Influence of Lysobacter enzymogenes Strain C3 on Nematodes

Influence of Lysobacter enzymogenes Strain C3on Nematodes J.C hen,1W.H.M oore,1G.Y.Y uen,2D.K obayashi,3E.P.C aswell-C hen1

Abstract:Chitinolytic micro?ora may contribute to biological control of plant-parasitic nematodes by causing decreased egg viability through degradation of egg shells.Here,the in?uence of Lysobacter enzymogenes strain C3on Caenorhabditis elegans,Heterodera schachtii,Meloidogyne javanica,Pratylenchus penetrans,and Aphelenchoides fragariae is described.Exposure of C.elegans to L.enzymogenes strain C3on agar resulted in almost complete elimination of egg production and death of94%of hatched juveniles after2d.Hatch of H.schachtii eggs was about50%on a lawn of L.enzymogenes strain C3on agar as compared to80%on a lawn of E.coli.Juveniles that hatched on a lawn of L.enzymogenes strain C3on agar died due to disintegration of the cuticle and body contents.Meloidogyne javanica juveniles died after4d exposure to a7-d-old chitin broth culture of L.enzymogenes strain C3.Immersion of A.fragariae,M. javanica,and P.penetrans juveniles and adults in a nutrient broth culture of L.enzymogenes strain C3led to rapid death and disintegration of the nematodes.Upon exposure to L.enzymogenes strain C3cultures in nutrient broth,H.schachtii juveniles were rapidly immobilized and then lysed after three days.The death and disintegration of the tested nematodes suggests that toxins and enzymes produced by this strain are active against a range of nematode species.

Key words:Aphelenchoides fragariae,biological control,Caenorhabditis elegans,chitinase-producing bacterium,Heterodera schachtii, Lysobacter enzymogenes,lytic,Meloidogyne javanica,Pratylenchus penetrans,nematode management,plant-parasitic nematodes

The egg shell of nematodes contains chitin (Maggenti,1981;Bird and Bird,1991),and it is pre-sumed that exposing eggs to chitinase can disrupt egg hatch.Both chitinase and chitinase-producing bacteria have been considered for use in reducing numbers of plant-parasitic nematodes in soil(Spiegel et al.,1986, 1987;Mercer et al.,1992;Cronin et al.,1997;Chen et al.,1999).

The genus Lysobacter is in the family Xanthomonada-ceae(?-subdivision of Proteobacteria)and is a genus of gliding and non-fruiting bacteria(Christensen and Cook,1978;Christensen,1984).Lysobacter species are typically found in soil and water habitats and are char-acterized by having gliding motility,high G+C content, and lytic activity against other microorganisms(Chris-tensen and Cook,1978).Lysobacter enzymogenes degrades chitin and produces lipases and proteases(Katznelson et al.,1964;Christensen and Cook,1978;Sullivan et al., 2003).“Myxobacteria”strains,which were later placed in L.enzymogenes(Christensen and Cook,1978),were reported to lyse bacteria-feeding nematodes but not the stylet-bearing nematodes Aphelenchus avena e and Het-erodera trifolii(Katznelson et al.,1964);the lytic activity, however,was not investigated further.Lysobacter enzymo-genes strain C3,which was repositioned from Stenotroph-omonas maltophilia(Sullivan et al.,2003),was found to be effective in the biological control of several fungal pathogens(Zhang and Yuen,1999;Yuen and Zhang, 2001;Yuen et al.,2001).Suppression of fungal patho-gens by L.enzymogenes strain C3was attributed in part to chitinase production(Zhang and Yuen,2000a,2000b; Zhang et al.,2001).The research described here was initiated to investigate the effect of L.enzymogenes strain C3on nematode eggs because the bacteria produce chitinases and other potentially antagonistic enzymes and compounds.Our observations led to an assessment of effects on vermiform stages of different nematode species.

Here we report on the effects of L.enzymogenes strain C3on the reproduction and viability of the bacteria-feeding nematode Caenorhabditis elegans.Caenorhabditis elegans has been used as a model system to identify bac-terial virulence factors(Kurz and Ewbank,2000).We also examined the in?uence of L.enzymogenes strain C3 on the survival of egg and juvenile stages of economi-cally important plant-parasitic nematodes,including the sugar-beet cyst nematode Heterodera schachtii,root-knot nematode Meloidogyne javanica,root-lesion nema-tode Pratylenchus penetrans,and foliar nematode Aphel-enchoides fragariae.

M aterials and M ethods

Bacteria:A spontaneous rifampicin-resistant deriva-tive of L.enzymogenes strain C3was used in these experi-ments(Giesler and Yuen,1998).Escherichia coli strain OP50,the uracil auxotroph used as food for rearing C. elegans,was obtained from the Caenorhabditis Genetic Center(CGC),University of Minnesota,St.Paul,and was included as a control treatment.Cultures of both bacteria were maintained as cryopreserved stock sus-pensions at-80°C(Stiernagle,1999).For these experi-ments,L.enzymogenes strain C3and E.coli strain OP50 were cultured using two different media,agar Nema-tode Growth Medium(NGM)(Brenner,1974)or liq-uid Nutrient Broth(NB)(Becton,Dickinson and Co., Sparks,MD).Lysobacter enzymogenes strain C3was also cultured in a chitin broth medium modi?ed from

Received for publication17November2005.

1Department of Nematology,University of California,Davis,CA95616.

2Department of Plant Pathology,University of Nebraska,Lincoln,NE68583. 3Department of Plant Biology&Pathology,Rutgers,New Brunswick,NJ 08901–8520

This research was supported by funding from the USDA(5306–22000–013–01S)and from a faculty research grant at the University of California,Davis. This manuscript is a contribution of the University of Nebraska Agricultural Research Division,Journal Series No.15008.The C.elegans N2strain and E.coli strain OP50used in this work were provided by the Caenorhabditis Genetics Center,which is funded by the NIH National Center for Research Resources (NCRR).Thanks to John Chitambar of the California Department of Food and Agriculture for nematode cultures,Jennifer Haynes and Kristi Sanchez for assistance in the laboratory,and Bruce Jaffee,Harry Kaya,and two anonymous reviewers for helpful suggestions on the manuscript.

E-mail:epcaswell@https://www.sodocs.net/doc/1c919095.html,

This manuscript was edited by Andrea Skantar Journal of Nematology38(2):233–239.2006.

?The Society of Nematologists2006.

233

Zhang and Yuen (2000b),which induces chitinase pro-duction in strain C3(Zhang and Yuen,2000a).

Chitin broth contained (NH 4)2SO 4at 1.0g/liter,MgSO 4.7H 2O at 0.3g/liter,and KH 2PO 4at 1.4g/liter.Practical-grade chitin (Sigma-Aldrich,St Louis,MO)was added to the solution at 10g/liter as a carbon source.Yeast extract solution (0.5g yeast extract in 50ml distilled water)(Sigma-Aldrich)was autoclaved separately and added to the sterilized broth to a con-centration of 0.5g/liter.

Bacterial lawns were created on NGM agar plates by pipetting 0.1ml thawed stock suspensions onto each petri dish and spreading the bacteria with a sterile glass pipet.Broth cultures were prepared by pipetting 0.1ml thawed stock suspension into 100-ml volumes of sterile broth in 500-ml Erlenmeyer ?asks,which were incu-bated at 26°C with shaking at 125rpm (Innova 4330Refrigerated incubator shaker,New Brunswick Scien-ti ?c,Edison,NJ).Chitin broth cultures of L.enzymogenes strain C3were inoculated 7d before the start of an experiment and were sieved through a 25-μm-pore sieve to remove larger chitin particles prior to use.Nematodes:The wild-type C.elegans strain N2(Bristol)was obtained from the Caenorhabditis Genetics Center.It was cryopreserved at ?80°C,and cultures were initi-ated for each experiment by pipetting 0.1ml cryopre-served culture onto NGM agar seeded with OP50.Nematode cultures were incubated in the dark for 3d,and the freshly laid eggs were transferred onto fresh NGM with OP50.The adults that subsequently devel-oped were used in the experiments.

An isolate of H.schachtii from Half Moon Bay,CA,was maintained on sugarbeets and cabbage in a green-house.Cysts were rinsed from roots,?oated from soil,and collected on sieves.When eggs were needed,they were removed from cysts by gently tearing open the cyst walls using forceps and immersing the cyst contents in 5%commercial bleach for 3min.Second-stage juve-niles were collected from cysts placed on Baermann funnels for 3d.

Cultures of A.fragariae and P.penetrans were obtained from the California Department of Food and Agricul-ture and maintained on carrot discs in jars.Juvenile and adult stages were recovered by adding water to the jar and swirling it around to collect nematodes loos-ened from the carrot surface.Juveniles of M.javanica were obtained from hydroponics cultures (Lambert et al.,1992).Eggs were obtained by bleach extraction (Barker,1985)of infected tomato roots obtained from plants grown in greenhouses.

Arti?cial tap water (ATW)(Greenaway,1970)was used to rinse nematodes and was included in some of the experiments as a control treatment.Petri dishes and cell culture plates containing bacteria and nema-todes were sealed with para?lm (Pechiney Plastic Pack-aging,Menasha,WI)to limit moisture loss and main-tained in a dark incubator at 25°C.Microscopic obser-vations of nematodes were made at x40magni?cation.Caenorhabditis elegans experiments:Adult C.elegans from NGM-E.coli OP50cultures were placed in ?lter-sterilized tap water on NGM to reduce bacteria adher-ing to the nematode cuticle.A young or gravid adult nematode was then transferred onto NGM on which C3had been cultured for 1,3,or https://www.sodocs.net/doc/1c919095.html,ing a separate set of L.enzymogenes strain C3cultures on NGM,bacterial numbers were determined by rinsing bacterial cells off the agar surface with ATW and plating dilutions of the rinse on NGM.Populations were expressed as log 10colony-forming units (CFU)per plate.Caenorhabditis el-egans were also transferred to 1-,3-and 5-d-old NGM cultures of E.coli OP50as the controls.The develop-ment,reproduction,and survival of C.elegans were de-termined daily.Nematode body length was measured to assist in the identi?cation of developmental stages of C.elegans .Each treatment was replicated three times,and the experiment was repeated.

A separate set of experiments was conducted as a comparative control for con?rming the effects of com-mercial chitinase on C.elegans.Chitinase (EC 3.2.1.14)from Serratia marcescens was obtained from Sigma-Aldrich (St Louis,MO).Chitinase prepared in 0.05M potassium phosphate buffer was added after the auto-claved NGM agar was allowed to cool to 55°C so that the chitinase would remain stable (Brurberg et al.,1996).Final chitinase concentrations were 0,0.021,and 0.21units/ml.Each agar-chitinase mix was pipetted into wells (0.8ml/well)of 48-well cell-culture plates and into 6-cm-diam.petri dishes (2ml/dish),and the hard-ened agar was inoculated with E.coli OP50.In one ex-periment,one newly hatched juvenile of C.elegans was transferred into each of 12replicate wells in a cell-culture plate containing 1-d-old cultures of E.coli OP50.The plates were incubated at 24°C ±1°C.The nema-todes were examined daily and the juveniles counted over 7d.When the progeny that arose from the origi-nal nematode placed in a well reached maturity,the original nematode was transferred into a fresh well con-taining the same chitinase concentration.In this man-ner,the total number of ?rst-generation progeny from the original nematode could be determined.In an-other experiment,a juvenile nematode was placed in a petri dish containing a 1-d-old culture of OP50.There were three replicate cultures per chitinase concentra-tion.These were incubated at 20°C ±1°C for 7d with-out any nematodes being removed.Progeny produc-tion and population numbers of C.elegans were assessed by suspending the nematodes on the plate in ATW,diluting the suspension to less than 200nematodes/ml,and counting the nematodes in 1ml of a subsample using a dissecting microscope.

Meloidogyne javanica in C3chitin broth cultures:The in?uence of L.enzymogenes strain C3grown in chitin broth on juveniles of M.javanica was evaluated with

234Journal of Nematology,Volume 38,No.2,June 2006

ATW as the control treatment.Aliquots of0.9ml of a 7-d-old L.enzymogenes strain C3culture and ATW were added to wells of48-well cell-culture plates.There were eight replicate wells for each treatment.Nematodes were added to each well in a0.1ml ATW suspension containing23to37nematodes.After the nematodes were incubated in the treatments for4d,the contents of each well were removed,diluted with9.0ml ATW, and incubated for an additional day in a60-×15-mm petri dish.Nematode survival was assessed based on the percentage of nematodes moving.The experiment was performed twice,once with a fresh culture of L.enzy-mogenes strain C3and the second time with the same culture of L.enzymogenes strain C3that had been stored for16d at4°C prior to use.

Plant-parasitic nematodes in C3NB cultures:These ex-periments tested the effects of L.enzymogenes strain C3 on various plant-parasitic nematodes by exposing nematodes to the following treatments:ATW;sterile NB;NB culture of E.coli OP50;NB culture of L.enzy-mogenes strain C3;and NB culture of C3?ltered using a 0.22-μm Millipore?lter to remove bacterial cells.Three day old bacterial cultures were used.Experiments were carried out in24-well cell-culture plates(Falcon),with 0.9ml aliquots of each treatment dispensed into each of?ve replicate wells.

Separate experiments were conducted twice for A. fragariae,P.penetrans,and M.javanica juveniles.For the experiments involving vermiform stages,approximately 10nematodes were transferred into each well.Nema-todes were counted daily and scored as active,inactive, or missing.The results of the two experiments were similar,so the data from the two experiments were combined for presentation.

Effects of L.enzymogenes C3on H.schachtii egg hatch:An experiment was conducted to investigate the in?uence of L.enzymogenes strain C3on H.schachtii egg hatch. Eggs suspended in?lter-sterilized tap water were pipet-ted onto the surface of NGM with established1-d-old lawns of L.enzymogenes C3or E.coli OP50.One and2ml ?lter-sterilized tap water was added to the NGM cul-tures on d0and5,respectively,to maintain the mois-ture level necessary for hatching.The effects of L.en-zymogenes strain C3on eggs and the numbers of eggs hatched were examined10d later.Each treatment was replicated three times and the experiment was con-ducted twice.

Effects of NB concentration on C3activity:Separate ex-periments were conducted to evaluate the effects of L. enzymogenes strain C3on juvenile stages of H.schachtii when the bacterium was grown in full-and half-strength NB.The half-strength medium was included to reduce the growth rate of L.enzymogenes strain C3.This experi-ment was performed twice,the?rst time using ATW and0.5×NB as control treatments.A mixture of anti-biotics(50units penicillin,0.05mg streptomycin,and 0.1mg neomycin,per ml,Sigma Chemical,St.Louis,MO)was added to restrict the growth of contaminating bacteria in NB.C3was cultured in full-strength and diluted NB for3d,at which time1ml of each treat-ment solution was pipetted into wells(?ve wells per treatment)of a24-well cell-culture plate.Five H. schachtii J2were transferred into each well.The number of active,inactive,or missing juveniles in each well was determined daily.This experiment was repeated.

R esults

Caenorhabditis elegans experiments:The reproduction of C.elegans was inhibited on NGM cultures of L.enzymo-genes C3,and the inhibition was greater in3-and5-d-old cultures than in1-d-old cultures(Fig.1).Shortly after transfer to L.enzymogenes strain C3cultures,the nematodes were observed actively feeding with vigorous metacorpal pumping.After1d of exposure to C3,how-ever,the rate of metacorpal pumping as observed at ×40magni?cation was reduced in adults exposed to C3 compared to adults exposed to OP50.After feeding for 1d on1-and3-d-old C3cultures,nematode adults produced35and3progeny/dish,respectively,com-pared to53and68in the E.coli OP50control.On 5-d-old cultures of C3,there was almost no progeny production,compared to61progeny produced on E. coli OP50.Numbers of C3at the beginning of the ex-periment were approximately107in1-d-old cultures and approximately1011in3-and5-d-old cultures.Cae-norhabditis elegans adults placed on1-d-old C3cultures did not produce eggs or survive into the second day (Table1).None of the juveniles that hatched after d1 developed into adults,and the number of juveniles present declined over time,such that fewer than two juveniles were alive after3d.About28%of adult C.elegans exposed to L.enzymogenes strain C3produced progeny internally,the phenomenon of facultative

vivipary F IG.1.Lysobacter enzymogenes strain C3(C3)bacterial population growth(without nematodes)curve vs.time(dotted line),and the in?uence of the age and population density of L.enzymogenes strain C3on Caenorhabditis elegans strain N2as compared to C.elegans grow-ing on E.coli OP50(solid lines).Numbers of nematode eggs and juveniles were counted1d after placing a single gravid adult on a1-, 3-,or5-d-old C3bacterial lawn.

Lysobacter enzymogenes and Nematodes:Chen et al.235

(Chen and Caswell-Chen,2003).Nematodes developed normally on the control E.coli OP50and produced 65progeny after 1d and too many progeny to count by d 3(data not shown).

While C.elegans juveniles with E.coli OP50on chiti-nase-amended NGM developed,matured,and survived,they produced fewer progeny than those in treatments without chitinase (Fig.2,Table 2).In the experiment conducted in wells of a cell-culture plate,the percent-age of hatched eggs and number of juveniles derived from a single nematode was decreased by chitinase (Fig.2).A similar effect of chitinase occurred in the petri dish experiment (Table 2).

Meloidogyne javanica in C3chitin broth cultures:No ju-veniles of M.javanica survived 4d exposure to L.enzy-mogenes strain C3cultured in chitin broth.There was no difference between broth that was freshly collected and broth that had been stored at 4°C for 16d (Table 3).Plant-parasitic nematodes in C3nutrient broth cultures:Lysobacter enzymogenes strain C3caused adults and juve-

niles of A.fragariae and P.penetrans and juveniles of M.javanica to become inactive and then dissolve (Figs.3,4).A small number of nematodes also became inac-tive in NB and E.coli OP50controls as compared to ATW,but none of the nematodes in the control treat-ments were lysed.The negative effect of NB and E.coli OP50on activity was likely due to bacterial contamina-tion that inevitably arose in the control wells.In a sepa-rate experiment,P.penetrans placed in nutrient broth amended with an antibiotic to prevent bacterial growth remained active longer than those in broth;further-more,nematodes inactivated by contaminating micro-organisms recovered activity when the contaminated broth was diluted 10-fold with water (data not shown).Effects of L.enzymogenes C3on H.schachtii egg hatch:Lysobacter enzymogenes C3growing on NGM agar re-duced hatch of H.schachtii eggs.Hatch of H.schachtii eggs after 10d exposure to C3and E.coli OP50was 49.9%(±3.7SE)and 79.3%(±6.7SE),respectively.Many of the eggs exposed to C3appeared abnormal,and the bodies of many H.schachtii juveniles were in poor condition with internal body organization unrec-ognizable.

Effects of NB concentration on C3activity:None of the H.schachtii juveniles placed in full-and half-strength NB cultures of L.enzymogenes C3were active after 1d (Fig.5).In contrast,nematodes in the ATW and the NB +antibiotics control treatments declined in activity dur-ing the experiment,but at least 20%of the nematodes remained active after 7d.Nematodes in the C3cultures started disintegrating by d 3,with disintegration occur-ring more rapidly in cultures of C3in full-strength NB.All of the nematodes in the controls remained intact throughout the experiments.

T ABLE 3.Percent survival (mean ±SE)of Meloidogyne javanica juveniles in Lysobacter enzymogenes strain C3a .

Treatment

Not stored

Stored for 16d

C3broth

0.0±0.00.0±0.0ATW b (control)

71.0±2.988.2±1.9

a

Nematodes were obtained from hydroponics cultures.Nematodes were ex-posed to broth from bacterial cultures of L.enzymogenes strain C3that was incubated at 26°C for 7d on a shaker (125rpm),with or without subsequent storage for 16d at 4°C.b

Arti ?cial tap water.

T ABLE 1.Reproduction and survival of Caenorhabditis elegans adults in the presence of Lysobacter enzymogenes strain C3.

Day 1a

Day 2

Day 3

L.enzymogenes strain C3Eggs 10.1±0.800Juveniles

18.9±2.6

5.6±1.2

1.3±0.1

Adult survival b +??Control

Eggs +Juveniles 64.5±4.3

NA c NA Adult survival

+

+

+

a

An adult was added to a 1-d-old L.enzymogenes culture dish on d 0(control was E.coli OP50).b

While there were adults alive (+)on d 1,all of them were dead (?)after 2d exposure to L.enzymogenes .c

Not available.Adults continued laying eggs after d 1,and by d 2and 3contained many nematodes of mixed stages that were too numerous to

count.

F I

G .2.Effects of chitinase on egg production and hatch of Cae-norhabditis elegans .One adult was inoculated into a well containing NGM and OP50E.coli with either 0.21,0.021,or 0units of chitinase per ml on d 0.The percentage of egg hatch and number of progeny juveniles per adult through d 5are plotted.Bars indicate standard error (n =12).

T ABLE 2.Effects of exogenous chitinase on reproduction and culture growth (mean ±SE)of Caenorhabditis elegans in vitro on d 7.

Chitinase concentration

Stage a

0.21unit/ml

0unit/ml b

Eggs 519±952,006±301Juveniles 3,413±8789,038±1,519Adults 713±146925±196

a

One newly hatched juvenile was placed into a petri dish on d 0,developed into an adult and produced progeny,resulting in the nematode culture on d 7containing the original adult and all subsequent generations,consisting of adults,eggs,and juveniles (n =3).

236Journal of Nematology,Volume 38,No.2,June 2006

D iscussion

This research was initiated to investigate the in ?u-ence of L.enzymogenes strain C3on nematodes,and the early emphasis was to determine whether it could kill nematode eggs via its production of chitinase.Lysobacter enzymogenes strain C3reduced the survival of both eggs and juveniles of the bacteria-feeding nematode C.el-

egans and the plant-parasitic nematode H.schachtii ,ju-veniles of M.javanica,and vermiform stages of P.pen-etrans and A.fragariae .It suppressed reproduction of C.elegans and killed adult and juvenile nematodes,and many eggs with abnormalities were observed.We dis-covered that the culture medium was important relative to nematicidal activity because L.enzymogenes strain C3grown in nutrient broth lysed juveniles of M.javanica and vermiform stages of P.penetrans and A.fragariae .It is evident that L.enzymogenes strain C3is capable of killing,disintegrating,and dissolving several species of plant-parasitic nematodes in vitro.

Strains of L.enzymogenes studied by Katznelson et al.(1964)killed bacteria-feeding nematodes but not stylet-bearing nematodes.The difference in results between studies could be related to differences among bacterial strains in production of speci ?c metabolites.Strain dif-ferences are known to occur in L.enzymogenes,such as in chitinase production (Folman et al.,2003)and in-duction of plant resistance to fungal pathogens (Kilic-Ekici and Yuen,2003).The effects of C3appeared to differ slightly among nematode species,with lysis by C3appearing most severe on M.javanica and least severe on P.penetrans .This may be due to differences in cuticle structure among nematode species or because our P.penetrans cultures contained both juveniles and adults.The suppression of nematode activity in control treatments of NB and of E.coli OP50was likely due to bacterial contamination that always occurred in

the

F I

G .3.Effects of Lysobacter enzymogenes strain C3grown in nutrient broth on (A)Aphelenchoides fragariae juveniles and adults;(B)Prat-ylenchus penetrans juveniles and adults;(C)Meloidogyne javanica juve-niles.Data presented are the number of nematodes that were not disintegrated after exposure to the treatments for 2d.ATW =arti ?-cial tap water;NB =nutrient broth;OP50=E.coli strain OP50;C3=Lysobacter enzymogenes strain C3;C3?lter =Lysobacter enzymogenes strain C3culture ?ltrate.Bars indicate standard

error.

F I

G .4.Photomicrograph of Pratylenchus penetrans placed in ATW for 48hr (A)and the lysed remains of P.penetrans following 48-hr exposure to ?uid from a NB culture of Lysobacter enzymogenes strain C3(B).

Lysobacter enzymogenes and Nematodes:Chen et al.237

control NB wells.Nematodes may lose activity tempo-rarily in contaminated NB and in E.coli OP50cultures,possibly due to stress,such as anoxia,or toxic com-pounds produced by contaminating microbes.The loss of nematode activity in C3cultures may also be in ?u-enced in part by anoxia,but nematodes disintegrated only in the presence of C3,indicating that the in ?u-ence of C3on nematode activity and viability is the result of toxic and lytic compounds.

In nematodes,chitin occurs as the second layer of the egg shell (Maggenti,1981;Bird and Bird,1991),so exposure of eggs to chitinase could be the mechanism for a decrease in egg viability as observed here.Al-though exogenous chitinase may in ?uence eggs depos-ited in the environment,it might also in ?uence egg production within the adult.Our observations that chitinase decreases C.elegans hatching and egg produc-tion suggest that C3might possibly exert greater in ?u-ence on eggs than revealed through assessment of egg hatch alone.

Although chitinase may act on nematode egg shells,

chitin is not a constituent of the nematode cuticle.Thus,the lethal effects of L.enzymogenes strain C3on all C.elegans stages,juveniles of H.schachtii and M.ja-vanica ,and vermiform stages of P.penetrans and A.fra-gariae indicate that other metabolites produced by L.enzymogenes strain C3,such as proteases and lipases (Zhang and Yuen,2000b),might also be responsible for the toxicity of L.enzymogenes strain C3to nematodes.The dramatic lysis of vermiform nematodes observed when L.enzymogenes strain C3was cultured in NB was not observed during culture in chitin broth.This sug-gests that manipulation of the resources supplied to L.enzymogenes strain C3can in ?uence its activity against nematodes.The activity of culture ?ltrates against M.javanica was not lost after storage for 16d at 4°C,an important consideration if C3were used to produce bioactive compounds that would be stored prior to use.In summary,the antagonistic effects of L.enzymogenes strain C3against nematodes in vitro include reduced reproduction and hatching in C.elegans and H.schachtii and,when cultured in nutrient broth,rapid lysis of vermiform stages of H.schachtii,M.javanica ,P.pen-etrans ,A.fragariae,and C.elegans .A rich NB culture medium for culturing C3resulted in a lytic capacity while other media did not,indicating that manipulat-ing bacterial nutrition may in ?uence L.enzymogenes ac-tivity against nematodes.It will be important to deter-mine the relevance of the observations described here to the more realistic conditions of nematodes infecting plants in soil.

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必须.一定（法律.法规等）动 词原形 2. Women who are exposed to second-hand smoke during their pregnancy can be at risk of abortion.能，会，可以(表示有能力或机会) 动词原形 二、常考情态动词的关键用法 (一) can 1. －Jim，it is time you went to bed．You need to get up early tomorrow. －It’s not f air，Mary can stay up till ten hut I have to go to bed at eight. 可以(表示允许) it were not for the fact that she can't sing，I would invite her to the party. 能，会，可以(表示有能力) 3. Luckily, iron can be reworked and mistakes don't have to be thrown away 能够，可以(表示某事物的特点) can be really difficult to get along with at times even though he’s a

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lie—lied—lied—lying （vt．＆．vi．）（撒谎）lie—lay—lain—lying （vi．）（躺下，位于）lay—laid—laid—laying （vt．＆vi．）（平放、产卵） 【口诀记忆】

撒谎lie,lied,lied,don't be a liar; 一“赖”到底是说谎（发音都是【lai】） 躺lie,lay,lain, lie in bed again; 三个不一样是平躺（原型，过去式，过去分词都不一样）

下蛋 lay,laid,laid,a hen laid an egg; 一“累”到底是下蛋（发音都是【lei】） 放置lay,laid,laid laid it in the bag. 下蛋不就是把蛋放置好嘛，所以变法跟下蛋完全一样。

tell a lie (opp) tell the truth lie to sb; lie on one's back How do they lie to each other? The book lay open on the desk.

A bright future lies ahead. He lay on his back. The trouble lies here. Japan lies to the east of China. An oil pipes is being laid between the two cities.

The hunters laid a trap for the tiger. Rainstorms have laid crops. Laying eggs is its full time job. She always lays her books on the shelf.

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比较ｍay和might 1）表示允许或请求;表示没有把握的推测;mａｙ放在句首,表示祝愿。 May Gｏd bｌｅss yｏu！ Ｈｅmiｇht be aｔhome． 注意：might 表示推测时,不表示时态。只是可能性比may 小。 2) 成语: may/might as well,后面接不带to 的不定式，意为"不妨"。 Ifｔhａt is thｅcase, we may ａs ｗell try. 典型例题 Ｐetｅｒ_＿＿come wｉｔh us tonighｔ，but ｈe iｓn't ｖery sｕrｅyet. A. mｕstB．ｍayC. canD. wｉll 答案Ｂ. 表可能性只能用may.此句意可从后半句推出。 比较hａｖe ｔo和mｕst 1)两词都是＇必须＇的意思，ｈave tｏ表示客观的需要，ｍｕｓｔ表示说话人主观上的看法，既主观上的必要。 My ｂroｔher ｗas ｖery ilｌ, ｓo Ｉhａd toｃaｌl tｈe docｔｏr iｎtｈｅmiddlｅof tｈｅniｇhｔ.我弟弟病得很厉害，我只得半夜里把医生请来。(客观上需要做这件事） He said that theyｍust wｏrｋｈａrd．他说他们必须努力工作。(主观上要做这件事) 2)hａve to有人称、数、时态的变化,而must只有一种形式。但must 可用于间接引语中表示过去的必要或义务。 Hｅｈaｄtｏlｏok after ｈｉｓｓisterｙｅsｔerday． 3) 在否定结构中：dｏn＇t hａvｅto表示"不必" mustｎ＇t表示"禁止"， You ｄｏn'ｔhａvｅto tell hiｍabout iｔ．你不一定要把此事告诉他。 Ｙｏu mustn＇ｔｔｅlｌhim aboｕt ｉt.你一定不要把这件事告诉他。 must表示推测 1)muｓｔ用在肯定句中表示较有把握的推测，意为"一定＂。

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