16S-23S rDNA Research Articles

Mikrocytos mytilicoli n.sp. (Cercozoa, Mikrocytida, Mikrocytiidae) infecting the copepod Mytilicola intestinalis (Arthropoda, Cyclopoida, Mytilicolidae), a symbiont of Mytilus galloprovincialis in Galicia (NW Spain)

During a histopathological survey of Mytilus galloprovincialis in Galicia (NW Spain), microcells were observed infecting several organs of the symbiont copepod Mytilicola intestinalis. Positive results of PCR assay with specific primers for genus Mikrocytos and a clear signal of in situ hybridization with MACKINI-1 digoxigenin- labelled DNA probe (DIG-ISH) indicated a protozoan parasite of Mikrocytos genus. The ultrastructural study revealed intra and extracellular locations, polymorphic nuclei, intracellular round vesicles in the cytoplasm and absence of mitochondria. The present paper reports the characterization of the Mikrocytos sp. infecting M. intestinalis and proposes a novel species in the genus: Mikrocytos mytilicoli n. sp. A sequence of 18S-28S rDNA was obtained with 95.6% maximum identity (query cover 100%) with Mikrocytos mackini. Phylogenetic analysis showed that M. mytilicoli n. sp. and M. mackini share a common ancestor. However, comparison of the ITS1 rDNA region showed low similarity (75.8%) with M. mackini, which, combined with differences in ultrastructural details, host and geographic location, support the designation of a new species. This is the first description of a microcytid parasite of the genus Mikrocytos from a non-bivalve host.

A New Species of Nematoda from the Bufo minshanicus in Southwestern China

A new nematode, Cosmocercoides minshan n. sp. (Cosmocercidae) is described from Bufo minshanicus in the Zoige grassland in southwestern China. The new species is characterized as a medium-sized worm (4.93–7.97 mm in length and 0.24–0.47 mm in width), but with a shorter spicule length (0.08–0.21 mm) than other Cosmocercoides, with lateral alae and 9 pairs of caudal papillae. Among the 21 recorded species in this genus, the morphology of the new species is different from all the other species previously reported. DNA sequences of C. minshan sp. showed that the species is almost identical to Cosmocercoides pulcher (99%), and Cosmocercoides tonkinensis (99%). However, there are differences in the morphology and structure of the rosetted papillae among these 3 species, as well as 4 nucleotide differences in the 18S–28S rDNA including the internal transcribed spacer region. In a neighbor-joining phylogenetic tree, C. tonkinensis, C. pulcher, and C. minshan formed a robust clade. Generally, the species is distinguished molecularly and morphologically; thus C. minshan is herein described as a new nematode species of Cosmocercoides.

Studies on Trueperella pyogenes isolated from an okapi (Okapia johnstoni) and a royal python (Python regius)

BackgroundThe present study was designed to characterize phenotypically and genotypically two Trueperella pyogenes strains isolated from an okapi (Okapia johnstoni) and a royal python (Python regius).Case presentationThe species identity could be confirmed by phenotypic properties, by MALDI-TOF MS analysis and by detection of T. pyogenes chaperonin-encoding gene cpn60 with a previously developed loop-mediated isothermal amplification (LAMP) assay. Furthermore, sequencing of the 16S ribosomal RNA (rRNA) gene, the 16S-23S rDNA intergenic spacer region (ISR), the target genes rpoB encoding the β-subunit of bacterial RNA polymerase, tuf encoding elongation factor tu and plo encoding the putative virulence factor pyolysin allowed the identification of both T. pyogenes isolates at species level.ConclusionsBoth strains could be clearly identified as T. pyogenes. The T. pyogenes strain isolated in high number from the vaginal discharge of an okapi seems to be of importance for the infectious process; the T. pyogenes strain from the royal python could be isolated from an apparently non-infectious process. However, both strains represent the first isolation of T. pyogenes from these animal species.

In silico structural homology modelling of EST073 motif coding protein of tea Camellia sinensis (L)

BackgroundTea (Camellia sinensis (L). O. Kuntze) is known as the oldest, mild stimulating caffeine containing non-alcoholic beverage. One of the major threats in south Asian tea industry is the blister blight leaf disease (BB), caused by the fungus Exobasidium vexans Masse. SSR DNA marker EST SSR 073 is used as a molecular marker to tag blister blight disease resistance trait of tea. The amino acid sequences were derived from cDNA sequences related to EST SSR 073 of BB susceptible (TRI 2023) and BB resistant (TRI 2043) cultivars. An attempt has been made to understand the structural characteristics and variations of EST SSR 073 locus that may reveal the factors influencing the BB resistance of tea with multiple bioinformatics tools such as ORF finder, ExPasy ProtParam tools, modeler V 9.17, Rampage server, UCSF-Chimera, and HADDOCK docking server. ResultsThe primary, secondary, and tertiary structures of EST SSR 073 coding protein were analyzed using the amino acid sequences of both BB resistant TRI 2043 and BB susceptible TRI 2023 tea cultivars. The coding amino acid sequences of both the cultivars were homologous to photosystem I subunit protein (PsaD I) of Pisum sativum. The predicted 3D structures of proteins were validated and considered as an acceptable overall stereochemical quality. The BB resistant protein showed CT repeat extension and did not involve in topology of the PsaD I subunit. The C terminal truncation of BB resistance caused the formation of hydrogen bonds interacting with PsaD I and other subunits of photosystem I in the modeled three-dimensional protein structure. ConclusionsCamellia sinensis EST 073 SSR motif coding protein was identified as the PsaD I subunit of photosystem I. The exact mechanism of PsaD I conferring the resistance for blister blight in tea needs to be further investigated.

Two new species of a new genus, Pseudorogneda, of Polycystididae discovered in Southern China.

Two new species of the new genus Pseudorogneda of Polycystididae were discovered in a brackish environment from Southern China. Pseudorogneda sinensis n. gen. n. sp., is distinguished by a semicircular double-walled prostate stylet type II that tapers in diameter from proximal to distal with a proximal-to-distal fold, and an inner wall with a funnel-shaped base originating at the midpoint of the outer wall; its flattened tubular prostate stylet type IV exhibits a horn-like base and a proximal-to-distal folded structure on its wall. For Pseudorogneda shenda n. gen. n. sp., its crescent-shaped prostate stylet type II is double-walled and the inner wall has a funnel-shaped base that starts at the proximal 2/3 position of the outer stylet and is fused with the outer stylet to form a sharp distal end; its tubular and slightly-curved prostate stylet type IV exhibits a slit and a distal fold. Phylogenetic analysis of 18S rDNA and 28S rDNA sequences shows that the two novel species are clustered to form an independent clade from other genera, which is consistent with morphological comparison in the establishment of two new species in a new genus.

Phylogenetic relationships of Strongyloides species in carnivore hosts.

Strongyloides stercoralis is a parasitic nematode and a major pathogen responsible for human strongyloidiasis. The presence of this species in the dog population has led to an interest in studying the phylogenetic relationships among Strongyloides spp. in carnivore hosts. In the present study, Strongyloides spp. from various carnivore hosts (raccoon, Japanese badger, Siberian weasel, raccoon dog, masked palm civet, and domestic cat) were sought. Except for civets, Strongyloides spp. were identified in all host species. Based on 18S rDNA sequences, nine OTUs (operational taxonomy units) were identified. Molecular phylogenetic analyses using 18S28S rDNA and mitochondrial cox1 (cytochrome c oxidase subunit 1) sequences clustered them into two groups. The first group (named the stercoralis/procyonis group) was comprised of six OTUs and occurred in cats, raccoon dogs, raccoons (S. procyonis), Siberian weasels, and Japanese badgers and included S. stercoralis from humans and dogs. The second group (named the planiceps group) was made up of Strongyloides spp. from raccoon dogs (two OTUs) and one OTU from Siberian weasels. Subsequent analysis using almost the full-length nucleotide sequences of protein-coding genes in their mitochondrial genomes placed Strongyloides spp. of cats in a sister taxon position to S. stercoralis, whereas S. procyonis from raccoons was more distantly related to them. The presence of Strongyloides spp. from various carnivore hosts, which are close relatives of S. stercoralis, suggests this group of Strongyloides (the stercoralis/procyonis group) essentially evolved as parasites of carnivores, although more data on Strongyloides spp. from primate hosts are needed.

A new species rises from beneath Florida: molecular phylogenetic analyses reveal cryptic diversity among the metapopulation of Crangonyx hobbsi Shoemaker, 1941 (Amphipoda: Crangonyctidae)

The phylogenetic structure of 23 populations of Crangonyx hobbsi, a stygobitic amphipod species endemic to the Floridan aquifer, is detailed. Molecular genetic analyses (based on three genes the nuclear 18S rDNA, 28S rDNA, and mitochondrial 16S rDNA) point to the presence of at least two phylogenetic clades within the aquifer. These clades align into two major zoogeographic regions, the Northern Suwannee River Basin and the Coastal and Central Lowlands in the Florida peninsula. Three species delimitation models indicate that the Northern Suwannee River Basin Clade represents a species level differentiation from C. hobbsi sensu stricto. Morphological analyses support the molecular analyses, revealing the presence of several taxonomically significant differences between the two species, including presence/absence of calceoli on the second antennae of males and differences in telson shape in both sexes. Additionally, the molecular diversity of C. hobbsi sensu stricto was shown to be surprisingly variable, suggesting the potential presence of additional diversity within the taxon.

Screening and molecular characterization of new thermo- and ethanol-tolerant Acetobacter malorum strains isolated from two biomes Moroccan cactus fruits.

Industrially, the sensitivity of acetic acid bacteria (AAB) to the high temperatures and the high ethanol concentrations is the major concerns for manufacturers. This study was conceived and designed to isolate and identify new thermo- and ethanol-tolerant AAB from Opuntia ficus-indica L. fruits. As a result, among 140 isolated bacterial strains, five selected strains (CR1, CR5, CR23, CZ2, and CZ15) exhibited important acetic acid production until 40°C. The use of 16S rDNA gene analysis was insufficient to identify selected bacteria. Indeed, except CR5 that presented 100% similarity to A. cerevisiae, the other strains presented similar homology rates simultaneously to the 16S rDNA sequences of A. cerevisiae and A. malorum. The reidentification by 16S-23S rDNA gene sequencing showed that CR1, CR23, and CZ15 were A. malorum, which were shown tolerance to the highest concentration of ethanol (12%) and produced elevated amount (40g/L) of acetic acid at 37°C. In summary, we showed the thermotolerance and ethanol tolerant character of new A. malorum strains, which can be used as a starter for vinegar production. Furthermore, during the molecular characterization of the isolated strains, we concluded that 16S-23S rDNA internal transcribed spacer sequence is of great importance for discriminating between AAB species as a complement to the identification by 16S rDNA sequencing.

Potential probiotic of Lactobacillus strains isolated from the intestinal tracts of pigs and feces of dogs with antibacterial activity against multidrug-resistant pathogenic bacteria.

The occurrence of multidrug-resistant pathogenic bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Acinetobacter baumannii (MDRAB), extended-spectrum β-lactamase (ESBL) Escherichia coli, and Pseudomonas aeruginosa, has become a serious problem in animals and public. The objective of this study was to identify and isolate lactic acid bacterial (LAB) strains from the intestinal tracts of pigs and feces of dogs and then characterize them as potential probiotics with antimicrobial activity against multidrug-resistant pathogenic bacteria. In a preliminary isolation screening, 45 of 1167 isolated LAB strains were found to have anti-S. aureus ATCC 27,735 activity. Using 16S rDNA and 16S-23S rDNA intergenic spacer region (ISR) sequences, five of these isolates were further identified as Lactobacillus animalis 30a-2, Lactobacillus reuteri 4-12E, Weissella cibaria C34, Lactococcus lactis 5-12H, and Lactococcus lactis 6-3H. Antimicrobial substance assays suggest that the L. lactis 5-12H, L. lactis 6-3H, L. animalis 30a-2, L. reuteri 4-12E, and W. cibaria C34 strains might produce bacteriocins and hydrogen peroxide (H2O2) as antimicrobial substances. The L. animalis 30a-2 and W. cibaria C34 strains were further characterized for probiotic properties and shown to have high acid and bile salt tolerance. Additionally, they have broad antimicrobial spectra, and can significantly repress the growth of all of the tested strains of MRSA isolates, some MDRAB, ESBL E. coli, and P. aeruginosa isolates, along with food-borne pathogenic bacteria such as Bacillus cereus ATCC 11778, Listeria monocytogens ATCC 19111, Salmonella spp., Shigella spp., and Yersinia enterocolitica BCRC 12986. This is the first report of H2O2-producing L. animalis 30a-2 and W. cibaria C34 isolated from the intestinal tracts of pigs and feces of dogs that have good antimicrobial activity against multidrug-resistant and food-borne pathogenic bacteria and have excellent probiotic properties.

Effects of Antibiotics on the Intestinal Microbiota of Mice.

Studies on human and mouse gastrointestinal microbiota have correlated the composition of the microbiota to a variety of diseases, as well as proved it vital to prevent colonization with resistant bacteria, a phenomenon known as colonization resistance. Antibiotics dramatically modify the gut community and there are examples of how antibiotic usage lead to colonization with resistant bacteria [e.g., dicloxacillin usage selecting for ESBL-producing E. coli carriage], as shown by Hertz et al. Here, we investigated the impact of five antibiotics [cefotaxime, cefuroxime, dicloxacillin, clindamycin, and ciprofloxacin] on the intestinal microbiota in mice. Five different antibiotics were each given to groups of five mice. The intestinal microbiotas were profiled by use of the IS-pro analysis; a 16S–23S rDNA interspace [IS]-region-based profiling method. For the mice receiving dicloxacillin and clindamycin, we observed dramatic shifts in dominating phyla from day 1 to day 5. Of note, diversity increased, but overall bacterial load decreased. For ciprofloxacin, cefotaxime, and cefuroxime there were few overall changes. We speculate that antibiotics with efficacy against the abundant anaerobes in the gut, particularly Bacteroidetes, can in fact be selected for resistant bacteria, disregarding the spectrum of activity.

Genetic Relatedness of NDM-Producing Klebsiella pneumoniae Co-Occurring VIM, KPC, and OXA-48 Enzymes from Surveillance Cultures from an Intensive Care Unit.

Klebsiella pneumoniae is an opportunistic bacterial pathogen most commonly associated with nosocomial infections, especially in intensive care unit (ICU) patients. Routine surveillance cultures for carbapenemase-producing Enterobacteriaceae have become a common practice for hospital infection prevention. The objective of this study was to investigate the genetic relatedness of carbapenem-resistant K. pneumoniae carrying blaNDM gene across multilocus sequence typing (MLST) scheme. Surveillance rectal swabs from 4,463 ICU patients admitted to the Rio de Janeiro hospital (March 2016-2017) were screened on CHROMagar mSuperCARBA. Of these, 631 isolates were subjected to VITEK 2 system for phenotypic microbial identification and antibiotic susceptibility testing. Out of 631 isolates, 108 were identified as K. pneumoniae, 103 of which were confirmed by PCR of 16S-23S rDNA internal transcribed spacer (ITS). Eleven blaNDM-positive isolates were subsequently screened for blaKPC, blaBKC, blaIMP, blaVIM, blaSPM, blaOXA-48, and mcr-1-8 genes. Twenty-seven percent (3/11) revealed co-occurrence with KPC, OXA-48, and VIM, 46% (5/11) with KPC and VIM, and 18% (2/11) with VIM type. No strains harbored the blaBKC, blaSPM, blaIMP, and mcr-1 to 8 resistance genes. All 11 isolates were resistant to β-lactams, ciprofloxacin 90%, tigecycline 82%, gentamicin 73%, and amikacin 18%, and were classified as multidrug resistant (MDR), extensively drug resistant (XDR), and pandrug resistant (PDR). Molecular epidemiology data based on MLST revealed 11 different STs, 8 of which were novel and 3 were previously described. Six out of the eight new STs were associated with MDR and PDR strains and two clonal complexes were reported, including CC258 and CC15. The coexistence of NDM-producing K. pneumoniae and other carbapenemase has been frequently described worldwide. Moreover, we report for the first time K. pneumoniae co-harboring up to four carbapenemases from active surveillance cultures.

Species-Specific Identification of Streptococcus based on DNA Marker in 16S-23S rDNA Internal Transcribed Spacer.

Streptococcus is closely correspondent to human. The accurate species-specific identification method of Streptococcus is important for the bacteria clinical diagnosis, molecular epidemiological analysis, and microecological study. In the last decades, DNA markers are widely utilized for identification of prokaryotic species. However, 16S rDNA, the most popular bacterial DNA marker, cannot properly distinguish closely related Streptococcus species. In present study, we employed 16S-23S rRNA gene internal transcribed spacer (ITS) sequence to explore the species-specific DNA marker. We predicted the secondary structure of Streptococcus ITS sequence transcribed products. Then we identified that the specific and consensus sequences in the primary structure can be found occupying an individual subunit in the secondary structure, which explained the foundation of the mosaic-like structure of ITS. We evaluated the specificity of ITS in Streptococcus, and found that the specificity can be detected by a further analysis of a BLAST result. Then, we developed an identification procedure based on the ITS sequence. We verified the procedure by 500 ITS sequence. The accuracy rate of this procedure was 100% for Streptococcus at genus level, and 99.3% at species level. It suggested that ITS can be utilized to accurately identify Streptococcus at the species level. This work suggests that further exploration of ITS could be applied in other bacterial genera for identification and classification, which may be a useful topic for future microbiology studies.

A new species of Isodictya (Porifera: Poecilosclerida) from the Southern Ocean

We discovered a new species of Porifera belonging to the genus Isodictya Bowerbank, 1864 during cruises aboard R/V Hesperides in Antarctica. Collected samples are mostly part of the surveys of the Spanish project BENTART whose main objective has been to study the benthic communities inhabiting sea bottoms of Livingston and Deception Island in the South Shetlands archipelago and the Antarctic Peninsula. Isodictya filiformis sp. nov., described here, is characterized by its fragile and thin morphology (very different from other known species in the area) and by having microxeas as additional microscleres. Three specimens were collected from Marguerite Bay, Low Island and Deception Island (Antarctic Peninsula) and one specimen at Peter I Island (Bellingshausen Sea). Its presence in Peter Island is quite relevant as this location is 390 km away from the nearest coast in the Bellingshausen Sea, an area that has scarcely been investigated in the past. However, results from the Bentart 03 Expedition seem to indicate that Peter I Island has a wide variety of benthic organisms, in contrast to the deep adjacent areas of Bellingshausen Sea. Apart from the morphological analyses, we place the new Isodictya species within its phylogenetic context using two nuclear markers (18S rDNA and 28S rDNA) and provide some information about the ecological preferences of the new species.

Novel insights into the enterotoxigenic potential and genomic background of Staphylococcus aureus isolated from raw milk

In this study, 53 Staphylococcus (S.) aureus strains were typed by 16S–23S rDNA intergenic spacer region (ISR) typing and staphylococcal enterotoxin gene (SEg) typing for all the staphylococcal enterotoxin (se) and staphylococcal enterotoxin-like toxin (sel) genes known to date, revealing a higher discriminatory power than that of multi locus sequence typing. Six strains, one of each ISR- and SEg-type, were genome sequenced and the ability to produce some classical and new SEs when growing in milk was investigated.The manual analysis of the six genomes allowed us to confirm, correct and expand the results of common available genomic data pipelines such as VirulenceFinder. Moreover, it enabled us to (i) investigate the actual location of se and sel genes, even for genes such as selY, whose location (in the core genome) was so far unknown, (ii) find novel allelic variants of se and sel genes and pseudogenes, (iii) correctly annotate se and sel genes and pseudogenes, and (iv) discover a novel type of enterotoxin gene cluster (egc), i.e. the egc type 5 in strains 356P and 364P, while S. argenteus MSHR1132 harbored the egc type 6.Four of the six S. aureus strains produced sufficient amounts of SEA, SEC, SED and SEH in milk to cause staphylococcal food poisoning (SFP), with S. aureus 372 P being the highest producer of SED in milk found to date, producing as much as ca. 47,300 ng/mL and 49,200 ng/mL of SED, after 24 and 48 h of incubation in milk at 37 °C, respectively. S. aureus 372 P released a low amount of SER in milk, most likely because the seR gene was present as a pseudogene, putatively encoding only 51 amino acids. These findings confirm that not only the classical SEs, but also the new ones can represent a potential hazard for the consumers’ health if produced in foods in sufficient amounts. Therefore, the detection of SEs in foods, especially if involved in SFP cases, should focus not only on classical, but also on all the new SEs and SEls known to date. Where reference methods are unavailable, the presence of the relevant genes, by using the conventional and real time PCR protocols we exhaustively provided herein, and their nucleotide sequences, should be investigated.

Burkholderia gladioli causes bacterial internal browning in sweetpotato of China

A new bacterial disease of sweetpotato was recently identified in Puning, Guangdong Province, China and is referred to bacterial internal browning of sweetpotato in this paper. One-third of the tubers of sweetpotato were infected in the field. These tubers in the field with bacterial inner brown displayed yellowing on the skin that turned into slightly roughened and cracked surface with progress in disease. Cross-sections of these roots revealed a yellowish brown or dark brown circle in the vascular cambium. Pathogenicity tests were conducted on different organs of two sweetpotato cultivars and revealed that the isolated bacteria are pathogenic on stems and roots. We confirmed the causal agent as Burkholderia gladioli based on biochemical and physiological characterization, carbon source utilization, and sequence analysis of partial 16S rDNA and 16S–23S rDNA internal transcribed spacer (ITS) region. To our knowledge, it is the first report on the occurrence of B. gladioli in sweetpotato as a pathogen.

Comparing conventional, biochemical and genotypic methods for accurate identification of Klebsiella pneumoniae in Sudan.

Klebsiella pneumoniae is recognized as one of the most important healthcare-associated pathogens worldwide due to its tendency to develop antibiotic resistance and cause fatal outcomes. Bacterial identification methods such as culture and biochemical tests are routinely used with limited accuracy in many low- and middle-income countries, including Sudan. The aim of this study was to test the accuracy of identification of K. pneumoniae in Khartoum, Sudan. Two hundred and fifty K. pneumoniae isolates were collected and identified using conventional phenotypic methods, biochemically using API 20E and genotypically by amplification of 16S−23S rDNA and sequencing of rpoB, gapA and pgi. Only 139 (55.6 %) of the isolates were confirmed as K. pneumoniae genotypically by PCR and 44.4 % were identified as non- K. pneumoniae . The results demonstrate that the identification panels used by the hospitals were inaccurately identifying K. pneumonia and led to overestimation of the prevalence of this organism. The current identification methods used in Khartoum hospitals are highly inaccurate, and therefore we recommend the use of a comprehensive biochemical panel or molecular methods, when possible, for accurate identification of K. pneumoniae .

First Report of the Potato Cyst Nematode, Globodera rostochiensis, on Potato in the Azores, Portugal

In Portugal, growing potatoes (Solanum tuberosum) has both social and economic importance. Because potato cyst nematodes (PCN) are major pests of potatoes, their presence in these areas could pose a risk to its production. Therefore, the National Survey Plan for PCN that was implemented in 2010 is still ongoing. This survey aims for the early detection of PCN to provide tools to control and manage initial PCN infestations below the damage threshold level, while avoiding their dispersal within both national and EU territories. During the 2018 National Survey in the potato production areas of the Sao Miguel Island (Azores), six bulk soil samples of 1 kg from a small field cultivated for household consumption in Nordeste County were investigated. A subsample of 500 g from the bulk sample was dried, and cysts were extracted using a Fenwick can. Dried cysts were hydrated and cut to release eggs, and hatched J2 were collected for morphometric analysis. The identification of PCN was based on a combination of n = 30 cysts and n = 90 J2 measurements. The cyst length excluding neck (L) was 493.658 ± 89.377 μm, width (W) was 474.090 ± 93.569 μm, the L/W ratio was 1.051 ± 0.115, and neck was 110.894 ± 26.353 μm. The number of cuticular ridges between the vulva and the anus was higher than 20. The distance from vulva to anus was 60.15 ± 6.7 µm with the Granek’s ratio ranging from 1.26 to 4.99. The vulva fenestrate was 25.13 ± 6.7 µm in diameter and anus visible in almost all specimens, with a V-shaped mark. The stylet length of J2 was 23.14 ± 1.8 µm with rounded knobs. Body length was 449.79 ± 30.3 µm, and average true and hyaline tail length were 50.92 ± 6.4 µm and 26.50 ± 4.3 µm, respectively. The overall morphometric values were within the range described in PM 7/40(4) (EPPO 2017) for Globodera rostochiensis. The above morphological characterization was also confirmed using a sequence-specific multiplex PCR (Bulman and Marshall 1997) described in PM7/40(4) (EPPO 2017) and conventional PCR of 18S rDNA (SSU), 28S rDNA (LSU), and cytochrome oxidase subunit I (COI) as described in PM7/129(1) (EPPO 2016). The nucleotide sequences (MN226947, MN238768, and MK798162) showed a similarity of 99 to 100% with available isolates of G. rostochiensis in NCBI database. A pathogenicity test was carried out in the greenhouse by planting tubers of the susceptible cultivar Desiree in four plastic pots of 15 cm diameter. Pots were inoculated with an initial population density of 5 J2 and eggs per gram of soil and harvested after 90 days. A final population (Pf) of mean of x¯ = 23.3 J2 or eggs per gram of soil was obtained, resulting in a reproduction factor (R=Pf/Pᵢ) of 4.66 ± 0.86. According to the European Commission Regulation EC 690/2008 updated by implementing regulation EU 2016/873, the Azores islands are recognized as protected zones for G. rostochiensis and G. pallida. This is the first report of the presence of G. rostochiensis in the Azores islands. Research will focus more on the spatial distribution of the nematodes, and based upon it integrated control methods will be devised including strict phytosanitary measures to prevent spread and ensure further the Azores as a PCN protected zone.

Bacterial endosymbionts of Placobdella (Annelida: Hirudinea: Glossiphoniidae): phylogeny, genetic distance, and vertical transmission

Blood-feeding leeches of the genus Placobdella have acquired intracellular alphaproteobacterial endosymbionts of the genus Reichenowia that potentially aid in the production of B vitamins, thereby ameliorating the lack of these essential nutrients in the diet of the hosts. For Placobdella associates, little is still known about the diversity, genetic makeup, and the mode of transmission of bacteria between leeches. We aimed to (i) place the bacterial symbionts in a phylogenetic context, (ii) compare patterns of cladogenesis between the bacteria and hosts to search for evidence of co-speciation, and (iii) explore the mode of bacterial transmission between leech parent and offspring. DNA sequencing of the bacterial 16S rDNA and 23S rDNA loci suggests that, whereas Reichenowia forms a monophyletic group within the alphaproteobacterial family Rhizobiaceae, no evidence for co-speciation between hosts and bacteria can be traced. Attempts at DNA amplification for ovarial tissues were negative for a range of species, but two 16S rDNA sequences retrieved from the testisacs of P. rugosa showed very high similarity with Reichenowia. Although we cannot rule out that this may be a contamination, or a different, potentially free-living species of bacteria, our results may indicate that Reichenowia is transferred from leech parent to offspring via the testisacs.

First report of the stubby-root nematode Nanidorus minor infecting Paspalum vaginatum, seashore paspalum grass in Georgia, USA

We found that Nanidorus spp. was pathogenic to seashore paspalum (Paspalum vaginatum) turfgrass as its population increased from 100 to 2,080 nematodes per pot 180 days after inoculation under greenhouse conditions. Morphological measurements of adult females were similar to those described for N. minor. Molecular analysis also confirmed the morphological identification by targeting three different regions of the genomic DNA. Three primer pairs targeting 18S rDNA (360F/932R), 28S rDNA (D2A/D3B) and ITS1 rDNA (BL18/5818) were used in singleplex PCR. Forward and reverse sequences of each individual primer set were then subjected to multiple alignment and the complimentary sequences were assembled into a consensus sequence. Upon nucleotide blast on the NCBI website, they were all confirmed to be N. minor. A one-step multiplex PCR method using specific primers and a fragment size of 190 bp also confirmed the identity of N. minor. To the best of our knowledge, this is the first report of N. minor infecting seashore paspalum turfgrass in Georgia.

First Report of the Root-Knot Nematode Meloidogyne incognita Infecting Salvia miltiorrhiza in Taiwan

The root-knot nematode Meloidogyne incognita is a serious and common pathogen that affects many economically important plants in Taiwan. In June 2012, Salvia miltiorrhiza Bge., which is a perennial herbaceous plant used for traditional Chinese medicine, exhibited stunting, leaf wilting, and root galls in Hualien County, Taiwan. Over 30% of plants in Zhuoxi Township, Hualien County, were diseased. Soil and root samples were collected from S. miltiorrhiza production areas. Second-stage juveniles (J2) of M. incognita were isolated from diseased roots, and morphological and morphometrical findings after examination of the species conformed with descriptions and measurements provided by Whitehead (1968). Molecular analyses were also conducted, and species were identified through polymerase chain reaction with species-specific primers Finc/Rinc, Inc-K14-F/Inc-K14-R, Fjav/Rjav, and Far/Rar (Randig et al. 2002; Zijlstra et al. 2000). Species-specific sequence characterized amplified region primers Finc/Rinc and IncK-14F/IncK-14R were used to determine whether these nematodes were M. incognita. The PCR produced expected amplification products of 1,200 and 399 bp, confirming the nematode to be M. incognita. Primers specific for M. arenaria (Far/Rar) and M. javanica (Fjav/Rjav) were used but failed to amplify fragments from the nematode DNA (Randig et al. 2002; Zijlstra et al. 2000). In addition, 5S-18S rDNA sequences were amplified using primer pair 194/195 (Blok et al. 1997). The obtained PCR fragments were sequenced and deposited in GenBank (MN729267 and MN729268). They showed 99.06 and 99.21% identity to the available GenBank M. incognita sequence (accession no. GU395506.1). A pathogenicity test was conducted in greenhouse conditions. Four 2-month-old S. miltiorrhiza plants were maintained in 5-dm³ pots, with each plant subsequently inoculated with 200 J2s. Four uninoculated plants were used as negative controls. After 8 weeks, all inoculated plants exhibited symptoms similar to those observed in the field, and 100% of root system tissues had galls. The nematodes were reisolated from root tissue and identified. No symptoms were observed in the control plants. To our knowledge, this is the first report of M. incognita infecting S. miltiorrhiza in Taiwan.