OBJECTIVETo study the cultural characteristics of Armillaria mellea (A. mellea ) on solid media.

METHODSA. mellea was cultured on semi-solid agar medium in dark conditions. Effects of different media, carbon sources, nitrogen sources, and temperature on growth and morphology of A. mellea were observed. The contents of polysaccharide, mannitol, glucose, and reducing sugars in A. mellea during different stages of development were determined.

RESULTSThe biomass and morphology of A. mellea were different in various media. Sugars were more effective carbon sources than the relevant sugar alcohols. Little molecular carbon sources such as alcohol and glycerol could be utilized by A. mellea, but starch only could be utilized slowly. Either organic or inorganic nitrogen sources could be uptaken and utilized effectively by A. mellea. No evidence was found that VitB1 affects the growth of A. mellea. The growth cycle on wort medium at 30 degrees C was shorter than that at 25 degrees C for 7 days. In logarithmic growth phase and stable phase, the polysaccharide contents of A. mellea were 9.24% and 4.70% respectively, while the mannitol contents were 10.08% and 10.58% respectively; glucose and reducing sugar contents remained low level in the whole growth stage.

CONCLUSIONSCarbon sources have a more remarked effect on the growth of A. mellea than the nitrogen sources do. Optimal temperature for the growth of A. mellea ranges 20-30 degrees C. Mannitol accumulates more than other little molecular carbohydrates in A. mellea.

Agaricales ; chemistry ; growth & development ; Culture Media ; Mannitol ; analysis

Gastrodia elata is a kind of precious traditional Chinese medicine. In the process of cultivation of G. elata, due to the influence of continuous cropping obstacles and other factors, the fungus materials and land that have been planted with G. elata are often abandoned, resulting in a great waste of resources. Based on the planting characteristics of G. elata and Phallus impudicus and the previous research experience in ecological agriculture, this paper analyzed the ecological adaptability characteristics of G. elata and P. impudicus, and summarized the key techniques of the G. elata-P. impudicus sequential planting pattern. Keeping track of the planting area, fungus-growing materials consumption and market sales of G. elata-P. impudicus sequential planting pattern, the ecological benefits of G. elata-P. impudicus sequential planting pattern from the aspects of utilization rate of fungus-growing materials were analyzed, the value of land resources per unit area, ecological environmental protection, labor cost and economic benefits were consi-dered. The technical principle of G. elata-P. impudicus sequential planting pattern was expounded according to their ecological habit, the season of harvest and planting, the difference of composition of fungus-growing materials, and the microbial ecology. The sequential planting pattern of G. elata-P. impudicus not only realized the double production of medicinal materials and edible fungi, reduced the waste of old fungus-growing materials, but also transformed the energy from nutrition-supplied fungi to edible and medicinal fungi, which guaranteed the ecological recycling and utilization of G. elata in the process of cultivation.
Agaricales/growth & development* ; Agriculture/methods* ; Gastrodia/growth & development* ; Medicine, Chinese Traditional ; Plants, Medicinal/growth & development*

The phenomenon that waste of fungus-growing materials in the planting process of Gastrodia elata is very common. It has been proved by practice that the used fungus-growing materials planted with G. elata can be used to plant Phallus impudicus. But the mechanism is unclear. In this study, we compared the different infested-capacity of Armillaria gallica and Phallus impudicus by morphological anatomy of the used fungus-growing materials. We also compared the differences on the two fungi consumed the main contents of fungus-growing materials, cellulose, lignin and hemicellulose, by using nitric acid-95% ethanol method, sulfuric acid method and tetrabromide method respectively, so that to explore the mechanism of A. gallica and P. impudicus recycle the fungus-growing materials, and to provide scientific basis for recycling the used fungus-growing materials of G. elata. The results showed that A. gallica had a strong ability to invade some parts outside the vascular cambium, but it had a weak ability to invade some parts inside the vascular cambium, while P. impudicus had a strong ability to invade the same parts. The contents of lignin and cellulose, which from inside and outside the vascular cambium of fungus-growing materials were significantly different. In the parts of outside the vascular cambium of fungus-growing materials, A. gallica degraded more lignin and cellulose, while P. impudicus degraded more hemicellulose. In the parts of inside the vascular cambium of fungus-growing materials, A. gallica degraded more cellulose, while P. impudicus degraded more hemicellulose. The present results suggested that A. gallica and P. impudicus made differential utilization of the carbon source in the fungus-growing materials to realize that P. impudicus recycle the used fungus-growing materials of G. elata. A. gallica used lignin and cellulose as the main carbon source, while P. impudicus used hemicellulose as the main carbon source.
Agaricales/growth & development* ; Armillaria/growth & development* ; Cellulose/metabolism* ; Lignin/metabolism* ; Polysaccharides/metabolism*

Gastrodia elata is a heterotrophic plant that needed to be symbiotic with Armillaria. The obstacle of continuous cropping in G. elata is serious during the G. elata cultivation, and the mechanism of obstacle in G. elata continuous cropping had not been solved. The planting of G. elata-Phallus impudicus is a new sequential planting pattern adopted in Guizhou province, but the effect of the cultivation on soil microbial community structure is still unclear. In this study, we collected four soil samples for the research including the soil without planted G. elata as control(CK), rhizosphere soil samples tightly adhering to the G. elata surface(GE), rhizosphere soil samples tightly adhering to Armillaria which was symbiotic with G. elata(AGE), the rhizosphere soil of P. impudicus planting after G. elata cultivation(PI). In order to explore the mechanism, the research study on the soil of G. elata-P. impudicus by using ITS and 16 S rDNA high-throughput sequencing technologies to detect soil microbial community structure including fungi and bacteria in the soil of CK, AGE, GE and PI. OTU clustering and PCA analysis of soil samples showed that the soil microbial diversity was relatively similar in AGE and GE. And the soil microbial in PI and CK clustered together. The results showed that AGE and GE had similar soil microbial diversity, as well as PI and CK. Compared with CK, the soil microbial diversity and abundance in AGE and GE were significantly increased. But the microbial diversity and abundance decreased in PI compared with AGE and GE. The annotation indicated that the abundance of Basidiomycota, Acidobacteria and Chloroflexi decreased, and that of Ascomycota, Zygomycota and Proteobacteria increased in AGE and GE compared with CK. In contrast to AGE and GE, PI was the opposite. The abundance of Basidiomycota, Acidobacteria and Chloroflexi increased in PI compared with AGE and GE. The abundance of microorganisms in the soil of PI and CK was similar. In addition, the co-culture of Armillaria and P. impudicus indicated that P. impudicus had obvious antagonistic effects on the growth of Armillaria. Therefore, it is speculated that the mechanism of G. elata-P. impudicus planting pattern related to the change of soil microbial. And we supposed that P. impudicus might inhibit the growth of Armillaria and change the soil microbial community structure and the abundance of soil microbial. And the soil microbial community structure was restored to a state close to that of uncultivated G. elata. Thus, the structure of soil microbial community planting G. elata could be restored by P. impudicus planting.
Agaricales/growth & development* ; Bacteria/classification* ; Fungi/classification* ; Gastrodia/microbiology* ; Microbiota ; Rhizosphere ; Soil Microbiology

The continuous cultivation of Rehmannia glutinosa causes the accumulation of phenolic acids in soil. It is supposed to be the reason of the so called "continuously cropping obstacle". In this study, phenolic acids (hydroxybenzoic acid, vanillic acid, eugenol, vanillin and ferulic acid) were degraded by the extracta of all the tested spent mushroom substrate (SMS) and the maximal degradation rate was 75.3%, contributed by extraction of SMS of Pleurotus eryngii. Pot experiment indicated that hydroxybenzoic acid and vanillin in soil were also degraded effectively by SMS of P. eryngii. The employment of SMS enhanced ecophysiology index to near the normal levels, such as crown width, leaves number, leaf length, leaf width and height. At the same time, the fresh and dry weight and total catalpol concentration of tuberous root weight of R. glutinosa was increased to 2.70, 3.66, 2.25 times by employment of SMS, respectively. The increase of bacteria, fungi and actinomycetes numbers in rhizosphere soil were observed after the employment of SMS by microbial counts. The employment of SMS also enhanced the enzyme activity in soils, such as sucrase, cellulase, phosphalase, urease and catelase. These results indicated that the employment of SMS alleviated the continuously cropping obstacle of R. glutinosa in some extent.
Agaricales ; chemistry ; metabolism ; Agriculture ; methods ; Biodegradation, Environmental ; Hydroxybenzoates ; analysis ; metabolism ; Rehmannia ; growth & development ; metabolism ; Soil ; chemistry ; Soil Microbiology

OBJECTIVETo study the effects of four species of endophytic fungi on the growth and polysaccharide and alkaloid contents of cultured Dendrobium nobile.

METHODEach fungal strain was cultured together with D. nobile, and the plant weight as well as the polysaccharide and alkaloid contents were measured.

RESULTCompared with the control group, Mycena sp. (MF23) was found to decrease the fresh weight of D. nobile by 24.9% (P < 0.01). All four species of fungi have no effect on the dry weight of this plant. On the other hand, Epulorhiza sp. (MF18) and MF23 could increase the percent of dry weight of seedlings (P < 0.05). Epulorhiza sp. (MF15) and MF24 increased the number of aerial roots by 4.25 times and 4.14 times respectively (P < 0.01). MF23 decreased the numbers of basal roots by 46.5% (P < 0.01). MF15, MF18, MF23 and MF24 were shown to increase the content of polysacchride by 153.4%, 52.1%, 18.5% and 76.7%, respectively. MF23 also increased the content of total alkaloid by 18.3%.

CONCLUSIONEndophytic fungi cultured together with D. nobile could affect the growth of this plant; they also increase total alkaloid and polysaccharide contents.

Agaricales ; physiology ; Alkaloids ; analysis ; Basidiomycota ; physiology ; Culture Media ; Culture Techniques ; Dendrobium ; chemistry ; growth & development ; microbiology ; Plants, Medicinal ; chemistry ; growth & development ; microbiology ; Polysaccharides ; analysis ; Symbiosis ; physiology

The production conditions of extracellular laccase from Armilliria mellea and the characteristics of the enzyme were studied. The experiment proved that initial pH5.5 of the culture medium and temperature at 25 degrees C were favorable for laccase synthesis. As carbon resources, cellobiose and raffinose were better in terms of productivity than maltose, sorbose and galactose. Organic nitrogen source was more suitable for Armilliria mellea to synthesize laccase than inorganic nitrogen source. Peat extract (PE) enhanced notably the yield of laccase; the maximal yield was 7 times as much as that of the control when PE concentration was 50%. Three isozymes were detected in culture supernatant named A, B and C respectively after their mobility on PAGE. After concentrated by (NH4)2SO4 precipitation, laccase A was further purified to homogeneity by preparative native PAGE and anion exchange column chromatography. The native enzyme was a single polypeptide with a molecular mass of approximately 59 kD estimated by SDS-PAGE, while 58 kD by gel filtration chromatography under non-denaturing conditions. Determined by IEF its isoelectric point was 4.0. The optimal pH value and temperature were 5.6 and 60 degrees C respectively in catalytic reaction of oxidizing guaiacol. At 60 degrees C and 65 degrees C, half-lives of laccase A were 45 min and 36.8 min, respectively. Enzyme activity was inhibited with 100 mmol/L Cl-, but was activated with 1 mmol/L SO4(2-). However, if the concentration of NaN3 was only 1 mmol/L, laccase A lost its activity completely. 10 mmol/L EDTA had no effect on laccase A, while 1 mmol/L Cu2+ could enhance its activity. Laccase A showed a good stability when the pH of the buffer varied from 5.2 to 7.2. Using guaiacol as the substrate, the Km was 1.026 mmol/L and the Vmax was 5 mumol/(min.mg); using ABTS instead, the Km was 0.22 mmol/L and Vmax was 69 mumol/(min.mg).
Agaricales ; drug effects ; enzymology ; growth & development ; Carbohydrates ; pharmacology ; Culture Media, Conditioned ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Enzyme Stability ; Hydrogen-Ion Concentration ; Laccase ; Oxidoreductases ; isolation & purification ; metabolism

The mitogen-activated protein kinase (MAPK) cascade, composed of MAPK kinase kinase (MAP3K), MAPK kinase (MAP2K), and MAPK, is abundantly conserved in all eukaryotes. MAPK along with MAPK cascade plays a vital regulatory role in the plant-arbuscular mycorrhiza/rhizobium nodule symbioses. However, the biological function of MAPK in orchid mycorrhiza (OM) symbiosis remains elusive. In the present study, a MAPK gene, designated as DoMPK1 (GenBank accession No. JX297594), was identified from D. officinale roots infected by an OM fungus-Mycena sp. using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The full length cDNA of DoMPK1 was 1 263 bp and encoded a 372 aa protein with a molecular weight of 42.61 kD and an isoelectric point (pI) of 6.07. The deduced DoMPK1 protein contained the conserved serine/threonine-protein kinase catalytic domain (39-325) and MAP kinase signature (77-177). Multiple sequence alignment and phylogenetic analysis demonstrated that DoMPK1 was highly homologous (71%-85%) to MAPK genes from various plant species and was closely related to those from monocots. Real time quantitative PCR (qPCR) analysis revealed that DoMPK1 was constitutively expressed in leaves, stems, roots and seeds, and the transcript abundance was not significantly different in the four included tissues. Furthermore, DoMPK1 transcript was markedly induced in roots at 30 d after fungal infection, with 7.91 fold compared to that of the mock inoculated roots, suggesting implication of DoMPK1 in the early D. officinale and Mycena sp. interaction and an essential role in the symbiosis. Our study characterized a MAPK gene associated with OM symbiosis for the first time, and will be helpful for further functional elucidation of DoMPK1 involving in D. officinale and Mycena sp. symbiotic interaction.
Agaricales ; growth & development ; Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; DNA, Complementary ; genetics ; Dendrobium ; enzymology ; genetics ; microbiology ; Gene Expression Regulation, Plant ; Mitogen-Activated Protein Kinases ; genetics ; metabolism ; Molecular Weight ; Phylogeny ; Plants, Medicinal ; enzymology ; genetics ; microbiology ; Sequence Alignment ; Symbiosis

Calcium-dependent protein kinases (CDPKs) play an important regulatory role in the plantarbuscular mycorrhiza/rhizobium nodule symbiosis. However, the biological action of CDPKs in orchid mycorrhiza (OM) symbiosis remains unclear. In the present study, a CDPK encoding gene, designated as DoCPK1 (GenBank accession No. JX193703), was identified from D. officinale roots infected by an OM fungus-Mycena sp. using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods, for the first time. The full length cDNA of DoCPK1 was 2137 bp in length and encoded a 534 aa protein with a molecular weight of 59.61 kD and an isoelectric point (pI) of 6.03. The deduced DoCPK1 protein contained the conserved serine/threonine-protein kinase catalytic domain and four Ca2+ binding EF hand motifs. Multiple sequence alignment demonstrated that DoCPK1 was highly homologous (85%) to the Panax ginseng PgCPK1 (ACY78680), followed by CDPKs genes from wheat, rice, and Arabidopsis (ABD98803, ADM14342, Q9ZSA2, respectively). Phylogenetic analysis showed that DoCPK1 was closely related to CDPKs genes from monocots, such as wheat, maize and rice. Real time quantitative PCR (qPCR) analysis revealed that DoCPK1 was constitutively expressed in the included tissues and the transcript levels were in the order of roots > stems > seeds > leaves. Furthermore, DoCPK1 transcripts were significantly accumulated in roots 30 d after fungal infection, with 5.16 fold compared to that of the mock roots, indicating involvement of DoCPK1 during the early interaction between D. officinale and Mycena sp., and a possible role in the symbiosis process. This study firstly provided important clues of a CDPK gene associated with OM symbiosis, and will be useful for further functional determination of the gene involving in D. officinale and Mycena sp. symbiosis.
Agaricales ; growth & development ; physiology ; Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; DNA, Complementary ; genetics ; Dendrobium ; enzymology ; genetics ; microbiology ; Gene Expression Regulation, Plant ; Molecular Weight ; Mycorrhizae ; growth & development ; physiology ; Phylogeny ; Plant Leaves ; enzymology ; genetics ; microbiology ; Plant Roots ; enzymology ; genetics ; microbiology ; Plant Stems ; enzymology ; genetics ; microbiology ; Plants, Medicinal ; enzymology ; genetics ; microbiology ; Protein Kinases ; genetics ; metabolism ; Seeds ; enzymology ; genetics ; microbiology ; Sequence Alignment ; Symbiosis