Identical ITS Sequences Research Articles

First Report of Erysiphe astragali Causing Powdery Mildew on Astragalus mongholicus in China.

Astragalus mongholicus Bge. [A. membranaceus Bge. var. mongholicus (Bge.) Hsiao] is a highly valuable perennial medicinal plant mainly distributed in China, whose dry roots are known as Huangqi in traditional Chinese medicine for reinforcing vital energy, strengthening superficial resistance, and promoting tissue regeneration (Lin et al. 2000). A. mongholicus roots of high quality are produced in Northwest and North China. Since July 2021, powdery mildew outbreaks happened annually on the leaves of A. mongholicus in a plantation (123° 56' 40'' E, 47° 22' 20'' N) in Qiqihar city, Heilongjiang Province, China. Disease incidence reached 100% by October (Fig. 1A-C), causing severe impairment of growth. Powdery mildew spots of circular or irregular shapes emerged on upper surface of leaf, resulting in plentiful lesion specks. Dense white hyphae appeared chaotically intertwined. Hyphae were hyaline and highly flexuous, 5.3 - 10.7 μm in diameter (n = 20). Chasmothecia were globose or slightly ovoid-shaped and turned dark brown when matured. Chasmothecia (diameter: 135.2 - 222.9 μm, n = 20) existed abundantly on the diseased leaves in the fields. Conidiophores were 89.0 - 129.9 μm in length (n = 20) and composed of one cylindrical, straight foot cell, followed by two cells and one to three conidia. Conidia were slim ellipsoid-shaped, occasionally ovoid-shaped, measuring 14.6 - 24.7 μm by 6.4 to10.4 μm, length/width ratio was 1.8 - 3.0 (n = 30). Hyphal appressoria were nipple-shaped and appeared in singular, occasionally in pairs. Unbranched germ tube emerged reaching out of the germinating conidia while forming an acute angle with the long axis. Comprehensively, the pathogen exhibited micro-morphology of the genus Erysiphe. For molecular identification, pathogen was carefully scraped off diseased leaves for DNA extraction. We used the DNA samples of three biological replicates for the sequencing of the ITS rDNA fragment (primers by (White et al. 1990). All the samples resulted in an identical ITS sequence (deposited in GenBank as OQ390098.1). It displayed 99.83% identity with OP806835.1 of an E. astragali voucher collected in Iran (Fig. 1D-M, O). Hence, our pathogen was identified as an E. astragali stain. Additionally, we amplified the Mcm7 sequence (using primers by (Ellingham et al. 2019), deposited as OQ397582.1). We propagated 40-day-old A. mongholicus plants via germinating seeds in pot soil and performed pathogenicity tests. Firstly, we incubated detached healthy leaves of propagated plants with severely symptomatic leaves collected from the fields in petri dishes under saturated moisture content and room temperature. Powdery mildew symptoms emerged on each healthy leaf (n = 5) after two weeks. Further, we infected healthy plants (n = 5) by gently pressing and rubbing symptomatic leaves on each healthy leaf, and kept them in a greenhouse (24 ℃, 80% humidity, 16/8-hour light/dark cycle). After a month, symptoms emerged on a number of leaves of each infected plant. We performed micromorphology observation (Fig. 1N-P) and ITS sequencing to confirm that the results fulfilled Koch's postulates. Powdery mildew caused by E. astragali on A. strictus in Tibet (Wang and Jiang 2023) and on A. scaberrimus in Inner Mongolia (Sun et al. 2023) have been reported. Here we report powdery mildew caused by E. astragali on Astragalus mongholicus for the first time. These Astragalus spp. are all acknowledged to have medicinal values in China but their usages are quite different.

A chromosome-level genome assembly of Zasmidium syzygii isolated from banana leaves.

Accurate taxonomic classification of samples from infected host material is essential for disease diagnostics and genome analyses. Despite the importance, diagnosis of fungal pathogens causing banana leaf diseases remains challenging. Foliar diseases of bananas are mainly caused by 3 Pseudocercospora species, of which the most predominant causal agent is Pseudocercospora fijiensis. Here, we sequenced and assembled four fungal isolates obtained from necrotic banana leaves in Bohol (Philippines) and obtained a high-quality genome assembly for one of these isolates. The samples were initially identified as P. fijiensis using PCR diagnostics; however, the assembly size was consistently 30 Mb smaller than expected. Based on the internal transcribed spacer (ITS) sequences, we identified the samples as Zasmidium syzygii (98.7% identity). The high-quality Zasmidium syzygii assembly is 42.5 Mb in size, comprising 16 contigs, of which 11 are most likely complete chromosomes. The genome contains 98.6% of the expected single-copy BUSCO genes and contains 14,789 genes and 10.3% repeats. The 3 short-read assemblies are less continuous but have similar genome sizes (40.4-42.4 Mb) and contain between 96.5 and 98.4% BUSCO genes. All 4 isolates have identical ITS sequences and are distinct from Zasmidium isolates that were previously sampled from banana leaves. We thus report the first continuous genome assembly of a member of the Zasmidium genus, forming an essential resource for further analysis to enhance our understanding of the diversity of pathogenic fungal isolates as well as fungal diversity.

Note on the genus Nemania (Xylariaceae) - first records and a new species of the genus from Iran.

In a survey of xylarialean fungi in northern Iran, some specimens attributable to the genus Nemania were collected, cultured and sequenced. Morphological evidence and phylogenetic analyses of a combined ITS, LSU, RPB2 and TUB2 gene dataset confirmed the presence of Nemaniadiffusa and N.serpens in Iran for the first time. Furthermore, the new species N.hyrcana, which shows similarities to N.subaenea and its putative synonym N.plumbea, but significantly differs from the latter in its DNA sequences, was encountered. All species are illustrated, described and discussed. In the phylogenetic analyses, for the first time, the overlooked ex-type ITS sequences of the neotype of the generic type, N.serpens and that of the holotype of N.prava, were added to a multi-gene matrix of Nemania. This revealed that the two accessions of N.serpens (HAST 235 and CBS 679.86), for which multigene data are available in GenBank, are misidentified, while the Iranian accession of N.serpens has an almost identical ITS sequence to the neotype, confirming its morphological species identification. The two previously accepted species of Euepixylon, E.udum and E.sphaeriostomum, are embedded within Nemania and are revealed as close relatives of N.serpens, supporting the inclusion of Euepixylon in Nemania.

Secondary DNA Barcodes (CAM, GAPDH, GS, and RpB2) to Characterize Species Complexes and Strengthen the Powdery Mildew Phylogeny

Powdery mildews are a group of economically and ecologically important plant pathogens. In the past 25 years the use of ribosomal DNA (rDNA) in the powdery mildews has led to major taxonomic revisions. However, the broad scale use of rDNA has also revealed multiple species complexes that cannot be differentiated based on ITS + LSU data alone. Currently, there are only two powdery mildew taxonomic studies that took a multi-locus approach to resolve a species complex. In the present study, we introduce primers to sequence four additional regions (CAM, GAPDH, GS, and RPB2) that have the potential to improve support values in both broad and fine scale phylogenetic analyses. The primers were applied to a broad set of powdery mildew genera in China and the United States, and phylogenetic analyses included some of the common complexes. In taxa with nearly identical ITS sequences the analyses revealed a great amount of diversity. In total 154 non-rDNA sequences from 11 different powdery mildew genera were deposited in NCBI’s GenBank, laying the foundation for secondary barcode databases for powdery mildews. The combined and single loci phylogenetic trees constructed generally followed the previously defined species/genus concepts for the powdery mildews. Future research can use these primers to conduct in depth phylogenetic, and taxonomic studies to elucidate the evolutionary relationships of species and genera within the powdery mildews.

New Cantharellus species from South Korea.

In this third contribution involving new Cantharellus species from South Korea, two new species are introduced. In addition, we document a first report of the recently described Japanese Cantharellus anzutake outside of Japan based on identical ITS sequence data. Cantharellus citrinussp. nov. is introduced as a new member of subgenus Cinnabarini, to which the closely related Korean C. albovenosus and Chinese C. phloginus also belong. Cantharellus curvatussp. nov. is introduced as a new member of subgenus Parvocantharellus, in which the Korean C. koreanus was recently placed. The respective placements of the new taxa are significantly supported by a phylogenetic analysis of sequences from the transcription elongation factor (tef-1).

Diversity and taxonomy of the Trifolium lupinaster polymorphic complex in Eastern Europe and Asia

The section Lupinaster of the genus Trifolium (Fabaceae) is characterized by taxonomic complexity due to the high level of polymorphism and polyploidy. Variants of T. baicalense, T. ciswolgense, T. pacificum, and T. spryginii are analyzed using morphological and molecular data to determine their taxonomic status. These species, which grow in Eastern Europe, Ural, Central and East Asia, and the Russian Far East, have been previously separated from T. lupinaster. ITS and trnL-trnF sequences show limited variation. All the investigated species of the T. lupinaster complex have identical ITS sequences, but slight differences due to single substitutions in the trnL-trnF region. The morphological variation of the leaflets and underground organs were characterized. According to these results, T. baicalense, T. ciswolgense, and T. spryginii, should be regarded as synonyms of T. lupinaster, whereas the Far Eastern T. lupinaster subsp. pacificum, distributed along the Pacific seashore of Eurasia (Russia and Japan), is considered as a subspecies of T. lupinaster. In order to discriminate T. lupinaster subsp. pacificum from T. lupinaster, we propose to use a coefficient which characterizes the obovateness of leaflet shape. Our clarification of the taxonomic status of narrow endemics and rare species from the genus Trifolium section Lupinaster has important implications for conservation purposes.

Genetic variability among native and introduced strains of the parasitic nematode Deladenus siricidicola

Sirex noctilio is an invasive Eurasian woodwasp that can kill pine (Pinus spp.) trees and has been introduced to areas of the Southern Hemisphere where plantations of introduced pines are grown. The main method of control of this invasive pest has been introduction and augmentation of a parasitic nematode, Deladenus siricidicola. The strain of D. siricidicola used for biological control of S. noctilio in the Southern Hemisphere originated in Sopron, Hungary. The genotype of D. siricidicola used for biological control sterilizes females of the strain of S. noctilio present in Australia. However, different strains of S. noctilio have been introduced to different geographic areas that have been invaded and different combinations of D. siricidicola and S. noctilio genotypes vary in whether these nematodes sterilize female S. noctilio. Moreover, even in the event of sterilization, partial sterilization can occur, where not all woodwasp eggs are compromised. Sirex noctilio has now invaded North America accidentally, putatively accompanying D. siricidicola, but these host/parasite pairings do not result in female sterilization. More information is needed about the genetic diversity of D. siricidicola both where it is native and introduced. In addition, the host range of these nematodes is necessary to understand to evaluate their potential use in areas where pine communities are native. We collected and evaluated Deladenus parasitizing S. noctilio, S. juvencus, and associated insects in Hungary, Denmark, Spain, and Italy, as well as in the United States. Phylogenetic analyses were unable to fully provide fine resolution, although some community structure was evident. Many D. siricidicola samples from Hungary had identical COI and ITS sequences to the strain of D. siricidicola accidentally introduced to North America putatively when S. noctilio invaded. The same or similar strains of D. siricidicola parasitize two different Sirex species that utilize pines as well as a Sirex parasitoid and a pine-boring beetle, demonstrating some limited variability in host specificity of this species. These results highlight the genetic diversity of Deladenus siricidicola in its native range in Europe.

First record of Erysiphe corylacearum on Corylus avellana in Switzerland and in central Europe

From July to November 2019, a previously unseen powdery mildew disease was found repeatedly on Corylus avellana (hazelnut) in the canton of Ticino in Switzerland. It occurred on leaves of C. avellana shrubs growing in hedges and deciduous forests dominated by Castanea sativa at six locations north of the city of Lugano around Sonvico, at altitudes ranging from 510 to 700 m. Morphological examination revealed small, white, effuse, amphigenous mycelial patches of 0.5-2 cm in diameter (Fig. 1). The ellipsoid to doliiform conidia (30-35 × 15-23 μm) were produced singly. They were mainly found in July and became rare in autumn. In contrast, the gregarious chasmothecia were common throughout the observation time. They measured 80-120 μm in diameter, had up to 14 appendices (60-100 μm long) with multiple dichotomously branched tips, and contained 2-5 obovoid asci (40-60 × 30-50 μm) with up to 8 ellipsoid ascospores (14-20 × 7-14 μm) (Fig. 2). These characters correspond to the description of E. corylacearum in Braun & Cook (3). The morphological identification was confirmed by sequencing the ITS-region of rDNA using three samples and performing a phylogenetic analysis as described in Beenken (2). PCR was done with the Erysiphales-specific primer pair PMITS1/PMITS2 (Cunnington et al., 4) to eliminate co-amplification of the fungus Ampellomyces quisqualis that parasitises E. corylacearum, as preliminary molecular and microscopic investigations on the Swiss material had shown. Obtained sequences were deposited in GenBank (Accession Nos. MN822721-MN822723). Voucher specimens were deposited in the fungal herbarium of ETH Zurich (ZT Myc 59971-ZT Myc 59973). The molecular phylogenetic analysis showed that the Swiss strains had identical ITS sequences to those of E. corylacearum from Azerbaijan, Georgia, Iran and Turkey and differed only in a few base pairs from those from China, Iran and Japan (Fig. 3). Erysiphe corylacearum is native to east Asia and has been reported from USA (Ohio) and Canada but until now it has been unknown from central Europe (Braun & Cook 3). Recently, Heluta et al. (5) reported its occurrence on C. avellana from Ukraine, but without molecular validation. The species has been reported as an invasive pathogen from hazelnut orchards in Turkey, Iran and Georgia where it caused serious damage (Arzanlou et al., 1, Meparishvili et al., 6). Despite active searching for E. corylacearum in Switzerland, it has not yet been found outside the one small area in the canton of Ticino. The pathogen differs from the native hazelnut powdery mildew Phyllactinia guttata, with which it sometimes co-occurs, by forming mycelia on the upper leaf surface and having smaller chasmothecia with branched appendices.

Taxonomy of the Verrucaria kalenskyi – V. xyloxena species complex in Finland

The taxonomy of species related to Verrucaria kalenskyi and V. xyloxena in Finland was studied using morphological characters and analyses of the ITS region. Nine species are accepted as part of the V. kalenskyi – V. xyloxena -complex: V. danica, V. inverecundula sp. nov., V. juankoskiensis sp. nov., V. kalenskyi, V. kiskoensis sp. nov., V. raesaenenii sp. nov., V. tallbackaensis sp. nov., V. aff. trabicola and V. xyloxena. The species group is characterised by relatively small perithecia and spores, a predominantly brown non-areolate thallus and their similarity in the nuclear ITS region. Only V. inverecundula differs from all other species in this group by a pale endolithic thallus and shorter involucrellum. All species are restricted to calcareous rocks and/or pebbles except V. xyloxena, which occurs mainly on calcareous soils, and V. aff. trabicola, which is epiphytic. Although morphologically similar V. modica sp. nov. is not part of the V. kalenskyi – V. xyloxena complex according to our ITS-genetree. In the ITS all other taxa of the complex form a strongly supported monophyletic clade. Verrucaria kalenskyi and V. xyloxena share almost identical ITS sequences, but are morphologically distinct. Most species treated in this study resemble V. kalenskyi but differ from that species in the following features: Verrucaria danica has a fimbriate prothallus, smaller perithecia and thinner involucrellum, V. juankoskiensis has broader spores, a poorly developed thallus and more sparsely occurring perithecia, V. kiskoensis has a thicker thallus and narrower spores, V. modica has a thicker involucrellum, V. raesaenenii has a fimbriate prothallus and perithecia often leaving pits in the substratum, and V. tallbackaensis has thalline covered, more sparsely occurring and slightly larger perithecia. Occurrences of V. xyloxena on calcareous pebbles are confirmed. Most studied species are very rare in Finland except for V. kalenskyi and V. xyloxena. Occurrences of V. danica, V. juankoskiensis, V. kalenskyi and V. raesaenenii are also confirmed from Norway. Verrucaria amylacea and V. anceps are excluded from the Finnish lichen biota. A lectotype is selected for V. danica.

The root-symbiotic Rhizoscyphus ericae aggregate and Hyaloscypha (Leotiomycetes) are congeneric: Phylogenetic and experimental evidence.

Data mining for a phylogenetic study including the prominent ericoid mycorrhizal fungus Rhizoscyphus ericae revealed nearly identical ITS sequences of the bryophilous Hyaloscypha hepaticicola suggesting they are conspecific. Additional genetic markers and a broader taxonomic sampling furthermore suggested that the sexual Hyaloscypha and the asexual Meliniomyces may be congeneric. In order to further elucidate these issues, type strains of all species traditionally treated as members of the Rhizoscyphus ericae aggregate (REA) and related taxa were subjected to phylogenetic analyses based on ITS, nrLSU, mtSSU, and rpb2 markers to produce comparable datasets while an in vitro re-synthesis experiment was conducted to examine the root-symbiotic potential of H. hepaticicola in the Ericaceae. Phylogenetic evidence demonstrates that sterile root-associated Meliniomyces, sexual Hyaloscypha and Rhizoscyphus, based on R. ericae, are indeed congeneric. To this monophylum also belongs the phialidic dematiaceous hyphomycetes Cadophora finlandica and Chloridium paucisporum. We provide a taxonomic revision of the REA; Meliniomyces and Rhizoscyphus are reduced to synonymy under Hyaloscypha. Pseudaegerita, typified by P. corticalis, an asexual morph of H. spiralis which is a core member of Hyaloscypha, is also transferred to the synonymy of the latter genus. Hyaloscypha melinii is introduced as a new root-symbiotic species from Central Europe. Cadophora finlandica and C. paucisporum are confirmed conspecific, and four new combinations in Hyaloscypha are proposed. Based on phylogenetic analyses, some sexually reproducing species can be attributed to their asexual counterparts for the first time whereas the majority is so far known only in the sexual or asexual state. Hyaloscypha bicolor sporulating in vitro is reported for the first time. Surprisingly, the mycological and mycorrhizal sides of the same coin have never been formally associated, mainly because the sexual and asexual morphs of these fungi have been studied in isolation by different research communities. Evaluating all these aspects allowed us to stabilize the taxonomy of a widespread and ecologically well-studied group of root-associated fungi and to link their various life-styles including saprobes, bryophilous fungi, root endophytes as well as fungi forming ericoid mycorrhizae and ectomycorrhizae.

Occurrence of anisakid parasites in marine fishes and whales off New Caledonia.

Anisakid nematodes are the most infamous parasites occurring in seafood with ability to infect humans. In the present study, the infective stages of five anisakid larval types, including Anisakis types I and III, Terranova types I and II and Contracaecum larval type, as well as adult Anisakis paggiae are reported from 16 host species from New Caledonian waters. The specific identity of the larval types was investigated using ITS sequence data. Anisakis larval types I and III were identified as Anisakis typica and Anisakis brevispiculata, respectively, based on identical ITS sequences. However, the specific identity of the Terranova larval types and Contracaecum larval type remains unknown until a matching ITS sequence from a well-identified adult is available. Several fish host species are reported for the first time for anisakid larval types found in this study. Considering that third-stage larvae of anisakids are known to be the infective stage of the parasite for humans and the popularity of seafood in New Caledonia, presence of these parasites in New Caledonian fish is of high importance in terms of public health and raising awareness among various stakeholders. Although adult nematodes in the present study were identified as Anisakis paggiae, the spicule length is shorter in our specimens and falls within the range reported for Anisakis oceanicus previously reported in Pacific waters from black fish (genus Globicephala) and later synonymised with Anisakis physeteris. However, our specimens are different from A. physeteris in morphology of ventriculus. Anisakis paggiae has been reported from whales in southern hemisphere and this is the first report from the Pacific regions.

Eucalypt powdery mildew caused by Podosphaera pannosa in Brazil

Eucalypt powdery mildew is an important disease in greenhouses and clonal hedges of Eucalyptus spp. in Brazil, which can cause leaf and shoot distortion, shoot discoloration, and growth reduction that results in production losses. Because reliable information regarding the causal agent of the disease is lacking, this study used ITS and 28S rDNA sequencing and morphological analyses to identify the powdery mildew pathogen that occurrs in eucalypt nurseries within different regions in Brazil. Based on the results of morphological characteristics and phylogenetic analyses, the pathogen isolates were identified as Podosphaera pannosa, also known as the rose powdery mildew pathogen. Cross inoculations with pathogen isolates from rose and eucalypt demonstrated that P. pannosa can infect both host species. The ITS sequence-based phylogeny showed that 42 sequences generated in this study were comprised within a single clade containing P. pannosa, which was supported by a posterior probability of 88%. Identical ITS sequences were obtained from all 42 pathogen isolates, which suggests a clonal population.

Phellinus noxius: molecular diversity among isolates from Taiwan and its phylogenetic relationship with other species of Phellinus based on sequences of the ITS region

BackgroundAnalysis of phylogenetic relationship of 91 isolates of Phellinus noxius obtained from 46 plant species in Taiwan did not show distinct grouping based on ITS sequences.Results However, the ITS nucleotides showed 20 different kinds of variations including single nucleotide polymorphisms, deletion and insertion in ITS1 and ITS2, but none in 5.8 S. The Taiwanese isolates of P. noxius were dividable into long (type L), median (type M) and short (type S) groups based on ITS sequence length. Two isolates with identical ITS sequence belonged to types L. Type M with 72 isolates was further divided into 33 subtypes, while types S with 17 isolates was further divided into two subtypes.ConclusionPhylogenetic analysis of ITS sequences among Phellinus species showed that isolates of P. noxius were in the same clade distinctly separated from other Phellinus species.

Occurrence of ascaridoid nematodes in selected edible fish from the Persian Gulf and description of Hysterothylacium larval type XV and Hysterothylacium persicum n. sp. (Nematoda: Raphidascarididae)

Despite several reports on the presence of the potentially zoonotic nematodes among edible fishes in the Persian Gulf, there is still no study on the specific identification of these parasites or their genetic characterisation. In the present study, a total of 600 fish belonging to five popular species of fish in the region, including Otolithes ruber, Psettodes erumei, Saurida tumbil, Scomberomorus commerson and Sphyraena jello were examined for infection with nematode parasites. Detailed microscopy of nematodes found in the present study followed by characterisation of the first and second internal transcribed spacers (ITS-1 and ITS-2, respectively) showed that they belong to five distinct taxa that could be potentially zoonotic. Anisakis type I was found in four species of fish, had identical ITS sequences as Anisakis typica previously reported in Australian waters and was different from those reported in the Nearctic. Hysterothylacium type VI in the present study was morphologically similar to those previously described from Australasian waters and ITS sequences were identical among Australian specimens and those found in the present study. Another Hysterothylacium larval type was also found in the present study which had identical ITS sequences and similar morphology to those previously reported and identified as H. amoyense in China Sea. Since no ITS sequence data from a well identified adult H. amoyense with an identifiable museum voucher number is yet available and due to some other issues discussed in the article we suggest assignment of this larval type from the China Sea and the Persian Gulf to H. amoyense is doubtful until future studies on a well identified male specimen of H. amoyense or other species reveals the specific identity of this larval type. We propose to refer to this larval type as Hysterothylacium larval type XV. In the present study we also describe a new species, Hysterothylacium persicum and discuss how to differentiate it from closely related species. We also found some adult females with distinct morphology and ITS sequence but due to lack of male specimens they have been referred as Hysterothylacium sp. in this paper. They had the same ITS sequence data as Hysterothylacium larval type VI. This study shows the presence of a relatively broad diversity of potentially zoonotic nematodes in edible fish of the Persian Gulf. Therefore educational campaigns for public and local health practitioners are suggested to protect consumers from becoming infected with these parasites.

Interactions between Karlodinium veneficum and Prorocentrum donghaiense from the East China Sea

The dinoflagellate Prorocentrum donghaiense is a dominant harmful algal bloom (HAB) species on the East China Sea (ECS) coast. The co-occurrence of Karlodinium veneficum with P. donghaiense is often observed and can later develop into dense blooms. However, the role of K. veneficum in P. donghaiense population dynamics is unknown. In the current study, three K. veneficum (GM1, GM2, and GM3) strains were isolated from the ECS with one (GM1) from a mixed, dense bloom of P. donghaiense and other HAB species. All three isolates had identical ITS sequences that were concordant with the species designation. Unique karlotoxin congeners were isolated from one strain (GM2). The sterol compositions of P. donghaiense and K. veneficum were consistent with sensitivity to karlotoxin in the former and insensitivity in the latter. Additional experimentation showed that: (1) in monocultures, higher growth rate of P. donghaiense than K. veneficum is observed in nutrient-enriched and nutrient-depleted media. In co-cultures, the growth of P. donghaiense is inhibited; (2) feeding on P. donghaiense by K. veneficum is clearly demonstrated by fluorescent dye tracking; and (3) the isolated karlotoxin is lethal to P. donghaiense in a concentration-dependent manner. From these studies we propose that K. veneficum may play a negative role in P. donghaiense bloom maintenance and that P. donghaiense may in turn be a bloom initiator as a prey item for K. veneficum.

Proteomic-based biotyping reveals hidden diversity within a microalgae culture collection: An example using Dunaliella

Accurate and defendable taxonomic identification of microalgae strains is vital for culture collections, industry and academia; particularly when addressing issues of intellectual property. We demonstrate the remarkable effectiveness of Matrix Assisted Laser Desorption Ionisation Time of Flight Mass Spectrometry (MALDI-TOF-MS) biotyping to deliver rapid and accurate strain separation, even in situations where standard molecular tools prove ineffective. Highly distinctive MALDI spectra were obtained for thirty two biotechnologically interesting Dunaliella strains plus strains of Arthrospira, Chlorella, Isochrysis, Tetraselmis and a range of culturable co-occurring bacteria. Spectra were directly compared with genomic DNA sequences (internal transcribed spacer, ITS). Within individual Dunaliella isolates MALDI discriminated between strains with identical ITS sequences, thereby emphasising and enhancing knowledge of the diversity within microalgae culture collections. Further, MALDI spectra did not vary with culture age or growth stage during the course of the experiment; therefore MALDI presents stable and accurate strain-specific signature spectra. Bacterial contamination did not affect MALDI’s discriminating power. Biotyping by MALDI-TOF-MS will prove effective in situations wherein precise strain identification is vital, for example in cases involving intellectual property disputes and in monitoring and safeguarding biosecurity. MALDI should be accepted as a biotyping tool to complement and enhance standard molecular taxonomy for microalgae.

Phytophthora Crown and Root Rot of Zucchini Squash in Almería, Spain.

Zucchini (Cucurbita pepo) is intensively cropped in approximately 4,500 ha of plastic houses in southern Spain. In 2008 to 2009, Consul, Cronos, and Tosca zucchini plants showed symptoms of leaf wilting, basal stem necrosis, and plant death. Incidences of dead plants were 20 to 30% and these plants were distributed in clusters. Phytophthora capsici Leonian was isolated from the basal stems of symptomatic plants, using PDA and cornmeal agar amended with a pimaricin, ampicillin, and rifampicin. Five resultant isolates (PCl-211, PCl-221, PCl-611, PCl-612, and PCl-811) on lima beans agar (LBA) produced white mycelia with lemon-shaped and papillate sporangia borne on long pedicels, but no oospores or chlamydospores. These isolates had an identical ribosomal DNA ITS sequence, matching with that of P. capsici in GenBank. The sequences of two representative isolates, PCl-211 and PCl-811, were deposited in GenBank with accession nos. KC662328 and KC688317, respectively. The pathogenicity of these five isolates was tested on zucchini cv. Consul in 1-liter containers filled with vermiculite in May and September of 2009. Plants were inoculated at the 2 to 3 true-leaf stage. Plates with LBA fully covered with colony of each isolate were separately blended and homogenized with 300 ml of sterile distilled water. Inocula were poured around stem at 50 ml per plant. Each experiment had three replicates and four plants per replicate. Treatments with different isolates were arranged in a randomized complete block design. In both experiments, 12 uninoculated plants served as controls. Test plants were maintained for a month following inoculation in a greenhouse with mean temperatures ranging from 21.9 to 27.9°C and from 20.7 to 24.6°C for the May and September experiments, respectively. The first wilting occurred 5 days after inoculation. At the end of the May experiment, all control plants and those inoculated with PCl-221 remained asymptomatic while 83.3% of those inoculated with PCl-211 and 100% of those with the other isolates were dead. Inoculated plants exhibited crown and root rots, excluding the secondary roots. In the September experiment, 83.3% and 33.3% of plants inoculated with PCl-211 and PCl-221, respectively, were symptomatic, while all plants inoculated with the other isolates were dead. The control plants remained healthy. The pathogen was consistently recovered from symptomatic plants in both experiments. Although P. capsici was reported in peppers (Capsicum annuum) in several provinces of Spain (1), to our knowledge, this is the first report of P. capsici as the causal agent of crown rot in zucchini plants in plastic houses in the Almería Province of Spain, one of the world's largest concentrations of greenhouses. Reference: (1) J. L. Andrés et al. Span J Agric Res 3:326, 2005.

First record of Pentapharsodinium (Peridiniales, Dinophyceae) in the China Sea, with description of Pentapharsodinium dalei var. aciculiferum

SummaryThe dinophyte genus Pentapharsodinium shares an identical plate formula with that of Ensiculifera, but lacks a spine on the first cingulum plate and a median sulcal plate (Sm). Herein we established eight strains by incubating single cysts of Pentapharsodinium dalei from the Chukchi Sea, Yellow Sea, and South China Sea. The six strains from the Chukchi Sea and Yellow Sea fit the description of P. dalei, and they share identical internal transcribed spacer region (ITS1, ITS2 and 5.8S rDNA) sequences. The two other strains (G96 and PDFC01) from the South China Sea are morphologically similar to P. dalei, but they possess a spine on the anterior sulcal plate and an additional Sm plate. These two strains were described as P. dalei var. aciculiferum. They share identical ITS sequences and differ from P. dalei strains PDCH01 (Yellow Sea) and SZN19 (Norway origin) at 20 and 26 positions (96.40% and 95.32% similarity) respectively. Maximum‐likelihood and Bayesian inference resolved a well supported clade consisting of P. dalei and P. dalei var. aciculiferum. Our results reveal that cysts of P. dalei might comprise several morphologically, genetically, and ecologically different varieties.

Morphology, phylogeny and toxin profiles of Gymnodinium inusitatum sp. nov., Gymnodinium catenatum and Gymnodinium microreticulatum (Dinophyceae) from the Yellow Sea, China

Four Gymnodinium species have previously been reported to produce microreticulate cysts. Worldwide, Gymnodinium catenatum strains are conservative in terms of larger subunit (LSU) rDNA and internal transcribed spacer region (ITS) sequences, but only limited information on the molecular sequences of other species is available. In the present study, we explored the diversity of Gymnodinium by incubating microreticulate cysts collected from the Yellow Sea off China. A total of 18 strains of Gymnodinium, from three species, were established. Two of these were identified as Gymnodinium catenatum and Gymnodinium microreticulatum, and the third was described as a new species, Gymnodinium inusitatum. Motile cells of G. inusitatum are similar to those of Gymnodinium trapeziforme, but they only share 82.52% similarity in LSU sequences. Cysts of G. inusitatum are polygonal in shape, with its microreticulate wall composed of approximately 14 concave sections. G. microreticulatum strains differ from each other at 69 positions (88.00% similarity) in terms of ITS sequences, whereas all G. catenatum strains share identical ITS sequences and belonged to the global populations. Phylogenetic analyses, based on LSU sequences, revealed that Gymnodinium species that produce microreticulate cysts are monophyletic. Nevertheless, the genus as a whole appears to be polyphyletic. Paralytic shellfish toxins (PSTs) were found in all G. catenatum strains tested (dominated by 11-hydroxysulfate benzoate analogs and N-sulfocarmaboyl analogs) but not in any of the G. microreticulatum and G. inusitatum strains. Our results support the premise that cyst morphology is taxonomically informative and is a potential feature for subdividing the genus Gymnodinium.

Characterization and virulence of Beauveria spp. recovered from emerald ash borer in southwestern Ontario, Canada

The emerald ash borer (EAB), Agrilus planipennis (Coleoptera: Buprestidae), is an invasive wood boring beetle that is decimating North America’s ash trees (Fraxinus spp.). To find effective and safe indigenous biocontrol agents to manage EAB, we conducted a survey in 2008–2009 of entomopathogenic fungi (EPF) infecting EAB in five outbreak sites in southwestern Ontario, Canada. A total of 78 Beauveria spp. isolates were retrieved from dead and mycosed EAB cadavers residing in the phloem tissues of dead ash barks, larval frass extracted from feeding galleries under the bark of dead trees. Molecular characterization using sequences of the ITS, 5′ end of EF1-α and intergenic Bloc region fragments revealed that Beauveria bassiana and Beauveria pseudobassiana were commonly associated with EAB in the sampled sites. Based on phylogenetic analysis inferred from ITS sequences, 17 of these isolates clustered with B. bassiana, which further grouped into three different sub-clades. However, the combined EF1-α and Bloc sequences detected five genotypes among the three sub-clades. The remaining 61 isolates clustered with B. pseudobassiana, which had identical ITS sequences but were further subdivided into two genotypes by variation in the EF1-α and Bloc regions. Initial virulence screening against EAB adults of 23 isolates representing the different clades yielded 8 that produced more than 90% mortality in a single concentration assay. These isolates differed in virulence based on LC50 values estimated from multiple concentration bioassay and based on mean survival times at a conidia concentration of 2×106 conidia/ml. B. bassiana isolate L49-1AA was significantly more virulent and produced more conidia on EAB cadavers compared to the other indigenous isolates and the commercial strain B. bassiana GHA suggesting that L49-1AA may have potential as a microbiological control agent against EAB.