Abstract

The reproduction toxicity of Pseudomonas fluorescens (PF) isolated from different locations of Tamil Nadu viz., PF 5, PF 18, PF 25 to earthworm, Eisenia fetida (Savigny, 1826) was estimated in a 56-day soil exposure based on OECD 222 (2016) and ISO 11268-2 (2012). Identical test concentration of 10⁶ cfu/kg dry soil was used for all the three strains of PF with four replicates. An untreated control (deionized water) with eight replicates was maintained for comparison. Adult earthworms were exposed to the treated soil and incubated under the test conditions for 28 days. Known quantity of cattle manure was added at weekly interval as food. Food consumption by adult worms per test container was observed and assessed up to 28 days. After 28 days, adult earthworms were sorted from the soil and assessed for mortality and weight loss (sub-lethal effects). Soil (without adult earthworms) was kept in the test container and juveniles/cocoons that produced by the earthworms were allowed to grow for further 28 days. The number of juveniles found in each container was counted at the end of test (56^th day). The results of the study indicated that all the validity criteria specified by the guideline were met and no mortality was observed in any of the treatment group and control. Food consumption of earthworms treated with PF strains were on par with control. Biomass change of earthworms and number of juveniles produced by the treatment groups were significantly increased compared to control.

1. Introduction

Earthworms are ecologically important and also an integral part of the food chain. They are ubiquitous in many soil types and a primary factor in enhancing soil fertility through their detritus feeding activities. Earthworms benefit the ecosystem by improving the physical structure and fertility of soil, plant growth and health, suppress weed growth and clean up dangerous chemicals in the environment. Earthworms are excellent model organisms in ecotoxicological studies of soil toxicity due to their exposure to soil contaminants via ingestion and passive absorption through their skin. ¹ Among different species of earthworms, Eisenia fetida and Eisenia andrei are recommended for toxicity testing ^{2, 3, 4} as they have relatively short life cycles, cocoon production, continuous breeding and easy culturing under laboratory conditions and cosmopolitan distribution. Eisenia fetida is an easily cultured earthworm species and amenable to good experimental design so they have been adopted as indicator organisms for the assessment of soil quality ^{2, 3}. Pseudomonas fluorescens (PF) strains are having plant growth-promoting properties and biocontrol properties, protecting the roots of some plant species against parasitic fungi as well as some phytophagous nematode. Bio control of plant pathogens by antagonistic microorganisms is a potential non-chemical means and is known to be a cheap and effective eco-friendly method for the management of crop diseases ¹. P. fluorescens have been successfully used for biological control of several plant pathogens ⁵ and biological control using plant growth promoting rhizobacteria strains especially from the genus Pseudomonas is an effective substitute for chemical pesticides to suppress plant diseases ⁶.

Pseudomonas fluorescens is proven to be an effective bio control agent to the root rot disease and antagonists against many fungal soil pathogens. During field application, PF isolate may leach to the soil profile where the earthworms present. In order to find out the long-term effect (in terms of reproduction) of PF isolates on earthworms, the present 56 day reproduction research was undertaken.

2. Materials and Methods

The experiments were conducted in the Department of Plant Pathology & Ecotoxicology, International Institute of Biotechnology and Toxicology (IIBAT), Padappai, Tamil Nadu, 601301, India. The 56 day test was conducted in the laboratory following the OECD guideline OECD 222, 2016 (Earthworm Reproduction Test, Eisenia fetida/Eisenia andrei) and ISO 11268-2, 2012. Pseudomonas fluorescens (PF5, 18, 25) is applied to earthworms by mixing into the soil in which they live.

P.fluorescens was Isolated from the rhizosphere soil of pulse crops field in various districts viz., Coimbatore, Kanchipuram, Cuddalore, Thiruvarur, Villupuram, Madurai and Tuticorin of Tamil Nadu. Pure cultures of selected Pf 5, Pf 18, Pf 25 colonies on King’s B agar plates were selected for the study and its host and location were tabulated below and studied for its shape, size and pigmentation. [Table 1]

Artificial soil was prepared according to OECD 207, 1984 with 10% Sphagnum-peat air-dried and finely ground (<2 mm) (Gramoflor GmbH, Vetchta, Germany), 20% Kaolin clay (Kaolinite content >30%) (ROMAC India Industrial minerals, Chennai), 70% fine quartz-sand (grain size with more than 50% by mass of particle size 50-200µ). The soil ingredients were homogenised for about 20 minutes using a homogenizer. From the bulk soil, a sample was taken and cow manure was added at 1% and its pH was checked. The mean pH of the artificial soil was observed as 6.25 (within 6 ± 0.5).

The maximum water holding capacity (MWHC) of the artificial soil was determined. Deionised water corresponding to 40-60% of the total water holding capacity was used to moisten the soil

The concentration of Pseudomonas fluorescens (PF 5, 18, 25) @10^-6 Colony Forming Units/ mL was used for the experiment. 0.5mL/replicate (500g) of the required dilution was added.

One day before the test item application, glass containers were filled with about 500 g dry artificial soil (one container per replicate) and moistened to approximately half of the final water content. On the day of the experiment, weighing and application of test item were carried out in two batches considering 2 replicates per batch (1kg dry soil/batch). Appropriate volume of the Pseudomonas fluorescens strains (PF 5, 18, 25) (calculated for two replicates) was added to the deionized water and applied to the pre moistened artificial soil. Each batch of soil was mixed thoroughly with a laboratory mixer. The soil was mixed over a period of approximately 5 minutes and the final test medium was ventilated before releasing the earthworms. Each batch was divided into 2 replicates, thus four replicates altogether at each concentration level. Similar procedure was followed for control soil without test item.

The soil moisture content of each container was checked periodically (once in a week) by weighing the test container. Based on the weight difference between initial weight water was added to compensate the evaporation loss. Soil moisture content and pH were determined for representative sample from each treatment group. (Table 1).

Temperature was in the range of 18-22ºC and light Intensity of 402 to 603 LUX with light regime 16 hours light and 8 hours darkness per day was maintained. Eight replications per control and 4 per each test item group were maintained.

10 per unit, i.e. 80 individuals per control and 40 individuals per test item group. Earthworms (Annelida: Oligochaeta), Eisenia fetida, Adults, approximately 6 – 7 months (all within 4 weeks of the same age), with well-developed clitellum was used. Body weight range of 305 to 594 mg, including gut contents was used. Earthworms were procured from a GLP accredited laboratory, Germany, confirmed by M/s. Ecoscience Research foundation, Taramani, Chennai, Tamil Nadu, India and bred under standardized conditions as per OECD 207 (1984) at IIBAT. Healthy adult earthworms were selected for the test. Earthworms were acclimatized for one day in artificial soil under test environmental conditions, fed with finely ground cow manure (ad libitum) sprinkled on the top of the soil. Earthworms were washed with tap water, blotted carefully with filter paper, individually weighed, and then released arbitrarily on the surface of the artificial soil after the soil was treated. After earthworm release, the perforated plastic lids were placed over the containers to prevent the test medium from drying and to avoid escape of the worms from the container.

Adults were exposed to treated soil for 4 weeks (28 days). After 4 weeks, adults were sorted from the soil and reweighed and discarded. The remaining soil was then returned to the containers with juveniles/cocoons and incubated under the same test conditions for an additional 4 weeks for reproduction assessment (56 days).

At the experiment start, 10 g/kg finely ground cow manure was mixed with the pre-moistened artificial soil before applying the test item. One day after application of Pseudomonas fluorescens (PF 5, 18, 25), 5 g (per container) of finely ground cow manure was mixed with 7 ml deionised water and scattered uniformly on the soil surface. Feed was added in the same way each week for the first four weeks of the experiment (i.e., day 1, day 8, day 15 & day 22). After the removal of the adult worms on day 28, 5g cow manure per container irrespective of the consumption was carefully mixed into the artificial soil to feed the juveniles. No additional food was given for the rest of the experiment.

The concentration of Pseudomonas fluorescens (PF 5, 18, 25) @10-6 Colony Forming Units/ mL was used for the experiment. 0.5mL/replicate (500g) of the required dilution was added.

The artificial soil was emptied from the containers and searched for earthworms on day 28 after application. The number of dead adult earthworms in each replicate was assessed. The earth worms that failed to respond to gentle stimulation were considered dead. Due to rapid decomposition under test conditions, missing earthworms were also considered dead.

Weights of all the live adult earthworms in each test container were taken at the test start (day 0) and at 28 days after application. The mean body weight change was calculated by comparing the mean earthworm body weight per replicate at start and end of the experiment.

After a total of 8 weeks (56 days), the experiment was terminated. The number of juveniles in each container was counted. Juveniles were collected from the soil by placing the containers in a water bath at approximately 60ºC. All emerging juveniles were removed and counted. Soil was then sorted manually to recover any remaining live young worms

After a total of 8 weeks (56 days), the experiment was terminated. The number of juveniles in each container was counted. Juveniles were collected from the soil by placing the containers in a water bath at approximately 60ºC. All emerging juveniles were removed and counted. Soil was then sorted manually to recover any remaining live young worms.

The software used to perform the statistical analysis was SAS 9.3 environment. Anova- Student-Newman-Keuls Test for post hoc comparison was done to compare the significant difference. No mortality and behavioral abnormality was observed in control and treatment group and analysis was not performed. The mean biomass changes (weight changes after exposure) in the control and the treatment were compared. Number of juveniles was analysed for each test treatment groups and control group. The data on food provision of the test item treated group and control groups was compared.

Adult worm mortality in the 28th day observation period did not exceed 10% (it was 0%).Mean loss of control earthworm biomass did not exceed 20% (was 2.62%). There were at least 30 juveniles per control unit containing 10 adults after the 8 weeks testing period (was 104 to 142 per replicate).The coefficient of variation for the mean number of juveniles in the untreated control did not exceed 30% (was 12.39%).Therefore, the test conditions are valid.

3. Results and Discussion

Pure cultures of selected Pf 5, Pf 18, Pf 25 colonies on King’s B agar plates were observed for shape, size and pigmentation. [Table 2]

No behavioral abnormality was observed in the treatments and control during the course of the experiment. It proves that PF does not affect its natural behavior. The mean biomass change of the worms exposed to Pseudomonas fluorescens (PF 5, 18, 25) was ranged from 5.93% to 6.32%. The mean body weight change of earthworms in the control was 2.62 %. (Table 3) Significant difference was observed in biomass change compared to control (One-way ANOVA). PF isolates increases the weight of the earthworms irrespective of all the three isolates tested. No mortality was observed in control and treatments with Pseudomonas fluorescens (PF 5, 18, 25)/kg dry soil. Hence the PF isolates donot have any lethal effect on earthworms.

The mean number of juveniles after exposed to Pseudomonas fluorescens (PF 5, 18, 25) was ranged from 142 to 166. The mean number of juveniles in the control was 117. The significant difference was observed in Pseudomonas fluorescens isolates (PF 5, 18, 25) compared to the control (One-way ANOVA). The increase in reproduction ranged from 99.98% (PF 25) to 100.80% (PF 18) over the control (Table 4). The increase in juvenile number was in the following order viz., PF 18> PF 5>PF 25.

The mean amount of food added to each treatment group and control during the first four weeks was ranged from 48.04 to 48.39 g. The amount of food added to the respective test containers over the entire test period was on par with the control (Table 5). Therefore no difference in their food habit is evident.

4. Conclusions

PF isolates (PF5, 18, 25) is not lethal to earthworms as no mortality was observed on 28-day observation. PF isolates (PF5, 18, 25) has not affected the reproduction rate of earthworms instead the treatment produced number of juveniles compared to control. Hence, it is recommended that PF5, 18, 25 can be used as ecofriendly biocontrol agent and growth promotor for crops. Further studies can be done with the isolates under field conditions to prove the suitability of the isolates (PF5, 18, 25) for field application.

Acknowledgements

The authors wish to thank the Management, Scientific Academic Board, IIBAT for providing the facility and inputs to conduct the experiment and Dr. R. Shanmugasundaram, Head, Department of Ecotoxicology and Entomology, Ross Life Sciences Private Limited., Pune for his support in this experiment.

References