Analysis on Bacterial Community Structure in Mushroom(Agaricus bisporus) Compost Using PCR-DGGE

This study aimed to investigate the bacterial communities in mushroom compost piles composed of rice straw, corn stover, and cow dung. Bacterial com- munities of samples at the beginning of composting, at the end of fermentation phase I and II were collected and analyzed using Polymerase Chain Reaction-De- naturing Gradient Gel Electrophoresis （PCR-DGGE） based on 16S rDNA universal primers from Escherichia coli. A total of 56 different clone sequences were obtained （GenBank accession number： KF630598-KF630653）. They were classified into seven phyla and 42 genera. Dominant microflora during composting belonged to phylum Proteobacteria, Firmicutes, and Actinobacteria, with the dominant genera of Bacillus, Paenibacillus, Thermomonospora, Thermasporomyces, Pseudomonas, and Cellvibrio. Bacterial diversity （Shannon index） analysis showed that bacterial species in com- post pile composed mainly of rice straw continuously increased during composting, while those in compost pile composed mainly of corn stover firstly increased and then reduced. Principal component analysis showed that corn stover compost sam- ples at the end of fermentation phase I and phase II were clustered into one group, suggesting that corn stover composted faster than anticipated. In general, rice straw compost has higher bacterial diversity but longer composting time period, while corn stover compost has lower bacterial diversity but shorter composting time period.

Research of Holotrichia diomphalia Larvae Cultivation Technology with Agaricus bisporus Residues

[Objective] The aim was to explore key technology to cultivate Holotdchia diomphalia larvae with Agaricus bisporus residues and to provide, technologicar sup- ports for processing of Agaricus bisporus residues. [Method] In the research, fodder thickness, population density and residues under different treatments were set to re- search effects on Holotrichia diomphalia larvae. [Result] The optimal thickness of fodder was 25 cm and the optimal feeding density was 44-56 larvae per hectare. The dry residues were more suitable, compared with decomposed residues and corn bran powders, for cultivation of Holotrichia diomphalia larvae. [Conclusion] Cultivation of Holotrichia diomphalia larvae with Agaricus bisporus residues is a new method to make use of Agaricus bisporus residues and of significance for extension of agricul- tural circulation chain, increase of economic benefits and ecological benefits.

Whole-year Cultivation Techniques of Agaricus bisporus in Lianyungang District

Conventional Agaricus bisporus production is mainly concentrated in au- tumn and winter. Thus the Agaricus bisporus supply is not available throughout the year. Even worse, the price of Agaricus bisporus is cheap in boom season, reduc- ing the economic benefits; but the product is unavailable off season. The whole-year production of Agaricus bisporus will help to solve the problem and increase the eco- nomic benefits of farmers. In this paper, the whole-year cultivation techniques of Agaricus bisporus were summarized. There are a total of 3 seasons of Agaricus bisporus （spring, summer, autumn-winter） throughout the year. And thus the whole- year supply of Agaricus bisporus is achieved, the utilization of mushroom house is improved and the income of farmers is increased.

Standardized Cultivation Technology of Agaricus bisporus

Agaricus bisporus is a complete nutrition food, widely cultivated, and the demand on cultivation is general y lower, which advances Agaricus bisporus industry to certain extent. In order to improve yield of Agaricus bisporus, the research ruled the standardized cultivation technology and increased the yield by 20%, which can be an effective way for improving farmers’ incomes.

Molecular Cloning and Expression of Polyphenoloxidase Genes from the Mushroom, Agaricus bisporus

The polyphenoloxidase（PPO） is the key enzyme considered to be responsible for mushroom browning.By using homology cloning and rapid amplification of cDNA ends（RACE）,two new PPO genes and the corresponding cDNA were identified from the fruit bodies of Agaricus bisporus（AbPPO3 and AbPPO4,GenBank accession nos.GU936494 and GU936493,respectively）.The genomic DNA sequences of AbPPO3 and AbPPO4 are 2 080 and 2 189 bp in length,respectively,encoding putative polypeptides of approximately 66 and 68 kDa.The deduced amino acid sequences show characteristic features of two copper-binding domains conserved in the type III copper proteins including fungal polyphenol oxidases.Sequence comparisons indicate that AbPPO3 and AbPPO4 present 55.3% similarity to each other（48% identity）.We also obtained more than 1.5-kb long sequences upstream of the start codon of the AbPPO3 and AbPPO4 and recognized them as their respective putative promoters.Analyses of the two PPO promoter regions show that they contain abundant cis-acting elements which are probably responsible for anaerobic induction,light,wound,stress,and auxin response.Semi-quantitative RT-PCR results indicate that AbPPO3 and AbPPO4 were highly expressed in the mature fruit bodies and up-regulated after 2-d storage of mushroom.These results suggest that AbPPO3 and AbPPO4 may play roles in A.bisporus browning and pigmentation during development and postharvest storage and the elements in promoters may act as regulatory elements for the inducible expression of AbPPO3 and AbPPO4.The successful cloning and expression analysis of AbPPO3 and AbPPO4 warrant a further investigation on the structure and function of A.bisporus PPO which points to the possible targets for genetic manipulation.

Identification of commercial cultivars of Agaricus bisporus in China using genome-wide microsatellite markers

Agaricus bisporus is one of the most widely cultivated mushrooms in the world. Commercial cultivars are usually phenotypically alike and easy to be copied by isolating tissue cultures. This brings great challenges to distinguish different cultivars and to protect new varieties. Thus, techniques for the accurate identification of cultivars are essentially required. In this study, we accurately identified 11 commercial cultivars of A. bisporus released in China by using microsatellite(SSR, simple sequence repeat) markers. SSR markers were developed by mining the genome sequence. A total of 3 134 SSRs were identified, of which 1 490 SSRs were distributed in gene models, and 1 644 in the intergenic regions. A total of 17 polymorphic primer pairs were developed and SSR fingerprints were constructed for all the commercial cultivars. These SSR markers generated a total of 73 alleles, with an average of 4.29 per locus. Specifically, the primer combination of AB_SSR_2341 and AB_SSR_2590 could distinguish all the 11 commercial cultivars. The similarity coefficients of the 11 commercial cultivars were between 0.56 and 0.95 indicating that some of them were close related. Our results provide an efficient technique for the identification of A. bisporus cultivars in China, which can also facilitate the marker-assisted breeding in the future.

Screening on Chemicals for Preventing Mycogone peniciosa Magn in Agaricus bisporus

[ Objective ] The aim was to screen out chemicals with good control effect against Mycogone peniciosa Magn in Agaricus bisporus. [ Method ] M. peniciosa ofA. b/sporus was taken as the detection bacteria, and nine kinds of chemicals were conducted plate efficacy test in the laboratory. Mushroom house validation was carried out for the screened chemicals with strong inhibition to M. peniciosa. [ Result] Nine kinds of chemicals had obvious difference in control effects on M. peniciosa and effects on growth ofA. bisporus; 2 400 and 3 200 times dilution of 78% Boer MZ WP and 2 400 times dilution of 69% Fulishuang WP after casing material processing had good control effects on M. peniciosa and had no apparent adverse effects on mycelial growth and fruiting development of A. bisporus. [ Conclusion] The 2 400 and 3 200 times dilution of 78% Boer MZ WP and 2 400 times dilution of 69% Fulishuang WP are worth popularizing.

Two Routes to Produce Chitosan from Agaricus bisporus

Two methods were used to produce chitosan by deacetylation of chitin which was extracted from Agaricus bisporus stipes.The first one gives chitosan 1 with low yield of 2.5%,degree of acetylation(DA)of 4%,molecular weight(MW)of 2.973×10^(5)(g/mol).The second route produces chitosan 2 with higher yield of 41%,degree of acetylation(DA)of 17.23%,molecular weight(MW)of 2.939×10^(5)(g/mol).Both chitosans were characterized by XRD,FTIR,^(1)H-NMR spectroscopy nuclear magnetic resonance of proton.The molecular weight(MW)was determined by size exclusion chromatography(SEC).Thermal analysis shows that both chitosans have moisture content lower than 10%.However chitosan 2 has the less ash%which is the quality grade for chitosan medical applications.Accordingly,fungal chitosan 2 could have potential medical and agricultural applications.

Effect of protease inhibitor from Agaricus bisporus on glucose uptake and oxidative stress in 3T3-L1 adipocytes

Objective:To explore the effect of the protease inhibitor from Agaricus bisporus(J.E.Lange)Imbach(AbPI)on glucose uptake and oxidative stress in 3 T3-L1 adipocytes.Methods:Adipocytes were differentiated and stained with OilRed-O staining to confirm adipogenesis.The toxic/protective effect of AbPI on the adipocytes was determined by MTT assay,intracellular reactive oxygen species generation through flow cytometry,and morphologically through confocal microscopy using propidium iodide,4,6-diamino-2-phenylindol dihydrochloride,and 2’,7’-dichlorofluorescein diacetate dyes.The uptake of fluorescent glucose analog,2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose by adipocytes was also studied through confocal microscopy.Results:MTT assay showed that the cell survival rate was(28.00±3.00)%,(92.33±2.60)%,and(71.34±2.10)%in the presence of 2 mM H2O2,AbPI alone,and AbPI and H2O2 both,respectively,in comparison to the control.Oil-Red-O staining indicated that Ab PI enhanced adipogenesis.AbPI stimulated the glucose uptake by adipocytes similar to the drug rosiglitazone,and showed insulinsensitizing effect in the presence of insulin,but failed to stimulate the uptake in the absence of insulin.Intracellular reactive oxygen species generation was reduced in differentiating adipocytes upon Ab PI treatment.Confocal microscopy showed that the damaged cell population rose to 3.50%,117.84%,and 261.50%in the presence of Ab PI alone,AbPI with H2O2,and H2O2 alone,respectively.Conclusions:The protease inhibitor enhances glucose uptake by adipocytes and exhibits a cytoprotective effect on them.

Morphological Features of Agaricus bisporus and Indoor Toxicity Test of Chemicals

Through separating and purifying diseased mushrooms infected by Mycogone perniciosa in Gaochun Area, Nanjing City, morphological features of the bacteria were identified, and toxicity of the bacterial strains was tested. The results showed that five bactericides in the experiment had obvious inhibitory effect on the growth of M. perniciosa, especially 50% Prochloraz manganese chloride complex WP 5 g/L had the best efficiency.

Preliminary Study on Mixed Use of Agaricus bisporus Residue and Microbial Agent to Control Cucumber Root-knot Nematode

Single or mixed use ofAgaricus bisporus residue and microbial agent had better control effect on cucumber root-knot nematode disease in greenhouse. The results showed that the control effect of single use of A. b/sporus residue was 32.9% - 34.7%, and the yield increasing rate was 2.1% - 8.7% ; the control effect of mixed use ofA. b/sporus residue and microbial agent was 59. 1% - 63. 6%, and yield increasing rate was 2.6% - 3.5%. The experimental dose had no ad- verse effects on soil physical and chemical properties. The mixed use of A. b/sporus residue and microbial agent was one of the effective ways to control root-knot nematode disease.

Bioinformatics analysis of metallothionein gene from <i>Agaricus bisporus, Ganoderma lucidum, Taiwanofungus camphorates</i>and <i>Paxillus involutus</i>

Metallothionein (MT) gene has an important role in the detoxification of toxic metals especially some heavy metals. Based on the published sequences of MT genes from Agaricus bisporus, Garnoderma lucidum, Taiwanofungus camphoratus and Paxillus involutus as a type of the edible or medical mushroom in NCBI database, their molecular structures, physiological functions and evolutionary relationship were analyzed using the bioinformatics methods. Results showed that AbMT, GlMT, TcMT and PiMT genes contained complete open reading frame (ORF) and their theoretical points of the last three encoding proteins were higher than 7.0. AbMT, GlMT, TcMT and PiMT were relatively rich in random coil and extended strand, but transmembrane helices and signal peptides were not found. 4 MTs were mainly localized in cell nucleus (over 60%) and their cellular functions might have some relation to central intermediary metabolism. Multiple sequence alignment indicated relatively high identity (more than 52%) and short genetic distances (lower than 0.900) among 4 MT nucleotide sequences. Abundant genetic diversity and strong codon bias were found based on the halotype diversity, average number of nucleotide differences, nucleotide diversity, effective number of codons, codon bias index and scaled chisquare. Simultaneously, we deduced that 4 MT genes during molecular evolution were under positive selection. The present study might provide basis for further investigation of MTs molecular mechanisms and genetic laws.

Accumulation Characteristics and Resistance to Heavy Metal Contamination of Agaricus bisporus Varieties in Shanxi Province,China

In order to evaluate the accumulation characteristics of heavy metals in the cultivars of Agaricus bisporus in Shanxi Province and to select the varieties with high resistances to heavy metal pollution, atomic absorption spectrophotometer and the atomic fluorescence spectrophotometer were used as the determination methods to compare the heavy metal contents and accumulation characteristics of 6 different varieties. The results showed that 3 heavy metal elements （lead, chromium and cadmium） were tested in the 6 varieties of A. bisporus , all of which were contaminated by heavy metals, but basically no beyond the national standards for food safety. In general, these varieties had less quality risk and could be eaten safely. The accumulation characteristics of heavy metals in these varieties were analyzed, and the varieties were screened according to the resistance to heavy metals pollution. The results showed that variety No. 1 and No. 6 were the varieties with strong comprehensive resistance to the 3 types of heavy metal pollution, and variety No. 3 was a species with weak comprehensive ability in resisting heavy metal pollution.

Study on the Accumulation of Heavy Metals in Varieties of Agaricus bisporus

The accumulation characteristics and resistance to heavy metal pollution of six varieties of Agar/cus b/spoms were studied. The results showed that arsenic, mercury and cadmium content could be detected in the six varieties, and all of the varieties were contaminated by heavy metals ,but they did not exceed the limits in the national standards for food safety. These varieties had a low quality risk and can be eaten safely. Varie-ties 1 and 6 were strongly resistant to the three kinds of heavy metals, while variety 4 was weakly resistant to heavy metal pollution.

The effects of dietary supplementation with mushroom or selenium enriched mushroom powders on the growth performance and intestinal health of post-weaned pigs

Background There is an urgent need to identify natural bioactive compounds that can enhance gastrointestinal health and promote pig growth performance in the absence of pharmacological levels of zinc oxide(ZnO).The objectives of this study were to:1)compare the effects of mushroom powder supplemented with inorganic selenium(inSeMP)to mushroom powder enriched with organic selenium(orgSeMP)to pharmacological levels of ZnO on growth performance and faecal scores(FS)for the first 21 d post-weaning(Period 1);and 2)compare the molecular and microbial effects of inSeMP and orgSeMP in these pigs on d 39 post-weaning(Period 2).Methods In Period 1,pigs(3 pigs/pen;8 pens/treatment)were assigned to:(1)basal diet(control);(2)basal diet+zinc oxide(ZnO)(3100 mg/kg d 1–14,1550 mg/kg d 15–21);(3)basal diet+mushroom powder supplemented with inorganic selenium(inSeMP)containing selenium(selenite)content of 0.3 mg/kg feed;(4)basal diet+mushroom powder enriched with organic selenium(orgSeMP)containing selenium(selenocysteine)content of 0.3 mg/kg feed.Mushroom powders were included at 6.5 g/kg of feed.Results In Period 1,there was no effect of diets on average daily gain(ADG)and gain:feed(G:F)ratio(P>0.05).The orgSeMP supplemented pigs had a lower average daily feed intake(ADFI)compared to all other groups(P<0.05).The ZnO supplemented pigs had reduced FS compared to the basal and mushroom group,while the orgSeMP supplemented pigs had lower FS compared to the basal group during the 21 d experimental period(P<0.05).In Period 2,there was no effect of diets on ADFI,ADG and G:F ratio(P>0.05).The orgSeMP supplementation increased the caecal abundance of bacterial members of the Firmicutes and Bacteroidetes phylum,including Lactobacillus,Agathobacter,Roseburia,and Prevotella and decreased the abundance of Sporobacter compared to the basal group,while inSeMP increased the caecal abundance of Prevotella and decreased the caecal abundance of Sporobacter compared to the basal group(P<0.05).Dietary supplementation with inSeMP increased expression of TLR4 and anti-inflammatory cytokine gene IL10 and decreased nutrient transporter gene FABP2 compared to the orgSeMP group(P<0.05).Conclusion OrgSeMP is a novel and sustainable way to incorporate selenium andβ-glucans into the diet of weaned pigs whilst improving FS and modulating the caecal microbiota.

Design and experiment of a picking robot for Agaricus bisporus based on machine vision

Harvesting represents the crucial stage in the cultivation process of Agaricus bisporus mushrooms.An important way for the production process of Agaricus bisporus to reduce costs and increase income is to ensure timely harvest of Agaricus bisporus,reduce harvesting costs,and improve harvesting efficiency.There are many disadvantages in manual picking,such as high labor intensity,time-consuming work and high cost.In this study,a set of mushroom picking platform including climbing mechanism,picking robot,and control system was designed and developed.The picking robot consisted of a truss mechanism,an image acquisition device,a mushroom collection device,and a picking actuator.The profile picking actuator could realize the function of constant force clamping.An online size detection algorithm for Agaricus bisporus based on deep image processing was proposed.The algorithm included removal of abnormal noise points,background segmentation,coordinate conversion,and diameter detection.The precision picking system for Agaricus bisporus with coordinate compensation function controlled by Industrial Personal Computer was designed,and the visual control interface was developed based on Labview.Through the performance test,the reliability of machine vision recognition and the overall operating stability of the picking platform were verified.The test results showed that in the process of machine vision recognition,the recognition accuracy rate was higher than 92.50%,the missed detection rate was lower than 4.95%,the false detection rate was lower than 2.15%,and the diameter measurement error was less than 4.50%.The image processing algorithm had high recognition rate and small diameter measurement error,which could meet the requirements of picking operation.The picking platform’s picking success rate was higher than 95.45%,the picking damage rate was lower than 3.57%,and the picking output rate was higher than 87.09%.Compared with manual picking,the recognition accuracy rate of the picking platform was increased by 6.70%,the picking output rate was increased by 1.51%.The overall performance of the picking platform was stable and practical.

双孢菇引进品种筛选试验

为筛选适宜湖北省种植的双孢菇(Agaricus bisporus)品种,以湖北省主栽品种As2796为对照,进行了江苏17号、蘑加1号、英秀1号、浙农2号引进品种的筛选试验。通过比较不同品种双孢菇生物学特性指标,综合考虑不同品种双孢菇的性状,浙农2号为较优的引进品种。

双孢菇培养料配方试验

利用农作物秸秆和鸡粪、猪粪等原料作为双孢菇(Agaricus bisporus)培养料,研究不同配方培养料对菌丝生长、菌丝满袋时间及双孢菇产量的影响,从而确定最优配方为稻草43.6%、牛粪35.7%、鸡粪15.9%、饼肥1.2%、尿素0.2%、石膏1.2%、过磷酸钙1.2%、石灰1.0%。

褐蘑菇水不溶性膳食纤维提取工艺优化

研究了化学提取法提取褐蘑菇(Agaricus bisporus)水不溶性膳食纤维的最优条件。结果表明,化学提取法提取褐蘑菇水不溶性膳食纤维的最佳工艺组合为料液比1∶10(m/V,g∶mL)、预处理时间0.5 h、提取时间2.0 h。此条件下水不溶性膳食纤维提取率为22.9%。

响应面优化双孢菇预煮液酶解工艺研究

以游离α-氨基氮为指标,对比了中性蛋白酶、木瓜蛋白酶、风味蛋白酶对双孢菇(Agaricus bisporus)预煮液的酶解效果,并采用Box-Behnken响应面法优化了中性蛋白酶的酶解工艺。结果表明,中性蛋白酶酶解效果优于风味蛋白酶和木瓜蛋白酶,其最优工艺为添加量0.41%,p H 6.5,55℃酶解2.95 h,在此条件下,游离α-氨基氮为419.6 mg/L。