Biocontrol mechanism of fungal pathogen through P. fluorescens ATCC 9028

Biocontrol mechanism of fungal pathogen through P. fluorescens ATCC 9028

Dr. Vishal Narayan Shinde*

Department of Botany, Late Annasaheb R D Deore Arts and Science college, Mhasadi,

Tal. Sakri, Dist:Dhule- 424304 (MS) India.

                       * Author for Correspondence: vishalshinde1001@gmail.com       

Abstract:

Biological control of plant pathogen by microorganism has been considered more natural and environmentally acceptable alternative to the existing chemical methods^[1]. Biological control has been developed as an alternative to synthetic fungicide treatment and considerable success had been achieved upon utilizing antagonistic microorganism to control both pre harvested and post harvested diseases^[2]. A variety of microbial antagonistic has ability to control several pathogens of various fruit and vegetables^[3].

            Antifungal assay using bacterial isolates such as P. fluorescens ATCC 9028 was tested against ten fungal pathogens of leafy vegetables. P. fluorescens ATCC 9028 was most effective with 58.19% fungitoxic activity against all tested fungal pathogens. Among the tested pathogen, F. moniliforme was highly susceptible with 65.78% inhibition and P. pullulans was highly resistant with 44.02% inhibition against all three antagonistic bacteria.

Keywords: Biological control, antagonistic bacteria., P. fluorescens etc.

Introduction:

On an average each crop plant can be affected by hundred or more than hundred diseases. The development of new physiological race pathogens to many of the systemic fungicides is gradually becoming ineffective. The biological control agents have enormous antimicrobial potential. They are effective in treatment of infectious diseases, simultaneously mitigating many of the side effects which are associated with pesticides. Therefore, there is growing realization in the people that biological control can be successfully exploited as an agricultural method for soil borne pathogens^[4].

            Beside this, Biological control of numerous crops by application of antagonistic bacterial isolates from suppressive soils has been accomplished during last two decades all over the world^[5]. The bacterium has been reported to be effective in controlling Phytopthora and Pythium amongseveral soil borne plant pathogens^[6]. Several studies have been demonstrated reduced incidence of disease in different crops after supplementing the soil with bacterial antagonists^[7]. Rhizosphere bacteria are excellent agents to control soil borne plant pathogens. Bacterial species like Bacillus, Pseudomanas, Serratia and Anthrobacter have been proved in controlling the fungal diseases^[14,15]. More recently an increasing number of reports have been focused on the potential of Bacillus subtilis as a biocontrol agent^[8]. A successful biocontrol agent efficiently suppresses the pathogen and reduces disease incidence. Biocontrol agent acts against pathogens by antagonism- competition, antibiosis and parasitism therefore in recent years a new biocontrol agent, Pseudomans flouresence have drawn attention due to the ability to produce secondary metabolites such as siderophore, antibiotic, volatile metabolites, HCN, enzyme and phytochrome which were highly antagonistc component to various phytopathogens^[9]. Pseudomonas flourescence is effective candidate for biological control of soil borne plant pathogens owing to their versatile nature, rhizophere competition and multiple mode of action^[10,11,12].

Material and method:

            Biological control of numerous crop diseases by application of antagonistic bacterial isolates from the soil has been accomplished during last two decades all over the world^[13,14,15]. Hence for the assessment of antifungal activity, the three bacterial isolates which has high antagonistic activity were procured from the Department of Microbiology, Government Institute of Science, Aurangabad as fallows,

            1)         Pseudomonas fluorescens ATCC 9028

            There after this bacterial cultures were transferred to fresh Nutrient agar slants in triplicates and were kept at 4⁰C in refrigerator for further studies.

Antifungal activity by antagonistic bacteria:

            The antifungal activity of three bacterial isolates was tested against ten pathogenic fungi of leafy vegetable by dual culture method^[16]. The antagonistic bacteria and targeted fungal pathogen were inoculated dually on PDA medium in sterile Petri dish 2-2.5 cm apart from each other. Whereas Petri plate without bacterial inoculation served as control and incubated at 37 ± 1 ^oC for 7 days. The inhibition of growing fungi by tested bacteria was quantified as distance of radial towards and away from bacteria in relation to control. The percent inhibition of mycelial growth of the fungi was calculated using formula,

                                             100 (R₁-R₂)

                               I  =

     R₁  

                                    Where              I           =          Inhibition of mycelial growth.

                                                            R₁        =          Mycelial growth in control

                                                            R₂        =          Mycelial growth in treated.

Result:

            The antagonistic effect of bacterial isolates was screened by dual culture method^[16]. The bacterial cultures, Pseudomonas fluorescens ATCC 9028 was tested against ten fungal pathogens of leafy vegetables. After a week of incubation, the growth of targeted fungal pathogens towards and away from the bacterial antagonistic isolate was recorded. The percent inhibition of mycelial growth over control was tabulated.

The bacterial antagonistic, P. fluorescens ATCC 9028 had significantly inhibited the radial growth of all tested fungal pathogen of leafy vegetables. Among tested pathogens, F.moniliforme and F. oxysporum were most sensitive and revealed 68.42% and 66.66% inhibition of mycelial over control (Table 1). On contrary, A. carthami and P. pullulans were most resistant and showed 50.90% and 49.18% inhibition respectively. While remaining pathogens namely C. lindemuthianum, F. roseum, A. brassicae, A. humicola, S. verruculosum and H. sativum showed 62.31%, 60%, 59.45%, 57.81%, 54.23% and 53.01% respectively inhibition (Table 1; fig. 1).

On average, F. moniliforme was found to be most sensitive with 65.78%  and P. pullulans as most resistant with 44.02% against all three bacterial antagonistic when compared to other tested fungal pathogens (Table 1).

Among the three tested antagonistic bacterial cultures, P. fluorescens ATCC 9028 was most effective and showed 58.19% fungitoxic activity (Fig 1).

Table No. 1: Antagonistic effect of P. fluorescens ATCC 9028 against ten fungal  pathogens of leafy vegetables.             

Pathogen	Mycelial growth in control (mm)	Mycelial growth of pathogen in presence P. fluorescens (mm)	% inhibition of  mycelial growth over control
A. brassicae	74	30	59.45 + 1.88
A. carthami	55	27	50.90 + 1.33
A. humicola	64	27	57.81 + 1.24
C. lindemuthianum	69	26	62.31 + 0.47
F. moniliforme	76	24	68.42 + 1.41
F. oxysporum	84	28	66.66 + 0.94
F. roseum	70	28	60.00 + 1.41
H. sativum	83	39	53.01 + 0.81
P. pullulans	61	31	49.18 + 1.63
S. verruculosum	59	27	54.23 + 1.88
C.V.			7.96%

Values expressed in mean + S.E.M. of triplicates.

Fig 1. Antagonistic effect of bacteria against ten fungal pathogens of leafy vegetables.

Discussion :

Antifungal activity of three bacterial isolates namely Pseudomonas fluorescens ATCC 9028 was tested against ten fungal pathogens of leafy vegetables by dual culture method. Similar work previously carried out by many workers and reported that bacterial isolates like Bacillus sp., Pseudomonas sp., Serratia sp. and Anthrobacter sp. have been proved their efficacy against many fungal diseases^[17,18]. In the present study among three tested antagonistic bacterial isolates, P.  fluorescens ATCC 9028 was most effective one and revealed 58.19% inhibition of mycelial growth all ten targeted fungal pathogens. Similar finding were reported by Moataza and Saad^[19] and mentioned that five isolates P.  fluorescens were effective and showed 56% inhibition of Phythopthora capsici and 58.08% inhibition of Rhizoctonia solani.  

Among the tested pathogens, F. moniliforme was most susceptible with 65.78% inhibition on contrary P. pullulans was most resistant with 44.02% inhibition against all three bacterial isolates. Antagonistic activity may be due to the production of secondary metabolites such as siderophore, antibiotic, volatile compounds, HCN, enzymes or may be due to phytochromes which were inhibitors of various phytopathogens^[18].

References:

1. Baker, R. and T. C. Paulitz. 1996. Theoretical basis for microbial interaction leading to biological control of soil borne plant pathogen In : Hall R., (ed). Principles and practice of managing soil borne plant pathogen. Am. Phytopathol. Soc. St. Paul. MN. pp. 50-79.
2. Janisiewicz, W. J. and L. Korsten. 2002. Biological control of post harvested diseases of fruits. Annu. Rev. Phytopathol. 40 : 411-441.
3. Mari, M. and M. Guizzardi. 1998. The post harvested phase: emerging technology for fungal disease. Phytoparacitica. 23 : 97-127.
4. Papavizas, G. C. and R. D. Lumsden. 1980. Biological control of soil borne fungal propogules. Annu. Rev. Phytopathol. 18 : 389-413.
5. Park, C. S., T. C. Paulitz and R. Baker. 1988. Biocontrol of fusarium wilt of cucumber resulting from interaction between Pseudomonas putida and non pathogenic isolates, Fusarium oxysporum. Phytopathol. 78 : 190-194
6. Shen, S. S., J. M. Kim and C. S. Park. 2002. Serratia plymuthica strain A21-4 : A potential biocontrol agent against phytopthora blight of pepper. Kor. J. Plant Pathol. 18 : 138-141.
7. Mukhopadhyay, A. N. 1987. Biological control of soil borne plant pathogen by Trichoderma spp. and Bacterium isolates. Indian J. Mycol. Plant Pathol. 17 : 1-9.
8. Weller, D. M., B. X. Zhang and R. J. Cook. 1985. Bacterial species and biopesticides in controlling fungal diseases. Plant Diseases. 69 : 710-713.
9. Ferreira, J. H.S., F. N. Mathee and A. C. Thomas. 1991. Biological control of Eutypa lata on grapevine by antagonistic strain of Bacillus subtilis. Phytopathol. 81 : 283-287.
10. Okigbo, R. N. and M. I. Osuinde. 2003. Fungal leaf spot disease of Mango (Mangifera indica L.) in Southeastern Nigeria and biological control with Bacillus subtilis. J. Plant Peotect. Sci. 39(2): 70-77.
11. Gupta, C. D., R. C. Dubey, S. C. Kang and D. K. Maheshwari. 2001. Antibiotic mediated necrophic effect on Pseudomonas GRC2 against two fungal plants pathogens. Current Sci.  81 : 91-94.
12. Kloepper, J. and M. Schroth. 1981. Relationship of in vitro antibiosis of plant growth promoting rhizobacteria and the displacement of root microflora. Phytopathol. 71 : 1020.
13. Waller, D., W. Howie and R. Cook. 1988. Relationship between in vitro inhibition of Gaenmannomyces graminis var. tritici and suppression of take all of wheat by Fluorescent psudomonads. Phytopathol. 78 : 1100.
14. Diby, P., K. Saju, Y. Jisha, A. Kumar, Y. Sharma and M. Anandaraj. 2005. Mycolytic enzyme produced by Pseudomonans fluorescens and Trichoderma spp. against Phytopthora capsici, (Pepper nigrum L.). Ind. Phytopathol. 58 : 10.
15. Park, C. S., T. C. Paulitz and R. Baker. 1988. Biocontrol of fusarium wilt of cucumber resulting from interaction between Pseudomonas putida and non pathogenic isolates, Fusarium oxysporum. Phytopathol. 78 : 190-194.
16. Leeman, M., F. M. De Quden, T. A. Van Pelt, C. Cornelissen, G. Matamala, P. A. H. M.  Bakker and B. Schippers. 1995. Suppersion of Fusarium wilt of raddish by co inoculation of Fluorescent pseudomonas spp. and root colonizing fungi. Eur. J. Plant Pathol. 102 : 21-31.
17. Larkin, R. P., D. L. Hopkins and F. N. Martin . 1996. Suppression of Fusarium wilt of watermelon by non pathogenic F. oxysporum and other microorganism recovered from a disease suppressive soil. Phytopathol. 86 : 812-286.
18. Skidmore, A. M. and C. H. Dickinson. 1976. Colony interaction of hypal interference between Septoria nodorum and Phylloplanefungi. Trans. Brit. Mycol. Soc. 66 : 57-64.
19. Moataza and M. Saad. 2006. Destruction of Rhizoctonia solani and Phytopthora capsici causing tomato root rot by Pseudomonas fluorescens lytic enzyme. Res. J. Agri. Bio. Sci. 2(6) : 274-281.

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