Molecular and morphological characterisation of <Emphasis Type="Italic">Aphelenchoides paraxui</Emphasis> n. sp. (Nematoda: Aphelenchoididae) isolated from <Emphasis Type="Italic">Quercus brantii</Emphasis> in western Iran

Aphelenchoides paraxui n. sp. is described and illustrated from bark samples of an oak tree (Quercus brantii L.) in Kermanshah province, western Iran. The new species is characterized by body length of 500–660 μm (females) and 630–665 μm (males), lip region set off from body contour, lateral fields with four lines, and total stylet 8–9 μm long with small basal swellings. The excretory pore is located ca one body diam. Posterior to metacorpus valve. The spicules are relatively large (29–33 μm in dorsal limb) with apex and rostrum rounded and well developed and the end of the dorsal limb clearly curved ventrad like a hook. The female tail is conical, the terminus having a complicated step-like projection, usually with many tiny nodular protuberances. Male tail bearing six (2 + 2 + 2) caudal papillae and a well-developed mucro. The new species belongs to the Group 2 category of Aphelenchoides species sensu Shahina (1996) in which eight known species among Group 2–4 sensu Shahina namely: A. arcticus, A. asteromucronatus, A. blastophthorus, A. lichenicola, A. saprophilus, A. seiachicus, A. silvester and A. xui, are the most closed species. Molecular analyses of the partial small subunit rDNA gene (SSU), D2/D3 expansion segments of the large subunit rDNA gene (LSU) and internal transcribed spacer (ITS) revealed this as a new species and supported the morphological results.     

Comparison of Chinese and Japanese A1 isolates of <Emphasis Type="Italic">Phytophthora infestans</Emphasis>

The mating type, glucose-6-phosphate isomerase (Gpi) and peptidase (Pep) genotypes, RG57 fingerprint, and mitochondrial DNA (mtDNA) haplotype of Chinese isolates of Phytophthora infestans collected in Hebei and Gansu in 1996 were compared with those of Japanese isolates collected during 1997–2000. The Chinese isolates were divided into four genotypes, one of which was identical to the dominant Japanese genotype, A1-A (mating type A1; Gpi 100/100; Pep 100/100; RG57 100010001100110100011001110: 1–25, 14a, and 24a; and mtDNA haplotype IIa). Comparison of the genotypes with reported data revealed that some completely and partially identical genotypes occur in Russia and parts of Europe. The other two A1 genotypes and one A2 genotype were also detected in Gansu (Gpi 100/100, Pep 100/100, and mtDNA haplotype Ia), which were regarded as unique to this region.     

Cytochrome c oxidase subunit 1 analysis of <Emphasis Type="Italic">Xiphinema diversicaudatum,X. pachtaicum,X. simile</Emphasis> and <Emphasis Type="Italic">X. vuittenezi</Emphasis> (Nematoda,Dorylaimida)

Genetic analyses using sequences of partial cytochrome c oxidase subunit 1 (cox1) gene of mitochondrial DNA were conducted to determine the extent of genetic variation within and among Xiphinema diversicaudatum, X. pachtaicum, X. simile and X. vuittenezi populations. Pairwise distance among the four species was 22.5 to 31.2%. Four different sequence variants of cox1 were determined among six populations of X. diversicaudatum and three variants among three populations of X. simile. Nucleotide variation was detected at 18 of 414 bp (1.9 to 2.7%) in X. diversicaudatum and 4 of 435 bp (0.2 to 0.9%) in X. simile. All changes were at silent sites. No nucleotide variation was detected within three populations of X. pachtaicum and within three populations of X. vuittenezi.     

<Emphasis Type="Italic">Aphelenchoides gorganensis</Emphasis> n. sp. (Nematoda: Aphelenchoididae)<Emphasis Type="Italic">,</Emphasis> a new species from Iran

Phytophthora capsici infection of chili pepper seedlings can cause substantial losses due to damping-off and collar rot diseases. Chemical control is no longer effective due to reported resistance development, on top of the related environmental concerns and the consumer demands for reduced use of fungicides. Biological control is a sustainable option, with several agents having been reported to be effective against this pathogen. This research focused on optimizing the application of strain THSW13 of Trichoderma hamatum and a bacterial isolate BJ10–86 with the objectives of improving chili pepper seed germination, reduce damping-off disease incidence, and improve the growth of the seedlings. Bacterial isolate BJ10–86 was subjected to molecular identification and found to be Pseudomonas aeruginosa. Chili pepper seeds treated with the biocontrol agents, individually or in combination, were seeded into commercial nursery media that had been pre-inoculated with P. capsici zoospores. Over a period of 35 days the chili pepper seed treatments significantly (P = 0.008) reduced the disease incidence of seedlings damping-off. Combined application of T. hamatum and P. aeruginosa was the best biocontrol treatment with an area under disease curve of only 36.61 units compared to 92.87 units for the control treatment. Similar results were observed in vitro where T. hamatum and P. aeruginosa synergistically inhibited P. capsici growth by 73.2 %. The inhibition activity of this treatment was similar to mefenoxam treatment, which implies that it is an effective and sustainable alternative for chili pepper seed treatment. The biocontrol seed treatment had no effect on seed germination and seedling growth.     

Fusarium rot of hyacinth caused by <Emphasis Type="Italic">Gibberella zeae</Emphasis> (anamorph: <Emphasis Type="Italic">Fusarium</Emphasis><Emphasis Type="Italic"> graminearum</Emphasis>)

Severe rot of leaves, peduncles and flowers caused by Gibberella zeae (anamorph: Fusarium graminearum) was found on potted plants of hyacinth (Hyacinthus orientalis), a liliaceous ornamental, in greenhouses in Kagawa Prefecture, Japan, in January 2001. This disease was named “Fusarium rot of hyacinth” as a new disease because only the anamorph, F. graminearum, was identified on the diseased host plant. The authors contributed equally to this work. The fungal isolate and its nucleotide sequence data obtained in this study were deposited in the Genebank, National Institute of Agrobiological Sciences and the DDBJ/EMBL/GenBank databases under the accession numbers MAFF239499 and AB366161, respectively.     

First report of blueberry red ringspot disease caused by <Emphasis Type="Italic">Blueberry</Emphasis><Emphasis Type="Italic">red</Emphasis><Emphasis Type="Italic">ringspot</Emphasis><Emphasis Type="Italic">virus</Emphasis> in Japan

Virus-like symptoms—red ringspots on stems and leaves, circular blotches or pale spots on fruit—were found on commercial highbush blueberry (Vaccinium corymbosum) cultivars Blueray, Weymouth, Duke and Sierra in Japan. In PCR testing, single DNA fragments were amplified from total nucleic acid samples of the diseased blueberry bushes using primers specific to Blueberry red ringspot virus (BRRV). Sequencing analysis of the amplified products revealed 95.7–97.7% nucleotide sequence identity with the BRRV genome. This paper is the first report of blueberry red ringspot disease caused by BRRV in Japan. The nucleotide sequence data reported in this paper are available in the GenBank/EMBL/DDBJ database as accessions AB469884 to AB469893 for BRRV isolates from Japan.     

Characterization of <Emphasis Type="Italic">Inago1</Emphasis> and <Emphasis Type="Italic">Inago2</Emphasis> retrotransposons in <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

From the genome of a Japanese field isolate of the rice blast fungus, Magnaporthe oryzae, we newly identified Inago1 and Inago2 LTR retrotransposons. Both elements were found to be Ty3/gypsy-like elements whose copies were dispersed within the genome of Magnaporthe spp. isolates infecting rice and other monocot plants. Southern hybridization patterns of nine re-isolates derived from conidia of the strain Ina168 produced after a methyl viologen treatment were not changed, indicating that the insertion pattern of Inago elements is relatively stable.     

Anthracnose of <Emphasis Type="Italic">Enkianthus campanulatus</Emphasis> and <Emphasis Type="Italic">Rhynchosia acuminatifolia</Emphasis> caused by <Emphasis Type="Italic">Colletotrichum gloeosporioides</Emphasis> (new occurrence)

In 2003–2004, anthracnoses of Enkianthus campanulatus and Rhynchosia acuminatifolia were found for the first time in Kanagawa Prefecture and Tokyo in Japan. These pathogens were identified as Colletotrichum gloeosporioides based on their pathogenicity, morphology and ribosomal DNA spacer sequences. Results were presented at the annual meeting of The Phytopathological Society of Japan in 2004.     

<Emphasis Type="Italic">Eremothecium coryli</Emphasis> and <Emphasis Type="Italic">E.</Emphasis><Emphasis Type="Italic">ashbyi</Emphasis> cause yeast spot of azuki bean

Yeast-like fungi were isolated from lesions on azuki bean (cv. Shin-Kyotodainagon) seeds that had been sucked by bean bugs in Kyoto Prefecture, Japan. On the basis of morphological and physiological characteristics and sequence data of the internal transcribed spacer (ITS) regions including the 5.8S rDNA, these yeasts were identified as Eremothecium coryli and E. ashbyi. Pathogenicity of those yeasts was confirmed by a reinoculation test. To our knowledge, this is the first report of the occurrence of yeast spot in azuki bean in Japan. The nucleotide sequence data reported are available in the GeneBank/EMBL/DDBJ database as accessions AB478291–AB478309 for E. coryli AZC1–19 and AB478310–AB478317 for E. ashbyi AZA1–8.     

Pathogenicity of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> against <Emphasis Type="Italic">Galleria mellonella</Emphasis>

The greater wax moth Galleria mellonella L. (Lepidoptera: Pyralidae) is occasionally found in beehives and is a major pest of stored wax. Entomopathogenic fungi have recently received attention as possible biocontrol elements for certain insect pests. In this study, 90 isolates of Beauveria bassiana and 15 isolates of Metarhizium anisopliae were screened for proteases and lipases production. The results showed significant variations in the enzymatic action between the isolates. In the bioassay, the selected isolates evinced high virulence against the 4th instar of the G. mellonella larvae. The isolates BbaAUMC3076, BbaAUMC3263 and ManAUMC3085 realized 100% mortality at concentrations of 5.5 × 10⁶ conidia ml⁻¹, 5.86 × 10⁵ conidia ml⁻¹, and 4.8 × 10⁶ conidia ml⁻¹, respectively. Strong enzymatic activities in vitro did not necessarily indicate high virulence against the tested insect pest. The cuticle of the infected larvae became dark and black-spotted, indicating direct attack of fungus on the defense system of the insects. The LC₅₀ values were 1.43 × 10³, 1.04 × 10⁵ and 5.06 × 10⁴ for Bba3263AUMC, Bba3076AUMC and Man3085AUMC, respectively, and their slopes were determined by computerized probit analysis program as 0.738 ± 0.008, 0.635 ± 0.007 and 1.120 ± 0.024, respectively.     

Genetic structure of <Emphasis Type="Italic">Pyrenophora teres</Emphasis> f. <Emphasis Type="Italic">teres</Emphasis> and <Emphasis Type="Italic">P. teres</Emphasis> f. <Emphasis Type="Italic">maculata</Emphasis> populations from western Canada

Infection by Pyrenophora teres f. teres (Ptt) or P. teres f. maculata (Ptm), the causal agents of the net and spot forms of net blotch of barley, respectively, can result in significant yield losses. The genetic structure of a collection of 128 Ptt and 92 Ptm isolates from the western Canadian provinces of Alberta (55 Ptt, 27 Ptm), Saskatchewan (58 Ptt, 46 Ptm) and Manitoba (15 Ptt, 19 Ptm) were analyzed by simple sequence repeat (SSR) marker analysis. Thirteen SSR loci were examined and found to be polymorphic within both Ptt and Ptm populations. In total, 110 distinct alleles were identified, with 19 of these shared between Ptt and Ptm, 75 specific to Ptt, and 16 specific to Ptm. Genotypic diversity was relatively high, with a clonal fraction of approximately 10 % within Ptt and Ptm populations. Significant genetic differentiation (PhiPT = 0.230, P = 0.001) was found among all populations; 77 % of genetic variation occurred within populations and 23 % between populations. Lower, but still significant genetic differentiation (PhiPT = 0.038, P = 0.001) was detected in Ptt, with 96 % of genetic variation occurring within populations. No significant genetic differentiation (PhiPT = 0.010, P = 0.177) was observed among Ptm populations. Isolates clustered in two distinct groups conforming to Ptt or Ptm, with no intermediate cluster. The high number of haplotypes observed, combined with an equal mating type ratio for both forms of the fungus, suggests that P. teres goes through regular cycles of sexual recombination in western Canada.     

Genetic diversity of <Emphasis Type="Italic">Rhizobium vitis</Emphasis> strains in Japan based on multilocus sequence analysis of <Emphasis Type="Italic">pyrG</Emphasis>, <Emphasis Type="Italic">recA</Emphasis> and <Emphasis Type="Italic">rpoD</Emphasis>

Forty-one strains of Rhizobium vitis, either tumorigenic (Ti) or nonpathogenic, were characterized using multilocus sequence analysis (MLSA) of the partial nucleotide sequences of pyrG, recA, and rpoD. The strains separated into seven clades. Rhizobium vitis (Ti) strains isolated from Japan were divided into five genetic groups (A to E), and nonpathogenic R. vitis strains were divided into two genetic groups (F and G). This result suggests that there are new genetic groups of R. vitis in Japan. Among these groups, members of A and B groups are widely distributed throughout Japan.     

Molecular characterization of <Emphasis Type="Italic">Xiphinema brevicollum</Emphasis> (Nematoda: Longidoridae) from the Czech Republic

Populations of Xiphinema brevicollum occurring in the Czech Republic were described morphologically and molecularly. Published species-specific primer set BL18 and BV3 was used to amplify three populations of X. brevicollum from the Czech Republic. These primers were tested against 9 species of Xiphinema and 11 species of Longidorus. Amplification was also observed for X. inaequale, X. italiae and X. lambertii. Three additional markers, mitochondrial cytochrome c oxidase subunit 1, ribosomal D2/D3 expansion segment of the 28S gene and 18S gene, were amplified and sequenced for X. brevicollum, X. inaequale and X. lambertii belonging to X. americanum-group. Comparison of cox1 sequences of X. brevicollum from the Czech Republic with X. taylori from the Slovak Republic (accession number AM086702) suggested that these populations represent the same species.     

Phylogeny of Egyptian isolates of <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> (CMV) and <Emphasis Type="Italic">Tomato mosaic virus</Emphasis> (ToMV) infecting <Emphasis Type="Italic">Solanum lycopersicum</Emphasis>

Four Cucumber mosaic virus (CMV) (CMV-HM 1–4) and nine Tomato mosaic virus (ToMV) (ToMV AH 1–9) isolates detected in tomato samples collected from different governorates in Egypt during 2014, were here characterized. According to the coat protein gene sequence and to the complete nucleotide sequence of total genomic RNA1, RNA2 and RNA3 of CMV-HM3 the new Egyptian isolates are related to members of the CMV subgroup IB. The nine ToMV Egyptian isolates were characterized by sequence analysis of the coat protein and the movement protein genes. All isolates were grouped within the same branch and showed high relatedness to all considered isolates (98–99%). Complete nucleotide sequence of total genomic RNA of ToMV AH4 isolate was obtained and its comparison showed a closer degree of relatedness to isolate 99–1 from the USA (99%). To our knowledge, this is the first report of CMV isolates from subgroup IB in Egypt and the first full length sequencing of an ToMV Egyptian isolate.     

<Emphasis Type="Italic">Citrus exocortis viroid</Emphasis> transmission through commercially-distributed seeds of <Emphasis Type="Italic">Impatiens</Emphasis> and <Emphasis Type="Italic">Verbena</Emphasis> plants

Surveys of Impatiens and Verbena species in local nurseries in Fredericton, Canada and Verbena species in New Delhi, India showed widespread infection of Citrus exocortis viroid (CEVd) in vegetatively-propagated and seed-grown plants. To determine viroid seed transmission, samples of eight varieties of Impatiens and 11 varieties of Verbena were obtained from four commercial sources. All 19 samples collected contained viroid infection irrespective of variety. The presence of viroid in non-germinated seed was 21%, while the transmission rate in seedlings was 66% in Impatiens walleriana in 2006. Following 2 years of seed storage, the respective figures were 6% and 26%. Similarly, in Verbena x hybrida the presence of viroid in seed was 13% in 2006 with a seed-transmission rate in seedlings of 28%, while the respective figures after 2 years of storage were 5% and 45%.     

Functional analysis of the melanin biosynthesis genes <Emphasis Type="Italic">ALM1</Emphasis> and <Emphasis Type="Italic">BRM2</Emphasis>-<Emphasis Type="Italic">1</Emphasis> in the tomato pathotype of <Emphasis Type="Italic">Alternaria alternata</Emphasis>

The tomato pathotype of Alternaria alternata (A. arborescens) produces the dark brown to black pigment melanin, which accumulates in the cell walls of hyphae and conidia. Melanin has been implicated as a pathogenicity factor in some phytopathogenic fungi. Here, two genes of the tomato pathotype for melanin biosynthesis, ALM1 and BRM2-1, which encode a polyketide synthetase and a 1,3,8-trihydroxynaphthalene (THN) reductase, respectively, have been cloned and disrupted in the pathogen. The gene-disrupted mutants, alm1 and brm2-1, had albino and brown phenotypes, respectively. The wild-type and the mutants caused the same necrotic lesions on the leaves after inoculation with spores. These results suggest that melanin is unlikely to play a direct role in pathogenicity in the tomato pathotype A. alternata. Scanning electron microscopy revealed that the conidia of both mutants have much smoother surfaces in comparison to the wild-type. The conidia of those mutants were more sensitive to UV light than those of the wild-type, demonstrating that melanin confers UV tolerance.     

Immunodetection of the replicative complex of <Emphasis Type="Italic">Barley yellow dwarf</Emphasis><Emphasis Type="Italic">virus</Emphasis>-PAV <Emphasis Type="Italic">in vivo</Emphasis>

The genomic fragments of two open reading frames (ORFs) 1 and 2 of German and Canadian PAV isolates of Barley yellow dwarf virus (BYDV-PAV) were sequenced. Sequences only slightly differed from previously published sequences of this virus. Two polyclonal antisera against proteins encoded by ORFs 1 and 2 of a German ASL-1 isolate were developed using recombinant antigens expressed in E. coli as a fusion either to His₆− or thioredoxin-tags. In Western blot analysis with total protein extracts from BYDV infected plants, antisera efficiently recognized the 99 kDa fusion protein expressed from ORF1 and ORF2 (P1–P2 protein). Later in infection the P1–P2 protein disappeared and two smaller proteins, revealing sizes of 39 and 60 kDa, could be detected.     

Structural conservation of the <Emphasis Type="Italic">hrp</Emphasis> gene cluster in <Emphasis Type="Italic">Xanthomons oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

The clustered hrp genes encoding the type III secretion system in the Japanese strains MAFF301237 and MAFF311018 of Xanthomonas oryzae pv. oryzae were sequenced and compared. The strains differ in their pathogenicity, location, and year of isolation. A 30-kbp sequence comprising 29 open reading frames (ORFs) was identical in its structural arrangement in both strains but differed from X. campestris pv. campestris, X. axonopodis pv. citri, and X. axonopodis pv. glycines in certain genes located between the hpaB-hrpF interspace region. The DNA sequence and the putative amino acid sequence in each ORF was also identical in both X. oryzae pv. oryzae strains as were the PIP boxes and the relative sequences. These facts clearly showed that the structure of the hrp gene cluster in X. oryzae pv. oryzae is unique.     

<Emphasis Type="Italic">Erwinia aphidicola</Emphasis> isolated from commercial bean seeds (<Emphasis Type="Italic">Phaseolus vulgaris)</Emphasis>

In late 2003, a new disease appeared in protected bean crops in southeastern Spain, causing a decrease of over 50% in production. Several samples of affected plants were collected and analyzed and the agent of this disease was identified as the bacterium Erwinia aphidicola, which had never been described as a pathogen previously. We attempted to determine the possible bacterium transmission through seeds, using 120 commercial bean seeds from the same batch as that used in an affected farm, and 120 seeds from the fruiting plants of the same farm. Seed coats, cotyledons and leaves of plants originating from them, were taken and analyzed. Several of the developed symptoms on plants from commercial and fruiting plant seeds were internervial chlorosis, necrotic pits and rough roots and they coincided with those observed on affected crops. Bacteria present in commercial seed cotyledons were isolated and analyzed by biochemical and molecular tests. Results confirmed the presence of Erwinia aphidicola in four analyzed seeds; moreover, Bacillus simplex/Bacillus muralis, Pseudomonas mendocina, Pseudomonas putida and Paenibacillus polymyxa were also identified.     

Partial characterisation of a novel <Emphasis Type="Italic">Tobamovirus</Emphasis> infecting <Emphasis Type="Italic">Actinidia chinensis</Emphasis> and <Emphasis Type="Italic">A. deliciosa</Emphasis> (Actinidiaceae) from China

Actinidia chinensis and A. deliciosa plants from China, showing a range of symptoms, including vein clearing, interveinal mottling, mosaics and chlorotic ring spots, were found to contain ~300 nm rod-shaped virus particles. The virus was mechanically transmitted to several herbaceous indicators causing systemic infections in Nicotiana benthamiana, N. clevelandii, and N. occidentalis, and local lesions in Chenopodium quinoa. Systemically- infected leaves reacted with a Tobacco mosaic virus polyclonal antibody in indirect ELISA. PCR using generic and specific Tobamovirus primers produced a 1,526 bp sequence spanning the coat protein (CP), movement protein (MP), and partial RNA replicase genes which showed a maximum nucleotide identity (88%) with Turnip vein clearing virus and Penstemon ringspot virus. However, when the CP sequence alone was considered the highest CP sequence identity (96% nt and 98% aa) was to Ribgrass mosaic virus strain Kons 1105. The morphological, transmission, serological and molecular properties indicate that the virus is a member of subgroup 3 of the genus Tobamovirus.