Characterization of first hemin-requiring Pseudomonas aeruginosa small-colony variants from the blood of an octogenarian male-patient with double pneumonitis.

A hemin-requiring Pseudomonas aeruginosa small-colony variant (SCV) was isolated from the blood of an octogenarian male-patient with double pneumonitis. The isolate was capable of growing on both sheep blood and chocolate agars but not on MacConkey agars without blood ingredient. Furthermore, the isolate revealed to grow only around the X-factor impregnated discs when examined using the X and V disc strips. However, not only RapID-NH system but also the VITEK2 system failed to identify the isolate. The isolate was finally identified as P. aeruginosa by the sequence of the 16S rRNA genes and the MALDI-TOF MS analysis. Interestingly, the isolate represented positive reaction for δ-aminolaevulinic acid (ALA)-test despite the requirement of hemin. Detailed analysis indicated that the isolate produced protoporphyrin IX from ALA. Therefore, the reason for the hemin dependence was deduced the dysfunction of hemH-encoded ferrochelatase behaving at the end of biosynthetic pathway of heme. However, the genetic analysis of hemH gene demonstrated no variations of both the DNA and the amino-acid sequences. To the best of our knowledge, this is the first clinical isolation of a hemin-dependent P. aeruginosa SCV from blood.

Fixed-route monitoring and a comparative study of the occurrence of herbicide-resistant oilseed rape (Brassica napus L.) along a Japanese roadside.

Previously, we conducted a roadside survey to reveal the occurrence of genetically modified (GM) oilseed rape along a Japanese roadside (Route 51). In this study, we performed successive and thorough fixed-route monitoring in 5 sections along another road (Route 23). Oilseed rape plants were detected on both sides of the road in each section between autumn 2009 and winter 2013, which included 3 flowering seasons. In four sections, more plants were found on the side of the road leading from the Yokkaichi port than on the opposite side. In the fifth section, the presence of clogged drains on the roadside, where juvenile plants concentrated, caused the opposite distribution: oilseed rape predominantly occurred along the inbound lanes (leading to the Yokkaichi port) in 2010 and 2012. Unlike in our previous survey, glyphosate- or glufosinate-resistant oilseed rape plants were abundant (>75% of analyzed plants over 3 years). Moreover, a few individuals bearing both herbicide resistance traits were also detected in some sections. The spillage of imported seeds may explain the occurrence of oilseed rape on the roadside. The abundance of herbicide-resistant oilseed rape plants may reflect the extent of contamination with GM oilseed rape seed within imports.

High-throughput capture of nucleotide sequence polymorphisms in three Brassica species (Brassicaceae) using DNA microarrays.

PREMISE OF THE STUDY: To capture molecular markers that are applicable to environmental risk assessment of genetically modified oilseed rape, and to streamline their development, we screened variations in nucleotide sequences of three Brassica species by DNA microarray analysis. METHODS AND RESULTS: Using the Affymetrix GeneChip Arabidopsis ATH1 Genome Array, we monitored gene expression at 22810 loci among the Brassica species and picked out 192 putative polymorphic loci. We sequenced 25 of these and successfully aligned them among all three species. All 25 loci possessed some interspecific and at times intraspecific nucleotide variation. CONCLUSIONS: DNA microarray analysis effectively detected a large number of nucleotide sequence variations among closely related Brassica species. The polymorphic regions will allow the subsequent development of functional gene markers.

Seeds of a possible natural hybrid between herbicide-resistant Brassica napus and Brassica rapa detected on a riverbank in Japan.

Transgenic herbicide-resistant varieties of Brassica napus, or oilseed rape, from which canola oil is obtained, are imported into Japan, where this plant is not commercially cultivated to a large extent. This study aimed to examine the distribution of herbicide-resistant B. napus and transgene flow to escaped populations of its closely related species, B. rapa and B. juncea. Samples were collected from 12 areas near major ports through which oilseed rape imports into Japan passed--Kashima, Chiba, Yokohama, Shimizu, Nagoya, Yokkaichi, Sakai-Senboku, Kobe, Uno, Mizushima, Kita-Kyushu, and Hakata--and the presence of glyphosate- and/or glufosinate-resistant B. napus was confirmed in all areas except Yokohama, Sakai-Senboku, Uno, and Kita-Kyushu. The Yokkaichi area was the focus because several herbicide-resistant B. napus plants were detected not only on the roadside where oilseed rape spilled during transportation but also on the riverbanks, where escaped populations of B. rapa and B. juncea grew. Samples of B. napus that were tolerant to both herbicides were detected in four continuous years (2005-2008) in this area, suggesting the possibility of intraspecific transgene flow within the escaped B. napus populations. Moreover, in 2008, seeds of a possible natural hybrid between herbicide-tolerant B. napus (2n = 38) and B. rapa (2n = 20) were detected; some seedlings derived from the seeds collected at a Yokkaichi site showed glyphosate resistance and had 2n = 29 chromosomes. This observation strongly suggests the occurrence of hybridization between herbicide-resistant B. napus and escaped B. rapa and the probability of introgression of a herbicide-resistance gene into related escaped species.

Development and characterization of a novel set of microsatellite markers for Arisaema serratum (Araceae).

PREMISE OF THE STUDY: We developed novel microsatellite markers in Arisaema serratum, a perennial herb that possesses pitfall flowers and exhibits labile sex expression, to facilitate research on parentage and pollination biology in this species. METHODS AND RESULTS: By using procedures for enrichment of desired microsatellite-containing fragments and PCR-based isolation of microsatellite arrays, we detected 18 novel microsatellite loci. Thirteen were highly polymorphic: the number of alleles per locus ranged from six to 46, the observed heterozygosities ranged from 0.320 to 0.940, and the expected heterozygosities ranged from 0.440 to 0.976. Nine of the 13 markers successfully amplified regions in congeneric species. CONCLUSIONS: These highly polymorphic markers will facilitate further studies on the mode of pollination and other aspects of reproductive biology in A. serratum.

Rapeseed species and environmental concerns related to loss of seeds of genetically modified oilseed rape in Japan.

Feral rapeseed in Japan consists of Brassica rapa, B. juncea and B. napus, mostly produced by escape from crops. Brassica rapa and B. juncea were introduced from abroad long ago as leaf and root vegetables and as an oil crop and breeders have developed various cultivars. Brassica napus was introduced in the late 1800s, mainly as an oil crop. Rapeseed production in Japan is low, and most demand is met by imports from Canada (94.4% of the 2009 trade volume). Recently, spontaneous B. napus, including genetically modified (GM) herbicide-resistant individuals, has been detected along Japanese roads, probably originating from seeds lost during transportation of imports. As GM oilseed production increases abroad, the probability of escape of GM oilseed rape in Japan will increase, raising environmental biosafety concerns related to the impact of feral rapeseed on heirloom brassicaceous crops. In this paper, we review the history of rapeseed introduction in Japan and future concerns.

Monitoring the occurrence of genetically modified oilseed rape growing along a Japanese roadside: 3-year observations.

Monitoring for escape of genetically modified (GM) oilseed rape (Brassica napus) during transport can be performed by means of roadside evaluations in areas where cultivation of this GM crop is not conducted, such as in Japan. We performed a survey of oilseed rape plants growing along a 20-km section of Japan's Route 51, one of the main land transportation routes in central Japan for imports of GM oilseed rape from the Port of Kashima into Keiyo District. Oilseed rape plants were found each year, but the number of plants varied substantially during the three years of our study: 2162 plants in 2005, 4066 in 2006, and only 278 in 2007. The low number in 2007 was probably caused by roadwork. Herbicide-resistant individuals were detected in the three consecutive years (26, 8, and 5 individuals with glyphosate resistance), but glufosinate-resistant plants (9 individuals) were detected only in 2005. The roadside plants occurred mainly along the inbound lane from Kashima to Narita. These plants are likely to have their origin in seeds spilled during transportation of cargo from the port, since there are no potential natural seed source plants for B. napus near Route 51. This is the first detailed report on the transition and distribution of herbicide-resistant oilseed rape plants following loss and spillage along Japanese roads.

Pollen movement in a natural population of Arisaema serratum (Araceae), a plant with a pitfall-trap flower pollination system.

Arisaema serratum possesses a pitfall-trap flower pollination system. However, little is known about the efficiency and pattern of pollen movement in A. serratum. Thus, the aims of this study are to (1) determine the paternal parents of the seeds and (2) elucidate pollen movement in a natural population. Paternity analysis using microsatellite markers was performed. Seeds were collected from a natural population of A. serratum in 2001 at Horigane, Japan. Small midges became trapped in female spathe tubes during the flowering period. We found that (1) seeds in a fruit were fertilized by multiple sires; (2) seeds sired by a paternal parent were either clumped, exclusively, or randomly distributed on the spadix, depending on the parent; (3) to a great extent, a few males contributed as sires; (4) distance from a female was not a factor in the inequality of reproductive success among males; (5) male reproductive success was not correlated with its size. We conclude that pollen carryover and the trap-flower pollination system are likely to result in multiple paternity and inequality in male success.