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">Trichoderma harzianum</Emphasis>- mediated reprogramming of oxidative stress response in root apoplast of sunflower enhances defence against <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

Trichoderma harzianum is an effective biocontrol agent against the devastating plant pathogen Rhizoctonia solani. Despite its wide application in agriculture, the mechanisms of biocontrol are not yet fully understood. Mycoparasitism and antibiosis are suggested, but may not be sole cause of disease reduction. In the present study, we investigated the role of oxidant-antioxidant metabolites in the root apoplast of sunflower challenged by R. solani in the presence/absence of T. harzianum NBRI-1055. Analysis of oxidative stress response revealed a reduction in hydroxyl radical concentration (^•OH; 3.6 times) at 9 days after pathogen inoculation (dapi), superoxide anion radical concentration (O₂^•−; 4.1 times) at 8 dapi and hydrogen peroxide concentration (H₂O₂; 2.7 times) levels at 7 dapi in plants treated with spent maize-cob formulation of T. harzianum NBRI-1055 (MCFT), as compared to pathogen-inoculated plants. The protection afforded by the biocontrol agent was associated with the accumulation of the ROS gene network: the catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and ascorbate peroxidase (APx), maximum activity of CAT (11.0 times) was observed at 8 dapi, SOD (7.0 times) at 7 dapi, GPx (5.4 times) and APx (8.1 times) at 7 dapi in MCFT-treated plants challenged with the pathogen. This was further supported by the inhibition of lipid and protein oxidation in Trichoderma-inoculated plants. MCFT stimulated the accumulation of secondary metabolites of phenolic nature that increased up to five-fold and also exhibited strong antioxidant activity at 8 dapi, eventually leading to the systemic accumulation of phytoalexins. These results suggest that T. harzianum–mediated biocontrol may be related to alleviating R. solani-induced oxidative stress.     

<Emphasis Type="Italic">Trichoderma harzianum</Emphasis> elicits defence response genes in roots of potato plantlets challenged by <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

Biological control of Rhizoctonia solani with Trichoderma harzianum has been demonstrated in several studies. However, none have reported the dynamics of expression of defence response genes. Here we investigated the expression of these genes in potato roots challenged by R. solani in the presence/absence of T. harzianum Rifai MUCL 29707. Analysis of gene expression revealed an induction of PR1 at 168 h post-inoculation (hpi) and PAL at 96 hpi in the plants inoculated with T. harzianum Rifai MUCL 29707, an induction of PR1, PR2 and PAL at 48 hpi in the plants inoculated with R. solani and an induction of Lox at 24 hpi and PR1, PR2, PAL and GST1 at 72 hpi in the plants inoculated with both organisms. These results suggest that in the presence of T. harzianum Rifai MUCL 29707, the expression of Lox and GST1 genes are primed in potato plantlets infected with R. solani at an early stage of infection. Mycothèque de l’Université catholique de Louvain of S. Cranenbrouck's affiliation is part of the Belgian Coordinated Collections of Micro-organisms (BCCM).     

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.     

Chitinolytic <Emphasis Type="Italic">Streptomyces griseorubens</Emphasis> E44G enhances the biocontrol efficacy against <Emphasis Type="Italic">Fusarium</Emphasis> wilt disease of tomato

Streptomyces griseorubens E44G is a chitinolytic bacterium isolated from cultivated soil in Saudi Arabia (a hot, arid climatic region). In vitro, antifungal potential of S. griseorubens E44G was assessed against the phytopathogenic fungus, Fusarium oxysporum f. sp. lycopersici (the causative agent of the Fusarium wilt disease of tomato). An inhibition zone of 24 mm was recorded. The chitinolytic activity of S. griseorubens E44G was proved when the colloidal chitin agar plate method was used. A thermostable chitinase enzyme of 45 kDa molecular weight was purified using gel filtration chromatography. The optimum activity was obtained at 60 °C and pH 5.5. The purified enzyme has shown a very pronounced activity against the phytopathogenic fungus, F. oxysporum. The molecular characterization of the chitinase gene indicated that it consists of 1218 bp encoding 407 amino acids. The phylogentic analysis based on the nucleotide DNA sequence and the deduced amino acids sequence showed high similarity percentages with other chitinases isolated from different Streptomyces species. In the field evaluation, application of both S. griseorubens E44G treatments significantly increased all tested growth and yield parameters and decreased the disease severity compared with the infected-untreated tomato plants suggesting potential as a biocontrol agent.     

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.     

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.     

<Emphasis Type="Italic">Paecilomyces lilacinus</Emphasis>, a potential biocontrol agent on apple rust mite <Emphasis Type="Italic">Aculus,schlechtendali</Emphasis> and interactions with some fungicides <Emphasis Type="Italic">in vitro</Emphasis>

The apple rust mite Aculus schlechtendali (Nal.) (Acari: Eriophyidae), is a main pest in apple-growing areas in Ankara, Turkey, and chemical control applications have some limitations. Entomopathogenic fungi have a potential for biological control of mites. In this study, an entomopathogenic fungus, Paecilomyces lilacinus (Thom) Samson (Deuteromycota: Hyphomycetes), was first isolated from the mite cadavers on Japanese crab apple leaves and pathogenicity of the fungus was observed in different inoculum densities and relative humidities. The pathogen caused up to 98.22% mortality of the mite population. The effects of some fungicides on the entomopathogenic fungus were determined in in vitro studies. Carbendazim, penconazole and tebuconazole were the most effective fungicides on mycelial growth of P. lilacinus, with EC₅₀ values under 3 μg ml⁻¹. In spore germination tests, captan, mancozeb, propineb were the most effective fungicides, followed by tebuconazole, penconazole, nuarimol and chlorothalonil. Sulphur could not inhibit the conidia germination totally at 5,000 μg ml⁻¹. Copper oxychloride and fosetyl-al prevented conidia formation at concentrations above 1,000 μg ml⁻¹.     

Nonhost resistance of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> against <Emphasis Type="Italic">Colletotrichum</Emphasis> species

Arabidopsis thaliana exhibits a durable resistance called nonhost resistance against nonadapted fungal pathogens. A. thaliana activates preinvasive resistance and terminates entry attempts by nonadapted fungi belonging to the genus Colletotrichum, which cause anthracnose disease in many plants. In the interaction between A. thaliana and nonadapted C. tropicale, the preinvasive resistance involves the PENETRATION 2-related antifungal secondary metabolite pathway and the ENHANCED DISEASE RESISTANCE 1-dependent antifungal peptide pathway. The development of invasive hyphae by C. tropicale owing to the reduction of preinvasive resistance then triggers the blockage of further hyphal expansion via the activation of the second layer of resistance, i.e., postinvasive resistance, which guarantees the robustness of the nonhost resistance of A. thaliana against Colletotrichum pathogens. Both the tryptophan-derived metabolic pathway and glutathione synthesis play critical roles in the postinvasive resistance against C. tropicale, although the molecular mechanism of postinvasive resistance remains to be elucidated. In this review, we describe the current understanding of the molecular background of the Arabidopsis nonhost resistance against Colletotrichum fungi and discuss perspectives for future research on this durable resistance.     

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.     

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.     

Assessing synergism of combined applications of <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> and <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> to control blast and bacterial leaf blight of rice

Laimaphelenchus suberensis sp. nov. obtained from declining Quercus suber trees of Herdade da Gouveia de Baixo, Alentejo, Portugal, is described and illustrated based on morphological, biometrical and molecular characters. The diagnosis of Laimaphelenchus species has been commonly based on the presence or absence of a vulval flap and on the shape structure of the tail tip. The species described here has been included in the Laimaphelenchus group without vulval flap, and can be distinguished from morphologically similar species by its tail tip shape structure that has a stalk-like terminus and three diffuse tubercles with 4–6 finger-like protrusions. For the molecular analyses, the mitochondrial DNA region from the cytochrome oxidase subunit I (mtCOI), the D2-D3 expansion segments of the large subunit (LSU) and small subunit (SSU) of rRNA gene were amplified and sequenced. Sequences of L. suberensis sp. nov. clustered separately from all Laimaphelenchus spp. with available sequences in Genbank, confirming its identification as a new species. This is the second report of the genus Laimaphelenchus in Portugal, associated with Q. suber: L. heidelbergi and L. suberensis sp. nov.     

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.     

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.     

Overexpression of <Emphasis Type="Italic">SlARG2</Emphasis> or <Emphasis Type="Italic">SlTD2</Emphasis> in Arabidopsis enhances resistance against <Emphasis Type="Italic">Plutella xylostella</Emphasis> L.

Tomato (Solanum lycopersicum L.) ARGINASE2 (ARG2) and THREONINE DEAMINASE2 (TD2) are involved in plant defense. These enzymes act in the midgut of herbivores fed on tomato plants to degrade the essential amino acids Arg and Thr, respectively. Although it has been demonstrated that overexpression of the SlARG2 gene in tomato enhanced its resistance against M. sexta larvae, knock-down the expression of SlTD2 reduced the resistance of tomato to lepidopteran herbivores; it remains unclear whether overexpression of SlTD2 could enhance the resistance of the host plants to herbivores, or whether combined overexpression of SlARG2 and SlTD2 could lead to synergistically enhanced resistance to insects. Here, we generated transgenic Arabidopsis plants overexpressing SlARG2 (SlARG2 OE) and SlTD2 (SlTD2 OE) individually as well as in combination (SlARG2-SlTD2 OE). Overexpression of these genes did not affect Arabidopsis development, seed yield, or Arg and Thr content. Insect-feeding bioassay was performed by feeding diamondback moth (Plutella xylostella L.) larvae on detached leaves of wild-type, SlARG2 OE, SlTD2 OE, and SlARG2-SlTD2 OE plants. Larvae fed on SlARG2 OE leaves showed approximately 31% to 35% reduction in weight and 6% to 10% reduction in survival rate compared to those fed on wild-type leaves. Although larvae fed on SlTD2 OE leaves showed no reduction in survival rate, they gained less weight. Whereas larvae fed on SlARG2-SlTD2 OE leaves showed neither reduction in weight nor reduction in survival rate. We further investigated the arginase enzymatic activity of the SlARG2 OE and SlARG2-SlTD2 OE transgenic plants. The SlARG2 OE line most resistant to diamondback moth larvae displayed the highest arginase activity. Our data indicate that overexpression of SlARG2 or SlTD2 in Arabidopsis can enhance its resistance against diamondback moth, whereas combined overexpression of SlARG2 and SlTD2 did not generate synergistically increased resistance to diamondback moth.     

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.     

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.     

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

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