茄病镰刀菌感染致角膜溃疡1例

报告1例由茄病镰刀菌感染引起的角膜溃疡。患者男性,40岁,木工,左眼红、痛、流泪、视力下降10 d。经过角膜清创,系统性及局部抗真菌治疗后好转。致病菌株经真菌学鉴定为茄病镰刀菌,它是真菌性角膜炎的常见致病菌之一。真菌性角膜炎病程进展迅速,能否及早诊治关系预后。真菌学检查是确诊的依据,临床应予重视。     

茄病镰刀菌致小腿慢性溃疡1例报告及病原真菌超微结构的研究

报道1例55岁男性农民,因右小腿溃疡4个月余就诊。取其溃疡组织作真菌培养和病理检查,真菌培养在含氯霉素、放线菌酮和不含氯霉素、放线菌酮的沙堡弱培养基中27℃都长出白色棉絮状菌落,而在不含氯霉素、放线菌酮条件下生长更好;在马玲薯培养基中长出镰刀状大分生孢子,菌种鉴定为茄病镰刀菌。8d后从溃疡处再取标本培养仍为茄病镰刀菌生长。病理切片经银染色后发现念珠状分隔菌丝,用透射电镜对病变组织内的菌体形态进行了超微结构研究。予伊曲康唑口服治疗,但患者未复诊而失访。     

茄病镰刀菌致皮肤透明丝孢霉病1例

目的报告1例茄病镰刀霉引起的皮肤透明丝孢霉病。方法从患者皮损取材作真菌镜检、培养及组织病理学检查。结果直接镜检及组织病理切片均发现真菌菌丝,3次培养均为同一菌株生长。镜下可见大分生孢子基部细胞短、钝圆,小分生孢子呈假头状着生,可见厚壁孢子。根据以上形态学特征鉴定为茄病镰刀菌。结论对于透明丝孢霉病应早期诊断,并进行体外抗真菌药物的敏感试验,明确并有效控制基础疾病。     

温度对茄病镰刀菌生长情况及产孢量的影响

目的 探讨不同孵育温度对茄病镰刀菌生长状态的影响,为获得大量饱子寻找较好方案.方法 茄病镰刀菌按孵育温度不同分为6组,A组、B组、C组、D组、E组分别于20℃,25℃,30℃,35℃,37℃温箱黑暗孵育7d,F组于35℃温箱黑暗孵育3d再于25℃温箱黑暗孵育至7d,各组在培养24h,3d,5d,7d时分别用游标卡尺测量...     

RNA干扰茄病镰刀菌ALP基因及其基因沉默菌株的制备

目的构建RNA干扰质粒载体抑制茄病镰刀菌碱性丝氨酸蛋白酶(ALP)基因,通过检测ALP的表达及酶活性变化筛选出基因沉默菌株。方法构建2个ALP的干扰载体,转化茄病镰刀菌孢子,获得ALP基因沉默菌株△ALP1、△ALP2,通过RT-PCR检测△ALP1、△ALP2的ALP基因mRNA变化,并通过蛋白琼脂廓清率培养基检测ALP酶活性变化,对所得菌株的毒力进行判断。结果 RNA干扰后所获基因沉默菌株中,ALP的mRNA表达量显著低于标准茄病镰刀菌株(F=184.67,P<0.01),其中ΔALP2抑制效果较好、酶活性显著低于标准菌株及△ALP1(q=5.276、5.463,P<0.01)。结论通过LiAc转化法对茄病镰刀菌ALP进行RNA干扰,可有效抑制茄病镰刀菌ALP的表达;ALP基因沉默茄病镰刀菌株的获取,为进行动物体内实验、深入研究ALP在茄病镰刀菌性角膜炎发病机制中的作用、探索新型治疗方法提供思路。     

以大蜡螟幼虫为模型研究伏立康唑抗茄病镰刀菌效果

目的构建大蜡螟幼虫动物茄病镰刀菌感染模型并观察伏立康唑治疗效果。方法选用临床分离获得茄病镰刀菌1株,实验所用孢子悬液浓度梯度为1×10⁵~1×10⁸CFU/mL,根据死亡率筛选出最佳感染浓度,并以此浓度感染大蜡螟幼虫,用伏立康唑(1.5 mg/kg)治疗;同时设置未处理组和生理盐水对照组,实验过程中收集大蜡螟幼虫尸体做病理检测并记录5 d内死亡情况,每24 h记录1次。通过感染后病理组织切片和大蜡螟幼虫生存率两个方面进行评价。结果大蜡螟幼虫在1×10⁷CFU/mL茄病镰刀菌感染后,5 d死亡率达100%,1×10⁷CFU/mL为本次实验组最佳感染浓度;大蜡螟幼虫感染第2 d死亡虫体病理检测发现大量茄病镰刀菌菌丝和孢子,经伏立康唑治疗后,单个幼虫体内菌体减少,菌体内菌丝减少;大蜡螟幼虫生存曲线显示伏立康唑治疗组较单茄病镰刀菌感染组生存率显著增高(P<0.05)。结论大蜡螟幼虫能够作为茄病镰刀菌体内感染的动物模型,并可用于观察药物治疗效果。     

茄病镰刀菌致ICR小鼠局部皮肤感染的实验研究

目的建立茄病镰刀菌感染皮肤的ICR小鼠模型。方法将茄病镰刀菌三个不同浓度的菌悬液分别接种于正常和免疫抑制ICR小鼠受损及正常未受损的皮肤,于接种后第7、14、21、28天处死动物,取皮疹和各脏器进行真菌镜检、培养和组织病理学检查。结果正常和免疫抑制组皮肤受损的小鼠均出现皮疹,而免疫抑制组皮疹程度重,持续时间长,且真菌镜检、培养及组织病理学大都可见菌丝。皮肤未受损的小鼠不发生皮疹。实验小鼠均未出现系统播散。结论接种109CFU/mL或1010CFU/mL茄病镰刀菌的孢子悬液于免疫抑制ICR小鼠擦伤的皮肤,可有效造成ICR小鼠的茄病镰刀菌皮肤感染。     

茄病镰刀菌致足部透明丝孢霉病1例

目的报道1例茄病镰刀菌引起的足部透明丝孢霉病。方法询问病史及体检,取足部的皮损行皮肤病理检查、真菌培养及致病菌的形态学观察、鉴定。结果致病菌为茄病镰刀菌,给予局部病灶切除及伊曲康唑治疗,随访3个月,病灶明显缩小。结论本病例证实为茄病镰刀菌引起的足部透明丝孢霉病,经口服伊曲康唑及局部病灶切除治疗,疗效明显。     

14例茄病镰刀菌所致角膜溃疡临床分析

目的探讨真菌性角膜溃疡的病原学特点、临床表现及抗真菌综合治疗的方法。方法对2004年10月-2006年10月送检的疑似感染的角膜标本进行镜检、培养及菌种鉴定。对其中14例病历资料完整的茄病镰刀菌所致角膜溃疡患者进行临床分析。结果在33例送检标本中分离出茄病镰刀菌24株（72．7％）。上述14例患者中,有3例角膜穿孔合并眼内炎行眼球内容物除去术,3例行结膜瓣或羊膜移植,1例角膜移植,7例经非手术治疗保留较好视力。结论茄病镰刀菌是我国北方真菌性角膜炎的主要致病菌,可导致视力严重受损且治愈困难。早期诊断配合以抗真菌为主的综合治疗可阻止病情进展,明显改善视力。     

Purification and characterization of a nitrilase from Fusarium solani O1

An intracellular nitrilase was purified from a Fusarium solani O1 culture, in which the enzyme (up to 3000 U L⁻¹) was induced by 2-cyanopyridine. SDS-PAGE revealed one major band corresponding to a molecular weight of approximately 40 kDa. Peptide mass fingerprinting suggested a high similarity of the protein with the putative nitrilase from Gibberella moniliformis. Electron microscopy revealed that the enzyme molecules associated into extended rods. The enzyme showed high specific activities towards benzonitrile (156 U mg⁻¹) and 4-cyanopyridine (203 U mg⁻¹). Other aromatic nitriles (3-chlorobenzonitrile, 3-hydroxybenzonitrile) also served as good substrates for the enzyme. The rates of hydrolysis of aliphatic nitriles (methacrylonitrile, propionitrile, butyronitrile, valeronitrile) were 14–26% of that of benzonitrile. The nitrilase was active within pH 5–10 and at up to 50 °C with optima at pH 8.0 and 40–45 °C. Its activity was strongly inhibited by Hg²⁺ and Ag⁺ ions. More than half of the enzyme activity was preserved at up to 50% of n-hexane or n-heptane or at up to 15% of xylene or ethanol. Operational stability of the enzyme was examined by the conversion of 45 mM 4-cyanopyridine in a continuous and stirred ultrafiltration-membrane reactor. The nitrilase half-life was 277 and 10.5 h at 35 and 45 °C, respectively.     

茄病镰刀菌单克隆抗体的制备及胶体金免疫层析试纸条的研发*

Objective: The cell lines secreting specific monoclonal antibodies (McAbs) were prepared by using Fusarium solani, one of the pathogenic fungi causing root rot of Fritillaria thunbergii, and the colloidal gold immunochromatographic test strip based on McAbs was developed to provide scientific basis for detecting root rot of F. thunbergii. Methods: Hybridoma technology was used to obtain cell lines that could secrete specific McAbs against F. solani using the whole protein extract of F. solani as the antigen. The specificity, titer, sensitivity and binding protein of McAbs were detected by indirect ELISA and Western blot. Colloidal gold particles were prepared by trisodium citrate reduction method and McAbs were labeled to prepare colloidal gold immunochromatographic strip. Results: Three cell lines secreting specific McAbs against F. solani were obtained, which were named as FsA3, FsG6 and FsD4. The detection sensitivity of FsA3 was 24.41 ng / mL, and that of both FsG6 and FsD4 was 12.21 ng / mL. FsA3, FsG6 and FsD4 had strong reactions to F. solani, and had no cross-reaction to Alternaria tenuissima, A. alternata, Botrytis cinerea, F. equiseti, F. incarnatum, F. oxysporum, Phoma sp., and Phomopsis oblonga. The colloidal gold immunochromatographic strip based on FsG6 showed only a quality control line when detecting the tissue culture seedlings of F. thunbergii. When 100 ng F. solani antigen or the samples of F. thunbergii infested with root rot disease were detected, there were visible quality control lines and test lines. Conclusion: The specificity and sensitivity of the McAbs and test strip are sufficient to detect F. solani isolated from diseased strains of F. thunbergii, which provides the technical support for the rapid detection of root rot of F. thunbergii in the field.     

High-level expression and characterization of Fusarium solani cutinase in Pichia pastoris

High-level extracellular production of Fusarium solani cutinase was achieved using a Pichia pastoris expression system. The cutinase-encoding gene was cloned into pPICZαA with the Saccharomyces cerevisiae α-factor signal sequence and methanol-inducible alcohol oxidase promoter by two different ways. The additional sequences of the c-myc epitope and (His)₆-tag of the vector were fused to the C-terminus of cutinase, while the other expression vector was constructed without any additional sequence. P. pastoris expressing the non-tagged cutinase exhibited about two- and threefold higher values of protein amount and cutinase activity in the culture supernatant, respectively. After simple purification by diafiltration process, both cutinases were much the same in the specific activity and the biochemical properties such as the substrate specificity and the effects of temperature and pH. In conclusion, the high-level secretion of F. solani cutinase in P. pastoris was demonstrated for the first time and would be a promising alternative to many expression systems previously used for the large-scale production of F. solani cutinase in Saccharomyces cerevisiae as well as Escherichia coli.     

香芹酚和丁香酚对腐皮镰刀菌的抑菌活性及抑菌机理

【背景】腐皮镰刀菌(Fusarium solani)是世界上最具破坏性的土传病原菌之一,严重影响作物的产量及品质。因此寻找并开发广谱的可持续生物防治药剂迫在眉睫,而植物次级代谢物为自然界筛选生物源天然杀菌剂提供了丰富的原料。【目的】研究香芹酚和丁香酚对腐皮镰刀菌的抑菌活性,探究其可能涉及的抑菌机理。【方法】采用菌丝生长速率法、十字交叉法和孢子萌发法分析香芹酚和丁香酚对菌丝和孢子的抑制活性,通过扫描电镜(scanning electron microscope,SEM)观察菌丝微观形态的变化,利用荧光染料碘化丙啶(propidium iodide,PI)观察细胞膜的损伤情况,并测定其对腐皮镰刀菌细胞膜胞外电导率、蛋白质含量和麦角固醇合成的影响。【结果】香芹酚和丁香酚对腐皮镰刀菌菌丝生长和孢子萌发均有显著的抑制效果,并呈现剂量依赖效应,EC50值分别为92.39μL/L和263.00μL/L。SEM结果表明,2种精油处理腐皮镰刀菌后,其细胞壁和细胞膜均遭到破坏从而不能维持菌丝正常的线性形态,表现出不同程度的弯曲、褶皱和凹陷。PI染色结果发现,2种精油处理严重破坏了腐皮镰刀菌细胞膜的完整性和...     

腐皮镰刀菌对不同品种核桃抗性生理指标的影响

为分析腐皮镰刀菌(Fusarium solani)引起的核桃根腐病对寄主抗性生理指标的影响,该研究以铁核桃、绵阳早熟、新疆2号3个核桃品种为材料,研究各品种受腐皮镰刀菌侵染后,不同时期的发病情况以及丙二醛(MDA)、可溶性糖含量以及过氧化物酶(POD)、多酚氧化酶(PPO)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)的动态变化。结果表明：接种病原菌50 d 后,铁核桃100％发病,属高感品种,绵阳早熟和新疆2号发病率分别为23.33％和16.67％,属抗病品种;在50 d 监测范围内,各品种对照组各项叶部生理指标仅出现上下波动且差异不显著;然而,各品种核桃接入腐皮镰刀菌后,可溶性糖含量随时间迅速下降,MDA 含量和 POD 活性总体表现为先上升趋势,SOD、PPO 活性总体则表现为先上升后下降的变化趋势,并且5个指标品种间差异显著,而 CAT 活性变化无规律。相关性分析表明,可溶性糖、MDA 含量、SOD、POD、PPO 活性可以作为核桃品种抗性评价指标。该研究结果为深入研究由腐皮镰刀菌引起的核桃根腐病发病机制以及通过诱导等措施提高其抗性的研究提供了理论基础。     

马铃薯糖苷生物碱对腐皮镰刀菌呼吸作用及其活性氧代谢的影响

【背景】腐皮镰刀菌(Fusariumsolani)是一种分布较为广泛的致病性真菌,可引起多种植物的土传病害,是枸杞根腐病的主要致病菌之一。马铃薯糖苷生物碱(potatoglycosidealkaloids,PGA)为一类植物源提取物,其原材料种植广泛、成本低廉,对腐皮镰刀菌具有较强的抑菌活性。【目的】探究PGA对腐皮镰刀菌呼吸作用及活性氧(reactive oxygen species, ROS)代谢的影响,从能量代谢角度揭示其可能的抑菌机理。【方法】以马铃薯芽为原材料,采用乙酸-氨水沉淀法提取PGA,以腐皮镰刀菌为供试病原菌,通过PDA和PDB培养体系考察PGA对腐皮镰刀菌菌丝生长的抑制作用,并确定半最大效应浓度(EC50);采用氧电极仪检测PGA对腐皮镰刀菌呼吸作用的影响;并通过PDB液态培养试验体系,研究PGA对腐皮镰刀菌抗氧化酶系统、ROS及其代谢产物丙二醛(malondialdehyde, MDA)的影响。【结果】PGA处理下菌丝体呼吸速率明显下降,且随着PGA处理时间的延长,表现出一定的时间浓度效应。PGA处理使胞内过氧化氢(H2O2)和超氧阴离子(O2-)含量显著增高(P...     

Effect of the high polycyclic aromatic hydrocarbon, benzo[a]pyrene, on the lipid content of Fusarium solani

The purpose of the present paper was to study the effect of the high polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene, on the lipid [fatty acid (FA) and sterol] composition and content of the fungi Fusarium solani and F. oxysporum, respectively recognized as good and poor PAH degraders. The major FAs and the major sterol that characterized the tested Fusarium strains were C16:0, C18:1, C18:2, and ergosterol. Lipid profiles of F. solani remained unchanged with the addition of benzo[a]pyrene in the culture media at all concentrations and duration of treatment. However, in the presence of benzo[a]pyrene, significant decreases in FA content, which reached 18 % in young cultures and 28 % in mature colonies, were registered. Similarly, the sterol content of F. solani was reduced by 27 % in the presence of benzo[a]pyrene. In contrast, no modification in lipid profile and lipid content were observed with F. oxysporum, a strain recognized as a low benzo[a]pyrene degrader.     

Suppression of maize root diseases caused by Macrophomina phaseolina, Fusarium moniliforme and Fusarium graminearum by plant growth promoting rhizobacteria

A plant growth-promoting isolate of a fluorescent Pseudomonas sp. EM85 and two bacilli isolates MR-11(2) and MRF, isolated from maize rhizosphere, were found strongly antagonistic to Fusarium moniliforme, Fusarium graminearum and Macrophomina phaseolina, causal agents of foot rots and wilting, collar rots/stalk rots and root rots and wilting, and charcoal rots of maize, respectively. Pseudomonas sp. EM85 produced antifungal antibiotics (Afa⁺), siderophore (Sid⁺), HCN (HCN⁺) and fluorescent pigments (Flu⁺) besides exhibiting plant growth promoting traits like nitrogen fixation, phosphate solubilization, and production of organic acids and IAA. While MR-11(2) produced siderophore (Sid⁺), antibiotics (Afa⁺) and antifungal volatiles (Afv⁺), MRF exhibited the production of antifungal antibiotics (Afa⁺) and siderophores (Sid⁺). Bacillus spp. MRF was also found to produce organic acids and IAA, solubilized tri-calcium phosphate and fixed nitrogen from the atmosphere. All three isolates suppressed the diseases caused by Fusarium moniliforme, Fusarium graminearum and Macrophomina phaseolina in vitro. A Tn5:: lac Z induced isogenic mutant of the fluorescent Pseudomonas EM85, M23, along with the two bacilli were evaluated for in situ disease suppression of maize. Results indicated that combined application of the two bacilli significantly (P = 0.05) reduced the Macrophomina-induced charcoal rots of maize by 56.04%. Treatments with the MRF isolate of Bacillus spp. and Tn5:: lac Z mutant (M23) of fluorescent Pseudomonas sp. EM85 significantly reduced collar rots, root and foot rots, and wilting of maize caused by Fusarium moniliforme and F. graminearum (P = 0.05) compared to all other treatments. All these isolates were found very efficient in colonizing the rhizotic zones of maize after inoculation. Evaluation of the population dynamics of the fluorescent Pseudomonas sp. EM85 using the Tn5:: lac Z marker and of the Bacillus spp. MRF and MR-11(2) using an antibiotic resistance marker revealed that all the three isolates could proliferate successfully in the rhizosphere, rhizoplane and endorhizosphere of maize, both at 30 and 60 days after seeding. Four antifungal compounds from fluorescent Pseudomonas sp. EM85, one from Bacillus sp. MR-11(2) and three from Bacillus sp. MRF were isolated, purified and tested in vitro and in thin layer chromatography bioassays. All these compounds inhibited R. solani, M. phaseolina, F. moniliforme, F. graminearum and F. solani strongly. Results indicated that antifungal antibiotics and/or fluorescent pigment of fluorescent Pseudomonas sp. EM85, and antifungal antibiotics of the bacilli along with the successful colonization of all the isolates might be involved in the biological suppression of the maize root diseases.     

Biocatalytic application of nitrilases from Fusarium solani O1 and Aspergillus niger K10

The nitrilases from Fusarium solani O1 and Aspergillus niger K10 showed a broad substrate specificity for carbocyclic and nonaromatic heterocyclic amino nitriles, the preferred substrates being five-membered γ-amino nitrile (±)-1a, six-membered γ-amino nitriles (±)-3a, (±)-5a and (±)-6a, pyrrolidine-3-carbonitriles (±)-9a and (±)-10a as well as piperidine-4-carbonitriles 14a and 15a. Both enzymes showed a strong diastereopreference for cis- vs. trans-γ-amino nitriles. The electronic and steric effects of N-protecting groups affected the reactivity of the nitriles. Amides as by-products of the nitrilase-catalyzed reaction were produced from heterocyclic amino nitriles (±)-9a, (±)-10a, 14a and 15a by the A. niger enzyme but only from nitrile (±)-9a by the F. solani enzyme.     

Inheritance of resistance to Fusarium graminearum in wheat

To study the inheritance of resistance in wheat to Fusarium graminearum, six resistant cultivars from China were crossed to two susceptible cultivars. The parents and their progenies were evaluated in the greenhouse for resistance to the spread of scab within a spike. A central floret was inoculated by injecting a droplet of inoculum at the time of anthesis. Inoculated plants were kept in a moist chamber for three subsequent nights. The proportion of scabbed spikelets was recorded six-times from 3-days to 21-days after inoculation, and the area under the disease progress curve (AUDPC) was calculated from these proportions. One to three genes, depending on the cultivar, conditioned resistance to scab as reflected by the AUDPC. A simple additive-dominance effect model fitted the segregation data for 8 of the 11 crosses. Dominance and epistatic effects were significant in a few crosses. These effects increased resistance in some crosses but decreased resistance in others. However, relative to additive effects, dominant and epistatic effects accounted for only a small portion of the genetic effects in the populations evaluated. The importance of additive effects means that it should be possible to accumulate different genes to enhance resistance to scab in wheat. Received: 15 December 1998 / Accepted: 17 June 1999