| Since the 20th century, with the quick development of agroforestry (interplanting mode etc.) in the Engineering of Coverting Farmland to Forestland and the regulation of agricultural production structure, the interaction between plant species, especially the allelopathic effects of trees on the growth of neighbour plants get increasing concern. In recent years, the planting aera of walnut has greatly expanded in Sichuan Province. Despite the enormous economic benefit gradually realized, the allelopathic effect of decomposing residue of walnut on intercropping crops was paid more and more attention to. The aim of this experiment was to study the impact of decomposing walnut leaf litter on the growth of spinach interplanted under the canopy of walnut, and to understand the action mechanism. A pot experiment was used to simulate the natural decomposition process of walnut leaf litter in soil, and spinach was selected the target plant. In Test A, three leaf litter application rates were:Al(30 g·pot-1)、 A2(60 g·pot-1) and A3(90 g·pof-1) together with CK (0 g·pot-1), the leaf litter was mixed with 8 kg of soil, thereafter the seeds of spinach were sowed. In Test B, the walnut leaf litter application gradient and the operation were the same as Test A except that the leaf litter was steamed for 12 h. In Test C, the leaf litter was added at the same rates as Test A except that the leaf litter was steamed for 12 h, then extracted by 80% acetone and anhydrous ethanol for 24 h, respectively, and again steamed for 12 h. In Text D, the seeds produced by plants in each treatment of Test A were sowed, and observation was implemented on the emergence of these seeds and growth of their seedlings. Also, the tolerance of these progeny to the decomposing walnut leaf litter (60 g·pot-1) was investigated; To compare and find out the potential action chemicals responsible for the effect of decomposing walnut leaf litter, gas chromatography- mass spectrometry (GC-MS) was used to identify secondary metabolites exsited in three kinds of leaf litter samples used in Test A, B and C. The results were as follows:(1) In the first 75 d, the leaf litter of walnut suppressed the height, taproot length, shoot biomass and below-ground biomass, exhibiting a increasing inhibition rate with the increase of leaf litter; During 95 d-115 d, inhibitory effect from almost all leaf litter doses on the morphological indicators gradually decreased, even the taproot was promoted by exposure to the leaf litter during 95 d-135 d; After 135 d, the morphological indicators were promoted and with the increase of walnut leaf litter the promotion enhanced; The nutrient absorption in spinach might be affected by decomposing walnut leaf litter, and spinach elongated its root to compensate for the lack of nutrient absorption, with the extension of the decomposition duration, the release of nutrients from the leaf litter gradually played a dominant role, and the growth of spinach was promoted.(2) Walnut leaf litter decomposition exerted a significant effect on the resistance physiology of spinach. At 75 d, the SOD and POD activities in spinach leaves were both promoted by the application of walnut leaf litter, but the CAT activity and the MDA content were hardly altered; During 95 d-115 d, activities of SOD, POD and CAT were all suppressed exposed to walnut leaf litter, exhibiting a increasing inhibiton rate with the increase of the walnut leaf litter, simultaneously, the content of MDA was higher compared to CK. At 135 d, the responses of these three antioxidative enzymes and MDA were opposite with those during 95 d～ 115 d. In respect of osmolytes, leaf soluble protein content was decreased, while leaf soluble sugar and free proline contents were promoted by exposure to walnut leaf litter during 75 d-115 d; However, the responses of soluble protein and soluble sugar were reversed at 135 d, but the free proline content did not show significant change.(3) Photosynthetic characteristics in spinach were impacted by exposure to walnut leaf litter. During 75 d-115 d, the chlorophyll and carotenoid contents of spinach treated with walnut leaf litter were reduced significantly; Photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr) were inhibited, while the intercellular CO2 concentration (Ci) was higher in treatment A2 and A3 but was lower in treatment A1 than that of CK. The AQ Y, Pn max, LSP, LCP and Rd in spinach were all inhibited by exposure to the leaf litter. When the light intensity was low (<160 μmol·m-2·s-1), no significant differences were observed in Pn among the treatments, but when the light intensity reached 160～900 μmol·m-2·s-1, the Pn showed a trend of A3>A2>A1>CK. When the light intensity exceeded 1350 μmol·m-2·s-1, a trend of CK> 1≈A3 was observed in Pn.(4) Decomposing walnut leaf litter delayed the flowering process of spinach, but the 1000-Kernel weight of seeds produced by leaf litter-treated spinach was higher than that produced by spinach in CK, and significant increase was observed in treatment A1 than CK (RI=0.134)..The emergence of seeds did not show significant difference between each leaf litter treatment and CK, and the growth of the progeny of spinch is not imnpacted by decomposing leaf litter. However, they all showed a limited growth by exposure to decomposing walnut leaf litter (60 g·pot-1). These results indicated that the decomposing leaf litter of walnut would not affect the growth of the progeny of spinach, and the resistance of the progeny to the decomposing walnut leaf litter would not altered, either.(5) Compare with the original walnut leaf litter, the inhibition of the steamed leaf litter was more fierce on the growth of spinach. The RI value observed on each morphological index caused by steamed leaf litter was greater than that caused by the original leaf litter at the same time. Thus, It was suspected that steaming could not remove the action chemicals in walnut leaf litter effectively, and the release of nutrients from the leaf litter might be enhanced by this pretreatment method.(6) When walnut leaf litter was steamed and extracted by organic dissolvents, it had no significant impact on the growth of spinach at 75 d. The RI values observed on the height, toproot length, biomass were greatly reduced compared to the trial using original walnut leaf litter. This indicated that the combination of steaming and organic solvent extraction was effective in removing the action chemicals exsisted in walnut leaf litter, and the alteration of soil gas and wter permeability could not explain the inhibitory effect of decomposing walnut leaf litter.(7) There were 14 substances in walnut leaf litter featuring a relative content greater than 1% according to the analysis using GC-MS. Of them, vitamin E (relative content 30.82%), squalene (26.74%) and γ-sitosterol (13.52%) were dominate representing approximate 70% of the total secondary metabolites identified; most of low boiling and water soluble substances were removed from the walnut leaf litter by steaming. There were only six substances featuring a relative content of more than 1%. Of them, vitamin E (37.30%), squalene (38.03%) and y-sitosterol (15.66%) were still the dominants, but compared with the original leaf litter in respect of the absolute peak area, they decreased by 33.32%-45.69%; Being steamed and extracted with organic solvents, the leaf litter also contained 6 substances whose relative content exceeded 1%. Vitamin E (34.59%), squalene (30.81%) and sitosterol (13.93%) were still the main components, but compared with the original leaf litter, their absolute peak areas were reduced by nearly 90%. |
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