【作者】 范成明；

【导师】 何月秋；

【作者基本信息】 云南农业大学 ， 植物病理学， 2007， 博士

【摘要】 根肿病是由根肿菌(Plasmodiophora brassicae Woronin)引起的一种威胁十字花科作物生产的世界性土传病害,由于其休眠孢子可以在土壤中存活至少10年,致使其防治成为世界性难题。为了有效控制大白菜根肿病的危害和减少化学农药的污染,本文通过实验室、温室和田间试验,评价了以球茎甘蓝、紫茎泽兰与青蒿为材料的土壤生物消毒对根肿病的防治效果,提出了可能的作用机制。在PSA培养基上,芸苔属植物比非芸苔属植物有更好的生物熏蒸能力,其中以球茎甘蓝的抑制作用最好,且有剂量效应。10g鲜球茎甘蓝粉末对隶属于11个种的30株真菌菌丝生长的抑制效果在26.2%-100%之间。甘薯长喙壳菌、烟草疫霉和油菜菌核菌对球茎甘蓝最为敏感,而黄瓜腐霉菌最不敏感。紫茎泽兰和青蒿也分别对20和17个真菌菌株有生物薰蒸作用。HPLC分析球茎甘蓝、紫茎泽兰和青蒿的结果表明,这3种植物中相同的挥发性成份较少,但紫茎泽兰和青蒿的挥发成份要多于球茎甘蓝。GC-MS分析的结果揭示烯丙基-异硫氰酸酯仅在球茎甘蓝中,紫茎泽兰和青蒿多以芳香类化合物为主。从温室中的防治效果来看,在2个季生产中,球茎甘蓝对根肿病的防治效果及增产作用均不及紫茎泽兰和青蒿。综合病情指数和产量因素来看,营养袋育苗25d后移栽,利于白菜根肿病的防治。在温室中,来自大白菜根际的枯草芽孢杆菌XF1 (Bacillus subtilis XF1)和假单胞杆菌XF2 (Pseudomonas sp. XF2 )对根肿病的防治效果显著,以109CFU/mL在播种前施入土壤后,XF1的处理在直栽60d内的大白菜没有明显症状,XF2的处理在直播30d内没有明显症状。在收获期,两个菌株的防治效果分别达89.2%与46.0%,增产分别达219.5%和139.3%。石灰、青蒿和石灰混合、紫茎泽兰、紫茎泽兰和石灰混合、青蒿、球茎甘蓝和石灰混合及球茎甘蓝等7个处理在大田中3季的平均防治效果分别为:41.9%、41.8%、31.1%、28.6%、28.5%、27.6%和19.8%;平均增产率分别为:8.3%、44.9%、30.1%、29.2%、24.2%、16.1%和9.6%。从对土壤中有效营养元素的影响来看,石灰能够降低土壤中有效N、有效B、有效S、有效Cu、有效Zn和有效Mn浓度;提高有效P、有机质、pH和交换Ca浓度;而对有效K没有影响。从总体来看,紫茎泽兰对土壤的修复能力最强,青蒿和球茎甘蓝次之;对增产效果来说,所有植物材料均好于石灰,但石灰与植物材料的混合处理好于单用植物材料,在培养基上有很好薰蒸作用的球茎甘兰的防病效果及增产效果最差。13项土壤理化指标与防治效果的关联关系为:有效Fe >有效Mn >交换Ca >有效B >有机质>有效K >有效Cu >有效Zn > Ec值> pH >有效S >有效P >有效N。在施入石灰后,有效Fe下降47.7%。有效Fe与有效B、有效Cu和有效Mn呈极显著正相关,与有效Ca呈显著正相关,与pH呈极显著负相关。13项土壤理化指标与增产效果的关联关系为:有效K> pH >有机质>交换Ca >有效B >有效Fe >有效Mn >有效Zn>有效Cu >有效N > Ec值>有效P >有效S。有效K在青蒿与石灰处理下,增加率最高,达154.6%。同时有效K与有机质呈极显著正相关性,与有效P和有效Zn呈显著相关。利用平板培养法和变性梯度凝胶电泳(DGGE)分析了8个处理中土壤微生物的在3季中的变化。从平板法的结果来看,球茎甘蓝、青蒿及青蒿与石灰混合处理明显地增加土壤微生物数量,分别增加为80.9%、82.6%和81.6%;而石灰处理明显减少土壤中微生物数量。但从DGGE的分析结果来看,不同处理间微生物的种群存在着一定的差异,但差异不明显。用Biolog生态板分析了第3季不同处理土壤中的微生物种群落水平生理图谱(CLPP)的结果表明,青蒿、青蒿与石灰混合两处理的平均颜色变化率(AWCD)最高,均为0.952;而球茎甘蓝与石灰处理的AWCD最低,为0.662。对照的常见优势种群指数显著地高于其他处理;植物材料处理的物种丰富度指数要比石灰处理的有所增加;石灰处理的均匀度指数要低于其他处理。因子分析的结果表明,对氨基酸类和糖类碳源平均利用率是评价不同处理的土壤代谢能力的重要指标。据报道,黑芥子酶活性与生物熏蒸活性密切相关。采用十字花科植物黑芥子酶基因的简并引物(MyrBs: 5-ACAGRTTCTCCWTHGCGTGG-3/ MyrBa: 5-ATBTWCCHYTTGTTAGCGGC-3)调查18种植物、2种真菌和2种细菌同源片段。通过blastn比对发现,在11种十字花科、2种菊科(莴苣和紫茎泽兰)和1种茄科(烟草)植物中,扩增产物为黑芥子酶基因的部分片断,而在其他8种生物中所得片断与黑芥子酶基因家族的同源性比较低。这11种十字花科植物的黑芥子酶基因序列相对保守。土壤生物消毒控制大白菜根肿病的可能机制为:一方面植物源材料释放出挥发性抑菌物质钝化土壤微生物,另一方面植物残体在土壤中遗留非挥发性物质影响土壤理化性质,进而影响土壤微生物种群变化,改善了植物生长条件,提高了抗性,降低了病害严重度和提高了产量。更多还原

【Abstract】 Clubroot is a serious soilborne disease of cruciferous crops caused by Plasmodiophora brassicae Woronin worldwide and a difficult problem because its pathogen’s resting spores can live in soil for at least 10 years. To control club root of Chinese cabbage and reduce environment pollution by chemicals application, the mechanisms of disease control and yield increase were studied by biological soil disinfection with fresh Brassica oleracea var. caulorapa de Candolle, Artemisia apiacea and Eupatorium adenophorum in the laboratory, the greenhouse and the field.In PSA medium, Brassica plants had better biofumigation effect on fungi than non-Brassica plants with dosage effect, among which B. oleracea var. caulorapa showed best suppression. Ten gram of B. oleracea var. caulorapa ground powder perФ9 cm plate by adding the powder in the cover and updown culture suppressed 26.2%-100%of mycelial growth for 30 fungi belonging to 11 species. Ceratocystis fimbriata, Phytophthora parasitica and Sclerotinia sclerotiorum were most sensitive to the plant and Pythium aphanidermatum was most unsensitive. E. adenophorum and A. apiacea suppressed 20 and 17 isolates, respectively.B. oleracea var. caulorapa, E. adenophorum and A. apiacea had different volatile compounds and few common ones based on high performance liquid chromatography (HPLC) and gas chromatograph-mass spectrometer (GC-MS) analyses. A. apiacea and E. adenophorum had more volatile compounds than oleracea var. caulorapa. Only were volatile Allyl-ITCs found in B. oleracea var. caulorapa, A. apiacea and E. adenophorum contained mainly aromatic compounds.E. adenophorum and A. Apiacea yielded and reduced club root more than B. oleracea var. caulorapa in the greenhouse experiments in two years. Biological soil disinfection combined with plastic nutrient sac seedling when transplanted in 25 days could control club root and increase yield effectively.Bacillus subtilis XF1 and Pseudomonas sp. XF2 from rhizospheric soil of Chinese cabbage could control club root in the greenhouse very efficiently. No symptom appeared in 60 days and 30 days after XF1 and XF2 application with 109CFU/mL suspension, respectively. Both strains controlled disease with 89.2% and 46.0% and increased Chinease cabbage 219.5% and 139.3%, respectively, in harvest period.Disease index declined gradually in 3 continual field trials by biological soil disinfection. Control effects were 41.9%, 41.8%, 31.1%, 28.6%, 28.5%, 27.6% and 19.8%; yield increased 8.3%, 44.9%, 30.1%, 29.2%, 24.2%, 16.1% and 9.6% averagely, respectively, by liming, A. apiacea plus lime, E. adenophorum, E. adenophorum plus lime, A. apiacea, B. oleracea var. caulorapa plus lime and B. oleracea var.caulorapa. Based on the investigation on nutrient elements in the soil, liming reduced efficient nitrogen, boron, sulphur, copper, zinc and manganese, increased efficient phosphorus, organic matter, pH value and exchangeable calcium, and did not change efficient potassium status. For yield increase, all plant materials were better than liming, but the mix of liming and plants were better than single plant. B. oleracea var. caulorapa was the worst in disease control and yield increase compared with the other treatments even though it showed the best biofumigation effect in PSA medium.The relatedness between control effect and 13 factors were in the oder: Fe> Mn > Ca > B >organic matter > K > Cu > Zn > Ec value> pH > S > P > N in effecience. Efficient Fe reduced by 47.7% and showed most significantly positive correlation with efficient B, Cu, and Mn and significantly with Ca, but nagetive with pH value. The relateness between yield increase and the 13 factors followed the order: K> pH value >organic matter > Ca >B > Fe > Mn > Zn> Cu > N > Ec value > P > S in efficient status for all factors. Efficient K increased yield to 154.6% when it was mixed with lime. It correlated positively to organic matter most significantly, to efficient P and Zn. By plating method and denatured griendent gel electrophorosis (DGGE), culturable microbes in the soils increased 80.9%, 39.5%, -18.4%, 82.6%, 81.6%, 20.8% and 6.8% by the treatments with B. oleracea var. caulorapa, B. oleracea var. caulorapa plus lime, lime, A. apiacea, A. apiacea plus lime, E. adenophorum, and E. adenophorum plus lime in the 3 continual field trials compared with the treatment without any materials (CK), indicating that plants increased microbe amounts and only liming reduced the amounts. Species number of microbes showed some difference among the soils based on the band data of DGGE. Microbial community dynamics in the soils was analyzed in the third season through ECO Biolog microplates. Community level physiological profiling (CLPP) showed that average well colour developments (AWCD) of A. apiacea and A. apiacea plus lime were the highest (0.952), and AWCD of B+L was the lowest (0.662). From the community diversity index, Simpson index of CK was significantly higher than the other treatments; Shannon-Wiener index (H’)of the treatments with plant materials were higher than with liming; Shannon-Wiener (J’) of treatments with liming were lower than others. According to Factor analysis, the carbon-source utilization of amino acids and carbohydrates was important to access metallization of soil.It was reported that myrosinase affected biofumigation. Therefore, the degenerate primer pairs, MyrBs: 5-ACAGRTTCTCCWTHGCGTGG-3 and MyrBa: 5-ATBTWC- CHYTTGTTAGCGGC-3 were designed to investigate the distributions and sequences of myrosinase gene among 18 plants, 2 fungi (Trichoderma harzianum and Colletotrichum gloeosporioides) and 2 bacteria (Bacillus subtilis and Pseudomonas sp.). The sequences from 11 Cruciferae, 2 Compositae(Lactuca sativa and E.adenophorum) and 1 Solanaceae(Nicotiana tabacum), showed highly homologic to known myrosinase genes, but other 8 lowly based on blastn results at the internet database. The sequences from 11 Cruciferae conserved relatively,The possible mechnisms of biological soil disinfection to control club root of Chinese cabbage are: 1. the plants could release votalie compounds to inactivate microorganisms in soil; 2. the plant residues could inprove growth conditions, increase cabbage resistance, reduce disease severity and increase yield by affecting soil physical and chemical natures and population of soil microorganisms.更多还原