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August 1994
Sydney
Royal Botanic Gardens
and
Fusarium Research LaboratoryDepartment of Crop Sciences
University of Sydney
Kathryn P. Gott
David Backhouse
L-j, j,u If e4
Lester W. IB~ .
Brett A. Summerell
Suzanne Bullock
Third Edition
i
LABORATORY MANDAnFOR ~:~),
,..):,.
FUSARIUM RESEARCH
ISBN 0 86758 849 7
589.2'4
Laboratory manual for Fusarium research'. 3rd ed.
Burgess, Lester W. (Lester William), 1942-
Suggested Cataloguing
Previous editions © 1983, 1988 by University of Sydney
University of Sydney
© 1994 by University of Sydney
1. Fusarium - Identification 2. Fungal diseases of plants I Laboratorymanual 3. Plant diseases - Identification I. Summerell, Brett A. 1959-.
I_ II. Bullock, Suzanne 1948-. III. Gott, Kathryn P. 1953-. IVI. Backhouse;David 1957-. V. University of Sydney, Department of Crop Sciences.VI. Title.
All rights strictly reserved. The checklist on page 40 may bephotocopied. No other part of this book may be reproduced III any formwithout the permission in writing of the publisher.
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Professors P.E. Nelson and T.A. Tousson of the Fusarium Research Centerhave provided invall1~ble support and advice for which we are ilndebted. Wealso acknowledge the-~'assistance and helpful advice provided by\many othercolleagues, in particular Dr C.M. Liddell, Dr W.F.O. Marasas, Dr G.A. Neish,Dr Carol Windels, and Dr T. Kommedahl.
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We gratefully acknowledge the assistance of Fred Stoddard and VeronicaMoschione in revising and reformatting the text. The financial kssistance ofthe Grains Research and Development Corporation, Australikn ResearchCouncil, and the Australian International Development Assistance Bureau for
. research on many aspects of the biology and taxonomy of ~usarium isacknowledged with gratitude. Co-operative studies on the taxonomy anddistribution of Fusarium species involving staff of the Fusaritim ResearchCenter, Pennsylvania State University have also been funded unaer the U.S.Australian Science and TechnologyAgreement by the Department of Industry,Science and Technology (Australia) and the National Science Foundation(U.S.A.).
The first edition of this manual was prepared primarily for use b~1participa.ntsat the Fifth International Fusarium Workshop at the University of Sydney inAugust 1983. This, and the second edition of 1988, found extensive use asgeneral references in mycological and plant pathological researchl In addition,the manual has been used to introduce undergraduate and postgraduatestudents to the taxonomy of Fusarium species, as well as] illustratingtechniques for use in the isolation and identification of these fungi. Strong
Idemand for the Manual from throughout the world, as well as recentdevelopments in taxonomy, have stimulated the production of this fhird edition.
Changes since the previous edition include the recognition of subspecies withinF. acuminatum and F. avenaceum; the inclusion of F. dimerurn , F.polyphialidicum and F. 'babinda' in the keys to species; and synonymy of F.graminum with F. heterosporum. The use of sections within tIle genus hasbeen avoided, pending determination of the affinities of several n€'~ly-describedspecies and a reappraisal of the validity of the sections defiried by Wollenweberand others. All of the photographs are new. The descriptions of species havebeen updated, and are based on the examination of over 40 000 cultures ofFusarium. The chapter on techniques for studying the ecology bf Fusariumspecies, included in the first two editions, has been omitted, since thisinformation is now available elsewhere.
~:Preface
_ lV _
Taxonomy .. 29Key Characters Used For The Identification Of Species 31Teleomorphs .....................•..................................................... 37Practical Hints On Identification l 37
i~;t*~;~~~!~ilc:~::~e~~;;;;;;~;;.Sp~~;~~'OfF;;;~~i~;;i;;"'I'" 38Australasia 41Fusarium dimerum 46Fusarium meri smoides 47Fusarium lateritium 48Fusarium decemcellulare · ! 49Fusari um poae ! 52Fusarium tricinctum J •••••• 53Fusarium sporotrichioides 54Fusarium chlamydosporum 55Fusarium moniliforme 00Fusarium proliferatum 62Fusarium anthophilum : 64
Isolation Procedures ................................... ............................... . 19Isolation From Plants 19Isolation From Soil : l :..22Isolation From The Atmosphere ! 24
Path.ogenicityTests. ................................................................... . ~
Preservation of Cultures 17·Lyophilisation 17Long Term Preservation On Silica Gel 17Temporary Preservation l 18Preservation For Herbarium Records J.. 18
Culturing Procedures : 12Identification J2Degenerate Cultural Variants 14Culture Mites 1 15Induction Of Perithecia 15
Media 5General Purpose Media ·5Selective Media 7Medium For Natural Inoculum 11
In.trod.uction 1
Preface iii
Table of Contents
33
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Fusarium subglutinans 1 65Fusarium nygamai j 70Fusarium 'babinda' ! 72Fusarium oxysporum l 74Fusarium solani l 76Fusarium culmorum 82Fusarium sambucinum 85Fusarium crookwellense f{lFusarium graminearum . , 89Fusarium avenaceum ssp. avenaceum 00Fusarium avenaceum ssp. aywerte 00Fusarium avenaceum ssp. nurragi 99Fusarium heterosporum 100Fusarium acuminatum ssp. acuminatum 102Fusarium acuminatum ssp. armeniacum l04Fusarium longipes J 106Fusarium compactum 107Fusarium equiseti 109Fusarium scirpi : 113Fusarium polyphialidicum : 115Fusarium semitectum .1. 116Fusarium beomiforme J 118
IReferences 124
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Introduction
Chapter 1
Fusarium oxysporum is one of the most variable species within tlie genus. Itincludes populations ,~hich cause vascular wilt diseases (Beckrtan, 1987),.populations which cause root, crown, tuber, corm and bulb rots (Nelson et al.,
I1981b), and populations which are soil saprophytes. Some members of
Some species, for example F. oxysporum and F. equiseti, have a high degree ofvariability in their cultural morphology and physiological character-istics(Burgess et al., 1989b). This inherent variability has presumably ertabled suchspecies to occupy a diversity of ecological niches in many geograPlhic regions(Table 2). Other species which are less variable, such as F. decemcellulare,tend to be less widely distributed (Table 2).
Many Fusarium species are particularly common in soil, and persist aschlamydospores or as hyphae in plant residues and organic matter (Burgess,1981). Several species produce airborne conidia and are common colonisers ofstems, leaves and floral plant parts (Burgess, 1981). Consequently farmingpractices, such as conservation tillage, which involve the preservation ofinfested plant residues are likely to increase the level of inoculum of Fusariumplant pathogens (Summerell et al., 1989).
The genus has a widespread distribution and representatives 0lccur in allmajor geographic regions of the world (Burgess, 1981). Some species have acosmopolitan geographic distribution whereas others tendl to occurpredominantly in tropical and subtropical regions, or cool to warm temperateregions (Table 2). A few species and subspecies are restricted to very specificregions and appear to be intimately associated with particular plant species or.groups (e.g. Sangalang et al., in press; Summerell et al., in press).
The genus Fusarium is one of the most economically important genera of fungiand includes many pathogenic species which cause a wide range of plantdiseases (Table 1) (Nelson et al., 1981b). There are species which I are highlymycotoxigenic producing a range of toxins affecting wildlife, livFstock andhumans (Maras as et al., 1984; Burgess, 1985; Joffe, 1986; Marasas & Nelson,1987; Nelson et al.; 1990), and there are a number of species which can causeopportunistic infections of humans, especially the immuno-supp~essed, andother animals (Rebell, 1981). In addition, many species are common soilsaprophytes (Nelson et al.,.1981b).
Table 1 , Some examples of the plant diseases caused by Fusarium speci,s.
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Species Disease Ref~rence
oxysporum Vascular wilts Beckman, 1987Booth, 1971Nelson, 1981Nelson et al., 1981b
, I Crown rot of tomatoPloetz, 1990 I
Jarvis & Shoemaker, 1978,I
solani Root rot of legumes and Burkholder, 191i9other' crops Nelson et al., 1981b
Imoniliforme Stalk, root and cob rot of Marasas et al., 1~79
corn Nelson et al., 1981b! 1
Trimboli & Burdess, 1983,~; Stalk and root rot of, sorghum 1985 I
Leslie et al., 1991subglutinans Stalk and cob rot of corn Nelson et al., 1981b,
Leslie et al., 199b~ ! 1
, \, II' " Pitchcanker of pine Dwinell et al., 1981!It! " Ij;
.,Fruit rot of pineapple Bolkan et al., 19~9p'IJ, '
I culmorum Foot and root rot of Cook, 1980wheat Nelson et al., 1981b
Igraminearum-Gixsvui 1 Crown rot of wheat, Burgess et al., 1~81barley, triticale, oats Burgess et al., 1987band grasses
Igraminearum Group 2 Stalk and cob rot of corn Nelson et al., 1981b(Gibberella zeae)
Head scab of wheat Atanasoff, 1920Sutton, 1982
Stub dieback of Nelson et al., 19i75carnations
avenaceum Root rot ofmedics, and Lamprecht et al." 1984other legumes Burgess et al., 1973
Stem rot of carnations INelson et al., 1981a
lateritium Storey's bark disease, Siddiqui & corbitt, 1963scaly bark and collarrot of coffee
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The taxonomic approach used in this Manual is similar to that in 1j-Ielsonet al.(1983), an illustrated taxonomy of Fusarium species which contains a usefulsynoptic key. A comprehensive pictorial atlas ofFusarium has bee1npublished
. Iby Gerlach and Nirenberg (1982). The taxonomic systems adopted by Nelsonet al. (1983) and Gerlach and Nirenberg (1982) are based on the pioneering workof Wollenweber and :R~inking(1935). The two systems differ in detail rather
The shape of the macroconidium, and the shape and mode or formation ofmicroconidia are remarkably consistent characters, and are rlsed as the. primary criteria for the identification of Fusarium species. The Ipresence orabsence of chlamydospores has been used as a primary criterion foridentification but is less reliable than the above criteria and a specialisedmedium such as soil agar may need to be used. Colonymorphology and growthrates on PDA are useful secondary criteria for identification. These criteria areused in the descriptions of species included in the taxonomic section of thisManual.
In designing pathogenicity tests it is important to recognise the role ofenvironmental factors, such as the soil microflora and soil moisture, in diseasedevelopment. Soil moisture may affect the activity of a pathogenj directly, orindirectly through its effect on the susceptibility of the host. Some pathogens,for example F. graminearum Group 1 and F. moniliforme, uSFa:lly causesevere symptoms only in hosts subjected to moisture stress at cerltain growthstages (Trimboli& Burgess, 1983; Liddell et al., 1986; Liddell & Burgess, 1988).
A knowledge of the taxonomy ofFusarium, and the appropriate prlcedures foridentification is basic to most studies of the genus. Although I Fusariumincludes some populations which are quite variable, the identification of mostspecies is not difficult if consistent and appropriate mediJ, culturingprocedures and incubation conditions are adopted.
F. oxysporum also cause opportunistic infections of humans and other animals(Rebell, 1981). The populations which cause vascular wilts are known asformae speciales and each has a narrow host range, while the populationswhich cause root rot and rots of vegetative plant parts tend to have a lessspecialised host requirement. Saprophytic populations of F. oxysporum areusually aggressive secondary colonisers of diseased plant parts, !particularlyroots, and are indistinguishable from the primary colonisers (pathogens) on amorphological basis. Thus carefully designed pathogenicity tests are essentialfor determining those populations of F. oxysporum which are pathogenic toplants. Indeed many of the populations isolated from diseased ~oot tissuesappear to be secondary colonisers. The situation is further complicAtedbecausea pathogen of one host may only be a secondary coloniser of another host.
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than in substance. The historical aspects of Fusarium taxonomy have beenreviewed by Toussoun and Nelson (1975), who also discussed the problems ofvariation and speciation vin the genus in detail, and by Nelson (1991). Thepublications cited above are indispensable reading for the serious student ofFusarium taxonomy.
1Based on surveys cited by Gordon (1952, 1960), Kommedahl et al. (1975), B~rgeSS (1981),Marasas et al. (1988), Burgess et al. (1988); Burgess & Summerell (1992), Summerell et al.(1993a); Sangalang et al. (in press) and authors' unpublished data.
2Restricted to relatively wet tropical regions.
tricinctum.
sporotrichioides
subglutinans
solani
sambucinumsemitectum
graminearumpoae
longipeseculmorumoxysporum
crookwellense
avenaceum
beomifor1e2compactum
decemcellJlare2
chlamydosporum
equiseti
moniliforme
acuminatum
", ISpecies which occur'i-. I
mainly in subtropical andtropical rekions
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Species which occurmainly in temperate
regions
Species which occur inmost climatic regions
. JTable 2 Guide to the occurrence of some Fusarium species in relation to climate!
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CLA is a natural substrate medium (Snyder & Hansen, 1947; Fisher et al.,1982) prepared by placing sterile carnation leaf pieces (approximately 1 pieceper 2ml agar) in a Petri plate and then adding sterile 2%Water ATr.
The carnation leaf pi~ces are prepared from fresh carnation leaves free fromfungicide or insecticide residue. Immediately after collection the l~aves are cutinto 5-8m:n pi.ecesand d~ed in a forced-air ~ve~ at ap~roximatelYI70°C for ~-4hours until brittle. Leaf pieces can also be dned In a microwave oven. The driedleaf pieces are packaged in alunhnium or polycarbonate coritainers and
2. Carnation Leaf-Piece Agar (CLA)
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Water Agar (0.05%), 0.5g agar in 1L of water, is used in the preparation of soildihrtion series. The small quantity of agar slightly retards sedimentation ratesof fungal propagules. The agar is dissolved in water before being Idispensed inaliquots of 100ml into McCartney bottles. Bottles are capped lobsely duringsterilisation and caps are tightened when sterilisation is complete.
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Hyphal growth is sparse on this medium so it is suitable for cultures fromwhich single hyphal tips are to be taken for the initiation of new colonies (seepage 14). Sparse growth on Water Agar also facilitates the isolation ofFusarium species from plant material, particularly roots.
Water Agar (2%)consists of 20g agar in lL ofwater and is recommended as thesubstrate for the germination of conidia used to initiate Fusarium bultures (seepage 13).
1.Water Agar (WA),~!lil!
GENERALPURPOSEMED~
A range of media has been developed for the isolation, growth and sporulationof Fusarium species. Included in this chapter are descriptions for thepreparation of some general purpose media and some selective media.
Carnation leaf-piece agar (CLA),potato dextrose agar (PDA) and slil agar (SA)are the standard media used in the identification of Fusarium .s~ecies at theFusarium Research Laboratory.
Media
Chapter 2
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,I PDA is a carbohydrate rich medium which contains 20g dextrose, 210g agar andthe broth from 250g white potatoes made up to 1L with tap water. The potatoesare unpeeled but washed and diced before boiling until just softl The boiled
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potatoes are filtered through cheesecloth leaving some sediment in the broth.. I
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5. Potato Dextrose Agar (PDA)
The medium has been used to promote the production of perithecia by Nectriahaematococca.
A 30% (v/v) suspension of V8 Juice® (Campbell's Soups Pty. Ltd.) is made upwith 2%Water Agar. Before addition of the agar the pH of the juice should beadjusted to 5.5-6.5 using l.OMNaOH.
4.V8-Juice Agar
3. KCIAgar
The addition of 4-8gL-1KCl to WA or CLA enhances the number and length ofmicrocOl:idial chains in culture~ of specie~ with this characteristic. I Chains arealso easier to observe as there IS less moisture on the surface of the agar andfewer droplets of moisture in the aerial mycelium.
Gibberella zece (Schw.) Petch (anamorph = Fusarium graminearur Group 2)and homothallic cultures of Nectria haematococca Berk. & Br. (anamorph =Fusarium solani) form perithecia readily on CLA if incubated unde~ light.
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sterilised by gamma irradiation (2.5 megarads). Sterilised leaf pieces can ·bestored at 2-5°C for up to 12months before use.
Most species of Fusarium sporulate on CLA in 6-10 da~:~.Using this mediumconidial shapes are more uniform than when using carbohydrate Irich mediasuch as PDA. Macroconidia are formed mainly in sporodochia whlich usuallydevelop on the leaf pieces..Macroconidia formed in sporodochia are preferred inidentification as they are more consistent in shape and lJngth thanmacroconidia formed from solitary monophialides on hyphae oJ the agar.Microconidia are more common on hyphae growing on the agar,loften awayfrom the leaf pieces. The mode of formation of microconidia, the presence ofchains of microconidia, and the presence of chlamydospores can be Ideterminedby direct examination with a compound microscope when small plates of CLA(6cm diameter) are used for routine identification of Fusarium' cu]tures. CLAis also suitable for the production of large numbers of conidia for e*perimentalwork, and can be prepared for this purpose in large flat bottles or large Petriplates.
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Agar 20.0gPeptone 15.0gKH2P04 . l.OgMgS04.7H20 0.5gTerrachlor® l.Og (contains PCNB 75%w/w)
;lutoclaved and cooled to 55°C before addijg, in 10ml~The basal medium issterile water:
PPA consists of a basal medium to which antibiotics and fungicides are addedI
and enables the selective isolation of Fusarium species from soil dilutions(Nash & Snyder, 1962). It is highly inhibitory to most other fungi ahd bacteriabut allows slow growth of Fusarium which form small colonies bf 5-10mmdiameter after 5-7 days.Basal medium in 1Lwater:
1.Peptone PCNB Agar (PPAINash - Snyder Medium)
SELECTIVE MEDIA
.SA is prepared by adding 500g sieved dry soil and 15g agar to 1L water. Theamount of soil used can be varied with soil type. Abundant chlamydosporeformation by various species has been observed on SA prepared wit~ 250g blackclay soil. The autoclaved medium should be regularly agitated while plates arebeing poured to ensure the even distribution of the solids to all platesl.
Chlamydospore formation is enhanced on Soil Agar (Klotz et al., 1988) and sothis medium is a critical supplement in the identification of some species ofFusarium .
6. Soil Agar (SA)
Although PDA is useful for the isolation of Fusarium species from plantmaterials many saprophytic fungi and bacteria also grow on PD.iAand mayinhibit the recovery ofFusarium. It is recommended that the conc~ntration ofpotato and dextrose be reduced by 50-75%and appropriate antibioticsl be added.
Conidia formed on PDA are usually variable in shape and size anJ so are less.reliable for use in identification. However colony morphology, pilgmentationand growth rates ofFusarium species on PDA are reasonably consistent if themedium is prepared carefully and if the cultures are initiated frok standardinocula (such as germinated single spores) and incubated und~r standardconditions. These colony characteristics are useful secondary 6riteria foridentification.
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1 Streptomycin is effective against Gram negative bacteria, Neomycin against G1ram positive·bacteria.
2-Dichloran (2,6-dichloro-4-nitroanaline) O.0065gmay be substituted by Allisan® O.013g or by.the fungicide PCNB (pentachloronitrobenzene) at a concentration ofO.75gL-1,as Ig'Terrachlor®.
DCPA was developed for the selective isolation of Fusarium species anddematiaceous hyphomycetes from cereal grains (Andrews & Pitt, 19186).
3. Dichloran Chloramphenicol Peptone Agar (DCPA)
SFA permits the slow growth of Fusarium species from plant roots and soildebris, and is less inhibitory than PPA to most fungi. Colonies bf differentspecies developing from a single root fragment or piece of de~ris can bedifferentiated more efficiently than on PPA. SFA is not suitable for the isolationofFusarium species from soil dilutions.
O.lg0.013gO.Olg
The basal medium is autoclaved and allowed to cool to 55°C before antimicrobial agents are added:in 10ml sterile water: Streptomycin sulfate
Dichloran-, added as Allisan®Neomycin sulfate
20.0g20.0g0.5 g2.0 g0.5 gl.0 gl.0 ml
Basal medium in 1L water: AgarDextroseKH2P04NaN03MgS04.7H20Yeast extract1%FeS04.7H20 soln
2. Selective Fusarium Agar (SFA)
l.Og0.12g
Streptomycin sulfate!Neomycin sulfate!
Developed for the selective isolation ofFusarium species from soil debris, SFAis a modified Czapeck-Dox medium containing anti-microbial agent~ (Tio et al.,1977).
?~,~j,The prepared plates should be allowed to "dry" in a cool dark place bFfore use sothat the water in the soil suspension is rapidly absorbed. Most species of
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Fusarium do not form distinctive colonies on PPA, sporulation tS poor andconidial morphology abnormal. Colonies must be subcultured for
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identification. Fusarium cultures should not be maintained on PPA becausethe metabolism of peptone leads to the accumulation of toxic ammoJia.
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3 Chloramphenicol is a broad spectrum antibiotic that can be autoclaved.
Two pieces of sterile filter paper (Lcm-) placed on the agar surface when setassist in stimulating ~:~orulation. I
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SNA is prepared by autoclaving, in 1L distilled water:KH2P04 1.0gKN03 l.OgMgS04.7H20 O.5gKCI O.5gGlucose O.2gSucrose O,2gAgar 20.0g
SNA, also known as low nutrient agar (LNA), is a weak nutrient agar whichhas been used for the identification and maintenance of Fus-drium and_Cylindrocarpon isolates (Nirenberg, 1976). In addition to limitihg culturaldegeneration, this medium allows consistent microconidial development andproduction of chlamydospores (Burgess et al., 1991). Howe¥Jr, discretesporodochia are not produced on this medium so consistent identification ofmacroconidia is not possible (Burgess et al., 1991).
4. Spezieller Nahrstoffarmer Agar (SNA)
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The basal medium, made up with 1L distilled water, contains: IAgar 20.0g1
Peptone 15.0glK2HP04 l.O~
IMgS04.7H20 O.5gl_Chloramphenicolf O.2g1
-After autoclaving add, in 10ml ethanol: Dichloran O.002g
Fusarium species produce well formed colonies on DCPA. It has beln proposedas an alternative medium for use in identification of Fusariufn species
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(Hocking & Andrews, 1987), but aerial mycelium is more sparse on DCPA thanI
on CLA so microconidia are generally produced less abundantly.I
Chlamydospores are also less abundant on DCPA than CLA. Not all species ofFusarium can be accurately identified using DCPA; so CLA is recommended
Ifor taxonomic studies (Burgess et al., 1991). DCPA should not be used as amaintena~ce medium. becau~e the metabolism of ~eptonel leads toaccumulation of ammoma to tOXIClevels. Mucoraceous fungi are suppressed bydichloran, and the absence of a carbohydrate source is selective -againstAspergillus and Penicillium species.
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To the basal medium is added in 10ml sterile distilled water:';~~/
l.Og0.5g0.5gO.Olg2.0g20.0g15.0g
K2HP04KClMgS04.7H20F3NaEDTAL-AsparagineD-GaladoseAgar
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The basal medium contains the following constituents in 1L distilled water andis autoclaved (121°Cfor 15m) and cooledto 55°Cbefore anti-microbial agents areadded.
Komada's medium has been recommended for the selective isolation of F.oxysporum from soil (Komada, 1975). F. oxysporum colonies are pigmented.Other Fusarium species are suppressed. I
6. Komada's Medium
MPDA allows the formation of distinctive colonies ofF. graminearum Group 1,and suppresses the growth of mucoraceous fungi and Trichoderma species.
0.16g0.013g0.06g
in 10ml sterile water: Streptomycin sulfateDichloran (added as Allisan®)Neomycin sulfate
After the basal medium has been autoclaved and cooled to 55°C antibiotics areadded:
MPDA was developed for the selective isolation of fungal pathogens from thecrown region of wheat plants. Specifically targeted are the fungi causing crown
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rot, Fusarium graminearum Group 1 and common root rot, Bipolarissorokiniana. I
The basal medium is prepared as for PDAbut the concentrations of polato brothIand dextrose are halved. Dextrose, 109, and the broth from 125g potato areI
made up to 1L. The concentration of agar can be reduced to 15g per L to allowthe crowns to be pressed into the surface of the medium.
5.Modified Potato Dextrose Agar (MPDA)
As SNA is transparent, cultures can be viewed by direct examination Lnder themicroscope or small blocks can be mounted on a slide with a drop of ~ater and -cover slip for observation.
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Cereal chaff and ground cereal grain (ratio 5:1) are used together. The chaff-I
grain mixture is soaked in water overnight at 5°C to leach phenoliccompounds, then drained thoroughly before distributing to glass jars orpolyester oven bags. The containers are sealed with a large cotton wool plugand autoclaved for 15 min each on two successive days. Containers areinoculated with a conidial or mycelial suspension (2ml per 250mlj chaff-grainmixture) which· is thoroughly shaken through the medium then incubatedunder standard conditions. The mixture should be shaken rkgularly to
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encourage colonisation. When the medium has been thoroughly colonised it is,
air dried, crushed and sieved to the required size for addition to soil or may bestored for up to 12months at 2-5°C. I
Inoculum suitable for addition to soil in pathogenicity tests can be preparedusing colonised chaff-grain as a substrate. It is particularly appropriate for. species which do not form chlamydospores but which normally petsist in soilas hyphae in plant residues.
Chaff-Grain Medium
MEDIUM FOR NATURAL INOCULUM
The·pH is adjusted to 3.8±0.2 with 10%phosphoric acid.
l.Og0.5gl.Og0.3g
PCNB as Terrachlor®OxgallNa2B407.10H20Streptomycin sulfate
--.Il[(~
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-",...--
~;J.,.
1"
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Sporulation and pigmentation are favoured by light, including ultra-violetwavelengths, and fluctuating temperatures (Snyder & Hansen, 19~1b, 1947;Leach, 1962). All cult~Tes for identification at the Fusarium Research.Laboratory are incubated in an alternating temperature regime, 25°C Iday/20°Cnight, with a 12hr photoperiod. Cultures are incubated 40cm below a light bank .
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Incubation conditions
Identification of Fusarium species at the Fusarium Research Laboratoryinvolves the growth of cultures on CLA and PDA from germinated singleconidia under standardised environmental conditions. A germinated Iconidiurncut from a WA plate (see below) is placed adjacent to a leaf piece on CLA. As
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CLA plates are poured the leaf pieces float to the side of the plate. This allowsthe fungus to grow on the leaf pieces where sporodochia are formed I and overan open area of water agar where the conidiophores producing microconidiaproliferate. A germinated conidium is placed in the centre of the PDA plates orslopes so that a uniform colony can develop. ' I
Chlamydospore formation is an important taxonomic criterion for thedifferentiation of some Fusarium species. Chlamydospores normally formwithin two weeks on CLA but formation is enhanced on soil agar. This is an-advantage for species such as F. acuminatum, F. nygamai and F. bJomiformein which chlamydospore formation is slow. Cultures on SA are initiatbd from a
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mass transfer of mycelium on an agar plug removed from a culture on CLA.Chlamydospores form on SA within 10-14 days following incubation in the darkat 25°C.
IDENTIFICATION
Morphological characteristics are basic for the identification and taxcnomv ofFusarium. However species of Fusarium exhibit a considerable degree ofmorphological and physiological variability. This is a result of genetic!plasticityand the ability of the fungus-to vary morphologically in response to changes inthe environment. It is therefore essential that standard culturing proceduresbe used in taxonomic studies on Fusarium to minimise variation daused bychanges in environmental factors. Cultures should be initiated froinl standardinocula, grown on media prepared exactly as described, and incubated undercontrolled and reproducible conditions.
Culturing Procedures
Chapter 3
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Single germinated conidium transfer
Colonies initiated from single conidia or hyphal tips are uniform andconsistent in appearance and ensure pure cultures. The single germinatedconidium transfer procedure is also valuable for separating mixed cultures
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encountered in isolations from diseased plant material or from soil. Thetechnique, originally developed by Hansen and Smith (1932), initially requirespreparation of a suspension of conidia in 10ml distilled sterile water in a testtube. A small scrape of macroconidia from a sporodochium is preferred toobtain a concentration equivalent to 1-10 conidia in a drop viewed under a low
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power objective of a compound microscope. "Dry" WA plates (at least 7days old)are seeded by pou;~ng the conidial suspension over the surface Iand the excessshaken off immedidtely. The plates are incubated in an inclined position (30-40°) in the dark at 25°C for 18-20hr. I
Figure 1 Mobile frame with lightbanks for the incubation ofFusarium cultures.
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(75cm wide) containing four 40W cool white fluorescent tubes ind one 36Wblack light tube (Philips® TL 36W/80 RS F40 BLB). Two light banks and twoshelves for cultures are constructed on a mobile frame (Fig. ID including a20cm space between the lower light bank and the upper shelf to preventcultures being heated by the lights beneath.
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-14 -
The use of standard culturing procedures enables degenerate cultural variantsto be recognised quickly~;,~nddiscarded (Toussoun & Nelson, 1975). !Althoughcultural degeneration is"a major problem in studies on FusariurA little is
. I 'known of the basic mechanisms involved. Such variants do differ from wild- .
DEGENERATE CULTURAL VARIANTS
Hyphal tip transfer
This technique is used to initiate cultures of several species, n6tably F.longipes, which frequently develop degenerate cultural variants fromgerminated conidia. Mycelium from a non-degenerate colony on CLA is used to
I
. initiate a colony on WA.Water agar plates should be poured on a slight slope soI
that the agar is shallower (approximately Lmm) on one side of the plate wherehyphal growth will be more limited. A single hyphal tip is cut out using theprocedures outlined for transferring germinated single conidia.
WA may be amended with antibiotics if the original culture is conthminatedwith bacteria. Alternatively a drop of 25% lactic acid may be added to theconidial suspension to inhibit bacterial growth. The acidified I conidialsuspension.should be allowed to stand 10 min prior to pouring. Germination ofthe conidia may be delayed for 24hr.
Figure 2 Procedure for removing a germinated single conidium from a WA plateusing a flat transfer needle.
1r:
Plates are examined using a dissecting microscope. A single. germinated. . I
conidium is removed on a small square of agar using a transfer needle or wirewith a flattened tip and sharpened edges. The knife edge of the needle is used to -cut a grid around the conidium and the flat surface used to remove the blockand transfer it to the desired medium (Fig 2).
..,~,
-15 -
Heterothallic cultures of F. solani f. sp. cucurbitae race 2 (= N ectriahaematococca) have sproduced perithecia using the following procedure. Thefungus is grown on V8';agar in slopes incubated in the dark at 25°C for 14 days.The cultures are then incubated under light (including UV wavele:hgths) for 48hr and spermatised with conidia of]the, appropriate compatibility type taken
, I,
Many species of Fusarium do not produce perithecia in culture. Some speciesare heterothallic and require the presence of suitable rnat.irig types for
Iperithecial induction. Isolates of F. graminearum Group 2 and somehomothallic isolates of F. solani do produce perithecia abundantly on CLA.Natural substrates usually enhance the formation of perithecia. Carnation leafpieces and stems, and wheat stems sterilised by gamma irradiatio1n have beensuccessfully used as substrates, as has V8 agar.~:
INDUCTIONOF PERITHECIA
Culture mites can be excluded from slope cultures by a rice paper cap glued,,' I
over the mouth of the test tube (Snyder & Hansen, 1946). The glue consists ofI
20% gelatine (w/w) with 5% copper sulfate (w/w) which inhibits microbialgrowth on the glue. The mixture is heated to dissolve the gelatine then pouredinto Petri plates to set and stored at 5°C. A tube is capped by warming themouth in a gas flame, inserting it lightly in the glue and then I placing the 'inverted tube onto a small piece of rice paper. The glue is allowed to set and theexcess paper is touched to a flame to burn away, leaving a 'cap over the mouthofthe tube. I
CULTURE MITES
The occurrence of degenerate cultural variants is more frequent in somespecies than others. Such variation is promoted by frequent sub-oulturing oncarbohydrate rich media using mass transfers of mycelium. The occurr-ence ofdegenerate cultural variants can be minimised by using natural ~ubstrate orlow nutrient media such as CLA or WA and by initiating cult~res from asingle germinated conidium or hyphal tip.
There are two types of degenerate cultural variants. The pionnotal tyPe is a flatslimy colony lacking aerial mycelium and consisting ofl abundantmacroconidia which may be distorted. The mycelial type consists of sterile,usually white, mycelium. The wild-type culture cannot be recov~red from adegenerate variant.
type cultures both morphologically and physiologically. Degenerate cultural'variants of pathogenic species are often avirulent and should be avoided instudies on disease. In contrast, mycotoxin production may not be affected indegenerate cultures (Wing et al., in press).
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-16 -
Ali (1993) developed a technique at the Fusarium Research Laboratory usingwheat straws to induce perithecial formation by Gibberella gordonii Booth (F.heterosporum). Straws were cut, tied into bundles of six, and placed i~to 150mlflasks containing 20ml tap water, plugged with cotton wool and cov~red withaluminium foil caps. After autoclaving for 20 min, the bundles of st}aw wereinoculated with 5mm plugs cut from agar cultures, one plug at each lend. Thecultures were incubated at 25°C day/20°C night, with 12hr photoperiod, asdescribed above. Perithecia were produced In compatible crosses aifter 8-10weeks.
from cultures grown in the light. The spermatised cultures are then incubatedunder constant light with fluctuating temperatures (in the range 10J25°C) for -14 days or until perithecia appear.
- 17-
Silica gel is the preferred method of storage in some laboratories because it doesnot require expensive apparatus and it is easy to use. Cultures can berepeatedly taken from a single storage tube, although contamination must beavoided. The major disadvantage is a gradual decline in viability but this can beovercome by replacement with fresh material (Windels et al., 1993). S~rvival ofFusarium species preserved by this method depends upon an abundantproduction of conidia. I
Silica gel (non-indicating) is dry heat sterilised at 180°C for 1.5hr in Jcrew cap• I
culture tubes then pre-cooled in an ice bath before use. A concentrated conidialsuspension is prepared by adding several colonised carnation leaf ipieces totubes containing 2ml sterile Difco skim milk. An aliquot, 0.3rn!1, of thesuspension of conidia is distributed over 3cm3 of crystals to moisten them.Silica gel will fuse if tO~;3:muchmoisture is added. A vortex mixer i1sused toredistribute the conidia and then the culture tubes are placed in ari ice bathuntil they cool as heat is released in the reaction between moisture knd silica
I •
gel (Windels et al., 1988). Both conidia)and milk solids are absorbed onto the
LONG TERM PRESERVATION ON SILICA GEL
More than 12 000 isolates have been lyophilised at the Fusarium ResearchLaboratory since 1971 by drying colonised carnation leaf-pieces in smJll glassampoules under high vacuum (10-L 10-2Torr). The ampoules are preJared byinserting a small cotton wool plug and then autoclaving in a loosely Icoveredbeaker. Four leaf-pieces from a culture two to three weeks old and initiatedfrom a single conidium are aseptically transferred to the ampoule ~hich isreplugged, labelled then heated and drawn out to an hour-glass shape/ using agas torch. The lyophilising unit is equipped with a refrigerated Ivacuumchamber to enhance drying. After 18hr the ampoules are sealed under highvacuum and stored at 5°C. All common species of Fusarium ha~e been
I
successfully lyophilised using this technique and have retained viability after20 years storage. .. I
Cultures can be revived by aseptically plating the dried leaf pieces onto CLA.T~e ampoule is first surface sterilised before it is shattered to releasei the leafpieces.
LYOPHILISATION
Preservation of Cultures
Chapter 4
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-18 -
Cultures are initiated from single germinated conidia and grown understandard conditions of temperature and light for 2 to 3 weeks. I Cultures arethen killed by exposing the plates to formalin in a closed container for 3 days.Preservation of the culture is achieved using agar and glycerine. Agar, 3g isdissolved in 147ml water, which is then dispensed as 6ml aliquots into testtubes before autoclaving. The lid of the culture dish is inverted, 1.5-1.75mlglycerine is added and then the 6ml aliquot of hot agar. is poured over the
. I
glycerine. The culture is aseptically lifted from the Petri dish and floated on themixture in the lid. Cultures are then allowed to dry in a drawer for 3-5 days
I
covered with a sheet ofwhite paper. When dried, the culture is rubbery and canbe removed from the Petri dish for storage. .. I
This procedure was refined for use with Fusarium species at the FusariumResearch Center, Pennsylvania State University.
Holotype specimens grown on PDA are required when lodging a formaldescription of a species or subspecies.
PRESERVATION FOR HERBARIUM RECORDS
The dehydration of colonised leaf pieces over silica gel for 48hr can be used as aI .temporary method of storage or for culture transfer through the postal system.The colonised leaf-pieces are taken from cultures approximately 2 weeks oldand placed in small sterile (gamma irradiated) envelopes. Durin~ dehydrationover silica-gel the envelopes are left partly open. I
Cultures can be revived by placing the leaf pieces on CLA.Alternatively conidiain sporodochia on the dry leaf pieces can be used to initiate new Jultures usingthe germinated single conidium transfer technique. .
TEMPORARY PRESERVATION
I
silica gel surface but the fungus does not colonise the substrate and so thechance of degeneration is restricted. Windels et al. (1988, 1993) cdution againststorage in sterile soil using a similar technique because the fungus colonisesthe soil both before : and during storage, increasing the potential fordegeneration and the consequent loss of morphological characters.
. l
The tissue selected for plating should be typical of the diseased material beingI
studi~d. The ol~est ~~crotic tissue s,ho~ld be av?ided as it .is llikely to ~ecolomsed extensively by saprophytes. TOXICmetabohtes present In very necrotic
• I
tissues may also inhibit the recovery of the pathogen. Similarly tissue that hasbeen damaged by implements or insects should be avoided as isaprophytesreadily colonise wound sites. Ideally}recently infected tissue ShOUlrbe selected
- 19-
The choice of isolation procedure will depend on the nature and number of. I
plant samples, the Fusarium species involved, and the necessity to isolate otherI
fungal genera. If the isolation of other fungi is required, the choice ofprocedures, particularly the types ofmedia used, may be restricted.1If there areonly a few samples to assess then a range of procedures can be selected tomaximise recovery of the target fungi. In contrast, the largel number of
I
samples involved in extensive surveys usually precludes the use of a range ofprocedures. I
• I
The isolation of Fusarium species from plants is affected by the nature of thediseased tissues, the method of surface sterilisation, the plating procedures,the medium and the incubation conditions.
ISOIATION FROM PLANTS
The isolation techniques described below have been used routinely in theFusarium Research Laboratory. I
While the majority of Fusarium species are fast-growing and aggressivecolonisers of culture media, some pathogenic species, 'particularlyF. decemcellulare and F. Lateritiurn, are slow-growing. These ]species areconsequently difficult to isolate from necrotic tissues contaminated bysecondary colonisers.
..,-~
The genus Fusarium contains many soilborne species as well as some specieswhich are well adapted to aerial dispersal. Many of these species are commonprimary or secondary colonisers of aerial and subterranean Jlant parts.Indeed saprophytic Fusarium species are often mistakenly assumed to bepathogenic because of their frequent isolation from diseased tissue! Care must. be exercised to distinguish pathogenic species from the saprophytes whenattempting to diagnose the cause of a plant disease. This normally involves apathogenicity test as discussed in Chapter 6. .
Isolation Procedures
Chapter 5
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- 20-
The choice of medium depends largely on the nature of the tissue involved inI
the isolation exercise. Low nutrient media such as CLA, WA or half-strengthPDA are used routinely at the Fusarium Research Laboratory for th1eisolationofFusarium species fro~ plant stems (Burgess et al., 1975; Francis & Burgess,1975), petioles and gra1h (Burgess et al., 1987a). Antibiotics can be added tothese media if bacteria interfere with the recovery of the Fusariu'm 'species.
J .Carbohydrate rich media are generally avoided in isolation studies because
t I
The isolation of pathogenic species from diseased roots is difficult because ofthe wide range of saprophytes which normally colonise necrotic rbot tissue,particularly the cortex. Fusarium root rot pathogens normally cdlonise thecortex and stele (vascular tissues) of the root. The frequency of isolation of thesespecies may be enhanced by plating segments of the stele after remo~al of partor all of the cortex.
The plating of small pieces (1-2 x 1-2mm) of tissue reduces the numb1erof fungideveloping from each piece. This simplifies subculturing and there is lesschance that slow-growing pathogens will be overgrown by fast-growingsaprophytes. However in some studies large sections of tissue can be Iplated if aselective medium is used. Entire subcrown internodes and crowns of wheatplants, for example, are plated on MPDA to assess the incidence of i~fection byF. graminearurn. Group 1, at the Fusarium Research Laboratory. I
Whichever technique is selected it is recommended that samples be 'dried' onI
sterile paper tissues, under a filtered air flow after surface sterilisation orwashing. Drying inhibits the growth of bacteria from the tissue.
Surface sterilisation may not be suitable for use with fine roots. Partialdisinfestation of such roots can be achieved by washing in a fin~ spray offiltered tap water for 30-120 min, and subsequent rinsing in steril~ water. Afine nozzle is recommended for use in washing roots, soil debris, and planttissue in general.
The procedure adopted for surface sterilisation depends largely on the nature ofI
the tissue. One of the most common procedures involves immersion of theI
sample in 1% sodium hypochlorite in 10% ethanol, the latter acting as awetting agent, and subsequent rinsing in sterile water. However some tissuesare quite porous and absorb the surface sterilant which eliminates thepathogen as well as contaminants on the surface. Itmay. be more appropriateto swab porous tissues, such as sorghum stalks affected by stalk rot,1with 95%ethanol. Swabbing with 95% ethanol is a preferred procedure fbr surfacesterilising diseased woody plant parts.
I
for isolation studies. Note that samples should be maintained in a cool dry stateduring transit to the laboratory to minimise growth of saprophytic fungi andbacteria.
- 21-
Ascospores can be used to initiate pure cultures from samples on which thetarget Fusarium is producing fertile perithecia. A small piece of ~issue withperithecia is washed, and after excess water is removed it is placed on theinner side of the lid of an inverted shallow Petri dish containing WA or CLA.The tissue is held in place with petroleum jelly. The inverted plate ilsincubatedat-25°C inside a plastic bag to maintain high humidity. Ascospores Are released
, ..after 24-48 hr and are impacted on the agar surface where they germinate and
Idevelop colonies. The fungus can then be subcultured onto other media byhyphal tip or single-spore transfer for identification. I
'¥..Slow-growing Fusari~!ih species such as F. avenaceum ssp. aoenaceum maybe difficult to isolate directly from necrotic root tissue which is ~extensivelycolonised by other fungi and bacteria., However these slow-growingspecies can
, I,:l-
There are several other techniques for recovering Fusarium species, directly orindirectly, from plant samples, which do not involve plating tissue segments onagar media. Some species produce sporodochia on the surface of the diseasedtissue. Macroconidia can be taken from these and used to prepare a conidialsuspension which is plated on WA containing antibiotics. Germinlated singleconidia are later taken to initiate pure cultures for identification.
they favour fast-growing saprophytes such as the mucoraceous' fungi andTrichoderma. In addition, some pathogenic Fusarium species Idegeneraterapidly to avirulent forms on these media:
At the Fusarium Research Laboratory, PDA is used for the isolation ofF. lateritium and F. decemcellulare from twigs of mulberry (Mor~s alba L.),and lychee (Litchi chinensis Sonn.) respectively. These fungi may be difficult toisolate on weak media because of their slow growth. The twigs should beswabbed with ethanol before plating. Itmay be appropriate to remove some ofthe outer tissues first. ... I.Selective media are normally used for the isolation of Fusariurn species fromdiseased crown or root samples. A range of selective media are described inChapter 2. It should be emphasised that all media are selective as they willfavour the growth of certain fungi over others. In this manudl the termselective medium is used only for media containing anti-microbial Jgents.
Isolation plates are incubated under the standard conditions o~ light andtemperature described in Chapter 3. The colonies which derelop fromsegments of tissue should be subcultured to CLA and the conidia produced onthese plates can then be used to initiate cultures for identification Js described.in Chapter 3. It may be preferable to subculture from very young coloniesdeveloping from tissue segments with the aid of a dissecting microscope. Thisprocedure may reduce the difficulties caused by fast-growing speciesovergrowing slow-growing species.
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- 22-
Fusarium species can be isolated directly from soil using the dilution plateI
technique (Nash & Snyder, 1962), by plating debris on selective media (Tioet al.,I
1977; McMullen & Stack, 1983), or indirectly using living root or sterile strawbaiting techniques. The most appropriate technique for the is6laltion of aparticular Fusarium will depend on its mode of persistence in soil. Speciessuch as F. oxysporum, F. solani and F. culmorum which form abundantchlamydospores in soil can be isolated consistently from soil using the dilutionplate technique. In contrast, species which mainly persist in soil as Iiyphae inplant residues such as F. graminearum and F. avenaceum ssp. av1enaceummay be isolated more consistently by plating debris on selective media.1 .
The spectrum of Fusarium species isolated and the frequency of isolation isinfluenced by soil sampling procedures in the field, transit and storageconditions of samples, and the isolation technique used. Fuearitini species,particularly plant pathogens, are distributed irregularly in soil (Wlearing &Burgess, 1977). A pathogen is normally more abundant in the vicinity of
I
diseased plant rema:ins. Indeed, infested residues may be concentrated on theI
surface or in the upper 1-5 em of the soil profile in fields where residues areretained on the soil sur'(ace. The spatial distribution of pathogens, ieither inresidues or as chlamydospores,will also be affected by the frequency and type of
ISOLATION FROM SOIL
For large-scale screening for crown rot in cereals, isolations may be made on toselective media poured into trays. At the Fusarium Research L~boratory,stainless steel catering trays approximately 30 x 40 x 2 em deep are hsed. Lids
I
are cut from Perspex sheeting to the same outside dimensions and held on withspring (bulldog) clips. The tray and lid are thoroughly swabbed with alcoholbefore use. Wheat crowns with subcrown internodes are washed a:nd surface
I
sterilised, then placed on the medium, 50 per tray. The lids are replaced andthe trays incubated under light for 4-5 days. Fusarium graminearu, Group 1may be recognised by its distinctive colonymorphology on MPDA. Best resultsare obtained if the agar concentration is 1.5%or lower so that the cr'owris andsubcrown internodes can be pushed into the surface of the medium.
I
sometimes be isolated indirectly using a combination baiting/plating technique.The necrotic tissue is thoroughly washed and cut into small pieces Iwhicharemixed with steam-air treated soil. Surface-sterilised seed of the same cultivaris then sown into this mixture. The bait plants are incubated under ponditionswhich are thought to favour the disease. The roots are recovered as soon aslesions develop and segments are plated on suitable media after! thoroughwashing and, if appropriate, surface sterilisation. This technique has beenused to indirectly isolate F. avenaceum ssp. avenaceum from necrotic roots ofsubterranean clover (Burgess et al., 1973).
- 23-
This technique involve,,~plating washed pieces of soil debris on selective media.A soil sample is suspended in water which is poured through a nest of sieves,in order, 4.0 mm, 2.0 mm and 0.5 mm in mesh aperture. The first Isieveretainsgravel and large pieces of plant residue. This residue can be surface sterilisedand plated on selective media to recover Fusarium species. The s~all pieces of
Debris isolation technique
It is important to ensure that the soil is air-dried before plating to reducebacterial growth on the plates. The medium should also be allowe1dto 'dry' forat least five days before use for the same reason. Furthermore it is Inecessary touse pipettes of the same inlet-bore diameter for quantitative studies. Note thatestimates of propagule numbers derived from colony counts on dilution platesdo not necessarily provide an accurate estimate of inoculum pdtential. Thecolonies may originate from single-celled or multi-celled propagules or fromhyphal clumps, free or within very small pieces of organic debris', Propagules
,
within pieces of debris which are wider than the inlet bore of the pipette will beexcluded from the dilution plates.
Colonies of the various species are more easily differentiated and sporulatemore profusely if dilution plates are incubated in the light. Direct identificationof colonies on soil dilution plates is not recommended because mariy Fusariumspecies produce similar colonies on PPA and spore morphology is irregular.All colonies should be subcultured for identification.
This technique involves the uniform dispersion of 1ml of soil suspension(between 1:50 and 1:2000 dilution in 0.05% water agar) across a selectivemedium such as PPA. A 1:500 or 1:1000 dilution is usually suitable for
I
cultivated or pasture soils whereas a 1:50 dilution may be more appropriate fordesert or arid grassland soils. The soil sample is lightly ground in!a mortar ifnecessary and sieved before it is added to the water agar. Propagules in the soilsuspension usually germinate within 2-3 days on PPA and produce smallcoloriies by 5-7 days. The suspension can be uniformly dispers1ed over themedium by carefully pipetting 1ml of soil suspension onto the meaium on oneedge of the PPA. The plate is then held with a slight slope away from thesuspension and gently shaken at right angles to the slope. Thel suspensionslowly spreads across the plate with a uniform wetting front.
Soil dilution plate technique
It is preferable to transport and store soil samples in paper bags. Plasticcontainers should be avoided as they prevent drying and encourage Ian increasein bacterial numbers. Soil samples should be stored at low temperature (2-5°C)to prevent microbial activity.
cultivation (inversion tillage or sub-tillage). These factors should be taken int? .account when designing a soil sampling procedure.
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- 24-
Exposed Petri plate method. This is a simple and widely usedmethod (Gregory, 1973). Petri plates containing an agar medium are exposedfor 5-60 min depending on spore concentration and wind conditions. 'Fusariumspecies can be selectively isolated from the atmosphere with this method usingPPA plates. The Petri plate should be located in an open sided shelter ifcollection is to be made in direct sunlight or rain, and care should be taken notto overexpose the plate. This technique is qualitative, of variable efficiency andselective with respect to spore size. It does however allow recovery of the fungi
I .
for accurate identification, and is simple and inexpensive. The senior author1
has successfully used this technique in semi-arid and desert areas but, found1
that flies and other inaects are attracted to, and contaminate, the medium.Ants can also be a problem if the plate is exposed close to the ground.
None of these methods impair the viability of the trapped propagule andtherefore permit recovery and identification of the living fungus. ThJy have allbeen used to isolate Fusarium species but vary widely in efficiency, selectivityand complexity.
Reported Methods
Fusarium species can be isolated from the atmosphere by any method whichenables the collection of living microscopic propagules. A number of thesemethods have been used by aerobiologists for many years but they hkve rarelybeen applied to the study of Fusarium aerobiology. A brief description of themain methods that have been used to isolate airborne Fusarium Ispecies isincluded in this section together with a description of other potentially valuablemethods.
ISOlATION FROM THE ATMOSPHERE
debris retained on the other two sieves cannot be surface sterilised because they _are too small and porous. This debris is retained on the sieves and ~ashed toremove soil particles under a fine spray of filtered tap water for 12 hr. Thewashed debris is damp-dried on sterile paper tissues or towels, and then dried
I
over silica-gel for 24 to 48 hr before plating on a selective medium. Fusariumspecies can be isolated from debris using, for example, PPA, SFA lor DCPA.The authors have isolated a wide range of Fusarium species from pasture andgrassland soils using this technique. McMullen and Stack (1983) evaluated thedilution plate, debris isolation and soil plate techniques and three selectivemedia for the isolation ofFusarium species from grassland soils. ThJ reader isreferred to the paper by McMullen and Stack (1983) for a useful dis1cussionofthe relative merits of each technique and medium.
ItI,.1
- 25-
A wide variety of spore traps and collectors have been designed, some of whichwould be suitable for the isolation ofFusarium species. However, k descriptionof these is beyond the scope of this section. Details of the theory arid practice ofaerobiology can be obtained from reviews by Davies (1971) and Gregory (1973).Two spore collectors are worthy of mention as they both have special features.
I
Ogawa and English (1955) described a cyclone collector that ,samples largevolumes of air into a liquid trap and is suited to low spore concentrations. Themost important feature of this collector is the principle of operation whichcould be readily adapted to suit many experimental situations. Collection can
. I
be made into a single volume of liquid or, more usefully, into graduatedaliquots that are changed according to a time base. Schwadbach (1979)
Idescribed a collector of very high efficiency that impacts spores onto Petri platesor living plant tissue. It could be valuable in conducting infectivity studiesusing field inocula. It is non-selective, small and simple to operate.
In any attempt to isolate Fusarium species from the atmosPhJre the samebasic rules apply as to the recovery of other fungi and bacteria.v.A methodshould be selected that suits the objectives of the experiment W11ithoutbeingoverspecified. In many studies. an exposed agar plate will be, perfectlysatisfactory but. a well designed collector such as those described should beemployed in quantitaeive studies. It is necessary to be aware of tlie limitationsand benefits of each d'~sign.
Other Methods
The Hirst Spore Trap (Hirst, 1952) is designed to impact airborne ~.articles ontoa sticky microscope slide. Isolations can be made from the catch if the slide iscovered with acetate tape or agar which is subsequently plated. This trap alsohas the advantage that the catch can be closely examined under thb microscopebefore propagules are cultured. It is volumetric, non-selective and very efficientunder most conditions. Lukezic and Kaiser (1966) used a Hirst Ttap to detectconidia of F. semitectum.
I . .'Spore traps. Several spore traps have been developed to compensate for theshortcomings of the exposed Petri plate and two of these hav~ been usedspecifically to trap Fusarium species.
The Anderson Spore Sampler (Anderson, 1958) has many advantages over theexposed Petri plate. It is non-selective with respect to spore sike and it isdesigned to sort the propagules according to size. It is volumetric, ~fficient and
I
permits collection directly onto agar plates. The main disadvantage is cost.Ooka and Kommedahl (1977) used an Anderson Sampler fitted witll. PDA platesto trap propagules ofF. moniliforme.
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- 26-
Various simple techniques have been used to test the pathogenicity ofFusarium species to above-ground plant parts (Purss, 1971; Nelson et si; 1975).Conidia are normally used as inoculum in these tests and can be prdduced onnatural substrate media";~uchas cereal chaff-grain medium or CLA.
Some pathogens only cause severe disease in plants which have been Isubjectedto stress. Thus the technique selected for the pathogenicity test must enable theapplication of stress if it is thought to be a key factor predisposing th~ plant tothe disease. Stress, for example, may be caused by inadequate soil hlOisture,extremes of temperature or herbicides.
The cultivars used in the pathogenicity test should be identical .toI those onwhich the disease has been observed in the field. These cultivars should begrown under environmental conditions similar to those which prevail in thegrowi~g seaso~ in are~s where the disease occurs. Plants sh~uld be Igro~n incontainers which permit normal growth and development dunng the period ofllie~~. . I
The pathogenicity of an isolate of Fusarium should be assessed using atechnique which enables the reproduction of typical symptoms of the diseaseover the time-scale relevant to the commercial glasshouse or field [situation.The following guidelines are recommended for the design of meaningful testsof pathogenicity. I
Typical wild-type cultures should be used for the preparation of inoculum.Ideally these cultures should have been recently isolated from disedsed plantmaterial and maintained on a low nutrient medium such las CLA.Alternatively lyophilised cultures with a similar history can be used. Isolatesthat have degenerated (mutated), or that have been subcultured repeatedly onrich media should be avoided as they are likely to be avirulent (Nel~on et al.,1986). IThe nature and amount of inoculum used in pathogenicity tests should. I
conform to the inoculum causing the disease under field conditions. It may benecessary to make assumptions in this respect as pathogenicity ~tests areusually initiated prior to detailed studies on the mode of persistence anddispersal of the species in question. I
Pathogenicity Tests
Chapter 6
- 27-
The untreated field soil should be free from soilborne fungal and otherpathogens of the relevant host, and from residual herbicides. Such soil can beobtained from a field that has been planted to other crops, or that hJs been long-·fallowed. I
The microflora in untreated field soil inhibits (suppresses) the activity of asoilborne pathogen. The removal of this inhibitory effect by sterilisktion allowsmany ~oilborne fun~a~:ii'rathog~nssuch as Fusarium t? proli!erate regetativelyfollowirig the addit.ien of Inoculum to the ster-ile SOIL The pathogenconsequently causes greater disease in sterile than in untreated soil. Thisoccurs because the level of inoculum is higher following the vegetative growth,and there is no microbial inhibition "ofthe pre-penetration phase bf infection.
The soil (sterile, pasteurised or untreated) has a critical influence on theseverity of disease caused by Fusarium, and other soilborne pathogens, Thus
I
the soil selected for a pathogenicity test should be similar to that associatedwith the disease under investigation in respect of physical arid chemical
r Iproperties. A sterile soil should be used if the disease is normally restricted tosterile soils. Crown and root rot of tomato caused by F. oxysporum f.~sp. radicislycopersici, for example, is mainly a problem in fumigated soil (Jarvis &Shoemaker, 1978). Conversely an untreated field soil should be used in respectof field crop diseases such as crown and root rots ofwheat and stalk rots of cornand sorghum.
·The' design of pathogenicity tests of suspected soilborne pathogeps is mor~difficult because the nature. and amount of inoculum" and the soil have asignifican~ effect on i~fection and diseas.e developmen~. Cert~inl structuresproduced In culture which have the potential to act as soilborne Inoculum maynot persist in soil and are thus unsuitable for pathogenicity studies!. Conidia ofF. auenaceutri ssp. auenaceum , for example, are rapidly lysed in natural soil athigher temperatures (Wearing, 1976)~In contrast, chlamydospores kay persist~ .' .for long periods in soil and are the main' mode of survival for a number ofsoilborne Fusarium pathogens. Chlamydospores should normally be used asthe inoculum ofF. oxysporum and F. solani, The diseases caused by these twofungi can be reproduced using relatively low levels of propagules, if ~he isolatesare virulent. Inoculum densities as low as 50-100 propagules/g soil can be quitesatisfactory for reproducing typical symptoms of disease. Some Ipathogenicspecies ofFusarium mainly persist in soil as hyphae in host ~esiduks. Suitableinoculum of these species can be prepared from cultures grown on sterilenatural substrates such as wheat chaff or corn stalks (Nelson et al.) 1986). Theinfested tissue is dried and milled before addition to 1:10i1.Wheat, barley or oatchaff (with or without grain) is an appropriate substrate for F. gr~minearum·Group 1, F. culmorum. and F. avenaceum ssp. avenaceum. Corn stalks are anappropriate substrate for F. moniliforme. This type of inoculum can be mixedevenly through the soil (1-2% w/w) or added as a fine horizontal layer ofinoculum within the soil profile (Liddell et al., 1986).
iIl.\ .~~~
~~~~
@i[:G:i::
I- 28-
*Emergence expressed as percent of seed sown.
92
Soil infested with F. graminearum Group 1 100*
Uninfested soil 83
ISterile soil
IUntreated soil
Table 3 Emergence of wheat, cultivar Timgalen, 12 days after sowing in untreatedsoil and sterile soil, infested with Fusarium graminearum Group 1. The soilwas at a moisture potential of -0.5MPa.
The choice of soil for a pathogenicity text is therefore critical if the results are tobe meaningful. The reader is referred to Park (1963) and Marois ana. Mitchell(1981) for detailed discussions of the influence of the soil microflo~a on theactivity of a particular fungal species.
The influence of soil treatment on the pathogenicity (virulence) ofI
F. graminearum Group 1 to wheat seedlings (Table 3, after Liddell, 1985a)illustrates this effect.
- 29-
)'~
.I::::!I
Approximately 1000 Fusarium species had been described by 1900, basedlargely on examinations of fruiting structures (sporodochia) on plant material.It is not surprising that many of these species were synonymous given the
I
inherent variability in Fusarium, particularly when grown using differentsubstrates and environmental conditions. I
This large number of species was reduced by Wollenweber and Reinking (1935)to 65 species, 55 varieties, and 22 forms, through a process of consolidation andrejection of species names. This reduction was based on 40 years lof taxonomicresearch involving the use of pure cultures on a range of media. In thispioneering monograph, called 'Die Fusarien', Wollenweber arid Reinkingarranged the species in 16 sections, and all taxonomic systems p~oposed sincethen have been based on the system of Wollenweber and R~inking. The
I
historical aspects of Fusarium taxonomy have been discussed by INelson et al.,(1983). Some of the taxonomic systems proposed since 1935 are briefly discussedhere. I
Snyder and Hansen subsequently proposed that the number of species befurther reduced to nine, following intensive studies of variation using culturesderived from single conidia (Snyder & Hansen, 1940, 1941a, 1945; 1954). Theirproposal became known as the nine-species system and was later illustrated byToussoun and Nelson (1976). Although the nine-species system was adopted bymany plant pathologists in the U.S.A., it was not widely accepted elsewhere.The widespread use of informal 'Cultivars' (Snyder et al., 19571 within somespecies showed that the reduction in species by Snyder and Hansen was tooextreme. Their system is rarely used now. However their reduction of species
Iin Section Elegans to the one species F. oxysporum (Snyder & Hansen, 1940)and species in Section Martiella & Ventricosum to the one species F. solani
''If' ; I
(Snyder & Hansen, lQ:41a)has been widely accepted. Snyder and IHansen madea significant contribution to Fusarium taxonomy through their .studies onvariability using the single spore te~hnique, and their recognition jOfthe need toexclude degenerate cultural variants from taxonomic consideration.
The genus Fusarium Link ex Fr. is classified in the class Hyphomycetes of thesubdivision Deuteromycotina (Fungi Imperfecti). Fusarium incljudes specieswhich produce hyaline macroconidia which are septate, and characterised by afoot-shaped or notched base to the basal cell. Microconidia and chlamydosporesmay be present or absent. The perithecial states (teleomorphs) ate known forsome species and belong to the Hypocreales.
~...Taxonomy
Chapter 7
~.._
- 30-
I
The taxonomic approach to Fusarium used in this edition corresponds to thatadopted in previous editions of this manual and the approach of Nelson et al .(1983). However, in this edition we have placed less emphasis on the use ofsections because of the difficulties in differentiating the boundarie's 6f some ofthe sections following the recognition of a number of new species. In additionseveral subspecies taxa described recently following the guidelines ofHawksworth (1974) have been included. The subspecies taxon was introducedinitially by Burgess et al . (1993a) to differentiate between F. acuminatum ssp.acuminatum (formerly F. acuminatum) and F. acuminatum ssp.
Iarmeniacum (formerly F. "armeniacum" in the previous edition of thismanual). These subspecies differ in many important characteristick but theshape of the macro conidia is not sufficiently different to warrant thedescription of F. acuminatum ssp. armeniacum as a separate spJcies. Theshape of the macroconidia is one of the primary defining character-istics ofFusarium species. The use of subspecies taxa allows for the differentiation of
Ipopulations which differ in important ecological and physiologicalcharacteristics but which may not differ significantly in I primarymorphological characte'It~. The authors also have recently recognised threesubspecies populations within F. avenaceum, two from unique environmentswithin Australia. .: '
I.:"I!J
Gerlach and Nirenberg (1982) published a pictorial atlas on Fusarium speciesI
in which they recognised over 90 species. These authors assume quite narrowlimits to variation within a species, and thus differentiate populations thatsome other workers might consider belong to a single species. I
Nelson et al . (1983) recognised 30 species and in contrast to GeJlach andNirenberg, theyassumed relatively broad limits to variation within ~ species.They also emphasised the importance of basing decisions on species ]limits onthe analysis of variation in a large number of cultures from a wide range of
I
substrates and geographic sources, a philosophy promoted originally by Snyderand Hansen. . I
While there are differences in the taxonomic approaches taken [by theseauthors, all recognise the majority of important species, and the similarities ofthe taxonomic systems far outweigh the differences. It is however important toremember that whatever system is chosen for the identification of Fusariumspecies that the techniques used by those authors for identification are followed
I
throughout the identification process.
Booth (1971; 1977) in his monographs on Fusarium also reduced the rrumber ofspecies but less drastically than Snyder and Hansen. He focussed attention on -the value of the nature of the conidiogenous cell bearing microconidia as aprimary taxonomic criterion. He also. introduced the use of growth Irate as asecondary taxonomic criterion.
- 31-
IThe term 'shape' embraces a number of features. The ratio of length to widthdetermines, for example, whether a macroconidium is stout or slender (Fig. 3).It can be falcate or relatively straight (Fig. 3). The walls can be almost parallel
I
or curved to different degrees giving, in the extreme, the dorsi-ventralcurvature appearance of macroconidia in species such as F. equiseti and F.
I
longipes. The walls can appear thick or thin. The number of septa usuallyI
varies between isolates of the one species but in some species is relativelyuniform. The septa in-a few species are not very distinct. The apic~l cell can beshort and blunt (roci'tided) or hooked (beaked), papillate (dolphin-nosed),tapered to a point or very long (filamentous) and even curved or coiled (Fig. 3).
I
The base of the basal cell is distinctly; foot-shaped in many species put in others, 'i
1. The shape of the macroconidium is generally the most importantcharacter in the definition and identification of Fusarium species. Thedescriptions of macroconidia are based only on macroconidia produced insporodochia on CLA. Note that macro conidia produced in the aerial hyphae onCLA or on PDA are often highly variable and should not be used foridentification.
KEY CHARACTERS USED FOR THE IDENTIFICATION OF SPECIES
The important characters used in the identification ofFusarium sjecies are:1. shape of the macroconidium;2. presence or absence of microconidia;3. shape and mode of formation of microconidia;4. nature of the conidiogenous cell bearing microconidia;5. presence or absence of chlamydospores;6. colony diameters on PDA after dark incubation for 3 days .at 25°C and
30°C; .. I.7. colony morphology on PDA after incubation for 10-14 days at alternating
day and night temperatures of 25°C/20°C and a 12 hour photoperiod,I
The relative importance of each of these characters in identification variesbetween species. The nature of the macro conidia, microconidia, cdnidiogenouscells and the chlamydospores as described in this Manual are b~sed on CLA
I
cultures unless otherwise indicated. Descriptive terms are those used inI
Ainsworth & Bisby's Dictionary of the Fungi (Hawksworth et al., 1983).
IThis manual is not an exhaustive description of all species and populations of .Fusarium. We have tried to present a number of techniques which should.allow the identification of Fusarium species with reasonable ease provided thetechniques are closely followed. These techniques have been tised by theauthors for many years and have facilitated the consistent identifi~ation of allFusarium species currently recognised and the identification of fuany newlydescribed species and subspecies. I
b
s_-
'.\
E
,i{
,----..v<~L.Li:1((tIL;!.· ..
- 32-
, ."
1:Iij \.Id[i\iI·I: II '
I!I:\
o-r. Basal cells ofFusarium.o. Foot-shaped basal cell e.g. F. crookwellense.p. Foot-shaped elongated basal cell e.g. F. longipes.q. Distinctly notched basal cell e.g. F. avenaceum ssp. avenaceum.r. Barely notched basal cell e.g. F. solani.
k-n. Apical cells of Fusarium.k. Blunt apical cell e.g. F. culmorum.1. Papillate apical cell e.g. F. sambucinum.m. Hooked apical cell e.g. F. lateritium.n. Tapering apical cell e.g. F. equiseti.
g-i. Macroconidial shapes.g. Slender, straight, almost needle-like macroconidium e.g.
F. avenaceum ssp. avenaceum. Ih. Macroconidium with dorsiventral curvature typical of F. equiseti.1. Macroconidium with dorsal (upper) side more curved than tHe ventral
(lower) side e.g. F. crookwellense. IJ. Typical Fusarium macroconidium. Apical cell is on left-hand side
and basal cell on right-hand side. I
a-f Microconidial shapesa. oval. b. reniformc. obovoidwith truncate based. pyriforme. napiformf. globose.
IFigure 3
- 33-
~DI •
q r
pm
kD ~~
o IpI
->J.'
· 0 G 0~
a b ca
I·I~ Q 0 0·
-5
~"
d e fa
~·-~
~-~·-
9 h'I
-•·-'I
-~
- 34-
. r'"t
~'-
,} .
::11,.1
- 35-
I
(Scale in photographs a, d = 1 um. Scale in all other photographs = ~Oum),
I
a-c. Microconidia produced from monophialides.a. Scanning electron micrograph of single microconidium produced
from monophialide.b. Chain of microconidia produced from monophialide on CLA, in situ.c. False-head of microconidia produced from monophialide oh CLA, in
situ. I
d-f. Microconidia produced from polyphialides. Id. Scanning electron micrograph of microconidia produced from cross-
shaped polyphialide. . Ie, f. Microconidia produced from polyphialides on CLA, in situ. Note that
microconidia produced from more than one opening per cell.
g, h. Chlamydospores. I
Figure 4.
.~
J~
- 36-
6. Colony diameters have previously been used as taxonomic criteria forFusarium species at the specific and infra-specific levels (Booth, 1971; Francis& Burgess, 1977;Gerlach & Nirenberg, 1982;Burgess et al., 1993a). They can beextremely useful in distinguishing some populations but it is esserrtial that
I
standard procedures be adopted for the assessment of colony diameters if thiscriterion is to be practically useful. Growth is assessed at 25°C and 3(i)°Cat theFusarium Research Laboratory. The colony diameters quoted in this Manualwere derived form cultures grown on PDA in plates (10 em diam.) for 72 hr inthe dark. The cultures were initiated from germinated single conidia, seededon water agar 18-20 h:f'~,beforeuse and germinated at 25°0 in the dark.Macroconidia from CLA'~'re normally used. The range of diameters Iqu,otedis
5. The ability of a species to form chlamydospores is used as a taxonomiccriterion. It is however, a criterion which must be used with caution becausetheir formation can be quite variable between isolates of the one species andindeed between successive cultures of the one isolate. Thus, while theirpresence is a useful criterion, their absence is not. Chlamydospores formabundantly in the aerial hyphae of some species growing I on CLA(F. compactum, F. equiseti, F. sambucinum) while in others they I are moreobvious in the hyphae or macro conidia on or in the agar (F. solani,F. oxysporum). Typical chlamydospores are illustrated in Fig. 4.
is notched (Fig. 3). The length of the macroconidium is quite variable in somespecies and care must be taken in using length as a taxonomic criterion.
I2. About half the species described in this Manual produce microconidia.The abundance of microconidia may vary significantly between isol~tes withinthe one species. Thus the CLA culture needs to be scanned very carefully fortheir presence. I
3. Microconidia vary considerably in shape and are mostly 1 6r 2 celled(Fig. 3). The microconidia of a few species have a flattened blase as inF. moniliforme, others are papillate as in F. poae or apiculate as in the spindleshaped microconidia of F. subglutinans. Microconidia are formed either in
I
chains or false-heads (Fig. 4). Species which form microconidia in chains alsoI
produce them in false-heads, and the development of microconidial chains isinfluenced by water potential (Fisher et al., 1983). Microconidia alre formedmore abundantly on PDA than CLAby some species such as F. poae. i
4. Microconidia are formed from monophialidic or pol~phialidicconidiogenous cells (Fig. 4). An isolate that· produces polyphialides will alsoproduce monophialides. Monophialides are phialides (conidiogen!ous cells)with one opening while polyphialides have more than one opening per cell.Monophialides and polyphialides are illustrated in Fig. 4.
- 37-
PRACTICAL HINTS ON IDENTIFICATION
1. Grow cultures on CLA and PDA from germinated single coniJia.
2. Incubate cultures-sander the conditions of light and temperatu~e describedearlier. If artificial ligThtis not available grow cultures under diffuse sunlight(never direct sunlight). . Ii.
A significant amount of research on the nature and relationships of theI
teleomorphs of many Fusarium species has been completed in recent years andthe reader is referred to the work of Leslie (1991), Samuels et al.1(1993) andRossman (1983) for further information. . .
Some of the most important species ofFusarium such as F. oxysporum do notproduce a perfect state. Those species which do produce perfect states belong inthe order Hypocreales, subclass Ascomycotina, class Euascomycetes or.Pyrenomycetes. The genera Nectria, Gibberella and Colonectria haveFusarium anamorphs.
TELEOMORPHS
based on a large number of isolates for most species. Note that growth rateschange quite dramatically as Fusarium cultures degenerate. I -
7. Colony morphology provides a useful secondary criterion inl Fusariumtaxonomy. Descriptions of colony morphology are based on slope (slant)cultures grown on PDA for 10-14 days under the standard conditions describedin Chapter 3. These conditions involve temperatures which alt~rnate on aday/night basis (25°C and 20°C) and a photoperiod of 12 hr. Desbriptions ofcolour are based on the 'Methuen Handbook of Colour' by Korrierup and
I
Wanscher (1978). Colony pigmentation is uniform within some species while inI
others it is a highly variable character. While the descriptions in this manualI
.were prepared from cultures on PDA slopes, they also apply to colonies grownon PDA in small Petri plates. I
IDegenerate cultural variants are of two basic types. One is a white mycelialform which produces no conidia, and the other is a 'slimy' slow-growing formcalled the pionnotal type. The latter produces abundant macroconidia andshould not be confused with isolates of F. solani and F. lateritiutrl, wild-typecultures of which usually produce sparse. mycelium and I abundantsporodochia. Wing et al. (in press) made a study of the influence of culturaldegeneration on a number of physiological factors including toxinl productionwhich illustrates the importance of an understanding of the nature and effectsof this phenomenon in research on Fusarium.
I].
- 38-
INote that all of the macroconidia illustrated in this manual were obtained from sporodochiaformed on CLA.
All macroconidia were obtained from sporodochia formed on CLA.Microconidia were desived from colonies on CLA. Both types of conidia weremounted in 0.15% wa£~r agar and photographed through a green filter on an
, '
PHarooRAPIDC PROCEDURES
10. Finally, use the Key for Identification to ascertain the species! Collect and,record all relevant information before you start to use the Key. A master copy ofa checklist of characters is provided on page 40. Photocopies of this may bemade and used for recording.
9. Measure diameters of colonies grown on PDA from germinated singleconidia in the dark for 72 hr using the procedures described earlier.
8. Examine and note the morphology and pigmentation of the, colony grownunder. alternating conditions of light and dark on PDA. Note the pigmentation,if present, in the agar.
7. If chlamydospores are present examine and confirm under x40 objective.A word of caution. All spherical cells are not chlamydospore's. Typicallychlamydospores are characterised by a thick double wall which is oftenverrucose (warty) on its outer surface. The contents are granular and highlyrefringent; rarely are they vacuolate.
5. If sporodochia are present on the CLA make a slide preparation of theI
macroconidi a! in water, and examine under the x40 objective of the compoundmicroscope. Note shape, particularly the first impression. Ignore the few
I
macroconidia that are obviously different from the predominant type.I
6. If microconidia are present make a slide preparation which shouldinclude the hyphae and conidiogenous cells from the area, where themicroconidia are observed. Examine under x40 objective and note 'the shape ofthe microconidia and the nature of the conidiogenous cells.
I4. Examine the culture on CLA under the x10 objective of the compound
I
microscope. Look carefully for microconidia and, if present, note how they areformed (false-heads or chains) and whether there is an indici:ition of thepresence of polyphialides. Then scan the aerial hyphae and the hyphae on andin the agar for the presence of chlamydospores. Avoid thei mistake ofidentifying swollen cells as chlamydospores. Chlamydospores have thick wallswhich may be rough.
3. Examine cultures after they are two weeks old. It may be necessary !oexamine again, one or two weeks later for chlamydospores.
- 39-
Olympus BH-2 microscope using a x40 objective. In situ micrographs ofmicroconidia were photographed with a x20 objective. All micrographs weretaken on Kodak Technical Pan 35 mm film rated at 100 ASA. They weredeveloped in Kodak HC-110 at dilution 'D' for 8 min at 20°C and printed onIlford Multigrade III resin-coated paper.
! .
I I : II I II II
I
I
00
.~t:).,"\f~..
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......
I(.) ,<1.)0..
---,
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-
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I
(:)0... ......
"0 II:::2° u
-
00 0o '0 0.~~00 U~ .J::
1<1.)Q) ......o~~
I -......rJ.lI:::
==
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~u::: ......o lOW
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I
I
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~ cd:.a
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1° cd= 8 ..c:
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-
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~...........c 00:.2.J::~ £ e,~ g
I IQ)..= 1a
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cd I......] """"00
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- 41-
See Guide IISee Guide III
Colony diameters on PDA, after 3 days at 25°C,greater than 2.0 cm.1. Microconidia present and usually
abundant. Borne singly, in false-heads orchains.
2. Microconidia absent or very sparse.
B.
F. decemcellulare
2. Microconidia abundant, formed in chains ..Abundant yellow sporodochia present onCLA and PDA. Droplets of exudatepresent in sporodochia
F. lateritium
11. Macroconidia slender, often longwith a hooked apical cell. Colony onPDA often flat (almost slimy) or withfloccose mycelium.
Guide to Identification IA. Colony diameters on PDA, after 3 days at 25°C,
less than 2.0 cm.1. Microconidia absent
1. Flat, yeast-like or coralloid (almostslimy), cream, yellow or burgundy F. dimerumcolonies on PDA F. merismoides
KEY FOR IDENTIFICATION OF C0Ml\10N SPECIES OF FUSARIUM INAUSTRALASIA
~,• J.
- 43-
If IIlf''iIIn ril!I'I
Shape of microconidia Conidiogenous cells Conspicuous feature II
bearing microconidia ii,ii,
Monophialides Yellow sporodochia with droplets of . I'Oval :IIexudate II
IiGlobose to lemon- Monophialides Urn shaped monophialides; 'IIIshaped microconidia in clusters. il
Lemon-shaped to Monophialides Microconidia in clusters I
"
pyriform IiI
IIOval, pyriform or Polyphialides and Pyriform microconidia on PDA II
Iispindle-sha ped monophialides/'Obovoid Polyphialides and Bushy appearance of short,
monophialides branching polyphialides I>Oval to obovoid Monophialides Microconidia mostly with flattened
base
Oval to obovoid Polyphialides and . Microconidia mostly with flattenedmonophialides base :
Oval, pyriform or .Polyphialides and Globose microconidia !globose monophialides i:
I: iOval Polyphialides and f'monophialides
r.'
Oval to obovoid Monophialides Short chains; some microconidiaI,
with a flattened baser .1
:~'~Ii
Oval to obovoid Polyphialides and Highly proliferating polyphialides :~!monophialides U:
Oval to reniform Monophialides Microconidia on short illU
monophialides il.~Oval to reniform Monophialides Abundant cream or bluish green I·IIsporodochia IOval to cylindrical Polyphialides and Polyphialides short, often cross-(1, 2 or 3-septate) monophialides shaped !
IiOval to spindle- Polyphialides and Polyphialides and spindle-shapedshaped monophialides microconidia ~
Long spindle-shaped Polyphialides and Rabbit's ear appearance of !I~t(4-5 septate) '~~onophialides spindle-shaped conidia I!
,'i~,:·
Globose to napiform Monophialides- Large globose microconidiaOval to fusiform
'""',
F
~
'"Guide to Identification m .:\,; ~J.. ~. ..: ~; 'I" , _(.:..'."., ......,... .~
.~,III 1,Ii' g!l!
Species Page Colony Diameters Pigmentation in PDA -,
onPDA .~
25°C 30°Cic..
I Greyish rose / burgundy1
culmorum 82 5.5 - 6.8 1.5 - 2.5 .~._
I sambucinum 85 2.4 - 3.5 1.1 - 2.1 Greyish rose / burgundy Eti or non-pigmented g,! ' crookwellense 87 5.4 - 6.6 1.5 - 2.5 Greyish rose / burgundy r-, r (~l' -I -t !_l:,1 graminearum 89 3.9 - 6.1 1.0 - 2.5 Greyish rose / burgundyi II:1 '"-u:
heterosporum 100 2.8 - 3.8 0.8 - 2.1 No pigment / pale
I: -orange 'IL!
I -avenaceum ssp. 96 2.8 - 4.0 0.5 - 2.5 Greyish rose / burgundy iiiI -I
I avenaceum ~I a.:.i'avenaceum ssp. aywerte 98 4.0 - 4.5 3.2 - 4.2 Peach / pale orange / red -'6"-
tJ'-'avenaceum ssp. nurragi 99 3.2 - 4.0 0.6 - 2.2 Peach / orange J
t::J
-"acuminatum ssp. 102 2.5 - 3.5 0.5 - 2.8 Greyish rose / burgundy
I:.(-1acuminatum .L!.~
~1
::1acuminatum ssp. 104 4.4 - 5.8 3.7 - 5.4 Apricot / reddish orange
,-1:;1:
~armeniacum 1r-·1:,;
longipes 106 3.7-5.4 4.4 - 6.1 Greyish rose / burgundy JC1'..VG:
compactum 107 4.1 - 5.4 4.2 - 5.8 Greyish rose / brown )'1,t;::
ti.-equiseti 109 3.4 - 4.6 2.8 - 4.4 Brown '1:£::
b~"'~n
"':'fii~:,,_ ~;;
semitectum tas 3.5 - 4.5 1.6 - 3.3 Brown I~I~"Q:;!
I~
'0g..;..
- 44-Jj£fr<;~
- 45-
Spindle-shaped macroconidia onpolyphialides and monophialides
Dark brown flecks in agar on PDA
Dark brown flecks in agar on PDA
Macroconidia ooze from sporodochium into orange 'columella'
Bright apricot / reddish orangesporodochia
Very long macroconidia
Very long macroconidia
Macroconidia needle-like
Bright orange sporodochia withexudate
Mycelium fills tube of PDA slopeculture
Abundant sporodochia on PDA
Papillate apical cell onmacroconidium
Abundant sporodochia on PDA
!.
Conspicuous featureShape of Macro-conidium
- 46-
,_-
iI
I:1,II,II:!.1,I:1
,r :
Comments. Little is known of this species but the fungus has, for example,been frequently isolated from grassland soils in the Alice Springs (arid climate)and Ceduna (temperate climate) regions of Australia using the dilution platetechnique (Sangalang et al., in press).
Key Characters. The colonymorphology of this species is quite distinct andthe macroconidia quite different to all other Fusarium species.
Chlamydospores are present but may be rare.
Microconidia are absent.
Diagnostic Characters on CLA. This species does not form distinctsporodochia and macroconidia are formed from monophialides on branchedconidiophores. The macroconidia are very small, curved, usually 1 to 2 septate,with a hooked apical cell (Fig. 5a). The base of the basal cell is blunt or slightlynotched.
ColonyMorphology on PDA. Fusarium dimerum is slow-growing, forming ayeast-like colony on PDA with little visible mycelium. The colony varies fromwhite to orange in colour and the undersurface is generally the colour of themycelium.
ColonyDiametersonPDA. 0.4 - 1.0 em at 25°00.5 - 1.2 cm at 30°0
Fusarium dimerum Penzig
iiIIIIiII,Ii
IiI,IiI
- 47-
~
•~ "it-'~~f
~:l
3"I31
~
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Comments. Little is known of the ecologyof this species but the fungus has,for example, been isolated from grassland soils in the Alice Springs (aridclimate) and Ceduna (temperate climate) regions of Australia using thedilution plate technique (Sangalang et al., in press) and from plant debris intemperate rainforest forest soils in Victoria, Australia (Summerell et al.,1993b).
.Key Characters. The colonymorphology of this. species is quite distinct anddifferent from most other Fusarium species.
Chlamydospores are formed singly, in pairs or chains .
Microconidia are absent.
Diagnostic Characters on CLA. This species does not form distinctsporodochia and macroconidia are formed from monophialides on branchedconidiophores within the aerial mycelium of the colony. The macroconidia areslightly curved, usually 3 to 4 septate, with a slightly curved apica:l cell (Fig.5b). The base of the basal cell is not foot-shaped or notched.
Colony Morphology on PDA. Fusarium merismoides forms a slow-growing,flat, yeast-like or coralloid (almost slimy) colony on PDA. The colony variesfrom yellow to orange to burgundy in colour and does not produce pigment inthe agar.
ColonyDiameterson PDA. 0.4- 1.0cm at 25°C0.5- 1.2em at 30°C
Fusarium merismoides Corda
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- 48-
Isolates have been received from coffee from Papua New Guinea whichcorrespond to descriptions ofF. stilboides (Booth, 1971) which is, here, includedwithin F. lateritium. Other isolates from coffee do not agree with speciesdescribed in the literature. Some isolates from coffee are homothallic, othersare infertile. Genetic sttttlies by Lawrence et al. (1985a, b) support the use of theone species, F. lateritium, for the various overlapping species populations thathave been described. Further research on this species is needed.
Comments. This species is complex and highly variable and little is knownabout its occurrence in Australia. Isolates of the fungus have been recovered bythe authors from soils in temperate parts of Australia particularly in forestswhere rainfall is abundant (Summerell et al., 1993b; Sangalang et al., inpress). It has also been isolated from mulberry trees affected with branch dieback in the Sydney region. Abundant blue-back perithecia are produced on thediseased mulberry branches in cool wet weather. These correspond toGibberella baccata (Wallr.) SaccoIt has also been shown to cause stem rot ofcelosia in New South Wales (Trimboli, 1972).
Key Characters. Colony morphology, slow growth, and the shape of themacroconidium, particularly the apical cell.
Chlamydospores are present in some isolates but their formation is too variableto be of taxonomic use.
Microconidia are normally absent but are present in some isolates. They areelliptical, oval, spindle- or club-shaped.
Diagnostic Characters on CLA. Fusarium lateritium forms virtually noaerial mycelium on CLA. Macroconidia are formed in pale orangesporodochia. They are long and thin, falcate to almost straight, with parallelwalls, variable septation, and the apical cell has a distinct hook or beak(Fig. 5c). The base of the basal cell is foot-shaped or notched. The macroconidiaare formed from monophialides on branched conidiophores in sporodochia.
ColonyMorphology on PDA. Fusarium lateritium is a slow growing specieswhich exhibits considerable variation. It is accepted here as a complex speciessensu Snyder & Hansen. In culture it forms very sparse white, pale orange orpale pink mycelium. Abundant macroconidia are produced in pale orange,reddish orange or pale pink confluent sporodochia over the entire surface of thecolony. Some isolates produce a reddish orange pigment in the agar whereasothers are non-pigmented.
ColonyDiametersonPDA 0.8 - 2.0 em at 25°C0.5 - 1.5 em at 30°C
Fusarium lateritium Nees
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Comments. This species is easily distinguished by its striking morphologicalfeatures. The macroconidia are larger than those of any of the other commonFusarium species. It is restricted to tropical and sub-tropical areas ofAustralia and other countries, and has been consistently associated withbranch canker and die-back of certain cultivars of lychee in the Cairns area(Pont and Burgess, unpublished data). It causes gall formation on cacao inSouth America .
Key Characters. Colony morphology, very large macroconidia andmicroconidia in chains.
Chlamydospores are absent.
Microconidia are formed abundantly in chains (Fig.5e) and are oval shapedand usually 1 celled (Fig. 5D. They are produced from long monophialideswhich are formed on branched conidiophores (Fig. 5e) or are formed directly onthe hyphae.
Diagnostic Characters on CLA. Macroconidia are formed in abundant lightyellow sporodochia. The macroconidia are very large (long and wide), falcate toslightly curved, thick-walled, usually 5 to 8 septate with a short and usuallyhooked apical cell and a foot-shaped base to the basal cell (Fig. 5d). Themacroconidia are produced from monophialides on branched conidiophores insporodochia and to a minor extent from monophialides formed on the hyphae.
Colony Morphology on PDA. Fusarium decemcellulare forms a distinctiveslow-growing colony on PDA. It produces sparse greyish rose floccosemycelium and abundant light yellow sporodochia in a conspicuous centralspore mass. Droplets of watery exudate form 'on the sporodochia. Thesedroplets may coalesce and flow over the colony. Fusarium decemcellulareproduces a greyish rose to burgundy pigment in the agar.
ColonyDiameters on PDA 1.5 - 2.5 em at 25°C1.1 - 2.2 em at 30°C
Fusarium decemcellulare Brick
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- 49-
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- 50-
(Scale in all photographs = 20 urn)
f. Fusarium decemcellulare: microconidiai ;'i:
e. Fusarium decemcellulare: formation of microconidia on CLA, in situ
d. Fusarium decemcellulare: macroconidia
c. Fusarium lateritium: macroconidia
b. Fusarium merismoides: macroconidia
a. Fusarium dimerum: macroconidia
Figure 5
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Comments. Colonies of F. poae on PDA resemble those ofF. graminearum,There is no detailed information on the distribution of this species in Australia,but isolates have been recovered from soil debris in both tropical and temperateareas.
Key Characters. The abundant globose microconidia formed from urnshaped monophialides in clusters which resemble a 'bunch of grapes'.
Chlamydospores are absent. Note that swollen cells may occur in the hyphae ofold cultures.
Microconidia are abundant, mainly globose, but some tending to lemonshaped, both may have a distinct papilla (Fig. 6b). The microconidia are formedfrom distinctive urn-shaped monophialides (Fig. 7a) on compact branchedconidiophores which appear like a 'bunch of grapes' when examined in situunder the xlO or x20 objective of the compound microscope (Fig. 7a, b). The urn-shaped monophialides have a distinct collarette. . .
Diagnostic Characters on CLA. This species produces few macroconidia.They are relatively short, falcate, usually 3 septate with a short, curved andpointed apical cell (Fig. 6a). The base of the basal cell is foot-shaped or notched.The macroconidia are produced from monophialides on branchedconidiophores in sporodochia or from monophialides formed directly on thehyphae.
Colony Morphology on PDA. Fusarium poae produces dense floccosemycelium initially white but becoming pinkish white to brownish orange withage. Sporodochia are rare. Abundant microconidia are usually produced. Thisspecies usually produces greyish rose to burgundy pigment in the agar.
Fusarium poae (peck)Wollenw.
ColonyDiameterson PDA. 4.2 - 5.4 em at 25°C2.4 - 3.9 em at 30°C
I- 53-·
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There is no detailed information on the occurrence of F. tricinctum in easternAustralia. Fusarium tricinctum has been neotypified (Neish, 1987).
Comments. The formation of the two types ofmicroconidia distinguishes thisspecies from F. poae which forms only globose microconidia. Fusarium poaeproduces microconidia from distinctive urn-shaped phialides. Fusariumtricinctum does not form polyphialides and this distinguishes it fromF. chlamydosporum and F. sporotrichioides both of which form polyphialideson CLA.
r
Key Characters. The shape of the macroconidia and the two types ofmicroconidia.
The microconidia are lemon-shaped to pyriform or allantoid to near spindleshaped, 0 to 1 septate and often have a papilla at the base (Fig. 6d). If an isolateis thought to be F. tricinctum it may be necessary to examine the microconidiafrom PDA slope cultures as well as cultures on CLA to find both types ofmicroconidia. The lemon-shaped microconidia of F. tricinctum are sometimesformed in a cluster which appears like a 'bunch of grapes' and resemblesF. poae in this respect (Fig. Te, d). This feature however, is not as obvious inF. tricinctum as it is in F. poae. Chlamydospores are present but not abundant,and are formed singly or in chains.
Diagnostic Characters on CLA. Macroconidia are formed abundantly inorange sporodochia. They are medium in length, slender, falcate to crescentshaped, usually 3 but may be 4 to 5 septate with a curved, pointed apical celland a foot-shaped or notched base to the basal cell which is narrow and pointed(Fig. 6c). Thus they appear quite pointed at both ends. The macroconidia areproduced from monophialides formed on branched conidiophores in thesporodochia or rarely by monophialides formed directly on hyphae.
Colony Morphology on PDA.. Fusarium tricinctum forms floccosemyceliuminitially white becoming pinkish white to brownish orange. It forms greyishrose to burgundy pigment in the agar .. Orange or burgundy colouredsporodochia may be present.
ColonyDiameters onPDA 2.9- 3.9em at 25°C0.2- 1.5em at 30°C
Fusarium tricinctum (Corda)Sacco
- 54-! .
I
Some isolates from ot'h,ercountries (mainly cold temperate areas) have provedto be highly mycotoxi~enic under certain conditions (Vesonder & Hesseltine,1981).
This species has been isolated from eastern Australia but there is no detailed . i'
information on its distribution or importance.
The oval to pyriform microconidia are produced more abundantly on PDA.Colonies of F. sporotrichioides on PDA resemble colonies of F. graminearumon the same medium.
Comments. The formation of polyphialides distinguishes this species fromF. poae and F. tricinctum. The presence of two types of microconidiadistinguishes it from F. chlamydosporum which does not produce pyriformmicroconidia. It may be necessary to examine both CLA and PDA cultures tofind both types ofmicroconidia.
Key Characters. The shape of the macroconidia, the two types ofmicroconidia and the presence of polyphialides.
Chlamydospores are formed abundantly as cultures age, singly or in chains orclusters and may become pale brown when mature.
Microconidia are produced abundantly from polyphialides. They are oval topyriform or spindle-shaped and 0 to 1 septate often with a papilla at the base(Fig. 6f, g). The spindle-shaped conidia are similar to the spindle-shaped.conidia of F. semitectum. They are commonly formed in pairs from onepolyphialide giving a 'rabbit-ears' appearance when examined in situ (Fig. 7e,f) and in this respect resemble F. semitectum and F. subglutinans on CLA.
Diagnostic Characters on CLA. Macroconidiaare formed abundantly in paleorange sporodochia. They are medium in length, falcate, 3 to 5 septate with acurved, pointed apical cell and a notched or foot-shaped base to the basal cell(Fig. 6e). They are produced from monophialides formed on branchedconidiophores in sporodochia or rarely from monophialides formed directly onhyphae.
1
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Colony Morphology on PDA. Fusarium sporotrichioides produces.abundantfloccose mycelium, initially white but becoming pinkish white to brownishorange with age. Pale orange sporodochia may be found in older cultures. Agreyish rose to burgundy pigment is produced in the agar.
ColonyDiametersonPDA 5.1- 6.1 em at 25°C3.2 - 4.2 em at 30°C
Fusarium sporotrichioides Sherb.
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-55-
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Comments. This species is common in soil throughout the warmer areas ofeastern Australia. It was found to be particularly abundant in semi-arid andarid grassland soils of central Queensland, but infrequently isolated fromsubtropical moist grasslands at the same latitude (Burgess & Summerell,1992). The fungus was isolated frequently from the Darwin (tropical monsoonalclimate) and Alice Springs (arid climate) regions by Sangalang et al. (in press).It has also occasionally been isolated from a range of plant tissues which itpresumably had colonised as a secondary invader.
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Key Characters. Abundant obovoidmicroconidiawith pointed bases producedfrom polyphialides.
Chlamydospores are produced abundantly in older cultures. Thechlamydospores are verrucose and pale brown and are formed either singly ormore usually in chains or clusters.
Microconidia are formed abundantly from polyphialides (Fig. 7g, h). They arepredominantly straight or obovoidwith a pointed base (Fig. 6i). They are mainly1celled but some 1 to 3 septate microconidia are present in most cultures.
Diagnostic Characters on CLA. Macroconidia are formed in orangesporodochia in some isolates but are rare in others and may be obscured by themycelium. The macroconidia are medium in length, relatively wide, usually 3to 5 septate with a short, curved and pointed apical cell and a notched or footshaped base to the basal cell (Fig. 6h). They are produced from monophialidesformed on branched conidiophores in sporodochia or more rarely frommonophialides formed directly on the hyphae.
Colony Morphology on PDA. Fusarium chlamydosporum forms white,floccose mycelium. Sporodochia are rarely formed on PDA. Fusariumchlamydosporum forms a greyish rose to burgundy pigment in the agar inAustralian isolates but a yellow to pale brown pigmentation has been reportedby Nelson et al. (1983). The production of abundant pale brown chlamydosporescan give the mycelium a pale brown appearance in older cultures.
Fusarium chlamydosporum Wollenw.&Reinking
ColonyDiametersonPDA. 3.4- 4.6em at 25°C3.7- 5.5em at 30°C
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, I- 56-
1. Fusarium chlamydosporum: microconidia
h. Fusarium chlamydosporum: macro conidia
g. Fusarium sporotrichioides: pyriform microconidiaf
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f. Fusarium sporotrichioides: spindle-shaped microconidia
e. Fusarium sporotrichiodes: macroconidia
d. Fusarium tricinctum: microconidia
c. Fusarium tricinctum: macroconidia
h. Fusarium poae: microconidia
a. Fusarium poae: macroconidia
Figure 6
(Scale in all photographs = 20 um)
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- 58-
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(Scale in all photographs = 20 um)
g, h. Fusarium chlamydosporum: formation of microconidia on CLA, in situ
e, f. Fusarium sporotrichioides: formation of microconidia on CLA, in situ.Note presence of polyphialides.
c, d. Fusarium tricinctum: formation of microconidia on CLA, in situ
a, b. Fusarium poae: formation of microconidia on CLA, in situ
Figure 7
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A different taxonomic treatment of the section Liseola has been .proposed byNirenberg (1976) and the reader is referred to the monograph by Gerlach and
Comments. Fusarium moniliforme is similar to F. proliferatum but thelatter is distinguished by the formation of chains of microconidia frompolyphialides. Our experience with Australian isolates indicates that thechains formed by F. proliferatum are usually shorter than those ofF. moniliforme. Fusarium moniliforme is also superficially similar toF. nygamai which forms microconidia in short chains or false-heads frommonophialides (Burgess & Trimboli, 1986).Fusarium nygamai however formsabundant macroconi~ia· in sporodochia and chlamydospones in the aerialhyphae in older cultures.
Key Characters. Presence of long chains of microconidia produced frommonophialides. Absence of chlamydospores.
Chlamydospores are absent. Note that swollen cells are sometimes formed byF. moniliforme and can be mistaken for chlamydospores.
Microconidia are formed abundantly in chains from monophialides onbranched conidiophores or from monophialides formed directly on the hyphae(Fig. 9a, b). The microconidia are clavate, usually single celled and have aflattened base (Fig. 8b). Microconidia are also formed, but less commonly, infalse-heads on monophialides.
The macroconidia are produced from monophialides on branchedconidiophores in the sporodochia and rarely from monophialides on hyphae.
Diagnostic Characters on CLA. Macroconidia are produced in pale orangesporodochia which may be obscured by the mycelium and abundant chains ofmicroconidia. Many isolates form very few or no sporodochia. The ability toform sporodochia is usually reduced or lost following repeated sub-culturing.The macroconidia are long, slender, falcate to almost straight, usually 3 to 5septate, and thin walled (Fig. 8a). The apical cell is slightly curved and taperedto a point. The base of the basal cell is foot-shaped or notched.
Colony Morphology on PDA. Fusarium moniliforme forms white floccosemycelium which may become greyish violet or greyish magenta with age.Sporodochia are normally absent but, if present, are pale orange. Darkcoloured sclerotia develop in some isolates. Pigmentation in the agar is quitevariable, ranging from no pigmentation or greyish orange in some isolates toviolet grey, dark violet or dark magenta (almost black) in others.
ColonyDiametersonPDA. 2.9- 3.9em at 25°C3.0- 4.0em at 30°C
Fusarium nwniliforme Sheldon
- 61-
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F. moniliforme produces fumonisin (Gelderblom et al., 1988), which isresponsible for equine leukoencephalomalacia (Kellerman et al., 1990; Nelsonet al., 1993). This problem is also known as mouldy corn disease, or -blindstaggers, of horses.
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While F. moniliforme is widely distributed in eastern Australia it appears thatit is more abundant in the warmer subtropical areas. It causes a wide range ofdiseases. In Australia it has been associated with stalk rot and cob rot of maize,basal stalk rot and root rot of sorghum (Burgess et al., 1986), top rot of sugarcane, foot rot of rice and crown rot of asparagus. It is well adapted to airdispersal and can infest seed of some hosts such as sorghum and maize. Itpersists in host residues on the soil surface or in the soil following mechanicalincorporation and can survive for up to 900 days in cool,dry conditions (Liddell& Burgess, 1985). It is commonin cultivated soils and it has also been isolatedfrom some grassland soils of eastern Australia. Further information is beingsought in this regard.
-];
Nirenberg for detailed information (Gerlach & Nirenberg, 1982). This proposalhas been discussed by Nelson et al. (1983). Fusarium moniliforme as describedabove is equivalent to F. verticillioides (Sacc)Nirenberg (1982). The teleomorphof F. moniliforme is Gibberella fujikuroi (Sawada) Ito and six matingpopulations have been recognised within this species (Leslie, 1991). Differentmating populations correspond to different anamorph species within thesection Liseola. The reader is referred to the work of John Leslie and associatesfor further information.
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Comments. Fusarium proliferatum is similar to F. moniliforme In manyrespects. The formation of polyphialides by F. proliferatum is the primarycriterion for separating the two species. Our experience ,with Australian
',,;<, 'isolates indicates that~;the chains of microconidia formed by F. proliferatum areusually shorter than those of F. moniliforme. Fusarium proliferatum, in somerespects, is also similar to F. nygamai. However, P. nygamai forms
Key Characters. Presence of chains of microconidia produced frompolyphialides. The chains are often formed in pairs in the shape of a 'V'.Chlamydospores are absent.
Chlamydospores are absent. Note that some swollen cells may develop in thehyphae of this species and appear, superficially, like chlamydospores.
The chains of microconidia are usually shorter than those of F~moniliformeand are often formed in pairs from polyphialides giving a characteristic 'V'shape (Fig.. 9d) when seen in situ under the xlO or x20 objective of thecompound microscope.
Microconidia are formed abundantly in chains from polyphialides (Fig. 9c, d)which may proliferate, less often from monophialides. Microconidia also formin false-heads. The microconidia are clavate, usually 1 to 2 celled, and have aflattened base (Fig. 8d). A few pyriform (pear-shaped) microconidia are presentin some isolates.
Diagnostic Characters on CLA. Macroconidia are produced in pale orangesporodochia which may be obscured by the mycelium and chains ofmicroconidia. Many isolates form very few or no sporodochia and the ability toform sporodochia is reduced or lost following repeated subculturing. Themacroconidia are long, slender, falcate to almost straight, usually 3 to 5 septateand thin walled (Fig. 8c). The macroconidia are produced from monophialideson' branched conidiophores in the sporodochia and rarely from monophialideson hyphae.
Colony Morphology on PDA. Fusarium proliferatum is similar in colonymorphology to F., moniliforme. It forms white floccose mycelium which maybecome greyish violet or greyish magenta with age. Sporodochia are normallyabsent but, if present, are pale orange. Dark coloured sclerotia develop in someisolates. Pigmentation in the agar is quite variable, ranging from nopigmentation or greyish orange in some isolates to violet grey, dark violet ordark magenta (almost black) in others.
ColonyDiametersonPDA 2.5 - 3.5 em at 25°C2.5 - 3.2 emat 30°C
Fusarium proliferatum (Matsushima)Nirenberg
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Fusarium proliferatum produces fumoniains, the cause of equrneleukoencephalomalacia(Rosset al., 1990).
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Isolates of F. proliferatum have been obtained from Queensland, New South,Wales and Victoria. Isolates were also recovered from soil associated withLivistona palms in Finke GorgeNational Park in central Australia (Gott et al.,in press), but there is no detailed data on its distribution or economicimportance in Australia. The reader is referred to the monograph by Gerlach, and Nirenberg (1982)for further information.
polyphialides rarely, usually forms very short chains of 2 to 10 microconidia,and forms chlamydospores.
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- 64-
Fusarium anthophilum has not been shown to be pathogenic to plants and hasrarely been isolated in Australasia.
Comments. The globose microconidia are not common in some isolates.These microconidia and the pyriform microconidia are distinctive anddistinguish the species from F. subglutinans.
Key Characters. The globosemicroconidia, presence of polyphialides and theabsence of chlamydospores.
Chlamydospores are absent.
Microconidia are of several types. The most distinctive is a globose spore whichis papillate and normally 1 celled but may be 2 celled (Fig. 8£). In additionsmaller pyriform (pear-shaped), oval or allantoid microconidia are produced,quite abundantly in some isolates. These are usually 1celled. Microconidia areproduced from polyphialides or monophialides (Fig. ge, f),
Diagnostic Characters on CLA. Macroconidia are formed in pale orangesporodochia. They are long, slender, falcate to almost straight, usually 3 to 5septate and thin-walled (Fig. 8e). The apical cell is curved and tapered to apoint. The base of the basal cell is foot-shaped or notched. The macroconidia areproduced from monophialides on branched conidiophores in the sporodochiaand rarely from monophialides formed directly on the hyphae.
Colony Morphology on PDA. Fusarium anthophilum forms abundant whitefloccose mycelium which may become greyish violet or greyish magenta inolder cultures. Sporodochia are rarely formed. Pigmentation in the agar isquite variable ranging from no pigmentation to violet grey, dark violet or darkmagenta (almost black) in others.
ColonyDiameterson PDA 2.5- 4.0em at 25°C2.0- 4.5cm at 30°C
Fusarium anthophilum (A Braun) Wollenw.
I
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3
- 65-
Fusarium subglutinans is common in the cooler areas of the .subtropicalregions of eastern Australia and in the temperate areas. It is associated with
Fusarium subglutinans was raised to species status by Nelson et al. (1983). It isequivalent to F. sacchqri (Butler) W. Gams var. eubglutinane CWollenw.&Reinking). ~;~i
Comments. This species is often mistakenly identified as F. oxysporum.Fusarium oxysporum does not form polyphialides and forms chlamydospores(slowly in some isolates).
Key Characters. Formation of microconidia In false-heads frompolyphialides and absence of chlamydospores.
Chlamydospores are absent. Note that swollen cells occur in the hyphae ofsome isolates.
Microconidia are usually produced in abundance in false-heads mainly frompolyphialides but also from monophialides (Fig. 9g, h). The microconidia areusually oval, elliptical or allantoid and 0 to 1 septate (Fig. 8h). Some isolatesalso produce longer microconidia (transitional macroconidia?) which arespindle-shaped, 2 to 3 septate and which may be apiculate at each end andappear similar to the macroconidia. The spindle-shaped microconidiaresemble th€ spindle-shaped conidia produced by F. semitectum. They are alsocommonly formed in pairs from the one polyphialide giving ar'rabbit-ears'appearance when examined in situ, and in this respect resembleF. semitectum and F. sporotrichioides on CLA.
Diagnostic Characters on CLA. Macroconidia are formed in pale orangesporodochia. They are long, usually slender, falcate to almost straight, usually3 to 5 septate, and thin-walled (Fig. 8g). The apical cell is usually curved andtapered to a point. The base of the basal cell is foot-shaped. The macroconidia of .F. subglutinans range considerably in size (length and width). Themacroconidia are produced from monophialides on branched conidiophores insporodochia and rarely from monophialides formed directly on the hyphae.
Colony Morphology on PDA. Fusarium subglutinans forms white floccosemycelium which may become greyish violet or greyish magenta with age.Sporodochia are normally absent but, if present, are pale orange. Darkcoloured sclerotia develop in some isolates. Pigmentation in the agar is quitevariable ranging from no pigmentation or greyish orange in some isolates toviolet grey, dark violet or dark magenta (almost black) in others.
ColonyDiametersonPDA. 2.3 - 3.7 emat 25°C1.1- 3.4 cmat 30°C
Fusarium subglutinans (Wollenw.&Reinking)Nelson,Toussoun&Marasas
-66-
'I:'
a. Fusarium moniliforme: macroconidia
b. Fusarium moniliforme: microconidia
c. Fusarium proliferatum: macroconidia
d. Fusarium proliferatum: microconidia
e. Fusarium anthophilum: macroconidia
f. Fusarium anthophilum: microconidia
g. Fusarium subgl utinans: macroconidia
h. Fusarium subglutinans: microconidia
(Scale in all photographs = 20 urn)
Figure 8
stalk rot and cob rot of maize and can be seed-borne (Francis & Burgess, 1975;Burgess et al., 1981).Some isolates from eastern Australia are homothallic andproduce perithecia readily in culture within 3 to 4 weeks on PDA in the lightbut not on CLA. The perfect state is Gibberella subglutinans (Edwards) Nelsonet al.
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- 67-
- 68-
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I
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ill:
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- 69-
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...,:l!Ij
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(Scale in all photographs = 20 urn)
g, h. Fusarium subglutinans: formation of microconidia on CLA, in situ. Notepresence of polyphialides.
f. Fusarium anthophilum: formation of globose microconidia on CLA, ittsitu
e. Fusarium anthophilum: formation of oval-shaped microconidia' in falseheads on CLA, in situ
c, d. Fusarium proliferatum: formation of microconidia on CLA, in situ
a, b. Fusarium moniliforme: formation of microconidia on CLA, in situ
Figure 9
-70 -
Comments. In respect to morphological characteristics, F. nygamai isintermediate between F. moniliforme and F. oxysporum. Both F. moniliformeand F. oxysporum include representatives which produce violet pigmentationin colonies on PDA. The macroconidia of F. nygamai could be mistaken forthose of either of<:!t.theother two species but usually are more like the
,""'J\"macroconidia of F. m:bniliforme. Fusarium nygamai however, forms abundantsporodochia on CLA, a characteristic ofF. oxysporum but not ofF. moniliformewhich forms few sporodochia. The microconidia of F. nygamai resemble thoseofF. moniliforme. Fusarium nygamai cannot be included in the 'section Liseola
Key Characters. The short chains of microconidia, chlamydospores and theformation of abundant macroconidia in sporodochia.
Chlamydospores are formed abundantly in some isolates and are rare inothers. They form after 3 to 4 weeks and are usually more common in the aerialhyphae. The chlamydospores are rough walled.
Microconidia are formed abundantly from monophialides formed on branchedconidiophores or from monophialides formed directly on the hyphae, in falseheads or short chains (Fig. 11a, b). Polyphialides are formed occasionally inolder cultures of some isolates. The short chains usually contain 2 to 10microconidia; however, some isolates produce chains with 10 to 20 or moremicroconidia. Many isolates of F. nygamai form very few short chains andthese are detected only after a careful scan of the entire colony, especially theperiphery where they may be more common, under the x10 objective of thecompound microscope. The microconidia are small, usually 1 celled andgenerally oval or elliptical in shape (Fig. lOb). Some of the microconidia have aflattened base.
Diagnostic Characters on CLA. Macroconidia are formed abundantly ingreyish orange sporodochia. They are short to medium in length, slender,falcate to almost straight, usually 3 septate, thin walled with a short taperedapical cell and a notched or foot-shaped base to the basal cell (Fig. lOa). Themacroconidia of F. nygamai are similar in shape to those of F. moniliforme.The macroconidia are produced from monophialides formed on branchedconidiophores in the sporodochia and to a minor extent from monophialidesformed directly on the hyphae.
Colony Morphology on PDA. Fusarium nygamai forms floccosemycelium,initially white becoming dull violet to dark violet in older cultures. A greyishorange or dark violet central spore mass is present in colonies of most isolates.A violet grey to dark violet pigment is produced in the agar.
ColonyDiametersonPDA. 2.5 - 3.5 cmat 25°C3.2 - 4.2 cmat 30°C
,1,.jJ.
I:
ti1;'j'
Fusarium nygamai Burgess&Trimboli
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It is not known if F. nygamai is pathogenic to plants.
The species F. nygamai was first observed by Trimboli (unpublished data)during surveys of Fusarium spp. and other fungi associated with basal stalkrot and root rot of grain sorghum in New South Wales between 1977 and 1978(Trimboli & Burgess, 1985). Representatives were isolated from sorghum rootsand were identified as F. moniliforme because chlamydospores were notobserved. Subsequently in 1980 Burgess and Trimboli (unpublished data) madea systematic study of Fusarium species colonising roots of grain sorghumgrown in field soil in the glasshouse. Isolates of F. moniliforme andF. nygamai were isolated and a few of the latter readily producedchlamydospores, enabling positive differentiation of the population fromF. moniliforme. Since 1980 several hundred isolates of F. nygamai have beenrecovered from Queensland and New South .Wales from grassland andcultivated soils and plant roots. It has, for example, been isolated from theNarromine, Forbes, Narrabri and Walgett areas of New South Wales and theWarwick, Rockhampton, Emerald, Longreach and Ayr areas of Queensland.Survey data indicate that it is more abundant in hot dry areas and it has beenisolated from soils in the Alice Springs region (Sangalang et al., in press).
because it forms chlamydospores. It cannot be included in section Elegansbecause it forms chains of microconidia. The fungus was formally described byBurgess and Trimboli (1986). It has some affinity to species described fromSouth Africa by Marasas et al. (1985) and Marasas et al. (1987). The stability ofthe morphological features ofF. nygamai was studied by Burgess et al. (1989a)who showed that the key characteristics of the fungus are stable both in cultureand following long-term storage. The production of polyphialides and culturepigmentation were variable and were not considered reliable characters foridentification of the species.
-71-
-72 -
Comments. In 1989 the authors initially recovered a number of isolates ofthis population from soil and root debris from sampling sites at Mount Lewis innorth Queensland, Australia (Summerell et al., 1993a). The most distinctivecharacteristic of cultures of these isolates were the production of fusiformmicroconidia from elaborate proliferating polyphialides. The macroconidiaproduced by these isolates were typical ofFusarium species in section Liseola.However the isolates produced chlamydospores, a characteristic whichexcludes this fungus from section Liseola as it is currently defined (Nelson etal., 1983). F. 'babinda' also has some affinity with F. nygamai, F. napiformeand F. dlamini. The morphology of the macroconidia, the formation ofmicroconidia and the production of chlamydospores are common to all of thesespecies. This fungus could most easily be confused with F. subglutinans, but F.'babinda' produces chlamydospores.,~'*
~ ,
Cultures of F. 'babinda' have only been recovered from soils associated with!I
moist forest vegetation in mostly mountainous areas on the east coast o~Australia. This fungus has not been recovered during systematic surveys of
. Igrassland soils from the humid tropics, arid, semi-arid or temperate areas of
I:rIII.'hI! :nil I
ilH I I
III 'I' I,L.l
Key Characters. Proliferating polyphialides with microconidia formed singlyand in false-heads. Production of chlamydospores.
Chlamydospores, hyaline to pale brown, are produced on both CLA and SAafter 4-6 weeks and are formed singly, in pairs, clusters and in chains.
Microconidia are formed on monophialides and polyphialides producedlaterally on aerial hyphae or on branched conidiophores, hyaline, subulate(Fig. llc, d). An cultures that have been examined produce elaborateproliferating polyphialides from which fusiform microconidia are produced(Fig. llc). Fusiform microconidia are also produced from false-heads inmonophialides on CLA. Microconidia are hyaline, 0-1 septate, and are neverformed in chains on KCI or other media (Fig. lad).
Diagnostic Characters on CLAMedium. Sporodochia are pale orange,produced abundantly on carnation leaf-pieces in the CLA. Macroconidiaareproduced from monophialides on the aerial hyphae or branchedconidiophores in sporodochia, hyaline, (3-) 5 septate, falcate with pedicellatebasal cell and slightly curved to very curved apical cell (Fig. lac).
Colony Morphology on PDA. On PDA F. 'babinda' produces abundantfloccosemycelium, initially white becoming pale orange with a violet centre orviolet. The reverse of the colonies is pale orange pink with a violet centre.
ColonyDiametersonPDA 2.5,- 3.7cm at 25°C1.5- 2.2 cm at 300C
Fusarium 'babinda' Summerell,Rugg& Burgess
-73 -i
Australia (Burgess & Summerell, 1992; Sangalang et al., in press; Summerell_et al., 1993a, b). No information is available regarding the pathogenicity ormycotoxicologyofF. 'babinda'.
,',I
fiiii
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-74 -
Comments. Isolates of F. oxysporum can be difficult to distinguish fromF. solani and F. subglutinane, A key feature of F. solani is the formation ofmicroconidia in false-heads on very long monophialides formed on the hyphae.Fusarium subglutinane.is distinguished by the formation of microconidia frompolyphialides and the absence of chlamydospores. Polyphialides are, however,difficult .to find in some isolates ofF. subglutinans. Thus all characters' need tobe assessed carefully when identifying these species. The colony morphology of
Key Characters. The formation of microconidia in false-heads on shortphialides formed on the hyphae, chlamydospores and the shape of themacroconidia and microconidia all help to distinguish F. oxysporum.
Chlamydospores are formed abundantly in most isolates, especiallysaprophytic clones from soil, but may be slow (3 to 6 weeks) to form in someisolates. They are more obvious in hyphae on the surface of the agar of the CLAplate.
Microconidia are usually formed abundantly in false-heads on shortmonophialides on hyphae (Fig. Lle, D. The microconidia are usually nonseptate and are oval, elliptical or reniform (kidney-shaped) (F'ig. 10D. Somecultures do not form abundant microconidia.
The macroconidia are formed from monophialides on branched conidiophoresin sporodochia and to a minor extent from monophialides on hyphae.
Diagnostic Characters on CLA. Macroconidia are formed in pale orange,usually abundant, sporodochia. The macroconidia are short to medium inlength, falcate to almost straight, thin walled and usually 3 septate (Fig. 10e).They tend to be somewhat pointed or tapered at each end. The apical cell isshort and is slightly hooked in some isolates. 'I'he base of the basal cell isnotched or foot-shaped.
Colony Morphology on PDA. Fusarium oxysporum is highly variable inrespect to colonymorphology (Burgess et al., 1989). It produces floccose, sparseor abundant mycelium ranging in colour from white to pale violet. Abundantpale orange or pale violet macroconidia are produced in a central spore massin some isolates. Small pale brown, blue to blue-black or violet sclerotia arepresent, sometimes abundant, in a minority of isolates. Fusarium oxysporumusually produces a pale to dark violet or dark magenta pigment in the agar butsome isolates do not produce any pigment. Some isolates of F. oxysporumdegenerate readily to the pionnotal form or to a flat 'wet' mycelial colony.
ColonyDiameterson PDA 2.5- 4.0em at 25°C2.5- 4.0em at 30°C
Fusarium oxysporum Schlecht.emend.Snyder&Hansen
-75 -
•II!
Saprophytic members of F. oxysporum commonly colonise necrotic roots, assecondary invaders, and are readily isolated, and therefore often mistakenlyassumed to be the primary cause of necrosis. Isolates of F. oxysporum need tobe tested for pathogenicity before conclusions can be made about their role in adisease .
Fusarium oxysporum includes many representatives which are pathogenic toplants causing vascular wilt diseases (Beckman, 1987), damping-off problems(Nelson et al., 1981b), and crown and root rots (Jarvis & Shoemaker, 1978).Isolates which cause wilt diseases are usually host specific and over 100formae speciales and races have been shown to exist. Wilt diseases are a majorproblem in many vegetable and ornamental crops, bananas and palms (Nelsonet al., 1981b; Summerell & Rugg, 1992). Cereals and grasses are apparentlyunaffected by F. oxysporum.
Fusarium oxysporum is common in cultivated soils of temperate and tropicalareas of eastern Australia, and is common in soils from tropical, subtropicaland temperate forests (Summerell et al., 1993a, b). It is also common inimproved-pasture soils of the temperate areas but is not common in grasslandsoils ofwestern Queensland (Burgess & Summerell, 1992) and was not isolatedfrom soil from the Simpson Desert, Australia.
F. solani on PDA is quite characteristic and consistent so that this fungus ismore easily identified than the other two species, given some experience.
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Fusarium solani is a cosmopolitan soilborne fungus which is, common incultivated and grassland soils of eastern Australia. It is more abundant in
Some isolates of F. solani are homothallic and quite fertile and readily formreddish orange or white perithecia in cultures on CLA, and PDA. Theteleomorph is Nectria~aematococca Berk. & Br.
Comments. This species can be confused with F. oxysporum. The longmonophialides bearing microconidia found in F. solani are, however, quitedifferent from the relatively short monophialides bearing microconidia inF. oxysporum.
Key Characters. The shape of the macroconidia, the long phialides bearingmicroconidia and chlamydospores.
Chlamydospores are formed abundantly in most cultures within 2 to 3 weeks.They are usually most obvious in hyphae on the agar surface.
Microconidia are formed abundantly in false-heads on very long monophialides(Fig. 11g, h). They are 1 or 2 celled and are oval, ellipsoidal or, reniform (Fig.10i).
The macroconidia are formed from monophialides on branched conidiophoresin the sporodochia and to a minor extent from monophialides on hyphae.
Diagnostic Characters on CLA. Macroconidia are formed abundantly incream sporodochia. Some variation in macroconidial morphology has beenobserved by the authors in isolates from tropical and temperate regions. Themacroconidia from temperate isolates are relatively wide, slightly curved,usually 3 to 4 septate, thick-walled with a short, blunt (rounded) and sometimeshooked apical cell and a notched base to the basal cell (Fig. lag). 'They are oftenreferred to as 'sausage-shaped' because they are rounded at each end. In someisolates the notched base to the basal cell is barely discernible. The tropicalisolates form macroconidia which tend to be narrower and longer with a moredistinct basal cell than macroconidia formed by temperate isolates (Fig. 10h).
Colony Morphology on PDA. Fusarium solani produces white to cream,usually sparse, floccose mycelium. Abundant macroconidia are produced inconfluent cream or bluish green sporodochia which give the colony a distinctiveappearance. The majority of Australian isolates produce no pigment in theagar, however some isolates develop a pale violet or pale brown pigment. Smallsclerotial bodies are sometimes present.
ColonyDiameterson PDA 2.1- 2.9cmat 25°C2.6- 3.6emat 30°C
Fusarium solani (Mart.) Appel&Wollenw.emend.Snyder&Hansen
i d
-77 -
Some members ofF. solani cause severe crown and root rots of a wide range ofplants such as peas, beans and tomatoes, tuber roots and in the tropics it isoften associated with cankers and die-back problems of trees (Nelson et al.,1981b). It has also been isolated from human eyes and nails (Rebell, 1981).
higher rainfall areas or in irrigated soils. It has been found to be the dominantFusarium species in soil samples collected from forest vegetation in easternAustralia (Summerell et al., 1993a, b).
(Scale in all photographs = 20 urn)
1. Fusarium solani: microconidia
h. Fusarium solani: macroconidia of isolate from tropical region
g. Fusarium solani: macroconidia of isolate from temperate region
Figure 10
a. Fusarium tiygamat: macroconidia
b. Fusarium nygamai: microconidia
c. Fusarium 'babinda': macro conidia
d. Fusarium 'babinda': microconidia
e. Fusarium oxysporum: macroconidia
f. Fusarium oxysporum: microconidia
F..
-78 -
-79 -
If
- 80-
ii.'I::ii
- 81-
(Scale in all photographs = 20 11m) ,
g, h. Fusarium solani: formation of microconidia on CLA, in situ
e, f. Fusarium oxysporum: formation of microconidia on CLA, in situ
c, d. Fusarium 'babinda': formation of microconidia from polyphialides (c)and false-heads (d) on CLA, in situ
a, b. Fusarium nygamai: formation of microconidia on CLA, in situ
Figure 11
[
!L
- 82-
~The macroconidia of so@e isolates of F. culmorum resemble the very shortmacroconidia produced by some isolates ofF. compactum. However cultures ofthese two fungi on PDA are quite different. In addition, the "growth ofF. culmorum is severely inhibited at 30°C compared to 25°C whereas the
Comments. Fusarium culmorum is most likely to be confused withF. sambucinum or F. crookwellense. The rapid growth rate of F. culmorum(5.5-6.8 em at 25°C) distinguishes it from F. sambucinum which grows slowly(2.4-3.5 ern at 25°C). Fusarium culmorum is distinguished fromF. crookwellense by the shape of the macroconidium. The macro conidia ofF. crookuiellenee are longer and have a distinct foot-shaped base to the basalcell and a tapering apical cell (Fig. 12c). The key diagnostic features fordifferentiating F. culmorum from similar species are summarised in Table 4.
Key Characters. The shape of the macroconidium (short and stout), colonymorphology on PDA and fast growth rate.
Chlamydospores develop singly, in chains or clumps. Chlamydosporeformation is variable and is not a reliable taxonomic criterion ..
Microconidia are absent.
Diagnostic Characters on CLA. Macroconidia are formed abundantly onCLA in orange to golden yellow sporodochia. The .macroconidia are short and.stout (Fig. 12a). The apical cell is usually blunt (rounded) but in some isolates itis slightly papillate and thus similar to that ofF. sambucinum. The base of thefoot-cell is usually notched and lacks the distinctive foot-shaped appearance ofmacroconidia formed by F. crookwellense, a similar species in respect to colonymorphology on PDA. The macroconidia are formed from monophialides onbranched conidiophores in sporodochia and to a minor extent frommonophialides on the hyphae.
Colony Morphology on PDA. Fusarium culmorum grows rapidly on PDAforming floccose mycelium which is usually light yellow around the centralspore mass and white at the apex of the slope. Greyish rose to pink myceliumdevelops at the periphery of the colony and is also evident underneath the lightyellow mycelium. Abundant sporodochia form in a large central spore mass(1 to 2 em diam.), initially pale orange becoming violet brown to dark brownwith age. Annular zonations of spore masses are formed by some isolatesunder alternating conditions of light and temperature. This species formsgreyish rose to burgundy pigment in the agar. Cultures which develop olivebrown mycelium and olive brown pigment in the agar are occasionally isolated.
ColonyDiameterson PDA 5.5 - 6.8 em at 25°C1.5 - 2.5 em at 30°C
Fusarium culmorum (W.G.Smith) Sacco
- 83-
Fusarium culmorum is regarded as an important foot and root rot pathogen ofcereals (Cook, 1980;Nelson et al., 1981b)and a root rot pathogen of other plants,such as clovers (Burgess et al., 1973), in coolto cold temperate regions of northAmerica and Europe. It has been isolated, at low frequencies, from wheatcrowns from Queensland (Darling Downs), New South Wales and Victoria(Burgess et al., 1975; Burgess et al., 1981). It has also been isolated fromnecrotic roots of subterranean clover in Victoria but did not prove highlypathogenic in seedling tests (Burgess et al., 1973).Fusarium culrnorurn. is notconsidered to be an important pathogen in eastern Australia.
Fusarium culmorum is most common in the temperate areas of south-easternAustralia, and has been regularly isolated at a low frequency fromsubterranean clover - rye grass pasture soils.
growth ofF. compactum at 30°C is equal to or slightly greater than its growthat 25°C.
"'~
,.!
- 84-
a Colony diameters determined after 72 hr incubation in the dark, the inoculum being a singlegerminated macroconidium.
b F.graminearum Group 2 forms perithecia abundantly on CLA.
culmorum 5.5 - 6.8 +++ Short, stout Notched Blunt orslightlypapillate
sambucinum 2.4 - 3.5 +++ Short, relatively Notched Papillatenarrow
crookwellense 5.4 - 6.6 +++ Medium, Foot-shaped Taperedwidenedat mid-point
graminearum 3.9 - 5.1 + Mediumto long,.. Foot-shaped TaperedGroup 1 relatively
narrow
graminearum 4.7 - 6.1 Mediumto long, Foot-shaped TaperedGroup 2b relatively
narrow
Apical cellBase ofbasal cell
General shape
Colonydiameteron PDAa25°C
Centralspore massonPDAslope
MacroconidiurnSpecies
Table 4 Comparison of key diagnostic features of F. culmorum, F. sambucinum,F. crookwellense and F. graminearum Groups 1 and 2.
.) Ir:,I;
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Isolates of F. sambucinum which lack rose, red or brown pigmentation in themycelium or agar and which form microconidia could be confused with
Comments. Fusarium sambucinum is often confused with F. culmorumbecause their macroconidia show some similarity. However, the macroconidiaof F. culmorum are usually more stout (shorter and wider) than those ofF. sambucinum. In addition, there is a significant difference in their growthrates on PDA (Table 4). Olive brown isolates of F. sambucinum could beconfused with brown isolates ofF. compactum and isolates ofF. equiseti whichproduce short macroc(i)~idia. The distinctive apical cell and growth ratesdifferentiate F. eambucinum from these two species.
Key Characters. The papillate apical cell of the macroconidium, slow growthand colony morphology.
Chlamydospores are formed in chains or clusters in the aerial mycelium ofsome isolates within 2 to 4 weeks. They may become pale brown or goldenyellow at maturity. Note that chlamydospore formation is sparse. in someisolates.
Microconidia, 1 to 2 celled, are present in cultures of some isolates. Theseisolates are probably of the type referred to as F. bactridioides Wollenw. byWollenweber and Reinking (1935) but it is preferable to include them withinF. sambucitiurn: because they are similar in all other characteristics whichhave been examined.
Diagnostic Characters on CLA. This species forms abundant macroconidiain pale orange sporodochia. The macroconidia are relatively short, usually 4 to5 septate with thick walls (Fig. 12b). The papillate tip of the apical cell is adistinctive feature of the macroconidium. This tip has been describedcolloquially as nipple-like or dolphin-nosed, both appropriate terms:' The base ofthe basal cell is usually notched but may have a distinct foot-shape. Themacroconidia are produced from monophialides on branched conidiophores insporodochia and rarely from monophialides formed on hyphae.
Colony Morphology on PDA. Fusarium sambucinum is relatively slowgrowing and extremely variable in respect to pigmentation. It forms white,floccosemycelium which may become greyish rose with age. Abundant orangesporodochia develop in a central spore mass in some isolates, others producedark brown or grey to violet sporodochia. A greyish rose to burgundy pigment isproduced in the agar. Isolates ofF. sambucinum with white mycelium and anolive brown or coffeepigment in the agar have been recovered.
ColonyDiametersonPDA. 2.4 - 3.5 em at 25°C1.1 - 2.1 emat 30°C
Fusarium sambucinum Fuckel
- 86-
Fusarium sambucinum has been isolated from necrotic roots of subterraneanclover in Victoria (Burgess et al., 1973)and has been implicated in storage rot ofpotato tubers in Victoria, Australia (Harrison & Downie, 1960).
Fusarium sambucinum is common in grassland soils of south-easternAustralia. It was the most common species encountered in surveys of thealpine grassland soils of the Mt. Kosciusko area. Isolates of the fungus havebeen recovered from soils from temperate rainforests in Victoria (Summerell,unpublished). Studies in other countries also indicate that it is more commonin cool to cold temperate regions (Burgess, 1981). Sangalang et al. (in press)have shown that F. sambucinum survives only for relatively short periods insoil when exposed to high temperatures. This indicates that the fungus ispoorly adapted to the high temperatures experienced in hot arid regions.Fusarium sambucinum has been regularly isolated from radiata pineseedlings (Pinus radiata Don) affected by damping-off, and soil from pinenurseries at Bombala and Orange, New South Wales, Australia (Keirle& Forbes, personal communication). However, it was not isolated from slashpine (P. elliottii Engelm.) or loblolly pine (P. taeda L.) seedlings or soil from apine nursery at Coffs Harbour, New South Wales. Coffs Harbour has asubtropical climate whereas Orange and Bombala are characterised by warmto hot dry summers and coldwet winters.
F. oxysporum. However the latter usually forms more abundant microconidiain false-heads on CLA and these are reniform (kidney shaped) or oval shapedwhereas those ofF. sambucinum are oval shaped.
- 87-
Fusarium crookwellense is mostly confused with F. culmorum, as cultures onPDA slopes are similar and growth rates on PDA overlap. However, themacroconidia ofF. crookwellense are longer and not as wide or stout as those ofF. culmorum. In contrast to the conspicuous foot-shape of the basal cell ofF. crookwellense, that of F. culmorum is less obvious. The apical cell ofF. crookwellense is distinctly curved and tapers gradually to a narrow apexwhereas that of F. culmorum is usually blunt (rounded) but can be slightlypapillate. The original cultures of F. crookwellense were isolated from dimplelike lesions on potato tubers from Crookwell, New South Wales, Australia in1971 (Burgess et al., 1982). Since then representative isolates have beenrecovered from a wide rapge of plant species (mainly crown or root tissues) andfrom debris from grassla;1:Mand cultivated soils. Studies in Australia indicatethat the fungus is generally more abundant in temperate areas (Table 2).Isolates of F. crookwellense have also been found in the northern areas of the·U.S.A., South Africa, France, Columbia and China.
The key features which differentiate F. crookwellense from similar species(F. sambucinum, F. culmorum and F. graminearum) are summarised inTable 4.
Comments. This specieswas described by Burgess et al. (1982)and the readeris referred to this paper for detailed information on its taxonomic affinities anddistribution.
Key Characters. Shape of the macroconidia, colony morphology and theabsence of microconidia.
Diagnostic Characters on CLA. Abundant macroconidia are formed in paleorange to dark brown sporodochia. They are intermediate in length, thickwalled, falcate with the dorsal side slightly more curved than the ventral,usually 5 septate and are normally widest at the mid-point (Fig. 12c).
Colony Morphology on PDA. Fusarium crookwellense grows rapidly anddevelops abundant sporodochia in a conspicuous central spore mass,· initiallypale orange becoming reddish brown to dark brown with age. Annularzonations of spore masses occur under alternating conditions of light andtemperature. Mycelium formation is sparse in isolates with abundantsporodochia. White floccosemycelium develops toward the apex of the slope. Incontrast, the mycelium is pale yellow below the spore masses and greyish roseat the periphery. Plectenchymatous bodies (0.5- 1mm in diam.) develop in thebasal half of the colonyof some isolates. A greyish rose to burgundy pigment isformed in the agar.
ColonyDiameters onPDA. 5.4- 6.6em at 25°C1.5- 2.5cm at 30°C
Fusarium crookwellense Burgess,Nelson&Toussoun
- 88-
F. crookwellense has been shown to cause mild root rot and foot rot of wheat inglasshouse trials using pasteurised field soil (Liddell, 1985b) and to cause someroot and crown disease in durum wheat cultivars (Balmas et al., in press).
- 89-
Group 2. The shape of the macroconidium, the absence of microconidia,colonymorphology on PDA and the formation of perithecia on CLA.
Group 1. The shape of the macroconidia, the absence of microconidia and"'~~
colony morphology on P~A.
Key Characters.
. Chlamydospore formation is variable and is not a useful taxonomic criterion.
Microconidia are absent .
Diagnostic Characters on CLA. The two populations (Groups 1 and 2) cannotbe differentiated on the basis of the morphology of their macroconidia orconidiophores. Isolates of both populations produce macroconidia in paleorange sporodochia, however isolates of Group 2 produce Iess abundantsporodochia. The macroconidia of both populations are relatively slender,falcate to almost straight, usually 5 to 6 septate, with a tapered apical cell and adistinctly foot-shaped base to the basal cell (Fig. 12d, e). Some isolates mayproduce macroconidia up to twice the length of those illustrated however this isnot common. The macroconidia are produced from monophialides on branchedconidiophores in sporodochia and rarely from monophialides formed directlyon the hyphae.
Colony Morphology on PDA. In PDAslope cultures F. graminearum Group 1forms uniform dense mycelium which usually fills the tube. The mycelium ispredominantly light yellow, greyish rose at the periphery and white at the apexof the colony. Sporodochia may form in a small central spore mass but arenormally overgrown by mycelium. Fusarium graminearum Group 2 developsuneven floccose mycelium which does not fill the tube. The colour of themycelium varies from white to pale orange to apricot with greyish rosemycelium at the periphery. Cultures of both groups form greyish rose toburgundy pigment in the agar.
30°C1.0 - 2.5 em0.5 - 2.0 em
Colony Diameters on PDA. 25°CGroup 1 3.9 - 5.1 emGroup 2 4.7 - 6.1 cm
Two populations, designated Groups 1 and 2, have been distinguished withinF. graminearum (Francis & Burgess, 1977). Members of Group 1 rarely formperithecia in nature and do not form them in culture on CLA from a singlemacroconidium. They are apparently heterothallic. In contrast, members ofGroup 2 form abundant perithecia in nature and on CLA and are homothallic.The perithecia formed by each group are similar, and the teleomorph isGibberella zeae (Schw.) Petch.
Fusarium graminearum Schwabe
- 90-
Members of Group 2 cause head blight ofwheat and stalk rot of maize in humidand high rainfall areas. They are common in coastal N.S.W., and the Kingaroyand Atherton Tableland areas of Queensland. Head blight of wheat is notnormally a problem in the main Australian wheat areas presumably becausemaize is not grown in r~,tation with wheat and dry conditions usually prevailbetween anthesis and ha'¥Vest.Head blight and stalk rot of maize cause; seriouslosses in the wetter areas of Europe and North America, par ticularly whenwheat is grown in rotation with maize. Fusarium graminearum Group 2 can
In Australia, crown rot of wheat, caused by members of Group 1, is a seriousproblem in the central and northern wheat areas of New South Wales and thewestern wheat areas of southern Queensland. Crown rot is favoured byfarming practices, such as conservation tillage, in which infested residues areretained on the soil surface (Summerell et al., 1989, Burgess et al., 1993b). Anoutbreak of head blight of wheat was caused by members of the Group 1population in north-west New South Wales in 1983, an exceptionally wet season(Burgess et al., 1987a).
Members of Group 2 form perithecia abundantly in nature and are essentiallyairborne pathogens which cause diseases of aerial plant parts such as headblight (head scab) ofwheat, barley and oats, stalk and cob rot of maize and stubdieback of carnations (Francis & Burgess, 1977; Burgess et al., 1981). They arethe fungi traditionally regarded as members of Gibberella zeae (Schw.) Petch.and produce two important mycotoxins, zearalenone and vomitoxin, in infestedgrain under certain environmental conditions (Vesonder & Hesseltine, 1981;Marasas & Nelson, 1987).
Members of Group 1 are essentially soilborne pathogens which cause crown rotof a wide range of temperate cereals including wheat, barley, rye and triticaleand grasses such as Hordeum leporinum. Lind and Phalaris paradoxa L.(Francis & Burgess, 1977; Burgess et al., 1981; Nelson & Burgess, in press).They have been isolated from grassland soils prior to cultivation in easternAustralia. Members of Group 1 have also been isolated from temperate cerealsin California and Washington State, U.S.A. (Cook, 1980; Nelson et al., 1981b)and in Italy (Balmas, 1994).
The influence of water potential and temperature on the growth andreproduction of F. graminearum Groups 1 and 2 has been studied in detail by ,.gWearing and Burgess (1979) and Sung and Cook (1981).
Comments. Fusarium graminearum can easily be confused withF. crookwellense and F. culmorum. Key characters which help differentiatethe above species and F. sambucinum are summarised in Table 4. The absence.of microconidia distinguishes isolates of F. graminearum from isolates ofF. poae and F. sporotrichioides which form colonies on PDA similar to those ofF. graminearum.
- 91-
Itmust not be assumed that tolerance to crown rot is indicative of tolerance tohead blight. They are two entirely different disease syndromes! The control ofhead blight of wheat and stalk rot of maize is difficult. These crops should notbe grown in rotation in humid areas. Soybeans are a useful crop in rotationwith wheat or maize in humid areas to alleviate problems caused byF. graminearum. Group 2. This fungus is airborne and the occurrence of headblight in wheat crops where the primary inoculum (ascospores) originatedfrom perithecia on old maize stubble in an adjacent field has been observed.
While F. graminearum Group 1 occasionally causes some seedling death inwheat and barley in very dry seasons, the inoculum is soilborne arid stubbleborne rather than seedborne and the fungus is rarely isolated from seed.Members of Group 1 persist mainly as hyphae in crop residues in or on soil andcan be readily isolated from debris found in soil (Burgess et al., 1981; Wearing& Burgess, 1977; Summerell & Burgess, 1988). Chlamydospore formation hasbeen demonstrated but does not appear to be an important mode of survival(Sitton & Cook, 1981).Members of Group 2 persist as hyphae in residues and asperithecia on residues on the soil surface (Wearing & Burgess, 1978). They canbe isolated from debris from cultivated soil where the inoculum has beenmechanically incorporated. Crown rot of wheat is controlled by the use oftolerant cultivars, rotation to resistant crops such as grain legumes orsorghum, long fallow (no crops or weeds for 18 months), stubble burning afterharvest or the use of fire-harrows in autumn, prior to planting '(Summerellet al., 1989; Burgess et al., 1993b). The tolerant cultivars are susceptible toinfection but yield well by comparison with intolerant cultivars. For example,in a recent field trial the proportion of white-heads in the tolerant cultivar'Cook' was 15% whereas it was 60% in the durum wheat 'Durati', a verysusceptible cultivar.
it
be seed-borne in humid areas where blight and cob rot occur. Members ofGroup 2 may cause seedling blight in temperate cereals or maize under certainconditions, the inoculum being seed-borne or soilborne.
a. Fusarium culmorum: macro conidia
b. Fusarium sambucinum: macroconidia
c. Fusarium crookwellense: macroconidia
d. Fusarium graminearum Group 1: macroconidia
e. Fusarium graminearum Group 2: macro conidia
(Scale in all photographs = 20 urn)
Figure 12
- 92-
- 93-
~iI;iU"~
~,-q,,_,.u:,lBW.:.~--
~--.~C:_"t
- 94-
-~
(Scale in all photographs = 20 urn)
c. Fusarium avenaceum ssp. nurragi: macroconidia
d. Fusarium heterosporum: macroconidia
e. Fusarium acuminatum ssp. acuminatum: macroconidia
f. Fusarium acuminatum ssp. armeniacum: macro conidia
h. Fusarium avenaceum ssp. aywerte: macroconidia
a. Fusarium avenaceum ssp. avenaceum: macroconidia
Figure 13IlIII
Ii~IIli
- 95-
~~~-i
~.,iI-'
.,~-ig
Ii
-II '1lt-' 'i;~I.--I.-~
~
,Ii,..
- 96-
F. avenaceum ssp. avenaceum is commonly confused with F. acuminatumssp. acuminatum. Although the latter produces chlamydospores, theirformation is slow and they may be difficult to detect. Colony types and growthrates overlap and do not help distinguish the two species. Thus one is left withthe shape of the macroconidia as the only reliable distinguishing feature. Theparallel sides, t'$in walls and the notched basal cell distinguish themacroconidia of F."avenaceum ssp. avenaceum from those ofF. acuminatumssp. acuminatum which have a degree of dorsi-ventral curvature, thick wallsand usually a distinctly foot-shaped base to the basal cell.
Comments. Fusarium avenaceum has recently been split into threesubspecies following the discovery of two subspecies in unique environments inAustralia (Sangal ang et al., in press). F. avenaceum ssp. avenaceum isdifferentiated from the other two subspecies by shorter macroconidia and has amore widespread distribution. Past references to F. avenaceum in theliterature probably refer to this subspecies.
Key Characters. The needle-like appearance of the thin walled macroconidia,the absence of chlamydospores and colonymorphology.
Chlamydospores are absent.
Microconidia are produced sparsely In some isolates. They are variable In
shape and septation.
, \
I' II;
it
\! i
': .
Diagnostic Characters on CLA. Macroconidia are formed in pale orangesporodochia. They are long, slender, falcate to almost straight, thin-walled andusually 5 septate (Fig. 13a). The apical cell tapers gradually to a pointed endand this feature together with parallel sides and a slender form give themacroconidia a needle-like appearance. The base of the basal cell is usuallynotched but in some macroconidia it does have a distinct foot-shape. Themacroconidia are formed from monophialides on branched conidiophores inthe sporodochia.
i1_
Colony Morphology on PDA. Fusarium avenaceum ssp. avenaceum formsabundant floccosemycelium which varies from white to light yellow to greyishrose. Abundant pale orange to brown sporodochia form in a central sporemass. The pigment formed in the agar is greyish rose to burgundy but mayappear brownish because of the light reflected from the central spore mass. Itshould be noted that this species is highly variable in respect to colonymorphology. It also degenerates readily to the pionnotal and less often to thewhite mycelial form.
I'!I,I
ColonyDiameterson PDA 2.8- 4.0em at 25°C0.5- 2.5em at 30°C
Fusarium avenaceum (Fr.) Saccosubspeciesavenaceum Sangalang et al,
- 97-
This fungus survives in soil in debris. Thus it can be isolated more readily byplating the debris as outlined in Chapter 5.
Fusarium avenaceum ssp. avenaceum is a recognised root rot pathogen ofpasture legumes (Burgess et al., 1973) and a recognised cause of crown androot rot of carnations (Nelson et al., 1981a).Although it has been isolated fromwinter cereals in south-eastern Australia (Burgess et al., 1975), it is notregarded as an important pathogen of these crops (Burgess et al., 1981).
Fusarium avenaceum ssp. avenaceum is a soilborne species which is commonin temperate grassland soils of south-eastern Australia. It is particularlycommon in subterranean clover-dominant pasture soils. It has been recoveredfrom soils from temperate forests (Summerell et al., 1993b). It has not beenisolated from soils or plants from tropical or semi-arid areas of Australia.
•• "1,'
-98-
The shape of the macroconidia in these isolates is similar to the macroconidiaofF. avenaceum sensu lato but they are wider and much longer. Furthermorethe new population resembles F. aoenaceum. in that microconidia andchlamydospores are not produced in culture. However the colony appearanceand colony growth rates ofF. avenaceum ssp. aywerte on PDA are consistentlydissimilar to F. ave,!Jaceum sensu lato. Thus F. avenaceum ssp. aywerte isdesignated as a subspecies ofF. avenaceum rather than a new species becauseof the similarity of the shape of the macroconidia.
Comments. Cultures of this subspecies were recovered initially from sitesdominated by spinifex (Triodia basedowii E. Pritz.) in the Deep Well area southof Alice Springs and in the McGraths Creek area north of Alice Springs in theNorthern Territory, Australia (Sangalang et al., in press). Additional cultureshave been isolated subsequently from Finke Gorge National Park south west ofAlice Springs, and from Kununurra, Western Australia. Recovery of thefungus is always associated with the presence of Triodia spp.
Key Characters. The very long, thin-walled macroconidia, the absence ofchlamydospores and colony morphology.
Microconidia and chlamydospores are absent.
Diagnostic Characters on CLA. Macroconidia are formed In orangesporodochia on the leaf pieces and on the agar and are produced frommonophialides on branched conidiophores in the sporodochia. Most isolatesform droplets of watery exudate on the sporodochia which give the sporodochiaa gelatinous appearance. In some cultures the sporodochia develop in annularzonations in response to diurnal lighting. Macroconidia are relatively narrowwith thin walls, long to very long and slightly curved to very curved. The apicalcell is tapered and slightly hooked. Macroconidia are predominantly 5-7 septatewith dimensions of 3.8-5.1 urn x 76.8-116.5 urn (Fig. 13b). Some macroconidiaare longer and 8-9 septate (107.5-125.4 um in length). A few macroconidia are60.2-79.4urn long and are 3-4 septate.
Colony Morphology on PDA. On PDA F. avenaceum ssp. ayuierte formsdense aerial mycelium pale orange in colour becoming pinkish white to whiteas the culture ages. The reverse is peach or pale orange to orange white incolour. Cultures recovered from the Finke Gorge National Park, Australiaproduce a darker red mycelium and a red pigment in the agar. Orangesporodochial masses are produced in the majority of isolates.
ColonyDiametersonPDA. 4.0 - 4.5 emat 25°C3.2 - 4.2 cmat 30°C
Fusarium avenaceum (Fr.) Saccosubspeciesaywerte Sangalang&Burgess_
1,1'f
- 99-
F. avenaceum ssp. avenaceum has been recovered from regions in Victoriaclose to the sites from which Fusarium avenaceum ssp. nurragi was recoveredbut has not been recovered from heathland soils. The differences between thethree subspecies are not"~consideredof sufficient significance to differentiatethem at species level particularly given the similarity of the shape i of themacroconidia.
The shape of the macroconidia in these isolates is similar to the macroconidiaofF. avenaceum ssp. avenaceum and F. avenaceum ssp. aywerte but they aresignificantly longer than F. avenaceum ssp. avenaceum. Other similarities tothese subspecies include the absence of microconidia and chlamydospores inculture. The growth rate of this subspecies is similar to that of F. avenaceumssp. avenaceum but lower than the growth rate ofF. avenaceum ssp. aywerte.The colonymorphology most closely resembles F. avenaceum ssp. aywerte.
Comments. Cultures of this subspecies were recovered from heathland sitesdominated by' Kunzea ambigua (Sm.) Druce, Banksia serrata L. f. andAllocasuarina paradoxa (Macklin) L. Johnson in Wilsons PromontoryNational Park, Victoria, Australia (Sangalang et al., in press). '.Chisfungushas not been recovered from other heathland soils sampled in easternAustralia including samples from Port Campbell National Park, Victoriawhich is similar in latitude and climate.
Key Characters. The very long, thin-walled macroconidia, the absence ofchlamydospores and colony morphology.
Microconidia and chlamydospores are absent.
Diagnostic Characters on CLA. Macroconidia are formed in orangesporodochia on the leaf pieces and on the agar and are produced frommonophialides on branched conidiophores in the sporodochia. The sporodochiaare normally formed in annular zonations in response to diurnal lighting.Macroconidia are narrow with thin walls and can vary in length from 34 urn to118 urn, most are in the range 68 to 118 urn (Fig. 13c). The width varies from3.8-5.0 um, Macroconidia are almost straight to very curved. The apical cell istapered and slightly curved and the base of the basal cell IS notched.Macroconidia are 4 septate, occasionally 5 septate, never more.
Colony Morphology on PDA. On PDA F. avenaceum ssp. nurragi formsdense aerial mycelium which is pale orange to white in colour. The reverse ispeach to orange in colour.
ColonyDiameters onPDA 3.2 - 4.0 emat 25°C0.6 - 2.2 emat 30°C
Fusarium avenaceum (Fr.) Saccosubspecies nurragi Summerell & Burgess
-100 -
I
I'I:1
:\
Comments. We have elected to use the name F. heterosporum for this fungusrather than F. graminum Corda, which was used in previous editions.According to Wollenweber & Reinking (1935) and Gerlach & Nirenberg (1982)the species differ in that F. graminum has slightly longer macroconidia, andF. heterosporum produces chlamydospores. In all other respects they aresimilar. Isolates from Australia usually have long macroconidia and do notproduce chlamydospores. However Ali (1993) showed that these species cannotbe distinguished by conidial morphology, colony morphology, growth rate onPDA, or response to t,.ymperature or osmotic potential and that cultures ofF. heterosporum in tIle sense of Wollenweber & Reinking (1935) did notconsistently produce true chlamydospores. As a result of the considerablevariations in the length of macroconidia within isolates of these species,differentiation is not warranted and the existence of two distinct species is
Key Characters. Abundant orange sporodochia on PDA and CLA. Shape of.the macroconidia.
Chlamydospore production is variable.
Microconidia are absent.
The macroconidia are formed from monophialides on branched conidiophoresin the sporodochia and to a minor extent from monophialides on hyphae.
Diagnostic Characters on CLA. Macroconidia are formed abundantly inorange sporodochia. They are medium in length, slender, falcate to almoststraight, thin-walled and predominantly 3 septate (Fig. 13d). The base of thebasal cell of some macroconidia is distinctly foot-shaped; in others it is notched.The macroconidia are similar to, but not as long as, macroconidia ofF. avenaceum.
'ii
I I.,! I
ColonyMorphology on PDA. The presence ofabundant orange sporodochiainthe centre of the colony is a conspicuous feature of this species. Thesporodochia usually develop in annular zonations in newly isolated culturesgrown under alternating conditions of light and dark, and temperature.Droplets of watery exudate form on the sporodochia of most isolates. Themycelium is dense and white to pinkish-white in colour. Small tufts (mounds)of white to light yellow mycelium form in some cultures. The mycelium nearthe base of the slope may become light yellow to yellow with age. The fungus. does not produce obvious pigment in the agar but the under surface can appearbrownish orange to caramel in colour because of the reflected light from thesporodochia. The fungus degenerates readily to a white mycelial form.
I• I
ColonyDiameters on PDA. 2.8 - 4.1 emat 25°C0.8 - 3.0 emat 30°C
Fusarium heterosporum Nees ex Fr.
l'Ir!Ilf"f '~
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,\
1-~,t.J-I'
-101-
There have been no reports of F. heterosporum causing plant disease inAustralia. It is however a regular contaminant of seed ofP. dilatatum and mayreduce its viability.
F. heterosporum is found in association with Claviceps paspali Steven & Hallon inflorescences of Paspalum dilatatum Poir and other Paspalum spp. in thecoastal and tableland areas of eastern Australia. It is more common in wetsummers. It has also occasionally been isolated from the inflorescences ofLolium spp. infected with Claviceps spp. from the southern and centraltablelands of New South Wales, Australia. The orange sporodochia ofF. heterosporum formed on florets or spikelets infected with Clcuiiceps sp. areconspicuous to the naked eye. Isolation records indicate that it has rarely beenrecovered from grassland, wheat or maize soils, wheat stems, corn stalks orroots of subterranean clover (Burgess et al., 1973;Burgess et al., 1975; Francis& Burgess, 1975).
Fusarium heterosporum has nomenclatural priority over F. graminum. It isalso the name most widely used for cultures of this type isolated from ergots inNorth America and Europe. We have therefore concluded that F. heterosporumis the most appropriate name for this fungus.
regarded as doubtful. Wollenweber and Reinking (1935) reported that bothF. graminum and F. heterosporum were commonly associated with grassinflorescences infected by Claviceps spp. (pages 53 and 73 in 'Die Fusarien'(Wollenweber& Reinking, 1935)but failed to include F. graminum in the list ofFusaria found occurring on Claviceps spp. (page 303).
-102 -
Comments. Fusarium acuminatum has recently been split into twosubspecies, F. acuminatum ssp. acuminatum and F. acuminatum ssp.armeniacum (Burgess et al., 1993a). This differentiation was made on the basisthat the differences in the shape of the macroconidia were not sufficient towarrant description of F. acuminatum ssp. armeniacum as a species. Theshape of the macroconidia is considered to be the primary criterion indifferentiating Fusarium species. However, the macroconidia ofF. acuminatum ssp. armeniacum are usually longer and wider. and have amore distinct foot-shaped base to the basal cell. Chlamydospores normally formabundantly within two weeks in cultures of F. acuminatum ssp. armeniacumon CLA. In addition the growth rates of the two species are significantlydifferent (Table 5) and the cultures ofF. acuminatum ssp. armeniacum, withabundant bright apricot tm",reddishorange sporodochia, are quite 'distinctive onboth PDA and CLA. Fusd:lium acuminatum ssp. acuminatum is most likely tobe confused with isolates of F. auenaceum ssp. auenaceum or F. acumiiiatumssp. armeniacum. Fusarium acuminatum ssp. acuminatum can only bedifferentiated from F. auenaceum ssp. auenaceum by the shape of the
Key Characters. The shape of the macroconidia, pigmentation in the agar,and growth rate.
Chlamydospore formation is very slow in most isolates from eastern Australia.They are formed in chains or clusters. Their presence is not a reliabletaxonomic criterion.
Microconidia are usually absent but are formed sparsely by some isolates andare 1 to 2 celled.
Diagnostic Characters on CLA. Macroconidia are formed in pale orangesporodochia. They are relatively slender, thick-walled, usually 5 septate with along tapering apical cell and a foot-shaped base to the basal cell (Fig. 13e). Themacroconidia have a distinct dorsi-ventral curvature but it is not as obvious asin macroconidia of F. equiseti. They are formed from monophialides onbranched conidiophores in sporodochia and rarely from monophialides onhyphae.
Colony Morphology on PDA. Fusarium acuminatum ssp. acuminatum, a'relatively slow growing species, forms white floccose mycelium which isabundant in some isolates. The mycelium can be greyish rose at the periphery.Sporodochia form in the centre of the colonyin a small central spore mass andare pale orange to dark brown. A greyish rose to burgundy pigment is formedin the agar.
ColonyDiameterson PDA. 2.5- 3.5emat 25°C0.5 - 2.8 em at 30°C
Fusarium acuminatum Ell.&Ev.subspeciesacuminatum Burgess et ale
:1 I
t -I _
-103 -
Fusarium acuminatum ssp. acuminatum is more common in the temperateareas of south-eastern Australia where it is found in grassland and cultivatedsoils. It is less common in the tropical areas and has not been isolated fromgrassland soils of western Queensland or soil samples from the central aridwoodlands and the Simpson Desert, Australia. Sangalang et al. (in press) haveshown in survival studies that F. acuminatum ssp. acuminatum has a lowersurvival rate at high temperature than fungi such as F. equiseti andF. compactum which are common in soils from hot, dry regions. Rose orburgundy pigmented isolates of F. compactum have possibly been mistakenlyidentified as F. acuminatum ssp. acuminatum by some workers. AlthoughF. acuminatum ssp. acuminatum is generally regarded as a saprophyte and asecondary colonist of necrotic or senescent root and crown tissues, someisolates can cause severe root rot in some clover species.
The growth rates of the two species on PDA are similar. Fusarium avenaceumssp. avenaceum does not form chlamydospores but because they form slowly incultures of F. acuminatum ssp. acuminatum this difference is not alwaysuseful in identification.
macroconidia. Macroconidia of F. acuminatum ssp. acuminatum have some.dorsiventral curvature, thick walls and a distinct foot-shape to the base of thebasal cell. Macroconidia of F. avenaceum ssp. avenaceum have parallel sides,thin walls and a notched base to the basal cell. Unfortunately there are someisolates which are very difficult to identify because the macroconidia areintermediate in shape and are not typical of either species.
.,_,.,.
-104 -iI',I',
I:Ii!!l
,i,'II,I';I'iIi ['ii,,[
The morphology and growth rates of colonies of F. acuminatum ssp.armeniacum on PDA are quite different from those regarded as typical ofF. acuminatum ssp. acuminatum and the macroconidia of F. acuminatumssp. armeniacum are generally larger than those of F. acuminatum ssp.acuminatum. Chlamydospores are usually formed within two weeks incultures of F. acurn.ituitu m ssp. armeniacum on CLA whereas they formslowly or not at all in c~itures ofF. acuminatum ssp. acuminatum. The longerand more slender macroconidia of F. acuminatum ssp. armeniacum might bemistaken for shorter macro conidia of F. longipes. Colony diameters ofF. acuminatum, F. acuminatum ssp. armeniacum and F. longipes on PDA at
Comments. Representatives of this population were first isolated inAustralia in 1973 and identified as F. acuminatum, principally because themacroconidia were similar.
Key Characters. The distinctive bright apricot to reddish orange sporodochiaon CLAand PDA, growth rate, and the shape of the macroconidium.
Chlamydospores are usually formed singly or in clusters within 10 days.
Microconidia are absent.
Diagnostic Characters on CLA. Macroconidia are formed abundantly insporodochia, the bright apricot to reddish orange colour of which is quitedistinctive. The macroconidia are long, thick walled, usually 5 septate with along tapering apical cell and a distinct foot-shaped base to the basal cell (Fig.13£).The macroconidia have a distinct dorsi-ventral curvature. In a suspensionof macroconidia it is usual to see a few very long macroconidia which are oftenstrongly falcate (Fig. 13£)with a distorted base to the basal cell. It is also normalto observe some macroconidia with a short 'side branch' (germ tube?) from onecell. The macroconidia are produced from monophialides from branchedconidiophores in sporodochia and to a minor extent from monophialidesformed directly on the hyphae.
Colony Morphology on PDA. This species is relatively fast-growing andproduces white to salmon floccose mycelium. Abundant apricot to reddishorange sporodochia develop in a, conspicuous central spore mass. Thesesporodochia may become brown with age. Fusarium acuminatum ssp.armeniacum produces a greyish rose to reddish brown pigment in the agar.This species degenerates readily to the pionnotal form or a slow-growingmycelial form. ,"
f,I
ColonyDiameterson PDA. 4.4- 5.8ern at 25°C3.7- 5.4em at 30°C
Fusarium acuminatum Ell.& Ev. subspecies armeniacum Forbes, Windels&Burgess
,it
-105 -
Fusarium acuminatum ssp. armeniacum is considered to be a saprophyte.
Fusarium acuminatum ssp. armeniacum has been found in south-eastern.Queensland 'and New South Wales and appears to be more abundant in highrainfall areas or in clay soils on flood-plain country in drier areas. It isparticularly common in the Coffs Harbour area of New South Wales (Burgesset al., 1993a) which has a warm sub-tropical climate. In contrast,F. acuminatum ssp. acuminatum was not isolated during intensive surveys ofgrassland and pine nursery soils in the CoffsHarbour area.
Cultures of F. acuminatum ssp. armeniacum are usually more highlytoxigenic than those of F. acuminatum ssp. acuminatum, and produce adifferent spectrum of trichothecenes (Wing et al., 1993a).
0.5 - 2.83.7 - 5.44.4 - 6.1
2.5 - 3.54.4 - 5.83.7 - 5.4
acuminatumssp. acuminatumssp. armeruacurn.
longipes
Colony Diameters (cm)25°C 30°C
Species/subspecies
Table 5 Colonydiameters ofF. acuminatum ssp. acuminatum, F. acuminatum ssp.armeniacum and F. longipes on PDA after 3 days dark incubation at 25°Cand 30°C.
25°C and 30°C are compared in Table 5. The colony diameters ofF. acuminatum ssp. armeniacum are intermediate between those of the othertwo species.
-106 -
Fusarium longipes is a tropical species which is common in grassland soils in''!i!'
the north-eastern are~~ of Queensland, Australia (Cairns, Mareeba,Rockhampton; Burgess & Summerell, 1992) and from grassland and wetlandsoils in the Darwin region (Sangalang et al., in press). It has not been isolatedfrom temperate regions. It can be regarded as a saprophyte.
Comments. Some isolates of F. equiseti produce macroconidia whichresemble those of F. longipes. Isolates ofF. longipes are distinguished by theformation of greyish rose to burgundy pigment in agar. Some isolates ofF. compactum also produce macroconidia which resemble those of F. longipes.However, sporodochia of F. compactum are smaller than those of F. longipesand do not form columellae of macroconidia.
Key Characters. Distinctive shape of the macroconidia, colony pigmentationon PDA and growth rates.
Chlamydospores are formed in chains or clusters in most cultures. Theybecome pale brown and verrucose (rough walled) with age.
Microconidia are absent.
Diagnostic Characters on CLA. Fusarium longipes forms abundantmacroconidia in orange sporodochia from which the macroconidia ooze into acolumella up to 5-6 mm in height. The macroconidia are very long, slender,thick walled, usually 5 to 7 septate with a distinct dorsi-ventral curvature (Fig.14a, b). There is a very distinct foot-shaped base to the basal cell with 'a longinstep and pronounced heel'. The apical cell is filamentous (whip-like) andmay twist about or be curved in a hook or a circle. A very distinctive shape!Small numbers of macroconidia, which are extremely variable in shape, oftenshort, are formed in the aerial mycelium. Macroconidia are. formed frommonophialides on branched conidiophores in sporodochia or frommonophialides formed on the hyphae.
Fusarium longipes forms a greyish rose or burgundy pigment in contact withthe agar. Cultures ofF. longipes degenerate readily to the pionnotal form or toa white mycelial form.
Colony Morphology on PDA. This species grows rapidly on PDA formingfloccose mycelium, initially white becoming greyish rose with age. A fewobvious orange sporodochia develop in the centre of the colony. Abundantmacroconidia ooze from these sporodochia into a columella which may be 4-6 mm in height.
Colony Diameters on PDA 3.7 - 5.4 em at 25°C4.4 - 6.1 em at 30°C
Fusarium longipes Wollenw. &Reinking
I
III
. JIi!
I.!f·i
-107 -
Key Characters. The shape of the macroconidia and colony diameters onPDAat 30°Cand 35°C(Table 6). '
Chlamydospores are formed abundantly In chains or clusters. They areverrucose and become p~l,ebrown with age.
e:
Microconidia are absent.
Diagnostic Characters on CLA. Macroconidia are formed in orangesporodochia on the leaf pieces and on the agar. They are usually 5 septate andhave a strong dorsi-ventral curvature, tapering apical cell (rarely whip-like)and a distinct foot-shape to the base of the basal cell. They are thick walled andvariable in length (Fig. 14c, d). The macroconidia formed in sporodochia arerelatively uniform in length but those formed in spore masses in the hyphaeare extremely variable. Macroconidia of F. compactum are similar to those ofF. equiseti and the two species cannot be differentiated with confidence on thebasis of the morphology of the macroconidium. However the apical cell ofmacroconidia of F. compactum is more sharply pointed (needle-like) than thatofF. equiseti. Fusarium compactum normally forms sparse mycelium on theagar of CLA whereas most isolates of F. equiseti form relatively abundantfloccose mycelium on the agar between the leaf pieces. The macroconidia areproduced from monophialides on branched conidiophores in sporodochia andto a minor extent from monophialides formed directly on the hyphae.
Fusarium compactum degenerates readily to the pionnotal form andoccasionally to a white mycelial culture. Isolates of F. compactum tend todegenerate more readily than those ofF. equiseti.
Isolates ofF. compactum are quite variable in respect to pigment formation inthe agar. Some isolates produce a greyish rose, violet brown or wine redpigment. In contrast other isolates produce a light brown to dark brownpigment. Dark brown pigment flecks may form in the agar of either of the abovetypes of cultures. A few isolates produce no pigment in the agar. .
Orange to brownish orange sporodochia form in the centre of some cultures. Inothers the macroconidia are in orange or pale orange spore masses in theaerial mycelium.
Colony Morphology on PDA. Fusarium compactum forms uneven whitefloccose mycelium in which white tufts of hyphae may develop. Greyish rosemycelium develops at the periphery of colonies of some isolates which developgreyish rose, violet brown or burgundy pigment in the agar.
ColonyDiametersonPDA. 4.1- 5.4em at 25°C4.2 - 5.8 em at 30°C
Fusarium compactum (Wollenw.)Gordon
'. 1.
j:\'fII
-108 -
Fusarium compactum is regarded as a saprophyte. Most isolates of F.compactum are toxic and produce high levels of trichothecene mycotoxins(Winget al., 1993b).
Fusarium compactum is common in grassland soils of western Queensland(Burgess & Summerell, 1992; Summerell et al., 1993a) and was the mostcommon of the three species of Fusarium (F. compactum, F. equiseti,F. chlamydosporum) isolated from soil samples from the Simpson Desert,Australia. Fusarium compactum was also frequently isolated from soils in thecentral arid woodlands of Australia (Sangalang et al., in press). Studies of thesurvival of F. compactum have shown that the fungus is well adapted tosurviving exposure to elevated temperatures (Sangalang et al., in press). Thedesert isolates of F. compactum form relatively short macroconidia comparedwith those from subtropical areas (Fig. 14c, d).
4.1 - 5.4 4.2 - 5.8 0.5 - 1.53.4 - 4.6 2.8 - 4.4 0.0 - 0.5
F. compactumF. equiseti
ColonyDiameter (cm)25°C 30°C 35°C
SpeciesI1j"
t:i.:Ii'I
iifI,~,"I:;Iii~ I
Table 6 Colony diameters of F. compactum and F. equiseti on PDA at threetemperatures.
Comments. The brown or non-pigmented isolates of F. compactum aresimilar to those ofF. equiseti in respect to most morphological characteristics.Colony diameters at 30°C and 35°C provide simple and objective criteria fordifferentiating the two species (Table 6) (Singh & Burgess, unpublished data).
) .-.
it
lf·,,I~.
t'I ..·,'I,1'
-109 -
. Fusarium equiseti is a cosmopolitan soil inhabitant and has been recoveredfrom most grassland soils of eastern Australia and from sandy soil from the .Simpson Desert and the central arid woodlands of Australia (Burgess et al.,1988;Burgess & Summerell, 1992; Summerell et al., 1993a; Sangalang et al., inpress). It is a commo~coloniser of senescent or damaged plant tissue.
,!~:~,Fusarium equiseti i; not an important pathogen, however it has beenimplicated in a few diseases such as rots of cucurbit fruits in contact with soil.
Comments. This species is most easily confused with F. compactum (brownpigmented isolates), F. semitectum and F. scirpi. It is distinguished fromF. compactum mainly on the basis of growth rate on PDA (Table 6). Fusariums c ir p i . produces abundant microconidia on polyphialides whereasF. semitectum produces abundant spindle-shaped conidia from polyphialides .
Key Characters. The shape of the macroconidia,brown pigmentation on PDAand chlamydospores.
Chlamydospores are normally formed in abundance in chains or clusters inthe aerial hyphae and in the agar. They becomeverrucose and pale brown withage.
Microconidia are absent.
Diagnostic Characters on CLA. Abundant macroconidia are formed inorange sporodochia. The macroconidia have a strong dorsi-ventral curvature,are usually 5 to 7 septate, with a tapering apical cell and a distinct foot-shapedbase to the basal cell (Fig. 14e).They are thick walled and extremely variable in.length ranging from 25j.lm to over 120j.lm. Some isolates produce longmacroconidia which have a filamentous or whip-like apical cell and whichmay resemble the macroconidia formed by F. longipes. Macroconidia areproduced from monophialides on branched conidiophores in sporodochia.
ii-'
Colony Morphology on PDA. Fusarium equiseti forms uniform floccosemycelium initially white to salmon but changing to beige with age. Myceliumformation can be abundant and fill the tube..Abundant sporodochia form in acentral spore mass in many isolates but may be obscured by the mycelium. Thespore mass ranges in colour from pale orange to dark brown and annularzonations of spore masses may develop under alternating conditions of lightand temperature. This species forms a pale brown to dark brown pigment incontact with the agar. Dark brown flecks are also usually formed in the agar.
ColonyDiametersonPDA 3.4-4.6em at 25°C2.8 - 4.4em at 30°C
'Fusarium equiseti (Corda)Sacco
(Scale in all photographs = 20 um)
e. Fusarium equiseti: macro conidia
c, d. Fusarium compactum: short (c) and long (d) macroconidia
a, h. Fusarium longipes: macroconidia
Figure 14
-110 -
iIi
-111-
-113 -
Isolates of F. scirpi were recovered from rangeland soil from Fowler's Gapnear Broken Hill, New South Wales in 1975 and provisionally referred to asmicroconidial isolates ofF. equiseti (Burgess, Nelson & Toussoun, unpublisheddata). Later it was discovered that Wollenweber had described and illustratedFusarium isolates with macroconidia similar to F. equiseti and with clubshaped microconidia and cross-shaped polyphialides. Wollenweber (1916-1935)recorded these isolatesj as either F. chenopodinum Thuem. or F. equiseti(Corda) Sacco var buU(J}tum (Sherb.) Wollenw. Wollenweber & Reinkingincluded F. chenopodinum as a synonym of F. scirpi Lambotte & Fairtrey in'Die Fusarien' (1935). Their description of F. scirpi included mention of clubshaped microconidia but did not contain a reference to the polyphialides. They
Comments. Isolates ofF. scirpi are easily confused with those ofF. equisetibecause they are similar in respect to colonymorphology on PDA and shape ofthe macroconidia from CLA. The presence of microconidia formed fromdistinctive, short and often cross-shaped, polyphialides in the hyphae ofcolonies on CLA indicates F. scirpi.
Key Characters. The shape of the macroconidia, relatively abundantmicroconidia, short, cross-shaped polyphialides and colony pigm_entation onPDA.
Chlamydospores are formed by F. scirpi in clumps or chains.
Microconidia are formed from short, truncate, often cross-shapedpolyphialides (Fig. 16a, b) The microconidia are club-shaped to ellipsoidal andare 0 to 3 septate (Fig. 15b).
Diagnostic Characters on CLA. Macroconidia are formed in orangesporodochia. They are long, with a strong dorsi-ventral curvature, 6 to 7septate, thick walled, and have a long tapering apical cell (Fig. 15a). Thus theyresemble macroconidia of F. equiseti. Macroconidia develop mainly frommonophialides on branched conidiophores in sporodochia and to a minorextent from monophialides on hyphae.
Colony Morphology on PDA. Colonies of F. scirpi are similar to those ofF. equiseti. Fusarium scirpi forms uniform floccose mycelium, initially whiteto salmon but changing to beige with age. Mycelium formation can beabundant and fill the tube. A central pale orange or brown spore mass isformed by some isolates but may be obscured by the mycelium. Fusarium scirpiforms a pale brown to dark brown pigment in the agar. Dark brown flecks arealso usually formed in the agar.
ColonyDiametersonPDA. 3.6- 4.8cm at 25°C3.6- 4.9emat 30°C
Fusarium scirpi Lambotte&Faut. emendBurgess,Nelson&Tousson
-114 -
Within Australia representatives ofF. scirpi have been isolated from southern,central and western Queensland, most areas of New South Wales and northwestern Victoria. It was not isolated from soil samples from the SimpsonDesert (F. compactum, F. equiseti and F. chlamydosporum were recoveredfrom these samples). Isolation data from systematic surveys indicates thatF. scirpi is more abundant in the warmer semi-arid areas (Burgess et al., 1988;Burgess & Summerell, 1992; Sangalang et al., in press). Records indicate thatF. scirpi exists mainly as a saprophyte.
retained F. equiseti var bullatum as a distinct variety but did not mention _microconidia in its description. Thus the species name F. scirpi was adoptedfor isolates producing club-shaped microconidia on short, usually cross-shapedpolyphialides and long macroconidia similar to F. equiseti. A thorough study ofthe relevant literature and type material was undertaken. Further isolates ofF. scirpi have also been recovered from South Africa (Marasas et al., 1988). Adetailed account of the nomenclatural history of F. scirpi and an amendeddescription has been published by Burgess et al. (1985).
:i'II
iII!'I:1,J!:\I
.1'\·fIii\I:i!,
I
-115 -
Comments. Fusarium polyphialidicum was described by Marasas et al.(1986) and was isolated from plant debris in soils in South Africa (Marasaset al., 1988). This species most resembles F. semitectum and F. subglutinans.It differs from F. semitectum in that there are more abundant polyphialidesand that the sporodochial macroconidia are longer, wider and with lessprominent foot and apical cells. The fungus differs from F. subglutinans inthat it produces chlamydospores, it does not produce violet pigments, theproduction of spindle shaped microconidia and the macroconidia are thickerwalled. Ali et al. (1991) recovered F. polyphialidicum from mouldy sorghumgrain collected in northern New South Wales, Australia. Recently Gott et al, (inpress) recovered isolates of the fungus from the central arid region of Australiawhich differed from the descriptions of Marasas et al. (1986). The growth ofthese isolates was greater on PDA, being 3.0 - 4.0cm at 25°C and 2.7 - 3.8cm.at30°C compared with 2.3 - 3.2cm and 2.0 - 2.9cm at 25°C and 30°C respectively.
Key Characters. The prominent polyphialides, often with larger spindleshaped conidia, macroconidia and colony morphology on PDA.
Chlamydospores are formed in aerial and submerged hyphae in pairs, clumpsor chains.
Diagnostic Characters on CLA. Sporodochia are produced in the aerialmycelium on CLA, and are white to pale orange; in some isolates sporodochiaare not produced or occur as white flecks within or on the agar. Macroconidiaare thick walled, 3-7 septate, mostly 5 septate with a curved apical cell and afoot-shaped basal cell (Fig. 15c). Microconidia are produced from conspicuouspolyphialides often with numerous openings (Fig. 16c, d). The microconidia arefusiform or subclavate produced in pairs or in false-heads (Fig. 15d). Somelarger microconidia may be produced which are spindle shaped similar tothose produced by F. semitectum.
Colony Morphology on PDA. Fusarium polyphialidicum forms' abundantuniform white to pale orange mycelium on PDA. A white to yellow pigmentdevelops in the agar. Colony morphology is very similar to that ofF. semitectum.
ColonyDiameters on PDA 2.3 - 4.0 em at 25°e2.0 - 3.8 em at 300e
Fusarium polyphialidicum Marasas, Nelson,Toussoun &VanWyk
-116 -
Comments. Two other species, F. subglutinans and F. sporotrichioides,produce spindle-shaped conidia from polyphialides on CLA. These conidia areoften produced in pairs and appear like 'rabbit-ears' thus resembling theformation of the spindle-shaped conidia of F. semitectum on CLA.Fusariumsporotrichioides is distinguished by the formation of two types of microconidiaand greyish rose to burgundy pigment in the agar of PDA cultures. Fusariumsubglutinans is distinguished by the presence of small oval microconidia aswell as spindle-shaped conidia. Cultures of F. subglutinans on PDA usuallydevelop violet pigments.
Key Characters. The spindle-shaped conidia formed on polyphialides(sometimes in pairs having a 'rabbit-ear'-like appearance), macroconidia,colonymorphology on PDA.
Chlamydospore formation is variable.
A few 1 to 2 celled microconidia may be produced, presumably from thepolyphialides in the aerial hyphae.
Macroconidia similar to those of F. equiseti are formed in orange sporodochiaby some isolates (Fig 15e). These macroconidia conidia are produced frommonophialides by branched conidiophores in the sporodochia and rarely frommonophialides on hyphae. The sporodochia may be obscured by the denseaerial hyphae. Sporodochia are rare in some isolates ofF. semitectum.
Diagnostic Characters on CLA. Abundant, straight to slightly curved,usually 5 septate, spindle-shaped conidia (Fig. 15f) are formed frompolyphialides on the aerial hyphae (Fig. 16e, D. These conidia can be readilyobserved in situ under the x10 or x20 objective of the compound microscope andmay form in pairs having a 'rabbit-ear'-like appearance which is quitedistinctive (Fig. 16e, D. Many tropical and sub-tropical isolates produce spindleshaped conidia which are longer than those produced by isolates fromtemperate areas. Isolates producing either short, medium or long spindleshaped conidia have been recovered, which do not differ in respect to othercharacteristics such as colony morphology.
Colony Morphology on PDA. Fusarium semitectum forms abundant uniformmycelium on PDA, initially white to salmon becoming beige with age. It doesnot form a central spore mass. A pale to dark brown pigment develops in tileagar. Colonymorphology is similar to that ofF. equiseti but F. semitectum doesnot form dark brown flecks in the agar.
ColonyDiameters on PDA 3.5 - 4.5 emat 25°C1.6 - 3.3 emat 30°C
Fusarium semitectum Berk. & Rav.
-117 -
Fusarium semitectum is common in grassland and agricultural soilsthroughout the tropical and temperate areas of eastern Australia but has notbeen isolated from alpine grassland soils. It is also commonly isolated fromaerial plant parts in tropical and sub-tropical areas (e.g. banana fruit) and canbe isolated from the air. It has been shown to be well adapted to air dispersal(Lukezic & Kaiser, 1966). Fusarium semitectum is not regarded as animportant pathogen but has been implicated in storage rot problems of bananasand other fruits CLukezic& Kaiser, 1966).
The name F. semitectum has been retained for this species but Booth andSutton (1984)have proposed a change to the species epithet (F. pallidoroseum).They also argue that the production of spindle-shaped conidia in this species isblastic rather than phialidic.
-118 -
Comments. The affinities ofF. beomiforme with other Fusarium species arenot known (Nelson etal.., 1987). The fungus is generally found in areas withhigher rainfall and is common in soils in the Rockhampton area of Australia,the Markham Valley of Papua-New Guinea and has been isolated from soilfrom Natal in South Africa (Nelson et al., 1987). The fungus has been recoveredrecently from soil in the Chillagoe region, a relatively dry location in the tropicsof Queensland, Australia (Summerell et al., 1993a). The rain at this locationfalls in intense monsoonal storms which may provide conditions, at leastbriefly, similar to those of more continuously moist climates and suitable forthe activity of the fungus.
Key Characters. The presence ofvery large napiform to globose microconidiais the most conspicuous feature of this species. The orange-red to reddishbrown pigment formed in the agar by F. beomiforme is also characteristic andhas not been observed by the authors in cultures of any other Fusarium species.
Microconidia are of two types. The most distinctive microconidium is large,globose, to napiform to lemon-shaped, papillate and normally one celled butmay be two celled (Fig. 15i), and is formed singly from monophialides (Fig.16h). Small oval to fusiform microconidia, mainly one celled (Fig. 15h) butoccasionally two celled, are produced from monophialides in the aerial hyphae,usually in false-heads (Fig. 16g). Chlamydospores are formed slowly after 3 to 4weeks in hyphae on CLA, and are hyaline to pale brown, smooth- or roughwalled, solitary or in pairs, rarely in clumps or chains. Chlamydosporeformation is enhanced on SA, occurring within 1 to 2 weeks.
Diagnostic Characters on CLA. Macroconidia are formed mainly in pale tobright orange sporodochia. The macroconidia are long, falcate, usually 3 to 5septate and thick-walled (Fig. 15g). The apical cell is slightly curved and thebase of the basal cell is foot-shaped or notched. The macroconidia are producedmainly from monophialides on branched conidiophores in the sporodochia,and from monophialides on the aerial hyphae.
Fusarium beomiforme forms abundant floccose,on PDA. Pigmentation in the agar is bright
Colony Morphology on PDA.white to pale orange myceliumorange-red to reddish brown.
ColonyDiameters on PDA. 3.0 - 3.9 em at 25°C3.6 - 4.6 em at 30°C
Fusarium beomiforme Nelson, Toussoun &Burgess
(Scale in all photographs = 20 11m)
1. Fusarium beomiforme: globose microconidia
h. Fusarium beomiforme: oval microconidia
g. Fusarium beomiforme: macroconidia
f. Fusarium semitectum: spindle-shaped conidia
e. Fusarium semitectum: macroconidia
d. Fusarium polyphialidicum: microconidia
c. Fusarium polyphialidicum: macroconidia
b. Fusarium scirpi: microconidia
a. Fusarium sctrpt: macroconidia
Figure 15
-120 -
-121-
.,
~p,/;~ ~... ...... .....
. '11, ..; ;~~':
i. '
c
-122 -
-123 -
(Scale in all photographs = 20 urn)
g, h. Fusarium beomiforme: formation of microconidia in false-heads (g) andglobose microconidia (h) on CLA, in situ.
e, f. Fusarium semitectum: formation of spindle-shaped conidia on CLA, Insitu
c, d. Fusarium polyphialidicum: formation of microconidia on CLA, in situ
a, b. Fusarium scirpi: formation of microconidia on CLA, in situ. Note crossshaped polyphialides
Figure 16
-124 -
Burgess, L.W. (1981). General Ecology. Fusarium: Diseases, Biology and Taxonomy. (eds. P.E.Nelson, T.A. 'I'oussoun je R.J. Cook) pp.225-235. The Pennsylvania State University
~Press, University Park. "~'i
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Balmas, V., Burgess, L.W. & Summerell, B.A. (in press). Reaction of durum wheat cv. Yallaroi tocrown and root rot caused by Fusarium graminearum Group 1 and Fusarium crookwellense.Australasian Plant Pathology.
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Atanasoff, D. (1920). Fusarium blight (scab) of wheat and other cereals. Journal of AgriculturalResearch 20: 1-32.
Andrews, S. & Pitt, J.I. (1986). Selective medium for isolation of Fusarium species anddematiaceous hyphomycetes from cereals. Applied and Environmental Microbiology51: 1235-1238.
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"i\"
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<r..:.."~~:~.Lawrence, E.B., Nelson, P.E. & Toussoun, T.A (1985a). Inheritance of compatibility and sex in
Gibberella baccata.Phytopathology 75: 322-324.
Kornerup, A & Wanscher, J.H. (1978). Methuen Handbook of Colour, 3rd ed. Methuen & Co.Ltd., London.
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