Hideo Ishii

Induced Resistance of Acibenzolar-S-methyl (CGA 245704) to Cucumber and Japanese Pear Diseases

Antifungal activity of the novel compound acibenzolar-S-methyl (CGA245704: benzo[1,2,3]thiadiazole-7-carbothioic acid S-methyl ester) was examined in vitro. No remarkable activity was observed on mycelial growth and conidial germination of almost all fungi tested. Only melon isolates of Didymella bryoniae were sensitive to this compound. On potted plants, acibenzolar-S-methyl showed control efficacy on anthracnose and scab of cucumber and rust of Japanese pear but not on Fusarium wilt of cucumber. In field trials, the occurrence of both rust and scab on Japanese pear was suppressed with this compound. Based on these experiments, it was suggested that acibenzolar-S-methyl induced resistance to some but not all diseases on cucumber and Japanese pear. Induction of disease resistance in cucumber was rapidly triggered after treatment with acibenzolar-S-methyl.

Fungicide Sensitivity and Phylogenetic Relationship of Anthracnose Fungi Isolated from Various Fruit Crops in Japan

Anthracnose diseases of fruit crops are mainly caused by Colletotrichum gloeosporioides and C. acutatum. In these Colletotrichum species, intra- and interspecific variation in fungicide sensitivity has been reported; however, the relationship between fungicide sensitivity and molecular phylogeny has not been analyzed. Fifty-one isolates from 10 fruit crops, acacia, and tea were tested for their sensitivities to thiophanate-methyl, diethofencarb, and iminoctadine-triacetate, and their internal transcribed spacer (ITS) and 5.8S regions of rDNA were analyzed. C. gloeosporioides isolates were divided into sensitive, less sensitive, intermediate resistant, or resistant to the three fungicides. In contrast, C. acutatum isolates were all less sensitive. In molecular phylogenetic analyses, C. gloeosporioides isolates fell into the same genetic group, whereas C. acutatum isolates were placed into two genetic groups. Although phylogenetic relationship was not closely related to fungicide sensitivity, the isolates of C. gloeosporioides most resistant to iminoctadine-triacetate were found in the same phylogenetic subgroup.

Characterisation of QoI-resistant field isolates of Botrytis cinerea from citrus and strawberry

BACKGROUND In 2004, field isolates of Botrytis cinerea Pers. ex Fr., resistant to strobilurin fungicides (QoIs), were first found in commercial citrus orchards in Wakayama Prefecture, Japan. Subsequently, QoI-resistant isolates of this fungus were also detected in plastic strawberry greenhouses in Saga, Ibaraki and Chiba prefectures, Japan. Biological and molecular characterisation of resistant isolates was conducted in this study. RESULTS QoI-resistant isolates of B. cinerea grew well on PDA plates containing kresoxim-methyl or azoxystrobin at 1 mg L(-1), supplemented with 1 mM of n-propyl gallate, an inhibitor of alternative oxidase, whereas the growth of sensitive isolates was strongly suppressed. Results from this in vitro test were in good agreement with those of fungus inoculation tests in vivo. In resistant isolates, the mutation at amino acid position 143 of the cytochrome b gene, known to be the cause of high QoI resistance in various fungal pathogens, was found, but only occasionally. The heteroplasmy of cytochrome b gene was confirmed, and the wild-type sequence often present in the majority of resistant isolates, indicating that the proportion of mutated cytochrome b gene was very low. CONCLUSION The conventional RFLP and sequence analyses of PCR-amplified cytochrome b gene are insufficient for molecular identification of QoI resistance in B. cinerea.

Fungicide Resistance in Plant Pathogens

Fungicide resistance emerged as a practical disease control problem in the 1970s, but it was the outcome of two workshops in Wageningen in 1980 and 1981 that set the framework for research to tackle the problem. Some but not all fungicides quickly select already-existing resistant mutants from within target pathogen populations. Several mechanisms contribute to resistance, but where targetsite changes predominate, cross resistance does not extend to other modes of action. Field effi cacy and bioassay are key to confi rming resistance, but molecular techniques are increasingly used to detect resistance and to augment biochemistry to determine mechanisms. Resistance is not inevitable but depends on the impact of both pathogen and fungicide properties on pathogen populations. Some factors can be manipulated to minimise resistance risk, and a cornerstone of anti-resistance strategies combines treatments with more than one mode of action, either in mixtures or in alternation. Controlled release formulations may also help reduce selection. Resistance has a fi nancial cost to users and manufacturers and seriously reduces available modes of action. Consequently to combat resistance, fungicides should be embedded in integrated disease management systems.

Chemical control of plant diseases

As the world population increases, we also need to increase food production. Chemical control has been critical in preventing losses due to plant diseases, especially with the development of numerous specific-action fungicides since the 1960s. In Japan, a host-defense inducer has been used to control rice blast since the 1970s without any problems with resistance development in the pathogen. Leaf blast has been controlled using a labor-saving method such as the one-shot application of a granular mixture of fungicide and insecticide to nursery boxes, which became mainstream in the 2000s. However, the need for many choices of fungicides that have several modes of action was demonstrated by the development of resistance to cytalone dehydratase inhibitors. In Europe, many pathogens have threatened cereals since the great increase in cereal production in 1970s, creating a large market for broad-spectrum fungicides. In Brazil, Phakopsora pachyrhizi was distributed to large soybean acreages during 2000s, and the outbreak of soybean rust resulted in a large increase in fungicide use. While the importance of chemical control is recognized, fungicide resistance is an avoidable problem; published guidelines on countermeasure and manuals on testing sensitivity to fungicides are available. Since chemical regulations have become stricter, new fungicides are less likely to be developed. Our task is to maintain the effectiveness and diversity of the present modes of action for fungicides and implement countermeasures against the development of fungicide resistance.

First application of PCR-Luminex system for molecular diagnosis of fungicide resistance and species identification of fungal pathogens

Fungicide resistance in plant pathogens is often caused by a single point mutation in a gene encoding fungicide target proteins. Such is the case for resistance to MBI-D (inhibitors of scytalone dehydratase in melanin biosynthesis) fungicides in rice blast fungus (Magnaporthe oryzae), which is caused by a mutation in the scytalone dehydratase gene that results in a replacement of valine with methionine at codon 75 of the fungicide target protein. PCR-Luminex, a novel system developed for high-throughput analysis of single nucleotide polymorphisms (SNPs) was successfully introduced to diagnose MBI-D resistance using specific oligonucleotide probes coupled with fluorescent beads. The PCR-Luminex system was further tested for its potential in identifying species causing Fusarium head blight on wheat. Four major pathogens, Fusarium graminearum (=F. asiaticum), F. culmorum, F. avenaceum, and Microdochium nivale, known to cause the disease, were tested, and the species were identified using the PCR-Luminex method. So far, this report is the first on the application of the DNA-based PCR-Luminex system in the area of crop protection and/or agricultural sciences.

Cytological evaluation of the effect of azoxystrobin and alternative oxidase inhibitors in Botrytis cinerea

Azoxystrobin (AZ), a strobilurin-derived fungicide, is known to inhibit mitochondrial respiration in fungi by blocking the electron transport chain in the inner mitochondrial membrane. Germination was strongly inhibited when Botrytis cinerea spore suspension was treated with AZ and the alternative oxidase (AOX) inhibitors, salicylhydroxamic acid (SHAM) and n-propyl gallate. However, chemical death indicators trypan blue and propidium iodide showed that those spores were still alive. When the spore suspension in the AZ and SHAM solution was replaced with distilled water, the germination rate almost recovered, at least during the first 2 days of incubation with AZ and SHAM solution. No morphological alteration was detected in the cells treated with AZ and SHAM, especially in mitochondria, using transmission electron microscopy. Therefore, simultaneous application of AZ and AOX inhibitors has a fungistatic, rather than a fungicidal, action.

Genetic analysis and molecular characterisation of laboratory and field mutants of Botryotinia fuckeliana (Botrytis cinerea) resistant to QoI fungicides

BACKGROUND QoI fungicides, inhibitors of mitochondrial respiration, are considered to be at high risk of resistance development. In several phytopathogenic fungi, resistance is caused by mutations (most frequently G143A) in the mitochondrial cytochrome b (cytb) gene. The genetic and molecular basis of QoI resistance were investigated in laboratory and field mutants of Botryotinia fuckeliana (de Bary) Whetz. exhibiting in vitro reduced sensitivity to trifloxystrobin. RESULTS B. fuckeliana mutants highly resistant to trifloxystrobin were obtained in the laboratory by spontaneous mutations in wild-type strains, or from naturally infected plants on a medium amended with 1-3 mg L(-1) trifloxystrobin and 2 mM salicylhydroxamic acid, an inhibitor of alternative oxidase. No point mutations were detected, either in the complete nucleotide sequences of the cytb gene or in those of the aox and Rieske protein genes of laboratory mutants, whereas all field mutants carried the G143A mutation in the mitochondrial cytb gene. QoI resistance was always maternally inherited in ascospore progeny of sexual crosses of field mutants with sensitive reference strains. CONCLUSIONS The G143A mutation in cytb gene is confirmed to be responsible for field resistance to QoIs in B. fuckeliana. Maternal inheritance of resistance to QoIs in progeny of sexual crosses confirmed that it is caused by extranuclear genetic determinants. In laboratory mutants the heteroplasmic state of mutated mitochondria could likely hamper the G143A detection, otherwise other gene(s) underlying different mechanisms of resistance could be involved.

QoI Fungicide Resistance: Current Status and the Problems Associated with DNA-Based Monitoring

QoI fungicides which inhibit mitochondrial respiration at the ubiquinol oxidation centre (Qo site) of the cytochrome bc1 enzyme complex, are one of the most important class of agricultural fungicides. QoI fungicides generally carry a high risk of pathogen resistance development with resistance occurring in over 30 pathogen species, such as powdery mildews, downy mildews, anthracnose, Alternaria spp., scab, and grey mould. Molecular mechanisms of QoI resistance have been intensively studied; a single point mutation which causes an amino acid change in cytochrome b, G143A in particular, was described to govern the expression of high resistance. A range of molecular methods including PCR-RFLP, allele-specific PCR, quantitative real-time PCR and pyrosequencing have been developed, enabling the rapid detection and quantification of resistance. However, the status of heteroplasmy in the mitochondrial genome which contains the cytochrome b gene can cause instability over time, making it difficult to precisely monitor QoI resistance in some pathogens. The role of the alternative oxidase pathway in QoI resistance is not clear as yet, although this enzyme is very likely involved in resistance development of grey mould in particular. Novel QoI fungicides have been developed, some of which show differential patterns of cross-resistance to pre-existing QoI fungicides. This paper summarizes QoI resistance development over the last decade as well as future research prospects.

Ultrastructural Study on Acibenzolar-S-Methyl-Induced Scab Resistance in Epidermal Pectin Layers of Japanese Pear Leaves

The infection behavior of Japanese pear scab pathogen Venturia nashicola race 1 was studied ultrastructurally in acibenzolar-S-methyl (ASM)-pretreated susceptible Japanese pear (cv. Kousui) leaves to determine the mechanism of ASM-induced scab resistance. On ASM-pretreated leaf surfaces, the infection behavior (conidial germination and appressorial formation) was similar to that on distilled water (DW)-pretreated leaves prior to cuticle penetration by the pathogen. However, after penetration, differentiated behavior was found in epidermal pectin layers and middle lamellae of the ASM-pretreated leaves. Subcuticular hyphae in epidermal pectin layers and middle lamellae of ASM-pretreated pear leaves were observed at lower frequency than in DW-treated leaves. The results indicated that fungal growth was suppressed in ASM-pretreated pear leaves. In the pectin layers of ASM- and DW-pretreated leaves, some hyphae showed morphological modifications, which were used as criteria to judge collapse of hyphal cells, including plasmolysis, necrotic cytoplasm, and cell wall destruction. More hyphae had collapsed in ASM-pretreated leaves than in DW-treated ones. In addition, the cell walls of collapsed hyphae broke into numerous fibrous and amorphous pieces, suggesting that ASM-induced scab resistance might be associated with cell-wall-degrading enzymes from pear plants. In addition, results from morphometrical analysis suggested that the activity or production of pectin-degrading enzyme from hyphae were inhibited by ASM application when compared with DW treatment.