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One of the biggest ecological challenges facing microbiologists and plant pathologists in the near future is the development of environmentally friendly alternatives to the extensive use of chemical pesticides for combatting crop diseases. Slide 3: living organisms, or natural products derived from these organisms, that are used to suppress pest populations. Bio Pesticides These organisms include plants (genetically modified crops), insects, nematodes and microorganisms. These beneficial organisms, also termed biological control (or biocontrol) agents, represent an environmentally friendly alternative to chemicals and offer different modes of action for combatting pathogens. Slide 4: The use of beneficial microorganisms is considered one of the most promising methods for more rational and safe crop-management practices. Antagonistic microorganisms may interfere with the growth and/or survival of plant pathogens and thereby contribute to biological control. Biocontrol can thus be used in situations: Where conventional pesticides cannot be used owing to residue concerns. Where no control is currently available. In the rapidly growing sector of organic farming. Slide 5: Phytostimulation Bioremediation Biofertilization Biocontrol Nutrient Signals Microbial Cell Factory Production of IAA. Increase rooting . Reduce Nitrogen Uptake. Reduce Fertilizer Usage. Reduce nitrate pollution. Nitrogen Fixation. Phosphate Solubilization. Reduce Fertilizer Usage. Reduce Water Pollution. Antimicrobial Metabolites Reduced Pathogens. Reduced Disease. Reduced Fungicides Usage. Clean-Up of Contaminated Soils. Gene Expression. Primary Metabolite. Secondary Metabolite . Slide 7: Several modes of action have been suggested to explain the biocontrol activity of biopesticides: Competition for space. To compete successfully with pathogen biocontrol agents: Should be better adapted to various environmental and nutritional conditions than the pathogen. Should have the ability to grow more rapidly than the pathogen. Should have the ability to survive under conditions that are unfavorable to the pathogen. Slide 8: Penicillium expansum is the agent of blue mould (pome fruits, cherries, nectarines and peaches). Cryptoccoccus laurentii is yeast able to rapidly colonize wounds on fruits and thereby to limit P. expansum growth. The wound environment is characterised by the presence of oxidant stressors (i.e. hydrogen peroxide). In this stressful environment C. laurentii is able to grow rapidly due to superoxide dismutase (SOD) and catalase (CAT) activity (Tolaini et al., 2010). Slide 9: Competition for nutrients. The level of control provided highly dependent on: Initial concentration of the biocontrol agents. The ability of the antagonist to rapidly colonize the wound site. most effective concentration 107–108 CFU/ml Attachment of microbial antagonist to the pathogene hyphae?. Antagonist microbe take nutrient more rapidly than target pathogens and so prevent spore germination and growth of pathogens. Slide 10: Candida saitona was effective at a concentration of 107 CFU/ml for controlling Penicillium expansum on apples. In an another study, a concentration of 108 CFU/ml was reported to be better in controlling blue mold (Penicillium expansum) on apples. This qualitative relationshipis highly dependent on the ability of the antagonists to multiply and grow at the wound site. Slide 11: Production of antibiotics. Bacillus such as B. subtilis, B. licheniformis, B. cereus, B. mycoides and B. amyloliquefaciens can produce anti-fungal lipopeptides against fungi in the natural environment. The known anti-fungal mechanism of lipopeptide is to elicit pores on fungal cell membranes. Lipopeptides are classified as: Fengycin. Surfactin. Iturin. Bacillomycin. Staining fungal cells (Rhizoctonia solani )after treated by 100g/ml of WH1 fungin for 2 h,. The leaked cytoplasm from hyphae is labeled by arrows (Gaofu et al., 2010). : Staining fungal cells (Rhizoctonia solani )after treated by 100g/ml of WH1 fungin for 2 h,. The leaked cytoplasm from hyphae is labeled by arrows (Gaofu et al., 2010). optical microscope fluorescent microscope Fungin plays an anti-fungal role by two models: High concentration to elicit pores on cell membrane. low concentration to induce apoptosis. Slide 13: Bacillus subtilis and Pseudomonas cepacia Burkh are known to kill pathogens by producing the antibiotic iturin. Agrobacterium tumefaciens K84 protects plant wounds against infection partially because it produces the antibiotic agrocin 84, which has specific toxicity against sensitive strains of A. tumefaciens. Bacillus amyloliquefaciens PPCB004 produce lipopeptides iturin A, fengycin and surfactin →→→→ selected as a potential antagonist to control Botrytis cinerea, Penicillium expansum and Rhizopus stolonifer on peach fruit. Slide 14: Direct parasitism. Degradation of pathogen and utilizing nutrients present in its body (ex. Entomopathogenic fungi , Trichoderma spp.). Induced resistance. Endophytic fungi or bacteria can induce systemic resistance in plants against pathogens after actively penetrating and colonizing the host, promoting the synthesis of biologically active compounds or causing changes in plant morphology and/or physiology Dong et al. (2003) reported that dry mycelium of Penicillium chrysogenum contributed to induced resistance against V. dahliae in cotton. Microbes Used as Biopesticides : Microbes Used as Biopesticides Bacteria →→→→ Bacillus thuringiensis (Bt), B. sphaericus, B. papilliae, Serratia entomophila. Viruses →→→→ Nuclear polyhedrosis viruses, granulosis viruses, non-occluded baculoviruses. Fungi →→→→ Beauveria netahizium, Entomophaga zoopthora, Paecilomyces normuraea. Protozoa →→→→ Nosema, Thelohania vairimorpha. BACTERIA AS BIOPESTICIDES : Among the 14 registered bacterial biocontrol agents: Six are based on Bacillus. Five on Pseudomonas. Two on Agrobacterium. One on Streptomyces. BACTERIA AS BIOPESTICIDES Slide 17: Bacteria that used as biopesticides can be divided into 2 type: Nonspore-forming→→→→ species in the Pseudomonaeae and the Enterobacteriaceae Aspore-forming bacteria →→→→ bacteria belong to the Bacilliaceae. Slide 18: Xenorhabdus Entomopathogenic nematodes of the families Steinernematidae and Heterorhabdititae, in conjunction with bacteria of the genus Xenorhabdus, have been used successfully deployed as biopesticides. For example, BioVECTOR 4 are usually applied to control insects in cryptic and soil environments. The nematodes harbor the bacteria in their intestines. The infective third-stage larvae enter the host through natural openings and penetrate into the haemocoel. The bacteria are voided in the insect and cause septicemia, killing the insect in approximately 48 h. The nematodes feed upon the bacteria and liquefying insect; and mature into adults. Pseudomonaeae : Pseudomonaeae Pseudomonads occur commonly in the rhizosphere of plants and they are an important for the control of soil borne plant pathogens. Numerous compounds have been shown to be important for biocontrol activity. Inhibitory compounds include: Degradative enzymes such as: Protease. Cellulase. Chitinase. B-glucanase. Antibiotics such as: Hydrogen cyanide. Phloroglucinol. Pyoluteorin. Pyrrolnitrin. Phenazines. Wheat root-colonizing Pseudomonas fluorescens Slide 20: Antifungal activity of Pseudomonas sp. and derivative strains (mutant) against Sclerotinia sclerotiorum (in the centre)(Berry et al., 2010). DF41 (wild typ). DF41-1278 (lipopeptide synthesis) DF41-469 (gacS) DF41-469 (pUCP23-gacS). Slide 21: Members of the Bacillus genus are often considered microbial factories for the production of a vast array of biologically active molecules potentially inhibitory for phytopathogen growth. For example, B. subtilis, has an average of 4–5% of its genome devoted to antibiotic synthesis and has the potential to produce more than two dozen structurally diverse antimicrobial compounds Their spore-forming ability makes these bacteria some of the best candidates for developing efficient biopesticide . Bacillus spores have a high level of resistance to the dryness necessary for formulation into stable products. Bacilliaceae Slide 22: Bacillus thuringiensis (Bt) Occurs naturally in the soil and on plants. Produces a crystal protein that is toxic to specific groups of insects. The insecticidal activity of Bt was first discovered in 1911; however, a product containing this bacterium as the active ingredient was not commercially manufactured until the 1950s. Slide 23: Several strains of Bt have been identified that infect and kill insects. Bt used mainly to control larvae (caterpillars) of insects in the Lepidoptera order (butterflies and moths) and for the control of mosquito larvae. Bt should be applied to the underside of the leaves because: Most larvae feed on the underside of leaves. Bt breaks down faster in sunlight, which will reduce its effectiveness. Slide 24: Bt kills the caterpillars in a rather interesting way: The Bt spores must be eaten by the young caterpillar. The bacteria grows inside the caterpillar, reproduces and produces crystalline toxins. The toxins then attack the lining of the insect gut, rupturing the cell walls and allowing the gut's contents to spill into the insect's circulatory system. The caterpillar soon dies of starvation and/or tissue damage. This will take from 12 hours to five days, depending upon: How much Bt the caterpillar ate. The size and species of the caterpillar. The type of Bt used. Slide 25: Bt mode of action ruptures gut cell lining Bt spores Slide 26: Insect larvae can damage the plant and this allows fungal spores to enter corn tissue . These fungi may produce mycotoxins which can be fatal to some animals and are possibly related to human cancers. Using genetically modified hybrids allows to control the ability of insects and diseases to attack crops. Bt Corn and Reduction of Human Health Infections Bt gene for insect control : Bt gene for insect control Bt delta endotoxin Slide 28: Bacillus popilliae A naturally occurring bacteria that have been mass-produced for the control of Japanese beetle larvae in turf since the 1940s. Cause "milky disease" in the beetle larvae and establish a resident population capable of causing mortality over several seasons if soil conditions are appropriate. Bacillus popilliae was the first insect pathogen to be registered in the U.S. as a microbial control agent. FUNGI AS BIOPESTICIDES : Entomopathogenic fungi are important natural regulators of insect populations and have potential as mycoinsecticide agents against diverse insect pests in agriculture. These fungi infect their hosts by penetrating through the cuticle, gaining access to the hemolymph, producing toxins, and grow by utilizing nutrients present in the haemocoel. FUNGI AS BIOPESTICIDES Slide 30: Entomopathogenic fungi may be applied in the form of conidia or mycelium which sporulates after application. Entomopathogenic fungi have been used solely or in combination with various insecticides as a part of insecticide resistant management against important crop pests. Beauveria bassiana. Metarhizium anisopliae. Nomuraea rileyi. Paecilomyces spp. Lecanicillium lecanii Life cycle for three Beauveria bassiana single spore isolates on coffee berry borer. : Life cycle for three Beauveria bassiana single spore isolates on coffee berry borer. Trichoderma : Trichoderma Trichoderma spp. are considered as potential biocontrol and growth promoting agents for many crop plants. Trichoderma populations can be established relatively easily in different types of soil and can continue to persist at detectable levels for months. Trichoderma spp are more competitive for nutrients than many other soil microbes Trichoderma viride is a biocontrol agent against soil borne plant pathogens and it can easily colonize in plant rhizosphere and help to promote the plant growth. Slide 33: Biocontrol activity of Trichoderma is due to combination of : Ability to serve as antagonist. Plant growth promoter. Plant defense inducer. Rhizosphere colonizer. Neutralizer of pathogen’s activity favoring infection. Slide 34: During mycoparasitic interactions between Trichoderma and fungal pathogen, a diffusible factor released from the host before physical contact is responsible for induction of hydrolytic enzymes. During direct contact, lectins in the host’s cell wall can induce coiling of the Trichoderma around the host hyphae and mycoparasite can produce appressorium-like structures to destroy the pathogen. Normal degradation of pathogen mycelia would take place after penetrating with appressorium-like structures. Slide 35: John et al. (2010) tested the potential of T. viride ATCC 9275 as a biocontrol of two fungal Pathogens (Fusarium oxysporum f. sp. adzuki and Pythium arrhenomanes) and effect of pathogen and antagonist on growth and preliminary yield (fruit) on soybean plants. Fusarium Pythium Trichoderma Trichodermal growth and inhibition of Pythium growth after 24, 48 and 120 h respectively (arrow show: Trichodermal growth) : Trichodermal growth and inhibition of Pythium growth after 24, 48 and 120 h respectively (arrow show: Trichodermal growth) Trichodermal growth and inhibition of Fusarium growth after 24, 48 and 120 h respectively (arrow show Trichodermal growth). Plate assay for antagonistic activity of Trichoderma on fungal pathogens Slide 37: Plant dry weight after 12 weeks of plant growth (John et al., 2010). 194% and 141% more height than pathogens alone Slide 38: Fruit yield after 12 weeks of plant growth (John et al., 2010). 5 and 1.6 times higher VIRUSES AS BIOPESTICIDES : VIRUSES AS BIOPESTICIDES The Cydia pomonella granulovirus (famil Baculoviridae, CpGV) is used worldwide to control the codling moth. Slide 41: The larva has to feed on the virus during the first development stage?: Since this larval stage of the insect life cycle is the most susceptible to infection with baculoviruses. Larval stage is located outside of the plant fruit enabling direct contact with the virus. Slide 42: Advantage of using CpGV as a biocontrol agent: Absolute selectivity. High effectiveness towards the target organism. It does not harm beneficial organisms. It is assumed that already one CpGV particle is mortal. Disadvantages of the untreated CpGV: Strong UV sensitiveness, which results in frequent reapplications during the period of growth. A low uptake. Removal of the water soluble virus through rainfall from the trees (could be avoided by the encapsulation with organic materials). Enhancing the bioefficacy of biopesticides : Enhancing the bioefficacy of biopesticides Manipulations in the physical and chemical environment during storage: Microbial antagonists are screened for their ability to develop rapidly under the required storage conditions and only those that fulfill the basic requirements are selected. Modification in the storage environment can be a useful strategy for enhancing the efficacy of microbial antagonists, as it is possible to manipulate the physical and chemical environment to the advantage of microbial antagonistsin storage. Inhibition effect of Pichia guillermondii yeast strain Z1 (1×108 CFU/ml) against Penicillium italicum (1×105 spores/ml) which causes blue mould on Valencia late citrus fruit at different temperatures and different relative humidity (Lahlali et al., 2010). : Inhibition effect of Pichia guillermondii yeast strain Z1 (1×108 CFU/ml) against Penicillium italicum (1×105 spores/ml) which causes blue mould on Valencia late citrus fruit at different temperatures and different relative humidity (Lahlali et al., 2010). 10°C 15°C 20°C 25°C Slide 45: Use of mixed cultures Widening the spectrum of microbial activity resulting in the control of two or more crop diseases. Increasing the effectiveness under different situations such as cultivars, maturity stages, and locations. Enhancing the efficiency and reliability of biocontrol as the components of the mixtures act through different mechanisms like antagonism, parasitism, and induction of resistance in the host. Combination of different biocontrol traits without the transfer of alien genes through genetic transformation. Slide 46: The enhancement of bio-efficacy of microbial antagonists may be due to: Better utilization of substrate, resulting in: Acceleration of the growth rate. Removal of substances inhibitory to one organism by the other microbial agent. Production of nutrients by one microbe that may be used by another. Formation of more stable microbial community that may exclude other microbes, including pathogens. When selecting microbial mixtures, certain attributes have to be considered: Absence of antagonism between one microbial antagonist against another. Selection of components with positive interactions (mutualism) that allow more effective utilization of resources. Penetration of Radopholus similis into roots of ‘‘Grand Naine’’ banana plants 7 days after inoculation (Chaves et al.,2009). : Penetration of Radopholus similis into roots of ‘‘Grand Naine’’ banana plants 7 days after inoculation (Chaves et al.,2009). Radopholus similis, a migratory endoparasitic nematode of plant roots, penetrates the root system and feed on the cytoplasm of nearby cells, causing cavities and reducing the ability of the plant to take up water and nutrients. Slide 48: Addition of salt additives in the microbial cultures: Calcium chloride. Calcium propionate. Sodium carbonate. Sodium bicarbonate. Potassium metabisulphite. Ethanol. Ammonium molybdate. Slide 49: Addition of nutrients and plant products. Salicylic acid (SA) is a natural phenolic compound present in many plants and is an important component in the signal transduction pathway. Exogenous application of SA has been found to enhance the efficacy of the biocontrol yeast Cryptococcus laurentii in pear, apple and in cherry fruit. Combination of Rhodotorula glutinis yeast with SA provided a more effective control of postharvest gray spoilage and natural spoilage of strawberries than the application of R. glutinis or SA alone (Zhang et al., 2010). Factors involved in the development of a biocontrol product (Droby et al.,2009). : Isolation & Screening Identification & Characterization Mode of Action Bioassay In vitro, In vivo Pilot test Scale up & Formulation Selected Strain Toxicology Commercial test Patent Registration For Use Factors involved in the development of a biocontrol product (Droby et al.,2009). FERMENTATION : The main types of fermentation used for microbial pesticides are: Solid-state. Solid substrate. Submerged. Bi-phasic approaches. A range of substrates have been used in the production of bioherbicides including: Rice. Wheat. Barley. Maize. Millet. FERMENTATION Characteristics of an ideal antagonist for commercial development : Characteristics of an ideal antagonist for commercial development Genetically stable. Effective at low concentration. Not fastidious in its nutrient requirements. Ability to survive adverse environmental conditions. Effective against a wide range of pathogens on different commodities. Amenable to production on inexpensive growth media. Easy to dispense. Not detrimental to human health. Amenable to formulation with a long shelf-life. Resistant to chemicals used in the postharvest environment. Compatible with commercial processing procedures Slide 53: EPA requires the following for microbial pesticides: Product charter and keeping of specimens in a recognized culture collection. Track pathogenicity rather than toxicity (how long does it take for the microbe to clear from a test animal). More extensive non-target testing than for chemical pesticides because these are living organisms capable of reproducing in the field. For example, 30-day feeding studies to assess pathogenicity against ladybeetles, lacewings and bees are required. ADVANTAGES OF BIOPESTICIDES : ADVANTAGES OF BIOPESTICIDES Agents are: Narrowly selective and pose few problems to nontarget organisms including natural enemies . Usually inherently less toxic than conventional chemical pesticides. Decompose quickly, avoiding pollution. DISADVANTAGES OF BIOPESTICIDES : DISADVANTAGES OF BIOPESTICIDES Instability of the protection effect. Have a limited period of activity - and usually are used with normal pesticide application techniques. Difficulty in establishment of the biopesticide agents in the fields. Ambiguity of modes of protection. Low potency. High cost of production. Slide 56: (US $ Billion) (US $ Million) Microbial pest control products released (Bailey et al., 2010) : Microbial pest control products released (Bailey et al., 2010) Slide 60: Thank you You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.