isolation and characterization of pseudomonas aeruginosa ... - SIPaV

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Journal of Plant Pathology (2010), 92 (3), 653-660

Edizioni ETS Pisa, 2010

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ISOLATION AND CHARACTERIZATION OF PSEUDOMONAS AERUGINOSA WITH ANTAGONISTIC ACTIVITY AGAINST PYTHIUM APHANIDERMATUM A.H. Al-Hinai1, A.M. Al-Sadi2, S.N. Al-Bahry3, A.S. Mothershaw4, F.A. Al-Said2, S.A. Al-Harthi5 and M.L. Deadman2 1Center

for Environmental Studies and Research, Sultan Qaboos University, Al-Khod 123, Oman of Crop Sciences, College of Agricultural and Marine Sciences, Sultan Qaboos University, PO Box 34, Al-Khod 123, Oman 3Department of Biology, College of Science, Sultan Qaboos University, Al-Khod 123, Oman 4Department of Food Science and Nutrition, College of Agricultural and Marine Sciences, Sultan Qaboos University, Al-Khod 123, Oman 5Royal Court Affairs, Al-Khod 123, Oman 2Department

SUMMARY

A study was conducted to isolate and characterize bacterial strains from greenhouse soils with potential for suppression of Pythium damping-off of cucumber. Out of 100 bacterial isolates collected from greenhouse soils, 20 isolates were found to be antagonistic to Pythium aphanidermatum in in vitro studies. The size of the growth inhibition zone of P. aphanidermatum on potato dextrose agar varied from 4 to 9 mm, with an average of 6.2 mm. Identification of these bacterial isolates to the species level using API20NE as well as sequences of the 16S ribosomal RNA confirmed their identity as Pseudomonas aeruginosa. Bacterial isolates showed optimum growth at 30˚C, whereas a temperature of 37˚C favored bacterial antagonistic activity. Bacterial growth and antagonistic activity were not affected at high salinity levels (10 dS m-1) known to be stressful to cucumber production. This provides evidence for tolerance to salinity among the isolated bacteria. In vivo assays provided evidence for inhibition of Pythium damping-off at 28˚C, with no effect on growth of cucumber seedlings. This is the first report of occurrence of Ps. aeruginosa with antagonistic activity against P. aphanidermatum in greenhouse soils in Oman. Key words: antagonism, asparagine, Cucumis sativus, metalaxyl, salinity.

INTRODUCTION

Cucumber is an important crop worldwide. In Oman, cucumber production has recently increased substantially, with over 90% of greenhouses used exclusively for cucumber production with almost 95% using soil-based systems. However, this increase has been hampered by several biotic and abiotic stresses, with damping-off being the most limiting biotic factor.

Corresponding author: A.M. Al-Sadi Fax: +96824413418 E-mail: [email protected]

Seedling losses due to this disease in Oman and elsewhere were reported to be as high as 75% (Stanghellini and Phillips, 1975; Al-Kiyumi, 2006). Increasing salinity of irrigation water in different regions in Oman is a further limiting factor. Salinity levels of irrigation water were reported to exceed 1.7 dS m-1 in more than 30% of greenhouses in the main agricultural region in Oman (Al-Sadi et al., 2010a). These salinity levels are known to be stressful to cucumber (Ayers and Westcot, 1985), but have been shown not to affect growth and reproduction of the causal agent of damping-off, Pythium aphanidermatum (Al-Sadi et al., 2010a, 2010b). Studies on the causal agents of cucumber dampingoff in Oman have characterized Pythium species as the only pathogens associated with this disease, with P. aphanidermatum being the most common and widespread (Al-Sadi et al., 2007). Pythium species are considered the most common pathogens associated with damping-off in different parts of the world (Stanghellini and Phillips, 1975; Howard et al., 1994). They also cause serious diseases on other cucurbits and plant species (Davison and McKay, 1999; Martin and Loper, 1999; Al-Sadi et al., 2008a). Management of Pythium damping-off in Oman relies on the use of a number of chemical and cultural practices. Metalaxyl is amongst the most commonly used fungicides in Oman and elsewhere (Hickman and Michailides, 1998; Al-Kiyumi, 2006; Al-Sadi et al., 2008e). It is usually applied before planting or immediately after transplanting seedlings into greenhouse soils. In addition, growers use solarization especially in summer to reduce Pythium populations in soil (Deadman et al., 2006). Soil replacement is another common practice, where growers remove the top 30 to 60 cm of greenhouse soil and replace it with uncultivated soil (Al-Sadi et al., 2008d). Recent reports have indicated a reduction in the efficacy of Pythium damping-off management practices. Although no resistance to metalaxyl was reported among Pythium populations (Al-Sadi et al., 2008b, 2008c), failure was related to rapid degradation of the active ingredient in greenhouse soils following its repeated use (AlSadi et al., 2008e). Reduction in Pythium control using metalaxyl due to development of fungicide resistance


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Antagonism of Ps. aeruginosa against P. aphanidermatum

and rapid degradation in soil has been reported elsewhere (Davison and McKay, 1999; Moorman et al., 2002). Alternative methods by which Pythium damping-off might be managed include biological control. Suppression of damping-off disease by fungal biocontrol agents has been attained through the use of P. oligandrum, P. nunn and Trichoderma species (Martin and Loper, 1999). In addition, several studies have reported the use of bacteria and actinomycetes (Pandey et al., 2001; ElTarabily et al., 2009). Bacterial strains of Pseudomonas fluorescens and other species are the most commonly reported fluorescent pseudomonads used to suppress damping-off (Martin and Loper, 1999). Carisse et al. (2003) reported efficient control of Pythium dampingoff using Bacillus marinus, Ps. fluorescens and Ps.aeruginosa. Pseudomonas antagonists were found to be superior to Bacillus antagonists in the control of damping-off of cucumber and sugar beet (Georgakopoulos et al., 2002). Other studies reported successful use of pseudomonads as biocontrol agents in greenhouses (Rankin and Paulitz, 1994) and fields (Weller and Cook, 1986). In addition, Pseudomonas species were used successfully to control several diseases in crops other than cucumber (Hultberg et al., 2000; Pandey et al., 2001; Amein et al., 2008). In Oman, no studies have attempted to characterize biocontrol agents with antagonistic activity against P. aphanidermatum, so it is unclear whether antagonistic fluorescent Pseudomonas species could be present in greenhouse soils. This study was therefore established to isolate bacteria with antagonistic activity against P. aphanidermatum for potential use as biocontrol agents. Specific objectives included: 1- To isolate bacteria with antagonistic activity against P. aphanidermatum, with particular emphasis on Pseudomonas species. 2- To investigate the effect of temperature and salinity on growth and antagonistic activity of the isolated bacteria. 3- To investigate the effect of selected bacteria on Pythium damping-off control. Findings from this study could provide information valuable for use in integrated programs for damping-off management in Oman.

MATERIALS AND METHODS

Collection of soil samples. Twenty-seven soil samples were collected from nine different commercial cucumber greenhouses in Barka, 10 km north-west of the capital area of Oman. Planting line 4 was chosen for sample collection in each greenhouse in order to standardize sample collection. In addition, samples were collected from three different places in a greenhouse; near the en-


trance (E), from the middle of the greenhouse (M) and near the cooling pad (P). Samples were collected from the top 5 cm of soil around healthy cucumber plants standing adjacent to diseased plants showing dampingoff or wilt symptoms. Collected soils were sealed in sterile polyethylene bags. Isolation of bacteria. One gram from each soil sample was placed in 9 ml of asparagine broth enrichment medium consisting of 2 g l-1 asparagine L-monohydrate (Fluka, Switzerland), 1 g l-1 K 2 HPO 4 (BDH, England) and 0.5 g l-1 MgSO4⋅7H2O (BDH, UK) in order to enhance Pseudomonas growth. The samples were incubated for 48 h at 37ºC with vigorous shaking at 200 rpm to provide aeration for the bacteria. A loopful of the resulting bacterial suspension was streaked onto asparagine plates containing 1.5% agar (Oxoid, UK) and incubated at 37ºC until colonies developed. The bacteria were then transferred to fresh asparagine plates according to the morphological characteristics of colony: colour, shape and size. Selection of antagonistic bacteria. One hundred bacterial isolates obtained from soil were screened for antagonism against isolate P085 of P. aphanidermatum, previously isolated from cucumber seedlings showing damping-off symptoms (Al-Sadi et al., 2007). Antagonism of bacteria against P. aphanidermatum was examined using a modified method of Montealerge et al. (2003). A loopful from each purified bacterial isolate was inoculated into 5 ml of asparagine broth and incubated for 48 h at 37ºC. Subsequently, 100 µl of bacterial suspension of each isolate was placed on different 10 mm diameter sterile paper discs (Whatman, UK). Four different discs were spaced around a central 10 mm plug of 2-day-old P. aphanidermatum on 2.5% potato dextrose agar (PDA, Oxoid, UK). The plate was incubated for 24 h at 30oC and the size of the inhibition zone of hyphal growth was determined. Bacteria which showed no suppression of fungal growth were discarded. The inhibition test was replicated three times. The active bacterial isolates were preserved in 15% glycerol at -80oC. Twenty bacterial isolates which showed antagonistic activity in the pre-evaluation test were subjected to further evaluation. A 10 mm sterile paper disk impregnated with 100 µl of a 48 h bacterial suspension was placed 1 cm away from the edge of a PDA plate and a 10 mm diameter mycelial plug from a 2-day-old culture of P. aphanidermatum was placed at the opposite side of the plate. The plate was incubated for 48 h at 30°C followed by measuring the size of the inhibition zone. A 10 mm diameter sterile paper disk soaked with 100 µl of asparagine broth was used as a control. The inhibition test was repeated three times for each isolate. Standard plate counts were performed at the same time as the an-


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tagonism test in order to determine the cell count of each isolate after 48 h of incubation. Identification of antagonistic bacteria. Gram staining and oxidase tests were conducted for the 20 bacterial isolates which showed antagonistic activity. The antagonistic isolates were biochemically identified using API20NE according to the manufacturer’s protocol (BioMerieux. France). In addition, the identity of two bacterial isolates (B007 and B094) was confirmed using sequences of the 16S ribosomal RNA. DNA extraction, polymerase chain reaction (PCR) and sequencing were undertaken at Macrogen Inc. (Korea). The 16S ribosomal RNA region was sequenced using primers 518F and 800R and compared using BLAST with sequences deposited at the National Center for Biotechnology Information (NCBI). Alignment of sequences from this study and similar sequences from GenBank was obtained using Clustal X (Larkin et al., 2007). Effect of temperature and salinity on development and antagonism of bacteria. In order to find out the optimal conditions for growth and antagonism of the bacterial isolates, the effect of different temperatures and salinity levels on the development and antagonism of antagonistic bacteria was examined. Three temperatures (23, 30, and 37°C) and 7 salinity levels (0.25, 0.5, 1.0, 2.0, 5.0, 10.0 dS m-1 and 0.01 dS m-1 as a control) were used. A bacterial suspension (100 µl) was placed in a tube containing 5 ml asparagine broth. The tube was incubated at the predetermined temperatures for 48 h after which bacterial growth was determined by measuring the optical density at 600 nm. Antagonistic activity was measured as described earlier. Asparagine broth was used as a control. Three replicates were used for each of the seven isolates tested (B007, B021, B038, B048, B049, B094 and B095). In order to determine the effect of increasing salinity on bacterial yield, a tube containing 20 ml of asparagine broth, previously adjusted to the desired NaCl concentration was inoculated with bacteria. The tubes were then incubated for 48 h at 37ºC and bacterial growth was estimated as before. Antagonistic activity of isolates cultured at the differing salinity levels was also examined as described earlier. Three replicates were used for each isolate (B007 and B094) and the experiment was repeated twice. The antagonism of selected bacterial isolates (B007 and B094) grown in asparagine media but tested for antagonism at different salinity levels on PDA medium (0.01, 0.25, 0.50, 1.0, 2.0, 5.0 and 10.0 dS m-1) was also examined. Antagonistic activity was measured as described earlier except that PDA, which was used for the antagonistic test, was adjusted to salinity levels of 0.01, 0.25, 0.50, 1.0, 2.0, 5.0 and 10.0 dS m-1 using NaCl.

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Three plates were used for each bacterial isolate and the plates were incubated for 48 h at 30oC followed by measuring the size of the inhibition zone. Filter papers inoculated with asparagine media served as a control. The experiment was repeated twice. The effect of increasing salinity was also tested on P. aphanidermatum growth in vitro as described by Al-Sadi et al. (2010b). Two-day-old mycelial plugs taken from P. aphanidermatum cultures grown on 2.5% PDA were transferred to 1.7% corn meal agar plates (CMA, Oxoid, UK) supplemented with 7 different concentrations of NaCl (0.01, 0.25, 0.50, 1.0, 2.0, 5.0 and 10 dS m-1). The plates were incubated at 25˚C and linear growth of P. aphanidermatum was recorded after 24, 48, 72 and 96 h. The percent reduction in growth relative to the control (0.01 dS m-1) was determined. Four replicate plates were used and the experiment was repeated twice. Effect of Ps. aeruginosa on Pythium damping-off. In order to test the ability of two bacterial isolates (B007 and B094) to suppress Pythium damping-off of cucumber, three different treatments were used. In the first treatment, a 12-cm diameter pot was half filled with potting mix. A 50-mm plug of a 3-day-old P. aphanidermatum culture grown on PDA was placed on the surface of the potting mix and was covered with a 4-cm layer of potting mix. Seven cucumber seeds (Hamada, Chile) were sown in each pot. Pots then received 20 ml of 48 h bacterial suspensions grown in asparagine media for each isolate separately. In the second treatment, bacterial suspensions of the two isolates were added separately to the pots but without infesting the potting mix with P. aphanidermatum. The third treatment consisted of asparagine media added to soil in pots which were infested with Pythium. Control pots were also used where media was added to the pots but without infestation with Pythium. The percent seedlings surviving pre- and post-emergence damping-off after 3 weeks was determined. The test was carried out at 28±2˚C and repeated twice. Statistical analysis. Data were analyzed using SAS v8 (SAS institute, Cary, USA). Treatment means were compared using Tukey’s Studentized range test with P < 0.05 being accepted as significant.

RESULTS

Isolation and selection of antagonistic bacteria. A total of 100 bacterial isolates were obtained from the different soil samples. Most of the isolated bacteria developed pale green to dark green pigmentation on asparagine medium and released a sweet grape-like odour. This was an indication that the isolated bacteria were potentially pseudomonads.


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Table 1. Mean size of the inhibition zone (mm) and dose CFU for 48-h-old antagonistic bacterial isolates. Bacterial isolates a

Size of inhibition zone (mm) b

Mean CFU/ml × 107 c

B048 B094 B038 B095 B049 B021 B007 B098 B034 B093 B089 B009 B008 B036 B088 B037 B091 B056 B053 B011

9.0a 8.5a 8.3a 8.0ab 8.0ab 7.0bc 7.0bc 6.7dc 6.3dce 6.0dfce 6.0dfce 5.7dfge 5.7dfge 5.5hfge 5.0hfgi 5.0hfgi 4.5hi 4.0i 4.0i 4.0i

1.2 1.5 2.0 3.5 3.0 1.6 3.3 3.3 2.1 2.6 2.8 1.6 2.6 3.4 1.7 2.5 1.3 2.0 2.7 1.4

a

The isolates ranked in term of inhibition zone size Means with the same letter in the same column are not significantly different at P < 0.05 (Tukey's studentized range test). c CFU indicates bacterial colony forming units. No significant correlation was found between CFU and size of inhibition zone with the different bacterial isolates (r = 0.08; P = 0.740) b

Twenty of the isolates were found to inhibit mycelial growth of P. aphanidermatum on PDA plates in a triplicate assay. All antagonistic isolates produced an inhibition zone with a yellow margin at the point of contact with the pathogen. Pythium growth suppression varied between 4.0 to 9.0 mm, with a mean of 6.2 mm (Table 1). The antagonistic isolates came from seven different soil samples obtained from four different greenhouses surveyed. The mean number of bacteria produced in as-

paragines medium after 48 h incubation varied from 1.2x107 to 3.5x107 CFU ml-1 (mean 2.3x107 CFU ml-1). No significant correlation was observed between size of the inhibition zone and mean colony forming units (CFU) of the bacterial isolates (r = 0.08; P = 0.740). Identification of antagonistic Pseudomonads. The 20 bacterial isolates which demonstrated antagonism were found to be Gram negative rods and gave a positive oxidase reaction. The isolates produced a yellow-

Table 2. Mean bacterial yield (A) and inhibition zone (mm) at different temperatures.

B007 B095 B048 B094 B049 B021 B038 Average a a

Bacterial yield (A, 600 nm) 23˚C 30˚C 37˚C 0.45 0.46 0.22 0.23 0.41 0.29 0.42 0.56 0.22 0.27 0.38 0.26 0.19 0.50 0.26 0.16 0.38 0.24 0.24 0.29 0.11 0.28b 0.42a 0.23b

Size of inhibition zone (mm) 23˚C 30˚C 37˚C 4.7 4.4 7.7 5.6 4.5 7.3 5.0 6.3 7.0 5.0 5.3 7.7 5.3 7.6 7.7 4.0 4.0 8.0 3.6 3.3 6.0 4.8b 5.1b 7.3a

Means with the same letters in the same row (for each category) are not significantly different at P < 0.05 (Tukey’s studentized range test).


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green pigment in asparagine medium. According to API20NE, all selected bacteria were identified as Ps. aeruginosa. Further identification of two selected isolates by sequencing the 16S ribosomal RNA using primers 518F and 800R produced a fragment 1461 bp in length. The sequence was identical for the two isolates from Oman and was also found to share 100% nucleotide identity with several sequences of Ps. aeruginosa isolates deposited in GenBank, including FM209186, EU331416, CP000744, AY631241 and AF094718. The 16S rRNA sequence of isolate B094 from Oman was deposited in GenBank under the accession number FJ985806. Effect of temperature and salinity on development of antagonistic bacteria. Bacterial yield, expressed as absorbance at 600 nm (A), was significantly better at 30°C than at 23 or 37°C. As incubation temperature increased, antagonistic activity increased and the inhibition zone was enlarged for all isolates (Table 2). Increasing salinity of asparagine media from 0.01 to 10.0 dS m-1 did not result in a significant effect on antagonistic activity of the isolates B007 and B094. There were no significant differences in the of inhibition zone size between the lowest (0.01 dS m-1) and highest (10.0 dS m-1) salinity levels (P > 0.05; Table 3), and no correlation between salinity level and size of inhibition zone or bacterial yield (Fig. 1). The number of bacterial colonies per ml of the asparagine media after two days of incubation did not differ among the different salinity levels (mean 2.1×107 CFU ml-1; data not shown).

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Table 3. Influence of increasing salinity of asparagine culture medium on size of inhibition zone (mm). Salinity level (dS m-1) Control B007 B094 0.01 0a 5.7a 6.7a 1.00 0a 5.0a 7.3a 10.00 0a 6.7a 6.3a Means with the same letter in the same column are not significantly different at P < 0.05 (Tukey’s studentized range test).

Increasing salinity level of the PDA media on which the antagonistic test was conducted up to 10 dS m-1 showed no significant effect on antagonistic activity of the tested bacteria (P > 0.05; Table 4). In addition, no significant correlation was found between increasing salinity of the culture media and antagonistic activity (Fig. 2). There was also no significant difference between growth of Pythium at 0.01 dS m-1 and growth at 10 dS m-1.

Fig. 2. Influence of salinity level of PDA medium on antagonistic activity of two bacterial isolates against P. aphanidermatum.

Fig. 1. Relationship between salinity level of asparagine medium and bacterial yield (A) or antagonistic activity (B) of bacterial isolates.

Effect of Ps. aeruginosa on Pythium damping-off. Results of in vivo tests showed that the percent survival rate of cucumber seedlings treated with bacterial isolates alone was 95 and 100% for isolates B007 and B094, respectively, when compared to the control. Adding P. aphanidermatum resulted in 0% survival in the control group, but had no significant effect on cucumber seedlings treated with bacteria for three weeks following germination (Table 5). In addition, the selected antagonistic bacteria in the control did not cause any necrosis on the seedling roots or chlorosis in the seedling. There was also no effect of bacteria on germination and fresh and dry weight of cucumber (data not shown).


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Table 4. Influence of increasing salinity of PDA media on inhibition zone by Ps. aeruginosa (B007 and B094) and growth rate of Pythium a.

Salinity level (dS m-1) Control b B007 b B094 b Pythium growth (mm/day) b 0.01 0.0a 6.0a 5.0a 33.7a 1.00 0.0a 5.7a 6.0a 39.3a 10.00 0.0a 6.7a 6.7a 34.0a a The growth rate of P. aphanidermatum was determined separate from the antagonistic test. b Means with the same letter in the same column are not significantly different at P < 0.05 (Tukey's studentized range test)

Table 5. Percent cucumber seedling survival after treatment of P. aphanidermatum-inoculated cucumber seedlings with two different isolates of Ps. aeruginosa. Treatment % seedling survival compared to the control Control 100 a Pythium 0b B007 95 a B007 + Pythium 81 a B094 100 a B094 + Pythium 100 a Values with the same letter in the same column are not significantly different from each other at P < 0.05 (Tukey’s Studentized range test).

DISCUSSION

Of 100 bacterial isolates assessed for antagonistic activity against P. aphanidermatum, 20 were found to be antagonistic, all belonging to Ps. aeruginosa. Previous studies reported antagonistic activity of Ps. aeruginosa against some Pythium species including P. splendens, P. debaryanum and P. myriotylum (Anjaiah et al., 1998; Tambong and Hofte, 2001; Perneel et al., 2007, 2008) as well as some Fusarium species (Anjaiah et al., 1998). In addition, several other studies reported antagonistic activity among Pseudomonas species including Ps. fluoresecens and P. corrugate against Pythium species (Hultberg et al., 2000; Pandey et al., 2001; Schmidt et al., 2004a, 2004b). In this study, in vivo tests on two Ps. aeruginosa isolates gave similar results to the in vitro tests, which provides evidence for the efficacy of these isolates in suppressing Pythium damping-off in cucumber. This agrees with previous reports on antagonistic activities of Ps. aeruginosa against Pythium (Anjaiah et al., 1998; Tambong and Hofte, 2001; Perneel et al., 2008). This appears to be the first report of Ps. aeruginosa in greenhouses in Oman with antagonistic activity towards P. aphanidermatum. Besides the beneficial antagonistic effects of Ps. aeruginosa, it is important to note the opportunistic role of this bacterium in a number of human diseases (Köhler and van Delden, 2009) and some plant diseases (Rahme et al., 1995). This study provides evidence for the existence of in-

traspecific variation in antagonistic activity among Ps. aeruginosa isolates; there were significant differences among the 20 bacterial isolates in the size of the inhibition zone in in vitro studies. This may be related to differences in types or amounts of antagonistic products (antibiotics) produced because there was no correlation between antagonism and bacterial cell count. Previous studies indicated that Ps. aeruginosa produces different types of antibiotic such as phenazine-1-carboxylic acid and phenazine-1-carboximde and rhamnolipid-biosurfactants that play a major role in Pythium suppression (Tambong and Hofte, 2001; Perneel et al., 2008). In addition, control of pathogens using Pseudomonas species has been reported to be due to competition for iron, antibiosis and induced systemic resistance in the host (Pieterse et al., 2001; Berg et al., 2002). However, further studies will be required to investigate factors involved in suppression of P. aphanidermatum-induced damping-off using Ps. aeruginosa. Previous studies have shown that cucumber yield and growth are affected by salinity levels beyond 1.70 dS m-1, and a 50% reduction in yield of cucumber was reported to occur at 4.2 dS m-1 (Ayers and Westcot, 1985). However, little appears to have been done on sensitivity of Ps. aeruginosa to these salinity levels. Our study shows that the antagonistic activity and growth of P. aeruginosa were not affected even at salinity levels of 10 dS m-1. This was evident from normal growth of P. aeruginosa in asparagine cultures adjusted to 10 dS m-1 as well as the unaffected antagonistic activity at this salinity level. In Oman, salinity levels of irrigation water in greenhouses commonly range from 0.44 to 7.77 dS m-1, with about 30% of greenhouses having salinity levels above 1.70 dS m-1 (Al-Sadi et al., 2010a). This study makes it unlikely that increasing salinity will inhibit bacterial growth or antagonism at salinity levels commonly encountered in greenhouses in Oman. In addition, it provides evidence for possible inhibition of P. aphanidermatum even at salinity levels at which its growth and reproduction was found not to be affected (Al-Sadi et al., 2010a, 2010b). Findings from this study also provide evidence for optimal growth and antagonistic activity of Ps. aeruginosa at relatively high temperatures (30-37°C) which are common in Oman. Previous work has shown that salinity levels of 10 dS m-1 had no effect on growth of other an-


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tagonistic bacteria such as Bacillus lentimorbus and B. subtilis (Montealegre et al., 2003). This study reveals for the first time the presence of bacteria with antagonistic activity against P. aphanidermatum in Oman. It also provides essential information on tolerance of these bacterial isolates to high salinity levels as well as temperatures which are common in the country. The differences we found in the antagonistic activity of bacterial isolates may necessitate further studies to examine the mechanisms of action of these bacteria as well their role in the observed differences of antagonism. In addition, future studies may be required to confirm the antagonistic activity of Ps. aeruginosa under greenhouse conditions with salinity levels commonly encountered in greenhouses as well as the antagonistic effects on other diseases of economic importance in Oman (AlSadi et al., 2010c; Al-Sadi and Deadman, 2010).

ACKNOWLEDGEMENTS

We would like to acknowledge Dr. Rashid Al-Yahyai for help in statistical analysis and Assila Al-Harthi, Yousif Al-Maqbali, Issa Al-Mahmooli and Aisha Al-Ghaithi for technical support. This project was partially funded through the strategic project SR/AGR/PLNT/04/01 on improvement of vegetable production in Oman.

REFERENCES Al-Kiyumi K.S., 2006. Greenhouse cucumber production systems in Oman: A study on the effect of cultivation practices on crop diseases and crop yields. Ph.D. Thesis. University of Reading, UK. Al-Sadi A.M., Drenth A., Deadman M., de Cock A.W.A.M., Aitken E.A.B., 2007. Molecular characterization and pathogenicity of Pythium species associated with damping-off in greenhouse cucumber (Cucumis sativus L.) in Oman. Plant Pathology 56: 140-149. Al-Sadi A.M., Deadman M.L., Al-Said F.A., Khan I., Al-Azri M., Drenth A., Aitken E.A.B., 2008a. First report of Pythium splendens associated with severe wilt of muskmelon (Cucumis melo) in Oman. Plant Disease 92: 313. Al-Sadi A.M., Drenth A., Deadman M.L., Aitken E.A.B., 2008b. Genetic diversity, aggressiveness and metalaxyl sensitivity of Pythium aphanidermatum populations infecting cucumber in Oman. Plant Pathology 57: 45-56. Al-Sadi A.M., Drenth A., Deadman M.L., de Cock A.W.A.M., Al-Said F.A., Aitken E.A.B., 2008c. Genetic diversity, aggressiveness and metalaxyl sensitivity of Pythium spinosum infecting cucumber in Oman. Journal of Phytopathology 156: 29-35. Al-Sadi A.M., Drenth A., Deadman M.L., Al-Said F.A., Khan I., Aitken E.A.B., 2008d. Potential sources of Pythium inoculum into greenhouse soils with no previous history of cultivation. Journal of Phytopathology 156: 502-505.

Al-Hinai et al.

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Al-Sadi A.M., Drenth A., Deadman M.L., Al-Said F.A., Khan I., Aitken E.A.B., 2008e. Association of a second phase of seedling mortality in cucumber seedlings with a rapid rate of metalaxyl biodegradation in greenhouse soils. Crop Protection 27: 1110-1117. Al-Sadi A.M., Al-Habsi N., Al-Kiyumi K., Al-Said F.A., AlRawahy S.A., Ahmed M., Deadman M.L., 2010a. Effect of salinity on growth, reproduction and pectolytic enzyme production by Pythium aphanidermatum: a serious soil borne pathogen of vegetable crops in Oman. In: Ahmed M., Al-Rawahy S.A., Hussain N. (eds). A Monograph on Management of Salt-Affected Soils and Water for Sustainable Agriculture, pp. 95-98. SQU, Muscat, Oman. Al-Sadi A.M., Al-Masoudi R.S., Al-Habsi N., Al-Said F.A., AlRawahy S.A., Ahmed M., Deadman M.L., 2010b. Effect of salinity on Pythium damping-off of cucumber and on the tolerance of Pythium aphanidermatum. Plant Pathology 59: 112-120. Al-Sadi A.M., Al-Ouweisi F.A., Al-Shariani N.K., Kaplen E., Al-Adawi A.O., Deadman M.L., 2010c. Histological changes in mango seedlings following infection with Ceratocystis manginecans, the causal agent of mango decline. Journal of Phytopathology (in press) (DOI: 10.1111/j.14390434.2010.01691.x) Al-Sadi A.M., Deadman M.L., 2010. Influence of seedborne Cochliobolus sativus (anamorph: Bipolaris sorokiniana) on crown rot and root rot of barley and wheat. Journal of Phytopathology 158: 683-690. Amein T., Omer Z., Welch C., 2008. Application and evaluation of Pseudomonas strains for biocontrol of wheat seedling blight. Crop Protection 27: 532-536. Anjaiah V., Koedam N., Nowark-Thompson B., Loper J.E., Hofte M., Tambong J.T., Cornelis P., 1998. Involvement of phenazines and anthranilate in the antagonism of Pseudomonas aeruginosa PNA1 and Tn5 derivatives toward Fusarium spp. and Pythium spp. Molecular Plant-Microbe Interactions 11: 847-854. Ayers R.S., Westcot D.W., 1985. Water Quality for Agriculture. FAO Irrigation and Drainage paper No. 29. FAO Publication Division, Rome, Italy. Berg G., Roskot N., Steidle A., Eberl L., Zock A., Smalla K., 2002. Plant-dependent genotypic and phenotypic diversity of antagonistic rhizobacteria isolated from different Vericillium host plants. Applied Environmental Microbiology 68: 3328-3338. Carisse O., Bernier J., Benhamou N., 2003. Selection of biological agents from composts for control of damping-off of cucumber caused by Pythium ultimum. Canadian Journal of Plant Pathology 25: 258-267. Davison E.M., McKay A.G., 1999. Reduced persistence of metalaxyl in soil associated with its failure to control cavity spot of carrots. Plant Pathology 48: 830-835. Deadman M., Al-Hasani H., Al-Sadi A.M., 2006. Solarization and biofumigation reduce Pythium aphanidermatum induced damping-off and enhance vegetative growth of greenhouse cucumber in Oman. Journal of Plant Pathology 88: 333-335. El-Tarabily K.A., Nassar A.H., Hardy G.E., Sivasithamparam K., 2009. Plant growth promotion and biological control of


660

16-11-2010

15:20

Pagina 660


Pythium aphanidermatum, a pathogen of cucumber, by endophytic actinomycetes. Journal of Applied Microbiology 106: 13-26. Georgakopoulos D.G., Fiddaman P., Leifert C., Malathrakis N.E., 2002. Biological control of cucumber and sugar beet damping-off caused by Pythium ultimum with bacterial and fungal antagonists. Journal of Applied Microbiology 92: 1078-1086. Hickman G.W., Michailides T.J., 1998. Control options for greenhouse cucumber damping-off disease. Journal of Vegetable Crop Production 4: 45-48. Howard R.J., Garland J.A., Seaman W.L., 1994. Diseases and Pests of Vegetable Crops in Canada: An Illustrated Compendium. Canadian Phytopathological Society and Entomological Society of Canada, Ottawa, Ontario, Canada. Hultberg M., Alsanius B., Sundin P., 2000. In vivo and in vitro interaction between Pseudomonas fluorescens and Pythium ultimum in the suppression of damping-off in tomato seedlings. Biological Control 19: 1-8. Larkin M.A., Blackshields G., Brown N.P., Chenna R., McGettigan P.A., McWilliam H., Valentin F., Wallace I.M., Wilm A., Lopez R., Thompson J.D., Gibson T.J., Higgins D.G., 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947-2948. Martin F.N., Loper J.E., 1999. Soilborne plant diseases caused by Pythium spp.: ecology, epidemiology, and prospects for biological control. Critical Reviews in Plant Sciences 18: 111-181. Montealegre J.R., Reyes R., Pérez L.M. Herrera R., Silva P., Besoain X., 2003. Selection of bioantagonistic bacteria to be used in biological control of Rhizoctonia solani in tomato. Electronic Journal of Biotechnology 6: 116-127. Moorman G.W., Kang S., Geiser D.M., Kim S.H., 2002. Identification and characterization of Pythium species associated with greenhouse floral crops in Pennsylvania. Plant Disease 86: 1227-1231. Pandey A., Palni L.M.S., Hebbar K.P., 2001. Suppression of damping-off in maize seedlings by Pseudomonas corrugata. Microbiological Research 156: 191-194. Perneel M., D’hondt L., De Maeyer K., Adiobo A., Rabaey K., Hofte M., 2008. Phenazines and biosurfactants interact in the biological control of soil-borne diseases caused by Pythium spp. Environmental Microbiology 10: 778-788.

Received January 29, 2010 Accepted March 29, 2010

Journal of Plant Pathology (2010), 92 (3), 653-660 Perneel M., Heyrman J., Adiobo A., De Maeyer K., Raaijmakers J.M., De Vos P., Hofte M., 2007. Characterization of CMR5c and CMR12a, novel fluorescent Pseudomonas strains from the cocoyam rhizosphere with biocontrol activity. Journal of Applied Microbiology 103: 1007-1020. Pieterse C., Pelt J., Wees S., Ton J., Kloostererzziel K., Kerurentjes J., Verhagen B., Knoester M., Sluis I., Bakker P., van Loon L., 2001. Rhizobacteria-mediated induced systemic resistance: triggering, signaling, and expression. European Journal of Plant Pathology 107: 51-61. Rahme L.G., Stevens F.J., Wolfort S.F., Shao J., Tompkins R.G., Ausubel F.M., 1995. Common virulence factors for bacterial pathogenicity in plants and animals. Science 268: 1899-1902 Rankin L., Paulitz T.C., 1994. Evaluation of rhizosphere bacteria for biological control of Pythium root rot of greenhouse cucumbers in hydroponic culture. Plant Disease 78: 447-451. Schmidt C.S., Agostini F., Leifert C., Killham K., Mullins C.E., 2004a. Influence of soil temperature and matric potential on sugar beet seedling colonization and suppression of Pythium damping-off by the antagonistic bacteria Pseudomonas fluorescens and Bacillus subtilis. Phytopathology 94: 351-363. Schmidt C.S., Agostini F., Leifert C., Killham K., Mullins C.E., 2004b. Influence of inoculum density of the antagonistic bacteria Pseudomonas fluorescens and Pseudomonas corrugata on sugar beet seedling colonisation and suppression of Pythium damping-off. Plant and Soil 265: 111-122. Stanghellini M.E., Phillips J.M., 1975. Pythium aphanidermatum: its occurrence and control with pyroxychlor in the Arabian desert at Abu Dhabi. Plant Disease Report 59: 559-563. Tambong J.T., Hofte M., 2001. Phenazines are involved in biocontrol of Pythium myriotylum on cocoyam by Pseudomonas aeruginos PNA1. European Journal of Plant Pathology 107: 511-521. Köhler T., van Delden C., 2009. Pseudomonas aeruginosa infections: from bench to bedside. Revue Medicale Suisse 5: 732-734. Weller D.M., Cook R.J., 1986. Increased growth of wheat by seed treatments with fluorescent pseudomonads, and implications of Pythium control. Canadian Journal of Plant Pathology 8: 328-334.