Fungus Glomus fistulosum

Folia Microbiol. 42 (5), 489-494 (1997)

An Improved Procedure for Root Surface Disinfection Suitable for Observations of Proliferation of Intraradical Hyphae of Arbuscular Mycorrhizal Fungus Glomus fistulosum M. GRYNDLER,H. HP~ELOV,6,and I. CHVJ~TALOV,6, Institute of Microbiology, Academy of Sciences of the Czech Republic, 142 20 Prague Received February 10, 1997 Revised version May 12, 1997

ABSTRACT. A disinfection procedure for mycorrhizal root segment surface was improved using neomycin and polymyxin B besides sodium hypochlorite, streptomycin and penicillin G, to obtain clean material for observation of proliferation of hyphae of arbuscular mycorrhizal (AM) fungi. The procedure of surface disinfection is more efficient, in terms of incidence of contamination, compared to the original procedure involving only sodium hytx)chlorite, streptomycin and penicillin G. A further decrease of visually detectable contamination can be achieved using bacteriostatic concentrations of rolitetracycline which cause a decrease of hyphal growth of the AM fungus Giomus fistulosum. The technique was used in experiment studying the uptake of U-14C-glucose by proliferating hyphae. An active uptake of glucose occurred even in the presence of all four antibioties at concentrations of 500 mg/L.

Plant root surface disinfection is used to obtain clean mycorrhizal root segments which serve either as saprophyte-free material for further observation of the mycorrhizal fungus (Williams 1990; Gryndler and Vos~itka 1996; Gryndler et al. 1996) or as a starting inoculum for its monoxenic culture on the host plant in vitro (Strullu and Romand 1986). Short ( 1 - 3 mm in length) root segments are used for this purpose. The surface disinfection procedure includes a treatment with a nonselective disinfectant (e.g. sodium hypochlorite) and an antibiotic bath containing either 200 mg/L streptomycin (Strullu and Romand 1986) or a mixture of streptomycin and penicillin G, each at a concentration of 500 mg/L (Williams 1990). After the procedure of disinfection has been finished, the root segments are washed and transferred into an incubation medium suitable for growth of the mycorrhizal fungus. Unfortunately, the yield of visibly noncontaminated root segments obtained as a result of such disinfection procedure was not satisfactory (Gryndler and Vos~tka 1996). Penicillin G (benzylpenicillin) is almost ineffective against many Gram-negative bacterial strains (Iskhakova 1988) as well as against some Gram-positive microbes (Levina et al. 1985). A combination of penicillin G with streptomycin, also often ineffective against Gram-negative bacteria (MarLs 1991), leaves many bacterial strains unaffected. This observation induced us to include other antibiotics into the disinfection procedure. Neomycin, combined, e.g., with polymyxins, is a compound which is successfully used as a wide range antibiotic in medicine for treatment of patients with severe digestive tract infections (Van-der-Vaaij et al. 1986). It has been reported that neomycin is reversibly bound to physical surfaces, both microbial and non-microbial (Hazenberg et al. 1986), which might increase its effectivity in the surface decontamination procedure. Polymyxin B is usually effective against Gram-negative bacteria (Freche and Acar 1988). We tested the decontamination effects of these two antibiotics and the effect of rolitetracydine as a bacteriostatic component of the incubation medium.

MATERIALS AND METHODS

Source ofmycorrhizal roots. Maize plants were grown in a hydroponic culture based on Perlitesteamed soil (5 : 1) substratum. Pregerminated seeds were surface disinfected with 1 % hydrogen peroxide for 2 rain before planting. The plants were inoculated with 10 g of a soil inoculum (20 mm below the seed) containing 400 spores of Glomus fistulosum SKOU et JAKOBSEN (isolate BEG 23). The plants were cultivated in vertically oriented plastic tubes (180 mm in height and 50 mm in diameter) covered with canvas at the bottom to keep the substratum inside. Each tube was then put in a separate black vessel and supplied with distilled water for the first two weeks of growth. After this two-week period,

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400 mL of a P2N3 mineral nutrient solution (Gryndler et al. 1992) was added to each vessel and renewed once a week. The plants were harvested after 4 weeks. Surface disinfection and procedure. After cutting off the shoots, the root systems were washed with lukewarm tap water and yellow coloured roots -- mycorrhizae -- were carefully selected. In all experiments, the selected roots were shaken 5 times with sterile distilled water for 5 rain to remove any retained particles of hydroponic substratum and subsequently kept for 4 h in a volume of 100 mL of a solution containing i drop of Tween 80 (Lachema, Czech Republic) and the antibiotics (cf. below). The roots were then immersed in a 1 : 50 solution of sodium hypochlorite (5 % available chlorine, Sigma) containing 1 drop of Tween 80 for 4 rain and washed with 500 mL of distilled water. The washed roots were transferred to deionized water and cut with sterile surgical scissors into segments 1 - 2 mm long. Incubation of root segments. Each root segment was placed in a 30 ~tL drop of a freshly prepared, filter sterilized incubation medium. The incubation medium contained in all cases a buffering mixture consisting of a weak base and a weak acid; 1 mmol/L BIS-TRIS (bis[2-hydroxyethyl]iminotris[hydroxymethyl]methane, Sigma) and 0.9 mmol/L MES (2-[N-morpholino]ethanesulfonie acid, Sigma), pH 6.3. The drops of the incubation medium were put on the inside bottom of the cover of a polystyrene Petri dish. The dishes, each containing 16 drops of the medium, were incubated in the dark in a humid chamber for 5 d at 25 *C. Quantification of hyphal growth. After the incubation, the root segments placed in the drops of the incubation medium were observed under a microscope (ma,tnaification 63• with an eyepiece equipped with a grid net focal plate. Except for Experiment 1, only the noncontaminated (visually) root segments were taken into account. The intersections of grid net lines with mycorrhizal mycelium were counted as a measure of hyphal growth. Sometimes the position of a root segment in the drop of the medium prevented the estimation because of the "dark-field-effect", when some parts of the microscope's visual field were darkened by optical contours of the drop. In this case, thin cover glass was put from the bottom to the pending drop of the medium, which eliminated the curved surface of the drop and its undesirable optical effects. The data were expressed as a mean total amount of intersections per root segment. One intersection corresponded to a hyphal length of 59 Ixm.

Testing the continuity of growing hyphae with intraradical structures of arbuscular mycorrhizal lung/. Root segments showing the proliferation of coarse aseptate hyphae from cutting faces were cleaned with 10 % potassium hydroxide at 90 *C for 30 rain, washed, acidified with 2 % lactic acid, and stained in 0.1% Trypan blue in lactophenol for 15 rain at 90 ~ The stained segments were washed with water, put in glycerol and examined under a microscope. Effect of treatments with antibiotics and detection of contamination. Mycorrhizal roots were exposed to antibiotic baths containing: (i) 500 mg/L streptomycin and 500 mg/L penicillin G (bath no. 1), (ii) 500 mg/L streptomycin, 500 mg/L penicillin G, 50 mg/L polymyxin B and 50 mg/L neomycin (bath no. 2) or (iii) 500 mg/L streptomycin, 500 mg/L penicillin G, 500 mg/L polymyxin B and 500 mg/L neomycin (bath no. 3). All three baths also contained Tween 80 (one drop per 100 mL). A part of root segment surface disinfected in bath no. 1 was incubated in a medium containing rolitetraeydine or neomycin at concentrations of 5 mg/L (description see above). All chemicals used were purchased from Sigma. Five parallel Petri dishes were used per treatment. Contaminated root segments were detected using three different methods. The first method was visual, observing the incubated Petri dishes under a light microscope (matmification 63x), the other two methods consisted in microbiological tests where a solid nutrient medium in Petri dishes was inoculated with a small amount of the incubation medium separately from each drop, using autoclaved wooden toothpicks. The second and the third methods used 0.2 % yeast extract (Oxoid) in water and medium T3 (Taylor 1951) respectively;, both media were solidified with 1.5 % agar. Bacterial growth on the solid media was estimated after 7 d of incubation at 28 ~ Assessment of U-14C-glucose uptake by proliferating hyphae in the presence of antibiotics. Roots were surface-disinfected using bath no. 3 and incubated in a medium (see above) supplied with 0.1 mmol/L phosphate. Twenty Petri dishes were used. After estimating the proliferation of hyphae and excluding the contaminated root segments, forty noncontaminated root segments showing growing hyphae of AM fungus were chosen for the next procedure. Ten segments were transferred to drops of the fresh incubation medium in a Petri dish and exposed to formaldehyde vapors in a sealed vessel for 30 rain. The root segments (either exposed or nonexposed to formaldehyde) were then transferred to a filter-sterilized incubation medium (see above) also containing 500 mg/L streptomycin, 500 mg/L penicillin G, 500 mg/L polymyxin B, 500 mg/L neomycin and 0.49 MBq/mL U-14C-glucose (Amersham, specific activity 10.8 GBq/mmol). The root segments exposed to formaldehyde vapors