Isolation and characterization of deleterious Pseudomonas aeruginosa KC1 from rhizospheric soils and its interaction with weed seedlings

2.1 Sampling

The soil used for bacterial isolation was collected from the rhizosphere of castor plant (Ricinus communis) grown in agricultural fields of the Maliyabagh village (25°20′39″N latitude and 84°12′46″E longitude) in Rohtas district of Bihar. The sampling was carried when the prevailing atmospheric temperature was 26 °C. Randomly selected plants were uprooted carefully and the excess of soil was removed by gentle shaking and the soil adhering to the roots formed composite samples. The collected samples were placed in sterilized plastic bags and transferred to the laboratory under temperature controlled conditions.

2.2 Isolation

The soil adhering to the roots was removed by gentle agitation, serially diluted in physiological saline (0.85%, NaCl, w/v) containing quartz particles for 20 min, spread plated in triplicate on King’s B agar (KBA) medium (Atlas, 1995) and incubated at 30 °C for 48 h. Bacterial cultures were maintained on the respective slants. After incubation at 28–30 °C for 2–3 days, bacterial colonies were counted. Representative colonies were selected on the basis of distinct morphological characteristics, including pigments, colony form, elevation and margin; texture; and opacity. A predominant yellowish white colony that fluoresced under UV light was purified and maintained on nutrient agar slants at 4 °C. All the subsequent experiments were carried out after raising fresh cultures.

2.3 Phenotypic characterization

The isolate was characterized microscopically, along with different tests as described by Holt et al. (1994). All tests were performed using exponentially grown cultures, except for the spore test, which required cultures from the early stationary phase.

2.4 DNA extraction, 16S rRNA gene sequencing and phylogenetic analysis

Genomic DNA from the selected isolate was extracted from pure cultures (1.5–3 ml), previously grown in 10 ml of Luria Bertani broth (incubated at 37 °C for 24 h at 200 rpm) using Bacterial Genomic DNA Isolation Kit RKT09 (Chromous Biotech Pvt. Ltd., Bangalore, India) and visualized on 0.8% (w/v) agarose gel. The amplification of 16S rRNA gene was carried out by using a Thermal cycler (ABI 2720) in 100 μl reaction volume containing 2.5 mM each of four dNTP, 10× PCR buffer, 3U of Taq DNA polymerase, 10 ng template DNA and 400 ng each of primer (F) 5′-AGA GTR TGA TCM TYG CTW AC-3′ primer (R) 5′-CGY TAM CTT WTT ACG RCT-3′. The amplification program was set as an initial denaturation at 94 °C for 5 min., followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 2 min and a final extension at 72 °C for 5 min. The sequencing was done according to manufacturer’s protocol using Big Dye Terminator Cycle Sequencing Kit (v3.1, Applied Biosystems) and analyzed in an Applied Biosystems Analyzer. The sequence of 16S rDNA (1450 bp) was aligned by using the BLASTn program (Atschul et al., 1990) to identify the most similar sequence. The isolate showed 99% similar identity with the closest known species in the database. Henceforth, the isolate was identified as P. aeruginosa KC1 and has been submitted to the GeneBank under accession number HM195190. 16S rDNA sequences of different strains of P. aeruginosa and its phylogenetically related species and genera were downloaded from Genbank database (http://www.ncbi.nlm.nih.gov/entrez) and aligned to construct a neighbor-joining phylogenetic tree using Clustal W algorithm with the help of MEGA software version 4.1 (Tamura et al., 2007).

2.5 Estimation of HCN production

Qualitative estimation of HCN was done by using a modified method of Bakker and Schippers (1987) with and without glycine (4.4 g l⁻¹) on KBA medium. The isolates changing the color of the filter paper (treated with picric acid and sodium carbonate) from deep yellow to orange and finally to orange brown to dark brown were selected for quantification of cyanide.

The quantification of cyanide was done as per the method of Lambert et al. (1975). Briefly, the selected isolate was cultured on KB agar with and without glycine in plates. A piece of filter paper (Whatman No.1), saturated in 1 M NaOH was placed in the lid and finally plates were sealed with parafilm. After 48 h incubation at 28 °C, the filter papers were removed, extracted with 5.0 ml of 1 M NaOH and titrated with acetic acid. Cyanide extracted into NaOH was further quantified by reacting with barbituric acid and pyridine reagent and the absorbance was read at 575 nm spectrophotometrically (Systronics 119). The amount of cyanide (nmol l⁻¹ KCN) was calculated using a standard curve of KCN in NaOH.

Bacterial cultures on each plate were suspended in sterile water, extracted with trichloroacetic acid, resuspended in 0.1 M NaOH in 2% Na₂CO₃, and analyzed for protein by a modified Lowry procedure (Ohnishi and Barr, 1978).

2.6 Laboratory experiments

Pre-germinated seeds of wheat (Triticum aestivum), spiny amaranth (Amaranthus spinosus) and pigweed (Portulaca oleracea) were used for laboratory experiments. Seeds were surface sterilized by immersing them in 95% ethanol for a few seconds (3–4 s) and then in 0.2% solution of HgCl₂ for three min, followed by several rinses with sterilized distilled water to remove sterilant, as documented in other investigations as well (Russel et al., 1982). Only sterile and viable seeds were used for further experiments.

In order to study the effect of bacteria on root and shoot growth of wheat, spiny amaranth and pigweed seedlings, two sets of experiments were carried out for seed bacterization, viz. (i) Contact method and (ii) Paired plate assembly method. In the contact method, pre-germinated seeds of each species with uniform radicles were inoculated with approximately 10⁶–10⁸ cfu of a 48 h old bacterial suspension. Control seeds were without the inoculum. The treated seeds were placed on 1% agar plates, sealed with parafilm, incubated at 28 °C and after 5 days root and shoot lengths were measured.

In the paired plate assembly method, pre-germinated surface-sterilized seeds (five per plate) of each species with uniform radicle length were evenly distributed on the surface of plates containing 1% water agar. In the other plate, bacterial isolates were streaked on the KB agar. Both the plates were paired in such a way that the plate containing weed seeds was in the lower position and the bacterium in the upper one. The paired plate assemblies were sealed with parafilm and incubated at 28 °C in triplicate. After 5 days, the root and shoot lengths were measured. Results were compared with root and shoot lengths in paired plate assemblies containing a non-inoculated KB agar plate, treated as control.

2.7 Glasshouse experiments

Five surface sterilized seeds of wheat (T. aestivum), spiny amaranth (A. spinosus) and pigweed (P. oleracea) were planted into 120 mm pot containing sieved and sterilised agricultural soil (pH = 7.7 ± 0.3% carbon = 0.56 ± 0.14% nitrogen = 0.08 ± 0.4% phosphate = 0.09 ± 0.3; steam heated at 110 °C for 2.5 h for three consecutive days). Bacterial cultures were grown as described for the laboratory experiments. The five seedlings in each pot were inoculated with a 0.5 ml of suspension (10⁸ cfu) of strain. After inoculation a 1 cm layer of sterilised sand was placed on the surface of each pot to reduce evaporation and air borne contamination. The plants were grown in a temperature controlled glasshouse at 25 °C, and watered with sterile distilled water every second day. There were four replicates for each seedling treatment. After 6 weeks plants were harvested, roots were washed free of sand and soil, and shoot and root lengths were measured. The entire plant was dried in an oven at 72 °C for 72 h and dry weight was recorded. The experiment was repeated thrice.

2.8 Statistical analysis

Statistical analysis was performed with the Statistical Package for Social Sciences (SPSS 16.0) software, and treatment means were compared at 5% level of significance.

3.1 Phenotypic characterizations

The bacterial isolate formed yellowish white, mucoid colonies of 1–2.5 mm diameter with smooth, regular margins and fruity odor on KBA plates at 28 °C for 1–2 days. The colonies fluoresced under UV light. Phenotypic characterization revealed that the isolate was motile, small rod, (1.9 μm × 0.5 μm) and stained Gram negative. It grew at the temperatures ranging from 4 to 42 °C but maximum growth was recorded at 30 °C. Positive reactions were recorded for, citrate utilization, catalase activity, oxidase activity, gelatine hydrolysis and lysine decarboxylase enzyme. The bacterial isolate was also able to utilize glucose, galactose, fructose, mannitol, trehalose and glycerol as sole carbon sources. Negative reactions were recorded for indole test, Voges–Proskauer, casein hydrolysis, amylase and H₂S production and urease activity.

3.2 Genotypic characterizations

The 16S rRNA gene sequence of the isolate had 99% identity with the type strain of P. aeruginosa (Accession No. EU714901), available in the NCBI GeneBank. The phylogenetic analysis of strain KC1, showing sequence homology with P. aeruginosa, is represented as a phylogenetic tree (Fig. 1).

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Figure 1

Phylogenetic tree showing the genetic relationship of Pseudomonas aeruginosa with taxonomically similar strains, species, and genus based on 16S rDNA sequences. Genbank accession number of each isolate is given in parentheses. The bootstrap values (n = 1000) are displayed at nodes.

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3.3 HCN production

During qualitative analysis, the isolate KC1 changed the color of impregnated filter paper from yellow to reddish brown after 48 h of incubation, indicating its potent cyanogenic property. Results of indicator assay for cyanogenesis were further confirmed by quantitative estimation and it was found that the isolate produced a significant level of HCN (3.80–4.78 nmol l⁻¹) in broth cultures containing 1 × 10⁻⁷ ml⁻¹ bacterial cells, over a period of 36 h (Fig. 2). The production of HCN was increased up to 6.98 nmol l⁻¹ when glycine was added in the culture media over a period of 36 h. It is evident from Fig. 2 that cyanide production in the presence of glycine mediated medium is high and cyanide accumulation in the culture was highest in the early exponential phase followed by early stationary phase with gradual decline in late stationary phase.

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Figure 2

Time course of cyanide production by Pseudomonas aeruginosa strain KC1 (HM195190). Data are the mean ±SD of 3 replicates and the experiment was repeated on two independent occasions. The bars indicate standard error of mean.

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3.4 Laboratory experiments

In laboratory experiments, the growth reduction in root length (19.26–89.3%) and in shoot length (31.17–75.85%) was more in all the three seedlings in the paired plate assemblies as compared to the contact method (Figs. 3 and 4). The maximum growth inhibition was seen in pigweed followed by spiny amaranth and wheat. Results of in vitro tests marked greater growth inhibition by gaseous metabolites released in paired plate assemblies than by other metabolites on weeds. The remarkable feature of the in vitro test was that wheat seedlings were found to be less influenced than the weed seedlings.

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Figure 3

Percentage reduction in root length and shoot length of spiny amaranth, pigweed and wheat seedlings in paired plate assemblies (n = 3).

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Figure 4

Percentage reduction in root length and shoot length of spiny amaranth, pigweed and wheat seedlings in contact seedling assay (n = 3).

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3.5 Glasshouse experiments

The glass house experiments witnessed root growth reduction up to 51.6% for pigweed seedlings, followed by spiny amaranth (42.5%) and wheat seedlings (12.72%). KC1 inoculated spiny amaranth seedlings showed the highest reduction in shoot lengths (43.5%) as compared to control (Fig. 5). Highest biomass reduction was recorded for pigweed seedlings (77.4%), while wheat seedlings recorded lowest biomass reduction (58.8%) as compared to untreated seedlings. Glasshouse experiment results were in accordance with laboratory experiment results, where weed seedlings growth parameters were reduced to a higher extent than crop seedlings.

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Figure 5

Percentage reduction in growth parameters of spiny amaranth, pigweed and wheat seedlings in glasshouse experiments (n = 3).

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