Diagnostics of phytopathogen fungi <Emphasis Type="Italic">Septoria tritici</Emphasis> and <Emphasis Type="Italic">Stagonospora nodorum</Emphasis> by fluorescent amplification-based specific hybridization (FLASH) PCR

A PCR system in the fluorescent amplification-based specific hybridization (FLASH) format was developed for the detection and identification of two important wheat pathogenic fungi Septoria tritici (teleomorph of Mycosphaerella graminicola) and Stagonospora nodorum (teleomorph of Phaeosphaeria nodorum), which cause spots on leaves and glumes, respectively. The pathogen detection system is based on the amplification of a genome fragment in the internal transcribed spacer 1 (ITS1) region and a site encoding the 5.8S ribosomal RNA. The forward primers to ITS1 and a universal reverse primer and a beacon type probe to the 5.8S ribosomal RNA region were chosen to provide the detection of the products in the FLASH format. This system was tested on different isolates of the pathogens, and on infected soil, leaf, and seed samples.     

Taxonomic notes on some species of the Cercospora complex (VI)

The new species Cercosporella pergulariae sp. nov., Pseudocercospora catalpicola sp. nov., Sirosporium rhamnigenum sp. nov., and Spiropes desmodiicola sp. nov. are described and Cercospora commelinicola Chupp is validated. Cercostigmina curta (Syd.) comb, nov., Eriocercosporella vitis-heterophyllae (Henn.) comb, nov., Mycovellosiella lactucae (Henn.) comb, nov., M. trichostemmatis (Henn.) comb, nov., M. tylophorae (Hansf.) comb, nov., Passalora caespitosa (Ellis & Everh.) comb, nov., P. chionanthi (Ellis & Everh.) comb, nov., Prathigada condensata (Ellis & Kellerm.) comb, nov., Pseudocercospora carrii (Barthol.) comb, nov., P. glaucescens (G. Winter) comb, nov., P. pamelae-ellisiae (G.P. Agarwal & N.D. Sharma) comb, nov., Pseudophaeoramularia angolensis (T. Cavalho &O. Mendes) comb, nov., Sporidesmium seminale (Ellis & Everh.) comb, nov., and Stenella praelonga (Syd.) comb. nov. are introduced. Cercospora litseae Henn. is reduced to synonym with Mycovellosiella litseae Meenu et al., and a second collection of Cercosporella indica from Vietnam is recorded.     

Phytophthora ×stagnum nothosp. nov., a New Hybrid from Irrigation Reservoirs at Ornamental Plant Nurseries in Virginia

A novel Phytophthora species was frequently recovered from irrigation reservoirs at several ornamental plant production facilities in eastern Virginia. Initial sequencing of the internal transcribed spacer (ITS) region of this species generated unreadable sequences due to continual polymorphic positions. Cloning and sequencing the ITS region as well as sequencing the mitochondrially encoded cytochrome c oxidase 1 and beta-tubulin genes revealed that it is a hybrid between P. taxon PgChlamydo as its paternal parent and an unknown species genetically close to P. mississippiae as its maternal parent. This hybrid has some diagnostic morphological features of P. taxon PgChlamydo and P. mississippiae. It produces catenulate hyphal swellings, characteristic of P. mississippiae, and chlamydospores, typical of P. taxon PgChlamydo. It also produces both ornamented and relatively smooth-walled oogonia. Ornamented oogonia are another important diagnostic character of P. mississippiae. The relatively smooth-walled oogonia may be indicative of oogonial character of P. taxon PgChlamydo. The new hybrid is described here as Phytophthora ×stagnum.     

Analysis of internal transcribed spacer1 (ITS1) region of rDNA for genetic characterization of Paramphistomum sp.

Paramphistomosis is the most prevalent disease of domestic ruminants, causing heavy economic loss in many countries across the world. The morphological identification of these parasites is difficult, therefore molecular characterization is used to discriminate Paramphistomum species. The present study was conducted to identify Paramphistomum sp. at Mardan District, Khyber Pakhtunkhwa (KPK), Pakistan. All samples of these rumen flukes were collected from buffalo. The gDNA was isolated from the adult parasites and the ITS1 region was amplified for the sequence analysis. All flukes had 100% similarity and there was no intraspecific variation. The Blast results showed that all flukes were P. cervi as they form a single cluster with P. cervi reported from China. The results of the ITS1 sequences of the present study with reference sequencing from China showed eight specific SNPs. This was the first study in which P. cervi was genetically characterized through the ITS1 region of rDNA at District Mardan, Pakistan. It can also be used as a marker for the genetic identification of Paramphistomum species.     

Athelia termitophila sp. nov. is the teleomorph of the termite ball fungus Fibularhizoctonia sp.

A new species of Athelia, A. termitophila, from Japan is described and illustrated on the basis of morphological and phylogenetic analyses. Basidiomes of this species are characterized by having hyphae sometimes with clamp connections at the septa, basidia without clamp connections at the basal septa, and ellipsoid to ovoid basidiospores measuring 4.5–6 × 3–4.5 μm. In culture, mycelia produce pale brown, orange-brown to brown, globose sclerotia measuring 0.24–0.41 mm diam. The sclerotia are distinctly different in shape and size from those of other Athelia species, and are occasionally found inside the woody substrate beneath basidiomes. They are identical in shape and size to those of Fibularhizoctonia sp., also known as termite balls. Phylogenetic analysis using internal transcribed spacer (ITS) sequence data revealed that A. termitophila is the teleomorph of Fibularhizoctonia sp.     

Syncephalastrum contaminatum,a new species in the Mucorales from Australia

A new species is described in the Mucorales family Syncephalastraceae: Syncephalastrum contaminatum, isolated as an in vitro culture from a laboratory contaminant. The species has variable copies of the internal transcribed spacer (ITS) regions, requiring cloning of these regions prior to Sanger sequencing before subsequent use in phylogenetic comparisons with other fungi. The genome of the strain was sequenced using short paired-reads to yield a draft genome of 28.6 Mb. Syncephalastrum contaminatum is distinguished by diverse DNA sequences at several loci from the other species of Syncephalastrum, including only 81% sequence identity with its ITS regions to that of S. racemosum. Its merosporangium produces four or more asexual spores and the genome sequencing information suggests that the species is heterothallic. The identification of this species highlights the limited knowledge about the early lineages of fungi both in Australia and globally.     

Paradiscogaster flindersi and P. oxleyi n. sp. (Digenea: Faustulidae): overlapping host and geographical distributions in corallivore chaetodontid fishes in the tropical Indo-west Pacific

A total of 2,868 individuals of 47 species of chaetodontids were examined for faustulids at seven major localities in the Tropical Indo-West Pacific (TIWP). Combined morphological and molecular analyses allowed us to describe Paradiscogaster oxleyi n. sp. from three localities in the TIWP and in three host species, Chaetodon lunulatus Quoy & Gaimard (type-host), C. ornatissimus Cuvier and C. meyeri Bloch & Schneider. Molecular analysis of the ITS2 region of rDNA from two host species and three localities supports the morphology-based conclusion that P. oxleyi n. sp. is the same species at the three localities. Paradiscogaster flindersi Bray, Cribb & Barker, 1994 is reported from three new localities in the TIWP and is now known from 13 chaetodontid species. Sequences from samples consistent with P. flindersi differed from those from P. oxleyi n. sp. in 11–12 base pairs. The host ranges of the two species overlap broadly. Neither species was found in French Polynesia but both were found at Swain Reefs on the Great Barrier Reef. Only one of the two species was found at each of the five other sites. Both species occur almost exclusively in specialist corallivores allowing the inference that the metacercariae occur in corals. Finally, a key to the species of Paradiscogaster is provided.     

The influence of some symbionts on the shell-selection behaviour of the hermit crabs, Pagurus pollicarus and Pagurus longicarpus

The influence of some symbionts on the shell-selection by the hermit crabs Pagurus pollicarus and P. longicarpus was examined by placing individual hermit crabs with two similar shells in a choice situation and recording the shell occupied after 12 hr. One shell contained a symbiont species and the other did not. The results indicated that organisms normally found on or in empty shells influence the shell-section by these species of hermit crab. P. pollicarus preferred shells occupied by the sea anemone Calliactis tricolor or by the hydroid Hydractina echinata as opposed to bare shells. P. longicarpus also preferred shells with H. echinata. Both crab species rejected shells with the barnacle Balanus amphitrite. Shells containing the molluscs Crepidula fornicata or C. plana were rejected by the smaller hermit crab P. longicarpus. These molluscs appeared to exert no influence on P. pollicarpus unless they were large or abundant, at which point their weight or occlusion of available space possibly has negative effects on the crab.     

Genetic diversity of Pseudo-nitzschia brasiliana (Bacillariophyceae) from Malaysia

To clarify the genetic diversity of a potentially toxic pennate diatom, Pseudo-nitzschia brasiliana found in Malaysian waters, 30 strains of P. brasiliana were established into clonal culture since May 2008. The ultrastructure of these strains was examined for confirmation of species identification. The genetic marker, internal transcribed spacer (ITS) of nuclear-encoded ribosomal DNA was used to examine the genetic diversity of P. brasiliana isolated from different geographical localities. The ITS sequences of P. brasiliana were highly conserved in their secondary structures, with five helices in the first internal transcribe spacer (ITS1) and four universal helices in the second internal transcribe spacer (ITS2) with a pseudo-helix. No compensatory base change was observed among the strains examined. Genetic divergences among the Malaysian strains ranged from 0.07 to 0.54%. The present study revealed a high genetic homogeneity of Malaysian P. brasiliana strains.     

Morphological and molecular study of <Emphasis Type="Italic">Longicollum pagrosomi</Emphasis> Yamaguti, 1935 (Acanthocephala: Pomphorhynchidae) from the barred knifejaw <Emphasis Type="Italic">Oplegnathus fasciatus</Emphasis> (Temminck &amp; Schlegel) (Perciformes: Oplegnathidae) in the East China Sea

Longicollum pagrosomi Yamaguti, 1935 (Acanthocephala: Pomphorhynchidae) collected from the barred knifejaw Oplegnathus fasciatus (Temminck & Schlegel) (Perciformes: Oplegnathidae) in the East China Sea (off Zhoushan Islands) was studied using light and scanning electron microscopy. The SEM observations revealed for the first time the presence of about 28 well-developed sensory papillae arranged in a circle on the copulatory bursa. In addition, L. pagrosomi was characterised using molecular methods by sequencing of the internal transcribed spacer (ITS) of the ribosomal DNA based on the newly collected material. Longicollum pagrosomi is the first species of the genus with the ITS region sequenced for the purpose of species identification. These new morphological and molecular data contributed to a reliable and accurate specific identification and differentiation of species.     

Intraspecific variability of Pythium myriotylum isolated from cocoyam and other host crops

Intraspecific variability within 51 isolates of Pythium myriotylum from cocoyam (Xanthosoma sagittifolium) and other host crops was analysed using optimum growth temperature, esterase banding patterns, AFLPs, rDNA–ITS sequencing, and virulence to cocoyam. P. myriotylum isolates virulent to cocoyam could easily be differentiated from other isolates of P. myriotylum by their optimum growth temperature. Isolates from cocoyam grew best at 28 °C with no growth at 37 °C, while P. myriotylum isolates from other host crops had their optimum growth temperature at 37 °C. Esterases produced consistent zymograms with 18 discrete esterase markers, but no monomorphic markers were produced for isolates virulent to cocoyam. Isozyme profiles based on esterase analysis showed that isolates that infect cocoyam plantlets formed a related group, irrespective of their geographic origin. P. myriotylum isolates from other host plants also grouped together, but could clearly be distinguished from the cocoyam cluster. AFLPs produced 189 scorable bands for the cocoyam isolates, of which 77 % are monomorphic. Phenetic analysis of AFLP data grouped all isolates originating from cocoyam together except for the isolates C103-04, CMR17, CMR22, and CMR25. These isolates regrouped with isolates of Pythium myriotylum from other host crops or the outgroup and were found not to be pathogenic for cocoyam. ITS sequences of isolates of P. myriotylum from cocoyam were 99.1–99.7 % identical to sequences deposited in GenBank. However, alignments of ITS sequences revealed a base transition at position 824 from adenine in typical isolates of P. myriotylum to guanine in isolates that could infect cocoyam plantlets. In a limited pathogenicity test, all isolates from cocoyam having guanine at position 824 were able to infect tissue culture derived cocoyam but not those exhibiting adenine. This study demonstrates for the first time, molecular evidence that isolates of P. myriotylum that infect cocoyam are distinct from P. myriotylum isolates from other crops and have developed a certain degree of host adaptation.     

Putative natural hybrid between Puccinia lagenophorae and an unknown rust fungus on Senecio madagascariensis in KwaZulu-Natal,South Africa

Several specimens of an aecial rust fungus were collected on Senecio madagascariensis during a field survey carried out in KwaZulu-Natal, South Africa. As telia were not present in the specimens collected, DNA sequence analyses were undertaken to determine the identity of the rust species. ITS and β-tub1 sequencing confirmed that one of the isolates recovered is Puccinia lagenophorae sensu lato. On the other hand, sequencing and RFLP analysis revealed the presence of two divergent copies of ITS and β-tub1 in all the other six isolates investigated. In both phylogenetic trees, one copy of the gene region grouped within a well supported clade with sequences of P. lagenophorae accessions from different geographical origins and hosts, and the Australian rusts Puccinia saccardoi and Puccinia stylidii. The other copy of these gene regions grouped within a separate clade comprising European accessions of Puccinia dioicae (ITS) and Uromyces sommerfeltii (β-tub1) that occur on Asteraceae hosts. Multiple copies of these gene regions were not observed in Australian isolates of P. lagenophorae. Our study provides some evidence that an interspecific hybrid rust fungus, with P. lagenophorae as one of its parents, may occur on S. madagascariensis in South Africa. The identity of the other parent remains unknown.     

Population survey of phytoseiid mites and spider mites on peach leaves and wild plants in Japanese peach orchard

A population survey of phytoseiid mites and spider mites was conducted on peach leaves and wild plants in Japanese peach orchards having different pesticide practices. The phytoseiid mite species composition on peach leaves and wild plants, as estimated using quantitative sequencing, changed during the survey period. Moreover, it varied among study sites. The phytoseiid mite species compositions were similar between peach leaves and some wild plants, such as Veronica persica, Paederia foetida, Persicaria longiseta, and Oxalis corniculata with larger quantities of phytoseiid mites, especially after mid-summer. A PCR-based method to detect the ribosomal ITS sequences of Tetranychus kanzawai and Panonychus mori from phytoseiid mites was developed. Results showed that Euseius sojaensis (specialized pollen feeder/generalist predator) uses both spider mites as prey in the field.     

Phenology,pollination, and breeding system of the threatened tree Caesalpinia echinata Lam. (Fabaceae), and a review of studies on the reproductive biology in the genus

Caesalpinia echinata (brazilwood) is a threatened tree endemic to the Brazilian Atlantic forest with a global importance as it is worldwide used to manufacture the most desirable bows for violins, violas, and cellos. Although there is an international initiative for its conservation, there is a lack of studies on the reproductive biology of the species, as for the genus, a fundamental understanding for the conservation of any species. In this paper, we investigated the phenology, pollination, and breeding system of C. echinata, presenting some conservation guidelines for the species, together with a review of studies on the reproductive biology in Caesalpinia s.l. The genus has a complex taxonomic history with recent attempts to reassign its limits. Pending a final resolution of the placing of some species (including C. echinata) our revision covers the traditional Caesalpinia s.l. and includes annotations indicating to which segregate genus each species is now considered to belong, as well as the appropriate combination where this has already been published. Field work was undertaken from October/2004 to December/2006 at the Tapacurá Ecological Station, northeastern Brazil. Flowering occurred mainly in the dry season and the peak of seed dispersal was at the beginning of the wet season. Anthesis is diurnal, lasting 1 day. The flowers are zygomorphic, yellow, sweet-scented, and the effective pollinators were mainly medium-sized to large bees of the genera Centris and Xylocopa, together with the introduced Apis mellifera. Nectar volume and sugar concentration averaged 2.9±1.0 μL and 29.5±9.4%, respectively. The ovary has 1–4 ovules (2.35±0.58) and the pollen/ovule ratio was 5631.2; pollen viability was high (95.9±4.8%). Natural fruit set was low (9.2%) with mature fruits averaging 1.7±0.9 seeds. Results of the controlled pollinations and analysis of pollen tube growth revealed C. echinata presents late-acting self-incompatibility. The pollination biology and breeding system of C. echinata are discussed, together with available data on its genetics and physiology, in terms of best conservation practice for this endangered species. Data on the reproductive biology of the genus are scarce, revealing the predominance of bee pollination and SI system, with the occurrence of late-acting self-incompatibility mechanisms in some species.     

Aspergillus osmophilus sp. nov., and a new teleomorph for A. proliferans

A new species of Aspergillus and a new teleomorph for A. proliferans, both isolated from cereals in Iran, are described using morphological and molecular data. A combined sequence dataset of the ITS region, partial β-tubulin and partial calmodulin genes resolved the relationships of members of section Aspergillus largely in concordance with morphological traits of ascospores. Aspergillus osmophilus sp. nov. is differentiated from the closest species, A. xerophilus by possessing larger ascospores, conidia and associated fruiting bodies. Both species are strongly xerophilic and possess ascospores with lobate-reticulate convex surfaces. The newly discovered teleomorph for A. proliferans is characterized by delicately roughened ascospores with a shallow or distinct furrow and finely roughened to irregular equatorial crests.     

PCR-Based Strategy To Detect and Identify Species of Phaeoacremonium Causing Grapevine Diseases

Species of Phaeoacremonium (especially Phaeoacremonium aleophilum) are associated with two severe diseases in grapevines, Petri disease in young plants and Esca disease in adult plants. Phaeoacremonium species grow slowly on culture medium, and it is difficult to identify these species on the basis of morphological characteristics. Primers Pm1 and Pm2 were designed in the ribosomal DNA internal transcribed spacer (ITS) regions ITS1 and ITS2, respectively. They yielded a single amplicon of 415 bp for nine species of Phaeoacremonium that may occur in grapevines. A nested PCR (using general fungal primers ITS1F/ITS4 in the primary reaction) was developed to detect Phaeoacremonium directly in grapevine wood. Molecular detection was more sensitive than the traditional method of culturing in growth medium was. Identification of Phaeoacremonium species was achieved by digesting the PCR-amplified fragment with the restriction enzymes BssKI, EcoO109I, and HhaI. It was possible to distinguish these species by their restriction fragment length polymorphism patterns, except for Phaeoacremonium viticola and Phaeoacremonium angustius, which had 100% similarity in their ITS region sequences. A species-specific PCR amplification of the partial β-tubulin gene using the primer pair Pbr4_1/T1 and Pbr8/T1 was necessary to differentiate P. angustius from P. viticola, respectively. An easy and fast protocol was developed to detect and identify species of Phaeoacremonium in a few hours. Primers defined here can be used in a plant nursery sanitation program to produce plants free of Phaeoacremonium spp. Use of healthy grapevine plants in new plantations is the most effective measure to manage Petri disease.     

Phylogenetic relationships of Puccinia horiana and other rust pathogens of Chrysanthemum × morifolium based on rDNA ITS sequence analysis

Isolates of the most important Puccinia species that have been reported on Chrysanthemum × morifolium were collected and the sequences of their ribosomal DNA internal transcribed spacers ITS1 and ITS2 were determined and used as phylogenetic markers. The focus of this study was on Puccinia horiana, due to its quarantine status and its impact in commercial chrysanthemum production. Three technical adjustments were needed to reliably obtain the nucleotide sequences starting from fresh or dried samples. The complete rDNA ITS nucleotide sequences of P. horiana, Puccinia chrysanthemi, and Puccinia tanaceti isolates of varying age and geographic origin were determined. We also identified an as yet undescribed Puccinia species on six old herbarium samples from chrysanthemum. This new species is morphologically similar to P. chrysanthemi and near identical to recent rust samples from Artemisia tridentata. P. tanaceti could not be confirmed as a pathogen of chrysanthemum. Different rDNA ITS sequences were present in P. horiana, with intra-isolate and inter-isolate variability in the length of three nucleotide repeat regions in the different rDNA tandem copies. We also identified three ITS types within P. horiana, with the rarer types displaying up to 67 bp nucleotide sequence differences. These rarer ITS types were detected at low copy number in all isolates. In general, very little rDNA ITS sequence variation was observed between P. horiana isolates from 1903 and 2003, and among isolates from different continents. Phylogenetic analyses using distance, Maximum Likelihood and Bayesian methods confirmed P. horiana, P. chrysanthemi, and the new Puccinia sp. as well-resolved groups, with P. horiana clustering in the clade where the economically important rust species of the Poaceae are located, and P. chrysanthemi and the new Puccinia sp. clustering in the clade where the majority of the rust fungi with hosts in the Asteraceae is located.     

Molecular phylogenetic status of korean strain of Podosphaera xanthii, a causal pathogen of powdery mildew on Japanese thistle (Cirsium japonicum) in Korea

Powdery mildew diseases are sensitive to climate change and spread can be favored by increased temperature and low moisture. During 2011 to 2012, a powdery mildew disease by a Podosphaera species was observed on the leaves of Japanese thistle (Cirsium japonicum) in Korea. The initial sign of this disease included scattered superficial white mycelia on leaves. As the disease progressed, abundant necrotic black spots exhibiting chasmothecia were formed on the leaves. rDNA ITS and 28S homologies of the fungus (EML-CSPW1) showed 100% identity values with those regions from many strains of P. xanthii (syn. P. fusca) via NCBI BLASTN search.     

Morphological and molecular characterization of a new autochthonous Trichoderma sp. isolate and its biocontrol efficacy against Alternaria sp.

The development of agriculture requires the use of microorganisms in the management of phytopathogens as a way to compensate for the use of chemical pesticides, in order to produce healthy crops. The objective of this study was to characterize a new isolate of Trichoderma sp. based on morphological and molecular features, and its potential ability to control the pathogen Alternaria sp. The antagonistic isolate was isolated from soil samples of potato fields in Guasave Sinaloa, Mexico, whereas the pathogen was collected from infected apple leaves in the orchard “La Escondida” in Guerrero County, Chihuahua, Mexico. For morphological characterization both fungi were grown on solid PDA medium. DNA of Trichoderma sp. was isolated using the CTAB method and PCR analyses were done using ITS1, ITS4 primers resulting in amplified products of 600 bp. These were sequenced, submitted to Genbank (acc. no. MN950427) and used for further phylogenetic analysis through Bayesian inference approach. Five clades were identified and the polytome topography recovered from clade 4 indicates a high genetic similarity with T. asperellum. A BLAST examination of the resulting sequence in GenBank showed 98.11% similarity with T. asperellum. This result together with the morphological and the phylogenetic analyses indicates that the isolate belongs to Trichoderma asperellum Samuels, Lieckfeldt & Nirenberg. Biocontrol tests of this isolate showed inhibition of Alternaria sp. between 50% and 93%. These results are essential for biodiversity research and give some new possibilities for pest management.