ITS Gene Research Articles

Comparative analysis of gut microbiota in healthy and diarrheic foals.

Diarrhea presents a substantial risk of high morbidity and mortality among foals. Although studies have shown connections between gut microbiota and several gastrointestinal diseases, there is still inadequate information on gut microbial alterations in foals during diarrhea. In this study, we conducted 16S rRNA and ITS gene amplicon sequencing to investigate gut bacterial and fungal differences between healthy and diarrheic foals. The results unveiled significant reductions in gut bacterial and fungal diversities among foals experiencing diarrhea, accompanied by notable shifts in the composition of gut microbial communities. A considerable decrease was observed in the relative abundance of 30 bacterial and 34 fungal genera. Moreover, two bacterial and eight fungal genera were utterly undetectable in the gut microbiota of diarrheic foals. Some decreased genera, such as Bifidobacterium and Saccharomyces, were deemed beneficial and recognized as probiotics. The study revealed significant alterations in foals' gut bacterial and fungal communities during diarrhea, which enriched our comprehension of gut microbial dynamics in foals across varying health statuses. These findings offer valuable insights for managing diarrhea through gut microbiota modulation, suggesting that probiotics may be superior to antibiotics in preventing and controlling foal diarrhea.IMPORTANCEThis research advances the understanding of gut bacterial and fungal dynamics in foals, highlighting gut microbiota dysbiosis as a potential contributor to foal diarrhea. Additionally, we observed that many altered bacteria and fungi were downregulated during diarrhea, including some probiotic strains. Consequently, our findings provide evidence that probiotics may offer superior efficacy compared with antibiotics as potential candidates for preventing and treating foal diarrhea.

The potential application of Illumina MiSeq sequencing in herbivore-diet analysis: quantitative estimation of sheep diet in meadow steppe

Abstract DNA barcoding approaches have been successfully applied for estimating diet composition. However, an accurate quantification in the diets of herbivores remains to be achieved. In the current study, we present a novel methodology that reveals the relationship between the actual proportions (by mass) of each herbage species in the diets and the relative proportions of the ITS2 gene sequences obtained from faecal samples to evaluate the diet composition of sheep in a meadow steppe. Nine common and twelve rare species of plants were employed for formulating six diets, along with the addition of feed supplements for improving the growth performance of sheep. Faecal samples were collected for DNA analysis over the period spanning days 7–12. A significant positive correlation (Spearman’s ρ = 0.389) was obtained between the actual proportions (by mass) of the herbage in the diet provided and the relative abundance of ITS2 sequences obtained from the faecal samples. A significant regression coefficient was found between the relative abundance of all common species and their respective herbage mass proportions. The accuracy of the relation equations, evaluated by utilizing the similarity coefficient, showed 84.69% similarity between the actual diet composition and the correct percentage. Taken together, the current study has provided empirical evidence for the accuracy and applicability of ITS2 as a DNA barcode for obtaining quantitative information about the diet composition of sheep grazing in species-rich grasslands.

Historical Lead Contamination Linked to Atmospheric Deposition is Associated With Declines in Ectomycorrhizal Diversity and Shifts in Fungal Community Composition

ABSTRACTEctomycorrhizal and saprotrophic fungi respond differently to changing edaphic conditions caused by atmospheric deposition. Within each guild, responses can vary significantly, reflecting the diversity of species and their specific adaptations to environmental changes. Metal contaminants are often deposited onto earth's surface through atmospheric deposition, yet few studies have assessed the relationship between soil metal contamination and fungal communities. The goal of this study was to understand how soil metal contamination and other edaphic factors vary across the spruce‐fir ecosystem in the Southern Appalachians and influence fungal diversity and function. Here, we characterize soil fungal communities using high‐throughput sequencing of the ITS2 gene region and found that higher soil lead (Pb) concentrations were associated with lower fungal diversity. Ectomycorrhizal fungi were less diverse (specifically hydrophilic ectomycorrhizal functional types) at plots with elevated soil Pb concentrations, while saprotrophic fungi were less diverse at plots with elevated soil carbon:nitrogen ratios. Fungal community composition was significantly influenced by pH, Pb, and spatial factors. This study identifies important relationships between fungal diversity and soil Pb concentrations and indicates variable responses of genera within well‐defined ecological guilds. Our work highlights the need to characterize poorly understood taxonomic groups of fungi and their function prior to further environmental degradation.

Genetic identification of Stephanofilaria sp. isolated from ulcerative dermal lesions in black rhinoceros.

Stephanofilaria is a genus of nematodes that cause ulcerative dermal lesions in large mammals. However, there is a dearth of knowledge on the molecular genetics of Stephanofilaria species infecting critically endangered rhinoceros. This study employed genetic barcoding genes to identify Stephanofilaria species and to determine its genetic diversity and evolution. Phylogenetic analyses on partial genes of the second internal transcribed spacer Ribosomal DNA (ITS-2) and cytochrome c oxidase subunit 1 (Cox-1), revealed a 77% and 93% bootstrap support at the Cox-1 and ITS-2 loci respectively to a clade containing previously identified Stephanofilaria species. Morphological examination also confirmed features diagnostic of Stephanofilaria dinniki previously known to infect rhinoceros. Gene diversity of Cox-1 was 0.931 ± 0.030 and 0.579 ± 0.104 for the ITS-2, whereas nucleotide diversity was 0.008 ± 0.002 and 0.00197 ± 0.0016 for the Cox-1 and ITS-2 genes respectively. Neutrality tests (Fu and Li's D* and Fu and Li's F*) were significantly negative (p<0.05) at all loci, whereas Tajima D and Fu's FS were each statistically significant (p<0.05) at the Cox-1 and ITS-2 loci respectively. The high gene diversity, low nucleotide diversity and negative neutrality tests are consistent with positive selection at the Cox-1 gene. Stephanofilaria infection among rhinoceros is currently restricted to highland sanctuaries compared to a widespread distribution in both lowlands and highlands in the 1960s suggesting an adaptation to vectors thriving in cooler highland temperatures. This is the first genetic identification of S. dinniki, in rhinoceros and will aid in diagnosis, treatment, studies, and rhinoceros conservation.

First report of Diaporthe pescicola causing leaf spot on tea (Camellia sinensis) in China.

Duyun Maojian Tea mainly cultivated in Duyun City is one of the ten famous teas in China. A brown leaf spot was discovered in March 2023 in Qiannan Normal University for Nationalities' Maojian Tea experimental field (26.29°N, 107.52°E). The incidence of the disease is about 40% surveyed on about 300 m2 of tea plants. Infected leaves showed light brown, round or irregular lesions. With disease progression, they enlarged and merged, creating dark brown necrotic zones along leaf edges. To identify the responsible pathogen, three symptomatic leaves from the tea nursery base were collected. The samples were cut 3 mm x 3 mm tissues with 75% ethanol for 30 s and 5% sodium hypochlorite for 60 s, rinsing three times with sterile water. Tissues were taken to potato dextrose agar (PDA) medium and incubated dark at 28 °C for 3 days. Three isolates (TF1, TF3 and TF5) with similar morphology were obtained from three samples, respectively. The colony showed white, with short, villous hyphae expanding radially. Notably, mycelium near the disc was erect and sparse 2 cm away. After 30 days, conidia were observed. Alpha conidia (5.9 to 8.6 μm × 2.0 to 2.8 μm (n = 50)) were colorless and transparent, no septum, ellipsoid to spindle-shaped. Beta conidia (11.1 to 25.3 μm × 1.0 to 1.8 μm (n = 50)) were transparent, no septum, filiform, hooklike, tapering towards the ends. The isolates were identified as Diaporthe pescicola based on the morphology (Zhang et al. 2022). Genomic DNA was extracted, the internal transcribed spacer region (ITS), β-tubulin (tub2), and translation elongation factor 1-alpha (TEF) genes were amplified with primer pairs ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), and EF1-728F/EF1-986R (Carbone and Kohn 1999), respectively, and sequenced. The ITS (PQ163865, PQ685064 and PQ685065), tub2 (PQ197032, PV055438 and PV055439), and TEF (PQ197029, PV055440 and PV055441) sequences of isolates (TF1, TF3 and TF5) were deposited in GenBank, respectively. BLAST searches showed >97% nucleotide identity to sequences of D. pescicola MFLUCC 16-0105 (ITS, 98.40% to KU557555.1; tub2, 97.40% to KU557579.1; and TEF, 97.63% to KU557623.1). The phylogenetic tree of ITS, tub2, and TEF gene sequences was constructed using MEGA 6 software with the neighbour-joining method (NJ), and the bootstrap method was employed for 1,000 repeated tests. Isolates TF1, TF3, TF5, and D. pescicola DT 1-1 were grouped together in a node with a bootstrap value of 99. Isolates were identified as D. pescicola. Since large quantities of conidia are difficult to produce in cultures, pathogenicity on the wounded leaves of 5-year-old tea plants of Duyun Maojian Tea local population was conducted by inoculating 6 mm mycelium discs of TF1. The control group and the treatment group were inoculated with 3 tea plants, repeated 2 times. All tea plants were incubated at 25 ± 1 °C, 70% humidity, and natural daylight. The onset of symptoms appeared 7 days later. Lesions were brown, similar to field symptoms. In contrast, the control were healthy. A strain exhibiting the same morphology as TF1 was re-isolated from the diseased portion. ITS, tub2, and TEF gene sequences were consistent with TF1, completing Koch's postulates. D. pescicola as a pathogen has caused leaf spot on Kerria japonica (Zhang et al. 2022), dieback on Prunus persica (Dissanayake et al. 2017) and Vitis vinifera (Manawasinghe et al. 2019), and shoot canker on Pyrus plants (Guo et al. 2020). To our knowledge, this is the first report of tea leaf spot caused by D. pescicola in China and worldwide. It is imperative to develop and implement control measures to manage the disease.

Supplementary Diets Impact the Abundance of Core Bacteria in the Gut of a Managed Stingless Bee but Not Its Fat Body Size

ABSTRACTThe management of stingless bees is an expanding activity that requires technical knowledge in order to face a number of challenges, from malnutrition to disease. In the present study, we evaluate the effect of supplementary diets based on sucrose (100% sucrose syrup or 50% sucrose syrup + 50% dried spirulina) on fat body mass and on the gut‐associated microbiota of Melipona quadrifasciata, one of the most important species in Brazilian stingless bee farming. The study was performed over 5 months, from the end of spring until the end of summer, when outbreaks of an annual disease occur. Both the fungal and bacterial microbiota were metabarcoded using ITS2 and 16S gene markers, respectively. Our experimental design aimed at incorporating both seasonal and treatment effects of diet on forager gut microbial composition. Our analyses show that supplementing bees' diet with sucrose‐based feed does not affect the amount of fat body of forager bees. In contrast, it alters the composition of the gut microbiota characterised by an increase in the relative abundance of acetic acid bacteria and unclassified lactobacilli. The significance of such changes promoted by a sucrose‐rich gut environment concerning bee health needs to be further investigated and carefully considered for stingless bee management.

Identification of fungi causing pre-harvest anthracnose and anthracnose-like symptoms on mango fruits (Mangifera indica)

During field surveys in Uttar Pradesh, India between 2020–2022, we consistently encountered pre-harvest anthracnose and anthracnose-like symptoms on green mangoes, with incidence levels ranging from 20% to 40%, particularly during periods of heavy rainfall. This study aimed to identify the fungal pathogens associated with these symptoms using a polyphasic approach. Pathogenicity was confirmed on detached fruits of mango cv. Dashehari, with isolated pathogens including Colletotrichum asianum, C. siamense, Botryosphaeria dothidea, Diaporthe phoenicicola, Alternaria burnsii, Lasiodiplodia hormozganensis, and Parasympodiella eucalyptii. Colletotrichum asianum and C. siamense were identified as predominant through phylogenetic analysis of multiple genes (internal transcribed spacer [ITS], actin [ACT], chitin synthase [CHS-1], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], and Mat1–2 gene region [APMAT] genomic regions). Lasiodiplodia hormozganensis and A. burnsii were identified through phylogenetic analysis based on ITS and translation elongation factor-1alpha (TEF) genes. Diaporthe phoenicicola was identified using ITS, TEF, and β-tubulin (TUB2)-based phylogenetic analysis. To the best of our knowledge, association of D. phoenicicola and A. burnsii with mango fruit spots is being reported for the first time in this study. Lasiodiplodia hormozganensis was the most aggressive pathogen, followed by C. siamense, C. asianum, B. dothidea, A. burnsii, D. phoenicicola, and P. eucalypti. Although the pathogenic behaviour of P. eucalyptii has been demonstrated on detached leaves and fruits, its role remains questionable due to its rare isolation. This study underscores the involvement of multiple Colletotrichum species and other fungal pathogens in mango anthracnose and highlights the need for further research into the diversity and geographical distribution of Colletotrichum species causing mango anthracnose in India.

Molecular Detection of Hemoparasites in Hematophagous Insects Collected from Livestock Farms in Northeastern Thailand.

Vector-borne parasitic diseases (VBPDs) are diseases that have a major impact on the health and productivity of livestock. VBPDs, including trypanosomosis, theileriosis, and anaplasmosis, are caused by blood parasites and Rickettsiales infections that can be transmitted by biting insects. The control and elimination of VBPDs is difficult and challenging. This study contributed to better understanding the impact of hematophagous insects on carrying pathogens to livestock by investigating the detection of Trypanosoma evansi, Theileria spp. and Anaplasma spp. in hematophagous insects collected from five livestock farms in Northeastern Thailand using polymerase chain reaction (PCR). The Stomoxys and Tabanus samples were collected using Nzi and Vavoua traps. Morphological and molecular identifications of hematophagous insects were performed. The Stomoxys samples were dissected into head and abdomen parts, while the Tabanus samples were dissected into head, salivary glands, and abdomen parts before DNA extraction individually. The host blood meal in the DNA of each part of the hematophagous insects was identified using PCR targeting the prepronociceptin (PNOC) gene. PCR detection of T. evansi, Theileria spp., and Anaplasma spp. were performed by targeting the ITS2, 18s rRNA, and msp4 genes, respectively. In total, 131 insect samples were collected, with Stomoxys calcitrans being the most common species at 40 (30.53%), followed by Tabanus spp. at 14 (10.69%) and the other Stomoxys spp. at 7 (5.34%), respectively. The hemoparasite detection results identified Theileria spp. in the abdomen (21.43%; 3/14) of three Tabanus samples. Neither Anaplasma spp. nor T. evansi were detected in this study. Additionally, host blood meal DNA was found in the abdomen of Stomoxys calcitrans (4.17%; 1/24) and Stomoxys spp. (20%; 1/5). For the Tabanus spp., host blood meal DNA was detected in the head (7.14%; 1/14), salivary glands (7.14%; 1/14), and abdomen (14.29%; 2/14). These results highlighted that Tabanus spp. might play an important role in Theileria transmission.

Aspergillus aflatoxiformans (Aspergillaceae): a new record to Indian mycobiota from Kashmir Himalaya, India

The present study was carried out as part of a mycological survey in various regions of the Kashmir Himalaya, J and K, India. While studying rhizospheric fungal diversity of medicinal plant Swertia petiolata D. Don, we isolated and identified Aspergillus aflatoxiformans fungi for the first time from Kashmir Himalaya. The isolation and identification were done based on cultural, microscopic, and molecular characteristics using the ITS gene sequencing. The colonies appeared white and cottony on the upper side, while the reverse side ranged from light yellow to yellowish-orange. Based on the closest identification through BLAST analysis, the nucleotide sequences of 23 isolates were compared to those in the GenBank database, showing a match with varying levels of global similarity. After biodiversity literature studies, we concluded that the isolated fungi was Aspergillus aflatoxiformans and represents a new record for the Kashmir Himalaya.

Lepiotakeralensis, a new species with epithelial pileal covering

A new species, Lepiota keralensis, was collected from Kerala, India and was studied using morphological and molecular (ITS, LSU and rpb2 gene) characters. The species is characterized by medium‐sized basidiomata with innate squarrose brown coloured squamules on a white background, hyaline, dextrinoid, non‐metachromatic, smooth‐walled basidiospores, absence of cheilocystidia and pleurocystidia, and globose to ellipsoid cells on pileal covering. Morphological as well as phylogenetic analyses confirm the placement of the new species in Lepiota sect. Echinatae. The discovery of the new species adds to the existing data on tropical lepiotas.

Morphology and phylogeny reveal a novel species, Tolypocladium phycosomatis (Ophiocordycipitaceae, Hypocreales), from Yunnan, China

Tolypocladium is classified within family Ophiocordycipitaceae. We formally describe and illustrate a novel species, Tolypocladium phycosomatis using morphological features and phylogenetic analyses. This new species was discovered in Yunnan Province, China. Phylogenetic analysis based on ITS, nrSSU, nrLSU, tef-1α, rpb1, and rpb2 gene sequences revealed that this species clustered closely with T. ovalisporum, forming a sister relationship. A comprehensive phylogenetic tree of the genus Tolypocladium was reconstructed, providing a foundation for further phylogenetic studies of this genus and its related taxa. This discovery enhances our understanding of species diversity within the genus Tolypocladium and provides a robust taxonomic foundation for their rational development and sustainable utilization.

Comparative analysis of fungal and bacterial composition in natural wines and their closest pesticide-treated counterparts

Fermentation produces diverse consumables like bread, wine, beer, and cheese, with fungi playing a key role. This study sequenced fungal and bacteria DNA from 46 commercial wines, paired by vineyard proximity, harvest time, and grape variety. One in each pair was natural, while the other had pesticide treatments and microbial adjunctions. Bacteria profiles were studied using targeted sequencing of the V3-V4 region of the 16 S rRNA gene while fungal profiles were studied with ITS gene targeted sequencing. Significant variability was observed in the microbial content of these wines. We identified 19 unique fungal species in natural wines, absent in pesticide-treated ones. Some have known health roles. The most abundant was Penicillium jiangxiense in Lot-et-Garonne, known for its antitumor properties. Hyphopichia pseudoburtonii, with notable aromatic properties, was also detected, commonly found in insect and fish microbiota. Antrodia favescens, related to the medicinal Antrodia cinnamomea, was identified in another location, known for its role in Chinese herbal medicine against cancer and metabolic diseases. Ochrocladosporium elatum, recognized for its antioxidant and antibacterial activities, was found elsewhere. Additionally, natural wines exhibited greater bacterial biodiversity, contributing to unique tastes and potential health benefits.

Rapid and simple detection of Candida albicans using closed dumbbell-mediated isothermal amplification.

Candida albicans, a human fungal pathogen, multiplies to invade body cells and causes fungal diseases in the condition of insufficient body's immune function. Early detection of C. albicans is required to guide appropriate prevention and treatment. The purpose of this study was to establish a C. albicans assay based on newly developed closed dumbbell-mediated isothermal amplification (CDA) to achieve rapid and simple point of care diagnostic. The CDA technique was carried out by specific primers targeting at the conserved C. albicans ITS2 gene. All primers were selected and evaluated by real-time fluorescence monitoring and endpoint visual judgement indicated by hydroxy naphthol blue (HNB). Optimal primers and accelerate primers (out primers and loop primers) were designed and selected after confirmation of the fundamental CDA primers to achieve more efficient CDA reaction for C. albicans detection (CA-OL-CDA). After establishment of the assay, 9 non-Candida albicans strains, including 3 Candida species were tested to negative by adopting the established CA-OL-CDA assay, indicated high specificity. The limit of detection of Candida albicans DNA by CA-OL-CDA assay was 6.2×10-6 ng/μL of DNA (10 copies/μL), 10-fold more sensitive than real-time quantitative PCR (qPCR). The CA-OL-CDA assay exhibited advantages of high specificity, sensitivity, simpler and more efficient operation. In addition, the CA-OL-CDA method holds potential in on-site detection for C. albicans using color shift by adopting the reaction mixture based on HNB.

Phylogenetic studies and distinction of aflatoxin-producing Aspergillus species in section Flavi, Ochraceorosei and Nidulantes: A review.

Phylogenetic studies and distinction of aflatoxin-producing Aspergillus species in section Flavi, Ochraceorosei and Nidulantes: A review.

Fungal and bacterial diversity present on the rind and core of Natural Bloomy Rind Artisanal Minas Cheese from the Canastra region, Brazil.

Fungal and bacterial diversity present on the rind and core of Natural Bloomy Rind Artisanal Minas Cheese from the Canastra region, Brazil.

Colletotrichum siamense Causing Leaf Spot on Black Olive (Bucida buceras) in Mexico

ABSTRACTDuring the summer of 2019, spots were detected on the leaves of black olive (Bucida buceras L.) trees distributed in urban areas in Los Mochis, Sinaloa, Mexico. Two single‐spore isolates were obtained and identified as Colletotrichum siamense by phylogenetic analyses of concatenated sequences of the ITS, gapdh, tub2, act, chs‐1 and gs genes. The pathogenicity of the isolates was demonstrated through inoculations under controlled conditions. This is the first report of C. siamense causing leaf spot of black olive in Mexico and worldwide.

Two novel entomopathogenic fungal species of Lecanicillium isolated from soil in China

The genus Lecanicillium was established in 2001 based on the type species Lecanicillium lecani (former, Verticillium lecani), which is an important entomopathogenic fungus. To date, more than thirty species in the genus have been reported, but much more are waiting to discover. In this study, two novel species isolated from soil in east China were identified. They differ from known closely related species primarily in sporulation structure, conidia and colony morphological characteristics, and sequence variations in the LSU, SSU, ITS, and TEF gene regions of the ribosomal DNA. Multigene phylogenetic analysis has provided strong molecular evidence supporting the classification of the strains, AH13B2 and JX15A210 within the genus Lecanicillium. The new species are formally named as follows: L. anqingense sp. nov. and L. renii sp. nov., respectively. Bioassays conducted on the greater wax moth, Galleria mellonella and crucifer aphid, Lipaphis erysimi showed that both fungal strains exhibit virulence. When treating with 1 × 108 spores/mL, the two strains gave mortality of > 60% and > 80% to G. mellonella and L. erysimi, respectively. Moreover, the median lethal times (LT50) of L. renii JX15A210 and L. anqingense AH13B2 against G. mellonella and L. erysimi were recorded as 8.06 d and 4.18 d, and 10.63 d and 5.07 d, respectively. This research enhances the species diversity within the genus Lecanicillium and provides valuable genetic resources for the biological control of pests and the development of biocontrol agents.

Gut Microbiome Diversity in European Honeybees (Apis mellifera L.) from La Union, Northern Luzon, Philippines.

Insects often rely on symbiotic bacteria and fungi for various physiological processes, developmental stages, and defenses against parasites and diseases. Despite their significance, the associations between bacterial and fungal symbionts in Apis mellifera are not well studied, particularly in the Philippines. In this study, we collected A. mellifera from two different sites in the Municipality of Bacnotan, La Union, Philippines. A gut microbiome analysis was conducted using next-generation sequencing with the Illumina MiSeq platform. Bacterial and fungal community compositions were assessed using 16S rRNA and ITS gene sequences, respectively. Our findings confirm that adult worker bees of A. mellifera from the two locations possess distinct but comparably proportioned bacterial and fungal microbiomes. Key bacterial symbionts, including Lactobacillus, Bombilactobacillus, Bifidobacterium, Gilliamella, Snodgrassella, and Frischella, were identified. The fungal community was dominated by the yeasts Zygosaccharomyces and Priceomyces. Using the ENZYME nomenclature database and PICRUSt2 software version 2.5.2, a predicted functional enzyme analysis revealed the presence of β-glucosidase, catalase, glucose-6-phosphate dehydrogenase, glutathione transferase, and superoxide dismutase, which are involved in host defense, carbohydrate metabolism, and energy support. Additionally, we identified notable bacterial enzymes, including acetyl-CoA carboxylase and AMPs nucleosidase. Interestingly, the key bee symbionts were observed to have a negative correlation with other microbiota. These results provide a detailed characterization of the gut microbiota associated with A. mellifera in the Philippines and lay a foundation for further metagenomic studies of microbiomes in native or indigenous bee species in the region.

Internal transcribed spacers enable species-level Metataxonomic analysis of ciliated protozoa.

Traditional morphology-based ciliate classification is often time-consuming and inaccurate, necessitating molecular approaches. Although 18S rRNA gene sequencing is widely used for taxonomic analyses of ciliates, its high degree of conservation makes it challenging to achieve species-level resolution. This study explores the potential of internal transcribed spacers (ITS1 and ITS2) and the 28S rRNA gene to improve taxonomic resolution beyond that offered by 18S rRNA gene in free-living and host-associated ciliates. A comparative analysis of ITS, the 18S, and 28S rRNA gene sequences retrieved from public databases indicated that ITS regions exhibit greater inter- and intra-specific sequence dissimilarity compared to 18S rRNA gene, supporting existing literature. We then designed universal primers targeting the ITS and 28S rRNA gene for freshwater and rumen ciliates. These primers were rigorously evaluated for their inclusiveness, specificity, and amplification efficiency using in-silico PCR, experimental PCR, followed by sequencing and metataxonomic analyses of the ciliate communities. In-silico analyses revealed inclusiveness exceeding 80%, while experimental analyses validated their specificity. Metataxonomic analyses of ciliates demonstrated that the ITS and 28S rRNA gene captured significantly greater taxonomic diversity than 18S rRNA gene. Also, ITS1 offered superior taxonomic resolution by detecting the most ciliate species that went unnoticed by the 18S rRNA gene. These findings underscore the superiority of ITS1, and to a lesser extent ITS2, as taxonomic markers for enhancing the resolution of freshwater and rumen ciliate communities. We recommend ITS1 as an alternative marker to overcome the limitations of 18S rRNA gene-based approaches in free-living and host-associated ciliate taxonomy.

First Report of Root Rot Caused by Fusarium equiseti on Syrian rue (Peganum harmala) in China.

Syrian rue (Peganum harmala L) belongs to the family Zygophyllaceae and is mainly distributed in arid and semi-arid areas of Xinjiang, Gansu, Ningxia, Qinghai, and Inner Mongolia in China. It serves as a pioneer species in soil and water conservation, as well as in reclamation of wastelands, playing a crucial role in soil preservation and stabilization against sand encroachment. In 2023, a disease resembling root rot and wilt affected the quality and yield of Syrian rue in Xilingol (122.67°N, 42.78°E), Inner Mongolia, China. Symptoms ranged from yellowing to wilting, accompanied by darkening of the xylem in the roots. This disease was observed in approximately 50% of Syrian rue plants in desertification-prone grassland. A total number of 45 symptomatic root samples were collected from Syrian rue plants to isolate the pahogen. Small pieces of diseased tissue were surface sterilized with 75% alcohol for five seconds, rinsed twice with sterilized water, dried, and then placed on water agar and incubated for 72 h at 25℃ in darkness. A total of 45 isolates were obtained, showing identical morphology and sequences of the internal transcribed spacer region (ITS), translation elongation factor 1-alpha (EF1-α) and RNA polymerase II second largest subunit (RPB2) genes. Therefore, the isolate LTP-5 was used as the representative for further study. On potato dextrose agar (PDA), fungal colonies of LTP-5 exhibited yellow to light brown colore with white aerial mycelia and irregular growth after 7 days of incubation at 25℃. Conidia and chlamydospre development were observed in carboxymethylcellulose sodium medium. Chlamydospores were abundant, terminal or intergrown between hyphae, rough brown walls of 5.9 to 19.82 μm in diameter (n = 75) were observed in the culture. Macroconidia had 5 to 7 septa, thick in the middle and thin at both ends, measured 28.17 to 62.81 μm in length and 1.50 to 4.19 μm in width (n = 128). Microconidia were absent. The morphological characteristics were consistent with the descriptions of Fusarium equiseti (Nelson et al., 1983). The pathogen was further confirmed to be F. equiseti by sequence analysis of the ITS, EF1-α and RPB2 genes amplified using polymerase chain reaction (PCR) with the primers ITS4/ITS5 (White et al., 1990), EF-1/EF-2 (O'Donnell et al., 1998), and RPB2-5F2 /fRPB2-7cR (Zhen et al., 2017). The sequences showed 100% similarity to the F. equiseti strain in the NCBI GenBank with accession numbers MH054914.1, KJ396323.1, and KT213286.1 for ITS, EF1-α, and RPB2, respectively. The sequences of PCR products were submitted to GenBank with accession numbers PP814863.1 (ITS), PP831628.1 (EF1-α) and PP826937.1 (RPB2). Pathogenicity tests were conducted through a pot assay. Five Syrian rue seedlings were inoculated by immersing plant roots in a suspension of 1×107 spores/mL for 30 min and transplanting to pots containing autoclaved soil. Control plants were immersed in sterile water. The inoculated plants showed yellow and root rot but no symptoms were observed on control plants. The same fungus was successfully re-isolated and identified based on the morphological characterization and sequence analyses from the infected root tissue but not from non-inoculated plants. In addition, F. equiseti has been reported to cause disease on Sugar beet (Khan et al., 2021), Watermelon (Rahman et al., 2021), potato (Cui et al., 2021), and other plants. This is the first report confirming F. equiseti as causal agent of Syrian rue wilt and root rot in China.