Francisco Javier Flores

In February 2024, cauliflower plants (Brassica oleracea var. botrytis) showing symptoms of black rot were observed at the experimental farm of the Faculty of Agricultural Sciences, Central University of Ecuador, in Quito, Ecuador. Affected plants exhibited characteristic V-shaped chlorotic lesions on leaves. Disease incidence was approximately 15% across the field. Symptomatic leaf tissue (5 mm² fragments) was surface-sterilized with 0.5% sodium hypochlorite, rinsed with sterile distilled water, and plated on nutrient agar. After 48 h at 28°C, yellow, mucoid, convex colonies with defined borders were observed. Two colonies were identified as Xanthomonas campestris using the Biolog system with 94 phenotypic tests. Genomic DNA from strain XccEC_1 was extracted using the CTAB protocol (Doyle and Doyle 1987) and sequenced on an Illumina NovaSeq platform (PE150, 100× coverage) at Biomarker Technologies (Hong Kong). The genome assembly (SPAdes v3.15; Bankevich et al. 2012) yielded 5,147,842 bp in 230 contigs with an N50 of 75,866 bp and 64.87% GC content. Phylogenomic analysis based on 100 conserved orthologous genes was performed using the BV-BRC codon tree pipeline (Olson et al. 2023). Protein sequences were aligned using MAFFT, and a maximum-likelihood tree was inferred using RAxML v8.2.12 under the GTRCAT model with 100 rapid bootstrap replicates. The resulting tree grouped XccEC_1 with X. campestris pv. campestris str. ATCC 33913 (100% bootstrap support) (Fig. S1). The complete genome of strain XccEC_1 was deposited in GenBank (accession no. JBUBWA000000000). For pathogenicity tests, five healthy five-week-old cauliflower seedlings were infiltrated on four leaves each with a bacterial suspension of XccEC_1 (10⁸ CFU/ml in PBS) using a needleless syringe, and one control plant was infiltrated with sterile distilled water. Plants were incubated at 25°C and 68% relative humidity. At 7 days post-inoculation, all inoculated plants developed chlorotic to necrotic foliar lesions, and by 12 days, vascular tissue blackening was evident (Fig. S2). Control plants remained asymptomatic, and no suspected X. campestris colonies were isolated from these plants. The bacterium was reisolated from symptomatic plants, and species identity was confirmed by 16S rRNA and GyrB sequencing (GenBank accession no. PX498286, PZ417655). The pathogenicity assay was repeated once with identical results. The XccEC_1 isolate was stored with code MB_59 at the Pontificia Universidad Católica del Ecuador Culture Collection. To our knowledge, this is the first report of X. campestris pv. campestris causing black rot on cauliflower in Ecuador. This pathogen poses a significant threat to Brassica production in the region (Vicente and Holub 2013) and warrants implementation of integrated disease management strategies.

Heparosan is a natural polymer with unique chemical and biological properties, that holds great promise for biomedical applications. The molecular weight (Mw) and polydispersion index (PDI) are critical factors influencing the performance of heparosan-based materials. Achieving precise control over the synthesis process to consistently produce heparosan with low Mw and low PDI can be challenging, as it requires tight regulation of reaction conditions, enzyme activity, and precursor concentrations. We propose a novel approach utilizing synthetic biology principles to precisely control heparosan biosynthesis in bacteria. Our strategy involves designing a biomolecular controller that can regulate the expression of genes involved in heparosan biosynthesis. This controller is activated by biosensors that detect heparosan precursors, allowing for fine-tuned control of the polymerization process. Through this approach, we foresee the implementation of this synthetic device, demonstrating the potential to produce low Mw and low PDI heparosan in the probiotic E. coli Nissle 1917 as a biosafe and biosecure biofactory. This study represents a significant advancement in the field of heparosan production, offering new opportunities for the development and manufacturing of biomaterials with tailored properties for diverse biomedical applications.

Bananas are Ecuador's second largest non-oil export product, and the quality of its fruit has established a strong presence in international markets. One-third of the world's banana exports originate from Ecuador. The Ecuadorian banana market is diversified, exporting fruit to various countries worldwide, making it a vital socio-economic and food security support for the country. Between January 2022 and December 2023, symptomatic Cavendish banana plants cv. Williams were observed in commercial plantations in three cantons of the Los Ríos province (Buena Fe, Valencia, and Quevedo), with an incidence ranging from 2% to 8%. Initial symptoms appeared as a dark purple lesion on the pseudostem that extended to the entire internal tissue. In later stages, the internal tissue softened, leading to pseudostem rot with a foul odor. In each canton, 9 samples of symptomatic pseudostems were taken. For each sample, seven 2 cm² pieces of the lesion were surface-sterilized and macerated in 9 ml of sterile peptone water (0.1% w/v). The macerate was diluted twice in sterile peptone water, plated on nutrient agar, and incubated at 28°C for 24 hours. The most abundant colonies in all cultures were yellowish-white, with irregular edges, smooth surfaces, and a whitish elevated center. Three colonies were isolated per canton. The isolates were Gram-negative bacilli, oxidase and urease negative, catalase, indole, and citrate positive. One isolate from each canton was selected for molecular identification and pathogenicity tests (FP220002 - Buena Fe, FP221366 - Valencia, FP231258 - Quevedo). The 16S rRNA gene was amplified and sequenced using primers 27F and 1492R (Wilson et al., 1991), along with gyrB, atpD, rpoB, and infB, as described by Brady et al. (2008). The resulting sequences were deposited in GenBank with accession numbers PQ279912-PQ279912 and PQ304949-PQ304960. The three isolates had identical sequences for each gene. The percentage identity ranged from 98.5% for infB to 100% for 16S with the Dickeya fangzhongdai type strain PA1 (NZ_CP020872.1) (Zhang et al., 2018). A multilocus phylogenetic analysis conducted with RAxML revealed that all isolates formed a discrete group along with other D. fangzhongdai strains, with the closest strains being DSM 101947 and QZH3 from China (Tian et al., 2016). Pathogenicity tests were conducted by syringe infiltration with 1 ml of a 1 × 10^8 CFU/ml bacterial suspension. Each of the three characterized isolates was inoculated into the pseudostems of five healthy four-month-old banana plants of the Williams cultivar. Negative control plants were infiltrated with sterile distilled water. The plants were incubated at 25°C and 74% relative humidity. Black lesions began to appear 20 days after inoculation, and 5 weeks post-inoculation, the plants exhibited clear symptoms of pseudostem soft rot, including dark purple lesions and the foul odor associated with tissue decay. Bacteria that morphologically resembled the inoculated cultures were reisolated and molecularly identified as D. fangzhongdai with 16S sequencing. Control plants remained symptom-free. This is the first report of Dickeya fangzhongdai causing soft rot in bananas, a disease recently described in Ecuador with Pectobacterium brasiliense as causal agent (Toaza et al. 2024), and which is becoming increasingly prevalent in the country.

Munition disposal practices have significant effects on microbial composition and overall soil health. Explosive soil contamination can disrupt microbial communities, leading to microbial abundance and richness changes. This study investigates the microbial diversity of soils and roots from sites with a history of ammunition disposal, aiming to identify organisms that may play a role in bioremediation. Soil and root samples were collected from two types of ammunition disposal (through open burning and open detonation) and unpolluted sites in Machachi, Ecuador, over two years (2022 and 2023). High-throughput sequencing of the 16S rRNA gene (for bacteria) and the ITS region (for fungi and plants) was conducted to obtain taxonomic profiles. There were significant variations in the composition of bacteria, fungi, and plant communities between polluted and unpolluted sites. Bacterial genera such as Pseudarthrobacter, Pseudomonas, and Rhizobium were more abundant in roots, while Candidatus Udaeobacter dominated unpolluted soils. Fungal classes Dothideomycetes and Sordariomycetes were prevalent across most samples, while Leotiomycetes and Agaricomycetes were also highly abundant in unpolluted samples. Plant-associated reads showed a higher abundance of Poa and Trifolium in root samples, particularly at contaminated sites, and Alchemilla, Vaccinium, and Hypericum were abundant in unpolluted sites. Alpha diversity analysis indicated that bacterial diversity was significantly higher in unpolluted root and soil samples, whereas fungal diversity was not significantly different among sites. Redundancy analysis of beta diversity showed that site, year, and sample type significantly influenced microbial community structure, with the site being the most influential factor. Differentially abundant microbial taxa, including bacteria such as Pseudarthrobacter and fungi such as Paraleptosphaeria and Talaromyces, may contribute to natural attenuation processes in explosive-contaminated soils. This research highlights the potential of certain microbial taxa to restore environments contaminated by explosives.

Metagenomics analysis has enabled the measurement of the microbiome diversity in environmental samples without prior targeted enrichment. Functional and phylogenetic studies based on microbial diversity retrieved using HTS platforms have advanced from detecting known organisms and discovering unknown species to applications in disease diagnostics. Robust validation processes are essential for test reliability, requiring standard samples and databases deriving from real samples and in silico generated artificial controls. We propose a MeStanG as a resource for generating HTS Nanopore data sets to evaluate present and emerging bioinformatics pipelines. MeStanG allows samples to be designed with user-defined organism abundances expressed as number of reads, reference sequences, and predetermined or custom errors by sequencing profiles. The simulator pipeline was evaluated by analyzing its output mock metagenomic samples containing known read abundances using read mapping, genome assembly, and taxonomic classification on three scenarios: a bacterial community composed of nine different organisms, samples resembling pathogen-infected wheat plants, and a viral pathogen serial dilution sampling. The evaluation was able to report consistently the same organisms, and their read abundances as provided in the mock metagenomic sample design. Based on this performance and its novel capacity of generating exact number of reads, MeStanG can be used by scientists to develop mock metagenomic samples (artificial HTS data sets) to assess the diagnostic performance metrics of bioinformatic pipelines, allowing the user to choose predetermined or customized models for research and training.

A new fusagra-like virus infecting papaya (Carica papaya L.) was genetically characterized. The genome of the virus, provisionally named "papaya sticky fruit-associated virus" (PSFaV), is a single molecule of double-stranded RNA, 9,199 nucleotides (nt) in length, containing two discontinuous open reading frames. Pairwise sequence comparisons based on complete RNA-dependent-RNA-polymerase (RdRp) sequences revealed identity of 79.4% and 83.3% at the nt and amino acid (aa) level, respectively, to babaco meleira-like virus (BabMelV), an uncharacterized virus sequence discovered in babaco (Vasconcellea x heilbornii) in Ecuador. Additional plant-associated viruses with sequence identity in the 50% range included papaya meleira virus (PMeV) isolates from Brazil. Phylogenetic analysis based on the amino acid sequences of the capsid protein (CP), RdRp, and CP-RdRp fusion protein genes placed PSFaV in a group within a well-supported clade that shares a recent ancestor with Sclerotium rolfsii RNA virus 2 and Phlebiopsis gigantea mycovirus dsRNA 2, two fungus-associated fusagraviruses. Genomic features and phylogenetic relatedness suggest that PSFaV, along with its closest relative BabMelV, represent a species of novel plant-associated virus classified within the recently established family Fusagraviridae.

This study presents the complete genome sequence of a novel nege-like virus identified in whiteflies (Bemisia tabaci MEAM1), provisionally designated as whitefly negevirus 1 (WfNgV1). The virus possesses a single-stranded RNA genome comprising 11,848 nucleotides, organized into four open reading frames (ORFs). These ORFs encode the putative RNA-dependent-RNA-polymerase (RdRp, ORF 1), a glycoprotein (ORF 2), a structural protein with homology to those in the SP24 family, (ORF 3), and a protein of unknown function (ORF 4). Phylogenetic analysis focusing on RdRp and SP24 amino acid sequences revealed a close relationship between WfNgV1 and Bemisia tabaci negevirus 1, a negevirus sequence recently discovered in whiteflies from Israel. Both viruses form a clade sharing a most recent common ancestor with the proposed nelorpivirus and centivirus taxa. The putative glycoprotein from ORF 2 and SP24 (ORF 3) of WfNgV1 exhibit the characteristic topologies previously reported for negevirus counterparts. This marks the first reported negevirus-like sequence from whiteflies in the Americas.