Seed-coating fenugreek with Burkholderia rhizobacteria enhances yield in field trials and can combat Fusarium wilt

Rhizosphere 3 (2017) 92–99

Contents lists available at ScienceDirect

Rhizosphere journal homepage: www.elsevier.com/locate/rhisph

Seed-coating fenugreek with Burkholderia rhizobacteria enhances yield in field trials and can combat Fusarium wilt

MARK

⁎

Harish Kumara, , R.C. Dubeyb, D.K. Maheshwarib a b

Ipca Laboratories Ltd., 125, Kandivali Industrial Estate, Kandivali (W), Mumbai 400067 India Department of Botany and Microbiology, Gurukul Kangri University, Haridwar, Uttarakhand 249404, India

A R T I C L E I N F O

A BS T RAC T

Keywords: Burkholderia PGPR Antagonism Fusarium oxysporum Fenugreek (T. foenum- graecum L.)

Two efficient bacterial strains (RHT8 and RHT12) were isolated from root nodules of fenugreek (Trigonella foenum-graecum L.) and identified as Burkholderia sp. on the basis of morphological, physiological, biochemical and molecular character. Both strains produced IAA, Siderophore, solubilized insoluble phosphate, but none of them showed volatile cyanogens production. The isolates exhibited chitinase, β-1,3-glucanase and ACC deaminase activity along with the growth inhibition of Fusarium oxysporum. It also caused degradation and digestion of cell wall components, resulting in hyphal perforation, loss of structural integrity of the mycelium, along with degradation of conidia. Both isolates Burkholderia sp. RHT8 and Burkholderia sp. RHT12 led to synergism and aggressive root coloniztion of the roots and enhanced growth. Bacterial consortia (RHT8 and RHT12) effectively resulted in increased grain yield by 40% of fenugreek as compared to control in field trial. Maximum vigour index, nodule number, root and shoot biomass were recorded with RHT8+RHT12 consortia in comparison to single inoculation as well as control. This study reveals the potential of Burkholderia sp. RHT8 and Burkholderia sp. RHT12 to be used as a bioinoculant for growth promotion of fenugreek along with the protection from Fusarium wilt.

1. Introduction Fenugreek (Trigonella foenum-graceum L.) is one of the most important spice of which both seeds and leaves find an application in Indian cuisine. Fenugreek wilt caused by Fusarium oxysporum is one of the important diseases resulting in heavy losses (Shivpuri and Bansal, 1987). In current scenario use of chemical fertilizer for biocontrol of soilborne plant pathogens including F. oxysporum has been shifted to the option of green technology that have an agriculture importance. The concept of sustainable agriculture is of a recent origin and denotes the maintenance of crop productivity at level necessary to meet the requirements of increasing population, without deteriorating the environment and depleting the natural resources. Plant growth promotion due to certain traits showed by plant growth promoting rhizobacteria is a well documented phenomenon. There are a number of mechanisms used by PGPR for enhancing plant growth and development in diverse environment across the globe. Plant growth promoting rhizobacteria having direct and indirect mechanisms that facilitate nutrient uptake or increase nutrient availability by nitrogen fixation, solubilization of mineral nutrients, mineralize organic compounds and production of phytohormones. The

⁎

formation of inhibition zones might be due to the action of biologically active components, including chitinolytic enzymes, laminarinase, cellulose, HCN, antibiotics and siderophore. PGPR is a promising sustainable and eco-friendly approach to obtain sustainable fertility of the soil and plant growth indirectly. Suppression of the pathogen arises because iron deficiency causes growth inhibition, decrease in nucleic acid synthesis, inhibition of sporulation and causes changes in cell morphology (Mathiyazhagan et al., 2004). This approach inspire a wide range of exploitation of plant growth promoting rhizobacteria led to reducing the need for agrochemicals (fertilizers and pesticides) to improve soil fertility and biocontrol of phytopathogens (Gupta et al., 2015). The proposed study will provide invaluable resource for developing efficient bacterial inoculants with innate multifarious activities viz. plant growth promotion. The indigenous isolates with these acquired traits, under a natural selection process, could be utilized for developing bioinoculant specific to crop and agro-climatic conditions for application and demonstrations to the local farmers. The present study has been aimed to exploit the plant growth promotory bacteria associated with root nodules in enhancement of productivity of fenugreek during field trial and their antagonistic activity against F. oxysporum.

Corresponding author.

http://dx.doi.org/10.1016/j.rhisph.2017.01.004 Received 20 November 2016; Received in revised form 18 January 2017; Accepted 26 January 2017 Available online 31 January 2017 2452-2198/ © 2017 Elsevier B.V. All rights reserved.

Rhizosphere 3 (2017) 92–99

H. Kumar et al.

To observe IAA production exponentially grown cultures (108cells/ml) of the strains were incubated separately on broth for 24 h (Bric et al., 1991). Cyanogen production was determined by the modified method of Bakker and Schippers (1987). Chitinase activity was observed by the method of Renwick et al. (1991) in a defined medium having colloidal chitin as the sole carbon source. Similarly, β-1,3-glucanase activity was assessed using the same medium except colloidal chitin was replaced by laminarin (Rangel-Castro et al., 2002).

2. Material and methods 2.1. Seeds The certified seeds of fenugreek var. Pusa early Bunching was procured from G.B. Pant University of Agriculture and Technology, Pantnagar (Uttarakhand, India) and stored at room temperature. 2.2. Isolation of Fusarium oxysporum

2.7. Screening of ACC deaminase activity The fungal pathogen i.e. Fusarium oxysporum was isolated from diseased roots of fenugreek by using blotter technique (de Temp, 1963). The fungal culture was maintained by regular subculturing on Czapek-Dox agar at 4 °C for one year.

Screening of bacterial isolates to produce ACC deaminase activity was done on the basis of their ability to use ACC as a sole nitrogen source in the minimal medium (Glick et al., 2003). All the bacterial isolates were grown in YEM broth and cells from log phase culture were harvested. Pellets were collected by centrifugation at 8000 rpm for 10 min at 4 °C, washed with sterile distilled water and resuspend in 1 ml of sterile water and spot inoculated on Petri plates containing minimal medium supplemented with 3 mM ACC. Plates containing minimal medium without ACC served as negative control and those containing minimal medium with (NH4)2SO4 as a nitrogen source served as positive control. The plates were incubated for 3–4 days at 28 ± 1 °C (Honma and Shimomura, 1978).

2.3. Isolation of plant growth promoting bacteria Root nodules were collected from fenugreek growing at location Dwarahat of Uttarakhand (India). For the isolation of the root nodulating bacteria, method of Vincent (1970) was followed. Fenugreek plants were uprooted carefully and nodules were collected from the roots. The nodules were washed with sterile distilled water followed by surface treatment with 95% ethanol (2 ml) and again with sterile distilled water. Washed nodules were surface sterilized with 4% NaOCl for 2–3 min and again washed for at least 6 times with sterile distilled water so as to remove the traces of NaOCl. The nodules were crushed in a half filled culture tube of sterile distilled water with the help of sterile glass rod. A milky bacterial suspension was obtained and serially diluted. The suspension was streaked on congo red yeast extract mannitol (CrYEMA) agar plates (Vincent, 1970). Plates were incubated at 28 ± 1 °C for 4–5 days and observed for specific characteristics of rhizobia. The isolates were maintained on CrYEMA agar slants at 4 °C for further use (Somasegaran and Hoben, 1985).

2.8. Detection of melanin production Bacteria were grown on TY plates supplemented with L-tyrosine (600 µg/ml) and copper sulphate (40 µg/ml). Fully grown colonies were treated with 0.05 ml of 10% sodium dodecyl sulfate (SDS) in TBE buffer (pH 8.3). After 24 h of incubation at room temperature, the colonies producing a diffusible dark brown pigment, were scored as melanin producers (Mel+). In this way, it was possible to determine cooccupancy when cells obtained from one nodule showed Mel+ or Mel– colonies (Castro et al., 2000).

2.4. Other bacterial strains 2.9. Estimation of nitrate and nitrite reductases Sinorhizobium (Ensifer) meliloti MTCC100 and Rhizobium leguminosarium MTCC99 were procured from Microbial Type Culture Collection Center (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh (India). All these strains were maintained on the media recommended in MTCC catalogue. Burkholderia sp. MSSP was procured from the Department of Botany and Microbiology, Gurukul Kangri University, Haridwar (U.K.). Morphological, physiological and biochemical characterization of the bacterial isolates were carried out according to Bergey's Manual of Determinative Bacteriology (Holt et al., 1994) and comparing with standard isolates.

The culture isolates, singly or in consortia was inoculated in YEM broth and cells were harvested by centrifugation at 10,000 rpm for 15 min. The supernatant was discarded. Then 1 ml of 1 mM EDTA and 1 ml of 1% lysozyme was added to the pellet then incubated for 10 min. Again it was centrifuged at 10,000 rpm for 15 min. The supernatant was discarded and mili Q H2O (5 ml) was added to pellet and centrifuge at 10,000 rpm for 15 min. µM of nitrite formed (nitrate reductase) or nitrite reduced (nitrite reductase) was measured by the amount of nitrite in medium (Snell and Snell, 1949) in terms of min−1 mg−1 mg−1 protein. Samples were harvested at regular interval of 30 min upto 180 min. For nitrate reductase, 10 ml buffer and 2 ml KNO3 (0.1 M) were added to the pellets. 3 ml sample was taken from it and 7 ml chromotrophic acid reagent was added, mixed gently and cooled rapidly to 40 °C and incubated in water bath at 40 °C for 20 min. O.D. of growing cultures was measured using spectrophotometer (UV-1601, Shimadzu) by measuring absorbance at 430 nm. For measuring nitrite reductase the pellets were resuspended in 5 ml buffer and 1 ml of 0.1 M NaNO2 was added to it. 1 ml sample was taken and 1 ml of Griess Hosvay's reagent, 8 ml distilled water was added. The mixture was shaken well and incubated at room temperature for 30 min. O.D. was measured using spectrophotometer (UV-1601, Shimadzu) by measuring absorbance at 540 nm (Snell and Snell, 1949). Proteins were estimated according to Lowry et al. (1951).

2.5. 16S rDNA sequencing Partial 16S rDNA gene sequencing was performed after PCR amplification with the primer fD1 (5’-CCGAATTCGTCGACAACAGAGTTTGATCCTGGCTCAG-3’) and rD1 (5’-CCCGG GATCCAAGCTTAAGGAGGTGATCCAGCC-3’). Amplification was performed in a Gene amplification PCR system 9700 thermal cycler (Applied Biosystem, USA). The PCR product was sequenced using a Big Dye terminator cycle sequencing kit (V 3.1) in an ABI Prism 3730 Genetic Analyzer (Applied Biosystem, USA). The sequences were analyzed against the NCBI database (Kumar et al., 2010). 2.6. Plant growth promoting attributes Phosphate solubilization was detected by the formation of transparent zones surrounding bacterial colonies on a medium containing insoluble phosphate after 3 d incubation at 28 ± 1 °C (Pikovskaya, 1948). Siderophore production was determined on Chrome-azurol S (CAS) medium following the method of Schwyn and Neilands (1987).

2.10. In vitro antagonistic activity Antagonistic properties of isolates were tested against F. oxysporum on modified PDA plates using dual culture technique (Skidmore and Dickinson, 1976). Growth inhibition was calculated by measuring 93

Rhizosphere 3 (2017) 92–99

H. Kumar et al.

having 33% water holding capacity). Average temperature range from 4 to 18 °C during winter season less rain fall. Seeds of fenugreek var. Pusa Early Bunching of uniform shape and size were surface sterilized by treating with 95% ethanol for 1 min followed by 4% NaOCl treatment for 10 min. Seeds were washed several times with sterile distilled water to remove the traces of chemical. Seeds were allowed to dry overnight under sterile air. Method of Weller and Cook (1983) were adopted for seed bacterization. The bacterized seeds were allowed to dry for 1 h at room temperature. Total bacterial cell count on inoculated seeds was determined by transferring 20 bacterized seeds in 100 ml of sterile water and plating on YEMA. Bacterized (≈ 109 cfu seed−1) and non bacterized seeds were sown in sandy loam soil in a field of four sets of treatments. Treatment 1: Soil inoculated with fenugreek seeds bacterized with Burkholderia sp. RHT8; Treatment 2: Soil inoculated with fenugreek seeds bacterized with Burkholderia sp. RHT12; Treatment 3: Soil inoculated with fenugreek seeds bacterized with Burkholderia sp. RHT8 and Burkholderia sp. RHT12 (consortia) - bacterized fenugreek seeds; Treatment 4: control (seeds without bacterial inoculation). The experiments were conducted using a randomize plot design with three replicates of each treatment. The plot size was 25 m2 (5 m×5 m) in which bacterized seeds were sown by broadcasting method with an equidistance of 30 cm. The plots were irrigated as needed. Seed germination (%) was noted on 15th day of sowing. Seedling growth (shoot and root length, fresh shoot and root weight) total number of pod and grain yield was recorded. Ten plants from each plot were randomly selected for recording the data after every 30 days of intervals. Early as well as late vegetative and reproductive growth parameters were recorded after every 30 d of intervals from sowing till the harvesting of crop (125 DAS). The data were analyzed statistically by using Analysis of Variance (ANOVA) for individual parameters on the basis of mean values to find out the significance at 1 and 5% levels.

the distance between the edge of bacterial and fungal colonies, and percent inhibition was calculated by following formula: Growth inhibition=[(C-T)/ C] 100. where C=Radial growth of fungus in control, T=Radial growth of fungus in dual culture. For preparation of SEM samples, the mycelia were collected from the zone of interaction with bacteria for scanning electron micrograph (SEM). Samples were observed at 15 kV in a LEO 485 VP Scanning Electron Microscope. Photo micrograph were recorded by the same machine (Kumar et al., 2010). 2.11. In vitro antagonistic activity of culture filtrate Antagonistic bacteria were inoculated in their respective broth and incubated in a rotary incubator shaker at 150 rpm, and 28 ± 1 °C for 7 days. After incubation the broth cultures of the strains were collected by centrifugation at 10,000 rpm for 15 min at 4 °C and passed through a millipore filter (0.45 µm). Culture supernatants were poured in the wells of the fungal challenged plates. After incubation at 28 °C for 5–7 days, growth inhibition of F. oxysporum was recorded (Kumar et al., 2010). 2.12. In vitro antagonism in iron-deficient and iron-sufficient condition In vitro test for antagonism of Burkholderia sp. RHT8 and Burkholderia sp. RHT12 was done under iron-deficient and ironsufficient (30 and 60 µM Fe3+) conditions against F. oxysporum. Iron deficient medium was prepared by treating bacterial broth (250 ml) with 8-hydroxyquinoline solution in 100 ml chloroform (upto final concentration 0.1%), followed by vigorous shaking. Excess hydroxyquinoline was removed by chloroform extraction until the disappearance of yellow colour. The Medium was boiled to remove the smell of chloroform. Agar (2%) was added and the contents autoclaved at 15 lb/ inch2 (121 °C) for 20 min. Antagonism was tested by following methodology described as above. Bacterial broth supplemented with 30 and 60 µM Fe3+ was used to carry out in vitro fungal assay under iron-sufficient conditions. Methodology for examining antagonism was adopted as described above (Kumar et al., 2010).

2.16. Root colonization Root colonization of Burkholderia sp. RHT8 and Burkholderia sp. RHT12 were carried out by raising antibiotic resistant marker strains. Mithicillin (100 µg ml−1), and Nalidixic acid (100 µg ml−1) supplemented medium were used for assessment of populations of RHT8 and RHT12 in the rhizospheres of the plants, respectively. Fenugreek seeds bacterized with strains Burkholderia sp. RHT8 and Burkholderia sp. RHT12 were sampled after 30, 60, 90 and 125 DAS and bacterial population on the roots were measured. Plants were carefully removed with a shovel and soil particles adhering to roots were gently removed. Care was taken not to remove soil particles tightly adhered to the roots. The roots were then cut into 1 cm long segments and 1 g of root segments were dipped into 5 ml of sterilized distilled water and vortexed 4–5 times to release the rhizosphere bacteria into water. Dilutions of the bacterial suspensions were pour plated on YEM agar medium containing Mithicillin and Nalidixic acid (100 µg ml−1) separately for enumerating the introduced RHT8 and RHT12 and poured on nutrient agar medium to evaluate the population of aerobic bacteria. After 24 h of incubation at 28 ± 1 °C, cfu were counted (Kumar et al., 2010).

2.13. Amplification of nitrogen fixationa nif H and nodulation nod C gene For amplification of nif H region (781 bp), two primers, viz. nif H1 5’ CGT TTT ACG GCA AGG GCG GTA TCG GCA 3’ and nifH2 5’ TCC TCC AGC TCC TCC ATG GTG ATC GG 3’ were used; the reaction mixture and PCR conditions were followed as described by Kumar et al., 2006. nodC gene from genome and plasmids of root nodule isolates was amplified using the nodC gene primers, e.g. nod C1 5’ GCC ATA GTG GCA ACC GTC GT 3’ and nodC2 5’ TCA CTC GCC GCT GCA AGT C 3’ following procedure of Kumar et al. (2006). 2.14. Development of microbial consortia All strains were tested for their antagonistic behavior against each other for microbial consortia development following the method of Pierson and Weller (1994) and Kumar et al. (2010).

3. Results 3.1. Isolation and characterization of fenugreek isolates

2.15. Seed bacterization and Field experiment setup A total of 54 strains were isolated from the root nodules of fenugreek, out of which 2 bacterial isolates have been selected and named as RHT8 and RHT12. Results of their characterization are summarized in Table 1. On the basis of 16S r DNA analysis, both the isolates belongs to genera Burkholderia. The Burkholderia sp. RHT8 (GU182115) and RHT12 (GU182117) showed that it belongs to βsubclass of Proteobacteria not any of the four branches of α – subclass

To demonstrate the role of rhizobacteria as natural plant growth promoter, two best isolates Burkholderia sp. RHT8 and Burkholderia sp. RHT12 individually and its consortia forms were examined for its ability to enhance the overall growth of fenugreek. Field trials was carried out at Village Kota murad nagar, Haridwar, India in sandy loam soil (82.3% sand, 6.2% silt, 7.8 clay, total organic C 0.0876%, pH 6.8 94

Rhizosphere 3 (2017) 92–99

H. Kumar et al.

3.3. In vitro antagonistic activity

Table 1 Morphological, physiological and biochemical characters of fenugreek isolates. Characteristics

RHT8

RHT12

MTCC 100

MTCC 99

MSSP

Gram reaction Growth at 5 °C 28 °C 45 °C Motility Endospore Shape Capsule PHB accumulation Generation time Catalase Oxidase Urease H2S production Gelatin hydrolysis Starch hydrolysis Citrate utilization Growth on GPA Growth in HAB 2% NaCl tolerance 8% KNO3 tolerance Ppt. in Ca- glycerophosphate MR-VP test

+ +++ + + – rod + + 1.6 + + + – + + + + + + – – +

+ +++ + + – rod + + 1.6 + + + – + + + + + + – – +

+++ ++ + – rod – + 1.3 + + + – + – + – + + + + +

+++ + + – rod + + 2.6 + + + – + – + – + + + + –

+++ + + – rod + + 2.6 + + – – + + + + – + – – –

Utilization of Arabinose Cellobiose Dextrose Melibiose Glycerol m-Inositol Lactose Maltose

– + + + + – + +

+ + + + + – + +

+ + + – + + + +

+ + + + + + + +

+ + + – – – + +

Both isolates Burkholderia sp. RHT8 and Burkholderia sp. RHT12 inhibited the colony growth of F. oxysporum on modified PDA at 28 ± 1 °C. Burkholderia sp. RHT8 and Burkholderia sp. RHT12 caused 68.4% and by 67.2% inhibition of F. oxysporum in their growth respectively in comparison to control was recorded after 5 days of incubation in dual culture (Table 3). In another experiment, cell free culture filtrate of Burkholderia sp. RHT8 and Burkholderia sp. RHT12 caused 37% and 41% inhibition of F. oxysporum respectively in comparison to control. Culture filtrate inhibited the radial growth of F. oxysporum less in comparison to dual culture. Increase in fungal inhibition corresponded to incubation period. It was a common tendency of all the bacterial antagonists. Inhibition started after 48 h but maximum inhibition occurred after 5 days. Although, the fungal inhibition due to cell free culture filtrates was less pronounced in comparison to dual culture inhibition by using cell culture. The cell free-culture filtrate almost completely inhibited the conidia formation. Mycelial growth of the pathogens decreased significantly in dual cultures by the antagonists with respect to control in iron-deficient conditions. In iron-supplemented (30 µM Fe+3) medium growth inhibition of F. oxysporum was reduced. The mycelial growth decreased in dual culture by Burkholderia sp. RHT8 and Burkholderia sp. RHT12 as compared to control in iron-deficient conditions. In iron-supplemented (30 µM Fe+3) medium inhibition of F. oxysporum was reduced. Burkholderia sp. RHT8 and Burkholderia sp. RHT12 caused inhibition maximally; but growth inhibition got declined at 60 µM Fe+3 supplemented medium in comparison to normal conditions. Under iron-limiting condition, Burkholderia sp. RHT8 and Burkholderia sp. RHT12 inhibited the growth of the pathogen by 45%, 48%, respectively. On the other hand, 43%, 40% inhibition occurred under ironsufficient condition after 120 h of incubation. The cell-free culture filtrate of Burkholderia sp. RHT8 and Burkholderia sp. RHT12 also restricted hyphal growth and conidia formation. Iron-deficient conditions were more conducive to reduction of pathogen in vitro rather than iron sufficient as compared to growth of pathogen in control (Table 3). Scanning electron microscopy examination revealed the clear halo cell formation, vacuolation, squeezing, empty cell formation, swelling and lysis of mycelium. Further, increase of incubation corresponded to the fungal inhibition (Fig. 1).

Abbreviations: +, positive; -, negative; GPA- glucose peptone agar; HAB- Hoffer's alkaline broth; PHB- poly hydroxyl butyrate; MR, methyl red, VP, vogas proskaur; MTCC 100- Ensifer meliloti; MTCC99- Rhizobium leguminosarum; MSSP- Burkholderia sp.; RHT8 - Burkholderia sp; RHT12 Burkholderia sp.

of Proteobacteria.

3.2. Plant growth promoting attributes

3.4. Amplification of nif H and nod C gene

Isolates RHT8 and RHT12 were able to form clear halos around their spot inoculation on the Pikovskaya's agar plates by dissolving the tricalcium phosphate. It is notable that both isolates showed siderophore production forming orange halos around their spots of inoculation on the CAS agar, whereas the standard strains failed to produce siderophore except Burkholderia sp. MSSP. The isolates RHT8 and RHT12 secreted IAA that displayed pink color into culture filtrates with and without the addition of tryptophan. None of isolate produced HCN. The isolates RHT8 and RHT12 showed production of chitinase on the chitin minimal agar medium and β-1,3-glucanase activity on the plates supplemented with laminarin as a sole carbon source. The bacterial isolates RHT8 and RHT12 containing ACC deaminase ability to grow on medium containing ACC as a sole nitrogen source. None of the isolates RHT8 and RHT12 showed melanin production (Table 2). In case of consortia (RHT8+RHT12), RHT8 and RHT12 maximum nitrate reductase activity was recorded after 30 min viz. 1.14, 1.11, 1.16 µM. A similar pattern was observed in the samples at different durations, while measuring nitrite reductase activity. The rate of nitrite reductase activity in bacteria was the maximum after 120 min. in consortia and further increase in incubation period showed a decline pattern.

The positive amplification of 781 and 500 bp amplicons in our isolates confirmed the presence nif H and nodC genes for nitrogen fixation and nodulation respectively. 3.5. Effect of Seed bacterization on growth and yield On the basis of strong in vitro PGP attributes, Burkholderia sp. RHT8 and Burkholderia sp. RHT12 selected for seed bacterization and root coloniztion study. Burkholderia sp. RHT8 showed resistance to Mithicillin (100 µg ml−1), and Burkholderia sp. RHT12 to Nalidixic acid (100 µg ml−1). Fenugreek seeds bacterized with Burkholderia sp. RHT8 and Burkholderia sp. RHT12 and RHT8+RHT12 consortia showed enhanced seed germination as compared to non-bacterized seeds (control). The RHT8+RHT12 consortia resulted in 95.0% seed germination, while RHT8 and RHT12 individually caused 86.6% and 85.0% germination as compared to control (76.6%) (Table 5). Single inoculation and co-inoculation applied to seed showed enhanced seed germination. Such treated seeds produced seedlings with enhanced plant length, fresh weight, dry weight, no. of nodules, leaf area, no. of pods/plant, no. of seeds/pod, grain yield and stover yield. Maximum vigour index was recorded with RHT8+RHT12 consortia in compar95

Rhizosphere 3 (2017) 92–99

H. Kumar et al.

Table 2 Plant growth-promoting and antifungal properties of strains isolated from fenugreek. T. foenumgraecum isolates

IAA

Burkholderia sp. RHT8 Burkholderia sp. RHT12 Standard strains MTCC 100 MTCC 99 MSSP

Phosphate solubilizationB

HCNC

SiderophoreD

ACC deaminseE

Chitinase activityF

β-1,3glucanase activityG

Melanin productionH

Antagonism against F. oxysporumI

+++

+++

–

+++

+++

++

+++

–

+++

+++

++

–

+++

+++

++

+++

–

+++

+ + ++

+ + +

– – +

– – +

+ + +

– – +

– – –

+ + –

+ + –

A

Abbreviations: A -, IAA negative, +, IAA positive; B -, Phosphate solubilization negative; +, phosphate solubilization positive, C -, Absence of halo formation; +, small halos < 0.5 cm wide surrounding colonies; ++, medium halos > 0.5 cm wide surrounding colonies; +++, large halos > 1.0 cm wide surrounding colonies; D -, HCN negative, +, HCN positive; E -, chitinase negative, + chitinase positive small halos < 0.5 cm wide surrounding colonies; ++, medium halos > 0.5 cm wide surrounding colonies; F -, β-1,3-glucanase negative, +, β-1,3glucanase positive; G- ACC deaminase; H- Melanin production; I- Antagonism; MTCC 100, Enisfer meliloti; MTCC 99, Rhizobium leguminosarum; MSSP, Burkholderia sp; n.a. Not assessed. Table 3 Antagonistic effect of Burkholderia sp. RHT8 and RHT12 against F. oxysporum under dual culture, with cell free culture filtrate, in iron-sufficient and iron-deficient conditions. Bacterial Isolates

Incubation (h)

Growth in control (mm)

Growth inhibition in dual culture (%)

Growth inhibition in cell free culture filtrate (%)

Iron-sufficient (%)

Iron-deficient (%)

Burkholderia sp. RHT8

48 72 96 120 48 72 96 120

38.0 ± 0.06 43.4 ± 0.07 52.3 ± 0.09 72.5 ± 0.10 38.0 ± 0.06 43.4 ± 0.07 52.3 ± 0.09 72.5 ± 0.10

44.7 50.7 56.8 68.4 42.1 47.5 55.8 67.2

15.5 23.6 30.8 37.0 14.5 24.7 32.9 41.0

19.6 25.3 30.5 43.8 18.4 23.7 28.5 40.9

18.3 26.5 32.8 45.0 19.0 29.4 37.8 48.0

Burkholderia sp. RHT12

(Values are means of 5 replicates).

were fast grower with average mean generation time of 1.6 h and were positively showed catalase and oxidase activity. RHT8 and RHT12 isolates grow on GPA as well as on HAB and 2% NaCl and unable to tolerate 8 KNO3%. RHT8 utilized dextrose, cellobiose, melibiose, glycerol, lactose and maltose but did not utilized arabinose and mInositol while RHT12 utilized arabinose also along with the other (Kumar et al., 2010). Isolates RHT8 and RHT12 produced IAA in tryptophan containing medium but also produce IAA in absence of tryptophan that was confirmed by the development of pink color with and without addition of tryptophan into their culture filtrate. Patten and Glick (1996) reported that eighty percent of the rhizospheric bacteria including the members of Rhizobia, Azotobacter and Pseudomonads produced indole-3-acetic acid (IAA). Bioproduction of indole acetic acid by Rhizobium, Mesorhizobium, Burkholderia, Sinorhizobium, Bradyrhizobium has been shown by many workers (Annapurna et al., 2007; Kannan and Sureendar, 2009). Phosphate solubilization was reported by Burkholderia sp. RHT8 and Burkholderia sp. RHT12. Similarly, Pandey (2008) reported the highest phosphate solubilization index in S. meliloti PP3 followed by Burkholderia sp. MSSP. Burkholderia sp. RHT8 and Burkholderia sp. RHT12 were showed siderophore production on CAS agar medium. Siderophore production by root nodulating rhizobial strains have been well documented (Carson et al., 2000). Chitinase and β-1, 3-glucanase are key enzymes in the decomposition of fungal hyphal wall (Rangel-Castro et al., 2002). In the present study, both isolates RHT8, RHT12 produced the Chitinase as well as β-1, 3-glucanase activity. Recently, Pandey (2008) reported the release of chitinase, β-1,3-glucanase and protease from Burkholderia sp. MSSP, a key enzyme associated with the dissolution of cell wall of F. oxysporum and F. udum. None of the isolate showed melanin production. Castro et al. (2000) reported that 62% of 24 Sinorhizobia strains isolated from different regions and found that produced melanin. This marker was stable after 4 years of

ison to single inoculation as well as control (Table 5). The other vegetative parameters such as number of pods per plants, number of nodules, root length, shoot length, shoot weight and root weight were also increased in comparison to control after 125 days (Table 4). Twin bacterial consortium induced the vegetative growth of plant in comparison to that of single inoculation (Table 4). The maximum stover yield was produced under consortia RHT8+RHT12 and finally by individual bacteria inoculation as compared to control in field trials. Total grain yield got increased by 40.6% in RHT8+RHT12 consortium as compared to control which is double to the single inoculation (Table 5). 3.6. Root colonization Burkholderia sp. RHT8 and Burkholderia sp. RHT12 showed effective root colonization of fenugreek. In vitro population density of antibiotic maker strain and indigenous resident bacteria was estimated in the rhizosphere of fenugreek. In all cases, plant growth parameters inoculated with microbial consortium were always better than single inoculation and control. The significant root colonization was observed and a correlation of root colonization and enhanced growth parameters was seen (Table 6). 4. Discussion In present study, both the isolates were characterized on the basis of morphological, physiological, biochemical and by means of molecular techniques and compared with standard strains viz. Sinorhizobium meliloti MTCC 100, Rhizobium leguminosarum MTCC 99 and Burkholderia sp. MSSP (Holt et al., 1994). On the basis of 16S r DNA analysis, both the isolates belongs to genera Burkholderia. Both isolates were Gram-negative, non-spore forming and capsulated, motile with single sub-polar flagellum. Both of them 96

Rhizosphere 3 (2017) 92–99

H. Kumar et al.

Fig. 1. Scanning Electron micrograph of Fusarium oxysporum showing mycelial growth by antagonistic effects of Burkholderia sp. RHT8 and Burkholderia sp. RHT12 (Hyphal perforation and lysis (A & B), Swollen hyphae and fragmentation (C & D), Degradation of mycelia and cytoplasm leakage (E-F)). Table 4 Effect of different treatment on the growth of fenugreek (var. Pusa early Bunching) after 125 days of sowing. Treatments

Length (cm) Shoot

Burkholderia sp. RHT8 Burkholderia sp. RHT12 Consortia (RHT8 + RHT12) Control SEM CD 1% CD 5%

**

56.66 55.83** 80.16** 33.00 2.5230 10.892 7.7729

Root *

10.33 11.00* 11.66* 8.66 0.7273 3.1403 2.2409

Fresh weight (g)

Dry weight (g)

Shoot

Shoot

**

18.36 18.33** 24.66** 12.86 1.2854 5.5497 3.9602

Root *

4.100 4.166* 5.033* 3.700 0.2483 1.0723 0.7652

**

3.366 2.800** 4.933** 1.976 0.2161 0.9331 0.6658

No. of nodules

Leaf area (mm2)

No. of Pods/ plant

No. of seeds /pod

18.33** 19.33** 20.33** 11.66 0.4146 1.7902 1.2775

130.00** 131.66** 172.00** 96.00 3.9591 17.092 12.197

15.00** 16.66** 23.66** 11.33 0.9594 4.1424 2.9560

18.66* 18.33* 18.66* 14.00 0.9229 3.9846 2.8434

Root 0.706** 0.846** 1.466** 0.606 0.0897 0.3873 0.2764

Abbreviations: Values are mean of ten replicates; ** = Significant at 0.01 level of LSD as compared to control; Significant at 0.05 level of LSD as compared to control.

*

= Significant at 0.05 level of LSD as compared to control;

ns

= Not

41% inhibition of F. oxysporum in comparison to control. Culture filtrate inhibited less radial growth of F. oxysporum in comparison to dual culture interaction. Increase of the fungal inhibition corresponded to incubation period. It was a common tendency of all the bacterial antagonists. Burkholderia sp. RHT8 and Burkholderia sp. RHT12 imparted varying degrees of inhibition of growth and conidia germination of phytopathogenic fungi due to production of antibiotics and antifungal compounds in culture filtrates (Skidmore and Dickinson, 1976; Pandey, 2008). Microscopic examination of Fusarium oxysporum under the influence of Burkholderia sp. RHT8 and Burkholderia sp. RHT12 clearly

multiple replicas. Melanin could thus be a useful marker because it is stable. Kucuk et al. (2006) reported the melanin production by Rhizobium sp. isolated from bean. Burkholderia sp. RHT8 and Burkholderia sp. RHT12 strongly inhibited the growth of F. oxysporum in vitro. Significant radial growth inhibition was recorded. Pandey et al. (2005) reported the bacterial isolate Burkholderia sp. MSSP from root nodules of Mimosa pudica showed antifungal activity against many phytopathogens. Recently we have reported the strong inhibition of F. udum by S. fredii KCC5 in dual culture (Kumar et al., 2010). Cell free culture filtrate of Burkholderia sp. RHT8 and Burkholderia sp. RHT12 caused 37% and 97

Rhizosphere 3 (2017) 92–99

H. Kumar et al.

Table 5 Effect of seed bacterization of Burkholderia on seed germination and grain yield of fenugreek after 125 days of sowing. Treatment

Germination percentage (%)

Plant length (Root length + Shoot length)

Vigour Index

Grain yield (kg h−1)

Stover yield (kg h-1)

Biological yield (kg h-1)

Harvest index (%)

% increase over control

Burkholderia sp. RHT8 Burkholderia sp. RHT12 Consortia (RHT8 + RHT12) Control

86.6**

10.7**

926.60**

1158.66**

1680.00**

2838.66

40.81

15.09

**

**

977.50

**

1132.00**

1683.33**

2815.33

40.20

12.45

**

1416.66**

2080.00**

3496.66

40.51

40.66

1006.66

1593.33

2599.99

38.71

11.5

85.0

**

**

95.0

15.6

1482.0

76.6

6.4

490.2

Abbreviations: Values are the mean of ten replicates. ** = Significant at 0.01 level of LSD as compared to control; * = Significant at 0.05 level of LSD as compared to control; ns = Not Significant at 0.05 level of LSD as compared to control.

Table 6 Average root colonization of fenugreek inoculated with bacterial strains Burkholderia sp. RHT8 and RHT12 after 30, 60, 90 and 125 days after sowing. Treatments

Burkholderia sp RHT8 Burkholderia sp RHT12 Consortia RHT8 RHT12

30 DAS

60 DAS

90 DAS

125 DAS

Bacterial population

Indigenous population

Bacterial population

Indigenous population

Bacterial population

Indigenous population

Bacterial population

Indigenous population

4.21

4.65

5.15

5.53

6.05

6.41

6.23

6.62

4.35

4.72

5.28

5.78

6.15

6.54

6.13

6.75

4.30 4.45

5.20

5.45 5.60

5.38

5.68 5.77

5.42

5.79 5.95

5.62

Abbreviations: values (log10 cfu g-1) are mean of 10 randomly selected plants.

46.3% increased seed germination over the control. Rhizosphere colonization is important not only as the first step in pathogenesis of soil borne microorganism but also is crucial in the application of microorganism for beneficial purposes (Lugtenberg et al., 2001). Antibiotic markers were developed and used to monitor and re-isolate the introduced beneficial rhizobacteria from the experimental field (Gupta et al., 2002; Bhatia et al., 2005). The antibiotic resistant marker strains of Burkholderia sp. RHT8 and Burkholderia sp. RHT12 and indigenous resident bacteria was used to monitor root colonization of fenugreek root at different time intervals. The positive root colonization ability of Burkholderia sp. RHT8 and Burkholderia sp. RHT12 as consortia lies in it being the successful colonizer of the spermosphere, increased seed emergence and its establishment in the rhizosphere of fenugreek protecting agent of F. oxysporum resulting in enhanced yield. Arora et al. (2001) used 100 µl/ml of ampicillin to re-isolate the rhizobia RMP1amp+ from the uninoculated rhizosphere. Thus, it may be concluded that PGPR strains showing important plant growth promoting traits proved to be very efficient in promoting the early vegetative and late reproductive growth parameters of fenugreek with excellent root colonization ability and strongly inhibited the growth of F. oxysporum. The strains could be useful in the formulation of new bioinoculants for commercial production.

showed degeneration and degradation of the hyphae in vitro. Mycelial deformities and inhibition of conidiation in several plant pathogenic fungi including F. oxysporum and M. phaseolina by Burkholderia cepacia was observed by Upadhyay and Jayaswal (1992). Burkholderia sp. RHT8, Burkholderia sp. RHT12 were confirmed by amplification of nifH and nodC fragments with 781 bp and 500 bp respective amplicons. Many workers have reviewed the presence of nod and nif gene in various bacteria such as Sinorhizobium (Hukka et al., 1998; Wang et al., 2009); Mesorhizobium (Hukka et al., 1998; Wang et al., 2009), Burkholderia (Chen et al., 2003), Rhizobium (Wang et al., 2009). Pandey (2008) also reported that four strains Burkholderia sp. MSSP, B. subtilis B1, S. meliloti PP3 and R. leguminosarum Pcc respectively were able to grow in presence of each other in consortium. Substrate competition is one of the major evolutionary driving forces in the bacterial world, and numerous experimental data obtained under well controlled conditions revealed how different organisms may effectively out-compete others because of better utilization of a given energy source. Burkholderia sp. RHT8 and Burkholderia sp. RHT12 were successfully grown as mixed cultures. Both strains namely Burkholderia sp. RHT8 and Burkholderia sp. RHT12 showed similar type of IAA production, phosphate solubilization, siderophore production and chitinase activity. It was found that the secondary metabolites production was enhanced in all the combinations and in the consortium they were the highest. In the treatments where consortia were applied, the grain yield got enhanced by 40% as compared to control in field trials. The dependence of grain yield of fenugreek on various growth and yield parameters and interdependence among growth and yield parameters are evident from the positive and significant correlation. A positive correlation was observed between in vitro and in situ antagonism in field experiment. A similar result has also been reported by many workers (Arora et al., 2001; Gupta et al., 2002). Arora et al. (2001) observed similar results with groundnut seeds bacterized with siderophore producing R. meliloti RMP3 and RMP5 that showed 52.8% and

Acknowledgements The author thank the Head, Department of Botany and Microbiology for providing laboratory facilities. Financial support from the Uttarakhand Council for Science and Technology (CS & T/R & D/ LS-19/06 UCOST, DST, Govt. of Uttarakhand, Dehradun) and University Grants Commission (No. F. 4-3/2006 (BSR)/11-13/ 2008(BSR), UGC RFSMS, New Delhi) is gratefully acknowledged by one of us (HK). 98

H. Kumar et al.

Rhizosphere 3 (2017) 92–99

References

591–598. Kumar, B., Kumar, M.S., Annapurna, K., Maheshwari, D.K., 2006. Genetic diversity of plant growth promoting rhizobia isolated form a medicinal legume, Mucuna prurience Linn.. Curr. Sci. 91 (11), 1524–1529. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275. Lugtenberg, B.J.J., Dekkers, L.C., Bloemberg, G.V., 2001. Molecular determinants of rhizosphere colonization by Pseudomonas. Annu. Rev. Phytopathol. 39, 461–490. Mathiyazhagan, S., Kavitha, K., Nakkeeran, S., Chandrasekar, G., Manian, K., Renukadevi, P., Krishnamoorthy, A.S., Fernando, W.G.D., 2004. PGPR mediated management of stem blight of Phyllanthus amarus (Schum and Thonn) caused by Corynespora cassiicola (Berk and Curt) Wei. Arch. Phytopathol. Plant Protect. 33, 183–199. Patten, C.L., Glick, B.R., 1996. Bacterial biosynthesis of indole-3 acetic acid. Can. J. Microbiol. 42, 207–220. Pandey, P., 2008. Role of Rhizobacteria in Biocontrol and Growth promotion of Cajanus cajan (Ph.D. Thesis). Gurukul Kangri University, Haridwar, India. Pandey, P., Kang, S.C., Maheshwari, D.K., 2005. Isolation of endophytic plant growthpromoting Burkholderia sp. MSSP from root nodules of Mimosa pudica. Curr. Sci. 89, 177–180. Pikovskaya, R.I., 1948. Mobilization of phosphorus in soil in connection with the vital activity of some microbial species. Mikrobiology 17, 362–370. Pierson, E.A., Weller, D.M., 1994. Use of mixtures of fluorescent pseudomonads to suppress take all and improve the growth of wheat. Phytopathology 84, 940–947. Rangel-Castro, J.I., Levenfors, J.J., Danell, E., 2002. Physiological and genetic characterization of fluorescent pseudomonas associates with Cantharellius cibarius. Can. J. Microbiol. 48, 739–748. Renwick, A., Campbell, R., Coe, S., 1991. Assesment of In vivo screening systems for potential biocontrol agents of Gaeumannomyces graminis. Plant Pathol. 40, 524–532. Schwyn, B., Neilands, J.B., 1987. Universal assay for the detection and dertermination of siderophores. Anal. Biochem. 160, 47–56. Shivpuri, A., Bansal, R.K., 1987. Fusarium wilt of fenugreek (Trigonella foenum-graecum L.) A new record. Ind. Mycol. Plant Pathol. 17, 104. Skidmore, A.M., Dickinson, C.H., 1976. Colony interaction and hyphal interferance between Septoria nodorum and phylloplane fungi. Trans. Br. Mycol. Soc. 66, 57–74. Snell, F.D., Snell, C.T., 1949. Calorimetric Methods of Analysis 2. Van Nostrand, Princeton, 785–807. Somasegaran, P., Hoben, H.J., 1985. Methods in legume-Rhizobium technology. NifTAL Project and MIRCEN, University of Hawaii, HI. Upadhyay, R.S., Jayaswal, R.K., 1992. Pseudomonas cepacia causes mycelial deformities and inhibition of condition in phytopathogenic fungi. Curr. Microbiol. 24, 181–187. Vincent, J.M., 1970. A Manual for the Practical Study of Root Nodule Bacteria IBP Handbook No. 15. Blackwell, Edinburgh, U.K. Wang, H., Man, C.X., Wang, E.T., Chen, W.X., 2009. Diversity of rhizobia and interactions among the host legumes and rhizobial genotypes in an agriculturalforestry ecosystem. Plant Soil 314, 169–182. Weller, D.M., Cook, R.J., 1983. Suppression of take all the wheat by seed treatment with fluorescent pseudomonads. Phytopathology 23, 23–54.

Annapurna, K., Balakrishnan, N., Vital, L., 2007. Verification and rapid identification of soyabean rhizobia in Indian Soils. Curr. Microbiol. 54, 287–291. Arora, N.K., Kang, S.C., Maheshwari, D.K., 2001. Isolation of siderophore producing strains of Rhizobium meliloti and their biocontrol potential against Macrophomina phaseolina that causes charcoal rot of groundnut. Curr. Sci. 81, 673–677. Bakker, A.W., Schippers, B., 1987. Microbial cyanides production in the rhizosphere in relation to potato yield reduction and Pseudomonas spp. mediated plant growth stimulation. Soil Microbiol. Biochem. 19, 451–457. Bhatia, S., Dubey, R.C., Maheshwari, D.K., 2005. Enhancement of plant growth and suppression of collar rot of sunflower caused by Sclerotium rolferii through fluorescent pseudomonas. Ind. Phytopathol. 58, 17–24. Bric, J.M., Bustock, R.M., Silversone, S.E., 1991. Rapid in situ assay for indole acetic acid production by bacteria immobilization on a nitrocellulose membrane. Appl. Environ. Microbiol. 57, 535–538. Carson, K.C., Meyer, J.M., Dilworth, M.J., 2000. Hydroxamate siderophore of root nodule bacteria. Soil Biol. Biochem. 32, 11–21. Castro, S., Carrera, I., Martinez-Drets, G., 2000. Methods to evaluate nodulation competitiveness between Sinorhizobium meliloti strains using melanin production as a marker. J. Microbiol. Methods 41, 173–177. Chen, W.M., moulin, L., Bontemps, C., Vandamme, P., Bena, G., Mosson, C.B., 2003. Legume symbiotic nitrogen fixation by β-Proteobacteria is widespread in nature. J. Bacteriol. 185, 7206–7272. de Temp, J., 1963. The blotter method for seed health testing. Proc. Int. Seed Test Assoc. 28, 1933. Glick, B.R., Lucy, M., Reed, E., 2003. Applications of free living plant growth promoting bacteria. Antonie van Leeuenhoek. 86, 1–25. Gupta, C.P., Dubey, R.C., Maheshwari, D.K., 2002. Plant growth enhancement and suppression of Macrophomina phaseolina causing charcoal rot of peanut by fluorescent Pseudomonas. Biol. Fert. Soils 35, 399–405. Gupta, G., Parihar, S.S., Ahirwar, N.K., Snehi, S.K., Singh, V., 2015. Plant growth promoting rhizobacteria (PGPR): current and future prospects for development of sustainable agriculture. J. Microb. Biochem. Technol. 7 (2), 096–102. Holt, J.H., Kreig, N.R., Sneath, J.J., Williams, S.T., 1994. Bergey's Manual of Determinative Bacteriology 9th edition. Williams and Wilkins Publication, Baltimore, Maryland, USA. Honma, M., Shimomura, T., 1978. Metabolism of 1 aminocyclo-propane-1-carboxylic acid. Agric. Biol. Chem. 42, 1825–1831. Hukka, K., Lindstrom, K., Young, J.P.W., 1998. Three phylogenetic groups of nodA and nifH genes in Sinorhizobium and Mesorhizobium isolated from leguminous trees growing in Africa and Latin America. Appl. Environ. Microbiol. 64, 419–426. Kannan, V., Sureendar, R., 2009. Synergistic effect of beneficial rhizosphere microflora in biocontrol and plant growth promotion. J. Basic Microbiol. 49, 158–164. Kucuk, C., Kivanc, M., Kinaci, E., 2006. Characterization of Rhizobium sp. isolated from Bean. Turk. J. Biol. 30, 127–132. Kumar, H., Bajpai, V.K., Dubey, R.C., Maheshwari, D.K., Kang, S.C., 2010. Wilt disease management and enhancement of growth and yield of Cajanus cajan (L) var. Manak by bacterial combinations amended with chemical fertilizer. Crop Protection 29,

99