Hydrogen peroxide concentrations detected in Agaricus bisporus sporocarps and relation with their susceptibility to the pathogen Verticillium fungicola

A dry bubble is an undifferentiated structure that forms in place of mushrooms when cultures of Agaricus bisporus are contaminated by Verticillium fungicola. Hydrogen peroxide concentrations were measured in lyophilised samples of bubbles and healthy sporocarps from cultures of genetically related strains of A. bisporus. The strains the more resistant to the pathogen had the higher levels of H2O2 concentration measured in the bubbles, but the differences in the healthy sporocarps were not significant. That is an indication of a higher reaction to the pathogen in the forming sporocarps of A. bisporus strains associated with their partial resistance to V. fungicola.     

Relationship between the presence of wall mucilage and the cellular disruption method employed in Agaricus bisporus tertiary mycelium

Abstract Hyphal walls of Agaricus bisporus fruiting bodies were prepared by three different methods of breakage. The chemical and electron microscopic results obtained support the idea that most of the polysaccharide mucilage, loosely bound to the cell walls, remains attached to them when a mild method of cell disruption is used.     

双孢蘑菇基因组密码子的偏好性分析

双孢蘑菇Agaricus bisporus是世界上最广泛栽培的食用菌之一.本研究通过分析双孢蘑菇基因组密码子使用偏性,探讨密码子偏性的影响因素及其对基因表达的影响.以双孢蘑菇基因组和转录组数据为依据,分析了双孢蘑菇基因组基因、高表达基因(high expression gene,HEG)和低表达基因(low expre...     

Comparison of partial sequence of the cap binding protein (eIF4E) isolated from Agaricus bisporus and its pathogen Verticillium fungicola

The 3 regions of the gene encoding the cap binding protein eIF4E were successfully isolated from Agaricus bisporus and Verticillium fungicola using a degenerate primer within the eIF4E gene and an anchored oligo d(T) primer. The deduced amino acid sequences contained 173 residues for A. bisporus and 171 residues V. fungicola. Analysis of these sequences shows that despite conserved regions of homology, centering around tryptophan residues, A. bisporus and V. fungicola are very diverse at the amino acid and DNA level. Percentage homology between the two fungi is low at the nucleotide, 35%, and amino acid level, 29%. The highest degree of similarity between the A. bisporus sequence and other published sequences is with the Homo sapiens eIF4E sequence (32%). V. fungicola exhibited highest homology with the eIF4E sequence from Caenorhabditis elegans (34%). Southern analysis of genomic DNA indicated a single copy of the gene within the A. bisporus genome.This revised version was published online in October 2005 with corrections to the Cover Date.     

Comparison of the changes in mushroom (Agaricus bisporus) compost during windrow and bunker stages of phase I and II

The changes in thermophilic fungi and biochemical characteristics, during windrow and bunker stages of phase I and phase II composts, were compared in this investigation. Composts prepared by the two phase I systems differed in a number of key parameters including mean straw length, population of Scytalidium thermophilum, dry matter, conductivity, nitrogen dry matter, ammonia, fibre content and ash. S. thermophilum populations in phase I composts were significantly higher in windrow compared to bunker‐composted materials as a result of the larger high temperature (65‐80°C) core in bunker treatment, which inhibited microbial activity. Assessment of the composts for loss of matter during composting has revealed that the bunker system can conserve fresh matter better than the windrow production system, possibly due to lower microbial activities during bunker composting. The productivity of the phase II composts prepared from windrow and bunker systems was compared in trials using commercial growers.     

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.     

The comparative effects of three formulations of diazinon on cropping of a hybrid and a non-hybrid strain of the cultivated mushroom Agaricus bisporus

Three formulations of diazinon: flowable granules (FG); wettable powder (WP); and emulsifiable concentration (EC), which are used for the control of mushroom pests, were compared for toxicity towards a hybrid and non-hybrid strain of the cultivated mushroom, Agaricus bisporus. With all formulations, yield and number of mushrooms were reduced according to dosage (linear trend). The EC was the most toxic and persistent formulation – equally so to both mushroom strains – and produced the most severe responses in all the parameters considered. The WP was the least toxic formulation. The hybrid strain was more susceptible to diazinon than the non-hybrid, but not markedly so.     

Relative contribution of nematodes (Caenorhabditis elegans) and bacteria towards the disruption of flushing patterns and losses in yield and quality of mushrooms (Agaricus bisporus)

Laboratory tests of bacteria isolated from the body surface, or from the gut, of a saprophagous rhabditid nematode Caenorhabditis elegans infesting mushrooms (Agaricus bisporus) showed that some bacteria enhanced nematode reproduction and that others inhibited it. As some bacteria were shown to inhibit mycelial growth of the mushroom, the effects of Acinetobacter calcoaceticus var. anitratus, Enterobacter cloacae and Serratia liquefaciens, either alone or in combination with C. elegans, on the flushing patterns, quality and yield of A. bisporus (strain Horst U3) were studied. Bacteria alone had little effect on flushing patterns whereas C. elegans delayed the onset of mushroom production and significantly disrupted the growth pattern of crops, with mushrooms appearing more regularly and not within obvious flushes. Inoculation with bacteria resulted in ‘browning’ of mushrooms that was even more pronounced in C. elegans treatments. Characteristic distortion of sporophores was observed only in the presence of C. elegans. Nematodes commonly colonised sporophores. Bacteria affected the size of nematode populations both on the sporophores and in the casing. Significant yield loss occurred; up to 10% when bacteria were inoculated, up to 27.8% when C. elegans was inoculated, and up to 35% with both bacteria and nematodes. Synergism between C. elegans and A. calcoaceticus var. anitratus was observed; the combination resulted in significantly greater reduction in mushroom yield than any other treatment. It is concluded that bacteria contribute to yield loss and quality deterioration in A. bisporus but that the effects are far greater in the presence of C. elegans.     

Comparative effects of three insect growth regulator insecticides and a dipteran-active strain of Bacillus thuringiensis on cropping of the cultivated mushroom Agaricus bisporus

Three insect growth regulator insecticides and an entomopathogenic strain of Bacillus thuringiensis (GC327), products effective against the mushroom sciarid, Lycoriella auripila, were compared for their effect on mushroom cropping. Cyromazine and diflubenzuron were applied as a surface drench to mushroom compost before or after pasteurisation (at filling or spawning, respectively); admixed into casing material (at casing); or at a combination of these times. Hexaflumuron and GC327 were applied only at filling and casing, respectively. The presence of the target pest, L. auripila, had no effect on treatment trends, although it was accounted for in the analysis by use of a yield model. The trial was notable for the disparate effects that cyromazine and diflubenzuron casing treatments had on mushroom cropping. Cyromazine treatments that included application at casing resulted in increases in yield, compared to the untreated control whereas, with diflubenzuron, the opposite was true, with treatment at casing alone causing the greatest reduction overall (10%). GC327 applied at casing was also conspicuous for giving a 13% increase in yield. Treating the crop at casing with either cyromazine or GC327, therefore, resulted in a 15% or 24% increase in yield, respectively, compared to a similar treatment with diflubenzuron. Hexaflumuron applied at filling caused increases in yield compared to application of cyromazine at filling and cyromazine or diflubenzuron at spawning. There were also effects on crop timing. The addition of a cyromazine casing treatment normally caused the distinct flushes of mushrooms to be produced significantly earlier than the untreated control (up to 2.5 days), as did GC327. With diflubenzuron, the earlier flushes were only produced by those treatments that did not include a casing application. The combinations that included a casing treatment with diflubenzuron initially produced mushroom flushes earlier than the untreated control. They became either synchronous with the control or they were delayed. From the crop tolerance perspective, therefore, cyromazine and GC327 would be the sciarid control products of choice for a commercial mushroom grower.     

The use of chemicals, antagonists, repellents and physical barriers for the control of Lycoriella auripila (Diptera: Sciaridae), a pest of the cultivated mushroom Agaricus bisporus

The sciarid, Lycoriella auripila, is a serious pest of commercial mushroom production. A series of trials demonstrated that the use of early, specifically-targeted, treatments of insecticides and/or antagonists and repellents, which distance treatment time from crop harvest, have the potential to play a useful part in the control of initial and subsequent generations of this pest. Of the treatments examined, those involving a drench treatment of the compost at filling (before pasteurisation) proved to be the most effective. Cyromazine and diflubenzuron were the most active insecticides tested, with cyromazine achieving a superior level of control of the initial infestation. Repellents and antifeedants were also effective, with calcium oxalate and sinapic acid both achieving about 50% control when applied at filling. Treatments applied later during the production cycle, unless in combination with a treatment at filling, were progressively less effective at controlling both the initial sciarid infestation and later generations of larvae. Multiple treatments caused greater reductions in fly populations than did the single treatments and continued to do so throughout the cropping cycle, the greatest reduction in the initial generation (79%) occurring with a triple treatment of cyromazine. With the exception of some diflubenzuron treatments, those that were effective resulted in increases in yield. The use of a physical paper barrier caused significant increases in both fly numbers and total yield.     

Development of a highly efficient gene targeting system for Fusarium graminearum using the disruption of a polyketide synthase gene as a visible marker

We cloned a polyketide synthase gene (pks12) from Fusarium graminearum, a devastating fungal pathogen of cereals. Transformation-mediated gene disruption led to an easily detectable albino phenotype of the disruptants. We used the disruption of the pks12 gene as a visible marker for transformation-mediated homologous recombination and optimized the transformation procedure to achieve a high rate of homologous recombination. In combination with the published genomic sequence data and the generation of expressed sequence tags (ESTs) for F. graminearum, this is a useful tool to investigate this important plant pathogen on a molecular level. Optimized transformation of F. graminearum resulted in at least 93% homologous recombination events when the homologous genomic DNA fragment in the vector had a size of approximately 800bp and was linearized in the middle. Using a genomic sequence of approximately 500bp in the transformation vector, 70% of the transformants still exhibited homologous recombination. On the contrary, no more than 10% homologous recombination events were observed when less than 400bp DNA fragments were used. We co-transformed F. graminearum with two different vectors. One vector harboured a DNA insert homologous to the pks12 gene, while the other vector consisted of the same vector backbone carrying the selection marker specific for F. graminearum. About 70% of the transformants had a disrupted pks12 gene, and all of these showed an integration of the second vector into the pks disruption vector. Therefore, the time-consuming construction of a single transformation vector can be avoided; furthermore, it is now easily feasible to express a gene construct at a defined and mutated genomic site.     

Cloning and characterization of genes specifically expressed during infection stages in the rice blast fungus

We isolated promoters of 12 genes from the rice blast fungus based on the sequences of randomly selected expressed sequence tags (ESTs) (appressorium formation stage cDNA library of Magnaporthe available from GenBank). These promoters (and the 5' coding regions if any) were fused in frame with egfp, and their expression patterns were examined under the epifluorescence microscope. Among them, two turned out to be specifically active in structures necessary for infection, viz. a promoter of adenylate cyclase interacting protein 1-like gene expressed in conidia, germ tubes, and appressoria, and a promoter of putative membrane-associated or secreted protein gene specifically expressed in appressoria. Although targeted knockout mutants of either gene failed to show detectable phenotypic alterations under laboratory conditions, these ESTs should be useful for identification of genes expressed during infection stages.     

Targeted disruption of genes for gp138, a cell-fusion-related protein in Dictyostelium discoideum, revealed the existence of a third gene

The sexual cycle of the cellular slime mold, Dictyostelium discoideum , offers a suitable experimental system to analyze sexual cell interactions. We have been analyzing molecular mechanisms involved in sexual cell fusion using complementary heterothallic strains in D. discoideum and have identified several cell surface proteins involved in the process. One of them, gp138 is present in strains of both mating types and considered to be responsible for membrane fusion itself. Two genes with high mutual homology, GP 138 A and GP 138 B , have been identified so far as encoding this protein. Expression of antisense RNA for GP 138 B has been shown to suppress sexual cell fusion, confirming the critical importance of these genes in sexual cell fusion. However, neither the functional relationship of the two gp138 genes nor the possibility of the existence of more genes that encode gp138 has been determined yet. In the present study, GP 138 A and GP 138 B were disrupted by homologous recombination in an effort to clarify these points. Analysis of the double knock-out mutants suggested the presence of a third gene for gp138.     

Insights into a nonhomologous integration pathway in the dermatophyte Trichophyton mentagrophytes: efficient targeted gene disruption by use of mutants lacking ligase IV

Targeted gene disruption experiments in Trichophyton mentagrophytes are impeded by the dominant of repair of DNA double strand breaks through a nonhomologous end joining pathway (NHEJ). Inactivation of human DNA ligase IV homologs, which is involved in the final step of the NHEJ pathway, has been shown to enhance homologous recombination (HR) frequency in filamentous fungi. To improve the frequency of HR in T. mentagrophytes, the lig4 homolog (TmLIG4) was disrupted. T. mentagrophytes lacking TmLIG4 showed no discernable phenotypic differences when compared to wild-type controls. Both mutant and parent strains had almost identical growth ability, sporulation rate and sensitivity to DNA damaging agents. When four different loci were disrupted in the TMLIG4-deficient mutant, HR frequencies reached as high as 93% depending on the locus, whereas they ranged from 0%-40% in the wild-type. These results suggest that studies in strains lacking TmLIG4 would help to improve our understanding of dermatophytosis by facilitating the genetic manipulation of dermatophytes.     

Chromosomal assignment of the chicken NP220 gene encoding a large DNA-binding nuclear protein and its targeted disruption in chicken DT40 cells

NP220s form a family of DNA-binding nuclear proteinsoriginally found in human cell lines. Four isoformsof NP220 are produced in humans and mice probably byalternative splicing of two sequence units. Theyhave RNA-recognition and arginine/serine rich motivescommonly found in many nuclear proteins important forpre-mRNA processing. In order to analyze the functionof NP220s, its gene was disrupted in chicken cell lineDT40. For this, gemomic DNA clone of chicken NP220was isolated and the gene was localized tochromosome 4q2.7–q2.8. Chicken NP220 conserves MH1and zinc finger-like motives found in human and mouseNP220s. Despite its expression as a mRNA of 6 kb inwild type DT40 cells, disruption of the NP220gene did not impair the growth of DT40 cells.     

Cloning and expression of a member of the Aspergillus niger gene family encoding alpha-galactosidase.

Summary An enzyme with -galactosidase activity and an apparent molecular weight of 82 kDa was purified from culture medium of Aspergillus niger. The N-terminal amino acid sequence of the purified protein shows similarity to the N-terminal amino acid sequence of -galactosidases from several other organisms. Oligonucleotides, based on the N-terminal amino acid sequence, were used as probes to clone the corresponding gene from a EMBL3 gene library of A. niger. The cloned gene (aglA) was shown to be functional by demonstrating that the 82 kDa -galactosidase is absent from a strain with a disruption of the agIA gene, and is over-produced in strains containing multiple copies of the aglA gene. Enzyme activity assays revealed that the 82 kDa -galactosidase A represents a minor extracellular -galactosidase activity in A. niger.