Improved observation of colonized roots reveals the regulation of arbuscule development and senescence by drought stress in the arbuscular mycorrhizae of citrus

Citrus is the typical mycorrhizal fruit tree species establishing symbiosis with arbuscular mycorrhizal (AM) fungi. However, arbuscule development and senescence in colonized citrus roots, especially in response to drought stress, remain unclear, which is mainly due to the difficulty in clearing and staining lignified roots with the conventional method. Here, we improved the observation of colonized roots of citrus plants with the sectioning method, which enabled the clear observation of AM fungal structures. Furthermore, we investigated the effects of one week of drought stress on arbuscule development and senescence with the sectioning method. Microscopy observations indicated that drought stress significantly decreased mycorrhizal colonization (F%and M%) although it did not affect plant growth performance. Fluorescence probes (WGA 488 and/or Nile red) revealed that drought stress inhibited arbuscule development by increasing the percentage of arbuscules at the early stage and decreasing the percentages of arbuscules at the midterm and mature stages. Meanwhile, drought stress accelerated arbuscule senescence, which was characterized by the increased accumulation of neutral lipids. Overall, the sectioning method developed in this study enables the in-depth investigation of arbuscule status, and drought stress can inhibit arbuscule development but accelerate arbuscule senescence in the colonized roots of citrus plants. This study paves the way to elaborately dissecting the arbuscule dynamics in the roots of fruit tree species in response to diverse abiotic stresses.

Microbiologic risk factors of recurrent choledocholithiasis postendoscopic sphincterotomy

BACKGROUND Choledocholithiasis is a severe disorder that affects a significant portion of the world’s population.Treatment using endoscopic sphincterotomy(EST)has become widespread;however,recurrence post-EST is relatively common.The bile microbiome has a profound influence on the recurrence of choledocholithiasis in patients after EST;however,the key pathogens and their functions in the biliary tract remain unclear.AIM To investigate the biliary microbial characteristics of patients with recurrent choledocholithiasis post-EST,using next-generation sequencing.METHODS This cohort study included 43 patients,who presented with choledocholithiasis at the Guangdong Second Provincial General Hospital between May and June 2020.The patients had undergone EST or endoscopic papillary balloon dilation and were followed up for over a year.They were divided into either the stable or recurrent groups.We collected bile samples and extracted microbial DNA for analysis through next-generation sequencing.Resulting sequences were analyzed for core microbiome and statistical differences between the diagnosis groups;they were examined using the Kyoto Encyclopedia of Genes and Genomes pathway hierarchy level using analysis of variance.Correlation between the key genera and metabolic pathways in bile,were analyzed using Pearson’s correlation test.RESULTS The results revealed distinct clustering of biliary microbiota in recurrent choledocholithiasis.Higher relative abundances(RAs)of Fusobacterium and Neisseria(56.61%±14.81%vs 3.47%±1.10%,8.95%±3.42%vs 0.69%±0.32%,respectively)and the absence of Lactobacillus were observed in the bile of patients with recurrent disease,compared to that in stable patients.Construction of a microbiological co-occurrence network revealed a mutual relationship among Fusobacterium,Neisseria,and Leptotrichia,and an antagonistic relationship among Lactobacillales,Fusobacteriales,and Clostridiales.Functional prediction of biliary microbiome revealed that the loss of transcription and metabolic abilities may lead to recurrent choledocholithiasis.Furthermore,the prediction model based on the RA of Lactobacillales in the bile was effective in identifying the risk of recurrent choledocholithiasis(P=0.03).CONCLUSION We demonstrated differences in the bile microbiome of patients with recurrent choledocholithiasis compared to that in patients with stable disease,thereby adding to the current knowledge on its microbiologic etiology.