Ectomycorrhizae of <Emphasis Type="Italic">Tuber huidongense</Emphasis> and <Emphasis Type="Italic">T. liyuanum</Emphasis> with <Emphasis Type="Italic">Castanea mollissima</Emphasis> and <Emphasis Type="Italic">Pinus armandii</Emphasis>

Tuber huidongense and T. liyuanum are common commercial white truffles in China that belong to the Rufum and Puberulum groups of the genus Tuber, respectively. Their mycorrhizae were successfully synthesized with two native trees—Castanea mollissima and Pinus armandii—under greenhouse conditions. The identities of the mycorrhizae were confirmed through internal transcribed spacer (ITS) sequence analyses, and their morphological characteristics were described. All of the obtained mycorrhizae have an interlocking pseudoparenchymatous mantle, which is a typical feature of truffle mycorrhizae. The mycorrhizae of T. huidongense on the two trees have hyaline branched emanating hyphae, similar to the documented mycorrhizae of the Rufum group. The unramified, spiky, and hyaline cystidia on the mycorrhizae of T. liyuanum with both C. mollissima and P. armandii further confirmed that this characteristic is constant for the mycorrhizae of the Puberulum group. The successful mycorrhizal syntheses on the two nut-producing trees will be of economic importance in the cultivation of the two truffles.     

Taxonomic position of Afghan vole (Subgenus <Emphasis Type="Italic">Blanfordimys</Emphasis>) by the sequence of the mitochondrial <Emphasis Type="Italic">cytb</Emphasis> gene

It is rather difficult to construct a system of gray voles of the tribe Microtini by a set of morphological and karyological characters because form generation is mosaic at these organization levels. The sequence of the mitochondrial cytochrome b gene was used to study the phylogenetic relationships and taxonomic position of the Central Asian subgenus Blanfordimys. Afghan vole Microtus (Blanfordimys) afghanus and Bucharian vole M. (Blanfordimys) bucharensis clustered with Pamir vole M. (Neodon) juldaschi, which is conventionally assigned to another subgenus. The last two species proved to be significantly closer to each other than either of them was to M. (Blanfordimys) afghanus, which disagrees with the monophyletic origin accepted for Blanfordimys. The genetic distances between the species of the subgenus Blanfordmys and M. juldaschi were comparable with the distances between the sister subgenera Microtus s. str. and Sumeriomys or Pallasiinus and Alexandromys and with the basal divergence of supraspecific clades in the subgenus Terricola. It was assumed that a special Central Asian group of species exists within the tribe Microtini and includes species of the subgenus Blanfordimys and M. juldaschi and that the subgenera Neodon and Blanfordimys should be revised.     

Reproductive tables of predatory phytoseiid mites (<Emphasis Type="Italic">Phytoseiulus persimilis,Galendromus occidentalis</Emphasis>, and <Emphasis Type="Italic">Neoseiulus cucumeris</Emphasis>)

Feeding of predatory mites (Phytoseiulus persimilis, Galendromus occidentalis, and Neoseiulus cucumeris) on different life stages of Tetranychus atlanticus under optimal conditions was studied. Daily and total consumption of prey by predators and selection of prey of different life stages were studied for 5 days and for the entire feeding period. Average daily food consumption [number of individuals] for the entire life period of mature mite females constituted 0.43 females + 5.0 [nymphs and males] + 3.4 eggs of Tetranychus atlanticus in P. persimilis; 0.12 females + 3.70 [nymphs and males] + 3.10 eggs in G. occidentalis; and 0.19 females + 4.10 [nymphs and males] + 3.50 eggs in N. cucumeris. During the entire period of feeding, P. persimilis preferred large individuals and at the postembryonic stages selected prey to a greater extent than G. occidentalis and N. cucumeris (61.8 and 55.1%, respectively). The use of a 5-day express-method is possible for estimation of some biological characteristics of phytoseiids that previously consumed the same food for a long period. In other cases, analysis of characteristics for the entire life period is necessary.     

Screening and molecular identification of lactic acid bacteria from <Emphasis Type="Italic">gari</Emphasis> and <Emphasis Type="Italic">fufu</Emphasis> and <Emphasis Type="Italic">gari</Emphasis> effluents

Bacterial strains were isolated from cassava-derived food products and, for the first time, from cassava by-products, with a focus on gari, a flour-like product, and the effluents from the production processes for gari and fufu (a dough also made from cassava flour). A total of 47 strains were isolated, all of which were tested to determine their resistance to acidic pH and to bile salt environments. Four of the 47 isolates tested positive in both environments, and these four isolates also showed antibacterial behaviour towards both Gram-positive and Gram-negative microbial pathogens (i.e. Methicillin-resistance Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, Salmonella enteritidis, Escherichia coli, Escherichia coli (O157), Yersinia enterocolitica). In most cases, the antibacterial activity was related to bacteriocin production. Molecular identification analysis (16S rDNA and randomly amplified polymorphic DNA-PCR) revealed that the four isolates were different strains of the same species, Lactobacillus fermentum. These results demonstrate that bacteria isolated from cassava-derived food items and cassava by-products have interesting properties and could potentially be used as probiotics.     

Association between <Emphasis Type="Italic">cagA</Emphasis>, <Emphasis Type="Italic">vacAi</Emphasis>, and <Emphasis Type="Italic">dupA</Emphasis> genes of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> and gastroduodenal pathologies in Chilean patients

In addition to the already known cagA gene, novel genetic markers have been associated with Helicobacter pylori (H. pylori) virulence: the dupA and vacAi genes. These genes might play an important role as specific markers to determine the clinical outcome of the disease, especially the vacAi gene, which has been expected to be a good marker of severe pathologies like gastric adenocarcinoma. In the present study, the association of cagA, dupA, and vacAi genes with gastroduodenal pathologies in Chilean patients was studied. One hundred and thirty-two patients positive for H. pylori were divided into two groups—non-severe and severe gastric pathologies—and investigated for the presence of cagA, dupA, and vacAi H. pylori virulence genes by PCR. The cagA gene was detected in 20/132 patients (15.2%), the vacAi1 gene was detected in 54/132 patients (40.9%), the vacAi2 gene was detected in 26/132 patients (19.7%), and the dupA gene was detected in 50/132 (37.9%) patients. Logistic regression model analysis showed that the vacAi1 isoform gene in the infected strains and the severity of the diseases outcome were highly associated, causing severe gastric damage that may lead to gastric cancer (p < 0.0001; OR = 8.75; 95% CI 3.54–21.64). Conversely, cagA (p = 0.3507; OR = 1.62; 95% CI 0.59–4.45) and vacAi2 (p = 0.0114; OR = 3.09; 95% CI 1.26–7.60) genes were not associated with damage, while the dupA gene was associated significantly with non-severe clinical outcome (p = 0.0032; OR = 0.25; 95% CI 0.09–0.65). In addition, dupA gene exerts protection against severe gastric pathologies induced by vacAi1 by delaying the outcome of the disease by approximately 20 years.     

The <Emphasis Type="Italic">Adh</Emphasis> locus and adaptation of <Emphasis Type="Italic">cn</Emphasis> and <Emphasis Type="Italic">vg</Emphasis> mutants in experimental populations of <Emphasis Type="Italic">Drosophila melanogaster MEIG</Emphasis>

A complex study on the adaptation of cn and vn mutants and the allozymes of alcoholdehydrogenase (ADH) was carried out in initially pure lines, and their panmixia populations during exchange of the mutant genotype with that of wild-type flies (C-S) and D) through saturating crossings. The relative adaptation of the genotypes was estimated by their effect on reproductive efficiency in the experimentally obtained population. Fecundity, lifespan, and the resistance of the studied genotypes to hyperthermia were investigated individually. It was shown that the high level of adaptation of the cn mutants and the low level of adaptation of the vg mutants was correlated with the presence of different ADH allozymes. In the studied population, the F-allozyme of ADH accompanied the vg mutation, while the S-allozyme of the enzyme was detected in cn mutants. Saturating crossings of C-S(Adh ^S)×vg(Adh ^F) and D(Adh ^F) × cn(Adh ^S), along with the parallel determination of the allele composition of the Adh locus, demonstrated that the complete substitution of the F-allozyme of ADH in the vg mutants by the S-allozyme in D flies, as well as the substitution of the S-allozyme of ADH in the cn mutants by the F-allozyme in D flies was realized only after the 15th–20th backcrosses. These results favor the coadaptation of cn and vg marker genes with alleles of the Adh locus and indicate the important role of the latter in the adaptation of genotypes. In the studied population, selection acted primarily against the vg mutants, which were inferior to the cn mutants, and heterozygote genotypes in indices of the main adaptation components.     

<Emphasis Type="Italic">R</Emphasis>-acetoin accumulation and dissimilation in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

Klebsiella pneumoniae is a 2,3-butanediol producer, and R-acetoin is an intermediate of 2,3-butanediol production. R-acetoin accumulation and dissimilation in K. pneumoniae was studied here. A budC mutant, which has lost 2,3-butanediol dehydrogenase activity, accumulated high levels of R-acetoin in culture broth. However, after glucose was exhausted, the accumulated R-acetoin could be reused by the cells as a carbon source. Acetoin dehydrogenase enzyme system, encoded by acoABCD, was responsible for R-acetoin dissimilation. acoABCD mutants lost the ability to grow on acetoin as the sole carbon source, and the acetoin accumulated could not be dissimilated. However, in the presence of another carbon source, the acetoin accumulated in broth of acoABCD mutants was converted to 2,3-butanediol. Parameters of R-acetoin production by budC mutants were optimized in batch culture. Aerobic culture and mildly acidic conditions (pH 6–6.5) favored R-acetoin accumulation. At the optimized conditions, in fed-batch fermentation, 62.3 g/L R-acetoin was produced by budC and acoABCD double mutant in 57 h culture, with an optical purity of 98.0 %, and a substrate conversion ratio of 28.7 %.     

<Emphasis Type="Italic">SnRK2</Emphasis> Homologs in <Emphasis Type="Italic">Gossypium</Emphasis> and <Emphasis Type="Italic">GhSnRK2.6</Emphasis> Improved Salt Tolerance in Transgenic Upland Cotton and <Emphasis Type="Italic">Arabidopsis</Emphasis>

SnRK2s are a large family of plant-specific protein kinases, which play important roles in multiple abiotic stress responses in various plant species. But the family in Gossypium has not been well studied. Here, we identified 13, 10, and 13 members of the SnRK2 family from Gossypium raimondii, Gossypium arboreum, and Gossypium hirsutum, respectively, and analyzed the locations of SnRK2 homologs in chromosomes based on genome data of cotton species. Phylogenetic tree analysis of SnRK2 proteins showed that these families were classified into three groups. All SnRK2 genes were comprised of nine exons and eight introns, and the exon distributions and the intron phase of homolog genes among different cotton species were analogous. Moreover, GhSnRK2.6 was overexpressed in Arabidopsis and upland cotton, respectively. Under salt treatment, overexpressed Arabidopsis could maintain higher biomass accumulation than wild-type plants, and GhSnRK2.6 overexpression in cotton exhibited higher germination rate than the control. So, the gene GhSnRK2.6 could be utilized in cotton breeding for salt tolerance.     

<Emphasis Type="Italic">In vitro</Emphasis> Blastogenesis inhibited by <Emphasis Type="Italic">Erwinia carotovora</Emphasis><Emphasis Type="SmallCaps">L</Emphasis>-Asparaginase

Escherichia coliL-asparaginase, an antileukaemic agent in man¹, inhibits in vitro mitogen or antigen-induced blastogenesis in man^2,3 and in animals (M. Bennett, E. G. Mayhew and T. Han, unpublished data) and suppresses bone-marrow derived antibody precursor cells in the mouse⁴. We now report that another L-asparaginase preparation—from Erwinia carotovora—also possesses antileukaemic activity^5,6 and has a more pronounced immunosuppressive effect on in vitro blastogenesis than the E. coli enzyme.     

Ectopic expression of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) <Emphasis Type="Italic">CsTIR</Emphasis>/<Emphasis Type="Italic">AFB</Emphasis> genes enhance salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Auxin receptors TIR1/AFBs play an essential role in a series of signaling network cascades. These F-box proteins have also been identified to participate in different stress responses via the auxin signaling pathway in Arabidopsis. Cucumber (Cucumis sativus L.) is one of the most important crops worldwide, which is also a model plant for research. In the study herein, two cucumber homologous auxin receptor F-box genes CsTIR and CsAFB were cloned and studied for the first time. The deduced amino acid sequences showed a 78% identity between CsTIR and AtTIR1 and 76% between CsAFB and AtAFB2. All these proteins share similar characteristics of an F-box domain near the N-terminus, and several Leucine-rich repeat regions in the middle. Arabidopsis plants ectopically overexpressing CsTIR or CsAFB were obtained and verified. Shorter primary roots and more lateral roots were found in these transgenic lines with auxin signaling amplified. Results showed that expression of CsTIR/AFB genes in Arabidopsis could lead to higher seeds germination rates and plant survival rates than wild-type under salt stress. The enhanced salt tolerance in transgenic plants is probably caused by maintaining root growth and controlling water loss in seedlings, and by stabilizing life-sustaining substances as well as accumulating endogenous osmoregulation substances. We proposed that CsTIR/AFB-involved auxin signal regulation might trigger auxin mediated stress adaptation response and enhance the plant salt stress resistance by osmoregulation.     

Distribution and parameters of genetic polymorphism in northern red-backed vole (<Emphasis Type="Italic">Clethrionomys rutilus</Emphasis>) and bank vole (<Emphasis Type="Italic">Clethrionomys glareolus</Emphasis>) in West Siberia

This article presents data on the genetic variability of the northern red-backed vole and the bank vole that live sympatrically in West Siberia. The two species of voles have comparable, relatively high indices of genetic variability of inter simple sequences repeats DNA. The proportion of polymorphic DNA markers is 95–98%, and the Nei’s genetic diversity index is 0.33–0.35. A total of 47–58% of allozyme loci in the voles are polymorphic, and the average heterozygosity per locus is 0.058 in the northern red-backed vole and 0.054 in the bank vole. Interpopulation differentiation is less pronounced in the red-backed vole (F _ST 0.293) compared to the bank vole (F _ST 0.475). Individuals of the hybrid line of the bank vole with the mitochondrial haplotype of the red-backed vole have been found by PCR typing of cytochrome b gene fragment of mtDNA. The distribution boundary of the hybrid line of bank voles goes farther to the northeast than was shown in earlier works. The proportion of hybrid specimens range from 2 to 34%. The indices of genetic variability in the hybrid line of the bank vole are lower than those of the parental species.     

Role of Arabidopsis <Emphasis Type="Italic">ABF1</Emphasis>/<Emphasis Type="Italic">3</Emphasis>/<Emphasis Type="Italic">4</Emphasis> during <Emphasis Type="Italic">det1</Emphasis> germination in salt and osmotic stress conditions

Key message

Arabidopsis det1 mutants exhibit salt and osmotic stress resistant germination. This phenotype requires HY5, ABF1, ABF3, and ABF4.

Abstract

While DE-ETIOLATED 1 (DET1) is well known as a negative regulator of light development, here we describe how det1 mutants also exhibit altered responses to salt and osmotic stress, specifically salt and mannitol resistant germination. LONG HYPOCOTYL 5 (HY5) positively regulates both light and abscisic acid (ABA) signalling. We found that hy5 suppressed the det1 salt and mannitol resistant germination phenotype, thus, det1 stress resistant germination requires HY5. We then queried publically available microarray datasets to identify genes downstream of HY5 that were differentially expressed in det1 mutants. Our analysis revealed that ABA regulated genes, including ABA RESPONSIVE ELEMENT BINDING FACTOR 3 (ABF3), are downregulated in det1 seedlings. We found that ABF3 is induced by salt in wildtype seeds, while homologues ABF4 and ABF1 are repressed, and all three genes are underexpressed in det1 seeds. We then investigated the role of ABF3, ABF4, and ABF1 in det1 phenotypes. Double mutant analysis showed that abf3, abf4, and abf1 all suppress the det1 salt/osmotic stress resistant germination phenotype. In addition, abf1 suppressed det1 rapid water loss and open stomata phenotypes. Thus interactions between ABF genes contribute to det1 salt/osmotic stress response phenotypes.

     

<Emphasis Type="Italic">Candida krusei</Emphasis> and <Emphasis Type="Italic">Candida glabrata</Emphasis> reduce the filamentation of <Emphasis Type="Italic">Candida albicans</Emphasis> by downregulating expression of <Emphasis Type="Italic">HWP1</Emphasis> gene

Pathogenicity of Candida albicans is associated with its capacity switch from yeast-like to hyphal growth. The hyphal form is capable to penetrate the epithelial surfaces and to damage the host tissues. Therefore, many investigations have focused on mechanisms that control the morphological transitions of C. albicans. Recently, certain studies have showed that non-albicans Candida species can reduce the capacity of C. albicans to form biofilms and to develop candidiasis in animal models. Then, the objective of this study was to evaluate the effects of Candida krusei and Candida glabrata on the morphogenesis of C. albicans. Firstly, the capacity of reference and clinical strains of C. albicans in forming hyphae was tested in vitro. After that, the expression of HWP1 (hyphal wall protein 1) gene was determined by quantitative real-time PCR (polymerase chain reaction) assay. For both reference and clinical strains, a significant inhibition of the hyphae formation was observed when C. albicans was incubated in the presence of C. krusei or C. glabrata compared to the control group composed only by C. albicans. In addition, the culture mixed of C. albicans-C. krusei or C. albicans-C. glabrata reduced significantly the expression of HWP1 gene of C. albicans in relation to single cultures of this specie. In both filamentation and gene expression assays, C. krusei showed the higher inhibitory activity on the morphogenesis of C. albicans compared to C. glabrata. C. krusei and C. glabrata are capable to reduce the filamentation of C. albicans and consequently decrease the expression of the HWP1 gene.     

<Emphasis Type="Italic">Entransia</Emphasis> and <Emphasis Type="Italic">Hormidiella</Emphasis>, sister lineages of <Emphasis Type="Italic">Klebsormidium</Emphasis> (Streptophyta), respond differently to light,temperature, and desiccation stress

The green-algal class Klebsormidiophyceae (Streptophyta), which occurs worldwide, comprises the genera Klebsormidium, Interfilum, Entransia, and Hormidiella. Ecophysiological research has so far focused on the first two genera because they are abundant in biological soil crust communities. The present study investigated the photosynthetic performances of Hormidiella attenuata and two strains of Entransia fimbriata under light, temperature, and desiccation stress. Their ultrastructure was compared using transmission electron microscopy. The two Entransia strains showed similar physiological responses. They used light more efficiently than Hormidiella, as indicated by higher oxygen production and relative electron transport rate under low light conditions, lower light saturation and compensation points, and higher maximum oxygen production during light saturation. Their requirement for low light levels explains the restriction of Entransia to dim limnetic habitats. In contrast, Hormidiella, which prefers drier soil habitats, responded to light gradients similarly to other aero-terrestrial green algae. Compared to Entransia, Hormidiella was less affected by short-term desiccation, and rehydration allowed full recovery of the photosynthetic performance. Nevertheless, both strains of Entransia coped with low water availability better than other freshwater algae. Photosynthetic oxygen production in relation to respiratory consumption was higher in low temperatures (Entransia: 5 °C, Hormidiella: 10 °C) and the ratio decreased with increasing temperatures. Hormidiella exhibited conspicuous triangular spaces in the cell wall corners, which were filled either with undulating cell wall material or with various inclusions. These structures are commonly seen in various members of Klebsormidiophyceae. The data revealed significant differences between Hormidiella and Entransia, but appropriate adaptations to their respective habitats.     

Identification of <Emphasis Type="Italic">Pm58</Emphasis> from <Emphasis Type="Italic">Aegilops tauschii</Emphasis>

Key message

A novel powdery mildew-resistance gene, designated Pm58, was introgressed directly from Aegilops tauschii to hexaploid wheat, mapped to chromosome 2DS, and confirmed to be effective under field conditions. Selectable KASP? markers were developed for MAS.

Abstract

Powdery mildew caused by Blumeria graminis (DC.) f. sp. tritici (Bgt) remains a significant threat to wheat (Triticum aestivum L.) production. The rapid breakdown of race-specific resistance to Bgt reinforces the need to identify novel sources of resistance. The d-genome species, Aegilops tauschii, is an excellent source of disease resistance that is transferrable to T. aestivum. The powdery mildew-resistant Ae. tauschii accession TA1662 (2n?=?2x?=?DD) was crossed directly with the susceptible hard white wheat line KS05HW14 (2n?=?6x?=?AABBDD) followed by backcrossing to develop a population of 96 BC₂F₄ introgression lines (ILs). Genotyping-by-sequencing was used to develop a genome-wide genetic map that was anchored to the Ae. tauschii reference genome. A detached-leaf Bgt assay was used to screen BC₂F_4:6 ILs, and resistance was found to segregate as a single locus (χ?=?2.0, P value?=?0.157). The resistance gene, referred to as Pm58, mapped to chromosome 2DS. Pm58 was evaluated under field conditions in replicated trials in 2015 and 2016. In both years, a single QTL spanning the Pm58 locus was identified that reduced powdery mildew severity and explained 21% of field variation (P value?<?0.01). KASP? assays were developed from closely linked GBS-SNP markers, a refined genetic map was developed, and four markers that cosegregate with Pm58 were identified. This novel source of powdery mildew-resistance and closely linked genetic markers will support efforts to develop wheat varieties with powdery mildew resistance.

     

Alginate biosynthesis by <Emphasis Type="Italic">Azotobacter</Emphasis> bacteria

The ability of representatives of various species of the bacterial genus Azotobacter (A. chroococcum 7B, A. chroococcum 12B, A. chroococcum 12BS, A. agile 12, A. indicum 8, A. vinelandii 17, and A. vinelandii 5B) to alginate synthesis has been studied. It has been shown that all tested bacterial strains have this ability to different extents. Capsular alginate comprises from 2.6 to 32% of the total amount of synthesized alginate in various bacterial species. Strains that are able to active synthesis of alginate have been selected; the effect of the medium composition on their biosynthesis has been studied. The optimal conditions for alginate synthesis by the A. chroococcum 12BS producer strain include the presence of mannitol (40 g/L), yeast extract (1%), and low concentration of phosphates (KH₂PO₄—0.008 g/L, K₂HPO₄—0.032 g/L) in the medium; alginate production under these conditions is 4.5 g/L. The effect of aeration on polymer biosynthesis has been revealed: an increase in aeration causes an increase in alginate synthesis, while its decrease promotes the synthesis of poly-3-hydroxybutirate. It has been shown by IR spectroscopy that alginates obtained under various conditions of cultivation contain different ratios of residues of mannuronic and guluronic acids (M/G from 70/30 to 80/20) in the polymer chain and also differ in the amount of acetyl groups (from 10 to 25%) in the polyme structure.     

<Emphasis Type="Italic">Candida krusei</Emphasis> isolated from fruit juices ultrafiltration membranes promotes colonization of <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 and <Emphasis Type="Italic">Salmonella enterica</Emphasis> on stainless steel surfaces

To clarify the interactions between a common food spoilage yeast and two pathogenic bacteria involved in outbreaks associated with fruit juices, the present paper studies the effect of the interplay of Candida krusei, collected from UF membranes, with Escherichia coli O157:H7 and Salmonella enterica in the overall process of adhesion and colonization of abiotic surfaces. Two different cases were tested: a) co-adhesion by pathogenic bacteria and yeasts, and b) incorporation of bacteria to pre-adhered C. krusei cells. Cultures were made on stainless steel at 25°C using apple juice as culture medium. After 24 h of co-adhesion with C. krusei, both E. coli O157:H7 and S. enterica increased their counts 1.05 and 1.11 log CFU cm², respectively. Similar increases were obtained when incorporating bacteria to pre-adhered cells of Candida. Nevertheless C. krusei counts decreased in both experimental conditions, in a) 0.40 log CFU cm² and 0.55 log CFU cm² when exposed to E. coli O157:H7 and S. enterica and in b) 0.18 and 0.68 log CFU cm², respectively. This suggests that C. krusei, E. coli O157:H7, and S. enterica have a complex relationship involving physical and chemical interactions on food contact surfaces. This study supports the possibility that pathogen interactions with members of spoilage microbiota, such as C. krusei, might play an important role for the survival and dissemination of E. coli O157:H7 and Salmonella enterica in food-processing environments. Based on the data obtained from the present study, much more attention should be given to prevent the contamination of these pathogens in acidic drinks.