Fine-scale genomic analysis of the tree endophyte Annulohypoxylon sp. FPYF3050 producing monoterpene 1,8-cineole.

1,8-Cineole has a potential in control crop pests and biofuels. The endophytic fungus, Annulohypoxylon sp. FPYF3050 (Neolitsea pulchella), can produce over 90% 1,8-cineole of relative area in its natural volatiles, possessed nematicide properties. The annotated genome of this strain will provide insights into potential application in biofumigation and terpene-based advanced biofuel.

Identification of ophiostomatalean fungi associated with Tomicus pilifer infesting Pinus koraiensis in Northeastern China.

Ophiostomatalean fungi usually facilitate bark beetles to infest tree hosts and seriously endanger the health of coniferous forests. Tomicus pilifer Spessivtsev is a common endemic bark beetle in Asia and primarily threatens Pinus koraiensis. Tomicus species have similar morphology; however, they can be differentiated by their genetic characteristics through phylogenetic analyses. To date, the 28S rDNA sequence of T. pilifer and the diversity of ophiostomatalean fungi associated with T. pilifer have not been reported. In this study, we aimed to clarify the taxonomic status of T. pilifer and identify ophiostomatalean fungi associated with T. pilifer infesting P. koraiensis in northeastern China. In total, 315 ophiostomatalean fungal strains were isolated from 62 adults of T. pilifer and 220 tissue samples from T. pilifer galleries in Jilin Province. Thirty-five representative strains were further identified by comparing their morphological and physiological characteristics and conducting the phylogenetic analysis of ITS, ITS2-LSU, TUB2, and TEF1-α. We identified nine species of ophiostomatalean fungi belonging to four genera, which included six novel species (Ceratocystiopsis changbaiensis sp. nov., Leptographium linjiangense sp. nov., Leptographium qieshaoense sp. nov., Ophiostoma piliferi sp. nov., Ophiostoma tonghuaense sp. nov., and Ophiostoma yaluense sp. nov.), two previously described species (Graphilbum interstitiale and Ophiostoma fuscum), and one undefined specie (Ceratocystiopsis sp. 1). To the best of our knowledge, this is the first report of G. interstitiale and O. fuscum in China and the fungal diversity of ophiostomatalean in T. pilifer. The dominant species were O. piliferi and L. qieshaoense, representing 39.37% and 35.87% of the isolates, respectively. The results of this study provide valuable information on the symbiotic relationship between bark beetles and ophiostomatalean fungi.

PeTGA1 enhances disease resistance against Colletotrichum gloeosporioides through directly regulating PeSARD1 in poplar.

Basic leucine zipper (bZIP) proteins play important roles in responding to biotic and abiotic stresses in plants. However, the molecular mechanisms of plant resistance to pathogens remain largely unclear in poplar. The present study isolated a TGACG-binding (TGA) transcription factor, PeTGA1, from Populus euphratica. PeTGA1 belongs to subgroup D of the bZIP family and was localized to the nucleus. To study the role PeTGA1 plays in response to Colletotrichum gloeosporioides, transgenic triploid white poplars overexpressing PeTGA1 were generated. Results showed that poplars with overexpressed PeTGA1 showed a higher effective defense response to C. gloeosporioides than the wild-type plants. A yeast one-hybrid assay and an electrophoretic mobility shift assay revealed that PeTGA1 could directly bind to the PeSARD1 (P. euphratica SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1) promoter, an important regulator for salicylic acid biosynthesis. The transactivation assays indicated that PeTGA1 activated the expression of PeSARD1, and PR1 (PATHOGENESIS-RELATED 1), a SA marker gene involved in SA signaling. Subsequently, we observed that the PeTGA1 overexpression lines showed elevated SA levels, thereby resulting in the increased resistance to C. gloeosporioides. Taken together, our results indicated that PeTGA1 may exert a key role in plant immunity not only by targeting PeSARD1 thus participating in the SA biosynthesis pathway but also by involving in SA signaling via activating the expression of PR1.

Bacterial and fungal communities of traditional fermented Chinese soybean paste (Doujiang) and their properties.

Soybean paste (Doujiang) is one of the traditional fermented foods from China, fermented by various microorganisms. However, the microflora of Doujiang keeps little known. In this study, the microbial communities of seven kinds of representative Doujiang samples were investigated by both culture-independent and culture-dependent methods. We found that core OTUs among seven Doujiang samples were mainly from Bacillus, Pseudomonas, Candida, and Aspergillus according to Illumina sequencing. Every type of Doujiang sample harbored a different composition of microbial community. Doujiang LSJ and LBJ had the highest bacterial and fungal richness and diversity, respectively. The structure of microbial community was remarkably correlated with Doujiang properties-pH, and the content of total protein, soluble protein, amino acid, and total sugar (p < .05). Bacillus spp. were most frequently isolated bacterial species. Fungi of Monascus, Candida, and Aspergillus were also isolated. Eleven microbial strains showed high protease activities to degrade corn proteins, which can form obvious transparent hydrolytic circles in corn gluten meal medium plates. Therefore, microbial communities were supposed to tightly connect to Doujiang type and properties. It is possible to apply potential protein-degrading microbial strains to corn byproducts for protein production in the future study.

Chinese chestnut yellow crinkle disease influence microbiota composition of chestnut trees.

CONTEXT: Candidatus-phytoplasma castaneae has been found as the causal agent of the Chinese chestnut yellow crinkle disease. However, the ecological impact of the disease on microbiota of chestnut trees is unknown. AIMS: The study aim was to clarify difference attributes in microbial community structure of asymptomatic and symptomatic chestnut leaves and twigs for chestnut tree health in orchard. METHODS: Sample collections were conducted with both symptomatic and asymptomatic chestnut trees. Total DNA was extracted. Fungal ITS rDNA and bacterial 16S rDNA were amplified. The PCR products were sequenced with Illumina HiSeq. Platform. RESULTS: A total number of 852 fungal and 1156 bacterial OTUs (operational taxonomic units) were detected. The asymptomatic samples had a higher fungal and bacterial diversity than symptomatic ones. Non-metric multidimensional scaling (NMDS) analysis showed microbial communities among symptomatic and asymptomatic leaves and twigs samples formed individual cluster. Overall, Ascomycota and Proteobacteria were the most abundant fungal and bacterial phyla, respectively. Significantly different taxa playing key roles for each microbiota structure were identified. In symptomatic trees, microbial groups of plant pathogens were more abundant. CONCLUSION: Our results demonstrated that the phytoplasma pathogen may exert significant influence on the microbial community structure. The study will provide further fundamental clues for the little studied phytoplasma pathogens effects on host microbiota, phytoplasma pathogen control strategies.

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Drought is a main factor affecting the growth and yield of Chinese chestnut trees in Yan-shan Mountains. To investigate the responses of chestnut seedlings to drought stress, the growth and physiological indices, including photosynthetic characteristics, biomass, proline, malondialdehyde, carbon and nitrogen contents were measured in roots, stems, and leaves after the Chinese chestnut 'Yanshanzaofeng' seedlings in the pots were treated by simulating drought for 22 days. The results showed that, compared with the normal irrigation, water contents in the roots, stems and leaves were decreased by 18.3%, 29.0% and 62.8%, respectively, accompanied by the considerable increases in the contents of proline (355.0%-1586.7%) and malondialdehyde except in the stems (41.1%-81.3%). The non-photochemical quenching coefficiency and net photosynthetic rate in the leaves were significantly decreased by 49.4% and 77.4%, respectively. The contents of non-structural carbohydrates were increased by 21.4% in stems and 69.5% in leaves, but that in roots did not change. The contents of nitrate were increased by 28.9% in stems and 26.8% in leaves, but that in roots did not change. Ammonium nitrogen was increased by 16.2%, 12.9% and 217.6% in roots, stems, and leaves, but being statistically significant in the leaves. These results indicated that drought stress led to serious damage to 'Yanshanzaofeng' chestnut seedlings, which inhibited photosynthetic performance, but they could improve their adaptation to drought stress by enhancing carbon and nitrogen metabolism. Our results provide a reference for the breeding and cultivation of drought resistance of the local Chinese chestnut resources.

Distinctive Gene Expression Profiles and Effectors Consistent With Host Specificity in Two Formae Speciales of Marssonina brunnea.

The knowledge on the host specificity of a pathogen underlying an interaction is becoming an urgent necessity for global warming. In this study, the gene expression profiles and the roles of effectors in host specificity were integrally characterized with two formae speciales, multigermtubi and monogermtubi, of a hemibiotrophic pathogen Marssonina brunnea when they were infecting respective susceptible poplar hosts. With a functional genome comparison referring to a de novo transcriptome of M. brunnea and Pathogen-Host Interaction database functional annotations, the multigermtubi strain showed abundant and significant differentially expressed unigenes (DEGs) (more than 40%) in colonizing the initial invasion stage and in the necrotrophic stage. The monogermtubi strain induced less than 10% of DEGs in the initial invasion stage but which abruptly increased to more than 80% DEGs in the necrotrophic stage. Both strains induced the least DEGs in the biotrophic stage compared to the initial invasion and necrotrophic stages. The orthologs of the effector genes Ecp6, PemG1, XEG1, ACE1, and Mg3LysM were exclusively induced by one of the two formae speciales depending on the infection stages. Some unigenes homologous to carbohydrate lytic enzyme genes, especially pectate lyases, were notably induced with multigermtubi forma specialis infection but not expressed in the monogermtubi forma specialis at an earlier infection stage. The extraordinary differences in the functional genome level between the two formae speciales of M. brunnea could be fundamental to exploring their host specificity determinant and evolution. This study also firstly provided the fungal transcriptome of the monogermtubi forma specialis for M. brunnea.

An Endophytic Diaporthe apiculatum Produces Monoterpenes with Inhibitory Activity against Phytopathogenic Fungi.

Volatile organic compounds (VOCs) from endophytic fungi are becoming a potential antibiotic resource. The inhibitive effects of VOCs produced by an endophytic fungus in Leucaena leucocephala were investigated on plant pathogens in this study. Using standard morphological methods and multigene phylogeny, the fungus was identified as Diaporthe apiculatum strain FPYF 3052. Utilizing a two- compartment Petri plate bioassay method, the VOCs from this fungus showed bioactivity ranging from 23.8% to 66.7% inhibition on eight plant pathogens within 24 hours. The SPME-GC/MS technique identified fifteen volatile compounds with dominant terpenoids γ-terpinene (39.8%), α-terpinene (17.2%), and (-)-4-terpineol (8.4%) from the VOCs. Commercial α-terpinene, γ-terpinene, and (-)-4-terpineol demonstrated inhibition on the tested pathogens at concentrations from 0.2 to 1.0 µl/ml within 72 h in the bioassay system. The inhibition rates were from 28% to 100% percent using 1.0 µl/ml within 48 h. (-)-4-Terpineol was the most active of the terpenoids causing up to 100% inhibition. The data illustrate that these monoterpenes play an important role in the inhibitive bioactivity of the VOCs of D. apiculatum FPYF 3052. Most importantly, (-)-4-terpineol is now for the first time, reported to have capability of strong antifungal activity and could be developed as an antibiotic substance.

Organs, Cultivars, Soil, and Fruit Properties Affect Structure of Endophytic Mycobiota of Pinggu Peach Trees.

Pinggu peach (Prunus persica (L.)) has great economic and ecological value in north China. As a plant, the peach is naturally colonized by a variety of endophytic fungi, which are very important for tree growth and health. However, the mycobiota composition and their affecting factors of the peach trees are still unknown. In our study, the fungal communities in flowers, leaves, stems, and roots of the three cultivars (Dajiubao, Qingfeng, and Jingyan) of Pinggu peach trees and in the rhizosphere soils were investigated by both Illumina Miseq sequencing of ITS rDNA and traditional culturing methods. For organs, except for roots, flowers had the highest fungal richness and diversity, while the leaves had the lowest richness and diversity. Ascomycota and Basidiomycota were the most abundant phyla among samples. The fungal assemblage composition of each organ was distinctive. Fungal communities of the three cultivars also differed from each other. The fungal community structure significantly correlated with soil pH, soil K, fruit soluble solid content, and fruit titratable acidity with the redundancy analysis (RDA). Most isolated fungal strains can be found within high-throughput sequencing identified taxa. This study indicates that plant organs, the cultivars, the soil, and fruit properties may have profound effects on the endophytic fungal community structure associated with Pinggu peach trees. With this study, microbiota-mediated pathogen protection and fruit quality promotion associated with peach trees could be further studied.

Endophytic bacterial communities of Jingbai Pear trees in north China analyzed with Illumina sequencing of 16S rDNA.

Plant endophytes play a crucial role in plant growth, health and ecological function. Jingbai pear (the best quality cultivar of Pyrus ussuriensi Maxim. ex Rupr.) has important ecological and economic value in north China. Conversation of its genetics has great meanings to pear genus (Pyrus L.). However, the bacterial community associated with the cultivar remains unknown. In this study, the structure of endophytic bacterial communities associated with different tissues and soil of Jingbai Pear trees was analyzed with Illumina Miseq sequencing of bacterial 16S rDNA. This is the first report on bacterial microbiome associated with Jingbai pear. Our results demonstrated that different tissues harbored a unique bacterial assemblage. Interestingly, Cyanobacteria was the most abundant phylum, followed by Proteobacteria and Actinobacteria. Samples from three different sites (soils) had significant differences in microbial communities structure. Redundancy analysis (RDA) showed that the bacterial community structure correlated significantly with soil properties-temperature, pH, nitrogen and carbon contents. The conclusion could facilitate to understand the interaction and ecological function of endophytic bacteria with host Jingbai pear trees, so as to benefit the pear variety genetic resource conservation and protection.

Antifungal Activities of Volatile Secondary Metabolites of Four Diaporthe Strains Isolated from Catharanthus roseus.

Four endophytic fungi were isolated from the medicinal plant, Catharanthus roseus, and were identified as Diaporthe spp. with partial translation elongation factor 1-alpha (TEF1), beta-tubulin (TUB), histone H3 (HIS), calmodulin (CAL) genes, and rDNA internal transcribed spacer (ITS) region (TEF1-TUB-HIS--CAL-ITS) multigene phylogeny suggested for species delimitation in the Diaporthe genus. Each fungus produces a unique mixture of volatile organic compounds (VOCs) with an abundant mixture of terpenoids analyzed by headspace solid-phase microextraction (SPME) fiber-GC/MS. These tentatively-detected terpenes included α-muurolene, ß-phellandrene, γ-terpinene, and α-thujene, as well as other minor terpenoids, including caryophyllene, patchoulene, cedrene, 2-carene, and thujone. The volatile metabolites of each isolate showed antifungal properties against a wide range of plant pathogenic test fungi and oomycetes, including Alternaria alternata, Botrytis cinerea, Colletotrichum gloeosporioides, Fusarium graminearum, and Phytophthora cinnamomi. The growth inhibition of the pathogens varied between 10% and 60% within 72 h of exposure. To our knowledge, the endophytic Diaporthe-like strains are first reported from Catharanthus roseus. VOCs produced by each strain of the endophytic Diaporthe fungi were unique components with dominant monoterpenes comparing to known Diaporthe fungal VOCs. A discussion is presented on the inhibitive bioactivities of secondary metabolites among endophytic Diaporthe fungi and this medicinal plant.

Genome-Wide Transcriptome Analysis Reveals the Comprehensive Response of Two Susceptible Poplar Sections to Marssonina brunnea Infection.

Marssonina leaf spot disease of poplar (MLDP), caused by the hemibiotrophic pathogen Marssonina brunnea, frequently results in damage to many poplar species. In nature, two formae speciales of M. brunnea exist that are susceptible to different poplar subgenera. Marssonina brunnea f. sp. monogermtubi infects poplar hosts from Populus sect. Aigeiros (Aig), while M. brunnea f. sp. multigermtubi always infects poplar hosts from Populus sect. Leuce Duby (Leu). Based on the fungal penetration structures, a comprehensive transcriptomic approach was used to investigate the gene expression patterns of these two poplar subgenera at three crucial infection stages. MLDP significantly altered the expression patterns of many genes involved in mitogen activated protein kinase (MAPKs) and calcium signaling, transcription factors, primary and secondary metabolism, and other processes in both poplar subgenera. However, major differences in gene expression were also observed between the two poplar subgenera. Aig was most responsive at the initial infection stage, while Leu largely interacted with M. brunnea at the necrotrophic phase. Furthermore, the differentially expressed genes (DEGs) involved in pathways related to biotic stress also differed substantially between the two poplar subgenera. Further analysis indicated that the genes involved in cell wall metabolism and phenylpropanoid metabolism were differentially expressed in the progression of the disease. By examining the expression patterns of genes related to the defense against disease, we found that several genes annotated with causing hypersensitive cell death were upregulated at the necrotrophic phase of MLDP, inferring that plant immune response potentially happened at this infection stage. The present research elucidated the potential molecular differences between the two susceptible interaction systems in MLDP and provided novel insight into the temporal regulation of genes during the susceptible response. To the best of our knowledge, this study also constitutes the first to reveal the molecular mechanisms of poplar in response to the transition of hemibiotrophic fungal pathogens from the biotrophic phase to the necrotrophic phase.

ITS2 sequence-structure phylogeny reveals diverse endophytic Pseudocercospora fungi on poplars.

For matching the new fungal nomenclature to abolish pleomorphic names for a fungus, a genus Pseudocercospora s. str. was suggested to host holomorphic Pseudocercosproa fungi. But the Pseudocercosproa fungi need extra phylogenetic loci to clarify their taxonomy and diversity for their existing and coming species. Internal transcribed spacer 2 (ITS2) secondary structures have been promising in charactering species phylogeny in plants, animals and fungi. In present study, a conserved model of ITS2 secondary structures was confirmed on fungi in Pseudocercospora s. str. genus using RNAshape program. The model has a typical eukaryotic four-helix ITS2 secondary structure. But a single U base occurred in conserved motif of U-U mismatch in Helix 2, and a UG emerged in UGGU motif in Helix 3 to Pseudocercospora fungi. The phylogeny analyses based on the ITS2 sequence-secondary structures with compensatory base change characterizations are able to delimit more species for Pseudocercospora s. str. than phylogenic inferences of traditional multi-loci alignments do. The model was employed to explore the diversity of endophytic Pseudocercospora fungi in poplar trees. The analysis results also showed that endophytic Pseudocercospora fungi were diverse in species and evolved a specific lineage in poplar trees. This work suggested that ITS2 sequence-structures could become as additionally significant loci for species phylogenetic and taxonomic studies on Pseudocerospora fungi, and that Pseudocercospora endophytes could be important roles to Pseudocercospora fungi's evolution and function in ecology.

Inhibitory Effect of Vaginal Lactobacillus Supernatants on Cervical Cancer Cells.

Lactobacilli have been shown to inhibit the proliferation of several types of cancer cells, but the effects of vaginal Lactobacilli on cervical cancer cells have seldom been reported. We incubated Caski cells with supernatants of predominant strains in the vagina and investigated their effects on cell growth and the possible mechanisms. Cell-free supernatants of Lactobacillus crispatus, L. jensenii, and L. gasseri were prepared and purified. Caski cells were treated with various concentrations of Lactobacillus supernatants (LS). The effect of LS on cell growth was investigated using MTT assays. The influence of LS on the cell cycle and expression of human papillomavirus (HPV) E6 and E7 oncogenes was determined by flow cytometry and RT-PCR, respectively. LS-inhibited Caski cell proliferation caused morphological changes in a pH-independent manner. Flow cytometric analysis revealed that cells exposed to LS exhibited a significant increase of cell number in S phase and a strong decrease of cell number in G2/M phase. Expression of HPV E6 and E7 oncogenes, as well as CDK2 and cyclin A was decreased after treatment with LS, while expression of p21 was increased. Supernatants of L. crispatus, L. jensenii, and L. gasseri have inhibitory effects on the viability of cervical cancer cells via regulation of HPV oncogenes and cell cycle-related genes. Lactobacillus, as a promising treatment for cancer, is being assessed for its effect, and these results provide further evidence in this respect.

Genome-wide transcriptional response of Populus euphratica to long-term drought stress.

Populus euphratica is native to semi-arid regions of the Xinjiang Uyghur Autonomous Region of China, and studying its drought responses will greatly increase the understanding of how trees acclimate to drought. Water was withheld for seven weeks in four different drought stress treatments, with regime 1 being the least drought stressed and regime 4 being the most, and the poplar's transcriptional profiles examined with Affymetrix Poplar GeneChip microarrays. The number of significantly up or down transcriptional changes increased with the severity of drought stress, with regime 1, 2, 3 and 4 showing 952, 1354, 2138 and 2360 altered transcripts, respectively. Only 277 of these were found in common across all four regimes, while 1938 transcripts were found to be unique to the individual treatments. Genes with altered transcript abundance included members of the transcription factor families AP2/EREPB, bZIP, NAC, NF-Y, WRKY, MYB and Homeobox, as well as genes for the small HSP, HSP70 and HSP90 heat shock protein families. Analysis of the transcript data from these experiments indicated that P. euphratica activates specific regulatory pathways according to the degree of drought stress it receives. These results provide important insights into the molecular mechanisms underpinning the drought stress responses of poplar, as well as providing candidates for future experimentation.

Comparison of main lactobacillus species between healthy women and women with bacterial vaginosis.

BACKGROUND: The normal microbial flora of the vagina plays an important role in preventing genital and urinary tract infections in women. Thus an accurate understanding of the composition and ecology of the ecosystem is important to understanding the etiology of these diseases. This study aimed to compare the characteristics of main lactobacillus species between healthy women and women with bacterial vaginosis (BV) by quantitative culture and PCR methods. Main lactobacillus species include L. crispatus, L. gasseri, L. jensenii and L. iners. METHODS: A total of 150 women attending Gynecology Outpatient Clinic of Beijing Friendship Hospital, were diagnosed as having BV because three or more of the following criteria were met (standard of Amsel's composite criteria): homogeneous discharge, elevated vaginal pH (pH > 4.5), production of amines, and presence of "clue" cells. Those with less than three of the criteria were considered as healthy. Simultaneously, smears were made of vaginal fluid and Gram stained, then were assessed qualitatively as normal (grade I), intermediate (grade II), or consistent with BV (grade III). Gardnerella vaginalis were identified by using Vitek 2 Compact and PCR methods. Lactobacillus species were identified by PCR methods. Gardnerella vaginalis and lactobacilli colony counts were determined by calculating the most number of colonies of each species in the appropriate plates (colonies between 10 and 300), corrected by the dilution of the sample in the plates, and multiplied by 10 (to account for plating 100 microl), in order to get colony forming units per milliliter of vaginal secretion. RESULTS: BV was diagnosed in 31% (46/150) patients using the composite criteria, the remainder being regarded as healthy. The majority of patients with BV had a smear assessed as grade III (91%, 42/46) and minority of them had a smear assessed as grade II (9%, 4/46). The majority of healthy women had a smear assessed as grade I (64%, 67/104). Smears assessed as grade II were found (36%, 37/104) among patients diagnosed as healthy, with less than three of the composite criteria. L. crispatus was cultured from 94% of healthy women and 83% of women with BV, with the former colonies count average value of 10(6) and the latter of 10(3). L. gasseri, L. iners, and L. jensenii were cultured from 85%, 68% and 43% of healthy women; and 28%, 89% and 44% of BV women, respectively. CONCLUSIONS: The quantities of four lactobacillus species except L. jensenii had a significant difference between healthy women and women with BV. Our results provide support for the negative association between L. iners and L. gasseri. Although L. crispatus were existent both in healthy and BV positive women's vagina, the numbers of L. crispatus were significantly different for the dominant number in healthy women. Smears of vaginal fluid and Gram stain play an important guiding role in bacteria culture.