柑橘内生燕麦镰刀菌Gds-1对柑橘青霉病的防治研究

柑橘青霉病是柑橘果实采后危害最大的病害。为得到一株可有效应用于柑橘青霉病防治的柑橘内生真菌菌株,进行了柑橘果实内生菌分离、离体与活体试验筛选、菌株形态学与多基因位点分子系统发育鉴定、菌株活体应用效果与作用特点以及菌株代谢产物稳定性与应用防效试验。结果表明:筛选所得的柑橘内生真菌Gds-1在离体与活体条件下均对柑橘青霉菌有较好的抑制效果;经多基因位点联合系统发育分析,将Gds-1鉴定为燕麦镰刀菌Fusarium avenaceum;提前48 h施用Gds-1菌体,对柑橘青霉病的预防效果最好 (防治效果为90.67%);Gds-1菌丝不会损害健康柑橘果实表皮,但可以在柑橘果实表皮伤口和被柑橘青霉菌感染的组织部位发挥作用,从而抑制柑橘青霉菌;当温度在 ?80~100 ℃和pH=5~10时,Gds-1无菌发酵液对柑橘青霉菌的拮抗作用高效稳定;单独施用Gds-1无菌发酵滤液28 d和56 d时,对柑橘青霉病的防效与100 μg/mL的抑霉唑相当,显著高于清水对照。本研究首次报道了生防镰刀菌F. avenaceum Gds-1对柑橘青霉病的防治作用,为柑橘果实采后生物源防腐剂的选择提供了新来源,为镰刀菌生物防治机理研究提供了新思路。     

Fluorescence microscope studies of Ustilago maydis and Penicillium italicum after treatment with imazalil or fenpropimorph

Imazalil and fenpropimorph caused morphological changes in sporidia of Ustilago maydis and in germinating conidia of Penicillium italicum, as observed by fluorescence microscopy using an optical brightener. Sporidia of U. maydis appeared swollen, distorted, multicellular and, sometimes, branched; conidia of P. italicum swelled in size, and extension of the germ tubes was strongly inhibited. Mycelium of P. italicum, treated with fenpropimorph, showed much enlarged hyphal diameters and relatively short distances between septa. Imazalil and fenpropimorph also caused an irregular deposition of β–1,3 and β-1,4 polysaccharides, probably chitin, in U. maydis and P. italicum. The latter phenomenon is discussed in relation to the following observed effects of fungicides that inhibit ergosterol biosynthesis: differences in effect on the morphology of budding and filamentous fungi; preferential inhibition of yeast-hypha conversion in dimorphic fungi; disorganisation of septum formation in budding fungi; and inhibition of spheroplast formation from budding fungi.     

Sensitivity of Penicillium digitatum and P. italicum to Postharvest Citrus Fungicides in California

ABSTRACT Penicillium digitatum isolates (326), collected in California citrus groves and packinghouses, were assayed qualitatively for their sensitivity to imazalil, thiabendazole, and o-phenylphenol. Eighteen typical triple-resistant isolates, acquired in each of 3 years (1988, 1990, and 1994), were assayed quantitatively for their sensitivity to each of the three fungicides. No significant differences were found in the mean sensitivity of the isolates collected in different years. However, the proportion of isolates that were resistant to all three fungicides increased from 43% in 1988 to 77% in 1990 and 74% in 1994. Imazalil-resistant biotypes of P. digitatum were isolated frequently in California packinghouses, while resistant P. italicum was rare. No fungicide-resistant biotypes of either species were collected from citrus groves. Wild-type P. italicum was slightly less sensitive than wild-type P. digitatum to all three fungicides. The concentration of imazalil producing 50% growth inhibition (EC(50)) was three times greater when the age of the P. digitatum assay inoculum was increased from 12 to 24 h. Activity of imazalil increased with pH of the assay medium in the range pH 5.1 to 5.9, reflecting the greater concentration of dissociated imazalil at the higher pH value.     

Effects of imazalil on sterol composition of sensitive and DMI-resistant isolates of Penicillium italicum

Imazalil differentially inhibited dry weight increase of 10-hour-old germlings of wild-type and DMI-resistant isolates ofPenicillium italicum in liquid malt cultures. EC₅₀ values ranged from 0.005 to 0.27 μg ml^?1. In all isolates ergosterol constituted the major sterol (over 95% of total sterols) in the absence of the fungicide. Therefore, DMI-resistance cannot be associated to a deficiency of the C-14 demethylation enzyme in the ergosterol biosynthetic pathway. Imazalil treatment at concentrations around EC₅₀ values for inhibition of mycelial growth resulted in a decrease in ergosterol content and a simultaneous increase in 24-methylene-24,25-dihydrolanosterol content in all isolates. A correlation existed between the imazalil concentration necessary to induce such changes in sterol composition and the EC₅₀ values for inhibition of mycelial growth of the different isolates. The reason for the differential effects of imazalil on sterol composition in the variousP. italicum isolates may be due to decreased accumulation of the fungicide in the mycelium and to other yet non-identified mechanisms of resistance.     

Effects of imazalil on a wild-type and fungicide-resistant strain of Aspergillus nidulans

The effects of different incubation conditions on toxicity and uptake of imazalil by mycelium of a wild-type (003) and fungicide-resistant (R264) strain of Aspergillus nidulans were determined. In agar plate and liquid shake culture, imazalil was 200 and 40 times less toxic to the R264 strain, respectively, than to the wild-type strain. Inhibition of C-4 desmethyl sterol (ergosterol) biosynthesis occurred rapidly in mycelium of both strains at minimum growth inhibitory concentrations. Imazalil was neither detoxified nor converted into a toxic compound by mycelial suspension. Increased uptake of imazalil by the two strains occurred as concentrations of the fungicide were increased. However, the percentage uptake of imazalil by the wild-type strain was highest at the lowest concentration. These results suggest that binding in the wild-type strain involved a small number of high-affinity sites which became saturated as fungicide concentrations increased; and that at higher concentrations considerable nonspecific binding occurred in both strains. Uptake of imazalil during the initial 10 min of incubation was considerably lower in resistant than in the wild-type strain. However, upon prolonged incubation, both strains took up near equal amounts of fungicide. Uptake of fungicide by both strains was not inhibited by incubation at low temperature but was stimulated by respiratory inhibitors. These data support, in part, the hypothesis that resistance to imazalil, as reported previously for fenarimol (M. A. de Waard and J. G. M. van Nistelrooy, Pestic. Biochem. Physiol. 13, 255 (1980)), is based on reduced uptake of fungicide by mycelium of the resistant mutant.     

Effect of fenpropimorph and imazalil on sterol biosynthesis in penicillium italicum

Fenpropimorph was found to be highly active against Penicillium italicum (EC₅₀ 0.01/μg ml^?1). Conidia of P. italicum, treated with low concentrations of fenpropimorph, swelled in size and showed distorted germ tubes. During the initial stages of mycelial growth, fenpropimorph had little or no effect on the dry weight increase, which became strongly inhibited within 24 h after addition of the toxicant (0.05, 0.1 and 0.2 μg ml^?1). Irregular deposition of β–1,3 and β–1, 4 polysaccharides, probably chitin, was observed after treatment with fenpropimorph or imazalil. Fenpropimorph (0.05 and 0.2 μ ml^?1) caused the accumulation of a major demethyl-sterol that was different from ergosterol. It was identified as ergosta-8, 14, 24(28)-trien-3β-ol by mass, infrared, ultraviolet and proton nuclear magnetic resonance, spectrometric procedures. At both concentrations, the accumulation was already detected after incubation for 2 h. In contrast, imazalil (0.1 μg ml^?1) caused the accumulation of several methyl- and dimethyl-sterols which were tentatively identified as eburicol (24-methylene-24, 25-dihydrolanosterol), 4, 14α-dimethylergosta-8, 24(28)-dien-3-one, 14α-methylergosta-8, 24(28)-dien-3-one and obtusifoliol (4, 14α-dimethylergosta-8, 24(28)-dien-3α-ol). The accumulation of ergosta-8, 14,24(28)-trien-3β-ol indicates inhibition of the Δ¹⁴-reductase in P. italicum in a similar manner to that found previously in Ustilago maydis.     

Antifungal mode of action of imazalil

Imazalil had no effect on the initial growth of mycelia of Penicillium italicum (for 10 hr) or Aspergillus nidulans (for 2 hr). In P. italicum during this period neither respiration nor cell permeability was affected, but uptake of [³²P]phosphate, [¹⁴C]leucine, or [¹⁴C]uridine was partially inhibited. The initial (5 hr) inhibition of substrate uptake coincided with a 50% reduction in ergosterol content. Within 0.5 hr, incorporation of [¹⁴C]acetate into C-4-desmethyl sterols was strongly inhibited in mycelia of A. nidulans treated with 0.5 μg/ml of imazalil. However, radioactivity in C-4-methyl and dimethyl sterols exceeded that of control cultures. Concentrations of imazalil as low as 0.005 μg/ml caused short-term (1 hr) declines of incorporation into desmethyl sterols and increases into the C-4-methyl and dimethyl sterols. Incorporation into phospholipids, triglycerides, and free fatty acids was not affected. These data suggest that the primary antifungal action of imazalil is inhibition of demethylation in the biosynthesis of ergosterol.     

Genetic aspects of resistance to imazalil in Aspergillus nidulans

Mutant strains of Aspergillus nidulans have been isolated which display a low level of resistance to imazalil, a recently developed systemic fungicide. Agar growth tests showed that A. nidulans is about three times as sensitive to imazalil when growing on supplemented minimal medium (SM) as compared with complete medium. This effect was reduced by adding glutamic acid to the SM.Imazalil resistance was found to be based on a multigenic system; 21 single gene mutations define 8 loci which were allocated to 6 different linkage groups. Mutations at different loci lead pleiotropically to one or more of the following properties: hypersensitivity or resistance to acriflavin, cycloheximide and neomycin, resistance to chloramphenicol and fenarimol, and to cold sensitivity. Of 120 cycloheximide-resistant strains isolated, 98 were also imazalil-resistant.Recombination analysis of different imazalil-resistant strains with mutations at three loci resulted in additive effects, giving strains with a high level of resistance to imazalil.The results indicate that imazalil may interfere either with protein synthesis like cycloheximide, chloramphenicol and neomycin or with synthesis or function of cell membranes. Interference with cell membrane synthesis might lead to altered sterol composition, resulting in selective permeability to different compounds.     

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。     

Characterisation of energy-dependent efflux of imazalil and fenarimol in isolates of Penicillium italicum with a low,medium and high degree of resistance to DMI-fungicides

Differential accumulation of [¹⁴C]imazalil and [¹⁴C]fenarimol by germlings of wild-type and DMI-resistant isolates ofPenicillium italicum was studied at various pH values. At pH 7 and 8 the low-resistant isolate E_300–3 accumulated 22% and 35%, respectively, less imazalil than the wild-type isolate W₅. Imazalil accumulation at pH 5 and 6 was similar. Isolate E_300–3 also accumulated less fenarimol as compared with the wild-type isolate. This difference was much more obvious than for imazalil and was observed at all pH values tested. Differences in accumulation of both imazalil and fenarimol between low (E_300–3), medium (H₁₇) and high resistant (I₃₃) isolates were not observed. These results suggest that decreased accumulation of DMIs is responsible for a low level of resistance only and that additional mechanisms of resistance might operate in isolates with a medium and high degree of resistance. With all isolates fenarimol accumulation was energy-dependent. This was not obvious for imazalil.The wild-type and DMI-resistant isolates had a similar plasma membrane potential as determined with the probe [¹⁴C]tetraphenylphosphonium bromide ([¹⁴C]TPP⁺). Various test compounds, among which ATPase inhibitors, ionophoric antibiotics and calmodulin antagonists, affected the accumulation of [¹⁴C]TPP⁺, [¹⁴C]imazalil and [¹⁴C]fenarimol. No obvious correlation between the effects of the test compounds on accumulation levels of the fungicides and [¹⁴C]TPP⁺ could be observed. These results indicate that the plasma membrane potential does not mediate the efflux of DMI fungicides byP. italicum.     

Effects of imazalil on sterol composition of sensitive and DMI-resistant isolates of Penicillium italicum

Imazalil differentially inhibited dry weight increase of 10-hour-old germlings of wild-type and DMI-resistant isolates ofPenicillium italicum in liquid malt cultures. EC₅₀ values ranged from 0.005 to 0.27 g ml^–1. In all isolates ergosterol constituted the major sterol (over 95% of total sterols) in the absence of the fungicide. Therefore, DMI-resistance cannot be associated to a deficiency of the C-14 demethylation enzyme in the ergosterol biosynthetic pathway. Imazalil treatment at concentrations around EC₅₀ values for inhibition of mycelial growth resulted in a decrease in ergosterol content and a simultaneous increase in 24-methylene-24,25-dihydrolanosterol content in all isolates. A correlation existed between the imazalil concentration necessary to induce such changes in sterol composition and the EC₅₀ values for inhibition of mycelial growth of the different isolates. The reason for the differential effects of imazalil on sterol composition in the variousP. italicum isolates may be due to decreased accumulation of the fungicide in the mycelium and to other yet non-identified mechanisms of resistance.Imazalil remt differentieel de toename in drooggewicht van 10-uur-oude gekiemde sporen van wild-type en DMI-resistente isolaten vanPenicillium italicum in vloeistofcultures van moutextract. De EC₅₀ waarden voor groei van de verschillende isolaten lopen uiteen van 0,005 tot 0,27 g ml^–1. In afwezigheid van het fungicide is in alle isolaten ergosterol het belangrijkste sterol (meer dan 95% van het totaal). DMI-resistentie kan daarom niet in verband staan met deficiëntie van het C-14 demethyleringsenzym in de ergosterol biosynthese. Imazalilbehandeling van mycelium bij concentraties rond de EC₅₀ waarde voor groeiremming, resulteerde bij alle isolaten in een afname van het ergosterolgehalte en een gelijktijdige toename van het gehalte aan 24-methyleen-24,25-dihydrolanosterol. Er bestaat dus een nauwe correlatie tussen de imazalilconcentratie die noodzakelijk is om vergelijkbare veranderingen in sterolsamenstelling te induceren en de EC₅₀ waarde voor remming van myceliumgroei van de verschillende isolaten. De differentiële effecten van imazalil op de sterolsamenstelling van de verschillendeP. italicum isolaten kunnen worden veroorzaakt door verminderde accumulatie van het fungicide in het mycelium en door andere, nog niet geïdentificeerde resistentiemechanismen.     

交枝顶孢霉杀柑橘全爪螨活性及其生物学特性研究

为了明确交枝顶孢霉Acremonium implicatum CQBBW8的杀螨活性及其生物学特性,采用触杀毒力法检测CQBBW8菌株对柑橘全爪螨的致死率,通过单因素试验测试不同培养基、碳源、氮源、温度、pH及紫外线对CQBBW8菌株生长、产孢及其分生孢子萌发的影响。结果表明,CQBBW8菌株对柑橘全爪螨Panonychus citri(McGregor)的校正致死率为54.42%,LC_(50)为1.2×10~6个/mL,LT_(50)值为5.175 9d。最佳生长和产孢培养基分别为PDA和SEA;菌丝生长最适碳源和氮源为甘露醇和蛋白胨,产孢最适碳源和氮源为可溶性淀粉和氯化铵;最适生长温度为25℃,最适产孢和分生孢子萌发温度为30℃;最适生长和产孢pH为6.0,最适萌发pH为7.0;紫外线对菌丝生长影响不明显,对产孢影响较大,紫外线照射时间越长,孢子萌发率越高。综上,CQBBW8菌株对柑橘全爪螨有较强的毒力,对营养需求不高,环境适应性较强,具有开发为杀螨真菌制剂的潜力。     

13种常用农药和玫烟色棒束孢SCAU-IFCF01的相容性研究

为了研究常用农药和生防真菌玫烟色棒束孢Isaria fumosorosea之间的相容性,本文测定了13种常用农药对玫烟色棒束孢孢子萌发、菌丝生长及产孢量的影响。试验结果表明,杀虫剂与玫烟色棒束孢的相容性优于杀菌剂。其中,高效氯氰菊酯与玫烟色棒束孢相容性最好,在常规浓度下,对玫烟色棒束孢孢子萌发和菌丝生长的抑制率分别只有2.42%和1.62%,对产孢基本无抑制作用,苏云金芽胞杆菌和氯氰菊酯与玫烟色棒束孢也有较好的相容性,二者在常规使用浓度下对孢子萌发、菌丝生长、产孢量的抑制率分别为6.05%、2.83%、22.41%和16.3%、7.81%、16.67%。高效氯氟氰菊酯、氟啶脲、丙溴磷与玫烟色棒束孢有一定的相容性。丙环唑、百菌清、戊唑醇、木霉菌、阿维菌素、甲维盐与玫烟色棒束孢的相容性则相对较差。     

Development of resistance to thiabendazole in Helminthosporium solani (silver scurf) as a result of potato seed tuber treatment

Applying thiabendazole to potato seed tubers affected with silver scurf caused by Helminthosporium solani sensitive to thiabendazole decreased the severity of disease on progeny tubers at harvest, but about 50% of the isolates from these were resistant to the fungicide. The disease was not decreased when samples of the progeny tubers were treated with thiabendazole and planted in the following year, and the incidence of resistant isolates increased. Resistant isolates continued to be present when tubers were planted in the next 2 years without fungicide treatment. Treatment with a mixture of thiabendazole and imazalil also decreased the disease and fewer isolates were resistant than when treated with thiabendazole alone, although the proportion increased after treatment with the mixture in the following year. When seed tubers were infected with thiabendazole-resistant H. solani , silver scurf on progeny tubers was not affected by thiabendazole applied to the seed tubers but was decreased by the mixture of thiabendazole and imazalil. Imazalil was equally effective against H. solani sensitive or resistant to thiabendazole.
Some isolates of H. solani had grey aerial mycelium and of 516 of these isolates obtained in 4 years 29% were resistant to the fungicide. Other isolates produced small, black colonies and their frequency increased with thiabendazole treatment of seed tubers. Of 244 of these isolates, 62% were resistant.     

The determination of imazalil on potatoes and its use in controlling potato storage diseases

Methods are described for the extraction and analysis by gas-liquid and high-pressure liquid chromatography of the fungicide imazalil, 1-(β-allyloxy-2, 4-dishlorophenethyl) imidazole, on potatoes. Before storage, over 80% was recovered from potatoes treated with 0.01–3.0 mg imazalil kg^?1, with a detection limit of 2 μg kg^?1. Imazalil applied to potatoes at 10 g t^?1 before storage decreased the incidence of gangrene (Phoma exigua), silver scurf (Helminthosporium solani), skin spot (Polyscytalum pustulans) and black scurf (Rhizoctonia solani), and was at least as effective as thiabendazole applied at 40 g t^?1. At 1 g t^?1 it also decreased skin spot and silver scurf. Incidence of black dot (Colletotrichum coccodes) was unaffected by these fungicide treatments.     

Metabolism of imazalil by wild-type and DMI-resistant isolates of Penicillium italicum

Metabolism of imazalil (1-[2-(2,4-dichlorophenyl)-2-(2-propenyloxy)ethyl]-1H-imidazole) inPenicillium italicum isolates with a wild-type sensitivity and with various degrees of resistance to sterol demethylation inhibitors was studied in liquid cultures. The metabolite 1-[2(2,4-dichlorophenyl)-2-(2,3-dihydroxypropyloxy)ethyl]-1H-imidazole (R42243) was detected in the culture filtrate after prolonged incubation. The metabolism occurred in the propenyl side chain of imazalil probably through epoxidation and hydratation. This is the first report of such a conversion of imazalil in fungi. R42243 was much less toxic toP. italicum than imazalil. Therefore, the metabolism can be regarded as a detoxification step. Both wild-type and resistant isolates metabolized imazalil, but metabolism by resistant isolates was faster than by the wild-type isolate. This is probably caused by a relatively strong inhibition of growth of the wild-type isolate by the fungicide. Results indicate that the detoxification of imazalil does not operate as a mechanism of resistance. This conclusion was confirmed by the fact that resistant isolates showed cross-resistance to miconazole and R42243, which had a similar structure as imazalil except for the propenyl side chain.     

南瓜叶枯病病原菌生物学特性研究

本文对南瓜叶枯病病原菌瓜链格孢（Alternaria cucumerina）进行了生物学特性研究,结果表明：温度、pH、光照对其生长和产孢都有一定的影响。该病原菌菌丝生长、产孢和萌发的适宜温度为28 ℃,pH为7时菌丝生长最快,pH为8时最易产孢。25 ℃下连续黑暗培养产孢最多。碳、氮源对菌丝生长和产孢量有一定的影响,碳源中甘露醇组菌丝生长最快,山梨醇处理产孢量最多。氮源中牛肉浸膏最适宜菌丝生长和产孢,硫酸铵最差。该菌致死温度为50 ℃。     

Techniques and parameters used in compatibility tests between Beauveria bassiana (Bals) Vuill and in vitro phytosanitary products

The objective of this work was to test and compare different techniques used in tests for compatibility between the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuill and phytosanitary products, in order to develop a protocol for in vitro tests. Four modes of contact were studied between B bassiana (CG432) and the fungicides iprodione (Rovral) 500 g litre(-1) SC) and azoxystrobin (Amistar) 500 g kg(-1) WG), and the insecticide endosulfan (Thiodan) 350 g litre(-1) EC), at three rates. The techniques consisted in incorporating the products into the culture medium (IM), combining the conidia into the products mix (MP) and spraying the products before (SB) and after (SA) inoculation of the fungus on Petri dishes. The fungitoxic effect of the products was studied on the basis of parameters such as germination, colony-forming units (CFUs), vegetative growth and sporulation. The effect of azoxystrobin on conidial germination was significantly higher in the IM technique than in the other techniques. With regard to CFUs, the IM and SB techniques showed the greatest differences relative to the control. Vegetative growth and sporulation were more affected when azoxystrobin was sprayed before the fungus was applied. At the commercial rate, iprodione had a greater effect on the CFU parameter in the IM and MP techniques, and on vegetative growth in the IM technique, than the other techniques used; however, there was no significant difference occurred between the techniques at the commercial rate with respect to germination and sporulation. Endosulfan was more toxic to germination in techniques SB and SA, and to the CFUs parameter in techniques IM and MP. As to vegetative growth and sporulation, regardless of rate, a more pronounced effect was observed in IM than in the other techniques. It can be inferred that there are differences between techniques and that a standardization of the compatibility tests is necessary. Another inference is that these techniques should reflect realistic exposure of the fungus to chemical formulations under field conditions.     

Real-time PCR法定量检测柑橘绿霉病菌对抑霉唑的抗性频率

 抑霉唑被广泛用来防治由指状青霉菌(Penicillium digitatum)引起的柑橘绿霉病。已有研究表明,柑橘绿霉病菌对抑霉唑的抗性由CYP51基因启动子区5个126-bp转录增强子的简单串联重复和126-bp转录增强子上199-bp的特异性片段插入所引起。基于这2种抗性分子机制,通过设计特异引物和优化条件,建立real-time PCR高通量分子检测技术,用于快速检测柑橘包装贮藏库中绿霉病菌群体对抑霉唑的抗性频率F_R,指导科学用药。     

芒果疮痂病菌生物学特性研究

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