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BIOLOGICAL CONTROLThe major methods of pest control can be grouped into three categories of (1) physical control, (2) chemical control and (3) biological control. Biological control is the control of one organism by another (Beirner, 1967). This control may be expressed as either a longer population of the pest (DeBach, 1964) or as a restriction or prevention of the severity or incidence of pest damage without regard to the pest population (Cook and Baker, 1983). Biological control depends on knowledge of biological interactions at the ecosystem, organism, cellular, and molecular levers and often is more complicated to manage compared with physical and chemical methods. Biological control is also likely to be less spectacular than most physical or chemical controls but is usually also more stable and longer lasting (Baker and Cook, 1974). In spite of biological controls having been used in agriculture for centuries, as an industry biological control is still in its infancy. Biological control is now being considered for an increasing number of crops and managed ecosystems as the primary method of pest control.Biological control was discovered by trial and error and then practiced in agriculture long before the term itself came into use (Baker and Cook, 1974). One example is the ancient practice of not growing the same crop species in the same field more frequently than every second or third year or even longer. Such crop rotation allows time for the pest or pathogen population in soil to decrease below some economic threshold because of the predatory, competitive, and other antagonistic effects imposedby the associated microflora and fauna. In other words, crop rotation allows time for the natural soil microbiota to sanitize the soil, especially with regard to the more specialized plant parasites and insect pests that are highly dependent on their host cropto maintain their populations. The era of modern biological control, involving the deliberate transfer andintroduction of natural enemies of insect pests, was launched 100 years ego with the highly successful introduction of the vadalia beetle from Australia to California in 1888 to control the cottony cushion scale of citrus. In 1914, the German plant pathologist C. F. von Tubuef wrote a somewhat speculative article entitled"Biologische Bekampfung von Pilzkrankheiten der Pflanzen." This is apparently the first reference in the scientific literature to the term "Biologische Bekampfung" or "biological control" (Baker, 1987).DeBach (1964) defined biological control as "the action of parasites, predators, or pathogens in maintaining another organism's population density at a longer average than would occur in their absence." This definition covers some highly successful biological controls of insect pests with natural enemies, but it does not accommodate some other highly successful controls accepted in other disciplines as examples ofbiological control. For example, citrus tristeza virus is controlled in Brazil by inoculating the citrus trees with a mild virus, which then protects the trees against the more severe strains (Costa and Muller, 1980). "Cross protection" was first shown by H. H. McKinney in 1929 to have potential for biological control of plant viruses. Plant pathologists refer to cross protection for control of plant viruses as biological controlMerits of Biocontrol Agents(1) Biological control is less costly and cheaper than any other methods.(2) Biocontrol agents give protection to the crop throughout the crop period.(3) They do not cause toxicity to the plants.(4) Application of biocontrol agents is safer to the environment and to the personwho applies them.(5) They multiply easily in the soil and leave no residual problem.(6) Biocontrol agents not only control the disease but also enhance the root andplant growth by way of encouraging the beneficial soil micro flora. It increasesthe crop yield also.(7) Biocontrol agents are very easy to handle and apply to the target.(8) Biocontrol agent can be combined with bio-fertilizers.

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(9) They are easy to manufacture.(10) It is harmless to human beings and animals (Environmentally safe.)Lytic enzymes: Lysis is the complete or partial destruction of a cell by enzymes.Lysis may be distinguished into two types, endolysis and exolysis. Endolysis(autolysis) is the breakdown of the cytoplasm of a cell by the cell’s own enzymesfollowing death, which may be caused by nutrient starvation or by antibiosis or othertoxins. Endolysis does not usually involve the destruction of the cell wall. Exolysis(heterolysis) is the destruction of cell by the enzymes another organism. Typicallyexolysis is the destruction of the walls of an organism by chitinases, cellulases etc. andthis frequently results in the death of the attacked cell.Hydrogen cyanide: Many rhizobacteria produce hydrogen cyanide and this hasbeen shown to play direct as well as indirect role in biological control of plant diseasesand increasing the yields. The florescent pseudomonas themselves produce HCN andare able to suppress the pathogens.Induced Systemic Resistance (ISR): ISR is the ability of an agent (a fungus, bacteria, virus, chemical etc.) to induce plant defense mechanisms that lead to systemic resistance to a number of pathogens. Inoculation of plants with weak pathogens or non- pathogens leads to induced systemic plant resistance against subsequent challenge by pathogens. The mechanisms remain largely unknown but typically the induced resistance operates against a wide range of pathogens and can persist for 3-6 weeks. The biocontrol agents bring about induced systemic resistance (ISR) through fortifying the physical and mechanical strength of cell wall as well as changing physiological and biochemical reaction of host leading to the synthesis of defense chemicals againstchallenge inoculation of pathogens. Defense reaction occurs due to accumulation of PR proteins (chitinase, B-1, 3 glucanse), chalcone synthase, phenylalanine ammonia lyase, peroxidase, phenolics, callose, lignin and phytoalexins.Plant growth promotion: Biocontrol agents also produce growth hormones like, Auxins, Cytokinin, Gibberellins etc. These hormones suppress the deleterious pathogens and promote the growth of plants and simultaneously increase the yield. The studies on mechanism of growth promotion indicated that PGPR promotes plant growth directly by production of plant growth regulators or indirectly by stimulating nutrient uptake, by producing siderophores or antibiotics to protect plant from soil borne pathogens or deleterious rhizosphere organisms. Pseudomonas spp. May increase plant growth by producing gibberellins- like substances, mineralizing phosphates.

Mode of Action of Biocontrol AgentsCompetition: Microorganism competes for space, minerals and organic nutrients toproliferate and survive in their natural habitats. This has been reported in both rhizosphere as well as phyllosphere. Competition has been suggested to a play a role in the biocontrol of species of Fusarium and Pythium by some strains of fluorescent pseudomonas. Competition for substrates is the most important factor for heterotrophic soil fungi. Success in saprophytic ability (CSA) and inoculums potential of that species. Those fungi with highest number of propagules or the greatest mass of mycelia growth have the greatest competitive advantage. Competitive saprophytic ability is the summation of physiological characteristics that make for success in competitive colonization of dead organic substrates.Antibiosis: Antibiosis is defined as antagonism mediated by specific or non – specific metabolites of microbial origin, by lytic agents, enzymes, volatile compounds or other toxic substances. Antibiosis plays an important role in biological control. Antibiosis is a situation where the metabolites are secreted by underground parts of plants, soil microorganism, plant residues etc. It occurs when the pathogen is inhibited or killed by metabolic

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products of the antagonists. The products include the lyric agents, enzymes, volatile compounds and other toxic substances.Mycoparasitism / Hyperparasitism: Mycoparasitism or hyperparasitism occurs when the antagonist invades the pathogens by secreting enzymes such as chitinases, celluloses, glucanases and other lytic enzymes. Mycoparasitism is the phenomenon of one fungus being parasitic on another fungus. The parasiting fungus is called hyperparasite and the parasitized fungus as hypoparasite. In mycoparasitism, two mechanisms operate among involved species of fungi. This may be hyphal of interfungus interaction i.e., fungus-fungus interaction, several events take place which lead to predation viz., coiling, penetration, branching and sporulation, resting body production, barrier formation and lyses.

CONTROL THROUGH USE OF TRANSGENIC BIOCONTROL MICROORGANISMSAlthough the mechanisms by which biocontrol organisms affect the pathogens against which they are usedare not well understood, it has become apparent that at least some of them produce antibiotics toxic to thepathogens, some produce enzymes that attack structural features, e.g., the cell wall of pathogens, some compete with the pathogen for space, nutrients, and water, and so on. Genetic engineering techniques have been used to add new genes or to enhance the genetic makeup of the biocontrol organism so that it may better attack the pathogen. Such genes include plant or microbe genes that code for toxins, enzymes, and other compounds that affect the pathogen adversely, or regulator genes that overexpress appropriate biocontrol genes already present in that organism.

DIRECT PROTECTION OF PLANTS FROM PATHOGENSDirect protection of plants from pathogens can be achieved in a few cases by biological controls (fungal and bacterial antagonists).Biological control practices for direct protection of plants from pathogens involve the deployment of antagonistic microorganisms at the infection court before or after infection takes place. The mechanisms employed by biocontrol organisms in weakening or destroying the plant pathogens they attack are primarily their ability to parasitize the pathogens directly, production of antibiotics (toxins) against the pathogens, their ability to compete for space and nutrients and to survive in the presence of other microorganisms, production of enzymes that attack the cell components of the pathogens, induction of defense responses in the plants they surround, metabolism of plant produced stimulants of pathogen spore germination, and possibly others. Although thousands of microorganisms have been shown to interfere with the growth of plant pathogens in the laboratory, greenhouse, or field and to provide some protection from the diseases caused by them, strains of relatively few microorganisms have been registered and are available commercially for use so far. The most commonly used microorganisms include three fungi: Gliocladium virens, sold as GlioGard for the control of seedling diseases of ornamental and bedding plants; Trichoderma harzianum, sold as F-Stop, for the control of several soilborne plant pathogenic fungi; and Trichoderma harzianum/T. polysporum, sold as BINAB T, for the control of wood decays. The other three commercially available microorganisms are bacteria: Agrobacterium radiobacter K-84, sold as Gallex or Galltrol for use against crown gall; Pseudomonas fluorescens, sold as Dagger G for use against Rhizoctonia and Pythium damping-off of cotton; and Baccillus subtilis, sold as Kodiak and used as a seed treatment. Although the actual use of these biological control products is rather limited, it is expected that these and other such products will find wide acceptance and will fill a real need in the not too distant future.

Fungal Antagonists-Biocontrol of Heterobasidion (Fomes) annosum by Phleviopsis (Peniophora) giganteaHeterobasidion annosum, the cause of root and butt rot of conifer trees infects freshly cut pinestumps and then spreads into the roots. Through the root contacts, it then spreads into the roots of standing trees, which it kills. If the stump surface is inoculated with oidia of the fungus Phleviopsis (Peniophora)

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gigantean immediately after the tree is felled, Phleviopsis occupies the cut surface and spreads through the stump into the lateral roots. There, it competes with successfully and excludes or replaces the pathogenicHeterobasidion in the stump, thereby protecting nearby trees. Oidia are applied to the cut surface either as a water suspension or as a powder or they are added to the lubricating oil placed on the chain saw andare thus deposited on the surface as it is cut

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Biocontrol of Chestnut Blight with HypovirulentStrains of the PathogenChestnut blight, caused by the fungus Cryphonectria (Endothia) parasitica, is controlled naturally in Italy andartificially in France through inoculation of cankers caused by the normal pathogenic strains of the fungus, with hypovirulent strains of the same fungus. The hypovirulent strains carry virus-like double-strandedRNAs (dsRNAs) that apparently limit the pathogenicity of the virulent strains. The dsRNAs apparently passthrough mycelial anastomoses (fusions) from the hypovirulent to the virulent strains, the latter are renderedhypovirulent, and the development of the canker slows down or stops. Hypovirulent strains are also beingtested in the United States, but so far the control of chestnut blight has been limited to experimental trees.It appears that U.S. strains are more variable than European ones and require multiple hypovirulence factors,therefore, the interaction is more strain specific.

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Biological Control of Soilborne DiseasesPrincipal fungi used as biological control agents against soilborne diseases include the two mentionedearlier as being used commercially, namely Gliocladium virens and Trichoderma harzianum. They are used inpotting mixes, are mixed with soil, or are used as solid matrix in seed-priming treatments. They are effectiveagainst damping-off diseases of ornamentals and vegetables caused by the oomycetes Pythium and Phytophthora, by the fungi Botrytis and Sclerotium, and some other fungi. In addition, Sporidesmium sclerotivorum, Coniothyrium minitants, Talaromyces flavus, and others give experimental control of some diseases caused by Sclerotinia, Rhizoctonia, and Verticillium.Some species of Pythium, such as Pythium nunn and P. oligandrum, protect potted ornamentals and vegetables from plant pathogenic species of Pythium, whereas T. flavus and binucleate Rhizoctonias protectplants from the pathogenic Rhizoctonia solani.

Fusarium wilts of several crops, such as celery, cucumber, and sweet potato, caused by the respective formae specialis of Fusarium oxysporum have been reported to be controlled successfully by inoculating transplants or cuttings with nonpathogenic strains of the same fungus. Some of these strains have been isolated from the vascular tissue of host plants that remained healthy while nearby plants had been killed by the wilt-inducingstrains of the fungus. It is believed that the nonpathogenic strains not only compete with the pathogenic ones

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in the rhizosphere and for infection sites, but they also enhance the resistance of the host toward the pathogenic strains.Mycorrhizae colonize roots intercellularly (ectomycorrhizae) or intracellularly (endomycorrhizae). Although mycorrhizae obtain organic nutrients from the plant, they benefit the plant by promoting nutrient uptake andenhancing water transport by the plant, thus increasing growth and yield, and sometimes by providing the plant with considerable protection against several soilborne pathogens. Mycorrhizae have been shown to provide considerable protection to pine seedlings from Phytophthora cinnamomi, to tomato and to cotton from Verticillium wilt and the root-knot nematode, and to soybean from Phytophthora megasperma and Fusarium solani. Commercial preparations of certain mycorrhizal fungi are available for promoting growth of the host plants; however, problems of production, specificity, and application of mycorrhizae to plants remain and, therefore, they are not used with the goal of protecting plants fromtheir pathogens.Biological Control of Diseases of Aerial Plant Partswith FungiMany filamentous fungi and yeasts have been shown to be effective antagonists of fungi infecting the aerialparts of plants. For example, inoculation of postbloom, dead tomato flowers with conidia of Cladosporiumherbarum or Penicillium sp. almost completely suppressed the subsequent infection of developing fruits byBotrytis cinerea. Similarly, spraying spores of common bark saprophytes, such as Cladosporium sp. and Epicoccum sp., and of the soil fungus Trichoderma on pruning cuts of fruit trees has prevented infection bycanker-causing pathogens such as Nectria galligena and Leucostoma (Cytospora) sp. Sprays with Trichodermain the field also reduced Botrytis rot of strawberries and of grapes at the time of harvest and in storage. Sclerotinia head rot of sunflower was reduced significantly by releasing into the field honeybees that had been previously contaminated heavily with spores of the biocontrol fungi Trichoderma spp., which the honeybees delivered promptly to the flowers. Several foliar diseases have also been reduced significantly (by more than 50%) when the leaves were sprayed with spores of common phylloplane fungi, e.g., Alternaria, Cochliobolus, Septoria, Colletotrichum, and Phoma or with spores of hyperparasites, e.g., the cucumber powdery mildew fungus Sphaerotheca fuliginea with spores of Ampelomyces quisqualis or Tilletiopsis, the wheat leaf rust fungus Puccinia triticina with spores of Darluca filum, and the carnation rust fungus with Verticillium lecanii. None of the aforementioned nor any of the numerous other known cases of fungal antagonism by fungi are used as yet for the practical control of any disease of aerial plant parts.Biological Control of Postharvest DiseasesPostharvest rots of several fruits could be reduced considerably by spraying the fruit with spores of antagonistic fungi and saprophytic yeasts at different stages of fruit development, or by dipping the harvested fruit in the inoculum. For example, yeast treatments reduced postharvest rotting of peach and apple. Also, significant reduction of citrus green mold (caused by Penicillium digitatum) was obtained by treating the fruit with antagonistic yeasts or the fungal antagonist Trichodermaviride, whereas preharvest and postharvest Botrytis rot of strawberries was reduced by several sprays of Trichoderma spores on strawberry blossoms and young fruit. Penicillium rot of pineapple wasreduced considerably by spraying the fruit with nonpathogenic strains of the pathogen. Similarly, severalantagonistic yeasts protected grapes and tomatoes from Botrytis, Penicillium and Rhizoctonia rots. One such yeast, Candida saitoana, controlled postharvest decay of apple fruit by inducing systemic resistancein apple fruit while at the same time increasing chitinase and b-1,3-glucanase activities in the fruit. In addition, the yeast Candida oleophila was approved for postharvest decay control in citrus and apples under the trade name Aspire.

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Bacterial AntagonistsBiocontrol of Soilborne DiseasesCrown gall of pome, stone, and several small fruits (grapes, raspberries) and ornamentals (rose and euonymous) is caused by the bacterium Agrobacterium tumefaciens. Crown gall can be controlled commercially by treating the seeds, seedlings, and cuttings with Galltrol, a suspension of strain K84 of the related but nonpathogenic bacterium Agrobacterium radiobacter. Control is based on production by strain K84 of a bacteriocin, called agrocin 84, which is an antibiotic specific against related bacteria. The bacteriocin selectively inhibits most pathogenic agrobacteria that arrive at surfaces occupied by strain 84. Because strains of A. tumefaciens insensitive to agrocin 84 were produced as a result of the natural transfer of the K84 gene for resistance to agrocin 84, a new strain (K-1026) was produced from K84 through genetic engineering so that the latter (K- 1026) lacks the ability to transfer its resistance gene to pathogenic Agrobacterium strains.Treatment of seeds such as cereals, sweet corn, and carrots with water suspensions, slurries, or powders containing the bacteria Bacillus subtilis strain A13 or Streptomyces sp. has protected the plants against rootpathogens and has resulted in better growth and yield of these crops. Pseudomonas rhizobacteria, primarily of the P. fluorescens, P. putida, P. cepacia, and P. aureofaciens groups, applied to seeds, seed pieces, and roots of plants have resulted in less damping-off, less soft rot, and consistent increases in growth and yield in

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several crops. Formulations of two of the aforementioned bacteria are sold commercially: B. subtilis is sold as Kodiak and P. fluorescens is sold as Dagger G. All have given good results in experimental trials but have, in general, given inconsistent results in large-scale trials.Biological Control of Diseases of Aerial Plant Parts with BacteriaNumerous bacteria, most of them saprophytic gramnegative bacteria of the genera Erwinia, Pseudomonas,and Xanthomonas and a few of the gram-positive genera Bacillus, Lactobacillus, and Corynebacterium, are found on aerial plant surfaces, particularly early in the growing season. Some pathogenic bacteria, such as Pseudomonas syringae pv. syringae, P. syringae pv. morsprunorum, P. syringae pv. glycinea, Erwiniaamylovora, and E. carotovora, also live epiphytically (on the surface) on leaves, buds, and so on before they infect and cause disease. In several cases, spraying leaf surfaces with preparations of saprophytic bacteria orwith avirulent strains of pathogenic bacteria has reduced considerably the number of infections caused by bacterial and fungal pathogens. For example, fire blight of apple blossoms, caused by E. amylovora, was partially controlled with sprays of Erwinia herbicola; and bacterial leaf streak of rice, caused by Xanthomonas translucens ssp. oryzicola, was reduced with sprays of isolates of Erwinia and of Pseudomonas. Several cases of added epiphytic bacteria inhibiting plant infections by fungi are known. For example, spraying grass plants with Pseudomonas fluorescens reduced infection by Drechslera (Helminthosporium) dictyoides, and spraying with Bacillus subtilis reduced infection of apple leaf scars by Nectria galligena and of grape by Eutypa lata. Similarly, spraying of peanut or tobacco plants with Pseudomonas cepacia or Bacillus sp. Reduced Cercospora and Alternaria leaf spot on these hosts, respectively. None of these biological controls is used in practice to control any disease of aerial plant parts so far.Biocontrol of Postharvest DiseasesPseudomonas bacteria protected lemons from Penicillium green mold and pear from various storage rots.Two Pseudomonas syringae strains have been approved for postharvest decay control in citrus, apples (Fig. 9- 22B), and pears under the trade name Bio-Save. When several kinds of stone fruits, namely peaches, nectarines, apricots, and plums, were treated after harvest with suspensions of the antagonistic bacterium Bacillus subtilis, they remained free of brown rot, caused by the fungus Monilinia fructicola, for at least nine days (Fig. 9-22C). Bacillus subtilis also protected avocado fruit from storage rots.Biocontrol with Bacteria of Bacteria-Mediated Frost InjuryFrost-sensitive plants are injured when temperatures drop below 0°C because ice forms within their tissues.Small volumes of pure water can be supercooled to -10°C or below without ice formation, provided no catalyst centers or nuclei are present to influence ice formation. It has been shown, however, that certainstrains of at least three species of epiphytic bacteria (P. syringae, P. fluorescens, and E. herbicola), which arepresent on many plants, serve as ice nucleation-active catalysts for ice formation at temperatures as high as-1°C. Such bacteria usually make up a small proportion (0.1–10%) of the bacteria found on leaf surfaces. Byisolating, culturing, mass producing, and applying non-ice nucleation-active bacteria antagonistic to icenucleation- active bacteria on the plant surfaces, it has been possible to reduce and replace large numbers of ice nucleation-active bacteria on treated plant surfaces with non-ice nucleation-active bacteria. This treatment protects frost-sensitive plants from injury at temperatures at which untreated plants may be severely injured.

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