Akira Uchino

Isolation and Expression of Genes for Acetolactate synthase and Acetyl - CoA carboxylase in Echinochloa phyllopogon, a Polyploid Weed species

BACKGROUND Target-site resistance is the major cause of herbicide resistance to acetolactate synthase (ALS)- and acetyl-CoA carboxylase (ACCase)-inhibiting herbicides in arable weeds, whereas non-target-site resistance is rarely reported. In the Echinochloa phyllopogon biotypes resistant to these herbicides, target-site resistance has not been reported, and non-target-site resistance is assumed to be the basis for resistance. To explore why target-site resistance had not occurred, the target-site genes for these herbicides were isolated from E. phyllopogon, and their expression levels in a resistant biotype were determined. RESULTS Two complete ALS genes and the carboxyltransferase domain of four ACCase genes were isolated. The expression levels of ALS and ACCase genes were higher in organs containing metabolically active meristems, except for ACC4, which was not expressed in any organ. The differential expression among examined organs was more prominent for ALS2 and ACC2 and less evident for ALS1, ACC1 and ACC3. CONCLUSION E. phyllopogon has multiple copies of the ALS and ACCase genes, and different expression patterns were observed among the copies. The existence of three active ACCase genes and the difference in their relative expression levels could influence the occurrence of target-site resistance to ACCase inhibitors in E. phyllopogon.

Cytochrome P450 CYP81A12 and CYP81A21 Are Associated with Resistance to Two Acetolactate Synthase Inhibitors in Echinochloa phyllopogon

Resistance to two herbicides in Echinochloa phyllopogon is associated with overexpression of two cytochrome P450s that are simultaneously controlled by a putative single genetic element. Previous studies have demonstrated multiple herbicide resistance in California populations of Echinochloa phyllopogon, a noxious weed in rice (Oryza sativa) fields. It was suggested that the resistance to two classes of acetolactate synthase-inhibiting herbicides, bensulfuron-methyl (BSM) and penoxsulam (PX), may be caused by enhanced activities of herbicide-metabolizing cytochrome P450. We investigated BSM metabolism in the resistant (R) and susceptible (S) lines of E. phyllopogon, which were originally collected from different areas in California. R plants metabolized BSM through O-demethylation more rapidly than S plants. Based on available information about BSM tolerance in rice, we isolated and analyzed P450 genes of the CYP81A subfamily in E. phyllopogon. Two genes, CYP81A12 and CYP81A21, were more actively transcribed in R plants compared with S plants. Transgenic Arabidopsis (Arabidopsis thaliana) expressing either of the two genes survived in media containing BSM or PX at levels at which the wild type stopped growing. Segregation of resistances in the F2 generation from crosses of R and S plants suggested that the resistance to BSM and PX were each under the control of a single regulatory element. In F6 recombinant inbred lines, BSM and PX resistances cosegregated with increased transcript levels of CYP81A12 and CYP81A21. Heterologously produced CYP81A12 and CYP81A21 proteins in yeast (Saccharomyces cerevisiae) metabolized BSM through O-demethylation. Our results suggest that overexpression of the two P450 genes confers resistance to two classes of acetolactate synthase inhibitors to E. phyllopogon. The overexpression of the two genes could be regulated simultaneously by a single trans-acting element in the R line of E. phyllopogon.

Cytochrome P450 genes induced by bispyribac‐sodium treatment in a multiple‐herbicide‐resistant biotype of Echinochloa phyllopogon

BACKGROUND Incremental herbicide metabolism by cytochrome P450 monooxygenases (P450s) has been proposed as the basis for resistance to bispyribac-sodium (bispyribac) in a multiple-herbicide-resistant biotype of Echinochloa phyllopogon. Upon exposure to bispyribac, strong induction of bispyribac-metabolising P450 activity has been reported in the resistant line, indicating that P450s induced by bispyribac are involved in the bispyribac resistance. RESULTS A polymerase chain reaction (PCR)-based cloning strategy was used to isolate 39 putative P450 genes from the bispyribac-resistant line of E. phyllopogon. Expression analysis by real-time PCR revealed that seven of the isolated genes were upregulated in response to bispyribac treatment of seedlings at the three-leaf stage. The transcript levels and protein sequences of the seven genes were compared between the bispyribac-resistant line and a susceptible line. CYP71AK2 and CYP72A254 were transcribed prominently in the bispyribac-resistant line. Amino acid polymorphisms were found in three genes, including CYP72A254. CONCLUSION Upregulated expression of these genes is consistent with the inducible herbicide-metabolising P450 activity under bispyribac stress that was reported in a previous study. This is the first study to compare P450 genes in arable weed species in order to elucidate the mechanism for P450-mediated herbicide resistance.

Copy Number Variation in Acetolactate Synthase Genes of Thifensulfuron-Methyl Resistant Alopecurus aequalis (Shortawn Foxtail) Accessions in Japan

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.

Suppressive effect of rice bran incorporation in paddy soil on germination ofMonochoria vaginalisand its relationship with electric conductivity

Laboratory experiments were conducted to investigate (1) the effects of the addition of rice (Oryza sativa. L.) bran to paddy soil on the germination of Monochoria vaginalis (Burm. f.) Kunth, and (2) the relationship between the electric conductivity (EC) of the soil solution and germination. Soil samples were collected at 4 sites in Japan. After flooded soils with rice bran had been incubated for 7 d at 30°C, the soil solution was collected using a porous cup and the EC of the soil solution was measured. The amounts of rice bran added to the soil were 0%, 0.3%, 0.6% and 0.9% (weight(w)/w). In the soil solution obtained, seeds of M. vaginalis were incubated for 3 d at 30°C, and the germination percentage was then analyzed. The addition of rice bran suppressed germination, and the degree of suppression increased with increasing content of rice bran. Although the same amount of rice bran was applied to each soil, the degree of growth suppression by rice bran as well as the EC of the soil solution differed among the soils. In each soil, there was a positive correlation between the amount of rice bran and EC, and the degree of growth suppression significantly increased with an increase in EC. When EC was higher than 150 mS m−1, seeds of M. vaginalis hardly germinated. There was no significant correlation between the oxidation-reduction potential (Eh) of soil and seed germination, suggesting that EC is a more reliable and convenient indicator than Eh for evaluating the relationship between the addition of organic material and seed germination. In conclusion, the addition of rice bran to soil increases the EC of the soil solution, and EC is one of the factors that suppress the germination of M. vaginalis. The suppressive effect of rice bran on germination is different among soils. This fact is attributed to the difference in EC due to the addition of rice bran. Thus, it is expected that EC can be used as an indicator for determining how much rice bran to add.

Tolerance of Rice (Oryza sativa L.) and Echinochloa Weeds to Growth Suppression by Rice Straw Added to Paddy Soil in Relation to Iron Toxicity

Abstract The effects of addition of rice straw to submerged soil on the emergence and growth of rice (Oryza sativa L.) and two paddy weeds (Echinochloa oryzicola Vasing. and Echinochloa crus-galli (L.) Beauv. var. crus-galli) were investigated. Rice straw suppressed both the emergence and growth of transplanted plants depending on the amount of rice straw added (0%, 0.3%, 0.6% and 0.9% (w/w)) in the order of E. crus-galli > E. oryzicola > rice. The severe suppression of emergence and growth of E. crus-galli in the presence of 0.9% rice straw in hydroponic culture was thought to be due to high Fe content of the shoots. Since the difference in tolerance for the toxicity of rice straw is an important factor, the addition of organic materials into soil may help to suppress Echinochloa weeds selectively.

Relationship between physical property of soil and growth of Monochoria vaginalis under paddy condition of organic farming—analysis using settled soil volume in water of superficial layer.

ABSTRACT Field experiments were conducted to analyze the relationship between the settled soil volume in water (SSVW) and the growth of Monochoria vaginalis (Burm. f.) Kunth under organic farming conditions. SSVW corresponds to the mud volume per dry matter weight. Soil was sampled from the superficial layer of the topsoil (<10 mm), which was of a finer texture than the rest of the topsoil. Without the application of rice bran, there was a negative correlation between SSVW and the number of individuals of M. vaginalis. This finding suggests that SSVW is useful as a physical indicator for the growth suppression of M. vaginalis. The application of rice bran dramatically reduced the number of M. vaginalis. The values of SSVW with rice bran were greater than those without rice bran. The analysis of SSVW indicates that the change in soil physical properties following the application of rice bran was one of the factors responsible for the suppression of M. vaginalis growth.

Promotive effect of soil solution on germination of Monochoria vaginalis under paddy conditions

ABSTRACT Growth of the weed Monochoria vaginalis (Burm. f.) Kunth under the conditions of organic rice production is a serious problem. The reason for the growth of M. vaginalis being dominant, especially in organic rice production, is not fully understood. In this study, laboratory experiments were conducted to analyze soil and seed factors in relation to the promotion of germination. (1) After incubation of flooded soil with or without the addition of rice bran (0.3%, 0.6%, and 0.9% in an air-dried soil basis), soil solutions were recovered and seeds of M. vaginalis were incubated in the soil solutions. Germination in the soils solutions without and with 0.3% rice bran was greater than that in distilled water. However, germination was suppressed in the presence of 0.6% and 0.9% of rice bran. These findings indicate that the solution from the soils with rice bran has different effects that may either increase or decrease germination. (2) A mixture of air-dried soil and distilled water was filtered to obtain a soil solution. Seeds were incubated in the soil solution (same as above). Environmental and physiological factors affected germination: exposure of seeds to light was an environmental factor and high germination activity and shallow dormancy of seed were physiological. The recovered soil solution promoted germination when these factors were not optimized. (3) There was a negative and significant correlation between dissolved oxygen (DO) in the soil solution and germination, indicating that a low content of DO was a promotive factor for germination. (4) Based on an experiment using pH buffers, germination increased with decrease in pH of soil solution, as long as the pH ranged from 4.0 to 7.0. This finding indicates that pH is also a factor that promotes germination.