Horizontal transmission and host-virulence attenuation of totivirus in violet root rot fungus Helicobasidium mompa

Monokaryotic strains of Helicobasidium mompa were used as vectors of a mycovirus between various H. mompa isolates to examine the transmissibility of one of the mycoviruses, totivirus (HmTV1–17 virus) in the hypovirulent isolate V17 of H. mompa. The isolates that acquired HmTV1–17 virus were also examined for any alteration in the virulence. Twelve dikaryotic isolates of H. mompa, belonging to 11 mycelial compatibility groups (MCGs) and being mycelially incompatible with isolate V17, were used as recipients of HmTV1–17 virus. Two monokaryotic isolates that were mycelially incompatible with isolate V17 and all of the recipients were also used as vectors of HmTV1-17 virus between isolate V17 and the recipients. When isolate V17 and recipients were directly paired on plate media, HmTV1-17 virus was transmitted from isolate V17 into 2 of the 12 recipients (i.e., 2 of the 11 MCGs). Two monokaryotic strains were paired with isolate V17, and the monokaryotic strains that acquired HmTV1-17 virus were then used as new virus donors. When the monokaryotic strains containing HmTV1-17 virus were paired with the 12 recipients, HmTV1-17 virus was transmitted into 7 of the 12 recipients from the monokaryotic strains (i.e., 7 of 11 MCGs). Based on these results, we concluded that monokaryotic strains could act as vectors to transmit HmTV1-17 virus into H. mompa isolates. When four of the H. mompa isolates that acquired HmTV1-17 virus were used to inoculate apple rootstock Malus prunifolia, the virulence of all of the isolates was attenuated from that of their parental isolates. Moreover, because the DNA fingerprints of the fungal isolates that acquired HmTV1-17 virus were the same as those of their parental isolates, the infection with HmTV1-17 virus is considered the cause of virulence attenuation of H. mompa.     

<Emphasis Type="Italic">Helicobasidium mompa</Emphasis> isolates from sweet potato in continuous monoculture fields

 Isolates of the violet root rot fungus Helicobasidium mompa were collected from herbaceous and tree plants. Their host preference was studied by inoculation experiments using carrots, sweet potatoes, and apple stocks. It was found that sweet potato isolates from Kyushu produced infection cushions on carrots and sweet potatoes but not on apple stocks. Other isolates did not show host preference. Sweet potato isolates were also characterized by ready hyphal mass (sclerotium) production. They were thought to have adapted to the habitat with high disturbance by annual tillage. Received: July 15, 2002 / Accepted: September 26, 2002     

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.     

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.     

Genetic variation among <Emphasis Type="Italic">Fusarium</Emphasis> isolates from onion,and resistance to <Emphasis Type="Italic">Fusarium</Emphasis> basal rot in related <Emphasis Type="Italic">Allium</Emphasis> species

The aim of this research was to study levels of resistance to Fusarium basal rot in onion cultivars and related Allium species, by using genetically different Fusarium isolates. In order to select genetically different isolates for disease testing, a collection of 61 Fusarium isolates, 43 of them from onion (Allium cepa), was analysed using amplified fragment length polymorphism (AFLP) markers. Onion isolates were collected in The Netherlands (15 isolates) and Uruguay (9 isolates), and received from other countries and fungal collections (19 isolates). From these isolates, 29 were identified as F. oxysporum, 10 as F. proliferatum, whereas the remaining four isolates belonged to F. avenaceum and F. culmorum. The taxonomic status of the species was confirmed by morphological examination, by DNA sequencing of the elongation factor 1-α gene, and by the use of species-specific primers for Fusarium oxysporum, F. proliferatum, and F. culmorum. Within F. oxysporum, isolates clustered in two clades suggesting different origins of F. oxysporum forms pathogenic to onion. These clades were present in each sampled region. Onion and six related Allium species were screened for resistance to Fusarium basal rot using one F. oxysporum isolate from each clade, and one F. proliferatum isolate. High levels of resistance to each isolate were found in Allium fistulosum and A. schoenoprasum accessions, whereas A. pskemense, A. roylei and A. galanthum showed intermediate levels of resistance. Among five A. cepa cultivars, ‘Rossa Savonese’ was also intermediately resistant. Regarding the current feasibility for introgression, A. fistulosum, A. roylei and A. galanthum were identified as potential sources for the transfer of resistance to Fusarium into onion.     

Cell interactions between a nonpathogenic <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> strain and root tissues of <Emphasis Type="Italic">Eucalyptus viminalis</Emphasis>

Nonpathogenic isolates of Fusarium oxysporum can be successful antagonists of pathogenic forms of the same fungal species that commonly attacks crop plants. The characteristics that distinguish nonpathogenic from pathogenic forms are not well understood. In this study, the mode of root colonization of Eucalyptus viminalis seedlings by a nonpathogenic F. oxysporum strain is described at the ultrastructural level. Root systems of E. viminalis plants were inoculated with nonpathogenic F. oxysporum strain Fo47 in an in vitro model system. Changes in the occurrence of nonesterified and methyl-esterified pectins in colonized E. viminalis roots were evaluated by in situ immunolabeling using two monoclonal antibodies, JIM 5 and JIM 7. Modes of penetration and root colonization patterns in E. viminalis seedlings by the nonpathogenic fungus were similar to those described for pathogenic forms of F. oxysporum. However, root interactions differed in that the nonpathogenic fungus did not induce host tissue damage. No papilla-like appositions were observed in host cells in response to invading hyphae, which did not disrupt the host plasma membrane in many cases, suggesting that a biotrophic relationship was established. Root colonization by the nonpathogenic strain did not induce alteration in JIM 7 labeling of methyl-esterified pectin in E. viminalis cell walls, whereas nonesterified pectin was detected to a significantly greater extent in cell walls of roots colonized by the fungus. Pectin components decreased slightly only at points of hyphal contact with host cells. Because nonpathogenic strains utilize pectin in pure culture, host control over enzyme activity or production by the fungi may at least partly explain their compatible interactions with host tissues.     

Pythium rot of figmarigold (<Emphasis Type="Italic">Lampranthus spectabile</Emphasis>) caused by <Emphasis Type="Italic">Pythium aphanidermatum</Emphasis>

A new leaf rot disease was found on the leaves of figmarigold (Lampranthus spectabile). The causal organism, identified as Pythium aphanidermatum was found to cause the same symptoms after artificial inoculation and was then reisolated from the inoculated plants. We propose to name the disease Pythium rot of figmarigold.     

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.     

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">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.     

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.     

Intraspecific diversity within avocado field isolates of <Emphasis Type="Italic">Rosellinia necatrix</Emphasis> from south-east Spain

Fifty-five isolates of Rosellinia necatrix, the cause of common avocado white root rot disease, were collected from south-east Spain and characterised according to their virulence behaviour and their molecular patterns to assess broader levels of genetic diversity. Virulence properties were revealed by in vitro inoculation on avocado plants. Differences in reaction types showed variability among these isolates. No sequence differences were observed when the internal transcribed spacer 1 (ITS1) and ITS2 regions and DNA fragments of the β-tubulin, adenosine triphosphatase and translation elongation factor 1 genes were explored in representive isolates from five virulence groups. Random amplified polymorphic DNA (RAPD) amplifications were also performed for each isolate using 19 random primers. Four of these primers revealed polymorphism among isolates and repetitive and discriminative bands were used to build an unweighted pair group with arithmetic mean tree. However, RAPD clustering showed low stability, and no correlation between RAPD and virulence groups was observed, possibly indicating high levels of sexual recombination.     

Potential inhibitors against <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>, produced by the fungus <Emphasis Type="Italic">Myrothecium</Emphasis> sp. associated with the marine sponge <Emphasis Type="Italic">Axinella</Emphasis> sp.

Sclerotinia sclerotiorum is a worldwide ascomycete fungal plant pathogen, which causes enormous yield losses on major economic crops such as crucifers, grain legumes and several other plant families. The objective of this research was to isolate and characterise some bioactive products from cultures of fungi associated with the marine sponge Axinella sp. In total, nine fungal isolates were obtained from the marine sponge Axinella sp. collected from the South China Sea. A group of test strains, including two G⁺ strains (Bacillus subtilis and Staphylococcus aureus), two G⁻ strains (Escherichia coli and Pseudomonas aeruginosa) and three fungi including two plant pathogenic fungi Sclerotinia sclerotiorum and Magnaporthe grisea and Saccharomyces cerevisiae, were employed as the indicator organisms for bioactivity screening. Using antagonistic tests and bioactive screening of the ethyl acetate (EtOAc) extracts of the corresponding cultures, fungal isolate JS9 showed the stronger efficacy against the test indicator strains, especially the indicator fungal pathogens. Isolate JS9 was further identified as Myrothecium sp. by a combination of morphological features and 18S rDNA BLAST on GenBank. Two macrocyclic trichothecenes, roridin A (compound 1) and roridin D (compound 2) were purified by tracking the activity of the EtOAc extract fractions and characterised with spectral analyses including MS, ¹H-NMR, ¹³C-NMR and disortionless enhancement by polarization transfer (DEPT). In vitro antifungal tests showed that the two macrocyclic trichothecenes were bioactive against S. cerevisiae, M. grisea and S. sclerotiorum with minimal inhibitory concentrations of 31.25, 125 and 31.25 μg ml⁻¹ for roridin A, and 62.5, 250 and 31.25 μg ml⁻¹ for roridin D, respectively. The present investigation demonstrated that two antifungal trichothecenes including roridin A and roridin D produced by the fungus Myrothecium sp. isolated from the marine sponge Axinella sp. could be potential inhibitors against the plant pathogen S. sclerotiorum. Lian Wu Xie and Shu Mei Jiang contributed equally to this work.     

Internal fruit rot of netted melon caused by <Emphasis Type="Italic">Pantoea ananatis</Emphasis> (=<Emphasis Type="Italic">Erwinia ananas</Emphasis>) in Japan

An internal fruit rot with a malodor was found in netted melons (Cucumis melo L.) in commercial greenhouses in Kochi Prefecture, Japan, in 1998, despite their healthy appearance and lack of water-soaking or brown spots on the surface. A yellow bacterium was consistently isolated from the affected fruits. To confirm the pathogenicity of eight representative isolates of the yellow bacterium, we stub-inoculated ovaries (immature-fruits) 5–7 days after artificial pollination, with a pin smeared with bacteria. After the melon fruits had grown for 60 more days, an internal fruit rot resembling the natural infection appeared, and the inoculated bacterium was reisolated. The melon isolates had properties identical with Pantoea ananatis, such as gram-negative staining, facultative anaerobic growth, indole production, phenylalanine deaminase absence, and acid production from melibiose, sorbitol, glycerol, and inositol. Phylogenetic analysis based on 16S rDNA sequences showed that the melon bacterium positioned closely with known P. ananatis strains. The melon bacterium had indole acetic acid (IAA) biosynthesis genes (iaaM and iaaH) and a cytokinin biosynthesis gene (etz). The bacterium could be distinguished from the other ‘Pantoea’ group strains by rep-PCR genomic fingerprinting. From these results, the causal agent of internal fruit rot was identified as a strain of P.ananatis [Serrano in (Philipp J Sci 36:271–305, 1928); Mergaert et al. in (Int J Syst Bacteriol 43:162–173, 1993)]. The nucleotide sequence data reported are available in the DDBJ database under accessions AB297969, AB373739, AB373740, AB373741, AB373742, AB373743 and AB373744.     

Resistance to <Emphasis Type="Italic">Penicillium</Emphasis><Emphasis Type="Italic">hirsutum</Emphasis> Dierckx in garlic accessions

Among the factors affecting the quality and yield of garlic production, blue mold caused by -- Penicillium spp. -- is responsible for economical losses in many countries. Allicin, present in garlic bulbs, has been suggested as having antifungal activity against some Penicillium species. This study was conducted to evaluate the response of garlic accessions against Penicillium hirsutum infection and to compare this response with bulb allicin content. Twelve garlic accessions were inoculated with P. hirsutum, and assayed in greenhouse and growth chamber experiments. Plant growth parameters and the fungal production of conidia were evaluated. Significant differences were found among the accessions. Accessions Castaño and Morado were most resistant whereas AR-I-125 and Fuego were always severely affected by the disease. A low correlation was found (r = 0.17) between allicin content and tolerance, indicating that allicin is not the main factor involved in the resistance against P. hirsutum.     

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.     

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.     

Relationships of <Emphasis Type="Italic">Barley yellow dwarf virus</Emphasis>-PAV and <Emphasis Type="Italic">Cereal yellow dwarf virus</Emphasis>-RPV from Iran with viruses of the family <Emphasis Type="Italic">Luteoviridae</Emphasis>

Barley yellow dwarf disease is one of the most important problems confronting cereal production in Iran. Barley yellow dwarf virus-PAV (BYDV-PAV) and Cereal yellow dwarf virus-RPV (CYDV-RPV) are the predominant viruses associated with the disease. One isolate of BYDV-PAV from wheat (PAV-IR) and one isolate of CYDV-RPV from barley (RPV-IR) were selected for molecular characterisations. A genome segment of each isolate was amplified by PCR. The PAV-IR fragment (1264 nt) covered a region containing partial genes for coat protein (CP), read through protein (RTP) and movement protein (MP). PAV-IR showed a high sequence identity to PAV isolates from USA, France and Japan (96–97%). In a phylogenetic analysis it was placed into PAV group I together with PAV isolates from barley and oats. The fragment of RPV-IR (719 nt) contained partial genes for CP, RTP and MP. The sequence information confirmed its identity as CYDV. However, RPV-IR showed 90–91% identity with both RPV and Cereal yellow dwarf virus-RPS (CYDV-RPS). Phylogenetic analyses suggested that it was more closely related to RPS. These data comprise the first attempt to characterise BYD-causing viruses in Iran and southwest Asia. The nucleotide sequence data reported appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers AY450425 and AY450454