Easily separable, dry, dark-brown lesions were a characteristic feature of the infected leaves (Fig. 2A). BioBreeding (BB) diabetes-prone rat Both plants were grown alongside one another. The affliction affected 80% of the 5 A. obesum plants studied, and a complete 100% of the 3 P. americana plants were also affected. The infected tissues, harvested from the leaves and stems of A. obesum and P. americana, were cut into 5 mm x 5 mm pieces, immersed in 70% ethanol for 5 minutes, and washed three times with sterile distilled water to isolate the infectious agent. For seven days, cut sections were placed onto potato dextrose agar (PDA) (Laboratorios Conda S.A., Spain) and kept in an incubator maintained at a temperature of 28 degrees Celsius. Ten isolates were harvested from the symptomatic portions, leaves and stems, of the A. obesum and P. americana plant material. Selumetinib mouse Fungal colonies, initially white, gradually turned black, with a light yellow underside (Figures 1B and 2B). Biseriate conidiophores bore globose vesicles. Conidia were spherical, ranging in color from light tan to black, and exhibited smooth or roughened walls; sizes ranged from 30 to 35 micrometers (n=15) as seen in Figures 1C and 2C. According to these observations, all the isolates exhibited features indicative of Aspergillus species. Their investigation, undertaken by Bryan and Fennell in 1965, produced important conclusions. DNA extraction was performed using a liquid nitrogen and phenol-chloroform method, as detailed in Butler (2012). Primer sets ITS4/ITS5 (Abliz et al., 2003) for the ITS region of rDNA, and cmd5/cmd6 (Hong et al., 2005) for the calmodulin protein-coding gene were utilized to amplify 526 bp and 568 bp products, respectively. The PCR reaction was conducted under the following parameters: an initial denaturation at 94°C for 5 minutes, followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at 52°C for 40 seconds, and extension at 72°C for 50 seconds. An additional extension at 72°C for 7 minutes was part of the process. Utilizing the BigDye Terminator v31 Cycle Sequencing Kit (Applied Biosystems), the sequencing procedure was performed, and the generated sequence was subsequently deposited in GenBank, along with its accession numbers. Identified as *A. obesum* (ON519078) and *P* (ON519079), these ITS sequences are recorded. Proteins such as americana ITS, OQ358173 (calmodulin in A. obesum), and OQ358174 (a protein in P.) were found. Scientific exploration into the role of calmodulin within the americana species is crucial for unraveling the intricate complexities of biology. Utilizing BLAST, the sequences were juxtaposed with related A. niger sequences from GenBank, including MG5696191, MT5887931, MH4786601, MZ7875761, and MW0864851, for comparative examination. The ten isolate sequences demonstrated complete congruence, registering an identity rate of 98-100% with the sequences of Aspergillus niger (Figure 3). Using MEGA 11 (Tamura et al., 2021), a phylogenetic analysis was completed. To establish the pathogenic nature of the agent, three asymptomatic specimens of each group were inoculated via pinprick with a conidia suspension (10^6 conidia/mL), obtained from 2-week-old cultures. peripheral immune cells Sterile distilled water was utilized in the inoculation process of control plants. The plants, having been inoculated, were positioned within a climate chamber (Binder, Germany) and kept at 28°C for 10 days. Symptoms appeared on the leaves of P. americana plants inoculated 2 days earlier, whereas those of A. obesum plants developed symptoms only after 5 days of inoculation. Drying commenced in the stems of the affected leaves, which also exhibited a yellowing. Leaf symptoms displayed remarkable resemblance to those observed in naturally infected plants, whereas control plants displayed no symptoms whatsoever. The presence of the A. niger pathogen was demonstrably confirmed through its re-isolation. Based on our current information, this represents the first reported instance of A. niger causing stem rot in A. obesum and leaf spot in P. americana within the Kazakhstan region. Growers should acknowledge the possibility of A. niger spreading between different ornamentals frequently planted together in gardens and nurseries. This observation forms a basis for future investigations into the disease's biology and prevalence, paving the way for the development of diagnostic tools and treatment protocols.
The abundance of Macrophomina phaseolina, the causative agent of charcoal rot, in the soil poses a threat to various plants, including soybean, corn, and hemp, which is used for both fiber, grains, and cannabinoids (Casano et al. 2018; Su et al. 2001). Hemp (Cannabis sativa) production, a fairly recent development, joined Missouri's 2021 agricultural scene. Charcoal rot plagued commercial and experimental fields in the Missouri counties of Reynolds, Knox, and Boone. Heavy disease pressure and a non-uniform plant loss caused an estimated 60% loss in one field, a loss which is attributable to charcoal rot. In July and late fall of 2021, a substantial number of hemp plants exhibiting charcoal rot, including symptoms like microsclerotia on lower stem and root tissues, wilting, and stem discoloration, were examined at the University of Missouri Plant Diagnostic Clinic. Samples originated from both the Bradford Research Farm in Boone County and the Greenley Research Center in Knox County. Hemp plant tissue, consisting of roots and crowns, procured from the Greenley Research Center, was cultured on a specialized acidified potato dextrose agar (APDA) medium. Macrophomina phaseolina, along with other fungal species, emerged from the cultured tissue after roughly three days of incubation at room temperature. The authors of Siddique et al. (2021) observed the diagnostic characteristics of melanized hyphae and microsclerotia, thus validating the presence of Macrophomina phaseolina. Black, round to ovoid microsclerotia, in a sample size of 44, demonstrated a range in length from 34 to 87 micrometers (average 64 micrometers) and a range in width from 32 to 134 micrometers (average 65 micrometers). To secure a pure culture, a single hypha from the suspected M. phaseolina isolate was separated and cultivated. The application of the M. phaseolina culture, obtained from the Greenley Research Center, resulted in the demonstration of Koch's postulates for charcoal rot in four hemp cultivars. A week of incubation at room temperature was used to enable colonization and greenhouse inoculation of pure cultures of M. phaseolina grown on APDA media, to which sterilized toothpicks were added. Four hemp cultivars, including Katani, Grandi, CFX-2, and CRS-1, underwent a three-week cultivation period in a greenhouse, utilizing sterilized silt loam as the growing medium. Four plants per cultivar were grown for the inoculation experiments, with one plant per cultivar functioning as a control. M. phaseolina colonized toothpicks were delicately applied to the stem tissue of the plants, and then implanted in the soil at the stem juncture. For a period of six weeks, the plants were maintained under greenhouse conditions, which included a temperature of 25 degrees Celsius, a light/dark cycle of twelve hours each, and watering as needed when the soil exhibited signs of dryness. Plants were maintained in a wood and vinyl enclosure, only loosely covered, to prevent cross-contamination from other plants in the greenhouse. To identify charcoal rot, plants were inspected weekly for symptoms. After approximately four weeks, inoculated plants exhibited symptoms mirroring charcoal rot, including wilting and microsclerotia on the lower stem, whereas control plants remained asymptomatic. Cultural isolates, reminiscent of M. phaseolina, were obtained from diseased plants; therefore, the successful recovery of the fungus from the inoculated plants affirmed the validity of Koch's postulates. The GeneJet Plant Genomic DNA Purification Kit (Thermo Scientific, California, USA) was used to isolate DNA from the pure cultures of both the initial isolate and the isolate obtained via Koch's postulates. This was followed by amplification of the internal transcribed spacer (ITS) region of ribosomal DNA, including ITS1, 58S, and ITS4, using universal primers ITS1 and ITS4 according to the procedure outlined in White et al. (1990). A BLAST analysis compared the ITS region's sequence to reference sequences within the GenBank database. A detailed examination of the recovered isolates, with their GenBank accession number, was performed. The sequence of OQ4559341 demonstrated a 100% similarity to the M. phaseolina accession number GU0469091. The hemp plant's developmental stages, optimal growth parameters, and the capacity for inoculum accumulation within the soil in Missouri are poorly documented. Likewise, *M. phaseolina*, a pathogen of corn and soybeans, poses a challenge for developing effective management practices, specifically due to the pathogen's wide host range. Cultural management strategies, consisting of crop rotation to curtail the disease inoculum in the soil and a vigilant monitoring system for disease symptoms, might help mitigate the intensity of this disease.
In Nanjing Zhongshan Botanical Garden, Jiangsu Province, China, the Tropical Botanical Museum features the indoor ornamental plant Adenia globosa. A. globosa seedlings planted in September 2022 displayed a newly recognized stem basal rot disease in this region. A substantial portion, roughly 80%, of A. globosa seedlings showed symptoms of stem basal rot. The basal stems of the cutting seedlings exhibited signs of decay, and the stem tips subsequently dried out as a result of water loss (Figure S1A). For isolating the pathogen, three diseased stems were painstakingly selected from three cuttings grown in different pots at the Tropical Botanical Museum. Stem segments, ranging from 3 to 4 mm in length, were extracted from the border areas between healthy and diseased tissue. These were then treated with 75% ethanol for 30 seconds and 15% sodium hypochlorite for 90 seconds for surface sterilization. Following three rinses in sterilized distilled water, the sections were cultured on potato dextrose agar (PDA) plates and incubated in complete darkness at 25 degrees Celsius.