Research Journal of Agriculture and Forestry Sciences __________________________________ ISSN 2320-6063 Vol. 1(9), 19-25, October (2013) Res. J. Agriculture and Forestry Sci. International Science Congress Association 19 Review Paper Pseudomonads: Potential Biocontrol agents of Rice Diseases Roy Manidipa, Sharma Gauri Dutta and Ramana Ch.Venkata Department of Life Science and Bioinformatics, Assam University, Silchar-788011, INDIA Vice Chancellor, Bilaspur University, Chattisgarh-495009, INDIA Department of Plant Sciences, University of Hyderabad, Hyderabad-500046, INDIAAvailable online at: www.isca.in, www.isca.me Received 29th August 2013, revised 16th September 2013, accepted 6th October 2013Abstract Biocontrol mechanism to suppress fungal and bacterial pathogens of rice cropby Pseudomonas sp. generally involves the production of antibiotics, siderophores, volatile compounds, hydrocyanic acid (HCN), enzymes and phytohormones. Rice is the staple food of over half of the world population. Diseases are the most important factors that affect rice production causing annual yield losses. Biological control is an eco-friendly, cost effective and sustainable alternative method in disease management. Keywords: Biocontrol, fungal and bacterial pathogen, Pseudomonas. Introduction Sustainable agriculture depends on the use of chemical fungicides, pesticides, herbicides and fertilizers. Repeated use of these chemicals is causing severe concern from the health and environmental point of view. In view of these the development of biologically based control method of diseases is now viewed not only asan eco-friendly but also sustainable agriculture. Research on biological control of rice diseases started mainly in the 1980. It is mainly concentrated on the identification, evaluation and formulation of potential biocontrol agents for deployment.Rice (Oryza sativa L.) is the most widely cultivated food crop in the world. Global rice production was 468.1M.T (million tons) during the year 2010 and it rose to a record 480 M.T in 2011. According to Agriculture ministry, India harvested a record 103.41 M.T of rice in the 2011-2012 crop years (July- June) as against 95.98M.Tin the previous year. More than 90% of rice produced in Asia,where China and India being the lead producer. Diseases are the significant limiting factors that affect rice production causing annual yield losses conservatively estimated 5%. More than 70 diseases are caused by fungi,bacteria, viruses or nematodes on rice among which rice blast (Magnaporthe grisea) is the most serious one. Table-1 List of rice diseases: (Fungal and Bacterial)5,6FUNGAL Aggregate sheath spot Ceratobasidium oryzae-sativae Gunnel and Webster (anamorph: Rhizoctonia oryzae-sativae (Sawada) Mordue Narrow brown leaf spot Cercospora janseana (Racib) O.Const. = C.oryzae Miyake (telemorph: Sphaerulina oryzina K.Hara) Black kernel Curvularia lunata (Wakk) Boedijn (telemorph: Cochliobolus lunatusR. R.Nelson &Haasis) Peckey rice (Kernel spotting) Damage by many fungi including Cochliobolus miyabeanus (Ito and Kuribayashi) Drechs. ex Dastur, Curvularia sp., Fusarium sp. Microdochium oryzae(Hashioka and Yokogi) Samuels and I.C. Halett, Sarocladium oryzae (Sawada) W. Gams and D. Hawksworth Blast (leaf, neck [rotten neck], nodal and collar Pyricularia grisea Sacc. = P.oryzae Cavara (teleomorph: Magnaporthe grisea (Hebert) Barr) Root rots Fusarium sp.Pythium sp.P.dissotocum Drechs. P.spinosum Sawada Brown spot Cochliobolus miyabeanus (Ito and Kuribayashi) Drechs. ex Dastur (anamorph: Bipolaris oryzae (Breda de Seedling blight Cochlio bolus miyabeanus (Ito and Kuribayashi) Drechs.ex Dastur, Curvularia spp.,Fusarium spp.,Rhizoctonia solani Research Journal of Agriculture and Forestry Sciences _______________________________________________ ISSN 2320-6063Vol. 1(9), 19-25, October (2013) Res. J. Agriculture and Forestry Sci. International Science Congress Association 20 Haan)Shoemaker Kuhn,Sclerotium rolfsii Sacc.(teleomorph: Athelia rolfsii(Curzi) Tu and Kimbrough) Crown sheath rot Gaeumannomyces graminis (Sacc) Arx and D.Olivier Sheath blight Thanatephorus cucumeris (A.B.Frank) Donk (anamorph: Rhizoctonia solani Kuhn) Downy mildew Sclerophthora macrospora (Sacc) Thirumalachar et al.Sheath spot Rhizoctonia oryzae Ryker and Gooch Eye spot Drechslera gigantea (Heald and F.A.Wolf) Ito Sheath rot Sarocladium oryzae (Sawada) W. Gams and D. Hawksworth=Acrocylindrium oryzae Sawada False smut Ustilaginoidea virens (Cooke) Takah. Stackburn (Alternaria leaf spot) Alternaria padwickii (Ganguli) M.B.Ellis Kernel smut Tilletia barclayana (Bref.)Sacc. and Syd. in Sacc. Neovossia horrida (Takah) Padwick and A. Khan Stem rot Magnaporthe salvinii (Cattaneo) R. Krause and Webster (synamorphs: Sclerotium oryzae Cattaneo, Nakataea sigmoidae(Cavara) K.Hara) Leaf smut Entyloma oryzae Syd. and P.Syd. Water-mold(seed-rot and seedling disease) Achlya conspicua Coker A.klebsiana Pieters Fusarium spp. Pythium spp.P.dissotocum Drechs P.spinosum Sawada Leaf scald Microdochium oryzae (Hashioka and Yokogi) Samuels and aI.C.Hallett= Rhynchosporium oryzae Hashioka & Yokogi Black sheath spot Curvularia fallax B.D.Gao et al BACTERIAL Bacterial blight Xanthomonas oryzae pv. Oryzae (Ishiyama)Swings et al. =X.campestris pv.oryzae (Ishiyama) Dye Grain rot Pseudomonas glumae Kurita and Tabei Bacterial leaf streak Xanthomonas oryzae pv. oryzicola(Ishiyama) Swings et al. Sheath brown rot Pseudomonas fuscovaginae (ex Tanii et al.) Miyajima et al. Foot rot Erwinia chrysanthemi Burkholder et al Pseudomonasisa genus of gammaproteobacteria belonging to the family Pseudomonadaceae and is gram negative rod shaped having one or more flagella, motile, non-spore forming aerobicsome strains also have anaerobic respiration with nitrate as the terminal electron acceptor and 58-69% GC content. It is a very large and important family of gram negative bacteria and is chemoheterotrophic with versatile function predominantly present in soil. They have the ability to colonize rhizosphere of a wide variety of crops including cereals, pulses, oilseeds and vegetables8,9 and can be used as plant growth promoters and biocontrol agents10-12. The genus Pseudomonas includes both fluorescent and non-fluorescent species. The fluorescent species produce water soluble yellow green pigments and fluorescence under low wave length UV radiation13. Although some Pseudomonads are well known plant pathogen (Pseudomonas syringae) but some members of this group are beneficial to plants. They are known to produce secondary metabolites like siderophores, antibiotics, HCN etc14 and enzymes like proteases and gluconases15,16 which made them the most promising group of plant growth promoting rhizobacteria involved in the biocontrol of plant diseases17-19. Bacterial antagonists have twin advantage of faster multiplication and higher rhizosphere competence hence Pseudomonas fluorescens has successfully used for biological control of several plant pathogens20 and its application as biocontrol agents has drawn wide attention because of the production of secondary metabolites such as siderophores, antibiotics, volatile compounds, HCN, enzymes and phytohormones21,22. Research Journal of Agriculture and Forestry Sciences _______________________________________________ ISSN 2320-6063Vol. 1(9), 19-25, October (2013) Res. J. Agriculture and Forestry Sci. International Science Congress Association 21 Table-2 Pseudomonas as a biocontrol agent for major rice diseases Disease Pathogen (Causal agent) Biological control agent developed Reference Blast (Bl) Pyricularia oryzae (Telimorph: Magnaporthe grisea)Pseudomonas fluuorescens23, 24,25 Sheath blight(ShB) Rhizoctonia solani (Telimorph:Thanetophorus cucumeris) Pseudomonas fluuorescensPseudomonasputida 26,27,28,29 Sheath-rot(Sh-R) Sarocladium oryzae Pseudomonas fluuorescens30,31,32 Bacterial Blight (BB) Xanthomonas oryzae pv.oryzae Pseudomonas fluuorescens33,34,35 Mechanism of Pathogen Suppression Antibiotic mediated suppression: Pseudomonas fluorescens has a gene cluster that produces antibiotics including compounds such as 2, 4-diacetylphloroglucinol (DAPG), phenazine, pyrrolnitrin, pyoluteorin and biosurfactant antibiotics36. Pseudomonas fluorescens is uniquely capable of synthesizing many of these antibiotics not only to enhance its own fitness but also to help in the maintenance of soil health and bioprotection of crop from pathogens37,38. Strains of plant associated Pseudomonas Pseudomonas fluorescens produce DAPG and was detected by PCR–based screening method that used primers Phl2a and Phl2b and amplified 745-bp fragment characteristics of DAPG. HPLC, 1H NMR and IR analysis provided further evidence for its production and showed that compound (DAPG) suppress the growth of Xanthomonas oryzae pv. Oryzaea causal agent of the bacterial blight of rice in vitro assay suppress up to 59%-64% in net house and in vivoi.e.in field experiments 39,40 . P.aeruginosa (PUPa3) isolated from rhizospheric soil of rice produce Phenazine-1-Carboxamide (PCN) control rice disease caused by Sarocladium oryzae and Rhizoctonia solani 41. Phenazine-1-Carboxylic acid (PCA) produced by P.fluorescens suppress the leaf and neck blast of rice42. Sarocladium oryzae (Sawada) the causative agent of rice sheath rot is highly sensitive to P.fluorescens due to the production of antibiotics 43-48. HCN mediated suppression: Hydrocyanic acid production plays a major role in suppression of the growth of phytopathogen. HCN inhibits the electron transport thereby the energy supply to the cell is disrupted leading to the death of the organism. It inhibits the proper functioning of enzymes and natural receptors by reversible mechanism of inhibition49. It is also known to inhibit the action of cytochrome oxidase50. HCN is a volatile compound which on interaction with fungi can easily degrade its cell wall51,52 presented evidence that HCN is involved in biological control by Pseudomonas fluorescens strain CHAO. HCN production by Pseudomonas isolated from Potato and wheat rhizosphere53. Plant associated Pseudomonas fluorescens produce HCN, a broad-spectrum antimicrobial compound involved in the biological control of root diseases where HCN synthase is encoded by three biosynthetic genes hcnA, hcnB and hcnC54. Siderophores mediated suppression: Siderophores are iron binding extracellular compound with low molecular weight and high affinity for ferric iron that are secreted by microorganisms to take up iron from the environment55. The ability of sequester iron provides a competitive advantage to microorganisms. Siderophore chelate ferric ions with a high specific activity and serves as vehicles for the transport of ferric iron in to the microbial cell56. Transport of iron in to the cell is mediated by a membrane receptor that specifically recognizes ferric-siderophore complex57. Since iron is an essential element and a cofactor in various oxidoreductive enzymatic reaction binding of this element to siderophore create an artificial deficiency in the soil and their mode of action in suppression of disease were thought to be solely based on competition for iron with the pathogen58,59. Fluorescent Pseudomonads are characterized by the production of yellow-green pigments termed pyoverdines which fluoresce under UV light and function as siderophores60. The role of siderophores produced by fluorescent Pseudomonas in plant growth promotion was first reported by Kloepper J.W. and Schroth M.N.61 and was later reported to be implicated in the suppression of plant pathogens62,63. Competition for iron between pathogens and siderophores of fluorescent pseudomonas has been implicated in the biocontrol of wilt diseases caused by Fusarium oxyporum64,65. Pyoverdines chelate iron in the rhizosphere and deprive pathogens of iron which is required for (Pseudobactin and ferrooxamine B) that chelate the scarcely available iron and thereby prevent pathogens from acquiring iron66. Siderophore production by plant growth promoting fluorescent Pseudomonas sp.RB13 was effective against several fungal and bacterial pathogens67. Siderophore mutant of fluorescent Pseudomonas have been used to elucidate the role of siderophores in biocontrol. Mutants were obtained by exposure to UV68. NTG and Tn5 transposon mutagenesis69-71 were compared with their wild types with respect to the suppression of diseases. The wild type strains were more effective in suppressing the diseases than Sid-mutants.Similarly the wild strain of Pseudomonas putida significantly suppressed chlamydospore of Fusarium oxysporum72 whereas the sid mutant fails to do so.Several strains of siderophore producing P.fluorescens have been shown to inhibit Fusarium oxysporum, Rhizoctonia solani and Acrocladium oryzae73. Siderophores produced by Pseudomonas showed good antifungal activity against plant deletorius fungi, viz. Aspergillus niger, A.flavus, A.oryzae, F.oxysporum and Sclerotium rolfsii74. The iron concentration in soil was lowered by the addition of an iron chelator75. Fluorescent pseudomonas when grown on casamino acid medium under iron deficiency trihydroxamate type pyoviridines forming hexadentate ligands with Fe3+ ions were Research Journal of Agriculture and Forestry Sciences _______________________________________________ ISSN 2320-6063Vol. 1(9), 19-25, October (2013) Res. J. Agriculture and Forestry Sci. International Science Congress Association 22 found and these siderophores were antagonistic to fungal pathogens like Fusarium oxysporum .Alternaria and Colletotrichum capsici76. Recently,the pseudobactin siderophore of P.fluorescens WCS374r was found to be an important determinant of ISR against blast disease of rice77. Enzymes and phytohormones mediated suppression: Chitinase, P-1,3 gluconase and cellulase are especially important fungus controlling enzymes due to their ability to degrade the fungal cell wall components such as chitin,P1,3 glucan and glucosidic bonds78-80. Pseudomonas fluorescens produce chitinase which involved in lysis and fragmentation of fungal cell wall and suppression of phytopathogenic fungi81. Chitinase excreting microorganisms have been reported as efficient biocontrol agents82-84. Fluorescent Pseudomonas produce plant growth promoting hormones and enzymes which suppress the growth of phytopathogenic fungi85,86. Pseudomonas fluorescens (AUPF25) produce protease, IAA and siderophore showed inhibition of mycellial growth of Pyricularia oryzae a causal organisms of blast disease of rice87 . Conclusion Chemical pesticides harm the environment and host-plant resistant,which is based on a single gene,may not be durable in the field leading to frequent resistance breakdowns.It is imperative to develop environment-friendly and sustainable control strategies.Biological control is an ecology-conscious, cost effective and sustainable alternative method in disease management. As reported by many researchers that Pseudomonas produce different antibiotics which suppress the rice disease causing pathogens it is important to find out these antibiotics are active against other diseases of rice or not. 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