Comparative Methods for Quantification of Sulfate-Reducing Bacteria in Environmental and Engineered Sludge Samples.

This study aimed to compare microscopic counting, culture, and quantitative or real-time PCR (qPCR) to quantify sulfate-reducing bacteria in environmental and engineered sludge samples. Four sets of primers that amplified the dsrA and apsA gene encoding the two key enzymes of the sulfate-reduction pathway were initially tested. qPCR standard curves were constructed using genomic DNA from an SRB suspension and dilutions of an enriched sulfate-reducing sludge. According to specificity and reproducibility, the DSR1F/RH3-dsr-R primer set ensured a good quantification based on dsrA gene amplification; however, it exhibited inconsistencies at low and high levels of SRB concentrations in environmental and sulfate-reducing sludge samples. Ultimately, we conducted a qPCR method normalized to dsrA gene copies, using a synthetic double-stranded DNA fragment as a calibrator. This method fulfilled all validation criteria and proved to be specific, accurate, and precise. The enumeration of metabolically active SRB populations through culture methods differed from dsrA gene copies but showed a plausible positive correlation. Conversely, microscopic counting had limitations due to distinguishing densely clustered organisms, impacting precision. Hence, this study proves that a qPCR-based method optimized with dsrA gene copies as a calibrator is a sensitive molecular tool for the absolute enumeration of SRB populations in engineered and environmental sludge samples.

First Report of Alternaria alternata Causing Leaf Spot on Broccoli in Ecuador.

In Ecuador, broccoli (Brassica oleracea var. italica) production is located in the Andean region, specifically Cotopaxi-Ecuador (INEC, 2019). A leaf pathogen has been constantly observed in this area, showing brown circular necrosis surrounded by yellowish halo-like spots causing leaf death (Fig. 1a). This pathogen was believed to be Alternaria sp.; however, the species was not determined either using classical or molecular tools. In 2021, ten leaves showing similar symptoms were collected in Cotopaxi and sent for pathogen identification. Here, leaf explants (0.25 cm2) showing disease symptoms were surface sterilized with 2% sodium hypochlorite (NaClO) and 70% ethanol (C2H6O), rinsed with sterile water, and transferred to Potato Dextrose Agar (PDA) media. Petri dishes were incubated in darkness at 25°C for five days. The single hyphal tip method was used to purify the cultures on PDA. Fifteen pure isolates were obtained after incubating for 14 days. Isolates were incubated under blacklight for two days to induce fungal sporulation. All isolates presented early white cotton-like mycelium that later became dark green (Fig 1b). Under the microscope, we observed straight primary conidia in simple or branched chains. Also, the conidia were obclavate, long ellipsoids, moderate in size (19.5-43.9 µm in length, 7.1-17.2 µm in width), and septate with few longitudinal septa. Lastly, the conidium body can narrow itself into a secondary conidia (Fig 1c) (Woudenberg et al., 2013). According to colony and conidia morphology, isolates were identified as Alternaria sp. (Woudenberg et al., 2013). Five isolates were randomly selected for DNA extraction and sequencing of ITS (internal transcribed spacer; Chou, H.H. and Wu, W.S. 2002), TEF (translation elongation factor; O'Donnell et al., 1998), and RPB2 (RNA polymerase II second largest subunit; Liu et al., 1999) gene regions. DNA sequences obtained from each marker were identical for all isolates. Consensus sequences and alignment were built using ClustalX in MEGA X (Kumar et al., 2018). Consensus sequences were deposited in GenBank with the following accession numbers: ITS, ON982232; TEF, ON983964; RPB2, ON983963. A multilocus Bayesian inference phylogenetic tree was constructed in Beast software (version 1.8.4) using the concatenated sequences (Drummond et al., 2012; Maharachchikumbura et al., 2014). The isolates in our study clustered with isolates of Alternaria alternata, confirming their identity (Figure 2). For Koch's postulates, healthy broccoli plants were grown in sterile soil for six weeks. The fungal conidia were suspended in sterile distilled water (1×106 conidia/ml), and the leaves were inoculated by spraying the spore solution. The control treatment was sprayed with sterile distilled water alone. Plants were maintained at 28°C and had more than 85% relative humidity (Sigillo et al., 2020). Seven days after inoculation, plants showed chlorosis and necrosis. Ten days later, 100% of the treated leaves presented brown circular necrosis (Fig. 1d). Control plants showed no disease symptoms. Re-isolation of the pathogen from the diseased leaf tissue was performed as previously described. The isolates presented the exact morphology of pure cultures obtained from field-diseased leaves. The pathogenicity test was performed twice. To our knowledge, this is the first report on A. alternata being the causal agent of leaf spot on broccoli in Ecuador. Disease diagnosis contributes to providing strategies against this pathogen. Further investigations are needed to find biological/chemical techniques or cultivar resistance to control this pathogen in broccoli.

Influence of Grafting on Rootstock Rhizosphere Microbiome Assembly in Rosa sp. 'Natal Brier'.

The root microbiome is vital in plant development and health and is highly influenced by crop cultural practices. Rose (Rosa sp.) is the most popular cut flower worldwide. Grafting in rose production is a standard practice to increase yield, improve flower quality, or reduce root-associated pests and diseases. 'Natal Brier' is a standard rootstock used in most commercial operations in Ecuador and Colombia, leading countries in producing and exporting ornamentals. It is known that the rose scion genotype affects root biomass and the root exudate profile of grafted plants. However, little is known about the influence of the rose scion genotype on the rhizosphere microbiome. We examined the influence of grafting and scion genotype on the rhizosphere microbiome of the rootstock 'Natal Brier'. The microbiomes of the non-grafted rootstock and the rootstock grafted with two red rose cultivars were assessed using 16S rRNA and ITS sequencing. Grafting changed microbial community structure and function. Further, analysis of grafted plant samples revealed that the scion genotype highly influences the rootstock microbiome. Under the presented experimental conditions, the rootstock 'Natal Brier' core microbiome consisted of 16 bacterial and 40 fungal taxa. Our results highlight that the scion genotype influences root microbe's recruitment, which might also influence the functionality of assembled microbiomes.

Microclimate is a strong predictor of the native and invasive plant-associated soil microbiome on San Cristóbal Island, Galápagos archipelago.

Understanding the drivers that affect soil bacterial and fungal communities is essential to understanding and mitigating the impacts of human activity on vulnerable ecosystems like those on the Galápagos Islands. The volcanic slopes of these Islands lead to steep elevation gradients that generate distinct microclimates across small spatial scales. Although much is known about the impacts of invasive plant species on the above-ground biodiversity of the Galápagos Islands, little is known about their resident soil microbial communities and the factors shaping them. Here, we investigate the bacterial and fungal soil communities associated with invasive and native plant species across three distinct microclimates on San Cristóbal Island (arid, transition zone and humid). At each site, we collected soil at three depths (rhizosphere, 5 cm and 15 cm) from multiple plants. Sampling location was the strongest driver of both bacterial and fungal communities, explaining 73% and 43% of variation in the bacterial and fungal community structure, respectively, with additional minor but significant impacts from soil depth and plant type (invasive vs. native). This study highlights the continued need to explore microbial communities across diverse environments and demonstrates how both abiotic and biotic factors impact soil microbial communities in the Galápagos archipelago.

Saponin determination, expression analysis and functional characterization of saponin biosynthetic genes in Chenopodium quinoa leaves.

Quinoa (Chenopodium quinoa Willd.) is a highly nutritious pseudocereal with an outstanding protein, vitamin, mineral and nutraceutical content. The leaves, flowers and seed coat of quinoa contain triterpenoid saponins, which impart bitterness to the grain and make them unpalatable without postharvest removal of the saponins. In this study, we quantified saponin content in quinoa leaves from Ecuadorian sweet and bitter genotypes and assessed the expression of saponin biosynthetic genes in leaf samples elicited with methyl jasmonate. We found saponin accumulation in leaves after MeJA treatment in both ecotypes tested. As no reference genes were available to perform qPCR in quinoa, we mined publicly available RNA-Seq data for orthologs of 22 genes known to be stably expressed in Arabidopsis thaliana using geNorm, NormFinder and BestKeeper algorithms. The quinoa ortholog of At2g28390 (Monensin Sensitivity 1, MON1) was stably expressed and chosen as a suitable reference gene for qPCR analysis. Candidate saponin biosynthesis genes were screened in the quinoa RNA-Seq data and subsequent functional characterization in yeast led to the identification of CqbAS1, CqCYP716A78 and CqCYP716A79. These genes were found to be induced by MeJA, suggesting this phytohormone might also modulate saponin biosynthesis in quinoa leaves. Knowledge of the saponin biosynthesis and its regulation in quinoa may aid the further development of sweet cultivars that do not require postharvest processing.