Analyze Of Reactive Oxygen Species (ROS) And Related Signalling At Stages Of Sclerotial Differentiation Of Rhizoctonia Spp.

Rhizoctonia spp. is worldwide pathogenic fungus. It has broad host and can infect hundreds of species of plants. Sclerotium is dark and formed by tangled mycelium, is considered to be dormant structures to survive long periods of unfavorable environmental conditions. These structures are of great significance in survival and disease cycle of pathogen under stress conditions. Studies on Rhizoctonia spp. at home mainly involved in pathogens, biology, genetic diversity, pathogenic mechanisms and so on, but the sclerotia differentiation mechanism is relatively small. Research on forming conditions, physiological and biochemical characteristics and molecular mechanisms of sclerotium can help us to understand of development mechanism of sclerotium and provide new theoretical basis for controlling these diseases.In this work, as the material of three strains including Rhizoctonia solani AG-1-IA isolate of maize, Rhizoctonia cerealis AG-D isolate of wheat and binucleate Rhizoctonia AG-A isolate of maize, we made a preliminary study on conditions affecting sclerotium formation, variations of signal molecules in every stages of sclerotia differentiation and characteristics of exogenous signal molecules. The results are as follows:1. Effect of environmental conditions on the development of sclerotialFirst, we analyzed the environmental conditions affecting sclerotium formation in 5 aspects, and found the optimum temperature of R. solani for sclerotial formation is 35℃, and optimum pH is 5, alternating light and dark light conditions is the favorable environment for it, and the optimum carbon source and nitrogen source are glucose and potassium nitrate; for R. cerealis the optimum temperature is 20℃, and others are consistent with R. solani.2. Reactive oxygen species in different stages of sclerotium differentiationOxidative burst is one of the rapid responses to external stimuli in eukaryotes, involved in a variety of processes of cell development. In order to understand whether there is involvement of reactive oxygen species in different stages of Rhizoctonia spp. sclerotium, we observed the production rate of O₂^·ˉ and found that it showed similar characteristics in three Rhizoctonia spp. fungi. The maximum value of O₂^·ˉ was in undifferentiated stage of sclerotium, and then decreased gradually to the lowest in the mature sclerotium.H₂O₂ is another reactive oxygen species more stable in eukaryotes. We observed the concentration of H₂O₂ at different stages of sclerotium differentiation and found that it was low at undifferentiated stage, increased rapidly in sclerotium initial stage, reached the maximum at developing stage. It is suggested that the oxidative burst such as O₂^·ˉ and H₂O₂ are playing an important role in sclerotium differentiation.3. Variation of Enzymes related with active oxygen metabolism in different stages of sclerotia differentiationThe variations of Enzymes related with active oxygen metabolism were analyzed in four aspects. The results showed that, they closely to be related with sclerotium differentiation. On the determination of lipid peroxidation that it was significantly diversities at different stages particularly the binucleate Rhizoctonia AG-A, it was low in undifferentiated stage and highest in mature stage (respectively 17 times and 7 times the undifferentiated stage and initial stage). The level of lipid peroxidation of others showed the similar trends.To determinate the Xanthine oxidase (XO) activity we found that it is significantly different at different stages, R. cerealis AG-D showed distinct changes in XO activity among the three fungi. It is low at undifferentiated stage and increased gradually to highest at mature stage.Superoxide dismutase (SOD) is an important active oxygen scavenging enzyme, its activity was measured at different stages and showed that highest at initiation stage and lower at undifferentiated and mature stages. And catalase (CAT) activity in different, it is highest at mature stage and lowest at undifferentiated stage. SOD and CAT activities in these there fungi showed the similar trends. In addition, these three strains are all contain three SOD isozymes and one CAT.4. The impact of exogenous signaling molecules on sclerotium differentiationThe impacts of exogenous signal molecules on sclerotium differentiation were analyzed in two aspects. We studied the sclerotium differentiation by exogenous cyclic adenosine monophosphate (cAMP). The results showed that R. solani AG-1-IA treated with different concentrations of cAMP were extremely significant differences in the number of sclerotium. the number of sclerotium increased as the increase of cAMP concentration at 0.0125～0.0375 mg/mL, but significantly reduced without cAMP. R. cerealis AG-D showed similar characteristics. At the same time, deformity of mycelial growth occurred because of the addition of cAMP.Oxalic acid (OA) is another important signal component in higher organisms, can involve in pH-mediated singal transduction pathway by binding with intracellular Ca²⁺. This study showed that, the numbers of sclerotium of R. solani AG-1-IA are different in HCl and OA under the same pH. At pH 7.0, the two treatments are close. However, at pH 4 6, the number of sclerotium in medium with OA significantly increased. Especially at pH 4.0 reached a significant level.