Translocation of Fungicides and Their Efficacy in Controlling Phellinus noxius, the Cause of Brown Root Rot Disease.

Brown root rot disease (BRRD), caused by Phellinus noxius, is an important tree disease in tropical and subtropical areas. To improve chemical control of BRRD and deter emergence of fungicide resistance in P. noxius, this study investigated control efficacies and systemic activities of fungicides with different modes of action. Fourteen fungicides with 11 different modes of action were tested for inhibitory effects in vitro on 39 P. noxius isolates from Taiwan, Hong Kong, Malaysia, Australia, and Pacific Islands. Cyproconazole, epoxiconazole, and tebuconazole (Fungicide Resistance Action Committee [FRAC] 3, target-site G1) inhibited colony growth of P. noxius by 99.9 to 100% at 10 ppm and 97.7 to 99.8% at 1 ppm. The other effective fungicide was cyprodinil + fludioxonil (FRAC 9 + 12, target-site D1 + E2), which showed growth inhibition of 96.9% at 10 ppm and 88.6% at 1 ppm. Acropetal translocation of six selected fungicides was evaluated in bishop wood (Bischofia javanica) seedlings by immersion of the root tips in each fungicide at 100 ppm, followed by liquid or gas chromatography tandem mass spectrometry analyses of consecutive segments of root, stem, and leaf tissues at 7 and 21 days posttreatment. Bidirectional translocation of the fungicides was also evaluated by stem injection of fungicide stock solutions. Cyproconazole and tebuconazole were the most readily absorbed by roots and efficiently transported acropetally. Greenhouse experiments suggested that cyproconazole, tebuconazole, and epoxiconazole have a slightly higher potential for controlling BRRD than mepronil, prochloraz, and cyprodinil + fludioxonil. Because all tested fungicides lacked basipetal translocation, soil drenching should be considered instead of trunk injection for their use in BRRD control.

Development and application of a method to classify airborne pollen taxa concentration using light scattering data.

Although automated pollen monitoring networks using laser optics are well-established in Japan, it is thought that these methods cannot distinguish between pollen counts when evaluating various pollen taxa. However, a method for distinguishing the pollen counts of two pollen taxa was recently developed. In this study, we applied such a method to field evaluate the data of the two main allergens in Japan, Chamaecyparis obtusa and Cryptomeria japonica. We showed that the method can distinguish between the pollen counts of these two species even when they are simultaneously present in the atmosphere. This result indicates that a method for automated and simple two pollen taxa monitoring with high spatial density can be developed using the existing pollen network.

[LONG-TERM RESULTS OF AIRBORNE POLLEN SURVEYS IN THE JAPANESE ARCHIPELAGO FROM THE PERSPECTIVE OF POLLEN ANTIGEN].

BACKGROUND: In 1986, the Ministry of Health and Welfare started an airborne pollen survey as part of measures against JC pollinosis. We reported the important tree pollen antigens in 2016. We have now estimated the longitudinal investigated results for successful prevention and treatment for allergic symptoms related to grass and weed pollen in Japan. METHOD: Since July 1986 we have monitored airborne pollen, year- round, using a gravitational pollen sampler (Durham's sampler), at more than 20 locations across Japan. Specimens were mailed to our facility, where they were stained with Calberla solution, counted under an optical microscope, and converted to the number of pollen per square centimeter. For convenience the number of collected pollen were compiled every six months, with the January to June samples classified as spring pollen and the July to December as autumn pollen even same family. RESULT: Total pollen counts at each location were extremely small compared to tree-pollen, averaging 73~650 pollen grains per year. The Sagamihara location had the greatest count. Unlike cedar and cypress there were no significant annual fluctuations, but grass and Ambrosia pollen counts are increasing in some regions. Spring grass pollen gave the largest count, at 30% of the total collected. CONCLUSION: This indicated we need to examine the rinoconjuctivitis and oral allergy syndrome related to herbaceous pollen carefully.The importance of airborne pollen surveys for the treatment of the patients with pollen allergies was suggested.

Comparative and population genomic landscape of Phellinus noxius: A hypervariable fungus causing root rot in trees.

The order Hymenochaetales of white rot fungi contain some of the most aggressive wood decayers causing tree deaths around the world. Despite their ecological importance and the impact of diseases they cause, little is known about the evolution and transmission patterns of these pathogens. Here, we sequenced and undertook comparative genomic analyses of Hymenochaetales genomes using brown root rot fungus Phellinus noxius, wood-decomposing fungus Phellinus lamaensis, laminated root rot fungus Phellinus sulphurascens and trunk pathogen Porodaedalea pini. Many gene families of lignin-degrading enzymes were identified from these fungi, reflecting their ability as white rot fungi. Comparing against distant fungi highlighted the expansion of 1,3-beta-glucan synthases in P. noxius, which may account for its fast-growing attribute. We identified 13 linkage groups conserved within Agaricomycetes, suggesting the evolution of stable karyotypes. We determined that P. noxius has a bipolar heterothallic mating system, with unusual highly expanded ~60 kb A locus as a result of accumulating gene transposition. We investigated the population genomics of 60 P. noxius isolates across multiple islands of the Asia Pacific region. Whole-genome sequencing showed this multinucleate species contains abundant poly-allelic single nucleotide polymorphisms with atypical allele frequencies. Different patterns of intra-isolate polymorphism reflect mono-/heterokaryotic states which are both prevalent in nature. We have shown two genetically separated lineages with one spanning across many islands despite the geographical barriers. Both populations possess extraordinary genetic diversity and show contrasting evolutionary scenarios. These results provide a framework to further investigate the genetic basis underlying the fitness and virulence of white rot fungi.

LONGITUDINAL MONITORING OF TREE AIRBORNE POLLEN IN JAPAN.

RATIONAL: In Japan patients with Japanese Cedar (JC) pollinosis have increased nation widely since the latter of 1970's. The Ministry of Health and Welfare of Japanese Governments has begun to take preventive measures against JC pollinosis and airborne pollen monitoring has begun to investigate as a causative agent since 1986. We have estimated the longitudinal investigation result for successful prevention and treatment against pollinosis in Japan. METHOD: We have monitored airborne pollen all year around since July 1986 by gravitational pollen sampler, Durham's sampler, at more than 20 locations in the Japanese Islands. Pollen samples were sent to our hospital and counted pollen number per cm2 after stained by Calberla solution and then classified main pollen grains as a causative agent of pollinosis. RESULT AND DISCUSSION: JC pollen number was the most of all, more than 40%, next cypress family, about 20%. They were occupied of more than 60% of all and they increased with the remarkable annual fluctuation as the allergen of JC pollinosis. Beech family pollen counting were also increasing and occupied about 10% of all pollen counts. In Hokkaido the prevalence of birch family pollen count was larger than that in other districts. There is cross-reactivity between beech and birch family which related with oral allergic syndrome.Perspective and Conclusion: In future new occurrences of oral allergy syndrome due to increasing allergic tree pollen grains would be appeared. The contentious pollen research should be important for patients with pollinosis in Japan.

Genetic Differentiation and Spatial Structure of Phellinus noxius, the Causal Agent of Brown Root Rot of Woody Plants in Japan.

Phellinus noxius is a pathogenic fungus that causes brown root rot disease in a variety of tree species. This fungus is distributed in tropical and sub-tropical regions of Southeast and East Asia, Oceania, Australia, Central America and Africa. In Japan, it was first discovered on Ishigaki Island in Okinawa Prefecture in 1988; since then, it has been found on several of the Ryukyu Islands. Recently, this fungus was identified from the Ogasawara (Bonin) Islands, where it has killed trees, including rare endemic tree species. For effective control or quarantine methods, it is important to clarify whether the Japanese populations of P. noxius are indigenous to the area or if they have been introduced from other areas. We developed 20 microsatellite markers from genome assembly of P. noxius and genotyped 128 isolates from 12 of the Ryukyu Islands and 3 of the Ogasawara Islands. All isolates had unique genotypes, indicating that basidiospore infection is a primary dissemination method for the formation of new disease foci. Genetic structure analyses strongly supported genetic differentiation between the Ryukyu populations and the Ogasawara populations of P. noxius. High polymorphism of microsatellite loci suggests that Japanese populations are indigenous or were introduced a very long time ago. We discuss differences in invasion patterns between the Ryukyu Islands and the Ogasawara Islands.

Comparative genomics of Taphrina fungi causing varying degrees of tumorous deformity in plants.

Taphrina fungi are biotrophic plant pathogens that cause plant deformity diseases. We sequenced the genomes of four Taphrina species-Taphrina wiesneri, T. deformans, T. flavorubra, and T. populina-which parasitize Prunus, Cerasus, and Populus hosts with varying severity of disease symptoms. High levels of gene synteny within Taphrina species were observed, and our comparative analysis further revealed that these fungi may utilize multiple strategies in coping with the host environment that are also found in some specialized dimorphic species. These include species-specific aneuploidy and clusters of highly diverged secreted proteins located at subtelomeres. We also identified species differences in plant hormone biosynthesis pathways, which may contribute to varying degree of disease symptoms. The genomes provide a rich resource for investigation into Taphrina biology and evolutionary studies across the basal ascomycetes clade.

Virulence of Bursaphelenchus xylophilus Isolated from Naturally Infested Pine Forests to Five Resistant Families of Pinus thunbergii.

Pine wilt disease is one of the most serious epidemic tree diseases in Japan, and resistant pine trees have been developed through a breeding program. To evaluate resistance of resistant families of Japanese black pine, Pinus thunbergii, to the pinewood nematode, Bursaphelenchus xylophilus, isolated from the field, and to determine whether differentiation of pathogenicity to resistant pine families appears in the nematode isolates, seedlings of five resistant pine families were inoculated with 25 nematode isolates. Disease incidence 18 weeks after inoculation was significantly different among nematode isolates and among pine families but there was no interaction effect between nematode isolate and pine family. This indicates that nematode isolates did not have differential host specificity to resistant families of P. thunbergii. Isolate Shimabara, a test isolate of the breeding program, showed the same degree of virulence as the highly virulent isolates frequently used in experiments. However, more virulent isolates than Shimabara were found among the isolates collected from natural pine forest. This indicated that B. xylophilus populations with higher virulence than Shimabara exist in the natural population. These findings are important in development of more efficient breeding procedures for resistant pine trees.

Genomic insights into the origin of parasitism in the emerging plant pathogen Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus is the nematode responsible for a devastating epidemic of pine wilt disease in Asia and Europe, and represents a recent, independent origin of plant parasitism in nematodes, ecologically and taxonomically distinct from other nematodes for which genomic data is available. As well as being an important pathogen, the B. xylophilus genome thus provides a unique opportunity to study the evolution and mechanism of plant parasitism. Here, we present a high-quality draft genome sequence from an inbred line of B. xylophilus, and use this to investigate the biological basis of its complex ecology which combines fungal feeding, plant parasitic and insect-associated stages. We focus particularly on putative parasitism genes as well as those linked to other key biological processes and demonstrate that B. xylophilus is well endowed with RNA interference effectors, peptidergic neurotransmitters (including the first description of ins genes in a parasite) stress response and developmental genes and has a contracted set of chemosensory receptors. B. xylophilus has the largest number of digestive proteases known for any nematode and displays expanded families of lysosome pathway genes, ABC transporters and cytochrome P450 pathway genes. This expansion in digestive and detoxification proteins may reflect the unusual diversity in foods it exploits and environments it encounters during its life cycle. In addition, B. xylophilus possesses a unique complement of plant cell wall modifying proteins acquired by horizontal gene transfer, underscoring the impact of this process on the evolution of plant parasitism by nematodes. Together with the lack of proteins homologous to effectors from other plant parasitic nematodes, this confirms the distinctive molecular basis of plant parasitism in the Bursaphelenchus lineage. The genome sequence of B. xylophilus adds to the diversity of genomic data for nematodes, and will be an important resource in understanding the biology of this unusual parasite.

Cross Inoculation Tests with Phellinus noxius Isolates from Nine Different Host Plants in the Ryukyu Islands, Southwestern Japan.

Brown root rot, caused by Phellinus noxius, leads to problems in management of shade, ornamental, and windbreak trees in subtropical regions of the world, and it has been causing serious problems in Japan since 1988. To identify the pathogenicity, host specialization, and virulence of the pathogen, cross inoculation tests were carried out using isolates of the fungus obtained from nine different tree species. P. noxius was pathogenic to all of the nine tree species tested, and it killed inoculated seedlings. Among the 810 trees inoculated with the fungus, a total of 141 trees (17.4%), including all nine species, were dead within 110 days after inoculation. The first symptom of infection was rapid wilt with discoloration of leaves 20 to 30 days after inoculation, and then the plants quickly declined. Only one isolate of P. noxius that was tested caused significantly higher mortality in its original host than in other species, suggesting that the pathogen has little to no host specificity. However, the fungus did show variation in virulence, with the isolates originating in Bischofia javanica and Casuarina equisetifolia causing significantly higher mortality than those collected from other host species. More isolates of P. noxius from the same host species and/or from various countries should be studied to understand host specialization and virulence of the pathogen.

Quantitative analysis of antigen specific IgE in tears in comparison to serum samples.

We determined pollen specific IgE in tears and compared these results to the concentration of specific IgE in serum samples. We obtained tears (using Schirmer strips) and serum samples from subjects with Japanese cedar (Cryptomeria japonica) pollinosis, and tested for C. japonica pollen specific IgE using a quantitative ELISA. Time kinetic analyses through the pollen season showed that specific IgE levels in tears were found to increase earlier than those in sera and reached their maximum at the end of or after the pollen season, from March to early June. In the C. japonica pollen free season, July to December, the specific IgE levels in tears decreased, although the serum levels remained relatively high. These results indicate that the quantitative assay for specific IgE in tears might be useful to identify specific eye allergens.

Erythritol-based dry powder of glucagon for pulmonary administration.

Glucagon, a key regulatory element of glycogen metabolism, is known to be effective in the clinical treatment of hypoglycemia and the maintenance of normal circulating glucose levels in patients with total pancreatectomy, however the clinical use of this gut hormone has been restricted to parenteral administration. In this investigation, we prepared dry powder dosage forms of glucagon, which were formulated by mixing micronized glucagon particles and excipients with larger carrier particles. To achieve alveolar deposition for subsequent systemic absorption, a dry powder inhalant (DPI) of glucagon was size-reduced to a mass median diameter between 1 and 6 microm, as measured by laser diffraction analysis. The use of erythritol as both excipient and carrier in DPI of glucagon resulted in high and reproducible flowability and dispersibility of the powder mixtures, and therefore it provided a low dosing of the active substances. Distinct transpulmonary absorption of glucagon was confirmed after intratracheal administration of the glucagon dry powder to anesthetized rats, as evidenced by the increase in the blood glucagon and blood sugar levels. These results suggested the usefulness of an erythritol-based powder form of glucagon for systemic administration.