CONTROL OF FUSARIUM WILT DISEASE IN COWPEA PLANT (VIGNA UNGUICULATA L.). USING SECONDARY METABOLITES PRODUCED IN BRADYRHIZOBIUM JAPONICUM

Rhizobium known for its nitrogen fixation and plant growth promoting capabilities which is symbiotically associative with legume plants. So forth Rhizobium used as biofertilizers in the agriculture. The ability of controlling plant diseases by using Rhizobium produced secondary metabolites as biocontrol agent is the current open area in the agriculture research. The nodules inhabited Rhizobium strains were selected for the production of secondary metabolites and the ability of controlling Fusarium was evaluated preliminarily by agar well diffusion assay. Four different Rhizobium were isolated, among that S1 cannot showed any inhibition, whereas S2, S3 and S4 were showed 11, 15 and 19mm of inhibition respectively. Among that S4 selected further and DNA isolated and identified using 16S rDNA gene sequencing. The sequences were submitted in genbank and got accession number MH165175. This organism was found to be Bradyrhizobium japonicum and mass cultured for compound extraction using organic solvents. The extracted secondary metabolite were purified using different chromatography techniques. The purified fractions were analyzed for the biocontrol of Fusarium sp., isolated from infected cowpea and results showed fraction 4 showed 21mm zone of inhibition. Further the selected fractions were analytically characterized to know the compounds present. Finally the purified compounds were evaluated for its biocontrol behavior against Fusaium sp., and plant growth promotion in in vitro conditions.


INTRODUCTION
Cowpea (Vigna unguiculata L.), is one of the most important legume crop grown in the tropical belt. This crop provides food, animal feed and cash for the rural population in addition to benefits to farmlands through in situ decay of root residues and ground cover due to the spreading habit of the plant. In addition, cowpea grain provides a cheap and nutritious food for relatively poor urban communities. Cowpea's high protein content, its adaptability to different types of soil and intercropping systems, its resistance to drought, and its ability to improve soil fertility and prevent erosion makes it an important economic crop in many developing countries (1). Cowpea due to its wide adaptation across the regions and soils harbours a number of diseases which considerably influence growth and productivity potential. One of the prime cowpea disease is Fusarium wilt caused by Fusarium oxysporum Schlechtend f.sp. ciceris is a major constraint to cowpea production throughout the world and particularly in the Indian subcontinent and the Mediterranean basin (2). Fusarium oxysporum can survive in soil several years by means of chlamydospores, which markedly reduce the potential of crop rotation as a disease management strategy. Efforts must be addressed toward developing new alternatives for more effective disease management. In current scenario use of chemical fertilizer for biocontrol of soilborne plant pathogens including F. oxysporum has been shifted to the option of green technology that have an agriculture importance.
Biological control agents for plant diseases are currently being examined as alternatives to synthetic pesticides due to their perceived level of safety and minimal environmental impacts. Strains of several bacterial species such as Bacillus, Pseudomonas and recently the Rhizobium group used as the biocontrol agents (1,3). As compared to the other biocontrol agents, Rhizobia offer the great advantage of symbiotic nitrogen fixation by association with legumes. Among the Rhizobium group, Rhizobium leguminosarum, Sinorhizobium meliloti and Bradyrhizobium japonicum have been used successfully against fungal pathogens belonging to the genera Macrophomina, Rhizoctonia and Fusarium. (4)(5)(6).
Induction of host defenses can be local and or systemic in nature, depending on the type, source, and amount of stimuli. Induced systemic resistance is one of the mechanisms of disease suppression by fungal pathogens by many microorganisms (7,8). Rhizobia have several mechanisms of action that allow them to control pathogens. In general, competition for nutrients, niche exclusion, induced systemic resistance and antifungal metabolites production are the chief modes of biocontrol activity in PGPR. The aim of the present work is to characterize and select Rhizobium isolates with antagonistic activity against wilt disease in cowpea as well as extraction of their secondary metabolites from selected rhizobia and their effects on Fusarium and disease development in Cowpea were assessed in vitro nursery conditions.

Sample Collection
Root from young and healthy seedling of cowpea (Vigna unguiculata L.) plants was collected from Viralimalai village, Pudukkottai district of Tamil Nadu, India. The collected samples were kept in plastic bags without affecting the root with nodules, brought to the laboratory under sterile conditions and stored in refrigerator for further processing.

Isolation and Identification of Rhizobium
Healthy cowpea nodules were detached from the root and further isolation of root nodulating rhizobia was carried out. The detached root nodules were washed in tap water to remove the adhering soil particles from nodule surface. Nodules were dipped in 0.1% mercuric chloride (HgCl2) solution for 30 sec and later washed successively ten times with sterilized distilled water to remove the traces of toxic HgCl2. Surface sterilized nodules were transferred in test tube containing 5 ml sterilized distilled water. These nodules were crushed with the help of sterilized glass rod to obtain a milky suspension of bacteroides. These were streaked on YEMA medium containing congo red. The plates were sealed by parafilm to avoid contamination and incubated at 28ºC for 7 days and make daily observations for the appearance of typical colonies of rhizobia (9). After the incubation period from the pool of well grown colonies were picked up and selected four different colonies named as S1, S2, S3 and S4. Further the cultures subjected to gram staining for the shape, size, and arrangement of bacterial isolates (10).

Biochemical Characterization and Molecular Identification
Various biochemical tests were carried out were indole test, methyl red test, Voges Proskauer test and citrate utilization tests based on standard microbiological manual in order to confirm primarily by the Rhizobium genus for all the four isolates. In order to confirm the Rhizobium genus secondarily by subjected to Bromothymol blue test and Ketolactose test, in which the Yeast Extract Mannitol was enriched with 1% (w/v) Bromothymol blue to selectively identify fast Rhizobium. All the four samples S1, S2, S3 and S4 were subjected to grow on BTB added YEM media for 48 hrs at 28 o C. Positive sample showed yellow color to acid production after incubation. Whereas in Ketolactose test, a loopful of the inoculums from a fully grown culture slant (7 days old culture) was transferred to a petri plate containing the ketolactose agar medium. After incubation for 5 days at 27 ο C the plates were flooded with a shallow layer of Benedict's solution. After pouring the plates were incubated for one hour at 30 ο C without any disturbance. The absence of yellow coloration around bacterial colonies confirms the presence of Rhizobium (11).

Molecular Identification
Overnight fresh bacterial broth cultures (2 ml) were taken and centrifuged at 10000 rpm for 5 minutes and the supernatant was discarded. To the pellet, 570 μl of TE buffer, 30 μl of 10% SDS were added, mixed thoroughly and incubated at 37°C for 1 hour. After incubation, 5M NaCl was added and mixed thoroughly by vortex. 80 μl of CTAB/NaCl solution was added, mixed thoroughly and incubated at 65°C for 10 min. Equal volume (0.7 -0.8ml) of chloroform or isoamyl alcohol was added, mixed and centrifuged at 10000 rpm for 5 min. The viscous supernatant was transferred to a fresh centrifuge tube (without disturbing the interface); equal volume of phenol/chloroform or isoamyl alcohol was added and spun at 10000 rpm for 5 min. The aqueous supernatant was transferred to a fresh centrifuge tube and 0.6 volume of ice cold isopropanol was added and incubated for 20 min at -20°C. Later, the tubes were centrifuged at 10000 rpm for 5 minutes. To the pellet, 70% of ethanol was added and centrifuged. The pellet was stored at -20°C after adding TE buffer or sterile mQ water. For running the sample, 0.8% of agarose was prepared in TE buffer. To 50 ml of agarose 3 μl of ethidium bromide was added and casted in the boat which contains comb. After cooling, the comb was removed and the sample was loaded. To 5 μl of the sample 2 μl of the gel loading dye was added and loaded in the respective well, and electrophoresis was done it 50 V (12).

16S rRNA PCR Amplification
Amplification of the 16S rRNA gene was conducted using universal primers: 27F(5'-GAGAGTTTGATCCTGGCTCAG-3'), 1495R (5'-CTACGGCTACCTTGTTACGA-3'). The final products were analyzed through electrophoresis on 1.2% agarose gel and stained with 0.5 g ml −1 ethidium bromide. The PCR products obtained were purified and sequenced using Sanger's dideoxynucleotide chain termination method and were sequences at Amnion sequencing Pvt. Ltd. India (Bangalore). The sequences obtained were aligned with previously published sequences available in NCBI using BLAST (13). The phylogenetic and evolutionary analyses were conducted using MEGA 5 software (14).

Isolation and Identification of Pathogenic Fungi
Infected stem (Brick red tissue in stem indicates wilt disease) region of cowpea plants were collected and submerged in 5% sodium hypochlorite for five minutes. After this treatment, they were extensively washed with sterile distilled water and placed on Petri dishes containing potato dextrose agar (PDA, Difco) and incubated at 22°C for 48 hrs. Morphological identification are done according to the standard taxonomic key included colony diameter, texture, colour identified in their sporulation state by staining with lactophenol cotton blue.

Identification of Potential Rhizobium 2.6.1. Antifungal Activity of Rhizobium Extracts against Fungal Pathogen
The Rhizobium strain S1, S2, S3, S4 were subjected to antifungal activity using agar well diffusion method against the Fusarium sp. on Muller Hinton Agar plates. The test fungi was spread over the agar plate using the sterile cotton swab and well were created on the agar. 100µl bacterial culture was added to into the well and incubated for 4 days at room temperature. Finally plates are observed for zones of inhibition and their diameter was measured with the help of antibiotic zone scale. Among the four isolate S3 and S4 showed better inhibition than the other isolates.

Extraction and Purification of Secondary Metabolites from Rhizobium Extracts
The Rhizobium isolates S4 was mass cultivated in Yeast Extract Mannitol broth and incubated at 37°C for 10 days with periodical shaking at 150 rpm. After the incubation period, the cultures are taken out and then centrifuged at 5000 rpm for 10 mins, the cell free supernatant was transferred to another flask. To this equal volume of different solvents such as ethyl acetate, diethyl ether, chloroform, and hexane was added and the compound was extracted using solvent extractor. The crude extracts were tested for their antimicrobial activity against Fusarium sp. The extracted compound was separated by column chromatography using silica gel as the packing material. The fraction was eluted by using chloroform: methanol: water (5:6:4) as the solvent system. Different fractions were eluted from column chromatography and checked for antifungal activity (15).

Characterization of Pure Compound
The collected fraction from Rhizobium isolate (S4) with higher antifungal activity were subjected to FT-IR (Perkin Elmer, Spectrum MRX-1 model) and also analyzed using GC-MS (model Q-Mass 910, Perkin Elmer) (16).

In vitro Seed Germination
The viability of the seeds were tested by using Tetrazollium chloride test (TZ test). The seeds were soaked in 0.1 % of 2, 3, 5 triphenyl tetrazollium chloride for few minutes and the seeds were evaluated for the viability. Sound tissues produce a normal red colour and resist the penetration of tetrazollium, and those seeds were said to be viable. After the viability test cowpea seeds were sterilized by using 70% ethanol followed by 0.1% mercuric chloride before rinsing with sterilized water. The experiments were carried out in a positive, negative control and preventive method. Positive control (cowpea seed+ pathogen + carboxy methyl cellulose), negative control (cowpea seed+ distilled water + carboxy methyl cellulose) and preventive test using 5 dose of fractions from nodule compound as well as from the Rhizobium culture compound (10, 20, 30, 40, and 50 µl) on seed (cowpea seed + pathogen + compound doses + carboxy methyl cellulose). The seeds were coated with respective materials and air dried overnight. The seed were placed on a sterile filter paper in petriplates. Water is sprinkled at regular intervals and kept at a place receiving sunlight for 7 days. The germination rate was assessed in triplicates after 7 days of incubation using the formula below (17).

Collection of Nodule and Isolation of Rhizobium
Healthy cowpea (Vigna unguiculata) plants were carefully uprooted from the cowpea field, Viralimalai village, Pudhukottai district of Tamil Nadu, India. The roots along with nodules were stored in sterile polythene bags at 4 ο C for further analysis. The surface sterilized root nodules were crushed and the suspension was streaked over the YEMA medium with the addition of congo red. Growth of large opaque mucoid elevated glistening colonies in the medium shows the presence of Rhizobium. Four different colonies S1, S2, S3 and S4 were selected for further study (Fig. 1).

Identification of Rhizobium 3.2.1. Morphology of Isolates
All the four isolates S1, S2, S3 and S4 were found to be circular, mucoid and raised colonies. The shapes of the isolates were identified by the staining techniques. Colony size, shape, margin, elevation and opacity of all the four bacterial isolates were tabulated (Table 1).

Biochemical characterization
The biochemical characterization is carried out to identify the genus of the isolated bacteria and along with two confirmatory tests ( Table 2). All isolates were streaked on Bromothymol blue added YEM selective media for further confirmation. Strains S1, S3, S4 showed growth in 2 days and turned YEM media from blue to yellow confirming their nature of being fast growers and acid producers whereas strain S2 was a slow grower and acid producer (Fig. 2). Whereas in the ketolactose test the plates were flooded with Benedict's reagent and incubated for one hour. The excess reagent is drained off and the plates were checked for colour change. All the four isolates used lactose from the medium and showed growth. No yellow colourations were found around the colonies after adding Benedict's reagent. Absence of yellow colouration around the colonies denotes the presence of Rhizobium.

Molecular Identification
The Rhizobium isolates having higher antagonistic S3 and S4 were found to be the potential isolates for acting against the fungal pathogen. Hence the genomic DNA was isolated for those particular two isolates (S3 and S4) by using C-TAB method. The isolated DNA was identified using 0.8% agarose gel followed by observation on ultraviolet trans illuminator which revealed sharp high molecular weight bands of DNA. This indicated that the DNA was of good quality and suitable for PCR analysis.

Fig. 2. Bromothymol blue test and Ketolactose test Notes:
In the first plate colour change of the medium from blue to yellow shows the presence of fast growing acid producers and in the second plate addition of Benedict's reagent does not cause any change in the medium.
The DNA of the bacterial isolates S3 and S4 were amplified by performing 16S rRNA gene of the isolated DNA using forward primer 27F (5'-GAGAGTTTGATCCTGGCTCAG-3') and the reverse primer 1495R (5'-CTACGGCTACCTTGTTACGA-3'). The conformation of amplification was done by agarose gel electrophoresis by running 5μl of PCR reaction mixture on 1% agarose gel. The amplified product was sequenced in AMNION Pvt. Ltd., Bangalore. The obtained sequences were compared with NCBI databases through BLAST analysis. 16S rRNA gene of the bacterial isolate S4 showed 99.9% similarity with the Rhizobium genus. Then the sequence was submitted in Gene Bank for the accession number MH165175 and the organism found to be as Bradyrhizobium japonicum.

Isolation and Identification of Pathogenic Fungi
The wilted stem of cowpea plant showed the presence fungal growth in the PDA plate. The fungal colony is macroscopically and microscopically identified (Fig. 3). Macroscopical colony morphology shows cottony mycelium with pale brown to yellowish brown zonation. The isolated fungi subjected to lactophenol cotton blue staining were observed under microscope. The spore formation and hyphal structures were identified and confirmed to be Fusarium strain. The staining shows the presence of sickle shaped conidiospores and septate spores which is the characteristic feature of Fusarium sp. The isolated Fusarium strains were stored at 5°C in tubes containing PDA.

Identification of Potential Rhizobium Antifungal Activity by Agar Well Diffusion Method
All the isolates were cultured in YEM broth and the supernatant was collected by centrifugation. The supernatant was then tested for antifungal activity against Fusarium sp., (Fig. 4.). The zone of inhibition was found around the wells containing the supernatants. There was no zone clearance found around the S1 supernatant, whereas the other three cultures showed zone of inhibition against the pathogen. Among them S2 showed 11mm, S3 a b c showed 15mm and S4 showed 19mm of inhibition against Fusarium sp., Fig. 4. Antifungal test of the culture supernatants, extraction from Bradyrhizobium japanicum against Fusarium sp. Notes: a. S1, S2, S3 and S4-Culture supernatants, b. F1-F5-Rhizobium Fractions, C-Ethyl acetate

Extraction of Secondary Metabolites from Bradyrhizobium japanicum
The collected supernatant from the isolates S3 and S4 were extracted with equal volume of different solvents such as water, ethyl acetate, diethyl ether, chloroform, and hexane. The crude extract is tested for antifungal activity by agar well diffusion method. Among the two isolates the crude extract of S4 isolate obtained from ethyl acetate extraction showed maximum inhibition of 19mm and S3 by 12 mm of inhibition than in the other solvents used (Fig.4). So the ethyl acetate extract of S4 only taken for further purification of the secondary metabolite using column chromatography.

Purification of the Secondary Metabolite from Bradyrhizobium japanicum by Column Chromatography
In column chromatography 10 different fractions were collected at regular time intervals to get pure compounds. All the obtained fractions were tested for antifungal activity by agar well diffusion method in MHA plates and among them, fractions F4 and F5 showed inhibition against Fusarium. The zones of inhibition were measured to be 21 and 18mm for F4 and F5. The pure extract F4 were dried and further subjected for structure elucidation.

Characterization of extracted compounds
Purified compounds further characterized using FT-IR, IR spectrum and The FT-IR spectrum showed various peaks at different wavelengths which indicate the presence of various chemical groups in the extracted sample (Fig. 5). Several chemical groups also existed in the extracts from  (Fig.3). GC-MS results of bacterial extract compound showed that major peak formed at the retention time 4.235, 17.2 and 22.5 were Propenemide , Hexadecanoic acid methyl ester and 1,2 benzenedicarboxylic respectively which has responsible for antimicrobial and antifungal character (Fig.6).

In vitro Seed Germination in cowpea seeds
Before the seed germination test the seeds subjected to Tetrazolium chloride test (TZ test) to check the viability of the seeds. Color change of seeds from normal to red colour indicates that the seed is viable various color change into brown indicates dead tissue. After 7 days of incubation, the plant indexes such as root length, shoot height, and germination percentage have been observed and compared with positive and negative control (Fig. 7) ( Table 3).

. In vitro seed germination in cowpea seeds
Notes : a -Tetrazollium chloride seed viability test, b -Seeds treated with different concentration (10,20,30,40, and 50 µl) of the compound obtained from Rhizobium. c -Positive control (seed + pathogen) and negative control (seed alone), d -Germinated seeds In comparison it is clearly seen that the compound untreated seeds show less or no growth as they cannot combat with the pathogen. But the compound treated seeds had grown well combating the pathogen. Moreover as the concentration of the