<meta charset="utf8"></head><body>Explorando recursos de fungos entomopatogênicos de brocas de madeira florestal

<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>Autor em destaque: Avaliação de fungos entomopatogênicos em populações selvagens de Monochamus alternatus para aplicações de biocontrole em brocas florest

Isolamento, Identificação Comportamental e Avaliação de Patogenicidade de Fungos Entomopatogênicos de uma Broca da Madeira Florestal

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

516 visualizações.  Zhejiang A&F University. Exploramos recursos fúngicos entomopatogênicos de populações selvagens de Monochamus alternatus. Tomando esta espécie como exemplo, poderíamos enriquecer a diversidade de fungos entomopatogênicos de outras brocas florestais para futuras aplicações de biocontrole. Recentemente, obtivemos três espécies de fungos certificados como fungos entomopatogênicos parasitas de Monochamus alternatus, duas das quais não são bem conhecidas, mas provaram ser po...

Vídeo: Autor em destaque: Avaliação de fungos entomopatogênicos em populações selvagens de Monochamus alternatus para aplicações de biocontrole em brocas florest

Forest wood borers (FWB) cause severe tree damage and economic losses worldwide. The release of entomopathogenic fungi (EPF) during the FWB emergence period is considered an acceptable alternative to chemical control. However, EPF resources have been significantly less explored for FWBs, in contrast to agricultural insect pests. This paper presents a protocol for exploring EPF resources from FWBs using wild Monochamus alternatus populations as an example. In this protocol, the assignment of traps baited with M. alternatus attractants to different populations guaranteed the collection of adequate samples with natural infection symptoms, during the emergence periods of the beetle. Following finely dissecting integuments and placing them onto a selective medium, fungal species were isolated from each part of beetle bodies and identified based on both molecular and morphological traits.
Several fungal species were certified as parasitic EPFs via re-infection of healthy M. alternatus with spore suspensions. Their behavioral phenotypes on M. alternatus were observed using scanning electron microscopy and further compared with those on the Coleopteran model insect Tribolium castaneum. For EPFs that present consistent parasitism phenotypes on both beetle species, evaluation of their activities on T. castaneum provided valuable information on lethality for future study on M. alternatus. This protocol helped the discovery of EPF newly reported on M. alternatus populations in China, which could be applied as an efficient approach to explore more EPF resources from other FWBs.

Here we present a protocol for obtaining entomopathogenic fungi from a forest wood borer and a substitutive way to evaluate their entomopathogenic activities using a Coleopteran model insect. This method is efficient and convenient for exploring entomopathogenic fungal resources from wood-boring insect pests in natural forests.

Biologia

Isolation and Behavioral Identification of Fungal Pathogens from Monochamus alternatus

After collecting the pine sawyer, Monochamus alternatus beetles, place the beetle into a sterile tube containing fresh twigs. Rear the beetles in a non-humidified incubator at 25 degrees Celsius. After incubation, record beetles showing decreased feeding and mobility.Transfer the stiff dead beetles to wet chambers. Dissect the beetles with fungal infections into main positions such as antennae, head, thorax, abdomen, wings, and legs. Using scissors, cut the integuments of each position into small pieces.Gently press the outer surface of these pieces onto the PDA plate containing antibiotics. Seal the plate with parafilm and incubate at 25 degrees Celsius until the tissues are entirely covered by mycelium. Then transfer the single colony according to phenotypic properties onto a new PDA plate.Extract the genomic DNA using the fungi genomic DNA isolation kit. Prepare PCR mixture with primers ITS1 and ITS4 to amplify the rDNA-ITS region of the DNA. Use a camera to capture mature, fungal pure colony morphology on the front and back sides of the PDA plate.Pluck conidia from the pure fungal colonies with an inoculation needle and transfer them to a glass slide with a drop of sterile water. Then place a cover slip over the specimen without introducing bubbles. Cut a five millimeter agar block with a scalpel from the edge of the fungal colony and transfer it to a clean glass slide with a drop of sterile water.Using a fine needle, carefully separate the fungi from the agar to observe specific structures. Under an optical microscope, observe the asexual morph of the fungi, including the posture of the hyphae, conidiophores, phialides, and conidia.

Isolamento e Identificação Comportamental de Patógenos Fúngicos de Monochamus alternatus

Studying Fungal Infection and Entomopathogenic Activity in Monochamus alternatus and Model Beetle Tribolium castaneum

To begin, sterilize the surface of a 24 hour starved Monochamus alternatus and Tribolium castaneum beetles with bleach, ethanol, and distilled water. To induce the fungal infection, first, immerse the pine twigs or wheat bran into the conidial suspension for 10 seconds. Then dip the beetles in the conidial suspension.After drying, place one beetle and one twig into a sterile 50 milliliter tube. Rear the beetles in a non-humidified incubator at 25 degrees Celsius. After incubation, transfer the dead beetles into a new 50 milliliter tube, and place a piece of sterile, moist cotton within the tube.Dissect the infected beetle bodies with fungal infections into positions like an antennae, head, thorax, abdomen, wings, and legs. Cut five millimeter disc plugs of mature fungal colonies. Place the infected beetle tissues into pre-chilled 2.5%glutaraldehyde at four degrees Celsius for two days.Next, wash the samples thrice in 0.1%phosphate buffer for five minutes and dehydrate in increasing ethanol concentrations for 10 minutes each. Dry the samples in a vacuum freeze dryer, and then observe the fungal structures under a scanning electron microscope. Treat the wheat bran with a conidial suspension for 10 seconds and after drying, transfer them into a Petri dish.Immersed T.castaneum beetles in the conidial suspension for 10 seconds before placing them into the Petri dish containing wheat bran. After incubation, count the dead beetles from the plate. Extract genomic DNA of parasitic entomopathogenic fungi using the fungi genomic DNA isolation kit.Using the given sets of primers, prepare a PCR reaction mixture for gene-specific amplification. After sequencing, align the nucleotide sequence using ClustalX 2. Finally, perform phylogenetic analysis using MEGA 6 based on the maximum likelihood method.Parasitic fungi like Aspergillus austwickii, Akanthomyces attenuatus, and Scopulariopsis alboflavescens caused significant infection symptoms in M.alternatus. SEM observations further supported the morphological characteristics of these parasitic fungi on the surface of the beetles. Entomopathogenic activity showed significantly higher mortality rates in parasitic fungi compared to the control group.The multi-gene phylogenetic tree demonstrated genetic distances between the three parasitic fungal species and other species within their respective genera.