The fungicide benomyl inhibited spore germination and hyphal length of the arbuscular mycorrhizal fungus Glomus mosseae when applied at doses of 21.25 μg/ml (agronomic dose), 10.62 μg/ml and 10 μg/ml. G. mosseae was able to germinate in the presence of 2.12 μg/ml of benomyl, and the percentage of spore germination was unaffected by dosis of 0.1, 0.01 and 0.001 μg/ml of the fungicide. However, all doses of fungicide tested in this study decreased the hyphal length. When ungerminated G. mosseae spores previously exposed to benomyl were transferred to water-agar medium without benomyl, the maximum germination was 16%. Small spores of G. mosseae were more resistant to benomyl than the larger ones. Our results show some of the factors which can explain the variability of the effect of benomyl on arbuscular mycorrhizal fungi

Alicia E. Martinez

Alicia Godeas

Ana Menendez Ana Menendez

Juan A. Ocampo

Nadia Venedikian

Viviana Chiocchio

Chiocchio, Viviana

Venedikian, Nadia

Martinez, Alicia E.

Ana Menendez, Ana Menendez

Ocampo, Juan A.

Godeas, Alicia

Hemeroteca Cientifica Catalana

173IntroductionArbuscular mycorrhizal (AM) symbioses are essentialcomponents of most plant systems, and the beneficial role thatAM fungi play in agricultural production is well known [2].Although the use of pesticides is fundamental for soybeancultivation, there are not many studies about their effects onplant growth and on nontarget microorganisms such as AMfungi [15]. However, benomyl, like many other fungicides, hasbeen reported to reduce AM fungi under some conditions. Theeffects of fungicides on AM symbiosis depend on environmentalfactors such as soil, crop system, climate, and also on the AMfungal strains themselves. This difficults the study of the effectof fungicides on AM fungus formation and function [16]. AsAM fungi cannot be cultured axenically in the absence of theplant host, the spore germination test has been used to studythe direct effect of pesticides on AM fungi [15].Systemic fungicides can be applied to soil around plantsand are absorbed by roots and translocated to other parts of theplant [11]. These kinds of fungicides are of great interest dueto their persistence in the plant and their action on the AMfungi, either on their vegetative or on their resistance structures[12]. Benomyl is among the most frequently used systemicfungicides against pathogenic fungi of cereals and oleaginousplants [10]. It has been found to affect negatively AM symbiosesby delaying or preventing the formation of AM symbiosisbetween fungi and roots and by decreasing plant P-uptake [8,13, 19]. Benomyl suppresses mycelial growth by preventingnuclear division. In fact, this fungicide inhibits mitosis byblocking the formation of β-tubuline and microtubules whenchromosomes are separated [6, 9, 11]. This work aims to findout some possible factors implicated in the sensitivity andresistance of spores of Glomus mosseae to benomyl.Materials and methodsSporocarps and spores were isolated by the wet-sievingtechnique [5] and identified as Glomus mosseae [4]. Onehundred G. mosseae spores were inoculated to alfalfa plantpots. These plants were cultured for four months until theirroots became well colonized (80% root length) and new G. mosseae spores were developed (41 spores/g of soil).Influence of benomyl on spore germination The effect of thefungicide benomyl [methyl 1-(butylcarbamoyl)-2-benzimidazo-lecarbamate, C14H18N4O3] from Dupont (Benlate, 50% activeViviana Chiocchio1Nadia Venedikian1Alicia E. Martinez1Ana Menendez1Juan A. Ocampo2Alicia Godeas11Department of Biology, Faculty of BiologicalSciences, University of Buenos Aires, Argentina2Department of Microbiology, ZaidínExperimental Station, CSIC, Granada, SpainReceived 15 April 2000Accepted 2 July 2000Correspondence to: Juan A. Ocampo. Departamento deMicrobiología. Estación Experimental del Zaidín.Profesor Albareda, 1. 18008 Granada. SpainTel.:+34-958121011 Fax:+34-958129600 E-mail:jocampo@eez.csic.esRESEARCH ARTICLEINTERNATL MICROBIOL (2000) 3:173–175© Springer-Verlag Ibérica 2000Effect of the fungicide benomyl onspore germination and hyphal lengthof the arbuscular mycorrhizal fungusGlomus mosseaeSummary The fungicide benomyl inhibited spore germination and hyphal lengthof the arbuscular mycorrhizal fungus Glomus mosseae when applied at doses of21.25 µg/ml (agronomic dose), 10.62 µg/ml and 10 µg/ml. G. mosseae was able togerminate in the presence of 2.12 µg/ml of benomyl, and the percentage of sporegermination was unaffected by dosis of 0.1, 0.01 and 0.001 µg/ml of the fungicide.However, all doses of fungicide tested in this study decreased the hyphal length.When ungerminated G. mosseae spores previously exposed to benomyl weretransferred to water-agar medium without benomyl, the maximum germination was16%. Small spores of G. mosseae were more resistant to benomyl than the largerones. Our results show some of the factors which can explain the variability of theeffect of benomyl on arbuscular mycorrhizal fungi.Key words Glomus mosseae · Arbuscular mycorrhizae · Benomyl · Spore germination ·Fungicideingredient) on the germination of G. mosseae spores was testedin vitro on 1% sterile water agar (Difco-Bacto). We added 10mM 2-(N-morpholin)ethane sulfonic acid (MES) to 1% wateragar to maintain the pH of the medium at 7 throughout theexperiment. The fungicide was added to the agar of each Petridish at the concentration of 21.25 µg/ml (equivalent toagronomic dose), 10.62, 2.12, 0.1, 0.01 and 0.001 µg/ml. Fiveto eight surface-sterilized spores were placed in each Petri dish[14]. Five replicates of each fungicide doses and controls wereused. The plates were incubated at 25ºC and spore germinationwas checked under a light microscope after 6 days until 19 daysof incubation. After 19 days hyphal length was also measuredby using the gridline intersect method [7]. The diameters of G. mosseae spores treated with 0, 0.1 and0.01 µg/ml. were measured under light microscope.Sensitivity of spores to benomyl Ungerminated spores exposedto fungicides were washed with sterile water and transferredto freshly-prepared plates of water-agar without fungicides.These were incubated as above and the percentage ofgermination was checked after seven days.Statistical analysis Data obtained were subjected to analysisof variance (ANOVA). Data obtained were arcsine-transformedbefore analysis. The means were compared by Tukey, Duncanand Scheffé multiple range tests at the 5% level.Results and Discussion Influence of benomyl on spore germination Table 1 showsthat spore germination in the controls was 57%. The germinationof G. mosseae spores was completely inhibited on mediacontaining 10, 10.62 and 21.25 µg/ml of benomyl. In the presenceof 1 and 2.12 µg/ml of benomyl the percentage of sporegermination were 12 and 17% respectively. At concentrationsof 0.1, 0.01 and 0.001 µg/ml of benomyl the percentage of sporegermination increased to 64, 67 and 72%, respectively. ANOVAshowed significant results between treatments and the Tukey,Duncan and Scheffé tests showed the same results. Table 1 Effect of benomyl on the percentage of spore germination and hyphallength of the germ tube of Glomus mosseaeAmount of benomyl Percent spore Hyphalapplied (µl/ml) germination* length* (mm)0 57 (c) 74.28 (c)0.001 72 (c) 12.95 (b)0.01 68 (c) 12.66 (b)0.1 64 (c) 12.15 (b)1 17 (b) 0 (a)2.12 12 (b) 0 (a)10 0 (a) 0 (a)10.62 0 (a) 0 (a)21.25 0 (a) 0 (a)*Column values followed by the same letter are not significant as Tukey,Duncan and Scheffé tests (P = 0.05). The regression line between percentage of spore germinationand benomyl concentration (C) showed the equation y = 23.04 – 19 logCr = –0.9547, P = 0.0002This equation indicates that benomyl concentrations equalor higher than 1 µg/ml decreased significantly the percentageof spore germination. Concentrations of benomyl equal to orlower than 0.1 µg/ml did not affect the percentage of sporegermination. All doses of fungicide tested decreasedsignificantly the length of the germ tube.At 0.1 and 0.01 µg/ml benomyl concentrations, the largestspores (with a diameter of more than 40 µm) showed a lowerrate of germination, than the small ones (21–30 and 31–40 µmdiameter) (Table 2).Table 2 Effect of benomyl on the percentage of germination of Glomusmosseae spores with different diametersDiameter of Concentration of benomyl applied (µl/ml)*spores (µm) 0 0.1 0.0121–30 33.3 (c) 43.7 (c)  35.3 (c)31–40 50.1 (c) 50.2 (c) 58.8 (c)41–50 16.7 (b) 6.2 (a) 5.8 (a)*Column values followed by the same letter are not significant as Tukey,Duncan and Scheffé tests (P = 0.05).Sensitivity of spores to benomyl Spores exposed to 21.25 and10.62 µg/ml of benomyl did not germinate when they weretransferred to water-agar plates. The spores exposed to 10, 2.12and 1 µg/ml of benomyl reached the percentage of 7, 11.7 and16.6% germination, but they did not develop mycelia whenthey were transferred to agar-water medium (Table 3).Table 3 Effect of benomyl on the percentage of germination and hyphal lengthof the germ tube of Glomus mosseae spores transferred to water-agarAmount of benomyl Percent spore Hyphalapplied (µl/ml) germination* length1 16.6 (b) 02.12 11.7 (b) 010 7 (b) 010.62 0 (a) 021.25 0 (a) 0*Column values followed by the same letter are not significant as Tukey,Duncan and Scheffé tests (P = 0.05).The use of spores allows to study the direct effect offungicides on AM fungi in the absence of plants. Our resultsshowed that 0.1, 0.01 and 0.001 µg/ml of benomyl did notinhibit the germination of G. mosseae spores, but inhibitedthe hyphal length of the germ tube. The walls of the germtube are more permeable than the multistratified walls of thespores. This suggests that the fungicide benomyl acted whenthe spore walls opened as the germ tube emerged from thespore. The germ tube is the most sensitive AM fungalstructure [18]. The sensitivity of AM fungi to fungicides isvariable [15]. In fact the effect of benomyl on G. mosseae174 INTERNATL MICROBIOL Vol. 3, 2000 Chiocchio et al.spores differs from that on Glomus caledonicum spores [3].10 ng/ml benomyl doses inhibit spore germination and hyphallength of G. caledonicum. However, our results showed thatG. mosseae was able to germinate in the presence of 2.12µg/ml of benomyl. Spore germination of G. mosseae wasinhibited in the presence of 10 µg/ml of benomyl, but whenthese spores were transferred to Petri dish with agar-waterwithout herbicide, the negative effect of benomyl decreased.However, this negative effect of benomyl on the hyphal lengthof the transferred spores was found even when the sporeswere exposed to the lowest dose of benomyl (1 µg/ml) usedin our study. Nevertheless, the spores of G. caledonicumexposed to 0.1 and 10 µg of benomyl reached 95%germination and hyphal lengths similar to those of the con-trols when they were transferred to water-agar medium [3].These results show that fungicidal or fungistatic effects ofbenomyl may vary in different AM fungi. Besides, our resultsalso indicate that the sensitivity of AM fungi depends notonly on fungal strains but also on the stage of fungaldevelopment. The size of the spores is another factor to be taken intoaccount in studies on the effect of fungicides on AM fungi. Ourresults show that smaller spores were more resistant than biggerones to benomyl. This fact might be due to the modification ofspore walls during their maturity allowing substances to enterthe spores, which can affect and modify their metabolism [1,17].Acknowledgments Financial support for this study was provided by thePrograma de Cooperación Científica con Iberoamérica (Joint projects Spain-Argentina), Ministerio de Educación y Cultura, Spain.References1. Bentinvenga SR, Morton JB (1994) Systematics of glomaleanendomycorrhizal fungi. Current views and future directions. In: PflegerFL, Linderman RG (eds) Mycorrhizae and plant health. APS Press, St.Paul, Minnesota, pp 283–3082. Bethlenfalvay GJ, Linderman RG (1992) Preface: mycorrhizae insustainable agriculture. ASA Special Publication no. 54. American Societyfor Agriculture, Madison, WI 3. Carr GR, Hinkley MA (1985) Germination and hyphal growth of Glomuscaledonicum on water agar containing benomyl. Soil Biol Biochem17:313–3164. Fracchia S, Mujica MT, García-Romera I, García-Garrido JM, Martín J,Ocampo JA, Godeas A (1998) Interactions between Glomus mosseae andarbuscular mycorrhizal sporocarp-associated saprophytic fungi. PlantSoil 200:131–1375. Gerdemann JW, Nicolson JH (1963) Spores of mycorrhizal Endogonespecies extracted from soil by wet sieving and decanting. Trans Br MycolSoc 46:235–2446. Hammersachlag RS, Sisler HD (1973) Benomyl and methyl-2-benzimidazolecarbamate (MBC): biochemical cytological and chemicalaspects of toxicity to Ustilago maydis and Saccharomyces cerevisiae.Pestic Biochem Physiol 3:42–547. Hepper CM, Jacobsen I (1983) Hyphal growth from spores of themycorrhizal fungus Glomus caledonicum: effect of amino acids. SoilBiol Biochem 15:55–588. Hetrick BAD, Wilson GT, Kitt DG, Schvab AP (1988) Effects of soilmicroorganisms on mycorrhizal contribution to growth of big bluestemgrass in non fertile soil. Soil Biol Biochem 20:501–5079. Howard RJ, Aist JR (1980) Cytoplasmic microtubules and fungalmorphogenesis: ultrastructural effects of methyl benzimidazole-2-ylcarbamate determined by freeze- substitution of hyphal tip cells. J CellBiol 87:55–6410. INTA (1997) Guía práctica para el cultivo de soja. Campaña 1997. InstitutoNacional de Tecnología Agropecuaria, Buenos Aires, pp 12511. Isaac S (1992) Plant-fungal interactions. Chapman and Hall, London12. Kurle EJ, Pfleger FL (1994) The effects of cultural practices and pesticideson VAM fungi. In: Pfleger FL, Linderman RG (eds) Mycorrhizae andplant health. APS Press, St. Paul, Minnesota, pp 101–131 13. Menge JA (1982) Effect of soil fumigants and fungicides on vesicular-arbuscular fungi. Phytopathology 72:1125–113214. Mosse B (1962) The establishment of vesicular arbuscular mycorrhizaunder aseptic conditions. J Gen Microbiol 27:509–52015. Ocampo JA (1993) Influence of pesticides on VA mycorrhizae. In: AltmanJ (ed) Pesticide interactions in crop production, CRC, Boca Raton, Florida,pp 214–22616. Perrin R, Plenchette C (1993) Effects of some fungicides applied as soildrenches on the mycorrhizal infectivity of two cultivated soils and theirreceptiveness to Glomus intraradices. Crop Protection 12:127–13317. Stürmer SL, Morton JB (1997) Developmental patterns definingmorphological characters in spores of four species in Glomus. Mycologia89:72–8118. Tommerup IC, Briggs GC (1981) Influence of agricultural chemicals ongermination of vesicular-arbuscular endophyte spores. Trans Br MycolSoc 76:326–32819. Verkade SD, Hamilton DF (1983) Effects of benomyl on growth ofLiriodendron tulipifera L. seedlings inoculated with the vesicular-arbuscular fungus, Glomus fasciculatus. Sci Horticult 21:253–260175Efect of benomyl on G. mosseae INTERNATL MICROBIOL Vol. 3, 2000

Effect of the fungicide benomyl on spore germination and hyphal length of the arbuscular mycorrhizal fungus Glomus mosseae

http://revistes.iec.cat/index.php/IM/article/download/9272/9270

Effect of the fungicide benomyl on spore germination and hyphal length of the arbuscular mycorrhizal fungus Glomus mosseae

Abstract

Similar works

Full text

Available Versions

Hemeroteca Cientifica Catalana