Establishment of a Chilli Thrips (Scirtothrips dorsalis Hood) Rearing System for Virulence Screening of Entomopathogenic Fungi

Summary

A detached-leaf bioassay protocol was established for mass-rearing chilli thrips using a modified detached-leaf method in agar-containing glass containers, ensuring high survival and synchronized development. This system enables efficient screening of entomopathogenic fungi virulence against 2^nd instar larvae and provides a reliable thrips supply for laboratory, greenhouse, and field evaluations.

Abstract

The chilli thrips (Scirtothrips dorsalis Hood) is an economically significant pest of ornamental and food crops in Taiwan. Entomopathogenic fungi (EPF) are working as an eco-friendly biological control agent for managing thrips. It is crucial to screen the virulence of EPF isolates against chilli thrips under laboratory conditions before field application. However, limited data on the rearing method of chilli thrips pose challenges for large-scale laboratory screening. To address this issue, we developed a modified laboratory-rearing method for the mass production of S. dorsalis.

The method utilizes a detached-leaf rearing system using an agar-containing glass container that is suitable for assessing EPF against chilli thrips. This optimized detached-leaf bioassay system is suitable for assessing the efficacy of EPF against chilli thrips. The system has the capacity to produce large numbers of thrips at the same developmental stage for virulence screening. According to the test, the result showed that Cordyceps cateniannulata (Cc-NCHU-213) isolate showed the highest virulence (53.6%) against 2^nd instar larvae of chilli thrips at 7 days post inoculation (d.p.i.). Moreover, the agar-embedded leaf discs remained fresh for over 7 days, allowing thrips to survive (90% survival rate of control treatment) throughout the observation period, indicating the stability of the rearing system. This protocol demonstrates a stable rearing system for mass production and for evaluating the virulence of EPF isolates against chilli thrips and that may have utility for other thrips, enabling screening for potential EPF isolates for controlling thrips in the field.

Introduction

The chilli thrips (Scirtothrips dorsalis Hood; Thysanoptera: Thripidae) is a polyphagous pest infesting over 100 plant species across 40 different families of plants, including vegetables and ornamental and fruit crops¹. It is native to tropical and subtropical Asia-Southeast Asia, India, Japan, and Taiwan². To our knowledge, the chilli thrips is the pest of agricultural and ornamental crops, including mango, tea, blueberry, and lotus in Taiwan. During the past two decades, chilli thrips has become a significant economic pest in several countries because of increased globalization and open agricultural trade. Ad....

Protocol

NOTE: The complete experimental flowchart is shown in Figure 1.

1. Chilli thrips field collection and colony maintenance

1. Collection of chilli thrips
   1. Visually examine both sides of leaves and within flowers for infestation of chilli thrips on blueberry plants in an affected blueberry farm.
   2. Record the details of each sample (e.g., part of the plant and severity of the infestation), and confirm the production practices with farmers, such as conventional farming or organic farming, including application of mineral oil and natural enemies.
   3. Cut the leaves....

Results

The experimental flowchart presented in this study demonstrated that the stable rearing system of the chilli thrips could be established, starting from the field collection to the screening of virulence of EPF isolates (Figure 1). Though morphological observation can distinguish chilli thrips from other flower thrips, the use of the partial COI sequence as a molecular marker, including PCR amplicon size and COI sequencing, can further confirm the identificati.......

Discussion

Chilli thrips is a globally significant pest affecting numerous economically important crops such as corn, peanut, pepper, soybean, strawberry, sweet potato, and tomato^1,11,19. Current control strategies rely on integrated approaches involving cultural practices, biological control agents, and chemical treatments, including the use of natural enemies, nematodes, and EPFs^{11,14<.......}

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
#00 insect pin	Entosphinx	DMM0220	Made in UK
1.5 mL centrifuge tube	Corning incorporated	MCT-175-C	Made in USA
10 μL Inoculating Loop	NEST Scientific	718201	Made in USA
100 bp DNA Ladder III	Geneaid	DL007	Made in Taiwan.
2x SuperRed PCR Master Mix	Biotools	TE-SR01	Made in Taiwan
6 cm Petri dish	Alpha Plus Scientific	16021	Made in Taiwan
agar	Bioman Scientific	AGR001.1	Microbiology grade
agarose	Bioman Scientific	PB1200	Made in Taiwan
BioGreen Safe DNA Gel Buffer	Bioman Scientific	SDB001T	Made in Taiwan
Easy DNA High-Speed Extraction Tissue Kit	Fisher Biotec	ET-100	https://www.fisherbiotec.com.au/wp-content/uploads/2021/11/FB-EDNA-DNA-Kits-flyer-11Nov21.pdf (Made in Australia)
Forward primer: LCO 1490	Genomics	PO-PR220919G6	Sequence: 5’-GGT CAA CAA ATC ATA AAG ATA TTG G-3’
GenepHlow Gel/PCR Kit	Geneaid	DFH300	http://www.protech-bio.com/UserFiles/file/Gene-Spin%20Genomic%20DNA%20Kit.pdf
glass rearing container	Sunshine Lab Equipment company		Size: 35 mm in height, 45 mm in inner diameter. Can be repalce by any simular size of plastic or glass cup.
hemocytometer	Paul Marienfeld	640030	Made in Germany
incubator	Hipoint	F740	Made in Taiwan
leather cutter	Real Handmade Workshop		https://shopee.tw/jj891004? categoryId=100636& entryPoint=ShopByPDP& itemId=16023929406
MiniAmp Thermal Cycler	Thermo Fisher Scientific	A37834	Made in USA
optical microscope	Nikon	Ci-L	Made in Japan
painting brush	Tian Cheng brush company	4716608400352	Can be replace by fine-tipped watercolor brush, equivalent to a size 0 or 1 round brush.
paper towel	may flower	BLS007-8	Made in Taiwan
Parafilm M	Bemis	PM-996	Made in USA
pellet pestle	Bioman Scientific	GT100R	Made in Taiwan
Reverse primer: HCO 2198	Genomics	PO-PR220919G6	Sequence: 5’-TAA ACT TCA GGG TGA CCA AAA AAT CA-3’
Sabouraud Dextrose Broth	HiMedia	MH033-500G	Made in India
stereomicroscope	Soptop	SZN71	Made in Taiwan
TAE buffer 50X	Bioman Scientific	TAE501000	Made in Taiwan
Tween 80	PanReac AppliChem	142050.1661	Made in Taiwan
zipper bag	wenchen plastic company	1809000061	size: 12 cm in wide and 17 centimeters in long. (Made in Taiwan)