42

The instivigations for evualting the efficacy of different isolates of Metarhizium

anisopliae (Metc.) against mahogany borer, Hypsipyla robusta and the arboreal termites,

Odontotermes spp., forest nurseries and plantations were surveyed in different districts

of Karnataka and detailed field and laboratory trials were conducted during 2007-2010.

The details of the methodology followed is elaborated in the chapter.

3.1 Incidence of shoot borer and termites

3.1.1. Study area

The damages caused by shoot borer Hypsipyla robusta and termite Odontotermes

obesus and O. redemanni were surveyed in the forest nurseries and plantations of

Karnataka. The forest divisions and ranges surveyed were: Bangalore rural-, Hosakote,

Devanahalli, Doddaballapur [920 m; 969 mm; 18.6 ºC - 29.0 ºC], Ramanagaram-[668 m;

766 mm; 22.2 ºC - 36.0 ºC], Mandya - Maddur [678 m; 766 mm; 20.2 ºC - 38.0 ºC],

Mysore- Hunsur, Priyapatna [770 m; 782 mm; 11.6 ºC - 38.0 ºC], Coorg- Madikeri,

Titimati [1525 m; 1892 mm; 11.0 ºC - 27.0 ºC], Hassan- Sakleshpur, Belur, Alur (943 m;

1200 mm; 18.2 ºC - 29.8 ºC], Chikmagalur- Sringeri, Mudigere, Kadur [1037 m; 1990

mm; 16.0 ºC - 32.8 ºC], Mangalore- Sullya, Puttur, Bantval (99 m; 3479 mm; 23.5 ºC -

30.0 ºC], Shimoga- Bhadravathi, Tirthahalli, Sagar [640 m; 1900 mm; 26.5 ºC - 40.0 ºC]

and Uttar Kannada- Yellapur, Mundgod [586 m; 1479 mm; 20.3 ºC - 33.0 ºC] (Plate-1).

The altitude, average rainfall and temperature of all the surveyed sites were also

recorded. Vegetation of the surveyed areas were composed of high and dense natural and

plantation forests comprising the species, Eucalyptus spp., Santalum album, Acacia spp.,

43

Albizia spp., Azadirachta indica, Casuarina equisetifolia, Dalbergia sissoo, Gmelina

arborea,. Prosopis spp., Shorea robusta, Terminalia spp. Tectona grandis Swietenia

mahagoni etc..

3.1.2 Incidence of H. robusta in Mahogany

A field survey was conducted in the nurseries of mahogany growing area

throughout the year (2008) in monthly intervals. The saplings of mahogany were

observed for signs of the borer attack such as recent exit holes, residues of larval feeding

and fresh larval frass. For determination of borer density, every plant was observed by

direct visual counting. The abundance of borer attack was expressed as number of

infested branch or lavae per plant. Height and Diameter at Breast height (DBH) were

taken for every plant in the study plots.

3.1.3 Incidence of Odontotermes spp. on plantation trees

A field survey was conducted in the plantation of teak, eucalyptus and sandal for

the presence of Odontotermes infestation. The numbers of trees infected with the mud

galleries on the stem were counted. The soldiers were collected from the galleries inside

mound and identified in the laboratory based on the termite identifying keys.

3.2 Collection, isolation and preservation of M. anisopliae isolates

3.2.1 Collection of cadavers

Different places in South India viz., Mysore, Yellapur, Mangalore, Madikere,

Sullya, Puttur of Karnataka, Pallakad and Manadwadi of Kerala and Udakamandalam,

Madurai of Tamil Nadu were surveyed for collection of infected insect specimens. Dead

44

insects were collected from plantations of teak, mahogany, coffee, sandal, and eucalyptus

during the time of pest attacks. Cadavers were collected in sterile glass vials and

processed in the laboratory for isolation of fungal pathogens.

3.2.2 Isolation of Metarhizium anisopliae

The cadavers were surface disinfected with 1% sodium hypochlorite solution and

placed on water agar medium amended with antibacterial agents. The plates were sealed

and incubated at 26 ± 0.3 ºC for seven to fourteen days. The cadavers with hyphae were

then transferred to selective medium (dextrose- 1 %; peptone- 1 %; oxgall- 1.5 %; agar-

3.2 %; dodine- 10 µg/ ml; cyclohexamide 250 µg/ ml and chloramphenicol- 500 µg/ ml)

(Veen and Ferron, 1966 and Liu et al., 1993) for the isolation of M. anisopliae. The

isolated colonies were grown on Sabouraud dextrose medium (Hi-Media) fortified with 1

% yeast extract at 26 ± 0.3 ºC in dark for seven days.

3.2.3 Identification of isolates (Humber, 1997)

Slide culture of the fungal isolates was carried out to critically observe the

conidiogenesis. Small blocks of agar medium (~ 1cm2) were placed on the sterile glass

cover slips (22 x 30 mm) which were placed in 2% water agar medium in a Petri dish.

The agar blocks were inoculated with 5 µl of culture and cover slips were placed over the

agar blocks and incubated at 26 ± 0.3 ºC in dark. After 12-14 hrs of incubation the agar

blocks were observed under the microscope for presence of philades using identification

keys.

3.2.4 Preservation of isolates

45

For the long-term storage The isolated and identified pure cultures were stored in

mineral oil. Sterile mineral oil was added to the vigorously growing culture slants in glass

culture tubes by covering the culture slants to the depth of 1 cm. The tubes were sealed

and stored upright in racks in deep freezer at -20 ºC.A batch of purified isolates were

maintained on on Sabouraud Dextrose Agar supplemented with yeast extract (SDAY)

and stored at 4 ºC for routine bioassay .

3.2.5 Viability of spores (Milner et al., 1991 and Inglish et al., 1996)

The viability of the fungal spores was assessed by direct enumeration of the

spores germinated. Conidial suspension of 100 µl (1 x 106 conidia ml-1) was spread on

the germination medium [yeast extract- 0.1%, chloranphenicol- 0.1%, tween 80- 0.01%

and 0.001-0.005% benlato (wp)] in petri plates. The plates were incubated at 26 ± 0.2º C

in the dark for 18-24 h and a drop of fixative (Lactophenol) was added on the surface of

the medium and cover slip was placed over the area. The number of spores showing germ

tube above 90% per microscopic field were considered as a viable culture.

Percentage of germination was calculated by the formula

% germination= (a/ a + b) x 100 where a-spores germinated, b-non-germinated spores

3.3 Rearing and maintenance of pests in laboratory

3.3.1 Rearing of Mahogany shoot borer

H. robusta larvae were collected from the nurseries in South Canara district of

Karnataka and reared in an environmental chamber under a photoperiod of 10:14 h (L: D)

at >85% RH and a temperature of 26 ± 0.2ºC. The larvae were reared on a modified

46

semi-synthetic diet (Ramareshiah and Shankaran, 1994). The larvae were reared on diet

served in petri plates or in plastic tubes. A new method, which emulated the natural

habitat of the pest, is also developed for rearing the borer larva. Bamboo sticks with

hollow cavities were cut into pieces (approx: Length=15cm x Diameter=8mm) and

packed with the diet. Before filling the diet, the pieces were disinfected by dipping in 3%

sodium hypochlorite solution for 2hrs and drying in oven. Individual larva was

transferred to the sticks filled with diet and plugged with cotton wool to prevent larva

from escaping. After the complete consumption of the diet, the larvae were transferred to

new sticks filled with fresh diet. This procedure of rearing was adopted considering the

boring behavior and cryptic nature of the larva in the field. After pupation, cotton plugs

were removed and the sticks were kept in cages with potted mahogany plants for insect

emergence, mating and egg lying. Leaves with eggs were transferred to glass bottles and

incubated at 26 ± 0.5ºC & 70-75% RH to facilitate hatching (Plate-2).

3.3.2 Collection and maintenance of termite culture

3.3.2.1 Collection of termites from field

Workers and soldiers of O. obesus and O. redemanni along with some portion of

termite comb and mound material were collected directly from mound in a separately 25

x 15cm (Lx W) plastic buckets. The buckets were covered with black plastic sheets and

brought to the laboratory. Similarly, Odontotermes sp. were collected by installing stakes

of the rubber wood (Hevea brasiliensis). The stakes measuring about one foot were

buried half in the termite infested area of plantations of teak and sandalwood. The

infested stakes were brought to the laboratory in plastic buckets

47

3.3.2.2 Maintenance of termite culture in laboratory (Verma, 1986)

Workers and soliders of Odontotermes sp. along with soil from mud galleries on

termite-infested trees and termite mounds were collected in plastic boxes 10 cm x 5 cm)

and brought to the laboratory. They were kept in glass jar and maintained in a

biochemical oxygen demand incubator at 25 ± 1°C. A glass jar (18 cm x 18 cm) was

filled with soil upto 4 cm height. Soils from the inner nest wall was used. Two small

pieces of rubber wood were partially buried into the soil for food. The fungus combs

together with termites and nest debris were added and the container was closed with a

perforated lid. To each jar was about 5-6 grams of termites were incorporated in addition

to the young larvae which usually adhere to the fungus combs. At the time of setting up

the culture the soils in the glass jar was watered slightly.

3.4 Pathogenecity tests

3.4.1 Preparation of spore concentration

Eighteen isolates of M. anisopliae collected from different insect species ( table 3)

along with four standard isolates from ARSEF (ARSEF 7413, ARSEF 1498, ARSEF

3290 and ARSEF 3603) were maintained in the laboratory and used in this study. About

100µl of spore suspension (1 x 106 conidia ml-1) were spread plated on SDAY medium

and incubated in dark at 26 ± 0.3 ºC for seven days. The plates were removed and aerial

conidia were harvested by flooding cultures with sterile water containing 0.05% Tween-

80. The conidia suspension was then vortexed at 2000 rpm for 20 min to detach conidia

form the conidiophores and filtered using two layer of cheesecloth. The viability of the

cultures were studied by the method described in the session 3.2.5. The fungal cultures

48

that showed above 90% germination were vortexed and enumerated using

haemocytometer. The suspension wasdiluted to obtain the desired concentrations of 1 x

104, 1 x 105, 1 x 106, 1 x 107 and 1 x 108 conidia ml-1 by following the methodology

described in www.lubilose.org/ laboratory techniques/ section-iii/ page-13 and 14.

3.4.2 Bioassay against H. robusta

3.4.2.1 Single concentration assay

The different larval instars (1st, 2nd, 3rd, 4th and 5th) of the shoot borer were

collected separately from a single batch of eggs hatched in the insectaria. A single

concentration of 108 conidia ml-1 was prepared and larvae were dipped separately in

conidial suspension for 5 seconds and transferred to bamboo sticks packed with artificial

diet. Care have been taken for collection of larval stages after molting. The control larvae

were treated with 0.05% Tween 80. Five replicates of five larvae each for each treatment

were maintained. The sticks were incubated for twelve days at 26 ± 0.3 °C. The mortality

and mycosis were recorded at 24 hrs intervals for 12 days.

3.4.2.2 Multiple concentration assays

In the multiple concentration assays, four concentrations viz., 1 x 104, 1 x 105, 1 x

106 and 1 x 107 conidia ml-1 were prepared separately from the eighteen isolates of M.

anisopliae. The second instar larvae with uniform size (≈ 6 mm) were collected and

dipped in the suspension for 5 sec. The larvae were transferred to bamboo sticks packed

with artificial diet and incubated for twelve days at 26 ± 0.3 °C to observe the mortality

and mycosis. The control larvae were treated with 0.05% Tween 80. Five replicates of 5

larvae each for each treatment were maintained.

49

3.4.2.3 Estimation of sporulation on the cadavers

To assess sporulation, cadavers from each treatment was individually transferred

to 1.5% water-agar medium and incubated for 7 and 15 days at 25 ± 0.5°C and >85%

RH. Following incubation, cadavers from each dose were transferred individually to 10

ml of sterile 0.05% Tween-80 and shaken to facilitate dispersion of conidia. The

suspension was then filtered through a sterile Whattman No.1 filter paper to remove

larger mycelial particles and serially diluted and spores counted under phase contrast

optics in an improved Neubauer Haemocytometer. The average conidial yields at

different doses and days were estimated

3.4.3 Bioassay against Odontotermes sp.

3.4.3.1 Single concentration assay against O. redemannai (Khan et al., 1991)

Conidial suspension of l ml (108 conidia/ml-1) were prepared separately from

twenty two fungal isolates. The conidial suspensions were poured in sterile petriplates

and allowed to dry partially. Termite workers were allowed to walk on the partially dried

fungal suspension for 1 min. The contaminated workers were transferred aseptically to

sterile petriplates that contained small pieces of rubber wood and pieces of fresh fungal

combs. The plates with treated workers were placed in plastic bags containing moist cloth

and incubated at 26 ± 0.3 ºC in dark. For each isolate, five replicates, each with 20

individual workers, were maintained and mortality was recorded at 24 hr intervals for up

to 10 days. A batch of 100 workers were treated with 0.05% Tween 80 and maintained as

control.

3.4.3.2 Single concentration assay against O. obesus

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Single concentration assay for the twenty two isolates were performed against O.

obesus also as per the method described in 3.4.3.1..

3.4.3.3 Multiple concentration assay against O. obesus

A detailed pathogencity test was conducted for eight M. anisopliae isolates that

produced higher mortality and sporulation against the workers of O. obesus. Four

concentrations viz., 1 x 104, 1 x 105, 1 x 106 and 1 x 108 conidia ml-1 were prepared and

bioassay was conducted using the method described in the section 3.4.3.1. The

experiments were repeated three times.

3.4.4 Horizontal transmission of fungal infection among O. obesus

The horizontal transfer of fungal infection among the termites was studied for five

isolates of M. anisopliae, where twenty workers and twenty soldiers were allowed to

walk over the partially dried fungal suspensions of 108 conidia/ml for 1 min. The treated

twenty workers and twenty soldiers were transferred separately to plastic trays of size 60

cm x 30 cm x 15 cm containing either eighty workers (untreated),or eighty soldiers

(untreated), small pieces of rubber wood and pieces of fresh fungal combs. The trays

were covered with dark moist cloth and incubated at 26 ± 0. 3 ºC in dark. For each

isolate, five replicates, each with twenty (treated) and eighty (untreated) individual

workers and soldiers were maintained separately, and mortality was recorded at 24 hr

intervals for 10 days. A batch of 100 workers and soldiers of infected termites was

maintained separately as control. Dead insects were incubated in a humid chamber to

confirm growth of the fungus on cadavers.

3.4.5 Repellency of termites to dry conidia and conidia mixed with attractant

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Repellency of the workers and soldiers of O. obesus to the five highly virulent

isolates of M. anisopliae was evaluated by modified method of Mburu et al. (2009) and

Hussain et al. (2010). Plastic trays (size: 3 x 2 x 1 ft) were filled with mound soil (up to

1.5 feet). Each tray was separated equally into two compartments A and B with nylon

gauze (60-mesh size) were the compartment A served as the releasing site for the

termites. In the treatments, first set contains (A- termite comb and rubber wood and B-

dry conidia 0.1 g, termite comb and rubber wood), second set contains ( A- termite comb

and rubber wood and B- only attractant, termite comb and rubber wood) and third set

contains (A- termites comb and rubber wood and B- attractant mixed with 0.1 g of

conidia, termite comb and rubber wood ).

About 100 workers and 50 soldiers were released in the compartment A and was

illuminated with florescent bulb (220 V, 13A, AC) for 3 min. The remaining portion of

the trays were shielded with a dark moistened cotton cloth. The combination of

brightness and darkness acted as a “push-pull” set of visual stimuli to induce the termites

to move away from the release area towards the treated compartments. The number of

termites foraging in the treated and control compartment together with those in their

respective arms were recorded at an interval of 10 min upto 90 min to give nine readings

for each replicates and mortality and mycosis was evaluated after 10 days of incubation at

26 ± 0.3 ºC.

3.4.6 Data analysis

Mortality observed in the controls was used to correct mortality in the treated

groups using Abbott’s formula (P= C-T/ C x 100) (Abbott, 1925). The cumulative

52

mortality response for each concentration of the isolates across the assessment period was

analyzed by Kaplan–Meier survival analysis using Probit model. The data from the

mortality, mycosis and sporulation estimates were subjected to arc sine transformation

and analyzed using analysis of variance. The least significant difference test was used to

compare the means. All the analyses were carried out using SPSS 11.0 for Windows. The

relative potency of each isolate was calculated by dividing the LC50 value that of each test

isolate by that of standard (Houping et al., 2002)..

3.5 Toxicity Test

3.5.1 Extraction of crude soluble protein

Conidia were harvested from seven day old culture plates by flooding with sterile

0.05 % Tween-80. The suspensions were filtered through Whattman No.1 filter paper and

10 ml of the filtered suspension was made into a concentration of 108 conidia/ ml-1 using

Neubauer Haemocytometer. About 10 ml of suspension was inoculated into one liter of

medium (dextrose- 40 g/ lit; yeast extract- 20 g/ lit, corn steep liquor- 30 g/ lit). The flask

were incubated as stationary culture for 3 days at 26 ± 0.3 °C and >85 % RH in dark. The

mycelia matt were filtered through Whattman No.3 filter paper and the suspension was

further passed through 0.22µm filter (Millipore). The resultant filtrate was subjected to

precipitation with 90% saturated ammonium sulphate.

The precipitated crude soluble proteins were collected by centrifuging at 10000 g

for 15 min at 4°C. The pellets were re-suspended in 20 mM Tris-HCL buffer (pH-7.5)

containing 1 mM phenyl methyl- sulfonylflouride (PMSF) and 1 mM EDTA. The

solution was desalted in a molecular porous membrane (Spectra Pro®1 Membrane) with a

53

cut off of 6000-8000 Dalton. The desalted fraction was then concentrated at 4°C by

embedding the membrane in polyethylene glycol. The proteins are finally purified with

pre-packed column of SephadexTM G-25 gel filtration media (HiPrepTM Desalting 26/10)

with typical flow velocity of 150 cm/h. The concentrations of soluble proteins were

determined by Lowry’s assay using bovine serum albumin as standard at 280 nm

absorbance in spectrophotometer.

3.5.2 Mycotoxic effects on H. robusta

The toxicity effects of Crude soluble protein extract (CSPE) were studied by

incorporating a standard concentration of 5 mg/ ml viz., 1 to 5 % in to the artificial diet

packed in bamboo sticks. The second instar larvae were transferred to treated artificial

diet and maintained at 26 ± 0.3 °C and >85 % RH in dark. The control larva was

incorporated with sterile buffer (20mM Tris-HCL (pH-7.5); 1 mM phenylmethylsulfonyl

flouride (PMSF) & 1mM EDTA) with different concentration as stated above. Mortality

was recorded at 24 h intervals for 10 days. Four replicates of five larvae each were

maintained for each treatment.

3.5.3 Mycotoxic effects on O. obesus

A 1 ml suspension of each concentration of toxins (1 to 5%) was poured into a

sterile Petri plate and allowed to dry partially. The workers of O. obesus were allowed to

walk over the dried suspension for 1 min. The treated workers were transferred

aseptically to sterile Petri plates with pieces of rubber wood and fungal combs. The plates

were placed in plastic bags containing moist cloth and incubated at 26 ± 0.3 ºC in dark

and the morality were observed at 24 hrs intervals for 10 days. Five replicates of each

54

isolate, each with 20 individual workers, were maintained. A batch 100 workers treated

with sterile buffer (20mM Tris-HCL (pH-7.5); 1 mM phenylmethylsulfonyl flouride

(PMSF) & 1mM EDTA) with different concentration was maintained as control.

3.5.4 Data analysis

The cumulative mortality response for each concentration of the isolates across

the doses used, was analyzed by Kaplan–Meier survival analysis. The LD50 data were

subjected to probit analysis. The analyses were carried out using SPSS 11.0 for Windows.

The relative potency for each isolates was calculated by dividing the LD50 value of each

test isolate by that of standard (Houping et al., 2002).

3.6 Field evaluation of the effective isolates

3.6.1 Field test on the use of M. anisopliae against H. robusta

3.6.1.1 Nursery spray

The nursery study was conducted in May 2010 at Sullya (North Karnataka) forest

range. Four plots of 50 mahogany plants (200 plants/treatments) each of 1 to 2 meters

height were selected. The spore concentration of 2.8 x 109 conidia / ml were mixed with 2

liters of 0.08% Tween 80 and sprayed using a hand sprayer. The control plots were

sprayed with 0.08% Tween 80 and maintained separately. As the infestation in nurseries

start by May each year, the spraying was done in early May and repeated on 7th ,42nd and

49th day which falls by June end when the shoot infestation is in peak. The reduction

percentage was evaluated by observing the mortality and mycosis.

55

3.6.1.2 Apical shoot injection method

The spore concentration of 2.8 x 109 conidia ml-1 was prepared and 0.1ml of spore

was injected in to the infested apical stem. Mortality of the larvae was recorded after

seven days of treatment for a period of 3 months. The control plants were inoculated

with 0.1 ml of 0.08% Tween 80.

3.6.2 Field test on the use of M. anisopliae against O. obesus

3.6.2.1 Trial site/Isolates

The experiment were carried out in the sandal plots at Bangalore rural area

(Gotipura and Nallal). The most promising isolates IWST-Ma13 and ARSEF 7413 were

used in the field study.

3.6.2.2 Harvesting of the fungal conidia

The two effective fungal isolates IWST-Ma13 and ARSEF 7413 were mass

multiplied on the solid substrate using rice grains., About 1 kg of broken rice (variety IR-

20 and IR-50) were soaked in distilled water upto 12 hours and 100g of dried rice were

transferred separately in to ten polythene bags (HiDispo BagTM; size- 40 cm h x 20 cm b).

The mouth of the bags were covered by inserting the tip of the bags in to plastic pipes ( 5

cm h x 3 cm b).The neck of the bags were plugged with cotton and autoclaved. A starter

culture of 10 ml were inoculate in to the rice bags separately and incubated at 26 ± 0.3

ºC in the dark. After twelve days of incubation, the sporulated grains were removed from

the bags and dried in the sterile air station for 18-24 hrs to remove excess moisture. The

56

dry spores were harvested in sterile glass vials using a MycoHarvester (Model-MH5;

Make-UK) and stored at -20 ºC.

3.6.2.3 Preparation of dust and bait mixture

About 10 grams of harvested spores were mixed in 100 gram of mixture

containing sugarcane bagasse (50 gram), cardboard (20 grams) and sawdust (30 grams).

To this mixture, 30 ml of starch water was added and mixed in a Speed Mixer at 3000

rpm for 5-10 min to get a semi-solid paste. The mixture was poured in to the ice-cubes

trays for the preparation of baits and dried in the trays for dust preparations.

3.6.2.4 Stake Test

The field-testing of fungal conidia was conducted using rubber wood as carrier.

The healthy stakes of Hevea brasiliensis (rubber wood) of 30.5 cm long and 3.18 cm

square in cross-section as per Indian Standard (IS: 4833-1968); were treated with the dry

conidia, conidia mixed with attractant and attractant alone by spreading the mixture over

the stakes. Six testing sites in the plantation of teak that was infected by O. obesus were

selected for laying out the specimen for tests. Six replication of each treatment and in

each samples along with three controls (untreated) were buried half below and half

exposed above the ground in horizontal and vertical rows of 60 cm apart. The stakes were

inspected for five months by monthly intervals.

3.6.2.5 Tree treatments

The control of O. obesus attack in heavily infested sandal tree plots. The plant

stem covered with mud galleries were selected around the plots. The experiments have

57

set upped as follows; the mud galleries were gently removed upto 30 cm diameter in the

middle of the infected stem (from base two feet height) leaving the other part intact. The

gunny bags (30 cm) dusted with dry conidia, conidia mixed with attractant and only

attractant were tied separately over the 30 cm area between the mud plasters on the stem.

The mortality, mycosis and width of galleries roofed over the gunny bags were observed

in an interval of (4, 7, 14, 28, 42, 56, 70, 84 and 98 days) for a period of three months.

The control trees were treated with gunny bags containing attractants only. A five

replicate of twenty-five trees each for each treatment were maintained.

3.6.2.6 Soil treatments

The soils were removed up to 15 cm depth around the stem (30 cm diameter) of

sandal tree. The mean average height of the mud galleries on each branches of stem were

measured before starting the experiments. A quantity of eight baits cubes were buried

around the stem of each plant. The width of the mud plaster covered the branches were

observed in a interval of (4, 7, 14, 28, 42, 56, 70, 84 and 98 days) for a period of three

months.. The mortality, mycosis and width of the mud galleries covered over the

branches were observed. The control trees were treated with only bait ie., without

conidia. A five replicate of twenty-five trees each for each treatment were maintained.

3.6.2.7 Mound treatments

3.6.2.7.1 Mound selection

Two to three years old active colonizes of O. obesus was selected before fifteen

days of the experiment by asking the farmers/ forest guards about the age of each mound

in eucalyptus plantations.. The activity of each mound was determined at the time of

58

selection using a “hole repair method” were, rectangular holes at the side of the active

mounds were dug down to the level of the termite comb. The volume of the mound

material removed was measured. The size of the hole was measured after 24 hours.

Mounds with holes sealed within 48 hours time were considered as active mounds.

3.6.2.7.2 Dusting inside the mounds

Conidial dust of (100 g) mixed with attractant were applied by blowing them into

the ventilations holes at the centre (top) of the mound, from a small container connected

to a bicycle pump through a rubber hose. During the operation, a cardboard was used to

cover the hole to prevent the spores dust from puffing out of the artificial hole. After

applications, the holes were covered with plastic sheets to prevent rainwater entering the

holes. The Activity of termite colonies in the mound was measured by AED -2000L

(Acoustic Emission Consulting) Insect pest detection kit at an interval of 0, 1st, 3rd and

end of 5th months. The mounds were cut open to observe mortality and mycosis after five

months of treatments.

3.7 Phenotypic characterization of M. anisopliae isolates

3.7.1 Morphological characteristics

Morphological features like color, size, shape of conidia and media pigmentation

were recorded. The color of the mycelium and media pigmentation on agar plates and

slants were recorded, shape of the conidia were observed using microscope and the size

measured using microscope and stage micrometer.

3.7.2 Cultural characteristics

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3.7.2.1 Effect of temperature on germination of conidia

Effect of temperature on germination was tested for twenty two isolates by

inoculating 2 µl of conidial suspension containing 2 x 108 conidia ml-1 on SDAY

(Sabouraud Dextrose Agar Yeast extract) medium in cavity slides and incubated at 20,

22, 24, 26, 28 and 30 ºC in complete darkness. At 14 hrs post inoculation, 1 ml of

formaldehyde (0.5%) was transferred onto each slide to halt germination. Percentage of

germination was then determined by counting number of spores germinated for each slide

at 40X magnification. Each slide serves as replicate with five replications per treatment.

3.7.2.2 Effect of RH on germination of conidia

Effect of relative humidity on germination was studied for twenty two isolates by

inoculating 2 µl of conidial suspension containing 2 x 108 conidia ml-1 on SDAY medium

in cavity slides. These cavity slides were incubated in dark at different humiditys, viz.,

12%, 33%, 43%, 52%, 78%, 85% and 96% obtained using chemicals (LiCl2 H2O MgCl2,

6H2O; K2CO3, 2H2O; Mg(NO3)2,6H2O; NaCl; KCl and K2SO4). At 14 hrs post

inoculation, 1ml of formaldehyde (0.5%) was transferred onto each slide to halt

germination. Percentage of germination was then determined by counting number of

spores germinated for each slide at 40X magnification. Each slide serves as replicate with

five replications per treatment.

3.7.2.3 Effect of pH on germination of conidia

Effect of pH on germination was studied for twenty two isolates with a pH of 4, 5,

6, 7 and 8). The pH of the medium were adjusted with 0.1N HCL and 0.1N NaOH after

autoclaving. A 2 µl of conidial suspension containing 2 x 108 conidia ml-1 was

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inoculated on SDAY medium in cavity slides and incubated in dark. At 14 hrs post

inoculation, 1ml of formaldehyde (0.5%) was transferred onto each slide to halt

germination. Percentage of germination was then determined by counting number of

spores germinated for each slide at 40X magnification. Each slide serves as replicate with

five replications per treatment.

3.7.2.4 Radial growth

Radial growth was studied for twenty two isolates by spread plating 0.1 ml

conidial suspension containing 2 x 108 conidia ml-1 on SDAY plates. Plates were

incubated at 26 ± 0.3 °C for four days in order to obtain mycelial matts. Mycelial mats

were cut from culture plates into round agar plugs using an 5-mm diameter cork borer.

Each agar plug of 5-mm thick was then transferred onto center of fresh SDAY plates.

Plates were sealed with Parafilm and incubated in complete darkness at 26 ± 0.3 °C.

Radial growth was recorded daily for seven days. Each plates served as replicate with

five replications per treatment.

3.7.2.5 Biomass

Biomass for 25 isolates were determined using 50ml of PDBY (Potato Dextrose

Broth with Yeast Extract) which was placed in 250ml conical flask and autoclaved at

121° C for 20min.The pH of the media was 5.9-6.0 after autoclaving. A suspension of

approximately 3x106 conidia m-1 of each of the 18 isolates was prepared in 0.05% sterile

Tween 80 in distilled water from 2 to 3 weeks old culture on PDAY. One ml of

suspension was used to inoculate each of four flasks per treatment and these were

incubated at room temperature while continuously shaking on a rotary shaker at 150rpm.

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Biomass was determined after 7 days fermentation. The content of the four replicate

flasks under each treatment were distributed into pre-weighed filter paper and filtered.

The pellets/residue were washed in distilled water to remove any remaining traces of

broth. The filter papers with the residue were placed in an oven at 80º until the samples

reached constant weight and total biomass determined by dry weight estimation.

3.7.3 Data analysis

The data were subjected to arc sine transformation and analyzed using analysis of

variance. The least significant difference test was used to compare the means. All the

analyses were carried out using SPSS 11.0 for Windows.

3.8 Enzymatic characterization of M. anisopliae isolates

3.8.1 Chitinase assay (Tikhonov et al., 2002)

Chitinolytic activity was determined by measuring the release of reducing

saccharides from colloidal chitin.. A reaction mixture containing 1 ml of crude soluble

proteins, 0.3 ml of 1M sodium acetate buffer pH 4.7 and 0.2 ml of colloidal chitin was

incubated at 40 ºC for 6- 24 hrs and then centrifuged at 12, 225g for 5 min at 6 ºC. After

centrifugation, an aliquot of 0.75 ml of the supernatant, 0.25 ml of 1% solution of

dinitrosalycilic acid in 0.7M NaOH and 0.1 ml of 10M NaOH were mixed in 1.5 ml

eppendorf tubes and heated at 100 ºC for 5 min. Absorbance of the reaction mixture at

582 nm (A582) was measured after cooling at room temperature. Each tube served as

replicate with three replications per treatment. Calibration curves with N-acetyl D-

glucosamine as standard were used to determine reducing sugar concentration. One unit

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of enzyme activity was defined as the amount of enzyme that released 1µmole of N-

acetyl D-glucosamine(NAGA) per minute.

3.8.2 Chitin deacetylase assay (CDA) (Kulkarani et al., 2008).

Chitin deacetylase activity was measured using acetylated ethylene glycol

chitosan as a substrate. For preparation of the substrate, ethylene glycol chitosan (40 mg)

was treated at 48 °C with 400 mg of NaHCO3 and 200 µmol of acetic anhydride in a total

volume of 4.5 ml and kept at 48 °C. After 24 hrs, 200 µl of acetic anhydride were added

and the mixture was allowed to stand for further 24 hrs at 48 °C .After dialysis with

molecular porous membrane with a cut off of (6000-8000 kDa) the product, acetylated

ethylene glycol chitosan (1 mg/mL) was used as a substrate for the assay of CDA.

The assay for CDA with 100 ml of 50 mM sodium tetraborate buffer, pH 8.5, 100

µl of 1 mg/ ml acetylated ethylene glycol chitosan, and 50 µl of crude soluble protein

incubated at 37 °C for 30 min. The reaction was terminated with addition of 250 µl of 5%

(w/v) KHSO4. For color development, 250 µl of 5% (w/v) NaNO2 was added and

allowed to stand for 15 min, and then 250 µl of 12.5% (w/v) ammonium sulfamate

(N2H6SO3) was added. After 5 min, 250 µl freshly prepared 0.5% (w/v) 3-methyl-2-

benzothiazoline hydrazone (MBTH) was added and the mixture was heated in a boiling

water bath for 3 min. The tubes were cooled under tap water and 250 µl of freshly

prepared 0.5% (w/v) FeCl3 was added and estimated spectrophotometrically at 650 nm.

One unit of enzyme released 1 µmol of glucosamine from acetylated ethylene glycol

chitosan per min ute.

3.8.3 Protease assay (Hossain et al., 2006)

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Protease activity was assayed using casein as substrate. The reaction mixture

containing 3ml of 1% (w/v) Hammerstein casein in 3ml 0.1M citrate phosphate buffer,

pH 7.0 and 3 ml of crude soluble proteins was incubated at 40± 1°C for one hour. The

reaction was stopped by the addition of 5 ml 20% (w/v) TCA and the absorbance was

measured at 650 nm in a spectrophotometer. Each tube served as replicate with three

replications per treatment. The amount of amino acids released was calculated from a

standard curve plotted against a range of known concentrations of tyrosine. One unit of

enzyme was defined as the amount of enzyme that released 1µg of tyrosine ml-1 of crude

enzyme/hr.

3.8.4 Lipase assay (Pignede et al., 2000).

The substrate emulsion was prepared with olive oil (50 ml) and gum arabic (50

ml, 10% w/v). The reaction mixture contained 1 ml crude soluble proteins, 5 ml substrate

emulsion, and 2 mL of 50 mM phosphate buffer, pH 6.8, and was incubated at 37°C for

one hour under shaking at 80 rpm. The reaction was stopped with 4 ml of acetone-ethanol

(1:1) containing 0.09% phenolphthalein as an indicator. Enzyme activity was determined

by titration with 50 mM NaOH of the release of fatty acids. One unit of lipase is the

amount of enzyme that released 1 µmol of fatty acids per min.

3.7.5 Data analysis

The data were subjected to arc sine transformation and analyzed using analysis of

variance. The least significant difference test was used to compare the means. All the

analyses were carried out using SPSS 11.0 for Windows.

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3.9 Phylogenetic analysis of M. anisopliae isolates

3.9.1 DNA extraction

Mycelia and conidia from each isolate were placed on SDA and single spore

colony was grown on Sabouraud dextrose broth (SDB), incubated on shaker (150 rpm) at

room temperature for 5-7 days. Mycelium was recovered by centrifugation and filtration

through Whatman No. 1 filter paper, washed twice with sterilized water, adding liquid

nitrogen and ground until a powder mycelium was obtained.

The powder was extracted by DNeasy Plant Mini Kit® (QIAGEN) following the

manufacturer’s instructions and stored genomic DNA at 4 °C. For each fungal isolate,

about 50 mg of mycelium was homogenized in DNA extraction buffer (100 mM Trizma;

1.4 M NaCl; 20 mM EDTA; 1% polyvinylpyrrolidone; 2% cetyltrimethylammonium

bromide (CTAB); 1% mercaptoethanol; 10 mg/mL proteinase K; pH 8.0) and incubated

for 45 min at 65 °C. After incubation the mixture was extracted twice with 24:1

chloroform:isoamyl alcohol, centrifuged at 3500 rpm min-1 for 15 min and the DNA

precipitated with isopropanol and stored at -20 °C overnight, after which the DNA was

washed with 70% and 95% alcohol, dried at room temperature and re-suspended in 150

µL of TE buffer pH 8.0. The DNA concentration was measured with a fluorometer and

the DNA stock solutions kept at -80 °C until needed.

3.9.2 PCR amplification

The ITS1-5.8s-ITS4 region of rDNA was amplified using the universal primers,

ITS-1 (5’-TCGGTAGGTGAACCTGCGG-3’) and ITS-4 (5’-

TCCTCCGCTTATTGATATGC-3’). Amplification reactions were performed at a total

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volume 25 µl, consisted of template DNA, 200 µMol dNTPs, 0.8 pMol each primer, 10X

PCR buffer and 1U Taq DNA polymerase (Biolabs, England). The condition of

temperature in thermal cycling was one cycle of initial denaturation at 95 °C for 5

minutes, followed by 35 cycles with denaturation at 94 °C for 1 minute 30 seconds,

annealing at 55 °C for 2 minutes, and extension at 72 °C for 3 minutes and a final

extension at 72 °C for 5 minutes. PCR products were separated by electrophoresis in 1%

agarose gels by comparison with 100 bp DNA Ladder.

3.9.3 Phylogenetic analysis

The ITS1 – 5.8S – ITS4 amplified products (about 540 bp) were purified with the

GFX PCR DNA and Gel Band Purification kit (Amersham Biosciences) and sequenced

in an automated system with services provided by Bangalore Gene. The sequences were

aligned using the ClustalW (http://www.ebi.ac.uk/clustalw) program and compared with

those available in the GenBank data base for M. anisopliae and M. flavoviridae using the

Genetic Data Environment (GDE) software and phylogenetic trees constructed using the

Neighbor- joining method (gopher://megasun.bch. umontreal.ca: 70/11/GDE) by 1000

bootstrap resembling. Phylogenetic inferences were performed and exposed using

TreeView.

3.9.4 Sequence submission

The sequence was deposited in GeneBank (NCBI) and Accession Numbers were

received.