【作者】 杨剑；

【导师】 卢昌义；

【作者基本信息】 厦门大学 ， 环境工程， 2008， 博士

【摘要】 杂草危害是农业生态系统和城市草坪管理中面临的主要问题之一。杂草的化学防治一直是杂草控制的主要手段,但化学除草剂的过度使用所带来的环境安全问题已日益受到公众的关注,而寻求天然无公害的除草剂被认为是解决这一弊端的有效途径之一。豆粕是大豆经过提取豆油后得到的一种副产品,含有40%～48%蛋白质,是一种常见的动物饲料。本文研究了豆粕及其水解物对植物种子萌发的抑制活性(以下称为萌前除草活性)及作用机制,评价了豆粕对成熟草坪植物、草坪质量及草坪土壤微生物多样性的影响。此外,还比较研究了与豆粕组成相似的玉米蛋白粉的萌前除草活性机制。上述研究为阐明豆粕乃至其他天然蛋白原料及其水解物的除草活性机制,为豆粕开发成天然萌前除草剂提供理论依据。主要研究结果如下。(1)通过培养箱及温室内植物种子发芽实验,系统地研究了不同施用量的豆粕对8种植物的萌前除草活性。结果表明:无论是在培养箱实验条件还是温室盆栽条件下,豆粕均能有效抑制植物种子萌发时根的生长发育并降低植物种子相对发芽率。豆粕的萌前除草活性具有种间差异,总体上看,豆粕对双子叶植物具有更强的萌前除草活性。培养箱实验条件下,豆粕施用量为7 mg/mL时,豆粕对野菊(Dendranthema indicum)、胜红蓟(Ageratum conyzoides)、水蜈蚣(Kyllingabrevifolia)、三叶鬼针草(Bidens pilosa)等杂草根长抑制率达到100%,而对多年生黑麦草(Lolium perenne)、一年生黑麦草(Lolium multiflorum)、杂三叶(Trifolium hybridum)等草坪植物种子萌发的根长抑制率为86%、52%和79%。无菌条件下,豆粕的萌前除草活性明显下降,当豆粕施用量达到10 mg/mL时,对多年生黑麦草和红三叶(Trifolium pratense)的根长抑制率(＜32%)远远低于非无菌条件下对这2种植物的根长抑制率(＞90%),从而证实了微生物在豆粕的萌前除草活性中起重要作用。培养箱实验条件下,各施用量豆粕液萌前除草活性实验前的pH为6.63-7.10,氨氮含量为1.07-1.63 mg/L之间,萌前除草活性实验后pH上升到8.26-8.86,氨氮含量上升到25.98-232.47 mg/L,而硝态氮及亚硝态氮含量少,除草活性实验前后变化很小。分析认为,豆粕液经过微生物分解所产生的游离氨是豆粕释放的除草活性物质。温室盆栽条件下,豆粕表施和混施2种施用方式对8种植物均有不同程度的萌前除草活性,混施比表施对植物根的生长有更强的抑制活性。(2)用碱性蛋白酶水解豆粕制备出豆粕水解物,分析了其萌前除草活性及作用机制。结果显示:在培养箱及温室条件下,豆粕水解物比豆粕有更高的除草活性。无菌条件下不同浓度的豆粕水解物(0.5 meg/mL、1mg/mL、1.5 mg/mL、2.0 mg,/mL)使多年生黑麦草根长增加了14%～17%,芽长增加了12%～14%,说明无菌条件下豆粕水解物没有除草活性。豆粕水解物除草活性前后pH及氨氮变化趋势与豆粕液的非常相似,说明豆粕水解物与豆粕具有相同的萌前除草活性机制,即豆粕水解物在微生物作用下所释放的游离氨是除草活性物质。(3)用超滤法制备不同相对分子质量分别为＞10000 Dal、5000-10000 Dal、3000-5000 Dal、1000-3000 Dal、＜1000 Dal的豆粕水解肽组分,发现不同相对分子质量的豆粕水解肽在相同氮含量条件下的除草活性无显著差异,并从相对分子质量小于1000 Dal的豆粕水解肽组分中分离纯化出一个6肽单体(Tyr-Ser-Tyr-Pro-Pro-Arg),分离过程中各多肽组分对多年生黑麦草的根抑制活性无显著差异,这些结果证实了豆粕及其水解物除草活性来源于其中的蛋白质或多肽部分,但豆粕水解肽的除草活性与其相对分子质量没有相关性。(4)通过培养箱发芽实验进一步比较玉米蛋白粉与豆粕的除草机制。结果表明:无菌条件下,玉米蛋白粉的除草活性急剧下降,玉米蛋白粉水解物还促进了多年生黑麦草的种子萌发及幼苗生长。无菌条件下,玉米蛋白粉及其水解物培养液的pH及氨氮基本上没有变化,而非无菌培养条件下,pH及氨氮急剧上升。此外,相同氮含量条件下,不同相对分子质量多肽的萌前除草活性没有显著差异。此项结果证明了豆粕、玉米蛋白粉等天然蛋白原料具有相同的萌前除草机制。(5)豆粕胁迫条件下,多年生黑麦草种子萌发及幼苗生长过程中SOD、POD、CAT活性及MDA、可溶性蛋白、可溶性糖含量表现出先升高后下降的趋势,各处理的SOD、POD、CAT活性均高于对照,不同处理SOD活性最高值发生在第10天至12天,POD活性最高值发生在第7天,CAT活性最高值发生在第7天至第10天。(6)以华南地区广泛栽培的草坪草——细叶结缕草(Zoysia tenuifolia)为研究对象,研究不同的豆粕施用量对细叶结缕草生长、草坪质量及光合作用特性的影响。结果表明:豆粕含有7%左右的氮,为秋冬季细叶结缕草草坪的生长提供了丰富的氮肥源,提高了植物体内硝酸还原酶活性,增加了氮素的吸收,使草坪草的光合能力增强,促进了草坪的生长,增加了草坪的生物量,有效地改善了草坪颜色、均一度,延长了细叶结缕草草坪绿期。(7)通过土壤酶活性分析,传统的微生物培养法以及PCR-DGGE、RAPD等分子生物学技术研究豆粕对草坪土壤微生物多样性的影响。结果显示:豆粕对多数土壤酶活性影响不大,特别是到实验后期,多数土壤酶活性与对照没有显著差异,只有高施用量(300 g/m~2)豆粕能较长时间地提高蛋白酶活性;豆粕只能在一定时间内增加土壤微生物的数量,随着豆粕的分解,养分被草坪植物吸收及渗漏流失,豆粕对土壤微生物的影响逐步减弱,加之土壤微生物的自身调节能力,豆粕处理后期对土壤微生物的数量总体影响比较小;PCR-DGGE指纹图谱分析显示出,高施用量的豆粕在施用后14天内一定程度上增加了草坪土壤细菌的丰富度、多样性指数及均匀度,但第28天时这些指标与对照无显著差异,而低用量的豆粕对草坪土壤细菌的多样性影响不大。土壤微生物RAPD指纹图谱分析表明,用豆粕一定时间内使土壤微生物的多样性增加,但实验末期(第28天)豆粕对土壤微生物的多样性的影响非常微弱。更多还原

【Abstract】 Synthetic herbicides are often used for weed control in agriculture ecosystem and turfgrass management. Public awareness of the widespread use of herbicides and the possible negative effects of their residues on the environment have led to increased efforts in the search for natural herbicidal active products. Soybean meal (SBM) is a byproduct of soybean oil extrcation, it contains 40%-48% protein, and is commonly used as an animal feed.In this paper, SMB and soybean meal hydrolysate (SBH) were studied for inhibitory effect on the germination of plant seed (also called preemergence herbicidal activity) on various weeds and turf plants in the growth chamber and in the greenhouse, and the effects of SBM on mature turf plant, turf quality and soil microbial diversity were also investigated. Besides, the preemergence herbicidal machnism of corn gluten meal (CGM) with similar composition to SBM was explored in growth chamber experiment. The above-mentioned studies are trying to illustrate the preemergence herbicidal mechanism of SBM and other protein, and provide theory basises for developing SBM as a natural preemergence herbicide. The main research results were as follows.(1) The preemergence herbicidal activity of SBM was evaluated on 8 plants in growth chamber and in greenhouse. The results showed that the root growth of germinating seeds can be inhibited effectively by SBM, and the seeds germination rate had been reduced. There were differences among species in their response to SBM treatment. In general, monocotyledonous plants were not susceptible to SBM as dicotyledonous plants. Weeds, i.e. Chrysanthemum indicum, Ageratum conyzoides, Kyllinga brevifolia and Bidens pilosa produced no roots with SBM at 7 mg/mL in growth chamber experiment, whereas reductions in root length of turf plants, i.e. Lolium perenne, Lolium muliiflorum and Trifolium hybridum had 86%, 52% and 79%, respectively. The preemergence herbicidal activity of SBM significantly decreased under the sterile condition. For example, L. muliiflorum and Trifolium pretense exhibited only < 32% reduction in root length with SBM at 10 mg/mL under the sterile condition, but (> 90%) reduction at the same SBM level under nonsterile condition, suggesting that the microbes play a very important role in the herbicidal activity of SBM. The pH of SBM media with various application rates were between 6.63 to 7.10 and the ammonia-nitrogen contents were between 1.07 to 1.63 mg/mL before the experiment. After cultured for 7 days under nonsterile condition, the pH rose to 8.26-8.86, and the ammonia-nitrogen contents reached 25.98 to 232.47 mg/L, whereas the nitric nitrogen and the nitrite nitrogen contents were very low and remained similar level before and after the experiment, proposing that free ammonia produced by the raised pH and ammonia-nitrogen of SBM after the cultivation were the main active herbacidal material. Herbicidal efficiency of SBM was compared between two application methods in the greenhouse. In general, the preplant-incorporated (PPI) treatments were more effective than soil-surfaced applications (PRE).(2) SBH were prepared by Alcalase 2.4 L, and herbicadal activity on L. perenne was tested in the growth chamber and greenhouse condition. Results showed that SBH exhibited a stronger herbicidal activity than SBM. SBH, however, prolonged L. perenne root length and shoot length at 14 to 17% and 12 to 14%, respectively, under sterile condition, while the germination did not change significantly, showing that SBH had no inhibitory effect on seed germination under sterile condition. This also suggested that microbes played an improtant role in the preemergence herbicidal activity of SBH. The similar changing trends of pH and ammonia-nitrogen content between SBH and SBM demonstrated that they had a similar preemergence herbicidal mechanism: free ammonia releasing from decomposed SBH by microbes was the herbicidal activity substance.(3) A series of hydrolyzhed soybean meal peptides (HSP) with different molecular weight (MW) (> 10000 Dal, 5000-10000 Dal, 3000-5000 Dal, 1000-3000 Dal, < 1000 Dal) were prepared by ultrafiltration, and then were subjected to L perenne bioassay. The results indicated that there was no significant difference among these different HSP at the same N content. Subsequently, a hexapeptide (Tyr-Ser-Tyr-Pro-Pro-Arg) was separated and purified from the HSP fraction with WM < 1000 Dal, and all the HSP fractions with different WM had no significant difference on the perennial ryegrass root inhibition, further confirming that the herbicidal activity was resulted from the proteins or peptides in SBM and SBH, and the herbicidal activity of SBH was not related to peptide’s MW. (4) Growth chamber germination experiment was conducted to study the preemergence herbicial activity of CGM and CGH. Results proposed that on the sterile condition, the herbicidal activity of CGM was dramatically decreased, and CGH simultaneously promoted the root and shoot growth of germinating L perenne. pH and ammomia-nitrogen content in CGM and CGH media did not change greatly under the sterile condition, but there was a dramatically increasing under the nonsterile condition. In addition, the preemergence herbicidal activity did not related to MW of hydrolyzed corn gluten peptides. These results concluded that there was a similar herbicidal mechanism between CGM and SBM.(5) SBM stress stimulated the activities of SOD, CAT, POD in the period of seed germinating and early seedling development of perennial ryegrass, and the peak of antioxidant enzymes activity occurred at 7-12 d. So did the content of soluble protein, soluble sugar and MDA.(6) SBM contained about 7% N by weight and provided an additional N source to turfgrass with well-developed root systems in autumn and winter. Nitrate reeducates activity, net photosynthetic rate, turf biomass, total nitrogen and chlorophyll content of Zoysia tenuifolia were enhanced by SBM treatments. The coloration and equitability of the turf were therefore improved, and the green stage also prolonged.(7) The impact of SBM on soil microbial diversity was studied by analyzing the soil enzyme activity and using the traditional incubation method and the molecular biological technology, such as PCR-DGGE and RAPD. Most soil enzyme activities did not change sigficantly, especially during the later experimental period, there was no obvious difference between SBM treatments and control, except that the high rate of SBM treatment (300 g/m~2) for a long time promoted the protein enzyme activity. The number of soil microorganism was increased by SBM treatments in a certain period. The impact of SBM on soil microorganism reduced gradually with decomposition, absorption and seepage nutrient of SBM. During the later period along with autoregulatory ability of soil microorganism, the number of soil microorganism was similar between the SBM treatment and the control. The analysis of PCR-DGGE fingerprinting revealed that the richness, diversity and uniformity index of lawn soil bacteria was enhanced by the high rate of SBM application within 14 days, but after 28 days there was no obvious difference of these targets between all treatments and control. SBM at low amount played little effect on the diversity of soil bacteria. The RAPD fingerprinting analysis demonstrated that the diversity of soil microorganism could be increased in a certain time after the treatments of SBM, but there was no obvious difference of soil microorganism diversity among all treatments and control after 28 days.更多还原