ITS Sequence Similarity Research Articles

Unveiling the airborne microbial menace: Novel insights into pathogenic bacteria and fungi in bioaerosols from nursery schools to universities

Bacterial and fungal aerosol pollution is widespread in indoor school environments, and poses potential health risks to students and staff. Understanding the distribution and diversity of microbial communities within aerosols is crucial to mitigate their adverse effects. Existing knowledge regarding the composition of bacterial and fungal aerosols, particularly the presence of potential pathogenic microorganisms in fine particulate matter (PM2.5) from nursery schools to universities, is limited. To bridge this knowledge gap, in the present study, we collected PM2.5 samples from five types of schools (i.e., nursery schools, primary schools, junior schools, and high schools and universities) in China. We used advanced single-molecule real-time sequencing to analyze the species-level diversity of bacterial and fungal components in PM2.5 samples based on 16S and ITS ribosomal genes, respectively. We found significant differences in microbial diversity and community composition among the samples obtained from different educational institutions and pollution levels. In particularly, junior schools exhibited higher PM2.5 concentrations (62.2–86.6 μg/m3) than other schools (14.4–48.4 μg/m3). Moreover, microbial variations in PM2.5 samples were associated with institution type. Notably, the prevailing pathogenic microorganisms included Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Streptococcus pneumoniae, and Schizophyllum commune, all of which were identified as Class II Pathogenic Microorganisms in school settings. Four potentially novel strains of S. commune were identified in PM2.5 samples collected from the university; the four strains showed 92.4 %–94.1 % ITS sequence similarity to known Schizophyllum isolates. To the best of our knowledge, this is the first study to explore bacterial and fungal diversity within PM2.5 samples from nursery schools to universities. Overall, these findings contribute to the existing knowledge of school environmental microbiology to ensure the health and safety of students and staff and impacting public health.

Morpho-phylogenetic characterizations reveal a new species of Coccomyces on Cunninghamia lanceolata from China

Specimen of Coccomyces collected on Cunninghamia lanceolata, from Huangshan Mountain in Anhui Province, China, was identified as a new species, named here as Coccomyces cunninghamiae. Although multi-gene (ITS, nrLSU, mtSSU) phylogenetic analysis showed that the new species was closely related to C. anhuiensis, it differs significantly from C. anhuiensis in morphology by intraepidermal or subepidermal ascomata, well-developed paraphyses forming epithecium and shape of ascospores. Moreover, the ITS sequence similarity between them is only 87.86%. Specimens studied are deposited in the Reference Collection of Forest Fungi of Anhui Agricultural University, China (AAUF).

Pathogenic bacteria and fungi in bioaerosols from specialized hospitals in Shandong province, East China

Bacteria and fungi are abundant and ubiquitous in bioaerosols in hospital environments. Understanding the distribution and diversity of microbial communities within bioaerosols is critical for mitigating their detrimental effects. Our knowledge on the composition of bacteria or fungi in bioaerosols is limited, especially the potential pathogens present in fine particulate matter (PM2.5) from specialized hospitals. Thirty p.m.2.5 filter samples were collected from five hospitals (i.e., oral, dermatology, chest, eye, and general hospitals) in Shandong Province, East China. The diversity of bacteria and fungi was analyzed at the species level using single-molecule real-time sequencing of the 16 S and internal transcribed spacer 1 (ITS) ribosomal genes, respectively. Significant differences were detected across sampling sites in terms of microbial diversity and community composition in PM2.5 as well as pollution concentrations. The range of PM2.5 concentrations observed in hospital halls was higher, ranging from 39.0 to 46.2 μg/m3, compared to the wards where the concentrations ranged from 10.7 to 25.2 μg/m3. Furthermore, microbial variations in PM2.5 bioaerosols were associated with hospital type. The most dominant pathogens identified were Vibrio metschnikovii, Staphylococcus epidermidis, Staphylococcus haemolyticus, Fusarium pseudensiforme, and Aspergillus ruber. Among these, A. ruber was identified as an opportunistic fungus in a hospital setting for the first time. Nine potentially novel strains of F. pseudensiforme, showing 84.5%–92.0% ITS sequence similarity to known Fusarium isolates, were identified in PM2.5 samples from all hospitals (excluding an eye hospital). This study highlights the importance of hospital environments in shaping microbial aerosol communities. To the best of our knowledge, this is the first study to provide insights into the bacterial and fungal biodiversity of PM2.5 in specialized hospitals, enriching research in healthcare environmental microbiology and carrying significant public health implications.

First report of Fusarium falciforme causing basal rot of onion (Allium cepa) in India.

Onion (Allium cepa, L) is a very important vegetable crop in India. India is the second largest producer of onion in the world and the crop is grown on more than 1.22 million hectares. Fusarium Basal Rot (FBR) is an economically important diseasef onion that causes considerable losses in onion production up to 50% in field and 30-40% during post-harvest storage of bulbs (Gupta and Gupta 2013; Rajamohan et al. 2019). Onion plants showing chlorosis, twisting, wilting, necrosis, bulb discoloration, rot in the basal parts of bulb and roots typical to FBR were observed, in a field trial of 36 onion cultivars during October 2020 in Bangalore, Karnataka. FBR incidence varied from 30-100% in this field (Fig 1 a-d). Symptomatic bulbs were washed with water, basal plate and fleshy leaves cut into 0.5 to 1 cm-size, surface disinfected with 1.5% sodium hypochlorite (NaOCl) for 3 min, and rinsed with sterile distilled water. Twenty pieces were placed on potato dextrose agar (PDA) in Petri plates and incubated at 25°C for 7 days. Colonies from single-spore isolates on PDA showed abundant white aerial mycelium. Colonies showed light pink or purple coloration on the reverse side of the culture plate with brown center (Fig 1e-f). Macroconidia were 19.13 to 28.35 (mean= 24.2) × 4.29 to 6.06 (mean= 5.05) µm, hyaline, falcate, with slightly curved apexes, and three to five septa. Microconidia were cylindrical to ellipsoid, aseptate, hyaline 8.20 to 12 (mean=10.0) × 3.55 to 4.79 (mean= 4.29) µm (Fig 1g). Chlamydospores were round, intercalary, hyaline, single or in chains (Fig 1h). Two isolates (IBFF-09 & IBFF-10) were analyzed for internal transcribed spacer-ITS (White et al. 1990) and translation elongation factor 1-α (tef1) gene (O'Donnell et al. 1998) by polymerase chain reaction (PCR) and sequenced. ITS and partial tef1 gene sequences of isolates IBFF-09 and IBFF-10 were submitted to the NCBI database (GenBank accession # ON394614&ON026859; # ON409480, ON093166 respectively). Phylogenetic analysis of tef1 gene placed the isolates with F. falciforme (Fig 1i). A pathogenicity test was performed by dipping roots of 28 days old healthy onion seedlings of a susceptible genotype 16/7Y GR3 into a conidial suspension (1 × 104 conidia/ml) of isolate IBFF-10 for 15 min and then transplanting the plants into pots containing sterilized potting mix. Inoculated plants developed typical symptoms of FBR and were all dead by 20 days post inoculation (Fig 1j) while the non-inoculated controls remained healthy. Pathogen was re-isolated from infected plants and showed the same morphology, ITS and tef1 sequence similarity as the original isolate, thus fulfilling Koch's postulates. Basal rot of onion by F. falciforme is reported from Mexico (Tirado-Ramírez et al. 2021). Till date, only F .oxysporum, F. proliferatum and F. solani have been implicated in onion FBR in India (Lee et al. 2021; Rathore and Patil, 2019). F. falciforme however, i prevalent in India and is reported to infect other crops (Gangaraj et al. 2022; Gupta et al. 2019; Homa et al. 2018). There is a high probability that this pathogen is contributing significantly to basal rot disease but it has not been reported yet. To our knowledge, this is the first report of F. falciforme infecting onions in India. In order to develop FBR resistant onion cultivars it is critical to identify and study the response of onion genotypes to different Fusarium spp causing the disease.

First Report of Stemphylium eturmiunum Causing Leaf Spot and Black Spot on Apple in Zhaotong, Yunnan, China.

Apple is the largest fruit tree crop in the world, and China is the largest apple-producing County in the world. Zhaotong, Yunnan Province is a typical cold and mountainous apple-producing area in China. However, apple production is threatened by diseases during the entire growing season, and among them, apple leaf spot and fruit black spot are severe. In previous reports, the main pathogen causing apple leaf spot and fruit black spot was Alternaria sp. (Lior, et al, 2017), while different pathogens were identified. In the current study, seven red Fuji apple fruit with typical black spot samples were collected randomly in Dongda company orchard, Sujiayuan town, Zhaotong, Yunnan on March 25, 2021. The spots on the surface of these apples appear rounded, the diseased parts turn brown or black in colour and the flesh became soften and rotten. The tissues of fruit epidermis at the edge between diseased and healthy parts were cut, soaked in 75% alcohol for 30 s, washed with sterile water three times, and air-dried. Five pieces of tissue were placed on PDA medium amended with rifampicin (50 mg/ml) and incubated in the dark at 25 ℃ for 3-5 days. After colonies grew, mycelial clumps were picked out from the edges of the colonies, transferred to new PDA plates, and incubated at 25 ℃ for 6 days. The diameter of the colonies reached up to 5.7 cm. A representative isolate was retained for further work and was named P6-3-1. The hyphae were white and dense at an early stage, the culture medium on the underside became yellow and the middle parts of the colonies were darker. With maturity, hyphae were clumped, became red with other colors interspersed, and the medium became dark red. Light brown spores were produced, with more vertical septa and fewer transverse septa. Two to three transverse septa were generally observed with obvious constriction at the transverse septa. Average spore size was 22.83 µm ± 2.04 µm × 14.58 µm ± 1.97 µm. DNA was extracted from mycelium, purified and amplified with two pairs of primers, ITS1/ITS4 (White et al. 1990) and gpdF/gpdR (Marcos P. S. Câmara, et al. 2002). The PCR products were sequenced and deposited in GenBank (accession NO.OK560128 and OK627661 ). The similarity of ITS sequences between the isolate and MH843733 (Stemphylium eturmiunum strain ST14) was 100%, and that of gpd sequences between the isolate and MH843728 (Stemphylium eturmiunum strain ST20) was 100%. The maximum parsimony method of Mega7.0 was used and demonstrated that the studied isolate converged to the same branch as Stemphylium eturmiunum. Koch's postulates was applied to identify the pathogenicity of this isolate. A disc of P6-3-1-culture on PDA (5 mm in diameter) was placed on apple leaves and fruit wounds. Sterile PDA was used as a control. All plants were kept in a growth chamber at 25-30 ℃. Four days after inoculation, the disease spot was observed on the inoculated sites and fruit, and with the extension of incubation time, the diseased spots continue to grow, and the leaf spots were not limited by the veins. The pathogen was re-isolated from the inoculated leaves and fruit, satisfying Koch's postulates. This pathogen can also cause postharvest rot of sweet cherry (Alice Spadoni, et al, 2020), postharvest rot on tomato (Prencipe Simona, et al, 2021), etc. This is the first report that Stemphylium eturmiunum can cause apple leaf spot and fruit black spot in Yunnan province, China. The apple black spot caused by Stemphylium eturmiunum was accurately identified. By distinguishing between the two similar diseases mentioned above, resistance to the host and management practices can be accrued based on the characteristics of the pathogen, its epidemiological pattern and the choice of an effective chemical fungicide.

First report of Rhizoctonia solani causing Banded leaf and sheath blight disease in Sugarcane (Saccharum officinarum) from Ghazipur district of Uttar Pradesh, India

During August 2016, banded leaf and sheath blight symptoms were observed on sugarcane (cv. BO-91) in the Village Bhawanipur of Ghazipur district of Uttar Pradesh (India). A survey was done to assess the prevalence of and collect the specimens of banded leaf and sheath blight disease from sugarcane fields in the Ghazipur district during 2016–17. A total of 15 fungal isolates were collected from different parts of the district and pathogenicity testing of these isolates was done following Koch postulates. All the 15 isolates were able to produce disease in the controlled laboratory conditions as well as in the field. Further, these isolates were morphologically characterized as Rhizoctonia solani. To confirm the identity at molecular level, ITS sequencing of two most virulent isolates was done. Based on the ITS sequence similarity, these two isolates, viz. SCRS-2018-1A and SCRS-2018-1B were identified as R. solani. As per the existing records and our knowledge based on the extensive survey of pertinent literature, this is the first report of R. solani causing banded leaf and sheath blight disease in Sugarcane from Ghazipur district of Uttar Pradesh, India.

Insights into the bacterial and fungal communities and microbiome that causes a microbe outbreak on ancient wall paintings in the Maijishan Grottoes

The Maijishan Grottoes, located on the ancient Silk Road in West China, was constructed from the 4th centuries onward for more than 1500 years and became a UNESCO World Cultural Heritage in 2014. A severe microbe outbreak occurred on the wall paintings in the caves of the Maijishan Grottoes in 2018. To prevent and control microbial damage to the wall paintings, we analyzed the bacterial and fungal communities in the normal wall samples and those with microbial plaques using high-throughput sequencing and the environmental factors associated with the microbe outbreak. The results showed that the normal wall samples were dominated by bacterial phyla Actinobacteria (71.7%), Proteobacteria (17.8%), Firmicutes (6.5%), Chloroflexi (2.4%), and Bacteroidetes (0.9%), and fungal families Trichocomaceae (43.4%), unclassified Capnodiales (23.2%), unclassified Ascomycota (12.1%), Teratosphaeriaceae (10.2%), and unclassified Eurotiomycetes (6.2%). The wall samples with microbial plaques were dominated by bacterial Actinobacteria (92.9%), Proteobacteria (5.3%), Firmicutes (1.1%), and Chloroflexi (0.3%) and fungal families unclassified Eurotiomycetes (93.3%), unclassified Ascomycota (3.3%), and Microascaceae (2.9%). These results indicated the great changes in bacterial and fungal communities during the microbe outbreak. The dominant fungus in the wall samples with microbial plaques was isolated and identified as a potential novel species which has 92% ITS sequence similarity with Arachnomyces sp. Canonical correspondence analysis (CCA) revealed that the microbe outbreak was primarily attributed to the excessive air humidity inside and outside of the caves. Thus, air humidity management is important for protecting the ancient wall paintings from microbial colonization and attack.

Diversity of Cultivable Microbes From Soil of the Fildes Peninsula, Antarctica, and Their Potential Application.

To explore the diversity and application potential of Antarctic microorganisms, 1208 strains bacteria and fungi were isolated from 5 samples collected from the Fildes Peninsula during China’s 27th and 31st Antarctic expeditions. By using 16S and ITS sequence similarity alignment, 83 strains bacteria belonging to 20 genera and 30 strains fungi belonging to 7 genera were identified. Among them, 1 strains bacteria and 6 strains fungi showed low sequence similarity to the database, suggesting that they might be novel species. Physiological-biochemical characteristics showed that the identified bacteria could utilize many kinds of carbohydrates and that the identified fungi could produce several kinds of extracellular enzymes. The fungal strain MS-19, identified as Aspergillus sydowii, possesses the potential to produce antifungal activity agents based on an activity-guided approach. Further isolation yielded four polyketones: versicone A (1), versicone B (2), 4-methyl-5,6-dihydro-2H-pyran-2-one (3), and (R)-(+)-sydowic acid (4). It should be noted that 1 displayed strong activity against Candida albicans, with an MIC value of 3.91 μg/mL.

Isolation, diversity, and antimicrobial activity of fungal endophytes from Rohdea chinensis (Baker) N.Tanaka (synonym Tupistra chinensis Baker) of Qinling Mountains, China.

Endophytic fungi have been emerged as fruitful resources for producing structurally fascinating and biologically active secondary metabolites. However, endophytic fungi from medicinal plants of Qinling Mountains–the most important natural climatic boundary between the subtropical and warm temperate zones of China with an astonishingly high level of biodiversity–have rarely been explored as potential sources of novel fungal species and active secondary metabolites. In this study, a total of 371 fungal colonies were successfully isolated from 510 tissue segments of the medicinal Tupistra chinensis Baker collected from Qinling Mountains, China. Roots of T. chinensis Baker are used as a folk medicine to ameliorate pharyngitis and treat rheumatic diseases. A total of 100 representative morphotype strains were identified according to ITS rDNA sequence analyses and were grouped into three phyla (Ascomycota, Basidiomycota, Mucoromycota), seven classes (Dothideomycetes, Sordariomycetes, Eurotiomycetes, Microbotryomycetes, Agaricomycetes, Leotiomycetes, Mortierellomycetes), and at least 35 genera. The genera of Collectotrichum (IF, 29.92%), Fusarium (IF, 8.36%), Aspergillus (IF, 8.09%), and Dactylonectria (IF, 5.39%) were most frequently isolated from the tissues of T. chinensis Baker. The Species Richness Index (S, 65) and the Shannon-Wiener Index (H′, 3.7914) indicated that T. chinensis Baker harbored abundant fungal resources. Moreover, five isolates were potential new taxa because of low similarity of ITS sequences ranged from 95.09%∼96.61%. Fifteen out of 100 endophytic fungal ethyl acetate extracts exhibited inhibitory activities against at least one pathogenic bacterium or fungus. Two important lead compounds produced by two stains (F8047 and F8075) with high antimicrobial activities were identified using high performance liquid chromatography (HPLC) and ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC–QTOF MS) analyses. In addition, it was noteworthy that the strain F8001, which may be a potential new species, showed antimicrobial activity and should be investigated further. Overall, these results indicated that the endophytic fungi from T. chinensis Baker could be exploited as a novel source of bioactive compounds.

Ectomycorrhizal fungal communities of secondary tropical forests dominated by Tristaniopsis in Bangka Island, Indonesia.

In Southeast Asia, primary tropical rainforests are usually dominated by ectomycorrhizal (ECM) trees belonging to Dipterocarpaceae, although arbuscular mycorrhizal trees often outcompete them after disturbances such as forest fires and clear-cutting, thus preventing dipterocarp regeneration. In some secondary tropical forests, however, potentially ECM trees belonging to Tristaniopsis (Myrtaceae) become dominant and may help ECM dipterocarp forests to recover. However, we have no information about their mycorrhizal status in these settings. In this study, we analyzed ECM fungal communities in tropical secondary forests dominated by Tristaniopsis and investigated which ECM fungal species are shared with other tropical or temperate areas. In total, 100 samples were collected from four secondary forests dominated by Tristaniopsis on Bangka Island. ECM tips in the soil samples were subjected to molecular analyses to identify both ECM and host species. Based on a >97% ITS sequence similarity threshold, we identified 56 ECM fungal species dominated by Thelephoraceae, Russulaceae, and Clavulinaceae. Some of the ECM fungal species were shared between dominant Tristaniopsis and coexisting Eucalyptus or Quercus trees, including 5 common to ECM fungi recorded in a primary mixed dipterocarp forest at Lambir Hill, Malaysia. In contrast, no ECM fungal species were shared with other geographical regions, even with Tristaniopsis in New Caledonia. These results imply that secondary tropical forests dominated by Tristaniopsis harbor diverse ECM fungi, including those that inhabit primary dipterocarp forests in the same geographical region. They may function as refugia for ECM fungi, given that dipterocarp forests are disappearing quickly due to human activity.

The signal crayfish (Pacifastacus leniusculus) in Lake Tahoe (USA) hosts multiple Aphanomyces species

The genus Aphanomyces (Oomycetes) comprises approximately 50 known species of water molds in three lineages. One of the most notorious is Aphanomyces astaci, the causative agent of crayfish plague. In this study, fresh isolates of Aphanomyces were collected from 20 live specimens of the signal crayfish Pacifastacus leniusculus (Dana, 1852) from Lake Tahoe, California, providing 35 axenic cultures of A. astaci as well as two apparently undescribed Aphanomyces spp. isolates. Based on the results of ITS-, chitinase-, mitochondrial rnnS- and rnnL-sequences and microsatellite markers combined, the Lake Tahoe A. astaci isolates were identical to isolates of A. astaci B-haplogroup commonly detected in Europe, and infection experiments confirmed their high virulence towards noble crayfish. One of the two undescribed Aphanomyces spp. isolates was highly similar to an Aphanomyces lineage detected previously in crustacean zooplankton (Daphnia) in Central Europe, while the other was distinct and most closely related (ITS sequence similarity of 93%) to either A. astaci or to Aphanomyces fennicus isolated recently from Astacus astacus in Finland. Neither of the two Aphanomyces spp. isolates caused crayfish mortality under experimental conditions. Our results indicate that the populations of North American signal crayfish can act as carriers of both pathogenic and non-pathogenic Aphanomyces at the same time. Furthermore, considering that a limited number of crayfish individuals from a single location yielded multiple distinct Aphanomyces isolates, our results suggest that substantial species diversity within this genus remains undescribed.

Development of seven novel specific SCAR markers for rapid identification of Phytophthora sojae: the cause of root- and stem-rot disease of soybean

Phytophthora sojae is a devastating pathogen that causes soybean Phytophthora root and stem rot. In this study, we developed seven pairs of polymerase chain reaction primers derived from sequence-characterized amplified regions (SCAR). These seven SCAR markers allowed discrimination of P. sojae from 17 different Phytophthora species and three other soilborne pathogens (Pythium ultimum, Fusarium solani and Rhizoctonia sp.) which also induce root rot in soybean. Among those 17 Phytophthora species, P. melonis has approximately 98% similarity in ITS sequences; P. drechsleri requires an annealing temperature up to 66 °C with an ITS-targeting diagnostic marker (PS primers) developed by Wang et al. (2006) for P. sojae; and P. sansomeana is a newly described soybean-infecting Phytophthora species. These three Phytophthora species could be specifically distinguished against P. sojae by these seven SCAR markers. After screening 100 random amplified polymorphic DNA (RAPD) primers, eight primers clearly produced specific bands only for P. sojae rather than other Phytophthora species tested. Subsequently, seven of eight P. sojae-specific RAPD markers were successfully converted into SCAR markers, namely, Scar276, Scar304, Scar333, Scar37, Scar519, Scar57 and Scar78. These SCAR markers were used to detect 75 isolates of P. sojae specifically, while no products were obtained for 29 additional isolates representing 17 other Phytophthora species and three other soilborne pathogens. Furthermore, Scar333 successfully allowed the detection with a sensitivity of 100 pg from genomic DNA of P. sojae, Scar276 had a higher sensitivity of 10 pg, and four specific SCAR primers (Scar304, Scar37, Scar519 and Scar78) had a sensitivity of 100 fg, which is the highest for detecting P. sojae until now. Six of the seven SCAR markers, with the exception of Scar57, were also used to detected P. sojae in artificial or naturally infected soybean seedlings and infested soil. Our findings demonstrate that SCAR markers provide a rapid and sensitive molecular diagnostic tool for the detection of P. sojae in plants, and will play a key role in effective management of the disease.

Calcarisporium cordycipiticola sp. nov., an important fungal pathogen of Cordyceps militaris

Production of the medicinal and edible fungus Cordyceps militaris has dramatically increased in China because of its alleged medicinal properties. During industrial production, a fungal parasite was found to infect the fruiting bodies causing significant quality and yield losses. Two isolates were obtained from fruiting bodies of C. militaris in Beijing and Shanghai mushroom farms and were characterized by morphological and phylogenetic analyses of SSU and ITS sequence data. The phylogenetic analyses showed that they belong to the genus Calcarisporium and are related to the type species, C. arbuscula with 91% ITS sequence similarity. We introduce this taxon as Calcarisporium cordycipiticola sp. nov., and compare it morphologically with other taxa in the genus. The interaction between this taxon, its host, and methods to control this fungicolous taxon need further studies.

Sources of varieties and quality of circular Fructus Ligustri Lucidi

This study aimed to trace sources and quantitatively analyze the specnuezhenide content of circular Fructus Ligustri Lucidi for clinical use. Different specifications of Fructus Ligustri Lucidi were identified using DNA barcoding technology and the specnuezhenide content was analyzed by High Performance Liquid Chromatography (HPLC). The ITS sequence of circular Fructus Ligustri Lucidi was identical to that of standard privet, which was determined through botanical identification. ITS sequence similarity between circular Fructus Ligustri Lucidi and Fructus Ligustri Lucidi which was registered in NCBI ranged from 99.5% to 100%. The sequences of circular and other Fructus Ligustri Lucidi were clustered in a Neighbor-Joining tree with bootstrap value of 95, and these sequences could be distinguished from adulterants. Conforming to pharmacopoeia standard, the average specnuezhenide content of circular Fructus Ligustri Lucidi was higher than that of chicken waist Fructus Ligustri Lucidi. Circular Fructus Ligustri Lucidi derived from Ligustrum lucidum Ait. and the specnuezhenide content was higher in circular Fructus Ligustri Lucidi than that in chicken waist Fructus Ligustri Lucidi.

Distinct phylogeographic structure recognized within Desmazierella acicola

Desmazierella acicola (anamorph Verticicladium trifidum, Chorioactidaceae) represents a frequent colonizer of pine needles in litter. Considering the global diversity and distribution of pine species, we expected different phylogenetic lineages to exist in different geographical and climatic areas inhabited by these hosts. We compared DNA sequence data with phenotypic characteristics (morphology of the anamorph and growth at three different temperatures) of 43 strains isolated mostly from pine and also spruce needle litter sampled in various geographical areas. Analyses of ITS rDNA recovered eight geographically structured lineages. Fragments of genes for the translation elongation factor 1-α, and the second largest subunit of RNA polymerase II reproduced similar lineages, although not all of them were monophyletic. The similarity in ITS sequences among the clade with samples from Continental-Atlantic Europe and four other clades was lower than 95%. Several lineages exhibit also a tendency toward host specificity to a particular pine species. Growth tests at different temperatures indicated a different tolerance to specific climatic conditions in different geographic areas. However, the surveyed phenotypic characteristics also showed high variation within lineages, most evident in the morphology of the anamorph. Until a morphological study of the teleomorph is carried out, all of these lineages should be treated as distinct populations within a single species.

Discovery of Thecaphora schwarzmaniana on Rheum ribes in Iran and Turkey: implications for the diversity and phylogeny of leaf smuts on rhubarbs

A leaf smut discovered on wild rhubarb Rheum ribes in Iran and Turkey is evaluated to identify the species. Based on morphological analyses using light and scanning electron microscopy and molecular phylogenetic analyses using ITS and LSU ribosomal DNA sequences, it is shown that the smut fungus belongs to Thecaphora schwarzmaniana, a rare species previously known on Rheum cordatum, R. lobatum, R. macrocarpum, and R. turkestanicum in some central Asian countries. This is the first documented case of Thecaphora schwarzmaniana on Rheum ribes and significantly expands the range of the species on the Asian continent. The ITS and LSU sequences of the Iranian and Turkish material differed from sequences of Thecaphora schwarzmaniana from Rheum palmatum from China. The re-study of the morphology of the respective specimen revealed morphological differences allowing the conclusion that Thecaphora on Rheum palmatum belongs to a distinct species. The new species is not described here due to the paucity of available material. Molecular phylogenetic analyses, based on ITS and LSU sequences, recovered Thecaphora on Rheum spp. in a clade including species on host plants from diverse unrelated families, indicating that multiple inter-family host jumps took place in the course of evolution of Thecaphora species. Vast similarity of ITS and LSU sequences of Thecaphora spp. on Rheum spp. point to their close relation suggesting that co-evolution could shape the diversification of Thecaphora species on Polygonaceae following an initial host jump from another host plant family.

Fungi ahoy! Diversity on marine wooden substrata in the high North

Marine fungi are severely understudied in the polar regions. We used molecularly identified cultures to study fungi inhabiting 50 intertidal and sea-floor logs along the North Norwegian coast. The aim was to explore the taxonomic and ecological diversity and to examine factors shaping the marine wood-inhabiting fungal communities. The 577 pure cultures analyzed clustered into 147 operational taxonomic units (OTUs) based on 97 % ITS sequence similarity. Ascomycota dominated, but OTUs belonging to Basidiomycota, Mucoromycotina and Chytridiomycota were also isolated. Nine OTUs could not be assigned to any fungal phylum. Almost half of the OTUs were considered non-marine. The western and eastern part of the Norwegian Barents Sea coast hosted different communities. Geography, substratum and site level variables contributed to shaping these communities. We characterized a previously overlooked fungal community in a poorly studied area, discovered high diversity and report many taxa for the first time from the marine environment.

Seasonal variation of dominant free-floating and attached Ulva species in Rudong coastal area, China

In this paper, species compositions and seasonal variations of attached Ulva species on Porphyra aquaculture rafts and free floating Ulva species at Rudong coastal area, Jiangsu Province of China were investigated during 2010–2011. Based on the sequences analysis of nuclear-encoded ITS (including 5.8S rDNA regions) and 5S rDNA spacer regions, dominant species of both attached and free-floating Ulva samples were identified as Ulva compressa, Ulva linza, Ulva prolifera and Ulva flexuosa. Phylogenetic tree based on sequences of ITS and 5S rDNA spacer regions for attached and free-floating Ulva species was constructed, respectively. Species compositions of the Ulva population attached on aquaculture rafts varied with seasons, and U. prolifera was only found on aquaculture rafts in March 2011 during the 2010–2011 Porphyra yezoensis cultivation season, which had the same sequences of ITS and 5S rDNA spacer regions as that of the dominant species bloomed in the Yellow Sea of China in 2008. Dominant species of the free-floating Ulva population at the early stage of the green tide were U. compressa, U. flexuosa, and U. linza. Free-floating U. prolifera appeared in the middle of May, 2011. ITS sequence similarity rates of U. compressa and U. flexuosa between the attached and free-floating species were 100%. And ITS and 5S rDNA spacer sequences of the attached and the free-floating U. prolifera population also showed no differences. Further study showed that there were two types of free-floating U. prolifera population (Type 5S-A and Type 5S-B) based on 5S rDNA spacer sequences. The present study would provide some useful information for clarifying the outbreak mechanism of green tides occurred in the Yellow Sea, China.

IAA-Producing Penicillium sp. NICS01 Triggers Plant Growth and Suppresses Fusarium sp.-Induced Oxidative Stress in Sesame (Sesamum indicum L.)

Application of rhizospheric fungi is an effective and environmentally friendly method of improving plant growth and controlling many plant diseases. The current study was aimed to identify phytohormone-producing fungi from soil, to understand their roles in sesame plant growth, and to control Fusarium disease. Three predominant fungi (PNF1, PNF2, and PNF3) isolated from the rhizospheric soil of peanut plants were screened for their growth-promoting efficiency on sesame seedlings. Among these isolates, PNF2 significantly increased the shoot length and fresh weight of seedlings compared with controls. Analysis of the fungal culture filtrate showed a higher concentration of indole acetic acid in PNF2 than in the other isolates. PNF2 was identified as Penicillium sp. on the basis of phylogenetic analysis of ITS sequence similarity. The in vitro biocontrol activity of Penicillium sp. against Fusarium sp. was exhibited by a 49% inhibition of mycelial growth in a dual culture bioassay and by hyphal injuries as observed by scanning electron microscopy. In addition, greenhouse experiments revealed that Fusarium inhibited growth in sesame plants by damaging lipid membranes and reducing protein content. Co-cultivation with Penicillium sp. mitigated Fusarium-induced oxidative stress in sesame plants by limiting membrane lipid peroxidation, and by increasing the protein concentration, levels of antioxidants such as total polyphenols, and peroxidase and polyphenoloxidase activities. Thus, our findings suggest that Penicillium sp. is a potent plant growthpromoting fungus that has the ability to ameliorate damage caused by Fusarium infection in sesame cultivation.

Microfungi associated with withering willow wood in ground contact near Syowa Station, East Antarctica for 40 years

Data are rather lacking on the diversity of microfungi associated with exotic plant substrates transported to continental Antarctica. We examined the diversity and species composition of microfungi associated with withering woody shoots of saplings of Salix spp. (willows) transplanted and in ground contact near Syowa Station, East Antarctica for more than 40 years. The willow saplings originated from Hokkaido, Northern Japan, and were experimentally transplanted in 1967–1968, but died within a few years. Dead willow shoots, unbranched and standing on bare ground for approximately 50 years, were used for the isolation of fungi with the surface disinfection method. A total of 43 isolates were retrieved from 32 (78 %) of the 41 shoots tested. The fungal isolates were classified into 18 molecular operational taxonomic units (MOTUs) based on the similarity of rDNA ITS sequences at the 97 % criterion. Leotiomycetes was the most common class in terms of the number of isolates and MOTUs, followed by Dothideomycetes, Sordariomycetes, and Eurotiomycetes. Molecular phylogenetic affinities suggested that the closest relatives of the MOTUs were saprobic and root-associated fungi. The result of the present study suggested that Cadophora luteo-olivacea is widespread in soils throughout Antarctica and likely indigenous.