Morphological, Pathogenic, Genetic And Molecular Variabilities Of Fusarium Spp., The Pathogens Of Asparagus Crown And Root Rot In Malaysia And Brunei Darussalam

MORPHOLOGICAL, PATHOGENIC,

^{GENETIC AND MOLECULAR} VARIABILITIES OF Fusarium spp., THE PATHOGENS OF ASPARAGUS

CROWN AND ROOT ROT IN MALAYSIA AND BRUNEI DARUSSALAM

.by

MOHAMEDOTHMANSAEEDAL�Morn

Thesis submitted in fulfillment of the

requirements

for the

degree

^of

Doctor of

Philosophy

April2006

ACKNOWLEDGEMENTS

First of

all,

^I render my thanks and

praise

^{to the}

Almighty ^Allah,

^who

offered me the

strength

^to

accomplish

^{this work.}

I would like to express my

deep gratitude

and sincere

appreciation

^to my

supervisor

Professor Dr. Baharuddin

Salleh,

^{School of}

Biological ^Sciences,

Universiti Sains

Malaysia

for his invaluable and sound

guidance,

^continued

encouragement,

enthusiasm and tireless efforts without which this thesis would not have been

possible.

^{I am}

deeply grateful

^{to him for}

taking

^{so much of his} valuable time to discuss the finer

points

^of the thesis with ^me in order to

complete

^{this work in the}

present

^form.

I am

grateful

^{to Dr. Latiffah}

Zakaria,

^a lecturer in Plant

Pathology

^at

School of

Biological ^Sciences,

Universiti Sains

Malaysia

for her generous

help

and

guidance especially

^{on molecular}

techniques.

I am

exceedingly grateful

to the Yemen

Government, University

^of

Hadhramout,

^Yemen

Embassy

^{in Kuala}

Lumpur,

for their financial

support

^and without their

help

^the

present project

would have been a mere dream.

Also,

^with

deep

^{sense of} honor I wish to extend my sincere

gratitude

^to

University

^Sains

Malaysia,

^{School of}

Biological

Sciences for their assistance that ^makes my

study

successful.

Sincere thanks are also due to the

following:

^{Mr. Kamarudin Maidin and}

Mrs. Wan Faridah

Mydin

^{for their technical assistance and Mr. Johari for}

photographic

assistance.

Thanks ^are also extended to all

post graduate

students in the Fusarium Research

Laboratory

^and

special

^{thanks are due to} PhD students Mr. Azmi

11

Abd Razak and Mrs. Nur Ain

Izzati,

and Msc student Mrs Mariam Abdullah for their advice and assistance.

I take this

opportunity

^to express my

deepest gratitude

^{to my}

family

^for

exhibiting great patience, goodwill, encouragement,

^{love and}

understanding throughout

^the

period

^{of my}

study.

Last but not

least,

^{sincere thanks are extended to} the staff and

post­

graduate

students in the

Department

^{of Plant}

pathology,

^{School of}

Biological Sciences,

^{Universiti Sains}

Malaysia

for their kindness and

continuing

^interest.

III

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ⁱⁱ

TABLE OF CONTENTS iv

LIST OF TABLES vii

LIST OF FIGURES ^ix

LIST OF PLATES xi

LIST OF ABBREVIATIONS Xiii

ABSTRAK Xv

ABSTRACT xvii

CHPTER 1 ^- INTRODUCTION ¹

CHAPTER 2 ^- LITERATURE REVIEW 8

2. 1

Taxonomy

and Classification of Fusarium spp. 8

2. 1. 1

Morphological

Characteristics 10

2. 1. 2 Molecular and Genetic Characteristics 13

2. 2 Disease

History

and Distribution 27

2. 3

Pathogenicity

of Fusarium

Species

that Cause the Crown and Root

Rot of

Asparagus

²⁸

2. 3. 1 The

Pathogen

²⁸

2. 3. 2

Pathogenicity

^{Test 29}

2. 3. 3 Inoculum Concentration and Media 31

2.4 Disease

Symptoms

^and

Histopathology

³¹

2. 5 Disease

Epidemiology

^{and Host}

Range

³³

2. 6 Distinction Between Fusarium Crown and Root

Rot,

Dead Stem and

Spear Spot

^{Rot 35}

2. 6. 1 Fusarium Crown and Root Rot 35

2.6.2 Dead Stem 36

2. 6. 3

Spear Spot

^and

Spear

^{Rot 37}

IV

4. 3. 3 Root

Dry

^Mass

4. 3. 4 Plants

Height

4. 3. 5 Plants Tillers

4. 4 Discussion and Conclusion

82 84 85 87

CHAPTER 5 ^- VEGETATIVE COMPATIBILITY GROUPS OF F.

proliferatum

^AND F. oxysporum

5. 1 Introduction 94

5. 2 Materials and Methods 100

5. 2. 1

Fungal

^{Isolates 100}

5. 2. 2 Media 101

5. 2. 3

Recovery

of Nit-Mutants 101

94

5.2.4 Generation ofNitrate

Non-Utilizing (Nit)

^{Mutants 101}

5.2. 5 Identification of Nit-Mutants

Phenotypes

¹⁰²

5. 2. 6

Complementary

^Test-for

vegetative Compatibility

¹⁰³

5.2.7 Statistical

Analyses

¹⁰⁴

5. 3 Results 105

5. 3. 1

Sectoring

and Nit-Mutant

Frequency

^10S

5. 3. 2 Identification of Nit-Mutant

Phenotypes

¹⁰⁶

S. 3. 3

Complementary

^{Test for}

vegetative Compatibility Grouping

¹¹¹

5.3.4

Complementation

^Testfor

Vegetative Compatibility Grouping

¹¹⁷

5. 3. 5

Heterokaryon Self-incompatibility

¹¹⁹

5. 4 Discussion and conclusion 119

CHAPTER 6^- RANDOM AMPLIFIED POL

Y:MORPHIC

^DNA

(RAPD)

¹³²

OF F.

proliferatum

^{AND F.} oxysporum 6. 1 Introduction

6. 2 Materials and Methods 6. 2. 1

Fungal

^Isolates

6. 2. 2 Genomic DNA Extraction

6.2.3 Quantification of DNA

Samples

6. 2. 4 PCR-RAPD

Analysis

6. 2. 5

Analyses

^{of Data}

132 134 134 137 138 139 142

Vl

6.3 Results

6. 3. 1 DNA Extraction for RAPD

Analyses

6. 3. 2 PCR-RAPD

Analysis

6.3.3 Random

Amplified Polymorphic

^DNA

(RAPD) Banding

Pattems for Fusarium spp. ¹⁵⁰

6. 3. 4 Data

Analysis

of RAPD Bands 159

143 143 144

6. 4 Discussion and Conclusion 163

6.4. 1

Sample

^{Size 163}

6. 4. 2 Primer

Screening

¹⁶³

6. 4.3

Optimization

^{of the RAPD-PCR}

Assay

¹⁶⁴

6. 4. 4

Optimization

^of

Template

^DNA Concentration 164 6. 4. 4

Optimization

^of

MgCI2

Concentration 165 6. 4. 6

Optimization

^of

(dNTPs)

Mix Concentration 166

6. 4. 7

Optimization

^of

Taq Polymerase

¹⁶⁶

6. 4. 8

Optimization

^{of Primer}Concentration 167 6. 4. 9. Random

Amplified Polymorphic

^DNA

(RAPD) Banding

Patterns of F.

proliferatum

^{and F.} oxysporum ¹⁶⁷

CHAPTER 7 ^- GENERAL DISCUSSION 173

CHAPTER 8 ^- GENERAL CONCLUSION AND FUTURE RESEARCH 184

8. 1 Conclusion 184

8. 2 Future Research 186

BIBLIOGRAPHY 188

APPENDICES 216

LIST OF PUBLICATION 233

vii

LIST OF TABLES

Page

Table 3.1

Geographicalongln

^of isolates of F. oxysporum ^{and F. 40}

proliferatum

used in this

study

Table 3.2

Sampling

^{location and}

frequency

^{of Fusarium} spp. ^{on 48} diseased asparagus and soils in

Malaysia

^{and Brunei} Darussalam in

1990^- 1991 and 2001 ^- 2002

Table 4.1 Translation of Disease

Symptoms

^Index

(DSI)

^{15 weeks after 79}

inoculation

(Schreuder

^et

^aI., 1995)

Table 4.2 Disease

Symptom

^Index

(DSI)

^of asparagus

variety

^{UC157 80}

inoculated with isolates of F.

proliferatum

^{and F.} oxysporum ⁱⁿ

greenhouse

Table 5.1 Identification

(phenotyping)

^{of nitrate non}

utilizing (nit)

^{mutants 103}

by growth

^{on different}

nitrogen

^sources

Table 5.2

Complementation

^reaction between nitrate

nonutilizing (nit)

¹⁰⁴

mutants of F.

proliferatum

^{and F.} oxysporum.

Table 5.3 Cumulative data on

frequency

^{of sectors and}

phenotypes

^{of 108}

nitrate

non-utilizing (nit)

^mutants of F. oxysporum and F.

proliferatum

^on

two media with 2% and 3% chlorate concentrations

Table 5.4 Mean

frequency

^{of sectors per}

colony

^{of 75} isolates of F. 109

proliferatum

^{on PDC and MMCwith 2% or}3% chlorate concentrations

Table 5.5 Mean

frequency

^{of sectors} per

colony

of 16 isolates of F. 109 oxysporum^{on PDC and} MMC with 2% or3% chlorate concentrations

Table 5.6 Mean

frequency

of nit-mutants per

colony

^{of 75} isolates of F. 110

proliferatum

^{onPDC and MMC with 2% or 3%} chlorate concentrations

Table 5.7 Mean

frequency

of nit-mutants per

colony

^of 16 isolates of 110

F.oxysporum

^{on POC} and MMC with 2% or 3% chlorate concentrations

Table 5.8 Mean

frequency

of nit-mutants per

colony

of 75 isolates of F. 111

proliferatum

^on PDC and MMC with 2% ^or 3% chlorate concentrations

Table 5.9 Mean

frequency

of nit-mutants per

colony

of 16 isolates of F. III oxysporum^on POC and MMC with 2% ^or 3% chlorate concentrations

Table 5.10 Isolates of F.

proliferatum

^classified

by vegetative

¹¹⁴

compatibility

^{and their}

origin

Table 5.11 Isolates ^of F. oxysporum classified

by vegetative

¹¹⁷

viii

compatibility

^{and their}

origin

Table 5.12

Origin

^of

self-incompatible

isolates of F.

proliferatum

¹¹⁹

Table 6.1

Geographical origin

of isolates of ^F. oxysporum and F. 136

proliferafum

used in PCR

study

Table 6.2 The

code,

sequence, nucleotide

length

^{and G+C content of 140}

primers

used in RAPD

Analysis

Table 6.3

Summary

^of number and characteristics of

amplification

¹⁴⁴

products

^{obtained from}

screening

of 20 random

primers

^from

Operon

primer

^{Kit A}

IX

Figure

^{6.10 RAPD}

banding patterns

of F. oxysporum isolates ^{obtained 153} with

primer

OPA-10 from

debris,

soil and infected asparagus ^from

Penang, Pahang, Selangor,

^{Melaka and Brunei} Darussalam.

Figure

^{6.11 RAPD}

banding patterns

^{obtained with}

primer

^{OPA-02 of F. 154}

proliferatum

isolates from infected _asparagus from

Penang.

Figure

^{6.12 RAPD}

banding patterns

obtained with

primer

OPA-02 of F. 154

proliferafum

isolates from infected asparagus ^from

Perlis, Pahang, Selangor, ^Sabah,

^{Melaka and Brunei} Darussalam.

Figure

^{6.13 RAPD}

banding patterns

obtained with

primer

OPA-03 of F. 155

proliferafum

isolates from infected asparagus from

Penang.

Figure

^{6.14 RAPD}

banding patterns

obtained with

primer

^{OPA-03 of F. 156}

proliferatum

^isolates from infected asparagus ^from

Perlis, Pahang.

Selangor, ^Sabah,

Melaka and Brunei Darussalam.

Figure

^{6.15 RAPD}

banding patterns

obtained with OPA-04 of F. 157

proliferatum

isolates from infected asparagus from

Penang.

Figure

^{6.16 RAPD}

banding patterns

obtained with OPA-04 of F. ¹⁵⁷

proliferatum

isolates from infected asparagus ^from

Perlis, Pahang.

Selangor, Sabah,

^Melaka and Brunei Darussalam.

Figure

^{6.17 RAPD}

banding

Patterns obtained with Primer OPA-10 of F. 158

proliferafum

isolates from infected asparagus from

Penang.

Figure

^{6.18 RAPD}

banding patterns

obtained with Primer OPA-10 of F. 158

proliferatum

^{isolates from} infected asparagus from

Perlis, Pahang, Selangor, Sabah,

Melaka and Brunei Darussalam.

Figure

^6.19

Dendrogram

^{from UPGMA}

analysis using Simple Matching

¹⁶²

Coefficient based on RAPD bands of F.

proliferatum

^{and F.} oxysporum isolates from infected asparagus, debris and soils

.

Xl

LIST OF PLATES

Page

Plate 3.1 Overall foliar

symptoms

of Fusarium ^crown and root rot 50 Plate 3.2 Fusarium oxysporum.

Colony

^{on PDA after 7}

days

^{of 52}

incubation:

a) obverse, whitish-cream; b)

^reverse,

pale blue; c)

^{oval to}

kidney-shaped microconidia; d) macroconidia; e)

^{false heads of}

microconidia ^on

monophialidic conidiophores; f)

macroconidia borne on

branched

monophialides; g)

^short

monophialidic microconidiophores; h) intercalary chlamydospores

Plate 3.3 Fusarium

pro/if

^eratum.

Colony

^{on PDA after 7}

days

^{of 54}

incubation:

a) obverse, greyish

^{violet or}

greyish magenta; b)

^reverse,

dark violet ^or dark

magenta; c) microconidia; d) macroconidia; e)

^short

chains of microconidia on

polyphialidic conidiophores; f) polyphialidic conidiophores

Plate 3.4 Fusarium solani.

Colony

^on PDA after 7

days

^of incubation:

a)

⁵⁶

obverse,

^{white to} cream;

b)

^reverse,

pale

^{brown at the centre and}

pale

violet in

rings; c)

^reinform

microconidia; d)

microconidia

(ellipsoidal

^to

reniform)

^and macroconidia with foot

shaped

^{basal cell}

(sausage­

shaped); e)

^and

f)

microconidia borne in false head on

monophialidic

microcon id

iophores

Plate 3.5 Fusarium semitectum.

Colony

^{on PDA after 7}

days

^{of 58}

incubation:

a) ^obverse,

^{white to}

^salmon; b)

^reverse,

pale

^{to dark}

^brown;

c) macroconidia; d)

^{conidia borne on}

polyphialides; e) polyphialides; f) chlamydospores.

Plate 3.6 Fusarium

longipes. Colony

^on PDA after 7

days

of incubation: 60

a) ^obverse,

^{white to}

greyish; b)

^{reverse, rose to}

burgundy; c)

macroconidia, long,

^{slender thick}

walled, usually

^5�7

septate

^{with a}

distinct dorsi-ventral curvature.

Plate 4.1

Symptoms

of asparagus ^crown and root rot caused

by

^{F. 77}

oxysporum and F.

proliferatum.

^{A. Roots}

showing

^brownish

discoloration and shrivelled. B. Control

(Healthy roots).

Plate 4.2

Asparagus plants (var.

^ue

157)

grown ⁱⁿ

polythene bags.

⁷⁷

(Inoculated plants

^{in three}

bags

^{on the left were}

wilted,

stunted and

collapsed (I); Right

^{- Non}

seedlings

^var. Ue157 inoculated

healthy plant (e)

Plate 4.3 Brown discolorations ^on roots of inoculated

plants (A). Healthy

⁷⁸

and clear roots of control

plants (B).

Plate 5.1 The _appearance of

fasting growing ^fan-shaped

^{sectors from 106}

xu

the

initially

^restricted

colony

^{of F.}

proliferatum wild-type

^{isolate P1506A}

on poe

(3%)

Plate 5.2 Growth of

wild-type

isolate P1506A of F.

proliferatum

^{and 107}

three nitrate

nonutilizing (nit)

^mutant

phenotypes

from P1506Aon media with oneof four different

nitrogen

^sources

Plate 5.3

Pairing

^ofnit-1 and Nit-M mutants derived from

self-compatible

¹¹²

isolate

(P1506)

^ofF.

proliferatum

^{on MM.}

Plate 5.4

Multiple pairing

^{of nit-1 and Nit-M mutants derived from 113}

multiple compatible

^isolates

(P1721A

^and

P1722A)

^{of F.}

proliferatum

^on

MM

Xlll

1-19

1-11 I-IM

A

ANOVA B

BEA BM

bp

C cfu CLA.

cm

CRD

ddH20

DOA DMRT DNA dNTPs DSI EDTA EtBr f. _sp.

f.spp.

FA

FB1

Fo

Fp

FUP G 9 hr ha HX Kb

Kg

L M M MARDI min ml

mm

mM MMC MON

List ofAbbreviations

Microgram (10.3 gram)

Micro liter

(10.3 ^ml)

Micromolar

Asparagus

Analysis

^ofVariance

Selangor

^State

Beauvericin Basal Medium Base Pair

Cytosine

Colony Forming

^Unit

Carnation

Leaf-piece Agar

Centimeter

Complete

^Randomized

Design

Dionized Distilled Water

Department

^of

Agriculture

Duncan's

Multiple Range

^Test

Deoxyribonucleic

^Acid

Deoxyribonucleotide Triphosphates

Disease

Severity

^Index

Ethylene

Diaminetetraacetic Acid Ethidium Bromide

Forma

Specialis

Formae

Speciales

Fusaric Acid Fumonisin

B1

Fusarium oxysporum Fusarium

proliferatum Fusaproliferin

Guanine Gram Hour Hectare

Hypoxanthine

^Medium

Kilobase

Kilogram (103 gram)

Liter Molar

Melaka State

Malaysia Agriculture

^{Research and}

Development

^Institute

Minute Milliliter Millimeter Milimolar

Minimal agarMedium with Chlorate Moniliformin

xiv

ng

NH4 N02 N03

NPK

NTSYS-pc

oe OPA P

p.s.i

peR PDA PDAC PPA PSA R RAPD RDI RFLP rpm S SA SMC spp SPSS TBE TE U

UPGMA USM UV V v/v veGs W WA

Nanogram

Ammonium Medium Nitrite Medium Nitrate Medium

Nitrogen, Phosphorous,

^Potassium

Numerical

Taxonomy

^and Multivariate

Analysis System Degree Centigrade

Operon Technologies

Primer Series A

Probability

Per

Square

^Inch

Polymerase

Chain Reaction Potato Dextrose

Agar

Potato Dextrose

Agar

Medium with Chlorate

Peptone

Pentachloronitrobenzene

Agar

Potato Sucrose

Agar

Perlis State

Random

Amplified Polymorphic

^DNA

Root lesionswith vascular Discoloration in crown and root Index Restriction

Fragment Length Polymorphism

Revolution per min Sabah State

Soil

Agar

Simple Matching

Coefficient

Species

Statistical

Package

^{for Social Science}

Tris-Borate-EDTA Tris-EDTA

Unit

Un

weighted

^Paired

Group Matching Analysis

Universiti Sains

Malaysia

Ultraviolet

Light

Volt

VolumeNolume

Vegetative Compatibility Groups

Watt

Water

Agar

xv

KEVARIABELAN

MORFOLOGI, PATOGENIK,

GENETIK DAN MOLEKULAR Fusarium spp., PATOGEN PENYAKIT REPUT PANGKAL BATANG DAN

AKAR ASPARAGUS DI MALAYSIA DAN BRUNEI DARUSSALAM

ABSTRAK

Asparagus (Asparagus officinalis) menjadi

^semakin

penting

^{di Asia}

Tenggara (SEA)

^{dan dalam waktu} yang

singkat menjadi

sayuran

pilihan.

Semua varieti yang ^ditanam di seluruh SEA

menghadapi

^ancaman serangan

penyakit

yang

paling banyak

menimbulkan

kerosakan,

^iaitu

penyakit reput pangkal batang

^{dan akar. Sasaran utama} tesis ini adalah

mengumpul

^dan

memencilkan Fusarium spp.

daripada

asparagus yang

menunjukkan gejala reput pangkal batang

^{dan akardan}

juga daripada

^{tanah di}

Malaysia

^{dan Brunei}

Darussalam.

Objektif

lain adalah untuk menilai

kepatogenan

^dan

kepelbagaian genetik

Fusarium spp. yang

dipencilkan

^serta

kevariabelannya menggunakan

RAPD berasaskan PCR.

Sejumlah

¹¹⁰

pencilan

yang terdiri

daripada

^lima

spesies

^{Fusariumtelah}

diperolehi daripada

^{enam kawasan}

pensampelan

^di

Malaysia

dan satu di Brunei Darussalam. Lima

spesies

yang ^telah

dikenalpasti

^{ialah F.}

proliferatum,

^F.

oxysporum, F.

so/ani,

^{F. semitectum dan F.}

/ongipes,

berdasarkan ciri

morfologinya.

^F.

proliferatum

^{dan F.} oxysporum

merupakan spesies

yang

terbanyak dipencilkan (83%). Ujian kepatogenan pencilan

^F.

proliferatum

^dan

F. oxysporum yang dilakukan

dengan menginokulat

anak benih asparagus varieti UC 157 di rumah tanaman

mengesahkan

bahawa kesemua

pencilan

adalah

patogeni.

^Pada

awalnya, gejala penyakit

yang

diperhatikan

^adalah

kekuningan

^di

bahagian

^{daun dan}

cabang.

Pokok yang

terjangkit menjadi

terencat

dengan

^akar

menjadi perang-kemerahan

^dan

mengecut.

^Belahan

XVI

batang

^dan

pangkal

tisu yang

terjangkit jelas menunjukkan

^warna perang­

kemerahan. Tanaman yang

parah terjangkit akhirnya

^mati.

Tujuh puluh

^lima

pencilan

^F.

pro/iferatum

^{dan 16}

pencilan

F. oxysporum telah

digunakan

^untuk

menghasilkan

^mutan reduksi nitrat

(nit) sebagai

^sektor

rintang

^{klorat di atas media} agar-agar

kentang

^dekstrosa

(PDAC)

^{dan media}

minimum

(MMC),

yang ditambah

dengan

^{2.0% dan 3%}

KCI03.

^Frekuensi

purata sektor,

^{mutant nit-1 dan nit-3 di atas PDAC}

didapati

^lebih

tinggi

^dan

menunjukkan perbezaan

yang bererti

(P

^s

0.05) berbanding

^{di atas MMC}

bagi

kedua-dua

spesies.

^Frekuensi

purata

^Nit-M

setiap

^{koloni di atas MMC lebih}

tinggi

^dan

menunjukkan perbezaan

yang bererti

(P

^S

0.05) berbanding

^{di atas}

PDAC

bagi

^kedua-dua

spesies. Kemudiannya,

mutan nit yang

dijana

^telah

digunakan

^dalam

ujian komplementasi

^untuk

mengetahui

^keserasian

vegetatifnya. Sebanyak

²³

kumpulan

^keserasian

vegetatif (VCGs)

^talah

dikenalpasti

^{untuk F.}

proliferatum

^{dan enam untuk F.} oxysporum.

DNA

bagi

⁵⁰

pencilan

yang mewakili dua

spesies

Fusarium tersebut dianalisis

menggunakan empat primer

^{RAPD iaitu}

OPA-02, OPA-03,

^OPA-04 dan OPA-10. Primer

dipilih

berdasarkan

keupayaan

^mereka

menghasilkan jalur

yang

jelas. Keputusan daripada

^{analisis RAPD}

berupaya menunjukkan

kevariabelan di

kalangan

^{dan di antara kedua-dua}

spesies

Fusarium. Analisis kluster

menggunakan

UPGMA berdasarkan

Simple Matching

Coefficient

(SMC) menunjukkan pencilan-pencilan

^tersebut

tergolong

di dalam dua kluster

utama,

iaitu

spesies

dan lokasi yang ^sama

tergolong

dalam kluster yang ^sama.

Keseluruhan

kajian menunjukkan

^bahawa

kompleks penyakit reput pangkal batang

^{dan akar}

sangat penting pada

^{semua varieti} asparagus ^di

Malaysia

^dan Brunei Darussalam.

Patogennya

^telah

dikenalpasti sebagai

^F.

XVll

proliferatum

^{dan F.} oxysporum berdasarkan

kepada

^ciri

morfologi, genetik (VCG)

^dan teknik molekul.

xviii

MORPHOLOGICAL, PATHOGENIC,

GENETIC AND MOLECULAR VARIABILITIES OF Fusarium spp., ^THE PATHOGENS OF ASPARAGUS

CROWN AND ROOT ROT IN MALAYSIA AND BRUNEI DARUSSALAM

ABSTRACT

Asparagus (Asparagus officinalis)

^is

becoming

^more

important

^{in South}

East Asia

(SEA)

^and

quickly becoming

^a

preferred vegetable.

All varieties

planted throughout

SEA have been and now are still

facing

^{the most} destructive disease i.e. asparagus ^{crown and root rot.} The main aim of the thesis ^was to collect and isolate Fusarium spp. from asparagus

plants showing

^{crown and}

root rot

symptoms

and their soils in

Malaysia

and Brunei Darussalam. The other

objectives

^{were to evaluate}

pathogenicity

^and

genetic diversity

^{within the}

Fusarium spp. and their

variability using

peR-based RAPD.

A total of 110 isolates

comprising

^five

species

^of Fusarium have been isolated from six

sampling

^{areas in}

Malaysia

^{and one in Brunei} Darussalam.

The five

species

^{identified were F.}

proliferatum,

F. oxysporum, ^F.

solani,

^F.

semitectum and F.

/ongipes,

^{based on}

morphological

characteristics. F.

proliteretum

^{and F.} oxysporum

represented

^the

highest percentage (830/0).

Pathogenicity

^{tests of F.}

proiiteretum

and F. oxysporum isolates

by inoculating healthy

asparagus

seedlings

^{var. UC157 in the}

greenhouse

confirmed that all isolates tested were

pathogenic.

^The

typical symptoms

^were

initially

^observed

as

yellowing

of leaves and branches. Infected

plants

^were stunted with reddish- brown discoloration and shrivelled roots. Sliced crowns and stems

clearly

showed reddish-brown discolorations of the infected tissues.

Heavily

^infected

plants collapsed

^{and died.}

XIX

Seventy

five isolates of F.

proliferatum

and 16 isolates of F. oxysporum

were used to

generate

^{nitrate reduction}

(nit)

^{mutants as} chlorate resistant sectors on two media i.e.

potato

dextrose agar

(PDAC)

and minimal medium

(MMC),

^{each amended with 2.0% and 3.0%}

KCI03.

^Mean

frequencies

^of

sectors, ^{nit-1 and nit-3 mutants on PDAC were}

significantly higher (P

^Si

0.05)

than those on MMC for the two

species.

^Mean

frequencies

of Nit-M per

colony

on MMC was

significantly higher (P

^Si

0.05)

^{than those on} PDAC for the two

species. Later,

recovered nit-mutants were used in

complementation

^{tests for}

vegetative compatibility. Twenty

^{three and six}

vegetative compatibility

^groups

(VCGs)

^were identified from F.

proliferatum

^{and F.} oxysporum,

respectively.

DNA of 50 isolates

representing

^{the two Fusarium}

^species

^were

analysed by using

^{four RAPD}

primers

^i.e.

OPA-02, OPA-03,

OPA-04 and OPA- 10. The

primers

^were chosen based ^on their

ability

^to

produce

well-defined and

reproducible banding patterns.

^Results of the RAPD

analyses

^{were able to}

show variabilities within and between the two

species

of Fusarium. Cluster

analysis

^{with UPGMA}

by using Simple Matching

Coefficient

(SMC)

showed that the isolates were clustered into two main groups Le. the ^same

species

^and

location were foundto group

together

^{in the same cluster.}

The whole

experiments

showed that crown and root rot disease

complex

was the most

important

^{disease on all} varieties of asparagus in

Malaysia

^and

Brunei Darussalam. The most

prevalent pathogens

^{were identified as F.}

proliferatum

and F. oxysporum

by using morphological, genetical (VCG)

^and

molecular

techniques.

xx

CHAPTER 1

INTRODUCTION

Asparagus (Asparagus

officinalis

L.)

^{is a}

hardy perennial vegetable,

native to the coastal

region

^of

Europe

and eastern

Asia,

^where it has been cultivated for over

2,000

years

(Sandsted

^et

^al., 2001).

^{Itwas a} well-known and valued

vegetable

^to both the Greeks and Romans

(Sandsted

^et

al., 2001).

^The

word asparagus ^comes from the Greek asparagos,

meaning

^{shoot or}

sprout (Sandsted

^et

^ai., 2001).

The genus

Asparagus belongs

^{to the}

lily Family

^Le.

Liliaceae and includes ^over 25 cultivated

species,

^but

only

^A.

officina/is,

^the

garden

asparagus, is grown for food

(Bailey

^and

Bailey, 1976). Asparagus

^is

produced

ⁱⁿ

temperate

^and

tropical regions

that span ^over 50 countries. Fields

are established with

seeds, transplants

^{or crowns,} and the marketable spears

are cut when

they

^{are 18 - 22 cm}

long

after the second or third year. Growers

expect

their asparagus fields to remain

profitable

^{for 10 -} 15 years with ^most fields

reaching peak production

^{after 5 -} 8 years. In

1997,

asparagus ^was grown ^{on over}

215,000

ha world wide. When

compared

^to

1992,

this acreage

represented

^a 27% increase in land

cropped

with asparagus

(Nigh, 1999).

Nutritionally,

asparagus has ^a low content of both calorie and

sodium,

-

yet

^it

provides significant

^amount of vitamins A and C in the diet. It also

provides

^the vitamins such as

riboflavin, niacin,

^and thiamine and the minerals such as

iron, phosphorus,

^and

potassium (Sandsted

^et

^ai., 2001). Asparagus

^is

becoming

^more

important

in South East Asia

(SEA).

^{Most of} the varieties cultivated in this

region

^were introduced from

temperate

^or

semi-temperate

countries into SEA

(Salleh

^et

ai., 1996).

It is believed that the first asparagus

1

seeds ^were introduced to

Malaysia

^{from Taiwan} in the 1950s.

Although

^{it is a}

fairly

^new crop, asparagus ^has become a

preferred vegetable especially by Malaysian

^{in the}

higher

income groups

(Salleh

^et

^ai., 1996).

During

^{recent and} continuous disease surveys ^{on 24} farms in

Malaysia, Indonesia,

Thailand and Brunei Darussalam carried out

by

^Salleh

(1990)

^and

Salleh et al.

(2004),

^{the most} destructive disease was found to be crown and root rot

(Fusarium proliferatum),

^followed

by

^wilts

(F.

^{oxysporum f. sp.}

asparag�,

anthracnose

(Col/efotrichum gloesporioides),

^brown

spot (CufVu/aria spp.) (Salleh

^et

^aI., 1996), Phomopsis blight (Phomopsis asparagl),

^stem

canker

(Fusarium spp.), Phytophthora

^rot

(Phytophthora megasperma),

^rust

(Puccinia asparagl),

^crown

spot

^{and shoot die-back}

(Altemaria tenuissima),

gray mold

(shoot blight) (Botryfis cinerea), purple spot (Stemphylium vesicarium), Cercospora blight (Cercospora asparagl),

and several viral and bacterial diseases.

Elsewhere in the

temperate countries,

^{over 12}

species

of Fusarium are

found

colonizing

^crown tissues of asparagus.

However, only

three diseases are

recognized

i.e. Fusarium crown and root rot, ^{dead stem} and spear

spot

^and spear ^rot

(Blok

^and

Bollen, 1995;

^{Schreuder et}

aI.,

^{1995 Elmer et}

ai., 1996).

^It

seems that Fusarium crown and root rot of asparagus is the ^most

economically important

^disease of asparagus all ^over the world. The disease has been described under several names; these include dwarf asparagus

(Cooke, 1923),

wilt and root rot

(Cohen

^and

Heald, 1941), seedlings blight (Graham, 1955),

foot rot

(van

^{Bakel and}

^Kertsen, 1970),

^{crown rot}

complex (Endo

^and

Burkholder, 1971),

^{and stem and crown rot}

(Johnston

^et

ai., 1979).

^{It was also}

cited as the

primary

disease associated with asparagus decline and

replants

2

problems (Grogan

^and

^{Kimble, 1959;}

^{Elmer et}

^al., 1996),

^and

replant

^bound

early

^decline

(Blok

^and

Bollen, 1995).

^The

plethora

^{ofcommon names reflects}

the wide range of

opinions

^{as to} the manifestation of the

symptoms

^{of this}

disease at different

stages. Asparagus

^{decline was defined}

by Grogan

^and

Kimble

(1959)

^{as a} "slow decline in the

productively

old asparagus

plantings,

^to

the

point

^{where the}

plantings

^become

unprofitable

^to rnaintain''.

Damage

^from

asparagus decline includes ^a reduction in spear size and

number,

and eventual death of the crown. Additional loss is incurred if abandoned asparagus fields

are

replanted

with asparagus. In these

plantings, stunting, chlorosis,

^{wilt and} death appear ^to

prevent

stand establishment

(Grogan

^and

Kimble, 1959).

^The

replant problem

in asparagus has many similarities ^to asparagus decline.

Grogan

^{and Kimble}

(1959)

defined the

replant problem

^{as "the}

inability

^to

establish

productive planting

^{in the} field where

planting

^have declined".

Overall,

one of the most

devastating

^{diseases of} asparagus ^{is crown and root rot} caused

by

^F.

proliferatum

and F. oxysporum in United States of

America, Europe,

Africa and Canada

(Blok

^and

Bollen, 1995;

Schreuder et

al., 1995;

Eimer et

al., 1996; Eimer, 2001; Yergeau

^et

al., 2005).

The disease can be

devastating

^to

seedlings

and young

plants. Symptoms

^{noted were extensive}

rotting

^{of feeder and}

storage

^roots. Vascular discoloration is also observed in the crown and base of infected

stems,

followed

by

^fern

chlorosis,

^{wilt and}

death. Reddish brown lesions were also

present

^{on the exterior of stems and}

roots

(Schreuder

^et

^al., 1995).

In

Malaysia,

^the

species

of Fusarium associated with asparagus ^crown and root rot was identified as F.

proliferatum by

^Salleh

(1990),

and later the causal

organisms

^{of the disease}

complex

^{were identified as F.}

nygamai,

^F.

3

oxysporum and F.

proliferatum by Sapumohotti (1992).

^basedon

morphological

characteristics. The main characteristics used were the

shape

^of

macroconida,

presence ^or absence of

microconidia. shape

^{and mode} of formation of

microconidia,

^{nature of the}

conidiogenous

^cells

bearing microconidia,

presence

or absence of

chlamydospores

^{as described}

by

Nelson et al.

(1983), Burgess

and Liddell

(1983), Burgess

and Trimboli

(1986), Singh

^{et al.}

(1991),

^and

Burgess

^{et al.}

(1994).

Visual assessment of the disease caused

by

^Fusarium

species

^{is often}

insufficient to

diagnose

^{the causal}

agents

of the disease

particularly

^where

several

organisms

^{induced similar}

symptoms

^{within a disease niche.}

Conventional methods for the identification of Fusarium

species

ⁱⁿ

plant

tissues involve isolation of the

fungus

^into axenic cultures. The isolated

organisms

^are

generally

^{identified on the basis of}

morphological

characteristics of the

colony,

conidia and

conidiogenous

cells. Reliable identification to the

species

^level

requires

considerable

expertise

^{and is}

greatly complicated by plasticity

^and

instability

of Fusarium

species

ⁱⁿ culture. These features of Fusarium

species

have resulted in several classification

systems,

^with

widely differing

ⁱⁿ

species concepts,

^{and were}

proposed by

Wollenweber and

Reinking (1935),

^Raillo

(1935; 1950), Snyder

^{and Hansen}

(1940; ^1941; 1945),

^Gordon

(1952),

^Bilai

(1955; 1970), Snyder

^{et al.}

(1957),

Messian and Cassini

(1968;

1981),

^Booth

(1971).

^Matuo

(1972),

^Joffe

(1974),

Gerlach and

Nirenberg (1982),

Nelson et al.

(1983)

^and

Brayford (1993).

^The

morphological

^and

physiological

methods used for identification and classification of Fusarium

species

^have

proved problematic (Nelson, ^1991;

^{Summerell et}

^et., 2003).

Therefore recent use of molecular markers has revolutionized the

analysis

^of

4

identification and

population biology

^of

plant pathogens (Milgroom

^and

Fry, 1997).

The

development

^{of random}

amplified polymorphic

^DNA

(RAPO)

markers

(Welsh

^and

McClelland, 1990;

^{William et}

aI., 1990)

^has

provided

^a

powerful technique

^for

investigating intra-specific

^and

genetic

variations in

fungi.

^RAPD

analysis

^{has been}

particularly

^{useful for studies of Fusarium} spp., and it has

provided genetic

markers that facilitate

population

studies and identification of

species

^{such as F.} oxysporum

(Assigbetse

^et

^aI.,

^1994;

Crowhurst et aI.,

1995)

^{and F.}

proliferatum (Nicholson, 2001).

^PCR

(polymerase

^chain

reaction)-based

^markers,

especially

RAPD have become

more

popular

because of their technical

simplicity,

^and

potential

^for

rapid screening

^of

large

^{numbers of} individuals

using

^{minimal amount of DNA. This}

technique

^{has been}

successfully

^{used to assess}

genetic variability

within many

plant pathogenic fungi (Goodwin

^{and Annis,}

^1991;

^{Jones and}

^Dunkle, 1993;

Huff et

aI., 1994; Kellyet al., 1994) including

Fusarium spp. in the Section Liseola

(Amoah

^et

ai., 1995; 1996; Voigt

^et

ai., 1995;

^{MacDonald and}

Chapman, 1997).

^{In these}

^studies,

^{isolates from} different countries were

surveyed

^{and RAPD}

techniques

^were

successfully

^{used to}

distinguish

^between

mating populations

of Fusarium spp. in the Section

Liseola,

^{the most difficult} group ^to be

confidently

^identified

by using morphological

characteristics.

Vegetative compatibility

group

(VCG)

^{is another}

aspect

^{that can be used}

as a new

approach

^{to detect}

genetic

lines within the

population

^{of various}

species, particularfy

^{in asexual}

fungi.

^{VCG are} ideal markers for

population

studies because

they

^occur

naturally

^{and are} easy ^{to score}

using spontaneous

nit-mutants

{Puhalla, ^1985; Sidhu, 1986;

^{Correll et}

ai., 1986b; 1987a;

^Sosland

5

and

Williams, 1987;

^{Jacobson and}

Gordon, 1988). Today,

^most

population genetic

^{studies on Fusarium} spp. such as those in the Section Liseola have been conducted

using

^{the VCG as a marker for}

genotyping

^other

fungal

isolates

(Farrokh-Nejad

^and

Leslie, 1990; Campbell

^et

ai., 1992;

^{Kedera et}

ai., 1994).

Strains that are

vegetatively compatible

^Le.

belong

^{to the same}

VCG,

can form a stable

heterokaryon,

^{and share an identical set of alleles at about}

10 vic loci

(Leslie, 1993).

^{The VCG}

technique

^is

particularly

suitable for

population genetic studies,

^of

especially

^{on Fusarium} spp. in the Section

Liseola,

because field isolates of this

fungus normally belong

^to many VCGs

(Leslie

^et

ai., 1992).

The occurrence and distribution of the most

important

disease i.e. crown

and root rot in South East Asia

(SEA),

^its

epidemiological

^{factors and the}

magnitude

^{of losses} inflicted upon asparagus ^were

reported by

^Salleh

(1990)

and Salleh et al.

(2004).

The disease situation ^{seems to} be

aggravated by

^the

fact that many asparagus farmers in SEA ^{are now}

actively engaged

ⁱⁿ

production

of asparagus. The meager

knowledge

of this disease in SEA

(especially

ⁱⁿ

Malaysia

^{and Brunei}

Darussalam) coupled

^{with the}

potential major

constraints to asparagus

production

necessitate a full attention. The

present study

^is

^therefore,

^intended:

1. To isolate and

identify

Fusarium spp. from

soils,

^debris and infected asparagus

plants

^{from different locations in}

Malaysia

^{and Brunei}

Darussalam.

2. To reconfirm F. oxysporum and F.

proliferatum

^{as the causal}

agents

^of

asparagus ^crown and root rot

(Kochis Postulate).

6

3. ^To evaluate the

genetic diversity

within the two Fusarium

species

^that

caused crown and root rot of _asparagus and to

investigate

^the correlation between VCG and

geographic

distribution.

4. To

study

^the molecular characteristics

by

^Random

Amplified Polymorphic

DNA

(RAPO)

^{of the two Fusarium}

species

^and to determine their

genetic

relatedness.

7

CHAPTER 2

LITERATURE REVIEW

2.1

Taxonomy

^and Classification of Fusarium spp.

The

taxonomy

of Fusarium

began

^{with the}

description

of the genus

by

Link in 1809 based on the presence of fusiform

non-septate

spores, borne ^{on a} stroma. The

publication

of Die Fusarien

by

Wollenweber and

Reinking (1935)

became the foundation of the

present system

of classification.

Later,

^Booth

(1971)

^{introduced a}

system

of classification based ^on

morphology

^{of the}

conidiogenous

cells and conidium

ontogeny. However,

many

mycologists

^found

that the two classification

systems

^{were too detailed} and difficultto be followed.

Thus,

^{efforts are}

being

^made

by

Fusarium taxonomists all over the world to

simplify

the classification

systems.

^This

undoubtedly

^{has led to}the existence of several different

systems

of classification which

regretfully,

^{are still not}

satisfactory

for the identification of all Fusarium

species.

^{The most}

acceptable part

^{of the two} classification

systems

^{has been the}

separation

of the genus into Sections or groups which ^were defined

by

^Booth

(1971)

^as

"aggregations

^of

related

species". Later,

^{Nelson et al.}

(1983) separated

^{each Section based on}

1)

presence ^{or absence of}

microconidia, 2) shape

^{of the}

microconidia, 3)

presence ^or absence of

chlamydospores, 4)

^{location of}

chlamydospores;

intercalary

^or

^terminal, 5) shape

^of

macroconidia,

^and

6) shape

of basal cells ^or foot cells of macroconidia. One of the Sections

recognised by

all Fusarium taxonomists is Liseola.

8

Wollenweber and

Reinking (1935)

^{included three}

species

^{and three}

varieties in the Section Liseola.

Snyder

^{and Hansen}

(1945), ^however,

^reduced

the number to ^a

single species

^i.e. F. moniliforme Sheldon amended

Snyder

and Hansen. Booth

(1971) recognised

^one

species

^{i.e. F.} moniliforme with one

variety

^i.e.

subglutinans. According

^{to Booth}

(1971),

the characteristics of the

species

^{in this Section are based on}

1)

microconidia formed in chains or false

heads, 2)

microconidia

spindle

^{to ovoid in}

shape, 3)

macroconidia slender with constricted

apical

^{cell and}

pedicellate

^basal

cell, 4) chlamydospores absent,

and

5)

cultures brownish white to orange cinnamon.

Later,

Gerlach and

Nirenberg (1982)

increased the number to nine

species

and five varieties.

Nelson et al.

(1983), however, recognised only

^six

species.

An ideal taxonomic

system

should reflect the

genetic

relatedness of taxa^.. It should also

recognise,

^{at an}

appropriate ^level,

^{taxa which are}

distinguished by practical

^and

significant aspects

^{of their}

pathogenicity.

myocotoxicology

^or

ecology (Burgess

^et

aI., 1997).

^The

history

of Fusarium

systematics

^{has shown marked}

swings

^between

excessively

^narrow

species concepts

and those which are so broad that

practical

information such as

pathogenicity

^and

toxigenicity

has been lost. Recent studies on

biodiversity

ⁱⁿ

Fusarium are based on the examination of

large population

^ofisolates in which traditional

morphological

^{criteria are}

integrated

with detailed data on

pathogenic specialization,

^toxin

production

^and

ecology,

^{and more}

recently

with information derived from molecular taxonomic studies

(Burgess

^et

^ai., 1997). During

^the

first decades of taxonomic

research,

many scientists contributed ^to describe

over 1000

species,

^{varieties and} forms of Fusarium.

Appel

^and Wollenweber

(1910)

and Wollenweber

(1913) ^published

^{a series of}

^important

^{studies on this}

9

unique

genus. On this

basis,

^{the modern}

concept

^of the genus Fusarium ^was created in Eastern

Europe (Wollenweber

^and

Reinking, 1935).

The authors of this

monograph

^{reduced over 1100}

species

^{of Fusarium to 65}

species

^{and 22}

forms and varieties.

However,

^{a much}

simpler system

^with

only

^nine

species

was

published by Snyder

^{and Hansen}

(1940; 1941; 1945)

^{in the USA.}

Later,

several classification

systems

^were

developed by

Messiaen and Cassini

(1968;

1981),

Gerlach and

Nirenberg (1982),

Nelson et al.

(1983).

One of the most used

systems by

^Booth

(1971)

^{was based on}

descriptions

^{of 12}

^Sections,

⁴⁴

species

^{and 7 varieties} of Fusarium.

Recently, Brayford (1993),

a successorof Dr. Colin Booth at the Commonwealth

Mycological ^Institute,

considered 12 Sections with 52

species

^{and 4 varieties. This} classification and its

phylogenetic relationships

^{were varified}

by

molecular and

genetic

^criteria

(Logrieco

^et

^aI., 1997).

Taxonomically,

^{the genus Fusarium is classified in the class}

Hyphomycetes, belonging

to the Sub-division

Deuteromycotina. Teleomorphs

of Fusarium spp. have been

placed

in the genera Nectria and

Gibberella,

^order

Hyphocreals (Ascomycetes).

^Until

today,

^the

taxonomy

of the genus Fusarium is not settled and the number of

species

and Sections varies

(Zema'nkova

^and

Lebeda, 2001).

2.1.1

Morphological

Characteristics

The genus Fusarium is characterized

by usually

^fast

growing, pale

^or

bright-coloured

^{colonies with a}

felty

^aerial

mycelium

^{and diffused or}

sporodochial sporulation.

Fusarium spp,

produce fusiform, curved. multiseptate

10

macroconidia with a

pointed apical

^{cell and a}

pointed

basal cell that has the appearance of ^a

foot,

hence called foot cell. In ^some

species,

^{smaller O - 1}

septate

microconidia are formed. Thick-walled

chlamydospores

^{may be}

present, depending

^{on the}

species (Booth, 1971).

Most of the Fusarium

species

^{isolated from nature}

produce

^their

macroconidia on

sporodochia.

^These

sporodochial types

^{often mutate in}

culture, especially

^{on rich} media. Mutations

however,

may

rarely

^{occur in}

nature. The mutants

mostly

^{show loss of}

pathogenicity

^and

toxigenicity (Nelson

et

aI., 1983).

^Two

major types

^{of mutants arise from the}

sporodochial type

^are

the

pionnotal type

^{and the}

mycelial type.

^The

pionnotal type produces

^{little or}

no aerial

mycelium,

^mass of macroconidia on the surface of the

colony

^and

more intense.

pigmentation

of colonies than the

sporodochial type.

^The

characteristics of the

mycelial type

^{are the}

production

^{of abundant aerial}

mycelium

^with very few or no macroconidia and

frequently

^{a lack of}

sporodochia

^and

pigmentation

^{in culture}

(Nelson

^et

al., 1994).

Studies on

morphological

characteristics ^were used to determine whether

phenotypic

characters could be found and used to differentiate sub­

species categories

e.g.

Group

^{I and}

Group

² strains of F.

graminearum (Aoki

and

O'Donnell, 1999). Morphological species

under Linnaean definitions are

delimited with two

primary

^{criteria. These are:}

(i) within-species (morphological consistency,

^and

(ii) sharp

^{breaks in}

consistency

^between

species (Mayr, 1963).

For several purposes,

morphologically-based species concepts

^and

taxonomies ^are useful tools for

(at least)

initial classification of

biodiversity.

^As

noted

by Taylor

^et

^al. (2000),

^the

greatest strengths

^{of the}

morphological species concepts

^for

fungi

^{are its}

general applicability

to any

fungal

^{taxon and}

11

its

widespread

and historical use. The Gerlach and

Nirenberg (1982)

^and

Nelson et al.

(1983)

^{taxonomies are both}

morphological

^{in nature and}

phylogenetic species concepts

^are

being

^{tested and into which new}

species

^are

being grafted.

Both

physical

^and

physiological

^{characters have been used a}

morphological

^{characters to}

distinguish

^Fusarium

species.

^The

shape

^{of the}

macroconidia often is

giving

^the

greatest weighting

^when

defining species,

^but

differences in macroconidial

shape

^{and size can be}

confusing, subjective,

^and

dependent

upon the environment in which

they

^are

produced.

Other spores, e.g., microconidial and

chlamydospores,

^{also are}

important

ⁱⁿ

morphological species.

The value of

physiological

^characters

including growth

^rates,

mycotoxins production,

^and

secondary

metabolites

produced

in different media varies. At

present, growth

rates, ^most

commonly

^at

25°C,

^{sometimes are used}

_by

^some

researchers to

separate closely

^related

species,

^butthis character is never the

primary

character for a

species

^definition of Fusarium. The

production

^of

secondary metabolites, including mycotoxins,

^{also may be used as an}

important

character in Fusarium

taxonomy (Thrane, 2001),

^{but is}

technically

difficult and

requires equipment

^{and chemical}

expertise

that many

mycologists

and

plant pathologists

^{lack. It} is be used to define

species,

^even

though

^the

ability

^{of a}

species

^to

produce

^a

particular secondary

^metabolite

(s)

^{often is a}

character of critical

ecological

and economic

importance.

12