1株拮抗植物病原真菌的细菌筛选及其抑菌机理的初步研究

从上海市郊农作物根际土壤中共分离纯化得到276株细菌,利用平板对峙法筛选出1株对多种植物病原真菌具有较强拮抗作用的菌株。经过形态学观察、生理生化特征以及16S rDNA的同源性分析,初步鉴定该菌株为桔黄假单胞菌(Pseudomonas aurantiaca),该菌株的16S rDNA序列已在GenBank中注册,登录号为GQ358919。通过显微镜观察,发现JD37菌株的主要抑菌机理是通过产生拮抗物质造成病原真菌菌丝体断裂、扭曲、畸形等异常生长现象。该菌产生的拮抗物质对酸、碱较为稳定,但对高温敏感。     

一株具有杀虫活性真菌的鉴定

通过对分离的一株海绵共附生真菌F28的形态特征、生理生化特性的测定及ITS1-5.8S-ITS2序列分析,最终将其确定为米曲霉(Aspergillus oryzae)。该菌株具有生长快,易培养的特点。将该菌株进行发酵和杀虫活性物质提取分离后,通过杀卤虫(Brine Shrimp)活性测试,发现该真菌的代谢产物粗提物具有很强的杀虫活性。     

山苍子叶内生真菌的纯化与鉴定

本研究选用PDA培养基,通过组织块分离法从山苍子叶中分离得到两株内生真菌。对照真菌鉴定手册,根据菌株和菌丝体形态学特征,并给合ITS区段的碱基序列分析,鉴定两株内生分别属于顶孢霉属和芒果球座菌属。     

一株小麦赤霉病拮抗菌的筛选鉴定及防治效果分析

自小麦赤霉病发病田块土壤中,分离得到1株小麦赤霉病高效拮抗菌株。通过形态特征、生理生化特性和16S rDNA序列比对分析,将这株拮抗菌株鉴定为奇异变形杆菌(proteus mirabilis)。P. mirabilis DY05发酵液和无细胞上清均可显著抑制禾谷镰刀菌菌丝体生长(抑制率分别为79.50%和51.25%)和分生孢子萌发(抑制率均为100%),减少呕吐毒素产生(分别减少84.32%和82.82%)。田间赤霉病防治试验结果显示,接种DY05,可降低发病率52.13%,同时病情指数降低48.74%,显示出较好的防治效果。促生生理活性评价试验结果显示,菌株DY05可以产生铁载体和IAA,并具有溶磷作用和ACC脱氨酶活性,具有很好的促生长潜力。盆栽试验结果表明,菌株DY05对小麦植株生长具有显著的促进作用。与对照组相比,菌株DY05处理可以显著增加小麦幼苗茎高、根长、鲜重和干重,其中茎高、根长、鲜重和干重分别提高了22.21%、26.41%、44.77%和26.53%。分离得到的拮抗菌株DY05具有拮抗病原真菌和促进植物生长的双重功能,为开发禾谷镰刀菌生物防治制剂提供了菌种材料。     

青蒿内生真菌分离、分子鉴定及深绿木霉对青蒿的促生作用研究

为从青蒿(Artemisia annua L.)内生真菌中筛选有促生作用的菌株,用组织分离法从青蒿侧枝中分离内生真菌并进行分子鉴定,对其中1株真菌深绿木霉对青蒿幼苗的促生作用进行了研究。结果表明,从青蒿侧枝中获得23株内生真菌,鉴定出16种,以炭疽菌属占优势。深绿木霉发酵液对青蒿种子发芽和幼苗生长具有显著抑制作用;深绿木霉液体发酵菌丝体在4-5月对青蒿幼苗生长具有显著的促进作用,但7月以后没有明显促进作用。因此,深绿木霉可作为青蒿苗肥,与化肥配合可施用于成株。     

两性霉素B产生菌——链霉菌AS4．1006

链霉菌AS 4.1006是从我国西南采集的土样中分离出来的拮抗菌,它对真菌有很强的抑制作用。对此菌株的形态、培养特征和生理特性进行了系统研究,经鉴定命名为结链霉菌AS4．1006(Streptomyces nodosus AS 4．1006)。此菌株产生两种抗菌素,其中主要成分,经提纯精制并测定其理化性质,证实此抗菌素是两性霍素B．     

1株人工栽培蛹虫草病原真菌的分离鉴定及生物学特性研究

为明确蛹虫草（Cordyceps militaris）栽培过程中发现的一种病原真菌及其生长特性,采用组织分离法自发病部位分离获得1株真菌CCBH-L,经柯赫法则确定其致病性,通过形态学特征及ITS序列分析,确定菌株CCBH-L为轮枝样镰刀菌(Fusarium verticillioides)。该病原菌在感染初期,通过竞争生长空间和营养物质,影响蛹虫草菌丝体生长和原基分化,导致子实体畸形,后期菌丝体蔓延至已经形成的子实体,导致子实体生长受阻。该病原菌生长的适宜温度为25~30 ℃,可见,高温利于该病原菌的生长,其生长的适宜pH值为6.0~8.0,适宜含水量为60%~75%。因此,在蛹虫草栽培过程中,将温度控制在20 ℃以下,可抑制该病原菌的生长,降低感染率。     

与内生真菌共培养对葡萄果肉细胞生理生化影响的差异研究

根据与内生真菌共培养过程中对葡萄细胞生理生化的影响差异将内生真菌菌株归类,筛选出具有不同利用价值的内生真菌资源。以分离于玫瑰蜜、赤霞珠和夏黑葡萄叶片的18个属47株内生真菌和佳美葡萄(Vitis vinifera L. cultivar:Gamay)果肉愈伤组织为试验材料,构建内生真菌与葡萄细胞共培养体系,并探索这些内生真菌菌株对葡萄果肉细胞生长、花色苷含量、苯丙氨酸解氨酶(PAL)活性等的生理生化影响。结果表明,与不同内生真菌共培养对葡萄细胞生长、花色苷总量和PAL活性都有不同程度的影响。其中共筛选出20株对葡萄细胞伤害较小的内生真菌;6株显著促进葡萄细胞生长的内生真菌;11株显著促进葡萄细胞PAL活性的内生真菌;5株促进葡萄细胞花色苷总量的内生真菌。此次研究为发掘和利用内生真菌资源调控葡萄生化品质特征提供了技术参考和一定的物质基础。     

尼泊尔马桑根瘤内生菌纯培养物的质粒检测

采用胶内裂解法快速检测了21株马桑根瘤内生菌纯培养物和4株弗兰克氏菌参考菌株的质粒,其中有5株马桑分离菌株和1株参考菌株含有质粒。除马桑菌株和参考菌株各有1株携带2个质粒外,其它菌株均只含有1个质粒。这些质粒的分子量约为13～20kb。根据所含质粒的大小和数目,将21株马桑分离菌株划分成4个质粒类群。实验还对菌丝体生长,细胞酶解和裂解等条件对质粒检测效果的影响进行了探讨。     

铁皮石斛内生真菌的分离及其挥发性成分分析

铁皮石斛(Dendrobium officinale)是我国重要的濒危中药材,其药用价值和经济价值很高。以铁皮石斛为研究对象,采用组织块法,从铁皮石斛根、茎和叶中分离获得22株内生真菌。采用固相微萃取固定吸附(SPME),结合气相质谱(GCMS)解吸附分析技术对其中具有产香特性的3株内生真菌KLBMPD001、KLBMPD004和LBMPD009的挥发性成分进行分析。结果显示,这3株内生真菌均含有较高比例的棕榈酸,分别为37.94%、24.15%和48.55%,其中菌株KLBMPD001和KLBMPD009含有一定比例的硬脂酸,分别为12.64%和37.37%,只有菌株KLBMPD004含有8.74%的酞酸二丁酯。     

A tale of three acaropathogenic fungi in Israel: Hirsutella, Meira and Acaromyces

We review published and unpublished studies conducted in Israel with six acaropathogenic fungi, assayed in order to control the citrus rust mite, Phyllocoptruta oleivora (Ashmead) (CRM). Hirsutella thompsonii Fisher was introduced twice, killed 80-90% of the exposed mites, but due to its requirements for near-saturation humidities was deemed unsuitable for local outdoors conditions. Hirsutella kirchneri (Rostrup) Minter et al. and Hirsutella necatrix Minter et al. were also introduced and assayed against CRM and spider mites, but their efficacy was unsatisfactory. Three indigenous fungi found to be associated with mites, Meira geulakonigii, Meira argovae and Acaromyces ingoldii--all three recently described by Boekhout, Gerson, Scorzetti & Sztejnberg--were assayed against several mites. Meira geulakonigii killed 80-90% of several spider mites and of the CRM, and caused some mortality of Iphiseius degenerans (Berlese), one out of three phytoseiid predators assayed. Mortality was not due to parasitization; extracts from the media in which the fungi had developed caused considerable mite death, suggesting that it was a result of fungal toxins. Data from a field study indicated that spraying blastoconidia of M. geulakonigii on grapefruits infested by CRM significantly reduced pest-incurred damage from 23 to 13%. Applying qRT-PCR methodology indicated that M. geulakonigii was endophytic within sealed grapefruit flowers and in the flavedo of the fruits' peel. Neither in the laboratory nor in the field was any evidence ever obtained that this fungus damaged the plants, leading us to hypothesize that M. geulakonigii serves as a "body guard" of grapefruits (and perhaps other plants as well). All three fungi suffered very little mortality after being exposed to various insecticides and acaricides that are in current local use (with the exception of sulfur). The ability of M. geulakonigii to reduce mite numbers without affecting the host plant, the minimal fungal effect on some predatory mites, its endophytic nature along with the apparent tolerance of M. geulakonigii to many insecticides and acaricides, suggest that this fungus could be suitable for integrated pest management (IPM) program.     

Antagonistic effects of the endophytic fungus Meira geulakonigii on the citrus rust mite Phyllocoptruta oleivora

AIMS: The fungus Meira geulakonigii has been shown to reduce populations of citrus rust mite (CRM; Phyllocoptruta oleivora) on citrus leaves and fruits, in both the field and laboratory. However, attempts to isolate the fungus from leaves and fruits have been unsuccessful. The aims of this study were therefore to determine whether M. geulakonigii is a citrus endophyte, and to assess possible mechanisms involved in its mite-antagonist activity. METHODS AND RESULTS: A quantitative real-time PCR and regular PCR approaches were developed to detect M. geulakonigii in both the field and laboratory. The fungus was detected throughout. Different methods revealed that M. geulakonigii is an endophyte, which colonizes both the peel of grapefruits. Applications of conidia protected the grapefruits against CRM, and fungal secretions extracted from growth media caused 100% CRM mortality. CONCLUSIONS: Meira geulakonigii is a beneficial endophyte of grapefruits that colonizes the fruit's peel, and protects it from CRM. SIGNIFICANCE AND IMPACT OF THE STUDY: Findings from this study demonstrate the endophytic nature of M. geulakonigii in its interaction with grapefruits. In addition, a molecular approach was developed to specifically detect the fungus inside the grapefruit peel. This approach can be used to assess the natural occurrence of M. geulakonigii in grapefruit.     

从药用植物根际土壤中分离出的2个中国新记录种(英文)

在进行黑龙江省药用植物根际土壤真菌多样性的研究中,分离获得了2个中国新记录种:蕨枝顶孢[Acremonium pteridii(J.C.Frankland)W.Gams]和穴形节皮菌的金孢属无性型[Chrysosporium anamorph ofArthro-derma cuniculi(Dawson)C.A.N.van Oorschot]。蕨枝顶孢的主要形态特征为分生孢子梗单生或二次及三次分支,具有1~3个隔膜,顶端着生单个分生孢子;分生孢子以假头状着生,卵形或者近圆形,内含1个油球。穴形节皮菌的金孢属无性型主要形态特征为短的侧分支与可育菌丝呈直角伸出,分生孢子具柄,生于短的突起上或者直立的侧分支上;分生孢子卵形或棍棒形,顶端钝圆,基部平截,大多数单胞,极少数为由2~3个细胞组成。文中对它们进行了详细的形态特征描述,标本保存于大连民族学院菌种保藏中心。     

Meira nashicola sp. nov., a novel basidiomycetous, anamorphic yeastlike fungus isolated from Japanese pear fruit with reddish stain

Three undescribed strains of basidiomycetous, anamorphic yeastlike fungi were isolated from Japanese pear fruits with a reddish stain collected in Tottori Prefecture, Japan. The strains are classified in a single group and assigned to the genus Meira by conventional and chemotaxonomic studies. Sequence analyses of the D1/D2 domain of 26S rDNA and internal transcribed spacer (ITS) regions indicate that the strains represent a novel species with a close phylogenetic relationship to Meira geulakonigii and M. argovae. The name Meira nashicola sp. nov. is proposed for the strains (type strain PFS 002 = MAFF 230028 = CBS 117161).     

Isolation of basidiomycetous anamorphic yeast-like fungus <Emphasis Type="Italic">Meira argovae</Emphasis> found on Japanese bamboo

A basidiomycetous anamorphic yeast-like fungus, isolated from new bamboo shoots collected in Japan, was assigned to Meira argovae by comparison of conidial morphology, physiological characteristics, rDNA sequences, and DNA-DNA relatedness with the ex-type strains of Meira species. This is the first record of the finding of M. argovae from other than mite cadavers and in regions other than Israel. Phylogenetic analysis based on the D1-D2 domain demonstrated that Meira species and teleomorphic Dicellomyces species, which include a bamboo leaf parasite, D. gloeosporus, formed sister clades.     

Heterogeneity of fast-oxidative muscle fibers of chicken demonstrated by anti-myosin monoclonal antibodies

Summary The fiber type composition of two fast muscles of the chicken, namely, adductor superficialis (AS) and pectoralis major (PM) was examined by the histochemical myosin ATPase staining and immunochemical techniques using monoclonal antibodies (McAbs). Two new McAbs produced against the myosin of the anterior latissimus dorsi (ALD) muscle of the chicken and named ALD-122 and ALD-83 were characterized to be specific for myosin heavy chain (MHC) and for myosin light chain-1 respectively. They were used in conjunction with previously reported McAbs specific for slow MHC (ALD-47), fast MHC (MF-14) and fast light chain-2 (MF-5). By the histochemical ATPase test most muscle fibers of AS and PM muscles reacted as IIA and IIB respectively. By immunofluorescent staining with the anti-MHC McAbs, ALD-122, and MF-14, the fibers of AS, muscle showed remarkable heterogeneity whereas PM muscle fibers reacted, uniformly. Differences in the myosin light chain composition of AS and PM muscles were also found by SDS-gel electrophoresis and immunoblot analysis with the anti-light chain McAb, ALD-83. The study clearly indicated that the histochemically homogenous (type IIA) AS muscle is composed of several subpopulations of fibers which differ in their myosin composition and that this heterogeneity of the muscle is not simply due to presence of variable amounts of slow myosin in its fibers.     

Reactive oxygen species formation and cell death in catalase-deficient tobacco leaf disks exposed to cadmium

The physiological responses of tobacco (Nicotiana tabacum L.) to oxidative stress induced by cadmium were examined with respect to reactive oxygen species (ROS) formation, antioxidant enzymes activities, and cell death appearance in wild-type SR1 and catalase-deficient CAT1AS plants. Leaf disks treated with 100 or 500 µM CdCl₂ increased Evans blue staining and leakage of electrolytes in SR1 or CAT1AS plants, more pronouncedly in the transgenic cultivar, but without evidence of lipid peroxidation in any of the cultivars compared to controls. Cadmium significantly reduced the NADPH oxidase-dependent O ₂ ^? formation in a dose dependent manner in SR1 very strongly at 500 µM (to 5% of the activity in the nontreated SR1 leaf disks). In CAT1AS, the NADPH oxidase activity was constitutively reduced at 50% with respect to that of SR1, but the magnitude of the decay was less prominent in this cultivar, reaching an average of 64% of the C at 21 h, for both Cd concentrations. Hydrogen peroxide formation was only slightly increased in SR1 or CAT1AS leaf disks at 21 h of exposure compared to the respective controls. Cd increased superoxide dismutase activity more than six times at 21 h in CAT1AS, but not in SR1 and reduced catalase activity by 59% at 21 h of treatment only in SR1 plants. Despite that catalase expression was constitutively lower in CATAS1 compared to SR1 nontreated leaf disks, 500 µM CdCl₂ almost doubled it only in CAT1AS at 21 h. The mechanisms underlying Cd-induced cell death were possibly not related exclusively to ROS formation or detoxification in tobacco SR1 or CAT1AS plants.     

The formation of perinucleolar bodies is important for normal leaf development and requires the zinc‐finger DNA‐binding motif in Arabidopsis ASYMMETRIC LEAVES2

In Arabidopsis, the ASYMMETRIC LEAVES2 (AS2) protein plays a key role in the formation of flat symmetric leaves via direct repression of the abaxial gene ETT/ARF3. AS2 encodes a plant‐specific nuclear protein that contains the AS2/LOB domain, which includes a z inc‐f inger (ZF) motif that is conserved in the AS2/LOB family. We have shown that AS2 binds to the coding DNA of ETT/ARF3, which requires the ZF motif. AS2 is co‐localized with AS1 in perinucleolar bodies (AS2 bodies). To identify the amino acid signals in AS2 required for formation of AS2 bodies and function(s) in leaf formation, we constructed recombinant DNAs that encoded mutant AS2 proteins fused to yellow fluorescent protein. We examined the subcellular localization of these proteins in cells of cotyledons and leaf primordia of transgenic plants and cultured cells. The amino acid signals essential for formation of AS2 bodies were located within and adjacent to the ZF motif. Mutant AS2 that failed to form AS2 bodies also failed to rescue the as2‐1 mutation. Our results suggest the importance of the formation of AS2 bodies and the nature of interactions of AS2 with its target DNA and nucleolar factors including NUCLEOLIN1. The partial overlap of AS2 bodies with perinucleolar chromocenters with condensed ribosomal RNA genes implies a correlation between AS2 bodies and the chromatin state. Patterns of AS2 bodies in cells during interphase and mitosis in leaf primordia were distinct from those in cultured cells, suggesting that the formation and distribution of AS2 bodies are developmentally modulated in plants.     

Candida albicans GRX2, encoding a putative glutaredoxin, is required for virulence in a murine model

Resistance of Candida albicans to reactive oxygen species is thought to enhance its virulence in mammalian hosts. Genes such as SOD1, which encodes the anti-oxidant, superoxide dismutase, are known virulence factors. We disrupted the gene GRX2, which encodes a putative glutathione reductase (glutaredoxin) in C. albicans, and we compared the mutant with an sod1Deltamutant. In vitro, the grx2Deltastrain, but not the sod1Delta strain, was defective in hypha formation. The grx2Deltastrain, but not sod1Delta, was significantly more susceptible to killing by neutrophils. When exposed to two compounds that generate reactive oxygen species, both mutants were susceptible to 1 mM menadione, but grx2Deltanull alone was resistant to diamide. Both mutants were attenuated in a murine intravenous challenge model, and a GRX2 reintegrant regained partial virulence. Emphasis on the putative function of products of genes such as SOD1 and GRX2 in resistance to oxidative stress may oversimplify their functions in the virulence process, since the grx2Deltastrain also gave defective hypha formation. Both mutants were sensitive to menadione and were slow to form germ tubes, though growth rates matched controls once the lag phase was passed.     

An investigation into the involvement of defense signaling pathways in components of the nonhost resistance of Arabidopsis thaliana to rust fungi also reveals a model system for studying rust fungal compatibility

Seventeen accessions of Arabidopsis thaliana inoculated with the cowpea rust fungus Uromyces vignae exhibited a variety of expressions of nonhost resistance, although infection hypha growth typically ceased before the formation of the first haustorium, except in Ws-0. Compared with wild-type plants, there was no increased fungal growth in ndr1 or eds1 mutants defective in two of the signal cascades regulated by the major class of Arabidopsis host resistance genes. However, in the Col-0 background, infection hyphae of U. vignae and two other rust fungi were longer in sid2 mutants defective in an enzyme that synthesizes salicylic acid (SA), in npr1 mutants deficient in a regulator of the expression of SA-dependent pathogenesis related (PR) genes, and in NahG plants containing a bacterial salicylate hydroxylase. Infection hyphae of U. vignae and U. appendiculatus but not of Puccinia helianthi were also longer in jar1 mutants, which are defective in the jasmonic acid defense signaling pathway. Nevertheless, haustorium formation increased only for the Uromyces spp. and only in sid2 mutants or NahG plants. Rather than the hypersensitive cell death that usually accompanies haustorium formation in nonhost plants, Arabidopsis typically encased haustoria in calloselike material. Growing fungal colonies of both Uromyces spp., indicative of a successful biotrophic relationship between plant and fungus, formed in NahG plants, but only U. vignae formed growing colonies in the sid2 mutants and cycloheximide-treated wild-type plants. Growing colonies did not develop in NahG tobacco or tomato plants. These data suggest that nonhost resistance of Arabidopsis to rust fungi primarily involves the restriction of infection hypha growth as a result of defense gene expression. However, there is a subsequent involvement of SA but not SA-dependent PR genes in preventing the Uromyces spp. from forming the first haustorium and establishing a sufficient biotrophic relationship to support further fungal growth. The U. vignae-Arabidopsis combination could allow the application of the powerful genetic capabilities of this model plant to the study of compatibility as well as nonhost resistance to rust fungi.