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Biological Control of Watermelon Seedling Blight Caused By Plant Protect. Sci. Vol. 54, 2018, No. 2: 00– doi: 10.17221/168/2016-PPS Biological Control of Watermelon Seedling Blight Caused by Acidovorax citrulli Using Antagonistic Bacteria from the Genera Curtobacterium, Microbacterium and Pseudomonas Sumer HORUZ 1,2* and Yesim AysaN 1 1Plant Protection Department, Faculty of Agriculture, Cukurova University, Adana, Turkey; 2Plant Protection Department, Faculty of Agriculture, Erciyes University, Kayseri, Turkey *Corresponding author: [email protected] Abstract Horuz S., Aysan Y.: Biological control of watermelon seedling blight caused by Acidovorax citrulli using antago- nistic bacteria from the genera Curtobacterium, Microbacterium and Pseudomonas. Plant Protect. Sci. The biological control of the watermelon seedling blight and fruit blotch disease was investigated by screening the potential use of antagonistic bacteria. Between May and August 2012, totally 322 putative antagonistic bacteria were isolated from symptomless melon and watermelon plants grown in Adana, Hatay, and Osmaniye provinces of the Eastern Mediterranean Region of Turkey. In vitro dual culture tests showed that 54 out of 322 strains inhibited the Acidovorax citrulli (Ac) growth with an appearance of clear zones between 2.3 and 27.0 mm in diameter. However, the remaining 268 strains did not exhibit any antagonistic activity against Ac. Seed treatments with fourteen individual antagonistic bacteria resulted in a significant reduction in disease incidence (DI) and severity (DS) ranging between 14.06–79.47% and between 4.57–41.49%, respectively. The bacteria Pseudomonas oryzihabitans (Antg-12), Micro- bacterium oxydans (Antg-57), Curtobacterium flaccumfaciens (Antg-198), and Pseudomonas fluorescens (Antg-273) were the most potent antagonistic bacterial isolates which reduced DI and DS as compared to the untreated control. This study suggested the potential of bacterial antagonists Curtobacterium flaccumfaciens, Microbacterium oxydans, Pseudomonas oryzihabitans, and Pseudomonas fluorescens for the biocontrol of Ac-induced bacterial fruit blotch (BFB). Keywords: Acidovorax citrulli; cucurbist; biocontrol; seed treatment Bacterial fruit blotch (BFB) disease caused by the are not so characteristic. However, disease symptoms bacterium Acidovorax citrulli Schaad et al. 2009 on the fruit appear as water-soaked, dark and olive- (formerly A. avenae subsp. citrulli; Schaad et al. green stain or blotch lesions with irregular margins 1978; Willems et al. 1992) is a devastating disease that are rapidly enlarged with brown discoloration of cucurbits and poses a great threat threat to cucur- and brown cracks on the surface within 10 days af- bit seed and fruit production throughout the world ter bacterial contamination. In the mesocarp of the (Burdman et al. 2005; Wal c ot t 2005; Mirik et al. infected fruits, water-soaked and smooth spots also 2006; Ren et al. 2006; Feng et al. 2013). Apart from occur and these rottings make the fruits unmarket- fruit blotch, A. citrulli also causes leaf and seedling able (Latin & Hopkins 1995). blight and blossom rot in cucurbitaceous plants BFB is a seed transmittable disease (O’Brien & (Frankle et al. 1993; Lessl et al. 2007; Wang et al. Martin 1999), because the bacterium can survive 2009). The symptoms of BFB include water-soaked, on/in seeds over 30 years under suitable storage greasy or irregular brown spots on true leaves which conditions (Block & Shepherd 2008). Thus, seed This study is a part of PhD thesis and was supported by Cukurova University Scientific Research Projects De- partment, Grant No. ZF2012D16. 1 Vol. 54, 2018, No. 2: 00– Plant Protect. Sci. doi: 10.17221/168/2016-PPS treatments and using bacteria-free seeds must be of BFB under greenhouse conditions. The authors the primary measure for the disease control (Melo demonstrated that microbiolisation of artificially et al. 2014). Many attempts including chemical and infested seeds with Bacillus sp. yielded 93.7% disease physical seed treatments have been developed to get severity reduction (Oliveira et al. 2006). Some rid of seedborne A. citrulli. Chemical and biochemi- other scientists also reported the potential use of cal treatments and fermentation of cucurbitaceous Bacillus sp. in reducing the incidence and severity seeds with chitosan, streptomycin sulphate, sodium of BFB (Santos et al. 2006; Medeiros et al. 2009; hypochlorite, peroxyacetic acid, mercury chloride, hy- Jiang et al. 2015). The studies in the biocontrol of drochloric acid, calcium chloride, copper-containing BFB with antagonistic bacteria as seed treatment have bactericides reduced the incidence of the disease. a potential to get rid of unmarketable seedlings or However, since the pathogen can be localised under fruits. Since there is no available biological control the seed coat (Rane & Latin 1992), the chemicals product in the market for BFB management, more failed to eliminate the pathogen from seeds (Sowell researches should be conducted. & Schaad 1979; Rane & Latin 1992; Hopkins et The main objectives of this study were (i) to isolate al. 1996, 2003; Lovic & Hopkins 2003; Zhao et candidate antagonistic bacteria for the biological al. 2003; Feng et al. 2007; Selcuk 2014). Other control of BFB, (ii) to screen the in vitro antibiosis alternative disease management strategies such as ability of biocontrol agents, (iii) to evaluate the effi- physical seed treatments were employed alone or cacy of antagonistic bacteria in the biological control combined with chemicals and they all varied widely of BFB via seed treatment, and (iv) to identify the in efficacy, but they did not completely remove the most potent antagonistic bacteria. pathogen from the seeds (Nomura & Shirakawa 2001; Hopkins & Thompson 2006). Even though early reports were successful in re- MATERIAL AND METHODS porting some promising resistance genes in cucurbit cultivars (Goth & Webb 1981; Hopkins & Thomp- Pathogenic bacterium, media and preparation son 2002) and the authors could identify several of bacterial inoculum. The pathogen Tasci-1 of tolerant lines in melon, however, there are no avail- Acidovorax citrulli (Ac) obtained from the culture able BFB-resistant cultivars (Bahar et al. 2009). The collection of the Laboratory of Phytobacteriology identification of resistance is quite complicated, since (Cukurova University, Faculty of Agriculture, De- A. citrulli can infect in all growth stages of cucurbits partment of Plant Protection, Adana, Turkey) was and the bacterial strains and monitored plant stage isolated from a naturally contaminated watermelon may influence the level of resistance (Johnson et al. fruit grown in Cukurova Region, Turkey in 2009. The 2011). In the absence of resistance in watermelon, pathogenic bacterium was identified using WFB1/ there is a need to use other environmentally safe WFB2 primer pairs (Wal c ot t & Gitaitis 2000). components that could be integrated into a disease King’s Medium B (KB) (Lelliott & Stead 1987) management strategy of BFB on watermelon. was used for culturing the pathogen in Petri dishes. Biological control with antagonistic bacteria or To prepare A. citrulli inoculum from a routinely yeasts has been considered to be a friendly and useful grown culture on KB at 25°C for 48 h, a suspension approach for plant disease management including was prepared in saline buffer (NaCl 0.85%) and the BFB on cucurbits (Fessehaie & Wal c ot t 2005; concentration was adjusted to A600 0.2 OD with a Medeiros et al. 2009; Wang et al. 2009; Jiang et al. spectrophotometer (Shimadzu UV-120-01, Kyoto, 2015; Melo et al. 2015). Fessehaie and Wal c ot t Japan). The bacterial suspension was 10-fold serially (2005) reported that when the maize pathogenic diluted with saline buffer and aliquots of 100 µl were bacteria Acidovorax avenae subsp. avenae AAA99-2 inoculated onto KB. The concentration of the bacte- or Pseudomonas fluorescens A506 were used as seed rial inoculum was estimated to be 1.4 × 108 CFU/ml or blossom treatment in watermelon, bacterial blotch and this concentration was used in further studies. transmission to seedlings or seed infestation was Isolation of antagonists and culture preparation. highly reduced under greenhouse conditions. In Between May and August 2012, antagonistic bacterial that study, 96 strains of epiphytic and endophytic strains were isolated from symptomless melon and bacteria isolated from symptomless melon plants watermelon plants grown in crop fields of Adana, were tested as seed treatment for the biocontrol Hatay, and Osmaniye Provinces of the Eastern Medi- 2 Plant Protect. Sci. Vol. 54, 2018, No. 2: 00– doi: 10.17221/168/2016-PPS terranean Region of Turkey. The symptomless and erson Zwaan, Made, The Netherlands) were obtained fresh leaves and blossoms were used for isolations. as untreated with chemicals. The seeds were im- Each field was surveyed and samples were collected mersed into 200 ml of 1% carboxymethyl cellulose randomly to get a homogeneous collection. Soil sam- (C5678; Sigma Aldrich, Taufkirchen, Germany) and ples were collected from healthy plants to receive Tasci-1 of A. citrulli suspension (1.4 × 106 CFU/ml) beneficial root-colonising bacteria. Sampled blossoms for 30 min and filtered through double-layered were used directly, whereas leaves were cut into small cheesecloth to get rid of bacterial residues. Seeds pieces, and soil samples were sieved to avoid big were dried at room temperature (25 ± 2°C) for fragments. About 10 g of each sample were placed in 24 h (Wang et al. 2009) and divided into three lots
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