ORIGINAL PAPER
In vitro study of the growth,development and pathogenicity responses of Fusarium oxysporum to phthalic acid,an autotoxin from Lanzhou lily
Zhijiang Wu 1,2?Liu Yang 1,2?Ruoyu Wang 1?Yubao Zhang 1?
Qianhan Shang 1,2?Le Wang 1,2?Qin Ren 1,2?Zhongkui Xie 1
Received:23January 2015/Accepted:12May 2015/Published online:21May 2015óSpringer Science+Business Media Dordrecht 2015
Abstract Continuous monoculture of Lanzhou lily (Lilium davidii var.unicolor Cotton)results in frequent incidence of fusarium wilt caused by Fusarium oxysporum .Phthalic acid (PA),a principal autotoxin from root exu-dates of Lanzhou lily,is involved in soil sickness by in-ducing autotoxicity.The aim of this study was to evaluate the direct allelopathic effects of PA on the growth,devel-opment and pathogenicity of F.oxysporum in vitro based on an ecologically relevant soil concentration.The results showed that PA slightly but not signi?cantly inhibited the colony growth (mycelial growth)and fungal biomass of F.oxysporum at low concentrations ranging from 0.05to 0.5mM,and signi?cantly inhibited the colony growth at the highest concentration (1mM).None of the PA con-centrations tested signi?cantly inhibited the conidial ger-mination and sporulation of F.oxysporum in liquid medium.However,mycotoxin (fusaric acid)yield and pathogenesis-related hydrolytic enzyme (protease,pecti-nase,cellulase,and amylase)activities were signi?cantly stimulated in liquid cultures of F.oxysporum containing PA at C 0.25mM.We conclude that PA at a soil level (i.e.0.25mM)is involved in plant–pathogen allelopathy as a stimulator of mycotoxin production and hydrolytic enzyme activities in F.oxysporum ,which is possibly one of the
mechanisms responsible for promoting the wilt disease of lily.
Keywords Phthalic acid (PA)áFusarium oxysporum áFusaric acid áHydrolytic enzymes
Introduction
Lanzhou lily (Lilium davidii var.unicolor Cotton),an im-portant edible crop bulb that is mostly distributed in the middle area of Gansu Province in China (Wang et al.2010),often experiences soil sickness or replanting prob-lems that cause a severe decrease in the yield and quality of lily bulbs.Fusarium wilt of lily is caused by pathogenic fungi,especially Fusarium species (Shang et al.2014).Fusarium oxysporum was identi?ed as the principal pathogen and accumulated in continuously cultivated soil in our previous study (data unpublished);it is believed to be the major causal factor of soil sickness in lily.Phthalic acid (PA),identi?ed as the principal autotoxin from root exudates of lily (Wu et al.2015),is also involved in soil sickness by inducing autotoxicity.
Root exudates can initiate and manipulate biological and physical interactions between roots and other soil organ-isms,and thus play an active role in root–microbe com-munication (Bais et al.2006;Bertin et al.2003;Broeckling et al.2008).By applying natural blends of phytochemicals derived from root exudates of Arabidopsis to the soil,Badri et al.(2013)found that phenolic-related compounds pre-dominantly modulate the soil microbiome.Increasingly,studies have shown that particular autotoxins (phenolic-related compounds)from root exudates not only directly suppress the growth of the plant itself (Asao et al.2007;He et al.2009;Kong et al.2008;Li et al.2012;Mahall and
Zhijiang Wu and Liu Yang have contributed equally to this article.&Zhongkui Xie
wxhcas@https://www.doczj.com/doc/038528100.html,
1
Gaolan Station of Agricultural and Ecological Experiment,Cold and Arid Regions Environmental and Engineering Research Institute,Chinese Academy of Sciences,Lanzhou 730000,China
2
University of Chinese Academy of Sciences,Beijing 100049,China
World J Microbiol Biotechnol (2015)31:1227–1234DOI 10.1007/s11274-015-1872-8
Callaway1992;Pramanik et al.2001;Yu et al.2000),but also indirectly stimulate soil-borne pathogens,and thus enhance disease incidences(Manici et al.2004;Nicol et al. 2003;Ye et al.2004;Zhang et al.2010).Autotoxins might in?uence the progression of soil-borne pathogens by modulating the resistance system of plants to pathogen infection(Nigh1989).However,the precise mode of ac-tion of the allelopathy between autotoxins and pathogens remains poorly understood.To investigate whether soil levels of PA were suf?cient to account for the allelopathic effect of lily,we determined whether PA at various con-centrations had different effects(stimulation or inhibition) on the growth,development and pathogenicity of F. oxysporum in vitro based on an ecologically relevant soil PA concentration(Wu et al.2015).
Materials and methods
Fungal strain and chemicals
The F.oxysporum culture we used was isolated from dis-eased roots of Lanzhou lily obtained from continuously cropped lily?elds(Yuanjiawan,Caoyuan,Xiguoyuan,and Hutan villages of Lanzhou City,Gansu Province,China)in the early autumn of2013.The fungus was transferred to potato dextrose agar(PDA)medium and incubated at 28±2°C in darkness.Phthalic and fusaric acid(FA)used in this study were purchased from a commercial supplier (Sigma-Aldrich,St.Louis,MO,USA).
Preparation of conidial suspension and amended
PDA plates
To prepare a conidial suspension,a block of the stock culture was transferred to PDA plates for7days at 28±2°C in darkness.The mycelium was scraped from the Petri dishes,mixed in10ml sterile distilled water and then?ltered through a four-layer sterile200-mesh nylon mesh?lter(0.154mm mesh).The?ltrate was used as the stock conidial suspension,which was further centrifuged at 30009g for10min and adjusted to a?nal concentration of 29103conidia/ml in sterile deionized water using a he-mocytometer(Li et al.2013).The resulting diluted conidial suspension was used to estimate conidial germination.
To prepare amended PDA plates,PA was diluted with sdH2O and used to amend the PDA at?nal concentrations of0.01,0.05,0.25,0.5,and1mM,which were set ac-cording to the concentration of PA in the soil of succes-sively mono-cropped Lanzhou lily in our previous study (Wu et al.2015).Unamended PDA plates served as con-trols.Three Petri plates(replications)were established for each treatment in a completely randomized design.The experiments were repeated twice.
Measurement of colony growth and sporulation
For the colony growth experiment,15l l of stock conidial suspension was inoculated onto the center of85-mm di-ameter sterile disposable plastic Petri dishes with25ml of amended PDA.All of the PDA plates were placed in a constant temperature incubator for10days in darkness at 28°C.At the end of the experiment,colony diameter was measured and expressed as the average of the longest and shortest diameters(Rosado-A′lvareza et al.2014),and fungal sporulation in each plate was determined.The spore ?ltrate was obtained following the same method as de-scribed above for the preparation of the conidial suspen-sion.The?ltrate was serially diluted and conidia were counted using a hemocytometer.
Estimation of conidial germination
The assay was carried out according to previously pub-lished methods(Wu et al.2008b)with minor changes. Brie?y,100l l aliquots of diluted conidial suspension were inoculated onto amended PDA plates prepared as described above,spread evenly with a sterile L-shaped spreader and then incubated in darkness at28°C.After2days of in-cubation,the spore germination was determined by counting the number of colony-forming units(c.f.u.)ap-pearing on the plates.
Measurement of fungal biomass and pathogenesis-related hydrolytic enzyme activities
Potato Dextrose Broth(PDB)cultures were supplemented with PA at0(control),0.01,0.05,0.25,0.5,or1mM and then adjusted to pH4.5with2M HCl(Wu et al.2009a). Aliquots(100l l)of stock conidial suspension of F.oxys-porum were inoculated into100-ml?asks with25ml of sterile amended PDB medium.There were three replicates for each treatment,and the assay was repeated twice.The cultures were incubated at180rev min-1(Qi et al.2012) and28°C for7days.At the end of the experiment,the cultures were?ltrated with paper(Wu et al.2009a;quan-titative?lter paper,Aohua Co.,Wuhan,China).The fungal residues on the paper were dried at80°C until a constant weight was achieved and then the dry weight of fungal biomass(mainly mycelia)was determined.The?ltered broth was centrifuged at4000rev min-1at25°C for 10min and used to assay pathogenesis-related hydrolytic enzyme activities.
Protease activity was assayed following Drapeau et al. (1972).The unit of activity(U)was de?ned as the amount that yielded0.001OD280units of change per minute.
Pectinase activity(mainly polygalacturonase)was de-termined with a1%(w/v)solution of pectin(Sigma,USA) in0.2M sodium acetate buffer(pH 5.5)at50°C for 10min(Silva et al.2005).The content of galacturonic acid released by enzymatic action was quanti?ed by the3,5-dinitrosalicylic acid(DNS)method(Miller1959).One unit of enzyme activity(U)was de?ned as the amount of en-zyme releasing1l mol of galacturonic acid per minute.
Cellulase activity was determined following Stewart and Leatherwood(1976)with minor changes.The reaction mixture was composed of0.8ml of1%carboxymethyl-cellulose(CMC)in citrate–phosphate buffer(pH5.8)and 0.2ml of enzyme solution.After incubation at50°C for 30min(Eveleigh et al.2009),the amount of reducing sugar(glucose)was measured by the DNS method(Miller 1959).One unit(U)of enzyme activity was de?ned as the amount of catalyzed glucose equivalent to1l mol per min under the assay conditions.
Using the method of Bernfeld(1955)with minor modi?cation,amylase activity was determined at40°C by mixing the diluted enzyme with2%(w/v)soluble starch dissolved in0.1M sodium acetate buffer(pH6.0). After heating for5min in boiling water,the reducing sugars,in the form of glucose,were determined by the DNS method(Miller1959).One unit(U)of amylase ac-tivity was de?ned as the amount of enzyme that released 1l mol of reducing sugar as glucose per min under the assay conditions.
Extraction and quanti?cation of mycotoxin
The culture medium(Venter and Steyn1998)was sup-plemented with PA at0(control),0.01,0.05,0.25,0.5,and 1mM.Aliquots(100l l)of stock conidial suspension of F. oxysporum were inoculated into100-ml?asks with25ml of sterile amended culture medium,which was then incu-bated at180rev min-1(Qi et al.2012)and28°C for 35days.Each treatment included three replicates and the assay was repeated twice.
The mycotoxin(FA)was assayed according to a previ-ously published method(Bacon et al.1996;Venter and Steyn1998)with some modi?cation.Brie?y,the cultures were acidi?ed to pH2.0with2M HCl and extracted se-quentially three times with an equal volume of ethyl ac-etate.The organic extracts were pooled,centrifuged for 10min at5000rev min-1and the supernatant was reduced to dryness in a rotary evaporator under a vacuum at35°C. The dried residue was redissolved in5ml of ethyl acetate and quanti?ed at268nm by spectrophotometry.A standard curve was prepared with FA across a range of suitable concentrations.
Statistical analysis
Analysis of variance(ANOVA)was performed on IBM SPSS Statistics19.0.Means were separated at the5% level by Duncan’s multiple range test.
Results
Effect of PA on the colony growth and biomass of F. oxysporum
The colony growth(mycelial growth)of F.oxysporum on amended PDA plates showed a slight reduction at PA concentrations ranging from0.01to0.5mM,while a sharp and statistically signi?cant reduction was observed at 1mM(Fig.1),indicating that1mM of PA signi?cantly inhibited the colony growth of https://www.doczj.com/doc/038528100.html,pared with the control(without PA),the fungal biomass of F. oxysporum in liquid culures showed a slight rise at 0.01mM PA and then a decline at higher doses,but no signi?cant decline was recorded(Fig.1),which showed that PA at concentrations of C0.05mM slightly inhibited the fungal biomass of F.oxysporum.At the highest con-centration,PA resulted in the maximum inhibition of col-ony growth(10.5%below the control)and the dry fungal biomass(13.4%below the control)of F.oxysporum
. Fig.1Colony growth on amended PDA plates and dry fungal biomass of F.oxysporum in liquid culures at different PA concen-trations.Data are average values with bars representing standard errors of three replicates.Different letters above the bars for each index indicate signi?cant difference among different PA concentra-tions at P\0.05according to Duncan’s test.There is no signi?cant difference among values labeled‘ab’,‘a’and‘b’
Effect of PA on the conidial germination and sporulation of F.oxysporum
The conidial (spore)germination and sporulation of F.oxysporum on amended PDA plates showed no dose-de-pendent decline and no signi?cant differences among var-ious PA concentrations (Fig.2),which suggested that PA did not statistically signi?cantly inhibit the spore germi-nation and sporulation of F.oxysporum at any concentration.
Stimulatory effect of PA on the mycotoxin yield of F.oxysporum
The mycotoxin yield (FA)of F.oxysporum manifested a slight but insigni?cant rise until the addition of C 0.25mM of PA to the liquid medium (Fig.3).PA at concentrations of C 0.25mM signi?cantly stimulated the mycotoxin yield of F.oxysporum and the stimulation effect became stronger as the dose increased.The mycotoxin yield was stimulated by 86.3%at 1mM in comparison with the control (Fig.3).
Stimulatory effect of PA on the pathogenesis-related hydrolytic enzyme activities of F.oxysporum
A signi?cant increase in the activities of protease,pecti-nase,cellulase and amylase in the liquid cultures was ob-served after the addition of PA at concentrations of C 0.25mM and there were no signi?cant differences among the high concentrations ranging from 0.25to 1mM (Fig.4),indicating that PA at concentrations of 0.25mM
and above signi?cantly stimulated protease,pectinase,cellulase and amylase activities.The extent of stimulation showed no signi?cant increase as the dose increased from 0.25to 1mM.The extent of stimulation by PA on different enzyme activities differed among concentrations.Amylase activity showed the greatest stimulation (77.5%over the control),followed by protease activity (73.9%over the control),pectinase activity (53.3%over the control)and cellulase activity (18.0%over the control)(Fig.4).
Discussion
It is important to study the allelopathic effects of root exudates on soil biotic factors at ecologically relevant soil concentrations (Duke 2010;Zhang et al.2010;Zhou et al.2013).In our previous study,PA accumulated in the soil during the monoculture of lily and reached 27.73l g g -1(dry weight;around 0.25mM)after 3years of monocul-ture (Wu et al.2015).We selected concentrations around 0.25mM as ecologically relevant concentrations and de-termined the direct allelopathic effects of PA at 0,0.01,0.05,0.25,0.5and 1mM on the growth,development and pathogenicity of F.oxysporum in vitro.
Our results clearly indicated that PA slightly inhibited the colony growth (mycelial growth)and fungal biomass of F.oxysporum at low concentrations (0.05–0.5mM),but only signi?cantly inhibited the colony growth at the highest concentration (1mM;Fig.1).The ?ndings of this work were similar to those previously reported by EI Modafar and EI Boustani (2001),who found that cell wall-bound phenolic acids (p -hydroxybenzoic,p -coumaric,ferulic,and sinapic acids)strongly reduced the mycelial growth of
F.
Fig.2Conidial (spore)germination and sporulation of F.oxysporum on amended PDA plates at different PA concentrations.Data are average values with bars representing standard errors of three replicates.The same letters above the bars for each index indicate no signi?cant difference among different PA concentrations at P \0.05according to Duncan’s
test
Fig.3Effect of PA at various concentrations on the mycotoxin yield of F.oxysporum in liquid cultures.Data are average values with bars representing standard errors of three replicates.Different letters above the bars for each index indicate signi?cant difference among different PA concentrations at P \0.05according to Duncan’s test
oxysporum in vitro in a dose-dependent manner.Other studies also showed that the hyphal growth and biomass of F.oxysporum f.sp.Niveum were strongly inhibited by cinnamic (Wu et al.2008b ),p -hydroxybenzoic (Wu et al.2010b ),succinic (Wu et al.2011),ferulic (Wu et al.2010a ),and benzoic (Wu et al.2009c )acids from the root exudates and decaying residues of watermelon.However,some of these studies found that conidial germination on plates and conidial production in liquid cultures were completely inhibited at the highest concentrations of cin-namic (Wu et al.2008b ),p -hydroxybenzoic (Wu et al.2010b ),and benzoic (Wu et al.2009c )acids,which was not in agreement with the results of the present study,in which PA showed no statistically signi?cant inhibition of the conidial germination and sporulation of F.oxysporum ,even at the highest concentration (1mM;Fig.2).This may be because the ecologically relevant concentrations of PA were relatively low and insuf?cient to cause such inhibi-tion.Recent studies have shown that many plant species resist potential soil-borne pathogens by modifying the amounts and composition of organic acids in their root exudates (Hao et al.2010;Kamilova et al.2006;Li et al.2013;Ling et al.2011;Pan et al.2013).Consequently,we believe that decreasing the colony growth and biomass of F.oxysporum by PA would be one of the mechanisms by which lily plants resist this pathogen.
Plant–pathogenic fungi damage host plants through in-vasion of hyphae into host vascular tissues and secretion of mycotoxins and hydrolytic enzymes related to patho-genesis during the infection process (Desjardins and Hohn 1997;Kikot et al.2009;Wagacha and Muthomi 2007;Walter et al.2010).Mycotoxins are fatal pathogenic factors that cause plants to wilt (Li et al.2011;Logrieco et al.2003;Stankovic et al.2007)and are largely represented by low molecular-weight secondary metabolites capable of damaging the vital activities of plant cells or causing their death below 10mM (Berestetskiy 2008).FA,a well-known nonspeci?c mycotoxin produced by all Fusarium species (Gapillout et al.1996;Toyoda et al.1988),par-ticularly pathogenic strains of F.oxysporum ,is the cause
of
Fig.4Effect of PA at various concentrations on the pathogenesis-related hydrolytic enzyme activities of F.oxysporum in liquid cultures.Data are average values with bars representing standard errors of three replicates.Different letters above the bars for each index indicate signi?cant difference among different PA concentra-tions at P \0.05according to Duncan’s test
wilting diseases in a variety of crops(Capasso et al.1996; Dong et al.2012;Toyoda et al.1988;Venter and Steyn 1998;Wang and Ng1999).FA induces early hyperpolar-ization of plant cells followed by signi?cant depolarization of the transmembrane electrical potential(D’Alton and Etherton1984).Bouizgarne et al.(2006)also reported that FA played an important role in the early stage of date palm infection by F.oxysporum f.sp.Albedinis.In the present work,PA at concentrations of C0.25mM signi?cantly stimulated the FA yield of F.oxysporum and this stimulation effect became stronger as the dose increased (Fig.3),which increased the risk of lily plants being in-fected by F.oxysporum.Wu et al.reported similar re-sults—that FA production by F.oxysporum f.sp.Niveum was stimulated by cinnamic(2008b),sinapic(2009b),p-hydroxybenzoic(2010b),ferulic(2010a),salicylic(2008a), and benzoic(2009c)acids.Evidence exists that the con-centration of FA in diseased plants is positively correlated with the virulence of F.oxysporum(Tan et al.2011). Consequently,increasing the FA yield may be a response mechanism of F.oxysporum to PA to enhance its own virulence to plants.
Cell wall degrading enzymes(CWDEs)produced by plant pathogenic fungi are also considered important pathogenicity factors(Annis and Goodwin1997;Wanjiru et al.2002;Kikot et al.2009).Among CWDEs,pectinases are considered typical examples of‘basic’compatibility factors,meaning that they are thought to be required for pathogenicity but not to determine any host cultivar speci?city(Reignault and Sancholle2005).It has been reported that cellulase is involved in the microbial degradation of cellulose by catalyzing the bioconversion of cellulose to soluble sugars and glucose(Klechkovskaya et al.1998;Wood and Garcia-Campayo1991).Pekkari-nen et al.(2000)also identi?ed proteinases as factors for the pathogenesis of pathogens.Our research showed that pathogenesis-related hydrolytic enzyme(protease,pecti-nase,cellulase,and amylase)activities were strongly and signi?cantly stimulated in liquid cultures of F.oxysporum amended with PA at C0.25mM(Fig.4);this might aid infection by F.oxysporum and its spread in host tissues,as well as satisfying its nutritional requirements,as reported in previous research(Wanjiru et al.2002).An earlier work demonstrated that the degree of pathogen virulence on crops could be affected by differences in the produc-tion of extracellular enzymes(Kikot et al.2009).The increased hydrolytic enzyme activities induced by PA may be a mechanism for enhanced pathogenicity of F. oxysporum.Moreover,we found that the extent of stimulation showed no dose-dependent increase at con-centrations ranging from0.25to1mM,which was per-haps due to the restricted growth of F.oxysporum by PA at higher concentrations.
The phenomenon of pathogen growth inhibition and promotion of virulence factors by a pure active compound has been observed in bacteria and has been attributed to multiple global regulators.For example,the expression of the global regulator CsrA affects virulence-associated traits and growth in Acanthamoeba castellanii(Forsbach-Birk et al.2004).The production of virulence factors by bac-terial pathogens is controlled by multiple global regulators, such as SarH1(Tegmark et al.2000),CodY(Sonenshein 2005),PecS(Rouanet et al.2004),mgr(Luong et al.2003) and ppGpp(Magnusson et al.2005).We speculate that PA might induce global regulator changes in fungal cells as an environmental signal.Future work on the molecular mechanism by which PA affects fungal growth and viru-lence factors will help identify potential multiple global regulators that control this process.
A major limitation of this study is that other mycotoxins besides FA were not measured because of the limited in-formation we had and our experimental conditions.Some pathogenesis-related mycotoxins including deoxynivalenol (Wagacha and Muthomi2007),fumonisin B1(Stankovic et al.2007),and T-2toxin(Logrieco et al.2002)have been found in other Fusarium species.However,we could not determine whether FA is the predominant toxin because of a lack of study on other mycotoxins of F.oxysporum.Thus, we need to perform further studies to obtain information on other mycotoxins originating from the F.oxysporum as-sayed in the present work.
In summary,the growth and development of F.oxys-porum were slightly but not signi?cantly inhibited by PA at concentrations ranging from0.05to0.5mM,while the mycotoxin yield and pathogenesis-related hydrolytic en-zyme activities of F.oxysporum were signi?cantly stimulated by PA at C0.25mM.The distinct effects of PA on different physiological characteristics of F.oxysporum might be explained by different mechanisms involved in the interaction between the plant and pathogen.PA at a soil autotoxic concentration(i.e.0.25mM)is suf?cient to ac-count for the allelopathic effect by stimulating the myco-toxin production and activities of hydrolytic enzymes of F. oxysporum possibly responsible for promoting wilt disease. Genetic modi?cation of genes associated with PA synthesis in lily plants or the addition of PA-degrading bacterial strains to decrease autotoxin production in the soil may be promising strategies to control the wilt disease of lily. Acknowledgments This work was?nancially supported by the Ningxia Agricultural Comprehensive Development Of?ce (NTKJ2014091),National Natural Science Foundation of China (31370447)and Hundred Talents Program of CAS‘‘Molecular mechanism of biological control on plant diseases.’’
Con?ict of interest The authors declare that they have no con?ict of interest.
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The way 的用法 Ⅰ常见用法： 1)the way+ that 2)the way + in which（最为正式的用法） 3)the way + 省略（最为自然的用法） 举例：I like the way in which he talks. I like the way that he talks. I like the way he talks. Ⅱ习惯用法： 在当代美国英语中，the way用作为副词的对格，“the way+ 从句”实际上相当于一个状语从句来修饰整个句子。 1)The way =as I am talking to you just the way I’d talk to my own child. He did not do it the way his friends did. Most fruits are naturally sweet and we can eat them just the way they are—all we have to do is to clean and peel them. 2）The way= according to the way/ judging from the way The way you answer the question, you are an excellent student. The way most people look at you, you’d think trash man is a monster. 3）The way =how/ how much No one can imagine the way he missed her. 4）The way =because
The way的用法及其含义（二） 二、the way在句中的语法作用 the way在句中可以作主语、宾语或表语： 1.作主语 The way you are doing it is completely crazy.你这个干法简直发疯。 The way she puts on that accent really irritates me. 她故意操那种口音的样子实在令我恼火。The way she behaved towards him was utterly ruthless. 她对待他真是无情至极。 Words are important, but the way a person stands, folds his or her arms or moves his or her hands can also give us information about his or her feelings. 言语固然重要,但人的站姿,抱臂的方式和手势也回告诉我们他(她)的情感。 2.作宾语 I hate the way she stared at me.我讨厌她盯我看的样子。 We like the way that her hair hangs down.我们喜欢她的头发笔直地垂下来。 You could tell she was foreign by the way she was dressed. 从她的穿著就可以看出她是外国人。 She could not hide her amusement at the way he was dancing. 她见他跳舞的姿势，忍俊不禁。 3.作表语 This is the way the accident happened.这就是事故如何发生的。 Believe it or not, that's the way it is. 信不信由你, 反正事情就是这样。 That's the way I look at it, too. 我也是这么想。 That was the way minority nationalities were treated in old China. 那就是少数民族在旧中
定冠词the的用法： 定冠词the与指示代词this ,that同源,有“那(这)个”的意思,但较弱,可以和一个名词连用,来表示某个或某些特定的人或东西. (1)特指双方都明白的人或物 Take the medicine.把药吃了. (2)上文提到过的人或事 He bought a house.他买了幢房子. I've been to the house.我去过那幢房子. (3)指世界上独一无二的事物 the sun ,the sky ,the moon, the earth (4)单数名词连用表示一类事物 the dollar 美元 the fox 狐狸 或与形容词或分词连用,表示一类人 the rich 富人 the living 生者 (5)用在序数词和形容词最高级,及形容词等前面 Where do you live?你住在哪? I live on the second floor.我住在二楼. That's the very thing I've been looking for.那正是我要找的东西. (6)与复数名词连用,指整个群体 They are the teachers of this school.(指全体教师) They are teachers of this school.(指部分教师) (7)表示所有,相当于物主代词,用在表示身体部位的名词前 She caught me by the arm.她抓住了我的手臂. (8)用在某些有普通名词构成的国家名称,机关团体,阶级等专有名词前 the People's Republic of China 中华人民共和国 the United States 美国 (9)用在表示乐器的名词前 She plays the piano.她会弹钢琴. (10)用在姓氏的复数名词之前,表示一家人 the Greens 格林一家人(或格林夫妇) (11)用在惯用语中 in the day, in the morning... the day before yesterday, the next morning... in the sky... in the dark... in the end... on the whole, by the way...
“tｈｅwａｙ+从句”结构的意义及用法 首先让我们来看下面这个句子: Ｒead the foｌloｗｉnｇpassagｅａnd talkａbout ｉt wi ｔh your classmaｔes．Try tｏｔeｌl whaｔyｏu thiｎk ｏf Tom aｎd ｏｆｔhe ｗay the chiｌｄrenｔrｅated ｈim. 在这个句子中，ｔhe waｙ是先行词,后面是省略了关系副词that或in whｉch的定语从句。 下面我们将叙述“the ｗaｙ+从句”结构的用法。 1.tｈe wａy之后，引导定语从句的关系词是tｈat而不是hoｗ,因此，<<现代英语惯用法词典＞＞中所给出的下面两个句子是错误的：Ｔhｉs is ｔｈeｗayｈowｉtｈａppened. This is the way how he ａlwａｙs treats me. 2．在正式语体中,thaｔ可被in which所代替;在非正式语体中，ｔhat则往往省略。由此我们得到theｗay后接定语从句时的三种模式：1) tｈe ｗaｙ＋ｔhａt-从句２）the way +ｉn whiｃh－从句3) the ｗay +从句 例如：Ｔｈe way(in whiｃh ，thａt) ｔhｅｓｅｃomｒade ｓloｏｋａｔｐrobｌems is wrong．这些同志看问题的方法
不对。 Ｔｈｅｗａy(that ,ｉn ｗhiｃh)yoｕ’ｒe doinｇit is comple ｔeｌy crａzy.你这么个干法,简直发疯。 Wｅａdmiｒｅd him for thｅｗay ｉｎｗhｉch he fａｃeｓdiｆficultieｓ． Waｌlａｃe ａｎd Ｄarwiｎｇreed ｏn the way ｉｎwhi ｃh ｄｉfｆｅrｅnt forms ｏf life had ｂegｕn.华莱士和达尔文对不同类型的生物是如何起源的持相同的观点。 Thｉs is ｔｈe ｗaｙ（tｈａｔ) hｅdｉd it. I lｉkeｄｔhe waｙ(ｔhat) ｓｈeｏｒganized ｔhe ｍeetｉng． 3.theｗay(tｈat)有时可以与hｏw(作“如何”解）通用。例如： That’s tｈe ｗaｙ(tｈaｔ) shｅspoke. = Tｈat’s how shｅspoke.
表示“方式”、“方法”，注意以下用法： 1.表示用某种方法或按某种方式，通常用介词in(此介词有时可省略)。如： Do it (in) your own way. 按你自己的方法做吧。 Please do not talk (in) that way. 请不要那样说。 2.表示做某事的方式或方法，其后可接不定式或of doing sth。 如： It’s the best way of studying [to study] English. 这是学习英语的最好方法。 There are different ways to do [of doing] it. 做这事有不同的办法。 3.其后通常可直接跟一个定语从句(不用任何引导词)，也可跟由that 或in which 引导的定语从句，但是其后的从句不能由how 来引导。如： 我不喜欢他说话的态度。 正：I don’t like the way he spoke. 正：I don’t like the way that he spoke. 正：I don’t like the way in which he spoke. 误：I don’t like the way how he spoke. 4.注意以下各句the way 的用法： That’s the way (=how) he spoke. 那就是他说话的方式。 Nobody else loves you the way(=as) I do. 没有人像我这样爱你。 The way (=According as) you are studying now, you won’tmake much progress. 根据你现在学习情况来看，你不会有多大的进步。 2007年陕西省高考英语中有这样一道单项填空题： ——I think he is taking an active part insocial work. ——I agree with you_____. A、in a way B、on the way C、by the way D、in the way 此题答案选A。要想弄清为什么选A，而不选其他几项，则要弄清选项中含way的四个短语的不同意义和用法，下面我们就对此作一归纳和小结。 一、in a way的用法 表示：在一定程度上，从某方面说。如： In a way he was right.在某种程度上他是对的。注：in a way也可说成in one way。 二、on the way的用法 1、表示：即将来(去)，就要来(去)。如： Spring is on the way.春天快到了。 I'd better be on my way soon.我最好还是快点儿走。 Radio forecasts said a sixth-grade wind was on the way.无线电预报说将有六级大风。 2、表示：在路上，在行进中。如： He stopped for breakfast on the way.他中途停下吃早点。 We had some good laughs on the way.我们在路上好好笑了一阵子。 3、表示：(婴儿)尚未出生。如： She has two children with another one on the way.她有两个孩子，现在还怀着一个。 She's got five children，and another one is on the way.她已经有5个孩子了，另一个又快生了。 三、by the way的用法
The way的用法及其含义（一） 有这样一个句子：In 1770 the room was completed the way she wanted. 1770年，这间琥珀屋按照她的要求完成了。 the way在句中的语法作用是什么？其意义如何？在阅读时，学生经常会碰到一些含有the way 的句子，如：No one knows the way he invented the machine. He did not do the experiment the way his teacher told him.等等。他们对the way 的用法和含义比较模糊。在这几个句子中，the way之后的部分都是定语从句。第一句的意思是，“没人知道他是怎样发明这台机器的。”the way的意思相当于how；第二句的意思是，“他没有按照老师说的那样做实验。”the way 的意思相当于as。在In 1770 the room was completed the way she wanted.这句话中，the way也是as的含义。随着现代英语的发展，the way的用法已越来越普遍了。下面，我们从the way的语法作用和意义等方面做一考查和分析： 一、the way作先行词，后接定语从句 以下3种表达都是正确的。例如：“我喜欢她笑的样子。” 1. the way+ in which +从句 I like the way in which she smiles. 2. the way+ that +从句 I like the way that she smiles. 3. the way + 从句(省略了in which或that) I like the way she smiles. 又如：“火灾如何发生的，有好几种说法。” 1. There were several theories about the way in which the fire started. 2. There were several theories about the way that the fire started.
way 的用法 【语境展示】 1. Now I’ll show you how to do the experiment in a different way. 下面我来演示如何用一种不同的方法做这个实验。 2. The teacher had a strange way to make his classes lively and interesting. 这位老师有种奇怪的办法让他的课生动有趣。 3. Can you tell me the best way of working out this problem? 你能告诉我算出这道题的最好方法吗？ 4. I don’t know the way (that / in which) he helped her out. 我不知道他用什么方法帮助她摆脱困境的。 5. The way (that / which) he talked about to solve the problem was difficult to understand. 他所谈到的解决这个问题的方法难以理解。 6. I don’t like the way that / which is being widely used for saving water. 我不喜欢这种正在被广泛使用的节水方法。 7. They did not do it the way we do now. 他们以前的做法和我们现在不一样。 【归纳总结】 ●way作“方法，方式”讲时，如表示“以……方式”，前面常加介词in。如例1； ●way作“方法，方式”讲时，其后可接不定式to do sth.，也可接of doing sth. 作定语，表示做某事的方法。如例2，例3；
t h e-w a y-的用法
The way 的用法 "the way+从句"结构在英语教科书中出现的频率较高, the way 是先行词, 其后是定语从句.它有三种表达形式:1) the way+that 2)the way+ in which 3)the way + 从句(省略了that或in which),在通常情况下, 用in which 引导的定语从句最为正式,用that的次之,而省略了关系代词that 或 in which 的, 反而显得更自然,最为常用.如下面三句话所示,其意义相同. I like the way in which he talks. I like the way that he talks. I like the way he talks. 一.在当代美国英语中,the way用作为副词的对格,"the way+从句"实际上相当于一个状语从句来修饰全句. the way=as 1)I'm talking to you just the way I'd talk to a boy of my own. 我和你说话就象和自己孩子说话一样. 2)He did not do it the way his friend did. 他没有象他朋友那样去做此事. 3)Most fruits are naturally sweet and we can eat them just the way they are ----all we have to do is clean or peel them . 大部分水果天然甜润,可以直接食用,我们只需要把他们清洗一下或去皮.
way的用法总结大全 way的用法你知道多少，今天给大家带来way的用法，希望能够帮助到大家，下面就和大家分享，来欣赏一下吧。 way的用法总结大全 way的意思 n. 道路,方法,方向,某方面 adv. 远远地，大大地 way用法 way可以用作名词 way的基本意思是“路,道,街,径”,一般用来指具体的“路,道路”,也可指通向某地的“方向”“路线”或做某事所采用的手段,即“方式,方法”。way还可指“习俗,作风”“距离”“附近,周围”“某方面”等。 way作“方法,方式,手段”解时,前面常加介词in。如果way前有this, that等限定词,介词可省略,但如果放在句首,介词则不可省略。
way作“方式,方法”解时,其后可接of v -ing或to- v 作定语,也可接定语从句,引导从句的关系代词或关系副词常可省略。 way用作名词的用法例句 I am on my way to the grocery store.我正在去杂货店的路上。 We lost the way in the dark.我们在黑夜中迷路了。 He asked me the way to London.他问我去伦敦的路。 way可以用作副词 way用作副词时意思是“远远地,大大地”,通常指在程度或距离上有一定的差距。 way back表示“很久以前”。 way用作副词的用法例句 It seems like Im always way too busy with work.我工作总是太忙了。 His ideas were way ahead of his time.他的思想远远超越了他那个时代。 She finished the race way ahead of the other runners.她第一个跑到终点,远远领先于其他选手。 way用法例句
t h e_w a y的用法大全
The way 在the way+从句中, the way 是先行词, 其后是定语从句.它有三种表达形式:1) the way+that 2)the way+ in which 3)the way + 从句(省略了that或in which),在通常情况下, 用in which 引导的定语从句最为正式,用that的次之,而省略了关系代词that 或 in which 的, 反而显得更自然,最为常用.如下面三句话所示,其意义相同. I like the way in which he talks. I like the way that he talks. I like the way he talks. 如果怕弄混淆，下面的可以不看了 另外，在当代美国英语中,the way用作为副词的对格,"the way+从句"实际上相当于一个状语从句来修饰全句. the way=as 1)I'm talking to you just the way I'd talk to a boy of my own. 我和你说话就象和自己孩子说话一样. 2)He did not do it the way his friend did. 他没有象他朋友那样去做此事. 3)Most fruits are naturally sweet and we can eat them just the way they are ----all we have to do is clean or peel them . 大部分水果天然甜润,可以直接食用,我们只需要把他们清洗一下或去皮. the way=according to the way/judging from the way 4)The way you answer the qquestions, you must be an excellent student. 从你回答就知道,你是一个优秀的学生. 5)The way most people look at you, you'd think a trashman was a monster. 从大多数人看你的目光中,你就知道垃圾工在他们眼里是怪物. the way=how/how much 6)I know where you are from by the way you pronounce my name. 从你叫我名字的音调中,我知道你哪里人. 7)No one can imaine the way he misses her. 人们很想想象他是多么想念她. the way=because 8) No wonder that girls looks down upon me, the way you encourage her. 难怪那姑娘看不起我, 原来是你怂恿的
The way 的用法 "the way+从句"结构在英语教科书中出现的频率较高, the way 是先行词, 其后是定语从句.它有三种表达形式:1) the way+that 2)the way+ in which 3)the way + 从句(省略了that或in which),在通常情况下, 用in which 引导的定语从句最为正式,用that的次之,而省略了关系代词that 或in which 的, 反而显得更自然,最为常用.如下面三句话所示,其意义相同. I like the way in which he talks. I like the way that he talks. I like the way he talks. 一.在当代美国英语中,the way用作为副词的对格,"the way+从句"实际上相当于一个状语从句来修饰全句. the way=as 1)I'm talking to you just the way I'd talk to a boy of my own. 我和你说话就象和自己孩子说话一样. 2)He did not do it the way his friend did. 他没有象他朋友那样去做此事. 3)Most fruits are naturally sweet and we can eat them just the way they are ----all we have to do is clean or peel them . 大部分水果天然甜润,可以直接食用,我们只需要把他们清洗一下或去皮.
the way=according to the way/judging from the way 4)The way you answer the qquestions, you must be an excellent student. 从你回答就知道,你是一个优秀的学生. 5)The way most people look at you, you'd think a trashman was a monster. 从大多数人看你的目光中,你就知道垃圾工在他们眼里是怪物. the way=how/how much 6)I know where you are from by the way you pronounce my name. 从你叫我名字的音调中,我知道你哪里人. 7)No one can imaine the way he misses her. 人们很想想象他是多么想念她. the way=because 8) No wonder that girls looks down upon me, the way you encourage her. 难怪那姑娘看不起我, 原来是你怂恿的 the way =while/when(表示对比) 9)From that day on, they walked into the classroom carrying defeat on their shoulders the way other students carried textbooks under their arms. 从那天起,其他同学是夹着书本来上课,而他们却带着"失败"的思想负担来上课.
The way的用法及其含义（三） 三、the way的语义 1. the way=as（像） Please do it the way I’ve told you.请按照我告诉你的那样做。 I'm talking to you just the way I'd talk to a boy of my own.我和你说话就像和自己孩子说话一样。 Plant need water the way they need sun light. 植物需要水就像它们需要阳光一样。 2. the way=how（怎样，多么） No one can imagine the way he misses her.没人能够想象出他是多么想念她！ I want to find out the way a volcano has formed.我想弄清楚火山是怎样形成的。 He was filled with anger at the way he had been treated.他因遭受如此待遇而怒火满腔。That’s the way she speaks.她就是那样讲话的。 3. the way=according as （根据） The way you answer the questions, you must be an excellent student.从你回答问题来看，你一定是名优秀的学生。 The way most people look at you, you'd think a trash man was a monster.从大多数人看你的目光中,你就知道垃圾工在他们眼里是怪物。 The way I look at it, it’s not what you do that matters so much.依我看，重要的并不是你做什么。 I might have been his son the way he talked.根据他说话的样子，好像我是他的儿子一样。One would think these men owned the earth the way they behave.他们这样行动，人家竟会以为他们是地球的主人。
一．Way：“方式”、“方法” 1.表示用某种方法或按某种方式 Do it (in) your own way. Please do not talk (in) that way. 2.表示做某事的方式或方法 It’s the best way of studying [to study] English.。 There are different ways to do [of doing] it. 3.其后通常可直接跟一个定语从句(不用任何引导词)，也可跟由that 或in which 引导的定语从句 正：I don’t like the way he spoke. I don’t like the way that he spoke. I don’t like the way in which he spoke.误：I don’t like the way how he spoke. 4. the way 的从句 That’s the way (=how) he spoke. I know where you are from by the way you pronounce my name. That was the way minority nationalities were treated in old China. Nobody else loves you the way(=as) I do. He did not do it the way his friend did. 二．固定搭配 1. In a/one way：In a way he was right. 2. In the way /get in one’s way I'm afraid your car is in the way， If you are not going to help，at least don't get in the way. You'll have to move－you're in my way. 3. in no way Theory can in no way be separated from practice. 4. On the way (to……) Let’s wait a few moments. He is on the way Spring is on the way. Radio forecasts said a sixth-grade wind was on the way. She has two children with another one on the way. 5. By the way By the way，do you know where Mary lives? 6. By way of Learn English by way of watching US TV series. 8. under way 1. Elbow one’s way He elbowed his way to the front of the queue. 2. shoulder one’s way 3. feel one‘s way 摸索着向前走；We couldn’t see anything in the cave, so we had to feel our way out 4. fight/force one’s way 突破。。。而前进The surrounded soldiers fought their way out. 5.. push/thrust one‘s way（在人群中）挤出一条路He pushed his way through the crowd. 6. wind one’s way 蜿蜒前进 7. lead the way 带路，领路；示范 8. lose one‘s way 迷失方向 9. clear the way 排除障碍，开路迷路 10. make one’s way 前进，行进The team slowly made their way through the jungle.
在the way+从句中, the way 是先行词, 其后是定语从句.它有三种表达形式:1) the way+that 2)the way+ in which 3)the way + 从句(省略了that或in which),在通常情况下, 用in which 引导的定语从句最为正式,用that的次之,而省略了关系代词that 或in which 的, 反而显得更自然,最为常用.如下面三句话所示,其意义相同. I like the way in which he talks. I like the way that he talks. I like the way he talks. 如果怕弄混淆，下面的可以不看了 另外，在当代美国英语中,the way用作为副词的对格,"the way+从句"实际上相当于一个状语从句来修饰全句. the way=as 1)I'm talking to you just the way I'd talk to a boy of my own. 我和你说话就象和自己孩子说话一样. 2)He did not do it the way his friend did. 他没有象他朋友那样去做此事. 3)Most fruits are naturally sweet and we can eat them just the way they are ----all we have to do is clean or peel them . 大部分水果天然甜润,可以直接食用,我们只需要把他们清洗一下或去皮. the way=according to the way/judging from the way 4)The way you answer the qquestions, you must be an excellent student. 从你回答就知道,你是一个优秀的学生. 5)The way most people look at you, you'd think a trashman was a monster. 从大多数人看你的目光中,你就知道垃圾工在他们眼里是怪物. the way=how/how much 6)I know where you are from by the way you pronounce my name. 从你叫我名字的音调中,我知道你哪里人. 7)No one can imaine the way he misses her. 人们很想想象他是多么想念她. the way=because 8) No wonder that girls looks down upon me, the way you encourage her. 难怪那姑娘看不起我, 原来是你怂恿的 the way =while/when(表示对比) 9)From that day on, they walked into the classroom carrying defeat on their shoulders the way other students carried textbooks under their arms.
“the way+从句”结构的意义及用法 首先让我们来看下面这个句子： Read the following passage and talk about it with your classmates. Try to tell what you think of Tom and of the way the children treated him. 在这个句子中，the way是先行词，后面是省略了关系副词that 或in which的定语从句。 下面我们将叙述“the way+从句”结构的用法。 1．the way之后，引导定语从句的关系词是that而不是how,因此，<<现代英语惯用法词典>>中所给出的下面两个句子是错误的：This is the way how it happened. This is the way how he always treats me. 2. 在正式语体中，that可被in which所代替；在非正式语体中，that则往往省略。由此我们得到the way后接定语从句时的三种模式：1) the way +that-从句2) the way +in which-从句3) the way +从句 例如：The way(in which ,that) these comrades look at problems is wrong.这些同志看问题的方法不对。
The way(that ,in which)you’re doing it is completely crazy.你这么个干法，简直发疯。 We admired him for the way in which he faces difficulties. Wallace and Darwin greed on the way in which different forms of life had begun.华莱士和达尔文对不同类型的生物是如何起源的持相同的观点。 This is the way (that) he did it. I liked the way (that) she organized the meeting. 3.the way(that)有时可以与how(作“如何”解)通用。例如： That’s the way (that) she spoke. = That’s how she spoke. I should like to know the way/how you learned to master the fundamental technique within so short a time. 4．the way的其它用法：以上我们讲的都是用作先行词的the way，下面我们将叙述它的一些用法。
定冠词the的12种用法 定冠词the 的12 种用法，全知道?快来一起学习吧。下面就和大家分享，来欣赏一下吧。 定冠词the 的12 种用法，全知道? 定冠词the用在各种名词前面，目的是对这个名词做个记号，表示它的特指属性。所以在词汇表中，定冠词the 的词义是“这个，那个，这些，那些”，可见，the 即可以放在可数名词前，也可以修饰不可数名词，the 后面的名词可以是单数，也可以是复数。 定冠词的基本用法： (1) 表示对某人、某物进行特指，所谓的特指就是“不是别的，就是那个!”如： The girl with a red cap is Susan. 戴了个红帽子的女孩是苏珊。 (2) 一旦用到the，表示谈话的俩人都知道说的谁、说的啥。如：
The dog is sick. 狗狗病了。(双方都知道是哪一只狗) (3) 前面提到过的，后文又提到。如： There is a cat in the tree.Thecat is black. 树上有一只猫，猫是黑色的。 (4) 表示世界上唯一的事物。如： The Great Wall is a wonder.万里长城是个奇迹。(5) 方位名词前。如： thenorth of the Yangtze River 长江以北地区 (6) 在序数词和形容词最高级的前面。如： Who is the first?谁第一个? Sam is the tallest.山姆最高。 但是不能认为，最高级前必须加the，如： My best friend. 我最好的朋友。 (7) 在乐器前。如： play the flute 吹笛子
Way用法 A:I think you should phone Jenny and say sorry to her. B:_______. It was her fault. A. No way B. Not possible C. No chance D. Not at all 说明：正确答案是A. No way，意思是“别想！没门！决不！” 我认为你应该打电话给珍妮并向她道歉。 没门！这是她的错。 再看两个关于no way的例句： （1）Give up our tea break? NO way! 让我们放弃喝茶的休息时间？没门儿！ （2）No way will I go on working for that boss. 我决不再给那个老板干了。 way一词含义丰富，由它构成的短语用法也很灵活。为了便于同学们掌握和用好它，现结合实例将其用法归纳如下： 一、way的含义 1. 路线
He asked me the way to London. 他问我去伦敦的路。 We had to pick our way along the muddy track. 我们不得不在泥泞的小道上择路而行。 2. （沿某）方向 Look this way, please. 请往这边看。 Kindly step this way, ladies and gentlemen. 女士们、先生们，请这边走。 Look both ways before crossing the road. 过马路前向两边看一看。 Make sure that the sign is right way up. 一定要把符号的上下弄对。 3. 道、路、街，常用以构成复合词 a highway（公路），a waterway（水路），a railway（铁路），wayside（路边）
way/time的特殊用法 1、当先行词是way意思为”方式.方法”的时候,引导定语从句的关系词有下列3种形式： Way在从句中做宾语 The way that / which he explained to us is quite simple. Way在从句中做状语 The way t hat /in which he explained the sentence to us is quite simple. 2、当先行词是time时,若time表示次数时,应用关系代词that引导定语从句,that可以省略; 若time表示”一段时间”讲时,应用关系副词when或介词at/during + which引导定语从句 1.Is this factory _______ we visited last year? 2.Is this the factory-------we visited last year? A. where B in which C the one D which 3. This is the last time _________ I shall give you a lesson. A. when B that C which D in which 4.I don’t like the way ________ you laugh at her. A . that B on which C which D as 5.He didn’t understand the wa y ________ I worked out the problem. A which B in which C where D what 6.I could hardly remember how many times----I’ve failed. A that B which C in which D when 7.This is the second time--------the president has visited the country. A which B where C that D in which 8.This was at a time------there were no televisions, no computers or radios. A what B when C which D that