Antagonistic rhizoplane bacteria induce diverse morphological alterations in Peronosporomycete hyphae during in vitro interaction Abhinandan Deora, Yasuyuki Hashidoko * , Md. Tofazzal Islam and Satoshi Tahara Laboratory of Ecological Chemistry, Division of Applied Bioscience, Graduate School of Agriculture, Hok- kaido University, 060-8589, Kita-Ku, Sapporo, Japan; *Author for correspondence (Phone +81-11-706-3839; Fax +81-11-706-4182; E-mail: [email protected]) Accepted 31 March 2005 Key words: Aphanomyces cochlioides, bacterial metabolites, dual culture assay, hyphae, Pseudomonas sp., zoospores Abstract A total of 150 bacteria were isolated from rhizoplanes of the host and non-host plants of a phytopathogenic Peronosporomycete Aphanomyces cochlioides. Upon screening, 5% of the isolates were evaluated as antagonists as they inhibited radial growth of A. cochlioides AC-5 hyphae in a dual culture assay. In addition, those antagonistic bacteria also induced characteristic morphological alterations in the A. cochlioides AC-5 hyphae that grew towards bacterial colonies. Hyphal morphological alterations ob- served in AC-5 and other tested strains of Peronosporomycetes included excessive branching, curly growth, unusually longer and pointed tip formation and swelling; all of these were comparable to the alterations induced by known antimicrobial compounds. Among the antagonistic bacteria, Pseudomonas sp. strain EC- S101 induced a unique branching pattern (tree-like) in AC-5 hyphae by continuous apical bifurcation of successive hyphae, where increases in number of branches and hyphal area were linearly correlated with time up to 10 h. Our observations suggested that the pathogen might have lost its ability of normal branch production; however maintained the capability of self-branching. Soluble extracts from the culture fluids of Pseudomonas sp. strain EC-S101 and Stenotrophomonas maltophilia EC-S105 induced similar excessive branching and curly growth in A. cochlioides hyphae as the respective bacterium. These results revealed that bacterial metabolites appeared to be responsible for induction of morphological alterations. Interestingly, the antagonistic bacteria that induced hyphal morphological alterations, also efficiently suppressed in vivo damping-off disease caused by AC-5. We suggest that antagonistic rhizoplane bacteria have the capability to induce diverse morphological alterations in Peronosporomycetes hyphae during in vitro interactions. Hyphal morphological alterations associated with growth inhibition and the induction of characteristic morpho- logical changes indicate antagonistic activity against the Peronosporomycete. Introduction The rhizoplane is considered as a suitable source of antagonistic microorganisms for the biocontrol of soilborne phytopathogens. One of the bioas- says used for screening against the phytopatho- gens is conventional in vitro dual culture agar assay. Although synthetic antimicrobial com- pounds have been found to induce morphological alterations in the hyphae of fungal pathogens along with radial growth inhibition (Kang et al., 2001), no previous screening studies using dual culture assay have considered whether antago- nistic bacteria could alter the morphology of European Journal of Plant Pathology (2005) 112:311–322 Ó Springer 2005 DOI: 10.1007/s10658-005-4753-4
Transcript
Antagonistic rhizoplane bacteria induce diverse morphological alterations
in Peronosporomycete hyphae during in vitro interaction
Abhinandan Deora, Yasuyuki Hashidoko*, Md. Tofazzal Islam and Satoshi TaharaLaboratory of Ecological Chemistry, Division of Applied Bioscience, Graduate School of Agriculture, Hok-kaido University, 060-8589, Kita-Ku, Sapporo, Japan; *Author for correspondence(Phone +81-11-706-3839; Fax +81-11-706-4182; E-mail: [email protected])
A total of 150 bacteria were isolated from rhizoplanes of the host and non-host plants of a phytopathogenicPeronosporomycete Aphanomyces cochlioides. Upon screening, 5% of the isolates were evaluated asantagonists as they inhibited radial growth of A. cochlioides AC-5 hyphae in a dual culture assay. Inaddition, those antagonistic bacteria also induced characteristic morphological alterations in theA. cochlioides AC-5 hyphae that grew towards bacterial colonies. Hyphal morphological alterations ob-served in AC-5 and other tested strains of Peronosporomycetes included excessive branching, curly growth,unusually longer and pointed tip formation and swelling; all of these were comparable to the alterationsinduced by known antimicrobial compounds. Among the antagonistic bacteria, Pseudomonas sp. strain EC-S101 induced a unique branching pattern (tree-like) in AC-5 hyphae by continuous apical bifurcation ofsuccessive hyphae, where increases in number of branches and hyphal area were linearly correlated withtime up to 10 h. Our observations suggested that the pathogen might have lost its ability of normal branchproduction; however maintained the capability of self-branching. Soluble extracts from the culture fluids ofPseudomonas sp. strain EC-S101 and Stenotrophomonas maltophilia EC-S105 induced similar excessivebranching and curly growth in A. cochlioides hyphae as the respective bacterium. These results revealed thatbacterial metabolites appeared to be responsible for induction of morphological alterations. Interestingly, theantagonistic bacteria that induced hyphal morphological alterations, also efficiently suppressed in vivodamping-off disease caused by AC-5. We suggest that antagonistic rhizoplane bacteria have the capability toinduce diverse morphological alterations in Peronosporomycetes hyphae during in vitro interactions. Hyphalmorphological alterations associated with growth inhibition and the induction of characteristic morpho-logical changes indicate antagonistic activity against the Peronosporomycete.
Introduction
The rhizoplane is considered as a suitable sourceof antagonistic microorganisms for the biocontrolof soilborne phytopathogens. One of the bioas-says used for screening against the phytopatho-gens is conventional in vitro dual culture agar
assay. Although synthetic antimicrobial com-pounds have been found to induce morphologicalalterations in the hyphae of fungal pathogensalong with radial growth inhibition (Kang et al.,2001), no previous screening studies using dualculture assay have considered whether antago-nistic bacteria could alter the morphology of
European Journal of Plant Pathology (2005) 112:311–322 � Springer 2005DOI: 10.1007/s10658-005-4753-4
hyphae during growth inhibition. Therefore, wescreened antagonistic bacteria on the basis ofhyphal growth inhibition that is associated withinduction of morphological alterations in somePeronosporomycete pathogens during interac-tions in a dual culture agar assay.
The tested organism selected during initialscreening was a Peronosporomycete Aphanomycescochlioides AC-5, causing a damping-off diseasein spinach, sugar beet and some other membersof Chenopodiaceae and Amaranthaceae. Anadditional advantage of selecting A. cochlioides, asoilborne pathogen, is that the hyphae of thispathogen act as a zoosporangium, from whichzoospores are released. Upon germination, theencysted zoospores penetrate host roots directlyor via appressoria and cause infection (Islam andTahara, 2001). Considerable attention has beenpaid over the last two decades to the biologicalcontrol of the pathogenic Peronosporomycetes.For example, a number of plant extracts (Islamand Tahara, 2001) and bacterial metabolites (deSouza et al., 2003) have been screened for zoo-spore stimulants, repellents, and halting and lysisactivities, thereby targeting only the control ofmotile pathogenic zoospores. Despite the impor-tance of mycelia as the source of zoospore pro-duction, scant efforts have been made to evaluatebiological agents for attacking the mycelia ofPeronosporomycetes, particularly antagonisticbacteria (Thrane et al., 1999). We speculate thatantimicrobial compounds altering the morphol-ogy of the hyphae may also affect zoosporeproduction and vice-versa, thereby decreasingpathogenicity.
Our aims here were to observe induction ofmorphological alterations in the Peronosporomy-cetes associated with growth inhibition in a dualculture assay by antagonistic rhizoplane bacteriaand to compare their effects by using knownantimicrobial chemical compounds. We isolated150 rhizoplane bacteria both from the host andnon-host plants of A. cochlioides and screenedtheir ability to inhibit mycelial growth and alterthe morphology of A. cochlioides hyphae on agarplates. Active strains were further tested for theirantagonistic activity against other Peronosporo-mycetes. The results of in vitro inhibitory activitieswere verified by assessing the disease suppressionability in sugar beet and spinach of Pseudomonassp. strain EC-S101, isolated from spinach, and
Stenotrophomonas maltophilia EC-S105, isolated fromsugar beet under controlled in vivo conditions.
Materials and methods
Isolation of rhizoplane bacteria
Roots of sugar beet (Beta vulgaris) cv. Yuden,spinach (Spinacia oleracea) (unknown cv.),mustard (Brassica campestris) (unknown cv.) andweeds grown in the fields of these crops were col-lected in June, 2003 from Hokkaido Universityexperimental farm, Sapporo and in October, 2003from a vegetable farm at Eniwa-shi, Hokkaido,Japan. Weeds collected from the sugar beet fieldwere Chenopodium album, Equisetum arvense,Solanum nigrum, Polygonum nodosum, Fallopiadumetorum, Stellaria alsine and from the spinachfield were Portulaca oleracea, Capsella bursa-pas-toris, Spergula arvensis, Amaranthus lividus and A.retroflexus. Bacteria were isolated and purified onNutrient Broth (Difco Laboratories, Detroit, MI,USA) solidified with agar (i.e. NA) using aliquotsof root washings as described by Williams andAsher (1996), and were preserved in 20% glycerolat )80 �C. For seed-associated bacteria, spinachcv. Tonic, sugar beet cv. Abendrot, C. album andA. retroflexus seeds were kept under running waterfor 48 h. Imbibed seeds were surface-disinfected by70% EtOH for 1 min followed by 2.5% NaOClsolution for 10–15 min. Sterilized seeds (two seedsof sugar beet and spinach, 10 seeds of C. albumand A. retroflexus) were inoculated in a culturetube containing 10 ml of 1/5 Hoagland�s S mediumsupplemented with 0.3% gellan gum (Hoagland�s Smedium (g l)1): Ca(NO3)2Æ4H2O, 1.18; KNO3,0.505; MgSO4Æ7H2O, 0.493; KH2PO4, 0.272).Culture tubes were then kept in the phytotron(23 �C, 16 h light and 8 h dark). After one month,10 ll of gellan gum medium was taken out using asterile micro-tip from the rhizosphere of individualcrop seedlings and inoculated on NA plates,serially diluted by streak culture. Isolation,purification and preservation were done asmentioned above.
Observation of hyphal morphological alterations
Initially, all bacterial isolates were tested for theirantagonistic activity against A. cochlioides AC-5
on PDA (DifcoTM Potato Dextrose Agar, Becton,Dickinson and Company, Sparks, MD, USA). Theactive isolates selected were further tested to assesstheir specificity against A. cochlioides AC-D (F1),and A. euteiches AE-F3, both on CMA. Pythiumaphanidermatum PA-5, another Peronosporomyc-ete, was tested on PDA medium. Bacteria used indual culture assay were incubated for 24 h at 25 �Con NA. Dual culture assay was done using streak-cultured bacterial colonies and a 6 mm i.d. mycelialplug of the pathogen cut from the edge of an ac-tively growing culture plate with a sterile corkborer, and placed 3 cm apart on the agar plate.Both pathogens and bacteria were inoculated onthe agar plate at the same time, except forP. aphanidermatum, which was inoculated aftertwo days of incubation of the bacteria because ofits rapid growth. Inhibition zones, measured fromtips of approaching hyphae to the edge of thebacterial streak, were recorded after incubation at25 �C for 96 h and 36 h for AC-5 and PA-5,respectively. Both AC-D (F1) and AE-F3 wereincubated at 22 �C for 10 days. Morphologicalcharacters of approaching hyphae were observedevery day under a light microscope (400·, IX70-S1F2, Olympus Optical Co. Ltd., Tokyo, Japan)and images were recorded with a digital camera(CAMEDIA C-3040 Zoom, Olympus Optical Co.Ltd.).
Assay of known antimicrobial compounds
The effects of some commercially available fungi-cides, antibiotics and synthetic compounds weretested using paper discs charged with a series ofconcentrations against A. cochlioides AC-5 on agarplates. Sterile paper discs of 8 mm diameter and 1.5mm thickness (Advantec Toyo, Japan) were used.Cytochalasin A (Sigma-Aldrich, St. Louis, MO,USA), zarilamide (synthesized in the laboratory)and calcium ionophore A23187 (Calbiochem, LaJolla, CA, USA) were dissolved in acetone, whilehymexazol (Tachigaren�) (Hokkai Sankyo, Sapp-oro, Japan) was in acetone–water (1:1). Individualcompounds were charged at 0.025, 0.25, 2.5 and25 lg disc)1, respectively. Control discs werecharged with the solvent alone. Paper discs weredried by evaporating the solvents under vacuum.The resulting discs were then put onto Petri dishescontaining PDA inoculated with a 6 mm diammycelial plugs cut from the edge of an actively
growing A. cochlioides AC-5 colony, placed 2 cmapart and incubated at 25 �C. Culture fluids ofPseudomonas sp. strain EC-S101 and Stenotropho-monas maltophilia EC-S105 were passed through aC18 column (Cosmosil 75C18-PREP, Nacalai Tes-que Inc., Kyoto, Japan), and metabolic substancestrapped on the reversed column chromatographicadsorbent were extracted with 30%, 50%, 80% and100% MeOH (Methanol). All of the four crude ex-tracts obtained were concentrated by rotary evap-oration, loaded on the paper discs and tested againstA. cochlioides AC-5 as described above. Solventalone 30%, 50%, 80% and 100%MeOHwere kept ascontrols. The growth inhibition zone together withmorphological changes of the approaching hyphaewere observed and measured every day.
Identification of active rhizoplane bacteria
For the determination of 16S rDNA sequences ofactive isolates, chromosomal DNA extraction wasdone using Isoplant II (Wako Pure ChemicalIndustries) Kit and quantified using Genequant pro(BiochromLtd., Cambridge, U.K.). The 16S rDNAregion was amplified by PCR using a HotStarTaq(Qiagen, Hilden, Germany) kit and universalprimers 27F and 1525R for the 16S rRNA gene(Weisburg et al., 1991). Thermal cycling was per-formed with Mastercycler� Gradient (Eppendorf,Hamburg,Germany). The thermal profile usedwas:preheating for 15 min of taq polymerase activationat 95 �C, and 30 successive cycles consisting of1 min of denaturation at 94 �C, 1 min of annealingat 56 �C, and 1 min of extension at 72 �C. Aconditioning step of 1 cycle at 72 �C for 10 min wasperformed and finally the reactionmixture was keptafter cooling to 4 �C. PCR direct-sequencing wasdone using Big Dye Terminator v3.1 CycleSequencing Ready Reaction kit (Applied Biosys-tems, Foster City, CA, USA) for the 1.5 kb PCRproduct as the template with the universal primers(1080R, 1112F, 926F and 803R) under the follow-ing conditions: 1 cycle of 96 �C for 5 min, 25 cyclesof 96 �C for 30 s, 50 �C for 15 s and 60 �C for4 min. Labelled PCR-direct mixture were purifiedand analyzed by sequencer (ABI Prism� 310Genetic analyzer, Applied Biosystems) according tothe manufacturer�s instructions. The 16S rDNAsequences of the strains were aligned with referencesequences obtained from the BLASTN database inDDBJ (DNA Data Base of Japan) website.
Time-course observation of hyphal branchingin A. cochlioides AC-5 during interactionwith Pseudomonas sp. strain EC-S101
To record the increases in area and number of tipsper hyphae, we undertook a time-course observa-tion of the in vitro dual culture interaction (day 3)between AC-5 hyphae growing towards thePseudomonas sp. strain EC-S101 by arranging a Petridish under light microscope (400·). Photographsof changes in hyphal growth with a set timeinterval (2 h) were taken with a digital camera upto 10 h. Artifacts to the images were processed byomitting the background using Microsoft� Paint(version 5.1) for estimating the area covered bymycelia on the agar surface. Area of mycelialgrowth was analyzed using a software, AreaMeasure (A. Hongu, MYKA Lab, Ver. 1.00, 1995,Japan) and numbers of tips per hyphae werecounted by visual observations.
Culture of bacteria in liquid medium
Pseudomonas sp. strain EC-S101 andStenotrophomonas maltophilia EC-S105 werecultivated in a 500 ml flask containing 200 ml ofAB minimal medium (g l)1: K2HPO4, 3.0; NaH2
PO4, 1.0; NH4Cl, 1.0; MgSO4Æ7H20, 0.3; KCl, 0.15;CaCl2, 0.01; FeSO4Æ7H2O, 0.0025; saccharose, 5.0)incubated at 25 �C for two weeks with shaking at100 rpm. Culture fluids were centrifuged at12,000 · g for 15 min at 5 �C and the bacterialpellets were washed as described (Bacilio-Jimenezet al., 2003).
Influence of bacterial presence on zoospore release
Zoospores were obtained from A. cochlioides AC-5by washing the mycelia three times with sterileH2O (Islam and Tahara, 2001). At the finalwashing, six different inocula of Pseudomonas sp.EC-S101 were added as water suspensions to themycelial suspension. Bacterial populations addedwere counted by the serial dilution method on NAplates kept at 25 �C overnight. After 16 h ofincubation at 20 �C, the number of zoospores ml)1
of suspension was counted microscopically (Islamet al., 2004). Each treatment was replicated threetimes and data were expressed as the mean
value ± standard error. Data were subjected toanalysis of variance using SPSS version 10.0 (SPSSInc., Chicago, IL, USA). Pairwise significant dif-ferences were tested by Fisher�s LSD-test.
In vivo disease suppression
Bacterial inoculation of seeds, soil used, wateringof seedlings and disease suppression ability by thetested bacteria were conducted as described pre-viously (Islam et al., 2005). Briefly, sterilized seedsof sugar beet cv. Abendrot and spinach cv. Tonicwere coated with 108 CFU seed)1 of Pseudomonassp. strain EC-S101 and Stenotrophomonas malto-philia EC-S105, respectively (Nakayama et al.,1999). Partially dried seeds (four) were sown in acell (4.5 · 4.5 · 4.5 cm3) of a plastic pack (36cells), each containing 30 g of fertilized soil (Forex,Japan), purchased from a nursery. Soil used in theexperiment was sterilized in a hot-air oven (SanyoSterilizer MOV-2125, Japan) at 160 �C for 4 h.The soil was a mixture of litter compost and vol-canic gravel having high capacity to retain themoisture; the porous nature helped in proper res-piration. Seedlings were grown in a growthchamber under controlled conditions (23 �C, 16 hlight and 8 h dark). Seedlings (12 days old) wereinoculated with A. cochlioides AC-5 zoospores atdoses of 1 · 104, 1 · 103, 1 · 102 and 0 seedling)1,respectively. Flooded conditions (about 40 ml ofwater) were maintained immediately afterzoospore inoculation to stimulate infection, andplants were watered every day about 20 ml percell. To compare the efficiency of Pseudomonas sp.strain EC-S101 and Stenotrophomonas maltophiliaEC-S105 to control disease, seeds were treatedwith a conventional fungicide, hymexazol at a doseof 7.5 g kg)1 of seed and an unidentified bacte-rium isolated from C. album that showed no inhi-bition zone and morphological alterations in dualculture assay. The number of healthy seedlings wasrecorded under each treatment at weekly intervalafter two weeks of inoculation of zoospores. Eachtreatment was replicated three times and data wereexpressed as number of healthy seedlings ± stan-dard error. Data were subjected to analysis ofvariance using SPSS version 10.0 (SPSS Inc.,Chicago, IL, USA). Pairwise significant differenceswere tested by Fisher�s LSD-test.
Antagonistic activity against Aphanomycescochlioides AC-5
In the initial screening, 150 bacteria isolated fromthe rhizoplanes of 14 plant species grown in thefield were tested for their ability to alter hyphalmorphology along with growth inhibition ofA. cochlioides AC-5 hyphae in a dual culture assay.
Five percent of the bacterial isolates caused char-acteristic morphological alterations (Figure 1b) inthe AC-5 hyphae growing towards bacterial colo-nies compared to the untreated control (Figure 1t)along with development of clear inhibition zoneson the agar medium (Figure 1a). The 16S rDNAsequence of the active isolates viz. EC-S101,EC-S102, EC-S103, EC-S104, EC-S105, EC-S106,EC-S107 and EC-S108 has been deposited inDDBJ (DNA Database of Japan) under accession
Figure 1. Photomicrographs showing hyphal morphological alterations in Aphanomyces cochlioides hyphae caused by rhizoplane
bacteria/bacterial metabolites (a) to (t). Interactions were studied on PDA, unless medium not specified. (a) Pseudomonas sp. strain
(100%) solubles of Stenotrophomonas maltophilia EC-S105: inhibition zone; (h) Methanol (100%) solubles of Stenotrophomonas
maltophilia EC-S105: curling; (i) Pseudomonas sp. strain EC-S101: early stage; (j) Pseudomonas sp. strain EC-S101: later stage on
CMA; (k) Pseudomonas sp. strain EC-S102: early stage; (l) Pseudomonas sp. strain EC-S102: later stage; (m) Stenotrophomonas
maltophilia EC-S106: curling; (n) Bacillus subtilis EC-S108: gall-like appearance and decrease in normal branching; (o) Bacillus sub-
tilis EC-S108: necrosis and swelling of apex; (p) Bacillus subtilis EC-S108: dense colonization of bacteria to the hyphae; (q) Delftia
sp. EC-S107: longer and pointed tips with irregular growth; (r) Klebsiella oxytoca EC-S104: apical branching and extensive vacuo-
lation; (s) Klebsiella oxytoca EC-S104: apical branching; (t) control. Scale bar – 50 lm.
315
numbers AB190286, AB200255, AB200256,AB200254, AB200253, AB200258, AB200259 andAB200257, respectively. Interestingly, the morpho-logical alterations noted were initiated before thevisible zones of growth inhibition developed in aparticular interaction. For example, Pseudomonassp. strains, EC-S101 and EC-S102 (from spinachand cabbage, respectively) induced excessivebranching in AC-5 hyphae (Figures 1i and k) fromday 1 of the interaction. The relative radial growth ofAC-5 hyphae was lower as compared to untreatedAC-5 hyphae; however, a visible radial growthinhibition zone between AC-5 hyphae and EC-S101 was developed at day 3 (data not shown).
After testing all of four crude MeOH extractsobtained from culture fluids, 100% MeOH solublesof both Pseudomonas sp. strain EC-S101 andStenotrophomonas maltophilia EC-S105 (Fig-ures 1c and g, respectively) induced similar exces-sively branched and curled hyphae (Figures 1dand h, respectively) to that induced in dual cultureby the respective bacterium (Figure 1b and f) whentested towards A. cochlioides AC-5. In the presenceof EC-S101, the approaching hyphae displayed aremarkable increase in branch frequency per unitlength by decreasing the internodal distance (Fig-ure 1b) as compared to that of the control (Fig-ure 1t). On maturity of the mycelium, assemblages
of tree-like branches were observed (Figure 1j). Asimilar pattern of branching was also observed forstrain EC-S102 (Figure 1l).
Interactions with Stenotrophomonas maltophiliaEC-S105 and EC-S106 (from sugar beet andA. retroflexus, respectively), resulted most of thesurface hyphae losing their radial growth andcurling in an anti-clockwise direction (Figures 1fand m). Bacillus subtilis EC-S108 (from Spergulaarvensis) induced overlapping of the hyphae,giving a gall-like appearance (Figure 1n, arrows).The induction of apical branching with swellingof hyphal tips and necrosis (darkened hyphae)were commonly seen (Figure 1o, arrows). Hyphaldiameter frequently varied in the colony. Later,the swarming bacteria colonized thehyphoplane of approaching hyphae (Figure 1p),and finally caused lysis of hyphae (data notshown).
Delftia sp. strain EC-S107 (from spinach roots)caused an increase in branching of hyphae withunusual longer and pointed-tip formation (Fig-ure 1q). In addition, increase in diameter at thebase of emergence of secondary branches wasobserved along with irregular growth directions.At advance stages of interaction with bacteria,extensive vacuolation was evoked (data notshown). Increase in apical branching, mainly in theprimary hyphae, and extensive vacuolation wereobserved in AC-5 on interaction with Klebsiellaoxytoca EC-S103 (Figure 1r) and EC-S104 (Fig-ure 1s), (from A. retroflexus and C. album,respectively).
The morphological alterations in the hyphae ofA. cochlioides occurring during interactionwith bacterial isolates were comparable to thosecaused by antimicrobial compounds againstPeronosporomycetes. An increase in branchingwas observed in the approaching hyphae of AC-5towards the paper disc loaded with cytochalasin A(Figure 2a) and Ca-ionophore A23187 (Figure 2b)at 25 and 2.5 lg disc)1, respectively. In contrast tothe induction of excessive branching, a decrease innormal branching (Figure 2d) appeared in hyphaeclose to hymexazol (at 2.5 lg disc)1), and zarila-mide (at 2.5 lg disc)1). In addition, other inhibi-tory effects such as swelling and extensivevacuolation in the AC-5 hyphae caused by EC-S108 were also observed on treatment with zari-lamide (Figure 2c).
Table 1. Number of bacteria from different plant sources
found to be antagonists against Aphanomyces cochlioides
AC-5 in dual culture assay
Plant source Total number
of bacteria
isolated
Number of isolates
showing
antagonism (%)
Beta vulgaris 22 1 (4)
Spinacia oleracea 20 2 (10)
Brassica campestris 9 1 (11)
Chenopodium album 15 1 (7)
Equisetum arvense 9 0 (0)
Solanum nigrum 5 0 (0)
Polygonum nodosum 6 0 (0)
Fallopia dumetorum 8 0 (0)
Stellaria alsine 7 0 (0)
Portulaca oleracea 7 0 (0)
Capsella bursa-pastoris 5 0 (0)
Spergula arvensis 12 1 (8)
Amaranthus lividus 10 0 (0)
Amaranthus retroflexus 15 2 (13)
Values in the parenthesis indicate percentage of isolates from
each plant species showing antagonism against AC-5.
316
Antagonistic specificity of the active isolatesto A. cochlioides AC-5
Among the eight isolates displaying remarkableinhibitory activities against AC-5, three isolates(EC-S101, EC-S102 and EC-S108) inducedinhibition zones along with morphologicalalterations in Pythium aphanidermatum PA-5.Pseudomonas sp. strain EC-S101 caused an in-crease in branching after 24 h (Figure 2e), but thebranching pattern was less dense than that inducedin AC-5. Pseudomonas sp. strain EC-S102 inducedswelling of hyphae along the length and at tipsafter 24 h (Figure 2f) and extensive vacuolationafter 48 h (Figure 2g). Both excessive branchingand extensive vacuolation were also induced byPseudomonas sp. strain EC-S101 after 48 and 72 h,respectively. Along with bacterial colonization ofthe hyphae (Figure 2h), similar morphologicalalterations (Figure 2i) appeared in PA-5 due to
Bacillus subtilis EC-S108 as those that occurred inAC-5. Testing against A. euteiches AE-F1,EC-S108 caused extensive vacuolation of hyphae(Figure 2j). By comparison, Pseudomonas sp. strainEC-S101 induced excessive branching in AE-F1hyphae similar to that observed in AC-5 (Fig-ure 2k). A similar effect of Pseudomonas sp. strainEC-S101 could be seen on A. cochlioides AC-D (F3)(Figure 2l).
Time-course observation of hyphal branchingin A. cochlioides AC-5 during interactionwith Pseudomonas sp. strain EC-S101
The increase in branching and hyphal area in AC-5hyphae on interaction (day 3) with Pseudomonassp. strain EC-S101 was in a linear correlation withtime up to 10 h (Figure 3). Interestingly, inductionof excessive branching appeared to be in a uniquepattern as the apices of hyphae were bifurcated,
Figure 2. Photomicrographs showing hyphal morphological alterations in Peronosporomycetes caused by rhizoplane bacte-
ria/synthetic compounds. (a) to (d) A. cochlioides AC-5: (a) Cytochalasin A: excessive branching; (b) Calcium ionophore A23187:
excessive branching; (c) Zarilamide: swollen hyphae and suppression of branching; (d) Hymexazol: decrease in normal branching.
(e) to (i) P. aphanidermatum PA-5; (e) Pseudomonas sp. strain EC-S101: apical branching; (f) Pseudomonas sp. strain EC-S102:
swelling; (g) Pseudomonas sp. strain EC-S102: extensive vacuolation; (h) Bacillus subtilis EC-S108: dense colonization of bacteria to
the hyphae; (i) Bacillus subtilis EC-S108: decrease in normal branching, swollen hyphae and necrosis. (j) and (k) AE-F1; (j) Bacillus
unlike the control hyphae where apices did notdivide (Figure 1t). Hyphal branching continued byapical division of new hyphae and finally formed atree-like structure (Figure 4).
Influence of bacterial presence on zoospore release
The number of zoospores released fromA. cochlioides AC-5 mycelia was significantlyreduced (P < 0.001) in proportion to the presenceof different population sizes of Pseudomonas sp.strain EC-S101 (Figure 5) 16 h after the additionof bacterial cells. Remarkably, all of the zoosporeswere encysted in the presence of higher popula-tions of bacteria (6.4 · 108, 2.1 · 108 and7.2 · 107 CFU ml)1). Even in the presence oflower populations of bacteria, zoospores were notas active as those in the control.
In vivo disease suppression
Results of in vivo studies indicated that none of thebacteria had negative effects on seed germination.Additionally, damping-off caused by A. cochlioidesAC-5 infestation was significantly reduced (P <0.001) after treatment with Pseudomonas sp.EC-S101 and Stenotrophomonas maltophilia EC-S105 as the number of healthy seedlings in thebacterial seed treatment was higher as comparedwith the control under different doses of inoculatedzoospores (Table 4). Seedlings free from black andshrink to dark, slender thread hypocotyls or rootswere considered as the healthy seedlings. However,in comparison with the bacterial treatment, thecommercial fungicide hymexazol exerted a strongerdisease suppression in both spinach and sugar beet,especially at the higher doses of zoospores (Table 4).
R2 = 0.99
0
20
40
60
80
100
120
140
0 2 4 6 8 10 12
Incr
ease
in h
ypha
l are
a (m
m2 )
R2 = 0.98
0
20
40
60
80
100
120
0 2 4 6 8 10 12Time (h)
Incr
ease
innu
nber
of
tips
Figure 3. Increase in hyphal tips and area in the A. cochlioides
AC-5 hyphal interaction (day 3) with Pseudomonas sp. strain
EC-S101 (At 0 h, number of tips were 66 and hyphal area was
58.62 mm2).
Figure 4. Time-course changes in the differentiated hyphae of A. cochlioides AC-5 during interaction (day 3) with Pseudomonas sp.
EC-S101.
318
The number of healthy seedlings after treatmentwith unidentified non-antagonistic bacteria fromC.album, was lower (P < 0.001) compared to othertreatments including the control.
Discussion
In the present study, we observed that antagonisticrhizoplane bacteria exerted characteristic
morphological alterations in soilborne Peronosp-oromycetes along with inhibition zone in a dualculture assay. The morphological alterationsinduced by antagonistic bacteria were comparableto those induced by known antimicrobial chemicalcompounds. It appears that the sensitivity ofA. cochlioides to such rhizoplane bacteria is rela-tively higher than that of the other Peronosporo-mycetes tested (Table 3). Since most of theantagonistic bacterial isolates were from the host
Table 2. Classes of morphological alterations induced in A. cochlioides AC-5 hyphae during interaction in dual culture with
antagonisitc bacteria isolated from different plant sources
Bacterial isolate/chemical compound Plant source Class of morphological alterations induced
Branching No branching Swelling Curling Pointing tips
Figure 5. Number of zoospores released from A. cochlioides hyphae in presence of different populations of Pseudomonas sp. EC-
S101. Bar indicates standard error of values. Data points bearing different letters are significantly different at P < 0.001 by
Fisher�s pairwise comparison. Value in parenthesis (40 ± 3) corresponds to number of zoospores released in presence of EC-S101
at 6.4 · 108 CFU ml)1.
319
plants of A. cochlioides that were also nonhosts ofA. euteiches and P. aphanidermatum (Table 3), itcan be assumed that these host plants might have ahigher ability to harbour such antagonistic bacte-ria that can protect them from their pathogens.Interestingly, bacteria that induced morphologicalalterations also showed potent disease suppressionactivity against A. cochlioides.
Induction of excessive branching in AC-5 onencountering Pseudomonas sp. strain EC-S101 wasapparently similar to those caused by antimicro-bial compounds viscosinamide and phenazine-1-carboximide in other Peronosporomycetes andother fungi (Thrane et al., 1999; Bolwerk et al.,2003). Both of these compounds are secondarymetabolites produced by P. fluorescens strainsDR54 and WCS365, respectively (Chin-A-Woenget al., 1998; Nielsen et al., 1999). We also foundthat Methanol (MeOH) solubles obtained fromculture fluid of Pseudomonas sp. strain EC-S101induces excessive branching in the hyphae ofAC-5; this, indicates the involvement of secondarymetabolites produced by EC-S101 in the inductionphenomenon of excessive branching.
Similarly, the curly growth of AC-5 hyphae indual culture with Stenotrophomonas maltophiliaEC-S105 or EC-S106 and by MeOH solubles fromculture fluid of strain EC-S105 tested by paper
disc, was similar to that observed in A. cochlioidesAC-5 due to a biocontrol rhizoplane bacteriumbelonging to Lysobacter (Islam et al., 2005), a re-lated genus of Stenotrophomonas (Sullivan et al.,2003) that produces xanthobaccin A (Nakayamaet al., 1999). Curly growth of the Fusarium oxy-sporum f. sp. radicis-lycopersici hyphae was alsofound during interaction with P. fluorescensWCS365 (Bolwerk et al., 2003). By comparison,necrosis, bulbous structure formation, extensivevacuolation and varying diameter of AC-5 hyphaeby Bacillus sp. EC-S108 are similar to effects ofzarilamide. It was observed that bacteria travelledabout 1–2 cm towards growing hyphae within aweek of incubation and densely colonized the hy-phoplane (Figure 1q), resulting in necrosis of thehyphae (data not shown). The migration of thebacteria to the hyphae and local colonizationsuggest that chemotactic substances could be in-volved. However, it is not yet known whether thelethal action behaviour of Bacillus subtilis EC-S108 is due to toxin production or competitionwith the pathogen.
We found that the increase in branchingfrequency and area in A. cochlioides AC-5 hyphaeduring encounter with Pseudomonas sp. strain EC-S101 was linearly correlated with time up to 10 h.The branching pattern induced in AC-5 hyphae
Table 3. In vitro antagonism of rhizoplane bacteria against phytopathogenic Peronosporomycetes
Isolate Aphanomyces cochlioides A. euteiches Pythium aphanidermatum
Note: All bacteria isolates are from non-host plants of A. euteiches and P. aphanidermatum.
Among the compounds tested against AC-5; zarilamide, hymexazol, cytochalasin A and calcium ionophore A23187 caused inhibition
zone at 25 lg disc)1 as +++, ++++, +, ++ and at 2.5 lg disc)1 as ++, +, ), + respectively. Compounds that did not cause
growth inhibition up to 25 lg per disc were: nicotinamide, polyflavonoid tannin, commercial tannin, paclitaxel, dimethyl DES,
b-estradiol, benlate, MBC, cytochalasin B and colchicine.aFor growth inhibition zone, each treatment was averaged over two replicates and evaluated as: ++++, >20 mm; +++, >10–
20 mm; ++, 5–10 mm; +, 0–5 mm; ), no inhibition.bBacteria isolated from non-host plants of A. cochlioides.cSeed-associated bacteria.
seemed to be unique with the continuous bifurca-tion of the apex of each successive hyphae leadingto a tree-like structure. The visible decrease ininternodal distance in the affected hyphae com-pared to controls indicates that the pathogenmight have lost its ability to regulate the normalbranching process, but maintained capability ofself-branching. Time-course studies using theconfocal laser scanning microscope and transmis-sion electron microscope revealed that Pseudomo-nas sp. strain EC-S101 exerts physiological stressessuch as cell wall thickness, extensive vacuolationand change in shape of nuclei, mitochondria andlipid bodies in A. cochlioides AC-5 hyphae; finallyresulting in the death of the pathogen (unpub-lished data).
Reduction in zoospore release and the induc-tion of encystment in A. cochlioides AC-5 in thepresence of Pseudomonas sp. strain EC-S101were similar to that in Pythium ultimum zoosp-ores released from the hyphae treated with vi-scosinamide or viscosinamide-producing P.fluorescens DR54 (Thrane et al., 2000). However,AC-5 hyphae in dual culture assay with Pseu-domonas sp. strain EC-S101 showed no oosporeformation similar to the response shown in vi-scosinamide-treated P. ultimum hyphae. There-fore suppression of damping-off in vivo byPseudomonas sp. strain EC-S101 and the Steno-trophomonas maltophilia EC-S105 treatment arealso likely to be due to the presence of activeagents produced by them. In addition to anti-biosis, the efficient colonization ability of Pseu-domonas sp. strain EC-S101 over the host rootsurface (data not shown) might also be involvedin nutrient and niche competition with thepathogen (Lugtenberg et al., 2002).
In conclusion, our dual culture observationsshowed that antagonistic rhizoplane bacteria in-duced diverse characteristic morphological alter-ations in the hyphae of phytopathogenicPeronosporomycetes. Hyphal morphologicalalterations were associated with growth inhibi-tion and the induction of characteristic mor-phological changes and indicated antagonisticactivity against the Peronosporomycete. How-ever, it is necessary to carry out further studieson the isolation of the metabolites responsiblefor these changes and to understand their modeof action and their usefulness as biocontrolagents.
Acknowledgements
We are thankful to Prof. R. Yokosawa, Health Sci-ence University of Hokkaido, for kindly providingAphanomyces sp. strains. We are also grateful toDr R. K. Behl, CCS Haryana Agricultural Univer-sity, India, for his help and advice. The financialsupport by Grant-in-Aid for scientific research(No. 16208032 to Y.H. and No. 14206013 to S.T.)and the scholarship for the first author at Hokkai-do University from the Ministry of Education,Culture, Sports, Science and Technology, Japanand a postdoctoral fellowship to M.T.I. from theJSPS are gratefully acknowledged.
References
Bacilio-Jimenez M, Aguilar-Flores S, Ventura-Zapata E,
Perez-Campos E, Bouquelet S and Zenteno E (2003)
Chemical characterization of root exudates from rice
Table 4. In vivo damping-off disease suppression ability of
rhizoplane bacteria in sugar beet and spinach seedlings
Treatment Zoospores
inoculated
per seedling*
Number of healthy
seedlings
Sugar beet Spinach
Pseudomonas sp. 1 · 104 3 ± 0.6 bc 3 ± 0.0 bc
EC-S101x 1 · 103 9 ± 0.6 cd 6 ± 0.6 bc
1 · 102 12 ± 0 d 10 ± 0.3 cd
0 12 ± 0 d 12 ± 0.0 de
Stenotrophomonas 1 · 104 5 ± 0.3 bc 3 ± 0.6 bc
sp. EC-S105x 1 · 103 8 ± 0.9 c 5 ± 0.9 bc
1 · 102 12 ± 0.0 d 10 ± 0.3 cd
0 12 ± 0.0 d 12 ± 0.0 de
Unidentified strainx 1 · 104 1 ± 0.3 ab 0 ± 0.0 ab
1 · 103 1 ± 0.3 ab 2 ± 0.3 ab
1 · 102 2 ± 0.7 ab 4 ± 0.7 bc
0 12 ± 0.0 d 12 ± 0.0 de
Hymexazoly 1 · 104 5 ± 0.3 bc 11 ± 0.3 cd
1 · 103 10 ± 0.3 cd 12 ± 0.0 de
1 · 102 12 ± 0.0 d 12 ± 0.0 de
0 12 ± 0.0 d 12 ± 0.0 de
Controlz 1 · 104 0 ± 0.0 ab 0 ± 0.0 ab
1 · 103 2 ± 0.3 ab 3 ± 0.6 bc
1 · 102 5 ± 0.7 bc 5 ± 0.3 bc
0 12 ± 0.0 d 12 ± 0.0 de
Values of number of healthy seedlings ± standard error were
based on three replicates, each including four plants. Values
followed by same letters are not significantly different at P
<0.001 by Fisher�s pairwise comparison.xBacteria were pelleted to the seeds at 108 CFU seed)1.yHymexazol was applied at 7.5 g kg)1 of seed.zSeeds without bacteria or fungicide applied.
*Zoospores were inoculated on 12 day old seedlings.
