Differential susceptibility of Amblyomma maculatum and Amblyomma americanum (Acari:Ixodidea) to the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae

Nymph and adult ticks from Ambylomma americanum and Ambylomma maculatum were treated with conidia and blastospores of the entomopathogenic fungi Beauveria bassiana (90517) and Metarhizium anisopliae (20500). Fungal suspensions of conidia harvested from potato dextrose plates containing 10⁸ conidia/ml caused greater than 90% mortality in adult A. maculatum but less than 10% mortality in adult A. americanum over a 28 day time course. Similarly, infection with M. anisopliae (10⁸ conidia/ml) resulted in 60 and 15% mortality in A. maculatum and A. americanum, respectively. Nymphs of both tick species were more susceptible to fungal infection reaching mortality rates of almost 100% for A. maculatum and over 35% for A. americanum. Scanning electron microscopy of infected ticks showed rapid attachment, germination, and proliferation of fungal spores on A. maculatum cuticles, and to a much lesser extent on A. americanum cuticles. Pentane extracts of A. americanum cuticle hydrocarbons inhibited germination and hyphal growth of B. bassiana conidia, whereas no inhibition was observed using A. maculatum extracts. Significant mortality towards A. americanum was observed (>60%, 28 days) only when the ticks were treated with B. bassiana directly from the growth medium (10⁷ blastospores/ml, grown for 3–4 days in Sabouraud dextrose + 0.5% yeast extract liquid media). These results indicate tick species display differential susceptibility to the entomopathogenic fungi B. bassiana and M. anisopliae, and that the ability to overcome fungistatic compounds present in the tick epicuticle may determine the likelihood of successful infection and virulence.     

C-C bond formation and cleavage in radical enzymes, a theoretical perspective

Quantum chemical methods are today a viable tool in the study of enzyme catalysis. The development of new density functional techniques and the enormous advancement in computer power have made it possible to accurately describe active sites of enzymes. This review gives a brief account of the methods and models used in this field. Three specific enzymes are discussed: pyruvate-formate lyase (PFL), spore photoproduct lyase (SPL), and benzylsuccinate synthase (BSS). What these enzymes have in common is that they use radical chemistry to catalyze C-C bond formation or cleavage reactions.     

Antifungal effects of chitosan with different molecular weights on in vitro development of Rhizopus stolonifer (Ehrenb.:Fr.) Vuill

Determination of the molecular weight of three types of chitosan was carried out by HPSEC-RI. The effect of low, medium and high molecular weight chitosan was evaluated on development of three isolates of Rhizopus stolonifer. Image analysis and electronic microscopy observations were done in spores of this fungus. Germination of R. stolonifer in potato dextrose broth with chitosan was also evaluated. Results pointed out that the low molecular weight chitosan was more effective for inhibition of mycelial growth while the high molecular weight chitosan affected spore shape, sporulation and germination. Studies of scanning and transmission electron microscopy revealed numerous and deeper ridge ornamentations of the chitosan-treated spore.     

The arachiform vacuolar body: an overlooked shared character in the ascospores of a large monophyletic group within Parmeliaceae (Xanthoparmelia clade, Lecanorales)

Sections of apothecia were used to study the internal morphology of ascospores in the largest monophyletic clade within Parmeliaceae composed of Xanthoparmelia and related genera. The results were compared with fertile representative species of most other parmelioid clades. All the Xanthoparmelia species had spores with a single smooth vacuole, which was peanut-shaped, with different degrees of constriction in the equatorial plane. This differs from the ellipsoid vacuole of other parmelioids. In the Xanthoparmelia clade, sexual reproduction seems much more common than in other parmelioids. Thus, we suggest that the presence of this unique spore morphology might contribute to the evolutionary success of this monophyletic group. Further, the discovery of this useful ascospore character demonstrates that detailed ascospore morphological studies significantly enhance molecular phylogenetic analyses. Ascospore features may be more taxonomically significant in Parmeliaceae than hitherto considered.     

The halotolerant fungus Glomerobolus gelineus is a member of the Ostropales

Glomerobolus gelineus is a halotolerant species with a unique method of ballistic propagation. The absence of both sexual and asexual spores made reliable placement of this species, based on morphology alone, within the current fungal classification problematical. A phylogenetic analysis of the large and small nuclear ribosomal subunit and the second largest subunit of RNA polymerase II placed this fungus within the Ostropales, an order comprising lichenized and saprobic species, with good statistical support. Subsequently, a more detailed analysis that combined the nuc LSU rDNA and the mt SSU rDNA confirmed a close relationship to the Stictidaceae. The phylogenetic placement of G. gelineus is also supported by morphological characters. We postulate that the hyphoma lobes of Glomerobolus correspond to the periphysoidal layer in the apothecium of Stictis, and the propagule to the hymenium. Moreover, the presence of crystals in the outer lobes of G. gelineus is another indication of its relationship with Ostropales, which have characteristic crystalliferous hyphae. The placement of Glomerobolus within the Ostropales further expands the ecological diversity exhibited by this order. It also provides a phylogenetic hypothesis for assessing the homology of the enigmatic hyphomal morphology with apothecia-forming Ascomycota.     

Partial purification and characterization of an initiation protein for germination from Clostridium perfringens spores

An initiation protein which promoted genermination of dithiothreitol-sodium dodcyl suflate-trated spores of clostridium perfringens was purified from 7-h culture supernatant fluid of cell of Cl. perfringenes. A 3100-fold purification was obtained after cellulose-phosphate and Sephadex G-100 chromatography. The isolated product had an apparent molecular weight of 100 000, an isoelectric point of 7.9, was reversibly inhibited by HgCl₂ and lysed isolated cortical fragments from Cl. perfringens spores.     

Microresonator mass sensors for detection of Bacillus anthracis Sterne spores in air and water

Towards the goal of developing a real-time monitoring device for microorganisms, we demonstrate the use of microcantilevers as resonant mass sensors for detection of Bacillus anthracis Sterne spores in air and liquid. The detection scheme was based on measuring resonant frequency decrease driven by thermally induced oscillations, as a result of the added mass of the spores with the use of a laser Doppler vibrometer (LDV). Viscous effects were investigated by comparing measurements in air and deionized (DI) water along with theoretical values. Moreover, biological experiments were performed which involved suspending spores onto the cantilevers and performing mass detection in air and water. For detection of spores in water, the cantilevers were functionalized with antibodies in order to fix the spores onto the surface. We demonstrate that as few as 50 spores on the cantilever can be detected in water using the thermal noise as excitation source. Measurement sensitivity of 9.23 Hz/fg for air and 0.1 Hz/fg for water were obtained. These measurements were compared with theoretical values and sources of improvement in cantilever sensitivity in a viscous medium were also discussed. It is expected that by driving the cantilevers and using higher order modes, detection of a single spore in liquids should be achievable.     

Monoclonal antibodies for the detection of Puccinia striiformis urediniospores

The fungal pathogen Pst causes yellow rust disease in wheat plants leading to crop losses. The organism spreads by releasing wind-dispersed urediniospores from infected plants. In this study a library of novel monoclonal antibodies (mAbs) was developed against Pst urediniospores. Nine mAb-producing cell lines were cloned and their cross-reactivities characterised against a panel of airborne fungal spores representing genera commonly found in the same environment as Pst. Two specific mAbs were used to develop a competitive ELISA (Pst mAb4) and a subtractive inhibition ELISA (Pst mAb8). Standard curves for both assays had good intra- and interday reproducibility. The subtractive inhibition ELISA had greater sensitivity with a detection limit of 1.5 × 10⁵ spores ml⁻¹. Cross-reactivity studies of Pst mAb8 in the subtractive inhibition ELISA, showed reaction with other Puccinia spores only, suggesting that common epitopes exist within this genus. The biosensor-compatible Pst mAb8 assay principle developed in this study has the potential to be implemented in future ‘label-free’ in-the-field systems for Pst detection.     

Chemoselective Deprotection of Triethylsilyl Ethers

An efficient and selective method was developed for the deprotection of triethylsilyl (TES) ethers using formic acid in methanol (5–10%) or in methylene chloride 2–5%) with excellent yields. TES ethers are selectively deprotected to the corresponding alcohols in high yields using formic acid in methanol under mild reaction conditions. Other hydroxyl protecting groups like t-butyldimethylsilyl (TBDMS) remain unaffected.     

地衣芽孢杆菌BF-002高产芽孢的氮源流加工艺研究*

目的: 提高地衣芽孢杆菌BF-002的芽孢产量,实现氮源流加过程的自动化控制,降低生产成本,为其他芽孢杆菌提高芽孢产率的研究提供一种思路。方法: 通过摇瓶做单因素实验,筛选最佳温度和碳氮源,在此基础上进行5 L发酵罐实验。初始添加不同浓度的氮源,探索芽孢形成与氮源的关系。提出相对氨基氮的概念,通过恒速补料、间歇补料和基于尾气CO₂浓度反馈流加三个策略控制相对氨基氮浓度水平。采用Python语言编写计算机控制程序,实现基于尾气CO₂浓度反馈流加策略的自动化控制。结果: 摇瓶筛选最佳温度及碳氮源分别为:37℃、葡萄糖、鱼粉蛋白胨、豆粕。上罐结果表明,相对氨基氮浓度越低芽孢率越高,采用基于尾气CO₂浓度反馈流加能将相对氨基氮控制在8.42 mg/OD₆₀₀水平,芽孢量可达4.25×10⁹ cfu/mL。利用计算机程序自动控制低价氮源氯化铵的流加,可以使芽孢量达到1.87×10¹⁰ cfu/mL,是前期最优批次的4.4倍,同时降低原料成本。结论: 将相对氨基氮浓度控制在适宜水平可以得到芽孢量较高的培养液,自动流加氯化铵策略能降低生产成本并实现自动化控制,为研究芽孢杆菌产孢提供一种思路。     

梨蒴珠藓孢子萌发与原丝体发育特征研究

为了解梨蒴珠藓（Bartramia pomiformis）孢子萌发和原丝体发育特征,在显微镜下观察室内人工培养的梨蒴珠藓单倍配子体发育过程。结果表明,梨蒴珠藓孢子吸水膨胀5 d后,开始破壁萌发,原丝体系统以丝状绿丝体为主,轴丝体在绿丝体上分化产生。培养22 d后,配子体在轴丝体细胞上分化产生。参照Nishida的标准,梨蒴珠藓孢子萌发类型为真藓型（Bryum-type）。这为梨蒴珠藓的人工扩繁提供了发育学基础资料。     

Soil burrowing Muscina angustifrons (Diptera: Muscidae) larvae excrete spores capable of forming mycorrhizae underground

Muscina angustifrons (Diptera: Muscidae) is a mycophagous species that exploits a variety of fungi, including ectomycorrhizal fungi. Larvae of this species have been shown to feed on sporocarps (including spores), and full-grown larvae leave sporocarps and pupate 0–6?cm below the soil surface. In this study, we examined whether M. angustifrons larvae are capable of transporting ectomycorrhizal fungal spores and enhancing ectomycorrhiza growth on host-plant roots. Full-grown larvae were found to move horizontally 10–20?cm from their feeding sites and burrow underground. These wandering larvae retained ectomycorrhizal fungal spores in their intestines, which were excreted following relocation to underground pupation sites. Excreted spores retained germination and infection capacities to form ectomycorrhiza on host-plant roots. In the infection experiments, ectomycorrhizal fungal spores applied in the vicinity of underground host-plant roots were more effective in forming ectomycorrhiza than those applied to the ground surface, suggesting that belowground transportation of spores by M. angustifrons larvae could enhance ectomycorrhizal formation. These results suggested that M. angustifrons larvae act as a short-distance spore transporter of ectomycorrhizal fungi.     

Spore photoproduct within DNA is a surprisingly poor substrate for its designated repair enzyme—The spore photoproduct lyase

DNA repair enzymes typically recognize their substrate lesions with high affinity to ensure efficient lesion repair. In UV irradiated endospores, a special thymine dimer, 5-thyminyl-5,6-dihydrothymine, termed the spore photoproduct (SP), is the dominant DNA photolesion, which is rapidly repaired during spore outgrowth mainly by spore photoproduct lyase (SPL) using an unprecedented protein-harbored radical transfer process. Surprisingly, our in vitro studies using SP-containing short oligonucleotides, pUC 18 plasmid DNA, and E. coli genomic DNA found that they are all poor substrates for SPL in general, exhibiting turnover numbers of 0.01–0.2 min⁻¹. The faster turnover numbers are reached under single turnover conditions, and SPL activity is low with oligonucleotide substrates at higher concentrations. Moreover, SP-containing oligonucleotides do not go past one turnover. In contrast, the dinucleotide SP TpT exhibits a turnover number of 0.3–0.4 min⁻¹, and the reaction may reach up to 10 turnovers. These observations distinguish SPL from other specialized DNA repair enzymes. To the best of our knowledge, SPL represents an unprecedented example of a major DNA repair enzyme that cannot effectively repair its substrate lesion within the normal DNA conformation adopted in growing cells. Factors such as other DNA binding proteins, helicases or an altered DNA conformation may cooperate with SPL to enable efficient SP repair in germinating spores. Therefore, both SP formation and SP repair are likely to be tightly controlled by the unique cellular environment in dormant and outgrowing spore-forming bacteria, and thus SP repair may be extremely slow in non-spore-forming organisms.     

影响泽泻蕨孢子培养的环境因素研究

为了解环境因子对泽泻蕨(Hemionitis arifolia Moore.)培养的影响, 采用孢子培养技术研究温度、光照、pH、密度等对其有性繁殖的影响。结果表明, 泽泻蕨孢子为需光萌发, 孢子萌发和配子体发育的最适环境条件为:温度25℃, 日光灯光照强度为150 μmol m^-2s^-1, pH 为6.5, 播种密度为5~8 grains cm^-2, 幼苗管护简单, 成活率较高, 未见病虫害。这为保护和开发我国泽泻蕨资源提供了理论依据和技术支持。     

Germination and reproductive biology in salty conditions of Asplenium marinum (Aspleniaceae), a European coastal fern

Although there are several species of ferns that are important components of many coastal ecosystems, the adaptations that allow these species to live in salty conditions have been studied only in two Acrostichum species. We have studied the effects of salt on germination, development of the gametophyte and sexual expression of the fern Asplenium marinum, which lives on coastal cliffs of Europe. Cultures at four different levels of salt concentration, 0%, 0.98%, 1.96% and 3.26%, were established for this purpose. Salt caused a significant delay in spore germination, and prevented it completely at high concentrations (3.26%). However, spores were able to recover germination ability after a period of seawater incubation once salinity concentrations decreased. Variable salinity conditions are a typical feature of sea cliffs where halophytes can grow. A salty culture medium also affects the development of the prothalli. Higher salinity of the substrate produced a significant decrease in gametophyte sizes, and affected the development of gametangia as well. Only males were found in a 0.98%-salt medium, and no gametangia were observed in cultures at 1.96%-salt medium. Recovered spores after one and a half month in high salinity concentration, 3.26%, were able, when transplanted to 0%-salt medium, to develop normal sexual prothalli, and this occurred in a shorter time than spores without any prior treatment. A. marinum can be considered as a halophyte fern that is able to germinate and grow in salty conditions. But it takes advantage of rainy periods that cause decrease of salt concentration, which seems to be important in particular for gametangia development and fertilization.     

农药对生防菌厚孢普可尼亚菌ZK7生长发育的影响

以PDA平板和PDB摇瓶培养法测定了6种常用化学农药对烟草根结线虫生防菌厚孢普可尼亚菌ZK7菌株孢子萌发率、平板抑制作用和液体培养生物量的影响。结果表明,生防菌ZK7菌株对低浓度的6种药剂均存在一定抗性,对不同药剂的抗性表现出差异。其中多菌灵和甲基托布津对生防菌ZK7菌株的抑制作用最大,其次是辛硫磷和雷多米尔,克百威和涕灭威抑制作用最小。     

Gibberellin precursor is involved in spore germination in the moss Physcomitrella patens

Gibberellins are ent-kaurene derived phytohormones that are involved in seed germination, stem elongation, and flower induction in seed plants, as well as in antheridia formation and spore germination in ferns. Although ubiquitous in vascular plants, the occurrence and potential function(s) of gibberellins in bryophytes have not yet been resolved. To determine the potential role of gibberellin and/or gibberellin-like compounds in mosses, the effect of AMO-1618 on spores of Physcomitrella patens (Hedw.) B.S.G. was tested. AMO-1618, which inhibited ent-kaurene and gibberellin biosynthesis in angiosperms, also inhibited the bifunctional copalyl diphosphate synthase (E.C. 5.5.1.13)/ent-kaurene synthase (E.C. 4.2.3.19) of P. patens. AMO-1618 also caused a decrease in spore germination rates of P. patens, and this inhibitory effect was less pronounced in the presence of ent-kaurene. These results suggest that ent-kaurene biosynthesis is required by P. patens spores to germinate, implying the presence of gibberellin-like phytohormones in mosses.     

DNA-Binding Properties of the Antibody Specific for the Dewar Photoproduct of Thymidylyl-(3′′-5′′)-Thymidine

A monoclonal antibody (DEM-1) specific for the Dewar photoproduct is used for detection and quantification of photolesions in DNA. To help understand the molecular recognition of damaged DNA by the antibody protein, we have cloned and sequenced the variable region genes of DEM-1. We have also prepared Fab fragments of DEM-1 (DEM1Fab), and synthesized two kinds of 3′-biotinylated oligonucleotides of different lengths containing a central Dewar photoproduct of TpT to analyze the effects of the antigen size on the binding rates by means of surface plasmon resonance (SPR). Results obtained from SPR analyses suggest that DEM1Fab may recognize tetranucleotide unit as the epitope.     

Permeability of gentamicin into the inside of Bacillus subtilis spores

Abstract The penetration of gentamicin into the inside of Bacillus subtilis spores was examined by an immunoelectron microscopy method with colloidal gold-immunoglobulin G complex. The colloidal gold particles were located mainly in the coat regions of spores and were not observed in the cortex or core regions. This result suggests the existence of an outer membrane inside the coat region as the primary permeability barrier to gentamicin.     

Changes in the organisation of surface rodlets during germination ofSyncephalastrum racemosum sporangiospores

Summary Dormant sporangiospores ofSyncephalastrum racemosum have a regular pattern of rodlets on their surface. During the spherical growth phase of spore germination these rodlets become displaced and areas free from rodlets appear. At the time of germ tube emergence the rodlets remain on the spherical portion of the spore whilst the germ tube has a smooth surface.