Tropical Agricultural Research Vol. IZ : 163-176 (2000) Effect of Associative Nitrogen Fixing and Phosphate Solubilising Bioagents on Growth, Yield and Quality of Sugarcane C. Shankaraiah and G. Hunsigi University of Agricultural Sciences Regional Research Station, V.C. Farm, Mandya-571 405 Karnataka, India ABSTRACT. Field investigations were carried out at Regional Research Station, Visveswaraya Canal Farm, Mandya of Karnataka, India to study the effect of inoculation of nitrogen facing and phosphate solubilising microorganisms (PSM) on growth, yield and quality ofsugarcane. Soil inoculation of nitrogen fixers viz., Azotabacter in plant crop and Azospirillum in ratoon crop ensure increased cane yields of 10-15% as a result of perceptible improvement in various growth and yield parameters. Enhanced nutrients use efficiency as evident from their higher availability and uptake, especially of nitrogen, were suggestive ofeconomy in fertilizer nitrogen up to 20% or 50 kg ha'. The leaching loss of nitrate nitrogen (N03-N) and consequent pollution of ground water was greatly reduced due to inoculation by Azotobacter and Azospirillum. Azospirillum is more rewarding in compacted soils of ratoon crop while, Azotobacter in light textured soils. Soil inoculation of Agrobacteriun radiobacter and Bacillus megaterium in plant crop and Aspergillus awamori and Bacillus megaterium in ratoon crop exhibit 8-10% higher cane yields besides economy in fertilizer phosphorus up to 25%. Further, enhanced availability and uptake of phosphorus indicated better P use efficiency and P solubilising capacity of PSM. The effect due to PSM is more pronounced with soluble sources of phosphorus vjz., single super phosphate in conjunction with pressmud. The bacterial cultures yjg., Agrobacterium radiobacter and Bacillus megaterium were efficient solubilisers ofphosphorus in plant crop whilst, the fungus Aspergillus awamori in ratoon crop. Inoculation of Azotobacter and Azospirillum among nitrogen fixers and Agrobacterium radiobacter among PSM exhibited jaggery (Gur) of higher quality and grade. INTRODUCTION Fertilizer, management with an integrated and ecofriendly approach seems to be the desirable route to achieve the objective of high yield, cane quality with sustainable productivity of both cane and sugar. Nitrogen (N) nutrition assumes primacy in sugarcane production as it is instrumental in improving yield and quality of sugarcane. Reduced N use efficiency (NUE) due to leaching and volatilisation losses, increasing cost of N fertilizers have added to reduced cost: benefit ratios. The leaching loss of nitrate N (NO}- N) is said to be the potential threat to ground water pollution and World Health Organisation (WHO) standard for drinking water with nitrate content is 100 mg I'1 (Biswas et al., 1992). Thus, economic and environmental considerations have prompted the need to stimulate research on alternate but cheaper, renewable and ecofriendiy supplementer of Shankaraiah & Hunsigi N. The advent of acetylene reduction assay for measuring nitrogeriase enzyme activity has lead to the establishment of biological N fixation (BNF) in sugarcane rhizosphere (Dobereiner et al., 1972). Azotobacter a free-living soil bacteria and Azospirillum an associative, microaerophillic symbiotic soil bacterium offer a great potential as biological N fixers in sugarcane crop. Acetobacter diazotrophicus an obligate endophytic bacterium is capable of fixing 200-250 kg N ha'1 per season meeting substantial part of N demand of sugarcane crop (Lima et al., 1987; Dobereiner, 1990). Phosphorus (P) is the second most important but deficient nutrient in sugarcane production. As a consequence of high cost of water soluble chemically processed phosphatic fertilizers and reduced P use efficiency due to fixation as insoluble phosphates, imbalance in NPK ratios are anticipated which eventually leads to reduced productivity of sugarcane. Mobilisation of insoluble P in soils through microbially mediated system assumes practical importance in increasing P use efficiency and associated growth related parameters in sugarcane. The soil isolates such as Agrobacterium radiobacter, Bacillus megaterium var. phosphaticum, Aspergillus awamori etc., have been reported to be efficient phosphate solubilising microorganisms (PSM) even with low grade phosphates such as rock phosphate, slag, bonemeal etc. (Gaur, 1990: Marwaha, 1995). The reports on the capabilities of such organisms suffer from major drawback as they are mostly of in vitro experimentation and showed solubilisation under highly artificial conditions of culture media (Chhonkar, 1994). However, beneficial influence of artificial inoculation of N fixers and PSM better known as biofertilizers or biological software in sugarcane has been reported under diverse agroclimatic conditions with positive gain in crop yields and economy in fertilizer N and P. Therefore, their differential behaviour towards varying agroclimatic conditions, varieties etc., prompted a detailed location specific field study on the efficacy of some selected bioagents in sugarcane in the Cauvery basin in southern Karnataka. The objectives of the investigations were to study besides, growth, yield and quality parameters of sugarcane, availability and uptake of major nutrients, economy in fertilizer N and P and quality and grading of jaggery (Gur) as influenced by N fixers and PSM. The possible reduction in the leaching loss of NCyN due to N fixers and the utilization of natural and cheaper sources of P due to PSM were programmed as added objectives. MATERIAL AND METHODS Field investigations were carried out at Regional Research Station, Visveswaraya Canal Farm, Mandya, Karnataka, India to study the effect of inoculation of N fixers and PSM independently on growth, yield and quality of sugarcane plant and ratoon during 1995-98. The soils of the study area is classified as Ustalfs with a sandy clay loam texture. They are neutral in reaction with low organic carbon and available N, medium in available P and'low in available K. The four field experiments include two each on N fixers and PSM on both plant and ratoon sugarcane. Four N fixers viz., Azotobacter chroococcum, Azospirillum brasilense, Acetobacter diazotrophicus (local and Brazilian isolates) superimposed with three levels of N viz., 150,200 and 250 kg ha'1 were tried in split plot design. Azotobacter chroococcum and Azospirillum brasilense as carrier based inoculants were applied to soil at 2.5 kg ha'1 in two equal splits at 30 and 60 days after planting in 164 Effect of Associative Nitrogen Fixing and Phosphate Solubilising plant crop and IS and 45 days after stubble shaving in ratoon experiment. The liquid cultures of Acetobacter diazotrophicus (local and Brazilian isolates) were diluted with sugarcane juice and inoculated through set treatment in plant and drenching the stubbles in ratoon experiments. The levels of N as per the treatments were imposed in four splits in plant and three splits in ratoon experiments as per the recommendations of the region. The varieties used were cv. Co 7804 and cv. Co 419 in plant and ratoon experiments respectively. Simultaneously, the treatments were simulated in pot culture study for collection and analysis of leachate for NCyN. The studies on PSM include three species of PSM viz., Agrobacterium radiobacter, Bacillus megaterium and Aspergillus awamori superimposed with two levels of P i.e., 75 and 100% recommended P and three sources of P i.e., single superphosphate (SSP), SSP+mussorie rock phosphate (MRP) to supplement 50% P each, SSP+pressmud (a byproduct of sugar industry) to supplement 50% P each were tried in split-split-plot design in plant and ratoon sugarcane. The PSM were inoculated through soil as carrier based inoculants at 10 kg ha"1 at 30 days after planting of sugarcane and 15 days-after of stubble shaving in ratoon experiments. The levels and sources of P as per treatments were imposed in full at planting and 30 days after stubble shaving in ratoon experiments. The growth, yield and quality parameters of sugarcane were recorded at appropriate stages of growth adopting standard procedures commonly in all the experiments. The nutrient indices, uptake and availability of major nutrients were estimated adopting standard procedures (Jackson, 1973; Subbaiah and Asija, 1956). The jaggery samples prepared in the laboratory at harvest were analysed and graded according to net rendament (NR) values (Khanna and Chakravarthi, 1954). The leachate samples collected periodically from field and pot culture studies were analysed for NCyN by Devarda alloy method (Sankaran, 1966). The statistical analysis was done using Fisher's method of analysis of variance technique. RESULTS AND DISCUSSION Effect of N levels and N fixers on growth, yield and quality of Sugarcane- plant and ratoon Growth and yield parameters The N levels and the N fixers had no perceptible influence on initial growth parameters such as germination, tillering capacity, shoot population both in plant and ratoon crop experiments. The leaf area index (LAI) and leaf area duration (LAD) in general exhibited marked improvement due to inoculation of Azotobacter and Azospirillum at 6* month of growth and harvest stages in both plant and ratoon experiments. The higher LAI and LAD are attributable for increase in number and size of leaves, extended source capacity and stay green character (Arabidopsis) as a result of production of plant growth regulators (PGR) and biologically active metabolites by the bioagents (Marwaha, 1995). : The values of LAI and LAD were higher in plant crop than in ratoon and declined sharply7* towards harvest. The crop growth rate (CGR) responded positively to both N fertilization' and N fixers viz., Azotobacter and Azospirillum in plant crop argudly due to higher LAI and 165 Shankaraian & Hunsigi LAD during grand growth period. Higher dry matter production due to inoculation of Azotobacter followed by Azospirillum in plant crop arid Azospirillum in ratoon crop at 6 t h month are attributable for improvement in LAI, LAD and CGR. The height arid weight of millable cane expressed significant improvement due to addition of N fixers viz., Azotobacter followed by Azospirillum in plant crop. Whilst addition of N up to 200 kg Ha'1 and Azospirillum alone expressed higher height and weight of millable cane in ratoon cane (Table 1). Cane and sugar yield Inoculation of Azotobacter registered the highest cane yield followed by Azospirillum at the recommended N level of 250 kg ha'1 in plant crop. In ratoon crop, :Azospirillum excelled with the highest cane yield of 126 Mg ha'1 at the same level of N. Improvement in yield following the inoculation of N fixers is ascribed for increase in heightand weight of millable cane. The increase in cane yield was to a tune of 21.22 Mg ha'1 and 9.33 Mg ha'1 due to Azotobacter and Azospirillum respectively, over no inoculation in plant crop. Whilst, it was to a tune of 5.44 Mg ha'1 and 3.67 Mg ha'1 due to Azospirillum and Azotobacter respectively over no inoculation in ratoon crop (Table 1 and 2). This establishes the superiority of Azotobacter in plant crop and Azospirillum in ratoon crop. Azotobacter is reported to perform better in semi dry loamy and sandy soils which are less compacted with better aeration as observed under plant crop soil conditions (Srinivasaii and Naidu, 1987). Azospirillum being microaerophillic and known to maintain high nitrogenase. activity Lin low oxygen tension environment substantiates its effectiveness under compacted' soils of ratoon crop. Azospirillum expressed significantly the highest millable cane* population in ratoon crop which is attributable for higher survival rate due to the production of PGR. It is evident from the data that the yield levels either on par or higher than that obtainable at recommended N level could be maintained even at reduced N level of 200 kg ha'1 upon inoculation with Azotobacter and Azospirillum in plant crop and Azospirillum in ratoon crop. This suggests the possibility of saving fertilizer N to an extent of 20% or 50 kg ha'1 indicating better N use efficiency. The juice quality parameters were not influenced due to imposed treatments. This is anticipated since N fertilization at the recommended level (250 kg ha'1) had no deleterious effect on juice quality. The sugar yield improved significantly due to the inoculation of Azotobacter with N fertilization which is essentially due to improved cane yield rather than juice quality parameters (Table 2). Tissue indices and uptake of major nutrients The index leaf N (3-6 leaf blades) is of paramount importance since it reflects the physiological status of plants (Alexander, 1973; Clements, 1980). Application of Azotobacter expressed significant increase in N index at both the stages of growth in- plant crop. The N index declined from an average value of 2.39% at 6* month to 1.05% at harvest exhibiting negative correlation with age. Lakshmikantham et al. (1970) observed maximum leaf N at formative and- grand growth period and progressive fall with advancement in age. Azotobacter registered higher uptake of N of 377 kg ha'1 at harvest stage (Table 3). This is attributable for higher dry matter production coupled with higher 166 Table 1. Growth and yield parameters as influenced by N levels and N fixers. Character LAI LAD (days) Dry matter (t ha 1 ) CGR Height of Weight of at 6* month lO* month-harvst at 6* month (g.nr 'd 1 ) millable cane (m) millable cane (kg) Plant/Ratoon Plant Ratoon Plant Ratoon Plant Ratoon Plant Plant Ratoon Plant Ratoon N levels N fixers 150 kg ha"1 7.09 3.62 205 142 25.85 18.30 19.79 2.50 2.01 1.47 1.23 200 kg ha'1 7.47 4.00 207 160 27.83 18.63 25.63 2.72 2.14 1.62 1.32 250 kg ha"1 7.31 3.88 205 153 27.14 17.34 28.35 2.83 2.19 1.69 1.34 Azotobacter chroococcum 7.99 4.03 219 148 30.92 18.17 36.17 2.89 2.13 1.72 1.31 Azospirillum brasilense 7.74 3.98 216 166 28.35 20.19 25.10 2.70 2.16 1.61 1.33 Acetobacter diazotrophicus (Local) 6.89 3.86 192 139 23.94 18.00 20.41 2.64 2.11 1.57 1.30 Acetobacter diazotrophicus (Brazil) 6.79 3.94 204 158 26.26 18.17 19.61 2.60 2.10 1.55 1.29 Control 7.07 3.36 198 149 23.90 15.94 21.66 2.55 2.07 1.52 1.27 LSD (P=0.05) N levels NS . • NS NS NS NS NS 8.283 0.089 0.135 0.053 0.083 N fixers 0.785 0.481 19.648 19.303 3.159 2.697 6.801 0.075 0.069 0.045 0.042 LSD • Least significant difference NS - Non significant Cane yield and sugar yield as influenced by N levels and N fixers. .. Character Cane yield (Mg ha 1 ) Sugar Yield (Mg ha 1 ) Plant/ratoon Plant Ratoon Plant N ievels/N fixers ISO kg ha 1 200 250 kg ha'1 kg ha"' Mean ISO kg ha'1 200 kg ha"' 250 kg ha - 1 Mean 150 kg ha-' 200 kg ha 1 250 kg ha-' • Mean Azotobacter Chroococcum 166.67 181.67 189.00 179.11 114.67 119.00 112.67 118.78 23.92 25.79 26.77 25.49 Azospirillum brasilense 154.67 170.33 176.67 167.22 113.00 122.33 126.33 120.55 20.99 24.27 24.43 23.23 Acetobacter diazotrophicus (Local) 151.33 167.00 172.00 163.44 115.67 118.00 119.67 117.78 21.10 23.64 24.42 23.05 Acetobacter diazotrophicus (Brazil) 150.33 165.33 168.67 161.44 111.33 117.67 112.33 117.11 21.38 23.47. 23.65 22.83 Control 142.33 160.33 171.00 157.89 106.33 120.33 118:67 115.11 19.91 22.94 24.06 22.30 Mean 153.07 168.93 175.47 11220 .119.47 121.93 21.46 24.94 24.67 . LSD (P=0.05) . ~ . . - ' . , N levels 5.44 7.4;24 0.683 * N fixers 4.69 ... 3.791 1.026 N levels * N fixers NS NS • NS N fixers * N levels NS NS. NS LSD - Least significant difference N S - Non significant Table 3. N index, N uptake, soil available N, soil Azotobacter count, No3-N in leachate and cost: benefit ratio as influenced by N levels and N fixers. Character N index (%) N uptake (kg ha'1) Soil av. N (kg ha 1 ) No,-N in leachate (mg I'1) Soil Azotobacter Costibenefit at 6* month at harvest at 8* month count (Xx'000 g 1 ) ratio Plant/Ratoon Plant Ratoon Plant Ratoon Plant Field sampling Pot sampling Plant Plant Ratoon N levels 150 kg ha'1 2.28 1.79 195 198 258 46.25 56.25 6.00 2.30 2.38 200 kg ha'1 2.52 1.92 225 178 323 60.62 59.38 7.17 2.50 2.51 250 kg ha'1 2.38 2.06 284 192 342 73.75 61.25 10.83 2.57 2.53 "ixers Azotobacter chroococcum 2.60 2.25 377 207 359 30.62 50.62 12.78 2.63 2.45 Azospirillum brasilense 2.26 2.03 210 206 311 56.87 50.00 - 2.45 2.50 Acetobacter diazotrophicus (Local) 2.35 1.92 214 186 303 65.62 66.88 - 2.40 2.45 Acetobacter diazotrophicus (Brazil) 2.48 1.71 190 190 312 85.62 56.87 - 2.37 2.44 Control 2.29 1.70 183 158 253 62.50 70.00 2.41 2.38 2.49 LSD(P=0.05) N levels NS 0.152 17.725 NS 22.263 2.237 1.437 N fixers 0.182 0.225 25.832 36.434 43.410 4.970 6.06 LSD - Least significant difference NS - Non significant Shankaraian'& Hunsigi tissue N content. Although, similar trend was observed in ratoon:crop}Azospirillum was equally effective with higher-indices and uptake of N more conspicuously at lower N level of200 kg ha'1. The values however, were lower in ratoon than in plant crop as ratoons are less efficient users of N (Hunsigi, 1982). Although the indices of P. and K revealed no definite trend, their uptake increased significantly due to Azotobacter and Azospirillum by virtue of higher dry matter production. The mean K indices and uptake declined towards harvest exhibiting negative correlation with advancement in age. Available soil nutrients and microbial load The soil available N in general showed significant improvement due to inoculation of N fixers and the highest availability of 359 kg ha'1 was recorded under Azotobacter at 8 t h month in plant crop (Table 3). The soil available N at harvest however showed marginally higher values due to bioagents and the sharp fall towards harvest is probably due to removal and exhaustion of N. The soil available P at harvest declined due to N fertilization and improved marginally due to inoculation of Azotobacter and Azospirillum. The soil available K however, did not differ due to treatments. The status of soil available nutrients in ratoon were not influenced significantly due to treatments. The population of Azotobacter in the soil at 8 t h month increased significantly following its inoculation. The effect was more pronounced when Azotobacter was inoculated in higher doses of N (Table 3). Quality and grading of jaggery Inoculation of Azospirillum produce jaggery with higher sucrose and lower reducing sugars (RS) and NR values of above 65 and thus classified under "A," grade with "Excellent" quality in both plant and ratoon crop experiments. This is attributable for higher uptake of N and P for their proper balance of 1 : 1.5 in juice (Ranadive, 1985). Unlike in plant crop, sucrose in jaggery increased with N fertilization with parallel reduction in RS content in ratoon crop which is assignable for reduced N use efficiency leading to lower N uptake besides earliness in maturity of ratoon crop (data not shown). Economics of use of N fixers Inoculation of Azotobacter, in plant crop and Azospirillum in ratoon crop in conjunction with recommended N level of250 kg ha'1 expressed higher net returns and cost benefit (C: B) ratios as a result of significant improvement in cane yield and economy in fertilizer N. The C: B ratios were 1 :2.63 in plant crop and 1 :2:50 in ratoon crop due to the inoculation of respective N fixtures as against 1 :2.38 and 1 2 . 4 9 respectively under no inoculation (Table 3) (data not shown). 170 Effect of Associative Nitrogen Fixing and Phosphate Solubilising Effect of levels and sources of P and PSM on growth, yield and quality of sugarcane- plant and ratoon Growth and yield parameters The growth parameters such as germination, tillering, shoot population, LAI and -LAD were not influenced significantly due to treatments. The production of dry matter at harvest increased due to inoculation of Agrobacterium radiobacter and Bacillus megaterium in conjunction with SSP+PM and SSP alone. Marked improvement in crop growth rate (CGR) was observed due to inoculation of Agrobacterium radiobacter and Bacillus megaterium and the highest CGR of 32.54 g m ' 2 d ' was observed with Agrobacterium radiobacter. The positive influence due to bioagents is attributable for production of plant growth regulators (PGR) besides increased availability and uptake of P. In ratoon crop, the shoot population increased due to P applied through SSP and SSP+PM and-inoculation of Aspergillus awamori. The LAI and production of dry matter at harvest were