Largest Subunit Of RNA Polymerase Research Articles

First Report of Wilt Caused by Fusarium nanum (FIESC 25) on Mungbean (Vigna radiata) in Pakistan.

Mungbean, Vigna radia (L.) R. Wilczek, is ranked 2nd next to chickpea (Cicer arietinum) in total cultivation and production in Pakistan. In August of 2022 and 2023, mungbean plants (cv. PRI Mung-2018) were found wilting in a field at the Ayub Agricultural Research Institute, Faisalabad, Pakistan. Wilted leaves turned yellow, died, but remained attached to the stem. Vascular tissue at the base of the stem showed light to dark brown discoloration. Roots were stunted with purplish brown to black discoloration. Symptomatic mungbean plants were collected from fields at five different locations (20 samples/location). Disease incidence was similar among the five fields, ranging from 5 to 10% at each location depending upon type of germplasm and date of sowing. For fungal isolation and morphological identification, symptomatic stem and root tissues were cut into ~5 mm2 pieces with a sterilized blade. Tissues were surface-sterilized for one min in a 0.5% sodium hypochlorite solution, rinsed twice in sterilized water, air dried on sterilized filter paper, and aseptically placed on potato dextrose agar (PDA) containing 0.5 g/L-1 streptomycin sulphate. Plates were incubated for 3-4 days at 25 ± 2°C with a 12-h photoperiod. Single-spore cultures were used for morphological and molecular analyses. Isolates on PDA grew rapidly and produced abundant white aerial mycelium that turned off-white to beige with age. Macroconidia were hyaline, falcate, typically 3-to-6 septate with a pointed apical cell and a foot-shaped basal cell, measuring 24.5-49.5 x 2.7-4.7 μm (n = 40). Globose to obovate chlamydospores measuring 5.8 ± 0.5 μm (n = 40) were produced singly or in chains and were intercalary or terminal and possessed roughened walls. The morphological data indicated the isolates were members of the genus Fusarium (Leslie and Summerell 2006). To obtain a species-level identification, a portion of translation elongation factor 1-α (TEF1), the largest subunit of RNA polymerase (RPB1), and the second largest subunit of RNA polymerase (RPB2) region were PCR amplified and sequenced using EF1/EF2 (O'Donnell et al. 1998), Fa/G2R (Hofstetter et al. 2007), and 5f2/7cr (Liu et al. 1999) primers, respectively. DNA sequences of these genes were deposited in GenBank under accession numbers MW059021, MW059017 and MW059019, respectively. The partial TEF1, RPB1 and RPB2 sequences were queried against the Fusarium MLST database (https://fusarium.mycobank.org/page/Fusarium_identification), using the polyphasic identification tool. The BLASTn search revealed 99.9% identity of the isolate to F. nanum (Xia et al. 2019), formerly FIESC 25 of the F. incarnatum-equiseti species complex (MRC 2610, NRRL 54143; O'Donnell et al. 2018). To confirm pathogenicity, roots of 3-5 leaf stage mungbean seedlings were soaked in a 106 spores ml-1 conidial suspension of the fungus for 15 min and then planted in 10 cm pots containing sterilized soil. Mock-inoculated plants with sterile water served as a negative control. Twenty pots that were used for each inoculated and control treatment were maintained at 25 ± 2°C, 14:8 h photoperiod, and 80% relative humidity in a growth chamber. After 15 days, leaf yellowing, internal browning from the base of stems and root discoloration was observed in all the inoculated plants. The uninoculated negative control plants remained asymptomatic. Fusarium nanum was re-isolated from artificially inoculated plants and identified by colony growth, conidial characteristics on PDA and molecular analyses (TEF1). To our knowledge, this is the first report of wilt caused by F.nanum on mungbean in Pakistan. In Pakistan, mungbean cultivation in irrigated areas has increased in recent years. It has been introduced frequently in citrus orchards, crop rotation of maize and sesame, intercropping with sugarcane and as green manure. However, citrus, maize, sesame and sugarcane are also hosts of Fusarium spp. Therefore, this information warrants sustainable crop protection and may have an impact on further interaction of F. nanum with other wilt pathogens.

Bisifusarium lunatum causing cladode soft rot in pear cactus (Nopalea cochenillifera L. Salm-Dyck) in Brazil.

Cactus pear var. miúda (Nopalea cochenillifera L. Salm-Dyck) is an important crop for the Northeast region of Brazil, composing one of the main sources of animal feed. By April 2021, cladode rot caused death of several cactus pear plants in a production area located in Itaporanga, Paraíba state, Brazil (7°21'55.35" S and 38°11'38.68" W). The infected cladodes showed brown circular necrotic spots, and soft rot with perforations that extended throughout the cladode, followed by tipping over and death of the infected plants. The incidence of the disease ranged from 10 to 30% of the plants. Bisifusarium strains were isolated and cultured on potato dextrose agar (PDA) and syntetic-nutrient-poor-agar (SNA). The colonies showed purple color on PDA. On SNA, macroconidia (n = 100) were abundant, hyaline, slightly falcate, three-septate, measuring 11.0-23.1 x 2.3-4.1 μm. Microconidia (n = 100) were oval, generally aseptate, measuring 4.1-8.7 x 2.3-3.0 μm. Conidiogenic cells formed into short monophialides. Chlamydospores were not observed. According to these morphological features, the pathogen was initially identified as Bisifusarium lunatum (Gryzenhoutm et al. 2017). For further confirmation of the identification, the partial sequences of translation elongation factor 1-alpha (TEF1-α) and the second largest subunit of RNA polymerase II (RPB2) genes were sequenced for a representative isolate (CMA 34: GenBank accession no: TEF1-α: OR536502; and RPB2: OR553509) and compared to other Bisifusarium species from GenBank database. Subsequently, it was subjected to a phylogenetic analysis of maximum likelihood including previously published sequences. According to BLAST searches, the TEF1-α and RPB2 sequences were 99% (637/640 nt) and 100% (312/312 nt) similar to B. lunatum (COUFAL0213: TEF1-α (MK640219), and RPB2 (MK301291)), respectively. The isolate was also clustered in a clade containing the ex-type of B. lunatum with 100% support (SH-aLRT and UFboot), being confidently assigned to this species. The pathogenicity test was performed after Medeiros et al. (2015), by using healthy two months old cactus pear seedlings (n = 10) cultivated in a greenhouse. Sterile toothpicks were distributed over colonies of the representative isolate grown on PDA at 25 ± 2 °C for seven days. Seedling cladodes were stuck with the toothpicks, moistened with sterile water and covered with transparent plastic bags for 24h, thus simulating a humid chamber. Following three months, all control plants (stuck with sterile toothpicks) remained healthy, while those inoculated with the representative isolate exhibited rot symptoms. This test was performed twice. B. lunatum was reisolated from symptomatic cladodes and identified as previously described, thus fulfilling the Koch's postulates. To our best knowledge, this is the first report of B. lunatum causing soft rot on N. cochenillifera in Brazil. Besides N. cochenillifera, this species was also reported on Opuntia ficus-indica in India (Gryzenhoutm et al., 2017), which raises concern regarding its ability to infect other forage sources for cattle feed in Brazilian semiarid regions. The present study highlights that the precise identification of B. lunatum is a key factor to adjust control strategies and management of the disease to prevent the spread of this disease to prevent its spread to other crops.

Glomus rugosae, a new arbuscular mycorrhizal species in Glomeraceae (phylum Glomeromycota) from maritime sand dunes of Poland and an ash pond of Czech Republic

Preliminary morphological analyzes of an isolate producing glomoid spores in culture and comparison of its 45S nuc rDNA sequences (= 18S-ITS-28S) with sequences available in GenBank suggested that this isolate is an undescribed arbuscular mycorrhizal fungus of the genus Glomus in the family Glomeraceae (phylum Glomeromycota). This suggestion was confirmed by phylogenetic analyzes with sequences of 45S and the largest subunit of RNA polymerase II (rpb1) gene that placed this isolate in an autonomous clade sister to that with Glomus macrocarpum, the type species of Glomus and Glomeromycota. In the field, this species, here named G. rugosae sp. nov., was associated with roots of Rosa rugosa, which inhabited the coastal dunes of the Hel Peninsula in northern Poland. In single-species cultures, G. rugosae formed typical vesicular-arbuscular mycorrhiza. Phylogenetic analyzes with 45S sequences of Glomus species and environmental sequences with a similarity of >96% to the 45S sequences of G. rugosae showed that G. rugosae was previously detected in the Czech Republic.

Stress-Induced Changes in Nucleocytoplasmic Localization of Crucial Factors in Gene Expression Regulation.

This study investigates the toxic effect of harmful materials, unfiltered by the placenta, on neonatal umbilical cord (UC) vessels, focusing on stress-induced adaptations in transcriptional and translational processes. It aims to analyze changes in pathways related to mRNA condensate formation, transcriptional regulation, and DNA damage response under maternal smoking-induced stress. UC vessels from neonates born to smoking (Sm) and nonsmoking mothers (Ctr) were examined. Immunofluorescence staining and confocal microscopy assessed the localization of key markers, including Transcription Complex Subunit 1 (CNOT1) and the largest subunit of RNA polymerase II enzyme (RPB1). Additionally, markers of DNA damage response, such as Poly(ADP-ribose) polymerase-1, were evaluated. In Sm samples, dissolution of CNOT1 granules in UC vessels was observed, potentially aiding stalled translation and enhancing transcription via RPB1 assembly and translocation. Control vessels showed predominant cytoplasmic RPB1 localization. Despite adaptive responses, Sm endothelial cells exhibited significant damage, indicated by markers like Poly(ADP-ribose) polymerase-1. Ex vivo metal treatment on control vessels mirrored Sm sample alterations, emphasizing marker roles in cell survival under toxic exposure. Maternal smoking induces specific molecular adaptations in UC vessels, affecting mRNA condensate formation, transcriptional regulation, and DNA damage response pathways. Understanding these intricate molecular mechanisms could inform interventions to improve neonatal health outcomes and mitigate adverse effects of toxic exposure during pregnancy.

Fusarium amaranthuense sp. nov. from amaranth is an emergent species closely related to Fusarium circinatum.

Amaranth (Amaranthus spp. L) is not native to South Korea but cultivated in small scales for ornamental purposes as well as leafy vegetables and pseudo cereals. In this study, a new species within the genus Fusarium was isolated from amaranth, showing stem rot symptoms from a farmer field in Hwaseong, South Korea. The disease is characterized by dark-brown spots with black borders, leading to withering. Phylogenetic analysis based on concatenated sequences of translation elongation factor 1-alpha (TEF1), beta-tubulin (tub2), calmodulin (cmdA), RNA polymerase largest subunit (RPB1) and RNA polymerase II second largest subunit (RPB2) genes, revealed that the obtained isolates were formed a distinct clade within the Fusarium fujikuroi species complex and closely related to F. circinatum. Cultural and morphological characteristics and pathogenicity on healthy amaranth plants (stem and leaves) were examined. The isolates readily differentiated from F. circinatum based on 1-5 septate macroconidia and the absence of sterile hyphae. Based on molecular and morphological characteristics, this fungus is demonstrated to be a new species and is described here as Fusarium amaranthuense, the causal agent of stem rot of amaranth in South Korea.

Two new species of Mallocybe from Himalayan forests of Asia

The genus Mallocybe is poorly studied in Asia, with little known species diversity of the genus in Himalayan forests of Pakistan, a hotspot for biodiversity. Based on morphological characters and molecular data, two new species of Mallocybe from Himalayas of Asia are described. One species, Mallocybe himalayana has been collected from Himalayas of China and Azad Jammu & Kashmir, Pakistan. Mallocybe kashmiriana has been described from Himalayas of Azad Jammu and Kashmir, Pakistan. Detailed morphological descriptions, drawings of microstructures for novel taxa are provided. Phylogenetic analyses inferred from internal transcribed spacer (ITS), large subunit (LSU) and second largest subunit of RNA polymerase II (rpb2) sequences show the novelty of these taxa.

First Report of Leaf Spot on Taraxacum mongolicum Caused by Alternaria solani in China.

Taraxacum mongolicum is a perennial herbaceous plant in the family Asteraceae, with a high edible and medicinal value and is widely planted in China. In August 2022, leaf spots were found on T. mongolicum in Tianjiazhai Town, Xining City, Qinghai Province, China (36°27'17.65″N, 101°47'19.65E, elevation: 2,408 m). The plants exhibited round or irregular brown spots, and the centers of some of the spots were gray (Fig. S1A). An investigation was performed over a hectare area, and the incidence of leaf spot reached 15%-30%, seriously affecting the quality and yield of T. mongolicum. Eleven T. mongolicum leaf spot samples were collected. To isolate the pathogenic fungus, approximately 0.5 cm×0.5 cm pieces of tissues were obtained using sterile scissors from the junction of infected and healthy tissues. The symptomatic leaves were surface-disinfected with 3% NaClO for 1.5 min and washed three times with sterile water. The disinfected pieces were dried and placed on water agar plates in an incubator for 2 days at 25°C. Subsequently, the leaf surface exhibited conidiophores and conidia. Eleven isolates were obtained by single spore isolation. The sparse aerial mycelia were dark grey to black brown in color on potato dextrose agar (PDA) (Fig. S2A), and produced dark, multi-septate conidia with 7-11 transverse septa and 1-2 longitudinal septa (Fig. S2C). Conidia with one or two beaks were long-ovoid, with an average length and width of 103.4 × 21.2 μm, and 80.7 × 3.9 μm of the beaks. One hundred and ten conidia were measured. The identification of 11 isolates was confirmed by multilocus sequence analyses of the internal transcribed spacer of ribosomal DNA (rDNA ITS) (White et al. 1990), and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Xu et al. 2022), actin (ACT) (Yang et al. 2020), histone 3 (HIS3) (Zheng et al. 2015), translation elongation factor 1-α (TEF1-α) (Carbone. 1999), and the second largest subunit of RNA polymerase II (RPB2) (Liu et al. 1999) genes. The sequences of all the isolates were deposited in Genbank (NCBI Accession Nos. ITS: OR105029-OR105039, ACT: OR135220-OR135230, GAPDH: OR135231-OR135241, HIS3: OR122992-OR123002, TEF1-α: PP055972-PP055982, and RPB2: PP055983-PP055993), and the sequence similarity of ITS, ACT, GAPDH, HIS3，TEF1-α and RPB2 were 100%, 98%, 100%, 99%, 100%, and 99% to the sequences of Alternaria solani, respectively. Combined sequences of ITS, GAPDH, TEF1-α, and RPB2 genes were concatenated and a maximum parsimony tree was constructed with PAUP* v. 4.0 alpha. The results indicated that 11 isolates were clustered together with A. solani (Fig. S2D). Therefore, 11 isolates were identified as A. solani based on their morphological and molecular characteristics. Eleven isolates were inoculated on their host to perform Koch's postulates. The isolates were grown on PDA for six days. Healthy one month old T. mongolicum seedlings were planted in 10 cm flowerpots (Fig. S1B) or the seedlings were moved to Petri dish (Fig. S1C), and their leaves were inoculated with 5 mL of hyphae suspension by smearing method. In addition, seedlings of the same age were treated with sterile water to serve as the control. The inoculated seedlings were moved into an artificial climatic box at 25℃, relative humidity was 70%, with 12 h light/12 h dark condition. Totally 80 seedlings were inoculated with isolates and 15 were used as the control. After 7 days, similar symptoms were observed on the plants inoculated with isolates, while control plants did not produce symptoms. The assays were conducted three times. Furthermore, isolates were re-isolated from the symptomatic leaves, and the colonial morphology was the same as the original isolates (Fig S2 A and B). The recovered isolates were identified as A. solani by amplifying and sequencing a portion of the HIS3 gene. Alternaria solani has been previously reported to cause early blight of potato and other Solanum crops (van der Waals et al. 2004; Zheng et al. 2015). To our knowledge, this is the first report of A. solani causing leaf spot of T. mongolicum in China. This disease must be considered in management practices, and our finding provided a basis for disease prevention and management.

Occurrence of Fusarium fujikuroi Causing Fusarium Wilt on Carthusian Pink in China.

Carthusian pink (Dianthus carthusianorum) is native to Europe and is widely grown in China for landscaping. In September 2022, wilting symptoms of carthusian pink were found in Xixia City (33°18'31″ N, 111°29'45″ E), Henan Province, China, with a disease incidence of 65%. Approximately 100 plants were surveyed on the landscaping lawns of the park. Initial symptoms were yellow to brown lesions on the base of stems and leaves. Later, the lesions spread throughout the plants, turning leaves yellow, and leading to root and leaf rot. Eventually, the plants shriveled and died (Figure S1a). Thirty diseased tissues isolated from the roots and leaves were cut into 5×5 mm pieces, which were surface sterilized with 75% ethanol solution for 30 seconds and 1% NaClO solution for 1 minute, rinsed three times in sterilized water, placed on potato dextrose agar (PDA) plates supplemented with 50 μg ml-1 streptomycin, and incubated at 28°C for five days. A total of 25 purified fungal strains with similar phenotypic features were obtained. Three representative strains named OSZ-P1, OSZ-P2, and OSZ-P3 were selected for identification. Fungal colonies developed an abundant aerial mycelium, initially white, which subsequently developed red to purple pigments (Figure S1b). Macroconidia were slender, straight, and measured 12.74 to 49.39 × 2.07 to 4.39 μm (n=50), with two to five septa. Microconidia were clavate and measured 6.31 to 11.61 × 2.15 to 4.02 μm (n=50) (Figure S1c). These morphological characteristics were consistent with Fusarium spp.. The rDNA internal transcribed spacer (ITS), β-tubulin gene (tub2), translation elongation factor 1-alpha gene (tef1), calmodulin (cmdA), RNA polymerase largest subunit (rpb1), and RNA polymerase II second largest subunit (rpb2) were amplified with primers ITS1/ITS4, BT-2a/BT-2b, EF1/EF2, CL1/CL2A, Fa/G2R, and 5F2/7Cr, respectively, for further identification (Yilmaz et al. 2021, O'Donnell et al. 2022). ITS (OQ726389, OQ726390, OQ726391), tub2 (OQ730191, OQ789645, OQ789646), tef1 (OR088904, OR088905, OR088906), cmdA (OR133730, OR133731, OR133732), rpb1 (OR088907, OR088908, OR133729), and rpb2 (OR133733, OR133734, OR133735) nucleotide sequences of the strains OSZ-P1, OSZ-P2, and OSZ-P3 were submitted to GenBank. BLASTn analysis of OSZ-P1 sequences exhibited 99 to 100% similarity with Fusarium fujikuroi sequences (strains Augusto2, I1.3, and CSV1) CP023096, CP023108, CP023084 of cmdA, CP023089, CP023077 of rpb1, and CP023093, CP023105, CP023081 of rpb2. A Phylogenetic tree was constructed of combined genes (tub2, tef1, cmdA, rpb1, rpb2) of sequences, alongside the sequences of the type strains by the neighbor-joining method. The three strains formed a clade with the type strains CBS257.52 and Augusto2 of F. fujikuroi in phylogenetic trees, being clearly separated from other Fusarium spp. (Figure S2). The morphological features and molecular analyses supported the strains as members of F. fujikuroi. To verify the pathogenicity, aboveground parts of the plants of five healthy six-month-old potted plants were sprayed with 100 µl of conidial suspension per pot (106 conidia ml-1), and five similar plants were sprayed with sterilized water as a control. All plants were placed in a climate incubator at 28°C and 90% relative humidity. Seven days after inoculation, withered and yellowed lesions were observed, similar to the natural lesions (Figure S1e). No symptoms were observed on the control plants. The whole pathogenicity tests were performed thrice. Reisolation resulted in cultures that were morphologically and molecularly identical to the original isolates, fulfilling Koch's postulates. Fusarium wilt disease has been reported on other plants of the genus Dianthus. Vascular wilt on Dianthus caryophyllus (carnation) caused by Fusarium oxysporum is the most destructive disease of carnation crops worldwide (Ardila et al. 2014). Fusarium acuminatum causing Dianthus chinensis root rot and foliage blight has recently been reported in Nanjing, China (Xu et al. 2022). To our knowledge, this is the first report of F. fujikuroi causing Fusarium wilt on carthusian pink worldwide. The host range of F. fujikuroi still needs to be clarified for accurate disease management in the selection of plant species for landscape.

An iPSC model for POLR3A-associated spastic ataxia: Generation of three unrelated patient cell lines

Spastic Ataxias (SA) are a group of neurodegenerative disorders with combined pyramidal and cerebellar system affection, leading to an overlap phenotype between Hereditary Spastic Paraplegias (HSP) and Cerebellar Ataxias (CA). Here we describe the generation of iPSCs from three unrelated patients with an ultra-rare subtype of SA caused by compound heterozygous mutations in POLR3A, that encodes the largest subunit of RNA polymerase III. iPSCs were reprogrammed from normal human dermal fibroblasts (NHDFs) using episomal reprogramming with integration-free plasmid vectors: HIHRSi004-A, derived from a 44 year-old male carrying the mutations c.1909 + 22G > A/c.3944_3945delTG, HIHRSi005-A obtained from a 66 year-old male carrying the mutations c.1909 + 22G > A/c.1531C > T, and HIHRSi006-A from a 27 year-old male carrying the mutations c.1909 + 22G > A/c.2472_2472delC (ENST00000372371.8).

First report of leaf blight disease caused by Aspergillus welwitschiae on Persicaria lapathifolia var. salicifolia in China.

Persicaria lapathifolia var. salicifolia (Sibth.) Miyabe, has long been extensively utilized in traditional medicine for its significant medical values (Seimandi et al, 2021). Despite its extensive use, the leaf blight disease of this plant has never been documented in China. However, in September 2023, the symptoms of leaf blight disease were observed on P. lapathifolia var. salicifolia on the campus of Zhejiang Normal University (29°8'3″ N, 119°37'47″ E), Zhejiang province, China. The disease incidence was 40% on the 50 plants surveyed, according to the field survey. The progression of leaf pathogenesis is mainly divided into three stages. Early symptoms manifested as the light yellow spots on the edges or tips of the leaves, which subsequently developed into brown or yellow irregular lesions and eventually led to the curling and wilting leaves. Thus, the leaf tissues (5 × 5 mm) from the border of diseased and healthy areas were surface-sterilized in 1% sodium hypochlorite for 3 min, followed by 75% ethanol for 30 s, and rinsed three times with sterile water. After drying on sterile filter paper, the leaf tissues were put on PDA medium and cultured at 25°C for 3 days. Seven purified fungal isolates were obtained, and one representative strain was selected for further identification. After that, the isolate was identified by the combination of morphological studies and molecular analysis. The fungi exhibited rapid growth on PDA, attaining a diameter of 80 to 85 mm in 5 days. The colonies were black with a yellow margin, and the reverse sides were light yellow and partly colorless. Moreover, the conidia were brown to black, smooth to slightly rough, measuring 3.2 to 3.8 μm (n = 30) in diameter, with radiated conidial heads and expanded ampulliform phialides under the optical microscope. Therefore, the isolate's characteristics were consistent with the descriptions of Aspergillus welwitschiae (Bres.) Henn. (Gherbawy et al, 2021). To further identify the isolate, the internal transcribed spacer (ITS) region (Gardeset al, 1993) and the second largest RNA polymerase subunit (RPB2) (Liu et al, 1999) were employed for phylogenetic analysis. The obtained sequences were despot in GenBank (Acc. Nos. OR797058 for ITS, OR797058 for RPB2, respectively), and exhibited a high degree of sequence homology to A. welwitschiae (MK450815, MK450818, LC179911, and LC000572), with 99% to 100% identity. Besides, multilocus phylogenetic analysis showed that the isolate gathered into one clade with A. welwitschiae. Based on the integrated morphological and molecular results, the isolate was determined to be A. welwitschiae. Six healthy 1-year-old P. lapathifolia var. salicifolia were used to verify Koch's postulates. Three leaves were wounded with sterile pins and inoculated with the conidial suspension (107 conidia/mL) of isolates, while plants inoculated with sterile water were used as controls. After sealing with plastic wrap for 24 hours, the plants were cultivated at 25 °C and 85% relative humidity. Necrotic lesions were observed on leaves 10 days after inoculation, while the control leaves remained asymptomatic. The fungi were re-isolated from the diseased leaves and identified as the original ones through morphological and molecular identification, confirming Koch's postulates. While A. welwitschiae has been reported to cause the rot disease of Sisal Bole in Brazil (Duarte et al, 2018) and maize ear in Serbia (Nikolic et al., 2023), to our knowledge, this study marks the first report of A. welwitschiae causing leaf blight on P. lapathifolia var. salicifolia in China, extending the host range to A. welwitschiae.

RNA-directed DNA methylation mutants reduce histone at the paramutated maize booster1 enhancer.

Paramutation is the transfer of mitotically and meiotically heritable silencing information between two alleles. With paramutation at the maize (Zea mays) booster1 (b1) locus, the low-expressed B' epiallele heritably changes the high-expressed B-I epiallele into B' with 100% frequency. This requires specific tandem repeats and multiple components of the RNA-directed DNA methylation (RdDM) pathway, including the RNA-dependent RNA polymerase (RdRP, encoded by mediator of paramutation1, mop1), the second-largest subunit of RNA polymerase IV and V (NRP(D/E)2a, encoded by mop2), and the largest subunit of RNA Polymerase IV (NRPD1, encoded by mop3). Mutations in mop genes prevent paramutation and release silencing at the B' epiallele. In this study, we investigated the effect of mutations in mop1, mop2 and mop3 on chromatin structure and DNA methylation at the B' epiallele, and especially the regulatory hepta-repeat 100 kb upstream of the b1 gene. Mutations in mop1 and mop3 resulted in decreased repressive histone modifications H3K9me2 and H3K27me2 at the hepta-repeat. Associated with this decrease were partial activation of the hepta-repeat enhancer function, formation of a multi-loop structure, and elevated b1 expression. In mop2 mutants, which do not show elevated b1 expression, H3K9me2, H3K27me2 and a single-loop structure like in wild-type B' were retained. Surprisingly, high CG and CHG methylation levels at the B' hepta-repeat remained in all three mutants, and CHH methylation was low in both wild type and mutants. Our results raise the possibility of MOP factors mediating RNA-directed histone methylation rather than RNA-directed DNA methylation at the b1 locus.

Fusarium clavum causes sugar beet seedling root rot in Wyoming, USA

Seedling rot in sugar beet was observed at Snyder Farms, Wyoming, USA, in April 2022. The diseased seedlings exhibited water-soaked brown to blackish lesions in hypocotyls with poor root systems. Four fungal isolates were retrieved from the diseased samples, and their morphological features were found to be identical to those of previously reported Fusarium species. Artificial inoculation with isolates developed similar symptoms to those observed in the field samples. Based on morphological characteristics, pathogenicity assays, sequence homologies of the internal transcribed spacer, translation elongation factor (tef1α), largest subunit of RNA polymerase II (rpb2), β-tubulin (β-tub), and multilocus phylogenic analyses with tef1α and rpb2, the causal agent was identified as Fusarium clavum (F. incarnatum-equiseti species complex 5). This is the first worldwide report of sugar beet seedling rot caused by F. clavum, detected in Wyoming, USA. This finding will help to formulate effective management practices to manage the pathogen and prevent its widespread occurrence.

Three New Species of Russulaceae (Russulales, Basidiomycota) from Southern China.

The characterization of natural fungal diversity impacts our understanding of ecological and evolutionary processes and can lead to novel bioproduct discovery. Russula and Lactarius, both in the order Russulales, represent two large genera of ectomycorrhizal fungi that include edible as well as toxic varieties. Based on morphological and phylogenetic analyses, including nucleotide sequences of the internal transcribed spacer (ITS), the 28S large subunit of ribosomal RNA (LSU), the second largest subunit of RNA polymerase II (RPB2), the ribosomal mitochondrial small subunit (mtSSU), and the translation elongation factor 1-α (TEF1-α) gene sequences, we here describe and illustrate two new species of Russula and one new species of Lactarius from southern China. These three new species are: R. junzifengensis (R. subsect. Virescentinae), R. zonatus (R. subsect. Crassotunicatae), and L. jianyangensis (L. subsect. Zonarii).

ARMC5 controls the degradation of most Pol II subunits, and ARMC5 mutation increases neural tube defect risks in mice and humans

BackgroundNeural tube defects (NTDs) are caused by genetic and environmental factors. ARMC5 is part of a novel ubiquitin ligase specific for POLR2A, the largest subunit of RNA polymerase II (Pol II).ResultsWe find that ARMC5 knockout mice have increased incidence of NTDs, such as spina bifida and exencephaly. Surprisingly, the absence of ARMC5 causes the accumulation of not only POLR2A but also most of the other 11 Pol II subunits, indicating that the degradation of the whole Pol II complex is compromised. The enlarged Pol II pool does not lead to generalized Pol II stalling or a generalized decrease in mRNA transcription. In neural progenitor cells, ARMC5 knockout only dysregulates 106 genes, some of which are known to be involved in neural tube development. FOLH1, critical in folate uptake and hence neural tube development, is downregulated in the knockout intestine. We also identify nine deleterious mutations in the ARMC5 gene in 511 patients with myelomeningocele, a severe form of spina bifida. These mutations impair the interaction between ARMC5 and Pol II and reduce Pol II ubiquitination.ConclusionsMutations in ARMC5 increase the risk of NTDs in mice and humans. ARMC5 is part of an E3 controlling the degradation of all 12 subunits of Pol II under physiological conditions. The Pol II pool size might have effects on NTD pathogenesis, and some of the effects might be via the downregulation of FOLH1. Additional mechanistic work is needed to establish the causal effect of the findings on NTD pathogenesis.

Morphological and phylogenetic analyses reveal three new species of Fusarium (Hypocreales, Nectriaceae) associated with leaf blight on Cunninghamialanceolata in China.

Chinese fir (Cunninghamialanceolata) is a special fast-growing commercial tree species in China with high economic value. In recent years, leaf blight disease on C.lanceolata has been observed frequently. The diversity of Fusarium species associated with leaf blight on C.lanceolata in China (Fujian, Guangxi, Guizhou, and Hunan provinces) was evaluated using morphological study and molecular multi-locus analyses based on RNA polymerase second largest subunit (RPB2), translation elongation factor 1-alpha (TEF-1α), and RNA polymerase largest subunit (RPB1) genes/region as well as the pairwise homoplasy index tests. A total of five Fusarium species belonging to four Fusarium species complexes were recognized in this study. Two known species including Fusariumconcentricum and F.fujikuroi belonged to the F.fujikuroi species complex, and three new Fusarium species were described, i.e., F.fujianense belonged to the F.lateritium species complex, F.guizhouense belonged to the F.sambucinum species complex, and F.hunanense belonged to the F.solani species complex. To prove Koch's postulates, pathogenicity tests on C.lanceolata revealed a wide variation in pathogenicity and aggressiveness among the species, of which F.hunanense HN33-8-2 caused the most severe symptoms and F.fujianense LC14 led to the least severe symptoms. To our knowledge, this study also represented the first report of F.concentricum, F.fujianense, F.fujikuroi, F.guizhouense, and F.hunanense causing leaf blight on C.lanceolata in China.

First report of Fusarium cugenangense causing root rot of tea plants (Camellia sinensis) in China.

Root rot is an important disease of tea plants owing to its unobvious early symptoms and permanent damage (Huu et al. 2016). In 2019, 5% of tea plants displayed symptoms consistent with root rot in a tea plantation (28°09'N, 113°13'E) located in Changsha city, Hunan province of China. The symptoms of the diseased tea plants ranged from wilting leaves to entirely dead. The roots had black lesions and rot typical of this disease. Symptomatic roots were collected, washed with water and disinfected with 75% ethanol, then cut into pieces and sterilized with 0.1% mercuric chloride for 30 s, 75% ethanol for 1 min, and rinsed with sterile water five times. After drying on sterilized filter paper, root tissues were cultured on potato dextrose agar (PDA) medium at 25 oC for 7 days in the dark. Four isolates, CAGF1, CAGF2, CAGF3, and CAGF4 were purified by selecting single spores. All isolates were subjected to a pathogenicity test. A conidial suspension of each strain was collected at a concentration of 2×106 conidia/mL. For the pathogenicity test, two-year-old field grown tea plants were transplanted in plastic pots containing 240 g of the rice grain-bran mixture (inoculated with 4 mL of conidial suspension and cultured for 14 days) and 960 g of sterilized soil (Huu et al. 2016). The pots without inoculated mixture served as control group. All the pots were kept in illumination incubators at 25 oC and a 12L:12D photoperiod. The pathogenicity test for each strain was repeated three times with three repetitions. Only strain CAGF1 exhibited pathogenicity to tea plants. Symptoms appeared on the third day post inoculation (dpi) and gradually worsened by the 7 dpi. On the 14 dpi, most leaves had died and the roots were black and partially rotten, similar to field symptoms. The reisolated fungus from potted roots was identified as CAGF1 based on ITS region and colony morphology, while isolation was attempted, CAGF1 was not isolated from the control plants, which fulfilled Koch's postulates. On PDA, the colony center of CAGF1 was purple with white margin, while on carnation leaf agar (CLA) medium was white. On CLA medium, macroconidia have 0 to 3 septa, measured 19.1 μm to 41.2 μm × 4.2 μm to 5.4 μm (mean= 31.2 μm × 4.8 μm, n=30). The microconidia were measured as 6.7 μm to 12.8 μm × 2.4 μm to 4.9 μm (mean= 10.1 μm × 3.3 μm, n=30), with 0 to 1 septa. And the chlamydospores were measured as 6.0 to 9.7μm (mean= 7.7μm, n=30). Morphologically, strain CAGF1 was identified as Fusarium oxysporum (Leslie and Summerell 2006). Additionally, the genomic DNA of strain CAGF1 was extracted by cetyltrimethylammonium bromide (CTAB) method, the internal transcribed spacer (ITS), elongation factor 1 alpha (EF-1α) and second largest subunit of RNA polymerase II (RPB2) were amplified using the primers ITS1/ITS4 (White et al. 1990), EF-1/EF-2 (Geiser et al. 2004) and fRPB2-5F/fRPB2-7cR (Liu et al. 1999), respectively. Sequences were deposited in GenBank (ITS, OK178562.1; EF-1α, OK598121.1; RPB2, OP381476.1). BLASTn searches revealed that strain CAGF1 was 100% (ON075522.1 for ITS and JX885464.1 for RPB2) and 99.6% (JQ965440.1 for EF-1α) identical to Fusarium oxysporum species complex (FOSC). Based on phylogenetic analysis, the strain CAGF1 was identified as Fusarium cugenangense, belonging to FOSC. To our knowledge, this is the first report of F. cugenangense causing root rot of tea plants in China. The findings are important for the management of this root rot and the improvement of economic benefits of tea cultivation.

Kgaria (Boletaceae, Boletoideae) gen. nov. in Australia: Neither a Tylopilus nor a Porphyrellus.

Kgaria is described as a new porphyrellus-like genus of Boletaceae to accommodate Tylopilus cyanogranulifer, a dark brown to dull lilac/violet, or rarely, nearly black bolete with a series of oxidation reactions progressing from blue to red then nearly black and a dark brown spore deposit. Idiosyncratic blue-green pigment encrustations (cyanogranules) and a similarly colored reaction of the hyphae located on pileus and stipe surfaces are also diagnostic. Phylogenetic analyses of nuclear large-subunit rDNA (nrLSU), translation elongation factor 1-alpha (tef-1), and the second largest subunit of RNA polymerase II (rpb2) infer Kgaria as a unique generic lineage with two species, one of which is newly described (K. similis). Tylopilus olivaceoporus, originally described at the same time and as distinct from T. cyanogranulifer, appears to be conspecific with the latter. Some darkly pigmented taxa with similar oxidation reactions that were recently described from Brazil, Guyana, and China are further supported by morphology and molecular data as discrete lineages in separate genera in subfamily Boletoideae. Citation: Halling RE, Fechner NA, Holmes G, Davoodian N (2023). Kgaria (Boletaceae, Boletoideae) gen. nov. in Australia: Neither a Tylopilus nor a Porphyrellus. Fungal Systematics and Evolution 12: 31-45. doi: 10.3114/fuse.2023.12.02.

Predictive Potential of RNA Polymerase B (II) Subunit 1 (RPB1) Cytoplasmic Aggregation for Neoadjuvant Chemotherapy Failure

We aimed to investigate the contribution of co-translational protein aggregation to the chemotherapy resistance of tumor cells. Increased co-translational protein aggregation reflects altered translation regulation that may have the potential to buffer transcription under genotoxic stress. As an indicator for such an event, we followed the cytoplasmic aggregation of RPB1, the aggregation-prone largest subunit of RNA polymerase II, in biopsy samples taken from patients with invasive carcinoma of no special type. RPB1 frequently aggregates co-translationally in the absence of proper HSP90 chaperone function or in ribosome mutant cells as revealed formerly in yeast. We found that cytoplasmic foci of RPB1 occur in larger sizes in tumors that showed no regression after therapy. Based on these results, we propose that monitoring the cytoplasmic aggregation of RPB1 may be suitable for determining—from biopsy samples taken before treatment—the effectiveness of neoadjuvant chemotherapy.

Characterization of Trichoderma strains for novel species-specific markers by multiplex PCR and antagonistic property against Alternaria ricini in castor (Ricinus communis L.)

The study aimed at characterizing six Trichoderma strains isolated from the rhizospheric soil of castor growing region of Telangana, India. Microscopic examination and sequencing of ribosomal DNA (rDNA) using internal transcribed spacer (ITS) and D1-D2 region-specific primers revealed them as members of Trichoderma sp. Whereas the phylogenetic analysis revealed four of those strains as Trichoderma asperellum, two strains were confirmed as Trichoderma harzianum with a high sequence similarity of >99%. Further, multiplex PCR results with Trichoderma-specific primers; TEF1 (translation elongation factor 1) and rpb2 (the second largest subunit of RNA polymerase II) gene-specific primers confirmed the findings resulting from the phylogenetic analysis. To develop a set of sequence characterized amplified region (SCAR) markers, these strains were screened with 185 random amplified polymorphic DNA (RAPD) primers. A pairwise comparison of correlation of coefficient values determined from the RAPD-based genetic similarity analysis revealed that TV3 and TS4 strains were closely related to each other (0.98) confirming them as T. harzianum while N13, 7316, TV5, and TV2B16 strains formed a different cluster with T. asperellum strains. A set of novel SCAR markers were developed to distinguish these two species, T. asperellum and T. harzianum including the reported Trichoderma strains as Ex-type strains. T. harzianum strains had extremely low sporulation ability to be tested for biocontrol properties. Of the four T. asperellum strains, 7316 followed by N13 demonstrated high antagonistic ability against the fungal pathogen, Alternaria ricini indicating these strains as potential biocontrol agents to limit Alternaria infection in castor.

Morphological and molecular characterization of Fusarium incarnatum as a causal disease agent of pepper (Capsicum annuum) fruit rot

AbstractChilli pepper (Capsicum annuum L.) is one of the most important commercially cultivated and consumed vegetables in Turkey. During a disease survey, typical symptoms of fruit rot were observed on mature chilli pepper fruits in several surveyed fields and on dried pepper fruits obtained from local retailers/bazaars in Hatay Province, Turkey. Disease incidence varied from 15 to 45% of the plants in the inspected fields. Following standard isolation procedures, 40 fungal isolates were isolated, purified and single‐spore cultures were obtained from surface‐disinfected, rotted dried pepper tissue. Of these isolates, six fungal isolates with dense, cottony white aerial mycelia that became beige with age, were isolated on a potato sucrose agar. All isolates were found to be pathogenic on artificially inoculated chilli pepper fruit. Based on morphological characteristics, the isolates were initially identified as Fusarium incarnatum (Desm.) Sacc. 1886. Morphological identification of F. incarnatum isolates was further confirmed by MALDI‐TOF and molecular analyses using the sequences of the internal transcribed spacer (ITS), partial translation elongation factor‐1α (TEF‐1α) and second largest subunit of RNA polymerase II (RPB2) loci. Phylogenetic analysis based on ITS, TEF‐1α, RPB2 and concatenation of TEF‐1α, RPB2 loci sequences performed with several isolates of Fusarium spp. confirmed that representative fungal isolates (MKUZF1 and MKUZF4) belong to F. incarnatum. To our knowledge, this is the first report of F. incarnatum causing fruit rot in chilli peppers grown in Turkey.