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.     

Effect of carpropamid on secondary infection by rice blast fungus

Carpropamid (WIN™, KTU 3616) provides good control of leaf and panicle blast by ‘one-shot’ nursery-box treatment. It inhibits melanin biosynthesis in appressorial cells of Pyricularia oryzae, making them hyaline. Penetration by infection hyphae from the hyaline appressoria into rice epidermal cells is substantially hindered. In addition, the spread of rice blast spores from primary lesions to the other parts of the plant leading to secondary infection is largely prevented when the plants are treated with carpropamid by spray or water surface application. Secondary infection was simulated in a glass chamber fitted with an ultrasonic humidifier. On treated plants, many blast spores formed in the lesions, but the number of air spora that were dispersed from the lesions decreased significantly. A similar suppression of the spore liberation was observed in vitro when lesions on rice leaf segments, or discs from Pyricularia cultures on oatmeal agar were treated with the chemical. Spores from treated lesions or from the cultures on oatmeal agar amended with the chemical germinated normally and produced well-melanized appressoria on cellophane membranes. In addition, the spores proved to be fully pathogenic towards rice seedlings, producing normal disease symptoms. These results strongly suggest that carpropamid reduces the secondary infection of rice by Pyricularia by specifically hindering spore liberation. © 1999 Society of Chemical Industry     

Characterization of mutations in the two-component histidine kinase gene that confer fludioxonil resistance and osmotic sensitivity in the os-1 mutants of Neurospora crassa

Osmotic-sensitive (os-1) mutant alleles in Neurospora crassa exhibit resistance to dicarboximides, aromatic hydrocarbons and phenylpyrroles. We have previously reported that the os-1 mutants can be classified into two groups based on their resistance to fungicides and osmotic stress: type I, which are highly resistant to iprodione and fludioxonil but moderately sensitive to osmotic stress, and type II, which are highly sensitive to osmotic stress but moderately resistant to fungicides. To explain the mechanism of resistance to these fungicides, we cloned and sequenced the mutant os-1 genes that encode putative osmo-sensing histidine kinase. Within the os-1 gene product (Os1p), the type I strains, NM233t and Y256M209, carried a stop codon at amino acid position 308 and a frameshift at amino acid position 294, respectively. These mutation sites were located on the upstream of histidine kinase and the response regulator domains of Os1p, strongly suggesting that type I strains are null mutants. The null mutants, NM233t and Y256M209, were highly resistant to iprodione and fludioxonil; thus Os1p is essential for these fungicides to express their antifungal activity. The amino acid changes in Os1p, 625Pro from Leu, 578Val from Ala, and 580Arg from Gly were found in the type II strains, M16, M155-1 and P5990, respectively. Os1p is novel in having six tandem repeats of 90 amino acids in the N terminal. Each amino acid change of the type II strains was located on the fifth unit of six tandem repeats. Type II strains with single amino acid changes were more sensitive to osmotic stress than the null mutants (type I), indicating that the amino acid repeats of Os1p were responsible for an important function in osmo-regulation.     

Synthesis and antifungal activity of imidazole-1-carboxylates

A series of imidazole-1-carboxylates was prepared by reacting various alcohols with trichloromethyl chloroformate and imidazole or N,N'-carbonyl-diimidazole. They were tested for fungitoxic activity in vitro against two phytopathogenic fungi, Botrytis cinerea (grey mould) and Gibberella fujikuroi, and for preventive efficacy against grey mould on cucumber leaves. 1-(4-Substituted phenoxymethyl)-2,2-dimethylpropylimidazole-1-carboxylates showed excellent in-vitro activity against B. cinerea, and moderate activity against G. fujikuroi, and some of them also effectively controlled grey mould in vivo. A 1H-1,2,4-triazole derivative corresponding to an imidazole derivative did not have any activity, while a thiocarboxylate corresponding to an imidazole carboxylate showed excellent activity against both B. cinerea and G. fujikuroi.     

Effects of low root temperature on sap flow rate,soluble carbohydrates,nitrate contents and on cytokinin and gibberellin levels in root xylem exudate of sand‐grown tomato

Tomato (Lycopersicon esculuntum Mill.) grown in open fields in dry land areas or in non‐controlled greenhouses are subjected to substantial daily changes in root temperature. In the field, root‐zone temperatures fluctuate both diurnally and during the growing season. The purpose of this study was to monitor root‐zone temperature effects on tomato initial growth, transpiration, sap flow rate, leaf and air temperatures differences, nitrate accumulation, total nitrogen, and soluble carbohydrates in the shoot and roots as well as levels of endogenous cytokinins and gibberellins in xylem exudate. Tomato seedlings were grown in three growth cabinets with variable control of root temperatures. Three day/night root temperature regimes (12/12, 16/8 and 20/20°C) were employed. Low day root temperatures of 12 and 16°C reduced shoot dry weight by 47 and 26%, root dry weight by 36 and 14%, shoot nitrate by 79 and 50%, root nitrate by 49 and 16%, levels of cytokinins in root xylem exudate by 27 and 13% and gibberellins by 65 and 23%, in relation to the respective values of 20°C day root temperature. Soluble carbohydrates in the shoot and roots were increased significantly (18 and 111%) by 12°C root temperature. The main effects of low root temperatures on shoot growth stem from slow upward transport of plant hormones and nitrate rather than reduction in their rate of biosynthesis or entry to the root, respectively.     

Risk assessment of ozone impact on <Emphasis Type="Italic">Fagus crenata</Emphasis> in Japan: consideration of atmospheric nitrogen deposition

Tropospheric ozone (O₃) is considered to be the air pollutant relating to the decline of Fagus crenata forest in Japan. In the present study, we assessed a risk of O₃ impact on the growth of F. crenata in Japan, giving consideration to the effects associated with atmospheric nitrogen (N) deposition based on the experimental study, national monitoring data for oxidant concentration and atmospheric N deposition, and a national vegetation survey. The average and maximum O₃-induced relative growth reduction (RG_red) of F. crenata across Japan were estimated to be 3.2 and 9.7%, respectively. Current levels of atmospheric N deposition were found to significantly affect the sensitivity of F. crenata to O₃. When the N deposition was assumed as zero, the estimated average and maximum RG_red were 2.3% and 5.7%, respectively. The inclusion of atmospheric N deposition data thus increased the estimated values for average and maximum RG_red (by 38% and 71%, respectively). Our results demonstrate that a change in the sensitivity to O₃ associated with atmospheric N deposition is an important consideration in the risk assessment of O₃ impact on the growth of F. crenata in Japan.     

Yield estimation of 1-year-old asparagus grown using rootstock-planting forcing culture

Rootstock-planting forcing culture was developed in asparagus to harvest spears even during the seasons when the plants become dormant, but the demand for them high. In this study, cumulative hours during which the air temperature remained lower than 5°C, i.e. chilling hours (CHs), were calculated to determine dormancy breakage for asparagus cultures. We also measured CIELab colour values for cut stems immediately before rootstock digging, and determined whether they could be substituted and/or compensated for CHs while evaluating asparagus plant productivity in different low-temperature backgrounds, and obtained regression equations for yield estimation. Asparagus seedlings were cultivated in seven different regions across Japan and brought to the study site for harvesting. Our regression equation based on CHs and rootstock weight for yield estimation had relatively high fitness (adjusted R² = 0.5795). The colour values of cut stalks at rootstock digging can also be used to evaluate their productivity. These values can be useful in regions where CHs cannot be determined, although their effectiveness was slightly lower than that of CHs of areas adjacent to the study sites.     

Molecular basis of the shish-kebab morphology in polymer crystallization

In the rich and long-standing literature on the flow-induced formation of oriented precursors to polymer crystallization, it is often asserted that the longest, most extended chains are the dominant molecular species in the "shish" of the "shish-kebab" formation. We performed a critical examination of this widely held view, using deuterium labeling to distinguish different chain lengths within an overall distribution. Small-angle neutron-scattering patterns of the differently labeled materials showed that long chains are not overrepresented in the shish relative to their concentration in the material as a whole. We observed that the longest chains play a catalytic role, recruiting other chains adjacent to them into formation of the shish.     

Diversity and vertical distribution of indigenous arbuscular mycorrhizal fungi under two soybean rotational systems

To evaluate the importance of arbuscular mycorrhizal fungi (AMF) to crop production, it is imperative to move beyond the plow layer to include the full soil profile impacted by plant roots. To illustrate this, we investigated the vertical distribution of AMF biomass and community structure within the top 100 cm of soil in soybean (Glycine max (L.) Merr., cv: Enrei) rotational systems cropped to wheat (Triticuma estivum L. cv: Bandowase) or left fallow using fatty acid methyl ester (FAME) biomarkers and molecular analysis, respectively. AMF biomass, as measured by concentration of C16:1cis11, declined during fallow and with increasing soil depth. Greater than 50 % of the stored AMF biomass was found at depths below 35 cm. Phylogenetic analysis revealed 16 AMF phylotypes, including nine Glomus, two Gigaspora, two Scutellospora, and one each of Diversispora, Paraglomus, and an unknown glomeromycete, at different sampling depths in this study. Cluster analysis based on the number and abundance of each AMF phylotype formed two distinct clusters separating wheat from fallow rotations. There was no distinct relationship with soil depth beyond clustering AMF communities above and below 20 cm under wheat. Redundancy analysis (RDA) and hierarchical cluster analysis demonstrated that AMF communities by soil depth within each rotation were not significantly different. However, AMF communities were clearly influenced by crop rotation, where the distribution of specific AMF phylotypes responded to the presence of the wheat crop.