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Identification and Genetic Diversity of Pectolytic Phytopathogenic Bacteria of Mono- and Dicotyledonous Ornamental Plants in Iran

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Identification and Genetic Diversity of Pectolytic Phytopathogenic Bacteria of Mono- and Dicotyledonous Ornamental Plants in Iran Journal of Plant Pathology (2014), 96 (2), 271-279 Edizioni ETS Pisa, 2014 Dahaghin and Shams-Bakhsh 271 IDENTIFICATION AND GENETIC DIVERSITY OF PECTOLYTIC PHYTOPATHOGENIC BACTERIA OF MONO- AND DICOTYLEDONOUS ORNAMENTAL PLANTS IN IRAN L. Dahaghin and M. Shams-Bakhsh Plant Pathology Department, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran SUMMARY Clostridium, Dickeya and Pectobacterium (Pérombelon and Kelman, 1980; Liao and Wells, 1987; Krejzar et al., Bacterial soft rot can be a destructive disease of orna- 2008). Pectobacterium carotovorum subsp. carotovorum mental plants. To identify the pathogenic soft rot bacteria (Pcc), one of the most important members of the soft rot occurring in ornamental plants in Tehran and Markazi bacteria group, has a wide geographical distribution and provinces (Iran), 57 isolates were obtained from 12 dif- causes disease on numerous important crop and ornamen- ferent mono- and dicotyledonous hosts and investigated tal plants (Pérombelon and Kelman, 1980; Wright, 1998; with regard to phenotypic, genotypic and pathogenicity Avrova et al., 2002; Pérombelon, 2002; Ma et al., 2007). features. Based on phenotypic characteristics, the bacte- This sub-species has been reported as a pathogen of an rial strains were identified as Pectobacterium carotovorum number of ornamental hosts (Table 1). In addition to Pcc, subsp. carotovorum. This was confirmed by subspecies- some other soft rotting bacteria with pectolitic activity, specific primers using PCR. Inoculation of all isolates into such as Dickeya chrysanthemi, Dickeya dieffenbachiae, Dick- Aglaonema leaves confirmed the pathogenicity of the iso- eya dadantii, Dickeya dianthicola (Parkinson et al., 2009), lated strains. To assess the genetic diversity within Pecto- Pectobacterium atrosepticum, Pseudomonas marginalis, and bacterium carotovorum subsp. carotovorum populations by Pseudomonas veronii (Wright and Burge, 2000; Hahm et rep-PCR, 26 isolates were selected according to their host al., 2003; Krejzar et al., 2008; Mikiciński et al., 2010) have and geographic distribution as well as two strains from been reported on different ornamental plants. Guilan province. Cluster analysis was conducted using the In Iran, soft rot bacteria have previously been reported UPGMA method and revealed a possible close relation- from ornamental plants including Fritillaria imperialis in ship between DNA fingerprints and geographical origins Kermanshah and Isfahan (Mahmoudi et al., 2007) and Agl- of isolates. Our results showed significant genetic variation aonema sp., Dieffenbachia sp., Epipremnum aureum, Ficus among the populations of this pathogen. To the best of elastic, Hippeastrum sp., Iris sp., Lampranthus sp., Maranta our knowledge, this is the first report of Pectobacterium leuconeura, Peperomia sp., Philodendron spp., Sansevieria carotovorum subsp. carotovorum on Peperomia obtusifolia, trifasciata, Schlumbergera bridgesii, Spathiphyllum sp. and P. caperata, Pilea cadierei, Plectranthus australis, Saintpaulia Syngonium podophyllum from Mazandaran, Guilan, Goles- ionantha and Kalanchoe tubiflora in Iran. tan and Khorasan Razavi provinces (Baghaee-Ravari et al., 2010, 2011). However, there are no published data related Key words: Soft-rot bacteria, Pectobacterium caroto- to the distribution of bacterial soft rot on these plants in vorum subsp. carotovorum, ornamental plant, diagnosis, Tehran and Markazi provinces, which are the main orna- PCR, survey mental plant propagation regions in Iran. In this study, we aimed at isolating and identifying the bacteria associated with soft rot of ornamental plants in two major growing areas of Iran and to evaluate the diversity of these isolates. INTRODUCTION MATERIALS AND METHODS Ornamental plants are often vulnerable to attack by pathogens because of intensive cultivation during pro- Bacterial isolates and media. Fifty-seven isolates of duction and suitable conditions for pathogen growth in pectolytic bacteria were obtained from diseased ornamen- greenhouses. An important disease which reduces the pro- tal plants of two different geographical regions of Iran, i.e. duction efficiency and quality of these crops is bacterial Tehran and Markazi provinces in 2009 and 2010 (Table 2). soft rot (Agrios, 2005). Soft rot can be caused by several In addition, two Pcc strains previously isolated from Philo- bacterial genera including Pseudomonas, Xanthomonas, dendron scandens in Guilan province (Baghaee-Ravari et al., 2011) were provided by Dr. S. Baghaee-Ravari. Corresponding author: M. Shams-Bakhsh Fax: +98.2148292200 The type strains of Pcc (IBSBF-863 = ATCC15713), P. E-mail: [email protected] atrosepticum (IBSBF-1819 = ATCC33260), P. betavasculorum 272 Genetic diversity of pectolytic bacteria in Iran Journal of Plant Pathology (2014), 96 (2), 271-279 Table 1. Ornamental host species of Pectobacterium carotovorum subsp. carotovorum and their origins. Hosts Origins References Agave americana China Chen et al., 1994 Aglaonema modestum China Chen et al., 1994 Aglaonema spp. Jamaica, Iran Costa et al., 2006; Baghaee-Ravari et al., 2011 Aloe vera China Chen et al., 1994 Amomum villosum China Chen et al., 1994 Begonia hiemalis Korea Choi and Lee, 2000 Begonia semperflorens China Chen et al., 1994 Calanthe discolor China Chen et al., 1994 Chamaecereus silvestrii Korea Kim et al., 2007 Chlorophytum comosum China Chen et al., 1994 Clivia miniata Korea Choi and Lee, 2000 Crinum asiaticum China Chen et al., 1994 Cymbidium pendulum China Chen et al., 1994 Dianthus sp. China Chen et al., 1994 Dieffenbachiae amoena Turkey Cetinkaya-Yildiz et al., 2004 Dieffenbachiae picta China Chen et al., 1994 Disporum sp. China Chen et al., 1994 Dracaena spp. Puerto Rico Cortes Monllor, 1990 Epipremnum pinnatum Puerto Rico Cortes Monllor, 1990 Fritillaria imperialis Iran Mahmoudi et al., 2007 Gomphrena globosa China Chen et al., 1994 Hosta plantaginea China Chen et al., 1994 Iris spp. Iran Baghaee-Ravari et al., 2011 Kalanchoe blossfeldiana Florida Engelhard et al., 1986 Kalanchoe spp. Israel Costa et al., 2006 Lilium longflorum Korea Hahm et al., 2003 Musa nana China Chen et al., 1994 Ornithogalum arabicum Kenya Costa et al., 2006 Paphiopedilum spp. China Chen et al., 1994 Pelargonium graveolens China Chen et al., 1994 Philodendron scandens Iran Baghaee-Ravari et al., 2011 Polygonatum sibricum China Chen et al., 1994 Saintpaulia sp. Korea Choi and Lee, 2000 Schlumbergera bridgesii Iran Baghaee-Ravari et al., 2011 Spathiphyllum wallisii Argentina Alippi and López, 2009 Speirantha gardenii China Chen et al., 1994 Syngonium podophyllum Iran Baghaee-Ravari et al., 2011 Tulipa spp. Turkey Boyraz et al., 2006 Wedelia chinensis China Chen et al., 1994 Zantedeschia aethiopica Puerto Rico, Italy Cortes Monllor, 1990; Buonaurio et al., 2002 Zantedeschia elliottiana Taiwan Lee et al., 2002 Zantedeschia spp. New Zealand, Poland Wright, 1998; Mikiciński et al., 2010 (IBSBF-787 = ATCC43762), P. carotovorum subsp. odorife- in sterile Petri dishes with a sterile, moistened filter paper. rum (IBSBF-1814 = ICMP11533) Dickeya chrysanthemi pv. A bacterial cell suspension was applied into shallow pits chrysanthemi (IBSBF-231 = ATCC11663) obtained from the made in the centre of potato disks, and the dishes were Instituto Biológico, Seção de Bacteriologia Fitopatologia incubated at 28°C. The inoculated disks were examined at (São Paulo, Brazi)) and the reference strain of P. wasabiae 24 and 48 h for soft rot by probing the tissue surrounding (SCRI 488) obtained from Scottish Crop Research In- the inoculum site with a loop to assess the ability to mac- stitute (Invergowrie, UK) were included for comparison erate them at 28°C (Bradbury, 1970; Schaad et al., 2001). in different tests. All bacterial isolates and strains were grown on nutrient agar (NA) at 25°C for 48 h. After incu- Phenotypic tests. Isolates that were characterized as bation, representative single bacterial colonies were puri- positive for pectolytic activity were tested with conven- fied on King’s B medium and selected isolates were further tional biochemical and physiological tests including: Gram characterized. All isolates and strains were stored in sterile reaction (Suslow et al., 1982), oxidative and fermentative water and in 30% (v/v) glycerol at −80°C. metabolism of glucose (Hugh and Leifson, 1953), oxidase and catalase activity (Schaad et al., 2001), production of Pectolytic ability. Potato tubers were surface-sterilized levan from sucrose (Lelliott and Stead, 1987), fluorescent with 96% ethanol, flamed and peeled aseptically, then pigment production (King et al., 1954), hydrolysis of gela- sliced into disks about 7-8 mm thick. These were placed tine, lecithin and casein (Dickey and Kelman, 1988), indole Journal of Plant Pathology (2014), 96 (2), 271-279 Dahaghin and Shams-Bakhsh 273 Table 2. Bacterial isolates used in this study. Number of isolates (from an area per host, with their collection numbers) Geographical origin Host Plant part 2 (T-Za1*, T-Za2) Tehran-Pakdasht Zantedeschia sp. Stem 5 (M-Za3*, M-Za4, M-Za5, M-Za6, M-Za7*) Markazi-Mahallat Zantedeschia sp. Root and tuber 5 (T-Ag1, T-Ag2, T-Ag3*, T-Ag4, T-Ag5*) Tehran-Hashtgerd Aglaonema sp. Leaf and stem 1 (T-Ag6*) Tehran-Shahriyar Aglaonema sp. Leaf 4 (T-Ag7*, T-Ag8, T-Ag9, T-Ag10 ) Tehran Aglaonema sp. Leaf 6 (M-Ag11*, M-Ag12*, M-Ag13, M-Ag14*, M-Ag15*, M-Ag16* ) Markazi-Mahallat Aglaonema sp. Leaf 1 (T-Di1) Tehran-Hashtgerd Dieffenbachia sp. Leaf 2 (T-Di2, T-Di3 ) Tehran-Pakdasht Dieffenbachia sp. Leaf 1 (T-Di4*)
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