In this article we will discuss about the natural and induced biological control of plant diseases.
Biological control may be defined as any condition or practice whereby survival or activity of a pathogenesis is reduced through the agency of any other living organism except man himself, with the result that there is a reduction in the incidence of the disease caused by the pathogen. Garrett (1965) defined biological control of plant disease as the reduction of inoculum density or disease producing activities of a pathogen or parasite in its active or dormant state, by one or more organism accomplished naturally or through manipulation of the environment, host or antagonist or by mass introduction of one or more antagonists.
W. Roberts (1874) was the first to demonstrate the antagonism between Penicillium glaucum and bacteria which paved the way for biological control of plant pathogens. Demonstration of Hartley (1921), Sanford (1926), Millarrd and Taylor (1927), Grossback and Broadfact (1931) and Rishbeth (1970) clearly established the way for biocontrol of plant diseases.
Through successful competition, production of enzymes (glucanases proteases and chitinases) to lyse pathogen cell walls, antibiotic and inhibitory metabolite production indirect effect resulting in modification of the environment adverse to pathogen. Biological control of plant pathogen occurs as, both resident and introduced antagonists are able to diminish plant diseases, but its general utility was skeptically regarded.
The ecological balance of soil microorganisms can be manipulated by modifying the organic matter content, pH, temperature, by competition of microorganisms for limited nutrients, parasiting the pathogen by microorganisms, production of antibiotics by antagonists, suppressive soil to soil- borne pathogens, introduction of microorganism of suppressive soils to conducive soils, inoculation of plant with mild strain of virus can protect it against subsequent inoculation with a more virulent strain (cross protection).
Biocontrol agent may induce silent defense genes that make plant system resistant against plant pathogens, application of more than one biocontrol agent, genes from biocontrol agents can be a good source for improving plant resistance to pathogens, transformation of biocontrol agents with extra genes could increase the antimicrobial activity of the strain.
Though the term biological control was used for the first time in relation to plant pathogens by C.F. Von Tubeuf in 1914, it is in 1921 when C. Hartley introduced microorganisms into soil to control root disease. Subsequently several plant pathologists including Sanford (1926); Millard and Taylor (1927); Sanford and Broadfood (1931) contd. Researches from 1950 to 1970 clearly established possibility to control plant diseases through microorganisms specially fungi like species of Trichoderma and Gliocladium.
The mechanism of biological control operates in variety of ways such as both resident and introduced antagonists are able to diminish plant diseases. Ecological balance of soil microorganisms can be manipulated by modifying the organic content, temperature, pH of soil. Microorganisms compete for limited available nutrients, microorganisms may parasitize other microorganisms, and antagonists produce antibiotics that may play a part in biocontrol of pathogen.
Some soils are naturally suppressive to some soil-borne plant pathogens or become so following prolonged occurrence of disease caused by pathogen, inoculation of plant with a mild strain of virus can protect it against subsequent inoculation with a more virulent strain and/or biocontrol agents may induce silent defense genes that make plant system resistant against plant pathogen induced resistance.
Total microflora of some suppressive soils can be transferred to conducive Soils making them suppressive, application of more than one biocontrol agents (mixed formulation) could be reliable means of reducing variability and increasing the reliability of biological control, genes from biocontrol agents can be a good source for improving plant resistance to pathogens. Chaoube et al., (2003) discussed elegantly various aspects of biological control of plant pathogens.
A good and stable biocontrol is possible when a potential organism is employed.
The biocontrol organism should possess following characteristics:
1. Utilize available nutrients, increase in number and colonize the region in rapid fashion.
2. The ability to produce an antibiotic is a desirable character.
3. It should be able to elicit resistance response at infection site.
4. Prolonged survivability is a quality.
5. Antagonist must be compatible with chemical used.
6. Biological agents should not be adversely affected by the storage environment specially with reduced O2, elevated CO2 and nitrogen levels.
7. Antagonist should not be health hazardous.
The mechanism of protection of plants by biocontrol is either by direct action against pathogen or indirectly by reducing host susceptibility (Fig. 18.10).
Plant diseases are controlled by antagonists involving variety of mechanisms which varies both with the pathogen and host as precised in table 18.18.
A. Natural Biological Control:
The natural reduction in pathogens population and disease incidence occur due to:
(i) Suppressive soils, and
(ii) Monoculture.
(i) Suppressive Soils:
Suppressive soils are unfit for the development of certain diseases. In these soils the saprophytic growth, pathogenic activity and survival of pathogen are reduced, if pathogen establishes it cause little or no damage. When continuous cropping with susceptible crop results decrease in disease severity. This is also called induced natural suppression. The chlamydospore germination and germ tube growth are highly suppressed in suppressive soils than in conducive soils.
There are now several evidences that by the use of suppressive factors it may be possible to establish microbially based resistance in certain soils conductive for diseases. The mechanism of suppressiveness has not been fully resolved. However, the explanation given for the suppressive phenomenon includes antibiosis, competition, parasitism and predators.
Albonnette et al. (1979) have concluded that the suppressiveness is linked with microbial activity and thus it involves biological control. Take all, fusarial wilt, damping off and potato scab have been effectively controlled by this principle. Baker (1950) demonstrated that small quantities of wilt suppressive soil when added to steamed soil or natural soil provides suppressiveness to Fusarium wilt of carnation.
(ii) Monoculture:
Significance of monoculture for biological control of soil-borne plant pathogens has been extensively discussed by Cooke and Baker (1983). King (1923) reported for the first time the decline of cotton root-rot caused by P. amnivorum due to monoculture. All disease of wheat was also controlled by suppressive factor which was festered by wheat monoculture.
These changes are:
(i) Development of specific antagonism,
(ii) Change in pathogens population, and
(iii) Change in the microflora, which affect the pathogen. Soil factors help in building up of suppressiveness in soil due to monoculture.
B. Induced Biological Control:
1. Annulment of Fungistasis:
Soil-borne plant pathogens survive in the soil for long time mainly because of soil fungistasis which inhibits germination of spores, sclerotia and chlamydospores of pathogens. Germinating spores are highly susceptible to lysis due to other microbes. Hence, to induce lysis of pathogens propagules the fungistasis should be first annulled. Stimulation of fungal propagules to germinate by organic materials without supporting formation of new resistant propagules may provide the basis for biological control.
Sclerotia of the onion wilt, root pathogen (Sclerotum cepivorum), do not germinate in soil because of fungistasis. Volatiles such as organic sulfides are exuded by the roots of onion and they stimulate germination of sclerotia of S. cepivorum. The germlings die either from nutrient exhaust or of lysis by microorganisms.
Similarly volatile compounds liberated during alfalfa hay decomposition increase microbial activity in soil, stimulate germination and lysis of sclerotia of S. rolfsii. Sanford (1926) reported the control of apple scab by green manuring and heavy application of organic matter controlled Phymatotrichum omnivorum of cotton are some of the examples of annulment of fungistasis. It has several advantages such as it may have multiple pathogen suppression, lasting effect and involve less cost.
2. Elevation of Fungistasis:
Instead of annulling fungistasis it can also be elevated and control soil-borne diseases. Soil can be made increasingly fungistasis to chlamydospores of Thielaviopsis basicola by amending it with alfalfa hay and allowing decomposition. Similarly rye and corn residues enhance fungistasis to chlamydospores of Fusarium solani f. sp phaseoli. These treatments elevate the level of toxicity of soil to such an extent that the pathogens propagules become inactivated even in the presence of root exudates.
3. Inhibitory Volatiles:
Crucifers when added to the soil, many volatiles such as methanethiol, dimethyl sulfide and dimethyl disulfide are released. These compounds control pea root-rot caused by Aphanomyces euteiches by inhibiting growth, zoospore mortality and germination of fungal propagules.
4. Antagonist Organisms:
Introduction of antagonist into the soil to reduce the activity of pathogen has been most frequently used practice because of increase in population of antagonist. Seed treatment and pelleting of seeds with antagonist proved to be successful in the control of root rot of rye caused by R. solani. Bacillus subtilis, Trichoderma spp., Pencillium spp. and chaetomium globosum were considered highly effective for protection of corn, peas and soya bean then control of crown gall caused by Agrobacterium tumifaciens by A. radiabacter. Organic amendments with high C/N ratio increase antagonists, Streptomyces spp., and control root-rot of bean caused by Rhizoctonia solani.
5. Cross Protection:
When avirulent strain of the pathogen is inoculated into the soil, the disease caused by virulent strains is reduced. Verticillium wilt of cotton is controlled by incorporating avirulent strain of the pathogen in the soil. The phenomenon involves the prior colonization of infection count by the antagonists. This is also called as induced resistance. Similarly Verticillium dahliae causing wilt of mint can be controlled by use of V.nigricans.
However, drawbacks of this method are:
(i) Avirulent strain of pathogens may have negative effect on plant health and vigour.
(ii) The crop protecting strains may have pathogenic effect on other plants grown in the same field.
6. Mycorrhizal Fungi:
Ectomycorrhizal fungi like Boletus variegata produce volatiles which inhibit the growth of Phymatotrichum cinnamomi and H. annosus. Similarly Davis et al. (1979) have reported that Verticillium wilt of cotton was less severe in VA mycorrhiae colonized roots. Commercial soil inoculum of Glomus deserticola is now distributed in California for biological control. However, careful study is needed before considering these fungi for biological control.
7. Amoebae:
The soil amoebae feed on fungal spores and hyphae. The perforation and lysis of the spores have been described in Thielaviopsis basicola. Baltruochat and Shanbeck (1975) have recorded decrease in the population of T. basicola and Cochliobolus sativum by addition of cysts or active vanpyrellid amoebae into soil. Chakraborty and Warcup (1983) have also reported reduction of take-all by mycophagous amoebae.
8. Mycoparasites:
Ayens and Solams (1981) have listed mycoparasites which have been successfully employed in the control of soil-borne diseases. Trichodema spp. has been exploited for this purpose as they readily parasitize many soil-borne fungi. Four species of Trichoderma viz., T. viride, T. haziaanum, T. koeningii and T. hamatus are reported to be mycoparasites. T. hamatum is reported to be a hyperparasite of R. solani and protected the seeds of pea and radish.
Trutmonn has employed Gliocladium roseum, Myrothecium verrucaria in the biological control of Sclerotinia scleriotarum. Sporidesmium sclerotiorum has been reported to parasites the sclerotia of Sclerotinia minor and Sclerotium cepivorum. Biological control of Sclerotinia lettuce crop in the field by S. sclerotivorum and R. solani on sugar beet by Corticum sp. is due to the pathogenic activity on respective plant pathogens. The mycoparasites are generally isolated from the soil by introducing sclerotia of fungi or other fungal structures.
The fungi parasiting plant pathogens are called hyperparasites. These fungi are being exploited by applying spore suspension to diseased plant (Table 18.19). Mycoparasitic species of Trichoderma (T.harzianum and T.Viride) have been successfully developed as biocontrol agents. T.viride grow on wide variety of substrates and on selerotia of other fungi. Conidia of Trichoderma sp do not survive well in soil but Chlamydospores survive for long time. Trichoderma cannot compete with other saprophytes and it is only a secondary invader. Green manure, compost supports Trichoderma well.
In soil fumigated with carbon disulfide or formaldehyde or methyl bromide, Trichoderma multiples well. These chemicals are non-inhibitory to Trichoderma but inhibit other saprophytes. Treatment of soil with steam enhances Trichoderma in soil. Similarly soil solarization and sub-lethal heating of soil encourages the Trichoderma. Thiram, Captan, PCNB and chloronel are inhibitory to Trichoderma but suppress other saprophytes. However, benonyl, Carbondazin, thiophanate methyl and thiabendazole are inhibitory to Trichoderma. Trichoderma does not survive in the rhizosphere and anaerobic soils.
Characteristic features of Trichoderma are:
(i) Methods of Application:
Wheat bran plus peat, barley grain and composted wood bark are good substrates for production of Trichoderma. Alginate pellet with bran are useful for introduction of Trichodera into soil. Diatomaceous earth granules impregnated with molasses also serve as carrier of Trichoderma into the soil. The fungus survives well in spermosphere. Hence, seed treatment with the fungus controls many seed-rot and damping off diseases.
(ii) Mechanism of Action:
Trichoderma produces antibiotic (trichodermin, gliotoxin and viridins) in culture but not in soil. Trichoderma invades hyphae of Pythium and Rhzoctonia produce haustoria. It produces β (1-3)-glucanase and chitinase then degrades the glucans in the cell wall of Pythium sp. while chitin and glucans in the cell walls of Rhizoctonia.
(iii) Control of Soil-Borne Diseases by Trichoderma:
Treatment of seeds of peas, tomato and tobacco with Trichoderma, controls the damping off by Pythium and R. solani. It controls the root rot of ground nut (Sclerotium rolfsii).
(iv) Limitations:
1. Trichoderma does not survive in the rhizosphere soil as well as in unamended soil. The fungal inoculum should be in young mycelial stage and old cultures with spores will be inactive.
2. Trichoderma does not survive in the soil.
3. Trichoderma controls soil-borne diseases only in early stages of crop growth.
Other Fungi:
Gliocladium virens acts similar to Trichoderma as a biocontrol agent against Pythium, Sclerotium and Rhizoctonia diseases. Sporidesmium sclerotiorum, Sclerotinia minor and Coniothyrum minutans control Sclerotima sclerotium population in soil.
9. Bacteria:
Many bacteria produce siderophores. Siderophores are low molecular weight ferric ion transport agents and supply iron to the bacterial cell. Siderophores are relatively complex with iron of very high affinity and made it unavailable to other microorganisms including pathogens. Inspite of wide variation in structure of siderophore with the producing organism, it forms six coordinate octahedral complex with ferric iron and reported to play an important role with iron nutrition.
Siderophore chelate Fe(III) and microbial membrane receptor proteins specifically recognize and take up the Fe complex. This results in making Fe unavailable to rhizophere microorganisms including plant pathogens which produce less siderophores or different siderophores with lower binding co-efficiencies. The result is less pathogen infection and controls the disease. Pseudomonas florescens and P. putida produce siderophores control black-rot of potato caused by Erwinia cartovara.
The siderophores of Pseudomonas fluorescens are called Pseudobacterin has been found to control all disease of wheat, barley caused by Gaumanomyces var tritici and flax wilt disease caused by Fusarium oxysporum f. sp. lini. Addition of Pseudobacterin to the soil also controls wilt diseases. Pseudobacterin converts pathogen conducive soils into pathogen suppressive soils. It suggests that suppressiveness of the soil is mainly due to siderophores.
10. Other Approaches:
(i) Solar heating of soil by covering it with polythene sheet results in the change of microbial activity. This process leads to increased microbial activity of the soil against pathogen,
(ii) Decrease the survability of the pathogen, and
(iii) Increased susceptibility of pathogen to soil microorganisms. Solarization has been effectively used in the control of Verlicillium dahlae, Sclerotium rolfsii and R. solani.
(b) Flooding:
Flooding of soil has been found to eliminate or reduce the population of F. oxysporum f. sp. cubens, V. dahliae and Sclerotinia sclerotiorum.
(c) Crop Rotation:
Baker and Cork (1979) reported that crop rotation is one of the most-oldest approaches. Crop rotation for 1 to 3 years is adequate for the control of Gaumonomyces graminis V. tritici and Cercosporella herpotrichoides. However, Cook and Baker (1983) feel that this approach is being replaced by tillage, crop sanitation and changing planting dates.
11. Development of Mixed Formulations of Biocontrol Agent:
Most of the studies on biological control of plant diseases deal with single biocontrol agent as against a single pathogen. Due to variety of situations and host specificity even at subspecies level, most of the times inconsistent performance of biocontrol agent is reported. Thus single biocontrol agent is not likely to be active in all soil environments or against all pathogens that attack the host plant.
This situation may be achieved either by-
(i) Selecting a strain of biocontrol agent with wide host range,
(ii) Modify the genetics of the biocontrol agent to add mechanisms of disease suppression that are operable against more than one pathogen,
(iii) Alter the environment to favour the biocontrol agent and disfavour the competitive microflora or develop strain mixtures with superior bicontrol agent activity,
(iv) Several strategies for developing mixtures of biocontrol agents could be developed including mixtures of organisms with different plant colonization patterns, mixtures of antagonists with different mechanisms of disease suppression, mixtures of taxonomically different organisms.
Commercialization of Biocontrol Agent:
Commercialization of biocontrol agent is a multistep process involving wide range of activities as depicted in Fig. 18.11.
Screenings for pesticidal organisms involves selection of healthy plant from areas otherwise infested with targeted pathogen disease and isolate organisms from leaf surface and rhizosphere. Isolates are then screened for activity against the pathogen in the laboratory assay, green house condition and optimization of conditions.
It is then evaluated under field conditions. Potential biocontrol agents are maintained and supplied to target agencies. In India biocontrol agents are maintained at project directorate, biological control, Bangalore and G.B. Pant University of Agriculture and Technology, Pantnagar.
Mass Production:
One of the greatest obstacles to biological control by introduced antagonists has been scarcity of methods for mass culturing and delivering the biocontrol agents. The unique problem of biocontrol agent is that represents a living system which must be able to stand the process of formulation and should remain sufficiently viable for a period until it reaches farmer.
Despite the limited scope, scientists are engaged in developing effective experimental systems for growth and delivery of antagonist. Methods of Mass production of widely used antagonist, Trichoderma are precised in table 18.20. The unique problem in developing biopesticide, living system, must sustain the process of formulation and remain sufficiently viable till it reaches farmers field.
Development of safe, easy to handle, cost effective formulation of biocontrol agent is of paramount importance. Blending of active ingredients such as fungal spores with inert carriers such as diluents and surfactants in order to alter physical characteristics to a more desirable form. Formulation must have a minimum shelf life of 2 years at room temperature. At present, kaolin and bentonite are being used as carrier materials of T.harzianum.
Biocontrol agents which have an easier and quicker passage for registration are with indigenous microorganisms that are never recorded as being plant, animal or human pathogens and specific to a defined group of target pathogens. The information required for registration of any biocontrol agents are systematic name and common name, natural occurrence and morphological description, details of manufacturing process (active and inert ingredients of formulation), mammalian toxicity, environmental toxicity and residual analysis. Some of the commercial formulations of fungal control agents are listed in Table 18.21.
Improvement of Biocontrol Agents:
Once the suitable antagonist is obtained, it may be further improved for desirable characteristics like better antagonistic activity, wider host range, tolerance to pesticides, and survivability in the environment atmosphere competence, and tolerance to adverse environmental conditions, vigorous growth and its long life in the laboratory. The most important and challenging biological control is the methodology of preparing and delivering the antagonist into the soil.
Impregnation of clay granules or diatomaceous earth for introducing inoculum of the antagonist is one such attempt. Commercial inoculum of P. gigantea spores are suspended in strong sugar solution and are now distributed in sacheti of biological control. For application, the contents of sachets are mixed with 5 liters water. Strains of Trichoderna spp. mixed in beetle powder and given a trade name B1, NAB control in France. A. radiobacter strain K 84 is also being distributed by several companies in a finely ground peat preparation for control of crown gall pathogen.
Genetic manipulation of biocontrol agents can play a great role in improving their potential. To improve biocontrol agents for desirable characteristics like better antagonistic ability, wide host range, tolerance to pesticides, survival ability in the environment, rhizosphere competence, tolerance to adverse environmental conditions, vigorous growth and self-life.
Different strategies adapted for this purpose are described below:
(i) Mutation:
Ahmad and Baker (1988) and Carsolio et al. (1999) improved Trichoderma harzianum for rhizosphere competence and T.viride to hypersensitivity respectively. Mukerjee et al. (1997) and Starz et al. (1988) have developed vinclazolin and benomyt tolerant mutants of Trichoderma viride respectively through mutagenesis. Selvakumar et al., (2000) have developed carboxin tolerant mutants of Trichoderma viride by exposing the tolerant biotypes of T.viride developed by UV irradiation and ethyl methane sulfonate. Mukherjee and Mukhopadhya (1993) developed stable mutants of T.viride which differed from the wild type strain in phenotype growth rate, sporulation and antagonistic potential by exposing to r-rays.
(ii) Protoplast Fusion:
Protoplast fusion is one of the best techniques for improvement of strain. T.viride strains are well known producers of chitinases, glucanases, and celluloses and different other mycolytic enzyme combination of these desirable traits from different species or strains may give rise to superior strain. Protoplast fusion technique has advantage as the possibility of obtaining recombinants is more and also allows testing of large number of recombinants in a short time.
(iii) Genetic Engineering:
Transgenic microorganisms in which specific strains are constructed to express genes from another microorganism whose products are inhibitory to plant pathogens are likely to have high utility as biocontrol agents. Lo et al. (1998) have developed transformant of T.harzianum strain 1295-22 by integrating β-glucouridinase (GUS) and hygromycin (hygB), phospotransfererase genes that exhibited increased biocontrol activity against R.solani as compared with wild type.
12. Prospects:
Though the biocontrol way of plant protection is well established but it has yet to become an integral part of plant protection and needs further attention in the following areas:
1. Exploration of biodiversity and their conservation is an utmost important as, more potential organisms are likely to be discovered.
2. Genetic improvement of bio-agents through molecular techniques.
3. Improvement in mass production technology, shelf life and delivery system.
4. More understanding of relationship among plant, pathogen, fertilizers and biocontrol agent is of basic need. How and why they work and interact with each other must be understood to evolve and formulate strategies.
5. There is need to select not only strains of biocontrol agent but also good growth and plant defense inducers.
6. Integration of biocontrol with other management practices under field condition is need of the hour.
7. Bio-priming of seeds with biocontrol agents, bio-fertilizers and micronutrients must be explored and exploited.
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