Intergenic Spacer Research Articles

Strategies to Overcome Antimicrobial Resistance in Nosocomial Infections, A Review and Update.

Nosocomial infections, also known as healthcare-associated infections, are a significant global concern due to their strong association with high mortality and morbidity in both developed and developing countries. These infections are caused by a variety of pathogens, particularly the ESKAPE group of bacteria, which includes the six pathogens Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. These bacteria have demonstrated noteworthy resistance to different antibiotics. Antimicrobial resistance mechanisms can manifest in various forms, including restricting drug uptake, modifying drug targets, inactivating drugs, active drug efflux, and biofilm formation. Accordingly, various strategies have been developed to combat antibiotic-resistant bacteria. These strategies encompass the development of new antibiotics, the utilization of bacteriophages that specifically target these bacteria, antimicrobial combination therapy and the use of peptides or enzymes that target the genomes or essential proteins of resistant bacteria. Among promising approaches to overcome antibiotic resistance, the CRISPR/Cas system stands out and offers many advantages. This system enables precise and efficient editing of genetic material at specific locations in the genome. Functioning as a bacterial "adaptive immune system," the CRISPR/Cas system recognizes, degrades, and remembers foreign DNA sequences through the use of spacer DNA segments that are transcribed into CRISPR RNAs (crRNA). This paper has focused on nosocomial infections, specifically the pathogens involved in hospital infections, the mechanisms underlying bacterial resistance, and the strategies currently employed to address this issue. Special emphasis has been placed on the application of CRISPR/Cas technology for overcoming antimicrobial resistance.

Clinical Applicability of Microbiota Sampling in a Subfertile Population: Urine versus Vagina.

The urogenital microbiota is increasingly gaining recognition as a significant contributor to reproductive health. Recent studies suggest that microbiota can serve as predictors for fertility treatment outcomes. Our objective was to investigate the degree of similarity in microbial composition between patient-collected urine and vaginal samples in a subfertile population. We enrolled women of reproductive age (20-44 years) diagnosed with subfertility and requiring in vitro fertilization (IVF) or IVF with intracytoplasmic sperm injection (IVF-ICSI) treatment. They self-collected both mid-stream urine samples and vaginal swabs before commencing the IVF or IVF-ICSI procedure. All samples were analysed using the intergenic spacer profiling (IS-pro) technique, a rapid clinical microbiota analysis tool. The main outcome measures were the degree of similarity of microbial composition between the two different, but simultaneously collected, samples. Our findings revealed a high correlation (R squared of 0.78) in microbiota profiles between paired urine and vaginal samples from individual patients. Nevertheless, the urinary microbiota profiles contained fewer species compared to the vaginal microbiota, resulting in minor but distinguishable differences. Furthermore, different subfertility diagnoses appeared to be associated with differences in microbial profiles. A noteworthy observation was the exclusive presence of Escherichia coli (E. coli) in both samples of women diagnosed with male factor subfertility. In conclusion, since urinary microbiota profiles seem to represent a diluted version of the vaginal microbiota, vaginal microbiome sampling to predict fertility treatment outcome seems preferable. To enhance the success of fertility treatments, further research is needed to gain deeper insights into a putative causal role of microbiota in the mechanisms of subfertility.

First Report of Rugonectria rugulosa Causing Dieback of Prunus salicina "Banbianhong" in China.

Banbianhong (Prunus salicina) is a valuable fruit crop cultivated in the southwestern regions of China for its high quality fruit and substantial economic benefits (Wang et al. 2021). In July 2023, dieback disease was observed in Banbianhong in Suijiang County, Zhaotong, Yunnan Province, with an incidence of up to 10%. The disease caused brownish nodules in the cortex of the infected plant, leading to dry cracking, and exposing grayish-brown xylem, which caused the plant to wither and die. Upon longitudinal cutting of twigs, lesions were evident and characterized by browning and necrosis of the cauline bundle and pith within the tissue. The diseased tissues were sectioned into 2 mm2 pieces and surface disinfected with 70% ethanol and 1% NaClO and rinsed with double distilled water. The tissue fragments were plated onto potato dextrose agar (PDA), and incubated for 7 days at 28°C. A total of six isolates were obtained, and two isolates (WG2-1 and WG2-3) were selected for morphological, molecular identification and pathogenicity testing. On PDA, the colony diameter of WG2-1 and WG2-3 were recorded as 5.50±0.56 mm/day and 6.08±0.37 mm/day, respectively. The microconidia of both isolates were zero-septate, smooth, greenish, subglobose to ellipsoidal, and measured 5 to 10 × 1.5 to 3 µm (n = 50) in size. However, macroconidia were not observed. The internal transcribed spacer rDNA (ITS) and β-tubulin 2 (TUB2) genes were amplified using the primer pairs ITS1/4 (White et al. 1989) and Bt2a/2b (Glass and Donaldson 1995). BLAST analysis of the obtained ITS sequences (accession Nos. PP581792) and TUB2 sequences (accession Nos. PP2815212) sequences showed 100% identity with Rugonectria rugulosa (accession nos. MG991753 for ITS and KM232007 for TUB2). Based on these characteristics, the isolates have been identified as R. rugulosa and have been deposited in the Agricultural Environment and Resource Research Institute plant pathogen lab at Yunnan Academy of Agricultural Sciences. In this study, Pathogenicity tests were conducted on one-year-old Prunus salicina "Banbianhong" twigs to complete Koch's postulates. The twigs were wounded using a 1-mm sterile corking borer and 14-day-old mycelium plugs of isolate (WG2-1) were inoculated and covered with sealing films. Sterile PDA plugs were placed into the wounds of control twigs. Eighteen healthy twigs were inoculated with isolates or PDA plugs (n = 6 twigs/treatment, the size of the twigs: 8 cm length and diameter about 0.7 cm, with three replications) and were placed in a closed sterile tray with the wet filter papers in a thermostatic incubator (type, HYC-260) at 28°C, respectively. The twigs inoculated with the isolates exhibited brown lesions site of inoculation, whereas the controls remained asymptomatic. R. rugulosa was successfully reisolated from the lesions but not from these control wounds and was identified by the methods described above. R. rugulosa has been reported to be associated with the die-back disease of Falcataria moluccana in China (Wang et al. 2019). The occurrence and spread of R. rugulosa in growing areas of Prunus salicina "Banbianhong" in Suijiang county have caused severe economic losses. To our knowledge, this is the first report of the die-back disease of Prunus salicina "Banbianhong" caused by R. rugulosa. These results confirm the pathogenicity of R. rugulosa in Prunus salicina "Banbianhong" and provide valuable insights for developing disease management and prevention strategies.

Metarhizium caribense sp. nov., a Novel Species of Entomopathogenic Metarhizium Fungi Associated with Weevils Impairing Coffee, Sugar Cane and Sweet Potato Cultivation.

(1) Background: Insect pathogenic fungi of the genus Metarhizium are under study and in application as highly solicited, more eco-system friendly substitutes for chemical insecticides in many countries and in different agricultural contexts. In Cuba and Florida, Metarhizium strains have previously been isolated from economically important coffee and sugar cane pests. (2) Methods: Unambiguous species delineation within the Metarhizium anisopliae species complex is methodologically challenging. Recently, a species-discriminating PCR approach has been developed based on ribosomal intergenic spacer (rIGS) sequences that covered the prominent four "PARB" species within the complex. This approach is combined here with further genetic markers and is extended to a further species. (3) Results: Metarhizium isolates from Cuba, found to be more naturally associated with the coffee berry borer, Hypothenemus hampei, were morphologically, microscopically and molecular taxonomically characterized. Multilocus sequence analysis based on 5TEF, MzIGS3 and rIGS markers delineated these weevil-associated strains from all previously established Metarhizium species. (4) Conclusions: The isolates under study represent a new fungal taxon proposed to be designated Metarhizium caribense. The rIGS-based species-discriminating diagnostic PCR is a suitable tool for the identification of new Metarhizium species and can be productively combined to approaches using further genetic markers.

Unique findings of <i>Phoma</i>-like fungi associated with soybean

Ascochyta leaf blight of soybean is a widespread disease caused by several closely related Phoma-like species, this disease often leads to significant crop losses. Among Phoma-like species from Didymellaceae family, the most frequently associated with symptomatic soybean tissues are species of the genera Boeremia and Didymella. Currently reliable species identification in Didymellaceae relies on polyphasic approach based on consolidated species concept and combined molecular phylogenetic, micromorphological and cultural features. At least three loci are commonly used for reconstruction of the molecular phylogeny of Didymellaceae: internal transcribed spacer (ITS) of the ribosomal DNA, partial RNA-polymerase II gene (rpb2), and β-tubulin (tub2). As a result of long-term phytosanitary monitoring of soybean crops, soybean leaves with symptoms of Ascochyta blight were collected from major soybean producing areas of Russia. From surface sterilized plant tissues more than 100 isolates of Phoma-like fungi were obtained and stored in the collection of pure cultures of the Laboratory of Mycology and Phytopathology (MF, All-Russian Institute of Plant Protection). Most of them, as a result of multilocus phylogenetic analysis, were identified as Boeremia and Didymella species. Eight isolates were identified as species of other genera, suspected to be rare findings. The aim of this study was to identify these eight isolates based on multilocus phylogenetic analysis, as well micromorphological, cultural, and pathogenicity data. Multilocus phylogenetic analysis has resulted in identification of all eight isolates to species level. Single isolate from the Ryazan region was Neoascochyta graminicola. Three other from three different districts of the Amur region were Remotididymella capsici. Two isolates from the Primorskiy territory and Amur region were Stagonosporopsis heliopsidis. Another two from two districts of the Amur region were S. stuijvenbergii. Pathogenicity tests have resulted in conclusion, that all studied isolates were not pathogenic for soybean leaves. Probably, these Phoma-like species are associate with soybean as saprophytes or endophytes. For all these Phoma-like species Glycine max was detected as substrate for the first time. Neoascochyta graminicola is widespread in Europe in association with Poaceae plants. There are only two findings of Remotididymella capsici in the world, both from leaves of Capsicum annuum. First finding was made in the former USSR in 1977 and was identified based on only morphological features. Second findings was collected in the Fiji and verified with multilocus phylogenetic analysis. Stagonosporopsis heliopsidis isolates were revealed in the USA, Canada, Netherlands and Russia and this fungus was believed to be specific for Asteraceae plants. Isolates of Stagonosporopsis stuijvenbergii are known only from soil in the Netherlands. Thus, such species as Neoascochyta graminicola and Stagonosporopsis stuijvenbergii were revealed in the Russia for the first time. Studied Remotididymella capsici isolates were first confirmed findings of this fungus in Russia. Additionally to detailed phylogenetic data, the manuscript is supplement with a detailed description of the cultural and micromorphological features of all species.

Molecular evidence for the origin of the little-known hybrid Potamogeton ×vilnensis

A combined study of morphology, stem anatomy, and the genetic markers (ITS1-ITS4 region of nuclear ribosomal DNA and rpl20-rps12 intergenic spacer of chloroplast DNA) was used to verify the identity of hybrid Potamogeton ×vilnensis Galinis previously recognized as P. gramineus × P. praelongus. Authentic herbarium material and newly collected specimens morphologically similar to the hybrid from the locus classicus (SE Lithuania) were used for comparative morphological, anatomical and molecular analyses. The study proved the paternity of P. lucens and P. praelongus and excluded the involvement of P. gramineus in hybridization and identity with P. ×angustifolius. This hybrid formula is shared with plants recorded in the Czech Republic and Denmark under the name P. ×jutlandicus Zalewska-Galosz. The site in Lithuania, known since 1961, is the first natural habitat of long-lived hybrid between P. lucens and P. praelongus whose origin has been confirmed by molecular markers. The scarce records of this taxon indicate that the knowledge of the hybrid species of Potamogeton and their distribution is insufficient and that more extensive regional studies, including molecular methods, are needed.

Diversity of bartonellae in mites (Acari: Mesostigmata: Macronyssidae and Spinturnicidae) of boreal forest bats: Association of host specificity of mites and habitat selection of hosts with vector potential.

Research into various bacterial pathogens that can be transmitted between different animals and may have zoonotic potential has led to the discovery of different strains of Bartonella sp. in bats and their associated ectoparasites. Despite their enormous species diversity, only a few studies have focussed on the detection of bacterial pathogens in insectivorous bats of boreal forests and their associated Macronyssidae and Spinturnicidae mites. We collected and molecularly analysed mite samples from forest-dwelling bat species distributed all along the boreal belt of the Palearctic, from Central Europe to Far East. Ectoparasitic mites were pooled for DNA extraction and DNA amplification polymerase chain reaction (PCRs) were conducted to detect the presence of various bacterial (Anaplasmataceae, Bartonella sp., Rickettsia sp., Mycoplasma sp.) and protozoal (Hepatozoon sp.) pathogens. Bartonella sp. DNA was detected in four different mite species (Macronyssidae: Steatonyssus periblepharus and Spinturnicidae: Spinturnix acuminata, Sp. myoti and Sp. mystacinus), with different prevalences of the targeted gene (gltA, 16-23S ribosomal RNA intergenic spacer and ftsZ). Larger pools (>5 samples pooled) were more likely to harbour Bartonella sp. DNA, than smaller ones. In addition, cave-dwelling bat hosts and host generalist mite species are more associated with Bartonella spp. presence. Spinturnicidae mites may transmit several distinct Bartonella strains, which cluster phylogenetically close to Bartonella species known to cause diseases in humans and livestock. Mites with ubiquitous presence may facilitate the long-term maintenance (and even local recurrence) of Bartonella-infestations inside local bat populations, thus acting as continuous reservoirs for Bartonella spp in bats.

Prevalence and genetic diversity of Bartonella spp. in wild small mammals from South Africa.

Bartonella spp. are intracellular bacteria associated with several re-emerging human diseases. Small mammals play a significant role in the maintenance and spread of Bartonella spp. Despite the high small mammal biodiversity in South Africa, there is limited epidemiological information regarding Bartonella spp. in these mammals. The main aim of this study was to determine the prevalence and genetic diversity of Bartonella spp. from wild small mammals from 15 localities in 8 provinces of South Africa. Small mammals (n = 183) were trapped in the Eastern Cape, Free State, Gauteng, Limpopo, Mpumalanga, Northern Cape, North West, and Western Cape provinces of South Africa between 2010 and 2018. Heart, kidney, liver, lung, and spleen were harvested for Bartonella DNA screening, and prevalence was determined based on the PCR amplification of partial fragments of the 16S-23S rRNA intergenic spacer (ITS) region, gltA, and rpoB genes. Bartonella DNA was detected in Aethomys chrysophilus, Aethomys ineptus, Gerbillurus spp., Lemniscomys rosalia, Mastomys coucha, Micaelamys namaquensis, Rhabdomys pumilio, and Thallomys paedulcus. An overall prevalence of 16.9% (31/183, 95% CI: 12.2%-23%) was observed. Bartonella elizabethae, Bartonella grahamii, and Bartonella tribocorum were the zoonotic species identified, while the remaining sequences were aligned to uncultured Bartonella spp. with unknown zoonotic potential. Phylogenetic analyses confirmed five distinct Bartonella lineages (I-V), with lineage IV displaying strong M. coucha host specificity. Our results confirm that South African wild small mammals are natural reservoirs of a diverse assemblage of Bartonella spp., including some zoonotic species with high genetic diversity, although prevalence was relatively low.IMPORTANCESmall mammals play a significant role in the maintenance and spread of zoonotic pathogens such as Bartonella spp. Despite the high small mammal biodiversity in southern Africa including South Africa, there is limited epidemiological information regarding Bartonella spp. in these mammals across the country. Results from our study showed the liver and spleen had the highest positive cases for Bartonella spp. DNA among the tested organs. Bartonella elizabethae, B. grahamii, and B. tribocorum were the three zoonotic species identified and five distinct Bartonella lineages (I-V) were confirmed through phylogenetic analyses. To the best of our knowledge, this study presents the first extensive nuclear diversity investigation of Bartonella spp. in South African small mammals in South Africa.

Novel Approaches to Mortierella alpina Identification and Arachidonic Acid Production Optimization.

Arachidonic acid (ARA) is an integral constituent of cell structures and is instrumental for the nervous, muscular, and immune systems' functions. The sore need for this nutrient may be fulfilled via production based on the fungus Mortierella alpina. The identity of the M. alpina culture obtained from Assiut University, Egypt, was confirmed based on internal transcribed spacer DNA barcoding and elongation enzyme RNA sequencing. Liquid media glucose and peptone as carbon and nitrogen sources, respectively, and diverse micronutritional factors were adjusted for optimal biomass and ARA production. Shake flask cultivation at 25 °C for 7 days produced around 0.570 g of ARA per liter of culture. M. alpina treatment using mutagen 5-fluorouracil and octyl gallate-supplemented glucose-yeast-agar screening plates and shake-flask incubation at 25 °C, then at 20 °C, followed by aging at 10 °C, led to >3 g ARA/liter culture, a yield considered suitable for potential commercial production.

First Report of Powdery mildew caused by Golovinomyces ambrosiae on Bidens pilosa in Guangdong Province, China.

Bidens pilosa L., an annual herbaceous plant with a wide distribution, possesses novel medicinal properties. In January 2021, a powdery mildew disease outbreak was documented on B. pilosa plants located in the roadside areas in Shenzhen, Guangdong Province, China, with 60 to 80% disease incidence. Initial symptoms manifested as small, irregular white powdery patches, primarily on the adaxial surfaces of leaves. Subsequently, the colonies expanded, forming coalescent colonies that spread across the leaves, petioles, and stems, eventually leading to the distortion and senescence of leaves. Hyphae are hyaline, flexuous to straight, septate, with thin walls and a width ranging from 2 to 8 μm. Hyphal appressoria are nipple-shaped. Conidophores are erect or slightly flexuous, ranging from 80 to 150 µm in length and 12 to 18 µm in width (n = 30). Typically, these conidophores bear chains of 2 to 5 immature conidia, displaying a sinuate outline. Foot-cells, located at the base of conidophores, are cylindrical and erect, approximately 33 to 100 µm in length and 6 to 10 µm in width (n = 30). Conidia are hyaline, ellipsoid-ovoid to barrel-shaped, and lack fibrosin bodies. Primary conidia are ellipsoid-ovoid in shape, characterized by a rounded apex and a subtruncate base, 25 to 40 µm × 15 to 22 µm in width. Secondary conidia are barrel-shaped with truncate or subtruncate ends, 27 to 35 µm × 15 to 20 µm in width. Germ tubes exhibit a longitubus pattern and are prominently produced at the perihilar or apical region of the conidia. No chasmothecia were observed in the collected samples. In order to conduct a molecular-level identification, mycelium and conidia were collected from B. pilosa leaves. Genomic DNA was subsequently extracted from these samples. The internal transcribed spacer (ITS), intergenic spacer (IGS) and beta-tubulin (tub2) sequences were performed using primer pairs ITS1/ITS4, IGS-12a/NS1R, and tub2, respectively (Carbone and Kohn 1999; Scholin et al. 1994; White et al.,1990). A 568-bp ITS, a 366-bp IGS, and a 354-bp tub2 sequences (GenBank accession nos. OR647592, OR978282 and OR978283) were obtained. The ITS sequence exhibited over 99.6% similarity to Golovinomyces ambrosiae (MT929773) and G. cichoracearum (MH590731). The IGS sequence displayed 100% similarity to G. ambrosiae (MK383490) and G. ambrosiae (OK349420). The tub2 sequence displayed 100% similarity to G. ambrosiae (MW981257) and G. ambrosiae (MW981256). Phylogenetic analysis of IGS, ITS and tub2 also grouped obtained sequences within the G. ambrosiae complex. Based on the analysis of morphological characteristics and sequence identity, the pathogen was identified as G. ambrosiae. In order to satisfy Koch's postulates, an infected leaf was carefully pressed onto leaves of six healthy young B. pilosa plants, each grown in a separate pot. Additionally, a control group consisted of six non-inoculated plants. All plants were placed in a greenhouse: 25°C, 14/10-h light/dark photoperiod, and relative humidity 50%. After 10 days, the inoculated leaves exhibited powdery mildew colonies similar to those observed in the original infected plants. At 16 days, the inoculated leaves exhibited discoloration and premature leaf drop. The pathogenicity test was conducted twice. Microscopic observation and sequencing confirmed that isolated fungus was identical to the original pathogen. G. ambrosiae has previously been documented on B. pilosa in Fuzhou, Fujian Province, China (Mukhtar et al., 2022). However, to the best of our knowledge, this study represents the first report of powdery mildew caused by G. ambrosiae on B. pilosa in Shenzhen, Guangdong Province, China.

First report of anthracnose caused by Colletotrichum fructicola on winter jasmine (Jasminum nudiflorum) in China.

Winter jasmine (Jasminum nudiflorum Lindl.) is a medium-sized, deciduous shrub native to China that has become a popular choice among gardeners and landscapers. In 2020 to 2021, symptoms of anthracnose including brown necrotic spots, enlarged irregular lesions and leaf blight were observed on leaves of 20 winter jasmine shrubs in a public garden (22°34'58'' N; 113°56'23'' E) in Shenzhen, China, and with an estimated disease incidence of 65%. Tissues samples (6 × 6 mm2) surrounding the necrotic spots were surface sterilized with 75% ethanol for 30 s, followed by 2% NaClO for 1 min, then rinsed with sterile water for three times and dried with sterile filter paper. Tissues were placed on potato dextrose agar (PDA) medium and incubated at 25℃. After 3 to 7 d, pure cultures were obtained by transferring hyphal tips to new plates and 32 isolates producing Colletotrichum-like colonies were obtained from 40 tissues (isolation frequency=32/(4×10)=80%). Three representative isolates YCH09, YCH23 and YCH32 were selected for further study. Three selected isolates were identical in morphological characteristics. Colonies on PDA after 5 d at 25℃ were white to gray with cottony mycelia and grayish-white on the underside of the culture. Conidia (n = 60) measured 15.4 ± 1.1 µm (13.0 to 17.1 µm) in length and 5.4 ± 0.3 µm (4.9 to 6.0 µm) in width and were hyaline, single-celled, cylindrical with rounded ends. Appressoria (n = 15) measured 7.1 ± 0.1 µm (5.3 to 8.9 µm) in length and 5.2 ± 0.2 µm (4.1 to 6.2 µm) in width and were brown to dark brown, ovoid. These morphological features were aligned with those of Colletotrichum spp. (Weir et al. 2012). Sequences of five genetic markers of representative isolates YCH09, YCH23 and YCH32 including the rDNA internal transcribed spacer region, chitin synthase, partial actin, β-tubulin 2 and Apn2-Mat1-2 intergenic spacer and partial mating type (Mat1-2) region were 99.3 to 100% identical to the ex-type isolate of C. fructicola strain ICMP 18581 (Zhang et al., 2020). From the maximum likelihood phylogenetic tree which was constructed based on concatenated sequences, three representative isolates (YCH09, YCH23 and YCH32) were clustered with other isolates of C. fructicola. The above morphological and molecular characteristics suggest that causal agent was C. fructicola. Pathogenicity was tested using a whole-plant assay. Five healthy plants were inoculated by spraying a conidial suspension (1.5×104 conidia/ml; 20 ml per plant) of the isolate YCH23 onto the foliage (Marshall et al., 2023). Three noninoculated control plants were sprayed with sterile water. All plants were placed in a greenhouse at 25±2℃ with approximately 75% relative humidity. Yellow lesions appeared on leaves of inoculated plants as early as 4 days after inoculation (DAI), and irregularly shaped brown spots similar to those observed in the field were formed on 10 DAI. Noninoculated plants remained asymptomatic. Colletotrichum isolates resembling morphological characters of YCH23 were reisolated from all inoculated plants, then identified as C. fructicola by DNA sequence analysis. C. fructicola is a well-known fungus causing anthracnose on more than 63 plant species including agricultural and horticultural plants worldwide (Talhinhas and Baroncelli, 2021). To our knowledge, this is the first report of C. fructicola infecting J. nudiflorum plants in China. Since its potential risk to other horticultural plant species, precautions may be necessary to minimize the spread of this fungi.

Insights into early animal evolution from the genome of the xenacoelomorph worm Xenoturbella bocki.

The evolutionary origins of Bilateria remain enigmatic. One of the more enduring proposals highlights similarities between a cnidarian-like planula larva and simple acoel-like flatworms. This idea is based in part on the view of the Xenacoelomorpha as an outgroup to all other bilaterians which are themselves designated the Nephrozoa (protostomes and deuterostomes). Genome data can provide important comparative data and help understand the evolution and biology of enigmatic species better. Here, we assemble and analyze the genome of the simple, marine xenacoelomorph Xenoturbella bocki, a key species for our understanding of early bilaterian evolution. Our highly contiguous genome assembly of X. bocki has a size of ~111 Mbp in 18 chromosome-like scaffolds, with repeat content and intron, exon, and intergenic space comparable to other bilaterian invertebrates. We find X. bocki to have a similar number of genes to other bilaterians and to have retained ancestral metazoan synteny. Key bilaterian signaling pathways are also largely complete and most bilaterian miRNAs are present. Overall, we conclude that X. bocki has a complex genome typical of bilaterians, which does not reflect the apparent simplicity of its body plan that has been so important to proposals that the Xenacoelomorpha are the simple sister group of the rest of the Bilateria.

The complete chloroplast genome of Rhododendronambiguum and comparative genomics of related species.

Rhododendron Linnaeus, 1753, the largest genus of woody plants in the Northern Hemisphere, includes some of the most significant species in horticulture. Rhododendronambiguum Hemsl, 1911, a member of subsection Triflora Sleumer 1947, exemplifies typical alpine Rhododendron species. The analysis of the complete chloroplast genome of R.ambiguum offers new insights into the evolution of Rhododendron species and enhances the resolution of phylogenetic relationships. This genome is composed of 207,478 base pairs, including a pair of inverted repeats (IRs) of 47,249 bp each, separated by a large single-copy (LSC) region of 110,367 bp and a small single-copy (SSC) region of 2,613 bp. It contains 110 genes: 77 protein-coding genes, 29 tRNAs, four unique rRNAs (4.5S, 5S, 16S, and 23S), with 16 genes duplicated in the IRs. Comparative analyses reveal substantial diversity in the Rhododendron chloroplast genome structures, identifying a fourth variant pattern. Specifically, four highly divergent regions (trnI-rpoB, ndhE-psaC, rpl32-ndhF, rrn16S-trnI) were noted in the intergenic spacers. Additionally, 76 simple sequence repeats were identified. Positive selection signals were detected in four genes (cemA, rps4, rpl16, and rpl14), evidenced by high Ka/Ks ratios. Phylogenetic reconstruction based on two datasets (shared protein-coding genes and complete chloroplast genomes) suggests that R.ambiguum is closely related to R.concinnum Hemsley, 1889. However, the phylogenetic positions of subsection Triflora Pojarkova, 1952 species remain unresolved, indicating that the use of complete chloroplast genomes for phylogenetic research in Rhododendron requires careful consideration. Overall, our findings provide valuable genetic information that will enhance understanding of the evolution, molecular biology, and genetic improvement of Rhododendron spieces.

Phylogenetic and morphological re-evaluation of Camptophora.

The genetic variety and habitats of Camptophora species, generally known as black yeast, have not been clarified. In this study, we re-evaluated Camptophora based on morphological observations and phylogenetic analyses. Because prior investigations on Camptophora only included a few strains/specimens, 24 Camptophora-related strains were newly obtained from 13 leaf samples of various plant species to redefine the genetic and species concepts of Camptophora. Their molecular phylogenetic relationships were examined using small subunit nuclear ribosomal DNA (nSSU, 18S rDNA), the internal transcribed spacer (ITS) rDNA operon, the large subunit nuclear ribosomal DNA (LSU, 28S rDNA), β-tubulin, the second largest subunit of RNA polymerase II (rpb2), and mitochondrial small subunit DNA (mtSSU). Single- and multi-locus analyses using nSSU-ITS-LSU-rpb2-mtSSU revealed a robust phylogenetic relationship among Camptophora species within Chaetothyriaceae. Camptophora species could be distinguished from other chaetothyriaceous genera by their snake-shaped conidia with microcyclic conidiation and loosely interwoven mycelial masses. Based on the results of phylogenetic analyses, two undescribed lineages were recognized, and Ca. schimae was excluded from the genus. ITS sequence comparison with environmental DNA sequences revealed that the distribution of the genus is restricted to the Asia-Pacific region. Camptophora has been isolated or detected from abrupt sources, and this was attributed to its microcycle. The mechanisms driving genetic diversity within species are discussed with respect to their phyllosphere habitats.

Genetic comparison among some cultivars of Catharanthus roseus (L.) G. Don. using three intergenic spacers of the chloroplast genome

Abstract. Susanto AH, Dwiati M. 2024. Genetic comparison among some cultivars of Catharanthus roseus (L.) G. Don. using three intergenic spacers of the chloroplast genome. Biodiversitas 25: 2999-3007. Catharanthus roseus (L.) G. Don belongs to the Apocynaceae family and is recognized as both an ornamental plant and a medicinal herb. By means of both hybridization and mutation breeding, numerous cultivars of C. roseus have been developed over time. This encourages the necessity for molecular characterization to establish a DNA barcode for each cultivar, particularly in relation to its potential as a medicinal herb. Therefore, this study aimed to genetically compare some cultivars of C. roseus based on three Intergenic Spacers (IGS) of the chloroplast genome, namely trnQ-rps16, rps16-trnK, and trnL-rpl32. Ten samples of C. roseus were randomly collected from around Purwokerto City, Central Java, Indonesia, representing cultivars with various corolla and flower eye colors. Universal primers were used to amplify the three molecular markers. The results showed that some insertion-deletions (indels) with rps16-trnK and trnL-rpl32 were observed, indicating some potential barcodes for C. roseus cultivars, following pharmacological analysis. On the other hand, very little genetic difference among samples was found with trnQ-rps16. It can be concluded that some other molecular markers, specifically those more closely related to the expression of flower color in C. roseus, should be explored to obtain candidate DNA barcodes in assisting the authentication of medicinal herbs.

Response of Soil Fungal Community to Reforestation on Shifting Sand Dune in the Horqin Sandy Land, Northeast China.

Reforestation of native shrub on shifting sand dunes has been widely used for desertification control in semi-arid grassland in Northeast China. Previous studies have confirmed that plantation establishment facilitates fixing sand dunes, restoring vegetation, and improving soil properties, but very few have focused on the response of the soil fungal community. In this study, a chronosequence of Caragana microphylla (CM) shrub sand-fixation plantations (8-, 19-, and 33-year-old), non-vegetated shifting sand dunes (0 years), and adjacent natural CM forests (NCFs; 50-year-old) in the Horqin sandy land were selected as experimental sites. Soil properties including enzymatic activities were determined, and the composition and structure of the soil fungal community were investigated using the Illumina MiSeq sequencing technique based on the internal transcribed spacer (ITS) rDNA. This study aimed to (1) describe the response of the soil fungal community to revegetation onto a moving sand dune by planting a native shrub plantation; (2) determine the main soil factors driving the succession of the fungal community; and (3) discuss whether the soil fungal community can be restored to its original state by reforestation. The reforestation of CM significantly ameliorated soil properties, increased soil fungal diversity, and altered the composition and structure of the soil fungal community. Ascomycota, Basidiomycota, and Zoopagomycota were the dominant phyla in all sites. Ascomycota did not respond to plantation development, whereas the other two dominant phyla linearly increased or decreased with the plantation age. The relative abundance of dominant genera varied with sites and showed a waning and waxing characteristic. The composition and structure of the soil fungal community in the 33-year CM plantation were very close to that of the NCF, indicating the restorability of the soil fungal community. The succession of the soil fungal community was directly driven by soil properties, of which soil moisture, organic matter, total N, urease, and protease were the main affecting factors.

Structural and Mechanical Characterization of DNA-Tethered Membranes on Graphene.

Lipid membranes that are separated from the surface of graphene by DNA tethers were prepared by surface functionalization with pyrene coupled to single-stranded DNA (ssDNA), followed by self-assembly of the mixture of ssDNA-functionalized phospholipid and the matrix phospholipids. The formation of uniform membranes was confirmed by fluorescence microscopy, and the structures of the systems before and after hybridization in the direction perpendicular to the global plane of the membranes were investigated using high-energy X-ray reflectivity. The thickness values of the DNA spacers (15 and 37 bp) calculated from the best-fit results were less than the expected thicknesses of the double-stranded DNA (dsDNA) chains taking the upright conformation, indicating that the DNA spacers are tilted with respect to the direction normal to the surface. The Young's moduli of the DNA-tethered membranes obtained by AFM nanoindentation showed higher values than the membranes with no DNA tethers, which suggests that the DNA layer resists against the compression, lifting up the membrane. Intriguingly, the presence of DNA tethers caused no increase in the yield depth. The smaller thickness values as well as the unchanged yield depth suggest that the dsDNA chains can tilt and rotate, which can be attributed to the flexible pyrene-DNA junction.

Complete Plastid Genome Sequences of Four Salsoleae s.l. Species: Comparative and Phylogenetic Analyses.

The taxonomic classification of the genera Salsola L., Pyankovia Akhani and Roalson, and Xylosalsola Tzvelev within Chenopodiaceae Vent. (Amaranthaceae s.l.) remains controversial, with the precise number of species within these genera still unresolved. This study presents a comparative analysis of the complete plastid genomes of S. foliosa, S. tragus, P. affinis, and X. richteri species collected in Kazakhstan. The assembled plastid genomes varied in length, ranging from 151,177 bp to 152,969 bp for X. richteri and S. tragus. These genomes contained 133 genes, of which 114 were unique, including 80 protein-coding, 30 tRNA, and 4 rRNA genes. Thirteen regions, including ndhC-ndhD, rps16-psbK, petD, rpoC2, ndhA, petB, clpP, atpF, ycf3, accD, ndhF-ndhG, matK, and rpl20-rpl22, exhibited relatively high levels of nucleotide variation. A total of 987 SSRs were detected across the four analyzed plastid genomes, primarily located in the intergenic spacer regions. Additionally, 254 repeats were identified, including 92 tandem repeats, 88 forward repeats, 100 palindromic repeats, and only one reverse repeat. A phylogenetic analysis revealed clear clustering into four clusters corresponding to the Salsoleae and Caroxyloneae tribe clades. These nucleotide sequences obtained in this study represent a valuable resource for future phylogenetic analyses within the Salsoleae s.l. tribe.

First report of Alternaria alternata causing leaf spot on Polygonatum kingianum in China.

Polygonatum kingianum is a Chinese herbal medicine that belongs to the genus Polygonatum of the family Liliaceae. In June 2023, Polygonatum kingianum Coll. et Hemsl. in nurseries in Qujing, Yunnan Province, China, showed irregular brown spots on the leaves, whole leaf necrosis, and plant death in serious cases, with an incidence of 10-20% (Fig. S1). To identify the pathogens of P. kingianum, six diseased samples were collected from nurseries with 0.6 acre. These diseased sample leaves were soaked in 0.1% HgCl2 for 1 min and 75% ethanol for 2 min and then rinsed thrice with sterile water. Treated leaves were cut into small pieces (5×5 mm) and cultured on potato dextrose agar (PDA) for five days at 28°C. Total thirteen fungal strains were isolated from PDA medium. The nuclear ribosomal internal transcribed spacer of ribosomal DNA (ITS rDNA) region of these 13 strains was amplified by polymerase chain reaction (PCR) using universal primers ITSI/ITS4 (White et al. 1990). Sequencing and BLAST of the ITS region on NCBI showed that 11 out of 13 fungal strains belonged to the genus Alternaria, with an identity ≥99%. We selected one of the Alternaria strains, HJ-A1, for further study. The HJ-A1 colony appeared grayish brown white-to-gray with a flocculent texture on the front side and a dark gray underside on the PDA medium (Fig. S1). The conidiophores appeared brown, either single or branched, and produced numerous short conidial chains. The conidia were obclavate to obpyriform or ellipsoid in shape and contained 1-4 transverse septa and 0-2 oblique septa. The conidial diameter was 27.30µm in length and 12.27µm in width. (Fig. S1). To further determine the species of HJA1, the genomic DNA of HJ-A1 was extracted using the Lysis Buffer for PCR (AG, Hunan, China). Four Alternaria genomic DNA regions including the ITS, translation elongation factor 1-α gene (TEF1-α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and Alternaria major allergen gene (Alt a1) were amplified by PCR using the primers as previously reported (Woudenberg et al. 2013, Hong et al. 2005). Sequence analysis revealed that the ITS (484bp) of HJ-A1 (NCBI No. PP082633), TEF1-α (267bp) of HJ-A1 (NCBI No. PP419893), GAPDH (582bp) of HJ-A1 (NCBI No. PP419892), and Alt a1 (522bp) of HJ-A1 (NCBI No. PP228046) shared the highest identity with A. alternata respectively (99≥%). A maximum likelihood phylogenetic tree was constructed with the combined sequence data sets of ITS, GAPDH, TEF, and Alt a1 using MEGA 7. The results showed that HJ-A1 strain clustered with A. alternate (Fig. S2). The pathogenicity of HJ-A1 was tested according to Koch's postulates by inoculating HJ-A1 conidia suspension (2×105 conidia/mL) into leaves of 1-year-old P. kingianum, with sterile water as a control. Each treatment group included 3 plants with 3 replicates. The tested plants were planted in a phytotron at 28℃ and 90% humidity. Three days after inoculation, symptoms similar to those under natural conditions were observed in the HJ-A1-inoculated plants, whereas no symptoms were observed in the control plants (Fig. S1). The same fungal strains were re-isolated from inoculated leaves and identified by morphologically and sequence of ITS. Previous studies showed that Alternaria alternata funji cause many plant diseases, such as fig fruit rot (Latinović N et al. 2014)，daylily leaf spot (Huang D et al. 2022), fruit blight on sesame (Cheng H et al. 2021)，leaf spot of Cynanchum atratum Bunge (Sun H et al. 2021) and so on. To our knowledge, this is the first report of A. alternata causing P. kingianum leaf spot in China. The discovery of this pathogen will help to guide the protection and control of P. kingianum disease.

Real-time application of ITS and D1-D3 nanopore amplicon metagenomic sequencing in fungal infections: Enhancing fungal infection diagnostics

While fungal infections cause considerable morbidity and mortality, the performance of the current diagnostic tests for fungal infection is low. Even though fungal metagenomics or targeted next-generation sequencing have been investigated for various clinical samples, the real-time clinical utility of these methods still needs to be elucidated. In this study, we used internal transcribed spacer (ITS) and D1-D3 ribosomal DNA nanopore amplicon metagenomic sequencing to assess its utility in patients with fungal infections. Eighty-four samples from seventy-three patients were included and categorized into ‘Fungal infection,’ ‘Fungal colonization,’ and ‘Fungal contamination’ groups based on the judgement of infectious disease specialists. In the ‘Fungal infection’ group, forty-seven initial samples were obtained from forty-seven patients. Three fungal cases detected not by the sequencing but by conventional fungal assays were excluded from the analysis. In the remaining cases, the conventional fungal assay-negative/sequencing-positive group (n=11) and conventional fungal assay-positive/sequencing-positive group (n=33) were compared. Non-Candida and non-Aspergillus fungi infections were more frequent in the conventional-negative/sequencing-positive group (p-value = 0.031). We demonstrated the presence of rare human pathogens, such as Trichosporon asahii and Phycomyces blakesleeanus. In the ‘Fungal infection’ group and ‘Fungal colonization’ group, sequencing was faster than culturing (mean difference = 4.92 days, p-value < 0.001/ mean difference = 4.67, p-value <0.001). Compared to the conventional diagnostic methods including culture, nanopore amplicon sequencing showed a shorter turnaround time and a higher detection rate for uncommon fungal pathogens.