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Much work has been carried out on different aspects of pest management in

forestry, and still mahogany cannot be grown successfully due to the classic low-density

pest, Hypsipyla robusta Moore. Even one or two attacks on young trees may render their

future timber production uneconomic. Similarly, the effectiveness of termite control

using entomopathogens in forest pest management, particularly in plantations forestry is

yet to be explored and evaluated. In the present instivetigation, it was observed two

entomopathogenic fungi, that could effectively control the bore and arboreal termites.

Infact many work have been carried out in the management of these pest our work was

unique and supportive to the future IPM programme in the forestry for control of this

xylophagous pests.

5.1 Incidence of pest attack

Plantation forestry were posed for greater expansion in many tropical countries

particularly of exotic fast growing species for production of pulp wood, paper

manufacture and medium density fiber wood. More than a hundred tree species are raised

in different forest locations of Karnataka. However, the understanding of the incidences

of borers and termites on dominant high value timber species like teak, mahogany, sandal

and eucalyptus are necessary to develop effective strategies to eliminate or suppress the

pest population. In the present study, we have selected nine forest divisions of Karnataka

for survey of the mahogany shoot borer H. robusta and the termite Odontotermes sp.

In the nurseries of selected locations, the saplings of S. mahagoni were severely

affected by the borer, H. robusta and upto about 78% of the apical shoots of the saplings

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infested were in a plot of 1000 plants. Studies conducted by Varma (2001) on the

incidence of the borer H. robusta on S. macrophylla saplings in a 169 ha area showed

70% of the plant orchards were infected. In the present study also it was observed that

majority of mahogany (S. mahagoni) saplings with 2- 4 feet DBH were found to be more

susceptible to the borer attack. In contrast, once the plants cross the sapling stage (≈ 5- 6

feet DBH) the borer attack diminished and the plants was less susceptible to the attack.

The present observation also substantiate the results reported by Varma (2001)

and Misra et al. (2003) where the infestation was chronic on the saplings of S.

macrophylla having plant height of 3-6 feet. In the field studies conducted in China by

Chen and Cha (1998), about 60- 100% of S. macrophylla saplings were injured by the

attack of H. robusta. Verma and Nutul (2000) and (2001), reported that 2-3 m tall trees of

T. ciliata were mainly affected by shoot borer H. grandella where maximum shoot

infestation per tree was 66.5%. The shoot infestation was peak during the months of May

to September and larvae undergoe over wintering stages from October to late March.

Studies on the incidence patterns of mahogany shoot borer showed that with

higher rainfall, low altitude and moderate temperature the borer attack increased

considerably. In experimental cases though some area recorded high rainfall, the

infestation level of the borer was comparatively equal to that of low rainfall area. In

another study by Mo et al., (1997a) the pattern of infestation level was closely correlated

with rainfall, the larger the amount of rainfall, the higher the infestation levels.

Temperature did not appear to affect the general infestation levels, but low daily

minimum temperatures in the winter were always associated with low proportions of

attacked trees. In this study, the Odontotermes infestation on the teak, sandal and

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equylaptus orchard showed a high percentage of infestation irrespective of the tree

species in almost five districts survived.

Studies conducted by Mohan (1995) damage of 24-yr-old teak plantation in

Maharashtra reveled that two genera of Odontotermes, viz., O. parvidens and O. distans

were predominant and the mortality was mostly in the girth classes below 45 cm. Gowda

et al. (1995) in their studies recorded that infestation of a single species, O. obesus

(Rambur) was predominant in the month of April to November. He also observed that O.

obesus (Holmgren) and O. feae (Wasmann) was the most dominant, during the period of

February to March in Coffea arabica in Karnataka. From the present study it was

observed that O. obesus (Rambur) and O. feae (Wasmann) are the predominant species in

teak and equlyaptus plantations.

It was also found that the incidence of O. redemanni in sandal were abundant in

the winter season which was supported by the observation of Remadevi et al. (1998)

where the infestation was upto 50% in Bangalore rural districts. In Maharashtra, O.

obesus incidence in the forest plantation varied from 4 to 40% (Meshram et al., 2001).

Similarly, the diversity of termites in tropical forests of young eucalyptus plantations in

Kerala was also discussed by Varma and Swaran, (2007).

5.2 Isolation of entomopathogenic fungi

In the present study, M. anispliae were isolated from almost in all sites supporting

the cosmopolitan character of this fungus. However, the study showed different

distribution patterns and frequencies of occurrence among various regions and habitat of

different forest areas of South India. This seems to be related to different environmental

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conditions such as altitude, climate and soil properties. It is important to analyze the

relationship between fungal densities, environmental factors and combination of biotis

and abiotic conditions. This confirms results from Bidochka et al., (1998) who found that

M. anisopliae was more abdunt in agricultural than forest soils, whereas the contrary was

true for Beauveria bassiana. .

Entomopathogenic fungi may be widely distributed in forest soils and have been

isolated from the organic (Oe + Oa horizons) layer of soil (Hajek et al., 2000). Similar

observations from the studies on densities of M. anisopliae in different locations showed

less number of propgules isolated form agriculture lands (Keller et al., 2003). Large

number of M. anisopliae obtained were isolated from lepidopteran hosts when compared

related to the other insect orders. The density and diversity of M. anisopliae in soil differs

profoundly from that in cadavers (Sonia et al., 2005). This might change interaction

between potential host species and affect the growth and competition among

entomopathogenic fungi. Amy et al. (2009) in their study isolated species of potentially

entomopathogenic fungi found in the soil which can be readily cultured in medium. In

this study, plating insect cadavers to assess external fungal growth showed in 98% of

cadavers of Coleoptera, Hemiptera, Isoptera and Lepidopteran were infected with

entomopathogenic fungi.

An earlier study found the prevalence of entomopathogenic fungi on larvae,

nymphs, and females to be 0.2, 0.3, and 10.3%, respectively (Kalsbeek et al., 1995).

Subsequent work by Zhioua et al. (1999), when the spores were removed and plated on

Sabouraud's dextrose agar with yeast (SDAY) medium, found 86% of entomopathogenic

fungi were isolated from larvae, and nymphs, stages collected from different insects

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order. The present results also showed most of the dead cadavers plated on the SDAY

medium containing selective indegrediants could isolate colonies of M. anisopliae.

5.3 Pathogenicity of M. anisopliae against shoot borer

In this investigation laboratory trials were conducted to evaluate the virulence of

M. anisopliae isolates against this shoot borer. It was observed that the first and second

instar larvae were highly susceptible to the tested isolates of M. anisopliae. The third

instar larvae were moderately susceptible and the fourth and fifth were found to be

susceptible to the fungal infections. Inocula dose significantly influenced the sporulation

rate of M. anisopliae in infected H. robusta. The in vitro development rate of this

pathogen suggested that sporulation reflected dose-related vegetative growth of the

fungus. The estimated sporulation generally tends to be lesser than the actual sporulation

rate.

Doberski (1981) also reported that spores of entomogenous fungi adhere strongly

to the insect cuticle that the number of spores washed off must be regarded as a minimum

figure. The amount of conidia produced on individual larvae was variable, whichmay be

due to the differential fungal colonization and development because of insufficient

nutrients on host cuticle, molting of larvae, compact integuments and immune response

of the individual larva.

Studies by Salvatierra et al. (1972) reported 50% mortality of H. grandella larvae

exposed to M. anisopliae, when a concentration of 1.4 x 107 conidia/ml were applied over

larvae. This virulence and high sporulating ability can be of good advantage as it would

enhance the dispersion of fungus, when developing a commercial technology using this

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isolate. The effect B. bassiana was also evaluated on the H. grandella in a ten month old

Cedrela odorata plantation (Sanchez and Velazquez, 1998 and Goulet et al., 2005) B.

bassiana applied at the interval of three months achieved 71% mortality.

The integrated management of Toon shoot borer by use of the entomopathogen

such as viruses, bacteria, fungi, and reckettsiae have been discussed (Casanova et al.,

2001 and Misra et al., 2003). It has been hypothesized that most virulent fungal strains

are generally isolated from the test organism or a closely related species. However, no

apparent relationship between pathogenicity and host of isolates was observed in our

study. IWST-Ma7 and IWST-Ma 4 exhibited substantial variation in speed of kill though

they have been isolated from the same insect species, Paliga machoeralis. Similarly, M.

anisopliae isolated from Schistocerca. Gregaria (Orthoptera: Acrididae) caused 100%

mortality at 108conidia ml-1 against second instar larvae of Spodoptera litura (Anand et

al., 2009).

5.4 Pathogenicity of M anisopliae against Odontotermes spp.

The results of the pathogenicity test, horizontal transmission of fungal infection

and repellency test confirmed that IWST-Ma13 and ARSEF 7413 were pathogenic

against O. obesus workers in laboratory conditions. However, the mortality and mycosis

of termites treated with 1 x 108 conidia ml-1 of IWST-Ma1, IWST-Ma2, IWST-Ma13 and

ARSEF 7413 did not exhibit much difference. Hence a detailed pathogenicity test was

carried out to find out LC50, AST and MST values of these isolates against O. obesus.

Horizontal transmission of fungal infection and repellency was subsequently tested

providing a soil environment for the termites.

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In a group of 100 O. obesus maintained in a stand filled tray, our results showed

that when 20% of workers were infected with M. anisopliae the fungus from the infected

workers and soldiers spread to the termites and killed them completely. The grooming

behavior exhibited by the termite might have contributed to the fast spread of the fungus

among the individuals. It could be concluded that there was maximum transmission with

the case of highly virulent strains and observable diseases development in the native

termites under these conditions.

Milner and Staples (1996) tested over 90 isolates of M. anisopliae against

Nasutitermes exitiosus and Coptotermes spp. workers and found that many isolates

induced over 80% mortality. Khan (1991) reported that among several mycopathogens,

M. anisopliae was more virulent to O. brunneus. Gunner et al. (1994) tested 21 isolates

of M. anisopliae against R. flavipes workers and observed 80–100% mortality within 3

days of treatment. Changjin et al. (2009) found that conidia from M. anisopliae var.

dcjhyium were highly virulent to O. formosanus, causing approximately 100% mortality

after 3 days post-inoculation at a concentration of 3 x 108 conidia/ml-1.

It is generally considered that most virulent fungal strains are isolated from the

test organism or a closely related species (Latch, 1965 and 1976, Soares et al., 1983 and

Poprawski et al., 1985) However, no apparent relationship between pathogenicity and

origin of isolates was observed in the present study. The isolates tested in this study

performed well, although they were not originally isolated from Odontotermes sp. The

most pathogenic isolate, was from a mummified lepidopteran larva collected from a

forest locality.

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Jones et al. (1996) also observed similar LC50 values from three isolates of M.

anisopliae recovered from three different species of scarab larvae in Australia, assayed

against C. formosanus. Milner and Staples (1996) tested more than 90 isolates of M.

anisopliae against N. exitiosus and Coptotermes spp. workers, and found that many of the

isolates induced over 80% mortality. In a separate study, Milner et al. (1998) also

observed only small differences in the mortality caused by several isolates from different

sources in these two species.

5.5 Horizontal transmission of fungal infection among termites

Horizontal transmission is an important component to be considered in the

development of termite control strategies because in many species, most of the colony

and the nest is not accessible to treatment directly. In the present study, the potential for

the dissemination of fungi applied to workers and soldiers at feeding sites was very high.

Lai (1977) demonstrated that C. formosanus workers moved more than 110m through

their galleries in 48 h. Jones et al. (1996) found that in preliminary laboratory

experiments, 49-100% mortality was caused in groups of C. formosanus when spores of

three strains of B. bassiana and M. anisopliae were applied as dust to 20% of the

workers.

Rosengaus and Traniello (1997) showed that exposing groups of 10 Zootermopsis

angusticollis nymphs to groups of fifteen resulted in 100% mortality of the treated

termites and over 50% mortality of the exposed termites. In our experimental results, the

grooming termite becomes contaminated but probably only around the mouth parts which

may groom another indivudial causing transmission to spread in the colony. But the

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residual effectiveness of the fungus inoculum will depend on the efficiency of spore

dispersal through the frass and proctodeal trophollaxis. Though the treated workers were

attacked, dismembered or buried alive, the attacking termites would be likely to be

contaminated by the exposure to the treated termites and so some horizontal transmission

could occur.

5.6 Repellency of termites in the presence of fungal pathogens

In addition to direct mortality as a consequence of contact with the dry conidia of

M. anisopliae, the spores of some isolates were identified to be repellant to the workers.

However, repellent spores may have less horizontal transmission potential from infected

or contaminated termites to other nest mates. The nature of repellency may be due to the

conidia, the dead or mummified cadavers, or even the high level of foreign particulate

material associated with contamination of workers.

Kramm et al. (1982) found that healthy workers of R. flavipes actively avoid nest

mates which had died from M anisopliae. Rath and Tidbury (1996) showed that, the

workers of C. acinaciformis foraged less on the treated cardboard and only 2.3% of

workers were found to forage on the spore treated cardboard over a three day period,

compared to 33% of workers which foraged on the untreated cardboard. The results from

our study suggested that the workers and soldiers of O. obesus elicited different

avoidance response when the conidia were applied this could be because of the complex

social and defensive interaction between workers and soldiers in the colony. In our study

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it also found that the dry conidia of highly pathogenic strains were less foraged by the

workers.

Rath (2000) also described a similar response in R. flavipes but alarm behavior

was exhibited by untreated termites. M. anisopliae mycelium could either disguise the

spores or render them non-repellent or could be made attractive to termites. Keeping the

concept in our focus we have tried to render the spores in an attractive form by mixing

with the attractant such as sawdust, sugarcane baggasse and cardboard powder, which is

highly rich in cellouse materials. This mixture showed positive response with the termites

were maximum percentage of workers were attracted towards the treated area that

favored epizootic transmission of fungal infection among the population. Rath and

Tidbury (1996) showed that 15% more workers of C. acinaiformis foraged on wetted

cardboard treated with M. anisopliae and a proprietary masking component than on

treated wetted cardboard.

5.7 Mycotoxic effects of M. anisopliae

In this study five isolates of M. anisopliae, was selected for toxicity test of the

culture extract against H. robusta and O. obesus in comparison to other strains of M.

anisopliae, that these isolated proformed well in their mortality, mycosis, lowest spore

load and short duration for mortality. The culture extract of these two isolates was highly

toxic to shoot borer H. robusta and O. obesus. The three calculated LD50 values, from

separate lots of the extract, yielded a mean LC50 value with a coefficient of variation.

These data indicated that the two isolates (IWST- Ma7 and IWST- Ma13) cultures

produced equivalent amounts of toxic compounds. As reviewed by Roberts (1981),

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culture filtrates and solvent extracts of M. anisopliae were toxic to a wide variety of

insects. This could be the first report of the toxicity of M. anisopliae extracts to H.

robusta and O. obesus. Destruxins, the best studied toxins of M. anisopliae, are

secondary metabolites readily extracted from culture filtrates (Roberts, 1969). Destruxins

vary widely in their toxicity and effects on insects. Several studies have established their

role in insect pathogenesis (Samuels et al., 1988 and Gillespie and Claydon, 1989).

Destruxins appear to act on the cell membrane as a calcium channel activator.

Our preliminary tests with partially purified toxins demonstrated their toxicity to

shoot borer and bark feeder. However, toxicity data of partially separated fractions of the

culture extracts for H. robusta and O. obesus suggested that the toxin fraction alone was

not entirely responsible for the toxicity to these pests. As with other entomopathogens,

M. anisopliae most likely produced a spectrum of biologically active components such as

proteases and chitinases which secreted act synergistically to degrade insect cuticle (St.

Leger et al., 1996 and Fang et al., 2005) and these components were probably

responsible, in large part, for the observed toxicity.

The crude soluble protein extract of the entomopathogenic fungus M. anisopliae

was evaluated (Ortiz-Urquiza et al., 2009) showed insecticidal activity and antifeedant

effect in adult flies. Four monomeric proteins of 15 kDa and the 11 kDa proteins appear

to be mainly responsible for the observed insecticidal effect. The toxicity effects of the

crude soluble protein extract of M. anisopliae were studied by Ortiz-Urquiza et al. (2010)

also and the electrophoretic analysis suggested that the monomer of 11 kDa played an

important role in the insecticidal effects. Faster cuticle penetration by the fungus

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decreases the chance of exposure of the fungus to destructive ecological factors such as

UV and to constitutive and inducible insect defenses (Fang et al., 2009a).

The toxic compounds emitted by ungerminated conidia on the cuticle or in the gut

are involved in the activity of M. anisopliae against A. aegypti larvae (Silva, et al., 2005).

Thus accelerating fungal penetration of the cuticle before the host has time to mount a

defense of sufficient magnitude to block it, will potentially improve the utility of fungal

pathogens as biocontrol agents by reducing the time of exposure to potentially

debilitating environmental conditions such as UV, and to constitutive and inducible insect

defenses such as melanization

5.8 Management of borer attack in mahogany saplings

In this study one isolate IWST-Ma7 showed effective control when applied as a

foliar spray during the pest infestation period. Though this isolate could not completely

eliminate the attack of shoot borer, it reduced the damage to tolerable levels. It was also

observed that the infestation level was effectively reduced after the foliar spray

applications in the peak periods of plant susceptibility and pest monitoring. The

prophylactic sprays of M. anisopliae in nurseries against H. robusta larvae would have to

be repeated frequently on a rapid growing shoot. Though the relative potency indices

were superior when the spores were injected in to the shoot that may kill the internal

feeders, as the first and second stage of larvae feed on the external plant parts and topical

spray may be an effective for control method.

The effect of B. bassiana was also evaluated on the H. grandella in a ten months

old Cedrela odorata plantation (Sanchez and Velazquez, 1998 and Goulet et al., 2005).

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B. bassiana applied at the interval of three months achieved 71% of mortality. Though

chemical control of Hypsipyla spp. shoot borers was practiced for about eight decades in

more than twenty three countries throughout the tropics, there is still no chemical or

application technology which provides reliable, cost-effective and environmentally sound

protection for any of the high-value meliaceous tree species (Wylie, 2001). In contrast,

when two-year-old Swietenia humilis trees were treated with deltamethrin, provided

complete control against the H. grandella attacks (Goulet et al., 2005). The possibilities

in the use of the predatory ants O. smaragdina for control of H. robusta was examined as

the species is one of the dominant ant species on the mahogany trees. Whether this ant

species protects the trees from attack by the shoot borer needed to be investigated further

(Grace and Laurence, 200l, Peng and Christian, 2006 and Lim et al., 2008).

In artificial shade trial of the seedlings of S. macrophylla, it was found that plants

grown under open conditions are more vulnerable to attack by H. robusta. Hence

provision of high shade during early growing period may increase the ability of to

recover better after shoot-borer attack (Mahroof et al., 2001). In another study Perez and

Esquivel et al. (2008) found that the proportion of infected individuals of S. mahogoni by

H. robusta did not differ between trees, either planted as monocultures or in mixed

plantations. The application of the conidia of the effective isolates may provide longer

protection since they are borne by wind which increases scopes of transmission in low

density larval populations like H. robusta.

5.9 Management of termite attack in plantations

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A major limiting factor, in the control of termites in urban pest control

programmes is that we can not prove whether termites are controlled or their nest

eliminated when treatment has been undertaken. Widely used techniques which allowed

the estimation of the termite populations, such as mark release –recapture protocol have

been found to be inaccurate and unreliable (Evans et al., 1998). Lack of activity at

treatment points of monitoring stations is not proof of elimination or suppression of the

population. Untreated control sites are not used in the studies on termite control in urban

areas, as home owners pest control operators and pesticides companies are not willing to

have continued termite activity with liability of increased property damage.

In the present study we used four different methods to test the efficiacy of fungal

pathogen. Stake treatment and exposure to termites showed reduction of attack. However,

control stacks had severe infestation after five months which also signify that the termite

colonies donot avoid the treated area. Application of 0.5% Wettable powder is through

standard techniques such as drilling into galleries of infected timbers and applying the

liquid under low pressure has been occasionally suggested methods. Krueger et al. (1995)

stated that the reinsepction of the Bioblast TM treated sites in USA showed 60% of the

wooden structure were free of termites, 15% still had partial activity, 5% had no

reduction in activity and 20% were still to be inspected. Where ever the termites were

eliminated, the structures had remained uninfested for a period ranging from 6 to 15

months. Rath (1995) observed from his studies, that there was no indication whether the

success of the treatments was due to colony elimination or suppression, or due to active

avoidance of treated areas. However, in our study the good coverage of fungal conidia

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over the gunny bags was effective though the learned avoidance help the termite to avoid

the treated area and prevent infestation.

From the tree treatment and bait treatments showed effective suppression of the

termites population in the treated plots. In the bait treatments in the soil we could find

that the suppression of the population was consistently high. It was also observed that the

baits were completely eaten by the termite population because addition of baits (cellulose

materials like, cardboard powder, sawdust and sugare cane baggasse) could have made

the spore non- repellant. Field experiments with different formulations of insecticides

(fenvalerate 0.4% dust, malathion 10% dust and sugarcane pressmud) against O. obesus,

showed effectiveness with the malathion 10% formulation (Deka et al., 1999).

Field evaluation of neem-based formulations (Neemgourd, Nimbicidine,

Multineem, Vanguard, Nemactin, Rakshak) against O. obesus in the plots of mango

plants showed effectiveness with Nimbicidine and Nemactin up to two months whereas

Rakshak, Multineem, Neemgourd and Vanguard were effective up to one month (Singh

and Singh, 2002a).The antifeedent activity of Thiamethoxam formulation (ACTARA 25

WG) against the African termites viz., Trinervitermes trinervius and O. smeathmani,

showed that the products are consumed by the termites rather than repelled (Huang et al.,

2005).

The foraging behaviour of O. rmosanus was studied on pure baits such as pine

powder; sugarcane powder; millet powder; powder of log of cultivated Lentinula edodes,

Artemisia argyi powder and potato powder. The studies showed that the consumption

rate, mud sheet area and mud sheet covering rate in the case of millet powder was high

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and all the additives significantly improved the phagostimulating effect on O. formosanus

(Huang et al., 2006). Field studies on the use of attractive toxicants in controlling ground

and deadwood termites were effective in controlling O. formosanus populations in the

field (Huang et al., 2006a). Baiting systems may provide long lasting control by

eliminating or at least reducing termite activity. In the studies to test efficacy of different

bait materials in managing O. obesus proved that sugarcane bagasse was more attractive.

Management of O. obesus by baiting system may rendered the colony weak (Rajavel et

al., 2007).

The use of M. anisopliae as a repellant in the field has been proposed by Ko et al.

(1982), Gunner et al. (1994), Milner and Staples (1996) and Milner et al. (1997) who

found a correlation between the level of M. anisopliae present in soils and the mortality

of C. formosanus. Localities in which the fungi were commonly present had never

reported C. formosanus infestations. The author could conclude that the presence of the

fungi in soil was providing protection against termite attack. Milner et al. (1997) showed

that by treating soil with M. anisoplaie damage to wood can be lessened but not

eliminated. They suggested that a minimum of dose rate 1 x 108 spores g-1 was effective.

Also, Ko et al. (1992) found that soil with 9 x 105 spores g-1 resulted in 100% mortality

of C, formosanus after 14 days at 15 C in the laboratory. Milner and Staples (1996)

reported that their treatments repelled termites rather than killed them and treatments

gave up to 3 years protection of timber under cool dry conditions but less than 6 months

under tropical conditions. Gunner et al., 1994 reported that M. anisopliae growing on an

oat bran flour medium was repellant to termites and protected wood structures in the

laboratory. But the repellency was lost once the fungus stopped growing even though

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fresh spores were present in the soil. He also reported that forgaing termites started to die

from M. anisopliae spores picked up from the soil.

In the treatment, directly dusting of large quantities of conidia mixed with

attractants in to the galleries of the mound was tried to specifically eliminate the colonies.

We found that the activity of the termites reduced gradually over a five months period

when compared to that of control mounds. This could be due to the rapid transmission of

the fungal infection among the population of the termites in inside the treated galleries.

Our study also indicated that by direct dusting inside the mound galleries, the termites

population could be eliminated rather than suppressed which is observed in the case of

tree and bait treatments.

The patent of Milner et al. (1997) which coners the control of termites with M.

anisopliae described few field treatments. Their treatments appeared to have been

described specifically to eliminate colonies by direct nest treatments of mound building

and tree nesting termite species. They found that treatments which consisted of applying

small doses of conidia to termites repairing damaged part of mounds were unsuccessful

as treated region were walled off and the colonies continued to grow in other parts of the

nest. Milner and Staples (1996) found that colonies could only be killed when large

quantities of pure dry conidia were blown directly into the nursery region. They found

success in several hundred colonies of five different species of termites with nests in

mounds or trees.

Milner and Staple (1996) found that indirect control of colonies by treatment of

termite feeding sites had shown highly variable results. They belived that this was due to

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the ability of termites to avoid the fungus as discussed earlier. Fernandes and Alves

(1991) found that either M. anisopliae or B. bassiana resulted in 100% mortality of

Cornitermes cumulance colonies with in 10 days of application of 5 g of dust to nests.

Hanel and Watson (1983) treated both the mound nests and the feeding sites of

Nasutiteermes exitiosus with M. anisopliae. They were not as successful as Fernandes

and Alves (1991) but concluded that disease can spread through a field colony from

conidia applied by dusting or spraying on a few of its members in the mound or away

from it at feeding sites. At least in some cases it could render a colony moribund. Lai

(1977) used the trap-treat-release method in an attempt to eliminate C. formosanus

colonies. His study on three nests using B. bassiana and M anisoplaie failed showing that

an epizootic was induced in the nests; however little information is given on the sampling

regime or declines of the colony size.

The termites great defense mechanism may be their avoidance of

entomopathoigenic fingi as discussed previously. While it is debatable as to what

component of the fungus treatment the termites are avoiding, there appears little doubt

that termites respond to the presence of diseased cadavers. These cadavers tend to be

buried by healthy workers (Kramm et al., 1982) prior to sporulation of the fungus on the

external surface. In the field they have seen section of carton nests of C. acinaciformis,

which have been heavily treated with 50% dust formulation of M. anisopliae, become

termite graveyards with hundreds of dead termites. The reminder of the nest then seems

to function normally. Milner and Staples (1996) also claims that treatments of mound

nests by damaging small section and appling Metarhizium based dusts is ineffective as

the termites wall-off the treated area. In laboratory studies, Milner et al. (1998) found a

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distinct separation of killed cadavers, M anisopliae inoculum sites and live termites,

suggesting some avoidance of both the inoculam and the cadavers.

Soldiers are responsible for the primary defense of the colony from macro-

organism such as attacking ant species (Watson and Gray, 1991). Soldiers of the

Rhinotermitidae and Termitidae have well-developed frontal glands, the secretion of

which is excuated through a pore on the frontal region of head. The frontal glands

secretion may be toxic or repellant to invading insects or may be sticky and entangle their

legs and antennae. In some species, the secretion also function as alarm pheromones

(Valtervoa and Vrkoc, 1994). While these secretion and others are not specifically

designed to be antibiotic that it is likely to inhibit microbial growth (Rosengaus et al.,

1998).

Apart from primary defense by secretion, Beattie et al. (1994) believed that

control of microorganism by N. exitiosus is in part due to the construction of the mound

nest. They found that the outer layer of the mound are very low in organic material and

have a very low matrix potential both of which would limit microbial growth. However,

they believed that the nursery area was well suited for microbial growth and anti

microbial substance are produced to defend part of the nests. The termite Z. angusticollis

line the nest chambers and galleries with faecal pellets and these have been shown to

inhibit the germination of M. anisopliae spores (Rosengaus et al., 1998).

5.10 Enzymatic character

Chitinase activity of M. anisopliae in the chitin-containing medium was 0.01 U/

ml at 120 h. St. Leger et al. (1986) and (1986a) reported chitinase activity (0.027 U/ml)

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in M. anisopliae after 120 h. He also observed that the appearance of extracellular

enzymes in entomopathogenic fungi was paralleled by the sequential solubilization of

cuticle constituents. The production profile for the M. anisopliae strain used in this study

showed that CDA and chitosanase activities were high. The proteolytic activity appeared

comparatively less than that of lipolytic activity.

The M. anisopliae CDA activity was not inhibited in the presence of solubilized

synthetic melanin (Sigma) or of sodium acetate added during the reaction. It suggested

that melanin may either combine with the biodegradable polysaccharides or inhibit

enzymes participating in wall lysis by producing transient molecules. Furthermore,

Lemburg (1998) demonstrated that the sclerotized cuticle layer in fixed insect cuticle of

Halicryptus spinulosa and Priapulus caudatus was resistant to chitinase hydrolysis.

Tokuyasu et al. (1996) reported that extracellular CDA from plant pathogenic

fungus Colletotrichum lindemuthianum was not inhibited by sodium acetate, and

Kafetzopoulos et al. (1993) showed that intracellular CDA of dimorphic fungus Mucor

rouxii was inhibited. M. anisopliae extracellular CDA was not inhibited in the presence

of sodium acetate (1–5 mM). Chitin deacetylase of C. lindemuthianum had an optimum

pH of 8, whereas CDA from M. rouxii had an optimum pH of 5.5 (Kafetzopoulos et al.,

1993 and Tokuyasu et al., 1996). Extracellular chitin deacetylase from M. anisopliae

also showed activity in the pH range 7.5– 9.2. The optimum pH for the enzyme activity

was found to be 8.5–8.8. It could be interesting to determine whether localization of

enzyme had any effect on the optimum pH.

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Fungal pathogenesis is a complex process and depends on various factors

including degrading enzymes for successful penetration of hosts. In addition, the life

cycle of entomopathogenic fungi is associated with the synthesis and secretion of

different components including extracellular enzyme (proteases), proteins, and low

molecular weight compounds (toxins) (Bandani 2005). Insect cuticle represents the

primary site for the establishment of mycosis by entomopathogenic fungi. Hence,

overcoming this first barrier is important in pathogenesis of fungi against insects. Similar

to the majority of fungal pathogens, B. bassiana and M. anisopliae uses a combination of

enzymes to penetrate the cuticle and access the nutrient-rich host hemocoel.

Production of ammonia by M. anisopliae was strongly stimulated by low levels of

amino acids in the medium when cells were derepressed for nitrogen and carbon (St.

Leger et al., 1998). Ammonia production by M. anisopliae increased the pH of the

medium and allowed production of subtilisin proteases, whose activities are observed

only at basic pH. In contrast, protease production by the Acid(M) mutants of M.

anisopliae was greatly reduced because of the acidification of the medium (St. Leger et

al., 1998). This suggests that alkalinization by ammonia production is adaptive by

facilitating the utilization of proteinaceous nutrients (St. Leger et al., 1998).

5.11 Molecular phylogeny

The ITS regions and 5.8S rDNA of Metarhizium were amplified using the ITS1

and ITS4 primers that was a unique fragment of approximately 550 bp for all isolates.

Fouly et al. (1997) analyzed the same region with 540 bp fragment for M. anisopliae var.

anisopliae strain and 600 bp for M. anisopliae strain. The PCR products were sequenced

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and compared to others in GenBank using BlastN. Sequencing data confirmed that all

sample isolates were M. anisopliae. This study indicated that it was very similar and that

M. anisopliae was the predominant variety in this part of the world as stated by

Rakotonirainy et al. (1994), Driver et al. (2000) and Anderson et al. (2001) .

Genetic distances between isolates were presented by branch length. Phylogenetic

analysis showed other Metarhizium species or varieties and outgroup from GenBank

could be clearly differentiated in clade A. The data support the monophyly of the M.

anisopliae group except M. anisopliae var. acridum, and recognized four clades (clades B

to E) within it. The isolates within clade A were sub divided into two groups with M.

anisopliae var. anisopliae and M. anisopliae and were clearly distinguished from each

other. In this study, M. anisopliae var. acridum sequences were different from other M.

anisopliae that was in aggrement with the observation of Neuveglise et al. (1994), Diver

et al. (2000) and Vetake et al. (2002).

Based on the results of molecular data, from this study as confirmed by Diver et

al. (2000), the 5’ region of EF-1ά was to date the most informative region to use for

routine species identification with in this genus. M. anisopliae var. anisopliae is

pathogenic to innumerable insect species. Whereas, M. anisopliae var. acridum is much

more host specific only Orthoptera insects (Jensen et al., 2001 and Thomsen and Jensen,

2002). This study confirmed that rDNA sequence data can be used to resolve

evolutionary relationships within Metarhizium genus and M. anisopliae evolutionary

lines.

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The dendrogram generated reported the twenty two isolates into five main clades

(cladeA, B, C, D and E). This work confirmed the high variability of ITSs and 5.8S

rDNA within M. anisopliae. These results agreed with Hegedus and Khachatourians

(1996) and Do and Adams (1991) who found M. anisopliae to be extremely diverse using

RAPD analysis. The study indicated that M. anisopliae are highly divergent, whereas and

IWST-Ma2 and IWST-Ma5 showed as distint group. However, Bootstrap re sampling

that used to estimate reliability of the tree showed less than 50% frequency. Despite their

origin, the isolates collected from cadavers did not correlate together.

The most important taxonomic reviews of Metarhizium were the revisions by

Pfeifer and Khachatourians (1993) and Do and Adams (1991) who isolated that

Metarhizium comprises three species, e species, M. anisopliae, M. flavoviride and M.

album. M. anisopliae that was separated into two varieties; short spored M. anisopliae

var. anisopliae and the long spored M. anisopliae var. major. The great diversity in M.

anisopliae supported those of other workers demonstrated using both biochemical and

molecular markers (Diver et al., 2000). Using biochemical profiles, Do and Adams

(1991) could distinguish groups of var. anisopliae more divergent than var. majus. Using

allozyme profiles, Pfeifer and Khachatourians (1993) proposed that M. anisopliae may be

composed of at least five varieties. Nevertheless, ITS regions, data did not provide

sufficient resolution to clarify the relationships within isolate or variety.

Many researchers were interested in this fungus and many taxonomic studies were

made because of their importance and potential in the biological control agent of pests.

As the use of M. anisopliae as a biological control increases, a more adequate and

accurate identification and separation of varieties of M. anisopliae or genus Metarhizium

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become important to confirm species and to specify this fungus to colonize a specific

insect host. Our data revealed fundamental similarities between morphological and

molecular groupings. Morphological features of the groups correlated strongly with ITS

and 5.8SrDNA sequence identity of groups. The occurrence of M. anisopliae in nature

indicated that this fungus is an ubiquitous organism with a worldwide distribution and

most of them related to each other, though some different parts of the globe. Molecular

analysis may express distinction distinguishing between the isolates even similar in

morphology. Morphological characteristics are generally complex and many involve

genome expression. It is suggested that morphology and molecular phylogeny would be

studied together to confirm identification of this fungus and their biological properties.