马铃薯立枯丝核菌拮抗菌QHZ11的实时荧光定量PCR快速检测与应用

【背景】植物根际促生菌(plant growth-promoting rhizobacteria,PGPR)在根际的定殖是其发挥作用的基础,直观有效的跟踪技术和定量方法是研究PGPR在根际原位分布规律的重要工具。【目的】建立一种马铃薯黑痣病病原菌——立枯丝核菌拮抗菌QHZ11的实时荧光定量PCR快速检测体系,并检测拮抗菌QHZ11在马铃薯根际的动态变化。【方法】根据GenBank中登录的类芽孢杆菌及近源菌株gyrB基因序列差异筛选特异性引物,优化反应条件;通过盆栽试验对马铃薯根际拮抗菌进行快速检测。盆栽试验设3个处理,T1：对照(无菌水,CK);T2：QHZ11菌悬液灌土(QHZ11);T3：将功能菌在有机肥中进行二次固体发酵制成生物有机肥(BOF11)。【结果】筛选出拮抗菌QHZ11的专用引物为gyrB-F/gyrB-R;建立的拮抗菌QHZ11实时荧光定量PCR检测方法特异性好、灵敏度高且重复性较好,线性相关系数为0.999 8,检测组内变异系数均在1%以内,扩增效率为0.9,可检测出1×103?1×1010 copies/g-soil的拮抗菌,具有检出限低和扩增效率高的特点。盆栽试验结果发现,T3处理马铃薯根际拮抗菌QHZ11的数量从接种的第10天即高出T2处理一个数量级,并于马铃薯盛花期(接种后第60天)达到峰值,说明二次固体发酵增加了拮抗菌在根际土壤中的存活和繁殖。【结论】建立的实时荧光定量PCR快速检测体系灵敏、高效,可为研究拮抗菌在马铃薯根际原位分布以及与病原菌的互作方面提供便捷有效的方法。     

基于EMA-qPCR的茄科青枯菌活体检测技术的建立

【目的】利用特异性核酸染料叠氮溴乙锭(Ethidium monoazide bromide, EMA)与实时荧光定量PCR技术相结合, 建立一种能有效区分青枯菌死活细胞的检测方法。【方法】样品DNA制备前经EMA渗透预处理, 再进行实时荧光定量PCR特异扩增菌体DNA。【结果】终浓度为2.0 mg/L的EMA能有效排除1.0×107 CFU/mL灭活青枯菌细胞DNA的扩增, 对活细胞和不可培养状态(Viable but non-culturable, VBNC)活菌的DNA扩增均没有影响。当每个定量PCR反应体系中的活细胞在5.0×100?5.0×104 CFU范围内时, 扩增Ct值与定量PCR反应体系中活细胞CFU对数值呈良好的负相关性(R2=0.992 5)。比较EMA-qPCR法和平板计数法对经过不同温度短期保存的青枯菌检测结果发现, 待检样品可在24 °C与4 °C冷藏条件下短期保存。【结论】本研究建立的EMA-qPCR方法能有效检测青枯菌VBNC细胞和有效区分死活菌, 避免或减少青枯菌PCR检测的假阳性和假阴性。     

油棕（Elaeis guineensis）中果皮发育过程中miRNA的表达动态分析

以CTAB法提取油棕（Elaeis guineensis）中果皮5个不同发育时期（G1～G5）的小RNA。从前期研究获得的油棕小RNA测序数据库中筛选12个候选miRNA,实时荧光定量PCR法（qRT-PCR）检测其在果实发育过程中的表达量变化,并进一步对显著差异表达的miRNA进行靶基因预测。结果表明：中果皮5个不同发育时期小RNA的OD260/OD280比值在1.7～2.0之间;浓度分别是289、364、476、213、390 ng/μL;qRT-PCR检测结果显示,12个候选miRNA在5个发育时期均显著性差异表达,特别是在中果皮发育第4个时期（G4）和第5个时期（G5）表达量极显著增高,其中miR395和miR156在第4个时期表达量最高;miR395和miR528在发育第5时期表达量最高;靶基因预测结果显示差异表达的部分miRNA,其靶基因可能参与了脂肪酸代谢通路,如磷脂酸磷酸脂酶和磷脂酶D。本研究筛选的与脂肪酸代谢相关的miRNA为今后油棕脂肪酸代谢调控通路研究提供了可能的线索。     

Role of cuticular lipids and water-soluble compounds in tick susceptibility to Metarhizium infection

The arthropod cuticle acts as a physiochemical barrier protecting the organism from pathogens' entry. Entomopathogenic fungi actively penetrate the cuticles of arthropod hosts and are therefore directly affected by cuticle composition. Previously we have observed that Metarhizium spp. developing on resistant ticks ultimately die without penetrating tick's cuticle, suggesting that the cuticles of resistant ticks have antifungal compounds. In the present study, lipids and water-soluble cuticular components were extracted from engorged female tick cuticles, of one susceptible and one resistant tick species to Metarhizium spp. While conidia exposed to lipids from the susceptible tick, Rhipicephalus annulatus, germinated and differentiated into appressorium, conidia exposed to lipids from the resistant tick, Hyalomma excavatum, were inhibited. Soluble cuticular component extracts from both susceptible and resistant ticks stimulated conidial germination but not appressorium differentiation. A comparative analysis of the fatty acid profile in lipid extract of each tick exhibited similar compositions, but the relative abundance of C16:0, C18:0, C18:1ω9C and C20:0 was 2–5 times higher in the extracts from resistant ticks. All of these fatty acids inhibited conidial germination in vitro at 1% and 0.1% w/v concentration, but C20:0 stimulated appressorium differentiation at low concentration. This is the first report demonstrating a possible link between the presence of antifungal compounds in a specific concentration in tick cuticle and tick resistance to infection.     

Post-proline Dipeptidyl Amino-peptidase from Flavobacterium

Nitrogenase catalyzes not only the reduction of N₂ to NH₃ but also the reduction of C₂H₂ to C₂H₄ and H⁺ ion to H₂ gas, etc. The detailed mechanism of the nitrogenase reaction is not clear. We have prepared monoclonal antibodies against Component I nitrogenase of A. vinelandii and examined the effects of antibodies on the nitrogenase reactions. A monoclonal antibody designated MA-1 inhibited C₂H₂ reduction activity strongly but did not inhibit H₂ evolution activity. MA-2, on the contrary, inhibited only H₂ evolution activity. MA-8 inhibited both C₂H₂ reduction and H₂ evolution activity to the same extent.     

Combining ethidium monoazide treatment with real-time PCR selectively quantifies viable Batrachochytrium dendrobatidis cells

Detection of the lethal amphibian fungus Batrachochytrium dendrobatidis relies on PCR-based techniques. Although highly accurate and sensitive, these methods fail to distinguish between viable and dead cells. In this study a novel approach combining the DNA intercalating dye ethidium monoazide (EMA) and real-time PCR is presented that allows quantification of viable B. dendrobatidis cells without the need for culturing. The developed method is able to suppress real-time PCR signals of heat-killed B. dendrobatidis zoospores by 99.9 % and is able to discriminate viable from heat-killed B. dendrobatidis zoospores in mixed samples. Furthermore, the novel approach was applied to assess the antifungal activity of the veterinary antiseptic F10^® Antiseptic Solution. This disinfectant killed B. dendrobatidis zoospores effectively within 1 min at concentrations as low as 1:6400.     

Isothermal amplification detection of nucleic acids by a double-nicked beacon

Isothermal and rapid amplification detection of nucleic acids is an important technology in environmental monitoring, foodborne pathogen detection, and point-of-care clinical diagnostics. Here we have developed a novel method of isothermal signal amplification for single-stranded DNA (ssDNA) detection. The ssDNA target could be used as an initiator, coupled with a double-nicked molecular beacon, to originate amplification cycles, achieving cascade signal amplification. In addition, the method showed good specificity and strong anti-jamming capability. Overall, it is a one-pot and isothermal strand displacement amplification method without the requirement of a stepwise procedure, which greatly simplifies the experimental procedure and decreases the probability of contamination of samples. With its advantages, the method would be very useful to detect nucleic acids in point-of-care or field use.     

绿僵菌及其与dsRNA混合使用对褐飞虱的防治效果

褐飞虱Nilaparvata lugens Stl是我国重要的迁飞性水稻害虫,本文研究了金龟子绿僵菌Metarhizium anisopliae及其与dsRNA混合使用对褐飞虱的防治效果。绿僵菌悬浮液1.6×108孢子/m L至8×106孢子/m L对褐飞虱2龄、4龄和成虫进行喷药,发现1.6×107孢子/m L对各个虫态虫龄均有良好致死效果,并且成虫和4龄若虫均好于2龄若虫。在交配行为上来看,绿僵菌处理过的褐飞虱成虫活跃度非常低,从配对开始一直到交配结束的各个阶段都受到明显影响,处理组3 h的交配率只有3.70%,而对照组的交配率为24.44%。还把褐飞虱几丁质合成酶基因A的dsRNA与绿僵菌混合使用防治褐飞虱2龄和4龄若虫,结果表明0.5μg/μL ds CHSA与绿僵菌混合使用的防治效果最好,2龄若虫的死亡率为89.63%,4龄若虫的死亡率达到93.94%。而0.2μg/μL ds CHSA与绿僵菌的混合,对2龄和4龄若虫的致死率为65.56%-76.52%。研究结果为褐飞虱的生物防治提供了新的思路。     

A novel qPCR protocol for the specific detection and quantification of the fuel-deteriorating fungus Hormoconis resinae

A wide variety of fungi and bacteria are known to contaminate fuels and fuel systems. These microbial contaminants have been linked to fuel system fouling and corrosion. The fungus Hormoconis resinae, a common jet fuel contaminant, is used in this study as a model for developing innovative risk assessment methods. A novel qPCR protocol to detect and quantify H. resinae in, and together with, total fungal contamination of fuel systems is reported. Two primer sets, targeting the markers RPB2 and ITS, were selected for their remarkable specificity and sensitivity. These primers were successfully applied on fungal cultures and diesel samples demonstrating the validity and reliability of the established qPCR protocol. This novel tool allows clarification of the current role of H. resinae in fuel contamination cases, as well as providing a technique to detect fungal outbreaks in fuel systems. This tool can be expanded to other well-known fuel-deteriorating microorganisms.