RESEARCH ARTICLE
Dolores Bernardo1 Amelia Pérez Cabo1 Monique Novaes-Ledieu1 José Pardo2 Concepción García Mendoza1* 1Biological Research Center, CSIC, Madrid, Spain 2Center for Research, Experimentation and Services of Mushrooms, Quintanar del Rey, Cuenca, Spain	Comparative effect of the fungicide Prochloraz-Mn on Agaricus bisporus vegetative-mycelium and fruit-body cell walls   Summary. Fungicides to control mycopathogens of commercial Agaricus bisporus, a mushroom cultivated for human consumption, are a major field of study, since these chemicals are toxic to both the host and its fungal parasites. The fungicide Prochloraz-Mn, used at its LD50 for A. bisporus, partially inhibited protein biosynthesis in the vegetative mycelial cell walls of this mushroom and caused significant changes in cell-wall polysaccharide structure, as deduced by methylation analysis and gas liquid chromatography-mass spectrometry (GLC-MS). Furthermore, the aggregated mycelial walls showed distinct alterations in their overall chemical composition following the administration of Prochloraz-Mn at the LD50 and the LD50 ×1000. As expected, GLC-MS studies indicated that the latter dose caused more appreciable differences in polysaccharide structure. The decrease in mushroom crop yields obtained from industrial cultures treated with Prochloraz-Mn to control V. fungicola infection depended on the dose of the fungicide employed, whereas fruit-body morphology was only slightly affected at the highest Prochloraz-Mn concentration used. [Int Microbiol 2004; 7(4):277-281] Key words: Agaricus bisporus · Prochloraz-Mn · vegetative-mycelial cell walls · fruit-body cell walls · carbohydrate rearrangement
Received 14 June 2004 Accepted 23 September 2004   *Corresponding author: C. García Mendoza Centro de Investigaciones Biológicas-CSIC Ramiro de Maeztu, 9 28040 Madrid, Spain Tel. +34-918373112. Fax +34-915360432 E-mail: cgm@cib.csic.es

 

 

Estudios comparativos del efecto del fungicida Prochloraz-Mn sobre las paredes celulares del micelio vegetativo y de los cuerpos fructíferos de Agaricus bisporus Resumen. Los fungicidas para el control de micopatógenos de Agaricus bisporus, un hongo de cultivo comercial para consumo humano, representan un importante tema de estudio debido a que son tóxicos tanto para el huésped como para sus parásitos fúngicos. El fungicida Prochloraz-Mn, empleado a su LD50 para A. bisporus, inhibe parcialmente la biosíntesis de proteínas en las pared celular del micelio vegetativo de este hongo y provoca cambios significativos en la estructura polisacárida de la pared celular, tal como se observa mediante el análisis de metilación y la cromatografía líquida de gases-espectrometría de masa (GLC-MS). Además, las paredes agregadas del micelio presentan diferentes alteraciones en la composición química global después de la administración de Prochloraz-Mn a la LD50 y LD50 ×1000. Como cabría esperar, los estudios de GLC-MS, indican que la última dosis causa más diferencias apreciables en la estructura polisacárida. La disminución en la producción del hongo en los cultivos industriales tratados con Prochloraz-Mn para controlar la infección por V. fungicola, dependía de la dosis de fungicida empleada, mientras que la morfología del cuerpo fructífero sólo resulta ligeramente afectada a la concentración de Prochloraz-Mn más elevada. [Int Microbiol 2004; 7(4):277-281] Palabras clave: Agaricus bisporus · Prochloraz-Mn · paredes celulares del micelio vegetativo · paredes celulares de los cuerpos fructíferos · redisposición de carbohidratos

Estudos comparativos sobre o efeito do fungicida Prochloraz-Mn sobre as paredes celulares do micélio vegetativo e dos corpos de frutificação de Agaricus bisporus Resumo. Os fungicidas usados no controle de micopatógenos de Agaricus bisporus, um fungo de cultivo comercial para consumo humano, representam um importante tema de estudo devido ao fato de que são tóxicos tanto para o hospedeiro como para os parasitas fúngicos. O fungicida Prochloraz-Mn, empregado na sua dose LD50 para A. bisporus, inibe parcialmente a biossíntese de proteínas da parede celular do micélio vegetativo deste fungo e provoca mudanças significativas na estrutura polissacarídica da parede celular, tal como se observa mediante a análise de metilação e de cromatografia líquida de gases-espectrometria de massa (GLC-MS). Por outro lado, as paredes agregadas do micélio apresentam diferentes alterações na composição química global depois da administração de Prochloraz-Mn nas doses LD50 e LD50 ×1000. Como esperado, os estudos de GLC-MS, indicaram que a última dose causa diferenças mais significativas na estrutura polissacarídica. A diminuição na produção do fungo nos cultivos industriais tratados com Prochloraz-MN para controle da infeção por V. fungicola, foi dependente da dose de fungicida empregada, enquanto que a morfologia do corpo de frutificação resulta ligeiramente afetada somente quando foi usada uma concentração mais elevada de Prochloraz-Mn. [Int Microbiol 2004; 7(4):277-281] Palavras chave: Agaricus bisporus · Prochloraz-Mn · paredes celulares do micélio vegetativo · paredes celulares dos corpos de frutificação · reposicionamento de carbohidratos

 

Introduction

Verticillium disease, or dry bubble disease, caused by Verticillium fungicola, affects the cultivated white mushroom Agaricus bisporus and is a persistent problem for mushroom farmers. Until now, crop losses have been limited by means of sanitation, culture methods and chemical applications. Although Prochloraz-Mn has been fundamental to preventing V. fungicola infection of A. bisporus, during the last decade the mycoparasite has become less sensitive to this fungicide [5] due to the development of resistance. Thus far, there has been one study dealing with the effect of Prochloraz-Mn on the cell wall of V. fungicola [2], but there are no reports in the literature of studies on the effects of this fungicide on A. bisporus vegetative and fruit-body mycelia, which are also exposed to its actions. Moreover, mushroom crop yields of industrial cultures have been shown to be affected to a certain extent by the routine use of Prochloraz-Mn, which is not surprising since both the mycopathogen and the host are fungi.

Previous studies on the mode of action of Prochloraz-Mn [1,6,8,11] have shown sterol biosynthesis inhibition, suggesting at the same time that there is more than one mechanism of inhibition (multi-site fungicide). This hypothesis was recently confirmed by demonstrating partial protein inhibition together with polysaccharide rearrangement in the cell walls of the mycopathogen V. fungicola due to the fungicide [2]. The present work describes a new approach and is a complement to those studies in that it analyzes possible changes in the chemical structure of the cell walls of both vegetative and aggregated mycelia of A. bisporus in the presence of Prochloraz-Mn. The consequences that these changes might have on the agronomic characteristics of the mushroom and on crop yields in industrial cultures due to the routine use of this fungicide in the control of verticillium disease have also been evaluated.

Materials and methods

Organism, culture conditions and preparation of hyphal cell walls. A. bisporus fruit bodies (Fungisem H 25, a smooth white non-hybrid variety developed by Fungisem S.A., Autol, La Rioja, Spain) were supplied by the Centro de Investigación, Experimentación y Servicios del Champiñón (CIES, Quintanar del Rey, Cuenca, Spain). A. bisporus vegetative mycelium was obtained by growing this organism on Raper medium [10] for 2 weeks. When necessary, the fungicide Prochloraz-Mn complex 46% (Sporgon, Schering) was added to the Raper medium and used at the corresponding LD₅₀ value obtained (0.34 mg pure product/l). The LD₅₀ was measured as described previously [2].

A. bisporus fruit bodies were grown at the CIES in the presence or absence of the fungicide Prochloraz at the LD₅₀ calculated for the vegetative mycelium. In another set of experiments, the fruit bodies were grown at a fungicide concentration of LD₅₀ ×1000 (340 mg/l), which is close to the recommended agronomic dose (500 mg commercial product /l). In both cases, the aggregated mycelial cell walls were prepared in the same way.

For cell-wall preparations, both vegetative and aggregated mycelia were freeze-dried, pulverized in a Sorvall omnimixer and then disintegrated in a Fritsch 6 balls-mill. The hyphal wall fragments were purified by repeated washings with distilled water until the material was completely clean (as determined by phase-contrast microscopy and the absence of protein in the water washings) and stored freeze-dried for further analysis.

Fractionation of cell-wall material and analytical techniques. Cell-wall polysaccharides were fractionated as described previously [3], except that fraction FIV was not separated into subfractions a and b. All procedures for chemical analysis, monosaccharide identification and methylation analysis have been described before [2].

Results and Discussion

Before examining the effect of Prochloraz-Mn on the chemical structure of A. bisporus vegetative mycelium and fruit-body cell walls, it is important to consider that yields of the mushroom crop in industrial cultures were virtually unaffected by treatment with the LD₅₀ of the fungicide, but were somewhat inhibited (2.48-3.14%) by the LD₅₀ ×1000. Slight variations of the sporophore surface texture, from smooth to rough, were observed only in response to the highest dose of Prochloraz-Mn used.

Differences in the overall cell-wall composition between A. bisporus vegetative and aggregated mycelium were shown previously [3,4]. The presence of the fungicide LD₅₀ partially inhibited the synthesis of cell-wall proteins of the vegetative mycelium (Table 1); there was a slight increase in the hexosamine content but the levels of neutral sugars remained unchanged. By contrast, in the aggregated mycelial walls, protein synthesis was not inhibited at concentrations of LD₅₀ or even at LD₅₀ ×1000. Instead, the amount of protein tended to increase following application of Prochloraz-Mn, accompanied by a progressive decrease in the hexosamine content.

Cell-wall fractionation yielded six different fractions, mainly composed of neutral carbohydrates together with amino sugars and proteins. The percentages of dry weight and neutral carbohydrates of the fractions isolated from the two kinds of walls (Table 2) differed only slightly in their response to Prochloraz-Mn, as in the aggregated walls the small increase in the dry weight of fractions FIII, FIV and FV was offset by the decrease in total neutral sugars of fractions FI, FII and FIV. Carbohydrate analysis of A. bisporus vegetative mycelial walls (Table 3) revealed the major presence of glucose in all of the extracted fractions. Nonetheless, treatment with Prochloraz-Mn caused considerable changes in the percentages of monomers (Table 3). For example, a significant increase in the amounts of glucose and galactose and the disappearance of mannose and xylose were observed in fraction FII, whereas in fraction FIII the increase in the percentage of glucose was due entirely to the absence of xylose whereas galactose and mannose remained unchanged. The compositions of the other fractions were not affected by the fungicide.

Regarding aggregated mycelial walls (Table 3), significant increases in the amounts of some monosaccharides in the isolated cell-wall fractions were found following treatment with Prochloraz-Mn, such as mannose in fraction FI, xylose in fraction FII and galactose in fraction FIV. The monosaccharide compositions of the other fractions changed only slightly, but xylose decreased in FIV, and glucose in FII.

The results of methylation analysis of A. bisporus vegetative and aggregated mycelial walls are given in Table 4 and are in good agreement with previously reported data on the chemical structure of both types of cell walls [3,4], although significant differences were detected after fungicide treatment. Fraction FI of vegetative mycelial walls (Table 4) consists of mucilage, mainly corresponding to a linear α-(1-4)-linked glucan with low percentages of (1-3) mannose and (1-6) galactose. These monosaccharides became more branched in response to Prochloraz-Mn, with the glucose linkages increasing to (1-3,4) and (1-4,6). Fraction FII, a mostly linear α-(1-3) glucan, was clearly affected by the fungicide, which inhibited the formation of xylose and mannose residues, increased (1-3,6) glucose linkages, and caused the introduction of a small amount of galactose into the furanose form. In the presence of Prochloraz-Mn, fraction FIII, composed mainly of a (1-4)-linked glucan, the mannose content was maintained whereas the number of (1-3,4) and (1-4,6) glucose units in xylose increased; also, as in the previous fraction, there was a small amount of galactofuranose. The components of fraction FIV also became more branched, in the form of (1-3,6) glucose linkages, due to the action of the fungicide. Fractions FV and FVI were less affected, but there was also a tendency toward increased branching.

Table 4 shows also the methylation analysis of the fractions isolated from aggregated mycelial walls of A. bisporus in the presence and absence of the fungicide. Fraction FI (mucilage) is essentially composed of a linear α-(1-4)-linked glucan, as seen in vegetative mycelial walls, with significant percentages of (1-3) and (1-6) glucose, (1-6) galactose, and (1-3) mannose. These became somewhat more branched in the presence of Prochloraz-Mn, with glucose linkages increasing to (1-3,4) and (1-3,6) and the terminal xylose being eliminated. In fraction FII, comprising mainly α-(1-3)-linked glucosyl residues, the (1-3) and (1-3,4) mannose linkages increased and the (1-6) and (1-2,3) glucose residues decreased in response to the fungicide. Fraction FIII, consisting mainly of (1-6)- and (1-3)-linked glucosyl residues, showed a significant decrease in (1-6) galactose and (1-4) glucose units, which, in Prochhloraz-Mn-treated samples, became more branched due to increased (1-3,4) and (1-3,6) glucosyl linkages. The other three fractions, FIV, FV and FVI, were less affected by the fungicide and showed only small increases in (1-3) mannose (FV) and a slight decrease in (1-6) galactose (FVI).

Preliminary experiments carried out using the shadowing technique followed by transmission electron microscopy (TEM) of both types of cell walls in the presence and absence of Prochloraz-Mn did not suggest significant differences in their respective hyphal surfaces, as was reported for the mycoparasite V. fungicola treated with the same fungicide [2].

The different effects induced by Prochloraz-Mn on vegetative mycelium and fruit-body A. bisporus cell walls could be related to their distinct chemical structures [3,4]. This might explain the greater sensitivity of vegetative mycelial cell walls to the fungicide, and the observation that fruiting bodies only developed from surviving hyphal strands that were not affected by the fungicide. Taken together, the results show a restructuring of both A. bisporus vegetative and aggregated mycelial cell walls after exposure to Prochloraz-Mn. Rearrangement of the different carbohydrates led to partially modified chemical structures. However, these modifications apparently did not affect the main agronomic characteristics of the mushrooms. The significance of these changes requires further investigation in light of the fact that the fungicide shows some toxicity not only to the mycopathogen V. fungicola [2] but also to its host, A. bisporus, which is cultivated for human consumption. New strategies eliminating the use of this chemical must be developed in order to control verticillium disease while avoiding possible secondary effects on human consumers. Note, however, that oral administration of Prochloraz-Mn to rats results in the complete metabolism and excretion of the fungicide [7,9].

Acknowledgements. Financial support for this study was provided by the Dirección General de Investigación Científica y Técnica (CICYT, grant AGL-2001-1885)) and the Junta de Comunidades de Castilla-La Mancha (grant 188/CH-51). Thanks are also given to Dr. A. Prieto for her valuable technical assistance in methylation experiments.

References

1.Barbera C (1994) Principales grupos de fungicidas. Mecanismos de acción. Phytoma España 62:7-10        [ Links ]

2. Bernardo D, Novaes-Ledieu M, Pérez Cabo A, Gea Alegría FJ, García Mendoza C (2002) Effect of the fungicide Prochloraz-Mn on the cell wall structure of Verticillium fungicola. Int Microbiol 5:121-125        [ Links ]

3. Calonje M, García Mendoza C, Novaes-Ledieu M (1996) New contributions to the wall polysaccharide structure of vegetative mycelium and fruit body cell walls of Agaricus bisporus. Microbiología SEM 12:599-606        [ Links ]

4. García Mendoza C, Avellán MA, Sánchez E, Novaes-Ledieu M (1987) Differentiation and wall chemistry of Agaricus bisporus vegetative and aggregated mycelium. Arch Microbiol 148:68-71        [ Links ]

5. Gea FJ, Tello JC, Honrubia M (1996) In vitro sensitivity of Verticillium fungicola to selected fungicides. Mycopathologia 136:133-137        [ Links ]

6. Hassall KA (1990) The biochemistry and uses of pesticides, 2nd ed. VCH, Weinheim        [ Links ]

7. Laignelet L, Rivière JL, Lhuguenot JC (1992) Metabolism of an imidazole fungicide (Prochloraz) in the rat after oral administration. Food Chem Toxicol 30:575-583        [ Links ]

8. Leroux P (1991) Résistance des champignons phytopathogènes aux fongicides. Phytoma 434:20-26        [ Links ]

9. Nedham D, Challis IR (1991) The metabolism and excretion of prochloraz, an imidazole-based fungicide, in the rat. Xenobiotica 21:1473-1482        [ Links ]

10. Raper CA, Raper JR, Miller RE (1972) Genetic analysis of the life-cycle of Agaricus bisporus. Mycologia 64:1088-1117        [ Links ]

11. Siegel MR (1981) Sterol-inhibiting fungicides: Effects on sterol biosynthesis and sites of action. Plant Dis 65:986-989        [ Links ]