Australasian Plant Dis. Notes (2015) 10:23 DOI 10.1007/s13314-014-0151-0
Bacterial wilt of common bean (Phaseolus vulgaris) caused by Curtobacterium flaccumfaciens pv. flaccumfaciens in Iran Ebrahim Osdaghi 1 & Seyed Mohsen Taghavi 1 & Javad Hamedi 2 & Fatemeh Mohammadipanah 2
Received: 14 June 2014 / Accepted: 9 November 2014 # Australasian Plant Pathology Society Inc. 2015
Abstract Bacterial wilt of common bean (Phaseolus vulgaris L.) (cv. Talash and Akhtar) caused by a yellow variant of Curtobacterium flaccumfaciens pv. flaccumfaciens was identified in East Azerbaijan Province, northwestern Iran. Interveinal necrotic lesions surrounded by chlorotic margins leading to total wilt were observed with up to 50 % of plants infected in some fields. The causal agent was characterized using biochemical tests and confirmed by specific polymerase chain reaction primers. This is the first report of bacterial wilt of common bean in Iran. Keywords Phaseolus vulgaris . Bacterial wilt . Curtobacterium flaccumfaciens pv. flaccumfaciens Bacterial wilt caused by Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) is one of the most destructive bacterial diseases in legumes (Wood and Easdown 1990; Tegli et al.
* Ebrahim Osdaghi
[email protected] 1
Department of Plant Protection, College of Agriculture, Shiraz University, Zip Code 71441-65186 Shiraz, Iran
2
Microbial Biotechnology Laboratory, Department of Microbiology, School of Biology and Center of Excellence in Phylogeny of Living Organisms, College of Science, University of Tehran, P.O. Box: 141556455, Tehran, Iran
2002; González et al. 2005; EPPO 2011; Sammer and Reiher 2012; Soares et al. 2013; Urrea and Harveson 2014). The host range of the pathogen includes legumes such as common bean (Phaseolus vulgaris), cowpea (Vigna unguiculata), mungbean (Vigna radiata), soybean (Glycine max), and pea (Pisum sativum) (Wood and Easdown 1990; EPPO 2011; Sammer and Reiher 2012). Bacterial wilt was first identified from South Dakota (USA) in 1926 on Phaseolus vulgaris (Hedges 1926) and has subsequently been recorded in Mexico (Yerkes and Crispin 1956), Australia (Wood and Easdown 1990), South-Eastern Spain (González et al. 2005), Canada (Huang et al. 2006), South America and Tunisia (EPPO 2011), and Germany (Sammer and Reiher 2012). Due to its virulence and economic impact on legume production, Cff is considered as an A2 quarantine pest in European and Mediterranean countries and is subject to phytosanitary and quarantine regulations in these regions (EPPO 2011; Sammer and Reiher 2012). In summer of 2013, five common bean (cv. Talash and Akhtar) fields with suspected bacterial disease symptoms were observed in East Azerbaijan Province, southwestern Iran. Symptoms included interveinal necrotic lesions surrounded by chlorotic margins (Fig. 1). In the advanced stages of infection, systemic wilt and plant defoliation were also observed (Fig. 2). This research aims to identify the causal agent of a common bean disease suspected to be bacterial wilt in Iran.
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Australasian Plant Dis. Notes (2015) 10:23
Fig. 1 Interveinal necrosis with chlorotic margins on common bean (cv. Talash) leaves caused by Curtobacterium flaccumfaciens pv. flaccumfaciens
Fields in Marand, Bonab, Bangin, Sufian and Shabestar region (Table 1) were surveyed and up to 50 % of the plants inspected were showing symptoms similar to those described for bacterial wilt (EPPO 2011). Symptomatic leaves of the infected plants were sampled (approximately 50 leaves per field) from Marand, Bonab, Bangin, Sufian and Shabestar regions (Table 1). About 5 mm3 of infected tissues of each leaf were cut from the margin of a lesion and ground in 5 ml of sterile distilled water. The suspension was streaked onto Yeast Peptone Glucose Agar (YPGA) medium (EPPO 2011) and incubated at 25 °C. Yellow-pigmented colonies 1-2 mm in diameter were present on YPGA plates after 48 h (Fig. 3). Nine isolates (Cffcb21, Cffcb22, Cffcb23, Cffcb24, Cffcb25, Cffcb26, Cffcb27, Cffcb28, C f f c b 2 9 ) ( Ta b l e 1 ) w e r e s e l e c t e d f o r f u r t h e r characterization. The strains were deposited in the University of Tehran Microorganisms Collection (UTMC) (accession number: UTMC00162). Biochemical tests as described by Schaad et al. (2001) were used to characterize the bacterial strains. All isolates were gram positive, of oxidative but not
Fig. 2 Totally wilted common bean (cv. Talash) plants infected by bacterial wilt pathogen, Curtobacterium flaccumfaciens pv. flaccumfaciens
fermentative metabolism, and had the ability to grow at 37 °C. All hydrolyzed aesculin, casein, and gelatin, produced acid from inositol, mannose, and maltose but not from mannitol and erythritol, were found to be catalase and oxidase positive, but of negative urease and indole production (Schaad et al. 2001; EPPO 2011). Specific detection and identification of the isolates was conducted using the PCR primer pair CffFOR2 5′GTTATGACTGAACTTCACTCC-3′ and CffREV4 5′GATGTTCCCGGTGTTCGA-3′ (Tegli et al. 2002). The bacterial strains were grown on YPGA medium for 48 h at 25 °C and DNA extraction was carried out using the boiling cells method (EPPO 2011). With PCR reactions, Universal PCR Kit, Gene Pak® PCR MasterMix Core (ISOGENE Laboratory, Moscow, Russia) was used based on the manufacture recommendations. With each isolate, a 20 μl PCR reaction including PCR buffer (100 mM Tris–HCl, 500 mM KCl), 1.5 mM MgCl2, 100 μM of each dNTP, 1 U of Taq DNA Polymerase, 50 ng total DNA and 1 μl of each primer (10 pmol × μl -1 ) was used. Xanthomonas axonopodis pv. phaseoli Araxa1 strain provided by Department of Plant Pathology, Faculty of Agriculture, Tarbiat Modares University (Osdaghi et al. 2009) and Cff Cffcw13 strain sourced from Microbial Culture Collection, Central Laboratory, Ramin Agricultural and Natural Resources University (Osdaghi et al. 2014), were used as negative and positive controls respectively. Duplicate PCR tests were conducted for each isolate. The PCR conditions were an initial denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 30 s; annealing at 62 °C for 45 s, extension at 72 °C for 45 s and a final extension at 72 °C for 10 min. The amplified products were separated by electrophoresis in 1.2 % agarose gel in Tris-Borate-EDTA (TBE) buffer, stained with CinnaGen DNA safe Stain (Cat. No. PR881603, CinnaGen Co., Tehran, Iran) and visualized under UV light. Amplification of 306 bp fragment of isolates (Fig. 4), as well as the biochemical tests results confirmed the bacterial strains as Cff. This fragment was also
Australasian Plant Dis. Notes (2015) 10:23 Table 1
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Description of the geographic areas and host plants of Curtobacterium flaccumfaciens pv. flaccumfaciens strains characterized in this study
Sampling Region
Cff Strain(s)
Cultivar
Seed class
Growth habit
Sampling date
Marand, East Azerbaijan Bonab, East Azerbaijan Bangin, East Azerbaijan Sufian, East Azerbaijan Shabestar, East Azerbaijan
Cffcb21, Cffcb22, Cffcb29 Cffcb23, Cffcb24 Cffcb27 Cffcb25, Cffcb26 Cffcb28
Talash Talash Talash Akhtar Akhtar
Brown Pinto Brown Pinto Brown Pinto Red Kidney Red Kidney
Vining Vining Vining Bush Bush
07.25.2013 08.14.2013 07.23.2013 08.15.2013 08.15.2013
Fig. 3 Yellow pigmented and domed colonies of Curtobacterium flaccumfaciens pv. flaccumfaciens, 1–2 mm in diameter on Yeast Peptone Glucose Agar (YPGA) medium (48 h old)
Fig. 4 The PCR products obtained with the primers CffFOR2 and CffREV4. Wells No.1: Thermo Scientific GeneRuler 1 kb DNA ladder (#SM0311), 2–10: Nine candidate strains of Curtobacterium flaccumfaciens pv. flaccumfaciens Cffcb21, Cffcb22, Cffcb23, Cffcb24, Cffcb25, Cffcb26, Cffcb27, Cffcb28, Cffcb29 respectively. 11: Positive
amp lif ied fr om the Cffcw13 strain b ut no t in X. axonopodis pv. phaseoli Araxa1 strain or the Bnotemplate DNA^ control reactions (Fig. 4). Pathogenicity tests were conducted on 10 day old common bean (cv. Talash) plants (fifteen plants for each treatment) as recommended by Urrea and Harveson (2014). The bacterial suspension (of 1 × 108 CFU/ml) of Cffcb21 and Cffcb22 isolates was prepared from 48 h old culture on YPGA. A node infiltration method (Urrea and Harveson 2014) was used for the inoculation of common bean plants. The negative control plants were inoculated in the same manner with distilled water. Disease symptoms on inoculated plants appeared 10–12 days after inoculation. First trifoliate leaves of the inoculated plants exhibited symptoms of marginal necrosis and interveinal chlorosis (Fig. 5). Infected plants finally died 15–20 days after inoculation. Koch’s postulates were completed by randomly selecting five infected plants and re-isolating the bacteria onto YPGA and then using the primer pair CffFOR2 and CffREV4 to confirm the pathogen identity (Tegli et al. 2002). Although yellow variant of Cff has recently been characterized in Iran (Osdaghi et al. 2014) as the causal
control, (DNA template from Cff strain Cffcw13). 12: DNA extract from non-infected common bean plants. 13: No DNA template control. 14: Negative control (DNA template from Xanthomonas axonopodis pv. phaseoli strain Araxa1)
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Fig. 5 Bacterial wilt symptoms on common bean (cv. Talash) plants artificially inoculated by Curtobacterium flaccumfaciens pv. flaccumfaciens
agent of cowpea (Vigna unguiculata) bacterial wilt, this is the first report of the pathogen on common bean in Iran. In order to conduct appropriate quarantine procedures and to prevent the spread of the disease to other regions, characterization of the potential host range of the pathogen and an evaluation of Iranian common bean cultivars for their susceptibility to find sources of resistance are recommended.
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