Erlenmeyer flasks

Some of the first attempts at synthesizing ectomycorrhiza under sterile conditions were made by Melin (1921, 1922) using Erlenmeyer flasks with sand as a substrate for addition of nutrient solution and for root growth. Subsequently, Hacskaylo (1953) used vermiculite while Marx and Zak (1965) used finely ground peat moss mixed with vermiculite in a ratio of 1:15 as the substrate. The substrates provided better aeration for the root system and the addition of peat lowered the pH. Chakra-varty and Unestam (1987a,b) achieved excellent ectomycorrhiza formation with Pinus sylvestris using "Leca" brick pellets as the substrate. Recently, Chakravarty et al. (1990) have used Promix Bx, a commercial mix of vermiculite-peat moss-perlite (Les Tourbières Premier Ltd, Riviere-du-Loup, Quebec, Canada) as the substrate.

In setting up the system, any size of flask can be used and the amount of substrate and nutrient solution added can be adjusted accordingly. In earlier experiments (Marx and Zak, 1965), 2-litre flasks containing a ratio of 840 ml vermiculite: 60 ml ground peat moss moistened with 550 ml nutrient solution were used. In later experiments (Chakravarty et al., 1990), 250ml flasks containing 100ml Promix Bx moistened with 90 ml nutrient solution were used for 8-week experiments using Pinus resinosa Ait. seedlings.

Flasks of any size containing substrate and nutrient solution are plugged either with foam or absorbent cotton and capped with a small beaker or aluminium foil (see Fig. 1) before autoclaving at 120 °C, 1 atm, for at least 15 min.

After the substrate has cooled, one or more aseptic seeds or seedlings are introduced into each flask under sterile conditions. Fungal inoculum can be added as plugs of actively growing mycelium or as a slurry of fungal hyphae at this time or after seedling establishment, depending on the objective of the experiment.

1. Advantages

This method is very easy to set up so that a large number of replicates can be used. Sterility is easily maintained during all phases of establishing this culture system and, once set up, maintenance requires little effort. Root development and ectomycorrhiza formation are excellent in a substrate such as vermiculite-peat moss or Promix Bx that allows good aeration and a buffered acidic environment conducive to growth of conifer seedlings. This system has been used very effectively in studying interactions between ectomycorrhizal fungi and root pathogens.

Foam plug

Aluminium foil

Plant Root Airation System

Erlenmeyer flask

Substrate (Peat/vermiculite or Promix Bx)

Fig. 1. Erlenmeyer flask with substrate and sterile seedling.

Erlenmeyer flask

Substrate (Peat/vermiculite or Promix Bx)

Fig. 1. Erlenmeyer flask with substrate and sterile seedling.

2. Disadvantages

Probably the biggest disadvantage of this system is the artificial environment imposed on the shoot system because, in a closed system, C02 might be limiting and ethylene might be expected to accumulate. Similarly, root exudates would accumulate in the substrate. This system is also not suitable for developmental studies of ectomycorrhiza and the extramatrical mycelium, because the root system is difficult to monitor without sacrificing the seedling. The flasks also occupy a considerable surface area of a growth chamber.

B. Mason jars

Trappe (1967), realizing the potential problems in growing conifer seedlings in enclosed chambers, devised a method to keep the root system sterile yet allowing the shoot system to grow into the environment. The assembly (Fig. 2) consists of a wide-mouth quart mason jar (canning jar) filled with perlite sieved to include particles 1-8 mm diameter. Nutrient solution is added to the perlite to moisten it and to leave about 5 cm depth of excess solution at the bottom. A steel vent tube (2 cm diameter, 3 cm long) is inserted into the perlite so that the top of the tube is flush with the lip of the jar. Perlite is removed from the vent tube before the jar top is covered with lightweight aluminium foil. A hole is poked through the foil at the site of the vent tube and the tube sealed to the foil by rubber cement (sterile lanolin could be used after autoclaving the assembly). The foil is folded over the outside of the jar's lip and held in place by a mason jar ring of appropriate size screwed down tightly over it. The vent tube is plugged with absorbent cotton, a glass Petri plate is placed over the top of the jar, and the whole assembly is autoclaved.

After cooling, the Petri plate is removed and a small hole poked aseptically into the foil and into the perlite near the rim of the jar. A sterile seedling radicle is placed in the hole and the hypocotyl is sealed into the chamber. Although Trappe (1967) used rubber cement to seal the seedling, some necrosis occurred. Sterile lanolin should probably be used. Jars are wrapped in foil and placed in growth chambers for seedling growth.

A later modification of the system involved lining the jar with paper towelling to improve capillary movement of nutrient solution.

Seedlings are inoculated with test fungi by inserting the needle of a syringe containing a slurry of fungal mycelium through the foil and injecting the slurry into the perlite adjacent to the root system.

Genes Matraces Erlenmeyer

1. Advantages

The main advantages of this system are that the shoot system grows under ambient conditions, roots growing against the glass can be observed conveniently, and the fungus can be distributed to a large portion of the root system.

2. Disadvantages

These assemblies take some effort to set up, particularly when adding the vent system. The perlite substrate has poor capillarity and some fungal species do not recover very well after fragmentation. A simpler system involves using a mason jar filled with either peat moss-vermicu-lite or Promix Bx moistened with nutrients, leaving out the vent tube, and using the lid assembly of the jar with a hole punched in it (see Fig. 3). The top of the assembly can be covered with aluminium foil during

Plants Erlenmeyer

Sterile lanolin Lid of Mason jar

Peat/vermlcullte or Promix Bx

Mason jar

Sterile lanolin Lid of Mason jar

Peat/vermlcullte or Promix Bx

Mason jar

Fig. 3. Simpler mason jar method.

autoclaving which can be removed prior to inserting the seedling into the hole and sealing it in with autoclaved lanolin.

C. Leonard jars

Mullette (1976) adapted a method first used by Vincent (1970) in studying nodulation to synthesize ectomycorrhiza between Eucalyptus gummifera (Gaertn. & Hochr.) and Pisolithus tinctorius (Pers.) Coker & Couch under different phosphorus concentrations. The apparatus, illustrated in Fig. 4, consists of a pyrex jar containing nutrient solution into which an inverted bottle with its base removed and its neck stoppered with absorbent cotton is inserted. The inverted bottle contains crushed quartz (quartz sand) or soil overlain by coarse quartz. The entire assembly can be covered with aluminium foil and autoclaved. After cooling, the aluminium foil is removed from the top, a sterile seedling is placed in the quartz, and fungal inoculum placed in the quartz. Mullette

Leonard Jar Assembly

(1976) used crushed fruit bodies of Pisolithus tinctorius as inoculum. The cotton acts as a wick to provide nutrients to the developing root system.

1. Advantages

Mullette (1976) claims that it is easy to retain sterility during long-term experiments and since a quantity (e.g. 1 litre) of nutrient solution can be used there is very little maintenance of the system. Another advantage is that the shoot system is exposed to ambient conditions.

2. Disadvantages

The main disadvantages of the system are the glassware required, the time involved to set up the system and the space required to maintain a suitable number of replicates for experiments. Sterility may be a problem during long-term experiments.

D. Test tubes

Pachlewska (1968) and Pachlewski and Pachlewska (1974) used test tubes containing water agar plus thiamine but without minerals to synthesize ectomycorrhiza between Pinus sylvestris and fungal species. This method was modified by Mason (1975, 1980) by adding minerals and glucose in addition to thiamine to the agar medium. Molina (1979, 1981) used 300 x 38 mm glass test tubes containing vermiculite and peat moistened with nutrient solution (see Fig. 5) to test a wide range of ectomycorrhizal fungi on Alnus species. He reported excellent seedling growth under these conditions. Promix Bx could be used instead of vermiculite-peat. Sohn (1981) used a mixture of quartz sand and ion-exchange resins in a similar setup.

-Aluminium foil

_Vermiculite/peat as substrate w

Fig. 5. Test tube system using vermiculite-peat as substrate. After Molina

Sylvia and Sinclair (1983a) designed a wick-culture system to test the effects of Laccaria laccata (Scop.:Fr.) Berk & Br. on Pseudotsuga menziesii (Mirb.) Franco (Douglas fir) seedling growth. This system was used subsequently (Sylvia and Sinclair, 1983b) to study the interaction between L. laccata and root pathogens, again with Douglas fir. The wick-culture system (Fig. 6) is constructed by lining a 200 x 32 mm test tube with a layer of cellophane, Whatman No. 3 chromatography paper and polypropylene in that order. The chromatography paper acts as a wick to keep the root system moist while the cellophane and polypropylene allow the root system to be removed from the tube without damage. A stainless steel rod is placed between the cellophane and chromatography paper for subsequent insertion of the seedling radicle

Plants Erlenmeyer

and for adding the slurry of fungal hyphae. Nutrient solution (40 ml) is added, the tube plugged with a foam plug and the assembly autoclaved. A sterile seedling is added between the foam plug and the wall of the test tube so that the shoot system is exserted into the environment. The shoot system is enclosed within a plastic bag for 2 weeks to prevent desiccation.

Yang and Wilcox (1984) used an apparatus (Fig. 7) combining features of the simple test tube system used by Molina (1979, 1981) with some aspects of the system designed by Sylvia and Sinclair (1983a). These authors lined test tubes with chromatography paper and then

Erlenmeyer Bouchon

Absorbent ration Cork stopper

Inoculum

C h mm atoftrap hy paper

Glass tubing [6 mm)

Vermieulite/Peal moss

Absorbent ration Cork stopper

Inoculum

C h mm atoftrap hy paper

Glass tubing [6 mm)

Vermieulite/Peal moss

Fig. 7. Test tube system combining features shown in Figs 5 and 6. After Yang and Wilcox (1984).

filled the tube with a vermiculite-peat mixture moistened with sterile water. Tubes were plugged with a cork stopper in which a bent glass tube plugged with cotton was inserted for aeration. Another hole bored into the cork near the rim of the tube was plugged with cotton during autoclaving. After autoclaving, the cotton was removed from the latter hole and a sterile seedling was inserted so that the radicle was between the test tube wall and the chromatography paper. Nutrient solution and

0.5. glucose was added at this time, as were two agar plugs of mycelium, one at the base of the seedling and the other in the vermiculite-peat mixture. Although not mentioned in the description, the photograph of the system indicates that parafilm was wrapped around the base of the cork.

Duchesne et al. (1988a) devised a simple test tube system to study the short-term effects of the ectomycorrhiza fungus, Paxillus involutus Fr,. on the root-rot organism Fusarium oxysporum in combination with Pinus resinosa Ait. seedlings. The apparatus (Fig. 8) consists of 1.5 x 18 cm test tubes lined either completely or partially with Whatman No. 2 chromatography paper, a foam plug and either a plastic cap or aluminium foil. After autoclaving, 5 ml of sterile nutrient solution is added to each tube and a seedling placed between the filter paper and test tube wall. This apparatus was very useful for studying the effect of root exudates on anti-fungal compound secretion by P. involutus (Duchesne et al., 1988b) and for structural studies (Farquhar and Peterson, 1989).

1. Advantages

The test tube systems in which the shoot system is allowed to grow into the environment have the advantage that shoots are not stressed by high levels of accumulated ethylene or low levels of CO2. The test tubes can be maintained at constant temperature by immersing them in a water bath. All the test tube systems have the advantage that a large number of samples can be run for any experiment because of the small amount of space required and the root systems can be monitored as they grow against the test tube wall. The systems employing chromatography paper wicks are excellent for studies on root exudates and interactions between ectomycorrhizal fungi and pathogenic organisms.

2. Disadvantages

The test tube systems in which the shoot is enclosed suffer, like all similar apparatus with enclosed shoots, in that C02 may be limiting to

Agar Flask
al. (1988a).

growth and ethylene might accumulate. All of the systems have the potential for the accumulation of phytotoxic substances in the nutrient medium.

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