SIRJ-APBBP Volume 2 Issue 1 (2015) www.scrutinyjournals.com ISSN 2349 - 0128 Scrutiny International Research Journal of Agriculture, Plant Biotechnology and Bio Products (SIRJ-APBBP) Optimization of salicylic acid production by Pseudomonas fluorescens for the control of Alternaria alternata leaf spot in tomato plant J. Renga Ramanujam1*, S. Kulothungan2, E. Kumaran3, S. Senthil Prabhu4, P. Arun5 and V. Shanmugaraju6 1, 3-6 2 Department of Microbiology, Dr. N.G.P Arts and Science College, Coimbatore-48, Tamilnadu, India Department of Botany and Microbiology, A.V.V.M. Sri Pushpam College, Poondi, Tamilnadu, India Article history: Submitted 19 September 2014; Accepted 29 December 2014; Published 28 February 2015 Abstract Our research work was carried out to emphasize the importance of biocontrol agents rather than control of diseases in plants through the chemicals. The most important beneficial bacteria which was considered as a suitable biocontrol agents, not only in controlling diseases but also in the growth promoting activity was Pseudomonas fluorescens. We isolated Pseudomonas fluorescens from the rhizosphere soil of tomato and they were of 10 isolates named as P1-P10. Salicylic acid production of Pseudomonas fluorescens was elucidated and also they were assessed for the influence of sugars, iron and aminoacid over the production of salicylic acid. Among these ten strains two were selected based on their antifungal activity and salicylic acid production. They were tested for growth promoting activity as well as in their disease control property through the extracts of waste materials were used to grow Pseudomonas fluorescens and salicylic acid production was analyzed. This may be a basic platform in preparing biofertilizers with Pseudomonas fluorescens, which can be used in the field without creating any environmental hazards to induce systemic acquired resistance in controlling diseases of many commercial crops. Key words: Salicylic acid, systemic required resistance, siderophores, Pseudomonas fluorescens and Alternaria alternata Corresponding author J. Renga Ramanujam Associate Professor, Department of Microbiology, Dr.N.G.P. Arts and Science College, Coimbatore-48, Tamilnadu, India Introduction It is conservatively estimated that diseases, insects and weeds together anually interfere with the production of, or destroy between 31 to 42% of all crops produced www.scrutinyjournals.com Renga Ramanujam et al., / SIRJ-APBBP 2:1 (2015) worldwide. Sunflower, Soyabean, Groundnut, Sesame and caster belong to kharif, while canola rapeseeds and mustared sunflowere and linseed are Rabi oil seed crops, which are being used to fulfill local requirements. Oil seed crops are subjected to various mechanical, physiological and biochemical stresses in all stages of growth and in all natural environments that interfere with their normal growth and development. Weather toxicants, pollutants, insects, viruses, fungi, nematodes, bacteria and seeds are primary hazards to the production. The Lycopersicum esculatum (Tomato) is an herbaceous, usually sprawling plant in the night shade family that is typically cultivated for its edible fruit, a Savory in flavor. Tomato plants typically reached to 1-3 meters (3-10 ft) in height, and have a week, woody strength that often vindes over the plants. Most cultivated tomatoes require 75 days from transplanting to first harvest for several ways before production declines. The two strains of plant growth promoting rhizobacteria (PGPR), Bacillus pumilus SE34 and Pseudomonas fluorescens 89b61, elicit systemic production against late blight on tomato and reduces disease severity by a level equivalent to systemic acquired resistance induced by phytopthora infestans or induced local resistance by chemical inducer-amino butyric acid in green house assays (Yan et al., 2002). Integrated disease management program reduces the cost of healthy cropping and the farmers can easily apply them in the field (Watterson et al., 1986). Lesions produced in the groundnut leaf by Alternaria alternata are small, chlorotic, water soaked, that spread over surface of the leaf. The lesions become necrotic and brown and are round to irregular in shape. Lesions can coalesce; give the leaf a ragged and blighted appearance. The fungus Alternaria alternata that cause spots and chlorosis in plants of many species produces Tentoxin. Alternaria is a large genus of worldwide distribution. It is a polyphagous fungus and occurs most frequently as a saprobe on dead and decaying organic materials. The characteristics feature of the genus is the production of obclavate or beaked, pigmented conidia with relatively thin longitudinal septa. Plants have evolved very sophisticated physical and biochemical mechanisms against infection by pathogens. The mechanisms of plant response to infection occur at both cellular and sub cellular levels, the physiological and biochemical basis of plant resistance to fungal, bacterial and viral pathogens has been associated with both performed and infection induced antimicrobial compounds (Soner et al., 2002). Induced resistance in plants can be split broadly into systemic acquired resistance (SAR) and induced systemic resistance (ISR) (Walters et al., 2005). SAR developed locally or systemically in response to pathogen infection, a phenomenon where by a plants own defense mechanisms are induced prior treatment with either a biological or chemical agent. The induced systemic resistance (ISR) is phenotypically similar to pathogen-induced systemic acquired resistance (SAR) in that it confers an enhanced defensive capacity against diseases caused by fungi, bacteria, viruses and nematodes. SAR is associated with the accumulation of plant pathogenesis related proteins, some of which has been demonstrated to possess antimicrobial properties. Systemic acquired resistance (SAR) induced biologically and chemically in plant, is associated with an ability to resist pathogen attack by enhanced activation of cellular defense mechanisms in plants. Pseudomonas fluorescens is a root-colonizing biocontrol strain which suppress soil-borne plant diseases caused by pathogenic fungi. Pseudomonas fluorescens 24 www.scrutinyjournals.com Renga Ramanujam et al., / SIRJ-APBBP 2:1 (2015) are gram-negative, strictly aerobic, multi-flagellated rods. They are aggressive colonizers of the rhizosphere of various crop plants and have a broad spectrum antagonistic activity against plant pathogens, such as antibiosis siderophore production and nutrition or site competition, some can also produce levels of HCN that are toxic to certain pathogenic fungi. In many crop-pathogen systems, the primary mechanism of biocontrol by fluorescent Pseudomonas spp., in production of antibiotics such as 2,4-diacetyl phloroglucinol (PHL), pyoluteorin (PLT), pyrrdnitin and phenezine-1-carboxylate. Biocontrol agents could be an alternative to chemical pesticides, with benefits to consumers, growers and the environment. Due to high costs and difficulties in application and effectiveness, only a few biological agents are used successfully against disease (Musetti et al., 2006). Root colonizing bacteria, especially flouresent Pseudomonas spp., can efficienty control diseases incited by soil borne phytopathogens (Maurhofer et al., 1994). In approaching the diseases management strategy, various concerns about environment hazards by excessive usage of fungicides, development of fungicide-tolerant pathogen strains, non-availability of both fungicides and their application technology to resource poor farmers, necessitates the development of more economical and eco-friendly alternative components of disease managements. Plants can be induced to develop enhanced resistance to pathogen infection by treatment with a variety of abiotic and biotic inducers. Biotic inducers include infection by necrotizing pathogens and plant growth promoting rhizobacteria, and treatment with nonpathogens or cell wall fragments. Abiotic inducers include chemicals which act as various points in the signaling pathways involved in disease resistance (Walters et al., 2005). Salicylic acid is recognized as an inducer of pathogen related protein (PRP) accumulation and SAR resistance when sprayed of on the plants, and it fulfills all the criteria of an activator. Salicylic acid (SA) is a phytohormone and phenol, ubiquitous in plants generating a significant impact on plant growth and development, photosynthesis, transpiration, iron uptake and transport. It also induces specific changes in leaf anatomy and chloroplast structure. Salicylic acid is another siderophore produced by Pseudomonas fluorescens important in pathogen related SAR. Accumulation of SA in the plants is required for SAR, where interestingly, under conditions of iron limitation the bacterial strains, Pseudomonas fluorescens, can produce siderophore. Siderophores, particularly salicylic acid, have been implicated in the ability of certain strains to trigger induced resistance in plants. Plants produced several secondary metabolite compound including alkaloids, cyanogenic glycosides. Glycosinolates, flavonoids, saponins, steroids and terpenoids are product themselves from the continuous attack of naturally acquiring pathogen, insect pests and environmental stress. Alternaria alternata is our primitive pathogen reckoned among various other pathogens. The destructive impact due to sabotage caused by Alternaria alternata have to be minimized by orienting an alternative mode over chemical mode which will be an appropriate option that option is going to be Pseudomonas fluorescens. Retrieving back to conventional agronomical methods with trivial manipulations is going to be the fashion of modern agriculture. 25 www.scrutinyjournals.com Renga Ramanujam et al., / SIRJ-APBBP 2:1 (2015) Materials and Methods Isolation and identification of Pseudomonas fluorescens from the soil sample The tomato plant rhizosphere soil samples were collected from different locations of a field using sterile polythene bags. Serial dilutions were done. Suspected colonies with greenish nature were looked for further confirmation as Pseudomonas fluorescens. In microscopic examination, expecting negative rods showing motility using polar flagella, the Gram staining and motility tests were carried out. In Biochemical analysis, conventional tests like IMViC, Oxidase, Catalase, Nitrate reduction and carbohydrate fermentation were performed, expecting the cultures to be citrate, oxidase and catalase positive to conform as Pseudomonas fluorescens. Growth parameters All the isolates obtained were designated as P1, P2, P3, and P4…P10. They were scrutinized under various parameters. Impact of temperature Nutrient broth was prepared separately in five conical flasks with 50ml each. They were sterilized, inoculated with loopful of Pseudomonas fluorescens and incubated at different temperature like 25ºC, 30ºC, 35ºC, 40ºC and 45ºC for 24 Hrs and observed for evaluating optimum temperature. Effect of pH Nutrient broth was prepared in 6 conical flasks with 50ml each. The pH was adjusted from 4 to 9 in each flask. The media was sterilized and inoculated Pseudomonas fluorescens and incubated at 37ºC for 24 Hrs to assess the optimum pH. Influence of sugars Nutrient broth was prepared and sterilized in five different conical flasks with 100 ml each. To each flasks 1gm of sugars like sucrose, maltose, mannitol, mannose and glucose were added and inoculated with test organism Pseudomonas fluorescens. All the flasks were incubated at 37ºC for 24Hrs and observed for maximum growth. Sorting of nitrogen sources Nutrient broth was prepared along with 1gm of Tryptone, Peptone, Yeast extract and Beef extract in four different conical flasks with Pseudomonas fluorescens and incubated at 37ºC for 24 hrs and analysed for maximum growth. Role of amino acid Nutrient broth was prepared along with 1gm of aminoacid added with 100ml each. Valine, Histidine, Alanine, Asparticacid, Phenyl, Alanine, Methionine, Cysteine, Threonine and Glycine. All the ten flasks with different amino acids were inoculated with Pseudomonas fluorescens and incubated at 37ºC for 24 hrs. 26 www.scrutinyjournals.com Renga Ramanujam et al., / SIRJ-APBBP 2:1 (2015) Effect of water Nutrient broth was prepared using three different water like tap water, distilled water and drinking water. The Medias were sterilized, inoculated with Pseudomonas fluorescens and incubated at 37ºC for 24Hrs. Effect of chemical fertilizers on the growth of Pseudomonas fluorescens About 90% of the farmers are using chemical fertilizers in the field for high productivity and yield. Chemical fertilizers like Urea, DAP complex and Potash were used for analysis. Nutrient broth of 50ml was prepared and to that above fertilizers at different concentrations 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35 gm was added individually. All the flasks were inoculated with the Pseudomonas fluorescens and incubated at 37ºC for 24 Hrs. Impact of salicylic acid on the growth of Pseudomonas fluorescens Salicylic acid (SA) is an abiotic elicitor, which is used to elicit the systemic acquired resistance in plants. SA of different concentration from 0.01M to 0.09M was added to nutrient broth, inoculated with Pseudomonas fluorescens and incubated at 37ºC for 24Hrs. Isolation of Alternaria alternata from the infected leaf of Groundnut Infected leaf was detached from the plant and it was subjected to surface sterilization with 0.02% mercuric chloride. Then the leaf was again washed with distilled water to remove excess of mercuric chloride. Czapek Dox Agar (CDA) medium was prepared, sterilized and poured onto the petriplate. The leaf was placed over the media, impregnated and incubated at room temperature for 7-8 days. Based on macroscopic appearance, as well as by microscopic appearance of conidia and conidiophores Alternaria alternata can be identified. Invitro antifungal activity Antifungal activity of Pseudomonas fluorescens (P1 - P10) against the foliar pathogen Alternaria alternata was determined by dual-culture assay. Method I Bacteria (Pseudomonas fluorescens) were inoculated as a line on one edge of a 90 mm diameter petriplate containing CDA medium and incubated at 37ºC. After 24 hours, a 5mmdiameter actively growing Alternaria alternata was inoculated at the center. Dual inoculated plates, with fungus alone as control. The inhibition zone between the two cultures was measured after 8 days of incubation at room temperature. Method II Pseudomonas fluorescens was added to the Czapek Dox broth at concentration of 5%, 10%, 15%, 20% and 25% separately in five conical flasks after sterilization of the medium. To that each flask 5mm disc of Alternaria alternata was inoculated and incubated at room temperature for 7 days. After the incubation, the dry weight of the fungus was noted. The 27 www.scrutinyjournals.com Renga Ramanujam et al., / SIRJ-APBBP 2:1 (2015) mycelial mat was filtered through Whatmann No: 1 filter paper, the filter paper containing the mycelial mat was dried in the oven at 60ºC and weighed. Salicylic acid production by Pseudomonas fluorescens (P4 and P9) Salicylic acid production of the strain was determined as per the method of Meyer et al., 1992. The strains were grown in the standared succinate medium (SSM) at 28±2ºC for 48 hrs. Salicylic acid (SA) production by the Pseudomonas fluorescens was measured in a stationary phase shake culture (48 hrs, 27ºC) in SSM, pH 7.0 that was inoculated with washed cells from a 48 hrs old SSM shake culture (Leeman et al., 1996). Cells were collected by centrifugation at 6000rpm for 5min and were resuspended in 1ml of 0.1 M phosphate buffer. 4ml of the cell free culture filtrate was acetified with 1N HCL to pH 2.0 and SA were extracted in chloroform (2×2 ml) 4 ml of water and 5 ml of 2M ferric chloride was added to the pooled chloroform phases. The absorbance of the purple iron-Sa complex, which was developed in the aqueous phase, was read at 527 nm. Qualitative SA analysis of culture supernatant was performed with TLC after the chloroform extraction. Effect of iron, amino acid, sugars on the SA production by P4 and P9 The effect of iron on the production of SA by Pseudomonas fluorescens, filter sterilized FeCl3 was added to the SSM in a linear concentration range from 0-0.5 mg and incubated for 48 hrs. Bacterial growth was measured turbidometry at 650 nm with spectrophotometer. Salicylic acid production of the strain was determined. SSM was added with the amino acids like Phenylalanine, Tryptophan, Histidine, Valine, Threonine, Glycine, Methionine and Cystine at a concentration of 0.1 gm/ml separately. The effect of these on the salicylic acid production was determined. Sugars like Sucrose, Maltose, Arabinose, Ramnose, Mannitol, Dextrose, Mannose, Fructose and Galactose were added at a concentration of 0.1 gm/ml of SSM separately. The production of salicylic acid was determined as per the method of Meyer et al., 1992. Salicylic acid from waste materials Banana skin, onion skin and beet root skin were crushed and its aqueous extracts were used for the study. In standard succinate medium (SSM) Succinate acid was substituted with these aqueous extracts. Modified SSM was prepared separately in three different conical flasks and inoculated with Pseudomonas fluorescens (P4), which was grown in SSM at 28±2ºC for 48hrs. It was incubated at 27ºC for 48hrs to assess the SA production. Green house evaluation of plant growth promoting activity of Pseudomonas fluorescens (P4-P9) P4 and P9 were evaluated for the plant growth-promoting activity and disease control on groundnut in the green house. Seeds of groundnut were surface sterilized with 0.02% (W/V) HgCl2 and washed three times with sterile distilled water to remove traces of HgCl2. Pseudomonas fluorescens were grown as a lawn on nutrient agar in 90mm diameter. Petri plates for 48 hours at 30ºC. The cells were scraped into 20ml of 0.5% carboxyl methyl cellulose (CMC) and the surface sterilized seeds were suspended in this cell suspension for 30 minutes. Bacterial seeds were dried under a flow of sterile air in laminar flow for 4-5 28 www.scrutinyjournals.com Renga Ramanujam et al., / SIRJ-APBBP 2:1 (2015) hours before rowing. The viable cell count as determined by various dilutions. Bacterized seeds with 0.5% CMC treated seeds as control were planted in the pots filled with red alfisol sand and farmyard (3:1:1). Temperature in the greenhouse was maintained at 28±2ºC and the pots were adequately watered daily. The emergence of seedlings was recorded 7 days after sowing (Rabindran et al., 2003). Result and Discussion From the 25 different groundnut rhizosphere soil, 10 strains of Pseudomonas fluorescens were isolated and they were named as P1, P2, and P3…….P10. Biocontrol activity that reiterated in evaluating role of various parameters such as temperature, pH, Sugar, Nitrogen source, Amino acid and Water. 35ºC was more conducive for the growth of Pseudomonas fluorescens and the optimum pH was 7.0 Glucose, the Pseudomonas fluorescens showed a maximum growth. Among the 4 nitrogen sources, yeast extract produced maximum growth; best result was observed in the aminoacid Threonine for P4-P9 followed by the tap water. (Fig. 1 - 2) According to Gugi (Gugi et al., 1991) the optimal temperature assessed was 30ºC but our isolates had maximum growth at 35ºC because our isolates were indigenous of tropical country soil that favor the optimum to 35ºC but other parameters was in accordance to his findings. Figure No 1: Representing the influence of various parameters like temperature, sugar, pH, nitrogen source, amino acids and water on the growth of Pseudomonas fluorescens (P1-P5) Figure No 2: Representing the influence of various parameters like temperature, sugar, pH, nitrogen source, amino acids and water on the growth of Pseudomonas fluorescens (P6-P10) The nutrient broth was added with three different fertilizers before inoculation, P4 and P9 showed a maximum growth in the media containing DAP and complex, as the concentration was increasing, the growth of Pseudomonas fluorescens also increased but not beyord 7% (Fig. 3 - 4). 29 www.scrutinyjournals.com Renga Ramanujam et al., / SIRJ-APBBP 2:1 (2015) Figure No 3: Indicating the role of fertilizers like DAP, complex, potash and urea over the growth of Pseudomonas fluorescens at 7% concentration (P1-P5) Figure No 4: Indicating the role of fertilizers like DAP, complex, potash and urea over the growth of Pseudomonas fluorescens at 7% concentration (P6-P10) But according to Sayyed et al., 2005 urea was assessed to be more yielding, chemical fertilizer in contrast to our DAP and complex. The maximum growth was seen at 0.01 M concentrations in P4 and P9 isolates. Based on the study of Subba (Subba et al., 1996) which emphasized 0.01 M as optimal concentration to induce SAR, which signifies that 0.01 M of SA as optimal (Fig. 5 -6). Figure No 5: Impact of SA on the growth of Pseudomonas fluorescens at various concentrations from 0.01M to 0.05M (P1-P5) 30 www.scrutinyjournals.com Renga Ramanujam et al., / SIRJ-APBBP 2:1 (2015) Figure No 6: Impact of SA on the growth of Pseudomonas fluorescens at various concentrations from 0.01M to 0.05M (P6-P10) In CDA and SDA surface of the colony was first grayish white, wooly and later became greenish black or brown with a light border. It eventually became covered by short, grayish, aerial hyphae. The reverse of the petriplate appeared as black colour. In the microscopic appearance, of the hyphae were septate and dark. The conidiophores were septate and variable length. Individual conidiophores were formed with bushy heads and conidia were with the short conical beak at the tip. Thus with all these macroscopic and microscopic evidences, it was confirmed as Alternaria alternata (Mehrotra, 2003). Antagonistic activity of Pseudomonas fluorescens against Alternaria alternata was tested with the dual-culture methods. Among the 10 isolates of Pseudomonas fluorescens, p4 and P9 showed high inhibition of Alternaria alternata growth in two methods when compared to other isolates (Fig. 7). 50 48 46 44 42 P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 Zone of inhibition Figure No 7: Apraisal of antifungal activity by Pseudomonas fluorescens on Alternaria alternate by dual culture methods- Method I (P1-P10) Isolates Similarlly, Pseudomonas fluorescens were screened for its antimicrobial activity against fungi including Alternaria cajani by Srivastava (Srivastava et al., 2008). Among them, Pseudomonas fluorescens showed highest inhibition percentage (81% to 100%) against Alternaria cajani. Likewise, Yogesh (Yogesh et al., 2005) also showed the result that Pseudomonas fluorescens successfully inhibited the growth of Fusarium solani causal agent of root rot in pea. He also observed that inhibition of the fungal growth by culture filtrate of Pseudomonas fluorescens was significant (60-100%) compared to control (Fig. 8-9). Mycelial growth was completely inhibited when the volume of the culture filtrate increased to 50% in the broth. 31 www.scrutinyjournals.com Renga Ramanujam et al., / SIRJ-APBBP 2:1 (2015) Figure No 8: Apraisal of antifungal activity by Pseudomonas fluorescens on Alternaria alternata by dual culture Method II (P1-P5) Figure No 9: Appraisal of antifungal activity by Pseudomonas fluorescens on Alternaria alternata by dual culture Method II (P6-P10) In connection with these results Krishna (Krishna et al., 2005) have proved that Pseudomonas spp., were highly inhibitory against 8 fungal pathogens of groundnut. Pseudomonas spp., quite often emerged as potent antagnonists in several screening programs. Broad-spectrum activity of Pseudomonas spp., contributes to their invitro antifungal activity and invivo disease control. These results fix with the results obtained by Rosales (Rosales et al., 1995), which shows that Pseudomonas spp., including Pseudomonas fluorescens were clearly inhibitory to the fungus Rhizobacteria solani. Wide zones devoid of mycelial growth were observed around bacterial colonies. The amount of salicylic acid produced by P4 and P9 was 45 µg/ml and 41µg/ml respectively. Qualitative salicylic acid analysis of the culture suprenatent was performed with TLC. With the appearance of pink colour spots in the developed plates, it was identified as salicylic acid. More over the salicylic acid separation and this analysis was performed along with the chemical salicylic acid for conformations. Leeman (Leeman et al., 1996) observed that Pseudomonas fluorescens is able to produce salicylic acid in the in-vitro growth condition. Production of salicylic acid by pseudomonas fluorescens WCS374 and WCS417 was found to be 54.6µg/ml and 7.8µg/ml respectively. The amount of salicylic acid produced in P4 was 45µg/ml and for P9 it was 41µg/ml, but at 0.1 mg FeCl3 it was 39µg/ml for P4 and 33µg/ml for P9. At 0.5 mg, salicylic acid production was reduced to 16µg/ml in p4 and 9µg/ml in P9 (Fig. 10). Thus the amount of salicylic acid production was decreased with increase in concentrations of FeCl3. Pseudomonas fluoresces secretes large amounts of salicylic acid production under iron limiting conditions in culture (Kris et al., 2002). 32 www.scrutinyjournals.com Renga Ramanujam et al., / SIRJ-APBBP 2:1 (2015) Figure No 10: Effect of iron on SA production by P4 and P9 The effect of amino acid on the salicylic acid production by P4 and P9 was studied by using different amino acids. Production of SA by P4 and P9 the amount varied depending upon the amino acids used (Fig. 11). They showed maximum salicylic acid production with phenylalanine because salicylic acid is biosynthesized from this amino acid by shikimate pathway, P4 produced 48µg/ml and P9 produced 47µg/ml. Figure No 11: Diagrammatic representations on effect of amino acids (0.1g/ml) on SA production. Cysteine is one of the salicylic acids exudates by tomato roots. So it is likely that salicylic acid is efficiently concentrated to pyochelin in the rhizosphere of tomato in the presence of L-Cysteine. This similar phenomenon had been described for Pseudomonas fluorescens, which produces salicylic acid, by influence of aminoacid at iron limiting conditions (Kris et al., 2002). The various sugars were added with SSM to which the bacteria were grown. The amount of salicylic acid produced by P4 and P9 were similar for each sugar but it varied with the type of sugar added. It showed maximum production with glucose only (Fig. 12). P4 and P9 produced 40µg/ml of salicylic acid with the amendment of glucose (Brion et al., 1999). He also proved that the combination of each mineral with either glycerol or glucose generally increased salicylic acid production. The salicylic acid production by Pseudomonas fluorescens, showed in enhancement of long surveillance any physiological properties to be sustainable one. Thus limited ones like banana skin, onion skin and beet root skin were supplemented as a substrate substituting succinic acid in SSM and analysed for salicylic acid production. Among the three, banana skin produced maximum of 42µg/ml of salicylic acid in elocation with onion and beetroot. Even though various strategies and analytical methods were adopted in our research still it is a nascent one that needs renovations, which will end in benefits. 33 www.scrutinyjournals.com Renga Ramanujam et al., / SIRJ-APBBP 2:1 (2015) Figure No 12: Role of sugars on SA production by P4 and P9 (0.1g/ml) GLU- Glucose; MAL-Maltose; ARA-Arabinose; RAM-Ramnose; MAT-Mannitol; SUC-Sucrose; MAN-Mannose; GALGalactose The growth promoting activity and disease control on groundnut in greenhouse was evaluated for P4-P9. Out of two bacterial isolates pseudomonas fluorescens (P4-P9), P4 showed the maximum growth with increase in root length and shoot length. Seed treated plants showed increased shoot and root length of about 60% more when compared with the untreated control plant. The disease severity was also reduced when the seeds were bacterized with Pseudomonas fluorescens. Figure No. 13: Analytical graphs elucidating the level of SAR induction by salicylic acid resulting in production of various phytochemical compounds Figure No. 14: Analytical graphs elucidating the level of SAR induction by Pseudomonas fluorescens resulting in production of various phytochemical compounds. Krishna (Krishna et al., 2005) have also proved that groundnut associated bacteria isolated from the rhizosphere (Pseudomonas aeruginosa) promoted the early growth of groundnut in the green house. They also showed increased shoot length of treated 34 www.scrutinyjournals.com Renga Ramanujam et al., / SIRJ-APBBP 2:1 (2015) seedlings. Maximum root length (60% over control) was observed following seed treatment with Pseudomonas aeruginosa. They effectively increased the root and shoot length by >43 and >32% respectively. Seed treatment with pseudomonas fluorescens WCS374 significantly reduced the relative percentage of fusarium wilt disease by Fusarium oxysporum in radish cropped in green house conditions (Leeman et al., 1995). In agree with this result, Meena (Meena et al., 2006) have also proved that seed treatment with Pseudomonas fluorescens strain Pf1 recorded the highest germination percentage and the maximum plant height. They also controlled late leaf spot disease of groundnut and increased the pod yield. Fresh plant treated with SA directly resulted in Acetic acid production an inducer of resistance, propanamine (CAS) is a pesticide product, Decan-1-ol is a factor that prevents tumor and necrosis (Fig. 13). Similarly fresh plant treated with Pseudomonas fluorescens (Biotic) produced 9 compounds (Fig. 14) like cyclobutanol, glycin, etanamine, N- Ethyl-NMethyl, diethyl methylamine, methyldiethylamine, benzeneethanamine, alphamethyl phenethylamine, octadecane, N-octadecane, heptanamine, 5-methyl-(CAS), 2-amino-5methylheptane. In cyclobutanol is a secondary alcohol derived from cyclobutane. Glycine it serves as additive in pet food and animal feed and buffering agent in antacids, analgesics and cosmetics. Ethylamine is widely used in chemical industry and organic synthesis. Diethyl methylamine it serves as a buffering agent in the lumen of the chloroplast in plants. Methyldiethylamine is produced by catalytic reaction of methanol and ammonia at elevated temperatures and high pressure. N-octadecane they are chemically inactive. 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