Agricultural Research Management Information System

Home
Research Summary
Search
About Us
- ARMIS
- Brochure
Contact Us
Report
User Request
Data Input
Help
- Operation Manual
  - PDF
  - Video
- Program Area & Commodity
We have reached 37600 number of research entries at this moment.

- Logout

Research Detail

Home
Research
Detail

Molecular Identification of Oomycete Species Affecting Aquaculture in Bangladesh

Mohammad Nasif Sarowar
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Md Jakir Hossain
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Tahmina Nasrin
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Tania Naznin
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Zakir Hossain
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Mohammad Matiur Rahman
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

Fish mycotic disease outbreaks occur due to infections with oomycete pathogens such as Saprolegnia spp. and Aphanomyces invadans, and cause large-scale fish production losses. Despite its negative impact on aquaculture, little is known about the diversity of oomycete pathogens. The aim of this study was to identify the diversity of pathogenic oomycetes causing infections in major aquaculture zones of Mymensingh and Jessore regions in Bangladesh. A total of 449 water and infected fish samples were collected from 28 fish farms in both regions of which 29 samples were able to grow out with mycelia on the Potato Dextrose Agar (PDA)/Glucose Peptone Yeast Agar (GPYA) plates. Sequence database searches using the rRNA Internal Transcribed Spacer (ITS) region revealed that 15 belonged to Pythium spp., 12 were Aphanomyces invadans and two corresponded to Saprolegnia parasitica. Five isolates of Pythium spp. were identified to the species level: one was closely related to Pythium catenulatum, four to Pythium rhizo-oryzae, rest were identified up to genus. The Pythium spp. were only isolated from water samples whereas A. invadans and S. parasitica were found in fish lesions. Phylogenetic analysis revealed that a single A. invadans clone exists in the sampled area. The results obtained confirm the existence of pathogenic oomycetes in Bangladesh fish farms and this will pave future research on diversity, prevention and control measures.

Keywords: Oomycete, Saprolegnia, Aphanomyces invadans, Internal transcribed spacer (ITS), Phylogeny, Pythium

Location: Jessore and Mymensingh districts

Start Date: 15-11-2015

End Date: 09-02-2016

Program Area: Knowledge Management

Commodity: Aquatic animal

Objectives:

The objective of this study was to understand the diversity of oomycetes species causing infections in the aquaculture farms of Mymensingh and Jessore region of Bangladesh

Methodology:

Water samples and tissues from apparently infected fish were collected from different aquaculture farms located in Jessore and Mymensingh districts during the winter season from 15^th of November 2015 to 09^th of February 2016. Water temperature and pH of sampling sites were measured. All samples were collected from 28 commercial fish farms of which 16 were from Jessore and the remaining from Mymensingh region. The exact location of the farms are not disclosed due to the confidentiality agreement. A total of 449 samples were collected of which 248 samples (55.23%) were from Mymensingh and 201 (44.77%) from Jessore. There were 399 (88.86% out of 449 total samples) water samples (217 from Mymensingh and 182 from Jessore) collected from the farms and 50 (11.14%) samples (31 from Mymensingh and 19 from Jessore) were tissue taken from the apparently infected fish. Water samples (10 ml) were collected in 15 ml sterile falcon tubes. Each tube contained an autoclaved adult mosquito (Anopheles sp.) as bait. All water samples were kept at 18 °C temperature for seven days to enable the colonization of the baits. For tissue samples, the infected fish were euthanized using 1 g/L Tricaine Methanesulfonate (MS222; Sigma-Aldrich) bath until the opercula and bodily movements were ceased completely in order to comply with institutional experimental animal ethics. A small sample (around 0.5 cm³) of infected tissues or small portion of fin (0.5 cm) was cut with a sterile scalpel and scissor, and washed with autoclaved distilled water, to reduce the unwanted microorganisms load adhered to the surface. The collected samples were placed aseptically on Potato Dextrose Agar (39 g/L PDA in dH₂O and autoclaved) and GPYA plates (glucose 3 g/L, peptone 1 g/L, yeast 0.5 g/L, agar 0.5 g/L, Nacl 0.5 g/L) plates. All the collected tissue samples were incubated for a week at 18 °C. The samples that showed positive growth were used for further investigation. Laboratory culture of collected samples- The bait (mosquito) within the collected water samples were inoculated on Petri dishes (90 mm diameter) filled with 25 mL growth medium supplemented with antibiotics (100 mg/L Vancomycin, 500 mg/L Ampicillin and 20 mg/L Natamycin) to reduce bacterial growth. Two different growth media were used; Potato Dextrose Agar and GPYA. For inoculated fish tissues on plates, a small portion of mycelia growing out from the inoculum was aseptically inoculated on new PDA and GPYA plates supplemented with antibiotics. All the plates were sealed with parafilm and incubated at 18 °C. The isolates were re-inoculated several times to eliminate bacterial contamination. DNA extraction- A small agar plug with mycelia was aseptically placed in 20 mL Pea broth (124 g/L green pea autoclaved in dH₂O). The liquid media containing inoculum were incubated at 18 °C for 3 days to allow mycelia to grow. DNA was extracted from the grown mycelia using the Phenol: Chloroform: Isoamyl alcohol (with the ratio of 25:24:1) method according to quantified (Nanodrop, Thermo Scientific, USA) and stored at −20 °C for downstream analysis. Phylogenetic analysis- Phylogenetic analysis was carried out using the Maximum Likelihood (ML) and Bayesian inference (BI). The MEGA v7 program was used to align and construct the ML tree. The Aphanomyces and Saprolegnia analysis involved 25 and 31 nucleotide sequences (retrieved sequences available in Appendix A). The sequences were aligned with ClustalW in MEGA v7 and were manually adjusted and, positions containing gaps and missing data were eliminated. For the ML analysis the general time reversal (GTR) + Gamma (G) model was considered the best substitution model based on the lowest Bayesian information criterion (BIC) and Akaike information criterion value (AIC), and tree was built using 1000 bootstrap replications. For BI, the analysis was performed using MrBayes 3.2.6 using the same model as the ML tree. The BI analysis included four parallel running of one and half million generations carried out separately, for each run eight Markov chain Monte Carlo (MCMC) were done having one cold and seven hot chains (temperature 0.10) for both Aphanomyces and Saprolegnia sequences. The datasets were samples every 1000^th generation. The burn-in threshold was analyzed in the Tracer v1.5.0 (http:/tree.bio.ed.ac.uk/software/tracer/). The first 25% trees were discarded for each run, the remaining trees were used for estimating posterior probabilities (pp).

Source of Information: Aquaculture and Fisheries 4 (2019)105–113

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The results suggests that fish pathogenic oomycetes A. invadans and S. parasitica prevailed in fish farms during the winter and this indicates that fish mortality occurs due to EUS and saprolegniosis. Intense sampling could provide a clear idea on oomycetes diversity involved with fish infections throughout the year and would be helpful to identify pathogenic isolates from fishes that are commercially important.

Knowledge Management: Journal