Partial 16S Ribosomal DNA Research Articles

Isolation, identification and pathogenicity of local entomopathogenic bacteria as biological control agents against the wild cochineal Dactylopius opuntiae (Cockerell) on cactus pear in Morocco

The Opuntia ficus-indica (L.) cactus, a crucial crop in Morocco, is threatened by the wild cochineal, Dactylopius opuntiae (Cockerell). The aim of this research was to investigate the efficacy of nine bacterial strains against both D. opuntiae nymphs and adults females applied individually or after black soap in the laboratory, greenhouse, and field conditions. Using the partial 16S ribosomal DNA, the bacterial isolates were identified as Pseudomonas koreensis, Pseudomonas sp., Burkholderia sp. and Bacillus sp. Under laboratory conditions, the insecticidal activity of P. koreensis strain 66Ms.04 showed the level mortality (88%) of adult females’ at 108 CFU/mL, 7 days after application. At a concentration of 108 CFU/mL, P. koreensis strain 66Ms.04 and Pseudomonas sp. (strains 37 and 5) caused 100% nymphs mortality rate three days after application. Under greenhouse conditions, the use of P. koreensis strain 66Ms.04 at 108 CFU/mL following the application of black soap (60 g/L) demonstrated the maximum levels of females and nymphs’ mortalities with 80 and 91.25%, respectively, after 8 days of treatment. In field conditions, the combined application of the P. koreensis strain 66Ms.04 at 108 CFU/mL with black soap at 60 g/L, for an interval of 7 days, significantly increased the mortality of adult females to 93.33% at 7 days after the second application. These findings showed that the combined treatment of P. koreensis strain 66Ms.04 with black soap can be a potent and eco-friendly pesticide against D. opuntiae.

A new species of Siphoderina Manter, 1934 (Digenea: Cryptogonimidae) infecting the Dory Snapper Lutjanus fulviflamma (Teleostei: Lutjanidae) from the east coast of South Africa

Parasitological assessment of marine fishes at Sodwana Bay in the iSimangaliso Marine Protected Area on the east coast of South Africa revealed a new species of cryptogonimid trematode infecting the pyloric caeca of the Dory Snapper, Lutjanus fulviflamma (Forsskål) (Lutjanidae). The new species is morphologically consistent with the concept of the large genus Siphoderina Manter, 1934; its phylogenetic position within this genus was validated through molecular sequencing of the ITS2 and partial 28S ribosomal DNA sub-regions. We name this species Siphoderina nanan. sp. and comment on the current state of understanding for this genus of cryptogonimids.

Pseudaphelenchus chunanensis n. sp. (Tylenchina: Aphelenchoididae) found in Pinus massoniana from China

Summary Pseudaphelenchus chunanensis n. sp. was isolated in branches of Pinus massoniana from eastern China. The new species is characterised by very short males (402 (360-452) μm) and females (470 (397-540) μm), three incisures in the lateral field, slender stylet with small but conspicuous basal knobs, excretory pore located at the level of anterior margin of the metacorpus or more anterior. Male spicules paired, thorn-like, separate, condylus truncate, sometimes broadly rounded, rostrum conical or triangular with bluntly pointed tip, calomus smoothly tapering together with lamina, thin and strongly arcuate in middle then tapering towards bluntly pointed distal tip, cucullus absent. A long and narrow bursa surrounds the entire tail but inconspicuous in most males. Conical tail bluntly rounded but sometimes has a short mucron. Female tail conical, smoothly tapering and slightly ventrally bent in distal part of tail, terminus fine or bluntly pointed. It is typologically different from six known Pseudaphelenchus species, and detailed phylogenetic analysis based on partial 18S ribosomal DNA (rDNA), internal transcribed spacer (ITS) and the D2-D3 region of 28S rDNA sequences further confirm this nematode as a new species.

New species of the genus Spio (Annelida, Spionidae) from the southern and western coasts of Korea.

A new spionid polychaete, Spiopigmentatasp. nov., is described from the southern and western coasts of Korea. This new species differs from its congeners by the combination of the following morphological characteristics: the presence of orange-brown pigmentation on the anterior part of the prostomium, black pigmentation on the peristomium and along the body, U-shaped nuchal organs, a comparatively long extension of metameric dorsal ciliated organs, three pairs of white dots per chaetiger, two to three posterior abranchiate chaetigers, and the presence of tridentate neuropodial hooded hooks. The partial 16S ribosomal DNA (rDNA) and nuclear 18S rDNA sequences of the new species and Spio sp. 2 reported by Abe and Sato-Okoshi (2021) from Japan showed high similarity, indicating that these two specimens belong to the same species. A detailed description and illustrations of the new species, together with molecular information, are provided.

Molecular characterization of Fasciola jacksoni from wild elephants (Elephas maximus maximus) of Sri Lanka: a taxonomic evaluation.

Fasciola jacksoni is a significant contributor to the health and mortality of Asian elephants, particularly those in Sri Lanka. Despite the impact of fascioliasis on elephant populations, it is a neglected veterinary disease with limited taxonomic understanding. Molecular characterization and phylogenetic analysis of F. jacksoni were carried out to evaluate its suggested basal position in the Fasciolidae. Adult worms were collected during post-mortem of elephants, and eggs were collected from living elephants in National parks across Sri Lanka. Using the mitochondrial genes nicotinamide dehydrogenase subunit 1 (nad1) and cytochrome oxidase subunit 1 (cox1), and a partial 28S ribosomal DNA (28S rDNA), DNA sequences were generated from the F. jacksoni adult and egg material. Maximum likelihood (ML) phylogenetic analyses did not resolve F. jacksoni to be basal to the Fasciolidae. Furthermore, the ML analyses showed that the genus Fasciola was not monophyletic and that F. jacksoni was a sister species to the deer liver fluke Fascioloides magna. A clear framework is required to determine the taxonomic status of F. jacksoni and this current study provides the first detailed application of molecular techniques from multiple hosts across Sri Lanka with the production of reference DNA sequences for this important parasite.

Subcutaneous merocercoids of Clistobothrium sp. in two Cape fur seals (Arctocephalus pusillus pusillus).

Fur seals represent intermediate hosts of the cestode Clistobothrium. Large sharks are definitive hosts for these parasites. Two female, 25– and 27-year-old fur seals, caught in the 1980s at the South African coast, were examined pathomorphologically. Both animals showed multifocal, up to 1 cm in diameter large cavities of the thoracic and abdominal subcutaneous adipose tissue containing intraluminal metacestodes of tapeworms, which were surrounded by a locally extensive, pyogranulomatous panniculitis. The metacestodes (merocercoids) of one fur seal were isolated from the subcutaneous adipose tissue and characterized morphologically and for the first time from this host by molecular techniques. The morphometric data corresponded with ‘delphini'-morphotype merocercoids, but the sequence of the partial 28S ribosomal RNA gene identified them as conspecific with merocercoids of the morphotype ‘grimaldii’. These merocercoid types are morphologically Type XV metacestodes of marine tapeworms and represent different species of Clistobothrium. Sequence data were generated for 18S, ITS1, 5.8S, ITS2, partial 28S ribosomal DNA and partial mitochondrial cox1 gene and phylogenetic analysis of 18S rRNA and partial 28S rRNA genes identified the fur seal merocercoids as Clistobothrium species. However, it cannot yet be assigned to species level because of limited molecular data from adult stages. Most likely, both fur seals were infected as juveniles in their original habitat, the coastal regions of South Africa. The metacestode infection is probably an incidental finding, however, there is a chronic inflammatory reaction next to the subcutaneous merocercoids. It is noteworthy, that the merocercoids remain in a potentially infective stage even after more than 20 years.

Two new species of Haplorchoides Chen, 1949 (Digenea: Heterophyidae) infecting an Australian siluriform fish, Neoarius graeffei Kner & Steindachner.

Combined morphological and molecular analyses are used to characterise two new species of Haplorchoides Chen, 1949 (Digenea: Heterophyidae) from an Australian siluriform fish. Haplorchoides maiwariensis n. sp. and H. daguilarensis n. sp. are described from the intestine of the Blue salmon catfish, Neoarius graeffei (Kner & Steindachner) (Siluriformes: Ariidae) from tidal reaches of the Brisbane River and from a freshwater creek off Lake Wivenhoe, Kipper Creek, Australia. The two new species most obviously differ from all previously described species of Haplorchoides in possessing clearly and reliably tripartite seminal vesicles. The two new species differ in the size and distribution of vitelline follicles, the size of the pigment granules and the form of the ventral sucker. Previously described species of Haplorchoides have been reported from Africa and Asia, principally from bagrid, schilbeid, silurid and sisorid catfishes; these are the first species reported from an ariid catfish. Complete ITS2 and partial 28S ribosomal DNA data were generated for both new species. The two species differ from each other by eight base pairs in the ITS2 region, and by 13 bp for the 28S region. The 28S rDNA sequence of H. daguilarensis agrees with a previously reported sequence from an unidentified species of Haplorchoides collected from N. graeffei in Lake Wivenhoe, Australia; we identify this previous report as relating to H. daguilarensis.

A molecular phylogenetic appraisal of the acanthostomines Acanthostomum and Timoniella and their position within Cryptogonimidae (Trematoda: Opisthorchioidea).

The phylogenetic position of three taxa from two trematode genera, belonging to the subfamily Acanthostominae (Opisthorchioidea: Cryptogonimidae), were analysed using partial 28S ribosomal DNA (Domains 1–2) and internal transcribed spacers (ITS1–5.8S–ITS2). Bayesian inference and Maximum likelihood analyses of combined 28S rDNA and ITS1 + 5.8S + ITS2 sequences indicated the monophyly of the genus Acanthostomum (A. cf. americanum and A. burminis) and paraphyly of the Acanthostominae. These phylogenetic relationships were consistent in analyses of 28S alone and concatenated 28S + ITS1 + 5.8S + ITS2 sequences analyses. Based on molecular phylogenetic analyses, the subfamily Acanthostominae is therefore a paraphyletic taxon, in contrast with previous classifications based on morphological data. Phylogenetic patterns of host specificity inferred from adult stages of other cryptogonimid taxa are also well supported. However, analyses using additional genera and species are necessary to support the phylogenetic inferences from this study. Our molecular phylogenetic reconstruction linked two larval stages of A. cf. americanum cercariae and metacercariae. Here, we present the evolutionary and ecological implications of parasitic infections in freshwater and brackish environments.

Updated molecular phylogenetic data for Opisthorchis spp. (Trematoda: Opisthorchioidea) from ducks in Vietnam

BackgroundAn opisthorchiid liver fluke was recently reported from ducks (Anas platyrhynchos) in Binh Dinh Province of Central Vietnam, and referred to as “Opisthorchis viverrini-like”. This species uses common cyprinoid fishes as second intermediate hosts as does Opisthorchis viverrini, with which it is sympatric in this province. In this study, we refer to the liver fluke from ducks as “Opisthorchis sp. BD2013”, and provide new sequence data from the mitochondrial (mt) genome and the nuclear ribosomal transcription unit. A phylogenetic analysis was conducted to clarify the basal taxonomic position of this species from ducks within the genus Opisthorchis (Digenea: Opisthorchiidae).MethodsAdults and eggs of liver flukes were collected from ducks, metacercariae from fishes (Puntius brevis, Rasbora aurotaenia, Esomus metallicus) and cercariae from snails (Bithynia funiculata) in different localities in Binh Dinh Province. From four developmental life stage samples (adults, eggs, metacercariae and cercariae), the complete cytochrome b (cob), nicotinamide dehydrogenase subunit 1 (nad1) and cytochrome c oxidase subunit 1 (cox1) genes, and near-complete 18S and partial 28S ribosomal DNA (rDNA) sequences were obtained by PCR-coupled sequencing. The alignments of nucleotide sequences of concatenated cob + nad1 + cox1, and of concatenated 18S + 28S were separately subjected to phylogenetic analyses. Homologous sequences from other trematode species were included in each alignment.ResultsPhylogenetic trees were inferred from concatenated (cob + nad1 + cox1) nucleotide sequences and combined 18S + 28S nucleotide sequences of five Opisthorchis sp. BD2013 samples and additional reference taxa. Both trees demonstrated the anticipated clustering of taxa within the superfamily Opisthorchioidea, the paraphyly of the genus Opisthorchis and the sister-species relationship of Opisthorchis sp. BD2013 with O. viverrini.ConclusionsWhile it is likely that Opisthorchis sp. BD2013 is distinct from O. viverrini, it is clearly a sister taxon of O. viverrini within the limited number of Opisthorchis species for which appropriate sequence data are available. The new sequences provided here will assist the diagnosis and the taxonomic clarification of the opisthorchiid species.

Morphological and molecular characterization of Trophurus wuhuensis n. sp. (Nematoda: Telotylenchinae) from soil associated with Cinnamomum camphora in China.

A new plant nematode species, Trophurus wuhuensis n. sp., was collected from the soil associated with Cinnamomum camphora in Wuhu, Anhui Province, China. The new species is characterized by having a female with a slender body 660.5-801.5 μm in length, stylet 12-14 μm long, knobs directed laterad, lateral field marked by short and scattered grooves, post-vulval uterine sac shorter than vulval body diameter, post-rectal intestinal sac absent, tail cylindroid, terminus with deep wrinkles; and male with a pointed tail terminus and spicules 16-18 μm long. The internal transcribed spacer sequences of ribosomal DNA (ITS rDNA) and partial 18S ribosomal DNA (18S rDNA) from T. wuhuensis n. sp. were amplified and sequenced. A phylogenetic analysis based on sequences of 18S rDNA fragments is given in this study.

Two new and one known species of Tergestia Stossich, 1899 (Trematoda: Fellodistomidae) with novel molecular characterisation for the genus.

Combined morphological and molecular analyses are employed to characterise three species of Tergestia Stossich, 1899 (Digenea: Fellodistomidae) from fishes of Moreton Bay, Queensland, Australia. Tergestia clonacantha Manter, 1963 is reported here for the first time from the halfbeak (Beloniformes: Hemiramphidae) species Arrhamphus sclerolepis krefftii (Steindachner), Hyporhamphus australis (Steindachner), H. quoyi (Valenciennes) and H. regularis ardelio (Whitley). Two new species, both infecting trevally (Perciformes: Carangidae) species, are described: T. maryae n. sp. from Alepes apercna Grant and T. henryi n. sp. from Pantolabus radiatus (MacLeay). Complete ITS2 and partial 28S ribosomal DNA data were generated for each of the new taxa. The three species differ from each other by 47-58 base pairs (bp) in the ITS2 rDNA region. Phylogenetic analysis of 28S rDNA supports Tergestia as a reliable generic concept, with our analyses showing that some species of the genus form a well-supported clade to the exclusion of all other fellodistomids for which sequence data are available.

Evaluation of In Vitro Antiamoebic Activity of Antimicrobial Agents Against Clinical Acanthamoeba Isolates.

The aim of this study was to elucidate in vitro antiamoebic activity of antimicrobial agents at short exposure times similar to those used for actual treatment against Acanthamoeba strains isolated from patients with keratitis. The 5 clinical Acanthamoeba isolated in Japan were used in this study. Identification of genotypes for the Acanthamoeba isolates was performed using partial 18S ribosomal DNA (rDNA), including the ASA.S1 region sequences. Fluconazole, miconazole, itraconazole, voriconazole, amphotericin B, natamycin (pimaricin), and micafungin (antifungal agents), and chlorhexidine (a biguanide disinfectant), and sulfamethoxazole and paromomycin (antibacterial agents) were used to determine the antiamoebic activity against Acanthamoeba, which were determined by 50% and 90% growth inhibitory concentrations (IC50 and IC90) following exposing to each drug at 25°C for 7 days and 12 h. Among the tested antimicrobial agents, natamycin strongly inhibited the growth of all Acanthamoeba isolates at low concentration in both the 7-day (IC90 = 4.1 μg/mL) and 12-h (IC90 = 11.6 μg/mL) assays. Additionally, sulfamethoxazole exhibited strong antiamoebic activity (IC90 = 9.8 μg/mL) at low concentration in the 7-day assay. Our findings showed that natamycin ophthalmic solution might be an effective agent against Acanthamoeba keratitis. Additionally, frequent administration of sulfamethoxazole ophthalmic solution or systemic sulfamethoxazole-trimethoprim is also considered as an effective treatment for Acanthamoeba keratitis.

Polycladida phylogeny and evolution: integrating evidence from 28S rDNA and morphology

Polyclad flatworms have a troubled classification history, with two contradicting systems in use. They both rely on a ventral adhesive structure to define the suborders Acotylea and Cotylea, but superfamilies were defined according to eyespot arrangement (Prudhoe’s system) or prostatic vesicle characters (Faubel’s system). Molecular data available cover a very limited part of the known polyclad family diversity and have not allowed testing morphology-based classification systems on Polycladida yet. We thus sampled a suitable marker, partial 28S ribosomal DNA (rDNA), from Polycladida (19 families and 32 genera), generating 136 new sequences and the first comprehensive genetic dataset on polyclads. Our maximum likelihood (ML) analyses recovered Polycladida, but the traditional suborders were not monophyletic, as the supposedly acotyleans Cestoplana and Theama were nested within Cotylea; we suggest that these genera should be included in Cotylea. The partial 28S rDNA trees were generally well supported and robust but in conflict with both Faubel’s and Prudhoe’s superfamilies. Therefore, we compiled morphological and anatomical characters for all taxa used and examined their distribution on our molecular tree. Combining morphological and molecular evidence, we redefined polyclad superfamilies. Acotylea contain tentaculated and atentaculated groups and is now divided in three superfamilies. The suborder Cotylea can be divided in five superfamilies. In general, there is a trait of anteriorization of sensory structures, from the plesiomorphic acotylean body plan to the cotylean gross morphology. Traditionally used characters, such as prostatic vesicle, eyespot distribution, and type of pharynx, are all homoplastic and likely have misled polyclad systematics so far.

First Report of Aphelenchoides bicaudatus (Nematoda: Aphelenchoididae) from South Korea

The genus Aphelenchoides (Fischer, 1894) includes a diverse group of species, some of which are of economic importance. A. bicaudatus (Imamura, 1931) Filipjev and Schuurmans Stekhoven, 1941 is reported for the first time from South Korea, with a detailed redescription of the species. Specimens were collected from chrysanthemum (Chrysanthemum morifolium) leaves and shoot tips in South Korea. The species was identified by morphological traits and molecular sequencing. A bifurcated tail distinguishes A. bicaudatus from its congeneric species. To confirm species identification, we determined the partial 18S ribosomal DNA sequence of the specimens and compared with those obtained from other Aphelenchoides species available on GenBank.

Morphological and molecular characterization of Myxobolus mucosus sp. n. (Myxosporea: Myxobolidae) with basifilamental sporulation in two cyprinid fishes, Rutilus rutilus (L.) and Leuciscus leuciscus (L.) in Russia.

A new Myxobolus species, Myxobolus mucosus sp. n., was found in the basifilamental location of roach, Rutilus rutilus, and common dace, Leuciscus leuciscus, during the investigation of fish myxosporean fauna in Finnish Bay of Baltic Sea and Lake Ladoga in 2014. Plasmodia from the two hosts share similar morphometric and morphological features which are whitish and slightly elongated oval and measured 300-500 μm in diameter. Mature spores of M. mucosus sp. n. are ellipsoidal in frontal view and lemon shaped in sutural view, measuring 13.6 ± 0.62 (12.8-14.2) μm in length, 10.7 ± 0.51 (10.3-11.4) μm in width, 7.8 ± 0.41 (7.2-8.3) μm in thickness and 13.0 ± 0.89 (11.5-14.2) μm in length, 10.8 ± 0.77 (10.2-11.5) μm in width, 7.7 ± 0.52 (7.3-8.0) μm in thickness from roach and common dace, respectively. Polar capsules were pyriform, equal in size, converging anteriorly, with 5.9 ± 0.22 (5.7-6.3) μm in length and 3.9 ± 0.3 (3.5-4.1) μm in width and 5.6 ± 0.38 (5.1-6.2) μm in length and 3.8 ± 0.24 (3.5-4.0) μm in width from roach and common dace, respectively. Spore valves are relatively thin, symmetrical, and smooth. The most remarkable morphological character of this new Myxobolus species is the distinct mucous envelope, surrounding the full myxospore valves, up to 25 and 32 μm, respectively, in roach and common dace. The partial 18S ribosomal DNA (rDNA) sequence variation of myxospores from two hosts is very tiny (below 0.8 %) but significantly differing from all available sequences of myxosporeans in GenBank. Therefore, it can be concluded that this myxosporean from these two closely related cyprinids are conspecific by combining morphological characteristics, tissue specificity, and molecular data. Phylogenetic analysis showed that this newly described species cluster with several gill-infecting Myxobolus species from cyprinid fish inhabiting in Eastern Europe.

Inoculation with Metal-Mobilizing Plant-Growth-Promoting Rhizobacterium Bacillus sp. SC2b and Its Role in Rhizoremediation

A plant growth-promoting bacterial (PGPB) strain SC2b was isolated from the rhizosphere of Sedum plumbizincicola grown in lead (Pb)/zinc (Zn) mine soils and characterized as Bacillus sp. based on (1) morphological and biochemical characteristics and (2) partial 16S ribosomal DNA sequencing analysis. Strain SC2b exhibited high levels of resistance to cadmium (Cd) (300 mg/L), Zn (730 mg/L), and Pb (1400 mg/L). This strain also showed various plant growth-promoting (PGP) features such as utilization of 1-aminocyclopropane-1-carboxylate, solubilization of phosphate, and production of indole-3-acetic acid and siderophore. The strain mobilized high concentration of heavy metals from soils and exhibited different biosorption capacity toward the tested metal ions. Strain SC2b was further assessed for PGP activity by phytagar assay with a model plant Brassica napus. Inoculation of SC2b increased the biomass and vigor index of B. napus. Considering such potential, a pot experiment was conducted to assess the effects of inoculating the metal-resistant PGPB SC2b on growth and uptake of Cd, Zn and Pb by S. plumbizincicola in metal-contaminated agricultural soils. Inoculation with SC2b elevated the shoot and root biomass and leaf chlorophyll content of S. plumbizincicola. Similarly, plants inoculated with SC2b demonstrated markedly higher Cd and Zn accumulation in the root and shoot system, indicating that SC2b enhanced Cd and Zn uptake by S. plumbizincicola through metal mobilization or plant-microbial mediated changes in chemical or biological soil properties. Data demonstrated that the PGPB Bacillus sp. SC2b might serve as a future biofertilizer and an effective metal mobilizing bioinoculant for rhizoremediation of metal polluted soils.

A phylogeny of Sericini with particular reference to Chinese species using mitochondrial and ribosomal DNA (Coleoptera: Scarabaeidae)

Sericini chafer beetles are inconspicuous and morphologically rather uniform. Their morphological homogeneity has resulted in taxonomic difficulties particularly with the generic classification but also with the recognition of the species in general. Here, we present a phylogenetic analysis of Sericini using maximum likelihood and Bayesian tree inference based on partial 28S ribosomal DNA (rDNA), cytochrome oxidase I (cox 1), and 16S rDNA (rrnL) for 26 genera and 173 species of Sericini, with major focus on Chinese taxa. Chinese taxa were resolved within two major clades of the subtribe Sericina, while basal lineages of Sericini as well as Trochalina did not include species from China. Our results confirm previous findings in which species relationships were not consistent with the current genus-level classification, as well as in a previously hypothesized close phylogenetic link between southwestern Chinese and Himalayan species. Large genera such as Neoserica, Serica, and Maladera all resulted to be polyphyletic, being split in several distantly related branches. But also, less intensely sampled groups like Lasioserica and Microserica were non-monophyletic. The major implication from this analysis is that the existing taxonomic system needs to be significantly revised by integrating morphology carefully in the context of a robust backbone phylogeny to allow delineation of monophyletic but unambiguously diagnosable genera.

Isolation of a chitinolytic Bacillus licheniformis S213 strain exerting a biological control against Phoma medicaginis infection.

Among nine chitinase-producing strains isolated from Tunisian soil, one isolate called S213 exhibited a potent chitinolytic activity. S213 strain was identified as Bacillus licheniformis by API 50CH system and sequence analysis of its partial 16S ribosomal DNA. Chitinolytic activity was induced either by colloidal chitin or fungal cell walls, and the highest chitinase activity reached at the late stationary phase exhibiting optimal temperature and pH of 50-60 °C and pH 6.0, respectively. SDS-PAGE analysis of the secreted colloidal chitin-induced proteins showed a major protein of about 65 kDa. This protein was identified as chitinase on the basis of its peptide sequences which displayed high homology with chitinase sequence of B. licheniformis ATCC 14580. Moreover, chitinolytic activity containing supernatant inhibited the growth of several phytopathogenic fungi including Phoma medicaginis. Interestingly, S213 strain reduced efficiently the damping-off disease caused by P. medicaginis in Medicago truncatula and should be envisaged in enzyme-based biopesticides against phytopathogen application.

Plant genotype and nitrogen fertilization effects on abundance and diversity of diazotrophic bacteria associated with maize (Zea mays L.)

This research has investigated the effect of two genotypes of maize (Zea mays L.) on the number and diversity of diazotrophic bacteria under different N-fertilization rates. Cultivars NK940 and PAU871 were grown in soil:sand with 0, 40, and 80 kg N ha−1 added as NH4NO3 under controlled conditions for 60 days. The number of diazotrophic bacteria of rhizosphere (S), disinfected roots (R), and stems (T) was determined by most probable number (MPN) method. Diazotrophic bacteria were isolated by N-free media, confirmed by amplification of nifH gene fragments, characterized by amplified 16S rDNA restriction analysis (ARDRA) and (GTG)5 fingerprinting, and identified by partial 16S ribosomal DNA (rDNA) sequence analysis. The diversity of the diazotrophic community from S and R was determined by Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis considering the nifH gene. Maize genotype had a marked effect on the number and diversity of endophytic communities, the NK940 community being more abundant and diverse than that of PAU871. Additionally, N-fertilization increased the number and diversity of diazotrophs in endophytic communities but not in rhizosphere samples. One hundred and six diazotrophs were isolated from S, R, and T samples. Pseudomonas and Enterobacter were the dominant and ubiquitous genera isolated and detected by culture-independent method. T-RFLP showed that the N-fixing populations of the rhizosphere of both cultivars were more diverse than those of inside roots. Principal component analysis (PCA) separated the samples by cultivar and demonstrated a more marked effect of N-fertilization on the NK940 diazotrophic community than on PAU871. These results support the hypothesis that plant genotype and fertilization conditions should be taken into account when searching for N-fixer inocula.

A new species of Plectognathotrema Layman, 1930 (Trematoda: Zoogonidae) from an Australian monacanthid, with a molecular assessment of the phylogenetic position of the genus.

During helminthological examinations of teleost fishes of Ningaloo Reef, Western Australia, a new species of the enigmatic genus Plectognathotrema Layman, 1930 was discovered infecting the pot-bellied leatherjacket Pseudomonacanthus peroni (Hollard). The new species, Plectognathotrema kamegaii n. sp., is formally described, and the status of its four congeners is reviewed. Plectognathotrema hydrolagi Olson, Hanson & Pratt, 1970 is not recognised within the genus, as it differs greatly from its congeners in the form and arrangement of the vitellarium, testes and uterus, and in that it was described from a holocephalan. Plectognathotrema cephalopore Layman, 1930 and P. tsushimaense Kamegai, 1970 are morphologically similar, were described from the same host species and from close geographical localities and are considered synonymous. The new taxon is most similar to P. cephalopore, but differs from it in being much smaller and possessing a distinctly larger oral sucker relative to its body size. The generation of novel partial 28S ribosomal DNA sequences allowed a phylogenetic assessment of the genus and demonstrated that Plectognathotrema clearly belongs in the Zoogonidae Odhner, 1902, rather than in the Fellodistomidae Nicoll, 1909, where it is currently assigned. With the inclusion of Plectognathotrema in the Zoogonidae, there are now three zoogonid genera for which the species are restricted to monacanthid teleosts, i.e. Cephaloporus Yamaguti, 1934, Plectognathotrema and Yamagutiplectognathotrema Parukhin, 1977. Species of these genera share pronounced morphological and ecological affinities and a previously synonymised subfamily, the Cephaloporinae Yamaguti, 1934, is resurrected for them. The host specificity of these trematodes is commented upon.