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

Morphological and Molecular Identification of Fusarium Oxysporum Sch. Isolated from Guava Wilt in Bangladesh

M ZAKIR HUSSAIN
Program Officer, Higher Education Quality Enhancement Project, UGC

M A RAHMAN
Bangladesh Agricultural Research Institute, Gazipur

MOHAMMAD NURUL ISLAM
Department of Botany, University of Dhaka, Dhaka

M A LATIF
Bangladesh Rice Research Institute, Gazipur

M A BASHAR
Department of Botany, University of Dhaka, Dhaka

Abstract/ Executive Summary:

Wilt of guava plants (Psidium guajava L.) is a serious disease in Bangladesh. Sixteen isolates of Fusarium oxysporum Sch. were collected from the root and stem fragments of guava plants growing in six districts of Bangladesh. Species identity was based on the colony character, nature of conidiogenous cell, morphology of microconidia, macroconidia and chlamydospores. Eleven isolates were confirmed as F. oxysporum through polymerase chain reaction (PCR) using species specific primers designed from the conserved regions of 18S rRNA gene.

Keywords: Molecular identification, Guava wilt, Fusarium oxysporum, PCR

Location:

Start Date:

End Date:

Program Area: Pest Management

Commodity: Guava

Objectives:

In the present investigation, an attempt was made to characterize the wilt pathogen/s of guava occurring in Bangladesh on the basis of morphological characters and PCR analysis.

Methodology:

The fresh root and stem fragments from guava trees showing typical wilt symptoms were collected from Barisal, Brahmanbaria, Chittagong, Faridpur, Gazipur, Khagrachari, Pirojpur and Rangpur districts of Bangladesh. Collected samples were cut into 10 - 15 cm lengths washed in running tap water followed by sterile distilled water and then splited lengthwise and cut into thin small pieces (≤ 1 cm). The cut pieces were surface sterilized with 0.1% HgCl₂solution. The surface sterilized inocula were plated on half-strength PDA medium with three inocula per Petri plate (9 cm in dia.) (Burgess et al. 1975). Inoculated Petri plates were incubated at 26 ±1ºC for five days. Pure culture was obtained through culturing macroconidia in water agar (Burgess et al. 1994). Purified isolates were kept at 4ºC by culturing them on PDA slants and for long time preservation cultured on water soaked wheat bran medium. Well grown cultures in wheat bran were air dried, wrapped with brown paper and kept at 2 - 4ºC for further use. Morphological studies of the isolates were carried out by culturing them on PDA plates and wheat bran at 26 ± 1ºC for five days. Colony colour, radial growth and sporulation were recorded for all the isolates. Morphological characters were studied following Burgess et al. (1994) and Aneja (2003). Colony diameters of the isolates were measured after three days of incubation at 26 ±1ºC. The colony morphology was recorded on the 12th day of incubation at 25 and 20ºC during day and night, respectively. Genomic DNA of 16 isolates was extracted from mycelia grown in liquid medium. A modified DNA extraction method of Ali (2002) was followed in the present investigation and purified DNA samples were kept in – 20ºC freezer for further analysis. The PCR was initiated by a initial denaturation step at 94ºC for 5 minutes following 35 cycles of 94, 54 and 72ºC each for 30 sec, with a final extension step of 5 min at 72ºC and ended with 4ºC. The PCR was carried out in an Eppendorf Mastercycler with 25 micro tubes capacity. PCR amplified products were stored in – 20ºC freezer for analysis by resolving in 2% agarose gel. The gel was prepared with ethidium bromide (final concentration 0.5 μg/ml) and after resolving the PCR amplified DNA the gel was viewed in a gel documentation system (microDoc, Cleaver Scientific Ltd.) to take picture.

Source of Information: Bangladesh J. Bot. 41(1): 49-54, 2012 (June)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

In the present investigation, sometimes it was difficult to distinguish F. oxysporum from other species of Fusarium based on the morphological features. Therefore, molecular characterization of the F. oxysporum was conducted for proper identification using PCR analysis. The genomic DNA isolated from morphologically identified 16 F. oxysporum isolates was subjected to PCR amplification. It was expected to amplify a 118 bp size fragment of the F. oxysporum 18S rRNA gene in the PCR amplification reaction using gene specific primers designed from the conserved regions. After agarose gel electrophoresis of the PCR amplified DNA it was observed that the selected primer pair exclusively amplified the expected 18S rDNA band of 118 bp size in 11 morphologically identified isolates of F. oxysporum. On the other hand, no amplification was observed in case of the isolates 3, 8, 10, 12 and 15 and was not considered as F. oxysporum. Development of specific primers for F. moniliformae was also reported by other workers. Hence, the results described here proved that the primer pair allowed a fast, reliable and specific identification of Fusarium oxysporum isolates and could be suitable for early diagnosis of Fusarium wilt of guava by plant pathologists.

Knowledge Management: Journal