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augmented effect and several other advantages, such as non-toxic and bio- ... Resistance to phytopathogens can be obtained by genetic engineering, such as by ... microbial cells and enzymes, microbial entrapment, alginate formulations and ... pesticide poisonings is approximately 67 thousands and 0.5 million per year ...
Chapter 3851

BIOPOLYMER BASED BIOCONTROL STRATEGIES AGAINST PHYTOPATHOGENS: NEW DIMENSIONS TO AGRICULTURE Surinder Kaur1,2, Gurpreet Singh Dhillon2, Mausam Verma3, Satinder Kaur Brar2*, Vijay Bahadur Chauhan1, and Ramesh Chand1 Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras, Hindu University (BHU), Varanasi-221005, India1 INRS-ETE, Université du Québec, 490, Rue de la Couronne, Québec, Canada G1K 9A92 Institut de recherche et de développement en agroenvironnement inc. (IRDA), 2700 rue Einstein, Québec (Québec), G1P 3W8, Canada3

ABSTRACT Phytopathogenic fungi have always been a major concern to agriculture that has led to several epidemics in the past and cause continuing economic losses. In order to maintain the pressing demand of increasing population, the management of such phytopathogens becomes the priority to ensure increased yield. The use of agrochemicals from decades has led to environmental pollution, toxicity and biomagnification. Increasing number of pesticide and fungicide resistance strains has become a matter of serious concern. The focus on alternative and controlled release to such chemical is gaining substantial importance in perspective of cost-effective and sustainable agriculture. In the current scenario, biopolymers have gained widespread interest with the potential to revolutionize agriculture. Polymers of living origin are termed as biopolymers which include cellulose, starch, chitin, chitosan, oligomers of chitin and chitosan, laminarin, carrageenan, proteins and peptides. Biopolymer play diverse role, such as in metabolism, nutrition, parasitism and defence mechanism against phytopathogens. They act via different mechanisms, for example, activating plant innate immunity, enhancing signal perception and transduction,

*

Corresponding author: E-mail ID: [email protected]; Phone: 001-418-6543116)

2 Surinder Kaur, Gurpreet Singh Dhillon, Mausam Verma et al… expressing the defensive genes and inducing accumulation of secondary metabolites against phytopathogens. The combination of biopolymers with biocontrol agents is gaining interest due to augmented effect and several other advantages, such as non-toxic and bio-degradable nature, wide spectrum antimicrobial activity and stability, ease of manipulation, large scale production, storage and transportation. The combination of these environmental friendly properties makes them an ideal candidate against many phytopathogens and as an alternative to environmentally harmful agro-chemicals. Resistance to phytopathogens can be obtained by genetic engineering, such as by cloning genes specific for pathogen like chitinases or and β-glucanases or by amending the bio-formulations with naturally occurring biopolymers. Manufacturing different bioactive molecules from these biopolymers may aid in improving and strengthening plant defence. In this chapter we will discuss the efficacy of different biopolymers such as chitin, chitosan, laminarin, carageenan among others and their biocontrol mechanism. We will also discuss various modes of application of these biopolymers with various biocontrol agents in order to achieve best results. For instance, immobilization of microbial cells and enzymes, microbial entrapment, alginate formulations and encapsulation of bio-control agents shall be discussed. Keywords: agriculture, β-glucanase, biocontrol, bioformulations, biopolymers, chitin, chitosan, laminarin, phytopathogenic fungi.

INTRODUCTION There are approximately 50,000 species of plant pathogens, 9,000 species of insects and mites and 8,000 species of weeds that cause 13%, 14% and 13% loss to crops worldwide, respectively [1,2]. It has been estimated that pesticide free crop production would increase the loss in fruits, vegetables and cereals due to pests damage upto 78%, 54% and 32% respectively [3].The consumption of fungicides/bactericides on maize in United States rised $6 million to $130 million from 2005 -2007 [2]. Annual application of chemical pesticides has reached up to 4.6 million tons worldwide. If the amount of sprayed pesticide is 100%, than only 1% is effective while the remaining 99% escapes to the non-targeted parts of the ecosystem such as soils, water bodies and the atmosphere, and ultimately into the food chain [2]. A report from World Health Organisation (WHO) and United Nations Environment Programme (UNEP), states that human pesticide poisonings is more than 26 million causing the deaths of around 2,20,000 individuals per year [4]. In United States and China, human pesticide poisonings is approximately 67 thousands and 0.5 million per year respectively. The growth and development of human population has lead to improvement and advancement in agriculture as well as industrial revolution which in turns has invited several negative effects such as environmental pollution, loss of bio-diversity, surface run-off of chemical fertilizers and pesticides, leading to surface and groundwater contamination throughout the globe. In order, to fulfill the increasing demands of food and agricultural commodities, the use of chemicals and fertilizers has also increased that not only disrupts the food chain and cause environmental pollution but also lead to bio-magnification and harmful human health hazards such as change in endocrine functions and immune systems, organ failure, poisioning and genetic disorders in humans as well as animals.

3 Biopolymer Based Biocontrol Strategies against Phytopathogens: New Dimensions… The current agricultural practices for increasing the production, such as non-judicious application of chemical pesticides and fertilizers have imposed a long list of environmental and health hazards [5, 6]. Moreover, emergence, re-emergence and endemic plant pathogens pose a serious threat to agricultural crops [7]. It has been reported that, inspite of widespread exploitation of chemical biocides in crop production, the losses due to pests and diseases are noteworthy. This mandates the need of efficient ecologically compatible strategies in agriculture in order to manage the phytopathogens. Figure 1 describes various strategies that can be formulated in various traditional as well as advanced formulations. Besides being highly efficient, an ideal pesticide should be bio-degradable and ecofriendly. This implicates the importance of biopesticides in plant disease management in an eco-friendly manner. Biopesticides are of microbial origin and their metabolites inhibit the growth and development of plant pathogens. Several biopolymers, have gained considerable interest in various sectors such as biotechnology industry, medical technology, genetic engineering, biofertilizers, biocontrol agents (BCAs) against various phytopathogens, municipal waste treatments and many others. The development and advancement of microbiology, agricultural microbiology, genetic engineering, molecular tools, bioinformatics and metagenomics has lead to the utilization of biopolymers particularly of microbial origin. Table 1 describes some of the most important benefits in using biopolymers or biopolymer mediated biocontrol in plants. Various biopolymers have been discovered with the potential to mitigate the undesirable effects of phytopathogenic microorganisms in agriculture and forestry. The use of bio-control agents (BCAs) along with the biopolymers with some specifications has the strong potential to bring down their impact below threshold level. Biopolymer based pesticides and fungicides has been harmonizing or even substituting their chemical counterparts.

MICROORGANISMS

Biopolymers üchitin üchitosan ülignin üEPS ülaminarin ücarrageenans üulvan

Solid formulation üGranules üwettable ülyophillised powders üAlginate beads

Secondary metabolites/ Antibiotics

Enzymes üendochitinases üexochitinases üglucanases

FORMULATIONS

Liquid formulation üSuspensions ühydrogel üMicroencapsulation üemulsions

Figure1. Various strategies for biocontrol that can be formulated against phytopathogens.

4 Surinder Kaur, Gurpreet Singh Dhillon, Mausam Verma et al… Resistance development in many microorganisms against various agro-chemicals has prompted the search for other alternatives for disease management. The bio-degradable nature of these natural biopolymers such as chitin, laminarin, glucanases and chitosan makes them extremely suitable for agricultural applications. Substitute for synthetic chemicals with ecofriendly and degradable biocides have become a major concern worldwide. Table 1. Advantages of utilizing biopolymer based biocontrol. Used directly against phytopathogens. Can be mixed with different biocontrol agents, antibiotics, chemical fungicides (in particular azole and other cellmembrane affecting compounds) Can be used in combinations. Act as inducer of antagonistic mechanisms. Function as elicitor of plant defence system. Formulation production can be readily manipulated and regulated at an industrial level. Final product is comparatively stable, easy to store and transport. Much less restrictive as compared to live or whole organism for commercialisation and utilization. Major concerns are eliminated: Production of high quality of pure propagules. Ability to survive downstream manufacturing and processing (drying for formulation). Sufficient shelf-life. Resistance to variable environmental conditions in the field (temperature, water, pH, light etc.). Production can be selectively induced and enhanced by controlled variation of the culture growth conditions (substrate components, pH and temperature).

CHITIN Chitin is extensively distributed in nature and the most abundant biopolymer existing on the earth next to cellulose which is produced by the plants. It occurs predominantly in the form of structural polysaccharide in fungal cell wall, outer shell of crustaceans, exoskeleton of arthropods, and nematodes [8]. It is a natural bio-polymer of unbranched chains of β-(1,4)linked 2-acetamido-2-deoxy-Dglucose (GlcNAc; N-acetylglucosamine; NAG) that makes a major structural component of most fungal cell walls. GlcNAc is a component of an asparagine-linked oligosaccharide of glycoprotein [9]. Chitin comprises approximately 2058% of the dry weight of the 75% of the total weight of crustaceans (shrimp, crab and krill) which is generally considered as waste [10]. Fungi belonging to ascomycetes, zygomycetes, basidiomycetes and phycomycetes are rich in chitin which is widely distributed in their cell walls, structural membranes of mycelia, stalks, and spores. The amount of chitin present in fungi varies from traces up to 40-45% of the organic fraction, while the remaining mostly contains proteins, glucans, lipids, and mannans [11]. The variation in the amounts of chitin present in the microbial cells generally depends on physiological changes in the natural environment. Chitin is the chief component in primary septa between mother and daughter cells of S Saccharomyces cerevisiae and accounts for only 1-2% of dry cell weight of yeasts [12]. Degradation of chitin at the bud site leads to cell separation and involves an extremely glycosylated endochitinases with an apparent molecular mass of approximately 130 KDa [13].

5 Biopolymer Based Biocontrol Strategies against Phytopathogens: New Dimensions… Among fungi, zygomycetes and basidiomycetes posses maximum amount of chitin in their cell wall particularly the members of the family Mucoraceae that posses chitosan along with chitin in their cell walls. Fungal chitinases are known to play key roles in nutrition, autolysis and morphogenesis while viral chitinases are mostly involved in pathogenesis [14]. Bacillus, Pseudomonas, Streptomyces, Paenibacillus, Stenotrophomonas, Aeromonas, Serratia and fungi from the genera Gliocladium and Trichoderma are known for extensive chitinase production. Chitin exists in three polymorphic forms that differ in the arrangement of molecular chains within the crystal shell as revealed by X-ray diffraction studies. These are α-, β-, and γchitins where α-chitin chains are arranged in an antiparallel fashion while β-, and γ-chitins are arranged in parallel and mixed forms respectively [15]. Chitin is hydrolysed by two major categories of enzymes: Endochitinases (EC 3.2.1.14) that cleaves chitin randomly at internal sites, producing soluble low molecular weight multimers of GlcNAc, the dimer N,Ndiacetylchitobiose, chitotetraose and chitotriose. Another enzyme is exochitinases that is further sub-divided into chitobiosidases (EC 3.2.1.29) and β-(1, 4) N-acetylglucosaminidases (EC 3.2.1.30). Starting at the nonreducing end of chitin microfibril, chitobiosidases catalyzes the progressive release of diacetylchitobiose whereas; β-(1, 4) N-acetyl glucosaminidases cleaves the oligomeric products of endochitinases and chitobiosidases, generating monomers of GlcNAc [16]. During the last decade, interest in chitinases has gained attention because of their wide range of applications. They have been utilized in a wide range of applications in food, biochemical industry, wastewater treatment, drug delivery, wound healing, dietary fibre and several chemical industries. They are known for their anti-microbial, anti-cholesterol and anti-tumor activities [17, 14]. Chitinases play a crucial function in plant resistance against various fungal pathogens owing to their antifungal properties and resistance induction [18]. Microbial chitinases of different origin are commercially available while at the same time, the feasible in-house bioproduction of chitinases can be carried out by using various negative costs agro-industrial wastes. Chitin functions as fibrous-strengthening element in fungal cell wall via strong hydrogen bonding, thereby providing rigidity. The fungal cell wall is rich in chitin, polysaccharides, and thus, their glycosidic bonds can be targeted for its inhibition by using biopolymer based biocontrol strategy (BBBS) against phytopathogens. Disruption of the glycosidic bonds results in weakening of the cell wall and disruption of cellular integrity, ultimately leading to leakage of cellular components. Chitinases can thus be used against phytopathogens either directly, indirectly using purified protein or through gene manipulation [19, 20, 21, 22].

Chitin Biosynthesis During chitin biosynthesis, monomers of GlcNAc are coupled in a reaction catalyzed by the membrane-integral enzyme chitin synthase, a member of the family 2 of glycosyltransferases. The molecular weight (MW) of the chitin depends upon the chain length which is influenced by the activity of chitin synthase. The polymerization requires uridino-di phosphate (UDP) and GlcNAc as a substrate and divalent cations as co-factors. Chitin biosynthesis occurs in following three distinct steps: (1) the catalytic domain of chitin

6 Surinder Kaur, Gurpreet Singh Dhillon, Mausam Verma et al… synthase facing the cytoplasmic site forms the polymer; (2) the translocation of the nascent polymer across the membrane and its release into the extracellular space; and (3) single polymers spontaneously assemble to form crystalline microfibrils of varying diameter and length [23].

Microbial Chitinases Several strains of ecto and endophytic actinomycetes are known to produce chitinases, such as, Streptomyces aureofaciens, that functions as potential BCA against many phytopathogenic fungi [24, 25, 26]. The antagonistic potential of Streptomyces has been attributed to the production of extracellular hydrolytic enzymes and antifungal metabolites [27, 28, 29, 30]. Among different hydrolytic enzymes, the two most important are, chitinases and β-1, 3-glucanases that have been effectively used in fungal cell wall lysis, such as in, Fusarium oxysporum, Sclerotinia minor, and Sclerotium rolfsii [31, 32]. Despite the fact that different actinomycetes might posses an array of different chitinases, which can be used efficiently against various phytopathogens, data pertaining to this is very limited or rather inconsistent. In depth studies are needed to evaluate the role of actinomycetes in BBBS in order to attain efficient management of plant pathogens. Various strains of Enterobacter spp., such as Enterobacter cloacae, E. aerogenes and E. agglomerans have been described as efficient BCAs against many phytopathogenic bacteria and fungi [33, 34]. E. cloacae have been reported to posses the maximum potential of chitinolytic enzymes [35, 24]. They are known to control different plant diseases, such as, rots and pre-emergence damping-off, wilt, crown and root rot incited by Pythium spp., Fusarium spp., Phytophtora cactorum etc., in pea, beet, cotton, cucumber and apple etc [36, 37]. The potential of inhibiting different phytopathogenic fungi has been attributed to the competition for nutrients and rhizosphere colonization, production of different volatile and nonvolatile antifungal metabolites, antibiotic-like substance and hydroxamate type siderophores by different strains of Enterobacter like E. aerogenes B8, E. cloacae and E. agglomerans [33, 38, 34]. In addition to the above described application of chitin, they are also involved in the synthesis of chitosan and chitooligosaccharides that functions as important anti-microbial agents, elicitors of lysozyme inducers, and immune-enhancers [39]. Both chitin and chitosan are non-toxic, biocompatible, biodegradable, and induce defence responses in plants [40]. However, insolubility in neutral aqueous solution and their high viscosity hampers their efficient utilization.

CHITOSAN Chitosan, the deacetylated derivative of chitin is an important constituent of the cell wall at various times during the life cycle of some fungal species [41]. Chitosan is not directly synthesized rather the deacetylase enzyme convert chitin to chitosan. According to some researchers, chitosan is a polymer having atleast 60% of D-glucosamine residues and degree of deactylation usually exceeds to 80% [42]. Chitosan being polycationic, non-toxic, bio-

7 Biopolymer Based Biocontrol Strategies against Phytopathogens: New Dimensions… degradable, bio-compatible as well as anti-microbial, has many applications especially in the agriculture, food, biomedicine and pharmaceutical industries. It has been used in enzyme immobilization, wastewater treatment, as a food additive and anti-cholesterolemic, for wound healing, and in pharmaceuticals in several drug delivery systems [43, 44]. N-deacetylation is a common step in the modification of sugar moieties, which may confer resistance to lysozyme action [45]. Chitin deacetylases (EC 3.5.1.41) are thought to be in close proximity to the regions where chitin traverses the plasma membrane [46]. As chitin is synthesized, the deacetylase enzyme converts it to chitosan. Chitosan is a polycation with higher solubility than neutrally charged chitin.

Chitosan Biosynthesis The formation of chitosan in fungal cell walls occurs in a multifaceted synergistic manner of two enzymes: (1) chitin synthase and; (2) chitin deacetylase. In the first step, chain of chitin is synthesised by chitin synthase using the chitin precursor, UDP-Glc-NAc. In the second step, hydrolysis of acetic groups from N-acetylglucosamine (GlcNAc) by the enzyme chitin deacetylase thereby, transforming it into glucosamine (GlcN) and finally forming chitosan [46]. In fungal cells, chitin deacetylases plays an important function in the chitosan biosynthesis. The mycelium of several fungi, such as Mucor rouxii, Absidia glauca, Aspergillus niger, Gongronella butleri, Pleurotus sajor-caju, Rhizopus oryzae, Lentinus edodes and Trichoderma reesei have been considered as possible sources of chitin and chitosan due to their presence in the cell walls [47, 48, 49, 50] . Chitosan also occurs naturally in some fungi (Mucoraceae) [51]. Several fungi contain deacetylases such as A. nidulans, Colletotrichum lindemuthianum, M. rouxii and R. oryzae [52]. Fungi belonging to the class Mucorales synthesize chitosan during their vegetative growth phase [53], whereas S. cerevisiae synthesizes chitosan only during the sporulating stage [41]. The functioning of deacetylases is critically inhibited by the substrate (chitin) insolubility. In vitro attempts failed to isolate acid-soluble chitosan by using amorphous chitin containing high degree of acetylation (DA) as a substrate for the deacetylase enzyme [54]. This has imposed a practical barrier for the synthesis of chitosan using deacetylases where in vivo activity is already achieved. Recently, a new chitosan formulation has been developed against wide range of foliar pathogens. Iriti et al. (2011) [55] evaluated the efficacy of a new chitosan formulation (Kendal Cops® (Kc)) against powdery mildew infection in grapevine using different Kc dilutions or with Kc alternated or mixed with fungicides, or with fungicides alone (penconazole and methyldinocap). There results suggested the efficacy of Kc for the management of powdery mildew at lowest concentration of 0.1% even under the conditions of high disease severity. Total phenol content and antioxidant activity was significantly higher in all Kc-treated grape tissues as compared to untreated and fungicide-treated grapes. The chitosan mediated BBBS presents an efficient and reliable alternative to chemical based pesticides and fungicides. Besides acting as antimicrobial agent in plant disease management, chitosan have various applications in bloom and fruit-setting stimulation, frost protection, plant growth promotion, post-harvest protection of fruits and vegetables, and the

8 Surinder Kaur, Gurpreet Singh Dhillon, Mausam Verma et al… controlled release of fertilizers and nutrients in the soil [56, 57, 58]. Chitosan mediated induction of plant defence responses have been demonstrated in various economically important crops such as, tomato, cucumber, strawberry, chilli, rose, germinating peanut, legumes and solanaceous plants [59]. The principle mechanism involved in the induction of plant defence response is elicited by chitosan is the production of different hydrolytic enzymes such as chitinases, pectinases and glucanases, and the synthesis of phytoalexins [60, 61]. Figure 2 describes biopolymer mediated biocontrol mechanism in plant encountered by plant pathogens. PAMPs/ MAMPs

PATHOGEN

B I O P O L Y M E R S

Induction & modulation of key enzymes of phenylpropanoid pathway

Disruption of cellular integrity PCD Immunoenhancers

üAnti-viral üActivation of plant innate immunity (SAR, ROS, RNS, HR) üModulation of JA and SA pathways üResistance to secondary infections üOxidation of phenolic compounds üPrecursor of secondary metabolites

Elicitors Deposition of Ca ion dependent callose synthase

PLANT DEFENCE

Figure 2. Biopolymer mediated plant defence mechanisms challenged by the pathogen.

Chitosan have been reported to increase the key enzymes of the phenylpropanoid pathway (phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL)) in soybean leaves and sweet basil (Ocimum basilicum L.) [62, 61]. PAL and TAL enzymes are the precursors of secondary metabolites (phytoalexins, lignin and flavonoids) that play key role in plantpathogen interactions. Recently, Chitogel, a derivative of chitosan, has been demonstrated to exhibit positive effect on net photosynthesis and grapevine physiology. The results showed 2fold increase in the average O2 production and 1.5-fold increase in CO2 fixation in grapevine plantlets cultured on medium supplemented with 1.75 % Chitogel [63].

Chitosan as Anti-Viral The role of natural or synthetic compounds in the activation of the plant innate immunity is one of the most important non-genetic strategies mainly against viral diseases [64]. Recognition of pathogen/microbe associated molecular patterns (PAMPs/MAMPs) such as, proteins, lipopolysaccharides, flagellin, fungal cell wall fragments, peptidoglycans, and lipid derivatives (conserved products of basic pathogen metabolism) leads to the induction of plant innate immunity by binding to a plant specific pattern recognition receptors (PRRs) [65,

9 Biopolymer Based Biocontrol Strategies against Phytopathogens: New Dimensions… 66].The whole process lead to the activation of well renounced cascade of molecular signals such as Ca2+ influx, generation of reactive nitrogen species (RNS) and reactive oxygen species (ROS), activation of hypersensitive response (HR), induction of salicylic acid (SA) ultimately leading to systemic acquired resistance (SAR). The role of chitosan in the deposition of a Ca2+-dependent callose (β-1, 3-D-glucan) synthase that functions in restricting the virus is well established [67]. Callose apposition in pores of phloem sieves and extracellular deposition of callose around the plasmodesmata restrains the transportation of viral particles through phloem vessels and cell-to-cell transport of viral particles through plasmodesmata, respectively [68]. Exogenous application of absisic acid (ABA) induces plant defence mechanism and callose deposition. Callose apposition contributes in developing plant resistance and Chitosan-elicited callose apposition has been reported in various plant species and mechanistically correlated to the biological activity of different chitosan polymers [69, 70, 71]. In a study conducted by Iriti and Faoro (2008) [72] Phaseolus vulgaris- tobacco necrosis virus (TNV) pathosystem, the chitosan application elicited callose apposition and ABA accumulation. They also showed that the treatment with ABA inhibitor nordihydroguaiaretic acid (NDGA), before CHT application inhibited callose deposition and plant resistance to the TNV infection. However, callose deposition imparts only a partial barrier in virus spreading and therefore must be cumulated by other defence mechanisms.

Chitosan in the Induction of Defensive Genes The activation of defence genes involved in plant defence mechanisms, such as gene encoding PAL and protease inhibitors are induced by chitosan [73, 74]. Chitosan plays an important role in the induction and modulation of jasmonic acid (JA) pathways via activation of genes encoding PAL and protease inhibitors [75, 73]. Activation of defence genes has been demonstrated in various crops such as rice, slash pine, and tomato [76, 77]. The antifungal potential of chitosan can be attributed to its ability to disrupt the fungal cell wall biosynthesis; modulating the plant resistance and altering the ability of pathogens to cause infection. Application of chitosan reduces the oxidative stress and increases thein polyphenol oxidases that results in the oxidation of phenolic compounds and thereby, enhances resistance to pathogens [61, 78]. Preincubation of suspension-cultured wheat cells in a growth medium of Pantoea agglomerans with chitin or chitosan led to a strong increase in extracellular peroxidase activity [79].

Chitosan Acts as Microbial Associated Molecular Patterns (MAMPs) in the Search of Pathogenesis Related Proteins (PRR) Chitosan acts as general elicitor and results in the induction of non-host resistance and activation of SAR. SAR proteins include antifungal chitinases, β-1, 3-glucanases, PR-1 and PR-5 and confer resistance to plants against secondary infections [80]. PR proteins play an important role in plant defence systems by degrading cell wall of phytopathogenic fungi [81, 82]. The activity of chitin deacetylase influences the degree of acetylation (the content of

10 Surinder Kaur, Gurpreet Singh Dhillon, Mausam Verma et al… GlcNAc groups) in the chitosan chain. Chitin synthase and chitin deacetylase also affects the properties of nascent chitosan, such as the DA and molecular weight. The activity of these two enzymes are known to be largely influenced by the presence of various metals ions and compounds in the growth medium. Different derivatives of chitosan, for example, N,O-acyl and N-alkyl, N-aryl and thiourea posses insecticidal and fungicidal activity [83, 84, 85]. Chitosan exhibits anti-microbial activity against various microorganisms such as, fungi, yeast and bacteria [86, 87, 88].

GREEN SYNTHESIS OF CHITIN AND CHITOSAN Wide range of chitin/chitosan applications in plant defence, sustainable and economical bio-production of these biopolymers is sought rather than using from commercial sources. Conventionally, on an industrial scale, chitosan is mainly derived from the waste product of crustacean exoskeletons obtained after the industrial processing of seafood, such as shrimps, crabs, squids and lobsters shell by chemical deacetylation by using hot concentrated base solution (40-50% w/v) for several hours [11]. However, the chitosan obtained by such treatments suffers some inconsistencies, such as protein contamination, inconsistent levels of deacetylation and high molecular weight which results in variable physico-chemical characteristics. There are some additional problems, such as environmental issues due to the large amount of waste concentrated alkaline solution, seasonal limitation of seafood shell supply and high cost [89]. In this context, production and purification of chitin/chitosan from the cell walls of fungi grown under controlled conditions offers advantage of being environmental friendly and provides greater potential for consistent product. Fungal mycelium can be obtained by simple fermentation regardless of geographical location or season. The extraction of chitosan is achieved by mild alkaline and acidic treatments as compared to chemical extraction methods and this can be generally considered as green approach. Moreover, fungal mycelia have lower levels of inorganic materials compared to crustacean shells, and no demineralization treatment is required during processing [90].The physico-chemical properties and yields of chitin/chitosan isolated directly from a fungus can be optimized by controlling fermentation and processing parameters. Furthermore, β-glucan, that can be isolated from the mycelia chitosan-glucan complex, has important applications in biomedicine [91]. Fungal biomass can be produced by solid-state fermentation (SSF) and submerged fermentation (SmF). SmF has specific advantage as this fermentation method provides easier control of fermentation parameters, such as pH, temperature and nutrient concentration in the fermentation medium. However, SSF is known to produce larger quantities of biomass as compared to SmF. Generally, the media reported for chitin/chitosan production from fungi contains yeast extract, D-glucose, and peptone. Recently, studies have been focused on the utilization of inexpensive carbon sources, such as biowastes for culturing fungi for chitin/chitosan production [92, 93, 94, 95]. Fermentative production of chitin/chitosan by culturing fungi on inexpensive bio-waste is an infinite and economical source. Considering the significant amounts of fungal-based waste materials accumulated from biotechnological and pharmaceutical industries, and the cost involved in managing the wastes, extraction of

11 Biopolymer Based Biocontrol Strategies against Phytopathogens: New Dimensions… highly functional value-added products, such as chitosan may provide a lucrative solution to these industries.

LIGNIN Lignins are complex phenolic polymers that are obtained from abundant renewable resources present, such as plants, trees and agricultural crops. Lignin is one of the widely distributed natural non-carbohydrate organic compound present in fibrous materials and second most abundant biopolymer on earth. It is an important component of woody plants and cereals and is known to play an important function in plant growth and development, cell wall strengthening and protection against various abiotic and biotic stresses.

Structure of Lignin Lignin is a constituent of the cell wall of various cell types of plants, such as wood fibres, vessels, and tracheids and constitutes up to 20–30% of the total weight of wood. Lignin encrusts lignocellulosic biomass as an amorphic mass of the cellulose fibres. Lignification of the plant cell wall provides high mechanical strength to the cell and also increases the resistance to microbial degradation. Structurally, lignin is a three-dimensional phenylpropanoid polymer mainly linked by ether bonds between monomeric phenylpropane units most of which are not readily hydrolyzable [96].. Generally, lignin biosynthesis includes three primary precursors, i.e. trans-coniferyl, trans-sinapyl and trans-p-coumaryl alcohols [97]. They differ in molecular composition and linkage type between the phenylpropane monomers, p-hydroxyphenyl, guaiacyl, and syringyl units derived from coniferyl, sinapyl and coumaryl alcohol precursors, respectively. As compared to other naturally occurring polymers, lignin is extraordinary in terms of low degree of order and high degree of heterogeneity in structure not only among plants of different genetic origin but also between different tissues of the same plant [98].Heterogeneity in the structure of lignin has been attributed to the variations in the polymer composition, size, cross-linking and functional groups [99]. Difference in structure of lignins exists mainly due to the presence of three alcohol units in different proportions. Hardwood lignins are made up of coniferyl alcohol (56%), sinapyl alcohol (40%) and pcoumaryl alcohol (4%). In hardwoods and dicotyl crops, like flax and hemp, and monocots like grass and cereals straws various ratios of coniferyl/sinapyl and p-coumaryl units [100] . In softwood lignins, proportions of different alcohol are mainly derived from approximately 80% coniferyl, 14% p-coumaryl and 6% sinapyl alcohols.

Lignins as Allelochemicals Allelochemicals production is increased under the conditions of biotic and abiotic stress such as diseases, extreme temperature, moisture deficit and herbicides. The allelopathic inhibition usually occurs from a combination of allelochemicals that affect various physiological processes in receiving plant or microbes [101]. Allelochemicals are generally transmitted from one plant to another in a terrestrial community as aqueous leachates, volatile

12 Surinder Kaur, Gurpreet Singh Dhillon, Mausam Verma et al… compounds or various exudates. Volatile allelochemicals reach to the receiving species from a donor plant by different routes such as they may travel through the air, adsorbed on soil particles or in the solubilised form in the soil solution. Similarly, water-soluble allelochemicals are known to leach from shoot tissues to the soil matrix or in the form of exudates from roots. Hence, spatial movements of allelochemicals can be within a short distance. The soil acts as allelochemical reservoir, from where the roots of a plant take up allelochemicals or lipidsoluble compounds adsorbed on soil particles and they can partition directly into root tissue. Plant residues decomposing in the soil will result in localized regions of higher allelochemical concentrations. The impact of allelochemicals in the soil on a receiving plant often depends on the chance encounters of the root system with such region which are rich in allelochemicals. Mostly, the allelopathic agents reported from higher plants are secondary compounds that are formed from either the acetate or shikimate pathway, or their chemical skeleton comes from a combination of these two origins. Popa et al. (2008) [102] described the potential application of lignin and polyphenols as allelochemicals. The authors recommended using these compounds to enhance crop production and develop a sustainable agriculture, including weed and pest control through crop rotations, residue management and various biocontrol approaches. They concluded that flax lignin and ammonium lignosulphonate have a biostimulating effect on cell division (mitosis), in the radicular meristems of P. vulgaris. The authors mentioned the possibility of induction of this process in case of flax lignin, as a result of the improvement of micro-media conditions at plant root level, correlated with the beneficial influence of lignin on the flora present in soil. This effect was found to be doubled by the protective character, especially of ammonium lignosulphonate, materialized in the diminution of the anatelophase frequency at lower level against the control sample. These allelochemicals can be used as herbicides, pesticides and growth stimulants.

EXOPOLYSACCHARIDE BIOFILMS IN PLANT DISEASE CONTROL Several microbes exist as multicellular aggregates generally described as biofilms that are associated with solid surfaces and in close proximity contact with other microbial cells in their natural environment. A biofilm is an aggregate of microorganisms in which cells adhere to each other and/or to a surface, generally produced by archaea, bacteria, and eukaryotic microbes [103]. Generally, bacterial cells exist as biofilms in the environment that are highly structured, surface-attached communities of cells encased within a self-produced extracellular polymeric substance matrix [104, 105]. Biofilm formation is a dynamic process and different mechanisms are involved in their attachment and growth. Biofilms may form on living or non-living surfaces, and represent a prevalent mode of microbial life [106]. Extracellular polymeric substances (EPS), exopolysaccharides, proteins and DNA forms complex matrix that helps in cell adhesion to surface and other cells. Several microorganisms naturally produce variety of biopolymers, such as polysaccharides, polyesters, and polyamides. The physical and physiological properties of biofilms depend on their composition, molecular weight and the polymeric matrix. The primary step in the biofilm formation is the adhesion of microbes through the secretion of

13 Biopolymer Based Biocontrol Strategies against Phytopathogens: New Dimensions… exopolymers. Microorganisms form a biofilm in response to various factors, such as, nutritional cues, cellular recognition of specific or non-specific attachment sites on a surface, or sometimes, by exposure of planktonic cells to sub-inhibitory concentrations of antibiotics [107]. The microbial cells in a biofilm differ physiologically from their planktonic forms that are generally single celled and may float or swim in a liquid medium. Biofilm cells respond to nutrient and waste product, diffusion gradients, modulate their metabolism as a function of their position within the biofilm, contact adjacent cells, and engage in cell to cell communication.

Exopolysaccharides in Plant Disease Management The attachment and colonization of plant surfaces by BCAs is of primary importance in order to achieve efficient biocontrol efficacy. Extracellular polymeric substances or EPS play an important role in the attachment and colonization. Rhizospheric bacteria are generally associated with the root surface and may increase plant yield by different mechanisms such as increasing nutrient uptake, disease suppression and phytohormone production. Bacterial biofilms formed on the plant roots, protects the colonization sites of the plant pathogens. It also reduces the availability of root exudates as nutritional elements for pathogen stimulation or subsequent colonization on the root [108]. For example, Bacillus and pseudomonads are extensively studied as plant growth promoting rhizobacteria (PGPR), BCA and degradation of organic polymers from the soil [109, 110]. Recently, it has been reported that Pseudomonas putida and Bacillus subtilis responds rapidly to the presence of root exudates in soils and forms biofilms on inert surfaces through different transcription factors, thereby, converging at root colonization sites and establishing stable biofilms networks [111, 112]. Kinsinger et al. (2003) [113] and Bais et al. (2004) [114] have demonstrated the biocontrol potential of a wild-type B. subtilis strain 6051 using P. syringae infection model. 3-d structure of B. subtilis biofilm has been shown in an experiment conducted on Arabidopsis root surfaces by treating with B. Subtilis. Haggag et al. (2008) [115] investigated the role of biofilm forming two bacterial strains, Paenibacillus polymyxa in controlling crown root rot disease caused by A. niger. The studies highlighted the importance of biofilms for initiating biocontrol activity. The strain B5 produces strikingly larger amounts of extracellular polymers. Both strains were able to suppress the pathogen but the higher biofilm former, P. polymyxa B5 offers significantly better protection against crown rot. The authors used scanning electron microscopy and solid surface assay to study colonization and biofilm formation ability of the strains. Therefore, biofilms may be envisaged as a new model system for the development of microbial technology thereby, providing insights into microbial biology and ecology. Development of accurate and realistic models of natural communities in vitro provides excellent keys in studying complex biological systems which is presently being speculated. The application of novel probes and improved analytical methods will gradually expand our vision on biofilm structures and the extent to which they are determined by EPS. It will also enhance the knowledge on the interaction of such biofilms with other biopolymers and/or polysaccharides as well as with other macromolecules, cells, ions and low-molecular-mass solutes and provide a multitude of microenvironments within any biofilm.

14 Surinder Kaur, Gurpreet Singh Dhillon, Mausam Verma et al…

LAMINARIN Growing concern regarding the environmental risks has motivated the research for alternative strategies to control plant disease, such as, the induced systemic resistance (ISR). These inducers are known as elicitors, which mimic pathogen attack. Recognition of pathogen is mediated via perception of elicitor molecules that is released during the interaction. Consequently, it leads to the activation of defence cascade reactions, including both chemical and physical defence responses [116]. Different forms of defence reactions include the accumulation of host-synthesized phytoalexins, deposition of phenolics, lignin or callose-like materials, increased activity of PR proteins (e.g., chitinases and glucanases) and induction of HR. Recently, elicitors have also been reported from different biochemical families such as, carbohydrates, lipids, and proteins known as PAMPs or MAMPs. Oligosaccharides of biological origin such as those derived from microbes, algae, or plant cell walls are wellknown MAMPs [117]. Laminarin, a linear β-1, 3 glucan, has been reported as one of the potent elicitors in various plants [118]. It is a reserve carbohydrate source of brown alga Laminaria digitata and composed of β-1, 3 glucan backbone with an average degree of polymerization (DP) of 25 glucose units with 1-3 single β-glucose branches at position [119, 120]. Laminarin was thought to act as an elicitor of plant defence responses. The application of algal extracts rendered crop plants with increased resistance to pests and diseases. Studies have also established the role of fungal β-glucans as efficient elicitors of defence responses in different plant species [121, 122]. It is actively involved in the induction of SAR, activation of plant defence responses and protection against various plant pathogens. However, it doesn’t functions directly against pests and is not associated with any toxic mode of action. Laminarin can be considered as a safe in terms of environmental pollution, exposure to humans and development of resistance in pathogens owing to its nontoxic mode of action to the target pest, its natural occurrence in the environment, and its history of exposure to humans and the environment without known toxicity. The studies showed that laminarin stimulates defence responses in cell suspensions of rice, alfalfa, tobacco and grapevine [123, 124, 125, 126]. Klarzynski et al. (2000) [125] studied the elicitation of defence responses in tobacco against the soft rot pathogen Erwinia carotovora by laminarin (degree of polymerization approximately 33) extracted from the brown algae Laminaria digitata. Aziz et al. (2003) [126] found that the same glucan was an effective elicitor against downy mildew pathogen, Plasmopara viticola and gray mould pathogen, Botrytis cinerea in grapevine. Similarly, the role of endopolygalacturanase1 (BcPG1) of Botrytis cinerea have been reported to induce defence-related responses in grapevine. The activation of defence in grapevine was mediated by accumulation of defence genes transcripts, mitogen activated protein kinases (MAPK), Ca2+ influx, the oxidative burst (nitrogen oxide (NO) and H2O2 production) and phytoalexin synthesis [127, 128]. More recently it has also been shown that laminarin induces ethylene- but not salicylic acid (SA) dependent responses in tobacco and Arabidopsis thaliana [129]. Structural activity analysis revealed that the sulphate residues are vital for the induction of the SA signalling pathway and increased elicitor activity which cannot be replaced by other anionic groups. The oligo- and polysaccharides of laminarin carrying sulfate residues have

15 Biopolymer Based Biocontrol Strategies against Phytopathogens: New Dimensions… important biological function in plant and animal systems [130, 131, 132]. Refractory state analysis provided evidence for two distinct perception systems for laminarin and laminarin sulphate (PS3, with a degree of sulfation of 2.4) in tobacco [129]. The studies also demonstrated that as compared to laminarin alone, chemical sulfation of laminarin induces the salicyclic acid (SA) signaling pathway in tobacco infected with tobacco mosaic virus (TMV) and Arabidopsis as compared to the native compound. It also resulted in the activation of large set of defence responses including SA, scopoletin, and SA- and ethylene-dependent PR proteins. Trouvelot and Coworkers (2008) [133] described that PS3 induced resistance of grapevine against P. viticola. The authors concluded that the resistance was associated with H2O2 production, upregulation of defence-related genes, and cytological changes, including phenol deposition, cytoplasmic disorganization, and localized plant and pathogen cell death, occurring specifically in cells invaded by the parasite structures. PS3-induced defence were similar to those occurring in a naturally tolerant hybrid grapevine in response to P. viticola infection. Studies with specific inhibitors suggested that callose deposition and the jasmonic acid (JA) pathway contributes to PS3-induced resistance (PS3-IR). In tobacco, elicitins induce the HR [134], which is restricted to the sites of elicitin infiltration and establishment of SAR [135]. Further investigations conducted by Menard et al. (2005) [136] suggested that PS3 remains localized to its infiltration site and that the eliciting activities of PS3 remain localized to the treated tissues, i.e. PS3 failed to exhibit systemic eliciting activities. PS3 exhibits a strong eliciting activity in the infiltrated tissue, as strong as that triggered by elicitors of the HR. Laminarin was shown to stimulate phytoalexin (glyceollin) accumulation localised resistance in soybean cotyledons and seedlings in soybean against Phytophthora megasperma f. sp. glycinea [137, 138]. Laminarin is the main component of several commercial seaweed liquid fertilisers. They induce the formation of antifungal compounds in alfalfa cotyledons and elicit D-glycanases (β-1, 3-glucanase and α-amylase) in Rubus fruticosus cell suspension culture [139].

OTHER BIOPOLYMERS Carrageenans Carrageenan belongs to a family of semi sulphated linear galactans found in the cell walls of many red algae. They are comprised of repeating dimers of an α-1,4-linked D-galactose (λcarrageenan) or 3,6-anhydro-D-galactose residue (κ- or ι-carrageenan) and a β-1,3-linked Dgalactose residue. Commercially, κ, ι-, and λ-carrageenan show increasing sulphate contents, 22%, 32% and 38% (w/w), respectively [140]. Till date only few studies demonstrated the potential of carrageenans and laminarin to induce plant defence system. Carrageenans are sulphated linear galactans with varying amounts of sulphate half esters, while laminarin is a glucan mainly composed of β-1, 3-linked D-glucose residues. Patier and Coworkers (1995) [141] reported that κ-carrageenan elicits β1,3-glucanase activity in Rubus fruticosus cell suspension cultures, oligo-κ- carrageenans

16 Surinder Kaur, Gurpreet Singh Dhillon, Mausam Verma et al… being more efficient than the native polysaccharide. The effect of the algal polysaccharides, namely carrageenans and laminarin, on plant gene expression related to defence, signalling, and primary metabolism was studied by Laurence et al. (2001) [142]. κ-carrageenans and laminarin elicits D-glycanase and phytoalexin accumulation in soybean seedlings, cell suspension cultures and detached cotyledons. The action of carrageenan and laminarin on tobacco leaves was compared with the activity of an elicitor of Phytophthora parasitica var. nicotianae (Ppn) as it was only known for its effects on tobacco cell suspension cultures [143, 144]. The three classes of target genes that were retained encode: (1) defence molecules with antagonistic effects on microbial growth, structure, and pathogenicity; (2) key enzymes of the signalling pathways leading to ethylene and jasmonic acid metabolism and; (3) a protein typical of the primary metabolism. In addition, the concentration of salicylic acid, another signalling molecule, was also amplified in response to Ppn elicitor. Carrageenan, particularly, λ-carrageenan has proved most effective in inducing signalling and defence in tobacco plants, indicating a relationship between higher sulphate content and plant defence responses. The elicitor activity of λ-carrageenan was of the same order of magnitude as the activity of Ppn elicitor in the infiltrated zone I tissues. The transient expression of signalling genes does not preclude that derived signals (i.e. ethylene and jasmonic acid) are produced for a longer time as shown for salicylic acid, whose levels exhibited a 7.3-fold increase over the control at 168 hours post infilteration (hpi). This would be consistent with the more prolonged expression of defence markers, which was particularly observed in response to λ-carrageenan. The activity of λ-carrageenan was found to be similar to Ppn elicitor. Moreover, λcarrageenan showed milder side effects, particularly on necrosis and on RBCs gene expression which was retained as a marker of the primary metabolism. This indicates that signalling and defence are not proportional to the extent of cell death. λ-carrageenan elicited signalling and defence in a dose dependent manner, being active at 100 mg/ml on SA production and fluorescent compounds accumulation. However, the higher concentrations of 100 and 1000 mg/ml of λ-carrageenan were used in these studies as compared with Ppn elicitor (30 mg/ml) or to purified elicitor molecules used in other studies [145, 146]. Patier et al. (1995) [141] showed that oligo-κ-carrageenans were more active as elicitors of laminarase than the polymer itself on Rubus fruticosus cell suspension culture. However, the λ-carrageenan that was used in this study was of commercial origin, one may assume that its activity would increase upon fractionation and purification of derived oligomers. Similarly, it might be expected that oligoglucans are more active than laminarin, as already demonstrated by Kobayashi et al. (1993) and Patier et al. (1993) [139, 147]. The authors showed that elicitation of phytoalexins and D-glycanases is much higher in response to the oligosaccharides. The mechanisms by which plant cells perceive carrageenans, laminarin and oligosaccharides derived from these polymers are not well understood. The isolation of a soybean receptor for β-glucan elicitors from P. megasperma f. sp. glycinea suggests that signalling in response to these and other glycan elicitors might be initiated by receptors on the plant plasma membrane [148]. Carrageenans also act as signalling molecules in algae–algae interactions also supported this hypothesis [132]. Therefore, it might be implicated that λcarrageenan elicits an extended array of defence-related genes in tobacco plants, without

17 Biopolymer Based Biocontrol Strategies against Phytopathogens: New Dimensions… affecting the primary metabolism. The understanding the plant defence responses elicited by such polysaccharides is prerequisite to their potential use as plant protectants.

Ulvan Elicitors are generally classified as exogenous and endogenous elicitors on the basis of their origin. They may be originated from the pathogen (PAMPs⁄MAMPs) or from the host plant itself (pathogen- induced molecular patterns or PIMPs) accordingly, termed exogenous and endogenous elicitors, respectively. Exogenous elicitors of fungal origin are chitin fragments, oligomers and polymers of chitosan or oligoglucans [149, 79, 150, 151]. Endogenous elicitors are oligogalacturonans derived from pectins, or sometimes cellodextrins, xyloglucans or polyglucans derived from cellulose or hemicelluloses. Several studies have shown that the elicitor-active compounds are generally carbohydrates. Glycans obtained from red and brown marine macroalgae can also act as elicitors of defence responses to protect land plants against fungal, bacterial or viral infections by triggering a state of socalled induced resistance [125, 152, 126, 136, 153, 133]. New approaches for the management of plant disease are being explored. Priming, the induction of resistance in the host using natural signal molecules has proved to be effective way in plant disease management [154]. The priming effect of naturally sulphated polysaccharide ulvan, from the green alga Ulva fasciata, reported enhanced resistance in wheat and rice cells to powdery mildew attack [155]. Some specific bacterial components such as, lipopolysaccharides (LPS) of Gram-negative bacteria exhibits priming effect [156]. Exopolysaccharides (EPS) exerts priming effect like lipopolysaccharides (LPS) of Gram-negative bacteria. These biopolymers functions as signal perception generated by phytopathogens that are not directly involved in the mobilization of plant defence response. However, they enhance the speed and/or strength of various responses challenged with various biotic or abiotic stresses [156]. Wheat and rice cell cultures primed with EPS isolated from plant-growth-promoting rhizobacterium Pantoea agglomerans elicited plant defence response when triggered by chitin oligomers or chitosan polymers [79]. Although, direct application of EPS to the suspension-cultured cells did not induce the oxidative burst, pretreatment of the cells with EPS for few hours primed the chitin hexamerinduced burst [79].

SYNERGISTIC EFFECT OF DIFFERENT BIOPOLYMERS IN AUGMENTATION OF BIOCONTROL ACTIVITY The antifungal activity of Trichoderma cell wall degrading enzymes (CWDEs) was synergistically enhanced when combined with different biopolymers such as, endochitinase, exochitinase and β-1,3-glucanase against Botrytis spores (ED50=1 mg/l) [157]. Similarly, combining bacterial metabolites, such as lipodepsipeptides with Trichoderma CWDE synergistically increased the antifungal activity to different plant pathogens [158]. Woo et al. (1999) [159] suggested that the combination of chitinases (endochitinases) with Trichoderma is more effective against various plant pathogens as compared to whole, live microorganisms

18 Surinder Kaur, Gurpreet Singh Dhillon, Mausam Verma et al… alone. Antifungal effect of azole compounds was increased up to tenfold with the addition of endochitinases originating from T. harzianum [157].

IMMOBILIZATION OF DIFFERENT BIOPOLYMERS IN PLANT DEFENCE The increasing demand of biological control of phytopathogens from the perspective of environment safety has lead to the development of advance formulations of microbial origin such as different biopolymers (chitinase, chitosanase, laminarin, and exopolysaccharides) that are actively involved in plant defence mechanisms. Immobilization of microbial cells and/or enzymes has become one of the most valuable tools in the field of biotechnology. Alginate formulations of biocontrol agents (e.g. Trichoderma spp., Talaromyces spp., Gliocladium spp.) increases fungal growth and proliferation in the soil thereby enabling successful biological control of phytopathogenic fungi such as, Fusarium spp., Rhizoctonia spp. and Sclerotium spp. [160]. Microbial entrapment protects the organism from inhibitory compounds or metabolites and provides prolonged metabolic activity such as, secretion of different biopolymers (CWDEs), when microbial cells are reclaimed. The increased production of CWDEs due to repeated use might be due to the formation of fragile beads. It results in the production and release of more conidia thereby, resulting in enhanced biocontrol. Kennedy (1987) [161] has explained the degradation of pellets occurs due to the presence of certain ions in the medium affecting the stability of the gel. Alginate encapsulation of T. harzianum Rifai (T24) has been reported for enhanced chitinase production but reduced β-1, 3-glucanase [162]. Alginate immobilized cultures of Conidiobolus spp. and A. flavus, produced high level of alkaline proteases, while alginate immobilized cultures of S. craterifer produced lower levels of mannanase as compared to free cultures [163, 164]. Some inconsistencies, such as poor immobilization, encapsulation efficiency, unknown pore size of beads and substrate/product diffusion limitations, have been reported for the enzyme production by the encapsulation process. Encapsulation technique has been further refined by incorporating nutrient carriers (adjuvant), e.g. wheat bran, milled chitin, corn cobs, fish meal, soy fibres and peanut hulls into the biopolymers in order to provide a substrate required for proliferation of the BCAs [165]. In addition being an inducible enzyme, it increases the production of chitinases when added as an adjuvant. However, this is not the case with the constitutive β-1,3-glucanase. The biocontrol efficacy of such formulations depends directly on the pH and incubation temperature since the microbial immobilization changes the physiological state of entrapped microorganisms [166, 165].

FUTURE PERSPECTIVES AND CHALLENGES In recent years, the recognition of microbial inoculants has increased substantially in the market. The development of advanced techniques in microbiology, genomics, and bioinformatics has lead to extensive and systematic research and new plant protection strategies. Biotechnological and genetic applications play an important role in modulating

19 Biopolymer Based Biocontrol Strategies against Phytopathogens: New Dimensions… strain improvement for increased production of different biopolymers that exhibits multifunction in plant defence. Biopolymers that can fulfil various functions in plants, lead to promising solutions for a sustainable, environment friendly agriculture. Although various biocontrol agents are available in the market as plant growth promoters and biocontrol agents, the importance of bioploymers such as, chitinase and chitosanase, plays an important role in overcoming various abiotic and biotic (salinity, drought, water logging, heavy metals, and pathogenicity) stresses emerging due to climate change. These biopolymers functions as microbial mixtures as multitasking inoculants that might aid to stabilize the effect or can generate new targets for the inoculants. However, it necessitates the study of the interaction of different biopolymers with the plant as well as the pathogen. The possibility of such interactions must be considered in risk assessment analysis before commercializing. Further, extensive research is also required to progress the scale-up, bioprocess expansion and optimization for fermentation and formulation processes for microbe originated biopolymers. However, recent advancement in genomic technologies will definitely help to optimize process parameters.

Lack of awareness, education or experience

Real or perceived lack of efficacy and higher cost

Quality concerns (inconsistency of results, unintended side effects)

Bottlenecks in the development of biopolymer based biocontrol strategies Figure 3. Bottlenecks in the development and commercialisation of biopolymer based biocontrol strategies.

Beside these technicalities, the revision of legislative processes for the development and commercialization of biopolymer based microbial inoculants in an important step. Figure 3 outilines some of the lacunae in the development of biopolymer based biocontrol agents/ strategies. Standardized methods for risk evaluation of BBBS in needed. In order to attain success in the direction of biocontrol strategies, the mutuality approach is required that will include inventive business management, product marketing, extension education, and progress in research (Figure 4). Studies on biopolymers have yielded a large increase in knowledge regarding their role as biocontrol agents. However, this knowledge is not sufficient for the formulation development

20 Surinder Kaur, Gurpreet Singh Dhillon, Mausam Verma et al… that can work efficiently in all different environmental conditions. Knowledge of the environmental conditions in the area of application and statistical based formulation approach techniques might revolutionise BBBS. Moreover, extensive studies are required for the maximum utilization of agro-industrial and other industrial wastes for the production of different biopolymers such as, chitinases, chitosanases and β-1,3-glucanases. The mode of application depending on the nature of the disease and the pathogen as well as the crop and the environmental conditions also needs to be investigated in detail. Various biopolymers with anti-fungal and anti-microbial properties can be applied as foliar spray, soil or soil-less mix treatments, seed treatments, root dips, and postharvest applications to fruits as a drench, drip, or spray. Commercially available formulations include dusts, dry and wettable powders, and dry and water-dispersible granules and liquids. It is therefore plausible to develop appropriate formulations suitable for different application required in plant disease management.

Increased academic support, research & discussion papers

Increased external communications with consumers, producers and stakeholders

What is required for BBBS?

Establishing a strategic plan to coordinate and focus activities

Greater advocacy at ministerial level

Figure 4. Major concerns for the research and development of biopolymer based biocontrol strategies (BBBS).

Serious efforts are required to study the important parameters and adjuvants in order to increase the stability of active enzyme and hence the shelf-life of the formulation during storage and transportation. Computational and statistical approach should be followed to design novel biopolymer based formulations comprising biocontrol agents. However, it needs the conventional understanding of biopolymer-pathogen interactions and biotechnological aspects in order to formulate powerful and economically feasible product. Different biopolymers can be employed in various systems such as, a purified recombinant optimized enzyme, as a product expressed and secreted by engineered microorganisms, or expressed in transgenic plants that promises additional tools for the management phytopathogens. Genes encoding β-1,3- glucanases, chitinases, chitosanases, and specific antifungal peptides or lectins are worth investigating for novel biocontrol strategies. Engineering different biopolymers together with different biocontrol agents or chemical pesticides can be expected to lead to more stable and safer protection of plants against pathogenic microbes.

21 Biopolymer Based Biocontrol Strategies against Phytopathogens: New Dimensions…

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