11

India is blessed with a variety of timber yielding tree species and as many as 1500

species are commercially utilised for diverse purposes. Some of the important plantation

tree species grown in India are Tectona grandis, Eucalyptus spp., Acacia spp., Dalbergia

sissoo, Swietenia sp. Santalum album, Melia dubia, Ailanthus excelsa, Leucaena

leucocephala etc. Productivity of forests in general, and particularly that of commercial

forest plantations, is very much affected by frequent outbreak of pests and diseases,

besides human interventions and various natural calamities. Among the pests, largely

insects belonging to the orders Coleoptera, Lepidoptera, Isoptera and Homoptera are the

major ones of significant economic importance that attack trees, right from the seeds to

the final product. Wood deterioration by xylophagous insects leads to unhealthy tree

growth and malformation of wood in timber yielding forest trees. Some such major pests

belong to Lepidoptera, Coleoptera and Isoptera. The review of literature pertaining to the

attack of xylophagous insects and their management aspects are covered in this chapter.

2.1 Mahogany shoot borer

2.1.1 Incidence of Mahogany borer pest attacks in Tropical and Sub tropical

countries

Mahogany (Swietenia mahagoni Jacq.) is a large deciduous high timber value tree

used for making furniture, paneling, railway sleepers, industrial and domestic wood

wares, traditional medicine etc (Wylie, 2001; Lopez et al., 2008). Continuous supply of

this species is often constrained by low natural regeneration and difficulty in

establishment mainly due to the attack by the shoot borer, Hypsipyla robusta Moore and

12

Hypsipyla grandella Zeller in the tropical and sub tropical parts of the world

(Cunningham et al., 2005; Ofori et al., 2007).

In Nigeria, lack of knowledge on the ecology and taxonomy of the shoot-boring

caterpillars Hypsipyla spp. has resulted the foresters not attempting to grow mahogany

(Swietenia spp., Khaya spp., Cedrela odorata L. etc) in many locations within the natural

range of the species (Eidt, 1995). In Indonesia Mahogany trees were affected by H.

robusta and Xylosandrus compactus (Eichoff) (Suratmo, 1996). In Bangladesh, the trees

of Swietenia macrophylla King were affected by two major borer insects - the mahogany

shoot borer, H. robusta and the mahogany collar borer, Pagiophloeus longiclavis

Marshall (Baksha and Islam, 1997).

In the Atlantic region of Costa Rica 90% of Mahogany trees were affected by

attack of H. robusta and showed high shoot proliferation (Lopez et al., 1997). In the

Amazon Basin Roba-mahogany (Carapa guianensis Aubl. and C. procera) the natural

distribution, abundance and phenology were affected by the high seed predation of H.

grandella (Ferraz et al., 2002). In the four species of African mahogany viz., Khaya

anthotheca (Welw.) C.Dc., K. ivorensis, Entandrophragma angolense Tiama, E. utile the

relative susceptibility to H. robusta attack, after 24 months was high in all the tree

species (Opuni et al., 2008). In Sri Lanka, the native tree of Chukrasia velutina M. Roem.

and S. macrophylla were severely damaged by the attack of H. robusta (Edirisinghe and

Tilakaratna, 1997).

In Himachal Pradesh, the larvae of H. robusta feed on fruits and shoots of T.

ciliata, (Verma and Nutul, 2000) and almost 80% of flowering trees surveyed showed

13

infestation of the fruit borer in mahogany and cedar plantations in Punjab. In Kerala an

area of 169 ha of S. macrophylla, 70% plants were bored by H. robusta larvae. The attack

of H. robusta on Swietenia mahagoni Jacq. and Toona ciliata M. Roem. were also widely

observed (Varma, 2001; Misra et al., 2003).

2.1.2 Economic significance of Mahogany shoot borer

The productivity of C. odorata was severely reduced by the attacks of stem-borer,

H. grandella (Haggar and Ewel, 1995). In contrast exotic species of Cedrela (C. odorata

and C. fissilis) grown on the New South Wales mid-north coast of Australia were not

attacked by the shoot borer H. robusta (Bygrave and Bygrave, 2001). It was also found

that the 50-96% seed capsules of West Indies mahoganies (S. mahagoni) were damaged

by H. grandella in spring (Howard and Giblin, 1997).

Vennetier (1998) and Soda et al. (2000) atated that as the wood resources are

changing from natural forest to plantation forest, some exotic high price trees of

mahogany S. macrophylla, S. mahagoni and T. ciliata were planted more in Southeast

Asian countries. Kalil et al. (2000) observed that the two shoot borers H. grandella and

H. robusta which are causing greater losses in the South American mahogany by their

attack on young moncultures of the species cause a loss of apical dominance. One of the

most expensive woods in the world the Brazilian mahogany S. macrophylla were unable

to be grown in Central and South America due to the attack of shoot borer H. grandella

(Batista et al., 2007).

In China about 60 to 100% of trees were injured by the attack of H. robusta that

significantly reduced the timber value in the market due to forking of the branches (Chen,

14

and Cha, 1998). About 62.5% shoot infestation was recorded in 2-3 m tall trees of T.

ciliate where single larva bored to an average of 65.48 cm shoot length before becoming

fully fed (Verma and Nutul, 2000; Speight and Cory, 2001).

2.1.3 Other hosts of Hypsipyla spp.

Minor pests such as the stem boring moth Zeuzera coffeae Nietner, 1861 and

scolytid shot-hole borers were found to damage young mahogany seedlings (Chan-Hing

and Matsumoto, 2000). In India, about 97.7% shoots of Antelaea azadirachta Adelb.

were infested by Hypsipyla sp and damaging all plants parts (Choudhary, 2001). The

nature and extent of damage of shoot borer, H. robusta, were also observed on C.

velutina plantations and Xylocarpus granatum K.D. Koenig mangrove in all phenological

stages (Hossain et al., 2004a; Jugale and Bhosale, 2007).

2.1.4 Biology and life cycle of H. robusta

Mo et al. (1997a) and (1997b) stated that the temporal pattern of infestation levels

were closely correlated with the increase in the rainfall, the larger the amount of rainfall,

the higher the infestation levels. But the temperature pattern did not appear to affect the

general infestation levels. He also observed, shoots positioned at above 90% of tree

height were attacked twice as often as the lower-positioned shoots.

Floyd and Hauxwell (2001) stated that Hypsipyla spp. is strong flyer that can

locate female and distant host trees. Once they mated with female the female lay between

200 and 450 eggs over 5 to 8 nights (Griffiths, 2001) on terminal shoot. Mo et al. (1997a)

and (2001) found that terminal shoots would be among the first to be attacked once a tree

is infested. There was a gradual shift of feeding loci with respect to host tissues as the

15

larva become aged. Feeding by larvae of the first two instars was found most often in

terminal foliage or previously damaged tissues of shoots or tree stems.

Mo and Tanton, (1995) and (1996) studied the diel patterns of adult emergence,

female calling, mating, egg-laying and activity rhythms in the laboratory under controlled

temperature (26± 2° C) and reversed light cycles (14 h light:10 h dark) and found that

most moths (82%) emerged in the early hours of the dark period (0-2.5 h after light-off).

Pith-feeding (tunnelling) started at the later 2nd instar. Mo et al. (1998) and Verma and

Nutul (2001) in their studies on the biology of H. robusta showed, five larval instars and

the full-fed larva pupated in a cocoon after a brief prepupal stage. The pupa has a size of

14.2 mm x 4.07 and lasted for 10-11 days. The species completed four generations in a

year and over wintered as prepupa from October to mid March

2.1.5 Management of Hypsipyla spp.

2.1.5.1 Physical Methods

2.1.5.1.1 Silvicultural

In the field studies of uneven planting of neem (Suharti et al., 1995) and Acacia

mangium Willd. (Matsumoto et al., 1997), physical barrier around S. macrophylla

plantations demonstrated the most effective control of the H. grandella attack. The

improved silvicultural practices such as pruning, weed control, foliar and soil fertilization

showed reduced borer damage and also suggested to use this approach in the improved

integrated pest management programme (Briceno, 1997).

16

Mahroof et al. (2001) explained artificial shade trial of the seedlings of S.

macrophylla showed that when plants grown under open conditions were more

vulnerable to attack by H. robusta. The provision of high shade during early growing

period may increase the ability to recover better after shoot-borer attack The pruning of S.

macrophylla upto 29 months after planting, showed significantly better values for form

traits with no apparent difference in growth traits (Cornelius, 2001).

Hauxwell et al. (2001a) correlated the levels of attack with factors such as shade,

planting density, species mixtures, site characteristics, in the plantation of S.

macrophylla, Cedrela odorata and S. mahogoni and showed silvicultural techniques were

usually attributed to more than one mechanism and it is difficult to assess which is the

most important one for minimizing the impact of Hypsipyla spp. In another study Perez

and Esquivel (2008) found that the proportion of infected individuals of S. mahagoni by

H. robusta did not differ between the species used in plantations, either planted as

monocultures or in mixed plantations.

2.1.5.1.2 Using attractants

Field trapping with identified sex pheromones (Z, E)-9,12-tetradecadiene-1-ol-

acetate, (Z)-9-tetradecen-1-ol-acetate and a 5:3:2 mixture of (Z, E)-9,12-tetradecadiene-

1-ol-acetate, (Z)-9-tetradecen-1-ol-acetate and (Z)-11-hexadecen-1-ol- of the mahogany

shoot borer, H. robusta, impregnated with synthetic acetate captured no males, although

these compounds have been identified as sex pheromones of H. robusta (Nakamuta et al.,

2002).

17

2.1.5.1.3 By weed management

The use of weed row, weed-free, and maize taungya methods were evaluated in 1-

year-old plantations of the Pacific mahogany (Swietenia humilis Zuccarini) which

showed very less control of the attack of H. grandella (Goulet et al., 2005)

2.1.5.2 Chemical Methods - Insecticides

The efficacy of two systemic granular insecticides (carbofuran 3G and thimet 10G

[phorate]) were tested by Bhandari et al. (1999) against the borer H. robusta in 1-yr-old

agroforestry plantations which showed that the insecticides Thimet at 15-25 g per plant

and two organophosphate insecticides in the soil protected the plants best, reducing the

number of plants attacked at 30 days after treatment. Though the chemical control of

Hypsipyla spp. shoot borers in Meliaceae were attempted for about eight decades in more

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

technology which will provide reliable, cost-effective and environmentally sound

protection for any of the high-value meliaceous tree species to produce a marketable stem

(Wylie, 2001). In contrast, when two-year-old (Swietenia humilis) trees were treated with

deltamethrin, it provided complete control against the H. grandella attack (Goulet et al.,

2005).

2.1.5.3 Biological methods

2.1.5.3.1 Natural enemies

The liberation of egg parasites Anthocephalus renalis Wtstn., Tetrastichus

spirabilis Waterston., Phanerotoma sp., Trichogrammatoidea nana (Zehntner) and Tr.

18

robusta Nagaraja for management of the shoot borer H. robusta and H. grandella at high

pest population level showed that the egg parasitoid, Trichogramma minutum Riley

failed to control H. robusta when transferred from the same host but only Tr. nana and

Tr. robusta were effective (Briceno, 1997; Sands and Murphy, 2001). A parasitic

nematode Hexamermis sp. was found to cause mortality of the H. robusta larvae in the

field and have been frequently isolated from larvae in the field. (Hauxwell et al., 2001b).

The parasitoid Apanteles sp. and Brachymeria conica (Ashmead) were found to parasitize

on larvae and pupae of H. robusta (Blanco et al., 2001).

The nematode Steinernema carpocapsae L. (Misra and Khan, 2003) and twelve

species of Chalcidoid (Antrocephalus hakonensis (Ashmead), A. hypsiphylae,

Brachymeria euploeae, B. hearseyi, B. tachardiae, Kriechbaumerella destructor

(Waterston), Tetrastichus spirabilis Wales, Trichospilus diatraeae Cherian and

Margabandhu, Diomorus orientalis Masi) were found to paratize the eggs and larvae of

H. robusta, (Kazmi and Neena, 2003). The egg parasitoids Solenopsis germinate

(Fabricius), Pheidole radoszkowskii Mayr and Crematogaster spp. were observed on one

developmental stage of H. grandella (Varon et al., 2005).The possibilities of the use of

the predatory ant Oecophylla smaragdina (Fabricius) was explored by a few workers.

This ant species may protect the trees from attack by the shoot borer and are likely to be

effective in controlling the shoot borer H. robusta (Grace and Laurence, 200l; Peng and

Christian, 2006; Lim et al., 2008).

19

2.1.5.3.2 Botanicals

Suharti et al. (1995) in their laboratory study on the effect of extracts of leaves

and seeds of neem at concentrations of 5, 10 and 20% against H. robusta showed that all

the concentrations repelled and killed the insects, and acted as contact poison. Mancebo

et al. (2002) and Goulet et al. (2005) studied the activity of two neem products, Azatin

and Nim 80 on H. robusta. Azatin caused quick direct toxicity and Nim 80 acted as a

growth disruptant, especially at high concentrations in both cases. The antifeedant

activity of plant species, Quassia amara L., Ruta graveolens L., R. chalepensis and

Sechium pittieri (Cogn.) C. Jeffrey. on third instar larvae of H. grandella larvae showed

that the antifeedant activity was high in Q. amara and R. graveolens at 10%

concentration (Mancebo et al., 2000; 2001).

2.1.5.3.3 Entomopathogens

Myres (1935) isolated Cordyceps sp and Metarhizium spp., when the larvae of H.

robusta and H. grandella were incubated in soil samples. Kandasamy (1969), Yamazaki

et al. (1990), Misra (1993) and Hauxwell et al. (2001) isolated Beauveria tenella (Sacc.),

B. brongniartii and B. bassiana with H. robusta. The effect B. bassiana evaluated on H.

grandella in a ten month old Cedrela odorata plantation by Salvatierra and Palm (1972),

Li et al. (1991), Sanchez and Velazquez (1998) and Goulet et al. (2005) showed that B.

bassiana applied in the interval of three months achieved 71% of death of larvae and B.

thuringiensis showed 67% death. Casanova et al. (2001) and Misra et al. (2003)

discussed the integrated management of Toon shoot borer by use of the entomopathogens

such as viruses, bacteria, fungi, and reckettsiae.

20

2.1.5.3.4 Irrigation and fertilizer application

Castillo et al. (2008) studied the effects of irrigation, fertilizer application (50 g

NPK/ tree) on the growth of 10-month-old cedar trees, showed negative results with

irrigation alone; fertilizer application resulted in the greatest increase in tree height and

diameter, and treatment with fertilizer + irrigation + insecticide gave the effective control

of the infestation H. grandella.

2.2 Arboreal termite, Odontotermes spp.

2.2.1 Distribution of Odontotermes sp.

The distribution of Odontotermes sp. was studied by several workers in tropical

and sub-tropical parts of the world. In Zimbabwe the species of the genera Macrotermes

and Odontotermes were primarily foragers involved in the damage of forest tress

(Mukumbareza, 1994) and in China about ten species of three families of Isoptera and

five genera of Microcerotermes remotes Ping & Xu (basionym) and Odontotermes

hainanensis (Light.) were dominant in a forest area of 0.25 km (Xiao et al., 1996). The

highly adaptable pest species composition and adaptability of Isoptera (Macrotermes

barneyi Light, Odontotermes formosanus Shiraki. and Microtermes dimorphus Tsai &

Chen.) in hilly areas of the mid-subtropics, southern subtropics and northern tropics in

China, varied between the regions, altitudes and niches (Lin et al., 1996).

Parihar (1994 and 1997) studied the nature and extent of damage on the standing

tress in forest by the genera Microtermes, Odontotermes and Coptotermes were identified

in Rajasthan. Thakur (1996 and 1999)and Rathore (1999) observed in the areas of

agriculture, horticulture and forestry in the arid areas of Gujarat, Punjab, Haryana were

21

adversely affected by species of Anacanthotermes macrocephalus (Desneux, 1906),

Coptotermes heimi (Wasmann), Odontotermes obesus (Rambur), O. latiguloides

(Roonwal & Verma) and O. distans (Holmgren and Holmgren).

Meyer et al. (1999) observed that the genera of Macrotermes, Cubitermes,

Amitermes, Odontotermes and Trinervitermes were the predominant mound building

termite species in agriculture and forestry area. The diversity of termites in tropical

forests of young eucalyptus plantations in Kerala (Varma and Swaran, 2007) showed that

during the month of April-November, a single species, O. obesus (Rambur) was the most

abundant and O. obesus (Holmgren) and O. feae (Wasmann) were dominant, during

February-March.

2.2.2 Damage and economic loss

Gowda et al. (1995) studied the damage on Grevillea robusta A.Cunm. ex R.Br

and Erythrina lithosperma Miq. by the species of O. obesus, O. redemanni, O. horni,

Macrotermes estherae and Nasutitermes indicola (Holmgren and Holmgren) was a

predominant termite that feeds on the bark and living tissues of the plants in Karnataka.

Mohan (1995) studied the damage of 24-yr-old teak Tectona grandis L.f. plantation in

Maharashtra observed that the termites of O. parvidens and O. distans were mostly

affects the girth classes below 45 cm.

Ali and Chaturvedi (1996) and Han and Ndiaye (1997) observed that the

seedlings, saplings and plantations of Dalbergia sissoo, T. grandis, Moringa oleifera

Catechu, Gmelina arborea Roxb., Ziziphus jujube Mill., Pongamia pinnata (L.) Panigrabi

22

and Ricinus communis L. were severely infected by Microtermes spp. and Odontotermes

sp in the month of April to September, and high in November to February.

Meshram et al. (1998), Remadevi et al. (1998) and Munthali et al. (1999)

reported that the plantation of Eucalyptus species Eucalyptus tereticornis, E. grandis, E.

resinifera, E. camaldulensis, E. brassiana and Santalum album were infested by the

dominant termites species Odontotermes spp. that leads to the loss of bark, poor health

and infestation of stem boring insects. Das et al. (1999) and Thakur (1999) studied the

infestation by termites in some plantations of Poplars (Populus spp.), showed that the

termites O. feae, Coptotermes heimi and O. distans where the primary causative factors in

dieback and mortality in certain clones of the exotics.

Bajwa and Gul (2000) and Meshram et al. (2001) showed that the nurseries and

plantations of exotic Paulownia and Gmelina arborea were also affected by major

termite, O. obesus. Baksha (2000 and 2001) studied the description, host record, nature of

damage, biology and management of the common timber pests and forest pests such as

Coptotermes spp., Cryptotermes spp., Neotermes spp. Odontotermes spp. and

Microcerotermes spp.

Arshad and Muhammad (2001) studied the damage of forestry species viz.,

Populus euamericana Guinier, Albizia lebbeck (L.) Benth., Morus alba L., D. sissoo,

Ficus virens L., Pinus roxburghii Sargent, Callistemon lanceollatus (Sm.), Bombax ceiba

L., Mangifera indica, Syzygium cumini (L.) Skeels., Cassia fistula L., T. ciliata,

Terminalia arjuna (Roxb.) Wight & Arn., A. arabica, D. sissoo and Heterophragma

adenophyllum (Wall. Ex G. Don) by three termite species, viz. O. obesus, Coptotermes

23

heimi and Microtermes obesi and severe damage was observed at the stem surface of

these plants.

The plantations of arecanut, okra, linseed and cotton were severely affected by O.

obesus and O. guptai in region of Maharashtra, West Bengal, Himachal Pradesh and

Bahawalpur (More et al., 2002, Singh and Joshi, 2003, Satpathi, 2003; Akhtar and

Glaulam, 2003). Ring-barking and root debarking of the sapling of Shorea spp and

Dalbergia sissoo Roxb were mainly by the genera of Odontotermes and Microtermes in

an enrichment planting and found near the tap-roots of the saplings (Guo, 2005).

2.2.3 Structure of mound of Odontotermes sp.

Patil and Basalingappa (1994) studied the occurrence and structure of mounds of

O. brunneus which was normally associated with vegetation and was either conical,

cylindrical, or dome shaped. The height of the mounds were 0.12-2.4 m and mounds had

hard, compact outer walls beneath where ventilation shafts and several interconnected

vaults housing the fungus gardens is present. Shahid and Akhtar (1995) sturied the nest

density of the fungus-growing subterranean termites belonging to the genus

Odontotermes was estimated as 60.69 and 50.58/ha. O. obesus was abundant than O.

gurdaspurensis and O. guptai. The circumference of the chambers in different nests

various and royal chamber was located 35 - 75 cm below ground level.

Darlington and Bagine (1999) and Darlington (2000) detailed that the mound of

O. montanus contained a very large and active nest consisting of 1,106 chambers with

149 kg dry weight of fungus comb and 96 chambers inside the mound. Lendi and

Basalingappa (2000) observed that in the nests of O. obesus of different locations, the

24

total numbers of workers and soldiers contributed to different functional behavior among

the populations.

Jouquet et al. (2002) observed that the termite O. pauperans build mounds

enriched with fine particle by modified soil texture for different parts of their structure.

The building activity increased when presented with different soil types and a notable

selection was observed in the use of a given soil type for a specific part of the structure

built. Miranda and D-Cruz (2005) explained that the termites species O. brunneus were

widely distributed mound building termite in Kerala. Mounds varying from 2 to 27 cm in

height and 11.9 to 115 cm in circumference at the base, contained a population ranging

from 3,900 to 70,700 individuals.

2.2.4 Biology of the genera, Odontotermes

Muhammad and Farooq (1994) studied the sex-ratio, sexual-dimorphism, weight

and moisture-content of O. redemanni. Gangawar and Kumar (2001) observed that a total

population of 99, 033 individuals with soldier and worker ratio of 1:8 were present in

mounds. The major workers passed through 5 instars, the first three were larval and the

last two were worker instars. Minor workers also developed from 3rd-instar larvae and

after another moult changed into minor workers. Pre-soldiers occurred in the 3rd larval

instar and after another six moults pre-soldiers developed into the soldier caste.

Vasanthkumar et al. (1994) studied the population density of workers, soldiers

and nymphs of the termite O. brunneus were the percentage of major workers in

peripheral fungus garden and foraging covered runways was higher compared to the other

parts of the mound. Percentage of minor workers was greater in the fungus garden around

25

the royal chamber and in the royal chamber than other parts of the mound. A high

percentage of soldiers were found concentrated in the fungus garden around the royal

chamber.

Mainuddin (1996) observed that the swarming of O. feae was always preceded by

the accumulated factors of rainfall, temperature, humidity and the soil environment. It

generally occurred in clear weather, occasionally during cloudy weather. Hanif (1999)

explained about the population of termites O. obesus contains workers (1,989) and

soldiers (2,219) and O. lokanandi had a population of 14, 050 termites. Seema et al.

(2005) in their behavioral studies of alates and colony foundation under laboratory

conditions in O. distans Holmgren & Holmgren initially lays about 25-28 comma shaped

eggs, which hatches out after 48-52 days. The first batch of workers became active after

40-45 days and takes 124 days from the date of swarming to the first gallery formation by

the workers.

Huang et al. (2008) studied the feeding and trophallaxis of O. formosanus using

rubidium chloride, the worker, soldier, and fifth- and sixth-instar recipients paired with

Rb-fed worker donors had a significantly higher Rb content than those paired with

control donors. The morphological differentiation of the genera of Odontotermes was

studied using scanning electron microscopic, the serrated mandible of soldiers helped to

differentiate between M. obesi and Odontotermes (Choudhury et al., 2001).

Farkhanda and Akhtar (2002a), (2002b) and Singh and Singh (2002a and 2000b)

studied the Manhattan distance of the tooth from the tip of the left mandible, margin, and

labrum were used to differentiate between the species of O. giriensis, O. assmuthi, O.

26

obseus and O. formosanus. Davison et al. (2001) and Kumari et al. (2009) studied the

taxonomy of termites of the genus Odontotermes were studied by partial sequences of the

mitochondrial 16S ribosomal RNA gene the termites O. tanganicus, O. anceps, O.

zambesiensis, O. obesus, O. bhagwatii and O. horni have distinct mitochondrial

haplotypes, with high A + T content confirming that these are separate biological species.

2.2.5 Management of termites

2.2.5.1 Physical method- Silivicultural and Synthetic trail-pheromone

Okwakol (2000) studied that the forest clearance resulted in a drastic reduction of

the number of species including O. amanicus and Microtermes luteus to about 40% of

what existed in the natural forest. Deng et al. (2002) synthesized a kind of trail-

pheromone analogue ((Z,Z)-dodeca-3,6-dien-1-ol, DDE-OH) of the black-winged

subterranean termite, O. formosanus. Active concentrations (10-3-10 ng/cm) of this

pheromone elicited similar behaviour in worker termites as natural pheromone extract

and they found that there was no difference between the response of workers to synthetic

and natural pheromones

2.2.5.2 Chemical methods- Insecticides

Intari et al. (1995) studied the various combinations of insecticides such as

alphamethrin (alpha-cypermethrin), lindane, dazomet and fungicides (mancozeb,

captafol) when applied in the plantation of Dalbergia latifolia soil against O. grandiceps

controlled the pest effectively. Similarly, eleven camphor oxime ether analogues and

heptachlor effectively killed all the worker termites of O. obesus (Pawar et al., 1995;

Srivastava et al., 1997). Akhtar and Mushtaq (1997) studied the effectiveness of Tenekil

27

(1600 ppm), a polychlorinated petroleum hydrocarbon, when applied to soil against O.

obesus and Microcerotermes championi showed effective against O. obesus.

Remadevi et al. (1998) studied the comparative effectiveness of different

insecticides (chlorpyrifos, BHC [HCH], phorate, quinalphos and lime) in various

combinations and doses and showed the use of chlorpyrifos 20 EC (1.5%) which gave

adequate protection for up to 4 months. Chande and Jagtap (1997) and Vankudre et al.

(1998) stated that the toxicities of organophosphorus compounds (s-triazole and fused

ring heterocycles), aldrin and dieldrin were effective to both the population of O. obesus

and O. wallonensis. Similarly, the granular application of Lindane at 0.5, 1.0, 1.5 and 2.0

kg a.i./ha in the soil reduced the level of Odontotermes sp. (Pushpa and Singh, 1998;

Sharma and Raina, 1998).

The toxicity effects of new pesticides such as Dursban (chlorpyrifos), Silonen and

Sumialfa (esenvalerate), endosulfan 35 EC at 7.3 ml kg-1 and Imidacloprid 70 WS when

applied in soil were effective against the population of Coptotermes formosanus,

Reticulitermes spp., Microtermes obesi, O. obesus, O. assmuthi, O. wallonensis, O.

formosanus and Trinervitermes biformis (Song et al., 2000; Kumawat, 2001; Santharam

et al., 2002; Kakde et al., 2006). The difference in transmission efficiency to nine kinds

of pesticides were the transmission efficiencies of O. formosanus to phoxim, beta-

cypermethrin, abamectin, acetamiprid and methomyl were higher than those of R.

chinensis (Fang et al., 2007).

2.2.5.3 Biological methods

2.2.5.3.1 Natural enemies

28

Sahayaraj (1995) showed the potential of the reduviid predator Acanthaspis

pedestris Stal as a biological control agent that effectively reduced the population of the

groundnut pest O. assmuthi The predatory behavior of the terrestrial planarian

Microplana termitophaga Jones, Darlington & Newson, 1990 consecutively consumed

13 termites of both workers and soldiers of O. transvaalensis over 3 h 14 min (Jones and

Cumming, 1998; Wesoowska and Cumming, 1999).

The larvae of a scuttle fly were reported as endoparasites of alate termites of O.

amanicus (Disney and Darlington, 2000) and Termitivorous salticid, Microheros

termitophagus Wesolowska et Cumming, 1999. showed predatory effect on the O.

transvaalensis (Wesoowska and Cumming, 2002). Ambrose et al. (2003) and Daniel and

Kumar (2003) showed that Rhynocoris longifrons (Stal) and Rhynocoris fuscipes can be

effectively used as a potential biological control agent against O. obesus.

Haddad and Wesoowska (2006) explained that the two Stenaelurillus species

from Southern Africa Stenaelurillus natalensis Haddad & Wesolowska 2006 were

feeding on the population of O. badius Similarly, the populations of Heterorhabditids and

Steinernematids were found to parasite on O. obesus (Kaushal et al., 2000) the

Steinernematids (S. feltiae) showed poor control over O. brunneus and O. formosanus

(Razak and Sivakumar, 2001; Zhu, 2002).

2.2.5.3.2 Botanicals

Efficacy of Calotropis procera leaves and seed kernels of Azadirachta indica,

were effective against Odontotermes spp (Parihar, 1994) and Rhizome oil of

Homalomena aromatic Schott. calamus oil from Acorus calamus L., clove oil from

29

Eugenia sp. and karanja oil from Pongamia glabra Vent., also showed the higher

percentage of mortality against O. obesus (Gurdip et al., 2000; Bhonde et al., 2001).

Gurdip et al. (2000) showed that when O. obesus population exposed for 2 h to the

essential oils of Coriandrum sativum Cilantro, H.aromatica and a monoterpenoid

(linalool) produced 100% mortality.

Singh et al. (2002), Manager et al. (2002) and Ganapaty et al. (2004) showed that

the leaf essential oil of Coleus amboinicus Lour., Calotropis extract and root extract of

Diospyros sylvatica Roxb. caused 100% mortality against O. obesus. Similarly, the leaf

extracts of Persicaria hydropiper (L.) Delabre and Pogostemon parviflorus Benth. to tea

termite, O. assamensis and O. obesus also showed 100% mortality (Rahman et al.,

2005). The termiticidal value of hot water extracts of the leaves of A. indica, Carica

papaya L., Lantana camara L., Ocimum tenviflorum L., P. glabra, Ricinus communis L.,

and Tagetes patula L. (Bharadwaj et al., 2006) and seed extract of Withania somnifera

(L.) Dunal (Indian ginseng), Croton tiglium L. (jamalgoota) and Hygrophila auriculata

Schumach. (talimkhana) was studied which caused changes in tunneling behavior,

number of bacterial colonies in hindgut and activities of enzymes in midgut of O. obesus

(Ahmed et al., 2007)

2.2.5.3.3 Entomopathogens

The pathogenic effects of Beauveria bassiana were evaluated against workers of

the termite O. obesus (Rambur), termite mortality ranged from 41-90% in treatments

compared with 19-21% in untreated controls. Mycelial growth was recorded on the leg

joint, abdomen, antenna, thorax and head regions of treated cadavers after 24 h

30

(Gurusubramanian et al., 1999). Infectivity of the soil mixed conidia of the fungal

pathogen Metarhizium anisopliae var. major was evaluated against the termite, O. guptai

the LC50 value at 120 h was calculated as 9.07 x 106 conidia/g soil. The LT50 value for the

most effective dose was 86.84 h. (Swaran and Varma, 2003).

The efficacy of M. anisopliae against scavenging termite pests of tea,

Odontotermes spp., was evaluated in vitro. The mortality of termite increased with

increasing period of incubation. Total mortality was observed within 12 days under

prevailing laboratory conditions (Debnath, 2005). An in vitro study was conducted to

evaluate the efficacy of M. anisopliae and B. bassiana against workers of the

subterranean termite O. obesus showed 50% mortality in worker termite population

(Kakde et al., 2005).

Dong et al. (2007) studied the efficacy of a new virulent M. anisopliae variety (M.

anisopliae var. dcjhyium) obtained from O. formosanus in China against the subterranean

termite, O. formosanus, showed 100% mortality of termites 3 days post-inoculation at the

concentration of 3x108 conidia/ml. The HCN-producing bacteria such as Pseudomonas

fluorescens (Flugge 1886) effectively killed O. obesus in laboratory and inhibit

cytochrome c oxidase (Devi et al., 2007).

2.2.5.4 Formulations/ Baits

Deka et al. (1999) in their field experiments with different formulations of

insecticides (fenvalerate 0.4% dust, malathion 10% dust and sugarcane pressmud) against

O. obesus, showed good effectiveness with the formulation malathion 10%. The field

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

31

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

good 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).

Huang et al. (2005) studied the antifeedent activity of Thiamethoxam formulation

(ACTARA 25 WG) against the African termites viz., Trinervitermes trinervius and O.

smeathmani that showed the product was consumed by the termites rather than repelled.

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

powder; sugarcane powder; millet powder; powder of log cultivating Lentinula edodes

(Berk.) Pegler (1976); Artemisia argyi H. Lev. & Vaniot powder and potato powder,

showed that the consumption rate, mud sheet area and mud sheet covering rate of the

millet powder was high and all the additives significantly improved the phagostimulating

effect on O. formosanus (Huang et al., 2006a).

Huang et al. (2006b) in their field studies on the use of attractive toxicants in

controlling ground and deadwood termites was effective in controlling O. formosanus

populations in the field. Rajavel et al. (2007) explained that baiting systems may provide

longer lasting control by eliminating or at least reducing termite activity the efficacy of

different bait materials in monitoring O. obesus for five days sugarcane bagasse was

more attractive. Study on management of O. obesus by baiting system resulted in

collection of 3, 130 termites in 7 days and made the colony weak.

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2.3 Metarhizium anisopliae (Metsch.) Sorokin

The ability to isolate the fungus Metarhizium and other entomopathogenic fungi

(EF) directly from soil, water or insect cadavers without fungal and bacterial

contaminants is a very useful tool for monitoring the incidence and/or persistence of

natural or introduced EF populations (Inglis et al., 2008), as well as for the discovery of

new isolates (Luz et al., 2004; Meyling and Eilenberg, 2006). The two most commonly

used methods for isolating entomopathogenic fungi from the environment are (1) baiting

the soil with an insect host, e.g., Galleria mellonella, which often affords good isolation

rates; or (2) using specific selective media containing fungicides and antibiotics to reduce

growth of contaminating fungi and bacteria that normally occur in soil.

Beilharz et al. (1982) in their study the two effective selective media for isolation

of many EF species are prepared by the addition of the fungicide dodine to oatmeal agar

or potato-dextrose agar (PDA) medium. Liu et al. (1993) used oatmeal agar selective

medium with dodine (ODA) is reportedly excellent for selecting certain Metarhizium and

Beauveria isolates from other soil-inhabiting fungi. Sneh (1991) and Griffin (1996)

explain the negative side of the ODA, however, is its opacity, which interferes with

counting colony forming units (CFUs) when colonies are small.

Luz et al. (2007) in their recent search for selective chemicals for isolating EF

from mosquito habitats found dodine and thiabendazole to be the most promising of the

five antifungal compounds examined). The fungicide dodine (n-dodecylguanidine

acetate) has a basic guanidine group that binds to anionic groups (carboxyls and

33

phosphate esters) and a lipophilic hydrocarbon chain that binds to the cytoplasmic

membrane

2.3.1 Heat tolerance and cold activity of M. anisopliae conidia

Most entomopathogenic fungi are mesophilic, showing good growth between 10

and 40 °C and optimal temperature between 25 and 35 °C (Walstad et al., 1970; Roberts

and Campbell, 1977). Yip et al. (1992) observed that maximum germination temperature

of many isolates of M. anisopliae is about 37 °C. However, substantial variation exists.

Certain M. anisopliae isolates that grow at 37 °C do not grow at low temperatures (5° C),

while isolates that grow at 5° C do not grow at higher temperatures (37° C ).

Rangel et al. (2005) and Fernandes et al. (2008) in their study some isolates do

not grow at either 5 or 37° C, but they do at 25° C . Also, high variability in thermo

tolerance was observed among M. anisopliae isolates when conidial suspensions were

exposed to high and low temperatures. Studies by Bidochka et al. (2001) and Li and Feng

(2009) on temperature tolerances of entomopathogenic fungi, high and low temperatures

were investigated with the aim of selecting isolates with highest resistance to extreme

environmental temperatures that may be encountered when applied as biological control

agents of insects and other arthropods.

2.3.2 Host-parasitic Interaction

2.3.2.1Conidial surface proteins

During M. anisopliae host infection, several enzymatic activities related to

penetration, toxic effects, and pathogenicity have been characterized (St. Leger et al.,

34

1990; Charnley, 2003). The participation of proteases, mainly Pr1 proteases, and

chitinases in the infection process is evident and has been demonstrated (Freimoser et al.,

2005). Other enzymes already described from this fungus, such as lipase (Silva et al.,

2005), superoxide dismutases (Schrank et al., 1993) and trehalase (Xia et al., 2002) in

addition to other unknown enzymes could be also related with infection and associated

with conidial surface.

Clergeot et al. (2001) and Voigt et al. (2005) The importance of enzymatic

activities like lipolytic activity at the initial stages of fungal infection have been widely

reported and closely related to infection systems of several pathogens including enzymes

present in the spore surface of filamentous fungus, which have a crucial role in host

infection. Recently, Qazi and Khachatourians (2007) and Wang and St. Leger (2007)

found that protease isoforms in hydrated conidia characterized as metalloproteases;

however, neither Pr1 nor Pr2 activities were detected.

2.3.2.2 Production of enzymes that disrupts the physiological integrity of hosts

St Leger et al. (1996) in their study of entomopathogenic fungi M. anisopliae that

produce proteases which are fundamental for pathogenicity, stated that since proteins are

abundant molecules present in arthropod cuticle. Segers et al. (1999) and Wang et al.

(2005) found that M. anisopliae secretes a diverse array of proteases, particularly

subtilisins, which are fundamental to transpose insect cuticle, colonize and digest tissues,

to survive in diverse niches in the environment during saprophytic existence, and may

also influence virulence and/or host specificity.

35

Freimoser et al. (2005) identified different proteases, including the subtilisins Pr1I

and Pr1J, elastase-like serine protease, carboxypeptidase, chymotrypsin and trypsin, on

M. anisopliae and reinforces the importance of proteases and the ability of this isolate to

cross arthropod cuticle. Fang et al. (2009) and Zaugg et al. (2008) reported that the

protease chymotrypsin, also identified in this work for DP, is present in the infection of

locust and other insects by M. anisopliae, as well as carboxypeptidases produced during

infection process of several fungi, including M. anisopliae.

Trypsins were described in entomopathogenic fungi, like M. anisopliae, in

response to cockroach (Periplaneta Americana Linnaeus, 1758) (St Leger et al., 1996)

and Callosobruchus maculates (Fabricus, 1775) (Murad et al., 2008), and in Zoophthora

radicans (Brefeld Batko) in response to the butterfly Pieris brassicae L. (Xu et al., 2006),

reinforcing trypsin participation in pathogenicity Chitinases are hydrolytic enzymes

secreted by many entomo- and mycopathogenic fungi. For M. anisopliae, due to cuticle

composition, chitinases and proteases work synergistically to degrade host cuticles during

the penetration process (Silva et al., 2005; Pedrini et al., 2007).

2.3.2.3 Toxic extrolites of Metarhizium anisopliae

Amiri et al. (1999) and Krasnoff et al. (2007) in their studied observed that

isolates of M. anisopliae produces a number of toxic extrolites, such as destruxins,

swainsonine, serinocyclins and cytochalasins. Among those destruxins (dtx), chemically

cyclic hepsadepsipeptides, are the most abundant metabolites and predicted to have

implications in fungal virulence. Bailey et al. (1996), Han et al. (2001), Wang et al.

(2004) and Hutwimmer et al. (2008) explains that Metarhizium dtx are believed to be

36

synthesized by a non ribosomal protein synthetase. CD chromosomes have been found in

plant pathogenic fungi and often contain genes coding for virulence determinants. The

dtx synthesis genes are located on an extra chromosome, termed conditionally

dispensable (CD).

2.3.2.4 Pigmentation and tolerance to abiotic stresses

Suderman et al. (2006) found that laccases in insect cuticle and plant cell walls

are cross-link structures that increases the rigidity in the outer wall of insects. Where

MLAC1 could be similarly involved in stabilizing the cell walls by forming cross-links.

The reduced ability of the DMlac1 appressoria to resist turgor pressure is consistent with

a change in the cell walls rigidity and/or permeability. Fang et al. (2010) in is study, he

identified a laccase MLAC1 in the filamentous fungus M. anisopliae that contributes to

conidial pigmentation, tolerance to abiotic stresses and pathogenicity. MLAC1 is only

expressed during isotropic growth, i.e. in conidia (pigmented), blastospores, appressoria

and hyphal bodies produced in insecta.

Bell and Wheeler (1984) studied the expression of MLAC1 very late in

conidiation and impaired pigmentation when Mlac1 is disrupted suggest an involvement

in conidial pigmentation. Pigments enhance the survival and competitive abilities of

fungi in diverse environments DHN-melanin is probably absent in M. anisopliae. Roberts

and St. Leger (2004) found that M. anisopliae lacked scytalone dehydratase activity (a

central enzyme in the DHN-melanin synthesis pathway). Rangel et al. (2006) found that

M. anisopliae also differs from other fungi in the signal transduction pathways involved

in pigmentation to adapte to deal with a broad vista of environmental stresses.

37

2.3.2.5 In vitro sub-culturing on the virulence

Fransen et al. (1987) found that entomopathogenic fungi Aschersonia aleyrodis

Webber lost its virulence towards greenhouse whitefly, Trialeurodes vaporariorum

(West.) after 19 sub-cultures on artificial medium. Morrow et al. (1989) reported a

decrease in the ability of Nomuraea rileyi (Farlow) Samsonto produce yeast-like hyphal

bodies after six conidial transfers on Sabouraud maltose and yeast extract agar. Further

development of a virulent progeny towards velvetbean caterpillar, Anticarsia gemmatalis

Hubner, 1818 after 10 conidial transfers was also observed.

Brownbridge et al. (2001) observed that a strain of Beauveria bassiana had

decreasing virulence against Leptinotarsa decemlineata (Bellaows and Perring) after 16

conidial transfers on an artificial medium. He also reported that no decrease in the

virulence of B. bassiana toward Bemisia argentifolii following repeated in vitro transfers.

On the other hand, Vandenberg and Cantone (2004) noted that the virulence of

Paecilomyces fumosoroseus (Wize) A.H.S.Br. & G.Sm. (1957) towards Diuraphis noxia

Kurdjumor, 1913 or Plutella xylostella did not change even after 30 in vitro transfers.

Interestingly, they further reported that different host passages had varying effects on

virulence.

2.3.3 Molecular studies

The development of recombinant DNA techniques, however, has made it possible

to significantly improve the insecticidal efficacy of viruses, bacteria and fungi (Inceoglu

et al., 2006; Wang and St. Leger, 2007). These advances have been achieved by

combining new knowledge derived from basic studies of the molecular biology and

38

genomics of these pathogens with technical developments that enable increases in gene

expression and the use of genes from other organisms that encode insecticidal proteins to

improve efficacy (Federici et al., 2008).

2.3.3.1 RAPD/RFLP

Poprawski et al. (1989), Viaud et al. (1996) and Neuveglise et al. (1994) studied

the geographic distribution among the population of Beauveria spp. using RFLP and

internal transcribed spacer region analysis, showed molecular variation among Beauveria

isolates and was related to insect host. Williams et al. (1990) and Berretta et al. (1998)

studied the genotyping of B. bassiana using PCR–RAPD that indicated that isolates from

the sugar cane borer Diatraea saccharalis shared 80% of homology of 276 bands.

Piatti et al. (1998) studied the regional variation in PCR–RAPD marker data of

Beauveria brongniartii isolates from the European cockchafer, Melolontha spp., in

France found that a high degree of similarity was present. He also used PCR–RAPD

analysis to distinguish two separate genetic groups of B. bassiana that infected the rice

water weevil, Lissorhoptrus oryzophilus, in Louisiana that were 45% divergent at 172

polymorphic bands. Urtz and Rice (1997) also suggested that the two groups represented

different populations that existed sympatrically

Based on PCR–RAPD and RFLP, Maurer et al. (1997) showed that B. bassiana

isolates derived from coleopteran insect species showed a high level of genetic

differentiation. Bidochka et al. (1994) and Berretta et al. (1998) in their studies observed

that no evidence for host-range clustering was shown for the entomopathogenic fungi

Metarhizium anisopliae and Metarhizium flavoviridae when PCR–RAPD bands were

39

analyzed. More recently, microsatellite data from Aspergillus flavus reported a lack of

significant genetic similarity of infective types of geographic component of isolate

variation was also found not to contribute to isolate differentiation (St. Leger et al.,

2000).

2.3.3.2 ITS

The molecular variation found between Beauveria isolates is related to the insect

host range, and was assessed using ITSs by Neuveglise and Brigoo (1994) and Viaud et

al. (1996) and using isozymes by Poprawski et al. (1988) and Mugnai et al. (1989). Fouly

et al. (1997) and Argentina (1999) studied an alternative approach is to use ribosomal

DNA (rDNA), an important molecular marker in the identification and differentiation of

species. He also explains that the rDNA operon of eukaryotes can be present in multiple

copies per genome, with each unit consisting of regions coding for rRNA, 18S, 5.8S and

28S genes as well as the two internal spaces (ITS 1 and ITS 2) between these regions,

each rDNA unit being separated by one intergenic space (IGS).

Esteve-Zarzoso et al. (1999) stated that the rDNA 18S and 28S regions are the

most conserved units, and may be used in differentiating genera and species, while the

ITS and IGS spacer regions have accumulated more variability and are better used to

differentiate species or strains within the same species. In the taxonomic revision of the

Metarhizium spp., Driver et al. (2000) recognized ten distinct clades or lineage of

organisms from 123 isolates based on internal transcribed spacer (ITS) regions, 5.8S

rDNA and the D3 region of 28S (LSU) of rDNA. The characterization of B. bassiana was

carried out using different kinds of DNA markers ITSs (Coates et al., 2003),

40

2.3.3.3 Improving efficacy by genetic engineering

St. Leger et al. (1996) and Bagga et al. (2004) explains that the most attractive

initial candidate genes for this approach include cuticle-degrading enzymes and toxins

that are encoded by single genes as they are highly amenable to manipulation by gene

transfer. Many of the cuticle-degrading enzymes that act synergistically to solubilize

cuticles are multiple-gene products with distinctive activity profiles. St. Leger et al.

(1996) stated that the first example of a recombinant fungal pathogen with enhanced

virulence, additional copies of the gene encoding the regulated cuticle-degrading protease

Pr1 were inserted into the genome of M. anisopliae and constitutively over expressed.

The resultant strain showed a 25% mean reduced survival times (LT50) towards Manduca

sexta as compared to parent wild-type strain.

2.3.3.4 Selection of virulence genes for enhancing efficacy

Quesada-Moraga et al. (2006) stated that the selection of potent virulence genes is

a crucial step for effective genetic engineering. Apart from cuticle-degrading proteinase,

a strain of M. anisopliae has been identified that produces an acute protein toxin activeat

0.7 µg/100 mg, and other toxins from M. anisopliae and B. bassiana are being isolated.

Quesada-Moraga and Vey (2004) isolated a toxic protein from B. bassiana (bassiacridin)

had an LT50 of 3 µg per insect when injected into fourth instar locust nymphs Locusta

migratoria.

Gongora (2004), St. Leger and Wang (2009) and Lu et al. (2008) employed

various techniques such as microarray, gene knockout, RNA interference, etc. that

identified adhesins, extracellular enzymes, and systems for evading host immunity. These

41

can be used to create novel combinations of insect specificity and virulence by

recombining them in other fungi, bacteria, or viruses to produce improved pathogens.

Wang and St. Leger (2007a) identified some genes are highly adapted to the

specific needs of M. anisopliae separate adhesins (Mad1 and Mad2) allow it to stick to

insect cuticle and plant cells, respectively However, the properties that make pathogens

unique are often organism- or species-specific. Thus, Mcl1 (immune evasion) with its

collagen domain is so far unique to M. anisopliae (Wang and St. Leger, 2006).