X-ray CT Detection,Green Prevention And Biocontrol Mechanism Of Populus Seeding Root Rot

Tree root rot,also known as Armillaria root rot(ARR),is a prevalent global infectious disease that affects more than 500 species of hosts.The ARR of Populus davidiana × Populus alba var.pyramidalis Louche(Pd)seriously affects normal tree growth,resulting in large amounts of seedling death in nurseries,and leading to vast economic losses in Chinese forestry.Currently,there is a need to characterize the internal decay processes of the disease and establish suitable biological control mechanisms.Elucidation of internal and external phenotype-molecular levels of plants will inform on prevention and control of this disease.In this study,we investigated the optimal conditions for х-ray CT imaging of Pd roots by testing different culture substrates,and analyzed the architectural changes in the root system of Pd after Armillaria solidipes infection.The Illumina HiSeq 2,500 transcriptome and HPLC-MS metabolome platform were used to sequence the Bacillus velezensis BY6-induced wild-type Pd seedings.Key resistance genes were investigated by multi-omics integration analysis technology from omics data,after which a mutant plant of the resistance gene of Pd was created.We compared transgenic and wild-type plants infected with A.solidipes,assessed the phenotypic characteristics of internal scans and external symptoms under х-ray CT,and determined the burst of reactive oxygen species(ROS),antioxidant enzyme system and changes in expressions of pathway-related genes in Pd during disease resistance.The mechanism of wild-type Pd responses to A.solidipes infection after BY6 induction was preliminarily determined.The main findings of this study are:(1)The combination of vermiculite and 1/2 MS culture medium had the х-ray CT imaging effect on Pd roots.The combined matrix did not affect Pd growth.After three-dimensional reconstruction of the root system,it was found that Pd root tip is first infected,and gradually spreads upwards from the ground,then inside to the phloem and xylem from root epidermis.(2)Growth indices(dry mass,fresh mass,plant height)and physiological indices(soluble sugar and soluble protein)of Pd seedlings were significantly increased after BY6 induction.At the same time,BY6 increased CAT,PAL,POD and SOD activities in Pd.(3)Gene transcription levels in leaves of wild-type Pd were significantly changed after BY6 induction.Transcript levels for 56,748 differentially expressed genes were down regulated or upregulated by more than 1.5-fold,and multiple metabolic pathways,including phenylalanine biosynthesis,ribosome and amino acid biosynthesis were significantly down regulated or upregulated,too.(4)A more than 1.5 times fold change of 53 metabolites in Pd was detected by comparative metabolomics HPLC-MS-based technology.In the positive ion mode,multiple synthetic pathways,including indole alkaloid biosynthesis,isoflavone biosynthesis,and phenylalanine metabolism were found to be markedly affected by BY6 induction.In the negative ion mode,levels of small molecule metabolites in multiple pathways,including isoflavone biosynthesis and phenylalanine were found to be altered.(5)We performed bioinformatics analyses based on integrated results of transcriptome and metabolome data and established that 15 major metabolic pathways,including niacin and nicotinamide metabolism,indole alkaloid biosynthesis,and phenylalanine metabolism were markedly affected after BY6 induction.Among them,phenylalanine metabolism exhibited the most significant effects.(6)The function of the PdMYB108 gene was preliminarily predicted.In evolutionary relationships,the PdMYB108 gene is closely correlated with the XM002302751.3 gene of Pd.Five PdMYB108 transgenic plants were created via Agrobacterium mediated plant genetic transformation.RT-qPCR analysis revealed that PdMYB108 gene expressions were suppressed in mature leaves and elevated in the phloem.(7)Phenotypic analysis of PdMYB108 transgenic Pd after A.solidipes infection under simulated field environment indicated that: Disease symptoms of transgenic Pd overexpressing A1 line were significantly relieved,and the disease index significantly decreased.The 4 growth indices,including dry mass,fresh mass,plant height and root length of A1 line were significantly better than those of the WT line.Scanning of the infected tree roots layer via х-ray CT showed that the density of the epidermis in the A1 line was uniform from top to bottom,and the pith was clearly visible,consistent with AC positive control line.Physiological index analysis showed that the A1 line had increased activities of POD,SOD and PAL and decreased activities of CAT,thereby reducing the levels of ROS in cells,and enhancing or inhibiting the expressions of resistance genes during the responses of Pd to pathogenic stress.Moreover,the A1 line induced the expressions of downstream genes of the JA\SA pathway,improved the disease resistance of Pd.