Applications of Biological Control in Field

Practical application is the translation of in vitro experiment into in vivo conditions. Owing to variations in environmental factors, sometimes, antagonistic potential of certain biocontrol agents fails in in vivo conditions. Now-a-days researches on induction of antagonistic potential and production of mutants adjustable to stress conditions are being carried out in most of the countries.

It is obvious that a suitable antagonist, present in rhizosphere, fails to antagonise the pathogen and control the disease, possibly due to its low inoculum potential. Some of the methods are now available which (i) can decrease virulence of the pathogens, (ii) increase hosts resistance, and (iii) stimulate the antagonistic potentialities and intensify their activity. These may be accomplished by one or more methods (Table 13.3).

Crop rotation
Crop rotation is the oldest and best example of the known biological control methods, which is practiced by the farmers in India and other countries. On rotating a crop in a field, followed by other crop, alteration in gross microbial community in soil is done. By doing so, inoculum density of a pathogen is lowered. Sometimes crop rotation has a long term effect. Microbial shift results in increased number of beneficial microbes, and decreased number of pathogens. For example, rotation of wheat with leguminous crops affects take-all of wheat. Leaf spot of pea nut caused by Cercospora sp. is reduced by 88 per cent on rotation with maize or soybean.

Irrigation
Irrigation practice is a versatile mean of manipulation of microbial community in soil. Many microorganisms, at high moisture level do not grow due to oxygen stress in soil. Consequently, anaerobic bacteria rapidly grow and participate in the antagonism. For example, practice of raised soil beds for controlling verticillium wilt of cotton, and root knot of cotton (caused by nematodes) are done in some countries.

Table 13.3. Examples of biological control of soil-borne plant pathogens. Source : Various research and review papers of mycology and plant pathology.

Alteration of soil pH
Many antagonistic fungi luxuriantly grow at low soil pH 4.0 and many more⁵ do not. By addition of certain fertilizers, such as ammonium sulphate, sodium nitrate, calcium nitrate, ammonium chloride and lime, soil pH is considerably changed and there develops a chance for change in community size. For example, soil acidity favors Fusarium solanif. s. phaseoli and Rhizoctonia solani but suppresses S. scabies and Verticillium albo-atrum. Take-all of wheat is more severe at alkaline pH. Fertilizers applied on forage directly affect root exudation and in turn on rhizosphere microflora (Rovira, 1965).

Organic amendments
Organic materials viz., crop residue, chitin, chopped leguminous hay, green manures, oil cake powders, rice husk, wheat and paddy straw and compost manures, when applied in field soil, bring about changes in the spectrum of micropial community and intensify their activities. Baker and Nash (1965) noticed the control of F.solani f. phaseoli causing disease on bean root with a high C: N ratio. Take-all of wheat was controlled by amending soil with gelatin (Juan and Lamaire, 1974). Baker and Cook (1974) have discussed that the amendments and crop residues can and should be one of our best weapons for elevating soil-borne plant disease without seriously polluting the soil environment with pesticides.

Treatment of soil with selected chemicals
The idea of recolonization and survival of treated soil with some chemicals by microorganism was given by Bliss (1951). He found that in soil treated with carbon disulphide for the control of Annillaria mellea from infected citrus root, population of T. viride rapidly increased. Consequently, A. mellea was killed directly by fungicidal action of carbon disulphide and necrotrophic activity of T. viride.

Pathak and Dwivedi (1981) have reported that population of antagonistic fungi, such as Aspergillus terreus, Penicillium citrinum Cephalosporium roseogriseum, T. viride etc. increased in soil treated with fungicides, insecticides and herbicides; consequently the wilt of tomato caused by Fusarium oxysporumf. lycopersici decreased. Increase in numbers of Aspergillus, Penicillium and Trichoderma in soil treated with benomyl have also been reported by People (1974). However, in biocontrol systems, pesticide-stimulated antagonists in soil may help in the integrated system of disease control (Marois and Mitchel, 1981).

Introduction of antagonists
In this method aim lies in the introduction of such antagonists that can intensify microbial interaction resulting in control of disease and or disease causing organism. A potential antagonist is isolated from a specialized niche (a niche is the habitat of organisms where they live and function with respect to other organisms and the environment), artificially multiplied on nutrient media and introduced in the same habitat for microbial interactions and control of a particular disease. It is unlikely that an antagonist can be applied for a number of disease/pathogen in a varied habitats. The means of introducing antagonists are seed inoculation, vegetative part inoculation and soil inoculation.

In recent years, several commercial products from microorganisms such as Suppresivit, Tris 002, Ecofit, SoilGard, Protus, etc. (Table 13.4) have been prepared by several companies to use them for the control of plant pathogens and diseases.

Table 13.4. Some of the biocontrol products employed. * Wettable powder; G, granules.

(i) Seed inoculation. Nowadays seed inoculation technology is developed in India and other countries as well. Different strains of Rhizobium spp. are now available which have been developed in a view of increased nodulation, nitrogen fixation and yield (Bacterization). It helps in disease control also. However, seeds of barley, wheat and oat soaked in aqueous suspension of Bacillus subtilis (10⁶ - 10⁷ cell/ml) and grown in fields infested with R. solani, Pythium sp. and Fusarium sp. gave a good result; wheat and oat showed increased tillering and dry weight, and barley and oat gave up to 90 per cent increase in yield. Similarly water suspension of B. subtilis (10⁶-3 x 10⁷ cell) and Streptomyces sp. (5 x 10⁴-3 x 10⁵ cells) applied on seeds of oat resulted in an increased yield by 40 per cent and 45 per cent respectively (Merriman et al., 1974).

Moreover, activity of antagonists can be stimulated by adopting both seed dressing and chemical treatment methods where individually seed treatment (seed dressing with microorganism) or chemical treatment fails to control the disease.

(ii)Vegetative part inoculation* When the vegetative parts of plant are wounded or plant is felled, the fresh wound releases an adequate amount of nutrients which serve as substrate for the microorganisms coming to its contact. Thus, the wounds support for rapid growth and sporulation of pathogens. Also the antagonists colonize the wound in the same manner. Therefore, inoculation of wounds with antagonistic saprophytes for disease control are now been practiced in many countries. Some of the examples are discussed in this connection.

Research work of Risbeth (1963) gives a most successful example for control of invasion of vegetative parts of pine (freshly cut pine stumps) by Fomes annosus, by sprinkling pellets of spores of Peniophora gigantea. This method is widely practiced in England.

The freshly cut surfaces of carnation cuttings when transplanted into soil were infested by Fusarium roseum, f. cerealis, a major uncontrolled disease. The protection of carnation was provided to the growing plants by inoculating the freshly cut surfaces with suspension of B. subtilis (Aldrich and Baker, 1970).

Trichoderma viride and B. subtilis are used to protect pruning wounds on apple tree shoots from the invasion by Nectria galligena causing cankers on it (Cook and Hunter, 1978). Inoculation of drill wounds in red mapple with conidia of T. harzianum in glycerol gave the complete protection against invasion by hymenomycetes within 21 months after treatment, (Pottle, et al., 1977). Control of Agrobacterium tumifaciens causing crown gall on many plants was done by its another species, A. radiobacter on artificially made wounds on roots of tree seedlings (Moore, 1977). A. radiobacter produced an unusual antibiotic substance which selectively inhibited the stain of A. tumifaciens. This antibiotic is known as nucleotide bacteriocins. A bacteriocin can be defined as "an antibiotic produced by certain strains of bacteria which is active against other strains of the same or closely related species" (Kerr, 1982).

(iii) Soil inoculation. Soil is a unique habitat which harbors a vast majority of microorganisms in a continuous dynamic state by actions and interactions. Now-a-days researches on introduction of antagonists in soil for disease control are in progress. Damping off of seedlings caused by Pythium ultimum and R. solani was reduced by introduction of Bacillus or Streptomyces species into the steamed soil (Broadbent et al., 1971). Broadbent et al., (1974) made an extensive trial in New South Wales Nursery to determine the feasibility of inoculated soil with bacteria to increase the growth rate of plant seedlings. Consequently, germination of portulaca, delphinium, egg plant and cabbage seeds was increased by Bacillus sp.

Gindrat (1979) has discussed the satisfactory requirements for microbiological preparations at commercial levels as below: (i) a standardized and dosable content of antagonist, (ii) the antagonists must remain viable for several months, (iii) excessive contamination by foreign microorganisms should be avoided because of the possible influence on the activity of the antagonists and (iv) the antagonists must be absolutely non-pathogenic on plants.

Use of mycorrhizal fungi
Use of vesicular-arbuscular mycorrhizal (VAM) fungi in biocontrol of soil-borne plant pathogens has increased its significance in recent years. Ricard and Bollen (1968) designated the mycorrhizal fungi controlling diseases as "immunizing commensals ". A VAM fungus, Glomus mosseae, increased the resistance of¹ tobacco roots to infection by Thielaviopsis basicola by increasing the arginine contents in roots. This amino acid inturn inhibited chlamydospore formation by the pathogen (Baltruschat and Schoenbeck, 1975). Zambolim and Schenck (1983) noted that G.mosseae reduced the effects of three pathogens (e.g. Macrophomina phaseoli, R, solani and F. solani) individually on soybean in autoclaved soil.

VAM fungi reduced the severity of disease caused by Olpidium brassicae on lettuce and tobacco (Schoenbeck and Dehne, 1979), and Rhizoctonia solani on Brassica naprus (Iqbal et al., 1971). Glomus fasciculatus when added in soil reduced severity of Sclerotium rolfsii on peanut (Krishna and Bagyaraj, 1982). VAM fungi are known to reduce the inoculum multiplication and infection of nematodes, viruses and bacteria as well (Schoenbeck and Dehne, 1979). In addition to certain exceptions, VAM fungi increase the growth and yield of certain crop plants.

Ectomycorrhizal fungi also warrant the penetration of soil-borne pathogens in host roots. Several ectomycorrhizal fungi such as Lacaria laccata, Leucopaxillus cereails, Suillus luetus, etc. are known to escape infection by Phytophthora cinnamomi on roots of Pinus taeda (Marx 1969).

Mechanism of interactions and protection
VA mycorrhizal fungi retard the development of pathogens in root systems and increase disease severity in non-mycorrhizal roots. This systemic influence can be attributed to better nutrition, enhanced plant growth and physiological stimulation in mycorrhizal plants. Roots colonized by a VAM fungus exhibit high chitinolytic activities. These enzymes can be effective against the other fungal pathogens. Under direct influence of mycorrhizal fungi, root tissues become more resistant to pathogenic attack. This induced resistance is strictly limited to the site of host-endophyte interaction and will only affect soil-borne pathogen. Thus, application of selected VA mycorrhizal fungi offers the possibility of increasing resistance against soil-borne pathogens (Dehne, 1982).

Ectomycorrhizal fungi develop a mantle around the roots which is composed of tightly interwoven hyphae in several layers. It completely covers the root meristem and cortical tissues. Thus, the mantle provides physical protection against the pathogens. Thickness of mantle varies with fungus types.