无机盐、激素与真菌联合诱导土沉香抗逆能力的研究

为了探究无机盐、激素与真菌联合诱导土沉香(Aquilaria sinensis)抗逆性及其与结香前期木芯变色长度之间的相关关系,以10年生土沉香为材料,采用均匀试验设计,开展了3种无机盐、3种激素及3种真菌组合试验。结果表明:(1)土沉香树体POD和SOD活性和MDA含量呈先升高后降低趋势,CAT活性在诱导后1天最高,随后呈下降趋势;(2)无机盐与真菌处理下土沉香的抗逆能力大于激素与真菌处理,其中无机盐与真菌组合试验的处理4(1.0%CaCl2+0.5%FeSO4+2.0%NaCl+黑绿木霉∶腐皮镰孢∶龙眼焦腐(1∶1∶1))和激素与真菌组合试验的处理2(0.01%茉莉酸甲酯+0.1%乙烯利+0.2%水杨酸钠+黑绿木霉∶腐皮镰孢∶龙眼焦腐(1∶1∶1))分别为同类处理最高;(3)土沉香抗逆能力高低与木芯变色长度存在显著正相关性;(4)理论上,土沉香抗逆能力最强诱导组合分别为0.93%CaCl2+0.53%FeSO4+2.5%NaCl+黑绿木霉∶腐皮镰孢∶龙眼焦腐(1∶1∶1)和0.005%茉莉酸甲酯+0.006%乙烯利+0.2%水杨酸钠+黑绿木霉∶腐皮镰孢∶龙眼焦腐(1∶1∶1)。     

对马铃薯具促生抗病作用的砖红镰刀菌及其遗传转化体系构建

本研究从药用植物马比木Nothapodytes pittosporoides的花瓣中分离获得了1株真菌,经形态学与ITS分子共同鉴定为砖红镰刀菌Fusarium lateritium。使用该菌处理马铃薯后发现,显著增强了马铃薯对晚疫病菌的耐受性,处理组植株感染率为37.5%,相较对照组87.5%的感染率显著降低;植株生长测定发现,处理组的马铃薯生物量、株高、根系生物量和主根数相较于对照组分别提高了1.25、1.19、2.3、1.47倍,表明该真菌对马铃薯还具有促生作用。为探究砖红镰刀菌促生抗病的分子机理,检测了植物生长素合成和免疫防御相关基因的表达模式,结果表明处理组植株生长素合成相关基因（StYUC5）显著上调,而免疫相关激素茉莉酸和水杨酸合成相关基因（StPI-I、StPAL和StPR1A）也不同程度上调。由此推测,砖红镰刀菌通过调控植物激素相关基因的表达介导马铃薯的促生和抗病。为了进一步探究砖红镰刀菌对马铃薯促生抗病的分子基础,构建了其遗传转化体系,并进行了优化,获得了GFP标记菌株。     

Secondary Metabolites Produced by a Strain of Fusarium oxysporum Used for Striga Control in West Africa

The production of fusaric acid and other toxins by a strain of Fusarium oxysporum used for control of the weed Striga hermonthica (Del.) Benth. was investigated. Culturing of the strain under optimal conditions for toxin production produced small amounts of fusaric acid and dehydrofusaric acid but no other toxins reported to be produced by some strains of F. oxysporum. Culturing of the fungus under conditions similar to those that would be used in the field produced no detectable toxins of concern for human health.     

Selection of an Isolate of the Insect Pathogenic Fungus Metarhizium anisopliae Virulent to the Lettuce Root Aphid, Pemphigus bursarius

The virulence of hyphomycete entomopathogenic fungi was measured in laboratory bioassays against the lettuce root aphid, Pemphigus bursarius, a serious pest of field lettuce grown in the UK. Of 25 isolates of fungi examined, only one isolate, Metarhizium anisopliae 391.93, killed lettuce root aphids consistently. This fungus was isolated originally from the closely related saltmarsh aphid, P. trehernei. The median lethal concentration of conidia at 10 days post6- 1 inoculation estimated from five independent bioassays was 2.45 × 10⁶ conidia ml^-1. The fungus had no significant effect on the mean number of offspring/aphid produced, but it sporulated 6 profusely on host cadavers, producing approximately 4 × 10⁶ conidia/cadaver 14 days after treatment, and diseased aphids died attached to plant roots. It thus has the potential to spread through densely packed colonies of P. bursarius feeding on the roots of susceptible or partially resistant plants.     

基于重组毕赤酵母的fusaruside生物合成

目的:构建产fusaruside的毕赤酵母菌株,解决天然小分子免疫抑制剂fusaruside的来源问题。方法:从禾谷镰刀菌Fusarium graminearum PH-1中扩增获得合成fusaruside的相关基因-3位去饱和酶[Δ3(E)-SD]和10位去饱和酶[Δ10(E)-SD]基因;并通过2A肽策略构建两种基因的共表达载体,转化到毕赤酵母GS115中进行双酶的诱导表达;对诱导后的毕赤酵母菌体进行甲醇和二氯甲烷的处理后,经高效液相色谱质谱联用仪(HPLC-MS)检测其中产物变化。结果:3位去饱和酶和10位去饱和酶在毕赤酵母中成功共表达,SDS-PAGE显示3位去饱和酶分子量约为48kDa,10位去饱和酶分子量约为65kDa; HPLC-MS显示重组酵母可以产生fusaruside。结论:与fusaruside原产菌株镰刀菌相比,该酵母菌的发酵时间更短、产量更高,为fusaruside的进一步开发与应用奠定基础。     

The Common Black Ant, Lasius niger (Hymenoptera: Formicidae), as a Vector of the Entomopathogen Lecanicillium longisporum to Rosy Apple Aphid, Dysaphis plantaginea (Homoptera: Aphididae)

The potential for common black ant Lasius niger workers to vector conidia of the entomopathogenic fungus Lecanicillium longisporum to colonies of the rosy apple aphid Dysaphis plantaginea was assessed in laboratory and field experiments. Scanning electron microscope studies showed that L. niger workers which were artificially contaminated with L. longisporum conidia, carried conidia primarily on their tarsae but also on antennae and mandibles. Neither L. niger workers nor larvae were susceptible to infection with L. longisporum. Workers which were artificially contaminated with conidia of L. longisporum initiated infection in colonies of healthy D. plantaginea on apple. Mortality due to L. longisporum was recorded as 68.3, 30.8 and 3.7% of aphids under laboratory, semi-field and field conditions, respectively.     

低温茬口空闲期土壤强还原消毒对西瓜枯萎病的影响

为探明低温茬口空闲期土壤强还原消毒(RSD)对西瓜枯萎病的防控效果,设置对照(CK)、淹水对照(FCK)、淹水添加2%(w/w)苜蓿粉(AL)、0.25%(w/w)乙酸(AC)以及AL+AC的RSD处理进行西瓜盆栽试验,采用定量PCR和Illumina Miseq等测定真菌、尖孢镰刀菌数量及真菌群落组成,并统计发病率和产量。结果表明: 与CK相比,FCK和各RSD处理均能显著降低尖孢镰刀菌数量及其在真菌中的比例,杀菌效果达86.1%～94.6%;AL、AC以及AL+AC处理显著增加了西瓜产量,降低了枯萎病发病率,且枯萎病防控效果分别为63.2%、73.7%和94.7%,而FCK与CK无显著差异。与CK相比,AL和AC处理显著改变了土壤真菌群落组成,FCK处理对真菌群落无显著影响;RSD处理均显著增加了柄孢壳属、假散囊菌、地丝菌、粪盘菌、韦斯特壳属以及久浩酵母菌等优势属的相对丰度,且其中大部分与尖孢镰刀菌数量及其在真菌中的比例以及发病率呈显著负相关关系。综上,低温茬口空闲期的RSD处理可以通过重塑真菌群落组成有效防控西瓜枯萎病的发生。     

尖孢镰刀菌亚麻专化型Folprp4基因参与调控菌丝生长和分生孢子发生

目的: 通过对尖孢镰刀菌中Folprp4基因的鉴定,揭示其在尖孢镰刀菌中的功能及致病相关性。方法: 基于同源重组原理,根据测定出的Folprp4基因序列,应用Split-Marker重组技术构建含有潮霉素抗性基因(hph)的基因缺失盒。将基因缺失盒经PEG介导转化到野生型原生质体中,在含有潮霉素B的TCC培养基上筛选转化子,通过PCR正负筛查获得Folprp4基因缺失突变株(ΔFolprp4)。构建含有Folprp4基因的载体pZDH1,并将其转化到敲除突变体中进行互补测验。结果: 与野生型(hm)和异位插入突变体(ecFolprp4)相比,敲除突变体菌丝生长受到严重阻碍,当野生型和异位插入突变体长满整个平板时,敲除突变体菌落呈小点状。敲除突变体的另一个显著变化是ΔFolprp4的分生孢子产量显著下降。侵染实验表明,ΔFolprp4对亚麻幼苗的毒力显著降低。互补实验表明,该互补载体的回复子(Folprp4-C)在菌落形态、生长速率、分生孢子产量和毒力方面均恢复到了野生型菌株。结论: Folprp4基因与尖孢镰刀菌的菌丝生长、分生孢子发生和致病性有关。     

Effect of wheat resistance, the parasitoid Aphidius rhopalosiphi, and the entomopathogenic fungus Pandora neoaphidis, on population dynamics of the cereal aphid Sitobion avenae

The influence of wheat (Triticum aestivumL.) resistance, the parasitoid Aphidius rhopalosiphiDe Stephani-Perez (Hymenoptera: Braconidae) and the entomopathogenic fungus Pandora neoaphidis(Remaudière et Hennebert) Humber (Zygomycetes: Entomophthorales) on the density and population growth rate of the cereal aphid Sitobion avenae(F.) (Hemiptera: Aphididae) was studied under laboratory conditions. Partial wheat resistance was based on hydroxamic acids, a family of secondary metabolites characteristic of several cultivated cereals. The partial resistance of wheat cultivar Naofén, the action of the parasitoid and the joint action of the parasitoid and fungus, reduced aphid density. The lowest aphid densities were obtained with the combination of the parasitoid and the fungus, but wheat resistance under these circumstances did not improve aphid control. Significant reductions of population growth rate (PGR) of aphids were obtained with the joint action of wheat resistance and natural enemies. In particular, the combined effects of parasitoids and fungi showed significantly lower PGR than the control without natural enemies in both wheat cultivars. Our results support the hypothesis that wheat resistance and the utilization of biological control agents could be complementary strategies in an integrated pest management program against cereal aphids.     

防治苦瓜枯萎病的拮抗放线菌分离筛选及鉴定

以苦瓜枯萎病菌为靶标菌,通过对峙培养试验和发酵滤液抑菌试验对分离自苦瓜根际土壤的放线菌进行筛选。候选菌株0250具有广谱抗真菌活性,根据培养特征、生理生化特性以及与同源性相近的菌株进行平均核苷酸一致性分析,被鉴定为Streptomyces rhizosphaericus,并评估了该菌株在温室和田间对苦瓜的促生长和防治枯萎病效果。结果表明: 链霉菌菌株0250对苦瓜枯萎病菌的平板抑制率为69.2%,对17种植物病原真菌的平板抑制率达64.3%~85.6%;该菌株的菌悬液处理能促进盆栽和田间苦瓜植株根、茎生长发育,提升产量,对苦瓜枯萎病的防病效果分别为66.9%和61.5%。预先用菌株0250菌悬液处理土壤再接种病原菌,对土壤尖镰孢菌数量抑制率达62.1%,显著提高了苦瓜幼苗苯丙氨酸解氨酶、过氧化物酶和β-1,3-葡聚糖酶活性以及根系活力。总之,菌株0250是一株对苦瓜枯萎病具有巨大生防潜力的放线菌资源。     

Glyphosate and bioherbicide interaction for controlling kudzu (Pueraria lobata), redvine (Brunnichia ovata), and trumpetcreeper (Campsis radicans)

In controlled environment experiments, the bioherbicidal fungus Myrothecium verrucaria (Alb. & Schwein.) Ditmar:Fr. was tested alone, in combination with, prior to, and following treatment with glyphosate [N-(phosphonomethyl)glycine] for control of kudzu [Pueraria lobata (Willd.) Ohwi], redvine [Brunnichia ovata (Walt.) Shinners], and trumpetcreeper [Campsis radicans (L.) Seem. ex Bureau] at temperatures of 20, 30, and 40°C. At all temperatures, kudzu was most adversely affected by the fungus, followed by trumpetcreeper and redvine, as indicated by greater mortality and dry weight reductions. Trumpetcreeper and redvine mortalities and dry weight reductions significantly increased when the fungus was applied 2 days after the glyphosate treatment. Application of the fungus combined with or prior to glyphosate treatment resulted in reduced weed control. Although pathogenesis and mortality also occurred at 20°C, disease development was favored by higher temperatures (30 and 40°C). Infected weeds of each species exhibited similar disease symptomatology within 12 h following treatment at incubation temperatures of 30 and 40°C. Disease symptomatology was characterized by necrotic flecking on leaves that coalesced into large lesions. Symptoms progressed, initially infecting cotyledons and leaves, and later (within 48 h) producing stem lesions. The fungus sporulated profusely on infected tissue and was easily reisolated. These results suggest that timing of glyphosate application in relation to combined treatment with the bioherbicide M. verrucaria can improve the control of kudzu, redvine, and trumpetcreeper.     

Screening and catalytic activity in organic synthesis of novel fungal and yeast lipases

A total of 969 microbial strains were isolated from soil samples and tested to determine their lipolytic activity by employing screening techniques on solid and in liquid media. Ten lipase-producing microorganisms were selected and their taxonomic identification was carried out. From these strains Achremonium murorum, Monascus mucoroides, Arthroderma ciferri, Fusarium poae, Ovadendron sulphureo-ochraceum and Rhodotorula araucariae are described as lipase-producers for the first time. Hydrolysis activity of the crude lipases against both tributyrin and olive oil was measured. Heptyl oleate synthesis was carried out to test the activity of the selected lipases as biocatalysts in organic medium. All the selected lipases were tested as biocatalysts in several organic reactions using unnatural substrates. Lipases from the fungi Fusarium. oxysporum and O. sulphureo-ochraceum gave the best yields and enantioselectivities in the esterification of carboxylic acids. F. oxysporum and Penicillium chrysogenum lipases were the most active ones for the acylation of alcohols without steric hindrance. A. murorum lipase is very useful for the esterification of menthol. F. oxysporum and Fusarium. solani lipases were very stereoselective in the synthesis of carbamates.     

A Multiple-Pathogen System for Bioherbicidal Control of Several Weeds

A novel system using four host-specific fungal plant pathogens applied in a single, postemergent spray to control pigweed, sicklepod, and showy crotalaria was tested under greenhouse conditions. The four pathogens were Phomopsis amaranthicola (a pathogen of pigweed species), Alternaria cassiae (a pathogen of sicklepod and showy crotalaria), Colletotrichum dematium f. sp. crotalariae and Fusarium udum f. sp. crotalariae (pathogens of showy crotalaria). Spore suspensions of each pathogen alone (10⁶ spores mL^-1) or a mixture of the four pathogens (1:1:1:1, v/v, 2.5×10⁵ spores mL^-1 of each pathogen, total 10⁶ spores mL^-1) were tested on four- to six-leaf stage seedlings of the three weed species grown together in pots. One week after inoculation (WAI), all sicklepod and showy crotalaria seedlings were killed, and all pigweed seedlings were killed by 6 WAI when inoculated with their respective pathogen(s) alone or a mixture of the pathogens. None of the weeds inoculated with the root-infecting pathogen F. udum developed wilt disease by the time the experiment was completed (6WAI). The results demonstrate the feasibility to control three weeds simultaneously with different fungi without loss of efficacy or alterations in host-specificity of each fungus in the given mixture. Scanning electron microscopy showed visual differences in the appearance or germination and further development of conidia of each pathogen on its respective host leaf surface compared to nonhost leaf surfaces, whether the pathogen was applied alone or in a mixture with the other pathogens studied. Application of several host-specific fungal pathogens in a bioherbicide mixture as a multi-component bioherbicide system may be advantageous for further development of simultaneous, broad-spectrum weed control.     

药用杜仲树皮真菌群落的核心真菌组研究

微生物在生态系统中起着重要作用。最近的研究表明,微生物群落具有核心组(分类单元),这些类群对宿主的健康、生长和生产有着重要的影响。基于MetaCoMET与共存网络两种方法对采自湖南、四川和贵州的药用杜仲树皮真菌群落进行了核心真菌组分析。MetaCoMET结果显示,在OTU水平上,核心真菌组共有16个分类单元,优势菌是丛赤壳科一未定真菌,其次为Fusarium pseudensiforme、一种黄丝菌Cephalothecaceae sp.和一种镰刀菌Fusarium sp.等。共存网络分析揭示了11个中枢真菌分类单元。虽然两种方法的分析结果不完全吻合,但在11个中枢真菌上具有较好的一致性。整体而言,特定核心真菌组具有一定的稳定性。研究结果为进一步揭示植物微生物组的功能提供支撑。     

单端孢霉烯族毒素的产生及分子调控机制

禾谷镰刀菌复合种(Fusarium graminearum species complex,FGSC)引起的赤霉病是小麦生产上危害最为严重的病害之一。赤霉病除了造成减产外,感病籽粒中含有多种镰刀菌毒素,如单端孢霉烯族的呕吐毒素,可引起人畜中毒和重大疾病,给食品安全构成严重威胁。过去20年,随着禾谷镰刀菌全基因组序列的公布和遗传转化体系的成熟,禾谷镰刀菌Fusarium graminearum的功能基因组学的研究取得了较大进展,单端孢霉烯族毒素的产生、调控机制及网络研究成为热点。本文综述国内外单端孢霉烯族毒素的生物合成和分子调控机制,包括合成基因簇及决定不同产毒化学型的基因、产毒调控元件、环境因子调控产毒的分子机制,可为小麦抗赤霉病的育种提供新思路,为新型药剂的研发提供分子靶标,为赤霉病的持续防控和毒素污染的有效治理提供理论依据。     

A commercial iron fertilizer increases the survival of Fusarium oxysporum f. sp. orthoceras propagules in a wheat flour-kaolin formulation

Fusarium oxysporum f.sp. orthoceras, a potential biocontrol agent against the parasitic weed Orobanche cumana Wallr., has proven to be efficacous under greenhouse conditions when formulated as wheat-kaolin granules. To help minimize the loss of viable fungal propagules during the formulation process, the addition of a commercial fertilizer containing the iron chelate of EDDHA to the formulation was investigated. The proportion of surviving propagules was significantly increased after adding the fertilizer. However, growing conidia in fertilizer-amended liquid medium did not reduce losses in viability during formulation. The efficacy of the formulated fungus and the storability at room temperature for the first 3 months was not affected by the iron fertilizer. The protective effect could neither be obtained with the chelator EDDHA alone nor with FeEDTA or FeSO_4.     

Contamination of malt barley and wheat by Fusarium graminearum and Fusarium culmorum from the crop years 2001-2003 in eastern Croatia

This study investigated infection levels with Fusarium graminearum and Fusarium culmorum in malt barley and wheat in eastern Croatia. The contamination was surveyed over three consecutive crop years (2001–2003) on five locations for barley and three wheat cultivating locations. F. graminearum loads reached levels of potentially serious threat for the commercial production of malting raw materials in both cereals (up to 29.1%). On the other hand, the mean percentage of kernels infected with F. culmorum was low to medium (up to 6.1%). The fungal invasions for years and locations were affected by meteorologic and other environmental factors and the pattern seemed to be consistent with species-specific optimal conditions reported by other authors.     

Shoot-feeding Aphids Promote Development of Gremmeniella abietina, the Fungal Pathogen Causing Scleroderris Canker Disease in Conifers

We tested experimentally whether shoot feeding aphids Cinara pinea (Mordv.) can promote the development of Gremmeniella abietina (Lagerb.) Morelet, a fungus which causes Scleroderris canker disease in conifers. Pine seedlings were infested with aphids at two different times, and subsequently inoculated with conidia of G. abietina at two different times. The degree of infestation was classified into three groups based on the number of aphids/seedling: none, one (low level), and three (high level). Because of parthenogenetic reproduction, the number of aphids increased during the summer, so we also used aphid numbers to explain the development of the symptoms. Necrosis caused by G. abietina was more prevalent in seedlings infested by aphids in June, and inoculated with fungus in July. Expressed as percents of the total length of the shoots, the necrosis had advanced 50%, 70%, and 95% with no, low, and high aphid levels, respectively. Canker formation was significantly greater when G. abietina conidia were inoculated in July, compared to August. In August, there were no statistically significant differences in the intensity of disease between aphid infestation levels, but the mean number of aphids was related to disease intensity. In the spring following infection, seedlings with aphids had more dead and fewer healthy terminal buds compared to seedlings without aphids.     

Field Evaluation of Beauveria bassiana: Its Persistence and Effects on the Pea Aphid and a Non-target Coccinellid in Alfalfa

Several strains of the entomopathogenic fungus Beauveria bassiana have been considered for use as microbial insecticides. Experimental sprays were conducted in an alfalfa field with an aphid-derived strain of B. bassiana to determine its persistence and its effects on pea aphids, Acyrthosiphon pisum (Homoptera: Aphididae) and a non-target aphid predator, Hippodamia convergens (Coleoptera: Coccinellidae). B. bassiana conidia persisted in the field for at least 28 days, when approximately 10% of the original inoculum was still present. In the lower canopy, more conidia were present than on other plant parts and they persisted longer on the leaves in this location. However, conidia were still abundant in the upper canopy, where 97.9% of the aphids and 95.5% of H. convergens larvae were found. Thus, both insect species were exposed to the fungus for at least 1 month. However, pea aphid populations were not affected by the fungus. The predator's incidence was reduced by 75-93% (depending on application rate) early in the season, but was not affected later in the season. Insect life history patterns and weather conditions are likely causes for the differences seen in field effects.     

Effects of the fungus <Emphasis Type="Italic">Lecanicillium lecanii</Emphasis> on survival and reproduction of the aphid <Emphasis Type="Italic">Schizaphis graminum</Emphasis>

This study investigated the effects of a strain of the fungus Lecanicillium lecanii (Zimm.) Zare and Gams (Hypocreales: Ascomycota) on the aphid Schizaphis graminum (Rondani) (Hemiptera: Aphididae). The fungus was administered to fourth instar nymphs and to alate and apterous adult morphs as a ground rice-kernel formulation. This study showed that L. lecanii formulation affected the survival of the aphids and interacted differently with the studied morphs, the Lethal Time values being lower for alate compared to apterous morphs and nymphs. The treatment also caused a significant reduction in the fecundity of the three treated aphid groups. Histological analysis revealed that the hyphae invaded the host hemocoel of a limited number of alate and apterous morphs: the fungus only entered through the spiracles. Scanning Electron Microscope observations revealed that L. lecanii adhered to the body surface of both adults and nymphs without differentiation. In conclusion, the present study suggests that this strain might be a good candidate for a programme of biological control of S. graminum and other aphid species.