridae)',Archives Of Phytopathology And Plant Protection,42:5,424 — 435

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Distribution of Metarhizium anisopliae (Metsch.) Sorokin(Deuteromycotina: Hyphomycetes) in Tamil Nadu, India,its biocontrol potential on Dysdercus cingulatus (Fab.)(Hemiptera: Pyrrhocoridae)

K. SAHAYARAJ & J. FRANCIS BORGIO

Crop Protection Research Centre, Department of Advanced Zoology and Biotechnology, St. Xavier’s

College (autonomous), Palayamkottai - 627 002, Tamil Nadu, India

(Received 17 June 2006)

Abstract40 soil samples were collected from 13 cotton, 12 paddy, 3 coconut, 2 tomato and forest and each onefrom groundnut, coriander, jack fruit, zee mays, potato, blackgram and castor fields. The number offungal isolate population was highest (1.86107) and lowest (1.66107) in the samples 11 (S11) and 15(S18), which were collected from the coriander and paddy fields in Pavoorchathiram, Tirunelveli districtand Sathoor, Virudunagar districts, respectively. Among the 40 samples collected from different areas,21 samples consisted of Metarhizium anisopliae (Metsch.) Sorokin. Minimum M. anisopliae colonies(21 colonies) were recorded from the cotton field soil sample (S14) of Vilupuram District. But thehighest fungal population (4.86104) among cotton fields was also recorded from the samples ofThoothukudy district. M. anisopliae strain could not be isolated from the soil samples collected inTrichy, Madurai, Virudunagar, Kanchipuram, Tiruvallur, Dharmapuri, Salem, Namakkal, Chennai,Cuddalore, Pondicherry, Sivagangai, Dindhugal, Ramanathapuram and Karur Districts. The impact ofall the 21 M. anisopliae isolates and also the standard MTCC892 were tested against Dysdercus cingulatus(Fab.) (Hemiptera: Pyrrhocoridae) adults and the LC50 values ranged from 2.256105 to 3.666108

spores/ml. The lowest (2.256105) and the highest (3.666108) LC50 values were recorded for theisolates CPRC16, and CPRC17, respectively. 11 sporulated D. cingulatus cadavers were observed withthe isolate CPRC18.

Keywords: Metarhizium anisopliae, Dysdercus cingulatus, biological control potential

Introduction

Soil is the main reservoir of infective propagules of many entomopathogenic fungi (Vanninen

1995; Milner et al. 1998a; Abebe 2002). Entomopathogenic fungi may be isolated from the

soil through use of the insect bait method and via selective media. In general, entomo-

pathogenic Hyphomycetes are considered to be weak saprotrophs, but since they possess

Correspondence: K. Sahayaraj, Crop Protection Research Centre, Department of Advanced Zoology and Biotechnology, St. Xavier’s

College (autonomous), Palayamkottai - 627 002, Tamil Nadu, India. Tel: þ0462 2560744. Fax: þ0462 2561765.

E-mail: ttn_ksraj@sancharnet.in

Archives of Phytopathology and Plant Protection

May 2009; 42(5): 424 – 435

ISSN 0323-5408 print/ISSN 1477-2906 online ª 2009 Taylor & Francis

DOI: 10.1080/03235400601160065

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the ability to infect living insects, they can gain access to a living insect relatively free of

competitors (Goettel & Inglis 1997). Ecological studies on the distribution of Metarhizium

anisopliae (Metsch.) Sorokin (Deuteromycotina: Hyphomycetes) has been undertaken,

attempting to link the occurrence of the fungi to soil types, habitats and/or geographical

locations (Rath et al. 1992; Brownbridge et al. 1993; Vanninen 1995). The most virulent

isolates are usually those which cause natural epizootics in a particular host (Milner 2000a,

2000b). However, it is best to search in the environment of the target pest for suitable

isolation of M. anisopliae (Milner et al. 1998a, 1998b; Purwar & Sachan 2006). Brownbridge

et al. (1993) believe that indigenous strains should be preferably used because they are already

well adapted to survive and multiply in their native environment; moreover, using indigenous

strains avoids the introduction of exotic species that might disrupt the ecosystem. No

indigenous Metarhizium isolates have been obtained in Tamil Nadu from natural ecosystem.

Entomofungal pathogens are well-known biocontrol agents that kill their host insects through

invasion and profuse mycelial growth (Nirmala et al. 2005; Padmaja 2005). M. anisopliae was

recorded as a pathogenic fungi to sucking insects such as green stink bug Nezara viridula (L.)

(Hemiptera: Pentatomidae) (Borges et al. 1993; Sosa-Gomez et al. 1997; Daniel et al. 1998),

Chinch bugs of the genus Blissus (Hemiptera: Lygaeidae) (Samuels et al. 2002), plant bug,

Lygus lineolaris Palisot de Beauvois (Hemiptera: Miridae) (Liu et al. 2003) subterranean

burrower bug Cyrtomenus bergi Froeschner (Hemiptera: Cydnidae) (Jaramillo et al. 2005;

Jaramillo & Borgemeister 2006) and malaria mosquito Anopheles gambiae S.S. (Scholte et al.

2006).

Dysdercus cingulatus (Fab.) (Hemiptera: Pyrrhocoridae) is a serious pest of cotton (Leakey &

Peery 1966; David & Kumaraswami 1978), which infests cotton in all the cotton growing

regions of India (David & Ananthakrishnan 2004). Adults and nymphs suck the sap from the

tender parts of the plant and bolls. The plants lose their vigour and bolls open badly with

stained lint. They also suck the sap from seeds and make them unfit for oil extraction. No

reports were available except the work of Borgio and Sahayaraj (2005) on the efficacy of

M. anisopliae on D. cingulatus. They recently reported the ovicidal effect of M. anisopliae on

D. cingulatus eggs. The objective of this study was therefore to isolate M. anisopliae from

different agricultural fields of Tamil Nadu, India and evaluate the relative pathogenicity of

M. anisopliae on D. cingulatus.

Materials and methods

Study area

Soil samples were collected from different agriculture fields of all districts of Tamil Nadu,

India (see Figure 1).

Standard Metarhizium anisopliae MTCC892

Standard M. anisopliae MTCC892 was obtained from the Microbial Type Culture Collection

(MTCC) and Gene Bank, Institute of Microbial Technology, Chandigarh, India. The culture

was used as reference and subcultures were used as standard.

Sample collection

A total of 40 samples (three replicates) of soil from the different field were collected in all

districts of Tamil Nadu. Using a sterilized stainless steel spoon, about 100 g of soil per sample

Distribution of bioefficancy of Metarhizium anisopliae 425

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Figure 1. Map showing the soil sampling sites of Tamil Nadu state.

426 K. Sahayaraj & J. F. Borgio

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was collected in sterile plastic bags (10 cm615 cm) from cultivated and fallow land, forest

floor, and other plantations by removing the soil at the depths of 2 cm, because the majority of

fungal inocula are usually concentrated on the upper soil layer (Ignoffo et al. 1977; Storey

et al. 1989). The collections were carried out from September 2004 to September 2005. All

samples were brought to the Crop Protection Research Centre, Department of Advanced

Zoology and Biotechnology, St. Xavier’s College, Palayamkottai, Tamil Nadu, India and

stored in the refrigerator (LG) at 48C for further microbial analyses.

Total fungal population (TFP)

One-gram of soil sample from each sample site was suspended in 9 ml of sterile distilled

water. The resulting suspension was serially diluted (1071 and 1077) and plated on Potato

Dextrose Agar (PDA) media plates and spread using a sterile L-shaped glass rod. The plates

were then incubated at 268C+ 28C and observed for fungal growth for seven days. The

fungal growth was assessed to quantify the number of colony forming units.

Isolation and sub-culturing of Metarhizium anisopliae from the soil

Fungi which was suspected to be Metarhizium spp. was sub-cultured to obtain pure cultures

of the suspected entomopathogens. Spores were taken from the initial cultures, using a sterile

loop and streaked on PDA plates. The plates were then incubated at 268C+ 28C in a dark

incubator for seven days. Spores growing on these secondary cultures were sub-cultured again

for seven days using the same media to obtain pure cultures.

Storage of pathogens

Stocks of pure cultures of each fungal isolate were grown on sterile test tube slopes with screw

tops. Spores were removed from the sub-culture of each isolate that had been prepared after

the first isolation and re-streaked evenly on separate sterile test tube slopes of PDA. The test

tubes were kept at 268C+ 28C in a dark incubator for 21 days with the cotton pluck and allow

to grow until sporulation. The slope cultures were then maintained at 48C in a fridge.

Working cultures (on PDA plates) were prepared from these slopes for each isolate. Working

cultures were rejected when older than 15 days.

Preparation of conidial suspension

The isolated conidia were sub-cultured in petri plates with PDA medium. The fungal conidia

were collected from 14 day- old cultures incubated at 278C by scrapping off with a glass rod.

A homogeneous conidial suspension was prepared in sterile distilled water by adding few

drops of wetting agent castor oil (0.1%). The conidial concentration of the suspension was

determined using an improved Neubauer Haemo Cytometer. Stock solutions of 3.56106,

3.66107, 7.36107, 2.96107, 2.66107, 1.76107, 3.56107, 2.46107, 2.66107, 3.46107,

1.56107, 1.46108, 6.36107, 1.16108, 2.36107, 3.26108, 1.76108, 2.86107, 3.56108,

4.86107, 1.16107 and 1.96107 conidia/ml were prepared for CPRC1, CPRC2, CPRC3,

CPRC4, CPRC5, CPRC6, CPRC7, CPRC8, CPRC9, CPRC10, CPRC11, CPRC12,

CPRC13, CPRC14, CPRC15, CPRC16, CPRC17, CPRC19, CPRC20, CPRC21 and

MTCC 892, respectively. Four spore concentrations such as 107, 106, 105 and 104 spores/ml

were prepared from the stock cultures of all the isolates including standard and water was

used as untreated control.

Distribution of bioefficancy of Metarhizium anisopliae 427

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Sources of Dysdercus cingulatus

D. cingulatus were collected from the cotton field from different parts of Tamil Nadu, India

and were maintained in the cotton plants and also with soaked cotton seeds. Laboratory

emerged adults (46 hours) were used for the present study.

Laboratory bioassay

Three D. cingulatus adults were released into (500 ml capacity) the containers. Different

concentrations of fungal conidial suspensions separately were sprayed on the pests using a

hand sprayer (Amway product, USA). After 10 minutes they were transferred and released

into a clean plastic container (500 ml). Five replications were maintained for each con-

centration separately. Mycelial growth on the insects and mortality were recorded for every

24 hours until 120 hours.

Statistical analysis

Finney’s (1971) formulas were used to calculate corrected mortality. From the corrected

mortality data, the probability integral of the chi-square distribution and LC50 were calculated

in order to differentiate the difference in efficacy among the isolates.

Results

Distribution of Metarhizium anisopliae isolates from different fields, its sampling area and total

fungal population (TFP) are presented in Table I. 13 cotton field soil, 12 paddy field soil,

3 coconut field soil, 2 samples of tomato field and forest soil and each soil sample from

groundnut, coriander, jack fruit, zee mays, potato, blackgram and castor (Table I) were

collected from Tamil Nadu. The results revealed that the number of fungal populations was

highest (1.86107) and lowest (1.66102) in samples 11(S11) and (S18) collected from

coriander and paddy fields in Tirunelveli and Virudunagar districts, respectively.

Among the 40 samples analysed, 21 samples contain M. anisopliae strain, identified based on

the colonial morphology and sporulating structures (Matsushima 1975, 1993; Domsch et al.

1980; Pitt & Hocking 1997). The isolates were named as CPRC (Crop Protection Research

Centre) series. The highest number of M. anisopliae colonies (21 colonies) was recorded from

the cotton field sample (S14, Table I) of Vilupuram Districts, followed by 12 colonies from the

cotton field samples of Thoothukudy districts. The highest fungal population (4.86104)

among cotton fields was also recorded from the samples of Thoothukudy district. Even though

the highest fungal population was recorded among the paddy field, soil samples of Trichy

districts do not contain M. anisopliae. Similarly M. anisopliae strain was not isolated from

the soil samples collected in Trichy, Madurai, Virudunagar, Kanchipuram, Tiruvallur,

Dharmapuri, Salem, Namakkal, Chennai, Cuddalore, Pondicherry, Sivagangai, Dindhugal,

Ramanathapuram and Karur Districts (Table I).

The LC50 value of the 21 M. anisopliae isolates and the standard MTCC892 against

Dysdercus cingulatus adults are presented in Table II. The LC50 value of the 21 Metarhizium

anisopliae isolates and the standard MTCC892 against Dysdercus cingulatus adults ranged from

2.256105 to 3.666108 spores/ml. Among the isolates, the lowest LC50 value (2.256105)

was recorded by the isolate CPRC16 and the highest LC50 value of 3.666108 with the isolate.

Sporulated Dysdercus cingulatus adult cadavers treated with Metarhizium anisopliae isolates are

represented in Figure 2. CPRC1, CPRC4, CPRC6, CPRC8, CPRC9, CPRC10, CPRC12,

428 K. Sahayaraj & J. F. Borgio

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CPRC16, CPRC18 and MTCC 892 isolates were sporulated on D. cingulatus cadavers. The

highest number (11) of sporulated D. cingulatus cadavers were recorded in the CPRC isolates.

Discussion

From the 40 samples collected from different field soils, only 52.5% contain fungal isolates of

Metarhizium anisopliae. Of 419 soil samples collected from a storal zone in Tasmania,

Table I. Distribution of Metarhizium anisopliae isolates from different field, its sampling area and total fungal

population (TFP).

Sample

name District name Village name Nature of field

TFP

(CFU/g)

No. of

isolates

Isolate

name

S1 Tirunelveli Melapalayam Kitchen garden 6.76103 1 CPRC 1

S2 Kanyakumari Ethamozhli Coconut 3.66103 3 CPRC 2

S3 Tirunelveli Kallidaikurichi Paddy field 9.06103 1 CPRC 3

S4 Tirunelveli Seithunganallore Paddy field 5.26104 2 CPRC 4

S6 Villupuram Janagipuram Paddy field 7.16104 1 CPRC 5

S7 Tirunelveli Manimuthar Forest 3.56103 1 CPRC 6

S8 Ariyaloor Pichanalloore Groundnut 2.96106 1 CPRC 7

S9 Trichy Paulpannai Paddy field 1.96105 – –

S10 Madurai Mattuthavani Paddy field 4.96104 – –

S11 Tirunelveli Pavoorchathiram Coriondre 1.86107 – –

S12 Tiruvannamalai Cherinthal Paddy 2.36103 1 CPRC 8

S13 Perambalur Thuraimangalam Cotton 2.26103 1 CPRC 9

S14 Viluppuram Ulundurpet Cotton 3.96104 21 CPRC 10

S15 Vellore Sainathapuram Paddy 4.06102 1 CPRC 11

S16 Nagapathinam Mailaduthurai Coconut field 1.36103 3 CPRC 12

S18 Virudunagar Sathoor Paddy 1.66102 – –

S19 Tuticorin Santhosh Nagar Cotton 4.86104 12 CPRC 13

S21 Kanchipuram Collectrate Cotton 1.26103 – –

S22 Tiruvallur Puduvayal Paddy 1.76102 – –

S27 Erode Bavani Cotton 3.06103 1 CPRC 14

S28 Dharmapuri Bommiddi Paddy 4.26102 – –

S29 Salem Omalur Cotton 8.06102 1 –

S30 Namakkal Kalanagam Tomato 1.96103 – –

S31 Chennai Vadapalani Paddy 1.76103 – –

S32 Cuddalore Pudukoppam Coconut 4.16103 –

S33 Thanjavur Nacheeyarkovil Cotton 8.06102 5 CPRC 15

S34 Thiruvarur Melamnakkal Cotton 1.06103 4 CPRC 16

S35 Pudukkottai Adonakkottai Cotton 4.76103 3 CPRC 17

S36 Pondicherry Bahour Jackfruit 3.66103 – –

S37 Sivagangai Thirupallode Cotton 2.06102 – –

S39 Dindhugal Otanchathiram Zee mays 2.66103 – –

S40 Nilgiri Kottamanthu Potato 5.76103 2 CPRC 18

S41 Coimbatore Lawdi Road Cotton 5.16102 2 CPRC 19

S42 Theni KRR Nager Forest 8.46103 7 CPRC 20

S43 Nilgiri Kottamanthu Carrot 1.16103 3 CPRC 21

S44 Coimbatore Maruthamalai Cotton 4.16103 – –

S45 Theni Periakulam Black Gram 7.46103 – –

S46 Dindhugal Otanchathiram Castor 3.46103 – –

S47 Ramanathapuram Kamudi Paddy 6.36102 – –

S49 Karur Jegateswaram Cotton 2.16103 – –

– indicates absence of Metarhizium anisopliae isolates.

Distribution of bioefficancy of Metarhizium anisopliae 429

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Table II. Efficacy of Metarhizium anisopliae isolates against Dysdercus cingulatus adults.

Isolate

name

Con. of

spore

Actual

mortality

(%)

Corrected

mortality

(%) LC50

Fiducial limits

Lower Higher Slope Intercept Chi2

CPRC 1 2.96104 46.66 38.46 2.596106 2.416106 2.806106 18.10338 9.56746 6.05809

4.16105 53.33 46.15

4.76106 66.67 61.54

3.66107 80.00 76.92

Control 13.33 0

CPRC 2 1.76104 40.00 35.71 2.356105 2.206105 2.516105 18.41517 9.83713 4.21005

3.66105 80.00 78.57

1.96106 80.00 78.75

1.46107 93.33 92.86

Control 6.66 0

CPRC 3 4.16104 33.33 16.67 2.756106 2.576106 2.946106 22.5087 11.30330 4.15749

2.86105 33.33 16.67

3.16106 66.67 58.33

3.561087 86.67 83.33

Control 20.00 0

CPRC 4 5.26102 20.00 7.69 3.056105 2.756105 3.386105 16.90762 8.81792 1.69655

2.46103 20.00 7.69

1.26105 53.33 46.15

8.06106 66.67 61.54

Control 16.67 0

CPRC 5 4.36104 40.00 30.77 2.656106 2.466106 2.876106 18.11341 9.52336 4.11091

2.96105 46.67 38.46

4.76106 73.33 69.23

3.56107 73.33 69.23

Control 13.33 0

CPRC 6 2.76105 46.67 11.11 2.906108 2.736108 3.096108 28.60961 13.64351 14.04556

6.16106 53.33 22.22

3.16107 66.67 44.44

4.86108 80.00 66.67

Control 40.00 0

CPRC 7 2.46105 40.00 10.00 2.606106 2.486106 2.726106 31.6354 15.16898 4.77726

2.96106 66.67 50.00

3.06107 86.67 80.00

1.86108 93.33 90.00

Control 33.33 0

CPRC 8 1.16105 53.33 22.22 2.496106 2.396106 2.596106 36.68334 17.38785 17.76429

4.76106 86.67 77.78

2.36107 93.33 88.89

3.66108 100 99.99

Control 40.00 0

CPRC 9 4.36105 66.67 44.44 2.476106 2.376106 2.586106 33.87244 16.23769 31.2109

4.36106 86.67 77.78

4.16107 86.67 77.78

4.66108 100.00 99.99

Control 40.00 0

(continued)

430 K. Sahayaraj & J. F. Borgio

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Table II. (Continued).

Isolate

name

Con. of

spore

Actual

mortality

(%)

Corrected

mortality

(%) LC50

Fiducial limits

Lower Higher Slope Intercept Chi2

CPRC10 1.26105 46.67 33.33 2.596106 2.436106 2.776106 21.98937 11.17765 6.10858

2.36106 53.33 41.67

2.36107 73.33 66.67

5.16108 86.67 83.33

Control 20.00 0

CPRC11 1.06104 33.33 16.67 2.696106 2.526106 2.876106 23.29842 11.64468 2.60163

2.66105 40.00 25.00

4.46106 73.33 66.67

1.56107 86.67 83.33

Control 20.00 0

CPRC12 6.36105 33.33 11.11 2.656107 2.546107 2.766107 37.59495 17.57691 4.79184

1.26106 66.67 44.44

4.06107 93.33 88.89

1.46108 93.33 88.89

Control 40.00 0

CPRC13 4.36104 33.33 33.33 2.476106 2.306106 26.66106 17.59669 9.43837 1.25999

5.76105 46.67 46.67

4.96106 73.33 73.33

6.36107 86.67 86.67

Control 0 0

CPRC14 7.06105 46.67 20.00 2.486106 2.376106 2.606106 31.80865 15.36348 13.58827

4.56106 86.67 80.00

1.16107 93.33 90.00

1.16108 93.33 90.00

Control 33.33 0

CPRC15 2.16104 53.33 41.67 2.376105 2.326105 2.566105 24.21252 12.29404 11.1733

6.06105 86.67 83.33

7.16106 86.67 83.33

2.36107 93.33 91.67

Control 20.00 0

CPRC16 6.36105 53.33 46.15 2.256106 2.126106 2.386106 24.32524 12.47841 7.95032

5.06106 93.33 92.31

1.56107 100.00 99.99

3.26108 100.00 99.99

Control 13.33 0

CPRC17 1.16105 6.67 6.67 3.666108 2.946108 4.556108 9.86829 5.796213 3.42104

1.56106 6.67 6.67

1.36107 13.33 13.33

4.76108 53.33 53.33

Control 0 0

CPRC18 3.36104 53.33 41.67 2.256105 2.386105 2.736105 20.13633 10.43938 11.0791

2.56105 66.67 58.33

3.46106 73.33 66.67

2.86107 80.00 75.00

Control 20.00 0

(continued)

Distribution of bioefficancy of Metarhizium anisopliae 431

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Table II. (Continued).

Isolate

name

Con. of

spore

Actual

mortality

(%)

Corrected

mortality

(%) LC50

Fiducial limits

Lower Higher Slope Intercept Chi2

CPRC19 6.16105 46.67 46.67 2.296106 2.36106 2.476106 17.25636 9.42457 5.16675

3.86106 80.00 80.00

4.46107 80.00 80.00

3.56108 86.67 86.67

Control 0 0

CPRC20 2.76104 13.33 13.33 2.546105 2.366105 2.746105 18.47499 9.56126 2.45513

3.56105 60.00 60.00

5.36106 60.00 60.00

4.86107 86.67 86.00

Control 0 0

CPRC21 2.06104 33.33 23.08 2.716107 2.526107 2.926107 19.91275 10.23483 8.75928

1.26105 40.00 30.77

1.06106 46.67 38.46

4.16107 93.33 92.31

Control 13.33 0

MTCC892 5.86104 40.00 40.00 2.746107 2.456107 3.066107 11.83536 6.90486 4.67067

6.16105 40.00 40.00

5.26106 46.67 46.67

6.66107 66.67 66.67

Control 0 0

Figure 2. Sporulated Dysdercus cingulatus adult cadavers treated with Metarhizium anisopliae isolates.

432 K. Sahayaraj & J. F. Borgio

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Australia, 28% (119) contained M. anisopliae (Rath et al. 1992). Vanninen (1995) in Finland

obtained from a total of 590 soil samples, 117 (19.8%) and 92 (15.6%) M. anisopliae and

B. bassiana isolates, respectively. Possible reasons for the high success in isolating

entomopathogenic fungi during this present study might have been the use of new

media and/or the fact that most samples were collected from wet soils and in irrigated

zones. M. anisopliae is more often isolated from loamy soil, relative to other soil types

(Rath et al. 1992; Steenberg 1995). Its abundance is related to high rainfall (Milner et al.

1998a; Rath et al. 1992; Steenberg 1995; Vanninen 1995). From this study it is not possible to

relate the distribution of M. anisopliae to different soil types or different climatic zones,

because of the small size of samples. M. anisopliae is considered to be thermophilic as it is rare

in cold areas (Vanninen 1995). Vanninen (1995) and Steenberg (1995) reported that

M. anisopliae is less severely affected by cultivation. However, Milner (1992) indicated a

scarcity of M. anisopliae in frequently cultivated fields in relatively dry and hot climates.

Harney and Widden (1991) suggested that forest soil was a natural reservoir of a broad

spectrum of entomopathogenic fungi; this was later also supported by Brownbridge et al.

(1993).

Benzophenylureas (Chakraborti & Chatterjee 1999) and DDT 5% (David &

Ananthakrishnan 2004) were used for red cotton bug control, but these chemicals have

been causing plant damage (Maranpuri & Khachatourians 1996). Moreover, the synthetic

insecticides have led to development of resistance, outbreak and resurgence of the insect-pest,

pollution hazards and disruption of the eco-balance (Chakraborti & Chatterjee 1999). These

environmental concerns urge the replacement of existing insecticidal controls by biological

pest control methods. Among the biological control agents reported against insects,

entomopathogenic fungi are considered the most suitable (Nahar et al. 2004; ICAR 2004;

Padmaja 2005). The green muscardine soil inhabiting the fungus M. anisopliae was reported

to attack a wide range of insect hosts, including Orthoptera, Coleoptera, Lepidoptera,

Hemiptera, Hymenoptera, and also soil living Arachnida (Abebe 2002; Purwar & Sachan

2006).

LC50 values were calculated after converting the percentage into probit values and the

relative potency of different isolates was worked out using probit regression analysis (Finney

1971). Results of the pathogenicity tests showed that entomopathogenic fungi have potential

in controlling the sucking pests Dysdercus cingulatus. All the M. anisopliae isolates tested were

pathogenic to D. cingulatus adults. Furthermore, the percentage of mortality was directly

proportional to the spore concentrations. Recently Borgio and Sahayaraj (2005) reported the

ovicidal effect of entomopathogenic fungi M. anisopliae isolates on D. cingulatus eggs.

Easwaramoorthy et al. (2001) recorded 10.00 – 90.00% mortality by M. anisopliae isolate

against sugarcane internode borer, Chilo sacchariphagus. But we observed 6.67 – 99.99%

mortality in D. cingulatus adults by M. anisopliae. Rath et al. (1995) linked spore surface

antigens of M. anisopliae to pathogenicity. Some of the tested isolates also sporulated well on

D. cingulatus cadavers. Differences in virulence against D. cingulatus were observed between

the isolates. The present study favours the findings of Gindin et al. (2001) and Zimmerman

(1993) that the rate of infection varies from strain to strain. Entomopathogenic fungi enter

into their hosts by direct penetration of the cuticle that functions as a barrier against most

microbial attack. Consequently, fungal entomopathogens have a particularly high potential for

biological control of sap-sucking insects that are difficult to combat with synthetic insecticides

(Kang et al. 1999). The conidia of entomopathogenic fungi are slimy and attach to the

cuticles of insects. The fungi infect insects by producing hyphae from germinating spores that

penetrate insects’ integument, and then destroy the internal contents and the insect dies

(Johri & Johri 2003). From the LC50 results it is evident that soil fungi M. anisopliae can kill

Distribution of bioefficancy of Metarhizium anisopliae 433

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D. cingulatus. Further mass production and field efficacy studies are essential before

recommending this biological control agent.

Acknowledgements

Our sincere thanks to Rev. Dr. A. Antonysamy S. J., Principal and Prof. M. Thomas

Punithan, Head, Department of Advanced Zoology and Biotechnology, St. Xavier’s College,

Palayamkottai for the laboratory facilities.

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