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     

Differentiation between Commercial Strains of Oyster and Button Mushrooms Using Molecular Markers

Analysis of commercial strains of two edible mushrooms, Pleurotus ostreatus and Agaricus bisporus, using PCR and isozyme electrophoresis techniques allowed us to differentiate groups of genetically similar and distant strains. Among the commercial strains of P. ostreatus, the level of genetic variation was higher suggesting a broader genetic basis employed in breeding of this mushroom. The cultivars and hybrids of A. bisporusshowed a higher level of homology. The isozyme markers (nonspecific esterase, leucinaminopeptidase, and phosphoglucoisomerase) are recommended for identification of the commercial strains of edible mushrooms.     

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     

Chemical Analysis of the Lamella Walls of Agaricus bisporus Fruit Bodies

Purified lamella wall fragments of Agaricus bisporus fruit bodies were analyzed and shown to consist of neutral sugars (46.5%), hexosamines (31.7%), proteins (9.5%), some lipid material (10.0%), and ash (1.4%). The cell walls were fractionated on the basis of their polysaccharide solubility in water and alkaline solutions. The isolated fractions, using methylation analysis, exhibited striking chemical structural differences compared with the same fractions obtained from the corresponding vegetative cells and fruit bodies (stipe and pileus) walls. The structural differences detected in the wall seem to correspond to the ultimate differentiation of the mycelium inside the fruit body of A. bisporus. Received: 30 November 1998 / Accepted: 29 January 1999     

Assessment of genetic relatedness among commercial and wild strains of Agaricus bisporus using repetitive extragenic palindromic sequences and polymerase chain reaction

The genetic relatedness among 18 strains of Agaricus bisporus was assessed based on the fragment pattern analysis obtained by the amplification of genomic DNA by BOX, ERIC (ERICIR-I/ERIC2) and REP (REP1RI/REP2I) gene sequences. Based on the banding patterns of PCR-amplified products, eight putative groups among the 18 commercial and wild strains were recognized. REP-PCR generated multiple distinct products showing considerable variability among the strains with ERIC and REP elements successfully enabled detection of wild and commercial A. bisporus. Strains originating from the same geographical location were not always genetically related. To our knowledge, this was the first relevance study of biodiversity in commercial and native populations of A. bisporus by using the REP-PCR technique. The results confirmed the usefulness REP-PCR typing in intraspecific genetic variation assessments of the button mushroom. High level of Iranian wild strains distance with the commercial cultivars approves their importance as a promising new source of diversity in A. bisporus breeding program.     

Proteomic investigation of interhyphal interactions between strains of Agaricus bisporus

Hyphae of filamentous fungi undergo polar extension, bifurcation and hyphal fusion to form reticulating networks of mycelia. Hyphal fusion or anastomosis, a ubiquitous process among filamentous fungi, is a vital strategy for how fungi expand over their substrate and interact with or recognise self- and non-self hyphae of neighbouring mycelia in their environment. Morphological and genetic characterisation of anastomosis has been studied in many model fungal species, but little is known of the direct proteomic response of two interacting fungal isolates. Agaricus bisporus, the most widely cultivated edible mushroom crop worldwide, was used as an in vitro model to profile the proteomes of interacting cultures. The globally cultivated strain (A15) was paired with two distinct strains; a commercial hybrid strain and a wild isolate strain. Each co-culture presented a different interaction ranging from complete vegetative compatibility (self), lack of interactions, and antagonistic interactions. These incompatible strains are the focus of research into disease-resistance in commercial crops as the spread of intracellular pathogens, namely mycoviruses, is limited by the lack of interhyphal anastomosis. Unique proteomic responses were detected between all co-cultures. An array of cell wall modifying enzymes, plus fungal growth and morphogenesis proteins were found in significantly (P < 0.05) altered abundances. Nitrogen metabolism dominated in the intracellular proteome, with evidence of nitrogen starvation between competing, non-compatible cultures. Changes in key enzymes of A. bisporus morphogenesis were observed, particularly via increased abundance of glucanosyltransferase in competing interactions and certain chitinases in vegetative compatible interactions only. Carbohydrate-active enzyme arsenals are expanded in antagonistic interactions in A. bisporus. Pathways involved in carbohydrate metabolism and genetic information processing were higher in interacting cultures, most notably during self-recognition. New insights into the differential response of interacting strains of A. bisporus will enhance our understanding of potential barriers to viral transmission through vegetative incompatibility. Our results suggest that a differential proteomic response occurs between A. bisporus at strain-level and findings from this work may guide future proteomic investigation of fungal anastomosis.     

Novel microsatellite markers suitable for genetic studies in the white button mushroom <Emphasis Type="Italic">Agaricus bisporus</Emphasis>

Co-dominant microsatellite molecular markers were obtained from the Agaricus bisporus cultivated mushroom. Their potential for both the molecular characterisation of commercial strains and the monitoring of the intraspecific genetic variation was demonstrated. The analysis of 673 unique sequences issued from public database and 59 from an enriched A. bisporus genomic library resulted in the development of a total of 33 single sequence repeat or microsatellite (SSR) markers. Their usefulness for genetic analysis was assessed on 28 strains, which include six cultivars representative of traditional lineage, two hybrids and 20 strains originating from wild populations. A. bisporus SSR markers displayed each from two to ten alleles, with an average of 5.6 alleles per locus. The observed heterozygosity ranged from 0 to 0.88. Cluster analysis resulting from SSR fingerprintings was in agreement with published A. bisporus population structure. A combination of only three selected SSR markers was sufficient to discriminate unambiguously 27 out of 28 distinct genotypes. However, the two genetically related hybrids were not distinguishable. Multiplexing was tested, and up to seven loci could be genotyped simultaneously. We are therefore reporting the first development in A. bisporus of a set of microsatellite markers powerful and suitable for genetic analysis.     

Heterosis among lines of mice selected for body weight

Summary After treatment of one strain of A. bitorquis and 12 strains of A. bisporus in modified monokaryotization solution, three types of mycelia were received: one is the original di- or heterokaryon, the other two were proven to be neohaplonts in A. bitorquis and two strains of A. bisporus. In neohaplonts of good fruiting strains, homokaryotic fruiting was observed.     

The indigenous coastal Californian population of the mushroom Agaricus bisporus, a cultivated species, may be at risk of extinction

This study employed nuclear and mitochondrial markers to assess the present-day composition of the population of Agaricus bisporus in coastal California. Favourable weather in the fall, winter and spring of 1990–91 furnished an uncommon opportunity to collect and study field material of the ‘button mushroom’A. bisporus, a cultivated species, from the region. The previous such season occurred 13 years earlier. Ninety-five nonredundant cultures from field material were prepared and genotypically characterized. These data were combined with data from earlier studies. Multilocus nuclear and mitochondrial genotypes were determined for 123 individuals. Genotypes were compared in pairwise fashion both within the sample and between this sample and others of diverse geographical origin or commercial provenance. Using parametric analysis and cluster analysis of nuclear similarities, and also mitochondrial data, two elements – indigenous and European – were apparent within the sample. This was consistent with our earlier results on a much smaller sample. At least 10 mitochondrial haplotypes (MTs) were present; based on genotypic similarities of associated nuclei, five (or six) MTs were Californian, four were European, and one was ambiguous. Based on MT origins, 54% of the 121 classifiable individuals in California were of European ancestry; natives constituted a minority at 46%. Even in the indigenous Monterey cypress habitat, where 84% of all individuals from California were sampled, non-native A. bisporus appeared to have achieved parity (at 48–49%) with the native population. In all other habitats, which are far more extensive, European individuals outnumbered Californian natives by 4:1. Some evidence of hybridization between the two ancestral groups was found. European strains appear to have been resident in California for approximately one century. The extensive occupancy of the native habitat by non-native germ plasm, the apparent inability of native strains to occupy or compete in non-native habitats, and the disproportionately large inoculum reservoirs represented by non-native habitat and agronomic activities all suggest that the native population is under considerable competitive pressure in what may be a very dynamic situation. If this surmise is correct, the native population may be at serious risk of further contraction, irreversible dilution through interbreeding, and possibly even extinction.     

Studies of the cell walls of Schizophyllum commune

Mechanically isolated cell wall materials of eight strains of Schizophyllum commune were studied by chemical and enzymatic procedures. Isolated wall material of each strain was separated by chemical methods into three fractions: A (cold alkali-soluble, , amorphous), B (warm alkali-soluble, amorphous), and C (alkali-insoluble, retaining appearance of hyphal fragments). Chemical tests indicated the presence of chitin in Fraction C and the absence of cellulose, lignin and pectic substances from all fractions. Analyses of acid hydrolysates indicated the presence of glucose in Fractions A, B and C, of hexosamine in Fraction C and the absence of galactose, mannose, 6-deoxyhexoses, xylose and other pentoses from all fractions. Unfractionated material, Fraction A and Fraction B were slightly attacked by commercial cellulase whereas Fraction C was heavily attacked. Commercial chitinase by itself did not attack Fraction C or unfractionated material to a significant extent. In the presence of cellulase, it was active upon Fraction C. Qualitative differences in cell wall composition between strains were not detected; however, quantitative differences were observed in the proportion of Fraction A and Fraction C as well as in the amount of the various breakdown products in certain strains. It is visualized that the cell wall of this fungus consists of a core of chitin covered by or intermeshed with glucose-containing polymers.     

Comparative studies of the quality of fresh and stored mushrooms of Agaricus bisporus with two tropical Agaricus bitorquis strains

Three white strains of mushroom were grown for quality assessment tests, a commercial Agaricus bisporus strain SOMYCEL U3 currently popular in most major mushroom producing countries and two tropical Agaricus bitorquis strains, ATCC 32675 and AGC W20. Mushrooms were harvested as stage 2 mushrooms (closed buttons with universal veil intact) and stored at 18°C (± 0.5°C) for 5 days during which time colour development, the rate of fruitbody maturation and weight loss were assessed. Throughout the storage period, a reflectance colormeter was used to monitor colour changes on the tops and sides of mushroom fruitbodies. The tops of both A. bitorquis strains were significantly more yellow than the A. bisporus strain, whereas the sides were significantly less yellow. Overall, the A. birorquis strain AGC W20 was clearly the least discoloured and least yellow at the time of harvest. Although all the three strains tested gave similar fresh weight losses during storage, i.e. approximately 10% per day, ATCC 32675 exhibited a very slow maturation rate. Both U3 and AGC W20 matured at a similar much faster rate forming open cups within the 5 day storage period. ATCC 32675 also showed the least increase in the degree of discolouration with time, whether readings were taken from the sides or tops of mushrooms. A breeding programme to combine the most salient features of AGC W20 (an intensely white mushroom at harvest, high yielding with distinct flush pattern) and ATCC 32675 (very slow maturation rate during storage) is suggested.     

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.     

DNA polymorphisms in commercial and wild strains of the cultivated mushroom,Agaricus bisporus

Summary DNA from the cultivated mushroom, Agaricus bisporus, was cloned into the bacteriophage lambda vector EMBL3 creating a partial genomic library. Ten random clones from the library were used to probe for restriction fragment length polymorphisms (RFLPs). Six of the ten probes detected polymorphisms and were used to demonstrate variation in wild and cultivated strains of the mushroom. These results suggest that RFLPs could form a basis for genetic finger-printing and subsequent strain protection in A. bisporus. In single spore progeny, RFLPs were used to demonstrate normal meiotic segregation and to differentiate between homokaryons and heterokaryons. RFLPs therefore have great potential in the development of the genetics and breeding of this commercially important species.     

Tolerance to dry bubble disease (Lecanicillium fungicola) in Iranian wild germplasm of button mushroom (Agaricus bisporus)

Agaricus bisporus is the most widely cultivated mushroom. The mushroom crop is subjected to several fungal diseases. Dry bubble disease caused by Lecanicillium fungicola is among notorious diseases of A. bisporus. This study aimed to assess phenotypic resistance to dry bubble disease among A. bisporus wild strains, collected from Iran regions. The reliability of resistance evaluations regarding disease incidence and intensity was well documented. The extraordinary tolerance of some wild strains to even high degrees of inoculum concentrations (10⁷ and 10⁸ spore/m² mushroom growth bed) of the pathogen in compare to commercial cultivars approved potentials of the wild germplasm in breeding programs for resistance. Also, the potential of some Microsatellite loci for the molecular-based rapid screening of tolerance was established by attributing SSR loci of phenotypically tolerant strains to QTLs for dry-bubble resistance-related traits.     

Differentiation of commercial strains of <Emphasis Type="Italic">Agaricus</Emphasis> species in China with inter-simple sequence repeat marker

In recent years, Agaricus mushroom production has undergone an important increase in the world. However, there is no consensus over the taxonomy of some commercial strains. So rapid and reliable methods to identify Agaricus strains are urgently needed. In this paper, six inter-simple sequence repeat (ISSR) primers were screened from 20 primers to analyse the genetic diversities of the 12 main commercial strains of Agaricus in China. The results showed that a total of 124 bands were produced, among which 80.6% was polymorphic, the similarity coefficients ranged from 0.69 to 0.98. Twelve tested strains were divided into 2 groups on the similarity coefficient of 0.75. Three Agaricus bitorquis strains were clustered in one group, and seven Agaricus bisporus strains were clustered with Agaricus strains S1 and S2 in the other group. As comparison with ISSR, intergenic spacers combined with restriction fragment length polymorphisms (IGS-RFLP) was employed to analyse genetic diversities of the tested strains by digesting IGS amplified products with restriction enzymes Hae III, Alu I, Rsa I, respectively, and IGS-RFLP cluster results were similar with those of ISSR. However, ISSR was superior to IGS-RFLP in differentiating closely related strains. It is suggested that the ISSR marker is an alternative method to differentiate Agaricus strains.     

Comparative study of Aspergillus mycotoxin production on enriched media and construction material

Isolates of Aspergillus flavus and Aspergillus fumigatus from indoor air were compared with a known mycotoxin producer for their capacity to produce mycotoxins on a variety of enrichment media and with growth on indoor substrates such as ceiling tile and wall board. In enrichment media, four of seven isolates of A. flavus produced at least one aflatoxin and both isolates of A. fumigatus produced mycotoxins. The spectrum of mycotoxins and their concentrations varied with the strain and medium. When the mycotoxin-positive strains were grown to a dense concentration on indoor construction and finishing materials such as ceiling tile and wall boards, mycotoxins were not detected in extracts of the materials. Colonization of indoor surfaces by mycotoxin-producing strains of A. flavus and A. fumigatus may not necessarily expose inhabitants to mycotoxins or result in production of mycotoxins. Received 09 February 1999/ Accepted in revised form 28 June 1999     

Biological control of mushroom brown blotch disease using antagonistic bacteria

Button mushroom brown blotch disease is one of the most important and devastating diseases in Iran which is caused by Pseudomonas tolaasii. To screen antagonistic bacteria against this pathogen, major mushroom cultivation centres in Iran were surveyed and samples were taken from compost, soil cover and button caps with or without visible symptoms. In total, 120 bacteria were isolated on the basis of their morphology and pathology on excised tissue blocks of the fresh Agaricus bisporus. Among all tested bacteria, thirty-six isolates produced variable inhibition zones and reduced the symptom incidence on tissue blocks of A. bisporus. The most effective antagonistic bacteria caused almost 100% inhibition of pathogenic bacterium. To confirm the identification of antagonistic bacteria, RNA polymerase beta-subunit gene (rpoB) of five antagonistic strains (A1, A2, A3, A4 and A6) were amplified using primer sets of long amplicon primers (LAPS) and LAP27. The polymerase chain reaction products of the strains A1, A3 and A6 were sequenced. Based on phenotypic, biochemical and molecular characteristics, the bacterial antagonists were identified as P. putida (A1), P. reactants (A2 and A6), P. fluorescens (A3 and A4) and Bacillus subtilis (A5), respectively. In all four criteria including weight of the diseased and healthy caps, per cent disease severity and per cent disease incidence, the treatment 2 (P. fluorescens A4) was the most effective from among T1, T3, T4 and T5 treatments. Overall results of this study suggest that bacterial antagonists may be potential biocontrol agents for biological promotion of the health and growth of button mushroom.