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.     

Association of seedling and adult plant resistance to <Emphasis Type="Italic">Sclerotium rolfsii</Emphasis> in Jerusalem artichoke (<Emphasis Type="Italic">Helianthus tuberosus</Emphasis> L<Emphasis Type="Italic">.</Emphasis>) under field conditions

Stem rot caused by Sclerotium rolfsii is an important problem for Jerusalem artichoke production. Host plant resistance is the most promising method to control disease. If resistant genotypes can be identified in seedlings and this resistance is closely related to resistance at maturity, the evaluation of disease resistance in adult plants could be curtailed or omitted, increasing the speed and efficiency of screening. The objective of this study was to determine the relationship between resistance to S. rolfsii in Jerusalem artichoke in seedling and in adult stages under field conditions. Field experiments were set up in different soil fertility environments in the rainy season during July to October 2014. In each environment, 10 varieties of Jerusalem artichoke with differences in resistance to S. rolfsii were planted and inoculated either 15 or 45 days after transplanting. Higher disease incidence was observed on adult plant stage, but disease severity was similar for both plant stages. The correlations between seedling and adult responses were positive and significant for disease incidence, area under disease progress curve and severity index. Screening for resistance to S. rolfsii in Jerusalem artichoke can be carried out on seedlings, thus improving the efficiency of selection.     

Metabolic profiles of groundnut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.) genotypes differing in <Emphasis Type="Italic">Sclerotium rolfsii</Emphasis> reaction

A total of 60 compounds of known structure, comprising sugars, sugar alcohols, fatty acids, amino acids, organic acids, phenols and sterols were identified in stem extracts of groundnut using GC-MS. Sugars and fatty acids were predominant in stem extracts as compared to other metabolites. Distinguished metabolite patterns were observed in control and 96 h after infection (h.a.i.). Succinic acid, pentitol, scopolin, D-glucose and D-turanose, myo-inositol, fructose and mannitol were observed to be higher in control plants, whereas, D-ribopyranoside, thymol, pentadecanoic acid and octadecanoic acid increased at 24 hai than that of control. Interestingly, phenol related compounds such as phenol, hydroquinone, guaicol-.beta.-d-glucopyranoside, scopolin were also found lower in non-infected stems of TG37A. Moreover, tolerant genotypes (CS 319 and CS 19) had higher content of Thymol-.beta.-d-glucopyranoside, pentitol, D-glucose, D-turanose, scopolin and hydroquinone than that of moderately tolerant and susceptible genotypes. Sugar profiles using Ion chromatography revealed that glucose content decreased in moderately susceptible and susceptible genotype after S. rolfsii infection. Both constitutive and induced levels of cinnamic acid was observed higher in resistant genotypes than that of susceptible ones which was further supported by phenylalanine ammonia lyase activity. Thus, our study demonstrates the biological role of metabolites specifically sugars, phenolics and fatty acids in plant defense responses.     

Pathological studies on the southern blight of China aster (<Emphasis Type="Italic">Callistephus chinensis</Emphasis>) caused by <Emphasis Type="Italic">Sclerotium rolfsii</Emphasis>

A severe outbreak of southern blight disease of China aster was observed during the post rainy season (September–November 2015) in the Mysore and Mandya Districts of Karnataka, Southern India. The disease incidence ranged between 12 and 47%. The typical disease symptoms include water-soaked lesions on leaves, stems and on the lower stem surfaces followed by quick wilting of the whole plant with abundant production of sclerotia near the stem-soil interface. The associated fungal pathogen was isolated on potato dextrose agar (PDA) medium, on which numerous reddish-brown sclerotia were seen. A total of 26 fungal isolates were isolated and studied for the mycelial compatibility. Isolate SrCCM 1 was used for pathogenicity analysis. The results of the study showed that, there was no variation among the isolates tested. Molecular identification of the pathogen by ITS-rDNA sequences of S. rolfsii showed 100% similarity with reference sequences. Based on the cultural, morphological and molecular characteristics, the fungal pathogen was identified as Sclerotium rolfsii Sacc. (Sexual morph: Athelia rolfsii (Curzi) C.C. Tu & Kimbr). Pathogenicity tests were performed on healthy leaves, roots and stems. Typical disease symptoms on leaves, stems and roots were evident after 5, 8 and 10 days of post-inoculation. Sclerotium rolfsii is known to cause diseases in economically important crop plants. However, no reports are available on the occurrence of S. rolfsii on China aster in India.     

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 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.     

<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.     

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.     

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">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.     

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.     

Host-delivered RNAi-mediated root-knot nematode resistance in <Emphasis Type="Italic">Arabidopsis</Emphasis> by targeting <Emphasis Type="Italic">splicing factor</Emphasis> and <Emphasis Type="Italic">integrase</Emphasis> genes

Root-knot nematodes (RKNs) are one of the most important biotic factors limiting crop productivity in many crop plants. The major RKN control strategies include development of resistant cultivars, application of nematicides and crop rotation, but each has its own limitations. In recent years, RNA interference (RNAi) has become a powerful approach for developing nematode resistance. The two housekeeping genes, splicing factor and integrase, of Meloidogyne incognita were targeted for engineering nematode resistance using a host-delivered RNAi (HD-RNAi) approach. Splicing factor and integrase genes are essential for nematode development as they are involved in RNA metabolism. Stable homozygous transgenic Arabidopsis lines expressing dsRNA for both genes were generated. In RNAi lines of splicing factor gene, the number of galls, females and egg masses was reduced by 71.4, 74.5 and 86.6%, respectively, as compared with the empty vector controls. Similarly, in RNAi lines of the integrase gene, the number of galls, females and egg masses was reduced up to 59.5, 66.8 and 63.4%, respectively, compared with the empty vector controls. Expression analysis revealed a reduction in mRNA abundance of both targeted genes in female nematodes feeding on transgenic plants expressing dsRNA constructs. The silencing of housekeeping genes in the nematodes through HD-RNAi significantly reduced root-knot nematode infectivity and suggests that they will be useful in developing RKN resistance in crop plants.     

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.     

Leaf Smut of <Emphasis Type="Italic">Sagittaria latifolia </Emphasis>Caused by <Emphasis Type="Italic">Doassansia horiana</Emphasis>

A smut-like disease was found on the leaves of Sagittaria latifolia in Japan. Spore balls collected from the leaves of S. latifolia and S. trifolia var. edulis were used to cross-inoculate leaves of pathogen-free plants of the two species to identify the pathogen. Spots and swellings formed on leaves of the two species 10 days after inoculation. These symptoms were quite similar to those of the leaf smut disease of S. trifolia var. edulis caused by Doassansia horiana, and the spore balls were characteristic of the fungus. Therefore, the authors conclude that D. horiana caused leaf smut disease on S. latifolia. Received 18 January 2000/ Accepted in revised form 14 May 2000     

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">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%.     

Improved attachment and parasitism of <Emphasis Type="Italic">Trichoderma</Emphasis> on <Emphasis Type="Italic">Meloidogyne javanica</Emphasis> in vitro

Monoclonal and polyclonal antibodies that bind to eggs and/or second-stage juveniles of the nematode Meloidogyne javanica were tested for their effects on the parasitic interactions between this nematode and the fungus Trichoderma. Parasitism of Trichoderma asperellum-203 and Trichoderma atroviride on nematode egg masses, eggs and juveniles was enhanced when antibodies were incorporated into in vitro parasitism bioassays. Parasitism on separated eggs (without gelatinous matrix) and their hatched juveniles was also improved, compared to controls without antibodies that did not attach fungal conidia. Improved parasitism could be due to bilateral binding of the antibodies to the nematodes and conidia, enabling better conidial attachment to the nematodes. Enhanced germination of antibody-bound conidia further improved parasitism. Differences were observed among antibodies in their effects on fungal parasitism and their interaction with Trichoderma species. We focused mainly on the egg- and juvenile-binding monoclonal antibody MISC that exhibited a stronger reaction with T. asperellum-203 than with T. atroviride. Pretreatment of this antibody with fucose inhibited its binding to nematodes and conidial attachment to nematodes, as well as conidial agglutination in the presence of the antibody. Antibody binding to juveniles affected their movement and viability, especially gelatinous matrix-originated juveniles. The fucose-specific lectin Ulex europaeus-I enhanced conidial attachment to nematode life-stages, and conidial agglutination occurred in its presence. These phenomena were inhibited by preincubating lectin with fucose. Our results suggest that carbohydrate residues, such as fucose, on the surface of the nematode and fungal conidia are involved in the antibody- and lectin-mediated improved parasitism.     

<Emphasis Type="Italic">Grapevine virus A</Emphasis> transmission by larvae of <Emphasis Type="Italic">Parthenolecanium corni</Emphasis>

Grapevine virus A (GVA, Vitivirus) was transmitted experimentally by first and second instars of the scale insect Parthenolecanium corni from grapevine to grapevine and to the herbaceous host Nicotiana benthamiana. This is the first report of GVA transmission by P. corni. Grapevine leafroll-associated virus-1 (Ampelovirus) was always present in the donor grapevines and, in every case, GVA was transmitted simultaneously with this ampelovirus from grapevine to grapevine, suggesting possible interactions between the two viruses for transmission.