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

By S.V. Shobha¹ and Radha D. Kale²
January 2008

  1. Mount Carmel College, Department of Environmental Science, 58 Vasanthanagar, Bangalore   560 052, India – Contact
  2. University of Agricultural Sciences, Department of Zoology, GKVK Campus, Bangalore   560 065, India – Contact
A preliminary study was carried out using simple laboratory techniques to examine the influence of different preparations from the body of earthworm Eudriluseugeniae on selected soil borne bacterial and fungal pathogens. Since this test was the first of its kind, testing procedures were standardized using different concentrations of extracts following different methods. The experiments were based on the principle of agar diffusion, turbidity development and rate of germination. The experiment involved the recording of inhibition zones formed by various extracts, amount of turbidity and suppression of rate of germination by different extracts. The results showed that the gut and body wall extracts had both antibacterial and antifungal activities by forming total inhibition zones, whereas coelomic fluid was found to be only antibacterial at the used concentrations. Body wall and gut extracts were found to be inhibitory to Xanthomonascampestris, Ralstoniasolanacearum, Erwiniacarotovora, Fusariumoxysporum and Botryodiplodia theobromae. Colemic fluid was inhibitory to X.campestris and E.carotovora. There was no inhibitory effect recorded by any of the extracts at the used concentrations during this study on Rhizactoniasolani, Alternariasolani and Sclerotiumrolfsii. Only delayed sporulation was observed in all the cases, but this was only a visual observation. The mixed extract when tested on F.oxysporum has shown a strong clear inhibition zone.
The study has proved that earthworm extracts can be effectively used for suppression of soil borne pathogens and that it can evolve as potential biopesticide.

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).

Table 1 · Different Tests Adopted for Antimicrobial Potency Tests
Method Description Criteria for Confirming Antimicrobial potency Pathogens
Incubation method
  • 9:1 ratio of Media and extract
  • Poured into the plate
  • After solidifying microbial culture was swabbed on solid agar surface
  • Kept for incubation at 37°C
Density of Microbial growth – visual observation All the pathogens
Filter paper disc method
  • On Solid agar medium the culture suspension was swabbed uniformly
  • Filter paper impregnated with 0.1ml of the extract was placed on the agar surface
  • Kept for incubation at 37°C
Clear zone (inhibition) around the filter paper disc All the pathogens
Well method
  • On Solid agar medium the culture suspension was swabbed uniformly
  • Using sterile pipette uniform sized wells/grooves were made
  • 0.5 ml of the extract – well
  • Kept for incubation at 37°C
Clear zone (inhibition) around the well All the pathogens
Turbidity method
  • Nutrient broth and extract in 9:1, 8:2, 7:3 ratio were used
  • Two loopfuls of 18 hours fresh nutrient broth bacterial inoculum was added and mixed well.
  • Control: 10 ml of the nutrient broth with 2 loopfuls of the culture suspension
  • Incubated for 18hours
  • Measurement of turbidity.
Turbidity in test solutions.The Higher the turbidity readings, higher is the growth rate of bacteria. Bacterial pathogens
X.campestris, R.solanacearum, E.carotovora
Germination study
  • Sterile cavity slide with three drops of extract
  • Introduction of fungal conidium
  • Sealing of cavity slide with a sterile cover slip
  • Left in an inverted position for 24 to 48 hours.
  • Observation carried out under the microscope once in 3 hours over a period of 24 – 48 hours for germination of fungal spores. .
  • Control -Distilled water and 2 % sucrose solution
The amount of time taken for germination B. theobromae
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.

Table 2 · The Test Results of Antibacterial Potency of Earthworm Extracts on Bacterial Pathogens
Pathogen Methods Quantity of extract used (ml) Results for different substances tested
Gut Extract Body Wall Extract Coelomic fluid
Xanthomonas campestris Incubation method 1 - - -
Well method 0.5 + + +
13 mm 15 mm 4 mm
Filter paper disc method 0.1 + + -
2 mm 1 mm 0
Turbidity method
(Control: 160*)
1, 2, 3 + + +
1 65 84 139
2 53 70 134
3 45 63 129
Ralstonia solanacearum Incubation method 1 - - -
Well method 0.5 + + -
1.5 mm 1.5 mm 0
Filter paper disc method 0.1 - + -
0 0.5 mm 0
Turbidity method
(Control: 160*)
1, 2, 3 + + +
1 69 80 120
2 54 70 124
3 43 61 129
Erwinia carotovora Incubation method 1 - - -
Well method 0.5 + + +
0.5 mm 0.5 mm 0.5 mm
Filter paper disc method 0.1 + + -
0.5 mm 0.5 mm 0
Turbidity method
(Control: 160*)
1, 2, 3 + + +
1 70 82 120
2 58 60 105
3 41 59 93

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).

Table 3 · Test Results of Antifungal Potency of Earthworm Extracts on Fungal Pathogens
Pathogen Methods Quantity of extract used (ml) Results for different substances tested
Gut Extract Body Wall Extract Coelomic fluid
Botryodiplodia theobromae Incubation method 1 - - -
Well method 0.5 - - -
Filter paper disc method 0.1 + + -
Germination study method 3 drops + + -
Fusarium oxysporum Incubation method 1 - - -
Well method 0.5 + + -
7 mm 10 mm 0
Filter paper disc method 0.1 - + -
0 8 mm 0
Alternaria solani Incubation method 1 - - -
Well method 0.5 - - -
Filter paper disc method 0.1 - - -
Rhizactonia solani Incubation method 1 - - -
Well method 0.5 - - -
Filter paper disc method 0.1 - - -
Sclerotium rolfsii Incubation method 1 - - -
Well method 0.5 - - -
Filter paper disc method 0.1 - - -

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.


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


  1. K.M. Harinikumar, D.J. Bagyaraj and R.D. Kale, Vesicular mycorrhizal propagules in earthworm cast. inn: Advances in management and conservation of soil fauna. G.K. Veeresh, D. Rajgopal and C.A. Viraktamath (eds). Oxford and IBH, 1991. New Delhi.pp.657-664
  2. Hudson, Berman D. Soil organic matter and available water capacity. Journal of Soil and Water Conservation. 1994.March–April. p. 189-194.
  3. Joel Reiten & Cindy Salter, 2002. Compost tea for suppression of Xanthomonas in carrot production
  4. R.D. Kale, Earthworm – significant contributions to organic farming and sustainable agriculture. In : Organic farming and sustainable agriculture. G.K. Veeresh, K.shivashankar and M.A. Singlachar (eds.), Association for promotion of Organic Farming, 1997, Bangalore,pp. 52-57
  5. R.D. Kale, K.Bano, M.N. Sreenivasa, D.J. Bagyaraj. Influence of worm cast (vee Com E UAS 83) on the growth and mycorrhizal colonization of two ornamental plants. South Indian Horticulture, 35 (1987): 433-437.
  6. R.D. Kale,. B.C. Mallesh, K.Bano, and D.J. Bagyaraj, Influence of vermicompost application on the available macronutrients and selected microbial populations in a paddy field. Soil boil. Biochem., 24 (1992.) 1317-1320.
  7. K. Karuna, Patil, C.R., Narayanaswamy, P. and Kale, R.D., Stimulatory effect of eathworm body fluid (vermiwash) on crinkle red variety of Anthurium andreanum Lind. Crop Res.,17(1999.)253-257.
  8. O. Khalifa, Biological control of Fusarium wilt of peas by organic soil amendments. Ann. Appl. Biol., 56 (1965.): 129-137.
  9. S.K. Nair, A. Naseema, K.S. Meenakumari, P. Prabhakumari and C.K.Peethambaran, Microflora associated with earthworms and vermicompost.J.Trop.Agi.,35 (1997.): 68-70
  10. IA Rivai Bakti, Fithri Damayanti Suri, and Azwar Agoes,The Antibacterial potency of Pontoscolex corethrurus Fr mull earthworm extract on Staphylococcus aureus and Escherichia coli growth. The Journal of the Indonesian Medical Association. (4)1:April 2003: 444-454.
  11. Y.M. Somasekhara, Halakeri, A.V. and Kale, R.D., Effect of culture filtrates of vermicompost against pomegranate wilt pathogen, ceratocystic fimbriata EII. And Halst. Abst. Session5. Soil biota as biocontrol agents. In VII natl. Symp. Soil Biol.Ecol 2001.pp 76.
  12. N. Subhashini, Effects of Earthworm extracts on certain Human/Animal pathogens. Dissertation submitted to Bharathidasan University. 2005
  13. P. Valembois, Roch, P., Lassegues, M. and Cassand, P., Antibacterial activity of the hemolytic system from the earthworm Eisenia fetida andrei. J.Invertebr. pathol. 40 (1982): 21-27.


Copyright © 2008, ECO Services International