Sulfonylurea Resistance Research Articles

Sulfonylurea resistance in Amaranthus hybridus from southern Brazil

Amaranthus hybridus is a C4 broadleaf species widely spread across Brazilian agricultural territory. Recently, several herbicide resistance reports have been documented in southern Brazil, including the reports for enolpyruvilshikymate-3-phosphate (EPSPS)- and acetolactate-synthase (ALS)- inhibitors. The objective of this study was to confirm the existence of an ALS resistant (R) A. hybridus population from Paraná state. Dose-response experiments were conducted with R and a known susceptible (S) population with herbicides from three different chemical groups of ALS inhibitors. Biomass relative to untreated control was quantified and GR50 (dose for 50% of biomass reduction), GR90 (dose for 90% of biomass reduction) and resistant index (RI) were calculated based on non-linear regression analysis. The R population was 6.9-fold resistant to chlorimuron-ethyl and 6.5-fold resistant to metsulfuron-ethyl (sulfonylureas - SUL). Additionally, the recommended rates from each herbicide was not sufficient to reach 90% control to R based on GR90 parameter estimation. There was no resistance to imazethapyr (imidazolinone - IMI) and cloransulan-methyl (triazolopyrimidine - TRI) due to the low doses of GR90 and non-significant RIs. The R A. hybridus population investigated was resistant to ALS inhibitors chlorimuron-ethyl and metsulfuron-ethyl (SUL), but susceptible to IMI and TRI herbicides.

Development and molecular analysis of a novel acetohydroxyacid synthase rapeseed mutant with high resistance to sulfonylurea herbicides

With the increasing promotion of simplified rapeseed cultivation in recent years, the development of cultivars with high resistance to herbicides is urgently needed. We previously developed M342, which shows sulfonylurea herbicide resistance, by targeting acetohydroxyacid synthase (AHAS), a key enzyme in branched-chain amino acid synthesis. In the present study, we used a progeny line derived from M342 for an additional round of ethyl methane sulfonate mutagenesis, yielding the novel mutant DS3, which harbored two mutations in AHAS genes and showed high sulfonylurea resistance. One mutation was the substitution Trp574Leu, as in M342, according to Arabidopsis protein sequencing. The other site was a newly recognized substitution, Pro197Leu. A KASP marker targeting Pro197Leu was developed and reliably predicted the response to sulfonylurea herbicides in the F2 population. The combination of Trp574Leu and Pro197Leu in DS3 produced a synergistic effect that greatly increased herbicide resistance. Analysis of the protein structures of AHAS1 and AHAS3 in wild-type and single-gene mutant plants revealed three-dimensional protein conformational changes that could account for differences in herbicide resistance characteristics including toxicity tolerance, AHAS enzyme activity, and AHAS gene expression.

Synergistic mutations of two rapeseed AHAS genes confer high resistance to sulfonylurea herbicides for weed control.

A double mutant 5N of rapeseed was obtained with a synergistic effect of high resistance to sulfonylurea herbicide. Excellent weed control was observed in Ning R201 created by 5N resources. Sulfonylurea herbicides, which inhibit acetohydroxyacid synthase (AHAS), have become the most widely used herbicides worldwide. However, weed control in rapeseed crop production remains challenging in China due to the shortage of available herbicide-resistant cultivars. In this study, we developed a rapeseed line (PN19) with sulfonylurea herbicide resistance through seed mutagenesis. Molecular analysis revealed a Trp-574-Leu mutation in BnAHAS1-2R of PN19 according to the sequence of Arabidopsis thaliana, and an allele-specific cleaved amplified polymorphic sequence marker was developed to target the point mutation. A double mutant (5N) with very high sulfonylurea resistance was then created through pyramiding two mutant genes of PN19 and M342 by molecular marker-assisted selection. Herbicide resistance identification, toxicology testing, and an in vitro enzyme activity assay of AHAS in 5N indicated that each mutant was four and eight times more resistant to sulfonylurea than M342 and PN19, respectively. Protein structure analysis of AHAS1 demonstrated that the leucine of mutant Trp-574-Leu destroyed the original π-plane stacking effect of the local region for tribenuron-methyl binding, leading to herbicide tolerance. Isobole graph analysis showed a significant synergistic effect of the combination of two mutant genes in 5N for improved tolerance to sulfonylurea herbicides. Finally, we bred rapeseed variety Ning R201 using 5N herbicide resistance resources, and observed excellent weed control performance. Together, these results demonstrate the practical value of 5N application for optimizing and simplifying rapeseed cultivation in China.

Development of herbicides for paddy rice in Japan

The history of the development of herbicides for mechanized paddy rice production in Japan can be characterized by a combination of products with several ingredients, by large availability in formulation, and by application methods for labor saving in accordance with natural and social conditions of the country, for instance, around 40% of national land located in hilly and mountainous areas, small size paddy fields consolidated in approximately 0.3 ha on average and so on. As for combination products, one‐shot herbicides that can control both annual and perennial weeds, including grasses, sedges and broadleaved weeds, mainly with sulfonylureas have been a major means of rice production since the 1980s. One‐shot herbicides have been improved by using newly developed chemicals with excellent herbicidal efficacy, such as acetolactate synthase (ALS), 4‐hydroxyphenylpyruvate dioxygenase (4‐HPPD), protoporphyrinogen oxidase (PPO) and very‐long‐chain fatty acid elongase (VLCFAE) inhibitors, and by combining ingredients that are effective against sulfonylurea‐resistant (SU‐R) biotypes of lowland weeds. The latest type of one‐shot herbicides can control noxious species such as Eleocharis kuroguwai as well as other ordinal species. Regarding herbicide formulation, “1 kg granule,” “Jumbo,” “Flowable,” “Diffusion granule” and so on have been developed to save farmers the troubles of applying herbicides. As for application methods, “at‐transplanting application,” “at‐irrigation inlet application” and utilization of radio‐controlled helicopters or boats were put into practical use as labor‐saving technology. As a result, farmers were spared the severe hand‐weeding work under blazing heat during the summer season. Japan Association for Advancement of Phyto‐Regulators (JAPR) have promoted and contributed to the development of herbicides through collaboration with agrochemical companies and research organizations since it was founded in 1964. In this paper, I explain the development progress of herbicide using materials on the mode of action, the trend of one‐shot herbicides, the feature of a labor‐saving formulation and the working hours for weed management in rice production.

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.

Basis of ACCase and ALS inhibitor resistance in Hordeum glaucum Steud.

Acetyl coenzyme-A carboxylase (ACCase) and/or acetolactate synthase (ALS) inhibitor resistance has been identified by herbicide resistance screening in eight populations obtained from cropping regions of South Australia. This study aimed to quantify the level of resistance and characterise the molecular basis of resistance to ACCase and ALS inhibitors in these H. glaucum populations. H. glaucum populations from the Upper-North region were highly resistant (resistance index RI > 12) to the aryloxyphenoxypropionate (APP) herbicides quizalofop and haloxyfop and less resistant (RI = 2-12) to cyclohexanedione (CHD) herbicide clethodim, and some Mid-North populations had a low level of resistance (RI = 2-6) to the sulfonylurea (SU) herbicide mesosulfuron. Gene sequencing confirmed the presence of Ile-1781-Leu, Ile-2041-Asn and Gly-2096-Ala mutations in the ACCase gene, with no mutation found in the ALS gene. The use of the known metabolic inhibitor malathion in combination with mesosulfuron enhanced the activity of this herbicide. These populations were also susceptible to SU herbicide sulfometuron. This study has documented APP-to-CHD herbicide cross-resistance, the first case of ACCase inhibitor resistance due to Ile-2041-Asn mutation, and characterised the resistance to ALS inhibitors in H. glaucum. Resistance to ACCase inhibitors is due to a target-site mutation. The reversal of SU resistance by malathion and susceptibility to sulfometuron suggests that non-target-site mechanisms confer resistance to ALS inhibitors. © 2016 Society of Chemical Industry.

Screening of cultivated and wildHelianthusspecies reveals herbicide tolerance in wild sunflowers and allelic variation atAhasl1(acetohydroxyacid synthase 1 large subunit) locus

AbstractAcetohydroxyacid synthase (AHAS) inhibiting herbicides have played a significant role in effective weed control in the cultivation of sunflower since their discovery. The development of sunflower lines and hybrids resistant to these herbicides made their post-emergence application possible. In this study, wildHelianthusspecies were screened phenotypically for imidazolinone (IMI) and sulfonylurea (SU) resistance by herbicide spray and for allelic variations atAhasl1(Ahas1 large subunit) locus using simple sequence repeat and single nucleotide polymorphism markers.Helianthus praecoxaccession 1823 plants showed promising resistance to three classes of SU-based herbicides andHelianthus nuttalliiaccession NUT05 plants remained green for a longer time after imazethapyr spray. A total of 50 accessions belonging to 21 wildHelianthusspecies of different ploidy levels were analysed for allelic variation along with some parents of commercial sunflower hybrids. None of the wild species showed the resistant allele (Ahasl1-1) similar to SCG101 (IMI-resistant line), although it is present in some of the parental lines of hybrids. However, the parental lines havingAhasl1-1-type allele failed to survive the field dose of imazethapyr spray. Inter-species and inter-accessional allelic variation could be observed among the species. PRA1823 and NUT05 showed repeat length variations atAhasl1locus. Sequencing of full lengthAhasl1gene from both these accessions did not reveal any resistance mutations in the protein sequences. The molecular basis of the phenotypes identified in this study could be explored further and utilized in breeding programmes for imparting herbicide resistance in cultivated hybrids across sunflower growing regions of the world.

Exploitation of sulfonylurea resistance marker and non-homologous end joining mutants for functional analysis in Zymoseptoria tritici

The lack of techniques for rapid assembly of gene deletion vectors, paucity of selectable marker genes available for genetic manipulation and low frequency of homologous recombination are major constraints in construction of gene deletion mutants in Zymoseptoria tritici. To address these issues, we have constructed ternary vectors for Agrobacterium tumefaciens mediated transformation of Z. tritici, which enable the single step assembly of multiple fragments via yeast recombinational cloning. The sulfonylurea resistance gene, which is a mutated allele of the Magnaporthe oryzae ILV2 gene, was established as a new dominant selectable marker for Z. tritici. To increase the frequency of homologous recombination, we have constructed Z. tritici strains deficient in the non-homologous end joining pathway of DNA double stranded break repair by inactivating the KU70 and KU80 genes. Targeted gene deletion frequency increased to more than 85% in both Z. tritici ku70 and ku80 null strains, compared to ⩽10% seen in the wild type parental strain IPO323. The in vitro growth and in planta pathogenicity of the Z. tritici ku70 and ku80 null strains were comparable to strain IPO323. Together these molecular tools add significantly to the platform available for genomic analysis through targeted gene deletion or promoter replacements and will facilitate large-scale functional characterization projects in Z. tritici.

Evaluation of Tribenuron-Methyl on Sulfonylurea-Resistant Lettuce Germplasm

A sulfonylurea (SU) herbicide resistance allele discovered in prickly lettuce was previously transferred to domestic lettuce with the cultivar name ‘ID-BR1’. ID-BR1 was acquired, and the SU resistance allele was transferred through traditional breeding methods to five common commercial lettuce types: butterhead, crisphead, green leaf, red leaf, and romaine. Field trials were conducted at Salinas, CA during 2011 and 2012 to evaluate POST applications of tribenuron-methyl (tribenuron) on SU-susceptible and SU-resistant lettuce types. Treatments included a nontreated control, pronamide applied PRE at 1,340 g ai ha−1, and tribenuron at 4, 9, and 17 g ai ha−1applied POST. Data collected were: weed control, crop injury estimates (0 = safe, 100 = dead), stand counts, and lettuce yields. Injury to lettuce from tribenuron was high in SU-susceptible lettuce types and low in SU-resistant accessions. With the exceptions of a romaine lettuce line that still may have some susceptible individuals, tribenuron did not reduce yield of SU-resistant lettuce, but did reduce the yield of SU-susceptible lettuce. Suppression of weeds such as common groundsel and annual sowthistle was higher with tribenuron than with pronamide. Tribenuron should be considered for registration as a lettuce herbicide for SU-resistant lettuce to improve current weed management options for that crop.

Sulfonylurea resistance reconstitution as a novel strategy for ILV2-specific integration in Magnaporthe oryzae

A sulfonylurea-resistant allele of the ILV2 gene encoding an acetolactate synthase from the rice-blast fungus Magnaporthe oryzae has been extensively used in fungal transformation as a dominant selectable marker that confers resistance to chlorimuron ethyl. We devised a novel strategy for site-specific integration of foreign DNA via sulfonylurea resistance reconstitution (SRR) by replacing the native ILV2 with the sulfonylurea-resistant ILV2SUR variant. In contrast to random ectopic integration, SRR-based targeted incorporation at a defined locus eliminates position/orientation effects, unnecessary mutations and/or variation in gene expression. Independent transformants derived from the same SRR construct showed consistent and reproducible fluorescent signal in M. oryzae. Furthermore, the high frequency (>95%) of ILV2-specific targeted integration via SRR circumvents the need for a deficiency in non-homologous end joining (NHEJ) pathway in the recipient strain. Unlike the split-marker technique, which is particularly suitable for targeted gene replacement, the SRR strategy should prove useful for promoter analyses, gene tagging and/or complementation analyses in filamentous fungi.

Occurrence of sulfonylurea resistance in Sagittaria trifolia, a basal monocot species, based on target‐site and non‐target‐site resistance

Sagittaria trifolia L. is one of the most serious weeds in paddy fields in Japan. Since the late 1990s, severe infestations of S. trifolia have occurred following applications of sulfonylurea herbicides in Akita prefecture. In this study, two accessions of S. trifolia, R1 and R2, were collected from paddy fields with severe infestations and their resistance profiles were determined in comparison to a susceptible accession, S1. R1 and R2 were highly resistant to bensulfuron‐methyl. R1 was also highly resistant to pyrazosulfuron‐ethyl, but R2 was susceptible. Relative to S1, R1 had an amino acid substitution at the Pro197 residue of acetolactate synthase (ALS), a well‐known mutation that confers sulfonylurea resistance, suggesting that R1 has a target‐site‐based resistance (TSR) mechanism. The sequence of the ALS gene in R2 was identical to that in S1. A Southern blot analysis indicated that there was only one copy of the ALS gene in S1 and R2. These results suggest that R2 has a non‐target‐site‐based resistance (NTSR) mechanism. R2 was moderately resistant to imazosulfuron but susceptible to thifensulfuron‐methyl. R2 and S1 were susceptible to pretilachlor, benfuresate, MCPA‐ethyl and bentazon. The results reveal the occurrence of two sulfonylurea‐resistant biotypes of S. trifolia that show different mechanisms of cross‐resistance to sulfonylureas related to TSR in R1 and NTSR in R2.

Evolution and genetics of sulfonylurea resistance in the paddy weeds of Japan

Evolution and genetics of sulfonylurea resistance in the paddy weeds of Japan

Effect of Management of Sulfonylurea Resistant Stellaria media on Barley Yield

Sulfonylureas represent one of the largest herbicide groups that have been widely used since 1980s. Their continuous use has resulted in development of sulfonylurea resistance in weeds. The aim of this research was to investigate options to manage putative sulfonylurea-resistant chickweed in barley stands and to evaluate the effect of chickweed and its management on barley yield. A field experiment was arranged as a randomized complete block design and included 14 herbicide treatments applied at two different times. Tribenuron-methyl (sulfonylurea) affected minimal control of chickweed. A bromoxynil-ioxynil (photosystem II inhibitor) mix did not control chickweed efficiently. However, nearly total control was achieved with fluroxypyr, mecoprop, and their mixtures (synthetic auxins and photosystem II inhibitors). Chickweed had no effect on barley yield whether controlled or uncontrolled. Therefore, further evaluation of the chickweed management threshold would be needed. It seems that even in the boreal region, typified by a cold climate, limited solar radiation, a very short growing season, and relatively low-intensity cropping systems, unilateral use of sulfonylureas might lead to herbicide resistance. Although resistant weed populations can be controlled with herbicides of groups other than the sulfonylureas, this represents an increasing problem when planning weed management, especially when including sulfonylurea-resistant crops.

Molecular characterization of Als1, an acetohydroxyacid synthase mutation conferring resistance to sulfonylurea herbicides in soybean

The AHAS gene family in soybean was characterized. The locus Als1 for sulfonylurea resistance was mapped and the resistant allele was characterized at the molecular level. Sulfonylurea (SU) resistance in soybean is controlled by Als1, a semi-dominant allele obtained by EMS mutagenesis over the cultivar Williams 82. The overall objective of this research was to map Als1 in the soybean genome and to determine the nucleotidic changes conferring resistance to SU. Four nucleotide sequences (GmAhas1-4) showing high homology with the Arabidopsis thaliana acetohydroxyacid synthase (AHAS, EC 4.1.3.18) gene sequence were identified by in silico analysis, PCR-amplified from the SU-resistant line BTK323STS and sequenced. Expression analysis showed that GmAhas1, located on chromosome 4 by in silico analysis, is the most expressed sequence in true leaves. F2:3 families derived from the cross between susceptible and resistant lines were evaluated for SU resistance. Mapping results indicate that the locus als1 is located on chromosome 4. Sequence comparison of GmAhas1 between BTK323STS and Williams 82 showed a single nucleotide change from cytosine to thymine at position 532. This transversion generates an amino acid change from proline to serine at position 197 (A. thaliana nomenclature) of the AHAS catalytic subunit. An allele-specific marker developed for the GmAhas1 mutant sequence cosegregated with SU resistance in the F2 population. Taking together, the mapping, expression and sequencing results indicate that the GmAhas1 sequence corresponds to the Als1 gene sequence controlling SU resistance in soybean. The molecular breeding tools described herein create the basis to speed up the identification of new mutations in soybean AHAS leading to enhanced levels of resistance to SU or to other families of AHAS inhibitor herbicides.

Microsatellite variability of sulfonylurea‐resistant and susceptible populations of Schoenoplectus juncoides (Cyperaceae) in Kinki, Japan

SummarySchoenoplectus 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.

Rapid diagnosis of sulfonylurea‐resistant Schoenoplectus juncoides [Roxb.] Palla using polymerase chain reaction–restriction fragment length polymorphism and isogene‐specific direct sequencing

Rapid diagnostic methods to detect known mutations in acetolactate synthase (ALS) genes that confer sulfonylurea (SU) resistance to Schoenoplectus juncoides were developed in this study. By using 11 SU‐resistant accessions (nine accessions with a Pro197 substitution in ALS1 or ALS2, one accession with an Asp376Glu substitution in ALS2 and one accession with a Trp574Leu substitution in ALS2), polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis for DNA fragments that were amplified simultaneously from genomic ALS1 and ALS2 and PCR–RFLP analysis for DNA fragments that were amplified from either of the genomic ALS1 or ALS2 were carried out. In each of the two PCR–RFLP analyses, a common PCR product was digested separately with the restriction enzymes, BspLI, MboI and MunI, in order to detect Pro197 substitutions, an Asp376Glu substitution and a Trp574Leu substitution, respectively. In each of the lanes where the detection of SU‐resistant substitutions was aimed, a specific band to suggest the existence of the said substitutions was observed in theoretically assumable ways. Separately, a direct sequencing method also was established, which was able to selectively sequence ALS1 or ALS2 from common templates containing both ALS1 and ALS2 by the isogene‐selective primers that were designed to anneal either of the ALS genes. It is expected that these methods could be used for the genetic analysis of SU‐resistant S. juncoides by providing rapid and accurate diagnosis.

Permanent neonatal diabetes mellitus due to KCNJ11 mutation in a Portuguese family: transition from insulin to oral sulfonylureas

Permanent neonatal diabetes mellitus (PNDM) is a rare form of diabetes diagnosed within the first 6 months of life. Heterozygous activation mutations in KCNJ11, encoding the Kir6.2 subunit of the ATP-sensitive potassium (K(ATP)) channel, which acts as a key role in insulin secretion regulation, account for about half of the cases of PNDM. The majority of the patients represent isolated cases resulting from de novo mutations. Approximately 20% have associated neurologic features: the most severe form, which includes epilepsy and developmental delay, is called developmental delay, epilepsy, and neonatal diabetes (DEND) syndrome and the milder form, with less severe developmental delay and without epilepsy, is designated intermediate DEND syndrome. Individuals with KCNJ11 mutations have been successfully transitioned from insulin to sulfonylurea (SU) therapy. Furthermore, there have been cases reported with variable improvement in neurological function following a successful switching. We describe a 12-year-old Portuguese girl with PNDM due to the previously reported R201C mutation in the KCNJ11 gene. Her medical history includes prematurity and moderate developmental delay. The mutation was inherited from her mother who has isolated PNDM. The patient was successfully transferred from insulin to SU, whereas her mother showed SU resistance. Despite good glycemic control, no improvements in the cognitive performance were verified. We present our experience in switching treatment from insulin to oral SUs in this family, and also discuss whether or not the girl's developmental delay is related with the Kir6.2 mutation. To our knowledge, this is the first Portuguese patient reported with successful transition to SU treatment.

Evaluation of novel sulfonylurea derivatives with a fused heterocyclic moiety as paddy herbicides that control sulfonylurea‐resistant weeds

Herbicidal activity and acetolactate synthase (ALS) inhibition of sulfonylurea derivatives with a fused heterocyclic moiety bonded to a sulfonyl group were investigated. Some compounds that had an imidazo[1,2‐b]pyridazine moiety substituted at the 2‐position by chlorine or methyl controlled sulfonylurea‐resistant (SU‐R) weeds and showed inhibitory activity to ALS prepared from SU‐R Schoenoplectus juncoides shoot. There was a correlation between in vitro and whole‐plant herbicidal activity of the compounds mentioned above against SU‐R Schoenoplectus juncoides. Among them 1‐(2‐chloro‐6‐propylimidazo[1,2‐b]pyridazin‐3‐ylsulfonyl)‐3‐(4,6‐dimethoxypyrimidin‐2‐yl)urea, propyrisulfuron, was selected for further evaluation. Propyrisulfuron effectively controlled paddy weeds at doses of 70 and 140 g a.i. ha−1 with good rice selectivity in a field trial.

Gene inactivation in the citrus pathogenic fungus Alternaria alternata defect at the Ku70 locus associated with non-homologous end joining

Low frequency of targeted gene disruption in filamentous fungi has been attributed to the predominance of ectopic integration that is controlled by a non-homologous end-joining (NHEJ) DNA repair mechanism. It has been shown in a number of fungi that suppression of NHEJ by inactivating the yeast Ku70 or Ku80 homolog facilitates homologous recombination, thereby enhancing the frequency of gene targeting. A Ku70 gene homolog was cloned from the necrotrophic fungus Alternaria alternata, causing citrus brown spot. The cloned gene, designated AaKu70, was independently inactivated by insertion of an acetolactate synthase gene (SUR) conferring sulfonylurea resistance or of a bacterial phosphotransferase gene (HYG) cassette conferring hygromycin resistance. The AaKu70 deficient strain reduced conidial formation and pigmentation slightly, but unchanged in toxin production and pathogenicity. Unlike many fungal species, mutation of the AaKu70 gene by inserting either SUR or HYG did not facilitate gene disruption, as tested for disruption targeting an AaAP1gene encoding a redox responsive transcription regulator or an AaHSK1 gene encoding a histidine kinase. However, a split marker approach, using truncated DNA fragments overlapping within the HYG, but not SUR, selectable marker, enhanced the frequency of AaAP1 disruption considerably in the progenitor and the ∆AaKu70 strain. Compared to the disruption targeting the AaAP1 gene, the frequency for recovery of the AaHSK1 impaired mutant was much lower, indicating an allele-dependent disruption. The present study indicates that AaKu70 has little or no effect on gene disruption and homologous integration in A. alternata.

Cross‐resistance to three frequently used sulfonylurea herbicides in populations of Apera spica‐venti from the Czech Republic

Hamouzová K, Soukup J, Jursík M, Hamouz P, Venclová V & Tůmová P (2011). Cross‐resistance to three frequently used sulfonylurea herbicides in populations of Apera spica‐venti from the Czech Republic. Weed Research51, 113–122.SummaryWhole‐plant glasshouse bioassays, enzyme assays and field experiments were conducted to examine the sulfonylurea herbicide resistance pattern in Apera spica‐venti populations from the Czech Republic. Varying, but very high levels of resistance to chlorsulfuron, sulfosulfuron and iodosulfuron were found, and cross‐resistance to all three herbicides was confirmed. In dose–response glasshouse studies, resistance factors (GR50 ratios relative to a susceptible standard) were above 230 for chlorsulfuron, 115–270 for sulfosulfuron and 27–148 for iodosulfuron. A biotype originating from Lomnice that was cross resistant to the three herbicides was also highly resistant to sulfometuron‐methyl. Concentrations of herbicides required to inhibit acetolactate synthase (ALS) enzyme activity by 50% were substantially higher (up to 4000 times) for all three resistant populations, compared with the reference susceptible population, indicating that the A. spica‐venti populations have ALS enzymes that are highly insensitive to the tested sulfonylureas. The differences in the spectra of resistance suggest that the sulfonylurea resistance mechanisms may differ among biotypes. The field experiment was designed to compare the control effect of selected sulfonylureas (chlorsulfuron and iodosulfuron), isoproturon and fenoxaprop‐P‐ethyl on ALS‐resistant populations. Sulfonylurea herbicides failed to control resistant populations, but these were well controlled with alternative mode of action herbicides, thus demonstrating practical options for weed management.