The white-line-in-agar test is not specific for the two cultivated mushroom associated pseudomonads, Pseudomonas tolaasii and Pseudomonas "reactans"

A sharply defined white line in vitro forms between the pathogenic form of Pseudomonas tolaasii and another Pseudomonas bacterium, referred to as "reactans". This interaction has been considered as highly specific. However, results presented in this study rise doubt about the strict specificity of this interaction, as some other pseudomonads, associated with the cultivated mushroom Agaricus bisporus, also yielded a white line precipitate when they were streaked towards Pseudomonas tolaasii LMG 2342T. Moreover, some Finnish isolates inducing brown blotch symptoms on mushrooms like P. tolaasii(T), produced a typical white precipitate when streaked towards P. "reactans" LMG5329, even though phenotypical and genotypical features exclude these isolates from the species P. tolaasii. We propose that the white-line-in-agar (WLA) test should no longer be considered as an unequivocal diagnostic trait of P. tolaasii.     

大蒜浸出液对平菇细菌性褐斑病病原菌托拉斯假单胞杆菌的抑菌作用

【背景】由托拉斯假单胞杆菌(Pseudomonas tolaasii)引起的平菇细菌性褐斑病在国内外大面积发生,导致产量降低,并有潜在的安全风险,寻找安全有效的抑菌剂对产业发展具有重要意义。【目的】通过5种不同溶剂提取得到大蒜浸出液,测定其对平菇细菌性褐斑病病原菌托拉斯假单胞杆菌的抑制作用,同时检测其对平菇菌丝生长的作用。【方法】利用抑菌圈法测定5种不同的大蒜浸出液对托拉斯假单胞杆菌的抑菌作用,利用平板扩散法筛选能促进平菇菌丝生长的药剂及适宜的浓度。【结果】5种大蒜浸出液原液对托拉斯假单胞杆菌均有显著的抑菌活性,其中大蒜山杏壳木醋液浸出原液抑菌效果最强。不同浓度的大蒜浸出液抑菌作用比较发现,浓度为10%的大蒜山杏壳木醋液浸出液具有较好的抑菌效果,其抑菌效果与0.33 mg/mL的链霉素相当,并对平菇菌丝生长有显著的促进作用,菌丝生长速度显著大于对照,并且菌丝浓密,边缘整齐。【结论】本研究为大蒜与山杏壳木醋液复配药剂防治平菇细菌性褐斑病奠定了实验基础。     

Enhancement of the antioxidants ergothioneine and selenium in Pleurotus eryngii var. eryngii basidiomata through cultural practices

Antioxidants are molecules that may reverse, prevent or slow cellular damage caused by free radicals. Increasing dietary intake of antioxidants is thought to reduce oxidative stress that may contribute to the development of several diseases. Mushrooms are known to contain antioxidants such as selenium, ergothioneine and phenolics that may serve this role. Here we sought to enhance selenium and ergothioneine concentration in Pleurotus eryngii var. eryngii basidiomata by modifying the techniques used for their commercial cultivation. To enhance selenium content in mushrooms, substrates were supplemented with sodium selenite (Na₂SeO₃) to reach selenium concentrations of 5 and 10 μg/g. Basidiomata of one commercial isolate (WC888) accumulated selenium up to 4.6 and 9.3 μg/g (d.w.), respectively. Therefore, a serving size (85 g) of fresh P. eryngii mushrooms produced on substrates supplemented with 5 and 10 μg/g of Na₂SeO₃ would supply 70.4 and 116.3% of the daily value of selenium (DV = 70 μg), respectively. Since selenium-enriched mushrooms would supply more than 20% of the DV, they could be considered an excellent source of selenium. Ergothioneine concentration was enhanced in mushrooms produced on low-moisture (55%) substrate compared to the commonly used 60% (high-moisture) in commercial cultivation. Mushrooms produced on low-moisture substrate had ergothioneine concentrations of 3.0 mg/g, while mushrooms produced on high-moisture substrate contained 2.2 mg/g or less. Use of a casing overlay for mushroom production resulted in significant yield increases on low-moisture substrate but not on high-moisture substrate.     

Isolation of a gram-positive bacterium effective in suppression of brown blotch disease of cultivated mushrooms,Pleurotus ostreatus andAgaricus bisporus, caused byPseudomonas tolaasii

A Gram-positive bacterium was isolated from a rottingPleurotus ostreatus fruiting body that markedly reduced the level of extracellular toxins (i.e., tolaasins) produced byPseudomonas tolaasii, the most destructive pathogen of cultivated mushrooms. The isolated bacterium is saprophytic but not parasitic nor pathogenic toP. ostreatus. A low ratio, ca. 10⁻³ cells of the isolated bacterium for oneP. tolaasii cells, was sufficient for detoxification in vitro. Inoculation of the isolated bacterium prevents the development of bacterial disease inP. ostreatus andAgaricus bisporus. The suppression of the disease development, however requires the initial cell density equivalent to ca. 10⁻¹ cells of the isolated bacterium for one cells of the pathogen. The effects is ascribed to the inactivation of tolaasin by the live, suppressive bacterial cells, and not to metabolites secreted from the organism into culture media. Examination by conventional bacteriological tests and with testing kits, i.e., MicroStation^TMSystem Release 3.5 (Biolog Inc., Hayward, CA), ATB Expression (bioMerieux Inc. Japan) and VITEK (bioMerieux Inc. Japan), failed to assign the organism to any defined bacterial genus. The suppressive bacterium may be useful in future for the development of biocontrol system and/or the construction of genetically modified edible fungi resistant to the disease caused byP. tolaasii.     

Evidence for genotypic differences between the two siderovars of Pseudomonas tolaasii,cause of brown blotch disease of the cultivated mushroom Agaricus bisporus

Sixteen representative isolates of Pseudomonas tolaasii, the causal agent of brown blotch of the cultivated mushroom Agaricus bisporus, were previously assigned to two siderovars (sv1 and sv2) on the basis of pyoverdines synthesized. Each isolate was pathogenic and produced a typical white line precipitate when cultured adjacent to Pseudomonas "reactans" strain LMG 5329. These 16 isolates of P. tolaasii, representing sv1 and sv2, were further characterized using genotypic methods to examine the relationships between the isolates. Rep-PCR studies revealed two distinct patterns from these isolates, which were consistent with the siderovar grouping. Ribotyping differentiated P. tolaasii LMG 2342T (sv1) and PS 3a (sv2) into two distinct ribotypes. A pair of primers, targeted to a 2.1-kb fragment of tl1 (encoding a tolaasin peptide synthetase), yielded the same PCR product from P. tolaasii LMG 2342T (sv1) and PS 22.2 (sv1), but not from PS 3a (sv2). Southern blot analysis indicated that homologues of tl1 are present in PS 3a, but the pattern of hybridization differed from PS 22.2 and LMG 2342T. Sequence determination and analysis of the internally transcribed spacer region ITSI for P. tolaasii LMG 2342T, LMG 6641, and PS 3a strains further supported the presence of the two siderovars. It is concluded that considerable genotypic differences exist among Finnish isolates of P. tolaasii causing brown blotch disease on the cultivated mushroom, which is in agreement with the phenotypic diversity highlighted through previous siderotyping studies.     

rtpA, a gene encoding a bacterial two-component sensor kinase, determines pathogenic traits ofPseudomonas tolaasii, the causal agent of brown blotch disease of a cultivated mushroom,Pleurotus ostreatus

Pseudomonas tolaasii strain PT814 produces extracellular toxins, tolaasins, and a volatile toxin, tovsin, that are responsible for the induction of brown blotch and rotting, respectively, in a cultivated mushroom,Pleurotus ostreatus. Insertions of single transposon mini-Tn5Km 1 into the chromosome ofP. tolaasii strain PT814 generated mutants that are pleiotropically defective in tolaasin and protease production, and altered in colony morphology. The mutants, however, produce tovsin at the level of wild-type. Variants phenotypically similar to the pleiotropic mutants ofP. tolaasii strain PT814 spontaneously occurred inP. tolaasii strain S8501 at 22–30°C in vitro. The occurrence of variants was significantly reduced in the presence of extracts ofP ostreatus or at a temperature of 15–20°C. ThertpA gene (rtpA=regulator gene of tolaasin production and other pleiotropic traits) isolated from aP. tolaasii strain PT814 gene library restored the wild-type phenotype in both the mini-Tn5km 1 insertion and spontaneous mutants. mini-Tn5km 1 insertions were also located in the allele ofrtpA. Nucleotide sequencing of thertpA DNA revealed an open reading frame of 2,751 bp predicted to encode a protein consisting of 917 amino acid residues with a molecular mass of 100.6 kDa and displaying the conserved amino acid sequence of both sensor, and receiver domains of “bacterial two-component regulators”. The data suggest that the machinery responding to environmental stimuli is essential for the pathogenic interaction ofP. tolaasii with the mushroom.     

The structure of the phosphorylated carbohydrate backbone of the lipopolysaccharide of the phytopathogen bacterium Pseudomonas tolaasii

A novel core-lipid A backbone oligosaccharide was isolated and identified from the lipopolysaccharide fraction of the mushrooms pathogen bacterium Pseudomonas tolaasii. The oligosaccharide was obtained by alkaline treatment of the lipopolysaccharide fraction. Since the repeating unit of the O-antigen contained one residue of -->4)-alpha-l-GulpNAcAN, the hydrolysis was accompanied by beta-elimination on this residue and following depolymerization, producing a mixture of oligosaccharides. The complete structural elucidation showed the presence of a single core glycoform and was achieved by chemical analysis and by (1)H, (31)P, and (13)C NMR spectroscopy applying various 1D and 2D experiments. [structure: see text]. All sugars are alpha-d-pyranoses, if not stated otherwise. Hep is l-glycero-d-manno-heptose, Kdo is 3-deoxy-d-manno-oct-2-ulosonic acid, P is phosphate. QuiN and DeltaGulNA are present in nonstoichiometric amount.     

Characteristics of stress response in a mushroom-pathogenic bacterium,Pseudomonas tolaasii, during the interaction withPleurotus ostreatus and carbon/nitrogen starvation in vitro

A brown blotch bacterium,Pseudomonas tolaasii strain PT814, expresses a high degree of cross-protection against generalized stress imposed by physical/chemical treatment, H₂O₂, UV, high temperature, ethanol and NaCl during the interaction withPleurotus ostreatus. Stress resistance was also noted in the bacterium in vitro under limited carbon and nitrogen sources. In addition, changes in cell morphology from a “metabolically active” rod to an “energy-saving” spherical shape were detected during starvation and the interaction. All the changes under stress were reversible. A homologue ofrpoS (σ ^S), a regulator that controls such physiological status during starvation in other bacteria, was identified inP. tolaasii strain PT814. Data suggest that the bacterium is able to withstand a complex stress environment for its survival through changes in its metabolic pattern.     

Occurrence ofPseudomonas tolaasii on fruiting bodies ofLentinula edodes formed onQuercus logs

A bacterial disease occurred on fruiting bodies ofLentinula edodes that formed outdoors onQuercus bedlogs during winter. The pathogen was identified asPseudomonas tolaasii based on morphological and bacteriological characteristics. Symptoms exhibited by infected fruiting bodies ranged from mild browning to severe necrotic cavities that characteristically developed in the cap tissue along the periphery of the attachment area to the stalk. The mode of symptom development was greatly influenced by the internal tissue structure of fruiting bodies. Multiplication of bacterial cells within the fruiting bodies was strictly intercellular and thus differed from previously reported bacterial disease ofL. edodes incited by an unidentified rod-shaped bacterium. The present strain ofP. tolaasii was capable of attacking theL. edodes mycelium in the inner bark and outer sapwood regions and caused lysis of heavily infected hyphae.Paper No. 301 of the Tottori Mycological Institute.     

Molecular cloning ofglpFKRD of a mushroom-pathogenic bacterium,Pseudomonas tolaasii strain PT814: induction of an avirulent mutant carrying a single mini-Tn5km 1 insertion inglpD

Pseudomonas tolaasii strain PT 814 causes brown blotch disease in cultivated mushrooms. A pleiotropic avirulent mutant was isolated by mini-Tn5km 1 insertion mutagenesis. The insertion was localized in an open reading frame (ORF) predicted to encodesn-glycerol-3-phosphate dehydrogenase (glpD). ORFs that should encode its regulator, kinase, and facilitator were also identified as theglp gene cluster in the bacterium. The data suggest that theglp system may contribute to the ecology of this pathogen.     

Structural determination of the O-specific chain of the lipopolysaccharide from the mushrooms pathogenic bacterium Pseudomonas tolaasii

The complete structure of the O-specific polysaccharide of the lipopolysaccharide isolated from the cultivated mushrooms pathogen Pseudomonas tolaasii is described. The structural determination, achieved by chemical and spectroscopical analyses, indicates a novel tetrasaccharide repeating unit built up of two units of 2-acetamido-2,6-di-deoxy-glucopyranose (Quinovosamine, QuipNAc) and two units of 2-acetamido-2-deoxy-gulopyranuronamide (GulpNAcAN), one of which is acetylated at C-3 position:     

Cultivation of different strains of king oyster mushroom (Pleurotus eryngii) on saw dust and rice straw in Bangladesh

Pleurotus eryngii is a popular mushroom due to its excellent consistency of cap and stem, culinary qualities and longer shelf life. In Bangladesh, where Pleurotus mushrooms are very popular, P. eryngii may take position among the consumers, but currently this mushroom is not cultivated in large scale there. In this study, 3 strains of P. eryngii such as Pe-1 (native to Bangladesh), Pe-2 (germplasm collected from China) and Pe-3 (germplasm collected from Japan) were cultivated on saw dust and rice straw and their growth and yield parameters were investigated. Pe-1 on saw dust showed the highest biological yield and efficiency (73.5%) than other strains. Also, the mycelium run rate and number of fruiting bodies were higher in Pe-1 than other two strains. The quality of mushroom strains was near about similar. On saw dust, the yield and efficiency were better than those cultivated on rice straw, however, on straw; the mushroom fruiting bodies were larger in size. This study shows the prospects of P. eryngii cultivation in Bangladesh and suggests further study in controlled environment for higher yield and production.     

The structure of a pyoverdine produced by a Pseudomonas tolaasii-like isolate

Cultures of Agaricus bisporus, the most extensively cultivated mushroom, can be infected severely by Pseudomonas tolaasii. This pathogen is characterized by the so-called white line reaction, a precipitate formed on agar plates between its colonies and those of P. reactans, both belonging to the collective species P. fluorescens. A recent study has shown that a group of P. tolaasii isolates can be subdivided into two groups or 'siderovars', based on the pyoverdines they produce (Munsch et al. 2000). One group of strains is characterized by the pyoverdine described by Demange et al. (1990). A representative of the second group (strain Ps3a) was found to produce the same pyoverdine as a strain which had been classified before as P. aureofaciens. However, based mainly on 16S rRNA gene sequence comparisons and REP-PCR generated fingerprints, the two strains are not identical. They are also distinguishable from the P. tolaasii type strain.     

Pseudomonas tolaasii and tolaasin: comparison of symptom induction on a wide range of Agaricus bisporus strains

Abstract A wide range of Agaricus bisporus , including commercial, wild and hybrid strains, were tested for resistance to brown blotch disease caused by Pseudomonas tolaasii . Effects of toxin and living bacteria were compared. Wild and hybrid A. bisporus ranged in the same order from very poorly to highly susceptible whatever the inoculum type used, tolaasin or bacteria. Symptom aspects induced by both inocula were visually identical, but some differences occurred in response intensity. The data suggest that toxin is probably not the only factor involved in symptom development.     

Unusual partially 3-O-methylated alpha-galactan from mushrooms of the genus Pleurotus

Indistinguishable partially 3-O-methylated galactans were isolated from the edible basidiomycetes Pleurotus eryngii and Pleurotus ostreatoroseus. They were obtained via successive aqueous extraction, freeze-thawing, precipitation with Fehling solution of soluble material, and ultrafiltration. Mono- and bidimensional 13C and 1H-nuclear magnetic resonance spectroscopy (HMBC, HETEROTOCSY, COSY, and HMQC), and methylation analysis were used to determine their structures. They were linear, partially 3-O-methylated, (1-->6)-linked alpha-d-galactans containing Gal and 3-Me-Gal, in a 3:1 molar ratio (GC-MS of alditol acetates).     

Pseudomonas fluorescens NZI7 repels grazing by C. elegans,a natural predator

The bacteriovorous nematode Caenorhabditis elegans has been used to investigate many aspects of animal biology, including interactions with pathogenic bacteria. However, studies examining C. elegans interactions with bacteria isolated from environments in which it is found naturally are relatively scarce. C. elegans is frequently associated with cultivation of the edible mushroom Agaricus bisporus, and has been reported to increase the severity of bacterial blotch of mushrooms, a disease caused by bacteria from the Pseudomonas fluorescens complex. We observed that pseudomonads isolated from mushroom farms showed differential resistance to nematode predation. Under nutrient poor conditions, in which most pseudomonads were consumed, the mushroom pathogenic isolate P. fluorescens NZI7 was able to repel C. elegans without causing nematode death. A draft genome sequence of NZI7 showed it to be related to the biocontrol strain P. protegens Pf-5. To identify the genetic basis of nematode repellence in NZI7, we developed a grid-based screen for mutants that lacked the ability to repel C. elegans. The mutants isolated in this screen included strains with insertions in the global regulator GacS and in a previously undescribed GacS-regulated gene cluster, ‘EDB'' (‘edible''). Our results suggest that the product of the EDB cluster is a poorly diffusible or cell-associated factor that acts together with other features of NZI7 to provide a novel mechanism to deter nematode grazing. As nematodes interact with NZI7 colonies before being repelled, the EDB factor may enable NZI7 to come into contact with and be disseminated by C. elegans without being subject to intensive predation.     

人工疏蕾对杏鲍菇产量及品质的影响

【背景】工厂化栽培中,杏鲍菇是不定点出菇,通常采用人工疏蕾的方法来实现对子实体数目的控制,但目前关于人工疏蕾保留子实体的数目无具体的研究。【目的】通过人工疏蕾的方式,研究保留不同子实体数目对杏鲍菇产量及品质的影响。【方法】对各处理组进行基质利用情况测定,对采收后各组子实体进行产量、形态指标及质构特性的测定。【结果】保留3—4个子实体的基质利用率较高,在生产实际中对成本的浪费较少;保留不同子实体数目对子实体形态指标、单菇重量及单包产量都有显著影响,保留1个子实体时,单菇重量最大但单包产量最低,保留4个子实体时,单包产量较高且子实体形态一致;从质构特性来看,保留子实体数目为4个时,杏鲍菇子实体品质最好、口感最佳。【结论】在杏鲍菇工厂化生产的人工疏蕾环节,保留子实体数目为3—4个时可以减少对成本的浪费,获得产量较高、品质较好的产品。     

DNA-fingerprinting (AFLP and RFLP) for genotypic identification in species of the <Emphasis Type="Italic">Pleurotus eryngii</Emphasis> complex

Wild populations of edible species are important source of genetic variability for cultivated lines that can undergo a drastic loss of diversity resulting from man’s selection. The development of tools aimed at the clear-cut and safe identification and assessment of genetic variability of the wild and cultivated strains is thus a fundamental goal of molecular genetic research. In this study, we used two polymerase chain reaction (PCR)-based fingerprinting methods—amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) of laccase and manganese peroxidase genes—to assess genetic differences among strains and independently evolving lineages belonging to the Pleurotus eryngii complex. Both laccase RFLP and AFLP have been proved to distinguish unambiguously the three taxa studied: Pleurotus ferulae, P. eryngii, and P. eryngii var. nebrodensis. AFLP also showed enough sensitivity to detect polymorphisms among the strains, proving to be an efficient DNA fingerprinting tool in studies of strain assignment. The divergent RFLP laccase and manganese peroxidase patterns are also discussed in relation to the role played by these genes in the interaction between these fungi and their host plants.     

Intergeneric hybridization between Pleurotus ostreatus and Schizophyllum commune by PEG-induced protoplast fusion

Intergeneric hybridization between Pleurotus ostreatus and Schizophyllum commune was studied using PEG-induced fusion. The fusion of protoplasts from auxotrophic mutant strains resulted in the formation of fusion hybrids in the frequencies of 3.6 to 7.3×10^–5. Most of these fusion hybrids were monokaryotic and sterile and no heterokaryosis occurred. Most fusants showed a significantly higher nuclear DNA content when compared to parental strains and no diploids (parent 1 genome plus parent 2 genome) were found. Some fusion hybrids revealed both parental fragments in nuclear and mitochondrial rDNA PCR profiles. AP-PCR (Arbitrarily-primed Polymerase Chain Reaction) fingerprints also indicated that most of the fusion products were recombinant hybrids.