First record of the tick <Emphasis Type="Italic">Ixodes</Emphasis> (<Emphasis Type="Italic">Pholeoixodes</Emphasis>) <Emphasis Type="Italic">kaiseri</Emphasis> in Turkey

Nymphs and larvae belonging to Ixodes spp. were collected from a red fox in Turkey. The ticks were identified morphologically and molecularly (16S rDNA PCR and phylogenetic analysis) as I. kaiseri. Sequence and phylogenetic analyses show that our I. kaiseri isolate is very similar to I. kaiseri isolates collected from Germany, Serbia, Romania, and Hungary. Therefore, the existence of I. kaiseri has been demonstrated for the first time in Turkey. More studies relating to the regional distribution and vectorial competence of I. kaiseri are needed.     

Detection of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> virulence genes in <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> subsp. <Emphasis Type="Italic">equisimilis</Emphasis> from Vellore,India

Streptococcus dysgalactiae subsp. equisimilis (SDSE), belonging to the group C and G streptococci, are human pathogens reported to cause clinical manifestations similar to infections caused by Streptococcus pyogenes. To scrutinize the distribution of gene coding for S. pyogenes virulence factors in SDSE, 255 isolates were collected from humans infected with SDSE in Vellore, a region in southern India, with high incidence of SDSE infections. Initial evaluation indicated SDSE isolates comprising of 82.35% group G and 17.64% group C. A multiplex PCR system was used to detect 21 gene encoding virulence-associated factors of S. pyogenes, like superantigens, DNases, proteinases, and other immune modulatory toxins. As validated by DNA sequencing of the PCR products, sequences homologous to speC, speG, speH, speI, speL, ssa and smeZ of the family of superantigen coding genes and for DNases like sdaD and sdc were detected in the SDSE collection. Furthermore, there was high abundance (48.12% in group G and 86.6% in group C SDSE) of scpA, the gene coding for C5a peptidase in these isolates. Higher abundance of S. pyogenes virulence factor genes was observed in SDSE of Lancefield group C as compared to group G, even though the incidence rates in former were lower. This study not only substantiates detection of S. pyogenes virulence factor genes in whole genome sequenced SDSE but also makes significant contribution towards the understanding of SDSE and its increasing virulence potential.     

Taxonomic study on <Emphasis Type="Italic">Alternaria</Emphasis> sections <Emphasis Type="Italic">Infectoriae</Emphasis> and <Emphasis Type="Italic">Pseudoalternaria</Emphasis> associated with black (sooty) head mold of wheat and barley in Iran

During the spring and summer of 2014 and 2015, wheat and barley fields in the Iranian provinces of Golestan and Alborz showed a high incidence of symptoms of black (sooty) head mold of wheat and barley. The isolation results revealed that Alternaria was associated with these symptoms. One hundred and forty isolates were collected and morphologically characterized based on the development of conidial chains with primary, secondary, and tertiary branching patterns, consistent with the three-dimensional sporulation complexity of members of Alternaria in sections Infectoriae and Pseudoalternaria. Subsequently, 16 Alternaria isolates exhibiting high morphological diversity were characterized based on extensive morphological and molecular comparisons. Phylogenetic analyses of three loci [ITS, glyceraldehyde 3-phosphate dehydrogenase (gpd), and plasma membrane ATPase (ATPase)] revealed that 15 isolates belonged to section Infectoriae but could not be assigned to phylogenetic species and one isolate represents a new species, Alternaria kordkuyana sp. nov., in section Pseudoalternaria. Morphological assessments revealed a high degree of variation among section Infectoriae isolates and that A. kordkuyana has significant morphological differences as compared to the three other species currently described in section Pseudoalternaria.     

Interaction between toxin crystals and vegetative insecticidal proteins of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in lepidopteran larvae

Insecticides based on crystalline toxins of Bacillus thuringiensis are very good biological plant protection products. However, the spectrum of activity of some toxins is narrow or resistance among insects has been developed. We tested the insecticidal activity of crystals of the B. thuringiensis MPU B9 strain alone and supplemented with Vip3Aa proteins against important pests: Spodoptera exigua Hübner (Lepidoptera: Noctuidae), Cydia pomonella L. (Lepidoptera: Tortricidae) and Dendrolimus pini L. (Lepidoptera: Lasiocampidae). The Cry toxins were more active for D. pini but less active against S. exigua and C. pomonella than Vip3Aa. Supplementation of Cry toxins by small amounts of vegetative insecticidal proteins demonstrated synergistic effect and significantly enhanced the toxicity of the insecticide. The results indicate the utility of Cry and Vip3Aa toxins mixtures to control populations of crops and forests insect pests.     

Environmental risk assessment of the egg parasitoid <Emphasis Type="Italic">Anaphes inexpectatus</Emphasis> for classical biological control of the <Emphasis Type="Italic">Eucalyptus</Emphasis> snout beetle, <Emphasis Type="Italic">Gonipterus platensis</Emphasis>

Classical biological control is a valuable tool against invasive pests, but concerns about non-target effects requires risk assessment studies. Potential non-target effects of Anaphes inexpectatus Huber and Prinsloo (Hymenoptera: Mymaridae) were assessed for a classical biological control programme against the Eucalyptus snout beetle, Gonipterus platensis (Marelli) (Coleoptera: Curculionidae). No-choice tests were conducted with 17 non-target species to assess host specificity, including 11 curculionids. In behavioural observations, A. inexpectatus showed no interest in any of the non-target species, but two weevil species were parasitised within five days of exposure, although at significantly lower rates than G. platensis. In choice tests, only one non-target, Hypera postica (Gyllenhal) (Coleoptera: Curculionidae), was parasitised, at a rate of 0.6%, while 50.0% of G. platensis eggs were parasitised. Based on the host specificity test results and the potential host fauna found in the target area, the likelihood of non-target effects resulting from the release of A. inexpectatus is considered to be negligible.     

Biodegradation of phenol, <Emphasis Type="Italic">o-</Emphasis>cresol, <Emphasis Type="Italic">m-</Emphasis>cresol and <Emphasis Type="Italic">p-</Emphasis>cresol by indigenous soil fungi in soil contaminated with creosote

Seven non-basidiomycetic fungi, Aspergillus, Candida, Cladosporium, Fusarium, Monicillium, Trichoderma and Penicillium, and two basidiomycetic fungi, Pleurotus and Phanerochaete were isolated from a creosote-contaminated soil by using mineral salts medium and soil extract broth containing antibiotics. Soil contaminated with phenol, o-cresol, m-cresol and p-cresol was collected from the yard of a wood treatment plant in South Africa and inoculated with the strains of Aspergillus, Cladosporium, Fusarium, Monicillium, Penicillium and Phanerochaete, selected from the isolate. The soil in some of the treatment reactors was amended with nutrient supplements to give a C:N:P ratio of 25:5:1. A total of 18 duplicate treatments were established and incubated in the dark at 25°C for 70days. The soil in all the reactors was tilled weekly and moisture was maintained at 70% field capacity. Soil samples were collected every 2weeks for analysis of residual concentrations of the phenols tested, pH measurement and moisture content determination. The nutrient-supplemented treatments were more effective in degrading the phenols (between 84 and 100%) than those that were not supplemented. Barley, which was used as bulking agent enhanced the growth of the fungi and subsequently the degradation of the phenols. Inoculation with a mixture of the six fungal isolates promoted more phenol degradation than with single isolates.     

Reactive oxygen species (ROS) and antioxidative enzyme status in <Emphasis Type="Italic">Solanum lycopersicum</Emphasis> on priming with fluorescent <Emphasis Type="Italic">Pseudomonas</Emphasis> spp. against <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

In plants, ROS signaling and increase in activities of antioxidants are among defense responses. The present study describes the oxidative stress profiling in model host plant tomato (Solanum lycopersicum L.), during an invasion of the wilt pathogen Fusarium oxysporum f. sp. lycopersici with or without seed priming with Pseudomonas isolates M80, M96 and T109. Tomato seeds were primed with known Pseudomonas isolates M80, M96 and T109 and the forty-day- old plants were challenged with spores of F. oxysporum under greenhouse conditions. Leaf samples were collected at 0, 24, 48 72 and 96 h post fungal challenge and analysed for systemic level of oxidative stress parameters including total phenolics, proline, hydrogen peroxide, lipid peroxidation and enzymatic antioxidants. Disease incidence in the plants under greenhouse conditions was also calculated. Results revealed that priming with Pseudomonas isolates resulted in reduced oxidative stress in the host, during pathogen invasion. M80-priming showed highest antioxidative protection to the host plants during F. oxysporum invasion. The observed reduction in hydrogen peroxide and lipid peroxidation in primed plants was in agreement with the increased activities of the corresponding antioxidant enzymes. Greenhouse results showed that the highest wilt disease symptoms were with M80-priming followed by M96 and T109. The present study gives substantial evidences on the oxidative stress mitigation in response to Pseudomonas-priming on the model tomato-Fusarium interaction system.     

The <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> efficacy of fluconazole in combination with farnesol against <Emphasis Type="Italic">Candida albicans</Emphasis> isolates using a murine vulvovaginitis model

Farnesol is a quorum-sensing molecule that inhibits biofilm formation in Candida albicans. Previous in vitro data suggest that, in combination with certain antifungals, farnesol may have an adjuvant anti-biofilm agent. However, the in vivo efficacy of farnesol is very questionable. Therefore, the in vitro and in vivo activity of fluconazole combined with farnesol was evaluated against C. albicans biofilms using fractional inhibitory concentration index (FICI) determination, time-kill experiments and a murine vulvovaginitis model. The median biofilm MICs of fluconazole-sensitive C. albicans isolates ranged between 4 -> 512 mg/L and 150–300 μM for fluconazole and farnesol, respectively. These values were 512 -> 512 mg/L and > 300 μM for fluconazole-resistant clinical isolates. Farnesol decreased the median MICs of fluconazole by 2-64-fold for biofilms. Based on FICI, synergistic interaction was observed only in the case of the sessile SC5314 reference strain (FICIs: 0.16–0.27). In time-kill studies, only the 512 mg/L fluconazole and 512 mg/L fluconazole + 75 μM farnesol reduced biofilm mass significantly at each time point in the case of all isolates. The combination reduced the metabolic activity of biofilms for all isolates in a concentration- and time-dependent manner. Our findings revealed that farnesol alone was not protective in a murine vulvovaginitis model. Farnesol was not beneficial in combination with fluconazole for fluconazole-susceptible isolates, but partially increased fluconazole activity against one fluconazole-resistant isolate, but not the other one.     

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.     

Regulatory activity of heterologous gene-activator <Emphasis Type="Italic">xlnR</Emphasis> of <Emphasis Type="Italic">Aspergillus niger</Emphasis> in <Emphasis Type="Italic">Penicillium canescens</Emphasis>

The gene encoding the xlnR xylanolytic activator of the heterologous fungus Aspergillus niger was incorporated into the Penicillium canescens genome. Integration of the xlnR gene resulted in the increase in a number of activities, i.e. endoxylanase, β-xylosidase, α-L-arabinofuranosidase, α-galactosidase, and feruloyl esterase, compared to the host P. canescens PCA 10 strain, while β-galactosidase, β-glucosidase, endoglucanase, and CMCase activities remained constant. Two different expression constructs were developed. The first consisted of the nucleotide sequence containing the mature P. canescens phytase gene under control of the axhA promoter region gene encoding A. niger (1,4)-β-D-arabinoxylan-arabinofuranohydrolase. The second construct combined the P. canescens phytase gene and the bgaS promoter region encoding homologous β-galactosidase. Both expression cassettes were transformed into P. canescens host strain containing xlnR. Phytase synthesis was observed only for strains with the bgaS promoter on arabinose-containing culture media. In conclusion, the bgaS and axhA promoters were regulated by different inducers and activators in the P. canescens strain containing a structural tandem of the axhA promoter and the gene of the xlnR xylanolytic activator.     

Immunohistochemical Cross-Reactivity Between <Emphasis Type="Italic">Paracoccidioides</Emphasis> sp. from Dolphins and <Emphasis Type="Italic">Histoplasma capsulatum</Emphasis>

Paracoccidioidomycosis ceti is a cutaneous disease of cetaceans caused by uncultivated Paracoccidioides brasiliensis or Paracoccidioides spp. Serological cross-reactions between paracoccidioidomycosis ceti and paracoccidioidomycosis, paracoccidioidomycosis and histoplasmosis, and paracoccidioidomycosis and coccidioidomycosis have been reported before. The present study aimed to detect immunohistochemical cross-reaction between antibodies to Paracoccidioides sp. and Histoplasma capsulatum, and vice versa. Thirty murine sera, obtained from experimental infections of 6 isolates of H. capsulatum, were reacted with paraffin-embedded yeast-form cells of Paracoccidioides sp. derived from a case of paracoccidioidomycosis ceti in Japan. The murine sera were also reacted with human isolates of H. capsulatum yeast cells, with P. brasiliensis yeast cells, and with fungal cells of Coccidioides posadasii. Three dolphins’ sera from cases of paracoccidioidomycosis ceti, two human sera from patients with paracoccidioidomycosis, and a serum from a healthy person with a history of coccidioidomycosis were used in order to determine that the tested fungal cells reacted properly. Sera derived from mice infected with an isolate of H. capsulatum reacted positively against yeast cells of Paracoccidioides sp., yeast cells of P. brasiliensis, and fungal cells of C. posadasii, while those derived from other strains were negative. The present study recorded for the first time the cross-reaction between the yeast cells of H. capsulatum and antibodies against Paracoccidioides spp., the yeast cells of Paracoccidioides sp. and antibodies against H. capsulatum, the yeast cells of Paracoccidioides sp. and antibodies against Coccidioides sp., and fungal cells of C. posadasii and antibodies against Paracoccidioides spp.     

Molecular genetic characterization of avian <Emphasis Type="Italic">Chlamydophila psittaci</Emphasis> isolates

RFLP analysis of the omp1 gene was used to characterize 51 avian Chlamydophila psittaci isolates. The analysis confirmed the predominance of genotype A in parrot C. psittaci isolates and revealed new omp1 genotypes in corvid C. psittaci isolates. The corvid isolates proved to lack an extrachromosomal plasmid. The omp1 and rRNA IGS sequences were determined for the new isolates. Phylogenetic analysis showed that isolate 1V, obtained from a crow, is intermediate in several characters between C. abortus and C. psittaci. The results were compared with data on the phylogenetic relationships of earlier chlamydium isolates.     

<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.     

Allele-specific CAPS marker in a <Emphasis Type="Italic">Ve1</Emphasis> homolog of <Emphasis Type="Italic">Capsicum annuum</Emphasis> for improved selection of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> resistance

Verticillium wilt (Verticillium dahliae) is an economically important disease for many high-value crops. The pathogen is difficult to manage due to the long viability of its resting structures, wide host range, and the inability of fungicides to affect the pathogen once in the plant vascular system. In chile pepper (Capsicum annuum), breeding for resistance to Verticillium wilt is especially challenging due to the limited resistance sources. The dominant Ve locus in tomato (Solanum lycopersicum) contains two closely linked and inversely oriented genes, Ve1 and Ve2. Homologs of Ve1 have been characterized in diverse plant species, and interfamily transfer of Ve1 confers race-specific resistance. Queries in the chile pepper WGS database in NCBI with Ve1 and Ve2 sequences identified one open reading frame (ORF) with homology to the tomato Ve genes. Comparison of the candidate CaVe (Capsicum annuum Ve) gene sequences from susceptible and resistant accessions revealed 16 single nucleotide polymorphisms (SNPs) and several haplotypes. A homozygous haplotype was identified for the susceptible accessions and for resistant accessions. We developed a cleaved amplified polymorphic sequence (CAPS) molecular marker within the coding region of CaVe and screened diverse germplasm that has been previously reported as being resistant to Verticillium wilt in other regions. Based on our phenotyping using the New Mexico V. dahliae isolate, the marker could select resistance accessions with 48% accuracy. This molecular marker is a promising tool towards marker-assisted selection for Verticillium wilt resistance and has the potential to improve the efficacy of chile pepper breeding programs, but does not eliminate the need for a bioassay. Furthermore, this work provides a basis for future research in this important pathosystem.     

<Emphasis Type="Italic">A2144G</Emphasis> Is the Main Mutation in the <Emphasis Type="Italic">23S</Emphasis> rRNA Gene of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> Associated with Clarithromycin Resistance

To detect point mutations A2115C, A2143G/C, and A2144G in the 23S rRNA gene of Helicobacter pylori associated with resistance of the microorganism to clarithromycin, a new powerful way of analysis was used. This method involved the reaction of minisequencing followed by MALDI-TOF mass spectrometry of reaction products. In ten analyzed clarithromycin-resistant clinical isolates of H. pylori obtained in Russia, the resistance was found to be mediated only by mutation A2144G in the 23S rRNA gene.     

Traps and Baits for Luring <Emphasis Type="Italic">Grapholita molesta</Emphasis> (Busck) Adults in Mating Disruption-Treated Apple Orchards

Grapholita molesta (Busck) is one of the main pests in apple crops in Brazil, where it is controlled by mating disruption (MD) with the use of the synthetic sex pheromone. However, sex-pheromone-based monitoring is not effective in MD-treated areas and may result in losses in production. This work has defined a trap model and a bait for luring G. molesta adults in MD apple orchards. The experiments were conducted in commercial apple orchards located in São Joaquim, SC, Brazil. Three trap models—McPhail, Pot, and Ajar—and three baits—grape juice (25%) (GJ), sugarcane molasses (25%) (SM), and a solution containing brown sugar (8.69%) and terpinyl acetate (0.05%) (TAS)—were assessed for luring G. molesta adults in areas subjected to the mating disruption. The assessments were performed weekly by collecting the insects caught in the traps. In addition, time needed to replace traps was also assessed, as well as the selectivity of the trap/bait set. In the laboratory, G. molesta adults were sexed, and the females were dissected to confirm reproductive status. We discuss our results and sugarcane molasses (25%) captured the least number of G. molesta adults regardless of the tested traps. The Ajar/TAS, Pot/TAS, and McPhail/GJ captured the largest number of G. molesta adults. The Ajar/TAS was the most selective and easier to handle. TAS was efficient in catching G. molesta until 14 days after preparation of the solution. Ajar/TAS has potential to be used in the monitoring of G. molesta in apple orchards.     

Mapping of single-copy genes by TSA-FISH in the codling moth, <Emphasis Type="Italic">Cydia pomonella</Emphasis>

Background

We work on the development of transgenic sexing strains in the codling moth, Cydia pomonella (Tortricidae), which would enable to produce male-only progeny for the population control of this pest using sterile insect technique (SIT). To facilitate this research, we have developed a number of cytogenetic and molecular tools, including a physical map of the codling moth Z chromosome using BAC-FISH (fluorescence in situ hybridization with bacterial artificial chromosome probes). However, chromosomal localization of unique, single-copy sequences such as a transgene cassette by conventional FISH remains challenging. In this study, we adapted a FISH protocol with tyramide signal amplification (TSA-FISH) for detection of single-copy genes in Lepidoptera. We tested the protocol with probes prepared from partial sequences of Z-linked genes in the codling moth.

Results

Using a modified TSA-FISH protocol we successfully mapped a partial sequence of the Acetylcholinesterase 1 (Ace-1) gene to the Z chromosome and confirmed thus its Z-linkage. A subsequent combination of BAC-FISH with BAC probes containing anticipated neighbouring Z-linked genes and TSA-FISH with the Ace-1 probe allowed the integration of Ace-1 in the physical map of the codling moth Z chromosome. We also developed a two-colour TSA-FISH protocol which enabled us simultaneous localization of two Z-linked genes, Ace-1 and Notch, to the expected regions of the Z chromosome.

Conclusions

We showed that TSA-FISH represents a reliable technique for physical mapping of genes on chromosomes of moths and butterflies. Our results suggest that this technique can be combined with BAC-FISH and in the future used for physical localization of transgene cassettes on chromosomes of transgenic lines in the codling moth or other lepidopteran species. Furthermore, the developed protocol for two-colour TSA-FISH might become a powerful tool for synteny mapping in non-model organisms.

     

Electrophysiological and behavioral response of the oriental fruit moth <Emphasis Type="Italic">Grapholita</Emphasis><Emphasis Type="Italic">molesta</Emphasis> (Busck, 1916) (Lepidoptera: Tortricidae) to the volatiles of apple fruits at different stages of development

The oriental fruit moth, Grapholita molesta, occurs in Southern Brazil throughout the year, and migrates from peach to apple orchards. Because moths rely on volatile organic compounds (VOCs) during the host-location process, variations in the emission of these compounds during fruit maturation can influence the time of infestation and preference of the moths for a particular genotype. The aim of this work was to identify VOCs emitted by the apples “Eva” and “Gala” at different stages of development and to determine the behavioral and electrophysiological responses of G. molesta to these compounds. For this purpose, VOCs from immature, maturing, and mature fruits of both cultivars were collected and analyzed by gas chromatography and gas chromatography-mass spectroscopy. The response of the antennae of virgin males and females and mated females to volatiles released by the three fruit stages was registered by gas chromatography coupled to an electroantennography. A dual-choice behavioral test for the different combinations of insect groups and fruit stage was also performed. Amongst the volatiles released by mature fruits, twelve compounds elicited a response. The antennae of the oriental fruit moth did respond to isoamyl hexanoate and α-farnesene emitted by “Eva” maturing fruits. In general, virgin females did not respond to volatiles in olfactometer bioassays and mated females were attracted to volatiles released by mature fruits. Our results show that the variation in the emission during the maturation of fruits can influence the orientation of G. molesta.