【作者】 Muhammad Ibrahim；

【导师】 杜雄明；

【作者基本信息】 中国农业科学院 ， 植物病理学， 2013， 博士

【摘要】 众所周知,有着白色黄金美誉的棉花,是一种重要的经济作物,在人类经济和社会发展中扮演着举足轻重的角色。目前在中国,虽然导致棉花减产的因素很多,但是一种由土传病原菌引起,被称之为“棉花癌症”的黄萎病是导致中国棉花减产的最主要原因之一,必须严加防控。因此,本研究的主要目的是找出高效的病害防治措施来控制日益严重的棉花黄萎病。本研究的思路是基于过去三十年里反复试验提出的综合病害管理方法(IDM),它仍然是作物可持续生产、环保型病害管理方法之一。本研究通过一系列的实验旨在验证综合防治黄萎病(IVWMC)方法在棉花上的可行性。本研究发现几种真菌在土壤、根系周围以及植株中不同的出现频率与黄萎病存在关联,真菌多样性与黄萎病的相关性,为探讨黄萎病的生物防治提供了基础。本研究通过扩增真菌基因组rRNA的ITS区域,分别从土壤和植株中分离出30和23种真菌。菌落形成单位(CFU)在播种前后的干土壤中呈显著性差异,而播种后所有真菌在土壤中的整体出现频率要显著高于播种前。相对于土壤稀释法,土壤平板技术对于确定真菌出现频率更简便,不过后者更有利筛选单个孢子。直接从棉花植株中分离真菌是确定真菌与植株之间相互作用的最佳方法。大多数的生防菌种,包括绿木霉(Trichoderma virens和T. viride),康宁木霉(T. koningiopsis),钩状木霉(T. hamatum),深绿木霉(T. atroviride),长枝木霉(T. longibrachiatum),拟青霉(Paecilomyces sp.)和毛壳菌(Chaetomium sp.)都是从土壤中分离而来,然而,拟青霉和毛壳菌在土壤和植株中均有发现。本研究还对强致病黄萎病菌的最佳生长条件也进行了探讨,发现培养基类型、温度、光周期和pH值对菌丝生长影响都呈显著(P<0.05)差异。本研究鉴定出了一个在纤维品质和产量均表现优异的抗黄萎病材料,同时建立了一种快速鉴定棉花抗病性的的标准和方法。田间和室内鉴定结果表明全部所选材料的抗病性均表现出显著差异(P<0.05)。本研究还表明材料1421Bt-4133、澳Siv2. Arcot-1和GP93为高抗材料,而Arcot402bne、Arcot438、新陆早3号和冀棉11号为高感材料,中21371和豫棉2067抗性中等,其中1421Bt-4133的其他重要性状也表现优异。通过比较,发现浸根法是筛选抗病材料最快捷的方法,浸根法和茎注射法之间没有明显的差异,前者更省工省时。混土法1和灌根法更有利于研究植株生长的相关参数。农艺性状分析结果表明,除纤维伸长率以外,产量和纤维品质(如铃重、籽棉产量、衣分、纤维细度和纤维均匀度)均受黄萎病影响。本研究还探讨了不同的化学物质对棉花植株的辅助抗性,我们测试了10种常规性杀菌剂单独使用,或与丁基羟基甲苯(BHT)和海藻酸钠(SA)混合施用对黄萎病菌的抑制效果,4个单独使用的杀菌剂(多菌灵,甲基硫菌灵,恶霉灵和五氯硝基苯)和它们与BHT和SA混合使用均表现出了显著(P<0.05)的抑菌作用。多菌灵和甲基硫菌灵分别与BHT+SA混合,对黄萎病菌生长也表现出了强力的抑制作用。对这四种具有较强杀菌作用的化学物质进一步在温室进行单独或混合使用,结果表明,与对照相比,这些杀菌剂显著提高了高感黄萎病材料的抗病性,其中多菌灵和甲基硫菌灵混合物使黄萎病菌存活率显著降低,因而表现出最强的抑菌性。而通过分离多菌灵混合物处理的植株,也未发现黄萎病菌的感染,进一步证明了该混合物最强的抑菌性。相反,恶霉灵和五氯硝基苯的混合物及其单独处理,则效果较差,黄萎病菌存活率最高。14种不同的单一化合物及其与海藻酸钠(SA)、十二烷基硫酸钠(SDS)和吐温80分别混合后也表现出了不同的抑菌能力,而硫酸铜(CuSO4)、氯化铁(FeCl3)和SDS表现出显著抑菌性。与其它组合相比,所有14种化合物与SDS组合均表现出100%的抑菌性。在温室条件下,几个核心化合物的防治效果在不同处理间[T-1:CuSO4; T-2:FeCl3和T-3.茉莉酸(JA)]单独使用时表现出显著的差异。T-1表现出最强的抑菌能力,其次是T-2,在处理后的株高和植株鲜重方面都超过了对照。核心化合物(CuSO4和FeCl3)与SDS和柠檬酸钠混合后,显著降低了病菌的存活力,并增加了植株株高和鲜重,其防治能力为T-1(CuSO4+SDS)>T-9(FeCl3+柠檬酸钠)>T-2(FeCl3+SDS)>T-8(CuSO4+柠檬酸钠)。BHT和缩节胺(DPC)结合使用也有相似结果。本研究同时鉴定了这些化合物在温室条件下对植物的保护作用,当SDS和柠檬酸钠与两个核心化合物(CuSO4和FeCl3)结合使用时抑菌性也有所改善。本研究还探讨了单独使用生防菌(BCA)或将其与一些化学物质混用后,在离体培养和温室条件下对黄萎病的抑制作用。利用双重培养法(微寄生)和挥发性代谢物(抗生作用)法研究了13个不同的真菌和9个细菌对黄萎病的防治效果,两个方法间表现出显著的差异。真菌类BCA的离体抗菌试验显示,长枝木霉和粗糙链孢霉(Neurospora crassa)对于抑制黄萎病菌具有最强的潜力。而这些生防菌与BHT、KH2PO4和L-鼠李糖等化学物质在离体混合培养时,依然保持高效。但是,抑菌率(%)相对于BCA单独使用时稍低。在温室条件下,4个所选的真菌类BCAs(长枝木霉、绿木霉、粗糙链孢霉和拟青霉)的抑菌效果与离体条件下的结果呈相关性。然而,不同生防菌在不同处理方法下的抑菌效果有所差异。4个BCAs分别处理种子后,对病菌的抑制作用并不存在显著差异,不过,相比绿木霉和拟青霉而言,长枝木霉和粗糙链孢霉在不同处理方法中均表现出最强的抑菌能力。细菌的离体培养的抑菌测试表明,不同的细菌具备不同的抑菌能力,在双重培养法和挥发性代谢物法两种培养方法中,枯草杆菌(Bacillus subtilis sub sp. Subtilis)表现出最佳抑菌能力,其次是巨大芽孢杆菌(B. megaterium)、解淀粉芽孢杆菌(B. amyloliquefaciens)、铜绿假单胞菌(Pseudomonas aeruginosa)、荧光假单孢菌(P. fluorescens Migula)和根癌农杆菌(Agrobacterium tumefaciens)。而且,解淀粉芽孢杆菌、铜绿假单胞菌和荧光假单孢菌在两种方法之间均没有显著差异。上述提到的化合物与细菌BCAs配合使用能起到更好抑菌效果。因此,我们对这4个细菌BCAs(枯草杆菌、解淀粉芽孢杆菌、铜绿假单胞菌和荧光假单孢菌)在温室条件下的抑菌能力也进行了测试,枯草杆菌抑菌效果最好,其次是解淀粉芽孢杆菌和铜绿假单胞菌。单独使用这些BCAs或与化学物质混用时对黄萎病菌的抑制作用都强,不过,荧光假单孢菌的抑菌能力在温室条件和离体条件下差别不大,温室下只在使用种子处理时有一定效果。总之,无论是单独,还是混合使用BCAs,对株高、鲜重和叶片数的促进作用,枯草杆菌最好,其次是解淀粉芽孢杆菌和铜绿假单胞菌。本研究也选择了一些具有抑菌潜力的中药作为抑菌剂,在离体培养和温室条件下,测试其对黄萎病的抑菌能力,这将为棉花生产可持续发展和环境友好型黄萎病综合防治提供更多的方法。筛选出的26种中药对黄萎病菌生长抑制作用差异显著,表现出不同的杀菌潜力。无论是离体培养还是温室条件下,五倍子(Rhus chinensis Mill.),乌梅(Prunus mum),黄连(Croptis chinensis Franch)和黄柏(Cortex phellodendri chinensis)均表现出较强的抑菌能力。其中五倍子和乌梅对黄萎病菌抑制和植物生长的促进作用都较强。利用高抗(Arcot-1)、高感(Arcot-438)黄萎病的两个近等基因系进行接菌处理,通过RNA-seq技术,分析与黄萎病抗性相关的差异表达基因,为研究棉花黄萎病抗性形成的分子机理奠定基础。本研究利用Solexa技术共建立了4个RNA-seq测序库,总共获得64077732高质量片段(5961913746个碱基),平均长度为93.04bp。对4个库进行从头拼接后共获得327477个转录本,平均长度为200bp,进一步拼接获得141935个Unigene,在TREMBL、NR、PFAM等数据库进行功能注释后发现,共有22170个Unigene基因注释到25个KOG途径,53357个Unigene基因归类到63个GO注释条目下,然而大多数Unigene都与生物学过程有关,特别是代谢过程和细胞过程。有23569个Unigene注释到KEGG途径,其中1078个注释到不同的酶反应,301个不同的代谢途径中。比较两个抗感材料的接菌处理[Arcot-438(In)/Arcot-1(In)]发现了41061个差异表达基因(DEGs)与抗病性有关,而对抗性材料的诱导表达处理[Arcot-1(CK)/Arcot-1(In)]发现41237个差异表达基因与抗性的诱导有关。进一步比较两个结果发现,共有13427个(4346个上调,9081个下调)共同表达的基因,这些基因分布于122个(73个上调,95个下调)GO和22个(14个上调,8个下调) KEGG途径中,它们及其所属途径可能与棉花黄萎病抗性机制产生具有密切的关系。GO结果还表明,大多数上调基因都集中在生物过程中的代谢进程、细胞进程和生物调节等分支上。同样地,大部分上调的差异表达基因也集中于细胞组成中的细胞、细胞部分等分支中,以及分子功能的结合、催化活性、运输因子等分支中。KEGG结果表明有12个重要的途径可能与棉花黄萎病抗性有密切关系,其中半胱氨酸和蛋氨酸合成,类黄酮生物合成以及牛磺酸和亚牛磺酸代谢途径是最重要的3个途径。根据基因注释和表达量差异分析,共鉴定了15个上调,9个下调的显著差异差异基因。而且,一些重要的相关基因,如E1.14.17.4(K05933)、DNMT.Dcm(K00558)、metE(K00549)等都与半胱氨酸和蛋氨酸合成通路(ko00270)密切相关。而基因FLS2(K13420)、RPS5(K13460)和RPS2(K13459)与植物病理互作通路(ko04626)相关,棉花在黄萎病菌侵染后,与植株抗性相关的大量基因和代谢途径的发现,可用于将来进一步深入研究棉花黄萎病抗性产生的分子机制。综上所述,本研究获得的棉花病害综合防治方法(IVWMC),将为控制黄萎病提供一个有效的措施,最终使棉农、管理人员和研究人员从中受益。更多还原

【Abstract】 Cotton (Gossypium spp.), generally known as ’white gold’, is one of the most important cash crops globally and plays a key role in economic and social development of the people. There are several constraints attributed to low productivity of Chinese cotton. However, currently Verticillium wilt disease caused by soil borne pathogen Verticillium dahliae, which is called "cancer" disease in cotton need to be controlled efficiently. Thus, the main object of current research lines was to find out the most effective and valuable management tools for growing obstacle of cotton in China. The concept of current study is based on integrated disease management (IDM) that has been adopted over the past three decades as an eco-friendly disease management approach for sustainable production of crops. Series of experiments were conducted to validate the results of hypothesis regarding Integrated Verticillium Wilt Management of Cotton (IVWMC) for sustainable production.Preliminary studies found the associations of fungal diversity with Verticillium wilt and provided the baseline information regarding biological control of Verticillium wilt. Several fungi with varied frequency in the soil, rhizosphere and cotton plant were found associated. About30different fungi from soil and23from cotton plants were isolated and confirmed through molecular characterization using internal transcribed spacer (ITS) region of rDNA of fungi. The colony forming unit (CFU)/g dry soil of fungi before and after planting cotton showed significant (P<0.05) variation. The overall frequency of all fungi for soil after sowing was significantly higher than before sowing. Soil plating was comparatively easiest than soil dilution method for the determination of frequency percentage, regardless, later method is useful for the screening of single spore isolate. Direct isolation of fungi from the cotton plant parts is the best technique for the determination of interaction of fungi with plant. Most of the antagonistic species (Trichoderma virens, T. viride, T. koningiopsis, T. hamatum, T. atroviride, T. longibrachiatum, Paecilomyces sp. and Chaetomium sp.) were isolated from soil; nevertheless, Paecilomyces and Chaetomium spp. were screened from plant and soil.The effects of media, temperatures, light regimes and pH on radial mycelial growth produced significantly (P<0.05) difference response. The optimum growth conditions concerning to virulent V. dahliae strain were confirmed in the present study.Studies were subjected to determine the excellent germplasms with high resistance and desirable yield and fibre quality traits, in order to fulfill the dearth, for optimum production; and were to find out the standardized and rapid method for screening of resistance. All the germplasm under in vivo and in vitro studies revealed significant (P<0.05) variation in wilt assessment parameters. Germplasms1421Bt-413, O Siv2, Arcot-1and GP93as highly resistance, whereas, Arcot402bne, Arcot438, Xinluzao-3and Jimianll as high susceptible. The germplasm Zhong21371and Yumian2067evaluated as moderately resistant. The germplasm1421Bt-4133was screened as an excellent material with all desired characteristics. Root dipping was found to be the quickest and easiest methods for screening. No significant difference was observed between the root dipping and stems injecting, but the latter method is laborious and time consuming. Soil mixing-1and soil drenching were found to be the useful for the study of plant growth parameters. Yield and fibre quality parameter such as boll weight, seed cotton yield, lint percentage; fibre fineness and uniformity percentage were also influenced by the severity of Verticillium wilt except fibre elongation.The induced resistance in cotton plant through different chemical compounds were examined. Ten conventional fungicides alone and in combination with butylated hydroxytoluene (BHT) and sodium alginate (SA) were tested under laboratory conditions. Four fungicides (Carbendazim, Topsin-M, Hymexazol and Pentachloronitrobenzene, PCNB) alone and their combinations with BHT and SA showed significantly (P<0.05) highest efficacy.The combinations of Carbendazim and Topsin-M with BHT+SA, respectively, also decolorized the mycelial plug of V. dahliae, indicated their strongest efficacy. These four strongest combinations and fungicides alone, were tested under greenhouse conditions, showed the significant improvement in growth parameters of two cotton germplasm Arcot-402bne (High Susceptible) and Yumian-2067(Tolerant) compared to control-1(Inoculated and untreated). Carbendazim and Topsin-M combinations had the lowest wilt incidence (WI) percentage and showed high efficacy. In contrast, Hymexazol and PCNB combinations and fungicides alone were exhibited lower effectiveness, with the highest WI percentage. No infection (%) of V. dahliae was observed with re-isolation from the plants treated by Carbendazim combination, clearly proved the strongest efficacy.The antifungal efficacy of fourteen different pure chemical compounds tested alone and combinations of Sodium alginate (SA), Sodium dodecyl sulfate (SDS) and Tween80showed inhibitory effect. Copper sulphate and Ferric chloride followed by SDS, when used alone significantly (P<0.05) reduced (100%) radial mycelial colony growth for V. dahliae. All SDS combinations exhibited significantly strongest efficacy (100%) compared to others. The integrative potential of core chemical compounds under greenhouse indicates the significant difference among three treatments when used alone (T-1:GuSO4; T-2:FeCl3and T-3:Jasmonic acid, JA). T-1showed strongest efficacy against Verticillium wilt incidence (WI) followed by T-2and also increased plant height (cm) and fresh plant weight (g) over control.The response of core chemical (CuSO4and FeCl3) combined with SDS and Sodium Citrate significantly reduced the WI percentage and increase plant growth for T-1(CuSO4+SDS) followed by T-9(FeCl3+Sodium Citrate), T-2(FeCl3+SDS) and T-8(CuSO4+Sodium Citrate). Similar results were also obtained with incorporation of BHT and DPC. The present study also confirmed the phytotoxicity of chemical compounds under greenhouse conditions. The improvement observed in the condition of plants treated with SDS and sodium citrate combinations with two core chemicals (CuSO4and FeCl3). The potential of biological control agents (BCAs) alone and in combination with some safe chemicals under in vitro and greenhouse conditions against Verticillium wilt of cotton were studied. Thirteen different fungal and nine bacterial BCAs that were tested through dual culture (mycoparasitism) and volatile metabolites (antibiosis) displayed significantly (P<0.05) varied response. The in vitro antifungal test of fungal BCAs reveals that Trichoderma longibrachiatum and Neurospora crassa remained most effective as compared to other BCAs indicate their strongest potential as BCAs against V. dahliae. The incorporation of safe chemicals (butylated hydroxytoluene (BHT), potassium dihydrogen phosphate (KH2PO4) and L (+)-Rhamnose) with BCAs also remained effective under in vitro, however, inhibition (%) was little lower compared to BCAs alone. The efficacy of four selective potential fungal BCAs (T. longibrachiatum, T.viride, N. crassa and Paecilomyces sp.) tested under greenhouse conditions were correlated with in vitro tests. However, the incorporation of safe chemicals and method of application influenced their efficacy. There was no significant variation in the reduction of WI percentage with seed treatment (ST) for four BCAs, however, T. longibrachiatum and N. crassa compared to T. viride and Paecilomyces sp. had strongest efficacy with seed treatment (ST), spore drenching (SD) and solid culture mix (SCM).The in vitro antifungal test of bacterial BCAs also exhibit different response. The antagonistic potential of Bacillus subtilis sub sp. subtilis followed by B. megaterium, B. amyloliquefaciens, Pseudomonas aeruginosa, P. fluorescens Migula and Agrobacterium tumefaciens remained the most effective compared to other BCAs for dual culture and volatile metabolites methods. The performance of B. amyloliquefaciens, P. aeruginosa and P. fluorescens was observed parallel with no significant difference for dual culture and volatile metabolites methods. The incorporation of safe chemicals enhanced the efficacy of BCAs. The antifungal potential of four selected potential bacterial BCAs (B. subtilis, B. amyloliquefaciens, P. aeruginosa and P. fluorescens.) was also confirmed under greenhouse test. The overall efficacy of B. subtilis followed by B. amyloliquefaciens, P. aeruginosa observed stronger to reduce the Verticillium wilt severity when used alone and in combinations with safe chemical. However, the response of P. fluorescens under greenhouse conditions was correlated with in vitro results by only seed treatment methods compared to spore drenching. Maximum increase in plant height, fresh weight and number of leaves was observed for B. subtilis followed by B. amyloliquefaciens, P. aeruginosa when used alone and in combinations with safe chemical.The antifungal potential of some selective Chinese medicinal herbs were confirmed under in vitro and greenhouse conditions against V. dahliae for sustainable and eco-friendly integrated disease management of cotton. The preliminary screening of26Chinese medicinal herbs for antifungal potential have exhibited significantly (P<0.05) varied response to reduce the radial mycelial colony growth of V. dahliae and showed organic fungicidal potential. The most effective Chinese herbs were Rhus chinensis Mill., Prunus mum, Croptis chinensis Franch and Cortex phellodendri chinensis under in vitro and greenhouse. However, Prunus mum and Rhus chinensis Mill, have been observed the best Chinese medicinal herbs to reduced the development of Verticillium wilt severity and improved the plant growth.The transcriptome of highly resistant (Arcot-1) and highly susceptible (Arcot-438) germplasm after V. dahliae infection for differentially expressed genes (DEGs) involved in the defense mechanism of cotton plant were analyzsed using deep-sequencing technology (RNA-Seq). The sequencing was performed on Illumina HiSeq2000for inoculated (In) and non-inoculated (CK) germplasm. Four different libraries Arcot-1_In, Arcot-438_In, Arcot-1_CK and Arcot-438_CK have been constructed using Solexa paired-end RNA-seq. A total64077732of valid high-quality reads (5961913746bases) were generated with5.96G bases and average length of93.04bp. De novo assembly of four libraries generated a total of327477transcripts, with the length of more than200bp and314MB. Moreover, a total of141935Unigene were obtained with about95MB from four libraries. The functional annotation of most of the Unigenes matched from TREMBL, NCBI non-redundant (NR) and PFAM database. A total of22,170Unigene were annotated into25KOG,53,357annotated Unigenes categorized into63GO terms, however, majority of Unigenes were associated with biological process, especially in metabolic process and cellular. The KEGG annotation yielded a total of23,569annotated Unigenes,1078annotated different enzymes and301different mapped pathways. The total of41,061specific induced differentially expressed genes (DEGs) from Arcot-438(In)_Arcot-1(In) and41,237resistance related DEGs from Arcot-1(CK)_Arcot-1(In) were screened after V. dahliae infection. Further analysis of induced and resistance related DEGs yielded a total13,427(up:4346, down:9081) common DEGs,122(up:73and down:95) common GO and22(up:14and down:8) common ko terms closely related to the defence mechanism of cotton plant against V. dahliae. The majority of induced and resistance related up-regulated DEGs were associated with metabolic process, cellular process, biological regulation from biological process compared to down-regulated. Similarly, the high percentage up-regulated DEGs in cell, cell part and organelle from cellular component, and in binding, Catalytic activity and transporter from molecular process were observed in the present study. The KEGG analysis of DEGs following the V. dahliae infection revealed12most important pathways have close relationship the defence mechanism. However, the majority of DEGs have been associated with Cysteine and methionine metabolism, Flavonoid biosynthesis and Taurine and hypotaurine metabolism pathways. The total of15up-and9down-regulated DEGs were identified, which were significantly (FC>=1.5and FC<=-1.5) involved in the defence mechanism of cotton plant after V. dahliae infection. The total of12other DEGs based on gene definition and high expression ratio, have also been identified in the present study. However, some most important genes such as El.14.17.4(K05933), DNMT.Dcm (K00558) and metE (K00549) showed close association with Cysteine and methionine metabolism (ko00270) pathway; whereas FLS2(K13420), RPS5(K13460) and RPS2(K13459) with plant pathogen interaction (ko04626) pathway. A large number of DEGs which were closely related to the defence mechanism of cotton plant were identified present study.The results achieved in present study for IVWMC, will hopefully be proved as an effective and valuable tool for suppression of determinal disease, Verticilium wilt that will ultimately be beneficial for the end-users (i.e. growers, managers and researchers).更多还原