BIOCONTROL OF BACTERIA & PHYTOPATHOGENIC FUNGI

Despite the many achievements of modern agriculture, certain cultaral practices have actually enhanced 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 of nitrogenous fertilizers at concentrations that enhance disease suceptibilily. Plant disease control, therefore, has now become heavily dependent on fungicides to combat the wide variety of fungal diseases that threaten agricultural crops. A land-mark study published by the U.S.Environment Protection Agency (EPA) indicates that, in the U.S. alone, 3000-6000 cancer cases are induced annually by pesticide residues on foods, and another 50-100 by exposure to pesticides during application. This type of findings have made the governments of many countries increasingly aware 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 replacing pesticides with environmentally safer methods are safer methods are currently being conducted at many research centres. The heightened scientific interest is biological control of plant pathogens 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 intensive activity is currently being geared towards the introduction of an increasing number of biocontrol agents into the market. The performance of a bio-control agents into the market. The performance 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, in addition to their antagonistic activity are capable of inducing growth responses by either controlling minor pathogens or by producing growth-stimulating factors.

Before biocontrol can become important component of plant disease management, it must be effective, reliable, consistent and economical to meet these criteria, superior strains, together with delivery systems that enhance biocontrol activity, must be developed. Existing biological control attributes can be enhanced by improving existing, known biological agents, with genetic manipulation. Genetic manipulations of biocontrol agents not only can enhance their activity, but also can expand their spectrum.

The growing interest in biocontrol with micro-organisms is also a response to the new tools of biotechnology plants and micro-organisms can now be manipulated to deliver the same mechanism of biological control, as has been done for the production of the delta endotoxin encoding gene transferred from Bacillus thuringiensis to plants to control insect pests. We can now think of micro-organisms with inhibitary activity against plant pathogens as potential sources of 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 and pseudomonas. Good soil biocontrol systems have been reported for species of Fusarium, Sclerotium, Scierotinia, Pythium and Rhizoctonia. The following biocontrol agents have already been registered; Agrobacterium radiobactor against crown gall (USA, Australia, NZ); Bacillus subtilis for growth enhancement (USA); Pseudomonas fluorescens against bacterial blotch (Australia); Pseudomonas fluorescens for seedling diseases (USA); Peniophora gigantea against Fommes annosus (UK); Pythium Oligandrum against Pythium spp. (USSR); Trichoderma viride against timber pathogens (Europe); Trichoderma spp. For root diseases (USSR); Fusarium oxysporium against Fusarium oxysporum (Japan); Trichoderma harzianum against root diseases (USA); Gliocadium virens for seedling diseases (USA); Trichoderma harzianum/Polysporum against 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 such that following the inducing challenge de novo resistance to pathogens is shown in normally suceptible plants. Both localized and systemic induced resistance are nonspecific and can act against a whole range of pathogens, but whereas localized resistance occurs in many plant species, systemic resistance is limited to some plants. Cross-protection differs from induced resistance in that, following inoculation with avirulent strains of pathogens or other microorganisms, both inducing microorganisms and challenge pathogens occur on or within the protected tissue.

The most commonly reported examples of cross-protection involving fungi are probably those used 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 blight fungus) on European castanea sativa in Italy, where naturally occuring hypovirulent strains were able to reduce the effect of virulent ones. These slower growing hypovirulent strains contain a single cytoplasmic element of double-stranded RNA (ds RNA) similar to that found in mycoviruses, that was transmitted by anastomosis in compatible strains through natural virulent populations of C. Parasitica..

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

C. Competition.

Competition occurs between micro-organisms when space or nutrients (i.e. carbon, nitrogen and iron) are limiting, and its role in the biocontrol of plant pathogens has been studied for many years, with special emphasis on bacterial biocontrol agents. An important attribute of a successful rhizosphere biocontrol agent would be the ability to remain at high population density on the root surface, 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, decreasing the incidence of root disease.

D. Antibiosis

The production of antibiotics by actinomycetes, bacteria and fungi is very simply demonstrated in vivo. Numerous agar plate tests have been developed to detect volatile and non-volatile antibiotic production 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.

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

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

E. Mycroparasitism :

Mycoparasitism occurs when one fungus exists in intimate association with another from which it derives some or all its nutrients while conferring no benefit in return. Biotrophic mycoparasites have a persistant contact with or occupation of living cells, whereas necrotrophic mycoparasites kill the host cells, often in advance of contact & penetration. Mycoparasitism is a commonly observed phenomenon in vitro & in vivo, & its mode of action & its involvement in biological disease control has been reviewed.

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

F Biocontrol of Airborne Diseases :

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

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

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

Biocontrol agents must normally achieve a high population in the phyloshpere to control other strains, but colonization by the agent may be reduced by competition with the indigenous microflora. Integration of chemical pesticides & biocontrol agents have been reported with Trichoderma spp. & P. syringae pv. Biocontrol agents tolerant to specific pesticides could be constructed 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 corresponding gene thereby producing a biological control agent that could be applied simultaneously or in alternation 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 from reaching the pathogen. Biological control agents colonize the rhizosphere, the site requiring protection & leave no toxic residues, as opposed to chemicals.

Micro organisms have been used extensively for the biological control of soilborne plant diseases as well as for promoting plant growth. Fluroscent pseudomonas are the most frequently used bacteria for biological control & plant growth promotion, but bacillus & streptomyces species have also been commonly used. Trichoderma, Gliocadium, and coniothyrium are the most commonly used fungal biocontrol agents. Perhaps the most sucessful biocontrol agent of a soilborne pathogen 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 Plant pathogens as with the pathogens on the phylloplane. Naturally occuring, nonpathogenic strains of Fusarium Oxysporium have been used to control wilt diseases caused by pathogenic Fusarium Spp. Molecular techniques have been used to remove various delterious traits of soilborne Phytopathogenic bacteria to construct a competitive antagonist of the pathogen.

Molecular techniques have also facilitated the introduction of beneficial traits into rhizosphere competent 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 contain cellulose in their cell wall & no appreciable levels of chitin. Biological control of some soilborne fungal diseases has been correlated with chitinase production, bacterial producing chitinases or glucanases exhibit antaganosm in vitro against fungi. A recombinant Escherichia coli expressing the chi A gene from S marcescens was effective in reducing disease incidence caused by screrotium rolfsii & Rhizoctonia solani. In other studies, chitinase genes from S. marcescens have been expressed in Pseudomonas spp. & the plant symbiont Rhizobium meliloti. The modified Pseudomonas 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 tree species; and against rots on a wide range of crops, caused by fusarium, Rhizoctonia, and pythium, and sclerotium forming pathogens such as Sclerotinia & Sclerotium. In many, experiments, showing successful biological control, the antagonistic Trichoderma was mycoparasite.

A. Mechanism of Action :

Form recent work, it appears that mycoparasitism is a complex process, including several successive steps. The first detectable interaction shows that the hyphae of the mycoparasite grows directly towards its host. This phenomenon appears a chemotropic growth of Trichoderma in 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 by forming hook like structures. In this respect, production of appressoria at the tips of short branches has been described for T. hamatum & T. harzianum. The interaction of Trichoderma with its host is specific. The possible role of agglutinins in the recongnition process determining the 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 the host hyphae. R. solani carries a lectin that binds to galactose & fucose residues on the 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 prey recognition by the predator Moreover, because it does not distinguish among biological variants of the pathogen, it enables the Trichoderma species to attack different R. Solani isolates. The activity of a second lectin isolated from S. rolfsii was inhibited by d-glucose or d-mannose residues, 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 that Trichoderma spp. Produced & secreted mycolytic enzymes responsible for the partial degradation of the host's cell wall -

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

The level of hydrolytic enzymes produced differs from host-parasite interaction analyzed. This phenomenon correlates with the ability of each Trichoderma isolate to cotnrol a specific pathogen. It is considered that mycoparasitism is one of the main mechanisms involved in the antagonism of Trichoderma as a biocontrol agent.

The process apparently includes
1) chemotropic growth of Trichoderma,
2) recognition of the host by the mycoparasite
3) secretion of extracellular enzymes,
4) hyphae penetration, and
5) lysis of the host.