Detection of cyclodiene insecticide resistance-associated mutations by single-stranded conformational polymorphism analysis

Cyclodiene insecticide resistance is associated with replacements of a single amino acid within the putative lining of a δ-aminobutyric acid (GABA)-gated chloride ion channel gene Resistance to dieldrin (Rdl). Only two resistance-associated amino acid replacements have been identified; alanine to serine in Drosophila melanogaster, D. simulans, Aedes aegypti, and Tribolium castaneum and alanine to glycine as a second allele in D. simulans. Here we report that single stranded conformational polymorphism (SSCP) analysis of genomic DNA, amplified by the polymerase chain reaction (PCR) for exon 7 of the Rdl gene, can be used to genotype strains or individuals of all of these insects. This technique also appears simultaneously to distinguish between D. melanogaster and D. simulans, sibling species only reliably identifiable by examination of male genitalia. The relative advantages of this genotyping technique against other PCR-mediated techniques in monitoring for insecticide resistance are discussed.     

帚枝霉属内生真菌激发子蛋白SbES诱导辣椒抗棒孢叶斑病作用机理

为明确帚枝霉属Sarocladium内生生防真菌HND5菌株外泌激发子蛋白SbES的诱导辣椒抗病作用机理,通过构建SbES蛋白的毕赤酵母Pichia pastoris重组蛋白表达菌株,利用纯化后的SbES重组蛋白处理辣椒植株,检测辣椒对棒孢叶斑病的抗性,以及相关抗病反应与抗病基因表达的变化。结果表明,0.1 mg/mL SbES重组蛋白可有效诱导辣椒产生对棒孢叶斑病的抗性,可激发辣椒叶片活性氧爆发、微过敏反应和胼胝质积累等抗病反应;并能有效提高辣椒叶片中与活性氧爆发、过敏性反应、胼胝质合成和植保素合成等抗病反应相关基因,以及水杨酸、茉莉酸和乙烯信号传导关键基因的表达。推测帚枝霉属内生真菌激发子蛋白SbES可通过激活多种抗病信号传导途径来激发辣椒产生对棒孢叶斑病的抗性。     

Molecular basis of resistance to acetolactate synthase-inhibiting herbicides in Sisymbrium orientale and Brassica tournefortii

Three Australian Sisymbrium orientale and one Brassica tournefortii biotypes are resistant to acetolactate synthase (ALS)-inhibiting herbicides due to their possession of an ALS enzyme with decreased sensitivity to these herbicides. Enzyme kinetic studies revealed no interbiotypic differences within species in K_m (pyruvate) (the substrate concentration at which the reaction rate is half maximal) but a greater V_max (the rate when the enzyme is fully saturated with substrate) for two of the resistant S orientale biotypes over susceptible levels. F₁ hybrids from reciprocal crosses between resistant and susceptible biotypes of S orientale showed an intermediate response to chlorsulfuron compared to the parental plants. ALS herbicide resistance in S orientale segregated in a 3:1 (resistant:susceptible) ratio in F₂ plants with a single rate of chlorsulfuron, indicating that resistance is inherited as a single, incompletely dominant nuclear gene. Two regions of the ALS structural gene known to vary in ALS-resistant biotypes were amplified and sequenced. Resistant S orientale biotypes NS01 and SS03 contained a single nucleotide substitution in Domain B, predicting a Trp (in susceptible) to Leu (in resistant) amino acid change. Two adjacent nucleotide substitutions (CC T to AT T) predicting a Pro (in susceptible) to Ile (in resistant) change in the primary amino acid sequence were identified in Domain A of resistant S orientale biotype SS01. Likewise, a single nucleotide substitution at the same site in the resistant B tournefortii biotype predicts a Pro (in susceptible) to Ala (in resistant) substitution. No other interbiotypic nucleotide differences predicted amino acid changes in the sequenced regions, suggesting that the amino acid substitutions reported above are responsible for resistance to ALS-inhibiting herbicides in the respective biotypes. © 1999 Society of Chemical Industry     

A functional XopAG homologue in Xanthomonas fuscans pv. aurantifolii strain C limits host range

Citrus canker is caused by two Xanthomonas species, Xanthomonas citri, which has become the primary pathogen where citrus canker occurs (type A citrus canker, Xc‐A), and X. fuscans pv. aurantifolii (Xfa), which consists of strains B and C. The B strain is less pathogenic than the A strain, but produces symptoms in all citrus species. The C‐type cankers only infect Key lime (Citrus aurantifolia) and produce a hypersensitive reaction (HR) in grapefruit (Citrus paradisi) leaves. An avirulence gene, avrGf2, was identified in a C strain that was responsible for the HR in grapefruit. AvrGf2 is a member of XopAG effector family and shares 45% identity at amino acid level with another member of the same family, AvrGf1 from strain Xc‐A^w, which was previously shown to elicit an HR in grapefruit. AvrGf2 shares sequence identity with other XopAG effectors present in Xanthomonas vasculorum, Xanthomonas campestris pv. musacearum and Pseudomonas syringae pv. tomato. Mutagenesis of avrGf2 in C strain resulted in a compatible reaction in grapefruit. There was no observable effect on virulence when Xc‐A transconjugants containing either avirulence gene were inoculated on Key lime. Expression of avrGf1 or avrGf2 in Xc‐A resulted in a similar phenotype following infiltration into grapefruit leaves, although the avrGf2 transconjugant elicited a faster HR and lower populations than the transconjugant containing avrGf1. Also, it was shown that all Xfa‐B strains tested contain a transposon in avrGf2 that helps to explain the differences in host range between B and C strains.     

Survey of mutations of a histidine kinase gene BcOS1 in dicarboximide-resistant field isolates of Botrytis cinerea

Previously, we cloned a putative osmosensing histidine kinase gene (BcOS1) and revealed that a single amino acid substitution, isoleucine to serine at codon 365, conferred dicarboximide resistance in field isolates of Botrytis cinerea. This point mutation (type I) occurred within the restriction enzyme TaqI site of the wild-type BcOS1 gene. Thus, a procedure was developed for detecting the type I mutation of the BcOS1 gene using a polymerase chain reaction (PCR) in combination with restriction fragment-length polymorphism (RFLP). Diagnosis by PCR-RFLP was conducted on the 105 isolates isolated from 26 fields in Japan. All dicarboximide-sensitive isolates (49 isolates) had the wild-type BcOS1 gene, and the 43 isolates with the type I mutation were resistant to dicarboximides without exception. These data indicate that dicarboximide-resistant isolates with type I mutation are widespread throughout Japan. However, other types of dicarboximide resistance were detected among isolates from Osaka; among the 24 resistant isolates from Osaka, 12 had the BcOS1 gene without the type I mutation. BcOS1 gene sequencing of these resistant isolates classified them into two groups, type II and type III. The type II isolates have three amino acid substitutions within BcOS1p (³⁶⁸Val to Phe, ³⁶⁹Gln to His, and ⁴⁴⁷Thr to Ser). The type III isolates have two amino acid substitutions within BcOS1p (³⁶⁹Gln to Pro and ³⁷³Asn to Ser). These amino acid changes are located on the amino acid repeat domain in BcOS1p. The three types of resistant isolates were all moderately resistant to dicarboximides without significant osmotic sensitivity, and their pathogenicity on cucumber leaves was also very similar to that of the wild-type isolate.     

Distribution of herbicide‐resistant acetyl‐coenzyme A carboxylase alleles in Lolium rigidum across grain cropping areas of South Australia

Resistance to the acetyl‐coenzyme A carboxylase (ACCase)‐inhibiting herbicides in Lolium rigidum is widespread in grain cropping areas of South Australia. To better understand the occurrence and spread of resistance to these herbicides and how it has changed with time, the carboxyl transferase (CT) domain of the ACCase gene from resistant L. rigidum plants, collected from both random surveys of the mid‐north of Southern Australia over 10 years as well as stratified surveys in individual fields, was sequenced and target site mutations characterised. Amino acid substitutions occurring as a consequence of these target site mutations, at seven positions in the ACCase gene previously correlated with herbicide resistance, were identified in c. 80% of resistant individuals, indicating target site mutation is a common mechanism of resistance in L. rigidum to this herbicide mode of action. Individuals containing multiple amino acid substitutions (two, and in two cases, three substitutions) were also found. Substitutions at position 2041 occurred at the highest frequency in all years of the large area survey, while substitutions at position 2078 were most common in the single farm analysis. This study has shown that target site mutations leading to amino acid substitutions in ACCase of L. rigidum are widespread across South Australia and that these mutations have likely evolved independently in different locations. The results indicate that seed movement, both within and between fields, may contribute to the spread of resistance in a single field. However, over a large area, the independent appearance and selection of target site mutations conferring resistance through herbicide use is the most important factor.     

Fusaric acid: phytotoxicity and in vitro production by Fusarium oxysporum f.sp. lilii,the causal agent of basal rot in lilies

The production of phytotoxins byFusarium oxysporum f.sp.lilii, a pathogen causing bulb and scale rot in lilies, was investigated. To determine the toxic activity of culture filtrate, a bioassay with in vitro grown scale bulblets or with callus was set up. The fungus produces toxic components in different culture media. The highest toxicity was observed in Czapek Dox medium. HPLC and GC/MS analyses revealed the presence of fusaric acid at toxic concentrations in this medium. The production of fusaric acid in time coincides with the increase of toxic activity in the culture filtrate. It is concluded that at least part of this toxicity is due to fusaric acid.     

葡萄对霜霉病抗性机理初探

经田间自然鉴定、接种鉴定及室内离体接种鉴定结果表明：葡萄对霜霉病抗性与品种叶内的含水量、可滴定酸、还原糖、淀粉、水解氨基酸、游离氨基酸含量无显著相关，与叶背气孔密度、气孔开闭程度也无相关，而外施高浓度蔗糖及邻苯二酚却显著降低霜霉菌的致病力。     

Characterization of acetolactate synthase from sulfonylurea herbicide‐resistant Schoenoplectus juncoides

Ten accessions of sulfonylurea‐resistant Schoenoplectus juncoides were collected from paddy fields in Japan. In order to characterize acetolactate synthase from sulfonylurea‐resistant S. juncoides, acetolactate synthase amino acid substitutions, whole‐plant growth inhibition and acetolactate synthase enzyme inhibition were examined. Schoenoplectus juncoides has two acetolactate synthase genes (ALS1 and ALS2). The sulfonylurea‐resistant accessions harbored amino acid substitutions at Pro197 or Trp574 in either ALS1 or ALS2 (the amino acid number is standardized to the Arabidopsis thaliana sequence). The whole plants of all the sulfonylurea‐resistant accessions showed resistance to imazosulfuron. The resistance level depended on the altered amino acid residues in acetolactate synthase. The acetolactate synthase enzyme that was partially purified from all the sulfonylurea‐resistant accessions was less sensitive to imazosulfuron, compared to the susceptible accession, suggesting that the resistance is related to the altered acetolactate synthase enzyme. In addition, the concentration–response inhibition of acetolactate synthase activity by imazosulfuron in the sulfonylurea‐resistant accessions was remarkably different with the presence of an amino acid substitution in either ALS1 or ALS2. Furthermore, the concentration–response inhibition of acetolactate synthase activity in the sulfonylurea‐resistant accessions with a P197S, P197T or W574L mutation showed a double‐sigmoid curve. The regression analysis of enzyme inhibition suggested that the abundance ratio of ALS1 to ALS2 enzymes was approximately 70:30%, with a range of ±15%. Taken together, these results suggest that the resistance of sulfonylurea‐resistant accessions of S. juncoides is related to altered acetolactate synthase in either ALS1 or ALS2, although the abundance of the altered acetolactate synthase in the plants is different among the sulfonylurea‐resistant accessions.     

Occurrence of resistance‐breaking strains of Beet necrotic yellow vein virus in sugar beet in northwestern Europe and identification of a new variant of the viral pathogenicity factor P25

Rhizomania, one of the most devastating diseases in sugar beet production, is caused by Beet necrotic yellow vein virus (BNYVV) and transmitted by Polymyxa betae. Previously, disease control was possible by cultivation of sugar beet hybrids carrying a major resistance gene Rz1, which restricts virus accumulation in taproots and suppresses symptom development. Over the last few years, BNYVV strains with four RNA components have arisen, which are able to overcome Rz1‐mediated resistance. All strains described so far possess an A67V amino acid exchange within the RNA3‐encoded P25 pathogenicity factor. In this study, BNYVV was isolated from Rz1 plants, collected in the United Kingdom, the Netherlands and Germany, displaying patches of strong rhizomania symptoms. Sequencing of the coat protein and P25 gene of three isolates showed 100% nucleotide sequence identity and detected AYPR as the P25 tetrad amino acid composition. The ability of this strain to accumulate to higher levels in young plants of Rz1 resistant but not in Rz1 + Rz2 resistant genotypes was initially demonstrated in a greenhouse assay in natural field soil from the Netherlands. This strain was loaded into a virus‐free P. betae population and compared to reference strains. The AYPR strain retained its resistance‐breaking ability in the Rz1 genotypes and displayed replication at a higher rate compared to the Rz1‐resistance‐breaking P type. The strain origin is unclear and it remains speculative whether the occurrence at different geographic locations is the result of independent selection or displacement of infested soil.     

抗精噁唑禾草灵和甲基二磺隆看麦娘ACCase和ALS基因突变

为明确看麦娘Alopecurus aequalis抗性种群YL的靶标抗性机制,采用基因克隆法对看麦娘抗性和敏感种群间乙酰辅酶A羧化酶(ACCase)和乙酰乳酸合成酶(ALS)基因序列进行扩增、克隆和测序,比对二者ACCase和ALS基因序列的差异,探寻其产生抗药性突变的基因位点,同时测定该突变型抗性种群YL对不同ACCase和ALS抑制剂类除草剂的交互抗性。结果显示,与看麦娘敏感种群TL相比,抗性种群YL的ACCase基因CT区域第2 041位氨基酸由异亮氨酸(ATT)突变为天冬酰胺酸(AAT),ALS基因Domain A区域第197位氨基酸由脯氨酸(CCC)突变为精氨酸(CGC)。看麦娘抗性种群YL对ACCase抑制剂炔草酯产生了高水平抗性,抗性倍数为43.96,对高效氟吡甲禾灵和精喹禾灵产生了中等水平抗性,抗性倍数分别为18.33和15.87,对唑啉草酯、烯草酮和烯禾啶较敏感;对ALS抑制剂氟唑磺隆产生了低水平抗性,抗性倍数为8.39,对啶磺草胺和咪唑乙烟酸较敏感。表明ACCase基因第2 041位和ALS基因第197位氨基酸突变是导致看麦娘抗性种群YL对精噁唑禾草灵和甲基二磺隆同时产生抗性的重要原因之一。     

Increased intensity of tert-butoxyl radical emission in 4-chloro-2-methylphenoxyacetic acid (MCPA) synthesis

The important herbicide, 2-methyl-4-chlorophenoxyacetic acid (MCPA) was synthesized by the chlorination of 2-methylphenoxyacetic acid with tert-butyl hypochlorite in the presence of methyl N,N-dimethylglycinate as a catalyst, giving a high yield and regioselectivity. The reaction was investigated using the spin-trapping technique in electron paramagnetic resonance measurement conditions, with nitrosodurene as a spin trap. Increased intensity emission of the tert-butoxyl radical (2.9 times in relation to the starting level) was observed after the catalyst had been introduced into the reaction mixture, indicating a free radical mechanism for the reaction. © 1999 Society of Chemical Industry     

Identification of resistance to either paraquat or ALS-inhibiting herbicides in two Western Australian Hordeum leporinum biotypes

BACKGROUND: Hordeum populations are becoming increasingly difficult to control in cropping fields. Two herbicide‐resistant H. leporinum populations were identified during a random crop survey after herbicides were applied. The study aimed to determine the herbicide resistance profile of these H. leporinum biotypes to a range of herbicides used for their control. RESULTS: Based on dose–response studies, one H. leporinum population was very highly resistant to sulfosulfuron and sulfometuron (both sulfonylurea herbicides) and also displayed low‐level resistance to imazamox (an imidazolinone herbicide). Reduced sensitivity of the ALS enzyme was identified with in vitro activity assays. Gene sequence analysis revealed a proline‐to‐threonine substitution at amino acid position 197 of ALS, which is likely to be the molecular basis for resistance in this population. Herbicide screening also revealed a different H. leporinum population with resistance to the bipyridyl herbicide paraquat. CONCLUSION: This study established the first cases of (1) sulfonylurea‐to‐imidazolinone cross‐resistance and (2) field‐evolved paraquat resistance in a Hordeum species in Western Australia. Copyright © 2012 Society of Chemical Industry     

The pyrethrins and related compounds part XXXIX— structure-activity relationships of pyrethroidal esters with cyclic side chains in the alcohol component against resistant strains of housefly (Musca domestica)

Activities of a range of pyrethroidal esters, incorporating structural variations in all regions of the acid and alcohol components, have been measured against two fully characterised and homozygous resistant strains of Musca domestica L. (kdr and super-kdr). The results, limited in this paper to esters of alcohols with cyclic side chains, indicate uniform resistance to the kdr strain across the whole range of structural variations. Against the super-kdr strain, while variation in the acid component has little effect, the resistance factor is sensitive to the nature of the alcohol component, in particular on whether it contains an α-cyano substituent.     

Increased rhizosphere populations of Trichoderma asperellum strain T34 caused by secretion pattern of root exudates in tomato plants inoculated with Botrytis cinerea

Root exudates secreted from plants can modify rhizosphere microbiota by enhancing or inhibiting the growth of biological control agents (BCAs) and/or pathogens. Similarly, microorganisms can modify the secretion of plant root exudates. The aim of this study was to analyse the effect of a Botrytis cinerea leaf infection on the secretion of tomato root exudates and on the populations of the BCA Trichoderma asperellum strain T34 (T34). This study found that the secretion pattern of root exudates in tomato plants was influenced by B. cinerea infection in plant leaves. An increase in the levels of gluconic acid was observed, while levels of sucrose and inositol decreased. A decrease in the severity of B. cinerea by the induction of systemic resistance triggered by T34 was also observed. Tomato plants infected with B. cinerea maintained the populations of T34 in the roots, while populations of T34 decreased in plants not inoculated with the pathogen. Samples exposed to media containing gluconic acid (as the only carbon source or at the same concentration found in roots exudates) saw an increase in the in vitro growth of T34 compared to media without gluconic acid. In conclusion, a change in the secretion pattern of root exudates caused by B. cinerea, together with the enhanced growth of T34 in the presence of gluconic acid, indicates the existence of leaf to root communication. The result of this is enhanced populations of T34, and in turn induced disease resistance and a consequential reduction in disease severity.     

Enhanced tomato resistance to bacterial canker by application of turtle oil

Pretreatment with oil of sea turtle Caretta caretta protected tomato plants against bacterial canker caused by Clavibacter michiganensis subsp. michiganensis (Cmm). The turtle oil was ineffective in inhibiting Cmm in an agar diffusion test, suggesting a mechanism of induced resistance. Under controlled conditions in the greenhouse, turtle oil lowered the disease index and had reduced the growth of bacteria up to 50.4% by 7 days after inoculation. Applying turtle oil to the foliage of tomato plants increased per-oxidase and lipoxygenase activities. Gas chromatography analyses of turtle oil indicated the presence of polyunsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid. These fatty acids may lead to the higher activity of the enzymes and probably induced plant resistance against the pathogen.     

Differentially expressed genes in dicamba‐resistant and dicamba‐susceptible biotypes

In an ongoing effort to investigate the mechanism of auxinic herbicide resistance in Kochia scoparia (kochia), polymerase chain reaction‐based cDNA suppression subtractive hybridization was used to identify genes that are differentially expressed between dicamba‐resistant (HRd) and dicamba‐susceptible (S1) kochia biotypes in response to herbicide treatment. Both the HRd and S1 adaptor‐ligated cDNAs were used in separate hybridizations in order to generate biotype‐specific clones. A total of 710 cDNAs, representing putative differentially expressed mRNAs, were isolated and subjected to further screening. The false‐positive cDNAs were removed by conducting two colony hybridizations and at least one Northern hybridization. Differential or biotype‐specific expression was confirmed for six clones each from the HRd and S1 plants. The S1‐related genes were constitutively expressed at higher levels than in the HRd plants, but none had significant sequence similarity to known genes. Among the HRd‐related genes, HRd‐88 had 42% amino acid sequence identity to a conserved domain within thiol peptidases, which might be involved in auxin‐regulated gene expression. The constitutively expressed and inducible (by the dicamba treatment) HRd‐39 had 40% identity and 60% similarity to a domain from the Fe(II)/α‐ketoglutarate‐dependent hydroxylase superfamily. The HRd‐39 gene product had the characteristics of an enzyme that is able to detoxify dicamba via oxidative hydroxylation and thus its overexpression might confer the dicamba resistance phenotype.     

Induction of resistance against pine wilt disease caused by Bursaphelenchus xylophilus using selected pine endophytic bacteria

Pine wilt disease (PWD) is the most destructive disease threatening pine worldwide. The disease is mainly caused by the pine wood nematode, Bursaphelenchus xylophilus, which is vectored by pine sawyer longhorn-beetles, Monochamus spp. This study aimed to select resistance-inducing pine endophytic bacteria for management of PWD. To set up a defence-related genes expression pattern for screening, four chemical inducers (salicylic acid, γ-aminobutyric acid (GABA), β-aminobutyric acid and α-aminobutyric acid) were tested in vitro on pine calli and in vivo on pine seedlings. Treatment with GABA had the greatest reduction in PWD severity on pine seedlings. The pattern of defence-related gene expression in calli treated with GABA was used to select potential resistance-inducing bacterial strains. In addition, 92 bacterial strains were isolated from pine tree needles and stems and were tested for expression of defence-related genes in pine calli in vitro. Among the tested strains, 13 showed a similar pattern to GABA treatment in at least four tested defence-related genes and were selected for the seedling assay. From the seedling assay, three bacterial strains (16YSM-E48, 16YSM-P180 and 16YSM-P39) showed significant reduction in PWD severity compared to the untreated control. Moreover, among the selected strains, cell-free culture supernatant of strain 16YSM-P180 significantly reduced PWD severity in inoculated pine seedlings. The selected strains were identified based on the 16S rRNA sequence as Pseudomonas putida 16YSM-E48, Curtobacterium pusillum 16YSM-P180 and Stenotrophomonas rhizophila 16YSM-P39. These selected strains are suggested as potential alternatives for management of PWD by induction of systemic resistance.     

Pyrethroid resistance in the tomato red spider mite, Tetranychus evansi, is associated with mutation of the para-type sodium channel

BACKGROUND: The tomato red spider mite, Tetranychus evansi (Baker and Pritchard), is a serious pest of solanaceous crops in many African countries. In this study an investigation has been conducted to establish whether mutation of the para‐type sodium channel underlies pyrethroid resistance in T. evansi strains collected in Southern Malawi. RESULTS: Two T. evansi strains from Malawi showed tolerance to the organophosphate chlorpyrifos and resistance (20–40‐fold) to the pyrethroid bifenthrin, but were susceptible to two contemporary acaricides (abamectin and fenpyroximate) in insecticide bioassays. Cloning of a 3.1 kb fragment (domains IIS5 to IVS5) of the T. evansi para gene from pyrethroid‐resistant and pyrethroid‐susceptible strains revealed a single non‐synonymous mutation in the resistant strains that results in an amino acid substitution (M918T) within the domain II region of the channel. Although novel to mites, this mutation confers high levels of resistance to pyrethroids in several insect species where it has always been associated with another mutation (L1014F). This is the first report of the M918T mutation in the absence of L1014F in any arthropod species. Diagnostic tools were developed that allow sensitive detection of this mutation in individual mites. CONCLUSION: This is the first study of pyrethroid resistance in T. evansi and provides contemporary information for resistance management of this pest in Southern Malawi. Copyright © 2011 Society of Chemical Industry