Surface active compounds produced by microorganisms are divided into

5 Biocontrol activity of Biosurfactant 5.1 Preamble Surface active compounds produced by microorganisms are divided into two main types. Those that reduce surface tension at air water interface (biosurfactants) and those that reduce the interfacial tension between immiscible liquids or at the solid liquid interface (bioemulsifires). Some biosurfactants are a suitable alternative to synthetic medicines and antimicrobial agents and may be used as safe and effective therapeutic agents (Banat, 2000 and Singh and Cameotra, 2004). A common mechanism underlying the direct antagonistic activity towards soil pathogens by Pseudomonas is the production of antifungal compounds.these antifungal compounds include the secretion of antibiotics and siderophores (Dwivedi and Johri, 2003). In the present investigation, the role of biosurfactants on inhibition of fungi and insect control was studied. Pseudomonas sp. are reported as biocontrol agents against Verticillium microsclerotia, a causative agent of Verticillium wilt prevalent in solanaceae plants. Biosurfactants are surface active compounds produced by a variety of microorganisms. The mode of action of biosurfactants in biological control involves the formation of channels in the cell wall and perturbations of the cell surface of the pathogen (Raaijmakers et al., 2006). The best-known biosurfactants in biological control are cyclic lipopeptides (Raaijmakers et al., 2006) and rhamnolipids (Stanghellini and Miller,1997). For example, Pseudomonas CMR12a produced a cyclic lipopeptide responsible for the control of Pythium and Phytophtora spp. (Perneel 2006), while Ps. aeruginosa PNA1 produces rhamnolipids (Perneel, 2006).Antifungal properties of biosurfactant produced by strains of Pseudomonas fluorescens is well documented in literature (Nielsen and Sørensen, 2003). Hultberg et

al., 2008 have reported that fluorescent pseudomonads with the biosurfactant producing ability can inhibit the growth of fungal pathogens such as Pythium ultimum (causative agent of damping off and root rot of plants), Fusarium oxysporum (causes wilting in crop plants), and Phytophthora cryptogea (causes rotting of fruits and flowers). The biosurfactant produced by this Pseudomonas sp. is considered to play major role in inhibition of in vitro viability of Verticillium sp (Debode et al., 2007). Strains of Pseudomonas sp. terminate the growth of pathogenic fungus Rhizoctonia solani (causes several plant diseases) and Phythium ultimum (causes damping off and root rot of plants) by production of dual functioning compounds tensin, viscosin and viscosinamid. The dual function includes biosurfactant and antifungal activity (Andersen et al., 2003). Colletotrichum gloeosporioides, causative agent for anthracnose on papaya leaves is reported to be controlled by biosurfactant producing Bacillus subtilis isolated from soil (Kim et al., 2010). The biosurfactants which have antagonistic properties against phytopathogens may also affect the other flora of the system. Thus, to construct a potent green surfactant with specificity against the phytopathogens, the chemical composition of the biosurfactant may be varied by altering the production strategies. The concept was biosurfactants are involved in the biological control activity of other plant pathogens by the bacterial strains was also proved by the work done by (Anjaiah et al., 1998; Woeng et al., 1998 and Tambong and Hofte 2001). Surfactins and iturins are lipopeptides produced by Bacillus subtilis Cohn (Delcambe, 1965). The general structure is a peptide ring of seven amino acids linked to a fatty acid chain. The length of the fatty acid chain can vary from C-l3 to C-16 for surfactins

and from C-14 to C-17 for iturins, giving different homologous compounds and isomers (n, iso, anteiso) for each lipopetide (Besson et al.,1977 and Peypoux et al., 1973 and Peypoux et al., 1984 andpeypoux et al., 1991). Surfactins have high surface and interface activity (Deleu et al., 1999; Maget-Dana and Ptak, 1992 and Razafindralambo et al.,1998) and develop different important biological properties including antiviral, antibacterial, and hemolytic activities (Bernheimer and Avigad, 1970 and Vollenbroich et al., 1997). The biological activity of surfactin was studied by many researches (Gallet et al., 1999; Grau et al., 1999; Maget-Dana and Ptak, 1995; Sheppard et a1.,1991and Thimon et al., 1993). Likewise iturins were successfully tested in different veterinary applications (De Keyser et al., 1960) for their antimycotic properties. More recent studies (Pusey et a1., 1988 and Pusey and Wilson, 1984) showed that Bacillus subtilis could be a potential biocontrol agent of crop diseases produced by harmful fungi and bacteria. Gokte and Swarup (1988) also showed that cells and spores of B. subtilis were toxic to nematodes Meloidogyne incognita, Heterodera cajani, Heterodera avenae and Anguina tritici. Moreover, Lazare et a1. 1996 reported the potential insecticide activity of spores and metabolites of B. subtilis on Drosophila melanogaster and their oviposition reduction ability on Callosobruchus maculatus.. Their activity was compared to those of classical detergents in order to determine the relationship between the surface active properties and the insecticidal activity.

5.2 Materials and Methods 5.2.1 Microbial Cultures and Growth Conditions The biosurfactant was assayed for antifungal activity against the plant fungal strains obtained from TNAU, Coimbatore. This fungi were grown on PDA plate at 28 o C and maintained with periodic sub culturing at 4 o C. Composition of Potato Dextrose Agar (PDA) Potato - 250g Dextrose - 15g Agar - 18g Chloramphenical - 0.5mg Distilled water - 1000ml ph - 6.7 The potato tubers were peeled off and weighed for about 250g tubers were chopped in to small pieces in to the sterile conical flask. After boiling the supernatant were collected and dextrose (15g) with agar (18g) to dissolve the ingredients. The medium was mentioned and adjusted to 6.5pH. Finally the medium was sterilized in autoclaved for 20min. and ph was checked again and adjusted. 5.2.2 Antifungal activity test Antifungal activity was screened by agar well diffusion method (Perez et al., 1990), against eight plant fungal pathogens Macrophomia phaseolina, aspergillus flavus, Rhizoctonia solani, Alternaria alternate, Aspergillus fumigates, penicillium

oxallicum, Fusarium oxysporum and Verticillium microsclerotia. The PDA medium was poured in to the sterile petriplates and allowed to solidify. The test fungal culture was evenly spreeded over the media using sterile cotton swab.then wells (6 mm) were made in the medium using sterile cork borer. 50µl of each extract was transferred in to a well and. 50µl of sterile distilled water was added in to another well. The plates were incubated at 27 C for 48-72 hrs. After the incubation the plates were observed for formation of clear incubation zone around the well which indicated the presence of antifungal activity. The zone of inhibition was calculated 5.2.3 Minimum inhibitory concentration (MIC) Minimum inhibitory concentration of the biosurfactant obtained from Pseudomonas stutzeri was determined using agar well diffusion method. The biosurfactant was tested for the antifungal activity against phytopathogenic fungi on potato dextrose agar (PDA) plates. On agar surface 6mm wells were made and 20-80µg/ml range of biosurfactant dissolved in sterile distilled water was inoculated into separate wells. Antagonistic activity was detected, after an incubation period of 2-3 days at room temperature (28±2 ºC). The presence of zone of clearance on agar plates was used as an indicator for the antifungal activity. The MIC value refers the lowest concentration of the compound (biosurfactant) that completely inhibited the growth of the pathogenic fungi.

5.2.4 Toxicity Bioassay Rhamnolipid was dissoluted in bicarbonate buffer 0.1M and incorporated to the insect artificial diet at different concentrations. A control was performed with bicarbonate buffer. Thirty unsexed insect larvae were placed into each 50ml-jar containing 10 g of artificial diet mixed with purified rhamnolipid and closed with wadding. The effects of rhamnolipid on larval and adult mortality were observed. Larvae of Leucinodes orbonalis and adults of Henosepilachna vigintioctopunvtata were examined in the present investigation and the concentrations used were 20g, 40g, 60g and 20g/ml. After 48hrs number of insects killed was counted and percent mortality was calculated.

5.3 Results When tested against eight plant fungal pathogens antifungal activity of rhamnolipid biosurfactant was noted only in two pathogens namely Fusarium oxysporum and Verticillium microsclerotia (Fig.15 and Tab. 5). The minimum inhibitory concentration value was 60g/ml for both (Fig. 16 and Tab. 6). Rhamnolipid toxicity to Leucinodes orbonalis and Henosepilachna vigintioctopunctata at a concentration of 25 µl to 100 µl showed high toxicity on adult insect (Tab. 7 and 8). The Rhamnolipid 100 µl concentration resulted 67.8 % larvae of Leucinodes orbonalis mortality (Fig. 17). Whereas rhamnolipid 100 µl showed 48.5% adults Henosepilachna vigintioctopunctata mortality (Fig. 18). after 48 h r s exposure. It appeared that rha mn olipid inhibited significantly

Fig. 15 Effect of rhamnolipid on the plant fungal pathogens Macrophomia phaseolina Aspergillus flavus Rhizoctonia solani Alternaria alternate Aspergillus fumigates Penicillium oxallicum

Fusarium oxysporum Verticillium microsclerotia Fig. 16 MIC on the plant fungal pathogens Fusarium oxysporum Verticillium microsclerotia

Fig. 17 Effect of rhamnolipid on the Larvae of Leucinodes orbonalis T 1-20g/ml, T 2-40g/ml, T 3-60g/ml, T 4-80g/ml, T 5- Control

Fig. 18 Effect of rhamnolipid on the Henosepilachna vigintioctopunctata T 1-20g/ml, T 2-40g/ml, T 3-60g/ml, T 4-80g/ml, T 5- Control

Tab. 5 Effect of rhamnolipid on the plant fungal pathogens Plant Fungal pathogen Result Macrophomia phaseolina - Aspergillus flavus - Rhizoctonia solani - Alternaria alternate - Aspergillus fumigatus - Penicillium oxallicum - Fusarium udam + Verticillium microsclerotia + Tab. 6 MIC on the plant fungal pathogens of Fusarium oxysporum and Verticillium microsclerotia Concentration of rhamnolipid (g/ml) Fusarium oxysporu Verticillium microscleroti 20 - - 40 - - 60 12mm 9 mm 80 15 mm 11mm

Tab. 7 Effect of rhamnolipid on the Larvae of Leucinodes Orbonalis Treatments Concentrations of rhamnolipid (g/ml) Mortality Rate (%) T 1 20 12.8 T 2 40 36.6 T 3 60 55.2 T 4 80 67.8 T 5 Control - Tab. 8 Effect of rhamnolipid on the Henosepilachna vigintioctopunctata Treatments Concentrations of rhamnolipid (g/ml) Mortality (%) Rate T 1 20 13.3 T 2 40 24.6 T 3 60 37.2 T 4 80 48.5 T 5 control -

5.4 Discussion In the present study among plant fungi tested, two of them namely Fusarium oxysporum and Verticillium microsclerotia were found to be inhibited by the rhamnolipid biosurfactant produced by Pseudomonas stutzeri. The present study is important as it deals with the biocontrol of biosurfactant in which not many research studies are available. Though previous studies were on biocontrol of Pseudomonas spp., the biosurfactant mediated biocontrol is an emerging idea of using microbial product for biocontrol rather than using the organism itself. In this regard, the present study deserves appreciation. To the knowledge of the researcher, nobody had tried biosurfactant so for on Fusarium oxysporum,an important fungal pathogen causing wilt disease,especially in the plants belonged to the family Solanaceae. This fungal pathogen is soil born and capable of living in soil for many years without host. Disease development is favoured by warm temperature (27-28 0 C), dry weather and acidic soil (ph 5-5.6). The fungus can be disseminatd by infected seed or transplants grown in infested soil, the fungus can be introduced in to a field by a contaminated equipment,or through human beings especially through their shoes, cloths etc., Soil particles from infested fields may be blown in to disease free fields. Fusarium and Verticillium wilt are responsible for dramatic yield losses in many crops all over the world (Pegg and Brad,2002). Verticillium dahlia and V.longispourm produce melonized resting structures known as microsclerdia,which can survive for more than a decade in soil (debode et al.,2007). In the past, studies on biological control of Fussarium and Verticillium have focused on the use of antagonistic bacteria like Pseudomonas spp.,and fungus like Talaromyces flavus.

According to Fravel et al., 1987and Fahima et al., 1992,production of glucose oxidase by the antagonist converts glucose to hydrogen peroxide which kill the Verticillium microsclerotia in vitro condition.leben et al.,1987, Berg et al.,2001, and Mercado-Blanco et al., 2004 targeted Verticillium wilt,using Pseudomonas spp., and these studies have specifically targetd the suppression of hyphal growth, which in turn reduce the effect of disease damage. A common mechanism underlying the direct antagonistic activity towards soil pathogens by Pseudomonas spp is the production of antifungal compounds. These antifungal compounds include the secretion of antibiotics and siderophores (Dwivedi and Johri, 2003) and lytic enzymes (Velazhahan et al.,1999).to date nobody had tried biosurfactant mediated Fusarium control. However Debode et al., 2007 showed biosurfactant deficient mutant of Pseudomonas could not control Verticilium microsclerotia. Mule and Bharthena (2012) worked on the control as Aspergillus parasiticum using an uncharacterized biosurfactant extracted from Sternophononas maltophila In the present investigation, MIC estimation showed that both fungi were inhibited at the level of 60-80µg/ml of the rhamnolipid. Mule and Bharthena (2012) showed a direct biosurfactant application at 10% concentration eliminate Aspergillus parasiticium infection in potato.compared to this, the MIC value observed in the present study seemed to be meagre and hence the rhamnolipid obtained might be more potential. They observed spore lysis by the biosurfactant. However their study as well as the present investigation could not report the exact mechanism of biosurfactant action against pathogenic fungi. However the surface

tension reduction ability may result in dissolution of cell wall and leakage of intracellular materials leading to death of the cells. The inhibitory activity of biosurfactant may be due to its surface active nature active on cell surface. They can act on lipids and are able to form pores in the membrane layers (Kim et al., 2004). According to Gordee and Porter (1961) majority of the storage compounds in Verticillium microsclerotia are of lipid in nature, biosurfactant might have acted on those compounds and deplete their stored nutrients leading to death. Debode et al., 2007 observed reduced germination of spores which might be due to this reason. Regarding fungal control, in nature apart from biosurfactant many other secondary metabolites like antibiotics, pigments, lytic enzymes might be contributing and this might be the reason that among ten pathogens tested only two were inhibited by the biosurfactant produced by P.stutzeri. Perneel (2006) opined that in potato tubers phenazine might be acting synergistically with biosurfactant leading to control of wilt disease caused by of Fusasium and Verticillium the result observed in the present study are practically important as once the plant become affected by these pathagenes, nothing can be done (i.e) so far no control strategy is available. So far no chemical (or) natural plant product had been reported to be active on these pathogens. The results of the present study were encouraging in this regard. Apart from direct inhibitory activity, the biosurfactants may also enhance the solubility of other secondary metabolities and spread them on the surface of plants due to which the chance for availability of those substances to the affected plant or plant parts would be more. Due to their permeabilizing ability into the cells through

cell membranes the biosurfactant may facilitate other metabolites to enter in to the plant cells. Apart from this when the control organism is a biosurfactant producer, it many help the adherence of the organism on the surface of the plant as well as on the surface of the patheogens when they are selectively sprayed on the infected parts or parts. Being adhesive in nature, it may be carried through insects and facilitate the spreading of the biosurfactants, as well as the other metabolities soluble in them. One or more above mentioned mechanisms may be involved in the control of fungi by biosurfactant tested. However the selective inhibition of these two fungi alone, deserves further research to test the activity of biosurfactant on specific chemical molecular present in these study. Stanghellini and Miller (1997) showed that rhamnolipid produced by strains of Ps.aeruginosa was highly effective against plant pathogens including Pythicum aphanider matum, Plasmopara lactucalradicus and Phytopthora capsici. In the present investigation the rhamnolipid produced by P.stutzeri showed pronounced insecticidal effect against Leucinodes orbonalis larvae as well as on adults of Henosepilachna vigintioctopunctata. The mortality rate was seemed to be concentration dependent. When 20-80g/ml concentration of rhamnolipid was tested against Leucinodes orbonalis larvae it resulted in 12.8% to 67.8% of mortality. In Henosepilachaa vigintioctopunetata when the same concentrations were tested the motality rate was between 13.3% to 48.5% (i.e) Leucinodes orbonalis larvae were more affected compared to the adult Henosepilachna vigintioctopunctata as larvae might be more susceptible than adults.

Ghribi et al., 2011 showed the larvicidal potential of B.subtilis SPA1. biosurfactant against the olive moth Prays oleae larvae. The degeneration of gut cells led to the larval death. The present study clearely indicated the potential of rhamnolipid biosurfactant as a fungicide and insecticide. Leucinodes orbonalis is a Lepidopteran insect, a potential brinjal fruit and shoot borer. Henosepilachna vigintioctopunctata is a serious pest attacking solanaceae plants especially tomato and brinjal. Ghribi et al., 2011 showed that b.thuringiensis cry toxin and B. subtlis biosurfactant resulted in similar histopalogical effects on lepidopteron larvae. The concentration of biosurfactant used by them was comparable to the present study. To the knowledge of the researcher, so for nobody had worked on a coleopteran insect. The present study is the first report on effect of rhamnolipid biosurfactant on coleopteran insect.