Soil acidification reduces crop production and exacerbates soil-borne diseases. Root exudates play important roles in the interaction between plants and soil microorganisms, yet changes in root exudates under acidic soils and their influence on plant resistance are unknown. Here, root exudates secreted by tobacco plants grown under strongly acidic, weakly acidic or neutral conditions were investigated in relation to infection by Ralstonia pseudosolanacearum, which causes a soil-borne bacterial wilt disease resulting in economic losses worldwide. Root exudate profiles changed under acidic conditions, with weakly and strongly acidic treatments revealing 354 and 580 differential metabolites, respectively. Organic and phenolic acids accumulated under acidic conditions, including p-coumaric acid ethyl ester, hydroxycitric acid, 1-naphthyl acetate, trans-cinnamic acid and 3,4,5-trimethoxycinnamic acid, which attracted and promoted R. pseudosolanacearum root colonisation. Exogenous addition of p-coumaric acid ethyl ester, ethyl ferulate, 1-naphthyl acetate, trans-cinnamic acid and 3,4,5-trimethoxycinnamic acid acidified soil and inhibited seed germination and plant growth. Soil pH declined by 0.34-0.66 units when treated with trans-cinnamic acid, 3,4,5-trimethoxycinnamic acid, p-coumaric acid ethyl ester and 1-naphthyl acetate. The cell number of R. pseudosolanacearum in roots increased by 7.9-232.2 times when tobacco plants were treated with hydroxycitric acid, 1-naphthyl acetate, trans-cinnamic acid, 3,4,5-trimethoxycinnamic acid and p-coumaric acid ethyl ester. The decrease in plant hormones abscisic acid, indole-3-butyric acid, IAA-Asp and trans-zeatin-riboside may hinder plant growth and reduce plant resistance to pathogen attack. Overall, the up-regulated exudation of organic and phenolic acids aggravates soil acidification, promotes bacterial wilt disease and hinders plant growth in acidic soils.

BACKGROUNDPlant root exudates play crucial roles in maintaining the structure and function of the whole belowground ecosystem and regulating the interactions between roots and soil microorganisms. Ralstonia solanacearum causes bacterial wilt disease in many plants, while root exudate-mediated inhibition of pathogen infection is poorly understood. Here, we characterize the chemical divergence between root exudates of healthy and diseased tobacco plants and the effects of that variability on the rhizosphere microbial community and the occurrence of bacterial wilt.RESULTSCompared with the healthy plants, root exudates in diseased plants showed distinct exudation patterns and metabolite profiles including increased amounts of flavonoids, phenylpropanoids, terpenoids and defense-related hormones, as well as distinct bacterial community composition, as illustrated by an increased abundance of Ralstonia and decreased abundances of Bacillus and Streptomyces in diseased plants rhizosphere. Pathogen infection stimulated roots to secrete more defensive compounds to inhibit pathogen growth. Change of root exudates modulated rhizosphere microbial community. Specific root exudates could benefit plants by attracting antagonistic Bacillus amyloliquefaciens and inhibiting pathogens. Bacillus amyloliquefaciens could utilize specific root exudates as carbon sources. Benzyl cinnamatel promoted the biofilm formation and colonization of B. amyloliquefaciens on roots.CONCLUSIONTo defend against pathogen invasion, tobacco plants recruited antagonistic and plant growth-promoting rhizobacteria to the rhizosphere by modifying root exudate profiles. Specific signal molecules are recommended to recruit beneficial microorganisms for controlling bacterial wilt. The results provide insights concerning the metabolic divergence of root exudates integral to understanding root-microorganism interaction. (c) 2024 Society of Chemical Industry.

Plant bacterial wilt disease caused by Ralstonia solanacearum leads to huge economic losses worldwide. Endophytes play vital roles in promoting plant growth and health. It is hypothesized that the endophytic root microbiome and network structure are different in healthy and diseased plants. Here, the endophytic root microbiomes and network structures of healthy and diseased tobacco plants were investigated. Composition and network structures of endophytic root microbiomes were distinct between healthy and diseased plants. Healthy plants were enriched with more beneficial bacteria and bacteria with antagonistic activity against R. solanacearum. R. solanacearum was most abundant in diseased plants. Microbial networks in diseased plants had fewer modules and edges, lower connectivity, and fewer keystone microorganisms than those in healthy plants. Almost half of the nodes were unique in the two networks. Ralstonia was identified as a key microorganism of the diseased-plant network. In healthy plants, abundant bacteria and biomarkers (Pseudomonas and Streptomyces) and keystone microorganisms (Bacillus, Lysobacter, and Paenibacillus) were plant-beneficial bacteria and showed antibacterial and plant growth-promoting activities. The endophytic strain Bacillus velezensis E9 produced bacillaene to inhibit R. solanacearum. Consortia containing keystone microorganisms and beneficial endophytic bacteria significantly regulated the endophytic microbiome and attenuated bacterial wilt by inducing systemic resistance and producing antibiotic. Overall, the endophytic root microbiome and network structure in diseased plants were different from those in healthy plants. The endophytic root microbiome of diseased plants had low abundances of beneficial bacteria and an unstable network and lacked beneficial keystone microorganisms, which favored infection. Synthetic microbial consortia were effective measures for preventing R. solanacearum infection. IMPORTANCE Bacterial wilt disease causes heavy yield losses in many crops. Endophytic microbiomes play important roles in control of plant diseases. However, the role of the endophytic root microbiome in controlling bacterial wilt disease is poorly understood. Here, differences in endophytic root microbiomes and network structures between healthy and diseased tobacco plants are reported. A synthetic microbial consortium containing beneficial endophytic bacteria was used to regulate the endophytic microbiome and attenuate bacterial wilt disease. The results could be generally used to guide control of bacterial wilt disease.

土壤酸化是导致烟草青枯病发生和流行的重要因素.利用不同量的生石灰改良酸化植烟土壤,分析生石灰对酸化土壤细菌群落结构和代谢功能的影响以及土壤细菌群落与环境因子的关系.结果表明,适量生石灰(1200～1500 kg/hm2)可提高酸化土壤细菌群落的Sobs、Shannon、Ace和Chao1等多样性指数,提升土壤中放线菌门(Actinobacteria)、绿弯菌门(Chloroflexi)和黏球菌门(Myxococcota)的相对丰度,增加能量代谢、萜类和多酮类代谢、翻译、折叠、分类与降解功能的基因丰度,降低膜运输功能的基因丰度.因此,适量生石灰(1200～1500 kg/hm2)可改善酸化土壤细菌群落结构和代谢功能,确定了土壤pH、交换性钙、交换性镁和可交换铝含量是影响土壤细菌群落的重要因子.

In order to understand the relationship between the occurrence of cigar Fusarium root rot and soil factors, we studied the soil fungi diversity and structural composition, physical and chemical properties and their relationship in Laifeng cigar producing areas with occurrence of cigar root rot. The results indicated that compared with uninfected control, the occurrence of cigar root rot caused reduction in the number of fungal species, decreased Shannon, Simpson, Chao1, and Ace indexed and lower relative abundance of Pseudobensingtonia and increased relative abundance of Fusarium in the rhizosphere soil. The disease index of root rot was significantly positively correlated with soil water content, capillary porosity, available potassium and the relative abundance of Fusarium（p＜0.05）, and negatively correlated with soil aeration porosity and the relative abundance of Pseudobensingtonia（p＜0.01）.Canonical correlation analysis showed that that soil capillary porosity and aeration porosity were the main soil factors affecting the occurrence of cigar Fusarium root rot. Overall, reducing soil capillary porosity and the relative abundance of Fusarium, increasing soil aeration porosity and the relative abundance of Pseudobensingtonia were conducive to the prevention and control of cigar Fusarium root rot.

通过对烟草连作病害土壤、连作非病害土壤及新垦植烟土壤细菌群落结构间的差异性比较,为以微生物手段减少烟草连作病害提供一定的理论依据.采用宏基因组高通量测序技术,测定分析三类土壤的细菌群落结构.结果表明,连作病害土壤细菌总OTUs与独有OTUs最多,群落结构相似,同源性高;连作病害土壤中的蓝细菌门(Cyanobacteria)、拟杆菌门(Bacteroidetes)、假单胞菌属(Pseudomonas)、铁矿沙单孢菌属(Arenimonas)、厌氧绳菌属(Bellilinea)、节杆菌属(Arthrobacter)、黄杆菌属(Flavobacterium)、类诺卡氏菌属(Nocardioides)占比显著高于连作非病害土壤和新垦土壤.连作非病害土壤样本间细菌群落结构差异较大,同源性较低.新垦土壤细菌慢生根瘤菌属(Bradyrhizobium)、酸土单胞菌属(Aciditerrimonas)、分枝杆菌属(Mycobacterium)、鞘氨醇单胞菌属(Sphingomonas)、红游动菌属(Rhodoplanes)5个属所占比例较高,但硝化螺旋菌属(Nitrospira)占比较低,细菌群落结构各具自身特点.

[目的]为筛选出防治根结线虫病的有效农业废弃有机物.[方法]选择湖北宣恩烟区根结线虫病发生严重的烟田,以施化学肥料作CK(对照),选用稻壳、烟草秸秆、稻壳+谷糠、烟草秸秆生物有机肥、茶枯等5组不同农业废弃物还田(15000 kg/hm2)为处理,对根结线虫病防治效果及其土壤酶活性、根系微生物群落的影响进行了研究.[结果]与对照相比,农业废弃物还田后烟株发病率和病情指数均不同程度降低,下降幅度在15.38％～76.91％和70.83％～93.78％,其中处理T1(稻壳)和处理T3(稻壳∶谷糠=1∶1)的病情指数显著低于对照(P＜0.05).土壤保肥能力增强,有机质含量和碱解氮均呈现不同程度的增加.土壤蔗糖酶活性、脲酶活性显著增加,较对照分别提高了16.65％～73.38％、5.89％～105.88％;磷酸酶活性也有增加的趋势,处理T5(茶枯)增加幅度达25％.改变了土壤微生物群落结构和丰度,处理T4(烟草秸秆生物有机肥)微生物门类最丰富,放线菌门(Actinobacteria)在处理T3(稻壳∶谷糠=1∶1)、T2(烟草秸秆)中相对丰度较高;微生物群类LEFSE差异性分析结果表明,处理T1(稻壳)中特异微生物群类是根瘤菌科(Rhizobiaceae).[结论]说明5种农业废弃物还田均能降低根结线虫病的发生程度,使土壤保肥能力增强,较好的改善土壤酶活性和微生物群落结构,其中处理T1(稻壳)15000 kg/hm2还田防治效果最好.

Continuous cropping tobacco has led to soil degradation and caused soilborne bacterial wilt disease. Fulvic acid as a biostimulator was applied to restore soil and control bacterial wilt disease.

为探索改善连作烟田土壤酸化现象的最佳措施,降低烟草青枯病发病率,选择常年种植雪茄烟、土壤酸化现象严重且青枯病频发的烟田为试验地,分析增施石灰、油菜还田及增施石灰+油菜还田3种调酸处理对土壤化学性状、细菌群落结构及雪茄烟株青枯病发生的影响.研究结果表明,增施石灰处理能显著提升土壤pH,改善土壤酸化现象,而油菜还田处理可显著增加土壤速效钾含量.相比对照,增施石灰处理土壤的碱解氮、速效钾、有机质含量分别增加17.04%、17.65%、21.74%,油菜还田处理分别增加1.42%、27.38%、23.73%,增施石灰+油菜还田处理分别增加26.79%、47.66%、49.10%.16S rDNA扩增子测序结果表明,对酸化植烟土壤增施石灰能显著增加细菌群落的整体相对丰度.其中,有益菌属芽单胞菌属(Gemmatimonas)的相对丰度相比对照提高124.84%.此外,增施石灰+油菜还田处理能显著提高可降解环境有害物质的细菌—鞘氨醇单胞菌属(Sphingomonas)的相对丰度,降低致病菌青枯雷尔氏菌属(Ralstonia)的相对丰度.移栽后90d时增施石灰、油菜还田及增施石灰+油菜还田处理相比对照青枯病发病率分别降低37.11%、15.45%、46.01%.

为挖掘具有缓解土壤酸化能力的微生物菌株,开发新型酸性土壤改良剂,通过原位培养从恩施烟区酸化植烟土壤中筛选获得一株荧光假单胞菌SSW-11(Pseudomonas fluorescens SSW-11),并采用盆栽试验和大田试验评价菌株发酵液灌根对酸化植烟土壤的改良效果以及对烟草青枯病的防效.结果表明:①菌株SSW-11发酵液对复合有机酸和复合肥料造成的土壤酸化有一定的缓解作用,土壤pH平均提高0.18;②菌株SSW-11发酵液灌根处理后42 d内,旺长期烟株根区酸化土壤pH显著提高(平均提高0.47);③菌株SSW-11发酵液可显著降低成熟期烟草青枯病的发病率和病情指数,对烟草青枯病的防效达52.01％.荧光假单胞菌SSW-11对缓解植烟土壤酸化和防治烟草青枯病具有较好的效果.

为挖掘具有缓解植烟土壤酸化能力的微生物菌株,采用盆栽试验和大田试验相结合的方法,应用生物形态学和分子生物学筛选鉴定了γ-聚谷氨酸(γ-PGA)高产菌株并研究其对酸化植烟土壤的改良效果.结果表明:(1)从黄土高原干旱土壤中筛选得到一株谷氨酸非依赖型γ-PGA高产菌株副地衣芽孢杆菌285-3(Bacillus paralicheniformis);(2)菌株285-3可有效缓解根系分泌物和复合肥料导致的土壤酸化,土壤pH平均提高0.3个单位以上;(3)菌株285-3配施有机肥,可显著降低酸化植烟土壤可交换酸度,对烟草青枯病的防效达到47.05％,同时提高烟叶产质量.综上所述,副地衣芽孢杆菌285-3对酸化植烟土壤有较好的改良效果,与有机肥合用具有控病和提高烟叶产质量的作用.

为明确影响雪茄烟青枯病发生的关键微生物和土壤因素,采用扩增子测序法,研究了青枯病发病与未发病根际土壤细菌和真菌群落结构组成、土壤理化性状及相互间的关系.结果表明,相对于未发病根际土壤,发病土壤有提高微生物多样性和复杂程度的趋势,增加了假单胞菌(Pseudomonas)、肠杆菌(Enterobacter)、弹球菌(Sphaerobolus)、毛枝菌(Trichocladium)、镰刀菌(Fusarium)等的相对丰度,降低了朱氏杆菌(Chujaibacter)、寡养单胞菌(Stenotrophomonas)、罗河杆菌(Rhodanobacter)、被孢霉菌(Mortierella)、毛壳菌(Chaetomium)等的相对丰度.病情指数与土壤含水量(SAWC)、毛管孔隙度(SCM)、有效磷(AP)显著正相关;与土壤通气孔隙度(SAP)、pH、交换性钙(ECa)显著负相关.典型相关、主成分及最小数据集分析表明土壤SCM、AP和劳尔氏菌(Ralstonia)、Chujaibacter相对丰度是雪茄烟青枯病发病的关键土壤及微生物因子.

为筛选出可有效防治烟草根结线虫的微生物菌剂,选用3株不同种类的芽孢杆菌(B3蜡样芽孢杆菌、B9蜡样芽孢杆菌和S2蕈状芽孢杆菌)微生物菌剂施于烟田,利用宏基因组测序技术分析了不同芽孢杆菌对烟株根际土壤细菌群落的影响及其对根结线虫的防控效果.结果表明,芽孢杆菌可增加土壤中细菌种数,且施用B3蜡样芽孢杆菌的土壤中细菌种数最多.相比施用清水(对照),B3蜡样芽孢杆菌能增加土壤中有益菌门硝化螺旋菌门(Nitrospirae)、有益菌属贪噬菌属(Variovorax)和罗思河小杆菌属(Rhodanobacter)的丰度,B9蜡样芽孢杆菌能增加有益菌属亚硝化螺菌属(Nitrosospira)的丰度,S2蕈状芽孢杆菌能增加有益菌属红假单胞菌属(Rhodopseudomonas)、中慢生根瘤菌属(Mesorhizobium)的丰度.假单胞菌科(Pseudomonadaceae)对根结线虫的防治有益,且在B3蜡样芽孢杆菌处理中其丰度显著高于对照.3种芽孢杆菌对烟株根结线虫病害均有一定的防治效果,但B3蜡样芽孢杆菌的效果最好.

为探索腐殖酸在烟草育苗中的应用效果,采用托盘育苗的方法,研究基质不同腐殖酸添加量(0、0.5、1.0、1.5、2.0 g/株)对烟苗生长及根系形态特征的影响.结果表明,随着基质中腐殖酸用量的增加,烟苗成苗率、壮苗指数、生物量、株高、茎粗、叶长及叶宽呈先增加再降低的趋势,在1.0～1.5 g/株达到最大值,其中烟苗成苗率达到92.3％,较对照增加了3.6个百分点;壮苗指数达到1.05,较对照增加16.7％.适量腐殖酸提高了烟苗根系表面积、平均直径、根系体积及各级根系总长.综合比较,育苗基质中添加腐殖酸能有效改善根系形态特征、提高烟苗的整体素质,最适宜的腐殖酸添加量为1.0～1.5 g/株(基质腐殖酸总含量为28.8％～34.4％).

[目的]分析青枯病烟田和健康烟田土壤代谢物组成的差异,筛选青枯病烟田土壤代谢标志物.[方法]以青枯病烟田和健康烟田的烟株根围土壤为研究对象,采用非靶向代谢组学LC-MS/MS分析技术,对3个时期(未发病、发病中期、发病后期)土壤代谢物组成进行研究.[结果](1)在未发病时、发病中期和发病后期,青枯病烟田和健康烟田土壤代谢物有显著差异的代谢物分别为115种、159种和105种.(2)3个时期共同差异代谢物有63种,其中正离子模式代谢物有49种,负离子模式代谢物有14种.49种正离子模式代谢物可以聚为2个大类和7个小类.(3)筛选出8种代谢标志物,分别为环巴胺(Cyclopamine)、麦芽四糖(Maltotetraose)、麦黄酮(Tricine)、凯林(Khellin)、6-(alpha-D-glucosaminyl)-1 D-myo-inositol、茄啶(Solanidine)、贝磷地尔(belfosdil)、硅雄酮(Silandrone).[结论]青枯病烟田和健康烟田土壤代谢物组成上存在较大差异,筛选出8种代谢标志物可为判别土壤感烟草青枯病状况提供一定参考.

Root-knot nematode (RKN) disease is a destructive soil disease that affects crop health and causes huge losses in crop production. To explore the relationships between soil environments, rhizobacterial communities, and plant health, rhizosphere bacterial communities were analyzed using metagenomic sequencing in tobacco samples with different grades of RKN disease. The results showed that the community structure and function of the plant rhizosphere were significantly correlated to the RKN disease. RKN density and urease content were key factors affecting the rhizosphere bacterial community. Urease accelerated the catabolism of urea and led to the production of high concentrations of ammonia, which directly suppressed the development of RKNs or by improving the nutritional and growth status of microorganisms that were antagonistic to RKNs. Further experiments showed that the suppression role of ammonia should be attributed to the direct inhibition of NH3. The bacterial members that were positively correlated with RKN density, contained many plant cell wall degrading enzymes, which might destroy plant cell walls and promote the colonization of RKN in tobacco roots. The analysis of metatranscriptome and metabolism demonstrated the role of these cell wall degrading enzymes. This study offers a comprehensive insight into the relationships between RKNs, bacteria, and soil environmental factors and provides new ideas for the biological control of RKNs.

为明确影响烟草青枯病发生的关键土壤因子,在青枯病发生的典型烟区采集了24个不同发病程度的植烟土壤样品,并采用分组比较、典范对应分析、主成分分析和最小数据集等方法研究了不同发病程度的土壤理化性状与细菌群落结构间的差异.结果表明,随着青枯病病情指数的提高,土壤pH、交换性钙(ECa)、有效锰(AMn)和通气孔隙度(SAP)呈显著降低的趋势,土壤含水率(SWC)和毛管持水量(SCC)呈明显增加趋势.烟草青枯病的发生对植烟土壤主要门水平细菌相对丰度影响较小,对鞘氨醇单胞菌属(Sphingomonas)、土壤红杆菌属(Solirubrobacter)、根瘤菌属(Rhizobium)的OTUs数量有显著降低效果.综合分析得出,SCC、pH、AMn和鞘氨醇单胞菌属、根瘤菌属的OTUs数量与病情指数的相关性达到显著(P<0.05)水平,是影响烟草青枯病发生的关键因子.

为了解土壤理化性状及细菌群落结构与青枯病发生的关系,在湖北恩施典型烟区采集了160个植烟土壤(82个未发病土壤,78个发病土壤),检测了土壤理化指标,并采用16S rRNA基因测序技术分析了烟株发病与未发病根际土壤细菌群落结构的差异.结果表明:(1)与未发病土壤相比,发病土壤细菌群落结构多样性更高.(2)发病土壤劳尔氏菌属(Ralstonia solanacearum)相对丰度显著高于未发病土壤,而鞘氨醇单胞菌属(Sphingomonas)、节杆菌属(Arthrobacter)和假单胞菌属(Pseudomonas)相对丰度则相反.(3)青枯病的发生使土壤微生物网络结构更复杂而稳定性降低,并改变了部分核心点位细菌种类.(4)典范对应分析(CCA)表明,土壤pH和速效磷(AP)可能是影响烟草青枯病发生的重要土壤因子.综上所述,土壤pH、AP与细菌Sphingomonas、Arthrobacter、Pseudomonas相对丰度的增加和Ralstonia相对丰度的降低可能是减少烟田青枯病发病的关键因素.

为研究黄腐酸与微生物菌剂协同对烟草青枯病及根际土壤细菌群落的影响,采用随机区组试验,调查了烟草青枯病田间发病情况,并对根际土壤样品中细菌16S rRNA基因的V4区进行扩增和高通量测序,分析各处理细菌群落结构.结果表明:①移栽120 d后,黄腐酸与微生物菌剂协同处理烟草青枯病发病率和病情指数显著降低.②黄腐酸与微生物菌剂协同处理可显著提高根际土壤细菌群落观测物种数(Observed_species)、香农指数(Shannon index)和辛普森指数(Simpson index).③黄腐酸与微生物菌剂协同处理可改善根际土壤细菌群落结构,显著提高根际土壤中节杆菌属(Arthrobacter)、寡养单胞菌属(Stenotrophomonas)和Bryobacter属的相对丰度,同时显著降低未确定属的伯克氏菌科(unidentified_Burkholderiaceae)、链霉菌属(Streptomyces)、未确定属的芽单胞菌科(unidentified_Gemmatimonadaceae)和戴氏菌属(Dyella)的相对丰度.黄腐酸与微生物菌剂协同对烟草青枯病有较好的防治效果,根际土壤细菌群落多样性显著增加,细菌群落结构发生明显变化.

研究以烟草根结线虫发病等级不同的烟田为研究对象,对烟株根际土壤理化性状、酶活性等指标与烟草根结线虫发病等级的关系进行系统研究,用主成分分析法找出了影响烟草根结线虫发病等级的主要土壤因子.结果 表明,土壤碱解氮(AN)、有机质(SOM)、团聚体平均质量直径(MWD)以及土壤脲酶、过氧化氢酶活性与根结线虫发病等级呈极显著负相关,磷酸酶活性则与根结线虫发病等级呈显著正相关,而土壤脲酶活性、有机质、交换性镁、交换性钙含量通过建立最小数据集被确定为影响当地烟草根结线虫发病等级的关键土壤因子.本结果可为降低根结线虫对农作物的危害、改良土壤提供科学依据.