Sorghum (Sorghum bicolor L.), is an important rainfed crop grown world over on 42 million ha in 98 countries and Nigeria, India, USA, Mexico, Sudan, China and Argentina are the major producers [1]. In India, sorghum was planted in 7.7 million ha with production of 7.24 million tonnes and productivity of 940 kg. ha^-1 [2].

Soil microorganisms play important role in determining plant productivity. For successful functioning of introduced microbial bioinoculants, exhaustive efforts have been made to explore soil microbial diversity of indigenous community, their distribution and behaviour in soil habitats [3]. Soil microorganisms are directly responsible for recycling of nutrients [4].

Considering the ill-effects of inorganic fertilizers on soil health, adoption of integrated nutrient management (INM) has been advocated for sustainable agriculture. Efforts to supplement nutrients through biofertilizers as part of INM helped the rainfed farmers significantly [5]. Microorganisms that facilitate nutrients availability and use could form sustainable solutions for present and future agricultural practices [6]. Microbes that indirectly or directly promote plant growth are referred to as plant growth promoting rhizobacteria (PGPR) [7]. Species of Pseudomonas comprise a large portion of the total culturable bacterial population in the rhizosphere. Due to the ubiquity and versatility of pseudomonads, there is a considerable interest in exploiting these bacteria for diverse agricultural applications such as plant growth promotion and pest management etc., [8].

Information on fluorescent Pseudomonas spp. from diverse agroecosystems and their plant growth promoting potential particularly in sorghum is scanty. In this paper, we report isolation and variations among 75 isolates of Pseudomonas spp. from 23 different agroecological regions of India with respect to their ability to promote nutrient uptake and growth in sorghum.

Bacterial cultures and seed bacterization

Seventy-five soil samples of different crops representing 31 locations from 13 states of India were used for isolation of fluorescent Pseudomonas spp. [9]. Sorghum seeds of cv. CSV-15 procured from Directorate of Sorghum Research, Hyderabad, India were bacterized with Pseudomonas isolates as described by Dileep Kumar and Dube [10].

Physico-chemical characterization of soil samples

Physical characters like pH, electrical conductivity (EC), particle size and chemical characters like macronutrients (N, P, K) and organic carbon content were characterized for all the collected soil samples and similar characterization was also done for the soil used for plant experiments (Table 3) [11].

Screening for plant growth promoting Pseudomonas isolates

Preliminary screening of Pseudomonas spp. isolates for their plant growth promotion (PGP) by seed bacterization was done as explained by Ali et al. [12]. Three bacterized seeds were sown in each paper cup containing sterile soil and six replicates were maintained with untreated control. 15 days after sowing (DAS) root length, shoot length and dry mass of seedlings (by drying to constant weight at 65oC) were recorded and relative increase was calculated as against un-inoculated control

Pot experiments and nutrient analysis

Fourteen Pseudomonas spp. isolates viz, P1, P2, P4, P5, P13, P14, P17, P20, P21, P22, P23, P28, P29 and P35 shortlisted from previous experiment with >50% enhancement in dry mass of seedlings (Table 1) were selected for pot culture experiments. Pot experiments with bacterized seeds were conducted as described by Sindhu et al. [13]. After 30 DAS root volume, shoot length, leaf area (measured by LI 3100 Lincoln Nebraska USA leaf area meter), total chlorophyll (measured by Minolta Spad chlorophyll meter-502) and dry mass of root and shoot were recorded.

Analysis of the macro- (NPK) secondary- (Na, Ca) and micro- (Fe, Cu, Mn, Zn) nutrients in the experimental plants was carried out following the protocols of Tandon [11]. Total carbohydrate content was estimated by anthrone method [14].

Data obtained from all experiments were subjected to analysis of variance (ANOVA). Mean values between treatments were compared using Fisher’s least significant difference (L.S.D) test (P<0.05). All plant growth parameters were given equal importance to follow Z-score ranking and to identify the promising Pseudomonas isolate showing best plant growth promotion.

Isolation of fluorescent Pseudomonas spp.

Pseudomonas spp. isolated from different soil samples showing fluorescein production on King’s B medium were selected and purified. 75 fluorescent Pseudomonas spp. were isolated from soil samples obtained from 31 different locations representing 13 states of India (Figure 1). Isolates were designated as P1 to P75 and added to the culture collection of Central Research Institute for Dryland Agriculture, Hyderabad. All the isolates were stored as 30% glycerol stocks at -20°C and revived periodically for further studies.

Figure 1: India map showing soils samples from different agro-ecosystems. Figures in parentheses in legends represent number of samples collected.

Characterization of soil samples

Of the 31 soil samples characterized, five were from western plains, one from western Himalayas, six from northern plains, two from central highlands, 11 from Deccan plateau, four from eastern ghats, and one each from eastern plateau and Chattisgarh-Mahanadi basin agro-ecological regions of India. The annual mean rainfall of these regions ranged from 150-1450 mm (lowest in western plains and highest in Chattisgarh-Mahanadi basin). The annual mean maximum soil temperatures ranged between 28-47°C with lowest being in western Himalayas and highest in western plains (Table 1).

Table 1: Agro-ecological regions, climatic conditions and their soil types of India used for isolation of fluorescent Pseudomonas spp.

Eight samples showed acidic pH from 6.0-6.9 with Phulbani sample recording lowest pH. Four soil samples were neutral with pH 7.0-7.2 and 19 were alkaline (pH 7.3-8.6) with Bijapur sample showing highest pH 8.6. Electrical conductivity (EC) of the samples ranged between 0.02 (Phulbani, Orissa) and 1.79 dS.m^-1 (Hisar, Haryana). Organic carbon (OC) content ranged between 0.12 (Phulbani) and 0.5% (Rajendranagar, Andhra Pradesh). Total available nitrogen content ranged from 62 (Hayatnagar, Andhra Pradesh) to 183 kg.ha^-1 (Arjia, Rajasthan). Similarly phosphorus (P_i) content ranged from 6.3 (Akola, Maharashtra) to 20.1 kg.ha^-1 (Parbhani, Maharashtra). Potassium content varied widely across samples with the highest being in 500.4 kg.ha^-1 Solapur (Maharashtra) and lowest of 55.6 kg.ha^-1 in Baribrahmana (Table 2).

Table 2: Physico-chemical properties and macronutrient status of soil types used for the isolation of Pseudomonas spp.

Table 3: Physico-chemical characters of soil used for plant growth studies

Screening for plant growth promotion

Most of the Pseudomonas spp. isolates promoted growth of sorghum seedlings. While 69 isolates showed increase in root length, 70 isolates showed increase in shoot length and dry mass as compared to un-inoculated control (Table 4). All the isolates were grouped into three categories based on increase in dry mass viz, <25%, 25-50% and >50%. Of the 75 isolates, 45% of them showed <25% enhancement of dry mass, whereas 29% of them enhanced dry mass in the range of 25- 50% and 17% of them showed >50% increment in dry mass (Figure 2). Remaining 7% isolates showed dry mass of less than control and were considered as deleterious rhizobacteria (Figure 2). 14 isolates viz, P1, P2, P4, P5, P13, P14, P17, P20, P21, P22, P23, P28, P29 and P35 that showed more than 50% enhancement in dry mass of seedlings were considered as potential strains for further pot-culture studies.

Table 4: Control P17 Figure 3: Plant growth promotion of sorghum by Pseudomonas sp. P17 in pots (30 days after sowing). Relative percentage increase in root, shoot length and dry mass of sorghum seedlings on seed bacterization with Pseudomonas spp. (15 DAS).

Figure 2: Grouping pattern of plant growth promoting fluorescent Pseudo monas spp. on sorghum.

Pot experiments and nutrient analysis

Seed bacterization of sorghum with fluorescent Pseudomonas spp. enhanced sorghum plant growth significantly (Figure 3, Table 5). Root volume in control was 0.17 cm³ whereas, in treatments it ranged between 0.18 and 0.30 cm³. Inoculation of sorghum with P1 and P17 showed maximum root volume of 0.30 cm³ compared to other treatments. Significant increase in shoot length was observed with P17 (36.2 cm) inoculation followed by P1 (33.5 cm) and P22 (32.2 cm). Highest root dry mass was recorded in P2 (78.4 mg) followed by P17 (69.1 mg) and P35 (66.9 mg). Maximum shoot dry mass was recorded in plants treated with P17 (83.1 mg) followed by P1 (69 mg) and P22 (68 mg). Inoculated plants also showed higher leaf area compared to control. Leaf area of plants inoculated with P17 was 31.6 cm² followed by P22 (27 cm²) and P20 (22 cm²) than un-inoculated control plants (9.8 cm²). Similar results were also recorded in case of chlorophyll content. P17 treated plants recorded highest chlorophyll content of 23 spad units followed by P22 and P23 treatments which were 22 and 21 spad units respectively compared to control (8 spad units). Overall increase in plant dry mass was highest in P17 treated plants (152 mg) followed by P22 (132 mg) and P23 (128 mg) (Table 5).

Table 5: Plant growth of sorghum as influenced by seed bacterization with fluorescent Pseudomonas spp. (30 days after sowing).

Figure 3: Plant growth promotion of sorghum by Pseudomonas sp. P17 in pots (30 days after sowing).

Inoculation of sorghum with Pseudomonas spp. not only enhanced plant growth but also increased nutrient uptake significantly (Table 6,7). Total carbohydrates content of treated plants was in the range of 17.3 to 30.6% with highest by P17 where as untreated plants had 15.4%. Phosphorus uptake was also significantly higher in treated plants with Pseudomonas isolates in general and P1 and P17 (1.35%) in particular compared to control (0.38%). Nitrogen uptake was also significantly higher in P17 treatment (2.254%) followed by P22 and P23 (2.24% in both). Similarly, sodium uptake in plants increased on treatment with P22 (0.54%) followed by P17 (0.52%) and P23 (0.51%). However, potassium uptake was more in P17 treated plants (2.9%) followed by P22 (2.85%) and P1 (2.54%). P5, P13 and P35 treated plants showed significantly higher Ca uptake of 0.88%, 1.02% and 1.15% respectively (Table 6).

Table 6: Macro-nutrient uptake in sorghum as influenced by seed coating with fluorescent Pseudomonas spp. (30 days after sowing).

Seed bacterization of sorghum with Pseudomonas isolates significantly increased the uptake of micronutrients (Table 7). Inoculation with P17 and P35 enhanced the Cu content (14 ppm) followed by P1 and P5 (12 ppm) compared to control (7 ppm). Higher quantity of Fe was accumulated in plants inoculated with P17 (3500 ppm) followed by P35 (2901 ppm) and P22 treatments (2527 ppm). Mn uptake was maximum in P17 treatment (237 ppm) followed by P28 (227 ppm) P4 (183 ppm) compared to control (74 ppm). Zn uptake was more on inoculation with P22 (936 ppm) followed by P17 (433 ppm) and P13 (429 ppm) than control (159 ppm) (Table 7). Overall impact of P17 treatment on nutrient uptake and plant growth was 70-220% and 30-290% respectively (Figure 4,5).

Table 7: Micro-nutrient uptake in sorghum as influenced by seed bacterization with fluorescent Pseudomonas spp. (30 days after sowing).

The microbial biodiversity of a region is mainly determined by agro-ecological systems and constituents of plant root exudates that decide the type and density of microbial population in a given crop production system [15]. Efficient colonization and/or physiological adaptation to adverse soil conditions are the options for soil bacteria to survive [16]. In the present study, with an aim of obtaining isolates of Pseudomonas spp., different crop production systems of diverse agro-ecological regions were surveyed (Figure 1) and 75 isolates were obtained. Fourteen isolates were found to enhance >50% dry mass of sorghum seedlings (Table 4). Of these, 9 isolates were from semi-arid deep alfisols belonging to agro-ecological region, 1 isolate each from semi-arid medium deep inceptisols/vertisols (deccan plateau), semiarid medium deep vertisol (deccan plateau), semi-arid black medium deep vertic inceptisol (northern plain), sub-humid alluvial deep inceptisol (western himalayas) and semi-arid medium deep vertisol (western plain) (Table 1). This indicates the congenial conditions of the soil perhaps facilitated the plant growth promotion (PGP) activity of these isolates as soil nutritional conditions are reported to be influencing the performance of PGPRs [17].

Present experiments on sorghum clearly indicated that Pseudomonas spp. can be used to enhance the plant growth as reported earlier [18,19]. Bacterial inoculated seedlings of different crops showed enhanced plant growth as reported by Kloepper et al. [20], Glick [21] and Dey et al. [22]. In the present study, a significant increase (P<0.05) in root and shoot length and dry mass of sorghum seedlings was observed due to seed bacterization (Table 5). The plant growth promotion could be attributed to the exertion of direct and/or indirect action of PGP traits [23].

Seed bacterization of sorghum with Pseudomonas spp. also enhanced the uptake of essential macro and micro-nutrients resulting in overall increase of plant growth (Table 6,7). It is in concurrence with the observations of Paul, et al. [24] and Kourosh et al. [25] who reported enhanced uptake of nutrients in black pepper and sweet basil due to seed bacterization with Pseudomonas spp. Increased nutrient uptake by plants inoculated with plant growth promoting bacteria has been attributed to the production of plant growth regulators at the root interface, which stimulate root development and better absorption of water and nutrients from soil [26,27]. In the present study, we observed significant impact (P<0.05) of Pseudomonas spp. on plant growth promotion in various parameters like root volume, shoot length, dry mass, chlorophyll content, leaf area etc. and enhanced macro and micro-nutrients uptake. Besides plant growth, inoculated plants clearly showed increased accumulation of nitrogen which is in agreement with observations of Puente et al. [28]. Esitken et al. [29] demonstrated that root inoculation of Bacillus and Pseudomonas sp. increased nutrient content (P, Fe, Zn, K and Mg) and plant growth of strawberry. Rhizobacteria efficacy on sorghum growth promotion in green house conditions was shown by Idris et al. [30]. Present findings are co-inciding with earlier studies. The importance and role of PGPR traits of Pseudomonas spp. in growth promotion of sorghum was shown by Praveen Kumar, et al. [31].

Defreitas and Germide [32] demonstrated that seed treatment with Pseudomonas spp. significantly enhanced early growth of winter wheat in low fertility asquith soil. Observations in the present study with Pseudomonas sp. P17 strain showed good plant growth enhancement (Figure 3,4) and higher nutrient uptake (Figure 5). Z-score ranking also revealed that P17 ranked as the best isolate among the other Pseudomonas isolates studied.

Figure 4: Influence of seed bacterization with Pseudomonas sp. P17 on plant growth parameters of sorghum.

Figure 5: Uptake of macro- and micro-nutrients as influenced by inoculation with Pseudomonas sp. P17 in sorghum.

Present studies were carried out with the objective of assessing the plant growth promoting potential of Pseudomonas spp. towards S. bicolor from diverse rainfed agro-ecosystems. Sorghum is generally cultivated under rainfed conditions and after sowing, generally the crop suffers due to a dry spell. During this period, if the plant is protected with a better vigour, it can tide over the dry spell and grow normally with the resuming of monsoon. A low cost technology like seed bacterization has been found to promote plant growth during early phenophase of the crop that is most vulnerable to dryspells. Isolates obtained from semi-arid deep alfisols are efficient PGPRs than the other isolates which depicts that the origin of the PGPR also plays an important role in determining the behaviour and efficacy of PGPRs in increasing the plant growth. In conclusion, improvement in nutrient uptake and growth of sorghum plants were observed on inoculation with fluorescent Pseudomonas sp. P17. Further investigations on this PGPR for its efficiency under field conditions are in progress to promote it as ‘low cost’ input for improved productivity of rainfed agro-production systems.

Authors thank Indian Council of Agricultural Research (ICAR), Ministry of Agriculture, Government of India, New Delhi for financial support under the network project “Application of Microorganisms in Agriculture and Allied Sectors (AMAAS)- Nutrient Management & PGPR”.