Relative potential of rhizobium species to enhance the growth and yield attributes of cotton (Gossypium hirsutum L.)

159 Relative potential of rhizobium species to enhance the growth and yield attributes of cotton (Gossypium hirsutum L.) M. Amjad Qureshi a,*, Haroon Shahzad b, M. Sajjad Saeed c, Sana Ullah d, M. Asif Ali a, Fakhar Mujeeb a, M.A. Anjum a a Agricultural Biotechnology Research Institute, AARI Faisalabad, Pakistan b Arid Zone Research Centre, PARC, D. I. Khan, Pakistan c Pulses Research Institute, AARI, Faisalabad, Pakistan d Institute of Soil & Environmental Sciences, University of Agriculture Faisalabad, Pakistan


Introduction
Cotton is the major cash crop of the country. It requires balanced amount of nutrients for proper growth and development. Microbial inoculants have the potential to compensate the mineral fertilizer and to provide a balanced diet to cotton. Biofertilizer technology has taken a part to minimize production costs and at the same time, avoid the environmental hazards (Galal et al., 2001). The microbe responsible for formulations of nodules i.e. mini factories of nitrogen is Rhizobium. Different species of Rhizobium forms nodules on different legumes. More than 20,000 members of family Fabaceae form nodules with Rhizobium (Gepts et al., 2005). Specificity of Rhizobium species is due to the chemical signaling between host legume and Rhizobium sp. Rhizobium-legume symbiosis is a well distinguished feature of legumes. Legumes fulfill most of its nitrogen by these symbiotic relationships. Rhizobium inoculation has been carried out in our agricultural systems for more than a century. Rhizobium besides nitrogen fixation in legumes also involved in variety of functions like P-solubilization due to production of organic acids, growth hormone production like auxins, gibberellins etc. and antibiotics (Sessitsch et al., 2002;Cassán et al., 2009;Mehboob et al., 2009;Ullah et al., 2017a). Literature reported that rhizobium strains influenced the roots of cotton due to the production of auxins resulted in efficient nutrient uptake (Hafeez et al., 2004;Hossain and Mårtensson, 2008;Gopalakrishnan et al., 2015). Interactions between plants and microorganisms in the rhizosphere can clearly affect crop yields. Rhizobacteria that benefit plant growth and development are called PGPR. PGPR can directly stimulate plant growth by producing phytohormones and by increasing nutrient uptake (Lippmann et al., 1995;Egamberdieva et al., 2008;Cassán et al., 2009) or by inducing systemic plant resistance towards pathogenic micro-organisms Noreen et al., 2012;Kumar and Jagadeesh, 2016). PGPR can stimulate plant growth indirectly by inhibiting the root pathogens (Gopalakrishnan et al., 2015;Habig et al., 2015). Rhizobia as a PGPR should be able to colonize and survive in the rhizosphere of non-legume plants because root colonization is an important first step in the interaction of beneficial bacteria with plants (Kloepper and Beauchamp, 1992;Mehboob et al., 2009;Egamberdieva, 2011;Habig et al., 2015;Kumar and Jagadeesh, 2016;Ullah et al., 2017a). Root colonization is the most crucial step for successful use of PGPR (Lugtenberg et al., 2001). PGPR facilitated the plants to uptake nutrients present in soil environment (Glick, 1995;Mehboob et al., 2009;Habig et al., 2015). Microbial inoculants only exhibited marginal increase in yield when tested under ideal conditions due to inconsistent response of crops . Researchers reported that crops inoculated with rhizobia exhibited increased amount of nutrients than uninoculated control (Biswas et al., 2000;Egamberdieva, 2011). Rhizobia having the potential of Psolubilization also enhance plant growth by increasing the nutrient mobilization, enhancing the availability of other trace elements and by production of plant growth promoting substances (Gyaneshwar et al., 1998;Gepts et al., 2005;Mehboob et al., 2009;Egamberdieva, 2011;Kumar and Jagadeesh, 2016). Present study was conducted to assess the comparative potential of Rhizobium species (berseem, chickpea, lentil, vegetable pea and mung bean) on growth and yield of cotton.

Isolation and purification of rhizobia of different crops
Yeast Extract Mannitol Agar (YMA) medium is used for the isolation of the Rhizobium spp. having composition per liter (5% K2HPO4:10ml; 2% MgSO4.7H2O: 10ml; 1% NaCl: 10ml; mannitol: 10 g; yeast extract: 1 g; agar: 15.0 g) (Vincent, 1970). For the preparation of the medium, the salts specified above were weighed and dissolved in water in pan and heated to dissolve the contents then it is shifted to conical flask and sterilized by autoclaving for 15-30 minutes at 15-20 lb inch -2 pressure and 121 0 C. After sterilization the media was poured on Petri plates aseptically in a laminar air flowcabinet and allow to solidify. Then this media is further surface sterilized by putting these plates in laminar flow cabinet under UV light. Plants of berseem, chickpea, lentil, vegetable pea and mung bean are uprooted at nodulation stage. The roots are thoroughly washed with tap water to remove soil particles from roots. Now collect the healthy nodules from roots of each crop plant without damaging the nodule. Now these undamaged nodules are immersed in 95% ethanol for 4-5 minutes. Then rinse them with sterilized distilled water. Now nodules are surface sterilized with 0.1% HgCl2 followed by washing with sterilized distilled water. Now punctured the nodule with needle or crushed it with sterilized forceps. Immediately streak the juice of punctured nodule on solidified YMA media in Petri plates in air flow cabinet by surface sterilized streaking needle. Incubate the inoculated media for 48-72 hours. The prolific colonies can be seen on the plate media after 2 days as they don't attained congored color. These colonies are then further multiplied on fresh prepared Petri plates as well as purified in slants and kept at 51 0 C for further experimentation.

Determination of auxin biosynthesis status of these Rhizobia spp.
Isolates of Rhizobium sp berseem (Br1, Br2, Br3, Br4, Br5), chickpea(CP1, CP2, CP3, CP4, CP5), lentil (L1, L2, L3, L4, L5) mung (M1, M2, M3, M4, M5) and Vegetablepea (Vp1, Vp2, Vp3)werescreened for their auxin biosynthesis potential colorometrically. For this purpose the isolates were cultured on the autoclavedGPM broth in test tubes with and without L-Tryptophan. The test tubes were covered and incubated at 28±2°C for one week and well shaken regularly throughout the week. The Un-inoculated control was kept for comparison. After incubation the contents were centrifuged @1000 rpm for 10 minutes and filtered through whatman filter paper No.2. The auxin biosynthesis potential was determined as Indole-3-acetic acid (IAA) equivalents by spectrophotometer at 535 nm using Salkowski's reagent (2 mL of 0.5M FeCl3 +98 mL of 35% HClO4) as reported by Sarwar et al. (1992). Isolates exhibited the highest auxin biosynthesis (Br5, Cp5, L3, Vp2 and M3) were selected for the study as mentioned in the Table 1.  (Mungbean), Rhizobium sp (Vegetablepea). Fertilizer @ 100-60 kg NP ha -1 was applied to all treatments. Experiment was laid out in completely randomized design (CRD) with five replications. All phosphorus andhalf nitrogen were applied as basal and half nitrogen was applied after 30 days of sowing. The photosynthetic rate, transpiration rate and photoactive radiation were determined by using IRGA (CI-340). Two repeats of cotton were uprooted after 45 days by irrigating the pots to record root / shoot length and mass. Then after 30 minutes when the soil became soft water from pipe was poured on to the pot as soil is removed and plant of cotton along with roots without any damage are obtained. The remaining three repeats were kept until maturity and growth / yield parameters were recorded. Data were subjected to statistical analysis by analysis of variance following completely randomized design (CRD) (Steel et al., 1997) and differences among the means were compared by the Duncan's multiple range test (Duncan, 1955).

Lab analysis
Chlorophyll content of leaf of each replication is determined colorimetrically by crushing the leaves in acetone and by centrifuging @ 1000 rpm for 10 minutes then run on spectrophotometer (Arnon, 1949). After uprooting the plants physical parameters like root and shoot length is measured by measuring scale in centimeters while root and shoot mass is determined by using digital balance. Then two gram of each of the root and shoot of each repeat is taken that is dipped and ground in autoclaved 10mL GPM in test tube and covered with cork. Two GPM containing test tubes are kept as control. This is kept for one week and shaken well three times a day regularly. Then the contents are centrifuged @ 1000 rpm for 20 minutes. 5 standards of auxin (1, 2, 3, 4 and 5) ppm were made from 1000 ppm auxin stock solution. Three mL of supernatant and auxin standards are taken in test tubes and added 2 mL of Salkowski's reagent (98mL 35% HClO4+ 2mL0.5M FeCl3) to each tube. After 30 minutes a pink color ring is formed on top of GPM supernatant that indicated presence of auxin. The samples were run these samples on spectrophotometer @535nm spectrum and check the biosynthesis of auxin as IAA equivalents by plant shoot and root by comparing it with control.

Results and Discussion
Data regarding IAA production by different strain of Rhizobium isolated from root nodules of crops under study is presented in Table 1. Keeping in view the above data it can be observed that Rhizobium species isolated from nodules of berseem, chickpea, lentil, vegetable pea, mungbean (Br5, Cp5, L3, Vp2 and M3) produced higher auxin values in order of 4.48, 3.81, 4.47, 3.67 and 4.24 µg ml -1 and the values were more enhanced with the supplement of L-tryptophan i.e. 6.54, 5.95, 5.39, 4.76 and 4.91, respectively. It was observed that all tested isolates produced IAA equivalents and effect was more pronounced with the application of L-tryptophan. The biochemical tests such as Congo red test, bromothymol and gram reaction showed the characteristic features of the tested isolates. Use of beneficial rhizobacteria to attain sustainable agriculture and nutrient availability for crop plants has been becoming an emerging approach in conservation of agriculture (Habig et al., 2015). Moreover, Rhizobia are potential partner of legumes crops but also act as beneficial rhizobacteria for non-legume or cereals (rice, maize, wheat) without forming nodules through different plant growth mechanisms such as nutrient availability, release of growth hormone, production of siderophores and lumichrome, improved root morphology, more root adhering soil and defense action (biocontrol, induced systemic resistance) Kumar and Jagadeesh, 2016). Among the phytohormones (auxins, gibberellins, abscisic acid, ethylene), auxins play major role in plant-microbe interaction, and growth and development of plants (Boivin et al., 2016). In this experiment rhizobial species isolated from five different legumes were assayed for their IAA-producing efficiency then verified to affect the growth and yield attributes of cotton. In results, rhizobial isolates Br5 showed maximum IAA-potential hence better impact on cotton growth and yield than other isolates. Our observation regarding auxin production related to different studies which were conducted to investigate the potential of IAA-producing rhizobial species to enhance plant growth (Hussain et al., 2014;Parthiban et al., 2016). Data regarding shoot/root mass is presented in Figure 1 showed that isolates of rhizobium species enhanced the shoot/root mass considerably. Inoculation with isolate Br5 produced 60.94, 64.40 g shoot/root mass that is 16.70 and 23.80 % higher than control while other isolates showed percent increase compared to control viz. Cp5 (8.1, 20.3%), L3 (8, 18.0%), Vp2 (4.3, 12.6%), M3 (4.7, 9.4%), respectively. Br5 i.e.18.3, 24.8 % higher than that of control while Cp5 (9.8, 16.5%), L3 (14.9, 12.7%) and M3 (5.53, 21.5%) and isolate of Vp2 has no impact regarding shoot length of cotton while improved root length 19%. The IAA equivalents in shoot/root content of cotton seedlings are presented in Figure 3. Results clearly demonstrated that bacterial inoculation improved the IAA content in shoot/root content of cotton seedlings as compared to control and isolates showed variable response regarding shoot/root IAA content. Results showed that the highest value of shoot/root IAA content was obtained with Br5 i.e. 4.41 and 3.65 µg g -1 followed by Cp5 i.e. 4.41 and 3.49 as compared to control i.e. 2.35 and 1.95, respectively. Inoculation of cotton seeds with rhizobial isolate caused maximum increase in length and mass of root and shoot which might be due to production of lumichromes that help in CO2 assimilation by roots (Dakora, 2003), riboflavin which stimulate root respiration (Dakora et al., 2002) and auxins to promote cell elongation through their expansion action (Pacheco-Villalobos et al., 2016). Beneficial rhizobacteria also influence the cell division and differentiation of plant roots which caused improved growth of shoot system (Verbon et al., 2016). These improvements are in lined with the study of (Noreen et al., 2012) who observed 54, 45 and 185% increase in root, shoot length and seedling fresh weight of Vignamungo (L.) when inoculated with IAA-generating strain Pseudomonas aeruginosa As-17. (Asghar et al., 2015) also noted a significant improvement in weight and length of root and shoots of wheat plants due to inoculation with different rhizobial isolates. Data regarding physical parameters i.e. plant height, boll weight, boll plant -1 , cotton leaf curl virus percentage (CLCV) and seed cotton yield from remaining repeats is presented in regarding CLCV percentage, it was observed that the highest CLCV percentage was observed with control and the least was observed by inoculation with Br5. Results regarding seed cotton yield clearly demonstrated that bacterial inoculation improved the seed cotton yield (47.0 g) as compared to control (34.50), respectively. Increase in seed cotton yield due to bacterial inoculation (36, 27.5, 18.8, 12.3, and 5.8%) due to application of Rhizobium species i.e. Br5, Cp5, L3, Vp2 and M3 was observed, respectively. Rhizobial inoculum left potential impact on cotton plants and increased the biomass and yield parameters of cotton crop including number of bolls per plant, boll weight and seed cotton yield. It might be attributed to different rhizospheric-synergistic effects of rhizobia such as biocontrol against pathogens (Hossain and Mårtensson, 2008), cyanide production (Adnan et al., 2016), production of exopolysacharides, antioxidants, better colonization ability (Hussain et al., 2014), growth hormones (Qureshi et al., 2013) like IAA in the cotton rhizosphere which improved root morphology, root proliferation (Hussain et al., 2009) thus better root system for acquisition and uptake of mineral nutrients (N, P and K) as seen in case of rice seed and seedling inoculation with different Rhizobium spp (i.e. Bradyrhizobium sp IRBG271, R. leguminosarum bv. trifolii E11 and Rhizobium sp. IRBG74,) (Biswas et al., 2000) ultimately improving cotton yield. Our results relevant to a recent study conducted by (Arshad et al., 2016) who found a substantial increment (13%) in lint and seed yield of transgenic and non-transgenic cotton plants upon inoculation with IAA-producing rhizobacterial strainBrevibacillus spp. TN4-3NF in comparison to un-inoculated control plants.
Results regarding different parameters obtained with IRGA (CI340) like Photosynthetic rate, Transpiration rate, Photo active radiation (PAR) and total chlorophyll content obtained by spectrophotometer are presented in Table 2. Results showed that the maximum photosynthetic, transpiration and PAR values were observed with Rhizobium sp (Br5) i.e. 89.0 μmole m -2 s -1 , 8.2 mmole m -2 s -1 and 863 μmole m -2 s -1 and total chlorophyll content 1.40 mg g -1 , respectively. Seed inoculation of cotton with rhizobial isolates put positive influence on physiology of cotton plants, thus, enhancing the photosynthetic activity and transpiration rate. This improvement by rhizobia may be due to the production of siderophoresenhancing iron (part of chlorophyll) supply, growth hormones (Adnan et al., 2016) and exopolysaccharide which facilitate water and mineral supply throughimproved root system (Egamberdieva and Kucharova, 2009;Hussain et al., 2014) ensuring better plant physiology for normal growth and development of cotton plants. These finding of our experiment according to an experiment in which inoculation of maize seeds with two PGPR strains (B.phytofirmans PsJN and Enterobactersp. FD17) caused significant increase in physiological parameters (photosynthetic rate 75%, stomatal conductance 87%, transpiration rate 84%, chlorophyll contents 22% of plants (Naveed et al., 2014). Study results concluded that Rhizobium species are effective PGPR if screened thoroughly and by determining its growth hormone production potential. Present study also predicted that Rhizobium species have specific response on different crops also study concluded that Rhizobium species having growth hormone production potential should have root colonizing ability for successful utilization in non-legumes.