Real-Time PCR Detection of the Onion Downy Mildew Pathogen Peronospora destructor From Symptomless Onion Seedlings and Soils.

An outbreak of downy mildew disease of onion, caused by Peronospora destructor, in Japan in 2016 necessitated a reevaluation of the primary inoculum sources to optimize disease management. Detection of the P. destructor pathogen in plants with asymptomatic infection and in soil would guide the application of fungicides according to the extent of infection before disease development. Here, we detected P. destructor in both plants and soil using newly developed primer sets (Pd ITS and Pd ITS 614) by both conventional and real-time PCR. Validation by real-time PCR with Pd ITS 614 showed that P. destructor DNA was amplified from symptomless seedlings at 3.7 × 102 to 1.0 × 100 conidium cells/50 mg leaf tissue, suggesting the detection of asymptomatic infection. Real-time PCR with Pd ITS amplified pathogen DNA from field soils at 1.6 × 103 to 8.3 × 101 oospore cells/g of soil. This real-time PCR assay provides a useful tool for identifying and quantifying inoculum sources, which may be the foundation of the design of integrated disease management strategies.

Controlled-Release Fine Particles Prepared by Melt Adsorption for Orally Disintegrating Tablets.

Melt adsorption is a manufacturing method that offers precise control of particle size distribution of granules and circumvents the disadvantages of conventional melt granulation. However, drug release from particles adsorbed with hydrophobic materials has not been fully investigated, and there are missing details as to whether particles manufactured by this technique can be applied to orally disintegrating tablets (ODT). In this report, we aimed to optimize process parameters and formulation to manufacture ODT containing melt adsorption-particles with the specific characteristic of sustained release. Melt adsorption particles containing Neusilin US2 as the adsorbent were prepared by using various waxes to determine the most suitable material for controlled release formulation. Glycerol fatty acid ester (Poem TR-FB: TR-FB) was the optimal wax examined because of its drug release pattern and tabletability. We then optimized manufacturing conditions by examining granulation time, disintegrant amount per tablet and compression force on the tablet for ODT that meet the criteria of controlled drug release, tensile strength and disintegration of the tablet. Multiple regression analysis revealed the effect of process parameters on tablet properties and drug release with increasing the granulation time affording sustained release of the drug. The analysis also showed that a high compression force crushed the granules coated by TR-FB, which impaired sustained drug release. From the regression model the optimal manufacturing conditions were determined, and the tablet prepared under these conditions concurred with the predicted values and met all criteria. This new technique should contribute to the development of ODT to improve medication adherence.

RNA sequence analysis data of Peronospora destructor maintained on onions.

Peronospora destructor (Berk.) is an important biotrophic oomycete that causes downy mildew on onion (Allium Cepa L.) worldwide, especially in humid and temperate regions. The disease attacks bulb and seed production of onion, resulting in losses in yield and quality of bulbs. Epidemiological studies have increased our understanding and control of downy mildew on onion; however, little is known about the molecular aspects of P. destructor behavior during infection. Here, we isolated RNA from four samples of sporangia and sporangiophores of P. destructor, which were maintained by spore inoculation onto onions in a growth chamber. We then used an Ion PGM next generation sequencer to acquire and assemble the RNA sequences of P. destructor. By transcriptome shotgun assembly, we obtained 2335 contigs (N50, 884 nucleotides (nt); mean length, 881.6 nt). The data are accessible at NCBI (BioProject PRJNA391849). Our data resource will facilitate further studies of the molecular events during P. destructor infection.

Scale-Up of Lubricant Mixing Process by Using V-Type Blender Based on Discrete Element Method.

A method for scale-up of a lubricant mixing process in a V-type blender was proposed. Magnesium stearate was used for the lubricant, and the lubricant mixing experiment was conducted using three scales of V-type blenders (1.45, 21 and 130 L) under the same fill level and Froude (Fr) number. However, the properties of lubricated mixtures and tablets could not correspond with the mixing time or the total revolution number. To find the optimum scale-up factor, discrete element method (DEM) simulations of three scales of V-type blender mixing were conducted, and the total travel distance of particles under the different scales was calculated. The properties of the lubricated mixture and tablets obtained from the scale-up experiment were well correlated with the mixing time determined by the total travel distance. It was found that a scale-up simulation based on the travel distance of particles is valid for the lubricant mixing scale-up processes.

Population Genetic Structure of QoI-Resistant Pestalotiopsis longiseta Isolates Causing Tea Gray Blight.

Resistance to the quinone outside inhibitor (QoI) fungicides in the tea gray blight-causing fungus Pestalotiopsis longiseta is a serious problem in Japanese tea cultivation. We conducted a population genetic analysis of QoI-resistant P. longiseta isolates on the Makinohara Plateau, Shizuoka Prefecture, Japan's largest tea-growing area, to elucidate the disease's epidemiology and the spread of QoI resistance. Inter simple sequence repeat (ISSR) analysis of 1,083 isolates from 395 fields collected from 2009 to 2012 detected 42 ISSR types, designated as PL01 to PL42. A total of 18, seven, and 38 ISSR types were detected in highly resistant, moderately resistant, and sensitive isolates, respectively. No distinct phylogenetic relationship corresponding to QoI sensitivity or sampling location was observed. No annual changes in the population genetic structure of highly resistant isolates were observed during the study period. A different ISSR type was predominant among QoI-resistant isolates in each region. Analysis of molecular variance revealed significant genetic differentiation in populations of highly resistant isolates among regions (FCT = 0.213) and farmers (FCT = 0.071). Consequently, we speculate that QoI-resistant P. longiseta strains occurred in a number of clonal lineages and spread by both natural and artificial transmission, such as rain splash and plucking machines, throughout each region on the Makinohara Plateau.

One of two major paralogs of AVR-Pita1 is functional in Japanese rice blast isolates.

We analyzed the avirulence gene AVR-Pita1 in Japanese rice blast isolates to determine how they gain virulence toward rice cultivars containing the Pita resistance gene. An avirulent isolate, OS99-G-7a (G7a), from a Japanese commercial field contained two paralogs of AVR-Pita1, designated as AVR-Pita1(JA) and AVR-Pita1(JB). Analysis of virulent, independent mutants derived from G7a, a single avirulent progenitor strain, indicated that AVR-Pita1(JA) was functional but AVR-Pita1(JB) was nonfunctional. The most frequent mutation was loss of AVR-Pita1(JA). Analyses of field isolates collected from diverse areas in Japan revealed that most of the AVR-Pita1 genes carried by Japanese isolates were identical to AVR-Pita1(JA) or AVR-Pita1(JB). The relationship between these major paralogs in Japanese isolates and the virulence of the strains carrying them indicate that AVR-Pita1(JA) is functional but AVR-Pita1(JB) is not, as is the case in G7a. Isolates that show virulence toward rice cultivars containing the Pita gene are presumed to have evolved virulence from avirulent origins via loss of AVR-Pita1(JA), except for one case in which virulence resulted from a base substitution. In this study, we discuss the properties and specificities of Japanese rice blasts that relate to virulence against Pita-containing rice. Furthermore, we present a method to amplify AVR-Pita1(JA) and AVR-Pita1(JB) separately and, specifically, to monitor functional AVR-Pita1 in Japan.

Improvement of dissolution property of poorly water-soluble drug by supercritical freeze granulation.

The dissolution property of the poorly water-soluble drug, flurbiprofen (FP) was improved by a novel supercritical freeze granulation using supercritical carbon dioxide. Supercritical freeze granulation was defined as a production method of the granulated substances by using the dry ice to generate intentionally for the rapid atomization of the supercritical carbon dioxide to the atmospheric pressure. This process utilized a rapid expansion of supercritical solutions (RESS) process with the mixture of the drug and lactose. In the supercritical freeze granulation, needle-like FP fine particles were obtained which adhered to the surface of lactose particles, which did not dissolve in supercritical carbon dioxide. The number of FP particles that adhered to the surface of particles decreased with an increase in the ratio of lactose added, leading to markedly improve the dissolution rate. This improvement was caused not only by the increase in the specific surface area but also the improvement of the dispersibility of FP in water. It is thus concluded that the supercritical freeze granulation is a useful technique to improve the dissolution property of the poorly water-soluble flurbiprofen.

Improvement of dissolution property of poorly water-soluble drug by novel dry coating method using planetary ball mill.

The dissolution property of a poorly water-soluble drug, flurbiprofen (FP), was improved by a novel dry coating method using a planetary ball mill. Several mixtures composed of water-soluble additives (D-mannitol, lactose, and erythritol), light anhydrous silicic acid, and flurbiprofen were prepared. These mixtures and several starches were co-ground in a planetary ball mill, and the surface of the starches was dry coated with the mixtures. The size, appearance, yield, and drug dissolution property of the dry coated preparations were evaluated, and the optimal formulation which improved the dissolution property of poorly water-soluble flurbiprofen was determined. The dissolution rate of FP was increased by dry coating of the surface of starches with microparticulated FP. It was further increased by co-grinding of FP, starch, and a water-soluble additive, or dry coating of the starch surface with microparticulated FP and light anhydrous silicic acid, as a glidant. These co-ground and dry coated preparations could be recovered from the pot of the planetary ball mill readily without adhesion to the inside wall of the pot. These are considered to be novel, industrially applicable methods for improving the dissolution rate of poorly water-soluble drugs.

Preparation of rapidly disintegrating tablets containing itraconazole solid dispersions.

The disintegratability of tablets prepared from two types of solid dispersions containing the water-soluble polymer TC-5 and the enteric polymer HP-55 as an excipient were compared. The disintegratability was better in the tablets of solid dispersions containing non-water-soluble HP-55 than those containing TC-5. In consideration of the dissolubility of solid dispersions containing HP-55, the mean diameter of the solid dispersion (coating powder) must be controlled to 120 microm or less, but as this markedly increases the adhesion/aggregation tendency of the particles (angle of repose: 47 degrees ), control of the adhesion/aggregation tendency emerged as another problem. Therefore, surface-modification was performed in a high-speed agitating granulator using 0.1% light anhydrous silicic acid as a surface modifier, and marked improvement in the flowability was observed. This made continuous tableting using a rotary tablet machine possible even with the poorly flowable solid dispersions. Also, in tableting of the solid dispersions, no recrystallization of amorphous itraconazole by the tableting pressure was observed, and the tablets maintained satisfactory dissolubility. Moreover, it was possible to obtain the rapidly disintegrating tablets with very satisfactory properties, i.e., a tablet hardness of 30 N or higher and a disintegration time of 30 s or less, by the addition of croscarmellose as a disintegrant at 2% to the surface-modified solid dispersion and selection of the tableting pressure at 4.5 kN.