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The present study was designed to assess the treatment effect of arginase inhibitor, L-Norvaline in abnormalities associated with high fat diet (HFD) and fructose-induced metabolic syndrome. The HFD and fructose was fed to the rats for a period of 45 days. Animals having body weight of 350 g and fasting blood sugar level of more than 250 mg/dl were considered as hyperlipidemic diabetic rats (HDR) and selected for the study. The HDR were divided into three groups having six animals each. The HDR received L–Norvaline (10 mg/kg/day, i.p.) and standard drug, gemfibrozil (60 mg/kg/day, p.o.), for a period of 30 days. Various hormonal, biochemical and tissue parameters were evaluated at the end of the study. Both treatments significantly decreased body weight, BMI, fasting blood sugar and insulin level and improved insulin resistance in HDR as compared to the toxicant control group. A significant improvement was observed in the lipid profile, levels of nitrate, leptin, C-reactive protein and adiponectin in HDR. L-Norvaline also caused slight decrease in the malondialdehyde level, though, no prominent effect was observed on the level of superoxide dismutase and reduced glutathione in the pancreas of HDR, as compared to the toxicant control group. L-Norvaline treatment also improved the histo-architecture of pancreatic cells. Results of the present study concludes that L-Norvaline caused significant alleviation of the abnormalities of MetS indicating that it can be used as potential treatment strategy for managing the symptoms of metabolic syndrome.

Dickeya solani one of the most aggressive pectinolytic phytopathogens, causes blackleg disease in potatoes, resulting in significant economic losses and adversely impacting one of the world's most important food crops. The diagnostics methods are critical in monitoring the latent infection for international trade of potato seed tubers and in implementation of control strategies. Our study employed a whole-genome comparative approach, identifying unique target gene loci (LysR and TetR family of transcriptional regulators gene regions) and designing loop-mediated isothermal amplification (LAMP) and a multi-gene-based multiplex TaqMan qPCR assays for specific detection and differentiation of D. solani . Both methods underwent meticulous validation with extensive inclusivity and exclusivity panels, exhibiting 100% accuracy and no false positives or negatives. The LAMP method demonstrated the detection limit of 100 fg and 1 CFU per reaction using pure genomic DNA and crude bacterial cell lysate, respectively. The qPCR detection limit was 1 pg, 100 fg and 10 fg with quadplex, triplex, and singleplex, respectively. None of the assays were impacted by any inhibitory or competitive effects after adding host DNA (in qPCR) or crude lysate (in LAMP). The assays proved robust and reproducible in detecting the target pathogen in infected samples, with the LAMP assay being field-deployable due to its simplicity and rapid results acquisition within approximately 9 min. The reproducibility was confirmed by performing the assay in two independent laboratories. These assays offer a robust, rapid, and reliable solution for routine testing, with applications in phytosanitary inspection, disease diagnosis, and epidemiological studies.IMPORTANCE Dickeya solani , one of the most aggressive soft rot causing bacteria and a quarantine pathogen, poses a severe threat to food security by causing substantial economic losses to the potato industry. Accurate and timely detection of this bacterium is vital for monitoring latent infections, particularly for international trade of potato seed tubers, and for implementing effective control strategies. In this research, we have developed LAMP and multi-gene-based multiplex TaqMan qPCR assays for specific detection of D. solani . These assays, characterized by their precision, rapidity, and robustness, are crucial for distinguishing D. solani from related species. Offering unparalleled sensitivity and specificity, these assays are indispensable for phytosanitary inspection and epidemiological monitoring, providing a powerful tool enabling management of this threatening pathogen.

Recently, species clustering within Dickeya zeae has been identified as complex, encompassing validly published names, including D. oryzae and D. parazeae , with some strains potentially delineating new species. In this study, genomes of strains isolated from a bacterial heart rot outbreak in pineapple ( Ananas comosus var. comosus ) on Oahu, Hawaii, along with two strains from pineapple in Malaysia, were sequenced. Orthologous average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values among the sequenced genomes ranged from 98.93-99.9% and 91.8-99.9%, respectively, supporting the classification of seven strains within the same species. Comparisons of ANI and dDDH values between these seven strains and type strains of D. zeae, D. parazeae, and D. oryzae ranged from 94.4-95.9% and 57.2-66.5%, respectively. These values fall below the proposed boundaries for new species designation, supporting the delineation of a novel species. Phylogenetic analyses, including 16S rRNA, gapA , multi-locus sequence analysis (MLSA) of 10 housekeeping genes, whole-genome, and pangenome analyses, were concordant and revealed a distinct monophyletic clade, separating these strains from other members of the D. zeae complex, with D. oryzae as the closest relative. Notably, a nitrogen fixation gene cluster comprising 28 genes, similar to the Klebsiella spp. nitrogenase gene cluster, was found in the genome of the seven pineapple strains. Based on polyphasic approaches, including ANI, dDDH, biochemical, physiological, and phylogenomic analyses, we propose the reclassification in a new species of the five pineapple strains from Hawaii A5391, A5410 T , A5611, A6136, and A6137, together with the two pineapple strains from Malaysia CFBP 1272 and CFBP 1278, previously classified as D. zeae . We propose the name Dickeya ananae sp. nov. for this taxon, represented by the type strain A5410 T (= ICMP 25020 T = LMG 33197 T )., Competing Interests: Conflicts of interest The authors declare that there are no conflicts of interest.

Treatment of dye pollutants prior to their release into the environment remains a formidable challenge, persisting as a longstanding issue. This study focuses on the development of a multiwalled carbon nanotube-foam (MWCNT-foam) composite through low-temperature chemical fusion (LTFC), resulting in a composite with a remarkably high accessible surface area (>475 m 2 g -1 ). The MWCNT-foam composite exhibits a three-dimensional porous structure and demonstrates a notable affinity for organic dye adsorption. The efficacy of this composite was evaluated against various cationic dyes such as Methylene blue (MB) and Crystal Violet (CV) as well as anionic dyes such as Congo red (CR) and Eriochrome black T (EB), and the composite showed removal rates exceeding 99%. Furthermore, the study delved into the impact of the initial dye concentration, adsorbent dosage, kinetics, and other factors on the performance of the MWCNT-foam composite. The adsorption process achieved equilibrium in 10 min and strongly correlated with the pseudo-second-order kinetic model and Langmuir isotherm. The maximum adsorption capacity of MWCNT-foam for MB, CV, CR, and EB was found to be 168.63, 147.49, 99.50, and 93.11 mg g -1 , respectively. In order to showcase the potential of this material for continuous adsorption, a specialized cartridge was designed and employed to treat dye solutions, demonstrating the feasibility of continuous mode adsorption.

Xanthomonas and Stenotrophomonas are closely related genera in the family Lysobacteraceae. In our previous study of aroid-associated bacterial strains, most strains isolated from anthurium and other aroids were reclassified as X. phaseoli and other Xanthomonas species. However, two strains isolated from Spathiphyllum and Colocasia were phylogenetically distant from other strains in the Xanthomonas clade and two strains isolated from Anthurium clustered within the Stenotrophomonas clade. Phylogenetic trees based on 16S rRNA and nine housekeeping genes placed the former strains with the type strain of X. sacchari from sugarcane and the latter strains with the type strain of S. bentonitica from bentonite. In pairwise comparisons with type strains, the overall genomic relatedness indices required delineation of new species; digital DNA-DNA hybridization and average nucleotide identity values were lower than 70 and 95%, respectively. Hence, three new species are proposed: S. aracearum sp. nov. and S. oahuensis sp. nov. for two strains from anthurium and X. hawaiiensis sp. nov. for the strains from spathiphyllum and colocasia, respectively. The genome size of X. hawaiiensis sp. nov. is ~4.88 Mbp and higher than S. aracearum sp. nov. (4.33 Mbp) and S. oahuensis sp. nov. (4.68 Mbp). Gene content analysis revealed 425 and 576 core genes present in 40 xanthomonads and 25 stenotrophomonads, respectively. The average number of unique genes in Stenotrophomonas spp. was higher than in Xanthomonas spp., implying higher genetic diversity in Stenotrophomonas ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Chuang, Dobhal, Alvarez and Arif.)

Irrigation water is a common source of contamination that carries plant and foodborne human pathogens and provides a niche for proliferation and survival of microbes in agricultural settings. Bacterial communities and their functions in irrigation water were investigated by analyzing samples from wetland taro farms on Oahu, Hawaii using different DNA sequencing platforms. Irrigation water samples (stream, spring, and storage tank water) were collected from North, East, and West sides of Oahu and subjected to high quality DNA isolation, library preparation and sequencing of the V3-V4 region, full length 16S rRNA, and shotgun metagenome sequencing using Illumina iSeq100, Oxford Nanopore MinION and Illumina NovaSeq, respectively. Illumina reads provided the most comprehensive taxonomic classification at the phylum level where Proteobacteria was identified as the most abundant phylum in the stream source and associated water samples from wetland taro fields. Cyanobacteria was also a dominant phylum in samples from tank and spring water, whereas Bacteroidetes were most abundant in wetland taro fields irrigated with spring water. However, over 50% of the valid short amplicon reads remained unclassified and inconclusive at the species level. In contrast, Oxford Nanopore MinION was a better choice for microbe classification at the genus and species levels as indicated by samples sequenced for full length 16S rRNA. No reliable taxonomic classification results were obtained while using shotgun metagenome data. In functional analyzes, only 12% of the genes were shared by two consortia and 95 antibiotic resistant genes (ARGs) were detected with variable relative abundance. Full descriptions of microbial communities and their functions are essential for the development of better water management strategies aimed to produce safer fresh produce and to protect plant, animal, human and environmental health. Quantitative comparisons illustrated the importance of selecting the appropriate analytical method depending on the level of taxonomic delineation sought in each microbiome., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Klair, Dobhal, Ahmad, Hassan, Uyeda, Silva, Wang, Kim, Alvarez and Arif.)

Dickeya fangzhongdai, a bacterial pathogen of taro (Colocasia esculenta), onion (Allium sp.), and several species in the orchid family (Orchidaceae) causes soft rot and bleeding canker diseases. No field-deployable diagnostic tool is available for specific detection of this pathogen in different plant tissues. Therefore, we developed a field-deployable loop-mediated isothermal amplification (LAMP) assay using a unique genomic region, present exclusively in D. fangzhongdai. Multiple genomes of D. fangzhongdai, and other species of Dickeya, Pectobacterium and unrelated genera were used for comparative genomic analyses to identify an exclusive and conserved target sequence from the major facilitator superfamily (MFS) transporter gene region. This gene region had broad detection capability for D. fangzhongdai and thus was used to design primers for endpoint PCR and LAMP assays. In-silico validation showed high specificity with D. fangzhongdai genome sequences available in the NCBI GenBank genome database as well as the in-house sequenced genome. The specificity of the LAMP assay was determined with 96 strains that included all Dickeya species and Pectobacterium species as well as other closely related genera and 5 hosts; no false positives or false negatives were detected. The detection limit of the assay was determined by performing four sensitivity assays with tenfold serially diluted purified genomic DNA of D. fangzhongdai with and without the presence of crude host extract (taro, orchid, and onion). The detection limit for all sensitivity assays was 100 fg (18-20 genome copies) with no negative interference by host crude extracts. The assays were performed by five independent operators (blind test) and on three instruments (Rotor-Gene, thermocycler and dry bath); the assay results were concordant. The assay consistently detected the target pathogen from artificially inoculated and naturally infected host samples. The developed assay is highly specific for D. fangzhongdai and has applications in routine diagnostics, phytosanitary and seed certification programs, and epidemiological studies., (© 2022. The Author(s).)

Melia dubia is one of the most important industrial tree species in the South East Asia . In last few decades, the populations of M. dubia has rapidly expanded in the Indian sub-continents, leading to an increase in the genetic diversity of species. However, very less information is available on intra-specific variation in Melia under the Indian subcontinent. Therefore, a present investigation was undertaken, to assess the level of diversity in seed and saplings of the Melia populations (ecotypes) collected from three agro-ecological regions of India. Results revealed that the seed and saplings of all the ecotypes are significantly different for all the traits, except for number of branches per plant, and the maximum variability was recorded in germination percentage, seed weight, internodal length, and sapling height of the species. The high heritability for seed weight (0.99), length (0.99), and width (0.97), and germination percentage (0.99) indicated that selection and genetic gain for these traits would be effective during the commencement of improvement program. Trait association analysis explained that higher seed weight significantly reduced sapling height, collar diameter, number of leaves per plant, internodal length, petiole length, and germination percentage (r = -0.86; p  < 0.001) that ultimately reduced the seedling vigor in Melia dubia . Interestingly, the number of branches per plant were not associated with any of the morphological traits. The first principal component explained 50.09% of the entire variation and all the traits contributed greatly to the variation for this principal component, except for number of branches, leaf width and seed length. The clustering approach assorted geographic variation of M. dubia populations into three main sub-clusters i.e. South, North, and North East populations each consisting of five, seven and one populations (including cultivar), respectively. Among different ecotypes, Bahumukhi, Varsha and US Nagar seed sources outperformed all others in seedling vigour (sapling height) and rest of the growth parameters. Overall, findings explained that considerable scope exists for the development of superior planting material of M. dubia through exploration of seeds and selection at the early seedling stage., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Pectobacterium parmentieri (formerly Pectobacterium wasabiae), which causes soft rot disease in potatoes, is a newly established species of pectinolytic bacteria within the family Pectobacteriaceae. Despite serious damage caused to the potato industry worldwide, no field-deployable diagnostic tests are available to detect the pathogen in plant samples. In this study, we aimed to develop a reliable, rapid, field-deployable loop-mediated isothermal amplification (LAMP) assay for the specific detection of P. parmentieri. Specific LAMP primers targeting the petF1 gene region, found in P. parmentieri but no other Pectobacterium spp., were designed and validated in silico and in vitro using extensive inclusivity (15 strains of P. parmentieri) and exclusivity (94 strains including all other species in the genus Pectobacterium and host DNA) panels. No false positives or negatives were detected when the assay was tested directly with bacterial colonies, and with infected plant and soil samples. Sensitivity (analytical) assays using serially diluted bacterial cell lysate and purified genomic DNA established the detection limit at 10 CFU/mL and 100 fg (18-20 genome copies), respectively, even in the presence of host crude DNA. Consistent results obtained by multiple users/operators and field tests suggest the assay's applicability to routine diagnostics, seed certification programs, biosecurity, and epidemiological studies., (© 2021. The Author(s).)

Dickeya zeae , a bacterial plant pathogen of the family Pectobacteriaceae, is responsible for a wide range of diseases on potato, maize, rice, banana, pineapple, taro, and ornamentals and significantly reduces crop production. D. zeae causes the soft rot of taro ( Colocasia esculenta ) and the heart rot of pineapple ( Ananas comosus ). In this study, we used Pacific Biosciences single-molecule real-time (SMRT) sequencing to sequence two high-quality complete genomes of novel strains of D. zeae : PL65 (size: 4.74997 MB; depth: 701x; GC: 53.6%) and A5410 (size: 4.7792 MB; depth: 558x; GC: 53.5%) isolated from economically important Hawaiian crops, taro, and pineapple, respectively. Additional complete genomes of D. zeae representing three additional hosts (philodendron, rice, and banana) and other species used for a taxonomic comparison were retrieved from the NCBI GenBank genome database. Genomic analyses indicated the truncated type III and IV secretion systems (T3SS and T4SS) in the taro strain, which only harbored one and two genes of T3SS and T4SS, respectively, and showed high heterogeneity in the type VI secretion system (T6SS). Unlike strain EC1, which was isolated from rice and recently reclassified as D. oryzae , neither the genome PL65 nor A5410 harbors the zeamine biosynthesis gene cluster, which plays a key role in virulence of other Dickeya species. The percentages of average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between the two genomes were 94.47 and 57.00, respectively. In this study, we compared the major virulence factors [plant cell wall-degrading extracellular enzymes and protease (Prt)] produced by D. zeae strains and evaluated the virulence on taro corms and pineapple leaves. Both strains produced Prts, pectate lyases (Pels), and cellulases but no significant quantitative differences were observed ( p > 0.05) between the strains. All the strains produced symptoms on taro corms and pineapple leaves, but the strain PL65 produced symptoms more rapidly than others. Our study highlights the genetic constituents of pathogenicity determinants and genomic heterogeneity that will help to understand the virulence mechanisms and aggressiveness of this plant pathogen., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Boluk, Arizala, Dobhal, Zhang, Hu, Alvarez and Arif.)

Mizuna (Brassica rapa var. japonica), a member of family Brassicaceae, is a leafy vegetable having phenolic and other compounds beneficial to human health, such as natural antioxidants (Khanam et al. 2012). In October 2020, a field of mizuna (variety: Early) on Oahu island was observed having 20-30% diseased plants. Four randomly selected infected mizuna plants, showing the symptoms of wilt and stem rot (Figure 1A-D), were collected and isolations were made to determine the pathogen. Small sections of infected stems were cut, surface sterilized with 0.6% sodium hypochlorite solution for 30 sec, followed by three consecutive rinses in distilled water. The tissues were macerated in a sterile 1.5 ml centrifuge tube containing 100 μl sterile water-macerated tissues were streaked onto crystal violet pectate medium (CVP) (Hélias et al. 2011) and incubated at 26 ± 2°C for 48 h. Isolated bacterial colonies that formed pits on the CVP plates were re-streaked onto dextrose peptone agar: Peptone (10 g/L), Dextrose (5 g/L) and Agar (17 g/L) (DPA-without tetrazolium chloride; Norman and Alvarez 1989) to obtain purified colonies for DNA isolation using DNeasy Blood and Tissue Kit (Qiagen, Germantown, MA). The two housekeeping genes (dnaA and gapA) were amplified and sequenced following the protocols used by Dobhal et al. (2020) and Boluk et al. (2020), for identity confirmation and phylogenetic analysis. Cleaned PCR products were sent to the GENEWIZ facility (Genewiz, La Jolla, CA) for sequencing of sense and antisense strands. The obtained sequences were aligned, manually edited, and consensus sequences were analyzed with BLASTn using the NCBI GenBank nucleotide and genome databases for identity confirmation. The BLASTn results demonstrated 100% query coverage of all four strains (PL248-PL251); and showed 100% identity of PL248 and PL249, and 99% identity of PL250 and PL251 with Pectobacterium brasiliense. All the sequences were submitted to the NCBI GenBank database under the following accession numbers: dnaA gene MW560271 - MW560274 (PL248 - PL251); and gapA gene MW560275 - MW560278 (PL248 - PL251). Pathogenicity was assessed by artificially inoculating 100 µl bacterial suspension of each strain (PL248 - 1.12x 10⁸ CFU/ml; PL249 - 1.32x 10⁸ CFU/ml; PL 250 - 1.2x 10⁸ CFU/ml and PL251 - 1.15x 10⁸ CFU/ml) onto four-week-old mizuna (variety: Leafy Asian Greens) plants in three replicates, using sterile pipette tips, which was stabbed into stem halfway and wrapped with parafilm. The inoculated plants were well maintained under controlled greenhouse conditions. As negative controls, three plants were inoculated with 100 µl distilled water. Soft rot and wilt symptoms (Figure 1E-H) were observed 24 hours post inoculation. No symptoms were observed on control plants (Figure 1F). All four strains were re-isolated from the inoculated plants and confirmed as P. brasiliense based on resequencing of the dnaA region and 100% homology with the sequences of original strain. In the phylogenetic tree (Figure 2), based on two housekeeping genes (dnaA and gapA), the bacterial strains from mizuna grouped with other P. brasiliense retrieved from the NCBI GenBank database. To our knowledge, this is the first report of P. brasiliense infecting mizuna plants in Hawaii or in the USA and is important because this species is one of the most aggressive pectolytic pathogens in the genus Pectobacterium. Understanding the diversity of different pectolytic phytopathogens is essential to formulating risk mitigation strategies as P. brasiliense could potentially pose a threat to additional vegetable crops, especially the crucifers vegetables (Arizala et al. 2019; Klair et al, 2021).

Pak choi (Brassica rapa subsp. chinensis) is an important vegetable crop native to China, known for high water content and low caloric value, containing high quality of protein, carbohydrates, fiber, vitamins, minerals, and secondary plant metabolites (Acikgoz, 2016). A pak choi field (8,000 sq. ft.) on Oahu, Hawaii, was visited in May 2020. About 10% plants were infected and showed characteristic symptoms of soft rot, wet lesions, macerated infected stem and necrotic leaves (Figure1A-D); leading to the suspect of one of the most devastating bacterial pathogens within genus Pectobacterium (Boluk et al. 2020; Li et al. 2019; Arizala et al. 2020; Arizala and Arif, 2019). Four infected plants were collected from the field, and stems were surface sterilized with 0.6% sodium hypochlorite solution for 30 sec, followed by three consecutive rinses in distilled water. The stems were aseptically macerated, streaked on Crystal violet pectate medium (CVP) (Hélias et al. 2011), and incubated for 48 h at 26 ± 2°C. The peculiar morphological characteristic of pectolytic bacterial pathogen, forming pits on CVP, were observed (Meng et al. 2016) (Figure 1E). Purification of bacterial colonies were done by re-streaking of a single colony on dextrose peptone agar (DPA-without tetrazolium chloride; Norman and Alvarez 1989). DNA was isolated from bacterial cultures using the DNeasy Blood and Tissue Kit (Qiagen, Germantown, MA), respectively. Molecular identification of four strains (PL243-246) were performed by the sequencing region of the housekeeping gene dnaA (chromosomal replication initiation protein) using Pec. dnaA-F1/R1 primer set (Dobhal et al. 2020). The amplified PCR product was enzymatically cleaned using ExoSAP-ITTM (Affymetrix Inc, Santa Clara, CA), and sent for sequencing at the GENEWIZ facility (Genewiz, La Jolla, CA) using both forward and reverse primers. The dnaA gene sequences were aligned using Geneious, and manually edited to remove the errors. The consensus sequences were analyzed with the NCBI BLASTn tool and were deposited in the NCBI GenBank under the accession numbers MT899920-MT899923. The NCBI BLASTn report indicated that all the sequences shared 99-100% identity and query cover with Pectobacterium brasiliense accession numbers MN544627-29. A phylogenetic analysis, using Geneious, was performed with the dnaA sequences representing different Pectobacterium spp., all strains grouped within the clade of P. brasiliense (Figure 2; Arizala et al, 2020). A pathogenicity assay was carried out in three replications on pak choi grown in pots containing commercial pot mixture, and maintained in the controlled-greenhouse (temperature 26-30°C; relative humidity 50-58%). Three-weeks old plant stems were artificially inoculated with 100 µl bacterial suspensions of PL243 (1.3x 10⁸ CFU/ml), PL244 (1.2x 10⁸ CFU/ml), PL 245 (1.2x 10⁸ CFU/ml) and PL246 (1.1x 10⁸CFU/ml); control plants were inoculated with 100 µl of distilled water (Figure 1F). Two days after inoculation, the soft rot and wilting symptoms (Figure 1G-H), similar to the ones observed on the field, were developed for all four strains tested. Bacteria was successfully re-isolated from the inoculated plants; DNA was isolated, amplified, sequenced for dnaA region and analyzed for 100% homology with original strains, to fulfill Koch's postulates. Based on the molecular characteristics re-isolates were identical to the original strains. To the best of our knowledge, this is the first report of P. brasiliense on pak choi in the USA. Recent reports indicated that the pathogen could potentially pose a threat to cruciferous crops, therefore, highlighting a need to conduct a state-wide survey for pectinolytic bacteria, and implement better management strategies to combat the vegetable crop losses.

The bacterial wilt pathogen, first known as Bacillus solanacearum, has undergone numerous taxonomic changes since its first description in 1896. The history and significance of this pathogen is covered in this review with an emphasis on the advances in technology that were used to support each reclassification that finally led to the current separation of Ralstonia solanacearum into three genomic species. Frequent name changes occurred as methodology transitioned from phenotypic, biochemical, and molecular studies, to genomics and functional genomics. The diversity, wide host range, and geographical distribution of the bacterial wilt pathogen resulted in its division into three species as genomic analyses elucidated phylogenetic relationships among strains. Current advances in phylogenetics and functional genomics now open new avenues for research into epidemiology and control of the devastating bacterial wilt disease., Competing Interests: The authors declare no conflict of interest.

Dickeya spp. cause blackleg and soft rot diseases of potato and several other plant species worldwide, resulting in high economic losses. Rapid detection and identification of the pathogen is essential for facilitating efficient disease management. Our aim in this research was to develop a rapid and field-deployable recombinase polymerase amplification (RPA) assay coupled with a lateral flow device (LFD) that will accurately detect Dickeya spp. in infected plant tissues without the need for DNA isolation. A unique genomic region ( mgl A/ mgl C genes) conserved among Dickeya spp. was used to design highly specific robust primers and probes for an RPA assay. Assay specificity was validated with 34 representative strains from all Dickeya spp. and 24 strains from other genera and species; no false positives or negatives were detected. An RPA assay targeting the internal transcribed spacer region of the host genome was included to enhance the reliability and accuracy of the Dickeya assay. The detection limit of 1 fg was determined by both sensitivity and spiked sensitivity assays; no inhibitory effects were observed when 1 µl of host sap, macerated in Tris-EDTA buffer, was added to each reaction in the sensitivity tests. The developed RPA assay is rapid, highly accurate, sensitive, and fully field deployable. It has numerous applications in routine diagnostics, surveillance, biosecurity, and disease management.

Bacterial leaf spot of tomato and pepper (BLS), an economically important bacterial disease caused by four species of Xanthomonas ( X. euvesicatoria ( Xe ), X. vesicatoria ( Xv ), X. gardneri ( Xg ), and X. perforans ( Xp )), is a global problem and can cause over 50% crop loss under unfavorable conditions. Among the four species, Xe and Xv are prevalent worldwide. Characterization of the pathogens is crucial for disease management and regulatory purposes. In this study, we performed a multilocus sequence analysis (MLSA) with six genes ( hrcN , dnaA gyrB , gapA , pdg , and hmbs ) on BLS strains. Other Xanthomonas species were included to determine phylogenetic relationships within and among the tested strains. Four BLS species comprising 76 strains from different serological groups and diverse geographical locations were resolved into three major clades. BLS xanthomonads formed distinct clusters in the phylogenetic analyses. Three other xanthomonads, including X. albilineans , X. sacchari , and X. translucens pv . undolusa revealed less than 85%, 88%, and 89% average nucleotide identity (ANI), respectively, with the other species of Xanthomonas included in this study. Both antibody and MLSA data showed that Xv was clearly separated from Xe and that the latter strains were remarkably clonal, even though they originated from distant geographical locations. The Xe strains formed two separate phylogenetic groups; Xe group A1 consisted only of tomato strains, whereas Xe group A2 included strains from pepper and tomato . In contrast, the Xv group showed greater heterogeneity. Some Xv strains from South America were closely related to strains from California, while others grouped closer to a strain from Indiana and more distantly to a strain from Hawaii. Using this information molecular tests can now be devised to track distribution of clonal populations that may be introduced into new geographic areas through seeds and other infected plant materials., Competing Interests: Authors declare no competing interest.

Destructive maceration, a wide host range, and longevity in non-plant substrates has established Dickeya dianthicola (blackleg of potato) as a significant threat to potato industries worldwide. To protect these businesses, a specific and sensitive point-of-care D. dianthicola detection tool is necessary. We have developed a loop-mediated isothermal amplification (LAMP) assay for specific, sensitive, and rapid detection of D. dianthicola, which can be streamlined for point-of-care use. The developed LAMP assay targets a unique gene, alcohol dehydrogenase, of D. dianthicola. Assay specificity was assessed using strains present in inclusivity (16 D. dianthicola strains) and exclusivity panels (56 closely related, potato pathogenic, and other bacterial strains). Amplification with strains of inclusivity panel occurred, and cross-reactivity with non-target DNA was not observed. The limit of detection (LOD) was 10 CFU/ml when dilutions were made before isolating the genomic DNA; however, LOD was determined as 1 pg using 10-fold serially diluted D. dianthicola genomic DNA. Similar LOD of 1 pg was observed when serially diluted target genomic DNA was mixed with host genomic DNA. LOD (1 pg) was also calculated with 10-fold serially diluted synthetic DNA fragments containing primer target sites. Naturally and artificially inoculated plant samples were used for field adaptability tests with the field-deployable Optigene Plant Material Lysis Kit and a heat block (65°C); the results were obtained within 20 minutes. Despite the lack of method precision, no false positives or false negatives were observed. Therefore, with prepared reactions and a steady heat source, this assay can be used for rapid point-of-care detection, which is imperative for quarantine, eradication, disease management, and border protection., Competing Interests: The authors have declared that no competing interests exist.

In this article, we present a novel synthesis of mesoporous SiO 2 /Ag nanostructures for dye (methylene blue) adsorption and surface plasmon mediated photocatalysis. Mesoporous SiO 2 nanoparticles with a pore size of 3.2 nm were synthesized using cetyltrimethylammonium bromide as a structure directing agent and functionalized with (3-aminopropyl)trimethoxysilane to introduce amine groups. The adsorption behavior of non-porous SiO 2 nanoparticles was compared with that of the mesoporous silica nanoparticles. The large surface area and higher porosity of mesoporous SiO 2 facilitated better adsorption of the dye as compared to the non-porous silica. Ag decorated SiO 2 nanoparticles were synthesized by attaching silver (Ag) nanoparticles of different morphologies, i.e. spherical and triangular, on amine functionalized silica. The photocatalytic activity of the mesoporous SiO 2 /Ag was compared with that of non-porous SiO 2 /Ag nanoparticles and pristine Ag nanoparticles. Mesoporous SiO 2 nanoparticles ( k d = 31.3 × 10 -3 g mg -1 min -1 ) showed remarkable improvement in the rate of degradation of methylene blue as compared to non-porous SiO 2 ( k d = 25.1 × 10 -3 g mg -1 min -1 ) and pristine Ag nanoparticles ( k d = 19.3 × 10 -3 g mg -1 min -1 ). Blue Ag nanoparticles, owing to their better charge carrier generation and enhanced surface plasmon resonance, exhibited superior photocatalysis performance as compared to yellow Ag nanoparticles in all nanostructures., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Several species of the genus Liposcelis are common insect pests that cause serious qualitative and quantitative losses to various stored grains and processed grain products. They also can contaminate foods, transmit pathogenic microorganisms and cause allergies in humans. The common occurrence of multi-species infestations and the fact that it is difficult to identify and discriminate Liposcelis spp. make accurate, rapid detection and discriminatory tools absolutely necessary for confirmation of their identity. In this study, PCR primers and probes specific to different Liposcelis spp. were designed based on nucleotide sequences of the cytochrome oxidase 1 (CO1) gene. Primer sets ObsCo13F/13R, PeaCo15F/14R, BosCO7F/7R, BruCo5F/5R, and DecCo11F/11R were used to specifically detect Liposcelis obscura Broadhead, Liposcelis pearmani Lienhard, Liposcelis bostrychophila Badonnel, Liposcelis brunnea Motschulsky and Liposcelis decolor (Pearman) in multiplex endpoint PCRs, which amplified products of 438-, 351-, 191-, 140-, and 87-bp, respectively. In multiplex TaqMan qPCR assays, orange, yellow, red, crimson and green channels corresponding to reporter dyes 6-ROXN, HEX, Cy5, Quasar705 and 6-FAM specifically detected L. obscura, L. brunnea, L. bostrychophila, L. pearmani and L. decolor, respectively. All developed primer and probe sets allowed specific amplification of corresponding targeted Liposcelis species. The development of multiplex endpoint PCR and multiplex TaqMan qPCR will greatly facilitate psocid identification and their management. The use of APCs will streamline and standardize PCR assays. APC will also provide the opportunity to have all positive controls in a single tube, which reduces maintenance cost and labor, but increases the accuracy and reliability of the assays. These novel methods from our study will have applications in pest management, biosecurity, quarantine, food safety, and routine diagnostics.

Irinotecan loaded nanostructured lipid carrier (NLC-Ir) was surface decorated with hyaluronic acid graft polymer. Hyaluronic acid is a biocompatible, non-antigenic and hydrophilic, CD-44 ligand that can impart many useful features to the nanocarrier for anticancer drug delivery. The present investigation demonstrated that hyaluronic acid coated HA-NLC had significantly lower haemolytic potential as compared to uncoated NLC. Further, HA-NLC had a reduced plasma protein interaction and low macrophage uptake. The in vivo tumor targeting and pharmacodynamics efficacy of HA-NLC was studied in Ehrlich's Ascites Tumor (EAT) allograft model. Radio scintigraphic biodistribution studies revealed that HA-NLC carrier got accumulated in the tumor tissues in good proportion. Additionally, the content of radioactivity associated with tumor tissues remained constant at 2, 4 and 24 h (2.41, 2.48 and 2.47%, respectively), while it got reduced in other organs. Furthermore, tumor to muscle ratio of radioactivity suggested a better accumulation of HA-NLC in tumor tissues that was significantly enhanced (P<0.05) with time. In vivo antitumor activity of hyaluronan coated HA-NLC-Ir was 5.8 and 2.6 times higher as compared to control and free drug solution respectively. Furthermore, encapsulation of irinotecan in HA-NLC-Ir nanocarrier was found to have reduced the thrombocytopenia and neutropenia associated with free irinotecan. Thus, it can be inferred that the hyaluronic acid decorated nanocarrier can provide a haemo-compatible, non-toxic and target based delivery system for the effective management of cancer., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Over expression of P-glycoprotein (P-gp) in cancer cells often results in highly aggressive, multi-drug resistant (MDR) phenotype. Such tumors are very difficult to treat with conventional therapy and often lead to failure of the treatment. In this work, we fabricated surface engineered hybrid lipid nanoparticles grafted with novel AL-HA polymer by mineralization technique. AL-HA graft polymer was prepared by covalent conjugation of alendronate sodium and hyaluronic acid. Compritol ATO 888 and capmule MCM C8 hybrid lipid mix was employed to prepare irinotecan containing nanostructured lipid carrier (NLC) by using functional excipients with P-gp inhibition activity. AL-HA was successfully grafted over NLC-Ir (uncoated irinotecan loaded NLC) by calcium-assisted mineralization. HA-NLC-Ir (hyaluronic acid coated irinotecan loaded NLC) particles have a nanoscale size of 386±2.2 nm along with a zeta potential value of 19.7±1.2 mV. NLC-Ir as well as HA-NLC-Ir showed a slow and sustained drug release. In vitro cell line studies performed on HT-29 and Colo-320 colon cancer cells revealed a reduced IC50 even in MDR cells. Flowcytometry studies demonstrated the capability of the developed nanocarriers to deliver the P-gp substrate moieties in MDR cancer cells. Furthermore, the targeting potential of HA-NLC was confirmed by CLSM studies. The cell line studies also revealed that NLC formulation had a potential of inhibiting P-gp by affecting ATPase activity and MDR1 gene expression., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Outbreaks of foodborne illness attributed to the consumption of Salmonella-tainted cantaloupe have occurred repeatedly, but understanding of the ecology of Salmonella on cantaloupe fruit surfaces is limited. We investigated the interactions between Salmonella enterica Poona, the plant pathogenic bacterium Erwinia tracheiphila, and cantaloupe fruit. Fruit surfaces were inoculated at the natural cracking stage by spreading S. enterica and E. tracheiphila, 20 µl at 107 cfu/ml, independently or together, over a 2×2 cm rind area containing a crack. Microbial and microscopic analyses were performed at 0, 9 and 24 days post inoculation (DPI). Even at 24 DPI (fruit maturity) S. enterica was detected on 14% and 40% of the fruit inoculated with S. enterica alone and the two-pathogen mixture, respectively. However, the population of S. enterica declined gradually after initial inoculation. E. tracheiphila, inoculated alone or together with Salmonella, caused watersoaked lesions on cantaloupe fruit; but we could not conclude in this study that S. enterica survival on the fruit surface was enhanced by the presence of those lesions. Of fruit inoculated with E. tracheiphila alone and sampled at 24 DPI, 61% had watersoaked lesions on the surface. In nearly half of those symptomatic fruits the watersoaking extended into the sub-rind mesocarp, and E. tracheiphila was recovered from that tissue in 50% of the symptomatic fruit. In this work, E. tracheiphila internalized through natural cracks on developing fruits. S. enterica was never detected in the fruit interior (ca. 2-3 mm below rind surface) under the limited conditions of our experiments, but the possibility that it, or other human pathogens that contaminate fresh produce, might also do so should be investigated under a wider range of conditions and produce types.

Foodborne illnesses caused by Salmonella enterica and Escherichia coli O157:H7 are worldwide health concerns. Rapid, sensitive, and robust detection of these pathogens in foods and in clinical and environmental samples is essential for routine food quality testing, effective surveillance, and outbreak investigations. The aim of this study was to evaluate the effect on PCR sensitivity of adding a short, AT-rich overhanging nucleotide sequence (flap) to the 5' end of PCR primers specific for the detection of Salmonella and E. coli O157:H7. Primers targeting the invA gene of Salmonella and the rfbE gene of E. coli O157:H7 were synthesized with or without a 12-bp, AT-rich 5' flap (5'-AATAAATCATAA-3'). Singleplex PCR, multiplex PCR, and real-time PCR sensitivity assays were conducted using purified bacterial genomic DNA and crude cell lysates of bacterial cells. The effect of background flora on detection was evaluated by spiking tomato and jalapeno pepper surface washes with E. coli O157:H7 and Salmonella Saintpaul. When targeting individual pathogens, end-point PCR assays using flap-amended primers were more efficient than nonamended primers, with 20.4 and 23.5% increases in amplicon yield for Salmonella and E. coli O157:H7, respectively. In multiplex PCR assays, a 10- to 100-fold increase in detection sensitivity was observed when the primer flap sequence was incorporated. This improvement in both singleplex and multiplex PCR efficiency and sensitivity can lead to improved Salmonella and E. coli O157:H7 detection.