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CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources 2011 6, No. 056

Review

Microbial biopesticides: opportunities and challenges

Opender Koul*

Address: Insect Biopesticide Research Centre, 30- Parkash Nagar, Jalandhar 144003, India.

*Correspondence: Email: okoul©airtelmail.in; okoul©koulresearch.org

Received:Accepted:

10 October 201117 November 2011

doi: 10.1079/PAVSNNR20116056

The electronic version of this article is the definitive one. It is located here: http://www.cabi.org/cabreviews

© CAB International 2011 (Online ISSN 1749-8848)

Abstract

Pesticides based on microorganisms and their products have proven to be highly effective, speciesspecific and eco-friendly in nature, leading to their adoption in pest management strategies aroundthe world. The microbial biopesticide market constitutes about 90% of total biopesticides andthere is ample scope for further development in agriculture and public health, although there arechallenges as well. This article reviews the various microbial biopesticides that are commerciallyavailable, the different approaches for their production and development, the recent technologicaladvances and the challenges faced by the microbial biopesticide field in the future.

Keywords: Microbials, Bacteria, Viruses, Fungi, Nematodes, Biopesticides, Pest management,Commercialization

Introduction

The damage and destruction inflicted on crops by pestshave had a serious impact on farming and agriculturalpractices for a long time. These pests include insects,fungi, weeds, viruses, nematodes, animals and birds. It hasbeen estimated that nearly 10 000 species of insects,50 000 species of fungi, 1800 species of weeds and 15 000species of nematodes destroy food and fibre crops usedby millions of people worldwide. In India alone, 30% of thecrop yield potential is lost as a result of insects, diseaseand weeds, corresponding to 30 million tons of food grain.In an attempt to avoid such losses, the primary strategyemployed has been to eliminate the pests by usingchemical pesticides such as chlorinated hydrocarbons,organophosphates and carbamates. However, despitethe successes achieved, potential hazards or risks haveemerged that have had a substantial impact on theenvironment; compounded further by indiscriminate andexcessive use of the products. Consequently, beneficialspecies have been lost and residual problems haveincreased, with subsequent impact on the food chain,groundwater contamination and resistance in pests. Toovercome the hazards associated with chemical pesti-cides, the use of biopesticides (pesticides derived fromsuch natural materials as animals, plants, microorganisms

and certain minerals) is increasingly being adopted. NorthAmerica uses the largest percentage of the biopesticidemarket share at 44%, followed by the EU and Oceaniawith 20% each, South and Latin American countries with10% and about 6% in India and other Asian countries[1, 2]. In terms of sales of biopesticides, the global marketin 2007 was US$672 million and projected as US$1000million for 2010 (Figure 1). The current growth of thechemical pesticide market is about 1-2% per year, whilegrowth in microbial pest control is about 10% per year(with some estimates projecting growth as high as 20%)[3]. As of 2007/08, estimates of microbial biopesticidesales were US$396 million at the end-user level, althoughthese estimates are likely to be just a fraction of the totalusage of such products, owing to the lack of informationavailable on the non-commercial use of such products inthese regions. Such estimates could be more unreliableglobally, given unquantified sales in other parts of theworld. A recent survey has shown that Europe is a large,intense and diverse pesticide market valued at $12850million in 2008; approximately 31.7% of the world's total.The six largest national agrochemical markets are inFrance, Italy, Spain, UK, Germany and Turkey. Francealone accounts for 26.2% of agrochemical sales of thewider Europe, with Italy taking 19.8%. The Nordic coun-tries, on the other hand, consume comparatively small

http://www.cabi.org/cabreviews

2 Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources

1000

2007 2010

400

See

700fl S

0 600

vi..E

400

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100

ico 12D

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Glob Eurup, Asia LatinAmerica

Figure 1 Biopesticide market in different continents(Source: BCC Research Report 2010)

amounts; Denmark, Estonia, Finland, Lithuania, Norwayand Sweden combined use just 2.2% of the total [4].

Over the past 150 years, a great deal of knowledge hasbeen gathered on the use of microorganisms includingbacterial, fungal, viral, protozoan or nematode-basedpreparations as pest control agents (see Box 1). However,the widespread use of biopesticides has been restricted,owing to various constraints at the developmental,registration and production levels. Assessment of the useof microorganisms in pest management suggests thatadvances have been incremental, rather than transfor-mative. This is apparently the result of higher productioncosts in comparison with conventional chemical pesti-cides, narrow target-species ranges and inefficient deliverysystems. Although there have been ample advances interms of new discoveries of microbial isolates and anincreasing ability to genetically manipulate the microbialagents involved, concerns about pest resistance andenvironmental and human safety remain. Furthermore,increased adoption of microbial biopesticides has comeunder threat from the development of new biorationalpesticides (including pest control agents, and chemicalanalogues of naturally occurring biochemicals such aspheromones, insect growth regulators, etc., which aremore environment friendly than synthetic chemicalpesticides). Comparative analyses of conventional insec-ticides with microbial biopesticides (see Box 2) suggestthat there is still a lack of knowledge regarding theinteractions of microbial biopesticides with pests, naturalenemies and the wider ecosystem.

Microbial control agents, based on naturally occurringfungi, bacteria, viruses or nematodes have offered somerealistic alternatives to chemical pesticides when usedas part of an ecologically based integrated pest manage-ment (EBIPM) or area-wide pest management strategy(AWPM) [5, 6]. There are many reasons for the recentincreased interest in microbial biopesticides, including

the development of resistance to conventional syntheticpesticides, a decline in the rate of discovery of novelinsecticides, increased public perception of the dangersassociated with synthetic pesticides, host-specificity ofmicrobial pesticides and improvement in the productionand formulation technology of microbial biopesticides.

In view of various opportunities and challenges thatare associated with the development of microbial bio-pesticides, this article reviews the commercially availablemicrobial biopesticides, the different approaches for theirproduction and development, the technological advancesmade and constraints envisaged in future in the field ofmicrobial biopesticides.

Microbial Biopesticides in Pest Management

Out of all the biopesticides used today, microbial bio-pesticides constitute the largest group of broad-spectrumbiopesticides, which are pest specific (i.e., do not targetnon-pest species and are environmentally benign). Thereare at least 1500 naturally occurring insect-specificmicroorganisms, 100 of which are insecticidal [2]. Over200 microbial biopesticides are available in 30 countriesaffiliated to the Organization for Economic Co-operationand Development (OECD) [7]. There are 53 microbialbiopesticides registered in the USA, 22 in Canada and21 in the European Union (EU) [8, 9] although reports ofthe products registered for use in Asia are variable [10].Overall, microbial biopesticide registrations are increasingglobally, the expansion of various technologies has

increased the scope for more products and the change inthe trend to develop microbial products is definitely onthe rise [1, 11].

Bacterial Biopesticides

The bacteria that are used as biopesticides can be dividedinto four categories: crystalliferous spore formers (suchas Bacillus thuringiensis); obligate pathogens (such as Bacilluspopilliae); potential pathogens (such as Serratia marcesens);and facultative pathogens (such as Pseudomonas aeruginosa).

Out of these four, the spore formers have been mostwidely adopted for commercial use because of their safetyand effectiveness. The most commonly used bacteria areB. thuringiensis and Bacillus sphaericus. B. thuringiensis is aspecific, safe and effective tool for insect control [12]. Itis a Gram-positive, spore-forming, facultative bacterium,with nearly 100 subspecies and varieties divided into 70serotypes [13]. The insecticidal property of B. thuringiensisresides in the Cry family of crystalline proteins that areproduced in the parasporal crystals and are encoded bythe cry genes. The Cry proteins are globular molecules(65-145 kDa, depending on the strain) with three struc-tural domains connected by single linkers. The 200 Cryproteins belong to a single family that contain about


Opender Koul 3

Box 1 Historical Perspective

Diseases of honeybees and silkworm have been known inChina and India since ancient times. Observations of diseasedsilkworms were recorded in China as far back as 2700 B.C.The first documentation of insect diseases is usually attrib-uted to the descriptions of honeybee maladies recorded byAristotle somewhere between 330 and 323 B.C. Most ofthe earlier work on insect pathology is devoted to thesetwo domesticated insects, viz. the silkworm, Bombyx mori(Linnaeus) and the honeybee, Apis mellifera Linnaeus. In 1835,Agostino Maria Bassi (called the Father of Insect Pathology)published his work on white muscardine disease of the silk-worm and the fungus responsible for this disease was later onnamed as B. bassiana (Balsamo) Vuillemin [135].

The first published record of a diseased insect appears tobe that of the 'Chinese plant worm', described and illustratedby Rene-Antoine Ferchault de Reaumur in 1726. He under-stood the `stemlike vegetable growth', emerging from anoctuid larva to be the root of a plant. In 1749, a Franciscanfriar in Cuba described dead wasps with little trees growingout of their bellies. This was a fungus now known as amember of the genus Cordyceps [136]. During the mid-nineteenth to early-twentieth centuries, numerous scientistsreported on the biology and pathology of the entomo-pathogenic fungi who recommended study of fungal epizooticsof insect to determine the most effective means of introdu-cing and transmitting such pathogens.

A Russian entomologist, Metschnikoff, conducted thefirst systematic experiments on the control of injuriousinsects with microorganisms by infecting grubs of the grainbeetle, Anisoplia austriaca, with the green muscardine fungus,M. anisopliae (Metschnikoff) Sorokin, in 1879. The fungus wasfound to be even more effective against the sugar beetcurculio, Cleonus punctiventris (Germ.). A Japanese scientist,who named it Bacillus sotto, first discovered a bacterium insilkworms in 1901 but it was 10 years later that a German

scientist, Ernst Berliner, isolated the bacterium from diseasedflour moths and assumed it was responsible for the sudden-collapse disease, which was affecting the insect. Since he hadobtained the first sample from a mill in Thuringia, he calledthe bacterium Bacillus thuringiensis (Bt).

Field trials with Bt to control the European corn borerwere conducted as early as the late 1920s and in 1938 thefirst commercial Bt preparation (Sporeine) came on to themarket in France. However, it was not until the 1960s that itsuse became widespread. In 1964, Biospor became the first Btpreparation to be licensed as a pesticide in Germany. Anotherbreakthrough in the development of microbial control camewith the discovery and practical application of the milkydisease bacteria, Bacillus popilliae Dutky, for the control of theJapanese beetle, Popillia japonica Newman on turfs in USAduring 1940s [137]. But with the discovery in 1970 of theparticularly virulent Bt strain B. thuringiensis kurstaki, which iseffective against the larvae of certain butterflies and moths,and the discovery in 1983 of the B. thuringiensis tenebrionisstrain, which is effective against certain beetles, including theColorado potato beetle, the range of microbial agents avail-able has grown considerably.

Viruses as microbial pesticides have a history of slowdevelopment. A. Maestri and E. Cornalia made the firstobservations of viral infections in 1856. Hoffman reportedNPV of nun moth in 1891 and Bolle demonstrated viraltransmission from diseased to healthy insects in 1894. Thefirst description of OBs as causatives of infection camein 1906 and in 1918 the filterable and transmissible natureof viral infection was established. However, the first bio-chemical study of the OBs was published in 1934 byGlaser and Stanley and it was after 1947, Edward Steinhausin USA and Gernot Bergold in Canada undertook amajor effort to develop insect viruses as microbial insecticides[138].

50 subgroups [14]. This three-domain family is charac-terized by protoxins of two different lengths, one beinglonger with a C-terminal extension necessary for toxicity.This extension also has a characteristic role in crystalformation within the bacterium [15]. Cry proteins areresponsible for feeding cessation and death of the insectand their biology has been comprehensively described [2].Cry protoxins are ingested [13, 16] and then solubilized(some toxin families need more alkaline pH and othersrespond more to neutral pH), releasing a protease-resistant biologically active endotoxin, before being

digested by protease of the gut to remove amino acidsfrom its C- and N-terminal ends. The C-terminal domainof the active toxin binds to specific receptors on thebrush border membranes of the midgut followed by theinsertion of the hydrophobic region of the toxin intothe cell membrane [17]. This creates a disruption in theosmotic balance, because of the formation of trans-membrane pores and ultimately cell lysis occurs in the gutwall, leading to leakage of the gut contents (Figure 2).

This induces starvation and lethal septicaemia of thetarget pest.

Many details of this process are still not understoodand the action seems to be more complex as indicated bythe existence of novel receptors and signal transductionpathways induced within the host following intoxication[18]. This could lead to altered activation of midgutproteases, resulting in differences in the toxin structurethat could affect binding to the peritrophic membrane,thereby accounting for host specificity [19]. Subspeciesof B. thuringiensis that are used as biopesticides includeB. thuringiensis tenebrionis (targeting Colorado potatobeetle and elm leaf beetle larvae), B. thuringiensis kurstaki(targeting a variety of caterpillars), B. thuringiensis israe-lensis (targeting mosquito, black fly and fungus gnat larvae)and B. thuringiensis aizawai (targeting wax moth larvaeand various caterpillars, especially the diamondback mothcaterpillar). These bacteria are mass-produced througheither solid or liquid fermentation. The cost of onelitre of medium for the production of B. thuringiensis



Box 2 Advantages and Disadvantages of Microbial Versus Chemical Pesticides

Microbial PesticidesAdvantages

Microbial pesticides are non-toxic and non-pathogenic tonon-target organisms and the safety offered is theirgreatest strength.Action of microbials is specific to a single group or speciesof pests, therefore, do not affect directly beneficial animalssuch as predators and parasitoids.Microbial pesticides can be used in many habitats wherechemical pesticides have been prohibited. Such habitatsinclude recreational and urban areas, lake and streamborders of watersheds, and near homes and schools inagricultural settings.Residues of microbial pesticides are non-hazardous and aresafe all the time, even close to harvesting periods of thecrops.They have a potential to control vectors. Some pathogenicmicrobes can establish in a pest population or its habitatand provide control during subsequent seasons or pestgenerations.

Disadvantages

Owing to the specificity of the action, microbesmay control only a portion of the pests present in a fieldand may not control other type of pests present in treatedareas, which can cause continuous damage.As heat, UV light and desiccation reduces the efficacy ofmicrobial pesticides, the delivery systems become animportant factor.Special formulations and storage procedures are

necessary. Shelf life is a constraint, given their short shelflives.

Given their pest specificity, markets are limited.The development, registration and production costscannot be spread over a wide range of pest controlsales; for example, insect viruses are not widely avail-able.

Some insects develop resistance to several insect patho-gens. Resistance management will have to be practiced, asit is with chemical pesticides.

Chemical PesticidesAdvantages

Chemical pesticides are cost-effective and economical tocontrol pests. Low labour input is required and they allowlarge areas to be treated quickly and effectively. Estimatessuggest that 4-fold returns are expected after the use ofthese pesticides.Chemical pesticides are flexible in the sense that theycontrol all pests with variation in type, activity and per-sistence.They are easily available in large quantities, at high qualityand at reasonable price.

Pesticides are often used to stop the spread of pests inimports and exports, preventing weeds and protectinghouseholds from destruction.They have substantial application in protection of pets andhumans from pests.

Disadvantages

Reduction in beneficial insects due to the toxicity of thesepesticides to non-target pests can result in changes inbiodiversity of an area and affect natural biological balance.Drift of sprays and vapour of chemical pesticides can causesevere problems in different crops, waterways and generalenvironment.Chemical pesticides do leave residues in food, either bydirect application or by bio-magnification. Because of theirpersistent use in agriculture, chemicals can reach under-ground aquifers and contaminate water bodies.There are poisoning hazards for pesticide operators givenexcessive exposure; though it depends on dose, toxicity,sensitivity and duration of exposure.Overuse of chemical pesticides encourages resistance.

israelensis has been estimated as US$ 1.2 and 0.01 usingcommercial complex medium versus by-products of in-dustrial factories, respectively. Some common commer-cial products based on Bacillus thuringiensis are availableglobally (Table 1).

B. sphaericus is a Gram-positive strict aerobic bacter-ium, which produces round spores in a swollen club-liketerminal or subterminal sporangium [20]. B. sphaericusstrains were isolated in the mid-1960s from mosquitoes,blackflies and grasshoppers [21]. These bacteria producean intracellular protein toxin (5511-1) and a parasporalcrystalline toxin at the time of sporulation [22]. Themosquito-larvicidal binary toxin produced by B. sphaericusis composed of BinB and BinA, 51.4 and 41.9 kDa,respectively. Bin proteins in combination form a crystaland in solution can exist as an oligomer containing twocopies each of BinB and BinA [23, 24]. Some toxic strains

also produce 100 kDa toxins encoded by mtx genes [25].Bacillus-sphaericus-based products are commonly used formosquito control (Table 1).

Other species of bacteria have little impact on pestmanagement though some commercial products based onAgrobacterium radiobacter, B. popilliae, B. subtilis, P. seudo-monas cepacia, P. seudomonas chlororaphis, P. seudomonasflourescens, P. seudomonas solanacearum and P. seudomonas

syringae are available (Table 1).

Viral Biopesticides

Over 700 insect-infecting viruses have been isolated,mostly from Lepidoptera (560) followed by Hymenoptera(100), Coleoptera, Diptera and Orthoptera (40) [2].About a dozen of these viruses have been commercialized


for use as biopesticides (Table 1). The viruses used forinsect control are the DNA-containing baculoviruses(BVs), Nucleopolyhedrosis viruses (NPVs), granuloviruses(GVs), acoviruses, iridoviruses, parvoviruses, polydna-viruses, and poxviruses and the RNA-containing reo-viruses, cytoplasmic polyhedrosis viruses, nodaviruses,picrona-like viruses and tetraviruses. However, the maincategories used in pest management have been NPVs andGVs. These viruses are widely used for control of vege-table and field crop pests globally, and are effective againstplant-chewing insects. Their use has had a substantialimpact in forest habitats against gypsy moths, pinesawflies, Douglas fir tussock moths and pine caterpillars.Codling moth is controlled by Cydia pomonella GVs onfruit trees [26] and potato tuberworm by Phthorimaeaoperculella GVs in stored tubers [27]. Virus-based prod-ucts are also available for cabbage moths, corn earworms,cotton leafworms and bollworms, beet armyworms,celery loopers and tobacco budworms (Table 1).

The mechanism of viral pathogenesis is through repli-cation of the virus in the nuclei or in the cytoplasmof target cells. The expression of viral proteins occursin three phases. First is the early phase, i.e. 0-6 h post-infection, second is the late phase, i.e. 6-24 h post-infection and the third phase is very late phase, i.e. up to72 h post-infection. It is at the late phase that virionsassemble as the 29 kDa occlusion body protein is syn-thesized. Numerous virions of NPVs are occluded withineach occlusion body to develop polyhedra. However,the GV virion is occluded in a single small occlusion body,to generate granules (Figure 2). Infected nuclei can pro-duce hundreds of polyhedra and thousands of granulesper cell. These can create enzootics, deplete the pestpopulations, and ultimately create a significant impact onthe economic threshold of the pest.

The viral biopesticides are usually only active againsta narrow host spectrum and after their application toplant surfaces, and baculovirus occlusion bodies (OBs)are rapidly inactivated by solar ultraviolet (UV) radiation,particularly in the UV-B range of 280-320 nm [28].However, their efficacy can be improved by the use offormulations that include stilbene-derived optical bright-eners, which increase susceptibility to NPV infection bydisrupting the peritrophic membrane [29] or inhibitingsloughing [30] or virus-induced apoptosis of insect midgutcells [31]. UV inactivation could be controlled by creatingsystems, which can filter UV radiation, as has beendemonstrated by using plastic greenhouse structures thatreduced the intensity of incident UV-B (280-315 nm)readings by >90% compared with external readings leadingto an increase in the prevalence of infection in larvae [32].

Fungal Biopesticides

The pathogenic fungi are another group of microbial pestmanagement organisms [2] that grow in both aquatic aswell as terrestrial habitats and when specifically associated

Opender Koul 5

with insects are known as entomopathogenic fungi. Theseare obligate or facultative, commensals or symbionts ofinsects. The pathogenic action depends on contact andthey infect and kill sucking insect pests such as aphids,thrips, mealy bugs, whiteflies, scale insects, mosquitoesand all types of mites [33, 34]. Entomopathogenic fungiare promising microbial biopesticides that have a multi-plicity of mechanisms for pathogenesis. They belong to12 classes within six phyla and belong to four majorgroups; Laboulbeniales, Pyrenomycetes, Hyphomycetesand Zygomycetes. Some of the most widely usedspecies include Beauveria bassiana, Metarhizium anisopilae,Nomuraea rileyi, Paecilomyces farinosus and Verticillium lecanii.

Many of them have been commercialized globally (Table 1).These fungi attack the host via the integument or gutepithelium (Figure 2) and establish their conidia in thejoints and the integument [35]. Some species such asB. bassiana and M. anisipoliae cause muscardine insect dis-ease and after killing the host, cadavers become mum-mified or covered by mycelial growth [36]. Some fungi,primarily streptomycetes, also produce toxins that actagainst insects [37]. About 50 such compounds have beenreported as active against various insect species belongingto Lepidoptera, Homoptera, Coleoptera, Orthoptera andmites [38]. The most active toxins are actinomycin A,cycloheximide and novobiocin. Spinosyns are commer-cially available biopesticidal compounds that were origin-ally isolated from the actinomycete Saccharopolysporaspinosa [39] and are active against dipterans, hymeno-pterans, siphonaterans and thysanopterans but are lessactive against coleopterans, aphids and nematodes [39].

Nematode Biopesticides

Another group of microorganisms that can controlpests is the entomopathogenic nematodes, which controlweevils, gnats, white grubs and various species of theSesiidae family [40-43]. These fascinating organismssuppress insects in cryptic habitats (such as soil-bornepests and stem borers). Commonly used nematodes inpest management belong to the genera Steinernemaand Heterorhabditis, which attack the hosts as infectivejuveniles (IJs) [44, 45]. IJs are free-living organisms, whichenter the hosts through mouth, anus, spiracles or cuticle(Figure 2). They are able to release their bacterial sym-bionts in to the haemocoel of hosts, killing the host within24-48 h [46]. The nematodes can complete up to threegenerations within the host, after which the IJs leave thecadaver to find the new hosts [44]. Entomopathogenicnematodes (EPN) can be mass-produced in vivo and in vitroin solid media or liquid fermentation [47-49]. Nematodesthat have been successfully produced in fermenters(7500-80 000-litre capacity) include Steinernema carpo-capsae, S. riobrave, Steinernema glaseri, Steinernema scap-terisci, Heterorhabditis bacteriophora and Heterorhabditismegidis, with a yield capacity up to 250 000 IJs /ml [49, 50].



Figure 2 (Cont.)

Spore discharge

2.5 days/RP Nil

Spore formation

0.6 day\

*lb

41111.Inoculation

1.2 days

Time to kill 3.3 days

NAL

0.9 days

Insect covered with mycelium Mycelium formation

Saprophytic phase Pathogenic phase

Beauveria bassiana targeting coffee berry borer

Death

1. Infective juveniles (Ijs) search for insect host

2. Us penetrate the host cavity via mouth, anus, spiracles or intersegmental membranes

3. IJs release symbiotic bacteria (e.g.., Xenorhabdus, Photorahabdus) from their gut in host blood

4. Multiplying bacteria cause septicemia and kill the host

5. Nematodes feed on multiplying bacteria and mature into adults

6. Nematodes reproduce and emerge as infective juveniles (IJs) from the host cadaver

h ttp://www.cabi.org/cabreviews

61.

2 4

1. Larva eats Bt spore having toxic crystal protein2. High pH in midgut dissolves crystal protein and interact

with epithelial cell receptors3. Crystal proteins create pores4. Host organism dies from the invasion

Opender Koul 7

O Insect feeding on virus - contaminated fn I lege

CIO$O up Of CiCrliSiOn bO(firpS 01E10

O Lumen of digestive 11,nt taikalineommions)

Virus particles being released from 08s andattaching to brush border at gut cells

O liepliCalkin Of virus in insect cell

NPV life cycle

Virus

Occlusion body

Nucleus

Cytoplasm

1.1 e occel

Gut I

Plant

Figure 2 Cartoon representation of effects of a bacterial, viral, fungal and nematode type of microorganisms. (NPV lifecycle source http://en.wikipedia.org/wiki/File:Npv-life_cycle.jpg)

The use of nematodes is done using a curative ratherthan prophylactic approach [42], for instance, as demon-strated in the case of Synanthedon exitiosa, using S. carpo-capsae and H. bacteriophora nematode species to inducefield suppression of the pest in a curative manner [51];1 50 000- 300 000 Us/tree were used three times duringSeptember and October for three consecutive yearsin order to obtain as much control as was achievedwith chemical pesticides. Some commercial products areavailable based on Steinernema and Heterorhabditis nema-tode formulations (Table 1). However, extensive studiesare required to optimize application parameters anddevelop efficient strains to achieve significant control ofpests through nematodes.

Protozoan Biopesticides

Although they infect a wide range of pests naturally andinduce chronic and debilitating effects that reduce thetarget pest populations, the use of protozoan pathogens asbiopesticide agents has not been very successful. Protozoaare taxonomically subdivided into several phyla, some ofwhich contain entomogenous species. Microsporan pro-tozoans have been investigated extensively as possible

components of integrated pest management programmes.Microsporidia are ubiquitous, obligatory intracellularparasites that are disease agents for several insect species.Two genera, Nosema and Vairimorpha, have some potentialas they attack lepidopteran and orthopteran insects andseem to kill hoppers more than any other insect [52].A study of Nosema pyrausta, a microsporidium infectingthe European corn borer, Ostrinia nubilalis, suggests that ina horizontal transmission, a spore is eaten by a Europeancorn borer larva, which germinates in the midgut,extrudes a polar filament and injects sporaplasm into amidgut cell. The sporaplasm reproduces and then formsmore spores, which can infect other tissues. Spores ininfected midgut cells are sloughed into the gut lumen andare eliminated along with faeces to the maize plant. Thesespores remain viable and are consumed during larvalfeeding so that the infection cycle is repeated in midgutcells of the new host. If a female larva is infected, Nosemais passed to the filial generation by vertical transmission.As the infected larva develops through to an adult theovarial tissue and developing oocytes become infectedwith N. pyrausta. The embryo is infected within the yolkand when larvae hatch, they are infected with N. pyrausta.Both horizontal and vertical transmissions maintainN. pyrausta in natural populations of European corn borer.


Table 1 Commercial microbial biopesticides developed so far (*products discontinued; **products having EPA experimental use permit)

Micro-organism Target pest Action Brand name Producer

BacteriaA. radiobacter Crown galls Antagonist Galltrol-A AgBioChem

Dygall Agbioresearch LtdNorbac 84-C* New BioProductsNogall Becker Underwood

B. popilliae Larvae of various beetles Stomach poison Doom Fairfax BiologicalJapademic

B. pumilus Effective against rust, downy and Fungicide Ballad Agraquest Inc.powdery mildews Sonata AS

Yield Shield Gustafson LLCB. sphaericus Mosquitoes Stomach poison VectoLex Valent BiosciencesB. subtilis Effective against root rot caused by Fungicide and antagonist Serenade Agra Quest, Inc.

Rhizoctonia, Fusarium, Alternaria, Epic Gustafson, Inc.Aspergillus and Pythium. Also Kodiakeffective against some foliar MBI 600diseases Companion** Growth Products

Cillus Green Biotech, KoreaGreen-all GHi Stick N/T** Becker UnderwoodSubtilex

B. subtilis FZB24 Effective against Rhizoctonia, Fungicide Rhizo-Plus FZB Biotechnik,Fusarium, Alternaria, Verticillium Rhizo-Plus Konz GmbHand Streptomyces on vegetablesand ornamental plants

B. thuringiensis var. aizawai Effective against lepidopterans in Stomach poison Florbac Valent BioSciencesvegetables and maize XenTari

Agree CertisDesign*TurexMattch* EcogenSolbit Green Biotech, Korea

B. thuringiensis var. galleriae Effective against American bollworm Stomach poison Spicturin ISCB, Indiaon cotton, tobacco caterpillar onchillies, leaf folder of rice anddiamondback moth on vegetables

B. thuringiensis var. israelensis Effective against mosquitoes and Stomach poison Bactimos Valent Biosciencesblack fly larvae and midges Gnatrol*

SkeetalVectoBacAcrobe American CyanamideAquabac Becker MicrobialBMPBacticide Biotech IntlBactis CaffaroBioprotec AEF Global Inc

03

BTI granules Clarke Mos. Cont.Prehatch SG MeridianVectocide Nu-Gro GroupSummit Bactimos Summit chemicalsTeknar Valent Biosciences

B. thuringiensis var. kurstaki Effective against most lepidopteran Stomach poison Bactospeine* Solvay Dupharlarvae and some leaf beetles BioBit Valent Biosciences

DipelFlorbacCordalene AgrichemBonide Bonide products IncLipel SP Som PhytopharmaBMP 123 Becker MicrobialBiobest-BT Biobest, BelgiumScutelloBaturad Cequisa AgroWorm Wipper* Cape Fear ChemCollapse* CalliopeForay Valent BiosciencesBiolepCondor CertisCostarCutlass*FoilLepinoxCrymax Ecogen/CertisM-Peril*MVP II Dow Agro Sci.Raven*Ecotech BioEcotech Pro Ecogen/AgrEvoJackpotRapax Ecogen /IntrachemFonNabitBio-Worm Killer Forward IntlGuardjet Green Light CoMaatch Mycogen/KubotaBatik MycogenBactosid K NPP, FranceBactec BT 16 Nu-Gro GroupBactec BT 32 Plato IndustriesTuribelInsectobiol Probelte S.A.Soilsery BT SamabiolAgrobac Sil Sent IncAble* Tecomag SRLDeliver CertisDelfin

Table 1 (Cont.)


B. thuringiensis var. tenebrionis

Erwinia amylovora (HrpN harpinprotein)

P. cepacia

P. chlororaphis

P. flourescens

P. solanacearumP. syringae

Pseudomonas + Azospirillum

Altemaria destruensAmpelomyces quisqualis

B. bassiana

Burkholderia cepacia

Effective against coleopteranbeetles on vegetables

Stomach poison

Multi-spectrum Insecticide, fungicide andnematicide

Effective against soil pathogenic Toxicfungi

Effective against fungal pathogens Seed treatment controlof barley and oats

Effective against P. tolasi on Antibacterialmushrooms and Erwinia on fruitcrops

Bacterial control in vegetables AntibacterialEffective against post-harvest Antagonist

pathogens on apples, pears andcitrus

Effective against brown patch and Antagonistdollar spot soil pathogens

Cuscuta controlPowdery mildew Control and

damping off disease controlEffective against variety of insects

such as crickets, white grubs, fireants, flea beetles, whiteflies, plantbugs, grasshoppers, thrips,aphids, mites, mosquito larvaeand many others

Effective against soil fungalpathogens

FungiHerbicideHyperparasitic

Javelin WGThuricide*Vault*Larvo-BT*Troy-BT*Ringer BTSafer BTKHaltDiteraNovodorTridentM-TrakMessenger

Intercept

Cedomon

ConquerBlight Ban A506

PSSOLBio-Save

BioJet

Smolder GQ-FectGreen-all

Insect-specific fungal disease Racer BBinducers Ostrinil

B roca ri l

MycotrolMycotrol-0BotanigardBoverinNaturalis-LNaturalis-H&GNaturalis-T&O

Insecticide TrichobassControls fungi via seed treatment Deny

Certis/ValentValent BiosciencesTroy BioscienceVerdant IncWoodstreamCanadaWockhardt LtdValent Biosciences

Mycogen

Eden Biosciences

Soil Tech

BioAgri AB

Mauri FoodsNu Farm Inc

NPP, KoreaJet Harvest Solutions

Eco-Soil

Sylvan BioproductsGreen Biotech, Korea

Agri LifeNPP, FranceFutureco BioSciEmerald Bioagric.

NextBioTroy Bioscience

AMC Chem, SpainStine Microbial Products

Candida oleophila Effective against postharvestpathogens like Botrytis and

Colonization of diseased tissues Aspire Ecogen

PenicilliumConiothyrium minitans Effective against Sclerotinia species

on canola, sunflower, peanut,soyabean and vegetables

Mode-of-action not clear Contans WG

KONI

Prophyta Biologischer GmbH

BIOVED LtdFusarium oxysporum Effective against pathogenic Seed treatment and soil Biofox C

(non-pathogenic) Fusarium on basil, carnation,cyclamen, tomato

incorporation Fusaclean SIAPANPP, France

Gliocladium catenulatumGliocladium spp.

Effective against Pythium,Rhizoctona, Botrytis and

Mode-of-action not clear PrimastopGlioMix

Verdera Oy, Finland

Didymella species on greenhousecrops

Prestop Fargo, UK

Gliocladium virens Effective against soil pathogenscausing damping off and root rot

Antagonist Soil Guardl2G Certis LLC

Hirsutella thompsonii Effective against mites Stimulates premature fungalepizootics

Mycar* Formerly Abbot

Lagenidium giganteum Effective against mosquito larvaeand related dipterans

Kill through zoospores Laginex* Agra Quest, Inc

M. anisopilae Effective against range of pests. Disease-causing fungus Bio 1020* Bayer AGGreen Muscle is specific for Bio-Blast EcoSciencelocusts and grasshoppers Bio-Path EcoScience

Biotrol FMA* Nutrilite ProductsGreen Muscle Bio Control Prod.Metaquino CODECAPPacer MA Agri Life

Ticks Insecticide Tick-Ex Earth Biosci. Inc.Black vine weevil Insecticide Met52 Fargo, UK

Myrothecium vaerrcaria Effective against many nematodes Nematicidal Ditera Valent BiosciencesPaecilomyces fumosoroseus Effective against whiteflies in Hyperparasitic Preferal WG Biobest, Belgium

greenhouse PFR-97** CertisPaecilomyces lilacinus Effective against nematodes Antagonist Paecil/Bioact Tech. Innovation Corp/Prophyta

BiologischerPhelbia gigantea Effective against pine and spruce

rustBiofungicide Rotstop Kemira

Phytophthora palmivora Vine strangler Infects via roots De Vine AbbottPythium oligandrum Management of fungal pathogens in

various cropsSeed treatment or soil

incorporationPolyversum Biopreparaty Ltd., Czech

Streptomyces griseoviridis Effective against wilt, seed rot and Antagonist Mycostop Verdera Oy andstem rot Mycostop Mix Rincon Vitova

Streptomyces lydicus Effective against soil borne diseasesof turf, nursery crops

Fungicide Actinovate SP Natural Industries Inc

Talaromyces flavus V117b Effective against fungal pathogensof tomato, cucumber, strawberryand rape oilseeds

Antagonist Protus WG Peophyta BiolgischerPflanzenschutz

Trichoderma harzianum Effective against variety of soil Mycoparasitic, Antagonistic Root Shield BioWorks Incpathogens and wound pathogens BioTrek 22g Wilbur-Ellis

Supresivit Borregaard, Denmark

Table 1 (Cont.)


T. harzianum + T. viride

Trichoderma sp.

Trichoderma viride

V. lecanii

Granulosis virus

NPV for Anagrapha falciferaNPV for A. gemmatalis

NPV for Autographa calofornica

Effective against Armillaria andBotryoshaeria and others

Supresses root pathogens

Effective against rot diseases

Effective against other microbes,aphids, whiteflies and thrips

Effective against leafroller andcodling moth and otherlepidopterans

Indian meal mothEffective against lepidopteransEffective against velvetbean

caterpillar and sugarcane borerEffective against Alfalfa looper

NPV for H. zea and H. virescens Effective against bollworms

NPV for L. dispar Effective against Gypsy moth

Mycoparasitic

Mycoparasitic

Mycoparasitic

Antibiotic and insect eatingfungus

InsecticideVirusesDisease causing virus

InsecticideDisease-causing virusDisease-causing virus

Disease-causing virus



T-22GT-22HBTrianum PBinabTrichodexTrichopelTrichojetTrichodowelsTrichosealTrichodryTrichoflowTrichogrowTrichopelVinevaxEcosom TVTricon

TriecoMealikil VLVerticillinMycotalThriptalVertalec

CapexCarposinCyd-XCLV LCGranupomMadex 3Virin-EKSVirin-GYAPVirosoft CP4CarpovirusineNutguard-VAfMNVPMultigenPolygenVFN80*GusanoBiotrolGemstar LCElcarDispavirus*

Bioworks Inc, EU

Bio-lnnovationMakhteshim, IsraelAgrimm Tech

AgrimmTechnologies Ltd.,New Zealand

Agri LifeGreen MaxAgroTechEcosense labsAgri LifeKoppert, Netherland

Tate and LyleKoppert, Netherlands

AndermattAgrichemCertis

BioBest, BelgiumAndermattNPO Vector

Biotepp IncNPP, FranceAgriVir LLCCertisEMBRAPAAgroggenCertis

Certis

NovartisCCIP Corp.

It)

NPV for Mamestra brassicae

NPV for Neodiprion sertifer,N. lecontei and N. abietis

NPV for Orgyia pseudotsugata

NPV for Spodoptera exigua

NPV for Syngrapha falcif

H. bacteriophora

S. glaseri

H. megidis

P. hermaphroditaS. carpocapsae

S. feltiae

Effective against lepidopterans

Effective against sawfly larvae

Effective againstDouglas-fir tussock mothEffective against beet and lesser

army worms, pig weed caterpillarand mottled willow moth

Effective against Helicoverpa andCydia spp.

Effective against many lepidopteranlarvae, turf and Japanese beetlesand soil insects

Effective against root weevils,cutworms, fleas, borers andfungal gnats

Effective against black vine weevilsand soil insects

Effective against slugsEffective against black vine weevils,

strawberry root weevils,cutworms, cranberry girdler andtermites



Insecticide

Disease causing virus

Larval disease causing virus

NematodesEntomopathogen

Entomopathogen

Entomopathogen

Slug eating nematodeEntomopathogen

Effective against vine weevils, Entomopathogenfungus gnats, sciarid flies and soilinsects

GypcheckMamestrinVirin-EKSAbietivLeconteivirusMonisarmiovirusViroxVirtuss WP

Ness-ANess-EOtienem-SSpod-X LCGemstarNPVSf

Cruiser*HeteromaskNema-BITNema-top/-greenLawn PatrolGrubstakeLarvanemTerranemBioSafe WG

LarvanemDickmaulrussler-

nematodenNemaslugBio-Safe-NBiovector 25Savior WGEcomaskExhibit SC-WDGHortscanGuardianScanmaskTermaskMilleniumNematac CNo FleaEntonemExhibit SF-WDGNemasysNema-plus

US Forest ServiceNPP/CalliopeNPOVectorSylvar TechnologiesCanadian Forestry Ser KemiraOxford Virology

Terra Nostra, Canada

Applied Chemical

EcogenCertis

Certis

EcogenBioLogicBITe-nemaHydro-GardensIntegrated Biocontrol SystemsKoppert

SDS Biotech K.K.

Koppert Bio Syst.Andermatt Biocontrol AG

Becker Underwood IncCertis

BioLogicNovartis BCMBioLogicIPM labs/PraxisARBICOBioLogicCertisBecker UnderwoodIntegrated Biocontrol SystemsKoppertNovartis BCMBecker Underwoode-nema GmbH


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N. pyrausta suppresses populations of European cornborer by reducing oviposition, percentage hatch andsurvival of infected neonate larvae [53].

The only protozoan registered for use as a biopesticideis the microsporidian, Nosema locustae, which infectsgrasshoppers (Table 1). This organism is most effectivewhen ingested by nymphal stages of grasshoppers andkills them within three to 6 weeks post-infection [53].However, not all infected grasshoppers are killed by thisprotozoan infection.

Microbial Products in Biopesticides

In addition to the proteinaceous toxins, microorganismsare also known to produce anti-pest chemical com-pounds. Fermentation broths provide a readily screenablesource of bioactivity against organisms or targets ofagricultural interest. Antinsectan compounds derivedfrom nonfilamentous bacteria (e.g., aminolevulinic acid,thiolutin, thuringiensin, xenorhabdins); actinomycetes andsome fungi (e.g., actinomycin A, aplasmomycin, avermec-tins, citromycin, milbemycins, nikkomycin, piericidins,spinosyns, various cyclic peptides, etc.) are well known astoxins, growth inhibitors, antifeedants and physiologicaldisrupters against a variety of pests [37, 54, 55]. Some ofthese compounds have been commercialized, such asavermectins and spinosyns.

Some transgenic crops can be considered amongmicrobially based products. Since 1996, more than200 million ha of land has been planted with Bacillus-thuringiensis-based (Bt) genetically engineered crops [56].While 29 countries planted commercialized biotech cropsin 2010, an additional 32 countries, have granted regu-latory approval for biotech crops for import of foodand feed use and release into the environment since1996. Varieties of these crops produce 18 differentcombinations of 11 B. thuringiensis toxins, which kill lepi-dopteran and coleopteran insects. Bt cotton and maizehave been successful and other crops such as transgenicrice, soyabean and rapeseed are making some headway.Commercial growing was reported in 2009 of smalleramounts of genetically modified (GM) sugar beet, papaya,squash (zucchini), sweet pepper, tomato, petunia, carna-tions, rose and poplar [57]. Recently, some research anddevelopment has been targeted to enhance crops that arelocally important in developing countries, such as insect-resistant cowpea for Africa [58] and insect-resistantbrinjal (eggplant) for India [59]. The European Commis-sion has recently approved Annflora: a GM potato devel-oped by German chemical company BASF, which is thefirst GM crop to be approved for cultivation in the EU for12 years, after Monsanto's MON 810 maize, which isengineered to be resistant to the European corn borercaterpillar, was licensed in 1998 [60].

http://www.cabi.orgicabreviews

Genetic Improvement

Bacteria

The genetic improvement of microbial pathogens aims tomake them more effective by increasing their rate ofreproduction, speed of transmission and infective abilityor increasing the quantity of toxin produced. For example,genetic transformation of B. thuringiensis has produceda strain that displays insecticidal activity against bothcoleopteran and lepidopteran insects [61]. The activity ofB. thuringiensis on the crop foliage or applications viasoil can also be enhanced by genetic manipulation. Forinstance, B. thuringiensis crystal proteins of the Cry34 andCry35 classes function as binary toxins showing activity onthe western corn rootworm, Diabrotica virgifera virgifera.Cry34A/Cry35A pairs are more active than the Cry34B/Cry35B pairs. The binary Cry34/Cry35 B. thuringiensis

crystal proteins are closely related to each other, areenvironmentally ubiquitous and share sequence simila-rities consistent with activity through membrane disrup-tion in target organisms. Modified Cry35 proteins whosesegments, domains and motifs have been exchanged withother proteins to enhance insecticidal activity can provideexcellent control of plant pests and rootworms [62].Similarly, Cry8Bb1 toxin polypeptide from B. thuringiensishas been engineered to contain a proteolytic protectionsite, which makes it insensitive to a plant protease, helpingto protect the toxin from any proteolytic inactivation.Modified Cry8Bb1 has been used for controlling cornrootworms, wireworms, boll weevils, Colorado potatobeetles and the alfalfa weevils [63].

A recent study shows that B. cereus group genomeshave a Bacillus enhancin-like (bel) gene, which has potentialto increase the insecticidal activity of B. thuringiensis-basedbiopesticides and transgenic crops based on B. thuringiensisgenes [64]. Bel genes encode peptides, which have20-30% similarity with viral enhancin protein. Theseproteins are known to enhance viral infections as theydegrade the peritrophic matrix of insect midguts. Thecombination of Bel and Cry1Ac increased the mortalityrate 2.2-fold [64].

Viruses

Use of recombinant baculovirus technology has a poten-tial to produce economical substitutes. Recombinantbaculoviruses (vEV-Tox34) expressing the gene Tox-34from a mite, Pyemotes tritici, increased the speed of killof the corn earworm, Helicoverpa zea [65]. Similarly,two genetically enhanced isolates of the Autographa cali-fomica nuclear polyhedrosis virus (AcMNPV) expressinginsect-specific neurotoxin genes from the spiders Diguetiacanities and Tegenaria agrestis (designated vAcTaITX-1 andvAcDTX9.2, respectively) have been evaluated for theircommercial potential against lepidopteran insects. While

Opender Koul 15

vAcTaITX-1 kills faster than vAcDTX9.2, the latter is afaster feeding deterrent, suggesting that it would be moreuseful in reducing crop damage [66]. However, developingcost-effective methods for producing recombinant BVs isvery challenging because DNA preparations from theseviruses and their transfection are very labour intensiveand time consuming.

Lymantria dispar multicapsid nucleopolyhedrovirus(LdMNPV) is used on a limited basis as a gypsy moth(L. dispar) control agent. In an effort to improve the effi-cacy (i.e., killing speed) of the LdMNPV, a recombinantviral strain (vEGT-) that does not produce the enzymeecdysteroid UDP-glucosyltransferase (EGT) was devel-oped. LT50 values of fifth-instar larvae infected with vEGT-were 33% lower when compared with larvae infectedwith a wild virus strain. In addition, Buthus eupeusinsect toxin-1, the Manduca sexta diuretic hormone, theB. thuringiensis ssp. kurstaki HD-73 delta-endotoxin, theHeliothis virescens juvenile hormone esterase, the P. triticiTxP-I toxin, Androctonus australis neurotoxin, Doi m V geneand T-urf 13 genes have been inserted into BVs for thepurpose of developing viral pesticides [67].

Entomopathogenic Nematodes

In the case of entomopathogenic nematodes, artificialselection has been successful in increasing infectivity andnematicide resistance [68]. The strain selection has showna gain of fitness with regard to host penetration andreproductive potential. The recent discovery that maizeroots damaged by the western corn rootworm emit a keyattractant for insect-killing nematodes has opened theway to explore whether a selection strategy can improvethe control of root pests [69]. Salame et al. [70] breda heterogeneous population of Steinernema feltiae fordesiccation tolerance and host-seeking ability after 10 to25 selection cycles. However, artificial selection for onetrait may come at a cost for other important traits such asinfectiousness, establishment and/or persistence in thefield. Using information from the sequenced genomes ofEPN may enable the production of GM nematodes withhigher storage stability, higher resistance to environ-mental stresses and higher biological control potential tobe developed in the near future [71-73].

Entomopathogenic Fungi

Two commonly used entomopathogenic fungi, Metarhi-zium anisopliae and B. bassiana have been extensivelystudied for elucidation of pathogenic processes andmanipulation of the genes of the pathogens to improvebiocontrol performance [74]. Additional copies of thegene encoding the regulated cuticle-degrading proteasePr1 were inserted into the genome of M. anisopliae andoverexpressed. The resultant strain reduced survival time



Box 3 Bacterial Biopesticides: A Case Study

Use of B. thuringiensis bacteria for pest management has beengoing on for decades. Many studies have come to the fore interms of the development of effective strains, formulations,and subsequently the isolation of Cry proteins was achievedwhich ultimately lead to the development of transgenics.However, the case study presented here is different andrelated to the discovery and commercialization of a microbialbiopesticide for a grass grub, C. zealandica based on thebacterium S. entomophila [139]. These bacteria were firstobtained from sick grubs, cultured and discovered to causeamber disease in insects. Bacterial septicaemia is accompaniedby a rapid breakdown of the cadaver and release of bacteriaback into soil. The first simple identifications established thebacteria as non-spore-forming members of the genus Serratia(Enterobacteriaceae). Subsequently it was established thatS. entomophila and S. proteamaculans bacteria, both of whichcould occur in pathogenic and nonpathogenic forms, popu-lated New Zealand pasture soils. Unlike other soil bacteria,Serratia can grow on media rich in thalium salts, and caprylatethallous agar provides an excellent selective mediumfor isolation [140]. To develop these bacteria as biopesticides

is interesting, because they can be cultured through in vitrofermentation and can be applied back to soil causing disease[141]. In the 1980s, Monsanto initiated a programme forbiopesticide development with New Zealand group led byMurray Willocks to develop a S. entomophila as the productInvadeTM, which became Monsanto's first and probablyonly biopesticide launched on to the market [142] whereonly 1 litre of fermenter product was required to treat ahectare of pasture. Invade Tm was used for several hectaresannually, but a new approach was required to gain greatermarket acceptance and key was to develop stable formula-tions. It was in 2001 that Von Johnson developed a newflowable granular product with long shelf life in ambientconditions [143]. Encoate Ltd. and Balance Agri-nutrientsLtd. in New Zealand have now developed the newformulations in collaboration with AgResearch, Lincon. Theproduct BioshieldTM is produced on large scale in the form ofgranules (10 tonnes/h). It is now known that Serratia spp.are capable of holding a range of insect toxins, which mayprovide important opportunities for the control of otherpests [139].

in tobacco hornworm (M. sexta) by 25% as compared withthe parent wild-type strain [75]. The remarkable extent towhich virulence can be increased is shown in the case ofthe scorpion toxin (AaIT) expressed in the M. anisopliaestrain ARSEF 549. The modified fungus gave the samemortality rates in M. sexta at 22-fold lower spore dosesthan the wild type, and survival times at some doses werereduced by 40% [76]. Similar results have been withmosquitoes with a 9-fold reduction in LC50 and coffeeberry borer beetle with a 16-fold reduction in LC50 [77].

Production and Development

Some microbial biopesticides are easy to produce anddevelop and can be manufactured using simple and in-expensive technologies. The BVs and EPN can be pro-duced in vivo in insects and entomopathogenic fungi, suchas Beauveria and Metarhizium, are produced on grains.Such simple technologies are useful for developingcountries where a substantial demand exists for localproduction and distribution at the farmers' level. How-ever, production methods are only one aspect ofthe development of a new microbial biopesticide [78],and one has to solve potential problems associatedwith contamination, formulation potency, attenuation ofpesticidal activity and shelf life. All these aspects requireequipment, expertise, material and capital. As such, smallentrepreneurs, particularly in the developing world areoften not able to meet these requirements and to someextent, a similar situation persists in small productionfacilities in the developed world [79-82]. It is estimatedthat to develop a single product costs >US$25 million

from discovery to formulation development in order toreach a farmer for application.

In the microbial biopesticides field, the major emphasishas been on Bacillus thuringiensis. Frankenhuyzen [83] hasrecently documented that to date 125 of the 174 holotypeknown toxins have been tested against 163 test species.However, the products are classified according to theirformulation. These formulations are emulsions, encapsu-lations and granules (for agriculture and forestry); wet-table powders (for gardens); and briquettes (for aquaticsystems). There are now many formulations based onspore-crystal complexes, which need to be ingested bythe pests for toxic action. The spore-crystal complexesare required to be carried by suitable excipients thatwould protect the spore crystal and at the same should bepalatable to the insect, i.e. with increased feeding pre-ference. A good example of such a formulation is of Cryprotein from B. thuringiensis with an attractant glycopro-tein [84] for killing fire ants. Some designs are also knownfor beet armyworm, Spodoptera exigua [85]. However, aclassical case study is the discovery and commercializationof a bacterial product for a grass grub, Costelytra zealandicabased on the bacterium Serratia entomophila (see Box 3).Many biodegradable materials have been used to prepareformulations, including liquid or solid carriers, surfactants,adjuvants, adherents, dispersants, stabilizers, moisturizers,attractants and protective agents [86]. Long shelf lifeand reliable efficacy, which are affected by moisture, arethe two basic impediments for commercialization of abacterial biopesticide and strategies to develop deliverymaterials with dynamic vapour sorption properties havebeen worked out in a recent study where three bio-pesticide delivery systems, THE -G, PEC-G and PESTA,


Opender Koul 17

Box 4 Fungal Biopesticides: A Case Study

Locusts and grasshoppers are extremely damaging pests invarious parts of the world. The largest locust swarm wasreported in Kenya in 1954, covered more than 1000 km2,contained 40 000 million insects, and weighed 80000 tonnes.One tonne of locusts eats as much food in one day as about2500 people [144]. Similarly, grasshoppers do more cropdamage on an average. The main control measure againstlocusts and grasshoppers has been broad-spectrum pesti-cides. For instance, between 1986 and 1989, donorsand national governments spent US$200 million spraying10 million ha with 15 million litres of the broad spectruminsecticides fenitrothion and malathion [145]; the pesticideshaving hazardous environmental impacts. In view of suchhazards, the US Agency for International Development(USAID) began supporting a programme to develop a bio-pesticide against locusts based on the entomopathogenicfungus M. anisopliae var. acridum. The LUBILOSA (Lutte Bio-logique contre les Locustes et Sauteriaux) project was set up in1989 to develop a biological means of controlling locusts andgrasshoppers. At the beginning, the project involved the CABIBioscience in the UK, IITA, and Departement de Formationen Protection de Vegetaux (DFPV) in Niger. CABI has man-aged the project but the technical project leader has been anIITA employee based in Benin since 1992, when LUBILOSAbegan small-scale field trials. By the end of Phase 3, thenumber of collaborators had increased to include Comite

Inter-Etat de Lutte contre la Secheresse au Sahel (CILSS),Deutsche Gesellschaft fur Technische Zusammenarbeit(GTZ) in Germany, and two private companies, BiologicalControl Products (BCP) in South Africa and Natural PlantProtection (NPP) in France.

In the first 10 years, the LUBILOSA project spent US$15million and produced an environmentally benign alternative tochemical pesticides. Demonstration trials and farmer partici-patory trials have been conducted in most Sahelian countriesin collaboration with the national programmes [146].A project in Australia has used LUBILOSA research data todevelop a biopesticide against Australian locusts [147]. Theproduct kills 80% of insects within 1 to 3 weeks. A company islicensed to manufacture the biopesticide, which has beenregistered in South Africa under the name Green Muscle®.This consortium of donors has recently funded a fourth phaseto "steward" the LUBILOSA biopesticide to higher adoptionrates and greater impact.

As such, LUBILOSA may well be a template for much moreof the activities of the Consultative Group on InternationalAgricultural Research (CGIAR) in the future. Hence, ananalysis of the impacts that LUBILOSA has had, couldhave in the future, and how this impact has and can beachieved, can teach us a great deal about public-privatepartnerships and the management of impact-focused research[148].

were analysed by dynamic vapour sorption analysis. Theobjective of this study was to demonstrate the moisturesorption profile of each system in air at 25°C and arelative humidity (RH) ranging from 0 to 90%. Thesestudies have revealed that moisture loss retards theactivity in the range of 2.3-3.4 times [87], thus suggestingthe implications relative to moisture distribution.

In the case of fungal biopesticides, a system for massproduction of conidia has been standardized after evalu-ating different solid matrices such as rice, wheat bran andmijo grains, contained in both, high-density polyethylenebags and aluminium trays, supplemented with differentorganic nitrogen sources and inoculated with differentinoculum types [88]. Once the matrices are established,where the greatest conidia production per gram of sub-strate is obtained, the conidia are separated and used asan active starter for elaborating the biopesticide proto-types. Recently, a complex coacervate formulation wasdeveloped for Colletotrichum truncatum, a bioherbicidalfungus against scentless chamomile, and tested in thegreenhouse. A two-step process was developed to for-mulate C. truncatum conidia. Firstly, an invert emulsionpreparation of C. truncatum conidia in non-refined vege-table oil with the aid of a surfactant was prepared, fol-lowed by encapsulating the C. truncatum conidia invertemulsion by complex coacervation. Formulation ingre-dients included non-refined vegetable oils, surfactants,proteins and carbohydrates. Most formulation ingredients

considered and tested in this study were compatible withC. truncatum, with no significant reduction in conidialgermination and mycelial growth. The surfactant soyalecithin promoted the greatest retention of C. truncatumconidia (88%) in the invert emulsion. In greenhousestudies, scentless chamomile disease was controlled sig-nificantly [89]. This example implies that fungal microbialbiopesticides could be very useful in field situations ifappropriate formulations and specific delivery systemsare developed. However, specific example of successfulfungi-based product is the LUBILOSA programme (seeBox 4).

The use of wild-type biopesticides in terms of regis-tration could be more appropriate as they will require ademonstration of efficacy and safety issues will be of lessconcern. There is evidence to show the potential of wild-type BVs [90, 91]. For instance, process patent is availablefor production of BVs via fermentation in Australia [92],which targets the Helicoverpa pest species and accountsfor a US$3.2 billion per annum market. Production costssuggested allows to target Helicoverpa pest species inareas where this pest is resistant to most low-costchemical options (15 $/ha), and where only more expen-sive chemicals are in use (30-50 $/ha). These studiessuggest that wild-type products with improved yields cancompete on cost alone in all markets, including extensivemarkets in India and China. Therefore, bioreactor-basedproduction of BVs requires more focus, which may be



Box 5 Viral biopesticides: A case study

Control of the velvetbean caterpillar, A. gemmatalis, in soya-bean in Brazil is an interesting case study where one can takean advantage of a naturally occurring nucleopolyhedrosis virus(AgMNVP) against a pest. This virus is currently used onapproximately two million hectares of soyabeans in Brazil andis one of the largest programmes worldwide for the use of aviral pathogen to control a pest of a single crop [149].Although implementation of the programme began in the1982/83 season, it gained momentum with the developmentof a wettable powder formulation in 1986, which was thentaken up by private companies for commercial production;use of AgMNVP reached 1.5 million ha in 1995. Field pro-duction of the virus became a big business in Brazil, involvingdifferent persons and small companies specialized in sellingAgMNPV-killed caterpillars [150]. A breakthrough in com-mercial pilot laboratory production occurred at EmbrapaSoybean in Londrina in 2002. In the production system of thevirus, eggs are obtained daily in adult oviposition rooms, andlarvae reared in separate rooms up to fourth instar in 500 ml

cardboard cups containing an insect diet. Daily 3% of thelarvae are transferred to plastic trays with diet and vermi-culture to obtain pupae and maintain the insect colony. Therest (97%) of the fourthh instars are taken to the virus pro-duction laboratory, where they are transferred to plastictrays containing AgMNVP- treated diet. Seven days later, deadlarvae are collected into plastic bags with a modified handvacuum cleaner. The larvae are then taken to a storage roomfor further processing and formulation of the product. Thiscommercial laboratory production started in the end of 2004and produces 800 000-1 000 000 larvae/day, resulting in

agMNVP to treat 1.8-2.0 million ha/year [149]. It is expectedthat AgMNVP use may reach to 4.0 million ha/year by 2012.This programme in Brazil has been successful because of itsimplementation of a soyabean IPM programme, proactiveactivities of extension units, high virulence of pathogen tothe host and efficient horizontal transmission, continuedexposure of pest to AgMNVP and ability to produce largequantities of virus under field conditions at a very low cost.

Box 6 Nematode biopesticides: A case study

Molluscs are pests for which few biological control agents areavailable. Therefore, this is a case of a successful commercialdevelopment of a novel species of nematode with specificactivity against slugs. A nematode, P. hermaphrodita has beenisolated from a destructive grey field slug species D. reticu-latum [151], which develops a very characteristic swelling inthe rear half of their mantle at an early stage of infection, andfollowing death, many large nematodes of 3 mm size can beseen feeding on the cadaver. This nematode has a curiousability to adopt necromenic life cycle in larger slugs, in whichthe Us enter the slug's body cavity and remain dormant therewithout doing harm until the slug dies, when the juvenilesdevelop and reproduce, feeding on the cadaver. When thenematode enters smaller slugs, it develops directly, causingdisease and death of the host. An interesting thing about

nematodes such as EPN is their mutalistic symbiosis withGram-negative entomopathogenic bacteria such as Xenor-habdus and Photorhabdus, but this is not true in case ofP. hermaphrodita. This nematode is capable of growth on awide range of bacteria, but nematodes grown on differentbacteria differ dramatically in virulence. The reason for this isunknown [152]. However, the use of a bacterium, Moraxellaosloensis that produced consistently pathogenic nematodesfor mass production in the variety of field experiments hasbeen highly significant and economically viable. The productbased on this nematode is now making a transition from beinga garden and protected crop treatment to being used in fieldvegetables [153]. The very famous product Nemaslug is soldin UK and each commercial packet contains 12 million dauerjuveniles of P. hermaphrodita.

cost-effective and high yielding. However, control of thevelvetbean caterpillar, Anticarsia gemmatalis in soyabean inBrazil is an interesting case-study based on laboratoryproduction where one can take an advantage of a naturallyoccurring nucleopolyhedrosis virus (AgMNVP) againstpest (see Box 5).

EPN can be commercially mass-produced in vivo orin vitro, in solid or in liquid culture, each system having itsown advantages and disadvantages relative to costs ofproduction, investments, quality of products and tech-nology. One of the good examples is a successful com-mercial development of a novel species of nematodewith specific activity against slugs where a nematode,Phasmarhabditis hermaphrodita has been isolated from adestructive grey field slug species Deroceras reticulatum(see Box 6). However, there are many challenges inmaking production more reliable and economical [93] and

process parameters require more research, particularlythe ones that influence the bacteria as well as the nema-todes in liquid cultures. Another significant factor is phasevariation of the symbiotic bacteria, shifting from primaryto secondary forms that lead to unpredictable yields [94].As of today, a huge number of Us is required for pestmanagement within a location; if these numbers could bedecreased, market opportunities for EPNs will definitelyincrease.

In terms of the formulation development, the ability toformulate viruses for application with commonly availableimplements makes viruses more attractive as biologicalcontrol agents. Commercially, dried formulations have anadvantage over liquid formulations for storage and hand-ling. However, dry formulations have constraints such asdustiness, inhalation risk and storage. Lyophilization hasbeen the most common method for stabilizing viruses but


is an expensive procedure [95]. Another method has beenthe encapsulation with cornstarch to prepare granularformulations. The lignin formulations have demonstratedextended residual activity in both laboratory and fieldexperiments [96, 97]. Spray drying [98] has been used forB. bassiana to microencapsulate conidia of this entomo-pathogenic fungus at low temperature [99, 100]. In addi-tion, various polymers are used to prolong the activityof the conidia and improve their shelf life. The best-encapsulated product, composed of 10% dextrin, 10%skimmed milk and 5% polyvinylpyrrolidone K90 as thecoating material, had a shelf life of 6 months at 4°C [101].However, ingredient selection, processing techniques andmoisture content still hinder the development and pro-duction processes of virus-based microbial biopesticides.Some delivery systems through drip irrigation systemshave also been studied recently. The suspendible entomo-pathogenic fungi and EPN evaluated through drip lines area viable alternative for application of water-soluble andinsoluble materials; however, the discharge rates needto be determined for uniformity of the delivery [102].Nematode formulations, however, need to be tailored andrequire the studies in physiological chemistry of nematodeand its ecology and behaviour. A recent overview ofnematode-based formulations [103] advocates the use ofmany types of formulations, including water-dispersablegranules. However, water-dispersable granular formula-tions were not successful on a larger scale [104]:dominant and successful ones were clay- and gel-basedformulations.

Resistance to Microbials

Among the various groups of microbial pathogens,development of resistance has been most frequentlyreported in the case of B. thuringiensis. Within the last fewyears, at least 16 insect species have been identifiedthat exhibit resistance to B. thuringiensis 8-endotoxinsunder laboratory conditions and field-evolved resistancehas been documented in noctuids such as Spodopterafrugiperda, Busseo la fusca and H. zea [105]. Reportsof development of resistance in field populations ofPlutella xylostella are essentially from the countries whereBacillus thuringiensis is extensively used, i.e. China, Japan,Phillippines, Malaysia, India and North America. To avoidthis resistance problem, genetic engineering was con-sidered as a useful tool where microbial genes fromB. thuringiensis were transferred to plants to producetransgenics and today we have B. thuringiensis cotton andB. thuringiensis maize available in 13 and 9 countries,respectively, grown on 42.1 million ha of land [106]. Thedevelopment of such transgenics was seen as a panacea interms of microbial control of pests; however, field resis-tance in H. zea as a result of an increase in the frequencyof resistance alleles is alarming [107]. The field-evolvedinsect resistance to B. thuringiensis crops and various

Opender Koul 19

aspects related to resistance monitoring methods havebeen comprehensively reviewed recently [105]; obviouslymore prominent in lepidopterans [108, 109]. Factorsassociated with field resistance are the failure to use high-dose B. thuringiensis cultivars and lack of a sufficient refuge.While implementation of the high-dose/refuge insectresistance management strategy has been successful indelaying field resistance to Bt crops [109], gene pyramid-ing is another approach used to try and address theemerging resistance problem [110, 111]. Pyramidingmeans the stacking of multiple genes so that more thanone toxin is expressed in the transgenic plant. However,gene-pyramiding needs to be sustainable and no cross-or multiple resistances should occur. The problem ofdeveloping multiple resistance cannot be summarilyignored as in the end they would render such strate-gies ineffective. Asymmetrical cross-resistance betweenB. thuringiensis toxins Cry1Ac and Cry2Ab in pink boll-worm [112] suggests that it is important to incorporatethe potential effects of such cross-resistance in resistancemanagement plans so as to help to sustain the efficacyof pyramided B. thuringiensis crops. Current evidencesuggests that gene pyramiding may not be a sustainablestrategy per se; therefore, other management strategiessuch as refugia, use of predators and parasitoids and croprotation strategies need to be incorporated in the man-agement plans [110, 112]. Transgenic plants that controlinsects via RNA interference are going to be a realitysoon [113, 114], which will broaden further the scope oftransgenics and can help in minimizing the drawbacks ofresistance. Some recent studies have shown that toxin-binding proteins such as cadherin promote B. thuringiensistoxicity [115]. These binding proteins facilitate toxinoligomerization and thus modify the B. thuringiensis toxin,which can prevent the resistance in comparison with thestandard B. thuringiensis toxins. The studies demonstratethat the toxicity of B. thuringiensis toxin Cry1Ab is reducedby cadherin gene silencing with RNA interference inM. sexta. The toxins that had cadherin deletion mutationskilled cadherin-silenced M. sexta and B.-thuringiensis-

resistant Pectinophora gossypiella [115].Recently, resistance in a baculovirus in the field has

been found in Europe where Cydia pomnella GV is oneof the main components of the codling moth control.C. pomonella GV in apple orchards has led to a high degreeof resistance in some populations [116, 117]. This is thefirst documented instance of field resistance to a com-mercially applied baculovirus [118]. Apparently, this is

either the result of the overuse of the product or thepredominant control strategy applied. However, there donot seem to be any reported examples of field develop-ment of resistance to entomopathogenic fungi or nema-todes [119]. However, there is evidence to demonstratethe existence of natural resistance mechanisms in insectsagainst fungi [120, 121] and nematodes [122], suggestingthat resistance to these pathogens cannot be summarilyignored.



Future Perspectives

Owing to some of the early successes and the continuinggrowth of biopesticide market, expectations for theperformance of microbial biopesticides have been high.However, there are many challenges that will need to beovercome. Firstly, questions have been raised about thebarriers that research patents place on humanitarian usesof patented technologies as well as on the conduct andavailability of publicly funded research results [1 23, 1 24].Secondly, current regulatory guidelines are inadequate,while information on the uptake of microbial controlstrategies must be collated and shared with the rest ofthe field. Furthermore, to implement local productionschemes in developing countries, intervention at thenational and international level will be important.Thirdly, there is also a need to look into the ecologicalrelevance vis-à-vis the use of microbial biopesticides.Some recent studies reveal that pattern and impactof these toxins varies from species to species, dependingon the ecosystem, the route of exposure and thenon-Bt control against which effects are quantified[125]. As such, the effect of microbial biopesticideson microbial communities must be carefully monitored[126].

In fact, there is a need for well-defined selection criteriaand a complete process description for the developmentof a microbial pest control product. For a commercialmicrobial product, three specific criteria for selection arerequired, i.e. toxicity, production efficiency and safety ofthe product. That means while screening process toxicityof the product will be relative to dose rate, mode-of-action, speed of kill, host range, sensitivity to abiotic fac-tors and persistence. Secondly, mass production will becritical criteria and should be a high-yield-oriented pro-cess. Thirdly, safety of product will be essential in relationto registration requirements and the costs involved.An important question, however, is when are microbialbiopesticides appropriate? Generally, scientists and bio-control companies seem to develop their productswithout a well-developed plan, though the approachshould be to develop a product to solve a problem or tograsp an opportunity. It is essential to make a detailedcharacterization like which pest, crop, region, time ofthe problem, solutions available, acceptable costs andmarket potential [1 27]. If these aspects are consideredand details are provided, a potential microbial productcould be obtained. A good example is the Lubilosa (seeBox 3), which was a problem-solving project. Therefore,recommended steps to obtain a good microbial pestcontrol product would be: (i) collection of isolates andidentification of perfect isolate, (ii) laboratory screeningfor efficacy, (iii) assessment of production efficiency,(iv) mode-of-action and toxicological properties, (v) glass-house trials and (vi) evaluation of efficacy under com-mercial conditions. If all these factors are considered,success is inevitable, perseverance to develop such

products will be rendered less risky, and questions suchas, 'how to walk a tightrope' [128] or 'The long andwinding road - discovery to commercial product: are wethere yet?' [1 29] will be answered.

In order to increase the utility of microbial pathogensin EBIPM programmes, systematic surveys are requiredin different agroecological zones to identify naturallyoccurring pathogens. Detailed studies are necessary onthe properties, mode-of-action and pathogenicity of suchorganisms. Ecological studies on the dynamics of diseasesin insect populations are necessary because the environ-mental factors play a significant role in disease outbreaksand ultimate control of the pests. It is expected that withthe recent advancements in microbial research coupledwith dedicated efforts from extension specialists, farmers,pest management regulators and the general public,microbial biopesticides could play a prominent role infuture EBIPM and AWPM programmes. As mentionedabove, structured project plans are required toachieve the goal. The roadmap to successful developmentand commercialization of a microbial pest controlproduct is amply illustrated in new flow diagrams recently,which provide the details of various phases' involvedand output information leading to consecutive stepsfor decision making and ultimately the market potential[127].

With respect to the ecology of microbial controlagents, this has been a major concern over the years thathas remained little researched. So far, biotechnologyand genetics has (understandably) driven the progress inmicrobial control but there has been negligible interest inhow these organisms have evolved to survive in nature.For example, the means by which B. thuringiensis survivesin nature have yet to be proven [1 30], and the costs andbenefits of cry gene possession are unclear. It is evident,however, from the plethora of cry genes that exist thatgene duplication and exchange are commonplace. In fact,its spore-forming nature makes it uncertain with regard tohow much vegetative existence B. thuringiensis has and sohow much mutation and recombination can take place. Itcertainly has a vegetative existence in at least some insectspecies, in which it causes pathology through conjugation[1 31]. An interesting aspect would be to see if thesame lack of association between sequence types andepisomal factors are evident in other environments whereB. thuringiensis can be demonstrated to have a vegetativeexistence [1 32]. A recent study [1 33] with seedlingsof clover (Triflorium hybridum) shows colonization byB. thuringiensis when spores and seeds were co-inoculatedinto soil. Both a strain isolated in the vegetative form fromthe phylloplane of clover and a laboratory strain were ableto colonize clover to about 3 times higher density whenseeds were sown in sterile soil rather than in non-sterilesoil. A strain lacking the characteristic insecticidal crystalproteins produced a similar level of colonization overa 5-week period as the wild-type strain, indicating thatcrystal production was not a mitigating factor during


colonization. A small plasmid, pBC16, was transferredbetween strains of B. thuringiensis when donor and reci-pient strains were sprayed in vegetative form onto leavesof clover and pak choi (Brassica campestris var. chinensis).The rate of transfer was about 0.1 transconjugants/recipient and was dependent on the plant species. Thelevels of B. thuringiensis that naturally colonized leavesof pak choi produced negligible levels of mortality in thirdinstar larvae of Pieris brassicae feeding on the plants.Considerable multiplication occurred in the excretedfrass but not in the guts of living insects. Spores in thefrass could be a source of recolonization from the soil andbe transferred to other plants. These findings illustratea possible cycle, not dependent on insect pathology, bywhich B. thuringiensis diversifies and maintains itself innature. The majority of research that has been carried outon B. thuringiensis has related to its insecticidal toxins andits survival in nature may not always depend on insectpathology. It can colonize seedlings from spores in thesoil, exchange genetic information on the phylloplane, andan appreciable multiplication can occur in the frass ofinsects that it did not kill [133]. A cycle of transmissionand survival can thus be envisaged. The enigma, however,is the cost and benefit of crystal protein production in itsecology, particularly when in competition with non-crystalprotein producing bacteria such as B. cereus. Therefore,longer-term studies in nature or in microcosms andtheir survival in soil and plants in the presence of sus-ceptible and non-susceptible invertebrates are required[133].

The mechanism of resistance, specifically for Cryproteins, is a matter of concern. Recently, a databaseconsisting of 12519 high-quality sequences have beendeveloped from the larval gut of European corn borer.This obviously can provide basis for future research todevelop gut-specific DNA microarrays to analyse thechanges of gene expression in response to B. thuringiensisprotoxins/toxins and the genetic difference(s) betweenBt resistance and susceptible strains [134]. In fact, 52candidate genes have been identified that may be involvedin Bt toxicity and resistance. For instance, out of selectedgenes, five genes with decreased expression and tenwith increased expression in Cry1Ab-resistant strain ofEuropean corn borer may help in identifying the genesinvolved in Bt resistance that could provide new leads intothe mechanism of Cry1Ab resistance in these insects[134].

Commercialization is the final and most difficult step inthe development of a microbial product. The most criticalfactors are developmental cost and time to market. Costsamount to US$14 - 21 million for a new entrepreneurand the time to market including registration is no lessthan 5-7 years. Therefore, to examine all these criticalfactors in the successful commercialization of microbialpest control products is essential in the developmentalprocess of a product and these critical factors have beencomprehensively discussed recently [127].

Opender Koul 21

Acknowledgements

This review article contains information gathered fromnumerous published resources, and thus I would like toextend my appreciation to all authors of the referencesused in this manuscript.

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