唾液乳杆菌抑制镰孢霉的研究

目的 研究唾液乳杆菌抑制产毒镰孢霉的生物学性能,初步探索抑菌机制.方法 以禾谷镰孢霉和尖孢镰孢霉2种典型霉菌为指示菌,唾液乳杆菌为测试对象,对霉菌孢子萌芽、孢子生长和菌丝体生长3个生理阶段进行抑制效应观察.结果 10%的唾液乳杆菌耗尽上清就能抑制83%的禾谷镰孢霉孢子和50%尖孢镰孢霉孢子萌芽;耗尽上清24 h内能显著抑制镰孢霉孢子的生长;96 h内孢霉菌丝体的生长.结论 唾液乳杆菌产生的有机酸对禾谷镰孢霉和尖孢镰孢霉生长起主要抑制作用.     

Antagonism and Action Mechanism of Antifungal Metabolites from Streptomyces rimosus MY02

The genus of Streptomyces , a saprophytic Gram-positive bacterium, has properties, which make them useful as pharmaceutical and biocontrol agents. A streptomyces strain MY02 from soil samples showed significant antagonism against 14 plant pathogenic fungi including Fusarium oxysporum f. sp. cucumarinum . Antifungal metabolite(s) SN06 from the culture of the strain MY02 were extracted with n -butanol and purified by silica gel column chromatography. The minimum concentration of SN06 inhibiting any visible fungal growth of F. oxysporum f. sp. cucumarinum is 12.5 μg/ml by twofold serial dilutions method. The mycelia of F. oxysporum f. sp. cucumarinum treated with SN06 were observed under the normal optics microscope. The results showed that some cells of hyphae began to dilate and formed some strings of beads. The cytoplasm oozed out of the cells with the culture time and so most of the cells became empty. The hyphae broke into many segments and then collapsed after 48 h. After inoculated in potato dextrose medium for 48 h, the filtrate of mycelia treated with 1% NaCl containing 12.5 μg/ml SN06 was scanned using ultraviolet spectrophotometer and absorption peak at 260 nm showed that the mycelia cell membrane of F. oxysporum f. sp. cucumarinum was broken and that nucleic acid oozed out of the cell.     

Interaction between Orobanche crenata and its host legumes: unsuccessful haustorial penetration and necrosis of the developing parasite

BACKGROUND AND AIMS: Orobanche species represent major constraints to crop production in many parts of the world as they reduce yield and alter root/shoot allometry. Although much is known about the histology and effect of Orobanche spp. on susceptible hosts, less is known about the basis of host resistance to these parasites. In this work, histological aspects related to the resistance of some legumes to Orobanche crenata have been investigated in order to determine which types of resistance responses are involved in the unsuccessful penetration of O. crenata. METHODS: Samples of resistance reactions against O. crenata on different genotypes of resistant legumes were collected. The samples were fixed, sectioned and stained using different procedures. Sections were observed using a transmission light microscope and by epi-fluorescence. KEY RESULTS: Lignification of endodermal and pericycle host cells seems to prevent parasite intrusion into the root vascular cylinder at early infection stages. But in other cases, established tubercles became necrotic and died. Contrary to some previous studies, it was found that darkening at the infection site in these latter cases does not correspond to death of host tissues, but to the secretion of substances that fill the apoplast in the host-parasite interface and in much of the infected host tissues. The secretions block neighbouring host vessels. This may interfere with the nutrient flux between host and parasite, and may lead to necrosis and death of the developing parasite. CONCLUSIONS: The unsuccessful penetration of O. crenata seedlings into legume roots cannot be attributed to cell death in the host. It seems to be associated with lignification of host endodermis and pericycle cells at the penetration site. The accumulation of secretions at the infection site, may lead to the activation of xylem occlusion, another defence mechanism, which may cause further necrosis of established tubercles.     

蔓割病不同抗性甘薯品种的茎部细胞结构观察

蔓割病是我国南方薯区甘薯主要病害之一,本研究采用直接观察、显微和超微结构观测等方法,对高抗、中感和高感蔓割病的3个甘薯品种(高抗品种:金山57,中感品种:热薯1号,高感品种:新种花)接种蔓割病菌28 d植株充分发病后其茎基部细胞的侵染结构进行了观察。结果表明,在用清水作对照处理时,3个品种茎部组织的细胞形态和结构正常且完整,细胞代谢强。高抗品种金山57无论是接种组还是对照组,均未发现病原菌丝的存在,其茎下部、中部和上部细胞结构相对完整。中感品种热薯1号,蔓割病菌从其茎基部侵入后导致茎基部细胞破损坏死,而中部寄主-病原互作较为活跃和典型,造成养分运输受阻,茎基部接种后病原菌丝会沿着寄主茎部的维管束和其他组织一直向寄主的茎部末端蔓延,遭受侵染后的部位其细胞反应与高感品种新种花类似。高感品种新种花遭受蔓割病原菌侵染后,病原菌菌丝从茎基部新鲜剪口侵入后进入表皮细胞、皮层、维管束并在甘薯茎基部蔓延至中部、上部,直至寄主整株枯死,甘薯茎的木质部导管出现侵填体,质壁分离;与此同时,细胞壁的沉积物及乳突在病原菌的入侵处形成,各种无定型物质或纤丝构成的织网迅速包围入侵菌丝。     

应用PCR-RFLP和巢式PCR检测黄瓜尖镰孢菌

以3株黄瓜尖镰孢菌(Fusarium oxysporum f.sp.cucumarinum)、23株镰孢菌属(Fusariumspp.)真菌和分离自土壤的20株真菌、6株细菌和7株放线菌为材料,采用化学裂解法提取总DNA,进行PCR-RFLP和巢式PCR检测,试验证明PCR-RFLP程序不能完全区分Fusarium属内不同种,而巢式PCR对黄瓜尖镰孢菌具有特异性.运用优化的PCR-RFLP和巢式PCR检测程序对染病黄瓜组织进行了检测,结果表明,两种方法均可在接种发病早期(未显症时)检测出黄瓜枯萎病菌,PCR-RFLP在感病品种接种后3d即可检测到病原菌,而巢式PCR在接种后5d才能检测到病原菌.     

生防菌根系定殖竞争作用对西瓜枯萎病发病机理的影响

【目的】西瓜枯萎病是由西瓜专化型尖孢镰刀菌(Fusarium oxysporum f.sp.niveum)引起的一种常见的毁灭性土传病害,对镰刀菌同属非致病性菌株与致病性菌株存在的竞争作用进行研究,有助于获得新的具有生防效果的菌株,从而拓宽西瓜枯萎病生物防治的手段。【方法】利用选择性培养基和稀释平板计数法对温室盆栽试验中西瓜根际和非根际土壤及植物组织中非致病性轮枝镰刀菌菌株(Fusarium verticillioides XA)与致病性尖孢镰刀菌(Fusarium oxysporum LD)进行计数,确定其在西瓜植株根际和组织中的定殖情况。【结果】将从田间西瓜枯萎病发病植株根部分离获得的菌株XA和LD接入健康土壤中,接种菌株XA既不会引起西瓜枯萎病发病症状,也不会影响西瓜植株生物量,但接种菌株LD导致严重发病症状。与单接种LD处理相比较,双接种(XA+LD)处理地上部鲜重和地上部干重都分别增加了151.2%和110%。XA菌株能成功定殖于西瓜根系,但在茎基部没有检测到。在接种菌株LD的处理中植物组织和土壤中致病性镰刀菌的数量达到(1.58 4.85)×104CFU/g。与单接种LD处理相比,双接种菌株XA和LD处理植物茎基部、根系、根际土壤和土体土壤致病性镰刀菌的数量分别下降63.3%、66.1%、3.3%和24.4%,根系、根际土壤和土体土壤非致病性镰刀菌的数量增加到(0.35 3.84)×104CFU/g;双接种处理对西瓜枯萎病的防效达57.8%。【结论】非致病性轮枝镰刀菌菌株XA可有效降低致病性尖孢镰刀菌LD对西瓜植株的定殖侵染能力,对西瓜枯萎病具有一定的生防效果。     

月腺大戟根总黄酮对尖孢镰刀菌抑制作用的研究

采用生长速率法和孢子萌发法分别测定月腺大戟根总黄酮对尖孢镰刀菌菌丝生长和分生孢子萌发的抑制率,显微观察总黄酮处理尖孢镰刀菌后菌丝体形态结构的变化,并测定菌丝体相对电导率,菌丝体内超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性。结果表明:月腺大戟根总黄酮对尖孢镰刀菌菌丝生长和分生孢子萌发均有显著的抑制作用,抑制率随总黄酮浓度增加而增高,20 mg/L时抑制率达100%。总黄酮处理后的尖孢镰刀菌菌丝较细,分支减少,透明度差,液泡数量增多且形成较大的液泡;菌丝体细胞膜透性增加,SOD、CAT活性呈先上升后下降的趋势。以上实验结果可为植物病害生物防治及开发植物源农药方面提供理论依据和实验技术。     

尖孢镰刀菌生产蒽醌色素的液体发酵条件研究

优化了尖孢镰刀菌液体发酵生产蒽醌类红色素的发酵条件。通过单因素实验和正交优化实验,确定最佳产色素发酵培养基为：可溶性淀粉30％,(NH4)2SO4 3％,MgSO4 0.3％,KH2PO4 4％,pH 6.0。产色素最适培养条件为：初始pH6.0,装液量20％,接种量10％,吐温-80添加量1％,温度28℃,摇床转速200r/min,发酵周期120h。此条件下,色素效价即可达到8.184U/ml,比优化前提高了1.8倍。国内首次对尖孢镰刀菌所产蒽醌色素进行研究,为其进一步应用奠定基础。     

Specific detection of the toxigenic species Fusarium proliferatum and F. oxysporum from asparagus plants using primers based on calmodulin gene sequences

Fusarium proliferatum and Fusarium oxysporum are the causal agents of a destructive disease of asparagus called Fusarium crown and root rot. F. proliferatum from asparagus produces fumonisin B1 and B2, which have been detected as natural contaminants in infected asparagus plants. Polymerase chain reaction (PCR) assays were developed for the rapid identification of F. proliferatum and F. oxysporum in asparagus plants. The primer pairs are based on calmodulin gene sequences. The PCR products from F. proliferatum and F. oxysporum were 526 and 534 bp long, respectively. The assays were successfully applied to identify both species from the vegetative part of the plants.     

海南省香蕉枯萎病病原菌的鉴定

香蕉枯萎病在海南省为首次报道。在Komada改良培养基鉴定的基础上,用温室人工接种法对采自海南省各市县香蕉种植区的18个香蕉和粉蕉假茎分离物进行鉴定。结果表明香蕉枯萎病菌的两种分离物在培养特性和致病性上存在明显区别,分离自粉蕉的12个菌株为1号生理小种,而分离自香蕉的6个菌株为4号生理小种。     

石灰碳铵熏蒸与施用生物有机肥对连作黄瓜和西瓜枯萎病及生物量的影响

采用稀释涂布平板计数法,研究了石灰碳铵及碳铵熏蒸对黄瓜和西瓜连作土壤尖孢镰刀菌数量的影响,以及熏蒸后施用生物有机肥对黄瓜和西瓜枯萎病的防控效果及植株生长的影响.结果表明:与对照相比,石灰碳铵及碳铵熏蒸后,连作土壤中黄瓜尖孢镰刀菌的数量分别下降95.4%及71.4%,西瓜尖孢镰刀菌的数量分别下降87.2%及64.2%;多因素方差分析表明,熏蒸、施用有机肥及作物种类均对土壤中尖孢菌数量、枯萎病发病率、防控率及生物量有显著影响;与未熏蒸施用普通有机肥对照相比,石灰碳铵熏蒸后施用生物有机肥能显著减少后茬黄瓜或西瓜土壤中尖孢镰刀菌的数量并显著降低枯萎病发病率,防控率高达91.9%及92.5%,同时显著增加了植株的株高、茎粗、SPAD值及干质量.表明石灰碳铵熏蒸及施用生物有机肥能够降低土壤中尖孢镰刀菌数量,有效防控黄瓜和西瓜枯萎病的发生并促进其植株生长.     

Isolation and characterization of endophytic streptomycete antagonists of Fusarium wilt pathogen from surface-sterilized banana roots

A total of 131 endophytic actinomycete strains were successfully isolated from surface-sterilized banana roots. These isolates belonged to Streptomyces (n=99), Streptoverticillium (n=28), and Streptosporangium (n=2) spp. The remaining 2 isolates were not identified. About 18.3% of the isolates inhibited the growth of pathogenic Fusarium oxysporum f. sp. cubense on banana tissue extract medium. The most frequently isolated Streptomyces sp. strain S96 was similar to Streptomyces griseorubiginosus. About 37.5% of the S. griseorubiginosus strains were antagonistic to F. oxysporum f. sp. cubense. The antagonism of strain S96 was lost when FeCl(3) was introduced into the inhibition zone. In vivo biocontrol assays showed that the disease severity index (DSI) was significantly (P=0.05) reduced and mean fresh weight increased (P=0.001) in plantlets treated with strain S96 compared to those grown in the absence of the biocontrol strain. These findings indicate the potential of developing siderophore-producing Streptomyces endophytes for the biological control of fusarium wilt disease of banana.     

The determination of physiological race of Fusarium oxysporum f. sp. lycopersici of tomato in Zhejiang, China

A group of differential tomato lines was used to identify the races of Fusarium oxysporum f. sp. lycopersici in Zhejiang, China. Marmande verte carries no resistant genes and Marporum carries gene I-1. Both lines Motelle and Mogeor have Gene I-1 and I-2. Tomato seedlings of eighteen days after sowing were inoculated with an isolate of Fusarium oxysporum f. sp. lycopersici, No. 98-2 and kept in a growth chamber. The seedlings were evaluated at fourteen days after inoculation. Results showed that Marmande verte and Marporum were severely infected by the pathogen and established as susceptible. Motelle and Mogeor were not infected and established as resistant. These results indicated that the isolate No. 98-2 represented the race 2 of Fusarium oxysporum f. sp. lycopersici and gene I-2 is necessary for obtaining resistance to this pathogen in the Zhejiang region.     

Growth Characteristics of Cytomegalovirus in Human Fibroblasts with Demonstration of Protein Synthesis Early in Viral Replication

In high-multiplicity infection of human fibroblasts, human cytomegalovirus of WI-38 human diploid cells produced early cell rounding 6 to 24 h after inoculation. This early cell rounding was caused only by inoculation with infectious virions. Inhibitors of protein synthesis, but not DNA inhibitors, prevented this cytopathic effect. Apparently, a new protein is synthesized in infected fibroblasts from about 2 h postinoculation. Infectivity of cell-associated and supernatant infectious virus reached maximal levels at 5 to 7 and 10 days postinoculation, respectively. Synthesis of DNA, infectious virus, complement-fixing antigen, and precipitin antigen all began between 24 and 48 h, with the bulk of synthesis occurring 48 to 96 h postinoculation.     

绿色木霉TV41对尖孢镰刀菌FW0在西瓜植株空间分布和枯萎病防控效果的影响

【目的】为进一步探究盆栽试验条件下绿色木霉TV41 (Trichoderma viride TV41)对尖孢镰刀菌FW0 (Fusarium oxysporum FW0)在西瓜植株空间分布的影响以及对西瓜枯萎病的防控效果。【方法】通过定期检测不同处理西瓜根际/根表尖孢镰刀菌的数量、西瓜植株根内/茎内尖孢镰刀菌的数量以及植株侧根被侵染比例和尖孢镰刀菌在植株内的侵染进程,进行多次盆栽试验并统计发病率。【结果】当绿色木霉和尖孢镰刀菌接种量均为5×105孢子/g基质时,绿色木霉TV41在西瓜根际/根表的定殖数量明显高于尖孢镰刀菌FW0的数量,接种了绿色木霉TV41的处理,根际/根表尖孢镰刀菌的数量(103/g基质)显著低于仅接种FW0的对照(104/g基质);绿色木霉TV41不仅能够有效减缓尖孢镰刀菌在西瓜植株内的侵染进程,而且能够有效降低西瓜植株根内、茎内尖孢镰刀菌的数量。与对照(只接种FW0)相比,接种绿色木霉后西瓜枯萎病的发病率从66%降低到27%。【结论】绿色木霉TV41能够通过影响尖孢镰刀菌FW0在西瓜植株的空间分布,从而有效防控西瓜枯萎病的发生,防控效果达到60%。     

Response of embryo axes of germinating seeds of yellow lupine to Fusarium oxysporum.

Defence responses of embryo axes of Lupinus luteus L. cv. Polo were studied 48-96 h after inoculation with Fusarium oxysporum Schlecht f.sp. lupini. The infection restricted the growth of embryo axes, the lengths of infected embryo axes 72 and 96 h after inoculation were 11 and 12 mm less in the controls, respectively, while their masses c. 0.03 g less than in the controls. The concentration of H2O2 in embryo axes of inoculated germinating seeds was higher than in the control. This was probably a consequence of oxidative burst as well as H2O2 generation by the invading necrotrophic fungal pathogen. EPR-based analyses detected the presence of free radicals with g1 and g2 values of 2.0052 +/- 0.0004 and 2.0031 +/- 0.0005, respectively. Concentrations of the radicals 72 and 96 h after inoculation were 50% higher than in the control. The values of the spectroscopic splitting coefficients suggest that they are quinone radicals. However, inoculated embryo axes possess a number of adaptive mechanisms protecting them from oxidative damage. A twofold increase in catalase (CAT, EC 1.11.1.6) activity was evidenced in embryo axes infected with F. oxysporum Schlecht f. sp. lupini, as compared to the control 48-96 h after inoculation. Superoxide dismutase (SOD, EC 1.15.1.1) activity 96 h after inoculation was 80% higher than in the control. Furthermore, EPR-based analyses revealed a higher concentration of Mn2+ ions after 72 h for inoculated embryo axes, as compared to the control. On the other hand, no increase was detected in the concentration of thiobarbituric acid reactive substances (products of lipid peroxidation) in infected embryo axes. The protective mechanisms induced in lupine embryo axes in response to F. oxysporum Schlecht f.sp. lupini were compared with responses to infections with pathogenic fungi elicited in other plant families.     

The possible involvement of peroxidase in defense of yellow lupine embryo axes against Fusarium oxysporum

Peroxidase activity (EC 1.11.1.7) towards phenolic substrates, i.e. pyrogallol, syringaldazine and guaiacol, and ascorbate peroxidase activity (EC 1.11.1.11) were analyzed in embryo axes of Lupinus luteus L. cv. Polo cultured on Heller medium for 96h after inoculation with the necrotrophic fungus Fusarium oxysporum f.sp. Schlecht lupini. Four variants were compared: inoculated embryo axes cultured with 60mM sucrose (+Si) or without it (-Si), and non-inoculated embryo axes cultured with 60mM sucrose (+Sn) or without it (-Sn). Between 0 and 96h of culture, peroxidase activity towards the phenolic substrates increased in all variants except -Si, where a decrease was noted in peroxidase activity towards syringaldazine and guaiacol, but not towards pyrogallol. In +Si tissues, a considerable increase in enzyme activity towards these substrates was recorded starting from 72h of culture. Lignin content of +Si tissues increased already at the first stage of infection, i.e. 24h after inoculation. Additionally, in +Sn tissues, high ascorbate peroxidase activity was observed during the culture. Its activity increased in +Si tissues, beginning at 72h after inoculation. However, this was lower than in +Sn tissues. At 72h after inoculation, a considerably stronger development of the infection was observed in -Si than in +Si tissues during our earlier research [Morkunas, I. et al., 2005. Sucrose-stimulated accumulation of isoflavonoids as a defense response of lupine to Fusarium oxysporum. Plant Physiol Biochem 2005; 43: 363-73]. Both peroxidases assayed towards phenolic substrates and ascorbate peroxidase was less active in -Si tissues than in -Sn tissues. Hydrogen peroxide concentration was much higher in -Si than in +Si tissues. These results indicate that peroxidases may be some of the elements of the defense system that are stimulated by sucrose in yellow lupine embryo axes in response to infection caused by F. oxysporum.     

Biological Control of Fusarium oxysporum f.sp. asparagi by Applying Non-pathogenic Isolates of F . oxysporum

Root rot severity of asparagus plants grown in sterilized field soil inoculated with Fusarium oxysporum f . sp . asparagi (Foa) was reduced by more than 50% when the soil was precolo nized by each of 13 non - pathogenic (np) isolates of F. oxysporum originating from asparagus roots or field soils . In a greenhouse experiment , application of six np isolates to naturally infested field soil was followed by a 23 - 49% decrease of disease severity , depending on the isolate . One of them , Fo47 originating from Fusarium suppressive soil in France , was applied to field plots infested with Foa . Foa root rot was not suppressed in asparagus plants grown for 1 year in these plots . Pathogenic and np isolates extensively colonized the root surface and isolates of both types infected the roots of asparagus plants grown in sterilized field soil , with significant differences among the np isolates . Inoculation of sterilized field soil with np isolates reduced germination of Foa chlamydospores by 43 - 64% depending on the isolate used . It is concluded that np isolates of F. oxysporum can suppress asparagus root rot caused by Foa in naturally infested field soil . The differences for root colonization capacity among the np isolates imply that selection for this trait might reveal isolates that perform better under field conditions .     

新疆甜瓜Fom-2基因同源序列的克隆及分析

近年来甜瓜枯萎病蔓延迅速,危害严重,分离克隆甜瓜抗病基因,利用转基因技术改良甜瓜抗病品性是一种从根本上解决甜瓜枯萎病危害的新途径。根据已知Fom-2基因序列设计特异引物,采用RT-PCR技术从新疆甜瓜抗病性品种黄旦子中扩增出580bp的cDNA片段,序列分析表明它与已发表Fom-2基因具有高度同源性基因片段,命名为X-Fom-2。以X-Fom-2为探针对新疆甜瓜黄旦子、伽什瓜、红心脆、金丽-2号进行Southem blot和Northem blot,结果表明,X-Fom-2以多拷贝形式存在。X-Fom-2在抗病品种黄旦子、金丽2号有表达且在病原菌诱导后增强,而在感病品种伽什瓜、红心脆中没有表达。