An overview of fungal and bacterial biopesticides to control plant pathogens / diseases

Chemical agents are extensively used in all countries of the world in1960s, for controlling the agricultural pest and pathogens, it became apparent that agricultural chemicals were responsible for causing environmental pollution, they were present in the food chain, they were capable of inducing pest resistance, development of disease resistance, toxic hazards to man, plants, domestic animals and wild life, as a result they are regarded as ecologically unacceptable. Therefore, from the last two decades, scientists are looking for environmentally and toxicologically safe and more effective methods to control plant pathogens /pests and they increased social pressure to replace them gradually with biopesticides which are safe to humans and non-target to other beneficial organisms and cheaper than the chemicals. The isolates of the biocontrol agents are formulated by using different organic and inorganic carriers either through solid or liquid fermentation. They are applied as a seed treatment, biopriming, seedling dip, soil application, foliar spray, fruit spray, sucker treatment and sett treatment.


INTRODUCTION
Due to plant diseases, every year nearly 10-20% of the total world food production decreases and this lead to loss of billions of dollars.Agriculture has been facing the destructive activities of numerous pests and pathogens from early times which lead not only to reduction of the yield of the crop, but also lose in terms of money and reduce the aesthetic value.However, the extensive use of the synthetic chemicals during the last three decades has raised a number of ecological problems.In the recent years, scientists have diverted their attention towards the potential of beneficial microbes.The disease causes a significant reduction of seed germination, seed quality thereby limiting its potential yield.Biocontrol agents act through the mechanisms of antibiosis, secretion of volatile toxic metabolites, mycolytic enzymes, parasitism and through competition for space and nutrients.Among different biological approaches, use of the microbial antagonists like fungi, and bacteria is gaining popularity (Eckert and Ogawa, 1988;Droby et al., 1991;Wisniewski and Wilson, 1992;Droby, 2006;Korsten, 2006).Fungi belonging to the genus Trichoderma and bacteria such as Pseudomonas, Bacillus subtilis are the most promising bio control agent which acts against a wide range of plant pathogens.Trichoderma spp.are capable of controlling a number of diseases of plants; they control large number of foliar and soil borne diseases (Papavizas, 1985).*Corresponding author.E-mail: ritu45880@gmail.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Majority of the isolates were identified as Trichoderma viride and Trichoderma harzianum.Many species of Trichoderma namely T. harzianum, T. viride, Trichoderma virens etc, isolates from rhizosphere, were good antagonistic potential against many soil borne fungi, such as Fusarium oxysporum, Sclerotium rolfsii, Rizoctonia solani.Trichoderma spp.produce antibiotics and antifungal toxic metabolites viz trichodermin, viridin, etc and also inhibit pathogens by secreting enzymes like glucanase, cellulase, chitinase, protease, etc, which disintergrate the cell wall of pathogen.Pseudomonas spp.which survive in the rhizosphere of the crop are also known as plant growth promoting rhizobacteria (PGPR).Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas cepacia are the important biocontrol agents and they produce antibiotics like Pyrol, Nitrin, Oomycin-A etc and hormones like indol acetic acid, Giberllic acid, and Siderophores that inhibit the growth of pathogens.A combination of P. fluorescens and T. harzianum provides effective control of disease of vanilla crop.Trichoderma-based agriculture products can be found as registered in many countries, and are sold and applied to protect and improve yield of vegetables, ornamentals and fruit trees (Lortio, 2005)."Biological control of soil borne plant pathogens by Trichoderma spp.and other bioagents is a vital area of plant pathological research all over the world these days".Biological plant protection is an important component in the eco-friendly management of plant diseases all over the globe.

Advantages of biopesticides
1. Avoids environmental pollution (soil, air and water).2. Avoids adverse effects on beneficial organisms, that is, maintain healthy biological control balance.3. Less expensive than pesticides and avoids problems of resistance.4. Biopesticides are self maintaining in simple application and fungicide needs repeated application 5. Biopesticides are very effective for soil borne pathogen where fungicidal approach is not feasible.6. Biopesticides are eco-friendly, durable and long lasting.7. Very high control potential by integrating fungicide resistant antagonist.8. Biopesticides help in induced system resistance among the crop species.Eg: Trichoderma sp.resistant to fungicide like, Benomyl and Metalaxyl etc.

Identification
Trichoderma (Plate 1) is one of the common fungal biocontrol agent, and is used worldwide for suitable management of various foliar and soil borne plant pathogens.Biocontrol agents like Trichoderma spp.are acclaimed as effective, eco-friendly and cheap, nullifying the ill effects of chemicals.Therefore, these biocontrol agents are identified to act against on array of important soil borne plant pathogens causing serious diseases of crops (Khandelwal et al., 2012;Motlagh and Samimi, 2013;Babu and Pallavi, 2013).

Identification of bacterial antagonists
Identification was carried out as per Buchanan and Gibbons (eds) 1974 (Bergey's manual of systematic bacteriology).Fluorescent Pseudomonads (Plate 4) produce fluorescent pigment on King's B medium but not on FeCl 3 -amended King's B medium.All of them show positive reaction for Kovac's oxidase test, arginine dihydrolase and gelatin liquefaction except for P. putida which is negative for gelatin liquefaction.Identification can also be carried out at the Microbial Type Culture Collection Centre (MTCC) of Institute of Microbial Technology, Chandigarh.
Bacillus spp.are bacillus-shaped under the electron microscope, Gram-positive, and motile.Many strains of B. subtilis have been shown to be potential biocontrol agents against fungal pathogens.Evidence to date suggests that the principal mechanism of this antifungal action involves the production of antibiotics, especially within soil microsites (Fravel, 1988).However, it is likely that several mechanisms act in concert to achieve control, including the production of volatiles, which have a significant effect on soil microbiology (Kim et al., 2003).

Antibiosis
It occurs during interactions with other microorganisms involving low molecular or it is mediated by the specific or non-specific metabolite of microbial origin, by lytic enzyme, volatile compound or other toxic substance (Marfori et al., 2002;Howell, 2003;Limon et al., 2004;El-Ghaouth et al., 2004;Singh and Sharma, 2007).It produces: a) Antibiotic: These are the microbial toxin at low concentration, poison or kill other microorganisms.For example, Gliocladium virens producing Gilotoxin is responsible for the death of the Rhizoctonia solani on potato tubers.Colonization of pea seed by T. viride result in significant amount of antibiotic producing viridin in the seed controlling Pythium spp.Phenazine antibiotic (Phz) produced by Pseudomonas fluorescens strain 2-79 has been implicated in control of take-all of wheat caused by Gaeumannomyces graminis var.tritici.(Handelsman and Parke, 1989).b) Bacteriocin: These are the antibiotic like substance that help to control the crown gall by the related Agrobacterium radiobacter strain K84.c) Volatile compound and metabolite production: Microorganisms which produce metabolite that can interfere with the pathogen on growth and activities.For example, a volatile or water-soluble substance produced by the host fungus serves as a chemoattractant for parasites (Deacon and Berry, 1992).Trichoderma spp. is a strain which is known to produce a number of antibiotics such as trichodernin, trichodermol, harzianun and harzianolide (Kucuk and Kivanc, 2004).The combination of hydrolytic enzymes and antibiotics results in a higher level of antagonism than that obtained by either mechanism alone (Howell, 1998).

Hyperparasitisms/mycoparasitism
Direct parasitisms or lysis of the death pathogen by other microorganisms is known as a hyperparasitism.Fungi that are parasitic on other fungi are usually referred to as mycoparasites (Baker and Cook, 1974).For example, Weindling in 1932 observed Trichoderma lignorum (T.viride) parasitizing the hyphae of Rhizoctonia solani and also suggested that inoculating soil with Trichoderma spores helps to control damping off of citrus seedling (Lo, 1997.)Sporidesmium sclerotium, is a biotrophic parasite and is often found only on sclerotia of plant pathogenic fungi such as Sclerotinia minor and Sclerotium cepivorum (the causal agents of lettuce drop) (Adams and Ayers, 1983).The mycoparasitic ability of Trichoderma species against some economically important plant pathogens allows for the development of biocontrol strategies (Harman et al., 2004;Motlagh and Samimi, 2013).

Competition
Trichoderma species are generally considered to be aggressive competitors, grow very fast and rapidly colonize substrates to exclude pathogens such as Fusarium spp.(Papavizas, 1985).Iron competition in alkaline soils may be a limiting factor for microbial growth in such soils (Costa and Loper, 1994).A few studies have demonstrated that siderophore biosynthesis in P. fluorescens plays a role in pathogen suppression (Costa and Loper, 1994;Leong and Expert, 1989).Leeman et al. (1996) have reported that iron-chelating salicylic acid is produced by selected P. fluorscens strains at low iron availability and may be involved in the induction of systemic resistance to Fusarium wilt of radish.A significant amount of research on the use of the microbial antagonists has been reviewed by several workers (Wisniewski et al., 1989;Droby et al., 1992;Piano et al., 1997;Castoria et al., 1997;Kim et al., 1997;Arras et al., 1998;Kurtzman and Droby, 2001;El-Ghaouth et al., 2004;Grebenisan et al. 2008;Saravanakumar et al., 2008).

PSEUDOMONAS FLOURESCENS ACTS AS A PLANT GROWTH-PROMOTING RHIZOBACTERIA
Plant growth-promoting rhizobacterium (PGPR) is present near the roots and colonizes the plant roots, and it reduces the incidence of plant disease (Kloepper and Schroth, 1978) .The PGPR works as an aggressive colonization, plant growth stimulation and biocontrol (Kloepper, 1980;Défago and Haas, 1990;Weller et al., 2002;Vessey, 2003;Lucy et al., 2004).In the mechanisms of PGPR, the process includes nitrogen fixation, phosphate solubilization and the production of phytohormones (such as auxin and cytokinin) and volatile growth stimulants (such as ethylene and 2,3-butanediol) which help to control many pathogens.Pseudomonas sp. is ubiquitous bacterium in agricultural soils.Pseudomonas spp.controls the diseases by producing siderophore (pyochelin) which has been identified as an antifungal antibiotic in a screening program (Phoebe et al., 2001).The characteristics of Pseudomonads sppare: (i) It produces a wide spectrum of bioactive metabolites (antibiotics, siderophores, volatiles and growth-promo-ting substances) (ii) It competes aggressively with other micro-organisms and it adapts to the environmental stresses also.(iii) Pseudomonads are also responsible for suppression of soil borne pathogens (Weller et al., 2002).Raaijmakers et al. (2002) and Morrissey et al. (2004), reported that some strains of Pseudomonas spp.produce more than one antibiotic compounds but they are not related to siderophores.Invitro studies revealed that, these antibiotics can inhibit fungal pathogens, but they can also be active against many bacteria.PGPR can also provide protection against viral diseases.Today, the use of PGPR has become a common practice in many regions of the world.

Antifungal activity of PGPR
P. fluorescens produces 2,4-diacetyl phloroglucinol which inhibits the growth of phytopathogenic fungi (Nowak-Thompson et al., 1994).Some PGPR degrade the fusaric acid produced by Fusarium sp.causal agent of wilt, which helps to prevent the pathogenesis (Toyoda and Utsumi, 1991).Some PGPR can also produce enzymes that can lyse the fungal cells.For example, Pseudomonas stutzeri produces extracellular chitinase and laminarinase which lyses the mycelia of Fusarium solani.P. fluorescens protects the plants against a wide range of important agronomic fungal diseases such as black rootrot of tobacco (Voisard et al., 1989), root-rot of pea (Papavizas and Ayers, 1974) root-rot of wheat (Garagulia et al., 1974) and damping-off of sugar beet (Fenton et al., 1992).
Pseudomonas shows biocontrol potential against phytopathogenic fungi in vivo and in vitro conditions from chickpea rhizosphere (Saraf et al., 2008).P. putida has potential for the biocontrol of root-rot disease complex of chickpea by showing antifungal activity against Macrophomina phaseolina (Laville et al., 1998).

Mass multiplication of fungal and bacterial antagonist
Methods which arE used for mass multiplication: a) Solid state fermentation b) Liquid state fermentation

Substrate which are used for solid fermentation
Sorghum grain, wheat straw, wheat bran, wheat bransaw dust medium, sand and sorghum medium, tapioca rind, coffee husk, sand-corn meal medium, rice bran and vegetable waste.

Procedure: Solid fermentation
The waste substrate cut in the form of pieces is shade dried under the shade.The dried substrate is measured and a gram of flour and yeast extracts is added for nutritional purpose.The moisture level of that mixture is maintained up to 40%.150 g of substrate are taken (Rice bran, FYM) into polypropylene bag, heat sealed and sterilized at 15 lb of pressure for 2-4 h for 3 successive days.Each bag is inoculated with the mycelia disk/liquid broth of Trichoderma and incubated at 28°C for 7-10 days.Solid substrate with Trichoderma can be mixed with the sand (1:2) applied to the soil directly

Constraints in solid fermentation
1. Preparation is bulky.2. There is a greater risk of contamination.3. Require larger space for growing, incubation and storage.

Media used for liquid fermentation
Molasses-brewer's yeast medium, Richards medium, Czapex Dox broth, V-8 Juice, PDB, Molasses-soy medium and Jaggery medium.Trichoderma is generally mass multiplied on molasses yeast broth medium.

Procedure
The ingredients were mixed with the distilled water and poured in a conical flask/horlicks bottle.The flasks were plugged, covered and kept for sterilization in autoclave for 15 min at 15 lbs.After Cooling at room temperature, they were inoculated with the mycelia disk of trichoderma (from 5-6 days old culture).After inoculation, the flasks were incubated byusing two methods: a) Stationary culture: The flasks containing sterilized molasses and yeast broth are inoculated at room temperature for 10 days.The fungal culture broth is used for the preparation of the formulation.b) Shaker culture: The flasks containing sterilized molasses and yeast broth are inoculated with Trichoderma culture and kept on a rotatory shaker at 150-180°C rpm for 3-5 days.In shaker culture more conidia or chlamydospores are produced.Trichoderma could be mass multiplied on large scale in a short period using fermenter.

Methods for preparation of talc based formulation
Trichoderma is multiplied in molasses/yeast medium / nutrient broth.The biomass produced is homogenized in a mix.The homogenized mix of biomass is added to talc

Mass multiplication of Pseudomonas flourescens
Add 0.6-0.8g Pseudomonas agar media in 250 mL flask.
Then it was autoclaved and inoculated with the culture.
Then, it was kept on a rotary shaker for 48 h, mixed with 1:2 sterilized talc powder and calcium carbonate was added.It was mixed and packed aseptically.

Carriers used in the formulation
There are two types of carriers which are available in organic or non-organic form.They should be economical and easily available.The organic carriers are peat, talc, lignite, kaolinite, pyrophyllite, zeolite, montmorillonite, alginate, pressmud, sawdust and vermiculite, etc. Carriers increase the survival efficiency of bacteria (Heijnen et al., 1993).Survival of P. fluorescens (2-79RN10, W4F393) in montmorillonite, zeolite and vermiculite increases as compared to the phyllite and talc.

Talc based formulations
Krishnamurthy and Gnanamanickam (1998) developed talc based formulation of P. fluorescens for the management of rice blast caused by Pyricularia grisea, in which methyl cellulose and talc is mixed at 1: 4 ratio and it is used in the concentration of 10 10 cfu/mL.Talc based formulations were also effective against rice sheath blight (Nakkeeran et al., 2004).Survival period of P. putida strain is up to 6 months in talc based formulations (Bora et al., 2004) (Table 1).

Method of application
Time of application of the Trichoderma is also important.Trichoderma can't tolerate heavy pressure.Therefore, it may be used strictly as a preventive measure, it can't cure infection.Trichoderma is least effective against the systematic disease than against more superficial one.It can not control the existing disease.A combination of chemical treatment with Trichoderma will be highly effective.A single strain of Trichoderma may not be sufficient to be effective under all conditions and against all diseases.A mix of different biocontrol agents is effective against all the diseases.

Seed treatment
Seed treatment is the most effective method.In seed treatment, seed priming is the process in which hydration of seed is controlled to a level that permits pregerminative metabolic activity to take place without emergence of the radical.Treatment of pigeonpea seeds with talc based formulation of P. fluorescens (Pf1) effectively helps to control fusarium wilt of pigeonpea (Vidhyasekaran et al., 1997).

Product Bio agent Use
Antagon-TV T. viride As seed and soil treatment for control of R. solani and M. phaseolina in pulses and vegetables Biocon T T. .v vi ir ri id de e Available in broth and dust formulations and used for the control of root and stem diseases of tea.

T T. . v vi ir ri id de e + + T T. . h ha ar rz za an ni iu um m
As seed treatment, soil treatment, seedling dip and foliar spray against the fungal pathogens of vegetables, pulses and cereals, also in sugar crops.

T T. . v vi ir ri id de e
As seed and soil treatment for the control of seed and soil borne diseases of vegetables and pulses.

Pseudomonas fluorescens
As seed, soil and seedling dip against fungal pathogens of cereals, pulses and vegetables.

Bacillus subtilis
Available in broth formulation and used for the control of black rot disease of tea caused by Corticium invisum and C. theae.

Defence-SF T. viride
As seed and soil treatment for the management of seed and soil-borne diseases of crops It was reported that numerous strains of fungal and bacterial isolates have biocontrol activity against several plant pathogenic fungi (Tables 3 and 4).

Seedling dip
Application of P. fluorescens strain mixtures by dipping the seedling of rice in bucket of water containing talc based formulation containing mixture of (20 g/l) for 2h and later transplanting it in the field helps to control sheath blight of rice (Nandakumar et al., 2001).

Seed priming
PGPR increases germination and improve seedling establishment.Bio-priming of seeds with bacterial antagonists increase the population load of antagonist to a tune of 10 fold on the seeds thus protected rhizosphere from the ingress of plant pathogens (Callan et al., 1990).

Soil application
Actively growing population is applied in the soil.
Trichoderma can be applied as granule as well as drench.In transplanted crops granules are applied in nursery.Tomatoes were grown in a potting mix containing the granular formulation of T. Harzanium.Vidhyasekaran and Muthamilan (1995) reported that the soil application of peat based formulation with P. fluorescens (Pf1) at the rate of 2.5 kg of formulation mixed with 25 kg of well decomposed farm yard manure, helps to control the chickpea wilt caused by Fusarium oxysporum f.sp.ciceris.

Foliar spray
Delivering of Pseudomonas to beet leaves inhibits the amino acids on the leaf surface and inhibits spore germination of Botrytis cinerea, Cladosporium herbarum and Phoma betae (Blakeman and Brodie, 1977).Application of B. subtilis to bean leaves reduces the incidence of bean rust (Uromyces phaseoli).Seed treatment and foliar application of P. fluorescens reduces the severity of rust and leaf spot under field conditions.Some commercially available biocontrol agents are listed in Table 2.   Lemessa and Zeller, 2007;Aliye et al., 2008;Ji et al., 2008;Maketon et al., 2008;Ji et al., 2008;Chen et

CONCLUSION
Biological control of fungal diseases of plants is ecofriendly and is a potential component of integrated disease management.Biological control of foliar diseases has received less attention, owing to the poor establishment of the introduced biocontrol agents and resulting variations in disease control.Application of synthetic fungicides has been the traditional strategy for the management of plant diseases.The increasing concern for health hazards and environmental pollution due to chemical use has needed the development of alternative strategies for the control of plant diseases.Management of plant diseases by employing microbial agents has been demonstrated to be most suitable strategy to replace the chemicals which are either being banned or recommended for limited use.This review reported the success of some biocontrol agents under laboratory and commercial conditions, and some bioproducts that have been developed for commercial use.

Table 1 .
Shelf life of bacteria based on the formulations.

Table 2 .
Commercial formulations of biocontrol agents available in India.

Table 3 .
Fungal bioagent of various plant pathogens in India.

Table 4 .
Evidence for successful experimental use of biological control agents of various diseases.

Table 4 .
Contd.Trichoderma spp.achieves only transitory localized dominance of the rhizosphere and these are active in only some soil and some season.Trichoderma spores are quiescent in active soil.Trichoderma spp.are likely to be effective for seed and seedling diseases, but not against disease of mature crop.
CONSTRAINTS IN THE USE OF A TRICHODERMATrichoderma spp. is an effective biocontrol agent that effectively controls the soilborne fungal pathogens, but, it produces adverse effects on the fungus arbuscular mycorrhizal (AM).AM fungi is presented in the roots of most herbaceous plants.Another problem has been low field performance of Trichoderma as biocontrol agent.