Truffles (Tuber spp.) are precious ectomycorrhizal fungi associated with trees such as Pinaceae and Fagaceae and play an important role in forest ecosystem. Some species of Tuber are praised in international edible mushroom market due to the unique flavors of fruiting bodies. Tuber-associated microorganisms are closely related to the development of mycelium, ectomycorrhizae, ascocarps and also affect the accumulation of special flavors and active substances of truffles. With the development of molecular biology technology, especially the application of high-throughput sequencing technology in recent years, the research on the mechanism of the formation and development of Tuber ascocarps and microbiological ecology has made a great progress. This review summarizes the achievements in research on the composition, diversity and ecological functions of Tuber-associated microbes and predicts the orientation in development and prospects of future research in these fields.
AM fungal community plays an important role in improving soil structure, enhancing soil fertility, increasing crop yield and disease resistance in the agro-ecological system. However, the agro-ecological system is a semi-natural ecosystem disturbed by the fertilization, planting, tillage and pesticide application, which have certain effects on AM fungal colonization intensity, biomass, spore density and community diversity. This paper reviews the effects of farming practices in recent years on AM fungal community structure, in order to put forward reasonable cultivation management measures to improve the ecological effects of AM fungi on the productivity of agro-ecological system, and to establish the sustainable development of ecological, economic and social benefits of the agricultural modernization.
Based on high-throughout sequencing technology, the fungal community composition and the relative abundance of fungal species in a hospital grassplot soil were analyzed after adding sterile keratin-rich chicken feather powder. During degradation of sterile chicken feather powder in the soil, enriched fungal species were detected, belonging to 128 genera, 71 families, 39 orders, 15 classes and 6 phyla. The fungal communities greatly varied in three different periods (initial stage 3d, mid-term 30d and later stage 90d) after addition of the sterile chicken feather powder. The diversity of fungal community in the samples was much richer in the initial stage (YY1) than that in other stages, and Guehomyces pullulans was found to be dominant (57.24%). In the mid-term (YY2), the relative abundances of species in the fungal community decreased, and an unclassified species was dominant, with relative abundance of 77.57%. The relative abundances of some potential human pathogens increased in the later stage (YY3), the dominant species was Microsporum gypseum. The relative abundance of Guehomyces pullulans dominant in the initial stage obviously decreased in the later stage. The keratin-rich chicken feather powder had a significant regulatory effect on the composition and relative abundance of the fungi in hospital grassplot soil, especially on some human potential pathogenic fungi.
The diversity of colonized fungi of Heterodera avenae cyst was investigated in Qingdao. Rhizosphere soil samples of winter wheat infected with H. avenae were collected regularly in three plots from regreening stage to maturity stage of wheat. The colonized fungi were isolated and purified by cyst puncture method, single spore isolation method or single hypha isolation method. Morphological and molecular identification and diversity analysis of these fungi were carried out. A total of 1 072 strains of fungi were isolated, belonging to 19 orders (including three incertae sedis), 28 families (including 10 incertae sedis), 35 genera (including three incertae sedis). Verticillium is the dominant genus with the highest relative abundance of 25.2%, and the relative abundance of Fusarium, Periconia and Alternaria, were 14.9%, 13.5% and 13.4%, respectively. Diversity analysis showed the samples from Modern Agricultural Science and Technology Demonstration Garden of QAU (Demonstration garden) in Jiaozhou have the highest species richness index (4.30), Shannon-Wiener diversity index (2.516) and Pielou’s index (0.755). The samples from Hanlinyuan Residential Area in Chengyang (Residential Area) has the lowest species richness index (3.18). The samples from Gejiatun in Chengyang (Gejiatun) have the lowest Shannon‐Wiener diversity index and Pielou’s index, being 2.077 and 0.654 respectively. Population composition survey of colonzied fungi indicated that the Jaccard similarity index of the three plots ranged from 0.486 to 0.607.
Verticillium dahliae is a soil-borne pathogenic fungus that causes Verticillum wilt with a broad range of host plants. At present the molecular infection mechanisms of the pathogen are poorly understood. The authors explored the function of gene VdSCH9 encoding serine/threonine protein kinase in colony growth and pathogenicity by knocking out VdSCH9 in the fungus. The gene SCH9 is found to be involved in the cAMP-PKA and TOR pathway in the yeast. It also plays an important role in colony growth, stress response and longevity of yeast. The ΔVdSCH9 mutant was significantly reduced in growth rate, and it was also defective in aerial hyphal growth and hyphal branching. The cotton pathogenicity assay shows that the disease index of ΔVdSCH9 mutant was 56.6, significantly lesser than that of wild type and complement mutant, being 90.5 and 82.8 respectively. The similar result was acquired for the VdSCH9-deleted mutant in pathogenicity index on eggplant plants, being 65.9 for ΔVdSCH9 strain, and 91.1 and 89.8 for wild type and complement mutants. The gene-deleted mutants were more sensitive under stresses of oxidation, hyperosmotion, and cell wall and membrane integrity. These results suggest that the gene VdSCH9 is evidently involved in colony growth, stress response and pathogenesis in V. dahliae.
Wet bubble disease caused by Hypomyces perniciosus is a disease of mushroom Agaricus bisporus. The molecular pathogenesis mechanisms of the disease have not been investigated. Agrobacterium-mediated genetic transformation (ATMT) system is an efficient and stable method for fungal transformation and insertion-mutant library construction to determine the function of pathogenicity related genes. In this study, ATMT protocol for T-DNA insertional mutagenesis transformation of H. perniciosus was optimized. Agrobacterium tumefaciens AGL-1 strain containing the binary vector pBHt1 carrying hygromycin-resistance gene was used to generate H. perniciosus strain WH001 mutant library. Our results showed that the concentration of hygromycin sensitivity of H. perniciosus was 250ng/L. The optimum T-DNA insertion mutation library system for Agrobacterium-mediated transformation of H. perniciosus was OD600=1 of the bacterial concentration, 30min of infection duration, and 3 days of the co-cultivation period with the supplementation of 1.5mg/mL of acetosyringone (AS) concentration. A large number and variety of mutants were acquired, and important phenotype included reduction in growth rate, loss of conidiation and loss of pathogenicity. This study shows that ATMT is a highly efficient method for studying the function of pathogenicity genes of H. perniciosus and understanding the molecular pathogenesis of wet bubble disease.
The purpose of this study was to establish a technical scheme for rapid determination of mitochondrial genotypes of Cordyceps militaris strains, and to investigate the genetic stability of C. militaris mitochondria after successive subcultures. All known intron loci (eight in total) in C. militaris mitochondrial genomes were amplified from C. militaris strains with available mitochondrial genomes, and amplicons were mixed to establish two sets of DNA molecular weight standards (M-I and M-II). M-I consisted of eight fragments with each corresponding to one of the eight known introns; M-II consisted of six fragments with each missing one intron at six known intron loci. To test the effectiveness of the established detection system, the same (presumed) intron loci were amplified from C. militaris test strains, including three strains with available mitochondrial genomes and two strains without available mitochondrial genomes, and amplicons were compared with fragments in M-I and M-II by agarose gel electrophoreses. By this way, we could accurately determine the mitochondrial intron distribution pattern of a C. militaris strain, which verified the effectiveness of the established detection system. Finally, ten tissue strains and eight single spore strains of C. militaris were subcultured continuously for 15 generations, and distribution patterns of mitochondrial introns were not found to vary. This study successfully constructed the technique scheme for rapid detection of mitochondrial genotypes of C. militaris, and found that the mitochondria of C. militaris had high genetic stability, which laid a foundation for studying mitochondrial inheritance of the fungus.
Identification of seven wild cordycipitoid fungal strains was based on morphological characteristics and phylogenic analysis of ITS sequence; the inhibiting activity against HepG2 cells of their mycelium ethanol extract was analysed by MTT assay. The results showed that MF7, MF9 and MF14 were identified as Isaria tenuipes, MF11, MF12 and MF13 were Isaria cicadae, and MF10 was Beauveria bassiana. MTT assay results showed that the ethanol extract of three Isaria cicadae strains and three Isaria tenuipes strains could hardly inhibit HepG2 proliferation, and the IC50 was greater than 500μg/mL; Beauveria bassiana strain MF10 (IC50=221.6μg/mL) was stronger than Paecilomyces hepiali fermented mycelium product (Jinshuibao capsule) (IC50=364μg/mL) and Hirsutella sinensis fermented mycelium product (Bailing capsule), (IC50=268.7μg/mL). The ethanol extract of Cordyceps militaris strains (MF1, MF5 and MF15) using as controls were found to be stronger in inhibitory effect on HepG2 cells, especially MF15 with the IC50 value of 55.56μg/mL, while IC50 of MF1 and MF5 was 198.5μg/mL and 134.1μg/mL respectively, implying that Cordyceps militaris mycelium has significant research value.
Cordyceps militaris has become one of the well-known edible and medicinal fungi in China and Southeast Asia. Although the fungus has been successfully cultivated and commercialized, it has encountered many problems in industrialized production, e.g. fungal disease caused by Calcarisporium cordycipiticola. In this study, the biological characteristics, pathogenicity and infection characteristics of this parasite were investigated. It was found that the branchy mycelia could produce a large number of conidia in a short time. The optimum growth temperature was 25°C, favouring the rapid spread of the disease. The conidia of the pathogen were more resistant to UV than those of C. militaris. The disease occurs in the later stage of the growth and development of C. militaris, and it can infect the surface of the cultures, and the bottom, middle and top of the fruiting bodies. Inoculation of conidia revealed that the pathogen could infect C. militaris at any stage of growth. Co-cultivation test indicated that C. cordycipiticola could gradually grow onto the mycelium surface of C. militaris. However, intertwinement of the mycelia of both species was not found. This study provides a reference for early detection and prevention of this disease during mass production of C. militaris.
To study effects of ethanol extract (EE) of Taiwanofungus camphoratus fruiting body (TCF) endemic to Taiwan on the ovalbumin (OVA)-induced allergic pneumonia and explore its possible mechanism. In vivo experiments using ovalbumin (OVA) sensitized to prepare allergic pneumonia model, observed the pathological changes of lung tissue in each group, and detected secretion of proinflammatory cytokines in cells induced by lipopolysaccharide (LPS); in vitro experiments using enzyme-linked immunosorbent assay (ELISA) determined the secretion of pro-inflammatory cytokines (IL-1, IL-6 and TNF-α) from THP-1 cells stimulated by LPS. In vitro macrophage (THP-1) anti-inflammatory test results showed that different concentrations of TCFEE could significantly inhibit LPS-induced THP-1 cell IL-1 secretion (P<0.001), completely inhibit the secretion of IL-6 and TNF-α secretion at concentrations of 20 and 80μg/mL. In the animal model of OVA-induced allergic pneumonia, compared with the model group, the KBA-TCFEE can significantly improve the appearance of lung color, bronchiolar lymphocyte aggregation, alveolar wall proliferation and alveolar cavity and other inflammation. Our results inferred that oral KBA-TCFEE effectively improved the OVA-induced allergic pneumonia and exhibited the systemic anti-inflammatory effect. The mechanism may be to inhibit the secretion of IL-1, IL-6 and TNF-α in THP-1 cells. The results can be also used as an important reference to prevent and control the harmful effects of haze pneumonia.
High-concentration ammonia inducing coughing method, phenol red excretion method, hypoxia tolerance method under normal pressure, confront sodium nitrite toxic anoxic method and myocardial ischemic hypoxia method have been adopted for investigating pharmacological action of Trametes suaveolens fruiting body different polarity extract on relieving cough, eliminating sputum and hypoxic tolerance. The male mice were randomly grouped (10 in each group). Physiological saline was treated as negative control (NCG), and “gui long ke chuan ning” (a mixture of Chinese herbal medicine) (KCN) and codeine (CDI) were treated as positive control. The low dose [TSPEE(L) 0.025g/kg], moderate dose [TSPEE(M) 0.05g/kg] and high dose [TSPEE(H) 0.1g/kg] T. suaveolens petroleum extract treatment groups, and low dose [TSWE(L) 0.5g/kg], moderate dose [TSWE(M) 1.0g/kg] and high dose [TSWE(H) 2.0g/kg] T. suaveolens water extract treatment groups were set up. The measurement indicators of mice include the cough incubation period, frequency of cough, frequency of suppress cough, antitussive activity (%), excretion volume of phenol red, dissipating phlegm rate (%), survival period of hypoxia tolerance under normal pressure, survival period of nitrite poisoning, survival period of acute cerebral hypoxia and the content of SOD, MDA, IL-2, IL-4 and IgG in serum. The result showed that TSPEE(L) had no obvious effect; TSPEE (M) had significant dissipating phlegm effect (P<0.05); TSPEE(H) could prolong the survival period of acute cerebral ischemia (P<0.05), increase the content of SOD, IL-2, IL-4 and IgG, and decrease the content of MDA (P<0.05); TSWE(L) could increase the cough inhibition rate and cough relieving activity, and prolong the survival period of hypoxia under normal pressure (P<0.05); TSWE (M) had a significant effect except for toxic brain hypoxia test (P<0.05); TSWE(H) could enhance cough inhibition rate, improve antitussive activity, increase survival period of sodium nitrite poisoning (P<0.05), increase hypoxia survival period under normal pressure and acute cerebral ischemia (P<0.01), increase the content of SOD, IL-2, IL-4 and IgG, and reduce the content of MDA (P<0.01). In conclusion, both petroleum ether extract and water extract of fruiting body of T. suaveolens had effects of relieving cough, eliminating sputum and hypoxia tolerance.
Armillaria mellea is an edible and medicinal mushroom with some medicinal value. The anti-neuroinflammatory effects of n-hexane extract from A. mellea (Hex-AM) are studied through animal experiments. Parkinson’s disease mice model was established by using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The experimental treatment groups include blank group, model group, Hex-AM low does group (10mg/kg), and Hex-AM high dose group (20mg/kg). The mice were orally treated with Hex-AM for 12 days. The loss of body weight, pole test, striatal TH, striatal Iba-1, iNOS enzyme activity, and related expression of mRNA were examined. The body weight loss was alleviated in Hex-AM groups, with a significant improvement in pole test. The results of western blot showed the increase of TH protein and inhibition of the expression of Iba-1 in Hex-AM groups as compared with model group. The mice of Hex-AM groups showed a significant decrease of iNOS and related mRNA expression in striatum. Our data suggest that Hex-AM may exert its anti-neuroinflammatory effect by inhibiting the over-activation of microglial cells and the activity of iNOS.
The fruiting bodies of mushroom usually accumulate high amounts of polyol and trehalose during their growth. However, there are fewer reports about these data in Ganoderma lingzhi. In this research the carbohydrates in fruiting body of G. lingzhi strain SH during growth are tested using HPAEC-PAD method. Results indicate that the main polyol in G. lingzhi SH is arabinitol and mannitol. The content of mannitol peaks in pileus of mature fruiting bodies, and the content of arabinitol peaks in the fruiting bodies when the basidiospores are massively produced. The variation of the content of mannitol and arabinitol shows an adverse trend in brief. The accumulation of one of these two polyol generally results in the decrease of another one. The peak content of trehalose is detected at the base of young fruiting bodies and it maintains a low level in the rest time of growth. The expression level changes of enzymes involving the metabolism of mannitol, arabinitol and trehalose are detected using qRT-PCR. It is found that the expression levels of most of these enzymes are higher in the base of the fruiting bodies than those in other parts of fruiting bodies. This expression disparity is more and more notable with the growth of fruiting bodies. It seems that the polyol and trehalose are mainly synthesized in the mycelia of the base region and then translocate to other regions of the fruiting bodies. After accumulation and consumption during growth, the difference of distribution in different parts of the fruiting bodies occurs.
A comparative analysis was made between laccase activities of Pleurotus ostreatus cultivated on different conventional substrate, i.e. sawdust, corncob, and cottonseed hull, under solid-state and submerged fermentation conditions for the purposes of selecting most suitable cultivation substrate. The results demonstrated that various culture substrate remarkably affected the laccase activities of P. ostreatus (P<0.001). The laccase activities of P. ostreatus under different fermentation methods were significantly different (P<0.001), but no difference was found on the 2nd day of cultivation (P>0.05). Under the conditions of solid-state fermentation and submerged fermentation, the laccase activity detected in synthetic medium containing cottonseed hull was higher than that in synthetic medium containing sawdust or corncob, indicating that the cottonseed hull could be considered as a better inducer for enhancing the laccase activities of P. ostreatus. The synthetic medium containing cottonseed hull was in favour of speeding up the laccase secretion from P. ostreatus, and advancing the time of enzyme production.
