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Río Celeste (Sky-Blue River) in Tenorio National Park (Costa Rica), a river that derives from the confluence and mixing of two colorless streams—Río Buenavista (Buenavista River) and Quebrada Agria (Sour Creek)—is renowned in Costa Rica because it presents an atypical intense sky-blue color. Although various explanations have been proposed for this unusual hue of Río Celeste, no exhaustive tests have been undertaken; the reasons hence remain unclear. To understand this color phenomenon, we examined the physico-chemical properties of Río Celeste and of the two streams from which it is derived. Chemical analysis of those streams with ion-exchange chromatography (IC) and inductively coupled plasma atomic emission spectroscopy (ICP-OES) made us discard the hypothesis that the origin of the hue is due to colored chemical species. Our tests revealed that the origin of this coloration phenomenon is physical, due to suspended aluminosilicate particles (with diameters distributed around 566 nm according to a lognormal distribution) that produce Mie scattering. The color originates after mixing of two colorless streams because of the enlargement (by aggregation) of suspended aluminosilicate particles in the Río Buenavista stream due to a decrease of pH on mixing with the acidic Quebrada Agria. We postulate a chemical mechanism for this process, supported by experimental evidence of dynamic light scattering (DLS), zeta potential measurements, X-ray diffraction and scanning electron microscopy (SEM) with energy-dispersive spectra (EDS). Theoretical modeling of the Mie scattering yielded a strong coincidence between the observed color and the simulated one.

Total polyphenol content (TPC), total flavonoid content (TFC), total anthocyanin content (TAC), and proanthocyanidin (PAC) content were determined in fruit from three tropical Vaccinium species (Vaccinium consanguineum, Vaccinium floribundum, and Vaccinium poasanum) from Costa Rica sampled at three stages of fruit development. Results show that TAC increased as the fruit developed, while TPC, TFC, and PAC content decreased. Anthocyanin profiles were evaluated using electrospray ionization tandem mass spectrometry. Cyanidin and delphinidin glycosides were the predominant anthocyanins for the three tropical Vaccinium species. Proanthocyanidins were characterized using attenuated total reflection Fourier transform infrared spectroscopy, nuclear magnetic resonance, and matrix-assisted laser desorption/ionization time-offlight mass spectrometry. The presence of procyanidin structures with B-type interflavan bonds were observed, but deconvolution of mass spectrometry isotope patterns indicated that PACs with one or more A-type interflavan bonds accounted for more than 74% of the oligomers at each degree of polymerization. KEYWORDS: proanthocyanidins, anthocyanin, A-type interflavan bonds, MALDI−TOF MS of the National Fruit Growing Program by the Costa Rican Department of Agriculture. 1,4,8 Polyphenols found in Vaccinium species, such as cranberry, contain hydroxycinnamic acids, flavonols, anthocyanins, and proanthocyanidins, among others. 9,10 Anthocyanins are watersoluble pigments that vary by the number of methyl, methoxy, or hydroxyl group, the nature and number of sugars attached to the molecule, and the nature and number of aliphatic or aromatic acids attached to sugars in the molecule. 11,12 Proanthocyanidins (PACs) are oligomers of flavan-3-ols. PAC structures vary by the nature of the monomer unit, interflavan linkage, and degree of polymerization (DP). 13 PACs are classified by the type of flavan-3-ol monomers, such as properlagonidins, which are oligomers of (epi)afzelechin, procyanidins, which are oligomers of (epi)catechin, and prodelphinidins, which are oligomers of (epi)gallocatechin. 14 However, many types of PACs contain more than one type of monomeric flavan-3-ols, such as PACs in grape seeds. PACs can have B-and A-type bonds. B-type bonds are defined by the presence of C 4 −C 8 or C 4 −C 6 interflavan bonds, whereas Atype bonds have C 4 −C 8 bonds but also have an additional interflavan bond between C 2 −O−C 7 or C 2 −O−C 5 . 9,13,15

Spray-induced gene silencing (SIGS) using topical dsRNA applications has risen as a promising, targetspecific, and environmentally friendly disease management strategy against phytopathogenic fungi. However, dsRNA stability, efficacy, and scalability are still the main constraints facing SIGS broader application. Here we show that Escherichia coliderived anucleated minicells can be utilized as a cost-effective, scalable platform for dsRNA production and encapsulation. We demonstrated that minicell-encapsulated dsRNA (ME-dsRNA) was shielded from RNase degradation and stabilized on strawberry surfaces, allowing dsRNA persistence in fieldlike conditions. ME-dsRNAs targeting chitin synthase class III (Chs3a, Chs3b) and DICER-like proteins (DCL1 and DCL2) genes of Botryotinia fuckeliana selectively knocked-down the target genes and led to significant fungal growth inhibition in vitro. We also observed a compensatory relationship between DCL1 and DCL2 gene transcripts, where the silencing of one gene upregulated the expression of the other. Contrary to naked-dsRNAs, ME-dsRNAs halted disease progression in strawberries for 12 days under greenhouse conditions. These results elucidate the potential of ME-dsRNAs to enable the commercial application of RNAi-based, speciesspecific biocontrols comparable in efficacy to conventional synthetics. ME-dsRNAs offer a platform that can readily be translated to large-scale production and deployed in open-field applications to control grey mould in strawberries.