Compounds from Ageratum conyzoides: isolation, structural elucidation and insecticidal activity

This work aimed at identifying plant compounds with insecticidal activity against Diaphania hyalinata (L.) (Lepidoptera: Pyralidae), Musca domestica (L.) (Diptera: Muscidae), Periplaneta americana (L.) (Blattodea: Blattidae) and Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae). The plant species used were: basil (Ocimum selloi Benth.), rue (Ruta graveolens L.), lion's ear (Leonotis nepetaefolia L.), Jimson weed (Datura stramonium L.), 'baleeira' herb (Cordia verbenaceae L.), mint (Mentha piperita L.), wild balsam apple (Mormodica charantia L.) and billy goat weed (Ageratum conyzoides L.). Firstly, the insecticidal activities of hexane and ethanol plant extracts were evaluated against adults of R. dominica. Among them, only the hexane extract of A. conyzoides showed insecticidal activity. The hexane extract of this plant species was therefore fractionated by silica gel column chromatography to isolate and purify its bioactive chemical constituents. Three compounds were identified using IR spectra, (1)H NMR, (13)C NMR, HMBC and NOE after gel chromatography: 5,6,7,8,3', 4', 5'-heptamethoxyflavone, 5,6,7,8,3'-pentamethoxy-4', 5'-methylenedioxyflavone and coumarin. The complete assignment of (13)C NMR to 5,6,7,8,3'-pentamethoxy-4', 5'-methylenedioxyflavone was successfully made for the first time. 5,6,7,8,3'-Pentamethoxy-4', 5'-methylenedioxyflavone did not show any insecticidal activity against the four insect species tested. 5,6,7,8,3', 4', 5'-Heptamethoxyflavone showed low activity against D. hyalinata and R. dominica and was not toxic to M. domestica or P. americana. In contrast, coumarin showed insecticidal activity against all four insect pest species tested, with the following order of susceptibility: R. dominica < P. americana < D. hyalinata < M. domestica after 24 h exposure.     

热处理对2种潜伏炭疽菌生长和致病性的影响

 通过测定热处理对潜伏侵染于芒果果实中的胶胞炭疽菌(Colletotrichum gloeosporioides Penz.)和香蕉果实中的芭蕉炭疽菌[C.musae(Berk.& Curt.)Arx.]离体培养菌的生长、繁殖和致病性的影响,结果表明:当温度达55℃和60℃,时间20 min时,对菌体的生长和孢子萌发可起明显抑制或杀伤作用,并降低其致病性。2种炭疽菌中,芭蕉炭疽菌比胶胞炭疽菌对热更敏感。作者认为,果实采后热处理时,应根据果实种类和不同菌,采用不同的处理温度和时间,才能得到更好的效果。     

荔枝炭疽病菌生物学特性的研究

 本文主要研究了温度、湿度、pH值、光照、营养对荔枝炭疽病菌(Colletotrichum gloeosporioides Penz.)生长、产孢和孢子萌发的影响。该菌菌丝生长的温度范围为8~38℃,最适28℃;产生分生孢子的温度范围为12~36℃,最适28~32℃;分生孢子萌发的温度范围为8~38℃,最适28~32℃。在pH 3~10的范围内该菌均能生长和产孢,菌丝生长最适pH 5~6;产生分生孢子最适pH3~4,分生孢子萌发最适pH 6~7。分生孢子在饱和湿度或水滴中萌发快,相对湿度低于85%时不能萌发。光照处理对该菌生长发育无显著性影响。葡萄糖、蔗糖、麦芽糖、果糖对分生孢子萌发有促进作用。分生孢子的致死温度为50℃ 10 min,菌丝体的致死温度为60℃ 30 min。     

Efficacy of certain agrochemicals on Aspergillus spp. and subsequent aflatoxin production in rice

Efficacy of certain fungicides and non-conventional chemicals against Aspergillus spp. contamination and subsequent aflatoxin production in rice was investigated. Among the 10 fungicides tested, carbendazim, contaf plus, folicur, propiconazole and saaf completely inhibited the growth of all Aspergillus spp. and aflatoxin B₁ (AFB1) production at 1 g or ml/kg concentration. Of the five non-conventional chemicals tested, benzoic acid effectively inhibited the mycelial growth of Aspergillus flavus (72%) at 4 g/kg, completely inhibited the Aspergillus parasiticus and Aspergillus niger even at 1 g/kg and Aspergillus ochraceus at 4 g/kg concentration. Vanillin completely reduced the AFB1 production at 4 g/kg of seed followed by sodium chloride with out inhibiting the mycelial growth. This study reveals that fungicides and non-conventional chemicals had effectively inhibited the mycelial growth of Aspergillus spp. and AFB1 production in rice.     

杀菌剂对辣椒疫霉不同形态菌体的毒力差异

室内离体条件下测定了9种杀菌剂对辣椒疫霉各生长阶段的抑制作用。结果表明:烯酰吗啉和氟吗啉对所测定的菌株P1319 4个发育阶段均有显著的抑制作用,EC50值分别为0.14~0.61 μg/mL 和0.18~0.68 μg/mL,EC95值均小于3 μg/mL;甲霜灵对菌丝生长和孢子囊形成有显著的抑制作用,但对休止孢和孢子囊萌发基本无影响;嘧菌酯和百菌清强烈抑制孢子囊形成、休止孢和孢子囊萌发,EC50值分别为0.03~6.15 μg/mL和0.07~1.97 μg/mL;较高浓度的代森锰锌可以抑制辣椒疫霉的孢子囊形成、休止孢萌发和孢子囊直接萌发,但对菌丝生长无抑制作用;三乙膦酸铝、p(ο)-丁酰基苯酚和邻-烯丙基苯酚对P1319的4个发育阶段的抑制作用较弱。     

不同生育阶段黄瓜菌核病菌对几种三唑类杀菌剂的敏感性

采用菌丝生长速率法测定了6种三唑类杀菌剂和4种其他杀菌剂抑制黄瓜菌核病菌Sclerotinia sclerotiorum(Lib.)de Bary菌丝生长的毒力,同时测定了其对菌核形成及萌发的抑制作用。结果表明,甲基硫菌灵抑制菌丝生长的毒力最低,EC50值为65.75 μg/mL;己唑醇和戊唑醇的抑制活性较高, EC50值分别为0.09和0.16 μg/mL,毒力分别是甲基硫菌灵的763.7和423.4倍;其次是丙环唑、氟硅唑、腈菌唑,毒力是甲基硫菌灵的137.3~286.6倍;再次为三唑酮,毒力仅为甲 基硫菌灵的20.6倍;腐霉利、菌毒清抑制菌丝生长的毒力较低,EC50值均大于10 μg/mL。在EC50值 浓度下,所有供试药剂对菌核形成及其数量均没有影响,但三唑类杀菌剂对菌核单重影响较大,各处理所形成的菌核单重降低率均在70%以上;而当药剂浓度在10 μg/mL以下时,己唑醇等三唑类杀菌剂对菌核萌发均没有影响。     

白藜芦醇及其衍生物对植物病原真菌的抑菌活性

为了明确植物源活性成分芪类化合物在农业病害防治中的前景,在离体条件下分别测定了白藜芦醇(Ⅰ)及其衍生物(Ⅱ~Ⅴ)对植物病原真菌菌丝生长的抑制作用以及对菌丝形态和孢子萌发的影响,采用叶片法和温室盆栽法研究了白藜芦醇的防病作用原理。结果表明:5个供试化合物对6种供试病原菌的菌丝生长均有不同程度的抑制作用,但均对番茄早疫病菌 Alternaria solani 的抑制活性最高,其中又以3,5-二羟基-4'-甲氧基二苯乙烯(Ⅲ)的抑制活性最高;白藜芦醇可造成番茄早疫病菌菌丝体畸形,并可抑制该病菌孢子的萌发,但未引起孢子形态改变。叶片法和温室盆栽法试验的结论一致,即白藜芦醇能够抑制病原菌的侵染,对植株起到一定的保护作用。     

Spiral gradient dilution, a rapid method for determining growth responses and 50% effective concentration values in fungus-fungicide interactions

ABSTRACT A new technique, the spiral gradient dilution method, was evaluated for determining 50% effective concentrations (EC(50) values) of fungicides for the inhibition of mycelial growth and conidial germination of various fungi. In this method, an agar medium is plated with a fungicide solution by means of a spiral plater, which applies the fungicide in a 2.5-log dilution in a continuous radial concentration gradient. Fungal inoculum is then placed along radial lines across the gradient. After incubation of the plates, distinct growth shapes were observed in different fungus-fungicide interactions. Mycelial growth responses to increasing fungicide concentrations ranged from abrupt to gradual transitions. Conidial germination responses were similar; in addition, distinct zones of confluent growth, nonconfluent growth, and outlier colonies were also identified, depending on the fungus-fungicide interaction. The fungicide concentration at the radial distance at which 50% reduction of growth or spore germination occurred, compared with growth or germination on unamended media, was calculated by a computer program. EC(50) values were obtained for mycelial growth in 22 fungus-fungicide interactions and for conidial germination in five interactions. The fungi evaluated were members of the Zygomycota, Ascomycota, Basidiomycota, and Deuteromycota. Nine fungicides, belonging to six different chemical classes, were tested. EC(50) values were compared with those obtained by the traditional agar dilution method. In linear regression analyses of the two methods, the models were highly significant (P < 0.01), and coefficients of determination (r(2)) were 0.92 for the mycelial growth assays and 0.94 for the conidial germination assays. Regression slopes were not significantly different from 1 (P > 0.05) with optimal program settings in the software. Estimated bias, coefficients of variation, and actual confidence intervals for the regression slope were 13.5%, 6.5%, and 1.14 +/- 0.14 for the mycelial growth assays and 7.5%, 14.5%, and 1.08 +/- 0.37 for the conidial germination assays. These analyses indicate that the spiral gradient dilution method is accurate and precise compared with the agar dilution method for calculating EC(50) values of fungicides in continuous growth responses to fungicide concentration gradients.     

嫁接茄子根系分泌物对黄萎病菌的化感作用

研究了不同砧木、不同浓度嫁接茄子根系分泌物对茄子黄萎病菌（Verticillium dahliae）孢子萌发和菌丝生长的影响。结果表明,与自根茄相比,所有参试的嫁接茄根系分泌物都不同程度抑制了黄萎病菌孢子萌发和菌丝生长;并且随着根系分泌物浓度的增加,抑制作用增强。     

爵床提取物对辣椒炭疽病菌的抑制作用及其苗期防效

爵床(J usticia procumbens L.)是一种传统的中药材.为探明爵床提取物对辣椒炭疽病菌的抑制作用,采用生长速率法和孢子萌发法分别测定了爵床提取物对辣椒炭疽病菌菌丝生长、孢子产生和孢子萌发的抑制作用,采用苗期盆栽试验观察爵床提取物对辣椒炭疽病的防治效果.结果表明,爵床甲醇提取物可抑制辣椒炭疽病菌菌丝生长、孢子产生和孢子萌发,其活性随着浓度的提高而增强,抑制中浓度分别为4.23、2.16和2.98 mg/mL.通过对爵床提取物处理后病菌孢子萌发形态显微观察,发现病菌孢子芽管长度受到明显抑制甚至不能萌发.当提取物浓度为16mg/mL时对苗期辣椒炭疽病的防效达73.08％.     

咪鲜胺对水稻恶苗病菌及其抗药突变体生长发育的影响

采用菌丝生长速率法和显微镜观察,研究了咪鲜胺对水稻恶苗病菌Fusarium fujikuroi生长发育及菌丝形态的影响。结果表明:咪鲜胺对水稻恶苗病菌敏感菌株及其抗药性突变体的菌丝生长、孢子萌发、芽管伸长均有明显的抑制作用,而对产孢量无影响。当咪鲜胺浓度为0.5 μg/mL时,能够完全抑制敏感菌株菌丝的生长,而不能抑制不同抗性水平抗性菌株的菌丝生长。随抗性倍数从24倍增加到112倍,药剂对抗药突变体菌落扩展的影响逐渐减小。咪鲜胺对敏感菌株菌丝生长的EC50 值为0.047 μg/mL,最小抑制浓度(MIC) 为 0.5 μg/mL。 20.00 μg/mL咪鲜胺可完全抑制亲本敏感菌株的孢子萌发,而对不同抗药突变体孢子萌发和芽管伸长的抑制率分别为84.1% ~89.0%和58.5％ ~65.8%,表明抗药突变体在菌丝生长、孢子萌发、芽管伸长等阶段对药剂的敏感性与亲本菌株相比都有了不同程度的降低。10 μg/mL咪鲜胺处理菌丝12 h后,亲本菌株菌丝顶端膨大畸形,抗药突变体的菌丝顶端膨大,并在菌丝上长有许多细小分枝,表明药剂对抗药突变体次生菌丝生长的抑制作用不明显。     

草酸对重寄生真菌盾壳霉分生孢子萌发和菌丝生长的影响

 本文研究了草酸对核盘菌的重寄生真菌盾壳霉(Coniothyrium minitans)分生孢子萌发和菌丝生长的影响。结果表明:草酸对盾壳霉分生孢子萌发没有明显促进作用,在酸碱性非缓冲基质(水琼脂)和酸碱性缓冲基质中,抑制盾壳霉分生孢子萌发的最低浓度分别为150和700μg/mL。在马铃薯葡萄糖琼脂培养基中,当草酸浓度为100~2000μg/mL时,盾壳霉菌丝能够生长,且浓度为300~500μg/mL的草酸对盾壳霉的菌丝生长具有明显的促进作用。在以草酸为唯一碳源的合成培养基中,在酸碱性非缓冲的条件下,当草酸浓度为100~2000μg/mL时,盾壳霉菌丝能够生长,且草酸浓度为500μg/mL时对盾壳霉菌丝生长具有促进作用,而当草酸浓度为2500μg/mL时,盾壳霉菌丝则停止生长。在酸碱性缓冲的合成基质中,草酸浓度为100~4000μg/mL时,盾壳霉菌丝能够生长,且草酸浓度为1500~2500μg/mL时对盾壳霉菌丝生长具有促进作用。在含草酸钙的混浊培养基(以草酸为唯一碳源)上,盾壳霉菌落区域形成了透明圈。上述结果说明盾壳霉能忍耐一定浓度的草酸而进行分生孢子萌发及菌丝生长,且这种真菌可能对草酸分子具有分解作用。     

浙江省柑橘绿霉病菌对嘧菌酯的敏感性研究

采用孢子萌发法和生长速率法测定了2000~2006年间采自浙江衢州、杭州、金华、丽水的65个柑橘绿霉病病菌Penicillium digitatum菌株对嘧菌酯的敏感性。结果表明:嘧菌酯对供试菌株 孢子萌发和菌丝生长的EC50值均呈单峰分布,分别介于0.020 1 ~0.260 0 μg/mL和0.005 3 ~0.079 4 μg/mL 之间,平均值分别为0.042 6 μg/mL 和0.025 0 μg/mL。敏感性频次分析结果表明,该65个菌株孢子萌发和菌丝生长对嘧菌酯的敏感性频率分布均符合正态分布,其EC50平均值0.042 6±0.030 4 μg/mL 和0.025 0±0.012 9 μg/mL可分别作为柑橘绿霉病菌孢子萌发和菌丝生长对嘧菌酯的敏感基线。     

植物提取物、碳酸钠和碳酸氢钠室内对杨树溃疡病菌生长的抑制作用

 采用菌丝生长速率法用金银花的花、黄花蒿的地上部分、蓝桉果实和黄柏果实的乙醇提取物及其不同极性的组分对杨树溃疡病菌菌丝生长的抑制作用进行了测定。以黄柏果实的抗菌活性最强,其次是金银花的花。抗菌活性成分主要存在于金银花的正丁醇组分、黄花蒿的石油醚组分和乙酸乙酯组分、蓝桉的水部分、黄柏的正丁醇组分。当培养基中碳酸钠和碳酸氢钠的浓度分别为8g/L时(对应的pH值分别为10.24和7.71),菌丝生长抑制率分别为100.00%和79.68%。如果用1mol/L氢氧化钠溶液调节培养基的pH值至10.00,菌丝生长抑制率为40.58%。说明碳酸钠或碳酸氢钠对菌丝生长的抑制作用,一方面是由于改变了培养基的pH值,另一方面可能是由于碳酸根和碳酸氢根离子抑制了菌丝的生长。     

冬青卫矛内生放线菌Streptomyces flavofuscus G1 发酵液中抑菌活性成分研究

从冬青卫矛内生放线菌Streptomyces flavofuscus G1的发酵液中分离得到化合物G13和G19。经核磁共振波谱及质谱分析,并结合相关文献,两个化合物分别被鉴定为phencomycin(G13)和4'-deacetyl-(-)-griseusin A(G19)。抑菌活性测定结果表明:G19对水稻白叶枯病菌Xanthomonas oryzae pv.oryzae、铜绿假单孢菌Pseudomonas aeruginosa(1.203 1)、蜡状芽孢杆菌Bacillus cereus(1.184 6)及金黄色葡萄球菌Staphyloccocus aureus(1.008 9)等供试病原细菌具有强烈的抑制作用,其最低抑菌浓度(MIC)值均为1.56μg/m L;对苹果树腐烂病菌Valsa mali和小麦赤霉病菌Fusarium graminearum菌丝生长亦有明显的抑制作用,其IC50值分别为14.70和24.35μg/m L。而G13对植物病原真菌油菜菌核病菌Sclerotinia sclerotiorum和苹果树腐烂病菌V.mali菌丝生长有强烈的抑制作用,其IC50值分别为5.21和4.82μg/m L。     

18种化学农药与绿僵菌相容性研究

本文检测了18种常用低毒化学农药对生防真菌绿僵菌Metarhizium anisopliae孢子萌发、菌丝生长及产孢的影响,分析它们与绿僵菌的相容性。总体看,杀虫剂、植物生长调节剂和除草剂与绿僵菌的相容性好于杀菌剂。其中,氰戊菊酯与绿僵菌相容性最好,即使在10倍的推荐浓度下,对孢子萌发无影响,对菌丝生长速度和产孢量的抑制率只有25.13%和38.63%。高效氯氟氰菊酯和乐果与绿僵菌也有较好的相容性,在推荐的田间使用浓度下,孢子萌发抑制率为30%左右,对菌丝生长和产孢量基本没有抑制。溴氰菊酯、啶虫脒、吡虫啉、甲霜灵、氟乐灵、阿维菌素、敌百虫、毒死蜱和波尔多液与绿僵菌有一定的相容性。矮壮素、哒螨灵、多菌灵、甲基硫菌灵、代森锰锌和三唑酮与绿僵菌相容性很差。     

福建省番茄灰霉病菌对氟啶胺敏感基线建立及与不同杀菌剂的交互抗性

为明确中国福建省番茄灰霉病菌对氟啶胺的敏感性及其与不同杀菌剂的交互抗性,采用菌丝生长速率法测定了106株采自福建省主要番茄产区的番茄灰霉病菌对氟啶胺的敏感性。结果表明,氟啶胺对福建省番茄灰霉病菌菌丝生长的EC50值在0.0037～0.0452 μg/mL之间,平均值为（0.0221 ±0.0098）μg/mL,其敏感性频率分布呈连续单峰曲线,符合正态分布,因此可将该EC50平均值（0.0221±0.0098）μg/mL作为福建省番茄灰霉病菌对氟啶胺的敏感基线,用于其田间抗药性监测。从106株菌株中选择15株对氟啶胺敏感性不同的菌株,测定了其对嘧霉胺、异菌脲、腐霉利和啶氧菌酯的敏感性。结果表明,供试5种杀菌剂对15株番茄灰霉病菌菌丝生长的平均抑制活性依次为氟啶胺 >异菌脲 >腐霉利 >啶氧菌酯 >嘧霉胺,氟啶胺与异菌脲、腐霉利、啶氧菌酯和嘧霉胺之间均不存在交互抗性。     

紫外光诱导灰葡萄孢产生抗多菌灵菌株的研究

测得多菌灵对Botrytis cinerea野生菌株B_2的最低抑制生长浓度(MIC)值为0.5ppm。然而,经紫外光照射定向诱导,从B_2获得了突变频率为I/0.9—4.5×10~4的抗多菌灵突变菌株。这种抗性菌株在含多菌灵1000ppm的马铃薯蔗糖琼脂培养基上,菌丝生长很少受到抑制,在多菌灵2000ppm情况下仍能生长,难以测定MIC值。诱变抗性菌株(B-UV1)在无药培养基上连续培养8个月,抗性程度仍未下降,但生长速率降低。 B-UV1菌株对苯来持、托布津杀菌剂表现高度的交互抗性,但对克菌丹等4种测试药剂的敏感性,与野生菌株相似。接种草莓时,B-UV1菌株引起的发病率为野生菌株的77.8％,而且病情较轻。     

Effects of low temperature on mycelial growth and spore germination of Botrytis allii in culture and on its pathogenicity to stored garlic bulbs

Botrytis allii was incubated at 20, 10,4, 2,0, – 2 and –4° to investigate effects of temperature on growth, sporulation and germination on potato dextrose agar (PDA), and to estimate incidence and severity of disease on garlic bulbs inoculated with the pathogen during storage. B. allii-was capable of growing to a colony diameter of 17 mm after 20 weeks, sporulating and germinating on PDA at temperature as low as – 4°C. After 12 weeks at –4 and -2°C the mycelial growth was observed only on 45–54% of bulbs wound-inoculated with conidial suspension, and infection was limited to 5 mm in diameter on the surface of inoculation sites without producing any symptoms of the disease. Temperature responses were similar for mycelial growth, conidial germination and infection, but mycelial growth and sporulation was slower and later on garlic bulbs than in culture. Wounds were readily colonized by B. allii. No disease was found on unwounded bulbs that were inoculated with conidial suspensions and with mycelial plugs at various temperatures. Only 16–95% of cloves became infected by contact with other cloves within the same bulb after 12 weeks of storage at temperatures of 4–20 C.     

Inhibition of mycelial growth and sporidial formation in the wheat pathogenNeovossia indica by a polyamine biosynthetic inhibitor

DL-α-Difluoromethylornithine (DFMO), a specific suicide inhibitor of the polyamine biosynthetic enzyme ornithine decarboxylase (EC 4.1.1.17), strongly inhibited mycelial growth and sporidial formation of the wheat pathogen,Neovossia indica, in vitro, while DL-α-difluoromethylarginine (DFMA), the analogous suicide inhibitor of arginine decarboxylase (EC 4.1.1.19), did not. The inhibited mycelial growth and sporidial formation were not only restored by putrescine (polyamine) addition, but were actually enhanced in the putrescine + DFMO cultures. Besides altering mycelial growth and morphology, DFMO also reduced the cell size drastically. The inhibition of fungal polyamine biosynthesis is discussed in relation to selective control of plant disease.