Small Subunit Research Articles

Two novel members of Onygenales, Keratinophyton kautmanovae and K. keniense spp. nov. from soil

Two new Keratinophyton species, K. kautmanovae sp. nov. and K. keniense sp. nov., isolated from soil samples originating from two different geographical and environmental locations (Africa and Europe) are described and illustrated. Phylogenetically informative sequences obtained from the internal transcribed spacer (ITS) region and the nuclear large subunit (LSU) rDNA, as well as their unique phenotype, fully support novelty of these two fungi for this genus. Based on ITS and LSU combined phylogeny, both taxa are resolved in a cluster with eight accepted species, including K. alvearium, K. chongqingense, K. hubeiense, K. durum, K. lemmensii, K. siglerae, K. submersum, and K. sichuanense. The new taxon, K. kautmanovae, is characterized by clavate, smooth to coarsely verrucose conidia, absence of arthroconidia, slow growth at 25 °C, and no growth at 30 °C, while K. keniense is morphologically unique with a high diversity of conidial shapes (clavate, filiform, globose, cymbiform and rhomboid). Both species are described based on their asexual, a chrysosporium-like morph. While the majority of hitherto described Keratinophyton taxa came from Europe, India and China, the new species K. keniense represents the first reported taxonomic novelty for this genus from Africa.

First report of Colletotrichum orbiculare causing anthracnose of pointed gourd in India.

Pointed gourd (Trichosanthes dioica Roxb.), of the Cucurbitaceae family, is widely cultivated as a vegetable in many countries such as Bangladesh, India, Pakistan, Myanmar, Nepal and Sri Lanka. Over 800,000 metric tons of pointed gourds are produced annually in India, where cultivation is estimated to occupy over 33,000 hectares of land (MoA & FW, Government of India). In summer 2018, significant losses (approximately 15-20%) occurred in the sub-Himalayan region in West Bengal state of India (21.14-21.30° N, 78.82-79.02°E) due to a disease with typical anthracnose-like symptoms on the fruits. Light yellowish, small sized round to irregular spots were also apparent on the leaves. These spots gradually increased in size and turned into light brown and were surrounded by yellow halo. The lesions on the fruits were circular, yellow-brown, necrotic and sunken. A survey of four fields (1.5 ha) was conducted and a disease incidence of 30-40% was observed. Necrotic tissues from fruit as well as leaves were cut into approximately 5 mm2, surface sterilized with 0.1% HgCl2, plated in potato dextrose agar and incubated at 28ºC for 7 days in the dark. A total of 50 morphologically similar colonies were obtained from 20 sampled fruits and 10 sampled leaves. Fungal colonies were initially white, becoming gray as the cultures aged on PDA. The cultures developed black acervuli around the center of the colony. Setae were brown in colour, 1-5 septate, 40-100 µm long. Conidia were also observed through light and scanning electron microscopy and exhibit as (4-6 ×13-19 µm) hyaline, aseptate, cylindrical to oblong, with one end round and other truncate. The morphological characteristics were found similar to Colletotrichum orbiculare Damm, P.F. Cannon & Crous as reported by Damm et al. (2013). Ten isolates were obtained by transferring hyphal tips to new PDA plates and incubating under the same conditions. To confirm the identity of the pathogen, genomic DNA was extracted from five pure isolates (PG-Pha, PG-Pha-2, PG-Pha-3, PG-Pha-4, PG-Pha-5) with the cetyltrimethylammonium bromide (CTAB). Further, the ITS1-5.8S-ITS2 region, D1/D2 region of the 28S rRNA large subunit (LSU), Actin (ACT) gene and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene were amplified using specific primers, ITS1/ITS4 (White et al. 1990), NL1/NL4, ACT1/ACT2 and GDF1/GDR1 respectively and PCR conditions described in Damm et al. (2012). A GenBank BLAST search showed 99-100% identity to the Colletotrichum orbiculare (Acc. Nos. KP898988 for ITS1-5.8S-ITS2, Z18997 for 28S rRNA, AB778553 for ACT gene, and KF178482 for GAPDH). All obtained sequences were submitted to the GenBank (Acc. Nos. MN006616, OP811046-OP811049, [ITS1-5.8-ITS2], MN006684, PP391616-PP391619 [28S rRNA], MN168524, PP400822-PP400825 [ACT gene], OP627091, PP400826-PP400829 [GAPDH]). For phylogenetic analysis, MEGA version 11 (Tamura et al. 2021) was used to construct a maximum likelihood tree with 1000 bootstrap replicates, based on a concatenation alignment of three gene sequences (ITS, Actin and GAPDH) of the all the five C. orbiculare isolates as well as sequences of other Colletotrichum species obtained from GenBank. The cluster analysis revealed that, isolate PG-Pha form a cluster with other C. orbiculare isolates. Pathogenicity tests were conducted to confirm Koch's postulates. Pathogenicity tests were performed in mature fruits by inoculating them (n=8) with 10 µl of a 1×106 conidia/ml suspension at needle puncture wound sites. In control set up sterile distilled water was pipetted on fruits. Fruits were placed on sterile trays covered with glasses and incubated at humid chambers at 28±2ºC with 12 h of light. Healthy one-month old potted pointed gourd plants (n=15) were sprayed with conidial suspension until run-off. A set of 15 plants were sprayed with sterile distilled water and maintained as control. The plants were kept in a greenhouse at 25ºC, >75% relative humidity, and a 16/8 h day/night cycle for 15 days. Sterile distilled water was sprayed on the plants at one day interval to maintain the humidity. Inoculated fruits started showing yellowing symptoms one day post inoculation and gradually yellow-brown sunken spots became visible at the place of puncture, whereas control fruits remain symptomless even after 7 days of inoculation. Inoculated leaves showed disease symptoms similar to those observed in the field whereas leaves of control sets were symptomless even after 15 days. The pathogenicity test was repeated thrice under the same conditions mentioned before. C. orbiculare was successfully re-isolated from all the symptomatic tissues of leaves as well as fruits, completing Koch's postulates. Previously, the pathogen has been reported as an important anthracnose pathogen of Cucurbitaceae, especially of cucumber (Cucumis sativus), melons (Cucumis melo), watermelon (Citrullus lanatus), pumpkin (Cucurbita pepo) and squash (Cucurbita maxima) (Farr and Rossman 2020). To our knowledge, this is the first report of C. orbiculare causing anthracnose of pointed gourd. This disease represents a threat to producers in India and central Asia. Further research may contribute to the development of management strategies for this disease.

First report of Enterocytozoon bieneusi in Erinaceus europaeus and Hemiechinus auritus hedgehogs from recovery centers of Portugal.

Enterocytozoon bieneusi microsporidia are emerging pathogens infecting a wide range of vertebrate and invertebrate hosts, known to have zoonotic features since they infect both wild and domestic animals, and humans. Despite their significance, there is very limited epidemiological data on microsporidia in hedgehogs, especially European hedgehogs (Erinaceus europaeus) and long-eared hedgehogs (Hemiechinus auritus), the former known as synantropic hedgehogs, and the latter suited as pets. As such, the present study aimed to assess the presence of E. bieneusi in hedgehogs from Portugal. For this purpose, fecal samples from 110 hedgehogs of three species-E. europaeus (n=106), H. auritus (n=1), and Atelerix albiventris (n=3)-were collected and tested for E. bieneusi by PCR targeting the internal transcribed spacer region and the flanking small and large subunits of the rRNA. We found an overall occurrence of 22.7% (25/110; 95% confidence interval [CI]: 15.28-31.70), with 22.6% (24/106; 95% [CI]: 15.08-31.79) in E. europaeus, 100% (1/1) in H. auritus, and 0% in A. albiventris. Interestingly, three novel genotypes were identified, all belonging to the potentially zoonotic Group 1. Our findings highlight the importance of hedgehogs as potential reservoirs for E. bieneusi and emphasize the need for further research to understand their role in transmission dynamics and assess the associated risks to public and veterinary health.

Pathogenicity Differentiation of Fusarium spp. Causing Fusarium Basal Rot and Wilt Disease in Allium spp.

Here, 12 Fusarium strains, previously described as F. oxysporum f. sp. cepae (Foc), were examined via multi-locus sequencing of calmodulin (cmdA), RNA polymerase II second largest subunit (rpb2), and translation elongation factor 1-alpha (tef1), to verify the taxonomic position of Foc in the newly established epitype of F. oxysporum. The strains in this study were divided into two clades: F. nirenbergiae and Fusarium sp. To further determine the host specifications of the strains, inoculation tests were performed on onion bulbs and Welsh onion seedlings as potential hosts. Four strains (AC145, AP117, Ru-13, and TA) isolated from diseased onions commonly possessed the secreted in xylem (SIX)-3, 5, 7, 9, 10, 12, and 14 genes and were pathogenic and highly aggressive to onion bulbs, whereas all strains except for one strain (AF97) caused significant inhibition of Welsh onion growth. The inoculation test also revealed that the strains harboring the SIX9 gene were highly aggressive to both onion and Welsh onion and the gene was expressed during infection of both onions and Welsh onions, suggesting the important role of the SIX9 gene in pathogenicity. This study provides insights into the evolutionary pathogenicity differentiation of Fusarium strains causing Fusarium basal rot and wilt diseases in Allium species.

Triclosan Dioxygenase: A Novel Two-component Rieske Nonheme Iron Ring-hydroxylating Dioxygenase Initiates Triclosan Degradation.

The emerging contaminant triclosan (TCS) is widely distributed both in surface water and in wastewater and poses a threat to aquatic organisms and human health due to its resistance to degradation. The dioxygenase enzyme TcsAB has been speculated to perform the initial degradation of TCS, but its precise catalytic mechanism remains unclear. In this study, the function of TcsAB was elucidated using multiple biochemical and molecular biology methods. Escherichia coli BL21(DE3) heterologously expressing tcsAB from Sphingomonas sp. RD1 converted TCS to 2,4-dichlorophenol. TcsAB belongs to the group IA family of two-component Rieske nonheme iron ring-hydroxylating dioxygenases. The highest amino acid identity of TcsA and the large subunits of other dioxygenases in the same family was only 35.50%, indicating that TcsAB is a novel dioxygenase. Mutagenesis of residues near the substrate binding pocket decreased the TCS-degrading activity and narrowed the substrate spectrum, except for the TcsAF343A mutant. A meta-analysis of 1492 samples from wastewater treatment systems worldwide revealed that tcsA genes are widely distributed. This study is the first to report that the TCS-specific dioxygenase TcsAB is responsible for the initial degradation of TCS. Studying the microbial degradation mechanism of TCS is crucial for removing this pollutant from the environment.

Phylogenetic and Pathogenic Evidence Reveals Novel Host-Pathogen Interactions between Species of Lasiodiplodia and Citrus latifolia Dieback Disease in Southern Mexico.

Mexico ranks second in the world for Persian lime (Citrus latifolia) exports, making it the principal citrus exporter within the national citrus industry, exporting over 600,000 tons per year. However, diseases are the main factor reducing production, resulting in significant economic losses. Among these diseases, fungal diseases like dieback, caused by species of Lasiodiplodia, are an emerging issue in Persian lime. Symptoms include gummosis, twig and branch dieback, cankers, the necrosis of bark and wood, fruit mummification, and tree decline. The aim of this study was to investigate the occurrence and pathogenicity of the fungal species associated with twig and branch dieback, cankers, and decline of Persian lime trees in southern Mexico, and to elucidate the current status of the Lasiodiplodia species causing the disease in Mexico. During June, July, and August of 2023, a total of the 9229 Persian lime trees were inspected across 230 hectares of Persian lime orchards in southern Mexico, and symptoms of the disease were detected in 48.78% of the trees. Branches from 30 of these Persian lime trees were collected. Fungal isolates were obtained, resulting in a collection of 40 strains. The isolates were characterized molecularly and phylogenetically through the partial regions of four loci: the internal transcribed spacer region (ITS), the β-tubulin gene (tub2), the translation elongation factor 1-alpha gene (tef1-α), and the DNA-directed RNA polymerase II second largest subunit (rpb2). Additionally, pathogenicity was assessed, successfully completing Koch's postulates on both detached Persian lime branches and certified 18-month-old Persian lime plants. Through multilocus molecular phylogenetic identification, pathogenicity, and virulence tests, five species were identified as causal agents: L. iraniensis, L. lignicola, L. mexicanensis, L. pseudotheobromae, and L. theobromae. This study demonstrates that in southern Mexico, at least five species of the genus Lasiodiplodia are responsible for dieback in Persian lime. Additionally, this is the first report of L. lignicola and L. mexicanensis as causal agents of the disease in citrus, indicating novel host interactions between species of Lasiodiplodia and C. latifolia.

Co-chaperonin GroES subunit exchange as dependent on time, pH, protein concentration, and urea

The discovery of a subunit exchange in some oligomeric proteins, implying short-term dissociation of their oligomeric structure, requires new insights into the role of the quaternary structure in oligomeric protein stability and function. Here we demonstrate the effect of pH, protein concentration, and urea on the efficiency of GroES heptamer (GroES7) subunit exchange. A mixture of equimolar amounts of wild-type (WT) GroES7 and its Ala97Cys mutant modified with iodoacetic acid (97-carboxymethyl cysteine or CMC-GroES7) was incubated in various conditions and subjected to isoelectric focusing (IEF) in polyacrylamide gel. For each sample, there are eight Coomassie-stained electrophoretic bands showing different charges that result from a different number of included mutant subunits, each carrying an additional negative charge. The intensities of these bands serve to analyze the protein subunit exchange. The protein stability is evaluated using the transverse urea gradient gel electrophoresis (TUGGE). At pH 8.0, the intensities of the initial bands corresponding to WT-GroES7 and CMC-GroES7 are decreased with a half-time of (23 ± 2) min. The exchange decreases with decreasing pH and seems to be strongly hindered at pH 5.2 due to the protonation of groups with pK ∼ 6.3, which stabilizes the protein quaternary structure. The destabilization of the protein quaternary structure caused by increased pH, decreased protein concentration, or urea accelerates the GroES subunit exchange. This study allows visualizing the subunit exchange in oligomeric proteins and confirms its direct connection with the stability of the protein quaternary structure.

DNA barcoding of epiphytic orchids for species level identification

ABSTRACT DNA barcoding has proven to be an effective technique for molecular-level identification in plants, including orchids which exhibit a great degree of genetic variability but show almost identical morphological features in closely related genera. The present study was conducted to evaluate the effectiveness of universal chloroplast gene markers, matK (maturase K gene) and rbcL (ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit) as DNA barcodes to identify Aerides multiflora and Rhynchostylis retusa at species level. The DNA sequences from different genotypes of both the orchid species collected from natural populations were compared with database sequences of orchid species using BLAST and maximum likelihood phylogenetic tree. For A. multiflora samples, the phylogenetic analysis revealed a highest of 99.4% sequence percentage alignment (for matK region) and 99.39% percent identity with sequences belonging to A. multiflora accessions. In the case of R. retusa, the matK region showed a highest sequence percentage alignment of 99.87% with the accessions of R. retusa, while the highest per cent identity for the rbcL region was 100% that matched with accessions of R. retusa, C. peduncularis and A. praemorsa. The study confirms the reliability of matK and rbcL markers for authentication of the two orchid species under the current investigation.

Cryptosporidium Infections in Neonatal Calves on a Dairy Farm.

This study was conducted with the aim of the molecular identification of the protozoan parasite Cryptosporidium spp. in calves in the early stage of their development on a dairy farm in Eastern Slovakia. Twenty-five Holstein and Holstein cross calves were included in the study and monitored from their birth to the fifth week of life (1-5 weeks). Fresh fecal samples were collected from the same group of calves each week, except during the fourth week, and with the exception of Sample 8. All samples were analyzed using the Ziehl-Neelsen staining method and coproantigen was tested using the ELISA test as the screening method. Using the ELISA method, the highest incidence of cryptosporidiosis was observed in the second week of life of the calves, while the antigen was detected in 21 (91.6%) calves. Using the Ziehl-Neelsen staining method, the highest incidence was also observed in the second week, with an incidence rate of 62.5%. Positive isolates confirmed by the ELISA test were molecularly characterized. The species and subtypes of Cryptosporidium in the positive isolates were identified using PCR and the sequence analysis of the small subunit of the ribosomal 18S RNA (ssu rRNA) and the 60 kDa glycoprotein (gp60) genes of the parasite. The sequence analysis of 29 isolates at the 18S rRNA loci confirmed the presence of two species-Cryptosporidium parvum and Cryptosporidium ryanae. Out of 29 isolates, 25 were assigned to the species C. parvum, with the gp60 locus identified as genotype IIaA17G1R1. Among the individual animal groups, calves are the most common reservoirs of the C. parvum zoonotic species. This disease has significant public health implications as contact with livestock and their feces and working with barn manure are major sources of infection, not only for other animals but also for humans.

First report of the barley root-knot nematode, Meloidogyne naasi Franklin, 1965 from Pennsylvania, with new host report.

Meloidogyne naasi Franklin, 1965, the barley root-knot nematode, was originally found in field crops such as cereals, grasses, and sugar beet (Beta vulgaris L.) in England and Wales, (Franklin,1965). This nematode is one of the most significant root-knot nematodes impacting grains in European countries (Santos et al. 2020). Among root-knot nematode species, M. naasi, exhibits a distinct preference for grasses, with documented impacts on turfgrasses leading to reduced growth and vigor (Skantar et al., 2023; Cook and Yeates, 1993). In September 2022, root-knot nematode females and second-stage juveniles (J2) were recovered from roots of fowl manna grass, Glyceria striata (Lam.) Hitchc., during a nematode survey on natural vegetation at the Allegheny National Forest (41°30'13.8"N 79°09'46.2"W). Second-stage juvenile specimens were recovered from soil using sugar centrifugal flotation (Jenkins, 1964). Small galls with egg masses were dissected from fowl manna grass roots originally collected from the surveyed areas. In parallel, five plants of non-infected fowl manna grass were placed in a pot in the greenhouse using naturally nematode-infested soil collected from the same forested area. Small galls and female specimens recovered from these plants were dissected and processed for further analyses. Female and J2 were fixed in 3% formaldehyde solution and processed to glycerin (Golden, 1990; Hooper, 1970). The specimens were examined by light microscopy, morphometric measurements, and molecular markers, which included the D2-D3 region of the large ribosomal subunit 28S, and the rDNA internal transcribed spacer region (ITS). The perennial pattern of five females analyzed morphologically were consistent to the patterns observed for M. naasi. The perennial patterns had coarse ridges on the cuticle in dorsal region forming broken irregular lines around anal and phasmid area. We also noted a prominent fold that covered some of the anus and showed a curved line between vulval slit and phasmids, typical of M. naasi. The area around the vulval area had a few or no striae except for a few lines radiating from the vulval slit as in the original description. Measurements of ten J2 had a body length ranged between 380 and 410 µm, stylet 11-13 µm, tail 50-70 µm long with a hyaline tail terminus between 12-22 µm in length, 4 lines in the lateral field, a and c ratio between 29.23-35.91 and 5.79-7.9 fitting the original description by Franklin, 1965 and others populations found in the USA (Skantar et al., 2023). The matrix codes for the female specimes are A32, B324, C3, D3 and for J2's A2, B21, C123, D1, E3, F12 (Subbotin et al., 2021). The amplified DNA fragments were sequenced, resulting in an 726 bp fragment flanked by the D2-D3 primers (PP097762), while for the ITS primers an 634 bp fragment was obtained (PP092043). Both generated sequences for the specimens collected in Pennsylvania revealed >99% similarity to M. naasi sequences deposited at GenBank, and therefore, validating the morphological analyses. Based on both morphological and molecular analyses the specimens collected in the state of Pennsylvania were identified as M.naasi. To our knowledge, this is the first report of this species from this state and being associated with naturally infected fowl manna grass.

Comparative plastome and mitogenome analyses indicate that the marine prasinophyte green algae Pycnococcus provasolii and Pseudoscourfieldia marina (Pseudoscourfieldiophyceae class nov., Chlorophyta) represent morphotypes of the same species.

The marine prasinophyte green algae Pycnococcus provasolii and Pseudoscourfieldia marina represent the only extant genera and known species of the Pycnococcaceae. However, their taxonomic status needs to be reassessed, owing to the very close relationship inferred from previous sequence comparisons of individual genes. Although Py. provasolii and Ps. marina are morphologically different, their plastid rbcL and nuclear small subunit rRNA genes were observed to be nearly or entirely identical in sequence, thus leading to the hypothesis that they represent distinct growth forms or alternate life-cycle stages of the same organism. To evaluate this hypothesis, we used organelle genomes as molecular markers. The plastome and mitogenome of Ps. marina UIO 007 were sequenced and compared with those available for two isolates of Py. provasolii (CCMP 1203 and CCAP 190/2). The Ps. marina organelle genomes proved to be almost identical in size and had the same gene content and gene order as their Py. provasolii counterparts. Single nucleotide substitutions and insertions/deletions were localized using genome-scale sequence alignments. Over 99.70% sequence identities were observed in all pairwise comparisons of plastomes and mitogenomes. Alignments of both organelle genomes revealed that Ps. marina UIO 007 is closer to Py. provasolii CCAP 190/2 than are the two Py. provasolii strains to one another. Therefore, our results are not consistent with the placement of Ps. marina and Py. provasolii strains into distinct genera. We propose a taxonomic revision of the Pycnococcaceae and the erection of a new class of Chlorophyta, the Pseudoscourfieldiophyceae.

Preparation and immunological activity evaluation of an intranasal protein subunit vaccine against ancestral and mutant SARS-CoV-2 with curdlan sulfate/O-linked quaternized chitosan nanoparticles as carrier and adjuvant

Chitosan and its derivatives are ideal nasal vaccine adjuvant to deliver antigens to immune cells. Previously, we successfully used a chitosan derivative, O-(2-Hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (O-HTCC), and a β-glucan derivative, curdlan sulfate (CS), to prepare a nanoparticle adjuvant CS/O-HTCC which could deliver ovalbumin to antigen presenting cells (APCs) through nasal inhalation. In this article, we used SARS-CoV-2 spike receptor binding domain (S-RBD) as the antigen and CS/O-HTCC nanoparticles as the adjuvant to develop a nasal mucosal protein subunit vaccine, CS/S-RBD/O-HTCC. The humoral immunity, cell-mediated immunity and mucosal immunity induced by vaccines were evaluated. The results showed that CS/S-RBD/O-HTCC could induce desirable immunization with single or bivalent antigen through nasal inoculation, giving one booster vaccination with mutated S-RBD (beta) could bring about a broad cross reaction with ancestral and different mutated S-RBD, and vaccination of the BALB/c mice with CS/S-RBD/O-HTCC containing S-RBD mix antigens (ancestral and omicron) could induce the production of binding and neutralizing antibodies against both of the two antigens. Our results indicate that CS/O-HTCC is a promising nasal mucosal adjuvant to prepare protein subunit vaccine for both primary and booster immunization, and the adjuvant is suitable for loading more than one antigen for preparing multivalent vaccines.

Prevalence and subtypes of Blastocystis in wild rodents from three provinces in China.

Blastocystis is one of the most critical intestinal protozoans in various hosts, including humans and mice. To determine the status of Blastocystis infection in wild rodents in China. A total of 344 faecal samples were collected from seven wild rodent species from three provinces, and the small subunit ribosomal RNA (SSU rRNA) genes of Blastocystis were amplified to determine their prevalence and subtypes. Of the 344 samples, 54 (15.70%) were detected as Blastocystis-positive. The prevalence of Blastocystis was 26.14% (40/153), 7.95% (7/88), and 6.80% (7/103) in wild rodents from Hunan Province, Yunnan Province, and Guangxi Province, respectively. The prevalence of Blastocystis in different wild rodent species varied from 0.00% (0/13) in Mus musculus to 40.00% (2/5) in Rattus rattus sladeni. The prevalence of Blastocystis in samples from the lake beach area (27.40%, 40/146) was significantly higher than in those from the mountain (6.80%, 7/103) and field regions (7.37%, 7/95). The prevalence in different seasons was 26.14% in summer (40/153), 7.95% in autumn (7/88), and 6.80% in winter (7/103). Moreover, a total of two Blastocystis subtypes were identified in the investigated wild rodents, including ST4 and ST5. The present study discovered the existence of Blastocystis infection in Rattus favipectus, Microtus fortis, Apodemus agrarius, Bandicota indica, Rattus rattus sladeni, and Rattus losea, expanding the host range of this parasite. The findings also demonstrate that wild rodents may be an important potential infection source for Blastocystis infection in humans and other animals.

SUPPLEMENTAL DESCRIPTION OF CABALLEROTREMA ANNULATUM (DIESING, 1850) OSTROWSKI DE NÚÑEZ AND SATTMANN, 2002 (DIGENEA: CABALLEROTREMATIDAE) FROM A NEW HOST (ELECTROPHORUS CF. VARII) AND LOCALITY (AMAZON RIVER, COLOMBIA) WITH PHYLOGENETIC ANALYSIS AND EMENDED GENERIC DIAGNOSIS.

Herein, we provide a supplemental description of Caballerotrema annulatum (Diesing, 1850) Ostrowski de Núñez and Sattmann, 2002 (Digenea: Caballerotrematidae Tkach, Kudlai, and Kostadinova, 2016) based on specimens collected from the intestine of an electric eel, Electrophorus cf. varii (Gymnotiformes: Gymnotidae) captured in the Amazon River (Colombia). This caballerotrematid can be differentiated from its congeners by the following combination of morphological features: body surface spines forming contiguous transverse rows, concentric (wrapping dorso-ventrally around body), distributing into posterior body half (vs. restricted to anterior body half in Caballerotrema brasiliensePrudhoe, 1960; indeterminate for Caballerotrema aruanenseThatcher, 1980 and Caballerotrema piscicola [Stunkard, 1960] Kostadinova and Gibson, 2001); head collar lacking projections (vs. having them in C. brasiliense, C. aruanense, and C. piscicola), narrow (head collar more narrow than maximum body width vs. the head collar being obviously wider than the body in C. brasiliense, C. aruanense, and C. piscicola); corner spines clustered (vs. corner spines distributing as 2 separated pairs in C. brasiliense, C. aruanense, and C. piscicola); pharynx approximately at level of the corner spines (vs. pharynx far anterior to corner spines in C. brasiliense, C. aruanense, and C. piscicola); and testes ovoid and nonoverlapping (C. aruanense; vs. sinuous and overlapping in C. brasiliense and C. piscicola). Based on our results, we revise the diagnosis of CaballerotremaPrudhoe, 1960 to include features associated with the shape and distribution of body surface spines, orientation and position of head collar spines, cirrus sac, seminal vesicle, oviduct, Laurer's canal, oötype, vitellarium, and transverse vitelline ducts. We performed Bayesian inference analyses using the partial large subunit ribosomal (28S) DNA gene. Our 28S sequence of C. annulatum was recovered sister to that of Caballerotrema sp. (which is the only other caballerotrematid sequence available in GenBank) from an arapaima, Arapaima gigas (Schinz, 1822) (Osteoglossiformes: Arapaimidae) in the Peruvian Amazon. Our sequence of C. annulatum comprises the only caballerotrematid sequenced tethered to a morphological description and a voucher specimen in a lending museum. The present study is a new host record and new locality record for C. annulatum. The phylogeny comprises the most resolved and taxon-rich evolutionary hypothesis for Echinostomatoidea published to date.

Recommendations on approving the name " Entomosporium", with a new species, E.dichotomanthes from China (Leotiomycetes, Drepanopezizaceae).

The phytopathogenic genus, Entomosporium can cause serious leaf diseases worldwide. Entomosporium has long been regarded as a synonym of Diplocarpon. However, different morphologies between Entomosporium and Diplocarpon make this doubtful. Based on morpho-phylogenetic analyses, the placement of the genus was re-evaluated in this study. The combined the internal transcribed spacer gene region (ITS) and the 28S large subunit ribosomal RNA gene region (LSU) phylogenetic analysis shows that Entomosporium is an independent clade within Drepanopezizaceae and formed a sister clade to the generic type Diplocarpon. Moreover, Hymenula and Pseudopeziza do not cluster in Drepanopezizaceae. We propose to resurrect the name Entomosporium, and exclude Hymenulacerealis and Pseudopezizamedicaginis from Drepanopezizaceae and propose to treat them under Ploettnerulaceae. A new species, E.dichotomanthes is also introduced from China based on morpho-molecular analyses which is associated with Dichotomanthestristaniicarpa.

Post-translational modifications of p65: state of the art.

P65, a protein subunit of NF-κB, is a widely distributed transcription factor in eukaryotic cells and exerts diverse regulatory functions. Post-translational modifications such as phosphorylation, acetylation, methylation and ubiquitination modulate p65 transcriptional activity and function, impacting various physiological and pathological processes including inflammation, immune response, cell death, proliferation, differentiation and tumorigenesis. The intricate interplay between these modifications can be antagonistic or synergistic. Understanding p65 post-translational modifications not only elucidates NF-κB pathway regulation but also facilitates the identification of therapeutic targets and diagnostic markers for associated clinical conditions.

Integrating bioinformatics and multiple machine learning to identify mitophagy-related targets for the diagnosis and treatment of diabetic foot ulcers: evidence from transcriptome analysis and drug docking.

Diabetic foot ulcers are the most common and serious complication of diabetes mellitus, the high morbidity, mortality, and disability of which greatly diminish the quality of life of patients and impose a heavy socioeconomic burden. Thus, it is urgent to identify potential biomarkers and targeted drugs for diabetic foot ulcers. In this study, we downloaded datasets related to diabetic foot ulcers from gene expression omnibus. Dysregulation of mitophagy-related genes was identified by differential analysis and weighted gene co-expression network analysis. Multiple machine algorithms were utilized to identify hub mitophagy-related genes, and a novel artificial neural network model for assisting in the diagnosis of diabetic foot ulcers was constructed based on their transcriptome expression patterns. Finally, potential drugs that can target hub mitophagy-related genes were identified using the Enrichr platform and molecular docking methods. In this study, we identified 702 differentially expressed genes related to diabetic foot ulcers, and enrichment analysis showed that these genes were associated with mitochondria and energy metabolism. Subsequently, we identified hexokinase-2, small ribosomal subunit protein us3, and l-lactate dehydrogenase A chain as hub mitophagy-related genes of diabetic foot ulcers using multiple machine learning algorithms and validated their diagnostic performance in a validation cohort independent of the present study (The areas under roc curve of hexokinase-2, small ribosomal subunit protein us3, and l-lactate dehydrogenase A chain are 0.671, 0.870, and 0.739, respectively). Next, we constructed a novel artificial neural network model for the molecular diagnosis of diabetic foot ulcers, and the diagnostic performance of the training cohort and validation cohort was good, with areas under roc curve of 0.924 and 0.840, respectively. Finally, we identified retinoic acid and estradiol as promising anti-diabetic foot ulcers by targeting hexokinase-2 (-6.6 and -7.2kcal/mol), small ribosomal subunit protein us3 (-7.5 and -8.3kcal/mol), and l-lactate dehydrogenase A chain (-7.6 and -8.5kcal/mol). The present study identified hexokinase-2, small ribosomal subunit protein us3 and l-lactate dehydrogenase A chain, and emphasized their critical roles in the diagnosis and treatment of diabetic foot ulcers through multiple dimensions, providing promising diagnostic biomarkers and targeted drugs for diabetic foot ulcers.

Transcription factor OsbZIP10 modulates rice grain quality by regulating OsGIF1.

Understanding and optimizing the process of grain filling helps the quest to maximize rice (Oryza sativa L.) seed yield and quality, yet the intricate mechanisms at play remain fragmented. Transcription factors (TFs) are major players in the gene networks underlying the grain filling process. Here, we employed grain incomplete filling (OsGIF1)/cell wall invertase 2, a key gene involved in grain filling, to explore its upstream TFs and identified a bZIP family TF, OsbZIP10, to be a transcriptional activator of OsGIF1. Rice grains of the knockouts of OsbZIP10 showed increased white-core rates but lower amylose content (AC), leading to better eating and cooking qualities in all genetic backgrounds investigated, though the impact of mutations in OsbZIP10 on grain weight depended on genetic background. Multi-omics analyses suggested that, in addition to OsGIF1, multiple genes involved in different biological processes contributing to grain filling were targeted by OsbZIP10, including OsAGPS1, a gene encoding the ADP-Glc pyrophosphorylase (AGPase) small subunit, and genes contributing to homeostasis of reactive oxygen species. Distinct genetic make-up was observed in OsbZIP10 between japonica and indica rice varieties, with the majority varieties of each subspecies belonging to two different haplotypes that were closely associated with AC. Overexpressing the haplotype linked to high-AC in the low-AC genetic background increased AC. Overall, this study sheds crucial light on the significance of the OsbZIP10-OsGIF1 module in the determination of rice grain quality, offering a potential avenue for genetic engineering of rice to produce seeds with tailored attributes.

Fusarium kamalianum, a new species of Fusarium from India from ornamental Chamaedorea seifrizii

A novel species, Fusarium kamalianum, belonging to the Fusarium oxysporum species complex (FOSC), is established to accommodate a fungal pathogen that was isolated from infected Chamaedorea seifrizii (Bamboo palm). The identification of this pathogenic strain was determined based on the cultural features, asexual-morphs, and phylogenetic analysis based on translation elongation factor 1-alpha (tef-1α), RNA polymerase second largest subunit (rpb2), internal transcribed spacer region (ITS), 28S large subunit (LSU), and β-tubulin (tub2) sequence data. The detailed taxonomic descriptions, cultural, microscopic images, and phylogenetic trees are presented for the introduced taxon. The sequencing data of this isolate exhibited substantial distinctness from the previously discovered species of Fusarium and created a unique distinct clade. This is the first study that reports infection in Chamaedorea seifrizii plants caused by Fusarium species.

Sclerotinia rot of Zephyranthes candida caused by Sclerotinia sclerotiorum and Sclerotinia minor.

Sclerotinia rot is a serious disease that occurs on Zephyranthes candida. A thorough understanding of the pathogenic fungal species and understanding the biological characteristics are important for controlling sclerotinia. Fungal strains were isolated from the diseased leaves of Z. candida through tissue isolation. Koch's hypothesis screened pathogenic strains by pathogenicity of healthy leaves, including re-isolation and identification. A multigene phylogenetic tree was constructed by extracting genomic DNA from pathogenic strains and measuring the nucleotide sequences at four sites, including the internal transcribed spacer (ITS), RNA polymerase II second largest subunit (RPB2), glyceraldehyde-3-phosphate dehydrogenase (G3PDH), and heat shock protein 60 (HSP60). Morphological characteristics of the fungal structures were evaluated through microscopic analysis. The growth of pathogens was observed and recorded under different pH, different temperatures, different carbon sources and different nitrogen sources to clarify their biological characteristics. Representative strains D7, D13, X4, and X15 infected Z. candida and caused sclerotinia rot. At the beginning of the culture, white flocculent fungal hyphae appeared on the potato dextrose agar (PDA) medium, and black spherical to irregular-shaped sclerotia appeared at the edge of the colony after 7 days. The diameter of the sclerotia was 2.4-8.6 mm and 0.4-0.9 mm, respectively. One sclerotium was able to germinate from 1 to 5 apothecia. Ascus were cylindrical or spindle-shaped, with a size of 110.0-120.0 × 9.2-11.6 μm. One ascus contained eight colorless, oval ascospores, with a size of 8.4-12.0 × 4.5-5.5 μm. Based on the phylogenetic tree constructed with the gene sequences for ITS, G3PDH, HSP60, and RPB2, D7 and D13 shared 99% homology with sclerotinia sclerotiorum, whereas X4 and X15 shared 99% homology with sclerotinia minor. S. sclerotiorum growth was more suitable when the culture temperature was 15°C-25°C, pH 5.0, carbon source was maltose and nitrogen source was yeast powder. S. minor growth was more suitable when the culture temperature was 15°C, pH 5.0, the carbon source was glucose, and the nitrogen source was yeast powder. The results identified the pathogens as S. sclerotiorum and S. minor. To the best of our knowledge, this is the first report of S. sclerotiorum and S. minor causing sclerotinia rot on Z. candida. We herein aimed to identify the causal agent of sclerotinia rot of Z. candida in China based on morphological characteristics, molecular identification, and pathogenicity tests. Performed the experiments on the biological characteristics, to understand the law of disease occurrence. We also evaluated methods for the effective control of this disease. Our findings provide support for further studies on the pathogenesis mechanism of sclerotinia rot.