Largest Subunit Of RNA Polymerase Research Articles

Small molecule targeting of transcription-replication conflict for selective chemotherapy

Targeting transcription replication conflicts, a major source of endogenous DNA double-stranded breaks and genomic instability could have important anticancer therapeutic implications. Proliferating cell nuclear antigen (PCNA) is critical to DNA replication and repair processes. Through a rational drug design approach, we identified a small molecule PCNA inhibitor, AOH1996, which selectively kills cancer cells. AOH1996 enhances the interaction between PCNA and the largest subunit of RNA polymerase II, RPB1, and dissociates PCNA from actively transcribed chromatin regions, while inducing DNA double-stranded breaks in a transcription-dependent manner. Attenuation of RPB1 interaction with PCNA, by a point mutation in RPB1's PCNA-binding region, confers resistance to AOH1996. Orally administrable and metabolically stable, AOH1996 suppresses tumor growth as a monotherapy or as a combination treatment but causes no discernable side effects. Inhibitors of transcription replication conflict resolution may provide a new and unique therapeuticavenue for exploiting this cancer-selective vulnerability.

First report of Curvularia geniculata causing leave spot disease of Bletilla striata in Zhejiang of China.

Bletilla striata, a perennial herbaceous plant belonging to the family Orchidaceae, is native to China and is widely distributed in the Yangtze River basin. In China, B. striata is a popular medicinal plant that is typically used to reduce wound bleeding and inflammation. In September 2021, distinct leaf spot symptoms were observed in more than 50% of B. striata plants in a traditional Chinese medicine plantation (ca. 10 ha) in Xianju City, Zhejiang Province, China. Small, round, pale brown, necrotic spots were first observed on the leaves. Subsequently, these lesions became grayish brown in the center and dark brown with slight protuberances at the margins and eventually enlarged to 5-8 mm on the leaves. Over time, the small spots enlarged and coalesced into necrotic streaks (1-2 cm). Leaves with symptoms of disease were cut, surface-sterilized, and plated on potato dextrose agar (PDA). Fungal colonies (28×28 mm) with grayish-black mycelia from all tissues were produced after 3 days of incubation at 26 °C. The mature colonies eventually turned black in the center, with obvious rings appearing after 10 days of culture. Basal conidia ranged from pale to dark brown, whereas apical ones were pale brown, with central cells being larger and darker than basal cells. Conidia were smooth and either fusiform, cylindrical, or slightly curved with rounded tips. They ranged in length from 22.34 to 36.82 (mean = 28.63) μm with 2-4 septations and slight septal constrictions. Monospore isolation was performed to obtain a pure culture. Strain BJ2Y5 was subsequently stored in the strain Preservation Center of Wuhan University (Wuhan, China) and the strain preservation number CCTCC M 2023123 was obtained. Fresh mycelia and conidia that grew at 26 ℃ for 7 days were collected from PDA plates. DNA was extracted using the Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech Co., Shanghai, China). The phylogenetic position of isolate BJ2-Y5 was clarified based on DNA sequence analysis of three loci, namely glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Berbee et al. 1999), the internal transcribed spacer (ITS) region (White et al. 1990), and partial sequences of the second largest subunit of RNA polymerase II (RPB2; O'Donnell et al. 2007). A BLAST search (GenBank accession nos. OP913168, OP743380, and OP913171) showed 99% homology to the reference isolate CBS 220.52. Based on the morphological and molecular information presented in this study these isolates were identified as C. geniculata (Hosokawa et al. 2003). Furthermore, we evaluated the pathogenicity of B. striata leaves by smearing a conidial suspension (106 conidia/mL) on both sides of leaves with and without wounds. Five inoculated and three non-inoculated leaves (smeared with sterile distilled water as a negative control) were kept in a greenhouse at 26 °C under natural sunlight and covered with plastic bags for 72 h to maintain humidity. After 7 days, small round spots appeared on the wounds. Fifteen days later, the symptoms of disease on the wounded inoculated leaves were similar to those observed in the original sample, whereas the control plants remained healthy. No symptoms of infection were observed in unwounded inoculated leaves. C. geniculata was successfully re-isolated from all five inoculated leaves and was confirmed based on Koch's postulates. To the best of our knowledge, C. geniculata infection has not been previously reported in B. striata.

First Report of Leaf Spot of Sugar Beet Caused by Stemphylium vesicarium in Minnesota, USA.

In July 2021, sugar beet (Beta vulgaris L.) leaves with numerous tan to brown spots with white-bleached center and oval to irregularly shaped were collected from a field in Minnesota (MN) (46.2774° N, 96.3100° W), with 15% disease incidence and 30% disease severity. Leaves were washed with tap water then surface disinfected in 1% NaOCl aqueous solution for 1 min. Samples were rinsed thrice with sterile distilled water and dried in a laminar flow hood. A 2-cm leaf disc was plated on potato dextrose agar amended with streptomycin sulfate (200 mg/L) and incubated for four days at 25°C under 12-h light/dark cycle. Single spore cultures were obtained by suspending in sterile water spores harvested from a single colony. The suspension was streaked on a dish with V8 agar media and incubated as described. Five pure cultures were transferred to clarified V8 agar media for morphological feature observations. Colonies were uniform in appearance and developed light to olivaceous green mycelium. Conidia were dark brown to olivaceous green in color and measured 30 × 18 μm (n=20). They were oblong to broadly oval shaped muriform, and multiseptated (1 to 5 septa). Hyphae were septate and pale brown. Conidiophores were short, septate, and light to dark brown in color. Based on the morphological characteristics, isolates were identified as Stemphylium vesicarium (Simmons 1969). Genomic DNA of all five isolates were extracted using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). PCR amplification and sequencing of the internal transcribed spacer (ITS) region (ITS1/ITS4 primers), the largest subunit of RNA polymerase II (5F2/7cR primers) (O'Donnell et al. 2009), the plasma membrane ATPase (ATPD-F1/ATPD-R1) gene (Lawrence et al. 2013), glyceraldehyde-3-phosphate-dehydrogenase gene (GAPDH) (gpd1/gpd2) (Berbee et al. 1999), and β-tubulin gene (Bt2a/Bt2b primers) (Glass and Donaldson 1995) were done using standard procedures. Sequences were submitted to GenBank under accession numbers OP584331 (ITS), OP589289 (RPB2), OP589290 (ATPase), OP994239 (GAPDH) and OP382477 (β-tubulin). The BLASTN search of the sequences showed 100% similarity with MT629829 (ITS) (525/525 bp), KC584471 (RPB2) (859/859 bp), JQ671770 (ATPase) (794/794 bp), MK105974 (GAPDH) (519/519 bp) and MN410922 (β-tubulin) (320/320 bp) reference sequences of S. vesicarium. Pathogenicity tests were done using four cv. Maribo MA 504 plants. S. vesicarium spore suspensions (1 × 106/ml) were sprayed on three leaves from each plant. This trial was repeated with three replicates. A similar group of plants were sprayed with autoclaved distilled water to serve as non-inoculated control. All plants were incubated in the mist chamber for 5 days at 25°C, under daily 14/10 light-dark cycles, and >80% relative humidity, then transferred to the greenhouse kept at 23 ± 2°C and a 12-h photoperiod. Fifteen days post-inoculation, all inoculated plants had multiple lesions with dark brown margins with a grayish center, and non-inoculated control plants were asymptomatic. The re-isolated fungus was morphologically similar to isolates retrieved from the field. S. vesicarium was reported on sugar beet in Michigan (Metheny et al. 2022). This is the first report of S. vesicarium causing disease on sugar beet in MN. Stemphylium sp. is a major problem of sugar beet in the Netherlands (Hanse et al. 2015). Efforts should be made to prevent introduction of susceptible beet cultivars so that the disease does not become widespread in the USA.

RNA polymerase II CTD is dispensable for transcription and required for termination in human cells.

The largest subunit of RNA polymerase (Pol) II harbors an evolutionarily conserved C-terminal domain (CTD), composed of heptapeptide repeats, central to the transcriptional process. Here, we analyze the transcriptional phenotypes of a CTD-Δ5 mutant that carries a large CTD truncation in human cells. Our data show that this mutant can transcribe genes in living cells but displays a pervasive phenotype with impaired termination, similar to but more severe than previously characterized mutations of CTD tyrosine residues. The CTD-Δ5 mutant does not interact with the Mediator and Integrator complexes involved in the activation of transcription and processing of RNAs. Examination of long-distance interactions and CTCF-binding patterns in CTD-Δ5 mutant cells reveals no changes in TAD domains or borders. Our data demonstrate that the CTD is largely dispensable for the act of transcription in living cells. We propose a model in which CTD-depleted Pol II has a lower entry rate onto DNA but becomes pervasive once engaged in transcription, resulting in a defect in termination.

Structure and phase separation of the C-terminal domain of RNA polymerase II.

The repetitive heptads in the C-terminal domain (CTD) of RPB1, the largest subunit of RNA Polymerase II (Pol II), play a critical role in the regulation of Pol II-based transcription. Recent findings on the structure of the CTD in the pre-initiation complex determined by cryo-EM and thenovel phase separation properties of key transcription components offers an expanded mechanistic interpretation of the spatiotemporal distribution of Pol II during transcription. Current experimental evidence further suggests an exquisite balance between CTD's local structure and an array of multivalent interactions that drive phase separation of Pol II and thus shape its transcriptional activity.

First report of stem rot of Scrophularia ningpoensis caused by Fusarium commune in China.

First report of stem rot of Scrophularia ningpoensis caused by Fusarium commune in China.

Root Rot Disease of Torreya grandis Caused by Fusarium fujikuroi in China.

Torreya grandis is an evergreen plant endemic of China and widely grown in Southern China. Its fruit is a precious nut in China, rich in vitamins and minerals, can be directly eaten, can also be used as medicinal plants with functions of lowering blood lipids and softening blood vessels (Wang 2022). From 2018 to 2020, typical root rot symptoms of Torreya grandis was found in plantations in Huangshan and surrounding areas of Huangshan, Anhui province, China. About 15 to 32% of root rot disease incidence was recorded at the plantation. Diseased plants were observed with symptoms such as yellow to brownish leaves without lesions and later drying, and rotten roots looked dark brown while the roots of heathy plants showed white, and eventually leading to the death of the diseased plant. The root rot symptomatic plants were collected in June of 2020. Tissues were cut to the length of 0.3 to 0.5 cm, then surface sterilized by 2% sodium hypochlorite for 2 min and 75% alcohol for 1 min, rinsed three times in sterile distilled water, and placed on potato dextrose agar (PDA) and incubated at 25℃ for 5 to 9 days. Eight isolates with similar morphology were isolated from single spores. On PDA, the isolates produced abundant aerial white mycelia with septation and turned violet to dark pink on the reverse side of the culture. Morphological characteristic was determined using a pure culture grown on synthetic low nutrient agar (SNA). Two types of conidia, microconidia and macroconidia, were observed on SNA. Macroconidia were long and slender, usually 3 to 5 septate, measuring 2.7 to 4.3 × 22.3 to 49.6 μm (n=30), and narrowed at the both ends. Microconidia were abundant, oval, clavate or ovate, zero to one septate and measured 1.6 to 3.9 × 4.4 to 13.0 μm (n=50). According to the culture and conidial characteristics, the isolates were tentatively identified as Fusarium species (Leslie and Summerell 2006). Four isolates were random selected for molecular identification. The general primers ITS1/ITS4 for internal transcribed spacer (ITS) (White et al. 1990), EF1/EF2 for translation elongation factor (TEF1) (O'Donnell et al. 1998), 5F2/7cR for the second largest subunit of RNA polymerase Ⅱ(RPB2) (O'Donnell et al., 2007), H3-1a/H3-1b for Histone H3 (Jacobs et al., 2010), F5/R8 for subunits 1 of DNA-directed RNA polymerase Ⅱ (RPB1) (O'Donnell et al. 2010) and MS3F/MS3R for mitochondrial small subunit (mtSSU) (Stenglein et al. 2010) were amplified, respectively. The products were sequenced and deposited in GenBank with accession numbers of MW350689, MW029444, ON077156, ON077158, ON077157, ON054432, respectively. Blast analysis showed 99.40 to 100% sequence homology with known F. fujikuroi isolates. A phylogenetic analysis based on the concatenated sequences clustered from the combined datasets (TEF1, RPB2, Histone H3, RPB1 and mtSSU) revealed the isolate most closely related to the F. fujikuroi (100% bootstrap). Fifteen 2-year-old healthy plants of Torreya grandis were selected for the pathogenicity test. A conidial suspension (1×106 conidia/ml) was prepared by collecting spores from 10-day-old cultures on PDA. The root of each plants inoculated with 200 ml of a 106 conidia/ml suspension, and the five control plants inoculated with sterilized water. The plants were incubated in green house with 25℃ (14 h light)/22℃ (10 h dark) at 85% humidity. Two weeks later, 100% of artificially inoculated plants showed the same symptoms similar to those observed in the plantation, like yellow leaves, dark brown and rotten roots, meanwhile, the roots of control plants displayed healthy. From symptomatic roots, the pathogen was reisolated which satisfying Koch's postulates. F. fujikuroi causes root rot of soybean and Reineckia carnea (Detranaltes et al. 2021, Sun et al. 2018).To the best of our knowledge, this is the first report of F. fujikuroi causing root rot of Torreya grandis in China.

First report of Fusarium incarnatum-equiseti species complex as the causal agent of pod rot of black gram (Vigna mungo L.) in India.

Black gram (Vigna mungo L.) is important pulse crop and is the third-most cultivated pulse in India (Swaminathan et al. 2023). In August 2022, pod rot symptoms were observed on a black gram crop with 80 to 92% disease incidence in the Crop Research Center, Govind Ballabh Pant University of Agriculture & Technology, Pantnagar (29.0222° N, 79.4908° E), Uttarakhand, India. Disease symptoms included white to salmon pink fungal-like growth over the pods. The symptoms were more severe initially at the tip of the pods, which expanded at later stages to cover the entire pod. Seeds present in the symptomatic pods were severely shrivelled and non-viable. Ten plants from the field were sampled to identify the causal agent. Symptomatic pods were cut into pieces, surface-disinfested with 70% ethanol for 1 min to reduce contaminants, rinsed with sterilized water three times, dried on sterilized filter paper, and aseptically placed on potato dextrose agar (PDA) amended with 30 mg/liter streptomycin sulphate. After 7 days of incubation at 25°C, three Fusarium-like (FUSEQ1, FUSEQ2, and FUSEQ3) isolates were purified by single spore transfer and subcultured on PDA. Fungal colonies on PDA were initially white to light pink, aerial, and floccose, and later on, colonies turned ochre yellowish to buff brown. After transfer to carnation leaf agar (Choi et al. 2014), isolates formed hyaline, 3 to 5 septate macroconidia, 20.4 to 55.6 × 3.0 to 5.0 µm (n = 50) with tapered, elongated apical cells and prominent foot-shaped basal cells. Chlamydospores were thick, intercalary, globose, and abundant in chains. No microconidia were observed. Based on morphological characters, the isolates were identified as belonging to the Fusarium incarnatum-equiseti species complex (FIESC) (Leslie and Summerell 2006). For molecular identification of the three isolates, total genomic DNA was extracted using the PureLink® Plant Total DNA Purification Kit (Invitrogen™, ThermoFisher Scientific, Waltham, Massachusetts, United States) and used for amplification and sequencing of a portion of the internal transcribed spacer (ITS) region, the translation elongation factor-1 alpha (EF-1α) gene, and the second largest subunit of RNA polymerase (RPB2) gene (White et al. 1990; O'Donnell 2000). The sequences were deposited in the GenBank database (ITS: OP784766, OP784777, and OP785092; EF-1α: OP802797, OP802798, and OP802799; RPB2: OP799667, OP799668, and OP799669). Polyphasic identification carried out in fusarium.org indicated 98.72 % and 100% similarity of FUSEQ1 and FUSEQ2 respectively to F. clavum and FUSEQ3 had 98.72 % similarity to F. ipomoeae. Both of the identified species are members of the FIESC (Xia et al. 2019). Pathogenicity tests were carried out on 45-day-old potted plants of V. mungo with seed pods, which were kept in a greenhouse. Plants were sprayed with 10 ml of a conidial suspension of each isolate (107 conidia/ml). Control plants were sprayed with sterile distilled water. The plants were covered with sterilized plastic bags after inoculation to maintain humidity and kept in a greenhouse at 25 ± 2°C. Within ten days, all the inoculated plants showed symptoms similar to those observed in the field, whereas control plants were symptomless. The pathogenicity test was repeated twice. Fungi consistently reisolated from the symptomatic pods were confirmed by morphological characterization and molecular assays as described above to belong to the FIESC, whereas no fungus was isolated from control pods. Fusarium spp. causing pod rot in green gram (V. radiata L.) has also been reported from India (Buttar et al. 2022). To our knowledge, this is the first report of FIESC as a causal agent of pod rot of V. mungo in India. The pathogen has the potential to cause considerable economic and production losses in black gram, and for that reason, disease management strategies should be implemented.

Identification and pathogenicity analysis of leaf brown spot of Juglans regia in China

English walnut (Juglans regia), has high economic and ecological value. As an important tree species for eliminating poverty, it is planted in many Provinces of China. In 2021, new pathogenic fungi were observed in English walnut in Guangyuan City, Sichuan Province, China. The initial symptom of leaf infection is that the leaves are covered with small black spots, which gradually expand into larger brown spots. Most of the spots appeared at the edges of the leaves, and yellow whorls were observed at the junction between the spots and the healthy leaves. The pathogenic fungi were isoalted form collecting disease samples and purified by single-spore culturing. In vitro and field experiments showed that the pathogen could cause brown spots on walnut leaves. The inoculation experiment showed that the symptoms in the field experiment were the same as those observed on the spot; however, slight differences were observed in the in vitro experiment. Ten isolates were obtained from walnut leaves with brown spot symptoms, and these were further characterized based on morphology and DNA sequencing. ITS (internal transcribed spacer), LSU (large sub-unit rDNA), rpb2 (second largest subunit of RNA polymerase) and tub2 (beta-tubulin) gene regions were used to construct phylogenetic trees and determine the evolutionary relationships among the collected strains. The isolate was identified as Nothophoma quercina by morphological and polygene analyses. As far as we are aware, the brown spots on walnut leaves caused by N. quercina is the first report of its kind.

POLR1A variants underlie phenotypic heterogeneity in craniofacial, neural, and cardiac anomalies

Heterozygous pathogenic variants in POLR1A, which encodes the largest subunit of RNA Polymerase I, were previously identified as the cause of acrofacial dysostosis, Cincinnati-type. The predominant phenotypes observed in the cohort of 3 individuals were craniofacial anomalies reminiscent of Treacher Collins syndrome. We subsequently identified 17 additional individuals with 12 unique heterozygous variants in POLR1A and observed numerous additional phenotypes including neurodevelopmental abnormalities and structural cardiac defects, in combination with highly prevalent craniofacial anomalies and variable limb defects. To understand the pathogenesis of this pleiotropy, we modeled an allelic series of POLR1A variants invitro and invivo. Invitro assessments demonstrate variable effects of individual pathogenic variants on ribosomal RNA synthesis and nucleolar morphology, which supports the possibility of variant-specific phenotypic effects in affected individuals. To further explore variant-specific effects invivo, we used CRISPR-Cas9 gene editing to recapitulate two human variants in mice. Additionally, spatiotemporal requirements for Polr1a in developmental lineages contributing to congenital anomalies in affected individuals were examined via conditional mutagenesis in neural crest cells (face and heart), the second heart field (cardiac outflow tract and right ventricle), and forebrain precursors in mice. Consistent with its ubiquitous role in the essential function of ribosome biogenesis, we observed that loss of Polr1a in any of these lineages causes cell-autonomous apoptosis resulting in embryonic malformations. Altogether, our work greatly expands the phenotype of human POLR1A-related disorders and demonstrates variant-specific effects that provide insights into the underlying pathogenesis of ribosomopathies.

Endophytic Trichoderma species from rubber trees native to the Brazilian Amazon, including four new species.

Fungi belonging to the genus Trichoderma have been widely recognized as efficient controllers of plant diseases. Although the majority of isolates currently deployed, thus far, have been isolated from soil, endophytic Trichoderma spp. is considered to be a promising option for application in biocontrol. In this study, 30 endophytic Trichoderma isolates-obtained from the leaves, stems, and roots of wild Hevea spp. in the Brazilian Amazon-were analyzed using specific DNA barcodes: sequences of internal transcribed spacers 1 and 2 of rDNA (ITS region), genes encoding translation elongation factor 1-α (TEF1-α), and the second largest subunit of RNA polymerase II (RPB2). The genealogical concordance phylogenetic species recognition (GCPSR) concept was used for species delimitation. A phylogenetic analysis showed the occurrence of Trichoderma species, such as T. erinaceum, T. ovalisporum, T. koningiopsis, T. sparsum, T. lentiforme, T. virens, and T. spirale. Molecular and morphological features resulted in the discovery of four new species, such as T. acreanum sp. nov., T. ararianum sp. nov., T. heveae sp. nov., and T. brasiliensis sp. nov. The BI and ML analyses shared a similar topology, providing high support to the final trees. The phylograms show three distinct subclades, namely, T. acreanum and T. ararianum being paraphyletic with T. koningiopsis; T. heveae with T. subviride; and T. brasiliensis with T. brevicompactum. This study adds to our knowledge of the diversity of endophytic Trichoderma species in Neotropical forests and reveals new potential biocontrol agents for the management of plant diseases.

Abstract 4989: Discovery and evaluation of ISM6466A, a novel covalent CDK12 inhibitor for the treatment of cancer

Abstract Cyclin-dependent kinase 12 (CDK12) belongs to the cyclin-dependent kinase (CDK) family of serine/threonine protein kinases. The CDK12/cyclin K complex regulates the elongation step of RNA transcription by phosphorylating Ser2 on the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II, and selectively affects the expression of genes, such as BRCA1 and BRCA2, involved in DNA Damage Response (DDR). CDK12 mutations as well as overexpression have been reported in different types of malignancies, making it an attractive cancer target. In this study, a series of covalent CDK12 inhibitors were designed and profiled in biochemical and cellular assays. ISM6466A was identified as a lead compound, demonstrating good in vitro efficacy, ADME properties, safety pharmacology profile (e.g., CYP, hERG), and a reasonable in vivo PK profile. Biochemically, ISM6466A exhibited reasonable selectivity over other CDKs, and inhibited cell proliferation in a panel of tumor cell lines with IC50 values in the nanomolar range (e.g., IC50 of ~100nM in the TNBC and AML cell lines, SUM149PT and MV4-11, respectively). Cell-based assays showed that ISM6466A has a selective inhibitory action for the phosphorylation of Ser2 on the CTD, but not for that of Ser5 or Ser7. Notably, ISM6466A demonstrated excellent in vivo anti-tumor efficacy in the SUM149PT breast cancer CDX model (Tumor Growth Inhibition, TGI, of 114% at 60 mg/kg QD) and in the MV4-11 AML CDX model (TGI of 95% at 30 mg/kg QD), without body weight loss. Data from an endpoint PKPD study in the MV4-11 CDX model revealed that ISM6466A inhibited phosphorylation of Ser2 on the CTD, and also inhibited DDR genes such as BRCA1 and ATR. Results from a 28-day rat DRF study revealed that the compound did not show evident signs of toxicity at the tested doses, suggesting a reasonable safety window for ISM6466A.In summary, the novel, covalent, small-molecule CDK12 inhibitor, ISM6466A, demonstrated excellent preclinical efficacy along with favorable drug-like properties. Our results underscore the therapeutic potential of ISM6466A for the treatment of cancer. Citation Format: Lihua Min, Hongfu Lu, Yihong Zhang, Jianping Wu, Xin Cai, Min Zheng, Hui Cui, Junwen Qiao, Xiao Ding, Sujata Rao, Feng Ren, Alex Zhavoronkov. Discovery and evaluation of ISM6466A, a novel covalent CDK12 inhibitor for the treatment of cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4989.

Intraspecific genetic diversity of the fish-infecting microsporidian parasite Pseudokabatana alburnus (Microsporidia).

Pseudokabatana alburnus is a xenoma-forming fish microsporidium, firstly described from the liver of the Culter alburnus from Poyang Lake in China. In the present study, P. alburnus was firstly reported from the ovary of 6 other East Asian minnows, including Squaliobarbus curriculus, Hemiculter leucisculus, Cultrichthys erythropterus, Pseudolaubuca engraulis, Toxabramis swinhonis, and Elopichthys bambusa. Genetic analysis revealed high sequence diversity in the ribosomal internal transcribed spacer region (ITS) and the largest subunit of RNA polymerase II (Rpb1) loci of P. alburnus isolated from different hosts and locations. The variation of Rpb1 mainly occurred in the 1,477-1737 bp regions. The presence of a wide variety of Rpb1 haplotypes within a single fish host, together with evidence of genetic recombination suggested that P. alburnus may have the intergenomic variation and sexual reproduction might be present in other hosts (possibly freshwater shrimp). Phylogenetic analysis and population genetic analysis showed that there was no geographical population divergence for P. alburnus. Homogeneity and high variability of ITS sequences indicates that ITS may be a suitable molecular marker to distinguish different P. alburnus isolates. Our data confirm the broad geographical distribution and host range of P. alburnus in the middle and lower reaches of the Yangtze River. Additionally, we emendated the genus Pseudokabatana to exclude the infection site, liver as one of the taxonomic criteria, and proposed that fish ovary was be the general infection site of P. alburnus.

A contribution to Porogramme (Polyporaceae, Agaricomycetes) and related genera

The polypores with shallow pores from tropical Asia and America are studied. Our molecular phylogeny based on the internal transcribed spacer (ITS), the large subunit nuclear ribosomal RNA gene (nLSU), the translation elongation factor 1-α gene (TEF1), and the largest subunit of RNA polymerase II (RPB1) demonstrates six clades are formed among Porogramme and related genera. Two new genera, Cyanoporus and Pseudogrammothele, are established, and the six clades represent Porogramme, Cyanoporus, Grammothele, Epithele, Theleporus, and Pseudogrammothele, respectively. The molecular clock analyses estimate the divergence times of the six clades based on a dataset (ITS + LSU + TEF1 + RPB1 + RPB2), and we recognize the mean stem ages of the six genera are earlier than 50 Mya. Three new species in Porogramme were morphologically and phylogenetically confirmed, and they are described as P. austroasiana, P. cylindrica, and P. yunnanensis. Phylogenetic analysis shows that type species of Tinctoporellus and Porogramme are nested in the same clade, and Tinctoporellus is treated as a synonym of Porogramme. Based on our phylogeny, twelve new combinations are proposed, and the differences between the new species and similar or related species are discussed.

First Report of Ceratobasidium sp. (AG-A and AG-G) Causing Root Rot and Stem Canker of Common Bean in Mexico.

Common bean (Phaseolus vulgaris L.), is a grain legume widely cultivated worldwide for its edible dry seeds and pods. In February 2021, root rot symptoms were observed in two common bean (cv. Azufrado Higuera) fields located in Ahome (25º96´19¨N, 109º33´42¨W) and Guasave (25º71´85¨N, 108º78´50¨W) municipalities in Sinaloa, Mexico. Diseased plants showed reduced growth, dark brown canker at the base of the stem, root rot, as well as the absence of secondary roots. The disease incidence was estimated up to 35%. For fungal isolation, symptomatic roots were surface sterilized with 1% sodium hypochlorite for 2 min, rinsed with sterilized distilled water two times, and blotted dry on sterile filter paper. Small fragments of diseased roots were placed on PDA medium and incubated at 25°C in darkness for 3 days. Rhizoctonia-like colonies were consistently obtained and 10 isolates were purified by the hyphal-tip method. Colonies on PDA were white initially and then turned brown after 15 days of incubation. The septate hyphae were 3.9 to 6.3 μm in width and branched at right angles with a septum near the point of branching. Microscopic examination by Safranine-O staining showed two nuclei per cell. The morphological features of the isolates resembled those of Ceratobasidium (Sneh et al. 1991). The two Ceratobasidium isolates were selected for molecular analysis and pathogenicity tests. The isolates were deposited in the Culture Collection of Phytopathogenic Fungi of the Faculty of Agriculture of Fuerte Valley at the Sinaloa Autonomous University (Accession nos. FAVF395 and FAVF396). For molecular identification, genomic DNA from each of the two isolates was extracted, and the internal transcribed spacer (ITS) region and partial fragments of the second largest subunit of RNA polymerase II (rpb2) gene were amplified and sequenced with the primer pairs ITS5/ITS4 (White et al. 1990) and RBP2-980F/RPB2-7cR (Liu et al. 1999), respectively. The ITS sequences (accession nos. ON630914 and ON630915) showed 99.66 and 99.01% identity with Ceratobasidium sp. (AG-A) from the USA (OM045887) and Ceratobasidium sp. (AG-G) from China (HM623627), respectively. Whereas, the rpb2 sequences (OM258171 and OM258172) showed 94.10 and 95.74% identity with Ceratobasidium sp. (AG-A) from Serbia (MT1267888) and Ceratobasidium sp. (AG-G) from Japan (DQ301701), respectively. A phylogenetic tree based on Maximum Likelihood and including combined ITS and rpb2 sequences data for Ceratobasidium spp. was generated. The phylogenetic tree grouped the isolates FAVF395 and FAVF396 within the Ceratobasidium sp. AG-A and AG-G clades, respectively. Pathogenicity tests for each isolate were performed by inoculating 10 healthy common bean seedlings (15-day-old) grown in pots. A total of 50 ml of a mycelial suspension adjusted to a concentration of 1 × 105 mycelial fragments/ml were directly placed on the stem base of each plant. Five uninoculated common bean seedlings were used as control. All plants were kept in a greenhouse for 15 days at temperatures ranging from 22 to 32°C. Root rot and stem canker symptoms appeared on inoculated seedlings after 10 days, whereas control plants remained symptomless. Fungi were reisolated from the infected roots and found to be morphologically identical to the isolates used for inoculation, thus fulfilling Koch's postulates. Ceratobasidium sp. has been previously reported as the causal agent of root rot of watermelon in Sonora, Mexico (Meza-Moller et al. 2014; Farr and Rossman 2022). To our knowledge, this is the first report of Ceratobasidium sp. causing root rot of common bean in Mexico. Further monitoring should be performed to quantify yield impacts and develop effective management strategies for this disease.

Assessing the Biocontrol Potential of Clonostachys Species Isolated as Endophytes from Coffea Species and as Mycoparasites of Hemileia Rusts of Coffee in Africa.

During surveys conducted in South America and Africa to identify natural fungal enemies of coffee leaf rust (CLR), Hemileia vastatrix, over 1500 strains were isolated, either as endophytes from healthy tissues of Coffea species or as mycoparasites growing on rust pustules. Based on morphological data, eight isolates-three isolated from wild or semiwild coffee and five from Hemileia species on coffee, all from Africa-were provisionally assigned to the genus Clonostachys. A polyphasic study of their morphological, cultural and molecular characteristics-including the Tef1 (translation elongation factor 1 alpha), RPB1 (largest subunit of RNA polymerase II), TUB (β-tubulin) and ACL1 (ATP citrate lyase) regions-confirmed these isolates as belonging to three species of the genus Clonostachys: namely C. byssicola, C. rhizophaga and C. rosea f. rosea. Preliminary assays were also conducted to test the potential of the Clonostachys isolates to reduce CLR severity on coffee under greenhouse conditions. Foliar and soil applications indicated that seven of the isolates had a significant effect (p < 0.05) in reducing CLR severity. In parallel, in vitro tests that involved conidia suspensions of each of the isolates together with urediniospores of H. vastatrix resulted in high levels of inhibition of urediniospore germination. All eight isolates showed their ability to establish as endophytes in C. arabica during this study, and some proved to be mycoparasites of H. vastatrix. In addition to reporting the first records of Clonostachys associated with healthy coffee tissues and with Hemileia rusts of coffee, this work provides the first evidence that Clonostachys isolates have potential as biological control agents against CLR.

Climacocystaceae fam. nov. and Gloeoporellaceae fam. nov., two new families of Polyporales (Basidiomycota).

Polyporales is a diverse group of Agaricomycetes including more than 2,500 species belonging to 255 genera and 18 families. Recently, many studies focused on the classification of Polyporales, but the familial placements of some taxa remain uncertain. In this study, two new families, Climacocystaceae and Gloeoporellaceae of Polyporales, are proposed based on morphological characters and molecular data. Phylogenetic analyses of the two new families are inferred from the DNA sequences of the internal transcribed spacer regions (ITS), the large subunit of nuclear ribosomal RNA gene (nLSU), the largest subunit of RNA polymerase II gene (RPB1), the second largest subunit of RNA polymerase II gene (RPB2), and the translation elongation factor 1-α gene (TEF1). Furthermore, the divergence time of Polyporales was estimated as an additional taxonomic criterion based on the conserved regions of five DNA fragments (5.8S, nLSU, RPB1, RPB2, and TEF1). Bayesian evolutionary analysis revealed that the ancestor of Polyporales splits with a mean stem age of 136.53 Mya with a 95% highest posterior density (HPD) of 118.08-158.06 Mya. The mean stem ages of the families within Polyporales originated between 66.02 and 119.22 Mya, of which Climacocystaceae occurred in a mean stem age of 77.49 Mya with a 95% HPD of 61.45-93.16 Mya, and Gloeoporellaceae occurred in a mean stem age of 88.06 Mya with a 95% HPD of 67.15-107.76 Mya.

A new species of Mutinus (Phallaceae, Phallales) from Shandong Province, China

A new species, Mutinus taishanensis (Phallaceae, Phallales, Basidiomycota), was found growing in a forest of Phyllostachys flexuosa and Quercus palustris in Zhulin Temple, Mount Tai Scenic Area (Tai’an City, Shangdong Province, China). It is described as new based on morphological, molecular and ecological evidence. Our phylogenetic analyses based on DNA sequences of the large subunit of nuclear ribosomal DNA (LSU), subunit 6 of ATP synthase (atp6) and the second largest subunit of RNA polymerase II (RPB2) support the inclusion of this new species in the genus Mutinus as it forms an independent clade in the phylogenetic tree. Morphologically, M. taishanensis differs from any known species by the combination of light red to red pseudostipe, red conical fertile portion (covering about 1/4–1/5 of the total length of pseudostipe), and relatively small basidiospores. We present this new species with illustrated morphological description. phylogeny and comparison with morphologically similar or phylogenetically related species.

Single-molecule localization-integrated trajectory analysis for the dynamic interaction of chromatin structure and transcription machinery.

The localization and dynamics of individual proteins in a living cell are essential information for investigating protein functions. Live-cell single-molecule imaging has provided direct information on dynamics involved in biological processes. We have identified the characteristic movement of transcription-related factors in elongation sites and directly measured the viscoelastic properties of phase-separated nucleolus proteins using single-molecule trajectory analysis in living cells. However, the functional dynamics are difficult to identify because of the highly heterogeneous distribution and mobility of individual molecules. Here, we developed localization-integrated single-molecule trajectory analysis with machine-learning-based trajectory classification for simultaneous single-molecule imaging. We applied this method to the dynamics of the largest subunit of RNA polymerase II (Pol II) in a crowded chromatin structure visualized by histone H3.1 labeled with spontaneously blinking protein in a living cell nucleus. We identified distinct Pol II mobility features by calculating various mobility indicators, including diffusion coefficient, spring coefficient, and confinement radius, then applied machine-learning methods, including dimensionality reduction and clustering. We also developed a series of analysis methods to quantify the spatial characteristics of the point distribution adjacent to the trajectory. These methods reveal that the spatial probability of surrounding histone distribution depends on the mobility state of Pol II. The simultaneous imaging technique and the new analysis method developed in this study directly link the physical properties of the transcription machinery with the biological function of chromatin.

First Report of Epicoccum sorghinum Causing leaf spot on Bothriochloa ischaemum in Malaysia.

Bothriochloa ischaemum (family Poaceae) is a perennial weed that can be found in borders of agricultural fields, pastures and roadsides in Malaysia. B. ischaemum is an important phytoremediation species in copper tailings dams (Jia et al. 2020). In December 2021, chlorotic spots with brown halos were observed on leaf samples of B. ischaemum with an incidence of approximately 80% in Penampang, Sabah province (5°56'50.4"N, 116°04'32.8"E). On older leaves, the spots coalesced into larger chlorotic spots. Small pieces (5 x 5 mm) of infected leaves collected from three plants were excised, and then surface sterilized according to Khoo et al. (2022). The fungus was isolated (one isolate was obtained) and cultured on potato dextrose agar (PDA) at 25°C. After 3 days, the colony had cottony aerial mycelia with light purple concentric rings appearing on the underside of the colony. Chlamydospores were produced, either unicellular or multicellular. Conidia were unicellular, hyaline, oval, and were 3.7 to 5.1 x 1.8 to 2.6 μm (n=20). Pycnidia were spheroid, and were 66.4 to 115.3 x 43.1 to 87.4 μm (n=20). Genomic DNA was extracted from fresh mycelia of the fungus based on the extraction method described by Khoo et al. (2022). Amplification of the internal transcribed spacer (ITS) region and large subunit (LSU) of rDNA, and actin (ACT), tubulin (TUB) and RNA polymerase II second largest subunit (RPB2) genes was performed using ITS1/ITS4, LR0R/LR7, ACT512F/ACT783R, T10/Bt2b and RPB2-5F2/RPB2-7cR primers, respectively (O'Donnell and Cigelnik, 1997; Liu et al. 1999; Sung et al. 2007; Chen et al. 2021). The PCR products were sequenced at Apical Scientific Sdn. Bhd.. Sequences were deposited in GenBank as OM453926 (ITS), OM453925 (LSU), OM451236 (ACT), OM451237 (TUB) and OM863567 (RPB2). Sequences of our isolate had 100% homology to ITS of isolate UMS (OK626271) (507/507 bp), LSU of isolate UMS (OM238129) (1328/1328 bp), ACT of isolate CZ01 (MN956831) (275/275 bp), TUB of isolate BJ-F1 (MF987525) (556/556 bp) and RPB2 of isolate HYCX2 (MK836295) (596/596 bp) sequences. Phylogenetic analysis was performed using the maximum likelihood method based on the general time reversible model with a gamma distribution and invariant sites (GTR + G + I) generated from the combined ITS, TUB, LSU and RPB2 sequences, indicating that the isolates formed a supported clade to the related Epicoccum sorghinum type sequences. Morphological and molecular characterization matched the description of E. sorghinum (Li et al. 2020). Koch's postulates were performed by spray inoculation (106 spores/ml) on the leaves of three healthy B. ischaemum plants, using isolate BPL01, while sterilized water was sprayed on three additional B. ischaemum which served as the control. Symptoms similar to those occurred after 6 days post inoculation. No symptoms occurred on controls. The experiment was repeated two more times. The reisolated pathogen was morphologically and genetically identical to E. sorghinum. E. sorghinum was reported previously on Brassica parachinensis (Yu et al. 2019), Camellia sinensis (Bao et al. 2019), Myrica rubra (Li et al. 2020), Oryza sativa (Liu et al. 2020) and Zea mays (Chen et al. 2021) in China. To our knowledge, this is the first report of E. sorghinum causing leaf spot on B. ischaemum in Malaysia. Our findings expand the geographic range and host range of E. sorghinum in Malaysia. B. ischaemum which is a weed in agricultural fields is a host of the pathogen and therefore could be a potential threat to Brassica parachinensis, Camellia sinensis, Oryza sativa and Zea mays in Malaysia. Weed management could be an effective way to eliminate inoculum sources of E. sorghinum.