A method to evaluate percentage of cucurbit seeds infested with <Emphasis Type="Italic">Acidovorax avenae</Emphasis> subsp. <Emphasis Type="Italic">citrulli</Emphasis>, pathogen of bacterial fruit blotch

We developed a method to detect Acidovorax avenae subsp. citrulli from cucurbit seeds that is more sensitive than conventional growing-out methods. Seeds were cultivated in test tubes for 2 weeks, and the bacteria grew semi-selectively on the seeds and seedlings. The plants were then vigorously shaken in water to suspend bacterial cells, and the suspension was spread on a selective medium for identifying the bacterium by colony appearance. The method is suitable for research purposes to evaluate the percentage of seeds infested with the bacteria, but not to detect bacterial contamination in commercial lots with thousands of seeds.     

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.     

<Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. Sp. <Emphasis Type="Italic">melonis-</Emphasis>melon interaction<Emphasis Type="Italic">:</Emphasis> Effect of grafting combination on pathogen gene expression

Bread wheat (BW) and durum wheat (DW) are both strongly affected by Septoria tritici blotch caused by the hemibiotrophic fungus Zymoseptoria tritici. However, only the BW-Z. tritici pathosystem has been well studied so far. Here, we compared compatible interactions between Z. tritici and both BW and DW species at the cytological, biochemical and molecular levels. Fungal infection process investigations showed close spore germination and leaf penetration features in both interactions, although differences in the patterns of these events were observed. During the necrotrophic phase, disease severity and sporulation levels were associated in both interactions with increases of the two cell-wall degrading enzyme activities endo-β-1,4-xylanase and endo-β-1,3-glucanase as well as protease. An analysis of plant defense responses during the first five days post inoculation revealed inductions of GLUC, Chi4, POX and PAL and a repression of LOX gene expressions in both wheat species, although differences in kinetics and levels of induction or repression were observed. In addition, peroxidase, catalase, glucanase, phenylalanine ammonia-lyase and lipoxygenase activities were induced in both wheat species, while only weak accumulations of hydrogen peroxide and polyphenols were detected at the fungal penetration sites. Our study revealed overall a similarity in Z. tritici infection process and triggered wheat defense pathways on both pathosystems.     

Cobweb disease on <Emphasis Type="Italic">Agaricus bisporus</Emphasis> caused by <Emphasis Type="Italic">Cladobotryum mycophilum</Emphasis> in Korea

This report is the first of cobweb disease on Agaricus bisporus in Korea. Cobweb on both fruit bodies and casing soils were observed on several mushroom farms in Gyeongbuk Province, Korea. Classical and molecular characterization indicated that the causal agent is Cladobotryum mycophilum. The isolated fungus was used to inoculate fruiting bodies of A. bisporus and caused the same symptoms.     

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.     

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.     

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.     

A new leaf blight disease of <Emphasis Type="Italic">Trifolium dasyurum</Emphasis> caused by <Emphasis Type="Italic">Botrytis fabae</Emphasis>

A new disease was observed on Trifolium dasyurum, with symptoms beginning as a halo spot and developing into a leaf blight. The causal organism was identified by microscopy and DNA sequence studies as Botrytis fabae. This strain of B. fabae was also demonstrated to cause disease on foliage of a range of pulse crops, including Vicia faba, Pisum sativum, and Lens culinaris. This study demonstrates the potential of this strain of B. fabae to not only pose a significant threat to T. dasyurum but also to pulses grown in rotation with T. dasyurum that are susceptible to this strain of B. fabae.     

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.     

White root rot disease of the lacquer tree <Emphasis Type="Italic">Toxicodendron vernicifluum</Emphasis> caused by <Emphasis Type="Italic">Rosellinia necatrix</Emphasis>

In early August 2010, lacquer trees (Toxicodendron vernicifluum) severely damaged by a root rot disease were found on plantations in Iwate, Japan. The causal agent was a fungus identified as Rosellinia necatrix, based on morphology and the sequence of the ribosomal DNA internal transcribed spacer region. The fungus was clearly pathogenic on T. vernicifluum root plantings. This report is the first of white root rot on T. vernicifluum.     

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.     

Characterization of <Emphasis Type="Italic">Inago1</Emphasis> and <Emphasis Type="Italic">Inago2</Emphasis> retrotransposons in <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

From the genome of a Japanese field isolate of the rice blast fungus, Magnaporthe oryzae, we newly identified Inago1 and Inago2 LTR retrotransposons. Both elements were found to be Ty3/gypsy-like elements whose copies were dispersed within the genome of Magnaporthe spp. isolates infecting rice and other monocot plants. Southern hybridization patterns of nine re-isolates derived from conidia of the strain Ina168 produced after a methyl viologen treatment were not changed, indicating that the insertion pattern of Inago elements is relatively stable.     

Pathogenicity of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> against <Emphasis Type="Italic">Galleria mellonella</Emphasis>

The greater wax moth Galleria mellonella L. (Lepidoptera: Pyralidae) is occasionally found in beehives and is a major pest of stored wax. Entomopathogenic fungi have recently received attention as possible biocontrol elements for certain insect pests. In this study, 90 isolates of Beauveria bassiana and 15 isolates of Metarhizium anisopliae were screened for proteases and lipases production. The results showed significant variations in the enzymatic action between the isolates. In the bioassay, the selected isolates evinced high virulence against the 4th instar of the G. mellonella larvae. The isolates BbaAUMC3076, BbaAUMC3263 and ManAUMC3085 realized 100% mortality at concentrations of 5.5 × 10⁶ conidia ml⁻¹, 5.86 × 10⁵ conidia ml⁻¹, and 4.8 × 10⁶ conidia ml⁻¹, respectively. Strong enzymatic activities in vitro did not necessarily indicate high virulence against the tested insect pest. The cuticle of the infected larvae became dark and black-spotted, indicating direct attack of fungus on the defense system of the insects. The LC₅₀ values were 1.43 × 10³, 1.04 × 10⁵ and 5.06 × 10⁴ for Bba3263AUMC, Bba3076AUMC and Man3085AUMC, respectively, and their slopes were determined by computerized probit analysis program as 0.738 ± 0.008, 0.635 ± 0.007 and 1.120 ± 0.024, respectively.     

Breeding <Emphasis Type="Italic">Lupinus albus</Emphasis> for resistance to the root pathogen <Emphasis Type="Italic">Pleiochaeta setosa</Emphasis>

Pleiochaeta root rot (PRR) caused by Pleiochaeta setosa is a serious, widespread fungal disease in lupin crops, especially in Lupinus albus (broad-leaf lupin, or white lupin). PRR resistance is common in the gene pool of L. albus with various landraces from the Mediterranean region being the most resistant, and suitable for use in breeding new cultivars. Heritability of resistance is sufficient to make good gains from selection but only when controlled-environment (CE) screening is used. Field disease nurseries on loamy soil gave much lower heritability of resistance. Field disease nurseries had spatially variable spore counts despite continuous lupin cropping, and this was partly responsible (along with climatic conditions) for their reduced precision compared to tests conducted in a CE. Giving infected L. albus roots a single, most-severe-lesion score on a 0–9 scale was adequate for CE screening but not as precise or discriminating as the more time-consuming method of six scores per root. Replication in CE experiments was reduced to two pots of 16 seedlings each without sacrificing genotype discrimination.     

Bacterial brown stripe of rice in soil-less culture system caused by <Emphasis Type="Italic">Acidovorax avenae</Emphasis> subsp. <Emphasis Type="Italic">avenae</Emphasis> in China

In 2010, the outbreak of a disease with symptoms similar to bacterial brown stripe was observed in rice seedlings planted in a perlite culture system in China. The causal bacterium was identified as Acidovorax avenae subsp. avenae based on its biochemical and physiological characteristics, cellular fatty acid composition, Biolog data, specific PCR detection and 16S rRNA gene sequence analysis. The bacterial isolates caused similar symptoms after inoculation of rice seedlings. This report is the first of bacterial brown stripe of rice in a soil-less culture system caused by A. avenae subsp. avenae in China.     

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

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

Epidemiology of root rot caused by <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> in <Emphasis Type="Italic">Brassica napus</Emphasis> crops

The infection of above-ground tissues of Brassica napus by Leptosphaeria maculans is well understood. However, root infection (root rot) under field conditions, the development of root rot over time and its relationship to other disease symptoms caused by L. maculans has not been described. A survey of B. napus crops was conducted in Australia to investigate the incidence and severity of root rot. Additionally, the pathway of root infection was examined in field experiments. Root rot was present in 95% of the 127 crops surveyed. The severity and incidence of root rot was significantly correlated with that of crown canker; however, the strength of this relationship was dependent on the season. Root rot symptoms appeared before flowering and increased in severity during flowering and at maturity, a pattern similar to crown canker suggesting that the infection of the root is an extension of the crown canker phase of the L. maculans lifecycle. All isolates of L. maculans tested in glasshouse experiments caused root rot and crown canker in B. napus and Brassica juncea. In the field, the main pathway of root infection is via invasion of cotyledons or leaves by airborne ascospores, rather than from inoculum in the soil. Root rot was present in crops in fields that had never been sown to B. napus previously, in plants grown in fumigated fields, and in glasshouse-grown plants inoculated in the hypocotyl with L. maculans.     

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.     

<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 salt-wort (<Emphasis Type="Italic">Salsola komarovii</Emphasis>) caused by <Emphasis Type="Italic">Colletotrichum truncatum</Emphasis>

In July 2006, black rot was observed on the leaves of 4-leaf-stage seedlings of salt-wort (Salsola komarovii) in Yamagata Prefecture, Japan. We isolated two single-conidial isolates from the diseased leaves. Although colony appearance of the isolates was different from that of each other, both isolates were identified as Colletotrichum truncatum by morphology and molecular similarity. After inoculation of healthy salt-wort plants with the isolates, the isolates were reisolated from symptomatic plants. We thus propose a new disease, anthracnose of salt-wort.