C. Keswani • K. Bisen • V. Singh • B.K. Sarma • H.B. Singh (*) Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi 221005, India e-mail: [email protected] e-mail: [email protected]
Recent shift in trends of agricultural practices from application of synthetic fertilizers and pestici pesticides des to organi organicc farmin farming g has brough broughtt into into focus focus the use use of microo microorga rganis nisms ms those those carryout carryout analogous analogous functions functions.. Formulatio Formulations ns of rhizomicro rhizomicroorgan organisms isms available available in global global markets markets range from talc-based talc-based and liquid and secondary secondary metabolitemetabolite-based based formulation formulations. s. The ideal conditions required for development of high efﬁciency formulations of biopesticides include selection of potent strains, shelf life, storage, application technology, quality control, trol, biosaf biosafety ety,, and regist registrat ration ion.. In this this chapte chapter, r, we will discuss the constraints associated with deve develo lopm pmen entt and and comm commer erci cial aliz izat atio ion n of bioino bioinocul culant ants. s. Moreo Moreover ver,, specia speciall emphas emphasis is will be on the next generation of antimicrobial secondary metabolite formulations which will not only have a much longer shelf life but also a higher efﬁciency against soilborne rne phytopathogens particularly against bacteria; also, a consortium of antimicrobial meta metabo boli lite tess agai agains nstt indi indivi vidu dual al path pathog ogen enss could be formulated and used regardless of geogra geographi phicc locatio location n where where the incide incidence nce of that particular disease is high. This approach would be unsurpassed by current technology, as the formulation would speciﬁcally target a part partic icula ularr path pathog ogen en whil whilee remai remaini ning ng soil soil microbiota would remain unaffected.
# Springer India 2016
N.K. Arora et al. (eds.), Bioformulations: for Sustainable Agriculture , DOI 10.1007/978-81-322-2779-3_2
C. Keswani et al.
biop biopes esti tici cide dess was was arou around nd 0.2 0.2 % of the the tota totall pesticides’ market during the year 2000, and it Convention Conventional al farming farming around around the world world is primarprimar- ampliﬁed to 4.5 % by 2010. The market value is ily based on chemical fertilizers and pesticides for expected to reach around US$ 1 billion (Singh al. 2012 2012). ). In India, India, curren currently tly 34 microorg microorgani anisms sms plant plant nutriti nutrition on and diseas diseasee manageme management, nt, a practi practice ce et al. which pounded huge negative impacts on human have been included in the schedule of Gazette of and environment health. Globally, rising aware- India for registration as biopesticide with Central Insecticid icidee Board, Board, Farida Faridabad bad,, under under section sectionss 9 ness ness of the the haza hazarrdous dous effe effect ctss of synt synthe heti ticc Insect (3B) and and 9(3) 9(3) of the the Inse Insect ctic icid ides es Act, Act, 1968 1968 pest pestic icid ides es has has incr increa ease sed d the the dema demand nd for for safe safer r (3B) (Table 2.1)) (Keswani et al. 2013a al. 2013a). ). alternatives alternatives.. Various Various microorgani microorganisms sms are currently currently (Table 2.1 Variou Variouss clas classe sess of bioc biocon ontr trol ol agent agentss have have being explored and utilized as biological control shown n sign signiﬁ iﬁca cant nt anta antago goni nism sm to a rang rangee of agents agents (BCAs) (BCAs) or biopesti biopesticid cides. es. Popul Popular ar BCAs BCAs show include Trichoderma spp., Pseudomonas ﬂuores- phytopathogens in vitro, but generally they have cence, Bacillus spp., Ampelomyces Ampelomyces quisqualis quisqualis, irregular performance in ﬁeld conditions. Various factors are respon responsib sible le for inconsi inconsiste stent nt perfor perfor- Agrobacteriu Agrobacterium m radiobacter radiobacter , nonp nonpat atho hoge geni nicc factors Fusarium Fusarium, Coniothyrium, and atoxigen atoxigenic ic Aspergil Aspergil- mance which includes poor shelf life, susceptibillus niger (Singh 2006 (Singh 2006;; Keswani et al. 2014 al. 2014,, 2015; ity of microbial strain to various abiotic stresses, Mish Mishra ra et al. al. 2015 2015). ). Approx Approxima imatel tely, y, there there are 1400 1400 and low organic carbon content in the soil. Forbiop biopes esti tici cide de prod produc ucts ts bein being g sold sold worl worldw dwid idee mulation technologies are used for stabilizing the (NAAS 2013) 2013). The tot total market share of microorganisms during production, storage, and
Int Introdu oductio ction n
Microbes listed in the Gazette of India for production production of biopesticide biopesticidess and registration registration under sections sections 9 Table 2.1 Microbes (3B) and 9(3) of the Insecticides Act, 1968 Bacterial
Formulation Technology of Biocontrol Agents: Present Status tus and Future Prospects
dist distri ribu buti tion on,, aidi aiding ng in the the appl applic icat atio ion n and and hand handli ling ng,, prot protec ecti ting ng the the micro microor orga gani nism sm from from damaging environmental factors, and enhancing the activity of the organism (Jones and Burges 1997). 1997 ). In a micr microb obia iall form formul ulat atio ion, n, the the majo major r focus is to preserve microorganism for enhancing thei theirr anta antago goni nism sm agai agains nstt targ target et path pathog ogen ens. s. Potency Potency of microbi microbial al formul formulatio ation n is primaril primarily y dependant on the strain of microorganism used, thou though gh ther theree may may be cruc crucia iall phys physic ical al and and nutritional requirements of the microorganism to rema remain in acti active ve for for long longer er time time.. Beneﬁ eneﬁci cial al microorganis microorganisms ms are considered considered as eco-friendl eco-friendly, y, and it is mandatory that any additives in the formulation should be eco-friendly. Commercial success of these formulations is based on the capability of a microorganism to survi survive ve and prolif prolifera erate te in the ﬁeld ﬁeld condit condition ion,, shelf life, and efﬁciency to control pest and disease, market price, ease of handling, and application cation (Lisansky (Lisansky 1985). 1985). Decisi Decision on of select selecting ing formulation depends fundamentally on the target organism to be managed, as well as on the ecology and biology of the biocontrol agents and host plant (Jacobsen and Backman 1993 1993). ). Moreover, the best feature of this approach is that it can easily be integrated with different pest management modules. BCAs have been formulated in various ways such as wettable powders, liquid, and granules for application such as sprays, seed treatments, drenches, and dips and incorporation into soil and pot mix.
in wett wettab able le powd powdeer, liqui iquid, d, and granu anular formul formulatio ations ns (Singh (Singh et al. 2012 2012,, 2014 2014)) (Table 2.2 2.2). ). Dusts (DP) are formulated by adding an active ingredient on ﬁne solid mineral powder such as clay and talc with particle size ranging from 50 to 100 μm. Dusts are applied directly to the target, either manually or mechanically. Inert ingredients used for dust formulation are anticaking agents, ultraviolet protectants, and adhesive materials to enhance adsorption. Dusts usually contain <10 % of microorganisms by weight. Granule (GR) particles are heavier and larger compared to dust. Microgranules (100–600 μm) and and coar coarse se part partic icle less size size (100 (100–1 –100 000 0 μm) are made made from from mine minera rall mate materi rial alss such such as sili silica ca,, kaol kaolin in,, star starch ch,, atta attapu pulg lgit ite, e, poly polyme mers rs,, grou ground nd plan plantt residues, and dry fertilizers (Tadros 2005 2005). ). Concentration of microorganisms in granules ranges from 5 to 20 %. Three types of granule formul formulati ations ons are curren currently tly availab available: le: (1) the micromicroorganism is sprayed on a rotating granular carrier withou withoutt a sticke sticker, r, (2) the microo microorga rganis nism m is attach attached ed to the the oute outerr surf surfac acee of a gran granul ular ar carr carrie ierr by a sticker, and (3) the microorganism is incorporated into a carrier paste or powder as a matrix.
Wettab Wettable le Powder Powderss and and Liqui Liquids ds
BCAs such BCAs such as Bacillus Bacillus subtilis subtilis, Pseudomonas putida, and Trichoderma spp. are used to control various diseases and are generally applied as dip or dren drenche chess and and spra sprays ys to frui fruitt afte afterr harv harves estt (Tronsmo and Dennis 1983 1983;; Colyer and Mount 1984;; Pusey and Wilson 1984 1984 Wilson 1984;; Wilson and Pusey 2.2 2.2 Type Typess of Form Formul ulat atio ions ns 1985). 1985 ). Spraying of Penicillium sp. to pineapple fruit result resulted ed in reduce reduced d postha postharve rvest st diseas diseases es Although Although pestic pesticide idess are formul formulated ated in various various fruit ways ways includi including ng dry formulat formulation ionss such such as dusts dusts (Lim and Rohrbach 1980 Rohrbach 1980). ). (DP), (DP), granul granules es (GR), (GR), and microgr microgranu anules les (MG); (MG); seed dressing formulations such as powders for seed dressing (DS); dry formulations for dilution 2.2.2 2.2.2 Granu Granular lar Formul Formulati ations ons in water including dispersible granules (WG) and wettable powders (WP); liquid formulations for Lignite silage was applied to produce granules Trichod oderm erma a harzi harzian anum um dilution in water such as emulsions, suspension containing Trich and concentrates (SC), oil dispersions (OD), and cap- Gliocl Gliocladi adium um roseum roseum to cont contro roll Rhizoctonia sule sule susp suspen ensi sion onss (CS) (CS);; and and ultr ultralo alow w volu volume me solani in soil soil causin causing g dampin damping-o g-off ff of peanut peanut formulation formulationss (Knowles (Knowles 2005, 2005, 2006) 2006) howe howeve ver, r, (Jones et al. 1984 al. 1984). ). Lignite was grinded to proglobally biopesticides available in the market are duce granules of 425–2000 μm in diameter and
C. Keswani et al.
Table 2.2 Types of pesticide formulations formulations (Modified (Modified from Patanjali and Raza 2013 Raza 2013;; Jones and Burges 1997 Burges 1997)) Formulation
Formulations diluted in water Emulsiﬁable concentrate EC Water-in-oil emulsion EO Oil-in-water emulsion EW Suspension concentrate SC Capsule suspension CS Soluble concentrate SL Water-soluble powder SP Water-soluble granule SG Tablet TB Briquette BR Wettable powder WP Water-dispersible granule WG Formulations diluted with organic solvents Oil-miscible liquid OL Oil-miscible ﬂowable OF Oil-dispersible powder OP Formulations applied undiluted Dustable powder DP Encapsulated granule CG Microgranule MG Electro-chargeable liquid ED Spreading oil SO Ultralow volume liquid UL Ultralow volume suspension SU Granule GR Seed treatments
Powder for dry seed treatment Flowable concentrate Solution for seed treatment Coated seed Water-dispersible powder Miscellaneous Bait concentrate Bait
Fe F eatures Emulsion formed when added to spray tank Preformed emulsion Preformed emulsion Suspended insoluble AI AI contained in capsules Used for water-soluble AI Powder soluble, but may contain inert ingredients Used for water-soluble AI Used for portable water-soluble AI Controlled-release formulation Typically consist of AI, clay carrier, and surfactants AI dispersed, but not dissolved, in water AI dissolved in organic solvent Suspension in organic liquid Powder to be applied in oil AI carried on free-ﬂowing powder Controlled-release granule Diameter below O.6 mm Used with electrostatic spray equipment Applied to water surface Applied through UL V sprayers As above Applied to soil and water
DS FS LS PS SS
Bait diluted before application
then amended with the product of sorghum ferment mentat atio ion. n. Isol Isolat ates es of T. harz harzia ianu num m and G. rose roseum um were were allo allowe wed d grow growin ing g on thes thesee granules for 7 days. These granules were allowed to air-dry followed by incubation before application in R. solani -infested soil. Trapping of antagonis onisti ticc micr microo oorg rgan anis ism m in calc calciu ium m algi algina nate te gran granul ules es,, also also know known n as pril prill, l, has has been been used used widely widely used (Connick (Connick 1988). 1988). Though most of the commercially available alginates are derived from kelp, other organisms are also reported to prod produc ucee algi algina nate tes. s. Algi Algina nate tess prod produc uced ed by
Azotobacter vinelandii can be used in place of kelp kelp algina alginate te for the produc productio tion n of microb microbial ial formulat lation to control plant pathogens (De Lucca et al. 1990). 1990). Furthe Furtherr resear research ch may lead to less costly alginates. Sodium alginates showing great variation in viscosity and purity are are avai availa labl blee comm commer erci cial ally ly.. More More gran granula ular r sodium sodium algina alginates tes like like Kelgin Kelgin HV, Kelgin Kelgin,, and sodi sodium um algi algina nate te IG-3 IG-350 50 are are easi easier er to hand handle le than the more powdery alginic acids. Daigle and Cotty (1995 (1995)) reported that 5 % gluten from wheat grains improved the
Formulation Technology of Biocontrol Agents: Present Status tus and Future Prospects
performance of a toxigenic Aspergillus ﬂavus but that concentrations were very complex to process. cess. The ﬁnal ﬁnal formul formulated ated produc productt contai contained ned 5 % gluten, 1 % sodium alginate, and 5 % corn cob grits. Likewise, in case of Streptomyce Streptomyce s spp. formulation, clay was handy to keep in suspension, ion, form formul ulat ateed in algi algin nate ate with with carr carrie ier r polyamide.
Bioc Biocon ontr trol ol Prod Produc ucts ts Containing Fungi and Their Formulations
Plethora of antagonistic fungi and their products have been registered as commercial biopesticides glob global ally ly (Tab (Table le 2.3 2.3)). Different types of formulations of these antagonists are currently available in markets and are successfully used against against various various phytopath phytopathogen ogens. s. Trichoderma spp., Gliocladium spp., Coniothyrium , and nonpathogenic strains of fungal genus are available as different formulations. Vari Variou ouss biop biopes esti tici cide de prod produc ucts ts (Bin (Binab ab-T -T,, Bio-Fung Bio-Fungus, us, Supresivit Supresivit,, RootShield RootShield,, T-22HB, T-22HB, T-22 T-22G, G, Tric Tricho hode dex, x, Tric Tricho hose seal al,, Tric Tricho hope pel, l, Tricho Trichodow dowels els,, BioMax BioMax,, BioVam BioVam,, Tricho Trichojec ject, t, Trichoderma 2000) contain Trichoderma spp. to control a variety of pathogens, including Fusarium, Botrytis, Gaeumannomyces , Rhizoctonia , Pythium, Sclerotinia , Verticillium, Sclerotium , and wood-rot fungi. Trichoderma product formulati lation on vari varies es cons consid ider erab ably ly.. Co Comb mbina inati tion on of Tricho Trichoder derma ma viride viride and T. harz harzia ianu num m is formulated differently, for example, as a pellet for soil (Trichopel), as dowels for injecting in woods (Trichodowels), as a wettable powder for resusp resuspens ension ion to apply apply on wounds wounds with brush brush (Trichoseal), and as a wettable powder in syringe (Trichoject). Bio-Fungus is available as a granule, as crumbles for soil incorporation, and as wetta ettabl blee powde owderr impr impreg egna nate ted d in stick ticks. s. Gliocladiu Gliocladium m virens virens formulation is available in alginate alginate prill under under the trade name SoilGard SoilGard and effectively used against R. solani and Pythium spp. Nonpat pathogenic strain of Fusarium oxysporum has has been been used used agains againstt pathog pathogenic enic F. oxysporum and Fusarium Fusarium moniliforme moniliforme on
carnation, tomato, basil, and cyclamen. Commercial cially ly avai availa labl blee prod produc ucts ts of nonp nonpath athog ogen enic ic F. oxysporum , namely, Biofox C, are formulated as alginate prill or dust, and Fusaclean is available able as microg microgran ranule ule.. Aspire Aspire contai containin ning g yeast yeast Candida Candida oleophila oleophila, is form formul ulate ated d as wetta wettabl blee powder for postharvest application in citrus and pome fruit to control Penicillium spp. and Botrytis. Ampelomyces quisqualis , parasite of powdery mild mildew ew fung fungi, i, is comm commer erci cial ally ly avai availab lable le as water-disp water-dispersib ersible le granule granule (AQ10) (AQ10) and applied applied to the leaves leaves of apples apples,, grapes grapes,, strawb strawberr erries ies,, tomatoes, tomatoes, cucurbits, cucurbits, and ornamental ornamentals. s. Pythium oligandrum has been formulated as granule or powder for use in seed treatment for management of pathogenic Pythium spp. (Jones and Burges 1997). 1997 ). Lewis and Lumsden (2001 (2001)) prepared a solid matr matrix ix form ormula ulation tion of Gliocladium and Trichoderma using wheat bran and vermiculite effective against R. solani . Formulated product was applied at a rate of 1.0 % (w/w). The product was was tes tested, ted, and and a signi igniﬁ ﬁcant cant red reducti uction on in damping-off of pepper seedlings was observed. Efﬁcacy of Trichoderma Trichoderma spp. against Sclerotinia sclerotiorum causi causing ng sunﬂ sunﬂowe owerr head head rot rot was was evaluated in the ﬁeld. Trichoderma formulation contained viable hyphal fragments, Trichoderma conidia, milled corn kernels, and industrial talc. Sunﬂower heads were infected with S. sclerotiorum sclerotiorum after 2 days of the ﬁrst delivery Trichoderma formulation by honeybees. Head of Trichoderma rot incide incidence nce was reduce reduced d in sunﬂow sunﬂower er when when 100 g formulation was carried by honeybees in a 10 h per day period (Escande et al. 2002 al. 2002). ). An invert emulsion based on soybean and coconut oils offered the lowest viscosity (27 0.81 cps) and most stable emulsion layer (93 % v/v) for formulating T. harzianum conidia (Batta (Batta 2004). 2004). In this formulation, 36 months of shelf life of conidia was recorded with 50 % declined viability at 20 1 C after 5 months. Botrytis sporulati lation on on the the frui fruitt lesi lesion on surf surfac acee was was also also inhibited after 10 days of inoculation. Biocontrol potential potential of Trichoderma isolated isolated from rhizorhizosphe sphere re of Musa sp. sp. was was evalu evaluat ated ed agai agains nstt F. oxysporum in vitro. Among different isolates, T. harzianum Th-10 was found most signiﬁcant
C. Keswani et al.
Table 2.3 Some commercially available fungal biocontrol products Organism
Di Disease against used
Candida oleophila Trichoderma harzianum, T. polysporll
Dust or alginate granule Granules, wettable powders, sticks, and crumbles
Postharvest application to fruit as drench, drip, or spray Postharvest application to fruit as drench, drip, or spray
Seed treatment soil or incorporation Applied after fumigation, incorporated incorporated in soil sprayed or injected
Spray Wettable powder, emulsiﬁable liquid or granule Spores, microgranule Spores in inert powder Gran ranules ules Granule (1 10 6 cfu g1) Granules or dry powder (both at 1 107cfu g1) Wettable powder
R. solani , S. rolfsii, Pythium sp. Pythium sp., R. solani, S. rolfsii Wettable powder Pythium sp., R. solani, S. rolfsii Wettable powder
In drip to rock wool; incorporate in potting Spray, chain saw oil Mix Mix with with soil soil or pott pottin ing g medium Granules are incorporated in soil or soilless growing media prior to seedlings Granules Granules added in furrow with granular applicator, by broadcast application to turf, mixed with green house soil Spray
Incorporated into soil or potting medium Spray
Formulation Technology of Biocontrol Agents: Present Status tus and Future Prospects
in pathogen pathogen inhibition inhibition.. Five organic substrates substrates includ including ing rice rice chaffy chaffy grain, grain, rice rice bran, bran, banana banana pseudostem, farmyard manure, and dried banana leaf leaf were were tested tested for the mass mass produc productio tion, n, and drie dried d bana banana na leaf leaf was was foun found d the the best best carr carrie ier r material for T. T. harzianum growth. Dried banana leaves were colonized within a few days by strain Th-10 and produced propagules of high density (4.6 1032 cfu g1 of leaf). leaf). Furthermor Furthermore, e, addition of jaggery (10 % w/v) to the dried leaves enhances the growth of T. T. Harzianum, and more than 6 months of surviva ival was recorded (Thangavelu et al. 2004 al. 2004). ). Trichoderma Trichoderma atroviride atroviride isol isolat atee C52 was was observed on onion roots when inoculated in soil with various formulations (McLean et al. 2005 al. 2005). ). Pellet formulation product maintained the con Trichoderma up to 105 cfu g1soil centration of Trichoderma in comp compar aris ison on to 101 cfu cfu and and 104 g1 soil concen concentra tratio tions ns that that were were mainta maintaine ined d by seed seed coating and solid substrate formulations, respectively. Trichoderma isolate C52 was inoculated into Sclerotium cepivorum -infested soil as both solid substrate and pellet formulations, and no difference was observed in disease control, but more healthy plants were observed in the pellet treatment. Increased root and shoot lengths, plant height, and dry weight were recorded after treatment of plants with T. viride formulated in talc. Application of T. T. viride formulation also resulted in a signiﬁcant reduction of sheath blight caused by R. solani (Mathivanan et al. 2005 al. 2005). ). New carrier ier form formu ulati latio on was dev develop eloped ed by usin using g T. harzianum M1, and resistance to carbendazim show showed ed inhi inhibi bito tory ry effe effect ct agai agains nstt Pythium aphanidermatum. Different formulations including lignite, talc, wettable powder, lignite + ﬂy ash-based powder formulation, bentonite paste, gelatin-glycerin gel, and polyethylene and glycol paste were developed for seed treatment. Shelf life of the microbial formulations was assessed at 24 C for 9 months. Up to 74 % reduction in disease incidence was recorded when Trichoderma formul formulati ation on was applie applied d as seed seed treatment. Additionally, enhanced plant biomass under ﬁeld and greenhouse conditions was also reco ecorde rded (Jay Jayaraj araj et al. al. 2006). 2006). Pell Pellet etiz ized ed formulations of kaolin clay and wheat bran in
an algina alginate te gel contai containin ning g conidi conidia, a, fermen fermentor tor biomass, biomass, or chlamydosp chlamydospores ores of G. virens virens and Trichoderma spp. spp. were were prep prepare ared d (Lew (Lewis is and and Papavizas 2007 Papavizas 2007). ). Higher population densities of Trichoderma and Gliocladium were observed observed when soil is incorporated with alginate pellets containing containing chlamydosp chlamydospores ores rather rather than condia condia and bran rather than kaolin as the carriers. A new Trichoderma asperellum formulation was develo developed ped using using soybe soybean an oil disper dispersio sion. n. Complete inhibition of Phytophthora Phytophthora megakarya causing cacao black pod disease was recorded when when form formul ulat atio ion n was was appl applie ied d to the the pods pods.. Ninety percent prevention of infection in treated pods was recorded after 1 week, and 50 % reduction after 3 weeks was recorded when formulation was sprayed on cacao clones susceptible to P. megakarya. The formulations showed a signiﬁcant effect in disease management (Mbarga et al. 2014 al. 2014). ). Talc-based formulation of a novel T. virid viridee, BHU-29 BHU-2953, 53, succe success ssfull fully y contro controlled lled damping-off of chili caused by P. aphanidermatum and tomato wilt caused by F. oxysporum. Signiﬁcant reduction in diseases was observed when T. viride formulated as 2 % wettable powder was applied to the seeds and furr furrow ow (Sin (Singh gh et al. al. 2014). 2014). Formul Formulatio ations ns of Trichoderma with with mixt mixtur uree of 1 % w/v w/v Su Sure re-1 Jell, 1 % w/v PDB, and 0.3 ml L of the surfactant tant Break BreakThr Thru u 100SL 100SL (BT) (BT) and an invert invert oil emulsion of 50 % v/v corn oil, 2.5 % w/v lecithin, and 0.5 % w/v PDB (COP) were evaluated against frosty pod rot pathogen Moniliophthora roreri. T. harzianum DIS 219f and Trichoderma ovalisporum DIS DIS 70a 70a were were appli applied ed (180 (180 ml tree tree1, 2.46 107 conidia ml1) in the ﬁeld. COP/DIS70a formulation resulted in a maximum increase in yield as compared to other treatments (Crozier et al. 2015 al. 2015). ).
Bioc Biocon ontr trol ol Prod Produc ucts ts Containing Bacteria and Their Formulation
Seve Several ral bact bacter erial ial spec specie iess havi having ng bioc biocon ontr trol ol potential have been formulated and are commercial cially ly avail availab able le (Tab (Table le 2.4 2.4). ). Nonpat Nonpathog hogeni enicc
C. Keswani et al.
Table 2.4 Some commercially available bacterial biocontrol products Biocontrol agent
Pseudomonas syringae ESC 10
P. syringae ESC 11
P. ﬂuorescens A506 P. ﬂuorescens NCIB B. subtilis
Blight Ban A 506 Conquer
Pseudomonas cepacia A. radiobacter
Intercept R. solani, Fusarium spp., 14 Pythium sp. Nogall, A. tumefaciens Diegall 16 PSSOL 12 P. solanacearum
P. solanacearum (nonpathogenic) P. ﬂuorescens NCIB 12089 B. subtilis B. cepacia-type Wisconsin M36
B. cinerea, Penicillium spp., Mucor piriformis, Geotrichum candidum B. cinerea , Penicillium spp., M. piriformis, G. candidum
Frost, Erwinia amylovora
Postharvest application to fruit as drench, dip, or spray Postharvest application to fruit as drench, dip, or spray Drench dip or spray
R. solani, Fusarium, Alternaria spp., and Aspergillus spp. Crown gall disease A. tumefaciens
Petri dishes with pure culture grown on agar (1.2 1011 cfu plate1) Wettable powder
Spray Added to a slurry, mix with a chemical fungicide Root dips, drench
Drench dip or spray
Washed plates, culture suspension
R. solani, Fusarium spp.
S. rolfsii, S. sclerotiorum
Fusarium, Pythium, spiral, lesion, lance, and sting nematode
Peat Peat carr carrie ierr or liqu liquid id
Seed treatment in planter box Seed eed trea treatm tmen entt or drip irrigation
A. radiobacter is commercially available under to the chick pea seed, the total shelf life of the various trade names such as Nogall, Galltrol-A, bacteria was recorded at 180 days (Vidhyasekaran Diegall, Diegall, and Norbac 84C. Bacteria Bacteria are generally and Muthamilan 1995 Muthamilan 1995). ). Peat-based formulation of suspended in non-chlorinated water and applied P. ﬂuorescens strains PfALR2 was developed and as dips and sprays to cuttings and stems or as soil assessed for root treatment, seed treatment, foliar Pseudomonas syringae syringae formulate drench. Pseudomonas formulated d as spraying, spraying, and soil application. application. All four treatments wett wettab able le powd powder erss is comm commer erci ciall ally y avai availa labl blee in combin combinatio ation n result resulted ed in the signiﬁca signiﬁcant nt control control of under under trade trade names names Bio-Sa Bio-Save ve 10 and Bio-Sa Bio-Save ve sheath blight in greenhouse greenhouse condition (Rabindran (Rabindran Vidhyasekaran n 1996). 1996). P. ﬂuorescens ﬂuorescens strain 11 and used for postharvest application to citrus and Vidhyasekara and and pome pome frui fruitt for for mana manage geme ment nt of Botrytis, PF-1, isolated from rhizosphere of maize roots, Mucor , Penicillium, and Geotrichum. showed antagonistic potential against against R. solani solani P. ﬂuorescens strains strains isolat isolated ed from from rhizosp rhizospher heree f. sp. sp. sasakii causing banded banded leaf and sheath blight blight of various various crops crops with with antagoni antagonisti sticc potent potential ial agains againstt of maize. Among the different carriers, talc and Fusarium Fusarium spp. were formulated as talc-based and peat maintained the population at 18.3 107 and peat-based products. In talc-based and peat-based 19.5 107 cfu cfu g1 of the bacterium, bacterium, respectively respectively,, formulations, P. ﬂuorescens survived for a maxi- after 40 days. Signiﬁcant control of disease was mum of 240 days. When formulation was applied recorded after the seed treatment with peat-based
Formulation Technology of Biocontrol Agents: Present Status tus and Future Prospects
formul formulatio ation n (Sivak (Sivakuma umarr et al. 2000 2000). ). Formulation Formulationss of B. subtil subtilis is AF 1 show showed ed both both plan plantt grow growth th promotion promotion and biocontrol biocontrol potential potential when prepared prepared in peat. The formulation was supplemented with A. niger myce myceliu lium, m, 0.5 0.5 % chiti chitin, n, and and orga organi nicc comcompost from cultivation of Agaricus Agaricus bisporus . Biocont contro roll pote potenti ntial al of form formul ulat ated ed prod produc ucts ts was was evalua evaluated ted against against two pathoge pathogens ns on ground groundnut nut and pigeon pea. Chitin, A. niger mycelium, and A. bisporus bisporus compost were used as supplement for improving the growth rate of B. B. subtilis AF 1A. Peat formulation supplemented with chitin when used for seed treatment demonstrated better control of wilt in pigeon pea caused by Fusarium udum and A. niger responsible responsible for crown rot disease ease in groundn groundnut ut (Manju (Manjula la and Podile Podile 2001). 2001). P. ﬂuorescens ﬂuorescens strains FP7 and PF1 and their consortiu sortium m were were formul formulated ated as talc-ba talc-based sed produc productt and mixed both with and without chitin were assessed assessed individually against sheath blight of rice. Signiﬁcant reduction in disease incidence was recorded after after the applic applicatio ation n of formul formulated ated produc productt through through seed seed,, soil soil,, root root,, and and foli foliar ar spra spray. y. In ﬁeld ﬁeld as much much as 62.1 % reduction in sheath blight incidence was observed in the consortium treatment containing chiti chitin n (Com (Comma mare re et al. al. 2002). 2002). 0.1 0.1 % calc calciu ium m hydroxide hydroxide signiﬁcantly signiﬁcantly promoted the growth of B. amyloliquefa amyloliquefaciens ciens strain strain B190 B190 used agains againstt Botrytis Botrytis elliptica elliptica in in lily. Spraying B. amyloliquefac amyloliquefaciens iens B190 mixed with 0.05 % sodium sodium carbona carbonate, te, 0.025 0.025 % calcium calcium hydrox hydroxide ide,, or 0.025 0.025 % ammo ammoni nium um nitra nitrate te supp suppre ress ssed ed the the gray mold on lily. Concentration of adjuvant was kept below 0.1 % (v/v); carboxymethyl cellulose (CMC) and Tween 80 were effective to B. amyloliquefac amyloliquefaciens iens B190 formulation formulation against against lily gray mold (Chiou and Wu 2003 Wu 2003). ). The efﬁcacy viride de and of talctalc-ba base sed d form formul ulat atio ions ns of T. viri P. ﬂuorescens alone and in combination on sheath blight disease, crop growth, and yield in rice was stud studied ied in ﬁeld ﬁeld expe experi rime ments nts.. Appl Applic icat ation ion of form formul ulat ated ed prod produc uctt of P. ﬂuor ﬂuoresc escen enss and T. viride either alone or in combination resulted in increase in root and shoot lengths and plant height when compared with control. Signiﬁcant redu reduct ctio ion n in shea sheath th blig blight ht incid inciden ence ce was was also also recorded after application of P. ﬂuorescens ﬂuorescens and T. viride (Mathivanan et al. 2005 al. 2005). ).
Bacillus licheniformis strain N1 exhibiting the biocontrol activity against Botrytis cinerea was formulated formulated using fermentati fermentation on of the bacterial bacterial culture in Biji medium. Wettable powder formulation of antagonist based on olive oil corn and starch was selected for evaluation of the disease control. A dose of 100-fold-diluted B. licheniformis N1E was found to be the optimum spray formulation and signiﬁcantly reduced the the dise diseas ase. e. 90.5 90.5 % redu reducti ction on in dise diseas asee by formulated product was recorded in comparison to the the 77 % redu reduct ctio ion n by synt synthe heti ticc fung fungici icide de incl includ udin ing g carb carben enda dazim zim and and diet dietho hofe fenc ncar arb. b. Results of this study indicated that the olive oiland corn starch-based formulation of B. licheniformis using using liquid liquid fermentatio fermentation n will be effec effectiv tivee agai agains nstt toma tomato to gray gray mold mold (Lee (Lee et al. 2006). 2006). Ninete Nineteen en isolat isolates es of antago antagonis nistic tic Pseudomonas and twelve isolates of yeast were scre screen ened ed for for the the bioc biocon ontr trol ol acti activi vity ty agai agains nstt Colletotrich Colletotrichum um musae causing banana anthracnose. P. ﬂuorescens strain FP7 showed a maximum inhibition of C. musae musae mycelial mycelial growth. growth. Water-in-oil formulation of P. ﬂuorescens FP7 was formulated by adding various oils such as rice bran (28.50 %), coconut (28.50 %), and castor (28.50 %) separately to the bacterial culture and bacterial populations were reported to survive for 210 days of storage. The application of waterwater-inin-oil oil formul formulati ation on of bacter bacterium ium signiﬁ signiﬁ-cantly cantly reduce reduced d the diseas diseasee incide incidence nce (Peera (Peeran n et al. al. 2014). 2014). B. subtilis strain BY-2 was unable to colonize the lea leaf surface and stem in oilseed rape when applied as pellet. Populations of BY-2 declined from 10 8 CFU seed1 to 104 CFU g root1 and 1023 CFU g stem1 after 60 days. days. Signiﬁ Signiﬁcan cantt reduct reduction ion in diseas diseasee was observed when compared to control (Hu et al. 2014 al. 2014). ). Efﬁcacy of aqueous suspension (Serenade ASO or QRD 145) and foliar sprays of wettable powder formulation (Serenade MAX or QRD 141) of B. B. subtilis QST 713 alone and in comb combin inat atio ion n with with cop copper per hydr hydro oxid xide was was invest investiga igated ted agains againstt bacter bacterial ial spot spot diseas diseasee of tomato. tomato. The aqueous aqueous suspensio suspension n of B. subtilis subtilis QST 713 alone alone signiﬁ signiﬁcan cantly tly reduce reduced d bacter bacterial ial spot on tomato foliage when compared to control. trol. The wettab wettable le powder powder of B. subtili subtiliss QST
C. Keswani et al.
713 alone did not reduce bacterial spot, but in B. subtil subtilis is-based -based biopes biopestici ticide, de, was used used as seed seed combinatio combination n with copper hydroxide hydroxide it reduced reduced inoculant for peanut, cotton, and beans to control dise diseas asee seve severi rity ty and and enha enhanc nced ed the the tota totall frui fruitt the the root root dise diseas ases es caus caused ed by Fusariu Fusarium m and Rhizoct Rhizoctooyield (Abbasi and Weselowski 2015 Weselowski 2015). ). nia (Mahaf (Mahaffee fee and Backma Backman n 1993 1993). ). Quantum-400 Quantum-4000, 0, a B. subtilis strain A13-based product, is commercially available as seed inoculants for peanut, and another biopesticide based on strain GB 07 named 2.5 See Seed Trea reatmen tmentt Epic Epic is availab available le for cotton cotton.. Bacter Bacterial ial BCAs BCAs activel actively y The appl applic icat ation ion of micr microb obes es to seed seed surf surfac acee colonize the rhizosphere and compete with other requires few technical considerations. Signiﬁcant micr microo oorg rgan anis isms ms incl includ udin ing g path pathog ogen enss (Bis (Bisen en amou amount nt of the the inoc inocul ulum umss must must surv surviv ivee the the appl applic icaa- et al. 2015). 2015). Nons Nonspo pore re-f -for ormi ming ng bacte bacteri rium um Enterobac bacter ter cloacae cloacae showed antagonistic activity tion procedure and must have the capacity to grow Entero in vicinity vicinity of seed. seed. Since seeds are at low moisture moisture against Pythium spp. spp. caus causin ing g seed seed rot rot in cott cotton on leve levels ls for for most most of the the time time duri during ng stor storag age, e, seed seeds. s. Simp Simple le seed seed inoc inocul ulat atio ion n tech techni niqu quee with with microorganisms must have the ability to survive (CMC) as sticker in cotton seeds was carried out under low water activity. Microorganisms might successfull successfully y (Nelson (Nelson 1988). 1988). Liqu Liquid id cult cultur uree of like likewi wise se need need to be mixe mixed d with with othe otherr acti active ve E. cloaca cloacaee was applied by solid matrix priming to ingr ingred edie ien nts, ts, for for exam exampl ple, e, inse insecctici ticide dess and and tomato and cucumber seeds in combination with a fungicides. These aspects raise issues of formula- fungicide (Harman and Taylor 1988). 1988). tion stability and strain selection. Seed treatment Generally, in seed treatment, treatment, microbial formuwith beneﬁcial microorganisms has been a prime lation is applied to seed as powder or liquid. For ﬂuorescens and Burkholderi Burkholderia a cepacia cepacia area area of inve invest stig igat atio ion n for for many many year yearss (Bis (Bisen en example, P. ﬂuorescens et al. 2015). 2015). Micr Microo oorg rgan anis isms ms with with vari variou ouss were applied to pea seeds with or without captan properties have been applied to seeds to perform for control of Aphanomyces Aphanomyces root rot and Pythium damping-o g-off ff (Parke (Parke et al. 1991 1991). ). Pota Potato to tube tubers rs have have various various functions, functions, including including plant growth promopromo- dampin tion, nitrogen nitrogen ﬁxation, ﬁxation, phosphate phosphate solubilizat solubilization, ion, been been trea treated ted with with asco ascosp spor ores es of Talaromyces and biolog biological ical contro controll of plant plant pathoge pathogens ns (Keswa (Keswani ni ﬂavus in in a pyrop rophyllite lite carrier ier (Fravel vel al. 1985). ). In order to ensure the better adhesion et al. 2013b al. 2013b). ). BCAs are applied to the seeds for et al. 1985 protection of seed and seedlings from effects of of bioi bioino nocu culan lants ts on seed seed surf surfac ace, e, stick stickers ers are are various seed-borne and soilborne plant pathogens. added added to microbi microbial al formula formulatio tions ns for seed seed treattreatFor the succes successfu sfull biolog biological ical contro control, l, applied applied ment. To control Pythium ultimum attack in pea microorganism must grow and colonize the rhizo- and soybean seeds, PelGel has been used to treat sphe sphere re in orde orderr to prot protec ectt the the plan plant, t, thus thus the relea release se the seed with P. putida (Paulitz et al. 1992 1992). ). In of microbes from the formulation and prerequisite cucumber seed treatment, PelGel has also been harzianum (Harman 1991; condi conditio tion n for growth growth are of param paramoun ountt import importan ance. ce. used with T. harzianum 1991; Taylor The comm commer erci cial al wett wettab able le powd powder er prod produc uctt et al. 1991). 1991). Another sticker Polynox-N-I0 was used for for seed seed treatm treatmen entt with with shal shalee and and then then appl applied ied Mycostop (Kemira AgroOy, Finland), containing used 8 Streptomyces Streptomyces griseoviridis griseoviridis stra strain in K61, K61, with with 10 on bean seeds with conidia of T. harzianum, and 1 cfug is available for seed treatment. The powder efﬁcien efﬁcientt diseas diseasee contro controll was observ observed. ed. Various Various 1 concentrat tration ionss of plant plant gum, gum, methyl methyl cellulo cellulose, se, is usedat 5–8 g kg seeds seeds for contro controll of seed-b seed-born ornee concen and soilbo soilborne rne fungal fungal phytop phytopath athoge ogens ns in herbs, herbs, and xanthane gum with talc have been used for vegetables, vegetables, and ornamentals ornamentals (Tomlin 1994). 1994). The treatment of potato tuber with PGPR (Kloepper Schroth h 1981) 1981). At 40 C, popu popula lati tion on of shel shelff life life of form formul ulat atio ion n is esti estima mate ted d to be 6 mont months hs and Schrot at 8 C or for 12 months at 12 C and must be rhizobacteria in talc and 20 % xanthane gum did stored in airtight containers. Seed inoculation with not decline for 2 months and resulted in better bacte acteri riaa to pro protect tect the the seed seed from from soilb oilbor orn ne incr increa ease se in plan plantt deve develo lopm pmen entt in ﬁeld ﬁeld pota potato to.. path pathog ogen enss and and prom promot otee plan plantt has has been been well well However, rhizobacteria did not last longer in forinvest investiga igated ted (Merri (Merriman man et al. 1974). 1974). Kodi Kodiak ak,, a mula mulati tion on cont contai aini ning ng gum gum traga tragaca cant nth h or gum gum
Formulation Technology of Biocontrol Agents: Present Status tus and Future Prospects
kara karaya ya.. CMC CMC was was appl applie ied d with with micr microb obia iall agen agents ts to seeds to control R. solani. One percent CMC with clay carrier carrier was applied applied to Verticillium Verticillium biguttatum biguttatum and other biocontrol agents to potato tubers (Jager and Velvis 1985 Velvis 1985). ). Various antagonists have been applied to treat the sugar beet seeds using either gum xanthan or methylcellulose in combination with a neutralized neutralized talc or peat carrier (Suslow (Suslow and Schroth 1982 Schroth 1982). ). Similarly, to control take-all disease ease in wheat, wheat, surf surfac acee-di disi sinf nfes este ted d seed seedss were were coated with P. ﬂuorescens ﬂuorescens in combination with 1 % methylcellulose (Weller and Cook 1983 Cook 1983). ). In seed treatment of Chinese aster with T. ﬂavus, a polymer binder was used in quartz ﬂour to pellet seeds seeds (Nagtza (Nagtzaam am and Bollen Bollen 1994 1994), ), and antagon antagonist ist T. ﬂavus was isolated from seeds after 17 years. Strains Strains of E. E. cloacae cloacae and Trichoderma were delivdelivered to cucumber and tomato seeds through solid matrix matrix primin priming g (Harman (Harman and Taylor Taylor 1988 1988). ). During During priming seeds are brought to a certain moisture level just below the required level for germination and then mixed with moistened Trichoderma or E. cloacae cloacae, shale, sphagnum moss, or bituminous coal. The seeds and carriers are then mixed with water and incubated before planting.
of the the micr microo oorg rgan anis ism m prio priorr to inoc inocul ulat atio ion n in ﬁeld, (2) poor survival of the organism in the environment, and (3) low quality of the microbial product itself. Success of biopesticides depends on the delivery of viable, active microorganisms in high numbers to the ﬁeld which requires highqualit quality y inocul inoculant ants. s. The carrie carrierr substr substrate ate is the most critical part of the microbial formulation, and it must be capable of supporting high numbers of microbe. Carriers are inert ingredients, and they do not have biocontrol potential; however, they can affect the efﬁcacy of the product and shelf life of microorganism (Table 2.5 (Table 2.5). ). Better surviv survival al of Pseudomonas spp. spp. on minera minerals ls with small particle size, such as zeolite, montmorillonite, and vermiculite, than on minerals with larger particle size such as talc, pyrophyllite, and kaolinite was observed during storage at 20 C (Dan (Dandu dura rand nd et al. al. 1994) 1994). Backman and RodriguezRodriguez-Kabana Kabana (1975 1975)) compar compared ed diatom diatomaaceous earth and attapulgus clay for their various physic physical al proper propertie tiess includ including ing waterwater-hol holdin ding g cap capacit acity y and and stre trength ngth afte afterr autoc utocla lav ving. ing. Attapulgus clay granules swelled in water and lost lost their their integr integrity ity after after autocl autoclavin aving, g, wherea whereas, s, diatom diatomace aceous ous earth earth granul granules es did not swell swell in water and remained intact after autoclaving thus making it to absorb a molasses-based medium for 2.6 2.6 Carr Carrie iers rs and and Adju Adjuva vant nt Used Used delivery delivery of T. harzianum harzianum to soil. Wheat branin Microbial Formulations perlit perlitee mixtur mixturee and poplar poplar bark bark compos compostt have have been used as carrier for nonpathogenic Fusarium Vari Variat atio ions ns in eff effecti ective vene nesss of micro icrobi biaal strains mixed with soil that induced resistance to formul formulati ations ons from from beneﬁ beneﬁcial cial microo microorga rganis nisms ms F. oxysporum f. f. sp. dianthi (Garibaldi are credited to three main causes: (1) presence et al. 1987). 1987). Fine-g Fine-grou round nd tree tree barks barks have have also also
Table 2.5 Various adjuvants used in microbial formulations Adjuvant
Maintain AI in solution Prevent premature drying of deposit
C. Keswani et al.
been used as a carrier (Stack et al. 1988 al. 1988). ). Peat is generally generally used as carrier carrier for Rhizobium Rhizobium spp. and is also useful for soil applications and seed coating of biocontrol agents. Huber et al. (1989 ( 1989)) used ﬁne-ground peat with a methylcellulose sticker for wheat seed treatment with bacteria for control Gaeumannomyces graminis . Alder bark has of Gaeumannomyces been used as a carrier to apply T. ﬂavus to potato seed pieces (Keinath et al. 1990 al. 1990). ). One of themec the mechan hanis isms ms invol involved vedin in biocon biocontro troll is the production production of hydrolytic hydrolytic enzymes by the antagantagonistic microorganism. The amount and type of nutrients in the formulation must allow ample production of hydrolytic enzymes (Stack et al. 1988 1988). ). Increas Increased ed molar molar concentr concentratio ations ns of carbon carbon and nitronitrogen sources (0.02–0.18 M maltose and 0.006–0 0.006–0.024 .024 M arginine arginine)) and increas increased ed carbon/ carbon/ nitrogen ratios (12:1–80:1) enhanced the proliferaproliferaThielaviopsis basicola and Trichoderma tion tion of Thielaviopsis spp. on lignite granules (Stack et al. 1987 al. 1987). ). Likewise, T. ﬂavus was formulated with eight different orga organic nic carrie carriers rs and used used again against st Verticillium dahliae on eggplant, and a maximum inhibition of patho pathoge gen n was recor recorded ded in treatm treatmen ents ts with with the highest carbon/nitrogen carbon/nitrogen ratios (159:1 for pyrophylpyrophyllite lite,, 97:1 97:1 for for corn corn cobs cobs)) (Fra (Frave vell et al. al. 1985 1985)). As new information on mechanism of biocontrol revealed, it may be possible to express desirable biocontrol trait traitss by manipu manipulat latin ing g nutri nutrient ent comp compos ositi ition on of formulations. For example, biocontrol potential of G. virens virens GL-2 GL-21 1 depe depend ndss on the the form form of nitr nitrog ogen en in the formulation. Alginate prill of G. virens with wheat bran as a carrier resulted in signiﬁcant control of Sclerotiu Sclerotium m rolfsii rolfsii in compa comparis rison on to vermi vermicuculite plus wheat bran (Ristaino et al. 1994 al. 1994). ).
Basi Basicc Info Inform rmat atio ion n Requ Requir ired ed for Microbial Product Registration
Certain information about the various parameters is prerequisite for the biopesticide registration. These parameters include microorganism strain speciﬁcations, cfu count of microorganism used, target target microo microorga rganis nism, m, moistu moisture re conten contentt of the product, type of formulation, and technical bulleti letin/ n/pr prod oduc uctt proﬁ proﬁle le.. Stra Strain in spec speciﬁ iﬁca cati tion onss
include information on genus and species, rhizosphere competence, biological control potential, grow growth th promo romoti tion on poten otenti tial al,, and and growt rowth h parameters like pH and temperature.
Strain Strain Specif Specifica icatio tions ns
Selection of potential antagonist strain under ﬁeld and lab condi conditio tions ns ensur ensures es the the effec effectiv tivee and cons consisistent performance of bioagent in ﬁeld. Screening of effect effective ive strai strain n can be done done in differ different ent ways: ways: sele electi ction of poten otenttial ial strain ain in relati lation on to phytopa phytopathog thogens ens,, screenin screening g of isolates isolates with high biotechn biotechnolog ological ical applicati application, on, or search search for economeconomical viable substrates which are suitable for mass production of bioagent (Singh et al. 2003 al. 2003,, 2006 2006). ).
Shelf Shelf Lif Life e and Storag Storage e
For commercialization of a microbial product in the market, it is essential that it has long shelf life and can be stored at room temperature. It has been been sugg sugges este ted d that that shel shelff life life of a mini minimu mum m 18 month onthss is accep ccepta tabl blee for comm commer erccial ial microorganism-based product. Storage at room temp temper erat atur uree is an esse essent ntia iall cond conditi ition on as the the farmers cannot afford the equipment to keep the produc productt at any temper temperatu ature. re. Accord According ing to the rece recent nt guid guidel elin inee of the the Cent Centra rall Inse Insect ctic icid idee Boar Bo ard d and and Regi Registr strat atio ion n Co Comm mmit itte tee, e, the the data data requ requir ired ed for for clai claimi ming ng 1-ye 1-year ar shel shelff life life of the the product is for 15 months for talc-based formulation, i.e., the microbe should remain viable for 15 months.
2.7. 2.7.3 3
CFU CFU Coun Countt
According to the Central Insecticide Board and Regi Regist stra rati tion on Co Comm mmit ittee tee guid guideli eline ne,, colo colony ny-forming unit (cfu) count should not be less than 2 106 spores ml1 or g1 on selective media (SM) for antagonistic fungi, and CFU count on sele select ctiv ivee medi medium um shou should ld be a mini minimu mum m of 8 1 1 1 10 ml or g for antagonis antagonistic tic bacteria bacteria (Singh 2012 (Singh 2012). ).
Formulation Technology of Biocontrol Agents: Present Status tus and Future Prospects
faci facilit litie iess which which are are gene genera rally lly not not avail availab able le to most producers, sellers, and farmers. Shelf life is result of combini combining ng severa severall factors factors includin including g The pathog pathogeni enicc contam contamina inants nts such such as Salmo- a result production tion technol technology ogy,, materia materiall used used as carrier carrier nella, Shigella , and Vibrio should not be present. produc packaging, ing,and and transpo transport. rt. The mass mass produc production tion Other microbial contaminants must not to exceed and packag of signiﬁca signiﬁcant nt number numberss of viable, viable, efﬁcien efﬁcient, t, and 1 104 counts ml1or g1. stable propagules of the microorganism is a prerequ requis isit itee in biop biopes estic ticid idee deve develo lopm pmen entt (Sing (Singh h et al. al. 2004b). 2004b). Submerg Submerged ed fermenta fermentatio tion n system system 2.7.5 2.7.5 Moistu Moisture re Conten Contentt has been traditionally followed by the producers over over the the solid solid subs substr trate ate ferm fermen entat tatio ion n becau because se of its Maxim aximum um mois moistu ture re cont conten entt of the the prod produc uctt cost-effectiveness and easy technology (Churchill should not exceed more than 8 % for dry formu1982;; Stowell 1991). 1982 1991). However, new large-scale lati lation on of fung fungii and and 12 % for for bact bacter eria ia (www. production systems are required for bacteria and cibrc.nic.in/2.1.22011.doc). cibrc.nic.in/2.1.22011.doc ). fungi that do not like to produce spores in liquid media. media. Unfortun Unfortunatel ately y fermen fermentati tation on system systemss for mass production based on solid substrates are not freq freque uent ntly ly avai availa labl blee (Con (Conni nick ck et al. al. 1990). 1990). 2.8 2.8 Cons Constr trai aint ntss in the the Prod Produc ucti tion on Submerged Submerged fermentation fermentation methods methods are generally generally of Microorganism-Based well adopted adopted for the mass mass produc production tion of second secondary ary Biopesticides metabolites, metabolites, antibiotics, antibiotics, organic organic acids, and bacteria; howe howeve ver, r, it is not not suit suitab able le for for the the prod produc ucti tion on of The main reasons for slow growth of microbial- ria; based biopesticide biopesticide industry industry include include inconsisten inconsistentt viable ﬁlamentous fungi. Therefore, selection of performance of ﬁnal product in ﬁeld condition, the cost-effective fermentation technology for the short shelf life of microorganism in formulation, mass production viable and efﬁcient propagules is possibilities of contamination with other plant and a matter of concern. Choice of carriers and adjuhuman human pathoge pathogens, ns, lack of suitab suitable le applicat application ion vant used in the formulation is another technical problem lem in the the deve develop lopme ment nt of stab stable le and and effe effect ctiv ivee technology, small market size, and lack of proper prob knowle knowledge dge about about the biopes biopestici ticides des in farmers farmers.. biop biopes esti tici cid des. One One of the the majo majorr goals in Research should be focused on the development formul formulatin ating g biopes biopestici ticides des is to maintain maintain the viabilviabilof superior formulation to protect the reliability of ity and effectiveness of the active ingredient for a the product, because a single failure will jeopar- possib possible le durati duration, on, prefer preferably ably 2 years. years. After After the dize the whole trade’s reputation. Production of production production of microorganis microorganism, m, the main challenge biopes biopestici ticides des is a long long proces processs which which include includess faced by producers in formulation development is selection of suitable strain for formulation, mass the shelf life of microorgani microorganism sm during the storage production of selected strain, screening of micro- peri period od.. If the the prod produc uctt carr carrie iess less less numb number erss of activ activee organi organism sm for suitabl suitablee carrier carrier for formula formulatio tion, n, ingredients due to shorter shelf life, the overall assessment of the shelf life of microorganism in performance of the formulation will be affected. selected formulation, and product efﬁcacy in ﬁeld condition. Several reports on contamination and low population of microorganism in biopesticides 2.9 2.9 Futur uture e Pro Prospe spects cts being sold in the market were registered (Singh et al. al. 2004a 2004a;; Alam Alam 2000 2000;; Aror Aroraa et al. al. 2010 2010). ). Due Due to The necessi necessity ty for more safe safe produc products ts for plant low microb microbial ial count, count, it is obviou obviouss that that their their perfo perforr- diseas diseasee managem management ent prompt promptss an inclinat inclination ion of mance in the ﬁeld is inconsistent and poor. The microbial biopesticide formulations with efﬁcient unpr unpred edict ictab able le seas season onal al natu nature re of the the exis existin ting g antagonism and good stability. Biopesticides give demand needs capable storage for biopesticides. eco-friendly eco-friendly alternatives alternatives to synthetic synthetic pesticides, pesticides, The storag storagee requir requires es sophisti sophisticate cated d and specia speciall yet yet they they conf confro ront nt vario various us difﬁ difﬁcu culti lties es in their their
Pathogen Pathogenic ic Contamin Contamination ation
production production,, formulation, formulation, and application. application. Since biopes biopestici ticide de generall generally y contai contains ns live organi organism, sm, maxi maximu mum m care care is need needed ed to main mainta tain in the the micr microb obia iall population and efﬁcacy from beginning to the end use. use. Study Study of their their formul formulatio ation n and produc production tion could enormously help in the commercialization of biopesticides. It appears to be that biopesticides will will have have a more more exte extens nsiv ivee use use in the the futu future re as thei their r application techniques enhance as less expensive iner inertt mate materi rial alss are are reco recogn gniz ized ed for for diff differ eren entt form formul ulat atio ions ns.. Intr Introd oduc ucti tion on of new new adjuv adjuvan ants ts showed showed signiﬁca signiﬁcant nt increas increasee in activity activity of microbe microbess and proposed a new area of research. Selection of proper formulation may enhance the product stabili bility ty,, larg larger er shel shelff life life,, and and perf perfor orm mance ance of microbes in ﬁeld conditions. Biopesticides offer a more balanced plant protection product application, and in the future formulation products should have more balance between production cost and efﬁciency (El–Sayed 2005 (El–Sayed 2005;; Rao et al. 2007 al. 2007;; Glare et al. 2012 al. 2012;; Khater 2012). 2012). Development related to the formul formulatio ation n type type would would possib possibly ly shift shift from dusts to granules, from suspension concentrates and and wett wettab able le powd powder erss to wate waterr-di disp sper ersi sibl blee granules, granules, and from single microorgani microorganism-bas sm-based ed product to microbial consortium-based formulation. With the advances in nanotechnology science, different new microbial formulations such as nan nanosusp suspen enssion, on, nanoemu emulsion, and and nanocapsule suspension with superior efﬁciency will be released in the market (Rao et al. 2007 2007;; Ghormade et al. 2011 al. 2011;; Glare et al. 2012 al. 2012). ). SigniﬁSigniﬁcant cant adva advanc ncem emen entt has has been been made made in the the prod produc uctio tion n of new new form formul ulati ation on prod produc ucts ts and and appl applic icat ation ion methods; however, there is still much work to be done. For further research to improve production and and appl applic icat atio ion n tech techni niqu ques es,, scie scien ntist tist and and researchers are likely to provide safe and effective products for plant disease management. Eco-friendly and safer biopesticides play an important role in modern agriculture; however, thei theirr majo majorr draw drawba back ckss have have led led to the the use use of nano nanote tech chno nolo logy gy in agri agricu cultu lture re (Mis (Mishr hraa and and Singh 2015). 2015). Silver nanoparticles (AgNPs) are the most frequently used metallic nanoparticles in various sector including ing agricultu lture (Jo et al. 2009; 2009; Kim et al. 2012; 2012; Mish Mishra ra et al. 2014). 2014). Many workers have reported the
C. Keswani et al.
antimicrobial activity of AgNPs against a vast range range of phytop phytopath athoge ogens. ns. A nanosi nanosized zed silica silica-silver silver partic particle le formul formulatio ation n was develo developed ped and showed signiﬁcant antimicrobial activity against a wide wide rang rangee of phyt phytop opat atho hoge gens ns incl includ udin ing g Colletotrichum sp., Pythium sp., P. syring syringae ae, Xanthomonas compestris , etc. (Park et al. 2006 al. 2006). ). Biosynthesized AgNPs of bacterium Serratia sp. BHU-S4 showed signiﬁcant antifunBipolaris is soroki sorokinia niana na gal gal acti activi vity ty agai agains nstt Bipolar (Mishra et al. 2014 al. 2014). ). Recove Recoverin ring g the real real connec connectio tions ns of AgNPs AgNPs with agroecosy agroecosystems stems including including soil, soil biota, biota, and plants and their poisonous quality level can be infe inferr whet whethe herr AgNP AgNP appl applic icati ation on coul could d be helpfu helpfull for agroec agroecos osyst ystems ems.. Consid Consideri ering ng the elements deciding destiny, transport, portability, and and pois poison onou ouss quali quality ty of AgNP AgNPss in soil soil,, it is additionally accepted that the size of the poisonous ous qual qualit ity y of thes thesee part particl icles es coul could d real really ly be evad evaded ed by cont contro rolli lling ng them them and and henc hencef efor orth th requires more profound exploration. Speciﬁcally, rese resear arch ch conc concen entr trat atin ing g on this this meth method odol olog ogy y utiliz utilizing ing biosyn biosynthe thesiz sized ed AgNPs AgNPs ought ought to get more thoughtfulness regarding an efﬁcient comprehension of how incorporating biosynthesized AgNP AgNPss in agrar agraria ian n appl applic icat atio ions ns cont contrib ribut utes es towa toward rd the the farm farmer er’s ’s bene beneﬁt ﬁts. s. One One impo importa rtant nt poin pointt is that that bio biosynt synthe hesi sizzed AgNP AgNPss have have demo demons nstr trat ated ed thei theirr wort worth h for for agri agricu cult ltur ural al appl applic icati ation onss by perf perfor ormi ming ng two two note notewo wort rthy hy undertakin undertakings, gs, viz., plant development development upgrade upgrade and plant malady management. Hence, now it is high time when future studies must be coordinated toward upgrading the utility of biosynthesized AgNPs in regular environments keeping in mind the end goal to predict their future agricultural extension.
Conc Conclu lusi sion on
In recent recent past, past, diseas diseasee manage managemen mentt strate strategie giess have have been been inclin inclined ed to much much safer safer altern alternativ atives es due to concerns over hazardous effects of chemical cal pest pestic icid ides es on huma human n and and plan plantt heal health th.. Biological control of plant pathogens employing livi livin ng micro icroor orga gani nissms offe offerrs such uch saf safe
Formulation Technology of Biocontrol Agents: Present Status tus and Future Prospects
alternative alternative to the chemicals. chemicals. In order to improve the efﬁciency and shelf life of biocontrol agents, various formulations based on solid and liquid carriers have been developed. Maximum care is required in biopesticide production and formulation as it contains live organisms. It is quite a conﬁrmation that biopesticides will have a more extensive use and share greater market space in the future. Speciﬁc procedures and technologies have been recently developed that would signiﬁcantly affect biopesticide formulations. Selection of abiotic stress tolerance or rhizospheric competence could allow a wider range of applications (Keswani 2015 (Keswani 2015). ). Moreover, fungicide tolerance screen screening ing would would promot promotee integr integrate ated d manage manage-ment with reduced chemical inputs. grateful to the Department Department of Acknowledgments HBS is grateful Biotechn Biotechnolog ology y (BT/PR (BT/PR5990 5990/AG /AGR/5/ R/5/587/ 587/2012 2012), ), New Delhi, for providing ﬁnancial support. KB is grateful to BHU for ﬁnancial assistance. BKS and CK are thankful to ICAR-A ICAR-AMAA MAAS S (Project (Project No. P27/131) P27/131) for ﬁnancial ﬁnancial support. support.
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