18 clinical and environmental isolates of Stenotrophomonas maltophilia were used (Table 1). Burkholderia cenocepacia (K 56-2) and Escherichia coli (DH5α) were used as positive and negative controls respectively. The CDC S. maltophilia isolates were provided by Dr. Judith Noble-Wang while the ATCC isolates were purchased from the American Type Culture Collection, Manassas, VA, USA.
ATCC isolates. These were isolated from different environments. K 279a was isolated from the blood culture of a patient suffering from cancer (Crossman et al., 2008). D457 was isolated from bronchial aspirate, ATCC 17666 from contaminated tissue culture, ATCC 700475 from cerebrospinal fluid from an HIV-seropositive Rwandan refugee with primary meningoencephalitis, and ATCC 13637 from Oropharyngeal region of patient with mouth cancer (www.atcc.org). ATCC 13270 was isolated from soil environment (©Global Catalogue of Microorganisms).
The fungal pathogen used in this study was Leptosphaeria maculans, the causal agent of blackleg in canola. The isolate of L. maculans used in this study belongs to the pathogenicity group (PG) 4, a subdivision of pathotype A (the aggressive and highly virulent cause of stem cankers in canola). Westar, a commercially planted canola cultivar which is susceptible to blackleg was used in this study. Seeds of Westar and inoculum of L. maculans were kindly provided by Dr. Luis Del Rio Mendoza of the Plant Pathology Department of North Dakota State University.
V8 Medium and others. Agar was prepared according to Suckstoff and Berg (2003) [20g agar, 1L distilled water, pH = 6] with the following modifications. Agar was made up of 10g agar (Difco) in 1L of distilled water (1%) and autoclaved. 10ml of agar were dispensed into 15 ml test tubes and stored in a cool dry place until used for canola plant growth. L. maculans was grown on V8 medium (200ml V8 juice, 3g CaCO3, 15g Agar and 800ml tap water). Conidia were harvested after 3 days and suspended in sterile distilled water. The suspension was kept on ice until use. Bacterial cultures. Freezer stocks of bacterial isolates were streaked on Luria Bertani (LB) plates and incubated at 37ºC overnight. A colony from each isolate was inoculated in 5 ml LB broth and incubated at 37 ºC, and 230 rpm for 16 hours. After incubating, bacterial cultures were centrifuged at 5000 x g for 5 min. The supernatant was discarded and the pellets/cells were suspended in 5 ml distilled water to wash and centrifuged at 5000 x g for 5 min. Cells were then suspended in 5ml distilled water.
Germination and treatment of canola seedlings. Canola seeds were placed in a 9 cm sterile glass petri dish with layers of sterile tissue paper soaked with sterile distilled water for 4 days at 25 ºC in the dark for germination. For the non-stress experiment, 10 canola seedlings were placed in sterile petri plate for each isolate. The 5 ml bacterial suspensions, at 106cfu/ml as described above were thoroughly vortexed and added to the seedlings in the various petri dishes. All seedlings were carefully submerged in the bacterial suspension for 4 hours. Thereafter, the seedlings were washed in sterile distilled water and inoculated on agar in test tubes. For the stress experiment, canola seedlings were primed by transferring seedlings into a fresh sterile 9 cm glass petri dish with layers of sterile tissue paper soaked with moderate salinity rate of 6 decisiemens per meter (0.33%) NaCl (www.canolacouncil.org) for 24 hour at 25 ºC. The primed seedlings were rinsed in sterile distilled water and thereafter treated in the same way as the non-stress treatment described above. Growth parameters (number of root branches, root and stem length) were recorded after one week of incubation at 25 ºC and 16/8 photoperiod conditions as described for strawberry by Suckstorff and Berg (2003). Using a pair of forceps, canola plants were carefully removed from test tubes onto a clean paper towel. Root and stem lengths were measured by the use of a ruler and a pair of calipers. Each bacterial isolate was tested in triplicates.
Based on preliminary results from one run of non-stress condition, three isolates of S. malltophilia, namely, ATCC 13637, ATCC 13270 and CDC 2007-07-01 and the isolates, K279a and D457 which have whole genome sequences were assessed further for their suitability to protect against the bacterium Burkholderia cenocepacia and the fungus L. maculans using the cv. Westar of canola as a host plant. To accomplish this, four day old canola seedlings were inoculated with 5 ml of each of the five S. maltophila isolates for two hours after which 5 mls of either B. cenocepacia or L. maculans was applied for an additional two hours. This experiment was carried out under three separate conditions: 1.) canola seedlings were exposed to B. cenocepacia and L. maculans for four hours; 2.) canola seedlings were inoculated with 5 mls S. maltophilia initially and subsequently exposed to 5 ml of Burkholderia cenocepacia or L. maculans and 3.) canola seedlings were initially inoculated with 5 ml of Burkholderia cenocepacia or L. maculans and subsequently exposed to 5 ml of S. maltophila isolates for 2 hours. For each S. maltophilia isolate, 12 tubes were used and these were replicated three times. Here again, stem and root length as well as the number of root branches of canola seedlings were the parameters recorded.
Freezer stocks of bacterial isolates were streaked on Luria Bertani (LB) plates and incubated at 37ºC overnight. A single colony from each isolate plate was inoculated into a 50µl PCR reaction master mix that contained reagents as follows: 5X Go Taq Buffer, 10 mM dNTP, 25 mM MgCl2, XgyrB1F - 51- ACG AGT ACA ACC CGG ACA A - 31, a threefold XgyrB1R - 51- CCC ATC ARG GTG CTG AAG AT - 31 (Yamamoto et al., 2000), GoTaq Polymerase and distilled water). The reaction was run for 40 cycles under the following thermocycling conditions: denature at 95 ºC for 30 seconds, annealing at 55 ºC for 1 minute, and extension at 72 ºC for 2 minutes. Amplified PCR products were confirmed by running an agarose gel at 100 volts for 45 minutes. The gel images were visualized by Ultraviolet light using flourchem software (FluorChem™ Q System), Bio-Techne, USA. PCR products were purified using Exosap and incubated at 37oC for 15 minutes and 80oC minutes for 15 minutes and held at 4oC. PCR products were diluted and sent to Macrogen USA for sequencing. The products were sequenced by 3730xl DNA analyzer using the Sanger sequencing analysis (capillary gel electrophoresis).
Nucleotide sequences were analyzed using Geneious bioinformatics software 6.1 version (Biomatters Limited, Auckland, New Zealand). The sequences were edited to generate uniform lengths for each isolate for both forward and reverse sequences using the gyrBF and gyrBR primers. Other S. maltophilia and closely related genome sequences were searched in National Center for Biotechnology Information (NCBI). The K279a genome was used as a reference sequence. Concatenated isolate sequences were aligned to the reference sequence and edited to obtain the correct sequence length of 725 base pairs. The reference sequence was deleted after alignment. The forward and reverse sequences were aligned to generate each isolate sequence in whole. Related genome sequences were also concatenated to 725 base pairs. Sequences were aligned into a file. Cluster analyses (neighbor-joining) were performed.
Data analyses were done using the general linear model procedure (Proc GLM) in statistical analysis software (SAS) version 9.3. The data were analyzed as one-way Analysis of Variance (ANOVA) with the randomized complete block design (RCBD). All inferences were conducted at 5% significance level. Treatments means were compared using the Bonferroni (Dunn)’s and Tukey’s tests at P ≤ 0.05. Before analysis, the difference between the median for each isolate and the “no bacteria” control was found and expressed as percentage change for the canola growth promotion experiment. For experiments involving investigation of the protective potential of S. maltophilia against L. maculans and K 56-2, the raw data was analyzed without any prior manipulations.