Integrated processes for olive mill wastewater treatment and its revalorization for microalgae culture.

The olive oil industry generates 30 million cubic meters of olive mill wastewaters (OMWWs) annually. OMWWs are a major environmental concern in the Mediterranean region due to their high organic matter content, suspended solids, unpleasant odor, and dark color. The application of primary treatments such as coagulation-flocculation, adsorption, and hybrid systems combining coagulation-flocculation with adsorption has enabled to remove part of the organic matter, color, turbidity, and growth-inhibiting compounds from OMWWs. Among these methods, the hybrid system combining activated carbon and chitosan has proven to be the best removal efficiency. Subsequently, secondary treatment involving the cultivation of Chlorella sp. on OMWWs pretreated with chitosan achieved the highest maximal specific growth rate (0.513 ± 0.022 day⁻1) and biomass productivity (0.621 ± 0.021 g/L/day). Notably, the fatty acids (FA) profile produced by Chlorella sp. cells grown under these conditions differed, underscoring the potential of OMWWs as a microalgal growth medium. This innovative approach not only addresses environmental issues but also opens new avenues for sustainable bioproducts.

Development of a duplex q-PCR for the simultaneous detection of Parachlamydia acanthamoebae and Simkania negevensis in environmental and clinical samples.

Parachlamydia acanthamoebae and Simkania negevensis, two Chlamydia-like bacteria, have been recently recognized as emerging human respiratory pathogens. The prevalence and frequency of these bacteria in the environment and among atypical pneumonia patients are still underestimated by classical cultures, immunohistochemistry and serology which are non-specific, long and tedious methods. This study aims to develop a new duplex probe-based q-PCR assay for the simultaneous detection and quantification of P. acanthamoebae and S. negevensis. The selected hydrolysis probes displayed no cross-reaction with the closely related Chlamydia or the other tested waterborne pathogens. The assay achieved a large dynamic range for quantification (from 5 × 106 to 5 DNA copies/reaction). Efficiencies of FAM and JOE label probes weren't affected when they were combined. They were close to 100%, indicating the linear amplification. The application of this diagnostic tool resulted in 9/47 (19%) and 4/47 (8.5%) positive water samples for P. acanthamoebae and S. negevensis, respectively. P. acanthamoebae was also covered from 2/78 (2.5%) respiratory specimens and only one case (1/200 = 0.5%) of P. acanthamoebae and SARS-CoV-2 co-infection was noticed. While S. negevensis wasn't detected in clinical samples, the developed duplex q-PCR was shown to be an accurate, highly sensitive, and robust diagnostic tool for the detection and quantification of P. acanthamoebae and S. negevensis.

Development of a new TaqMan-based PCR assay for the specific detection and quantification of Simkania negevensis.

Simkania negevensis is an emerging Chlamydia-like bacterium related to human respiratory diseases. An early and accurate detection of this pathogen could be useful to monitor the potential infectious risks and to set suitable outbreak control measures. In Tunisia, distribution and abundance of S. negevensis remain until now largely unknown. In the present work, a qPCR assay, targeting the 16S rRNA gene, for fast detection and quantification of S. negevensis was developed and validated. A high specificity for S. negevensis detection displaying no cross-reaction with the closely related Chlamydia spp. or the other tested microorganisms was noticed. qPCR assay performance was considered very satisfying with detection limits of 5 DNA copies per reaction. qPCR assay validation was performed by screening 37 clinical specimens and 35 water samples. S. negevensis wasn't detected in respiratory samples, but it was found in four cases of water samples. We suggest that the qPCR assay developed in this study could be considered sufficiently characterized to initiate the quantification of S. negevensis in environmental samples.

Towards novel Cry toxins with enhanced toxicity/broader: a new chimeric Cry4Ba / Cry1Ac toxin.

Attempts have been made to express or to merge different Cry proteins in order to enhance toxic effects against various insects. Cry1A proteins of Bacillus thuringiensis form a typical bipyramidal parasporal crystal and their protoxins contain a highly conserved C-terminal region. A chimerical gene, called cry(4Ba-1Ac), formed by a fusion of the N-terminus part of cry4Ba and the C-terminus part of cry1Ac, was constructed. Its transformation to an acrystalliferous B. thuringiensis strain showed that it was expressed as a chimerical protein of 116 kDa, assembled in spherical to amorphous parasporal crystals. The chimerical gene cry(4Ba-1Ac) was introduced in a B. thuringiensis kurstaki strain. In the generated crystals of the recombinant strain, the presence of Cry(4Ba-1Ac) was evidenced by MALDI-TOF. The recombinant strain showed an important increase of the toxicity against Culex pipiens larvae (LC50 = 0.84 mg l-1 ± 0.08) compared to the wild type strain through the synergistic activity of Cry2Aa with Cry(4Ba-1Ac). The enhancement of toxicity of B. thuringiensis kurstaki expressing Cry(4Ba-1Ac) compared to that expressing the native toxin Cry4Ba, might be related to its a typical crystallization properties. The developed fusion protein could serve as a potent toxin against different pests of mosquitoes and major crop plants.

Characterisation of novel Bacillus thuringiensis isolates against Aedes aegypti (Diptera: Culicidae) and Ceratitis capitata (Diptera: Tephridae).

Bacillus thuringiensis is successfully used in pest management strategies as an eco-friendly bioinsecticide. Isolation and identification of new strains with a wide variety of target pests is an ever growing field. In this paper, new B. thuringiensis isolates were investigated to search for original strains active against diptera and able to produce novel toxins that could be used as an alternative for the commercial H14 strain. Biochemical and molecular characterization revealed a remarkable diversity among the studied strains. Using the PCR method, cry4C/Da1, cry30Ea, cry39A, cry40 and cry54 genes were detected in four isolates. Three strains, BLB355, BLB196 and BUPM109, showed feeble activities against Aedes aegypti larvae. Interestingly, spore-crystal mixtures of BLB361, BLB30 and BLB237 were found to be active against Ceratitis capitata with an LC50 value of about 65.375, 51.735 and 42.972 µg cm(-2), respectively. All the studied strains exhibited important mortality levels using culture supernatants against C. capitata larvae. This suggests that these strains produce a wide range of soluble factors active against C. capitata larvae.

Responses of Dunaliella sp. AL-1 to chromium and copper: Biochemical and physiological studies.

Microalgae have gained recognition as versatile candidates for the remediation of heavy metals (HMs). This study investigated the biosorption potential of Dunaliella sp. AL1 for copper (Cu(II)) and hexavalent chromium (Cr(VI)) in aqueous solutions. The marine microalga Dunaliella sp. AL1 was exposed to half-sublethal concentrations of both metals in single and bimetallic systems, and responses in algal growth, oxidative stress, photosynthetic pigment production, and photosynthetic performance were evaluated. Cu and/or Cr exposure increased the generation of reactive oxygen species (ROS) in microalgae cells but did not impact algal growth. In terms of photosynthesis, there was a decrease in chlorophylls and carotenoids production in the microalgae culture treated with Cr, either alone or in combination with Cu. The study recorded promising metal removal efficiencies: 26.67%-20.11% for Cu and 94.99%-95.51% for Cr, in single and bimetallic systems, respectively. FTIR analysis revealed an affinity of Cu and Cr ions towards aliphatic/aldehyde C-H, N-H bending, and phosphate groups, suggesting the formation of complex bonds. Biochemical analysis of microalgae biomass collected after the removal of Cr alone or in combination with Cu showed a significant decrease in total carbohydrate content and soluble protein levels. Meanwhile, higher lipid accumulation was recorded and evidenced by BODIPY 505/515 staining. Fatty acid composition analysis by GC revealed a modulation in lipid composition, with a decrease in the ratio of unsaturated fatty acids (UFA) to saturated fatty acids (SFA), in response to Cu, Cr, and Cu-Cr exposure, indicating the suitability of the biomass for sustainable biofuel production.

Lead removal from the aqueous solution by extracellular polymeric substances produced by the marine diatom Navicula salinicola.

Microbial extracellular polymeric substances (EPS) have recently emerged as significant contributors in diverse biotechnological applications. Extracellular polymeric substances (EPS), produced by a Navicula salinicola strain, have been studied for potential applications in a specific heavy metal (lead (Pb II)) removal from wastewater. The optimisation of operational parameters, mainly pH, Pb and EPS concentrations, using the Box-Behnken design (BBD) was undertaken to enhance lead uptake. The higher Pb adsorption capacity reached 2211.029 mg/g. Hydroxyl, carbonyl, carboxyl, phosphoric, and sulfhydryl groups were identified quantitatively as potential sites for Pb adsorption. EPS exhibited a notable flocculation rate of 70.20% in kaolin clay at a concentration of 15 mg/L. They demonstrated an emulsifying activity greater than 88%, showcasing their versatile potential for both sedimentation processes and stabilising liquid-liquid systems. EPS could be excellent nonconventional renewable biopolymers for treating water and wastewater.

Production and purification of fucoxanthins and ß-carotenes from Halopteris scoparia and their effects on digestive enzymes and harmful bacteria.

Algae constitute a significant part of marine biodiversity. They represent a renewable source of bioactive metabolites from drug development and therapeutic fields. Fucoxanthin and ß-carotene from the brown macroalgae Halopteris scoparia, were extracted using conventional organic solvent extraction, then purified, to homogeneity, based on various chromatographic principles. Their effects on digestive enzymes and harmful bacteria were investigated. The capacities of both purified pigments to inhibit α-amylase and trypsin enzymes were evaluated. Purified fucoxanthin and ß-carotene exhibited interesting α-amylase inhibition activities, with IC50 of 300 and 500 µg/mL, respectively. Moreover, trypsin inhibition activities were detected using purified these two pigments. The antibacterial potential of the purified pigments was evaluated. ß-carotene showed to be a great antibacterial natural compound against gram-positive and gram-negative bacteria such as Listeria monocytogenes, Staphylococcus aureus and Salmonella enterica with Minimal Inhibitory Concentration (MIC) of about 0.225, 0.1125, 0.225 µg/mL, respectively. Those findings are in favor of the exploitation of H. scoparia pigments in therapeutic fields as an antidiabetic source directly by the inhibition of α-amylase and trypsin as well as antibacterial agents against gastrointestinal infections.

Characterization of cellular toxicity induced by sub-lethal inorganic mercury in the marine microalgae Chlorococcum dorsiventrale isolated from a metal-polluted coastal site.

Mercury (Hg) is a global pollutant that affects numerous marine aquatic ecosystems. We isolated Chlorococcum dorsiventrale Ch-UB5 microalga from coastal areas of Tunisia suffering from metal pollution and analyzed its tolerance to Hg. This strain accumulated substantial amounts of Hg and was able to remove up to 95% of added metal after 24 and 72 h in axenic cultures. Mercury led to lesser biomass growth, higher cell aggregation, significant inhibition of photochemical activity, and appearance of oxidative stress and altered redox enzymatic activities, with proliferation of starch granules and neutral lipids vesicles. Such changes matched the biomolecular profile observed using Fourier Transformed Infrared spectroscopy, with remarkable spectral changes corresponding to lipids, proteins and carbohydrates. C. dorsiventrale accumulated the chloroplastic heat shock protein HSP70B and the autophagy-related ATG8 protein, probably to counteract the toxic effects of Hg. However, long-term treatments (72 h) usually resulted in poorer physiological and metabolic responses, associated with acute stress. C. dorsiventrale has potential use for Hg phycoremediation in marine ecosystems, with the ability to accumulating energetic reserves that could be used for biofuel production, supporting the notion of using of C. dorsiventrale for sustainable green chemistry in parallel to metal removal.

A new TaqMan real-time PCR assay to detect Parachlamydia acanthamoebae and to monitor its co-existence with SARS-COV-2 among COVID-19 patients.

Human respiratory infections caused by a large variety of microbial pathogens are the most common diseases responsible for hospitalization, morbidity and mortality. Parachlamydia acanthamoebae, a Chlamydia-related bacterium, has been found to be potentially associated with these diseases. An early and accurate diagnosis of this pathogen could be useful to avoid the potential respiratory complications linked especially to COVID-19 patients and to set suitable outbreak control measures. A TaqMan-PCR assay was developed to detect and quantify Parachlamydia acanthamoebae in environmental and clinical samples from patients of all ages with COVID-19. The selected hydrolysis probe displayed no cross-reaction with the closely related Chlamydia or the other tested pathogens. This q-PCR achieved good reproducibility and repeatability with a detection limit of about 5 DNA copies per reaction. Using this q-PCR assay, Parachlamydia acanthamoebae was detected in 2/78 respiratory specimens and 9/47 water samples. Only one case (1.3%) of Parachlamydia acanthamoebae and SARS-COV-2 co-infection was noticed. To our knowledge, the combination of these two respiratory pathogens has not been described yet. This new TaqMan-PCR assay represents an efficient diagnostic tool to survey Parachlamydia acanthamoebae on a large-scale screening programs and also during outbreaks.

Primary structural features, physicochemical and biological properties of two water-soluble polysaccharides extracted from the brown Tunisian seaweed Halopteris scoparia.

Marine algae are the most abundant resource in the marine environment and are still a promising source of bioactive compounds including hydrocolloids. This study contributes to the evaluation of the biological and biotechnological potentials of two water soluble polysaccharides, namely alginates (AHS) and fucoidan (FHS), extracted and purified from Halopteris scoparia, an abundant Tunisian brown macroalgae collected in Tunisia (Tabarka region). The total sugars, neutral monosaccharides, uronic acids, proteins, polyphenols, and sulfate groups contents were quantified for both fractions, as well as their functional groups and primary structural features by Fourier transform infrared spectroscopy, ionic and/or gas chromatography and nuclear magnetic resonance analyses. AHS and FHS showed significant anti-inflammatory (IC50 ≈ 1 mg/mL), anticoagulant (e.g., 27-61.7 for the activated partial thromboplastin time), antihyperglycemic (0.1-40 µg/mL) and anti-trypsin (IC50 ≈ 0.3-0.4 mg/mL) effects. FHS and a hydrolyzed fraction showed a very promising potential against herpes viruses (HSV-1) (IC50 < 28 µg/mL). Besides, AHS and two hydrolyzed fractions were able to stimulate the natural defenses of tomato seedlings, assessing their elicitor capacity, by increasing the activity of phenylalanine ammonia-lyase (66-422 %) but also significantly changing the polyphenol content in the leaves (121-243 %) and roots (30-104 %) of tomato plants.

A fast and accurate method for specific detection and quantification of the bloom-forming microalgae Karlodinium veneficum in the marine environment.

Karlodinium veneficum is a toxic benthic globally distributed dinoflagellate which has direct impacts on human health and the environment. Early and accurate detection of this harmful algal bloom-forming species could be useful for potential risks monitoring and management. In the present work, a real-time PCR targeting the internal transcribed spacer ribosomal DNA region for the specific detection and absolute quantification of K. veneficum was designed. Then, the assay conditions were adjusted and validated. The developed qPCR was highly specific for the target species and displayed no cross-reactivity with closely related dinoflagellates and/or other microalgal species commonly distributed along the Tunisian coast. Its lowest detection limit was 5 rDNA copies per reaction, which is often considered satisfying. qPCR assay enumeration accuracy was evaluated using artificially inoculated environmental samples. The comparison of the cell abundance estimates obtained by qPCR assay with the theoretical estimates showed no statistically significant difference across a range of concentrations. We suggest that the qPCR approach developed in the present study may be a valuable tool to investigate the distribution and seasonal dynamics of K. veneficum in marine environments.

Improvement of Biomass and Phycoerythrin Production by a Strain of Rhodomonas sp. Isolated from the Tunisian Coast of Sidi Mansour.

Microalgae are photoautotrophic microorganisms known as producers of a large variety of metabolites. The taxonomic diversity of these microorganisms has been poorly explored. In this study, a newly isolated strain was identified based on the 18S rRNA encoding gene. The phylogenetic analysis showed that the isolated strain was affiliated with the Rhodomonas genus. This genus has greatly attracted scientific attention according to its capacity to produce a large variety of metabolites, including phycoerythrin. Growth and phycoerythrin production conditions were optimized using a Plackett-Burman design and response surface methodology. An expression profile analysis of the cpeB gene, encoding the beta subunit of phycoerythrin, was performed by qRT-PCR under standard and optimized culture conditions. The optimization process showed that maximum cell abundance was achieved under the following conditions: CaCl2 = 2.1328 g/L, metal solution = 1 mL/L, pH = 7 and light intensity = 145 µmol photons/m2/s, whereas maximum phycoerythrin production level occurred when CaCl2 = 1.8467 g/L, metal solution = 1 mL/L, pH = 7 and light intensity = 157 µmol/m2/s. In agreement, positive transcriptional regulation of the cpeB gene was demonstrated using qRT-PCR. This study showed the successful optimization of abiotic conditions for highest growth and phycoerythrin production, making Rhodomonas sp. suitable for several biotechnological applications.

Development of a novel TaqMan qPCR assay for rapid detection and quantification of Gymnodinium catenatum for application to harmful algal bloom monitoring in coastal areas of Tunisia.

Gymnodinium catenatum is a dinoflagellate known to cause paralytic shellfish poisoning (PSP), commonly associated with human muscular paralysis, neurological symptoms, and, in extreme cases, death. In the present work, we developed a real-time PCR-based assay for the rapid detection of the toxic microalgal species, G. catenatum, in environmental bivalve mollusc samples as well as seawater samples. G. catenatum-specific primers and probe were designed on the ITS1-5.8S-ITS2 rDNA region. Hydrolysis probe qPCR assay was optimized. ITS1-5.8S-ITS2 rDNA region copy numbers per G. catenatum cell genome were estimated to be 122.73 ± 5.54 copies/cell, allowing cell quantification. The application of the optimized qPCR assay for G. catenatum detection and quantification in field samples has been conducted, revealing high sensitivity (detection of around 1.3105 cells/L of seawater samples. Thus, the designed hydrolysis probe qPCR assay could be considered an efficient tool for phytoplankton monitoring whilst ensuring accuracy and sensitivity and providing cost and time savings.

Influence of the sulfate content of the exopolysaccharides from Porphyridium sordidum on their elicitor activities on date palm vitroplants.

Given the increasing interest that is being paid to polysaccharides derived from algae as plant natural defense stimulators, the degree of sulfation of exopolysaccharides produced by P. sordidum for inducing defense responses in date palm vitroplants was investigated. Firstly, the culture parameters of P. sordidum were optimized to maximize the amount of sulfate in EPS using a Box-Behnken experimental design and the elicitor effects of two EPS which differ in the sulfation degrees were compared. Results demonstrated that the concentrations of NaCl, NaNO3 and MgSO4 set at 28, 0.54 and 16.31 g/L, respectively yielded the best sulfate contents. To elucidate defense-inducing activities in date palm vitroplants, EPS with the highest sulfate content (EPS1) were prepared for comparison with those obtained under standard conditions (EPS0). A fucoidan extracted from Cystoseira compressa was used as positive control and MgSO4 as negative control. Both EPS and the fucoidan displayed H2O2 accumulation and expression of PR1, SOD, PAL and WRKY genes. Interestingly, EPS1 was significantly more bioactive than EPS0 and the fucoidan suggesting that the elicitor activity is positively correlated with the sulfate groups content of this polysaccharide.

Potential of three local marine microalgae from Tunisian coasts for cadmium, lead and chromium removals.

Metal elements are widely used in various industrial activities and are considered as common water source contaminants. Thus, the development of cost-effective, simple design and efficient processes for trace metal elements removal from contaminated water sources is of great interest. The effects of cadmium, lead and chromium on growth, biomolecules accumulation and metabolic responses of Amphora coffaeiformis, Navicula salinicola and Dunaliella salina isolated from Tunisian coasts were tested. The bioremediation capacities of the three microalgae strains and the mechanisms involved in ions metal removal were also investigated. N. salinicola and D. salina seem to be better tolerating to Cr, while A. coffaeiformis and N. salinicola showed high resistance to Pb. The expression profile analyses by qRT-PCR of the antioxidant defense-related genes revealed that Cd, Pb and Cr treatments induce the up-regulation of catalase and superoxide dismutase coding genes for A. coffaeiformis and D. salina. Regarding N. salinicola, the catalase coding gene seems to be overexpressed after Cd, Pb and Cr exposure while only Cd and Cr induce superoxide dismutase gene overexpression. Moreover, the phytochelatin synthase (a metal chelator synthesis-related gene) was up-regulated in N. salinicola, A. coffaeiformis and D. salina after Cr exposure and also in A. coffaeiformis and D. salina after Cd exposure. While Pb treatments induce overexpression of phytochelatin synthase coding gene only for D. salina. Studied strains showed promising metal removal efficiencies for both Pb and Cr ions metals reached 95% for D. salina. Ion metal removal mechanisms study revealed that intracellular bioaccumulation process is used by D. salina for Cr up-taking. However, both intracellular and extracellular removal mechanisms are involved for Pb and Cr removal using A. coffaeiformis, N. salinicola and for Pb removal using D. salina. FTIR analysis demonstrated that several functional groups as carboxyl, hydroxyl, amino, phosphate and sulfate may participate in the bioadsorption process.

Zinc biosorption by Dunaliella sp. AL-1: Mechanism and effects on cell metabolism.

Phycoremediation is being considered as an eco-friendly and safe technology for toxics eradication from contaminated aquatic systems. The zinc biosorption capacity of Dunaliella sp. AL-1 was demonstrated. Zinc impacted cell growth and photosynthetic pigments accumulation showing exposure time and concentration-dependent effects. The investigation of the antioxidant protective response to zinc exposition proved a stimulation of guaiacol peroxidase (GPX) activity and an increased rate of total phenolics, flavonoids, condensed tannins and glutathione (GSH). The Box-Behnken design was used to optimize zinc removal conditions by Dunaliella sp. AL-1 strain. The maximum experimental zinc uptake was obtained when zinc concentration, algae dose, initial pH, and contact time were set at 25 mg/L, 0.5 g/L, 7.59 and 13 h 43 min, respectively. Under completely optimized conditions, the fraction of zinc removed intracellularly was much lower than the adsorbed on the cell surface. FTIR analysis Dunaliella sp. AL-1 biomass demonstrated that several functional groups as OH, CH2, CO, PO, COO and CO may participate in the biosorption process. A comparative proteomic analysis through nano-HPLC coupled to LC-MS/MS, was performed from pre- and post-zinc treatments cells. Among 199 identified proteins, 60 were differentially expressed of which 41 proteins were down-regulated against 19 up-regulated ones. Target proteins have been demonstrated to be implicated in different metabolic processes mainly photosynthesis and antioxidant defenses.

Optimization of Exopolysaccharides Production by Porphyridium sordidum and Their Potential to Induce Defense Responses in Arabidopsis thaliana against Fusarium oxysporum.

Polysaccharides from marine algae are one novel source of plant defense elicitors for alternative and eco-friendly plant protection against phytopathogens. The effect of exopolysaccharides (EPS) produced by Porphyridium sordidum on elicitation of Arabidopsis thaliana defense responses against Fusarium oxysporum was evaluated. Firstly, in order to enhance EPS production, a Box-Behnken experimental design was carried out to optimize NaCl, NaNO3 and MgSO4 concentrations in the culture medium of microalgae. A maximum EPS production (2.45 g/L) higher than that of the control (0.7 g/L) was observed for 41.62 g/L NaCl, 0.63 g/L NaNO3 and 7.2 g/L MgSO4 concentrations. Structurally, the EPS contained mainly galactose, xylose and glucose. Secondly, the elicitor effect of EPS was evaluated by investigating the plant defense-related signaling pathways that include activation of Salicylic or Jasmonic Acid-dependent pathway genes. A solution of 2 mg/mL of EPS has led to the control of fungal growth by the plant. Results showed that EPS foliar application induced phenylalaline ammonia lyase and H2O2 accumulation. Expression profile analysis of the defense-related genes using qRT-PCR revealed the up-regulation of Superoxide dismutases (SOD), Peroxidase (POD), Pathogenesis-related protein 1 (PR-1) and Cytochrome P450 monooxyge-nase (CYP), while Catalase (CAT) and Plant defensin 1.2 (PDF1.2) were not induced. Results suggest that EPS may induce the elicitation of A. thaliana's defense response against F. oxysporum, activating the Salicylic Acid pathway.

q-PCR-based assay for the toxic dinoflagellate Karenia selliformis monitoring along the Tunisian coasts.

Karenia selliformis is a marine dinoflagellate responsible for fish-kill events. Its presence has been reported along the Tunisian coasts (south-eastern Mediterranean Sea) since the 1990s. In the present study, a quantitative-PCR assay, based on the internal transcribed spacer (ITS) molecular marker, was developed to detect and quantify K. selliformis in environmental bivalve mollusk samples and in seawater samples. The assay was optimized, and its specificity was confirmed using cross-reactivity experiments against microalgal species commonly found on the Tunisian coasts and/or closely related to K. selliformis. Calibration curves were performed by tenfold dilutions of plasmid DNA harboring target sequence and genomic DNA, attaining a limit of detection of around 5 copies of target DNA per reaction, far below one K. selliformis cell per reaction. The field application of the developed assay showed a powerful detection capability. Thus, the designed assay could contribute to the deployment of in-field diagnostic tools for K. selliformis blooms monitoring.