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.     

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.     

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.     

Survival of <Emphasis Type="Italic">Curtobacterium flaccumfaciens</Emphasis> pv. <Emphasis Type="Italic">flaccumfaciens</Emphasis> in the soil under Brazilian conditions

Mustard clubroot, caused by Plasmodiophora brassicae, is a serious disease that affects Brassica juncea var. tumida Tsen, a mustard plant that is the raw material for a traditional fermented food manufactured in the Chongqing Municipality, People’s Republic of China. To find antagonistic bacteria for P. brassicae, 124 bacteria were obtained from the rhizosphere soil of B. juncea var. tumida grown in Fuling, Chongqing. Isolates were preliminarily chosen by evaluating the inhibition rate of the P. brassicae resting spore germination. The biocontrol effects of three antagonistic bacteria against clubroot on B. juncea var. tumida were evaluated in a greenhouse experiment. B18 showed the highest control efficiency, at 63.4% in the greenhouse test. In a field trial, B18 was also effective in controlling clubroot, but only at a 49.7% efficiency rate. According to 16S rDNA sequence analysis, strain B18 had a 100% sequence similarity with type strain Zhihengliuella aestuarii DY66^T (EU939716). Based on morphological, cultural, physiological and biochemical characteristics, the DNA G + C content, polar lipids, fatty acids, cell wall analysis, as well as DNA–DNA hybridization, strain B18 was identified as Z. aestuarii B18. Thus, the isolate B18 might have a potential biocontrol application for clubroot. We report for the first time that Z. aestuarii B18 can control clubroot.     

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.     

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.     

<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.     

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.     

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.     

Toxigenicity and pathogenicity of <Emphasis Type="Italic">Fusarium poae</Emphasis> and <Emphasis Type="Italic">Fusarium avenaceum</Emphasis> on wheat

In a field experiment between 2004 and 2006, 14 winter wheat varieties were inoculated with either a mixture of three isolates of F. poae or a mixture of three isolates of F. avenaceum. In a subsequent climate chamber experiment, the wheat variety Apogee was inoculated with individual single conidium isolates derived from the original poly conidium isolates used in the field. Disease symptoms on wheat heads were visually assessed, and the yield as well as the fungal incidence on harvested grains (field only) was determined. Furthermore, grains were analysed using LC-MS/MS to determine the content of Fusarium mycotoxins. In samples from field and climate chamber experiments, 60 to 4,860 μg kg⁻¹ nivalenol and 2,400 to 17,000 μg kg⁻¹ moniliformin were detected in grains infected with F. poae and F. avenaceum, respectively. Overall, isolate mixtures and individual isolates of F. avenaceum proved to be more pathogenic than those of F. poae, leading to a higher disease level, yield reductions up to 25%, and greater toxin contamination. For F. poae, all variables except for yield were strongly influenced by variety (field) and by isolate (climate chamber). For F. avenaceum, variety had a strong effect on all variables, but isolate effects on visual disease were not reflected in toxin production. Correlations between visual symptoms, fungal incidence, and toxin accumulation in grains are discussed.     

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.     

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.     

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">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%.     

Natural phenolic acids from wheat bran inhibit <Emphasis Type="Italic">Fusarium culmorum</Emphasis> trichothecene biosynthesis <Emphasis Type="Italic">in vitro</Emphasis> by repressing <Emphasis Type="Italic">Tri</Emphasis> gene expression

The effect of natural phenolic acids from wheat bran on type B trichothecene biosynthesis by Fusarium culmorum was investigated in vitro. Durum wheat bran contained various monomeric forms of phenolic acids, with ferulic acid being the most abundant. In addition, various oligomeric forms of ferulic acid and mainly dimeric forms were also detected. When liquid cultures of F. culmorum were supplemented with a natural wheat bran extract, trichothecene production was fully inhibited. The exact mechanism by which toxin synthesis is repressed remains to be clarified but we showed that the phenolic acid treatment resulted in a drastic reduction in the expression level of structural trichothecene biosynthetic genes. The inhibitory efficiency of the natural phenolic acid extract was significantly higher than that of a reconstituted mixture containing similar concentrations of monomeric forms. Thus, to elucidate the full repression of type B trichothecene production induced by the natural phenolic acid extract from wheat bran, two hypotheses can be raised: (i) a synergistic impact of monomeric and dimeric forms of phenolic acids, (ii) the occurrence of an unidentified oligomeric form able to efficiently repress toxin yield. As a first attempt to investigate the effect of oligomeric forms, one of the most abundant dimer of ferulic acid, the 8-5′-benzofuran dimer, has been synthesized in vitro and was shown to inhibit trichothecene biosynthesis to the same extent than the monomer of ferulic acid.     

Conidial sporulation of <Emphasis Type="Italic">Stemphylium solani</Emphasis> under laboratory conditions and infectivity of the inoculum produced <Emphasis Type="Italic">in vitro</Emphasis>

Potato virus Y (PVY) is the type-species of the genus Potyvirus, family Potyviridae, being reported as a major tomato (Solanum lycopersicum L.) pathogen in several regions of the world. Pepper yellow mosaic virus (PepYMV) was originally described as a resistance-breaking Potato virus Y (PVY) isolate on Capsicum annuum L. cultivars, and afterwards it was also reported infecting tomatoes in Brazil. In the present work, a search for sources of resistance to both PepYMV and PVY was conducted in a collection of 119 accessions belonging to seven Solanum (section Lycopersicon) species. This germplasm was initially evaluated to PepYMV reaction by mechanical inoculation followed by symptom observations and ELISA. Potential PepYMV resistance sources were identified for the first time in S. habrochaites, S. peruvianum, S. corneliomuelleri, S. chilense, S. pimpinellifolium, and one accession derived from an interspecific cross (S. lycopersicum x S. peruvianum). A sub-group of 24 accessions with negative serology for PepYMV was also challenged with a PVY isolate, followed by serological and molecular detection with universal primers. Solanum habrochaites ‘L.03683’ and ‘L.03684’ were the only accessions found with stable resistance to both viruses. These results confirm S. habrochaites as the most important source of multiple resistance factor(s) to distinct Potyvirus species.     

Cloning of the pathogenicity-related gene <Emphasis Type="Italic">FPD1</Emphasis> in <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis>

We selected a reduced-pathogenicity mutant of Fusarium oxysporum f. sp. lycopersici, a tomato wilt pathogen, from the transformants generated by restriction enzyme-mediated integration (REMI) transformation. The gene tagged with the plasmid in the mutant was predicted to encode a protein of 321 amino acids and was designated FPD1. Homology search showed its partial similarity to a chloride conductance regulatory protein of Xenopus, suggesting that FPD1 is a transmembrane protein. Although the function of FPD1 has not been identified, it does participate in the pathogenicity of F. oxysporum f. sp. lycopersici because FPD1-deficient mutants reproduced the reduced pathogenicity on tomato.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession number AB110097     

Concentration-dependent effect of salicylic acid application on wheat seedling resistance to take-all fungus, <Emphasis Type="Italic">Gaeumannomyces graminis</Emphasis> var. <Emphasis Type="Italic">tritici</Emphasis>

Application of 0.1 and 0.2 mM salicylic acid (SA) significantly reduced take-all disease caused by Gaeumannomyces graminis var. tritici (Ggt) and increased the root and shoot lengths and biomass, whereas 0.5 and 1 mM SA had no significant effect. The effective SA concentrations also increased the activities of soluble peroxidase (SPOX) and cell-wall-bound peroxidase (CWPOX) and the concentration of total phenolic compounds. SPOX activity was highest at days 4 and 3 in healthy roots and those inoculated with Ggt, respectively, and that of CWPOX at day 6 in both healthy and inoculated roots. The concentration of phenolic compound was also highest at day 3 in both healthy roots and those inoculated with Ggt. The results indicate that the protective effect of SA depends on certain concentrations which increase peroxidase activity and phenolic compounds accumulation in the wheat roots; higher SA concentrations did not differ from the controls.     

Differences between spring wheat cultivars in tolerance to <Emphasis Type="Italic">Fusarium</Emphasis> seedling blight under organic field conditions

Use of Fusarium-infected seed for cereal crops results in a reduced plant density due to seedling blight. This is especially a problem in organic agriculture, for which currently no practical seed disinfection methods are available. In the present paper we investigated whether spring wheat cultivars differ in tolerance to seedling blight in vivo, whether the possible differences could be linked to cultivar differences in initial growth rates, and whether differences in weed infestation were related to differences in emergence. Seed six spring wheat cultivars (Melon, Lavett, SW Kungsjett, Epos, Pasteur, Thasos), containing three Fusarium infection levels were obtained and sown in two field experiments in 2006 and 2007 and in an outdoor pot experiment in 2007. Results indicated that the six spring wheat cultivars differed in their tolerance to seedling blight, and consequently in the percentage of emergence of their seeds. The relative levels of tolerance to seedling blight of the six cultivars were robust in the three experiments performed. No clear relationship between initial growth rates and tolerance was found. In our experiments, no early and homogenous weed pressure was present, but in the 2007 field experiment a relationship between initial seedling emergence and weed infestation after anthesis was determined. Based on the presented results we suggest that additional to resistance to Fusarium head blight (FHB), differences in tolerance to seedling blight should also be considered during selection of wheat cultivars for organic agriculture.