<meta charset="utf8"></head><body>探索森林蛀木虫的昆虫病原真菌资源

<table><tbody><tr><td>1.5 mL, 2 mL centrifuge tubes</td><td>Biosharp</td><td>BS-15-M</td><td></td></tr><tr><td>10 &amp;micro;L pipet tips</td><td>Sangon Biotech</td><td>F601216</td><td></td></tr><tr><td>10 &amp;micro;L, 20 &amp;micro;L, 100 &amp;micro;L, 200 &amp;micro;L, 1,000 &amp;micro;L pipettes</td><td>Rainin</td><td></td><td></td></tr><tr><td>1,000 &amp;micro;L pipet tips</td><td>Sangon Biotech</td><td>F630102</td><td></td></tr><tr><td>2 mL cryogenic vials</td><td>Corning</td><td>430659</td><td></td></tr><tr><td>20x PBS buffer</td><td>Sangon Biotech</td><td>B548117-0500</td><td></td></tr><tr><td>200 &amp;micro;L pipet tips</td><td>Sangon Biotech</td><td>F601227</td><td></td></tr><tr><td>2,000 bp maker</td><td>TaKaRar</td><td>SD0531</td><td></td></tr><tr><td>50 mL tubes</td><td>Nest</td><td>602052</td><td></td></tr><tr><td>50% glutaraldehyde solution</td><td>Sangon Biotech</td><td>G916054</td><td></td></tr><tr><td>50x TAE buffer</td><td>Sangon Biotech</td><td>B548101</td><td></td></tr><tr><td>6x loading buffer</td><td>TaKaRar</td><td>SD0503</td><td></td></tr><tr><td>Agarose</td><td>Sangon Biotech</td><td>A610013</td><td></td></tr><tr><td>Anhydrous ethanol</td><td>Jkchemical</td><td>LB10V37</td><td></td></tr><tr><td>Biochemistry Cultivation Cabinet</td><td>Shanghaiyiheng</td><td>LRH-250F</td><td></td></tr><tr><td>Chloroform</td><td>Juhua</td><td>61553</td><td></td></tr><tr><td>Commercial beetle traps</td><td>FEIMENGDI</td><td>BF-8</td><td>www.yinyouji.com</td></tr><tr><td>Gel imager</td><td>Bio-Rad</td><td>GelDoc XR+</td><td></td></tr><tr><td>Glycerol</td><td>Sangon Biotech</td><td>A600232</td><td></td></tr><tr><td>High speed refrigerated centrifuge</td><td>Sigma</td><td>D-37520</td><td></td></tr><tr><td>High-Pressure Steam Sterilization Pot</td><td>Mettler Toledo</td><td>JA5003</td><td></td></tr><tr><td>Isopropyl alcohol</td><td>General-reagent</td><td>G75885B</td><td></td></tr><tr><td>Nucleic acid dye</td><td>Sangon Biotech</td><td>A616696</td><td></td></tr><tr><td>Optical Microscope, OM</td><td>Leica</td><td>DM2000</td><td></td></tr><tr><td>Parafilm</td><td>Parafilm</td><td>PM996</td><td></td></tr><tr><td>PCR meter</td><td>Heal Force</td><td>Trident960</td><td></td></tr><tr><td>Penicillin G</td><td>Marklin</td><td>GB15743</td><td></td></tr><tr><td>Potato dextrose agar, PDA</td><td>Oxoid</td><td>CM0139</td><td></td></tr><tr><td>Potato dextrose broth, PDB</td><td>Solarbio</td><td>P9240</td><td></td></tr><tr><td>Primers</td><td>Sangon Biotech</td><td>/</td><td></td></tr><tr><td>Primers Taq</td><td>TaKaRar</td><td>RR902A</td><td></td></tr><tr><td>Rapid Fungi Genomic DNA Isolation Kit</td><td>Sangon Biotech</td><td>B518229</td><td></td></tr><tr><td>Scanning Electron Microscope, SEM</td><td>Hitachi</td><td>S-3400N</td><td></td></tr><tr><td>Streptomycin</td><td>Marklin</td><td>S6153</td><td></td></tr><tr><td>Tetracycline</td><td>Marklin</td><td>T829835</td><td></td></tr><tr><td>Tween-80</td><td>Marklin</td><td>T6336</td><td></td></tr><tr><td>Vacuum freeze dryer</td><td>Yamato</td><td>DC801</td><td></td></tr><tr><td>Vortex Shaker</td><td>HLD</td><td>WH-861</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>Marklin</td><td>M6230</td><td></td></tr></tbody></table>

isolation, behavioral identification, and pathogenicity assessment of entomopathogenic fungi from a forest wood borer

Read this Scientific Article Protocol about Isolation, Behavioral Identification, and Pathogenicity Assessment of Entomopathogenic Fungi from a Forest Wood Borer at JoVE.com

<meta charset="utf8"></head><body>作者聚焦：野生 Monochamus alternatus 种群中昆虫病原真菌的评估，用于森林蛀木虫的生物防治应用

森林蛀木虫昆虫病原真菌的分离、行为鉴定和致病性评估

Forest wood borers (FWB) cause severe tree damage and economic losses worldwide. The release of entomopathogenic fungi (EPF) during the FWB emergence ...

isolation-behavioral-identification-pathogenicity-assessment

Research

JoVE Journal

Biology

Isolation, Behavioral Identification, and Pathogenicity Assessment of Entomopathogenic Fungi from a Forest Wood Borer

606 次观看.  Zhejiang A&F University. 我们探索了来自野生 Monochamus alternatus 种群的昆虫病原真菌资源。以该物种为例，我们可以丰富其他森林蛀木虫的昆虫病原真菌的多样性，用于未来的生物防治应用。我们最近获得了三种被认证为 Monochamus alternatus 寄生昆虫病原真菌的真菌物种，其中两种并不为人所知，但已被证明是昆虫的有效病原体。该协议有助于发现更多的 Monochamus alternatus ...

视频: 作者聚焦：野生 Monochamus alternatus 种群中昆虫病原真菌的评估，用于森林蛀木虫的生物防治应用

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

从胎盘中的造血干细胞的分离和分析

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

科研

生物学

Psychophysiological Stress Assessment Using Biofeedback 

In the last half century, research in biofeedback has shown the extent to which the human mind can influence the functioning of the autonomic nervous system, previously thought to be outside of conscious control. By letting people observe signals from their own bodies, biofeedback enables them to develop greater awareness of their physiological and psychological reactions, such as stress, and to learn to modify these reactions. Biofeedback practitioners can facilitate this process by assessing people s reactions to mildly stressful events and formulating a biofeedback-based treatment plan. During stress assessment the practitioner first records a baseline for physiological readings, and then presents the client with several mild stressors, such as a cognitive, physical and emotional stressor. Variety of stressors is presented in order to determine a person's stimulus-response specificity, or differences in each person's reaction to qualitatively different stimuli. This video will demonstrate the process of psychophysiological stress assessment using biofeedback and present general guidelines for treatment planning.

Psychophysiological Stress Assessment Using Biofeedback

In this video we will describe one method of assessing person's psychophysiological reaction to stress using biofeedback. We will also present general guidelines for treatment planning.

使用生物反馈的心理生理压力评估

In the last half century, research in biofeedback has shown the extent to which the human mind can influence the functioning of the autonomic nervous ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

吸血鬼从细胞外液分离<em&gt;秀丽隐杆线虫</em

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

体外评估对离体骨骼肌的收缩，疲劳性和交替“

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

隔离和线粒体从培养细胞和小鼠组织功能分析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. To improve the mating efficiency of B. Xylophilus and to increase the chances of mating observation, virgin adults were cultured and used for the investigation. Eggs were obtained by keeping the nematodes in water and allowing the females to lay eggs for 10 min. The second-stage juveniles (J2) were synchronized by incubating the eggs for 24 h at 25 &#176;C in the dark, and the early J4 were obtained by culturing the J2 with grey mold, Botrytis cinerea, for another 52 h. At this time point, most J4 nematodes could be clearly distinguished as being male or female using their genital morphology. The male and female J4 were collected and cultured separately in two different Petri dishes for 24 h to get virgin adult nematodes. A virgin male and a virgin female were paired in a drop of water in the well of a concave slide. The mating behavior was filmed with a video recorder under a stereomicroscope. The whole period of the mating process was 82.8 &#177;3.91 min (mean &#177;SE) and could be divided into 4 different phases: searching, contacting, copulating, and lingering. The mean minutes of duration were 21.8 &#177; 2.0, 28.0 &#177; 1.9, 25.8 &#177; 0.7 and 7.2 &#177; 0.5, respectively. Eleven sub-behaviors were described: cruising, approaching, encountering, touching, hooping, locating, attaching, ejaculating, separating, quiescence, and roaming. Interestingly, obvious intra-sexual competition was observed when one female was grouped with 3 males or one male with 3 females. This protocol is useful and valuable, not only in investigating the mating behavior of B. xylophilus, but also in acting as a reference for ethological studies of other nematodes.

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A protocol for investigating the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, is presented. Behavioral features of B. xylophilus are described in the mating process.

观察和交配行为的量化在我国松材线虫，<em&gt;松材线虫</em

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. ...

Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important to isolate and select indigenous EPF. Therefore, the soil bait method combined with the insect bait (mealworm, Tenebrio molitor) system was used in this study with some modifications. The isolated EPF were then subjected to the virulence test against the agricultural pest Spodoptera litura. Furthermore, the potential EPF strains were subjected to morphological and molecular identifications. In addition, the conidia production and thermotolerance assay were performed for the promising EPF strains and compared; these data were further substituted into the formula of effective conidia number (ECN) for laboratory ranking. The soil bait-mealworm system and the ECN formula can be improved by replacing insect species and integrating more stress factors for the evaluation of commercialization and field application. This protocol provides a quick and efficient approach for EPF selection and will improve the research on biological control agents.

Here we present a protocol based on the&#160;mealworm (Tenebrio molitor)-bait system that was used for isolating and selecting entomopathogenic fungi (EPF) from soil samples. An effective conidia number (ECN) formula is used to select high stress tolerant EPF based on physiological characteristics for pest microbial control in the field.

土壤样本中昆虫病原真菌的分离与选择及真菌对害虫的毒力评价

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important ...

Mass Production of Entomopathogenic Fungi, Metarhizium robertsii and Metarhizium pinghaense, for Commercial Application Against Insect Pests

Entomopathogenic fungi of the Metarhizium anisopliae species complex have gained importance as the biological control agents of agricultural insect pests. The increase in pest resistance to chemical insecticides, the growing concerns regarding the negative effects of insecticides on human health, and the environmental pollution from pesticides have led to a global drive to find novel sustainable strategies for crop protection and pest control. Previously, attempts to mass culture such entomopathogenic fungi (EPF) species as Beauveria bassiana have been conducted. However, only limited attempts have been conducted to mass culture Metarhizium robertsii and M. pinghaense for use against insect pests. This study aimed to mass-produce a sufficient number of resilient infective propagules of South African isolates of M. robertsii and M. pinghaense for commercial application. Three agricultural grain products, flaked oats, flaked barley, and rice, were used as the EPF solid fermentation substrates. Two inoculation methods, conidial suspensions and the liquid fungal culture of blastospores were used to inoculate the solid substrates. Inoculation using conidial suspensions was observed to be relatively less effective, as increased levels of contamination were observed on the solid substrates relative to when using the blastospore inoculation method. Flaked oats were found not to be a suitable growth substrate for both M. robertsii and M. pinghaense, as no dry conidia were harvested from the substrate. Flaked barley was found to favor the production of M. robertsii conidia over that of M. pinghaense, and an average of 1.83 g &plusmn; 1.47 g of dry M. robertsii conidia and zero grams of M. pinghaense conidia was harvested from the substrate. Rice grains were found to favor the conidial mass production of both M. pinghaense and M. robertsii isolates, with an average of 8.2 g &plusmn; 4.38 g and 6 g &plusmn; 2 g harvested from the substrate, respectively.

Mass Production of Entomopathogenic Fungi, Metarhizium robertsii and Metarhizium pinghaense, for Commercial Application Against Insect Pests

Entomopathogenic fungi have gained importance as the biological control agents of agricultural insect pests. In this study, the mass production of a sufficient number of resilient infective propagules of South African isolates of both Metarhizium robertsii and M. pinghaense for commercial application against insect pests was successfully conducted using agricultural grain products.

大规模生产昆虫致病真菌、 罗伯氏菌 和 异根真菌，用于昆虫害虫的商业应用

Entomopathogenic fungi of the Metarhizium anisopliae species complex have gained importance as the biological control agents of agricultural insect pests. ...

Comparison of Methods for Isolating Entomopathogenic Fungi from Soil Samples

The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating entomopathogenic fungi (EPF) from soil samples. The soil is a rich habitat for microorganisms, including EPF particularly belonging to the genera Metarhizium and Beauveria, which can regulate arthropod pests. Biological products based on fungi are available in the market mainly for agricultural arthropod pest control. Nevertheless, despite the high endemic biodiversity, only a few strains are used in commercial bioproducts worldwide. In the present study, 524 soil samples were cultured on potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide (CTC medium). The growth of fungal colonies was observed for 3 weeks. All Metarhizium and Beauveria EPF were morphologically identified at the genus level. Additionally, some isolates were molecularly identified at the species level. Twenty-four out of these 524 soil samples were also surveyed for EPF occurrence using the insect bait method (Galleria mellonella and Tenebrio molitor). A total of 51 EPF strains were isolated (41 Metarhizium spp. and 10 Beauveria spp.) from the 524 soil samples. All fungal strains were isolated either from croplands or grasslands. Of the 24 samples selected for comparison, 91.7% were positive for EPF using Galleria bait, 62.5% using Tenebrio bait, and 41.7% using CTC. Our results suggested that using insect baits to isolate the EPF from the soil is more efficient than using the CTC medium. The comparison of isolation methods in addition to the identification and conservation of EPF has a positive impact on the knowledge about biodiversity. The improvement of EPF collection supports scientific development and technological innovation.

Entomopathogenic fungal colonies are isolated from tropical soil samples using Tenebrio bait, Galleria bait, as well as selective artificial medium, i.e., potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide (CTC medium).

从土壤样品中分离昆虫致病真菌的方法比较

The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating entomopathogenic ...

Isolation and Screening from Soil Biodiversity for Fungi Involved in the Degradation of Recalcitrant Materials

Environmental pollution is an increasing problem, and identifying fungi involved in the bioremediation process is an essential task. Soil hosts an incredible diversity of microbial life and can be a good source of these bioremediative fungi. This work aims to search for soil fungi with bioremediation potential by using different screening tests. Mineral culture media supplemented with recalcitrant substances as the sole carbon source were used as growth tests. First, soil dilutions were plated on Petri dishes with mineral medium amended with humic acids or lignocellulose. The growing fungal colonies were isolated and tested on different substrates, such as complex mixtures of hydrocarbons (petrolatum and used motor oil) and powders of different plastic polymers (PET, PP, PS, PUR, PVC). Qualitative enzymatic tests were associated with the growth tests to investigate the production of esterases, laccases, peroxidases, and proteases. These enzymes are involved in the main degradation processes of recalcitrant material, and their constitutive secretion by the examined fungal strains could have the potential to be exploited for bioremediation. More than 100 strains were isolated and tested, and several isolates with good bioremediation potential were found. In conclusion, the described screening tests are an easy and low-cost method to identify fungal strains with bioremediation potential from the soil. In addition, it is possible to tailor the screening tests for different pollutants, according to requirements, by adding other recalcitrant substances to minimal culture media.

Here, we present a protocol for screening soil biodiversity to look for fungal strains involved in the degradation of recalcitrant materials. First, fungal strains able to grow on humic acids or lignocellulose are isolated. Their activity is then tested both in enzymatic assays and on pollutants such as hydrocarbons and plastics.

森林蛀木虫昆虫病原真菌的分离、行为鉴定和致病性评估

副本

摘要

探索更多视频

此视频中的章节

从胎盘中的造血干细胞的分离和分析

使用生物反馈的心理生理压力评估

吸血鬼从细胞外液分离

体外评估对离体骨骼肌的收缩，疲劳性和交替“

隔离和线粒体从培养细胞和小鼠组织功能分析

观察和交配行为的量化在我国松材线虫，

土壤样本中昆虫病原真菌的分离与选择及真菌对害虫的毒力评价

大规模生产昆虫致病真菌、罗伯氏菌和异根真菌，用于昆虫害虫的商业应用

从土壤样品中分离昆虫致病真菌的方法比较

土壤生物多样性中参与顽固性物质降解的真菌的分离与筛选

森林蛀木虫昆虫病原真菌的分离、行为鉴定和致病性评估

副本

摘要

探索更多视频

此视频中的章节

从胎盘中的造血干细胞的分离和分析

使用生物反馈的心理生理压力评估

吸血鬼从细胞外液分离

体外评估对离体骨骼肌的收缩，疲劳性和交替“

隔离和线粒体从培养细胞和小鼠组织功能分析

观察和交配行为的量化在我国松材线虫，

土壤样本中昆虫病原真菌的分离与选择及真菌对害虫的毒力评价

大规模生产昆虫致病真菌、 罗伯氏菌 和 异根真菌，用于昆虫害虫的商业应用

从土壤样品中分离昆虫致病真菌的方法比较

土壤生物多样性中参与顽固性物质降解的真菌的分离与筛选

大规模生产昆虫致病真菌、罗伯氏菌和异根真菌，用于昆虫害虫的商业应用