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Biological Control of Plant Pathogens using Biotechnological Aspects:- A Review

Research Article Open Access
MK Khokhar1*, Renu Gupta1 and Radheyshyam Sharma2
1Department of Plant Pathology, Rajasthan College of Agriculture, Maharana Pratap University of Agriculture and Technology, Udaipur-313001, India
2Department of Molecular Biology and Biotechnology Rajasthan College of Agriculture, Maharana Pratap University of Agriculture and Technology, Udaipur-313001, India
*Corresponding authors: MK Khokhar
Department of Plant Pathology
Rajasthan College of Agriculture
Maharana Pratap University of Agriculture and Technology
Udaipur-313001, India
E-mail: khokharmk3@gmail.com
 
Received August 24, 2012; Published August 30, 2012
 
Citation: Khokhar MK, Gupta R, Sharma R (2012) Biological Control of Plant Pathogens using Biotechnological Aspects:- A Review. 1:277. doi:10.4172/scientificreports.277
 
Copyright: © 2012 Khokhar MK. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
 
Abstract
 
Plant pathogens are the most important factors that cause major losses to agricultural products every year. To minimize these losses peoples are dramatically used pesticide and fungicide that will cause toxic effect of human health. Thus, the need of sustainable agriculture will increasingly rely on the integration of biotechnology with traditional agricultural practices. Most sustainable and environmentally acceptable control may be achieved using biocontrol agents due to the effort to reduce the use of agrochemicals and their residues in the environment and in food. These BCA controlling plant diseases through various mechanism viz., hyperparasitism, predation, antibiosis, cross protection, competition for site and nutrient and induced resistance. Identifying, understanding and utilizing microorganisms or microbial products to control of plant diseases and to enhance crop production are integral parts of sustainable agriculture. Biological control has the potential to control crop diseases while causing no or minimal detrimental environmental impact. In this review, different aspects of biological control of fungal plant diseases including modes of action, application strategies, modern tools of microbial genetics or molecular techniques, which can use to improve the biocontrol activity, metabolites and products that could have important environmental benefits and outlooks, will be discussed.
 
Keywords
 
Plant disease; Mechanism; Biological control; Biotechnology; Sustainable Agriculture
 
Introduction
 
Plant pests including insects, parasitic weeds and pathogens are the most important biotic agents causing serious losses and damages to agricultural products. Plant pests need to be controlled to ensure food, feed and fiber production quantitatively and qualitatively. Indiscriminate use of pesticide and fungicide lead to pollute the environment and cause serious effect to human health and non-target organisms [1]. Thus there is a need to reduction or elimination of synthetic pesticide applications in agriculture is highly desirable. One of the most promising means to achieve this goal is by the use of new tools based on biocontrol agents (BCAs) for pest and disease control alone or to integrate with reduced doses of chemicals in the control of plant pathogens resulting in minimal impact of the chemicals on the environment [2]. Biological control of plant diseases has been considered a viable alternative method to manage plant diseases [3]. Biological control refers to the purposeful utilization of introduced or resident living organisms, other than disease resistant host plants, to suppress the activities and populations of one or more plant pathogens or reproduction of one organism using another organism [4]. A variety of biological controls are available for use, but further development and effective adoption will require a greater understanding of the complex interactions among plants, people and the environment.
 
Interactions between Plants and Beneficial Microbes
 
Throughout the lifecycle, plants and pathogens interact with a wide variety of organisms. These interactions can significantly affect plant health in various ways [5,6]. Several forms of direct or indirect interactions between plants and microorganisms have been found such as mutualism, protocooperation, commensalisms, neutralism, competition, amensalism, parasitism and predation [7]. For the development of plant diseases it involves plants and microbes, the interactions that lead to biological control take place at multiple levels. From the plant’s point of view, biological control may be considered a positive result arising from different specific and non-specific interactions [8,9]. Mutualism is an association among several species where all of them are benefited from this association [10]. Sometimes, it can be an obligatory relation involving close physical and biochemical contact between two organisms, such as those between plants and mycorrhizal fungi [5,7]. Commensalism is also a symbiotic interaction between two living organisms, where one organism benefits and the other is neither harmed nor benefited [11]. Biological interactions in which the population density of one species has absolutely no effect on the other are called neutralism [12]. In contrast, antagonism between organisms results in a negative outcome for one or both. Competition within and between species caused a decreased growth, activity, and/ or fecundity of the interacting organisms [8]. Biocontrol can occur when non-pathogens compete with pathogens for nutrients and sites in host plant. Direct interactions that benefit one population at the expense of another also affect our understanding of biological control. Parasitism is also a symbiotic relation in which two organisms coexist over a prolonged period of time [7,8,13]. In this type of interaction, one organism, usually the physically smaller (parasite) benefits and the other (host) is harmed. Another interesting contribution to biocontrol is when host infection and parasitism by relatively avirulent pathogens may lead to biocontrol of more virulent pathogens through the stimulation of host defense systems. Finally predation refers to the hunting and killing of one organism by another for consumption and sustenance. While the term predator typically refers to animals that feed at higher trophic levels in the macroscopic world, it has also been applied to the actions of microorganisms such as protists and mesofauna, i.e. fungal feeding nematodes and microarthropods, that consume pathogen biomass for sustenance [8]. Biological control can result in various forms of these types of interactions, depending on the environmental conditions within which they occur.
 
Mechanism of Plant Disease Management
 
Hyperparasitism and predation
 
In this mechanism the pathogen is directly attacked by a specific BCA that kills it or its propagules. In general, there are four major classes of hyperparasites: obligate bacterial pathogens, hypoviruses, facultative parasites, and predators. A classical example of Pasteuria penetrans is an obligate bacterial pathogen of root-knot nematodes that is used as a BCA. Hypoviruses are hyperparasites, a classical example is the virus that infects Cryphonectria parasitica, a fungus causing chestnut blight, which causes hypovirulence, a reduction in disease-producing capacity of the pathogen. The phenomenon has controlled the chestnut blight in many places [14]. However, the interaction of virus, fungus, tree, and environment determines the success or failure of hypovirulence. There are several fungal parasites of plant pathogens, including those that attack sclerotia (i.e. Coniothyrium minitans) while others attack living hyphae (i.e. Pythium oligandrum) and, a single fungal pathogen can be attacked by multiple hyperparasites. For example, Acremonium alternatum, Acrodontium crateriforme, Ampelomyces quisqualis, Cladosporium oxysporum, and Gliocladium virens are just a few of the fungi that have the capacity to parasitize powdery mildew pathogens [3,15]. Other hyperparasites attack plant-pathogenic nematodes during different stages of their life cycles (i.e. Paecilomyces lilacinus and Dactylella oviparasitica). In contrast to hyperparasitism, microbial predation is more general and pathogen non-specific and generally provides less predictable levels of disease control. Some BCAs exhibit predatory behavior under nutrient-limited conditions. However, such activity generally is not expressed under typical growing conditions. For example, some species of Trichoderma produce a range of enzymes that are directed against cell walls of fungi. However, when fresh bark is used in composts, Trichoderma spp. do not directly attack the plant pathogen, Rhizoctonia solani. But in decomposing bark, the concentration of readily available cellulose decreases and this activates the chitinase genes of Trichoderma spp., which in turn produce chitinase to parasitize R. solan [16].
 
Antibiotic-mediated suppression
 
Antibiotics are microbial toxins that worked at low concentrations, poison or kill other microorganisms. Most microbes produce and secrete one or more compounds with antibiotic activity [17-19]. In some instances, antibiotics produced by microorganisms have been shown to be particularly effective at suppressing plant pathogens and the diseases they cause. In situ production of antibiotics by several different biocontrol agents has been measured [20]. However, the effective quantities are difficult to estimate because of the small quantities produced relative to the other, less toxic, organic compounds present in the phytosphere and several methods have been developed to ascertain when and where biocontrol agents may produce antibiotics detecting expression in the infection court is difficult because of the heterogenous distribution of plant-associated microbes and the potential sites of infection. In a few cases, the relative importance of antibiotic production by biocontrol bacteria has been demonstrated, where one or more genes responsible for biosynthesis of the antibiotics have been manipulated. For example, mutant strains incapable of producing phenazines [21] or phloroglucinols [22] have been shown to be equally capable of colonizing the rhizosphere but much less capable of suppressing soil borne root diseases than the corresponding wildtype and complemented mutant strains. Several biocontrol strains are known to produce multiple antibiotics which can suppress one or more pathogens. For example, Bacillus cereus strain UW85 is known to produce both zwittermycin and kanosamine. The ability to produce multiple classes of antibiotics, that differentially inhibit different pathogens, is likely to enhance biological control. Recently, Pseudomonas putida WCS358r strains genetically engineered to produce phenazine and DAPG displayed improved capacities to suppress plant diseases in field-grown wheat [23]. Selective examples of BCA’s given below are particularly effective. Pseudomonas fluorescens F113 produces 2,4-diacetyl-phloroglucinol against Pythium spp. Agrobacterium radiobacter produces agrocin 84, against Agrobacterium tumefaciens [24]. Bacillus subtilis QST713 produces iturin A against Botrytis cinerea and R. solani [25,26]. B. subtilis BBG100 produces mycosubtilin against Pythium aphanidermatum [18]. B. amyloliquefaciens FZB42 produces bacillomycin and fengycin against Fusarium oxysporum [27]. Pseudomonas fluorescens 2-79 and 30-84 produce phenazines against Gaeumannomyces graminis var. tritici, Trichoderma virens produces gliotoxin against Rhizoctonia solani [28].
 
Cell wall degrading enzymes
 
Several BCA’s produce enzymes able to hydrolyze chitin, proteins, cellulose, and hemicellulose, thus contributing to direct suppression of plant pathogens. There are selective examples of BCA’s able to produce enzymes, effective against certain plant pathogens. Serratia marcescens chitinases and genes encoding them have been shown to have biocontrol potential in a variety of experiments. A highly chitinolytic strain of S. marcescens was found to suppress the growth of Botrytis spp, Rhizoctonia solani, and Fusarium oxysporum [29]. However, such activities are rather indicative of the need to obtain carbon nutrition. Lysobacter and Myxobacteria are known to produce plentiful amounts of lytic enzymes, and some isolates have been shown to be effective at suppressing fungal plant pathogens [5]. So, the lines between competition, hyperparasitism, and antibiosis are generally disguised.
 
Competition
 
Although difficult to be proven directly, much indirect evidence suggests that competition between pathogens and non-pathogens for nutrient resources is important for restricting disease incidence and severity. Soil-borne pathogens, such as species of Fusarium and Pythium, infecting through mycelial contact, are more susceptible to competition by other soil and plant-associated microbes than by those germinating directly on plant surfaces which they invade through appressoria and infection pegs. Rhizosphere or phyllosphere BCA’s generally protect the plant by rapid colonization, thus consuming completely the limited available substrates so that none is left for pathogens to grow. For example, effective catabolism of nutrients in the spermosphere has been identified as a mechanism contributing to the suppression of Pythium ultimum by Enterobacter cloacae [30,31]. At the same time, these microbes produce metabolites that suppress pathogens. These microbes colonize the sites where water and carboncontaining nutrients are most readily available and utilize root mucilage. To survive in such an environment, microorganisms secrete iron-binding ligands called siderophores that sequester iron from the microenvironment. Biocontrol based on competition for essential micronutrients, such as iron, has also been examined [32] were the first to demonstrate the importance of siderophore production as a mechanism of biological control of Erwinia carotovora by several plant growth promoting Pseudomonas fluorescens strains.
 
Induced resistance
 
Plants respond to a variety of chemical stimuli produced by BCA’s, such stimuli can either induce host plant defences through biochemical changes expressing resistance mechanisms against subsequent infection by pathogens. Induction of host defences can be localised and/or systemic in nature. The determinants and pathways of induced resistance stimulated by BCA’s and other non-pathogenic microbes have being occasionally characterized. The first of these pathways, called systemic acquired resistance (SAR), is mediated by salicylic acid (SA), which typically leads to the expression of pathogenesis-related (PR) proteins including a variety of enzymes. A second case, referred to as induced systemic resistance (ISR), is mediated by jasmonic acid (JA) and/or ethylene, which are produced following applications of some non-pathogenic rhizobacteria. Some most striking examples of bacterial determinants and types of disease resistance (ISR) induced by BCA’s include a Bacillus mycoides strain able to produce peroxidase, chitinase and β-1,3-glucanase in sugar beet [33]. B. subtilis GB03 and IN937 producing 2,3-butanediol in Arabidopsis [34]. Pseudomonas putida strains producing a lipopolysaccharide in Arabidopsis [35]. Serratia marcescens 90-166 producing siderophore in cucumber [36]. A number of strains of root-colonizing microbes have been identified as potential elicitors of plant host defences. Some biocontrol strains of Pseudomonas sp. and Trichoderma sp. are known to strongly induce host plant defences. A number of chemical elicitors of SAR and ISR may be produced by the PGPR strains upon inoculation, including salicylic acid, siderophore, lipopolysaccharides, and 2,3-butanediol, and other volatile substances [34,37,38].
 
Plant Growth Promoting Rhizobacteria
 
Several PGPR bioinoculants are currently used commercially. They are called different names and operate through a range of mechanisms: (i) bioprotectants, through suppression of plant disease (ii) biofertilizers, through improved nutrient acquisition (iii) biostimulants, through phytohormone production. Bioinoculants include bacteria belonging in the genera Bacillus, Paenibacillus Streptomyces, Pseudomonas, Burkholderia and Agrobacterium, which are currently used as BCA’s also at commercial level. They suppress plant disease through induction of systemic resistance, production of siderophores or antibiotics. Biofertilizers are also available for increasing crop uptake of nitrogen from nitrogen- fixing bacteria (Azospirillium), and iron uptake from siderophore-producing bacteria (Pseudomonas). Species of Pseudomonas and Bacillus can produce as yet not well characterized phytohormones or growth regulators that cause extensive root growth, thus increasing the absorptive surface of plant roots. These PGPR are referred to as biostimulants and the phytohormones that they produce include indole-acetic acid, cytokinins, gibberellins and inhibitors of ethylene production. Current means of delivery of inoculants include peat, granular, liquid and wettable powder formulations. A major determinant of growth promotion is the magnitude of their ability to colonize rhizosphere. Several new studies have contributed to the development of new biofertilizer products that utilize natural antimicrobial compounds produced by diverse antagonists [39-42].
 
Siderophores
 
Siderophores are low-molecular-weight (0.5 to 1.5 KDa), high specificity Fe chelating agents [43]. They are produced under ironlimiting conditions by almost all aerobic and facultative anaerobic microorganisms examined. Siderophores have been demonstrated to play a major role in plant disease suppression by some bacterial biocontrol agents which inhibit the growth or the metabolic activity of plant pathogens by sequestering iron [44].
 
Volatile compounds
 
Volatile compounds from the biological control agents has an important part of the inhibitory mechanism, especially under closed storage conditions. Production of volatile ammonia has been implicated as a possible mechanism to control soil borne pathogens [45].
 
Alkaloids
 
The basic unit in the biogenesis of the true alkaloids are amino acids. Alkaloids are highly reactive substances with biological activity in low doses [46]. Several strains of microorganisms produce alkaloids. Alkaloids, elimoklavine, festuklavine produce by Trichoderma and agroklavine, ergometrine produce by Penicillium have antifungal activity against, Botrytis cinerea, Fusarium solani and Alternaria tenius as well as antibacterial action which depressed the growth of several pathogenic bacteria, cause the death of living cell.
 
Phenols
 
Phenols may also be involved in protection of plants from pathogens. Antagonism of P. fluorescens strain 2-79 (NRRL B-15132) suppressed take-all disease of wheat can be attributed to production of the 2-acetamidophenol (0.05 g/liter, in cultures) by the strain [47].
 
Application of Molecular Genetic to Improve Biocontrol Agents
 
Genetical improvement of biocontrol agents
 
Genetically improved antagonistic hyperparasitic microorganisms tend to increase their effectiveness as biological control agents. Effective performance requires either improvement of the environment to favour the biocontrol agent or genetic improvement of the agent [48]. Genetic manipulation will be done to enhance antifungal metabolites productivity of biocontrol agents, to improve antagonistic potential of biocontrol agents, to control a broad spectrum of phytopathogens and to develop biocontrol agents tolerance of some stress conditions. Thus it can be achieved by chemical and physical mutation, sexual hybrids, homokaryons, and genetic manipulation e.g, directed mutagenesis, protoplast fusion, recombination, transformation or isolation of useful genes from biocontrol fungi without functional sexual stages [49,50].
 
Mutation as tool for the improvement
 
Many authors were applied the mutagensis by either the physical or the chemical mutagens to generate new biotypes with improve the potentialities of biocontrol agents and / or antifungal metabolites producers. Induced mutation is one of the most commonly used routine to restrain the genetic construction of microorganisms.
 
Use of Protoplast Fusion for Genetic Improvement or Manipulation
 
Protoplast fusion is a quick and easy method for combining the advantageous properties of distinct promising strains. Protoplast fusion techniques were used to combine genetic traits desirable for improving biocontrol activity by T. harzianum and increased amounts of specific proteins. Fusion of protoplast derived from two efficient biocontrol strains of T. harzianum resulted in the recovery of a progeny strain with greatly improved biocontrol ability. After that, directed transfer of fungal genetic sequences encoding for factors important in biocontrol will be possible as soon as such sequences are available.
 
Improvement the bioagents via directed mutagenesis
 
Many authors were applied the mutagensis by either the physical mutagens (i.e. gamma irradiation or UV-irradiation) or the chemical mutagens to generate new biotypes with improve the potentialities of biocontrol agents and / or antifungal metabolites producers [51]. Haggag [44] used mutation technique by UV light and improved the production of important P. fluorescens of antibiotics, phenazine, pyrrolnitrin and phloroglucinol as well as siderophore pigment production against some tomato damping-off pathogens (Fusarium solani, Fusarium oxysporum f.sp. lycopersici and Rhizoctonia solani). These mutant strains increased the antibiosis and the fluorescens on King's medium comparing to the wild-type. A mitogen-activated protein kinase encoding gene, tvk1, from Trichoderma virens was cloned, and its role during the mycoparasitism, conidiation, and biocontrol was examined in tvk1 null mutants. These mutants showed, a clear increase in the level of the expression of mycoparasitism-related genes during direct confrontation with the plant pathogen Rhizoctonia solani [52]. The null mutants displayed an increased protein secretion phenotype as measured by the production of lytic enzymes in culture supernatant compared to the wild-type [53]. Consistently, biocontrol assays demonstrated that the null mutants were considerably more effective in disease control than the wildtype strain or a chemical fungicide. In addition, tvk1 gene disruptant strains sporulated abundantly in submerged cultures, a condition that is not conducive to sporulation in the wild type. These data suggest that Tvk1 acts as a negative modulator during host sensing and sporulation in T. virens [53].
 
 
Genetic modification and transformation
 
A genetic modification approach was used to further enhance the biocontrol ability of biocontrol agents [54]. Genetically modified (GM) was used in strains, P. fluorescens F113Rif (pCU8.3) and P. fluorescens F113Rif (pCUP9), were developed for enhanced phenazine-1- carboxylate (Phl) production and assessed for biocontrol efficacy and impact on sugar beet in microcosm experiments [55]. Pseudomonas aeruginosa PNA1, isolated from the rhizosphere of chickpeas in India. mutant (FM13) deficient in phenazine production were obtained following transposon mutagenesis of PNA1 [56]. Anthranilate, an intermediate in the tryptophan biosynthesis pathway, suppressed mycelial growth of Pythium spp. in culture and damping-off of P. vulgaris and lettuces. It is concluded that anthranilate, excreted by FM13 as a consequence of the trpC mutation, may have contributed to the suppression of Pythium damping-off by the mutant.
 
Methods of Application of Antagonists
 
Overall application
 
Successful application of biological control strategies requires more knowledge-intensive management. Understanding when and where biological control of plant pathogens can be profitable, requires an appreciation of its place within integrated pest management systems [57]. In general, the foundation of a sound pest and disease management program in an annual cropping system begins with cultural practices that alter the farm landscape to promote crop health [57]. These include crop rotations that limit the availability of host material used by plant pathogens [8]. Proper use of tillage can disrupt pathogen life cycles and prepare seed beds of optimal moisture and bulk density. In nurseries and greenhouses environmental control can be more tightly regulated in terms of temperature, light, moisture and soil composition, but the design of such systems cannot wholly eliminate disease problems [25].
 
The second layer of defense against pests consists of the quality of crop germplasm. Breeding for pathogen resistance including fungal pathogens contributes substantially to crop success in most regions [8]. Newer technologies that directly incorporate genes into crop genomes, commonly referred to as genetic modification or genetic engineering, are bringing new traits into crop. Other technologies, such as seed washing, testing for pathogens and treatments are also used to keep germplasm pathogen-free. In perennial cropping systems, such as orchards and forests, germplasm quality may be more important than cultural practices, because rotation and tillage cannot be used as regularly [8]. Upon these two layers, growers can further reduce pathogen pressure by considering both biological and chemical inputs. Biologically based inputs such as microbial fungicides can be used to interfere with pathogen activities. Registered biofungicides are generally labeled with short reentry intervals and pre-harvest intervals, giving greater flexibility to growers who need to balance their operational requirements and disease management goals. When living microorganisms are introduced, they may also augment natural beneficial populations to further reduce the damage caused by targeted pathogens [57,58].
 
Applying to the infection site
 
Application directly to the infection court at a high population level to swamp the pathogen (inundate application), seed coating and treatment with antagonistic fungi and bacteria, i.e., Trichoderma harzianum and Pseudomonas fluorescens [57,59], antagonists applied to fruit for protection in storage, i.e., Pseudomonas fluorescens [60,61] and application to soil at the site of seed placement [59]. These types of applications are the most commonly used procedures which have resulted in the successful control of several fungal plant pathogens.
 
One place application
 
Biocontrol microorganisms are applied at one place (each crop year), but at lower populations which then multiply and spread to other plant parts and give protection (augmentative application) against fungal pathogens. An Example of this method is Plant Growth Promoting Rhizobacteria (PGPR) and atoxigenic Aspergillus flavus on wheat seed scattered on the soil to spread to cotton flowers where they displace aflatoxin producing strains of A. flavus and fungal antagonists added to soil [17,32].
 
Occasional application
 
One time or occasional application maintains pathogen populations below threshold levels. In theory, parasites of the pathogen, or hypovirulent (disease carrying) strains of the pathogen, might be used and not require yearly repetition (i.e., hypovirulent strains of the chestnut blight pathogen) in which host plant is inoculated with attenuated strains of pathogenic that protects the host plant against the virulent strains of pathogen [62].
 
Figure 1: Lectin-carbohydrate interaction.
 
Future Outlook
 
Some of the research criteria that will advance our understanding of biological control and the conditions under which it can be most fruitfully applied. Ecological factors play very important roles in the performance and activity of biocontrol-active microorganisms. Application strategies still there are some areas which should be investigated and developed for the enhancement of the effectiveness of biocontrol microorganisms. Introducing new strains and mechanisms of fungal plant pathogens are very diverse and their pathogenicity is different on host plants, it is therefore very important to look for new and novel biocontrol microorganisms with different mechanisms.
 
 
References