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

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#181;L pipet tips</td>
			<td>Sangon Biotech</td>
			<td>F601216</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#181;L, 20 &#181;L, 100 &#181;L, 200 &#181;L, 1,000 &#181;L pipettes</td>
			<td>Rainin</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1,000 &#181;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 &#181;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>&#946;-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

577 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

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 ...

科研

生物学

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 ...

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.

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

Environmental pollution is an increasing problem, and identifying fungi involved in the bioremediation process is an essential task. Soil hosts an ...

脂肪组织来源的干细胞分离和表征

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

作者聚焦:分离和鉴定源自 Sprague Dawley 大鼠脂肪组织的间充质干细胞  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

斯普拉格道利大鼠脂肪组织间充质干细胞的分离和鉴定

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...

针对芥菜蚜虫的昆虫病原真菌的优化生物测定

Assessment of Aphidicidal Effect of Entomopathogenic Fungi against Parthenogenetic Insect, Mustard Aphid, Lipaphis erysimi (Kalt.)

The mustard aphid (L. erysimi) is a pest that infests various cruciferous crops and transmits plant viruses. To achieve eco-friendly pest management, entomopathogenic fungi (EPF) are potential microbial control agents for controlling this pest. Therefore, virulence screening of EPF isolates under Petri dish conditions is necessary before field application. However, the mustard aphid is a parthenogenetic insect, making it difficult to record data during Petri dish experiments. A modified system for detached-leaf bioassays was developed to address this issue, using a micro-sprayer to inoculate conidia onto aphids and prevent drowning by facilitating air-drying after spore suspension. The system maintained high relative humidity throughout the observation period, and the leaf disc remained fresh for over ten days, allowing parthenogenetic reproduction of the aphids. To prevent offspring buildup, a process of daily removal using a painting brush was implemented. This protocol demonstrates a stable system for evaluating the virulence of EPF isolates against mustard aphids or other aphids, enabling the selection of potential isolates for aphid control.

作者聚焦:使用昆虫病原真菌对抗芥菜蚜虫的环保害虫管理 

This protocol presents an optimized detached-leaf bioassay system for evaluating the effectiveness of entomopathogenic fungi (EPF) against the mustard aphid (Lipaphis erysimi (Kalt.)), a parthenogenetic insect. The method outlines the data collection process during Petri dish experiments, enabling researchers to consistently measure the virulence of EPF against mustard aphids and other parthenogenetic insects.

昆虫病原真菌对孤雌生殖昆虫、芥菜蚜虫、 Lipaphis erysimi （Kalt.） 的蚜虫作用评估

The mustard aphid (L. erysimi) is a pest that infests various cruciferous crops and transmits plant viruses. To achieve eco-friendly pest management, ...