Diverse ALS mutations and cross-and multiple-resistance to ALS and EPSPS inhibitors in flucarbazone­sodium-resistant Bromus japonicus populations from Hebei province, China.

Japanese brome (Bromus japonicus) has become one of the main weeds in wheat fields in Hebei province of China and causes a large decrease of wheat production. A total of 44 putative resistant and 2 susceptible Japanese brome populations were collected in the 2021/2022 crop season from Hebei province of China to determine resistance levels to flucarbazone­sodium and to investigate the diversity of acetolactate synthase (ALS) mutations, as well as to confirm the cross-and multiple-resistance levels to ALS and EPSPS (5-enolpyruvate shikimate-3-phosphate synthetase) inhibitors. Whole plant bioassay results showed that 15 out of 44 populations tested or 34% were resistant to flucarbazone­sodium. The resistance indices of Japanese brome to flucarbazone­sodium ranged from 43 to 1977. The resistant populations were mainly distributed in Baoding and Shijiazhuang districts, and there was only one resistant population in Langfang district. Resistant Japanese brome had diverse ALS mutations, including Pro-197-Ser, -Thr, -Arg and Asp-376-Glu. The incidence of Pro-197-Ser mutation was the highest at 68%. Application of the CYP450 inhibitor malathion suggested that CYP450 was involved in metabolic resistance in a population without an ALS mutation. The population with Pro-197-Thr mutation evolved weak cross-resistance to mesosulfuron-methyl and pyroxsulam, and it is in the process of evolving multiple-resistance to glyphosate.

Trp-574-Leu mutation and metabolic resistance by cytochrome P450 gene conferred high resistance to ALS-inhibiting herbicides in Descurainia sophia.

Descurainia sophia (flixweed) is a troublesome weed in winter wheat fields in North China. Resistant D. sophia populations with different acetolactate synthetase (ALS) mutations have been reported in recent years. In addition, metabolic resistance to ALS-inhibiting herbicides has also been identified. In this study, we collected and purified two resistant D. sophia populations (R1 and R2), which were collected from winter wheat fields where tribenuron-methyl provided no control of D. sophia at 30 g a.i. ha-1. Whole plant bioassay and ALS activity assay results showed the R1 and R2 populations had evolved high-level resistance to tribenuron-methyl and florasulam and cross-resistance to imazethapyr and pyrithiobac­sodium. The two ALS genes were cloned from the leaves of R1 and R2 populations, ALS1 (2004 bp) and ALS2 (1998 bp). A mutation of Trp 574 to Leu in ALS1 was present in both R1 and R2. ALS1 and ALS2 were cloned from R1 and R2 populations respectively and transferred into Arabidopsis thaliana. Homozygous T3 transgenic seedlings with ALS1 of R1 or R2 were resistant to ALS-inhibiting herbicides and the resistant levels were the same. Transgenic seedlings with ALS2 from R1 or R2 were susceptible to ALS-inhibiting herbicides. Treatment with cytochrome P450 inhibitor malathion decreased the resistant levels to tribenuron-methyl in R1 and R2. RNA-Seq was used to identify target cytochrome P450 genes possibly involved in resistance to ALS-inhibiting herbicides. There were five up-regulated differentially expressed cytochrome P450 genes: CYP72A15, CYP83B1, CYP81D8, CYP72A13 and CYP71A12. Among of them, CYP72A15 had the highest expression level in R1 and R2 populations. The R1 and R2 populations of D. sophia have evolved resistance to ALS-inhibiting herbicides due to Trp 574 Leu mutation in ALS1 and possibly other mechanisms. The resistant function of CYP72A15 needs further research.

Target-site mutation and enhanced metabolism endow resistance to nicosulfuron in a Digitaria sanguinalis population.

Digitaria sanguinalis is a competitive and annual grass weed that commonly infests crops across the world. In recent years, the control of D. sanguinalis by nicosulfuron has declined in Hebei Province, China. To determine the resistance mechanisms of D. sanguinalis to nicosulfuron, a population of D. sanguinalis where nicosulfuron had failed was collected from a maize field of Hebei Province, China. Whole-plant dose-response experiments demonstrated that the resistant population (HBMT-15) displayed 6.9-fold resistance to nicosulfuron compared with the susceptible population (HBMT-5). Addition of the glutathione S-transferase (GSTs) inhibitor 4-chloro-7-nitrobenzoxadiazole (NBD-Cl) significantly reduced the resistance level of the HBMT-15 population to nicosulfuron, and the GSTs activity of the HBMT-15 population was higher than the HBMT-5 population after nicosulfuron treatment. In vitro acetolactate synthase (ALS) enzyme experiments revealed that the nicosulfuron I50 value for the HBMT-15 population was 41 times higher than that of the HBMT-5 population. An Asp376 to Glu substitution in the ALS gene was identified in the HBMT-15 population. The HBMT-15 population had a moderate (2- to 4-fold) level of cross-resistance to three other ALS inhibitors (imazethapyr, pyroxsulam, and flucarbazone­sodium), but was susceptible to pyrithiobac­sodium. This study demonstrated that both an Asp376 to Glu substitution in the ALS gene and GSTs-involved metabolic resistance to ALS inhibitors coexisted in a D. sanguinalis population.