Induction of disease resistance in Norway spruce (Picea abies) by necrotizing fungi

Norway spruce trees ( Picea abies ) preinoculated with the root rot fungus Heterobasidion annosum , Nectria fuckeliana or a pathogenic strain of the blue-stain fungus Ceratocystis polonica were more efficiently protected against a subsequent massive inoculation with pathogenic C. polonica than trees pretreated with nonpathogenic C. polonica or sterile malt agar. Control trees that received no pretreatment were extensively colonized by the mass inoculation. There was a strong negative correlation between the length of the phloem necroses induced by the pretreatment inoculations and the extent of host symptoms caused by mass inoculation with pathogenic C. polonica . The degree of induced resistance in Norway spruce thus depended on the amount of host tissue destroyed by the pretreatment.     

Cytochrome P450 monooxygenase-mediated neonicotinoid resistance in the house fly Musca domestica L

Neonicotinoids play an essential role in the control of house flies Musca domestica. The development of neonicotinoid resistance was found in two field populations. 766b was 130- and 140-fold resistant to imidacloprid and 17- and 28-fold resistant to thiamethoxam in males and females, respectively. 791a was 22- and 20-fold resistant to imidacloprid and 9- and 23-fold resistant to thiamethoxam in males and females, respectively. Imidacloprid selection of 791a increased imidacloprid resistance to 75- and 150-fold in males and females, respectively, whereas selection with thiamethoxam had minimum impact. Neonicotinoid resistance was in all cases suppressed by PBO. The cytochrome P450 genes CYP6A1, CYP6D1 and CYP6D3 were constitutively over-expressed in resistant strains and CYP6D1 and CYP6D3 differentially expressed between sexes. The highest level of CYP6A1 expression was observed in both gender of the imidacloprid-selected strain after neonicotinoid exposure. CYP6D1 expression was increased after neonicotinoid exposure in resistant males. CYP6D3 expression was induced in both sexes upon neonicotinoid exposure but significantly higher in females.     

Detection of insecticide resistance-associated mutations in cat flea Rdl by TaqMan-allele specific amplification

We are interested in correlating the performance of fipronil in populations of cat fleas (Ctenocephalides felis) with potential cross-resistance conferred by point mutations in the Resistance to dieldrin gene, Rdl. Here we report the sequencing of exon 7 of the cat flea Rdl gene and the documentation of a putative resistance-associated mutation predicting the replacement of alanine302 with a serine. We describe two polymerase chain reaction (PCR) based diagnostics for the detection of this mutation. First, PCR mediated detection of a restriction endonuclease polymorphism (PCR REN) using a BsmAI site created by the resistance-associated mutation. Second, a TaqMan assay using allele specific fluorogenic probes and the TaqMan 5^′ specific nuclease. We describe how such diagnostics can be used in the diagnosis of resistance in the field and laboratory.     

Adult Drosophila melanogaster glutathione S-transferases: Effects of acute treatment with methyl parathion

The effect of acute treatment of methyl parathion (MP) on the expression of BSP/GSH-agarose purified glutathione S-transferases (GSTs) in Drosophila melanogaster was investigated. Using 2-D gel analysis of the identified Epsilon-class, only DmGSTE6 (100%) and DmGSTE7 (72%) demonstrated significant increases in expression, suggesting the possibility that both may be involved in MP metabolism. A smaller percentage increase was also observed in DmGSTD1 (18%), a known DDT dehydrochlorinase, DmGSTE3 and DmGSTE9 and a putative Epsilon-class GST, CG16936, were shown not to be responsive to the challenge. Our finding demonstrates that not all member of the Epsilon-class GST, which are known for their role in insecticide resistance, are immediately responsive to this toxic challenge.     

Molecular analysis of resistance in a deltamethrin-resistant strain of Musca domestica from China

We investigated the molecular basis of resistance in a strain of house fly (BJD) from Beijing, China. This strain showed 567-fold resistance to commonly used deltamethrin. Flies were 64-fold resistant to deltamethrin synergized by piperonyl butoxide (PBO). The 5′-flanking sequence of the cytochrome P450 gene CYP6D1 in BJD strain had a 15-bp insert as in the LPR strain. Two mutations (kdr, super-kdr) in the voltage sensitive sodium channel (VSSC) were also detected in the BJD strain. Our results showed that a combination of resistance alleles for CYP6D1 and VSSC existed in deltamethrin resistant house flies in China.     

Transformation a mutant Monochoria vaginalis acetolactate synthase (ALS) gene renders Arabidopsis thaliana resistant to ALS inhibitors

Acetolactate synthase (ALS) genes from Monochoria vaginalis resistant (R) and susceptible (S) biotypes against ALS inhibitors found in Korea revealed a single amino acid substitution of Proline (CCT), at 169th position based on the M. vaginalis ALS sequence numbering, to serine (TCT) in conserved domain A of the gene (equal to the proline 197 in Arabidopsis thaliana ALS gene sequence). A. thaliana plants transformed with the single mutated (Pro₁₆₉ to Ser) M. vaginalis ALS gene (including transit signal peptide) showed cross-resistance patterns to ALS-inhibiting herbicides, like as sulfonylurea-herbicide bensulfuron methyl (R/S factor of 9.5), imidazolinone-herbicide imazapyr (R/S factor of 5.1), and triazolopyrimidine-herbicide flumetsulam (R/S factor of 17.6) when measuring hypocotyls’ length of A. thaliana. The ALS activity from the transgenic A. thaliana plants confirmed the cross-resistance pattern to these herbicides like as R/S factor of 8.3 to bensulfuron methyl, 2.3 to imazapyr, and 13.2 to flumetsulam.     

Biochemical determination of acetylcholinesterase genotypes conferring resistance to the organophosphate insecticide chlorpyriphos in field populations of Bemisia tabaci from Benin, West Africa

Resistance to chlorpyriphos insecticide in Bemisia tabaci from a field population collected in Benin, West Africa was suggested with bioassay showing the presence of two sub-populations. Patterns of acetylcholinesterase (AChE) inhibition by the organophosphate chlorpyriphos-oxon were analyzed to estimate the number of possible genotypes with different sensitivity expected in three B. tabaci field populations collected in Benin. The analysis of inhibition patterns in these populations compared with four laboratory strains of B. tabaci using chlorpyriphos-oxon allowed the differentiation of three possible genotypes. In the reference strain SUD-S we detected two different acetylcholinesterases with different sensitivity to chlorpyriphos oxon suggesting the presence of two genes ace 1 and ace 2. The proportion of the insensitive enzyme (ace 2) was estimated to be 31%. In field populations we can detect two alleles at the same gene locus ace 1: one susceptible ace1S and one resistant ace1R. Both strains called Arizona University and Mexico-S2 have lost sensitive ace1S but the field populations from Benin clearly contained at least three genotypes confirming heterogeneous populations not completely resistant.     

亲和病原菌诱导黄瓜组织结构抗病性机制

炭疽病菌预先接种黄瓜可以诱导其产生对褐斑病的抗性.经炭疽病菌(1.0×106个孢子/mL)诱导48h后,黄瓜对褐斑病的抗性最大可达97.83%.在同种马铃薯葡萄糖液体培养基中培养,这两种病原菌的竞争力差异显著,黄瓜炭疽病菌能够获得更多的营养物质,生长旺盛,而褐斑病菌的生长则受到严重抑制.经苯胺兰染色发现,炭疽病菌诱导后黄瓜叶片中胼胝质的积累量显著增加.此外,黄瓜炭疽病菌与褐斑病菌的侵染方式存在一定的差异,前者可以通过气孔或直接穿透细胞壁侵入寄主.而后者只能通过直接穿透细胞壁侵入寄主.     

The clone of laccase gene and its potential function in cuticular penetration resistance of Culex pipiens pallens to fenvalerate

Decreased insecticides cuticular penetration, as one of resistant mechanisms in insect, has been extensively documented. Laccases, are enzymes with p-diphenol oxidase activity, was related to the cuticular tanning in insect. In this study, one laccase 2 gene (CpLac2) was cloned from Culex pipiens pallens. The CpLac2 contains an open reading frame (ORF) of 2289 bp and encodes a putative 762 amino acid protein. The deduced protein of CpLac2 was more similar to laccase 2 than other insect laccases, and shared the highest identity with laccases from the same family mosquito, Aedes aegypti and Anopheles gambiae. The developmental expression model of CpLac2 in C. pipiens pallens was measured by RT-PCR. The result showed the CpLaC2 was abundantly expressed in egg, the 4th instar larva and pupa, which suggested the role of CpLac2 for egg chorion tanning and cuticular sclerotization. Meanwhile, the expression of CpLac2 in fenvalerate-susceptible and -resistant strains of C. pipiens pallens was measured by real-time PCR. The result revealed the CpLac2 was significant higher expressed in resistant strain than in susceptible strain. The overexpression of CpLac2 in resistant strain suggested that resistance could derive from reinforcement of the cuticle, which decreased the penetration of insecticide in cuticle.     

几种代谢酶基因与柑橘全爪螨对双甲脒抗性的关系

为了明确羧酸酯酶(carboxylesterase,CarE)基因、谷胱甘肽S-转移酶(glutathione S-transferases,GST)基因和过氧化氢酶(catalase,CAT)基因与柑橘全爪螨Panonychus citri对双甲脒抗性的关系,通过BLAST检索,从柑橘全爪螨转录组数据库中对这3种代谢酶抗性相关基因进行鉴定,并采用RPKM法对双甲脒抗性品系和敏感品系代谢抗性相关基因进行表达差异分析,对差异较大的基因作定量PCR检测.基因差异性分析发现,抗性品系中有9条CarE基因、12条GST基因及6条CAT基因表达量发生上调,13条CarE基因、12条GST基因和3条CAT基因表达量发生下调;Pc29773nrt、Pcl7807nlg和Unigene31477为上调倍数最高的3个基因,其log2 ratio (RS/SS)分别为12.95、10.81、10.01.定量分析显示,Pc29773 nrt、Pcl7807nlg和Unigene31477的上调倍数分别为3.72、2.03和3.09,Pc29773 nrt和Unigene31477上调显著.研究表明柑橘全爪螨Pc29773nrt和Unigene31477上调与其对双甲脒的抗性相关.     

Resistance development in rice blast disease caused by Magnaporthe grisea to tricyclazole

Sensitivity to tricyclazole of 129 single-conidial isolates of rice blast fungus, Magnaporthe grisea, was determined. EC₅₀ values ranged from 0.06 to 1.12 mg/L with an average value of 0.46 ± 0.09 mg/L according to the detached leaf segment tests. No significant difference of sensitivity was observed between isolates from Guangdong and Jiangsu where decreased efficacy was reported, and from two other provinces where tricyclazole provided excellent disease control. In seedling tests, tricyclazole could control the most tolerant isolate GY-6 successfully with a efficacy of 81.5% at the concentration of 40 mg/L. Sensitivities of GY-6 and DY-2, the most sensitive isolate, to tricyclazole were both unstable in sub-cultured single-conidial offspring isolates, with respective mean EC₅₀ values of 5.40 ± 0.97 and 4.50 ± 0.88 mg/L calculated from seedling tests. There was no amino acid difference between them in the coding sequences of 1,3,6,8-tetrahydroxynaphthalene reductase and 1,3,8-trihydroxynaphthalene reductase. These results suggested that the decreased control reported in Guangdong and Jiangsu could not be attributed to the occurrence of resistance. When continuously “inoculated-reisolated-reinoculated” under the selection of tricyclazole in vivo, sensitivity of DY-2 decreased 10-fold after 20 generations, although the sensitivity of GY-6 did not shift significantly.     

Single nucleotide polymorphism detection at the Hypothenemus hampeiRdl gene by allele-specific PCR amplification with Tm-shift primers

The resistant Rdl allele for dieldrin insecticide was detected on the Hypothenemus hampei populations from Colombia using conventional PCR methods. Based on this sequence, a melting temperature (T_m) shift genotyping method that relies on allele-specific PCR is described for insecticide resistance-associated single nucleotide polymorphism (SNP) at the H. hampeiRdl gene. The method reported here uses GC-rich tails of unequal length attached to allele-specific primers containing 3′ terminal bases that correspond to SNP allelic variants. Specific PCR products are identified by inspection of a melting curve on a real-time PCR thermocycler using SYBR Green DNA binding dye. Resistant and susceptible alleles resulted in specific PCR products with T_m of 83.3 ± 0.1 °C and 86.0 ± 0.2 °C, respectively. The RdlT_m-shift genotyping method is a new method to identify the Rdl gene in the coffee berry borer H. hampei, the principal pest of coffee that in general show low genetic diversity and very few genetic strategies for control of this pest have been developed. The method supplies a high-throughput tool for dieldrin resistance-associated SNP diagnostic in the coffee berry borer which will be useful for resistance-management strategies and as genetic marker in the colombian insect populations for genetics research.     

香蕉中内源条斑病毒的基因型及其激活

为明确香蕉中不同基因型内源条斑病毒（endogenous Banana streak virus,eBSV）的激活及其机制,根据eBSOLV-GD（endogenous Banana streak OL virus Guangdong）的结构设计引物,采用内源条斑病毒基因型分析方法及RT-PCR检测法对其基因型、表达和激活进行了研究。结果表明,只含A基因组的香蕉不含eBSOLV-GD;含B基因组的香蕉均含eBSOLV-GD。在所检测的含B基因组的香蕉中eBSOLV-GD可分为6种基因型,部分为感染型的eBSOLV-GD。粉蕉和大蕉中eBSOLV-GD的片段Ⅱ和Ⅲ能表达,eBSOLV-GD经组织培养后可激活产生BSOLV-GD,引起香蕉发病,发病率为8.6%~18.0%,表明发病率的高低与香蕉的基因型有关,与eBSV基因型无关。     

Warfarin resistance in Rattus losea in Guangdong Province, China

Control of rodent populations is performed worldwide with coumarin derivatives, such as warfarin. After widespread use, their effect has been diminished by the rapid spread of resistant rodents. Warfarin resistance in Rattus loseas in Jiangmen and Zhanjiang City, Guangdong Province, was investigated by lethal feeding tests. Twenty-three of 30 R. loseas trapped in Jiangmen City were assayed as warfarin-resistant individuals, whereas only 1 of 30 rodents in Zhanjiang was resistant. These results emphasize the need for thorough resistance monitoring as a basis for adequate control measures to prevent the use of ineffective rodenticides in Jiangmen City. The resistance mechanism mainly involves VKORC1, the molecular target for coumarin drugs. VKORC1 mRNA expression in wild-caught resistant animals showed no difference compared with that in susceptible individuals. Mutation screening of VKORC1 was carried out and an Arg58Gly mutation was identified as the prevailing type in R. loseas from Jiangmen City, which may constitute the genetic basis of anticoagulation resistance in R. losea in this resistance region.