Appendix 2: PCR determination of Species of Colletotrichum
Nucleic acid detection methods such as PCR have become common tool for microbial
identification and diagnosis. PCR amplification can be performed directly for various
microbial cultures, for filamentous fungi and yeast, prior isolation of DNA is often
preferred. This method becomes an important role in ensuring consistent test results.
Material and method
Molecular characterization of single spore cultures of Colletotrichum species (C.
acutatum, C. gloeosporioides, C. capsici and C. boniense) were tested by determining
DNA characterization./DNA marking by using PCR.( polymerase chain reaction )
condition using ITS4 and ITS5 and Ca1NT2 primers and RFLP enzyme ( ALUI, BAMHI
and RSAI )
Rapid mini preparation of fungal DNA (Liu et al, 2000)
Key reagent preparation,
Lyses buffer: 400mM Tris-HCl (pH 8.0).60mM EDTA (ph 8.0).150mM NaCl.1% SDS.
Denature buffer: 100 ml solution made of 5M potassium acetate .11.5 ml of glacial acetic
acid and 28.5 ml of distilled water.
Protocol
1. A lump of mycelia obtained from cultural media was added into new 1.5 ml
eppendorf tube contained 500 ml lyses buffer.
2. Incubate at RT (room temperature) for 10 minutes.
3. Add 150 μl Denature buffer and vortex briefly.
4. Centrifuge (Cfg) 12000 rpm for 1 min. and transfer supernatant into new
eppendorf vial.
5. Repeat step 4 again.
6. Add the same volume of isopropanol and mix briefly (ca 600 μl) RT.20 min.
7. Cfg 12000 rpm for 2 min. and discard the supernatant.
8. Wash the pellet with 300 μl 70% alcohol and dry the pellet 30 min.
9. Re-suspense pellet in 50 μl TE buffer.
10. One μl was used for PCR.
Appendix 3 Methods for testing Pathotype of Phytophthora
capsici accessions
Inoculum preparation

Culture P. capsici on V8 juice agar medium. Transfer a 7 mm mycelia plug to the
center of each 9-cm Petri plate and incubated at 28 o C with continuous lighting for 4
days.

Cut each agar culture into four equal pieces with sterile knife: move each piece to
an empty sterile Petri plate and cut into approximately 0.5 cm2 blocks.

Add sterile water to cover the agar blocks and incubate at room temperature for one
hour: decant and replace with 18 ml sterile water should bring the level about the
upper surface of the agar block.

Incubate the flooded Petri dish for 24 hr at 28 oC with continuous lighting to produce
sporangia.

Transfer the plats to a 4 oC for I hr to initiate zoospore formation.

Return to the 28 oC lighted incubator for 1 hr zoospore release.

Decant the zoospore suspension, and if needed wash the agar blocks to get greater
recovery of zoospore.

Transferred representative sample of the suspension to a haemocytometer and heat
on a slide warmer at 40 oC for one min to immobilize the zoospore for counting.

The stock zoospore suspension should be kept cool and the appropriate dilution
made using chilled water immediately prior to inoculation.
Inoculation and incubation
Twenty eight to 35 day old seedlings were inoculated with 5 ml of inoculum (10 5
zoospores / ml) at the base of each plant, soon after watering. The inoculated plants
were incubated in incubation room with day time temperature was maintained 30 oC:
mist blower was on and adjusted to maintain high soil moisture.
Disease reaction categories
Observations were done at 1, 2, 3 and 4 weeks after inoculation. Based on the
percentage of surviving plants 30 dai, pepper line are categorized as follow:

Resistant = 80 – 100 % survival plants

Moderately resistant = 50 – 70 % survival plants

Moderately susceptible = 20 -40 % survival plants

Highly susceptible = 0 – 19 % survival plants.
Standard pattern of standard differential plants is mention in Table 2.
Table 2.Standard pattern of four standard differential plants
Lines/PI No
Resistance to
Pathotype -1
Pathotype-2
P-3athotype
Early Calwonder
S
S
S
PBC-137
R
S
S
PB C-602
R
R
S
PI-201234
R
R
R
Appendix 4: Use of Dot Immunobinding Assay (DIBA) for
detection of plant viruses
Sri Hendrastuti Hidayat1), Dedek Haryadi1), Endang Opriana1), Sri Sulandari2)
Department of Plant Protection, Faculty of Agriculture, Bogor Agricultural University
Jalan Kamper, Darmaga Campus, Bogor 16680
Department of Plant Pests and Diseases, Faculty of Agriculture, Gadjah Mada University
Jalan no.1, Flora Bulaksumur Campus, Yogyakarta 55281
INTRODUCTION
Methods developed for plant virology have been of central importance to other branches
of plant pathology. The requirement to work with viruses at the subcellular level drove
technology to develop new tools for their study. Since the 1930s, researchers in the field
of plant virology have contributed greatly to universally applicable methodologies. Until
now, methods based on the protein component of the viral particle have been mainly
used in plant virus detection. Enzyme-linked immunosorbent assay (ELISA) and Dot
immunobinding assay (DIBA) has become the most popular methods of serological
detection of viruses in plants, because of its simplicity and wide applicability. Detection
of plant viruses using DIBA has been reported among others for tobacco mosaic virus
(TMV) (Somowiyarjo et al., 1997), and begomoviruses (Sulandari et al., 2004). In our
study, we confirmed the application of DIBA for detecting pepper yellow leaf curl
Indonesia begomovirus (PYLCIV) and chilli veinal mottle potyvirus (ChiVMV).
MATERIALS AND METHODS
Plant samples involved in the study consist of tomato, eggplants, cucumber, chilli
pepper, weed (Ageratum conyzoides) artificially inoculated with PYLCIV; different
genotypes of chilli pepper artificially inoculated with ChiVMV; and viruliferous whiteflies
given acquisition feeding period on PYLCIV-infected plants.
Young, upper leaves of infected (symptomatic) and non infected plants were
harvested and crushed individually in microfuge tubes in tris buffer saline (Tris-HCl
0.02M, NaCl 0.15M, pH 7.5) with the ratio of 1:10 (b:v). Tubes were centrifuge at 10,000
g for 2 min, and 10 µl of the supernatant of each sample was spotted on nitrocellulose
membrane (Hybond–P, Amersham Biosciences, UK) for DIBA according to Mahmood et
al. (1997). Similar procedure was used for assay using viruliferous whiteflies.
Polyclonal antibody for PYLCIV used in the assay was produced specifically to
PYLCIV isolate Segunung (PYLCIV-Bgr) whereas antibody for ChiVMV was purchased
from DSMZ, Germany.
RESULTS AND DISCUSSION
Viral antigens were readily detected on membranes when plant sap was spotted.
Positive reaction was indicated by development of purple colours on the membrane at
the sample spot.
Antibody for PYLCIV-Bgr at a 1/1000 dilution gave strong reaction to chili pepper,
tomato, and A. conyzoides, but no reaction was noticed with eggplant and cucumber.
Positive reaction was observed on symptomatic as well as non symptomatic leaves
indicating that antibody for PYLCIV-Bgr has a good detection level. Polyclonal antibody
for PYLCIV was able to detect the virus from group as well as single viruliferous
whiteflies. This evidence showed the potential use of the assay to be used as a tool to
study the epidemiology of the diseases.
Similar results were observed when polyclonal antibody to ChiVMV was used to
detect the infection of ChiVMV in chilli pepper lines. Based on signal intensity on the
membrane it can be concluded that the ability of the virus to replicate and distribute
systemically is correlated to the host response. Strong positive signal was evidenced on
chilli pepper lines with susceptible response or when using virulent isolate. This assay
could be suggested as a useful tool for screening germplasm activity in breeding
program.
REFERENCES
Mahmood T, Hein GL, French RC. 1997. Development of serological procedures for
rapid and reliable detection of wheat streak mosaic virus in a single wheat curl mite.
Plant Dis 81:250-253.
Somowiyarjo S, Sumardiyono YB, Suharno. 1997. Pemanfaatan membran
nitroselulosa untuk pengiriman antigen uji dalam deteksi TMV dengan DIBA. J Perlind
Tan Indo 1:1-5.
Sulandari S, Rusmilah S, Sri HH, Soemartono S, Jumanto H. 2004. Pembuatan
antiserum dan kajian serologi virus penyebab penyakit daun keriting kuning cabai. . J
Perlind Tan Indo 10:42-52