Increased toxicity of Bacillus thuringiensis Cry3Aa against Crioceris quatuordecimpunctata,Phaedon brassicae and Colaphellus bowringi by a Tenebrio molitor cadherin fragment

BACKGROUND: Biopesticides containing Cry insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are effective against many lepidopteran pests, but there is a lack of Bt‐based pesticides for efficient control of important coleopteran pests. Based on the reported increase in Bt toxin oligomerization by a polypeptide from the Cry3Aa receptor cadherin in Tenebrio molitor (Coleoptera: Tenebrionidae), it was hypothesized that this cadherin peptide, rTmCad1p, would enhance Cry3Aa toxicity towards coleopteran larvae. To test this hypothesis, the relative toxicity of Cry3Aa, with or without rTmCad1p, against damaging chrysomelid vegetable pests of China was evaluated. RESULTS: Cry3Aa toxicity was evaluated in the spotted asparagus beetle (Crioceris quatuordecimpunctata), cabbage leaf beetle (Colaphellus bowringi) and daikon leaf beetle (Phaedon brassicae). To assess the effect of rTmCad1p on Cry3Aa toxicity, neonate larvae were fed Cry3Aa toxin alone or in combination with increasing amounts of rTmCad1p. The data demonstrated that Cry3Aa toxicity was significantly increased in all three vegetable pests, resulting in as much as a 15.3‐fold increase in larval mortality. CONCLUSION: The application of rTmCad1p to enhance Cry3Aa insecticidal activity has potential for use in increasing range and activity levels against coleopteran pests displaying low susceptibility to Bt‐based biopesticides. Copyright © 2011 Society of Chemical Industry     

对绿盲蝽具有杀虫活性Bt菌株的筛选及Cry15Aa蛋白活性的研究

Bt棉有效控制了棉田主要害虫棉铃虫(Helicoverpa armigera),然而原来处于次要地位的刺吸式口器害虫盲蝽(Heteroptera:Miridae)为害逐年加重,目前对绿盲蝽(Apolygus lucorum Meyer-Dür)有效的抗虫基因未见报道。本研究以本实验室保存的Bt杀虫基因和菌株为材料,对绿盲蝽进行杀虫活性筛选。利用本实验室先前克隆的Mtx类杀虫基因cry15Aa表达产物进行绿盲蝽杀虫活性测定,研究结果显示Cry15Aa蛋白对绿盲蝽具有一定的杀虫活性。通过杀虫活性测定,获得对绿盲蝽有效的Bt菌株20株;对这些菌株表达蛋白进行质谱鉴定,LC-MS/MS质谱鉴定结果显示,这些菌株可能含有Cry1Aa、Cry1Ab、Cry1Ac、Cry1Ae、Cry1Af、Cry1Ag、Cry1Ah、Cry1Ba、Cry1Be、Cry8Ha等十余种对鳞翅目、鞘翅目害虫有杀虫活性的蛋白片段,并且有4株Bt菌株样品中检出了Mtx类杀虫蛋白Cry15Aa的片段。但是进一步基因鉴定结果表明,这4株菌株并不含有cry15Aa全长基因,推测这些菌株中含有Cry15类的新基因。上述研究结果表明这些菌株中可能存在对盲蝽有效的新型杀虫蛋白。本研究在发现了cry15Aa基因所表达的蛋白对绿盲蝽具有杀虫活性的同时,获得了对绿盲蝽具有杀虫活性的Bt新菌株,对绿盲蝽的生物防治具有重要的意义。     

Beetle-specific Bacillus thuringiensis Cry3Aa toxin reduces larval growth and curbs reproduction in Spodoptera littoralis (Boisd.)

Application of Bacillus thuringiensis tenebrionis (Bt) and expression of the Bt protein Cry3Aa in genetically modified crops are used for targeted control of the Colorado potato beetle Leptinotarsa decemlineata (Say). The Cry3A proteins are selectively toxic for the beetles but the present study describes effects of Cry3Aa on the Egyptian armyworm, Spodoptera littoralis (Boisduval). Cry3Aa expressed in potatoes or added to an agar-base wheat-germ diet reduced the growth of S. littoralis caterpillars and the fertility of adults. The effect of 1.4 mg kg(-1) Cry3Aa in potato leaves was comparable with that of 3.3 mg kg(-1) in the diet. This difference in activity was correlated with better digestibility and higher conversion efficiency of the diet that also supported higher reproduction rate: S. littoralis grown on the potatoes reached a similar size to those on the diet but laid only 702 instead of 1077 eggs per female. Cry3Aa consumption reduced body growth as a consequence of lower food intake without significantly affecting food digestibility and the conversion efficiency of nutrients. The 11% and 5% body weight reductions caused by 1.4 mg kg(-1) Cry3Aa in potatoes and 3.3 mg kg(-1) in the diet, respectively, were associated with 74% and 65% reduction in the number of progeny; S littoralis grown on a diet with 9.1 mg kg(-1) Cry3Aa were 10% smaller and produced no viable progeny. These data suggest that the curtailment of reproduction was not caused by a general shortage of nutrient reserves but by a more direct Cry3Aa effect on the reproduction process.     

Efficacy of layer treatment with methoprene for control of Rhyzopertha dominica (Coleoptera: Bostrychidae) on wheat, rice and maize

BACKGROUND: Insect growth regulators are promising alternatives to traditional pesticides in stored grain. The efficacy of the juvenile hormone analogue methoprene was evaluated as a layer treatment in a laboratory experiment for control of Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae) in wheat, rice and maize. RESULTS: Adults of R. dominica were placed in vials containing 33, 26 and 29 g (to a depth of 6.5 cm) of wheat, rice and maize, respectively, that was entirely or partially treated with 1, 5 or 10 mg kg^?1 methoprene. In wheat and rice, the layer treatments were not as effective as the whole‐grain treatment, but there was decreased progeny production as the application rate increased. However, on maize the partial treatments were as effective as the whole‐grain treatment at 5 and 10 mg kg^?1. CONCLUSIONS: The results suggest that partial layer treatments with methoprene can be used to control R. dominica on maize but may not be effective for control of this species on wheat and rice. Copyright © 2011 Society of Chemical Industry     

PTD-Cry3Aa原核融合表达产物对甘薯小象甲的活性研究

为探讨TAT PTD对苏云金杆菌鞘翅目特效基因CryAa是否具有协同作用,本试验在实现了融合基因TAT PTD-Cry3Aa大肠杆菌原核表达的基础上,以甘薯小象甲为生测对象测定了原核表达产物PTD-Cry3Aa与Cry3Aa的毒力。结果表明,用大肠杆菌表达的Cry3A和PTD-Cry3Aa包涵体初提物均对甘薯小象甲成虫和幼虫有杀虫活性,且表现出一定毒力增效作用,对成虫的增效倍数为2.01倍,对幼虫的增效作用更加明显,达到3.19倍。这一研究结果对于甘薯小象甲的生物防治研究以及转基因抗虫甘薯的研究具有重要的指导作用。     

Interaction of Cry1Ac toxin (Bacillus thuringiensis) and proteinase inhibitors on the growth, development, and midgut proteinase activities of the bollworm, Helicoverpa zea

Potential resistance development to Bt cotton in certain lepidopterans has prompted research to develop strategies that will preserve this environmental-friendly biotechnology. Proteinase inhibitors are potential candidates for enhancing Bt toxicity against lepidopteran pests and for expanding the spectrum of control for other insects. Interactions of Bt toxin from Bacillus thuringiensis and proteinase inhibitors were investigated by monitoring growth, development, and gut proteinase activities of the bollworm, Helicoverpa zea. Several proteinase inhibitors were combined with Bt protoxin Cry1Ac in artificial diet and fed to newly molted 3rd-instar bollworm larvae to determine effects on larval body weight and length, pupation progress, and mortality rate. Major midgut proteinase activities, including caseinase, tryptic, and chymotrypsin activities, were examined after treatment. A concentration of Bt at a level causing minimal mortality (<10%), was mixed with the following proteinase inhibitors: benzamidine, phenylmethylsulfonyl fluoride (PMSF), and N-α-tosyl-l-lysine chloromethyl ketone (TLCK). When compared with controls, the synergistic effect of Bt toxin and proteinase inhibitors caused significant decreases in mean larval weight and length over time. Midgut samples tested against the substrates azocasein, α-benzoyl-dl-arginine-p-nitroanilide (BApNA), and N-succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (SAAPFpNA) showed significant decreases in the protease activity of larvae fed Bt plus inhibitor versus control. Interaction of Bt and proteinase inhibitors significantly retarded larval growth and resulted in developmental delay and up to 20% mortality.     

Purification, characterization and inhibition studies of α-amylase of Rhyzopertha dominica

Rhyzopertha dominica causes extensive damage to stored wheat grains. α-Amylase, the major insect digestive enzyme, can be an attractive candidate to control the insect damage by inhibiting the enzyme through α-amylase inhibitors. R. dominica α-amylase (RDA) was purified to homogeneity by differential ammonium sulphate fractionation, Sephadex G-25 and Sephadex G-100 column chromatography. The homogenous α-amylase has a molecular weight of 52 kDa. The pH optima was 7.0 and temperature optima was 40 °C. Activation energy of RDA was 3.9 Kcal mol⁻¹. The enzyme showed high activity with starch, amylose and amylopectin whereas dextrins were the poor substrates. The purified enzyme was identified to be α-amylase on the basis of products formed from starch. The enzyme showed Km of 0.98 mg ml⁻¹ for starch as a substrate. Citric acid, oxalic acid, salicylic acid, HgCl₂, tannic acid and α-amylase inhibitors from wheat were inhibitors whereas; Ca²⁺ and Mg²⁺ were the activators of RDA. Ki values calculated from Dixon graphs with salicylic acid, citric acid, oxalic acid and wheat α-amylase inhibitors were 6.9, 2.6-8.2, 3.2 mM and 0.013-0.018 μM, respectively. The Lineweaver-Burk plots with different inhibitors showed mixed type inhibition. Wheat α-amylase inhibitor showed mainly competitive inhibition with some non-competitive behaviour and other inhibitors showed mainly non-competitive inhibition with some un-competitive behaviour. Feeding trials with salicylic acid, citric acid, oxalic acid and wheat α-amylase inhibitor showed significant effect of salicylic acid and oxalic acid along with wheat α-amylase inhibitor in controlling the multiplication of R. dominica.     

Lethal doses of ozone for control of all stages of internal and external feeders in stored products

BACKGROUND: Gaseous ozone (O₃) has potential for control of insects in stored grain. Previous studies have focused on freely exposed insects. Immatures of internal pests (e.g. Sitophilus spp. and most stages of Rhyzopertha dominica F.) are protected within kernels and probably require higher doses and/or longer treatment times for full control. A laboratory study determined the doses of ozone necessary for full control of freely exposed and internal stages of eleven stored‐product pest species. Test insects were three species of Sitophilus, R. dominica, Tribolium confusum Jacquelin du Val, T. castaneum Herbst, Plodia interpunctella Hübner, Sitotroga cerealella Olivier, Oryzaephilus surinamensis L., Ephestia kuehniella Zeller and Stegobium paniceum L. Insects were exposed to continuous flows of ozone in doses of 10–135 ppm and exposure times of 5–8 days. Dose‐mortality bioassays were conducted on three species of Sitophilus and P. interpunctella. RESULTS: Freely exposed stages (with a few exceptions) were controlled with 35 ppm of ozone for 6 days. Full mortality of internal stages within kernels required exposure to 135 ppm for 8 days. CONCLUSION: This study confirms that higher doses and/or longer treatment times are necessary for control of internal stages of stored‐product pests. Copyright © 2012 Society of Chemical Industry     

Efficacy of vaporised ethyl formate/carbon dioxide formulation against stored‐grain insects: effect of fumigant concentration,exposure time and two grain temperatures

BACKGROUND: The ethyl formate/carbon dioxide (CO₂) formulation Vapormate? is a rapid‐acting fumigant being developed for the control of stored‐grain insects. The effects have been investigated of concentration, exposure times of 1, 3, 24 and 72 h and two grain temperatures, 15 and 25 °C, on its efficacy against mixed‐stage cultures of Sitophilus oryzae (L.) Tribolium castaneum (Herbst) and strongly phosphine‐resistant Rhyzopertha dominica (F.) strain QRD569. RESULTS: High mortalities (≥92%) of mixed‐stage cultures of all three species were obtained when grain was fumigated with the formulation (193 g m^?3 ethyl formate) for 1 h. Complete control of R. dominica QRD569 and T. castaneum was achieved with 63 and 76 g m^?3 ethyl formate respectively, with exposure for 24 h, whereas mean mortality of S. oryzae was 86% under the same conditions. Mortalities of S. oryzae juvenile stages were significantly lower than adults under the conditions tested, which was due to pronounced tolerance of mid‐stage pupae to the fumigant. Reducing grain temperature from 25 to 15 °C had no effect on insect mortality. CONCLUSION: Ethyl formate/CO₂ formulation is highly effective against stored‐grain insects over a range of concentrations and exposure times. Efficacious fumigations were conducted in as little as 1 h, and a strongly phosphine‐resistant R. dominica strain was readily controlled with the fumigant. Copyright © 2009 CSIRO, Australia. Published by John Wiley & Sons, Ltd     

苏云金芽孢杆菌Cry8Ca2蛋白的纯化与活性的研究

cry8 Ca2基因是本实验室自行分离克隆的一种新型苏云金芽孢杆菌(Bacillus thuringiensis,Bt)杀虫晶体蛋白基因,其表达130 kDa蛋白对铜绿异丽金龟(Anomala corpulenta)和黄褐异丽金龟(A.exoleta)幼虫具有较高的活性。本研究对Cry8Ca2蛋白原毒素及胰凝乳蛋白酶消化片断的纯化条件及活性进行研究,最适的消化条件为质量比1∶20,37℃,消化1 h。通过阴离子交换层析得到纯的活性毒素蛋白。用原毒素与纯化的毒素对铜绿丽金龟幼虫进行生测,Cry8Ca2原毒素蛋白的LC₅₀为0.47μg/g土,纯化的毒素的LC50为0.08μg/g土,毒素的杀虫活性可以达到原毒素的6倍。     

四种Bt毒素蛋白对二点委夜蛾幼虫毒力测定及中肠组织病理学变化

为更好地了解苏云金芽胞杆菌Bacillus thuringiensis毒素蛋白对二点委夜蛾Athetis lepigone的毒力以及作用机理,通过饲喂含有Cry1Ac、Cry1Ab、Cry2Ab和Vip3Aa四种不同Bt毒素蛋白饲料,测定Bt毒素蛋白对二点委夜蛾幼虫的毒力,并观察取食4种毒素蛋白后幼虫中肠组织的病理学变化。结果显示,二点委夜蛾幼虫取食毒素蛋白后72 h,Cry1Ab和Cry1Ac毒素蛋白对二点委夜蛾幼虫的杀虫活性较高,校正死亡率为84.7%和76.4%;Vip3Aa和Cry2Ab毒素蛋白的毒力较弱。二点委夜蛾幼虫取食4种Bt毒素蛋白后,中肠柱状细胞微绒毛脱落,杂乱地分散在肠腔内,杯状细胞变形和腔内微绒毛脱落,线粒体和内质网等变形破裂,细胞核的核膜消失、核质凝聚和形状发生变化,经Cry1Ab和Cry1Ac毒素蛋白处理后中肠细胞的病变症状和速度明显高于Cry2Ab和Vip3Aa毒素蛋白处理。表明Cry1Ab和Cry1Ac毒素蛋白对二点委夜蛾幼虫杀虫活性较高,显著高于Cry2Ab和Vip3Aa毒素蛋白,且对其中肠细胞的破坏作用也较强。     

Capture of stored-grain Coleoptera with WB Probe II Trap: influence of grain type

The commercially available trap WB Probe II Trap have been used in laboratory to monitor insects activity in grains at 24±1°C and 70±5% r.h.. The major coleopteran pests of cereals in Italy,Oryzaephilus surinamensis (L.),Rhyzopertha dominica (F.),Sitophilus oryzae (L.) andTribolium castaneum (Herbst), were added to the grain at about 0.9 insect per kg. Two tests were prepared: in the testA insect activity was observed in spring wheatTriticum aestivum, L.; in the testB were compared in oat grainAvena sativa L., spring wheatTriticum aestivum L. and maize grainZea mays L. Significant differences in the numbers of insects collected by the traps were observed, both when compared across traps and cereal types. In all cereals considered traps trapped moreS. oryzae thanT. castaneum andO. surinamenis; the species less trapped wereR. dominica.     

Evaluation of natural diatomaceous earth deposits from south‐eastern Europe for stored‐grain protection: the effect of particle size

BACKGROUND: The use of diatomaceous earths (DEs) provides a promising alternative to the use of contact insecticides in stored‐product IPM. Geographical origin and the physical properties of a given DE may affect its insecticidal activity. In the present study, DE samples were collected from different locations of south‐eastern Europe, and their efficacy was evaluated in the laboratory against Cryptolestes ferrugineus (Stephens) (Coleoptera: Cucujiidae), Sitophilus oryzae (L.) (Coleoptera: Curculionidae) and Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae). In addition, three fractions comprising particles of different size were obtained from each DE sample and assessed with regards to their effectiveness against the above stored‐product insect pests. RESULTS: DE from the Greek region of Elassona was the most effective against C. ferrugineus and S. oryzae, whereas the DE Kolubara 518, mined in the Serbian region of Kolubara, was the most effective against R. dominica. Smaller particles were more effective than larger particles against the three tested species, although significant differences in the efficacy of fractions containing particles of 0–150 µm and particles with sizes of < 45 µm were not always recorded. CONCLUSIONS: Deposits from south‐eastern Europe appeared to be very effective against the tested species, and therefore this region should be further evaluated as a source of development of commercial products. Particle size is a physical property that should always be taken into account during the DE manufacturing process, as it can strongly influence the insecticidal action of a given product. Copyright © 2009 Society of Chemical Industry     

Extraction of allyl isothiocyanate from horseradish (Armoracia rusticana) and its fumigant insecticidal activity on four stored‐product pests of paddy

BACKGROUND: Isothiocyanates (ITCs) extracted from Armoracia rusticana Gaertn., May & Scherb. have been shown previously to have insecticidal activity. Allyl isothiocyanate (AITC), a major component of ITCs with high volatility, was therefore extracted using different methods and tested as a fumigant against four major pest species of stored products, maize weevil Sitophilus zeamais (Motsch.), lesser grain borer Rhizopertha dominica (F.), Tribolium ferrugineum (F.) and book louse Liposcelis entomophila (Enderlein). RESULTS: Whereas there was no significant difference between hydrodistillation and supercritical carbon dioxide fluid extraction in extraction rate for AITC from A. rusticana, both methods resulted in higher extraction efficiency than water extraction. AITC fumigation showed strong toxicity to the four species of stored‐product pests. Adult mortality of 100% of all four pest species, recorded after 72 h exposure to AITC fumes at an atmospheric concentration of 3 µg mL^?1, showed no significant difference from that of insects exposed to phosphine at 5 µg mL^?1, the recommended dose for phosphine. CONCLUSIONS: The results suggest good insecticidal efficacy of AITC against the four stored‐product pests, with non‐gaseous residuals on stored products. AITC obtained from A. rusticana may be an alternative to phosphine and methyl bromide against the four pest species. Copyright © 2009 Society of Chemical Industry     

Development of nanoalumina dust as insecticide against Sitophilus oryzae and Rhyzopertha dominica

This study aims to characterize and improve the insecticidal activity of nanostructured alumina dusts. To accomplish these goals, multiple solution based synthesis routes utilizing standard aluminum salt precursors were utilized to synthesize three unique types of alumina dust. These were compared with regards to morphology, particle size and surface area using electron microscopy and dynamic light scattering particle size analysis. Insect toxicity of the various dusts was assessed using two insect species that are pests of stored grain, Sitophilus oryzae and Rhyzopertha dominica. The dust synthesized using a modified glycine-nitrate combustion process consistently yielded greater mortality rates, and all dust types were more effective on S. oryzae than on R. dominica, although the difference varied across dust types. The data show that insecticidal activity is dependent on particle size, particle morphology and surface area but also indicated that minimizing particle size and maximizing surface area are not the sole dominant factors influencing efficacy. This study does however suggest that pesticide dusts can be engineered through modified synthesis to better target different insect species.     

Insecticidal efficacy of fipronil against four stored‐product insect pests: influence of commodity,dose, exposure interval,relative humidity and temperature

BACKROUND: Fipronil is an insecticidal pyrazole that is commonly used as an insecticide in field crops, urban pesticide and veterinary medicine, but there are no reports of its evaluation against stored‐product insects. Three series of laboratory bioassays were conducted to assess fipronil as a potential grain protectant against adults of Sitophilus oryzae (L.), Tribolium confusum Jacquelin du Val, Rhyzopertha dominica (F.) and Prostephanus truncatus (Horn). Factors such as dose (0.01, 0.1, 1 and 10 mg fipronil kg^?1 grain), exposure interval (24 h, 48 h, 7 days and 14 days), temperature (20, 25 and 30 °C), relative humidity (RH; 55 and 65%) and commodity (wheat, maize, barley and paddy rice) were evaluated with regard to their impact on the insecticidal activity of fipronil. Progeny production was assessed after 64 days of exposure. RESULTS: At doses of < 1 mg fipronil kg^?1 grain, efficacy of fipronil was enhanced by an increase in temperature from 20 to 25 °C against S. oryzae or R. dominica for exposures of > 48 h and against T. confusum or P. truncatus for exposures of > 24 h. A further increase in temperature from 25 to 30 °C, although it enhanced efficacy at doses of < 1 mg fipronil kg^?1 grain against S. oryzae at all exposure intervals and against T. confusum after 7 days of exposure, it was negatively associated with efficacy against R. dominica or P. truncatus for exposures of > 24 h. By contrast, increase in RH did not have a significant impact on efficacy of fipronil. Although the performance of fipronil among the tested commodities was species dependent, this substance appeared to be less effective in paddy rice than in barley, maize and wheat. Regardless of the treated commodity, progeny production of all the tested species was almost suppressed with doses higher than 0.1 mg fipronil kg^?1 grain. CONCLUSIONS: Fipronil appeared to be a very effective alternative to the existing substances in stored‐grain protection at doses equal to or higher than 1 mg fipronil kg^?1 grain, and thus these doses require further evaluation in terms of safety for the consumer and residues on stored products. Copyright © 2010 Society of Chemical Industry     

Interaction of proteinase inhibitors with Cry1Ac toxicity and the presence of 15 chymotrypsin cDNAs in the midgut of the tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae)

BACKGROUND: The potential development of resistance to Bacillus thuringiensis (Bt) cotton and surging of non‐targeted insects is a major risk in the durability of Bt plant technology. Midgut proteinases are involved in Bt activation and degradation. Proteinase inhibitors may be used to control a wide range of insects and delay Bt resistance development. Proactive action to examine proteinase inhibitors for synergistic interaction with Bt toxin and cloning of proteinase cDNAs for RNAi is necessary to make transgenic cotton more versatile and durable. RESULTS: A sublethal dose (15 ppb) of Cry1Ac, 0.5% benzamidine and 0.02% phenylmethylsulfonyl fluoride significantly suppressed midgut azocaseinase, tryptic and chymotryptic activities, and resulted in reductions in larval and pupal length and mass of Heliothis virescens. The combination of proteinase inhibitor and Bt suppressed 20–37% more larval body mass and 26–80% more enzymatic activities than the inhibitor only or Bt only. To facilitate knockdown‐resistance‐related proteinase genes, 15 midgut chymotrypsin cDNAs were sequenced. Most predicted chymotrypsins contained the conserved N‐termini IVGG, three catalytic center residues (His, Asp and Ser), substrate specificity determinant (Ser or Gly) and cysteines for disulfide bridges. These putative chymotrypsins were separated into three distinct groups, indicating the diverse proteinases evolved in this polyphagous insect. CONCLUSION: H. virescens has evolved diverse midgut proteinase genes. Proteinase inhibitors have potential insecticidal activity, and the interaction of Bt with proteinase inhibitors is desirable for enhancing Bt toxicity and delaying resistance development. Intensive sequencing of chymotrypsin cDNAs will facilitate future functional examinations of individual roles in Bt toxicity and resistance development and facilitate targeted control using RNAi and/or proteinase inhibitors. Copyright © 2012 Society of Chemical Industry     

Involvement of a vascular hypersensitive response in quantitative resistance to Ralstonia solanacearum on tomato rootstock cultivar LS‐89

Ralstonia solanacearum causes bacterial wilt disease in Solanaceae spp. Expression of the Phytophthora inhibitor protease 1 (PIP1) gene, which encodes a papain‐like extracellular cysteine protease, is induced in R. solanacearum‐inoculated stem tissues of quantitatively resistant tomato cultivar LS‐89, but not in susceptible cultivar Ponderosa. Phytophthora inhibitor protease 1 is closely related to Rcr3, which is required for the Cf‐2‐mediated hypersensitive response (HR) to the leaf mould fungus Cladosporium fulvum and manifestation of HR cell death. However, up‐regulation of PIP1 in R. solanacearum‐inoculated LS‐89 stems was not accompanied by visible HR cell death. Nevertheless, upon electron microscopic examination of inoculated stem tissues of resistant cultivar LS‐89, several aggregated materials associated with HR cell death were observed in xylem parenchyma and pith cells surrounding xylem vessels. In addition, the accumulation of electron‐dense substances was observed within the xylem vessel lumen of inoculated stems. Moreover, when the leaves of LS‐89 or Ponderosa were infiltrated with 10⁶ cells mL^?1 R. solanacearum, cell death appeared in LS‐89 at 18 and 24 h after infiltration. The proliferation of bacteria in the infiltrated leaf tissues of LS‐89 was suppressed to approximately 10–30% of that in Ponderosa, and expression of the defence‐related gene PR‐2 and HR marker gene hsr203J was induced in the infiltrated tissues. These results indicated that the response of LS‐89 is a true HR, and induction of vascular HR in xylem parenchyma and pith cells surrounding xylem vessels seems to be associated with quantitative resistance of LS‐89 to R. solanacearum.     

Resistance to quinclorac and ALS-inhibitor herbicides in Galium spurium is conferred by two distinct genes

Classical Mendelian experiments were conducted to determine the genetics and inheritance of quinclorac and acetolactate synthase (ALS)‐inhibitor resistance in a biotype of Galium spurium. Plants were screened with the formulated product of either quinclorac or the ALS‐inhibitor, thifensulfuron, at the field dose of 125 or 6 g active ingredient (a.i.) ha^?1 respectively. Segregation in the F₂ generation indicated that quinclorac resistance was a single, recessive nuclear trait, based on a 1 : 3 segregation ratio [resistant : susceptible (R : S)]. Resistance to ALS inhibitors was due to a single, dominant nuclear trait, segregating in the F₂ generation in a 3 : 1 ratio (R : S). The genetic models were confirmed by herbicide screens of F₁ and backcrosses between the F₁ and the S parent. F₂ plants that survived quinclorac treatment set seed and the resulting F₃ progeny were screened with either herbicide. Quinclorac‐treated F₃ plants segregated in a 1 : 0 ratio (R : S), hence F₂ progenitors were homozygous for quinclorac resistance. In contrast, F₃ progeny segregated into three ratios: 1 : 0, 3 : 1 and 0 : 1 (R : S) in response to ALS‐inhibitor treatment. This segregation pattern indicates that their F₂ parents were either homozygous or heterozygous for ALS‐inhibitor resistance. Therefore, there were clearly two distinct resistance mechanisms encoded by two genes that were not tightly linked as demonstrated by segregation patterns of the F₃.