Inverse regulation of SOS1 and HKT1 protein localization and stability by SOS3/CBL4 in Arabidopsis thaliana.

To control net sodium (Na+) uptake, Arabidopsis plants utilize the plasma membrane (PM) Na+/H+ antiporter SOS1 to achieve Na+ efflux at the root and Na+ loading into the xylem, and the channel-like HKT1;1 protein that mediates the reverse flux of Na+ unloading off the xylem. Together, these opposing transport systems govern the partition of Na+ within the plant yet they must be finely co-regulated to prevent a futile cycle of xylem loading and unloading. Here, we show that the Arabidopsis SOS3 protein acts as the molecular switch governing these Na+ fluxes by favoring the recruitment of SOS1 to the PM and its subsequent activation by the SOS2/SOS3 kinase complex under salt stress, while commanding HKT1;1 protein degradation upon acute sodic stress. SOS3 achieves this role by direct and SOS2-independent binding to previously unrecognized functional domains of SOS1 and HKT1;1. These results indicate that roots first retain moderate amounts of salts to facilitate osmoregulation, yet when sodicity exceeds a set point, SOS3-dependent HKT1;1 degradation switches the balance toward Na+ export out of the root. Thus, SOS3 functionally links and co-regulates the two major Na+ transport systems operating in vascular plants controlling plant tolerance to salinity.

Genome-wide identification of heavy-metal ATPases genes in Areca catechu: investigating their functionality under heavy metal exposure.

Heavy-metal ATPases (HMAs) play a vital role in plants, helping to transport heavy metal ions across cell membranes.However, insufficient data exists concerning HMAs genes within the Arecaceae family.In this study, 12 AcHMA genes were identified within the genome of Areca catechu, grouped into two main clusters based on their phylogenetic relationships.Genomic distribution analysis reveals that the AcHMA genes were unevenly distributed across six chromosomes. We further analyzed their physicochemical properties, collinearity, and gene structure.Furthermore, RNA-seq data analysis exhibited varied expressions in different tissues of A. catechu and found that AcHMA1, AcHMA2, and AcHMA7 were highly expressed in roots, leaves, pericarp, and male/female flowers. A total of six AcHMA candidate genes were selected based on gene expression patterns, and their expression in the roots and leaves was determined using RT-qPCR under heavy metal stress. Results showed that the expression levels of AcHMA1 and AcHMA3 genes were significantly up-regulated under Cd2 + and Zn2 + stress. Similarly, in response to Cu2+, the AcHMA5 and AcHMA8 revealed the highest expression in roots and leaves, respectively. In conclusion, this study will offer a foundation for exploring the role of the HMAs gene family in dealing with heavy metal stress conditions in A. catechu.

ABA INSENSITIVE 2 promotes flowering by inhibiting OST1/ABI5-dependent FLOWERING LOCUS C transcription in Arabidopsis.

The plant hormone abscisic acid (ABA) is an important regulator of plant growth and development and plays a crucial role in both biotic and abiotic stress responses. ABA modulates flowering time, but the precise molecular mechanism remains poorly understood. Here we report that ABA INSENSITIVE 2 (ABI2) is the only phosphatase from the ABA-signaling core that positively regulates the transition to flowering in Arabidopsis. Loss-of-function abi2-2 mutant shows significantly delayed flowering both under long day and short day conditions. Expression of floral repressor genes such as FLOWERING LOCUS C (FLC) and CYCLING DOF FACTOR 1 (CDF1) was significantly up-regulated in abi2-2 plants while expression of the flowering promoting genes FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) was down-regulated. Through genetic interactions we further found that ost1-3 and abi5-1 mutations are epistatic to abi2-2, as both of them individually rescued the late flowering phenotype of abi2-2. Interestingly, phosphorylation and protein stability of ABA INSENSITIVE 5 (ABI5) were enhanced in abi2-2 plants suggesting that ABI2 dephosphorylates ABI5, thereby reducing protein stability and the capacity to induce FLC expression. Our findings uncovered the unexpected role of ABI2 in promoting flowering by inhibiting ABI5-mediated FLC expression in Arabidopsis.

Global Genetic Diversity of Tobacco Ringspot Virus Including Newly Reported Isolates from Cotton (Gossypium hirsutum) in Oklahoma.

Cotton is one of the most salient cash crops globally and in the United States. Lately, several virus-like diseases have been reported from cotton in the United States such as the tobacco ringspot virus (TRSV) in Oklahoma. TRSV has been reported from various hosts worldwide with minimal phylogenetic examination. In this study, complete genome sequences of four TRSV isolates from cotton were isolated, and the genetic diversity was investigated along with additional available TRSV isolates retrieved from GenBank. Phylogenetic analysis based on the complete RNA1 and RNA2 sequences distributed all TRSV isolates into three major phylogenetic clades exhibiting a differential clade composition depending on the segment. The TRSV cotton isolates exhibited differential grouping between the RNA1 and RNA2 analyses. Additionally, monophyletic subclades of isolates appeared to be conserved between both segments. Thirty-five recombination events in RNA1 and 23 in RNA2 were identified with implications in the variation of the phylogenetic analyses. Furthermore, multiple hypotheses of TRSV evolution were generated based on the phylogenetic analyses, but to test them, more complete genomes of TRSV will be needed. This study provides the first complete genome analysis of TRSV isolates infecting cotton in the United States and a detailed analysis of global TRSV isolates.

First report of the Horse nettle virus infecting tomato (Solanum lycopersicum L.) in the United States.

Tomato (Solanum lycopersicum L.), a member of the Solanaceae family, represents one of the most extensively cultivated vegetable species worldwide and traces its origin to western South America (Caruso et al. 2022). In a field survey conducted in 2023 in Bixby, Tulsa County, Oklahoma, distinct symptoms were noted in two plants: one exhibited mottling and cupping of leaves and brown discoloration on leaves, petioles, and stems, while the other displayed a downward curling of leaves. Leaf samples from both symptomatic tomato plants (labelled as K4 and K5) were collected, and total RNA was extracted individually via the TRI Reagent® method (Molecular Research Center Inc., Cincinnati, OH, USA). Subsequently, the RNA samples were pooled and subjected to high-throughput sequencing (HTS) on the NextSeq 500/550 high-output kit v2.5 (Illumina, U.S.A.) at the genomic facility, Oklahoma State University (Stillwater, OK). Total read count of 8,227,020 (average length =150.5 bp) was obtained, trimmed, and de novo assembled using CLC Genomics Workbench v22.0.1 (QIAGEN) and used for BLASTn and BLASTx analysis. Two contigs: 6,375 bp (average coverage 2,915.92, read count 142,538) and 3,564 bp (average coverage 3,035.91, read count 82,370) from the pooled sample showed 88.6% and 96.7% nucleotide identities with RNA 1 (OP292294) and RNA 2 (OP292295) of Horse nettle virus A (HNA-A) isolate MD-1, respectively. Sequences of both partial contigs (RNA 1, accession no. PP063196) and RNA 2, accession no. PP063197) were submitted to GenBank. The HTS data did not reveal any other viral or viroid sequences in these two tomato samples. To further confirm the presence of HNV-A, total RNA from K4 and K5 samples was tested individually by RT-PCR using HNV specific primers (Supplementary Table 1) based on the two partial contig sequences. The expected PCR products (491 bp and 451 bp) were obtained only from the K4 sample and none from the K5 sample. PCR products were extracted from an agarose gel, cloned into the pGEM®-T Easy vector (Promega), and transformed into Escherichia coli DH5α cells (New England Bio Labs). Two clones for each PCR product were sequenced by Sanger sequencing. Nucleotide sequence comparisons and BLASTn analysis of 491 bp and 451 bp showed 86% and 97% nucleotide identity with RNA 1 and RNA 2 of HNV-A isolate MD-1 (OP292294 and OP292295), respectively. Additionally, eight more leaf samples from eight different symptomatic tomato plants were collected in the same field and tested by RT-PCR as described above. All eight samples were positive by RT-PCR, but no PCR band was obtained in the total RNA from a healthy tomato leaf used as a control. Sequences from the PCR products were identical to the obtained HTS sequences. Our results confirmed for the first time that HNV-A can infect tomatoes. Currently, HNV-A has been reported to only infect a single weed (Horse nettle, Solanum carolinense) (Zhou et al. 2023). The identification of HNV-A in tomatoes in Oklahoma suggests a potential host shift is of concern for local growers as well as tomato growers worldwide. This shift underscores the urgency for an in-depth investigation into the transmission and host specificity of HNV-A. This is the first report in the United States and the world that HNV-A could infect tomatoes naturally in a grower field.

LC-ESI-QTOF-MS/MS Identification and Characterization of Phenolic Compounds from Leaves of Australian Myrtles and Their Antioxidant Activities.

Phenolic compounds, present in plants, provide substantial health advantages, such as antioxidant and anti-inflammatory properties, which enhance cardiovascular and cognitive well-being. Australia is enriched with a wide range of plants with phytopharmacological potential, which needs to be fully elucidated. In this context, we analyzed leaves of aniseed myrtle (Syzygium anisatum), lemon myrtle (Backhousia citriodora), and cinnamon myrtle (Backhousia myrtifolia) for their complex phytochemical profile and antioxidant potential. LC-ESI-QTOF-MS/MS was applied for screening and characterizing these Australian myrtles' phenolic compounds and the structure-function relation of phenolic compounds. This study identified 145 and quantified/semi-quantified 27 phenolic compounds in these Australian myrtles. Furthermore, phenolic contents (total phenolic content (TPC), total condensed tannins (TCT), and total flavonoids (TFC)) and antioxidant potential of phenolic extracts from the leaves of Australian myrtles were quantified. Aniseed myrtle was quantified with the highest TPC (52.49 ± 3.55 mg GAE/g) and total antioxidant potential than other selected myrtles. Catechin, epicatechin, isovitexin, cinnamic acid, and quercetin were quantified as Australian myrtles' most abundant phenolic compounds. Moreover, chemometric analysis further validated the results. This study provides a new insight into the novel potent bioactive phenolic compounds from Australian myrtles that could be potentially useful for functional, nutraceutical, and therapeutic applications.

Shotgun metagenomics and computational profiling of the plastisphere microbiome: unveiling the potential of enzymatic production and plastic degradation.

Plastic pollution is one of the most resilient types of pollution and is considered a global environmental threat, particularly in the marine environment. This study aimed to identify plastic-degrading bacteria from the plastisphere and their pharmaceutical and therapeutic potential. We collected samples from soil and aquatic plastisphere to identify the bacterial communities using shotgun metagenomic sequencing and bioinformatic tools. Results showed that the microbiome comprised 93% bacteria, 0.29% archaea, and 3.87% unidentified microbes. Of these 93% of bacteria, 54% were Proteobacteria, 23.9% were Firmicutes, 13% were Actinobacteria, and 2.1% were other phyla. We found that the plastisphere microbiome was involved in degrading synthetic and polyhydroxy alkanoate (PHA) plastic, biosurfactant production, and can thrive under high temperatures. However, no association existed between thermophiles, synthetic plastic or PHA degraders, and biosurfactant-producing bacterial species except for Pseudomonas. Other plastisphere inhabiting plastic degrading microbes include Streptomyces, Bacillus, Achromobacter, Azospirillum, Bacillus, Brevundimonas, Clostridium, Paenibacillus, Rhodococcus, Serratia, Staphylococcus, Thermobifida, and Thermomonospora. However, the plastisphere microbiome showed potential for producing secondary metabolites that were found to act as anticancer, antitumor, anti-inflammatory, antimicrobial, and enzyme stabilizers. These results revealed that the plastisphere microbiome upholds clinical and environmental significance as it can open future portals in a multi-directional way.

First report of Potato Yellow Dwarf Nucleorhabdovirus infecting pepper (Capsicum spp.) in Oklahoma.

Pepper (Capsicum spp.) is an economically valuable crop used for spice and fresh vegetable (Tripodi, and Kumar. 2019). The total acreage of peppers in Oklahoma is low (NASS, 2020), but there are numerous farms that are growing peppers for commercial production and provide fresh produce to local farmer's markets and processing industries. During a field survey in 2021, pepper plants with leaf distortion, mottling of the leaf, apical yellowing, and vein banding were observed in commercial pepper fields (Supplementary Figure 1). Seventeen leaf samples were collected from symptomatic pepper plants in Caddo County of Oklahoma. Total RNA was extracted from each sample using the Spectrum Plant total RNA kit, and screened by RT-PCR for Pepper mild mottle virus (PMMoV) as described previously (Ali and Ali, 2015). Total RNA from two negative PCR samples (named Caddo57 and Caddo64 respectively) were analyzed by high throughput sequencing. A total read count of 45,780,855 (average length = 73.51bp) and 21,163,567 (average length = 73.86 bp) for Caddo57 and Caddo64 respectively, were assembled in de novo using CLC genomic workbench (QIAGEN) and used for BLASTn and BLASTx analysis. Three contigs: 4,259bp (average coverage 31776.33X), 4,378 (average coverage 21773.09X), and 4,206bp (average coverage 57419.46X) for Caddo57 isolate showed 90-92% nucleotide (nt) identities with partial sequences of several genes (M, G, L, P, N, X, and Y genes) of PYDV isolate (KY549567). Similarly, four contigs: 4,204bp (average coverage 57446.92X), 2,738bp (average coverage 16192X), 4,257bp (average coverage 31791X), and 1,510bp (average coverage 33051.35X) were obtained for Caddo64 isolate and showed 90-92% nt identities with the same genes of PYDV isolate (KY549567). To further confirm the presence of PYDV, total RNA from Caddo57 and Caddo64 samples were tested by RT-PCR assays using newly designed primers (Table 1) based on the contigs sequences obtained above. The expected PCR products from both isolates were directly sequenced. Using BLASTn, nucleotide sequences of both L gene (OP805375), and N gene (OP805377) for Caddo57 isolate showed 92.82% and 92.392% identities respectively with PYDV isolate (KY549567). Similarly, nucleotide sequences of L gene (OP805376) and N gene (OP805378) of Caddo64 isolate showed 92.27% and 92.08% identities respectively with PYDV isolate (KY549567). The species demarcation criteria for nucelorhabdoviruses is 50% in cognate genes (Walker et al. 2018). These results demonstrate that our isolates align with PYDV species, and do not constitute evidence for a divergent nucleorhabdovirus member. The remaining 15 samples were negative by RT-PCR assay to PYDV. Our results confirmed the presence of PYDV infecting pepper in Oklahoma. Currently, PYDV has been reported infecting potato, tobacco, marigold, pepper, tomato, and white clover. The geographical distribution of PYDV appears to be limited to the US with detection in Wisconsin, Minnesota, California, New York, and Maryland (Walker et al. 1939, Lockhart, 1989, Falk et al. 1981, Chiu et al. 1970, Hammond et al. 2017). The PYDV is naturally transmitted by aphids and leafhoppers (Ghosh et al. 2008). The recognition of PYDV in Oklahoma is of notable concern for local growers. Further studies are needed to expand upon the relationship of these PYDV isolates to the currently reported isolates in the USA. This is the first report of potato yellow dwarf nucleorhabdovirus (PYDV) infecting pepper in Oklahoma.

Identification of Phenolics Profile in Freeze-Dried Apple Peel and Their Bioactivities during In Vitro Digestion and Colonic Fermentation.

Freeze-dried apple peel powder (Fd-APP) was subjected to in vitro digestion and colonic fermentation to evaluate the variations in its phenolic composition, bioactivities (antioxidant activity, α-amylase, and α-glucosidase inhibition), and fecal metabolic outputs. A total of 88 phenolics were tentatively identified, of which 51 phenolic compounds were quantitated in Fd-APP sample extracts before digestion, and 34 were released during subsequent phases of digestion. Among these, phenolic acids showed the highest bio accessibility index (BI) of 68%, followed by flavonoids (63%) and anthocyanins (52%). The inhibitory functions of Fd-APP extract against α-amylase and α-glucosidase pre- and post-digestion were moderate and ranged from 41.88 to 44.08% and 35.23 to 41.13%, respectively. Additionally, the antioxidant activities revealed a significant (p ≤ 0.05) decline during the in vitro digestion. However, the colonic fermentation stage presented different products where the intact parent phenolic compounds present in Fd-APP were utilized by gut microbes and produced various phenolic metabolites such as 3- hydroxyphenyl acetic acid (3-HPAA), ferulic acid (FA), 3-(4-hydroxyphenyl) propionic acid (3,4 HPPA) and 4- hydroxybenzoic acid (4-HBA). Furthermore, colonic fermentation of Fd-APP accelerated the production of short-chain fatty acids (SCFAs), with acetic acid being the most prevalent (97.53 ± 9.09 mM). The decrease in pH of fermentation media to 4.3 significantly (p ≤ 0.05) enhanced counts of Bifidobacterium (10.27 log CFU/mL), which demonstrated the potential prebiotic effects of Fd-APP. These findings indicated that the consumption of apple peel as a constituent of novel functional foods may support and protect the intestinal microbiota and consequently promote human health.

Probiotic Yoghurt Enriched with Mango Peel Powder: Biotransformation of Phenolics and Modulation of Metabolomic Outputs after In Vitro Digestion and Colonic Fermentation.

This study investigated the health-promoting effects and prebiotic functions of mango peel powder (MPP) both as a plain individual ingredient and when incorporated in yoghurt during simulated digestion and fermentation. The treatments included plain MPP, plain yoghurt (YA), yoghurt fortified with MPP (YB), and yoghurt fortified with MPP and lactic acid bacteria (YC), along with a blank (BL). The identification of polyphenols in the extracts of insoluble digesta and phenolic metabolites after the in vitro colonic fermentation were performed employing LC-ESI-QTOF-MS2. These extracts were also subjected to pH, microbial count, production of SCFA, and 16S rRNA analyses. The characterisation of phenolic profiles identified 62 phenolic compounds. Among these compounds, phenolic acids were the major compounds that underwent biotransformation via catabolic pathways such as ring fission, decarboxylation, and dehydroxylation. Changes in pH indicated that YC and MPP reduced the media pH from 6.27 and 6.33 to 4.50 and 4.53, respectively. This decline in pH was associated with significant increases in the LAB counts of these samples. The Bifidobacteria counts were 8.11 ± 0.89 and 8.02 ± 1.01 log CFU/g in YC and MPP, respectively, after 72 h of colonic fermentation. Results also showed that the presence of MPP imparted significant variations in the contents and profiles of individual short chain fatty acids (SCFA) with more predominant production of most SCFA in the MPP and YC treatments. The 16s rRNA sequencing data indicated a highly distinctive microbial population associated with YC in terms of relative abundance. These findings suggested MPP as a promising ingredient for utilisation in functional food formulations aiming to enhance gut health.

Optimized Extraction of Polyphenols from Unconventional Edible Plants: LC-MS/MS Profiling of Polyphenols, Biological Functions, Molecular Docking, and Pharmacokinetics Study.

Plant bioactive phenolic metabolites have recently attracted the attention of researchers due to their numerous health advantages. Therefore, this study aimed to investigate with advanced techniques the bioactive metabolites and antioxidant and antidiabetic capacity of four unconventional edible plant leaves: lemongrass (Cymbopogon citratus (DC.) Stapf), chicory (Cichorium intybus L.), moringa (Moringa oleifera Lam.), and ryegrass (Lolium perenne L.). The extraction process was optimized using different solvents. These plants' phenolic composition, identification, and characterization have been determined herein using LCESI-QTOF-MS/MS. This research identified 85 phenolic compounds, including 24 phenolic acids, 31 flavonoids, 7 stilbenes and lignans, and 17 other metabolites. Moreover, the study determined that moringa has the highest total phenolic content (TPC; 18.5 ± 1.01 mg GAE/g), whereas ryegrass has the lowest (3.54 ± 0.08 mg GAE/g) among the selected plants. It seems that, compared to other plants, moringa was found to have the highest antioxidant potential and antidiabetic potential. In addition, twenty-two phenolic compounds were quantified in these chosen edible plants. Rosmarinic acid, chlorogenic acid, chicoric acid, ferulic acid, protocatechuic acid, and caffeic acid were the most abundant phenolic acids. In silico molecular docking was also conducted to investigate the structure-function relationship of phenolic compounds to inhibit the alpha-glucosidase. Finally, the simulated pharmacokinetic characteristics of the most common substances were also predicted. In short, this investigation opens the way for further study into these plants' pharmaceutical and dietary potential.

Screening and Characterization of Phenolic Compounds from Selected Unripe Fruits and Their Antioxidant Potential.

The food sector's interest in sustainability and the demand for novel bioactive compounds are increasing. Many fruits are wasted every year before ripening due to various climatic conditions and harsh weather. Unripe mangoes, grapes, and black lemons could be rich sources of phenolic compounds that need to be fully elucidated. Using fruit waste as a source of bioactive chemicals has grown increasingly appealing as it may have significant economic benefits. Polyphenols are beneficial for human health to inhibit or minimize oxidative stress and can be used to develop functional and nutraceutical food products. In this context, this study aimed to characterize and screen unripe mangoes, grapes, and black lemons for phenolic compounds using LC-ESI-QTOF-MS/MS and their antioxidant activities. Unripe mangoes were quantified with higher total phenolic content (TPC, 58.01 ± 6.37 mg GAE/g) compared to black lemon (23.08 ± 2.28 mg GAE/g) and unripe grapes (19.42 ± 1.16 mg GAE/g). Furthermore, unripe mangoes were also measured with higher antioxidant potential than unripe grapes and black lemons. A total of 85 phenolic compounds (70 in black lemons, 49 in unripe grapes, and 68 in unripe mango) were identified, and 23 phenolic compounds were quantified using LC-MS/MS. Procyanidin B2, gallic acid, epicatechin, caffeic acid, quercetin, and chlorogenic acid were measured with higher concentration in these selected unripe fruits. A positive correlation was found between phenolic contents and the antioxidant activities of unripe fruits. Furthermore, chemometric analysis was conducted to validate the results. This study will explore the utilization of these unripe fruits to develop functional and therapeutic foods.

Comparison Of Different Methods Of Controlling Pain During Debonding Of Orthodontic Brackets.

Objective: To compare the efficacy of finger pressure and plastic wafers in terms of pain control during debonding. METHODS: This cross sectional study was conducted at the Department of Orthodontics, Armed Forces Institute of Dentistry, Rawalpindi, Pakistan, from January to June 2020, and comprised patients of either gender aged 13-21 years who had completed fixed orthodontic treatment with 0.022 brackets of Roth prescription and required debonding. The patients was divided into two equal groups. In group A, teeth were stabilised with finger pressure with cotton between the finger and teeth, and then debonding was done using open mouth technique. In group B, teeth were stabilised using a plastic wafer between maxillary and mandibular teeth, and then debonding was done using closed mouth technique. Pain was assessed using a visual analogue scale VAS. Data was analysed using SPSS 23. RESULTS: Of the 110 patients, each of the 2 groups had 55(50%) subjects. Overall, there were 35(32%) males and 75(68%) females. The mean age of the sample was 16±2.4 years. Mean pain scores among the males was 32.0±7.68 compared to 34.067±12.59 among the females (p>0.05). Subjects in group B had significantly less pain than those in group A (p<0.05). Conclusion: Plastic wafer was found to be more effective in terms of controlling pain during debonding compared to finger pressure.

Heterologous expression of bacterial dehydrin gene in Arabidopsis thaliana promotes abiotic stress tolerance.

Salinity, low temperature, and drought are major environmental factors in agriculture leading to reduced crop yield. Dehydrins (DHNs) are induced transcriptionally during cellular dehydration and accumulate in different tissues during abiotic stresses. Here we isolated and characterized a bacterial gene BG757 in Arabidopsis, encoding a putative dehydrin type protein. ABA induces the expression of various dehydrins in plants, therefore, to elucidate the potential role, ABA sensitivity was examined in Arabidopsis transgenic lines expressing BG757. Interestingly, BG757-expressing plants showed hypersensitivity towards NaCl and ABA during seed germination. In addition to germination, BG757-expressing plants also showed root growth retardation in the presence of ABA and NaCl when compared with wild type (WT), suggesting that BG757 positively regulate salt stress and ABA response. Furthermore, BG757-expressing plants showed significant drought tolerance compared with WT. Consistent with drought tolerance, expression levels of stress inducible genes (DREB2A, RD22, RD26, LEA7 and SOS1) were strongly upregulated in transgenic plants compared with WT. All together these results suggest that heterologous expression of bacterial gene, BG757 in plants promotes resistance to environmental stresses. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01358-w.

Novel HOXD13 variants in syndactyly type 1b and type 1c, and a new spectrum of TP63-related disorders.

Syndactyly is the most common limb defect depicting the bony and/or cutaneous fusion of digits. Syndactyly can be of various types depending on the digits involved in the fusion. To date, eight syndactyly-associated genes have been reported, of which HOXD13 and GJA1 have been explored in a few syndactyly but most of them have unknown underlying genetics. In the present study HOXD13, GJA1 and TP63 genes have been screened by resequencing in 24 unrelated sporadic cases with various syndactyly. The screening revealed two pathogenic HOXD13 variants, NM_000523:c.500 A > G [p.(Y167C)], and NM_000523:c.961 A > C [p.(T321P)] in syndactyly type 1b and type 1c, respectively. This is the first report to identify HOXD13 pathogenic variant in syndactyly type 1b and third report in syndactyly type 1c pathogenesis. Furthermore, this study also reports a TP63 pathogenic variant, NM_003722:c.953 G > A [p.(R318H)] in Ectrodactyly and Cleft lip and palate (ECLP). In conclusion, the current study expands the clinical spectrum of HOXD13 and TP63-related disorders.

Effectiveness of Village Health Volunteer Parallel Program for Proactive Action to Reduce Risk Factors for Cholangiocarcinoma in Two High-Risk Countries in the Greater Mekong Subregion.

Thailand and Laos were classified as risk areas for cholangiocarcinoma (CCA) in a 2017 assessment in the Greater Mekong Subregion. In 2019, the potential of village health volunteers (VHVs) in both risk areas was developed. The VHVs trained in 2014 (VHV-A) were mentors transferring knowledge of CCA prevention to the trainees (known as VHV-B) in a parallel manner. After that, VHV-Bs in each area educated people to change their behavior. Both parties worked in the same direction to reduce risk factors. In 2020, data were collected after the program was organized in the same populations. The people were aged 30-69 years, whose names were in the civil registration, and had lived in that area for at least five years. Afterward, no less than 172 participants from each location were randomly selected. The research tools used were intervention and questionnaires. Descriptive and inferential statistics were employed for data analysis. After the experiment, all the experimental group's risk factors were significantly different from those of the control group. This study's outcome was an effective program for proactive action in reducing risk factors in the risk areas. Therefore, it should be applied to reduce risk factors for CCA in other regions.

First Report of Tobacco Ringspot Virus Naturally Infecting Cotton (Gossypium hirsutum L.) in the United States.

Cotton (Gossypium hirsutum L.) is one of the major cash crops grown in the United States (U.S.) with a total acreage of over 11.5 million acres in 2021 (NASS, 2021). In Oklahoma, cotton represents an important economic crop and was grown on 490,000 acres during the 2021 growing season (NASS, 2021). In 2021, during a survey of a cotton field in Beckham County of Oklahoma, cotton plants showed typical virus-like symptoms including mosaic, yellow ring spots, discoloration and short internodes (Supplementary Fig. 1). Thirteen symptomatic and five asymptomatic samples were collected from cotton plants and brought to the University of Tulsa for further processing. Total RNA was extracted from all samples using the Spectrum Plant Total RNA Kit (Sigma-Aldrich). Total RNA from two symptomatic samples (named EC3 and EC4) were subjected to high-throughput sequencing (HTS) on the NextSeq 500/550 High-Output kit v2.5 (Illumina, USA) at the genomic facility, Oklahoma State University (Stillwater, OK, USA). A total of 17,542,322 and 22,572,118 trimmed pair-ends reads for both samples were assembled using CLC Genomics Workbench (v12.0.3) (Qiagen, Inc) and subjected to BLASTn analysis. Two contigs of 556 bp and 1062 bp (average coverage 2,799X) for sample EC3 showed 99% and 91% nucleotide (nt) identities with 3'-UTR, 5'-UTR and P1A gene respectively of the Tobacco ringspot virus (TRSV) RNA1 of isolates WA-AM1 (MW495243.1), and IA-1-2017 (MT563078.1) respectively. The other two contigs, 221 bp and 561 bp (average coverage 23,070X) for sample EC4 showed, 100% and 96% nt identities with 3'-UTR, protease, and RdRp genes of TRSV RNA1, isolates WA-AM1 and YW (MT042825.1) respectively. The HTS data did not reveal any other viral sequence in these two cotton samples. To further confirm the presence of TRSV in these samples, previously designed specific primers to TRSV (Forward: 5'-GGAAATTAACTGGGATGATTT-3' and Reverse: 5'-GAGCTCCAACCTTAAAACCA-3') for RNA1, targeting the P1A and helicase genes, and another primer pair (Forward: 5'-GCATCCTCCCATGTTTTCT-3' and Reverse: 5'-GGGACAAACACGACACTA-3') for RNA2, targeting the coat protein and 3'-untranslated region, of TRSV were tested by RT-PCR assay. The sizes of amplified PCR products obtained from both isolates (EC3 and EC4) on 1% agarose gel were approximately 1,000 bp for RNA1 and 1,100 bp for RNA2. The amplified PCR products were cloned and three independent recombinant clones for each primer set were analyzed by Sanger sequencing. The resulting consensus sequences were used in a BLASTn search against the Genbank and matched with TRSV sequences. Consensus nt sequences analysis specific to RNA1 of EC3 isolate (Accession no. OM563300) and EC4 isolate (Accession no OM563301) showed 97% nt identities with TRSV isolate IA-1-2017. Consensus nt sequences specific to RNA 2 of EC3 isolate (Accession no. OM630605) and EC4 isolate (Accession no OM5630606) showed 92% and 90% nt identities with the corresponding sequences of WA-AM1 and IA-1-2017 isolates respectively. Further screening of the remaining 11 symptomatic samples resulted in two more positive TRSV samples that were co-infected with Cotton leafroll dwarf virus (CLRDV), six were positive to only CLRDV, and three were negative to both viruses by RT-PCR assay using the above TRSV specific primers and CLRDV specific primers AL674F/1407R (Avelar et al. 2019). None of the asymptomatic cotton samples were positive by RT-PCR to TRSV or CLRDV. Our results confirmed the presence of TRSV infection in these symptomatic cotton plants. The presence of TRSV could pose a new threat to cotton crops in Oklahoma and the U.S. due to its wide host range (Adam and Antoniw, 2005), and transmission through many vectors including nematodes, (Keinath et al. 2017), and seed (Hill and Whitman, 2014). To the best of our knowledge, this is the first report of TRSV infecting cotton naturally in the U.S. and in the world.

First Report of Tomato Yellow Leaf Curl Virus Infecting Pepper and Tomato in Oklahoma.

Tomato yellow leaf curl virus (TYLCV), (genus Begomovirus and family Geminiviridae) is one of the devastating viruses infecting tomatoes (Solanum Lycopersicon) and could have a significant impact on tomato production worldwide (Moriones and Navas-Castillo, 2000; Rybicki, 2015; Perez-Padilla et al. 2019). TYLCV was first reported in the United States (US) in 1997 in Florida (Polston et al. 1999) and since then it has been reported in several other states but not in Oklahoma (de Sa et al. 2008). Both pepper (Capsicum spp.) and tomato are grown in >50 counties of Oklahoma for fresh produce in the local market, and for processing industries. During a survey in 2021, pepper (bell pepper cv SV3964) and tomato (cv grand marshall) plants grown in a commercial field in Bixby, Oklahoma, showed typical virus-like symptoms including yellowing, leaf curling, cupping, twisting, and mottling (Fig. 1). Estimated disease incidence was 10% in both crops. In addition, whiteflies (Bemisia tabaci) were also observed on these plants. Twenty symptomatic samples from pepper (n=16) and tomato (n=4) plants, and four asymptomatic from pepper (n=2) and tomato (n=2) plants were collected and brought to the Plant Virology Lab at the University of Tulsa for further analysis Total RNA was extracted from all samples using the Spectrum Plant Total RNA Kit (Sigma-Aldrich, LOT: SLCG7913). RNA from two symptomatic samples, one each from pepper and tomato (named BX6 and BX11, respectively) were subjected to high-throughput sequencing (HTS) on the NextSeq 500/550 high-output kit v2.5 (Illumina, USA) at the genomic facility, Oklahoma State University (Stillwater, OK, USA). A total of 22,385,404 (average length 73.6 bp) and 19,012,605 (average length 73.7 bp) trim pair-end for both samples (BX6 and BX11) were assembled using CLC Genomics Workbench (v12.0.3) (Qiagen, Inc) and subjected to BLASTn analysis. The three contigs: 393 bp (coverage 363.24X), 670 bp (coverage 489.92X), and 990 bp (coverage 112.96X) for BX6 isolate, and four contigs: 393 bp (coverage 341.64X), 668 bp (coverage 457.23X), 814 bp (coverage 107.86X), and 990 bp (coverage 93.71X), for BX11 isolate, showed from 96 to 99% nucleotide (nt) identities with movement protein, capsid protein, C3, V2, C4, AC1, AC2, AC3 protein genes of the TYLCV isolates from Australia, China, Iran, Mexico, South Korea, and the US. The HTS data did not reveal any other viral sequence in these two samples. To further confirm the presence of TYLCV in these samples, three overlapping TYLCV-specific primer pairs (Supplementary Table 1) were designed based on the alignment of complete genome sequences of TYLCV isolates present in the Genbank. Total DNA was extracted from fresh leaf tissues of BX6 and BX11 samples using the Plant DNA Kit (Omega BIO-TEK,) and used in the PCR assay. The expected PCR products (814 bp, 1,085 bp and 1,068 bp) were generated from both samples providing further evidence of TYLCV infection. To obtain the complete genome sequence, these PCR amplified DNA fragments of both isolates were gel extracted and cloned using the pGEM-T® Easy Vector system. Three independent clones of each fragment were sequenced and analyzed by Sanger sequencing. The resulting consensus sequences were used in a BLASTn search against the GenBank and had the highest identities (99-99.2%) with TYLCV sequences. The overlapping consensus nt sequence of the BX6 isolate was 2,782 nt (Accession no ON321843), whereas that of BX11 was 2,777 nt (Accession no ON785706) showed 99.2% and 99.0% nt identities with the corresponding sequence of TYLCV isolates (KX347141 and KX347142) respectively from Australia. The nt identity between BX6 and BX11 was 99.7%. Further screening by PCR of the remaining 18 symptomatic field samples (pepper, n=15 and tomatoes, n=3) revealed TYLCV infection in two pepper and all three tomato samples consistent with the greater susceptibility of tomato. None of the asymptomatic pepper and tomato samples were positive for TYLCV. DNA from both BX6 and BX11 samples was also used in the rolling circle amplification (RCA) assay (TempliPhi Amplification Kit, Cytiva) and the presence of circular TYLCV DNA was detected. Our results demonstrated the presence of TYLCV infection in these symptomatic pepper and tomato plants. This is the first report of TYLCV infecting pepper or tomato in open fields in Oklahoma. Further surveys are needed to determine the incidence of TYLCV and the presence of its vector in other counties throughout Oklahoma.

First report of Watermelon crinkle leaf-associated virus 2 infecting watermelon (Citrullus lanatus) in Oklahoma.

Watermelon (Citrullus lanatus) is one of the major cucurbit crops in Oklahoma, which is grown on >1,200 acres. It contributes $16 million to the state's economy (Shrefler et al., 2017; NASS, 2022). During the 2021 growing season, virus-like symptoms on watermelon plants were observed in a grower's field in Blaine County, Oklahoma. These symptoms included mottling, crinkled leaf edges, and a unique yellow mosaic pattern (Fig. 1). Leaf tissue samples were randomly collected from nine plants and total RNA was extracted using Spectrum Plant Total RNA Kit (Sigma-Aldrich). Total RNA from one sample (named BL13) was subjected to high-throughput sequencing (HTS) on the NextSeq 500/550 High-Output kit v2.5 (Illumina, USA) at the genomic facility of Oklahoma State University. A total of 21,52,786 trimmed pair-end reads were assembled using CLC Genomics Workbench (v12.0.3) (Qiagen, Inc.) and subjected to BLASTn analysis. Of these, three contigs of 6,626 bp, 1,383 bp and 1,233 bp (average coverage 20,658X) showed 99% nucleotide (nt) sequence identity with RNA1 of a watermelon crinkle leaf-associated virus (WCLaV-2) isolate from Brazil (LC636073). The HTS data for BL13 samples also revealed three contigs of 263 pb, 567 bp and 1,066 bp that matched with cucumis melo crytpic virus isolate CmCV-HLI but was not confirmed by RT-PCR. The presence of WCLaV-2 in the BL13 sample was confirmed by RT-PCR using primers (RNA1-F: 5'-CAAAACGCCCATAAG AATAG-3'and RNA1-R: 5'- CTCCATTAATGAGAACTAGGAG-3') which amplified a 1.2 kb segment of the RNA1. The PCR product was confirmed on 1% agarose gel, cloned and three independent recombinant clones Sanger sequenced (Eurofins Genomics LLC, Louisville, KY, USA). The resulting consensus sequence (1.2 Kb) (ON453838) was subjected to a BLASTn search against the Genbank databases, resulting in 99.76% nt identity with WCLaV-2 RNA1 (Accession no. LC636073). Screening of the remaining eight symptomatic samples by RT-PCR revealed another positive sample (named BL7) for WCLaV-2. Consensus nt sequence (1.2 Kb) analysis for the virus isolate from the BL7 sample (Accession no. ON453839) shared 99.84% nt identities with WCLaV-2 RNA1 (LC636073). Total RNA from BL13 and BL7 samples, as well as the remaining seven samples, were tested against three major potyviruses (PRSV-W, WMV, and ZYMV) by multiplex RT-PCR (Rajbanshi & Ali, 2019). Both BL13 and BL7 samples were negative to all three potyviruses but the other eight samples were positive to one or two of the potyviruses. WCLaV-2 is a negative-sense ssRNA virus (family Phenuiviridae) and has been recently reported from watermelon in China (Xin et al., 2017) and Brazil (Maeda et al. 2022). In the United States, it has been reported from Florida (Hendrick et al., 2021), Georgia (Adeleke et al., 2022), and Texas (Hernandez et al., 2021). The mode of transmission of WCLaV-2 and its direct effect on watermelon fruit is not known (Xin et al., 2017; Maeda et al., 2022). Our results report for the first time the occurrence of WCLaV-2 infecting watermelon naturally in Oklahoma and show a further potential threat to watermelon production in the State. Further studies are needed to determine the incidence of WCLaV-2 in other counties of Oklahoma.

First Report of Fusarium oxysporum f. sp. vasinfectum causing Fusarium Wilt of Cotton in Kansas, U.S.A.

Fusarium wilt is one of the most devastating diseases of cotton (Gossypium spp.). It is caused by the soil-borne pathogen Fusarium oxysporum Schlechtend. f. sp. vasinfectum (Atk.) Snyd. & Hans (Atkinson, 1892). To date, eight races of F. oxysporum f. sp. vasinfectum have been reported worldwide based on their diverse genetics and reactions to the host (Cianchetta, et al. 2015). Fusarium wilt symptoms appear at all developing stages. The fungus enters through the roots and colonizes the vascular system, leading to discoloration and wilting (Davis et al. 2006). During a survey in the July 2021 growing season, cotton plants showing typical wilting symptoms, stem discoloration, and root rot (Fig. 1A and B) were observed in two cotton fields in Sumner County, Kansas. In order to confirm the causal agent, root tissues and lower parts of the stems were collected from 25 diseased cotton plants and cleaned as previously described by Larren et al, (2001). Small segments (1-2 cm) of roots and stems were incubated on potato dextrose agar (PDA) at room temperature. The isolates were purified with the single spore method and 23 of 25 isolates (92%) showed typical morphology of F. oxysporum, with a white-peach pigmentation as reported previously (Leslie and Summerell 2008). Two isolates, CK13B and CK18A, one from each of the two fields were selected, and genomic DNA was extracted with E.Z.N.A Fungal DNA Mini kit (Omega Bio-tek, Norcross, GA). Polymerase chain reaction (PCR) assays were performed as reported previously (Ortiz, et al. 2017) to amplify and sequence a portion of three nuclear genes: translation elongation factor (EF-1α), phosphate: H* symporter (PHO), and ß-tubulin (BT). BLASTn analysis of CK13B sequences showed 93-98% identity with F. oxysporum f. sp. vasinfectum isolates originating from Australia, with 93.43% (1706/1826 pb) identity of EF-1α to isolate AuSeed14 (KT323873), 95.11% (1849/1944 bp) of PHO to isolate AuK24232 (KT323909), and 97.56% (1840/1886 bp) identity of BT to isolate AusSeed14 (KT323833). Sequences of CK13B for EF1-α, PHO, and BT genes were submitted to GenBank, accession no. of ON754247, ON754248, and ON754249, respectively. The CK18A isolate showed high identity with F. oxysporum f. sp. vasinfectum races 1, 2, and 6 lineage isolates (Ortiz et al., 2017), with 99.89% (1822/1824 bp) identity of the EF1-α gene to isolate CDR238 (KT323838) from Arkansas, 98.36% (1916/1948 bp) identity of the PHO gene to isolate CDR1131 (KT323887) from Louisiana and 98.78% (1864/1887 bp) identity of the BT gene to isolate ATCC36198 (KT323799) from Brazil. Sequences of the CK18A isolate were submitted to GenBank, accession no. ON651444, ON725043, and ON725044, respectively. Pathogenicity of both isolates was tested on G. hirsutum as previously described (Kim et al. 2005) with three replicates per isolate and a control treatment in a growth chamber. Briefly, cotton seedlings were maintained with 12 h light/dark cycle at 25-20 °C. At the first true leaf stage seedlings were uprooted, roots were cleaned by rinsing with sterile water then placed in a conidial suspension (1×106 conidia/ml) for 2 h while the healthy control seedlings were dipped in water only. Plants were put into sterile soil, and the results were observed and recorded every seven days for a period of 30 days. The inoculated plants showed the same symptoms as those observed in the fields, wilting, leaf chlorosis, and necrosis. Both isolates were re-isolated from the roots of the inoculated plants. DNA was extracted and used for PCR with the specific gene primers as listed above. The sequences matched those of the original isolates, completing the Koch's pustulates. To our knowledge, this is the first report of F. oxysporum f. sp. vasinfectum causing Fusarium wilt disease of cotton in Kansas. These results will help growers select cultivars and design disease control strategies accordingly (Davis, et al., 2006). Further work is needed to determine the specific races in the area.