Contribution of Biofertiliser ( Frateuria auranta ) in an Integrated Potassium Management Package on Growth of Apple Banana

Bananas demand high amounts of potassium for optimal growth and productivity, yet deficiencies are widespread amid the low input production strategy of smallholder farmers in Uganda. Of the potassium pool in the soil, 90% -98% is unavailable for plant uptake. Judicious application of fertilisers is required to alleviate soil fertility problems complemented with manures and biofertilisers in an integrated nutrient management (INM) package. Biofertilisers such as potassium solubilising bacteria (KSB) have potential to solubilise unavailable forms of K in soil to forms that are readily absorbed by the plants. However, the added value of each component in this integrated K management package in apple bananas is not known. Therefore, an experiment was set up to quantify the relative contribution of mineral K, manure and KSB on the growth of apple bananas. Potted tissue culture plantlets of apple banana (cv. Sukali ndiizi) were used. The treatments comprised of a full factorial combination of mineral fertiliser (Muriate of potash, 60% K 2 O), animal manure and KSB (Frateuria auranta). The manure and muriate of potash were applied to supply a total of 150 kg∙K∙ha − . Soil microbiological assays were run to evaluate the contribution of indigenous microbial K solubilising activity in the soil to the experimental INM package. Data on pseudostem height, girth at collar and 30-cm height, constrained smallholder farmers.


Introduction
Bananas demand a high amount of potassium yet smallholder farmers barely apply mineral fertilisers due to poor access and high prices [1]. Judicious use of mineral fertiliser is needed to overcome deficiencies [2] although it is reported to be costly financially and environmentally [3]. Application of manure can be helpful in alleviating K deficiencies [4] and minimising leaching losses but it is constrained by lack of access to adequate amounts for effective nutrient application rates. Using biofertilisers can reduce the need for external nutrient inputs [5] [6] which enable plants to exploit nutrient stocks from the soil more effectively. Out of the pools of K found in the soil, that in soil minerals make up 90% to 98% of the total content but it is unavailable for plant uptake [7].
Application of manure and biofertilisers in combination with inorganic fertilisers plays multiple important roles in growth and development of the plants as well as sustenance of soil fertility and environmental quality. This is the concept of Integrated Nutrient Management (INM). It has been shown to be effective in several horticultural crops [8]. It significantly increases the growth and yield of guavas [9], black gram [10] and banana cultivar Grande naine [8]. Integrated nutrient management lays emphasis on continuous improvement in soil productivity on long term basis through judicious use of mineral fertilisers, biofertilisers and manures and their proper management for optimum growth, yield and quality of crops in specific agro-ecological situation [11]. Manure is a broad term, which includes livestock excreta, left-over feed and other wastes from processing livestock products, household waste compost, crop residue composts and fresh plant materials applied to the soil for soil fertility management as green manures. Manure improves the soil physical, chemical and microbiological properties and ultimately the crop yield [5] [8].
Biofertilisers are preparations containing living or latent cells of efficient strains of microorganisms that facilitate crops' access to and/or uptake or assimilation of nutrients via their interactions with the crops in the rhizosphere [12] when applied on seed or directly to the soil. A wide range of bacteria namely: Bacillus mucilaginosus, Bacillus edaphicus, Bacillus circulans, Paenibacillus spp., Acidithiobacillus ferrooxidans, Pseudomonas sp., Burkholderia sp. [13] and Frateuria aurantia [14] have been reported to release potassium in available form from potassium-bearing minerals in soils. These KSB were found to dissolve potassium [15], silicon and aluminium from insoluble K-bearing minerals such as micas, illite and orthoclases, by secreting organic acids which either conditions. This study therefore was conceptualised to quantify the relative contribution of different K sources in an integrated nutrient management package (viz. K-solubilising bacteria, mineral K and organic manure) on the growth of apple bananas.

Objectives
The broad objective of this study was to develop an INM package for improving apple banana growth, sustaining soil fertility and maintaining environmental health via enhancement of the biological mechanisms for potassium dynamics from various pools in the soil.
Specific objectives 1) To assess the interactive effects of manure and inorganic K fertiliser on the diversity of indigenous K-solubilising microorganisms in the soil.
2) To assess the interactive effects of manure and inorganic K fertiliser on the activity and abundance of K-solubilising microorganisms in the soil.
3) To assess the relative contribution of manure, inorganic fertiliser K, manure and exotic KSB on the growth of apple bananas.

Study Area
The study was carried out at Kawanda National Agricultural Research Laborato-

Experimental Set Up and Data Collection
The experiment was set up in a screen house.

Soil and Foliar Analysis
Before application of inputs (i.e. manure, MOP or KSB) a soil and a manure sample was reserved for soil nutrients analysis. The samples were analysed for available N, P, K, Ca and soil organic carbon. Organic carbon was quantified calorimetrically at 600 nm after extraction with potassium dichromate and sulphuric acid [20]. Total nitrogen was extracted using sulphuric/selenium acid mixture at 330˚C and then quantified calorimetrically using Nessler and Phenate method. The exchangeable cations (K and Ca) and available P were extracted using Mehlich 3 solution and quantified using an emission flame photometer and atomic absorption spectrometer, respectively. Twenty weeks after planting, the third fully expanded leaf with its leaf stalk was collected per treatment per replicate for foliar analysis. The leaf sample was placed in paper bags and oven dried to constant weight. The samples were finely ground and analysed for N, P and K concentrations, the procedures of foliar analysis [21]. Soil pH was analysed using a pH meter at a ratio of 1:2.5, sample to water, and soil particle distribution done to determine the soil textural class [21].

Microbial Analysis
This was done to determine the abundance and activity of indigenous KSB in the soil. A soil sample per treatment per replicate was collected in sterilised collection bottles. The soil was then kept on ice to maintain microbial diversity until analysis. The samples were cultured using modified Aleksandrov medium containing 0.25% bromothymol blue indicator (BTB) incubated at 35˚C -37˚C for Open Journal of Soil Science The soil samples were serial diluted to 10 −5 and each dilution was incubated for 24 hours on selective Aleksandrov medium with BTB. All colonies that grew on media and formed a halo solubilisation zone were considered to be potassium solubilising organisms. The colonies were counted using a colony counter and different colonies distinguished based on their morphological characteristics as used by [23]. The colony diameters were estimated using a ruler. Different colonies were isolated their gram staining reaction determined along with their respective cell shapes under microscopic observation. The different colonies were also subjected to a catalase test using hydrogen peroxide; the occurrence of gas bubbles was scored positive for catalase activity by the organisms.
Quantitative determination of solubilised potassium was done using the protocol by [7]. To quantify how much potassium was solubilised by Frateuria auranta (an organism inoculated in the soil for K-source), pure culture containing only F. auranta were inoculated in Aleksandrov broth media and grown for 10 days with constant shaking. The sample was analysed for K according to the procedure by [7] using an emission flame photometer. The sample was compared with Aleksandrov broth media where no organism was grown as the control.

Data Analysis
Soil and foliar samples data were analysed using a one-way analysis of variance (ANOVA) and the means separated using least significance difference (LSD) at 95% confidence interval. Data on growth parameters were analysed using ANOVA using general treatment structure to separate the different factor effects and their means were also separated with LSD at 5%. To compare the accumulated biomass, data were first loge transformed to fit the equations used to calculate biomass; the data were then analysed using a one-way ANOVA and means separated with LSD at 5%. ANOVA was carried out to test the significance of the treatment differences in all analyses. All analyses were done using GenStat 12 edition software.

Analysis Results from Soil and Foliar Samples
Manure contained N, P, K levels that were greater than the soil critical values.
The Organic carbon content of 8.43% was also above the critical levels of 2.5% in soil. The soil sample used, however, was slightly higher than the soil critical values ( Table 2).
The nutrient inputs doubled the K concentration in the leaves relative to the control but MOP had to be combined with either KSB or manure or both for maximum K concentration in the leaves (Table 3). Manure was necessary for increasing foliar N concentration relative to the control. On their own, KSB and MOP had no significant effect on foliar N concentration ( Table 3). The trend in treatment effects on foliar N concentration was paralleled with that for foliar P (

Soil Microbial Analysis
The number of CFUs reduced with increasing dilution. At a given dilution, the control (no input) had the lowest number of CFUs (  (Table 4).
Combining manure with KSB gave more CFUs than combining manure with MOP across dilutions except at ×10 −4 where they gave similar number of CFUs.
All the different microbial colonies (cells) on the petri dishes were rod-shaped.
Their colony diameter ranged from 1.5 mm (small) to 6 mm (large). All the colonies reacted positively to the catalase test, but the Gram test showed both posi-

Effects of integrated potassium management on growth
Application of potassium significantly increased the growth parameters of apple bananas. Height was significantly increased by the K input, regardless of source, by between 14 and 23% relative to the control (Table 6). However, manure was   in AGB and TB in comparison to all other treatments ( Table 7).
As was observed in accumulated biomass, treatment T1 recorded significantly low (p < 0.001) leaf area than all the other treatments. Treatments T2 (MOP alone) and T5 (KSB alone) also were observed to record significantly low leaf area as compared to the rest of the treatments. Treatments which received manure as an input were observed to obtain higher leaf area although the difference were not statistically significant (Table 7).

Discussion
Results of soil analysis indicated that the exchangeable potassium (0.16 cmol/kg) in the soil was moderate comparing critical values of tropical soils, where less than 0.15 cmol/kg is below optimum [25]. However, this value was far below the critical required level of 1.3 cmol/kg required by bananas suggested by [26]. Nitrogen and organic matter were above the critical levels of 0.2% and 2.5% respectively Open Journal of Soil Science as quoted by [27].
Foliar analysis results, however, indicated that treatment with no input had significantly low K (2.3%) than all the other treatments and it was also below the critical level of 3.0% of bananas [28]. Treatments of manure and inorganic K (T4) and treatment with all inputs (T8) recorded the highest K content >6.0% and these were significantly higher than values obtained in treatment of MOP alone (5.22%) and KSB alone (4.97%). Nitrogen was below the critical levels of 2.6% according to [28] in all treatments except those treatments where manure was applied. It was highest in T8 (all inputs) of 2.9% followed by manure and MOP (T4) of 2.8 and then MOP and KSB (T7) of 2.7% (Table 3). This was an indicator that the manure supplied the N that supplemented that from the soil for the plant requirement for growth.
Application of manure also had a significant contribution to the soil microbial content (Table 4). This could be because most microbes are organotrophic and therefore their activity increased as a response to application of manure [29] [7].
Application of manure to the soil boosted the microbial growth hence the high numbers in manure treatments. Treatments T1 (no input) and T2 (MOP only) also had living organisms (Table 4) despite sterilisation. These organisms could have been introduced into the pots from the potting mixture, but did not increase.
The probable organisms found in the study were from Bacillus, Pseudomonas and Frateuria genera (Table 5). Frateuria was KSB from the inoculant. Pseudomonas and Bacillus spp could have been introduced with manure, because they play an important part in the process of decomposition [17] [30]. Among the soil bacterial communities, B. mucilaginosus, B. edaphicus and B. circulanscan Table 7. Above ground and total biomass as estimated using girth at 30cm and leaf area as estimated using allometric equations. Frateuria auranta used in this study was able to released potassium from potassium aluminium silicate mineral in Aleksandrov broth of 7.4 mg/l in a period of 10 days of inoculation ( Figure 1). This was higher than 4.2 mg/l reported by [31] using B. mucilaginosus in media supplemented with muscovite mica. The observed K solubilisation rate in this study was only 5% of that from bacterial strains isolated by [7], solubilised up to 400 mg/l K from potassium aluminium silicate in 28 days, although their strains were not taxonomically identified.
Potassium solubilising bacteria are able to solubilise insoluble K from forms such as micas, illites, quartz, orthoclases [32] through production of organic acids. Acid production was observed in this study by organisms turning BTB modified media from blue (pH 7.2 ± 0.2) to yellow, indicating pH change to acidic; although this study did not isolate the different acids produced. Microorganisms including KSB can have a considerable role in providing K to plant by storing K in their biomass (a significant quantity of fixed K), which is potentially available to plants [33]. All these mechanisms of K solubilisation could have contributed to the amount of K observed to have been released by F. auranta used in this study.
Potassium application whether from MOP, KSB or manure significantly increased growth (height, girth, leaf length and width) as compared to no input at all (Table 6; Figure 2 and Figure 3). This was in agreement to other fertiliser trials conducted on banana in many studies in the world [1] [27] [34] [35]. Single input application (MOP alone, Manure or KSB alone) also significantly (p < 0.001) increased the growth of apple bananas as compared to the control where no input was applied (Table 6) although MOP alone effect was highly masked by application of manure and KSB. This was an indication that sufficient application of manure or KSB caused as much effect on growth of apple bananas as where MOP was used. Use of alternative sources to MOP would be a beneficial factor to alleviating the cost effects of high prices which farmers cannot afford [2].
Integrating different K sources together (MOP × Manure, MOP × KSB, KSB × Manure) generated a much greater effect on the growth of bananas (Table 6). The results of the study showed that using KSB alone, did not have significant effects over the treatments where KSB was applied with Manure or MOP (Table   6; Figure 2 and Figure 3). This is in line with [7] [23], that KSB requires a carbon source for their own growth and biomass accumulation. Therefore, application of KSB in soils with low organic matter would not produce significant effects on growth. Therefore, application of KSB x Manure produced a greater impact on the growth parameters than where MOP was applied with manure ( Table 6). Application of manure has additional benefits to the soil such as supplying nitrogen, organic matter, buffering rapid changes in soil pH and increasing the diversity and activity of beneficial soil microorganisms responsible for growth in plants [4].
Common farmer practice is use of manure alone, although this study indicated that with manure alone plants are able to outperform sole mineral K ( Table 6), application of manure in their fields is usually below the required levels of 13 t/ha/yr [27] [36]. Therefore, augmenting application of manure with MOP or KSB shows potential for enhanced growth, and possibly higher yields due to additional nitrogen. [37] observed higher pseudo-stem height and girth in treatments with higher N and K input.
Integrated application of MOP × KSB × Manure (T8) resulted into higher significant accumulated biomass than no input or sole mineral K input application (Table 7). This is in agreement with the results obtained by [38] in Cotton where they used farm yard manure, biofertiliser and inorganic fertiliser to achieve high levels of dry matter accumulation. [39] observed that biomass accumulation in Sukali ndiizi (apple banana) was more influenced by available moisture content in the soil, explaining the 22.9 g and 40.4 g of AGB and TB respectively obtained in a treatment in their moisture stress study where there was no fertiliser input.
However, treatment T8 (combination of KSB, manure and MOP) in this study recorded, 36.1 g and 97.9 g for AGB and TB, respectively that translates into 57.5% and 142% increase over the control which this study attributes to effect of proper nutrition. This also agrees with [39] that proper nutrition of plants is manifested by the biomass accumulated by the plant.
Highest above ground and total biomass was obtained in treatments combinations with manure (T4-Manure × MOP, T7-KSB × Manure and T8-MOP × Manure × KSB). This could have been because manure provided nitrogen apart from K, thus promoting vegetative growth more vigorously than other treatments [40].
The same trends were observed with regard to leaf area; highest was recorded in T8 and this was statistically significant to no input and single input application.
Application of fertiliser (potassium) increased leaf area and biomass accumulation in apple bananas which allies with the findings of [41].

Conclusions
Application of manure, MOP or KSB increased the diversity of organisms capa-