Aspergillus sibiricus (Aspergillaceae, Eurotiales), a novel acid-tolerant species in Aspergillus section Fumigati

A novel species in Aspergillus section Fumigati is described as Aspergillus sibiricus. The new species was isolated from an open coal mine in Altai, Siberia, Russia and is represented by ex-type strain CBS 143307. We used a polyphasic taxonomic approach to characterise the species. A description based on morphological features is provided and shows that the new species differs morphologically from closely related species (A. assulatus, A. nishimurae and A. waksmanii). Partial sequences of the loci encoding β-tubulin (BenA), calmodulin (CaM), ribosomal polymerase II second largest subunit (RPB2) and internal transcribed spacer rDNA region (ITS1-5.8S-ITS2) were analyzed as well. Sequences data, macro- and micromorphological and physiological characteristics distinguish A. sibiricus from all known species in section Fumigati, series Unilaterales. A. sibiricus, isolated from an acidic habitat (pH 3.2–3.4), is able to grow at pH 2 and is considered an acid-resistant species.

A new species of Galerina (Hymenogastraceae, Agaricales) from northeast China

Galerina variibasidia, a new species in the Hymenogastraceae, was discovered in Arxan, Inner Mongolia Autonomous Region, China. This species is presented with morphological description, illustrations, and supported by phylogenetic analyses using Maximum Likelihood and Bayesian Inference analyses with the internal transcribed spacer rDNA sequences.

First Report of Soft Rot on Dendrobium officinale caused by Epicoccum sorghinum in China

In recent years, soft rot is one of the most serious diseases in the production of Dendrobium officinale. In this study, we took the diseased plants of Dendrobium officinale in Guizhou as samples, through Koch's rule and sequence analysis of rDNA internal transcribed spacer (rDNA-ITS), calmodulin (cmdA), the second largest subunit of RNA polymerase Ⅱ (RPB2), elongation factor EF-1 α and β-tubulin (β-Tub), it was determined that the pathogen of Dendrobium officinale soft rot was sorghum accessory cocci. This is our first report on the soft rot of Dendrobium officinale caused by Epicoccum sorghinum in China. The morphological characteristics of the pathogen shown in the study will have a certain reference value for the prevention and control of the soft rot of Dendrobium officinale in the future.

Taxonomy and Phylogeny of Four New Species in Absidia (Cunninghamellaceae, Mucorales) From China

Four new species within the genus Absidia, A. globospora, A. medulla, A. turgida, and A. zonata, are proposed based on a combination of morphological traits, physiological features, and molecular evidences. A. globospora is characterized by globose sporangiospores, a 1.0- to 3.5-μm-long papillary projection on columellae, and sympodial sporangiophores. A. medulla is characterized by cylindrical to oval sporangiospores, a 1.0- to 4.5-μm-long bacilliform projection on columellae, and spine-like rhizoids. A. turgida is characterized by variable sporangiospores, up to 9.5-μm-long clavate projections on columellae, and swollen top of the projection and inflated hyphae. A. zonata is characterized by cylindrical to oval sporangiospores, a 2.0- to 3.5-μm-long spinous projection on columellae, and as many as eight whorled sporangiophores. Phylogenetic analyses based on sequences of internal transcribed spacer rDNA and D1–D2 domains of LSU rDNA support the novelty of these four species within the Absidia. All new species are illustrated, and an identification key to all the known species of Absidia in China is included.

Species Diversity in Penicillium and Acaulium From Herbivore Dung in China, and Description of Acaulium stericum Sp. Nov.

Penicillium and Acaulium species are common in the fresh of herbivore dung and can produce abundant secondary metabolism, which play important roles as decomposers of organic materials, food industry, and enzyme factories. Besides, the well-characterized diversity of dung fungi offers accessible systems for dissecting the function of fungi in gut and for exploring potential to produce high cellulases in herbivorous animal. During a survey of intestinal fungi from herbivorous animal in China, more than 400 were isolated, 38 belonging to Penicillium and 4 belonging to Acaulium were obtained from 12 healthy animals including marmot and chinchilla and selected for detailed study. Putative taxa were characterized by a multi-gene sequencing analysis testing the partial β-tubulin (TUB), the internal transcribed spacer rDNA (ITS), calmodulin (CAM), and RPB2, and a detailed phenotypic study. Penicillium strains were identified as six sections, 12 known species. In addition, four Acaulium isolates were identified as Acaulium album and Acaulium stericum sp. nov. based on morphology and phylogeny of multi-gene sequences. This study shows that the species diversity of Penicillium on herbivore dung has not been widely studied and that seems to be a good source of offers opportunities for discovery of new cellulases from microbial communities.

Isolation and Molecular Characterisation of Ginger (Zingiber officinale. rosc.) Soft Rot Pathogenic Fungi from Champhai District of Mizoram, India

Ginger (Zingiber officinale Rosc.,Zingiberaceae) is an important crop grown in India known for its therapeutic uses. Wilting caused by bacteria, soft rot and yellow disease caused by fungi are the major diseases affecting Ginger production and its cultivation hampering its growth and development. The current study emphasize on the morphological and molecular identification of fungal pathogens causing soft rot disease in ginger that have become one of the major problem among farmers of Mizoram, India as a result of which resulted in a huge decline in rhizome yield and its production. Nevertheless, due to limited studies of the medicinal plants the causative agents associated with these plants are not available. The objective of this study is to investigate the diversity of fungi that cause soft rot in ginger. Twenty fungi were successfully isolated from four villages of Champha iDistrict, Mizoram. The fungi isolated from the infected rhizome were cultured and identification was carried out using morphological characteristics and molecular analysis of DNA sequence generated from Internal Transcribed Spacer rDNA region. Of the samples collected, F. solani,F. oxysporum, P. myriotylum were the most common causative agents. Much research work has yet to be undertaken explore the diversity of pathogrnic fungi causing soft rot in Mizoram which could be very significant in introducing competent and eco-friendly disease management programme.

A Two-Step PCR Protocol Enabling Flexible Primer Choice and High Sequencing Yield for Illumina MiSeq Meta-Barcoding

High-throughput amplicon sequencing that primarily targets the 16S ribosomal DNA (rDNA) (for bacteria and archaea) and the Internal Transcribed Spacer rDNA (for fungi) have facilitated microbial community discovery across diverse environments. A three-step PCR that utilizes flexible primer choices to construct the library for Illumina amplicon sequencing has been applied to several studies in forest and agricultural systems. The three-step PCR protocol, while producing high-quality reads, often yields a large number (up to 46%) of reads that are unable to be assigned to a specific sample according to its barcode. Here, we improve this technique through an optimized two-step PCR protocol. We tested and compared the improved two-step PCR meta-barcoding protocol against the three-step PCR protocol using four different primer pairs (fungal ITS: ITS1F-ITS2 and ITS1F-ITS4, and bacterial 16S: 515F-806R and 341F-806R). We demonstrate that the sequence quantity and recovery rate were significantly improved with the two-step PCR approach (fourfold more read counts per sample; determined reads ≈90% per run) while retaining high read quality (Q30 > 80%). Given that synthetic barcodes are incorporated independently from any specific primers, this two-step PCR protocol can be broadly adapted to different genomic regions and organisms of scientific interest.

Ampelomyces strains isolated from diverse powdery mildew hosts in Japan: Their phylogeny and mycoparasitic activity, including timing and quantifying mycoparasitism of Pseudoidium neolycopersici on tomato

A total of 26 Ampelomyces strains were isolated from mycelia of six different powdery mildew species that naturally infected their host plants in Japan. These were characterized based on morphological characteristics and sequences of ribosomal DNA internal transcribed spacer (rDNA-ITS) regions and actin gene (ACT) fragments. Collected strains represented six different genotypes and were accommodated in three different clades of the genus Ampelomyces. Morphology of the strains agreed with that of other Ampelomyces strains, but none of the examined characters were associated with any groups identified in the genetic analysis. Five powdery mildew species were inoculated with eight selected Ampelomyces strains to study their mycoparasitic activity. In the inoculation experiments, all Ampelomyces strains successfully infected all tested powdery mildew species, and showed no significant differences in their mycoparasitic activity as determined by the number of Ampelomyces pycnidia developed in powdery mildew colonies. The mycoparasitic interaction between the eight selected Ampelomyces strains and the tomato powdery mildew fungus (Pseudoidium neolycopersici strain KTP-03) was studied experimentally in the laboratory using digital microscopic technologies. It was documented that the spores of the mycoparasites germinated on tomato leaves and their hyphae penetrated the hyphae of Ps. neolycopersici. Ampelomyces hyphae continued their growth internally, which initiated the atrophy of the powdery mildew conidiophores 5 days post inoculation (dpi); caused atrophy 6 dpi; and complete collapse of the parasitized conidiphores 7 dpi. Ampelomyces strains produced new intracellular pycnidia in Ps. neolycopersici conidiophores ca. 8–10 dpi, when Ps. neolycopersici hyphae were successfully destroyed by the mycoparasitic strain. Mature pycnidia released spores ca. 10–14 dpi, which became the sources of subsequent infections of the intact powdery mildew hyphae. Mature pycnidia contained each ca. 200 to 1,500 spores depending on the mycohost species and Ampelomyces strain. This is the first detailed analysis of Ampelomyces strains isolated in Japan, and the first timing and quantification of mycoparasitism of Ps. neolycopersici on tomato by phylogenetically diverse Ampelomyces strains using digital microscopic technologies. The developed model system is useful for future biocontrol and ecological studies on Ampelomyces mycoparasites.

First report of Colletotrichum gloeosporioides causing leaf spot on Cyclobalanopsis glauca in China

Cyclobalanopsis glauca (Thunb.) Oerst. is one of the most widely distributed species of evergreen broad-leaved tree in subtropical areas of China. It is also grown in Korea, Japan, and India. Because of its beautiful shape, C. glauca is commonly used for greening gardens and walkways. In July 2018, leaf spots on C. glauca were observed in Zhejiang province (Lishui, N: 28°26’ 6.75”;E: 119°54’11.22), China. About 70% of the trees were found to be diseased, with approximately 50% of leaves showing symptoms. The symptoms on C. glauca leaves initially appeared as small brown-yellow spots which gradually expanded, developing a light brown central and dark brown to black margin. The spots ranged from 4 to 15 mm in diameter. Ten symptomatic fragments measuring approximately 5×5 mm from each leaf were surface disinfested with 70% ethanol for 30 s, and then they were rinsed in sterile distilled water and placed on potato dextrose agar (PDA) medium at 25 °C in the dark for five days. Segments of colony perimeters were then transferred to new plates. The colonies initially produced white mycelia that later turned gray-white with pink and occasionally black dots scattered on the surface of the mycelium. Spores were aseptate, cylindrical, 8 to 15 μm in length, and 3 to 5 μm wide, most with rounded ends, a few with one apex round and the other fusiform, as described for Colletotrichum gloeosporioides (Penz.) Sacc. (Agostini et al. 1993). The internal transcribed spacer rDNA (ITS: MK758005) and two nuclear protein-coding genes (CHS: MK784770, ACT: MK784769) were amplified with ITS1/ITS4, CHS-79F/CHS-345, and ACT-512F/ACT-783R, respectively (Weir et al. 2012). The sequence had 99.61% identity to GQ485605 for ITS, 99.56% to GQ856782 for ACT, and 100% to GQ856733 for CHS of C. gloeosporioides CBS 953.97 in GenBank, respectively. To fulfill Koch’s postulates, spores (1×108) of the isolate were sprayed onto leaves of twelve 2-year-old C. glauca plants (at least six leaves per plant). The fungus was inoculated on one side of each leaf, and distilled water was used as a mock inoculated control on the other side. The plants were cultivated in the greenhouse to maintain high humidity and a temperature near 25 °C. After 9 days, 100% of the leaf halves that had been inoculated had symptoms identical to those observed on affected C. glauca leaves in the field, while no symptoms were observed on the mock inoculated half of each leaf. The fungus was reisolated from the symptoms and identified as C. gloeosporioides using techniques previously described. To our knowledge, this is the first report of C. gloeosporioides infecting C. glauca in China. This study will establish a foundation for the further study of C. gloeosporioides to address the disease effectively. References: Agostini, J. P., et al. 1993. Phytopathology. 82:1177. Weir, B. S., et al. 2012. Stud. Mycol. 73:115. This work was supported by the National Science Foundation for Young Scientists of China (31800035).

Mastomys natalensis (Smith, 1834) as a natural host for Schistosoma haematobium (Bilharz, 1852) Weinland, 1858 x Schistosoma bovis Sonsino, 1876 introgressive hybrids

Cercarial emission of schistosomes is a determinant in the transmission to the definitive host and constitutes a good marker to identify which definitive host is responsible for transmission, mainly in introgressive hybridization situations. Our goal was to test the hypothesis that micro-mammals play a role in Schistosoma haematobium, S. bovis, and/or S. haematobium x S. bovis transmission. Small mammal sampling was conducted in seven semi-lacustrine villages of southern Benin. Among the 62 animals trapped, 50 individuals were investigated for Schistosoma adults and eggs: 37 Rattus rattus, 3 Rattus norvegicus, 9 Mastomys natalensis, and 1 Crocidura olivieri. Schistosoma adults were found in four R. rattus and two M. natalensis, with a local prevalence reaching 80% and 50%, respectively. Two cercarial chronotypes were found from Bulinus globosus experimentally infected with miracidia extracted from naturally infected M. natalensis: a late diurnal and nocturnal chronotype, and an early diurnal, late diurnal, and nocturnal chronotype. The cytochrome C oxidase subunit I mtDNA gene of the collected schistosomes (adults, miracidia, and cercariae) belonged to the S. bovis clade. Eleven internal transcribed spacer rDNA profiles were found; four belonged to S. bovis and seven to S. haematobium x S. bovis. These molecular results together with the observed multi-peak chronotypes add M. natalensis as a new host implicated in S. haematobium x S. bovis transmission. We discuss the origin of the new chronotypes which have become more complex with the appearance of several peaks in a 24-h day. We also discuss how the new populations of offspring may optimize intra-host ecological niche, host spectrum, and transmission time period.