| In this study, field experiment and pot experiment were used to reveal the mechanism of continuous cropping obstacle of Pseudostellariae heterophylla. The yield and quality of P. heterophylla were surveyed by field experiment. The microscopic structure of the leaf and root of P. heterophylla were surveyed with microscope through paraffin slice method. The relative chlorophyll content of leaf (SPAD) of P. heterophylla was measured with the SPAD-502 (made in Japan), the net photosynthetic rate of leaf was measured with Li-6400 (made in USA), the fluorescence kinetics parameters (F0,Fm,Fv,Fp,QY_max,Ft_Lss,NPQ_Lss,F0_Lss,Fm_Lss,qP_Lss,QY_Lss) were measured with FluorCam (made in Czech republic). The differential proteomics method was used to analyze the differential expression of proteins in P. heterophylla leaves under the circumstances of P. heterophylla-Rice-P. heterophylla crop rotation or P. heterophylla continuous cropping. The extract from the rhizospheric soil of P. heterophylla was identified by GC-MS after dissolved by methanol. The non-rhizospheric soil extract was used for control. The rhizospheric soil microbial flora and their variation trends under continuous cropping circumstance were surveyed through dilution plate method, microbe culture method and terminal restriction fragment length polymorphism (t-RFLP) technique. The citrate and SDS buffers sequential extraction method were respectively used to extract the soil protein from the rhizospheric soil and non-rhizospheric soil of P. heterophylla. By means of 2-dimensional electrophoresis and contrast with the dimensional gel map of total soil proteins, the data of differential proteins were searched through bioinformatics researching.The results could be summarized as follows: (1) the allelochemicals extremely significantly inhibited the growth of root and shoot of lettuce (P<0.01), and the inhibition degree was extremely significantly (P<0.01) proportional to the concentrations of allelochemicals. It was indicated that the allelochemicals from rhizospheric soil of P. heterophylla has strong allelopathic potential.The allelochemicals, which were extracted from the rhizospheric soil of P. heterophylla, were used to treat the normal growth P. heterophylla. Three weeks after treatment, the leaf tip and the leaf rim of P. heterophlla was withered, the withered area of the leaf was proportional to the concentration of extract. The allelochemicals, had autotoxic effect on photosynthetic system of P. heterophylla, and the toxic effect degree been proportional to the concentrations of extract. The pot experiment was conducted to simulate continuous cropping and"P. heterophylla-Rice-P. heterophylla"rotation cropping was taken as the control. The results showed that continuous cropping led to autotoxic effect on photosynthetic system of P. heterophlla.After planted 45 days, the transverse sections of leaf of P. heterophylla showed that continuous cropping led to decreased cell density in the leaf, compared with control (The P. heterophylla was planted in pot with non-rhizospheric soil); cellulas were absence in some areas under microscope, compared with control. The transverse sections of root showed that continuous cropping also led to decreased cell density in the root, and cellulas were also absence in some areas under microscope. It suggested that continuous cropping resulted in some cell death occurred in the leaf and root of P. heterophylla. That is, the continuous cropping obstacle of P. heterophylla was occurred at cellular level.Continuous cropping led to extremely significantly (P<0.01) yield decreasing, the yield of the continuous cropping was only one third of the control. Compared the continuous cropping with the control experiment, the root numbers of P. heterophylla was extreme significantly (P<0.01) reduced, and the root length of P. heterophylla was significantly (P<0.05) shortened, the root numbers of the continuous cropping was about half of the control, the root length of the continuous cropping declined by 8% from that of the control. The polysaccharide content of the root from continuous cropping reduced to 88.08% of the control; the Ginseng saponins Rb1 of the root from continuous cropping reduced to 44.33% of the control. It was suggested that continuous cropping also decreased the medicinal quality of P. heterophylla.(2) The senescence-associated similar protein, the Ras-related protein, the senescence-related receptor-like kinase, the PR-protein and the Ras-related protein RHN1 were all upregulated, these proteins are related to plant senescence or disease; the phospholipid hydroperoxide glutathione peroxidase, the plant-type serine-threonine protein kinase, the calcium-dependent protein kinase, the chalcone synthase and the thioredoxin-related protein were all up-regulatively expressed, the cell wall glycine-rich protein, the homologue to SKP1, the zinc finger protein, and the maturase K were all down-regulated, these proteins are related to plant resistance; both the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and the cytochrome b6 were down-regulatively expressed, the ribulose bisphosphate carboxylase small chain c and the cytochrome b6/f complex subunit VI were up-regulatively expressed, these proteins are related to photosynthesis; the sucrose-UDP glucosyltransferase and the NF-YB5 were down -regulatively expressed, the proteasome subunit beta type 1, the synthase beta subunit, the cytokinin histidine phosphotransfer protein 4 and the NAD-dependent glyceraldehyde-3-phosphate dehydrogenase were all up-regulatively expressed, these proteins are related to plant metabolism of energy or cell division. It was suggested that the P. heterophylla replanting disease resulted from up-regulated expression of the proteins associated with senescence or disease, and resulted in disturbed expressions of the proteins, which are related to plant resistance or energy metabolism or cell division. It was also suggested that the continuous planting of P. heterophylla resulted in down-regulated expression of the important photosynthesis related protein; this is the molecular base of the declined photosynthesis rate in the replanted P. heterophylla.(3) The relative content of phenol quinines, organic acids, esters and alcohols in the rhizospheric soil were higher than that in non-rhizospheric soil, respectively. And the relative content of aldehyde ketones, amines, benzenes, hydrocarbons, and heterocycles in the rhizospheric soil were lower than that in non-rhizospheric soil, respectively. It was concluded that the phenol quinines, the organic acids, the esters and the alcohols were mainly autotoxins of the P. heterophylla.(4) The results based on the dilution plate method showed that the soil bacteria numbers of Tr2 (continuous cropping) were extremely significant more than that of Tr1 (rotation of P. heterophylla-Rice-P. heterophylla), the soil bacteria numbers of the CK (non-rhizospheric soil never planted P. heterophylla) were least, they were extremely significantly less than that of the Tr2, but non-significant (P>0.05) less than that of Tr1. As far as fungi and actinomycetes, their variation trends were both extremely significant (P<0.01) increased as the continuous cropping years. It was concluded that the continuous cropping of P. heterophylla led to the soil turning from bacterial types into fungal types.The results based on the specialized culture method showed that the nitrobacteria in the soil was not influenced by continuous cropping of P. heterophylla; the aerobic nitrogen-fixing bacteria numbers increased in the first year of cropping of P. heterophylla, abrupt decreased as the continuous cropping of P. heterophylla, less than the numbers of CK; the aerobic cellulose decomposition bacteria numbers also increased in the first year of cropping of P. heterophylla, and extremely significanty (P<0.01) decreased as the continuous cropping of P. heterophylla; the anaerobic cellulose decomposition bacteria numbers extremely significanty increased as the continuous cropping of P. heterophylla. It was concluded that continuous cropping of P. heterophylla would destroy the balance of rhizosphere soil microbial flora.The field survey results based on t-RFLP technique showed that there were 45 genera of bacteria in the rhizospheric soil of P. heterophylla, and 23 genera of them belonged to pathogens; there were 50 genera of bacteria in the non-rhizospheric soil, and 19 genera of them belonged to pathogens; there were 28 genera of fungi in the rhizospheric soil of P. heteropylla, none of them belonged to pathogens exempt for the Saprotroph.The pot experiment survey results based on t-RFLP technique showed that there were 118 genera of bacteria in the Tr2 (continuous cropping rhizospheric soil of P. heterophylla), and 23 genera of them belonged to pathogens; there were 122 genera of bacteria in the Tr1 ("P. heterophylla-Rice-P. heterophylla"rotation cropping rhizospheric soil of P. heterophylla), and 11 genera of them belonged to pathogens; there were 84 genera of bacteria in the CK (non-rhizospheric soil never planted P. hetrophylla), and 36 genera of them belonged to pathogens; there were 28 genera of Streptomyces in the Tr2 were detected. The pot experiment survey results based on t-RFLP technique also showed that there were 24 genera of fungi in the CK, 34 genera of fungi in the Tr1 and 38 genera of fungi in the Tr2, that is, the fungi numbers increased with the continuous cropping of P. heterophylla. Neither there were pathogens in rhizospheric soil, nor pathogens in non-rhizospheric soil of P. heterophylla. The diversity of soil bacteria analyzed results based on t-RFLP technique showed that the bacteria diversity of Tr2 was worse than that of Tr1, but the bacteria diversity of CK was the worst. The diversity of soil fungi analyzed results based on t-RFLP technique showed that the fungi diversity of Tr2 was the worst, and the fungi diversity of CK was the best. In light of t-RFLP technique survey results, it also indicated that continuous cropping of P. heterophylla would destroy the balance of rhizosphere soil microbial flora.Which kinds of microbes were mainly related to the toxic potential of rhizospheric soil of P. heterophylla was an issue in this research. The principal component analysis was used to reveal this issue based on t-FRLP profiles digested by MspI enzyme of the bacterial flora. The results showed that the first principal component (the first main microbes) related to the toxic potential of rhizospheric soil of P. heterophylla had 56 genera, e.g. Acidosphaera rubrifaciens str., the second principal component (the second main microbes) related to the toxic potential of rhizospheric soil of P. heterophylla had 4 genera, e.g. Alicyclobacillus sp. str..(5) The Asparagine synthase and Glutamate synthase content of rhizospheric soil were both lower than that of non-rhizospheric soil, these enzymes are related to nitrogen cycle; the prolyl-tRNA synthetase, putative RNA polymerase sigma-24 subunit ECF subfamily, DNA polymerase III subunit alpha and transcriptional regulator content of rhizospheric soil were all lower than that of non-rhizospheric soil, DNA helicase II and elongation factor Tu content of rhizospheric soil were higher than that of non-rhizospheric soil, these proteins are related to cell division or protein synthesis. The results of these cell division or protein synthesis related protein might result in the decrease of bacteria numbers and its biodiversity. The results also showed that the zinc-containing alcohol dehydrogenase superfamily protein, polysaccharide deacetylase, 3,4-dihydroxy-2-butanone 4-phosphate synthase, response regulator and heat shock protein HtpG content of rhizospheric soil were all higher than that of non-rhizospheric soil, these proteins, which are related to resistance of organism, resulted from response of soil microbe to the accumulation of allelochemicals in rhizospheric soil of P. heterophylla. The results finally showed that the aspartate carbamoyltransferase catalytic subunit and acetate kinase content of rhizospheric soil are both higher than that of non-rhizospheric soil; it might be relative to the higher of organic acid in the rhizospheric soil.
|
PDF Full Text Request