MASTER’S SEMINAR

Presented byAnkita M.Sc (Plant Pathology)1st year2016-17

ROLE OF ENTOMOPATHOGENIC

FUNGI IN INSECT CONTROL

INTRODUCTION•An entomopathogenic fungus is a fungus that can act as a parasite of insects and kills or seriously disables them (Singkaravanit et al., 2010).•It is a form of microbial control •Here virulence is caused by contact and action is through penetration(Nadeau et al., 1996)•Main aim of insect control is to keep the population of insect below economic threshold level(ETL) •They are effective against eggs, larvae, intermediate stages and adults of a variety of insects including locusts, grasshoppers, mosquitoes, and others

Impact of Entomopathogeni

c fungi

ENTOMOPATHOGENIC FUNGI (Moore et al.,1996)

homoptera

Lepidoptera

Diptera

Hemiptera

Coleoptera

Orthoptera

Dermaptera

Hymenoptera

Deuteromycetes /fungi imperfecti

Zygomycetes Ascomycetes Basidiomycetes

Beauveria Coelomomyces Cordyceps Septobasidium

Metarhizium Entomophthora - -Lecanicillium Massospora - -Paecilomyces - - -Hirsutella - - -Nomurae - - -

EXAMPLES OF SOME IMPORTANT ENTOMOPATHOGENIC FUNGI

MODE OF ACTION OF FUNGUS• Entry of fungi is through the integuments. (Wang et al., 2005; Cho et al., 2006) •Infective unit- spore (conidium).•Germination of conidia and formation of appresoria.•Penetration of cuticle by enzymatic as well as mechanical action(Hajek and St.Leger,1996).•Complete invasion.•Production of conidiophores by erumpent of cuticle.•Death of the host by obliteration of tissues as well through the toxins produced.

Healthy insect’s cuticle

Mycelium formation

Insect covered with mycelium

Spore formation and death of the

insect

Spore discharge

conidium

Germination of conidiaAppresorium formation and formation of infection peg

mycelium LIFE CYCLE OF FUNGUS

SYMPTOMS SHOWN BY INSECTS ON

INFESTATION BY FUNGUS•Loss of appetite •Attempt to climb higher up•General/partial paralysis•Discolored patches on integuments•Body hardens and the insect is in upright on its leg at the time of death•Specifically we uses the term “Mycoses” for such changes in insects and can be seen in – Lepidoptera, Hemiptera, Hymenoptera , Coleoptera and Diptera

Enzymes produced

1.Chitinase2.Chitosanase3.Chitobiase

4.Lipases

The successfulness of infection was directly proportional to secretion of exoenzymes (Khachatourians, 1996).

5.Phospholipase6.Proteases7.Peptidases

PATHOGENECITY OF ENTOMOPATHOGENIC FUNGI

The extensive transcriptomic and genetic study (Freimoser et al., 2003; Cho et al., 2007) of entomopathogenic fungal infection process has revealed that the genes governing following characters has important role in pathogenecity -

•Chitinases(Fang et al., 2005), •Guanine nucleotide-binding proteins and its regulator(Fang et al.,2007),•Adhesin which helps in attachment of spore(Wang & St. Leger, 2007), •A perilipin-like protein(Wang & St. Leger, 2006). •Subtilisin protease(St. Leger et al., 1996)

TOXIN PRODUCED•Toxin produced are the by products of metabolism and are not primarily used by the producer for killing the insect .•Entomogenous fungi are known to produce “Destruxins” and “Aflatoxins” •They are the only mycotoxins detected in the insect body at advance stages of infection in sufficient quantities to cause death.

Tritrophic interaction•Tritrophic effects-reason behind the success of entomopathogens(Cory J,S.& Ericsson J,D.2009)•Plant-mediated effects on fungal entomopathogens. •Plants could affect fungal entomopathogenseither directly or indirectly-1. Direct effects means anything emanating from

or produced by the plant that influences fungal infection of the insect

2. Indirect effects could occur before or after exposure of the insect to fungus, and would include factors that alter insect condition.

•Potential direct plant-mediated effectsmight include: (1) Plant exudates affecting theconidia directly, (2) Herbivore-induced plant volatilesaffecting sporulation or germination, (3) Leaf topology and surface chemistry, influencing the rate of spore acquisition by the host insect, (4) Plant architecture altering spore persistence, and (5) Leaf modifications of microclimate and thus spore germination.

•Indirect effects- (1)Plant quality, either allelochemicals or nutrients, altering insect condition (e.g. immunity) and thus disease resistance, (2) nutritional quality altering insect morphology (e.g. cuticle depth) which would influence the infection process, (3) changes in insect growth rate, which might alter the exposure of the insect to fungal entomopathogens, and (4) Plant structure altering insect behaviour, and thus fungal encounter rate.E.g..- Leaf surface wax- Metarhizium anisopliae- Mustard beetles (Phaedon cochleariae) on various crucifers- Dewaxing and Surface leachates increased germination and virulence. (Inyang et al.,1999)

3.IMPORTANT ENTOMOPATHOGEN

IC FUNGI

1.Beauveria bassiana

Beauveria spp-

• White muscardine fungus•Discovered in 1835 as cause of the Muscardine Disease of domesticated Silkworms(Furlong & Pell, 2005; Zimmermann, 2007) . • Got high host specificity• Pest of agricultural and forest including the Colorado potato beetle, the codling moth, and several genera of termites, American bollworm (Thakur et al., 2010).•Can be isolated from insect cadavers or from soil in forested areas by using media as well as by baiting soil with insects(Beilharz et al., 1982).

AGAINST•Termites •Thrips• Whiteflies •Aphids •Grasshoppers•Beetles •Caterpillars•Silkworms

SILK WORM LARVA

GRASSHOPPER

Spodoptera litura

BEETLE CICADA

2.Metarhizium anisopliae

Metarhizium spp.-

•Earlier it was first isolated from infected larvae of the wheat cockchafer Anisopliae austriaca in 1879 and named as Entomphthora anisopliae . •Renamed as M. anisopliae by Sorokin in 1883 (Tulloch, 1976) •Causes green muscardine disease. •Pathogenic to a large number of agricultural and forest insect species.(Ferron, 1978)

•Grubs of Coconut rhinoceros beetle •Grasshopper •Rice BPH •Sugarcane Pyrilla •Bollworm

AGAINST

RHINOCEROS BEETLE GRUB

GRASS HOPPER

RICE BPH

3.Lecanicillium muscarium

Lecanicillium lecanii (Zimm.) Zare & W.Gams(2001)

•Previously known asVerticillium •The fungus appears to have been first observed in Ceylon (Sri Lanka) about 1861, on diseased Lecanium coffeae •Widely distributed fungus•Controls whitefly and several aphids species, including the green peach aphids (Myzus persicae) for use in the greenhouse chrysanthemums( Hamlen et al., 1979). •Fungus attacks nymphs and adults of white fly and stuck to the leaf underside by means of a filamentous mycelium (Nunez et al., 2008).

GREEN SCALE

Effective against-•Coffee green scale •Other Hemipterans

Nomurae rileyi

Nomuraea sp.-

• Nomuraea rileyi can cause epizootic death in insects. • Lepidopteran including Spodoptera litura and some belonging to Coleoptera are susceptible to it(Ignoff, 1981) .• The host specificity of N. rileyi and its ecofriendly nature encourage its use in insect pest management. •Although, its mode of infection and development have been reported for several insect hosts such as Trichoplusia ni, Heliothis zea, Bombyx mori, Pseudoplusia includans

Larva of Spodoptera infested with Nomurae

Paecilomyces fumosoreus

Paecilomyces sp.-

•Paecilomyces fumosoroseus also called “Yellow Muscardine” (Nunez et al., 2008) . •Effective over Bemisia and Trialeurodes spp. in both greenhouse and open field environments.•Grows extensively over the leaf surface under humid conditions that helps it to spread rapidly through whitefly populations( Wraight et al., 2000) . •Best for controlling the nymphs of whitefly (Kim et al.2002). •These fungi cover the body of whitefly with mycelial threads and stick them to the underside of the leaves.•The nymphs show a “feathery appearance” (Nunez et al., 2008) .

• Trichoplusia ni• Heliothis zea• Bagrada cruciferarum• Bombyx mori• Anticarsia gemmatalis.

AGAINST

Bagrada cruciferarum infested by Paecillomyces fumosoroseus

Hirsutella thomsoni

Hirsutella thompsonii – •Originally isolated from an eriophyid mite in Tamil nadu•Effective on Eriophyid mites, particularly the coconut mite (Aceria guerreronis Keifer)•Major crop use is in coconut plantations, but can be used in palmyrah palm and in arecanut. •Widespread in nature •Not pathogenic to non-target species.•Not shown any adverse effects on the environment

It is specific to the eriophyid mites 1.Coconut mite2.Citrus rust mite

AGAINST

CITRUS RUST MITE

Coconut mite infected with Hirsutella thomsonii

Formulation of fungal propagules and Usage

• They are being used in different forms like -a. Dust b. Mixed with waterc. Mixed with Oil• Best method for is application of active ingredient

with oil

The formulation of propagules of fungal entomopathogenic fungi are guided by

•Improved product shelf-life•Bio-control efficacy, •Physical characteristics of the product for applicationFor e.g.- Control of insect pests of the phylloplane - spore suspensions are applied as spray application

Entomopathogenic fungi product available in

market Product name Firm Bio-guard rich Plantrich chemicals &

biofertilizers ltd.Bio-power T.Stanes &company Ltd.Racer Agri lifebeavera Jai biotech industriesBrigade Kan biosys pvt Ltd.Mycojal Pest control pvt. Ltd.Bio-be-ba Microplex- hosted by nagarjuna

agro chemicalsBaba Multiplex bio tech pvt. Ltd.

Metabeave R b herbal agro

Beauveria bassiana

Product Firm

Bio-magic T.Stanes &company LtdBiomet rich Plantrich chemicals & biofertilizers

ltd.Pacer Agrilife , India

Kalichakra International panacea LtdCropmet Microplex- hosted by nagarjuna

agro chemicalsMetrocid Sri biotech laboratories India Ltd.Metaz Jai biotechMetarhizium Multiplex biotech pvt. Ltd.Jasmeta Shri ram solvent extraction pvt

Ltd.Biostorm Varsha bioscience & technology

Metarhizium anisopliae

Product Firm

Bio-catch T.Stanes &company LtdBiovert rich Plantrich chemicals &

biofertilizers ltd.Mealikil Agrilife , India Vertimust Jai biotech industriesBiograde - v Kan biosys pvt. Ltd.Vertifire - L International panacea Ltd.Cropfit Microplex- hosted by nagarjuna

agro chemicals

Lecanicillium muscarium

Product Firm Paci hit rich Plantrich chemicals &

biofertilizers Ltd.Mysis Varsha bioscience & technologyNematox Sri biotech laboratories India Ltd.

Paecillomyces spp.

•In India more than 60% of pesticide is used in agriculture•Among chemical pesticides(Wahab,2003,2005,2009,2015)1. Insecticides=60%2. Fungicides + bactericides=20%3. Herbicides =17%4. Others =3%• Majority of chemical pesticide not only kill the pest but also kill

many beneficial insects and non-target animals of ecosystem (Abhilash and Singh,2009)

• Posing serious threat to human health (Maroni et al., 2006, Wahab,2004)

• High risk to field workers and consumers• Resistance, resurgence and persistence of insect

PRESENT SCENARIO

USEFULNESS IN PRESENT SCENARIO

•By going for this method we can reduce our dependency on

chemicals.

•We can have favourable diversity of flora and fauna near

crop region by keeping non target organisms.

•No problem of biomagnification.

•No pollution of water table .

•They have shown induced systemic resistance in the host

plant (Vega, 2008 )

Fungus Pest & Crop Field efficacy ReferenceBeauveria bassiana

Coffee berry borer, Hypothenemus hampei

Spray of B. bassiana spore suspension(1×107

spores/ml) containing 0.1% sunflower oil and 0.1per cent wetting agent during monsoonreduced 50-60per cent berry borer incidence inCoorg, Karnataka

Anon. (2001)

Sunflower: Helicoverpaarmigera

Spray of oil suspension of B. bassiana (200mg/l) in Andhra Pradesh

Devi and Hari (2009)

Green gram:White grubs

Soil application @5×1013

conidia/ha effective control achieved in Assam

Bhattacharyya et al.(2008)

Biological Control of pests using entomopathogenic fungi in India

Fungus Crop and pest Field efficacy Reference

Metarhiziumanisopliae

Coconut: Rhinoceros beetle, Oryctes rhinoceros

Spraying of Spores in its breeding sites 5X1011

spores/m3 to the compost pits andmanure heaps

Anon. (2000)

Potato White grubBrahmina sp.

Soil application @ 5x1013

conidia/ha along with chlorpyrifos 20 EC@ 200 g a.i./ha resulted in thehighest tuber yield (155 q/ha) in HP.

Bhagat et al. (2003)

Soyabean: white grubHolotrichia longipennis

Soil application formulation applied @ 5x1013 conidia/ha, 61.50per cent reduction in grubPopulation in MP

Pandey (2010)

Sr. No Treatment Grub mortality (%)

1 Steinernema carpocapsae (80 IJ/cm²)

34.9

2 Heterorhabditis indica (80 IJ/cm²)

45.9

3 Beauveria bassiana (10 ⁶ conidia/cm2 )

34.0

4 Metarahizium anisopliae (10⁶ conidia/cm²)

74.4

5 Chlorpyriphos (0.06%) 86.46 Control( Untreated ) 8.5

Evaluation of entomopathogenic fungi and EPNs against Apple root borer Dorysthenes

hugelii at YSPUHF, Solan (Anonymous:Annual progress report (2012-13) of AICRP

on biological control of crop pest and weed)

Fungus Crop and pest

Field efficacy Reference

Lecanicilli-um lecanii

Coffee green scale,Coccus viridis

Spraying spores@ 16 X 106

spores/ml along withTween-80 twice at 2 weeks interval caused 97.6 per cent mortality of the pest

Jayaraj (1989

Citrus green scaleCoccus virids

Spraying of spore (2x106spores/ml) along with 0.005 per cent Quinalphos and 0.05per cent Teepol was found highly effective killing 95.58 percent and 97.55percent scales in coffee and citrusrespectively

Singh (1995)

Indian mustard andRapeseed: Mustard aphid, Lipaphis erysimi

Spray @ (106spores/ml). There was a significant reduction in aphid infestation at 10 DAS

Rana et al. (2002

Fungus Crop and pest Field efficacy Reference

Nomurae rileyi Castor: Spodoptera litura in AP

Spraying of spore (10x1010

spores/ml) along with 0.02per cent Tween-80

Vimala Devi and. Prasad (1997)

Soybean: Spodoptera litura,Helicoverpa armigera,Thysonoplusia orichalcea

N. rileyi spores spraying @ 2X106

/ml twice at10 days interval during Kharif in North Karnataka was cheaper than insecticidaltreatment and cost effective

Lingappa et al. (2002)

ADVANTAGES

Harmless to other forms of life

High degree of specificity

Compatible with many chemical insecticide

Resistance to microbes is less likely to develop

Self sustaining so economical

Easy applicationAesthetically acceptable

DISADVANTAGESSpecificity is disadvantageous as in some cases only single insect is not involved

Totally dependent on environment, so uncertainty is always there

Not quick results ,as establishment takes time

CONSTRAINTS •A hi-tech venture terms of safety and sustenance

•Viability and virulence of fungal inoculum are the pre-requisite threshold for their efficacy (Doust & Roberts, 1983).

•An array of abiotic stresses like temperature (Rangel et al., 2005a), UV radiations (Rangel et al., 2006a), humidity (Lazzarini, 2006), edaphic factors and nutrient source (Shah,2005) affect negatively the field use of entomopathogens as bio-control agents

•Soil temperature is a major factor, that affects the success or failure in the establishment and production of fungal inoculums (Thomas & Jenkins, 1997)

Eg. 1) M. anisopliae has temperature tolerance upper limit as 37-40ºC (Thomas & Jenkins, 1997). 2) B. bassiana on the other hand can survive up to a maximum temperature of 37 ºC (Fargues et al., 1997)..Environmental factors affect pathogenicity as well as mode of virulence of entomopathogenic fungi (Hasan, 2014).

•They should be tolerant to the soil temperature but also have to survive through thermoregulatory defense response of the host insect

•Dry heat exposure causes DNA damage leading to depurination resulting into mutation (Nicholson et al., 2000)

•Wet heat i.e. heat in conjunction with high humidity causes protein denaturation and membrane disorganization.

The application of entomopathogenic fungi for insect control is increasing largely because of •Greater environmental awareness•Food safety concerns •The failure of conventional chemicals due to an increasing number of insecticide resistant species.•They provides us significant and selective insect control.

CONCLUSION

THANK-YOU