Molecular characterization of <Emphasis Type="Italic">Phytophthora porri</Emphasis> and closely related species and their pathogenicity on leek (<Emphasis Type="Italic">Allium porrum</Emphasis>)

White tip, caused by Phytophthora porri, is a destructive disease in the cultivation of European leek (Allium porrum). P. porri and closely related species such as P. brassicae, P. primulae and P. syringae belong to the phylogenetic clade 8b within the genus Phytophthora. The objectives of this study were to establish the position of P. porri and closely related species within the Phytophthora clade 8b; to study genetic variation among P. porri isolates from leek and closely related species and to test the hypothesis that host-driven speciation has occurred within this clade. AFLP analysis could clearly make a distinction between isolates of P. porri from Allium species and related Phytophthora species such as P. brassicae, P. syringae and P. primulae. DNA similarity and cluster analysis based on 353 markers demonstrated little genetic diversity within the P. porri population from Allium species although Belgian and Dutch P. porri isolates from leek could be distinguished from Japanese P. porri isolates from other Allium species and the P. porri isolate from carrot. Our results point to incipient speciation within the P. porri isolates, which could have been driven by the host plant or by geographic isolation. ITS sequence analysis confirmed the results obtained by AFLP and showed a close relationship between P. porri isolates from Allium and P. primulae and between the P. porri isolate from carrot and P. brassicae. We hypothesize that interspecific hybridization has occurred within this clade.     

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.     

Real-time detection of <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis> and <Emphasis Type="Italic">P. citrophthora</Emphasis>in citrus roots and soil

Two primers, specific for Phytophthora nicotianae (Pn6) and P. citrophthora (Pc2B), were modified to obtain Scorpion primers for real-time identification and detection of both pathogens in citrus nursery soils and roots. Multiplex PCR with dual-labelled fluorogenic probes allowed concurrent identification of both species ofPhytophthora among 150 fungal isolates, including 14 species of Phytophthora. Using P. nicotianaespecific primers a delayed and lower fluorescence increase was also obtained from P. cactorumDNA. However, in separate real-time amplifications, the aspecific increase of fluorescence from P. cactorum was avoided by increasing the annealing temperature. In multiplex PCR, with a series of 10-fold DNA dilutions, the detection limit was 10 pg l^-1 for P. nicotianaeand 100 pg l^–1 for P. citrophthora, whereas in separate reaction DNA up to 1 pg l^-1 was detected for both pathogens.Simple and rapid procedures for direct DNA extraction from soil and roots were utilised to yield DNA whose purity and quality was suitable for PCR assays. By combining these protocols with a double amplification (nested Scorpion-PCR) using primers Ph2-ITS4 amplifying DNA from the main Phytophthora species (first round) and PnB5-Pn6 Scorpion and Pc2B Scorpion-Pc7 (second round), it was possible to achieve real-time detection of P. nicotianaeand P. citrophthora from roots and soil. The degree of sensitivity was similar to that of traditional detection methods based on the use of selective media. The analyses of artificially and naturally infested soil showed a high and significant correlation between the concentration of pathogen propagules and the real-time PCR cycle threshold.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation as a useful tool for the molecular genetic study of the phytopathogen <Emphasis Type="Italic">Curvularia lunata</Emphasis>

In order to explore the molecular mechanisms of virulence and genetic variance of Curvularia lunata in maize, an ATMT (Agrobacterium tumefaciens-mediated transformation) system was established in order to create a wide range of insertional transformants of C. lunata. Our results showed that the germinating conidia were the ideal starting material for transformation. Based on our optimised transformation condition, the transformation efficiency of C. lunata with T-DNA was improved greatly, and the average transformation frequency was as high as 84 ± 5 transformants per 1 × 10⁶ germlings. Southern blotting results of 39 randomly-selected transformants showed a unique hybridisation pattern and predominant single-copy insertions. An ATMT library containing approximate 3000 transformants was generated, and four types of transformants were screened in terms of growth rate, sporulation, mycelial pigmentation, and toxin production in vitro. This library will be used to identify genes involved in the virulence of the pathogen.     

Phytophthora blight of southern star (<Emphasis Type="Italic">Oxypetalum caeruleum</Emphasis>) caused by <Emphasis Type="Italic">Phytophthora palmivora</Emphasis> in Japan

In 2004, a damping-off symptom was found on southern star, Oxypetalum caeruleum, in Kochi Prefecture, Japan. Two Phytophthora strains with different colony patterns on potato dextrose agar were isolated, and their pathogenicity was confirmed by inoculation of southern star plants and their reisolation from symptomatic plants. Both fungi were identified as Phytophthora palmivora based on morphology, physiology, and sequence analysis of the internal transcribed spacers of nuclear ribosomal DNA. This is the first report of Phytophthora blight of southern star in the world.     

First report of phytophthora rot on <Emphasis Type="Italic">Wasabia japonica</Emphasis> caused by <Emphasis Type="Italic">Phytophthora drechsleri</Emphasis> in Japan

In September 2014, Phytophthora rot on wasabi plants [Wasabia japonica (Miq.) Matsum.] was found for the first time in the city of Okutama, Tokyo, Japan. A Phytophthora sp. strain was constantly isolated from brown stem bases and rhizomes of infected plants. The same symptoms as those observed in the field were produced in vitro through inoculation of test plants with the isolated Phytophthora sp. The fungus was identified as Phytophthora drechsleri based on morphological and DNA sequence comparison. Phytophthora rot, “eki-byo” in Japanese, is proposed for this disease common name.     

Conventional PCR and real time quantitative PCR detection of <Emphasis Type="Italic">Phytophthora cryptogea</Emphasis> on <Emphasis Type="Italic">Gerbera jamesonii</Emphasis>

A conventional PCR and a SYBR Green real-time PCR assays for the detection and quantification of Phytophthora cryptogea, an economically important pathogen, have been developed and tested. A conventional primer set (Cryp1 and Cryp2) was designed from the Ypt1 gene of P. cryptogea. A 369 bp product was amplified on DNA from 17 isolates of P. cryptogea. No product was amplified on DNA from 34 other Phytophthora spp., water moulds, true fungi and bacteria. In addition, Cryp1/Cryp2 primers were successfully adapted to real-time PCR. The conventional PCR and real-time PCR assays were compared. The PCR was able to detect the pathogen on naturally infected gerbera plants and on symptomatic artificially infected plants collected 21 days after pathogen inoculation. The detection limit was 5 × 10³ P. cryptogea zoospores and 16 fg of DNA. Real-time PCR showed a detection limit 100 times lower (50 zoospores, 160 ag of DNA) and the possibility of detecting the pathogen in symptomless artificially infected plants and in the re-circulating nutrient solution of closed soilless cultivation systems.     

<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.     

High incidence of <Emphasis Type="Italic">Pelargonium line pattern virus</Emphasis> infecting asymptomatic <Emphasis Type="Italic">Pelargonium</Emphasis> spp. in Spain

Geraniums (Pelargonium spp.) are traditional ornamental plants largely cultivated in Europe and northern America. Vegetative propagation makes them prone to viral infections, which have detrimental effects on crop production and quality. Asymptomatic samples collected in Spain were tested for a range of viruses using ELISA. The tobamovirus, Tobacco mosaic virus (TMV), the cucumovirus, Cucumber mosaic virus (CMV), and several viruses in the family Tombusviridae, namely, Pelargonium line pattern virus (PLPV), Pelargonium flower break virus (PFBV), and Pelargonium leaf curl virus (PLCV), were detected either singly or in combination in 59.2% of 800 samples. PLPV and PFBV infections were confirmed by dot-blot hybridisation. The most relevant viral infection found on Spanish asymptomatic geraniums was by Pelargonium line pattern virus (PLPV). Symptoms did not develop for 3 years on most of the PLPV infected geranium plants under greenhouse conditions.     

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.     

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.     

Resistance of <Emphasis Type="Italic">Solanum</Emphasis> species to <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> subgroup IA and its vector <Emphasis Type="Italic">Myzus persicae</Emphasis>

Sixty-nine tomato genotypes representing nine Solanum species were evaluated for resistance to Cucumber mosaic virus (CMV) subgroup IA and its aphid vector Myzus persicae. Resistance was assessed by visual scoring of symptoms in the field under natural conditions, and in the greenhouse by artificial inoculations through aphid M. persicae and mechanical transmissions in the year 2007 and 2009. Considerable variation in responses was observed among the evaluation methods used. Field evaluations were found liable to errors as different levels were observed for the same genotypes in the different years, however mechanical inoculation was found to be the most useful in identifying CMV subgroup IA resistance, in contrast aphid transmission was most useful in identifying insect transmission resistance. All genotypes observed as highly resistant to CMV subgroup IA in the field or through vector transmission became systemically infected through mechanical inoculations. Using mechanical inoculation, six genotypes (TMS-1 of S. lycopersicum, LA1963 and L06049 of S. chilense, LA1353, L06145 and L06223 of S. habrochaites) were found resistant and another six (L06188 and L06238 of S. neorickii, L06219 of S. habrochaites, L05763, L05776 and L06240 of S. pennellii) were found tolerant showing mild symptoms with severity index (SI) ranging 1-2 and with delayed disease development after a latent period (LP) of 18–30 days. However, these genotypes were found to be resistant to highly resistant in the field and through inoculation by M. persicae; and they also supported low population levels of M. persicae except TMS-1. Another nine genotypes (LA2184 of S. pimpinellifolium L., LA2727 of S. neorickii, LA0111, L06221, L06127 and L06231 of S. peruvianum L., LA1306, L06057 and L06208 of S. chmielewskii) showing a susceptible response after mechanical inoculation were highly resistant, resistant and tolerant after M. persicae transmission. The resistant genotypes, identified in the present study can be exploited in the breeding programmes aimed at developing tomato varieties resistant to CMV subgroup IA and broadening the genetic base of CMV-resistant germplasm. The differences observed between mechanical and aphid transmission suggests that one should consider both evaluation methods for tomato germplasm screening against CMV subgroup IA.     

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.     

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.     

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.     

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.     

Monitoring of <Emphasis Type="Italic">Phytophthora</Emphasis> species on fruit trees in Bulgaria

Disease on fruit trees in Bulgaria caused by Phytopthora cactorum and P. citrophthora was found in the period 1998–1999. Leaves of some trees become reddish during July, and later in the season fall off. Infected trees die during the same season, or the next season. Observations on symptom development and spread of Phytophthora root and crown rot of fruit trees was undertaken from 1999 to 2009. Disease incidence is between 2% and 14% in some gardens and nurseries. The disease was registered in the regions of Plovdiv, Kjustendil, Sliven, Yambol, Karnobat, Bourgas and Svishtov. Samples from infected plant tissues were taken and isolations were done on selective PARP media, or by applying a baiting bioassay. Based on morphological and cultural characteristics and temperature requirements the following Phytophthora species have been identified: Phytophthora cactorum, P. citrophthora, P. drechsleri, P. cryptogea, hybrid and Pythium. Pathogenicity of the isolates was tested on green apple fruits or one-year-old apple rootstocks. Laboratory studies of the effect of temperature on mycelia growth showed that most isolates can grow from 5° up to 30°C, with an optimum from 18° to 25°C. Only three strains grew at 35–36°C, two developed slowly, one grew well. The optimal pH for mycelia development was tested. Aiming at control of disease, in vivo pot trials have been carried out for studying resistance of rootstocks to P. cactorum. At the end of the growing season a good level of resistance has been shown in the rootstocks M29C, Gizela 6, and MAXMA 14.     

<Emphasis Type="Italic">Eutypa lata</Emphasis>, the causal agent of dieback in red currant (<Emphasis Type="Italic">Ribes rubrum</Emphasis>) and gooseberry (<Emphasis Type="Italic">R. uva-crispa</Emphasis>) in the Netherlands

Dieback of red currant (Ribes rubrum) and gooseberry (Ribes uva-crispa) is an increasing problem in commercial fields in the Netherlands. Field surveys were done in 2006–2007 and samples with dieback symptoms were analysed. In this study the causal agent was diagnosed as Eutypa lata, based on morphological characteristics and rDNA-ITS sequence data. The field surveys revealed the presence of the anamorph and teleomorph states of the fungus produced on dead infected currant wood. Eutypa lata is a vascular pathogen of many woody plants. Related fungi from the same family Diatrypaceae are difficult to distinguish from E. lata based on morphological features. The genetic variability of E. lata was compared by rDNA-ITS sequencing of isolates from different hosts and origins. Within the E. lata isolates little variability in the ITS sequences was observed. Phylogenetic analysis showed no clear subdivisions within the species. Eutypa lata strains isolated from the different hosts were closely related, indicating that there is no direct evidence for host specificity.     

<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.