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The Fusarium Laboratory Manual

This laboratory manual provides an overview of the biology of Fusarium and the techniques involved in the isolation, identification and characterization of individual species and the populations in which they occur. It is the first time that genetic, morphological and molecular approaches have been incorporated into a volume devoted to Fusarium identification. The authors include descriptions of species, both new and old, and provide protocols for genetic, morphological and molecular identification techniques. About the Author John F.

Leslie received his Ph. Table of contents Foreword. Techniques and Methods. Media — Recipes and Preparation. Techniques for Recovering Fusarium. Techniques for Growing and Maintaining Fusarium. Fertility Concepts. Nucleic Acid Analyses. Taxonomy and Identification of Fusarium.

Description

A Brief History of Fusarium Taxonomy. Usually one carnation leaf piece is added per 2 ml KH2PO4 1 gof medium. In 60 mm diameter Petri dishes, pieces KNO3 1 gper dish and in mm diameter dishes, Immediately after collection, the leaves are cut into Sucrose 0. The dried leaf dium has gelled can increase sporulation. Sterilized leaf pieces can be stored dry at room medium promotes sporulation and good conidiogeneoustemperature for up to 12 months before use.

Cultures grown onare more uniform in size and form, than do cultures SNA often are of value for examining microconidia, asgrown on carbohydrate-rich media such as PDA or this medium supports the formation of a range of differ-Czapek-Dox. Macroconidia form primarily in sporodo- ent microconidia. In some cases microconidia formed onchia, which usually develop on the leaf pieces. Macroco- SNA will differ morphologically from those seen onnidia formed in sporodochia are preferred for identifica- CLA.

Many strains also form chlamydospores more read-tion as they are more consistent in shape and length than ily on SNA than they do on CLA, so for species in which Media 7microconidial morphology or the presence of chlamy- tion series. The small amount of agar retards the sedi-dospores are important identification criteria, cultures mentation rates of fungal propagules. SNA is until the agar dissolves, and the heated medium distrib-transparent, so cultures can be viewed directly with a uted into flasks, or, more commonly, McCartney bottles. Soil Agar SA. PDA is a carbohydrate- on SA , making this medium useful for the identi-rich medium that contains 20 g dextrose, 20 g agar, and fication of some species of Fusarium.

SA is prepared bythe broth from g white potatoes made up to 1 L with placing g of sieved dry soil into a flask andtap water. The potatoes are unpeeled, washed, diced and bringing the total volume to 1 L with tap water and auto-boiled until soft actual time varies with the size of the claving for 15 min. After the first autoclaving, add 15 gpotato. Filter the boiled potatoes through a single layer of agar and autoclave for a second 15 min.

The amountof cheesecloth, which leaves some sediment in the broth. Abundant chlamy-Commercially available preparations of PDA, e. While pouringrations. However, colony morphology, pigmentation and CLA the fungal cultures produce more and longer micro-growth rates of cultures of most Fusarium species on conidial chains The chains also are easier to seePDA are reasonably consistent if the medium is prepared since there is less moisture on the agar surface and fewerin a consistent manner, and if the cultures are initiated moisture droplets in the aerial mycelium.

These colony characteristics often are useful sec- 2. PDA is used by some researchers for the isolation of A number of media have been developed for the specificFusarium species. We do not recommend this medium isolation of Fusarium spp. If PDA is used for the ferred for most other uses. MGA and RbGu media arerecovery of fungi from plant material, then the concentra- variations on PPA that have been widely used by sometion of potato and dextrose should be reduced by researchers.

New media are beingin section 2. L of distilled H2O. As hyphal growth is sparse on this me- , with modifications by Nelson et al. PPAdium, it is suitable for growing cultures from which indi- is composed of a sugar-free basal medium supplementedvidual hyphal tips are taken to initiate new colonies see with antibiotics and fungicides. The base medium con-section 4. In some instances, the sparse growth on WA tains:facilitates the isolation of Fusarium species from plantmaterial, particularly roots. Peptone 15 g WA 0. The pH is adjusted to 3.

The Fusarium Laboratory Manual

The streptomycin stock solution is 5 solutions. However, there issoil or diseased plant material is placed, or dispensed into enough overlap with some related species, e. Vials can the G. If plates are being used in for the recovery of Fusarium communities that containsoil dilutions see section 3. This mediumteria but allows slow growth of Fusarium. It enables the contains:selective isolation of Fusarium species from soil dilu- Peptone 15 gtions and from rotting plant material that often is infested KH2PO4 1 gwith other fast-growing microbes.

Often, Malachite green oxalate 2. Agar 20 gThis means that colonies usually must be subcultured be- H2O to 1 Lfore a definitive identification can be made. Although thesporulation is poor, there may be enough spores so that a After autoclaving, streptomycin and chloramphenicol aresubculture originating from a single spore can be gener- added as for PPA. This medium has not been used asated from colonies growing on PPA.

The only difference be-be left on PPA for more than weeks. This medium probably willgenerates relatively high levels of ammonia that eventu- become more important as the availability of PCNB dueally kill the colonies.

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MGA also is reported to be more inhibitory of common contaminants, e. The basal medium contains: PPA. The malachite green level in the medium is impor- D-Galactose 20 g tant, as the level of malachite green ppm used in L-Asparagine 2 g the Singh-Nene medium , from which this formu- KH2PO4 1 g lation was derived, prevents germination of many Fusa- KCl 0. This medium Distilled H2O to 1 L is particularly effective for selecting for maize pathogens Media 9such as F. SFA was developed easier to distinguish on this medium.

SSM was developeddebris. SFA is a modified Czapek-Dox medium see sec- for the selective isolation of Fusarium pseudograminea-tion 2. Commercially available prepara- Agar 20 g tions of PDA, e. The streptomycin and neomycin stocks are the stock solutions are added. This me-prior to autoclaving. Colonies of different species de- medium. In such studies, e. Although number of samples to be processed rapidly.

SFA was developed for isolation of Fusarium from soildebris, it is not suitable for the isolation of Fusarium spe- 2. This Chaff-Grain Medium. Inoculum suitable for addition tomedium was developed by van Wyk et al. Cereal chaff and grain L-Alanine 0. Rose Bengal 0. The medium is similar more than 24 hours, to leach phenolic compounds. After After min, wrap Synthetic and semi-synthetic media often are used forthe chaff-grain mixture in the cheesecloth and squeeze studying physiological characters, for isolating DNA, anduntil no more water can be released.

The media in this section are all based The drained mixture is distributed into glass jars or on the Czapek-Dox medium recipe, as modified by Cor-Erlenmeyer flasks, which are filled to a depth of ap- rell et al. The basal medium contains:proximately 5 cm.

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Containers are inoculated with a KCl 0. Distilled H2O to 1 L The inoculated material is shaken vigorously to It is critical to use distilled water in this medium to pre-evenly distribute the inoculum throughout the medium. The length of the incubation trace element levels. Agar should be at least of bacterio-depends on the rate of growth for the fungus. Usually 7- logical grade. Higher levels of agar purity occasionally14 days of incubation is sufficient. These cultures should are warranted, e.

Lower grades of agar maygins to decrease. Once the substrate is completely phic mutations clearly. For work with liquid cultures, thecolonized, it is removed from the container and air dried agar is omitted completely. When required for some enzyme activities, e. In other cases, e. This procedure has been used This recipe makes a nominal ml. The citric acidwith numerous Fusarium species, e.

Detecting Plant Diseases in the Lab

The solu-culmorum, F. It also is suitable for studies sary.


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More commonly this solution is stored in a cappedwith a number of fungal genera, e. The CHCl3 forms a small ball at the bottom of the container and it should be possible to detect its presence by smelling the solution in the bottle. If during storage the smell disappears or the ball of CHCl3 shrinks signifi- Media 11cantly or is no longer visible, then the stock solution is Inositol 4 gprobably no longer saturated with CHCl3, and additional Ca pantothenate mgCHCl3 should be added.

Thiamine mg Additional ingredients are added to the basal me- Pyridoxine 75 mgdium see below , depending upon the experimental pro- Nicotinamide 75 mgtocol, prior to autoclaving and use. Adding a drop or two Ascorbic acid 50 mgof vegetable-dye based food coloring, available at the lo- Riboflavin 30 mgcal grocery store, to media before autoclaving allows p-aminobenzoic acid 5 mgmedia types to be distinguished based on color rather Folic acid 5 mgthan by relying on stripes on the side of a plate or differ- Biotin 5 mgences in the plugs for slants to make these distinctions.

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For anyone who's studying, researching or even being an expert of Fusarium, this is the most detailed laboratory handbook. Not only describing Fusarium species in general, this book also explain briefly enough about Fusarium forma speciales Fusarium f. View 1 comment.

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About John F. John F.