Identification and fine mapping of <Emphasis Type="Italic">Pi33</Emphasis>, the rice resistance gene corresponding to the Magnaporthe grisea avirulence gene <Emphasis Type="Italic">ACE1</Emphasis>

Rice blast disease is a major constraint for rice breeding. Nevertheless, the genetic basis of resistance remains poorly understood for most rice varieties, and new resistance genes remain to be identified. We identified the resistance gene corresponding to the cloned avirulence gene ACE1 using pairs of isogenic strains of Magnaporthe grisea differing only by their ACE1 allele. This resistance gene was mapped on the short arm of rice chromosome 8 using progenies from the crosses IR64 (resistant) × Azucena (susceptible) and Azucena × Bala (resistant). The isogenic strains also permitted the detection of this resistance gene in several rice varieties, including the differential isogenic line C101LAC. Allelism tests permitted us to distinguish this gene from two other resistance genes [Pi11 and Pi-29(t)] that are present on the short arm of chromosome 8. Segregation analysis in F₂ populations was in agreement with the existence of a single dominant gene, designated as Pi33. Finally, Pi33 was finely mapped between two molecular markers of the rice genetic map that are separated by a distance of 1.6 cM. Detection of Pi33 in different semi-dwarf indica varieties indicated that this gene could originate from either one or a few varieties.Communicated by D.J. Mackill     

Pirimiphos-methyl resistance in two stored product mites, Acarus siro and Acarus farris, as detected by impregnated paper bioassay and esterase activity assays

The response to pirimiphos-methyl, in one strain of Acarus farris and two strains of Acarus siro, was assessed using an impregnated filter paper bioassay and by the selection of adults following exposure to pirimiphos-methyl. It was concluded that one of the strains of A. siro was resistant to pirimiphos-methyl and that a major resistance mechanism was involved. The second strain of A. siro gave a response similar to that of a laboratory strain unexposed to organophosphates and was considered to be susceptible. The A. farris strain responded to selection at the ED50 but not at the ED99, and it was concluded that a minor resistance mechanism is present in this strain. Assays of esterase activity were used to attempt to identify the biochemical mechanisms involved in the resistance detected by the bioassays. The A. farris and susceptible A. siro strains showed similar levels of esterase activity but the esterase activity of the resistant A. siro strain was significantly greater. An increase in esterase activity followed selection of both the A. farris strain and the resistant A. siro strain. An acetylcholinesterase assay showed no significant difference between the susceptible and pirimiphos-methyl selected strains of A. siro. The results suggest that esterases are involved in the resistance to pirimiphos-methyl found in A. siro and A. farris but that in A. siro, at least, other mechanisms may also be present.     

Changes in enzyme titres with age in four geographical strains of Aedes aegypti and their association with insecticide resistance

Abstract. The enzymes acetylcholinesterase, glutathione S-transferase (GST), glucose 6-phosphate dehydrogenase (G6PD), and general esterases were assayed in four strains of Aedes aegypti mosquitoes aged between 1 and 30 days. Microtitre plate methods were used to assay activity in the homogenates of individual mosquitoes. The levels of GST and G6PD declined with the age of the mosquitoes, while the activity for the other enzymes remained constant. Soluble protein content was also found to decline with mosquito age in all the strains. Insecticide bioassays showed that two strains (Trinidad and Virtudes) of Ae. aegypti were resistant to DDT, deltamethrin and malathion, whereas two other strains (Bangkok and Indian) were susceptible to all four classes of insecticides tested. Higher esterase activity levels in the resistant compared to the susceptible strains were assumed to be the cause of organophosphate resistance. The combination of DDT and deltamethrin resistance in two strains with normal GST and G6PD characteristics suggests that a kdr-type nerve insensitivity mechanism may be involved.     

Organophosphorus and carbamate resistance in the predacious miteTyphlodromus pyri due to insensitive acetylcholinesterase

The cause of parathion and propoxur resistance inTyphlodromus pyri was studied in a Dutch strain in which resistance was dependent on a semi-dominant gene. Activity of glutathione S-transferase and acetylcholinesterase and reaction rate of acetylcholinesterase with paraoxon and propoxur were measured in this resistant (R) and in a susceptible (S) strain. The R strain was 100-fold resistant to parathion and 2300-fold resistant to propoxur. A 36-fold reduction was found in rate of inhibition of acetylcholinesterase in the R strain for paraoxon, and a 14-fold reduction for propoxur. In combination with the monogenic nature of the resistance, this proves that the insensitivity of acetylcholinesterase is the cause of resistance. The rate constant of acetylcholinesterase inhibition at 25°C in the S and R strains was 1.5×10⁵ and 4.2×10³ M ^–1 min^–1 respectively for paraoxon, and 5.1×10⁴ and 3.6×10³ M ^–1 min^–1 for propoxur. There was no significant difference between the R and S strains in glutathione S-transferase activity. The R strain had a somewhat lower acetylcholinesterase activity than the S strain.     

RFLP mapping of resistance to the blackleg disease [causal agent,Leptosphaeria maculans (Desm.) Ces. et de Not.] in canola (Brassica napus L.)

We report the tagging of genes involved in blackleg resistance, present in the French cultivar Crésor of B. napus, with RFLP markers. A total of 218 cDNA probes were tested on the parental cultivars Crésor (resistant) and Westar (susceptible), and 141 polymorphic markers were used in a segregating population composed of 98 doubled-haploid lines (DH). A genetic map from this cross was constructed with 175 RFLP markers and allowed us to scan for specific chromosomal associations between response to blackleg infection and RFLP markers. Canola residues infested with virulent strains of Leptosphaeria maculans were used as inoculum and a suspension of pycnidiospores from cultures of L. maculans, including the highly virulent isolate Leroy, was sprayed to increase disease pressure. QTL mapping suggested that a single chromosomal region was responsible for resistance in each of the four environments tested. This QTL accounted for a high proportion of the variation of blackleg reaction in each of the assays. A second QTL, responsible for a small proportion of the variation of blackleg reaction, was present in one of four year-site assays. A Mendelian approach, using blackleg disease ratings for classifying DH lines as resistant or susceptible, also allowed us to map resistance in the region of the highly significant LOD scores observed in each environment by interval mapping. Results strongly support the presence of a single major gene, named LmFr ₁ controlling adult plant resistance to blackleg in spring oil-seed rape cultivar Crésor. Several RFLP markers were found associated with LmFr ₁.     

Acetylcholinesterase. Two types of modifications confer resistance to insecticide.

Quantitative and qualitative changes in acetylcholinesterase confer resistance to insecticides. We have constructed several Drosophila melanogaster strains producing various amounts of enzyme by P-mediated transformation. Toxicological analysis of these strains demonstrates that resistance to organophosphorus insecticides is correlated with the amount of acetylcholinesterase in the central nervous system. Resistance may also be qualitatively determined. Comparison of the Drosophila acetylcholinesterase gene between a resistant strain caught in the wild and a wild type susceptible strain only revealed one nucleotide transition resulting in the replacement of a phenylalanine by a tyrosine. Flies mutant for acetylcholinesterase and rescued with a minigene mutagenized for this same transition produced an altered enzyme which renders flies resistant to pesticides.     

Incidence of multiple antibiotic resistant Escherichia coli in the Bhavani River

Of 107 Escherichia coli strains isolated from the water, sediment and fish of the Bhavani River, all of which are considered potential causes of human enteric disease, 62% were resistant to more than four antibiotics. Levels of resistance to bacitracin, penicillin, and novobiocin were generally high whereas those to polymyxin-B and chloramphenicol were much lower. A high incidence of multiple antibiotic resistant E. coli was noted in all samples and the multiple antibiotic resistance index of the strains showed that 95% of the strains originated either from man or cattle.     

Linkage of acetylcholinesterase insensitivity to methyl parathion resistance inHeliothis virescens

Resistance to methyl parathion insecticide has evolved in the tobacco budworm,Heliothis virescens, and several biochemical mechanisms have been identified in various strains. Reduced sensitivity of acetylcholinesterase to inhibition by methyl paraoxon, the active metabolite of the insecticide, is controlled by a single autosomal locus,AceIn. We report thatAceIn is genetically linked to methyl parathion resistance, which is expressed as a dominant gene. Methyl parathionresistant and -susceptible strains were intercrossed and the resulting mixed colony was heterozygous atAceIn. Pair matings from the mixed colony were chosen, on the basis ofAceIn genotype only, to establish strains Ace-S and Ace-R, homozygous forAceIn ^SS andAceIn ^RR, respectively. The Ace-R strain was 15.9-fold resistant compared toAceIn ^SS, while hybrid progeny expressed 24.6-fold resistance, demonstrating dominant inheritance of resistance. When progeny of the backcross (Ace-S×Ace-R) to Ace-S were exposed to a discriminating dose of methyl parathion, 24.5% survived as predicted by the model of a single resistance gene. Survivors displayed only theAceIn ^RS genotype, demonstrating a linkage disequilibrium which was highly significant. Assuming that no other resistance genes are linked closely toAceIn, it would appear thatAceIn is a powerful gene for resistance, conferring a resistance proportional to the slower rate of inhibition in the resistant enzyme. The contribution ofAceIn to resistance relative to detoxicative genes and the possible interaction of resistance genes are discussed.     

Biochemical studies carried out on different groups ofCandida parapsilosis andCandida rhagii strains by comparing some cellular and mitochondrial activities

A comparative biochemical study was performed on some strains ofCandida rhagii and on strains belonging to different subgroups ofCandida parapsilosis. Measurements of alcohol dehydrogenase activity, resistance to drugs and occurrence of an alternative pathway enabled us to confirm the classification between several subgroups within theC. parapsilosis species.     

Mixtures of plant growth-promoting rhizobacteria for induction of systemic resistance against multiple plant diseases

Studies of induced systemic resistance using strains of plant growth-promoting rhizobacteria (PGPR) have concentrated on the use of individual PGPR as inducers against multiple diseases of a single crop. To date, few reports have examined the potential of PGPR strain mixtures to induce systemic resistance against diseases of several different plant hosts. The objective of this study was to select mixtures of compatible PGPR strains with the capacity to elicit induced systemic resistance in four hosts. The specific diseases and hosts tested in this study included: bacterial wilt of tomato (Lycopersicon esculentum) caused by Ralstonia solanacearum, anthracnose of long cayenne pepper (Capsicum annuum var. acuminatum) caused by Colletotrichum gloeosporioides, damping off of green kuang futsoi (Brassica chinensis var. parachinensis) caused by Rhizoctonia solani, and cucumber mosaic virus (CMV) on cucumber (Cucumis sativus). To examine compatibility, seven selected PGPR strains were individually tested for in vitro antibiosis against all other PGPR strains and against three of the tested pathogens (R. solanacearum, C. gloeosporioides, and R. solani). No in vitro antibiosis was observed among PGPR strains or against pathogens. Twenty-one combinations of PGPR and seven individual PGPR were tested in the greenhouse for induced resistance activity. Results indicated that four mixtures of PGPR and one individual strain treatment significantly reduced the severity of all four diseases compared to the nonbacterized control: 11 mixtures reduced CMV of cucumber, 16 mixtures reduced bacterial wilt of tomato, 18 mixtures reduced anthracnose of long cayenne pepper, and 7 mixtures reduced damping off of green kuang futsoi. Most mixtures of PGPR provided a greater disease suppression than individual PGPR strains. These results suggest that mixtures of PGPR can elicit induced systemic resistance to fungal, bacterial, and viral diseases in the four hosts tested.     

分离自母婴肠道的假小链双歧杆菌的耐药性分析

【背景】由于滥用抗生素导致细菌耐药性日益严重。对于双歧杆菌,人们往往注重其益生功能的挖掘而忽视了对其耐药性的研究,存在一定的安全隐患。【目的】检测母婴肠道中假小链双歧杆菌的耐药性,探究婴儿肠道中假小链双歧杆菌耐药性的来源。【方法】利用微量肉汤稀释法测定48株分离自母婴肠道的假小链双歧杆菌对14种抗生素的耐药性,比较分离自不同家庭母婴肠道中假小链双歧杆菌的耐药性。【结果】48株母婴肠道分离株对四环素、氯霉素、新霉素、环丙沙星100%耐药,对其余10种抗生素耐药率依次为：卡那霉素98%、利福平80%、克林霉素78%、甲氧苄啶63%、红霉素59%、庆大霉素43%、链霉素16%、万古霉素14%、氨苄西林6%、利奈唑胺2%。母婴肠道分离株的耐药性无显著差异,分离自同一家庭母婴肠道的菌株具有相似的耐药表型。【结论】分离自母婴肠道的假小链双歧杆菌对多种抗生素具有耐药性,婴儿肠道中假小链双歧杆菌的耐药性可能是由母亲肠道垂直传递而来。     

Microarray analysis of the interaction between the aphid <Emphasis Type="Italic">Rhopalosiphum padi</Emphasis> and host plants reveals both differences and similarities between susceptible and partially resistant barley lines

The bird cherry-oat aphid (Rhopalosiphum padi L.) is an important pest on cereals causing plant growth reduction without specific leaf symptoms. Breeding of barley (Hordeum vulgare L.) for R. padi resistance shows that there are several resistance genes, reducing aphid growth. To identify candidate sequences for resistance-related genes, we performed microarray analysis of gene expression after aphid infestation in two susceptible and two partially resistant barley genotypes. One of the four lines is a descendant of two of the other genotypes. There were large differences in gene induction between the four lines, indicating substantial variation in response even between closely related genotypes. Genes induced in aphid-infested tissue were mainly related to defence, primary metabolism and signalling. Only 24 genes were induced in all lines, none of them related to oxidative stress or secondary metabolism. Few genes were down-regulated, with none being common to all four lines. There were differences in aphid-induced gene regulation between resistant and susceptible lines. Results from control plants without aphids also revealed differences in constitutive gene expression between the two types of lines. Candidate sequences for induced and constitutive resistance factors have been identified, among them a proteinase inhibitor, a serine/threonine kinase and several thionins. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

大肠杆菌的耐酸机制及其改造研究进展

微生物细胞在自然环境或工业应用中经常受到酸胁迫,严重制约细胞生长性能和产物合成效率。为了在各种酸性环境中生存,耐酸细菌发展出多种保护机制来维持细胞内pH稳态,如氢离子消耗、细胞膜保护、代谢修饰等。因此,深入研究耐酸机制、改进菌株耐酸能力对于利用微生物发酵合成高附加值产品具有重要意义。作为模式微生物,大肠杆菌耐酸机制的研究较为透彻,近年来其耐酸性改造也取得了重大进展。本文主要总结了大肠杆菌的氧化或葡萄糖抑制系统(acid resistance system 1, AR1)、谷氨酸依赖型耐酸系统(acid resistance system 2, AR2)、精氨酸依赖型耐酸系统(acid resistance system 3, AR3)、赖氨酸依赖型耐酸系统(acid resistance system 4, AR4)和鸟氨酸依赖型耐酸系统(acid resistance system 5, AR5)、细胞膜保护以及生物大分子修复等方面的耐酸机制,并概述了利用传统代谢工程、全局转录工程和适应性实验室进化等方法构建大肠杆菌耐酸菌株的研究进展,同时展望了大肠杆菌耐酸机制及其改造的后续研究方向...     

Pleiotropy of insecticide resistance in the codling moth, Cydia pomonella

The codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), has developed resistance to various insecticides. Relative fitness of one susceptible strain (Sv) and two strains selected for resistance to diflubenzuron (Rt) and deltamethrin (Rv), respectively, was measured in the absence of insecticide selection pressure. Mating rate, fecundity, fertility, developmental time, fifth instar weight, and adult longevity were compared. Both resistant strains were less fecund and fertile, developed more slowly, weighed less, and had shorter life-spans than the susceptible strain. These results indicate that biological constraints are associated with insecticide resistance in the codling moth. We also found that fitness estimates of the Rv strain did not differ statistically from those of the Rt strain. Enhanced mixed-function oxidase and glutathione-S-transferase activities have been shown to be involved in insecticide resistance in both Rt and Rv strains. This suggests that the fitness cost described in both resistant strains was mainly associated to metabolic resistance. The impact of such deleterious pleiotropy of insecticide resistance in C. pomonella in terms of resistance management in the field is discussed.     

相对定量分析异烟肼、链霉素单耐药与多耐药结核分枝杆菌临床分离株的蛋白质组差异

【目的】探讨异烟肼(isoniazid,INH)、链霉素(streptomycin,SM)单耐药结核分枝杆菌(Mycobacterium tuberculosis,MTB)与INH/SM多耐药MTB蛋白质组差异。【方法】应用i TRAQ结合Nano LC-MS/MS定量蛋白质组学技术,分析临床分离INH、SM或INH/SM耐药MTB与H37Rv标准株间均表达差异蛋白;并以INH/SM耐药MTB与H37Rv比值为对照,相对定量分析单耐药与多耐药MTB蛋白表达差异倍数;运用DAVID 6.7分析差异蛋白生物功能;STITCH 5.0分析差异蛋白与INH和SM相互作用。【结果】与H37Rv标准株比较,58个蛋白在INH、SM耐药与INH/SM耐药MTB间均有表达差异,共同差异蛋白生物功能主要为氧化还原酶活性和转移酶活性;主要参与丙酸代谢信号通路。共同差异蛋白中,与INH/SM耐药MTB比较,Rv2986c和Rv1908c在INH、SM耐药MTB均表达上调1.25倍;Rv3133c和Rv0577则均表达下调0.7倍;生物信息学预测发现以上4种蛋白可直接或间接与INH、SM进行相互作用。【结论】INH、SM单耐药和INH/SM多耐药MTB蛋白表达谱有较大差异,蛋白Rv2986c、Rv1908c、Rv3133c和Rv0577表达水平及相互作用可能与INH和SM耐药有关。     

Variation of Dominance of Newly Arisen Adaptive Genes

Newly arisen adaptive alleles such as insecticide resistance genes represent a good opportunity to investigate the theories put forth to explain the molecular basis of dominance and its possible evolution. Dominance levels of insecticide resistance conferred by insensitive alleles of the acetylcholinesterase gene were analyzed in five resistant strains of the mosquito Culex pipiens. Dominance levels were found to differ between strains, varying from partial recessivity to complete dominance. This variation was not explained by differences in catalytic properties of the enzyme, since four of the five resistant strains had identical inhibition properties for the insensitive acetylcholinesterase. Among these four laboratory strains and in individuals collected from natural populations, we found a correlation between increased acetylcholinesterase activities and higher dominance levels. We propose a molecular explanation for how variation in acetylcholinesterase activity may result in variation of dominance level. We also conjecture that the four resistant strains did not differ in their amino acid sequence in the catalytically active regions of acetylcholinesterase, but that the expression of the gene was regulated by either neighboring or distant sites, thereby modifying the dominance level. Under this interpretation, dominance levels may evolve in this system, since heritable variation in acetylcholinesterase activity was found.     

Resistance to clofentezine and hexythiazox inPanonychus ulmi from apples in Australia

The ovicides clofentezine and hexythiazox have been used in Australia againstPanonychus ulmi (Koch) since 1984 and 1987, respectively. Failure of clofentezine in the field againstP. ulmi was first reported in early 1988. In 1988–89, laboratory bioassays were carried out against susceptible reference strains, and discriminating doses for clofentezine and hexythiazox established. Suspected resistant strains were tested, as were randomly collected strains.Panonychus ulmi from 23 apple orchards in eastern Australia were found to be resistant to clofentezine, and 19 of these strains were also resistant to hexythiazox. In all cases, a total of four or more ovicide sprays had been used. Of 15 samples ofP. ulmi from blocks where less than four ovicides had been used, none were resistant to either clofentezine or hexythiazox. A survey of orchards in the Orange district of New South Wales found that 30% had received four or more ovicide sprays and were likely to experience resistance problems. The control problems with clofentezine and hexythiazox means the few remaining acaricides will be subject to extreme selection pressure.     

Genetic analysis ofCochliobolus heterostrophus polyoxin-resistant mutants

Nine polyoxin-resistant mutants ofCochliobolus heterostrophus were isolated after ethyl methanesulphonate mutagenesis. All were highly resistant to polyoxin (MIC≥1,600 ppm). Crosses between the mutants and a wild-type strain revealed that the resistance trait was inherited to the offsprings in different fashions. Four of the mutant strains inherited polyoxin resistance in a 1∶1 segregation ratio, indicating that the phenotypes in these strains were due to alteration at a single locus. Allelism tests revealed four new loci,Pol1, Pol2, Pol3 andPol4, for polyoxin resistance in these mutant strains. The genes responsible for the phenotypes of the other five mutant strains were not determined, because of extremely slow growth of progenies in one cross, sterility in another cross, and inexplicable responses to polyoxin of the progenies in the other crosses. No linkage was detected between the genes for polyoxin resistance and mating type.     

Investigating the Molecular Mechanisms of Organophosphate and Pyrethroid Resistance in the Fall Armyworm Spodoptera frugiperda

The fall armyworm Spodoptera frugiperda is an economically important pest of small grain crops that occurs in all maize growing regions of the Americas. The intensive use of chemical pesticides for its control has led to the selection of resistant populations, however, to date, the molecular mechanisms underlying resistance have not been characterised. In this study the mechanisms involved in the resistance of two S. frugiperda strains collected in Brazil to chlorpyrifos (OP strain) or lambda-cyhalothrin (PYR strain) were investigated using molecular and genomic approaches. To examine the possible role of target-site insensitivity the genes encoding the organophosphate (acetylcholinesterase, AChE) and pyrethroid (voltage-gated sodium channel, VGSC) target-site proteins were PCR amplified. Sequencing of the S. frugiperda ace-1 gene identified several nucleotide changes in the OP strain when compared to a susceptible reference strain (SUS). These result in three amino acid substitutions, A201S, G227A and F290V, that have all been shown previously to confer organophosphate resistance in several other insect species. Sequencing of the gene encoding the VGSC in the PYR strain, identified mutations that result in three amino acid substitutions, T929I, L932F and L1014F, all of which have been shown previously to confer knockdown/super knockdown-type resistance in several arthropod species. To investigate the possible role of metabolic detoxification in the resistant phenotype of the OP and PYR stains all EST sequences available for S. frugiperda were used to design a gene-expression microarray. This was then used to compare gene expression in the resistant strains with the susceptible reference strain. Members of several gene families, previously implicated in metabolic resistance in other insects were found to be overexpressed in the resistant strains including glutathione S-transferases, cytochrome P450s and carboxylesterases. Taken together these results provide evidence that both target-site and metabolic mechanisms underlie the resistance of S. frugiperda to pyrethroids and organophosphates.     

The sensitivity of the pseudomurein-containing genus Methanobacterium to inhibitors of murein synthesis

The sensitivity to inhibitors of various steps of murein synthesis was studied with six strains of methanogenic bacteria. Four of the strains belong to the genus Methanobacterium, which contains pseudomurein in its cell walls. This polymer-as well as murein-is not present in the two control organisms, Methanosarcina barkeri and Methanospirillum hungatii, which were found to be resistant to all inhibitors of murein synthesis. The four strains of Methanobacterium were resistant to the antibiotics fosfomycin, D-cycloserine, vancomycin, penicillin G and cephalosporin C, all of which inhibit the synthesis or function of the peptide subunits of murein. On the other hand, the four strains were susceptible to bacitracin, nisin, gardimycin and enduracidin. It is therefore assumed that the biosynthesis of murein and pseudomurein, respectively, may have some reactions of the so-called lipid cycle and the polymerization of the heteroglycan strands in common.