Growth responses of mycorrhizal Boletus and Rhizopogon species to pesticides

Notes and brief articles

434

NAGANISHI, H. & KAWAKAMI, N. (1955). On Blakeslea Thaxter in Japan. (II) Bl. circinans sp.nov. Bulletin. Faculty of Engineering. Hiroshima University 4, 1831 87.

POITRAS, A. W. (1955). Observations on asexual and sexual reproductive structures of the Choanephoraceae. Mycologia 47, 702-713. SACCARDO, P. A. (1891). Sylloge Fungorum 9, 339. SCHIPPER, M. A. A., SAMSON, R. A. & STALPERS, J. A. (1975). Zygospore ornamentation in the genera Mucor and Zygorhynchus. Persoonia 8, 321-328.

THAXTER, R. (1903). Mycological notes. 1. A New England Choanephora. Rhodora 5, 97-102. THAXTER, R. (1914). New or peculiar Zygomycetes. 3. Blakeslea, Dissophora and Haplosporangium, nova genera. Botanical Gazette 58, 353-366. WEBER, G. F. & WOLF, F. A. (1927). Heterothallism in Blakeslea trispora. Mycologia 19, 302-307. WOLF, F. A. (1917). A squash disease caused by Choanephora cucurbitarum. Journal of Agricultural Research 8, 319-328.

GROWTH RESPONSES OF MYCORRHIZAL BOLETUS AND RHIZOPOGON SPECIES TO PESTICIDES E.

J. DASILVA,

L. E. HENRIKSSON* AND MILDA UDRIS

Institute of Physiological Botany, University of Uppsala, S-75121 Uppsala, Sweden The benefits of introducing mycorrhizal infection at the time of transplanting forestry stock in order to encourage the development of a sturdy root system are well known (Bjorkman, 1961; Marx, 1970, 1973; Iyer, Lipas & Chesters, 1971) and failure to establish native forest trees as well as exotic species is often attributed to the absence of suitable mycorrhizal fungi in the soil (Harley, 1969). In recent years the use of herbicides and other pesticides has been rapidly increasing and reduction in forest productivity has been blamed on the eradication of mycorrhizal-forming fungi in the rhizosphere (Iyer & Wilde, 1965). The appearance of symptoms of nutrient deficiencies in the foliage of trees lacking mycorrhizas has followed the application of organic fumigants which presumably disrupted the mycotrophic mechanism (Henderson & Stone, 1967). In an attempt to ascertain whether some of the common pesticides have a direct physiological effect on mycorrhizal fungi, laboratory cultures of three ectomycorrhizal species were tested with each of six pesticides. The species used in this investigation were eetomycorrhizal hymenomycetes and gasteromycetes: Boletus variegatus, B. luteus and Rhizopogon roseolus (provided by Prof. Elias Melin, University of Uppsala). They were maintained on Hagem's agar (Norkrans, 1950). For the experiments, they were grown in the dark in still cultures in 100 ml Erlenmeyer flasks with 10 ml of the slightly modified Norkrans (1950) medium II at 25°. The medium contained 20'0 g glucose, 0'5 g ammonium tartrate, 1'0 g KH 2POu 0'5 g MgS0 4.

* To whom correspondence should be directed. Trans. Br. mycol, Soc. 68 (3) (1977)

7H20, 4'4 mg ZnS04·7H20, 5'0 mg MnS0 4. 4H 20, and 5'0 mg ferric citrate in 1 1 distilled water (pH 5'2). For inoculation, portions of mycelium on Hagem agar, c. 2 mm square, were transferred to Erlenmeyer flasks containing 20 ml of the modified Norkrans medium II. After a growth period of 7-10 days, the mycelia were pooled and homogenized with glass beads (Wiken et al., 1951) in 100 ml sterile distilled water. From the resulting suspension, 0'5 ml was used to inoculate each of the 100 ml flasks (6 pesticides, 4-7 concentrations. g species, 3-4 replications) used in the screening test of the pesticides. Growth was measured as dry weight of the mycelium and pH measurements were taken following harvest of the mycelium. Three groups of pesticides were used in this study: (1) the phenoxyacetic group consisted of 2,4-D (Nutritional Biochemical Corporation, USA), 2,4,5-T (' Pestinal', Riedel de Haen, Germany), and MCPA (Farbenfabrik Bayer AG, Germany), (2) the miscellaneous herbicide group consisted of paraquat (Plant Protection Ltd, UK), and amitrole (Gullviks AB, Sweden), and (3) the insecticide malathion (Plantex AB, Sweden). These pesticides were selected because they have been widely used in agriculture and forestry. The same lots of the chemicals of the latter two groups have earlier been tested in respect to the effect of pesticides on blue-green algae and nitrogen fixation (DaSilva, Henriksson & Henriksson, 1975a, b). The two Boletus spp. (hymenomycetes) were stimulated by low concentrations of all three of the phenoxyacetic acid herbicides (Fig. 1). All three species were suppressed in growth by higher concentrations, but Rhizopogon roseolus

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Notes and briefarticles

435

50

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o 10 ppm

§ 100 ppm

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250 ppm

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B. var. B. lUI, R. ros.

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Effect of the phenoxyalkanoate group of pesticides - 2,4-D, 2,4,5- T and MCPA - on Bole tus oariegatus, B . luteus and R hizopogon roseolus after 12 days at 2 5 °C in the dark.

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Fig. 2 . Growth of Boletus uariega tus at 2S °C in modified Norkrans medium II in the dark with respect to different levels of the bipyridylium quaternary herbicide paraquat. Graphic representations are of determinations in triplicate. Fig. 3. Growth response at 12 days of three ectomycorrhi zal fungi to the insecticide malathion. Growth expressed as dry weight. Trans . Br , my col. S oc. 68 (3) (1977)

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Notes and brief articles Table 1. Effect of amitrole on the growth of thr ee ectomy corrhizal f ungi : determinations fro m experiments in quadruplicat es (Boletus ssp .) and triplicates (Rhizopogon sp,) Boletus variegatus

Growth Concentration Dry weight (mg) (days) (ppm) 6 l '7±O 'l °0'5 l '7 ±O'l 1'0 1"9±O'l l '4±O 'l 5'0 10'0 o'6 ±o'o 0'1 ± O'O 50'0 100'0 0'1 ± O'O 6,8±o'4 0 8 7'1 ±O'2 0'5 1'0 6'8±O 'l 6,8±O'1 5'0 10'0 6'2 ±O'3 50'0 1'4±O'2 100'0 o'8±o'o 10 0 17'5 ±O'7 0'5 17'7±o'7 1'0 17'O±O'3 5'0 1S'9±O'S 10'0 14'7 ±1 '1 6,6 ±o'l 50'0 100"0 3"9 ±O'3 12 0 29'2± O'6 29'2 ±O'8 0'5 1'0 29'3± O'9 28'6 ±o'7 5'0 28,6±1'1 10'0 14'3 ± 1'2 50 "0 100'0 l1 '2±O '7

pH 4"7 4'7 4'6 4'7 5'1 5'2 5'2

3'9 3'9 3"9 4'0 4'0 4'6 5'0

3'4 3'5 3'3 3'5

3'8 4'1 4"3 3'2 3'2 3'2 3'2 3'2 3'3 3'5

(gasteromycete) was suppressed at lower concentrations than the other species. The herbicides amitrole and paraquat both suppressed growth of the fungi at higher concentra tions, 1'0 ppm and 5'0 ppm, respectively (T able 1; Fig , 2). At concentrations of 1'0 ppm and above, paraquat was lethal to B. v ariegatus (Fig. 2), The insecticide malathion, tended to stimulate growth of all three species of fungi at th e concentrations used in this study (Fig. 3) although stimulation was least in B. uariegatus. While pH steadily decreases in time with fungal growth, there was no evidence that either the kind of pesticide or its concentration (T able 1) made any difference to the amount of acidity produced. Plants that are killed by the phenoxyacetic acid herbicides are generally stimulated in growth at low concentrations, A similar response was observed in the three mycorrhizal fun gi used in this study. Calanacea & Illyes (1954) reported an enhancement of growth in Aspergillus niger, Trans. s« mycol, Soc, 68 (3) (1977)

Boletus luteus

Dry weight (mg) 0"4±0 "1 0"4 ±O'l 0"4±O'l O'S±O'l O'3 ±O'l O'2 ±O'l 0'1 ± O'l l '9 ±o 'l l'9 ±0 "0 l'9±o'2 l'S±O'l l'4±o'2 l '2±O'l O'7±O'l 7'7±o'l 7'5 ±O'3 7'S±O'2

7'6 ±o'l 6'1 ± O'2 3'7±o'3 2'3 ±o'2 24'1 ± 0"9 25'1 ± O'3 2S'7 ±l'l

27'2 ± o'S 24'9±O 'S 18"9 ± 2'O 13'2 ± O,6

Rhizopogon roseolus

pH 5"2 5'2 5'2 5'2 5'2 5'2 5'2

4'8 4'8 4'8 4'9 4'9 4'9 5'1

4'2 4'2 4'2 4'2 4'4 4'5

4'8 3'1 3'1 3'1 3'0 3'1 3'3 3'5

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21'S±3,8

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following appli cation oh,4-D, Alexander (1969) has reviewed the selective inhibitory and stimulatory effects of pesticides on micro-organisms. Whereas the concentration of paraquat emp loyed in thi s study was about the same as that used by farm ers and foresters in normal field app lications , the concentrations of the other herbicides were many time s great er than that normally used, Since the phenoxyacetic acid-containing herbicides were slightly stimulatory to the mycorrhizal fungi at low concentrations, and since these herbicides are highly biodegradable under normal field conditions, there is no evidence from this study that these substances would have an adverse effect on mycorrhizas. Amitrole is known to be rapidly degraded under field cond itions and at low concentrations appeared to be harmless, Of the herbicides tested, paraquat is the only one that could be expected on the basis of the recorded data to have advers e ecological effects under field cond itions , In laboratory cultures, abundant phosphorus

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Notes and briefarticles and potassium were always present, but under field conditions these elements are often very limited in supply. Henderson & Stone (1967) and Lipas (1968) have shown a reduction in the uptake of these elements in biocide-treated soils. If any of these pesticides have such an effect, the stimulation of growth observed in the laboratory cultures would be less likely in field conditions. If any of these pesticides interfere with the establishment of symbiosis between the ectotroph and its host, the former would soon be eliminated from the ecosystem. Since eetomycorrhizas are essential in the rearing of forest nurseries and the ma intenance of the rh izosphere environment this would also have an adverse effect on the trees. Therefore, even though it is known that these pesticides, apart from paraquat, do not adversely affect growth in culture of the three fungi investigated, further ecological studies are clearly needed before concluding that they are harmless under field conditions. This study demonstrates that the six pesticides tested have distinct and significant effects on the metabolism of the mycorrhizal fungi studied. Since the tested pesticides were randomly chosen, it may be assumed that most herbicides and possibly other pest icides will affect the metabolism of mycorrhizal fungi. Further studies are needed to evaluate the ecological consequences, if any, resulting from use of pesticides in general, and to ascertain the precise metabolic responses of the ectomycorrhizas. The authors are indebted to Prof. Elias Melin and Docent Elisabet Henriksson, University of Uppsala, Sweden, to Prof. L. C. Pearson, Ricks College, USA, and Prof. Sagar Krupa, University of Minnesota, USA, for stimulating discussions and helpful criticism. We also thank Prof. Nils Fries, Institute of Physiological Botany, for the facilities extended. A UNESCO fellowship (to E .].DS.) helped finance the research and the authors gratefully acknowledge it. REFERENCES ALEXANDER, M. (1969). Microbial degradation and biological effects of pesticides in soil. Soil Biology, pp. 209-236. Paris: Unesco.

Tran s. Br, mycol. Soc. 68 (3) (1977)

437

BJORKMAN, E. (1961) . The influenceof ectomycorrhizae

on the development of forest trees after planting. Proceedings XIIth I.U.F.R .a. Congress, Wien 24, 1-3·

CALANACHA, L. & ILLYHS, G. (1954) . The effect of dichlorophenoxyacetic acid (2,40-0) and trichlorophenoxyacetic acid (2,40,5- T) on the production of citric acid by Aspergillus niger. Studii cerecetdri ~tiin{ijice. Filiala Cluj, Academia RPR ser, II, 5, 239-246.

DASILVA, E. J., HENRIKSSON, L. E. & HENRIKSSON, E. (1975 a). Effect of pesticides on blue-green algae and nitrogen-fixation. Archives of Environmental Contaminat ion and Toxicology 3, 193-204.

DASILVA, E. J., HENRlKSSON, L. E. & HENRIKSSON, E. (1975b). Effect of pesticides on nitrogen-fixation by blue-green algae. Revista de Microbiologia, S. Paulo 5,73-74·

HARLEY, J. L. (1969 ). The biology of the mycorrhizae, znd ed. London: Leonard Hill. HENDERSON, G. A. & STONE, E. L. (1967). Interactions of phosphorus availability, mycorrhizae and soil fumigation in coniferous seedlings. Agronomy Abstracts, p. 134.

IYHR, J. G. & WILDE, S. A. (1965). Effect of Vapam biocide on the growth of red pine seedlings.Journal of Forestry 63, 703-704. IYHR, J. G., LIPAS, E. & CHHSTHRS, G. (1971) . Correction of mycotrophic deficiencies of tree nursery stock produced on biocide-treated soil. Mycorrhizae. Proceedings of the First North American Conferenceon Mycorrhizae, 1969. Miscellaneous Publication, U.S. Department of Agriculture (ed. E. Hacskaylo), p. 255. LIPAS, E. J. (1968). Dynamics of nutrient elements in

soils of Wisconsin forest nurseries. M.S. thesis, University of Wisconsin. MARx D. H. (1970). The influence of ectomycorrhizal fungi on the resistance of pine roots to pathogenic infections. V. Resistance of mycorrhizae to infection by Phytophthora cinnamomi. Phytopathology 60,14721473·

MARX, D. H. (1973). Ectomycorrhizae as biological deterrents to pathogenic root infections. Annual R eview of Phytopathology

10,

429-454.

NORKRANs, B. (1950). Studies in growth and cellulolytic enzymes of Tricholoma. Symbolae botanicae upsalienses 11, 1-126. WIKEN, T., KELLER, H. G., SCHELLING, C. L. & STOCKLI, A. (1951). Uber die Verwendung von Myze1suspensionen als Impfmaterial in Wachstumsversuchen mit Pilzen. Experientia 7, 237-239.

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