Isolation and partial characterization of an antifungal protein from the endophytic Bacillus subtilis strain EDR4

An antifungal protein E2, from the culture filtrate of the endophytic Bacillus subtilis strain EDR4 of wheat with a high activity against numerous fungal species in vitro and take-all in wheat caused by Gaeumannomyces graminis var. tritici in vivo, was purified by (NH₄)₂SO₄ precipitation, hydrophobic-interaction chromatography, anion-exchange chromatography and polyacrylamide gel electrophoresis (PAGE). The molecular mass of the protein was about 377.0 kDa determined by gel permeation chromatography (GPC) using a Superdex 200 10/300 GL pre-packed column and the pI value of the protein detected by isoelectric focusing PAGE was 6.59. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the antifungal protein showed a band with a molecular mass of 39.1 kDa, which suggest that the native protein consists of multi-subunits. The amino acid sequences of three peptides from the antifungal protein were obtained by using a nano-ESI-MS/MS (Q-TOF2) System. The protein isolated may be regarded as a new protein according to amino acid sequences of three peptides. The purified protein exhibited inhibitory activity on mycelium growth of e.g. Fusarium graminearum, Macrophoma kuwatsukai, Rhizoctonia cerealis, Fusarium oxysporum f.sp. vasinfectum, Botrytis cinerea and G. graminis var. tritici (Ggt). Scanning electron microscopy showed that hyphae of Ggt treated with the antifungal protein were severely deformed. The antifungal protein E2 exhibited ribonuclease and hemagglutinating activities as well as a trifle protease activity. However, no β-1,3-glucanase, β-1,4-glucanase, chitinase or protease inhibitory activities were detected.     

Hyper-susceptibility to deltamethrin in para DDT resistant Drosophila melanogaster

The extensively studied para gene encodes a α-subunit of the voltage-activated sodium channel in Drosophila melanogaster, which is the documented target site of DDT and pyrethroid insecticides. The para^ts-1 fruit fly line carries a recessive sex-linked insecticide-resistance trait (para^ts-1 allele) that has been defined on the basis of the behavioral phenotype of temperature-sensitive paralysis. We have determined that para^ts-1 confers hyper-susceptibility to deltamethrin in addition to the previously annotated resistance to DDT, revealing the presence of negative cross-resistance. We investigated the potential use of negative cross-resistance shifting para^ts-1 gene frequencies in D. melanogaster populations. After five generations of selection, the para^ts-1 allele, respectively, became more or less frequent whether Drosophila populations were selected with DDT or deltamethrin.     

Isolation of a novel Carica papaya α-amylase inhibitor with deleterious activity toward Callosobruchus maculatus

Cowpea (Vigna unguiculata) is a subsistence crop for small and poor farmers from Latin America and Africa. This culture is commonly damaged by cowpea weevil (Callosobruchus maculatus), which burrow into stored seeds to fed on. Due to impact of larval predation, several plant defense studies have been developed, indicating that α-amylase inhibitors are able to impede and/or reduce bruchids digestive process. In this report, a novel α-amylase inhibitor from papaya seeds (Carica papaya) with activity against cowpea weevil enzymes was purified and biochemical characterized. Peeled seeds were macerated and extracted with a 0.6 M NaCl and 0.1% HCl solution. Crude extract was precipitated with ammonium sulphate (100%). After dialyses, this rich fraction was applied onto a CM-Cellulose column and retained peak was submitted to an analytic reversed-phase column HPLC (Vydac C-18TP) yielding several peaks. Only one fraction, with molecular mass of 4562 Da, showed significant inhibitory activity against C. maculatus α-amylases. Otherwise, no inhibitory activities against mammalian α-amylases were observed. Bioassays using artificial seeds containing C. papaya α-amylase inhibitor rich fraction (0.5% and 1.0%) were also conduced showing that α-amylase inhibitors were able to increase larval mortality (50%) and also decrease insect fecundity and adult longevity. These results showed the presence of an α-amylase inhibitor from C. papaya seeds with high specificity to insect enzymes, indicating that this inhibitor probably could be used, through genetic engineering, in the construction of transgenic plants with enhanced resistance toward cowpea weevil.     

拮抗性链霉菌对大丽轮枝菌微菌核形成与萌发的影响

为探索拮抗性链霉菌对棉花黄萎病病原大丽轮枝菌Verticillium dahliae Kleb.的抑菌机制,采用菌丝生长速率法、微菌核萌发法研究了6株拮抗链霉菌无菌发酵滤液对大丽轮枝菌生长、微菌核形成与萌发的影响。链霉菌无菌发酵滤液对大丽轮枝菌菌落生长、菌核形成和微菌核萌发有明显抑制作用。其中菌株B49的抑菌效果最好,5倍稀释发酵液培养14天时对菌落生长的抑菌率达69.7%;菌株B49、D184和Act12的5倍稀释发酵液对微菌核形成的抑制率达100%;将经B49、D184和Act12发酵液处理后丧失形成微菌核能力的大丽轮枝菌菌株转接至不含发酵液的PDA培养基,连续传代至第5代,其仍然不能恢复形成微菌核的能力;微菌核在含有菌株D184 5倍稀释发酵液的培养基上培养168 h时,萌发率仅为38.3%。     

葡萄应答霜霉病过程中多磷酸肌醇激酶基因与过氧化氢的作用机制

以对霜霉病不同抗性的葡萄品种左优红和霞多丽为材料,利用分子生物学和植物生理学试验手段,结合药理学试验,探讨葡萄在应答霜霉病过程中葡萄多磷酸肌醇激酶基因（VvIPK2）和H2O2的作用机制。接种霜霉病菌后15 h葡萄叶片VvIPK2表达量是正常水平的12倍,接种后3 hH2O2含量达最大值,同时苯丙氨酸解氨酶和几丁质酶活性升高;多磷酸肌醇激酶（IPK2）抑制剂、外源H2O2及H2O2清除剂均能改变霜霉病菌所引起的抗性葡萄品种左优红叶片PAL和几丁质酶活性的变化,同时可以影响不同抗性品种叶片的感病情况;IPK2抑制剂对葡萄霜霉病菌引起的H2O2水平变化没有影响;清除H2O2可减弱葡萄霜霉病菌对VvIPK2表达量的诱导效应。研究表明H2O2位于IPK2的上游,通过调控PAL和几丁质酶活性参与葡萄应答霜霉病过程。     

Effect of chitosan on growth and plasma membrane properties of Rhizopus stolonifer (Ehrenb.:Fr.) Vuill

The effect of chitosan (0.5, 1.0 and 2.0 mg ml⁻¹) on mycelium growth, potassium efflux, pH of the incubation medium, and activity of plasma membrane H⁺-ATPase of Rhizopus stolonifer was evaluated. The mycelium growth of R. stolonifer was reduced on media amended with chitosan at 1.0 and 2.0 mg ml⁻¹. The highest antifungal index (65%) was obtained on media containing chitosan at 2.0 mg ml⁻¹. Potassium efflux induced by the addition of the three chitosan concentrations was demonstrated. The highest potassium effluxes were observed with addition of chitosan at 2.0 mg ml⁻¹. R. stolonifer grown 72 h on Potato Dextrose Broth without chitosan generated a decrease in the medium’s pH from 5.4 to 4.8. However, the addition of chitosan at all concentrations caused increases on pH of medium cultures. The highest pH increases were observed on media amended chitosan at 2.0 mg ml⁻¹ resulting in the raising of pH level to 6.9. Additionally, there was an important inhibitory effect of chitosan (1.0 and 2.0 mg ml⁻¹) on H⁺-ATPase activity in the plasma membrane of R. stolonifer (32.70%).     

20-(S)-喜树碱7-C-取代衍生物的合成及杀松材线虫活性研究

为研究喜树碱(CPT)类化合物的杀线虫活性,以喜树碱为原料,经烷基化、氧化、酯化等步骤合成了13个7-C-取代的20-(S)-喜树碱衍生物,其中化合物 14 未见文献报道,所有衍生物的结构经红外光谱(FT-IR)、核磁共振氢谱(1H NMR)和液-质联用(LC-MS)等分析手段进行了表征。采用浸渍法测定了化合物对松材线虫Bursaphelenchus xylophilus的毒杀活性。结果表明:与母体化合物喜树碱相比,7-C-取代的20-(S)-喜树碱衍生物具有更强的杀线虫活性,其中化合物7-苄基喜树碱、7-甲酰基喜树碱、7-苯甲酰氧甲基喜树碱在24 h的致死中浓度(LC50值)分别为2.28、2.21和1.37 mg/L,明显高于母体化合物喜树碱的LC50值12.18 mg /L。     

Methoxyfenozide and pyriproxifen alter the cellular immune reactions of Eurygaster integriceps Puton (Hemiptera: Scutelleridae) against Beauveria bassiana

Effects of the two insect growth regulators (IGRs) methoxyfenozide, 20-hydroxyecdysone (20E) agonist, and pyriproxifen, Juvenile hormone (JH) agonist, were examined on the cellular immune responses of the Sunn pest, Eurygaster integriceps versus the entomopathogenic fungus Beauveria bassiana. The simultaneous treatment with the IGRs and the fungal spores altered haemocyte count (total and differentiate), nodulation response and phenoloxidase (PO) activity in a dose- and time-dependent manner. It was observed that different concentrations of methoxyfenozide increased total and differentiate haemocyte numbers as well as B. bassiana-induced nodulation response. In contrast with the JH agonist, pyriproxifen significantly decreased total and differentiate haemocyte numbers and inhibited nodule formation in E. integriceps adults. The 20E agonist displayed major effects when injected at the doses 2.79 and 5.59 μg/mg adult. In contrast, injecting adults by pyriproxifen significantly impaired their ability to raise an efficacious response against the fungal spores. The ability of the two IGR analogues to interfere with activity of the PO system in haemolymph of E. integriceps adults was also investigated 6 h after injection by fungal spores. Methoxyfenozide had an excitatory effect on PO activity when the 5.59 μg/mg concentration was used against adults. Conversely, pyriproxifen had an inhibitory effect on PO activity when used at 1.49 μg/mg adult concentration. These findings demonstrate that pyriproxifen may interfere with cell-mediated immunity of E. integriceps. So, pyriproxifen could be a good candidate for the integrated control of the Sunn pest.     

渐狭蜡蚧菌Lecanicillium attenuatum产几丁质酶的活性及对南方根结线虫卵孵化的抑制作用

为探究卵寄生真菌产生的几丁质酶对南方根结线虫卵孵化的抑制作用,采用分离自黑龙江大豆孢囊线虫孢囊上的菌株CGMCC5328,利用NAG和pNP法测定其几丁质降解酶系和外切酶的活性,分析几丁质酶粗酶液对南方根结线虫卵的孵化抑制效果.结果表明,菌株CGMCC5328经形态学和ITS序列分析鉴定为渐狭蜡蚧菌Lecanicillium attenuatum;在几丁质酶诱导培养基中培养第4天时其分泌的几丁质降解酶系达酶活高峰,为16.90 U/mL;第6天外切酶达酶活高峰,为0.59 U/mL,之后酶活趋于稳定持续至第14天;几丁质酶分子量分别为79.6、65.6、54.1、42.1和32.9 kDa;其中培养第7天的几丁质酶粗酶原液对南方根结线虫卵孵化的抑制率为83.17%;第6天菌株CGMCC5328对卵的寄生率为85.10%.表明高效产几丁质酶的卵寄生真菌渐狭蜡蚧菌菌株CGMCC5328对南方根结线虫卵孵化具有明显的抑制效果.     

Enzymatic dynamics of catechol oxidase from Gastrolina depressa

Properties of the phenoloxidase (PO) from adult of Gastrolina depressa Baly (Coleoptera: Chrysomelidae) as well as effects of some metal ions and inhibitors on the activity of PO purified by (NH₄)₂SO₄ were determined. The optimal pH and temperature of the enzyme for the oxidation of catechol were determined to be at pH 7.5 and at 40 °C, respectively. The kinetic parameters for the oxidation of L-DOPA and catechol by the PO were 15.01 and 9.17 mM, respectively. The PO activity was strongly inhibited by Zn²⁺ and Cu²⁺, different to Mg²⁺ slightly. Both ascorbic acid and cysteine exhibited competitive inhibition and the inhibitory constants (K_i) were determined to be 2.22 mM and 0.40 mM, respectively.     

Chiral β-arylalkyl-1H-1,2,4-triazoles as demethylase inhibitors: Biological evaluation and its stereoselective interaction with sterol 14α-demethylase from Penicillium digitatum

A series of chiral β-arylalkyl-1H-1,2,4-triazole derivatives were prepared and both their in vitro antifungal activities against Penicillium digitatum and binding activity toward CYP51 protein of P. digitatum (PdCYP51) were tested. In general, the in vitro inhibitory activities of R- and S-enantiomers were in good agreement with the corresponding binding activities in cultured cells. Furthermore, the preliminary molecular docking modeling of representative compounds are described to provide more insight into their stereoselective interaction, as well as further rationalize the observations of the activities of chiral azoles as demethylase inhibitors for defense against postharvest pathogens.     

Biological and biochemical responses of Biomphalaria alexandrina to some extracts of the plants Solanum siniacum and Artemisia judaica L.

The molluscicidal activity of cold water, boiled water, methanol, ethanol, acetone and chloroform extracts of Solanum siniacum and Artemisia judaica L. plants against Biomphalaria alexandrina snails was carried out. The tests revealed plant’s ethanol extract was more toxic to the snails than the other tested extracts. Therefore, it was tested against snails’ fecundity (M_x), reproduction rate (R₀) and their infection with Schistosoma mansoni miracidia. In addition, biochemical parameters and the activities of some enzymes in tissues of snails treated with the two tested plants were determined. As well, glucose concentration in snails’ hemolymph was evaluated. Exposure of B. alexandrina snails to plant’s ethanol extract led to a significant reduction in their survival and snails’ fecundity, reproduction rate. In addition, it caused a considerable reduction in the infectivity of S. mansoni miracidia to the snails. Also, it caused a reduction in number of cercariae per snail during the patent period and in the period of cercarial shedding. The results revealed that the glucose concentration in hemolymph and Lactate level in soft tissues of treated snails were increased (P < 0.001) while glycogen, total protein, the lipid content and the pyruvate level in snail’s tissues decreased (P < 0.001). The activities of lactic dehydrogenase (LDH), pyruvatekinase (PK) and cytochrome oxidase (CY) enzymes in homogenate of snail’s tissues were reduced (P < 0.001) in response to treatment with the two tested plants while protease (PR) activity increased (P < 0.001). It is concluded that the application of LC₂₅ of ethanol extract of S. siniacum and A. judaica L. may be helpful in snail control as it interferes with the snails’ biology and physiology.     

Mechanism of resistance to quinclorac in smooth crabgrass (Digitaria ischaemum)

The mechanism of resistance to quinclorac was investigated in a smooth crabgrass biotype [Digitaria ischaemum (Schreb. ex Schweig) Schreb. ex Muhl] from Tulare County, California. Quinclorac (8.96 kg a.i. ha⁻¹) had no effect (P = 0.18) on the resistant (R) biotype, but reduced fresh weight of a susceptible (S) biotype by 93%. After treatment with 4.48 kg a.i. quinclorac ha⁻¹, the S biotype produced about three times more ethylene than the R biotype and accumulated cyanide in tissues. Similar amounts of endogenous cyanide resulting from treatment with KCN reproduced quinclorac phytotoxicity. Pre-treatment with the ACC synthase inhibitor AVG reduced quinclorac phytotoxicity by 37% and ethylene production by 89%. These data suggest a target site-based mechanism of resistance involving stimulation of ACC synthesis and accumulation of cyanide. Also, the R biotype had four times more β-cyanoalanine synthase activity than the S biotype, suggesting a higher ability to detoxify cyanide.     

Biological and biochemical parameters of Biomphalaria alexandrina snails exposed to the plants Datura stramonium and Sesbania sesban as water suspensions of their dry powder

Four plant species, as a dry powder of their leaves, were tested against Biomphalaria alexandrina snails, the intermediate host of Schistosoma mansoni. The bioassay tests revealed the plants Datura stramonium and Sesbania sesban to be more toxic to the snails than the other two ones. Therefore, they were tested against snails’ fecundity (M_x), reproduction rate (R_o) and their infection with S. mansoni miracidia. In addition, total protein concentration and the activities of the transaminases (AsT and AlT) and phosphatases (AcP and AkP) enzymes in hemolymph and tissues of snails treated with these plants were determined. As well, glucose concentration in snails’ hemolymph was evaluated.Exposure of snails for 4 weeks to LC₁₀ and LC₂₅ of the plants D. stramonium and S. sesban dry powder markly suppressed their M_x and R_o. The reduction rates of R_o for snails exposed to LC₂₅ of these plants were 62.1% and 76.4%, respectively. As well, a considerable reduction in the infection rates of snails exposed to these plants either during, pre- or post-miracidial exposure was recorded. Thus, infection rates of snails treated during miracidial exposure with LC₁₀ of D. stramonium and S. sesban were 41.7% and 52.2%, respectively, compared to 92.6% for control group (P < 0.01). These plants, also, reduced the duration of cercarial shedding and cercarial production/snail. So, snails exposed to LC₂₅ of these plants shed 372.8 and 223.2 cercariae/snail, respectively, compared to 766.3 cercariae/infected control snail (P < 0.01).The results, also, revealed that glucose and total protein concentrations in hemolymph of snails treated with LC₁₀ and LC₂₅ of these plants were decreased, meanwhile, the activities of the enzymes AsT, AlT, AcP and AkP were elevated (P < 0.01). However, the activity of AcP in tissues of treated snails was decreased compared to that of control ones. It is concluded that LC₂₅ of the plants D. stramonium and S. sesban negatively interferes with biological and physiological activities of B. alexandrina snails, consequently it could be effective in interrupting and minimizing the transmission of S. mansoni.     

Lead optimization and anti-plant pathogenic fungi activities of daphneolone analogues from Stellera chamaejasme L.

In order to find the biorational pesticides, we have synthesized two series of daphneolone analogues including 5-methylfuryl chalcones and 5-nitrofuryl chalcones by optimizing daphneolone analogues from Stellera chamaejasme L. with the good insecticidal properties of Qinghai-Tibet Plateau. All the synthesized compounds have been evaluated for anti-plant pathogenic fungi activities. The bioactivity assay showed that some of these daphneolone analogues were potentially active against plant pathogenic fungi, Rhizoctonia solani, Gibberella zeae, Bipolaris maydis, Sclerotia sclerotium and Botrytis cirerea, while the most potent 1-(2,4-dichlorophenyl)-3-(5-methylfuran-2-yl)propenone (compound 7) in this study showed good inhibitory activity against R. solani at 200 mg L⁻¹ with ca. 100% inhibition.     

无机化合物对南方根结线虫行为的影响

利用体外生物测定方法,研究常见无机化合物对南方根结线虫Meloidogyne incognita 2龄幼虫(J_2)存活、趋性以及卵囊内卵孵化的影响.结果表明,不同化合物不同浓度处理对M.incognita的影响差异显著.随着无机化合物浓度的增加,J_2死亡率上升,NH_4HCO_3、Na_2SO_4、CuCl_2·2H_2O、CuSO_4·5H_2O和FeCl_3·6H_2O浓度分别为O.03、0.03、0.0003、0.0004和0.0007 mol/L即可抑制其存活.除Cu~(2+)化合物外,其它无机化合物在供试浓度下均对卵囊内卵的孵化有明显抑制作用.含Ca~(2+)化合物均对南方根结线虫J_2有吸引作用,而含NH_4~+的NH_4HCO_3和NH_4NO_3对J_2排斥.除碳酸氢铵LC_(90)抑制种子萌发外,其它化合物对番茄种子萌发无抑制作用,但对后期幼根的伸长有抑制作用.盆栽试验结果显示,CuSO_4·5H_2O防治效果最佳,在LC_(50)处理下与对照相比防效可达70.8%.