Invitro Studies on Control of Soil-Borne Plant Pathogens by Earthworm Eudrilus Eugeniae Exudates

Key words: Antibacterial – Eudrilus eugeniae, aqueous extracts, soil-borne plant pathogens.

1. Introduction

Use of vermicompost as an organic amendment to agricultural lands is practiced by many Indian farmers and they opine that the application of vermicompost brings down the incidence of many soil borne diseases in crops. The earthworm secretions and microbes harbored in the vermicompost act as plant growth stimulators. Experimental results have shown improved active nodulation in legumes (Kale, 1997), increased symbiotic mycorrhizal association with the roots (Kale et al, 1987; Harinikumar et al, 1991). An increase in total microbial activity has been reported in the fields receiving vermicompost (Kale et al, 1992; Nair et al 1997). Karuna et al. (1999) reported the use of earthworm body fluid (Vermiwash) as a spray to the tissue cultured crinckle red variety of Anthurium andreanum. The suppression of disease caused by soil borne pathogens on application of vermicompost has been reported (Somshekara et al, 2001). The coelomic fluid of the Earthworm, Eisenia fetida andrei (Savigny) was demonstrated to possess an antimicrobial activity directed against earthworm pathogenic bacteria – namely; Gram-negative Aeromonas hydrophila {(Chester) Stainer} and gram-positive Bacillus megaterium (de Bary) by Valembois et al (1982). Rivai Bakti et al, (2003) studied the antimicrobial activity of earthworm extract (Pontoscolex corethrurus Fr.Mull) in different solvents such as n-hexane, chloroform and methanol. They reported the antibacterial activity of the extracts on Staphylococcus aureus and Escherichia coli, by recording the inhibition zones.

Though many researchers have reported the suppression of diseases on application of vermicompost and compost tea there has been no work carried out on using earthworm and its secretions directly as antimicrobial agents. The aim of the study was to find out the effect of aqueous earthworm extracts (body wall extract, gut extract and coelomic fluid) of the earthworm Eudrilus eugeniae on selected soil-borne plant pathogens using simple laboratory techniques.

2. Materials and Methods

2.1 Earthworm Species

For the purpose of this study Earthworm species used was Eudrilus Eugeniae, a native of Nigerian forests but that has found a mention about its distribution in coastal region of south India in Fauna of British India. It is a popular earthworm used for waste management.

2.2 Collection of Earthworm Extracts

2.2.1 Coelomic fluid extract

Fresh earthworms from the compost pits were taken in batches of 4-5 and placed on a petri plate held in slanting position. The base of a beaker with ice cubes was run over the worms to give cold shocks, so that coelomic fluid oozed out from the dorsal pores of the earthworms. The coelomic fluid was collected using sterile dropper into a sterile test tube. About 15 – 20 ml of the fluid was collected from 100 earthworms.
The earthworm Gut was cleared of the organic waste by allowing them to feed on wet ordinary filter paper for 48 hours by keeping them in a box with pinholes for proper aeration inside the box.

2.2.2 Gut and body wall extracts

The filter paper fed earthworms were narcotized after collection of coelomic fluid by swabbing with 70% alcohol and then dissected to separate body wall and gut. Gut and body wall were macerated separately and homogenized using a sterile glass homogeniser, a pinch of sterile sand and sterile distilled water to get 10% aqueous homogenate. The homogenates were centrifuged at 5000 rpm separately and supernatant was transferred into sterile test tubes. About 6 ml of Gut extract and 12 ml of body wall extract were collected from 10 earthworms.

2.2.3 Processing of extracts

The extracts collected were filtered using membrane filter technique. The filtered extracts were stored in the refrigerator at 4°C until use. The frozen extracts were thawed slowly at room temperature and then used for the experiments. Fresh extracts and stored extracts were used to test the effect of storage on the pathogens.

2.3 Pathogen Cultures

Pure cultures of plant pathogens were obtained from Plant Pathology Department, University of Agricultural Sciences, Bangalore. Following is the list of Pathogens used:

A. Bacterial pathogens

1. Xanthomonas campestris
2. Ralstonia solanacearum
3. Erwinia carotovora

B. Fungal pathogens

1. Botryodiplodia theobromae
2. Rhizactonia solani
3. Alternaria solani
4. Fusarium oxysporium
5. Sclerotium rolfsi i

2.3.1 Maintenance of stock cultures

The pure cultures were stored in the refrigerator and were sub cultured often so as to maintain purity and viability of cells. Bacteria were sub cultured once in 3-4 days by streak plate method, while fungi were sub cultured once in 8-10 days.

2.3.2 Preparation of culture suspension

Uniform and even distribution of culture could not be achieved by taking the culture directly from the plate. Hence culture suspensions were prepared. Only fresh culture suspensions were used for the study.

1. Bacterial culture suspension
   Nutrient broth was used for the preparation of bacterial culture suspension. Using a sterile loop, the bacterial cells were transferred from the stock culture plate into a small quantity of nutrient broth taken in a sterile test tube. It was used for studies only after 18 hours of incubation at room temperature.
2. Fungal culture suspensions
   Fungal culture suspensions were prepared using 0.1% saline (Sodium Chloride) solution as media. Using a sterile loop the fungal spores were carefully transferred into a small quantity of saline solution taken in a test tube and used immediately.

2.4 Antimicrobial Potency Tests

Since the target organisms were both bacteria and fungi, a single common method could not be adopted. Therefore, to test the influence of extracts directly on the pathogens three methods were experimented for both bacteria and Fungi.

1. Incubation method
2. Agar diffusion method – Filter paper Disc method
3. Agar diffusion method – Well method

Along with these three methods, Turbidity method for bacteria and germination study for fungi was also conducted (Table 1).

2.4.1 Antifungal property of mixed extract

A combination of gut extract, body wall extract and coelomic fluid in a ratio of 1:2:4ml was prepared and tested on F.oxysporum inoculated plate by placing the filter paper disc impregnated with 0.2 ml of the mixed extract. In the same plate a groove was also made and 0.5ml of the extract was added. The purpose of experimenting both filter paper disc method and groove method was to demonstrate the concentration effect of the extract on pathogen inhibition.

3. Results

In this study simple laboratory techniques were used to test the antimicrobial potency of the earthworm extracts on certain soil borne plant pathogens. Since this was a preliminary study, the standardized methodology was not available from the literature. Hence different approaches were used to study the influence of worm extracts on pathogens. Most of them were variations of culture plate technique. Among them Well method and turbidity method, gave better results at the used concentrations of the extracts. Though the concentration of extracts used was one ml in incubation method and similar to that of turbidity method, it was ineffective and this could have been due to the small quantity of the extract (compared to the media and culture) present, which was probably not sufficient to express the inhibitory effect. Whereas in case of turbidity method the extract was in direct contact with the pathogen culture and hence the inhibitory effect or suppressiveness was more evident.

From the study it was observed that, the concentration of the extract used for testing was very critical for the antimicrobial potency to be expressed. In filter paper disc method, the amount of the extract was very little and did not diffuse widely into the media (the extract could have got exhausted quickly on the surface itself) and the possibility of the non availability of the extract to the pathogen was high as in case of incubation method. In well method the concentration of the extract was 0.5 ml and probably it was sufficient enough for the extract to express inhibitory effect. The extract diffused slowly into the media so that the antimicrobial activity was successfully expressed.

Only fresh extracts have been proved to be inhibitory whereas the extracts after 48 hours of storage had no such inhibitory effect and no zone of inhibition was observed.

3.1 Antibacterial Potency of Earthworm Extracts

All the three extracts have shown inhibition zones by well method for X.campestris and E.carotovora whereas in case of R.solnacearum only body wall and gut extracts have shown antibacterial activity at the used concentrations. This has been supported by the turbidity method. With increasing concentrations gut and body wall extracts have recorded reduced turbidity levels whereas in case of coelomic fluid even increase in concentration of extract has not shown significant effect on growth of any of the bacteria. The results have been summarized in Table 2.

3.2 Antifungal Potency of Earthworm Extracts

Body wall and gut extracts were highly inhibitory to the fungal pathogens F.oxysporum and B.theobromae germination study has shown delayed sporulation in case of B.theobromae in body wall and gut extracts (Table 3).

In case of A.solani, R.solani and S.rolfsii no inhibition zones were recorded but delayed sporulation was observed. This was only a visual observation.

When a mixed extract was used to test the effect on F.oxysporum, very interestingly strong zone of inhibition was obtained, indicating that a combination of extracts will have a better effect on fungal pathogens rather than individual extracts. Probably this is the reason why vermicompost is so very successful in fungal disease suppression. The earthworms present in the soil release coelomic fluid form the dorsal pores, the body wall which is in direct contact with the soil and the excreta from the gut of the worm, all act together and result in antimicrobial activity thus suppressing several soil-borne plant diseases.

4. Discussion

From the study it is evident that the earthworm extracts possess antimicrobial properties. The suppressive effect or inhibitory effect is highly concentration specific and the study on fungal pathogens indicates that the extracts can be effectively used to control the sporulation of fungal pathogens. Several studies have also reported the inhibition of plant pathogens by earthworm secretions. Reiten & Salter (2002) have reported strong inhibition for X.campestris pv.Carotovora culture plate method using compost tea. Their studies indicate that compost tea can be used to control X.campestris pv.Carotovora both in the laboratory studies as well as in the field. Our studies have proved inhibition of X.campestris, E.carotovora and R.solanacearum by the earthworm extracts but extracts were tested on the pathogens directly and at laboratory level. Hudson and Berman (1994) have reported strong suppression of Rhizactonia, on application of compost to the soil. Khalifa (1965) has reported suppression and control of Fusarium using compost. Body wall and gut extracts have shown inhibitory effect on F.oxysporum in our studies. Apart from the soil borne plant pathogens, animal fungal pathogens Candida albicans, Cryptococcus neoformans and Trycophytan metagrophyte(Subhashini, 2005), were also found to be inhibited by the body wall, gut and coelomic extracts.

This being a preliminary study, complete biochemical characterization of the extracts was not carried out. However, coelomic fluid is found to contain the free amino acid Tryptophan, which possesses antibiotic property. Body wall and gut extracts have not shown the presence of any free amino acids. Further studies need to be carried out to establish concentration levels.

From the experiment it is evident that earthworm extracts have direct influence on the soil borne plant pathogens. Though this study was carried out at laboratory level, it has given promising results that can be used to carry out more detailed research in the field of plant pathogen control on using vermicompost or the secretions of earthworms wherever it is possible.

Acknowledgements

This study was carried out in partial fulfillment of the requirements for M.Phil programme from Bharathidasan University, Trichy, India. We would like to thank Rev Sr.Jesuina and Sr. Albina for permitting to use the laboratory facilities at Mount Camel College. This study could not have been possible without the help rendered by Dr.Chandrashekar, Professor, Plant pathology department, UAS, GKVK campus, Bangalore. India

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