Species-specific Sequence Characterized Amplified Region Research Articles

Discrimination of lethal Russula subnigricans from wild edible and morphologically similar mushrooms in the genus Russula using SCAR markers

Russula is a diverse genus of mushrooms that is consumed worldwide. Russula subnigricans is a lethal species that causes myotoxicity upon ingestion. It is morphologically similar to wild foraged species, including R. nigricans and R. densifolia. Misidentification of these mushrooms can lead to severe food poisoning. Several PCR-based approaches have been used to overcome this problem. In this study, 22 samples of wild edible Russula species were collected, and five clinical samples of R. subnigricans leftovers were obtained. Based on the sequence data of the internal transcribed spacer, the phylogenetic trees revealed that these 27 samples could be classified into well-established clades of six subgenera. Fifty arbitrary primers were used in high annealing temperature rapid amplified polymorphic DNA (HAT-RAPD) technique to screen polymorphic bands among R. subnigricans, R. nigricans, R. densifolia, and other edible species. Distinctive RAPD fragments were obtained from 11 primers and used in sequence characterized amplified region (SCAR) technique. Three pairs of species-specific SCAR primers were selected to distinguish lethal R. subnigricans, R. nigricans, and R. densifolia with SCAR markers of 213, 420, and 609 bp, respectively. Ten-fold sensitivity test revealed that the SCAR marker of R. subnigricans could be detected at DNA template concentrations as low as 30 pg/μL. This marker could detect the presence of lethal R. subnigricans even at 1% in a cooked sample of mixed mushroom remnants. The developed SCAR markers ensured rapid and accurate diagnosis of suspected myotoxic mushrooms, thereby reducing the risk of severe foodborne illness and death.

The weed Amaranthus palmeri is a host of Meloidogyne incognita in Uruguay

Amaranthus palmeri plants showing symptoms caused by root-knot nematode infection were detected in the municipality of Palmita, Soriano, Uruguay. Based on morphology, esterase phenotypes, and species-specific sequence-characterized amplified region (SCAR) sequences, the causal agent was identified as Meloidogyne incognita. Pathogenicity was confirmed using a modified version of Koch's postulates. To our knowledge, this is the first report of M. incognita infecting the roots of the weed A. palmeri in Uruguay.

Genetic intraspecific diversity of Meloidogyne javanica parasitizing vegetables in southern Iran.

In order to investigate different species of root-knot nematodes associated with vegetable production in southern regions of Iran, 37 populations of the most predominant species, Meloidogyne javanica, were recovered. Morphological and morphometric studies were carried on the characters of females, males, J2s and perineal patterns. Species-specific Sequence Characterized Amplified Region (SCAR) primers confirmed morphological studies, and all these populations produced specific band in 670 bp using Fjav and Rjav primers. Genetic diversity of different populations was studied by Inter Simple Sequence Repeats (ISSR) and Random Amplified Polymorphic DNA (RAPD) markers implementing 10 primers for each approach. Results revealed a relatively low genetic diversity (the percentage of polymorphic bands were 19.08 and 24.60 for ISSR and RAPD, respectively). The analyses of molecular variance indicated that the variation resulted from genotypic variations within region and variances among regions are 81% and 19% for RAPD, and 86% and 14% for ISSR, respectively. On the other hand, FST and Nm values are 0.140 and 1.535 for ISSR while these values are 0.188 and 1.079 for RAPD. So it can be concluded that there is a great deal of gene flow between populations due to the movement of plant material contaminated with nematodes, which results in high mixing between populations. ISSR and RAPD datasets failed to group populations according to their geographic region. There were no pathotypes or other intraspecific biological entities observed in the species.

Detection of interspecific hybrids between creeping bentgrass and colonial bentgrass using DNA marker analysis

AbstractBentgrasses (Agrostis spp.) are cool‐season turfgrasses that have utility on golf courses or other turf applications requiring low mowing heights. Interspecific hybridization of Agrostis species is a strategy that has been proposed to enhance biotic and abiotic stress tolerance in this genus. Non‐transgenic, efficient, and cost‐effective methods for interspecific hybrid detection are needed. In this study two bentgrass species creeping bentgrass (A. stolonifera L.), and colonial bentgrass (A. capillaris L.) were allowed to intercross under field conditions. Progeny plant DNA was extracted and stored using Flinders Technology Associates (FTA) PlantSaver cards followed by primer specific polymerase chain reaction (PCR) and high‐resolution melt analysis. Species identifying PCR primers were optimized for real‐time PCR through fragment resequencing of species‐specific sequence characterized amplified region (SCAR) markers. A total of 2289 potential interspecific hybrid bentgrass progeny were screened with 99.4% of the DNA extractions successful. The percent interspecific hybridization, on an individual plant basis, ranged from 1 to 85% and a total of 524 interspecific‐hybrid bentgrasses were identified representing 23% of plants tested. Interspecific hybridization is common between these bentgrass species and the proposed detection method provides a non‐transgenic, efficient, and cost‐effective method for interspecific hybrid identification.

Occurrence of Meloidogyne javanica as a Pathogen of Cockscomb (Celosia cristata) in Brazil

Cockscomb (Celosia cristata L., family Amaranthaceae) is an ornamental plant species cultivated in pots or used as cut flowers in floral arrangements. In April 2018, cockscomb ‘Comum’ exhibiting many galls on the roots was detected and sampled from several areas (27°21′34″S, 53°23′37″W) of Frederico Westphalen county, Rio Grande do Sul State, Brazil. Identification of Meloidogyne sp. (Nematoda: Meloidogynidae) was carried out by observations of perineal patterns (n = 30) and esterase phenotypes (n = 30), morphological measurements of second-stage juveniles (J2) (n = 50), and amplification and sequencing of the ITS1-5.8S-ITS2 rRNA region (primer set: forward 5′-TTGATTACGTCCCTGCCCTTT-3′ and reverse 5′-TCCTCCGCTAAATGATATG-3′) and the D2 to D3 fragment of the 28S rRNA gene (primer set: forward 5′-ACAAGTACCGTGAGGGAAAGTTG-3′ and reverse 5′-TCGGAAGGAACCAGCTACTA-3′). In addition, cockscomb roots were processed to determine the number of J2 and eggs of Meloidogyne sp. Nematode population density observed in the sample was 2,100 J2 per gram of cockscomb roots. The J2 had the following morphometric characteristics: length (L) = 405.5 ± 61.0 (368.5 to 415.6) µm, a = 29.6 ± 2.5 (26.8 to 33.4), c = 8.00 ± 0.5 (6.0 to 9.6), stylet = 13.5 ± 0.5 (11.3 to 15.9) µm, dorsal esophageal gland orifice (DGO) = 3.20 ± 0.4 (2.9 to 3.9) µm, tail length = 55.5.5 ± 4.0 (54.1 to 59.5) µm, and hyaline tail terminus = 10.3 ± 0.8 (9.0 to 11.5) µm. Female perineal patterns included a low trapezoidal dorsal arch and smooth striae interrupted by a pair of incisions on both sides. These lateral ridges that divide the dorsal and ventral striae are unique to Meloidogyne javanica (Treub, 1885) Chitwood, 1949 (Rammah and Hirschmann 1990). The polymorphisms in the esterase bands observed during electrophoresis revealed the typical phenotype of J3 (Rm = 1.00,1.25, 1.40) M. javanica populations (Carneiro et al. 2000). Sequence homologies were determined by comparing sequences of polymerase chain reaction (PCR) amplicons of D2 to D3 of 28S and ITS1-5.8S-ITS2 rRNA region that were noted to be 516 bp (GenBank MK072827) and 554 bp (MK072828) in length, respectively. The sequences of both markers exhibited 99 and 100% identity, respectively, with sequences being more similar to those of M. javanica isolates from Brazil, Vietnam, and China. The confirmation of nematode species identity was performed by PCR species-specific sequence characterized amplified region (SCAR) using a primer set composed of Fjav (5′-GGTGCGCGATTGAACTGAGC-3′) and Rjav (5′-CAGGCCCTTCAGTGGAACTATAC-3′). The PCR amplification using SCAR produced a specific fragment of expected size (∼670 bp) for M. javanica (Donkers-Venne et al. 2000). In greenhouse tests, cockscomb Comum plants being maintained in pots containing sterilized soil were inoculated with 5,000 eggs and J2 of the original M. javanica population; a noninoculated control was included, and treatments were replicated 10 times. After 60 days, all inoculated plants showed reduced growth compared with the control. Galling symptoms on the roots were similar to those observed in field-grown plants, and the nematode reproduction factor (final population/initial population) was 20.5. The noninoculated plants did not present gall formation in the roots, and their development was not affected either. The morphological and molecular characteristics of reisolated root-knot nematodes were identical to those of M. javanica. These results confirmed the nematode’s pathogenicity to cockscomb. To our knowledge, this is the first report on M. javanica parasitizing cockscomb in Brazil; therefore, our findings are of great importance to Brazilian flower growers to help them establish management measures M. javanica.

First Report of Meloidogyne graminicola (Nematoda: Meloidogynidae) on Barley (Hordeum vulgare) in Brazil

Winter barley (Hordeum vulgare L.) has become a growing addition to cereal crops in Brazil and, until recently, the root-knot nematode (Meloidogyne sp.) has not been reported to infect barley in this country. Meloidogyne graminicola is a major pathogen of rice (Oryza sativa L.) and causes significant yield losses in both upland and flooded fields in Rio Grande do Sul, Brazil (Negretti et al. 2017). In September 2017, many nematode galls were observed on the roots of barley plants (cv. BRS Brau), and samples were taken from areas in Santa Maria county (29°45′S; 53°38′W), Rio Grande do Sul State, Brazil. Meloidogyne sp. was identified using esterase phenotypes (n = 40), perineal patterns (n = 40), morphological measurements of second-stage juveniles (J2) (n = 50), and amplification and sequencing of the ITS1-5.8S-ITS2 rRNA region and the D2–D3 fragment of the 28S ribosomal RNA gene. The barley root systems were also processed to determine the number of J2s and eggs of Meloidogyne sp. The nematode population density observed in the samples was 2,585 eggs plus J2s per gram of barley root. The J2s had the following morphometric characteristics: length (L) = 458.2 ± 38.0 (390.0 to 476.0) µm, a = 26.6 ± 1.5 (24.9 to 29.4), c = 6.15 ± 0.4 (5.1 to 7.6), stylet = 14.2 ± 0.5 (13.4 to 15.0) µm, dorsal esophageal gland orifice (DGO) = 3.9 ± 0.4 (3.4 to 4.6) µm, tail length = 70.5 ± 4.7 (63.1 to 75.3) µm, and hyaline tail terminus = 19.5 ± 1.8 (14.5 to 25.5) µm. The female’s perineal patterns were oval with a low dorsal arch without a lateral line. The cuticular striations were smooth and thick in the dorsal region of the vulva, and the phasmids were close together (13.1 to 17.9 μm). These morphological characteristics matched the original description of M. graminicola (Golden and Birchfield 1968). The polymorphisms of esterase bands seen using electrophoresis revealed the phenotype VS-1 (Rm = 0.71) typical of M. graminicola. The sequence homologies were determined by comparing them with those from GenBank. PCR amplicons of D2–D3 of the 28S and ITS1-5.8S-ITS2 rRNA regions were 531 bp (GenBank MH707259) and 440 bp (MH703577), respectively. The sequences of both markers exhibited 99 and 100% identity, respectively, with sequences corresponding to M. graminicola isolates (D2–D3: MH843665 and KY660544; ITS: MH842692 and KY346887) from Brazil and China. Identification of the nematode species was confirmed by PCR using the species-specific sequence-characterized amplified region (SCAR) with primer sets GRAJ17-1F and GRAJ17-1R. PCR amplification using SCAR produced a specific fragment of the expected size (∼230 bp) in M. graminicola (Mattos et al. 2019). In greenhouse tests, barley plantlets from cv. BRS Brau that were maintained in pots with sterilized soil were inoculated with 5,000 eggs plus J2s of the original population of M. graminicola using 10 replicates; a noninoculated control was included in the test. After 60 days, the growth of all the inoculated plants was reduced compared with that of the control. Galling symptoms on the roots were similar to those in the field, with a nematode reproduction factor (final population/initial population) of 28.5. There were no galls on the roots of noninoculated plants, and plant development was not affected. The morphological and molecular characteristics of the reisolated root-knot nematode were identical to those of M. graminicola. M. graminicola has been reported on barley in India (Vaish et al. 2012). To our knowledge, this is the first report on M. graminicola parasitizing barley in Brazil. This finding is of great importance for Brazilian barley production because this nematode can severely damage barley plants and become a major problem for the cultivation of this crop.

First report of Meloidogyne javanica (Nematoda: Meloidogynidae) infecting Scoparia dulcis, a medicinal plant in Brazil.

Medicinal plants Scoparia dulcis showing symptoms caused by root-knot nematodes were detected in the municipality of Cachoeira do Sul, Rio Grande do Sul State, Brazil. Based on the morphological, esterase phenotypes, and molecular analyses of the mitochondrial DNA region between the cytochome oxidase subunit II and 16S rRNA genes (mtDNA) and species-specific sequence characterized amplified region (SCAR), the causal agent of the observed symptoms was identified as Meloidogyne javanica. Pathogenicity was confirmed by fulfilling a modified version of Koch’s postulates. To our knowledge, this is the first report of M. javanica infecting S. dulcis in Brazil.

Establishment of a PCR Assay for the Detection and Discrimination of Authentic Cordyceps and Adulterant Species in Food and Herbal Medicines.

Accurate detection and differentiation of adulterants in food ingredients and herbal medicines are crucial for the safety and basic quality control of these products. Ophiocordyceps sinensis is described as the only fungal source for the authentic medicinal ingredient used in the herbal medicine “Cordyceps”, and two other fungal species, Cordyceps militaris and Isaria tenuipes, are the authentic fungal sources for food ingredients in Korea. However, substitution of these three species, and adulteration of herbal material and dietary supplements originating from Cordyceps pruinosa or Isaria cicadae, seriously affects the safety and reduces the therapeutic efficacy of these products. Distinguishing between these species based on their morphological features is very difficult, especially in commercially processed products. In this study, we employed DNA barcode-based species-specific sequence characterized amplified region (SCAR) markers to discriminate authentic herbal Cordyceps medicines and Cordyceps-derived dietary supplements from related but inauthentic species. The reliable authentication tool exploited the internal transcribed spacer (ITS) region of a nuclear ribosomal RNA gene (nrDNA). We used comparative nrDNA-ITS sequence analysis of the five fungal species to design two sets of SCAR markers. Furthermore, we used a set of species-specific SCAR markers to establish a real-time polymerase chain reaction (PCR) assay for the detection of species, contamination, and degree of adulteration. We confirmed the discriminability and reproducibility of the SCAR marker analysis and the real-time PCR assay using commercially processed food ingredients and herbal medicines. The developed SCAR markers may be used to efficiently differentiate authentic material from their related adulterants on a species level. The ITS-based SCAR markers and the real-time PCR assay constitute a useful genetic tool for preventing the adulteration of Cordyceps and Cordyceps-related dietary supplements.

Development of Species-Specific SCAR Markers, Based on a SCoT Analysis, to Authenticate Physalis (Solanaceae) Species.

Physalis is an important genus in the Solanaceae family. It includes many species of significant medicinal value, edible value, and ornamental value. However, many Physalis species are easily confused because of their similar morphological traits, which hinder the utilization and protection of Physalis resources. Therefore, it is necessary to create fast, sensitive, and reliable methods for the Physalis species authentication. Intended for that, in this study, species-specific sequence-characterized amplified region (SCAR) markers were developed for accurate identification of the closely related Physalis species P. angulata, P. minima, P. pubescens, and P. alkekengi var. franchetii, based on a simple and novel marker system, start codon targeted (SCoT) marker. A total of 34 selected SCoT primers yielded 289 reliable SCoT loci, of which 265 were polymorphic. Four species-specific SCoT fragments (SCoT3-1404, SCoT3-1589, SCoT5-550, and SCoT36-520) from Physalis species were successfully identified, cloned, and sequenced. Based on these selected specific DNA fragments, four SCAR primers pairs were developed and named ST3KZ, ST3MSJ, ST5SJ, and ST36XSJ. PCR analysis of each of these primer pairs clearly demonstrated a specific amplified band in all samples of the target Physalis species, but no amplification was observed in other Physalis species. Therefore, the species-specific SCAR primer pairs developed in this study could be used as powerful tools that can rapidly, effectively, and reliably identify and differentiate Physalis species.

Short Communication: Rapid and accurate genetic authentication of Penthorum chinense by improved RAPD-derived species-specific SCAR markers

Mei Z, Khan MA, Zhang X, Fu J. 2017. Short Communication: Rapid and accurate genetic authentication of Penthorum chinense by improved RAPD-derived species-specific SCAR markers. Biodiversitas 18: 1243-1249. For the genetic identification and authentication of living organisms, development of Sequence-Characterized Amplified Region (SCAR) markers from Random Amplified Polymorphic DNA (RAPD) fragments is a valuable molecular approach. By using SCAR markers, molecular analysis is simplified to a Polymerase Chain Reaction (PCR) analysis using PCR primers designed from specific sequences of the RAPD amplicons. In this study, RAPD fragments from improved RAPD amplification of a perennial herb Penthorum chinense Pursh from China were cloned into a T-vector, and positive clones were identified by PCR amplification, and sequenced with the Sanger sequencing method for the SCAR marker development. Five SCAR markers were developed that were very specific to P. chinense, and deposited in GenBank (accession numbers: KX671029, KX671030, KX671031, KX671032 and KX671033). BLAST searches of these five nucleotide sequences in the GenBank database showed no identity with markers from other species. This study was developing five specific SCAR markers, has enabled reliable genetic identification of the herbal plant species Penthorum chinense Pursh, useful for authenticating future samples of this important medicinal herb.

Development of molecular markers for authentication of the medicinal plant species Patrinia by random amplified polymorphic DNA (RAPD) analysis and multiplex-PCR

The authentication of components from plant species is vital for the quality control and standardization of herbal medicines. Due to morphological similarities when dried, it is very difficult to distinguish authentic medicinal plants Patrinia villosa and P. scabiosifolia from adulterants of P. rupestris and P. saniculifolia in the herbal medicine Patriniae Radix. To establish a reliable authentication tool distinguishing authentic Patriniae Radix species from adulterants, we used random amplified polymorphic DNA (RAPD) genomic profiling and obtained several species-specific DNA fragments for each of the four Patrinia species. Based on the sequences of these amplicons, we developed sequence characterized amplified region (SCAR) markers as stable molecular authentication tools. SCAR markers to identify the authentic Patriniae Radix species, P. villosa and P. scabiosifolia, were developed. To accurately identify adulterants, we also developed SCAR markers able to identify the inauthentic P. rupestris and P. saniculifolia species. Furthermore, we established multiplex-PCR SCAR assays to simultaneously distinguish all four species using combinations of species-specific SCAR marker primer sets. These efforts resulted in reliable DNA-based molecular markers to identify authentic Patriniae Radix species from their adulterants and a rapid authentication assay for the efficient identification of Patrinia species. These markers will facilitate the standardization of Patriniae Radix preparations and therefore prevent the distribution of adulterants.

Designing a SCAR molecular marker for monitoring Trichoderma cf. harzianum in experimental communities.

Several species of the fungal genus Trichoderma establish biological interactions with various micro- and macro-organisms. Some of these interactions are relevant in ecological terms and in biotechnological applications, such as biocontrol, where Trichoderma could be considered as an invasive species that colonizes a recipient community. The success of this invasion depends on multiple factors, which can be assayed using experimental communities as study models. Therefore, the aim of this work is to develop a species-specific sequence-characterized amplified region (SCAR) marker to monitor the colonization and growth of T. cf. harzianum when it invades experimental communities. For this study, 16 randomly amplified polymorphic DNA (RAPD) primers of 10-mer were used to generate polymorphic patterns, one of which generated a band present only in strains of T. cf. harzianum. This band was cloned, sequenced, and five primers of 20-23 mer were designed. Primer pairs 2F2/2R2 and 2F2/2R3 successfully and specifically amplified fragments of 278 and 448 bp from the T. cf. harzianum BpT10a strain DNA, respectively. Both primer pairs were also tested against the DNA from 14 strains of T. cf. harzianum and several strains of different fungal genera as specificity controls. Only the DNA from the strains of T. cf. harzianum was successfully amplified. Moreover, primer pair 2F2/2R2 was assessed by quantitative real-time polymerase chain reaction (PCR) using fungal DNA mixtures and DNA extracted from fungal experimental communities as templates. T. cf. harzianum was detectable even when as few as 100 copies of the SCAR marker were available or even when its population represented only 0.1% of the whole community.

Molecular identification of lactic acid bacteria in Chinese rice wine using species-specific multiplex PCR

Chinese rice wine (CRW), a national unique and traditional food, has more than 4,000 years of history and is very popular in China. Lactic acid bacteria (LAB) play important roles in the fermentation of CRW because of its strong lactic acid production and high acid tolerance. It would be of practical interest to isolate the LAB species from CRW and to develop a simple and effective method for its accurate identification. In this study, 11 CRW-related LAB strains, frequently occurring in the fermentation of CRW, were isolated or collected and analyzed with the nested specifically amplified polymorphic DNA (nSAPD)-PCR technique. Eleven species-specific sequence characterized amplified region (SCAR) markers were generated from 11 polymorphic nSAPD bands. Three multiplex PCR assays were further established for the joint use of the 11 species-specific SCAR markers. Thus, a simple rapid molecular method was developed based on the application of nSAPD, SCAR, and multiplex PCR techniques, which allowed a rapid and simultaneous detection of 11 CRW-related LAB species. The simple rapid PCR-based molecular method can be used as a useful tool for the identification of LAB species in the traditionally fermented foods, especially in the rapid developing CRW industry in China.

Identification and authentication of Rosa species through development of species-specific SCAR marker(s).

Roses (Rosa indica) belong to one of the most crucial groups of plants in the floriculture industry. Rosa species have special fragrances of interest to the perfume and pharmaceutical industries. The genetic diversity of plants based on morphological characteristics is difficult to measure under natural conditions due to the influence of environmental factors, which is why a reliable fingerprinting method was developed to overcome this problem. The development of molecular markers will enable the identification of Rosa species. In the present study, randomly amplified polymorphic DNA (RAPD) analysis was done on four Rosa species, Rosa gruss-an-teplitz (Surkha), Rosa bourboniana, Rosa centifolia, and Rosa damascena. A polymorphic RAPD fragment of 391 bp was detected in R. bourboniana, which was cloned, purified, sequenced, and used to design a pair of species-specific sequence-characterized amplified region (SCAR) primers (forward and reverse). These SCAR primers were used to amplify the specific regions of the rose genome. These PCR amplifications with specific primers are less sensitive to reaction conditions, and due to their high reproducibility, these species-specific SCAR primers can be used for marker-assisted selection and identification of Rosa species.

Molecular characterization of Eimeria spp. from chicken by Polymerase Chain Reaction based on species-specific SCAR markers

Coccidiosis in chickens is one of the major problems of poultry industry that is caused by protozoan parasites of genus Eimeria . Present study was conducted to characterize the Eimeria species infecting poultry in different regions of Chittagong district of Bangladesh. Seven species of Eimeria including E. tenella, E. necatrix, E. acervulina, E. maxima, E. brunetti, E. mitis, and E. praecox have been characterized using conventional PCR analysis through amplifying unique single copy sequences derived from sequence characterized amplified region (SCAR) markers. The present study is the first of its type to use molecular tools to identify Eimeria spp. infecting chicken in Bangladesh. The study indicates that the modern molecular technique involving PCR to detect the Eimeria species are more reliable and accurate than the traditional morphology-based technique.

Molecular identification of root-knot nematode Meloidogyne incognita from kiwi fruit orchards in Ordu province, Turkey

Root-knot nematodes (Meloidogyne spp.) are one of the major groups of nematodes identified on kiwi fruit (Actinidia deliciosa Chev.). This investigation was conducted to identify root-knot nematodes encountered the in kiwi fruit orchards in the Ordu province of Turkey by means of Polymerase Chain Reaction (PCR). For this purpose, infected plant root samples were collected from 17 kiwi fruit orchards located in Center, Persembe, Unye, Gulyali, Ikizce and Ulubey districtsof the province during the years 2011-2012.Females directly obtained from the infected roots of orchard populations were used for the molecular identifications. It was carried out with an identification key using ribosomal DNA (rDNA) region, Intergenic Spacer (IGS-2) region between 5S-18S genes, and the speciesspecific Sequence Characterized Amplified Region (SCAR) markers. All samples from the districts of Ordu province were identified as Meloidogyne incognita by the exact size of 502 bp amplicons by SEC1F/SEC1R scar primers. This study was the first detailed investigation both in the province and country on kiwi fruit

First Report of the Giant Stem Nematode, Ditylenchus gigas, from Broad Bean in Iran.

The giant stem nematode, Ditylenchus gigas (Nematoda: Tylenchida) has been recorded from several European and African countries mainly bordering the Mediterranean Sea (2). This nematode causes considerable yield loss of broad bean, Vicia faba, and it may induce more severe damage than the typical faba bean race of D. dipsaci. Spread of infestation through seed limits export of broad bean and has made these nematodes quarantine pests in many countries (2). Broad bean is cultivated in the north, west, southwest, and central parts of Iran. Although D. dipsaci has been reported from different crops and regions in Iran, there is no record of broad bean infection by this nematode. A survey of broad bean fields was conducted in the north and west provinces in a continuation of a study on different populations of D. dipsaci in Iran in May to July of 2007 and 2008 and resampling from some farms in June 2012. The sampling was performed at flowering stage and after. The aboveground plant samples were collected and cut into pieces of 2 to 3 cm, then incubated for 5 to 6 h in Whitehead trays. Morphological and molecular analysis of isolated nematodes from Kermanshah and Lorestan provinces revealed the presence of D. gigas in the samples. Of the 23 plant samples of cv. Barekat collected from Mazandaran and Golestan provinces in the north, 47.8% were infected with stem nematode, mostly with high population density of over 20,000 nematodes per 5 plant stems. The percentage of infected samples of broad bean cv. Shakhbozy collected in Lorestan and Kermanshah provinces in the west was 76.5%. The symptoms of infection were observed as necrotic lesions on the stem surface and reduction of internode distances in severe infection. The giant stem nematode population from Kermanshah showed the following characters: females (n = 20), L = 1,650 ± 140 (1,270 to 1,875) μm; b' = 8.6 ± 0.6 (7.7 to 10.0), c = 19.0 ± 1.3 (19.2 to 21.2), c' = 4.7 ± 0.5 (1.1 to 5.3), stylet = 11.6 ± 0.5 (11 to 12) μm; post vulval sac = 96 ± 16 (58 to 140) μm; vulval-anus distance = 217.0 ± 21.0 (178 to 272) μm, tail = 86.4 ± 9.4 (66 to 102) μm; males (n = 10), L = 1,495 ± 148 (1,236 to 1,636) μm; b' = 7.7 ± 0.3 (7.3 to 8.1), c = 17.3 ± 0.7 (16.3 to 18.6), stylet = 11.3 ± 0.5 (11 to 12) μm, tail = 86.5 ± 8.5 (71 to 95) μm, spicules = 24.8 ± 1.7 (23 to 28) μm. The morphological and morpohometric features were generally in agreement with those published for D. gigas (2). The morphological identification of D. gigas from Iran was supported by the analyses of the ITS rRNA and the D2-D3 expansion segments of the 28S rRNA gene sequences. The rRNA gene of D. gigas from broad bean and D. dipsaci from garlic were amplified and sequenced using two primer sets: (i) the TW81 and AB28 for the ITS-rRNA and (ii) D2A and D3B for partly 28S rRNA gene, as described by (2). New sequences were deposited in the GenBank under accession numbers KC310732 through KC310735. The Iranian D. gigas sequences showed 100% similarity with those of the Italian D. gigas isolates (ITS rRNA: HQ219231, HQ219232; D2-D3 of 28S rRNA: HQ219217 and HQ219216). The identification was further supported by PCR with species specific SCAR (sequence characterized amplified region) primers for this species (1). The specimens from broad bean generated a specific fragment ∼200 bp for D. gigas, whereas the samples with D. dipsaci from garlic and alfalfa produced one fragment ∼250 bp specific for this species. To our knowledge, this is the first report of D. gigas from broad bean in Iran.

The development of species-specific SCAR markers for delimitation of Calypogeia species

The genus Calypogeia belongs to one of the most difficult groups of leafy liverworts, with about 90 species. Nine species of the genus are currently known from Europe: C. azurea, C. integristipula, C. neesiana, C. suecica, C. muelleriana, C. sphagnicola, C. fissa, C. arguta, and C. azorica. Recently, another species, morphologically resembling C. muelleriana but genetically distinct from it, was detected using isozyme markers. In the present study, C. muelleriana and C. sp. nov. were further characterized by 13 and 14 specific inter-simple sequence repeat band patterns, respectively. Nei’s genetic distance between the two species was 0·61. Three species-specific bands were converted into sequence characterized amplified region (SCAR) markers. The first SCAR primer pair amplified a product of 360 and 600 bp in C. muelleriana and C. sp. nov., respectively. The two other SCAR markers were diagnostic for other European Calypogeia species.

Molecular Fingerprinting and Population Dynamics of Brevipalpus Mites on Texas Citrus

Abstract Three species of Brevipalpus mites are known potential vectors of the citrus leprosis virus (family Rhabdoviridae, genus Cyto- and Nucleorhabdovirus, CiLV). Species identification of these mites is difficult because of their small size and morphological similarities. The objective of this study was to develop an accurate and rapid molecular fingerprinting method for identifying B. phoenicis and B. californicus on Texas citrus. Iso-colonies of the two Brevipalpus species were cultured on immature citrus fruit and identified using a dichotomous key. Whole genome amplification was used to produce DNA template from single mites to identify the two species by amplified fragment length polymorphism and sequence-characterized amplified region (SCAR). Population dynamics of Brevipalpus were monitored in four citrus orchards during the 2007 growing season. Subsamples of mites were identified morphologically or by species-specific SCAR markers. Molecular fingerprinting was very efficient in identifying the two species of mites in subsamples, even in cases where the morphological identification did not discriminate between species. Brevipalpus spp. density per fruit was significantly affected by the host plant species, with sweet orange fruit hosting significantly higher density than grapefruit, Citrus x paradisi Macfad. The percentage of fruit infested with Brevipalpus mites varied significantly with time but not with host plant and the host plant by sampling date interaction. Comparison of molecular fingerprinting and morphological identification provided a high percentage of match >85% between the two methods, suggesting that molecular fingerprinting could facilitate studies on population dynamics of Brevipalpus in citrus.

Development of SCAR Markers for Species Identification of the Genus Nepenthes (Nepenthaceae)

Nepenthes species in Thailand, namely N. mirabilis Druce, N. gracilis Korth., N. smilesii Hemsl., N. ampullaria Jack and N. kampotiana Lecomte, were collected for development of Sequence Characterized Amplified Region (SCAR) marker, a genotype identification tool. Forty Random Amplified Polymorphic DNA (RAPD) primers were screened and three successful primers produced different banding patterns including five candidate species-specific markers. The candidate markers were cloned and sequenced. The marker sequences are 602, 379, 420, 473 and 1017 bp for N. mirabilis, N. gracilis, N. smilesii, N. ampullaria and N. kampotiana, respectively. Then the sequences were used to design primers for development of a species-specific band being a SCAR marker, including Mir 1, Mir 2 and Mir 3 for N. mirabilis; Gra 1 and Gra 2 for N. gracilis; Smi 1, Smi 2 and Smi 3 for N. smilesii; Amp 1 and Amp 2 for N. ampullaria and Kam 1 and Kam 2 of N. kampotiana. The primers were evaluated with each other Nepenthes species. Finally, species-specific SCAR markers were successfully developed for N. gracilis, N. ampullaria and N. kampotiana. Application of these markers is feasible for identification of Nepenthes species in Thailand.