Real-time PCR-based Y-specific sperm quantification assay in Queensland fruit fly: Insights to patterns of sperm storage.

Studies of reproductive biology in insects often require quantification of sperm production, transfer or storage. Here, we develop a quantitative real-time PCR-based assay using a Y-specific marker for quantification of sperm from spermathecae of female Queensland fruit fly ('Q-fly'), overcoming constraints typical of traditional sperm quantification methods. The assay enables accurate and reliable quantification of as few as 50 sperms and provides a means to analyse large numbers of samples with flexible timing. The real-time PCR method enables revised understanding of how many sperms are stored by female Q-flies, the distribution of storage between the two spermathecae and the relationship between copula duration and sperm storage. Real-time PCR assays based on Y-specific markers provide an effective solution for sperm quantification in tephritid flies, as well as in other insects and potentially other animals with sperm storage organs.

New and Interesting Fungi. 3.

Seven new genera, 26 new species, 10 new combinations, two epitypes, one new name, and 20 interesting new host and / or geographical records are introduced in this study. New genera are: Italiofungus (based on Italiofungus phillyreae) on leaves of Phillyrea latifolia (Italy); Neolamproconium (based on Neolamproconium silvestre) on branch of Tilia sp. (Ukraine); Neosorocybe (based on Neosorocybe pini) on trunk of Pinus sylvestris (Ukraine); Nothoseptoria (based on Nothoseptoria caraganae) on leaves of Caragana arborescens (Russia); Pruniphilomyces (based on Pruniphilomyces circumscissus) on Prunus cerasus (Russia); Vesiculozygosporium (based on Vesiculozygosporium echinosporum) on leaves of Muntingia calabura (Malaysia); Longiseptatispora (based on Longiseptatispora curvata) on leaves of Lonicera tatarica (Russia). New species are: Barrmaelia serenoae on leaf of Serenoa repens (USA); Chaetopsina gautengina on leaves of unidentified grass (South Africa); Chloridium pini on fallen trunk of Pinus sylvestris (Ukraine); Cadophora fallopiae on stems of Reynoutria sachalinensis (Poland); Coleophoma eucalyptigena on leaf litter of Eucalyptus sp. (Spain); Cylindrium corymbiae on leaves of Corymbia maculata (Australia); Diaporthe tarchonanthi on leaves of Tarchonanthus littoralis (South Africa); Elsinoe eucalyptorum on leaves of Eucalyptus propinqua (Australia); Exophiala quercina on dead wood of Quercus sp., (Germany); Fusarium californicum on cambium of budwood of Prunus dulcis (USA); Hypomyces gamsii on wood of Alnus glutinosa (Ukraine); Kalmusia araucariae on leaves of Araucaria bidwillii (USA); Lectera sambuci on leaves of Sambucus nigra (Russia); Melanomma populicola on fallen twig of Populus canadensis (Netherlands), Neocladosporium syringae on branches of Syringa vulgarishorus (Ukraine); Paraconiothyrium iridis on leaves of Iris pseudacorus (Ukraine); Pararoussoella quercina on branch of Quercus robur (Ukraine); Phialemonium pulveris from bore dust of deathwatch beetle (France); Polyscytalum pinicola on needles of Pinus tecunumanii (Malaysia); Acervuloseptoria fraxini on Fraxinus pennsylvanica (Russia); Roussoella arundinacea on culms of Arundo donax (Spain); Sphaerulina neoaceris on leaves of Acer negundo (Russia); Sphaerulina salicicola on leaves of Salix fragilis (Russia); Trichomerium syzygii on leaves of Syzygium cordatum (South Africa); Uzbekistanica vitis-viniferae on dead stem of Vitis vinifera (Ukraine); Vermiculariopsiella eucalyptigena on leaves of Eucalyptus sp. (Australia).

Genera of phytopathogenic fungi: GOPHY 3.

This paper represents the third contribution in the Genera of Phytopathogenic Fungi (GOPHY) series. The series provides morphological descriptions, information about the pathology, distribution, hosts and disease symptoms for the treated genera, as well as primary and secondary DNA barcodes for the currently accepted species included in these. This third paper in the GOPHY series treats 21 genera of phytopathogenic fungi and their relatives including: Allophoma, Alternaria, Brunneosphaerella, Elsinoe, Exserohilum, Neosetophoma, Neostagonospora, Nothophoma, Parastagonospora, Phaeosphaeriopsis, Pleiocarpon, Pyrenophora, Ramichloridium, Seifertia, Seiridium, Septoriella, Setophoma, Stagonosporopsis, Stemphylium, Tubakia and Zasmidium. This study includes three new genera, 42 new species, 23 new combinations, four new names, and three typifications of older names.

Foliar pathogens of eucalypts.

Species of eucalypts are commonly cultivated for solid wood and pulp products. The expansion of commercially managed eucalypt plantations has chiefly been driven by their rapid growth and suitability for propagation across a very wide variety of sites and climatic conditions. Infection of foliar fungal pathogens of eucalypts is resulting in increasingly negative impacts on commercial forest industries globally. To assist in evaluating this threat, the present study provides a global perspective on foliar pathogens of eucalypts. We treat 110 different genera including species associated with foliar disease symptoms of these hosts. The vast majority of these fungi have been grown in axenic culture, and subjected to DNA sequence analysis, resolving their phylogeny. During the course of this study several new genera and species were encountered, and these are described. New genera include: Lembosiniella (L. eucalyptorum on E. dunnii, Australia), Neosonderhenia (N. eucalypti on E. costata, Australia), Neothyriopsis (N. sphaerospora on E. camaldulensis, South Africa), Neotrichosphaeria (N. eucalypticola on E. deglupta, Australia), Nothotrimmatostroma (N. bifarium on E. dalrympleana, Australia), Nowamyces (incl. Nowamycetaceae fam. nov., N. globulus on E. globulus, Australia), and Walkaminomyces (W. medusae on E. alba, Australia). New species include (all from Australia): Disculoides fraxinoides on E. fraxinoides, Elsinoe piperitae on E. piperita, Fusculina regnans on E. regnans, Marthamyces johnstonii on E. dunnii, Neofusicoccum corticosae on E. corticosa, Neotrimmatostroma dalrympleanae on E. dalrympleana, Nowamyces piperitae on E. piperita, Phaeothyriolum dunnii on E. dunnii, Pseudophloeospora eucalyptigena on E. obliqua, Pseudophloeospora jollyi on Eucalyptus sp., Quambalaria tasmaniae on Eucalyptus sp., Q. rugosae on E. rugosa, Sonderhenia radiata on E. radiata, Teratosphaeria pseudonubilosa on E. globulus and Thyrinula dunnii on E. dunnii. A new name is also proposed for Heteroconium eucalypti as Thyrinula uruguayensis on E. dunnii, Uruguay. Although many of these genera and species are commonly associated with disease problems, several appear to be opportunists developing on stressed or dying tissues. For the majority of these fungi, pathogenicity remains to be determined. This represents an important goal for forest pathologists and biologists in the future. Consequently, this study will promote renewed interest in foliar pathogens of eucalypts, leading to investigations that will provide an improved understanding of the biology of these fungi.

Genera of phytopathogenic fungi: GOPHY 2.

This paper represents the second contribution in the Genera of Phytopathogenic Fungi (GOPHY) series. The series provides morphological descriptions and information regarding the pathology, distribution, hosts and disease symptoms for the treated genera. In addition, primary and secondary DNA barcodes for the currently accepted species are included. This second paper in the GOPHY series treats 20 genera of phytopathogenic fungi and their relatives including: Allantophomopsiella, Apoharknessia, Cylindrocladiella, Diaporthe, Dichotomophthora, Gaeumannomyces, Harknessia, Huntiella, Macgarvieomyces, Metulocladosporiella, Microdochium, Oculimacula, Paraphoma, Phaeoacremonium, Phyllosticta, Proxypiricularia, Pyricularia, Stenocarpella, Utrechtiana and Wojnowiciella. This study includes the new genus Pyriculariomyces, 20 new species, five new combinations, and six typifications for older names.

Fungal Planet description sheets: 400-468.

Novel species of fungi described in the present study include the following from Australia: Vermiculariopsiella eucalypti, Mulderomyces natalis (incl. Mulderomyces gen. nov.), Fusicladium paraamoenum, Neotrimmatostroma paraexcentricum, and Pseudophloeospora eucalyptorum on leaves of Eucalyptus spp., Anungitea grevilleae (on leaves of Grevillea sp.), Pyrenochaeta acaciae (on leaves of Acacia sp.), and Brunneocarpos banksiae (incl. Brunneocarpos gen. nov.) on cones of Banksia attenuata. Novel foliicolous taxa from South Africa include Neosulcatispora strelitziae (on Strelitzia nicolai), Colletotrichum ledebouriae (on Ledebouria floridunda), Cylindrosympodioides brabejum (incl. Cylindrosympodioides gen. nov.) on Brabejum stellatifolium, Sclerostagonospora ericae (on Erica sp.), Setophoma cyperi (on Cyperus sphaerocephala), and Phaeosphaeria breonadiae (on Breonadia microcephala). Novelties described from Robben Island (South Africa) include Wojnowiciella cissampeli and Diaporthe cissampeli (both on Cissampelos capensis), Phaeotheca salicorniae (on Salicornia meyeriana), Paracylindrocarpon aloicola (incl. Paracylindrocarpon gen. nov.) on Aloe sp., and Libertasomyces myopori (incl. Libertasomyces gen. nov.) on Myoporum serratum. Several novelties are recorded from La Réunion (France), namely Phaeosphaeriopsis agapanthi (on Agapanthus sp.), Roussoella solani (on Solanum mauritianum), Vermiculariopsiella acaciae (on Acacia heterophylla), Dothiorella acacicola (on Acacia mearnsii), Chalara clidemiae (on Clidemia hirta), Cytospora tibouchinae (on Tibouchina semidecandra), Diaporthe ocoteae (on Ocotea obtusata), Castanediella eucalypticola, Phaeophleospora eucalypticola and Fusicladium eucalypticola (on Eucalyptus robusta), Lareunionomyces syzygii (incl. Lareunionomyces gen. nov.) and Parawiesneriomyces syzygii (incl. Parawiesneriomyces gen. nov.) on leaves of Syzygium jambos. Novel taxa from the USA include Meristemomyces arctostaphylos (on Arctostaphylos patula), Ochroconis dracaenae (on Dracaena reflexa), Rasamsonia columbiensis (air of a hotel conference room), Paecilomyces tabacinus (on Nicotiana tabacum), Toxicocladosporium hominis (from human broncoalveolar lavage fluid), Nothophoma macrospora (from respiratory secretion of a patient with pneumonia), and Penidiellopsis radicularis (incl. Penidiellopsis gen. nov.) from a human nail. Novel taxa described from Malaysia include Prosopidicola albizziae (on Albizzia falcataria), Proxipyricularia asari (on Asarum sp.), Diaporthe passifloricola (on Passiflora foetida), Paramycoleptodiscus albizziae (incl. Paramycoleptodiscus gen. nov.) on Albizzia falcataria, and Malaysiasca phaii (incl. Malaysiasca gen. nov.) on Phaius reflexipetalus. Two species are newly described from human patients in the Czech Republic, namely Microascus longicollis (from toenails of patient with suspected onychomycosis), and Chrysosporium echinulatum (from sole skin of patient). Furthermore, Alternaria quercicola is described on leaves of Quercus brantii (Iran), Stemphylium beticola on leaves of Beta vulgaris (The Netherlands), Scleroderma capeverdeanum on soil (Cape Verde Islands), Scleroderma dunensis on soil, and Blastobotrys meliponae from bee honey (Brazil), Ganoderma mbrekobenum on angiosperms (Ghana), Geoglossum raitviirii and Entoloma kruticianum on soil (Russia), Priceomyces vitoshaensis on Pterostichus melas (Carabidae) (Bulgaria) is the only one for which the family is listed, Ganoderma ecuadoriense on decaying wood (Ecuador), Thyrostroma cornicola on Cornus officinalis (Korea), Cercophora vinosa on decorticated branch of Salix sp. (France), Coprinus pinetorum, Coprinus littoralis and Xerocomellus poederi on soil (Spain). Two new genera from Colombia include Helminthosporiella and Uwemyces on leaves of Elaeis oleifera. Two species are described from India, namely Russula intervenosa (ectomycorrhizal with Shorea robusta), and Crinipellis odorata (on bark of Mytragyna parviflora). Novelties from Thailand include Cyphellophora gamsii (on leaf litter), Pisolithus aureosericeus and Corynascus citrinus (on soil). Two species are newly described from Citrus in Italy, namely Dendryphiella paravinosa on Citrus sinensis, and Ramularia citricola on Citrus floridana. Morphological and culture characteristics along with ITS nrDNA barcodes are provided for all taxa.

Evaluating irradiation dose for sterility induction and quality control of mass-produced fruit fly Bactrocera tryoni (Diptera: Tephritidae).

The sterile insect technique has been routinely used to eradicate fruit fly Bactrocera tryoni (Froggatt) incursions. This study considers whether fly quality in a mass-rearing facility can be improved by reducing irradiation doses, without sacrificing reproductive sterility. Pupae were exposed to one of five target irradiation dose ranges: 0, 40-45, 50-55, 60-65, and 70-75 Gy. Pupae were then assessed using routine quality control measures: flight ability, sex ratio, longevity under nutritional stress, emergence, and reproductive sterility. Irradiation did not have a significant effect on flight ability or sex ratio tests. Longevity under nutritional stress was significantly increased at 70-75 Gy, but no other doses differed from 0 Gy. Emergence was slightly reduced in the 50-55, 60-65, and 70-75 Gy treatments, but 40-45 Gy treatments did not differ from 0 Gy, though confounding temporal factors complicate interpretation. Reproductive sterility remained acceptable (> 99.5%) for all doses--40-45 Gy (99.78%), 50-55 Gy (100%), 60-65 Gy (100%), and 70-75 Gy (99.99%). We recommend that B. tryoni used in sterile insect technique releases be irradiated at a target dose of 50-55 Gy, providing improved quality and undiminished sterility in comparison with the current 70-75 Gy standard while also providing a substantial buffer against risk of under dosing.

Yeast hydrolysate supplementation increases field abundance and persistence of sexually mature sterile Queensland fruit fly, Bactrocera tryoni (Froggatt).

The sterile insect technique (SIT) is a non-chemical approach used to control major pests from several insect families, including Tephritidae, and entails the mass-release of sterile insects that reduce fertility of wild populations. For SIT to succeed, released sterile males must mature and compete with wild males to mate with wild females. To reach sexual maturity, the Queensland fruit fly, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), must obtain adequate nutrition after adult emergence; however, in current SIT programs sterile B. tryoni receive a pre-release diet that lacks key nutrients required to sustain sexual development. The chief objective of this study was to determine whether pre-release yeast hydrolysate (YH) supplements affect the persistence and abundance of sexually mature sterile male B. tryoni under field conditions. Experiments were run in outdoor cages under conditions of low and high environmental stress that differed markedly in temperature and humidity, and in the field. Under low environmental stress conditions, survival of sterile B. tryoni was monitored in cages under three diet treatments: (i) sugar only, (ii) sugar plus YH or (iii) sugar plus YH for 48 h and sugar only thereafter. Under high environmental stress conditions survival of sterile B. tryoni was monitored in cages under four diet treatments: (i) white sugar only, (ii) brown sugar only, (iii) white sugar plus YH and (iv) brown sugar plus YH. In a replicated field study, we released colour-marked sterile B. tryoni from two diet regimes, YH-supplemented or YH-deprived, and monitored abundance of sexually mature males. In the low-stress cage study, there was no effect of diet, although overall females lived longer than males. In the high stress cage study, mortality was lower for YH-fed flies than YH-deprived flies and females lived longer than males. In the field, YH supplementation resulted in higher abundance of sexually mature sterile males, with 1.2 YH-fed flies trapped for every YH-deprived fly trapped. Under field conditions, YH supplementation can increase over-flooding ratios and hence may improve the effectiveness of SIT programmes.

Desiccation resistance of wild and mass-reared Bactrocera tryoni (Diptera: Tephritidae).

In pest management programmes that incorporate the sterile insect technique (SIT), the ability of mass-reared insects to tolerate dry conditions may influence their survival after release in the field. In the present study, desiccation resistance of adult mass-reared Queensland fruit flies, Bactrocera tryoni (Frogatt) (Diptera: Tephritidae), that are routinely released in SIT programmes was compared with that of wild flies at 1, 10 and 20 days after adult eclosion. Under dry conditions without access to food or water, longevity of mass-reared B. tryoni was significantly less than that of their wild counterparts. Desiccation resistance of mass-reared flies declined monotonically with age, but this was not the case for wild flies. The sharp decline in desiccation resistance of mass-reared flies as they aged was likely explained by decreased dehydration tolerance. As in an earlier study, desiccation resistance of females was significantly lower than that of males but this was particularly pronounced in mass-reared females. Female susceptibility to dry conditions corresponded with declining dehydration tolerance with age and associated patterns of reproductive development, which suggests that water content of their oocyte load is not available for survival during periods of water stress.

The use of a real-time PCR primer/probe set to observe infectivity of Yersinia ruckeri in Chinook salmon, Oncorhynchus tshawytscha (Walbaum), and steelhead trout, Oncorhynchus mykiss (Walbaum).

Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM), a common pathogen affecting aquaculture facilities and implicated in large losses of cultured fish. Fisheries scientists continue to gain a greater understanding of the disease and the pathogen by investigating methods of identification and pre- and post-infection treatment. In this study, a real-time PCR probe set for Y. ruckeri was developed to detect daily changes in the bacterial load during pathogen challenges. Two species of fish, Chinook salmon, Oncorhynchus tshawytscha, and steelhead trout, Oncorhynchus mykiss, were exposed to two strains of Y. ruckeri (Hag and SC) during bath challenges. A subset of fish was killed daily for 14 days, and the kidney tissue was biopsied to enumerate copies of pathogen DNA per gram of tissue. While Chinook exposed to either the Hag or SC strains exhibited similar pathogen loads, those exposed to the Hag strain displayed higher mortality (∼66%) than fish exposed to the SC strain (∼24% mortality). Steelhead exposed to the Hag strain exhibited a greater pathogen load and higher mortality (∼42%) than those exposed to the SC strain (<1% mortality). Steelhead challenged with either strain showed lower pathogen loads than Chinook. The study illustrates the efficacy of the probe set to enumerate Y. ruckeri bacterial growth in the kidneys of fish. Also, strains of Y. ruckeri display species-specific growth patterns that result in differential mortality and pathogen load.

Bacteriophage-derived enzyme that depolymerizes the alginic acid capsule associated with cystic fibrosis isolates of Pseudomonas aeruginosa.

AIMS: To identify enzymes associated with bacteriophages infecting cystic fibrosis (CF) strains of Pseudomonas aeruginosa that are able to degrade extracellular alginic acids elaborated by the host bacterium. METHODS AND RESULTS: Plaques produced by 21 Ps. aeruginosa-specific phages were screened for the presence of haloes, an indicator of capsule hydrolytic activity. Four phages produced haloed plaques, and one (PT-6) was investigated further. PT-6 was shown by electron microscopy to belong to Podoviridae family C1, to reduce the viscosity of four alginate preparations using a rolling ball viscometer and to release uronic acid-containing fragments from the polymers, as judged by spectrophotometry and thin layer chromatography. The alginase was partially purified by gel filtration chromatography and shown to be a 37 kDa polypeptide. CONCLUSIONS: Infection of CF strains of Ps. aeruginosa by phage PT-6 involves hydrolysis of the exopolysaccharide secreted by the host. SIGNIFICANCE AND IMPACT OF THE STUDY: The alginase produced by PT-6 has the potential to increase the well-being of CF suffers by improving the surface properties of sputum, accelerating phagocytic uptake of bacteria and perturbing bacterial growth in biofilms.

Optimizing irradiation dose for sterility induction and quality of Bactrocera tryoni.

The current study is an important step toward calibrating, validating, and improving irradiation methods used for Bactrocera tryoni (Froggatt) sterile insect technique (SIT). We used routine International Atomic Energy Agency/U.S. Department of Agriculture/Food and Agriculture Organization quality control tests assessing percentage of emergence, flight ability, sex ratio, mortality under stress, reproductive sterility, and sexual competitiveness, as well as a nonstandard test of longevity under nutritional stress to assess the impact of a range of target irradiation doses (60, 65, 70, 75, and 80 Gy) on the product quality of mass reared B. tryoni used in SIT. Sterility induction remained adequate (>99.5%) for sterile male-fertile female crosses, and 100% sterility was achieved in fertile male-sterile female crosses and sterile male-sterile female crosses for each irradiation doses tested. There was significant increase in mortality under stress as irradiation dose increased, and reduced participation in mating by males irradiated at higher doses. The current target-sterilizing dose for SIT of 70-75 Gy is associated with significant reduction in fly product "quality". Our data suggest that adequate sterility and improved fly quality could be achieved through a small reduction in target sterilizing dose.

Identification of two new genes conferring resistance to Colletotrichum acutatum in Capsicum baccatum.

Resistance to anthracnose, caused by Colletotrichum capsici and C. acutatum, was investigated in Capsicum baccatum PBC80 and PBC1422 and C. chinense PBC932. Mature green and ripe fruit were inoculated with 13 isolates of the two Colletotrichum species PBC80 contained the broadest spectrum of resistance to both Colletotrichum species because none of the isolates were able to infect the genotype. At both fruit maturity stages, PBC1422 was infected by only Colletotrichum acutatum. PBC932 at ripe fruit stage was infected by both C. capsici and C. acutatum, except for one isolate, 158ci, that did not infect PBC932. PBC932 at the mature green fruit stage was infected by only C. acutatum. An intraspecific cross between PBC80 and PBC1422 was developed to determine inheritance of resistance to C. acutatum. Anthracnose resistance was assessed at mature green and ripe fruit stages using 0 to 9 disease severity scores. Frequency distribution of the disease scores in the F(2) and BC(1) populations suggested a single recessive gene responsible for the resistance at mature green fruit stage and a single dominant gene for the resistance at ripe fruit stage. Linkage analysis between the two genes identified in both fruit maturity stages showed the genes to be independent. Based on phenotypic data, the two newly identified genes, co4 and Co5, from PBC80 appeared to be different loci from the co1 and co2 previously identified from PBC932 and will be valuable sources of resistance to anthracnose in chili breeding programs.

Components derived from Pelargonium stimulate macrophage killing of Mycobacterium species.

AIMS: To determine the capacity of extracts of Pelargonium reniforme and Pelargonium sidoides, plants of the Geraniaceae family, to stimulate the uptake and killing of mycobacteria by murine macrophages and to identify the constituents that are responsible. METHODS AND RESULTS: Bioassay-guided fractionation of aqueous P. reniforme extracts yielded five chemically distinct structures with the capacity to increase the rate of intracellular killing by macrophages. These were: gallic acid, methyl gallate, myricetin and quercitin-3-O-beta-d-glucoside, in addition to the previously unrecognized constituent 1-O-(2-(4-methoxyphenyl)ethyl-6-O-galloyl-glucopyranoside. Kinetics of intracellular accumulation of Mycobacterium tuberculosis and Mycobacterium fortuitum by macrophages were indistinguishable; pure preparations of the four previously known plant constituents stimulated macrophage killing, but not uptake, of M. tuberculosis and M. fortuitum equally well. CONCLUSIONS: A number of distinct molecular species are present in the medicinal plant P. reniforme that stimulate the killing of the intracellular pathogen M. tuberculosis. SIGNIFICANCE AND IMPACT OF THE STUDY: These observations support the view that Pelargonium extracts may have utility in the treatment of tuberculosis.

Pathotypes of Colletotrichum capsici, the Causal Agent of Chili Anthracnose, in Thailand.

Eleven isolates of Colletotrichum capsici were screened on nine chili genotypes derived from four cultivated species of Capsicum: Capsicum annuum, C. baccatum, C. chinense, and C. frutescens. Host reactions were assessed 9 days after inoculation by microinjection of spores into the pericarp of red fruit. A set of disease scales, with 0 to 9 scores, were developed for anthracnose infection of each Capsicum sp. based on percent lesion size in relation to fruit size, appearance of necrotic or water-soaked tissue, and presence of acervuli. Three pathotypes, PCc1, PCc2, and PCc3, were identified according to differential qualitative infection of fruit of C. chinense genotypes PBC932 and C04714. PCc1 was the most virulent pathotype, infecting all genotypes of C. annuum, C. chinense, and C. frutescens, whereas PCc3 was the least virulent pathotype, infecting only the genotypes C. annuum and C. frutescens. Quantitative infection occurred in all chili genotypes except for genotypes of C. baccatum, where no infection occurred, demonstrating various levels of aggressiveness of isolates within pathotypes.

Development and optimization of sequence-tagged microsatellite site markers to detect genetic diversity within Colletotrichum capsici, a causal agent of chilli pepper anthracnose disease.

Genomic libraries enriched for microsatellites from Colletotrichum capsici, one of the major causal agents of anthracnose disease in chilli pepper (Capsicum spp.), were developed using a modified hybridization procedure. Twenty-seven robust primer pairs were designed from microsatellite flanking sequences and were characterized using 52 isolates from three countries India, Sri Lanka and Thailand. Highest gene diversity of 0.857 was observed at the CCSSR1 with up to 18 alleles among all the isolates whereas the differentiation ranged from 0.05 to 0.45. The sequence-tagged microsatellite site markers developed in this study will be useful for genetic analyses of C. capsici populations.

Investigation of the antibacterial activity of 3-O-octanoyl-(-)-epicatechin.

AIMS: To measure antibacterial activity of the semi-synthetic flavonoid 3-O-octanoyl-(-)-epicatechin and investigate the mechanism of action. METHODS AND RESULTS: MICs determined by the broth microdilution method were 50 microg ml(-1) for beta-lactam sensitive and resistant Staphylococcus aureus, and 100 microg ml(-1) for vancomycin sensitive and resistant enterococci. In time-kill studies, 100 microg ml(-1) 3-O-octanoyl-(-)-epicatechin reduced colony forming unit numbers of antibiotic sensitive and methicillin-resistant Staph. aureus below detectable levels within 120 min. Bacterial aggregation was not observed when cells exposed to 3-O-octanoyl-(-)-epicatechin were examined by light microscopy. It was also shown that 50 microg ml(-1) 3-O-octanoyl-(-)-epicatechin is capable of reducing colony forming unit numbers of high cell density Staph. aureus populations by 80-fold within 60 min incubation, and inducing leakage of 50% of their internal potassium within just 10 min. CONCLUSIONS: 3-O-Octanoyl-(-)-epicatechin is active against Gram-positive bacteria, has bactericidal activity against both antibiotic sensitive and resistant strains, and is likely to exert its primary antibacterial effect by damaging the cytoplasmic membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: 3-O-Octanoyl-(-)-epicatechin has significant antibacterial activity and additional structural modification and/or formulation studies may allow this to be potentiated.

The polyphenol (-)-epicatechin gallate disrupts the secretion of virulence-related proteins by Staphylococcus aureus.

AIM: (-)-epicatechin gallate (ECg) modifies the morphology, cell wall architecture and beta-lactam antibiotic susceptibility of Staphylococcus aureus. As these effects result primarily from intercalation into the bacterial cytoplasmic membrane, the capacity of ECg to modulate the secretion of two key staphylococcal virulence factors, coagulase and alpha-toxin, was examined. METHODS AND RESULTS: Bioassays were used to determine coagulase and haemolysin activity in culture supernatants of a number of S. aureus isolates grown in the presence and absence of ECg; alpha-toxin secretion was also evaluated by immunoblotting. Growth in ECg reduced the levels of activity of both proteins in culture supernatants; the effects could only be partly explained by ECg-mediated inhibition of bioactivity and by induction of secreted proteases. CONCLUSION: ECg suppresses the secretion of coagulase and alpha-toxin by clinical isolates of S. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: The observation that secretion of key components of staphylococcal virulence can be compromised by a naturally occurring polyphenol supports the notion that ECg and related compounds may have therapeutic utility for the control of infections that are currently difficult to treat due to the propensity of methicillin-resistant S. aureus to accumulate antibiotic resistance genes.

Aggregation of Staphylococcus aureus following treatment with the antibacterial flavonol galangin.

AIM: The flavonol galangin, an antimicrobial constituent of the traditional medicines propolis and Helichrysum aureonitens, is being assessed as part of an ongoing investigation into the antibacterial activity of flavonoids. The present study sought to establish whether galangin has any aggregatory effect on bacterial cells. METHODS AND RESULTS: In preparatory time-kill assays, 50 microg ml(-1) of galangin was found to reduce colony counts of c. 5 x 10(7) CFU ml(-1)Staphylococcus aureus NCTC 6571 by approximately 15 000-fold during 60 min of incubation. Subsequent light microscopy studies demonstrated significant increases in the number of large clusters of bacterial cells in populations treated with the flavonol. CONCLUSION: Data presented here show that galangin causes aggregation of bacterial cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The finding that galangin causes bacterial cells to clump together may implicate the cytoplasmic membrane as a target site for this compound's activity. More importantly, this observation indicates that decreases in CFU numbers detected in time-kill and minimum bactericidal concentration (MBC) assays in previous investigations were at least partially attributable to this aggregatory effect. This raises the possibility that galangin is not genuinely bactericidal in action, and calls into question the suitability of time-kill and MBC assays for determining the nature of activity of naturally occurring flavonoids.

Broadcasts of wing-fanning vibrations recorded from calling male Ceratitis capitata (Diptera: Tephritidae) increase captures of females in traps.

Female Mediterranean fruit flies, Ceratitis capitata (Wiedemann), from the sterile-male rearing facility in El Pino, Guatemala, were exposed to broadcasts of wing-fanning vibrations recorded from males engaged in calling behavior to investigate the feasibility of developing a female-selective acoustic trap. The recorded signals had frequent amplitude fluctuations and peak frequencies approximately 350 Hz, typical of signals observed in previous studies of Mediterranean fruit fly acoustic behavior. Females did not exhibit long-distance phonotaxis, but remained near a speaker significantly longer when the sounds were broadcast at 103-107 dB than when the speaker was silent. In addition, significantly higher percentages of females were captured by yellow adhesive traps next to a broadcasting speaker than by traps next to a silent mimic. Additional bioassays were conducted with synthetic, 350-Hz tones produced by a thermoacoustic tube as well as with silent mimics of the different sound sources to examine the relative responsiveness of female Mediterranean fruit flies to traps with different acoustic and visual features. The visual attributes of the different sound source assemblies significantly affected capture rates. The range over which the broadcast significantly increased the percentage of female captures was <0.5 m, which may limit the utility of these acoustic cues in large-scale trapping programs. However, the findings of this study do justify further testing of whether optimized short-range acoustic signals could be used to augment longer range pheromonal and visual cues to improve the efficacy of female-selective traps.