Introduction

Fusarium oxysporum was considered as the most important phytopathogenic and toxigenic genus of filamentous fungi worldwide (O’Donnell, 1996; Langseth et al.,   1999; Eskola et al., 2001; Kosiak et al., 2003; Zhang et al., 2007; Suga et al., 2008). Fusarium wilt caused by Fusarium oxysporum (Schlecht. Emend.Snyder& Hansen) leads to significant loss to crop yield and quality of nuts in groundnut plants. Cellulose    is the main component of the plant cell wall. It is synthesized by plasma membrane protein complexes. It is deposited directly on the wall in a directional way (Taylor N.G 2008). It is a linear homopolymer    composed of β (1→4)   linked d-glucose units.  Cellobiose is a glucose dimer linked by β (1→4) and found as repeating unit of cellulose. It is found that plant pathogens produce the exocellular enzymes like cellulolytic, hemi cellulolytic, pectinolytic and proteolytic enzymes which degrade the major components of Cellwall (Wheeler, 1975; Riou,1991). Most pathogens capable of producing more cellulolytic than pectolytic enzymes (Sadik et al., 1983). The chemistry of   cellwall affects by enzymes produced by pathogens and further leads to cellwall degradationI (Albersheim and Jones 1969; Miettinen- Oinnonen and Suominen 2002). The production of cellulolytic enzymes and degradation of   cellulose by several fungi has been reported by many workers, (Basu and Ghose 1960; Bateman 1964; Sohaila et al., 2011).  The studies reported that most of the plant pathogenic organisms are capable of degrading cellulose by producing a cellulose complex which involves the synergistic action of three main enzymatic complexes.¹ endo-β1-4-glucanases (endo-β1-4-d-glucan 4-glucanohydrolase,² exo-β1-4-glucanase or cellobiohydrolase (exo- β1-4-d-glucan 4-cellobiohydrolase, and³ β-glucosidase or cellobiase.  These components act synergistically to hydrolyze cellulose to glucose. Endo-glucanase hydrolyzes internal β-1,4 linkages of cellulose chains and creates new reducing and non-reducing ends. Exoglucanase cleaves disaccharide cellobiose from the non-reducing end (cellobiohydrolase). In some cases, it cleaves from the reducing end (cellobiosidase) of the cellulose chain. These cellobiose units and short-chain cello dextrins are hydrolyzed by β-glycosidase into individual monomeric units of glucose (Nelson 1994; Moreria 2005; Beguin and Aubert 1994).  Cellulases is associated with pathogenicity of number of microorganisms (Jan and Chen 2003).  There are many studies on biological control of soil borne diseases caused especially by Fusarium oxysporum  (Marois et al., 1981; Sivan and Chet, 1986).  It was successfully demonstrated that biological control of Fusarium wilt by Trichoderma spp and Pseudomonas spp Saravanan et al., 2003; Tu and Chang 1983; Duijff   et al., 1999). Combined application of different biocontrol agents has improved disease protection (Jetiyanon and Kloepper 2002). Studies on banana demonstrated that biocontrol agents in different combinations have improved plant growth parameters than individual treatments by reducing the Fusarium oxysporum f.sp. cubense infection in field conditions. (Sukhada et al., 2010). Abeysinghe (2009) suggested that   combination of B. subtilis with   Pseudomondas strains can lead to greater plant protection against R. solani and S. rolfsii than individual application. Combining   antagonists plays   more effective biological control treatments of infection rather than individual antagonists (Janiesiewicz 1996). Weller (1994) found that combinations of several fluorescent Pseudomonads have greater   biocontrol activity against take all of wheat as compared to application of single strains. Wahid (2006) reported that combination of biocontrol agents gave better results than using them singly.  Talaviya   and Jadega (2015) established that combined application of T.viride+T.harzianum+  Pseudomonas fluorescens was found  most effective in controlling  cumin wilt disease and also had highest seed yield. There seems to be no report on the control of Fusarium wilt with the combinations of biocontrol agents on Arachis hypogaea.L. The present study was taken to determine the significant interactive effects of Trichoderma spp and Pseudomonas fluorescens   in biocontrol of Fusarium wilt of   Arachis hypogaea L. in comparison with application of single strains.

Materials and Methods

Microbial Cultures

The bacterial and fungal cultures used in the study were obtained from Institute of Microbial Technology (IMTECH), Chandigarh. Fusarium oxysporum was cultured on Potato Sucrose Agar(PSA) for 30 days. Trichoderma viride and Trichoderma harzianum were cultured on Malt Extract Agar(MA) and Pseudomonas fluorescens   on Antartic biotic Medium(ABM) for 30 days. All these cultures further   grown on Czapek’s medium separately for 7 days at 28°±0.2C. The culture filtrates were taken after centrifuged.

Enzyme Production

For the assay of 1,4 β – endoglucanases, Czapek’s broth was supplemented with carboxy methyl Cellulose and for 1,4- β exo glucanases microcrystalline cellulose was used. Culture filtrates of Tv+Pf (1+2%), Th+Pf (1.5+2%) Tv+Th (1+1.5%) Tv+Th (1+1.5 %) in their OIC (Optimum Inhibitory Concentration) were amended to 50ml Czapek’s liquid media separately. The two discs of 9 mm of F. oxysporum was inoculated in each flask and incubated in the BOD incubator at 28°± 0.2C for 7 days. The control (with Fusarium oxysporum) and treated flasks (Tv+Pf, Th+Pf, Tv+Th) were all maintained in triplicates. After incubation, the fungal mat and the liquid media were separated by Whatman No.1 filter paper.  The filtrates were further centrifuged at 5000 rpm for 10 min and the supernatant was used as the enzyme source.

Assay of 1,4 -β – Endoglucanase  (C_x) Activity

The activity of   1,4 endoglucanases was assayed as per the method described by Mahadevan and Sridhar (1986) and the enzyme activity was calculated as percent reduction in viscosity of the substrate. The reducing sugars released by the enzyme sources in the same reaction mixture was estimated according to Wang et al., (1997).

Substrate Preparation

Carboxyl Methyl Cellulose (0.5g) was dissolved in 100 ml of sodium acetate-acetic acid buffer with pH 5.2 and kept in water bath at 50-60°C for 5-10 min.  The   substrate was   filtered   and   stored at 4°C with a layer of toluene.

Ostwald viscometer 150 size was used to determine the viscosity loss of cellulose substrate.

4 ml of carboxyl methyl cellulose, 1 ml of the buffer and 2 ml of enzyme was pipetted out into the viscometer. The contents were mixed well and the efflux time of the mixture was determined at every 30-min interval for 3 h incubation   and   the enzyme   activity   was   calculated   from  the  following   formula.

T₀-T₁

V=     ______× 100

T₀-T_W

Where V = percent loss in viscosity          T_o = flow time of reaction mixture at 0 minute

T₁= flow time of reaction mixture at a particular time interval   T_w = flow time of distilled water 

Assay of    1,4  -β – Exoglucanase  activity

1,4- β-Exo glucanase activity was measured by estimating the reducing sugars released by the breakdown of avicel   with   anthrone reagent.  To 1 ml of enzyme source, 1 ml of buffer and 0.5 ml of substrate were added in a test tube and incubated at room temperature for 2 h. The reaction mixture was mixed well and centrifuged.  To 2 ml of the above supernatant, 3 ml of orcinol reagent was taken in the test tubes and 10 ml   of anthrone reagent was added on ice. The tubes were mixed well and heated in a water bath at 80°C exactly for 20 minutes and immediately cooled under running tap water. The colour developed was read at 485 nm in Systronics Spectrophotometer. A blank was prepared with 2% H₂S0₄ instead of orcinol.  Control was maintained with boiled enzyme reaction mixture and with zero-time reaction mixture.

Estimation of Cellobiase

To estimate the cellobiase enzyme, the mixture of 1.5 ml of the buffer, 2.5 ml of 5 mM cellobiose   and 1 ml of the enzyme was incubated at 30°C for 2 h. Then the reaction was terminated. By using DNS (dinitro salicyclic acid) reagent, the amount of glucose liberated by the enzyme was measured at 575 nm in   Systronic   Spectrophotometer. Glucose was used as standard.

Statistical Analysis

The data of experiment was statistically analyzed according to ANOVA and significance with in the column with Tukey HSD multiple range test (TMRT) at 5% level of significance (n=3)

Results and Discussion

The 1,4 -β – Endoglucanase of control reduced the viscosity of the substrate to 80% at 180min. The lowest activity was observed in the enzyme source obtained from the culture treated with Trichoderma viride + Pseudomonas fluorescens (Tv+Pf) (13.3% viscosity loss at 180 min) followed by Trichoderma harzianum + Pseudomonas fluorescens (Th+Pf) (17.39%) and Trichoderma viride + Trichoderma harzianum(Tv+Th) (21.05%). (Fig.1)

The activity of 1,4 -β – Exoglucanase was expressed in specific activity units (SAU). All the treatments inhibited the activity of 1,4 -β –Exo-glucanase at varying degree. Higher amount of monogalacturonic units was released in the case of enzyme source obtained from the control (487.53 SAU), followed by   Tv+Th (Trichoderma viride+ Trichoderma harzianum) (234.00 SAU) and Th+Pf (Trichoderma harzianum + Pseudomonas fluorescens) (165.05 SAU). Among the treatments the least amount of sugar was liberated in the case of enzyme source obtained from treatment Tv+Pf (Trichoderma viride + Pseudomonas fluorescens) (95.14 SAU). (Table 1).

The   Cellobiase activity was observed in enzyme source of control (297.36 SAU). The lowest rate of enzyme activity was observed in Tv+Pf (Trichoderma viride + Pseudomonas fluorescens)-treated enzyme source (91.50 SAU) followed by   Trichoderma harzianum + Pseudomonas fluorescens Th+ Pf (155.12 SAU) and Trichoderma viride + Trichoderma harzianum and (Tv+Th) (195.18 SAU). (Fig.2)

Figure 1: Effect of culture filtrates of Trichoderma viride + Pseudomonas fluorescens (Tv + Pf), Trichoderma harzianum + Pseudomonas fluorescens (Th + Pf), Trichoderma viride+ Trichoderma harzianum (Tv+ Th) on the  activity  1,4 -β – Endoglucanases of  Fusarium oxysporum  in vitro   Click here to View figure

Table 1: Effect of culture filtrates of Trichoderma viride + Pseudomonas fluorescens (Tv + Pf), Trichoderma harzianum + Pseudomonas fluorescens (Th + Pf), Trichoderma viride+ Trichoderma harzianum (Tv+ Th) on  the  activity  1,4 -β – Exoglucanases of  Fusarium oxysporum  in vitro

^ap< 0.001 as compared to control SAU= micro g of maltose equivalent liberated /h

The values within a column followed by different letters are significantly different according to Tukey’s HSD multiple range test (TMRT) at 5% level of significance(n=3)

The  Cellobiase activity was observed in enzyme source of control (297.36 SAU). The lowest rate of enzyme activity was observed in Tv+Pf (Trichoderma viride + Pseudomonas fluorescens)-treated enzyme source (91.50 SAU) followed by those treated with Trichoderma harzianum + Pseudomonas fluorescens Th+ Pf (155.12 SAU) and Trichoderma viride + Trichoderma harzianum and (Tv+Th) (195.18 SAU).(Fig.2)

Figure 2: Effect of culture filtrates of Trichoderma viride + Pseudomonas fluorescens (Tv + Pf), Trichoderma harzianum + Pseudomonas fluorescens (Th + Pf), Trichoderma viride + Trichoderma harzianum (Tv+ Th) on the  activity   of cellobioses – of  Fusarium oxysporum  in vitro   Click here to View figure

^ap< 0.001 as compared to control SAU= micro g of maltose equivalent liberated /h

The values within a column followed by different letters are significantly different according to Tukey’s HSD multiple range test (TMRT) at 5% level of significance(n=3)

Maximum inhibiton of 1,4 β Endoglucanases, 1,4 Exoglucanases, and Cellobiase enzyme activity was recorded in Tv+Pf (Trichoderma viride + Pseudomonas fluorescens) treated culture. Our results are in agreement with the earlier studies recorded by Karthikeyan et al. (2006) that   T. viride, P. fluorescens, neem cake, T. viride + neem cake, P. fluorescens + neem cake   that applied to soil has considerably reduced the stem rot incidence compared to control in groundnut plants 60 days after sowing. Khan et al. (2004) reported that combination of Trichoderma and Pseudomonas has better efficacies in control of Fusarium infection on chickpea   than using them singly.

Salman et al. (2017) concluded   that application of Pseudomonas to field crops such as watermelon and similar crops that   are   affected by Fusarium spp   improve their   productivity and yields of such crops.  The mixtures of bioagents   also acts a PGPR. The    phytohormones    viz. auxins, gibberellins, cytokinins and ethylene produced by bacteria play a major role in growth promotion.  The same is reported by Lehar et al. (2016) stating that administration of biological agents of T. viride combined with P. fluorescens and Streptomyces sp. produce growth hormone or PGPR which stimulates better plant growth and thereby increasing yield in potato and ability to control disease caused by Phytophthora infenstan and Ralstonia solanacearu.

Our results indicated that the inhibition of 1,4 β Exoglucanases, 1,4 β Endoglucanases, and Cellobiases was recorded in Th+Pf (Trichoderma harzianum + Pseudomonas fluorescens) treated culture. Our results substantiate earlier reports that in management of plant diseases   the use of biocontrol agents in combinations were more effective than individual agents (Thilagavathi et al., 2007; Ganeshmoorthi et al., 2008). The application of T.harzianum, captan and neem seed extract two days after pathogen inoculation significantly reduced damping off disease caused by S. rolfsii in greenhouse grown tomato plants   (Okereke and Wokocha 2006). Thangavelu and Gopi (2015) found that the combination of bacterial antagonists could provide sustainable management of Fusarium wilt of banana under field conditions.

Our results also revealed that   inhibition of 1,4 Exoglucanases 1,4 β Endoglucanases and Cellobiases was recorded in Tv+ Th (Trichoderma viride + Trichoderma harzianum) treated culture. Similar observation were recorded by Rini and Sulochana (2007) that T.viride  and T. harzianum, with P . fluorescens were found to be compatible and more effective in suppressing the seedling disease of chilli and tomato.

Bosah et al.  (2010) reported that Trichoderma spp. proved to be the most effective biocontrol   agent against S. rolfsii in inhibiting the growth of the pathogen by 80% under in vitro conditions. In greenhouse trial, inoculation of mint plants with either T. harzianum or T. virens significantly re-duced the collar rot caused by S. rolfsii and was accompanied by significant increase in herb and oil yield (Singh and Singh 2004).

Conclusion

Results from the present study revealed that cellulolytic enzymes (Exo and endo Glucanases, and Cellobiases) produced by Fusarium oxysporum were inhibited by culture filtrate of Trichoderma viride+Pseudomonas fluorescens (1+2%) followed by Trichoderma harzianum+ Pseudomonas fluorescens (1.5+2%) and Trichoderma viride +Trichoderma harzianum(1+1.5%). The different disease suppression mechanisms together lead to enhanced disease suppression by the combined application of   above strains.   The reasons for the reduced wilt incidence and severity   may attributed to various mechanisms of Trichoderma spp. viz. mycoparasitism, spatial and nutrient competition, antibiosis by enzymes and secondary metabolites, and induction of plant defence system. The Pseudomonas produced secondary metionabolites like phenazine, 2,4-diacetylphloroglucinol, pyocyanine, pyoluteorin and pyrrolnitrin which were involved in enhancing plant growth by suppressing the disease caused   by Fusarium oxysporum.  In this study combinations of   biocontrol agents   showed significant reduction in Fusarium wilt disease. The application of fungal and bacterial combination, i.e.   T. viride + P. fluorescens (1+2%) has proved to be more effective in managing the Fusarium wilt in Arachis hypogaea L as compared to other combinations and single strains.

Acknowledgements

We are thankful to UGC University Grant Commission (India) for funding and to pursue the research under the category of Post-Doctoral Fellowship for Women.

Conflict of Interest

The co-author Prof. Rupam Kapoor is the mentor/ Research supervisor for my research project. The authors have no conflict of interest. On behalf of both authors, I am endorsing the same.

Funding Source

The study was funded by UGC(India). The corresponding author Dr. P. Rajeswari has received research grant for Post-doctoral research project titled Biochemical effects of combinations of Trichoderma spp  and Pseudomonas fluorescens against Fusarium wilt of Arachis hypogaea.L(Groundnut)under the category of Post-Doctoral Fellowships for Women(F.15-1/2015-17/PDFWM-2015-17-AND-34147(SA-II)

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