Molecular detection of Sigatoka disease complex of banana in Cavite State University, Indang, Cavite / by Lois Daniel M. Jacela.

Thesis (Bachelor of Science in Agriculture Major in Crop Science) Cavite State University.

Includes bibliographical references.

College of Agriculture, Food, Environment and Natural Resources (CAFENR), Department of Crop Science

JACELA, LOIS DANIEL M. Molecular Detection of Sigatoka Disease Complex of
Banana at Cavite State University, Indang, Cavite. Undergraduate Thesis. Bachelor of
Science in Agriculture (Major in Crop Science). Cavite State University, Indang, Cavite.
January 2019. Adviser: Dr. Hosea dL. Matel.

This study was conducted from January to October 2018 at the Research Center and
Central Experiment Station of Cavite State University Indang, Cavite. This study aimed to (1)
assess disease severity of banana infected with Sigatoka Disease Complex (SDC) in Cavite
State University Indang, Cavite, (2) isolate fungal cultures from banana leaves with SDC and
determine fungal morphology through microscopic evaluation, (3) determine the
symptomatology of SDC in the locality, and (4) identify through PCR based detection the
presence of (a) Pseudocercospora fijiensis, (b) Pseudocercospora musae and (c)
Pseudocercospora eumusae.
Different areas planted with banana in CVSU, Indang, Cavite were assessed for the
disease severity of SDC: Central Experiment Station (CES), SAKA, EBA farm and Coffee Gene
Bank. Disease severity in the CES was 59%, 40% for SAKA, 31% for EBA farm and 30% for
Coffee Gene Bank.
Three microbial isolates were purified from 3 mm SDC-infected banana leaf sections by
sub-culturing on PDA plates. Fungal morphologies were observed through binocular
microscope, from slide culture preparation and after heat-fixing and staining of the cultures
using lactophenol cotton blue and crystal violet. Two of the isolates showed septate conidia with
clear color, while the ascospore was clear in color and have only one septum. The fungal
morphology for these two isolates resembles that of P.fijiensis. The other isolate showed
absence of septum in the conidia.
DNA was extracted from three purified isolates and subjected to Polymerase Chain
Reaction (PCR). Gradient PCR was used to optimize the annealing temperature of the primer
that was previously used for the detection of the SDC. Annealing temperature of 61 oc and 63
0C were suitable in producing good PCR product. Through PCR, 500bp amplicon size was

observed for gene specific primer pair ACTR/MFactF, which was the expected amplicon size
for P. fijiensis.
One of the isolates was further confirmed as P. fijiensis through demonstration of Koch's
postulate. Inoculation of healthy banana leaves with the isolate showed symptoms associated
with SDC after fourteen days of inoculation. The disease progressed to late stage after fifty
days of inoculation showing large black lesions surrounded by yellow halo. These similar
symptoms were observed in the field among SDC-infected bananas.
PCR based method confirmed the presence of Black Sigatoka Disease in banana growing
areas in Cavite State University, Indang Cavite.

Submitted to the University Library 02/11/2020 T-8517