Detection of symbionts and virus in the whitefly <Emphasis Type="Italic">Bemisia tabaci</Emphasis> (Hemiptera: Aleyrodidae), vector of the <Emphasis Type="Italic">Mungbean yellow mosaic India virus</Emphasis> in Central India

Legume crops in Central India, the main soybean production area of the country, may suffer from yellow mosaic disease caused by the Mungbean yellow mosaic India virus (MYMIV). MYMIV is transmitted by the sweet potato whitefly, Bemisia tabaci (Gennadius), which is a species complex composed of various genetic groups. This vector species harbors different endosymbionts among regional strains and among individuals. To elucidate fundamental aspects of this virus vector in the state of Madhya Pradesh, the infection status of the symbionts and the virus in whiteflies was studied. A polymerase chain reaction (PCR) survey of the whiteflies collected in Madhya Pradesh found four secondary endosymbionts, Arsenophonus, Hemipteriphilus, Wolbachia, and Cardinium, in addition to the primary endosymbiont Portiera. Arsenophonus and Hemipteriphilus were highly infected but the infection rates of Wolbachia and Cardinium were low. MYMIV was detected in whitefly populations collected from various host plants in Madhya Pradesh. The whitefly populations belonged to the Asia I and II genetic groups; several different Asia II populations were also distributed. Specific relations were not observed among symbiont infection status, virus infection, and the whitefly genetic groups in the populations of Madhya Pradesh, though Cardinium was highly detected in the Asia II-1 group. New primers, which can be used for PCR template validation and for discriminating two phylogenetically close endosymbionts, were designed.     

Non-destructive direct polymerase chain reaction (direct PCR) greatly facilitates molecular identification of spider mites (Acari: Tetranychidae)

Spider mites (Acari: Tetranychidae) include serious agricultural pests and some species have spread globally as invasive species.. For this reason, rapid and simple identification of spider mite species is necessary for agricultural field and plant quarantine inspection. DNA sequence-based molecular techniques can rapidly identify spider mites. However, extracting DNA from minute invertebrates is difficult, expensive and time consuming. Here, we describe a polymerase chain reaction (PCR) technique in which the whole body of a spider mite adult is non-destructively soaked in PCR solution. The mite is then removed intact and can be used as a voucher specimen, leaving the PCR solution as the template and avoiding the need for a DNA extraction kit. For this study, we used six common spider mite species from four genera [Tetranychus urticae Koch (red form), Tetranychus kanzawai Kishida, Tetranychus parakanzawai Ehara, Oligonychus gotohi Ehara, Eotetranychus smithi Pritchard & Baker and Panonychus citri (McGregor)]. A portion of the mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified with a universal primer pair from all individuals examined and sequenced. This method shortened the time for molecular identification from 9 to 5 h and eliminated the cost of commercial kits for DNA extraction [ca. 600 yen (~5.3 USD) per sample].     

Polymorphism of the mitochondrial DNA control region in eight sturgeon species and development of a system for DNA-based species identification

Intraspecific and interspecific nucleotide sequence variations of the mtDNA control region (D-loop) were studied with mtDNAs isolated from tissue specimens of more than 1400 sturgeons of nine species: Russian sturgeon Acipenser gueldenstaedtii, Persian sturgeon A. persicus, Siberian sturgeon A. baerii, Amur sturgeon A. schrenkii, Fringebarbel sturgeon A. nudiventris, sterlet A. ruthenus, stellate sturgeon A. stellatus, beluga Huso huso, and kaluga H. dauricus. The results were used to analyze the interspecific variation of the mtDNA control region in the given set of species and to develop a test system of ten species-specific primers, which allowed species identification from noninvasive tissue samples, spawn, and food products of eight species. The system proved suitable for multiplex PCR. A method was developed for the first time to reliably differentiate the A. baerii mitotype and the baerii-like mitotype of A. gueldenstaedtii. It was found that, although genetically separate, A. gueldenstaedtii and A. persicus are relatively young species and have common mitochondrial haplotypes, precluding their identification via mtDNA analysis alone. To develop a system for species identification of A. gueldenstaedtii and A. persicus, it is necessary to study the polymorphism of nuclear markers.     

Rapid and simultaneous detection of <Emphasis Type="Italic">Salmonella</Emphasis> spp., <Emphasis Type="Italic">Escherichia coli</Emphasis> O157, and <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> by magnetic capture hybridization and multiplex real-time PCR

The application of rapid, specific, and sensitive methods for pathogen detection and quantification is very advantageous in diagnosis of human pathogens in several applications, including food analysis. The aim of this study was the evaluation of a method for the multiplexed detection and quantification of three significant foodborne pathogenic species (Escherichia coli O157, Salmonella spp., and Listeria monocytogenes). The assay combines specific DNA extraction by multiplex magnetic capture hybridization (mMCH) with multiplex real-time PCR. The amplification assay showed linearity in the range 10⁶–10 genomic units (GU)/PCR for each co-amplified species. The sensitivity corresponded to 1 GU/PCR for E. coli O157 and L. monocytogenes, and 10 GU/PCR for Salmonella spp. The immobilization process and the hybrid capture of the MCH showed good efficiency and reproducibility for all targets, allowing the combination in equal amounts of the different nanoparticle types in mMCH. MCH and mMCH efficiencies were similar. The detection limit of the method was 10 CFU in samples with individual pathogens and 10² CFU in samples with combination of the three pathogens in unequal amounts (amount’s differences of 2 or 3 log). In conclusion, this multiplex molecular platform can be applied to determine the presence of target species in food samples after culture enrichment. In this way, this method could be a time-saving and sensitive tool to be used in routine diagnosis.     

Species composition and arthropod pest feeding of phytoseiid mites in a Japanese pear greenhouse

A population survey of spider mites and phytoseiid mites was conducted on Japanese pear leaves in a greenhouse. For the survey, the method to estimate phytoseiid mite species composition using quantitative sequencing was modified to be applicable for phytoseiid mite species inhabiting in the greenhouse. Results show the dominant appearance of Neoseiulus californicus (McGregor), Neoseiulus womersleyi (Schicha), and Neoseiulus makuwa (Ehara) from the end of June to late September and their contribution in spider mite control. PCR-based method to detect the ribosomal internal transcribed spacer (ITS) sequences of spider mites from phytoseiid mites was developed. The method shows sensitivity to detect the ITS sequences of Tetranychus urticae Koch from single N. californicus adult at 168 h after ingestion of the spider mite. PCR-based method to detect the cytochrome c oxidase subunit I sequences of several arthropod pests belonging to Hemiptera, Thysanoptera, and Acari from phytoseiid mites was also developed. Results show that phytoseiid mites prey on Eriophyes chibaensis (Kadono) and Aphis gossypii (Glover), in addition to spider mites.     

The high diversity of <Emphasis Type="Italic">Lotus corniculatus</Emphasis> endosymbionts in soils of northwest Spain

The diversity of rhizobia that establish symbiosis with Lotus corniculatus has scarcely been studied. Several species of Mesorhizobium are endosymbionts of this legume, including Mesorhizobium loti, the type species of this genus. We analysed the genetic diversity of strains nodulating Lotus corniculatus in Northwest Spain and ten different RAPD patterns were identified among 22 isolates. The phylogenetic analysis of the 16S rRNA gene showed that the isolated strains belong to four divergent phylogenetic groups within the genus Mesorhizobium. These phylogenetic groups are widely distributed worldwide and the strains nodulate L. corniculatus in several countries of Europe, America and Asia. Three of the groups include the currently described Mesorhizobium species M. loti, M. erdmanii and M. jarvisii which are L. corniculatus endosymbionts. An analysis of the recA and atpD genes showed that our strains belong to several clusters, one of them very closely related to M. jarvisii and the remanining ones phylogenetically divergent from all currently described Mesorhizobium species. Some of these clusters include L. corniculatus nodulating strains isolated in Europe, America and Asia, although the recA and atpD genes have been sequenced in only a few L. corniculatus endosymbionts. The results of this study revealed great phylogenetic diversity of strains nodulating L. corniculatus, allowing us to predict that even more diversity will be discovered as further ecosystems are investigated.     

mtDNA-based identification of two widespread roach species (<Emphasis Type="Italic">Rutilus</Emphasis>, Cyprinidae) characterized by sympatric zone

Test systems using multiplex PCR and restriction analysis have been designed for the simple and rapid identification of two widespread species of roach, Rutilus rutilus and R. lacustris, characterized by the sympatric zone. This method is based on the variation of nucleotide sequences of the mitochondrial markers (cytochrome oxidase I subunit and cytochrome b); it allows identifying the species omitting the DNA sequencing.     

Multiplex real-time PCR assay for detection of <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 and screening for non-O157 Shiga toxin-producing <Emphasis Type="Italic">E. coli</Emphasis>

Background

Shiga toxin-producing Escherichia coli (STEC), including E. coli O157:H7, are responsible for numerous foodborne outbreaks annually worldwide. E. coli O157:H7, as well as pathogenic non-O157:H7 STECs, can cause life-threating complications, such as bloody diarrhea (hemolytic colitis) and hemolytic-uremic syndrome (HUS). Previously, we developed a real-time PCR assay to detect E. coli O157:H7 in foods by targeting a unique putative fimbriae protein Z3276. To extend the detection spectrum of the assay, we report a multiplex real-time PCR assay to specifically detect E. coli O157:H7 and screen for non-O157 STEC by targeting Z3276 and Shiga toxin genes (stx1 and stx2). Also, an internal amplification control (IAC) was incorporated into the assay to monitor the amplification efficiency.

Methods

The multiplex real-time PCR assay was developed using the Life Technology ABI 7500 System platform and the standard chemistry. The optimal amplification mixture of the assay contains 12.5 μl of 2 × Universal Master Mix (Life Technology), 200 nM forward and reverse primers, appropriate concentrations of four probes [(Z3276 (80 nM), stx1 (80 nM), stx2 (20 nM), and IAC (40 nM)], 2 μl of template DNA, and water (to make up to 25 μl in total volume). The amplification conditions of the assay were set as follows: activation of TaqMan at 95 °C for 10 min, then 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 60 s.

Results

The multiplex assay was optimized for amplification conditions. The limit of detection (LOD) for the multiplex assay was determined to be 200 fg of bacterial DNA, which is equivalent to 40 CFU per reaction which is similar to the LOD generated in single targeted PCRs. Inclusivity and exclusivity determinants were performed with 196 bacterial strains. All E. coli O157:H7 (n = 135) were detected as positive and all STEC strains (n = 33) were positive for stx1, or stx2, or stx1 and stx2 (Table 1). No cross reactivity was detected with Salmonella enterica, Shigella strains, or any other pathogenic strains tested.

Conclusions

A multiplex real-time PCR assay that can rapidly and simultaneously detect E. coli O157:H7 and screen for non-O157 STEC strains has been developed and assessed for efficacy. The inclusivity and exclusivity tests demonstrated high sensitivity and specificity of the multiplex real-time PCR assay. In addition, this multiplex assay was shown to be effective for the detection of E. coli O157:H7 from two common food matrices, beef and spinach, and may be applied for detection of E. coli O157:H7 and screening for non-O157 STEC strains from other food matrices as well.

     

Molecular marker analysis of <Emphasis Type="Italic">Cynanchum wilfordii</Emphasis> and <Emphasis Type="Italic">C. auriculatum</Emphasis> using the simple ARMS-PCR method with mismatched primers

SNPs can be used to discriminate allele-specific genotypes based on the 3′-terminal nucleotide of a primer corresponding to a specific SNP site. However, reliable discrimination between alleles in closely related species is not sufficient for molecular genetic identification. To overcome this problem, the present study evaluated the amplification refractory mutation system (ARMS)-PCR analysis method to discriminate closely related species, which utilizes specific primers that have a base pair change within three bases closest to the SNP site. The aim of this study was to develop a simple and precise DNA-based method for molecular authentication of C. wilfordii when compared with the highly similar species C. auriculatum. Molecular authentication of C. wilfordii and C. auriculatum was achieved using specific modified primers for DNA sequences of chloroplast TrnL-F and nuclear internal transcribed spacer (ITS) regions. A species-specific SNP was detected for each of the two species, and mismatched ARMS-PCR was conducted to specifically identify the SNP site. Plant samples collected from different locations were used to validate the selectively modified SNP markers, and the established method was determined to be effective. Therefore, this study provides a rapid and reliable method for specific identification of the medicinal plants, C. wilfordii and C. auriculatum.     

Molecular diagnostics of the R81T mutation on the D-loop region of the <Emphasis Type="Italic">β</Emphasis>1 subunit of the nicotinic acetylcholine receptor gene conferring resistance to neonicotinoids in the cotton aphid, <Emphasis Type="Italic">Aphis gossypii</Emphasis> (Hemiptera: Aphididae)

Aphis gossypii Glover is a cosmopolitan and polyphagous aphid species. When neonicotinoid-resistant clones of A. gossypii were recently found in Japan, it was subsequently discovered that a point mutation (R81T) in the D-loop region of the β1 subunit of the nicotinic acetylcholine receptor gene had conferred this resistance. To quickly identify this neonicotinoid-resistant allele, we developed a molecular diagnostics test using a multiplex polymerase chain reaction method that targeted the R81T mutation.     

Rearing system for the predatory phytoseiid <Emphasis Type="Italic">Euseius concordis</Emphasis> (Acari: Phytoseiidae)

Several species of predatory mites, especially those of the family Phytoseiidae, are potentially useful for the control of pest mites and insects. Among the phytoseiids, Euseius concordis (Chant), a species commonly found in South America, has been studied for possible use as biological control agent of pest mites. Given that Euseius species are known to feed on plant leaves and on pollen, the objective of this study was to determine plant species and pollen sources suitable to establish a pilot method of production of that species, based on a set of comparisons in the laboratory. Out of four plant species evaluated in the study, higher survivorship of E. concordis females in the absence of supplementary food was observed on leaflets of Canavalia ensiformis L. Out of two pollen types, higher survivorship was obtained on pollen of Typha domingensis L. In a subsequent step, the population of E. concordis increased 19.3× within 21 days when that pollen of T. domingensis was offered to the predator on plantlets of C. ensiformis, at 25.0 ± 1 °C, 70.0 ± 10% RH and 12 h photophase. Future studies may confirm the economic viability of this setup for the mass production of E. concordis.     

Larval aquatic and terrestrial mites infesting parthenogenetic <Emphasis Type="Italic">Ischnura hastata</Emphasis> (Odonata: Coenagrionidae) from the Azores islands

We report here the prevalence of parasitism by water mites (Arrenurus sp.) and terrestrial mites (Leptus killingtoni) on parthenogenetic Ischnura hastata (Odonata: Coenagrionidae) from the Azores islands. Leptus killingtoni was only found on the island of Pico, and the prevalence of infestation was highly variable among the different ponds studied, ranging from 0 to 41%. Leptus killingtoni was observed on three of the four odonate species from the archipelago: I. hastata, I. pumilio, and Sympetrum fonscolombii, all of them new hosts for this species. Aquatic mites have been found parasitizing I. hastata females on the island of São Miguel. The prevalence of mite parasitism by Arrenurus sp. on I. hastata was very low, ranging from 12% (2003) to 1% (2008), and in most of the studied ponds, no mites were found attached to females. Although I. hastata coexists with a sexual congener species in the Azores (I. pumilio), they are syntopic in only a small fraction of ponds. Therefore, a comparison between I. hastata and I. pumilio was insufficient to test the predictions of the Red Queen Hypothesis, and further research on parasitism rates in both species needs to be done. In any case, the low prevalence of mite parasitism found in the Azores, coupled with the fact that most of the populations in the archipelago are almost free from competitors and predators, could explain the persistence of these I. hastata parthenogenetic populations, despite their low levels of genetic variation.     

Gamasid mites of small mammals in the semi-desert territories of Saratov Trans-Volga region

New data on the species composition and comparative analysis of gamasid mites occurring on small mammals in the semi-desert territories of Saratov Trans-Volga region are reported. In all, 18 species of gamasid mites (9 genera, 5 families) were found, including 9 species which had not been previously recorded in the region: Hypoaspis (Stratiolaelaps) miles Berlese, 1882, H. (Geolaelaps) heselhausi Oudemans, 1912, H. (G.) lubrica Oudemans et Voigts, 1904, Laelaps multispinosus Banks, 1909, L. jettmari Vitzthum, 1930, Cyrtolaelaps mucronatus G. et. R. Canestrini, 1881, Haemogamasus citelli Bregetova et Nelzina, 1952, Hirstionyssus eusoricis Bregetova, 1956, and Hi. ellobii Bregetova, 1956; 5 of these species are new to Saratov Province.     

Characterization of the genome of <Emphasis Type="Italic">Saccharomyces</Emphasis> yeasts from red berry wines

Using restriction analysis of noncoding rDNA regions, multiplex PCR, and molecular karyotyping, we have examined Saccharomyces strains isolated from red berry wine materials in Russia, Belarus, and Ukraine. According to the molecular analysis, all strains belong to the species S. cerevisiae. A correlation was revealed between microsatellite fingerprints of the strains and the source of their isolation. The strains isolated from juices and from the surface of different berries showed distinct PCR profiles. The genome compositions of interspecific Saccharomyces hybrids of natural and laboratory origin were studied.     

DNA-Based Identification of <Emphasis Type="Italic">Valeriana officinalis</Emphasis> s.l.: a Multiplexed Amplification Refractory Mutation System (MARMS) and High Resolution Melting Curve Analysis (HRMA)

The wide distribution of Valeriana officinalis as a herbal remedy as well as the considerably higher concentration of putative mutagenic valepotriate metabolites in other drug-delivering valerian species like Valeriana procera Kunth and Valeriana jatamansi Jones ex Roxb. illustrate the necessity of secure authentication of roots of Valeriana officinalis s.l., especially as the morphologically similar roots of the acutely toxic Veratrum album can be mistaken for those of Valeriana officinalis. We developed two DNA-based systems, a multiplex amplification refractory mutation system (MARMS), and a high-resolution melting curve analysis (HRMA) assay, both based on a sequence mutation within the atpB-rbcL region. With both methods, identification of Valeriana officinalis s.l. was possible. With the HRMA, the characteristic melting curve of 33 samples of Valeriana officinalis s.l. and of two commercial samples of Valerianae radix was distinct from the melting curves of all other Valeriana species (60 accessions), and from the closely related genera Centranthus and Valerianella. Since adulteration of Valeriana with toxic Veratrum species was reported previously, Veratrum primers were included in a multiplex PCR-HRM analysis. This system allowed the detection of a Veratrum admixture down to the level of 0.01 %. Although the advantages, in terms of sensitivity, specificity and practicality of the HRM for analysis of degraded plant material were superior to the MARMS assay, both methods are suitable for routine analysis. The results demonstrated the general ability of HRMA to detect specific (toxic) adulterations in drugs in a semiquantitative way.     

Mesostigmatic mites associated with birds and mammals in Iran. A review

There are diverse relationships between mites and birds or mammals. These mites may play an important role in epizootics and in the perpetuation of some significant diseases. The purpose of this study is to revise the current knowledge of mesostigmatic mites occurring on birds, including their nests and mammals and their substrates, in Iran and to compare the results with other regions of the Palearctic ecozone. This study presents a revised list of 38 species of mesostigmatic mite occuring on birds (17 species) and mammals (24 species) or in their nests/substrates in Iran. Dermanyssus gallinae, Ornithonyssus sylviarum and Parasitus hyalinus were found on both birds and mammals. The species composition of mites reported in Iran was compared with other regions of the Palearctic ecozone. Parasites, specifically those from genera Dermanyssus, Ornithonyssus and Liponyssoides, may be hazardous to human health. Species from these genera were predominant among the reported mites.     

Identification of <Emphasis Type="Italic">Bacillus</Emphasis> Probiotics Isolated from Soil Rhizosphere Using 16S rRNA, <Emphasis Type="Italic">recA</Emphasis>, <Emphasis Type="Italic">rpoB</Emphasis> Gene Sequencing and RAPD-PCR

Some Bacillus species, especially Bacillus subtilis and Bacillus pumilus groups, have highly similar 16S rRNA gene sequences, which are hard to identify based on 16S rDNA sequence analysis. To conquer this drawback, rpoB, recA sequence analysis along with randomly amplified polymorphic (RAPD) fingerprinting was examined as an alternative method for differentiating Bacillus species. The 16S rRNA, rpoB and recA genes were amplified via a polymerase chain reaction using their specific primers. The resulted PCR amplicons were sequenced, and phylogenetic analysis was employed by MEGA 6 software. Identification based on 16S rRNA gene sequencing was underpinned by rpoB and recA gene sequencing as well as RAPD-PCR technique. Subsequently, concatenation and phylogenetic analysis showed that extent of diversity and similarity were better obtained by rpoB and recA primers, which are also reinforced by RAPD-PCR methods. However, in one case, these approaches failed to identify one isolate, which in combination with the phenotypical method offsets this issue. Overall, RAPD fingerprinting, rpoB and recA along with concatenated genes sequence analysis discriminated closely related Bacillus species, which highlights the significance of the multigenic method in more precisely distinguishing Bacillus strains. This research emphasizes the benefit of RAPD fingerprinting, rpoB and recA sequence analysis superior to 16S rRNA gene sequence analysis for suitable and effective identification of Bacillus species as recommended for probiotic products.     

A novel real-time TaqMan™ PCR assay for simultaneous detection of Neotropical fox species using noninvasive samples based on <Emphasis Type="Italic">cytochrome c oxidase subunit II</Emphasis>

Strategies to evaluate and monitor elusive mammal species require the development of genetic techniques and their application to unambiguous biological material for ecological and genetic studies. In order to assess cytochrome c oxidase subunit II gene inter- and intraspecific variations, we compared sequences from different Neotropical canids and domestic dogs. We developed a primer pair to amplify a 154-bp fragment of this gene and a species-specific multiplex TaqMan? assay for accurate identification of two native fox species occurring in sympatry in South America, the crab-eating fox (Cerdocyon thous) and the pampas fox (Lycalopex gymnocercus). The assays can also distinguish domestic dogs (Canis lupus familiaris) from both wild foxes. The use of different fluorescent reporter dyes for species identification in a multiplex probe PCR-RT assay reduces labor and costs. The methodology presented in this study demonstrates an efficient approach to enable high-performance analysis and represents a reliable cost-effective tool for molecular ecology research to monitor the wild canid populations by noninvasive genetic sampling. This standardized assay will allow large-scale high-throughput analyses in a routine and reliable way.     

Multiplex Genotyping of Allelic Variants of Genes Involved in Metabolizing Antileukemic Drugs

A biochip, primer set, and genotyping protocol were developed to simultaneously address 16 single nucleotide polymorphisms in antileukemic drug metabolism genes, including TPMT, ITPA, MTHFR, SLCO1B1, SLC19A1, NR3C1, GRIA1, ASNS, MTRR, and ABCB1. The genotyping procedure included a one-round multiplex polymerase chain reaction (PCR) with simultaneous incorporation of a fluorescent label into the PCR product and subsequent hybridization on a biochip with immobilized probes. The method was used to test 65 DNA samples of leukemia patients. Fluorescence signal intensity ratios in pairs of wildtype and respective mutant sequence probes were analyzed for all polymorphic markers and demonstrated high accuracy of genotyping. The reliability of genotype determination using the biochip was confirmed by direct Sanger sequencing.