Selenium volatilization by Mortierella species

Notes and brief articles Table

1.

177

Measurements of 40 conidia of Corynespora acaciae on two Acacia hosts (in pm)

Acacia pycnantha(HJS 72'02) No. of septa per conidium No. of conidia in category Average Range Acacia obliquinervia (HJS 81'07) No. or septa per conidium No. of conidia in category Average Range

1 1 14 x6 14 x 6

2 8 16'9 x 6'5 14-20 x 6-7

3 14 21' 1 X7'1 17-27 x 6-8

4 13 26'5 x 7'1 23-29 x 6-9

5 4 28'2 x 7'5 24-30 x 7-8

1 2 12'5 x 6'5 12-13 x 6-7

2 15 17·6 x 6'5 15-19 x 6-8

3 18 22·8 x 6·8 15-30 x 6-8

4 5 24 x 7'4 22-26 x 7-8

5 0

SELENIUM VOLATILIZATION BY MORTIERELLA SPECIES BY R. ZIEVE, P.

J. ANSELL,

T. W. K. YOUNG AND P.

J. PETERSON

Department of Biological Sciences, Chelsea College, University of London, Hortensia Road, London SWIO oQR Eight species of Mortierella were grown in pure culture and were shown to volatilize 75 selenium from inorganic selenite-supplemented media. No volatile selenium compounds were detected from control flasks of sterile medium or from cultures of M. roseonana. Concentrations of sodium selenite of 100 mg 1-' and above were inhibitory to growth and the colonies appeared pink/orange, indicating the deposition of elemental selenium. The role of micro-organisms in the methylation of inorganic selenium compounds was discovered by Challenger & North (1934), who found that Scopulariopsis brevicaulis (Sacc.) Bain. could convert both selenite and selenate to dimethylselenide, which has a garlic-like odour. The production of volatile selenium compounds by micro-organisms appears to be a phenomenon which is not restricted to a single group (Doran, 1982), although Fleming & Alexander (1972), Barkes & Fleming (1974), and Doran (1982) reported that fungal species predominate in this activity. In most previous investigations (Doran, 1982) high levels of selenite (460-2900 mg 1-') have been added to media from which selenium methylating organisms were isolated. Studies on selenium volatilization by microorganisms known to produce either volatile sulphur compounds or a garlic-like odour do not appear to have been reported. Most of the members of the Mortierellaceae, a family of Mucorales containing about 75 described species isolated from soils, have been reported to produce a garlic-like odour in culture, although the composition of the volatile substance(s) has not been determined. There are three subgenera of Mortierella; Micromucor, Mortierella (Garns, 1977) and Gamsiella (Benjamin, 1978), together with a few species which have not been assigned to any of the subgenera. A range of Mortierella species was investigated in order to determine their ability to volatilize added Trans. Br. mycol. Soc. 84 (1), (1985)

selenium and to establish whether or not this ability coincides with the garlic-like odour produced by control cultures. Fungi were obtained from either the Centraal Bureau voor Schimmeicultures, (CBS), Baarn, Netherlands or the Commonwealth Mycological Institute (CMI), Kew, U.K. The following species were examined. Subgenus Gamsiella: Mortierella multidivaricata Benjamin CBS 227.78. Subgenus Micromucor: Mortierella roseonanaGarris & Gleeson CBS 473 .74, M ortierella ramanniana var. angulispora (Naumov) Linnem. IMI 189782, Mortierella isabellina Oudem. IMI 192527. Subgenus Mortierella: Mortierella decipiens (Thaxter) Bjorling CBS 263.61, Mortierella stylospora Dixon-Stewart IMI 148605. Species not assigned to a subgenus: Mortierella chlamydospora Chesters CBS 529.75, Mortierella indohii Chien CBS 720.71, Mortierella indohii Chien CBS 460.75. Each species was grown on 2 % malt agar plates at room temperature, from which plugs of agar (8 mm") were used to inoculate 100 ml sterile malt extract-peptone broth (30 g 1-' malt extract, 5 g I-I mycological peptone, Oxoid reagents), contained in the 250 ml conical flasks described by Zieve & Peterson (1981). During incubation, the glass stoppers were replaced by sterile cotton wool plugs. Cultures were incubated for seven days on a rotary shaker at 100 rev min" at 22°C prior to the addition of 75S e-selenite. Each treatment was

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Notes and brief articles duplicated. At day 7, 30-60 {kg 1-\ selenium (unless otherwise stated) as sodium selenite or sodium selenate containing 25 {kCi 75selenium were added to each replicate flask. For studies of selenium volatilization, the cotton wool plug was removed from each flask and replaced with a glass stopper bearing an inlet tube, the side arm of the flask acting as an outlet. Air was flushed through the experimental flasks at approximately 5 ml min-i, and passed through a 10 ml concentrated nitric acid trap. Traps designed to fit a well-type crystal counter (Nuclear Enterprises PSR6) were assayed daily for radioactivity. The effect of selenium on radial growth rate was determined by placing a plug of inoculum (8 mm") in the centre of a 2 % malt agar plate containing the appropriate concentration of sodium selenite. All plates were duplicated and incubated at room temperaure for 10 days. Daily measurements of radial growth of the colonies, colony morphology and colour were noted. With the exception of M. roseonana, all species converted "selenium from 75selenite to the volatile form. M. roseonana, however, grew more slowly than the other species (Table 1) and it is possible that volatile selenium production was below the detection limit of 10- 5 {kg Se collected in the trap. The effect of sodium selenite on radial growth rate is shown in Table 1. Above 10 mg 1-\ sodium

Table 1. Effect of sodium selenite additions to media on radial growth rate (mm day:') Concn of sodium selenite (mg r ')

Species M. M. M. M.

0

roseonana

1'4

stylospora

4'0

chlamydospora indohii CBS 720.71

5'2 5'1

1'3 3'9 5'1 5'1

10

100

1000

1'3 3'9 5'1 5'1

1'1

0'0 1'3

3'0

4'7 2'1

0,8

selenite, the colony radial growth rate was reduced. Only the growth of M. roseonana was completely inhibited at 1 g 1-\. At a concentration of 0'1 g 1-\ a pink/orange colour was observed at the site of the inoculum of M. indohii 720.71, M. stylospora and M. roseonana, whereas at 1 g 1-\ the entire colony appeared pink/orange in all four species, indicating that these fungi can convert selenite to elemental selenium (Lapage & Bascomb, 1968). M. stylospora was tested for its capability to metabolize selenate, as well as selenite, to volatile selenium (Table 2). 75Selenium was evolved from cultures supplied with selenite or selenate, and for both forms of selenium the amount evolved was greater at 0'1 g 1-\ than at the lower concentration. More 75selenium was evolved from cultures supplied with selenite than with selenate. This result can be compared with those for Penicillium spp. where cultures grown in the presence of selenite released up to 16 times more volatile selenium than when grown in the presence of selenate (Barkes & Fleming, 1974). At 0'1 g 1-\ selenite or selenate the quantity of 75selenium evolved was of the same order of magnitude as that reported for species of Fusarium, Penicillium and Scopulariopsis (Barkes & Fleming, 1974). The results presented in this communication provide additional evidence that fungi are important in the selenium cycle by reducing inorganic forms to elemental and volatile forms, although the nature of the volatile compounds has not been defined. The results of tests with nitric acid and silver nitrate, however, are consistent with the conclusion that the compound evolved from selenite-enriched media is dimethylselenide, rather than hydrogen selenide. This conclusion is in agreement with recent work (Chocat et al., 1983) on the presence of seienocysteine lyase, a novel enzyme catalysing the conversion of selenocysteine into alanine and H 2 Se, in various bacteria, but no significant activity was found in yeasts and other fungi.

1'4 REFERENCES

Table 2. "rSelenium volatilization by M. stylospora cultures supplied with 75selenite or 75selenate over a z-day period Selenium addition to media (mg I-I)

pg of 75$e evolved

Selenite 0'06 100

Selenate 0'°3 100

Trans. Br. mycol. Soc. 84 (1), (1985)

BARKES, L. & FLEMING, R. W. (1974). Production of dimethylselenide gas from inorganic selenium by eleven soil fungi. Bulletin of Environmental Toxicology 12, 308-311.

BENJAMIN, R. K. (1978). Gamsiella, a new subgenus of Mortierella (Mucorales: Mortierellaceae). Aliso 9, 157-170. CHALLENGER, F. & NORTH, H. E. (1934). The production of organometalloidal compounds by micro-organisms. II. Dimethyl selenide. Journal of the Chemical Society 68-71. CHOCAT, P., ESAKI, N., NAKAMURA, T., TANAKA, H. & SODA, K. (1983). Microbial distribution of selenocysteine lyase. Journal of Bacteriology 156,455-457·

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Notes and brief articles DORAN, J. W. (1982). Micro-organisms and the biological cycling of selenium. In Advances in Microbial Ecology vol. 6 (ed. K. C. Marshall), pp. 1-32. New York: Plenum. FLEMING, R. W. & ALEXANDER, M. (1972). Dimethylselenide and dimethyitelluride formation by a strain of Penicillium. Applied Microbiology 24, 424-429. GAMS, w. (1977). A key to the species of Mortierella. Persoonia 9, 381-391.

179

LAPAGE, S. P. & BASCOMB, S. (1968). Use of selenite reduction in bacterial classification. Journal of Applied Bacteriology 31, 568-580. ZIEVE, R. & PETERSON, P. J. (1981). Factors influencing the volatilization of selenium from soil. Science of the Total Environment 19, 277-284.

EFFECT OF STILL AND MOVING MOISTURE-SATURATED AIR ON SPORULATION OF DRECHSLERA AND PERONOSPORA BY C. M. LEACH

Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97330, U.S.A Drechslera turcica and Peronospora destructor sporulated in profusion on infected leaves incubated at constant temperature in moisture-saturated air that was still, but not when saturated air was in motion at velocities from 0'3 to 1"5 ta]«. Atmospheric humidity is a limiting factor in the development offoliar plant diseases caused by fungi (Colhoun, 1973). It affects sporulation, spore discharge and spore germination. Although the importance of humidity in the development ofplant diseases has been recognized for more than a century, the fundamental nature of its effect on sporulation is not fully understood (Griffin, 1982; Hawker, 1957). Most foliar pathogens require humidities near saturation to sporulate (Colhoun, 1973), including the two fungi selected for this study, Drechslera turcica (Pass.) Subram. & Jain, the cause of Northern Leaf Blight of maize and Peronospora destructor (Berk.) Casp., the cause of downy mildew of onions. D. turcica produces conidia only at relative humidities (r.h.) above 86%, and maximum sporulation is observed at 92 % and higher (Leach, Fullerton & Young, 1977); P. destructor behaves similarly (Yarwood, 1943) and produces sporangia only at r.h. 90 % and higher. Why do D. turcica and P. destructor require high humidity to sporulate? A possible clue was encountered during an investigation of the mechanism of active spore discharge by foliar pathogens. It was discovered that the surfaces of leaves detached from naturally grown plants were elec- . trically charged under certain conditions of humidity and light (Leach & Apple, 1984); field intensities were generally in the 0-500 V cm' range. When detached leaves were placed in humidity chambers at r.h. 100%, they became charged but only when the air was still, not if the saturated air was moved at velocities of 0'3 mls and higher (Leach, 1984). Electrophysiological procesTrans. Br. mycol. Soc. 84 (1), (1985)

ses have been implicated in plant, animal (Jaffe & Nuccicitelli, 1977) and fungal development (Harold, 1977; Stump et al., 1980) and perhaps sporulation of foliar fungi may also involve electrophysiological events. Ifleaf surface electrical fields are important in sporulation, then their reduction or elimination may reduce or prevent sporulation. To test this possibility, experiments were conducted to determine whether moisturesaturated air in motion would reduce or prevent sporulation of D. turcica and P. destructor growing on infected leaves. The effect of moving saturated air on sporulation was studied in two ways. One apparatus (Figs 1,2) was constructed for use with leaves of maize infected with D. turcica and the other (Fig. 3) for onion leaves systemically infected withP. destructor. All experiments were performed in a constanttemperature incubator in darkness. Air velocities over specimens were measured with an anemometer (type 8500, 'thermo-anemometer', Alnor Instrument Co., Niles, Illinois, U.S.A.). Air velocity was controlled in the air circulation chamber (Fig. 1) by means of a small fan located within the flexible tubing. The fan speed was adjusted with a rheostat. Air speeds in the wind tunnel (Fig. 3) were controlled by a regulator valve in a compressed-air supply line. Temperature was monitored continuously in all experiments with thermocouples and a recorder; humidity was also monitored continuously in all experiments (HM 111 Relative Humidity Indicator, Weathermeasure, P.O. Box 41257, Sacramento, CA 95841, U.S.A.). The possibility that air temperature within the air circulation chamber (Fig. 1) might be influenced by

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