Effects of conidial densities and spray volume of Metarhizium anisopliae and Beauveria bassiana fungal suspensions on conidial viability,droplet size and deposition coverage in bioassay using a novel bioassay spray system

A newly developed spray tower was used to characterise droplet distribution and coverage of conidial suspensions of Metarhizium anisopliae ATCC 62176 and Beauveria bassiana NI8 with different spray volumes. ATCC 62176 and NI8 had different spray models which could be caused by the surface physicochemical characteristics of the strains and conidia.     

球孢白僵菌两种施用方式对侵染桔小实蝇的影响及田间的防治效果

室内研究了球孢白僵菌Beauveria bassiana不同施用方式对侵染桔小实蝇Bactrocera dorsalis的影响,以及田间喷施球孢白僵菌防治桔小实蝇的效果。结果表明,两种诱集感染方式桔小实蝇成虫存活率分别为40%、41.7%,差异不显著;添加性引诱剂在白僵菌侵染桔小实蝇的过程中没有起到明显地促进感病的作用,只是缩短了病程,IIPC为0.41-0.44,说明两种诱集感染方式对桔实蝇成虫有一定的控制作用。三种施菌方式的桔小实蝇羽化率、成虫死亡率与对照差异均显著;以IIPC为评价指标,施菌方式对桔小实蝇各因子的影响大小顺序为：成虫存活率 >羽化率>化蛹率,其中以对成虫存活率的影响最大。应用化学防治和球孢白僵菌田间防治桔小实蝇的效果表明,施用2500倍40%毒死蜱效果最好,控制作用达到71.9%,其次是喷施2.0×108孢子/mL球孢白僵菌孢子悬浮液,防效达46.9%,施用2500倍4.5%高效氯氰菊酯防治效果达最差, 防效仅37.5%。     

Mass production of Beauveria bassiana for regulation of Leptinotarsa decemlineata populations

A simple liquid medium which enhanced the production of conidiospores by an isolate of the entomophagous fungus Beauveria bassiana is described. Spore production was attained using cultures floating in inflated sections of plastic tubing (“polyethylene cushions”) and glass bottles. A method is described for determining lethal doses and lethal times for second- and third-instar Leptinotarsa decemlineata larvae.     

Comparison of the organic compounds composition of Hylobius abietis males and females before and after exposure to Beauveria bassiana infection

In the present study, the lipid composition from male and female Hylobius abietis is analyzed pre- and post-infection with Beauveria bassiana. The compounds are analyzed by gas chromatography-mass spectrometry. In the lipids of males and females fatty acids, fatty acid methyl esters, alcohols, amino acids and other organic compounds are identified. The lipids of males and females after B. bassiana exposure contain 31 and 33 organic compounds, respectively, whereas the lipids of non-infected males and females contain 30 and 37 compounds, respectively. The most abundant compounds in infected and non-infected males are fatty acids: C16:0, C18:0, C18:1 and C18:2. Contents of dominant compounds from the lipids of infected and non-infected males are similar to those extracted from the lipids of infected and non-infected females. It should be noted that the total mass of organic compounds is higher in non-infected insects than infected insects.     

变温干燥固体发酵产物对球孢白僵菌分生孢子性能的影响

[目的]评价球孢白僵菌固体发酵产物的干燥温度对产后分生孢子性能的影响.[方法]采用28℃2和35℃组合的7种恒温或变温处理干燥发酵产物,分析收获的分生孢子质量.[结果]变温干燥可显著降低产后孢子粉的杂菌污染.干燥温度对活孢率和孢子萌发速度影响不一致.35℃恒温干燥5 h后活孢率与新鲜孢子无明显差异,但萌发中时缩短了9.3%.干燥处理提高了孢子对高温和紫外辐射的耐受性.适当的变温干燥比恒温干燥有利于增强孢子抗逆性.干燥温度影响分生孢子胞内海藻糖积累,但其含量与抗逆性无直接相关性.优化干燥温度可提高产后分生孢子毒力.在370～450孢子/mm2剂量下,经28℃ 24 h后升至35℃干燥2 h或35℃恒温干燥5 h的分生孢子对桃蚜的致死中时分别比新鲜孢子缩短了10.6 h和7.5 h.[结论]球孢白僵菌固体发酵产物的干燥温度是影响产后孢子粉杂菌污染、孢子活力、抗逆性和毒力的重要因素.     

采用两种不同施药方法评价八种药剂对韭蛆的防治效果

【目的】为筛选有效防治韭蛆Bradysia odoriphaga Yang et Zhang的药剂及科学合理的施药方法。【方法】本试验充分抓住韭蛆喜湿这一生物学特性,采用两种不同的施药方法(单次定点淋根法和二次灌根法)评价8种药剂对韭蛆的防治效果。【结果】同种药剂不同的施药方法,二次灌根法在韭蛆防效、韭菜保苗率和韭菜增产率三方面均明显高于单次定点淋根法。同时,噻虫胺、噻虫嗪和吡虫啉3种药剂单独使用,或分别与辣根素混配,对韭蛆均表现出最佳的防治效果。【结论】本研究提出了一种科学合理的施药方法,对防治韭蛆具有重要的意义。     

Pre-harvest retting of flax with glyphosate: effects of novel methods of spray application on penetration of the canopy and uniformity of deposition

Four methods of spray application were compared in 1986 with the recommended method of glyphosate application for pre-harvest retting of flax (Linum usitatissimum). Sampling individual plants immediately after application of glyphosate and dye demonstrated that spray penetration through the crop canopy, measured as dye deposition on the lower stem as a percentage of total deposition per plant, was the same for all methods of application. None of the methods of application improved the uniformity of dye deposition.
The following year, effects of deseeding prior to spraying and of using an air-assisted sprayer were investigated. Deseeding increased dye deposition on the lower stem but did not improve the efficacy of glyphosate. Application with an air-assisted sprayer increased the proportion of spray deposited on the lower stem but not the absolute amount deposited or the efficacy of glyphosate. Dye deposition on individual plants varied greatly for all methods of application and, when glyphosate was applied using the same methods, undesiccated plants occurred in all plots.
Considerable improvements in spray penetration and uniformity of spray deposition appear necessary for successful pre-harvest retting of flax with glyphosate.     

四种小鼠肠道微生物DNA提取方法比较

采用异硫氰酸胍(guandine thiocyanate,GITC)法、Tiangen DNA提取试剂盒、Omega DNA提取试剂盒和广泛应用的十六烷基三甲基溴化铵(hexadecyl trimethyl ammonium bromide,CTAB)法提取小鼠粪便微生物总DNA,通过比较所提取DNA的浓度和纯度,发现粪便DNA提取试剂盒提取的DNA纯度最高但浓度最低;CTAB法所得的DNA浓度最高但纯度最低;GITC法所得DNA的浓度高于粪便DNA提取试剂盒,纯度高于CTAB法。通过变性梯度凝胶电泳(16S r DNA-PCR-DGGE)指纹图谱分析技术进一步比较了各种提取方法所代表微生物群落的丰富度和多样性。结果表明,GITC法提取得到的DNA所代表细菌的丰富度和多样性显著高于其他3种方法。本实验所建立的GITC法可更全面地反映肠道微生物的多样性和群落结构,是一种较为理想的粪便微生物DNA提取方法。     

Comparison of DNA extraction methods for human gut microbial community profiling

The human gut harbors a vast range of microbes that have significant impact on health and disease. Therefore, gut microbiome profiling holds promise for use in early diagnosis and precision medicine development. Accurate profiling of the highly complex gut microbiome requires DNA extraction methods that provide sufficient coverage of the original community as well as adequate quality and quantity. We tested nine different DNA extraction methods using three commercial kits (TianLong Stool DNA/RNA Extraction Kit (TS), QIAamp DNA Stool Mini Kit (QS), and QIAamp PowerFecal DNA Kit (QP)) with or without additional bead-beating step using manual or automated methods and compared them in terms of DNA extraction ability from human fecal sample. All methods produced DNA in sufficient concentration and quality for use in sequencing, and the samples were clustered according to the DNA extraction method. Inclusion of bead-beating step especially resulted in higher degrees of microbial diversity and had the greatest effect on gut microbiome composition. Among the samples subjected to bead-beating method, TS kit samples were more similar to QP kit samples than QS kit samples. Our results emphasize the importance of mechanical disruption step for a more comprehensive profiling of the human gut microbiome.     

Comparison of two methods of preparing enzyme-antibody conjugates: application of these conjugates for enzyme immunoassay.

Two methods of preparing enzyme-antibody conjugates were evaluated. High yields of conjugate were obtained with both methods. The first procedure utilizes the homobifunctional crosslinking reagent N,N′-o-phenylenedimaleimide. Sulfydryl residues were introduced into second antibodies by reaction with methyl-mercaptobutyrimidate. The modified antibodies were reacted with N,N′-o-phenylenedimaleimide, excess reagent was removed by gel filtration, and the activated antibodies were cross-linked to β-galactosidase. Up to 80% of the enzyme was conjugated to immunologically active antibody with approximately 90% retention of enzyme activity. The second method utilizes the heterobifunctional meta-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). The antibodies were reacted with MBS, excess reagent was removed by gel filtration and the activated antibodies were crosslinked to β-galactosidase. Typically, approximately 80% of the enzyme was conjugated to immunologically active antibody with approximately 90% retention of both enzyme and antibody activity. Conjugates prepared using these two procedures were used as labels in an immunoassay system and were able to detect approximately 5 to 10 ng of first antibodies. The MBS procedure was simpler to perform, could more easily be adapted to large-scale work, and gave more reproducible results, and the conjugates produced were able to detect slightly lower concentrations of first antibody.     

Comparison of two methods for designing calorimeters using stirred tank reactors

Calorimetry is a robust method for online monitoring and controlling bioprocesses in stirred tank reactors. Up to now, reactor calorimeters have not been optimally constructed for pilot scale applications. Thus, the objective of this paper is to compare two different ways for designing reactor calorimeters and validate them. The “heat capacity” method based on the mass flow of the cooling liquid in the jacket was compared with the “heat transfer” method based on the heat transfer coefficient continuously measured in the cultivation of Escherichia coli VH33 in a 50 L stirred tank reactor. It was found that the values of the “heat transfer” method agreed very well with the calculated values from the oxygen consumption. By contrast, the curve of the “heat capacity” method deviated from that of the oxygen consumption calculated with the oxycaloric equivalent. In conclusion, the “heat transfer” method has been proven to have a higher degree of validity than the “heat capacity” method. Thus, it is a better and more robust means to measure heat generation of fermentations in stirred tank bioreactors on a pilot scale. Biotechnol. Bioeng. 2013; 110: 180–190. © 2012 Wiley Periodicals, Inc.     

Assessment of two biomass estimation methods for aquatic vegetation growing on the Amazon Floodplain

Studies of macrophyte productivity in the Amazon region are limited by accessibility and costs; hence, they may suffer from reduced sample size and representation. The present study compares a phenometric (indirect) method and a subsampling (direct) method in terms of accuracy and applicability to estimation of aquatic macrophyte biomass in the Amazon. The results show that phenometric models were not as effective as selective subsampling for the estimation of macrophyte biomass under the studied conditions. Phenometric models performed more acceptably for predicting emergent biomass, and less for submerged and total biomass (r² = 0.77, p < 0.05, RMSE = 200-600 g/m² dry mass). Improvements in r² by using species-specific phenometric models were mostly not significant. Phenotypic variation across the studied region was large enough to preclude the generalization of phenometric relationships into accurate numeric models, while the direct subsampling method was able to account for this variation (RMSE < 500 g/m² dry mass). Subsampling also allowed a significant reduction on the physical effort of biomass sampling, which directly translated into wider and more complete sampling. We suggest that direct subsampling presents the best trade-off between accuracy and coverage for macrophyte biomass measurement in the Amazon floodplain.     

Comparison of two PCR methods for rapid identification of Leptospira genospecies interrogans

Based on (i) an analysis of Leptospira 16S rDNA sequences determined by us and of those from databases and (ii) a previously published finding that restriction fragment length polymorphisms (RFLPs) within the Leptospira 16S and 23S rDNA were detected by nine restriction enzymes and these RFLPs allowed categorisation of Leptospira into eight genospecies, we predicted that one particular Dde I restriction site polymorphism within 16S rDNA could be independently used for identifications of Leptospira strains belonging to the genospecies interrogans . Two PCR-based methods, namely allele-specific amplification (ASA) and PCR-RFLP, were tested for the rapid detection of the Dde I restriction site polymorphism. One or two representative strains from each of nine genospecies were tested by ASA, whereas 73 strains from nine genospecies and two field isolates were tested by PCR-RFLP. Our experiments showed that the ASA method was not as specific as intended, but the PCR-RFLP method was useful for rapid identifications of the genospecies interrogans . We have not only confirmed a previous finding and extended the number of samples particularly from the genospecies biflexa , weilii , and inadai , but also simplified a previous PCR-RFLP protocol.     

Comparison of two methods for in vivo estimation of the glenohumeral joint rotation center (GH-JRC) of the patients with shoulder hemiarthroplasty

Determination of an accurate glenohumeral-joint rotation center (GH-JRC) from marker data is essential for kinematic and dynamic analysis of shoulder motions. Previous studies have focused on the evaluation of the different functional methods for the estimation of the GH-JRC for healthy subjects. The goal of this paper is to compare two widely used functional methods, namely the instantaneous helical axis (IHA) and symmetrical center of rotation (SCoRE) methods, for estimating the GH-JRC in vivo for patients with implanted shoulder hemiarthroplasty. The motion data of five patients were recorded while performing three different dynamic motions (circumduction, abduction, and forward flexion). The GH-JRC was determined using the CT-images of the subjects (geometric GH-JRC) and was also estimated using the two IHA and SCoRE methods. The rotation centers determined using the IHA and SCoRE methods were on average 1.47±0.62 cm and 2.07±0.55 cm away from geometric GH-JRC, respectively. The two methods differed significantly (two-tailed p-value from paired t-Test ～0.02, post-hoc power ～0.30). The SCoRE method showed a significant lower (two-tailed p-value from paired t-Test ～0.03, post-hoc power ～0.68) repeatability error calculated between the different trials of each motion and each subject and averaged across all measured subjects (0.62±0.10 cm for IHA vs. 0.43±0.12 cm for SCoRE). It is concluded that the SCoRE appeared to be a more repeatable method whereas the IHA method resulted in a more accurate estimation of the GH-JRC for patients with endoprostheses.     

落叶松（Larix gemelinii）苗挥发物两种收集方法的对比分析

采用预浓缩和固相微萃取(SPME)2种方法对2年生兴安落叶松苗挥发物成分进行采集并做GC-MS分析.结果表明,落叶松苗挥发物主要成分为萜烯类化合物,其中α-蒎烯、4(10)-侧柏烯、莰烯、β-蒎烯、D-柠檬烯和β-水芹烯所占比例较高;整株、针叶和树皮挥发物的组成和含量存在差异,针叶中饱和烷烃含量较高,树皮中倍半萜烯、醇、酮和酯类挥发物含量较高,并且这2种组织经SPME采集检测的挥发物中石竹烯和α-蛇麻烯含量增加幅度较大,它们可能在昆虫近距离寄主定位中起作用.对2种气体收集方法的分析比较,认为预浓缩结果接近落叶松挥发物组分和含量的自然特征,SPME受萃取头纤维特性的影响,对倍半萜稀类化合物吸附作用强,对烷烃吸附差,并且不同化合物的同时存在可能影响其吸附效率,导致检测量准确度下降,因此预浓缩法更适合植物挥发物定性和定量分析.     

Comparison of two 15N labelling methods for assessing nitrogen rhizodeposition of pea

Two ¹⁵N labelling methods for assessing net rhizodeposition of nitrogen (N) in pea crop (Pisum sativum L.) were compared in the greenhouse and in the field: the cotton-wick (CW) and the split-root (SR) methods. Rhizodeposition is defined as the organic material lost from roots during their growth through the soil. CW is a method in which ¹⁵N urea was supplied to the plant in pulses via a wick threaded through the stem. In SR, the root system was divided between a hydroponic labelling compartment (LC) containing the labelling nutrient solution (1 or 5 mM ¹⁵NO₃–¹⁵NH₄) and a compartment filled with soil in which the amount of ¹⁵N rhizodeposition was assessed. The percentage of N derived from rhizodeposition (%Ndfr), was used to calculate the amount of N rhizodeposition which was obtained from the ratio of atom % ¹⁵N excess of the soil : atom % ¹⁵N excess of the roots. Above ground parts in the field accumulated markedly more dry matter and N than in the greenhouse, regardless of the labelling method. ¹⁵N enrichments of above ground parts were higher than those of roots recovered from the soil. Results indicated that amount of ¹⁵N applied to plants were lower in SR than in CW. Additionally, LC roots of SR tended to retain large amounts of ¹⁵N. As a consequence, atom % ¹⁵N excess of roots was less than 1% in SR, whereas most values varied from 1% to 4% in CW. However, relationships between enrichments of the soil and of the roots were different in SR and CW. It was not possible to compare the Ndfr:root-N ratio between the two methods, but the ratio of Ndfr:plant-N was found to be 10% higher in SR than in CW. Finally, relative to total plant-N, the total contribution of below ground parts to the N pool of the soil reached 22–25% at maturity for the two methods. From our experiments, we could not conclude that one method is better than the other for estimating either net rhizodeposition of N or the contribution of a pea plant to the soil N pool. However, CW is easier to adapt and monitor under field conditions than SR.     

两种抽提乙型肝炎病毒共价闭合环状DNA方法的效果比较

乙型肝炎病毒(hepatitis B virus,HBV)作为一种嗜肝DNA病毒,在感染肝细胞后会在细胞核中形成病毒转录复制的模板和基因储存库--共价闭合环状DNA(covalently closed circular DNA, cccDNA),其持续存在是乙型肝炎慢性化和难以治愈的核心,也是此研究领域内的重点。从细胞样品中稳定抽提获取cccDNA对于保证cccDNA检测的准确性至关重要。Hirt法是一种抽提真核细胞染色体外DNA的方法,被用于HBV cccDNA的抽提,但存在操作复杂和耗时长等问题。为简化操作,有研究对Hirt法进行改良,结合硅胶膜离心柱来抽提染色体外DNA,但尚不清楚该法用于HBV cccDNA抽提与传统Hirt法的效果差异。本研究基于HBV cccDNA细胞转染系统、HBV复制细胞系及感染系统,以DNA印迹(Southern blot)和定量聚合酶链式反应(quantitative polymerase chain reaction, qPCR)作为检测评价手段,平行比较了传统Hirt-酚/氯仿法与改良Hirt-过柱法抽提HBV cccDNA的效果。结果表明,两种方法具有相当的抽提效率和抽提特异性,而改良Hirt-过柱法耗时更短,提示在进行细胞HBV cccDNA抽提时可选择改良Hirt-过柱法以提高实验效率。     

Comparison of DNA and RNA quantification methods suitable for parameter estimation in metabolic modeling of microorganisms

Recent developments in cellular and molecular biology require the accurate quantification of DNA and RNA in large numbers of samples at a sensitivity that enables determination on small quantities. In this study, five current methods for nucleic acid quantification were compared: (i) UV absorbance spectroscopy at 260 nm, (ii) colorimetric reaction with orcinol reagent, (iii) colorimetric reaction based on diphenylamine, (iv) fluorescence detection with Hoechst 33258 reagent, and (v) fluorescence detection with thiazole orange reagent. Genomic DNA of three different microbial species (with widely different G+C content) was used, as were two different types of yeast RNA and a mixture of equal quantities of DNA and RNA. We can conclude that for nucleic acid quantification, a standard curve with DNA of the microbial strain under study is the best reference. Fluorescence detection with Hoechst 33258 reagent is a sensitive and precise method for DNA quantification if the G+C content is less than 50%. In addition, this method allows quantification of very low levels of DNA (nanogram scale). Moreover, the samples can be crude cell extracts. Also, UV absorbance at 260 nm and fluorescence detection with thiazole orange reagent are sensitive methods for nucleic acid detection, but only if purified nucleic acids need to be measured.     

2种石房蛤毒素完全抗原交联方法的比较及多克隆抗体的制备

[目的]探究石房蛤毒素(STX)完全抗原制备方法和STX多克隆抗体免疫方案。[方法]通过碳二亚胺法(EDC)和高碘酸盐法(periodate reaction)2种交联方法,将小分子石房蛤毒素与牛血清白蛋白(BSA)、鸡卵清蛋白(OVA)和孔血蓝蛋白(KLH)分别进行交联,制备了6种形式STX完全抗原,并对交联物进行琼脂糖凝胶电泳鉴定和紫外吸收峰迁移变化鉴定。分别将EDC法和高碘酸盐法交联的STX-BSA、STX-KLH 4种完全抗原作为免疫原,对Balb/c小鼠进行免疫,获得STX多克隆抗体。通过间接ELISA法,对不同方法制备的多克隆抗体进行分析比较。[结果]在石房蛤毒素完全抗原的制备中,在交联方法的选择上,EDC法较高碘酸盐法更具优势;而在免疫原的选择上,STX-BSA完全抗原效果最好。[结论]本研究探究了2种制备STX完全抗原的方法,为今后多克隆抗体生产以及特异性单克隆抗体筛选提供数据支撑。