New (9βH)‐Lanostanes and Lanostanes from Mikania aff. jeffreyi (Asteraceae)

Four new (9βH)‐lanostanes, i.e., (9βH)‐3β‐acetoxylanosta‐7,24‐diene, (9βH)‐3‐oxolanosta‐7,24‐diene, (9βH,24R)‐3β‐acetoxy‐24‐hydroxylanosta‐7,25‐diene, and (9βH,24S)‐3β‐acetoxy‐24‐hydroxylanosta‐7,25‐diene, two new lanostanes, i.e., (24R)‐3β‐acetoxy‐24‐hydroxylanosta‐8,25‐diene and (24S)‐3β‐acetoxy‐24‐hydroxylanosta‐8,25‐diene, and two known lanostanes, i.e., 3β‐acetoxylanosta‐8,24‐diene and 3‐oxolanosta‐8,24‐diene, were obtained from a new Mikania species (Asteraceae) besides pentacyclic triterpenes, steroids, and diterpenes. The structures of the compounds were determined by spectroscopic methods. This is the second study about acetyl‐lanosterols from higher plants. Moreover, (9βH)‐lanostanes are very rare metabolites from dicotyledone angiosperms. The occurrence of these terpenes together in the same plant makes the species a good source for lanostane‐ and (9βH)‐lanostane‐biosynthesis studies.     

Genetic Diversity of Fusarium oxysporum Populations Isolated from Tomato Plants in Tunisia

Fusarium crown and root rot of tomato (Lycopersicon esculentum) caused by Fusarium oxysporum f. sp. radicis‐lycopersici is a new devastative disease of tomato greenhouse crops in Tunisia. Nothing is known neither about the population of this pathogen in this region, nor about the population of F. oxysporum f. sp. lycopersici the causal agent of Fusarium wilt of tomato. In order to examine the genetic relatedness among the F. oxysporum isolates by intergenic spacer restriction fragment length polymorphism (IGS‐RFLP) analysis and to elucidate the origin of the formae specialesradicis‐lycopersici in Tunisia by looking for genetic similarity of Tunisians isolates with isolates from a foreign source, the genetic diversity among F. oxysporum f. sp. radicis‐lycopersici and F. oxysporum f. sp. lycopersici populations was investigated. A total of 62 isolates of F. oxysporum, obtained from symptomless tomato plants, were characterized using IGS typing and pathogenicity tests on tomato plants. All Fusarium isolates were highly pathogenic on tomato. Fusarium oxysporum f. sp. radicis‐lycopersici isolates were separated into five IGS types. From the 53 F. oxysporum f. sp. radicis‐lycopersici isolates, 34 isolates have the same IGS types (IGS type 25), and the remaining 19 isolates were distributed into four IGS types. However, the only nine isolates of F. oxysporum f. sp. lycopersici have six different IGS types. This difference of diversity between the two formae speciales suggests that F. oxysporum f. sp. radicis‐lycopersici isolates have a foreign origin and may have been accidentally introduced into Tunisia.     

Bacterial and plant natriuretic peptides improve plant defence responses against pathogens

Plant natriuretic peptides (PNPs) have been implicated in the regulation of ions and water homeostasis, and their participation in the plant immune response has also been proposed. Xanthomonas citri ssp. citri contains a gene encoding a PNP‐like protein (XacPNP) which has no homologues in other bacteria. XacPNP mimics its Arabidopsis thaliana homologue AtPNP‐A by modifying host responses to create favourable conditions for pathogen survival. However, the ability of XacPNP to induce plant defence responses has not been investigated. In order to study further the role of XacPNP in vivo, A. thaliana lines over‐expressing XacPNP, lines over‐expressing AtPNP‐A and AtPNP‐A‐deficient plants were generated. Plants over‐expressing XacPNP or AtPNP‐A showed larger stomatal aperture and were more resistant to saline or oxidative stress than were PNP‐deficient lines. In order to study further the role of PNP in biotic stress responses, A. thaliana leaves were infiltrated with pure recombinant XacPNP, and showed enhanced expression of genes related to the defence response and a higher resistance to pathogen infections. Moreover, AtPNP‐A expression increased in A. thaliana on Pseudomonas syringae pv. tomato (Pst) infection. This evidence led us to analyse the responses of the transgenic plants to pathogens. Plants over‐expressing XacPNP or AtPNP‐A were more resistant to Pst infection than control plants, whereas PNP‐deficient plants were more susceptible and showed a stronger hypersensitive response when challenged with non‐host bacteria. Therefore, XacPNP, acquired by horizontal gene transfer, is able to mimic PNP functions, even with an increase in plant defence responses.     

Different mutations in the oafA gene lead to loss of O5-antigen expression in Salmonella enterica serovar Typhimurium

Aims: To analyse genetic changes in the oafA gene explaining the loss of O5‐antigen expression in Salmonella Typhimurium and Salm. 4,[5],12:i:‐. Methods and Results: The oafA gene in 52 O5‐antigen‐negative and 77 O5‐antigen‐positive Salm. Typhimurium (N = 47) and Salm. 4,[5],12:i:‐ (monophasic Salm. Typhimurium strains, N = 82) was investigated by a combination of PCR screening and DNA sequencing to identify mutations leading to the suppression of the O5‐antigen. Various DNA sequence changes within the open reading frame (ORF) of oafA in O5‐antigen‐negative strains could be identified. In 77% of the O5‐antigen‐negative strains, a 7‐bp deletion of a duplicated sequence within the functional oafA gene led to a frameshift in the ORF. In four strains, an IS4 element and in two, an IS1 element was inserted at different positions. Four other strains carried at different positions single base pair substitutions causing a premature stop codon. Finally, in two strains, a deletion of the oafA 3′end of undetermined size was responsible for the lack of O5‐antigen expression. In none of the strains investigated, the complete ORF of oafA was deleted. Primers were designed and used to detect the most prominent variants. Conclusions: O5‐antigen‐negative Salm. Typhimurium and Salm. 4,[5],12:i:‐ strains carry an oafA pseudogene caused by different genetic events indicating that there is a selection for oafA mutations leading to the loss of O5‐antigen expression. Significance and Impact of the Study: The loss of O5‐antigen expression may be an example of a common evolutionary mechanism to escape host defence or to adapt to environmental changes. The data are the basis for the development of diagnostic PCR assays for the differentiation of O5‐antigen‐positive and O5‐antigen‐negative Salm. Typhimurium and its monophasic (Salm. 4,[5],12:i‐) strains.     

Triterpenoids from the Roots of Craibiodendron henryi W. W. Smith

A new triterpenoid, 11a.O.trans-p-coumaroyltaraxerol （1）, along with 11 known triterpenoids, taraxerone （2）, taraxerol （3）, 2α,3β,23,24-tetrahydroxyolean-12-en-28-oic acid （4）, oleanolic acid （5）, β-amyrin （6）, 3β,23-dihydroxylursan-12-en- 28-oic acid （7）, 2α,3β-dihydroxyursan-12-en-28-oic acid （8）, 2α,3β,23-trihydroxyursan-12-en-28-oic acid （9）, 2α,3β,24- trihydroxyursan-12-en-28-oic acid （10）, u rsolic acid （11）, and 3-O-acetylursolic acid （12）, was isolated from Craibiodendron henryi W. W. Smith （Ericaceae）. The structures of these compounds were elucidated on the basis of spectral evidence. Antioxidant activity and vasodilator effect of compound 1 were assessed.     

Novel multiplex polymerase chain reaction primer set for identification of Lactococcus species

Aims: The gram‐positive bacterial genus Lactococcus has been taxonomically classified into seven species (Lactococcus lactis, Lactococcus garvieae, Lactococcus piscium, Lactococcus plantarum, Lactococcus raffinolactis, Lactococcus chungangensis and Lactococcus fujiensis). This study aimed to develop a novel multiplex polymerase chain reaction (PCR) primer set for the identification of the seven lactococcal species, as well as to differentiate the two industrially important dairy subspecies, L. lactis subsp. lactis and L. lactis subsp. cremoris. Methods and Results: A multiplex PCR primer set was designed based on the nucleotide sequences of the 16S rRNA gene of the seven lactococcal species. The specificity of the established one‐step multiplex PCR scheme was verified using more than 200 bacterial strains, in which a complete sequence match was confirmed by partial sequencing of their 16S rRNA gene. Conclusions: The one‐step multiplex PCR enables the identification and speciation of bacterial strains belonging to the genus Lactococcus and the differentiation of strains of L. lactis subsp. lactis and L. lactis subsp. cremoris. Significance and Impact of the Study: This work provides an efficient method for identification of lactococcal strains of industrial importance.     

Identification of I‐7 expands the repertoire of genes for resistance to Fusarium wilt in tomato to three resistance gene classes

The tomato I‐3 and I‐7 genes confer resistance to Fusarium oxysporum f. sp. lycopersici (Fol) race 3 and were introgressed into the cultivated tomato, Solanum lycopersicum, from the wild relative Solanum pennellii. I‐3 has been identified previously on chromosome 7 and encodes an S‐receptor‐like kinase, but little is known about I‐7. Molecular markers have been developed for the marker‐assisted breeding of I‐3, but none are available for I‐7. We used an RNA‐seq and single nucleotide polymorphism (SNP) analysis approach to map I‐7 to a small introgression of S. pennellii DNA (c. 210 kb) on chromosome 8, and identified I‐7 as a gene encoding a leucine‐rich repeat receptor‐like protein (LRR‐RLP), thereby expanding the repertoire of resistance protein classes conferring resistance to Fol. Using an eds1 mutant of tomato, we showed that I‐7, like many other LRR‐RLPs conferring pathogen resistance in tomato, is EDS1 (Enhanced Disease Susceptibility 1) dependent. Using transgenic tomato plants carrying only the I‐7 gene for Fol resistance, we found that I‐7 also confers resistance to Fol races 1 and 2. Given that Fol race 1 carries Avr1, resistance to Fol race 1 indicates that I‐7‐mediated resistance, unlike I‐2‐ or I‐3‐mediated resistance, is not suppressed by Avr1. This suggests that Avr1 is not a general suppressor of Fol resistance in tomato, leading us to hypothesize that Avr1 may be acting against an EDS1‐independent pathway for resistance activation. The identification of I‐7 has allowed us to develop molecular markers for marker‐assisted breeding of both genes currently known to confer Fol race 3 resistance (I‐3 and I‐7). Given that I‐7‐mediated resistance is not suppressed by Avr1, I‐7 may be a useful addition to I‐3 in the tomato breeder's toolbox.     

Assessment of ATR‐NIR and ATR‐MIR spectroscopy as an analytical tool for the quantification of the total polyphenols in Dendrobium huoshanense

In this study, the potentiality of applying attenuated total reflectance near‐infrared (ATR‐NIR) and attenuated total reflectance mid‐infrared (ATR‐MIR) techniques combined with a partial least squares (PLS) regression technology to quantify the total polyphenols (TPs) in Dendrobium huoshanense (DHS) was investigated and compared. The real TP contents in the DHS samples were analysed using methods of reference. The capability of the two IR spectroscopic techniques to quantify the TPs in DHS was assessed by the root‐mean‐square error of calibration (RMSEC) and determination coefficients (R²). The results showed that both NIR and MIR might be used as a fast and simple tool to replace traditional chemical assays for the determination of the TP contents in DHS, and the best NIR model showed slightly better prediction performance [root‐mean‐square error of prediction (RMSEP): 0.307, R²: 0.9122, ratio performance deviation (RPD): 4.43] than the best MIR model (RMSEP: 0.440, R²: 0.9069, RPD: 3.09). Results from this study indicated that both the NIR and MIR models could be used to quantify the TP in DHS, and ATR‐NIR appeared to be the more predominant and more robust technique for the quantification of the TP in DHS.     

Two New Triterpenoid Saponins from Akebia quinata （Thunb.） Decne.

Two new triterpenoid saponins, hederagenin 3-O-α-L-arabinopyranosyl-（1→〉2）-α-L-arabinopyranoside named akeboside La （compound 1）, oleanolic acid 3-O-α-L-arabinopyranosyl-（1→〉2）-β-D-glucopyranoside named akeboside Lb （compound 2）, along with five known saponins, oleanolic acid 3-O-α-L-rhamnopyranosyl-（1→〉2）-α-L- arabinopyranoside （compound 3）, hederagenin 3-O-α-L-rhamnopyranosyl-（1→〉2）-α-L-arabinopyranoside （compound 4）, oleanolic acid 3-O-β-D-xylopyranosyl-（1→〉3）-α-L-rhamnopyranosyl-（1→〉2）-α-L-arabinopyranoside （compound 5）, 3-O-α-L-rhamnopyranosyl-（1→〉2）-α-L-arabinopyranosyl oleanolic acid 28-O-α-L-rhamnopyranosyl-（1→〉4）-α-D- glucopyranosyl-（1→〉6）-β-D-glucopyranoside （compound 6）, 3-O-α-L-rhamnopyranosyl-（1→〉2）-α-L-arabinopyranosyl hederagenin 28-α-L-rhamnopyranosyl-（1→〉4）-β-D-glucopyranosyl-（1→〉6）-β-D-glucopyranoside （compound 7） were isolated from the n-butanol part of the 80% ethanol extracts of the dried stems of Akebia quinata （Thunb.） Decne. Compound 5 was isolated from plants of genus Akebia for the first time. The structures were elucidated on the basis of physicochemical properties and spectral data.     

New variants of polar glycopeptidolipids detected in Mycobacterium simiae, including 'habana' strains, as evidenced by electrospray ionization-ion trap-mass spectrometry

Aims: To determine the composition of polar glycopeptidolipids (pGPLs) of Mycobacterium simiae and, particularly, those of ‘habana’ strains, in a search for specific markers given the immunogenic potential of ‘habana’ TMC 5135 in experimental tuberculosis. Methods and Results: pGPLs were determined in free lipid extracts using electrospray ionization‐ion trap‐mass spectrometry (ESI‐IT‐MS), working in both negative‐ and positive‐ion mode. In the case of TMC 5135, the presence of the previously characterized GPL‐II (containing 2,4‐di‐O‐CH₃ glucuronic acid as distal sugar in the oligosaccharide antigenic moiety) and GPL‐III (containing 4‐O‐CH₃ glucuronic acid as distal sugar) was confirmed using MS/MS and MS/MS/MS approaches. Interestingly, some ‘habana’ strains presented variants of GPL‐II, designated GPL‐II′‐A and GPL‐II′‐B. A di‐O‐CH₃‐deoxy‐hexose (tentatively, 2,3‐di‐O‐CH₃‐fucose) was identified as the penultimate sugar in the oligosaccharide moiety of GPL‐II′‐A, whereas in GPL‐II′‐B the penultimate sugar was fucose (tentative identification). On the contrary, the distal sugar of the oligosaccharide chain of pGPLs of Myco. simiae ATCC 25275^T was identified as tri‐O‐CH₃‐glucuronic acid (designated GPL‐sim^T‐I, with two variants: GPL‐sim^T‐I‐A and GPL‐sim^T‐I‐B), O‐CH₃‐glucuronic acid (designated GPL‐sim^T‐II) and di‐O‐CH₃‐glucuronic acid (GPL‐II′‐A and GPL‐II′‐B). The penultimate sugar of the oligosaccharide chain of GPL‐sim^T‐I‐A and GPL‐sim^T‐II was identified as di‐O‐CH₃‐deoxy‐hexose (tentatively, 2,3‐di‐O‐CH₃ fucose), and that of GPL‐sim^T‐I‐B as deoxy‐hexose (tentatively, fucose). In all strains studied, each [M‐H]^? and [M+Na]⁺ ion was revealed as a mixture of homologous compounds varying in the number of –O‐CH₃ groups present in the oligosaccharide moiety and in the length of the fatty acyl linked to the peptide. Conclusions: The present work indicates that, within a similar general pattern of pGPLs, different strains of Myco. simiae present some variations, so that new compounds (GPL‐II′‐A, GPL‐II′‐B, GPL‐sim^T‐I‐A, GPL‐sim^T‐I‐B and GPL‐sim^T‐II) were defined. Noteworthy was the fact that the ‘habana’ strains clearly differed from the type strain of Myco. simiae. Significance and Impact of the Study: The data obtained can be used in the delineation of the ‘habana’ group of Myco. simiae, including the quality control of the immunogenic strain ‘habana’ TMC 5135.     

Genotyping the Heading Date of Male-Sterile Rice Line II-32A

II-32A, an elite male-sterile line of rice （Oryza sativa L.）, has been widely used for the production of hybrid rice seed In China. Heading date In most combinations using II-32A shows transgressive Inheritance or similarity to the latter parent, but the genotype of II-32A with respect to major genes for heading time Is unknown. This limits the further exploitation of this sterile line In breeding and hybrid seed production. Using a number of major gene heading date Isogenlc lines and heading date QTL near-lsogenic lines, we genetically analyzed II-32B under both long- and short-day conditions. We show that II-32B carries two photoperlod-sensltlve genes, E1 and E3, a recessive late-heading gene, ef-l, and a photoperlod-sensltlve allele, Se-1^u. In addition we Identified In II- 32B a recessive Inhibitor for E1 or Se-1^n and other modified photoperlod-sensltlve genes. The heading-date constitution of II-32A was determined to be E1e2E3Se-1^uef-li-Se-1.     

In vitro Antibiosis by Pseudomonas syringae Pss22d,Acting Against the Bacterial Blight Pathogen of Soybean Plants,Does Not Influence In planta Biocontrol

The epiphyte Pseudomonas syringae pv. syringae 22d / 93 (Pss22d), isolated from soybean leaves, had been characterized as a promising and species‐specific biocontrol strain in vitro and in planta against the plant pathogen P. syringae pv. glycinea (Psg), which causes bacterial blight of soybean. Three toxins are known to be produced by Pss22d: syringomycin, syringopeptin and 3‐methylarginine (MeArg). In contrast to syringopeptin and syringomycin, MeArg inhibited the growth of Psg in vitro. To examine if the toxins produced by Pss22d are responsible for antagonistic effects in planta, the pathogen Psg was co‐inoculated with either Pss22d wild‐type, a syringopeptin/syringomycin‐negative double mutant (Pss22d.ΔsypA/syrE), or a MeArg‐negative mutant (Pss22d.1) into wounds of pin‐pricked leaves of greenhouse‐grown soybean plants, respectively. In all three cases, the wild‐type Pss22d and its toxin‐deficient mutants prevented development of disease symptoms normally caused by Psg. These results indicated that neither syringopeptin, nor syringomycin, nor MeArg was required for Pss22d’s antagonistic activity in planta. Consequently, factors other than the three toxins may contribute to the intra‐species antagonism in planta.     

Identification of candidate genes for key fibre‐related QTLs and derivation of favourable alleles in Gossypium hirsutum recombinant inbred lines with G. barbadense introgressions

Fine mapping QTLs and identifying candidate genes for cotton fibre‐quality and yield traits would be beneficial to cotton breeding. Here, we constructed a high‐density genetic map by specific‐locus amplified fragment sequencing (SLAF‐seq) to identify QTLs associated with fibre‐quality and yield traits using 239 recombinant inbred lines (RILs), which was developed from LMY22 (a high‐yield Gossypium hirsutumL. cultivar) × LY343 (a superior fibre‐quality germplasm with G. barbadenseL. introgressions). The genetic map spanned 3426.57 cM, including 3556 SLAF‐based SNPs and 199 SSR marker loci. A total of 104 QTLs, including 67 QTLs for fibre quality and 37 QTLs for yield traits, were identified with phenotypic data collected from 7 environments. Among these, 66 QTLs were co‐located in 19 QTL clusters on 12 chromosomes, and 24 QTLs were detected in three or more environments and determined to be stable. We also investigated the genomic components of LY343 and their contributions to fibre‐related traits by deep sequencing the whole genome of LY343, and we found that genomic components from G. hirsutum races (which entered LY343 via its G. barbadense parent) contributed more favourable alleles than those from G. barbadense. We further identified six putative candidate genes for stable QTLs, including Gh_A03G1147 (GhPEL6), Gh_D07G1598 (GhCSLC6) and Gh_D13G1921 (GhTBL5) for fibre‐length QTLs and Gh_D03G0919 (GhCOBL4), Gh_D09G1659 (GhMYB4) and Gh_D09G1690 (GhMYB85) for lint‐percentage QTLs. Our results provide comprehensive insight into the genetic basis of the formation of fibre‐related traits and would be helpful for cloning fibre‐development‐related genes as well as for marker‐assisted genetic improvement in cotton.     

Investigation of the diversity of effector genes in the banana pathogen,Fusarium oxysporum f. sp. cubense,reveals evidence of horizontal gene transfer

It is hypothesized that the virulence of phytopathogenic fungi is mediated through the secretion of small effector proteins that interfere with the defence responses of the host plant. In Fusarium oxysporum, one family of effectors, the Secreted In Xylem (SIX) genes, has been identified. We sought to characterize the diversity and evolution of the SIX genes in the banana‐infecting lineages of F. oxysporum f. sp. cubense (Foc). Whole‐genome sequencing data were generated for the 23 genetic lineages of Foc, which were subsequently queried for the 14 known SIX genes (SIX1–SIX14). The sequences of the identified SIX genes were confirmed in a larger collection of Foc isolates. Genealogies were generated for each of the SIX genes identified in Foc to further investigate the evolution of the SIX genes in Foc. Within Foc, variation of the SIX gene profile, including the presence of specific SIX homologues, correlated with the pathogenic race structure of Foc. Furthermore, the topologies of the SIX gene trees were discordant with the topology of an infraspecies phylogeny inferred from EF‐1α/RPB1/RPB2 (translation elongation factor‐1α/RNA polymerase II subunit I/RNA polymerase II subunit II). A series of topological constraint models provided strong evidence for the horizontal transmission of SIX genes in Foc. The horizontal inheritance of pathogenicity genes in Foc counters previous assumptions that convergent evolution has driven the polyphyletic phylogeny of Foc. This work has significant implications for the management of Foc, including the improvement of diagnostics and breeding programmes.     

Geography of Pinus elliottii Engelm. and Pinus palustris Mill. leaf life-spans in the southeastern U.S.A.

Aim Past studies have investigated differences in leaf life‐spans between deciduous and evergreen species. Environmental controls such as light, temperature, and nutrient and moisture availability explain differences in leaf life‐spans between species. This study examined intraspecific leaf life‐spans across climate and nutrient gradients within the geographical range of Pinus palustris Engelm (longleaf pine) and Pinus elliottii Mill. (slash pine). Location Five study areas in the southeastern United States were selected along the north–south geographical range of Pinus elliottii and Pinus palustris. Methods Leaf life‐span was calculated based on stand inventories and annual litterfall totals for each site, and allometric relationships between d.b.h. and foliar biomass. Results Leaf life‐span of P. elliottii ranged from 1.28 to 1.95 years between sites. Leaf life‐span of P.palustris varied by nearly a factor of 5 between the study site with the lowest and highest value (0.58–2.49 years). anova indicated that leaf life‐spans of P. elliottii were not significantly different among sites. In contrast, anova indicated a significant difference for P. palustris leaf life‐spans among sites (P < 0.05). The Tukey multiple comparisons tests showed that 2 study areas were the only pair of P. palustris sites with a significant difference in leaf life‐spans. Main conclusions The geographical variation in leaf life‐spans between two species illustrates the different phenotypic responses to environmental controls. The variation in leaf life‐spans by individuals of P. palustris across a geographical range illustrated in this study suggests that P. palustris may exhibit a greater phenotypic plasticity than P. elliottii.     

Water-Soluble Constituents of Cudrania tricuspidata （Carr.） Bur.

In order to find new structural and biologically active compounds, the constituents of the bark of Cudrania tricuspidata （Carr.） Bur. were investigated and a new 6-p-hydroxybenzyltaxifolin glucoside, named tricusposide （compound 1）, together with 16 known compounds, was isolated by solvent partition, macroporous adsorption resin AB-8, silica gel, Sephadex LH-20 chromatography. Using spectroscopic methods, the structures of the compounds were elucidated as 6-p-hydroxybenzyl taxifolin-7-O-β-D-glucoside （compound 1）, dihydroquerctin-7-O-β-D-glucoside （compound 2）, dihydrokaempferol-3-O-β-D-glucoside （compound 3）, dihydroquercetin （compound 4）, peonoside （compound 5）, sphaerobioside （compound 6）, quercimeritrin （compound 7）, genistein （compound 8）, aromadendrin （compound 9）, kaempferol （compound 10）, genistin （compound 11）, 3,4-dihydroxystyryl alcohol （compound 12）, sucrose （compound 13）, 1,3,5,6-tetrahydroxyxanthone （compound 14）, gericudranin E （compound 15）, gericudranin C （compound 16）, and orobol （compound 17）. Compounds 2-6, 8, 9, 12-14, and 17 were isolated from this genus for the first time.     

Untreated and enzyme‐modified bovine whey products reduce association of Salmonella Typhimurium,Escherichia coli O157:H7 and Cronobacter malonaticus (formerly Enterobacter sakazakii) to CaCo‐2 cells

Aims: Adhesion of a micro‐organism to a cell surface is often considered to be the first step in pathogenesis. Inhibiting this process may have therapeutic effects in vivo. This study investigates the inhibitory effects of various bovine whey products on the association of Salm. Typhimurium, E. coli O157:H7 and C. malonaticus (formerly Enterobacter sakazakii) to the human CaCo‐2 cell line. Invasion of CaCo‐2 cells by Salm. Typhimurium and C. malonaticus was also examined. Methods and Results: Infection assays were performed by incubating pathogenic acteria with CaCo‐2 cells in the presence of untreated (UT) or enzyme‐modified (EM) whey products. Associated micro‐organisms were directly quantified by plate counts. Invasion of CaCo‐2 cells by Salm. Typhimurium and C. malonaticus in the presence/absence of test materials was also quantified using gentamicin protection assays. At a concentration of 40 mg ml^?1, some UT whey products reduced association and invasion, but this effect was enhanced following hydrolysis with porcine pancreatic lipase. Conclusions: Both UT and EM sweet whey protein concentrates (WPCs) were found to be particularly effective inhibitors of association and invasion. All EM whey products significantly (P < 0·05) inhibited invasion of C. malonaticus into epithelial cells, causing a 2‐log reduction in the quantity of these micro‐organisms internalized. Significance and Impact of the Study: The present study suggests that whey products can inhibit association to and invasion of CaCo‐2 cells by selected micro‐organisms and may be useful in the treatment and/or prevention of foodborne infections.     

MASS SEXUAL REPRODUCTION IN THE TOXIGENIC DIATOMS PSEUDO‐NITZSCHIA AUSTRALIS AND P. PUNGENS (BACILLARIOPHYCEAE) ON THE WASHINGTON COAST,USA1

Sexual reproduction is documented for the first time in field populations of the pennate diatoms Pseudo‐nitzschia australis Freng. and P. pungens (Grunow ex Cleve) Hasle (var. cingulata Villac and hybrids between var. cingulata and var. pungens). A bloom dominated by these species began on June 26, 2006, along Kalaloch Beach, Washington, USA, coincident with a drop in the Si(OH)₄:NO₃ ratio to below two. Multimodal size distributions were detected for both species, and synchronous auxosporulation occurred within the smallest size class during a 3‐week window. Auxospores and initial cells created a new class of large cells, and cells in the intermediate size classes increased in abundance during auxosporulation. Mating cells of both species were attached to colonies of surf‐zone diatoms. Paired gametangia, gametes, zygotes, auxospores, and large initial cells were found. Auxosporulation began first for P. pungens (June 30), apparently once a critical, high cell concentration was reached, followed by P. australis (July 5), when the total Pseudo‐nitzschia cell concentration reached 929,000 cells · L^?1. Low frequencies of auxosporulation occurred throughout the bloom but increased 4‐fold for P. australis and 3‐fold for P. pungens when macronutrients were reduced to low levels on July 11. A 2‐year life cycle was estimated for P. australis and 3 years for P. pungens, both with annual auxosporulation. Domoic acid (DA) in razor clams reached a maximum of 38 μg DA · g^?1 on July 18. A significant relationship existed between the percent of cells within the new size range and DA concentrations in razor clams on the same beach.