Research On The The Physiological And Molecular Mechanism Of Tobacco Resistance To Meloidogyne Incognita

Tobacco root-knot nematode disease is a soil-borne disease around the world,especially Meloidogyneincognita,whice is harmful to tobacco production and causing huge economic losses annually.In production,resistant breeding is the most economical and effective approach for preventing root knot nematode.Therefore,identification the disease resistance of tobacco to Meoidogyne incognita,and research its resistance mechanism had important theoretical significance and application value.Therefore,the research was on the base of identification of tobacco(G28,resistant;susceptible,Long bohuang)to Meloidogyne incognita resistance level,systematically studied related physiological and biochemical indexes,histopathological response,small RNA Sequencing,metabolome analysis,and revealed the resistance mechanism of tobacco to Meloidogyne incognita.It provides a scientific basis in control of root-knot nematodes.The main results of this study are as follows:1.G28 and Long bohuang showed significant different resistance to Meloidogyne incognita.After inoculation with Meloidogyne incognita,the morbidity rate and disease index were 0 in G28;100 and 81.75 were obtained for Long bohuang,respectively.The susceptible tobacco of Long bohuang was infected by Meloidogyne incognita,produced a large number of root knots,fibrous root growth significantly reduced,seriously affected the absorption of water and nutrients,resulting in reduced yield and quality of tobacco.However,resistant tobacco of G28 had no root knot and fibrous root grow well.Therefore,Long bohuang was considered susceptible and G28 was considered resistant genotypes.Histopathological observation showed that ultrastructure of root cells was remarkablely changed after Meloidogyne incognita invading.Nematode juveniles penetrated the roots of Long bohuang,established feeding sites and induced formation of large multinucleate giant cells with deep cytoplasm staining and vigorous cell metabolism.In the root of G28 plants,hypersensitive response and vacuoles were produced after Meloidogyne incognita penetrates the root.2.Reactive oxygen metabolism and phenylaprapanoid metabolism play an important role in tobacco under Meloidogyne incognita stress.The activities of SOD,POD and CAT in G28 were significantly higher than those in Long bohuang,and the content of MDA was significantly lower than that in Long bohuang.The results showed that the increase of SOD,POD and CAT activity of G28 could rapidly scavenging the accumulation of reactive oxygen species and reduce the degree of membrane lipid peroxidation.Phenylaprapanoid metabolism enzymes PPO,PAL,and TAL were significantly increased after Meloidogyne incognita infection,and the content in G28 was significantly higher than in Long bohuang.These results suggested that phenylaprapanoid metabolism can accelerate the synthesis of phenolic substances,lignin,plant protectin and other disease-resistant secondary substances in plants,enhance the disease resistance of host plants to Meloidogyne incognita.3.To identify nematode-responsive miRNAs,small RNA sequencing technique were performed with G28 and Long bohuang as material.A total of 30 682 255(G28-CK),24 066 860(G28-RKN),26 047 385(Long-CK),and 21 909 545(Long-RKN)clean reads were generated from the four sRNA libraries.The length distribution of small RNA sequences ranged from 17 nt to 35 nt,with most reads having a length of 24 nt.Clean reads mapped to the reference genome,we identified 164 conserved miRNAs belonging to 53 families,and 1011 novel miRNAs seemed to be specific to tobacco.4.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that the targets genes of these miRNAs are mainly involved in plant growth and development,transcription factors,phytohormone signaling,plant-pathogen interaction.nine significant differentially expressed miRNAs were selected to verify the authenticity of small RNA sequencing results by qRT-PCR.Five candidate miRNAs,miR398,miR399a,miR397,miR408、miR477a and miR827 in G28 were significant differentially expressed,which could be used for the study of resistant to Meloidogyne incognita and play an important role in defense against Meloidogyne incognita infection in tobacco.5.To clarify the response of host,metabolic changes of Long bohuang and G28 during infection with Meloidogyne incognitawere conducted by liquid chromatography-mass spectrometry(LC-MS).Multivariate statistical methods of PCA and OPLS-DA were used for pattern recognition and candidate biomarker screening.After inoculation with nematodes,77 and 87 possible endogenous metabolites in G28 were identified in G28 and Long bohuang,respectively.The results showed a significant difference of metabolites,which indicated that tobacco showed strong response toMeloidogyne incognita.6.Those identified as candidate biomarkers included terpenoids,flavonoids,alkaloids,aromatic amino acid and phospholipids that were related to resistance to biotic and abiotic stresses.This result revealed that host plant could activate a positive resistant response at both primary and secondry metabolism to promote the synthesis of resistance-related substances,once host plant recognized the nematode infection.Pathway analysis revealed that pathways related to the metabolism of amino acid,phytohormone,phenylpropanoids,shikimic acid,oxidative phosphorylation and TCA cycle were significantly changed in resistant tobacco G28 under Meloidogyne incognita stress,played a key role in host-nematode interactions.