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The Potential of Bacillus spp. and Pseudomonas spp. as Biological Control Against Pantoea sp. (or Biocontrol Agents to) Cokorda Javandira1*), Luqman Qurata Aini2) dan Abdul Latief Abadi2) Graduate School of Plant Science, Faculty of Agriculture, Brawijaya University, Jl. Veteran, Malang 65145, East Java Indonesia. 2) Faculty of Agriculture, Brawijaya University, Jl. Veteran, Malang 65145, East Java Indonesia. *)Corresponding author phone: +62-819-16180186, Email :cokordajavandira@yahoo.co.id ABSTRACT One of new biotic constraints in corn production in Indonesia is leaf blight disease caused by Pantoea spp. which is needed to be controlled. The purpose of this research is to study the potential of Bacillus spp. and Pseudomonas spp. as biological control (biocontrol agents) of corn leaf blight caused by Pantoea spp. The results showed that all bacterial strains of Bacillus and Pseudomonas have potential in inhibiting the growth of Pantoea spp. by showing the clear zone on the agar plate. The antibiosis types are bactericide or bacteriostatic. On pot experiment all bacterial strains showed the reduction of the disease incidence at the same level compared with that of bactericide streptomycin suphate 20%. All bacterial strains as well as bactericide could reduce the disease incidence at 20-25% compared with that of control (aquades treatment only). Thus the results suggest that all bacterial strains are potential as biological control agents against leaf blight disease on corn leaf caused by Pantoea spp. Keywords: Biological control, Bacillus sp., Pseudomonas sp. and Pantoea sp. INTRODUCTION Corn (Zea mays L.) is one of the commodities of potential as a source of carbohydrate, food indutries, food livestock and important on Indonesia's economy. One of the new constraint biotic factor of corn production is leaf blight disease caused by Pantoea spp. Damage this pathogen can impact on corn physiological thus influence to corn productivity. Case study in Brazil, Pantoea spp. damage loss productivity until 60% (Cota et al., 2010). Pantoea spp. are one Organisme Quarantine in Indonesia Quarantine Departement. Based data from Indonesia Statistics Agency (BPS) year 2012, Province of East Java is one potential site corn producer with corn production 5, 995, 001 ton. Based last research, Suryani (2012), find leaf blight diseases on corn in Kediri regency that supposedly caused by bacteria Pantoea spp. Futher reported that Pantoea spp. has characteristic of shaped is oval with size 0,4-2,2 m, shaped somewhat flattened round with the edge and colored yellow. This bacterial can be spread through water, seed and insect vector. A host of this bacteria are broad, among other corn, cane, sorghum, rice, tomatoes, wheat, cotton and melon (Cota et al., 2010). Pataky (2004) say to manage of disease can be integrated, including host resistance, protection/ eradication and exclusion. This research want to (escape) studies (study) as bacterial biological agens can control the pathogens. (bacterial as biological agents to the pathogens)
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Bacillus spesies and Pseudomonas spesies known have potential as biological control agents to inhibited many plant pathogens (Cook and Baker, 1996). The ability of Bacillus spp. and Pseudomonas spp. can compete (?) to used nutritions, producing of secondary metabolite as antibiotic, sidefor and extracellular enzyme. The aim of this research to studies the potential of strains Bacillus spp. and Pseudomonas spp. as biological control leaf blight disease caused by Pantoea spp. MATERIALS AND METHODS The research held (was done) in bacteriology laboratory and in glass house of Agriculture Faculty, University of Brawijaya from september 2012 until february 2013. Material that used in this research among (are) culture of bacteria Pantoea spp., Bacillus spp. strains UB-ABS1, UB-ABS4, UB-ABS5 and Pseudomonas spp. strains UB-PF1, UB-PF3, UB-PF4 and all of them is collection of disease laboratory, Faculty of Agriculture University of Brawijaya, sweet corn seed Jago strains (East West Seed, Indonesia), nutrient agar and kings B medium, bactericide streptomycin sulphate 20% and chlorofoam. An instrument used to research are petridish, analytic scales, hand sprayer, spectrophotometer 21 (Milton Roy Company, USA), vortex, rotary shaker and micropipet. Antagonism Assays Bacillus spp. dan Pseudomonas spp. against Pantoea spp. in Petridish The antagonism assay should be conducted by a spray inoculation methods (Kawaguchi et al., 2008). Cell suspension all strains of Bacillus spp. and Pseudomonas spp. prepared from 48 hours culture on nutrient agar medium, adjusted to OD660 = 1.0 to about 109 Colony Forming Unit (CFU)/ml for inoculation. Placing (dipped) a sterile paper disk (5-mm diameter) to suspension for 1 minute and then dried off for two hours, put to petridish with nutrient agar plate and incubating (incubate) it for 2 days at 27oC. All strains Bacillus spp. and Pseudomonas spp. were killed with chlorofoam on each plate, the plates were misted with a dilute suspension of Pantoea spp (to about 109 CFU)/ml. The plates were photographed 2 days after misting, and the diameter of clear zone of (as) inhibition indicative (?) of antibiosis, was measured. Characteristic Antagonism Assays from Bacterial Biological Control Mode of action antagonism assay Bacillus spp. and Pseudomonas spp. against Pantoea spp. is known by take part of the clear zone then be included to test tube (containing 10 mL of 0.5 % pepton solution, after that shaker during 24 hours. If the pepton solution becomes pure it showed bactericidal, whereas the solution is muddy it showed bacteristatic action (Djatmiko et al., 2007). Antibiosis Supernatant Activity Test of Biological Control Against Pantoea spp. For the production of bacteriocins, all strains Bacillus spp. and Pseudomonas spp. was grown in 10 mL Luria Bertani (LB) medium at 27oC in rotary shaker for 48 h. the cells were harvested by centrifugation at 10,000 rpm for 10 minute and the culture supernatant was sterilized by filtration with 0.45 m milipore membranes. To examination of potential antibiosis activity, supernatant suspension entered on the wellbore in nutrient agar plate using a cork borer (diameter 0.5 mm). Wellbore observation after 48 h.
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Potential of Bacterial Bacillus spp. and Pseudomonas spp. decrease leaf blight disease syndrome on corn leaf caused Pantoea spp. Plants were used as seedling at approximately 14 days after cropping and leafy strands 4 was inoculated with Bacillus spp. and Pseudomonas spp. by sprayed approximately 5 mL/plants using hand sprayer. A day later the leaves sprayed with suspension pathogen Pantoea spp (Beattie and Marcell, 2002). After inoculation, the plants were incubated in an enclosed plastict tent for 12 h at 23oC and 90% RH, after that moved that plants to glass house at 27oC and 60% RH (Valenzuela et.al, 2007). The intensity of disease damage evaluation after 7 days inoculation pathogens, intensity disease damage counting each plant in five scoring level (Lee and Hong, 2010). Evaluation rapid adherence population of bacteria biological control on corn leaves Potential of colonization bacteria Bacillus spp. and Pseudomonas spp. was done with spread method (viable plate count) modified from Beattie and Marcell (2002). A few corn leaves harvested after 7 days inoculation. The corn leaves is taken example used cork borer (diameter 0.8 mm) in three sector, upper, center and down. Enter into a test tube and 10 mL 0.1 buffer potassium phosphates, pH 7.0. To separate that bacteria on leaf surface, the suspension last vortex for 10 min after that spread on agar plate. Statistics Analysis Data from observations of antagonism in petridish, intensity leaf blight disease on corn, estimates of population adherent bacterial were analysed as log-transformed data then analyzed by analysis of variance (ANOVA) and Duncan multiple range test (DMRT) on 0.05 level. RESULTS AND DISCUSSION The Potential of Bacillus spp. and Pseudomonas spp. against Pantoea spp. in Petridish Based on (x) The result of the test on nutrient agar plate on a petridish (figure 1) all treatment with Bacillus species, Pseudomonas species and bactericide streptomycin sulphate 20% influence to inhibit the growth of Pantoea spp. In control treatment (x), aquades only there is no clear zone. Treatment with Bacillus spp. and Pseudomonas spp. give same influence compared than bactericide streptomycin sulphate 20% by produce clear zone (figure 3) (Fig 3). Clear zone are indicator activity mechanism of biological control, such as antibiotics, bacteriocin and enzyme (Sood et al., 2007). Bacterium Bacillus species and Pseudomonas species capable of controlling gram negative pathogenic, Pantoea species are gram negative bacteria. Antibiotic or bacteriocin compound are result of secondary metabolism, that can damage function and protection of membrane cell of Pantoea species.

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Tabel 1. The Antibiosis type of bacterial strains Bacillus sp. and Pseudomonas sp. T1 T2 T3 T4 T5 T6 T7 T8 Treatment Aquades steril only Bactericide Streptomycin sulfat 20% Bacillus spp. strain UB-ABS4 Bacillus spp. strain UB-ABS5 Bacillus spp. strain UB-ABS1 Pseudomonas spp. strain UB-PF3 Pseudomonas spp. strain UB-PF4 Pseudomonas spp. strain UB-PF1 Type of action Empty Bactericidal Bactericidal Bacteristatic Bactericidal Bacteristatic Bacteristatic Bacteristatic

Figure1. Clear zone index of antagonistics against Pantoea sp. Number followed by the same letters are not significantly different at p=0.05 according DMRT

B
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Figure 2. Antagonistic type produce from bacterial Bacillus spp. dan Pseudomonas spp. Bagian ini berisi foto,A. sehingga ukuran file lebih dari 25 MB. Jadi saya hilangkan Bacteristatic, B. Bactericidal.
sementara. Jika pas nanti ketemu akan saya tunjukkan foto yang saya maksud ini. Trims

Figure 3. In vitro antibiosis against leaf blight disease Pantoea sp. Paper disk with control steril aquades (A), streptomycin sulphate 20% (B), bacteria Bacillus spp. (C,D, E) and bacteria Pseudomonas spp. (F, G, H). Two days after misting, a halo of growth inhibition was seen around the paper disk.

Figure 4. Leaf blight damage on corn leaf. Number followed by the same letters are not significantly different at p=0.05 according DMRT.

Figure 5. Adherent population of bacterial on corn leaf. Number followed by the same letters are not significantly different at p=0.05 according DMRT.
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Antagonist Characteristic of Bacterial Biological Control Bacteria Bacillus species and Pseudomonas species showed potential as candidate for biological agens plant pathogens, because that ability to produce bactericidal or bacteristatic (figure 2). Observation from table 1, one Bacillus species have ability to produce bactericidal which is almost same with chemical bactericide (streptomycin sulphate 20%), but two strains of Bacillus species produce bacteristatic, whereas all Psedomonas species showed antagonistics by bacteristati (bacteriostatic, check). A group of Bacillus species known largely capable of antimicrobial compound, in higher concentration may be killed plant pathogens than in lower concentration just inhibit vegetative growth of pathogens. Djatmiko et al. (2007) says, bacteria Psedomonas fluorescens can control plant pathogens Ralstonia solanacearum caused licat disease by bacteristatic. Activity of Supernatant of Biological Control against Pantoea spp. Antimicrobial compounds as an bacteriocin group because the supernatant just consist on 27oC and 70 oC, after that if their bacteriocin can consist on heat treatment like 100 oC and 121oC the supernatant compounds are antibiotic because suciffiecient imprevious like chemical bactericide streptomycin sulphate 20%. Supernatant produce by Bacillus species and Pseudomonas species their ability nearly same as chemical bactericide. According Sutoyo et al. (1998) antimicrobe compounds produce by all strains Bacillus species and Pseudomonas species enough potential accordance with characteristic of bacteriocin. Bacteriocin antimicrobe is a compound of protein that could inhibit pathogens and other microorganism caused disease. The bacteriocin is produce at the exponential growth phase. Bacteriocins as an alternative to classical antibiotics produced by ribosomal synthesis. Antimicrobial peptide may exert in some case a narrow or in the cases a relatively broad spectrum of antimicrobial activity. Many antimicrobial peptide produce by Bacillus spp. are a potential for biological control of phytipathogens and as antibiotic precursors. Several factor influence the bacteriocin activity on the target bacterial cell. They include the structure and amount of the substance, the composition of the cytoplasmic membrane, the structure and the expression level of a protein with an immunity function, and the chemical composition of the environment. Bacillus species produce bacteriocins with different mode of action. Tochicin, lichenin and thuricin have a established bactericidal effect, then subtilisin inhibit vegetative cell by bacteristatic mechanism (Motta et al., 2008). Influence of bacterial Bacillus spp. and Pseudomonas spp. against Pantoea spp. on corn plants Ability of all strains Bacillus spp., Pseudomonas spp. and bactericide streptomycin 20% on corn plants can reduce intensity of leaf bligth disease caused pathogens Pantoea spp (figure 4). Bacteria biological agents Bacillus spp. and Pseudomonas spp can reduce 20%25% of leaf blight disease density. Corn plant still sickness becaused pathogen capable infecting host, although on leaf surface has been there are antagonist bacterium. According to Agrios (2005) plant pathogens can infect a plant by penetration the lining of the cuticule and cell walls on leaf tissue. Plant pathogenic bacteria Pantoea spp. known capable produce extracellular pollysaccharide, that can be extracellular polysaccharides can help pathogens
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survive on leaves surfaces. Microcolony forming a dense population then penetrate in to leaf tissue. Therefore, pathogens can infection and cause disease occurs as a result of contact and interaction between host and plant pathogen. If at the moment occurs in contact with environment condition that favor the pathogen can infect plants easier and disese will develop quickly (Beattie and Lindow, 1999). Effect of Leaf Surface waxes on bacterial adhesion Based on the result of potential adherent of Bacillus species and Pseudomonas species to corn leaves colonization. Population levels of Bacillus spp. and Pseudomonas spp. have a significant differences than control aquades only. The bacterial Bacillus spp. could survive with spores thus enhance epiphytic fitness (Jacobs and Sundin, 2001). If compare to control aquades only and bactericide the population Several bacterial adherence may be mediated by pili, carbohydrates, glycoproteins, hydrophobic interactions and even a localized positive charge (Romantschuk, 1992). Survival is also predicated the ability of organisms to cope with the varied environmental stress conditios, including fluctuating water ability, heat, osmotic stress and exposure to solar ultraviolet radiation. Bacterial Psedomonas spp. and Pantoea spp. is the most ubiquitous patricipants of phyllosphere communities (Lindow and Brandl, 2003). CONCLUSION Based from antagonism assay in petridish, supernatant activity assay know all strain bacterial Bacillus spp. and Pseudomonas spp. has potential to inhibit Pantoea spp., characteristic antagonisms is bactericide or bacteristatic. Thus antagonism assay on corn plant, bacterial Bacillus spp. and Pseudomonas spp. can decreased intensity of leaf blight disease damage caused Pantoea spp. 20-25%. ACKNOWLEDGEMENTS Thank you for BPKLN DIKTI that fund the research the is research was funded by BPKLN DIKTI. REFERENCES Agrios, G. N. 2005. Plant Phatology 5 Edition. Elsevier Academi Press. United State of America. Page 616-686. Arwiyanto, T., Y. M.S. Maryudani and N. N. Azizah. 2007. Sifat-Sifat Fenotipik Pseudomonas fluorescens, Agensia Pengendalian Hayati Penyakit Lincat pada Tembakau Temanggung. Biodiversitas. 8(2):147-151. Beattie, G. A. and S. E. Lindow. 1999. Bacterial Colonization of Leaves : A Spectrum of Strategies. Phytopathology. 89(5):353-359. Beattie, G. A and L. M. Marcell. 2002. Comparative Dynamics of Adherent and Nonadherent Bacterial Population on Maize Leaves. Phytopathology 92(9):1015-1023.

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Cook, R. J. AND K. F. BAKER. 1996. THE NATURE AND PRACTICE OF BIOLOGICAL CONTROL OF PLANT PATHOGENS. THE AMERICAN PHYTOPATHOLOGICAL SOCIETY PRESS. UNITES STATE OF AMERICA . 539 PP. Cota, L. V., R. V. Costa. D. D. Silva, D. F. Parreira, U. G. P. Lana and C. R. Casela. 2010. First report of pathogenecity of Pantoea ananatis in sorghum in Brazil. Australasian Plant Diseases Notes. 5:120-122. Djatmiko, H. A., T. Arwiyanto, B. Hadisutrisno AND B. H. SUNARMINTO . 2007. POTENTION OF THREE GENERA BACTERIA FROM THREE CROP RHIZOSPHERE AS B IOLOGICAL CONTROL AGENT OF THE LINCAT DISEASE . J. I NDONESIAN AGRICULTURAL SCIENCE . 9(1):40-47. Jacobs, J. L. and G. W. Sundin. 2001. Effect of Solar UV-B Radiation on a Phyllosphere Bacterial Community. Applied and Environmental Microbiology. 67(12):5488-5496. Kawaguchi, A., K. Inoue and Y. Ichinose. 2008. Biological Control of Crown Gall of Grapevine, Rose, Tomato by Nonpathogenic Agrobacterium vitis Strain VAR03-1. Phytopathology. 98(11):1218-1225. Lee, H. B. and J. P. Hong. 2010. First Report of Leaf Blight caused by Pantoea agglomerans on Rice in Korea. Plant Disease. 94(11):1372. Lindow, S. E. and M. T. Brandl. 2003. Microbiology of the Phyllosphere. Applied and Environmental Microbiology. 64(4):1875-1883. Motta, A. S., F. S. Flores, A. A. Souto and A. Brandelli. 2008. Antibacterial activity of a bacteriocin-like substance produce by Bacillus sp. P34 that targets the bacterial cell envelope. Antonie van Leeuwenhoek. 93:275-284. Pataky, J. K. 2004. Stewarts wilt of corn. The Plant Health Instructor. DOI:10.1094/PHI-I2004-0113-01 Schaad, N., J. Jones and W. Chun. 2001. Laboratory Guide for the Identification of Plant Pathogenic Bacteria, 3rd Edition. APS Press. America. Page 1-71. Sood, A., Shivesh S., K. Viviek dan L. T. Ram. 2007. Antagonism of Dominant Bacteria in Tea Rhizosphere of Indian Himalayan Regions. Journal Appl. Science Environment Management. 11(4):63-66. Suryani, L. 2012. Karakterisasi Bakteri Patogen Penyebab Penyakit Layu dan Hawar Daun pada Tanaman Jagung. Tesis. PPS Universitas Brawijaya. Malang. Suryani, L., L. Q. Aini., A. N. Sugiharto and A. L. Abadi. 2012. Characterization of Bacterial Pathogen Causing Wilt and Leaf Blight on Corn (Zea mays) by Physiological, Biochemical and Molecular Methods. Agrivita. 34(3): 286-295. Sutoyo, L. I. Sudirman, S. B. Poerwanto, Y. Widyastuti and I. Sudirman. 1998. Screening of Lactic Acid Bacteria from Many Sources of Animal and Plant for the Production of Bacteriocin. Proceeding of Life Science Research. IPB. September, 03 1998. Bogor.
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Valenzuela, G. M., H. V. S. Rojas and D. O. Martinez. 2007. First Report of Pantoea agglomerans causing Leaf Blight and Vascular Wilt in Maize and Sorghum in Mexico. Plant Disease. 91(10):1365