Two infected plant tissues, each measuring 5 millimeters by 5 millimeters, were treated sequentially with 95% ethanol for one minute, followed by 70% ethanol for one minute and finally 1% sodium hypochlorite for one minute, to isolate the causative pathogen. The samples were rinsed thrice with distilled water and then dried using sterile filter paper. Subsequently, the samples were transferred to a medium containing 15% water agar and 100 ppm streptomycin, and incubated at 25 degrees Celsius in the dark. Using potato dextrose agar (PDA, Sparks, MD 21152, USA), three independent isolates (HNO-1, HNO-2, HNO-3) from Haenam tissues and three others (KJO1-1, KJO1-2, KJO1-3) from Ganjin tissues were developed after single-hypha-tip purification of hyphae emerging from randomly chosen independent tissue samples at each site. Initially, the PDA colonies displayed a white pigmentation, subsequently changing to a light brown after fourteen days. Two weeks' incubation on PDA resulted in all collected isolates developing globose and irregular sclerotia that were a dark brown to black color. White to dark brown binuclear hyphae, branching at right angles with a septum near each branch, and multinucleate cells, strongly suggest that these isolates are Ceratobasidium cereale, as reported by Boerema et al. (1977), Burpee (1980), and Sharon et al. (2008). Molecular identification of the organism hinges on the ITS sequence (GenBank accession numbers provided). Using primer pairs ITS4/5 (White et al., 1990), LROR/LR5 (Vilgalys and Hester, 1990), bRPB2-6F/bRPB2-71R (Matheny, 2005; Reeb et al., 2004), TEF1-F/TEF1-R (Litvintseva et al., 2006), and ATP61/ATP62 (Kretzer and Bruns, 1999), respectively, the amplification of MW691851-53 (HNO-1 to HNO-3), MW691857-59 (KJO1-1 to KJO1-3), LSU (OQ397530-35), rpb2 (OQ409878-83), tef1 (OQ409884-89), and atp6 (OQ409890-95) regions from six isolates was carried out. The ITS region sequence analysis revealed 99.7% identity for C. cereale strain WK137-56 (KY379365) and 99.8% for Ceratobasidium sp. Medicina defensiva For the record, AG-D is linked to KP171639. A maximum likelihood phylogenetic analysis, performed with the MEGA X software (Kumar et al., 2018), classified the six isolates within a clade containing C. cereale, supported by analyses of concatenated ITS-LSU, rpb2, tef1, and atp6 sequences (Gonzalez et al., 2016; Ji et al., 2017; Tomioka et al., 2021; Li et al., 2014). Deposited in the Korean Agriculture Culture Collection were the representative isolates HNO-1, assigned accession number KACC 49887, and KJO1-1, with accession number KACC 410268. Six isolates were cultivated for pathogenicity assessment using sterilized ray grains at 25°C in darkness, allowing them to grow for three weeks to serve as the inoculum. Cultivars five oats ( Choyang seeds were planted in receptacles, each holding 80 grams of infected ray grains, 150 grams of composite soil, and 150 milliliters of water from (Baroker Garden Soil, Seoul Bio Co., LTD). A mixture of 80 grams sterilized ray grains, 150 grams of composite soil, and 150 milliliters of water was used to treat the control. In the controlled environment of a 20°C growth chamber, inoculated and control pots were positioned to experience a 12-hour photoperiod and 65% humidity. The symptoms of sharp eyespots, recognizable on the oat sheaths of seedlings, appeared three weeks after the inoculation. No symptoms were noted in the control plants. Each of the three infection assays produced analogous results. The re-isolation of the pathogen was followed by confirmation of its identity through morphological and molecular analyses. Oats, less economically viable than barley and wheat in Korea, have garnered limited etiological research. Previous reports documented sharp eyespot disease, caused by C. cereale, in barley and wheat (Kim et al., 1991); this study, however, presents the first case of this disease affecting oats within Korea.
The oomycete Phytopythium vexans, identified by de Bary, Abad, de Cock, Bala, Robideau, A. M. Lodhi, and Levesque, is a prevalent waterborne and soil-inhabiting pathogen, causing root and crown rot in a wide array of plants, encompassing many woody ornamentals, fruits, and forest trees. Early and accurate Phytophthora detection in nursery environments is of paramount importance, given the pathogen's rapid spread to neighboring healthy plants via the irrigation network. Unfortunately, conventional strategies for the detection of this pathogen are frequently characterized by time-consuming procedures, ambiguous outcomes, and substantial financial burdens. Thus, a precise, sensitive, and quick molecular diagnostic method is required to overcome the impediments presented by traditional identification techniques. To identify *P. vexans*, a loop-mediated isothermal amplification (LAMP) assay was designed and implemented in this study. While designing and screening several sets of LAMP primers, PVLSU2 demonstrated specificity for P. vexans, failing to amplify other closely related oomycetes, fungi, and bacteria. The developed assays, moreover, were sufficiently sensitive to amplify DNA quantities up to 102 femtograms per reaction. In detecting infected plant specimens, the real-time LAMP assay demonstrated a greater sensitivity than traditional PCR and culture-based methodologies. Additionally, the sensitivity of both LAMP assays enabled detection of as few as 100 zoospores when dispersed in 100 milliliters of water. Disease diagnostic labs and research institutions are expected to experience time savings in P. vexans detection thanks to the anticipated implementation of LAMP assays, allowing for earlier preparedness during disease outbreaks.
The devastating powdery mildew is caused by the specific fungal strain Blumeria graminis f. sp. The tritici (Bgt) strain is a growing concern for wheat production's future in China. The initial work in breeding mildew-resistant cultivars comprises mapping quantitative trait loci (QTL) for resistance to powdery mildew and generating markers readily adopted by breeders. From a cross of Jingdong 8 and Aikang 58, a population of 254 recombinant inbred lines (RILs) yielded the identification of an all-stage resistance gene and multiple QTLs. The population's resistance to powdery mildew was evaluated across six field environments over three successive growing seasons, employing two distinct mixtures of Bgt isolates, #Bgt-HB and #Bgt-BJ. Seven consistently observed QTLs were mapped to chromosome arms 1DL, 2AL, 2DS, 4DL, 5AL, 6BL.1, and 6BL.2 by employing the genotypic data from the Wheat TraitBreed 50K SNP array. The QTL situated on chromosome 2AL displayed resistance to all stages of Bgt race E20, evidenced in greenhouse tests and correlating with up to 52% of the observed phenotypic variance in field trials, though only showing resistance against #Bgt-HB. Based on its genomic location and DNA sequence, the gene responsible for this QTL was anticipated to be Pm4a. The entity QPmja.caas-1DL presents a multifaceted challenge. Research highlighted QPmja.caas-4DL and QPmja.caas-6BL.1 as possible new QTL influencing powdery mildew resistance. QPmja.caas-2DS and QPmja.caas-6BL.1 demonstrated activity against the diverse range of Bgt mixtures, implying a broad-spectrum resistant nature. A KASP marker, exhibiting close linkage to QPmja.caas-2DS, was created and verified across a group of 286 wheat cultivars. The leading cultivars, Jingdong 8 and Aikang 58, having served as pivotal breeding parents, underscore the value of the reported QTL and markers for wheat research and breeding efforts.
In the Yangtze River basin, the perennial herbaceous plant Bletilla striata, belonging to the Orchidaceae family, is a common native of China. sternal wound infection B. striata, a medicinal plant, serves as a conventional remedy for wound bleeding and inflammation in China. During September 2021, a substantial portion (over 50%) of B. striata plants within a 10-hectare traditional Chinese medicine plantation in Xianju City, Zhejiang Province, China, exhibited noticeable leaf spot symptoms. Necrotic spots, small, round, and pale brown, appeared initially on the leaves. Afterward, the lesions' central areas assumed a grayish-brown color. Their edges turned dark brown with slight protuberances, eventually reaching 5-8 mm in size on the leaves. Over the course of time, the small spots increased in size and joined, developing into necrotic streaks (1-2 cm) in length. Leaves demonstrating disease characteristics were collected, surface-sterilized, and cultivated on plates containing potato dextrose agar (PDA). Following 3 days of incubation at 26 degrees Celsius, colonies of fungi (2828 mm) were observed, characterized by grayish-black mycelia that spread through all tissues. Basal conidia displayed a range of colors from pale to dark brown, in sharp contrast to the uniform pale brown pigmentation of apical conidia. Central cells of apical conidia were significantly larger and darker in shade compared to their counterparts in the basal conidia. Conidia, characterized by smooth surfaces and rounded tips, presented as fusiform, cylindrical, or subtly curved morphologies. Length measurements spanned the range of 2234 to 3682 meters, with a mean of 2863 meters, and included 2 to 4 septations that had subtle constrictions. Monospore isolation was employed with the aim of achieving a pure culture sample. Subsequently, the BJ2Y5 strain was preserved within the strain preservation facilities at Wuhan University (Wuhan, China), receiving the preservation identifier CCTCC M 2023123. From PDA plates incubated at 26 degrees Celsius for a period of seven days, the newly grown mycelia and conidia were gathered. The Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech Co., Shanghai, China) was employed to extract the DNA. Menadione manufacturer The phylogenetic position of isolate BJ2-Y5 was elucidated through DNA sequencing analysis of three genetic markers: glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the internal transcribed spacer region (ITS), and a portion of the second largest subunit of RNA polymerase II (RPB2). A BLAST search, employing GenBank accession numbers, produces. Comparatively, isolates OP913168, OP743380, and OP913171 demonstrated a high degree of homology (99%) to the reference isolate CBS 22052.