Improvement on genetic transformation in the nematode-trapping fungus Arthrobotrys oligospora and its quantification on dung samples

An improved DNA-mediated transformation system for nematode-trapping fungus Arthrobotrys oligospora based on hygromycin B resistance was developed. The transformation frequency varied between 34 and 175 transformants per μg linearized DNA and 93% of the transformants were stable for drug resistance when tested 100 randomly selected transformants. More than 2000 transformants were obtained by transformation of the fungus with pBChygro in the presence of HindIII and among them, one, YMF1.00110, which lost its ability of forming predacious structure, was isolated. Southern analysis showed that the plasmid DNA had integrated into the genome of all tested transformants (including YMF 1.00110) except one. The transformant tagged with hph gene could be re-isolated and quantified from dung samples based on the resistance of hygromycin B. All the results suggested that the method of restriction enzyme mediated integration (REMI) should facilitate not only the insertional mutagenesis for tagging and analysis genes of interest but also the ecological investigation of tagged fungi in a given environment.     

Significance of electron dense microbodies in trap cells of the nematophagous fungus Arthrobotrys oligospora

We have studied the fate of electron dense microbodies in nematode-trapping organs (traps) of the fungus A. oligospora during the initial hours following nematode capture. The interaction studies were performed with isolated traps which had captured a nematode under conditions where the fungal cells had no access to external energy sources. Video enhanced contrast microscopy showed that under these conditions the number of dense bodies present in the trap cell that formed the penetration tube, rapidly decreased. During subsequent penetration and development of the infection bulb this decrease continued while at this time common cell organelles such as mitochondria and vacuoles were formed. This was confirmed by electron microscopy which also revealed that the dense bodies were degraded by means of an autophagic process. The organelles were degraded individually and finally turned into compartments which, based on ultrastructural criteria, were considered vacuoles. Fusion of such vacuoles into larger organelles frequently occurred. The degradation process was initiated early in the interaction since initial stages were already evident within 15 min after capture. Generally it took 1–2 h before the infection bulb had fully developed and trophic hyphae formation started. During this time the original trap cell, characterized by numerous dense bodies, was transformed into an active vegetative hyphal cell containing typical cell organelles such as nuclei, mitochondria, a strongly proliferated endoplasmic reticulum, vacuoles and normal microbodies but lacked dense bodies. This disappearance of dense bodies was confined to the cell that penetrated the nematode and—less frequently—its two neighbouring cells in the hyphal loop. In the other cells, constituting the trap, the dense bodies remained unaffected. As will be discussed, the present results support our current view that traps of A. oligospora contribute to the survival of the organism in its natural environment.     

Alginate films for delivery of root-knot nematode inoculum and evaluation of microbial interactions

A method was developed for utilizing alginate films to deliver inoculum into soil and evaluate microbial antagonistic activity against nematode eggs. Eggs of Meloidogyne incognita were harvested from galled tomato roots (Lycopersicon esculentum), surface disinfested, suspended in 2% (w/v) aqueous sodium alginate, and applied to 2.5 × 5.0 cm polyvinyl chloride coated fiberglass screens (1.5 mm² mesh size) at a uniform thickness of 0.5 mm. The alginate solution was gelled by dipping in 0.25 M CaCl₂. Films containing eggs were observed in vitro and egg development was evaluated. The number of immature eggs and eggs with first stage juveniles declined linearly over time while the number of empty eggs shells, and hatched juveniles increased over time, indicating that the alginate gel did not inhibit development and motility of M. incognita juveniles. In a greenhouse experiment using cucumber (Cucumis sativus) the number of galls g^-1 root was correlated with the number of eggs in alginate films placed in each pot at planting. Films containing M. incognita eggs were buried in field soil containing organic amendments, incubated, removed from soil, rinsed with water, and observed. The number of immature eggs in grids from soil amended with chitin or flax seed meal were lower than in untreated soil; percent parasitized eggs was also greater in films from amended soil than from untreated soil.     

An improved method for rearing Halotydeus destructor (Acari: Penthaleidae) in the laboratory

We developed an improved method for rearing Halotydeus destructor in the laboratory. Mite numbers increased rapidly over the summer of 1992–1993 when fed on vetch, Vicia sativa cv Blanchefleur; in contrast mite numbers were low and fell over the second half of the summer of 1990–91 when fed on subterranean clover, Trifolium subterraneum cv Junee. On V. sativa at a fluctuating temperature from 11 to 18°C 200 mites produced a mean of 1308 and 1455 progeny per transfer in 2 years, a 6–7-fold rate of increase per transfer. The mean transfer time was 35 days and using the interval between transfers as a measure of generation time, the mites completed six to seven generations with no evidence for development of diapause eggs from August until April. There was considerable variability in the numbers of mites produced per pot, associated in part with the presence of Verticillium sp. fungus in 35% of the pots during transfers. Improvement in H. destructor rearing resulted from the use of V. sativa cv Blanchefleur as a food source, maintenance of high humidity and adequate ventilation within cages, and the transfer of mites to fresh food sources at early nymphal stages, which reduced the spread of fungal infections.     

Response of nematode-trapping fungi to organic substrates in a coastal grassland soil

To understand why Arthrobotrys oligospora and other nematode-trapping fungi are common and sometimes abundant in the coastal grassland soils of the Bodega Marine Reserve (BMR, Sonoma County, CA), we examined how resident trapping fungi responded to the addition of eight organic substrates (lupine leaves, grass leaves, dead isopods, dead moth larvae, isopod faeces, deer faeces, shrimp shells, and powdered chitin). We were especially interested in the effects of dead isopods because isopods are abundant at BMR and because previous studies had documented strong responses of A. oligospora to other arthropods (dead moth larvae). Soil from BMR was packed into vials (40 g dry mass equivalent per vial with water potential at −230 kPa and bulk density at 0.9 g cm⁻³), and one substrate or no substrate was added to the soil surface. After 30 d at 20 °C, trapping fungi were quantified by dilution plating and most probable number procedures. The response of A. oligospora was inversely related to substrate carbon:nitrogen (C:N) ratio: substrates with low C:N ratios (dead isopods, lupine leaves, dead moth larvae) usually caused large increases in A. oligospora whereas those with higher C:N ratios (isopod faeces, deer faeces, grass leaves) did not. An exception was chitin powder, which had a low C:N ratio, but which did not cause A. oligospora to proliferate. Responses of A. oligospora were directly related to the quantity of nitrogen added with each substrate, and those substrates that caused large increases in resident nematodes usually caused large increases in A. oligospora. Other trapping fungi did not respond as strongly as A. oligospora.     

Hyphal fusion during initial stages of trap formation in Arthrobotrys oligospora

Hyphal fusion during initial stages of trap formation by Arthrobotrys oligospora was studied by video-enhanced contrast and electron microscopy. Trap initials grew perpendicularly to the parent hypha, then curved around and anastomosed with a peg that developed on the hypha. Trap initials usually developed 40–140 m apart while the anastomosis occurred 20–25 m from the initial. Vigorous cytoplasmic movements in trap initials and developed traps corresponded to intense staining with fluorescein diacetate (FDA) of these cells. In addition, bundles of microfilaments were seen in developing loops of traps. On fusion organelle migration took place from the tip cell of the trap into the peg. Later on a septum was formed at the site of fusion.     

念珠菌—上皮细胞互作研究进展

念珠菌作为共生菌定植于机体黏膜表面,一般情况下并不引起感染,但当机体出现免疫力下降或微生态失衡等状况时,可引发口咽念珠菌病、外阴阴道念珠菌病等黏膜感染。对于念珠菌黏膜感染的治疗,虽然抗菌药物是不可或缺的因素,但宿主自身的免疫力,尤其是黏膜上皮细胞作为抵御念珠菌感染的第一道防线,发挥着重要作用。本文将念珠菌—上皮细胞相互作用研究进展作一综述。     

Photosynthetic electron transport is differentially affected during early stages of cultivar/race-specific interactions between potato andPhytophthora infestans

The influence of the early stages of fungal infection on chloroplast metabolism was studied in cultivar/race-specific interactions between potato (Solanum tuberosum L. cv. Datura) and the late-blight fungusPhytophthora infestans. The accumulation of several mRNAs encoding components of the photosynthetic apparatus was not affected, either in compatible or in incompatible interactions. However, within 3 h after inoculation of potato leaves with fungal spores, a change in the photochemistry of photosystem II was detectable by measuring chlorophylla fluorescence. Characteristic fluorescence parameters, such as maximum fluorescence yield (F_m), variable fluorescence yield (F_v) and photochemical efficiency (F_v/F_m), were specifically reduced in the compatible host/pathogen interaction. Analyses of photochemical and nonphotochemical fluorescence quenching showed an increase in the photochemical fraction. The amounts of two selected thylakoid membrane proteins and of total chlorophyll remained unchanged during this process, suggesting that the functional modification of the electron-transport system was not correlated with a change in the composition of the photosynthetic apparatus. The alterations of photosynthetic electron transport represent a rapidly detectable and sensitive physiological marker for compatible interactions in the potato/Phytophthora infestans pathosystem.     

An improved method for screening Frankia viability in strain CcI3

An improved method for determining the viability of the nitrogen-fixing actinomycete Frankia is presented. This method uses tetrazolium red as a vital stain, which proved more effective than a previously used method of acridine orange staining.     

An improved method of measuring photosynthetic electron transport in Euglena under in vivo conditions

A method is described to measure photochemical activity in intact cells of Euglena under in vivo conditions. The method employs a cell wall digesting enzyme (cellulysin) to induce enough permeability in the cell walls and membranes in order to allow dyes, commonly used to investigate light-dependent electron transport reactions to enter, but without inducing a concomittant efflux of metabolites. Between 1 and 2 h of incubation in 5% (w/v) cellulysin provided conditions which allowed measurement of photosystem I-, II- and I+II-dependent electron transport with rates up to 600% higher than in control cells; whereas other cell wall degrading enzymes (cellulase and pectinase) still did not increase the entry of the dyes. Cellulysin up to 2 h of incubation had little or no effect on whole cell respiration, photosynthetic O₂ evolution, or the export of potassium and (¹⁴C) labeled compounds out of cells; therefore cellulysin obviously did not change the normal habit or physiology of Euglena. Cellulysin (4 h digestion), cellulase and pectinase (2–4 h of incubation) on the other hand led to a lowering of respiration and light-dependent O₂ evolution, and increased the efflux of K⁺, but apparently decreased that of (¹⁴C)labeled fixation products.Abbreviations DBMIB dibromothymoquinone - DCPIP 2,6-dichlorophenol-indophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMMIB 2,3-dimethyl-5,6-methylenedioxy-p-benzoquinone - MV methylviologen - PSI photosystem I - PS II photosystem II     

An improved method for callus proliferation and regeneration of Fuchsia hybrida

Best callus initiation was obtained when single-node explants of Fuchsia hybrida were incubated in the light on Gamborg B5 medium containing 5×10^-6 M indoleacetic acid and benzylaminopurine at 5×10^-7 M or 10^-6 M. Healthy callus proliferation was maintained in darkness on full-strength B5 medium supplemented with 5×10^-6 M IAA and 5×10^-7 M BAP. Regeneration from callus was obtained in 3 to 6 weeks, using half-strength hormone-free Campbell & Durzan medium.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4 dichlorophenoxyacetic acid - IAA indoleacetic acid - IBA indolebutyric acid - NAA -naphthaleneacetic acid - SE standard error     

Bsr-REMI: An improved method for gene tagging using a new vector inDictyostelium

Using a plasmid pBsr2 which carries a blasticidin S-resistant gene, we have improved the method of REMI (restriction enzyme-mediated integration) provided for insertional mutagenesis inDictyostelium discoideum (bsr-REMI). To confirm usefulness of thebsr-REMI, transformation efficiency, copy number of integrated DNA, and randomness of integration into genome were examined.     

辽宁碱蓬根际土壤真菌多样性的季节变化及其耐盐性

采用稀释平板法和形态学鉴定法进行了辽宁碱蓬(Suaeda liaotungensis)根际土壤真菌的分离鉴定及季节、盐度对真菌多样性的影响分析。结果表明:春、夏、秋、冬四季辽宁碱蓬根际土壤中的真菌菌落数分别为6410、4180、5730和3340,种类分别为6属13种、9属16种、11属31种、6属12种,共分离鉴定出13属42种真菌;其根际土壤真菌的多样性指数、丰富度指数和均匀度指数,从大到小的次序均为秋夏春冬,3种指数均在秋季达到峰值;多样性指数和丰富度指数差异较大,均匀度指数波动较小,春季和冬季的均匀度指数相近。辽宁碱蓬根际土壤真菌种类明显多于无植被土壤,是无植被土壤的7倍,多样性指数和均匀度指数分别是无植被土壤的4—8倍和1—1.2倍;其根际土壤真菌优势种群包括青霉属(Penicillium)、葡萄穗霉属(Stachybotrys)、枝孢属(Cladosporium)、木霉属(Trichoderma)、曲霉属(Aspergillus)和镰孢属(Fusarium);4个季节的优势菌不尽相同,但均有一个共同的优势菌属青霉属。以Na Cl浓度梯度法制造盐胁迫生境,检测辽宁碱蓬根际土壤真菌对盐胁迫的响应,结果表明:60%左右的菌种能耐受5%以下盐度、15%左右的菌种能耐10%—20%盐度;筛选出了6株高度耐盐菌:细交链孢霉、草酸青霉、产黄青霉、烟曲霉、细极链格孢和赭曲霉,其中最高耐盐菌种赭曲霉可耐20%盐度。创新之处在于以盐度和季节为变量检测辽宁碱蓬根际土壤真菌的种群构成,为其深入研究奠定了理论基础。     

锦鸡儿属灌丛化对草原化荒漠区土壤线虫群落组成和代谢足迹的影响

调查了内蒙古草原化荒漠区3种锦鸡儿[垫状锦鸡儿(Caragana tibetica)、荒漠锦鸡儿(C.roborovskyi)和狭叶锦鸡儿(C.stenophylla)]灌丛内、外土壤线虫群落多样性、组成和代谢足迹,以及相关的土壤理化性质和植物群落特征,旨在探讨锦鸡儿属灌丛对土壤线虫群落的影响,并分析这种影响是否具有灌木种间差异,同时从线虫功能团水平上探究灌丛如何通过非生物因素和生物因素影响线虫群落组成。结果表明：锦鸡儿属灌丛对土壤线虫多度、丰富度和多样性无显著影响,但却显著影响土壤线虫群落组成。原因是灌丛对土壤线虫群落具有物种选择性;灌丛可能主要是通过根系分泌物、凋落物质量等因素,而不是通过土壤理化性质和林下植物影响线虫群落组成。土壤线虫组成的变化引起线虫代谢足迹发生明显变化。灌丛内土壤线虫功能代谢足迹大于灌丛外(除狭叶锦鸡儿外),说明灌丛内土壤线虫群落对碳的利用率更高。3种锦鸡儿属灌木中,狭叶锦鸡儿的结构代谢足迹最大,表明狭叶锦鸡儿对捕食杂食类线虫代谢活性的促进作用更强。土壤线虫组成的变化通过线虫代谢足迹导致土壤食物网结构发生显著性变化。狭叶锦鸡儿灌丛土壤干扰程度低,营养富集状况好...     

基于根际与凋落物际评价转Bt水稻对土壤线虫群落的影响

转基因作物在商业化推广前,有必要评价其对非靶标土壤生物的生态影响。根系和凋落物与土壤的界面是代表植物直接影响土壤的两个典型活性微域,即根际和凋落物际。基于室内盆栽实验构建水稻根际和凋落物际,比较了这两个高活性微域中转Bt水稻克螟稻(KMD)与亲本水稻秀水11(XSD)对土壤可利用资源、微生物学性质和线虫群落的影响。结果表明:转Bt水稻对土壤性质的影响依赖于水稻生育期和微域的变化。与XSD相比,KMD在苗期和拔节期显著提高了凋落物际土壤可溶性有机氮含量(P0.05);在苗期显著降低了凋落物际土壤铵态氮含量,但在成熟期显著提高了凋落物际与交互微域土壤的铵态氮含量(P0.05)。与亲本水稻相比,转Bt水稻分别在苗期和成熟期显著提高了凋落物际土壤微生物生物量碳和活性,而在拔节期和成熟期显著降低了根际土壤微生物生物量氮含量(P0.05)。微域间的交互作用显著影响土壤微生物生物量、可溶性有机碳和氮、线虫总数和各食性线虫的数量(P0.05)。转Bt水稻总体上降低了土壤线虫总数,尤其在苗期和拔节期的凋落物际土壤中达到显著水平。重复测量方差分析表明转Bt水稻显著影响土壤食细菌(P0.01)和食真菌(P0.05)线虫数量,及线虫群落的通道指数和富集指数。水稻收获后KMD秸秆的Bt蛋白含量显著高于亲本,全部KMD处理的土壤样品中Bt蛋白含量无显著变化,因而转Bt水稻对土壤生物学性质的影响可能并非来源于Bt蛋白的作用,而更可能来自于水稻生长性状和凋落物性质的差异。     

Microcalorimetric studies of the interactions between cytochromes c and c1 and of their interactions with phospholipids

Thermotropic properties of purified cytochrome c₁ and cytochrome c have been studied by differential scanning calorimetry under various conditions. Both cytochromes exhibit a single endothermodenaturation peak in the differential scanning calorimetric thermogram. Thermodenaturation temperatures are ionic strength, pH, and redox state dependent. The ferrocytochromes are more stable toward thermodenaturation than the ferricytochromes. The enthalpy changes of thermodenaturation of ferro- and ferricytochrome c₁ are markedly dependent on the ionic strength of the solution. The effect of the ionic strength of solution on the enthalpy change of thermodenaturation of cytochrome c is rather insignificant. The formation of a complex between cytochromes c and c₁ at lower ionic strength causes a significant destabilization of the former and a slight stabilization of the latter. The destabilization of cytochrome c upon mixing with cytochrome c₁ was also observed at high ionic strength, under which conditions no stable complex was detected by physical separation. This suggests formation of a transient complex between these two cytochromes. When cytochrome c was complexed with phospholipids, no change in the thermodenaturation temperature was observed, but a great increase in the enthalpy change of thermodenaturation resulted.     

An improved breeding method for solitarious locusts

In order to unravel the physiological, endocrine, and behavioral differences between gregarious and solitarious forms of the desert locust, Schistocerca gregaria (Forsk.) (Orthoptera, Acrididae), a constant supply of rather large numbers of solitary individuals has to be guaranteed. This represents a bottleneck, mainly because of the intensity of the labor involved and limited laboratory accommodation. The method we describe here substantially reduces the space and manpower needed. The survival rate we obtained in the solitarised population was relatively high, reaching about 55%. The optimal rearing temperature proved to be 32–36 °C. Cabbage leaves and oat flakes sufficed for feeding all year round. Special racks have been designed that enable high density stacking and easy handling. The solitarisation process was monitored over ten consecutive generations. Changes in morphometrics, eye stripes, color, and behavior were recorded, of which only morphometrics, temperature related development, and mortality are discussed. A shift towards the solitarious phase was recorded, with clear differences between gregarious, 1^st generation and 7^th to 10^th generation solitarious locusts.     

An enzymic method for the determination of chitin and chitosan in fungal cell walls

The free and N-acetyl glucosamine contents, serving as a measure of the amounts of chitosan and chitin respectively, were determined in the chitinase hydrolysates of the cell wall of a wild strain ofNeurospora crassa. Chitinase, obtained from cultures ofSerratia marcescens, could hydrolyse the cell wall completely apart from being capable of hydrolysing preparations of chitin and chitosan. The free and N-acetyl glucosamines, released by chitinase hydrolysis, were determined by a modified Morgan-Elson reaction carried out in the presence and absence of acetic anhydride. The method is capable of estimating chitin and chitosan contents in as little as 100 μg of cell wall material.