Identification and Characterization of Virulence Factor of Several Indonesian Xanthomonas oryzae oryzaepv. FATIMAH , APON ZAENAL MUSTOPA , IQBAL KUSNANDARSYAH1* 2 1AND 1Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development (ICABIOGRAD) Jl. Tentara Pelajar No.3A Cimanggu, Bogor 16111, Indonesia; 2Research Center for Biotechnology, Indonesian Institute of Sciences (LIPI) Jalan Raya Bogor Km 46. Cibinong, Bogor 16911, Indonesia Xanthomonas oryzae oryzae Xoo Oryza sativa pv. ( ) is the bacterial causative agent of leaf blight in rice ( L.), the most serious bacterial disease of rice in many rice growing areas worldwide. This study aimed to identify and characterize several virulence factors of seven isolates from Yogyakarta, West Java, and West Sumatera. The Xoo identification of using 16S rRNA confirmed high homology to pv. PXO99 and Xoo Xanthomonas oryzae oryzae A revealed three groups. The first group was Xoo93229, the second group containing Xoo1110, Xoo1122, Xoo1130, Xoo7624 and Xoo8024 as the same cluster with PXO99 and the third group was KACC10331 and A MAFF311018. The amounts of exopolysaccharide (EPS) and cellulase produced were varying depending on the Xoo isolates. The EPS were produced more by isolate Xoo1130, Xoo1122 and Xoo8024. All tested isolates revealed similar cellulase activity except for isolate Xoo8024. The pathogenicity assay among the isolate Xoo showed that all tested isolates were virulent except Xoo7624. The assay revealed that the tested isolates in planta have multiplied and continued increasing the population size except for Xoo1110 and Xoo7624. High yield of EPS, cellulase activity, more virulence, and increasing population size revealed from isolate Xoo1130 and Xoo1122. Key words: pv. 16 rRNA, virulence factorXanthomonas oryzae oryzae, pv. ( ) merupakan bakteri penyebab penyakit hawar daun padi ( Xanthomonas oryzae oryzae Xoo Oryza sativa L.) dan merupakan penyakit yang serius di daerah pertanaman padi. Penelitian ini bertujuan untuk mengidentifikasi, mengkarakterisasi dan mengukur aktivitas fenotipe beberapa faktor virulen pada tujuh isolat bakteri yang diisolasi dari Yogyakarta, Jawa Barat, dan Sumatera Barat. Identifikasi bakteri Xoo Xoo menggunakan 16S rRNA diperoleh homologi yang tinggi dengan pv. PXO99 dan Xanthomonas oryzae oryzae A hasil pengelompokan didapatkan tiga grup. Pada grup pertama adalah bakteri Xoo93229, grup kedua adalah bakteri Xoo1110, Xoo1122, Xoo1130, Xoo7624 dan Xoo8024 bersama PXO99 dan pada grup ketiga adalah A KACC10331 dan MAFF311018. Besarnya eksopolisakarida (EPS) dan faktor virulen lainnya bervariasi bergantung pada isolat yang digunakan. EPS pada isolat Xoo1130, Xoo1122 dan Xoo8024 diproduksi lebih banyak. Aktivitas cellulase memberikan hasil yang sama pada semua isolat kecuali pada isolat Xoo8024. Uji patogenisitas dan menunjukkan bahwa enam isolat yang diujikan adalah virulen kecuali Xoo7624 in planta kurang virulen pada varietas IR24, peningkatan jumlah populasi bakteri pada semua isolat kecuali pada isolat Xoo1110 dan Xoo7624. Isolat Xoo1130 dan Xoo1122 memberikan nilai yang tinggi pada produksi EPS, aktivitas cellulase, virulensi, dan jumlah populasi bakteri. Kata kunci : pv. 16 rRNA, faktor virulensiXanthomonas oryzae oryzae, Vol.8, No.3, September 2014, p 103-111 DOI: 10.5454/mi.8.3.3 *Corresponding author; Phone: + , Fax: 62-251-8337975 + ; email: fatimahsuw@gmail.com62-251-8338820 B a c te r ia l le a f b li gh t (B LB ) c a us e d by Xanthomonas oryzae oryzae Xoopv. ( ) is the most devastating bacterial disease of rice production worldwide. in the fourth position of top 10 Xoo bacterial plant pathogen after , Pseudomonas syringae Ralstonia solanacearum Agrobac te rium a nd tumefacient et al Xoo (Mansfield . 2012). infects the rice leaf typically through hydathodes at the leaf tip, broken trichomes, leaf margins and wounds in the leaves or roots, multiplies in the intercellular spaces and enters into xylem vessels (Ou 1985; Nozue . et al 2011). After sufficient multiplication in the intercellular spaces of the underlying epitheme, the bacteria enter the xylem and spread in the vascular system (Mew . 1993; Liu . 2006).et al et al produces a range of virulence factors, Xoo including EPS, extracellular enzymes, and type III effectors (Liu . 2014). EPS can play a critical role et al in facilitating adhesion of bacteria to the host surface during initial stages of plantpathogen interactions and disease development (Subramoni 2006). Cell-et al. mailto:fatimahsuw@gmail.com wall degrading enzymes, such as cellulases, pectinases, xylanases and proteases, are secreted by plant pathogens cells to break down the components of host cell walls and may play a crucial role in virulence and bacterial nutrition (Temujin . 2011). Many et al xanthomonads require a type III secretion system (T3SS) for pathogenicity on plant hosts, and the requirement reflects the utilization of T3 effectors to mediate the processes of pathogen adaptation to specific host tissues, species and genotypes (Buttner and He 2009; White . 2009; Verdier . 2012).et al et al This study aimed to identify and characterize several virulence factors of seven from Indonesia Xoo and provided new insight towards Indonesian Xoo isolates as general feature of for the development of effective disease control methods. MATERIALS AND METHODS Identification of pv using 16S X. oryzae oryzae rRNA. Genomic DNA of Xoo was extracted from 5 ml NB cultures grown overnight. The bacterial cells were pelleted and lysed in 650 µL extraction buffer (100 mM Tris pH 8, 100 mM EDTA, 250 mM NaCl, 15% SDS (w/v), 1% PVP-40 (w/v) at 65 C for 30 minutes. DNA o was isolated using a modified method with 100 µL of 5 M potassium acetate and precipitated with isopropanol (George . 1997). et al For 16S rDNA sequencing, primers 8F (5'- AGAGTTTGATCATGGCTCAG-3' and 15R (5'- AAGGAGGTGATCCAACCGCA-3') were used to amplify the full length of bacterial 16s rDNA (Chao . 2008). CR mixture contained 10 mM et al Each 25 μl P Tris-HCl (pH 8.3), 50mM KCl, 1.5mM MgCl2, 200μM of each dNTP, 400nM of each primer, 1 U of Taq polymerase, and 10 ng of the DNA template. The PCR conditions were 96 °C for 5 min; 35 cycles consisting of 96 °C for 1 min, 55 °C for 3 min, and 72 °C for 1 min; and 72 °C for 7 min. The PCR products were subjected to gel electrophoresis in 1.5% agarose gel, followed by ethidium bromide staining. The PCR product was sequenced (Macrogen). Sequence assembly was performed with DNA Baser software. Similarity searches with sequences were performed by online BLAST analysis. For phylogenetic analysis, sequences were aligned by using the ClustalW software. The Measurement of Exopolysaccharide (EPS). measurement of exopolysaccharide (EPS) was conducted as described by Jeong . (2008). A single et al colony of each Xoo strain was inoculated in 40 ml of NB medium and incubated for 72 h at 28 C with o agitation. The optical density of the bacterial cultures was adjusted to 1.0 at 600 nm with NB. The culture supernatants were transferred into new 50-ml tubes and supplemented with 1.0% potassium chloride (w/v; final concentration). Two volumes of absolute ethanol were added to each solution, and the tubes were placed at -20 C overnight. The precipitated crude EPS was o collected by centrifugation for 30 min at 83,000×g. The EPS pellets were dried at 55 C for 12 h and the dry o weight of each was measured. Cellulase activities were assayed Cellulase Assay. as described (Jeong . 2008). Xoo strains were et al cultured in NB medium for 72 h, after which the optical density of the cultures was adjusted to 1.0 at 600 nm with NB. Thirty microliters of culture supernatant were placed in a hole in the assay agar medium, which contained 0.1% carboxymethyl cellulose, 50 mM sodium phosphate (pH 7.0), 0.8% agarose, and 0.02% sodium azide, and the plate was incubated for 20 h at 28 C. The incubated plates were stained with 0.1% o Congo Red for 10 min and then washed several times with 1 M NaCl. After washing, the cellulase activity was determined by measuring the diameter of the clear zone around the hole. Pathogenicity and Assays. In Planta The Xoo strains were grown on NB medium for 2 days at 28 °C. The bacterial cells were resuspended in sterilized water at an optical density of 600 nm (OD 600) (about 10 cfu -8 ml ). Bacterial blight inoculation was carried out on 6--1 week-old on resistance rice varieties IRBB7 and Code and susceptible rice varieties IR24 and Kencana Bali using the leaf-clipping method (Kaufmann 1973). Experiments were conducted under greenhouse conditions. For pathogenicity assay, the lesion length was measured in 7, 14 and 21 days after inoculation with 10 leaves for each strain. In planta assay was carried out on susceptible rice variety IR24. It determined Xoo strains multiplication in planta at six time points after infection by leaf clipping on 3, 6, 9,12, 15 and 18 days after inoculation. The in planta was assayed as described by Hu . (2007). The leaf et al pieces were then ground in 10mM MgCl2. The leaf homogenate was diluted in 10mM MgCl solution.2 Serial dilutions were made and spread onto PSA agar plates. The plates were incubated at 28 °C until the number of colony-forming units (cfu) per leaf was counted. 104 F ATIMAH ET AL . Microbiol Indones RESULTS Growth Curve of . Xoo Population growth is studied by analyzing the growth curve of a microbial culture. The bacterial growth can be plotted as the logarithm of the number of viable cells versus the incubation time. The resulting curve has four distinct phases. The cell density of isolates increased Xoo from 0.1 to 2.7 (OD ) during 48 h of growth at 600nm 28 C (Fig 1). The growth cell started at the early o exponential until 18 h and reached its maximum at the early stationary phase on 24 h then remained stable until 48 h during stationary phase. Xoo1122 Isolate showed increasing in its growth rate on exponential phase (18-24 h) however Xoo7624 growing slow compare to other isolates. Identification of using 16S rRNA and Xoo Phylogenetic Analysis. The partial 16S rRNA gene sequences (1490 bp) of all strains were determined. Then, the sequences were compared with related bacteria in GenBank and sequence similarities were determined using the BLAST program. The result confirmed that all isolates showed high homology (99-100%) to pv. PXO99 Xanthomonas oryzae oryzae A (Table 2). The Clustalw analysis of the 16S rRNA gene sequences revealed three groups. The first group containing one isolate, Xoo93229, the second group containing. Xoo1110, Xoo1122, Xoo1130, Xoo7624 and Xoo8024 as the same cluster with PXO99 , and the A third group KACC10331 and MAFF311018 were Xoo clustered together (Fig 2). Fig 1 Optical density at 600 nm of seven Indonesian Xoo Strains. Table 1 Bacterial strains used in this study Strain Collection site Cultivar source Source Xoo93229 Harjobinangun, Yogyakarta Cisadane ICABIOGRAD Xoo7624 Bogor,West Java Local Variety ICABIOGRAD Xoo8024 Cianjur, West Java Local variety ICABIOGRAD Xoo11-010 Cianjur West Java Ciherang ICABIOGRAD Xoo11-022 Maninjau, West Sumatera Kuriak Putiah ICABIOGRAD Xoo11-028 Maninjau, West Sumatera Kuriak Putiah ICABIOGRAD Xoo11-030 Maninjau, West Sumatera Kuriak Putiah ICABIOGRAD 3,5 3 2,5 2 1,5 1 0,5 0 12 18 24 30 36 42 48 Time Course (h) X00 1130 X00 1122 X00 7624 X00 8024 X0093229 X00 1110 X00 1128 Volume 8, 2014 Microbiol Indones 105 Measurement of Exopolysaccharide (EPS). The seven tested colonies produced EPS with different Xoo yield of production. Isolate Xoo1130, Xoo1122 and Xoo8024 giving more production of dry weight up to 80 mg however Xoo1110, Xoo7624, Xoo1128 and Xoo93229 showed less production around 40-50 mg (Fig 3). Cellulase Activities. Cellulase produced by the tested on medium agar plates showed the clear Xoo zones around the wells refer to cellulase activities was measured (Fig 4). All isolates revealed similar Xoo cellulase activity (17-18 mm) except for strain Xoo8024 (12 mm). Fig 2 ClustalW analysis of 16S rRNA sequence of Indonesian Isolates Xoo93229, Xoo7624, Xoo8024, Xoo1110, Xoo Xoo1122, and Xoo1130 compared with PXO99A (CP000967), MAFF311018 (AP008229), and KACC10331 (Ae013598). Table 2 Identity of Indonesian pv isolatesXanthomonas oryzae oryzae Isolate Species Identiity (%) Accession Number Xoo93229 Xanthomonas oryzae pv. oryzae PXO99A 100 CP000967.1 Xoo7624 Xanthomonas oryzae pv. oryzae PXO99A 99 CP000967.1 Xoo8024 Xanthomonas oryzae pv. oryzae PXO99A 100 CP000967.1 Xoo11-010 Xanthomonas oryzae pv. oryzae PXO99A 99 CP000967.1 Xoo11-022 Xanthomonas oryzae pv. oryzae PXO99A 100 CP000967.1 Xoo11-030 Xanthomonas oryzae pv. oryzae PXO99A 99 CP000967.1 Xoo93229 Xoo7624 Xoo8024 Xoo11010 Xoo1122 Xoo1130 Xo99A MAFF311018 KACCI10331 106 F ATIMAH ET AL . Microbiol Indones Pathogenicity Assay. All tested strains caused leaf blight on the surface of rice cultivar IR24 on 5 days after inoculation. The development of bacterial leaf blight symptoms in rice plants inoculated with Xoo strains observed on lesions leaf of resistant cv. IRBB7 and Code and susceptible cv. IR24 and Kencana Bali on 21 d post inoculation. Symptoms of BLB appeared on leaves as pale-green to grey-green water-soaked streaks near the leaf tip and margin. These lesions coalesced and became yellowish-white with wavy edges. On leaf sheath of susceptible cultivars, the affected leaves will turn yellow, roll up and wilt rapidly and systemic infection that produces tannish- grey to white lesions along the vein under greenhouse inoculation (Fig 5). Xoo Differences in virulence among the strains were quantified according to the lesion length of the necrotic area. Xoo93229, Xoo1122, Xoo1128, Xoo1130, Xoo1110 and Xoo8024 strains were shown to cause symptoms at day 7 after inoculation. It also showed the development of disease and increased virulence to susceptible rice cultivar IR24 at 21 d post inoculation. The average lesion length was 30, 31, 32, 32, 27, and 26 cm, respectively. Strain Xoo7624 showed the lowest lesion length at day 7 post inoculation and displayed the same pattern of lesion length until 21 d post inoculation (Fig 6). Fig 3 EPS dry weights of seven Indonesian Xoo Strain. Fig 4 Cellulase produced by the seven pv. on medium agar plates. The clear zones around the Xanthomonas oryzae oryzae wells refer to cellulase activities of strain (a) Xoo1130, (b) Xoo93229, (c) Xoo110, (d) Xoo1122, (e) Xoo8024, (f) Xoo Xoo7624, and (g)Xoo1128. a b c d e f g Volume 8, 2014 Microbiol Indones 107 100 90 80 70 60 50 40 30 20 10 0 Xoo Strain Xoo 93 229 Xoo 1110 Xoo 1128Xoo 1130 Xoo 1122 Xoo 8024 Xoo 7624 A 1 2 3 B 1 2 3 1 2 3 4 1 2 3 4 Fig 5 Leaf phenotype of IRBB7 and IR24 as an example showing increased susceptibility to pathogen strain (A) Xoo93229 and B) Xoo1122. Leaf 1) IRBB7, leaf 2) and 3) IR24. Leaves were photographed 5 days after inoculation. Arrow ( indicates position of clip-inoculation of the leaf tips. (C) Lesions leaves of 21 days after inoculated with Xoo93229 (left) and Xoo1122 (right). Resistant cultivar: Leaf 1) IRBB7 2). Code and Susceptible cultivar: 3) IR24, and 4) Kencana Bali. Fig 6 Pathogenicity assay of strains on the susceptible rice cultivar IR24. Length of lesions was measured in 7, 14, and 21 Xoo DAI with 10 leaves for each strain. Fig 7 Growth curve of isolates on rice leaves after inoculation. Mean CFU per leaf was calculated from two Xoo independent experiments with three leaves for each strain. 108 F ATIMAH ET AL . Microbiol Indones 45 40 35 30 25 20 15 10 5 0 Days After Inoculation Xoo1122 Xoo1130 Xoo1110 Xoo93229 Xoo7624 Xoo8024 Xoo1128 7 14 21 1,00E+12 1,00E+11 1,00E+10 1,00E+09 1,00E+08 1,00E+07 1,00E+06 1,00E+05 1,00E+04 Days After Inoculation 0 63 9 12 15 21 Xoo93229 Xoo1122 Xoo1110 Xoo1130 Xoo7624 Xoo8024 Xoo1128 In Planta Assay. Similar amounts of bacteria (2.8x10 cfu) were inoculated on rice by leaf clipping. 4 The multiplied at six time points Xoo strains in planta after infection. Initially, the distinct difference was observed in 3 days after inoculation (DAI) however Xoo1110 and Xoo7624 strain were less in population size (3x10 and 2x10 , respectively). Xoo93229, 6 6 Xoo1122, Xoo1128 and Xo1130 strain multiplied continued increasing until it reached a maximal population in 10 cfu and Xoo1110 and Xoo7624 strain 11 reached a maximal population in 10 cfu within 15 days 9 after inoculation (Fig 7). DISCUSSION The identification of 16 rRNA sequence, characterization of its growth curve and the important virulence factors such as cellulase and extracellular polysaccharide (EPS), pathogenicity assay and in planta assay have been examines in this study using seven Indonesian isolates. The amounts of EPS Xoo and cellulase produced were varying depending on the Xoo strain. The identification of 16 rRNA gene sequence was used to confirm the identity of strains. The 16S rRNA gene is most typically used for classification of bacteria. In this study, the 16S rRNA gene sequenced of all Xoo isolates used confirmed high homology to Xanthomonas oryzae pv. oryzae PXO99 (Salzberg A 2008). A dendrogram depicting the estimated et al. phylogenetic relationship was based on comparisons of the available 16s rRNA sequence data for the Xanthomonas oryzae pv oryzae. Indonesian Xoo isolates was clustered as the same cluster with PXO99 A (Salzberg . 2008) however the isolate Xoo93229 et al was in different cluster. Hauben . (1997) reported et al that a very small degree of divergence of 16S rRNA gene sequences among the Xanthomonas. Lang . et al (2010) reported the high degree of diversity of Xanthomonas isolates within and between countries. In this study, the third cluster consisted of Korean strain KACC10331 (Lee . 2005) and the Japanese strain et al MAFF311018 (Ochiai . 2005). It was similar with et al Salzberg . (2008) which used the phylogenetic et al relationships among PXO99 and the complete A genome of the genomes of MAFF311018, and KA CC103 31 ge ner ate d a c lad ogra m whe re MAFF311018 and KACC10331 was grouped together. The Xoo1130, Xoo1122 and Xoo8024 isolates produced high EPS compare to other isolates. Based on their colony morphology, these three isolates were mucoid and shiny when compared with the other isolates (data not shown). This phenotype results from the production of copious amounts of the extracellular polysaccharide (EPS), known as xanthan gum. Thein and Prathuangwong (2010) reported that colonies of mutants were smaller when compared with the wild type that resulted from less production of EPS. In this study, we used Jeong . (2008) method which the et al NB medium does not contain sucrose, results in a more consistent optical density for Xoo, and was used for EPS assays. He . (2006) mentioned that high et al concentrations of sucrose in the medium have been reported to result in high background in assays of some extracellular enzyme activities. However it should be noted that Lan . (2007) observed in long-term et al storage and culture will lead to spontaneous loss of virulence and associated with reduction of EPS production. Partially purified EPS preparations have been found to induce rice leaf wilting. This may be due to cell membrane leakage caused by EPS (Yang and White 2004). The diameter of the zone of clearance indicates the ability of the bacteria to hydrolyze cellulase. The capacity of the bacterial isolates to degrade cellulase was indirectly determined using the degradation of c a r bo xy me th y l- c e llu lo se (C MC) , ind ic a t ing endoglucolytic activity (Soares . 2012). In this et al study, all of the Xoo tested isolates giving similar cellulase activity except for isolate Xoo8024. However the variation of cellulase activity using plate assay method was not related with the virulence ability. Hu . (2007) reported that cellulase plate assay et al method failed to discriminate the difference of enzyme activity between mutants and wild type also mentioned by Jeong . (2008) that the cellulase activities of the et al rpf mutants and the complementation strains were similar to those of the wild-type. Efficient methods for recovering bacterial cells directly from plant tissues permit analyses of in vivo expression in plant-pathogen interactions (Mehta 2003) and may help in the early detection of genes involved in pathogenicity (Thwaites 2004). Hu et al et al. (2007) showed that the expression of the gene can only be detected when Xoo grows in planta but cannot be detected when it grows on PSA agar. In this study, the difference of population size and the increasing of multiplication related with the ability to colonize rice Volume 8, 2014 Microbiol Indones 109 seedlings have associated with the virulence ability. The pathogenicity assay among the Xoo isolate showed severely increased virulence to susceptible rice cultivar IR24 except for Xoo7624. However it assumed that our result comes from wild type compare to mutant type generated by study of Feng . (2009) reported et al that the xrvA mutant GXN1280 and the xrvA overexpression strain GXO3098 showed a significant reduction in lesion length compared to the wild-type strain but the bacterial populations of these mutants in rice leaves were not significantly different from that of the wild-type. In this study, isolate Xoo1130 and Xoo1122 revealed high yield of EPS, cellulase activity, virulence, and increasing population size. In contrary with Xoo7624 revealed low yield of EPS, less virulence, and low number of population size but high cellulase activity. Xoo93229 revealed high cellulase activity, more virulence, and increasing population size but low yield of EPS production. Xoo8024 with high yield production of EPS, more virulence and increasing population size was low in cellulase activity. Initially it was presumed that all the tested isolates will have association between the virulence factor and pathogenicity. Regarding phenotypic characterization, recent molecular characterization of Xoo, the availability of genome sequence for rice and Xoo may facilitate our research for identification of many new pathogen-associated molecular patterns (PAMPs) and avirulence and virulence effectors (Liu . 2014).et al ACKNOWLEDGMENT This work was financially supported by Indonesian Toray Science Foundation Science and Technology Research Grant (ITSF-STRG). 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Transcription activator-like (TAL) effectors targeting OsSWEET genes enhance virulence on diverse rice ( ) Oryza sativa varieties when expressed individually in a TAL effector-deficient strain of . New Xanthomonas oryzae Phytologist. doi: 10.1111/j.1469-8137.2012. 04367. White FF Potnis N. Jones JB. and Kloebnik R. 2009. The type III effectors of Xanthomonas. Mol Plant Pathol. 10: 749-766. doi: 10.1111/J.1364-3703.2009.00590. Yang B, White F. 2004. Diverse members of the AvrBs3/PthA family of type III effectors are major virulence determinants in bacterial blight disease of rice Mol Plant Microbe Interact, 17:1192-1200. . doi.org/10.1094/MPMI.2004. 17.11.1192. Volume 8, 2014 Microbiol Indones 111