THE UPTAKE OF PARAQUAT BY SOIL FUNGI

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1 New Phytol. (1976) 76, THE UPTAKE OF PARAQUAT BY SOIL FUNGI BY S. N. SMITH AND A. J. E. LYON Department of Botany, Sheffield {Received 21 November 1975) SUMMARY The mucoraceous fungi, Mucor hiemalis and Zygorhynchus heterogamous, are considerably more susceptible to paraquat than the imperfect species, Aspergillus niger and Penicillium frequentans. The inhibitory effect of increasing levels of paraquat on hyphal extension, dry matter production and spore germination are much more marked in Mucor and Zygorhynchus. When grown on media containing paraquat, the mycelia of these species contain approximately twice as much of the herbicide as those oi Aspergillus or Penicillium. Feeding (^'*C-methyl)-paraquat confirms that twice as much activity is accumulated by the susceptible species. It is suggested that susceptibility to paraquat is associated with the degree to which the herbicide accumulates in the mycelium. INTRODUCTION The herbicide, paraquat dichloride (i,i'-dimethyl-4,4'-bipyridylium), has a marked effect on the growth rate (Wilkinson and Lucas, 1969a), respiration rate (Wilkinson and Lucas, 1969b), sporulation (Gareth Jones and Williams, 1971) and spore germination (Wilkinson and Lucas, 1969c; S. N. Smith, unpublished) of certain fungi. Wilkinson and Lucas (i969a,b) have shown that fungal species differ in their susceptibility to paraquat dichloride. The experiments described here examined the physiological basis for such differences. Preliminary experiments had indicated that Penicillium frequentans and Aspergillus niger were tolerant of comparatively high concentrations of paraquat dichloride in their substrate, whereas Mucor hiemalis and Zygorhynchus heterogamous were susceptible to much lower levels of the herbicide. These four species were therefore selected for this study. MATERIALS AND METHODS,,. Species. Aspergillus niger Van Tiegh., Penicillium frequentans Wes., Mucor hiemalis Wehmer, Zygorhynchus heterogamous Vuillcmin, isolated from soil obtained from Tapton Gardens, University of Sheffield, were maintained on Oxoid malt extract agar. Paraquat. Pure paraquat dichloride for calibration of the assay was donated by M. J. Edwards, Jealotts Hill Research Station, Imperial Chemical Industries (I.C.I.) Ltd. The commercial formulation of paraquat, 'Gramoxone W', (I.C.I. Ltd.) was used in fungal growth media. C^C-Mcthyl) paraquat dichloride was obtained from the Radiochemical Centre, Amersham. Other chemicals. Hyflo Super Cel was obtained from Koch Light Ltd.; Zerolit 225 cation exchange resin mesh size' from B.D.H. and Lissapol NDB from LCI. Ltd

2 480 S. N. SMITH AND A. J. E. LYON Determination of growth rate. Measurement of growth rate was by radial extension and colony dry weight. To measure radial extension, plug cultures were inoculated on to plates of Oxoid malt extract agar containing different concentrations of paraquat up to 2000 mg/1. The colony diameters were measured along two axes at right angles to each other. To measure colony dry weight, spore suspensions were inoculated into ioo-ml Erlenmeyer flasks, each containing 20 ml of 2% malt extract medium with an appropriate concentration of paraquat. In this case, the maximum concentration of paraquat used was 500 mg/1, since, above this, spores failed to germinate. The cultures were shaken at room temperature for 7 days, harvested, dried overnight at 90 C and weighed. Spore germination. The mycelium from which the spores were derived was grown on malt extract agar + an appropriate concentration of paraquat. Mucor hiemalis and Zygorhynchus heterogamus were grown on media containing 500 mg paraquat/1; Aspergillus niger and Penicillium frequentans on media containing 1000 nig/1. After 2 weeks at laboratory temperature, the spores were removed by washing with sterile water. The concentration of spores in the suspension was determined with a haemocytometer and adjusted to a uniform level with sterile distilled water. Aliquots of spore suspension were then plated on to malt extract agar with different concentrations of paraquat and incubated at laboratory temperature. The percentage of spores showing a distinct germ tube was determined 18 and 36 h later. Preparation and extraction of mycelium. The mycelial tissue for extraction was grown in a modified 2-I Penicillin flask with continuous bubbling of sterile air through the medium. Each vessel contained i 1 of autoclaved (15 p.s.i. for 20 min) medium consisting of 2% malt extract, i % yeast extract and paraquat to a concentration of 50 mg/1. It was inoculated with a spore suspension of the appropriate species and incubated at room temperature for 7 days. The mycelial mat was then removed by flltration using a Buchner funnel, washed with 250 ml of distilled water and divided into 5 g wet weight portions. Each was suspended in 15 ml of distilled water and homogenized for i min using a 'Polytron' ultrasonic homogenizer, the blades of which were theti washed with a further 10 ml of distilled water. These were added to the homogenate in an extraction vessel and made up to 35 ml with distilled water. Paraquat assay procedure. The method follows that of Calderbank and Yuen (1964) with minor modifications to accommodate the smaller volumes involved. The homogenate was refluxed for 5 h with 5 ml of 18 N sulphuric acid. After refluxing, 10 ml of distilled water was added through the condenser before removal of the extraction vessel. The homogenate was filtered through a layer of 'Hyflo' Super Cel on a Buchner funnel and washed with two ioo-ml portions of distilled water. The resulting filtrate was transferred to a separating funnel and passed through a column of Zerolit 225 cation exchange resin prepared by the method outlined by Calderbank and Yuen (1964). The paraquat residue was recovered by washing with saturated ammonium chloride solution. The resulting paraquat was reduced by sodium dithionite to give a blue colour the optical density of which was determined spectrophotometrically at 600 nm. The extraction procedure was calibrated by addition of a known amount of pure paraquat to mycelium grown in the absence of the herbicide to assess for losses during extraction. Uptake of {^'^C-Methyl) paraqitat. Each fungus was inoculated into 20 ml of sterile medium consisting of 2% malt extract, 0.5% yeast extract and 50 mg/1 pure paraquat dichloride, containing 0.5 ^Ci (^""'C-methyl) paraquat dichloride (i.i x io*^ dpm). The flasks also contained different concentrations of the wetting agent, Lissapol NDB. Cultures were incubated on a shaker for 5 days at room temperature. The mycelia were

3 uptake of paraquat by fungi 481 removed from the flasks by Buchner filtration using a hardened filter paper and washed with distilled water. Each mycelium was placed in a Marshall vial with ro ml of distilled water and homogenized for i min with the 'Polytron'. A I-ml aliquot of the homogenate was transferred to an oven-dried weighed planchet. The planchets and homogenate were dried in a 90 C oven overnight, allowed to cool and reweighed. The amount of activity on each planchet was determined with a Nuclear Chicago planchet counter and corrected for self-absorbtion. RESULTS Growth rate Fig. I shows the effect of different concentrations of paraquat on hyphal extension rate. When grown on media devoid of paraquat, the two mucoraceous fungi, Zygorhynchus heterogamous and Mucor hiemalis, have much higher linear extension rates than the imperfect fungi, Penicillium frequentans and Aspergillus niger. Increasing concentra Paraquat concentrations (mg/t) iraquat concenfrations imq/i) Fig- I Fig. 2 Fig. I. The effeet of paraquat on the linear extension rate of four speeies of fungi. A, Zygorhynchus heterogamous; A, Mucor hiemalis;, Penicillium frequentans; O, Aspergiltus niger. Fig. 2. The effect of paraquat on dry matter produetion by four speeies of fungi. A, Zygorhynchus heterogamous; A, Mucor hiemalis;, Penicillium frequentans; O, Aspergillus niger (±95% eonfidence limits). 500 tions of paraquat up to 2000 mg/1 have virtually no effect on the extension rates of the two imperfect fungi, whereas the growth rate of Zygorhynchus and Mucor is dramatically reduced with increasing herbicide levels such that they fail to extend at 2000 mg/1. In contrast to their differing extension rates, all four species have similar rates of increase in mycelial dry matter when grown in the absence of paraquat (Fig. 2). However, increasing levels of paraquat reduce the growth rates of Mucor and Zygorhynchus much more than those of Penicillium and Aspergillus. Dry matter production by Mucor and Zygorhynchus is reduced by 50% by a paraquat level of 130 mg/1, whereas the same

4 482 S. N. SMITH AND A. J. E. LYON ir (a) looir (c) (d) g Paraquat concentrations (mg/0 _L I _L Paraquat concentrotions (mg/1) Fig. 3. The effect of paraquat on the germination of spores of Mucor hiemalis (a), Zygorhynchus heterogamous (b), Penicillium frequentans (c) and Aspergillus niger (d). n. Spores from mycelium grown in the absence of paraquat sampled after 18 h; O, spores from mycelium grown on 500 mg/i paraquat sampled after 18 h;, spores from mycelium grown in the absence of paraquat sampled after 36 h; -, spores from mycelium growh on 500 mg/1 paraquat sampled after 36 h (For explanation of limits, see text) ;. - ' " :., j.! : '.. ''' "! :; '"" Lissapol NDB concentration (mg/1) Fig. 4. Levels of '*C in mycelia of four fungi fed with C'C-methyl) paraquat in the presence of different concentrations of Lissapol NDB. A, Zygorhynchus heterogamous; A, Mucor hiemalis;, Penicillium frequentans; O, Aspergillus niger (±95% confidence limits).

5 uptake of paraquat by fungi 483 reduction in Penicillium and Aspergillus is not achieved until the concentration reaches 270 mg/1 and 450 mg/1 respectively. The mucoraceous fungi fail to grow at a concentration of 250 mg/1, whereas the imperfect species show appreciable growth at 500 mg/1. Spore germination Fig. 3 represents the percentage germination of spores of each species studied at each concentration of paraquat. These data were subjected to an angular transformation (Snedecor and Cochran, 1967) to permit the calculation of 95% confidence limits. The limits were then retransformed to percentages and plotted against their respective points. The response of all species show reduced spore germination with increasing herbicide concentrations. This is more marked for Mucor hiemalis and Zygorhynchus heterogamous. Spores derived from colonies grown on agar containing paraquat germinate more readily in the presence of paraquat than those derived from colonies on agar alone. However, when spores are plated on to agar without paraquat, those from colonies grown in the absence of the herbicide germinate more readily than those from colonies grown in its presence. Uptake of paraquat Table i shows the levels of paraquat found in the mycelia of the four species after incubation for 7 days in a medium containing the herbicide. Colonies of Zygorhynchus and Mucor contain approximately twice as much paraquat as those of Penicillium and Table i. Levels of paraquat in mycelia grown for 7 days in medium containing 50 mg paraquat/i Paraquat level ' '.,, :.. Species (mg/g fresh wt of mycelium±95% confidence limits) Aspergillus niger ± 5.37 Penicillium freguentans, Zygorhynchus heterogamous _ Mucor hiemalis Aspergillus. This difference in behaviour is confirmed by the results of an experiment in which colonies of the four fungi were incubated with (^"^C-methyl) paraquat for 5 days in the presence of different levels of the wetting agent Lissapol NDB (Fig. 4). Colonies of Mucor and Zygorhynchus contain considerably higher levels of activity than those of Penicillium and Aspergillus. At low concentrations of Lissapol NDB, the apparent rise in the level of activity is not significant. DISCUSSION Our results confirm those of previous workers who have found that paraquat has an inhibitory effect on mycelial growth and activity. We have found that Mucor hiemalis and Zygorhynchus heterogamous are considerably more susceptible to paraquat than Penicillium f'requentans and Aspergillus niger. Mucor and Zygorhynchus show a reduction in both hyphal elongation and dry matter production with increasing concentrations of paraquat. Colonies of these species ceased to extend at 2000 mg/1 of paraquat, whereas no increase in dry weight was found above 200 mg/1. This difference is due partly to the greater susceptibility to paraquat of the spore inocula used in the dry weight determinations compared with mycelial disc inocula..

6 484 S. N. SMITH AND A. J. E. LYON By contrast, paraquat has a much lower inhibitory effect on the rate of increase of dry weight of Aspergillus and Penicillium. These species grew at concentrations in excess of 500 mg/1. Their hyphal extension rate was unaffected by paraquat up to 2000 mg/1. These results are of particular significance when compared with those of Tu and Bollen (1968), who showed an increase in the proportion of colonies of Penicillia and a decrease in those of the Mucorales when Gramoxone was applied under field conditions. These differences between the mucoraceous and imperfect fungi are consistent with the behaviour of their germinating spores. Spores of Mucor and Zygorhynchus fail to germinate at much lower concentrations than those of Aspergillus and Penicillium. In addition our results indicate that mycelium grown on medium containing paraquat yields spores which have a higher germination rate in the presence of the herbicide. However, when such spores are allowed to germinate in the absence of paraquat they do so less readily than spores from mycelium grown on medium devoid of the herbicide. It therefore seems likely that paraquat-grown spores show some degree of 'adaptation' to growth in the presence of the herbicide. It must be emphasized that these results have been obtained after only 2 weeks' exposure of the spore-producing mycelium to paraquat. Our unpublished data indicate that repeated subculturing on paraquat-containing medium eventually results in the production of spores which show greatly reduced viability on both paraquat and control media. These differing susceptibilities of mucoraceous and imperfect fungi may be partially explained by their abilities to absorb paraquat. Colonies of Mucor and Zygorhynchus grown on paraquat medium contain twice as much herbicide as those of Penicillium and Aspergillus. When colonies are fed with ^''^C-paraquat, the levels of activity confirm that the mucoraceous fungi absorb more than twice as much as the imperfect species. We therefore suggest that susceptibility to paraquat is strongly correlated with the accumulation of the herbicide in the mycelium. Such accumulation will depend not only on the rate at which paraquat is transported into the hyphae but also on the rate at which it is degraded within the mycelium. A subsequent paper will show that susceptible and tolerant fungi differ markedly in their ability to degrade paraquat. The results of exposing colonies to (^*C-methyl) paraquat in the presence of different concentrations of the wetting agent Lissapol NDB do not support the suggestion (Wilkinson and Lucas, 1969) that the wetting agent increases permeation of paraquat dichloride into the mycelium. ; ACKNOWLEDGMENTS..., Acknowledgment is made to the S.R.C. for a research studentship to S.N.S. and to I.C.I. Plant Protection Ltd., Jealotts Hill who supplied analytical paraquat dichloride. REFERENCES. CALDERBANK, A. & YUEN, S. H. (1964). An ion-exchange method for determining paraquat residues in food crops. Analyst, 90, 99. GARETH JONES, D. & WILLIAMS, J. R. (1971). Effect of paraquat on growth and sporulation of Septoria nodorum and Septoria tritici. Trans. Brit, mycol. Soc, 57, 351. SNEDECOR, G. W. & CocHRAN, W. G. (1967). Statistical Methods. 6th Ed. Iowa State Univ. Press. Tu, C. M. & BOLLEN, W. B. (1968). Effects of paraquat on microbial activities in soils. Weed Research, 8, 28. WILKINSON, V. & LUCAS, R. L. (1969a). Effects of herbicides on the growth of soil fungi. New PhytoL, 68, 709. WILKINSON, V. & LUCAS, R. L. (1969b). Effects of constituents of Gramoxone on rates of respiration of soil fungi. Weed Research, 9, 288. WILKINSON, V. & LUCAS, R. L. (1969c). Graxoxone W: its effect on spores and mycelia of Rhizopus stolonifer. Trans. Brit, mycol. Soc. 53,

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