Determination of Desirable Properties of Bacteria, Fungi and Their Biofilm Associated with Rubber Rhizosphere

: Rhizosphere microbes and their community structure ‘biofilm’ play a significant role in maintaining the dynamic of soil fertility. In this study, isolated bacteria and fungi from rubber root rhizosphere in red yellow podzolic soils and their biofilm structures were formulated under laboratory conditions. They were evaluated for their effectiveness on solubilization of insoluble inorganic compounds, calcium hydrogen phosphate (CaHPO 4 ), higher grade Eppawala rock phosphate (HERP) and Eppawala rock phosphate (ERP)) in liquid medium, production of indole acetic acids (IAA) and capacity of fixing atmospheric nitrogen using acetylene reduction assay (ARA). The relationships between variables were identified using regression analysis. The solubilization of CaHPO 4 in liquid medium by different bacterial strains was accompanied by a drop in pH (4.2-6.1) from an initial pH of 6.8-7.0, which followed a polynomial relationship between the pH of the medium and the amount of soluble phosphorus. The medium with HERP showed less relationship compared to CaHPO 4 between the above parameters. No correlation was found between pH and the amount of P solubilized of the culture medium containing ERP. Out of 30 bacterial isolates, five isolates formed proper biofilm community structure. The biofilm solubilized significantly high amount of phosphorus in liquid medium containing CaHPO 4 compared to their bacterial and fungal counterparts and was observed synergistic effect for ARA. Production of IAA of biofilm was higher than that of bacteria alone cultures. Thus the biofilm formation of rhizosphere microbes seems to be very important for improved soil fertility.


INTRODUCTION
Rubber plantations were first established in Sri Lanka at the beginning of the 20 th century. Since then, many individual plantations have undergone 3 or 4 planting cycles. Depleted soil nutrients must be replenished through balanced and efficient organic and inorganic fertilizers and through improved soil management practices. Among the organic sources, biofertlizers have been recognized as economical alternatives for imported fertilizers.
Soil is an excellent niche of microorganisms, i.e. protozoa, fungi, bacteria and viruses. Some soil bacteria that are able to colonize surrounding the plant roots is called rhizobacteria, and play a significant role in maintaining the dynamic of soil fertility and plant growth (Kloepper, 2003;Bakker et al., 2007). Rhizobacteria which provides beneficial effects on the host plant growth via direct and indirect mechanisms, are referred to as plant growth promoting bacteria (PGPB) (Kloepper and Schrot, 1981). These bacteria support fixation of atmospheric nitrogen, solubilization of phosphatic minerals and secretion of stimulating hormones, like auxin also known as Indole 3 Acetic Acid (IAA) (Huddedar et al., 2002;Chopade et al., 2008;Bashan and de-Bashan, 2010). Nitrogen fixing capabilities of these strains had been quantified using the nitrogenase enzyme, which reduces acetylene to ethylene, and the production of ethylene was measured using gas chromatography (Dilworth, 1966;Stewart et al., 1967;Staal et al., 2001). Solubilization of insoluble inorganic aluminum and iron phosphate compounds is an important strategy for increasing availability of phosphorus in the soil (Barber, 1984;Tan, 1993;Marschner, 1995). Some soil bacterial isolates have an ability to solubilize inorganic low soluble phosphates Wakelin et al., 2004;Chen et al., 2006;Song et al., 2008).
Solubilization of insoluble inorganic compounds (CaHPO 4 , HERP & ERP) in solid and liquid medium has been widely used as the isolation methodology of phosphate solubilizing microorganisms (Pikovaskaya, 1948). Plant growth promoting rhizobacteria can also promote plant growth through secretion of plant growth regulators; e.g. IAA (Lee et al., 2004) and can be quantified by following the protocol adapted by Gordon and Weber, 1951. Isolation of indigenous microbes capable of solubilizing phosphorus, fixing of atmospheric nitrogen and secretion of plant growth promoting hormones is an important procedure because of their superior adaptability to the isolated environment than the introduced strains.
Community of microbes consists of microbial cells with extracellular biopolymers are named as a biofilm (Seneviratne, 2003 andSeneviratne et al., 2008b). Further, biofilms are more efficient than their monocultures or mixed cultures of effective microbes and studies so far gave encouraging results on soil fertility (Seneviratne et al., 2008(Seneviratne et al., , 2009(Seneviratne et al., and 2011. Considerable attention has therefore been focused recently on microbial interference on biofilm formation in the environment and their potential to increase nutrient availabilities in the soil. The objective of this study were to (i) measure the phosphate solubilizing, nitrogen fixing and IAA production capacities of some indigenous bacterial fungal species isolated from rhizosphere of Rubber plants (Hevea brasiliensis), (ii) select promising strains for the formation of biofilm and evaluate their effectiveness under in-vitro conditions.

Isolation of indigenous rhyzosphere micro organisms
Root samples were collected from a mature rubber field at Dartonfield Estate, Agalawatta. They were placed on petri dishes containing PDA and morphologically distinct fungal colonies were identified and sub-cultured on same medium to purify. Root samples were shaken in 50 ml sterilized distilled water for 30 minutes to extract rhizosphere bacteria. Samples were then serially diluted in sterilized distilled water and suspensions were spread on nutrient agar (NA) plates. Cultures were incubated at 28 ± 2°C for 24 to 48 hours.
Morphologically different colonies were isolated by sub culturing of single colonies on NA plates (Parkinson et al., 1971).

Quantitative estimation of phosphorus solubilization
Thirty bacterial isolates were grown in NA broths were transferred into 20 ml of PYK broth supplemented with 0.4 % of CaHPO 4 . They were incubated on a rotary shaker (60 rpm) with continuous shaking at 28 ± 2°C for 6 days. Each incubated broth cultures with four replicates were sampled periodically at one day interval and the supernatant of the culture was separated after centrifuging at 6000 rpm for 15 minutes. Quantitative analysis of phosphorus was done by the molybdenum-blue method (Murphy and Riley, 1962) with Skalar Auto Analyzer and pH of supernatant was determined using Jenway pH meter with glass electrode. Based on the clustering pattern of P solubilization, isolates which were unable to solubilize more P (less than 100ppm) sorted out under one cluster and those isolates were not further evaluated. Twenty two bacterial isolates that resulted high P solubilization (higher than 100ppm) were grouped under two clusters were further assessed for their P solubilization with HERP and ERP. Rock phosphates (RP) were obtained from a locally available RP deposit at Eppawala (ERP), which has 38 % and 30% of total P 2 O 5 in HERP and ERP, respectively.

Quantification of indole acetic acid (IAA) production
The second screenings of isolated bacterial cultures were done on the basis of IAA production. Selected twenty two single bacterial cultures were incubated for 14 days in Tris-YMRT medium and centrifuged at 3000 rpm for 15 minutes. One milliliter aliquot of the supernatant was vigorously mixed with 4ml of Salkowski's reagent. Absorbance of each culture medium was measured at 535 nm and concentrations were determined with the calibration curve prepared with analytical grade indole-3-acetic acid (Gordon and Weber, 1951).

Acetylene reduction assay (ARA) of bacteria
The twenty two isolates were further characterized on their ability of nitrogen fixation or acetylene reduction assay. The starting medium for acetylene reduction assay was the semisolid glucose + malate medium as described by Patriquin et al. (1980). This medium was modified to optimize ethylene production and was then named ML medium (Penido et al., 1985;Park et al., 2005). Autoclaved medium was cooled to 50°C and 4ml of aliquots were transformed into 18 ml vials. Flasks containing 20 ml of combine carbon medium (CCM) broth were inoculated with 10 µl of a broth culture of each bacteria in late lag phase, and incubated for maximum growth at 32°C with mild shaking. Each of these cultures containing 70 µl were then transferred separately to the ARA assay vials, which containing 4 ml of ML medium. These vials were incubated aerobically, for 24 hours at 32°C. After incubation, cotton plugs were replaced with a rubber stopper and 1.8ml of acetylene was injected in to each sample. After incubation for 1 hour at 30°C, 0.3 ml of sample from the gas was withdrawn and injected to a gas chromatography (Porapak N column at 110°C and N 2 as the carrier gas at a flux of 120 ml/minute) (Rodrigues et al., 2008).

Effectiveness of biofilms
Selected twenty two bacterial isolates and most common fungi Aspergillus were maintained in yeast manitol broth (YMB) separately. They were co-cultured for the formation of biofilm according to the methodology developed by . They were evaluated for their effectiveness on P solubilization in broth medium with 1% of CaHPO 4 , acetylene reduction assay and indole acetic acid production.

Statistical analysis
Mean values and standard deviations were calculated to represent P solubilization with CaHPO 4 . Moreover, hierarchical clustering analysis was done employing the statistical package GenStat 17 to categorize the isolates based on their P solubilization with CaHPO 4. Regression analysis were came out to derive the relationship between solubilized phosphorus and their growth medium acidity with three different P sources CaHPO 4 , HERP and ERP. Effectiveness of phosphorus solubilization in biofilm was observed using two orthogonal comparisons (a) bacterial cultures with fungal only and biofilm (b) fungal only cultures with biofilm. Analysis of variance was done to compare nitrogenase activity (ARA) and Indole acitic acid (IAA) production in between bacteria and their biofilm community.

Qualitative estimation of phosphorus solubilization
Out of 30 bacterial isolates, ten isolates were able to solubilize CaHPO 4 and resulted halos on CaHPO 4 inoculated PYK solid medium (Figure 1a), whilst other bacteria did not produce solubilization halo around their colonies (figure 1b).

Quantitative estimation of phosphorus solubilization
In liquid medium with CaHPO 4, out of 30 bacterial isolates, 22 were capable of solubilizing phosphorus higher than 100ppm (Table 1) and were clustered in two different regions ( Figure 2). The isolates which were able to solubilize phosphorus less than 100ppm were clustered separate to others (Figure 2). Bacterial isolates those have an ability to solubilize phosphorus more than 100ppm of the medium with CaHPO 4 were further evaluated for their phosphorus solubilization of the medium with HERP and ERP. For the medium with HERP, the strains coded as B3, B5, B6, B7, B9, B17, B23 and B25 increased the soluble phosphorus content more than 20 ppm at the end of the incubation period (mean value = 33.55ppm). In the medium containing ERP, 5 strains coded as B5, B6, B17, B23 and B25 out of 22 strains increased the soluble phosphorus content more than 20ppm at the end of the incubation period (mean value = 34.1ppm). In general, strains B5, B6, B17, B23 and B25 exhibited higher solubilizing capability for the three sparingly soluble inorganic phosphates in liquid medium than the other strains and it was evidenced by the clustering pattern of the dendrogram based on phosphorus solubilization in the medium with CaHPO 4 (Figure 2).

Quantitative estimation of phosphorus solubilization with biofilm
Out of 30 bacterial isolates, 14 bacterial isolates had an ability to colonize on fungal mycelia and formed biofilm community structure (Figure 3). The rest did not form biofilm.

Fig. 3. A microscopic view of fungal filaments attached by bacteria for the formation of fungal bacterial biofilm Magnification: 2,000 x.
Fast growing fungi formed mat like structure when they were grown on liquid culture medium. The fungi those were co-cultured with bacteria for the formation of biofilm was also observed the same mat structure on the top of the medium. Part of the soluble phosphorus in liquid culture medium, produced by fungal alone culture or biofilm could be deposited in that mat like structure. In order to evaluate the effectiveness of phosphorus solubilization in biofilm, orthogonal comparison was made to compare bacterial broth Similarity Axis containing soluble P (variable 1) with fungi alone (variable 2) and their biofilm culture (variable 3) ( Table 2). This contrast effect was significant at the probability level p< 0.0001.

Phosphorus solubilization and growth medium acidity (pH)
The majority of bacterial strains acidified liquid medium containing three insoluble inorganic phosphates. The solubilization of CaHPO 4 in the liquid medium by different strains was accompanied by a drop in pH (4.2 to 6.1) from an initial pH of 6.8-7.0 after the incubation period of 72 hours. There was an inverse correlation between the pH of the medium and the released of P content in the supernatant of the culture medium containing CaHPO 4 and HERP as a sole source (Figure 4a and 4b). No correlation was found between pH and the amount of P released in the culture medium containing ERP (Figure5).

Indole acetic acid production (IAA) of isolates
Out of 22 bacterial isolates which are capable of CaHPO 4 solubilizing higher 100ppm, 9 isolates were screened based on indole acetic acids (IAA) production ability. Those isolates were coded as B2, B3, B4, B6, B7, B12, B19, B23 and B25. There were significant differences (P<0.0001) among the nine isolates on the production of IAA which ranged from 0.3-4.8µg/ml, with mean value of 3.0122 ± 1.26. Same pattern was observed on the production of IAA among the biofilm community structures and it ranged 0.7-7.9 mg/ml, with the mean value of 4.9348 ± 1.98. Furthermore, a significant difference ((P<0.0001) in the production of IAA was recorded in the presence of particular biofilm community structures (B2F2, B3F2, B6F2, B23F2, B25F2) compared to their bacteria alone counterpart ( Figure 6).

Fig. 6. Indole acetic acid production of medium containing different bacteria and their biofilm community structures
Nitrogenase activity was observed with 3 bacterial isolates which were coded as B3, B19 and B25 out of total 30 bacterial isolates and it was ranged from 3-8 µmol C 2 H 4 /hr. Moreover, some bacterial isolates that exhibited either no or low nitogenase activity, showed significantly (p<0.0001) higher nitrogenase activity with their biofilm community structure (Table 3).

DISCUSSION
Most of the selected morphologically different bacterial isolates showed rapid growth on culture medium. Phosphorus solubilization in solid medium was observed from the production of halo with some bacterial isolates in the media containing CaHPO 4 . However, there were no any solubilization halos in solid medium containing HERP and ERP sources. Similar observations with CaHPO 4 and Al(H 2 PO 4 ) 3 and FePO 4 .2H 2 O had been reported (Marra et al., 2012;Ogut et al., 2010 andQin et al., 2011). According to these studies, halos appeared in sparsely soluble CaHPO 4 and no halos appeared in water insoluble Al and FePO 4 . With liquid medium, all strains were capable of solubilizing CaHPO 4 , HERP and ERP with varying degrees. Many of the strains which did not display a solubilization halo in solid medium were capable of solubilizing phosphorus in liquid medium. All isolated bacterial strains were capable of solubilizing CaHPO 4 in liquid medium and similar results were observed in a study evaluating the solubility of CaHPO 4 in liquid PYK medium by bacteria from the rhizosphere of Allium fistolusum L., Capsicum annum L., Sesamum indicum L., and Oryza sativa L. (Chung et al., 2005). Lower solubilization index with solid medium without formation of solubilization halo around the bacterial isolates was promoted in liquid medium with favorable conditions for the diffusion of organic acids (Marra et al., 2012). Biological, chemical and physical properties may engage with the ability of soil microorganisms to solubilize insoluble inorganic phosphate. In many cases, acidification is the key process involved in phosphate solubilization. A significant negative correlation between the pH of the culture medium and phosphorus solubilization by several genera and species were observed by many authors Chen et al., 2006;Marra et al., 2011). There was an inverse correlation between pH of the liquid culture medium and the released of P content in the supernatant of the culture medium containing CaHPO 4 and HERP as a sole sources. The organic acids released by phosphorus solubilizing bacteria could drop pH of the growth medium (Maliha et al., 2004;Pradhan & Sukla, 2005). No correlation was found between pH and the amount of P solubilized of the culture medium containing ERP. The reduction of pH in some cases does not correlate with solubilized phosphorus (Chen et al., 2006;Khan et al., 2007b). Alternative possibilities other than organic acids for inorganic P solubilization have been reported. For example, extrusion of proton accompanied with the cation uptake (Khan et al., 2007b), formation of exopolysaccharide (Yi et al., 2008), production of chelating substances (Whitelaw, 2002;Kucey, 1988;Welch et al., 2002) and through exchange reactions (Gahoonia et al., 1992;Jones, 1998;Trolove et al., 2003) can be highlighted. Present study indicated that, the formation of biofilm with effective microbes has an ability to solubilized sparingly soluble CaHPO 4 more than their bacterial and fungal counterparts. Similar observation has been made by the others (Seneviratne and Indrasena, 2006); Jayasinghearachchi and Seneviratne, 2006) where 8-13 fold increment of phosphorus solubilization was observed with biofilm application. This could be due to high level of organic acids production associated with biofilm community with high level of gene expression and it was different from their individual microbes present in the environment (Vilain and Brozel, 2006).
Some bacterial isolates B2, B3, B6, B7, B12, B17, B23 and B25 the secreted IAA ranging from 2.5-5ppm than the other bacterial isolates. Moreover, the IAA production of biofilm was ranging from 3-7ppm and higher than their mono cultures. There is evidence that the capacity of producing IAA was higher with biofilm compared to their bacterial only cultures (Bandara et al., 2006;Seneviratne et al., 2009).
Most of the bacterial isolates associated with rubber root rhizosphere had the ability to grow in nitrogen free medium. But three bacterial isolates coded B3, B19 and B25 showed high nitrogenase activity compared to other isolates. The nitrogenase activity of the biofilms was significantly higher than that of their monocultures (Seneviratne and Jayasinghearachchi, 2005;Seneviratne et al., 2008). Several studies have reported the benefits of nitrogen fixing prokaryotes in growth and nutrition of many plant species; According to the observation of Jayasinghe et al. (1989) and Jayasinghe and Wettasinghe (1991), the nitrogen fixing ability of leguminous cover crop in rubber plantations was severely affected with high nitrogen level of the environment where the leguminous counterpart in rubber plantations is fertilized with high nitrogen fertilizers. However, the ability of the rhizosphere microorganisms of rubber tree to fix N under such conditions had not been investigated.
Research should be done in the future in order to evaluate whether the biofilms could overcome failures, if any, associated with rubber root rhizosphere, especially for nitrogen fixing capability.
The isolation and screening of IAA producing, nitrogen fixing and P solubilizing bacterial isolates associated with rubber root rhizosphere is important for the development of biotechnological product that can be used by growers as partial substitute for costly inorganic fertilizers.

CONCLUSION
We were able to isolate 14 rhizobactera spp. (coded as B1, B2, B3, B4, B6, B7, B9, B10, B12, B19, B20, B21, B23, B25) associated with rubber root rhizosphere that are not only capable of P solubilization, N fixation and IAA secretion, but also form biofilms with some Aspergillus sp isolated from rubber rhizosphere. It was revealed that the fungal bacteria biofilm communities are more effective in their biological performances than their monoculture counterparts.

ACKNOWLEDGEMENT
Author greatly acknowledge the financial support provided by research facilitation fund of Postgraduate Institute of Agriculture, University of Peradeniya.