Effects of garlic (<Emphasis Type="Italic">Allium sativum</Emphasis>) juice containing allicin on <Emphasis Type="Italic">Phytophthora infestans</Emphasis> and downy mildew of cucumber caused by <Emphasis Type="Italic">Pseudoperonospora cubensis</Emphasis>

The volatile antimicrobial substance allicin (diallylthiosulphinate) is produced in garlic when the tissues are damaged and the substrate allicin (S-allyl-l-cysteine sulphoxide) mixes with the enzyme alliin-lyase (E.C.4.4.1.4). Allicin undergoes thiol-disulphide exchange reactions with free thiol groups in proteins and it is thought that this is the basis of its antimicrobial action. At 50 μg ml^-1, allicin in garlic juice inhibited the germination of sporangia and cysts and subsequent germ tube growth by Phytophthora infestans both in vitro and in vivo on the leaf surface. Disease severity in P. infestans-infected tomato seedlings was also reduced by spraying leaves with garlic juice containing allicin over the range tested (55–110 μg ml⁻¹) with an effectiveness ranging from approximately 45–100%. Similarly, in growth room experiments at concentrations from 50–1,000 μg ml⁻¹, allicin in garlic juice reduced the severity of cucumber downy mildew caused by Pseudoperonospora cubensis by approximately 50–100%. These results suggest a potential for developing preparations from garlic for use in specialised aspects of organic farming, e.g. for reducing pathogen inoculum potential and perhaps for use under glass in horticulture.     

A new pathotype of<Emphasis Type="Italic">Pseudoperonospora cubensis</Emphasis> causing downy mildew in cucurbits in Israel

Downy mildew, incited by the comycetePseudoperonospora cubensis (Berk.et Curt.) Rost., was recorded in Israel during the years 1979–2001 on cucumber (Cucumis sativus) and melon (Cucumis melo) and classified as pathotype 3. In July 2002 severe outbreaks of downy mildew occurred on pumpkin (Cucurbita moschata) and summer squash (Cucurbita pepo subsp.pepo). Host range pathogenicity studies revealed high compatibility of the new population with cucumber, melon, pumpkin and summer squash but low compatibility with watermelon. This new population was therefore designated as pathotype 6. The possible origin of this new pathotype in Israel is discussed. http://www.phytoparasitica.org posting Oct. 9, 2003.     

Mating type and sexual reproduction of <Emphasis Type="Italic">Pseudoperonospora cubensis</Emphasis>, the downy mildew agent of cucurbits

The oomycete Pseudoperonospora cubensis is a leaf pathogen causing severe damage to members of the Cucurbitaceae, especially cucumber and melon. It propagates clonally by sporangia. Oospores of P. cubensis were previously observed in nature but their formation in the laboratory was never reported nor their germination or infection. Here we report on the sexual reproduction of P. cubensis under controlled conditions in the laboratory. When field isolates were inoculated singly onto detached leaves of cucurbits in growth chambers no oospores were produced. However, when pairs of selected isolates were mixed and inoculated onto detached leaves, oospores were formed in the mesophyll within 6–11 days, suggesting that P. cubensis is heterothallic, having two opposite mating types, A1 and A2. Isolates belonging to pathotype 3 were all A1 whereas isolates belonging to the new pathotype 6 were either A1 or A2. Oospores were spherical, ~40 μm in diameter, hyaline to red-brown in color. Oospores were produced regularly, in large numbers, in Cucumis sativum and Cucumis melo, very seldom and in very small numbers in Cucurbita pepo, Cucurbita maxima and Citrullus lanatus, and not in Cucurbita moschata. Oospores were formed at 12.5–21°C but not at 25°C. Under moisture-saturated atmosphere oospores were also produced in leaves of intact plants. Oospores inoculated onto detached leaves in growth chambers produced F1 downy mildew lesions at 6–21 days after inoculation, many in Cucumis sativum, Cucumis melo and Cucurbita moschata, very few in Cucurbita pepo or Citrullus lanatus, and none in Cucurbita maxima. This report shows that P. cubensis is heterothallic, having A1 and A2 mating types which can cross and enable sexual reproduction in cucurbits. A preliminary report on part of the results has been published earlier.     

A potential perennial host for <Emphasis Type="Italic">Pseudoperonospora cubensis</Emphasis> in temperate regions

Pseudoperonospora cubensis causes great losses in cucurbitaceous crops worldwide. In cool temperate climates of northern Europe or North America overwintering as active mycelium is not possible, because all hosts so far reported there are summer annuals. Oospores have not yet been found in these regions under field conditions. The only perennial member of the Cucurbitaceae found naturally in central and northern Europe is Bryonia dioica. To date this plant has not been recorded as a host for downy mildews, but our infection trials demonstrate that P. cubensis is able to infest this plant. Amplification and sequencing of the ITS rDNA confirmed the observed downy mildew disease on B. dioica as P. cubensis. From these findings, the possibility that P. cubensis may be able to overwinter on this perennial host cannot be excluded. Whether or not B. dioica plays a part in the epidemology of P. cubensis in Europe requires evaluation by further studies.     

Host matrix has major impact on the morphology of <Emphasis Type="Italic">Pseudoperonospora cubensis</Emphasis>

Oomycetes contain some of the economically most important pathogens of flowering plants. Most have a rather narrow host range, often being restricted to single host species. In downy mildews and other obligate biotrophic plant parasites, like powdery mildews and rusts, delimitating species on grounds of morphological characteristics is often hardly possible and thus often based on only subtle differences. This has led to the widespread application of a broad species concept for these organisms. Consequently, despite the fact that morphological differences were reported for Pseudoperonospora cubensis from different host species, the corresponding new pathogen species were not accepted as being independent, and the host range of Pseudoperonospora cubensis is reported to encompass more than 50 host species in the Cucurbitaceae in temperate to tropical climates. However, recent studies have reported narrow host ranges for other downy mildew genera and advocated a narrow species concept. Here, we report successful colonisation of five different tribes of the Cucurbitaceae by a strain of Pseudoperonospora cubensis and demonstrate that the host matrix has a major impact on the morphology of the pathogen. On the basis of five morphological criteria significant differences could be found for all hosts. These differences were more pronounced in phylogenetically unrelated than in related hosts. Our results provide evidence for a broad host range of Pseudoperonospora cubensis and demonstrate that species delimitation based on morphological characters is not feasible in Pseudoperonospora on Cucurbitaceae. Also in other biotrophic plant pathogens, the situation could be similar, thus necessitating thorough morphological, molecular phylogenetic and cross inoculation experiments for species recognition.     

Synergistic interaction between BABA and mancozeb in controlling<Emphasis Type="Italic">Phytophthora infestans</Emphasis> in potato and tomato and<Emphasis Type="Italic">Pseudoperonospora cubensis</Emphasis> in cucumber

Spray mixtures consisting of the plant activator BABA (DL-3-aminobutyric acid) and the protectant fungicide mancozeb were significantly more effective than BABA or mancozeb alone in controlling late blight (Phytophthora infestans) in potato and tomato and downy mildew (Pseudoperonospora cubensis) in cucumber. A mixture composed of 5 parts BABA and 1 part mancozeb (w/w, a.i.) exhibited a higher synergy factor than the 1+1 or the 1+5 (BABA + mancozeb) mixtures. No synergistic interaction was detected between BABA plus mancozeb in controlling sporangial or cystospore germination, nor mycelial growth ofP. infestans in vitro. The results showed enhanced effect of mancozeb in BABA-induced plants, suggesting, therefore, that lower dosages of this fungicide may be sufficient to control late blight or downy mildew under field conditions. http://www.phytoparasitica.org posting July 15, 2003.     

Identification of <Emphasis Type="Italic">Pseudoperonospora cubensis</Emphasis> using real-time PCR and high resolution melting (HRM) analysis

To develop a molecular method to identify Pseudoperonospora cubensis in cucumber leaves with signs of downy mildew, we compared the nucleotide sequences of ribosomal DNA-internal transcribed spacer, cytochrome oxidase II, and β-tubulin genes of P. cubensis and P. humuli isolates. Seven single nucleotide polymorphisms (SNPs) distinguished P. cubensis and P. humuli based on variations in β-tubulin sequences, and one specific primer set was designed for further analysis. Real-time PCR and high resolution melting analysis showed that the primer set can be used to specifically identify P. cubensis in cucumber leaves with downy mildew.     

Rice <Emphasis Type="Italic">terpene synthase 18</Emphasis> (<Emphasis Type="Italic">OsTPS18</Emphasis>) encodes a sesquiterpene synthase that produces an antibacterial (<Emphasis Type="Italic">E</Emphasis>)-nerolidol against a bacterial pathogen of rice

Plant volatile compounds, including terpenes, are known to be involved in the rice defense system. In the present analysis of a terpene synthase, OsTPS18, in rice, we found that OsTPS18 was localized in the cytoplasm and synthesized the sesquiterpenes (E)-nerolidol and (E)-β-farnesene. The amounts of (E)-nerolidol and (E)-β-farnesene increased after jasmonic acid (JA) treatment. (E)-Nerolidol had significant antibacterial activity against Xanthomonas oryzae pv. oryzae (Xoo). These results suggest that (E)-nerolidol plays an important role in JA-induced resistance against Xoo and that it functions as an antibacterial compound in rice.     

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.     

Recent races of spinach downy mildew pathogen <Emphasis Type="Italic">Peronospora farinosa</Emphasis> f. sp. <Emphasis Type="Italic">spinaciae</Emphasis> in Japan

Isolates of spinach downy mildew fungus, Peronospora farinosa f. sp. spinaciae, collected at seven sites in Japan from December 2013 to March 2016, were identified as belonging to races 8, 10, 12 and 13, and one was unauthorized. Races 10, 12, 13 and the unauthorized were first found in Japan.     

Morphological and molecular characterization of <Emphasis Type="Italic">Oidium</Emphasis> subgenus <Emphasis Type="Italic">Reticuloidium</Emphasis> (powdery mildew) newly occurred on cucumber in Japan

In 2002, a powdery mildew with catenate conidia lacking fibrosin bodies was found on cucumber in a greenhouse in Kanagawa Prefecture, Japan. Morphological observation revealed that the fungus belongs to Oidium subgenus Reticuloidium, anamorph of the genus Golovinomyces. Molecular phylogenetic analyses of the nucleotide sequences of the rDNA ITS regions and D1/D2 domains of the 28S rDNA indicated that the fungus belongs to the clade of G. orontii with other Golovinomyces fungi from a wide range of host plants, suggesting that the fungus was newly transported from abroad. Because there has been no prior report of cucumber powdery mildew caused by Reticuloidium, further research on the physiology, epidemiology, control and resistant cucumber varieties is required.     

Efficacy of a new slow-release volatile system against <Emphasis Type="Italic">Aphis gossypii</Emphasis> Glover (Hemiptera: Aphididae) on cucumber, <Emphasis Type="Italic">Cucumis sativus</Emphasis> L.

Many arthropod agricultural pests have become resistant to pesticides through inappropriate usage. A consequence of this resistance is often cross-resistance with other pesticides with similar or related modes of actions. As a result of increasing pesticide resistance, a movement to alternative, ‘green’ botanical pesticides has been growing. A new formulation of volatile oils, extracted from a variety of plants allows for slow-release and longer duration in the field. In this publication we present results of the efficacy of one of these products against the cotton aphid Aphis gossypii on cucumber plants in replicated choice tests. The slow release Ap2, a Bio-Pel product from Newseal Mineral and Coatings Ltd. (hereafter Newseal), significantly reduced the population of aphids on test plants and prevented their movement upwards on the plant. Additionally, four Newseal products caused no mortality to the aphid parasitoid Aphidius colemani or to the generalist predator, Orius laevigatus.     

Temperature and plant age drive downy mildew disease epidemics on oilseed <Emphasis Type="Italic">Brassica napus</Emphasis> and <Emphasis Type="Italic">B. juncea</Emphasis>

Studies were undertaken on the effects of temperature (14/10 °C and 22/17 °C day/night) and plant age (15, 23, 31 and 40 day-old-plants) on the severity of downy mildew (Hyaloperonospora parasitica) on oilseed Brassica cultivars (temperature: Brassica juncea Montara, B. napus Atomic, ATR-Hyden, Hyola 432, Hyola 450 TT, Thunder TT; plant age: B. juncea Dune, B. napus Surpass 402 and Hyola 450 TT). For temperature studies, there were significant (P?<?0.001) effects of temperature, cultivar, and cultivar x temperature interaction. On cotyledons of susceptible cultivars (B. napus Hyola 450 TT and Thunder TT), plants were symptomatic at 22/17 °C by 48 h post inoculation (hpi) and with abundant sporulation evident by 72 hpi, and with all cotyledons of B. napus Thunder TT collapsed by 7 days post inoculation (dpi). However, at 14/10 °C, there were no symptoms on the same cultivars until 5 dpi, and sporulation only observed at 7 dpi. Percent disease index values (DI%) at 22/17 °C of B. juncea Montara and B. napus ATR-Hyden, Hyola 432, Atomic, Hyola 450 TT and Thunder TT were 4.5, 49.0, 51.4, 65.8, 86.3 and 96.0, respectively, with all except B. juncea Montara having significantly lower (P?<?0.001) disease at 14/10 °C with DI% values of 2.8, 30.4, 27.9, 31.1, 44.4 and 76.4, respectively. For plant age studies, there were significant (P?<?0.001) effects of plant age, cultivar, and cultivar x plant age interaction. DI% was significantly higher at 15 compared to 40 day-old-plants (dop) across all cultivars. B. juncea Dune showed greatest resistance, particularly on 40 dop, with DI% values of 25.8, 24.6, 22.9 and 7.5, for 15, 23, 31 and 40 dop, respectively. B. napus Surpass 402 showed high susceptibility on cotyledons of 15 dop but moderate resistance on leaves of other ages, with DI% values of 59.0, 31.2, 27.1 and 26.2 for 15, 23, 31 and 40 dop, respectively. B. napus Hyola 450 TT showed very high susceptibility at the cotyledon stage on 15 dop, but some resistance on 23 dop and more so on 31 and 40 dop, with DI% values of 84.0, 41.2, 35.4 and 32.9 for 15, 23, 31 and 40 dop, respectively. Together, these findings explain for the first time why development of downy mildew epidemics on susceptible cultivars occurs early in the growing season when warmer seasonal temperatures in autumn coincide with presence of seedlings; in contrast to later in the growing season on less susceptible older plants coinciding with cooler and less favourable winter temperatures. Increasing maximum and minimum temperatures associated with climate change have likely fostered the increased severity of downy mildew over the past 15 years.     

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.     

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.     

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.     

Rice <Emphasis Type="Italic">OsPBL1</Emphasis> (ORYZA SATIVA ARABIDOPSIS PBS1-LIKE 1) enhanced defense of <Emphasis Type="Italic">Arabidopsis</Emphasis> against <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> DC3000

The receptor-like cytoplasmic kinases (RLCK family VII) are required for plant defense against various pathogens. Previously, OsPBL1 (ORYZA SATIVA ARABIDOPSIS PBS1-LIKE 1) was isolated from rice as a potential RSV (rice stripe virus) resistant factor, but its physiological roles in plant defense are yet to be investigated. In this study, we demonstrated that OsPBL1increased defense against P. syringae in transgenic Arabidopsis. To ascertain the role of OsPBL1 gene in plant defense, OsPBL1 tagged with HA (i.e. Hemagglutinin) was overexpressed in Arabidopsis and examined for the resistance against Pseudomonas syringae pv. tomato DC3000 (i.e. Pst DC3000). At 3 dpi of Pst DC3000, transgenic Arabidopsis lines exhibited the reduced chlorotic lesion and propagation of P. syringae, compared to wild type. Elevated pathogen resistance of transgenic lines was correlated with increased H₂O₂ accumulation and callose deposition on the infected leaves. It was also revealed that expression levels of salicylic acid dependent genes such as PR1, PR2, and PR5, were induced higher in transgenic lines than wild type. Taken together, our data suggested that OsPBL1 exerted the role in defense against pathogen attacks in plant via mainly facilitating salicylic acid dependent pathway.     

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.     

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.     

<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.