Dry, dark-brown lesions, readily detaching from the infected leaves, were evident (Fig. 2A). Selleckchem Nirmatrelvir In a contiguous manner, both plants were cultivated. For the A. obesum species, 80% (out of 5 plants) were found to be affected, and all 3 P. americana specimens examined were 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. The excised fragments were positioned on potato dextrose agar (PDA) media (Laboratorios Conda S.A., Spain) and maintained in an incubator set to 28 degrees Celsius for seven days. Ten isolates were derived from the symptomatic samples of A. obesum and P. americana foliage and stems. qatar biobank Beginning as white, fungal colonies transitioned to black, displaying a light yellow reverse side (Figures 1B and 2B). Their conidiophores were biseriate and bore globose vesicles; conidia were spherical, light tan to black in color, featuring smooth or roughened walls and sizes ranging from 30 to 35 µm (n = 15) (Figures 1C and 2C). All the isolates displayed characteristics consistent with those of Aspergillus species, based on these observations. In 1965, Bryan and Fennell's research delivered a substantial contribution to the field. The liquid nitrogen and phenol-chloroform extraction method, as reported in Butler (2012), was used to extract the DNA sample. Using the primer pairs ITS4/ITS5 (Abliz et al., 2003) and cmd5/cmd6 (Hong et al., 2005), respectively, a 526-base-pair product from the ITS region of rDNA and a 568-base-pair product from the calmodulin protein-coding gene were amplified. The PCR process was carried out under these conditions: initial denaturation at 94°C for 5 minutes, then 35 cycles consisting of denaturation at 95°C for 30 seconds, annealing at 52°C for 40 seconds, and finally extension at 72°C for 50 seconds. The protocol included a 7-minute extension at 72°C as a concluding phase. Employing the BigDye Terminator v31 Cycle Sequencing Kit (Applied Biosystems), the sequencing of the sample was carried out, and the obtained sequence was submitted to GenBank with its corresponding accession numbers. Concerning *A. obesum* (ON519078) and *P* (ON519079), their respective ITS sequences are documented. The list of proteins includes americana ITS, OQ358173 (calmodulin from A. obesum), and OQ358174 (a protein from the species P.). Intriguing insights into the functioning of calmodulin, observed within the americana species, are constantly being revealed. Comparative analysis of these sequences against other A. niger sequences in GenBank was performed using BLAST, encompassing accession numbers MG5696191, MT5887931, MH4786601, MZ7875761, and MW0864851. A consistent pattern emerged across the sequences of ten isolates, displaying a 98-100% similarity to the Aspergillus niger sequences (Figure 3). To conduct the phylogenetic analysis, MEGA 11 (Tamura et al., 2021) was used. To confirm the infectious nature of the organism, three asymptomatic plants each were injected with a conidia suspension (10^6 conidia/mL), produced from 2-week-old cultures, using a pinprick method. infant immunization Sterile distilled water was applied to the control plants for inoculation purposes. After inoculation, plants were placed in a Binder climate chamber (Germany) and held at 28°C for a duration of 10 days. Symptoms were noticed in inoculated plants of P. americana after 2 days, while inoculated A. obesum plants took 5 days for symptoms to appear on their leaves. Drying commenced in the stems of the affected leaves, which also exhibited a yellowing. Leaf symptoms displayed a pattern akin to those found in naturally infected plants, while the control plants remained entirely without any symptoms. The A. niger pathogen's presence was confirmed through its re-isolation. Our current knowledge indicates that this is the initial report highlighting A. niger's contribution to stem rot in A. obesum and leaf spot in P. americana, observed within Kazakhstan. Since ornamental plants are frequently intermixed in gardens and nurseries, growers need to be cognizant of the potential for A. niger to spread amongst them. This finding provides a springboard for further study into the biological and epidemiological nature of this illness, spurring the development of diagnostic tools and appropriate management strategies.

Charcoal rot, a soil-borne disease triggered by Macrophomina phaseolina, is prevalent and has been documented as affecting soybean, corn, and numerous other plants, including hemp used for fiber, grain, and cannabinoid production (Casano et al., 2018; Su et al., 2001). Hemp (Cannabis sativa) production in Missouri during 2021 represented a relatively recent entry into the state's agricultural scene. Commercial and experimental fields in Reynolds, Knox, and Boone counties of Missouri experienced reports of charcoal rot. Due to a severe disease outbreak and a non-uniform plant loss, one field under scrutiny saw roughly 60% of its yield affected, a loss directly attributable to charcoal rot. During July and late fall of 2021, a considerable number of hemp plants displayed symptoms consistent with charcoal rot. These included microsclerotia on lower stem and root tissue, wilting, and stem discoloration. These specimens were received at the University of Missouri Plant Diagnostic Clinic from the Bradford Research Farm in Boone County and the Greenley Research Center in Knox County. Plant tissues, including roots and crowns, collected from hemp plants at the Greenley Research Center, were placed onto a medium of acidified potato dextrose agar (APDA). Macrophomina phaseolina, as well as various other fungi, demonstrated growth from the plated tissue after about 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. Forty-four microsclerotia were found to be black, characterized by a round to ovoid shape, and exhibited a length varying from 34 to 87 micrometers (average 64 micrometers) and a width varying from 32 to 134 micrometers (average 65 micrometers). A pure culture of the M. phaseolina isolate was prepared by isolating a single hypha from the putative sample. Four hemp cultivars were assessed for charcoal rot, utilizing the M. phaseolina culture from the Greenley Research Center to verify Koch's postulates. Following the addition of sterilized toothpicks, pure cultures of M. phaseolina on APDA plates were incubated at room temperature for one week to enable colonization, making them suitable for greenhouse inoculation. Utilizing sterilized silt loam, four hemp cultivars, Katani, Grandi, CFX-2, and CRS-1, were cultivated in a greenhouse for a duration of three weeks. To enable inoculation, four plants were cultivated for each cultivar, and one plant per cultivar acted as a control. The plants' stem tissue was inoculated with M. phaseolina-colonized toothpicks, gently rubbed onto the stem and then inserted into the soil at the stem's location. For six weeks, the plants were cultivated under regulated greenhouse conditions—25 degrees Celsius, a 12-hour light/dark cycle, and watering on an as-needed basis as determined by the dryness of the soil. To prevent cross-contamination with other greenhouse plants, wooden and vinyl-coated containers, only loosely sealed, held the plants. Plants were routinely examined weekly for indications of charcoal rot. Symptoms of charcoal rot, including wilting and the presence of microsclerotia on the lower stem, appeared on the inoculated plants after about four weeks, while the control plants displayed no such symptoms. From diseased plants, isolates with characteristics strikingly similar to M. phaseolina were obtained; consequently, the recovery of the fungus from inoculated plants confirmed the satisfactory fulfillment of Koch's postulates. The GeneJet Plant Genomic DNA Purification Kit (Thermo Scientific, California, USA) was employed to extract DNA from pure cultures of both the original isolate and the isolate derived through Koch's postulates. Subsequently, the internal transcribed spacer (ITS) region of ribosomal DNA, encompassing ITS1, 58S, and ITS4 segments, was amplified using universal primers ITS1 and ITS4 (White et al., 1990). By employing BLAST analysis, the ITS region's sequence was sequenced and compared to reference sequences in GenBank. Further research included a detailed examination of the recovered isolates, indicated by their GenBank accession number. Sequence OQ4559341 demonstrated a complete (100%) match to the M. phaseolina accession number GU0469091. Soil inoculum buildup in Missouri hemp, along with its growth conditions and life cycle, is poorly understood. Moreover, the corn and soybean crops are susceptible to *M. phaseolina*, a known pathogen, and implementing successful management strategies proves challenging owing to the pathogen's extensive host range. Agricultural practices focused on cultural management, including the use of crop rotation to decrease the concentration of disease agents in the soil and diligent monitoring for symptoms, might effectively lessen the impact of this disease.

The Tropical Botanical Museum, situated in Nanjing Zhongshan Botanical Garden, Jiangsu Province, China, proudly displays Adenia globosa, an exquisite indoor ornamental plant. A new stem basal rot disease was observed on A. globosa seedlings, which were planted in the area during September of 2022. Stem basal rot affected an estimated 80% of the A. globosa seedlings. Decaying basal stems of the cutting seedlings, accompanied by eventual drying of the stem tips because of water loss, are detailed in Figure S1A. To ascertain the pathogen, three cuttings, exhibiting disease symptoms, were harvested from separate pots within the Tropical Botanical Museum's collection. Stem portions, measuring 3 to 4 millimeters, were taken from the boundary region between healthy and diseased tissue. Surface sterilization was performed using 75% ethanol for 30 seconds, followed by 15% sodium hypochlorite for 90 seconds. Subsequent rinsing with sterile distilled water was done three times. The segments were then plated onto potato dextrose agar (PDA) plates and kept in the dark for incubation at 25 degrees Celsius.