Population dynamics of Tricentrus sp.and detection of jujube witches'-broom phytoplasma in a Shandong jujube orchard

LI Yong; WEI Jingwei; YU Guoyue; TAN Jing; PEI Guangying; LI Shu; YANG Fan

doi:10.13925/j.cnki.gsxb.20250656

Abstract：[Objective]China's jujube industry has achieved an annual output value exceeding 100 bil-lion RMB,serving as a crucial economic source for approximately 20 million farmers in saline,sandy,and arid regions.Shandong Province,one of China's major jujube-producing areas,features plantations concentrated in the saline-alkali regions of northwest Shandong and the barren mountainous areas of central and southern Shandong.The jujube industry plays a critical role in supporting local forestry and fruit production.However,in recent years,the rapid spread of jujube witches' broom disease in Shan-dong has caused widespread jujube tree mortality,threatening the sustainability of the industry.This dis-ease,caused by phytoplasma,is primarily transmitted by vector insects.Understanding the composition and distribution of these vectors is essential for developing targeted and effective management strate-gies.To address this,the current study identified dominant pest species in a severely affected jujube or-chard in Feixian County,Shandong Province,and analyzed their role in the transmission of jujube witches' broom phytoplasma.[Methods]Between April and November 2024,sticky traps were used to monitor the population dynamics of insect pests in a jujube orchard.The orchard was divided into three monitoring sites:the upper,middle,and lower sections,all located in areas severely affected by witch-es'broom disease.Notably,the upper and lower sections had more abundant weed growth compared to the middle section.The most abundant pest species were identified using morphological and molecular techniques.DNA was extracted using the DP304 DNA extraction kit,following the manufacturer's in-structions.PCR amplification of the mitochondrial COI gene was performed using universal primers LCO1490 and HCO2198.The reaction was set up in a total volume of 30 μL,containing 0.5 μL of each primer,0.5 μL of dNTPs,0.3 μL of LA Taq polymerase(TaKaRa),3.0 μL of 10× LA buffer,1 μL of DNA template,and 24.2 μL of ddH2O.The PCR conditions included an initial denaturation at 94 ℃ for 5 minutes,followed by 39 cycles at 94 ℃ for 30 seconds,50 ℃ for 30 seconds,and 72 ℃ for 1 minute,with a final extension at 72 ℃ for 7 minutes.PCR amplification success was confirmed by observing～650 bp bands on 2％agarose gel electrophoresis at 145 V.The successful PCR products were sent for se-quencing.Based on BLAST results and references,mitochondrial sequences from 16 Membracidae spe-cies and 4 outgroup species were downloaded from the NCBI database.The sequences were aligned and trimmed to a uniform length of 658 bp using BioEdit software.A Maximum Likelihood(ML)phy-logenetic tree was constructed in MEGA 11 using ultrafast bootstraps(10 000 replicates)and SH-aLRT(1000 replicates).Nested PCR was employed to detect the presence of jujube witches'broom phyto-plasma in the extracted DNA,following laboratory protocols.The presence of phytoplasma was con-firmed by observing target-specific bands in the second round of amplification.[Results]Morphologi-cal identification confirmed the species as Tricentrus sp.Phylogenetic analysis grouped the collected specimens(Specimens 1,2,3,6,7,and 9)with Tricentrus davidi(Fallou)in the same clade,suggesting close phylogenetic relationships.Due to the lack of relevant reference sequences in the NCBI database,the specimens were preliminarily assigned to the genus Tricentrus,pending further species-level identi-fication.The population dynamics of Tricentrus sp.exhibited significant temporal and spatial variation.Temporal analysis showed significant changes over time(x2=6.524,df=1,p=0.011),while spatial analysis revealed differences across orchard sections(x2=39.412,df=2,p＜0.001).Population densi-ties in the upper and middle sections were significantly higher than those in the lower section(p＜0.001).In the lower section,individuals were only detected on September 23 and 30,with average counts of 1.2±1.2 and 0.4±0.4,respectively.Populations began to rise on August 26,peaking on Septem-ber 30,with mean counts of 10.4±6.1 in the upper section and 12.0±10.6 in the middle section.Num-bers declined rapidly thereafter,with no individuals detected after October 28,when daily maximum temperatures fell below 17 ℃.However,a slight resurgence was observed in November when tempera-tures rose above 17 ℃.A total of 36 Tricentrus sp.individuals were collected from the orchard.Nested PCR analysis detected jujube witches'broom phytoplasma in 8 of these specimens,yielding an infec-tion rate of 22.22％.[Conclusion]This study identified Tricentrus sp.as a dominant pest species in a ju-jube orchard and monitored its population dynamics during the fruit ripening period.The pest popula-tion peaked in late September,followed by a rapid decline,with a slight resurgence observed in Novem-ber.A close association was observed between Tricentrus sp.populations and weed abundance in the or-chard,suggesting that weeds may play a role in shaping pest dynamics.Strengthening orchard manage-ment practices,including targeted pest control and weed removal,is recommended to reduce pest popu-lations and potentially mitigate the spread of jujube witches' broom disease.