Brown root rot disease (BRRD) is a highly destructive tree disease. Early diagnosis of BRRD has been challenging because the first symptoms and signs are often observed after extensive tissue colonization. Existing molecular detection methods, all based on the internal transcribed spacer (ITS) region, were developed without testing against global Phellinus noxius isolates, other wood decay fungi, or host plant tissues. This study developed SYBR Green real-time quantitative PCR (qPCR) assays for P. noxius. The primer pair Pn_ITS_F/Pn_ITS_R targets the ITS, and the primer pair Pn_NLR_F/Pn_NLR_R targets a P. noxius-unique group of homologous genes identified through a comparative genomics analysis. The homologous genes belong to the nucleotide-binding-oligomerization-domain-like receptor (NLR) superfamily. The new primer pairs and a previous primer pair G1F/G1R were optimized for qPCR conditions and tested for specificity using 61 global P. noxius isolates, five other Phellinus species, and 22 non-Phellinus wood decay fungal species. While all three primer pairs could detect as little as 100 fg (about 2.99 copies) of P. noxius genomic DNA, G1F/G1R had the highest specificity and Pn_NLR_F/Pn_NLR_R had the highest efficiency. To avoid false positives, the cutoff Cq values were determined as 34 for G1F/G1R, 29 for Pn_ITS_F/Pn_ITS_R, and 32 for Pn_NLR_F/Pn_NLR_R. We further validated these qPCR assays using Ficus benjamina seedlings artificially inoculated with P. noxius, six tree species naturally infected by P. noxius, rhizosphere soil, and bulk soil. The newly developed qPCR assays provide sensitive detection and quantification of P. noxius, which is useful for long-term monitoring of BRRD status.

Mushroom poisoning contributes significantly to global foodborne diseases and related fatalities. Amanita mushrooms frequently cause such poisonings; however, identifying these toxic species is challenging due to the unavailability of fresh and intact samples. It is often necessary to analyze residues, vomitus, or stomach extracts to obtain DNA sequences for the identification of species responsible for causing food poisoning. This usually proves challenging to obtain usable DNA sequences that can be analyzed using conventional molecular biology techniques. Therefore, this study aimed to develop a DNA mini-barcoding method for the identification of Amanita species. Following the evaluation and optimization of universal primers for DNA mini-barcoding in Amanita mushrooms, we found that the internal transcribed spacer (ITS) gene sequence primer ITS-a was the most suitable DNA barcode primer for identifying Amanita species. Forty-three Amanita samples were subsequently amplified and sequenced. The sequences obtained were analyzed for intra- and inter-species genetic distances, and a phylogenetic tree was constructed. The findings indicated that the designed primers had strong universality among the Amanita samples and could accurately identify the target gene fragment with a length of 290 bp. Notably, the DNA mini-barcode accurately identified the 43 Amanita samples, demonstrating high consistency with the conventional DNA barcode. Furthermore, it effectively identified DNA from digested samples. In summary, this DNA mini-barcode is a promising tool for detecting accidental ingestion of toxic Amanita mushrooms. It may be used as an optimal barcode for species identification and traceability in events of Amanita-induced mushroom poisoning. KEY POINTS: • Development of a DNA mini-barcoding method for Amanita species identification without fresh samples. • The ITS-a primer set was optimized for robust universality in Amanita samples. • The mini-barcode is suitable for screening toxic mushroom species in mushroom poisoning cases.

Leaf-blight disease caused by the Fusarium oxysporum is an emerging problem in Dendrobium chrysotoxum production in China. Symptoms of leaf blight were observed on seedlings of D. chrysotoxum cultivated in a nursery in Ruili City, Yunnan Province, China. In this study, we isolated the Fusarium sp. associated with leaf-blight disease of D. chrysotoxum from the diseased seedlings. A pathogenicity test was performed to fulfill Koch\'s postulates to confirm the pathogenicity of isolated strains and identified using morphological and molecular techniques. The results revealed that all four isolated Fusarium sp. isolates (DHRL-01~04) produced typical blight symptoms followed by marginal necrosis of leaves on the D. chrysotoxum plants. On the PDA medium, the fungal colony appeared as a white to purplish color with cottony mycelium growth. Microconidia are oval-shaped, whereas macroconidia are sickle-shaped, tapering at both ends with 2-4 septations. The phylogenetic trees were construed based on internal transcribed spacer (ITS), translation elongation factor (EF-1α), and RNA polymerase subunit genes RPB1 and RPB2 genes, respectively, and blasted against the NCBI database for species confirmation. Based on the NCBI database\'s blast results, the isolates showed that more than 99% identify with Fusarium oxysporum. To our knowledge, this is the first comprehensive report on the identification of Fusarium oxysporum as the causal agent of Dendrobium chrysotoxum leaf blight in Yunnan Province, China, based on morphological and molecular characteristics.

This molecular-epidemiological study was conducted in several locations in Cairo and Giza Governorates in domestic cats (Felis catus) to detect the most common intestinal helminths in feces and molecularly characterize this nematode. So, three hundred domestic cats were admitted to different clinics around Cairo and Giza Governorates with severe diarrhea, even watery, between January 2023 and April 2023. The ages of the cats ranged from 1 to 2.5 years old. Blood, sera, and urine samples were collected for further investigation of the health condition of the animals. Toxascaris leonina was the major intestinal parasite found in cat stools, with a prevalence rate of 5% (15 cats). Toxascaris leonina (T. leonina) eggs had oval elliptical surfaces and thick cuticles. An embryo was located inside the smooth outer shell wall of the shell. The animals suffer from normocytic normochromic anemia with leukocytosis, relative lymphocytosis, and thrombocytopenia. The amplification of the ITS-rDNA region from the ascaridoid nematodes was successfully performed using NC5 and NC2 primers. The PCR product of the ITS-rDNA fragment was sequenced and yielded 860 bp. The accession number of the sequenced ITS-rDNA region was OQ735413, submitted to Gene Bank, and based on the blast analysis of NCBI, the current ascaridoid nematode proved to be genetically related to the family Ascarididae and identified as T. leonina.

Enterocytozoon bieneusi, Encephalitozoon spp., Cryptosporidium spp., and Giardia duodenalis (G. intestinalis) are enteric pathogens that cause diarrhea in pigs. This study aimed to determine the prevalence of these enteric parasites and their coinfection with E. bieneusi in diarrheic pigs in Southwest China (Chongqing and Sichuan) using nested polymerase chain reaction (nPCR) based methods.
A total of 514 fecal samples were collected from diarrheic pigs from 14 pig farms in Chongqing (five farms) and Sichuan (nine farms) Provinces. The prevalence of Encephalitozoon spp., Cryptosporidium spp. and G. duodenalis was 16.14% (83/514), 0% (0/514), and 8.95% (46/514), respectively. Nested PCR revealed 305 mono-infections of E. bieneusi, six of E. cuniculi, two of E. hellem, and nine of G. duodenalis and 106 concurrent infections of E. bieneusi with the other enteric pathogens. No infections of E. intestinalis and Cryptosporidium species were detected. The highest coinfection was detected between E. bieneusi and E. cuniculi (10.5%, 54/514), followed by E. bieneusi and G. duodenalis (5.8%, 30/514) and E. bieneusi and E. hellem (2.9%, 15/514). E. bieneusi was the most frequently detected enteric pathogen, followed by E. cuniculi, G. duodenalis and E. hellem. There was a significant age-related difference in the prevalence of E. cuniculi in fattening pigs (χ2 = 15.266, df = 3, P = 0.002) and G. duodenalis in suckling pigs (χ2 = 11.92, df = 3, P = 0.008) compared with the other age groups. Sequence analysis of the ITS region of Encephalitozoon species showed two genotypes (II and III) for E. cuniculi and one (TURK1B) for E. hellem. Only G. duodenalis assemblage A was identified in all nested PCR-positive samples. E. bieneusi was found more often than other enteric pathogens.
This study showed that E. bieneusi, Encephalitozoon spp. [E. cuniculi and E. hellem] and G. duodenalis were common enteric parasites in diarrheic pigs in Chongqing and Sichuan Provinces. In case of both mono-infection and coinfection, E. bieneusi was the most common enteric pathogen in diarrheic pigs. Thus, it may be a significant cause of diarrhea in pigs. Precautions should be taken to prevent the spread of these enteric parasites.

BACKGROUND: Microbial communities have occasionally been observed in part of the ephemeric reproductive structure of floral stigmas, but their prevalence, phylogenetic diversity and ecological roles are understudied. This report describes the first study of bacterial and fungal communities in immature and mature stigma tissue of the endangered holoparasitic plant Phelipanche arenaria. Culture-dependent methods coupled with next-generation sequencing indicated that a small surface of the flower stigma was an unexpectedly rich and diverse microhabitat for colonization of microbial. We also compared the enzymatic activity of the bacterial communities between immature and mature stigmas samples.
RESULTS: Using high-throughput sequencing methods, we identified and classified 39 to over 51 OTUs per sample for bacterial OTUs represented by Pantoea agglomerans and P. ananatis, comprising 50.6%, followed by Pseudomonas, Luteibacter spp., Sphingomonas spp. with 17% of total frequency. The bacterial profile of immature stigmas of P. arenaria contained unique microorganisms (21 of the most numerous OTUs) that were not confirmed in mature stigmas. However, the enzymatic activity of bacteria in mature stigmas of P. arenaria showed more activity than observed in immature stigmas. In the fungal profile, we recorded even 80 OTUs in mature stigmas, consisting of Capnodiales 45.03% of the total abundance with 28.27% of frequency was created by Alternaria eichhorniae (10.55%), Mycosphaerella tassiana (9.69%), and Aureobasidium pullulans (8.03%). Additionally, numerous putative plant growth-promoting bacteria, fungal pathogens and pathogen-antagonistic yeasts were also detected.
CONCLUSIONS: Our study uncovered that P. arenaria stigmas host diverse bacterial and fungal communities. These microorganisms are well known and have been described as beneficial for biotechnological and environmental applications (e.g., production of different enzymes and antimicrobial compounds). This research provided valuable insight into the parasitic plant-microbe interactions.

In the present study, we evaluated the genetic variability of the internal transcribed spacer (ITS) region and the pyruvate:ferredoxin oxidoreductase (pfor) A gene of Trichomonas vaginalis from female patients and its possible implications in the host-parasite relationship. Phylogenetic and genetics of populations analyses were performed by analyzing sequences of the ITS region and partial pfor A gene of clinical samples with T. vaginalis, as previously documented. Alignments of protein sequences and prediction of three-dimensional structure were also performed. Although no correlation between the main clinical characteristics of the samples and the results of phylogeny was found, a median-joining analysis of ITS haplotypes showed two main clusters. Also, pfor A, due to its phylogenetic divergence, could be used as a marker to confirm the genus and species of trichomonads. Alignment of protein sequences and prediction of three-dimensional structure showed that PFOR A had a highly conserved structure with two synonymous mutations in the PFOR domain, substituting a V for a G or a S for a P. Our results suggest that the role of genetic variability of PFOR and ITS may not be significant in the symptomatology of this pathogen; however, their utility as genus and species markers in trichomonads is promising.

In the realm of applied phycology, algal physiology, and biochemistry publications, the absence of proper identification and documentation of microalgae is a common concern. This poses a significant challenge for non-specialists who struggle to identify numerous eukaryotic microalgae. However, a promising solution lies in employing an appropriate DNA barcoding technique and establishing comprehensive databases of reference sequences. To address this issue, we conducted a study focusing on the molecular characterization and strain identification of Tetraselmis and Chlorella species, utilizing the internal transcribed spacer (ITS) barcode approach. By analyzing the full nuclear ITS region through the Sanger sequencing approach, we obtained ITS barcodes that were subsequently compared with other ITS sequences of various Tetraselmis and Chlorella species. To ensure the reliability of our identification procedure, we conducted a meticulous comparison of the DNA alignment, constructed a phylogenetic tree, and determined the percentage of identical nucleotides. The findings of our study reveal the significant value of the ITS genomic region as a tool for distinguishing and identifying morphologically similar chlorophyta. Moreover, our results demonstrate that both the ITS1 and ITS2 regions are capable of effectively discriminating isolates from one another; however, ITS2 is preferred due to its greater intraspecific variation. These results underscore the indispensability of employing ITS barcoding in microalgae identification, highlighting the limitations of relying solely on morphological characterization.

Among molecular-based techniques for fungal identification, Sanger sequencing of the primary universal fungal DNA barcode, the internal transcribed spacer (ITS) region (ITS1, 5.8S, ITS2), is commonly used in clinical routine laboratories due to its simplicity, universality, efficacy, and affordability for fungal species identification. However, Sanger sequencing fails to identify mixed ITS sequences in the case of mixed infections. To overcome this limitation, different high-throughput sequencing technologies have been explored. The nanopore-based technology is now one of the most promising long-read sequencing technologies on the market as it has the potential to sequence the full-length ITS region in a single read. In this study, we established a workflow for species identification using the sequences of the entire ITS region generated by nanopore sequencing of both pure yeast isolates and mocked mixed species reads generated with different scenarios. The species used in this study included Candida albicans (n = 2), Candida tropicalis (n = 1), Nakaseomyces glabratus (formerly Candida glabrata) (n = 1), Trichosporon asahii (n = 2), Pichia kudriavzevii (formerly Candida krusei) (n = 1), and Cryptococcus neoformans (n = 1). Comparing various methods to generate the consensus sequence for fungal species identification, the results from this study indicate that read clustering using a modified version of the NanoCLUST pipeline is more sensitive than Canu or VSEARCH, as it classified species accurately with a lower abundance cluster of reads (3% abundance compared to 10% with VSEARCH). The modified NanoCLUST also reduced the number of classified clusters compared to VSEARCH, making the subsequent BLAST+ analysis faster. Subsampling of the datasets, which reduces the size of the datasets by approximately tenfold, did not significantly affect the identification results in terms of the identified species name, percent identity, query coverage, percentage of reads in the classified cluster, and the number of clusters. The ability of the method to distinguish mixed species within sub-populations of large datasets has the potential to aid computer analysis by reducing the required processing power. The herein presented new sequence analysis pipeline will facilitate better interpretation of fungal sequence data for species identification.

Prescribed burn is a management tool that influences the physical structure and composition of forest plant communities and their associated microorganisms. Plant-associated microorganisms aid in host plant disease tolerance and increase nutrient availability. The effects of prescribed burn on microorganisms associated with native ecologically and economically important tree species, such as Cornus florida L. (flowering dogwood), are not well understood, particularly in aboveground plant tissues (e.g., leaf, stem, and bark tissues). The objective of this study was to use 16S rRNA gene and ITS2 region sequencing to evaluate changes in bacterial and fungal communities of five different flowering dogwood-associated niches (soil, roots, bark, stem, and leaves) five months following a prescribed burn treatment. The alpha- and beta-diversity of root bacterial/archaeal communities differed significantly between prescribed burn and unburned control-treated trees. In these bacterial/archaeal root communities, we also detected a significantly higher relative abundance of sequences identified as Acidothermaceae, a family of thermophilic bacteria. No significant differences were detected between prescribed burn-treated and unburned control trees in bulk soils or bark, stem, or leaf tissues. The findings of our study suggest that prescribed burn does not significantly alter the aboveground plant-associated microbial communities of flowering dogwood trees five months following the prescribed burn application. Further studies are required to better understand the short- and long-term effects of prescribed burns on the microbial communities of forest trees.