Biocontrol efficiency of native plant growth promoting rhizobacteria against rhizome rot disease of turmeric

Biological Control 129 (2019) 55–64

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Biocontrol efficiency of native plant growth promoting rhizobacteria against rhizome rot disease of turmeric

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C. Chenniappana, M. Narayanasamya, , G.M. Daniela, G.B. Ramarajb, P. Ponnusamyb, J. Sekarc, P. Vaiyapuri Ramalingamc a

Biocontrol and Microbial Metabolites Lab, Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai 600 025, Tamil Nadu, India Department of Biotechnology, K.S. Rangasamy College of Technology, Tiruchengode 637 215, Namakkal District, Tamil Nadu, India c Microbiology Lab, M.S. Swaminathan Research Foundation, 3rd Cross Street, Taramani Institutional Area, Taramani, Chennai 600 113, India b

A R T I C LE I N FO

A B S T R A C T

Keywords: Biological control Turmeric Rhizome rot Fengycin DAPG Pseudomonas Bacillus

This study was intended to evaluate the biocontrol efficacy of rhizobacteria isolated from turmeric fields against fungal pathogens, which are associated with the rhizome rot of turmeric. The antagonistic potential of 157 rhizobacterial isolates obtained from the turmeric fields was assessed against seven rhizome rot fungal pathogens; namely, Rhizoctonia solani MML4001, Fusarium solani MML4002, Schizophyllum commune MML4003, Macrophomina phaseolina MML4004, Fusarium graminearum MML4005, Fusarium solani MML4006 and Fusarium solani MML4007. Sixteen rhizobacterial isolates exhibited consistent antagonistic activity against all seven tested rhizome rot fungal pathogens. Experiments were done to assess various biocontrol mechanisms and plant growth promoting traits exhibited by these 16 selected rhizobacterial isolates. Polyphasic taxonomical identification revealed them as Pseudomonas aeruginosa MML2424, P. aeruginosa MML2515, P. aeruginosa MML2519, Bacillus amyloliquefaciens MML2522, B. amyloliquefaciens MML2547, Bacillus tequilensis MML2476, Bacillus cereus MML2533, Bacillus subtilis MML2406, B. subtilis MML2411, B. subtilis MML2415, B. subtilis MML2451, B. subtilis MML2458, B. subtilis MML2473, B. subtilis MML2483, B. subtilis MML2490 and B. subtilis MML2518 (GenBank Accession numbers: KJ655538 to KJ655553). Detection of genes encoding for different biocontrol traits viz., production of antibiotics; i.e., 2,4-diacetylphloroglucinol, pyrrolnitrin, pyoluteorin, bacillomycin D, fengycin, hydrogen cyanide, and lytic enzyme, cellulase in these strains was carried out by polymerase chain reaction using gene-specific primers. Furthermore, field experiments were conducted using bioformulations of eight selected promising biocontrol-PGPR. Results of this study revealed that most of the PGPR, particularly P. aeruginosa MML2424 and B. subtilis MML2490, appeared promising for commercialization, which can be used for plant growth promotion and management of turmeric rhizome rot disease.

1. Introduction India is the leading producer, consumer, and exporter of turmeric (Curcuma longa L.) in the world. In the international market, Indian turmeric varieties are highly recognized for their superior quality with high curcumin content. India has 172,000 ha under turmeric cultivation, with a total production of 851,000 metric tonnes (Muthusamy, 2013). However, turmeric production in many areas is limited by its diseases caused by various pathogens. Among all diseases, rhizome rot is considered to be the major constraint for successful turmeric cultivation because of its attribution towards severe decline in yield, and sometimes total loss of the crop. Moreover, disease management practices followed by farmers in many turmeric fields are still insufficient

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for satisfactory control of rhizome rot disease. Furthermore, there are no disease resistant varieties/germplasms of turmeric available in India (Singhal, 2003). Although fungicides such as Carbendazim and Bordeaux mixtures have shown promising results in controlling the disease, phytotoxicity and fungicide residues are currently the major environmental pollution issue leading to human health hazards. Furthermore, fungicide resistance developed by fungal pathogens discourages their use for disease control (Whipps and Lumsden, 1991). In this regard, biological control is an environmentally sound, viable, and sustainable crop protecting alternative for reducing the use of these agrochemicals (De Weger et al., 1995; Gerhardson, 2002; Mathivanan and Manibhusahan rao, 2004). Biocontrol of soil-borne diseases, such as rhizome rot of turmeric, is particularly complex

Corresponding author. E-mail address: [email protected] (M. Narayanasamy).

https://doi.org/10.1016/j.biocontrol.2018.07.002 Received 19 November 2017; Received in revised form 30 May 2018; Accepted 6 July 2018 1049-9644/ © 2018 Elsevier Inc. All rights reserved.

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2. Materials and methods

because these diseases occur in the dynamic environment at the interface of root and soil, known as the rhizosphere (Handelsman, and Stabb, 1996). The rhizosphere is the largest ecosystem on Earth with enormous energy flux and abundant microorganisms, such as bacteria and fungi (Barriuso et al., 2008). Among these microorganisms, free living soil bacteria inhabiting the rhizosphere, which have the ability to colonize plant root systems can stimulate plant growth (plant growthpromoting rhizobacteria; PGPR). They can also protect plants against pathogen infections (biocontrol strains) (Kloepper et al., 1989; Berg and Smalla, 2009). These PGPR can promote plant growth directly by interacting with the metabolism of their host plants, providing in-demand substances such as nitrogen, phosphorus, iron, and some plant hormones; indirectly through antagonistic activity against plant pathogens, via production of antibiotics, lytic enzymes, hydrogen cyanide, catalase, and siderophore; and through competition for nutrients and space (Bashan and de-Bashan, 2005). Utilization of such PGPR with biocontrol activity is a promising approach for the control of rhizome rot in turmeric, as well as for the improvement of plant growth and rhizome yield. Several investigations were carried out to suppress rhizome rot using the fungal antagonists belonging to the genus Trichoderma (Jagtap et al., 2011; Anoop and Suseela Bhai, 2014). However, only a few attempts have been made to utilize the enormous potential of these rhizobacteria for the management of rhizome rot of turmeric. Moreover, collecting suitable native antagonistic rhizobacteria from the turmeric rhizosphere soil would enhance the control of rhizome rot disease, since the native strains show better performance in the rhizosphere soil than the exotic strains. Among the various rhizospheric bacteria, species belonging to Bacillus and Pseudomonas have been studied for their biocontrol activities for many years. These bacterial strains have their own mode of either one or more biocontrol mechanisms to compete their antagonist. However, it is likely that the most effective PGPR strains will act via multiple mechanisms to control their pathogens. Vassilev et al. (2006) reviewed the importance of the presence of multiple plant growth promoting mechanisms in the microorganisms. Additionally, the discovery of biosynthetic genes for common, wellcharacterized antibiotics and for other PGPR traits has resulted in a better understanding of the performance of bioinoculants in the field, and also provides the opportunity to enhance the beneficial effects of PGPR strains by genetic modification (Bloemberg and Lugtenberg, 2001). Screening candidate strains for particular antibiotic-encoding sequences represents a prompt approach, while ensuring the production of particular antibiotics (Ramarathnam et al., 2007) such as the antifungal metabolites pyoluteorin (PLT), pyrrolnitrin (PRN), phenazines, 2,4-di-acetyl phloroglucinol (2, 4-DAPG), fengycin, bacillomycin, and iturin. Therefore, in the present study, a fusion of conventional techniques together with molecular genetics is brought forward to identify the most promising PGPR with manifold mechanisms for the development of bioformulations to control the fungal pathogens of turmeric rhizome rot. While critical considerations to designing successful formulations of PGPR microbial biomass from the lab to the field are many-fold; the foremost one is selection of a suitable bacterial biomass carrier. Among the various organic carriers, talc (magnesium silicate) proved to be the best in terms of its easy application, storage, long shelf life, and quality of formulation (Chaube et al., 2003; Mathivanan et al., 2000, 2005; Sallam et al., 2013). Moreover, talc based formulations of rhizobacteria are successful against soil-borne plant diseases (Krishnamurthy and Gnanamanickam, 1998; Nandakumar et al., 2001). Considering these potential benefits, the current study was carried out to evaluate the efficiency of talc based powder formulations of the selected PGPR under field conditions for the management of rhizome rot fungal pathogens and plant growth promotion of turmeric.

2.1. Isolation of rhizobacteria from turmeric rhizosphere soil samples Rhizosphere soil samples were collected from farmers’ fields in turmeric growing districts of Tamil Nadu such as Erode, Namakkal, and Tiruppur. The intact root systems with rhizomes were dug out and loosely adhering soil was dislodged from the root zone by gentle shaking. These soil samples were collected in sterile polythene bags and brought to the laboratory for isolation of rhizobacteria. Ten grams of each turmeric rhizosphere soil sample were suspended in 95 mL of sterile distilled water in a conical flask and kept on a rotary shaker at 120 rpm for 30 min. The resulting suspension was serially diluted and spread plated onto Nutrient Agar (NA) and King’s B Agar (KBA) medium. The isolation plates were incubated at room temperature (28 ± 2 °C) for 48 h. Distinct single colonies of fluorescent pseudomonads (FPs) and other rhizobacterial isolates were picked and subcultured to purity on NA/KBA medium. They were maintained on NA/ KBA medium, in glycerol stocks (840 µL broth cultures with 500 µL of 80% glycerol at −20 °C) and in sterilized native soil at −20 °C. 2.2. Turmeric rhizome rot fungal pathogens The seven most infective rhizome rot fungal pathogens namely, R. solani MML4001, F. solani MML4002, S. commune MML4003, M. phaseolina MML4004, F. graminearum MML4005, F. solani MML4006, and F. solani MML4007 which showed the highest percentage of infection in the greenhouse study conducted by the authors earlier at the Centre for Advanced Studies in Botany, University of Madras were subjected for antagonistic activity assay. 2.3. Antagonistic activity of rhizobacteria against rhizome rot pathogens Classical dual culture plate assay (Fokkema, 1978) was performed to screen the rhizobacteria against rhizome rot pathogens. The mycelial disc of each pathogen measuring 8 mm was placed in the center of a PDA plate and four different rhizobacteria were placed equidistantly in the periphery of the plate, leaving a1 cm gap from the plate rim. The plates were incubated for two to five days at room temperature (28 ± 2 °C), after which, the antagonistic activity of rhizobacteria against rhizome rot pathogens was observed as zone of inhibition (ZOI), if any. Among the 157 rhizobacterial isolates, sixteen showed promising antagonistic activity against all seven tested fungal pathogens were selected for further studies. 2.3.1. Mechanisms of biocontrol and plant growth promotion traits exercised by selected rhizobacteria The mechanisms of biocontrol activity, including the plant growth promoting traits exercised by the selected 16 rhizobacterial isolates on suppression of turmeric rhizome rot fungal pathogens, were determined by various in vitro experiments. The competition for nutrition, production of lytic enzymes, antibiotic metabolites, IAA, ammonia, and phosphate solubilization was investigated. 2.3.2. Siderophores production Siderophores production was determined by the chrome azurol S (CAS) assay (Schwyn and Neilands, 1987). The CAS PIPES agar plates streaked with the rhizobacterial isolates were incubated in dark at 28 ± 2 °C for five days. Plates were then observed for a change in the color of the CAS PIPES agar medium from blue to brownish orange, which indicated the production of siderophores by rhizobacterial isolates. 2.3.3. Production of lytic enzymes Nutrient agar (NA)/King’s B Agar (KBA) medium was supplemented with the respective substrate (0.5% gelatin, 0.5% carboxymethyl 56

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carried out as per the method of Cappuccinio and Sherman (2004), and the results were interpreted with the key provided in the Bergy’s Manual of Determinative Bacteriology (1994). Further molecular characterization was done by isolating the genomic DNA of the 16 rhizobacteria based on the method of Arun and Jas Preet (2005), followed by 16S rRNA sequencing to confirm their identity. The 16S rRNA genes of the genomic DNA of the rhizobacterial isolates were amplified with the bacterial universal primers: 27F: 5′AGAGTTTGATCCTGGCT CAG 3′ and 1492 R: 5′ GGTTACCTTGTTACGACTT 3′, which were performed in 25 μL reaction using the following conditions: initial denaturation at 94 °C for 4 min, followed by 35 cycles of denaturation at 94 °C for 1 min, then annealing at 55 °C for 1 min, and finally an extension at 72 °C for 2 min in an automated MyGene™ Peltier Thermal Cycler (MG96G). PCR products were sequenced after purification with the support of a service provider, Eurofins Genomics India Pvt Ltd. Bangalore, India.

cellulose (CMC), 0.1% chitin, and 0.1% laminarin) for the assay of lytic enzymes viz., protease, cellulase, chitinase, and β-1, 3 glucanase. The substrate colloidal chitin for chitinase was prepared by the method of Skujins et al. 1965. The rhizobacterial isolates were streaked separately on respective medium and incubated for two days at room temperature (28 ± 2 °C). Cellulase, chitinase, and β-1, 3 glucanase production was visualized by a translucent zone around the rhizobacterial colony after staining with 0.3% Congo red and destained with 1 N NaCl for 15 min each. Protease activity was visualized as a clear zone around the rhizobacterial colony, after the plates were flooded with saturated ammonium sulfate solution prepared in 0.1 N HCl. 2.3.4. Production of volatile metabolite; hydrogen cyanide (HCN) Nutrient sucrose agar (NSA) medium was used to detect the production of HCN by antagonistic rhizobacteria, as described by Lorck (1948). Filter paper strips saturated with 0.5% picric acid in 2% aqueous sodium carbonate solution were placed on the lid of the test isolate inoculated NSA plates. After two days of incubation, the production of HCN was determined by the change of color of the filter paper from yellow to reddish brown.

2.6. GenBank, NCBI deposit of rhizobacterial sequences The obtained sequences were compared with other bacterial DNA sequences in the National Centre for Biotechnology Information’s (NCBI) GenBank database using the Basic Local Alignment Search Tool (BLAST). The sequences of rhizobacteria were deposited in the GenBank database of NCBI.

2.3.5. Production of antibiotic metabolites All the 16 selected rhizobacterial isolates were grown in 250 mL Nutrient broth at room temperature for two days to obtain the cell-free culture filtrates. The culture filtrates were then extracted with equal volume of ethyl acetate. Extraction of secondary metabolites using methanol from all 16 selected rhizobacterial isolates was done based on the method of Kim et al. (2004). The metabolites present in cell-free filtrates was precipitated by adding 3 N HCl to a final pH of 2.0 and further dissolved in chloroform/methanol solvent system. The ethyl acetate and methanolic crude extracts of antibiotic metabolite thus obtained were loaded separately in the plates using sterile disc method at different concentrations ranging from 50, 100, 150, and 200 µL against the seven fungal pathogens. After 3–7 days, a clear zone of inhibition was observed around the discs, if any.

2.7. Detection of known antibiotic coding genes using PCR The total genomic DNA isolated from the 16 rhizobacteria were used for the detection of biosynthetic genes that encode for the production of antibiotics such as, 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin (PRN), pyoluteorin (PLT), bacillomycin D, fengycin, and the volatile metabolite, HCN. The gene responsible for the production of cellulase enzyme was also analyzed in all the antagonists. Genespecific primers were used to detect the biocontrol related antibiotic biosynthetic genes using PCR. Oligonucleotide primers were synthesized by VBC – Biotech Service GmbH (Brehmstraβe 14 A, A – 1110 Vienna). The primer sets and PCR amplification conditions are given in Table 1. The reference strain, Pseudomonas protegens CHA0, was used as a positive control for the detection of DAPG, PRN, PLT, and HCN antibiotic genes. All the PCR amplifications were performed in a 25 µL reaction mixture containing 1 μL (20 ng) of template DNA, 1 μL (20 pmol) of each primer, 12.5 μL of premix (2× master mix red) containing 2.5 U Taq DNA polymerase, PCR buffer, 1.5 mM MgCl2, and 200 μM dNTPs (Ampliqon, Denmark), as well as 9.5 μL of sterile double distilled water. PCR reactions were performed in an automated MyGene TM Peltier Thermal Cycler (MG96G).

2.4. Plant growth promotion traits 2.4.1. Production of indole acetic acid (IAA) The rhizobacterial isolates were tested for the production of IAA (Gupta et al., 2002). Two drops of o-phosphoric acid were added to 2 mL of each cell free culture supernatant and the development of color was observed. The presence of a pink color showed the production of IAA. 2.4.2. Phosphate solubilization Modified Basal Sperber Medium (MBSM) was prepared (Sperber, 1958), sterilized and poured into sterile Petriplates. The rhizobacteria were streaked on the solidified MBSM and incubated for two days at room temperature. Production of a halo zone around the rhizobacterial growth indicated the phosphate solubilizing ability of the respective bacterium.

2.8. Selection of efficient biocontrol-PGPR for bioformulations Among the 16 rhizobacterial isolates, eight efficient biocontrol PGPR viz., Pseudomonas aeruginosa MML2424, P. aeruginosa MML2515, B. amyloliquefaciens MML2547; Bacillus tequilensis MML2476, B. subtilis MML2406, B. subtilis MML2451, B. subtilis MML2458 and B. subtilis MML2490 were selected for developing talc formulations based on the results of various in vitro experiments regarding their antagonistic activity, biocontrol mechanisms, and plant growth promoting traits.

2.4.3. Production of ammonia All the rhizobacterial isolates were tested for the production of ammonia in peptone water (Cappuccinio and Sherman, 1992). Ammonia production was confirmed by the development of brown to yellow colour by the addition of Nessler’s reagent in each tube after incubation at 36 ± 2 °C for 48–72 h.

2.9. Mass production and preparation of selected PGPR talc formulations The selected eight PGPR were grown in NB/KBB for 28–36 h according to different PGPR at 30 °C. Their uniform growth was maintained and ensured by checking the OD value and was used as bacterial inoculum. Then, the PGPR were grown separately in Hopkins’ flasks containing 2500 mL of NB/KBB for mass production and incubated at room temperature at 220 rpm on a rotary shaker. The PGPR cultures were harvested after 28–36 h, mixed with pre-sterile talc powder at 1:3

2.5. Characterization of rhizobacterial isolates Morphological and physicochemical characterizations of all 16 rhizobacterial isolates were done. Gram staining and different physicochemical tests such as motility, indole, methyl red, Voge’s Proskauer, citrate utilization, oxidase, catalase, and carbohydrate utilization were 57

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Table 1 The primers and the PCR conditions for amplification of genes encode for biocontrol traits. Biocontrol gene

Primers

Sequence and amplification conditions

Cycle (Nos)

References

DAPG

Phl2a Phl2b

5′-GAGGACGTCGAAGACCACCA-3′ 5′-ACC GCAGCATCGTGTATGAG-3′ Initial denaturation: 94 °C for 90 sec 94 °C for 35 sec Renaturation: 53 °C for 30 sec 72 °C for 45 sec Final extension: 72 °C for 10 min

35

Mavrodi et al. (2001)

PLT

PltBf PltBr

5′-CGGAGCATGGACCCCCAGC-3′ 5′GTGCCCGATATTGGTCTTGACCGAG-3′ Initial denaturation: 94 °C for 2 min 94 °C for 1 min Renaturation: 58 °C for 45 sec 72 °C for 1 min Final extension: 72 °C for 10 min

29

Mavrodi et al. (2001)

PRN

Prncf Prncr

5′-CCACAA GCCCGGCCAGGAGC-3′ 5′-GAGAAGAGCGGGTC ATGAAGCC-3 Initial denaturation: 94 °C for 2 min 94 °C for 1 min Renaturation: 58 °C for 45 sec 72 °C for 1 min Final extension : 72 °C for 10 min

30

Mavrodi et al. (2001)

HCN

PM2 PM7-26R

5′-TGCGGCATGGGCGTGTGCCATTGCTGCCTGG-3′ 5′-CCGCTCTTGATCTG-CAATTGCAGGCC-3′ Initial denaturation: 94 °C for 2 min 94 °C for 30 sec Renaturation: 57 °C for 30 sec 72 °C for 60 sec Final extension: 72 °C for 10 min

35

Svercel et al. (2007)

Bacillomycin

BACC1F BACC1R

5′-GAAGGACACGGCAGAGAGTC 5′-CGCTGATGACTGTTCATGCT Initial denaturation : 94 °C for 3 min 94 °C for 1 min Renaturation: 60 °C for 30 sec 72 °C for 1 min 45 sec Final extension: 72 °C for 10 min

35

Ramarathnam et al. (2007)

Fengycin

FEND1F FEND1R

5′-TTTGGCAGCAGGAGAAGTTT 5′-GCTGTCCGTTCTGCTTTTTC Initial denaturation: 94 °C for 3 min 94 °C for 1 min Renaturation: 62 °C for 1 min 72 °C for 1 min 45 sec Final extension: 72 °C for 10 min

45

Ramarathnam et al. (2007)

Cellulase

CelBF CelBR

5′-CCATGGATCATGAGGATGTGAAAACTC 5′-CTCGAGTGAATTGGTTGTCTGAGCTG Initial denaturation: 94 °C for 3 min 94 °C for 30 sec Renaturation: 51 °C for 30 sec 72 °C for 1 min Final extension: 72 °C for 10 min

30

Zafar et al. (2011)

dung manure were used as commercial fungicide, commercial bioproduct and FYM (control), respectively. Each field measured 0.5 acre with 30 cm spacing between rows and 15 cm between plants. Talc formulation of each PGPR was mixed thoroughly with turmeric seed rhizome at 2 kg/ton prior to sowing. Talc formulation of each PGPR at 1 kg/acre was mixed with 100 kg of farm yard manure (FYM) and applied in the soil of respective plot before sowing. Second soil application of talc formulation PGPR was done after 30 days of sowing. All the agronomical practices such as irrigation and weeding were adapted in raising the crop.

(v/w), and air dried aseptically for 24 h. When the moisture content was reduced to approximately 20%, the dried formulation was sieved using a 125 μ test sieve. Carboxymethyl cellulose (CMC) at 10 g/kg was added to the formulation as a sticking agent. The formulations were packed separately in milky propylene bags and labeled with their respective names and stored at room temperature. 2.9.1. Evaluation of talc formulations of PGPR under field conditions The field experiments to study the efficacy of the eight selected PGPR talc formulations for the management of turmeric rhizome rot disease as well as the turmeric plant growth were conducted over two years. The first experiment took place from July 2012 to April 2013, and the second took place from July 2013 to April 2014 in two locations, in farmers’ fields at Vellianaikattur and Salaipudur in Kodumudi taluk, Erode district of Tamil Nadu, India, as they are hotspots for turmeric rhizome rot using randomized block design (RBD) with 12 treatments and three replications. Ridomil Gold, RhizoMats and cow

2.10. Experimental observations Five plants were tagged randomly in each plot to assess the plant growth parameters and score the disease incidence. Growth parameters viz., leaf length, leaf area, shoot length, and shoot diameter were recorded. The rhizome rot incidence was recorded after harvest of the 58

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antagonists to solubilize tricalcium phosphate. All 16 rhizobacterial isolates produced brown color when Nessler’s reagent was added, which indicated that all the selected antagonists were able to produce ammonia (Table 2).

turmeric crop using the modified scale of Bowen (2007). The crop was harvested after maturity; rhizomes were separated and cleaned; and the fresh weight was recorded. The dry weight of the rhizome was also estimated after proper drying. Curcumin is a potent component of turmeric and the rhizome quality is to be determined based on the curcumin content. Hence, the extraction and quantification of curcumin from the turmeric rhizomes of different treatments was done according to the methods of Bagchi (2012) and Kumar et al. (2014).

3.4. Characterization of selected rhizobacterial isolates Most of the isolates (13) are Gram positive rod-shaped motile bacteria, whereas the isolates MML2424, MML2515, and MML2519 are Gram negative rod-shaped motile bacteria. All 16 rhizobacterial isolates showed positive reactions for the catalase test and all the antagonists not including MML2533 showed a positive reaction for the oxidase test. Fourteen rhizobacterial isolates utilized citrate as a sole source of carbon. Different types of carbohydrates utilized by the antagonistic rhizobacteria were documented. The genomic DNA of the 16 antagonistic bacteria were obtained and visualized. The PCR products approximately ranging from 1380 to 1450 bp were amplified for all 16 bacterial isolates from their 16S rRNA gene regions. The rhizobacterial 16S rRNA sequences obtained were deposited in the GenBank database of NCBI with the accession numbers from KJ655538 to KJ655553. Among the 16 rhizobacteria, MML2424, MML2515, and MML2519 exhibited higher similarity to Pseudomonas aeruginosa. Two of them, MML2522 and MML2547 showed maximum similarity to Bacillus amyloliquefaciens. The sequence of the rhizobacterial isolate MML2476 was identical to Bacillus tequilensis. The isolate MML2533 had a highly similar sequence to that of Bacillus cereus. The other nine isolates were found to be identical with Bacillus subtilis.

2.11. Statistical analyses The field experimental data were tabulated and analyzed for variance (one way ANOVA) using ASSP (Agres Statistical Software Package) software. The least significant difference (LSD) analysis was performed to separate the group mean when ANOVAs were significant at p = 0.05. 3. Results 3.1. Isolation and morphological characterization of rhizobacteria A total of 157 rhizobacterial isolates were isolated from turmeric rhizosphere soil samples using Nutrient agar and King’s B agar media as pure cultures. The population of rhizobacteria in turmeric rhizosphere soils ranged from 1.7 × 103 cfu/g to 6.7 × 103 cfu/g. The soil pH of the collected samples ranged from 5.8 to 7.2. Some fluorescent pseudomonads (FPs) and many non-fluorescent rhizobacteria were isolated from the turmeric rhizosphere soils. All 157 rhizobacterial isolates were designated sequentially from MML2401 to MML2557 and their morphological characteristics were recorded.

3.5. Detection of known antibiotic coding genes using PCR The Phl2a/Phl2b primers specific for the DAPG biosynthetic gene amplified the expected 745 bp DAPG gene in the DNA of the PGPR strain, P. aeruginosa MML2519, as that of the positive control strain, P. protegens CHA0. The PLT biosynthetic gene specific primers amplified the expected 773 bp product of the positive control strain, P. protegens CHA0. However, none of the PGPR strains showed amplification for the fragment of pltB gene. The gene-specific primers PrnCf and PrnCr amplified the expected 719-bp fragment of prnC from the DNA of the PGPR strain, P. aeruginosa MML2515, as that of the positive control, P. protegens CHA0. The PM2/PM7-26R primers specific for HCN amplified the expected 570 bp fragment of hcnAB gene in the DNA of the PGPR strain, P. aeruginosa MML2424 and in the positive control, P. protegens CHA0. For bacillomycin D, the PGPR strains, B. subtilis MML2406 and B. amyloliquefaciens MML2547, amplified the specific 875 bp product of the bacillomycin D biosynthetic gene. The amplification product of the isolates B. subtilis MML2406 and B. amyloliquefaciens MML2547, when sequenced and blast-searched, yielded very high similarity to the sequence of bacillomycin D operon of B. subtilis (GenBank accession No.: AY137375). The fengycin biosynthetic gene cluster primers FEND1F/ FEND1R amplified the expected 964 bp biosynthetic gene in the DNA of the PGPR strain, B. subtilis MML2490. The amplified 964 bp product of B. subtilis MML2490 was found to be homologous to the fenD gene of B. subtilis (GenBank accession No.: AJ011849) when sequenced and searched in the GenBank. The CelBF and CelBR primers specific for the cellulase gene amplified the expected 1650 bp biosynthetic gene in the DNA of the PGPR strains, B. subtilis MML2415, P. aeruginosa MML2424, B. subtilis MML2451, B. subtilis MML2473, B. subtilis MML2490, and P. aeruginosa MML2515.

3.2. Antagonistic activity and biocontrol mechanisms of rhizobacteria against turmeric rhizome rot pathogens The zone of inhibition (ZOI) against rhizome rot fungal pathogens ranged between 1 and 15 mm. Most of the rhizobacteria showed antagonistic activity against at least any one or more of the fungal pathogens. Among the 157 rhizobacteria tested, 51 rhizobacteria did not show antagonistic activity were not antagonistic to any of the turmeric rhizome rot fungal pathogens while 16 exhibited antagonistic activity to all seven rhizome rot fungal pathogens and were selected for further studies (Fig. 1). Among the 16 rhizobacterial antagonists tested, six rhizobacteria (38%) (MML2515, MML2518, MML2519, MML2522, MML2533, and MML2547) produced siderophores as they changed from the blue color CAS PIPES agar medium to light brownish orange. Fourteen isolates, not including MML2515 and MML2519, produced proteolytic activity in gelatin amended agar medium. Thirteen isolates, not including MML2424, MML2515, and MML2519 produced cellulase in Carboxymethyl cellulose (CMC) amended agar medium. Eight rhizobacterial isolates produced measurable chitinase in colloidal chitin amended agar medium. None of the antagonists produced measurable β-1, 3 glucanase in laminarin amended agar medium. Among the 16 rhizobacterial antagonists tested, only one antagonist, MML2424, has cyanogenic ability to produce HCN (Table 2). Antifungal activity of the ethyl acetate and methanolic extracts of 16 rhizobacteria produced varied ranges of zone of inhibition (ZOI) against the seven tested fungal pathogens in disc diffusion assay. 3.3. Plant growth promotion traits

3.6. Selection of efficient biocontrol-PGPR for bioformulations Five isolates namely, MML2424, MML2515, MML2518, MML2522, and MML2547 were able to produce the growth promoting phytohormone IAA. Among the 16 rhizobacterial antagonists tested, three isolates, MML2424, MML2515, and MML2519 produced a halo zone in the Modified Basal Sperber medium, which indicated the ability of the

Among the eight selected isolates, P. aeruginosa MML2424 and P. aeruginosa MML2515 are good producers of IAA and ammonia, and are able to solubilize phosphate. B. tequilensis MML2476 and three B. subtilis strains (i.e. MML2406, MML2451, and MML2490) produced lytic 59

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Fig. 1. Antagonistic activity of rhizobacteria against turmeric rhizome rot pathogens. Inhibition of turmeric rhizome rot pathogens growth by PGPR. Four PGPR were streaked in the periphery of each plate against a rhizome rot pathogen in centre. A-E: Rhizome rot pathogens (A: R. solani MML4001; B: F. solani MML4002; C: S. commune MML4003; D: M. phaseolina MML4004; E: F. graminearum MML4005; F: F. solani MML4006; G: F. solani MML4007).

FYM control in field-1. In field-2 also, T2 significantly (p = 0.05) increased the shoot length (100.61%), shoot diameter (68.75%), leaf length (41.64%), and leaf area (50.1%) compared to FYM control followed by B. subtilis MML2490 and B. amyloliquefaciens MML2547 (data not shown). All the PGPR treatments significantly (p = 0.05) reduced the rhizome rot disease incidence in turmeric, compared to farmers’ practice and FYM control. Notably, among the PGPR formulations, the formulation of P. aeruginosa MML2424 showed the least disease score (10.5%) followed by B. subtilis MML2490 (17.0%) and B. amyloliquefaciens MML2547 (17.5%) in field-1. Similarly, in field-2, formulation of P. aeruginosa MML2424 showed least rhizome rot disease (10.53%) followed by B. subtilis MML2490 (13.8%) and B. amyloliquefaciens MML2547 (18.43%) (Table 3). Among the PGPR treatments, T2 (P. aeruginosa MML2424) significantly (p = 0.05) increased the turmeric yield parameters followed by T6 (B. subtilis MML2490) and T8 (B. amyloliquefaciens MML2547) (Table 4). The curcumin content was also increased to 42.35% and 66.99% in the rhizomes of T2 (P. aeruginosa MML2424) treatment

enzymes and showed excellent antagonistic activity against all seven fungal pathogens. The methanolic extracts of B. subtilis MML2458 showed better antifungal activity against the fungal pathogens. B. amyloliquefaciens MML2547 was a producer of siderophore, protease, cellulase, and the ethyl acetate extracts showed better inhibition of the fungal pathogens in the disc diffusion assay. All the above PGPR strains have been selected, mass multiplied and developed talc based bioformulations as described in the Materials and Methods. The talc formulations of all eight selected PGPR exhibited shelf life of more than 90 days when stored at room temperature. 3.7. Efficacy of talc formulations of PGPR under field conditions Application of PGPR talc formulations significantly improved the plant growth in turmeric, compared to FYM control in field conditions. Among the treatments evaluated, T2 (P. aeruginosa MML2424) significantly (p = 0.05) increased the shoot length (14.7%), shoot diameter (68.60%), leaf length (41.96%), and leaf area (50%) compared to Table 2 Biocontrol and PGP traits in the antagonistic rhizobacterial isolates. Antagonist

MML2406 MML2411 MML2415 MML2424 MML2451 MML2458 MML2473 MML2476 MML2483 MML2490 MML2515 MML2518 MML2519 MML2522 MML2533 MML2547

Zone of clearance (mm) Protease

Cellulase

Chitinase

β-1,3 glucanase

7 6 10 11 4 14 10 10 3 6 − 5 − 3 7 10

5 3 3 − 5 6 6 3 5 6 − 7 − 3 5 10

3 2 3 − 3 − 3 2 3 2 − − − − − −

− − − − − − − − − − − − − − − −

60

HCN

Siderophore

IAA

Phosphate solubilization

Ammonia

− − − + − − − − − − − − − − − −

− − − − − − − − − − + + + + + +

− − − + − − − − − − + + − + − +

− − − + − − − − − − + − + − − −

+ + + + + + + + + + + + + + + +

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Table 3 Effect of talc formulations of PGPR on rhizome rot disease reduction in turmeric. Treatment No.

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 CD (p = 0.05)

Treatment

B. subtilis MML2406 P. aeruginosa MML2424 B. subtilis MML2451 B. subtilis MML2458 B. tequilensis MML2476 B. subtilis MML2490 P. aeruginosa MML2515 B. amyloliquefaciens MML2547 Commercial fungicides Commercial bioproduct Farmer’s practice FYM (control)

Disease incidence (%)

Disease reduction compared to Farmers’ practice (%)

Disease reduction compared to FYM (%)

Field-1

Field-2

Field-1

Field-2

Field-1

Field-2

17.83b ± 2.25 10.5a ± 1.30 23.9c ± 2.76 20.03c ± 1.73 26.6d ± 3.17 17.0b ± 0.86 29.33d ± 2.75 17.5b ± 1.00 21.93c ± 2.00 15.36b ± 1.76 33.1d ± 3.00 40.26e ± 4.02 2.577

23.3b ± 2.25 10.53a ± 1.78 26.53c ± 2.50 25.0c ± 2.29 28.13c ± 2.01 13.8a ± 1.75 30.1b ± 2.81 18.43b ± 1.50 22.5c ± 2.50 17.5b ± 1.95 39.83d ± 4.64 43.43d ± 3.76 3.440

46.12 68.28 27.79 39.48 19.64 48.64 11.38 47.13 33.74 53.58 − −21.65 –

41.50 73.55 33.38 37.23 29.37 65.35 24.43 53.72 43.51 56.06 − −9.05 –

55.71 73.92 40.65 50.25 33.94 57.78 27.15 56.54 45.53 61.84 17. 80 – –

46.35 75.75 38.91 42.44 35.22 68.22 30.69 57.56 48.19 59.71 8.28 – –

Values on disease incidence are mean of three replicates with SD. Values with different alphabetical letters in superscript are significant at p = 0.05.

compared to Farmers’ practice and FYM control, respectively in field-1 (Table 5). Similar results were also recorded in field-2.

investigation, some bacterial isolates showed high inhibition on fungal pathogens, whereas others showed less activity in the dual culture plate assay. Among the 157 rhizobacteria, 16 were antagonistic to all seven rhizome rot fungal pathogens, whereas seven rhizobacteria were antagonistic to six fungal pathogens and these bacteria were unable to control any one of the seven fungal pathogens. Some of the rhizobacteria were antagonistic to any one or up to five of the fungal pathogens. Similar results have previously been reported by other researchers (Eredogan and Benlioglu, 2010; Zheng et al., 2011). In most of the studies, it has been observed that a significant difference existed among different strains of the same genera isolated from the same ecological niche in terms of their antagonistic behavior against a particular pathogen (Prashar et al., 2013). As the seven turmeric rhizome rot fungal pathogens belong to different genera or different strains of the same genera, obviously there might be divergence in the antagonistic property of the rhizobacterial isolates towards these varied ranges of rhizome rot fungal pathogens. The expression of different biocontrol mechanisms such as lytic enzymes or other effective antifungal compounds may explain this disparity among the antagonistic rhizobacterial isolates. The 16 selected turmeric rhizosphere antagonists exerted different mechanisms of biocontrol to control their rhizome rot fungal pathogens along with plant growth promotion traits. Production of cell wall degrading enzymes is one of the major mechanisms used by biocontrol agents to control fungal phytopathogens (Chet et al., 1990; Mathivanan

4. Discussion Recently, several reviews have discussed the plant-driven selection of bacteria as an important issue (Hartmann et al., 2009; Doornbos et al., 2012; Drogue et al., 2013; Bulgarelli et al., 2013). Since root exudate composition changes along the root system, according to the stages of plant development and to plant genotypes, the rhizo-microbiome composition also differs (Berg and Smalla, 2009; Aira et al., 2010; Bulgarelli et al., 2013; Chaparro et al., 2013). Accordingly, in this study, the enormous untapped potential of the PGPR from the vicinity of the turmeric roots or the rhizosphere was explored in order to control the rhizome rot fungal pathogens and to improvise the plant growth. Isolation of rhizobacteria from the turmeric rhizosphere is also in accordance with Pliego et al. (2011), who suggested that it may be practical to isolate microorganisms from agroecosystems under various farm management practices. This finding may aid in choosing microorganisms that are compatible with certain farm management practices such as fertilizer and pesticide applications and tillage practices. An in vitro pre-screening test of dual culture allowed Cazorla et al. (2007) to select four B. subtilis strains, PLC1605, PCL1608, PCL1610, and PCL1612, with noticeable antifungal activity against Rosellinia necatrix and other soil-borne phytopathogenic fungi. In the present Table 4 Effect of talc formulations of PGPR on rhizome yield in turmeric. Treatment No.

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 CD (p = 0.05)

Treatment

B. subtilis MML2406 P. aeruginosa MML2424 B. subtilis MML2451 B. subtilis MML2458 B. tequilensis MML2476 B. subtilis MML2490 P. aeruginosa MML2515 B. amyloliquefaciens MML2547 Commercial fungicides Commercial bioproduct Farmer’s practice FYM (control)

Rhizome yield (t/acre)

Yield increase compared to Farmers’ practice (%)

Yield increase compared to FYM (%)

Field-1

Field-2

Field-1

Field-2

Field-1

Field-2

5.27d ± 0.18 7.70a ± 0.12 4.45e ± 0.45 4.45e ± 0.46 4.17f ± 0.21 7.06b ± 0.17 4.25f ± 0.22 6.11c ± 0.13 4.46e ± 0.18 6.13c ± 0.21 4.08f ± 0.24 3.19f ± 0.36 0.464

33.73 48.59 16.53 24.78 13.87 41.10 6.13 37.02 4.97 41.48 – −5.06 –

29.17 88.62 9.01 8.99 2.28 73.10 4.26 49.70 9.39 50.31 – −21.76 –

40.86 56.51 22.74 31.44 19.94 48.63 11.79 44.33 10.56 49.02 5.33 – –

65.10 141.09 39.33 39.30 30.73 121.26 33.26 91.34 39.82 92.12 27.82 – –

6.25b ± 6.95a ± 5.45c ± 5.83c ± 5.32d ± 6.60a ± 4.96d ± 6.40a ± 4.91d ± 6.61a ± 4.67e ± 4.44e ± 0.468

0.27 0.16 0.31 0.40 0.31 0.10 0.16 0.10 0.21 0.31 0.30 0.41

Values on rhizome yield are mean of three replicates with SD. Values with different alphabetical letters in superscript are significant at p = 0.05. 61

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Table 5 Effect of talc formulations of PGPR on curcumin content in turmeric. Treatment No.

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 CD (p = 0.05)

Treatment

B. subtilis MML2406 P. aeruginosa MML2424 B. subtilis MML2451 B. subtilis MML2458 B. tequilensis MML2476 B. subtilis MML2490 P. aeruginosa MML2515 B. amyloliquefaciens MML2547 Commercial fungicides Commercial bioproduct Farmer’s practice FYM (control)

Curcumin content (%)

Curcumin content increase compared to Farmers’ practice (%)

Curcumin content increase compared to FYM (%)

Field-1

Field-1

Field-2

Field-1

Field-2

3.28a 3.47a 3.11b 3.25a 3.09b 3.39a 2.82b 3.36a

Field-2

± ± ± ± ± ± ± ±

0.163 0.149 0.196 0.212 0.261 0.117 0.265 0.159

3.23a 3.38a 3.16a 3.35a 3.28a 3.27a 2.86b 3.32a

± ± ± ± ± ± ± ±

0.143 0.055 0.162 0.153 0.250 0.081 0.230 0.128

34.60 42.35 27.73 33.32 26.71 39.07 15.85 37.83

16.29 21.82 13.79 20.50 18.11 17.63 2.88 19.59

57.90 66.99 49.84 56.39 48.64 63.14 35.90 61.68

17.99 23.60 15.45 22.26 19.82 19.34 4.38 21.34

3.15a ± 3.08b ± 2.44c ± 2.08c ± 0.332

0.293 0.195 0.160 0.217

3.08b ± 3.27a ± 2.78c ± 2.74c ± 0.273

0.140 0.156 0.230 0.290

29.10 26.37 – −14.75 –

10.91 17.79 – −1.44 –

51.44 48.24 17.31 – –

12.53 19.51 1.46 – –

Values on curcumin content are mean of three replicates with SD. Values with different alphabetical letters in superscript are significant at p = 0.05.

concluded that the 16 rhizobacterial isolates from the turmeric rhizosphere might be a pool of various secondary metabolites, to be further explored in detail. P-solubilizing bacteria (PSB) have already been applied in agronomic practices as potential bioinoculants to increase crop productivity. In India, a consortium, Microphos culture, was developed by the Indian Agricultural Research Institute (IARI), which contains two very efficient P-solubilizing bacteria (Pseudomonas striata and Bacillus polymyxa) (Gaur, 1990). Three of our antagonists, MML2424, MML2515, and MML2519, were able to solubilize tricalcium phosphate. Generally, IAA secreted by rhizobacteria increases root surface area and length, and thereby provides the plant greater access to soil nutrients. Also, rhizobacterial IAA loosens plant cell walls, and as a result facilitates an increasing amount of root exudation that provides additional nutrients to support the growth of rhizosphere bacteria (Glick, 2012). In this study, five antagonists produced different shades of pink color which indicated the production of phytohormone, IAA. Ammonia production is another important trait of PGPR that indirectly influences the plant growth. There is indirect evidence of usefulness of free-living N2-fixing bacteria in crop improvement under tropical and subtropical conditions, especially with strains excreting high amounts of ammonia, in addition to a variety of growth promoting factors in the presence of carbon rich root exudates (Narula and Gupta, 1987). Interestingly, in this study, all the selected antagonists were able to produce ammonia. Similarly, ammonia production by all the tested rhizobacterial isolates was reported by Malleswari and Bagyanarayana (2013) and Bumunang and Babalola (2014). Most of the selected turmeric rhizosphere isolates of the current study were identified as Bacillus spp., except P. aeruginosa. The microbial populations interact with each other and their host plant through the actions of secreted metabolites (Kim et al., 2011). Therefore, in this study, the higher population of Bacillus spp. in the turmeric rhizosphere suggested that the turmeric root exudates may favour the growth of Bacillus spp. due to various secondary metabolites of them or vice versa. However, the impact of these secreted metabolites of Bacillus spp. on the turmeric plant health and growth and the multifactorial nature of these kinds of interactions must be studied elaborately. Despite the importance of antibiosis in biological control, little is known about the genes involved in antifungal activity. PCR based amplification using gene-specific primers of biosynthetic genes for various antibiotics is highly valuable in assessing whether a given strain produces a particular antibiotic or not (Ramarathnam et al., 2007). The biocontrol agents, best characterized at the molecular level belong to the genus Pseudomonas (Bloemberg and Lugtenberg, 2001). In this

et al., 1998; Kobayashi et al., 2002). The complexity of the fungal cell wall makes it a formidable challenge as a primary target for bacterial attack, as bacteria would need to rapidly produce a wide variety of exoenzymes to degrade cell wall components to the level needed to compromise structural integrity (de Boer et al., 2005) and enhance biocontrol efficacy (Someya et al., 2007). Rhizospheric bacterial community associated with GM and non-GM maize crops was studied for plant growth promoting (PGP) characteristics and biocontrol characteristics, especially fungal cell wall hydrolyzing enzymes such as cellulase, chitinase, protease, pectinase, and lipase by Ahmad et al. (2013). In the present study also, production of these cell wall-degrading enzymes has been considered as an important criterion in the selection of efficient PGPR and interestingly, among the 16 antagonists, 88% showed protease production, 81% showed cellulase production, and 50% showed chitinase production. However, in the present study, none of the 16 antagonists produced β-1,3 glucanase in laminarin amended agar medium. This result was similar to with the findings of Ruchi et al. (2012), who isolated 26 Pseudomonas strains from the rhizosphere of apple and pear, and screened for the production of various PGPR activities and lytic enzymes production viz., protease, chitinase, and glucanase. Out of 26 isolates, none of them showed β-1, 3 glucanase activities. Siderophores help the microorganisms to compete against fungal pathogens for available iron and siderophore-mediated Fe uptake by the plants as a result of siderophore producing rhizobacterial inoculations have been well documented (Rajkumar et al., 2010). In the present study, among the 16 rhizobacterial antagonists tested, six antagonists were siderophore producers and hence, competitors for iron in their environment. However, only one antagonist, MML2424, has the ability to produce HCN, which is believed to account for a substantial part of the growth inhibition or death of the pathogen/pest caused by HCN producing organisms (Blumer and Haas, 2000). However, there were investigations reporting harmful effects of HCN on plants, such as inhibition of energy metabolism of potato root cells (Bakker and Schippers, 1987), and reduced root growth in lettuce (Alström and Burns, 1989). Hence, it is necessary to study the effect of HCN production of the isolate MML2424 upon the growth of turmeric plants in the future. The bio-efficacy of antifungal metabolites of rhizobacteria isolated from the turmeric ecosystem depicts that the ethyl acetate extracts of most of the antagonists were ineffective when compared to the methanolic extracts of the antagonists. In contrast to these results, the ethyl acetate extracts of four bacterial antagonists, namely MML2519, MML2522, MML2533, and MML2547, were able to highly reduce the mycelial growth of F. solani MML4007. Hence, it can be 62

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tomatoes (Kandan et al., 2005). In addition to plant growth promotion, rhizome rot disease control, and rhizome yield, the treatments of different PGPR strains have significantly increased the curcumin content in turmeric compared to FYM treated control. Importantly, curcumin is a potent component of turmeric and rhizome quality is determined based on the curcumin content. A similar increase of curcumin content was recorded following the treatment of Azotobacter chroococcum in turmeric (Kumar et al., 2014). The reason for curcumin content increase due to treatment of PGPR has not been studied in the present investigation. However, Kumar et al. (2014) hypothesized that the turmeric rhizome contains a number of phenolic compounds, such as curcuminoids and sesquiterpenoids, and these exudates likely attract beneficial bacteria towards turmeric roots to colonize effectively, resulting in the enhancement of curcumin synthesis. In light of the results obtained from this study, most of the PGPR, particularly P. aeruginosa MML2424 and B. subtilis MML2490, appear promising, as do other PGPR strains for commercialization, since they significantly improved all the plant growth parameters and reduced the rhizome rot disease percentage. Furthermore, the PGPR enhance the rhizome yield and curcumin content compared to farmers’ current practices and FYM control. Therefore, their talc formulations would be ideal biocontrol-PGPR products, which can be used for plant growth promotion and management of turmeric rhizome rot disease.

study, the biosynthetic genes that encode for the production of the most crucial anti-fungal metabolites, 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin, pyoluteorin, and the volatile antifungal compound HCN, were analyzed in three P. aeruginosa PGPR strains isolated from the turmeric rhizosphere. In this study, the gene-specific primers Phl2a/ Phl2b, PrnCf/PrnCr, and PM2/PM7-26R exhibited the presence of known antibiotic genes DAPG in the PGPR strain, P. aeruginosa MML2519; PRN in P. aeruginosa MML2515; and HCN in P. aeruginosa MML2424 respectively, as that of the positive control strain, P. protegens CHA0. However, none of the three tested PGPR strains in this study showed amplification for the fragment of pltB gene (PLT) with the specific PltBf/PltBr primers. This finding is similar to the results of Zhang et al. (2006) for the tested strain P. chlororaphis PA23. Bacillomycin and fengycin were shown to act against phytopathogenic fungi in a synergistic manner (Chen et al., 2009). In the present study, the BACC1F/BACC1R primers identified the PGPR strains, B. subtilis MML2406 and B. amyloliquefaciens MML2547, as potential bacillomycin D producers among the thirteen Bacillus strains. Similarly, the gene-specific primers FEND1F and FEND1R amplified the expected 964 bp fengycin biosynthetic gene in the DNA of B. subtilis MML2490 only. Additionally in the present study, the PCR amplification of cellulase gene identified six potential cellulolytic bacteria. Even though 13 Bacillus spp. were obtained in this study from the turmeric rhizosphere, only a few possess the fengycin and bacillomycin D gene. This result supports earlier findings of Athukorala et al. (2009). They stated that the very low percentage of fengycin and bacillomycin D producing bacteria among the studied isolates indicated that a comparatively low percentage of bacteria in the environment have the ability to produce these antibiotics and, within the same species, the capability is rather variable. Another reasonable explanation for this observation among the Pseudomonas or Bacillus PGPR strains might be due to the failure of the PCR method or the primers. However, this study can be considered to provide valid data confirming the presence of antibiotic coding genes of some efficient PGPR, antagonistic to fungal pathogens of turmeric rhizosphere. Bacterial plant growth promotion is a well-established and complex phenomenon that is often achieved by the activities of more than one PGP trait exhibited by plant associated bacteria. Many isolates from this study were able to exhibit more than three PGP traits, which may promote plant growth directly, indirectly, or synergistically. Similar to this finding, the existence of multiple biocontrol mechanisms among PGPR have been reported by several other researchers. However, such findings on indigenous isolates of India are less commonly explored (Farah et al., 2006; Ahmad et al., 2008). Therefore, in the present investigation, attempts were made to select strains exhibiting the highest number of traits associated with the PGP ability of the target crop, turmeric under in vitro conditions, as a prelude to their use at field level. Hence, eight efficient biocontrol-PGPR were selected for developing bioformulations based on the results of various in vitro experiments. Different genera of bacteria have been studied as PGPR; however, investments in the research and development of bioproducts have been higher in projects on Pseudomonas and Bacillus (Figueiredo et al., 2010). This explains their potentiality as successful biocontrol products in the field. The eight selected PGPR in this study also belong to the same group and might be considered as the ideal choice for the control of rhizome rot in the field as bioformulations. Researchers have already established that the strains of Bacillus and Pseudomonas improved plant growth in different crops (Ryu et al., 2005; Weller and Cook, 1983). Accordingly, in this study, P. aeruginosa MML2424, B. subtilis MML2490, and B. amyloliquefaciens MML2547 significantly improved the plant growth and reduced the disease incidence in turmeric compared to farmers’ practice and FYM control in field conditions. The yield parameters in turmeric were also increased noticeably by these treatments. Similarly, Bacillus and Pseudomonas spp. have been reported to increase the plant growth parameters in sunflowers (Srinivasan et al., 2009), rice (Shanmugaiah et al., 2006) and

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