<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

The devastation caused by insect pests has led to great ecological and economic losses in both forest and agricultural ecosystems. Most agricultural pests expose themselves to natural enemies or artificial control agents while damaging host plants. Instead, forest wood borers (FWB) nearly complete their whole developmental cycles inside host tree trunks1, which raises large challenges to explore efficient biocontrol organisms from FWB in the wild field. What is even worse is that FWBs carry a great number of phytopathogens2 or have an intimate relationship with these pathogens as their potential vectors3,4, dramatically amplifying the negative effects of FWB on forest health. Excessive use of chemical insecticides can alleviate FWB severity, but the emergence of insecticidal resistance5,6 limits their environmental application. In certain cases, insect parasitoids, predatory arthropods as well as entomopathogenic microbes were released as biocontrol agents to the distribution areas of FWB7 and were proven to be efficient and economically acceptable alternatives to chemical control8,9,10.
Entomopathogenic fungi (EPF) are regarded to have the advantage in controlling FWB over most other microbial groups. Their spores can be carried by insect hosts and stably fixed on body surfaces via penetration into the cuticle or integument8,11. EPF also present excellent adaptability to environmental stresses and some species colonize well in the tissue of trees as endophytes12,13, facilitating their growth, survival, and transmission. However, compared to that in agricultural industries, the species diversity of EPF used in natural forest ecosystems is remarkably restricted14,15,16. Beauveria bassiana (strain PPRI 5339) was evidenced as the most promising strain to promote an IPM program to Eucalyptus weevils in South Africa17 and the combination of two promising isolates of B. bassiana provided an opportunity for the practical microbial control of red palm weevil, Rhynchophorus ferrugineus, at different life stages in palm tree fields18. In addition to Beauveria and the well-known Metarhizium, other EPF genera of the order Hypocreales, especially species of Lecanicillium (many of which are now classified into the genus Akanthomyces19,20), showed strong pathogenicity and high potential in management of forest pests, such as the Cypress aphid in Chile21.
The pine sawyer beetle Monochamus alternatus is a notorious pine forest pest in China and neighboring countries, which burrows into branches and trunks of pine trees to impede the transportation of nutrients and water22,23,24. Moreover, M. alternatus also promotes the invasion of the plant-parasitic pine wood nematode (Bursaphelenchus xylophilus, PWN) as its main vector beetle. Another congeneric species of the beetle, M. galloprovincialis, has spread PWN in several countries in Europe in recent years25. Previous research reported several genera of natural EPFs from Monochamus spp., such as Beauveria, Metarhizium, and Lecanicillium (Verticillium, an even former name of Lecanicillium), in Spain, Japan, and the Anhui/Zhejiang Provinces of China26,27,28,29. Nevertheless, these collections of EPFs seem to be commonly restricted in a certain location, compared to the wide occurrence of Monochamus beetles in natural fields. As the M. alternatus beetle has a wide geographical distribution in China, it could be regarded as a representative wood borer to explore more potential EPFs across different populations.
In the present protocol, we introduce a specific procedure exploring EPFs from several geographical populations of M. alternatus in southern China. This protocol uses a model Coleopteran beetle as a substitute to perform entomopathogenicity assays, under the condition that the tested fungal species has a consistent behavioral phenotype on both beetle species. This protocol can also provide insights into EPF exploration for other forest wood borers, in which the diversity of their entomopathogenic fungal species is underestimated or less investigated.

<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

We explore entomopathogenic fungal resources from wild Monochamus alternatus populations. Taking this species as an example, we could enrich the diversity of entomopathogenic fungi of other forest wood borers for future bio-control applications. We have recently obtained three fungal species certified as parasitical entomopathogenic fungi of Monochamus alternatus, two of which are not well-known, but have proven to be potent pathogens of insects.This protocol helps the discovery of more EPFS of Monochamus alternatus, and performs a substitute way to evaluate their entomopathogenic activities, which can also provide insights into entomopathogenic fungal exploration for other forest wood borers.

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

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

502 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 ...