Introduction

Sugarcane (Saccharum species hybrids) is an exhaustive crop that can uptake great amount of soil nutrients for its biomass production. In addition to micronutrient exportation, about 65 kg N, 90 kg P2O5 and 170 kg K2O are taken up for a target yield of 50 t/ha (Kathiresan 2008). The use efficiency of N fertilizers in sugarcane applied with recommended dose of N in the range of 250 to 400 kg/ha is only 20–30 %, and hence, at every harvest of the crop, soil suffers a net loss of 50–100 kg N/ha. Similarly, out of the total phosphorus fertilizers applied to the crop, only 15–20 % can be used, and the rest is fixed in the soil as phosphates of Ca, Al or Fe depending on the soil reaction. A permanent manurial trial, conducted for 33 years at Anakpalle (Andhra Pradesh), revealed that sugarcane crop without addition of fertilizers yielded about 40 t/ha of cane annually. The soil nitrogen reserve under this crop, however, increased by 50 % of the initial value which clearly indicated that the root-associated diazotrophs contribute significant quantity of nitrogen for sustaining the production of sugarcane (Suman 2003).

Inoculation of N-fixing microbes to sugarcane has increased the cane yield by 5–15 %, saved 25 kg fertilizer N ha−1 and also improved the juice quality parameter, viz sucrose and purity (Hari 1995; Srinivasan and Naidu 1987). Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium highly specific to sugar-rich crops such as sugarcane, sweet potato, pineapple and sugar beet. It was found to occur in the roots, stems, leaves (Cavalcante and Dobereiner 1988; Li and MacRae 1991; Reis et al. 1994), rhizosphere soil and even in cane juice (Muthukumarasamy et al. 1994) in appreciable number in the intercellular spaces of parenchyma and is considered as an obligate endophyte (Tejera et al. 2003). It can excrete about half of its fixed nitrogen in a form that plants can use; excess nitrogen fertilization decreases the population of G. diazotrophicus associated with sugarcane (Fuentez-Ramirez et al. 1999). It has also been reported that besides N fixation, all the strains of G. diazotrophicus produced indole acetic acid in a culture medium supplemented with tryptophan in the range of 0.14 to 2.42 μg/ml ((Fuentez-Ramirez et al. 1993). Furthermore, it has been reported its ability to solubilize inorganic phosphates from the soil and make available P for the inoculated crops. Hence, Gluconacetobacter inoculation to sugarcane significantly increased the cane girth, chlorophyll content, total nitrogen, cane length, and number of millable canes, resulting in the cane yield increase by 42 % over control (Chauhan et al. 2010). Moreover, there is need to study the survival and activity of Acetobacter in sugarcane cuttings from previous-year inoculated crop. With this view, a field experiment was conducted to study the effect of G. diazotrophicus and phosphate-solubilizing bacteria (PSB) set treatment with varying N and P levels on sugarcane seed yield, quality and possibility of saving of chemical fertilizer nitrogen and phosphorus and the survival and activity of Acetobacter in the cuttings from previous-year inoculated crop.

Materials and Methods

A field experiment was conducted in a 3 × 3 × 2 factorial randomized block design (FRBD) with three replications on sugarcane variety CoM 0265 during Suru season (January planting) of 2012–13, 2013–14 and 2014–15 at Central Sugarcane Research Station, Padegaon, Tal. Phaltan, Dist. Satara (Maharashtra State, India) to study the effect of G. diazotrophicus and PSB set treatment with varying N and P levels on sugarcane seed yield, quality and possibility of saving chemical fertilizer nitrogen and phosphorus. The plot size employed was 6.0 × 6.0 m (5 rows of 6 m). The biofertilizers, viz. G. diazotrophicus and PSB, were from Biological Nitrogen Fixation Scheme, College of Agriculture, Pune. For treatment of sugarcane bud-sets, the two eye bud-sets were dipped in the biofertilizer suspension prepared by mixing 10 kg G. diazotrophicus and 1.25 kg PSB in 100 l water per hectare for 30 min prior to planting. The crop was fertilized as per the treatment details given as under.

Treatment Details

Factor A::

N levels (% of recommended dose, i.e., 600 kg/ha):

(a) 50 % recommended dose

(b) 75 % recommended dose

(c) 100 % recommended dose

Factor B::

P2O5 levels (% of recommended dose, i.e., 230 kg/ha):

(a) 50 % recommended dose

(b) 75 % recommended dose

(c) 100 % recommended dose

Factor C::

Biofertilizer treatments (for first year):

(a) Uninoculated control

(b) Set treatment with G. diazotrophicus 10 kg + PSB 1.25 kg in 100 lit water/ha for 30 min.

Biofertilizer treatments (for second and third years):

(a) Planting material from previous-year Acetobacter inoculated plot

(b) Fresh planting material inoculation with Acetobacter.

Recommended K2O (115 kg/ha), FYM/Compost (20 t/ha) and pretreatment of sets with dimethoate + carbendazim were common to all the treatments.

The observation on germination at 30 days after planting was recorded. The data on cane height, internode length, girth, number of millable canes (NMC), two eye bud-set yield, commercial sugar (CCS %) and sucrose content in seed cane was recorded at 10 months. Moreover, cutting germination in coco pith trays was recorded at 10 months. The population of G. diazotrophicus in cane juice was determined at 10 months by using serial dilution and plating technique (Aneja 2003). The soil samples were also analyzed for the available N and P after harvest as per the standard procedure described by Jackson (1967). The data were subjected to statistical analysis by employing the standard methods of analysis of variance (Panse and Sukhatme 1985) using MSTATC package.

Results and Discussion

The pooled results on growth parameters, two eye bud-set yield and quality parameters of sugarcane seed plot as influenced by microbial inoculation are presented in Figs. 1, 2, 3 and 4. The fertilizer levels (N and P levels), inoculation treatments and the interaction of N levels and inoculation treatments as well as P levels and inoculation treatments had a significant influence in growth and yield parameters, Acetobacter population in cane and the soil N and P status at harvest.

Fig. 1
figure 1

Effect of G. diazotrophicus and PSB set treatment with varying N and P levels on growth parameters of sugarcane seed plot (Pooled: Suru 2012–13, 2013–14 and 2014–15)

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Fig. 2
figure 2

Effect of G. diazotrophicus and PSB set treatment with varying N and P levels on sugarcane two bud-set yield and NMC (Pooled: Suru 2012–13, 2013–14 and 2014–15)

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Fig. 3
figure 3

Effect of G. diazotrophicus and PSB set treatment with varying N and P levels on Acetobacter count in cuttings (×104) (Pooled: Suru 2012–13, 2013–14 and 2014–15)

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Fig. 4
figure 4

Effect of G. diazotrophicus and PSB set treatment with varying N and P levels on quality parameters of sugarcane (Pooled: Suru 2012–13, 2013–14 and 2014–15)

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Growth and Yield Parameters of Sugarcane

The effect of G. diazotrophicus and PSB set treatment with varying N and P levels on growth parameters and two eye bud-set yield of sugarcane seed plot are presented in Figs. 1 and 2, respectively. Among ‘N’ levels, 100 % N recorded the highest germination (58.01 %), cane height (201.4 cm), internode length (13.51 cm), NMC (84,844/ha), no. of eye buds (18.99/cane) and two eye bud-set yield (806.22 thousands/ha). However, it was at par with 75 % N in respect of germination (56.30 %), cane height (198.8 cm), internode length (13.39 cm), NMC (84,461/ha), no. of eye buds (18.83/cane) and two eye bud-set yield (797.06 thousands/ha).

Among ‘P’ levels, 100 % P recorded the highest germination (56.72 %), cane height (200.3 cm), NMC (84,595/ha), no. of eye buds (18.87/cane) and two eye bud-set yield (799.29 thousands/ha). However, it was at par with 75 % P in respect of germination (56.19 %), cane height (199.1 cm), NMC (84,328/ha), no. of eye buds (18.74/cane) and two eye bud-set yield (797.06 thousands/ha).

Between the inoculation treatments, fresh planting material inoculated with Acetobacter recorded significantly higher germination at 30 DAP (56.41 %), cane height (198.5 cm), internode length (13.34 cm), cane girth (11.76 cm), NMC (84,328/ha), no. of eye buds (18.74/cane) and two eye bud-set yield (791.73 thousands/ha) than the uninoculated planting material from previous-year Acetobacter inoculated plot.

Among the interactions, 75 % N coupled with fresh planting material inoculated with Acetobacter recorded the highest germination (58.36 %), cane height (202.6 cm), cane girth (12.24 cm), NMC (86,830/ha) and two eye bud-set yield (830.63 thousands/ha). However, it was identical to 100 % N coupled with uninoculated planting material from previous-year Acetobacter inoculated plot in respect of germination (58.11 %), cane height (201.3 cm), cane girth (11.90 cm) and two eye bud-set yield (805.91 thousands/ha), indicating the survival and activity of Acetobacter in the cuttings from the previous-year inoculated crop.

Moreover, the interaction of 75 % P coupled with fresh planting material inoculated with Acetobacter recorded the highest germination (59.02 %), cane height (203.4 cm), internode length (13.63 cm), cane girth (12.28 cm), NMC (86,511/ha), no. of eye buds (19.30/cane) and two eye bud-set yield (836.09 thousands/ha). However, it was identical to 100 % P coupled with fresh planting material inoculated with Acetobacter in respect of germination (57.70 %), cane height (202.69 cm), internode length (13.47 cm), NMC (85,462/ha), no. of eye buds (19.11/cane) and two eye bud-set yield (817.65 thousands/ha).

Gluconacetobacter inoculation to sugarcane has been reported to significantly increase the plant height, cane length and NMC over control, resulting in the increased cane yield (Chauhan et al. 2010). Moreover, the nitrogen-fixing bacterial inoculations to sugarcane have also been able to improve the cane girth (Hari 1995; Srinivasan and Naidu 1987). Over 40 % of atmospheric nitrogen fixed was contributed by Acetobacter diazotrophicus besides increased cane yield by 10–25 % (Bhor et al. 2006). The improvement in sugarcane yield to the tune of 7–10 t/acre and in sugar recovery by 0.5–1.0 % with 50 % reduction in the recommended dose of chemical nitrogen by use of the nitrogen-fixing Acetobacter diazotrophicus has been reported by Muthukumarasamy et al. (2002).

Cutting Germination in Coco Pith

The germination in coco pith trays at 10 months was significantly influenced by different fertilizer levels (N and P levels) and inoculation treatments, whereas various interactions showed nonsignificant effect. The overall germination in coco pith trays ranged from 72.89 to 86.44 %. Among N levels, 75 % N recorded the highest germination (80.98 %), but it was at par with 100 % N (80.30 %). Among P levels, 75 % P recorded the highest germination (80.76 %); however, it was at par with 100 % P (80.11 %). Between the inoculation treatments, fresh planting material inoculated with Acetobacter recorded significantly highest germination (79.83 %) than the uninoculated planting material from last-year Acetobacter inoculated plot (77.75 %).

Acetobacter Count in Cuttings

The effect of G. diazotrophicus and PSB set treatment with varying N and P levels on Acetobacter count in cuttings at 10 months is presented in Fig. 3. The Acetobacter count at 10 months was significantly influenced by different fertilizer levels (N and P levels) and inoculation treatments. Among various interactions, the interaction of N and P levels as well as interaction of N levels and inoculation treatments showed significant effect on the Acetobacter count. The overall Acetobacter count at 10 months varied from 4.08 to 8.03 × 104. Among N levels, 75 % N recorded significantly higher Acetobacter count in cuttings (6.85 × 104) followed by 50 % N (6.18 × 104) and 100 % N (4.82 × 104). Among the P levels, 75 % P recorded significantly higher Acetobacter count (6.26 × 104) followed by 50 % P (5.82 × 104). Between the inoculation treatments, fresh planting material inoculated with Acetobacter recorded significantly highest Acetobacter count (6.27 × 104) than the uninoculated planting material from previous-year Acetobacter inoculated plot (5.63 × 104). Among the interactions, 75 % N coupled with fresh planting material inoculated with Acetobacter recorded significantly highest Acetobacter count (7.38 × 104). The interaction of 75 % N coupled with 75 % P recorded the highest Acetobacter count (7.32 × 104); however, it was identical with 75 % N coupled with 100 % P (6.86 × 104).

It has been reported that the number and activity of endophytic bacteria is reduced when the sugarcane crop is grown under high or optimal nitrogen input levels (Fuentez-Ramirez et al. 1999; Muthukumarasamy et al. 2002; Suman 2003).

CCS and Sucrose Content in Cuttings

The effect of G. diazotrophicus and PSB set treatment with varying N and P levels on quality parameters of sugarcane is presented in Fig. 4. The commercial cane sugar (CCS) and sucrose content in seed cane at 10 months were not significantly influenced by N levels, P levels, inoculation treatments and their interactions as well. The CCS in seed cane ranged from 9.71 to 10.68 %, whereas the sucrose content varied from 14.00 to 15.27 %.

Available Soil N and P at Harvest

The available N status of soil at harvest was significantly influenced by different fertilizer levels (N and P levels), inoculation treatments and their interactions as well. The overall available soil N ranged from 174.67 to 199.00 kg/ha. Among the N levels, 100 % N recorded significantly highest available soil N at harvest (193.17 kg/ha). Among the P levels, 100 % P recorded the highest available soil N at harvest (189.42 kg/ha). However, it was identical with 75 % P (187.94 kg/ha). Between the inoculation treatments, fresh planting material inoculated with Acetobacter showed significantly higher available soil N at harvest (190.80 kg/ha) than the uninoculated planting material from previous-year Acetobacter inoculated plot (183.11 kg/ha). Among the interactions, 100 % N coupled with fresh planting material inoculated with Acetobacter recorded the highest available soil N at harvest (194.78 kg/ha); however, it was identical to 75 % N coupled with fresh planting material inoculated with Acetobacter (193.72 kg/ha) and 100 % N coupled with uninoculated planting material from previous-year Acetobacter inoculated plot (191.56 kg/ha). Similarly, the interaction of 75 % P coupled with fresh planting material inoculated with Acetobacter recorded the highest available soil N at harvest (194.11 kg/ha); however, it was identical to 100 % P coupled with fresh planting material inoculated with Acetobacter (192.17 kg/ha). The increase in soil nitrogen reserve under sugarcane crop by 50 % of the initial value due to the nitrogen fixation by root-associated diazotrophs helping sustained production of sugarcane has been reported by Suman (2003).

Moreover, the available P status of soil at harvest was significantly influenced by different fertilizer levels (N and P levels) and inoculation treatments; however, their interactions were nonsignificant. The overall available soil P ranged from 131.8 to 20.47 kg/ha. Among the N levels, 100 % N recorded significantly highest available soil P at harvest (19.80 kg/ha). Among the P levels, 100 % P recorded the highest available soil P at harvest (18.55 kg/ha); however, it was identical to 75 % P (17.98 kg/ha). Between the inoculation treatments, fresh planting material inoculated with Acetobacter showed significantly higher available soil P at harvest (18.76 kg/ha) than the uninoculated planting material from previous-year Acetobacter inoculated plot (16.61 kg/ha).

Cost–benefit analysis

The economics of G. diazotrophicus and PSB set treatment with varying N and P levels on sugarcane seed yield and quality is depicted in Table 1. The cost–benefit analysis of different inoculation treatments (Table 2) showed that the fresh planting material inoculated with Acetobacter recorded highest C:B ratio (4.06) followed by planting material from previous-year Acetobacter inoculated plot (3.89). Among the fertilizer levels, 75 % of recommended N and P2O5 dose recorded highest C:B ratio (4.27). The interaction of fertilizer levels and inoculation treatments showed that 75 % of recommended N and P2O5 dose coupled with the fresh planting material inoculated with Acetobacter showed the highest C:B ratio (4.63).

Table 1 Economics of G. diazotrophicus and PSB set treatment with varying N and P levels on sugarcane seed yield and quality (Pooled: 2012–13, 2013–14 and 2014–15)
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Table 2 Cost–benefit analysis of inoculation treatments and fertilizer levels
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Conclusion

It is recommended to treat the sugarcane sets with 10 kg Gluconacetobacter diazotrophicus + 1.25 kg PSB in 100 l water/ha for 30 min before planting for saving of 25 % N and 25 % P for sugarcane seed plot besides improved growth, two eye bud-set yield, Acetobacter population in cane and soil N and P status at harvest.