Isolation and Screening of Silicate Bacteria from Various Habitats for Biological Control of Phytopathogenic Fungi *

Silicate solubilizing bacteria (SSB) can play an efficient role in soil by solubilizing insoluble forms of silicates. In addition to this some SSB can also solubilize potassium and phosphates, hence increasing soil fertility and enhancing plant defense mechanisms. A total of 111 bacterial strains were isolated from various habitats of Pakistan and screened for solubilization of silicate, phosphate and potassium on respective media. Out of these, 35 bacterial isolates were capable of solubilizing either silicate, phosphate or potassium. Amongst these 7 bacterial isolates were capable of solubilizing all three minerals tested. The highest silicate (zone diameter 54 mm) and phosphate solubilization (zone diameter 55 mm) was observed for bacterial isolate NR-2 while the highest potassium solubilization was observed for NE-4b (zone diameter 11 mm). Dual culture antagonistic assays were carried out by using these bacterial isolates against four plant pathogenic fungi Magnaporthae grisae, Rhizoctonia solani, Altarnaria alternata and Macrophomina pheasolina. Mean zone of inhibition of these bacterial isolates against the four pathogenic fungi ranged between 4 mm to 39 mm. The largest zone of inhibition against all four bacterial strains was recorded for bacterial isolate NR-2 followed by NE-4b. These strains will be further investigated for their plant growth promoting activities in the future.


Introduction
Silicon (Si) enhances the growth, development and yield of many plants and is reported to decrease the incidence of many fungal diseases in different pathosystems by strengthening the cell walls and especially the outer membrane of epidermal cells in leaves thus increasing resistance to the penetration of pathogenic fungi [1] [2].Silicon benefits the plants in several other ways by accelerating growth, conferring rigidity to leaves thus maximizing leaf surface area for photosynthesis and mitigating the effects of abiotic stresses like drought, salt and metal toxicity in several plants including wheat, rice, sugarcane, cucumber, tomato, citrus and barley [3]- [8].Despite of its abundance in earth's crust it is mostly present in insoluble forms that cannot be readily absorbed by plant roots [9] [10].It remains in insoluble form unless solubilized by weathering action of rocks or biological activity of plant roots and microorganisms.Silicate solubilizing bacteria (SSB) can play an efficient role here by solubilizing insoluble forms of silicates hence increasing soil fertility and enhancing plant defense mechanisms [9].The solubilized Si in the form of orthosilicic acid (H 4 SiO 4 ) is absorbed along with water [10].Si is accumulated in the form of silica gel and is deposited in epidermal cells, scelerenchyma, vascular bundles and in florescence brackets in cereals [11].The accumulated Si not only improves growth and yield of these plants but is also involved in induction of systemic resistance (ISR) against pests and diseases [12].
The present study was carried out to isolate and characterize silicate solubilizing bacteria from various habitats and evaluation of antagonistic activity against four major crop pathogens Magnaporthae grisae, Rhizoctonia solani, Altarnaria alternata and Macrophomina pheasolina.

Collection of Soil Samples from Different Locations
Soil samples were collected from various habitats in Pakistan (Table 1).Soil samples were also collected from fields which had been under routine cultivation of rice, wheat and sugarcane (Table 1) as these crops are high silicate accumulators.Half Kg of each soil sample was collected at a depth of 10 cm from top soil.The samples were then air dried and ten gram of each air-dried soil sample was transferred to 100 mL of sterile distilled water in a 250 ml Erlenmeyer flask separately.The samples were than incubated on a rotary shaker (120 rpm) for 30 min at room temperature as described by [13].Serial dilutions of each soil sample, ranging from 10 −1 to 10 −5 was prepared in sterile water.One mL aliquot of the appropriate dilution (10 −5 ) was spread on plates containing Luria Bertani (LB) agar.Plates were incubated at 30˚C ± 1˚C for 24 -48 h and colonies were examined.Morphologically distinct colonies were counted, selected, purified and maintained in LB broth amended with glycerol [14].

Morphological Characterization
Selected bacterial isolates were purified and plated on LB agar plates.Morphological characteristics were studied for pure single colonies [14].

Silicate Solubilization
Bacterial isolates from different soil samples were then spread on agar plates containing silicate medium with 0.25% insoluble magnesium trisilicate [15].Plates were incubated at a temperature of 30˚C ± 1˚C for 36 -72 h.After the incubation period, the bacterial colonies exhibiting clear zones around them were selected for further investigations.
Percentage of silicate solubilizing bacteria was checked for each soil sample and calculated as follows.

Phosphate Solubilization
All of the isolated bacterial isolates were checked for phosphate solubilization on Pikoviskaya agar plates [16]- [18].

Semi Quantitative Screening of the Bacterial Isolates
All of the selected bacterial isolates were subjected to semi quantitative mineral (Si, P and K) solubilization assay in in-vitro conditions.Each bacterial isolates was inoculated on three different bacterial agar media as above and after the incubation period (36 -72 h) at a temperature of 30˚C ± 1˚C, the area of clearing zones was calculated and recorded in mm.Each experiment considering a single bacterial isolate was repeated at least three times.

Acid Production by Bacterial Isolates
Freshly prepared broth cultures of selected bacterial isolates were spread on LB agar plates amended with bromophenol blue and incubated for 24 -72 h at a temperature of 30˚C ± 1˚C.Acid production was detected as blue media turned yellow around bacterial colonies.

Antagonistic Assays
Antagonistic activity of selected bacterial isolates was checked against selected pathogenic fungi including Magnaporthae grisae, Rhizoctonia solani, Altarnaria alternata and Macrophomina pheasolina using dual culture assays as described by  [18].Briefly, a 10 mm disc of an actively growing pure culture of pathogenic fungi grown on potato dextrose agar (PDA) was placed at the centre of a Petri dish containing appropriate test medium (rice flour agar RFA; PDA; Tomato juice agar etc.).A circular inoculum of selected bacterial isolates was made with a 6 cm diameter Petri dish dipped in a suspension of bacterial broth culture (5 × 10 9 -cfu•mL −1 ) and placed around the fungal disc.Plates were incubated for one week at 30˚C ± 1˚C and mean zone of inhibition was recorded after 7 days.Each experiment considering a single bacterial isolate was repeated at least three times.

Statistical Analysis
Each experiment was run thrice and means were calculated.

Results
Soil samples were collected during April to June 2011 from different habitats in Pakistan including Narowal, Khushab, Kallar Kahar, Chakwal, Bochaal, Miyani, Rawalpindi, Murree, Jhika Gali, Nathia Gali, Ayubia, Barian and Morkallan (Mansehra).Samples were collected either from fields that had been long under cultivation of rice, wheat or sugar cane or from mountainous regions which are rich in minerals.A total of 111 bacterial isolates were isolated from these soil samples and maintained as pure cultures.Each bacterial strain was checked for silicate, phosphate and potash solubilization on respective medium.Out of 111 bacterial isolates 35 bacterial isolates that showed solubilization of any of the three minerals were selected for further studies.Twenty-nine bacterial isolates were capable of solubilizing magnesium trisilicate, 28 were capable of solubilizing phosphates and 12 were observed to solubilize potash (Table 1).Amongst these 7 bacterial isolates were capable of solubilizing all three minerals tested.The highest silicate (zone diameter 54mm) and phosphate solubilization (zone diameter 55 mm) were observed for bacterial isolate NR-2 while highest potassium solubilization was observed for NE-4b (zone diameter 11 mm).The highest percentage of silicate solubilizing bacteria was observed in soil samples collected from Ayubia and Nathia Gali mountains where 100% bacterial isolates were silicate solubilizers while 64% of the total bacterial population was silicate solubilizers in soil samples obtained from Narowal fields which had been under routine cultivation of wheat and rice (Figure 1).A few silicate solubilizers were seen in samples from potato field (Nathia Gali) and wild Mint plant rhizosphere and endosphere which were taken from Morkallan village near Mansehra (Figure 2, Figure 3).
Similarly the highest percentage of phosphate solubilizing bacteria was seen in soil samples from Ayubia and Nathia Gali Mountains and almost all of the silicate solubilizers in these samples were phosphate solubilizers (Figure 3).The highest population of potash solubilizers was seen in soil samples taken from Ayubia Mountains followed by wheat rhizosphere samples taken from Narowal and a few potash solubilizers were seen in soil samples taken from Rawalpindi.
The selected 35 bacterial isolates were checked for antagonistic activity against plant pathogenic fungi such as Magnaporthae grisae, Rhizoctonia solani, Altarnaria alternata and Macrophomina pheasolina and zone of inhibition recorded (Table 2).Almost all of the selected isolates exhibited at least some antagonistic activities against the four pathogens.Mean zone of inhibition of these bacterial isolates against the four pathogenic fungi ranged between 4 mm to 39 mm.Highest antagonistic activity was observed in case of bacterial strain NR-2, isolated from wheat rhizosphere, against all fungal pathogen followed by NE-4b, isolated from wheat root from Narowal region.

Discussion
Bacterial isolates can solubilize insoluble minerals such as silicates, phosphates and potash into soluble form by production of organic acids such as 2 keto-gluconic acid, alkalis and polysaccharides [20].Bacteria have been reported to accelerate the dissolution of silicates by producing excess proton, organic ligands, hydroxyl anion, extra cellular polysaccharides (EPS) and enzymes [21]- [24].Despite abundance of microorganisms in soil only a few are capable of solubilizing silicates and our results indicated that out of a total of 111 bacterial isolates only 29 were capable of solubilizing silicates.Similarly, phosphate solubilization by bacterial isolates is also attributed to production of gluconic acid, 2 keto-gluconic acid and phosphatases.Our results indicated that almost all of the bacterial isolates capable of solubilizing phosphates and silicates produced acid as detected by yellow halo formation on media containing bromophenol blue and low pH in broth cultures.
Efficient silicate solubilizing bacteria can help release other essential nutrients in soil [25].This can be directly due to solubilization of other minerals by silicate solubilizing bacteria or indirectly due to solubilized silicon.We also observed that most of the silicate solubilizers were able to solubilize phosphates with a few exceptions such as bacterial strain NE-1, R1, R2 and AYM-1 which solubilized silicates but not phosphates.It has been previous reported that the solubilized Si improves availability of phosphorus to plants by competing with P fixation sites in soil [25].Thus Si acts as a substitute for P in plant system.Silicon is reported to increase the availability of Phosphorus indirectly by decreasing the availability of Fe and Mn in plants [26].
Potassium is also one of the major micronutrients required by the plant for optimum growth and yield.Potassium plays a regulatory role in plant being a constituent in 60 different enzymes in plants involved in drought tolerance and water use efficiency.Recent studies have shown that besides drought tolerance potassium plays an  important role in various metabolic processes and disease resistance in plants, almost all crops require potassium for optimum growth [27].Deficiency of potassium leads to symptoms like chlorosis and leaf fall, unhealthy root system and then reduction in nutrient uptake and reduced cytokinin production in roots.This deficiency of potassium in soil can be successfully managed by application of microbial fertilizers based on bacterial isolates that can solubilize potassium in soil.Potassium is mostly present in soil in the form of silicates hence silicate solubilizing bacteria can play an efficient role here by liberating soluble potassium in soil.Our results indicated that some of silicate and phosphate solubilizing bacterial isolates were capable of solubilizing potassium as well while a few, such as R1 and R2, were found to solubilize silicates and potassium but not phosphate as detected by plate assays (Table 1, Figure 3).In addition to increasing the availability of Si, P and K in soil, the silicate solubilizing bacteria provide a very efficient system of biological control of plant pathogenic fungi.These bacteria can not only directly combat phyto-pathogenic fungi but indirectly by release of Si in soil which in turn induces disease resistance in plants either by acting as a physical barrier or by acting as a modulator of host resistance to pathogen.Si is deposited beneath the cuticle to form a cuticle-Si double layer which mechanically impedes penetration of fungi and thus disrupts the infection process [26]- [30].
Almost all of the selected bacterial isolates showed at least some antagonistic activities against the four fungal pathogens used.Highest antagonistic activity was observed in case of bacterial isolate NR-2 as shown by maximum zone of inhibition observed against all of the fungal pathogens tested followed by bacterial isolate NE-4b and R-6 respectively.There are several ways by which these silicate solubilizing bacteria can antagonize fungal pathogens.These include production of hydrolytic enzymes, siderophores, HCN and antibiotics [13] [14] [18].The antagonistic potential of these bacteria is an important ability of practical utility in development of biopesticides against the fungal pathogens.These bacterial isolates will be further investigated for combating plant pathogens in green house experiment for a possible development of bioformulations for commercial purpose.

Figure 1 .
Figure 1.Percentage of silicate, phosphate and potash solubilizing bacteria in plant root and soil samples collected from different area of Pakistan.

Figure 3 .
Figure 3. Venn diagram illustrating solubilization of Si, P and K by 35 selected bacterial isolates Values represent actual numbers of bacterial isolates.

Table 1 .
Clear zone diameter was recorded after 24 to 72 h.Morphological characterization, Si, P and K solubilization of 35 bacterial isolates from various habitats in Pakistan.

Table 2 .
Inhibition of phytopathogenic fungi by selected Si, P and K solubilizing bacterial isolates.