ITS Fragments Research Articles

Bjerkandera adusta Collected from Niğde: Analysis of Total Phenolic Compound, Antioxidant, vnd Antimicrobial Properties

Macrofungi have long been used as food and medicinal purposes by humankind. This study aimed to identify a macrofungus sample collected from Niğde, Türkiye in 2019 via molecular techniques and determine the content of total phenolic compounds, antioxidant and antimicrobial activities. According to data obtained from the sequence analysis of ITS fragment of rDNA the macrofungus sample was identified as Bjerkandera adusta (Willd.) P. Karst (1880). The sample was extracted with ethanol and methanol. Total phenolic compound content, antioxidant activity via DPPH scavenging method and antimicrobial activity via disc diffusion method of B. adusta was determined by using these extracts. The amount of total phenolic compounds was found as 772.28 µg GAE/mL for a methanolic extract of B. adusta. The ethanol extract of B. adusta showed 79.66% scavenging activity of 0.1 mM DPPH solution. The highest inhibition zone diameter was measured as 28±1 mm against P. aeruginosa by ethanolic extract, while the lowest antimicrobial activity was found in 15 µL methanol extract against S. typhimurium with an inhibition zone diameter of 8.7±1.2 mm.

First Molecular Detection of Bartonella bovis and Bartonella schoenbuchensis in European Bison (Bison bonasus)

Bartonella bacteria infect the erythrocytes and endothelial cells of mammalians. The spread of the Bartonella infection occurs mainly via bloodsucking arthropod vectors. Studies on Bartonella infection in European bison, the largest wild ruminant in Europe, are lacking. They are needed to clarify their role in the maintenance and transmission of Bartonella spp. The aim of this study was to investigate the presence of the Bartonella pathogen in European bison and their ticks in Lithuania. A total of 38 spleen samples from bison and 258 ticks belonging to the Ixodes ricinus and Dermacentor reticulatus species were examined. The bison and tick samples were subjected to ssrA, 16S-23S rRNA ITS, gltA, and rpoB partial gene fragment amplification using various variants of PCR. Bartonella DNA was detected in 7.9% of the tissue samples of European bison. All tick samples were negative for Bartonella spp. The phylogenetic analysis of 16S-23S rRNA ITS, gltA, and rpoB partial gene fragment revealed that European bison were infected by B. bovis (2.6%) and B. schoenbuchensis (5.3%). This is the first report addressing the occurrence of B. bovis and B. schoenbuchensis in European bison in Europe.

The Analysis of the Mycobiota in Plastic Polluted Soil Reveals a Reduction in Metabolic Ability.

Plastic pollution is a growing environmental issue that results in its accumulation and persistence in soil for many decades, with possible effects on soil quality and ecosystem services. Microorganisms, and especially fungi, are a keystone of soil biodiversity and soil metabolic capacity. The aim of this research was to study soil fungal biodiversity and soil microbial metabolic profiles in three different sites in northern Italy, where macro- and microplastic concentration in soil was measured. The metabolic analyses of soil microorganisms were performed by Biolog EcoPlates, while the ITS1 fragment of the 18S ribosomal cDNA was used as a target for the metabarcoding of fungal communities. The results showed an intense and significant decrease in soil microbial metabolic ability in the site with the highest concentration of microplastics. Moreover, the soil fungal community composition was significantly different in the most pristine site when compared with the other two sites. The metabarcoding of soil samples revealed a general dominance of Mortierellomycota followed by Ascomycota in all sampled soils. Moreover, a dominance of fungi involved in the degradation of plant residues was observed in all three sites. In conclusion, this study lays the foundation for further research into the effect of plastics on soil microbial communities and their activities.

The efficacy of AuNP-probe conjugate nanobiosensor in non-amplification and amplification forms for the diagnosis of leishmaniasis

Nanobiosensor platforms have emerged as convenient and promising approaches with remarkable efficacy for the diagnosis of infectious diseases. Gold nanoparticles (AuNPs) have been widely used due to numerous advantageous properties such as optical, electrical, physicochemical and great biomolecules binding capabilities. This study aimed to apply AuNP-Probe Conjugate for the detection of Leishmania spp., using colorimetric and amplification methods targeting parasitic ITS2 fragment. The first method was carried out by hybridization of 10µL of DNA with 4 µL of probe and addition of 5 µL of 0.2 N HCl (non-amplification method). Second method was followed by polymerase chain reaction (PCR) amplification using thiolated primer, 5 µL of AuNP and 5 µL of 0.2 N HCl. The appearance of red and purple colors indicated positive and negative results, respectively. The minimum of detection for non-amplification and amplification methods for three strains of Leishmania namely L. major, L. tropica and L. infantum were determined to be 32 fg/µL and 16 fg/µL, respectively. Sensitivity for detection of visceral leishmaniasis (VL) for non-amplification and amplification methods included 96% and 100%, respectively and for cutaneous leishmaniasis (CL) included 98% and 100%, respectively. The results of this investigation revealed that sensitivity of amplification method was the same as RT-qPCR, while that of non-amplification method was lower. However, this method was promising because of no need for any equipment, high specificity, enough sensitivity, low cost and rapidity (less than 30 min) to complete after genomic DNA extraction.

The genetic characteristics of Sarcoptes scabiei from Chinese serow (Capricornis milneedwardsii) and goral (Naemorhedus goral arnouxianus) compared with other mites from different hosts and geographic locations using ITS2 and cox1 sequences.

Scabies is a common parasitic disease in many mammalian species, caused by the infestation of Sarcoptes scabiei. There is no consistent conclusion on whether Sarcoptes mites from different hosts or geographic locations have apparent genetic divergence. In this study, we collected and morphologically identified S. scabiei from Chinese serow and goral, and we described the genetic diversity of S. scabiei and other mites based on phylogenetic analyses of the ITS2 and cox1 sequence fragments, including data available in GenBank. The mites isolated from Chinese serow and goral were S. scabiei, and they were morphologically similar. The phylogenetic trees and haplotype networks showed that S. scabiei from other locations worldwide did not cluster according to host divergence or geographical distribution. Additionally, the Fst values were - 0.224 to 0.136 and - 0.045 to 1 between S. scabiei from different hosts, including humans and domestic and wild animals, based on partial ITS and cox1 sequences. Worldwide S. scabiei samples formed three clusters (with H2, H5, and H12 at their centers) in the ITS and one cluster (with C9 at the center) in the cox1 haplotype phylogenetic network. The S. scabiei collected from Chinese serow and goral were morphologically similar and had the same genotype. A study on the genetic characteristics of S. scabiei from Chinese serow and goral together with other mites from different hosts and geographic locations around the world showed no obvious divergence. These findings indicated that scabies likely is a zoonotic disease and that the global prevalence of scabies is probably related to the worldwide trade of domestic animals.

Molecular Identification And Phylogenetic Analysis of Fasciola Species Isolated from Sheep And Cattle Using The Its2 Rdna Marker in Duhok Province, Kurdistan Region, Iraq

The aims of the current study were molecular identification and phylogenetic analysis of Fasciola spp. isolated from infected cattle and sheep using the ITS2 region of rDNA as a molecular marker in Duhok province, Kurdistan region, Iraq. DNA was extracted from 40 adult flukes isolated from the livers of infected sheep (23) and cattle (17) slaughtered at the abattoirs of Duhok Province. The amplification of the rDNA ITS2 region of these 40 specimens was done by PCR using primers (FITS2-F and FITS2-R) designed for this study by the authors. ITS2 fragments (approximately 314 bp) of the rDNA were successfully amplified, and produced monoclear bands, that were confirmed by gel electrophoresis. From these isolates, 5 amplicons of ITS2 (2 sheep and 3 cattle) were selected for sequencing, and then the obtained sequences were compared to those in NCBI-GenBank for genotyping and phylogenetic analysis. The blast alignment of NCBI-GenBank revealed four sequences belonging to Fasciola hepatica and one belonging to Fasciola gigantica, both with 100% similarity. The genetic distance between F. hepatica and F. gigantica was 0.0127. Phylogenetic analysis revealed that the sequences in this study were clustered into two main clades (F. hepatica and F. gigantica), with other sequences available in GenBank. The study concluded that ITS2 rDNA sequences were appropriate to identify both Fasciola species isolated from the infected sheep and cattle in Duhok province, which were identical to those of neighboring countries (Iran and Turkey). The primers of ITS2 rDNA region that designed for the current study can be useful for the diagnosis of animals and humans fasciolosis

Morphological variation in Northwest Pacific Devaleraea mollis and description of D. inkyuleei sp. nov. (Palmariaceae, Rhodophyta)

ABSTRACT We assessed the range of morphological variation in Devaleraea mollis from the Northwest Pacific. Genetic analyses based on the 5' end of the genes coding for cytochrome c oxidase I (COI-5'), cytochrome oxidase b (cob) and photosystem I reaction centre apoprotein A1 (psaA), as well as the ITS1–5.8S–ITS2 (ITS) fragment of rDNA, revealed that the morphospecies D. mollis includes two species. The alga from Japan previously identified as ‘Palmaria palmata’ resolved as sister to D. stenogona and is here described as D. inkyuleei sp. nov. The species is morphologically similar to D. mollis, but it has a more southern range. Devaleraea inkyuleei is only known from Japan (Honshu and Hokkaido) and from Aniva Bay (Sakhalin Island, Russia).

Soil texture is a stronger driver of the maize rhizosphere microbiome and extracellular enzyme activities than soil depth or the presence of root hairs

AimsDifferent drivers are known to shape rhizosphere microbiome assembly. How soil texture (Texture) and presence or lack of root hairs (Root Hair) of plants affect the rhizosphere microbiome assembly and soil potential extracellular enzyme activities (EEA) at defined rooting depth (Depth) is still a knowledge gap. We investigated effects of these drivers on microbial assembly in rhizosphere and on potential EEA in root-affected soil of maize.MethodsSamples were taken from three depths of root hair defective mutant rth3 and wild-type WT maize planted on loam and sand in soil columns after 22 days. Rhizosphere bacterial, archaeal, fungal and cercozoan communities were analysed by sequencing of 16S rRNA gene, ITS and 18S rRNA gene fragments. Soil potential EEA of ß-glucosidase, acid phosphatase and chitinase were estimated using fluorogenic substrates.ResultsThe bacterial, archaeal and cercozoan alpha- and beta-diversities were significantly and strongly altered by Texture, followed by Depth and Root Hair. Texture and Depth had a small impact on fungal assembly, and only fungal beta-diversity was significantly affected. Significant impacts by Depth and Root Hair on beta-diversity and relative abundances at taxonomic levels of bacteria, archaea, fungi and cercozoa were dependent on Texture. Likewise, the patterns of potential EEA followed the trends of microbial communities, and the potential EEA correlated with the relative abundances of several taxa.ConclusionsTexture was the strongest driver of rhizosphere microbiome and of soil potential EEA, followed by Depth and Root Hair, similarly to findings in maize root architecture and plant gene expression studies.

Bees under interactive stressors: the novel insecticides flupyradifurone and sulfoxaflor along with the fungicide azoxystrobin disrupt the gut microbiota of honey bees and increase opportunistic bacterial pathogens

The gut microbiome plays an important role in bee health and disease. But it can be disrupted by pesticides and in-hive chemicals, putting honey bee health in danger. We used a controlled and fully crossed laboratory experimental design to test the effects of a 10-day period of chronic exposure to field-realistic sublethal concentrations of two nicotinic acetylcholine receptor agonist insecticides (nACHRs), namely flupyradifurone (FPF) and sulfoxaflor (Sulf), and a fungicide, azoxystrobin (Azoxy), individually and in combination, on the survival of individual honey bee workers and the composition of their gut microbiota (fungal and bacterial diversity). Metabarcoding was used to examine the gut microbiota on days 0, 5, and 10 of pesticide exposure to determine how the microbial response varies over time; to do so, the fungal ITS2 fragment and the V4 region of the bacterial 16S rRNA were targeted. We found that FPF has a negative impact on honey bee survival, but interactive (additive or synergistic) effects between either insecticide and the fungicide on honey bee survival were not statistically significant. Pesticide treatments significantly impacted the microbial community composition. The fungicide Azoxy substantially reduced the Shannon diversity of fungi after chronic exposure for 10 days. The relative abundance of the top 10 genera of the bee gut microbiota was also differentially affected by the fungicide, insecticides, and fungicide-insecticide combinations. Gut microbiota dysbiosis was associated with an increase in the relative abundance of opportunistic pathogens such as Serratia spp. (e.g. S. marcescens), which can have devastating consequences for host health such as increased susceptibility to infection and reduced lifespan. Our findings raise concerns about the long-term impact of novel nACHR insecticides, particularly FPF, on pollinator health and recommend a novel methodology for a refined risk assessment that includes the potential effects of agrochemicals on the gut microbiome of bees.

Molecular Characterization of Animal Fasciola Spp. Isolates from Lorestan Province, Western Iran.

Background:We aimed to detect the genetic diversity of samples identified morphologically as Fasciola spp. from sheep, cattle and goat from Lorestan Province, western Iran using PCR-RFLP method. Besides, we evaluated the genetic diversity indices, sequencing and phylogenetic analysis using mitochondrial gene (ND1 and CO1).Methods:PCR-RFLP analysis of ribosomal ITS1 fragment by RsaI restriction enzyme to investigate the genetic characteristics of Fasciola species obtained from different hosts (18 sheep, 21 cattle, and 17goats) was conducted. The samples were sequenced. Sequences were evaluated using BLAST software and the parasite species were identified with similarity percentage and overlap with the species registered in the gene bank. Then similarity and diversity of intra-species and intra-species diversity of Fasciola species were calculated.Results:In Lorestan, based on RFLP pattern, 93% (52) of the Fasciola spp. isolates had a RFLP pattern related to F. hepatica and 7% (4) were F. gigantica. No hybrid forms were detected. The CO1 gene could clarify 19 haplotypes against ND1 gene that found 22 haplotypes among livestock. Sequencing results of the mtDNA showed intra-species identity 98. 5%–100% and Intra-species-diversity: 0–1.5% compared to the GenBank sequences.Conclusion:Using PCR-RFLP method, two species of F. hepatica and F. gigantica, were present in Lorestan Province, but F. hepatica was more prevalent. Mitochondrial genes could better test variability indices in different hosts than ribosomal genes, consequently among mitochondrial genes, the ND1 gene could better examine differences and similarities than CO1.

Morphological and Genetic Variability in Radix auricularia (Mollusca: Gastropoda: Lymnaeidae) of Lake Baikal, Siberia: The Story of an Unfinished Invasion into the Ancient Deepest Lake

This article aims to reconstruct the invasion of the ear-shaped pond snail, Radix auricularia (Linnaeus, 1758), to Lake Baikal, East Siberia. This species is widely distributed in the Palaearctic and Northern America, and since the early 20th century has formed abundant and sustained populations in Lake Baikal. The data provided on the morphological and genetic variability of R. auricularia help to better describe and delineate the species. With an integrative approach involving morphological and molecular data, we improved the knowledge of the intraspecific variability of R. auricularia in the most important characteristics used for its determination. Molecular sequences of nuclear spacer fragment ITS-2 and mitochondrial gene fragment cyt–b were obtained from 32 individuals of Radix (including seven outgroup Radix species) collected from various parts of Lake Baikal and adjacent waterbodies and compared with sequences of 32 individuals of R. auricularia from different regions of the Palaearctic as well as with individuals determinated as R. intercisa from Lake Baikal, R. iturupica from the Kurile Islands, R. ussuriensis from the Khabarovsk region, R. narzykulovi from Tajikistan, and R. schubinae from the Amur region. Molecular genetic analyses revealed that all specimens collected from Lake Baikal belong to R. auricularia. There are no genetically distinct groups of snails that would correspond to two morphospecies previously recorded in Lake Baikal (e.g., R. auricularia s. str. and R. intercisa). Variability of the characteristics that are commonly used for species identification (shell morphology, mantle pigmentation, shape and position of the bursa copulatrix, length and position of the bursa duct, length ratio of preputium to penial sheath) were found in individuals analysed with molecular genetics to be broader than recognised in the current literature. Some shells of R. auricularia collected from Lake Baikal resemble shells of another lymnaeid species, R. balthica, and without molecular assessment can be confused with the latter. Geometric morphometric analysis of more than 250 shells revealed no observed hiatus between Baikalian and non-Baikalian R. auricularia. The probable stages and pathways of R. auricularia invasion to Lake Baikal’s ecosystem are outlined and discussed. Factors such as global climate warming and human activity stimulated and facilitated the ongoing dispersal of ear pond snails within Lake Baikal.

Red Foxes (Vulpes vulpes) Are Exposed to High Diversity of Borrelia burgdorferi Sensu Lato Species Infecting Fox-Derived Ixodes Ticks in West-Central Poland.

The role of red fox, Vulpes vulpes, and its associated ticks in maintaining Borrelia burgdorferi sensu lato (s.l.) was studied. A total of 1583 ticks were removed from ears of 120 infested animals and were identified as species using a nested PCR targeting the ITS2 and coxI fragments of Ixodes DNA. Ixodes kaiseri prevailed (76%), followed by I. canisuga, I. ricinus, and I. hexagonus. In total, 32.4% of 943 ticks revealed Borrelia DNA and 10 species of B. burgdorferi s.l. complex were identified. Borrelia garinii and B. afzelii comprised 70% of all infections. The other eight species included B. americana, B. bissettiae, B. burgdorferi sensu stricto (s.s.), B. californiensis, B. carolinensis, B. lanei, B. spielmanii, and B. valaisiana. Analysis of tissues from 243 foxes showed that 23.5% were infected with B. burgdorferi s.l. Borrelia garinii was detected in 91% of the infected animals, including 31% of mixed infections with B. afzelii, the second most prevalent species, followed by B. spielmanii. The predominance of B. garinii in PCR-positive animals and infected larval ticks (38.1%), suggests that this spirochete and B. afzelii are preferentially associated with foxes. Although red foxes are exposed to a high diversity of B. burgdorferi s.l. species found in engorged Ixodes ticks, their reservoir competence for most of them appears to be low.

Long-amplicon MinION-based sequencing study in a salt-contaminated twelfth century granite-built chapel

The irregular damp dark staining on the stonework of a salt-contaminated twelfth century granite-built chapel is thought to be related to a non-homogeneous distribution of salts and microbial communities. To enhance understanding of the role of microorganisms in the presence of salt and damp stains, we determined the salt content and identified the microbial ecosystem in several paving slabs and inner wall slabs (untreated and previously bio-desalinated) and in the exterior surrounding soil. Soluble salt analysis and culture-dependent approaches combined with archaeal and bacterial 16S rRNA and fungal ITS fragment as well as with the functional genes nirK, dsr, and soxB long-amplicon MinION-based sequencing were performed. State-of-the-art technology was used for microbial identification, providing information about the microbial diversity and phylogenetic groups present and enabling us to gain some insight into the biological cycles occurring in the community key genes involved in the different geomicrobiological cycles. A well-defined relationship between microbial data and soluble salts was identified, suggesting that poorly soluble salts (CaSO4) could fill the pores in the stone and lead to condensation and dissolution of highly soluble salts (Ca(NO3)2 and Mg(NO3)2) in the thin layer of water formed on the stonework. By contrast, no direct relationship between the damp staining and the salt content or related microbiota was established. Further analysis regarding organic matter and recalcitrant elements in the stonework should be carried out.Key points• Poorly (CaSO4) and highly (Ca(NO3)2, Mg(NO3)2) soluble salts were detected• Halophilic and mineral weathering microorganisms reveal ecological impacts of salts• Microbial communities involved in nitrate and sulfate cycles were detected

Evaluation of Four Commonly Used DNA Barcoding Loci for Ardisia Species Identification.

Ardisia plants have been used as medicinal plants for a long time in China. Traditional techniques such as morphological, microscopic, and chemical identification methods all have limitations in the species identification of Ardisia. For the sake of drug safety, four DNA barcodes (psbA-trnH, ITS, rbcL, and matK) were assessed for Chinese Ardisia plants using a total of 121 individuals from 33 species. Four criteria (The success rates of PCR amplification, DNA barcoding gap, DNA sequence similarity analysis and NJ tree clustering analysis) were used to evaluate the species identification ability of these four DNA barcodes. The results show that ITS had the highest efficiency in terms of PCR and sequencing and exhibited the most apparent inter- and intra-specific divergences and the highest species identification efficiency. There was no significant increase in species identification after combining the three cpDNA fragments with the ITS fragment. Considering the cost and experimental effectiveness, we recommend ITS as the core barcode for identifying Chinese Ardisia plants.

Molecular Analysis of the Heterakis dispar Population in Domestic Geese Based on the ITS1-5.8rRNA-ITS2 Fragment.

Simple SummaryHeterakis is a nematode which infects the ceca of birds. In geese, Heterakis dispar is the most common species. Due to the scarcity of data concerning the H. dispar population, we are providing this analysis based on the ITS1-5.8rRNA-ITS2 region, one of the most frequently used fragments in genetic analysis. We analyzed 71 H. dispar specimens isolated from 20 geese flocks. In the analyzed fragment, four nucleotide differences were noted, resulting in six types of sequences (A, B, C, D, E, and F). The most frequently noted type was type A (45%), followed by type B (18.3%), type C and D (11.3%), type E (8.5%), and finally, type F (5.6%). Infection with nematodes from different types of groups was noted in 30% of flocks, with type A being the most prevalent, followed by types B, D, or E to make up the remaining 100%. This study represents the first H. dispar population analysis based on the ITS1-5.8rRNA-ITS2 fragment.Heterakidosis is a parasitic infection in birds caused by the cecal parasite Heterakis spp. The most common species in geese is H. dispar, the largest avian heterakids species. Because of a scarcity of data concerning the H. dispar population, the aim of this study was the genetic analysis of Heterakis dispar isolated from geese flocks based on the ITS1-5.8rRNA-ITS2 fragment. Among the 71 H. dispar specimens isolated from 20 geese flocks, six haplotypes were determined (A, B, C, D, E, and F). The four nucleotide substitutions were noted in both ITS fragments, and all of them were transitions between adenine and guanine, or thymine and cytosine. The most frequently noted haplotype was type A (45%), followed by type B (18.3%), type C and D (11.3%), type E (8.5%), and F (5.6%). Infection with nematodes from different haplotype groups was noted in 30% of the flocks, with type A being the most prevalent, followed by types B, D, or E to make up 100%. This study represents the first H. dispar population analysis based on the ITS1-5.8rRNA-ITS2 fragment.

First Report of Southern Blight Caused by Sclerotium delphinii on Euonymus fortunei in China.

Euonymus fortunei is an evergreen shrub-vine in the family Celastraceae, widely used as a groundcover or a vine to climb walls, or traditional herbal medicine in China. In August 2019, typical southern blight symptoms that included basal stem rot and the presence of sclerotia in rotted tissue were observed on E. fortunei in Kunshan city, Jiangsu province, China. Disease incidence was estimated at approximately 15 to 20%; meanwhile, approximately 30 to 40% of diseased plants died. The infected plants showed brown to dark stem necrosis near the base, leaf yellowing and wilting. White mycelia and white to dark reddish-brown sclerotia were observed at the base of the stem and rotten tissue. To isolate the causal organism, infected stem tissue and sclerotia collected from diseased plants in a median strip in Kunshan (31°23'40"N, 120°54'57"E) were disinfected with 70% ethanol for 2 to 3 sec, followed by 2 min in 5% NaClO, rinsed three times with sterile water, then plated on potato dextrose agar (PDA) medium, and incubated at 25°C. Isolated colonies were subcultured by needle tip transfer 3 days later. Isolates had white mycelia on PDA, with a radial growth rate of 15.2 to 18.7 mm/day. White and orange sclerotia were developed after 5 to 8 days and eventually turned dark reddish-brown. The sclerotia were globoid or irregular with surface markings (1.4 to 4.3 mm diam.; mean = 2.59 mm; n = 50) on PDA, and the average number of sclerotia produced per Petri dish ranged from 35 to 85 (mean = 52; n = 10). Microscopic observations found septal hyphae and clamp connections. These morphological features were identical to the description of Sclerotium delphinii (syn. Sclerotium rolfsii var. delphinii) (Mukherjee et al. 2015; Punja and Damiani 1996; Stevens 1931). A representative isolate YKY2020.01 was stored in the Key laboratory of National Forestry and Grassland Administration on Ecological Landscaping of challenging Urban Sites in Shanghai. For molecular identification, DNA of the isolate YKY2020.01 was extracted using the Fungal DNA Kit (OMEGA bio-tek, China). The internal transcribed spacer region (ITS fragment including ITS1, 5.8S rDNA, and ITS2 region) was amplified with primers ITS1/ITS4 (White et al. 1990), and then sequenced by Sangon Biotech (Shanghai, China). BLAST analysis in NCBI found the ITS sequence of YKY2020.01 (MW916955) was 99.84% similar to S. delphinii strain CBS272.30 (MH855140). Phylogenetic analysis using maximum likelihood (ML) method placed isolate YKY2020.01 in the same clade as S. delphinii. To evaluate pathogenicity, hyphal blocks (0.7 cm diam.) were placed at the base of the stem of healthy E. fortunei (n = 5 plants). Five healthy plants were inoculated by uncolonized agar blocks as controls. All plants were kept in a greenhouse with a temperature range from 21 to 25.6°C (mean = 24.9°C) and relative humidity of 50%. Inoculated plants were symptomatic after 3 days and wilted after 12 days. Symptoms in inoculated plants were similar to those observed under natural conditions, whereas the control group remained asymptomatic. The fungal pathogen was reisolated from symptomatic tissue and confirmed as S. delphinii. To the best of our knowledge, this is the first report of S. delphinii causing southern blight on E. fortunei in China and worldwide. This finding provides concise and practical information on the newly emerged disease of E. fortunei, which is beneficial for future disease management.

Dynamic Characteristics of Soil Microorganism in Rhizosphere Soils of Tobacco Over Different Growth Stages

To explore the composition and changing patterns of microbes in the rhizosphere soils of tobacco over different growth stages, we extracted samples from rhizosphere soils at the resettling stage, vigorous growing stage, budding stage, mature stage and the decaying stage in the tobacco lifecycle. The V4–V5 fragments of the 16S rDNA and the ITS1 fragments of the 18S rRNA have been sequenced by Illumina MiSeq, a high-flux sequencer. A total of 8031 bacterial OTUs and a total of 1983 fungal OTUs have been tested. Both the Chao1 index and Shannon index of bacteria in the rhizosphere soil samples change in a V-shaped pattern over the five growth stages, which peak at the budding stage and increase at the decaying stage after a period of falling. In the growing cycle of tobacco, the dominant bacteria include γ-Metamorphomycetes, α-Metamorphomycetes, Actinomycetes and Acidobacteria, and the dominant fungal phyla include Crustacea, Ascomycetes, Agaricomycetes, IS-s-Retroconis sp XAE_090. The sufficiency and diversity of microbial communities in the rhizosphere soil samples range greatly over the five growth stages. This study lays a theoretical foundation supporting further explorations into the changing rules of microbial communities in the rhizosphere soil of tobacco growth and the relationship between microbial communities and tobacco soil-borne diseases.

Changes in Bacterial and Fungal Community of Soil under Treatment of Pesticides

Experiments were carried out in soil microcosms with the treatment of pesticide formulations—imidacloprid, benomyl, and metribuzin in single and tenfold application rates. For additional stimulation of microorganisms, a starch–mineral mixture was added to some variants. For all samples, high-throughput sequencing on the Illumina MiSeq platform of the V4 (16S rRNA) and ITS1 (18S rRNA) fragments was carried out. As a result, it was possible to establish the characteristic changes in the structure of the soil fungal and bacterial communities under pesticides application. The application of pesticides was accompanied by dramatic shifts in alfa-diversity of the fungal community. The phylum Basidiomycota was likely to be involved in the degradation of pesticides. The changes in the relative abundance of the genera Terrabacter, Kitasatospora, Streptomyces, Sphingomonas, Apiotrichum, Solicoccozyma, Gamsia, and Humicola can be proposed as an indicator of pesticide contamination. It is suggested to use these markers for large-scale assessment of the effect of pesticides on soil microbial communities instead of classical integral methods, including within the framework of state registration of pesticides. It is also recommended to research the effect of pesticides on the soil microbiome during artificially initiated successions using the additional source of carbon.

Trichostrongyloid nematodes in ruminants of northern Iran: prevalence and molecular analysis

BackgroundThis study was carried out to investigate the prevalence and analyze the molecular characteristics based on the internal transcribed spacer (ITS) 2 region of the ribosomal RNA (RNA) gene of trichostrongylid nematodes in different ruminants from Guilan province, northern of Iran.MethodsThe gastrointestinal tracts of 144 ruminants including 72 cattle, 59 sheep, and 13 goats were collected from an abattoir in Guilan province during July to September 2018. After isolation the helminths, male specimens were identified based on morphological parameters. PCR and partial sequencing of the ITS2 fragment were conducted. After phylogenetic analysis, the intraspecific and interspecific differences were calculated.ResultsThe prevalence of total infections with the nematodes was 38.9, 74.6 and 84.6% among cattle, sheep and goats, respectively. Eleven species of trichostrongylid nematodes including Haemonchus contortus, Marshallagia marshalli, Trichostrongylus axei, T. colubriformis, T. vitrinus, Ostertagia trifurcata, Teladorsagia circumcincta, Marshallagia occidentalis, O. lyrata, O. ostertagi, and Cooperia punctate were recovered from the ruminants. The most prevalent trichostrongyloid nematodes in cattle, sheep and goats were O. ostertagi (26.4%), M. marshalli (64.4%) and T. circumcincta (69.2%), respectively. Phylogenetic tree was discriminative for Trichostrongylidae family, while phylogenetic analysis of the ITS2 gene represented low variations and no species identification of Haemonchidae and Cooperiidae families.ConclusionsThis study suggests the high prevalence and species diversity of trichostrongyloid nematodes in different ruminants, indicating the importance of implement antiparasitic strategies in north regions of Iran. As well, this study showed that the ITS2 fragment is not a discriminative marker for Haemonchidae and Cooperiidae families, and investigation of other genetic markers such as mitochondrial genes would be more valuable for better understanding of their phylogenetic relationships.

Assessing candidate DNA barcodes for Chinese and internationally traded timber species.

Accurate identification of species from timber is an essential step to help control illegal logging and forest loss. However, current approaches to timber identification based on morphological and anatomical characteristics have limited species resolution. DNA barcoding is a proven tool for plant species identification, but there is a need to build reliable reference data across broad taxonomic and spatial scales. Here, we construct a species barcoding library consisting of 1550 taxonomically diverse timber species from 656 genera and 124 families, representing a comprehensive genetic reference data set for Chinese timber species and international commercial traded timber species, using four barcodes (rbcL, matK, trnH-psbA, and ITS2). The ITS2 fragment was found to be the most efficient locus for Chinese timber species identification among the four barcodes tested, both at the species and genus level, despite its low recovery rate. Nevertheless, the barcode combination matK+trnH-psbA+ITS2 was required as a complementary barcode to distinguish closely related species in complex data sets involving internationally traded timber species. Comparative analyses of family-level discrimination and species/genus ratios indicated that the inclusion of closely related species is an important factor affecting the resolution ability of barcodes for timber species verification. Our study indicates that although nuclear ITS2 is the most efficient single barcode for timber species authentication in China, complementary combinations like matK+trnH-psbA+ITS2 are required to provide broader discrimination power. These newly-generated sequences enrich the existing publicly available databases, especially for tropical and subtropical evergreen timber trees and this current timber species barcode reference library can serve as an important genetic resource for forestry monitoring, illegal logging prosecution and biodiversity projects.