ETIOLOGICAL STUDY OF BLACK SPOT DISEASE ON ROSE LEAVES FROM KOTA

SAMARAHAN, SARAWAK

FATIN FATANAH BINTI RAMLE

Bachelor of Science with Honours

(Resource Biotechnology)

2010

Faculty of Resource Science and Technology

ETIOLOGICAL STUDY OF BLACK SPOT DISEASE ON ROSE LEAVES FROM

KOTA SAMARAHAN, SARAWAK

FATIN FATANAH BINTI RAMLE

This project is submitted in partial fulfillment of the requirements for the degree of

Bachelor of Science with Honours

(Resource Biotechnology)

Faculty of Resource Science and Technology

UNIVERSITI MALAYSIA SARAWAK

2010

DECLARATION

I hereby declare that no portion of this dissertation has been submitted in support of an

application for another degree of qualification of this or any other university or institution of

higher learning.

............................................................

(FATIN FATANAH BINTI RAMLE)

Resource Biotechnology Programme

Department of Molecular Biology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak.

i

Acknowledgement

Praised be to Allah, who has blessing me with the strength to accomplish this

project promptly. Special thanks goes to my supervisor, Prof Dr Ismail Ahmad, who is

most responsible for helping me to complete the writing of dissertation, had confidence in

me when I doubted myself, and brought out the good ideas through this challenging

research. He showed me different ways to approach a research problem and the need to be

persistent to accomplish any goal. Besides, I would like to thank my laboratory mates who

gave insightful comments and reviewed my work. Heartfelt thanks are also for my family

and fellow course mates for their help and encouragement that make this project possible.

Above all, i want to express my deep gratitude to everyone who has contributed directly or

indirectly in the completion of this project, all your kindness and patience are highly

appreciated.

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TABLE OF CONTENT

ACKNOWLEDGEMENT i

TABLE OF CONTENT ii

LIST OF ABBREVIATIONS v

LIST OF TABLES

LIST OF FIGURES

ABTRACT

vi

vii

1

1.0 INTRODUCTION & OBJECTIVES

2

2.0 LITERATURE REVIEW

2.1. Roses and their Significance

2.2. Symptoms of Common Diseases on Rose Leaves and their Implications

2.2.1. Powdery Mildew

2.2.2. Black Spots

2.2.3. Rust

2.2.4. Virus Infection

2.2.5. Downy Mildew

2.3. Diagnosis and Its Importance

2.4. Isolation and Identification of Etiological Agent

2.4.1. Koch’s Postulates

2.4.2. Leaf Inoculation Methods

2.5. General Methods of Controlling Rose Diseases

2.6. Chemical Fungicide Applications

2.7. Chemical Control of Rose Fungal Growth

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3.0 MATERIALS & METHODS

3.1. Sampling

3.2. Isolation and Subculture

3.3. Detached-leaf Inoculation

3.4. Shoot Inoculation

3.5. Assessment of Rose Leaf Infection

3.5.1. Mycelium Growth

3.5.2. Conidia and Ascocarps Formation

3.6. Identification of Fungus

3.6.1. Slide Culture Method

3.7. Fungicide Test

3.7.1. Measurement of Fungal Growth

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4.0 RESULTS

4.1. Isolation of Fungi from Infected Rose Leaves

4.2. Results of Inoculation

4.2.1. Detached-leaf (in vitro) Inoculation

4.2.2. Shoot Inoculation

4.3. Identification of Isolate 10.1

4.3.1. Disease Symptoms Assessments

4.3.2. Etiological Agent Morphology

4.4. Result of Slide Culture

4.5. Effect of Fungicide on Colony Growth

5.0 DISCUSSION

5.1. Isolation of Fungi from Infected Rose Leaves

5.2. Detached-leaf Inoculation and Re-isolation

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5.3. Shoot Inoculation

5.4. Identification of Etiological Agents of Black Spot Disease

5.4.1. Mycelium Growth of Fungus

5.4.2. Development of Ascocarps

5.4.3. Identification using Slide Culture

5.7. Effect of Fungicide on Fungal Growth

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6.0 CONCLUSION & RECOMMENDATION

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REFERENCES

APPENDIX A

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v

LIST OF ABBREVIATIONS

a.i. active ingredient

cm centimeter

i.i. inert ingredient

mg miligram

mL millilitre

mm millimeter

PDA potato dextrose agar

RMV rose mosaic virus

w/w weight over weight

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LIST OF TABLES

Page

Table 4.1 Detached-leaf inoculation of 25 isolates through 3 series of

inoculations

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Table 4.2 Means of fungal growth measurement from 4 replicates 20

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LIST OF FIGURES

Page

Figure 4.1 Symptom of necrosis (black area) was observed on the upper

surface of rose leaf during the 7th

days of shoot inoculation.

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Figure 4.2 Observation under light microscope with 60X magnification.

Detection of fungal growths indicating the dark color of

mycelia which were fully covered the inoculation site on rose

leaf after 3 days of in-vitro inoculation.

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Figure 4.3

Observation under light microscope with 60X magnification.

Growth of fungal mycelia on the surface of rose leaf. Color

of the mycelia represents dark grey or almost black.

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Figure 4.4

Compound microscope photographs (60X magnification) of

black spots symptoms on different inoculated leaves.

Different stages of disease symptom development on rose

leaves infected with the same isolate (10.1). (a) Circular

shape of dark lesion appeared on the upper surface of leaf;

(b) and (c) irregular shape of black spots observed on the

upper surface of infected leaves.

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Figure 4.5 Observation under compound microscope with 60X

magnification. Several elliptical hyaline conidia found

adjacent the masses of mycelia which composed by a

network of septate hyphae.

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Figure 4.6 Compound microscope photographs of ascocarps with 60X

magnification. Cleistothecia were found after 2 weeks in

vitro inoculation. The appendages are non-branching and

black in color. (d) Four cleistothecia observed on glass slide

taken from infected leaf 2 weeks after inoculation; (e)

ruptured cleistothecium releasing ascospores.

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Figure 4.7 Means of fungal growth from 4 replicates were plotted on a

graph where fungal growth measurement (cm) versus period

of fungicide test (day).

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1

Etiological Study on Black Spot Disease of Rose Leaves from Kota Samarahan, Sarawak

Fatin Fatanah Binti Ramle

Resource Biotechnology

Department of Molecular Biology

Faculty of Resource Science and Technology

University Malaysia Sarawak

ABSTRACT

Rose (Rosa sp.) is an ornamental plant of choice for home gardening, landscaping and commercial

growing for many years. Unfortunately, several prevalent diseases including the black spot disease have

been devastating the health, aesthetic value and its marketability. Hence, before an efficient control

method can be applied to overcome the problems, this study aims to identify the etiological agent on the

black spot disease on rose leaf by means of Koch’s Postulate. Isolated pathogens from infected rose

leaves were inoculated via in vitro leaf inoculation and shoot inoculation. Infected leaves were then re-

isolated and identified the etiological agent using slide culture method. Further fungicide test was done

on the growth of the fungal pathogen by utilizing chemical fungicide. A total of 25 isolates were

completely used as inoculum source to infect the young rose leaves. However, only 1 isolate was

showed the black spot symptom at the end of the both inoculation experiments. Since the reproductive

structures developed in 2 different representative species, which are Uncinula sp. in teleomorph and

Marssonina rosae in anamorph, these have made the study unable to determine the actual causal agent

of black spot disease on rose leaf. Thus, further research need to be carried out to investigate the

presence of mixed pathogens on the disease. Nevertheless, both of the causal agents gradually reduced

in their length of colony growth when chemical fungicide was applied.

Keywords: etiological agent, Rose, black spot

ABSTRAK

Ros (Rosa sp.) adalah tumbuhan perhiasan yang telah menjadi pilihan selama bertahun-tahun bagi

tanaman rumah, landskap dan tanaman komersial. Malangnya, beberapa penularan penyakit termasuk

penyakit tompok hitam telah memusnahkan kesihatan, sifat semulajadi dan pasarannya. Maka, sebelum

kawalan yang efisyen diaplikasikan untuk mengawal masalah, kajian ini bertujuan mengenal pasti agen

yang menjadi punca pada penyakit tompok hitam berdasarkan peraturan Koch. Patogen yang telah

dipencilkan daripada daun ros yang berpenyakit telah diinokulasikan melalui inokulasi daun in vitro

dan inokulasi pucuk. Daun yang telah dijangkiti penyakit kemudian dipencilkan sekali lagi and

mengenal pasti punca penyakit dengan meanggunakan kaedah kultur slaid. Ujian racun kulat

kemudiannya telah dijalankan terhadap pertumbuhan patogen kulat dengan menggunakan racun kulat

kimia. Sejumlah 25 pencilan telah digunakan sepenuhnya sebagai sumber inokulasi untuk menghasilkan

penyakit pada daun muda ros. Bagaimanapun, hanya 1 pencilan telah menunjukkan tanda penyakit

tompok hitam di akhir kedua-dua eksperimen inokulasi. Disebabkan struktur pembiakan dihasilkan

daripada 2 spesis yang berlainan, iaitu Uncinula sp. pada peringkat teleomorph dan Marssonina rosae

pada peringkat anamorph, kajian ini tidak dapat mengenal pasti punca agen yang sebenar pada

penyakit tompok hitam ke atas daun ros. Jadi, kajian lanjut perlu dijalankan untuk menyisat kehadiran

patogen yang bercampur pada penyakit. Walau bagaimanapun, kedua-dua agen telah menunjukkan

pengurangan dalam ukuran pertumbuhan koloni apabila racun kulat kimia diaplikasikan.

Kata Kunci: punca agen, ros, tompok hitam,

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1.0 INTRODUCTION

Rose (Rosa sp.) is an ornamental plant of choice for home gardening, landscaping and

commercial growing for many years (Nameth & Chatfield 1996). Due to the admiration of

delightful properties on roses throughout the world, roses have been classified as the queen

of flowers (West, 2004). Unfortunately, this plant is prone to infection by several plant

pathogens which cause diseases and gradually destroy its health, aesthetic value and

marketability.

Several prevalent diseases on rose leaf including black spot (Leahy, 1990),

powdery mildew (Sharma, 2006), rust (Hoffer et al., 2000), virus infection (Hagan and

Mullen, 2007) and downy mildew (Pirone, 1978), have been identified. These diseases can

cause damages to the whole or may be parts of the rose plant. Accordingly, strategies in

coping with the crisis need to be considered in order to overcome the problems. However,

before control strategies can be efficiency applied, isolation and identification of the

etiological agents must first be carried out.

Recently, garden rose plants in of a resident from Kota Samarahan were observed

to come down with symptoms of leaf spots. The leaves became yellowish and then fall to

the ground. The disease also affected the flowers and caused the petals of the flowers to

lose their shape and beauty. Hence, before control method can be applied, this study aims

to isolate and identify the causal agent of the disease.

Objectives:

1. To identify the etiological agent/agents of rose diseases on selected leaves from

Kota Samarahan, Sarawak by using Koch’s postulates.

2. To determine the effects of fungicide on the growth of fungal pathogen.

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2.0 LITERATURE REVIEW

2.1 Roses and their Significance

Hawke (1997) maintains that, the popularity of roses is centuries old as the beauty of the

flower has been used extensively in gardens, landscapes, and in cut flower arrangements

(Hameed et al., 2006). Besides, Hofer et al., (2000) claims that, roses are the most popular

perennial flowering plant in the United States and in fact, they grow well in the pacific

Northwest. They are grown commercially in containers, as field liners and in greenhouses.

In addition, each of rose species has their own unique fragrance, form and color.

Accordingly, the extraordinary characteristics on roses have been considered as a premier

flowering woody ornamental plant (Leahy, 1990). However, in order to grow roses

successfully and to preserve the great beauty of roses, the health maintenance of roses

should be considered seriously (Hawke, 1997).

2.2 Symptoms of Common Diseases on Rose Leaves and their Implications

2.2.1 Powdery Mildew

Bilgrami and Dube (1993) stated that, powdery mildew is easily recognized by the white

powdery coating on the plants, which consist of enormous conidia formed by the fungus.

Fungal pathogen that caused powdery mildew on rose leaf is known as Sphaerotheca

pannosa (Nameth & Chatfield, 1996). When the powdery mildew fungus infects young,

expanding leaves and shoots, twisting and distortion will often occur in addition to the

development of the white, powdery fungal growth. Leaf shedding occurs when plants are

severely infected. Besides, repeated powdery mildew outbreaks can reduce plant vigor and

possibly bud set (Hagan and Mullen, 2007).

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2.2.2 Black Spots

Rose black spot is one of the most severe diseases on rose leaves (Wallis and

Lewandowski 2008; Malek and Debener, 1998). The disease symptoms signing with small

dark lesions; vary in size from 2-12 mm and surrounded by yellow tissue (Mangandi and

Peres, 2009; Patridge 2008). According to Patridge (2008), the disease is caused by

Diplocarpon rosae (sexual stage) and Marssonina rosae (asexual stage). In severe cases,

leaf spots tend to coalesce and cause extensive necrosis and abscise prematurely (Horst,

1983; Coyier, 1985).

2.2.3 Rust

Several rust fungi of the genus Phragmidium are detected to be the causal agent of rust

disease on rose leaf (Hoffer et al., 2000). According to Bilgrami and Dube (1993), rust

diseases have attracted maximum attention from the plant pathologist due to the

destructiveness and vexed biology. Orange appearance or rust colored growth on the

underside of the leaves is usually a symptom of rust disease on rose plant. Unlike infection

of black spot disease, younger leaves tend to show symptoms after the older leaves. Under

favorable conditions, rust can cover the entire leaf of the rose plant and sometimes cause

premature defoliation (Nameth and Chatfield, 1996).

2.2.4 Virus Infection

The effects of viruses in rose plants often appear as yellow ring spots, erratic white to

yellow line patterns, and yellow-green mottle on the leaves (Pirone, 1978; Hagan and

Mullen, 2007). They are marked with a pattern of light and dark areas forming a “mosaic”.

Such symptoms are frequently associated with curling of the leaves and growth other

abnormalities of growth. Hagan and Mullen (2007) claim that, the most obviously

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symptoms of virus infection are during periods of rapid growth in the spring and fall.

Therefore, results in the reduction of plant vigor and become more susceptible to the

damage caused by extreme drought or low temperatures (Hagan and Mullen, 2007). The

most common virus that causes rose mosaic disease is Rose Mosaic Virus (RMV)

(Goldberg, 2006).

2.2.5 Downy Mildew

Downy mildew disease is usually characterized by large red to brown angular spots

sometimes surrounded by a yellow halo appear on the leaves (Bilgrami and Dube, 1993).

The yellow blotches typically develop before the diseased leaves are shed. Besides, other

properties of downy mildew include elongated, discolored blotches in about 0.5-1 inch in

length. Moreover, under favorable conditions, sparse, gray spore masses are sometimes

seen on lesions on the lower leaf surface (Hagan and Mullen, 2007). The disease is caused

by Peronospora sparsa (Hagan & Mullen, 2007).

2.3 Diagnosis and Its Importance

Diagnosis of etiology of a disease infecting a plant is utmost important to prevent plant

productiveness and strategies in the control procedure. Exact species identification of

fungal pathogens is essential in plant pathology since misidentification may lead to

erroneous management advice (Feau et al., 2009). Accordingly, reliable pathogen

identification is critical first step in assessing potential impact, disease risk and

determining management options (Desprez-Loustau et al., 2007).

Fungal identification is traditionally accomplished using dichotomous keys, though

synoptic, multiple entry and probabilistic keys are sometimes used. Dichotomous keys, and

to a lesser extent the others, can be very sensitive to individual characters, and characters

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which cannot be determined, are typical or wrongly determined can result in no

identification or misidentification (Bridge et al., 1994; Morris & Boddy, 1995). More

often, a book a manual pictorial or diagrammatic representation of the different genera

(Barnet & Hunter, 1972) can assist identification to genera level or event to species level.

This is usually not a problem for experts in a particular group, but it is for non-

experts, and there is an urgent need for tools which assist accurate identification by the

latter. Expert systems are possible solution and some have been constructed for

identification of fungi and fungal diseases (Petrini & Rusca, 1989; Rose, 1992). Obtaining

and encoding the appropriate knowledge base is, however, no easy task (Morris & Boddy,

1992; Bridge et al., 1994).

Artificial neural networks (ANNs) are another possible solution. They do not

require expert taxonomic knowledge for their implementation, only a large accurate set of

data and once implemented, can cope with incomplete, partly contradictory ` fuzzy' date

(Morris & Boddy, 1995; Morgan et al., 1998)

2.4 Isolation and Identification of Etiological Agent

Successful control of plant diseases depend on accurate identification of the pathogen and

the disease (Burgess et al., 2008). Before identification can be done, the probable of

etiological agents must first be isolated. A number of studies have been conducted to

determine the etiological agent of rose diseases (Kiss et al., 2001; Debener & Linde, 2003;

Heffer et al., 2000). Proper technique of isolation intergrated with a precise identification

of the causal agents may lead to a better result and achieve the best solution to overcome

the disease problem.

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Gachomo and Kotchoni (2010) stated that one of the most important factors that

influence the effectiveness of an intergrated plant disease management program on rose

plant is the variation of the causal agents. For this reason, it is very essential to recognize

and characterize the causal agent appropriately in order to design a suitable method of

controlling the disease.

2.4.1 Koch’s Postulates

In order to verify a causal agent on a particular disease, a scientific rule should be used as a

basic rule and a guidance to firmly establish the link between germ and the disease

(Bilgrami and Dube, 1993). Koch’s postulates are a set of criteria for experimentally

determining whether a particular pathogen is the cause of a particular disease. Based on the

Koch’s postulate, there are four requirements that must be accomplished for an organism to

be verified as a cause of a disease.

In this context, firstly, the organism must be present in all cases of the black spot

disease. Secondly, the organism must be able to be isolated from the host which is rose leaf

and grown artificially in a pure culture in controlled conditions. Besides, it is necessary for

the organism to be reproduced and caused the same disease (black spots) when introduced

into another healthy rose leaf. Lastly, the same organism must be able to be recovered and

purified from the rose leaf that was experimentally infected.

2.4.2 Leaf Inoculation Methods

Detached-leaf or in vitro leaf inoculation is an efficient method to screen the virulence of

pathogenicity of isolates. A study by Yokomi and Gottwald (1988) proved that the

technique was excellently utilized in order to determine the pathogenicity of the causal

agent in aphids. Detached-leaf inoculation method was claimed to be a rapid technique.

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Besides, the disease symptoms can be measured precisely due to the high degree of

replication of the causal agent (Benedikz et al., 1981).

Another leaf inoculation method includes the shoot inoculation technique.

Essentially, shoot meristems surrounded by a few leaf primordia, are cut and selected as

the inoculation materials (Covert et al., 2002). These shoots are inoculated with inoculum

by means of natural conditions including temperature and moisture to stimulate a better

plant inoculation.

2.5 General Methods of Controlling Rose Diseases

Cultural practices are now being considered as essential back up procedures for

management of resistance varieties. However, due to the meteorological and biotic factors,

specific cultural practices are often locally restricted in their application (Singh, 1984). The

same practice may have an entirely different effect under different climate regimes, on

different host plant, and of course against pathogen differing in their modes of action and

survival (Palti and Rotem, 1983).

Each cultural practice has a direct and indirect effect on the development of rose

plant diseases (Palti and Rotem, 1983; Pataky, 1987). For instance, Leahy (1990)

maintained that planting resistant varieties is one of the most common cultural controls

used to avoid many rose plant diseases (Leahy, 1990). Nevertheless, Hoffer et al., 2000

argued that it might be hard to find a cultivar of disease resistance with the color and

fragrance desired at the same time (Hoffer et al., 2000). This is further strengthened by

Singh (1984) which suggested that successful use of cultural practices for disease control

can be made only when a complete knowledge of the nature of the pathogen and its

behavior are available.

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The availability of various cultural control strategies in the management of rose

diseases is of utmost important. In order to increase their effectiveness, fungicide

application should be integrated with the cultural practices. Only then maintenance of plant

health and best results of plant growth can be achieved (Leahy, 1990). The efficacy of

fungicide used in the field depends on a number of factors that operate independently or

jointly. These factors may be the characters of the fungicide, prevailing environment at the

time of application, timing and method of application of the fungicide (Singh, 1984).

2.6 Chemical Fungicide Applications

The application of chemical fungicides to the growing ornamental plant, especially on rose

plant has been for about twenty years a principle means of disease control or prevention

(Bain et al., 1989). Successful control of plant diseases by chemicals depends upon the

correct usage of fungicide. Furthermore, for reasons of safety and the prevention of

environmental side effects, chemicals should be always used carefully (Booth and Burden,

1983).

2.7 Chemical Control of Rose Fungal Growth

Chemical controls are successful as long as cultural and sanitation practices are followed.

In order to work effectively, applications must be made preventively, providing a

protective fungicide barrier and simultaneously kills germinating fungal spores that land on

uninfected leaves (Wallis and Lewandowski, 2008). Therefore, by knowing the actual

etiological pathogen of the disease, compatibility and simplicity of fungicides could be

utilized as one of the alternative methods to determine their effectiveness on pathogens

growth.

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Some examples of fungicides that are marketed toward the homeowner market and

labeled for the control of black spot of rose include Captan, Chlorothalonil Mancozeb,

Myclobutanil, potassium bicarbonate, propiconazole, thiophanate-methyl, copper

hydroxide, copper salts, lime sulfur, neem oil, and sulfur (Wallis and Lewandowski, 2008).

Besides, zineb is one of the organic sulphur compounds which also called

dithiocarbamates. Organic sulphur compounds are the most important, most versatile and

most widely used group of modern fungicides. Zineb is sold as Dithane Z-78 and is

excellent, safe multipurpose foliar and soil fungicide for the control of leaf spots, blights,

fruit rots of vegetables, flowers, fruit, tree and shrubs (Pirone, 1978).

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3.0 MATERIALS & METHODS

3.1 Sampling

Diseased leave samples were obtained from rose plants from home gardener in Kota

Samarahan, Sarawak. Several infected leaves were randomly collected from unidentified

rose cultivars and brought to the laboratory in a clean container.

3.2 Isolation and Subculture

Isolation of fungi from rose leaves was done according to isolation method by Choi et al.,

(1999) with some modifications. First of all, disease symptoms of rose leaves were

carefully examined including the shape and the size of lesions. After properly cleaned the

working bench area with 70% ethanol, the infected leaves area (black spots), were cut into

small pieces from the edge of the lesions, approximately 2-4mm per by means of a sterile

surgical blade.

For surface sterilization, the cut tissue pieces were soaked in a beaker containing

sodium hypochlorite for approximately 5 minutes before drenched into 70% ethanol for 3

minutes. After blotting the tissues on sterile filter paper, the surface-sterilized samples

were aseptically placed on potato dextrose agar (PDA). The plates were incubated at room

temperature (25⁰C).

The plates were examined after 2-4 days regularly to avoid overgrowth of fungus

within the same plate. After 4 days of incubation fungal mycelium were transferred into

new PDA plate. Agar slants on PDA were also prepared for longer period of storage.

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3.3 Detached-leaf Inoculation

Detached-leaf inoculation technique is a modification of in vitro methods described by

Yokomi and Gottwald (1988). Prior to the inoculation process, each leaf was surface

sterilized by spraying with 70% ethanol and immediately followed by washing with sterile

distilled water. Subsequently the leaves were then artificially injured by using sterile

needle and placed in sterile petri dishes. Sterile tooth picks were used to prevent the leaves

for touching the lower surface of the petri dishes.

In this method, the inoculum agar discs containing fungal mycelia were cut from

the culture aseptically. Finally, fungal isolates were placed onto the injured surface of a

sterilized young leaf. Other inoculums were similarly placed onto the young leaves

aseptically. All the petri dishes were sealed with parafilm and incubated at room

temperature (25⁰C) for one week before observation under light microscope. Experiment

of inoculation was repeated three times following similar techniques as completed before.

3. 4 Shoot Inoculations

Fungal pathogen selected from positive results in the third in vitro inoculation was further

inoculated with a slightly different condition. In the first post-inoculation, a single leaf was

inoculated by a single type of suspected fungal pathogen and was incubated in petri dish.

Final inoculation was done by inoculating the actual fungal pathogen on a leaf rose of a cut

shoot with a natural growth condition.

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3.5 Assessment of Rose Leaf Infection

3.5.1 Mycelium Growth

One week after, observations of all inoculated pathogens (detached-leaf inoculation and

shoot inoculation) on healthy young leaves were done. Agar discs containing the mycelium

of fungal growth were removed from the rose leaves surface aseptically by using sterile

loop. The leaves were then examined under light microscope with the different

magnifications at 4X, 10X and 60X. The presence of the mycelia from the injury holes

were used as indicator of infection which is positive result. Besides, the color of the leaf

and color of the mycelia were studying and described.

3.5.2 Conidia and Ascocarps Formation

Second observation was done two weeks later from the date of inoculation. Conidiophores

and ascocarps found were examined under different magnifications of light microscope. In

order to examine the morphology of the ascocarps, method proposed by Zaracovitis (1965)

was done with some modifications. Several different types of ascocarps were picked off

from the leaf surface and put on a glass slide with a wet mount preparation using sterile

distilled water. After covering the sample with a cover slip, the prepared slides were then

observed under microscope with the aid of photographic facility. Photos were captured and

the structures of conidiophores and ascocarps were described.

3.6 Identification of Fungus

3.6.1 Slide Culture Method

Slide culture method was done according to Harris (1986), with several modifications. A

portion of PDA containing fungal growth was transferred onto the surface of glass slide

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and covered with cover slip. The cover slip was attached to the glass slide with the aid of

plasticine to prevent the destruction of its natural structure and arrangement of conidia on

the conidiophores. Prepared culture slides were then put inside the petri dish and sealed

with parafilm. The petri dishes were incubated at room temperature and examined after one

week. Observation was focused on the color, shape of the mycelia and structure of conidia.

3.7 Fungicide Test

Perozin 800 (MASPRO RESOURCE SDN. BHD) which containing zineb 80% w/w (a.i)

and 20% w/w (i.i) were utilized as fungicide in order to test the effects of several different

concentration against the fungal pathogens. Preparation of PDA media containing

fungicide solution was done from 2 mg/50ml stock solution of fungicide (Appendix A).

Fungal growth on the fungicide plates were recorded in a period of one week. The

colony diameter of the mycelium growth was measured daily. The data was analyzed and a

graph was constructed to interpret the final result of fungicide applications on the fungal

pathogen.