Identification and characterization of a potyvirus causing chlorotic spots on <Emphasis Type="Italic">Phalaenopsis</Emphasis> orchids

A putative virus-induced disease showing chlorotic spots on leaves of Phalaenopsis orchids was observed in central Taiwan. A virus culture, phalaenopsis isolate 7-2, was isolated from a diseased Phalaenopsis orchid and established in Chenopodium quinoa and Nicotiana benthamiana. The virus reacted with the monoclonal antibody (POTY) against the potyvirus group. Potyvirus-like long flexuous filament particles around 12–15 × 750–800 nm were observed in the crude sap and purified virus preparations, and pinwheel inclusion bodies were observed in the infected cells. The conserved region of the viral RNA was amplified using the degenerate primers for the potyviruses and sequence analysis of the virus isolate 7-2 showed 56.6–63.1% nucleotide and 44.8–65.1% amino acid identities with those of Bean yellow mosaic virus (BYMV), Beet mosaic virus (BtMV), Turnip mosaic virus (TuMV) and Bean common mosaic virus (BCMV). The coat protein (CP) gene of isolate 7-2 was amplified, sequenced and found to have 280 amino acids. A homology search in GenBank indicated that the virus is a potyvirus but no highly homologous sequence was found. The virus was designated as Phalaenopsis chlorotic spot virus (PhCSV) in early 2006. Subsequently, a potyvirus, named Basella rugose mosaic virus isolated from malabar spinach was reported in December 2006. It was found to share 96.8% amino acid identity with the CP of PhCSV. Back-inoculation with the isolated virus was conducted to confirm that PhCSV is the causal agent of chlorotic spot disease of Phalaenopsis orchids in Taiwan. This is the first report of a potyvirus causing a disease on Phalaenopsis orchids.     

<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.     

<Emphasis Type="Italic">Turnip ringspot virus</Emphasis> recognised on Chinese cabbage in Russia

The nucleotide sequence of the 3′-terminal part of the RNA1 genome segment of the M12 isolate of comovirus Turnip ringspot virus (TuRSV) was established. This isolate originated in 1989 in Moscow (Russia) from Chinese cabbage with Radish mosaic virus-like symptoms. Comparison of the M12 RNA polymerase amino acid sequence with that of Radish mosaic virus (RaMV) revealed significant differences; these proteins are of different length and are only about 75% identical. On the other hand, the amino acid sequence of the M12 RNA polymerase was more than 94% identical with that of TuRSV recently described in Toledo (USA). We conclude that TuRSV occurs in Europe as well as in America and probably represents a new species of the genus Comovirus.     

<Emphasis Type="Italic">Odontoglossum ringspot virus</Emphasis> causing flower crinkle in <Emphasis Type="Italic">Phalaenopsis</Emphasis> hybrids

A new disorder exhibiting flower crinkle on Phalaenopsis orchids bearing white flowers has been observed in Taiwan, China and Japan for several years. This disorder decreased the flower longevity and was considered as a physiological syndrome. The objective of this study was to identify and characterize the real causal agent of this new Phalaenopsis disorder. Five plants of Phalaenopsis hybrids “V3” (Phal. Yukimai × Phal. Taisuco Kochdian) with flower crinkle symptoms were collected and tested by enzyme-linked immunosorbent assay with antisera against 18 viruses. The extract of leaves and flowers from one diseased plant (96-Ph-16) reacted positively only to antiserum against Odontoglossum ringspot virus (ORSV), while those from the other four plants (96-Ph-7, 96-Ph-17, 96-Ph-18 and 96-Ph-19) reacted positively to the antisera against ORSV and Cymbidium mosaic virus (CymMV). Five ORSV isolates, one each from flowers of those five diseased Phalaenopsis orchids, were established in Chenopodium quinoa. A CymMV culture was isolated from the flowers of one of the ORSV/CymMV mix-infected Phalaenopsis orchids (96-Ph-19). To determine the causal agent of the flower crinkle disease, healthy Phalaenopsis seedlings were singly or doubly inoculated with the isolated ORSV and/or CymMV. Results of back inoculation indicated that ORSV is the sole causal agent of the crinkle symptom on petals of Phalaenopsis orchid. The CP gene of the ORSV isolates from this study shared 97.3–100% nucleotide identity and 96.2–100% amino acid identity with those of 41 ORSV isolates available in GenBank. This is the first report demonstrating ORSV as the sole virus causing flower crinkle disease on Phalaenopsis orchids.     

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.     

Identification and characterization of <Emphasis Type="Italic">Choanephora</Emphasis> spp. causing Choanephora flower rot on <Emphasis Type="Italic">Hibiscus syriacus</Emphasis>

Hibiscus syriacus, as a national flower of Korea, is most popularly used for ornamental purposes and includes numerous cultivars, and it is widely planted in temperate zones that feature hot summers. We investigated Choanephora flower rot on H. syriacus from 2012 to 2014 in Korea and Japan and confirmed Choanephora infection in several localities in both countries. Here, our objectives were to identify the main causal agent of Choanephora flower rot on H. syriacus and describe its morphological and molecular characteristics. We identified 44 out of 50 isolates as Choanephora cucurbitarum and the remainder as C. infundibulifera based on morphological characterization and phylogenetic analysis. The sequences of the internal transcribed spacer region (ITS) of ribosomal DNA and the D1/D2 region of the large subunit (LSU) rDNA of examined isolates were compared with sequences obtained from GenBank, and the analysis of the results revealed 100 % identity with the corresponding sequences of C. cucurbitarum and C. infundibulifera strains. Classification of the Choanephora species performed here according to the key described by Kirk (1984) corresponded with the results of the phylogenetic analysis of this study. Through intraspecific and interspecific mating tests, the characteristics of zygospore were described in details. Pathogenicity tests using both species showed the same symptoms, causing blossom blight and soft rot on the flowers, which were identical to those observed in the field. All identified causal agents of Choanephora rot were indeed Choanephora species, where C. cucurbitarum was identified in the majority, while the others were in the minority of examined samples.     

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.     

Identification and characterization of <Emphasis Type="Italic">Alternaria alternata</Emphasis> (Fr.) Keissler causing <Emphasis Type="Italic">Ceratonia</Emphasis> Blight disease of carob (<Emphasis Type="Italic">Ceratonia siliqua</Emphasis> L.) in Turkey

A new dagger nematode, Xiphinema tica n. sp., is described and illustrated from several populations extracted from soil associated with several crops and wild plants in Costa Rica. The new dagger nematode is characterised by a moderate body size (3276–4240 μm), a rounded lip region, ca 13.5 μm wide, separated from body contour by a shallow depression, amphidial fovea large, stirrup-shaped, a moderately long odontostyle ca 135 μm long, stylet guiding ring located at ca 122 μm from anterior end, vulva almost equatorial (50–54%), well-developed Z-organ, with heavy muscularised wall containing in the most of specimens observed two moderately refractive inclusions variable in shape (from round to star-shaped), with uterine spines and crystalloid bodies; female tail short, dorsally convex-conoid, with rounded end and a small peg, with a c’ ratio ca 0.8, bearing two or three pairs of caudal pores and male absent. The unique and novel uterine differentiation based on the coexistence of a well-developed Z-organ mixed with uterine spines and crystalloid bodies in Xiphinema prompted us to update and include this combination of characters in the polytomous key of Loof and Luc (1990). Integrative diagnosis was completed with molecular data obtained, using D2-D3 expansion segments of 28S rDNA, ITS1-rDNA, partial 18S–rDNA and the partial mitochondrial gene cytochrome c oxidase subunit 1 (coxI). The phylogenetic relationships of this species with other Xiphinema spp. indicated that X. tica n. sp. was monophyletic to the other species from the morphospecies Group 4, Xiphinema oleae.     

Anthracnose of <Emphasis Type="Italic">Polygonatum falcatum</Emphasis> caused by <Emphasis Type="Italic">Colletotrichum dematium</Emphasis>

Severe spotting, blight and drop of leaves caused by Colletotrichum dematium were found on potted plants of Polygonatum falcatum, a liliaceous ornamental, in open fields in Kagawa Prefecture, Japan, in May 2001. This new disease was named anthracnose of P. falcatum. Keisuke Tomioka, Jouji Moriwaki, Toyozo Sato contributed equally to this work. The fungal isolate and its nucleotide sequence data obtained in this study were deposited in Genebank, National Institute of Agrobiological Sciences and the DDBJ/EMBL/GenBank databases under accessions MAFF239500 and AB334523, respectively.     

Characterization of <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">carotovorum</Emphasis> causing soft-rot disease on <Emphasis Type="Italic">Pinellia ternata</Emphasis> in China

Pinellia ternata is a traditional Chinese herb which has been used in China for over 1,000 years. A soft-rot disease characterized by water-soaked lesions and soft-rot symptoms with a stinking odour was commonly observed in cultivated fields of this plant, and Pectobacterium-like bacteria were consistently isolated from the infected tissues. Two typical strains (SXR1 and ZJR1), isolated from Shanxi and Zhejiang, respectively, were identified. Pathogenicity tests revealed that these strains were virulent to P. ternata and induced the same symptoms as observed in the field. Characterization involving fatty acid profile, metabolic and physiological properties, 16S rDNA sequence and PCR-RFLP identified both isolates as P. carotovorum subsp. carotovorum (Pcc). The 16S rDNA of both isolates shared 97–99% sequence similarity with that of Pcc strains. The phylogenetic trees showed that both isolates were clustered in the group of Pcc and P. carotovorum subsp. odorifera and both PCR-RFLP profiles were consistent with the pattern E produced by the minority of Pcc strains. Thus, isolates SXR1 and ZJR1 were characterized as Pcc in spite of some differences. This is the first report that Pcc has been proven as a causal agent of soft-rot disease on P. ternata.     

Identification and characterization of the <Emphasis Type="Italic">Enterobacter</Emphasis> complex causing mulberry (<Emphasis Type="Italic">Morus alba</Emphasis>) wilt disease in China

Mulberry wilt disease (MWD) was recently identified in Hangzhou, Zhejiang province, China. Typical symptoms of the disease are browning of vascular tissues, leaf wilt, defoliation, and tree decline. Unlike the symptoms of bacterial wilt disease caused by Ralstonia solanacearum, symptoms of MWD generally started from the bottom of the plants and moved upward. In inoculation experiments, four selected MWD strains caused mulberry shoot leaf wilt, discoloration, and defoliation. They also induced whole plant leaf wilt, defoliation and dark brown discoloration of vascular tissue. Based on Biolog metabolic profiles, fatty acid methyl ester analysis (FAME) and sequence analysis of the partial 16S rDNA and rpoB genes four MWD strains were identified as members of the genus Enterobacter. The 16S rDNA and rpoB gene sequences revealed a close relationship among two isolates, R2-2 and R6-2, and the E. asburiae type strain JCM6051. The isolates showed >98% similarity to E. asburiae JCM6051 in their rpoB gene. These results indicated that isolates R2-2 and R6-2 belonged to E. asburiae. No similarity in 16S rDNA sequences above 97% was found between either of the remaining isolates, R11-2 or R18-2, and any recognized Enterobacter species, suggesting that the two isolates may represent novel Enterobacter species. rpoB gene similarity values between the isolates and Enterobacter spp. type strains were <98%, providing further evidence that the two isolates may represent a novel species within the Enterobacter. The causal agent for MWD was previously reported to be E. cloacae, however, this study found that other Enterobacter spp. (E. asburiae and Enterobacter sp.) also cause MWD.     

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.     

Identification and characterization of <Emphasis Type="Italic">Apple stem grooving virus</Emphasis> causing leaf distortion on pear (<Emphasis Type="Italic">Pyrus pyrifolia</Emphasis>) in Taiwan

A putative virus-induced disease of pear (Pyrus pyrifolia var. Hengshen) showing symptoms of reduced size of foliage and leaf distortion was observed in orchards in central Taiwan in 2004. The sap of symptomatic leaf samples reacted positively to an antiserum against Apple stem grooving virus (ASGV). Two virus cultures, designated as TS1 and TS2, were isolated from symptomatic pears. Flexuous filamentous virions of ∼ 12 × 600 nm were observed in symptomatic pear leaves and purified virus preparations. Results of back inoculation of pear seedlings with TS1 revealed that ASGV was the causal agent of the disease. Sequence analyses of the cloned coat protein (CP) genes of TS1 and TS2 shared 88–92.4% nucleotide and 90.7–97.1% amino acid identities with those of other ASGV isolates available in GenBank. The polyclonal antibody generated against ASGV TS1 has been routinely used for the detection of the ASGV-infection in the imported pear scions for quarantine purpose via enzyme-linked immunosorbent assays (ELISAs). One of 1,199 samples of pear scions imported from Japan during 2005–2007 was identified as ASGV-positive and the virus was designated as AGJP-22. The CP gene amplified from this AGJP-22 shared 97.9–98.3% amino acid identities to those of the domestic isolates and they were closely related phylogenetically. To date, these data present for the first time conclusive evidence revealing that ASGV is indeed the causal agent of the pear disease displaying symptoms of reduced size of foliage and leaf distortion in Taiwan.     

Bacterial black spot caused by <Emphasis Type="Italic">Burkholderia andropogonis</Emphasis> on <Emphasis Type="Italic">Odontoglossum</Emphasis> and intergeneric hybrid orchids

A new bacterial black spot disease was observed on Odontoglossum, Odontioda, Odontocidium, and Vuylstekeara orchids in Japan. Typical symptoms on the leaves were dark or black spots (or both) with a yellow halo. The causal agent was identified as Burkholderia andropogonis (Smith 1911) Gillis, Van Van, Bardin, Goor, Hebbar, Willems, Segers, Kersters, Heulin and Fernandez 1995. The isolates were pathogenic on four original host orchids, Phalaenopsis orchid, and tulip; they were not pathogenic on white clover or corn after needle stab inoculation. An antibiotic bactericide (oxytetracycline/streptomycin mixture WP) was most effective for controlling the disease.     

Reaction of <Emphasis Type="Italic">Leersia</Emphasis> grasses to <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> collected from Japan and Asian countries

The present study was conducted to determine if there is specificity in the host-pathogen relationship between the isolates of Xanthomonas oryzae pv. oryzae, the causal bacterium for rice blight and Leersia grasses, the alternative weed hosts of the disease. Plants of three species of Leersia, namely, L. sayanuka, L. oryzoides and L. japonica, were collected from various parts of Japan and were inoculated with the X. oryzae pv. oryzae isolates obtained from various locations in Japan and from 11 Asian countries. Four L. sayanuka plants were found susceptible to all Race II isolates and some Race I isolates, but were resistant to all Race III isolates. Race III is known to have a wider range pathogenicity to rice cultivar groups compared with Race I and II. Although the reactions of two L. oryzoides plants to Race I and II isolates were similar to that of L. sayanuka, the L. oryzoides plant collected from Niigata Prefecture showed a susceptible reaction to some Race III isolates. On the other hand, L. japonica plants gave reactions different those of L. sayanuka and L. oryzoides, with two plants of L. japonica found to be resistant to all test isolates collected from Japan. The Asian isolates exhibited a wide host range against the international differential rice cultivars, but almost all of them were avirulent to Leersia plants. These results indicate that the relationship between the pathogenicity of the causal bacterium and the resistance of host plants is very complex, and suggest that pathogenic diversity of X. oryzae pv. oryzae might be related to the resistance of Leersia spp.     

White rust of <Emphasis Type="Italic">Ipomoea</Emphasis> caused by <Emphasis Type="Italic">Albugo ipomoeae-panduratae</Emphasis> and <Emphasis Type="Italic">A. ipomoeae-hardwickii</Emphasis> and their host specificity

In some areas of Japan, yellow spots with white pustules on leaves, stems, petioles, peduncles and calyces were found on Ipomoea nil, I. triloba, I. lacunosa and I. hederacea var. integriuscula. We demonstrated that the diseases on I. nil, I. triloba and I. lacunosa were caused by host-specific strains of Albugo ipomoeae-panduratae and defined three forma speciales of the fungus, respectively, for the three Ipomoea species: “f. sp. nile”, “f. sp. trilobae” and “f. sp. lacunosae”. Because the diseases were new to Japan, we coined the Japanese name “shirosabi-byo”, which means white rust. We also showed that the disease on I. hederacea var. integriuscula was caused by A. ipomoeae-hardwickii. We named this new disease “white rust (shirosabi-byo in Japanese)”.     

Suppression of <Emphasis Type="Italic">Papaya ringspot virus</Emphasis> infection in <Emphasis Type="Italic">Carica papaya</Emphasis> with CAP-34, a systemic antiviral resistance inducing protein from <Emphasis Type="Italic">Clerodendrum aculeatum</Emphasis>

CAP-34, a protein from Clerodendrum aculeatum inducing systemic antiviral resistance was evaluated for control of Papaya ringspot virus (PRSV) infection in Carica papaya. In control plants (treated with CAP-34 extraction buffer) systemic mosaic became visible around 20 days that intensified up to 30 days in 56% plants. During this period, CAP-34-treated papaya did not show any symptoms. Between 30 and 60 days, 95% control plants exhibited symptoms ranging from mosaic to filiformy. In the treated set during the same period, symptoms appeared in only 10% plants, but were restricted to mild mosaic. Presence of PRSV was determined in induced-resistant papaya at the respective observation times by bioassay, plate ELISA, immunoblot and RT-PCR. Back-inoculation with sap from inoculated resistant plants onto Chenopodium quinoa did not show presence of virus. The difference between control and treated sets was also evident in plate-ELISA and immunoblot using antiserum raised against PRSV. PRSV RNA was not detectable in treated plants that did not show symptoms by RT-PCR. Control plants at the same time showed a high intensity band similar to the positive control. We therefore suggest that the absence/delayed appearance of symptoms in treated plants could be due to suppressed virus replication.     

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.     

Morphological and molecular characterization of <Emphasis Type="Italic">Diaporthe (</Emphasis>anamorph <Emphasis Type="Italic">Phomopsis)</Emphasis> complex and pathogenicity of <Emphasis Type="Italic">Diaporthe aspalathi</Emphasis> isolates causing stem canker in soybean

The complex of Diaporthe (asexual morph) species occurring on soybean constitutes an important pathogenic group associated with diseases such as pod and stem blight, seed decay and stem canker. Stem canker, caused by Diaporthe aspalathi, has been reported as the most aggressive form of canker and its occurrence has limited soybean crop productivity in the southern United States. The main form of pathogen control is the use of stem canker resistant soybean varieties. In this study, strains of Diaporthe and Phomopsis were isolated from stem and seeds of soybean in different locations in South America during the years 1989–2014. Genomic DNA from 26 isolates were analyzed by PCR-restriction fragment length polymorphism (RFLP) and Amplified Fragment Length Polymorphism (AFLP) techniques, and sequencing of internal transcribed spacer (ITS) regions of ribosomal DNA. The molecular analysis of ITS sequences by alignment with those of ex-type strains deposited in GenBank and morphological characteristics allowed the identification of Phomopsis longicolla, D. phaseolorum var. sojae, D. caulivora and D. aspalathi. An analysis of the pathogenicity of 13 isolates of D. aspalathi inoculated in soybean genotypes carrying different resistance genes to stem canker (Rdm1, Rdm2, Rdm3, Rdm4, Rdm5 and Rdm?) enabled us to identify the occurrence of at least three races of D. aspalathi occurring in Brazil. Among the isolates identified as D. aspalathi, both molecular and phenotypic analyses showed clustering depending on the date of collection and pathogenicity, which revealed the existence of variability of the pathogen.