Phylogenetic and Morphological Evidence for Three New Species of Diaporthales (Ascomycota) from Fujian Province, China.

Members of the fungal order Diaporthales are sac fungi that include plant pathogens (the notorious chestnut blight fungus), as well as saprobes and endophytes, and are capable of colonizing a wide variety of substrates in different ecosystems, habitats, and hosts worldwide. However, many Diaporthales species remain unidentified, and various inconsistencies within its taxonomic category remain to be resolved. Here, we aimed to identify and classify new species of Diaporthales by using combined morphological and molecular characterization and coupling this information to expand our current phylogenetic understanding of this order. Fungal samples were obtained from dead branches and diseasedleaves of Camellia (Theaceae) and Castanopsis (Fagaceae) in Fujian Province, China. Based on morphological characteristics and molecular phylogenetic analyses derived from the combined nucleotide sequences of loci of the internal transcribed spacer regions with the intervening 5.8S nrRNA gene (ITS), the 28S large subunit of nuclear ribosomal RNA gene (LSU), the translation elongation factor 1-α gene (tef1), the partial beta-tubulin gene (tub2), and partial RNA polymerase II second-largest subunit gene (rpb2), three new species of Diaporthales were identified and characterized. They are as follows: Chrysofolia camelliae sp. nov., Dendrostoma castanopsidis sp. nov., and Pseudoplagiostoma wuyishanense sp. nov. They are described and illustrated. This study extends our understanding of species diversity within the Diaporthales.

Understanding and exploring the diversity of soil microorganisms in tea (Camellia sinensis) gardens: toward sustainable tea production.

Leaves of Camellia sinensis plants are used to produce tea, one of the most consumed beverages worldwide, containing a wide variety of bioactive compounds that help to promote human health. Tea cultivation is economically important, and its sustainable production can have significant consequences in providing agricultural opportunities and lowering extreme poverty. Soil parameters are well known to affect the quality of the resultant leaves and consequently, the understanding of the diversity and functions of soil microorganisms in tea gardens will provide insight to harnessing soil microbial communities to improve tea yield and quality. Current analyses indicate that tea garden soils possess a rich composition of diverse microorganisms (bacteria and fungi) of which the bacterial Proteobacteria, Actinobacteria, Acidobacteria, Firmicutes and Chloroflexi and fungal Ascomycota, Basidiomycota, Glomeromycota are the prominent groups. When optimized, these microbes' function in keeping garden soil ecosystems balanced by acting on nutrient cycling processes, biofertilizers, biocontrol of pests and pathogens, and bioremediation of persistent organic chemicals. Here, we summarize research on the activities of (tea garden) soil microorganisms as biofertilizers, biological control agents and as bioremediators to improve soil health and consequently, tea yield and quality, focusing mainly on bacterial and fungal members. Recent advances in molecular techniques that characterize the diverse microorganisms in tea gardens are examined. In terms of viruses there is a paucity of information regarding any beneficial functions of soil viruses in tea gardens, although in some instances insect pathogenic viruses have been used to control tea pests. The potential of soil microorganisms is reported here, as well as recent techniques used to study microbial diversity and their genetic manipulation, aimed at improving the yield and quality of tea plants for sustainable production.

Three New Species of Russulaceae (Russulales, Basidiomycota) from Southern China.

The characterization of natural fungal diversity impacts our understanding of ecological and evolutionary processes and can lead to novel bioproduct discovery. Russula and Lactarius, both in the order Russulales, represent two large genera of ectomycorrhizal fungi that include edible as well as toxic varieties. Based on morphological and phylogenetic analyses, including nucleotide sequences of the internal transcribed spacer (ITS), the 28S large subunit of ribosomal RNA (LSU), the second largest subunit of RNA polymerase II (RPB2), the ribosomal mitochondrial small subunit (mtSSU), and the translation elongation factor 1-α (TEF1-α) gene sequences, we here describe and illustrate two new species of Russula and one new species of Lactarius from southern China. These three new species are: R. junzifengensis (R. subsect. Virescentinae), R. zonatus (R. subsect. Crassotunicatae), and L. jianyangensis (L. subsect. Zonarii).

Assessing the structure and diversity of fungal community in plant soil under different climatic and vegetation conditions.

Introduction: Understanding microbial communities in diverse ecosystems is crucial for unraveling the intricate relationships among microorganisms, their environment, and ecosystem processes. In this study, we investigated differences in the fungal community structure and diversity in soils from two contrasting climatic and vegetation conditions: the Xinjiang western China plateau and the Fujian southeastern coastal province. Methods: A total of 36 soil samples collected from two climatic regions were subjected to high-throughput ITS gene sequencing for fungal community analysis. In conjunction soil physicochemical properties were assessed and compared. Analyses included an examination of the relationship of fungal community structure to environmental factors and functional profiling of the community structure was using the FUNGuild pipeline. Results: Our data revealed rich fungal diversity, with a total of 11 fungal phyla, 31 classes, 86 orders, 200 families, 388 genera, and 515 species identified in the soil samples. Distinct variations in the physicochemical properties of the soil and fungal community structure were seen in relation to climate and surface vegetation. Notably, despite a colder climate, the rhizosphere soil of Xinjiang exhibited higher fungal (α-)diversity compared to the rhizosphere soil of Fujian. ß-diversity analyses indicated that soil heterogeneity and differences in fungal community structure were primarily influenced by spatial distance limitations and vegetation type. Furthermore, we identified dominant fungal phyla with significant roles in energy cycling and organic matter degradation, including members of the Sordariomycetes, Leotiomycetes, Archaeosporomycetes, and Agaricomycetes. Functional analyses of soil fungal communities highlighted distinct microbial ecological functions in Xinjiang and Fujian soils. Xinjiang soil was characterized by a focus on wood and plant saprotrophy, and endophytes, whereas in Fujian soil the fungal community was mainly associated with ectomycorrhizal interactions, fungal parasitism, and wood saprotrophy. Discussion: Our findings suggest fungal communities in different climatic conditions adapt along distinct patterns with, plants to cope with environmental stress and contribute significantly to energy metabolism and material cycling within soil-plant systems. This study provides valuable insights into the ecological diversity of fungal communities driven by geological and environmental factors.

Characterization of Terpenoids from the Ambrosia Beetle Symbiont and Laurel Wilt Pathogen Harringtonia lauricola.

Little is known concerning terpenoids produced by members of the fungal order Ophiostomales, with the member Harringtonia lauricola having the unique lifestyle of being a beetle symbiont but potentially devastating tree pathogen. Nine known terpenoids, including six labdane diterpenoids (1-6) and three hopane triterpenes (7-9), were isolated from H. lauricola ethyl acetate (EtOAc) extracts for the first time. All compounds were tested for various in vitro bioactivities. Six compounds, 2, 4, 5, 6, 7, and 9, are described functionally. Compounds 2, 4, 5, and 9 expressed potent antiproliferative activity against the MCF-7, HepG2 and A549 cancer cell lines, with half-maximal inhibitory concentrations (IC50s) ~12.54-26.06 µM. Antimicrobial activity bioassays revealed that compounds 4, 5, and 9 exhibited substantial effects against Gram-negative bacteria (Escherichia coli and Ralstonia solanacearum) with minimum inhibitory concentration (MIC) values between 3.13 and 12.50 µg/mL. Little activity was seen towards Gram-positive bacteria for any of the compounds, whereas compounds 2, 4, 7, and 9 expressed antifungal activities (Fusarium oxysporum) with MIC values ranging from 6.25 to 25.00 µg/mL. Compounds 4, 5, and 9 also displayed free radical scavenging abilities towards 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide (O2-), with IC50 values of compounds 2, 4, and 6 ~3.45-14.04 µg/mL and 22.87-53.31 µg/mL towards DPPH and O2-, respectively. These data provide an insight into the biopharmaceutical potential of terpenoids from this group of fungal insect symbionts and plant pathogens.

Characterization of an α-Amylase from the Honeybee Chalk Brood Pathogen Ascosphaera apis.

The insect pathogenic fungus, Ascosphaera apis, is the causative agent of honeybee chalk brood disease. Amylases are secreted by many plant pathogenic fungi to access host nutrients through the metabolism of starch, and the identification of new amylases can have important biotechnological applications. Production of amylase by A. apis in submerged culture was optimized using the response surface method (RSM). Media composition was modeled using Box-Behnken design (BBD) at three levels of three variables, and the model was experimentally validated to predict amylase activity (R2 = 0.9528). Amylase activity was highest (45.28 ± 1.16 U/mL, mean ± SE) in media composed of 46 g/L maltose and1.51 g/L CaCl2 at a pH of 6.6, where total activity was ~11-fold greater as compared to standard basal media. The enzyme was purified to homogeneity with a 2.5% yield and 14-fold purification. The purified enzyme had a molecular weight of 75 kDa and was thermostable and active in a broad pH range (> 80% activity at a pH range of 7-10), with optimal activity at 55 °C and pH = 7.5. Kinetic analyses revealed a Km of 6.22 mmol/L and a Vmax of 4.21 µmol/mL·min using soluble starch as the substrate. Activity was significantly stimulated by Fe2+ and completely inhibited by Cu2+, Mn2+, and Ba2+ (10 mM). Ethanol and chloroform (10% v/v) also caused significant levels of inhibition. The purified amylase essentially exhibited activity only on hydrolyzed soluble starch, producing mainly glucose and maltose, indicating that it is an endo-amylase (α-amylase). Amylase activity peaked at 99.38 U/mL fermented in a 3.7 L-bioreactor (2.15-fold greater than what was observed in flask cultures). These data provide a strategy for optimizing the production of enzymes from fungi and provide insight into the α-amylase of A. apis.

Morphological and phylogenetic analyzes reveal two new species of Melanconiella from Fujian Province, China.

Introduction: Species of Melanconiella include a diverse array of plant pathogens as well as endophytic fungi. Members of this genus have been frequently collected from the family Betulaceae (birches) in Europe and North America. Little, however, if known concerning the distribution of Melanconiella and/or their potential as pathogens of other plant hosts. Methods: Fungi were noted and isolated from diseased leaves of Loropetalum chinense (Chinese fringe flower) and Camellia sinensis (tea) in Fujian Province, China. Genomic DNA was extracted from fungal isolates and the nucleotide sequences of four loci were determined and sued to construct phylogenetic trees. Morphological characteristics of fungal structures were determined via microscopic analyses. Results: Four strains and two new species of Melanconiella were isolated from infected leaves of L. chinense and C. sinensis in Fujian Province, China. Based on morphology and a multi-gene phylogeny of the internal transcribed spacer regions with the intervening 5.8S nrRNA gene (ITS), the 28S large subunit of nuclear ribosomal RNA (LSU), the second largest subunit of RNA polymerase II (RPB2), and the translation elongation factor 1-α gene (TEF1-α), Melanconiellaloropetali sp. nov. and Melanconiellacamelliae sp. nov. were identified and described herein. Detailed descriptions, illustrations, and a key to the known species of Melanconiella are provided. Discussion: These data identify new species of Melanconiella, expanding the potential range and distribution of these dark septate fungi. The developed keys provide a reference source for further characterization of these fungi.

Isolation of a highly virulent Metarhizium strain targeting the tea pest, Ectropis obliqua.

Introduction: Tea is one of the most widely consumed beverages around the world. Larvae of the moth, Ectropis obliqua Prout (Geometridae, Lepidoptera), are one of the most destructive insect pests of tea in China. E. obliqua is a polyphagus insect that is of increasing concern due to the development of populations resistant to certain chemical insecticides. Microbial biological control agents offer an environmentally friendly and effective means for insect control that can be compatible with "green" and organic farming practices. Methods: To identify novel E. obliqua biological control agents, soil and inset cadaver samples were collected from tea growing regions in the Fujian province, China. Isolates were analyzed morphologically and via molecular characterization to identity them at the species level. Laboratory and greenhouse insect bioassays were used to determine the effectiveness of the isolates for E. obliqua control. Results: Eleven isolates corresponding to ten different species of Metarhizium were identified according to morphological and molecular analyses from soil and/or insect cadavers found on tea plants and/or in the surrounding soil sampled from eight different regions within the Fujian province, China. Four species of Metarhizium including M. clavatum, M. indigoticum, M. pemphigi, and M. phasmatodeae were documented for the first time in China, and the other species were identified as M. anisopliae, M. brunneum, M. lepidiotae, M. majus, M. pinghaense, and M. robertsii. Insect bioassays of the eleven isolates of Metarhizium revealed significant variation in the efficacy of each isolate to infect and kill E. obliqua. Metarhizium pingshaense (MaFZ-13) showed the highest virulence reaching a host target mortality rate of 93% in laboratory bioassays. The median lethal concentration (LC50) and median lethal time (LT50) values of M. pingshaense MaFZ-13 were 9.6 × 104 conidia/mL and 4.8 days, respectively. Greenhouse experiments and a time-dose-mortality (TDM) models were used to further evaluate and confirm the fungal pathogenic potential of M. pingshaense MaFZ-13 against E. obliqua larvae. Discussion: Isolation of indigenous microbial biological control agents targeting specific pests is an effective approach for collecting resources that can be exploited for pest control with lowered obstacles to approval and commercialization. Our data show the presence of four different previously unreported Metarhizium species in China. Bioassays of the eleven different Metarhizium strains isolated revealed that each could infect and kill E. obliqua to different degrees with the newly isolated M. pingshaense MaFZ-13 strain representing a particularly highly virulent isolate potentially applicable for the control of E. obliqua larvae.

Co-expression network analysis uncovers key candidate genes related to the regulation of volatile esters accumulation in Woodland strawberry.

MAIN CONCLUSION: FveERF (FvH4_5g04470.1), FveAP2 (FvH4_1g16370.1) and FveWRKY (FvH4_6g42870.1) might be involved in fruit maturation of strawberry. Overexpression of FveERF could activate the expression of AAT gene and ester accumulation. Volatile esters play an important role in the aroma of strawberry fruits, whose flavor is the result of a complex mixture of various esters. The accumulation of these volatiles is closely tied to changes in metabolism during fruit ripening. Acyltransferase (AAT) is recognized as having a significant effect in ester formation. However, there is little knowledge about the regulation network of AAT. Here, we collected the data of RNA-seq and headspace GC-MS at five time points during fruit maturation of Hawaii4 and Ruegen strawberry varieties. A total of 106 volatile compounds were identified in the fruit of woodland strawberries, including 58 esters, which occupied 41.09% (Hawaii4) or 33.40% (Ruegen) of total volatile concentration. Transcriptome analysis revealed eight transcription factors highly associated with AAT genes. Through the changes in esters and the weight co-expression network analysis (WGCNA), a detailed gene network was established. This demonstrated that ERF gene (FvH4_5g04470.1), AP2 gene (FvH4_1g16370.1) and one WRKY gene (FvH4_6g42870.1) might be involved in expression of AAT genes, especially ERF genes. Overexpression of FveERF (FvH4_5g04470.1) does activate expression of AAT genes and ester accumulation in fruits of strawberry. Our findings provide valuable clues to gain better insight into the ester formation process of numerous fruits.

Rapid evolutionary dynamics of pepper mild mottle virus.

Pepper mild mottle virus (PMMoV) is one of the most destructive pathogens of pepper crops and has major impacts on global crop yields. Some aspects of the molecular biology of PMMoV have been studied intensively, but estimates of its evolutionary rate have shown considerable variation. We investigated the phylodynamics of PMMoV by analysing 171 nucleotide sequences of the coat protein gene, sampled between 1980 and 2016. Our Bayesian phylogenetic analyses, using the structured coalescent, dated the crown group to 1949 (95% credibility interval 1935-1962). We reveal that PMMoV has been evolving at a rate of 9.363 × 10-4 substitutions/site/year (95% credibility interval 7.362 × 10-4-1.138 × 10-3). This is similar to evolutionary rates estimated for animal RNA viruses, indicating that PMMoV has been undergoing rapid evolutionary dynamics.

Overexpression of an EaZIP gene devoid of transit peptide sequence induced leaf variegation in tobacco.

Leaf variegation is an ornamental trait that is not only biologically but also economically important. In our previous study, a Mg-protoporphyrin IX monomethyl ester cyclase homologue, EaZIP (Epipremnum aureum leucine zipper) was found to be associated with leaf variegation in Epipremnum aureum (Linden & Andre) G.S. Bunting. The protein product of this nuclear-encoded gene is targeted back to chloroplast involving in chlorophyll biosynthesis. Based on a web-based homology analysis, the EaZIP was found to lack a chloroplast transit peptide (cTP) sequence. In the present study, we tested if overexpression of the EaZIP cDNA with or without the cTP sequence could affect leaf variegation. Transgenic tobacco plants overexpressing EaZIP genes with (EaZIPwcTP) and without (EaZIPwocTP) cTP sequence were generated. Many plant lines harboring EaZIPwocTP showed variegated leaves, while none of the plant lines with EaZIPwcTP produced such a phenotype. Molecular analysis of T0 plants and selfed T1 progeny, as well as observations of tagged marker GFP (green fluorescent protein) did not show any other difference in patterns of gene integrity and expression. Results from this study indicate that transgenic approach for expressing EaZIPwocTP could be a novel method of generating variegated plants even through the underlying mechanisms remain to be elucidated.