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. 2017 Apr 11;7(1):27. doi: 10.1007/s13205-017-0602-3

Crop specific plant growth promoting effects of ACCd enzyme and siderophore producing and cynogenic fluorescent Pseudomonas

Priyanka 1, Toshy Agrawal 2, Anil S Kotasthane 2,✉, Ashok Kosharia 1, Renu Kushwah 2, Najam Waris Zaidi 3, U S Singh 3
PMCID: PMC5388656  PMID: 28401463

Abstract

Fluorescent Pseudomonas, aerobic, Gram-negative bacteria possess many traits that make them well suited as biocontrol and growth promoting agents. Our study revealed that isolates vary in mechanisms involved in the antagonist interactions against pathogen and growth stimulatory effects on host plant. Most of the potential antagonistic fluorescent Pseudomonas identified were avid iron chelators (P233, P201, 176, P76 and, P76). Wide variation in ACCd enzyme production was observed. ACCd enzyme assay tested P141 > P247 > P126, as potential ACCd enzyme producer. Cynogenic fluorescent Pseudomonas isolates P76 and P124 exerted strong inhibitory against S. rolfsii. However, another cynogenic fluorescent Pseudomonas P179 had no influence against R solani and S. rolfsii which remains unexplained. Noticeable crop specific plant growth stimulation exerted by different fluorescent Pseudomonas was observed on wheat (P124), chickpea (P72), lathyrus (P85, P216), greengram (P11), blackgram (P99, P233); bottlegourd (P248, P167); rice (P176, P247).

Keywords: ACCd enzyme, Confrontation assay, Fluorescent Pseudomonas, HCN, PGPR, Siderophore

Introduction

Aerobic gram negative Fluorescent Pseudomonas spp. have emerged as the largest and potentially most promising group of plant growth promoting rhizobacteria involved in the bio-control of plant diseases (Weller et al. 2002; Fravel 2005). A large number of secondary metabolites, growth hormones, antibiotics and chelating compounds such as siderophores (Choudhary et al. 2009; Beneduzi et al. 2012) are known to be released by these fluorescent pseudomonads. They maintain soil health by employing a wide variety of mechanisms including nitrogen fixation, enhanced solubilization of phosphate and phytohormone production (such as auxin and cytokinin). Plant growth-promoting rhizobacteria (PGPR) competitively colonize plant roots and stimulate plant growth and/or reduce the incidence of plant disease. Fluorescent Pseudomonas applied to seed or soil provides excellent control against plant pathogens (De La Fuente et al. 2006; Lagzian et al. 2013). Pseudomonas spp. produce an arsenal of antimicrobials (including hydrogen cyanide, HCN), pyoluteorin, phenazines, pyrrolnitrin, siderophores, cyclic lipopeptides and 2,4-diacetylphloroglucinol (DAPG) (Thomashow and Weller 1991; Weller 2007). This is considered as an indirect strategy to promote plant growth as well as the ability to induce systemic resistance in plants (Santoyo et al. 2012; Glick 2014). In this study, we evaluated the fluorescent isolates for siderophore and HCN production. PGPR and their interactions with plants are exploited commercially (Podile and Kishore 2007) and hold great promise for sustainable agriculture. Applications of these associations were investigated in Wheat (Triticum aestivum), Chickpea (Cicer arientinum), Lathyrus (Lathyrus sativus), Greengram (Vigna radiata), Blackgram (Vigna mungo), Bottlegourd (Lagenaria siceraria) and Rice (Oryza sativa) through seed bacterization.

Materials and methods

Microorganisms and culture conditions

The experimental material consisted of purified twenty-four isolates (Table 1) of fluorescent Pseudomonas spp. from soils (rhizospheric and non-rhizospheric) of different geographical locations of Chhattisgarh. Isolation of fluorescent pseudomonads was done by adopting serial dilution method on King’s B (KB) medium. Isolates were characterized on the basis of biochemical tests as per the procedures outlined in Bergey’s Manual of Systematic Bacteriology (Sneath 1986). Glycerol stock of isolates were maintained in the culture collections of the Department of Plant Molecular Biology and Biotechnology, Indira Gandhi Krishi Vishwavidyalaya, Raipur, Chhattisgarh, India and revived on KMB slants when required. Fungal pathogens Rhizoctonia solani and Sclerotium rolfsii were isolated from naturally infected sick soils of rice and chickpea and maintained on PDA slants.

Table 1.

Fluorescent Pseudomonas spp. isolates used in the present study

S. no. Isolates Origin/location
1 P5 Fluoresecent Pseudomonas Fenugreek soil, IGKV Horticulture, Raipur
2 P6 Pseudomonas putida Cashew tree soil, IGKV Horticulture, Raipur
3 P11 Pseudomonas putida Brinjal plant soil, IGKV Horticulture, Raipur
4 P67 Pseudomonas fluorescens Charama
5 P72 Pseudomonas putida Chhati
6 P76 Pseudomonas putida Mustard field soil, chhati
7 P85 Pseudomonas aeruginosa Cow dung soil, Darba
8 P99 Fluoresecent Pseudomonas Jagtara
9 P124 Fluoresecent Pseudomonas Kanker forest-3
10 P126 Pseudomonas putida Kanker forest soil
11 P129 Pseudomonas putida Kanker forest soil
12 P141 Pseudomonas putida Kirda
13 P143 Pseudomonas putida Kodebor
14 P151 Pseudomonas putida Kurud
15 P161 Pseudomonas putida VIP nursery soil, Raipur
16 P167 Pseudomonas putida Purur
17 P176 Pseudomonas putida Rice field, fallow land soil, Raipur
18 P179 Fluoresecent Pseudomonas Rice-lathyrus field soil, Raipur
19 P201 Pseudomonas putida VIP road, Raipur
20 P205 Pseudomonas putida Babool tree soil, VIP road, Raipur
21 P216 Fluoresecent Pseudomonas Bamboo tree soil, VIP road, Raipur
22 P233 Fluoresecent Pseudomonas Badi, Tekabheta
23 P247 Fluoresecent Pseudomonas Laichopi, Raweli
24 P248 Pseudomonas aeruginosa Maize, Parsada
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Siderophore production

Qualitative and quantitative estimation of siderophore production was done by CAS assay (Schwyn and Neilands 1987). Specific tests were carried out for the identification of hydroxamate and catecholate types of siderophores following the standard methods (Arnow 1937). For qualitative estimation, chrome azurol S solution was prepared and added to melted King’s B agar medium in the ratio 1:15. Spot inoculation at the centre of the CAS plate was done from actively growing cultures of Pseudomonas. Colonies exhibiting an orange halo after 3 days incubation (28 ± 2 °C) were considered positive for siderophore production and the diameter of the orange halo was measured. Simultaneously succinate medium (broth) was also used for qualitative estimation of siderophore production on the basis of fluorescence observed after 3 days incubation (28 ± 2 °C).

Quantitative spectrophotometric assay for siderophore production (liquid assay)

For siderophore quantification, actively growing cultures of Pseudomonas was inoculated to 20 mL King’s B broth in 100 mL flasks and incubated for 3 days at 28 ± 2 °C. The bacterial cells were removed by centrifugation at 3000 rpm for 5 min. 0.5 mL of the culture supernatant was then mixed with 0.5 mL of CAS solution and 10 µl shuttling reagent (sulfosalicyclic acid). After 20 min of incubation, the absorbance of colour obtained was determined using spectrophotometer at 630 nm. Un-inoculated King’s B broth was used as blank while reference solution was prepared by adding CAS dye and shuttle solution to King’s B and absorbance was recorded. Values of siderophore released in King’s B was expressed as percent siderophore units and calculated using the formula: (A r − A s)/A r × 100; where A r is the absorbance of reference solution and A s is the absorbance of samples.

Hydroxyquinoline mediated siderophore test

Isolates were inoculated on King’s B medium supplemented with a strong iron chelater 8- Hydroxyquinoline (50 mg/L) (De Brito et al. 1995) and incubated at 28 ± 2 °C for 48–72 h. Only those bacteria that produce a more avid iron chelator will grow.

Arnow’s assay

Arnow’s assay was used for qualitative determination of catechol type of siderophore. Actively growing cultures of Pseudomonas were inoculated to 20 mL King’s B broth in 50 mL tubes and incubated for 3 days at 28 ± 2 °C. The bacterial cells were removed by centrifugation at 3000 rpm for 5 min. Three milliliter of the culture supernatant was then mixed with 0.3 mL of 5 N HCl solution, 1.5 mL of Arnow’s reagent (10 g NaNO2, 10 g Na2MoO4·2H2O dissolved in 50 mL distilled water) and 0.3 mL of 10 N NaOH. After 10 min the presence or absence of pink colour was observed and noted.

Tetrazolium test

This test is based on the capacity of hydroxamic acid to reduce tetrazolium salt by hydrolysis of hydroxymate groups using a strong alkali. The reduction and release of alkali shows red colour to a pinch of tetrazolium salt when 1–2 drops of 2 N NaOH and 0.1 mL of test sample is added. Instant appearance of a deep red colour indicated the presence of hydroxamate siderophore.

FeCl3 test

One milliliter of the culture supernatant was mixed with freshly prepared 0.5 mL of 2% aqueous FeCl3 and observed for the presence and absence of deep red colour.

Confrontation assay

Fluorescent Pseudomonas isolates were multiplied on King’s B broth and incubated for two days at 28 °C till the fluorescent pigment appeared in the broth. Petri-plates containing pre-sterilized potato dextrose agar (PDA) medium was inoculated with plant pathogenic fungi Sclerotium rolfsii or Rhizoctonia solani (in the center) and incubated at 25 °C for three days till the fungus completely covered the entire plate. Bipartite interactions were performed following a simple confrontation assay technique as proposed by Kotasthane et al. (unpublished results), wherein edge of glass funnel was deployed for deposition of bio-agent surrounding pre-inoculated fungal pathogen.

HCN production

The production of HCN was estimated by method of (Wei et al. 1991). The cultures were grown on KM plates supplemented with 4.4 g/L glycine as a precursor and the filter paper strips soaked in saturated picric acid solution were exposed to the growing Pseudomonas isolates. The plates were incubated for 7 days at 28 ± 2 °C and observations were recorded as change in the colour of filter paper to brown as positive indicator for HCN production.

Quantitative estimation of ACC deaminase activity

ACC deaminase activity was determined by measuring the production of α-ketobutyrate and ammonia generated by the cleavage of ACC by ACC deaminase (Honma and Shimomura 1978; Penrose and Glick 2003). Pseudomonas isolates were grown in 5 mL of trypticase soya broth at 28 °C until they reached stationary phase. The cells were collected by centrifugation, washed twice with 0.1 M Tris–HCl (pH 7.5), re-suspended in 2 mL of modified DF minimal medium supplemented with 2 mM final concentration of ACC. Incubated at 28 °C with shaking for another 36–72 h. The induced bacterial cells were harvested by centrifugation at 3000g for 5 min, washed twice with 0.1 M Tris–HCl (pH 7.5), and re-suspended in 200 μL of 0.1 M Tris–HCl (pH 8.5). The cells were labilized by adding 5% toluene (v/v) and then vortexed at the highest speed for 30 s. Fifty microlitre of labilized cell suspension was incubated with 5 μL of 0.3 M ACC in an microcentrifuge tube at 28 ± 2 °C for 30 min. The negative control for this assay consisted of 50 μL of labilized cell suspension without ACC, while the blank consisted of 50 μL of 0.1 M Tris–HCl (pH 8.5) with 5 μL of 0.3 M ACC. The samples were then mixed thoroughly with 500 μL of 0.56 N HCl by vortexing and the cell debris was removed by centrifugation at 12,000g for 5 min. A 500 μL aliquot of the supernatant was transferred to a glass test tube, mixed with 400 μL of 0.56 N HCl and 150 μL of DNF solution (0.1 g 2,4-dinitrophenylhydrazine dissolved in 100 mL of 2 N HCl); and incubated at 28 °C for 30 min. One milliliter of 2 N NaOH was added to the sample before the absorbance at 540 nm was measured. The concentration of α-ketobutyrate in each sample was determined by comparison with a standard curve generated as follows: A stock solution of 100 mmol/L α-ketobutyrate (Sigma-Aldrich Co., Mumbai, India) was prepared in 0.1 mol/L Tris–HCl (pH 8.5) and stored at 4 °C. Just prior to use stock solution is diluted with same buffer to make a 10 mmol/L solution from which a standard concentration curve is generated. Each 500 μL α-ketobutyrate solutions of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 µM (prepared from stock solution) were mixed, respectively, with 400 μL of 0.56 N HCl and 150 μL DNF solution. One milliliter of 2 N NaOH was added and the absorbance at 540 nm was determined as described above. The values for absorbance versus α-ketobutyrate concentration (µM) were used to prepare a standard curve.

Estimation of PGPR activity

The cultures of fluorescent Pseudomonas spp. were inoculated in 100 mL conical flask containing 25 mL King’s B broth and incubated at 28 ± 2 °C for 48 h. For field trials seed bacterization was done with stock cultures (used for confrontation assays) of selected fluorescent Pseudomonas isolate. Slurry for seed bacterization was prepared @ 5 mL of bacterial culture +3 g of talcum powder/kg of seed. Care was taken for uniform coating of all the seeds, which were dried in shade. Seeds of each of Wheat (GW-272), Chickpea, Lathyrus (KH-014), Greengram (puspa vishal), Blackgram (T-U-94-2), Bottlegourd (Lagenaria siceraria), Rice (swarna) were planted in pots containing soil mixed with sand and compost in the ratio of 3:1:1. Plants growth were measured for root and shoot length.

Statistical analysis

The design of all phenotype assays and plant growth experiments was a completely randomized block with three replications per treatment. Data of all biochemical tests and plant growth experiments were subsequently analyzed by ANOVA followed by Duncan’s test using WASP (Web Agri Stat Package) software (http://icargoa.res.in/wasp/index.php). Critical difference at 0.05 level of significance was calculated for the observed values along with average and standard deviation. Duncan’s test controls the Type I comparison wise error rate and as per Duncan’s grouping, means with the same letter are not significantly different. Duncan’s test can be used irrespective of whether F is significant or not and compares all possible pairs of treatment means.

Results

Isolates were characterized on the basis of biochemical tests as per the procedures outlined in Bergey’s Manual of Systematic Bacteriology (Sneath 1986) and tests reported by (Blazevic et al. 1973) that specifically differentiate the fluorescent Pseudomonas into P. aeruginosa, P. putida and P. fluorescence (Table 1).

Qualitative and quantitative assay for siderophore production

Twenty-four isolates of fluorescent Pseudomonas were screened by different siderophore assay. All fluorescent Pseudomonas isolates produced siderophore on iron deficient succinate medium with variable chromogenic response and were also +ve for siderophore production on CAS agar plate assay and hydroxamate type of siderophore assay (tetrazolium and FeCl3 test). Siderophores are also viewed as contingent antibiotics, the selective advantage is easily verified by placing a bacterial culture in a medium containing a strong iron chelator 8-hydroxyquinoline (50 mg/L); only those bacteria that produce a more avid iron chelator grow. All fluorescent Pseudomonas isolates except P5, P11, P67, P72, P99, P126, P161, P167, P179 and P205 were +ve in HQ test. Fluorescent pseudomonas isolates P6, P85 and P233 produce a more avid iron chelator and produced high % siderophore units in quantitative test. Arnow’s assay (test for catechol type of siderophore) tested +ve for 22 isolates except P85 and P216. Fluorescent pseudomonas isolates P233 > P176 > P141 > P76 > P201 were all avid iron chelators and tested +ve for all siderophore tests (Fig. 1; Table 2).

Fig. 1.

Fig. 1

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Percent Siderophore units and ACCd enzyme producing ability of different fluorescent Pseudomonas

Table 2.

Siderophore, ACCd enzyme, HCN producing ability and inhibitory effect of fluorescent Pseudomonas isolates against R solani and S. rolfsii

Treatment Siderophore ACC (α ketobutyrate/mg protein/h) HCN Confrontation assay (% inhibition)
% Siderophore units HQ Test R. solani S. rolfsii
1 P5 33.54bcdefg ± 10.21 – 1.48 ± 0.23 – 38.90abc ± 11.10 19.45ef ± 2.75
2 P6 45.1abcde ± 1.77 *** 2.9 ± 0.15 – 51.10ab ± 1.10 35.55abcde ± 6.65
3 P11 28.67cdefgh ± 11.97 – 13.11 ± 1.29 – 34.45abc ± 12.75 27.25bcdef ± 9.45
4 P67 9.48gh ± 6.15 – 10.51 ± 0.06 – 37.25abc ± 9.45 27.80bcdef ± 0.05
5 P72 17.40fgh ± 10.74 – 4.42 ± 0.07 – 42.20abc ± 3.30 26.10cdef ± 6.60
6 P76 54.79ab ± 1.46 * 16.24 ± 0.05 +++ 49.45ab ± 7.25 49.40a ± 5.00
7 P85 51.15abcd ± 14.48 *** nd – 40.00abc ± 6.70 43.35abc ± 4.45
8 P99 15.93gh ± 5.93 – 19.75 ± 0.21 – 29.45bcd ± 7.25 33.85abcde ± 8.35
9 P124 11.98gh ± 3.65 * 20.37 ± 0.25 +++ 43.85abc ± 11.65 35.00abcde ± 7.20
10 P126 46.77abcde ± 0.10 – 31.05 ± 0.046 – 36.10abc ± 8.30 17.21ef ± 15.0
11 P129 49.9abcd ± 3.23 * 23.94 ± 0.32 – 42.20abc ± 2.20 8.33f ± 2.78
12 P141 56.46ab ± 0.21 * 40.87 ± 0.08 – 40.00abc ± 6.70 23.30cdef ± 7.80
13 P143 45abcde ± 5.00 * 16.27 ± 0.14 – 42.75abc ± 1.65 27.80bcdef ± 11.10
14 P151 45abcde ± 5.00 * 28.64 ± 0.26 – 50.00ab ± 0.05 21.65def ± 10.55
15 P161 12.71gh ± 6.05 – 25.61 ± 0.05 – 22.20cd ± 0.05 10.00f ± 1.10
16 P167 7.92h ± 4.49 – nd – 35.00abc ± 5.00 41.65abcd ± 2.75
17 P176 61.36a ± 1.98 * 25.89 ± 0.21 – 52.75ab ± 8.35 32.20abcde ± 7.70
18 P179 21.56efgh ± 12.81 – 21.61 ± 0.27 +++ 4.44d ± 2.22 10.01f ± 7.79
19 P201 52.82abc ± 12.82 * 25.02 ± 0.37 – 55.00a ± 5.00 25.00cdef ± 7.20
20 P205 17.39fgh ± 10.73 – 28.56 ± 0.02 – 4.44d ± 2.22 22.20def ± 0.05
21 P216 41.98abcdef ± 1.35 ** nd – 44.45abc ± 5.55 47.20ab ± 8.30
22 P233 62.92a ± 0.42 *** 26.75 ± 0.05 – 45.55abc ± 12.55 32.80abcde ± 6.10
23 P247 33.34bcdefg ± 16.67 ** 32.61 ± 1.22 – 41.65abc ± 8.35 22.80def ± 5.00
24 P248 26.875defgh ± 16.88 ** 23.55 ± 0.272 – 36.65abc ± 25.55 47.20ab ± 2.80
Max. 62.915a ± 0.42 55.00a ± 5.00 49.40a ± 5.00
Min. 9.48gh ± 6.15 4.44d ± 2.22 8.33f ± 2.78
CD (0.01%) 34.198 – 27.70
CD (0.05%) 25.230 25.39 20.45
CV 4.247 32.08 34.60
Fcal 2.17 2.03
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Values are average of three replications; values after ± represents standard error; CV, coefficient of variance; CD, critical difference; Values are significant at 1 and 5% levels; As per Duncan’s grouping means with the same letter are not significantly different; HQ hydroxyquinoline test; *** Luxuriant/high growth; ** medium growth; * low growth; –, no growth; nd, not determined

Screening of 1-aminocyclopropane-1-carboxylic acid deaminase (ACC Deaminase) containing fluorescent Pseudomonas isolates

Test for ACC deaminase activity revealed wide variation in quantified amount of α-ketobutyrate produced by different fluorescent Pseudomonas isolates (Fig. 1; Table 2) which allowed us to classify them as high, medium and low ACC deaminase enzyme producing groups. Group of fluorescent Pseudomonas isolates produced µmol α ketobutyrate/mg protein/h in the range of 40.87 ± 0.08 to 25.02 ± 0.37 and 23.94 ± 0.32 to 10.51 ± 0.06 were placed in high and medium ACC deaminase producing groups. Isolate P141 was the highest enzyme producer followed by P247 and P126 (Fig. 1; Table 2). Three isolates P216, P5, P72 were identified as low ACC deaminase producers.

Hydrogen cyanide production by isolates of fluorescent Pseudomonas spp.

Out of 24 Pseudomonas isolates only three isolates P67, P124 and P179 turned the strip brown confirming +ve for HCN production.

In vitro antagonistic activity of fluorescent Pseudomonasisolates againstR. solaniandS. rolfsii

Confrontation assays were performed to assess antagonistic potential of 24 fluorescent Pseudomonas isolates in vitro against R. solani and S. rolfsii (Fig. 2; Table 2). Differences in growth inhibitions of R. solani and S. rolfsii ranged from 4.44 to 55 and 8.325 to 49.4%, respectively. Confrontation assays revealed isolate P76 exerting antagonism against both R solani and S rolfsii, where as fluorescent Pseudomonas isolates (P201, 176, P6, P151 and P248, P216, P85, P167) exerted pathogen specific antagonism against R. solani and S. rolfsii, respectively. Isolate P205 and P129 showed the lowest inhibitory effect on R. solani and S. rolfsii, respectively. Variation in quantitative inhibitory data was significant at 0.01 and 0.05% level for S. rolfsii and 0.05% for R. solani.

Fig. 2.

Fig. 2

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Inhibitory effect of fluorescent Pseudomonas isolates against R solani and S. rolfsii

Correlation between siderophore production and in vitro antagonistic activity of fluorescent Pseudomonas isolates against R. solani and S. rolfsii

Some correlation between inhibitory effects and the ability to produce siderophore (quantitative assay) was observed. All potential fluorescent pseudomonas isolates identified following confrontation assays (P201, 176, P6, P151, P76 and P248, P216, P85, P167, P76 effective against R. solani and S. rolfsii, respectively) were high siderophore producers except P167 and P248. Fluorescent pseudomonas isolates P6, P85 and P233 produce a more avid iron chelator and produced high % siderophore units in quantitative tests of which P6 and P233 exhibited antagonistic activity against R solani. Similarly some correlation was also observed between antagonism and ability to produce HCN by fluorescent Pseudomonas. Isolates P76, P124, 179 were cynogenic of which P76 and P124 exerted strong inhibitory effects during confrontation assays against S. rolfsii, whereas P179 expressed no influence against these two soil borne fungal pathogens which remains unexplained.

Plant growth promoting response of rice, wheat, greengram, blackgram, lathyrus, chickpea, bottlegourd following seed bacterization with fluorescent Pseudomonas isolates

From pot experiments, plant growth-attributing characters such as root and shoot lengths were recorded for seven crops (rice, wheat, bottlegourd, lathyrus, chickpea, greengram, blackgram). Significantly greater amount of root and coleoptile growth stimulation was recorded in the seedlings of seven different crops derived following seed bacterization with 24 different fluorescent Pseudomonas isolates as compared to untreated control (Figs. 3, 4; Tables 3, 4). Effects amongst fluorescent Pseudomonas treatments to stimulate plant growth also varied. Plants of seven crop derived from bacterized seed had more stimulatory effects on coleoptile elongation than root length. Fluorescent Pseudomonas isolates P176 stimulated coleoptiles elongation on all seven crops tested. Stimulation of coleoptile elongation by fluorescent Pseudomonas isolates was more predominant in wheat followed by chickpea, lathyrus, greengram, blackgram, bottlegourd and rice.

Fig. 3.

Fig. 3

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Plant growth promoting expression of bottle gourd seedling following seed bacterization with fluorescent Pseudomonas isolate

Fig. 4.

Fig. 4

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Plant growth promoting expression of bottle gourd seedling following seed bacterization with fluorescent Pseudomonas isolate black gram, chick pea and lathyrus seedling following seed bacterization with fluorescent Pseudomonas isolate

Table 3.

Plant growth promoting response of rice, wheat and bottle gourd seedlings following seed bacterization with fluorescent Pseudomonas isolates

S. no. Treatment Rice Wheat Bottlegourd
Root length (cm) Shoot length (cm) Root length (cm) Shoot length (cm) Root length (cm) Shoot length (cm)
1 Control 9.325efg ± 1.135 20.3hijk ± 1.498 30.2bcdefg ± 0.742 31.73jk ± 0.4107 43.9efg ± 12.27 17.6efgh ± 1.254
2 P5 12.2abcdef ± 1.301 20.775ghijk ± 0.771 28.42defgh ± 1.369 33.04ij ± 0.6838 45.7defg ± 6.74 14.33fgh ± 0.794
3 P6 10.25cdefg ± 0.487 21.65fghij ± 1.713 27.68efgh ± 1.685 30.77k ± 1.0157 42.83efgh ± 0.82 16.4efgh ± 0.589
4 P11 8.5fg ± 0.951 21.225fghijk ± 0.684 31.86bcdef ± 1.999 33.78hi ± 0.6555 44.5efg ± 6.01 11.1fgh ± 0.733
5 P67 14.5ab ± 2.480 23.225abcdefg ± 0.390 27.24fgh ± 1.259 36.04efg ± 0.5142 54cdefg ± 8.01 12.75fgh ± 0.777
6 P72 10defg ± 0.970 19.25jk ± 0.811 27.56fgh ± 1.755 36.35defg ± 0.9332 83.3b ± 1.15 15fgh ± 1.780
7 P76 8.525fg ± 0.581 21.85efghi ± 0.835 30.66bcdefg ± 0.897 36.75cdef ± 0.8353 70.3bc ± 2.77 36.73bcd ± 5.981
8 P85 12.175abcdef ± 1.035 24.45abcd ± 0.362 27.19gh ± 1.119 37.25bcde ± 0.8825 41.83fgh ± 12.44 40.68bc ± 7.366
9 P99 9.2efg ± 0.426 23.6abcdef ± 0.942 27.69efgh ± 1.102 38.84ab ± 0.4621 70.8bc ± 2.64 56.73a ± 5.496
10 P124 10.3cdefg ± 2.358 22.775abcdefgh ± 0.536 36.52a ± 2.766 38.18abcd ± 1.1289 87.7b ± 7.23 58.93a ± 9.433
11 P129 10.825bcdefg ± 0.409 20.135ijk ± 0.613 28.12efgh ± 1.907 38.55abc ± 0.5677 69.88bc ± 9.66 24.53def ± 4.514
12 P126 9.775efg ± 0.559 22.35bcdefghi ± 0.598 30.07bcdefg ± 1.316 36.89bcdef ± 0.8538 64.95bcdef ± 8.54 50.65ab ± 12.99
13 P141 7.15g ± 0.891 22.15cdefghi ± 0.366 26.56gh ± 1.240 39.78a ± 0.9069 67.83bcd ± 11.11 11.4fgh ± 1.023
14 P143 7.625g ± 1.004 19.025k ± 1.424 31.14bcdefg ± 1.775 35.9efg ± 0.2994 59.18cdefg ± 4.64 37.4bcd ± 3.447
15 P151 13.625abcd ± 2.755 24.725ab ± 0.201 30.86bcdefg ± 1.150 35.58efgh ± 0.6486 75.63bc ± 6.82 20.85efg ± 0.296
16 P161 12.5abcde ± 0.631 23.325abcdefg ± 0.239 32.28abcde ± 1.513 35.83efg ± 0.4839 42.4efgh ± 9.24 12.58fgh ± 0.862
17 P167 9.8defg ± 0.715 23.15abcdefg ± 0.744 24.38h ± 1.906 34.45ghi ± 0.4381 73.5bc ± 16.98 63.75a ± 5.883
18 P176 10defg ± 1.772 24.95a ± 0.833 34.51ab ± 1.602 38.27abcd ± 0.6350 65.6bcde ± 10.00 37.5bcd ± 11.76
19 P179 7.8g ± 0.356 22defghi ± 0.082 28.61defgh ± 1.749 36.09efg ± 0.3703 20.33h ± 1.13 3.95h ± 0.466
20 P201 8.625fg ± 2.138 21.4fghijk ± 1.485 29.44cdefg ± 1.610 38.59abc ± 0.9865 52.88cdefg ± 7.83 15.4fgh ± 1.362
21 P205 7.55g ± 0.144 22.325bcdefghi ± 0.725 29.75cdefg ± 1.447 36.81cdef ± 0.3945 42.7efgh ± 5.66 14.175fgh ± 0.312
22 P216 8.7efg ± 1.079 24.675abc ± 1.071 27.41fgh ± 1.529 35.16fgh ± 0.2973 60cdefg ± 9.37 11.55fgh ± 0.689
23 P233 13.9abc ± 2.200 24.325abcde ± 0.531 32.82abcd ± 1.641 35.86efg ± 0.3638 41.58gh ± 4.30 30.98cde ± 12.056
24 P247 16a ± 1.567 22.3bcdefghi ± 1.219 33.79abc ± 3.066 37.28bcde ± 0.4847 44.5efg ± 11.85 7.14gh ± 0.865
25 P248 10defg ± 0.799 20.825 ghijk ± 1.314 29.48cdefg ± 1.389 38.12abcd ± 0.5951 112.73a ± 4.10 36.7bcd ± 3.604
Max. 16a ± 1.567 24.95a ± 0.833 36.52a ± 2.766 39.78a ± 0.9069 112.73a ± 4.10 63.75a ± 5.883
Min. 7.15g ± 0.891 19.025k ± 1.424 24.38h ± 1.906 30.77k ± 1.0157 20.33h ± 1.13 3.95h ± 0.466
CD 0.01 5.077 3.390 6.165 2.636 30.907 20.522
CD 0.05 3.825 2.568 4.652 1.987 23.295 15.464
C V 26.234 8.167 12.461 4.372 27.961 41.673
F.cal** 2.987 3.412 2.814 9.65 5.559 10.136
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Values are average of three replications; values after ± represents standard deviation; CV, coefficient of variance; CD, critical difference; ** Values are significant at 1 and 5% levels; As per Duncan’s grouping means with the same letter are not significantly different

Table 4.

Plant growth promoting response of lathyrus, chickpea, green gram and black gram seedlings following seed bacterization with fluorescent Pseudomonas isolates

S. no. Treat Lathyrus Chickpea Greengram Blackgram
Root length (cm) Shoot length (cm) Root length (cm) Shoot length (cm) Root length (cm) Shoot length (cm) Root length (cm) Shoot length (cm)
1 Control 18.185bcdefg ± 0.679 13.365i ± 1.094 28.333bcdef ± 0.726 16.333mn ± 0.498 17.28bcde ± 2.10 13.7h ± 1.747 16.435ef ± 0.733 12.625mnop ± 0.3637
2 P5 20.565abcde ± 0.957 16.635cdefg ± 0.835 29bcdef ± 6.449 14.933n ± 0.536 15.39de ± 2.58 14.665fgh ± 0.830 18.565bcdef ± 1.115 15.815defgh ± 0.3044
3 P6 19.415abcde ± 0.853 19.135abc ± 0.382 20.533fg ± 0.088 21.066defghi ± 0.636 18.04bcd ± 1.80 18.225abcd ± 1.19 20.985abcd ± 1.401 18.025ab ± 0.5456
4 P11 22.575ab ± 1.424 19.835ab ± 0.521 29.833bcde ± 0.441 22.866bcde ± 0.754 18.03bcd ± 0.44 20.385a ± 0.987 19.925bcde ± 1.858 16defg ± 0.3969
5 P67 19.385bcdef ± 2.496 13.885hi ± 0.922 27.333cdef ± 1.202 21.5cdefg ± 1.510 14.72de ± 0.48 17.625bcde ± 0.468 20.215bcde ± 1.540 13.375jklmnop ± 0.742
6 P72 19.085bcdef ± 1.464 16.2defgh ± 0.450 38.85a ± 6.438 25.7a ± 0.115 17.47bcde ± 1.12 16.1defgh ± 1.022 20.225bcde ± 2.244 13.165lmnop ± 0.4625
7 P76 12.785 g ± 0.585 13.15i ± 0.157 36.833ab ± 2.920 21.266defgh ± 0.504 13.99e ± 2.29 14.8fgh ± 0.452 19.15bcde ± 1.187 14.575hijk ± 0.3172
8 P85 18.285bcdef ± 2.483 20.815a ± 0.780 28.833bcdef ± 0.833 23.233bcd ± 0.536 16.22cde ± 0.68 14.985fgh ± 1.236 20.665abcde ± 0.959 16.8bcde ± 0.3007
9 P99 22.35ab ± 3.126 17.765bcdef ± 0.677 34.166abc ± 2.351 21.7cdefg ± 0.751 15.82cde ± 1.29 19.125abc ± 0.859 19.765bcde ± 0.251 18.615a ± 0.3912
10 P124 21.375abcd ± 3.196 20.2ab ± 0.774 26.833cdef ± 4.086 23.833abc ± 0.167 17.95bcd ± 0.77 17.615bcde ± 0.750 18.635bcdef ± 0.884 15.235fghi ± 0.6759
11 P126 16.375defg ± 1.830 18.1bcde ± 0.174 23defg ± 2.021 20.75efghi ± 1.010 15.99cde ± 1.49 15.325efgh ± 0.820 17.425def ± 0.574 12.5nop ± 0.3048
12 P129 16.465defg ± 1.723 18.015bcde ± 0.923 27.666cdef ± 0.882 25.066ab ± 0.924 15.69cde ± 2.80 16.865cdefg ± 1.170 19.715bcde ± 1.178 14.025ijklm ± 0.4498
13 P141 15.665efg ± 2.070 14.8ghi ± 1.093 33.166abc ± 2.242 18.933hijkl ± 0.470 22.15a ± 0.76 16.4defg ± 0.524 19.65bcde ± 0.991 17.515abc ± 0.4502
14 P143 17.55bcdefg ± 0.484 15.285fghi ± 0.739 33.65abc ± 3.839 21defghi ± 1.732 18.49abcd ± 0.84 19.175abc ± 1.301 22.135abc ± 2.148 17.685abc ± 0.4719
15 P151 13.965fg ± 0.380 16.8cdefg ± 1.365 31.433abcd ± 2.599 21.066defghi ± 0.581 19.59abc ± 1.48 20.235a ± 1.473 16.465ef ± 0.802 12.335op ± 0.4575
16 P161 15.775efg ± 1.730 13.115i ± 0.984 30.5abcde ± 2.180 22.166cdef ± 0.928 15.94cde ± 0.38 16.185defg ± 0.648 16.785def ± 1.574 13.275klmnop ± 0.2087
17 P167 18.735bcdef ± 0.671 16.85cdefg ± 1.646 22efg ± 4.726 16.833lmn ± 0.899 18.63abcd ± 1.27 16.465defg ± 0.390 17.835def ± 1.218 13.885ijklmn ± 0.3319
18 P176 20.525abcde ± 2.771 18.785abcd ± 1.573 24.25defg ± 3.320 19.5ghijk ± 1.155 17.08bcde ± 0.23 19.675ab ± 0.338 22.7ab ± 2.257 17.165bcd ± 0.6932
19 P179 16.7cdefg ± 1.198 13.365i ± 0.546 16.75g ± 1.299 18.25jklm ± 0.722 20.37ab ± 0.17 16.065defgh ± 0.228 17.75def ± 2.027 16.5cdef ± 0.3857
20 P201 19.915bcde ± 3.689 16.065efgh ± 0.933 24.4defg ± 3.114 19.733ghijk ± 0.536 16.47bcde ± 1.08 16.935cdef ± 0.388 19.885bcde ± 1.499 16.75bcde ± 0.4392
21 P205 18.615bcdef ± 0.742 16.525defg ± 0.545 23.933defg ± 0.788 20.166fghij ± 0.601 16.60bcde ± 1.80 14.45gh ± 0.149 16.95def ± 1.551 14.175ijkl ± 0.3099
22 P216 25.7a ± 2.957 15.185fghi ± 0.508 29.866bcde ± 4.332 20.766efghi ± 0.865 18.00bcd ± 1.41 16.965cdef ± 0.889 19.675bcde ± 2.523 14.7ghij ± 0.8147
23 P233 15.715efg ± 1.047 14.65ghi ± 0.899 28.666bcdef ± 0.441 22.5cdef ± 1.155 17.84bcde ± 1.07 18.065abcd ± 0.268 24.825a ± 2.510 15.585efgh ± 0.8166
24 P247 19.315bcdef ± 1.818 19.825ab ± 1.567 31.4abcd ± 2.358 18.733ijkl ± 0.617 16.00cde ± 0.97 16.65defg ± 0.388 18.125cdef ± 1.196 15.7efgh ± 0.4449
25 P248 21.93abc ± 2.049 20.705a ± 0.755 26.033cdef ± 2.338 17.366klm ± 0.857 16.09cde ± 1.12 15.2efgh ± 0.228 14.585f ± 0.716 16.8bcde ± 0.6334
Max. 25.7a ± 2.957 20.815a ± 0.780 38.85a ± 6.438 25.7a ± 0.115 22.15a ± 0.76 20.385a ± 0.987 24.825a ± 2.510 18.615a ± 0.3912
Min. 12.785g ± 0.585 13.115i ± 0.984 16.75g ± 1.299 14.933n ± 0.536 13.99e ± 2.29 14.665fgh ± 0.830 14.585f ± 0.716 12.335op ± 0.4575
CD 0.01 7.243 3.447 11.443 3.190 – 3.268 5.700 1.857
CD 0.05 5.467 2.590 8.584 2.391 3.923 2.464 4.298 1.401
C V 20.760 10.994 18.490 7.075 16.217 10.361 15.917 6.495
Fcal** 2.274 7.27 2.835 9.781 1.688 4.488 2.115 14.069
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Values are average of three replications; values after ± represents standard deviation; CV, coefficient of variance; CD, critical difference; ** Values are significant at 1 and 5% levels; as per Duncan’s grouping means with the same letter are not significantly different

Potential isolates stimulating plant growth (coleoptiles elongation and/or root length) in different crops were as follows

Wheat (GW-272): P124 stimulated coleoptiles elongation by 20.23% and root length by 17.30%; Chickpea (Cicer arientinum): P72 stimulated coleoptiles elongation 36.45% and root length by 27.08%; Lathyrus (KH-014): P85 stimulated coleoptiles elongation by 35.78% and P216 stimulated root length by 29.22%; Greengram (puspa vishal): P11 stimulated coleoptiles elongation by 32.81%; Blackgram (T-U-94-2): P99 stimulated coleoptiles elongation 32.16% and P233 stimulated root length by 33.8%; Bottlegourd (Lagenaria siceraria): P248 stimulated coleoptiles elongation 72.39% and P167 root length by 68.83%; Rice (swarna): P176 stimulated coleoptiles elongation 16.56% and P247 stimulated root length by 41.69%. Fluorescent Pseudomonas isolates stimulating both coleoptiles elongation and/or root length were P141 on greengram; P6, P143, P176 and P233 on blackgram; P76, P99, P124 and P167 on bottlegourd; and P151, P233 on rice. Fluorescent Pseudomonas isolates stimulating only root length were: P72, P129, P141 and P151 on bottlegourd; and P67 and P247 on rice.

Discussion

Understanding the mechanisms involved in the antagonist interactions between bacteria, pathogen and host plant is important for efficient utilization of these natural resources in crop health management (Thomashow and Weller 1991). In soil, plant roots normally coexist with bacteria and fungi which may produce siderophores capable of sequestering the available soluble iron and hence interfere with plant growth and function. Siderophore production confers competitive advantages to PGPR that can colonize roots and exclude other microorganisms from this ecological niche (Haas and Défago 2005). Under highly competitive conditions, the ability to acquire iron via siderophores may determine the outcome of competition for different carbon sources that are available as a result of root exudation or rhizo deposition (Crowley 2006). Siderophores production by strains of Pseudomonas spp. for plant disease control is of great interest because of its possibilities in the substitution of chemical pesticides. In this study, we have compared the ability of several fluorescent Pseudomonads to produce suderophores, cyanogenesis and antagonism in plate assay. All potential fluorescent Pseudomonas isolates identified following confrontation assays were high siderophore producers except P167, P248. Of the potential antagonistic fluorescent Pseudomonas isolates P233, P201, P176, were effective against R. solani where as P76 against both R solani and S. rolfsii were avid iron chelators and high siderophore producers. Similarly, microbial cyanogenesis has been demonstrated in a few bacterial species (belonging to the genera Pseudomonas, Chromobacterium, Rhizobium and several cyanobacteria (Blumer and Haas 2000). Glycine has generally been used as a precursor of cyanide in fungi and bacteria (Brysk et al. 1969; Wissing 1974) and cyanogenesis is one of the mechanisms of antagonism and biocontrol properties (Haas and Défago 2005; Lanteigne et al. 2012). In this investigation identified cynogenic isolates P76 and P124 exerted strong inhibition against S. rolfsii where as cynogenic P179 was ineffective against R solani and S. rolfsii remains unexplained. Our study revealed that isolates vary in the mechanisms and ability to inhibit pathogens. Plant growth-promoting bacteria use a number of different mechanisms to promote the growth of plants (Glick 2012), but enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase producing strains are the key bacterial trait which relives plants from deleterious effects of ethylene by cleaving ACC, into ammonia and –ketobutyrate (Honma and Shimomura 1978) and also facilitates plant growth (Glick et al. 2007; Glick 2014). ACC deaminase activity revealed wide variation in ACCd enzyme production in the range of 40.87 ± 0.08 to 25.02 ± 0.37 and 23.94 ± 0.32 to 10.51 ± 0.06 µmol α ketobutyrate/mg protein/h and P141 > P247 > P126, were potential ACCd enzyme producer. Fluorescent pseudomonas are one of the most abundant bacteria in the rhizosphere of many plants (Botelho and Mendonça-Hagler 2006), have large capacity to produce phytohormones, mainly auxins (Patten and Glick 2002a, b; Khalid et al. 2005) and secondary metabolites, such as antibiotics (Bergsma-Vlami et al. 2005), thus they are able to improve plant growth and plant health (Belimov et al. 2009a, b). Seed (of crops) biopriming with different isolates of fluorescent pseudomonas with ability to produce different levels of ACCd enzyme and siderophore were correlated with plant growth promoting effects. More stimulatory effects on coleoptile elongation than root length were observed. Our combined in vitro and pot experiment show the potential of isolate P176 to be developed as a commercial bio-inoculant as it stimulated coleoptiles elongation on all seven crops tested. Noticeable effects of plant growth stimulation were observed more on legume crops than on cereals. Potential isolates stimulating plant growth (coleoptiles elongation and or root length) specific to different crops were as follows: Both coleoptile elongation and root length:- Wheat (P124), Chickpea (P72); Only coleoptile elongation:- Greengram (P11), Lathyrus (P85), Blackgram (P99), Bottlegourd (P248), Rice (P176); Only root length:- Lathyrus (P216), Blackgram (P233), Bottlegourd (P167), Rice (P247). Nonetheless, this study and the results are particularly useful for identifying likely candidates for bio-control and for making educated guesses concerning the mechanisms by which they induce plant growth.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest in the publication.

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