Production and use of biological pest control agents

TIBTECH - MAY 1986

Production and use of biological pest control agents George

G. K h a c h a t o u r i a n s

Biological pest control agents are gaining prominence for the control of insect pests in agriculture and forestry. The shift from chemical control has been due to environmental concerns and recent innovations in biotechnology. Production and use of biological insect control agents is the challenge of the future for pest management. Biological pest control agents are gaining in prominence for the control of insect pests in agriculture and forestry 1-3. The growth of interest in biological pest control agents occurred in part because of public awareness of the dangers of indiscriminate use of chemical pesticides, especially after the publication of Silent Spring by Rachel Carson, in 1962. Additionally, research published in the last two decades on the relationship between pesticides and mutation, cancer, human disease, damage to wildlife, and toxicity to plants has pointed towards a shift away from chemical control of pests. In the last decade developments in biotechnology are generating interest and the means necessary for displacement of synthetic chemical insecticides which currently dominate the marketplace 4'5. Various estimates indicate that biological pest control agents, which currently represent 1% of the annual $10 × 10 9 worldwide market for insecticides will increase to become 10% or more by the end of the decade. The potential for widespread use of biological pest control agents~ is timely because of their relative host specificity and ecologically non-disruptive nature. Biological pest control agents are naturally occurring organisms, including viruses, bacteria, fungi, protozoa and nematodes. Viruses G. G. KhachatouriansisattheDepartment of Applied Microbiology and Food Science, and Bioinsecticide Laboratory, College of Agriculture, University of Saskatchewan, Saskatoon, S7N OWO Canada, (~ 1986, Elsevier Science Publishers 13.V., Amsterdam

have an interesting ecological specificity and unique location within the biological world. For example, the insect viruses which are members of the family Baculoviridae (BV) are responsible for diseases of insects and mites 6 and do not contain structural or biochemical similarities to viruses of the vertebrates or higher plants. A substantial portion of the bacteria and fungi are facultative pathogens of insects affecting the host by a number of mechanisms such as invasion of tissues and/or production of toxins. The bacteria can be divided into three ecological types: (1) those which reproduce within the susceptible host system; (2) those which do not reproduce, necessitating repeat application; and (3) those of either type (1) or (2), depending on the circumstances 7. Because of these unique properties a number of these microorganisms have been commercially produced and are replacing existing chemical pesticides.

Production and use ofbioinsecticides Viruses There are about 650 viruses that have been isolated from insects. About 540 were obtained from lepidopteran, 90 from hymenopteran and 20 from orthopteran, coleopteran and dipteran insects. During the 1970s the first commercial viral pesticide was registered. Since then the potential of these agents has been realized (Table 1) and tremendous use of viruses such as baculoviruses, has been made for control of insect populations a. Insect virus infections of susceptible larvae result in pathological symptoms and, ultimately,

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death. About 100 insects are susceptible to baculoviruses. Certain baculoviruses produce granulosis disease. In general, the mass production of many of the insect viruses involves incubation of infected insects, and harvesting of diseased larvae or adult insects. The subsequent purification of the viral preparation is time consuming and labor-intensive, the expense of which is added to the cost of viral insecticide production increasing the production costs 4-5fold. The obstacles to commercial production and use are numerous 8 and include: (1) the development of efficient processes for large scale production; (2) marketability of the products especially where the selective host ranges are required; (3) development of procedures for scheduling and monitoring of field applications; and (4) stabilization of the product under field conditions, e.g. sunlight and dessiccation. Bacteria Many bacteria cause disease and death in insects. Four groups have been recognized based on their pathogenicity to insects: (1) obligate pathogens (e.g. Bacillus popilliae, B. larvae); (2) crystalliferous sporeforming bacilli (e.g.B. thuringiensis); (3) facultative pathogens (e.g. B. sphaericus, Pseudomonas aeruginosa, Achromobacter spp.); and (4) potential pathogens (e.g. Serratia marcescens) 9. The best example of bacterial insecticides and one that has been investigated for almost 80 years is that of the members of the genus Bacillus. Four species are considered as primary insecticides: B. thuringiensis, B. popilliae, B. moritai and B. sphaericus. The greatest commercially used species are B. thuringiensis which is cultured & vitro, and B. popilliae, which is grown in rive on the Japanese beetle (Popilliae japonica). B. thuringiensis produces entomocidal toxins, ~- and [5-exotoxins and 6-endotoxin. The latter, also called insecticidal crystal protein (ICP); is synthesized during sporulation, and accounts for the commercial value of B. thuringiensis as a biological pest control agent 4. Oral administration of ICP to lepidopteran larvae causes cellular damage to the

TIBTECH - MAY 1986

- Table

I

Viruses in commercial and experimental production a Virus

Target

Product

Manufacturer/Country

Heliothis NPV

Heliothiszea

Elcar

Zoecan (Sandoz) Corp./USA

Biotrol-VHZ

Nutrile Products Inc./USA

Gypchek Virin-ENSh Virin-Diprion NPV Biocontrol-1

US Forest Service USSR USSR Kemira Oy Co./Finland US Forest Service

CPV

Japan

(corn earworm)

Heliothis virescens (tobacco budworm)

Lymantria dispar N PV

Lymantria dispar (gypsy moth)

Neodiprion sertifer N PV

Neodiprion sertifer

Orgyia pseudotsugata N PV

Orgyia pseudotsugata

Dendrolimus spectabilis

Dendrolimus spectabilis

(European pine sawfly) (Douglas fir tussock moth) CPV aAdapted from Refs 2 and 6.

(pine caterpillar)

gut epithelium and cessation of turers for Bacillus products worldfeeding. With sub-lethal doses an wide are shown in Table 2. acute phase followed by recovery occurs 1°. Larger doses cause patho- Fungi logical changes resulting in larval There are several entomopathodeath 11'12. The commercial produc- genic and saprophytic fungi which tion, targeted hosts and manufac- have been used for the control of Table

insect populations 13'14. For the commercial production of these fungi, ingredients, concentrations, quantity and quality of the fermentation methods used are virtually identical to those required for the synthesis of either fungalbiomass or metabolites 15

2

Commercial Bacillus products a Species

C o m m e r c i a l name

Target

Manufacturer/Country

Bacillus moritai

Serotype/variety --

Lavillus M

Diptera

Bacillus popilliae

--

Doom, Japidemic

Coleopteran larvae

Sumitomo Chemical Co./ Japan Fairfax Biological Lab./ USA Reuter Laboratory, Inc./ USA Abbott Laboratories/USA Sandoz-Wander, Inc./ USA Zoecan (Sandoz) Corp./ USA Thompson-Hayward Chemical Co. (distributor)/USA Roger Bellon/France Procida France Biochem. Products/ France LIBEC/France Farbwerbe Hoechst/ West Germany ChemapoI-Biokrma/ Czechoslovakia Serum zavod Kalinovica/Yugoslavia Glavmikrobioprom/USSR Glavmikrobioprom/USSR Glavmikrobioprom/USSR Agricultural Microbiology/USSR Glavmikrobioprom/USSR Glavmikrobioprom/USSR Biochem Products/France Zoecan (Sandoz) Corp./ USA

Milky spore disease

Bacillus thuringiensis (B. thuringiensis

H3(HD-1)/Kurtsaki H3(HD-1)/Kurtsaki

5-endotoxin + spores)

exotoxin)

Bacillus thuringiensis

Numerous Lepidoptera

Certan --

Bacillus thuringiensis (B. thuringiensis

Dipel Thuricide

Bactur

H-1 thuringiensis H3(HD-1 )/Kurtsaki H-3(HD-1 )Kurtsaki

Bactospeine Plantibac BugTime

H-3(HD-1 )Kurtsaki H-3(HD-1 )/Kurtsaki

Sporeine Biospor

H-1/thuringiensis

Bathurin

H-I(HD-1 )/

Baktukal

H4/dendrolimus H-5/galleriae H-1/insectus Hl/thuringiensis

Dendrobacillin Entobacterin-3 Insektin Biotoksybacillin

--israelenis H-14

Eksotokin Toxobaktedn Bactmos, Vectobac Teknar

aAdapted from Refs 2, 3 and 4.

Culex spp. Aedes spp. Anopheles

TIBTECH - MAY 1986

by fermentation technology. T h e r e are several entomopathogenic fungi which do not growin vitro or require very complex media. For example, Entomophthora gry]li is cultivated in vivo, using either fieldcollected diseased insects or healthy insects infected in the laboratory 16. Fungal formulations currently being evaluated contain a mixture of fungal mycelial fragments and conidia or blastospores. Active ingredients are formulated in a car~ier base which often is made of bentonite, kaolin clay, or a mixture of various agricultural by-products. Commercial products are formulated either as wettable powders or as dust formulations and to these, antioxidant and UV-screening materials are added.

Nematodes and Protozoa Very few nematodes and protozoa have been mass produced industrially or used as biological pest control agents in large scale field trials (Table 4). Nosema ]ocustae is an obligate parasite and is cultured in the host insect, e.g. grasshoppers 2, necessitating extraction from ground whole insects by filtration and centrifugation, to arrive at a pure preparation. The major obstacle for commercialization has been production of these organisms.

Biotechnology and biological pest control agent production Biotechnology can affect industrial production and cost effectiveness of biological pest control agents. The cost of gross input for in vitro production of bacteria and fungi includes the use of media, energy (both for culture growth and sterilization of the equipment), time and labour. Biotechnological innovations will lead to improvements in the process technology 2. Cost of production could be reduced by using cheaper fermentation media and shorter fermentation cycles. A great many biotechnological advances for the production of such insecticides are aimed at genetic engineering of the insecticides or their toxins to develop more potent and broader host range toxigenic bacteria, exclude other toxins and metabolites from the preparation and maximize the parameters that govern fungal sporulation15.17. Where insecticidal crystal protein (ICP) production is desired, research has been directed to the production of B. thuringiensis asporogenous mutants ~8"~9, and hence an improved diversion of cell biomass to the production of the ICP. It can be estimated that such improvements would increase the productivity 3-5-

fold. There are important differences between ICPs and the plasmids which encode them vary depending on the strain of B. thuringiensis from which they are derived. Toxicity and host range of the ICPs vary for these toxins 2°. These are two characteristics which might be altered effectively by genetic manipulations. The serological and molecular characteristics of the ICPs from various isolates of B. thuringiensis have been analysed 17. The respective DNAs have been sequenced 21 and cloned into E. coil and B. subti]is zz'23. In spite of major accomplishments in molecular studies the widespread use of this insecticide is still very limited. The main method for the commercial production of B. thuringiensis has been submerged batch fermentation. We are examining continuous phase-production or continuous culturing and harvesting of the ICP-containing culture material. The end product from the batch fermentation process contains cells, spores, extracellular enzymes and proteins, other low molecular weight material, and ICPs. These are harvested by high speed continuous flow centrifugation and acetone precipitation into a thick paste containing all of the sedimentable material. During formulation the paste is mixed with

-- Table 3

Entomopathogenic fungi in commercial and experimental production a Fungus

Target

Product

Manufacturer/Country

Beauveria bassiana

Colorado potato beetle codling moth European corn borer pine caterpillar mosquito larvae citrus rust mite spittle bug sugarcane frog hopper lepidopteran larvae aphids coffee green bug greenhouse whitefly thrips Northern Joint-vetch water hyacinths H. annosum of conifers tree roots apple canker disease

Boverin

USSR

Culicinomyces clavisporus Hirsutella thompsonii Metarhizium anisopliae Nomuraea rileyi Verticillium lecanii

Colletotrichum gloeosporioides Cercospora rodrnanfi Peniophora gigantea Trichoderma virdae

People's Republic of China

EAO b Mycar Metaquinoc

USA Abbott Laboratories/USA Brazil

EAO Vertalec

USA Tate and Lylein Ltd/UK

Mycotal Thriptal -EAO --

Tate and Lylein Ltd/UK Tate and Lylein Ltd/UK USA Abbott Laboratories/USA Bio-Basic Ltd./UK

--

aAdapted from Refs I and 29. bEAO, experimental application only. c Marketed under tradenames Biocontrol ® , Biomax ® , Combio ® , Metabiol ® and Metapol ®.

--

TIBTECH - MAY 1986

_

Table

4

Entomopathogenic protozoa in experimental production a Protozoa

Target

Remarks

Nosema Iocustae Nosema pyrausta Nosema fumiferanae Vairimorpha necatrix

grasshoppers European corn borer spruce budworm cabbage looper corn earworm tobacco budworm

Peaceful Valiey Farm Supply/USA

aAdapted from various authors in Ref. 2.

various adjuvants, wetting agents, and sticky material (stickers) and is spray-dried for final commercial packaging 24. The quality of the material is determined using standardized bioassays and/or monoclonal antibody assays. Production of fungi or their metabolites offers the greatest prospects in this area, especially when the techniques for genetic manipulation of antibiotic producers become readily available. The major obstacles in the area of fungal bioinsecticides are the genetic instability of the fungi, identifying the mechanism of action of toxic material or secondary metabolites, and understanding the molecular genetics of pathogenicity. Production of metabolites toxic to nontargeted insects or the environment could be reduced or prevented by genetic manipulation. For Micromonosporidiae (e.g. Nosema) and viruses, productivity could be increased substantially if in vitro tissue culture systems were available. Some media for in vitro cultivation of insect viruses need animal sera, which are expensive and may contain animal viruses. Use of fermentation technology for culturing insect cells and biotechnologically derived animal sera substitutes should be of help here. Equally helpful would be the cloning of insect virus material into soil bacteria such as B. thuringiensis, or into plants. The economics o f bioinsecticide production and use It has been suggested that the cost of research development and obtaining US Environmental Protection Agency approval for a new bioinsecticide will limit introduction of products with potential markets of less than $10 million. In fact, most companies

involved in biotechnology would want to aim at a market of $50 million or more 25. It is suggested that as a result of this, bioinsecticides are not likely to be developed for many low volume applications. However, bioinsecticides may still sell significantly. The cost of research, development and product safety analysis for a new chemical pesticide (i.e. $15 million or more) means that equivalent work on bioinsecticides becomes an attractive alternative (i.e. under $1 million). Additionally, the quantity of bacterial or viral insecticides needed for treating certain agricultural acreages or forests may be significantly lower (0.025-0.25 kg ha -1) compared with that of chemical insecticides. This must be related to the costs and economics of production. The cost of production of 10 litres of B. thuringiensis israelensis (BTi) culture using complex and defined liquid salts media is $7-12 compared with $0.02-0.03 if produced from by-products of industrial factories 26, Another example of costeffective production data is from the study of Herflin eta]. 27. They showed that mosquito larvicidal strains of B. sphaericus 1593 and BTi H14 grow on commercial powders of soy products, fish meal, dried milk products, raw sewage, blood and serum from animal, bone meal, chicken parts, animal dung and agricultural waste material, yielding sufficient culturebiomass with toxic properties. In the case of viral insecticides the successful adoption of any biological pest control agent technology would be judged by the reduction of the production costs. Work at US Department of Agriculture by Bell and Shapiro 28 has resulted in a doubling of the active life of Gypcheck (see Table 1) and a drop in the production cost from $75 to $5 per hectare.

Conclusions The present advantages of biological pest control agents are: (1) their high degree of specificity for pest control; (2) little or no effect on nontargeted and beneficial insects and man; (3) absence of insect resistance; (4) absence of residue build-up in the environment; and (5) potential impact from biotechnologicat research and development. However, biological pest control agents have a number of limitations: (1) of under 100 known agents only 20 are EPA registered; (2) killing time may be slow; (3) some are environmentally unstable although some can recycle; and (4) their production is expensive. It is hoped that biotechnology and a responsible environmental outlook will help us change the limitations and make biological pest control agents products for the agriculture of today. For this, future research and development in the areas of identification of new etiological agents of insect disease, and mechanism and genetic basis of bioinsecticide-induced insect disease are needed. It can be concluded that biotechnology has a significant role in the production and manufacturing of biological pest control agents and ultimately lowering the input costs of agriculture. The challenge in this area has not been fully met. However, it is anticipated that with many firms producing or developing bioinsecticides there will be greater activity in the area. Ultimately the production of effective bioinsecticides in sufficient numbers will displace the synthetic chemical insecticides and the associated ecological disadvantages. References 1 Papavizas, G.C. (ed) (1981). Biological Control in Crop Production, Granada Publishing

TIBTECH - MAY 1986

2 Burgess, H. D. (ed) (1981). Microbial Control o f Pests and Plant Diseases, 1970-1980, Academic Press 3 Kurtsak, E. (ed) (1982). Microbial and Viral Pesticides, Marcel Dekker 4 Miller, L. K., Lingg, A. J. and Bulla, L. A. Jr (1983) Science 219, 715-721 5 Kirschbaum, J. B. (1985) Annu. Rev. Entomol. 30, 51-70 6 Payne, C. C. (1982) Parasitology 85, 35-77 7 Burgess, H. D. (1982) Parasitology 84, 79-118 8 Tweeten, K. A., Bulla, L.A. and Conoiglo, R. A. (198~)Microbio]. Rev. 45, 379-408 9 Bucher, G. E. (1960)]. InsectPatho]. 2, 172-195 10 Spikes, A. G. and Spence, K. D. (1985) Tissue and Ce]] 17, 379-394 11 Fast, P. G. and Morrison, I. K. (1977) ]. Invert. Pathol. 30, 208-211 12 Endo, Y. and Nishiitsutsuji-Owo, J. (1980) ]. Invert. Pathol. 36, 90-103 13 Lisansky, S. G. and Hall, R. A. (1983) in Fungal Technology, Vol. 4 Filam ell to us Fungi, pp. 327-345, Edward Arnold 14 Ignoffo, C. M. (ed.) (1978) Proceedings []

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Benefits of Rhizobium to agriculture Janet I. Sprent Crops fixing nitrogen by means of endosymbiotic rhizobia are a major world source of protein and soil nitrogen. Interactions between the bacteria and host plant are currently being unravelled. This will enable current rhizobial biotechnology and developing plant biotechnology to be targetted towards more efficient nitrogen fixation and new nodulated, nitrogen fixing crops. Nitrogen fixation research celebrates its centenary this year. In 1886 it was first s h o w n c o n c l u s i v e l y that pea plants grow better in nitrogen deficient soil if they have n o d u l e s on their plant roots I (Fig. 1). A bacterium, t h e n called B a c i l l u s radicicola but n o w k n o w n as R h i z o b i u m t e g u m & o s a r u m , was subsequently isolated from and found to stimulate

]. I. Sprent is at the Department o f Biological Sciences, University o f Dundee, Dundee DD1 4HN, UK. ~) 1986, Elsevier Science Publishers B.V., Amsterdarn

Environ. Microbiol. 50, 623-628 21 Schnepf, E. H., Wong, H.C. and Whiteley, H. R. (1985) ]. Biol. Chem. 260, 6264-6272 22 Klier, A., Fargette, F., Ribier, J. and Rapoport, G. (1982) EMBO]. 1, 791-799 23 Schnepf, H. E. and Whiteley, H.R. (1981) Prec. Natl Acad. Sci., USA 78, 2893-2897 24 Couch, T. L. and Ignoffo, C. M. (1981) in Microbial Control o f Pests and Plant Diseases, 1970-1980 (Burgess, H. D., ed.), pp. 621-634, Academic Press 25 Genetic Technology News (1985) February, p. 6 26 Dharmsthiti, S. C., Pantuwatana, S. and Bhumiratana, A. (1985)]. Invert. Pathol. 46, 231-238 27 Hertlein, B.C., Hornby, J., Levy, R. and Miller, T. W. (1981) Soc. Indust. Microbio]. Prec. 22, 53-60 28 Biotechnology News (1982) 2, 2 29 Jaques, R. P. (1983) Agric. Ecosyst. Environ. 10, 101-126 30 Brooks, W. M. (1980) Biotechnol. Bioeng. 22, 1415-1440 31 Fuxa, J. R. and Brooks, W. M. (1979) ]. Invert. Path. 33, 86-94

of the l st ]oin t USA/USSR Conference on Production, Selection, and Standardization o f Entomopathogenic Fungi, American Society for Microbiology Kenney, D. S. and Couch, T. L. (1981) in Biological Control in Crop Production, Beltsville Symposia on Agricultural Research, pp. 143-150 Soper, R. S. and Ward, M. G. (1981) in Biological Control in Crop Production, Beltsville Symposia on Agricultural Research, pp. 161-180, Granada Publishing Dulmage, H. T. (1981) in Biological Control in Crop Production, Beltsville Symposia on Agricultural Research, pp. 129-141, Granada Publishing Johnson, D. E., Niezgadski, D. M. and Twaddle, G. M. (1980) Can. ]. Microbiol. 26, 486-491 Wakisaka, Y., Masaki, E., Koizumi, K., Nishimoto, Y., Endo, Y., Nishimura, M.S. and Nishiitsutsuji-Uwo, J. (1982) Appl. Environ. Microbiol. 43, 1498-1500 MacLinden, J. H., Sabourin, J.R., Clark, B. D., Gensler, D. R., Workman, W.E. and Dean, D.H. (1985) App].

p r o d u c t i o n of these root nodules. Inside the nodule, R h i z o b i u m reduces atmospheric nitrogen to amm o n i a w h i c h the host l e g u m e incorporates into organic c o m p o u n d s 2. Searches were soon u n d e r w a y for strains of R h i z o b i u m to give i m p r o v e d nitrogen fixation and in 1895 a patent leading to the biotechnology of R h i z o b i u m i n o c u l a n t p r o d u c t i o n was taken out 3. Strains of rhizobia for use w i t h particular legumes (e.g. clover, peas and lentils) were grown in bulk, added to a suitable carrier (sterile ground peat being the most common),

0166-9430/86/$02.00

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packaged and used either as a seed dressing or incorporated into drills at sowing.

The contribution of Rhizobium Properly managed, legumes can fix all the nitrogen for their o w n needs and m a y enrich soil nitrogen for associated or subsequent crops. Before the mineral boom, the e c o n o m i e s of N e w Zealand and Australia relied almost entirely on legume-based agriculture, using introduced legumes such as w h i t e clover (Trif o l i u m repens), and subterranean clover ( T r i f o l i u m subterraneum), respectively, together with suitable rhizobia. Even at a fairly conservative estimate of 50 kg nitrogen fixed ha -1 year -1, the 108 ha of l e g u m e pasture in Australia w o u l d save $2.7 x 109 at 1986 prices for nitrogen as urea. This assumes that all of the fertilizer nitrogen w o u l d be incorporated into the plant, w h i c h it is not (see also Table 1). The true value is more likely to be at least $3.7 x 109. The central highlands of Brazil, covering about 2 x 108 ha, are nutrient poor but because there is reasonable rainfall, plant yields of grain legumes such as dry bean (Phaseolus vulgaris) a n d