Dual Activity of a Viral Lysozyme with High Efficiency for Growth Inhibition of Erwinia amylovora

ABSTRACT The lysozyme from Erwinia amylovora phage PhiEa1h was investigated for its ability to inhibit growth of bacteria and compared with the lysozyme from Escherichia coli phage T4. The assays to measure lysozyme activity included cell lysis and growth inhibition of bacteria. Bacterial strains with kanamycin resistance were not affected by lysates containing the PhiEa1h-enzyme. The titer of Micrococcus luteus but not of Erwinia amylovora was diminished by cell extracts containing T4 lysozyme. In contrast, PhiEa1h lysozyme preferentially inhibited E. amylovora, exceeding the T4 lysozyme activity at least one million-fold. Spherical cells were formed after application to E. amylovora similar to lyz-gene expression in Escherichia coli. Heating of cell extracts destroyed the murami-dase activity, but retained an antibacterial activity. Other plant-associated bacteria related to Erwinia amylovora also were inhibited for growth when cell extracts with PhiEa1h lysozyme were applied to soak pear slices and potato slices. Ooze formation and soft rot caused by E. amylovora or E. carotovora subsp. atroseptica, respectively, were strongly reduced and the PhiEa1h lysozyme was more efficient compared with extracts containing T4 lysozyme.     

Viral inclusions in monocotyledons infected by maize streak and related geminiviruses

Isolates of maize streak virus (MSV) were examined by thin-section electron microscopy in plants, assessed for characteristic features of infection and compared with other related geminiviruses infecting monocotyledons from Africa, islands in the Indian Ocean, and the Pacific Island of Vanuatu. Arrays of virus particles, often crystalline, were most often seen in the nucleus. The morphology of the nuclear crystalline arrays was characteristic of certain isolates or groups of isolates (strains). Infected nuclei could be seen in cells from the phloem parenchyma, vascular bundle sheath and mesophyll tissue, and also in epidermal guard cells of plants infected with the maize strain of MSV. The particle arrays varied in morphology from regular rows of virions forming distinctive blocks, to randomly arranged aggregates in certain areas of the nucleus. We consistently failed to find viral crystalline arrays associated with infection of panicum streak virus (PSV) and sugar cane streak virus (SSV) isolates either in these hosts or in maize. Occasionally arrays of MSV particles were found outside the nuclear envelope in physiologically active cells. Accumulations or sheets of MSV particles were seen lining the walls of some phloem companion cells. Crystalline aggregates of particles were frequently observed in the cell vacuole, after lysis of the nuclear membrane of dead cells which made up the chlorotic lesions, the typical symptom of virus infection. Virus preparations from all hosts contained typical geminate particles regardless of the morphology of the virion arrays. The effect on chloroplasts appeared to vary between isolates and this is discussed in relation to lesion colour. The arrangement of virions in the nucleus as a taxonomic character is diagnostic for MSV. Inclusions with crystalline structure found in sieve elements of infected plants were not immunogold labelled when thin sections were probed using antiserum to the virus particles.     

信息化合物对寄生峰寄主定向与定位行为的调控

本文从植物源和昆虫源信息化合物对寄生蜂寄主定向、定位行为的调控方面进行了综述，并对其在害虫生物防治中的应用进行了讨论。     

Biological Control of Sclerotium rolfsii and Verticillium dahliae by Talaromyces flavus Is Mediated by Different Mechanisms

ABSTRACT Ten wild-type strains and two benomyl-resistant mutants of Talaromyces flavus were examined for their ability to secrete the cell wall-degrading enzymes chitinase, beta-1,3-glucanase, and cellulase, to parasitize sclerotia of Sclerotium rolfsii, to reduce bean stem rot caused by S. rolfsii, and to secrete antifungal substance(s) active against Verticillium dahliae. The benomyl-resistant mutant Ben(R)TF1-R6 overproduced extracellular enzymes and exhibited enhanced antagonistic activity against S. rolfsii and V. dahliae compared to the wild-type strains and other mu tants. Correlation analyses between the extracellular enzymatic activities of different isolates of T. flavus and their ability to antagonize S. rolfsii indicated that mycoparasitism by T. flavus and biological control of S rolfsii were related to the chitinase activity of T. flavus. On the other hand, production of antifungal compounds and glucose-oxidase activity may play a role in antagonism of V. dahliae by retardation of germination and hyphal growth and melanization of newly formed microsclerotia.     

Inhibition of acetylcholinesterases by pulegone-1,2-epoxide

Toxicological observations indicate that pulegone-1,2-epoxide, isolated from the medicinal plant, poleo (Lippia stoechadifolia), is an insect neurotoxin. These observations prompted studies of the action of pulegone-1,2-epoxide on acetylcholinesterase activity. Kinetic analyses of the hydrolysis of acetylthiocholine by eel acetylcholinesterase in the presence of pulegone-1,2-epoxide shows an irreversible inhibition of the enzyme in a manner similar to that shown for carbamates. Five other monoterpenoids were similarly tested for their effects on acetylcholinesterase, and comparison of the inhibitions suggests that both the epoxy and keto groups are required for irreversible inhibition. Although pulegone-1,2-epoxide inhibits both house fly and Madagascar roach (Gromphadorhina portentosa) acetylcholinesterases in vitro, no correlation between inhibition of roach acetylcholinesterases in vivo and the onset of symptoms preceding death was shown.     

天然产物对植物病毒的抑制作用

本文简要介绍了自然界中含有病毒抑制剂的植物和微生物,以及天然产物对植物病毒产生抑制作用的有效成发和作用机理。     

15种植物的单宁提取物对烟草花叶病毒(TMV)的抑制作用

 测定了15种植物的乙醇粗提物对TMV的体外抑制作用,其中11种具有明显效果,这11种植物粗提物经水/氯仿萃取,水萃取物具有明显抑制TMV效果,而氯仿萃取物抑制效果差。水萃取物用明胶去除单宁后,基本失去抑制TMV的能力,表明单宁在其中起着重要作用。从杠板归与假槟榔种子中提取的总单宁对TMV具有明显的抑制作用;病毒粒体样品在分别加入这2种植物总单宁后,电镜下表现为聚集结合;TMV分别与这2种植物的单宁混合后,通过透析,可恢复大部分侵染力,表明单宁对病毒并无直接毒害作用;TMV外壳蛋白(CP)与单宁混合后,在蛋白质的聚丙烯酰胺凝胶电泳中表现为结合现象;单宁对TMV体内抑制复制效果不明显;单宁在接种TMV前后施用并没有降低TMV侵染力;这些结果表明这2种植物单宁对TMV抑制作用可能是与TMV-CP结合而引起病毒侵染力下降或影响病毒CP对侵染位点的识别。     

Inhibition of aflatoxin production in Aspergillus flavus by pyrazolines and pyrazoles

A new series of 1,3,5-triarylpyrazolines, obtained from 3, 4-dichloroacetophenone, were readily dehydrogenated by tetrachloro-o-benzoquinone at room temperature to the pyrazoles. The spectra and fluorescence of the compounds are discussed. The pyrazolines and pyrazoles did not significantly affect the mycelial growth of 19 fungal isolates, but at low concentrations, they inhibited aflatoxin production by Aspergillus flavus.     

格氏线虫表皮蛋白和分泌蛋白抑制昆虫免疫活性的比较

格氏线虫表皮蛋白和分泌蛋白都具有抑制昆虫免疫反应的能力,能够帮助侵染期线虫侵染寄主,但两者所含蛋白组分并不相同,从而导致蛋白活性也有所差异。本研究采用乙醇萃取和昆虫匀浆诱导,分别从侵染期的格氏线虫体表及分泌物中得到了表皮蛋白和分泌蛋白,并比较了这两者之间的异同。结果表明,无论是在昆虫体内还是体外,侵染期线虫表皮蛋白和分泌蛋白都具有抑制昆虫免疫反应的生物活性,并且分泌蛋白的活性更强。本研究为进一步研究线虫侵染与昆虫免疫间的相互关系和线虫抑制昆虫免疫反应的作用机理提供了科学佐证。     

Inhibition of calmodulin-dependent enzymes in rat brain by hexachlorocyclohexane

Ca²⁺ homeostasis is one of the major regulatory mechanisms operating in the nervous system, with calmodulin translating the Ca²⁺ message into cellular response. To check if hexachlorocyclohexane (HCH) acts as a calmodulin antagonist in the nervous system of rats, the in-vitro effect of HCH on calmodulin-dependent Ca²⁺-ATPase and cAMP-phosphodiesterase (PDE) in rat brain has been studied. In the membrane fraction from rat brain, a basal activity of Ca²⁺-ATPase was obtained in the absence of Ca²⁺. Inclusion of Ca²⁺ (1 mM) increased the enzyme activity by 70%. Further, addition of fluphenazine, a potent calmodulin antagonist, inhibited the Ca²⁺-dependent enzyme activity (IC₅₀ = 85 μM), demonstrating the calmodulin dependence of the enzyme activity. The Ca²⁺- and calmodulin-dependent Ca²⁺-ATPase was inhibited by HCH in a dose-dependent manner (IC₅₀ = 80–90 μM). Ca²⁺- and calmodulin-dependent cAMP-PDE from the cytosolic fraction of rat brain was inhibited by HCH (340 μM) by 79%. Addition of excess calmodulin reversed the inhibitory effects of HCH or fluphenazine on Ca²⁺-ATPase and cAMP-PDE, suggesting their direct interaction with calmodulin. By fluorescence interaction studies it has been shown that HCH interacts directly with calmodulin. These studies show that HCH may modulate the intracellular concentration of Ca²⁺ and cAMP, by decreasing the effectiveness of calmodulin towards its effector enzymes, resulting in an altered signal transduction in the nervous system.     

Inhibition of ergosterol biosynthesis by triadimenol in Ustilago avenae

In Ustilago avenae sporidia, following the first doubling period of about 4 h, triadimenol (2 μg ml^?1) affected sporidial multiplication more severely than other growth processes; daughter cells failed to separate from the parent sporidia resulting in chains of interconnected cells. Triadimenol incubated with the fungus for 8 h interfered neither with respiration nor with protein and nucleic acid synthesis but after 6 h the toxicant had induced a higher content of free fatty acids. Triadimenol markedly altered, both quantitatively and qualitatively, the sterols in sporidia of U. avenae. Incorporation of [¹⁴C]acetate (in the form of sodium acetate) into lipid fractions for a period of 2 h revealed that the toxicant powerfully inhibited the synthesis of the 4-demethyl sterol fraction (predominantly ergosterol), whilst the 4,4-dimethyl sterol fraction rapidly accumulated. This was confirmed by g.1.c. analysis of the sterols after 6 and 8 h incubation which showed that the amount of ergosterol, the major sterol in untreated sporidia, was diminished while simultaneously 4,4-dimethyl, 4-methyl and 14-methyl sterols increased. The accumulation of 14-methyl sterols suggests that triadimenol acts as a potent inhibitor of one of the metabolic steps involved in the demethylation at the 14-position during ergosterol biosynthesis.     

Inhibition of insect pest α-amylases by little and finger millet inhibitors

The inhibitory effect of three proteinaceous inhibitors isolated from little and finger millet was examined on gut α-amylases for four stored grain and four phytophagous insect-pests. Additionally, using native PAGE, several α-amylases isozymes were observed in all insect-pests studied. Furthermore, thermostabilities and the pH optimum for insect-pests α-amylases, which varied from acidic to alkaline, were also determined. On the other hand, proteinaceous inhibitors from little millet seeds Panicum sumatrense (LMCO3) and from finger millet (FMCO11 and FMCO13) inhibited insect-pests α-amylases with different proportions. The highest inhibition percent was recorded for LMCO3 and FMCO13 against Callosobruchus chinensis α-amylase, where the inhibition percent was approximately 70 and 50%, respectively. Furthermore, millet α-amylase inhibitors also reduced significantly digestive α-amylolytic activities of Acaea janata, C. cephalonica, Sitophilus oryzae, and Tribolium castaneum, indicating that these α-amylase inhibitors could be used toward crop insect-pests.     

利用小RNA深度测序技术从新疆葡萄上检出8种病毒

正Kreuze et al(.2009)发现病毒特异的小RNA(small RNA,sRNA)为多拷贝的重叠序列,推测可通过深度测序技术获得病毒的大量sRNA序列,经拼接和比对分析后能高效鉴定病毒种类。目前,利用sRNA深度测序技术已在作物和昆虫上发现多种病毒(Liu et al.,2011;He et al.,2015)。2013年本课题组在葡萄病害调查过程中发现,伊犁州部分地区的葡萄在种植4~5年后,叶片黄化、变小,整个植株矮化,座果     

Inhibition by aluminum of mycelial growth and of sporangial production and germination inPhytophthora infestans

Mycelial growth on clarified V8 agar of the potato late blight pathogenPhytophthora infestans was inhibited when either aluminum chloride (AlCl₃, 6 H₂O) or aluminum sulfate (Al₂(SO₄)₃, 18 H₂O) was added to the culture medium at concentrations of 2.5–100 mg.l^–1 Al³⁺. Toxicity of Al³⁺ varied among the fiveP. infestans isolates tested, but toxicity of sulfate and chloride salts was similar for a given isolate. Overall sporangial production was affected in all five isolates by both Al³⁺ forms. Al³⁺ also decreased sporangial germination at concentrations equal to or greater than 10 mg.l^–1 in two isolates. These data support the hypothesis of aluminum toxicity as a major factor in soil suppressiveness toP. infestans.     

Inhibition of seed germination and early stem elongation of Cuscuta australis by ethofumesate

Hmergence of dodder (Cuseuta australis R. Br.) was unexpecledly prevented in plois of a sugarbeet field where ethofumesate had been applied at 1.6 Kg/ha. Appropriate laboratory tesls have shown that ethofumesate is a potent inhibitor of dodder seed germination (I D₅₀= 10 μg/ml) and of early stem elongalion (ID₅₀= 73μg/ ml). Wheat. a standard susceptible species to field applications of ethofumesate, is equally susceptible to inhibition of stem elongation (ID₅₀=68 μg/ml), but it is far less susceptible to inhibition of germination (I D₅₀=188 μg/ml). Compared to propyzamide. ethofiimesate is a stronger inhibitor of germination. Metolachlor and Hercules 22234 [eihyl N-chloroacetyl-N-(2,6-diethylphenyl) aminoacetate] are also active in inhibiting dodder germination and stem elongation, but at higher concentrations than ethofumesate. Current methods of controlling dodder in sugarheets may be improved with inclusion of pre-emergence use of ethofumesate.     

The Analysis of Fruit Protection Mechanisms Provided by Reduced-Pathogenicity Mutants of Colletotrichum gloeosporioides Obtained by Restriction Enzyme Mediated Integration

ABSTRACT Colletotrichum gloeosporioides is an important postharvest pathogen that attacks ripe avocado fruit. Two reduced-pathogenicity mutants, Cg-M-142 and Cg-M-1150, previously obtained by restriction enzyme mediated integration, were used for the sequential analysis of the induction of biocontrol in avocado fruit. Plant biochemical indicators, such as H(+)-ATPase activity and levels of reactive oxygen species, phenylalanine ammonia lyase, epicatechin, and an antifungal diene, were investigated. The main difference between Cg-M-142 and Cg-M-1150 was the lack of appressorium formation by the latter. Preinoculation of avocado fruit with Cg-M-142 enhanced H(+)-ATPase activity and the production of reactive oxygen species. These early signaling events were followed by higher phenylalanine ammonia lyase activity and higher levels of epicatechin and the antifungal diene, and decay was delayed. Unlike Cg-M-142, Cg-M-1150 did not activate early signaling events related to fruit resistance. We suggest that the initiation of early signaling events affecting fruit resistance is determined by the capability of the pathogen to interact with the fruit during appressorium formation. Furthermore, the intensity of the fruit defense response determines the level of resistance during fruit storage.     

Inhibition of neurotoxic esterase and acetylcholinesterase by organophosphorus compounds in selected ectothermic vertebrates

The sensitivity of brain acetylcholinesterase and neurotoxic esterase to inhibition by several organophosphorus compounds was studied in selected ectothermic vertebrates. These enzymes are associated with organophosphorus compound acute and delayed toxicity, respectively. In addition, the susceptibility of several of these species to delayed neurotoxicity induced by organophosphorus compounds was studied. Larvae of the gray treefrog, Southern leopard frog, and narrow-mouthed toad were exposed dermally to tri-o-tolyl phosphate or phenyl saliginen cyclic phosphate (PSCP); no symptoms of delayed neurotoxicity were observed in any of these animals up to 2 weeks after metamorphosis. No symptoms of delayed neurotoxicity were seen in juvenile bullfrogs exposed to multiple ip doses of PSCP. The specific activity of neurotoxic esterase was highest in the larval bullfrog, with juvenile channel catfish and adult mosquitofish demonstrating intermediate levels. The larval Southern leopard frog, adult Northern leopard frog, juvenile green treefrog, and adult marine toad exhibited extremely low activities. The specific activity of acetylcholinesterase was highest in the juvenile channel catfish. Neurotoxic esterase in the larval bullfrog was more sensitive to organophosphate inhibition than that in either fish. PSCP was a more potent neurotoxic esterase inhibitor than leptophos-oxon. The juvenile channel catfish had the acetylcholinesterase most sensitive to organophosphate inhibition. Under the conditions tested, no evidence of in vivo sensitivity to the organophosphate-induced delayed neurotoxicity phenomenon was observed.     

Inhibition of two family 18 chitinases by various allosamidin derivatives

The inhibitory activities of several allosamidin derivatives on two family 18 chitinases, an insect enzyme from the epithelial cell line from Chironomus tentans, and a bacterial enzyme, chitinase A from Serratia marcescens, were evaluated. The following structural requirements are necessary for inhibition of the Chironomus enzyme: 1. One N-acetylallosamine residue can be omitted without impairment of enzyme inhibition. 2. At least one N-acetylallosamine sugar must be present. 3. Glucosamine can replace the allosamine moiety without a negative effect on the inhibitory activity. 4. The spatial arrangement of the allosamizoline moiety is important for inhibition. 5. If one sugar is omitted and the arrangement of the cyclitol residue is changed, the inhibitory effect is diminished further. For purified chitinase A from Serratia marcescens the arrangement of the aglycone moiety is equally important, but recognition of the sugar is different: 1. Omission of one allosamine residue decreases the inhibitory activity considerably. 2. Inhibition is improved if the remaining N-acetylallosamine is replaced by the epimer N-acetylglucosamine. Only endochitinase activity is affected, since chitin formation (up to 10^-4 M ) and N-acetylglucosaminidase activity (up to 10^-3 M ) are not impaired, at least in Chironomus cells. © 1998 SCI.     

Inhibition of ecdysis in Oncopeltus fasciatus by 2-acetylpyridine thiosemicarbazones

[1-(2-Pyridinyl)ethylidene]hydrazide of 1-pyrrolidinecarbothioic acid (AI3-63967) and seven similar thiosemicarbazones applied topically to Oncopeltus fasciatus were toxic or prevented ecdysis in fifth instars without the appearance of supernumerary nymphs. Treatments with AI3-63967 did not affect weight gain of the nymphs but delayed by 2 days the rise in hemolymph ecdysteroids and reduced their maximal titer by ca. 40%. Restoration of the ecdysteroid titer by injections of makisterone A or 20-hydroxyecdysone did not prevent the molting aberrations. Treated fourth instars exhibited incomplete ecdysis followed by death and treated adults died within 9 days post-treatment. Apparently, this group of thiosemicarbazones acts by a mechanism different from that of juvenile hormone mimics or chitin synthesis inhibitors.