Chemical and structural differences in cell wall polysaccharides of two monokaryotic strains and their resulting dikaryon of Agaricus bisporus

Compatible monokaryotic strains of Agaricus bisporus ATCC 36975 and 36976 and the resulting dikaryon of their mating were grown in a liquid medium, and their respective cell walls were prepared. Significant differences were not found in the gross chemical composition of the three hyphal types. However, the neutral carbohydrate composition of the complete walls and their fractions was found to be somewhat different in each strain. More consistent differences were encountered in the chemical structure of the distinct polysaccharidic wall fractions in the three types of organisms. Some of these structural wall differences can be considered as characteristic markers for differentiating the mono- and dikaryotic types of A. bisporus. Received: 16 February 1996 / Accepted: 14 March 1996     

Some significant differences in wall chemistry among four commercial Agaricus bisporus strains

Significant differences in gross wall chemical composition were detected in four commercial Agaricus bisporus strains. All were grown under the same conditions and their walls prepared by a mild method of breakage. A more detailed analysis of the wall fractions, isolated by means of their distinct solubilities, also showed striking structural differences among the four strains studied. The detected differences, not only in the overall composition of the wall but also in the polysaccharide structure, could assist in the characterization of strains and/or varieties of the commercial basidiomycete A. bisporus.     

Differentiation and wall chemistry of Agaricus bisporus vegetative and aggregated micelia

Changes in the chemical composition of isolated cell walls and fractions were encountered during the differentiation of vegetative and aggregated mycelia of Agaricus bisporus.Differentiation was accompanied by quantitative variations of the wall polysaccharidic components. Neutral carbohydrates were composed of glucose, galactose, mannose and xylose and glucosamine as the only amino sugar. Differences in wall chemistry were correlated to the secondary and tertiary mycelial forms.     

Effect of the fungicide Prochloraz-Mn on the cell wall structure of <Emphasis Type="Italic">Verticillium fungicola</Emphasis>

The chemical structure of the cell wall of two isolates of Verticillium fungicola collected from diseased fruit bodies of the commercial mushroom Agaricus bisporus treated with the fungicide Prochloraz-Mn was analyzed. The isolates were obtained during different periods of time and grown in the absence and presence of the LD₅₀ values of the fungicide for V. fungicola. In addition, another V. fungicola isolate collected previous to the routine utilization of Prochloraz-Mn but grown under the same conditions was also analyzed. The overall chemical composition of the cell wall from the three isolates showed detectable differences in their basic components, with a significant decrease in the protein content in fungicide-treated cells. This inhibitory effect was partially compensated by an increase in neutral and/or aminated carbohydrates and was accompanied by appreciable modifications of polysaccharide structure, as deduced after methylation analysis and gas-liquid chromatography-mass spectrometry (GLC-MS). Moreover, differences in hyphal morphology caused by the fungicide were observed by transmission electron microscopy (TEM). Accepted 2 May 2002 Electronic Publication     

Cell wall composition of the yeast and mycelial forms of Yarrowia lipolytica

The cell walls of the yeast and mycelial forms of Yarrowia lipolytica were isolated and purified. Electron microscopy studies showed no differences between both types of cell walls. Chemical analysis revealed that the yeast cell wall contained 70% neutral carbohydrate, 7% amino sugars, 15% protein, 5% lipids and 0.8% phosphorus. Mycelial cell walls contained 70% carbohydrate, 14% aminosugars, 6% protein, 5% lipids and 0.6% phosphorus. Three polysaccharides: -glucan, mannan and chitin were detected. Proteins were solubilized from both cell wall fractions and separated by polyacrylamide gel electrophoresis. About 50 protein bands were detected, four of them corresponding to glycoproteins. The cell walls of the yeast and mycelial forms of Y. lipolytica were qualitatively similar and only quantitative differences were found.Abbreviations GlcNAc N-acetylglucosamine - FITC-WGA fluorescein isothiocyanate-wheat germ agglutinin - PAS periodic acid Schiff     

双孢蘑菇疣孢霉病的发病过程及病原菌的核相研究

【目的】确定有害疣孢霉的传播途径,明确双孢蘑菇受有害疣孢霉侵染后发病症状和微观形态变化,以及有害疣孢霉的核相。【方法】将有害疣孢霉喷施于培养料及覆土材料的不同深度,观察记录双孢蘑菇的发病情况;将有害疣孢霉接种于不同生长阶段的双孢蘑菇子实体,观察记录其发病情况;使用光学显微镜及扫描电镜观察双孢蘑菇子实体受有害疣孢霉侵染前后的形态变化;通过DAPI(4′,6-二脒基-2-苯基吲哚)染色的方法对有害疣孢霉核相进行观察。【结果】将有害疣孢霉接种于培养料及覆土层的不同深度得到双孢蘑菇发病率如下:覆土层表面覆土层中间覆土与培养料交界处培养料中间层;有害疣孢霉可以侵染双孢蘑菇的任意阶段,将其接种于原基直径小于3 mm子实体表面时,得到不能正常分化的"马勃状"组织;对有害疣孢霉的侵染过程进行观察得到:其孢子可粘附于双孢蘑菇表面,并萌发长出芽管,接种处双孢蘑菇表面产生褐色病斑,双孢蘑菇菌丝体发生质壁分离,最后菌丝体膨大,细胞壁变薄甚至溢裂,菌丝体内部中空;有害疣孢霉产生两种类型的分生孢子,Ⅰ类无隔膜含1个细胞核;Ⅱ类具1隔膜含2个细胞核,2个细胞核被隔膜分开;细胞核的第1次有丝分裂发生于分生孢子母细胞中;厚垣孢子由上下2个细胞构成,上胞中含有2个细胞核。下胞含1–2个细胞核。有害疣孢霉的厚垣孢子萌发可产生1–2个芽管,芽管中细胞核的数目不断变化,一般0–2个细胞核。【结论】双孢蘑菇受其侵染后发生显著的细胞学变化;我们对有害疣孢霉做遗传分析时,进行单孢分离需挑取无隔膜的分生孢子为实验材料进行遗传分析。     

Occurrence of Verticillium fungicola var. fungicola on Agaricus bitorquis Mushroom Crops in Spain

Diseased fruit bodies of Agaricus bitorquis, with similar symptoms to those caused by dry bubble on Agaricus bisporus, were observed in some Spanish crops during summer 1999. Isolates of Verticillium fungicola from A. bitorquis and A. bisporus were submitted to different temperatures and to prochloraz–Mn sensitivity tests. All the isolates collected from A. bitorquis and A. bisporus were identified as V. fungicola var. fungicola. Artificial infections of A. bisporus and A. bitorquis with V. fungicola var. fungicola are also described in the present study. The appearance of natural infections of V. fungicola var. fungicola in A. bitorquis crops could well be due to the growing temperatures used in Spain, which are considerably below those used in other countries.     

Antibodies against the fungal enzyme mannitol dehydrogenase

Affinity-purified, electrophoretically homogeneous NADP⁺-mannitol dehydrogenase (MtDH; EC 1.1.1.138), isolated from fruit bodies ofAgaricus bisporus (Lange) Sing., was used to produce polyclonal antibodies. Antiserum against MtDH and the affinity-purified antibody inhibited enzyme activity in a dose-dependent manner, with about 15 mol of purified antibody required for 50% inhibition of 1 mol MtDH. Immunological specificity of the antisera was demonstrated by double immunodiffusion, the dotimmunobinding assay, and immunoblotting. Controls (preimmune sera and preadsorbed antisera) were negative. An enzyme-linked immunosorbent assay (ELISA) was established to quantitate immunobinding in various cell fractions of the fungus. The bound protein was found predominantly in the soluble fraction (150,000g), where it contributed 5% to the total protein of the fruit body and 1% to that of the mycelium. Though only to a minor extent, the anti-MtDH antiserum also bound to the supernatants obtained following treatment of a “mixed membrane fraction” and the cell wall fraction (54,000g and 4,000g sediments, respectively; both rigorously washed with buffer) with digitonin or sodium dodecyl sulfate/heat. In sporocarp extracts, the antibody bound specifically to a protein of M_r 30K (corresponding to the subunit molecular weight of MtDH) as shown by immunoblotting, whereas mycelial extracts contained three (30K, 26K, 24K) protein bands, which cross-reacted with the anti-MtDH antiserum.     

Purine degradation in the edible mushroom Agaricus bisporus

Agaricus bisporus is able to use urate, allantoin, allantoate, urea and alloxanate as nitrogen sources for growth. The presence of urate oxidase, allantoinase, ureidoglycolase and urease activities, both in fruit bodies and mycelia, points to a degradative pathway for urate similar to that found in various microorganisms. So far all efforts, to demonstrate the enzyme responsible for allantoate degradation failed. A urease inhibitor appeared to be present in cell-free extracts, from fruit bodies.     

Electron-energy-loss spectroscopic imaging of calcium and nitrogen in the cell walls of apple fruits

Changes in texture are an integral part of ripening in most fleshy fruits and these changes are thought to be determined, primarily, by alterations in cell wall structure. Electron energy loss spectroscopy (EELS) imaging was used to obtain quantitative information on the levels of calcium and nitrogen in the cell walls of apple (Malus domestica Borkh. cv. Cox's Orange Pippin) fruits. Samples of fruit cortex were prepared for EELS by high-pressure freezing and molecular distillation drying to minimize loss and redistribution of soluble cell wall components such as calcium. The EELS imaging successfully resolved calcium and nitrogen levels in the middle lamella and primary cell wall. When the elemental compositions of the cell walls of Cox's apples from two sites in the UK were compared at harvest or after 6 months storage, the orchard which always produced consistently firmer fruit had significantly lower levels of cell wall calcium and higher levels of cell wall nitrogen. This result was unexpected since firm texture in apples and other fruits has been commonly associated with elevated levels of fruit calcium. The nitrogen-rich material in the sections used for EELS was insoluble in acidified methanol, indicating that it represented a high-molecular-weight component in the cell wall. Furthermore, total tissue hydroxyproline levels were greatest in material with elevated cell wall nitrogen, suggesting enhanced levels of wall structural proteins in the tissue. These data indicate a correlation between increased amounts of cell wall nitrogen and firm fruit texture. The possible role of cell wall proteins in determining the textural properties of fruit tissue is discussed. Received: 19 November 1998 / Accepted: 28 January 1999     

Subcellular distribution of aluminum, bismuth, cadmium, chromium, copper, iron, manganese, nickel, and lead in cultivated mushrooms (Agaricus bisporus and Pleurotus ostreatus)

In this work, the distribution of nine metals in two types of cultivated mushroom had been investigated. For Agaricus bisporus, the biomass was separated into caps and stalks, and for Pleurotus ostreatus, the entire mushrooms were taken for analysis. Electrothermal atomic absorption spectrometry was used for total element determination in acid digests. For accuracy checking, the certified reference material (NIST 1571, citrus leaves) was analyzed. The results obtained for the two fungi species were within the ranges of concentration reported previously by other authors. Subcellular fractionation was accomplished by centrifugation of cell homogenates, which has been suspended in Tris-HCl buffer. In the first centrifugation (7300g, 4°C, 10 min), cell walls were separated (pellet I), and the second centrifugation (147,000g, 4°C, 60 min) yielded mixed membrane fraction (pellet II) and cytosol (supernatant II). Recoveries of the fractionation procedure were in the range 70–100% (with the exception of Fe). For all elements studied, the highest relative contributions were found in cytosol fractions of the fruiting bodies (63–72%, 49–76%, 44–93%, 26–87pc, 55–85%, 50–68%, 41–78%, 39–78%, 54–67% respectively for Al, Bi, Cd, Cr, Cu, Fe, Mn, Ni, and Pb. Lower contributions were found in cell walls (respectively 22–32%, 24–44%, 6.1–47%, 12–52%, 7.3–37%, 7.9–32%, 19–52%, 20–42%, and 25–38%) and only minute amounts in the mixed membrane fraction (3.0–5.8%, 0.7–7.0%, 0.7–8.3%, 1.0–22%, 7.5–14%, 16–24%, 1.1–19%, and 5.1–7.7%). The results obtained indicate that small water-soluble molecules were the primary forms of nine elements in two mushroom species studied. On the other hand, the evidence has been provided on elements binding to larger, water-insoluble molecules contained in the structures of cell wall and membranes. The relative distribution was both element and fungi dependent. Thus, in P. ostreatus, total element levels were higher than in A. bisporus, with the preference for their accumulation in cytosol. On the contrary, total element content in the latter fungi was lower; however, a clear tendency toward more efficient element incorporation to the water-insoluble structures was observed (no apparent differences between stalks and caps). These findings might contribute in a better understanding of the accumulation of metals in mushrooms.     

Cell wall metabolism during fruiting of the basidiomycete Schizophyllum commune

During the development of fruit bodies of the basidiomycete Schizophyllum commune, the alkali-insoluble (R glucan) and alkali-soluble (S glucan) cell wall fractions are synthesized during the entire course of morphogenesis. The water soluble glucan (WSG) is not synthesized after an early stage. There is also a relative increase in the proportion of S glucan during development which appears related to a change in the proportion of the components synthesized. Data are also presented to show that several fruiting mutants also have specific cell wall differences, and that there is a significant contribution to cell wall structure by genes which do not cause a macroscopically observable change in phenotype.     

Peroxidase isozyme patterns in the skin of maturing tomato fruit

The cessation of tomato fruit growth is thought to be induced by an increase in the activity of enzymes which rigidify cell walls in the fruit skin. Peroxidase could catalyse such wall‐stiffening reactions, and marked rises in peroxidase activity were recently reported in skin cell walls towards fruit maturity. Peroxidase isoforms in the fruit are here analysed using native gel electrophoresis. New isoforms of apparent M_r 44, 48 and 53 kDa are shown to appear in cell walls of the fruit skin at around the time of cessation of growth. It is inferred that these isozymes are present in the cell wall in vivo. Fruit from a range of non‐ripening mutants were also examined. Some of these do not soften or ripen for many weeks after achieving their final size. The new isozymes were found in skin cell walls of mature fruit in each of these mutants, as in the wild‐type and commercial varieties. It is concluded that the late‐appearing isozymes are not associated with fruit ripening or softening, and are probably not ethylene‐induced. They may act to control fruit growth by cross‐linking wall polymers within the fruit skin, thus mechanically stiffening the walls and terminating growth.     

ENDOSPERM DEVELOPMENT IN THE DATE PALM (PHOENIX DACTYLIFERA) (ARECACEAE)

Date seeds were sampled at regular intervals from pollination (March) to mature fruit (September) and processed for light microscopy and SDS-PAGE. Seed fresh weight rose until early June and then declined slightly through September due to a continuous decrease in water content. Cell wall formation started in May in the free nuclear endosperm and proceeded centripetally from the inner integument to the seed center. Wall thickening in each cell started in cell corners and showed a layered appearance with calcofluor white staining. It started in early June in the center of the seed and proceeded centrifugally such that the outer cells showed cell wall thickening in late June. Thickened cell walls were soft and PAS positive at inception, but staining disappeared and hardness increased during wall maturation. Cell elongation in the radial direction accompanied wall thickening. Protein body formation started after cell wall thickening and followed the same centrifugal developmental pattern. Mature protein bodies occurred in even the outermost cells by early July. No further structural changes occurred after this time. The high molecular weight storage proteins appeared in late June, which is when protein bodies had formed in all but the outer endosperm cells; however, these proteins did not appear simultaneously and minor changes in protein bands continued until maturation. α-Galactosidase activity was present in the developing endosperm and peaked at 13 wk after pollination. The data suggest that the thickened wall is deposited as a highly substituted galactomannan, but that most of the galactose side branches are clipped off presumably by α-galactosidase during cell wall polymerization.     

Expression of the urease gene of Agaricus bisporus: a tool for studying fruit body formation and post-harvest development

Fruit body initials of Agaricus bisporus contain high levels of urea, which decrease in the following developmental stages until stage 4 (harvest) when urea levels increase again. At storage, the high urea content may affect the quality of the mushroom, i.e. by the formation of ammonia from urea through the action of urease (EC 3.5.1.5). Despite the abundance of urea in the edible mushroom A. bisporus, little is known about its physiological role. The urease gene of A. bisporus and its promoter region were identified and cloned. The coding part of the genomic DNA was interrupted by nine introns as confirmed by cDNA analysis. The first full homobasidiomycete urease protein sequence obtained comprised 838 amino acids (molecular mass 90,694 Da, pI 5.8). An alignment with fungal, plant and bacterial ureases revealed a high conservation. The expression of the urease gene, measured by Northern analyses, was studied both during normal development of fruit bodies and during post-harvest senescence. Expression in normal development was significantly up-regulated in developmental stages 5 and 6. During post-harvest senescence, the expression of urease was mainly observed in the stipe tissue; expression decreased on the first day and remained at a basal level through the remaining sampling period.     

Ultrastructure at the Host-Parasite Interface of Phytophthora capsici in Roots and Stems of Capsicum annuum

The infecting hyphae of Phytophthora capsici grew intercellularly in infected tissues of roots and stems of pepper (Capsicum annuum). The vascular tissues were not markedly disorganized even when heavily infected. Intercellularly growing hyphae penetrated the host cells by forming haustorium-like bodies. The consistent features of ultrastructural changes in infected tissues of pepper roots and stems were degeneration of cell organelles and dissolution of host cell walls. The cytoplasm detached from the cell wall aggregated abundantly around some haustorium-like bodies or the penetration sites of fungal hyphae. The host cell walls were palely stained, thinned and swollen, possibly being biochemically altered by the action of fungal macerating enzymes. Electron-dense, wall-like material was apposed on the outer wall of xylem vessel contacted by fungal hyphae. The infecting hyphae were also surrounded by granular, dark-staining cytoplasm. Characteristics of host cell responses to the invading P. capsici were the deposition of papilla-like material on host cell walls next to hyphae and the encasement of haustorium-like bodies with wall appositions.     

Peroxidase catalysed cross-linking of an intrinsically unstructured protein via dityrosine bonds in the oocyst wall of the apicomplexan parasite, Eimeria maxima

Apicomplexan parasites such as Eimeria maxima possess a resilient oocyst wall that protects them upon excretion in host faeces and in the outside world, allowing them to survive between hosts. The wall is formed from the contents of specialised organelles – wall-forming bodies – found in macrogametes of the parasites. The presence of dityrosine in the oocyst wall suggests that peroxidase-catalysed dityrosine cross-linking of tyrosine-rich proteins from wall-forming bodies forms a matrix that is a crucial component of oocyst walls. Bioinformatic analyses showed that one of these tyrosine-rich proteins, EmGAM56, is an intrinsically unstructured protein, dominated by random coil (52–70%), with some α-helix (28–43%) but a relatively low percentage of β-sheet (1–11%); this was confirmed by nuclear magnetic resonance and circular dichroism. Furthermore, the structural integrity of EmGAM56 under extreme temperatures and pH indicated its disordered nature. The intrinsic lack of structure in EmGAM56 could facilitate its incorporation into the oocyst wall in two ways: first, intrinsically unstructured proteins are highly susceptible to proteolysis, explaining the several differently-sized oocyst wall proteins derived from EmGAM56; and, second, its flexibility could facilitate cross-linking between these tyrosine-rich derivatives. An in vitro cross-linking assay was developed using a recombinant 42 kDa truncation of EmGAM56. Peroxides, in combination with plant or fungal peroxidases, catalysed the rapid formation of dityrosine cross-linked polymers of the truncated EmGAM56, as determined by western blotting and HPLC, confirming this protein’s propensity to form dityrosine bonds.     

Polygalacturonase isozyme 2 binding and catalysis in cell walls from tomato fruit: pH and β-subunit effects

The catalytic activity of endopolygalacturonase (PG, EC 3.2.1.15) against pectic polymers in vitro is typically not expressed in vivo. In the present study, the binding and catalytic properties of PG isozyme 2 and the influence of the β-subunit protein were investigated in cell walls prepared from tomato fruit expressing an antisense gene to the β-subunit protein. Cell walls prepared from mature-green fruit were employed for binding and assay of PG2. Walls were provided with rate-limiting quantities of purified PG2 and incubated at 100 mM KCl, pH 4.5, or 25 mM KCl, pH 6.0. Cell walls of both β-subunit antisense and wild-type fruit retained comparable quantities of added PG2. The release of pectin from PG2-loaded walls was proportional to the quantity of added enzyme, consistent with a finite catalytic capacity of individual PG proteins. β-Subunit-antisense cell walls released 2- to 3-fold higher levels of pectin in response to PG2 than did wild-type walls. Cell walls incubated at pH 6.0 released lower quantities and showed less extensive depolymerization of pectins than did walls incubated at pH 4.5. Pectins recovered from ripe fruit were similar in size distribution to polymers released by PG2 at pH 6.0, indicating that pH can influence both quantitative and qualitative aspects of pectin metabolism and may be responsible for the restricted hydrolysis of pectins in vivo. Molecular mass differences were not evident in the polymers rendered freely soluble in response to PG2-mediated hydrolysis of β-subunit-antisense compared with wild-type cell walls. The solubilization of pectin from cell walls was not the sole indicator of the extent of PG-mediated cell wall hydrolysis. Hydrolytic modifications were also evident in a pectic fraction extracted from postcatalytic cell walls with 50 mM CDTA (trans-1,2-cyclohexanediamine-N,N,N′,N′-tetraacetic acid), and were more extensive for the β-subunit-antisense cell walls compared with the wild-type walls. Pectic polymers derived from ethanol insoluble-powders showed molecular mass downshifts during ripening but differences between the β-subunit-antisense and wild-type fruits were not observed.     

Natural Abundance 13C Nuclear Magnetic Resonance Spectra of Intact Fungal Mycelia

We examined the change of the composition of the cell wall polysaccharides prepared from cells of the salt-tolerant yeast Zygosaccharomyces rouxii grown in two media containing 20% NaCl and 0% NaCl. Comparative analysis of their walls showed that the wall obtained from salt-free medium had greater quantities of alkali-insoluble fraction and smaller quantities of mannan than the walls obtained from 20% NaCl medium. The alkali-insoluble fractions from the cell walls obtained from salt-free medium contained a large amount of glucosamine and a smaller amount of linear β-1,3-glucosidic linkage than the fractions from the cell walls obtained from 20% NaCl medium. Structural analyses showed that the mannans from each cell wall contained an α-1,6-mannbsidic linked backbone to which single mannose and mannobiose units were connected as side chains by α-1,2-mannosidic linkages. However, when cells were grown in the presence of 20% NaCl, the side chains of the mannans consisting of a mannobiose unit were largely reduced.

These results indicated that the structure of alkali-insoluble glucan and mannan were greatly affected by the presence of NaCl in the final medium.