Isolation, Characterization of Phosphatase Producing Bacteria and Fungi-Their Effect on Enhancing the Growth of Vigna radiata

The mung bean (Vigna radiata), is known as the green gram, belongs to legume family. The mung bean is commonly cultivated in Indian subcontinent. It is used as an ingredient in savory and sweet dishes.
       
The soil-plant-microbe interactions has got much importance in the recent decades. The plant nutrition and growth is promoted by the application of microbial inoculants Bashan and Holguin (1997) and Sullivan (2001). For plant development major essential macronutrients is phosphorous Dave and Patel (1999). The phosphorous present in soils is not utilized by plants this may be due to presence of inorganic phosphate and it is not present in free available form. Readily soluble or water soluble P, when applied in acid soils is rapidly fixed to unavailable forms and accounts for low phosphate use efficiency Sarkar and Uppal (1994).
       
Phosphate-solubilizing microorganisms (PSM) render insoluble phosphate into soluble form through the process of acidification, chelation and exchange reactions. These organisms would replace expensive fertilizers and also catalyze the soil fertilizers Rodrigue and Fraga (1999) that can be readily utilized by plants. Reserachers investigated availability of phosphate fraction by plants in the presence of phosphate solubilizing microorganisms (PSMs) like Agrobacterium sp, Bacillus sp, Pseudomonas sp, Bacteroides sp. and Xanthomonas sp and Fungi like Aspergillus sp, Penicillium sp, Mucor sp and Rhizopus sp. The phosphatase enzyme plays a key role in transforming organic forms of phosphate to plant available inorganic form, enzyme may therefore be very important in phosphorus nutrition of plants Tarafdar et al., (1992). Phosphate solubilizing fungi (PSF) have been reported to have greater ability to solubilize insoluble phosphate than bacteria Nahas (1996). The effect of PSB on yield, by green gram was studied by Karnavat et al., (2018). PSB application reduced the adsorption and fixation of exogenous P sources and improved the utilization rate of P fertilizer in reclaimed soil (Xiao-Kai et al., 2017). 
       
The previous studies were related to the isolation of bacteria as phosphate solubilizer and the little information is available about the parallel study of bacteria and fungi as phosphate solubilizer. The present paper reveals the comparative study of bacteria and fungi as phosphate solubilizing microorganisms and determine their effect on the growth of Vigna radiata.

Sample collection
 
Samples of dung (cow and buffalo), garden rhizosphere soil, water from putrefactive lakes (Tank Bund and Shamirpet) were used for study during 2017-2018 at St. Francis College for Women, Begumpet Hyderabad. The serially diluted
samples were inoculated on Pikovskaya (PVK) medium (Two sets of plates were prepared).
 
Isolation and identification of PSB (phosphate solubilizing bacteria)
 
The first set of plates were incubated at 37^oC, 24-48 hrs, clear halo zones were observed around colonies. The isolates were characterized based on Gram staining, cell morphology, colony morphology and biochemical properties (Indole, Methyl Red, Voges Proskauer, Citrate utilization, Urease). The Bergey’s Manual of Determinative Bacteriology was used to identify isolates.
 
Carbohydrate fermentation
 
The Arabinose, Sucrose and Mannitol were used to evaluate fermentation property of bacterial isolates.
 
Isolation and identification of PSF (phosphate solubilizing fungi)
 
The second  set of plates were incubated at 27°C, 24-48 hrs, clear halo zones were observed around colonies. Based on colony morphology and microscopically identification of spore structure by Lactophenol cotton blue staining method isolates were identified.
 
Estimation of phosphatase
 
The isolates (bacteria, incubated at 37°C, 24 hrs; fungi, incubated at 27°C, 24 hrs culture  (PVK broth) supernatant was estimated for phosphatase (Lowry 1951).
 
Effect of pH on phosphatase production
 
The cultures were incubated at 37°C (bacteria) and 27°C (fungi) for 48 hrs at pH 1, 5, 7, 10, 14, supernatant was estimated for phosphatase.
 
Effect of temperature on phosphatase production
 
The optimum temperature for phosphatase production was estimated in 48 hrs culture supernatant, of bacterial (incubated at 37°C) and fungal (incubated at 27°C) isolates.
 
Phosphate estimation
 
The culture supernatant of isolates was analysed for phosphate content by Chlorostannou Reduced Molybdo- phosphoric Blue Colour Method Jackson (1973).
 
Effect of carbon source on phosphate production
 
The PVK broth containing Glucose, Lactose, Sucrose and Mannitol was estimated (in supernatant) for phosphate production by isolates.
 
Effect of nitrogen source on phosphate production
 
The Ammonium sulpate, Sodium nitrate, Potassium nitrate and Urea on phosphate production by bacterial and fungal isolates were estimated in culture supernatants.
 
Acid phosphatase assay
 
Acid phosphatase assay was performed using para-nitrophenyl phosphate (PNP-P) as substrate Ingham et al., (1979).
 
Pot experiment
 
The green gram (Vigna radiata) seeds were collected from local market and were soaked for 30 min in respective bacterial and fungal isolate cultures then sowed in pots. The garden rhizosphere soil was collected and mixed with sand in 1:1 ratio. The soil was separated and Tricalcium phosphate (TCP) was added at the rate of 200 mg/kg of soil. The soil was autoclaved. The pots with five treatments (T₀, T₁, T₂, T₃ and T₄) were performed. T₀, T₁ and T₂ were not exposed to cultures of isolates. T₃ and T₄ were exposed to respective cultures of bacterial and fungal isolates. T₂ and T₄ contain TCP. The pots were regularly irrigated with sterile water and kept in sunlight. The respective O/N (overnight) culture (2 ml) was added to pots T₃ and T₄. The germination time for seeds was 4-5 days. After 7 days shoot length and increase in number of roots of plants, with different treatments were measured and compared.

Sample collection
 
The bacterial A, B, C, D, E; and fungal isolates F1, F2, F3, F4 were isolated from different sources (Table 1). Microorganisms enhance P (Phosphorous) availability to plants by mineralizing organic P in soil was reported by Suhana et al., (2019).
 

 
Isolation and identification of PSB
 
The pure culture, A, B, C, D and E (Table 2) were sub-cultured on PVK agar; presence of small, round, powdery appearance, opaque, dry, slightly raised creamish white colonies on PVK agar showed zone of clearance (Fig 1) indicating phosphate solubilizing property of isolates. Qian and Shanjiang (2019) identified phosphate dissolving microorganism in soil.
 

 

 
Carbohydrate fermentation
 
The bacterial isolates (Table 3) were identified as Bacteroides sp. Song et al., (2005) identified Bacteroides sp based on biochemical scheme.
 

 
Isolation and identification of PSF
 
The pure cultures F1, F2, F3 and F4 (Table 4) were identified as Aspergillus sp and Mucor sp, colonies on PVK agar showed zone of clearance (Fig 1). Agnihotri (1970) isolated Aspergillus sp showing phosphate solubilizing activity. Onyia et al., (2015) isolated fungi showing phosphate solubilizing property.
 

 
Estimation of phosphatase
 
The B and F2, (Table 5) showed highest phosphatase production. Rudek and Haque (1976) reported extracellular production of enzyme phosphatase by Bacteroides sp. Porschen and Spaulding, (1974) reported Bacteroides species produced phosphatase with strongest activity.
 

 
Effect of pH on phosphatase production
 
The maximum production of phosphatase was at pH 7 and pH 5 with bacterial (Table 6) and fungal isolates; highest concentration was between pH 5-7 (acidic range), considered as acid phosphatases. Arcand and Schneider (2006) reported, in plants and microorganisms, primary mechanism of P (phosphate) solubilisation is acid phosphatase biosynthesis.
 

 
 
At 37°C, bacterial; and 27°C, fungal (Table 7) isolates showed maximum phosphatase production. Rosso et al., (1995) reported optimal conditions for growth of Mucor racemosus, was at 22°C; optimum growth of Bacteroides sp.was at 37°C-39°C, investigated by Skerman (1975). Behra et al., (2017) reported maximum acid phosphatase production at 45°C.
 

 
Phosphate estimation
 
The B and F2 showed highest phosphate production (Table 8). Ponmurugan and Gopi (2006) stated that there was a positive correlation between phosphate solubilizing capacity and phosphatase enzyme activity.
 

 
Effect of carbon source on phosphate production
 
The B showed maximum phosphate production with Glucose, Lactose, Mannitol; F2 showed maximum phophate production with Sucrose (Table 9). Chen et al., (2016b) investigated the effect of carbon sources on the insoluble phosphate solubilization by Pseudomonas sp PSB12. Nautiyal et al., (2000) reported that the glucose and lactose were the best carbon source and sucrose, sorbitol were identified as poor carbon source for phosphate solubilization.
 

 
Effect of nitrogen source on phosphate production
 
The A, B C, F2 and F4 with Ammonium sulphate; D, F1 and F3 with urea (Table 10) showed maximum production of phosphate. Tingting et al., (2019) reported NH₄Cl induced phosphate solubilization was best source of nitrogen.
 

 
Acid phosphatase assay
 
The B and F2 showed highest enzyme activity (µg/ml of enzyme liberated per min). The maximum enzyme activity was observed by all fungal than bacterial isolates (Table 11). Seshagiri and Tallapragada (2016) reported maximum phosphatase activity in P.indica. Behra et al., (2017) investigated Phosphate solubilization and acid phosphatase activity of Serratia sp.
 

 
Pot experiment
 
The effect of five treatments (T₀, T₁, T₂, T₃ and T₄) on growth of Vigna radiata were observed after a period of seven days. T₄ resulted in significant increase in shoot length with all bacterial and fungal isolates than T₃ (Table 12). T₄ also resulted in increase in number and length of roots with B, C, D, E, F1, F2, F3 and F4. In presence of phosphate solubilizing bacterial and fungal isolates insoluble phosphate TCP was solubilized. The soluble phosphate in soil resulted in increase in shoot length, increase in number and length of roots (Fig 2). The presence of native micro flora (T₀), available nutrients in soil (T₁) and presence of TCP (T₂) resulted in small increase in shoot length. T₃ resulted in small increase in shoot length with B, D and negligible growth with fungal isolates. Kim et al., (1997) reported Glomus etunicatum and Enterobacter agglomerans, able to solubilize insoluble phosphate. Lal (2002) observed, seed treatment with PSB such as Pseudomonas florescens and Bacillus megaterium resulted in enhancement of seedling length of Cicer arietinum. The microorganisms showing phosphate solubilizing property were isolated from different sources and their uses were investigated by Tyagi et al., (2003); Mohinder Kaur et al., (2011); Zahoor Ahmad Baba et al., (2014) and Behera et al., (2017).
 

 

The present study indicates bacterial isolate B and fungal isolate F2 were found to be potent phosphate solubilizing microorganisms (PSM). The application of PSM as inoculants to Vigna radiata, enhanced the plant growth, indicated by increase in the shoot length and in number of roots. The isolates with phosphate solubilizing and acid phosphatase production property may have probable use in agricultural application.

The authors appreciate the Principal and Head of the Department of Microbiology, St. Francis College for Women, Begumpet, Hyderabad for providing laboratory facilities to carry out this study.