INTRODUCTION:

Bananas are staple food for many tropical populations ^[1]. It is originated from South and Southeast Asia and Pacific countries and Musa acuminata and Musa balbisiana are recognized as the major progenitors of present day bananas^[2]. Currently, banana is grown in and around 150 countries across the world on an area of 10.1 million haproducing 121.5 million tons^[3,4]. India alone accounts for 27.43% of global production with about 30 million tons per annum with an area of 8.3 lakh ha [FAO STAT 2011]^[5,6]. As a dessert, banana is more filling, easy to digest, fat free rich source of carbohydrate with a calorific value of 90 Kcal per 100g of fruit and free from sodium, making it a salt free food fruit^[7,8].

The banana plant is a largest herbaceous flowering plant which has a very large mat with different microorganisms which includes fungi, bacteria, actinomycetes and etc., is helpful in improving the physical properties, water holding capacity and reduces the other pathogenic soil microorganism^[9,10]. Phosphorus solubilizing fungi play role in phosphorus nutrition by enhancing its availability to plants through release from inorganic and organic soil P pools by solubilization and mineralization^[11,12]. Use of phosphorus solubilizing fungi as inoculants increase P uptake^[13,14]. Therefore, utilization of phosphate solublizing microorganisms is considered to be a sound strategy in improving the productivity of lands that are currently under crop production^[15,16]. However, the studies under isolating the rhizosphere phosphate solubilizing fungi in banana were not much familiar.

MATERIALS AND METHODS:

Field Location:

The present study was carried out in the research farm of National Research Center for Banana [ICAR], Podhavur village, Tiruchirapalli, Tamil Nadu.

Treatment Details:

The details of the treatments are as follows:

Treatment 1:

Pot mixture alone without application of phosphate

solubilizing fungi (PSF)

Treatment 2:

Pot mixture + application of 10g PSF plant^‑1

Treatment 3:

Pot mixture + application of 20g PSF plant^‑1

Treatment 4:

Pot mixture+ application of 30g PSF plant^‑1

Treatment 5:

Pot mixture+ application of 40g PSF plant^‑1

Treatment 6:

Pot mixture+ application of 50g PSF plant^‑1

Treatment 7:

Control (Soil alone without manures & bio fertilizers)

Pot mixture for T 1 to T 6 consists of sand, soil and organic manures in the ratio of 1:2:1.

Collection of soil samples:

The soil samples were collected from the selected rhizosphere of the banana at a depth of 15cm. The collected soil samples must be kept in polythene bag with suitable description and identification marks. The samples were immediately transported to the laboratory for further studies.

Isolation of fungi:

One gram (1g) of moist soil sample was dispersed in 9ml of autoclaved distilled water and was thoroughly shaken. 1ml of the above solution was again transferred to 9ml of sterile distilled water to form 10³, 10⁴, 10⁵, 10⁶ and 10⁷ serials for each soil sample. 0.1ml of each dilution was dispensed on Rose Bengal agar (RBA) medium and incubated at 27-30ᵒC for 3 days.

Screening for phosphorus solubilizing activity on solid media:

Preliminary screening for phosphate solubilization was done by a plate assay method using Pikovskaya’s (PVK) medium supplemented with tricalcium phosphate [TCP]. After solidification of the media, a pinpoint inoculation of fungal strains was made onto the plates under aseptic conditions. The plates were incubated at 30ᵒC for 7 days with continuous observation of the colony diameter. The appearance of a transparent halo zone around the fungal colony indicated the phosphate solubilizing activity of the fungus.

Efficiency of phosphate solubilization:

The isolates were studied for phosphate solubilizing ability on Pikovskaya’s (PVK) medium. The halo zone was measured at regular intervals. The efficiency was higher in the fungal species those can able to produce the maximum diameter of the transparent halo zone.

Growth rate of the fungus on solid media:

The growth rate of the fungal isolates was observed on potato dextrose agar, Rose Bengal agar and pikovskaya’s agar medium. Then the plates were incubated at room temperature. The diameter of the colony was measured after 48 hours of incubation for 7 days.

Growth rate on liquid media:

The growth rate of the fungal isolates was observed in three different enrichment medium, with glucose, sucrose and maltose as a carbon source. 250 ml of sterile media was taken in a 500ml of Erlenmeyer flask and it was sterilized. To this medium two loop full of fungal pure culture was inoculated and incubated at room temperature under shaking condition for 15 days, the fungal mat were carefully taken and the fresh weight (wet weight) were taken. Then the fungal mat was kept in hot air oven at 80ᵒC for three days till the mat became dry. The dry weight of the fungal mat was measured.

Screening for organic acid production:

Phosphate solubilization of the fungi was achieved by the production of organic and inorganic acids. Screening of organic acid production was done by using phenol red as pH indicator. Fungal isolates were inoculated to the respective plates. The plates were incubated at room temperature for 4-5 days. Control plates were maintained. After incubation the change in media colour from red to yellow indicated positive for organic acid production.

Screening for the production of extracellular polysaccharide [EPS]:

Extraction of EPS:

Shake flask culture method was carried out for screening of extracellular polysaccharide (EPS) producing organism. The flasks were then incubated in rotary shaker for 2 weeks at150 rpm. The fungal biomass was separated from the liquid medium by centrifugation (4000 rpm, 15 min) and the supernatant was filtered and 95% ethanol (v/v) was added and left overnight at 4ᵒC for polysaccharide precipitation. The precipitate was collected as the crude EPS fraction.

Soil experiment for pot culture analysis:

35 tissue culture Grand Naine plants were planted in the pot culture. The identified Phosphate solubilizing fungal strains was mass cultivated and it was mixed with talcum powder and it was applied to tissue culture plants at different ratio. After the application of phosphate solubilizing fungi the growth of the plant and the soil nutrient content was measured at regular intervals.

Plant growth analysis:

Initially the height and girth of the TC plants were noted. After planting every 15 days the height, girth, number of new leaves raised and length, girth of the new leaves was measured and it was tabulated. Finally the length of the root was measured by uprooting the plant and the length was compared with the root of the control plant.

Soil nutrient analysis:

The soil sample was collected from the pot culture. It is preferable to air dry soils at 20-25ᵒC and 20 to 60% relative humidity. After air drying soil samples are crushed gently in pestle and mortar and sieved through 2mm sieve.

Estimation of calcium (ca):

Five grams of the soil sample was taken a clean air dried container and 25ml of 1M ammonia acetate was added and the mixture was kept in a shaker for 5 minutes for proper mixing. Then it was filtered through whatman filter paper no: 42. From this 5ml of aliquot was taken in 100 ml beaker and one calcium hardness tablet and 5 ml of 10% NaOH solution was added (pink colour was developed). The contents in the beaker were titrated against 0.02N EDTA till the development of purple colour. The Ca content was calculated as per the formula (Ca%=Titre value x 0.04%).

Estimation of magnesium (Mg):

Five ml of the soil aliquot was taken in a container add total hardness tablet and 5ml of ammonia buffer solution. Titrate against 0.02N EDTA until the development of ash colour, and the reading was noted down. The percentage of Mg present in the sample was calculated by the following method (Mg%=(Titre value of Ca-Titre value of Mg) x 0.0243%).

Estimation of available nitrogen (N):

Easily oxidizable organic carbon and mineralizable N are largely used as a measure of available N. 0.5 gram of sieved (2mm) air dried soil was taken in a 500ml conical flask. Add 10ml of potassium dichromate and 20ml of concentrated sulphuric acid. After 30 minutes add 200ml of tap water and 10ml of orthophosphoric acid solution with two drops of diphenylamine indicator titrate the contents against 0.5N ferrous ammonium sulphate till the appearance of end point i.e., apple green colour. The percentage of N present in the sample was calculated by the following method (Organic carbon (%) B=10(B-T) x 0.6).

Estimation of available phosphorus (P):

2.5g of sieved (2mm) air dried soil was added into a plastic container.Add a little of Dargo G 60 (or) equivalent grade P free activated charcoal and 50ml of sodium bi carbonate solution (Olsen's reagent) and it was kept on the reciprocating shaker for 30 minutes. Similarly a blank was maintained without soil. Filtrate was taken through Whatman no. 40 or 42. From this five ml of aliquot was taken in a 25ml volumetric flask it was then acidified with 2.5M sulphuric acid to pH 5. Add 20 ml of distilled water and four ml of reagent B (ammonium molybdate). After 30 minutes, the intensity of blue colour on a spectrophotometer was read at 882 nm. The reading was noted down and the amount of P present in the soil was calculated by the given formula:

Calculation of phosphorus (P):

Soil P%=0.008 X Reading value = x

x X 5 ml aliquot=y

y X 50ml NaHCO3=z ppm

2.5 g soil

Kg/ha=z X 20,000=Kg/ha.

Estimation of Available Potassium (K):

Available potassium or exchangeable potassium, along with calcium and magnesium are usually determined in neutral normal ammonium acetate. Extraction is carried out by shaking followed by filtration or centrifugation. The potassium is estimated by using a flame photometer and Ca and Mg either by EDTA-titration or by using atomic absorption spectrophotometer. In soils with appreciable amount of soluble K, Ca and Mg these cations are estimated in a saturation extract. 5gm of air dried, soil samples were taken and stored into a plastic container. 25ml of ammonium acetate solution was added and kept for 5 minutes in shaker. Filtrate was taken through whatman filter paper No.1 into a clean and air dried beaker. The flask was shaken immediately before the suspension is poured into the funnel and his readings were recorded using flame photometer.

The percentage of K present in the sample was calculated by the following method

Total volume of aliquot (ml) x Metre value

K=----------------------------------------------------- X 100

Aliquot taken (ml)

RESULT AND DISSCUSION:

Isolation and identification of fungus:

Four morphologically different fungal isolates were selected based on the zone formation in the pikovskayas agar medium the isolates shows maximum zone around the colony was chosen for further studies refer Figure 1.

Figure 1: Microscopic observation of A. Aspergillus awamori, B. Aspergillus niger, C. Arthrographics cuboidea and D. Paecilomyces lilacinus.

Isolate I showed mycelial, or thread like hyphae are divided by a septum. Isolate II showed conidial heads are large, globose, dark brown becoming radiate and tending to split into several loose columns with age. Isolate III showed conidiophores generally short and can be branched or unbranched. The conidia which are produced from the conidiophores are smooth single celled and hyline. Isolate IV showed branched conidiophores which give rise to clusters of phialides.

Efficiency of P solubilization:

The efficiency of P solubilization was studied based on the zone formation in PVK. A. niger was identified as best P solubilizer among the 4 isolates. It forms 3.1 cm zone around the colony at the fifth day of incubation. Followed by A. awamori, Peciliomyces lilacinus, Arthrographics cuboidae.

Growth rate of fungi on solid media:

After 48 hours of incubation the diameter of the colonies were measured at every 24 hours for 5 days. Growth was seen in the Isolate I, II, IV after 48 hours of incubation, but the isolate III shows visible colonies only after 72 hours of incubation.

Growth on Rose Bengal agar Medium:

Figure 2: Represent the growth on rose bengal agar medium

Growth on Potato Dextrose agar:

Figure 3: Represent the growth on potato dextrose agar

Growth on PVK:

Figure 4: Represent the growth on potato dextrose agar

Growth rate of fungi on liquid media:

Growth rate on liquid media was determined by measuring the wet weight and dry weight biomass of the fungal genera. The growth was observed in three different enrichment media having different carbon sources. The dry weight biomass was maximum in sucrose enriched medium for Aspergillus awamori, Aspergillus niger and Arthrographics cuboidea compared to Paecilomyces lilacinus refer Table 1.

pH of the culture media:

All the four culture showed decreased in pH with time refer figure 5. Minimum pH was observed after 60 hours. The pH was lowered down due to the liberation of organic acids in liquid media. The minimum pH of 3 was shown by isolate V after 60 hours. The pH of the isolates I, II and III were 3.3, 3.96 and 4.12 respectively after 60 hours of growth.

Figure 6: Colour change observed in PDA medium

Initial observation of TC plants:

Table 2: Initial observation of TC plants

Treatments	R1		R2		R3		R4		R5		Mean
Treatments	length	girth	length	girth	length	girth	length	girth	length	girth	length	girth
T1	16	4	15	4	11.5	4.2	12.5	4	13	3.8	13.6	4
T2	16	4.5	13.8	4.5	12.4	4	12	3.2	10.2	3.2	12.8	3.8
T3	13	3.8	10.2	4.5	13	4	14	4.5	13.2	3.2	12.6	4
T4	12	4	13	4	14	4	15.8	4.3	16.2	4.2	14.2	4.1
T5	15	3.8	14	4.5	16	4.5	14.5	4.2	15	4.5	14.9	4.3
T6	11.3	4	12.8	3.2	12	4.5	15.5	5	14.5	4	13.2	4.1
T7	14	4.5	15	4.5	12.5	3.8	13	4	13.5	3.8	13.6	4.1

15 days after PSF application:

Table 3: 15 days after PSF application

Treatments	R1		R2		R3		R4		R5		Mean
Treatments	length	girth	length	girth	length	girth	length	girth	length	girth	length	girth
T1	16.2	5.2	16	4.8	12.2	5.2	13.2	4.7	14.1	4.3	14.3	4.8
T2	17.8	5.2	14	4.3	13.1	4.8	13.2	5	10.6	4.2	13.7	4.7
T3	13.4	4.8	16	4.9	13	5.2	14.5	4.7	15.1	4.8	14.4	4.8
T4	15.1	4.6	14	5	14.5	5	16.2	5	15.5	5.7	15.06	5
T5	15.2	5.3	14	4.7	16.3	5.4	14.2	4.9	15.2	5.6	14.9	5.1
T6	13	4.6	11	3.8	12.5	4.8	16	5	14.8	4.9	13.4	4.6
T7	16.5	4.5	16	4.5	14	4.2	14.2	4.2	13.3	4.2	14.8	4.3

30 days after PSF application:

Table 4: 30 days after PSF application

Treatments	R1		R2		R3		R4		R5		Mean
Treatments	length	girth	length	girth	length	girth	length	girth	length	girth	length	girth
T1	15	8.3	15.5	7.1	17	7.5	14	6.6	10	4.2	14.3	6.7
T2	15	6.8	13.2	5.7	13.4	6.7	14	11.2	13	6.6	13.7	7.4
T3	17	8.2	16.1	6.6	17.2	7.1	15.2	6.8	14.8	6.6	16.06	7
T4	16.5	6.8	18.1	7.6	15.7	7.2	17.8	6.8	22.5	8.8	18.1	7.4
T5	17.2	8	19.1	9	18	7.9	16	7.3	17.3	8.9	17.5	8.2
T6	14.2	6.1	11.5	5.3	13	6.2	15.1	7.5	17	8	14.1	6.6
T7	14.2	5.2	13.2	5.2	13.5	5.5	12.8	5.5	11.3	5.5	13	5.3

Figure 7: Growth of treated plants in pot

Root length measurement:

The tissue cultured plants were uprooted carefully and the length of the root was measured. Compare to other treatments T6 shows the maximum root development refer figure 8 and table 5.

Figure 8:Uprooted plants for root length measurement

Measurement of root length (cm):

Table 5: Measurement of root length

Treatments	R1	R2	R3	R4	R5
T1	54.6	51.2	28.5	18.8	17.6
T2	56.1	53	34	27.8	22.4
T3	54.5	45.5	38.2	33.7	26.4
T4	39.5	28.3	22.6	18.3	16.8
T5	66.2	52.6	39.2	23.5	19.2
T6	65.7	59.5	47.2	34.6	28.4
T7	48.2	35.1	31.2	28.6	24.6

Soil nutrient analysis:

The result pretaining to the effect on soil nutrients are presented in table 6 and it was found that application of PSF was significantly increased the nutrient content in the soil which favours the growth of the plant. Especially in case of phosphate the available P was increased based on graded dose of the PSF application.

Table 6: Effect of PSF on Primary nutrients contents in the rhizosphere soil of banana

Treatments	Initial	Two weeks	Four weeks	Six weeks
T1-Only manures without application of (PSF)	1.9	1.5	3.1	1.9
T2-Application of 10 grams of PSF	1.5	1.8	1.9	1.2
T3-Application of 20 grams of PSF	1.7	1.3	0.93	0.7
T4-Application of 30 grams of PSF	1.6	1.1	0.92	0.41
T5-Application of 40 grams of PSF	2.8	2.4	0.67	1.2
T6-Application of 50 grams of PSF	1.9	1.6	0.28	1.3
T7-Control soil alone	1.8	2.2	0.65	0.39

CONCLUSION:

In this work 4 fungal isolates I, II, III & IV were isolated and screened for their capacity of P solubilization.All the 4 cultures shows maximum growth at 120 hours (in soil media). There was a significant decrease in the pH of the media due to the production of organic acids.The isolate I, II, IV showed maximum solubilization in PVK. In field studies T6 which contains the maximum dose of PSF gaves a better results as compare to other treatments. Additional dosage may require for the best results. However the identified PSF can be mass cultivated and it can be used as a biofertilizer. According to the laboratory experiment of P solubilization it was found that the fungi have more efficiency to solubilize the P than bacteria. Hence there is need to develop the strain of fungi as P fertilizer, also the application of the biofertilizer prepared by fungi should be helpful to reduce the salinity of soil neutralization phenomenon, because these microorganisms release the acids in very minute quantity in P solubilization.

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Received on 16.03.2018 Modified on 06.05.2018

Research J. Pharm. and Tech 2018; 11(11): 4953-4959.

DOI: 10.5958/0974-360X.2018.00902.2

S. No	Fungal isolates	Glucose		sucrose		Mannitol
S. No	Fungal isolates	Fresh wt	Dry wt	Fresh wt	Dry wt	Fresh wt	Dry wt
1	Aspergillus awamori	16.76	3.9	17.06	5.3	18.37	3.2
2	Aspergillus niger	45.98	5.02	40.99	4.9	45.04	4.9
3	Arthrographics cuboidea	27.06	4.3	27.88	4.2	20.44	3.06
4	Paecilomyces lilacinus	27.61	2.9	25.64	3.5	28.97	3.4
	Mean values	4.03		4.4		3.6