First detection and complete genome sequence of a new tobamovirus naturally infecting Hibiscus rosa-sinensis in Hawaii.

High-throughput sequencing was used to analyze Hibiscus rosa-sinensis (family Malvaceae) plants with virus-like symptoms in Hawaii. Bioinformatic and phylogenetic analysis revealed the presence of two tobamoviruses, hibiscus latent Fort Pierce virus (HLFPV) and a new tobamovirus with the proposed name "hibiscus latent Hawaii virus" (HLHV). This is the first report of the complete sequence, genome organization, and phylogenetic characterization of a tobamovirus infecting hibiscus in Hawaii. RT-PCR with virus-specific primers and Sanger sequencing further confirmed the presence of these viruses. Inoculation experiments showed that HLFPV could be mechanically transmitted to Nicotiana benthamiana and N. tabacum, while HLHV could only be mechanically transmitted to N. benthamiana.

Survey of Viruses Infecting Basella alba in Hawaii.

Malabar spinach plants (Basella alba, Basellaceae) with leaves exhibiting symptoms of mosaic, rugosity, and malformation were found in a community garden on Oahu, HI in 2018. Preliminary studies using enzyme-linked immunosorbent assay and reverse-transcription (RT)-PCR identified Basella rugose mosaic virus (BaRMV) in symptomatic plants. However, nucleotide sequence analysis of RT-PCR amplicons indicated that additional potyviruses were also present in the symptomatic Malabar spinach. High-throughput sequencing (HTS) analysis was conducted on ribosomal RNA-depleted composite RNA samples of potyvirus-positive plants from three locations. Assembled contigs shared sequences similar to BaRMV, chilli veinal mottle virus (ChiVMV), Alternanthera mosaic virus (AltMV), Basella alba endornavirus (BaEV), broad bean wilt virus 2 (BBWV2), and Iresine viroid 1. Virus- and viroid-specific primers were designed based on HTS sequencing results and used in RT-PCR and Sanger sequencing to confirm the presence of these viruses and the viroid. We tested 63 additional samples from six community gardens for a survey of viruses in Malabar spinach and found that 21 of them were positive for BaRMV, 57 for ChiVMV, 21 for AltMV, 19 for BaEV, and 14 for BBWV2. This is the first characterization of the virome from B. alba.

Genome sequence of pineapple secovirus B, a second sadwavirus reported infecting Ananas comosus.

The complete genome sequence of pineapple secovirus B (PSV-B), a new virus infecting pineapple (Ananas comosus) on the island of Oahu, Hawaii, was determined by high-throughput sequencing (HTS). The genome comprises two RNAs that are 5,956 and 3,808 nt long, excluding the 3'-end poly-A tails, both coding for a single large polyprotein. The RNA1 polyprotein contains five conserved domains associated with replication, while the RNA2 polyprotein is cleaved into the movement protein and coat protein. PSV-B is representative of a new species in the subgenus Cholivirus (genus Sadwavirus; family Secoviridae), as the level of amino acid sequence identity to recognized members of this subgenus in the Pro-Pol and coat protein regions is below currently valid species demarcation thresholds.

Complete genome organization and characterization of Hippeastrum latent virus.

The complete genome sequences of two carlaviruses were determined by high-throughput sequencing of RNA extracted from ringspot and mosaic, disease symptoms on leaves of spider lily plants (Crinum asiaticum, family Amaryllidaceae) growing as landscape plants in Hawaii. One, named Nerine latent virus (NeLV)-Hawaii with a genome of 8281 nucleotide exhibited the highest nucleotide identity and amino acid similarity of 95.5% and 96.0%, respectively, to the genome sequence of an isolate of NeLV from Narcissus sp. in Australia (JQ395044). The second, named Hippeastrum latent virus (HiLV)-Hawaii with a genome of 8497 nucleotides exhibited the highest nucleotide identity and amino acid similarity, 84.3% and 88.7%, respectively, to the sequence of a previously uncharacterized HiLV isolate from a potted flowering plant, Amaryllis (Hippeastrum hybridum Hort) in Taiwan (DQ098905). The amino acid sequence similarities of replicase (Rep) and coat protein (CP) between HiLV-Hawaii and NeLV-Hawaii were 44.8% and 38.4%, respectively. Results of viral protein Rep and CP amino acid sequence comparisons from various carlaviruses provide evidence that HiLV and NeLV, previously classified as synonymous viruses are in fact unique viruses. This is the first report for the complete sequence, organization, and phylogenetic characterization of HiLV and the first detection of HiLV both in C. asiaticum and in the USA.

A novel ampelovirus associated with mealybug wilt of pineapple (Ananas comosus).

Mealybug wilt of pineapple (MWP) is the most important and complex viral disease affecting pineapple worldwide. High-throughput sequencing was conducted to characterize a new virus identified only in symptomatic pineapple plants and tentatively named pineapple mealybug wilt-associated virus 6 (PMWaV-6). Data analyses revealed a genome of 17,854 nucleotides with an organization resembling members of the genus Ampelovirus, family Closteroviridae. Encoded proteins shared sequence identity with the corresponding proteins of grapevine leafroll-associated virus 3, blackberry vein banding-associated virus, and PMWaV-2. The present study reports the discovery of PMWaV-6, a putative and distinct new member of the genus Ampelovirus, subgroup I, its potential involvement in MWP, and the development of PMWaV-6-specific RT-PCR assays to detect and monitor this virus in field samples.

Genome characterization of fig umbra-like virus.

The complete genome of a new umbra-like virus from edible fig (Ficus carica) was identified by high-throughput sequencing. Based on its similarity to umbra-like virus genome sequences available in GenBank, the proposed name of this new virus is "fig umbra-like virus" (FULV). The genome of full-length FULV-1 consists of 3049 nucleotides organized into three open reading frames (ORFs). Pairwise comparisons showed that the complete nucleotide sequence of the virus had the highest identity (71.3%) to citrus yellow vein-associated virus (CYVaV). In addition, phylogenetic trees based on whole-genome nucleotide sequences and amino acid sequences of the RNA-dependent RNA polymerase showed that FULV forms a monophyletic lineage with CYVaV and other umbra-like viruses. Based on the demarcation criteria of the genus Umbravirus, and lack of two umbravirus ORFs, we propose that FULV is a putative new member of the umbra-like virus clade within the family Tombusviridae.

Roles of transcription factor SQUAMOSA promoter binding protein-like gene family in papaya (Carica papaya) development and ripening.

SQUAMOSA promoter binding protein-like (SPL) family plays vital regulatory roles in plant growth and development. The SPL family in climacteric fruit Carica papaya has not been reported. This study identified 14 papaya SPLs (CpSPL) from papaya genome and analyzed their sequence features, phylogeny, intron/exon structure, conserved motif, miR156-mediated posttranscriptional regulation, and expression patterns. 14 CpSPLs were clustered into 8 groups, and two distinct expression patterns were revealed for miR156-targeted and nontargeted CpSPLs in different tissues and fruit development stages. The expression changes of CpSPLs in ethephon and 1-MCP treated fruit during ripening suggested that the CpSPLs guided by CpmiR156 play crucial roles in ethylene signaling pathway. This study sheds light on the new function of SPL family in fruit development and ripening, providing insights on understanding evolutionary divergence of the members of SPL family among plant species.

Identification and complete genomic sequence of a novel sadwavirus discovered in pineapple (Ananas comosus).

The complete genomic sequence of a putative novel member of the family Secoviridae was determined by high-throughput sequencing of a pineapple accession obtained from the National Plant Germplasm Repository in Hilo, Hawaii. The predicted genome of the putative virus was composed of two RNA molecules of 6,128 and 4,161 nucleotides in length, excluding the poly-A tails. Each genome segment contained one large open reading frame (ORF) that shares homology and phylogenetic identity with members of the family Secoviridae. The presence of this new virus in pineapple was confirmed using RT-PCR and Sanger sequencing from six samples collected in Oahu, Hawaii. The name "pineapple secovirus A" (PSVA) is proposed for this putative new sadwavirus.

Occurrence of tomato leaf curl Bangladesh virus and associated subviral DNA molecules in papaya in Bangladesh: molecular detection and characterization.

Forty-five papaya samples showing severe leaf curl symptoms were tested by PCR with a degenerate primer set for virus species in the genus Begomovirus. Of these, 29 were positive for tomato leaf curl Bangladesh virus (ToLCBV). The complete genome sequences of ToLCBV (GenBank accession no. MH380003) and its associated tomato leaf curl betasatellite (ToLCB) (MH397223) from papaya isolate Gaz17-Pap were determined and characterized. Defective betasatellites were found in ToLCBV-positive papaya isolates Gaz19-Pap, Gaz20-Pap and Gaz21-Pap. This study confirmed that papaya is a host of ToLCBV, ToLCB, and other defective and recombinant DNA satellites in Bangladesh.

Papaya Ringspot Virus Isolates From Papaya in Bangladesh: Detection, Characterization, and Distribution.

Papaya ringspot virus (PRSV) is the major constraint to papaya (Carica papaya) production in Bangladesh. Disease symptoms occurred in 90 to 100% of the plants surveyed. Full-length genomes of PRSV strains from severely infected papaya plants were determined using the Illumina NextSeq 500 platform, followed by Sanger DNA sequencing of viral genomes obtained by reverse-transcription PCR(RT-PCR). The genome sequences of two distinct PRSV strains, PRSV BD-1 (10,300 bp) and PRSV BD-2 (10,325 bp) were 74 and 83% identical to each other, respectively, at the nucleotide and amino acid levels. PRSV BD-1 and PRSV BD-2 were 74 to 75% and 79 to 88% identical, respectively, to other full-length PRSV sequences at the nucleotide level. Based on phylogenetic analysis, PRSV BD-2 was most closely related to PRSV-Meghalaya (MF356497) from papaya in India. PRSV BD-1 formed a branch distinct from the other PRSV sequences based on nucleotide and amino acid sequence comparisons. Comparisons of the genome sequences of these two strains with other sequenced PRSV genomes indicated two putative recombination events in PRSV BD-2. One recombinant event contained a 2,766-nucleotide fragment highly identical to PRSV-Meghalaya (MF356497). The other recombinant event contained a 5,105-nucleotide fragment highly identical to PRSV-China (KY933061). The occurrence rates of PRSV BD-1 and PRSV BD-2 in the sampled areas of Bangladesh were approximately 19 and 69%, respectively. Plants infected with both strains (11%) exhibited more severe symptoms than plants infected with either strain alone. The full-length genome sequences of these new PRSV strains and their distribution provide important information regarding the dynamics of papaya ringspot virus infections in papaya in Bangladesh.

Analysis of volatile profiles of green Hawai'ian coffee beans damaged by the coffee berry borer (Hypothenemus hampei).

BACKGROUND: The coffee berry borer (CBB), Hypothenemus hampei, is the most destructive insect pest of coffee globally, causing significant losses in yield and leading to 'off' flavors in damaged beans. Automated headspace sampling (AHS) and gas chromatography-mass spectrometry (GC-MS) were used to investigate changes in the volatile profiles of CBB-damaged green coffee beans. Green coffee from three coffee farms on the island of Hawai'i were sorted into three levels of CBB damage: non-damaged, slightly damaged (1-2 pinholes/bean), and heavily damaged (> 2 pinholes/bean). RESULTS: Distinct differences were found between green coffee bean samples based on the amounts of eight prominent volatiles. The amount of CBB damage was particularly correlated with the amount of both hexanal and 2-pentylfuran. Principal component analysis showed clustering of non-damaged green beans, which did not overlap with the slightly or heavily damaged clusters. Good separation was also found between a mixture of 50% slightly damaged and non-damaged coffee. However, 20% slightly damaged and non-damaged coffee clusters showed strong overlap. CONCLUSION: Understanding the effects of CBB damage on coffee flavor profiles is critical to quality control for this valuable agricultural product. The results of this study show that the volatile profiles of green coffee beans vary with CBB damage. With specific volatile profiles for CBB-damaged coffee identified, coffee samples can be tested in the lab, or potentially on the farm or in coffee mills, to identify high levels of CBB damage that may lead to off flavors and a reduction in product quality and value. © 2018 Society of Chemical Industry.

Volatile changes in Hawaiian noni fruit, Morinda citrifolia L., during ripening and fermentation.

BACKGROUND: Noni fruit (Morinda citrifolia L., Rubiaceae) has been used in traditional medicine throughout the tropics and subtropics and is now attracting interest in western medicine. Fermented noni juice is of particular interest for its promising antitumor activity. The present study collected and analyzed volatiles released at nine time intervals by noni fruit during ripening and fermentation using headspace autosampling coupled to gas chromatography-mass spectrometry. RESULTS: Twenty-three noni volatiles were identified and relatively quantified. In addition to volatiles previously identified in noni, four novel volatile 3-methyl-2/3-butenyl esters were identified via the synthesis of reference compounds. Principle component analysis (PCA) and canonical discriminant analysis (CDA) were used to facilitate multidimensional pattern recognition. PCA showed that ripening noni fruit cluster into three groups, pre-ripe, fully ripe (translucent) and fermented, based on released volatiles. CDA could 83.8% correctly classify noni samples when all ripeness stages were analyzed and 100% when samples were classified into the three PCA groupings. CONCLUSION: The results of the present study confirm the identities of 3-methyl-2/3-butenyl esters, both novel and previously identified, through the synthesis of reference compounds. These esters constitute a large percentage of the volatiles released by fully ripe and fermented noni and likely produced from the decomposition of noniosides, a group of unique glucosides present in the fruit. © 2017 Society of Chemical Industry.

Withanolides derived from Physalis peruviana (Poha) with potential anti-inflammatory activity.

Three new withanolides, physaperuvin G (1), with physaperuvins I (2), and J (3), along with seven known derivatives (4-10), were isolated from the aerial parts of Physalis peruviana. The structures of 1-3 were determined by NMR, X-ray diffraction, and mass spectrometry. Compounds 1-10 were evaluated in lipopolysaccharide (LPS)-activated murine macrophage RAW 264.7 cells. Compounds 4, 5, and 10 with potent nitric oxide inhibitory activity in LPS-activated RAW 264.7 cells, with IC50 values in the range of 0.32-7.8µM. In addition, all compounds were evaluated for potential to inhibit tumor necrosis factor-alpha (TNF-α)-activated nuclear factor-kappa B (NF-κB) activity with transfected human embryonic kidney cells 293. Compounds 4-7 inhibited TNF-α-induced NF-κB activity with IC50 values in the range of 0.04-5.6µM.

Anti-inflammatory and Quinone Reductase Inducing Compounds from Fermented Noni (Morinda citrifolia) Juice Exudates.

A new fatty acid ester disaccharide, 2-O-(ß-d-glucopyranosyl)-1-O-(2E,4Z,7Z)-deca-2,4,7-trienoyl-ß-d-glucopyranose (1), a new ascorbic acid derivative, 2-caffeoyl-3-ketohexulofuranosonic acid γ-lactone (2), and a new iridoid glycoside, 10-dimethoxyfermiloside (3), were isolated along with 13 known compounds (4-16) from fermented noni fruit juice (Morinda citrifolia). The structures of the new compounds, together with 4 and 5, were determined by 1D and 2D NMR experiments, as well as comparison with published values. Compounds 2 and 7 showed moderate inhibitory activities in a TNF-α-induced NF-κB assay, and compounds 4 and 6 exhibited considerable quinone reductase-1 (QR1) inducing effects.

Determination of the absolute configuration of chaetoviridins and other bioactive azaphilones from the endophytic fungus Chaetomium globosum.

Chemical investigation of an endophytic fungus Chaetomium globosum isolated from leaves of Wikstroemia uva-ursi led to the isolation of two new azaphilones, chaetoviridins J and K (1 and 3), along with five known derivatives (2 and 4-7). The structures of azaphilones were determined by NMR, X-ray diffraction, Mosher's method, and CD analysis. The isolated compounds were evaluated for their cancer chemopreventive-potential based on their abilities to inhibit tumor necrosis factor alpha (TNF-α)-induced nuclear factor-kappa B (NF-κB). Compounds 4, 5, 7, and synthetic 8 and 9 inhibit nitric oxide (NO) production with IC50 values in the range of 0.3-5.8 µM. Compounds 4, 5, and 9 also displayed (TNF-α)-induced NF-κB activity with IC50 values in the range of 0.9-5.1 µM.

Supplementation of papaya leaf juice has beneficial effects on glucose homeostasis in high fat/high sugar-induced obese and prediabetic adult mice.

Prediabetes is characterized by a cluster of glycemic parameters higher than normal but below the threshold of type 2 diabetes mellitus (T2DM). In recent years, phytochemical-rich plant extracts have gained popularity as therapeutic agents for metabolic disorders. This study investigated the effects of papaya leaf (PL) juice supplementation on blood glucose levels in diet-induced obese and prediabetic adult mice. B65JL F1 mice (n = 20) at 12-14 months old were fed a high fat/sugar diet (HFHS) for 120 days. Mice were switched to restricted rodent chow of 3 g feed/30 g body weight/day, supplemented with 3 g/100 mL PL juice for 30 days. HFHS diet remarkably increased fasting plasma glucose levels from 114 ± 6.54 mg/dL to 192.7 ± 10.1 mg/dL and body weight from 32.5 ± 1.6 to 50.3 ± 4.1 g. HFHS diet results in hyperglycemia, insulin resistance, hyperlipidemia, and liver steatosis. The combination of PL juice and restricted diet significantly reduced body weight and fasting blood glucose levels to 43.75 ± 1.4 g and 126.25 ± 3.2 mg/dl, respectively. Moreover, PL juice with a restricted diet significantly improved lipid profile: cholesterol from 204 to 150 mg/dL, LDL-c from 110.4 to 50 mg/dL, and triglyceride from 93.7 to 60 mg/dL. Additionally, PL juice combined with a restricted diet significantly reduced adiposity, reversed fatty liver, and restored skeletal muscle Glut4 and phosphorylated (p-AKT (ser473). This study demonstrated that supplementation of PL juice with a restricted diet was more effective than a restricted diet alone in reversing major symptoms related to prediabetic and obesity conditions.

Physical, Chemical, and Sensory Properties of a Turmeric-Fortified Pineapple Juice Beverage.

Beverage mixtures based on pineapple (Ananas comosus) and turmeric (Curcuma longa) juice as a ready-to-drink product were developed, and their physicochemical, nutritional, and sensory properties were evaluated. Four different concentrations of turmeric juice (5%, 10%, 15%, and 20% (v/v)) were added to pineapple juice to make turmeric-fortified pineapple (TFP) juice samples. Pineapple juice without turmeric was the control. The L*, a*, b*, titratable acidity (TA), total antioxidant capacity, and %DPPH scavenging values, as well as the concentrations of the phenolic compounds curcumin and demethoxycurcumin, were significantly increased with increasing turmeric concentration. Thirty volatile compounds were detected in the mixed juice samples with turmeric. Most of the turmeric-specific compounds, including monoterpenes, sesquiterpenes and turmerones, were detected in the TFP juice samples. While the antioxidant activity of the juice samples increased with increasing turmeric concentration, the pineapple juice fortified with 10% turmeric (10%T) had the best overall quality as determined by panelists. Greater concentrations of turmeric were associated with decreased palatability due to reduced mouthfeel and sweetness and increased aftertaste and sourness. These results suggest that the 10%T juice could be developed into a commercial functional beverage with increased overall flavor and nutritional quality.

Selective Detection of Escherichia coli K12 and Staphylococcus aureus in Mixed Bacterial Communities Using a Single-Walled Carbon Nanotube (SWCNT)-Functionalized Electrochemical Immunosensor with Dielectrophoretic Concentration.

An electrochemical immunosensor has been developed for the rapid detection and identification of potentially harmful bacteria in food and environmental samples. This study aimed to fabricate a microwire-based electrochemical immunosensor (MEI sensor) for selective detection of Escherichia coli and Staphylococcus aureus in microbial cocktail samples using dielectrophoresis (DEP)-based cell concentration. A gold-coated tungsten microwire was functionalized by coating polyethylenimine, single-walled carbon nanotube (SWCNT) suspension, streptavidin, biotinylated antibodies, and then bovine serum albumin (BSA) solutions. Double-layered SWCNTs and 5% BSA solution were found to be optimized for enhanced signal enhancement and nonspecific binding barrier. The selective capture of E. coli K12 or S. aureus cells was achieved when the electric field in the bacterial sample solution was generated at a frequency of 3 MHz and 20 Vpp. A linear trend of the change in the electron transfer resistance was observed as E. coli concentrations increased from 5.32 × 102 to 1.30 × 108 CFU/mL (R2 = 0.976). The S. aureus MEI sensor fabricated with the anti-S. aureus antibodies also showed an increase in resistance with concentrations of S. aureus (8.90 × 102-3.45 × 107 CFU/mL) with a correlation of R2 = 0.983. Salmonella typhimurium and Listeria monocytogenes were used to evaluate the specificity of the MEI sensors. The functionalization process developed for the MEI sensor is expected to contribute to the sensitive and selective detection of other harmful microorganisms in food and environmental industries.

A single-walled carbon nanotubes-based electrochemical impedance immunosensor for on-site detection of Listeria monocytogenes.

Real-time and sensitive detection of pathogenic bacteria in food is in high demand to ensure food safety. In this study, a single-walled carbon nanotubes (SWCNTs)-based electrochemical impedance immunosensor for on-site detection of Listeria monocytogenes (L. monocytogenes) was developed. A gold-plated wire was functionalized using polyethylenimine (PEI), SWCNTs, streptavidin, biotinylated L. monocytogenes antibodies, and bovine serum albumin (BSA). A linear relationship (R2  = 0.982) between the electron transfer resistance measurements and concentrations of L. monocytogenes within the range of 103 -108 CFU/ml was observed. In addition, the sensor demonstrated high selectivity towards the target in the presence of other bacterial cells such as Salmonella Typhimurium and Escherichia coli O157:H7. To facilitate the demand for on-site detection, the sensor was integrated into a smartphone-controlled biosensor platform, consisting of a compact potentiostat device and a smartphone. The signals from the proposed platform were compared with a conventional potentiostat using the immunosensor interacted with L. monocytogenes (103 -105 CFU/ml). The signals obtained with both instruments showed high consistency. Recovery percentages of lettuce homogenate spiked with 103 , 104 , and 105 CFU/ml of L. monocytogenes obtained with the portable platform were 90.21, 90.44, and 93.69, respectively. The presented on-site applicable SWCNT-based immunosensor platform was shown to have a high potential to be used in field settings for food and agricultural applications. PRACTICAL APPLICATION: The developed immunosensor was developed for on-site detection of L. monocytogenes. The limit of detection of the sensor was 103 CFU/ml with a detection time of 10 min. In order to facilitate the requirements for effective on-site screening for food safety, the sensor was integrated into a smartphone-controlled platform, so that the bio-molecular interactions were converted into impedance signals and transmitted wirelessly to a smartphone by a hand-held EIS transducer.

Preserving plant samples from remote locations for detection of RNA and DNA viruses.

Viral diseases in plants have a significant impact on agricultural productivity. Effective detection is needed to facilitate accurate diagnosis and characterization of virus infections essential for crop protection and disease management. For sensitive polymerase chain reaction (PCR)-based methods, it is important to preserve the integrity of nucleic acids in plant tissue samples. This is especially critical when samples are collected from isolated areas, regions distant from a laboratory, or in developing countries that lack appropriate facilities or equipment for diagnostic analyses. RNAlater ® provides effective, reliable sample storage by stabilizing both RNA and DNA in plant tissue samples. Our work indicated that total RNA or DNA extracted from virus-infected leaf samples preserved in RNAlater ® was suitable for reverse transcription polymerase chain reaction (RT-PCR), PCR, Sanger sequencing, high-throughput sequencing (HTS), and enzyme-linked immunosorbent assay (ELISA)-based diagnostic analyses. We demonstrated the effectiveness of this technology using leaf tissue samples from plants with virus symptoms grown in farmers' fields in Bangladesh. The results revealed that RNAlater ® technology was effective for detection and characterization of viruses from samples collected from remote areas and stored for extended periods. Adoption of this technology by developing countries with limited laboratory facilities could greatly increase their capacity to detect and diagnose viral infections in crop plants using modern analytical techniques.