Introduction to Fusarium taxonomy

Teleomorphs of FusariumGibberella – the majority of Fusarium speciesHaematonectria – Fusarium solani complexAlbonectria – Fusarium decemcellulareTeleomorph morphology usually is not sufficientlyfor identification purposes.Gibberella zeae

Key characters for identifying Fusarium speciesMacroconidiaSizelarge versus smallNumber of septarange and averageShapeelongate versus squatdegree of curvature

Key characters for identifying Fusarium speciesDorsal sideVentral sideDorsal sideVentral sideMacroconidiaApical cellsBasal cells

Apical cells shapes:A1.Basal cells shapes:B1.A2.A3.B2.B3.A4.B4.A1. BluntA2. PapillateA3.TaperingA4. HookedB1. Foot-shapedB2. Elongated foot-shapedB3. Distinctly notchedB4. Barely notched

Key characters for identifying Fusarium speciesMicroconidiaShape and SizeNumber of cellsFalse heads or/and chains

Key characters for identifying Fusarium speciesConidiophores are special structures of hyphae whichfinished in fertile cells - phialides and provide conidiawith nutrients.- unbranched, sparsely or densely branched.Conidogenous cellsMonophialides – having only one opening per cellPolyphialides – having more than one opening per cell (but notnecessarily for all conidiogenous cells)

Key characters for identifying Fusarium speciesMonophialidesPolyphialides

Length of the Conidiogenous cellLong (F.solani)Short (F.poae)

Key characters for identifying Fusarium speciesMesoconidiaSpindle-shaped conidia that are typically produced in F.sporotrichioides, F. chlamydosporum, F. semitectum, F.camptoceras and in some isolates of F. avenaceum and F.subglutinans.These spores are not produced in sporodochia, only in the aerialmycelium.F. sporotrichioides

Key characters for identifying Fusarium speciesChlamydosporesThe presence of chlamydospores is the diagnostic character.Their absents is not informative character.They can take long period of time to form (up to 6 weeks).Formed singly, doubles, in clumps or in chains.May be found in aerial mycelia or embedded in the agar.

Key characters for identifying Fusarium speciesColony morphologyAbundance and color of aerial myceliumPigmentationGrowth rates, especially at 25°C and 30°C

ReverseSurface

Beginning the identification processThe majority of isolates are not difficult toidentify if you use:•Controlled temperature and light conditions•Standard media•Cultures initiated from single spores

Growth ConditionsRemember, try to use the conditions outlined inthe identification guide you are following!Temperature – generally 25°C or fluctuating25/20°C day/night cycle.LightGood light conditions will help to stimulate conidiaformation.Artificial daylight (cool white tubes) on a 12 hrlight:12 hr dark cycle necessary to maximizeproduction of sporodochia.Black light (near-UF, emission ca. 310-360 nm) maybe essential for teleomorph development.

Standard media for growth of Fusarium fungiMedia for Fusarium isolation:Potato dextrose agar (PDA)Czapek-Dox agarThe media are supplemented with antibiotics and fungicides after theagar is melted and the temperature is not higher than 55 degrees C.

Antibiotic solutionsAntibiotic Stock solution, g Workingconcentration,ml/L of mediumStreptomycin 5 /100ml H 2O 20 G-EffectiveagainsbacteriaNeomycin 1/ 100ml H 2O 12 G+Chlotetracyclinehydrochloride0.5/ 100ml H 2O 10 G- and G+Chloramphenicol 0.5/10 ml ethanol (96%) 1 G- and G+Fungicides prevent growth of saprophytesPCNB (Pentachloronitrobenzene): 750mg/L. PCNB is usuallyadded as 1 g of Terrachlor, which contained 75% PCNB (w/w).Dichloran: 200 mg in 100 ml ethanol (96%) (1 ml is added to1000 ml agar before autoclaving).Propiconazole: 0.375 mg /1L after the agar is melted and thetemperature is below 55 degrees C.The linear growth of fungi may be suppressed by Triton X-100,stock solution 2% is autoclaved and used at the rate of 10 ml/Lof medium.

Weak nutrient mediaCarnation Leaf-Piece Agar (CLA). CLA is a natural substratemedium prepared by aseptically placing sterile carnation leaf pieces,3-5 mm, into a Petry dish and adding sterile 2% water agar (5-6 leafpieces is dishes). Water agar (WA) consist of 20 g agar in 1 L ofdistilled H 2 O.Spezieiller Nährstoffarmer Agar (SNA). In 1 L of distilled H 2 O:KH 2 PO4 1 g; KNO 3 1 g; MgSO 4 x 7H 2 O 0.5 g; KCl 0.5 g; Glucose 0.2 g; Sucrose 0.2 g; Agar 20 g.Placing 1-2 pieces of sterile filter paper (approximately 1cm 2 ) on the agarsurface after the medium hag gelled can increase sporulation.Media promotes sporulation and good conidiogeneous cell development.Media transparent, so cultures can be viewed directly with microscope (x100).CLA- surface CLA- reverse SNA- surface

Criteria for identificationPDA medium:Colony morphologyPigmentation – hyphae and in agarGrowth ratesCLA and SNA media:Presence/absence of conidiaShape and formation of conidiaNature of the conidiogenous cellsPresence/absence of chlamydospores

Single spore isolationCan be used for macroconidia, microconidia and ascospores.If you want to be sure in identification, use singlespore cultures!Never use cultures before single sporing in the further studies(genetics, molecular, pathogenicity, etc.)! More than onespecies/strain of fungi or Fusarium from the same substrate.

Protocol:Dilution Plate• Prepare sterile water in a vial• Transfer sporodochial conidia from culture with a fine needlein a water.• Check the concentration and dilute (5-10 conidia/drop) undera low power (10x) microscope.• A few drops of the suspension spread over 2% dry and thinWA plates.• Leave plates for 6-15 hr.• Check plates for germinating conidium along under a lowpower (10x).• Mark with a fine marker.• Pick small squares of agar around a germinating conidiumusing a fine sterile needle and transfer to maintenance media.

* If the culture is not form conidia then pick a hyphal tipseparated from others by spreading on WA plate and transferto maintenance media.

Fusarium genusMain species:F. graminearum (Gibberella zeae) complexF. culmorumSection DiscolorFusarium head blight

Section DiscolorColony morphology- fast growing (5 to 7 cm >growth in 7 days)- red/pink pigmentsMacroconidia- relatively robust and thick-walled- typically formed in sporodochia- less often on hyphaeMicroconidia- absent or rareConidiophores- monophialidesChlamydospores- present in conidia and hyphae- single, chains, clumps- inconsistently produced

Section DiscolorColony appearanceF. graminearumsurfaceF. culmorumreversesurfacereverse

Section DiscolorMacroconidiaFusariumSize Cells Surface Septate Sporodochialength ratio* apical basal ventral dorsalF. graminearum 40-60 11 tapered footshapedstraightsmoothlyarched5-6 paleorangeF. culmorum 25-35 6 blunt,roundedfootshape-notchedstraight curved 3-4 brick redto brown* approximate ratio = length/ width of macroconidiaF. graminearum F. culmorum

Section RoseumMain species:F. avenaceum (Gibberella avenacea)Cosmopolitan, especially temperate zones.Wide range of plants:seedling disease, crown & root rot.Colony morphology- highly variable- surface: floccose, grey rose, yellow, white.- growth rate varies from moderate to rapidPigment - grey rose to burgundy, brownishConidiophores- branched and unbranched monophialidesSporodochia: Usually bright orangeChlamydospores - Absent

Section RoseumF. avenaceumMacroconidia formed on hyphae and in sporodochiaMacroconidia in sporodochia– long, slender, thin-walled, usually 3-5 septate– falcate to almost straight "needle-like"Apical cell – elongate, bentBasal cell – notched or foot-shapedMacroconidia &Mesoconidia in aerial mycelium– often abundant– fusiform, comma-shaped, 0-3 septate

Section SporotrichiellaMicroconidia pyriform, globular to spindle-shaped, or lemon-shaped.Main species:F. sporotrichioides F. poaeF. tricinctum(Gibberella tricincta)F. langsethiae

Section SporotrichiellaF. sporotrichioidesCosmopolitan.Occurs on a wide variety of plants & soil.Colony morphology- rapid growth, abundant floccose mycelium- sporodochia may occur in older culturesSurface: white, pinkish to brownish redPigment: rose to burgundyMacroconidia: abundant- on hyphae and sporodochia- falcate, 3-5 septateApical cell: curved, pointedBasal cell: notched or foot-shapedMicroconidia: abundant- oval to pyriform (pear-shaped), spindle-shaped- 0-1 septate, often with papillaConidiophores- monophialides & polyphialidesChlamydospores - Abudant

Section SporotrichiellaF. poaeCosmopolitanMore common on gramineous hostsColony morphology- rapid growth, abundant floccose mycelium- sporodochia rareSurface: white, pinkish whitePigment: rose to burgundyMacroconidia: generally rare- on hyphae and sporodochia- falcate, 3-5 septateApical cell: short, curved, pointedBasal cell: foot-shaped or notchedMicroconidia: abundant- globose to pyriform, often with papilla- 0-1 septate,- produced in clusters, "bunch of grapes"Conidiophores- unbranched & branched monophialidesChlamydospores - AbsentSome isolates has fruity odor

Section SporotrichiellaF. langsethiaeUp to now found only in EuropeDetected in small cereals grainColony morphology- slow growth, nearly lack of aerial myceliumSurface: white to peach, pinkish white, powderPigment: peach to violetSporodochia absentMicro morphology similar to F. poaeMacroconidia: absentMicroconidia: abundant- globose to pyriform, often with papilla- 0-1 septate,- produced in clusters, "bunch of grapes"Conidiophores- unbranched & branched monophialidesChlamydospores - AbsentNo odor

Section SporotrichiellaF. tricinctumCosmopolitan, more common in temperate areasColony morphology- moderate rapid growth,- mycelium as compact cushin- sporodochia may occur in older culturesSurface: red to vinaceous or purplePigment: red to vinaceous or purpleMacroconidia:- usually form in sporodochia- falcate to crescent-shaped, slender- 3-5 septateApical cell: curved, pointedBasal cell: notched or foot-shaped, narrow andpointedMicroconidia: abundant- lemon-shaped to pyriform, ellipsoidal- 0-1 septateConidiophores:monophialides, richly branched, slenderChlamydospores: formed singly or in chains

Section GibbosumMain species:F. equiseti (Gibberella intricans)F. acuminatum (Gibberella acuminata)Weak pathogens of wide range of plants, in soil.MacroconidiaUsually strongly curved on both ventral and dorsal sides of conidiastriking foot-shaped basal cellOften quite largeNo microconidiaChlamydospores presentPigment: red or brown

Section GibbosumF. equisetiCulture morphology:- fast growing- abundant myceliumSurface: white to beige or brownPigment: pale to dark brown with brown flecksMacroconidia- various sized - occasionally very long- 5-septate, with strong curvatureApical cell: tapering cellBasal cell: foot shapedChlamydospores abundant in hyphae, brownReverseSurface

Section GibbosumF. acuminatumCulture morphology:- slow-middle growing- floccose myceliumSurface: peach-beigePigment: carmine redSporodochia blight orangeMacroconidia- slender, sickle-shaped- 3-5-septateApical cell: tapering cellBasal cell: pedicellate shapedChlamydospores in hyphaeSurfaceReverse

Section LiseolaGibberella fujikuroi species complexF. proliferatumThe tester strains of G.fujikuroi complexWorldwide distributed on a wide variety of plantsColony morphology- rapid growth, abundant floccose mycelium- sporodochia may occurSurface: white, becoming purple-violet with agePigment: varies in violet intensityFertile perithecia G.fujikuroi after makingcrosses with the tester strain F.proliferatumReverse

Section LiseolaGibberella fujikuroi species complexF. proliferatumMacroconidia: usually abundant- on hyphae and sporodochia- slender, thin-walled, relatively straight, 3-5septateApical cell: curvedBasal cell: poorly developedMicroconidia: abundant- oval with flattened base, pyriform also mayoccur- 0 septate- in short chains and in false headsConidiophores- monophialides & polyphialidesChlamydospores - absent

Section ElegansF.oxysporum species complexWorldwide distributed on a wide variety of plants. Animportant vascular wilt and root rot pathogens.Many isolates are host specific and differentiated as formaespeciales (f.sp.) – infraspecific subdivision.Colony morphology varies widely!!!-may be floccose, sparse, abundant- sporodochia may occur, pale orangeSurface: white, becoming purple-violet with agePigment: unpigmented to dark violetMacroconidia: usually abundant- on hyphae and sporodochia- short to medium lenght- straight, to slightly curved- thin walled, usually 3- septateApical cell: tapered and curvedBasal cell: foot shaped to pointedMicroconidia: abundant- oval, elliptical- 0 septate- in false headsConidiophores- Short monophialidesChlamydospores– usually formed abundantly and quickly-singly or in pairsSurface