Inheritance of resistance in <Emphasis Type="Italic">Solanum nigrum</Emphasis> to <Emphasis Type="Italic">Phytophthora infestans</Emphasis>

Solanum nigrum, black nightshade, is a wild non-tuber bearing hexaploid species with a high level of resistance to Phytophthora infestans (Colon et al. 1993), the causal agent of potato late blight, the most devastating disease in potato production. However, the genetic mode of resistance in S. nigrum is still poorly understood. In the present study, two S. nigrum accessions, 984750019 (N19) and #13, resistant (R) and susceptible (S), respectively, to three different isolates of P. infestans, were sexually crossed. The various kinds of progeny including F1, F2, F3, and backcross populations (BC₁; F1 × S), as well as two populations produced by self-pollinating the R parent and S parent, were each screened for susceptibility to P. infestans isolate MP 324 using detached leaf assays. Fifty seedling plant individuals of the F1 progeny were each resistant to this specific isolate, similarly to the seedling plants resulting from self-pollination of the resistant R parent. Thirty seedling plants obtained from self-pollination of the S parent were susceptible. Among a total of 180 F2 plants, the segregation ratio between resistant and susceptible plants was approximately 3: 1. Among the 66 seedling plants of the BC₁ progeny originating from crossing an F1 plant with the susceptible S parent, there were 26 susceptible and 40 resistant plants to P. infestans. The segregation patterns obtained indicated monogenic dominant inheritance of resistance to P. infestans isolate MP 324 in S. nigrum acc. 984750019. This gene, conferring resistance to P. infestans, may be useful for the transformation of potato cultivars susceptible to late blight.     

<Emphasis Type="Italic">Corythucha ciliata</Emphasis>, a new <Emphasis Type="Italic">Platanus</Emphasis> pest in Turkey

An invasive Tingidae, the platanus lace bug Corythucha ciliata (Say, 1832) (Hemiptera: Tingidae), which specializes on Platanus spp., was found for the first time in Turkey in 2007; it was recorded from a 120 km² area in the northwestern part of the country. Infestations occurred in an area between Taşkesti and Abant in Bolu Province, which is located near major cities and two main motorways. The pest species is newly spreading in Turkey, causing noticeable damage to Platanus orientalis trees.     

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.     

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.     

A new phenotype of <Emphasis Type="Italic">Polymyxa betae</Emphasis> in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The understanding of the molecular biology of Polymyxa betae, the protist vector of Beet necrotic yellow vein virus, remains limited because of the obligate nature of this root endoparasite and the limited data on the genome of Beta vulgaris, its most common host plant. The aim of this work was to assess the infection of P. betae in Arabidopsis thaliana in order to learn more about the P. betae genome and its interaction with the host. The susceptibility of a set of ecotypes of various origins to a monosporosorus and aviruliferous isolate of P. betae was analyzed in a series of bioassays conducted under controlled conditions. P. betae was detected in roots of A. thaliana using light microscopy and PCR. The infection severity was relatively low in this species compared with B. vulgaris, but the different stages of the life cycle were present. The phenotype of P. betae in A. thaliana root cells differed from the phenotype in B. vulgaris: the spore-forming phase was more prevalent in comparison with the sporangial phase, and the sporosori contained a lower number of spores. The compatible interaction between P. betae and A. thaliana obtained after the inoculation of zoospores and optimal conditions for the development of P. betae provide a new model system that can be used to improve the knowledge on the P. betae genome and on the mechanisms of the spore-forming phase of P. betae.     

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.     

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.     

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.     

<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.     

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.     

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.     

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">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%.     

Identification and characterization of pathotypes in <Emphasis Type="Italic">Puccinia horiana</Emphasis>, a rust pathogen of <Emphasis Type="Italic">Chrysanthemum</Emphasis> x <Emphasis Type="Italic">morifolium</Emphasis>

Puccinia horiana is the causal agent of chrysanthemum white rust or Japanese rust. This microcyclic autoecious rust has a quarantine status and can cause major damage in the commercial production of Chrysanthemum x morifolium. Given the international and often trans-continental production of planting material and cut flowers of chrysanthemum and the decreasing availability of registered fungicides in specific regions, breeding for resistance against P. horiana will gain importance and will need to involve the appropriate resistance genes for the pathotypes that may be present. As pathotypes have not been well characterized in this system, the main objective was to build an international collection of isolates and screen these on a large collection of cultivars to identify different pathotypes. Using a robust and high throughput bioassay, we tested 36 selected cultivars with 22 individual single-pustule isolates of P. horiana. The isolates originated from three different continents over 4 different collection years and included some isolates from cultivars previously reported as resistant. In most cases the bioassays resulted in a clear scoring of interaction phenotypes as susceptible or resistant, while in several cases consistent intermediate phenotypes were found, often on specific cultivars. Twenty-four of the cultivars gave a differential interaction phenotype profile. All isolates produced a unique profile, infecting a minimum of 4 and a maximum of 19 differential cultivars. Based on the Person analysis of these profiles, this pathosystem contains at least seven resistance genes (and seven avirulence genes), demonstrating the highly complex race structure in this pathosystem.     

<Emphasis Type="Italic">Fusarium ershadii</Emphasis> sp. nov., a Pathogen on <Emphasis Type="Italic">Asparagus officinalis</Emphasis> and <Emphasis Type="Italic">Musa acuminata</Emphasis>

Two Fusarium strains, isolated from Asparagus in Italy and Musa in Vietnam respectively, proved to be members of an undescribed clade within the Fusarium solani species complex based on phylogenetic species recognition on ITS, partial RPB2 and EF-1α gene fragments. Macro- and micro-morphological investigations followed with physiological studies done on this new species: Fusarium ershadii sp. nov can be distinguished by its conidial morphology. Both isolates of Fusarium ershadii were shown to be pathogenic to the monocot Asparagus officinalis when inoculated on roots and induced hollow root symptoms within two weeks in Asparagus officinalis seedlings. In comparison mild disease symptoms were observed by the same strains on Musa acuminata seedlings.     

<Emphasis Type="Italic">Lupinus luteus</Emphasis>, a new host of <Emphasis Type="Italic">Phytophthora cinnamomi</Emphasis> in Spanish oak-rangeland ecosystems

Phytophthora cinnamomi is an aggressive pathogen on Lupinus luteus (yellow lupin), causing root rot, wilting and death of this crop, common in oak-rangeland ecosystems ('dehesas') in south-western Spain. The oomycete, the main cause of Quercus decline in the region, was isolated from roots of wilted lupins in the field. Artificial inoculations on four cultivars of L. luteus reproduced the symptoms of the disease, both in pre- and post-emergence stages, recovering the pathogen from necrotic roots. These results suggest the potential of yellow lupin as inoculum reservoir for the infection of Quercus roots. This is the first report of P. cinnamomi as root pathogen of L. luteus.     

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.     

The chemotaxis regulator <Emphasis Type="Italic">pilG</Emphasis> of <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> is required for virulence in <Emphasis Type="Italic">Vitis vinifera</Emphasis> grapevines

Type IV pili of X. fastidiosa are regulated by pilG, a response regulator protein putatively involved in chemotaxis-like operon sensing stimuli through signal transduction pathways. To elucidate the roles of pilG in pathogenicity of X. fastidiosa, the pilG-deletion mutant XfΔpilG and complemented strain XfΔpilG-C were generated. While all strains had similar growth curves in vitro, XfΔpliG showed significant reduction in cell-matrix adherence and biofilm production compared with wild-type X. fastidiosa and XfΔpilG-C. The genes pilE, pilU, pilT, and pilS were down-regulated in XfΔpliG when compared with its complemented strain and wild-type X. fastidiosa. Finally, no Pierce’s disease symptoms were observed in grapevines inoculated with XfΔpilG, whereas grapevines inoculated with the wild-type X. fastidiosa and complemented strain of XfΔpilG-C developed typical Pierce’s Disease (PD) symptoms. The results indicate that pilG has a role in X. fastidiosa virulence in grapevines.     

Competence of <Emphasis Type="Italic">Frankliniella occidentalis</Emphasis> and <Emphasis Type="Italic">Frankliniella intonsa</Emphasis> strains as vectors for <Emphasis Type="Italic">Chrysanthemum stem necrosis virus</Emphasis>

The vector competence of Frankliniella occidentalis for Chrysanthemum stem necrosis virus (CSNV) was evaluated. Three vector strains with distinct competences for Tomato spotted wilt virus (TSWV) transmission were investigated, including an artificially selected strain (TsH) that has a particularly high competence (>90 %). Newly hatched larvae of F. occidentalis were given an acquisition access period of 5 days on CSNV-infected D. stramonium leaves, and reared to maturity. Their transmission efficiencies were examined using a leaf disk assay using Petunia x hybrida leaves. Following the leaf disk assay, the virus accumulation in the vectors was examined via a double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) of their bodies. The results showed that the CSNV acquisition and transmission efficiency of the TsH strain did not differ from those of the others, indicating that the competence of F. occidentalis as a vector for CSNV is not related to that for TSWV. The CSNV transmission and acquisition efficiencies of two F. intonsa strains (Hiroshima and Fukuoka) were also evaluated. In Hiroshima strain, 35 % of adults were viruliferous, but only two transmitters (3 %) were observed. In Fukuoka strain, 6 % were viruliferous, and no transmitters were observed. These results indicate that F. intonsa cannot be a major vector for CSNV. The accumulation of CSNV in the adults of F. occidentalis and F. intonsa evaluated using DAS-ELISA showed a significant difference in ELISA values among transmitter, viruliferous non-transmitter, and non-viruliferous individuals. These results clearly demonstrated that only transmitters that accumulated a threshold quantity of virus can transmit CSNV to plants.