Resistance evolution and mechanisms to ALS-inhibiting herbicides in Capsella bursa-pastoris populations from China.

Capsella bursa-pastoris is a serious broadleaf weed in winter wheat fields in China. It has evolved high levels of resistance to acetolactate synthase (ALS) inhibiting herbicides and has caused substantial losses of wheat yield in recent years. We monitored the herbicide resistance of Capsella bursa-pastoris collected from 18 regions of Shandong Province in 2009, 2013 and 2017, respectively. Compared with the 2009 populations, the number of populations resistant to florasulam had increased in 2013 and 2017. Resistance to tribenuron-methyl increased in 2013, but decreased in 2017. The 2009 and 2013 populations developed resistance only to tribenuron-methyl, but some 2017 populations developed cross-resistance to imazethapyr and florasulam as well. Mutations in ALS (Pro-197-Thr/Ser/His/Arg/Leu/Gln) were identified in the 2009 and 2013 populations; however, two ALS mutations (Pro197 and/or Trp574) were identified in 2017 plants. Meanwhile, plants containing both point mutations (Pro197 + Trp574) were identified in the 2017 populations. This study demonstrated that target site gene mutations were the main reason for Capsella bursa-pastoris resistance to ALS-inhibiting herbicides. Although target-site mutation is the reason for resistance to ALS-inhibiting herbicides in Capsella bursa-pastoris, the resistance patterns and mutations identified have changed over time.

Isolation and expression of acetolactate synthase genes that have a rare mutation in shepherd's purse (Capsella bursa-pastoris (L.) Medik.).

Acetolactate synthase (ALS) inhibitor-resistant biotypes are the fastest growing class of herbicide-resistant weeds. Shepherd's purse (Capsella bursa-pastoris (L.) Medik.), a tetraploid species and one of the most troublesome weeds in wheat production, has evolved ALS inhibitor resistance. To confirm and characterize the resistance of shepherd's purse populations to ALS-inhibiting herbicides, whole-plant bioassays were conducted. To investigate the molecular basis of resistance in shepherd's purse, the ALS gene was sequenced and compared between susceptible (S) and resistant (R) biotypes. Two partial intronless ALS genes (ALS-1 and ALS-2) were identified, and two heterozygous mutations (CCT to TCT in ALS-1 and CCT to CAT in ALS-2) at position 197 (Pro197Ser and Pro197His) providing resistance were simultaneously found in a single plant in a resistant population. Our results confirmed that the resistant shepherd's purse population showed high-level resistance to tribenuron-methyl (RI = 59.8), pyroxsulam (RI = 38.7) and flucarbazone-Na (RI = 88.0). Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) results suggested that the difference in ALS gene expression was small between S and R populations, which may be insufficient to cause herbicide resistance, and according to the results of in vitro ALS activity, insensitivity of ALS may be the main mechanism of high resistance to tribenuron-methyl in resistant populations.

Tribenuron-methyl resistance and mutation diversity of the AHAS gene in shepherd's purse (Capsella bursa-pastoris (L.) Medik.) in Henan Province, China.

Shepherd's purse is a troublesome dicot weed that occurs in the major wheat-producing areas in China. Twenty-eight shepherd's purse populations were collected from winter wheat-planting areas in Henan Province and used to evaluate tribenuron-methyl resistance and acetohydroxyacid synthase (AHAS) gene-mutation diversity. The results indicate that all 28 shepherd's purse populations were resistant to tribenuron-methyl at different levels compared with the susceptible population. Mutation of the 197 codon (CCT) changed proline (Pro) into tyrosine (Tyr), histidine (His), leucine (Leu), serine (Ser), arginine (Arg), alanine (Ala) and threonine (Thr), whereas mutation of the 574 codon (TGG) changed tryptophan (Trp) into leucine (Leu). Among these amino acid changes, a co-concurrence of Pro197Leu and Trp574Leu substitutions was identified for the first time in resistant weed species. Furthermore, Pro197Tyr, Pro197Arg and Pro197Ala substitutions have not been previously reported in shepherd's purse. The results of the in vitro AHAS assay suggest that an insensitive AHAS is likely involved in the resistance to tribenuron-methyl in the R populations with AHAS gene mutations, and the non-target-site based resistance might exist in some populations.

Characterization of the variable 3' UTR and expression of the two intron-containing KIN transcripts from Capsella bursa-pastoris.

KIN genes are crucial members of the cold-regulated (COR) gene family, and are exclusively involved in normal developmental processes in many organs and respond to a variety of abiotic stresses in plants. Here, we cloned and sequenced not only two completely-spliced KIN transcripts (CbKIN1-S and CbKIN2-S), but also two intron-containing KIN transcripts (CbKIN1-U and CbKIN2-U), from Capsella bursa-pastoris, a widespread plant of the Brassicaceae family. The CbKIN1-U and CbKIN2-U transcripts each contained one additional intron in the coding region compared to the corresponding CbKIN1-S and CbKIN2-S transcripts. In addition, the two intron-containing KIN transcripts were found by rapid amplification of cDNA 3' ends (3' RACE) analysis with specific primers to have variable 3' untranslated regions (3' UTRs). We also analyzed CbKIN1-U and CbKIN2-U levels in different organs and embryonic stages by quantitative polymerase chain reaction (qPCR). They were found to be expressed in middle-stage embryos and flowers. After abscisic acid (ABA) treatment, CbKIN1-U and CbKIN2-U showed strong responses in young leaves and weak responses in flowers. Levels of the two intron-containing KIN transcripts were markedly increased in young leaves when plants were exposed to cold and heat stress. Both of them showed stronger responses to ABA treatment and cold stress than that to heat stress. CbKIN1-U and CbKIN2-U share similar gene expression profiles in development and in response to exposure to different stresses, suggesting that they probably play similar biological roles in C. bursa-pastoris.

Comparative analysis of the variable 3' UTR and gene expression of the KIN and KIN-homologous LEA genes in Capsella bursa-pastoris.

As the crucial members of the cold-regulated (COR) gene family, KIN genes are involved in diverse abiotic stress responses in plants. In the present study, KIN genes from the widespread plant Capsella bursa-pastoris were identified and analyzed to better understand the powerful adaptation of this species. Two KIN genes were cloned and sequenced by 3' RACE. As some COR genes are homologous to LEA genes, three KIN-homologous LEA genes were also identified. We deduced the amino acid sequences of the five proteins to estimate their phylogenetic relationships, and grouped them into three subfamilies (CI, CII, and CIII). Variable 3' UTRs were found in CI, CII, and CIII genes. Using qPCR, we evaluated the transcriptional levels of the five genes in different organs and embryonic stages. Two CI genes were exclusively expressed in early embryos and flowers. The CII and CIII genes showed obvious up-regulation in young leaves after heat stress, cold stress, and ABA treatment. Two of the CI genes, however, rarely responded to those stresses in young leaves. In contrast, all five genes showed differential responses in flowers when C. bursa-pastoris plants were sprayed with ABA. Furthermore, the expression of these genes in C. bursa-pastoris was compared to that of the corresponding Arabidopsis genes, and similar gene expression profiles were found in both species. Our findings suggest that these five genes play different roles in development and the responses to abiotic stresses in C. bursa-pastoris. Key message We characterized two KIN and three KIN-homologous LEA genes, and analyzed their variable 3'UTR and organ-specific, embryo-developmental, stress-induced gene expression in Capsella bursa-pastoris.

Herbicidal activity of curvuiinic acid isolated from Nimbya alternantherae.

A phytotoxic compound, identified as 2-(2-acetyl-3,5-dihydroxyphenyl) acetic acid (curvulinic acid), was isolated from liquid cultures of the phytopathogenic fungus Nimbya alternantherae. The activity of curvulinic acid on seed germination and seedling growth of Capsella bursa-pastoris was evaluated. Percentage seed germination of C. bursa-pastoris was decreased with increasing concentrations of curvulinic acid. The compound had stronger inhibitory effects on root length than shoot length. At a concentration of 600 microg x mL(-1), curvulinic acid caused 73.5% and 66.7% growth inhibition on roots and shoots with IC50 values of 204.7 and 281.1 microg x mL(-1), respectively. The finding of curvulinic acid in N. alternantherae and its herbicidal activity are reported here for the first time.

Expression analysis and functional characterization of a novel cold-responsive gene CbCOR15a from Capsella bursa-pastoris.

The cold-responsive (COR) genes involved in C-repeat binding factor signaling pathway function essentially in cold acclimation of higher plants. A novel COR gene CbCOR15a from shepherd's purse (Capsella bursa-pastoris) was predicted to be a homolog of COR15 in Arabidopsis. The analysis of tissue specific expression pattern as well as characterization of the CbCOR15a promoter revealed that the expression of CbCOR15a was induced by coldness not only in leaves and stem but also in roots. Sequence analysis showed that a 909 bp promoter region of CbCOR15a contained two CRT/DRE elements, two ABRE elements, one auxin-responsive TGA-element and one MeJA-responsive CGTCA-motif. In young seedlings the expression of CbCOR15a could be apparently increased by SA, ABA, MeJA and IAA, and transiently increased by GA(3) accompanied by obvious feedback suppression. According to the altered physiological index values in tobacco under cold treatments, the overexpression of CbCOR15a significantly increased the cold tolerance of transgenic tobacco plants. It can be suggested that CbCOR15a was involved in cold response of Capsella bursa-pastoris associated with SA, ABA, MeJA, IAA and GA(3) regulation and confers enhanced cold acclimation in transgenic plants.

Modeling effects of herbicide drift on the competitive interactions between weeds.

Herbicides may drift onto road verges or natural areas adjacent to arable fields and affect nontarget plants. The effect of low doses of mecoprop-P on the competitive interactions and plant community dynamics was investigated in a model system using Capsella bursa-pastoris and Geranium dissectum as test plants. Dose-response experiments on single species showed that compared to G. dissectum, C. bursa-pastoris was more affected by mecoprop-P. Consequently, we expected that G. dissectum would outcompete C. bursa-pastoris when mecoprop-P was applied at a low dose in the competition experiment. Indeed, mecoprop-P had a significant effect on the interspecific competitive ability of both C. bursa-pastoris and G. dissectum. Our previous expectation, however, was not met: The interspecific competitive ability of both species increased significantly with the dose of the herbicide, and it was predicted that C. bursa-pastoris and G. dissectum are more likely to coexist in natural habitats with low concentrations of the herbicide compared to natural habitats with relatively high concentrations. The results from the dose-response experiments on the single species and the more laborious competition experiment approach, which is assumed to mimic the dynamics of plant communities more closely, show considerable discrepancies even though the experiments were performed at the same time and in the same greenhouse. This finding generally reduces the credibility of using single-species tests in ecological risk assessment of herbicide use.