CHARACTERIZATION of BACTERIAL PATHOGEN CAUSING WILT and LEAF BLIGHT on CORN (Zea Mays) by PHYSIOLOGICAL, BIOCHEMICAL and MOLECULAR METHODS
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286 AGRIVITA VOLUME 34 No. 3 OCTOBER - 2012 ISSN : 0126-0537 CHARACTERIZATION OF BACTERIAL PATHOGEN 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 Pantoea 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 bacteria 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 pathogens 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.