Keywords

13.1 Introduction

Plant diseases are caused by various biotic and abiotic factors viz. fungi, bacteria, viruses, viroids, phanerogamic parasites, protozoans, and nematodes are taking heavy toll of crops. These pathogens are causing substantial losses in different crops and therefore need to be managed. Several chemicals have been used in the past to manage the diseases caused by various pathogens. No doubt, some degree of control was achieved, but it posed new problems of residual toxicity and development of resistant strains of the pathogens. Delivery of microbial inoculants is being a very attractive option since it would substantially reduce the use of agrochemicals (Berg 2009). Microorganisms play a vital role in cropping systems, particularly plant-growth-promoting microorganisms (PGPMs). Soil or seed inoculation with microbial inoculants may lead to changes in the structure of the indigenous microbial population, which is important with regard to the safety of introduction of microbes in plant microenvironment (Trabelsi and Mhamdi 2013). Many reports indicate that the application of microbial inoculants can influence, at least temporarily, the resident microbial communities; therefore, screening of biocontrol agents (BCAs) with broad-spectrum activity and capacity to elicit systemic resistance in plants and offer protection against a wide range of pathogens needs to be done. Success in the identification of new microbial inoculants that exhibit significant control of various root and foliar diseases in the past few decades has contributed to the rising interest in the biological control of various phytopathogens. Further, an in-depth study of the action of BCAs is needed before using them on a large scale. End number of formulations approved by regulatory authorities around the globe are available for use in disease management. Therefore, biocontrol agents or antagonists as a means of plant disease control has gained importance in recent years. The biocontrol agent multiplies in the soil and remains near the root zone of plants and offer protection even at later stages of crop growth. The antagonistic activity of biocontrol agents against plant pathogens is highly specific against a particular pathogen and/or different races of the pathogen. Delivery system of BCAs mainly depends on the type of pathogen to be managed, the stage of the crop to be protected, the nature and severity of the disease, and the climatic conditions of the region (Desai et al. 2000). For application of a good formulation, a proper delivery method of microbial inoculants is essential. Since bioformulation incorporated in the soil have high densities of viable and efficient microbes for a rapid colonization of host rhizosphere, it may induce at least a transient perturbation of the equilibrium of soil microbial community. However, a modification in the microbial community structure caused by inoculation could be buffered by ecosystem resilience, which is driven by the level of diversity and interactions of the plant–soil biota (Kennedy 1999).

Seed treatment is a practical method of delivery system for both fungal and bacterial biocontrol agents. Biological control agents applied to seed have been shown to protect the seed against many seed-borne pathogens of crops, as well as increase plant growth and vigor (Jambhulkar and Sharma 2013). The use of biological agents as seed treatment is a valuable and an equally effective protection as chemical seed treatment. Physiological seed treatment such as seed priming has been used to quicken seed germination and improve the survival of seedlings (Burelle 2000). Bioformulation may directly be applied to plant roots in the form of root dip, spray, drip, or flood application for the management of soil-borne pathogens (Gasic and Tanovic 2013). Commercialization of good biocontrol agents becomes difficult due to impractical dosage recommendations, limited or inconsistent control efficacy, and improper delivery system. A better understanding of the ecological and epidemiological relationship between microorganisms and suitable delivery systems that will carry fungal strains with enhanced fungicide resistance will help to reduce the gap between experimental results and commercial use of biopesticides. Developing accurate delivery system is foremost important to assure effectiveness of bioformulation under field conditions. Unlike agrochemicals, in which chemical is dissolved in a solvent, most microbial inoculants are particulate suspensions. Problems with suspensions include settling of the microbial pesticides, nozzle blockages, stress affecting the viability of spores, inappropriate droplet size, large number of infective spores packed to a droplet, etc. (Bateman et al. 2007). Researches are in progress to optimize the delivery system for each group of biopesticides. The application of fluorescent pseudomonads by seed treatment (Niranjana et al. 2009), seedling root dip (Verma 2009), and soil drenching (Jeyalakshmi et al. 2010) has been attempted my many workers to control phytopathogens in various crops. Jambhulkar and Sharma 2013 reported the efficacy of various carrier formulations of Pseudomonas fluorescens through seed treatment, seedling root dip and soil drenching in unity and in combination. Work on droplet size revealed that smaller droplets would enhance effectiveness of microbial inoculants (Alves and Bateman 2013), whereas larger droplets are suitable for entomopathogenic nematodes (Bateman et al. 2007). Stable, effective formulations and appropriate delivery system are needed to convince farmers to adopt bioformulations.

13.2 Microbial Inoculants as Biocontrol Agents

Indiscriminate use of chemical compounds as pesticides damages the entire agroecosystem, which encourages the use of biopesticides. In fact, there is great potential of biopesticides in organic and conventional agriculture. Biological control by antagonistic microbial inoculants is a potential nonchemical means for crop protection, which is seen as a very attractive plant protection measure as it would substantially reduce the use of chemical pesticides and fungicides, and there are now an increasing number of inoculants being commercialized for various crops (Berg 2009). There is immense role of microorganisms in agricultural ecosystem, particularly plant-growth-promoting microorganisms (PGPMs). Plant growth benefits are mainly attributed to three major mechanisms: (1) PGPMs acting as biofertilizers (such as nitrogen-fixing bacteria and phosphate-solubilizing bacteria) assist uptake of plant nutrients by providing fixed nitrogen and other nutrients; (2) phytostimulators (microbes expressing phytohormones such as Azospirillum) can directly promote the growth of plants usually by producing phytohormones, and (3) biological control agents (such as Trichoderma, Pseudomonas, Bacillus, etc.) protect plants against phytopathogenic organisms (Mohiddin et al. 2010; Dawar et al. 2010). Microbial inoculants have various benefits over chemical pesticides: they (a) are more safe, (b) show reduced environmental damage, (c) show more targeted activity, (d) are effective in smaller quantities, (e) are able to multiply but are also controlled by the plant and indigenous microbes, (f) have quicker decomposition procedures, (g) are less likely to induce resistance by the pathogens and pests, and finally (g) can be used either in organic and conventional agriculture (Berg 2009).

Bacillus is a genus of bacteria known to elicit induced systemic resistance (ISR) in plants. In addition, Bacillus spp. have reduced incidence of viral diseases, for example, cucumber mosaic virus on tomato. On plants that are not challenged with pathogens, it has been reported that Bacillus can increase fresh weight and number of fruits and flowers (Kloepper et al. 2004). Pseudomonas, a genus of bacteria that can colonize plant roots and suppress pathogens through the production of antibiotics, is a genus that can elicit ISR as well (Kloepper et al. 2004). Bacteria in this genus have a strong potential as biocontrol and growth-promoting agents due to the following characteristics: a) rapid growth in vitro; b) rapid utilization of seed and root exudates; c) ability to colonize and multiply in the rhizosphere and the spermosphere, as well as inside the plants; d) production of metabolites like antibiotics, siderophores, and growth promoters; e) competition with other microorganisms; and, finally, f) ability to adapt to environmental stress (Weller 2007). In early attempts, products made of this Bacillus spp. failed due to the instability of the culture and lack of long-term viability (Kloepper et al. 2004). It is known that the majority of bacteria that promote plant growth are rhizosphere inhabitants; they have been designated as plant-growth-promoting rhizobacteria (PGPR). The most promising group of PGPR for biocontrol of plant diseases is fluorescent pseudomonads. Fluorescent pseudomonads associated with plants include Pseudomonas fluorescens, P. putida, P. aeruginosa, and P. aureofaciens. Plant-growth-promoting rhizobacteria may stimulate the production of biochemical compounds associated with host defense; massive accumulation of phytoalexins and phenolic compounds; increase in the activities of PR proteins, defense enzymes, and transcripts; and enhanced lignification. The induction of SAR using various ISR inducers has been of recent interest with quite reasonable success (Meena 2014).

Another group of microbes corresponds to some types of algae that have biotechnological potential as soil fertilizers for plant production and some macroscopic marine algae (Eklonia maxima) that improve the growth and yield of plants (Reisser 2010; Crouch and van Staden 1992). Finally, several genera and species of PGPR and different microbes are used as inoculants; the diversity represents an opportunity to start research in this area and provide new solutions for the current necessities of agriculture.

Soil microorganisms form a very complex and dynamic community between different compartments and levels. Microorganisms can survive in the spermosphere, a zone influenced by the seeds, which is full of nutrients that support their growth. In addition, they can inhabit the phyllosphere, a zone that comprises the above-ground parts of the plants, whose most relevant characteristic is the fact that it is in constant fluctuation related to external facts as temperature, radiation, and water availability. Other zones correspond to the vascular tissue, the rhizosphere, and the endophytic sites. Several strains of Trichoderma have been described as antagonistic fungi that are able to attack a wide range of phytopathogenic fungi. The production and secretion of fungal-cell-wall-degrading enzymes and compounds affecting the integrity of fungal membrane and cell walls are considered as the key steps in the antagonistic process by Trichoderma (Chet et al. 1998). Antagonism may be accomplished by competition, parasitism, and antibiotics or by a combination of these. Parasitism involves the production of several hydrolytic enzymes that degrades cell wall of pathogenic fungi. β-1,3 glucanase and chitinase are the key enzymes responsible for fungal cell and sclerotial cell wall lysis. These enzymes are produced by several fungi and bacteria and may be an important factor in biological control. Moreover, in the rhizosphere, region that includes plant roots and surrounding soil, intensive interactions between plants, soil, and microfauna take place due to its high energy and carbon content. This accumulation in the rhizosphere corresponds to all the compounds produced by plant roots, most of which are organic derived from photosynthesis and other plant processes (Pinton et al. 2001). Different and varied biochemical signal exchanges take place between these communities and their host plants; indeed, a wide diversity of bacteria and plant-associated microbes can interact with plants in a beneficial way, either by enhancing their growth and/or controlling phytopathogens (Beattie 2006; Nihorimbere et al. 2011).

13.3 Formulations Used for Inoculation of Microbial Inoculants

Development of a bioformulation is necessary to commercialize biocontrol technologies by industries. The commercial use of plant-growth-promoting rhizobacteria requires inoculum that retains high cell viability and can easily be transported and applied to seed. It needs extensive studies for large-scale multiplication of a biocontrol agent (BCA), which include suitable and inexpensive medium, method of fermentation (solid or liquid), type of formulation (wettable powder, liquid, granular), nature of filler material, delivery systems, optimum shelf life, and storage conditions. Application guidelines are set by considering all these aspects of a bioformulation. Delivery of free cell form is usually impractical to achieve satisfactory bioremediative effect because microbes are encumbered by biotic and abiotic stresses from the environment (Ting et al. 2010). The aims of formulating viable cells are to ensure that adequate cell viability is sustained to increase the efficacy of the cells and to facilitate the delivery and handling processes (Filho et al. 2001). A bioformulation can improve product stability and shelf life and also protect microbial inoculants against different environmental conditions and provide initial food source (Jambhulkar and Sharma 2014). Application of microbial inoculants either to increase crop health or to manage plant diseases depends on the development of commercial formulations with suitable carriers that support the survival of microorganism for considerable length of time (Aeron et al. 2011). A formulated microbial product is a product composed of one or more biological control agents mixed with ingredients that will improve its survival and effectiveness (Schisler et al. 2004). Those microbial inoculants formulated for delivery to soil are of especial importance due to their specific court of action, which is the rhizosphere. Microbial inoculants can be applied to the soil as fluid suspensions, as powder formulations, and as granules for soil and spray application. Fluid suspensions are prepared based on culture concentrates diluted in water or a buffer solution prior to application. They can also be prepared as dormant aqueous suspensions, obtained after harvesting the bacteria from a liquid culture, washed free of the spent medium and stored at a specific concentration in sterile water at room temperature (Miranda 2012). Microbial inoculants are formulated as dry formulation for direct application dusts (DP), seed dressing formulations-powders for seed dressing (DS), granules (GR), microgranules (MG), dry formulations for dilution in water-water dispersal granules (WG), and wettable powders (WP); liquid formulations for dilution in water emulsions, suspension concentrates (SC), oil dispersion (OD), suspoemulsion (SE), capsule suspension (CS), ultra low volume formulations (Knowles 2005, 2006). Powdered formulations are more commonly used. They consist of organisms concentrated into dry or wet powders. Depending on the composition of the powders, they can be applied directly to the soil, suspended in water, or dusted onto seeds. The commonest method to formulate granular products is to mix the organism and the ingredients with the granules (Burges 1998). In general, product formed from solid or semi solid-state fermentation does not require sophisticated formulation procedures prior to use. For example, grain or other types of organic matter upon which antagonists are grown are simply dried ground and added to the area to be treated. There are several problems with solid-state fermentation, which may make the system inappropriate for commercial product development. The preparations are bulky, they may be subject to a greater risk of contamination, and they may require extensive space for processing, incubation, and storage. The liquid-state fermentation is devoid of such problems, and large quantities of biomass can be produced within a few days. Either the biomass can be separated from medium and concentrated or the entire biomass with medium can be incorporated into dusts, granules, pellets, wettable powders, or emulsifiable liquids. The carrier material may be inert or a food base or a combination of both. Inoculant formulations depend directly on the carrier used for the delivery of the products because target microorganisms are mixed with it before being applied. Carriers are the inert ingredients that hold or dilute the microorganism to the desired concentration and improve coverage and distribution (Burges 1998). Commercial application of PGPR either to increase crop health or to manage plant diseases depends on the development of commercial formulations with suitable carriers that support the survival of bacteria for a considerable length of time. Carriers constitute the key for the effective release of the different products; they need to be effectively chosen due to their diversity (e.g., water, vermiculite, calcium sulphate, mineral soil and sand, vegetable oil, corn cob) that starts from classic ones to new and unconventional ones (Bashan 1998; Burges 1998). Certain specific conditions might increase the efficacy of a formulation. Addition of organic acids to T. koningii formulations and polysaccharides and polyhydroxyl alcohols to T. harzianum increases the activity of BCAs (Connick et al. 1991). The carrier represents the principal portion of inoculants. The materials from which they are made define their effectiveness. Moreover, they have to fill certain requirements in order to be efficient. First, they need to have the capacity to deliver the correct concentration of viable cells at the time they are needed. The reason is because there are certain ranges of concentrations that can be inoculated in certain crops. In addition, as inoculants should be sterile; carriers should be chemically consistent and able to provide enough water-holding capacity for microbial growth (Bashan 1998). Moreover, carriers need to be easily available and able to be mixed with other compounds like nutrients in order to provide a good environment for the live cells. In fact, they need to be easy to mix and easy to fabricate as they are intended to be used massively. Furthermore, they need to be easy to handle and have longer shelf life because they will be used by farmers who will use them periodically and will need to have reservoirs of the products for rapid use (Bashan 1998). Foliar application of fluorescent pseudomonads was attempted by few workers (Gnanamanickam and Mew 1992; Bahadur et al. 2007; Prathuangwong et al. 2013), all of whom used bacterial cell suspension for seed treatment, soil application, or foliar sprays. Use of bacterial suspensions is impractical for large-scale application to control foliar diseases in the field. A powder formulation with longer shelf life would be beneficial (Tables 13.1 and 13.2).

Table 13.1 Commercial formulations of biocontrol agents available in India
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Table 13.2 Commercial formulations of biocontrol agents available worldwide
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13.4 Delivery Systems

Plant-growth-promoting rhizobacteria are delivered through several means based on the survival nature and mode of infection of the pathogen. It is generally delivered through seed treatment, root dip, soil application, and irrigation water. An ideal formulation is expected to facilitate the delivery of the living biocontrol agents in its active state, at the right place, at the right time. While the formulated microbial products must be effective at the site of action and compatible with agronomic practices, they should be easy to apply to and adhere to plant parts such as seeds, tubers, cuttings, seedlings, transplants, and mature plants or be available in the soil medium.

13.4.1 Seed Treatment

Biological formulations applied to seeds greatly help to deliver the agents to the spermosphere of plants, where, in general, extremely conducive environments prevail. The BCAs are therefore provided an excellent opportunity to survive, multiply, persist, and exercise control of soil-borne phytopathogens (Cook and Baker 1983). Seed treatment has the potential to deliver microbial agents “in the right amount, at the right place and at the right time.” With increasing public awareness of the potential environmental and health hazards of both agrochemicals and the advances in biotechnology to improve the performance in microbial products, the application of microbial inoculants to seeds (Chandra and Greep 2010; Chandra et al. 2006) is likely to increase in the future. With an aim to deliver the active ingredients as close to the target as possible, this approach continues to receive considerable attention from end users. Significant advances in seed treatment technology has been achieved due to consistent work done around the globe, and this approach is an attractive means for introducing biological control agents into the soil–plant environment, as these introduced organisms are offered the selective advantage to be the first colonizers of plant roots. At the time of planting seedlings, the formulated products can be used directly (powders, liquids) without stickers. Powders for seed treatment are formulated by mixing an active ingredient, inert carrier to facilitate product adherence to seeds by mixing seeds with formulated product (Woods 2003). Additives such as gum arabic and xanthan gum are used to prolong the survival of microbial agents applied to seeds. Alginate hydrogel, used as a seed encapsulation material, maintains the entity in a viable state and protects it from other stresses. Seed priming, in which seeds are mixed with an organic carrier and then moisture content, is brought to a level just below that required for seed treatment which has been used to deliver T. harzianum to control Pythium-induced damping-off on cucumber (Callan et al. 1990). In another process of seed treatment, an industrial film-coating process which was developed for the application of chemicals and biological crop protection agents is being utilized for the application of Trichoderma spp. on radish and cucumber seeds through a film coating and was shown to be effective against damping-off (Cliquet and Scheffer 1997). Prathuangwong et al. (2013) applied kaolin-based formulation of Pseudomonas fluorescens SP007s as seed treatment and spray to reduce fungal population in rice plants.

13.4.2 Seed Biopriming

Biopriming, a seed treatment system that integrates the biological and physiological aspects of disease control, involves coating the seed with fungal or bacterial biocontrol agents (El-Mougy and Abdel Kader 2008). It is a method of treating seeds with microbial inoculants and incubating under warm and moist conditions until just prior to radical emergence. Priming is one of the simple techniques which improve the vigor, seedling establishment, and plant efficiency in the field. There are three main large-scale priming approaches using different methods to regulate water potential, which are quite popular in European countries: (1) Osmoconditioning: seeds are incubated in an aerated solution of an osmoticum such as polyethylene glycol, or an inorganic salt such as potassium nitrate or phosphate, using high liquid–seed ratio (e.g., 10:1) in stirred bioreactors of various designs. At the end of the process, seeds are rinsed before further processing. (2) Solid-matrix priming technique: seeds are mixed with equivalent quantity of friable, nonclumping, inert material, e.g., a carbonaceous, preferably ligneous shale or coal, with osmotic component at least 90 % of the equilibrium water potential, moistened sufficiently to equilibrate seeds to the correct water content. Extraneous solid material is sieved off after incubation. (3) Basic priming method: incubate damp seeds and bring the seed directly to predetermined moisture content by various means, without using external matrix or osmotic agent to regulate seed water potential (McQuilken et al. 1998). Priming allows the early DNA transcription and RNA and protein synthase which repair the damaged parts of the seeds and reduce metabolic exudation (Entesari et al. 2013). These agents thus improve the seed germination characteristics and the seedling emergence under unfavorable conditions and priming results in a stronger plant. Seed priming can improve the physiological responses and increase seed tolerance to environmental stress (Khan et al. 2008).

This technique is more useful over simple coating of seeds as it results in rapid and uniform seedling emergence. Trichoderma conidia germinate on seed surface and form a layer around bioprimed seeds. These bioprimed seeds tolerate adverse soil conditions better. Biopriming may also reduce the amount of biocontrol agents that is applied to seeds (Ramanujam et al. 2010). Enhancements of seed inoculation with biological agents in combination with priming which will stabilize the efficiency of biological agents have been reported by previous workers (Callan et al. 1990; Warren and Bennett 1999). Nayaka et al. (2008) bioprimed maize seeds with conidial suspension of T. harzianum for the control of F. verticillioides and fumonisins in maize. It was found that the pure culture of T. harzianum was more effective in reducing the F. verticillioides and fumonisin incidence, followed by talc formulation. Biopriming with microbial inoculants is potentially able to promote rapid and uniform seed germination and plant growth. Moeinzadeh et al. (2010) reported the application of UTPf76 and UTPf86 strains of Pseudomonas fluorescens on improving sunflower seed germination and promotion of seedling growth. These bioprimed strains enhanced seed factors such as germination index, germination percentage, germination rate and vigor index, and also seedling growth indices, including root length, shoot height, dry and wet weight of seedlings, and numbers of lateral roots. In biopriming, the selected strains were applied to the seed during osmopriming with NaCl.

13.4.3 Seed Encapsulation

The reproducible results following introduction of microbial inoculants into soil relies on the survival rate of the inoculated microbes in heterogenous soil environment, and it can be achieved by improved encapsulation technique (Young et al. 2006). It is a specialized seed-coating process which involves enveloping the seed, microbes, and possibly few other components such as pesticides or micronutrients, in a gelatinous or polymer gel matrix, thereby prolonging the survival of microbial inoculants on seed. A gel-encapsulation system developed with hard alginate prill to coat or pellet seeds for making formulation of biocontrol fungi (Lumsden and Lewis 1989). The gel-like matrix allows the cell to remain viable with its catalytic ability for longer duration. Alginate forms microbeads immediately in the presence of polyvalent cations by binding the cation to guluronic acid units (Witter 1996) in a single step with a sufficient mechanical strength. Digat (1991) has patented a process to produce granules of up to about 8-mm diameter, with a core containing liquid microbial inoculants and an outer protective coating layer. GEL COAT is an example of seed encapsulation which is an alginate hydrogel product patented as a delivery system for entomopathogenic nematodes (Boyetchko et al. 1999). This method of delivery system has a distinct advantage of being user friendly and environmentally safe, since the active ingredients are effectively sealed until they are released during germination. Major factors that need to be taken care of while adopting this technique are seed inoculum density, coating stability, both for microbes viability and coat integrity, in association with user feasibility and cost of production. Pseudomonas putida CC-FR2-4 and Bacillus subtilis CC-pg104 encapsulated in alginate supplemented with humic acid and inoculated to Lactuca sativa L. seedlings and observed significant plant growth by Rekha et al. (2007). Encapsulation of Bacillus megaterium was attempted by Sivakumar et al. (2014), with bacterial alginate by enriching the bead microenvironment with humic acid, and high viability of encapsulated bacteria with minimum cell loss after 5 months of storage was observed, thereby achieving successful plant-growth promotion of rice seedlings. This novel technique clearly demonstrates that inoculation of encapsulated microbial inoculants promotes plant growth and is feasible for application in agricultural industry.

13.5 Soil Application

Soil treatment is preferred when biocontrol agents are too sensitive to desiccation (Warrior et al. 2002). The biocontrol agent (BCA) establishes a high population in the soil, making them suppressive to the disease. Niche exclusion also becomes operative in such cases, as the increase in number of the introduced microbes renders essential nutrients unavailable to soil pathogens and other less beneficial microflora (Lumsden et al. 1995). Soil acts as repertoire of both beneficial and pathogenic microbes; delivering of microbial inoculants to soil will increase the population dynamics of augmented bacterial antagonists and thereby suppress the establishment of pathogenic microbes on to the infection court. Many species of Trichoderma have also been formulated extensively, using cellulosic carriers and binders and modern thin-film coating techniques, in an attempt to introduce them into the rhizosphere regions of seedlings to protect them from diseases such as Rhizoctonia solani and Pythium ultimum. However, the major limitation of fungi as seed coatings remains; so they do not colonize the rhizosphere as readily as the bacterial agents (Warrior et al. 2002). Numerous attempts have been made to control several soil-borne pathogens by incorporating natural substrates colonized by antagonists of pathogen into soil (Sesan and Csep 1992). Though drenching of soil with aqueous suspensions of bioagent propagules was carried out, there will not be any even distribution of bioagents in the soil. Bankole and Adebanjo (1998) reported that soil drenching with suspension of T. viride was very effective in reducing infection from cow pea seeds infected with Colletotrichum truncatum (brown blotch). Soil drenching with T. harzianum has given good control of stem rot of groundnut caused by S. rolfsii (Kulkarni and Anahosur 1994). An aqueous drench containing conidia of T. harzianum controlled wilt of chrysanthemum by preventing reinvasion by F. oxysporum.

Weststeijn (1990) found that root rot in tulip caused by P. ultimum was reduced by mixing Pseudomonas suspensions thoroughly through the soil to a concentration of 108 cells per gram dry soil before planting the bulbs. Wilt disease of sunflower was found to be suppressed when P. cepacia strain N24 was applied to the seedbeds at the rate of 500 ml per m2 under greenhouse conditions (Hebber et al. 1991).

A technique of enrichment of farmyard manure (FYM) with Trichoderma culture for soil and nursery bed application is widely accepted and appreciated by farmers for soil treatment against soil-borne pathogens. This technique involves less labor and time to multiply Trichoderma culture to manifold for soil application. Vidhyasekaran and Muthamilan (1995) stated that soil application of peat-based formulation of P. fluorescens (Pf1) at the rate of 2.5 Kg of formulation mixed with 25 Kg of well-decomposed farm yard manure, in combination with seed treatment, increased rhizosphere colonization of Pf1 and suppressed chickpea wilt caused by Fusarium oxysporum f.sp. ciceris. Application of Trichoderma harzianum Th3-enriched farm yard manure in soil, along with seed treatment, before sowing of chickpea to ward off against root rot caused by Rhizoctonia solani exceptionally reduce the disease and increased yield (Jambhulkar et al. 2015).

13.6 Foliar Application

Liquid formulations are being commonly applied on foliar parts of the plants for control of foliar pathogens. The efficacy of the foliar application mainly governs by the microclimate of the crop canopy. The crop canopy has varied concentration of nutrients like amino acids, organic acids, and sugars exuded through stomata, lenticels, hydathodes, and wounds. It affects the efficacy and survival of antagonists in phylloplane.

Kelly Cartwright (1995) reported that three spray applications of Pseudomonas cepacia to cuttings during a 2-week period were more effective than either one or two bacterial sprays in the control of Rhizoctonia stem rot of poinsettia. Rice blast (P. oryzae) can be effectively controlled by foliar spray of talc-based powder formulation of P. fluorescens strain Pf1 (1 kg ha−1). The effectiveness of spraying persisted up to 2 weeks. When the bacterial product was sprayed on plants grown from treated seed, the effectiveness was higher than when spraying was carried out without any prior seed treatment (Vidhyasekaran et al. 1997). Foliar application of Pseudomonas chlororaphis (PA-23), Bacillus amyloliquifaciens (BS6), Pseudomonas sp (DF41), and B. amyloliquifaciens (E16) was found very effective against causal agent of stem rot of canola, Sclerotinia sclerotiorum in field (Fernando et al. 2007). Trichoderma species can be applied as foliar spray to control diseases affecting above-ground parts. Biological control of foliar diseases is not so developed as biocontrol of soil-borne diseases. The reasons for the paucity of examples of biocontrol of foliar diseases may be the availability of cheap and effective chemical fungicides and the ease of application to the foliage, and results obtained with biocontrol agents were not so good as those obtained with common fungicides (Elad and Kirshner 1992).

The dosage and frequency of application have to be standardized based on the crop value, which could be a reliable and practical approach. Selected strains from many genera of bacteria isolated from these suppressive soils have the potential to reduce plant diseases when applied to the plant root environment (Weller et al. 2002). Today liquid bioformulations with high potency, cost-effective with good suspension properties, and good stability are available and being successfully adopted globally. Additives are important for application in monocots which facilitates adhesion of microorganisms on plant tissues. Additives such as stickers, spreaders, adjuvants, and emulsifiers in foliar sprays facilitate adhesion of microorganisms on plant tissues (Harvey 1991).

13.7 Root Dip

The nature of pathogen may be seed borne or soil borne; it may establish host parasite relationships by entering through the root. Hence, protection of the rhizosphere region by prior colonization with PGPR will prevent the establishment of host–parasite relationship. Seedling roots can be treated with spore or cell suspension of antagonists either by drenching the bioagents in nursery bed or by dipping roots in microbial inoculant suspension before transplanting. This method is suitable for the vegetable crops and rice where transplanting is practiced (Singh and Zaidi 2002). In an experiment, Jambhulkar and Sharma (2013) dipped paddy seedlings in suspension of talc-based bioformulation of Pseudomonas fluorescens for 2 h before transplanting, and it showed reduction in bacterial leaf blight of rice. Similarly, dipping of rice seedlings in talc-based formulation of P. fluorescens (PfALR1) prior to transplanting reduced sheath blight severity and increased yield in Tamil Nadu, India (Rabindran and Vidhyasekaran 1996). Nandakumar et al. (2001) reported that P. fluorescens strain mixtures by dipping the rice seedlings in bundles in water containing talc-based formulation of strain mixtures (20 g/l) for 2 h and later transplanting it to the main field suppressed sheath blight incidence.

13.8 Fluid Drilling Technology

Fluid drilling, also referred to as fluid sowing or gel seeding, is the technology of sowing seeds that have been germinated, using a gel to suspend and transfer them to the seedbed. This delivery system involves the incorporation of biocontrol agents into fluid drill gels. The major advantage of sowing germinated seed compared to dry seed is earlier and more uniform emergence. The gel protects the exposed radicle from mechanical damage and also provides the growing seedling with an initial water source. Unfortunately, the gel tends to attract microorganisms, including soil-borne pathogens, which may result in an increased incidence of disease. Conway 1986 has used fungicides as adjuvant to the gel matrix to decrease damping-off disease caused by R. solani in chili peppers. Fluid drilling offers an ideal system for the delivery of a biocontrol agent such as Trichoderma for the control of soil-borne disease problems (Fisher et al. 1983). In one study, vegetable or fruit tree seedlings were dipped into gels incorporated with antagonists so that the root area was surrounded by a thin layer of gel before the seedlings were planted. Fluid-drilling gels have been used to deliver T. harzianum for the control of R. solani and S. rolfsii on apple (Conway 1986). This innovative approach, utilizing the benefits derived from fluid drill technology, offers considerable promise for the formulation and application of biocontrol microorganisms. But in future, the technique of fluid drilling will be successful only if sowing of primed seeds rather than germinated seeds are used in carrier gel (Pill 1991). The positional advantage due to additive incorporation in the fluid-drilling gel shows an efficient, cost-effective, and environmentally sound application method for bioagents.

13.9 Microbigation

Applying microbial biocontrol agents to control weeds, soil pathogens, and soil insect through drip irrigation system is called “microbigation” (Boari et al. 2008). The uniform and precise application of microbial particles close to the target organism and to the plant to be protected can increase the success of a biological control treatment. To make acceptability of biocontrol application among farmers, use of systems or technologies that are usually available in agriculture can be modified and enlarge the market. An exploratory drip irrigation system was carried out by Boari et al. 2013, using dripper lines, drippers, filters, and other tools commonly used in irrigation and precision agriculture in the greenhouse to evaluate their suitability for applying microbial biocontrol agents. Conidial suspensions of marketed or marketable agents were used, i.e., Fusarium oxysporum, F. solani, T. harzianum, and Paecilomyces lilacinus. They demonstrated that conidial suspensions (106 conidia ml−1) can pass through the drippers without causing clogging, regardless of their size, and remained viable. A further advantage could be the limitation of the applied doses to the crop root zone and not the whole field, and therefore a reduction of the costs for treatment. Several biocontrol agents could be applied at the soil level through this system, such as mycoherbicides (Charudattan 2001), antagonists (Whipps and Lumsden 2001), and biopesticides (Copping 1999). They can be applied at plant transplanting or through soil drenching or root dip (Alabouvette et al. 1993).

13.10 Coaggregation Assay

Microbial inoculant formulation has a very important effect in the inoculation process as it determines the potential of the bioagents (Bashan et al. 1984). Poor performance of bioformulations in agriculture was reviewed by Van Veen et al. (1997), and suggested to use multiple microbial consortia for multipronged attack against phytopathogens and to thrive together in unique ecological niches in ideal proportions, instead of using a single strain, for a single trait. Coaggregation is a bacteria–bacteria, fungus–fungus, or fungus–bacteria interaction, and the interactions are highly specific that only few or certain species are consortial partners.

Coaggregation was first reported by Gibbons and Nygard (1970), who referred to it as interbacterial aggregation, and it was readily observed with naked eyes (Cisar et al. 1979). Coaggregation is effective only when equal numbers of partners are present and genetic stability of coaggregation is mediated by surface components that recognize a carbohydrate on the cell of the partner (Kolenbrander and Phucus 1984). Coaggregation has been reported earlier among certain bacterial species. Bougeu and Mc Bride (1976) and Kolenbrander and Phucus (1984) reported that Actinomyces viscosus T14V and Streptococcus sanguis 34 co-aggregated by a mechanism which is not inhibited by 1 M NaCl and is independent of dextran, requires calcium and pH in the range of 8.0 to 8.5. Recently, Sivakumar and Joe (2008) attempted coaggregation of Azorhizobium caulinodans with A. brasilense, A. chroococcum, Bacillus megatherium, and Pseudomonas fluorescens to develop coaggregates with multiple benefits using seed powders of many plants, viz., Moringa oleifera, Strychnos potatorum, and Sappindus emaignatus. There is wide scope of using coaggregates to deliver microbial inoculants for obtaining multiple benefits in different crops against various soil-borne pathogens. Studies on coaggregates open up the possibilities for further investigation of the genetic basis of effective coaggregation and also the nature of cellular mechanism.

13.11 Consortia Application

Judicious use of microbial inoculants as biocontrol agent (BCA) is a potentially important component of sustainable agriculture. The principal biocontrol mechanism involved includes mycoparasitism, antibiosis, competition, and induced resistance; additional mechanisms are hypovirulence mediated through fungal viruses and inhibition of enzymes involved in plant pathogenicity (Kapat et al. 1998). Individual biocontrol mechanism could be predominant for some BCAs, but there are also many instances where more than one mechanism may operate in a given BCA isolate. The ecological processes determining the fate of such biological control are complex. Thus, it is not surprising that, in addition to variable control efficacies, biocontrol success in field crops has been limited despite much research effort. Most biocontrol success has been achieved in greenhouse cultivation (Paulitz and Belanger 2001), where ecological parameters are less variable. Because of the inconsistent or limited biocontrol achieved in the field, BCAs have also been used in combination with fungicides or cultural practices (Shtienberg and Elad 1997).

Use of mixtures of cultivars (Mundt 2002) or fungicides (Brent and Hollomon 2007) has been successfully adopted in many crops to increase and maintain disease control efficacy when individual cultivars or fungicides may not be able to control disease effectively. To improve biocontrol efficacies achieved through the use of a single BCA, there has been increasing interest recently among researchers in using mixtures of BCAs to exploit potential synergistic effect among them (Xu et al. 2011). Number of biocontrol mechanism may operate in mixed BCA populations, and we need to consider both direct and indirect interactions between different BCA populations. Compared with the more efficacious BCA, combined use of two or more BCAs may lead to increased, reduced, or similar biocontrol efficacy (Xu et al. 2011).

Biocontrol agents applied individually are not likely to perform consistently against all pathogens of the crop or under diverse crop conditions. A combination of biocontrol agents is more likely to have a greater variety of traits responsible for the suppression of one or more pathogens, and also it is likely to have these traits expressed over a wide range of environmental conditions (Crump 1998). Numerous studies (Meyer and Roberts 2002; Roberts et al. 2005) have reported increased performance in the suppression of pathogens or disease by combinations of biocontrol agents. Incompatibility among microbes combined in biocontrol preparations is possible since biocontrol agents are typically selected based on their antagonistic behavior towards other microbes (Leeman et al. 1996; Meyer and Roberts 2002). Several researchers have indicated that strains combined in biocontrol preparations must be compatible for increased disease suppression to occur (Raupach and Kloepper 1998; Roberts et al. 2005). Accumulating evidence from literature has shown that compatible multiple strains appear to be an important prerequisite for the desired effectiveness of strains and more consistent disease suppression (Ganeshmoorthi et al. 2008). Compatible strains of P. fluorescens (Pf1, Py15 and Fp7) and Bacillus subtilis strains (EPCO 16 and EPC 5) were found to effectively inhibit the growth of Alternaria solani in tomato crop (Sundaramoorthy and Balabaskar 2012). Similarly, experiments for the biological control of the bacterial blight pathogen revealed that different species of Bacillus applied to rice plants as a seed treatment before sowing, a root dip prior to transplantation, and two foliar sprays prior to inoculation could afford up to 59 % suppression of the disease. These treatments could also bring about a twofold increase in plant height and grain yield (Vasudevan and Gnanamanickam 2000). Efforts are in progress, including formulation of synergy hypothesis in relation to biocontrol mechanism to exploit microbial mixture for uses in biocontrol of plant diseases.

13.12 Conclusion

Today in the light of the growing concern towards environmental safety, suppression of plant diseases through biocontrol agents is gaining ground as an alternative to traditional disease management strategies. Now it is necessary to focus on the challenges involved in testing, formulating, and delivering newer potential biocontrol agents within the context of integrated disease management. Undoubtedly choosing correct microbial inoculants is the foremost factor governing the success of biocontrol program. But making it reach to the field with a suitable delivery method maintaining consistent performance is the next most important challenge. Thus, the major challenge for plant pathologists is to develop a specific delivery system for a particular bioagent against a specific pathogen. There is a trend prevalent among farmers to use or apply a single biocontrol agent, but research in the area of consortia application with multiple mechanism of disease control against more than one pathogen is the need of the hour. Also, future research strategies should emphasize on achieving viable and stable biological product. In addition, future research should be attempted to develop a systematic approach to select a suitable method of delivery system based on the characteristics of the microbial inoculants. Another research area in the wake of less labor and increasing mechanization in agriculture, a model of pilot irrigation system such as microbigation, needs to be invented for applying microbial inoculants in field level to the diseased plant in the form of viable spores or fungal conidia through drippers. Overall, a multifaceted management program will require helping the end user to grow a disease-free crop.