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BIOCONTROL OF BACTERIA & PHYTOPATHOGENIC FUNGI

Despite the many achievements of modern agriculture, certain cultaral practices have actuallyenhanced the destructive potential of diseases. These practices include use of genetically similar

crop plants in continuous monoculture, use of plant cultivars suceptible to pathogens, and use ofnitrogenous fertilizers at concentrations that enhance disease suceptibilily. Plant disease control,therefore, has now become heavily dependent on fungicides to combat the wide variety of fungaldiseases that threaten agricultural crops. A land-mark study published by the U.S.EnvironmentProtection Agency (EPA) indicates that, in the U.S. alone, 3000-6000 cancer cases are inducedannually by pesticide residues on foods, and another 50-100 by exposure to pesticides duringapplication. This type of findings have made the governments of many countries increasinglyaware of the drawbacks of many chemical pesticides, in terms of their effect on the environment,as well as on the growers & consumers of agricultural products. Studies aimed at replacingpesticides with environmentally safer methods are safer methods are currently being conductedat many research centres. The heightened scientific interest is biological control of plantpathogens is partly a response to growing public concerns over chemical pesticides.

Biological control is a potent means of reducing the damage caused by plant pathogens.Commercialized systems for the biological control of plant diseases are few. Although intensiveactivity is currently being geared towards the introduction of an increasing number of biocontrolagents into the market. The performance of a bio-control agents into the market. Theperformance of a biocontrol agent cannot be expected to equal that of an excellent fungicide;although some biocontrol agents have been reported to be as effective as fungicide control.

Potential agents for biocontrol activity are rhizosphere-competent fungi & bacteria which, inaddition to their antagonistic activity are capable of inducing growth responses by eithercontrolling minor pathogens or by producing growth-stimulating factors.

Before biocontrol can become important component of plant disease management, it must beeffective, reliable, consistent and economical to meet these criteria, superior strains, together with

delivery systems that enhance biocontrol activity, must be developed. Existing biological controlattributes can be enhanced by improving existing, known biological agents, with geneticmanipulation. Genetic manipulations of biocontrol agents not only can enhance their activity, butalso can expand their spectrum.

The growing interest in biocontrol with micro-organisms is also a response to the new tools ofbiotechnology plants and micro-organisms can now be manipulated to deliver the samemechanism of biological control, as has been done for the production of the delta endotoxinencoding gene transferred from Bacillus thuringiensis to plants to control insect pests. We cannow think of micro-organisms with inhibitary activity against plant pathogens as potential sourcesof genes for disease resistance.

The successful control by biological means in the phylophane that have been reported involve

mainly rusts powdery mildews and diseases caused by following genera of pathogens :Alternaria, Epicoccum, Sclerotinia, Spetoria, Drechisera, Venturia, Plasmopara, Erwinia andpseudomonas. Good soil biocontrol systems have been reported for species of Fusarium,Sclerotium, Scierotinia, Pythium and Rhizoctonia. The following biocontrol agents have alreadybeen registered; Agrobacterium radiobactor against crown gall (USA, Australia, NZ); Bacillussubtilis for growth enhancement (USA); Pseudomonas fluorescens against bacterial blotch(Australia); Pseudomonas fluorescens for seedling diseases (USA); Peniophora gigantea againstFommes annosus (UK); Pythium Oligandrum against Pythium spp. (USSR); Trichoderma virideagainst timber pathogens (Europe); Trichoderma spp. For root diseases (USSR); Fusarium

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oxysporium against Fusarium oxysporum (Japan); Trichoderma harzianum against root diseases(USA); Gliocadium virens for seedling diseases (USA); Trichoderma harzianum/Polysporumagainst wood decay (USA).

I) Mechanism of Biological Control of Plant Diseases.

A. Induced Resistance and cross-protection.

Induced resistance is a plant response to challenge by microorganisms or abiotic agents suchthat following the inducing challenge de novo resistance to pathogens is shown in normallysuceptible plants. Both localized and systemic induced resistance are nonspecific and can actagainst a whole range of pathogens, but whereas localized resistance occurs in many plantspecies, systemic resistance is limited to some plants. Cross-protection differs from inducedresistance in that, following inoculation with avirulent strains of pathogens or othermicroorganisms, both inducing microorganisms and challenge pathogens occur on or within theprotected tissue.

The most commonly reported examples of cross-protection involving fungi are probably thoseused against vascular wilts. Inoculation with nonpathogenic formae speciales of Fusarium and

vernullum species, or with other fungi or bacteria, all have shown different levels of cross-protection.

B. Hypovirulence.

Hypovirulence is a term used to describe reduced virulence found in some strains of pathogens.This phenomenon was first observed in Cryphonectria (Endothia) parasitica (chestnut blightfungus) on European castanea sativa in Italy, where naturally occuring hypovirulent strains wereable to reduce the effect of virulent ones. These slower growing hypovirulent strains contain asingle cytoplasmic element of double-stranded RNA (ds RNA) similar to that found inmycoviruses, that was transmitted by anastomosis in compatible strains through natural virulentpopulations of C. Parasitica..

Hypovirulence has also been reported in many other pathogens, including Rhizoctonia Solani,Gaeumannomyces gramini var. tritici & ophiostoma ulmi, but the transmissible elementsresponsible for hypovirulence or reduced vigor of the fungi are subjected to debate and may bedue to ds RNAs, Plasmids, or viruses.

C. Competition.

Competition occurs between micro-organisms when space or nutrients (i.e. carbon, nitrogen andiron) are limiting, and its role in the biocontrol of plant pathogens has been studied for manyyears, with special emphasis on bacterial biocontrol agents. An important attribute of a successfulrhizosphere biocontrol agent would be the ability to remain at high population density on the rootsurface, providing protection of the whole root for the duration of its life. Mycorrhizal fungi can

also be considered to act as a sophasticated form of competition or cross-protection, decreasingthe incidence of root disease.

D. Antibiosis

The production of antibiotics by actinomycetes, bacteria and fungi is very simply demonstrated invivo. Numerous agar plate tests have been developed to detect volatile and non-volatile antibioticproduction by putative biocontrol agents and to quantity their effects on pathogens. In general,however, the role of antibiotic production in biological control in vitro remains unproved.

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Species of Gliocadium and Trichoderma are well-known biological control agents that produce arange of antibiotics that are active against pathogens in vitro and, consequently, antibioticproduction has commonly been suggested as a more of action for these fungi.

Within bacterial biocontrol agents several species of the genus, Pseudomonas produceantibiotics involved in their ability to control plant pathogens.

E. Mycroparasitism :

Mycoparasitism occurs when one fungus exists in intimate association with another from which itderives some or all its nutrients while conferring no benefit in return. Biotrophic mycoparasiteshave a persistant contact with or occupation of living cells, whereas necrotrophic mycoparasiteskill the host cells, often in advance of contact & penetration. Mycoparasitism is a commonlyobserved phenomenon in vitro & in vivo, & its mode of action & its involvement in biologicaldisease control has been reviewed.

The most common example of mycoparasitism is that of Trichoderma SSP. Which attack a greatvariety of phytopathogenic fungi responsible for the most important diseases suffered by crops ofmajor economic importance worldwide.

F Biocontrol of Airborne Diseases :

Many naturally occuring microorganisms have been used to control diseases on the aerialsurfaces of plants. The most common bacterial species that have been used for the control ofdiseases in the phylloshpere include Pseudomonas syringae, P. fluorescens, P. cepacia, Erwiniaherbicola, and Bacillus subtilis, Fungal genera that have been used for the control of air bornediseases include Trichoderma Ampelomyces, and the yeasts Tilletiopsis & sporobolomyces.

Phytopathogenic bacteria possess serveral genes that encode phenotypes that allow them toparasitize plants & overcome defense responses elicited by the plant. In addition,phytopathogenic bacterial possess pathogenicity genes such as hrp. Isogenic avirulent mutantscan be produced by insertional inactivation of genes involved in pathogenicity. Nonpathogenicmutants of Erwinia amylovora, produced by transposon mutagenesis, have also been used in thebiological control of fire blight.

Antibiosis has been proposed as the mechanism of control of serveral bacterial & fungal diseasesin the phyllosphere. Molecular biology techniques could be used to enhance the efficacy ofbiocontrol agents that use antibiosis as a more of action.

Biocontrol agents must normally achieve a high population in the phyloshpere to control otherstrains, but colonization by the agent may be reduced by competition with the indigenousmicroflora. Integration of chemical pesticides & biocontrol agents have been reported withTrichoderma spp. & P. syringae pv. Biocontrol agents tolerant to specific pesticides could beconstructed using molecular techniques. Resistance to the fungicide benomyl is conferred by a

single amino acid substitution in one of the B-tubulins of Trichoderma viridae. The correspondinggene thereby producing a biological control agent that could be applied simultaneously or inalternation with the Fungicide.

G. Biocontrol of Soil borne Disease

Chemical control of soil borne plant diseases is frequently ineffective because of the physical &chemical heterogeneity of the soil , which may prevent effective concentrations of the chemical

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from reaching the pathogen. Biological control agents colonize the rhizosphere, the site requiringprotection & leave no toxic residues, as opposed to chemicals.

Micro organisms have been used extensively for the biological control of soilborne plant diseasesas well as for promoting plant growth. Fluroscent pseudomonas are the most frequently usedbacteria for biological control & plant growth promotion, but bacillus & streptomyces species have

also been commonly used. Trichoderma, Gliocadium, and coniothyrium are the most commonlyused fungal biocontrol agents. Perhaps the most sucessful biocontrol agent of a soilbornepathogen is Agrobacterium radiobactor strain K84, used against crown gall disease caused by A-tumefaciens:

Competition as a mechanism of biological control has been exploited with soil borne Plantpathogens as with the pathogens on the phylloplane. Naturally occuring, nonpathogenic strains ofFusarium Oxysporium have been used to control wilt diseases caused by pathogenic FusariumSpp. Molecular techniques have been used to remove various delterious traits of soilbornePhytopathogenic bacteria to construct a competitive antagonist of the pathogen.

Molecular techniques have also facilitated the introduction of beneficial traits into rhizospherecompetent organisms to produce potential biocontrol agents. Chitin & b -(1,3) - glucan are the two

major structural components of many plant Pathogenic fungi, except by oomycetes, which containcellulose in their cell wall & no appreciable levels of chitin. Biological control of some soilbornefungal diseases has been correlated with chitinase production, bacterial producing chitinases orglucanases exhibit antaganosm in vitro against fungi. A recombinant Escherichia coli expressingthe chi A gene from S marcescens was effective in reducing disease incidence caused byscrerotium rolfsii & Rhizoctonia solani. In other studies, chitinase genes from S. marcescens havebeen expressed in Pseudomonas spp. & the plant symbiont Rhizobium meliloti. The modifiedPseudomonas strain controlled the pathogen F. Oxysporium f. species rodelens &Gauemannomyces graminis var. tritici.

III) The Trichoderma system -

Trichoderma spp. act against a range of economically important aerial & soilborne plant

pathogens. They have been used in the field & greenhouse against silver leaf on plum, peach &nectarine; Dutch elm disease on elm's honey fungus (Armillaria mellea) on a range of treespecies; and against rots on a wide range of crops, caused by fusarium, Rhizoctonia, andpythium, and sclerotium forming pathogens such as Sclerotinia & Sclerotium. In many,experiments, showing successful biological control, the antagonistic Trichoderma wasmycoparasite.

A. Mechanism of Action :

Form recent work, it appears that mycoparasitism is a complex process, including severalsuccessive steps. The first detectable interaction shows that the hyphae of the mycoparasitegrows directly towards its host. This phenomenon appears a chemotropic growth of Trichodermain response to some stumuli in the hosts's hyphae or toward a gradient of chemicals produces by

the host.

When the mycoparasite reaches the host, its hyphae often coil around it or are attached to it byforming hook like structures. In this respect, production of appressoria at the tips of shortbranches has been described for T. hamatum & T. harzianum. The interaction of Trichodermawith its host is specific. The possible role of agglutinins in the recongnition process determiningthe fungal specificity has been recently examined. Indeed, recognition between T. harzianum &two of its major hosts, R. solani & S. rolfsii, was controlled by two different lectins present on thehost hyphae. R. solani carries a lectin that binds to galactose & fucose residues on the

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Trichoderma cell walls. This lectin agglutinates conidia of a mycoparasitic strain of T. harzianum,but did not agglutinate two non-parasitic strains. This agglutinin may play a role in preyrecognition by the predator Moreover, because it does not distinguish among biological variantsof the pathogen, it enables the Trichoderma species to attack different R. Solani isolates. Theactivity of a second lectin isolated from S. rolfsii was inhibited by d-glucose or d-mannoseresidues, apparently present on the cell walls of T. harzianum.

Following these interactions the mycoparasite sometime penetrates the host mycelium,apparently by partially degrading its cell wall Microscopic observations led to the suggestion thatTrichoderma spp. Produced & secreted mycolytic enzymes responsible for the partial degradationof the host's cell wall -

The Complexing & diversity of the chitinolitic system of T. harzianum involves the complementarymodes of action of six enzymes, all of which might be required for maximum efficiency against abroad spectrum of chitin-containing plant pathogenic fungi.

The level of hydrolytic enzymes produced differs from host-parasite interaction analyzed. Thisphenomenon correlates with the ability of each Trichoderma isolate to cotnrol a specificpathogen. It is considered that mycoparasitism is one of the main mechanisms involved in the

antagonism of Trichoderma as a biocontrol agent.

The process apparently includes1) chemotropic growth of Trichoderma,2) recognition of the host by the mycoparasite3) secretion of extracellular enzymes,4) hyphae penetration, and5) lysis of the host.

The involvement of volatile & nonvolatile antibiotics in the antagonism by Trichoderma has beenproposed. Indeed some isolates of Trichoderma excrete growth-inhibitary substances. Thus, thebiocontrol ability of Trichoderma strains is most likely conferred by more than one exclusivemechanism. In fact, it seems advantageous for a biocontrol agent to supress a plant pathogen

using multiple mechanisms.