Isolation, screening, characterization, and selection of superior rhizobacterial strains as bioinoculants for seedling emergence and growth promotion of Mandarin orange (Citrus reticulata Blanco).

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Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Laval on 06/17/14 For personal use only.

Isolation, screening, characterization, and selection of superior rhizobacterial strains as bioinoculants for seedling emergence and growth promotion of Mandarin orange (Citrus reticulata Blanco) Elizabeth Thokchom, Mohan Chandra Kalita, and Narayan Chandra Talukdar

Abstract: Mandarin orange (MO) is an important fruit crop of tropical and subtropical regions of the world. A total of 217 morphologically distinct rhizobacteria from MO orchards in 3 states of northeastern India were isolated and analyzed for 4 plant-growth-promoting (PGP) attributes: nitrogen fixation, production of indole acetic acid like substances, solubilization of phosphate, and ability to antagonize pathogenic fungi. Isolates were ranked based on in-vitro-assayed PGP attributes, and 10 superior isolates were selected to test their effect on seedling emergence and seedling growth in a completely randomized pot experiment. These 10 isolates increased seedling emergence over a noninoculated control within 45 days after sowing. Five isolates, namely RCE1, RCE2, RCE3, RCE5, and RCE7, significantly increased shoot length, shoot dry biomass, and root dry biomass of 120-day-old seedlings over the noninoculated control. The beneficial effects of 4 selected strains, namely Enterobacter hormaechei RCE-1, Enterobacter asburiae RCE-2, Enterobacter ludwigii RCE-5, and Klebsiella pneumoniae RCE-7, on growth of the seedlings were visible up to 1 year after their transfer to 8 kg capacity pots. These strains were superior both in terms of in-vitro-assayed PGP attributes and of their beneficial effect in low phosphorus soil and, thus, may be promising bioinoculants for promoting early emergence and growth of MO seedlings. Key words: mandarin orange rhizosphere, PGPR, 16S rRNA gene, Enterobacter, P solubilization. Résumé : La mandarine est un important fruit cultivé dans les zones tropicales et subtropicales. On a isolé un total de 217 rhizobactéries morphologiquement distinctes de mandarineraies situées dans trois états du nord-est de l’Inde, et l’on a analysé chez elles 4 attributs de stimulation de la croissance végétale (SCV), soit la fixation de l’azote, la production de substances apparentées a` l’acide indole-acétique, la solubilisation de phosphates et la faculté de contrer les champignons pathogènes. Les isolats ont été classés selon leurs attributs SVC analysés in vitro et les 10 isolats les plus performants ont été retenus pour que l’on établisse leur impact sur l’émergence et la croissance des plantules dans une expérience en pot complètement randomisée. Ces 10 isolats ont accéléré l’émergence des plantules par rapport au témoin non inoculé dans les 45 jours qui suivirent l’ensemencement. Cinq isolats, a` savoir RCE1, RCE2, RCE3, RCE5 et RCE7, ont su accroître la longueur de la pousse et la biomasse sèche des pousses et des racines de plantules de 120 jours dans une proportion significative par rapport au témoin non inoculé. Les bienfaits de 4 souches sélectionnées, a` savoir Enterobacter hormaechei RCE-1, Enterobacter asburiae RCE-2, Enterobacter ludwigii RCE-5 et Klebsiella pneumoniae RCE-7 sur la croissance des plantules sont demeurés visibles jusqu’a` 1 an après leur transplantation dans des pots d’une capacité de 8 kg. Ces souches étaient supérieures tant au chapitre de leurs attributs SCV analysés in vitro que de leurs effets bénéfiques en sol pauvre en phosphore, et seraient donc des inoculants biologiques prometteurs aptes a` accélérer l’émergence et la croissance des plantules de mandariniers. [Traduit par la Rédaction] Mots-clés : rhizosphère de la mandarine, RSCV, gène de l’ARNr 16S, Enterobacter, solubilisation du P.

Introduction Mandarin orange (MO) (Citrus reticulata Blanco) is an important fruit, commercially cultivated in subtropical and tropical regions of the world including the northeastern states of India. Most MO orchards of the northeastern states are of nucellar seed origin and generally bear fruits for a long time, up to 100 years. Although grafted MO plants are more productive, farmers are reluctant to shift to grafted plant orchards, as grafted trees bear fruits for a short duration (20–25 years). However, perennial orchards suffer gradual yield decline for several reasons, with poor nutrient management being a major one (Ghosh 2007; Srivastava et al. 2010).

Organic nutrient management or integrated nutrient management is recommended for maintaining soil and environment quality and also for obtaining better fruit quality of MO orchards (Abd El Migeed et al. 2007; Medhi et al. 2007; Srivastava et al. 2010; Abdel-Hak et al. 2012). In organic nutrient management or integrated nutrient management, inocula of plant-growth-promoting rhizobacteria (PGPR) may be an important component for nutrient management in both seed origin nurseries and perennial orchards. PGPR promote the growth of plants by multiple mechanisms, such as nutrient solubilization and fixation, stress relief, production of plant hormones and antibiotics, and suppression of plant

Received 19 August 2013. Revision received 18 December 2013. Accepted 18 December 2013. E. Thokchom and N.C. Talukdar. Institute of Bioresources and Sustainable Development, Takyelpat Institutional Area, Imphal-795001, Manipur, India. M.C. Kalita. Department of Biotechnology, Gauhati University, Guwahati-781 014, Assam, India. Corresponding author: N.C. Talukdar (e-mail: [email protected]). Can. J. Microbiol. 60: 85–92 (2014) dx.doi.org/10.1139/cjm-2013-0573

Published at www.nrcresearchpress.com/cjm on 20 December 2013.

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Table 1. Soil chemical characteristics of Mandarin orange orchards of 3 sites. Physicochemical properties Site

C:N ratio

pH

TOC (g·kg−1)

TN (ppm)

TP (ppm)

TK (ppm)

AvlP (ppm)

I (Tamenglong) II (Tinsukia) III (Shillong)

9.2±1.1 11.5±2.1 6.2±0.6

6.3±0.3 6.2±0.1 6.1±0.07

23.3±3.3 13.7±1.0 15.3±2.1

2556.7±388.7 1292.9±336.2 2481.7±142.5

110.0±50.0 150.0±30.0 170.0±50.0

296.8±98.1 296.1±18.6 314.5±36.4

17.8±3.0 10.0±3.0 9.0±1.0


Note: Values are mean of 3 replicates ± standard error. TOC, total organic carbon; TN, total nitrogen; TP, total phosphorus; TK, total potassium; AvlP, available phosphorus.

pathogens by induction of plant defenses and (or) out-competition of pathogens (Kloepper et al. 1989; Lugtenberg and Kamilova 2009). PGPR inoculants are receiving increased attention for use in agriculture (Pandey et al. 1998; Nelson 2004; Choudhary and Johri 2008). Rhizobacteria of cereal crops have been studied extensively, and inocula of efficient strains are commercially exploited (Thakuria et al. 2003; Khalid et al. 2004; Park et al. 2005; Anderson and Habiger 2012). However, studies on rhizobacteria and the endophytic bacterial community of perennial crops, including fruit tree species, have been limited. Some recent studies reported on the rhizobacteria and endophytic community of grapes (Aballay et al. 2011), strawberry (Berg et al. 2002), potato (Sessitsch et al. 2004), banana (Thomas and Soly 2009), tea (Mazumdar et al. 2007), avocado tree (Nadeem et al. 2012), a few forest species (Izumi et al. 2008; Procopio et al. 2009; Taghavi et al. 2009), and a few species of citrus (Araujo et al. 2002; Trivedi et al. 2011). So far, there is no report on the isolation of indigenous PGPR from MO rhizospheres (MORs). The objective of this study was to isolate and characterize beneficial bacteria that are present in MORs and to screen efficient PGPR isolates for enhancement of emergence and growth of MO seedlings in pot experiments. We focused on the identification of potential PGPR isolates by using selective culture media, by characterizing their salient metabolic features, and by determining the identities of selected bacteria using 16S rRNA gene sequencing. Our approach was to first screen the novel plant-growth-promoting (PGP) isolates of MOR soils by conducting in vitro qualitative and quantitative assays for traits related to (i) mineral nutrition, including phosphate (P) solubilization and nitrogen (N) fixation, (ii) the production of growth hormones like indole acetic acid (IAA), and (iii) antagonism to soil-borne fungal plant pathogens. Subsequently, we tested a few efficient strains in pot experiments conducted in a greenhouse using seeds of MO as the test crop and natural soil as substrate for bioinoculum development.

Materials and methods Soil sample collection Roots and rhizosphere soil samples were collected from 3 MO (Citrus reticulata Blanco) orchard locations: Thangal in the Tamenglong district of Manipur (India), Borgaon in the Tinsukia district of Assam (India), and Umiam in the Ri Bhoi district of Shillong in Meghalaya (India), which are some of the main orange growing areas in the northeastern states of India. The orchard trees were 10–20 years old and of nucellar seed origin. The trees were planted in rows spaced 5–6 m apart with a distance between trees of 5–6 m; trees were of uniform growth and vigour. As per the USDA 7th approximation soil classification (USDA 1960), the soil of Thangal was classified as red and yellow soils (Ultisols), of Tinsukia as recent alluvial soil (Entisols), and of Ri Bhoi (Shillong) as red loamy soil (Alfisols). The soil chemical characteristics were determined by standard methods (Table 1). Rhizospheric soils along with lateral roots branching away from the main radial roots at a depth of about 5 to 15 cm from the surface were collected by carefully uprooting the root system. Roots and rhizosphere soils were carried to the laboratory in a cool box and stored at 4 °C.

Isolation and in vitro PGP screening of rhizobacteria from MOR Loosely adhered soils were removed and 1 g of the roots with tightly adhered soil was ground in a mortar and pestle. The mixture was serially diluted and plated separately on Pikovskaya agar, King’s B agar, Rojo Congo agar (Rodriguez-Caceres 1982), and nutrient agar. The morphologically different colonies were purified, stored in 30% (m/v) glycerol at −80 °C Deep Freezer. Two hundred and seventeen pure isolates obtained from the 3 selective media were subjected to in vitro PGP screening. Pure isolates were first screened for N fixation assay by stabbed inoculation in N-free bromothymol semisolid medium as per the protocol of Dobereiner et al. (1995). Qualitative screening for P solubilization was done using NBRI-BPB (National Botanical Research Institute - Bromophenol Blue) medium, since this medium is more specific than Pikovskaya agar (Nautiyal 1999), which was used during isolation. Qualitative screening for IAA production was done as per the protocol of Bric et al (1991). Of the isolates, 101 showed pellicle growth and colour change in N-free bromothymol semisolid medium; 119 were P solubilizers showing halo-zone formation in NBRI-BPB agar medium, with a zone size of ≥8 mm; and 34 reacted with Salkowski reagent, indicating production of IAA-like substances. All together 32 isolates were positive for all 3 PGP traits and were further screened for quantitative production of IAA-like substances using the Loper and Schroth (1986) method and quantitative determination of P solubilization as per the protocols of Nautiyal (1999) and Fiske and Subbarow (1925). Ultimately, the 32 isolates were tested for in vitro antifungal–antagonistic activity against the fungal pathogens Macrophomina phaseolina and Rhizoctonia solani by using dual-culture technique as an indirect PGP screening method. Finally, we adopted a method of ranking the 32 isolates for selection of superior isolates for plant bioassay experiment. In this ranking method, 32 strains with multiple PGP functions, i.e., P solubilization, IAA-like substances production, N fixation, and biocontrol (antifungal) activity, were allotted ranks. The highest value of an attribute, i.e., the isolate producing highest quantity of a PGP attribute, received a rank value of 32 (total isolates 32), and the lowest a value of 1. For N fixation and antifungal activity, isolates positive for N fixation were given rank 1 and those negative rank 0. The ranks of the attributes were added up. On the basis of total rank value, 10 strains were selected for screening in the bioassay experiment. The 10 high ranking isolates were identified by Microbial Type Culture Collection (IMTECH, Chandigarh, India) based on morphological, physiological, and biochemical characteristics. Determination of ACC deaminase activity The 1-aminocyclopropane-1-carboxylate (ACC) deaminase activities of the 10 strains were determined following the procedures described by Penrose and Glick (2003) for both cell culture and measurements of enzyme activity. In brief, bacterial isolates used for measurement of ACC deaminase activity were grown in liquid DF medium to late log phase, after which they were separated from the spent medium by centrifugation. The cells were washed by suspending the pellets in 1 mL of 0.1 mol·L−1 Tris–HCl, pH 7.6, recentrifuging at 13 500g for 5 min, and decanting to remove the washed supernatant. The washed cells were then resuspended in Published by NRC Research Press


Thokchom et al.

600 ␮L of 0.1 mol·L−1 Tris–HCl, pH 8.5, and 30 ␮L of toluene was added. The suspensions were vortexed, and 100 ␮L aliquots of the toluenized cells were set aside at 4 °C for protein determination. The remaining cells were immediately assayed for ACC deaminase activity by measuring the rate of formation of ␣-ketobutyrate, which is the degradation product of ACC. For these measurements, 200 ␮L suspensions of toluenized cells were placed into centrifuge tubes to which was added 20 ␮L of 0.5 mol·L−1 ACC. The cell suspensions were vortexed and placed in an incubator for 15 min at 30 °C. After incubation, 1 mL of 0.5 mol·L−1 HCl was added, after which the solutions were vortexed and then centrifuged for 5 min at 13 500g. One millilitre aliquots of the supernatants were placed in glass tubes containing 800 ␮L of 0.5 mol·L−1 HCl. Finally, 300 ␮L of 2,4-dinitrophenylhydrazine reagent was added. The contents were vortexed and incubated for 30 min at 30 °C. After incubation, 2 mL of 2 mol·L−1 NaOH was added, and the absorbance of the mixture was measured at 540 nm. All measurements were carried out in triplicate. ACC deaminase activity was calculated as micromoles of ␣-ketobutyrate per milligram of protein per hour. 16S rRNA gene amplification and sequencing Genomic DNA was obtained by using the modified protocol of Sambrook et al. (1989). Universal bacteria specific primers were used for PCR amplification of the 16S rRNA gene. The primer sequences are as follows: forward primer — GM3F-5=-AGAGTTTGATCMTGG-3=, and reverse primer — GM4R-5=-TACCTTGTTACGACTT-3=. PCR cocktails (50 ␮L) contained 50 nmol·L−1 primers, 200 ␮mol·L−1 dNTPs (Promega Co., Southampton, England), 1× Taq polymerase buffer, 1 U of Taq polymerase (Merck Co.), and 50 ng of template DNA. Thermocycling conditions consisted of an initial denaturation step at 95 °C for 5 min; 30 amplification cycles of 94 °C for 1 min, 58 °C for 1 min, and 72 °C for 3 min; and a final extension step of 72 °C for 10 min (1000R Thermocycler, BioRad). A 5 ␮L aliquot of each amplification product was electrophoresed on 0.8% agarose gel in 0.5× TBE buffer at 80 V for 45 min and stained with ethidium bromide, and the PCR products were visualized with a UV transilluminator. The sequencing reactions were performed using the same primers used for amplification in an automated ABI3100 genetic analyser (Applied Biosystems) in GeNei, Bangalore. The sequence electropherogram data were validated using Chromas LITE version 2.02 software (www.technelysium. com.au), and the sequence similarity matching was carried out using Ribosomal Database Project and NCBI database (www.ncbi.nlm. nih.gov). Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 4 (Tamura et al. 2007). The sequences were aligned and the consensus sequence was computed using Clustal W software. An evolutionary distance matrix was generated following the Kimura 2 parameter distance model (Kimura 1980). Evolutionary trees for the data sets were inferred by the neighbor-joining method of Saitou and Nei (1987) by using the neighbor-joining program, MEGA version 4. The nucleotide sequences of 16S rRNA gene were deposited in GenBank. The accession numbers of the 16S rRNA nucleotide sequences of the 10 selected strains are shown in Fig. 1. Bioassay using MO seeds Bacterial inoculants The 10 strains that were selected after screening in in vitro PGPR assays were used as bacterial inoculants. Bacterial cultures were incubated for 48 h with shaking (120 r·min−1) at 30 °C. One hundred millilitres of each culture broth was mixed with 180 g of sterilized compost to form a slurry. This slurry was used to introduce the bacteria as coatings on MO seeds. To obtain sterile compost, the required amount of compost was autoclaved 3 times in a tyndallization approach.

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Pot soil type The pot experiment was conducted in the Institute’s Polyhouse. The pot soil was air-dried, pulverized, and sieved through a 3.2 mm screen and not sterilized (natural soil). Three hundred and fifty grams of the soil and 5 g of sterile compost were taken in each small pot of diameter 10 cm and 80% moisture was maintained by applying tap water and mixed properly. The pots were kept in a naturally ventilated polyhouse covered with shade net (75%). The pot soil contains 7100 ppm total organic carbon (C), 400 ppm total N, 100 ppm available N, 300 ppm total P, 15.8 ppm available P, 540 ppm total potassium (K), and 169 ppm available K. Total organic C and total N were determined by the wet-oxidation method and the Kjeldhal technique (Kelpus Elite Ex, Pelican Equipments), respectively. Phosphorus was determined by the vanadomolybdophosphoric acid yellow color method using a spectrophotometer (UV Pharma Spec – 1700, SHIMADZU), and total K by atomic absorption spectroscopy (AAnalyst 200, Perkin Elmer). The available N content was determined by alkaline permanganate oxidation method, available P content by stannous chloride blue color method, and available K content by ammonium acetate method. The soil pH was 6.5. Experimental setup The experiment had a completely randomized block design with 11 treatments including the control, and each treatment had 10 replications. Orange seeds were removed from unripe oranges, washed several times with sterile distilled water to remove the mucilaginous substances, and surfaced sterilized. MO seeds were treated separately with the cultures of 10 selected strains, and the treated seeds were sown in the pots. The control pot received only Luria–Bertani broth medium. Each inoculum and control treatment had 10 replicate pots and each pot received 3 seeds. For the control, the orange seeds were coated with noninoculated medium mixed with a similar quantity of sterile compost. The pot positions were rearranged everyday so as to render equal exposure to sunlight and were watered to constant weight. Plant growth parameters Seedling emergence was recorded from the 15th day after sowing and at an interval of 15 to 30, 30 to 45, and 45 to 60 days after sowing and cumulative emergence up to 30, 45, and 60 days was calculated. After 4 months, half of the orange seedlings from each treatment were carefully removed from the pot, and shoot length, root length, shoot fresh biomass, and root fresh biomass were measured. Shoot and root dry biomasses were determined by oven-drying at 65 °C for 3 days. The seedlings of the remaining pots of 4 selected treatments were transferred to 8 kg capacity plastic pots, and growth parameters were assessed for a period of 1 year. After 1 year, the number of branches and the length of individual branches were recorded and totalled to get the total branch length; plant height and girth diameter were also recorded. Girth diameter is the main stem diameter taken at 5 cm above the top soil. Statistical analysis Results of the measurements were subjected to analysis of variance (ANOVA) and additionally Duncan’s Multiple Range Test (P ≤ 0.05) for the bioassay results using SPSS Statistical Software package, version 2.1.

Results and discussion The rhizosphere of the selected MO orchards of northeast India was found to harbor at least 217 isolates and some of these isolates showed multiple PGP attributes. We followed a stepwise qualitative and quantitative screening method to initially select 10 superior strains, based on the magnitude of 4 PGP attributes, to inoculate seeds of MO in an in vivo experiment. Associative and free-living microorganisms may contribute to the nutrition of Published by NRC Research Press

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Fig. 1. Phylogenetic tree of partial 16S rRNA gene sequences of the 10 isolated strains (with accession number given in parentheses) showing the relationships with reference type (T) strains obtained from NCBI and RDP databases. The tree was constructed by using the MEGA4 software after aligning the sequences with ClustalW and generating evolutionary distance matrix inferred by the neighbor-joining method using Kimura parameter 2. The numbers at the nodes indicate the levels of bootstrap support based on data for 1000 replicates; values inferred greater than 50% are only presented. The scale bar indicates 0.002 substitutions per nucleotide position.

plants through a variety of PGP mechanisms (Ahmad et al. 2008; Raaijmakers et al. 2009). A particular bacterium may affect plant growth using any one or more of these mechanisms. Although, microbial inocula have been used in MO orchards to enhance plant growth and yield (Abd El Migeed et al 2007; Medhi et al 2007; Abdel-Hak et al 2012), the inocula did not originate from the MO rhizosphere. It is important to select superior inocula from rhizobacteria isolated from the MO rhizosphere. We have detected rhizobacteria of MO belonging to the genera Enterobacter, Pantoea, Achromobacter, Pseudomonas, Citrobacter, Klebsiella, and Ochrobactrum using 16S rRNA gene sequencing (Supplementary Table 11). These genera belong to the Gammaproteobacteria family. Previously, Trivedi et al. (2011) reported the dominance of bacterial species of Enterobacter, Klebsiella, Achromobacter, and Pantoea with PGP attributes on the roots of Valencia orange (Citrus sinensis) trees. Isola-

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tion of species of these genera from cereal crops and their PGP and biocontrol abilities have also been reported earlier (Cattelan et al. 1999; Khalid et al. 2004; Park et al. 2005; Anderson and Habiger 2012). Thus, our results on dominance of Gammaproteobacteria, based on molecular identification, and of genera on Valencia orange and cereal crops suggest a universal occurrence of members of Gammaproteobacteria with PGP attributes in plant rhizospheres. Additionally, we isolated species of PGPR belonging to the genera Ochrobactrum and Citrobacter (Supplementary Table 11), which have not been previously isolated from other citrus rhizospheres, although Gardner et al. (1985) reported the isolation of Citrobacter species from the xylem of lemon roots. The rhizobacteria of MO were obtained by plating on 3 selective media, and the isolates of each selective medium were cross streaked in other media, which was useful in determining the

Supplementary data are available with the article through the journal Web site at http://nrcresearchpress.com/doi/suppl/10.1139/cjm-2013-0573. Published by NRC Research Press

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Fig. 2. Seedling emergence of Mandarin orange (Citrus reticulata Blanco) at 30, 45, and 60 days after sowing of all the treatments (Control, RCE1, RCE2, RCE3, RCE4, RCE5, RCE6, RCE7, RCE8, RCE9, RCE10). Each bar represents the mean ± SD (n = 3). Bars marked without a common letter within each interval (Days after Innoculation) are significantly different (P < 0.05).

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Table 2. Phosphate solubilization, IAA-like substances production, ACC deaminase activity, nitrogen fixation assay, and antifungal activity by 10 selected rhizobacterial strains of Mandarin orange rhizosphere.


Antifungal activity‡

Strain*

Phosphate solubilization (␮g·mL−1)

IAA-like substances production (␮g·mL−1)

ACCd activity (␮mol ␣-ketobutyrate· (mg protein)−1·h−1)

Nitrogen fixation†

Rhizoctonia solani

Macrophomina phaseolina

RCE1 RCE2 RCE3 RCE4 RCE5 RCE6 RCE7 RCE8 RCE9 RCE10 MTCC 4218 MTCC 4714 MTCC 102 MTCC 103 MTCC 4684

213.8±15.9 123.7±5.9 130.7±22.4 126.8±11.7 109.3±17.6 173.4±7.8 173.2±17.3 102.0±14.2 267.9±24.1 129.0±11.3 6.2±1.1 83.6±6.6 36.5±7.4 68.4±8.8 ND

3.0±0.6 3.3±0.3 5.8±0.5 3.1±0.6 3.6±1.1 7.0±1.9 12.0±2.3 5.2±0.5 1.8±0.2 3.1±0.4 2.1±0.3 0.5±0.1 3.1±0.2 1.2±0.3 ND

14.7±2.4 5.1±0.7 20.4±2.1 82.4±7.5 9.8±1.5 58.7±3.4 53.4±9.4 2.3±1.8 2.7±0.7 2.3±0.2 ND ND ND ND 23±1.2

+ + + + + + + + + + + + + + ND

+ — + + + + — — + + + + + + ND

+ — — — + + — — + — — — — — ND

Note: ND, not done; IAA, indole acetic acid; ACCd, 1-aminocyclopropane-1-carboxylate deaminase. Values for phosphate solubilization, IAA-like substances production, and ACCd activity are the means of 3 replicates ± standard error. *MTCC 4218, Azospirillum amazonense; MTCC 4714, Bacillus megaterium; MTCC 102, Pseudomonas putida; MTCC 103, Pseudomonas fluorescens; MTCC 4684, Burkholderia cepacia. †Nitrogen fixation assay was based on the color change in nitrogen-free bromothymol blue medium. +, color change from yellow to green–blue; —, no color change. ‡Antifungal activity was based on inhibition zone formation after dual inoculation of test strain and the pathogenic fungus. +, inhibition zone formation; —, no inhibition zone formation. Rhizoctonia solani and Macrophomina phaseolina are pathogenic fungi.

proportion of them with dominant PGP attributes. PGP attributes such as P solubilization, N fixation, and IAA production of species in the genera Enterobacter, Pantoea, Pseudomonas, Achromobacter, and Klebsiella were reported by earlier workers (Jha and Kumar 2009; Trivedi et al. 2011; Farina et al 2012; Ribeiro and Cardoso 2012), and thus, their growth on cross-streaking plates and the varying magnitude of these attributes in quantitative assays were expected. Several previous studies also attributed the beneficial effect of these rhizobacteria on cereal (Park et al. 2005; Montanez et al. 2012) and a perennial fruit crop (Aballay et al. 2011) to their PGP traits. In this study, 14.7% of the rhizobacterial isolates possessed multiple PGP attributes, and more than half of the total isolates (54.8%) could solubilize insoluble P. The total and available P contents of the rhizospheric soil samples of the study sites were low (Table 1). Previous workers (Singh et al 2006; Srivastava et al. 2010) also showed that the total soluble P content of MO orchard soils of northeast India was low. We speculate that the low available P content in soil may have favored a large number of P solubilizers. Similarly, N contents of these soils are also low, and N-fixing bacteria may have a role in fixation and in increasing the availability of N to MO roots. Quantitative assays of the isolates showed production of soluble P of 8.0 to 267.9 ␮g·mL−1 and IAA-like substances of 0.8– 12.0 ␮g·mL−1 of the culture supernatant. The highest amount of soluble P was produced by RCE9 (267.9 ␮g·mL−1) followed by RCE1 (213.8 ␮g·mL−1), and IAA production was found to be highest in the case of strains RCE7 (12.0 ␮g·mL−1) and RCE6 (7.1 ␮g·mL−1) (Table 2). The quantities of IAA-like substances produced in vitro with 50 ␮g·mL−1 tryptophan in the culture broth by some of the isolates of this study were much higher than quantities reported earlier (Ahmad et al. 2008). Of the 10 strains, 7 were antagonistic to Rhizoctonia solani and 4 were antagonistic to Macrophomina phaseolina. Only 4 strains namely RCE1, RCE5, RCE6, and RCE9 were antagonistic to both pathogenic fungi. Antagonism against fungal pathogens is one of the measures of indirect PGP mechanisms (Guinazu et al. 2013).

Table 3. Effect of 10 rhizobacterial strains on different growth parameters of Mandarin orange seedlings grown in natural soil in small pots for 4 months after sowing. Shoot parameter

Root parameter

Rhizobacterial strain

Length (cm)

Dry biomass (mg·plant−1)

Length (cm)

Dry biomass (mg·plant−1)

Control RCE1 RCE2 RCE3 RCE4 RCE5 RCE6 RCE7 RCE8 RCE9 RCE10 LSD (0.05)

9.2a 17.3d 15.3cd 12.9bc 12.2b 13.0bc 12.2b 16.9d 13.4bc 12.4b 13.4bc 2.8

208.7a 382.5bc 335.0bc 332.5bc 332.5bc 312.5abc 300.0ab 420.0c 297.5ab 305.0abc 325.0bc 101.6

17.4a 22.1abc 19.7ab 24.2bcd 19.6ab 28.0d 20.8abc 26.2cd 20.2ab 17.7a 19.3ab 5.1

200.0a 332.5bc 440.0cd 470.0d 310.0ab 392.5bcd 365.0bcd 350.0bc 315.0ab 297.5ab 297.5ab 105.6

Note: Values followed by different letters indicate a significant difference (P = 0.05) between treatments as compared with the control, by Duncan's Multiple Range Test.

Inoculation with the 10 strains stimulated emergence of seedlings of MO seeds in the presence of the indigenous population to a different extent. Inoculation of strains RCE2, RCE6, and RCE7 were found to be statistically superior in stimulating seedling emergence than the noninoculated control within 15–30 days after sowing (Fig. 2). However, after 45 days of sowing, the effect of inoculation among the strains was not statistically significant, although there was a significant effect as compared with the noninoculated control (Fig. 2). By 60 days, all of the seedlings emerged in all the treatments. These data indicate that inoculation of MO seeds with efficient strains can hasten seedling emergence in nursery, which is advantageous for obtaining seedlings of perennial crops in a shorter time for out-planting in orchards. This stimulation of emergence of MO seeds could be due to the effect of Published by NRC Research Press

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Table 4. Plant growth parameters of 1-year-old Mandarin orange seedlings in 8 kg capacity pots to which 4-month-old seedlings from the 4 treatments (RCE1, RCE2, RCE5, and RCE7) and control treatment were transferred. Rhizobacterial isolate

Plant height (cm)

No. of primary branches

Total length of primary branch (cm)

No. of secondary branches

Total length of secondary branch (cm)

Girth diameter* (mm)

Control RCE1 RCE2 RCE5 RCE7 LSD (0.05)

31.1b 50.1a 40.8ab 55.0a 55.4a 15.3

6.6a 6.2a 9.4ab 9.4ab 10.8b 3.3

51.5a 59.8a 118.8b 80.6ab 79.8ab 45.0

2.0b 7.8a 10.2a 8.2a 7.2a 5.7

2.6b 39.0a 75.1c 44.4a 38.1a 22.8

6.7a 8.4b 8.5b 8.4b 7.5ab 1.23

Note: Data are mean of 5 replicates of plants. Values followed by different letters indicate a significant difference (P = 0.05) between treatments as compared with the control, by Duncan's Multiple Range Test. *Girth diameter is derived from the value of circumference of stem measured at 5 cm above soil level.

any PGP attributes determined in vitro. IAA is a plant hormone that stimulates seedling emergence and development of the root system, and has also been speculated to improve the fitness of plant–microbe interactions (Brandl and Lindow 1998; Patten and Glick 2002). Although, it is possible that the enhancement of seedling emergence observed with RCE7, RCE6, and RCE8 may be an effect of the higher amounts of IAA produced by these isolates, it does not explain the enhancement observed with the low-IAAproducing strain RCE2. This strain may produce other hormones such as gibberellic acid and ethylene, which also stimulate emergence and germination (Kucera et al. 2005), but we did not carry out any assays to confirm this. However, all the 10 selected strains used in the in vivo pot experiment possess ACC deaminase activity in the range of 2.3–82.4 ␮mol ␣-ketobutyrate·(mg protein)−1·h−1. Penrose and Glick (2003) earlier showed ACC deaminase of PGPR strains as an easily measured and useful trait in evaluating the effect of PGPR strains on root growth. Therefore, it is possible that ACC deaminase along with other PGP traits may play a role in stimulating root growth. Our data also showed that aggregate ranking of PGP traits of strains may be useful for identifying strains with the ability to enhance plant growth. The effect of inoculation of the 10 strains on the shoot length, root length, shoot dry biomass, and root dry biomass of the 120-day-old seedlings was found to be different (Table 3). Shoot length of MO seedlings of all the inoculated treatments was statistically significantly different (at P < 0.05) from that of the noninoculated control, with strains RCE1 and RCE7 producing a significantly higher effect on the shoot length. On the other hand, strains RCE3, RCE5, and RCE7 increased root length significantly over that of the noninoculated control. Similarly, dry shoot and root biomasses of 6 inoculated treatments increased significantly over that of the noninoculated treatment (Table 3). Overall analysis and comparison of effect of the strains showed that RCE1, RCE2, RCE3, RCE5, and RCE7 produced a statistically significant effect on at least 3 plant growth parameters. The superior beneficial effect of 4 of these strains was also evident up to 365 days after transfer to bigger pots of 8 kg capacity, as the different growth parameters of MO seedlings, except the total number and length of primary branches, were found to be statistically significant different from the noninoculated control (Table 4). Strain RCE3 inoculated seedlings could not be transferred to the 8 kg capacity pots. Based on this result, we suggest that vegetative traits such as plant height, number of secondary branches, total length of secondary branches, and girth diameter are important parameters to consider in evaluating the effect of PGPR strains on MO plants for at least up to 1 year from the time of sowing. Solubilization of insoluble phosphorous compounds in the rhizosphere by microorganisms is an important means of achieving PGP (Nautiyal 1999), and such PGP bacteria could enhance the availability of the limiting nutrient, such as phosphorus, to the host (Cattelan et al. 1999). The experimental soil contained a low level of available P (15.8 ppm), and better growth of MO seedlings

in inoculated treatments than in the noninoculated control may be due to the release of soluble P continuously for absorption by the plant over a period of 1 year. Enhanced root and shoot growth of inoculated MO seedlings in the first year of growth may be correlated with PGP traits such as ACC deaminase activity and production of IAA-like substances. Thus, an apparent conclusion can be drawn from our results that MOR carries PGPR strains with multiple PGP attributes and their inocula may be useful in growth enhancement of MO. The presence of specific PGP traits suggests that these rhizobacteria can promote plant growth by more than one mechanism and that some of these strains should be tested further in field inoculation experiments.

Acknowledgement The senior author is supported by an Inspire Fellowship Programme, Department of Science and Technology, Goverment of India. The authors express gratitude to the Curator of the Microbial Type Culture Collection, Chandigarh, India. for biochemical characterization of the isolates, and the Director of the Institute of Bioresources and Sustainable Development, Manipur, India, for providing laboratory facilities to carry out this research. The senior author is thankful to K. Chandradev for his valuable technical help during the experiment. The authors thank the reviewer for critical reviews, which helped improvement of the manuscript.

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