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Biocontrol Potential Of Pseudomonas Species Against Phytopathogens

Rupali Kamble1, Prerna Jadhav2, Manaswi Gurjaraffiliated to pune university

Fergusson college pune, pune, Maharashtra, India.

Abstract: The occurrence and spread of plant diseases may cause severe loss to the yield of crop and economical loss also. These plant diseases can be treated by chemical fertilizers, and may effective against disease causing agent. But the use of chemical fertilizers may lead to cause accumulation of residue, which ultimately cause to reduce the fertility of the crop field. So now-a-days there is increasing commercial and environmental demand in the use of microbe-based agents (bio-control agents) as alternative to, or in combination with, chemicals for controlling the spread and severity of a range of crop diseases. Many microbes of various genera have been proved the ability to produce secondary metabolites which could control plant diseases. Among them, fluorescent pseudomonads are currently considered as the most effective bacteria for biological control of soil and foliar diseases. Fluorescent pseudomonads enhance the plant growth parameters, and hence, they are called plant growth-promoting rhizobacteria (PGPR).All these biological properties madethe way for the use of secondary metabolites from pseudomonads as bio-control agents in agriculture. Total 24 bacterial and 4 fungal plant pathogens were obtained from infected parts of different plants for this study. Four species of Pseudomonas were isolated from rhizosphere soil of various plants, one from departmental laboratory and one from MCC- NCCS, Pune. The ability of six Pseudomonas speciesto produce secondary metabolites namely the iron-chelating agent (siderophore), volatiles (HCN) and antibiotic (Phenazine-1-carbixylic acid) acid pigmented compounds (Fluorescein and pyocyanin) and plant growth promoting hormone(IAA) were studied. Extraction and purification of metabolites was performed using TLC and liquid-liquid extraction with various solvents, quantitation of some extracted products estimated colorimetrically. All the extracted metabolites were assayed for antimicrobial and antifungal activity against isolated plant pathogens. Statistical comparison of results were done.

Keyword: Pseudomonads, PGPR, secondary metabolites, biological control, plant diseases.

I. INTRODUCTION

The ability of microorganisms to suppress plant disease have been well studied already by many workers against various plant diseases [20]. The natural examples of biological control have been attributed to the indogenous beneficial rhizosphere microflora, especially fluorescent pseudomonads. Numerous mechanisms may account for these biocontrol properties, including the production of inhibitory compounds or metabolites. Microbial metabolites such as siderophores and secondary metabolites with antimicrobial properties are considered to play a major role in disease suppression. Metabolites with Biocontrol properties have been reported from diverse members of the beneficial rhizosphere flora. however, those produced by the fluorescent pseudomonads have received the most attention. This is probably due to the abundance of this diverse group of bacteria and their obvious importance in the rhizosphere, coupled with the relative ease with which they can be genetically manipulated.

Fluorescent pseudomonads produce a variety of metabolites, many of which are inhibitory to other microorganisms and some of which are implicated in the biological control of plant pathogens. Pseudomonas sp. possesses many traits that make them well suited as biocontrol and growth-promoting agents. These include the ability to-

Grow rapidly in vitro and to be mass produced. Rapidly utilize seed and root exudates. Colonize and multiply in the rhizosphere and spermosphere environments and in the interior of the plants. Produce a wide spectrum of bioactive metabolites. Compete aggressively with other microorganisms. Adapt to environmental stresses and, InexpensiveThe identification of a link between a metabolites and the suppression of a particular disease is an on-going

goal of many research which the use of recombinant DNA technology. Some of criteria used to indicate that particular metabolite has a primary role in biological control are shown below:

Mutants defective in metabolites are unable to show inhibition of the pathogen in the laboratory. The biocontrol ability of the mutants reduced in the field. Complementation of the mutant with wild-type DNA sequences restores biocontrol ability. The purified metabolite shows fungicidal or antimicrobial properties. The metabolite may be detected in the rhizosherei. in situ when producing strains are present [29]. Aim of this paper is to evaluate the ability of metabolites produced by Pseudomonas strains in biocontrol of

phytopathogens, Extraction of secondary metabolites from Pseudomonas species, Effect of secondary metabolites on phytopathogens, Antibacterial activity and Antifungal activity.

The introduction of paper contains the nature of research work, purpose of work, and the contribution of this paper. It contains the references of the previous work done. This template is in Word document, provides authors with most of the formatting specifications required by the author for preparation of their research paper.

II MATERIALS AND METHODS

2.1 Isolation of plant pathogens:

Plant pathogens were isolated from various infected plant parts. The parts of plant which was infected by fungus or bacteria were collected and removed the infected area. approximately 1cm. Surface sterilized by using 1% HgCl 2

and washed with sterile saline. Infected area was scratched or scraped by sterile forceps or scalpel into the approximately 3ml sterile saline. Suspension was made and allowed it to stand for 24 hours. Serial dilution of suspension was carried out.

2.2 Purification of pathogens:

Pathogens from diluted suspension which was 24hr old were plated onto the Starr’s agar medium for isolation of pathogens. Pure colonies of plant pathogens were transferred onto Starr’s agar slants for further use.

2.3 Isolation and Identification of Pseudomonas species:

Four isolates of Pseudomonas were isolated from rhizosphere soil of various plants. Rhizosphere soil was collected from various plants and was used to make fine powder. Finely minced and air dried soil was used to make suspension. 1 g of soil was added into 9 ml of sterile saline. 1g of air dried, powered soil was added to 9ml of distilled water to make 10 Dilution was vigorously shaken on a magnetic shaker for 20-30 minutes to obtain uniform suspension. Serial dilutions were prepared from 10-1 to 10-9. Cetrimide agar medium was used for isolation of Pseudomonas strains. 1 ml of soil suspension from aliquot dilution (10 -1, 10-3 , 10-5 10-7, 10-9) was transferred to cetrimide agar medium and incubated at 28 + 20C for 24 hours. Individual colonies with yellow, blue-green, and brownish pigments were detected and marked. Individual colony was picked up and was transferred into fresh nutrient broth medium. Single colony was transferred of to cetrimide agar or nutrient agar medium slants to obtain pure culture and was stored in refrigerator at 40C.

Pseudomonas putida species was obtained from departmental laboratory and Pseudomonas chlororaphis was obtained from MCC-NCCS, Pune.

2.4 Detection of siderophore:

Production of siderophores by Pseudomonas sp. was assayed by the plate assay method as described by Schwyn and Neilands [15]. The tertiary complex chromeazurol S (CAS) served as an indicator. To prepare one liter of the Blue agar, 60.5 mg CAS was dissolved in 50 ml water and mixed with 10 ml Fe3+ solution. A forty-eight hour-old culture of fluorescent pseudomonads was streaked onto the succinate medium. amended with the indicator and incubated for three days. Production of siderophore was estimated calorimetrically, by taking optical density at 515nm (λmax). Un-inoculated broth with siderophore reagent served as blank.

2.5 Detection of hydrogen cyanide:

Pseudomonas sp. were grown on Tryptic-soy-agar (TSA) for production of hydrogen cyanide. Whatman filter paper strips or discs were soaked in a picric acid solution were placed in the lid of each Petri-plate. Petri-plates were then sealed with parafilm and incubated at 28°C for 48 hours. A color change was observed. Change in the color of filter paper from yellow to light brown, brown or reddish brown of the strips was served as an indication of weak, moderate or strong production of HCN.

2.6 Detection of fluorescein:

Fluorescein is water soluble, chloroform insoluble fluorescent pigment by many of Pseudomonas sp.

Pseudomonas agar F favors the formation of fluorescein. Spot inoculation was done for detection of fluorescein production for all Pseudomonas sp. tests were done in duplicates. Resulting fluorescence due to fluorescein production was observed under UV transilluminator from one plate, another plate from duplicates was used for purification by TLC with ethyl acetate: chloroform (5:5; v/v). Silica contained visibly brown colored spots were removed from the plate and were eluted using 200μl of methanol. 100μl sample was used for antifungal and antibacterial activity.

2.7Detection of pyocyanin:

Pseudomonas agar P was used for production of pyocyanin which reduces fluorescein formation [25, 17]. Spot inoculation was done for detection of pyocyanin production for all Pseudomonas sp., tests were done in duplicates. Resulting fluorescence due to pyocyanin production was observed under UV transilluminator from one plate, another plate from duplicates was used for purification by TLC with ethyl acetate: chloroform (5:5; v/v). Silica contained visibly yellow colored spots were removed from the plate and were eluted using 200μl of methanol. 100μl sample was used for antifungal and antibacterial activity.

2.8 Detection of Phenazine-1-Carboxylic Acid (PCA):

All six Pseudomonas sp. were tested for production of PCA. Pseudomonas sp. were streaked on Luria-Bertani (LB) agar plates and incubated at room temperature for 24 - 48 hours. The single colony from all Pseudomonas

strains on LB agar plate was transferred into 100 ml of modified King’s A broth (KA) and incubated at room temperature with shaker incubator (20000 rpm) for 24 hours[5].

For purification of phenazine, crude phenazine was treated in 2 steps. Initially the pH of the crude phenazine solution was adjusted to 2.5 and residues removed by centrifugation at 3,500 rpm for 15 min. After that, this solution was separated by a liquid-liquid extraction with dichloromethane. The extracted phenazine was then purified on a silica gel column,equilibrated with dichloromethane. The optimum solvent system for the silica gel column was 90 % (v/v) dichloromethane in ethyl acetate[5].

2.9 Detection of IAA:

IAA production by different Pseudomonas isolates was determined using Salkowski’s reagent. The purified and freshly grown cultures on Luria-Bertani (LB) medium were transferred into tubes containing 5 ml LB broth supplemented with 100μg L-tryptophan and were incubated at 28±1°C for 2 days. The broth was then centrifuged for 5 min at 10,000 rpm and in the supernatant equal volume of Salkowski’s reagent was added. The contents were mixed and allowed to stand in the dark at room temperature for 30 min to develop color. The Production of IAA was estimated colorimetrically, optical density was recorded at 465nm (λmax). Un-inoculated broth served as control.

Extracted IAA was then used for detection of antibacterial and antifungal activity against isolated plant pathogens.

2.10 Antibacterial activity of metabolites

2.10.a. Well diffusion method:

Petri plates containing 20ml Muller Hinton medium were inoculated or seeded with 24hr culture of bacterial strains. 100μl of the Pseudomonas extracts which were treated to get the metabolites such as siderophore, indole acetic acid were added to the wells. The plates were then incubated at 37°C for 24 hours. The antibacterial activity was assayed by measuring the diameter of the inhibition zone formed around the well. Sterile distilled water was served as control.

2.10.b. Disc diffusion method:

Muller Hinton Agar plates were prepared and the test microorganisms were inoculated by the spread plate method. Filter paper discs were soaked with the metabolites produced by six Pseudomonas sp. such as Phenazine-1- carboxylic acid, pyocyanin and fluorescein and were placed in the previously prepared agar plates. The agar plates were then incubated at 37ºC for 24hr. After incubation, each plate was examined. The resulting zones of inhibition were uniformly circular with a confluent lawn of growth. The diameters of the zones of complete inhibition were measured and recorded and sterile distilled water was used as control.

2.11 Extraction of metabolites for antifungal activity:

A bacterial culture was grown in 200 ml of LB broth for 90 hours at 30oC. The culture was harvested at 15000 rpm foe 35 minutes. The pH of the cell free supernatant was adjusted to 9.0 with 1N NaOH and extracted with 30 ml of chloroform. The pH of the supernatant was then adjusted to 3.0 with 1N HCl and extracted for times with a total volume of 120 ml of chloroform i.e. 30ml chloroform per extraction. The organic fraction was washed with water, dehydrated with unhydrous sodium sulfate, and evaporated to dryness. The residue was re-dissolved in 500μL of methanol. 20μL sample was applied to a silica gel plate and chromatography was carried out with chloroform: acetic acid (49: 1; V/V). The silica containing visibly color yellow and orange spots were carefully removed from the silica plate and the compounds were eluted using 200μl of methanol. The silica was removed using centrifugation, and 10μl of the clear supernatant was used to check the antifungal activity [24].

2.12 Antifungal activity of metabolites

2.12.a. Well diffusion method:

Pseudomonas strains which were treated for antifungal metabolites synthesis were tested for their antagonistic activity against fungal plant pathogens. Fungal isolates were inoculated into the Potato dextrose broth for enrichment. Seven day old culture of fungal isolates were taken and plated onto the Saboroud’s agar medium by spread plate method. 100μl of the Pseudomonas extracts were added into the wells. The plates were then incubated at 37°C for 48 hours. The antifungal activity was observed. Sterile distilled water was served as control.

2.12.b. Disc diffusion method:

Potato Dextrose Agar plates were prepared and the test fungi were inoculated by the spread plate method. Filter paper discs were soaked with the secondary metabolites produced by six Pseudomonas sp. such as siderophores, IAA, Phenazine-1- carboxylic acid, pyocyanin and fluorescein and were placed in the previously prepared agar plates. The agar plates were then incubated at 37ºC for 48hr. After incubation, each plate was examined. Sterile distilled water was used as control

III RESULT

3.1 Collection and Isolation of plant pathogens:

Twenty four bacterial and 4 fungal pathogens were isolated from infected parts of different plant.(Fig 1 A and B)

A B

Fig.1: A) collection of infected plant parts, B) Isolation of plant pathogens from infected plant parts

Fig.2: Isolated twenty plant pathogens

3.2 Isolation and collection of Pseudomonas species: Four Pseudomonasspecies i.e. P. fluorescence, P. infestans, P. synxantha, P. aerofaciens were obtained from rhizosphere soil of different plants from working Institute. P. putida was obtained from departmental laboratory and P. chlororaphis was obtained from MCC-NCCS, Pune.(Fig 3)

Fig.3:Isolation of 4 Pseudomonas sp.

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3.3 Detection of siderophore, its antimicrobial and antifungal activity:

Qualitative and quantitative estimation of siderophore was observed from all species of Pseudomonas.

Fig 4:Detection of siderophore.

Estimation of siderophore by colorimeter revealed that all the six isolate were produced siderophore. The highest concentration of siderophore was obtained by P. aurofaciens and P. fluorescens. (Fig 4)

Fig.5: antimicrobial activity of siderophore tested by agar well diffusion method produced by all Pseudomonas species against plant pathogens. Four representative images have been shown above which indicates that only some species are able to inhibit plant pathogens by the action of siderophore which indicates no significant results.

3.4 Detection of Hydrogen cyanide:

In the present study qualitative estimation revealed that Pseudomonas putidashowed strong production of HCN, isolate 3 shows moderate production of HCN while remaining isolates shows less to no production of HCN.(Fig 6)

Fig.6: In the present study qualitative estimation was observed which revealed that Pseudomonas putida(B&D) shows strong production of HCN, Pseudomonassynxantha (C) shows moderate production of HCN while one isolate shows no production of HCN (A: P. chlororaphis).

3.5 Fluorescein production, antimicrobial and antifungal activity:

i) ii)

Fig.7 i) : Detection of fluorescent spots by the production of fluorescien under UV transilluminator. ii) A and C shows Thin layer chromatogram by the flourescein and B shows sample containing fluorescein after scrapping the pigment from chromatogram in methanol.

B C D

BA C

A


A B.

020406080

100120140160180200

P. chloro-raphis

P. putida

P. fluo-rescens

P. synxan-thap. infes-tans

z o n e o f i n h i b i t i o n ( m m )

Fig. 8 A :Antimicrobial activity of Fluorescein B.: Graph showing antimicrobial activity of fluorescein produced by six different Pseudomonas strains. Results revealed that all plant pathogens were inhibited by fluorescein. Pathogen 9 showing maximum inhibition followed by pathogen 23, 21 and 7

respectively, while pathogen 12 showing less inhibition

0

20

40

60

80

100

120P. chlororaphis

P. putida

P.fluorescens

P.synxantha

P.infestans

P. aureofaciens

Fungal pathogens

z h o n e o f i n h i b i t i o n ( m m )

Fig.9: i) Antifungal activity of Fluorescein against fungal pathogens A) A. niger,B) A. flavus, C) M. anisopilae D) F.oxysporum respectively. F.oxysporum shows more inhibition by fluorescein, while A. flavus shows less. ii)Graph showing antifungal activity of fluorescein produced by six different Pseudomonas strains. Results revealed that all plant pathogens were inhibited by fluorescein. Fusarium oxysporum showing maximum inhibition, while A. flavus showing less inhibition.

3.6 Detection of pyocyanin, its antimicrobial and antifungal activity:

i) ii)

Fig.10: i) Detection of fluorescent spots by the production of fluorescien under UV transilluminator. ii) A and C shows Thin layer chromatogram by the pyocyanin and B shows sample containing fluorescein after scrapping the pigment from chromatogram in methanol.

i) ii)

0

50

100

150

200P. chlororaphis

P. putida

P. fluorescens

P. synxantha

P. infestans

P. aurofaciens

z o n e o f i n h i b i t i o n ( m m )

Fig.11 i) :Antimicrobial activity of pyocyanin. ii) Graph showing antimicrobial activity of pyocyanin produced by six different Pseudomonas strains. Results revealed that all plant pathogens were inhibited by pyocyanin. Pathogen 15 showing maximum inhibition followed by pathogen 23, 14 and 19 respectively, while pathogen 1 showing less inhibition.

A B C D

E

BA C A B C


3.7 Detection of phenazine-1-carboxylic acid, its antimicrobial and antifungal activity:

i) ii)

02 04 06 08 010 0

12 0

Ph yt op ath og en s

zone

of i

nhib

ition

(mm

)

Fig.17: i) Antimicrobial activity of PCA. ii) Graph showing antimicrobial activity of PCA produced by six different Pseudomonas strains. Results revealed that all plant pathogens were inhibited by PCA. Pathogen 11 and 15 showing maximum inhibition followed by pathogen 19, 14 and 9 respectively, while pathogen 6 showing less inhibition

3.8 Detection of IAA, antimicrobial and antifungal activity:

i) ii)

Fig.13 i): Detection of IAA. Quantitation of IAA. Result revealed that all the six isolate were produced IAA. The highest concentration of siderophore was obtained P.aurofaciensand P. synxantha. ii):Antimicrobial activity of IAA produced by six Pseudomonas s

050

100150200250300350 P. chlororaphis

P. putida

P. fluorescens

P. synxantha

P. in festans

P.aureofaciens

z o n e o f i n h i b it i o n ( m m )

Phytopathogens

i) ii)

020406080

100120

P. chloro-raphisP. putida

P.fluorescens

P.synxantha

P.infestans

P. aureo-faciens

Z o n e o i n h i b i t i o n ( m m )

Fungal pathogens

Fig. N - i):Antfungalal assay of IAA against plant pathogens. A) Fusarium, B) M. anisopliae, C)A. niger. ii): Graph showing antifungal activity of IAA produced by six different Pseudomonas strains. Results revealed that all plant pathogens were inhibited by IAA. A. niger showing maximum inhibition, while M. anisopliae showing less inhibition.

BCA

Fig.14 Graph showing antimicrobial activity of IAA produced by six different Pseudomonas strains. Results revealed that all the six isolates grown in culture medium containing soyflour as tryptophan source produced IAA. The highest concentration of IAA was obtained from Pseudomonas aereofaciens


DISCUSSION:

All the isolated bacterial and fungal plant pathogens were used and the activity of metabolites extracted from six Pseudomonas species have been assayed. Detection of siderophore was done, estimation of siderophore by colorimeter revealed that all the six isolate were produced sideophore. The highest concentration of siderophore was obtained by P. aurofaciens and P. fluorescens.andlowest concentration shown by P.fluorescens and P. putida. Detection of hydrogen cyanide was done qualitatively by observing the color change of filter paper strip from yellow to brown and red brown which indicates moderate to strong production of HCN. P. putidashowed strong while P. syxanthashowsmiderate production of HCN. Flourescein and pyocyanin which are water soluble and chloroform insoluble pigments, production of these pigments were observed by all pseudomonads, fluorescent spots were detected under UV transilluminator. Purification was done by TLC and detected brown and yellow spots were scrapped and dissolved and used for antibacterial and antifungal activity, results showed strong inhibition of plant pathogens. Phenazine-1-carboxylic acid showed good antimicrobial activity of PCA which may be due to acidity because PCA contains a carboxylic group in the structure. IAA production of have been detected with Salkowaski’s reagent. Quantification of siderophore was done colorimetrically. Result revealed that all the six isolate were produced IAA. The highest concentration of siderophore was obtained P. aurofaciens and P. synxantha.

CONCLUSION:

Environmental concern have focused interest on the development of biological control agents as an alternative to chemical agent, environmental-friendly strategy for the protection of agricultural and horticultural crops from phytopathogens.Pseudomonads are such proven biological control agent. All the Pseudomonas species are able to produce various metabolites such as siderophore, HCN, fluorescein, pyocyanin, Phenazine-1-carboxylic acid, IAA. These metabolites have been recorded to control wide range of phytopathogens i.e. bacteria and fungi. These having multidisciplinary approaches which may beneficial to become a bio-control agent of plant pathogens.It may be potential tool for sustainable agriculture and option for chemical fertilizers.

FUTURE PROSPECTS

Identification of isolated phytopathogenshave to be done upto genus level. Effeciency of extracted metabolites from Pseudomonas species have to be checked directly onto the living

plants. Lytic enzyme assays have to be performed for all species of Pseudomonas that have been used in present study. Formulation of Pseudomonas with its metabolites have to be made.

AKNOWLEDGEMENTThrough this acknowledgement we express our sincere gratitude to all those who have been directly or indirectly associated with this project, and have helped us make it a worthwhile experience. Our heartfelt thanks is extended to the Principal of Fergusson College and to all of our teachers, for their co-operation throughout the project.

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