Diversity and antagonistic potential of apoplastic bacteria against Ralstonia pseudosolanacearum race 4 causing bacterial wilt of ginger


  • ICAR-Indian Institute of Spices Research, Division of Crop Protection, Kozhikode, Kerala, 673012, India


Bacterial wilt caused by Ralstonia pseudosolanacearum race 4 is a devastating disease of ginger, for which almost all control measures met with limited success. In this study, 150 bacteria isolated from the apoplastic fluid of ginger were screened for antagonism against R. pseudosolanacearum both in vitro and in planta and shortlisted six isolates which were further characterized for biocontrol and plant growth promoting traits. The promising isolates were identified as Bacillus subtilis (IISRGAB 5), B. marisflavi (IISRGAB 43), B. licheniformis (IISRGAB 107), Agrobacterium tumefaciens (IISRGAB24), Micrococcus luteus (IISRGAB 48) and Staphylococcus haemolyticus (IISRGAB 146). Green house evaluation against R. pseudosolanacearum, by seed priming and soil drenching showed that B. licheniformis strain GAP107–MTCC 12725, was able to reduce bacterial wilt incidence up to 67%. Hence, this bacterium was identified as a suitable candidate for developing a potential biocide for the management of bacterial wilt in ginger.


Apoplastic Bacillus licheniformis, bacterial wilt, ginger, Ralstonia pseudosolanacearum race 4

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Achari GA, Ramesh R. 2014. Diversity, biocontrol, and plant growth promoting abilities of xylem residing bacteria from solanaceous crops. Int J Microbiol. 14: doi: 10.1155/2014/296521 https://doi.org/10.1155/2014/296521 PMid:24963298 PMCid:PMC4055287

Amaresan N, Jayakumar V, Thajuddin N. 2014. Isolation and characterization of endophytic bacteria associated with chilli (Capsicum annuum) grown in coastal agricultural ecosystem. Indian J Biotechnol.13: 247-255

Amaresan N, Jayakumar V, Kumar K, Thajuddin N. 2012. Endophytic bacteria from tomato and chilli, their diversity and antagonistic potential against Ralstonia solanacearum. Arch Phytopathol Plant Prot. 45 (3): 344-355 https://doi.org/10.1080/03235408.2011.5872 73

Aneja KR. 2003. Experiments in microbiology, plant pathology and biotechnology. New Age International Publishers

Apun K, Jong BC, Salleh MA. 2000. Screening and isolation of a cellulolytic and amylolytic Bacillus from sago pith waste. J Gen Appl Microbiol. 46(5): 263-267 https://doi.org/10.2323/jgam.46.263 PMid:12483578

Anthony KJP, Murugan M, Gurunathan S. 2014. Biosynthesis of silver nanoparticles from the culture supernatant of Bacillus marisflavi and their potential antibacterial activity. J Indust Eng Chem. 20(4):1505-1510 https://doi.org/10.1016/j.jiec.2013.07.039

Aravind R, Antony D, Eapen, SJ, Kumar A, Ramana KV, 2009. Isolation and evaluation of endophytic bacteria against plant parasitic nematodes infesting black pepper (Piper nigrum L.). Indian J Nematol. 39(2):211-217.

Bacon CW and Hinton DM. 2006. Bacterial endophytes: The endophytic niche, its occupants, and its utility. pp. 155– 194. In: Gnanamanickam SS (Eds.). Plant-Associated Bacteria. Springer; Netherlands.

Bacon CW, Hinton DM. 2002. Endophytic and biological control potential of Bacillus mojavensis and related species. Biol Control. 23(3): 274-284 https://doi.org/10.1006/bcon.2001.1016

Bell CR., Dickie GA, Harvey WLG, Chan JWYF. 1995. Endophytic bacteria in grapevine. Can J Microbiol.

(1): 46-53. https://doi.org/10.1139/m95-006

Barretti PB, de Souza RM, Pozza EA, de Souza JT (2012) Combination of endophytic bacteria and resistant cultivars improves control of Ralstonia wilt of tomato. Australas Plant Pathol. 41(2): 189-195 https://doi.org/10.1007/s13313-011-0107-1

Belimov AA, Dodd IC, Safronova VI, Shaposhnikov AI, Azarova TS, Makarova NM, Davies WJ, Tikhonovich, IA. 2015. Rhizobacteria that produce auxins and contain 1 amino cyclopropane 1 carboxylic acid deaminase decrease amino acid concentrations in the rhizosphere and improve growth and yield of well watered and water limited potato (Solanum tuberosum). Ann Appl Biol. 67(1):11-25 https://doi.org/10.1111/aab.12203

Cappuccino JC, Sherman N. 1992 Microbiology: A Laboratory Manual. 3rd ed. Benjamin/cummings Pub.

Co. pp. New York: 125-179.

Cappuccino JG, Sherman N. 2005. Microbiology: A Laboratory Manual. 7th ed. Benjamin Cummings: Pearson Education, Inc. New York

Casida Jr LE, Klein DA, Santoro T. 1964. Soil dehydrogenase activity. Soil Sci. 98(6): 371-376 https://doi.org/10.1097/00010694-196412000-00004

Cawoy H, Bettiol W, Fickers P, Ongena M. 2011. Bacillusbased biological control of plant diseases. pp. 273-302, In: Stoytcheva M (Ed.). Pesticides in the modern worldpesticides use and management. InTech, Rijeka, Croatia. https://doi.org/10.5772/17184

Cheng FY, Burkey KO, Robinson JM, Booker FL. 2007. Leaf extracellular ascorbate in relation to O3 tolerance of two soybean cultivars. Environ Pollut. 150(3):355362. https://doi.org/10.1016/j.envpol.2007.01.022 PMid:17442469

Clarke PH, Cowan ST. 1952. Biochemical methods for bacteriology. Microbiology 6(1-2): 187-197 https://doi.

org/10.1099/00221287-6-1-2-187 PMid:14927866

Collins CH, Lyne PM, Grange JM. 1995. Collins and Lyne’s Microbiological Methods, 7th ed. ButterworthHeinemann, UK, pp. 114

De Boer SH, Copeman RJ, 1974. Endophytic bacterial flora in Solanum tuberosum and its significance in bacterial ring rot disease. Can J Plant Sci. 54: 115- 122 https://doi.org/10.4141/cjps74-019

Dong Z, Canny MJ, McCully ME, Roboredo MR, Cabadilla CF, Ortega E. Rodes R. 1994. A nitrogen-fixing endophyte of sugarcane stems (a new role for the apoplast). Plant Physiol. 105(4): 1139-1147 https://doi.org/10.1104/ pp.105.4.1139 PMid:12232271 PMCid:PMC159442

Engelbrecht MC. 1994. Modification of a semi-selective medium for the isolation and quantification of Pseudomonas solanacearum. ACIAR Bacterial Wilt Newsletter. 10: 3-5

Feng H, Li Y, Liu Q. 2013. Endophytic bacterial communities in tomato plants with differential resistance to Ralstonia solanacearum. Afr J Microbiol Res. 7(15): 1311-1318 https://doi.org/10.5897/AJMR12.375

Gagne S, Richard C, Rousseau H, Antoun H. 1987. Xylemresiding bacteria in alfalfa roots. Can J Microbiol.

(11): 996-1000. https://doi.org/10.1139/m87-175

Gardner JM, Feldman AW, Zablotowicz RM. 1982. Identity and behavior of xylem-residing bacteria in rough lemon roots of Florida citrus trees. App Environ Microbiol. 43(6): 1335-1342.

Gaur AC. 1990. Physiological functions of phosphate solubilising microorganisms. pp. 16-72. In: Gaur AC (Ed.). Phosphate solubilising microorganisms as biofertilizers. Omega Scientific publishers. New Delhi,

Hallmann J, Quadt-Hallmann A, Mahaffee WF, Kloepper JW. 1997. Bacterial endophytes in agricultural crops. Can J Microbiol. 43(10): 895-914 https://doi.org/10.1139/m97-131

Hallmann J, Kloepper JW, Rodriguez-Kabana R. 1997a. Application of the Scholander pressure bomb to studies on endophytic bacteria of plants. Can J Microbiol. 43(5): 411-416. https://doi.org/10.1139/m97-058

Jasim B, Joseph AA, John CJ, Mathew J, Radhakrishnan EK. 2014. Isolation and characterization of plant growth promoting endophytic bacteria from the rhizome of Zingiber officinale. 3 Biotech 4(2): 197-204 https:// doi.org/10.1007/s13205-013-0143-3 PMid:28324450 PMCid:PMC3964247

Ji X, Lu G, Gai Y, Zheng C, Mu Z. 2008. Biological control against bacterial wilt and colonization of mulberry by an endophytic Bacillus subtilis strain. FEMS Microbiol Ecol. 65(3): 565-573 https://doi.org/10.1111/j.15746941.2008.00543.x PMid:18631174

Kim JH, Lee SH, Kim CS, Lim EK, Choi KH, Kong HG, Kim DW, Lee SW, Moon BJ. 2007. Biological control of strawberry gray mold caused by Botrytis cinerea using Bacillus licheniformis N1 formulation. J Microbiol Biotechnol. 17(3): 438-444.

Kim YC, Leveau J, Gardener BBM, Pierson EA, Pierson LS, Ryu CM. 2011. The multifactorial basis for plant health promotion by plant-associated bacteria. Appl Environ Microbiol. 77(5): 1548-1555 https://doi.org/10.1128/ AEM.01867-10 PMid:21216911 PMCid:PMC3067257

Klement Z. 1965. Method of obtaining fluid from the intercellular spaces of foliage and the fluid’s merit as substrate for phytobacterials pathogens. Phytopathology 55: 1033-1034.

Kloepper JW, Reddy MS, Rodríguez-Kabana R, Kenney DS, Kokalis-Burelle N, Martinez-Ochoa N, Vavrina CS. 2004. Application for rhizobacteria in transplant production and yield enhancement. Acta Hortic. 217230 https://doi.org/10.17660/ActaHortic.2004.631.28

Kong HG, Kim JC, Choi GJ, Lee KY, Kim HJ, Hwang EC, Moon BJ, Lee SW. 2010. Production of surfactin and iturin by Bacillus licheniformis N1 responsible for plant disease control activity. The Plant Pathol J. 26(2):170177 https://doi.org/10.5423/PPJ.2010.26.2.170

Kumar A, Hayward AC. 2005. Bacterial diseases of ginger and their control. pp 341-366. In: Ravindran PN, Babu KN (Eds.). Monograph on Ginger. CRC Press, Boca Raton, FL, USA,

Lalande R, Bissonnette N, Coutlée D, Antoun H. 1989. Identification of rhizobacteria from maize and determination of their plant-growth promoting potential. Plant Soil 115(1): 7-11. https://doi.org/10.1007/ BF02220688

Lee JP, Lee SW, Kim CS, Son JH, Song JH, Lee KW, Kim HJ, Jung SJ, Moon BJ. 2006. Evaluation of formulations of Bacillus licheniformis for the biological control tomato gray mold caused by Botrytis cinerea. Biol Control. 37(3): 329-337 https://doi.org/10.1016/j.biocontrol.2006.01.001

Lemessa F, Zeller W. 2007. Screening rhizobacteria for biological control of Ralstonia solanacearum in Ethiopia. Biol Control. 42(3): 336-344 https://doi.org/10.1016/j.biocontrol.2007.05.014

Lemos ML, Toranzo AE, Barja JL. 1985. Antibiotic activity of epiphytic bacteria isolated from intertidal seaweeds.Microb Ecol. 11(2): 149-163 https://doi.org/10.1007/BF02010487 PMid:24221303

Lorck H. 1948. Production of hydrocyanic acid by bacteria. Plant Physiol. 1(2): 142-146 https://doi.org/10.1111/j.1399-3054.1948.tb07118.x

Luwe MW, Takahama U, Heber U. 1993. Role of ascorbate in detoxifying ozone in the apoplast of spinach (Spinacia oleracea L.) leaves. Plant physiol. 101(3):969-976. https://doi.org/10.1104/pp.101.3.969 PMid:12231749 PMCid:PMC158714

Lyons T , Ollerenshaw JH , Barnes JD. 1999. Impacts of ozone on Plantago major: apoplastic and symplastic antioxidant status. New Phytol. 141(2): 253-263 https:// doi.org/10.1046/j.1469-8137.1999.00338.x

Maketon M, Apisitsantikul J, Siriraweekul C. 2008. Greenhouse evaluation of Bacillus subtilis AP-01 and Trichoderma harzianum AP-001 in controlling tobacco diseases. Braz J Microbiol. 39(2): 296-300 https://doi.org/10.1590/S1517-83822008000200018 PMid:24031219 PMCid:PMC3768408

Mendes R, Pizzirani-Kleiner AA, Araujo WL, Raaijmakers JM. 2007. Diversity of cultivated endophytic bacteria from sugarcane: genetic and biochemical characterization of Burkholderia cepacia complex isolates. Appl Environ Microbiol. 73(22): 7259-7267 https://doi.org/10.1128/ AEM.01222-07 PMid:17905875 PMCid:PMC2168197

Misaghi IJ, Donndelinger,CR. 1990. Endophytic bacteria in symptom-free cotton plants. Phytopathology 80(9): 808-811. https://doi.org/10.1094/Phyto-80-808

Nabti EH, Mokrane N, Ghoul M, Manyani H, Dary M, Megias MG. 2013. Isolation and characterization of two halophilic Bacillus (Bacillus licheniformis and Bacillus sp.) with antifungal activity. J Ecol Health Environ. 1: 13-17 https://doi.org/10.12785/jehe/010102

Nouchi I, Hayashi K, Hiradate S, Ishikawa S, Fukuoka M, Chen CP, Kobayashi K. 2012. Overcoming the difficulties in collecting apoplastic fluid from rice leaves by the infiltration-centrifugation method. Plant Cell Physiol. 53(9):1659-1668. https://doi.org/10.1093/pcp/pcs102 PMid:22813544

Prameela TP. 2016. Studies on biovar specific diagnostics for Ralstonia solanacearum Yabuuchi (Smith) infecting ginger (Zingiber officinale Rosc.) and evaluation of apoplastic microbes for biocontrol. Ph.D. Thesis.

Submitted to Mangalore University, Karnataka.

Ramesh R, Joshi AA. Ghanekar MP. 2009. Pseudomonads: major antagonistic endophytic bacteria to suppress bacterial wilt pathogen, Ralstonia solanacearum in the eggplant (Solanum melongena L.). World J Microbiol Biotechnol. 25(1): 47-55 https://doi.org/10.1007/s11274-008-9859-3

Roberts PD, Momol MT, Ritchie L, Olson SM, Jones JB, Balogh B. 2008. Evaluation of spray programs containing famoxadone plus cymoxanil, acibenzolarS-methyl, and Bacillus subtilis compared to copper sprays for management of bacterial spot on tomato. Crop Prot. 27(12): 1519-1526 https://doi.org/10.1016/j.cropro.2008.06.007

Rudrappa T, Biedrzycki ML, Kunjeti SG, Donofrio NM, Czymmek KJ, Paul WP, Bais HP. 2010. The rhizobacterial elicitor acetoin induces systemic resistance in Arabidopsis thaliana. Commun Integr Biol.

(2): 130-138 https://doi.org/10.4161/cib.3.2.10584 PMid:20585504 PMCid:PMC2889968

Saddler GS. 2005. Management of bacterial wilt disease. pp. 121-132. In: Allen C, Prior P, Hayward AC (Eds.). Bacterial wilt disease and the Ralstonia solanacearum species complex. APS press.

Sarwar M, Kremer RJ. 1995. Determination of bacterially derived auxins using a microplate method. Lett Appl Microbiol. 20(5): 282-285. https://doi.org/10.1111/j.1472-765X.1995.tb00446.x

Schwyn B, Neilands JB. 1987. Universal chemical assay for the detection and determination of siderophores. Anal Biochem. 160(1): 47-56. https://doi.org/10.1016/00032697(87)90612-9

Upreti R, Thomas P. 2015. Root-associated bacterial endophytes from Ralstonia solanacearum resistant and susceptible tomato cultivars and their pathogen antagonistic effects. Front Microbiol. 6: 255. https:// doi.org/10.3389/fmicb.2015.00255 PMid:25926818 PMCid:PMC4396348

Van Peer R, Niemann GJ, Schippers B. 1991. Induced resistance and phytoalexin accumulation in biological control of Fusarium wilt of carnation by Pseudomonas sp. strain WCS417r. Phytopathology 81(7): 728-734.


Vermelho AB, Meirelles MNL, Lopes A, Petinate SDG, Chaia AA, Branquinha MH. 1996. Detection of extracellular proteases from microorganisms on agar plates. Mem Inst Oswaldo Cruz. 91(6): 755-760. https://doi.org/10.1590/ S0074-02761996000600020 PMid:9283660

Wang XB, Luo YM, Liu WX, Li ZG. 2011. Identification, antimicrobial activity and field control efficacy of an endophytic bacteria strain against peanut bacterial wilt. Chin J Biol Control 27(1): 88-92.

Yamada T, Kawasaki T, Nagata S, Fujiwara A, Usami S, Fujie M. 2007. New bacteriophages that infect the phytopathogen Ralstonia solanacearum. Microbiology. 153 (8) : 2630-2639. https://doi.org/10.1099/mic.0.2006/001453-0 PMid:17660427

Yoon JH, Kim IG, Kang KH, Oh TK, Park YH. 2003. Bacillus marisflavi sp. nov. and Bacillus aquimaris sp. nov., isolated from sea water of a tidal flat of the Yellow Sea in Korea. Int J Syst Evol Microbiol. 153(8): 2630-2639.


Zinniel DK, Lambrecht P, Harris NB, Feng Z, Kuczmarski D, Higley P, Ishimaru CA, Arunakumari A, Barletta RG, Vidaver AK. 2002. Isolation and characterization of endophytic colonizing bacteria from agronomic crops and prairie plants. Appl Environ Microbio. 68(5): 2198-2208. https://doi.org/10.1128/AEM.68.5.21982208.2002 PMid:11976089 PMCid:PMC127535


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