The Mechanism Of Bacteria Strain Klebsiella Penumoniae Induced Soybean Resistance To Atrazine

Atrazine is a photosystem-II (PSII)-inhibiting herbicide that interferes with photosynthetic electron transport, resulting in oxidative stress. Soybean (Glycine max (L.) Merrill) is an atrazine-sensitive crop, and its productivity is severely impacted by soils containing atrazine residues. Our previous study indicated that the bacteria Klebsiella pneumoniae strain SnebYK can induce resistance to atrazine in soybean, both before and after pasteurization. Based on SnebYK and P-SnebYK induction effect, the physiological and biochemical reaction, proteomics and molecular mechanism were detected. The results were as follows:1. The evaluation of safety:The SnebYK and P-SnebYK showed some inhibition on the germination rate of wheat seeds; but they could promote the soybean seed germination and the growth of embryo and radicle. There were no significant effects on the rice and peanut seed germination. Observation of the growth condition, SnebYK and P-SnebYK had certain influence on the rice、wheat and soybean growth, but no significant effect on peanut growth.2. Further to determine the induced effect of bacterial strain SnebYK and P-SnebYK fifty different soybean varieties were selected. The results showed that soybean can grow normally in the soil which atrazine effective component is greater than 500 g/hm2 after SnebYK and P-SnebYK treatment, dry shoot weight production of soybean increased relatively to control; plant height, root length, fresh shoot weight and fresh root weight were analyzed, there was no significant difference compared to control; injury response of shoot and root were both above 80%; blossom and fruit was not affected by atrazine. In the mature period, plant height, pod number per plant and hundred-grain weight were determined, there was no significant difference compared with control. This suggested that bacteria SnebYK and P-SnebYK can effectively increase the tolerance of soybean of atrazine, for soybean and corn crop rotation and soybean fields application of atrazine provide the possibility.3. The physiological and biochemical mechanisms of soybean resistance to atrazine induced by exogenous SnebYK and P-SnebYK were studied. SnebYK and P-SnebYK could increase the CO2 assimilation rate, the stomatal conductance and the intercellular CO2 concentration, reduced stomatal restrictions, increased the soybean photosynthetic rate, remitted of atrazine damage to soybean plants. Atrazine could significantly inhibit the photosynthesis of soybean plants, after sprayed 48h nearly to death. Seeds treated with SnebYK and P-SnebYK atrazine could alleviate the destruction of chlorophyll and root activity inhibition. SnebYK and P-SnebYK could increase the glutathione-S-transferase (GST) and glutathione reductase (GR) activity, but had no significant effect on the activity of glutathione peroxidase (GPX), peroxide enzyme (POD) and polyphenol oxidase (PPO). After spraying atrazine, these enzyme activity increased significantly, which indicated that these enzymes were involved in the metabolic degradation of atrazine in soybean and clear oxygen free radicals caused by atrazine stress, protected cells from reactive oxygen species and chloroplast damage.4. In order to study the molecular mechanisms of this induced resistance, protein change in soybean leaves induced by SnebYK and P-SnebYK was investigated using two-dimensional gel electrophoresis. Differentially expressed proteins (relative to a non-induced control) that changes in protein abundance in more than 3 fold and good repeatability were successfully identified by MALDI-TOF MS. Quantitative image analysis revealed that ten protein spots exhibited significant changes in intensity in SnebYK treatment, relative to the control, two proteins were observed to be increased, whereas one was decreased. In addition, SnebYK induced the appearance of five new proteins, whereas two disappeared. Thirty-three spots were detected as significantly different in P-SnebYK, relatively to the control. Fourteen proteins were increased, whereas five were decreased. Furthermore, nine special expressed proteins were detected, while five others disappeared. These identified proteins were predicted to be involved in photosynthesis, energy metabolism, signal transduction/transcription, detoxification, antioxidant and others.5. With actin gene as the inner control, the genes expression of 2-Cys peroxiredoxin BAS1-like, Acyl coenzyme A oxidase, Gamma glutamyl hydrolase, Lycopene epsilon cyclase, Ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit, P450, GST, GR, GPX and MRPs were detected by quantitative RT-PCR. These genes were induced by SnebYK and P-SnebYK or spraying atrazin after SnebYK and P-SnebYK treatment. The results showed that the Ribulose、Lyc、2-Cys Prx、acetyl-CoA oxidase and GGH genes were increased in different degrees during 30 days after SnebYK and pasteurization SnebYK treatment. SnebYK and P-SnebYK could induce GST and GR genes expression. After spraying atrazine, the genes of P450, GST, GR, GPX and MRPs were up-regulate.6. Through the different components separation of SnebYK fermented liquid, and detected the different components effect on the induction of soybean resistance to atrazine.The results showed that ammonium sulfate precipitation extract has certain effect on the induction, preliminary identified that the effective active ingredient was ammonium sulfate precipitation extract, which provided some foundation for further purification.