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.

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.

Blister blight (BB) disease is caused by the obligate biotrophic fungal pathogen Exobasidium vexans Massee and seriously affects the yield and quality of Camellia sinensis. The use of chemical pesticides on tea leaves substantially increases the toxic risks of tea consumption. Botanic fungicide isobavachalcone (IBC) has the potential to control fungal diseases on many crops but has not been used on tea plants. In this study, the field control effects of IBC were evaluated by comparison and in combination with natural elicitor chitosan oligosaccharides (COSs) and the chemical pesticide pyraclostrobin (Py), and the preliminary action mode of IBC was also investigated. The bioassay results for IBC or its combination with COSs showed a remarkable control effect against BB (61.72% and 70.46%). IBC, like COSs, could improve the disease resistance of tea plants by enhancing the activity of tea-plant-related defense enzymes, including polyphenol oxidase (PPO), catalase (CAT), phenylalanine aminolase (PAL), peroxidase (POD), superoxide dismutase (SOD), β-1,3-glucanase (Glu), and chitinase enzymes. The fungal community structure and diversity of the diseased tea leaves were examined using Illumina MiSeq sequencing of the internal transcribed spacer (ITS) region of the ribosomal rDNA genes. It was obvious that IBC could significantly alter the species\' richness and the diversity of the fungal community in affected plant sites. This study broadens the application range of IBC and provides an important strategy for the control of BB disease.

A new species, Wikstroemiafragrans (Thymelaeaceae, Daphneae), from Danxiashan National Park, Shaoguan, Guangdong of China is described and illustrated. It is similar to the sympatric W.trichotoma, but can be differentiated easily from the latter by its shorter racemose inflorescences, yellowish green calyx tube, and smaller leaves. It also resembles the allopatric W.fargesii, but differs from it by its strigose-pubescent ovary and disk scale that is 2- or 3-dentate apically. Phylogenetic analysis using the nuclear DNA internal transcribed spacer (ITS) region revealed that W.fragrans falls within the Wikstroemia clade; based on current sampling, W.fragrans is closely-related to W.capitata. It is also the first species of Wikstroemia known to be endemic to the Danxia landform and is classified provisionally as Critically Endangered according to the IUCN Red List Categories and Criteria.

The internal transcribed spacer (ITS) is one of the most extensively sequenced molecular markers in plant systematics due to its generally concerted evolution. While non-concerted evolution has been found in some plant taxa, such information is missing in Lycium. Molecular studies of six species and two variants of the genus Lycium revealed high levels of intra- and inter-individual polymorphism in the ITS, indicating non-concerted evolution. All genomic DNA ITS paralogues were identified as putative pseudogenes or functional paralogues through a series of comparisons of sequence features, including length and substitution variation, GC content, secondary structure stability, and the presence of conserved motifs in the 5.8S gene, and the rate of evolution. Approximately, 60% of ITS pseudogenes could be easily detected. Based on phylogenetic analysis, all pseudogenes were highly distinct from their corresponding functional copies, tended to evolve neutrally, and clustered randomly together in the evolutionary tree. The results probably suggest that this ITS non-concerted evolution is related to the recent divergence between tandem repeats within the Lycium genome and hybridization between species. Our study complements those of pseudogenes in plant taxa and provides a theoretical basis for the phylogeny and genetic origin of the genus Lycium while having important implications for the use of ITS molecular markers for phylogenetic reconstruction.

In the present research, challenges arose when many reports have been published on the poisoning of humans due to the ingestion of crops of Crataegus plants contaminated with cyanobacterial toxins. The discovery of several poisonings around agricultural zones prompted us to study the toxic compounds in a strain of Neowestiellopsis which is the most abundant in the agricultural zones of Kermanshah province of Iran, using a polyphasic approach. Molecular procedure was followed to study these strains deeply.
To elucidate their systematic position, besides the 16S rRNA gene, the analyses of molecular toxicity markers, namely nos, mcy G, mcy D and internal transcribed spacer (ITS), were also used.
Based on the results, for the first time, we record the presence of a gene cluster coding for the biosynthesis of a bioactive compound (Nostopeptolides) that is very rare in this family and the presence of toxic compounds (microcystin), which might account for the poisoning of humans.
This case is the first observation of a toxic soil strain from the genus Neowestiellopsis from agricultural fields in Iran.

In recent years, trafficking and abuse of hallucinogenic mushrooms have become a serious social problem. It is therefore imperative to identify hallucinogenic mushrooms of the genus Psilocybe for national drug control legislation. An internal transcribed spacer (ITS) is a DNA barcoding tool utilized for species identification. Many methods have been used to discriminate the ITS region, but they are often limited by having a low resolution. In this study, we sought to analyze the ITS and its fragments, ITS1 and ITS2, by using high-resolution melting (HRM) analysis, which is a rapid and sensitive method for evaluating sequence variation within PCR amplicons. The ITS HRM assay was tested for specificity, reproducibility, sensitivity, and the capacity to analyze mixture samples. It was shown that the melting temperatures of the ITS, ITS1, and ITS2 of Psilocybe cubensis were 83.72 ± 0.01, 80.98 ± 0.06, and 83.46 ± 0.08 °C, and for other species, we also obtained species-specific results. Finally, we performed ITS sequencing to validate the presumptive taxonomic identity of our samples, and the sequencing output significantly supported our HRM data. Taken together, these results indicate that the HRM method can quickly distinguish the DNA barcoding of Psilocybe cubensis and other fungi, which can be utilized for drug trafficking cases and forensic science.

Dumasia taxonomy and classification have long been problematic. Species within this genus have few morphological differences and plants without flowers or fruits are difficult to accurately identify. In this study, we evaluated the ability of six DNA barcoding sequences, one nuclear (ITS) and five chloroplast regions (trnH-psbA, matK, rbcL, trnL-trnF, psbB-psbF), to efficiently identify Dumasia species. Most single markers or their combinations identify obvious barcoding gaps between intraspecific and interspecific genetic variation. Most combined analyses including ITS showed good species resolution and identification efficiency. We therefore suggest that ITS alone or a combination of ITS with any cpDNA marker are most suitable for DNA barcoding of Dumasia. The phylogenetic analyses clearly indicated that Dumasia yunnanensis is not monophyletic and is separated as two independent branches, which may result from cryptic differentiation. Our results demonstrate that molecular data can deepen the comprehension of taxonomy of Dumasia and provide an efficient approach for identification of the species.