Cowpea lipid transfer protein 1 regulates plant defense by inhibiting the cysteine protease of cowpea mosaic virus.

Many virus genomes encode proteases that facilitate infection. The molecular mechanism of plant recognition of viral proteases is largely unexplored. Using the system of Vigna unguiculata and cowpea mosaic virus (CPMV), we identified a cowpea lipid transfer protein (LTP1) which interacts with CPMV-encoded 24KPro, a cysteine protease, but not with the enzymatically inactive mutant 24KPro(C166A). Biochemical assays showed that LTP1 inhibited 24KPro proteolytic cleavage of the coat protein precursor large coat protein-small coat protein. Transient overexpression of LTP1 in cowpea reduced CPMV infection, whereas RNA interference-mediated LTP1 silencing increased CPMV accumulation in cowpea. LTP1 is mainly localized in the apoplast of uninfected plant cells, and after CPMV infection, most of the LTP1 is relocated to intracellular compartments, including chloroplast. Moreover, in stable LTP1-transgenic Nicotiana benthamiana plants, LTP1 repressed soybean mosaic virus (SMV) nuclear inclusion a protease activity, and accumulation of SMV was significantly reduced. We propose that cowpea LTP1 suppresses CPMV and SMV accumulation by directly inhibiting viral cysteine protease activity.

Synergistic combination therapy using cowpea mosaic virus intratumoral immunotherapy and Lag-3 checkpoint blockade.

Immune checkpoint therapy (ICT) for cancer can yield dramatic clinical responses; however, these may only be observed in a minority of patients. These responses can be further limited by subsequent disease recurrence and resistance. Combination immunotherapy strategies are being developed to overcome these limitations. We have previously reported enhanced efficacy of combined intratumoral cowpea mosaic virus immunotherapy (CPMV IIT) and ICT approaches. Lymphocyte-activation gene-3 (LAG-3) is a next-generation inhibitory immune checkpoint with broad expression across multiple immune cell subsets. Its expression increases on activated T cells and contributes to T cell exhaustion. We observed heightened efficacy of a combined CPMV IIT and anti-LAG-3 treatment in a mouse model of melanoma. Further, LAG-3 expression was found to be increased within the TME following intratumoral CPMV administration. The integration of CPMV IIT with LAG-3 inhibition holds significant potential to improve treatment outcomes by concurrently inducing a comprehensive anti-tumor immune response, enhancing local immune activation, and mitigating T cell exhaustion.

Enhancing cowpea wilt resistance: insights from gene coexpression network analysis with exogenous melatonin treatment.

BACKGROUND: Cowpea wilt is a harmful disease caused by Fusarium oxysporum, leading to substantial losses in cowpea production. Melatonin reportedly regulates plant immunity to pathogens; however the specific regulatory mechanism underlying the protective effect of melatonin pretreated of cowpea against Fusarium oxysporum remains known. Accordingly, the study sought to evaluate changes in the physiological and biochemical indices of cowpea following melatonin treated to facilitate Fusarium oxysporum resistance and elucidate the associated molecular mechanism using a weighted gene coexpression network. RESULTS: Treatment with 100 µM melatonin was effective in increasing cowpea resistance to Fusarium oxysporum. Glutathione peroxidase (GSH-PX), catalase (CAT), and salicylic acid (SA) levels were significantly upregulated, and hydrogen peroxide (H2O2) levels were significantly downregulated in melatonin treated samples in roots. Weighted gene coexpression network analysis of melatonin- and Fusarium oxysporum-treated samples identified six expression modules comprising 2266 genes; the number of genes per module ranged from 9 to 895. In particular, 17 redox genes and 32 transcription factors within the blue module formed a complex interconnected expression network. KEGG analysis revealed that the associated pathways were enriched in secondary metabolism, peroxisomes, phenylalanine metabolism, flavonoids, and flavonol biosynthesis. More specifically, genes involved in lignin synthesis, catalase, superoxide dismutase, and peroxidase were upregulated. Additionally, exogenous melatonin induced activation of transcription factors, such as WRKY and MYB. CONCLUSIONS: The study elucidated changes in the expression of genes associated with the response of cowpea to Fusarium oxysporum under melatonin treated. Specifically, multiple defence mechanisms were initiated to improve cowpea resistance to Fusarium oxysporum.

In Vivo Delivery of Spherical and Cylindrical In Vitro Reconstituted Virus-like Particles Containing the Same Self-Amplifying mRNA.

The dramatic effectiveness of recent mRNA (mRNA)-based COVID vaccines delivered in lipid nanoparticles has highlighted the promise of mRNA therapeutics in general. In this report, we extend our earlier work on self-amplifying mRNAs delivered in spherical in vitro reconstituted virus-like particles (VLPs), and on drug delivery using cylindrical virus particles. In particular, we carry out separate in vitro assemblies of a self-amplifying mRNA gene in two different virus-like particles: one spherical, formed with the capsid protein of cowpea chlorotic mottle virus (CCMV), and the other cylindrical, formed from the capsid protein of tobacco mosaic virus (TMV). The mRNA gene is rendered self-amplifying by genetically fusing it to the RNA-dependent RNA polymerase (RdRp) of Nodamura virus, and the relative efficacies of cell uptake and downstream protein expression resulting from their CCMV- and TMV-packaged forms are compared directly. This comparison is carried out by their transfections into cells in culture: expressions of two self-amplifying genes, enhanced yellow fluorescent protein (EYFP) and Renilla luciferase (Luc), packaged alternately in CCMV and TMV VLPs, are quantified by fluorescence and chemiluminescence levels, respectively, and relative numbers of the delivered mRNAs are measured by quantitative real-time PCR. The cellular uptake of both forms of these VLPs is further confirmed by confocal microscopy of transfected cells. Finally, VLP-mediated delivery of the self-amplifying-mRNA in mice following footpad injection is shown by in vivo fluorescence imaging to result in robust expression of EYFP in the draining lymph nodes, suggesting the potential of these plant virus-like particles as a promising mRNA gene and vaccine delivery modality. These results establish that both CCMV and TMV VLPs can deliver their in vitro packaged mRNA genes to immune cells and that their self-amplifying forms significantly enhance in situ expression. Choice of one VLP (CCMV or TMV) over the other will depend on which geometry of nucleocapsid is self-assembled more efficiently for a given length and sequence of RNA, and suggests that these plant VLP gene delivery systems will prove useful in a wide variety of medical applications, both preventive and therapeutic.

Hollow Mesoporous Silica Nanoparticles as a New Nanoscale Resistance Inducer for Fusarium Wilt Control: Size Effects and Mechanism of Action.

In agriculture, soil-borne fungal pathogens, especially Fusarium oxysporum strains, are posing a serious threat to efforts to achieve global food security. In the search for safer agrochemicals, silica nanoparticles (SiO2NPs) have recently been proposed as a new tool to alleviate pathogen damage including Fusarium wilt. Hollow mesoporous silica nanoparticles (HMSNs), a unique class of SiO2NPs, have been widely accepted as desirable carriers for pesticides. However, their roles in enhancing disease resistance in plants and the specific mechanism remain unknown. In this study, three sizes of HMSNs (19, 96, and 406 nm as HMSNs-19, HMSNs-96, and HMSNs-406, respectively) were synthesized and characterized to determine their effects on seed germination, seedling growth, and Fusarium oxysporum f. sp. phaseoli (FOP) suppression. The three HMSNs exhibited no side effects on cowpea seed germination and seedling growth at concentrations ranging from 100 to 1500 mg/L. The inhibitory effects of the three HMSNs on FOP mycelial growth were very weak, showing inhibition ratios of less than 20% even at 2000 mg/L. Foliar application of HMSNs, however, was demonstrated to reduce the FOP severity in cowpea roots in a size- and concentration-dependent manner. The three HMSNs at a low concentration of 100 mg/L, as well as HMSNs-19 at a high concentration of 1000 mg/L, were observed to have little effect on alleviating the disease incidence. HMSNs-406 were most effective at a concentration of 1000 mg/L, showing an up to 40.00% decline in the disease severity with significant growth-promoting effects on cowpea plants. Moreover, foliar application of HMSNs-406 (1000 mg/L) increased the salicylic acid (SA) content in cowpea roots by 4.3-fold, as well as the expression levels of SA marker genes of PR-1 (by 1.97-fold) and PR-5 (by 9.38-fold), and its receptor gene of NPR-1 (by 1.62-fold), as compared with the FOP infected control plants. Meanwhile, another resistance-related gene of PAL was also upregulated by 8.54-fold. Three defense-responsive enzymes of POD, PAL, and PPO were also involved in the HMSNs-enhanced disease resistance in cowpea roots, with varying degrees of reduction in activity. These results provide substantial evidence that HMSNs exert their Fusarium wilt suppression in cowpea plants by activating SA-dependent SAR (systemic acquired resistance) responses rather than directly suppressing FOP growth. Overall, for the first time, our results indicate a new role of HMSNs as a potent resistance inducer to serve as a low-cost, highly efficient, safe and sustainable alternative for plant disease protection.

Greenhouse cultivation enhances pesticide bioaccumulation in cowpeas following repeated spraying.

Understanding pesticide residue patterns in crops is important for ensuring human health. However, data on residue accumulation and distribution in cowpeas grown in the greenhouse and open field are lacking. Our results suggest that acetamiprid, chlorantraniliprole, cyromazine, and thiamethoxam residues in greenhouse cowpeas were 1.03-15.32 times higher than those in open field cowpeas. Moreover, repeated spraying contributed to the accumulation of pesticide residues in cowpeas. Clothianidin, a thiamethoxam metabolite, was detected at 1.04-86.00 µg/kg in cowpeas. Pesticide residues in old cowpeas were higher than those in tender cowpeas, and the lower half of the plants had higher pesticide residues than did the upper half. Moreover, pesticide residues differed between the upper and lower halves of the same cowpea pod. Chronic and acute dietary risk assessments indicated that the human health risk was within acceptable levels of cowpea consumption. Given their high residue levels and potential accumulation, pesticides in cowpeas should be continuously assessed.

Signatures of plant defense response specificity mediated by herbivore-associated molecular patterns in legumes.

Chewing herbivores activate plant defense responses through a combination of mechanical wounding and elicitation by herbivore-associated molecular patterns (HAMPs). HAMPs are wound response amplifiers; however, specific defense outputs may also exist that strictly require HAMP-mediated defense signaling. To investigate HAMP-mediated signaling and defense responses, we characterized cowpea (Vigna unguiculata) transcriptome changes following elicitation by inceptin, a peptide HAMP common in Lepidoptera larvae oral secretions. Following inceptin treatment, we observed large-scale reprogramming of the transcriptome consistent with three different response categories: (i) amplification of mechanical wound responses, (ii) temporal extension through accelerated or prolonged responses, and (iii) examples of inceptin-specific elicitation and suppression. At both early and late timepoints, namely 1 and 6 h, large sets of transcripts specifically accumulated following inceptin elicitation. Further early inceptin-regulated transcripts were classified as reversing changes induced by wounding alone. Within key signaling- and defense-related gene families, inceptin-elicited responses included target subsets of wound-induced transcripts. Transcripts displaying the largest inceptin-elicited fold changes included transcripts encoding terpene synthases (TPSs) and peroxidases (POXs) that correspond with induced volatile production and increased POX activity in cowpea. Characterization of inceptin-elicited cowpea defenses via heterologous expression in Nicotiana benthamiana demonstrated that specific cowpea TPSs and POXs were able to confer terpene emission and the reduced growth of beet armyworm (Spodoptera exigua) herbivores, respectively. Collectively, our present findings in cowpea support a model where HAMP elicitation both amplifies concurrent wound responses and specifically contributes to the activation of selective outputs associated with direct and indirect antiherbivore defenses.

Assessing the effects of 24-epibrassinolide and yeast extract at various levels on cowpea's morphophysiological and biochemical responses under water deficit stress.

BACKGROUND: Due to the factor of water deficit, which has placed human food security at risk by causing a 20% annual reduction in agricultural products, addressing this growing peril necessitates the adoption of inventive strategies aimed at enhancing plant tolerance. One such promising approach is employing elicitors such as 24-epibrassinolide (EBR) and yeast extract, which are potent agents capable of triggering robust defense responses in plants. By employing these elicitors, crops can develop enhanced adaptive mechanisms to combat water deficit and improve their ability to withstand drought condition. This study investigates the impact of different levels of EBR (0, 5, 10 µm) and yeast extract (0 and 12 g/l) on enhancing the tolerance of cowpea to water deficit stress over two growing seasons. RESULTS: The findings of this study demonstrate that, the combined application of EBR (especially 10 µm) and yeast extract (12 g/l) can increase seed yield (18%), 20-pod weight (16%), the number of pods per plant (18%), total chlorophyll content (90%), and decrease malondialdehyde content (45%) in cowpea, compared to plants grown under water deficit stress without these treatments. Upon implementing these treatments, impressive results were obtained, with the highest recorded values observed for the seed yield (1867.55 kg/ha), 20-pod weight (16.29 g), pods number per plant (9), and total chlorophyll content (19.88 mg g-1 FW). The correlation analysis indicated a significant relationship between the seed yield, and total chlorophyll (0.74**), carotenoids (0.82**), weight of 20 seeds (0.67**), and number of pods (0.90**). These traits should be prioritized in cowpea breeding programs focusing on water deficit stress. CONCLUSIONS: The comprehensive exploration of the effects of EBR and yeast extract across various levels on cowpea plants facing water deficit stress presents a pivotal contribution to the agricultural domain. This research illuminates a promising trajectory for future agricultural practices and users seeking sustainable solutions to enhance crops tolerance. Overall, the implications drawn from this study contribute significantly towards advancing our understanding of plant responses to water deficit stress while providing actionable recommendations for optimizing crop production under challenging environmental conditions.

Inactivated Cowpea Mosaic Virus for In Situ Vaccination: Differential Efficacy of Formalin vs UV-Inactivated Formulations.

Cowpea mosaic virus (CPMV) has been developed as a promising nanoplatform technology for cancer immunotherapy; when applied as in situ vaccine, CPMV exhibits potent, systemic, and durable efficacy. While CPMV is not infectious to mammals, it is infectious to legumes; therefore, agronomic safety needs to be addressed to broaden the translational application of CPMV. RNA-containing formulations are preferred over RNA-free virus-like particles because the RNA and protein, each, contribute to CPMV's potent antitumor efficacy. We have previously optimized inactivation methods to develop CPMV that contains RNA but is not infectious to plants. We established that inactivated CPMV has reduced efficacy compared to untreated, native CPMV. However, a systematic comparison between native CPMV and different inactivated forms of CPMV was not done. Therefore, in this study, we directly compared the therapeutic efficacies and mechanisms of immune activation of CPMV, ultraviolet- (UV-), and formalin (Form)-inactivated CPMV to explain the differential efficacies. In a B16F10 melanoma mouse tumor model, Form-CPMV suppressed the tumor growth with prolonged survival (there were no statistical differences comparing CPMV and Form-CPMV). In comparison, UV-CPMV inhibited tumor growth significantly but not as well as Form-CPMV or CPMV. The reduced therapeutic efficacy of UV-CPMV is explained by the degree of cross-linking and aggregated state of the RNA, which renders it inaccessible for sensing by Toll-like receptor (TLR) 7/8 to activate immune responses. The mechanistic studies showed that the highly aggregated state of UV-CPMV inhibited TLR7 signaling more so than for the Form-CPMV formulation, reducing the secretion of interleukin-6 (IL-6) and interferon-α (IFN-α), cytokines associated with TLR7 signaling. These findings support the translational development of Form-CPMV as a noninfectious immunotherapeutic agent.

The Potency of Cowpea Mosaic Virus Particles for Cancer In Situ Vaccination Is Unaffected by the Specific Encapsidated Viral RNA.

Plant virus nanoparticles can be used as drug carriers, imaging reagents, vaccine carriers, and immune adjuvants in the formulation of intratumoral in situ cancer vaccines. One example is the cowpea mosaic virus (CPMV), a nonenveloped virus with a bipartite positive-strand RNA genome with each RNA packaged separately into identical protein capsids. Based on differences in their densities, the components carrying RNA-1 (6 kb) denoted as the bottom (B) component or carrying RNA-2 (3.5 kb) denoted as the middle (M) component can be separated from each other and from a top (T) component, which is devoid of any RNA. Previous preclinical mouse studies and canine cancer trials used mixed populations of CPMV (containing B, M, and T components), so it is unclear whether the particle types differ in their efficacies. It is known that the CPMV RNA genome contributes to immunostimulation by activation of TLR7. To determine whether the two RNA genomes that have different sizes and unrelated sequences cause different immune stimulation, we compared the therapeutic efficacies of B and M components and unfractionated CPMV in vitro and in mouse cancer models. We found that separated B and M particles behaved similarly to the mixed CPMV, activating innate immune cells to induce the secretion of pro-inflammatory cytokines such as IFNα, IFNγ, IL-6, and IL-12, while inhibiting immunosuppressive cytokines such as TGF-ß and IL-10. In murine models of melanoma and colon cancer, the mixed and separated CPMV particles all significantly reduced tumor growth and prolonged survival with no significant difference. This shows that the specific RNA genomes similarly stimulate the immune system even though B particles have 40% more RNA than M particles; each CPMV particle type can be used as an effective adjuvant against cancer with the same efficacy as native mixed CPMV. From a translational point of view, the use of either B or M component vs the mixed CPMV formulation offers the advantage that separated B or M alone is noninfectious toward plants and thus provides agronomic safety.

Comparison of the biology of Frankliniella intonsa and Megalurothrips usitatus on cowpea pods under natural regimes through an age-stage, two-sex life table approach.

Two thrips, Megalurothrips usitatus (Bagnall) and Frankliniella intonsa (Trybom) are major pests of cowpea in South China. To realistically compare the growth, development and reproductive characteristics of these two thrips species, we compared their age-stage, two-sex life tables on cowpea pods under summer and winter natural environmental regimes. The results showed that the total preadult period of M. usitatus was 8.09 days, which was significantly longer than that of F. intonsa (7.06 days), while the adult female longevity of M. usitatus (21.14 days) was significantly shorter than that of F. intonsa (25.77 days). Significant differences were showed in male adult longevity (10.68 days for F. intonsa and 16.95 days for M. usitatus) and the female ratio of offspring (0.67 for F. intonsa and 0.51 for M. usitatus), and the total preadult period of M. usitatus (16.20 days) was significantly longer than that of F. intonsa (13.66 days) in the winter regime. The net reproductive rate (summer: R0 = 85.62, winter: R0 = 105.22), intrinsic rate of increase (summer: r = 0.3020 day-1, winter: r = 0.2115 day-1), finite rate of increase (summer: λ = 1.3526 day-1, winter: λ = 1.2356 day-1) and gross reproduction rate (summer: GRR = 139.34, winter: GRR = 159.88) of F. intonsa were higher than those of M. usitatus (summer: R0 = 82.91, r = 0.2741, λ = 1.3155, GRR = 135.71; winter: R0 = 80.62, r = 0.1672, λ = 1.1820, GRR = 131.26), and the mean generation times (summer: T = 14.73 days, winter: T = 22.01 days) of F. intonsa were significantly shorter than those of M. usitatus (summer: T = 16.11 days, winter: T = 26.25 days). These results may contribute to a better understanding of the bioecology of different thrips species, especially the interspecific competition between two economically important cowpea thrips with the same ecological niche in a changing environment.

GC/MS and LC/MS Phytochemical Analysis of Vigna unguiculata L. Walp Pod.

Vigna unguiculata (L. Walp) or Cowpea pod methanolic extracts phytochemical analysis, total phenolic content (TPC), and secondary metabolite profiling were determined using gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-mass spectrometry (LC/MS) analysis. GC/MS analysis revealed twenty compounds in the extract, while LC/MS analysis identified twenty-four compounds. GC/MS chromatogram analysis suggested the presence of opioid α-N-Normethadol a major constituent found in methanolic extract and fatty acid esters carotenoid is found second major constituent. LC/MS chromatogram and the mass spectral analysis demonstrated the presence of flavonoids, carotenoids, and alkaloids as major phytochemicals. We investigated the antibacterial, anti-fungal, and anti-oxidant activity of pod methanolic extract. The extract was found equally effective against E. coli, S. pyogenes, and P. aeruginosa with MIC 100 µg/mL similar to the standard Ampicillin (MIC 100 µg/mL). C. albicans were found to be most susceptible to Vign unguiculata pods methanolic extract with a MIC of 250 µg/mL. The pod extract showed significant DPPH scavenging activity (IC50 =78.38±0.15) which suggests its antioxidant potential.

Photorhabdus-Derived Secondary Metabolites Reduce Root Infection by Meloidogyne incognita in Cowpea.

Root-knot nematodes (RKNs) cause significant economic damage to crop plants, spurring demand for safe, affordable, and sustainable nematicides. A previous study by our research team showed that the combination of two nematicidal secondary metabolites (SMs) derived from Photorhabdus bacteria, trans-cinnamic acid (t-CA), and (4E)-5-phenylpent-4-enoic acid (PPA) have a synergistic effect against RKNs in vitro. In this study, we considered in planta assays to assess the effects of this SM mixture on the virulence and reproductive fitness of the RKN Meloidogyne incognita in a cowpea. Factorial combinations of five t-CA + PPA concentrations (0, 9.0, 22.9, 57.8, and 91.0 µg/ml) and two nematode inoculation conditions (presence or absence) were evaluated in 6-week growth chamber experiments. Results from this study showed that a single root application of the t-CA + PPA mixture significantly reduced the penetration of M. incognita infective juveniles (J2s) into the cowpea roots. The potential toxicity of t-CA + PPA on RKN-susceptible cowpea seedlings was also investigated. The effect of t-CA + PPA × nematode inoculation interactions and the t-CA + PPA mixture did not show significant phytotoxic effects, nor did it adversely affect plant growth parameters or alter leaf chlorophyll content. Total leaf chlorophyll and chlorophyll b content were significantly reduced (by 15 and 22%, respectively) only by the nematode inoculum and not by any of the SM treatments. Our results suggest that a single root application of a mixture of t-CA and PPA reduces M. incognita J2's ability to infect the roots without impairing plant growth or chlorophyll content.

First report of Agroathelia rolfsii causing southern blight on cowpea in China.

Cowpea (Vigna unguiculata L.) is a legume consumed as a high-quality plant protein source in many parts of the world. In August 2023, it was observed that a plant disease affected cowpea growth in Yiyang (28.34°N, 112.55°E), China. The average disease incidence was 10%, resulting in 8.5% economic losses in approximately 3,000 m2. The symptoms initially appeared as brown lesions near the stem-soil interface and the lesions were colonized by white mycelia. As the disease progressed, the disease symptoms included constriction and brown staining at the base of the stem, covered with a small amount of white mycelia. Eventually, the entire plants withered and collapsed and many sclerotia were scattered on the ground around the diseased stem. Twenty samples (10 sclerotia and 10 diseased tissue fragments) were collected from symptomatic plants for causal agent isolation. Samples were disinfected with 70% ethanol for 30 s, 5% NaClO for 1 min, rinsed three times with sterile water, dried and placed on potato dextrose agar (PDA) plates at 28℃ in the dark. In total, 20 isolates were obtained by the hyphal tip method (Terrones et al. 2022) and showed a consistent phenotype of white cottony mycelia on PDA with an growth rate of 12.9 to 21.3 mm/day (n = 20). Sclerotia formed at five to eight days post inoculation, were initially whitish, turning beige and eventually dark brown. The diameter of mature sclerotia ranged from 0.89 to 2.13 mm (mean = 1.64±0.29 mm; n =50). For pathogen identification, ITS1/ITS4 (White et al. 1990) and EF1-983F/EF1-2218R (Rehner and Buckley 2005) primers were used to amplify the internal transcribed spacer regions (ITS) and translation elongation factor-1 alpha gene (TEF-1α), respectively. The sequences of all 20 isolates showed 99% to 100% similarity with Agroathelia rolfsii sequences from GenBank by BLAST analysis. The sequences of two representative strains, ID1 and ID4, were deposited in GenBank. The ITS sequences of ID1 (OR689482) and ID4 (OR689481) were ＞99% similar to A. rolfsii strain QJ7 (593/596 bp; MZ750983) and A. rolfsii strain Kale078 (565/568 bp; MN872304), respectively. Also, TEF-1α sequences of ID1 (OR713735) and ID4 (OR713736) were ＞99% similar to the sequences of A. rolfsii strain HS-Sr (1073/1073 bp; OL416131) and A. rolfsii strain MSB1-2 (1070/1075 bp; MN702790), respectively. Phylogenetic analysis based on ITS and TEF1-α sequences indicated that ID1 and ID4 clustered into the A. rolfsii clade. Based on morphology and sequence analyses, the isolates ID1 and ID4 were identified as A. rolfsii (anamorph Sclerotium rolfsii). Pathogenicity tests were conducted three times on healthy 30-day-old cowpea seedlings. Five plants were inoculated with 6-day-old mycelial discs (6 mm) of ID1 or ID4 at the base of the seedlings (n = 30) while four plants were inoculated with a sterile PDA disc as a control (n = 12). All seedlings were cultivated in a greenhouse with a temperature of 26°C to 28°C and relative humidity 60% to 80% with a 14/10 h light/dark photoperiod. Eight days later, all the fungal inoculated seedlings showed symptoms including brown necrosis and collapse of the stems, and eventual withering of the seedlings. Control plants remained asymptomatic. The causal pathogens were reisolated in PDA plates and identified by ITS sequence analysis, completing Koch's postulates. To our knowledge, this is the first report of A. rolfsii causing southern blight on cowpea in China. Early accurate diagnosis will help farmers to adopt suitable practices to control disease outbreaks and reduce losses.

Identification and Virulence of Five Isolates of Root-Knot Nematode Meloidogyne floridensis on Vegetables.

Meloidogyne floridensis is of particular concern because it reproduces on tomato, pepper, corn, and tobacco cultivars that have resistance to the common tropical root-knot nematode (RKN) species (i.e., Meloidogyne incognita, M. arenaria, and M. javanica). During a survey of 436 randomly selected vegetable fields in Georgia in 2018, 6 M. floridensis-infested fields were found and cultured from single egg-mass isolates on a susceptible tomato (cultivar Rutgers), and speciated using molecular analyses. Five isolates of M. floridensis were identified from collard, cowpea, cucumber, watermelon, and tomato fields by DNA sequence-based identification targeting mitochondrial genes (cytochrome c oxidase subunit II, transfer RNAHis, large subunit ribosomal RNA, and NADH dehydrogenase subunit 5). Two greenhouse trials determined the host preference and reproduction level for each M. floridensis isolate. Evaluations were conducted on Rutgers tomato, a resistant tomato (cultivar Skyway, carrying the Mi-1.2 gene), and vegetable crops associated with the origin of M. floridensis populations. This study confirmed that most associated vegetables, except collards, were good hosts to M. floridensis, having a reproduction factor >1. All isolates were able to reproduce aggressively on the resistant tomato. We found variations among M. floridensis isolates in pathogenicity and reproduction levels on the vegetable crops tested which should be considered when using or developing host resistance.

Cowpea Mosaic Virus and Natural Killer Cell Agonism for In Situ Cancer Vaccination.

We have previously shown the plant virus Cowpea mosaic virus (CPMV) to be an efficacious in situ cancer vaccine, providing elimination of tumors and tumor-specific immune memory. Additionally, we have shown that CPMV recruits Natural Killer (NK) cells within the tumor microenvironment. Here we aimed to determine whether a combination of CPMV and anti-4-1BB monoclonal antibody agonist to stimulate tumor-resident and CPMV-recruited NK cells is an effective dual therapy approach to improve NK cell function and in situ cancer vaccination efficacy. Using murine models of metastatic colon carcinomatosis and intradermal melanoma, intratumorally administered CPMV + anti-4-1BB dual therapy provided a robust antitumor response, improved elimination of primary tumors, and reduced mortality compared to CPMV and anti-4-1BB monotherapies. Additionally, on tumor rechallenge there was significant delay/prevention of tumor development and improved survival, highlighting that the CPMV + anti-4-1BB dual therapy enables potent and durable antitumor efficacy.

Cowpea isoflavones enhance the osteoblast differentiation and antioxidant capacity in synergy with vitamin D and ß-carotene: A mechanistic in vitro study.

Background: Osteoporosis (OS) is a pathological condition that makes bones susceptible to fractures by affecting the balance between bone formation and resorption. Recent literature uncovered the possible potential of bioactive compounds with antioxidant mechanisms to counter the issue. Cowpea (CP) isoflavones based on our previous study, vitamin D and natural antioxidant ß-carotene for its pleotropic protective effects were assessed alone and in combination. Aim: The study aims to assess the antioxidant and osteoblast differentiation abilities of cowpea isoflavones alone and in combination of vitamin D (VD) and ß-carotene (BC) in the human osteosarcoma cell line Saos2. Methods: Saos2 cells were maintained in cell culture conditions and concentrations of CP extract (genistein + daidzein), BC and VD required to increase cell proliferation were estimated using MTT assay. Upon treating cells with the EC50 concentrations, lysates were prepared and levels of alkaline phosphatase (ALP) and osteocalcin were evaluated using ELISA. Oxidative stress parameters and osteoblast differentiation markers were evaluated. Results: CP extract (genistein + daidzein), BC and VD concentrations which enhanced the cell proliferation rate were determined and elevated levels of ALP and osteocalcin upon treatment was observed. Anti-oxidant stress parameters studied showed an increase in cells upon treatment compared to control. Significant alterations in levels of protein involved in osteoblast differentiation are observed upon treatment. Conclusion: Cowpea isoflavones has shown a significant activity against OS by elevating antioxidant parameters and inducing osteoblast differentiation in the present study.

Genetic Architecture of Salt Tolerance in Cowpea (Vigna unguiculata (L.) Walp.) at Seedling Stage Using a Whole Genome Resequencing Approach.

Cowpea (Vigna unguiculata (L.) Walp.) is a diploid legume crop used for human consumption, feed for livestock, and cover crops. Earlier reports have shown that salinity has been a growing threat to cowpea cultivation. The objectives of this study were to conduct a genome-wide association study (GWAS) to identify SNP markers and to investigate candidate genes for salt tolerance in cowpea. A total of 331 cowpea genotypes were evaluated for salt tolerance by supplying a solution of 200 mM NaCl in our previous work. The cowpea panel was genotyped using a whole genome resequencing approach, generating 14,465,516 SNPs. Moreover, 5,884,299 SNPs were used after SNP filtering. GWAS was conducted on a total of 296 cowpea genotypes that have high-quality SNPs. BLINK was used for conducting GWAS. Results showed (1) a strong GWAS peak on an 890-bk region of chromosome 2 for leaf SPAD chlorophyll under salt stress in cowpea and harboring a significant cluster of nicotinamide adenine dinucleotide (NAD) dependent epimerase/dehydratase genes such as Vigun02g128900.1, Vigun02g129000.1, Vigun02g129100.1, Vigun02g129200.1, and Vigun02g129500.1; (2) two GWAS peaks associated with relative tolerance index for chlorophyll were identified on chromosomes 1 and 2. The peak on chromosome 1 was defined by a cluster of 10 significant SNPs mapped on a 5 kb region and was located in the vicinity of Vigun01g086000.1, encoding for a GATA transcription factor. The GWAS peak on chromosome 2 was defined by a cluster of 53 significant SNPs and mapped on a 68 bk region of chromosome 2, and (3) the highest GWAS peak was identified on chromosome 3, and this locus was associated with leaf score injury. This peak was within the structure of a potassium channel gene (Vigun03g144700.1). To the best of our knowledge, this is one the earliest reports on the salt tolerance study of cowpea using whole genome resequencing data.

Transcriptome Analysis of Differentially Expressed Genes Associated with Salt Stress in Cowpea (Vigna unguiculata L.) during the Early Vegetative Stage.

Cowpea (Vigna unguiculata (L.), 2n = 22) is a tropical crop grown in arid and semiarid regions that is tolerant to abiotic stresses such as heat and drought. However, in these regions, salt in the soil is generally not eluted by rainwater, leading to salt stress for a variety of plant species. This study was conducted to identify genes related to salt stress using the comparative transcriptome analysis of cowpea germplasms with contrasting salt tolerance. Using the Illumina Novaseq 6000 platform, 1.1 billion high-quality short reads, with a total length of over 98.6 billion bp, were obtained from four cowpea germplasms. Of the differentially expressed genes identified for each salt tolerance type following RNA sequencing, 27 were shown to exhibit significant expression levels. These candidate genes were subsequently narrowed down using reference-sequencing analysis, and two salt stress-related genes (Vigun_02G076100 and Vigun_08G125100) with single-nucleotide polymorphism (SNP) variation were selected. Of the five SNPs identified in Vigun_02G076100, one that caused significant amino acid variation was identified, while all nucleotide variations in Vigun_08G125100 was classified as missing in the salt-resistant germplasms. The candidate genes and their variation, identified in this study provide, useful information for the development of molecular markers for cowpea breeding programs.

The Genome of Fusarium oxysporum f. sp. phaseoli Provides Insight into the Evolution of Genomes and Effectors of Fusarium oxysporum Species.

Fusarium oxysporum f. sp. phaseoli, the causal agent of cowpea fusarium wilt, is a serious threat to cowpea production in China. In this study, a sample of cowpea fusarium wilt was identified as Fusarium oxysporum f. sp. phaseoli using the methods of morphological characters and molecular detection. We further reported the first genome assembly for Fusarium oxysporum f. sp. phaseoli, with 53.7 Mb genome sequence comprising 14,694 genes. Comparative genomic analysis among five Fusarium oxysporum genomes showed that four accessory chromosomes in the five Fusarium oxysporum display similar characteristics, with low sequence similarity (55.35%, vs. overall average of 81.76%), low gene density (2.18 genes/10 kb vs. 3.02 genes/Mb) and highly transposable element density (TEs) (15.01/100 kb vs. 4.89/100 kb), indicating that variable accessory chromosomes are the main source of Fusarium oxysporum evolution. We identified a total of 100 Fusarium oxysporum f. sp. phaseoli-specific effectors in the genome and found 13 specific effector genes located in large insertion or deletion regions, suggesting that insertion or deletion events can cause the emergence of species-specific effectors in Fusarium oxysporum. Our genome assembly of Fusarium oxysporum f. sp. phaseoli provides a valuable resource for the study of cowpea fusarium wilt, and the comparative genomic study of Fusarium oxysporum could contribute to the knowledge of genome and effector-associated pathogenicity evolution in Fusarium oxysporum study.