杀菌剂对斜纹夜蛾SL细胞系和幼虫的生物活性

以MTT法筛选了19种杀菌剂对斜纹夜蛾Spodoptera litura SL细胞的毒杀活性,并以该方法研究了三唑类杀菌剂对SL细胞的毒力。结果表明: 福美双、烯唑醇、己唑醇、氟硅唑、苯霜灵、苯醚甲环唑、戊唑醇和腈菌唑等杀菌剂对SL细胞具有优异的毒杀活性。三唑类杀菌剂腈菌唑、烯唑醇、己唑醇和戊唑醇处理SL细胞48 h后,LC₅₀值分别为21.94 μg/mL、 23.80 μg/mL、 33.16 μg/mL和47.63 μg/mL。以考马斯亮蓝G₂₅₀法研究了腈菌唑对SL细胞中蛋白质含量和乳酸脱氢酶(LDH)漏出率的影响, 20 μg/mL腈菌唑处理12 h、24 h、48 h和72 h后,SL细胞中蛋白质含量分别降低8.55%、25.95%、42.95%和67.05%;处理24 h和48 h后,SL细胞的LDH漏出率分别为30.66%和32.05%。以浸叶喂食法处理斜纹夜蛾3龄幼虫,三唑类杀菌剂可显著抑制试虫体重增长。以0.5 μg/头、 1.0 μg/头和2.0 μg/头剂量的腈菌唑注射处理72 h后,斜纹夜蛾4龄幼虫血细胞数量分别降低12.31%、 25.96%和25.73%;腈菌唑注射处理48 h和72 h后,对斜纹夜蛾幼虫的LD₅₀值分别为1.59 μg/头和1.53 μg/头。结果显示以离体培养细胞为对象,从现有杀菌剂中寻找新的杀虫剂先导化合物具有良好的研究潜力。     

腈菌唑导致的细胞膜穿孔对斜纹夜蛾SL细胞膜通透性的影响

研究了腈菌唑导致的细胞膜穿孔对离体培养斜纹夜蛾Spodoptera litura卵巢细胞(SL细胞)膜通透性的影响。结果表明: 腈菌唑可使不能穿过SL细胞膜的大分子物质荧光染料PI大量穿过细胞膜进入胞内,20和40 μg/mL腈菌唑处理后12 h,SL细胞荧光强度增加率分别为11.95%和25.80%;处理后24 h,SL细胞荧光强度增加率分别为27.77%和57.49%。腈菌唑也可明显提高SL细胞内大分子物质乳酸脱氢酶的漏出率,20和40 μg/mL腈菌唑处理SL细胞24 h后,胞内乳酸脱氢酶漏出率分别为30.66%和43.93%;处理后48 h,漏出率分别为41.22%和57.91%。腈菌唑可以明显提高SL细胞胞内钙离子含量,20和40 μg/mL腈菌唑处理SL细胞24和48 h,胞内钙荧光强度与对照差异显著。处理后24 h,腈菌唑对SL细胞的LC₅₀值为35.84 μg/mL,明显高于典型细胞毒剂鱼藤酮的活性。当质量比为1∶1和2∶1时,腈菌唑与鱼藤酮联用对SL细胞处理后24 h的LC₅₀值为43.92和26.09 μg/mL,共毒系数分别为137.60和188.49,增效作用显著,随着腈菌唑的含量增加,增效作用增加。腈菌唑对SL细胞具有良好的抑制作用,可以打通限制物质穿透的细胞膜屏障,提高农药活性成分的有效利用率。     

产芳香腈水解酶的恶臭假单胞菌Pseudomonas putida CGMCC3830的筛选、鉴定及发酵优化(英文)

近年来微生物腈水解酶水解腈类化合物制备有机酸已逐步受到关注。本研究分离到一株表现出较高腈水解酶活力的细菌菌株,通过形态学、生理生化实验以及16S rRNA基因序列分析将其鉴定为恶臭假单胞菌Pseudomonas putida CGMCC3830。结合单因素及响应面法对该菌株产腈水解酶的发酵条件进行了优化,获得最适培养条件为:甘油13.54 g/L,胰蛋白胨11.59 g/L,酵母粉5.21 g/L,KH2PO4 1 g/L,NaCl 1 g/L,脲1 g/L,初始pH 6.0及培养温度30℃。通过优化,酶活由2.02 U/mL提升至36.12 U/mL。对该菌株底物特异性的考察结果表明,恶臭假单胞菌腈水解酶对芳香族腈类化合物具有较高的水解活力。将其应用于烟酸的生物合成中,2 mg/mL游离细胞能90 min内将20.8 g/L 3-氰基吡啶彻底转化,制备得到相应烟酸。这些结果表明恶臭假单胞菌P.putida CGMCC3830在烟酸的规模化生产中具有一定的应用潜力。     

苹果套袋果实黑点病菌杀菌剂室内测定研究

为筛选有效抑制套袋苹果黑点病多种病原的杀菌剂,选择25%吡唑醚菌酯悬浮剂、250 g/L嘧菌酯悬浮剂、10%苯醚甲环唑水分散粒剂、430 g/L戊唑醇悬浮剂、30%氟菌唑可湿性粉剂和80%克菌丹水分散粒剂6种杀菌剂,采用菌丝生长速率法,对引起套袋苹果黑点病的6种主要病原菌进行室内抑菌活性测定,获得了对多种黑点病病原具有抑制活性的杀菌剂。嘧菌酯、吡唑醚菌酯、戊唑醇和苯醚甲环唑4种杀菌剂对6种病原具有较好的抑菌活性,EC₅₀均小于20μg/mL,具较好田间应用前景。结果说明甲氧基丙烯酸酯类杀菌剂嘧菌酯、吡唑醚菌酯和三唑类杀菌剂戊唑醇、苯醚甲环唑有望作为交替使用防治套袋苹果黑点病的候选农药。     

6种抗真菌药物对皮肤癣菌体外抗真菌活性评价

目的评价6种抗真菌药物对皮肤癣菌体外抗真菌活性。方法采用CLSI推荐的M-38P方案对分离自足癣和体、股癣的皮肤癣菌进行联苯苄唑、硝酸舍他康唑、硝酸异康唑、盐酸布替萘芬、阿莫洛芬、利拉萘酯6种抗真菌药物敏感性测定。结果联苯苄唑MIC范围为0．03—4mg／L,MIC50为1mg／L,MIC90为2mg／L。硝酸舍他康唑分别为0．06—16mg／L、0．5mg／L和2mg／L。硝酸异康唑分别为0．03～2mg／L、0．25mg／L和0．5mg／L。盐酸布替萘芬分别为0．0025～0．04mg／L、0．01mg／L和0．02mg／L。阿莫罗芬分别为0．01～〉0．08mg／L、0．02mg／L和0．04mg／L。利拉萘酯分别为0．004—0．625mg／L、0．039mg／L和0．312mg／L。结论6种抗真菌药物对皮肤癣菌均有强的抗菌活性,由强到弱依次为布替萘芬、阿莫罗芬、利拉萘酯和咪唑类药物。     

秦冠苹果品种对苹果黑星病的组织学抗性研究

为揭示苹果抗病品种秦冠在组织细胞学水平上抗苹果黑星病的特征,本研究采用扫描和透射电镜技术,将苹果黑星菌Venturia inaequalis接种侵染寄主后,系统观察抗病品种秦冠和感病品种嘎啦的叶片组织和细胞结构变化。扫描电镜观察结果表明,黑星菌分生孢子悬浮液接种秦冠和嘎啦叶片48 h后,病菌沿叶脉生长扩展,其菌丝可从叶片气孔或直接侵入。透射电镜观察结果表明,秦冠叶片的角质层厚度明显高于嘎啦,其中秦冠角质层平均厚度为1.75 μm,嘎啦为1.06 μm。透射电镜观察结果表明,黑星菌菌丝在寄主叶肉细胞间扩展,导致嘎啦栅栏组织细胞萎缩,排列松散,叶绿体变形受损,细胞内出现较大淀粉粒和胞内物质外渗流失,并在后期发展成大量细胞坏死;而秦冠虽症状类似,但受损程度明显小于嘎啦。以上结果表明,秦冠在组织细胞学上对苹果黑星病具有抗侵染、抗扩展和延缓病程发展的作用,可作为苹果黑星病抗性育种材料加以利用。     

苹果轮纹病病害名称之规范

苹果轮纹病(apple ring rot)为苹果重要病害,严重危害果实和枝干,甚至造成幼树枯死等。苹果轮纹病可引起果实腐烂、枝干疣突、溃疡、粗皮等症状,导致苹果轮纹病病害汉语名称使用混乱,如苹果轮纹病、苹果干腐病、苹果果实轮纹病、苹果枝干轮纹病、轮纹烂果病等。病原拉丁学名的使用亦相当混乱,葡萄座腔菌Botryosphaeria dothidea、贝林格葡萄座腔菌B. berengeriana、贝林格葡萄座腔菌梨生专化型B. berengeriana f. sp. pyricola、七叶树壳梭孢Fusicoccum aesculi、锹冢大茎点霉Macrophoma kuwatsukai、梨生囊孢Physalospora pyricola和梨生球座菌Guignardia pyricola等学名都在被使用。本文根据近些年来国内外的研究新进展,认为苹果轮纹病是一类复合病害,与欧美国家发生的白腐病为同一种病害,其病原包括葡萄座腔菌B. dothidea和锹冢葡萄座腔菌B. kuwatsukai。建议将该复合病害汉语统称为苹果轮纹病,英文采用apple ring rot。鉴于两种病原在我国苹果产区的普发性,建议在病害发生规律及抗病育种等研究中,对于葡萄座腔菌和锹冢葡萄座腔菌均需充分重视。     

火龙果溃疡病防治药剂的筛选

火龙果溃疡病是火龙果生产中常见的病害之一,对火龙果产量和品质的影响较大,近年来火龙果溃疡病的发生频率逐年增加.本研究通过菌丝生长抑制法测定9种杀菌剂对火龙果溃疡病病菌的杀菌活性.结果表明,戊唑·咪鲜胺、肟菌·戊唑醇、吡唑·毒氟磷有较强的抑菌作用,EC50分别为0.0209,0.0762,2.0358mg/L,本研究为生产上防治火龙果溃疡病药剂的筛选提供了参考.     

毛豆中腈菌唑残留量的气相色谱法测定

采用气相色谱法定量分析毛豆上腈菌唑的微量残留量。样品经乙腈提取,液液分配,中性氧化铝柱层析净化后,以气相色谱电子俘获检测器法(GC-ECD)测定,DB-1701毛细管柱、氮气为载气,柱温150℃20℃/min 260℃(10 min),气化室温度240℃,检测器温度300℃,外标法定量。该方法快速、准确,在0.05~2.00 mg/L范围内线性相关系数r²=0.9998,平均回收率91.1%~99.0%,变异系数1.22%~2.94%,最小检测量1.0×10^-12 g,最低检出浓度5.0×10^-4 mg/kg。     

海南省火龙果炭疽菌病病原鉴定及有效药剂筛选

从海南省火龙果主要种植区采集病样,分离纯化出多个炭疽病菌菌株,对其进行了形态学、分子生物学和致病性的测定。同时,采用菌丝生长速率法测定了生产上常用药剂对该病菌的毒力。结果表明:该病菌的病原为胶胞炭疽菌(Colletotrichum gloeosporioides)。在供试的6种药剂中,25%吡唑醚菌酯EC、50%咪鲜胺锰盐WP、10%苯醚甲环唑WG和430 g/L戊唑醇SC对火龙果炭疽病菌的抑制效果较好,EC50依次为0.007 5 mg/L、0.109 3 mg/L、2.651 7 mg/L和5.406 0 mg/L。50%多菌灵WP和70%甲基硫菌灵WP抑制效果较差。经回归曲线比较,病菌对25%吡唑醚菌酯EC敏感性最高,对50%多菌灵WP敏感性最低。     

Assessment of Sensitivities to Anilinopyrimidine- and Strobilurin-fungicides in Populations of the Apple Scab Fungus Venturia inaequalis

The sensitivity of Venturia inaequalis populations to the anilinopyrimidine fungicides pyrimethanil and cyprodinil was analysed by microscopic in vivo analysis of conidiophore formation. The sensitivity to the strobilurin kresoxim-methyl was analysed using an in vitro germination assay and by determination of the diseased leaf area and conidia produced in vivo. Baseline sensitivities were determined with V. inaequalis populations from control orchards that had never been treated with fungicides. Comparison of the baseline sensitivities with sensitivities of populations obtained from orchards that had received 43 anilinopyrimidine treatments over 4 years, or from an orchard with 54 kresoxim-methyl treatments over 6 years indicated that no resistance to these fungicides has developed at the sites sampled.     

Venturia inaequalis: the causal agent of apple scab

The fungus Venturia inaequalis infects members of the Maloideae, and causes the disease apple scab, the most important disease of apple worldwide. The early elucidation of the gene-for-gene relationship between V. inaequalis and its host Malus has intrigued plant pathologists ever since, with the identification of 17 resistance (R)-avirulence (Avr) gene pairings. The Avr gene products are presumably a subset of the total effector arsenal of V. inaequalis (predominantly proteins secreted in planta assumed to facilitate infection). The supposition that effectors from V. inaequalis act as suppressors of plant defence is supported by the ability of the pathogen to penetrate the cuticle and differentiate into large pseudoparenchymatous structures, termed stromata, in the subcuticular space, without the initiation of an effective plant defence response. If effectors can be identified that are essential for pathogenicity, the corresponding R genes will be durable and would add significant value to breeding programmes. An R gene cluster in Malus has been cloned, but no V. inaequalis effectors have been characterized at the molecular level. However, the identification of effectors is likely to be facilitated by the resolution of the whole genome sequence of V. inaequalis. TAXONOMY: Teleomorph: Venturia inaequalis Cooke (Wint.); Kingdom Fungi; Phylum Ascomycota; Subphylum Euascomycota; Class Dothideomycetes; Family Venturiaceae; genus Venturia; species inaequalis. Anamorph: Fusicladium pomi (Fr.) Lind or Spilocaea pomi (Fr.). LIFE CYCLE: V. inaequalis is a hemibiotroph and overwinters as pseudothecia (sexual fruiting bodies) following a phase of saprobic growth in fallen leaf tissues. The primary inoculum consists of ascospores, which germinate and penetrate the cuticle. Stromata are formed above the epidermal cells but do not penetrate them. Cell wall-degrading enzymes are only produced late in the infection cycle, raising the as yet unanswered question as to how V. inaequalis gains nutrients from the host. Conidia (secondary inoculum) arise from the upper surface of the stromata, and are produced throughout the growing season, initiating multiple rounds of infection. VENTURIA INAEQUALIS AS A MODEL PATHOGEN OF A WOODY HOST: V. inaequalis can be cultured and is amenable to crossing in vitro, enabling map-based cloning strategies. It can be transformed readily, and functional analyses can be conducted by gene silencing. Expressed sequence tag collections are available to aid in gene identification. These will be complemented by the whole genome sequence, which, in turn, will contribute to the comparative analysis of different races of V. inaequalis and plant pathogens within the Dothideomycetes.     

The role of melanin in the antagonistic interaction between the apple scab pathogen Venturia inaequalis and Microsphaeropsis ochracea

The objective of this study was to examine the role of melanin in the interaction between the mycoparasite Microsphaeropsis ochracea and the apple scab pathogen Venturia inaequalis. Melanin was extracted from the cell wall of the pathogen and its chemical and physical properties determined on the basis of biochemical tests and visible and infrared spectra. The physical and chemical characteristics of V inaequalis melanin were similar to the those of synthetic dihydroxyphenylalanine (DOPA) melanin. Precursors of the four known melanin biosynthetic pathways were tested for their ability to restore the pigmentation of an albino strain of V inaequalis. Scytalone, an intermediate of the 1,8-dihydroxynaphthalene (DHN) pathway, was the only precursor to restore the dark-brown pigmentation. Tricyclazole and pyroquilon, two antipenetrant fungicides, specific inhibitors of DHN melanin synthesis in Pyricularia oryzae, were used to confirm the melanin pathway in V. inaequalis wild type. A reddish-brown pigment was obtained due to the accumulation of shunt products of the DHN melanin pathway instead of a dark-brown pigment, suggesting that the melanin extracted from V inaequalis was a DHN melanin. Furthermore, growth of an albino mutant of V. inaequalis on scytalone-amended medium resulted in the formation of dark granules similar to those seen in wild-type isolates. Transmission electron microscopic observations of M. ochracea grown in the presence of melanin showed that the granules accumulated gradually along fungal cell walls to form a uniform dark coating.     

The Vh8 locus of a new gene-for-gene interaction between Venturia inaequalis and the wild apple Malus sieversii is closely linked to the Vh2 locus in Malus pumila R12740-7A

The wild apple (Malus sieversii) is a large-fruited species from Central Asia, which is used as a source of scab resistance in cultivar breeding. Phytopathological tests with races of Venturia inaequalis were performed to differentiate scab-resistance genes in Malus as well as an avirulence gene in the pathogen. A novel gene-for-gene interaction between V. inaequalis and Malus was identified. The locus of the scab-resistance gene Vh8 is linked with, or possibly allelic to, that of the Vh2 gene in Malus pumila Russian apple R12740-7A, at the lower end of linkage group 2 of Malus. Race 8 isolate NZ188B.2 is compatible with Vh8, suggesting the loss or modification of the complementary AvrVh8 gene, while isolate 1639 overcomes both Vh2 and Vh8, but is incompatible with at least one other gene not detected by any of the other race isolates tested. Our research is the first to differentiate scab-resistance genes in a putative gene cluster in apple with the aid of races of V. inaequalis.     

Localization of fungal fimbriae by immunocytochemistry in pathogenic and nonpathogenic isolates of Venturia inaequalis

Pathogenic and nonpathogenic isolates of Venturia inaequalis were grown in liquid culture. Hyphae were treated with two types of fimbrial antiserum (AU- and AV-1) and examined by immunofluorescent microscopy, in order to establish the distribution of fimbrial epitopes in whole cell mounts. The AV-1 antiserum was specific for the glycoprotein subunits while the AU-antiserum was specific for the protein moieties present on the fimbriae of Mycobotryum violaceum. The use of fimbrial antiserum with immunocytochemistry and transmission electron microscopy demonstrated a clear distinction between pathogenic and nonpathogenic isolates of V. inaequalis, based on the appearance of the fungal cell wall and the distribution of fimbrial epitopes labeled with AV-1 antiserum and immunogold complex. In actively growing hyphae of the pathogenic isolate, characterized by distinct cellular organelles, small vacuoles, and lipid bodies, fimbrial epitopes were concentrated in the fungal cell wall and were present minimally on the outer surface. In contrast, actively growing hyphae of the nonpathogenic isolate of V. inaequalis had extensive fine hair-like protrusions in the fungal cell wall which labeled with the AV-1 antiserum and immunogold. The distribution of fimbrial epitopes in V. inaequalis was highly dependent on the developmental growth stage of the fungal mycelium. Aging mycelia in both the pathogenic and nonpathogenic isolates of V. inaequalis were characterized by a large central vacuole and no label. In the pathogenic and nonpathogenic isolates of V. inaequalis grown in vitro, the distribution of fimbrial glycoprotein epitopes provided a more complex profile than that seen in M. violaceum.     

Evaluation of microcapsule trunk injections for the control of apple scab and powdery mildew

A 3‐year field trial was conducted using established apple cv. Crown Gold and English oak (Quercus robur L.) to assess the efficacy of eight fungicides applied via microcapsule trunk injection against the foliar pathogens apple scab (Venturia inaequalis) and powdery mildew (Phyllactinia sp). In both apple cv. Crown Gold and English oak, the fungicide myclobutanil was not taken up when microcapsules were inserted into the tree vascular system at the root flare. Disease severity in injected trees, excluding myclobutanil, was lower over the following two growing seasons compared to water‐injected controls indicating seven of the eight fungicides used in this study provided a significant degree of protection against scab and powdery mildew infection. A difference in the magnitude of pathogen control achieved was recorded between fungicides. Of the fungicides tested, penconazole, pyrifenox and carbendazim significantly reduced disease severity and significantly increased leaf chlorophyll (Fv/Fm) and SPAD values as a measure of tree vitality and chlorophyll content, respectively, in both apple cv. Crown Gold and English oak over two growing seasons after microcapsule injection. Based on the results of this investigation, it is suggested that these three fungicides be used in preference to thiabendazole, fosetyl‐aluminium, triadimefon and propiconazole for the control of apple scab and powdery mildew where outbreaks of these foliar pathogens are problematic.     

Sensitivity of Venturia inaequalis to Trifloxystrobin and Difenoconazole in Uruguay

The sensitivity of Venturia inaequalis to trifloxystrobin and difenoconazole was studied in Uruguay. Populations of V. inaequalis were collected from apple orchards with different histories of trifloxystrobin use. Sensitivity of populations to trifloxystrobin was analysed using a method for testing spore germination published by FRAC, using a discriminatory concentration of 2.0 mg a.i./l. Resistance to trifloxystrobin was widespread in the region of commercial apple production with resistance detected in all orchards examined, the incidences ranging from 3% to 95%. The highest frequencies were found in orchards with the most intensive use of Quinone outside inhibitors (Q_oI) fungicides. Sensitivities of isolates of V. inaequalis to difenoconazole were assessed at five concentrations using a mycelial growth assay on isolates (33 isolates per orchard) from one non‐commercial (baseline orchard) and two commercial orchards having differing histories of difenoconazole use. Populations in both commercial orchards exhibited reduced sensitivities to difenoconazole compared to the baseline orchard. Resistance factor (RF) values of 6.6 and 11.74 were measured in the orchards with moderate (up to 4 sprays per season) and intensive use (more than 5 sprays per season) of difenoconazole, respectively. A single‐assessment concentration (SAC) was identified for assessing difenoconazole sensitivity of V. inaequalis isolates by fitting linear regressions between log₁₀ EC₅₀ and relative growth (RG) of the isolates at each fungicide concentration testing, and comparing the goodness‐of‐fit of the regression lines. Comparable results were obtained based on EC₅₀ values and RG values at a SAC of 0.05 mg of active ingredient per litre (a.i. per l). Populations from both commercial orchards differed from the baseline population, in that isolates with RG ≥70 were present at substantial levels in the former but absent from the latter.     

Inheritance studies of apple scab resistance and identification of Rvi14, a new major gene that acts together with other broad-spectrum QTL

Scab, caused by the fungal pathogen Venturia inaequalis, is the most common disease of cultivated apple (Malus xdomestica). The fungal races 6 and 7 have now overcome the major resistance gene Vf, which is widely used in apple breeding programmes. New breeding strategies to achieve durable resistance are thus necessary. The aim of this study was to determine the genetic basis of quantitative resistance of the apple cultivar 'Dülmener Rosenapfel', known to be scab resistant under different environmental conditions. An F1 progeny derived from the cross between the susceptible cultivar 'Gala' and 'Dülmener Rosenapfel' was tested in a greenhouse with a multi-isolate inoculum of V. inaequalis. Rvi14, a new major gene that conditions a chlorotic-type reaction, was mapped on linkage group (LG) 6 in a genomic region not known to be involved in disease resistance. A further three quantitative trait loci (QTL) for resistance were identified. One co-localized with Rvi14 on LG6, whereas the remaining two were detected on LG11 and LG17, in genomic regions already reported to carry broad-spectrum QTL in other genetic backgrounds. Since a selective genotyping approach was used to detect QTL, an expectation-maximization (EM) computation was used to estimate the corrected QTL contributions to phenotypic variation and was validated by entire progeny genotyping.     

Somatic variation plays a key role in the evolution of the Vf gene family residing in the Vf locus that confers resistance to apple scab disease

A cluster of four receptor-like genes has been previously identified in the Vf locus of the crabapple Malus floribunda clone 821 that confers resistance to five races of the fungal pathogen Venturia inaequalis, the casual agent of apple scab disease. Pairwise comparisons of the four Vf paralogs in both promoter and coding regions reveal their timeline evolutionary history. The four Vf paralogs have evolved from four ancient Vf members resulting from two sequential duplication events of a single Vf progenitor initially present in the Malus genome. The coding sequences of the four Vf paralogs are characterized with high numbers of unique polymorphic nucleotides, a number of short duplications/deletions, various deletions of complete LRR copy units, and a casual insert of a transposon-like element. Significant high ratios of nonsynonymous to synonymous substitutions, Ka/Ks, are observed in the putative ligand binding residues in the LRR domains. No sequence exchange between the four Vf paralogs is observed. Compared with promoter regions, only nucleotide substitutions are dramatically elevated in the coding regions. The results presented in this study strongly indicate that the Vf locus is under strong and steady horizontal selective pressures imposed by the fungal pathogen V. inaequalis, and divergent selection on somatic variations plays a key role in shaping the resistance specificity.