The Biological Function Of Oxalic Acid In The Interaction Between Sclerotinia Sclerotiorum And Host Plants

Sclerotinia sclerotiorum is a necrotrophic Ascomycota pathogen, its host range includes over.400species of75families. In the infection process, it secretes cell wall degradation enzymes (CWDEs) and oxalic acid (OA). CWDEs can disrupt the structure of cells and degrade host substances to obtain the nutrients for its growth. OA functions in many ways for pathogenecity:adjustment of local pH to activate pH related genes and improve CWDEs activity; chelation of Ca2+to affect Ca2+concentration and signalling; suppression of reactive oxygen species (ROS) and direct toxity to plant cells. OA exists not only commonly in many fungi such as Sclerotinia sclerotiorum, but also in plants, although its content varies in different plant species. Current researches on OA functional mechanism mainly focus on OA secreted from pathogens, while that from plant side is neglected, thus does not reflect complete roles of OA in the interactions between plants and pathogens.In this study, effect of OA on growth and pathogenecity of S. sclerotiorum mycelia was analyzed. Effect of exogenous OA treatment on disease resistance in plant species of various OA content was evaluated. Finally functional mechanisms of OA were dissected from the view of effect on hormones and ROS accumulation. The main results are as follows:(1)Effect of OA on growth and pathogenecity of S. sclerotiorum mycelia was clarified. Growth and pathogenecity of S. sclerotiorum mycelia reduced with incresment of mycelium age, and the old mycelia exhibited significant lower growth and pathogenecity in comparison with the young mycelia. This difference might be related with the growth of aerial mycelia. Data of exogenous OA treatment experiments revealed that effect of OA on growth and pathogenecity of S. sclerotiorum mycelia was realted with OA concentration, concentration higher than10mM showed strong inhibiting effect, indicating that OA acts as a growth regulator in addition to as a pathogenecity factor of S. sclerotiorum. Additionally, growth of S. sclerotiorum required an enviorment with a pH of lower than5.5, thus secretion of OA by S. sclerotiorum can maintain a pH beneficial to fungal growth.(2)Effect of plant endogenous OA and exogenous OA treatment on plant disease resistance was made clear. Plants containing varied OA, including spinach, rape, tobacco, tomato and Nicotiana benthamiana were investigated for their resistance. Results showed that spinach, which contains high OA was resistant, while tomato and N. benthamiana, which contain low OA were highly susceptible to5. sclerotiorum, thus the content of OA in plant species is positively correlated with disease resistance to OA-secreting pathogens. Exogenous OA experiments demonstrated that OA could induce resistance and the concentration of OA required for resistance induction varied in different plant species,1mM to10mM for N. benthamiana, while over20mM for rape, demonstrating a positive correlation between the required OA concentration and the OA content of plant species. In addition to concentration, pH and treatment method of OA also affected the role of OA. OA with a pH of5.5, which is similar to pH in the extracellular space of plant cells, exhibited higher resistance inducing ability when compared to pH3.5. Infiltration of OA resulted in lower resistance inducing ability in comparison with spraying of OA.(3) Role of content and signaling pathway of phytohormones including abscisic acid (ABA), jasmonic acid (JA), ethylene, salicylic acid (SA), auxin, cytokinin gibberellins (GA) and brassinolide (BR) in resistance to S. sclerotiorum was revealed, and interactions between hormones and OA in resistance was unveiled. Mutant analyses demonstrated that ABA, JA and ethylene pathways positively while SA pathway negatively regulated resistance to S. sclerotiorum. However, exogenous hormone experiments showed that SA treatment effectively induced resistance. S. sclerotiorum inoculation enhanced content of ABA, JA and auxin, induced early but reduced later SA content, while reduced BR content. Compared with pathogen inoculation, OA (10mM) treatment caused similar change of content of hormones such as ABA, JA, SA and BR, but resulted in different alteration of auxin and cytokinin content. OA with different pH showed different effect effect on hormone content, OA with pH5.5exhibited more significant activity when compared with OA with pH3.5. Detection of OA content in various hormone mutants demonstrated that content of hormones affected OA accumulation. Muants inhibiting biosynthesis of ABA, ethylene and GA and mutant of JA receptor COI1accumulated higher amount of OA while mutants blocking SA biosynthesis accumulated less OA in comparison with wild-type plants, which coincided with profiles of OA content after pathogen inoculation and resistance of the mutants to S. sclerotiorum. DAB staining analysis revealed that some hormone mutants altered in ROS accumulation. However, OA treatment did not affect this hormone-dependent regulation of ROS accumulation. Level of ROS negatively correlated with ABA content, but not with ABA signaling pathway components including ABU, ABI2and ABI4. Additionaly, the mutant eto2-1, which causes high level of ethylene, accumulated high amount of ROS. However, the remaining mutants affecting hormone biosynthesis and signaling did not exhibit significant change of ROS accumulation when compared to wild-type plants.