Evolving techniques in high-throughput (HTP) mass spectrometry (MS) are key to satisfying the ever-increasing sample analysis rates. For a complete analysis using techniques such as AEMS and IR-MALDESI MS, a substantial volume of 20 to 50 liters of sample is indispensable. Liquid atmospheric pressure matrix-assisted laser desorption/ionization (LAP-MALDI) MS is proposed as an alternative for ultra-high-throughput protein analysis, specifically requiring only femtomole quantities within 0.5 liters of solution. A high-speed XY-stage actuator allows for the movement of a 384-well microtiter sample plate, which has facilitated sample acquisition rates of up to 10 samples per second and a resulting data acquisition rate of 200 spectra per scan. MK-28 manufacturer Experimental results indicate that protein mixtures with a concentration of 2 molar can be analyzed efficiently at this rate, whereas individual proteins require a significantly lower concentration of 0.2 molar. This showcases the LAP-MALDI MS method's considerable promise in high-throughput, multiplexed protein analysis.
Squash of the straightneck variety (Cucurbita pepo var.), exhibits a noticeable straight neck structure. Florida farmers rely heavily on the recticollis cucurbit crop for their yield. In the region of Northwest Florida, within a ~15-hectare straightneck squash field, an incident of virus-like symptoms was noted on straightneck squash during the early fall of 2022. Visible symptoms included yellowing, gentle leaf crinkling (as detailed in Supplementary Figure 1), peculiar mosaic patterns, and deformations on the fruit's surface (as illustrated in Supplementary Figure 2). The presence of the disease affected approximately 30% of the plants in the field. Due to the distinct and pronounced symptoms, a theory of multiple viral infections was proposed. Seventeen plants, chosen at random, were subjected to testing. MK-28 manufacturer The plants' freedom from infection with zucchini yellow mosaic virus, cucumber mosaic virus, and squash mosaic virus was verified via Agdia ImmunoStrips (USA). Using the Quick-RNA Mini Prep kit (Cat No. 11-327, Zymo Research, USA), 17 squash plants served as the source material for the total RNA extraction process. Plant samples were analyzed for the presence of cucurbit chlorotic yellows virus (CCYV) (Jailani et al., 2021a) and watermelon crinkle leaf-associated virus (WCLaV-1) and WCLaV-2 (Hernandez et al., 2021), using a conventional OneTaq RT-PCR Kit (Cat No. E5310S, NEB, USA). Specific primers targeting both RNA-dependent RNA polymerase (RdRP) and movement protein (MP) genes were used to test for WCLaV-1 and WCLaV-2 (genus Coguvirus, family Phenuiviridae), revealing 12 out of 17 plants to be positive in Hernandez et al.'s (2021) study, and no positive tests for CCYV. Furthermore, twelve straightneck squash plants exhibited a positive reaction to watermelon mosaic potyvirus (WMV) detection via RT-PCR and sequencing, as detailed in Jailani et al. (2021b). The partial RdRP sequences of WCLaV-1 (OP389252) and WCLaV-2 (OP389254) showed 99% and 976% nucleotide identity, respectively, with the isolates KY781184 and KY781187 from China. In addition, the detection or non-detection of WCLaV-1 and WCLaV-2 was further confirmed through a SYBR Green-based real-time RT-PCR assay. This assay utilized distinct MP primers for WCLaV-1 (Adeleke et al., 2022) and uniquely designed MP primers for WCLaV-2 (WCLaV-2FP TTTGAACCAACTAAGGCAACATA/WCLaV-2RP-CCAACATCAGACCAGGGATTTA). Both viruses were identified in 12 of the 17 straightneck squash plants, thus confirming the accuracy of the initial RT-PCR results. The combined presence of WCLaV-1, WCLaV-2, and WMV resulted in a heightened severity of symptoms manifesting on both the leaves and fruits. Previous research indicated the first appearance of both viruses in the United States within watermelon crops of Texas, Florida, and Oklahoma, and Georgia, along with zucchini plants in Florida, as detailed in the literature (Hernandez et al., 2021; Hendricks et al., 2021; Gilford and Ali, 2022; Adeleke et al., 2022; Iriarte et al., 2023). Straightneck squash in the United States has, for the first time, been found to be affected by WCLaV-1 and WCLaV-2, according to this report. These findings demonstrate the effective dissemination of WCLaV-1 and WCLaV-2, whether in isolated or mixed infections, to cucurbit species other than watermelon in Florida. The significance of evaluating the modes of viral transmission is escalating, to enable development of superior management practices.
In apple orchards of the Eastern United States, bitter rot, a severe summer rot disease, emerges from the presence of Colletotrichum species. Given the disparities in virulence and sensitivity to fungicides between organisms in the acutatum species complex (CASC) and the gloeosporioides species complex (CGSC), the importance of tracking their diversity, geographical distribution, and frequency percentage for successful bitter rot disease control cannot be overstated. A collection of 662 isolates from apple orchards in Virginia demonstrated the superior representation of CGSC isolates, at 655%, compared to the 345% representation of CASC isolates. By analyzing 82 representative isolates using morphological and multi-locus phylogenetic methods, we ascertained the presence of C. fructicola (262%), C. chrysophilum (156%), C. siamense (8%), and C. theobromicola (8%) from the CGSC collection, and C. fioriniae (221%) and C. nymphaeae (16%) from the CASC collection. In terms of abundance, the species C. fructicola ranked highest, followed by C. chrysophilum and, lastly, C. fioriniae. In the context of our virulence tests, 'Honeycrisp' fruit inoculated with C. siamense and C. theobromicola exhibited the most substantial rot lesions, both in size and depth. Fruit from 9 apple cultivars and 1 wild accession of Malus sylvestris, harvested during early and late seasons, were evaluated under controlled conditions for their susceptibility to C. fioriniae and C. chrysophilum. Every cultivated variety displayed susceptibility to both representative bitter rot species, with the Honeycrisp variety proving the most susceptible and Malus sylvestris, accession PI 369855, the most resistant. The Mid-Atlantic region sees substantial variability in the presence and number of Colletotrichum species, with this study offering location-specific insights into apple cultivars' vulnerability. The successful management of bitter rot, an emerging and persistent issue in apple production, both pre- and postharvest, necessitates our findings.
The third most cultivated pulse in India is black gram (Vigna mungo L.), a crucial crop, as stated by Swaminathan et al. (2023). In August 2022, a black gram crop at the Crop Research Center, Govind Ballabh Pant University of Agriculture & Technology, Pantnagar (29°02'22″ N, 79°49'08″ E), Uttarakhand, India, exhibited pod rot symptoms with a disease incidence ranging from 80% to 92%. The presence of a fungal-like growth, showcasing a color gradient from white to salmon pink, indicated disease on the pods. The severity of the symptoms began at the pod tips and then spread to encompass the whole of the pod, in later stages. Pods displaying symptoms housed seeds that were extremely shriveled and lacked viability. Ten plants, part of a comprehensive field study, were selected to determine the pathogenic agent. Following the division of symptomatic pods, their surfaces were disinfected with 70% ethanol for a minute to reduce contamination, followed by triple rinsing with sterile water and thorough air drying on sterilized filter paper. Subsequently, they were aseptically transferred to potato dextrose agar (PDA) plates amended with 30 mg/liter streptomycin sulfate. Following 7 days at 25°C of incubation, three Fusarium-like isolates (FUSEQ1, FUSEQ2, and FUSEQ3) underwent purification via single-spore transfer and were then subcultured on PDA agar. MK-28 manufacturer Floccose, aerial, and initially white to light pink fungal colonies cultivated on PDA later developed an ochre yellowish to buff brown coloration. Transferring isolates to carnation leaf agar (Choi et al., 2014) resulted in the growth of hyaline macroconidia, which exhibited 3 to 5 septa and dimensions of 204 to 556 µm in length and 30 to 50 µm in width (n = 50). These macroconidia were distinguished by tapered, elongated apical cells and prominent foot-shaped basal cells. Chains contained thick, globose, and intercalary chlamydospores in large numbers. A search for microconidia proved unsuccessful. Analysis of morphological features placed the isolates definitively within the Fusarium incarnatum-equiseti species complex (FIESC), according to Leslie and Summerell (2006). To ascertain the molecular identities of the three isolates, genomic DNA was extracted from each using the PureLink Plant Total DNA Purification Kit (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA). This extracted DNA served as the template for amplification and sequencing of the internal transcribed spacer (ITS) region, the translation elongation factor-1 alpha (EF-1) gene, and the RNA polymerase second largest subunit (RPB2) gene, following methods established by White et al. (1990) and O'Donnell (2000). GenBank's repository now includes sequences for the following: ITS (OP784766, OP784777, OP785092); EF-1 (OP802797, OP802798, OP802799); and RPB2 (OP799667, OP799668, OP799669). Fusarium.org is where the polyphasic identification experiments were executed. 98.72% similarity was found between FUSEQ1 and F. clavum. FUSEQ2 and F. clavum exhibited a 100% matching similarity. Meanwhile, FUSEQ3 shared a 98.72% degree of similarity with F. ipomoeae. According to Xia et al. (2019), both of the species identified belong to the FIESC group. Seed pod-bearing potted Vigna mungo plants, aged 45 days, were evaluated for pathogenicity within the confines of a greenhouse. The plants were sprayed with a conidial suspension from each isolate (at 107 conidia per ml), using a volume of 10 ml per plant. A spray of sterile distilled water was administered to the control plants. The inoculated plants were placed inside a greenhouse where the temperature was held at 25 degrees Celsius, and then covered with sterilized plastic bags to maintain humidity levels. After just ten days, the inoculated plants demonstrated symptoms resembling those found in the field, whereas the control plants displayed no symptoms.