Evaluating Efficacy of Biosurfactants from Bacterial Isolates in Conferring Protection against Rhizoctonia and Sclerotium Infection in Wheat and Peanut Plants

This work out in collaboration between both the authors. Author SP conceived and designed the study. Author JF carried out the experiments and author SP performed the analysis of data. Author JF wrote the first draft of the manuscript and managed the literature searches. Author SP edited and proofread the final manuscript. Both authors read approved the final ABSTRACT Aims: To isolate biosurfactant producers from natural habitat and to test the antimicrobial activity of the extracted biosurfactant against fungal plant pathogens. activity. Potent biosurfactant producing isolates were biochemically characterized and identified up to genus level using Bergey’s manual. Biosurfactant was extracted by chloroform: Methanol method. Characterization of extracted biosurfactant was done using blue agar plate and orcinol assay. Agar well diffusion method was used to test antimicrobial activity of biosurfactants. Ability of the biosurfactants to provide protection against fungal plant pathogens was demonstrated in vivo using wheat and peanut plant seedlings. Results: Three isolates BMW1, BMW2 and BPS1 showing good biosurfactant activity were selected for biosurfactant production. They belonged to genus Pseudomonas, Bacillus and Micrococcus . Extraction of culture supernatant gave white residue which was used in further studies as biosurfactant. The biosurfactant produced by isolates BMW1 and BPS1 was glycolipid anionic biosurfactant while CTAB medium indicated non-ionic nature of biosurfactant from BMW2. Biosurfactant extracted from all three isolates showed good antimicrobial activity. Biosurfactant produced by BMW1 and BPS1 most effectively protected peanut plantlets from Sclerotium rolfsii infection and wheat plantlets from Rhizoctonia solani infection respectively. Conclusion: Our study suggested a strategy for eliminating plant pathogenic fungi by using microbial biosurfactants.


INTRODUCTION
Enhancing agricultural productivity to meet demands of growing human population is a matter of great concern for all countries. Plants are constantly exposed and threatened by a variety of pathogenic microorganisms. Globally, about 15% of crop harvests are lost each year due to diseases caused by plant pathogens and about 85% of these diseases are caused by fungi. Rhizoctonia solani is one such pathogenic fungus responsible for a wide range of commercially significant plant diseases. It is the major fungus responsible for brown patch (a turfgrass disease), as well as black scurf of potatoes, bare patch of cereals, root rot of sugar beet, belly rot of cucumber, sheath blight of rice, peanut and many other pathogenic conditions [1]. Root rot is an important disease in wheat and barley caused by Rhizoctonia solani. Because of its wide host range, R. solani also causes root rot of peas, canola, and many other broadleaf crops. The most common symptom of Rhizoctonia is "damping-off" characterized by non-germination of severely infected seed.
Sclerotium rolfsii, is another soil-borne pathogen causing root rot, stem rot, wilt and foot rot on more than 500 plant species including almost all important agricultural (sweet potato, pumpkin, corn, wheat and peanut) and horticultural (Narcissus, Iris, Lilium, Zinnia, and Chrysanthemum) crops [2]. Peanut crops sustain higher losses than any other agricultural crop worldwide. The wide host range, prolific growth, and ability to produce persistent sclerotia contribute to the large economic losses associated with this pathogen. Currently available antifungal agents for controlling these fungi are highly toxic, non-biodegradable and their indiscriminate use causes environmental pollution. Some researchers have focused their attention on developing alternatives to chemical pesticides for controlling agriculturally important pests and diseases in recent years. One of these alternatives is biosurfactant.
Biosurfactants are surface-active agents produced by microorganisms such as bacteria and microscopic fungi, at the microbial cell surface or excreted extracellularly [3,4]. Different types of biosurfactants (glycolipids, lipopolysaccharides, lipopeptides) are produced by microorganisms [5][6][7]. Biosurfactants are superior to the chemical surfactants with respect to their biocompatibility, lower toxicity, higher biodegradability, higher stability, ability to act in extreme temperature and pH conditions [8][9][10]. Biosurfactants have several applications in pharmaceutical, cosmetics, petroleum, and food industry [4,[11][12][13][14]. Extracted biosurfactants have also been reported to inhibit the aflatoxin production by Aspergillus sp. which infects crops such as cottonseed, peanuts and corn during storage as well as at agricultural field [20]. In one recent study, rhamnolipids inhibited zoospore forming plant pathogens that have acquired resistance to commercial chemical pesticides [21]. Biosurfactants produced by rhizobacteria are known to have antagonist properties [22]. Another investigation has shown that rhamnolipid can stimulate plant immunity which is considered as an alternative strategy to reduce the infection by plant pathogens [23]. Thus the microbial biosurfactants play diverse roles in plant pathogen elimination directly or indirectly. Their probably low phytotoxicities, and natural compositions make them attractive candidates for future antimicrobials in agriculture.
Several reports have highlighted the advantages and antimicrobial properties of biosurfactants in vitro but there are fewer reports stating the in vivo application of biosurfactants for protecting plants from pathogenic microorganisms in agriculture.
Evaluating the efficacy of biosurfactants in controlling fungal pathogens may thus prove to be an important strategy in reducing chemical fungicide usage and thereby reducing the environmental pollution.
The present study attempted to isolate and screen for potent biosurfactant producing bacterial strains from natural habitats and then explored the potential of the extracted biosurfactant in controlling plant pathogenic fungi (Sclerotium and Rhizoctonia) in commercially important food crops.

Sample Collection
Soil samples were collected from near a petrol pump and seawater from Worli sea face located in Mumbai. All samples were collected in clean plastic bags/bottles. The samples were used immediately to prevent any deterioration.

Isolation and Enrichment of Biosurfactant
Producing Microorganisms 1g of soil sample /10 ml of seawater sample was taken and serially diluted up to 10 -6 dilution in 0.85% sterile saline. 0.1 ml of each dilution was spread on Cetrimide agar. This medium is selective for Pseudomonas species (as many species are good biosurfactant producers). Marine agar plates were used to cultivate marine bacteria. Plates were incubated at room temperature for 72 hours. After incubation, isolated colonies with morphologically different bacteria were selected and purified on the same media three times using Streaking method. Pure cultures of different isolates thus obtained, were inoculated in 100 ml of Mineral salt medium containing 2 drops of edible oil + kerosene + diesel in 1:1:1 ratio as carbon source [24]. Flasks were incubated with continuous shaking at 200 rpm for 7 days at room temperature. Cultures producing biosurfactant is evidenced by appearance of foam and formation of emulsion. The culture medium was centrifuged to remove cell debris and the supernatant was collected and tested for presence of surfactant by performing screening tests.

Screening for Potent Biosurfactant Producer
Biosurfactants are capable of decreasing surface and interfacial tensions, as well as form and stabilize oil-in-water or water-in-oil emulsions. The isolates obtained were tested for their biosurfactant production capacity by the following methods:

Blood haemolysis test
A fresh single colony from the isolated plates was picked using sterile nichrome loop and streaked on blood agar plates containing 5% (v/v) blood and incubated for 48 hours at 37°C [25]. Presence of clear zone around the colonies indicated the presence of possible biosurfactant producer as many biosurfactant producing bacteria have ability to haemolyse blood [26].

Drop-collapse test
Two micro-litre of crude oil was applied to each cavity of a glass cavity slide. The slide was equilibrated for 1h at room temperature and then 5 μl of the cultural supernatant was added to the surface of oil (test). In the control, uninoculated medium was added instead of culture supernatant. The shape of the drop on the oil surface was inspected after 1 min. Biosurfactant producing cultures giving flat/ less convex drops were scored as positive '+'. Those cultures that gave rounded convex drops were scored as negative '-', indicative of the lack of biosurfactant production [27]. This assay relies on the destabilization of liquid droplets by surfactants [28].

Penetration assay
This assay relies on the phenomenon of silica gel entering the hydrophilic phase from hydrophobic paste much more quickly in the presence of biosurfactants which leads to a colour change [29]. In this assay, the cavities of a 96 well microtiter plate were filled with 150 μl of a hydrophobic paste made up of oil and silica gel.
The paste was covered with 20 μl of oil. 10 μl of 1% safranin was added to 90 μl of the culture supernatant. In the control, uninoculated medium was added instead of culture supernatant. This coloured supernatant was then placed on the surface of the paste. The upper phase changes from clear red to cloudy white within 15 minutes if biosurfactant is present. Biosurfactant free supernatant will turn cloudy but stay red.
Based on the results of qualitative screening tests, isolates showing good surfactant activity were selected for further studies.

Identification of Biosurfactant Producing Isolates
The isolates that showed good emulsification / biosurfactant activity were identified up to Genus level by using Bergey's manual of determinative bacteriology.

Production of Biosurfactants
The selected isolates were individually inoculated in mineral salt medium containing mixtures of oils (edible oil + kerosene + diesel) in 1:1:1 ratio and incubated on shaker at 160 rpm at room temperature for 7 days. Flasks were inoculated in duplicate and uninoculated flasks served as control. After incubation, bacterial cells were removed by centrifugation at 5000 rpm for 30 minutes.

Extraction and Purification of Biosurfactant
To the supernatant thus obtained, 6N HCl was added to adjust the pH of 2.0 and then the solution was kept at 4°C for 24 h. Equal volumes of chloroform: methanol was added in the ratio of 2:1. This mixture was shaken well to ensure proper mixing and then left overnight. White oily residue seen at the interface between the two liquids indicates the presence of biosurfactant. The residue was collected after centrifugation at 5000 for 30 min. The crude biosurfactant pellet formed was carefully removed with the help of micropipette and kept in test tubes for evaporation [31]. 1 ml methanol was added to the partially purified biosurfactant. After filtration, the fraction was evaporated to dryness. The dry biosurfactant thus obtained was dissolved in phosphate-buffered saline (PBS), pH 7.0 at a concentration of 10 mg/mL and used for further experiments.

Emulsification activity measurement
Emulsification activity correlates to the concentration of biosurfactant [32]. The emulsification activity was determined by adding 2 ml of kerosene to 2 ml of biosurfactant in a tube. The control test tube contained water instead of biosurfactant. The sample was homogenized in a vortex at high speed for 2 min and allowed to settle for 24 hrs. The emulsification index was then calculated by using given formula.
Emulsification activity (%) = Height of emulsion layer X 100 Total height

Blue agar plate method
It is a semi quantitative method used for detection of extracellular anionic biosurfactant [33]. Mineral salt agar medium supplemented with glucose as carbon source (2%) and cetyltrimethyl ammonium bromide (CTAB: 0.5 mg/mL) and methylene blue (MB: 0.2 mg/mL) was used for the detection. 30 ul of biosurfactant was loaded into the each well made in methylene blue agar plate using a sterile cork borer (4 mm). Control well was loaded with distilled water. The plate was then incubated at 37°C for 48 hours. A dark blue halo zone around the well is indicative of anionic nature of biosurfactant.

Orcinol assay for determination of glycolipids
The orcinol assay is used for the direct assessment of the amount of glycolipids present in the extracted sample [34]. In this method, to 0.1 ml of each sample extract, 0.9 ml of distilled water was added to make up the volume to 1 ml. To this 3 ml of a solution containing 0.19% orcinol (in 53% H 2 SO 4 ) was added. After heating for 30 min at 80°C, the samples were cooled at room temperature and the absorbance at 420 nm was measured. Blank was prepared using distilled water. The rhamnose containing glycolipids concentration was then calculated from a standard curve prepared with standard Lrhamnose (100 μg/ml) and expressed as rhamnose equivalents (mg/ml). Rhamnolipid concentration can be calculated based on the assumption that 1 μg of rhamnose corresponds to 2.5 μg of rhamnolipid.

Evaluation of the ability of biosurfactant to protect crop plantlets prone to fungal infection in vivo
Two day germinated wheat and peanut seedlings were transplanted into 10 cm diameter plastic pots containing sterile 200 g of soil. The soil in the pots for wheat seedlings was infected with Rhizoctonia solani and for peanut seedling was infected with Sclerotium rolfsii by mixing 5 ml saline suspension of 1 week old slant of these fungi (OD-0.018). In TEST pots, the germinated seedlings were precoated with biosurfactant from Isolates 1 (BMW1), 2 (BMW2) and 3 (BPS1) by dipping the seeds in the solution prior to sowing. In each of the TEST cups, 8 such germinated coated seedlings were sown. Two controls were maintained a) CONTROL 1 (uninfected seedlings) b) CONTROL 2 (artificially infected seeds without biosurfactant). All TESTS and both CONTROLS were prepared in triplicates. Plants were grown at 28 +/-2°C with a 12 hour photoperiod and watered by spraying 10ml of water twice a day for a month. The effect on growth of the plantlets was recorded in terms of the number of plantlets growing, colour of leaves, health of plant, length of growth. The health of plants was evaluated using following scale (good = no wilting symptoms in 95% plants, average = slight or partial wilting of 5-20% plants, poor = general plant wilting and lesions in more than 20% plants, very poor = permanent wilting in 95% plants, and Dead = dried brown plantlets).

Statistics
All tests were conducted in triplicate as mentioned above and the plant growth was expressed as mean length±std. deviation in cm.

Isolation of Biosurfactant Producing Microorganisms
A total of twenty isolates with different colony morphologies were obtained on cetrimide agar and Marine agar plates. All the twenty isolated strains were screened for biosurfactant production.

Screening for Potent Biosurfactant Producer
Eleven among the twenty isolates were found to produce biosurfactants. To select the potent biosurfactant producer more than one screening tests i.e. blood haemolysis test, drop collapse test, penetration test were employed.

Blood haemolysis test
Blood agar haemolysis test was used as primary screening technique to detect biosurfactant secretion. Only five out of twenty isolates showed positive results for haemolytic activity i.e. formation of a clear zone around the colonies (Fig. 3C). Isolates BMW1, BMW2 (weak) and BPS1 were able to cause haemolysis of blood.

Drop-collapse test
Biosurfactants have surface activity which reduces the interfacial tension between the liquid drop and the hydrophobic surface and makes the drop of biosurfactant collapse. The cell free culture supernatants containing surfactant showed collapsed or less convex droplet compared to control ( Fig. 2A) and were scored as positive (+ve). Eleven different isolates including BMW1, BMW2 and BPS1 were found to have such surface tension reduction property.

Penetration assay
Biosurfactants are known to have good penetration ability [29]. Evidence shows that cultures producing biosurfactant display emulsification of oil. The colored culture supernatant placed on the surface of the hydrophobic paste, resulted in color change in upper phase from red to cloudy white which was noted as positive test. No change in color or appearance of red color wells was taken as negative test. Eleven out of the twenty isolates which also gave positive drop collapse test, showed penetration activity. Isolates BMW1, BMW2 and BPS1 were found to have better surface tension reduction and penetration ability compared to others and were chosen to obtain biosurfactant.

Identification of Isolates
The 3 isolates ( Fig. 1; top row) named BMW1, BMW2 and BPS1 showed good results for biosurfactant production. These isolates were identified using morphological (Macroscopic Colony characteristics, Gram's staining) and biochemical properties. BMW1 was found to be Gram -ve rods in singles. BMW2 was found to be Gram +ve rods in singles and BPS1 was found to be Gram +ve cocci (Fig. 1). BMW1 showed similarity to Pseudomonas, BMW2 with Bacillus and BPS1 with Micrococcus (Table 1).

Production of Biosurfactants
All the three selected bacterial isolates were cultured in Mineral salt medium containing 1% of edible oil + kerosene + diesel (P+K+D) in 1:1:1 ratio as carbon source.

Extraction and Purification of Biosurfactant
Extraction was done in chloroform: methanol mixture using culture supernatant. White emulsion was seen for all three cultures and was dried by evaporating the solvents.

Emulsification Activity Measurement
Biosurfactant is known to have emulsification property [37]. The extracted biosurfactants were subjected to quantitative testing for their abilities to emulsify crude oil i.e. kerosene in this case. Control tubes which contained water instead of culture supernatant did not show any emulsification (Fig. 2B). The emulsification activities were calculated in terms of percentage using the formula given in methods. Extracted biosurfactant from all three isolates (BMW1, BMW2 and BPS1) showed high emulsification activity (Table 1).

Characterization of Biosurfactant
Biosurfactants extracted from the three different isolates were characterized to find out the nature of biosurfactant and the amount of glycolipids present.

Blue agar plate method for detection of anionic biosurfactant
A dark blue halo zone around the well was indicative of anionic nature of biosurfactant. Pseudomonas (BMW1) and Micrococcus (BPS1) showed distinct dark blue halo zone indicating that these two isolates produce an anionic biosurfactant (Fig. 2D). Pseudomonas spps and Micrococcus spps are known to produce glycolipid i.e. rhamnolipid which is an anionic biosurfactant [38]. BMW2 showed very weak blue halo zone which may be due to the isolate belonging to genus Bacillus, which is known to produce non-ionic biosurfactant i.e. lipopeptides [39].

Orcinol assay for determination of glycolipids
Bacterial biosurfactant can be anionic (rhamnolipids) or non-ionic (lipopeptides) type. The orcinol assay was used for direct assessment of the amount of glycolipids present in the biosurfactant using L-rhamnose standard graph. The maximum glycolipid content was found in Pseudomonas spps (BMW1) and Micrococcus spps (BPS1) ( Table 1). This confirmed that biosurfactants isolated from BMW1 and BPS1 is a rhamnolipid. The biosurfactant from BMW2 was probably a lipopeptides hence showed negligible absorbance in orcinol assay.

Antimicrobial activity of the extracted biosurfactants against fungal plant pathogens
Biosurfactant extracted from Pseudomonas spps (BMW1) showed antifungal activity against both Sclerotium rolfsii and Rhizoctonia solani while Micrococcus spps (BPS1) showed better antimicrobial activity against Sclerotium than against Rhizoctonia solani. Bacillus spps (BMW2) was found to have better antimicrobial activity against Sclerotium compared to Rhizoctonia solani (Fig. 3).    being soil-borne fungi; their control using chemical pesticides is quite difficult and expensive. The possibility of pest management in crops via biosurfactants with antimicrobial properties provides a reasonable perspective and was evaluated further using vivo studies.

In vivo evaluation of the ability of biosurfactant to protect crop plantlets prone to fungal infection
The ability of the extracted biosurfactant to control fungal pathogens was assessed using wheat and peanut plants infected with fungal pathogens i.e. Rhizoctonia Sclerotium rolfsii respectively. infection of the cereal plant is largely confined to the juvenile tissues of seedlings; hence two day germinated wheat seedlings were used in the experiment. Three readings of the clear zone diameter were taken for each well and the mean was calculated to determine biosurfactants with antimicrobial properties provides a reasonable perspective and was evaluated further using in

evaluation of the ability of biosurfactant to protect crop plantlets
The ability of the extracted biosurfactant to control fungal pathogens was assessed in vivo using wheat and peanut plants infected with Rhizoctonia solani and . Rhizoctonia is largely confined to the juvenile tissues of seedlings; hence two day germinated wheat seedlings were used in the The effect of biosurfactant on the growth of plant infected with the pathogens was monitored over a period of a month (Tables 3, 4). A preliminary test conducted with 20 seeds coated with biosurfactants at 1 mg/ml, had shown that, all seeds germinated and no toxicity was observed (data not shown here). The uninfected seedlings (control 1) of both plants grew vigorously, free of stress into healthy plantlets, while seedlings infected with pathogens (control 2) had few smaller leaves, which turned yellow/brown and wilted over time (Fig. 4).
In case of wheat, the most common symptom of Rhizoctonia disease "damping-off" i.e. non germination of severely infected seed was seen in control 2. Of the infected seedlings, 66% were killed as they emerged from soil. Infected seedlings not killed by the fungus had cankers (reddish-brown lesions). The white vegetative mycelia of R. solani were seen growing at the roots. In the TEST treated with biosurfactant from Pseudomonas spp. (BMW1), a few wheat plantlets grew initially but most of the leaves turned brown within 14 days and eventually died (same as observed in case of infected control 2). Thus biosurfactant from isolated strain of Pseudomonas spp. (BMW1) was ineffective in protecting wheat plant against Rhizoctonia infection in vivo. The TEST plantlets treated with Bacillus spp. (BMW2) biosurfactant showed a lower than average number plantlets surviving (60%) compared to control 1, stunted growth and signs of infections were visible even after 21 days whereas the biosurfactant obtained from Micrococcus spp. (BPS1) was most effective in promoting normal growth in the treated wheat plantlets (91% survivors) in spite of being infected initially by Rhizoctonia solani (Table 3). The signs of infection were also less pronounced in these plantlets.  In case of the peanut plantlets infected with Sclerotium, symptoms of infection such as yellowing and wilting of the branches, main stem, or the entire plant were distinctly visible in the control 2 plantlets and a mere 13% of the total germinated seedlings sown (which had not received any biosurfactant treatment) survived the fungal infection. Growth of the plantlets was slow for first few days in all TEST pots compared to that of uninfected CONTROL 1. But the Test plantlets receiving Pseudomonas (BMW1) biosurfactant treatment showed improvement within 7 days and healthy plant growth resumed at end of 14 days (more than 75% survivors) indicating elimination of the pathogen. When compared with control 1, Bacillus (BMW2) and Micrococcus (BPS1) biosurfactants treatment showed of less number of plants (60%) growing throughout the observation period though they were as healthy as the plantlets in uninfected control 1 (Table 4).
Similar protective antifungal properties of biosurfactants produced by strains of Pseudomonas fluorescens and other bacteria are well documented in literature. Rhamnolipids were found to decrease the incidence of water-borne damping-off disease caused by Phytophthora and Pythium in chili pepper and tomato [40]. Rhamnolipids act against zoospore producing plant pathogens by direct lysis of zoospores via the intercalation of rhamnolipids within plasma membranes of the zoospore which are not protected by a cell wall. Recently rhamnolipid and lipopeptides have proven to be efficient in protecting other in vivo plant systems like pepper plants from Phytophthora blight disease and Colletotrichum orbiculare infection on leaves of cucumber plants [41]. The lipopeptide produced by B. subtilis has also been reported to inhibit the other phytopathogenic fungi, including F. graminearum, Pythium irregulare and Cladosporium fulvum [42].
Results of our in-vivo study revealed that the extracted the biosurfactants from isolate BMW1 and BPS1 were able to protect peanut and wheat plants from fungal pathogens i.e. Sclerotium rolfsii and Rhizoctonia solani respectively and allowed healthy plant growth. Thus these phytopathogens can be controlled through direct seed treatment with biosurfactant in a single