Molecular and serological approaches to detection of Ralstonia solanacearum (E.F. Smith) Yabuuchi in Banana (Musa spp.)

Date

3-1998

Abstract

This study devised a detection method for monitoring the strains of the bacterial wilt organism , Ralstonia solanacearum in banana. The method made use of monoclonal antibodies which were generated using a DNA PCR product for enhanced specificity. The DNA was conjugated with methylated bovine serum albumin (MBSA), a carrier protein and was used as antigen in producing the antibodies.

Fourteen monoclonal antibody cell lines were produced from the fusion experiment between myeloma cells and antigen-immunized spleen cells of mice.

Monoclonality of the hybridomas was ensured by cloning twice by the limiting dilution technique. Specific antibody-producing monoclonal cell lines were selected by the enzyme linked immunosorbent assay (ELISA) technique. Two monoclones were used successfully in mass producing the antibodies in mice.

Test of sensitivity of the produced banana monoclonal antibodies showed that the detection limit was at 1 x 107 cells/ml for whole cells in culture and in soil and 1 x 105 cells/ml for lysed cells.

Immunomagnetic separation (IMS) which consisted of using both polyclonal and monoclonal antibodies was tried in detecting the wilt organisms from the soil. However, there was no success in the test done due to the problem of high adherence of soil particles on the magnetic bead surface. Nevertheless, this technique offers a good promise when fully refined.

The banana antibodies were also applied in the detection of R. solanacearum from 75 laboratory cultures (maintained as water stocks) of different banana and non banana isolates of the organism and 131 field collected banana, non banana and soil samples. Four tomato strains from these laboratory isolates, which may actually be banana strains reacted with the banana antibody. On the other hand, a large proportion of the banana samples screened showed positive detection of the wilt organism especially those collected from the Mindanao area. Screening of soil samples showed varied distances where organisms was detected. The antibodies were also applied in indexing 208 banana plants from the orchard nursery of the Dept. of Horticulture, UPLB, Cavite and Quezon. Some plants registered positive reactions.

The activity and specificity of the banana antibodies were confirmed through cross reactivity tests againsts 12 different genera of bacterial pathogens and 10 species of Pseudomonas. None of the bacterial genera tested cross reacted with the M114 antibodies, however, one species of Pseudomonas, P. maltophilia, cross reacted with the R. solanacearum antibodies. No PCR amplification of the DNA of this organism was observed using M1114 primers.

This is pioneering work on the use of DNA as immunogen. The conventional method utilizes whole bacterial cells or other bacterial antigens in producing antibodies for the detection of a plant panthogen. This study also combines both serological and molecular approaches in generating reagent for the banana strain of the bacterial wilt organism.

Document Type

Dissertation

Degree

Doctor of Philosophy in Microbiology

College

Graduate School (GS)

Adviser/Committee Chair

Asuncion K. Raymundo

Committee Member

Ida F. Dalmacio, Adelina A. Barrion, Marina P. Natural, Rita P. Laude,

Language

English

LC Subject

Banana--Diseases and pests, Banana wilt

Location

UPLB Main Library Special Collections Section (USCS)

Call Number

LG 996 1998 M5 E92

Notes

Doctor of Philosophy ( Microbiology)

This document is currently not available here.

Share

COinS