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Molecular Characterization Reveals the Presence of Plant Pathogenic Pythium Spp. Around Bangladesh Agricultural University Campus, Mymensingh, Bangladesh

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

Mohammad 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

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

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

Abstract/ Executive Summary:

Oomycetes (water molds) are ubiquitous organism thriving in moist or aquatic environment. They can cause devastating infections to animals and plants. Farmers, both crop and fish, count huge losses every year due to outbreak of oomycete infections. However, the lack of study in Bangladesh makes it harder to address the problem in the country. This study was conducted to investigate the diversity of oomycetes in the surrounding crop fields and water bodies of Bangladesh Agricultural University campus, Mymensingh, Bangladesh. The sampling took place during the winter season. A total of 356 water samples were collected out of which only seven came positive with oomycete growth. The seven isolates were grown in potato dextrose agar (PDA) plates. The isolates were identified using molecular methods that included DNA extraction, PCR amplification and subsequent sequencing of the internal transcribed spacer (ITS) region of the genomic DNA of the samples. The BLAST analysis of the retrieved sequences to GenBank revealed that four of the isolates were Pythium catenulatum, two were Pythium rhizo-oryzae and the remaining isolate was Pythium torulosum strain. Pythium spp. are known to be plant pathogens of both crops and vegetables. The results suggest the evidence of plant pathogenic oomycetes around the study area that were causing damage to the crops and extend understanding of the diversity of the genus Pythium.

Keywords: Pythium, Internal transcribed spacer, Plant pathogenic, Oomycete, Polymerase chain reaction

Location: BAU campus, Mymensingh, Bangladesh.

Start Date: 27-11-2015

End Date: 15-02-2016

Program Area: Knowledge Management

Commodity: Aquatic animal

Objectives:

To study the diversity of oomycetes around Bangladesh Agricultural University (BAU) campus, through molecular characterization of ITS region for better understanding of the species/strains present compared to already reported species worldwide.

Methodology:

Sample collection: A total of 356 water samples were collected from 20 water bodies such as small ponds, ditches around the crop fields of BAU campus, Mymensingh, Bangladesh. To meet the research objective of the study, 9 samplings were carried out within the span of 79 days between 27th November, 2015 and 15th February, 2016. Water samples (10 mL) were collected during each sampling in individual 15 mL falcon tubes containing a sterile (autoclaved) mosquito as bait. Water samples with the pond water in falcon tube were kept in room temperature for 2 days. Mycelia culture in laboratory: All of the samples were cultured in a vertical laminar air flow cabinet (cleaned with 70% ethanol and treated in UV for 15 min before each use). Inoculation of colonized baits from water samples were carried out by placing it on 9 cm diameter petri dish of 25 mL potato dextrose agar (PDA, 39 g in 1 L dH₂O and autoclaved) medium supplemented with Vancomycin (200 mg L^–1), Ampicillin (500 mg L^–1) and Pimaricin (50 mg L^–1) and sealed with parafilm before being incubated at 18°C. Agar plug with mycelial growth were re-inoculated repeatedly on new plates still they were 100% bacteria free. DNA extraction: Once a bacteria free culture was attained, genomic DNA from the mycelium was extracted for further identification. Genomic DNA from the mycelia samples was extracted using a Wizard^® Genomic DNA purification kit following manufacturer’s instructions with some minor modification. The mycelium were grown in from GPY (GPY, glucose 3 g L^–1, yeast 0.5 mg L^–1, peptone 1 g L^–1, agar 0.5 mg L^–1, NaCl 0.5 mg L^–1) broth for 1 day at 18°C in incubator. The mycelia were completely dried on sterile filter paper inside laminar air flow cabinet. The dried mycelium was homogenized in 500 μL nuclei lysis solution supplied with the kit in a sterile tissue homogenizer and transferred into a 1.5 mL flip cap tube aseptically. The RNase A solution supplied in the kit was added in the tube and inverted several times. The mixture was incubated at 65°C for 45 min in heat block, with inversions every 15 min. After being cooled down to room temperature, 200 μL of mixture sample was transferred to a new 1.5 mL sterile microfuge tube and 400 μL of protein precipitation solution supplied with the kit was added. The mixture was vortexed vigorously for 5 min to emulsify the 2 phases. The tube was centrifuged (Centrifuge 5415R, eppendorf) at 16,000 rpm for 5 min. Five hundred microliters supernatant containing the DNA was carefully pipetted into a new 1.5 mL sterile microfuge tube that already contained 500 μL isopropanol. The tube was inverted until thread like strand of DNA appeared and incubated at 65°C for 5 min, before being centrifuged at 4°C at 16,000 rpm for 10 min. The supernatant was decanted carefully and the pellet was washed with 300 μL 70% ethanol several times. The pellet along with 70% ethanol was centrifuged at 13,000 rpm for 2 min. The ethanol was carefully air-dried and the pellet was re-suspended in 30 μL of DNA rehydration solution supplied in the kit. The tube was finally incubated at 65°C for 20 min, with gently tapping every 10 min. Each extracted sample was diluted as necessary to obtain 50 ng μL^–1 DNA concentration and checked further for quality by electrophoresis on 1% agarose gel. PCR amplification and band visualization: The PCR reaction was carried out in a thermal cycler (Eppendorf Mastercycler Gradient) to amplify the ITS region of the genomic DNA. To perform the PCR, 5 μL 2x GoTaq^® G2 Colorless Master Mix (Promega^®, contains DNA Polymerase, dNTPs, MgCl₂ and reaction buffer) was added with 1 μL template DNA (~50 ng μL^–1), 0.3 μL (10 μM) forward primer (ITS 5 alt: 5"TGA AAA GTC GTA ACA AGG TT 3"), 0.3 μL (10 μM) reverse primer (ITS 4 alt: 5"TCC TCC GCT TAT TGA TAT G 3") and 3.4 μL nuclease free water to make the final reaction volume of 10 μL. The 0.2 mL sterile PCR tubes were placed in a Mastercycler Gradient and the PCR reaction was run under the following conditions: 1 cycle at 95°C for 5 min followed by 30 cycles each having 95°C for 30 sec, 57°C for 30 sec and 73°C for 1 min finishing with a final elongation cycle at 73°C for 7 min. The reaction mixture was cooled down at 4°C for 5 min to end the reaction cycle. About 50 mL of 1% agarose gel containing 3 μL ethidium bromide (Promega^®, conc. 100 μg mL^–1) was prepared to test the quality of the DNA. Six microliters of a mixture of PCR sample (5 μL PCR product and 1 μL 6×loading dye added together) was loaded into each well of the gel with a micropipette. A 1 kbp DNA marker (Promega, USA) was loaded into 1st well in the gel to study the band size. The agarose gel was flooded with 600 mL TAE buffer in a gel running chamber and was run for 70 min at 120 V before photographing under UV light (High Performance Ultraviolet Transilluminator) to visualize DNA bands. DNA sequencing: The PCR amplicons were prepared according to the instruction of the sequencing company and sent off for sequencing to Bioneer, Korea. The PCR samples were cleaned up and diluted down to 40 ng μL^–1 that was a requirement outlined by the sequencing company. The generated sequences (fasta file) were aligned with the Sequence Alignment Editor Software (BioEdit) and consensual sequences introduced in Basic Local Alignment Search Tool in order to find matching species. The sequences obtained were compared with homologous sequences in the GenBank.

Source of Information: International Journal of Agricultural Research, 12: 199-205.

Article Link (Online Journal): https://scialert.net/abstract/?doi=ijar.2017.199.205

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Pythium spp. are devastating pathogens of plant roots and vegetables. Despite its huge economic importance, these oomycete pathogens are widely neglected. The lack of studies into the diversity of the pathogen have been one of the major issues contributing to the present sceranrio. Therefore, findings of study confirms the presence of Pythium spp. in the study area. Hence, it will contribute to future research on finding specific prevention and control measure of Pythium spp. in the crop field.

Knowledge Management: Journal