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The international grain trade is a major pathway for the introduction of alien plants because grain commodities can be contaminated with various weed seeds. To evaluate how alien weed seeds derived from imported grain commodities affect local vegetation in international trading ports, we conducted a vegetation survey on a set of grain landing ports and non-grain landing ports in nine regions throughout Japan to compare the vegetation between these two types of ports. We also surveyed weed seed contamination of wheat imported into Japan, and the contamination rate was calculated for each species based on our survey and previous studies on weed seed contamination. The vegetation clearly differed between the grain landing ports and the non-grain ports. In the grain landing ports, alien species were more abundant than in non-grain landing ports. All indicator species of the grain landing ports were reported to be contaminants in grain commodities. Furthermore, there was a tendency for the more abundant species at the grain landing ports to show higher contamination rates in grain commodities. These results indicate that contaminant seeds spill from imported grain in grain landing ports, and highly contaminated species are likely to become established. We clearly show that weed seed contamination in grain commodities plays an important role in propagule pressure. Gathering information about the prevalence of weeds in grain-exporting countries and monitoring the weed species composition in imported grain commodities is becoming increasingly important for predicting the unintentional introduction of troublesome weeds and identifying effective weed management options.

The genetic and molecular basis of resistance evolution in weeds to multiple herbicides remains unclear despite being a great threat to agriculture. A population of late watergrass [Echinochloa phyllopogon (Stapf.) Koso-Pol.] was reported to exhibit resistance to ≥15 herbicides from six sites of action, including thiobencarb (TB). While previous studies disclosed that the resistance to a majority of herbicides such as acetolactate synthase (ALS) and acetyl-CoA carboxylase inhibitors is caused by the overexpression of herbicide-metabolizing cytochrome P450s (CYP81A12 and CYP81A21), the resistance mechanisms to some herbicides remain unknown. Here, we analyzed the resistance segregation in the progenies between resistant and sensitive populations and performed a transgenic plant sensitivity assay to resolve whether TB resistance is endowed by the same CYP81A12/21-based cross-resistance mechanism or other unknown multiple-resistance mechanisms. In the F6 progenies, resistance to the ALS inhibitor bensulfuron-methyl cosegregated with the resistances to many other herbicides under the CYP81A12/21-based cross-resistance mechanism; however, TB resistance segregated independently. Furthermore, CYP81A12/21 failed to confer TB resistance in transgenic Arabidopsis thaliana L. Heynh, thus confirming that TB resistance in resistant E. phyllopogon is not endowed by the two P450s that are responsible for the metabolism-based cross-resistance. This study provides evidence that resistance in E. phyllopogon to herbicides with multiple sites of action is endowed by both P450-based and other uncharacterized non–target site based mechanisms. Our findings add another layer in the understanding of resistance evolution to multiple herbicides in E. phyllopogon. Identification of the key genes endowing TB resistance will be the future direction of this research.

The evolution of herbicide resistance in weeds is an example of parallel evolution, through which genes encoding herbicide target proteins are repeatedly represented as evolutionary targets. The number of herbicide target-site genes differs among species, and little is known regarding the effects of duplicate gene copies on the evolution of herbicide resistance. We investigated the evolution of herbicide resistance in Monochoria vaginalis, which carries five copies of sulfonylurea target-site acetolactate synthase (ALS) genes. Suspected resistant populations collected across Japan were investigated for herbicide sensitivity and ALS gene sequences, followed by functional characterisation and ALS gene expression analysis. We identified over 60 resistant populations, all of which carried resistance-conferring amino acid substitutions exclusively in MvALS1 or MvALS3. All MvALS4 alleles carried a loss-of-function mutation. Although the enzymatic properties of ALS encoded by these genes were not markedly different, the expression of MvALS1 and MvALS3 was prominently higher among all ALS genes. The higher expression of MvALS1 and MvALS3 is the driving force of the biased representation of genes during the evolution of herbicide resistance in M. vaginalis. Our findings highlight that gene expression is a key factor in creating evolutionary hotspots.

CYP81A P450s armor Echinochloa phyllopogon against diverse and several herbicide chemistries. CYP81A substrate preferences can be a basis for cross-resistance prediction and management in E. phyllopogon and other related species. Metabolism-based herbicide resistance is a major threat to agriculture, as it is unpredictable and could extend resistance to different chemical groups and modes of action, encompassing existing, novel and to-be-discovered herbicides. Limited information on the enzymes involved in herbicide metabolism has hindered the prediction of cross-resistance in weeds. Members of CYP81A subfamily in multiple herbicide resistant (MHR) Echinochloa phyllopogon were previously identified for conferring cross-resistance to six unrelated herbicide classes. This suggests a critical role of CYP81As in endowing unpredictable cross-resistances in E. phyllopogon, thus the functions of all its nine putative functional CYP81A genes to 33 herbicides from 24 chemical groups were characterized. Ectopic expression in Arabidopsis thaliana identified the CYP81As that can confer resistance to multiple and diverse herbicides. The CYP81As were further characterized for their enzymatic functions in Escherichia coli. CYP81A expression in E. coli was optimized via modification of the N-terminus, co-expression with HemA gene and culture at optimal temperature. CYP81As metabolized its herbicide substrates into hydroxylated, N-/O-demethylated or both products. The cross-resistance pattern conferred by CYP81As is geared towards all chemical groups of acetolactate synthase inhibitors and is expanded to herbicides inhibiting photosystem II, phytoene desaturase, protoporphyrinogen oxidase, 4-hydroxyphenylpyruvate dioxygenase, and 1-deoxy-D-xylulose 5-phosphate synthase. Cross-resistance to herbicides pyrimisulfan, propyrisulfuron, and mesotrione was predicted and confirmed in MHR E. phyllopogon. This study demonstrated that the functional characterization of the key enzymes for herbicide metabolism could disclose the cross-resistance pattern and identify appropriate chemical options to manage the existing and unexpected cross-resistances in E. phyllopogon.

SummaryPrevious research unveiled that the overexpression of catalytically promiscuous CYP81A cytochrome P450s underlies the multiple-herbicide resistance (MHR) in a Californian population of Echinochloa phyllopogon. However, it does not fully accommodate the resistance to diverse herbicides in MHR E. phyllopogon although the genetic inheritance of MHR was suggested as under a single gene control.We investigated the high-level resistance to diclofop-methyl in MHR E. phyllopogon. Detailed diclofop-methyl metabolism was analyzed, followed by gene expression study and functional characterization of P450 genes. The generality of the MHR mechanism was investigated using another MHR line.The MHR line rapidly produced two distinct hydroxylated-diclofop-acid, only one of which was the major metabolite produced by CYP81A12/21. Gene expression study identified the genetically linked overexpression of a novel gene CYP709C69 with CYP81A12/21 in the MHR line. The gene conferred diclofop-methyl resistance in plants and produced another hydroxylated-diclofop-acid in yeast. The activity was observed in some CYP709C in plants. Unlike the broad substrate-specificity in CYP81As, CYP709C69 showed narrow substrate-specificity. The overexpression of the CYP81A and CYP709C69 was also observed in another MHR line.The present findings establish a novel concept that genetically-linked simultaneous overexpression of herbicide-metabolizing genes enhances and broadens the profile of metabolic resistance in weeds.

Understanding the characteristics of alien species is a prerequisite for any biological study or anti‐invasion management strategy. Lolium rigidum (Poaceae) is an alien species that has become naturalized on the sandy coasts of Japan; however, it exhibits extensive morphological variation, leading to speculation that several taxa of Lolium have become naturalized. Here, we compared the morphology, reproductive biology and genetic structure of this Lolium species by growing individuals from different locations in the same environment to clarify whether the observed morphological variation is genetically based or is caused by intraspecific variation as a result of environmental differences. Principle component analyses of 11 morphological traits separated the study species into two types. Bagging experiments showed that one type exhibited an outcrossing life history, whereas the other type selfed exclusively. Nuclear DNA microsatellite analyses supported this distinction between these morphological types, with no intermediate individuals being found. The outcrossing type exhibited high genetic diversity, whereas the selfing type exhibited almost no polymorphism, reflecting the differences in their breeding systems. Moreover, both types differed from the economically important outcrossing species Lolium multiflorum and Lolium perenne. These two types of Lolium are expected to have different introduction histories and invasive potential on the coasts of Japan. In conclusion, it is important to distinguish between these types through additional biological studies to design effective control measures.

Californian populations of Echinochloa phyllopogon have evolved multiple-herbicide resistance (MHR), posing a threat to rice production in California. Previously, we identified two CYP81A cytochrome P450 genes whose overexpression is associated with resistance to acetolactate synthase (ALS) inhibitors from two chemical groups. Resistance mechanisms to other herbicides remain unknown. We analyzed the sensitivity of an MHR line to acetyl-CoA carboxylase (ACCase) inhibitors from three chemical groups, followed by an analysis of herbicide metabolism and segregation of resistance of the progenies in sensitive (S) and MHR lines. ACCase herbicide metabolizing function was investigated in the two previously identified P450s. MHR plants exhibited resistance to all the ACCase inhibitors by enhanced herbicide metabolism. Resistance to the ACCase inhibitors segregated in a 3 : 1 ratio in the F2 generation and completely co-segregated with ALS inhibitor resistance in F6 lines. Expression of the respective P450 genes conferred resistance to the three herbicides in rice, which is in line with the detection of hydroxylated herbicide metabolites in vivo in transformed yeast. CYP81As are super P450s that metabolize multiple herbicides from five chemical classes, and concurrent overexpression of the P450s induces metabolism-based resistance to the three ACCase inhibitors in MHR E. phyllopogon, as it does to ALS inhibitors.

Reproductive isolation plays an important role in population differentiation and speciation, thus enhancing biodiversity in wild plants. Hybridisation sometimes involves rapid reproductive isolation between parents and their hybrids through the novel traits of hybrids derived from a new combination of genomes. Here, we report how a hybrids’ new phenotype contributes to rapid reproductive isolation between two ecotypes ofImperata cylindrica. The two ecotypes differ in their flowering phenology and habitats. An analysis with genetic markers revealed that hybrid populations consisted of only F1individuals. Both parental ecotypes flowered in spring, but F1s flowered in fall. This drastic shift in flowering phenology prevented backcrossing parental ecotypes to F1. F1s flowered in fall and dispersed seeds in winter. The germination percentage of seeds set on F1was extremely low in their habitats, and seedlings did not survive due to the low temperatures in winter, resulting in the absence of a F2generation. In conclusion, flowering phenology mismatch promotes reproductive isolation between parents and F1, resulting in a hybrid population consisting of only F1s.

Although an increasing number of investigations have been made into the evolution of alien species once introduced, few studies have identified the invasion routes of these introduced species. Because multiple introductions are common in invasive species, failing to take into account the introduced lineages can be misleading when studying evolutionary change in alien species after they begin to extend their ranges. In Japan, diverse lineages of ryegrasses (Lolium spp.) were introduced as forage crops and contaminants in trading grain and have expanded to sandy coasts. We studied the expansion route of populations established along the coasts of three geographic regions within Japan by comparing variations in morphology and nuclear microsatellite and chloroplast DNA in the two habitats where ryegrasses were first introduced: croplands and international seaports. Chloroplast DNA haplotypes did not differ significantly among habitats and regions, but the coastal and seaport populations displayed similar microsatellite genetic compositions and morphological characteristics. Our results revealed that coastal populations originated from seaport populations derived from contaminants. Selective forces from the past, including domestication and naturalization, may have assisted the introduced lineages in colonizing new habitats.Nomenclature: Italian ryegrass, Lolium multiflorum L.; rigid ryegrass, Lolium rigidum Gaudin.

The rice production system in Japan is changing due to the aging of rice farmers, shortage of labor, mechanization, and increase of large-scale rice fields and increased application of direct sowing, all of which contribute to the lack of sophisticated weed management practices. Because the changing production system has not improved and likely worsened weed management practices, weedy rice (Oryza sativa L.) has become a serious threat to rice production in Japan. We analyzed weedy rice accessions from Nagano, the central part of Japan, and other rice accessions to elucidate the origin of this weedy rice using a whole-genome single nucleotide polymorphism (SNP) array. For developing sustainable weedy rice management practices, the elucidation of the origin of re-emerging weedy rice is crucial. Population genetic analysis indicated that weedy rice in Nagano was phylogenetically independent from the local red rice cultivar with low genetic diversity. Past and recently re-emerging weedy rice ecotypes in Nagano shared a similar genetic background which implies the evolution of weedy rice by severe natural selection. Window-based FST and selection sweep analysis revealed the divergence of some of the known key domestication-related genes, such as shattering gene sh4 and qsh1, of weedy rice from domesticated rice cultivars.

This review states the outline of field crop production of Japan and weed problems to be solved. Field crop production of Japan is characterized by rice cultivation and small size farming. Most major field crops other than rice are imported, and various kinds and a very large amount of living weed seeds contaminate the imported grains. These alien weeds sometimes invade agricultural fields and cause serious losses of crop yields and reductions in the quality of agricultural products. Emergence of herbicide resistant biotypes of weeds, especially paddy weeds, is also issues to be solved. Weedy rice is recently emerging again due to the change of rice production system and the origin of recently emerging weedy rice is not clarified yet.

Monochoria vaginalis and M. korsakowii are allotetraploid noxious weeds in rice cultivation. Occurrences of resistance to acetolactate synthase (ALS)-inhibiting herbicides have been reported in these weeds in Japan since the 1990s. The existence of multiple copies of ALS genes in both species has hindered and complicated the detailed study of molecular mechanisms in them. To determine the copy number and full-length of ALS genes in both species, we first amplified partial sequences of ALS genes and separated them by cloning. Five and three distinct sequences were identified in M. vaginalis and M. korsakowii, respectively. RACE and TAIL PCR successfully isolated full-length ALS genes, revealing that one copy of ALS genes in both species is a pseudogene formed by a frameshift mutation. Interestingly, one of the four putative functional ALS genes in M. vaginalis contains an intron in the 3'-untranslated region. Amplification and sequencing of the full-length ALS genes in sensitive and suspected resistant lines revealed a non-synonymous point mutation at codon Pro197, resulting in amino acid substitutions (Leu, Ser, or Ala) well known to endow ALS inhibitor resistance. Importantly, codon Pro197 of the M. korsakowii pseudogene encodes leucine (Leu) both in resistant and sensitive plants, which is also known to confer ALS inhibitor resistance when ALS genes are functional. Dose responses to imazosulfuron of the lines analyzed for ALS genes were in agreement with the existence of the mutations. These results suggest that some caution is needed when diagnosing molecular resistance in M. korsakowii. The information of copy number and full-length sequences will help diagnose ALS resistance and make a basis for the study of the evolution of ALS resistance in Monochoria spp.

Summary Imperata cylindrica (cogongrass) is one of the most invasive grass weeds found worldwide. In Japan, this grass grows as three climate types: cool temperate (CT), common (CM) and subtropical (ST). An early flowering (E) type has been also reported. Among them, the CM type is large and most invasive. Recent global warming may cause the extension of distribution of the CM type to northern parts of Japan and may allow hybrids to form between the CM and CT or E types. The hybrids sometimes show heterosis and this leads to new weed problems. Distinguishing between the hybrids and parents on a morphological basis is difficult. We investigated polymorphisms of eight cpDNAs to enable distinction between the ecotypes and the hybrids. Fourteen haplotypes were detected in 33 Japanese clones, based on variations in eight cpDNA regions. Based on three substitutions in the psbA–matK spacer, the ORF170 intron1, the trnL(UAA) intron–trnF(GAA) spacer and indels in the trnL(UAA) intron–trnF(GAA) spacer, the haplotypes were clustered into three phylogenetic groups: I, IIa and IIb, which corresponded with the CM and ST, E and CT types, respectively. The polymorphism in the cpDNA therefore enables us to distinguish among the ecotypes and is useful for prediction of the range expansion of the CM type.

Severe infestations of Alopecurus aequalis (shortawn foxtail), a noxious weed in wheat and barley cropping systems in Japan, can occur even after application of thifensulfuron-methyl, a sulfonylurea herbicide. In the present study, nine accessions of A. aequalis growing in a single wheat field were tested for sensitivity to thifensulfuron-methyl. Seven of the nine accessions survived application of standard field rates of thifensulfuron-methyl, indicating that severe infestations likely result from herbicide resistance. Acetolactate synthase (ALS) is the target enzyme of sulfonylurea herbicides. Full-length genes encoding ALS were therefore isolated to determine the mechanism of sulfonylurea resistance. As a result, differences in ALS gene copy numbers among accessions were revealed. Two copies, ALS1 and ALS2, were conserved in all accessions, while some carried two additional copies, ALS3 and ALS4. A single-base deletion in ALS3 and ALS4 further indicated that they represent pseudogenes. No differences in ploidy level were observed between accessions with two or four copies of the ALS gene, suggesting that copy number varies. Resistant plants were found to carry a mutation in either the ALS1 or ALS2 gene, with all mutations causing an amino acid substitution at the Pro197 residue, which is known to confer sulfonylurea resistance. Transcription of each ALS gene copy was confirmed by reverse transcription PCR, supporting involvement of these mutations in sulfonylurea resistance. The information on the copy number and full-length sequences of ALS genes in A. aequalis will aid future analysis of the mechanism of resistance.

Summary Compared with natural seed dispersal, human-mediated seed dispersal could spread herbicide resistance genes on a much larger scale. Herbicide-resistant weed seeds have been reported as contaminants in commercial grain. We investigated the contamination of seeds of Lolium species with target-site mutations conferring resistance to acetolactate synthase (ALS)-inhibiting herbicides in wheat imported from the USA, Canada and Australia into Japan. We also investigated the establishment of ALS-inhibiting herbicide-resistant Lolium species in 12 seaports in Japan that are major entry points for international commodities. We found herbicide-resistant Lolium spp. seeds from all classes of wheat samples. Resistant individuals became established at six of eight ports where more than 50 kt of imported wheat is unloaded every year. The establishment of resistant Lolium spp. individuals was common at major grain landing ports. Monitoring over 3 years at one port revealed that the frequency of resistant individuals did not fluctuate between years. Many resistant individuals were distributed in front of the entrance of a fodder company, but a few resistant individuals were found in areas 2 km away from the port. The results indicate that gene flow is rare through pollen or seed movement from resistant plants to peripheral populations. Further extensive and long-term monitoring is necessary to perform a comprehensive risk assessment of herbicide-resistant plants entering Japan through major commercial ports.

In order to clarify the growth and reproductive success of seed-derived Sagittaria trifolia individuals emerging at different times within a season, seeds were sown on May 16, June 16 and July 22 2010 and allowed to grow in pots under shaded and unshaded conditions. The timing of when the first inflorescence of each seedling appeared was recorded, as well as the number of seeds and tubers produced and the dry weight of each organ. In total, 220 seedlings were used, 23–59 in each treatment. As the sowing time was delayed, so too was the appearance of the first inflorescence. Additionally, the number of inflorescences, fruits and seeds produced by the unshaded plants significantly decreased with the sowing time. The total dry weight was significantly greater in the unshaded than in the shaded plants: the July-sown, shaded plants grew very poorly. The relative resource allocation to the tubers was greater in the late-emerging, shaded seedlings. Even in the shaded groups, except for the July-sown plants, most of the plants set seed. Therefore, when addressing weed control in paddy fields, one should keep seedlings in mind, in addition to plants that have grown from tubers.

The grassland of Inner Mongolia, China, has experienced a decrease in area. To provide useful information for artificial grassland restoration, the germination characteristics of four native grass species, Leymus chinensis (Trin.) Tzvel., Agropyron cristatum (L.) Gaertn., Bromus inermis Leyss. and Elymus dahuricus Turcz., were compared under laboratory conditions. The seeds of A. cristatum and B. inermis showed >70% germination at a wide range of temperatures from 10 to 30°C with or without light. Seeds of E. dahuricus did not show clear dormancy, but required a rather high temperature (20–30°C) for germination. When the temperature was alternated between day and night, it was the higher of the temperatures (the daytime temperature) that largely determined the percentage of germination. The germination of E. dahuricus was higher (max. 87%) in darkness than in light (max. 58%). Intact seeds of L. chinensis showed considerable germination (47%) only under a widely fluctuating temperature (30/10°C). Additionally, the dehusking of L. chinensis seeds improved the germinability at a constant temperature. These results are suggestive of a different regeneration niche among the four species, which should be taken into account when selecting the species and method for grassland restoration.

Severe infestations of Alopecurus aequalis (shortawn foxtail), a noxious weed in wheat and barley cropping systems in Japan, can occur even after application of thifensulfuron-methyl, a sulfonylurea (SU) herbicide. In the present study, nine accessions of A. aequalis growing in a single wheat field were tested for sensitivity to thifensulfuron-methyl. Seven of the nine accessions survived application of standard field rates of thifensulfuron-methyl, indicating that severe infestations likely result from herbicide resistance. Acetolactate synthase (ALS) is the target enzyme of SU herbicides. Full-length genes encoding ALS were therefore isolated to determine the mechanism of SU resistance. As a result, differences in ALS gene copy numbers among accessions were revealed. Two copies, ALS1 and ALS2, were conserved in all accessions, while some carried two additional copies, ALS3 and ALS4. A single-base deletion in ALS3 and ALS4 further indicated that they represent pseudogenes. No differences in ploidy level were observed between accessions with two or four copies of the ALS gene, suggesting that copy number varies. Resistant plants were found to carry a mutation in either the ALS1 or ALS2 gene, with all mutations causing an amino acid substitution at the Pro197 residue, which is known to confer SU resistance. Transcription of each ALS gene copy was confirmed by reverse transcription PCR, supporting involvement of these mutations in SU resistance. The information on the copy number and full-length sequences of ALS genes in A. aequalis will aid future analysis of the mechanism of resistance.

Summary Schoenoplectus juncoides is one of the most harmful weeds found in East Asian paddy fields. Recent emergence of biotypes that are resistant to the herbicide sulfonylurea (SU) has made weed control difficult. To examine the effect of the evolution of this herbicide resistance on genetic diversity within local populations, we investigated microsatellite variability within and among paddy field populations of S. juncoides in Kinki, Japan. In vivo assay of acetolactate synthase activity and root elongation assay in the presence of SU revealed that of 21 populations, five were sulfonylurea-susceptible (SU-S) and eight were completely sulfonylurea-resistant (SU-R). The remaining eight populations were a mixture of SU-S and SU-R individuals. The average gene diversity for SU-R populations (HS = 0.168) was lower than those for SU-S (HS = 0.256) and mixed (HS = 0.209) populations, but the difference was not significant. This indicates that positive selection for SU-R phenotype did not cause a genome-wide reduction in genetic diversity. Genetic differentiation among S. juncoides populations was higher than that observed for most weed species studied previously. Although populations in neighbouring paddy fields showed a high level of differentiation, Bayesian clustering analyses suggested that some level of gene flow occurs among them and that the genetic exchange or colonisation between neighbouring populations could contribute to the geographical expansion of the resistant allele.

Vincetoxicum pycnostelma Kitag. (Apocynaceae) is a perennial herb that grows in grassland habitats in East Asia and the western part of Amur. Urbanization, land development, abandonment of traditional management of grasslands, or any combination of these factors are rapidly reducing grassland habitats, and V. pycnostelma is now ranked as a “Near Threatened” species on the Red List of Japan. Identification and characterization of pollinators of this endangered species are important in its conservation. We observed and collected insect visitors to the flowers at night and in the morning in the populations of V. pycnostelma on the banks of the Kidzu River, Kyoto, Japan from June to August in 2011. Pollinators were characterized as those insects on which flower pollinaria were attached. Twenty-seven species (9 families; 3 orders) were observed visiting the flowers; individuals of 14 lepidopteran species (6 families) had pollinaria attached to their bodies and most visited the flowers at night. Adult ...

Summary Mazus pumilus is an annual self-pollinating weed that is commonly found in arable land, vegetable gardens and roadsides. This weed harbours insects and pathogens that attack vegetables. The mechanism of resistance to paraquat of M. pumilus found in Ohita, Japan, was studied. Whole plant bioassays revealed that the resistant (R) biotypes were four to six times less susceptible than controls. Chlorophyll destruction of leaf discs by paraquat treatment in R biotypes was 4–20 times lower than those of susceptible (S) biotypes. Ferric reducing antioxidant power (FRAP) values in R biotypes were higher than those of S biotypes before and after paraquat treatments. The activity of superoxide dismutase (SOD) was also higher in R biotypes than those of S biotypes before and after treatment with paraquat, but the activities of ascorbate peroxidase (APX) and catalase (CAT) were not different between R and S biotypes. Change of ascorbate (AsA) contents before and after paraquat treatment was equivalent in both biotypes. These results indicate that the increased SOD activity and antioxidant capacity in R biotypes contribute to the resistance to paraquat of M. pumilus.

Lindernia procumbens and L . dubia are common annual weeds in flooded rice fields of Japan. Two subspecies of L. dubia , subsp. major and subsp. dubia , are usually recognized in Japan but they are both regarded as synonyms of L. dubia elsewhere. In a cluster analysis based on AFLP, most L. dubia subsp. major formed a separate cluster from L. dubia subsp. dubia although 11% of haplotypes classified using AFLP were not coincident with classification using the shape of leaf bases, which is the commonly used identification trait. Artificial F 1 plants between L. procumbens and L. dubia subsp. major , and between L. procumbens and L. dubia subsp. dubia did not produce seed. Forty percent of capsules produced by F 1 plants from these two subspecies were slimmer and 80% pollen were sterile in slimmer capsules. However, seed number of most F 1 capsules was not different from that of self-fertilized plants, suggesting that there was no complete reproductive isolation between the subspecies. Natural hybridization of these subspecies may have occurred but we are not aware of it because F 1 plants are rare and F 2 plants are indistinguishable from these subspecies.

The association of the micro-scale distribution of six plant species in a lawn, including the lawn grass, Zoysia japonica, with the soil's environmental factors was studied in an urban park in Kyoto, Japan. The cover of each plant species was scored in July and August. The relative elevation, depth of waterlogging after a shower, soil water content, and soil hardness were measured in 144 quadrats in a 12 m × 12 m plot. The subsurface soil was sampled at every fourth quadrat and was analyzed for the percentage of fine earth and the levels of total carbon, total nitrogen, and available phosphate. Three correlation criteria, namely the Pearson's, Mantel, and partial Mantel correlations, showed considerable disagreement in indicating whether or not there was a significant correlation between each plant species and environmental variable. Although the reason for the disagreement was not always clear, some unimodal or inverted unimodal responses of a plant species against an environmental factor, which was not detectable by the Pearson's correlation coefficient, were indicated to be significant by the Mantel or partial Mantel tests. There were four plant–environment pairs that had a significant correlation in all three criteria: Juncus tenuis in August had a positive correlation with the soil water content in July and August and a negative correlation with soil hardness and Z. japonica showed a negative correlation with the water content in August. The results suggest that the small patches of lawn with poor drainage are the sites of invasion by J. tenuis.

在来種を緑化資材として利用する際，当該地域の生物多様性を保全する配慮が必要で，遺伝的攪乱を生じさせない手法の確立が求められている。しかし，現在，ほとんどすべての在来種について，緑化に利用するための移動許容範囲に関する情報は蓄積されていない。本研究では，緑化資材として有用なチガヤについて，近畿地方における移動許容範囲を提示することを目的として，近畿地方由来の111 系統とそれ以外の地方から採集した9 系統を加えた合計120 系統についてAFLP 解析をおこなった。その結果，近畿地方におけるチガヤの遺伝的分化の程度は低く，明瞭な地域集団の分化は認められなかった。しかし，自生地の緯度にともなう変異が存在する可能性があった。これらの結果から，近畿地方でチガヤを緑化資材として利用する際には，その移動範囲はほぼ100 km を目安とすることが適切であろうと考えられた。

ミズアオイ属水生雑草の野外集団およびさく葉標本について外部形態に関する32形質を調査し，個体内，集団内個体間および種内集団間の各レベルでその変異を検討した。1）個体内における形態の表現型可塑性の大きさは形質によって異なった。同じ形質であってもその可塑性は分類群によって異なっていた。また可塑性をもつ形質のほとんどは量的形質であり，可塑性の程度は遺伝的に制御され，栄養器官であるか生殖器官であるかには関係がなかった。さらに個体内に形態的奇形現象が認められた。例えば，コナギの花は総状花序であり，正常な花序には分枝がないが，分枝をもつ花序を有する個体が見いだされた。ミズアオイはミズアオイ属の中で唯一花序に分枝をもつ種であり，この奇形現象はコナギがミズアオイと極めて近縁であることを示唆している。2）ミズアオイの形態形質の集団内個体間変異は生育環境に強く影響されていることが認められた。特に花序あたりの花数、葉幅、葉の長さと花序の有花部分の長さは水深、他種との競合、土壌の肥沃度、遮光の有無などによって大きく影響を受けた。3）ミズアオイの種内集団間変異は集団内に見られる形質の可塑性よりも明らかに大きかった。コナギの集団間変異は局所的な生育環境に対応する変異とは考えられず，広い地域間に見られる温度や日長の差異など緯度の違いに対応した南北に傾度をもつ極めて大きな変異を示した。

The root and rhizome systems of dominant perennial grasses in Inner Mongolian grassland were clarified. We surveyed the vertical distribution of root and rhizome biomass in the natural stands, and the changes of under-ground biomass and the branching pattern of rhizomes for transplanted plants in a container experiment. Most roots of Leymus chinensis, Bromus inermis, Elymus dahuricus and Agropyron cristatum were distributed in the soil depth of 0–10 cm. Roots of E. dahuricus and A. cristatum were distributed in a shallower soil layer, but those of L. chinensis and B. inermis were distributed in a deeper soil layer. Biomass of above-ground parts increased with growth, resulting in a decreasing ratio of under-ground parts to total biomass. Rhizomes of L. chinensis and B. inermis were distributed in the soil depth of 0–10 cm, but E. dahuricus and A. cristatum did not have rhizomes. L. chinensis had longer rhizomes and new ramets were produced away from their mother plant. B. inermis had many short rhizomes and produced daughter plants near their mother plant.

All living organisms spontaneously generate ultraweak photon emissions, which originate from biochemical reactions in cells. Current research uses the ultraweak photon emission from organisms as a novel indicator in nondestructive analyses of an organisms living state. This study indicates that ultraweak photon emissions from Monochoria vaginalis are different between resistant biotypes (R) to sulfonylurea (SU) and susceptible biotypes (S). In SU-R biotypes, distinct increases in photon emissions were observed, but there was little increase in SU-S biotypes. In addition, photon emissions from the resistant biotypes of M. vaginalis were suppressed by treatment with P450 inhibitors. This suggests that cytochrome P450 monooxygenase, which plays a crucial role in the metabolic detoxification of SUs, could be associated with the generation of ultraweak photon emissions. Ultraweak photon emissions have a potential use in a novel diagnosis system as an indicator in a nondestructive testing of weeds resistant to SUs.

Monochoria vaginalis is one of the most serious weeds of rice fields in Asia. The species has both chasmogamous (CH) and cleistogamous (CL) flowers on individual plants. The objective of this study is to clarify the reproductive characteristics that affect the selfing rate of M. vaginalis. An emasculation experiment revealed that the CH flowers emasculated just after flower opening produced seeds. The seed production in the flowers emasculated just after flower opening was less than that of the flowers emasculated and hand-pollinated with pollen from another plant. These results indicate that, although self-pollination before anthesis occurs, cross-pollination also can produce seeds. An experimental manipulation of light and the emergence time of the plants revealed that more than half of the flowers produced were CL flowers and the CH/CL ratio decreased with reduced light availability. The plants that emerged later produced as many CH flowers as those that emerged earlier. There was no significant difference between the CH and CL flowers in the seed number per fruit and the seed mass. These results suggest that M. vaginalis has reproductive characteristics that make the selfing rate high; however, outcrossing also can occur. Under favorable light intensity conditions, such as on the edges of paddy fields or in fallow fields, M. vaginalis will produce more CH flowers and will have a higher outcrossing rate than in shaded conditions.

The severe degradation of grasslands caused by overgrazing and other diverse human operations has become widespread in Inner Mongolia, China. In this study, the vegetations of several grasslands under different land-use conditions were compared to examine the relationships between the vegetation and the disturbance of the grassland. Floristic and life-form compositions of the stands representing the six different sites were researched by the quadrat method: a non-grazed grassland (G1), a lightly grazed grassland (G2), a path (P), an abandoned field (AF) and two artificial meadows (M1 and M2). P, AF, M1 and M2 were located in a part of the grazed grassland. AF, M1 and M2 had been tilled when planted. The dominant species found in G1 and G2 were Potentilla fragarioides, Poa annua, and Cleistogenes squarrosa. Artemisia annua, Chenopodium album, Cannabis sativa f. ruderalis and Calystegia hederacea were found in P and AF, being scarce in the grassland areas. Amaranthus retroflexus, Kochia scoparia and Setaria viridis, which are cosmopolitan weeds, were dominant only in AF. Leymus chinensis, Agropyron cristatum, Potentilla bifurca, and Potentilla tanacetifolia were widely observed across all the surveyed sites. The land-use change from grassland to path caused a decrease of perennial species and an invasion of annual species by heavy trampling. A similar vegetational change occurred with the land-use change from grassland to abandoned field due to the destruction of original vegetation by tillage. Human disturbance such as tillage and trampling also resulted in a decrease in the species with the tufted- and branched-form and an increase in the species with the erect-form. Tillage could produce a more serious impact on the grassland vegetation as compared to grazing.

Summary The inheritance of sulfonylurea (SU) resistance in Monochoria vaginalis was investigated based on the bensulfuron-methyl response phenotypes of F1 plants between SU-resistant (R) and -susceptible (S) and segregation analysis in F2 progenies. Differences of SU resistance between SU-R biotypes and F1 plants at the recommended field dose were also investigated by comparing shoot dry weight. All F1 plants survived the treatment with 25 g a.i. ha−1 bensulfuron-methyl, one-third of the recommended field dose, and showed similar responses to SU-R plants. Conversely, all F1 plants died or showed extreme necrosis at 225 g a.i. ha−1, three times the recommended field dose, as SU-S plants. F2 plants were classified as either R or S phenotype. Segregation for resistance to bensulfuron-methyl in F2 families did not differ from the expected 3:1 (R:S) ratio at 25 g a.i. ha−1. At 225 g a.i. ha−1, the F2 families segregated in a 1:3 (R:S) ratio. These results suggest that SU resistance in M. vaginalis is controlled by a single nuclear allele with resistance being dominant at low dose and susceptibility dominant at high dose. Moreover, F1 plants died or were extremely injured after application of bensulfuron-methyl at the recommended field dose, although SU-R biotypes grew normally.

TILLING (Targeting Induced Local Lesions IN Genomes) is a low-cost, high-throughput reverse genetic technique that employs a mismatch-specific endonuclease CEL-1 to discover induced point mutations in the genes of interest. The use of the TILLING technique to survey natural variation in genes is called EcoTILLING. Here, we report a modified EcoTILLING method for the discovery of mutations in multigene family, which we coin “Self-EcoTILLING” by using an allotetraploid Monochoria vaginalis ALS multigene family as an example. The mutations could be detected by TILLING of PCR products resulting from the primers specific to both Als1 and Als3 without involving the experimental step of mixture of reference and query DNA. Either of the two co-amplified loci could serve as reference DNA to the other. We demonstrate with this example that Self-EcoTILLING is a fast, reliable and economical technique of detecting single-nucleotide mutations in polyploid plants containing multigene family.

SDS-PAGE was used to determine the variation in high molecular weight glutenin subunits (HMW-GS) alleles present in 170 landraces of common wheat from Sindh, Balochistan and NWFP in Pakistan. Three alleles for Glu-A1, six alleles for Glu-B1 and four alleles for Glu-D1 were detected. These included three new alleles, namely Glu-B1e, Glu-B1h and Glu-D1n, which had not previously been found in landraces of Pakistani wheat. Furthermore, Glu-D1m and Glu-D1n, which are considered to be endemic alleles in Pakistan and Afghanistan, were both detected from Balochistan. Twenty genotypes were identified based on combinations of alleles at the three Glu-1 loci. The present study showed that Pakistani landraces of common wheat have maintained a broad diversity of HMW-GS alleles, and that they may serve as genetic resources to improve wheat varieties adapted to Pakistani climatic and edaphic conditions.

Summary Monochoria vaginalis is one of the most serious weeds of rice fields in Asia. The species is predominantly selfing. To reveal the potential for multiple mutational events, outcrossing and gene flow in the sulfonylurea-resistant (SU-R) M. vaginalis populations, we investigated (i) if each SU-R population was a single SU-R biotype or a mixture of several SU-R biotypes using restriction analysis or direct sequencing of acetolacatate synthase (ALS) genes and (ii) genetic diversity of SU-R and -susceptible (S) populations using amplified fragment length polymorphism (AFLP) analysis. Nineteen or 20 individuals were sampled from four SU-R and five SU-S populations respectively. Amino acid substitutions conferring resistance in the SU-R populations were Pro197Ser in the ALS1 or ALS3, or Asp376Glu in the ALS1 and each SU-R population was composed of a single SU-R biotype. In cluster analysis each SU-R individual formed a cluster, whereas the individuals from a SU-S population belonged to different clusters. Some SU-R populations showed polymorphic AFLP loci. The results indicated that these SU-R biotypes emerged from a single mutational event and any gene flow of SU-R genes from adjacent populations did not occur. A low level of outcrossing and recombinations of SU-R genes occurred within some SU-R populations of M. vaginalis.

All living organisms spontaneously generate ultraweak photon emissions, which originate from biochemical reactions in cells. Current research uses the ultraweak photon emissions from organisms as a novel tool to investigate the physiological states of plants. In this study, we found ultraweak photon emissions from leaf segments of rice and several paddy weed species treated with a sulfonylurea herbicide. There is a definite difference in photon emissions among plant species, and rice (Oryza sativa), barnyardgrass (Echinochloa crus-galli) and Cyperus serotinus showed extremely strong enhancement of photon emissions. Photon emissions from these three species treated with sulfonylurea herbicide were suppressed when the leaf segments were treated with the cytochrome P450 monooxygenase (P450) inhibitors, piperonyl butoxide and malathion. These results suggest that P450 inhibitors affect the ultraweak photon emissions from plants.

TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful reverse genetic technique that employs a mismatch-specific endonuclease to discover induced point mutations in genes of interest. The use of the TILLING technique to survey natural variation in genes is called Ecotilling. We report an adaptation of Ecotilling for rapid detection of single-nucleotide mutations in the acetolactate synthase (ALS) genes of sulfonylurea (SU)-resistant (R) Monochoria vaginalis (Pontederiaceae), a paddy weed, in Japan. Genomic DNA of a SU-R plant (target DNA) was mixed with the DNA of a SU-susceptible (S) plant (reference DNA). Ecotilling detected two nucleotide mutations in the ALS gene of SU-R M. vaginalis. These 2 mutations were confirmed by DNA sequencing. A single nucleotide mutation (C to A), in the codon CCT to CAT and another mutation (C to T), in the codon CCT to TCT were identified by sequencing. Both mutations result in the disruption of a Pro codon in the conserved Domain A region with the consequent substitution of a His residue in the first mutation and a Ser residue in the second. Substitution of the Pro residue in Domain A of the ALS gene has been reported to result in insensitivity to SUs in many weed biotypes. This study demonstrates that Ecotilling is a fast, reliable, economical method for detecting single-nucleotide mutations in genes arising from herbicide selection.