Molecular basis of multiple resistance to ACCase- and ALS-inhibiting herbicides in Alopecurus japonicus from China.

Fenoxaprop-P-ethyl-resistant Alopecurus japonicus has become a recurring problem in winter wheat fields in eastern China. Growers have resorted to using mesosulfuron-methyl, an acetolactate synthase (ALS)-inhibiting herbicide, to control this weed. A single A. japonicus population (AH-15) resistant to fenoxaprop-P-ethyl and mesosulfuron-methyl was found in Anhui Province, China. The results of whole-plant dose-response experiments showed that AH-15 has evolved high-level resistance to fenoxaprop-P-ethyl (95.96-fold) and mesosulfuron-methyl (39.87-fold). It was shown via molecular analysis that resistance to both fenoxaprop-P-ethyl and mesosulfuron-methyl was due to an amino acid substitution of Ile1781 to Leu in acetyl-CoA carboxylase (ACCase) and a substitution of Trp 574 to Leu in ALS, respectively. Whole-plant bioassays indicated that the AH-15 population was resistant to the ACCase herbicides clodinafop-propargyl, clethodim, sethoxydim and pinoxaden as well as the ALS herbicides pyroxsulam, flucarbazone-Na and imazethapyr, but susceptible to the ACCase herbicide haloxyfop-R-methyl. This work reports for the first time that A. japonicus has developed resistance to ACCase- and ALS-inhibiting herbicides due to target site mutations in the ACCase and ALS genes.

Molecular basis for resistance to ACCase-inhibiting herbicides in Pseudosclerochloa kengiana populations.

Pseudosclerochloa kengiana is a troublesome annual grass weed of wheat fields in the rice-wheat double cropping areas in China. Resistance has evolved in P. kengiana under continuously selective pressure of ACCase-inhibiting herbicides. Whole-plant experiments showed that two suspected resistant populations 12-SD-12 and 12-SD-13 were highly resistant to fenoxaprop-P-ethyl (69.9- and 57.2-fold); moderately resistant to clodinafop-propargyl (5.9- and 4.1-fold) and pinoxaden (4.4- and 3.5-fold); lowly resistant to fluazifop-P-butyl (2.2- and 2.0-fold) and sethoxydim (1.8- and 1.6-fold), but were sensitive to clethodim (1.0- and 0.9-fold) and mesosulfuron-methyl (1.1- and 0.9-fold). Molecular analyses confirmed that a Trp1999 to Ser mutation was present in the resistant populations. Two dCAPS markers were also developed to positively determine the wild type Trp and mutant Ser alleles at ACCase position 1999. All 350 individual plants of 12-SD-12 population analysed were heterozygous mutants. Meanwhile, 318 mutant heterozygotes and 32 wild types were detected from the 12-SD-13 population. In addition, the analysis of plant genotype and phenotype showed that all wild type plants were killed after treatment with any one of the ACCase-inhibiting herbicides used, while individual plants carrying the W1999S mutation survived except when treated with clethodim. To our knowledge, this is the first report of pinoxaden resistance and a Trp-1999-Ser mutation in the plastid ACCase for P. kengiana.

Multiple resistance to ACCase and AHAS-inhibiting herbicides in shortawn foxtail (Alopecurus aequalis Sobol.) from China.

Shortawn foxtail (Alopecurus aequalis) is a troublesome grass weed infesting winter wheat and oilseed rape productions in China. Fenoxaprop-p-ethyl and mesosulfuron-methyl failed to control shortawn foxtail of AHSX-1 population collected from a wheat field in Shou County, Anhui province. Molecular analyses revealed that Asp2078Gly mutation of ACCase and Trp574Leu mutation of AHAS were present in plants of the AHSX-1 population. The homozygous plants were isolated and cultured until seed maturity. Whole-plant herbicide bioassays were conducted in the greenhouse using the purified seeds of F1 generation. Dose-response experiments showed that the AHSX-1 population has evolved a very high level resistance to fenoxaprop-p-ethyl (RI = 275) and mesosulfuron-methyl (RI = 788). To determine the sensitivity to other herbicides, assays were conducted at the single recommended rate of each herbicide. Based on the results, the AHSX-1 population was considered to be highly resistant to clodinafop-propargyl, pyroxsulam and flucarbazone-sodium, moderately or highly resistant to quizalofop-p-ethyl, clethodim, sethoxydim and pinoxaden, and susceptible to isoproturon and chlorotoluron. This is the first report of Asp2078Gly mutation in shortawn foxtail and the two robust dCAPS markers designed could quickly detect Asp2078 and Trp574 mutations in ACCase and AHAS gene of shortawn foxtail, respectively.

A novel Pro197Glu substitution in acetolactate synthase (ALS) confers broad-spectrum resistance across ALS inhibitors.

Water chickweed (Myosoton aquaticum L.), a competitive broadleaf weed, is widespread in wheat fields in China. Tribenuron and pyroxsulam failed to control water chickweed in the same field in Qiaotian Village in 2011 and 2012, respectively. An initial tribenuron resistance confirmation test identified a resistant population (AH02). ALS gene sequencing revealed a previously unreported substitution of Glu for Pro at amino acid position 197 in resistant individuals. A purified subpopulation (WRR04) that was individually homozygous for the Pro197Glu substitution was generated and characterized in terms of its response to different classes of ALS inhibitors. A whole-plant experiment showed that the WRR04 population exhibited broad-spectrum resistance to tribenuron (SU, 318-fold), pyrithiobac sodium (PTB, > 197-fold), pyroxsulam (TP, 81-fold), florasulam (TP, > 36-fold) and imazethapyr (IMI, 11-fold). An in vitro ALS assay confirmed that the ALS from WRR04 showed high resistance to all the tested ALS inhibitors. These results established that the Pro197Glu substitution endows broad-spectrum resistance across ALS inhibitors in water chickweed. In addition, molecular markers were developed to rapidly identify the Pro197Glu mutation.

Target-site basis for resistance to acetolactate synthase inhibitor in Water chickweed (Myosoton aquaticum L.).

Water chickweed is a widespread and competitive winter annual or biennial weed of wheat in China. One Water chickweed population (HN02) resistant to several acetolactate synthase (ALS) inhibitors was found in Henan province of China. Whole-plant bioassays showed that HN02 was high resistance to tribenuron (292.05-flod). In vitro ALS assays revealed that resistance was due to reduced sensitivity of the ALS enzyme to tribenuron. The I50 value for HN02 was 85.53 times greater respectively than that of susceptible population (SD05). This altered ALS sensitivity in the resistant population was due to a mutation in the ALS gene resulting in a Pro197 to Ser substitution. Cross-resistance experiments indicated that HN02 exhibited various resistance patterns to pyrithiobac-sodium, florasulam and pyroxsulam, without resistance to imazethapyr. This is the first report of tribenuron-resistant Water chickweed in Henan province of China, target-site based resistance was established as being due to an insensitive form of ALS, resulting from a Pro to Ser substitution at amino acid position 197 in the ALS gene.

Inhibition of Fusarium oxysporum by AgNPs biosynthesised using Cinnamomum camphora fruit extract.

Cinnamomum camphora fruit extract was used to biosynthesise silver nanoparticles (AgNPs), and the optimised synthesis system was ascertained through solution colour change and ultraviolet-visible absorption spectra. It contained 20 ml of fruit extract, 4 mM Ag nitrate, and pH 7. AgNPs obtained based on such conditions were spherical and finely dispersed, with an average size of 20.3 nm. As-synthesised AgNPs exhibited excellent antifungal effect against Fusarium oxysporum. At a dose of 400 µg/ml of AgNPs, the inhibition rate of colony growth reached 61.00% and an IC50 value of 154.39 µg/ml. In addition, the conidia germination was totally inhibited at 100 µg/ml of AgNPs. Results of this study provide a new approach for biological control of plant pathogenic fungi, and it makes that possible for developing a brand new fungistat.

Biosynthesis of AgNPs by B. maydis and its antifungal effect against Exserohilum turcicum.

In the present study, Bipolaris maydis was used to synthesise silver nanoparticles (AgNPs). Several parameters that influence the synthesis of AgNPs such as fungus age, the concentration of Ag nitrate (AgNO3), and incubation time were explored to find the optimum synthesis condition. Furthermore, the antifungal activity of AgNPs against Exserohilum turcicum was determined by measuring inhibition zone diameter, colony formation, and conidia germination. The optimal biosynthesis system included fungus age of 7 days, 8 mM AgNO3, and an incubation time of 120 h. Under these conditions, synthesised NPs were near round, and the average particle size was about 21 nm. At the experiment, the diameter of the inhibition zone reached a maximum of 8 mM AgNO3 and 72 h. In addition, the inhibition rate of colony and conidia reached 83.39 and 100%, respectively, with 200 µg/ml AgNPs. The results offer a novel pathway for phytopathogen control and make it likely to develop new eco-friendly antimicrobial.