286

AGRIVITA VOLUME 34 No. 3 OCTOBER - 2012 ISSN : 0126-0537

CHARACTERIZATION OF BACTERIAL CAUSING WILT AND LEAF BLIGHT ON CORN (Zea mays) BY PHYSIOLOGICAL, BIOCHEMICAL AND MOLECULAR METHODS

Lilis Suryani 1*), Luqman Qurata Aini 2,3), Arifin Noor Sugiharto 2,3) and Abdul Latief Abadi 2)

1) Agricultural Quarantine Agency Class I Banjarmasin Jl. Sutoyo S. No. 1134, Banjarmasin South Kalimantan Indonesia 2) Faculty of Agriculture, University of Brawijaya, Jl. Veteran Malang 65145 East Java Indonesia 3) Maize Research Center, Faculty of Agriculture, University of Brawijaya Indonesia Jl. Veteran Malang 65145 East Java Indonesia *) Corresponding author Phone: +62-511-3353980 E-mail:[email protected]

Received: May 29, 2012/ Accepted: September 12, 2012

ABSTRACT materials for food industry (Akil and Dahlan, 2010). More than 55% of domestic corn production is In 2011, we found a new bacterial disease cha- estimated to be used for livestock feed, 30% for racterized by wilt, dwarf and blight symptoms on food consumption, and the rest is for seed and sweet corn in Batu, East Java, Indonesia. The other industrial needs (Kasryno et al., 2010). objective of this study is to characterize the Production and harvest area of corn in Indonesia causal agent of the disease. In this study, several are arising during the last five years. In 2011, the assays were conducted, including hypersensitive corn production reached 17,629,033 tons yielded response, pathogenicity, physiological and from 3.861.433 ha. East Java province is known to biochemical characteristics, PCR detection using be the largest corn producer in Indonesia. In 2011, two specific species primer pairs for the corn production in East Java province was stewartii pv. stewartii, and homology analysis of 5,443,705 tons yielded from 1,204,063 ha of 16S rRNA gene sequence. Four Gram-negative, production area (BPS, 2011). non-motile, facultative anaerobic were Several plant diseases caused by fungal isolated from the diseased tissue. Only two and bacterial pathogen have been reported strains, BD1 and BB2, gave positive result in causing losses of corn production in Indonesia. hypersensitive reaction, pathogenicity, and Recently, a new bacterial disease on corn found in Koch’s postulate assays. BB2 and BD1 strains several corn production area is wilt disease also showed positive results in the PCR accompanied by leaf blight symptom. In 2009, this amplification using specific primers derived from disease was reported in West Sumatra with the the P. stewartii subsp. stewartii 16-23S gene disease incidence ranging from 1%-15%. Based region but showed negative result when using on the symptom and several physiological primers derived from P. stewartii subsp. stewartii characteristic, the disease was predicted to be hrpS gene region. Sequence of partial 16S rRNA Stewart’s wilt caused by Pantoea stewartii (Rahma gene of BD1 and BB2 showed highest homology and Armansyah, 2009). Recently, we found the at 96% to P. stewartii subsp. stewartii strain similar bacterial wilt and leaf blight disease on ATCC 8199 (NR. 044800.1). This results suggest sweet corn planted in Batu, East Java with the that bacterial isolated from sweet corn disease incidence ranging from 1% to 10%. in Batu were strains of Pantoea spp. The most widely known of wilt and leaf blight disease of corn is Stewart’s disease (Pataky, Keywords: Zea mays, Pantoea spp, wilt, leaf blight, 2003). The causal agent of the disease is patho- PCR genic bacteria known as Bacillus stewartii, Bacterium stewartii, Erwinia stewartii, Pseudo- INTRODUCTION monas stewartii, Pseudobacterium stewartii, In Indonesia, corn has been an important Phytobacterium stewartii or Xanthomonas stewartii crops for both the major feed for livestock and raw (Brenner et al., 1984). In 1993, the bacteria were

Accredited SK No.: 81/DIKTI/Kep/2011 http://dx.doi.org/10.17503/Agrivita-2012-34-3-p286-295

287

Lilis Suryani et al.: Characterization of Bacterial Pathogen Causing Wilt and Leaf……………………………………. transfered to the genus of Pantoea to be Pantoea stewartii subsp. stewartii (Mergaert et al., 1993). Isolation and Identification of Bacterial The corn flea beetle Chaetocnema pulicaria Pathogen from Diseased Corn Plant Melsheimer (order: Coleoptera, family: Chrysome- Bacterial pathogens were isolated from lidae) is known to be the primary vector for P. plant leaves and stalks showing wilt or blight stewartii subsp. stewartii (Menelas et al., 2006). symptoms collected on January 2011 from corn The potential risk of seed transmission is fields in Batu. Small sections of stalk or leaf tissues considered very important in international shipment with indicated symptoms were cut aseptically from of corn seeds hence more than 50 countries ban the margins of lesions and macerated in 1 ml of the importation of corn seeds unless it has been sterile distilled water for 30 minutes. Plant extracts certified free of P. stewartii subsp. stewartii (Coplin were streaked onto Nutrient Agar (NA), incubated et al., 2002; Michener et al., 2002). at 28ºC, and examined after 2 to 5 days for Two phases of Stewart's wilt disease can colonies appearance. Initial identification was occur on corn plants. The seedling wilt phase performed i.e. the Gram staining, Hugh-Leifson occurs when the young seedling plants are assay, pigment production on YDC, and infected systemically, whereas the leaf blight hypersensitive reaction (HR) on tobacco. phase occurs when the the infection occurs after seedling stage. The blight symptom appeared Pathogenicity and Koch’s Postulate Assays pale-green to yellow linear streaks with irregular or Pathogenicity assay on corn plants was wavy margins develop parallel to leaf veins. These performed by inoculation on sweet corn plants lessions become necrotic by age and on cultivar Jambore. Each bacterial strain was grown susceptible cultivars the lessions may extend to on NA plates for 48 h at 28ºC, and then each the entire length of the leaf. The leaves on the bacterial strain was suspended in sterile distilled infected young plants usually show striped white or water to obtain approximately 108 CFU/ml. Ten yellow (Lipps et al, 2003; Pataky, 2003). Systemic days old plants were inoculated using two infection on corn seedling can cause high yield methods: (i) suspensions of pathogenic bacteria losses (Freeman and Pataky, 2001). were infiltrated into leaf tissues with a needle-less The study of the disease is still limited in syringe and (ii) the suspensions were injected into Indonesia. Since its ability to infect corn plant in plant stems. Inoculated plants were put in a seedling stage, the disease is potential to be very chamber and maintained in room temperature with destructive such as downy mildew caused by 90% relative humidity. After 2 days, the plants Peronosclerospora maydis which has been were transferred to greenhouse. Bacteria were re- causing great loss of corn production in Indonesia. isolated from symptomatic leaves or stems tissues East Java province is the largest corn producer as and confirmed by observation of colony well as the center of corn seeds production in morphology on YDC, Gram staining, and Hugh- Indonesia. Therefore, the presence of this disease Leifson assay. can be a serious problem not only for corn Physiological and Biochemical Assays production but also for healthy seed production. Isolates were assayed as described by This study aimed to characterize the Schaad et al. (2000) i.e. growth at 37°C; nitrate bacterial pathogen causing wilt and leaf blight on reduction; gelatin liquification; indole production; corn found in Batu, East Java. motility; utilization of citrate, malonate and tartrate;

acid production from glycerol, lactose, maltose, MATERIALS AND METHODS mannitol, sorbitol, meso inositol, cellobiose and

sucrose. The study was conducted at Plant Patho- logy laboratory of Plant Protection Department, DNA Isolation Faculty of Agriculture, Brawijaya University, Genomic DNA was isolated from bacterial Malang, Indonesia and at laboratory of Mole-cular cells using alkali lysis method (Ausubel et al., Biology of Biology Department, Faculty of Sience, 1996) with minor modification. The 24 hours Islamic State University, Malang, Indonesia, from cultured bacterial cells were harvested by September 2011 to March 2012. centrifugation at 10.000 g for 5 minutes and re-

288

Lilis Suryani et al.: Characterization of Bacterial Pathogen Causing Wilt and Leaf……………………………………. suspended in 525 µl TE buffer. Bacterial cells were AAGGAGGTGATCCAGCCGCA-3′) (Weisburg et then disrupted by adding 60 µl of 20% SDS and al., 1991). DNA was amplified in 25 μl reaction Proteinase K (200 µg/ml), and then incubated at volumes containing 4 μl template DNA, 12,5 μl Go 37°C for 60 minutes. The suspension was mixed Taq® Green Master Mix 2X from Promega, 2,5 μL with 100 µl 5N NaCl and 80 µl CTAB and of 100 pmol of each primer, and 3,5 μL DDW. The incubated at 65°C for 30 minutes. 750 µl conditions of PCR amplification were 1 cycle at Chloroform Isoamyl Alcohol (CIAA) was added in 95°C for 3 min; 40 cycles at 95°C for 1 min, at the suspension, mixed thoroughly, and separated 55°C for 1 min, and at 72°C for 2 min; post by centrifugation at 11.000 g for 10 minutes. extension at 72°C for 3 min. For the confirmation, Supernatant was transferred into new tubes and the PCR product was separated on 1.5% agarose the equal volume of Phenol Chloroform Isoamyl- gel at 1.5 V/cm. The DNA fragment was stained in Alcohol (PCI) was added, mixed thoroughly and ethidium bromide solution (0.5 μg/mL in TAE separated by centrifugation at 11.000 g for 10 buffer) for 15 min and subjected to Gel-Doc minutes. The DNA from the supernatant was observation. The size of the expected amplicon precipitated using equal volume of isopropanol and was 1.5 kb. centrifugated at 12.000 g for 2 minutes. DNA The amplified PCR products were purified precipitate was washed with 70% ethanol, re- using ethanol/EDTA precipitation method. The centrifuged at 12.000 g for 2 minutes, dried on sequencing was performed using BigDye® room temperature and re-suspended with 30µl TE Terminator v3.1 Cycle Sequencing Kit (Applied buffer containing RNAase. Biosystems, USA). Sequences were analyzed using Sequencing Analysis Software v5.31 PCR Assay (Applied Biosystems, USA). The GenBank/EMBL For the determination of pathogenic Pantoea, databases were used for homology search using two specific primer pairs designed for the the BLAST program that available in the website: detection of P. stewartii subsp. stewartii (Coplin http://blast.ncbi.nlm.nih.gov/Blast.cgi (National et al., 2002) were used. Primers ES16 (5’-GCG Center for Biotechnology Information, USA). AACTTGGCAGAG AT-3’) and ESIG2c (5’- Phylogenetic and molecular evolutionary analyses GCGCTTGCGTGTTATGAG-3’) were derived were conducted using MEGA version 5 (Tamura et from the sequence of 16S-23S rRNA/ITS region of al., 2001). P. stewartii subsp. stewartii whereas HRP1d (5’- GCACTCATTCCGACCAC-3’) and HRP3r (5’- RESULTS AND DISCUSSION CGGCATACCTAACTCC-3’) were derived from hrpS gene region of P. stewartii subsp. stewartii. In this study we characterized the patho- The condition of PCR amplification were: 1 cycle genic bacterium causing wilt accompanied with at 95°C for 1 min, 55 cycles of: 30 s at 95°C, 30 s blight symptoms on corn found in Batu area, East at 55°C, 60 s at 72°C, and 1 cycle of post Java. The disease intensity varied from 1-10% of extension at 72°C for 3 min. The PCR product was the plant population. The leaf blight symptom was separated on 1.5% agarose gel at 1.5 V/cm, typically light brown with irregular edges (Figure 1). stained in ethidium bromide solution (0.5 μg/mL in Dwarf symptoms were found less than 5%, and TAE buffer) for 15 min, and subjected to Gel-Doc the wilt symptoms were found on mature plants at observation. The size of the expected amplicons approximately 50 days old. Blight symptoms were were 0.92 kb (ES16/ESIG2c) and 0.9 kb also found in younger plants. The wilted corn (HRP1d/HRP3r) . plants released bacterial mass from the cutting of rotted stalk. The characteristic of the symptoms Analysis of Partial 16S rRNA Gene Sequence mentioned above appeared to resemble the The partial 16S rRNA gene fragment was symptoms of Stewart's wilt disease of corn amplified using the universal primers fD1(5′- (Pataky, 2003). AGAGTTGATCCTGGCTCAG-3′) and rD1(5′-

289

Lilis Suryani et al.: Characterization of Bacterial Pathogen Causing Wilt and Leaf…………………………………….

Figure 1. Disease symptoms on corn observed in the field. A, leaf blight; B, stalk rot; C, dwarf

Bacterial Pathogen Isolation and Hypersensitive Physiological and Biochemical Characteristics Assay The results of physiological and bio- Four bacterial strains i.e. BB1, BD1, BB2, chemical tests showed that strain BD1 and BB2 BB3 were isolated from leaves and stalks of corn isolated from leaves and stem respectively had showing disease symptoms. The bacteria were nearly similar characteristics, with the exception able to grow on NA medium in 24 hours at room on the use of carbon from meso inositol assay. temperatures. Colonies were circular, 1-2 mm, Characteristics of BD1 and BB2 strains somewhat convex with flat edges. All isolates resembled more to P. ananatis, but they had were Gram-negative rod, facultative an-aerobic unique main characters similar to P. stewartii and showed yellow colonies in YDC medium. All subsp. stewartii i.e. non-motile and were unable bacterial strains produced HR in tobacco leaves to produce indole. These results showed both at 48 hours after infiltration. BD1 and BB2 strains have physiological and biochemical characteristics quite differently from Pathogenicity Test P. stewartii described by Schaad et al. (2000). When 10 days old seedlings were infiltrated with Both BD1 and BB2 strains showed non- suspension of all bacterial strains, water soaked motile suggesting that these strains are resem- emerged in 2 days after inoculation. The bled to P. stewartii subsp. stewartii. However, symptoms changed into blight with irregular other biochemical characteristics showed variable edges two days later. When the bacterial compared with those of P. stewartii subsp. suspensions were injected into the stems of 10 stewartii. P. stewartii subsp. stewartii are histori- days old corn plants, only BD1 and BB2 strains cally described as aflagellated and non-motile produced wilt symptoms within a week (Figure 2). bacteria (Pepper, 1967), the most distinguishing These strains induced consistent symptom in character of P. stewartii subsp. stewartii from corn plants similar to those observed in the field. other Pantoea. Transmission of P. stewartii Control plants inoculated with water did not subsp. stewartii in the field requires the feeding develop any symptoms. Bacterial pathogens were action of the pathogen bearing corn flea beetle, successfully reisolated from the symptom of Chaetocnema pulicaria, which serves as plants inoculated with strain BD1 and BB2. overwintering host for P. stewartii subsp. stewartii Colonies recovered diseased plants were also (Pataky 2003). The existence of the corn flea yellow, Gram negative, and facultative anaerobic. beetle in Indonesia is still unknown.

290

Lilis Suryani et al.: Characterization of Bacterial Pathogen Causing Wilt and Leaf…………………………………….

Figure 2. Symptoms on the pathogenicity test, A. wilt symptom (right) compared to control (left), B. watersoak appeared on stalk, C. water soak appeared on leaf, D. leaf blight

Detection of Bacterial Strains using Two The ability of plant-pathogenic bacteria to Species-Specific Primer Pairs For P. stewartii elicit the hypersensitive response (HR) in subsp. stewartii resistant or non-host plants is correlated with Primers ES16/ESIG2c were designed for their ability to cause disease in susceptible the detection of P. stewartii subsp. stewartii to hosts. These capabilities are determined by amplify the unique sequence of 16S to 23S hypersensitive response and pathogenicity (hrp) rRNA intergenic transcribed spacer (ITS) region genes, which encode components of a protein (Coplin et al.,2002). It is known that DNA secretion pathway, regulatory factors, and a sequences in the 16S to 23S intergenic spacer number of secreted effector proteins. P. stewartii region exhibit a great deal of sequence and subsp. stewartii posses hrpS, one of hrp genes length variation. The variations in this region which involved in the HR elicitation in non host have been shown to be useful for differentiating plants as well as pathogenicity in host plants species of prokaryotes (Barry et al., 1991). (Lindgren, 1997) Electrophoresis product using primer ES16/ESIG2c (Figure 3) indicated that 0,92 kb Partial 16S rRNA Gene Sequence Analysis amplicons were detected on the BD1 and BB2 The 16S rRNA gene sequence analysis is strains, but no amplicons were detected on the a powerful and accurate method for determining BB1 and BB3 strains. Positive control of phylogenetic relationships (Barry et al., 1991). genomic DNA of P. stewartii subsp. stewartii BLAST search using partial 16S rRNA gene DM5262 strain also showed the amplicon, sequences showed that BD1 and BB2 strains suggesting the positive result of both BD1 and had the highest homology at 96% to P. stewartii BB2 strains. subsp. stewartii strain ATCC 8199 (accession The PCR assay using HRP1d and HRP3r number NR_044800.1). These results suggest primers derived from P. stewartii subsp. stewartii that both BD1 and BB2 strains are closed to hrpS gene region showed that no amplicon was species of P. stewartii subsp. stewartii. shown in all of 4 bacterial strains. Positive control However, there is a consensus that two bacteria of genomic DNA of P. stewartii subsp. stewartii can be determined as same species if they DM5262 strain showed the amplicon, suggesting shared as high as or more than 97% homology the negative result of the existence of P. of 16S rRNA gene sequence. Thus, the BD1 stewartii subsp. stewartii hrpS gene in all 4 and BB2 strains could not be considered as P. bacterial strains. stewartii subsp. stewartii

291

Lilis Suryani et al.: Characterization of Bacterial Pathogen Causing Wilt and Leaf…………………………………….

Table 1. Physiological and biochemical characteristics of the bacterial strains isolated from corn, and published strains of Pantoea stewartii subsp. stewartii, Pantoea ananatis and Pantoea agglomerans (Schaad et al., 2000)

Characteristic BD1 BB2 P.stewartii P.ananatis P. agglomerans subsp. stewartii (herbicola strains) Growth at 37°C + + - + + Yellow pigmen on YDC + + + + + Indole production - - - + V Nitrate reduction + + - V + Gelatin liquification + + - + + Motility - - - + + Utilization of: * Citrate + + - + + * Malonate - - - - + * Tartrate + + - + - Acid production from: * Gliserol + + - + - * Lactose + + - + V * Mannitol + + + + + * Sorbitol - - V + - * Cellobiose + + - + V * Maltose + + - + + * Meso inositol + - - + - * Sucrose + + + + + V, between 21-79% of strains positive

Figure 3. Gel electrophoresis visualization of PCR amplified bacterial DNA using primers ES16/ESIG2c (left) and hrpS (right); 1. 1kb DNA ladder; 2. DM5262; 3. BB1; 4. BD1; 5. BB3; 6. BB2

292

Lilis Suryani et al.: Characterization of Bacterial Pathogen Causing Wilt and Leaf…………………………………….

293

Lilis Suryani et al.: Characterization of Bacterial Pathogen Causing Wilt and Leaf…………………………………….

Figure 4. Sequence alignment of the 16S rRNA genes of P. stewartii subsp. stewartii (Pstw), Pantoea ananatis (Pan), Pantoea agglomerans (Pag), strain BD1, and strain BB2. Identical nucleotides are indicated by dotes, and deletions are indicated by dashes.

Multiple alignments of nucleotide sequences Overall, the results showed that BD1 and were performed using Clustal W program. The BB2 strain were non-motile similar to P. stewartii sequences were align with the nucleotide sequen- subsp. stewartii. However, physiological and ce of P. stewartii subsp. stewartii strain ATCC biochemical traits of BD1 and BB2 strains 8199 as well as those of P. ananatis and P. showed variable to those of P. stewartii subsp. agglomerans which has close relationship to P. stewartii. In addition, PCR amplification assay stewartii subsp. stewartii. The BD1 and BB2 with two types of specific species primer pairs showed high similarity to P. stewartii subsp. for the detection of P. stewartii subsp. stewartii stewartii, although several bases have been showed inconsistency. Homology of 16S rRNA changed (Figure 4). gene sequences of BD1 and BB2 strains to P. The phylogenetic relationship derived stewartii subsp. stewartii were below 97%, a from a neighbor-joining analysis of the pairwise consensus value for the two bacteria considered comparison among the partial 16S rRNA gene same species. The ELISA test using AGDIA Kit sequences of both BD1 and BB2 strains with six for P. stewartii subsp. stewartii also showed sequences of well described species of the negative result for both bacterial strains (data genus Pantoea is shown in Figure 5. Escherichia not shown). Taken together the results were not coli, Erwinia chrysantemi, and Ralstonia solana- consistent to verify that BD1 and BB2 bacterial cearum were used as the out group taxons. The strains found in Batu is P. stewartii subsp. sequences of P. ananatis, P. agglomerans, P. stewartii. Thus, we conclude that the bacterial stewartii, E. coli, E. chrysantemi, R. solanacea- strains are the unique strain of Pantoea spp. rum were obtained from the GenBank/EMBL The surveillance and further researches on the databases. The tree diagram demonstrated that disease are necessary to develop strategies to the sequences of BD1 and BB2 are clustered in manage the disease. a group with P. stewartii subsp. stewartii supported by high bootstrap value of 100.

294

Lilis Suryani et al.: Characterization of Bacterial Pathogen Causing Wilt and Leaf…………………………………….

Figure 5. Phylogenetic tree showing the relationship among selected partial 16S rRNA gene sequences from Pantoea spp. and strains isolated from corn. The numbers at the nodes indicate the levels of bootstrap support based on data for 1,000 replicates. Accession numbers were showed in brackets. Bar represents genetic distance. Pan = P. ananatis, Pag = P. agglomerans, Pstw = P. stewartii, E. coli = Escherichia coli, Echryst = E. chrysantemi, Rs = R. solanacearum

CONCLUSION REFERENCES

Based on all tests performed, bacterial Akil, M. and H.A. Dahlan. 2010. Budidaya jagung pathogen causing wilt and leaf blight on corn dan diseminasi teknologi. Dalam Jagung: found in Batu was Pantoea spp. The bacteria teknik produksi dan pengem-bangan. had physiological and biochemical characteris- Litbang Deptan. Jakarta. p.192-204. tics quite different from other published strains of Ausubel F.M., R. Brent, R.E. Kingston, D.D. P. stewartii subsp. stewartii, indicated that BD1 Moore, J.G. Seidman, J.A. Smith and K. and BB2 are possibly unique strains of Pantoea Struhl. 1996. Current protocols in mole- spp. Further research on the disease is cular biology. John Wiley and Sons, New necessary to develop strategies to manage the York. disease as well as to prevent greater losses. Barry, T., G. Colleran, M. Glennon, L. Dunican and F. Gannon. 1991. The 16S/23S ribo- ACKNOWLEDGEMENTS somal spacer as a target for DNA probes to identify eubacteria. PCR Methods Appl. We would like to thank Drs. David L. p.151-156. Coplin and Doris R. Majerczak from Ohio BPS. 2011. Tanaman pangan. http://www.bps. University for their kindness to provide DNA go.id/tnmn_pgn.php sample of P. stewartii subsp. stewartii DM5262 Brenner, D.J., G.R. Fanning, J.K. Leete Knutson, strain as positive control. This research was A. G. Steigerwalt and M. I. Krichevsky. 1984. funded by Dit.Litabmas Dikti, through Grand Attempts to classify herbicola group- Research of Decentralized University received agglomerans strains by by Dr. Arifin Noor Sugiharto. deoxyribonucleic acid hybridization and phenotypic tests. Int. J. Syst. Bacteriol. 34: 45-55.

295

Lilis Suryani et al.: Characterization of Bacterial Pathogen Causing Wilt and Leaf…………………………………….

Coplin, D.L., D.R. Majerczak, Y. Zhang, W.S. Michener, P. M., J. K. Pataky and D.G. White. Kim, S. Jock and K. Geider. 2002. Identi- 2002. Rates of transmitting Erwinia fication of Pantoea stewartii subsp. stewartii from seed to seedlings of a stewartii by PCR and strain differentiation sweet corn hybrid susceptible to Stewart’s by PFGE. Plant Dis. 86: 304-311. wilt. Plant Dis. 86: 1031-1035. Freeman, N.D. and J.K. Pataky, J.K. 2001. Pataky, J.K. 2003. Stewart’s wilt of corn. Levels of Stewart’s wilt resistance ne- APSnet, American Phytopathological So- cessary to prevent reductions in yield of ciety, St. Paul. MN, U.S.A. Published on- sweet corn hybrids. Plant Dis. 85: 1278- line. 1284. Pepper, E. H. 1967. Stewart’s bacterial wilt of Kasryno, F., E. Pasandaran, Suyamto, M.O. corn. Monograph 4. American Phyto- Adnyana. 2010. Gambaran umum eko- pathological Society, St. Paul, MN, U.S.A nomi jagung Indonesia. Dalam Jagung: Rahma, H. and Armansyah. 2009. Penyebaran teknik produksi dan pengembangan. penyakit Stewart oleh bakteri Pantoea Litbang Deptan. Jakarta. p. 474-497. stewartii sebagai penyakit baru pada Lindgren P.B. 1997. The role of hrp genes tanaman jagung (Zea mays) studi kasus during plant bacterial interactions. Annual di Pasaman Barat. Working Paper. Fakul- Review of Phytopath. 35: 129-152. tas Pertanian. Univ. Andalas. http://lp. Lipps, P.E., A.E. Dorrance and D.R. Mills. 2003. unand.ac.id/?pModule=penelitian&pSub= Stewart's bacterial wilt and leaf blight of penelitian&pAct=detail&id=9&bi=2 corn. Ohio State University Fact Sheet. Schaad, N.W., J.B. Jones and W. Chun . 2000. http://ohioline.osu.edu. Laboratory guide for identification of plant Menelas, B., C.C. Block, P.D. Esker and F.W. pathogenic bacteria. Third Edition. Gail Jr. Nutter. 2006. Quantifying the feeding Hoover. Maryland. pp.372. periods required by corn flea beetles to Tamura, K., D. Peterson, N. Peterson, G. acquire and transmit Pantoea stewartii. Stecher, M. Nei and S. Kumar. 2011. Plant Dis. 90: 319-324. MEGA5: Molecular evolutionary genetics Mergaert, J., L. Verdonck and K. Kersters. 1993. analysis using maximum likelihood, Transfer of Erwinia ananas (synonym, evolutionary distance, and maximum Erwinia uredovora) and Erwinia stewartii parsimony methods. molecular biology to the Genus Pantoea emend. as Pantoea and evolution (submitted). ananas (Serrano 1928) comb. nov. and Weisburg, W.G., S.M. Barns, D.A. Pelletier and Pantoea stewartii (Smith 1898) comb, D.J. Lane. 1991. 16S Ribosomal DNA nov., Respectively, and description of amplification for phylogenetic study. J. Pantoea stewartii subsp. indologenes Bacteriol. 173(2): 697-703. subsp. nov. Int. J. Sistematic Bacteriol. 43(1): 162-172.