Inducing the Entry of Third Stage Dispersal Juveniles of Bursaphelenchus xylophilus into Cryptobiosis through Osmotic Regulation

Summary

The present protocol describes a method for inducing the dispersal of third-stage juveniles (pre-dauer juveniles, J_Ⅲ) of Bursaphelenchus xylophilus into cryptobiosis through osmotic regulation of potassium chloride (KCl) ions. This method can provide technical support for research on the stress resistance mechanisms of B. xylophilus.

Abstract

Cryptobiosis is a state where organisms lose nearly all their internal water and enter anhydrobiosis under extreme environmental stress. The dispersal third-stage juveniles (pre-dauer juveniles, ) of Bursaphelenchus xylophilus can enter cryptobiosis through dehydration and revive upon rehydration when environmental conditions improve. Osmotic regulation is crucial for their survival in this process. In this study, specimens of B. xylophilus were collected from dead Pinus massoniana due to pine wilt disease in Ningbo, Zhejiang Province, China. Following immersion in 8% potassium chloride (KCl) solution, B. xylophilus entered a cryptobiotic state after gradual dehydration due to increased external osmotic pressure by natural water evaporation. B. xylophilus could resist low-temperature stress at -20 °C. B. xylophilus could revive upon rehydration, with a survival rate of 92.1%. This process can regulate the entry of B. xylophilus into cryptobiosis, enabling them to resist extreme environments. This method described in our study is simple and reliable, providing technical support for studying the stress resistance mechanisms of B. xylophilus.

Introduction

Pine wilt disease is one of the most severe forest diseases in China¹. The pinewood nematode Bursaphelenchus xylophilus (B. xylophilus) poses a significant threat to pine species such as Pinus massoniana (P. massoniana) by reproducing within the host, leading to rapid wilting and death. Under unfavorable environmental conditions after the death of the host, the reproductive juveniles of B. xylophilus may enter pre-dauer juveniles (). These juveni....

Protocol

1. Collection of B. xylophilus

1. Collection of infected disks of P. massoniana
   NOTE: All procedures complied with the Administrative Measures for Pine Wilt Disease Epidemic Area and Infected Wood and other applicable regulations. A P. massoniana forest infected by B. xylophilus in Xiangshan County, Ningbo City, Zhejiang Province, was selected as the sample site. .......

Discussion

In this study, B. xylophilus was collected from dead P. massoniana in Ningbo, Zhejiang Province. It should be noted that B. xylophilus was selected from a mixed population with different instars. Specifically, round logs with a high density of B. xylophilus were chosen, followed by the screening of

Materials

Name	Company	Catalog Number	Comments
Glass slide	Bioland	GS7101-7525	75 mm × 25 mm × 1 mm
Long necked glass funnel	Nanjing Kangluoda Experimental Technology	N/A	150 mm
Microscope	Phenix	XSP-35-1600X	PH100-2B41L-IPL
Petri dish	Nanjing Kangluoda Experimental Technology	TB-624935792745	50 mm × 50 mm
Pinchcock	Nanjing Kangluoda Experimental Technology	TB-624973311743	36 mm × 11 mm  × 55 mm
Pipette	Eppendorf	3123000012	10–100 µL
Potassium chloride (KCL)	Solarbio Life Science	P9921	500 g
Rubber tube	Nanjing Kangluoda Experimental Technology	TB-624923457094	7 mm × 5 mm
Sterilizer	BinJiang	https://detail.1688.com/offer/644766597053.html?_t=1730892390541&spm= a2615.7691456.co_0_0_wangpu _score_0_0_0_0_1_0_0000_0.0	75L
Tissue paper	Heart to heart imprint	M3	22.5 cm × 21.5 cm