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Soil Science Society of America Journal 78:538-545 doi:10.2136/sssaj2013.07.0315. Drought is major constraint to the productivity of cereals in arid and semiarid areas like Pakistan. Several approaches have been reported in the literature to mitigate the drought-induced impacts on plant growth.
Soil Science Society of America Journal 78:538-545 doi:10.2136/sssaj2013.07.0315. Drought is major constraint to the productivity of cereals in arid and semiarid areas like Pakistan. Several approaches have been reported in the literature to mitigate the drought-induced impacts on plant growth.
Soil Science Society of America Journal 78:538-545 doi:10.2136/sssaj2013.07.0315. Drought is major constraint to the productivity of cereals in arid and semiarid areas like Pakistan. Several approaches have been reported in the literature to mitigate the drought-induced impacts on plant growth.
doi:10.2136/sssaj2013.07.0315 Received 30 July 2013 *Corresponding author zazahir@yahoo.com Soil Science Society of America, 5585 Guilford Rd., Madison WI 53711 USA All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher. Scrutinizing Rhizobia to Rescue Maize Growth under Reduced Water Conditions Soil Biology & Biochemistry D rought is a major constraint to the productivity of cereals (reducing yield up to 40.8%) in arid and semiarid areas (Boyer, 1982) like Pakistan. Change in climate due to global warming is resulting in irregular rainfall, fash fooding, increased salinity, high temperature, and drought in the country, which contributes substantially to reduced growth and yield of these crops. Pakistan has been facing water defcit situations for many years due to reduced river fow, low rainfall, and high salinity (Economic Survey of Pakistan, 2011). Terefore, several approaches have been reported in the literature to mitigate the drought-induced im- pacts on plant growth. Mulching, bed planting, deep tillage, cover cropping (physi- cal methods), and foliar sprays of chemicals such as plant growth regulators (indole acetic acid, zeatin, and abscisic acid) and osmo-protectants (silver, silicon, proline, glycine betaine, salicylic acid, and jasmonic acid) have been adopted to ameliorate the impact of drought stress (Hussain et al., 2011). However, conventional breeding and selection of drought tolerant genotypes on the basis of osmoregulation have also been adopted (Hussain et al., 1986; Ahmad et al., 1987) but this is a slow and lengthy process. On the other hand, rhizobiaa group of N fxing bacteria (in symbiosis Muhammad Baqir Hussain Zahir Ahmad Zahir* Hafz Naeem Asghar Sajid Mahmood Institute of Soil & Environ. Sciences University of Agriculture Faisalabad, (38040) Pakistan Availability of fresh water for crop production is a major concern these days. Therefore inoculation biotechnology has been tried as a tool to support crop growth and yield under water defcit conditions. Thirty rhizobia were isolated from the nodules of three legumes (Lens culinaris, Vigna radiata, and Cicer arietinum) cultivated in arid and semiarid regions of Punjab (Pakistan). Nine fast growing isolates were evaluated for drought tolerance capability with PEG-6000-induced water stress. These nine isolates were assessed for plant- growth-promoting-activity on maize under drought in gnotobiotic conditions by planting inoculated seeds between flter sheets soaked with each respec- tive inoculum. Drought was imposed by applying PEG solutions of -0.61 and -1.2 MPa osmotic potential and isolates rhizobial strain (RS)-1, RS-3, RS-8, and RS-12 were selected as prominent growth promoters. Selected iso- lates were evaluated in a pot trial under natural conditions where irrigation was delayed at the vegetative and reproductive growth stages of the maize crop to develop water defcit stress. Improvement in fresh cob yield, root/ shoot length, shoot fresh weight and drought tolerance index was observed. The isolates displayed P-solubilization (RS-1 and RS-12), siderophores (RS-3 and RS-8), oxidase (RS-1), indole acetic acid, catalases, and exopolysaccha- rides production ability (RS-1, RS-3, RS-8, and RS-12). Isolates RS-1 and RS-3 were identifed as Rhizobium phaseoli whereas RS-8 and RS-12 were Mesorhizobium ciceri. Results suggested rhizobia as potential inoculants to maize for improving growth and productivity under drought. However, evalu- ation in feld conditions is required to confrm their capability. Abbreviations: RS, rhizobial strain; YEM, yeast extract mannitol. Published April 8, 2014 Published March 21, 2014 www.soils.org/publications/sssaj 539 Soil Biology & Biochemistry with legumes) have been reported to improve growth and produc- tivity of non-legumes through mechanisms other than biological N 2 fxation (Noel et al., 1996; Hussain et al., 2009). Rhizobia may perform as bio-fertilizers (improving nutrition and nutrient-use efciency; Zaidi et al., 2009), bio-stimulators (plant growth regu- lator production; Etesami et al., 2009) or bio-protectants (antibio- sis, chitinase, osmolytes, and 1-aminocyclopropane-1-carboxylate [ACC] deaminase production; Zahir et al., 2008). Plant growth promotion under normal conditions has been tested in rhizobi- um-inoculated rice (Oryza sativa L.), maize (Zea mays L.), and wheat (Triticum aestivum L.; Yanni et al., 1997, 2001; Gutirrez- Zamora and Martnez-Romero, 2001; Mehboob et al., 2011). However, information regarding plant growth promotion efects of rhizobial inoculation on non-legumes exposed to drought is scarce. Rhizobia are capable of surviving in soils with limiting moisture levels; however, population densities tend to be lower un- der desiccated conditions and increase as the moisture stress is re- lieved (Fuhrmann et al., 1986). Athar and Johnson (1997), Athar (1998), and Elboutahiri et al. (2010) observed enormous ability of rhizobia to survive under water potentials up to -1.5 MPa. Rhizobia can alleviate the efect of water stress in plants by altering leaf stomatal conductance, transpiration, photosynthetic capacity, and root morphology in inoculated non-legumes (Chi et al., 2005). Terefore, Alami et al. (2000) evaluated an exopoly- saccharide producing strain, YAS34, belonging to Rhizobium species for ameliorating the impact of drought and improving water and nutrient-use efciency in sunfower (Helianthus ann- uus L.). Tey recorded a signifcant increase in N uptake and im- proved root and shoot dry biomass under normal and water lim- ited conditions. Similarly, rhizobial strain KYGT207 (isolated from the drying soil) improved the growth of wheat under water defcit conditions due to the improved aggregation of rhizo- sphere soil (Kaci et al., 2005). Following the literature reviewed, we found a room for rhizobial inoculation to non-legumes under drought conditions as scanty information is available in this area. We hypothesized that the rhizobial isolates capable for surviv- al under drought conditions and having the characteristics for improving plant growth of maize under drought could be good criteria for selecting potential rhizobial inoculants for improving growth and yield of maize under drought. MATERIALS AND METHODS Isolation, Purifcation, and Preservation of Rhizobia Numerous rhizobia were isolated from the root nodules of three legumes (Lens culinaris, Vigna radiata, and Cicer arietinum) cultivated in arid (Chakwal) and semiarid (Faisalabad) areas of Punjab (Pakistan). Te plants were dug up with non-rhizosphere soil to avoid damage to the roots and nodules. Tese plants were transferred to the laboratory in polythene bags. Non-rhizosphere soil was removed by gentle shaking and further adhering soil was removed by washing the roots with tap water. Nodules were cut from the roots with a sterilized razor blade and collected in sepa- rate Petri dishes. Disinfestation of nodules was done by dipping in ethanol (95%, v/v) for 10 s and 5% (v/v) sodium hypochlorite for 10 min followed by thorough rinsing with sterilized water (Abd-Alla et al., 2012). Sterilized nodules were rolled on the Petri dishes containing autoclaved yeast extract mannitol (YEM) agar medium and incubated at 28 1C for 72 h to confrm the sterilization (Muzzamal et al., 2012). Tese surface-sterilized nodules were crushed with a sterilized glass rod in separate steril- ized test tubes containing sterilized distilled water. Homogenate suspensions were streaked in Petri dishes containing autoclaved YEM agar medium and incubated at 28 1C. Further streaking for isolation was performed three to four times to obtain pure cultures of the diferent isolates. Nine fast growing isolates were collected, coded as rhizobial strain (RS) with numbers and pre- served in glycerol at -20 1C. Tese isolates were confrmed as rhizobia by streaking on congo-red agar plate (Grams test) and by studying their colony morphology. Drought Tolerance Assay Rhizobial isolates (RS-1, RS-2, RS-3, RS-6, RS-7, RS-8, RS- 10, RS-12, and RS-13) were tested for drought/desiccation tol- erance capability using PEG-6000 in YEM broth medium (Busse and Bottomley, 1989). Inoculum for each isolate was prepared in a sterilized 50-mL conical fask containing 20 mL of YEM broth and incubated for 72 h at 28 1C, shaking at 100 rpm. Cells were harvested by centrifugation at 4000 g at 4C for 10 min. For inoculation, an inoculum of optical density (OD) 0.5 McFarland units (i.e., 10 8 cells mL -1 ) was developed by us- ing densitometer (Den-1 Densitometer, McFarland, UK) in ster- ilized YEM liquid medium (Shakeri et al., 2011). Un-inoculated control medium was maintained to subtract its OD from the OD of the inoculum. Test tubes containing 7 mL of YEM broth with diferent drought levels (No PEG, 15, 25, and 35% PEG having osmotic potentials -0.04, -0.69, -1.65, and -2.15 MPa, re- spectively, measured by Cryoscopic Osmometer [OSMOMAT- 030-D, Gonotec, Germany]) were autoclaved. Afer autoclaving, osmotic potential was measured again to check any change in the developed potentials, but there was no signifcant change. For each isolate triplet test tubes at each drought level were inocu- lated with 0.5 mL inoculum of OD 0.5 (10 8 cell mL -1 ). A trip- licate set of un-inoculated control test tubes was also maintained. Rhizobia show maximum growth afer 96-h incubation at 28 1C and 100 rpm which was confrmed in few samples using di- lution plate technique (data not shown). Terefore, OD afer the 96-h incubation in an orbital shaking incubator at 28 1C and 100 rpm was measured using densitometer and the correspond- ing population was counted by dilution plating technique (data not shown; Wollum, 1982). Rhizobial isolates showing high OD under drought were considered as drought tolerant. Screening of Rhizobia for Plant Growth Promoting Activity under Drought All nine rhizobial isolates used in the drought tolerance as- say were further tested for plant growth promoting activity un- der drought using maize (Zea mays L.) cv. Neelum as the test plant. Inoculum was prepared as described above. Afer achiev- 540 Soil Science Society of America Journal ing uniform cell density, two sterilized flter paper sheets were saturated by pouring on the respective inoculum. Tree surface sterilized (by dipping in 5% sodium hypochlorite for 10 min) pre-germinated seeds (germinated with hydro-priming on fl- ter paper sheets) were dipped in the inoculum of 0.5 OD (i.e., 10 8 cells mL -1 ) for 10 min and placed between the sheets soaked in the respective inoculum. Te flter paper sheets were rolled and put in sterilized glass jars as described by Asghar et al. (2002). Sterilized broth was used for the control treatment. Each treatment was replicated thrice. Diferent drought poten- tials (No PEG, 15 and 25% PEG having potentials -0.02, -0.61, and -1.20 MPa, respectively) were maintained in sterilized Hoagland solution (1/2 strength) by using PEG-6000. Light (275 mol m -2 s 1 ) and dark periods were adjusted at 10 and 14 h, respectively, and a suitable temperature (2530C) was maintained. Five days afer sowing, drought treatments were applied by irrigating plants with PEG solutions. Te trial was harvested afer 15 d of drought treatment and data regarding root/shoot length (longest root/shoot), and dry biomass were recorded. Isolates showing signifcant improvement in results were considered as efcient plant growth promoters. Pot Trial Selected isolates from the screening trial (RS-1, RS-3, RS- 8, and RS-12) were evaluated for their capability to improve maize growth and reduce the impact of water defcit stress in pots (pot dimensions: height of 30 cm, diameter of 23 cm, con- tained 12 kg sieved sandy loam soil [pH, 7.3; EC e , 2.17 dS m -1 ; OM, 0.84%; Total N, 0.04%; available P, 7.1 mg kg -1 ; extract- able K, 107 mg kg -1 ; saturation percentage, 37%] collected from the experimental feld of the Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad) in greenhouse. For the purpose, inoculum was prepared in the laboratory as described for the screening trial above. Maize seeds were coated with the inoculum prepared by mixing each bacterial culture with autoclaved peat: clay (3:1) and a 15% (w/v) table sugar so- lution. Inoculated seeds were dried under shade. Five seeds per pot were sown and thinned to one seedling afer 1 wk of ger- mination. Water stress was applied either at the vegetative (fve leaf ) or the reproductive (silking) growth stage of the crop by delaying irrigation. Te irrigation was delayed until the appear- ance of wilting symptoms in maize leaves (9 d for vegetative or 7 d for reproductive stage water stress). Plants having drought at the vegetative stage were not water stressed at the reproductive stage and vice versa. A control treatment with normal irrigation was also maintained. Recommended doses of NPK (180, 140, 90 kg ha -1 , calculated according to the weight of soil) were ap- plied using urea, diammonium phosphate (DAP), and sulfate of potash (SOP) fertilizers. A completely randomized design was followed with three replications. Plants were grown to maturity and at harvest (96 d afer sowing), diferent growth and yield parameters (fresh cob yield, longest root and shoot length, and shoot fresh weight) were recorded. Drought Tolerance Index (DTI) A drought (stress) tolerance index was calculated by dividing the shoot or root length of inoculated seedlings under stress (SLI or RLI and St) with the shoot or root length of un-inoculated and unstressed seedlings (SLUI or RLUI and USt) (modifed from Shetty et al., 1995). (SLI or RLI and St) DTI (SLUI or RLUI and USt) = where SLI, shoot length of inoculated seeding; RLI, root length of inoculated seedling; SLUI, shoot length of un-inoculated seedling; RLUI, root length of un-inoculated seedling; St = stressed seedling; USt, un-stressed seedling. Identifcation and Characterization of Selected Rhizobial Isolates Te selected rhizobial isolates were identifed by using the BIOLOG identifcation system (Microlog System release 4.2, Biolog Inc., USA). Te confrmation of the rhizobial isolates was performed by isolation of the rhizobia from the nodules of Vigna radiata and Cicer arietinum, and on re-inoculation, these isolates nodulated respective legume hosts. Te N fxation activ- ity of the nodules was confrmed with the pinkish appearance in the nodules. Te selected isolates of rhizobia were character- ized for exopolysaccharides synthesis, following the method de- scribed by Ashraf et al. (2004); catalase, by adopting MacFaddin (1980); oxidase activity, following Steel (1961); and IAA (auxin) production was recorded in the presence or absence of its pre- cursor L-tryptophan by implementing the protocol of Sarwar et al. (1992). Organic acid production was assessed by growing iso- lates on YEM with bromothymol blue (orange/yellow growth is a positive reaction; Vincent, 1970), siderophores by the method of Schwyn and Neilands (1987). Mehta and Nautiyal (2001) scheme was adopted to measure the solubilization of inorganic P in the medium. Statistical Analysis Data were analyzed by analysis of variance (ANOVA; Steel et al., 1997) and means were compared using Tukeys test. Sofware used for the analysis was Statistix 8.1 (Analytical Sofware, USA). RESULTS Drought Tolerance Assay Optical density of the isolates decreased with increase in the PEG concentration (No PEG, -0.04 MPa; 15% PEG, -0.69 MPa; 25% PEG, -1.65 MPa; and 35% PEG, -2.15 MPa) of the medium (Table 1). Te decrease in cell number with increas- ing PEG concentration was also evident using dilution plate technique (data not shown). Under normal conditions (No PEG), rhizobial isolate RS-6 produced the maximum OD, fol- lowed by RS-7. However, isolate RS-8 had the lowest OD un- der normal conditions. Isolates RS-13 and RS-12 were the low performers at 15% PEG induced drought stress, but RS-7 was www.soils.org/publications/sssaj 541 prominent among the isolates followed by RS-6. At 25% PEG induced drought, RS-3 performed best among the isolates fol- lowed by RS-8 and RS-2, respectively. Isolate RS-13 remained the last among the isolates at 25% PEG induced drought as was in 15% PEG induced drought. Te trend changed at 35% PEG induced drought, where isolate RS-8 became most efcient in drought tolerance followed by isolates RS-1. Isolate RS-6 which was prominent among the isolates at 15% PEG induced drought and in normal conditions, became most sensitive to drought at 35% PEG induced drought. Sensitivity of rhizobial isolates to drought stress was calculated by dividing their OD at drought with their OD under normal condition. Isolate RS-6 was most sensitive at -2.15 MPa osmotic potential, followed by RS-13 at both -1.65 and -0.69 MPa osmotic potentials. However, lowest sensitivity was observed by RS-7, RS-3, and RS-8 (-30.4, -52.5, and -63.5%) at -0.69, -1.65, and -2.15 MPa osmotic potentials, respectively, as compared with all other isolates. Screening on the Basis of Plant Growth Promoting Activity under Drought Performance of the inoculated maize seedlings was signif- cantly diferent from the un-inoculated control for some isolates but not for others (Table 2). Isolates RS-1, RS-3, RS-8, and RS- 12 were the most prominent in plant growth promotion among the isolates tested. Inoculation of rhizobial isolates RS-3, RS-8, and RS-12 sig- nifcantly improved the root length of the maize seedlings as com- pared with un-inoculated control under normal conditions where- as other isolates could not show signifcant diferences (Table 2). At 15% PEG induced drought, isolates RS-3, RS-7, RS-8, and RS- 13 inoculation gave signifcantly high root length with respect to un-inoculated control. Isolates RS-3 and RS-13 inoculation gave signifcant improvement in the root length of the seedlings over control at 25% PEG induced drought. However, isolate RS-3 sub- stantially improved root length at all water defcit conditions (No PEG, 15% PEG, and 25% PEG) compared with the other isolates and the un-inoculated control. Rhizobial inoculation gave signifcant increase in the shoot length of the maize seedlings as com- pared with un-inoculated control under normal conditions (Table 2). Similarly, inoculation pro- duced signifcant improvement in the shoot length over un-inoculated control at 15 and 25% PEG in- duced drought except RS-10 inoculated seedlings which showed statistically similar shoot length as compared with un-inoculated control. Isolate RS-8 showed highest shoot length among the isolates un- der normal and 15% PEG induced drought. But iso- late RS-1 gave highest shoot length at 25% PEG in- duced drought as compared with other inoculants. Root dry weight was signifcantly improved with the inoculation of rhizobia over un-inocu- lated control except RS-13 inoculated seedlings under normal conditions (Table 2). At 15% PEG induced drought, rhizobial inoculation signif- cantly increased the root dry weight with respect to un-inoculated control. Inoculation of rhizobia also signifcantly improved root dry weight at 25% PEG induced drought over un-inoculated control except RS-2 which showed statistically similar root dry weight with un-inoculated control. Under nor- mal and 15% PEG induced drought conditions, isolate RS-8 showed highest increase in the root Table 1. Optical density (OD) of rhizobia at densitometer under normal (No PEG), 15, 25 or 35% PEG-induced drought in yeast extract mannitol (YEM) broth after a 96-h incubation at 28 1C and 100 rpm. Isolate No PEG 15% PEG 25% PEG 35% PEG RS-1 0.94 c 0.37 d 0.21 b 0.17 ab RS-2 0.73 e 0.30 f 0.24 b 0.12 ef RS-3 0.61 f 0.40 c 0.29 a 0.13 d-f RS-6 1.11 a 0.47 b 0.22 b 0.07 g RS-7 1.02 b 0.70 a 0.23 b 0.14 c-e RS-8 0.52 g 0.33 e 0.24 b 0.19 a RS-10 0.87 d 0.31 ef 0.20 bc 0.16 bc RS-12 0.73 e 0.25 g 0.17 c 0.15 b-d RS-13 0.86 d 0.25 g 0.13 d 0.11 f HSD 0.008 0.007 0.011 0.007 Means sharing similar letters are statistically similar to each other at p = 0.05. HSD shows honestly signifcance difference. The osmotic potential of the growth media were as No PEG, -0.04 MPa; 15% PEG, -0.69 MPa; 25% PEG, -1.65 MPa; and 35% PEG, -2.15 MPa. Table 2. Screening of rhizobia for plant growth promotion of maize under nor- mal, 15 or 25% PEG-induced drought in gnotobiotic conditions. Root length (cm) (n = 3) Shoot length (cm) (n = 3) Isolates No PEG 15% PEG 25% PEG No PEG 15% PEG 25% PEG Un-inoculated 13.9 bc 12.7 d 10.7 c 33.2 d 29.2 c 26.4 c RS-1 17.8 ab 15.0 a-d 14.3 a-c 46.0 ab 44.0 ab 40.5 a RS-2 15.0 bc 15.0 a-d 11.7 bc 41.2 bc 40.2 b 37.8 ab RS-3 20.4 a 17.6 a 17.3 a 46.8 a 40.0 ab 39.4 a RS-6 17.5 a-c 14.2 b-d 13.2 a-c 44.6 a-c 40.0 b 38.1 ab RS-7 17.2 a-c 16.5 ab 15.6 a-c 47.3 a 40.3 b 38.7 ab RS-8 19.5 a 16.2 a-c 15.2 a-c 48.0 a 46.3 a 39.5 a RS-10 13.3 c 13.2 cd 12.5 a-c 41.3 bc 29.8 c 28.8 c RS-12 19.3 a 15.6 a-d 12.6 a-c 46.3 a 44.9 ab 39.8 a RS-13 17.0 a-c 16.7 ab 16.3 ab 40.7 c 40.8 ab 34.0 b HSD 1.19 0.94 1.42 1.41 1.58 1.41 Root dry weight (g plant -1 ) (n = 3) Shoot dry weight (g plant -1 ) (n = 3) Un-inoculated 0.33 d 0.16 d 0.06 e 0.20 e 0.07 f 0.04 e RS-1 0.66 b 0.55 a 0.49 a 0.42 a-c 0.40 a 0.17 b-d RS-2 0.50 c 0.42 b 0.17 de 0.44 ab 0.35 a-c 0.25 ab RS-3 0.44 c 0.40 bc 0.23 d 0.47 a 0.30 bc 0.26 a RS-6 0.47 c 0.38 bc 0.25 cd 0.40 a-c 0.15 ef 0.12 de RS-7 0.49 c 0.41 bc 0.36 bc 0.46 ab 0.43 a 0.23 a-c RS-8 0.82 a 0.56 a 0.42 ab 0.39 bc 0.36 ab 0.27 a RS-10 0.48 c 0.29 c 0.25 cd 0.35 cd 0.21 de 0.11 de RS-12 0.50 c 0.42 b 0.29 cd 0.47 a 0.38 ab 0.16 cd RS-13 0.43 cd 0.32 bc 0.28 cd 0.29 d 0.27 cd 0.26 a HSD 0.03 0.04 0.04 0.02 0.02 0.02 Means sharing similar letters are statistically similar to each other at p = 0.05. HSD shows honestly signifcance difference. No PEG, 15 and 25% PEG solutions were having potentials -0.02, -0.61 and -1.20 MPa, respectively. 542 Soil Science Society of America Journal dry weight whereas, at 25% PEG in- duced drought, RS-1 showed high- est increase in the root dry weight. But variable results were observed in shoot dry weight, where most of the inoculated treatments remained statistically similar to each other, yet signifcantly diferent from respec- tive un-inoculated control except RS-6 (at 15 and 25% PEG induced drought), and RS-10 (at 25% PEG induced drought), which were at par with their respective un-inoculated controls (Table 2). However, RS-3 and RS-12 gave highest increase in shoot dry weight over control under normal conditions. At 15% PEG in- duced drought, RS-7 gave highest signifcant increase over con- trol and at 25% PEG induced drought, RS-8 inoculated seed- lings gave highest shoot dry weight. Among the plants inoculated with diferent rhizobial iso- lates, a decrease in drought bearing capability was observed with an increase in PEG concentration (Fig. 1). However, isolates RS- 1, RS-3, RS-8, and RS-12 improved tolerance index up to 51, 57, 59, and 53%, respectively, over the un-inoculated control at 15% PEG induced drought (-0.61 MPa, osmotic potential). Whereas, at 25% PEG level (osmotic potential, -1.20 MPa), isolates RS-1, RS-3, RS-8, and RS-12 showed 53, 49, 49, and 50% increase in drought tolerance index, respectively, over un-inoculated con- trol. Te un-inoculated seedlings showed 12 and 20% reduction in drought tolerance index (at 15 and 25% PEG, respectively) with respect to the un-inoculated and unstressed control. Pot Trial Rhizobial isolates RS-1, RS-3, RS-8, and RS-12 were evalu- ated for their ability to promote the growth of maize under drought stress in pots under green- house conditions. Data presented in Table 3 dem- onstrate the signifcant ability of these rhizobia to enhance the fresh cob yield, root length and shoot fresh weight of maize. Inoculation and drought could not make signifcant diferences for the shoot length of plants. Inoculation with RS-3, RS-8, and RS-12 gave signifcant improvement in the root length of the maize plants compared with un-inoculated control in normal conditions. However, RS-3 was the only inoculant to show signifcant improvement for root length among the treatments when drought was applied at vege- tative growth stage of the crop. Whereas, drought at reproductive growth stage was signifcantly relieved by RS-1 and RS-12 inoculation through signifcantly improving root length as compared with un-inoculated control. As far as, shoot fresh weight is concerned, RS-12 inoculation signifcantly improved it over control in normal conditions. Inoculation of RS-1, RS-8, and RS-12 showed signifcant improvement in the shoot fresh weight as compared with un-inoculated control where drought was created at reproductive growth stage of the crop. Under drought at vegetative growth stage of the crop, isolate RS-1 in- oculation gave signifcant improvement in the shoot fresh weight with respect to un-inoculated control. Among the isolates, RS-3 showed the highest fresh cob yield compared with all other iso- lates with 37% increase over the un-inoculated control. Isolate RS-1 was least efcient among the isolates tested with 27% in- crease in fresh cob yield over un-inoculated control. Drought at reproductive stage was more injurious than that at vegetative growth stage. Te drought tolerance index (Fig. 2) demonstrated that rhi- zobial inoculation was a good strategy for reducing the deleteri- ous impact of drought on maize growth and productivity. Water defcit stress imposed at the vegetative or reproductive stages resulted in 7 or 20% reductions in the drought tolerance index, Table 3. Growth and yield parameters of maize infuenced by rhizobial inocula- tion in pot conditions where irrigation was skipped at vegetative (D1) or repro- ductive (D2) growth stages of the crop or irrigated normally (D0). Shoot length (cm) (n = 3) nonsignifcant Root length (cm) (n = 3) Isolates D0 D1 D2 D0 D1 D2 Un-inoculated 111.7 102.0 98.7 68.7 b 64.0 b 55.0 c RS-1 120.0 118.3 115.0 71.7 b 67.0 b 65.7 a RS-3 117.7 110.3 105.7 80.3 a 78.0 a 58.0 bc RS-8 129.0 126.7 115.7 79.0 a 68.0 b 61.0 b RS-12 123.3 112.7 101.0 82.0 a 67.3 b 66.7 a HSD 19.3 25.9 18.4 4.0 4.0 4.6 Shoot fresh weight (g pot -1 ) (n = 3) Fresh cob yield (g pot -1 ) (n = 3) Un-inoculated 136.3 b 127.3 b 103.7 b 76.3 d 66.7 d 57.7 c RS-1 144.3 ab 143.0 a 120.0 a 93.0 b 85.3 b 77.0 a RS-3 141.3 ab 129.0 b 116.3 ab 100.7 a 92.0 a 82.3 a RS-8 144.7 ab 140.7 ab 124.3 a 95.7 b 88.7 ab 79.0 a RS-12 153.7 a 136.3 ab 117.7 a 85.3 c 76.0 c 69.7 b HSD 15.0 13.9 12.9 4.5 4.2 5.4 Means sharing similar letters are statistically similar to each other at p = 0.05. HSD shows honestly signifcance difference. Fig. 1. Drought tolerance index of maize seedlings (on the basis of shoot length) due to rhizobial inoculation at 15 or 25% PEG-induced drought in gnotobiotic conditions. www.soils.org/publications/sssaj 543 respectively. Inoculation increased the drought tolerance index of maize plants; RS-3 was the most promising isolate for increasing the drought tolerance index in maize plants under pot conditions. Identifcation and Benefcial Characteristics of Selected Rhizobial Isolate Selected isolates were identifed as Rhizobium phaseoli (RS-1 and RS-3), and Mesorhizobium ciceri (RS-8 and RS- 12), respectively, by use of the BIOLOG identifcation system. Tese identifcations were confrmed when their respective hosts produced efective nodules (pink colored) afer re-inoculation. Characteristics such as catalase and exopolysaccharides pro- duction were observed for all four isolates (Table 4). Rhizobial strain-1 (RS-1) was the only isolate positive for oxidase produc- tion. Further, phosphate solubilization activity and organic acid production were found in RS-1 and RS-12, but RS-3 and RS-8 did not have these properties. Results for siderophores produc- tion showed that rhizobial isolates RS-3 and RS-8 had the ability to synthesize siderophores however, RS-1 and RS-12 were non- producers. Indole-acetic acid (IAA) production was recorded for all the isolates in the presence or absence of L-tryptophan. Isolate RS-1 produced highest IAA, with or without L-tryptophan (3.95, 30.58 mg L -1 ), respectively, followed by isolate RS-12 and RS-8. Lowest IAA among the isolates was pro- duced by isolate RS-3. DISCUSSION Survival of inoculated rhizobia in soil is a major concern and depends on the severity of the environment. Ofen, inoculant bacte- ria added to soil sufer from desiccation and high temperature stresses. Rhizobia have a re- markable ability to survive under severe water stress, as indicated by the drought tolerance test of the isolates presented here (Table 1). However, drought decreases the rhizobial cell numbers with increase in the PEG concentra- tion. Findings of our study are in line with the results of Athar and Johnson (1997), Athar (1998), and Elboutahiri et al. (2010) who re- ported survival capability of rhizobia up to -1.5 MPa water potential. Teir ability to live under severe water def- cit conditions might be due to exopolysaccharides production and catalase or oxidase activity (Goyal et al., 1986; Fox, 2005). As bacterially produced exopolysaccharides enhance the nutrient and water holding capacity in the micro-environment to prolong their existence. Catalases are involved in scavenging the stress in- duced H 2 O 2 to reduce the cell damage and oxidases are helpful in maintaining cell metabolism under stress. About 82.2% of 157 Sinorhizobium melilotii (isolated from arid and semiarid areas of Morocco) isolates showed tolerance up to -1.5 MPa (Tami- Alami et al., 2010). Drought tolerant rhizobia accumulated osmolytes, and changed their cell morphology to survive under water defcit stress condition (Busse and Bottomley, 1989). One most important adaptation of exopolysaccharides producing Rhizobium leguminosarum bv. viciae 3841 is the development of bioflm for survival under desiccated conditions (Vanderlinde et al., 2010). Tose drought tolerant rhizobia could be helpful in rescuing the plant growth under drought conditions due to pro- longed existence in the rhizosphere. Drought tolerance is the ability of a plant to grow; fower and yield under suboptimal water supply (Farooq et al., 2009). Root length is a very critical plant growth parameter especially under reduced water conditions. In our study, inoculation with drought tolerant rhizobial isolates resulted in remarkable improvement in maize growth as shown by improved root or shoots length, root or shoots dry weight (Table 2) and drought tolerance index (Fig. 1) in pot trial in axenic condition. Aferward, in- oculation of selected rhizobia RS-1, RS-3, RS-8, and RS-12 improved the cob yield whether drought was applied (at vege- tative or reproductive stage) or not (Table 3). Similar improve- ments in the sorghum root or shoots dry weight was observed with the inoculation of Rhizobium or Bradyrhizobium under drought (Rashad et al., 2002). Tese improvements in the growth of sorghum might be due to the ability of Rhizobium or Bradyrhizobium to produce phytohormones, particularly auxin or cytokinin which are more important during drought Fig. 2. Drought tolerance index of maize plants (on the basis of root length) due to rhizobial inoculation under drought at vegetative or reproductive growth stage of the crop in pots. Table 4. Identifcation and plant growth promoting characteristics of selected rhizobial isolates. Rhizobium phaseoli Mesorhizobium ciceri RS-1 RS-3 RS-8 RS-12 Oxidase + Catalase + + + + Exopolysaccharides + + + + P-solubilization + + Siderophores + + Organic acid + + IAA production, mg L -1 Without L-TRP 3.95 0.92 1.35 3.27 With L-TRP 30.58 12.68 19.98 28.30 Plus sign represents the presence and minus sign shows the absence of the characteristics. 544 Soil Science Society of America Journal stress. Nitrogen nutrition, water uptake, and dry biomass of sun- fower were also enhanced due to the inoculation of exopolysac- charide producing Rhizobium strain, YAS34, under water stress (Alami et al., 2000). Moreover, exopolysaccharides producing rhizobial strain KYGT207 improved the rhizosphere soil aggrega- tion (Kaci et al., 2005). Fitouri et al. (2012) measured signifcant improvement in shoot dry weight of Hedysarum when inoculated with a moderately drought tolerant Rhizobium sullae strain iso- lated from a semiarid region of Tunisia. In our study, inoculation with rhizobia improved the drought tolerance index of maize, which might be due to their ability to produce exopolysaccharides, antioxidants (catalase), or phytohormones (IAA; Table 4). Rhizobial production of exopolysaccharides might have altered the maize rhizosphere by creating a bioflm on the root surface that might have enhanced water and nutrient availability to the host. Te increased root growth observed in inoculated maize could be due to indole- 3-acetic acid production by rhizobial isolates. Te better maize shoot growth observed, also suggests the involvement of mi- crobial assisted nutrition with their phosphate solubilization and siderophores activity (enhanced the iron availability and uptake). Whereas, oxidase and catalase production might have assisted rhizobial survival under water stress. It has been recognized that the plants show variable re- sponses to water defcit stress at diferent developmental stages (Mogensen et al., 1985; Gupta et al., 2001). Tese unfavorable conditions also adversely impact the development of reproduc- tive organs and grain yields (Mogensen et al., 1985; Gupta et al., 2001; Kettlewell et al., 2010). In our study, the drought impact was more severe at reproductive stage of maize as compared with drought at vegetative growth stage. However, rhizobial inocula- tion might have improved the rate of photosynthesis and ulti- mately the cob yield under drought. Similar, fndings have been reported by diferent researchers due to the inoculation of non- legumes with benefcial bacteria under drought (Vardharajula et al., 2011; Yandigeri et al., 2012). Afer the identifcation and confrmation of the rhizobial isolates it can be argued that the rhizobial isolates were selec- tive in host type and environmental conditions. Rhizobium and Mesorhizobium are those bacteria which nodulate mung bean and chickpea (less water consuming crops), respectively. Tey are also good producers of plant growth regulators such as sidero- phores, exopolysaccharides, and organic acids (Table 4). From the fndings of these experiments, it can be inferred that rhizobial inoculation to maize under reduced water condi- tions could be an opportunity to reduce drought stress impact on maize growth. However, isolates of Rhizobium phaseoli (RS- 1, RS-3), and Mesorhizobium ciceri (RS-8, RS-12) were most profcient. Inoculation of these selected rhizobial isolates could be helpful for improving maize growth and yield under reduced water conditions. Increases in the yield of maize with the inoc- ulation of indigenous rhizobial isolates in arid (Chakwal) and semiarid (Faisalabad) regions of Punjab (Pakistan) could be sup- portive in poverty alleviation. Adoption of inoculation biotech- nology of efcient rhizobia for non-legumes may reduce the pro- duction costs by reducing the use of chemicals such as fertilizers and pesticides, with environmental safety as an additional ben- eft. Moreover, selected isolates need to be evaluated in natural feld conditions for improving growth and yield of maize under reduced water conditions before their recommendation for large scale application. ACKNOWLEDGMENTS We are grateful to the Higher Education Commission (HEC) of Pakistan for fnancial assistance. Funding was provided by HEC under Indigenous 5000 PhD Fellowship Program (Batch V). 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