Powdery Mildew of Prairie Gentian: Characteristics,Molecular Phylogeny and Pathogenicity

In March 1999, we found prairie gentian (Eustoma grandiflorum) infected with powdery mildew in a greenhouse in Oita Prefecture, Japan. Morphological observation revealed that the causal fungus belongs to the mitosporic genus Oidium subgenus Pseudoidium [teleomorph: Erysiphe sensu Braun and Takamatsu (2000)]. Precise taxonomic position of the fungus, however, is uncertain due to lack of the perfect stage. We determined the nucleotide sequence of the rDNA ITS region of the fungus. Comparison of the sequence with those obtained from DNA databases of this fungal group revealed that the sequence is identical to those of powdery mildews from garden four-o'clock (Mirabilis jalapa) and broad bean (Vicia faba). Inoculation of an isolate from garden four-o'clock caused mildew on prairie gentian and broad bean, suggesting that the prairie gentian mildew originates from garden four-o'clock or broad bean. Molecular phylogenetic analysis indicated a close relationship of this fungus to Erysiphe baeumleri on Vicia spp. and E. trifolii on Trifolium pratense. From these results, we propose that prairie gentian mildew diverged from a Fabaceae-parasitic ancestor. Received 14 March 2002/ Accepted in revised form 28 May 2002     

Suppurative meningoencephalitis and perineuritis caused by Streptococcus gallolyticus in a Japanese Black calf

A 179-day-old calf, which was weak and stunted, showed neurological signs and was euthanized. Postmortem examination revealed extensive and severe cloudy area in the meninges, and pleural pneumonia. Gram-positive cocci were isolated from systemic organs. Biochemical and 16S rRNA gene sequence analyses identified the isolate as Streptococcus gallolyticus, and its subspecies was suggested to be gallolyticus (SGG). The isolate was classified as a novel sequence type (ST115) by the multilocus sequence typing scheme for SGG and showed susceptibility to penicillin, ampicillin, amoxicillin, florfenicol, sulfamethoxazole-trimethoprim, and chloramphenicol. Histopathologically, suppurative meningoencephalitis and perineuritis were detected. As SGG has been isolated solely from a cow with mastitis in Japan, this is the first SGG infection in a calf with suppurative meningoencephalitis and perineuritis in this country.     

Phytogeny and Cross-infectivity of Powdery Mildew Isolates (Podosphaera fuliginea s. lat.) on Cosmos and Cucumber

To clarify the relationship between the phylogeny and infectivity of isolates of Podosphaera fuliginea s. lat. (= Sphaerotheca fuliginea s. lat.) from cosmos and cucumber, more than 50 powdery mildew isolates from these two plants were subjected to nucleotide sequencing or PCR-RFLP analysis of the rDNA internal transcribed spacer (ITS) region and cross-inoculation tests. The isolates from both cosmos and cucumber are genetically monotypic, and there are six nucleotide substitutions in the rDNA ITS region between isolates from cosmos and cucumber. Cross-inoculation tests of these isolates revealed that isolates from cosmos are not pathogenic on cucumber. Although isolates from cucumber produced conidia on leaves of cosmos in the laboratory, the conidial density was much lower than that from isolates from cosmos. This result, as well as the fact that the cucumber strain was not isolated from cosmos in fields, suggests that isolates from cucumber do not infect cosmos in the field. Therefore, powdery mildews on cosmos and cucumber can be regarded to have become specialized for their hosts both genetically and pathogenically. The present study reconfirms the close relationship between phylogeny and infectivity of powdery mildew fungi. Host specialization may be a trigger that causes genetic divergence of powdery mildew fungi. Received 28 June 2000/ Accepted in revised form 4 September 2000     

Determination of DTNB-reactive sulphhydryl groups in soil humic acids: their enrichment in humic acids from volcanic acid soils

Thirteen acid soils were collected from typical volcanic regions in Japan (S content: 0.9–4.1, mean = 2.2g kg^?1; pH (H₂O): 2.81–3.93, mean = 3.33), as well as nine reference soils (S: 0.6–1.7, mean= 1.1 gkg^?1; pH(H₂O): 4.10–4.74, mean = 4.47). Humic acids were extracted from the soils separately with 0.1 M NaOH and precipitated by acidification (pH = 2, HCl). After purification, the humic acids were subjected to colorimetric analysis using a DTNB reagent [5,5′-dithiobis(2–nitrobenzoic acid] for the active -SH group. Since humic acids have significant absorption at 4I2 nm, the coloured compound (5–mercapto-2–nitrobenzoic acid) was separated from the humic acids by ultrafiltration or solvent extraction prior to the colorimetric measurement. Humic acids also caused discoloration of the coloured compound when they coexisted in the reaction solutions. Thus, the reproducible determination of -SH was accomplished by employing a standard addition technique (-SH standard: cysteine). Although -SH contents obtained by the ultrafiltration method were considerably higher than those by the solvent-extraction method, probably due to the denaturation of humic acids by the higher buffer concentration used in the ultrafiltration method, they correlated well with each other. The humic acids from acid soils contained apparently higher concentrations of -SH (120–510, mean = 270mg S kg^?1 by the ultrafiltration method; 8–110, mean = 38mg S kg^?1 by the solvent-extraction method) compared to those from reference soils [20–260, mean = 90mg S kg^?1 by the former; not detectable (<5)-34, mean = 11 mg S kg^?1 by the latter]. This -SH enrichment in the humic acids from acid soils may result from the degradation and subsequent humification of S-rich debris of plants and micro-organisms and/or direct incorporation of volcanic acid gas (H₂S) into the humic acids.     

Disinfectant resistance of Salmonella in in vitro contaminated poultry house models and investigation of efficient disinfection methods using these models

Salmonellaenterica subsp. enterica (Salmonella) shows disinfectant resistance by forming biofilms on solid surfaces. However, efficient disinfection methods to eliminate Salmonella biofilms from farms have not yet been examined in detail. In this study, more than 80% of Salmonella strains from farms in Yamagata prefecture, Japan, were biofilm producers. Regardless of the extent of their biofilm formation ability, their biofilms were highly resistant to hypochlorous acid on plastic surfaces. To establish efficient disinfection methods in farms, we developed in vitro Salmonella-contaminated poultry house models by depositing dust on ceramic and stainless-steel carriers in poultry houses for one month and culturing a representative Salmonella strain on the carriers. Biofilm-like structures, including Salmonella-like cells, were observed on the models by scanning electron microscopy. Salmonella was not efficiently removed from the models even by cleaning with a surfactant at 25/65°C and disinfection with quaternary ammonium compound or hypochlorous acid at 25°C; on the contrary, viable Salmonella cells increased in some tests under these conditions, suggesting that these models successfully simulate the highly persistent characteristics of Salmonella in farms. However, the persistent bacterial cells were markedly decreased by soaking in 65°C surfactant followed by rinsing with 80°C water, additional cleaning using chlorine dioxide or disinfection with dolomitic lime, suggesting the effectiveness of these methods against Salmonella in farms. Since many different disinfection conditions may be easily tested in laboratories, our models will be useful tools for establishing effective and practical disinfection methods in farms.     

Microbial biomass and nitrogen transformations in surface soils strongly acidified by volcanic hydrogen sulfide deposition in Osorezan, Japan

Volcanic acidification has created unique ecosystems that have had to adapt to the acidic environments in volcanic regions. To characterize the primary microbial properties of strongly acidified soils in such environments, we investigated microbial biomass, nitrogen transformations and other relevant chemical properties in the surface soils of solfatara and forests from Osorezan, a typical volcanic region in Japan, and compared the results to common Japanese forest soils. Soil microbial biomass C (MBC) and N (MBN) were determined using the chloroform fumigation–extraction method. Potential net N mineralization and net nitrification were measured in aerobic laboratory incubations. Long-term acidification in the Osorezan soils by volcanic hydrogen sulfide deposition caused low soil pH (3.0–3.8), base cation deficiency and increased concentrations of toxic ions such as Al³⁺. The proportions of MBC to total carbon (MBC/TC ratio) and MBN to total nitrogen (MBN/TN ratio) were lower than those in common Japanese forest soils. The extreme acidic conditions may have inhibited microbial survival in the Osorezan acid soils. Net N mineralization occurred at rates comparable to those in common Cryptomeria japonica forest soils, probably because of the presence of acid-tolerant soil microorganisms. Net nitrification was completely inhibited and autotrophic ammonia oxidizers were not detected by the MPN method. The inhibition of nitrification prevents nitrogen leaching from the soils, thus maintaining a nitrogen cycle in the volcanic acid region in which     (and NH₃) is recycled among microorganisms and plants.     

A novel multiplex PCR assay to detect and distinguish between different types of Paenibacillus larvae and Melissococcus plutonius,and a survey of foulbrood pathogen contamination in Japanese honey

Paenibacillus larvae and Melissococcus plutonius are the causative agents of American and European foulbroods of honey bees, respectively. Since their virulence and resistance to disinfectants differ depending on the genotypes/phenotypes of the strains, the discrimination of strain types is important for the effective control of these diseases. Methods to detect and differentiate pathogens in honey are useful for surveying the contamination status of beehives/apiaries. In the present study, we selected a sequence (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FI763267","term_id":"224923825"}}FI763267) as the specific target for enterobacterial repetitive intergenic consensus (ERIC) II-type P. larvae strains for the first time and developed a novel multiplex PCR assay that precisely distinguishes between the major types of foulbrood pathogens (ERIC I and II P. larvae and typical and atypical M. plutonius) in one reaction. In addition, we found that commercially available kits designed for DNA extraction from Mycobacterium in feces efficiently extracted DNA from foulbrood pathogens in honey. Using the multiplex PCR assay and DNA extraction kits, all the targeted types of P. larvae and M. plutonius were detected in honey spiked with the pathogens at a concentration of 100 bacterial cells/strain/ml. Moreover, 94% of the Japanese honey samples examined in the present study were contaminated with one or more types of the foulbrood pathogens. These results indicate that the newly developed methods are useful for detecting foulbrood pathogens in honey. The epidemiological information obtained by these methods will contribute to the effective control of foulbroods in apiaries.     

Infection of Melissococcus plutonius clonal complex 12 strain in European honeybee larvae is essentially confined to the digestive tract

Melissococcus plutonius is an important pathogen that causes European foulbrood (EFB) in honeybee larvae. Recently, we discovered a group of M. plutonius strains that are phenotypically and genetically distinct from other strains. These strains belong to clonal complex (CC) 12, as determined by multilocus sequence typing analysis, and show atypical cultural and biochemical characteristics in vitro compared with strains of other CCs tested. Although EFB is considered to be a purely intestinal infection according to early studies, it is unknown whether the recently found CC12 strains cause EFB by the same pathomechanism. In this study, to obtain a better understanding of EFB, we infected European honeybee (Apis mellifera) larvae per os with a well-characterized CC12 strain, DAT561, and analyzed the larvae histopathologically. Ingested DAT561 was mainly localized in the midgut lumen surrounded by the peritrophic matrix (PM) in the larvae. In badly affected larvae, the PM and midgut epithelial cells degenerated, and some bacterial cells were detected outside of the midgut. However, they did not proliferate in the deep tissues actively. By immunohistochemical analysis, the PM was stained with anti-M. plutonius serum in most of the DAT561-infected larvae. In some larvae, luminal surfaces of the PM were more strongly stained than the inside. These results suggest that infection of CC12 strain in honeybee larvae is essentially confined to the intestine. Moreover, our results imply the presence of M. plutonius-derived substances diffusing into the larval tissues in the course of infection.     

Detection of macrolide resistance genes,ermC and ermB,in Japanese honey using real-time PCR assays

American foulbrood (AFB) is a honeybee disease caused by Paenibacillus larvae, and tylosin is used as the prophylactic in Japan. Honey contains macrolide-resistant bacteria that are a potential source of genes that may confer tylosin resistance to P. larvae. To investigate the potential risk of such genes in Japanese honey, we developed real-time PCR assays for the detection of important macrolide resistance genes, ermC and ermB, and analyzed 116 Japanese honey samples with known contamination status of P. larvae. Consequently, 91.38% of samples contained ermC and/or ermB, and 71.55% of samples contained both ermC and P. larvae, suggesting the possible emergence of tylosin-resistant P. larvae in Japan. Therefore, judicious use of the prophylactic is essential in maintaining its effectiveness.