EFFECTS OF HERBICIDES ON THE GROWTH OF SOIL FUNGI

Transcription

1 New Phytol. (1969) 68, EFFECTS OF HERBICIDES ON THE GROWTH OF SOIL FUNGI BY VALERIE WILKINSON AND R. L. LUCAS Department of Agricultural Science, University of Oxford {Received 5 February 1969) SUMMARY Three methods have been used to investigate the effects of a number of commercial herbicides on the growth of certain soil fungi: measurements of hyphal extension across agar plates; measurements of hyphal extension along sterilized plant material; and manometric techniques. Three points, in particular, emerged from these studies. First, that there was no stimulation of fungal growth. Herbicide interference in growth included suppression of spore germination, inhibition of the rate of linear extension of the mycelia, and abnormalities in growth habit and in patterns of spore production. Secondly, that some herbicides (e.g. linuron and paraquat) were more fungitoxic than others (e.g. MCPA and simazine) to a range of organisms. Thirdly, that there were differences between fungi in their sensitivity to individual herbicides. All three methods have shown consistent differences between fungi in their ability to tolerate paraquat. Trichoderma viride, in particular, has been found to be sensitive to paraquat. The inhibitory effects were observed at concentrations well within the range likely to be experienced in the field. INTRODUCTION Many of the studies on the interaction of herbicides and soil microorganisms have been concerned with herbicide persistence. They have dealt with the breakdown of herbicidal compounds by soil microorganisms, and in this respect bacteria appear to be more active than fungi. Implicit in the knowledge that soil microorganisms vary in their ability to metabolize herbicides is the fact that herbicides exert a selective effect on microorganisms, and in consequence they may produce qualitative changes in the soil populations. Previous studies of the effects of herbicides on soil microorganisms have been primarily concerned with quantitative microbial activity or specific microbiological processes, such as nitrification, which again involves bacteria in particular. Details of these and other investigations involving a number of herbicides are given in the extensive reviews by Bollen (1961) and Fletcher (i96, 1966). The present paper describes a series of experiments which investigate and compare the effects of five herbicides, each representative of a different group of compounds, on certain soil fungi. The purposes of this survey were to determine general differences between these herbicides in fungitoxicity and to distinguish between fungi in their tolerance of individual compounds. MATERIALS Organisms Helminthosporium sativum P., K. & B. was obtained from culture stocks at Cambridge University, and Mucor hiemalis Wehmer and Fusarium culmorum (W. G. Smith) Sacc. from the Commonwealth Mycological Institute. 79

2 VALERIE WILKINSON AND R. L. LUCAS Ophiobolus graminis Sacc. was isolated from the roots of infected wheat plants and Rhizopus stolonifer (Ehrenb. ex Fries.) Lind., Trichoderma viride Pers. ex Fries., Eurotium sp. and Cliaetomium sp. were isolated from a sandy loam soil. Media Except for Ophiobolus graminis, the fungi were grown on potato dextrose agar (PDA) and Czapek-Dox agar (Dox). The ph at inoculation was 7. and 6.8 respectively. O. graminis was grown on these media supplemented with both i % malt extract and i % yeast extract. Liquid medium (Bianchi, 1964) was also used. It consisted of the following: ammonium tartrate, 5. g; NH4NO3, i.o g; KH2PO4, i.o g; MgS4. 7H2O,.5 g; NaCl, o.i g; CaClj. 2H2O, O.I g; sucrose, 1 g; distilled water, i 1. Biotin and thiamine were added at rates of 1 ^g/1 and 5 ^g/1 respectively. Both agar and liquid media were sterilized in an autoclave. Herbicides These were the commercial formulations of MCPA (4-chloro-2-methylphenoxyacetic acid), linuron (iv'-[3,4-dichlorophenyl]-iv-methoxy-a^-methylurea), simazine (2-chloro- 4,6-bisethylamino-i,3,s-triazine), tri-allate (5-2,3,3-trichloroallyl-ArA/'-diisopropylthiocarbamate) and paraquat (i,i'-dimethyl-4,4'-bipyridylium dichloride). The commercial formulations were 'MCPA 25' (supplied by Boots Pure Drug Company Ltd) 'Linuron 5' (Du Pont Company Ltd) 'Weedex' (Fison's Pest Control Ltd) 'Avadex BW (Monsanto Chemicals Ltd) and 'Gramoxone W (Imperial Chemical Industries Ltd) respectively. These five herbicides will subsequently be referred to by the abbreviated name of their phytotoxic principle and not by their trade name, although in every case the complete commercial formulation was used. Spectrophotometric tests showed that MCPA, simazine and paraquat remained stable after sterilization by autoclaving, but that tri-allate was completely decomposed by this treatment and linuron was partly decomposed. Consequently, dilutions of the first three were made up and sterilized by autoclaving, and dilutions of linuron and tri-allate, which are self-sterile at high concentrations, were made up in sterile distilled water. METHODS Fungal growth measured as linear extension on agar Petri dishes, 8.5 cm in diameter and containing 25 ml of medium incorporating the herbicides at concentrations of o-iooo ppm, were inoculated centrally. Inoculations of Ophiobolus graminis, Helminthosporium sativum, Trichoderma viride, Fusarium culmorum and Furotium sp. were made with a disc, 4. mm in diameter, cut from the margin of young colonies. Inoculations of Rhizopus stolonifer were made with a spore suspension. Replication was four-fold. The plates were incubated at 25 C and fungal growth rates were determined by measurements of colony diameter. Colonies of Ophiobolus graminis, Helminthosporium sativum and Eurotium sp. were measured at 24-hour intervals, those of Fusarium culmorum, Trichoderma viride and Rhizopus stolonifer twice daily. Two measurements, at right angles to one another, were made each time. Measurements were continued until the fungus had colonized the plate or for a maximum of 14 days. Extension growth was adopted at the criterion for comparison rather than increase in mycelial weights (Eno,

3 Effects of herbicides on soil fungi 1962; Rodriguez-Kabana, Curl and Funderburk, 1966) because it allows measurements to be made more readily on organisms growing on solid media and because it enables a series of measurements to be made on the same material. Fungal growth measured as linear extension along straw segments Plant material was used so that patterns of interference in fungal growth caused by herbicides incorporated into agar media could be compared with those which might occur in decaying material in the field. Straw was selected because it is easy to handle, and with the recent expansion of cereal cultivation, it is probably the most common residue ploughed into agricultural soils. Experiments were confined to MCPA and paraquat. The fungi cultured on treated straw segments were Ophiobolus graminis, Fusarium culmorum, Eurotium sp. and Trichoderma viride. Internodal segments of wheat straw, each approximately 1 cm long and not less than 3 mm in diameter, were soaked overnight in either (a) distilled water, (b) a 1 ppm or (c) a 1 ppm solution of one of the herbicides, and then sterilized. Five millilitre aliquots of PDA at the bottom of 6 x i in. boiling tubes were inoculated with fungal discs. When the fungus had colonized the agar a length of straw was introduced into each tube. One end was pushed into the agar to hold the straw upright and away from the walls. Replication was four-fold and the tubes were incubated at 25 C. Growth rates were determined by 24-hour measurements of fungal extension along the straw surface. Respiration studies The effects of paraquat at concentrations of -4 ppm on rates of oxygen uptake by fungi were studied, using standard Warburg techniques. Conical flasks containing 2 ml of liquid medium were inoculated with either Fusarium culmorum, Trichoderma viride, Mucor hiemalis, Eurotium sp. or a sporing culture of Chaetomium sp. and incubated at 25 C on a shaker. Suspensions of the first three fungi could readily be transferred to Warburg flasks with a pipette. Forceps were used for colonies of Eurotium sp. and Chaetomium sp. ju RESULTS Effects of herbicides on fungal growth Linear extension on agar. Table i shows the rates of fungal growth on agar containing herbicides. Three points, in particular, emerged from this survey. First, there was no significant stimulation of fungal growth by any of these herbicides. Second, the fungi showed similar responses to individual herbicides, with a few exceptions. In order to demonstrate this point, values for the rates of fungal growth on PDA incorporating linuron and simazine from Table i are shown in Fig. i. Third, grovi^th from spores of Rhizopus stolonifer was completely inhibited by paraquat. The effects of paraquat on germination of spores of R. stolonifer were subsequently examined further and these results are described elsewhere (Wilkinson and Lucas, 1969). Of the five herbicides, simazine was the least effective fungitoxin. There was little depression of fungal growth at a concentration of 1 ppm and appreciable growth even at 1 ppm (see Fig. i). Tri-allate and MCPA showed marked inhibition only at the higher concentrations. The majority of fungi grew at a concentration of 1 ppm although growth was only slow. With the exception of R. stolonifer fungi tolerated

4 712 VALERIE WILKINSON AND R. L. LUCAS Table i. Growth rates of fungi on agar media incorporating herbicides Herbicide concentration MCPA (ppm) PDA DOX o IO 5 O o IO Soo O IO 5 O 1 soo O 1 soo O 1 soo O O 57-O S5-O S3-O 24-S S-S O Simazine PDA DOX 93-O O IIO.O S Herbicides Linuron PDA DOX S O S Tri-allate PDA DOX O 9S-O o S-S 44-S O Paraquat PDA DOX S 9S-O S O O Fungus Eurotium sp. Helminthosporium sativum Rhizopus stolonifer Ophiobolus graminis Fusarium culmorum Trichoderma viride Average colony diameters were plotted against time, to give a series of sigmoid curves. A value for fungal growth rate under eacb herbicide regime was determined using the tangent of the angle made by the linear part of the curve with the horizontal axis. Such values were then expressed as a percentage of the figure for the control, untreated colonies. concentrations of up to 1 ppm of paraquat but at higher concentrations growth was severely inhibited. Fusarium culmorum was one of the fungi most resistant to paraquat. Linuron produced the greatest inhibitory effect, in some cases reducing growth rate by 5% at 1 ppm (Fig. i). As well as affecting the rate of fungal growth some herbicides promoted abnormalities in growth habit and pigmentation. Ophiobolus graminis growing on both PDA and Dox containing 5 ppm and 1 ppm of MCPA produced colonies which had an unusual feathery appearance. Colonies of Eurotium sp. on media incorporating 1 ppm of paraquat were pale pink-brown in contrast to deep sulphur yellow on untreated media. Tri-allate and linuron affected spore production by Trichoderma viride (see Plate, i). The dark areas on the plates correspond to formations of spores. Both herbicides reduced sporulation and resulted in spores being produced in concentric rings. The inhibitory effect of linuron at a concentration of 1 ppm was greater than that of tri-allate at 5 ppm. Linear extension along straws. Results are presented in Table 2. They show that paraquat was more inhibitory than MCPA to the fungi, which agrees with above observations. MCPA had little effect on fungal growth at the concentrations used. The inhibitory effect of paraquat incorporated into straws on growth of Fusarium culmorum and Furotium sp. was similar to its depressant effect on both fungi on agar media. But

5 THE NEW PHYTOLOGIST, 68, 3 PLATE I The effects of linuron and tri-allate on patterns of sporulation by Trichoderma viride growing on (a) PDA; (b) PDA+ ioo ppm of linuron; (c) PDA+ 5 ppm of tri-allate. VALERIE WILKINSON AND R. L. J^UCAS EFFECTS OF HERBICIDES ON SOIL FUNGI (facing page 712)

6 Effects of herbicides on soil fungi 713 (a) (b) (f) 1 5 _L I I I 1 Herbicide concentration {ppm) Fig. I. Growth rates of fungi on agar medium containing simazine (O) and linuron ( ). (a) Ophiobolus graminis; (b) Fusarium culmorum; (c) Trichoderma viride; (d) Eurotium sp.; (e) Helminthosporium sativum; (f) Rhizopus stolonifer. Table 2. Growth rates of fungi along straw segments treated with herbicides Herbicide concentration (ppm) Herbicide MCPA Paraquat Fungus Ophiobolus graminis Eurotium sp. Trichoderma viride Fusarium culmorum The values for growth rate have been calculated from graphs of fungal extension in the manner already described for Table i, so that comparisons can be made between effects of herbicides in both types of substrate.

7 VALERIE WILKINSON AND R. L. LUCAS in the case of Ophiobolus graminis and Trichoderma viride, paraquat added to straws had a greater depressing effect than it had when incorporated into agar. T. viride produced a sparse mycelial network over the walls of the boiling tubes, and in tubes containing straws treated with ioo ppm of paraquat this fungus spread further up the glass than the straw. This indicates that paraquat suppressed fungal extension even when this could take place to a limited extent away from a substrate. Effects of paraquat on oxygen uptake Rates of oxygen uptake by cultures of Fusarium culmorum, Trichoderma viride, Mucor hiemalis, Eurotium sp. and Chaetomium sp. treated with paraquat are shown in Figs In all cases oxygen uptake was reduced, but these fungi differed in their 2- o 1- Time (hours) Fig. 2. Rate of oxygen uptake by Fusarium culmorum treated vith paraquat. D, Control; L, IO ppm; #, ppm; O, 2 ppm;, 4 ppm. tolerance of paraquat. Fusarium culmorum, Eurotium sp. and Chaetomium sp. were little affected by concentrations up to and including ioo ppm. Concentrations of 2 ppm and 4 ppm were more inhibitory to Fusarium culmorum and Eurotium sp. than to Chaetomium sp. By contrast to these three fungi, both Trichoderma viride and Mucor liiemalis were inhibited by lower concentrations of paraquat. Trichoderma viride, in particular, showed sensitivity to paraquat. The rate of oxygen uptake by this fungus was depressed at a concentration of 1 ppm. DISCUSSION Experiments in which fungal extension across agar was measured show that some herbicides (e.g. paraquat and linuron) are more inhibitory to fungi than others (e.g.

8 Effects of herbicides on soil fungi 715 Herbicide addition 5 I Time (hours) Fig. 3. Rate of oxj'gen uptake by Eurotium sp. treated with paraquat. D, Control; A, 1 ppm;, 1 ppm; 1, 2 ppm;, 4 ppm. 4 ~ Time (hours) Fig. 4. Rate of oxygen uptake by Chaetomium sp. treated with paraquat. D, Control; A, 1 ppm;, 1 ppm; O, 2 ppm;, 4 ppm.

9 VALERIE WILKINSON AND R. L. LUCAS 1- Herbicide addition 5 O i Time (hours) Fig. 5. Rate of oxygen uptake by Trichoderma viride treated with paraquat. D, Control; A, 1 ppm;, 1 ppm; O, 2 ppm;, 4 ppm. 2 r 15 Herbicide addition Time (tiours) Fig. 6. Rate of oxygen uptake by Mucor hiemalis treated with paraquat. D, Control; A, 1 ppm;, 1 ppm; O, 2 ppm;, 4 ppm.

10 Effects of herbicides on soil fungi MCPA and simazine). Such results are in agreement with other work on these herhicides (Wilkinson, 1967). Differences between a number of other herbicides in their fungitoxicity have also been reported (Bain, 1961; Chappell and Miller, 1956; Davis and Dimond, 1953; Shennan and Eletcher, 1965; Smith and Fletcher, 1964). The results of investigations into interference with fungal growth by herbicides incorporated into straw support those obtained from agar culture. In some cases, however, inhibition caused by paraquat was more severe on straw. This suggests that measurements of herbicide interference in fungal growth by methods using a relatively rich medium may underestimate inhibition which might occur in nutritionally poorer substrates in the field. Measurements of fungal growth on treated straw segments revealed that Trichoderma viride, in particular, was sensitive to paraquat. This effect was confirmed by respiration studies. Selectivity by a herbicide against an organism such as T. viride might have important ecological consequences in view of its known antagonism against a number of plant pathogens such as Armillaria mellea (Bliss, 1951; Garrett, 1958), Fomes annosus (Rishbeth, 195, 1951), Rhizoctonia solani (Weindling, 1932, 1934; Daines, 1937) and Sclerotium rolfsii (Rodriguez-Kabana et al., 1967; Curl, Rodriguez-Kabana and Funderburk, 1968). Applications of herbicides at normal field rates are not reported to cause marked changes in the total number of soil microorganisms or in levels of microbial activity (see reviews by Audus, 1964; Bollen, 1961; Fletcher, i96, 1966). While there may be no permanent quantitative changes in the activity of soil populations there may be significant qualitative changes within these populations following herbicide treatments. Kaufmann (1964) reported that applications of simazine, atrazine, linuron and diuron to soil stimulated fungi known to be antagonistic to Fusarium species. Following treatment of tomato plants with 2,4-D Warren, Graham and Gale (1951) observed an increase in activity in the soil of an actinomycete with strong anti-fungal properties. This organism proved inhibitory to Rhizoctonia solani, Alternaria solani, Sclerotium rolfsii, Venturia inaequalis and a number of other fungi. A number of herbicides have been found to inhibit particular fungi, some of them pathogenic organisms (Bain, 1961; Bever and Slife, 1948; Chappell and Miller, 1956; Hsai and Christensen, 1951; Lebed, 1964; Nair, 1957) but little ecological significance has been attached to such results. It has been suggested that laboratory investigations show that fungi are tolerant of concentrations of herbicides higher than those occurring in the field. Calculations of concentrations of herbicide present in the soil indicate that there is less than 1 ppm in the top 6 in. (Bollen, 1961; Fletcher, 1956; Newman and Downing, 1958). Such calculations, however, assume uniform spread of the herbicide throughout the specified layer, a situation not found in practice. Applications of herbicide will result in localized 'pockets' in the soil, containing a comparatively high concentration of the chemical. Perhaps of greater importance is accumulation of herbicides by plants, because following the death of the plant its tissues become subject to microbial decomposition. Calderbank and Tomlinson (1968) found that immediately after applying paraquat at a rate of 5 lb/ac to a grass sward the herbage contained 124 ppm of paraquat. Even 4 months after treatment they detected residues of 136 ppm of paraquat. At such concentrations herbicides have significant selective effects on fungi. Measurements of both growth and respiration of fungi have shown consistent differences between them in their ability to tolerate the herbicide paraquat. This suggests that the presence of a herbicide such as paraquat in plant tissues may alter the outcome of competition between fungi competing to colonize the tissues. A qualitative change in the

11 7i8 VALERIE WILKINSON AND R. L. LUCAS species capable of colonizing and decomposing plant material might subsequently affect the persistence of pathogenic organisms capable of saprophytic survival in these tissues, either through direct antagonism or through changes in the rate of decomposition of the infected material. ACKNOWLEDGMENT One of us (V.W.) would like to thank the Ministry of Agriculture for a post-graduate Research Studentship which enabled this work to be carried out. REFERENCES AUDUS, L. J. (1964). Herbicide behaviour in the soil. In: The Physiology and Biochemistry of Herbicides (Ed. by L. J. Audus), p London and New York. BAIN, D. C. (1961). Effect of various herbicides on some soil fungi in culture. PL Dis. Reptr, 45, 814. BEVER, W. M. & SLIFE, F. W. (1948). Effect of 2,4-D in culture medium on the growth of three pathogenic fungi. Phytopathology, 38, 137. BiANCHi, D. E. (1964). An endogenous circadian rhythmn in Neurospora crassa. J. gen. Microbiol., 35,437. BLISS, D. E. (1951). The destruction oi Armillaria mellea in citrus soils. Phytopathology, 41, 665. BOLLEN, W. B. (1961). Interactions between pesticides and soil micro-organisms. A. Rev. Microbiol., 15, 69. CALDERBANK, A. & ToMLiNSON, T. E. (1968). The fate of paraquat in soils. Outl. Agric, 5, 252. CHAPPELL, W. E. & MILLER, L. I. (1956). The effects of certain herbicides on plant pathogens. PI. Dis. Reptr, 4, 52. CURL, E. A., RODRIGUEZ-KABANA, R. & FXINDERBURK, H. H. (1968). Influence of atrazine and varied carbon and nitrogen amendments on growth of Sclerotium rolfsii and Trichoderma viride in soil. Phytopathology, 58, 323. DAINES, R. H. (1937). Antagonistic action of Trichoderma on Actinomyces scabies and Rhizoctonia solani. Am. Potato J., 14, 85. DAVIS, D. & DIMOND, A. E. (1953). Inducing disease resistance with plant growth-regulators. Phytopathology, 43, 137. ENO, C. F. (1962). The effect of simazine and atrazine on certain of the soil microflora and their metabolic processes. Proc. Soil Crop Sci. Soc. Fla, 22, 49. FLETCHER, W. W. (1956). Effect of hormone herbicides on the growth of Rhizobium trifolii. Nature, Lond., 177, FLETCHER, W. W. (i96). The effect of herbicides on soil micro-organisms. In: Herbicides and the Soil (Ed. by E. K. Woodford & G. R. Sagar), p. 2. Oxford. FLETCHER, W. W. (1966). The effect of herbicides on soil micro-organisms. Proc. 8th Br. Weed Control Conf., 3, 896. GARSETT, S. D. (1958). Inoculum potential as a factor limiting lethal action by Trichoderma viride Fr. on Armillaria mellea (Fr.) Qu^l. Trans. Br. mycol. Soc, 41, 157. HsAi, Y. & CHRISTENSEN, J. J. (1951). Effect of 2,4-D on seedling blight of wheat caused by Helminthosporium sativum. Phytopathology, 41, ioii. KAUFMANN, D. D. (1964). Effect of s-triazine and phenylurea herbicides on soil fungi in corn- and soybeancropped soil. Abstr. in Phytopathology, 54, 897. LEBED, E. S. (1964). The effect of some herbicides on soil microflora. Nauch. Dokl. vyssh. Shk., 3, 171. NAIR, P. N. (1957). Factors affecting resistance of flax to Fusarium lini. Diss. Abstr., 17, 942. NEWMAN, A. S. & DOWNING, C. R. (1958). Herbicides and the soil. J. agric. Fd Chem., 6, 352. RISHBETH, J. (195). Observations on the biology of Fomes annosus, with particular reference to East Anglian pine plantations. I. The outbreaks of disease and ecological status of the fungus. Ann. Bot., 14, 365. RISHBETH, J. (1951). Observations on the biology of Fomes annosus, with particular reference to East Anglian pine plantations. III. Natural and experimental infection of pines, and some factors affecting severity of the disease. Ann. Bot., 15, 221. RODRIGUEZ-KABANA, R., CURL, E. A. & FUNDERBURK, H. H. (1966). Effect of four herbicides on growth of Rhizoctonia solani. Phytopathology, 56, RODRIGUEZ-KABANA, R., CURL, E. A. & FUNDERBURK, H. H. (1967). Effect of atrazine on growth response of Sclerotium rolfsii and Trichoderma viride. Can. J. Microbiol., 13, SHENNAN, J. L. & FLETCHER, W. W. (1965). The growth in vitro of micro-organisms in the presence of substituted phenoxyacetic and phenoxybutyric acids. Weed Res., 5, 266. SMITH, J. E. & FLETCHER, W. W. (1964). 3:5-Dihalogeno-4-hydroxybenzonitriles and soil micro-organisms. Hort. Res., 4, 6. WARREN, J. R., GRAHAM, F. & GALE, G. (1951). Dominance of an actinomycete in a soil microflora after 2,4-D treatment of plants. Phytopathology, 41, 137. WEINDLING, R. (1932). Trichoderma lignorum as a parasite of other soil fungi. Phytopathology, 22, 837.

12 Effects of herbicides on soil fungi 719 WEINDLING, R. (1934). Studies on a lethal principle effective in the parasitic action of Trichoderma lignorum on Rhizoctonia solani and other soil fungi. Phytopathology, 24, WILKINSON, V. (1967). Factors affecting the growth of soil fungi. D.Phil thesis (unpublished), University of Oxford. WILKINSON, V. & LUCAS, R. L. (1969). Gramoxone W: its effects on spores and mycelia of Rhizopus stolonifer. (In press.)

13