Variation of Soil Bacterial Communities in Forest Soil Contaminated with Chainsaw Lubricants.

Pollutants can exist in the soil for a long time and alter the bacterial community. Using lubricants to prevent the wear of chainsaw blades is necessary for thinning activities and wood harvesting. We investigated the influences of soil contamination with chainsaw lubricants on soil bacterial communities. Bio-oil, mineral oil, and recycled oil were scattered on each treatment to investigate variations in soil bacterial structure during treated periods using the Illumina MiSeq sequencing platform. The results obtained were 5943 ASVs, 5112 ASVs, and 6136 ASVs after treatment at one month, six months, and twelve months, respectively. There was a significant difference in Shannon and Simpson indices between treatments and controls. A total of 46 bacterial genera with an average relative abundance of more than 1.0% were detected in all soil samples. Massilia was the most common genus detected in control at one month, with an average relative abundance of 14.99%, while Chthoniobacter was the most abundant genus detected in bio-oil, mineral oil, and recycled oil treatments at one month, with an average relative abundance of 13.39%, 14.32%, and 10.47%, respectively. Among the three chainsaw lubricants, bio-oil and mineral oil had fewer impacts than recycled oil. The abundances of several functional bacteria groups in the bio-oil treatment were higher than in other treatments and controls. Our results indicated that different chainsaw lubricants and their time of application affected the soil bacterial community composition.

First Report of Anthracnose Caused by Colletotrichum siamense on Machilus thunbergii Siebold & Zucc. in South Korea.

Machilus thunbergii Siebold & Zucc., known as Japanese bay tree, is an evergreen tree distributed widely in East Asia, including South Korea, where the species is of ecological importance. Machilus thunbergii provides habitat for wildlife species and is a common urban tree. In September 2022, anthracnose symptoms on leaves were observed in Jeju (33°26'02.4"N, 126°19'48.8"E) and Tongyeong (34°49'27.1"N, 128°24'01.8"E) in South Korea. Disease incidence on leaves of each affected tree, naturally growing in an urban forest area covering approximately 0.5 ha was approximately ~ 70 % in each study area. Anthracnose symptoms that were observed on 70 to 80% leaves per tree in each study area included orbicular or irregular, whitish-grey spots on leaves that were 1.5 to 3.0 cm in diam. In some cases where leaves were severely affected, larger blotches were formed, leading to bleaching symptoms and eventually defoliation. For pathogen isolation, two or three leaves showing anthracnose symptoms from each of the 15 trees were randomly selected and brought to the laboratory. Fungal isolations were then directly made by transferring spores from acervuli that developed on diseased leaves onto potato dextrose agar (PDA) media. Cushion shaped acervuli filled with salmon to orange-colored conidial masses were produced on media approximately two weeks after the incubation at 25 ± 1°C with a photoperiod of 12 h. Conidia were single celled, hyaline, cylindrical with rounded ends, smooth walls, 13.7 to 18.1 µm long and 3.1 to 4.5 µm wide (n=30). Among 15 cultures that were successfully isolated, 10 isolates were retained based on culture characteristics, and two randomly selected monoconidial cultures were deposited in the culture collection (CDH) of the Chungnam National University, Republic of Korea (Accession No. CDH057-58). Two isolates selected, CDH057 and CDH058, were subjected to identification, and this was achieved based on multiplesequence comparisons using on internal transcribed spacer regions of rDNA (ITS1 and ITS2), partial sequences of actin (ACT) and ß-tubulin (TUB2) gene regions amplified using ITS1F / ITS4, ACT-512F / ACT-783R and T1 / Bt2b, respectively (Weir et al. 2012). The representative sequence data were deposited in GenBank under the accession numbers OR473277 and OR473278 for the ITS, OR480772 and OR480773 for ACT, and OR480774 and OR480775 for TUB2. The resulting sequences were further used for a phylogenetic analysis based on the maximum likelihood method using a concatenated dataset of the ITS, ACT and TUB2 gene sequences for Colletotrichum species in the C. gloeosporioides clade. The results showed that the pathogen isolated in this study clustered with Colletotrichum siamense (Vouchered specimens: MFLU 090230, COUFPI291, and COUFPI294) (Prihastuti et al. 2009). Sequence comparisons revealed that the isolates obtained in this study differed from the type species of C. siamense (MFLU 090230; FJ972613 for ITS, FJ 907423 for ACT, FJ907438 for TUB2) at 2 of 258 bp (∼0.8%) and 6 of 387 bp (∼1.6%) in the ACT and TUB2 sequences, respectively, while the ITS was identical to the type species. For pathogenicity tests, a total of ten three-year-old seedlings of M. thunbergii were used. The leaves of each tree were sprayed with 5 ml of conidial suspension (105 conidia/ml, isolate CDH057). Three control plants were sprayed with sterile water. After being sprayed, treated areas were sealed with a plastic bag for 24 hours to preserve humidity. Anthracnose symptoms, identical to those observed in the field, appeared five to seven days after the inoculations, while no symptoms were observed on control plants. The isolates used in the pathogenicity test were reisolated from 90% of lesions, and their identity was confirmed based on sequence comparisons, thus fulfilling Koch's postulates. Species of the C. gloeosporioides species complex include important plant pathogens, particularly C. siamense, which cause significant losses of economic and ecological relevance on a wide range of hosts (~ 100 hosts) (Talhinhas and Baroncelli 2021). Although C. fioriniae in the C. acutatum species complex, was found on M. thunbergii in South Korea (Thao et al. 2023), anthracnose associated with C. siamense on M. thunbergii has not been reported in the country. In this regard, this is the first report of anthracnose caused by C. siamense on M. thunbergii in South Korea. To effectively control the disease, more attention should be paid on the host range of the pathogen and other regions where the disease caused by the pathogen might occur in the country.

The complete mitochondrial genome of a wood-decaying fungus Vanderbylia fraxinea (Polyporaceae, Polyporales).

Vanderbylia fraxinea (Bull.) D.A. Reid, 1973 is an important wood-inhabiting fungus that plays a significant role in nutrient recycling in most forest ecosystems. In this study, the complete mitochondrial genome of V. fraxinea was characterized through de novo assembly using Illumina sequencing data and genome annotation. The mitochondrial genome is a circular molecule of 115,473 bp with a GC content of 28.66%. It comprises a total of 62 genes. Among these, 36 are protein-coding genes including 21 free-standing open reading frames (ORFs), 24 transfer RNA genes, and two ribosomal RNA genes. Core gene set commonly found in fungal mitochondrial genomes is also present in this genome, such as the apocytochrome b (cob), three subunits of the cytochrome c oxidase (cox1, cox2, and cox3), seven subunits of the NADH dehydrogenase (nad1, nad2, nad3, nad4, nad4L, nad5, and nad6), and three subunits of the ATP synthase (atp6, atp8, and atp9), as well as ribosomal RNA subunits (rns and rnl) and a set of transfer RNA genes. Phylogenetic analysis of the protein-coding sequences from the mitochondrial genome revealed a close relationship between V. fraxinea and the Ganoderma species within the Polyporaceae family.

The Presence or Absence of Alkaline Phosphatase Activity to Discriminate Pluripotency Characteristics in Porcine Epiblast Stem Cell-Like Cells.

Porcine embryonic stem cells (pESCs) would provide potentials for agricultural- and biotechnological-related applications. However, authentic pESCs have not been established yet because standards for porcine stem cell-specific markers and culture conditions are not clear. Therefore, the present study reports attempts to derive pluripotent epiblast stem cells either from in vitro or in vivo derived porcine embryos. Nine epiblast cell lines (seven lines from Berkshire and two lines from Duroc) could only be isolated from day 9- to 9.5-old in vivo derived early conceptuses. Pluripotency features were analyzed in relation to the presence or absence of alkaline phosphatase (AP) activity. Interestingly, the mRNA expression of several marker genes for pluripotency or epiblast was different between putative epiblast stem cells of the two groups [AP-positive (+) pEpiSC-like cell 2 line and AP-negative (-) pEpiSC-like cell 8 line]. For example, expressions of OCT-3/4, NANOG, SOX2, c-MYC, FGF2, and NODAL in AP-negative (-) porcine epiblast stem cell (pEpiSC)-like cells were higher than those in AP-positive (+) pEpiSC-like cells. Expression of surface markers differed between the two groups to some extent. SSEA-1 was strongly expressed only in AP-negative (-) pEpiSC-like cells, whereas AP-positive (+) pEpiSC-like cells did not express. In addition, we report to have some differences in the in vitro differentiation capacity between AP-positive (+) and AP-negative (-) epiblast cell lines. Primary embryonic germ layer markers (cardiac actin, nestin, and GATA 6) and primordial germ cell markers (Dazl and Vasa) were strongly expressed in embryoid bodies (EBs) aggregated from AP-negative (-) pEpiSC-like cells, whereas EBs aggregated from AP-positive (+) pEpiSCs did not show expression of primary embryonic germ layers and primordial germ cell markers except GATA 6. These results indicate that pEpiSC-like cells display different pluripotency characteristics in relation to AP activity.

Endothelial Cells Differentiated from Porcine Epiblast Stem Cells.

Pluripotent stem cells (PSCs) have the ability of self-renewal that can retain the characteristics of the mother cell, and of pluripotency that can differentiate into several body types. PSCs typically include embryonic stem cells (ESCs) derived from the inner cell mass of the preimplantation embryo, and epiblast stem cells (EpiSCs) derived from the epiblast of postimplantation embryo. Although PSCs are able to be used by differentiation into endothelial cells as a potential treatment for vascular diseases, human ESCs and induced PSCs (iPSCs) are followed by ethical and safety issues. Pigs are anatomically and physiologically similar to humans. Therefore, the goal of this study was to establish an efficient protocol that differentiates porcine EpiSCs (pEpiSCs) into the endothelial cells for applying the treatment of human vascular diseases. As a result, alkaline phosphatase (AP)-negative (-) pEpiSCs cultured in endothelial cell growth basal medium-2 (EBM-2) differentiation medium in association with 50 ng/mL of vascular endothelial growth factor (VEGF) for 8 days were changed morphologically like the feature of endothelial cells, and expression of pluripotency-associated markers (OCT-3/4, NANOG, SOX2, and C-MYC) in porcine differentiated cells was significantly decreased (p < 0.05). Additionally, when pEpiSCs were cultured in EBM-2 + 50 ng/mL of VEGF, porcine differentiated cells represented a common endothelial cell marker positive (CD31+) but monocytes and lymphocytes marker negative (CD45-). Therefore, these results indicated that pEpiSCs cultured in EBM-2 + 50 ng/mL of VEGF culture condition were efficiently differentiated into endothelial cells for the treatment of blood vessel diseases.

Characterization of Diaporthe spp. Associated With Spruce Decline on Colorado Blue Spruce in Michigan.

Since 2006 there has been a decline in Colorado blue spruce (CBS; Picea pungens) planted as landscape trees and for Christmas tree production throughout the Lower Peninsula of Michigan. This decline is characterized by a slow loss of needles in the lower portion of the tree starting at branch tips, followed by entire branch dieback, which progresses upward over several years. This dieback has been linked to shallow branch cankers visible in the phloem when the bark layer is removed. Isolates in the fungal genus Diaporthe have been consistently isolated from lesion margins on symptomatic branches. Before the initial reports of declining CBS in landscape and Christmas trees, Diaporthe was known only as a nursery disease of CBS. To determine the species of Diaporthe linked to the decline of CBS in Michigan, seven gene regions were sequenced from a collection of Diaporthe isolates collected in 2011 through 2018 from CBS and other coniferous hosts. Subsequent phylogenetic analyses indicated that Diaporthe eres and a novel Diaporthe clade were present on symptomatic CBS in Michigan. The new species D. brevicancria nov. is described, and Koch's postulates were confirmed for D. brevicancria nov. and D. eres. D. brevicancria nov. produced the largest cankers in greenhouse pathogenicity trials, and dual inoculations of D. brevicancria nov. and D. eres produced intermediate cankers.

Penicillin Trunk Injection Affects Bacterial Community Structure in Citrus Trees.

Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas), an uncultured α-proteobacterium, is the most destructive disease of citrus trees worldwide. In previous studies, trunk injections of penicillin reduced CLas titers and HLB symptoms in citrus. However, antibiotic effects on the whole plant microbial community, which include effects on taxa that interact with CLas, have not yet been addressed. In this study, we investigated the effects of penicillin injection (0, 1000, and 6000 mg L-1) on rhizospheric and endophytic bacterial communities of grapefruit trees in field and greenhouse experiments through culture-independent high-throughput sequencing. DNA extractions from petioles and roots were subjected to 16S rRNA high-throughput sequencing, and reads were clustered by sequence similarity into operational taxonomic units (OTUs). Principal coordinates analysis based on weighted-UniFrac distances did not reveal differences in bacterial communities among treatments in any of the sample sources. However, pairwise linear discriminant analysis indicated significant differences in relative abundance of some taxa (including CLas) among treatments. Network analysis showed that penicillin produced major changes in root bacterial community structure by affecting interspecific microbial associations. This study provides new knowledge of the effect of antimicrobial treatments on interspecific relationships in citrus microbial communities.

A Novel Fusarium Species Causes a Canker Disease of the Critically Endangered Conifer, Torreya taxifolia.

A canker disease of Florida torreya (Torreya taxifolia) has been implicated in the decline of this critically endangered species in its native range of northern Florida and southeastern Georgia. In surveys of eight Florida torreya sites, cankers were present on all dead trees and 71 to 100% of living trees, suggesting that a fungal pathogen might be the causal agent. To identify the causal agent, nuclear ribosomal internal transcribed spacer region (ITS rDNA) sequences were determined for 115 fungi isolated from cankers on 46 symptomatic trees sampled at three sites in northern Florida. BLASTn searches of the GenBank nucleotide database, using the ITS rDNA sequences as the query, indicated that a novel Fusarium species designated Fsp-1 might be the etiological agent. Molecular phylogenetic analyses of partial translation elongation factor 1-alpha (EF-1) and RNA polymerase second largest subunit (RPB2) gene sequences indicate that Fsp-1 represents a novel species representing one of the earliest divergences within the Gibberella clade of Fusarium. Results of pathogenicity experiments established that the four isolates of Fsp-1 tested could induce canker symptoms on cultivated Florida torreya in a growth chamber. Koch's postulates were completed by the recovery and identification of Fsp-1 from cankers of the inoculated plants.