Full-length ITS Research Articles

An updated phylogeny and adaptive evolution within Amaranthaceae s.l. inferred from multiple phylogenomic datasets.

Amaranthaceae s.l. is a widely distributed family consisting of over 170 genera and 2000 species. Previous molecular phylogenetic studies have shown that Amaranthaceae s.s. and traditional Chenopodiaceae form a monophyletic group (Amaranthaceae s.l.), however, the relationships within this evolutionary branch have yet to be fully resolved. In this study, we assembled the complete plastomes and full-length ITS of 21 Amaranthaceae s.l. individuals and compared them with 38 species of Amaranthaceae s.l. Through plastome structure and sequence alignment analysis, we identified a reverse complementary region approximately 5200 bp long in the genera Atriplex and Chenopodium. Adaptive evolution analysis revealed significant positive selection in eight genes, which likely played a driving role in the evolution of Amaranthaceae s.l., as demonstrated by partitioned evolutionary analysis. Furthermore, we found that about two-thirds of the examined species lack the ycf15 gene, potentially associated with natural selection pressures from their adapted habitats. The phylogenetic tree indicated that some genera (Chenopodium, Halogeton, and Subtr. Salsolinae) are paraphyletic lineages. Our results strongly support the clustering of Amaranthaceae s.l. with monophyletic traditional Chenopodiaceae (Clades I and II) and Amaranthaceae s.s. After a comprehensive analysis, we determined that cytonuclear conflict, gene selection by adapted habitats, and incomplete lineage sorting (ILS) events were the primary reasons for the inconsistent phylogeny of Amaranthaceae s.l. During the last glacial period, certain species within Amaranthaceae s.l. underwent adaptations to different environments and began to differentiate rapidly. Since then, these species may have experienced morphological and genetic changes distinct from those of other genera due to intense selection pressure.

Species discrimination power of ITS DNA barcoding on Tristaniopsis species on Bangka Island, Indonesia

The rDNA ITS barcoding marker has been widely used for plant identification, along with other primers for the chloroplast sequence region. This study applied the ITS primer to discriminate Tristaniopsis spp. on Bangka Island. Twelve leaf samples were collected from five community forests in Bangka district. The pairwise distance between sequences calculated using Kimura’s two parameters in MEGA11 revealed that pelawan air is different from other pelawan specimens (1.58–2.1%), while pelawan orange and pelawan merah specimens showed low variation (0–0.78%). There is a barcoding gap in the application of the ITS markers (full-length ITS including 5.8s, ITS1, and ITS2) for Tristaniopsis, which is shown by the higher value of minimum interspecific divergence than the value of maximum interspecific divergence for all three regions. Phylogeny trees constructed from the nucleotide sequence of 12 pelawan and other related taxa clustered pelawan merah and pelawan orange together and separated them from pelawan air.

Different roles of host and habitat in determining the microbial communities of plant-feeding true bugs

BackgroundThe true bugs (Heteroptera) occupy nearly all of the known ecological niches of insects. Among them, as a group containing more than 30,000 species, the phytophagous true bugs are making increasing impacts on agricultural and forestry ecosystems. Previous studies proved that symbiotic bacteria play important roles in these insects in fitting various habitats. However, it is still obscure about the evolutionary and ecological patterns of the microorganisms of phytophagous true bugs as a whole with comprehensive taxon sampling.ResultsHere, in order to explore the symbiotic patterns between plant-feeding true bugs and their symbiotic microorganisms, 209 species belonging to 32 families of 9 superfamilies had been sampled, which covered all the major phytophagous families of true bugs. The symbiotic microbial communities were surveyed by full-length 16S rRNA gene and ITS amplicons respectively for bacteria and fungi using the PacBio platform. We revealed that hosts mainly affect the dominant bacteria of symbiotic microbial communities, while habitats generally influence the subordinate ones. Thereafter, we carried out the ancestral state reconstruction of the dominant bacteria and found that dramatic replacements of dominant bacteria occurred in the early Cretaceous and formed newly stable symbiotic relationships accompanying the radiation of insect families. In contrast, the symbiotic fungi were revealed to be horizontally transmitted, which makes fungal communities distinctive in different habitats but not significantly related to hosts.ConclusionsHost and habitat determine microbial communities of plant-feeding true bugs in different roles. The symbiotic bacterial communities are both shaped by host and habitat but in different ways. Nevertheless, the symbiotic fungal communities are mainly influenced by habitat but not host. These findings shed light on a general framework for future microbiome research of phytophagous insects.DpRsUfddvNEYPX-MDhm9d6Video

Best practices in metabarcoding of fungi: From experimental design to results.

The development of high-throughput sequencing (HTS) technologies has greatly improved our capacity to identify fungi and unveil their ecological roles across a variety of ecosystems. Here we provide an overview of current best practices in metabarcoding analysis of fungal communities, from experimental design through molecular and computational analyses. By reanalysing published data sets, we demonstrate that operational taxonomic units (OTUs) outperform amplified sequence variants (ASVs) in recovering fungal diversity, a finding that is particularly evident for long markers. Additionally, analysis of the full-length ITS region allows more accurate taxonomic placement of fungi and other eukaryotes compared to the ITS2subregion. Finally, we show that specific methods for compositional data analyses provide more reliable estimates of shifts in community structure. We conclude that metabarcoding analyses of fungi are especially promising for integrating fungi into the full microbiome and broader ecosystem functioning context, recovery of novel fungal lineages and ancient organisms as well as barcoding of old specimens including type material.

The curse of the uncultured fungus.

The international DNA sequence databases abound in fungal sequences not annotated beyond the kingdom level, typically bearing names such as “uncultured fungus”. These sequences beget low-resolution mycological results and invite further deposition of similarly poorly annotated entries. What do these sequences represent? This study uses a 767,918-sequence corpus of public full-length fungal ITS sequences to estimate what proportion of the 95,055 “uncultured fungus” sequences that represent truly unidentifiable fungal taxa – and what proportion of them that would have been straightforward to annotate to some more meaningful taxonomic level at the time of sequence deposition. Our results suggest that more than 70% of these sequences would have been trivial to identify to at least the order/family level at the time of sequence deposition, hinting that factors other than poor availability of relevant reference sequences explain the low-resolution names. We speculate that researchers’ perceived lack of time and lack of insight into the ramifications of this problem are the main explanations for the low-resolution names. We were surprised to find that more than a fifth of these sequences seem to have been deposited by mycologists rather than researchers unfamiliar with the consequences of poorly annotated fungal sequences in molecular repositories. The proportion of these needlessly poorly annotated sequences does not decline over time, suggesting that this problem must not be left unchecked.

The Global Soil Mycobiome consortium dataset for boosting fungal diversity research

Fungi are highly important biotic components of terrestrial ecosystems, but we still have a very limited understanding about their diversity and distribution. This data article releases a global soil fungal dataset of the Global Soil Mycobiome consortium (GSMc) to boost further research in fungal diversity, biogeography and macroecology. The dataset comprises 722,682 fungal operational taxonomic units (OTUs) derived from PacBio sequencing of full-length ITS and 18S-V9 variable regions from 3200 plots in 108 countries on all continents. The plots are supplied with geographical and edaphic metadata. The OTUs are taxonomically and functionally assigned to guilds and other functional groups. The entire dataset has been corrected by excluding chimeras, index-switch artefacts and potential contamination. The dataset is more inclusive in terms of geographical breadth and phylogenetic diversity of fungi than previously published data. The GSMc dataset is available over the PlutoF repository.

Characterization of fungal communities on shared bicycles in Southwest China

BackgroundThe widespread use of shared bicycles has increased the demand and sanitary requirements for shared bicycles. Previous studies have identified potentially pathogenic bacteria on the surfaces of shared bicycles, but fungal communities have not been investigated.MethodsWe sampled shared-bicycle handles and saddles from five selected locations in a metropolis (Chengdu, China, n = 98) and used surrounding air deposition samples as controls (n = 12). Full-length ITS sequencing and multiple bioinformatic analyses were utilized to reveal fungal community structures and differences.ResultsAspergillus was dominant on both the handles and saddles of shared bicycles, and Alternaria and Cladosporium were the most abundant families in the air samples. Significant differences in fungal community structures were found among the three groups. The handle samples contained higher abundances of Aureobasidium melanogenum and Filobasidium magnum than the saddle and air samples. The saddle samples had a higher abundance of Cladosporium tenuissimum than the other two sample types (P < 0·05). A higher abundance of fungal animal pathogens on shared-bicycle surfaces than in air by FUNGuild (P < 0·05). Moreover, the co-occurrence network of fungi on handles was more stable than that on saddles.ConclusionThere were more potential pathogens, including Aspergillus pseudoglaucus, Aureobasidium melanogenum, Kazachstania pintolopesii, Filobasidium magnum, Candida tropicalis, and Malassezia globose were found on shared bicycles than in air, suggesting that hands should not contact mucous membrane after cycling, especially in susceptible individuals, and hygiene management of shared bicycles should be given more attention by relevant organizations worldwide.

Taxonomic profiling of Symbiodiniaceae and bacterial communities associated with Indo-Pacific corals in the Gulf of Thailand using PacBio sequencing of full-length ITS and 16S rRNA genes

Corals live with complex assemblages of microbes including bacteria, the dinoflagellate Symbiodiniaceae, fungi and viruses in a coral holobiont. These coral-associated microorganisms play an important role in their host fitness and survival. Here, we investigated the structure and diversity of algal and bacterial communities associated with five Indo-Pacific coral species, using full-length 16S rRNA and internal transcribed spacer sequences. While the dinoflagellate communities associated with Poriteslutea were dominated with Symbiodiniaceae genus Cladocopium, the other four coral hosts were associated mainly with members of the Durusdinium genus, suggesting that host species was one of the underlying factors influencing the structure and composition of dinoflagellate communities associated with corals in the Gulf of Thailand. Alphaproteobacteria dominated the microbiomes of Pocillopora spp. while Pavonafrondifera and P. lutea were associated primarily with Gammaproteobacteria. Finally, we demonstrated a superior performance of full-length 16S rRNA sequences in achieving species-resolution taxonomic classification of coral-associated microbiota.

Molecular detection and genetic characteristics of Babesia gibsoni in dogs in Shaanxi Province, China

BackgroundSeveral members of genus Babesia are important pathogens causing babesiosis in dogs. In China, at least five Babesia species have been described in dogs or ticks. This study sought to determine the prevalence and molecular characteristics of various Babesia spp. in dogs in cities in Shaanxi Province in China, including Xi’an and Hanzhong.MethodsA total of 371 blood samples were collected from pet dogs presenting to veterinary clinics in the cities of Xi’an and Hanzhong in Shaanxi, China. Babesia spp. DNA was detected via amplification of partial 18S rRNA genes by semi-nested PCR. Almost full-length 18S rRNA, ITS, partial TRAP and complete cytb genes were recovered for analysis of the genetic characteristics and relationships with known isolates.ResultsA single species, Babesia gibsoni, was identified in dogs in Xi’an and Hanzhong. Consistently, B. gibsoni was also detected in 14 ticks collected from positive dogs. Sequence similarities and phylogenetic analysis suggested that the isolates identified herein showed a closer genetic relationship with isolates from East Asian countries rather than India, Bangladesh, or the USA. Sequence analysis based on tandem repeat analysis of the TRAP gene further revealed that specific haplotypes were circulating in both Xi’an and Hanzhong, with no specific regionality. In addition, 10.9% of all isolates with atovaquone (ATV)-resistance were identified because of M121I mutation in the deduced cytb protein.ConclusionsThis study revealed a high prevalence rate of Babesia infection. Babesia gibsoni was the only Babesia species identified in cases of canine babesiosis in the cities of Xi’an and Hanzhong cities in Shaanxi, China. In addition, the TRAP gene presented high genetic diversity across isolates. Such information is useful for elucidating the epidemiological characteristics of canine babesiosis, as well as the overall genetic diversity of Babesia spp. circulating in dog populations in Shaanxi Province.

First Report of Leaf Spot of Maize (Zea mays) Caused by Bipolaris setariae in China

In September 2016, leaf spots were observed on maize (Zea mays L.) cultivar Tieyan 58 at the Zhangzhuang farm near TieLing in Liaoning province in China. Symptoms of early infection on leaves were small, water-soaked, translucent circular to oval spots that later enlarged, with the centers turning gray color. Spots were elliptic to round shaped with a chlorotic halo. Fifteen percent incidence was observed in this farm. The incidence of this disease ranged from 5 to 20% in 15 counties of Liaoning province in 2017. Yield losses of maize attributed to leaf spot varied from 4 to 11%, with estimated losses as high as 30% when severe epidemics occurred. The causal agent was recovered from symptomatic leaf samples using standard phytopathological procedure. Leaf pieces were placed on potato dextrose agar (PDA) plates that were incubated at 24 to 26°C in the dark for 5 to 6 days. Single spores of six colonies were transferred to fresh PDA and incubated in the dark for 7 to 8 days for morphological identification. Colony margins were irregular to circular and were gray to black in color on PDA. Conidiophores were single or in clusters, geniculate, slightly flexuous or straight, and two- or three-septate. Conidia were slightly curved, truncated, spindle-shaped to fusiform, dark olive, four- to 10-septate, and 8 to 16 × 37 to 106 µm in size, with a slightly protuberant hilum. Conidia with a distinct hilum are the characteristic of Bipolaris maydis. Based on these cultural and morphological characteristics, the fungus was identified as Bipolaris setariae (Saw.) Shoem. (da Cunha et al. 2012). To confirm the morphological identification, mycelia were scraped from PDA plates, and genomic DNA was isolated by kit (Aidlab Biotech, Beijing, China). The internal transcribed spacer regions (ITS1-5.8S-ITS2) and partial translation elongation factor 1-alpha (TEF1-alpha) gene of six isolates were amplified using the primer pairs ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999). The PCR products were cloned into pMD-19T Cloning Vector (Sangon Biotech, Shanghai, China). The clones were purified with the TIANprep Mini Plasmid Kit (Tiangen Biotech, Beijing, China) to get the full-length ITS sequence and partial TEF1-alpha sequence. BLAST analysis indicated 100% nucleotide sequence identity with B. setariae for six isolates (GenBank accession nos. CBS141.31 and EF452444). Sequences were submitted to GenBank (accession nos. MK002698 and MK439841). Pathogenicity of isolates was tested by spraying conidial suspension of six isolates (1 × 10⁶ conidia/ml). Leaves of two maize plants, hybrid Tieyan 58 (seven/eight-leaf stage) were inoculated for one isolate. Twelve plants sprayed with sterile water served as the noninoculated controls. Plants were maintained at 25 to 27°C in the greenhouse for 10 to 12 days. Small, translucent circular to oval lesions appeared in all inoculated plants. Lesions were similar to leaf spots found in the field, whereas noninoculated control plants were asymptomatic. According to Zhang’s method, disease intensity index ranged 31.5 to 33.3 for six isolates, with no significant difference (Zhang et al. 2019). Koch’s postulates were fulfilled by reisolation of B. setariae from inoculated plants. This fungus was previously described as a pathogen of coconut in China (Niu et al. 2014), but this is the first record of B. setariae on maize in China.

Towards PacBio-based pan-eukaryote metabarcoding using full-length ITS sequences.

Development of high-throughput sequencing techniques has greatly benefited our understanding about microbial ecology, yet the methods producing short reads suffer from species-level resolution and uncertainty of identification. Here, we optimize Pacific Biosciences-based metabarcoding protocols covering the internal transcribed spacer (ITS region) and partial small subunit of the rRNA gene for species-level identification of all eukaryotes, with a specific focus on Fungi (including Glomeromycota) and Stramenopila (particularly Oomycota). Based on tests on composite soil samples and mock communities, we propose best suitable degenerate primers, ITS9munngs + ITS4ngsUni for eukaryotes and selected groups therein and discuss the pros and cons of long read-based identification of eukaryotes.

PacBio amplicon sequencing for metabarcoding of mixed DNA samples from lichen herbarium specimens

The detection and identification of species of fungi in the environment using molecular methods heavily depends on reliable reference sequence databases. However, these databases are largely incomplete in terms of taxon coverage, and a significant effort is required from herbaria and living fungal collections for the mass-barcoding of well-identified and well-curated fungal specimens or strains. Here, a PacBio amplicon sequencing approach is applied to recent lichen herbarium specimens for the sequencing of the fungal ITS barcode, allowing a higher throughput sample processing than Sanger sequencing, which often required the use of cloning. Out of 96 multiplexed samples, a full-length ITS sequence of the target lichenised fungal species was recovered for 85 specimens. In addition, sequences obtained for co-amplified fungi gave an interesting insight into the diversity of endolichenic fungi. Challenges encountered at both the laboratory and bioinformatic stages are discussed, and cost and quality are compared with Sanger sequencing. With increasing data output and reducing sequencing cost, PacBio amplicon sequencing is seen as a promising approach for the generation of reference sequences for lichenised fungi as well as the characterisation of lichen-associated fungal communities.

Genetic relatedness of a new Japanese isolates of Alexandrium ostenfeldii bloom population with global isolates

In recent years, blooms of toxic Alexandrium ostenfeldii strains have been reported from around the world. In 2013, the species formed a red tide in a shallow lagoon in western Japan, which was the first report of the species in the area. To investigate the genetic relatedness of Japanese A. ostenfeldii and global isolates, the full-length SSU, ITS and LSU sequences were determined, and phylogenetic analyses were conducted for isolates from western and northern Japan and from the Baltic Sea. Genotyping and microsatellite sequence comparison were performed to estimate the divergence and connectivity between the populations from western Japan and the Baltic Sea. In all phylogenetic analyses, the isolates from western Japan grouped together with global isolates from shallow and low saline areas, such as the Baltic Sea, estuaries on the east coast of U.S.A. and from the Bohai Sea, China. In contrast, the isolates from northern Japan formed a well-supported separate group in the ITS and LSU phylogenies, indicating differentiation between the Japanese populations. This was further supported by the notable differentiation between the sequences of western and northern Japanese isolates, whereas the lowest differentiation was found between the western Japanese and Chinese isolates. Microsatellite genotyping revealed low genetic diversity in the western Japanese population, possibly explained by a recent introduction to the lagoon from where it was detected. The red tide recorded in the shallow lagoon followed notable changes in the salinity of the waterbody and phytoplankton composition, potentially facilitating the bloom of A. ostenfeldii.

Bursaphelenchus pterocarpi n. sp. (Tylenchina: Aphelenchoididae) found in Pterocarpus sp. imported into China from Ghana

Summary Bursaphelenchus pterocarpi n. sp. isolated from Pterocarpus sp. logs imported from Ghana to Ningbo, China, is described and illustrated. The new species is characterised by a female body length of 630-946 μm, lateral field with four lines, a short 12.6-13.9 μm long stylet with small basal swellings, excretory pore located posterior to nerve ring, outstretched reproductive system with amoeboid sperm inside the spermatheca, a well-developed vulval flap, tail conical and straight with a 1.9-4.8 μm long mucron at the tip, and lacking males in both the wild type and fungal-cultured populations. In a concatenated tree, reconstructed on the basis of the partial 18S, full length ITS and partial 28S rDNA D2-D3 sequences, the new species occupied a basal placement to the clade of xylophilus-group and africanus-group species.

New occurrences of corticioid and poroid fungi (Basidiomycota) in Kedrovaya Pad Nature Reserve, Primorye Territory, Russian Far East

New data on corticioid and poroid basidiomycetes from Kedrovaya Pad Nature Reserve, Primorye Territory, Russian Far East, is presented. Altogether there are 182 known species from the reserve; 76 are reported as new to the reserve and two as new to Russia. The material was collected in July of 2016 from 9 study plots located in forests of different types. Findings of some rarely collected species such as Botryobasidium botryoideum, Cerinomyces aff. aculeatus, Junghuhnia aurantilaeta, Phanerochaete robusta and Tyromyces wynneae are briefly discussed. The full-length ITS sequences of Junghuhnia aurantilaeta, Phanerochaete robusta, Pyrrhoderma cf. sendaiense and Tyromyces wynneae were obtained and deposited in the GenBank. Junghuhnia aurantilaeta is proposed to be included in the new edition of the Red Data Book of Primorye Territory. The paper expands the knowledge of distribution of many species and increases the total number of species reported for the reserve.

Using the ITS2 sequence-structure as a DNA mini-barcode: A case study in authenticating the traditional medicine“Fang Feng”

An ideal DNA barcode should be short enough to be easily amplified. However, barcodes that are too short may lack discriminatory efficacy, and how additional phylogenetic information can be obtained and used without the addition of nucleotides is a major concern. In this study, we selected 135 individuals that represented 97 species from 21 genera to explore the possibility of employing the short ITS2 fragment as a barcode to identify the traditional medicine “Fang Feng” using sequence and structure information. The results revealed that all of the ITS2 sequences had a common four-helix folding structure and some shared motifs. This conserved structure facilitated the alignment of ambiguous sequences from highly divergent taxa. The sequence-structure data yielded a maximum parsimony (MP) tree in which the species referred to as “Fang Feng” was successfully identified. Additionally, we compared the species-level discriminatory powers of the sequence-structure ITS2 and the full-length ITS (ITS1, 5.8S, ITS2), and found that their efficacies were very similar. These findings support the possibility of incorporating the ITS2 secondary structure into DNA barcode analysis. Therefore, we propose that ITS2 sequence-structure information could be used as an alternative mini-barcode when a full-length ITS is not present.

First Report of Leaf Spot of maize (Zea mays) Caused by Bipolaris spicifera in China

In September 2016, leaf spots were observed on maize (Zea mays L.) cultivar Tieyan 58 at the Zhangzhuang farm near TieLing in Liaoning province in China. Symptoms of early infection on leaves were small, water-soaked, translucent circular to oval spots that later enlarged, with the centers turning gray color. Spots were elliptic to round shaped with a chlorotic halo. Fifteen percent incidence was observed in this farm. The incidence of this disease ranged from 5 to 20% in 15 counties of Liaoning province in 2017. Yield losses of maize attributed to leaf spot varied from 4 to 11%, with estimated losses as high as 30% when severe epidemics occurred. The causal agent was recovered from symptomatic leaf samples using standard phytopathological procedure. Leaf pieces were placed on potato dextrose agar (PDA) plates that were incubated at 24 to 26°C in the dark for 5 to 6 days. Single spores of six colonies were transferred to fresh PDA and incubated in the dark for 7 to 8 days for morphological identification. Colony margins were irregular to circular and were gray to black in color on PDA. Conidiophores were single or in clusters, geniculate, slightly flexuous or straight, and two- or three-septate. Conidia were slightly curved, truncated, spindle-shaped to fusiform, dark olive, four- to 10-septate, and 8 to 16 × 37 to 106 µm in size, with a slightly protuberant hilum. Conidia with a distinct hilum are the characteristic of Bipolaris maydis. Based on these cultural and morphological characteristics, the fungus was identified as Bipolaris setariae (Saw.) Shoem. (da Cunha et al. 2012). To confirm the morphological identification, mycelia were scraped from PDA plates, and genomic DNA was isolated by kit (Aidlab Biotech, Beijing, China). The internal transcribed spacer regions (ITS1-5.8S-ITS2) and partial translation elongation factor 1-alpha (TEF1-alpha) gene of six isolates were amplified using the primer pairs ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999). The PCR products were cloned into pMD-19T Cloning Vector (Sangon Biotech, Shanghai, China). The clones were purified with the TIANprep Mini Plasmid Kit (Tiangen Biotech, Beijing, China) to get the full-length ITS sequence and partial TEF1-alpha sequence. BLAST analysis indicated 100% nucleotide sequence identity with B. setariae for six isolates (GenBank accession nos. CBS141.31 and EF452444). Sequences were submitted to GenBank (accession nos. MK002698 and MK439841). Pathogenicity of isolates was tested by spraying conidial suspension of six isolates (1 × 10⁶ conidia/ml). Leaves of two maize plants, hybrid Tieyan 58 (seven/eight-leaf stage) were inoculated for one isolate. Twelve plants sprayed with sterile water served as the noninoculated controls. Plants were maintained at 25 to 27°C in the greenhouse for 10 to 12 days. Small, translucent circular to oval lesions appeared in all inoculated plants. Lesions were similar to leaf spots found in the field, whereas noninoculated control plants were asymptomatic. According to Zhang’s method, disease intensity index ranged 31.5 to 33.3 for six isolates, with no significant difference (Zhang et al. 2019). Koch’s postulates were fulfilled by reisolation of B. setariae from inoculated plants. This fungus was previously described as a pathogen of coconut in China (Niu et al. 2014), but this is the first record of B. setariae on maize in China.

A cost-effective and efficient strategy for Illumina sequencing of fungal communities: A case study of beech endophytes identified elevation as main explanatory factor for diversity and community composition

Abstract We describe an accurate and efficient workflow for highly multiplexed paired-end Illumina sequencing of fungal full-length ITS amplicons. The impact of habitat and substratum conditions on leaf-inhabiting fungal communities was analysed. Fully vital and clearly senescent leaves of European beech (Fagus sylvatica) were sampled along an elevation gradient of about 1000 m in the Bavarian Alps, Germany, in autumn 2013. Surface-sterilised leaves were used for genomic DNA extraction, tagging-by-amplification and high-throughput sequencing. Significant correlation of community composition with elevation was observed. The mycobiome was little affected by the physiological state of the leaves, because only a partial shift of taxonomic composition was observed from vital towards clearly senescent leaves.

First Report of Phomopsis longicolla Causing Leaf Spot on Soybean in China.

During July 2012, leaf spots affecting 60% of the leaves were observed on soybean cultivar He Feng 60 in fields near Shenyang City, Liaoning Province, leading to 5 to 10% yield loss. The leaf spots were associated with the leaf margins and were irregularly shaped, with brown to black margins and surrounded by a thin, yellow halo. Often, several spots merged to form large necrotic areas, which contained numerous pycnidia on the underside of the leaf. Small pieces (5 mm2) were excised from the margin of diseased and healthy tissue, surface-sterilized in 70% ethanol solution for 30 s and 0.1% mercuric chloride solution for 1 min, washed in three changes of sterile distilled water, and transferred to plates containing potato dextrose agar (PDA). Cultures were maintained in an incubator at 25°C with a 12 h dark/light photoperiod for 5 to 7 days. On PDA, colonies were white with yellow areas, floccose, dense, and moderately fast growing, attaining a diameter of 3.9 mm after 5 days and 9.0 mm after 14 days. Finally, large black stromata appeared after 28 days at 25°C. The conidiomata pycnidia were black, stomatic, globose, length 83.6 to 232 μm, width 37.9 to 146.3 μm and produced α-conidia that were unicellular, hyaline, sometimes two-guttulate, length 4.75 to 8.25 μm, width 1.50 to 3.00 μm. β-Conidia were not observed. To confirm the morphological identification, the ribosomal internal transcribed spacers (ITS1-5.8S-ITS2) from isolates were sequenced (GenBank Accession No. KC460334). The PCR products were cloned into a pMD-19T Cloning Vector (Sangon Biotech, Shanghai, China). The clones were purified with TIANprep Mini Plasmid Kit (Tiangen Biotech, Beijing, China) to get the full-length ITS sequence. BLAST analysis of the isolates showed 100% nucleotide sequence identity with Phomopsis longicolla (AY745021). Four additional primer pairs-large subunit (NL1/NL4), beta-tublin gene (Bt2a/Bt2b), translation elongation factor 1α gene(EF1-728F/EF1-986R), and act gene(ACT-512F/ACT-783R) (1,2)-were amplified and sequenced as described above. The large subunit gene, β-tubulin gene, and translation elongation factor 1α gene from isolates were sequenced (Sangon Biotech). BLAST analysis indicated that the isolates had 100% nucleotide sequence identity with P. longicolla (AB107259, HQ333514, and AF398896). Because the act gene sequence of P. longicolla was not in the NCBI database, this sequence had 94% nucleotide sequence identity with P. cuppatea (JN230389). To fulfill Koch's postulates, five leaves on five healthy soybean plants were inoculated with a conidial suspension (106/ml). Plants inoculated with sterile water served as the noninoculated controls. Plants were incubated in the greenhouse at 25°C. All the inoculated leaves developed pinhead spots on the leaves, gradually increasing to large brown spots. Spots were irregularly shaped, brown and necrotic in the center, and surrounded by a yellow halo. Black pycnidia appeared after 10 days, whereas the noninoculated control plants remained asymptomatic. P. longicolla was consistently recovered from all inoculated plants, except the control. Morphological description of isolates was similar to that of Hobbs (3). However, as described by Hobbs and others, P. longicolla conidiomata pycnidia have prominent necks more than 200 μm long, opening by apical ostioles; locules are uniostiolate or multiostiolate, globose, up to 500 μm wide. The pycnidia size of isolates by frozen section method was smaller than that of Hobbs. Based on morphological and sequence comparisons, the pathogen of leaf spot disease is caused by P. longicolla. This is the first reported leaf spot caused by P. longicolla on soybean in China.

Phylogeny of Theileria buffeli genotypes identified in the South African buffalo (Syncerus caffer) population

Theileria buffeli/orientalis is a group of benign and mildly pathogenic species of cattle and buffalo in various parts of the world. In a previous study, we identified T. buffeli in blood samples originating from the African buffalo (Syncerus caffer) in the Hluhluwe–iMfolozi Game Park (HIP) and the Addo Elephant Game Park (AEGP) in South Africa. The aim of this study was to characterise the 18S rRNA gene and complete internal transcribed spacer (ITS1-5.8S-ITS2) region of T. buffeli samples, and to establish the phylogenetic position of this species based on these loci. The 18S rRNA gene and the complete ITS region were amplified from DNA extracted from blood samples originating from buffalo in HIP and AEGP. The PCR products were cloned and the resulting recombinants sequenced. We identified novel T. buffeli-like 18S rRNA and ITS genotypes from buffalo in the AEGP, and novel Theileria sinensis-like 18S rRNA genotypes from buffalo in the HIP. Phylogenetic analyses indicated that the T. buffeli-like sequences were similar to T. buffeli sequences from cattle and buffalo in China and India, and the T. sinensis-like sequences were similar to T. sinensis 18S rRNA sequences of cattle and yak in China. There was extensive sequence variation between the novel T. buffeli genotypes of the African buffalo and previously described T. buffeli and T. sinensis genotypes. The presence of organisms with T. buffeli-like and T. sinensis-like genotypes in the African buffalo could be of significant importance, particularly to the cattle industry in South Africa as these animals might act as sources of infections to naïve cattle. This is the first report on the characterisation of the full-length 18S rRNA gene and ITS region of T. buffeli and T. sinensis genotypes in South Africa. Our study provides invaluable information towards the classification of this complex group of benign and mildly pathogenic species.