Mode of action of the postharvest biocontrol yeast, Pichia guilliermondii. I. Characterization of attachment to Botrytis cinerea

Physiological and Molecular Plant Pathology )1991) 39, 245-258

245

Mode of action of the postharvest biocontrol yeast, Pichia guilliermondii . I . Characterization of attachment to Botrytis cinerea . MICHAEL WISNIEWSKI~', CHARLES BILES+, SAMIR DROBY§, RANDY MCLAUGHLINt, CHARLES WILSONt and EDO CHALUTZ§ +United States Department of Agriculture . Agricultural Research Service, Appalachian Fruit Research Station, 45 117ltshire Road, Kearneysville, West Firginia 25430, U.S.A . ; and $,new Mexico State University, Department of Entomology, Plant Pathology and Weed Science, Las Cruces, .Aeu Mexico ; §Agricultural Research Organisation, The Folcani Center, Bet Dagan, Israel 50250 Accepted fir publication August 1991'

An isolate (87) of the yeast Pichia guilliermondii, protects apples from postharvest fruit rotting fungi Botryttis cinerea and Penicillium expansum . In order to examine the yeast-pathogen interaction . B . cinerea was grown on agar plates overlayed with cellophane . Effective and non-effective yeast isolates were applied near the young hyphal growth . Samples were taken 24 It later from the area where the fungi and yeast had intersected . Light microscopy revealed a general attachment of the effective biocontrol agent P. guilliermondii (isolate 87 ; and a non-effective isolate ('1171 of Debaryomyces hansenii . Low temperature scanning electron microscopy 1 LTSEM) indicated that both species of yeast attached to the fungal hyphae, but the 87 isolate attached fastidiously . Twenty-four hours after applying the 87 isolate to B . cinerea, pitting and collapse of the hyphae were observed . These observations were confirmed using transmission electron microscopy . These features were not observed with the ineffective isolate of D . hansenii . Further experiments indicated that attachment of P . guilliermondii to hyphae of B. cinerea could be blocked by agents that alter protein integrity (salts, proteases, etc .) and certain sugars . Isolates of both species produced i)-(1-3) glucanase when cultured in various carbon sources and on cell walls of fruit rotting pathogens . Culture supernatants from P. guilliermondii, however, yielded two- to five-fold more /3-)1-3 glucanase activity compared with 1) . hansenii . Data indicate that tenacious attachment, along with secretion of cell wall degrading enzymes, may play a role in the hiocontrol activity of this yeast antagonist .

INTRODUCTION As an alternative to the use of conventional fungicides, biological control of postharvest diseases of fruit is an area of great potential [18] . In this regard, the use of isolate 87 of Pichia guilliermondii Wickerham has shown broad spectrum activity in the control of a number of postharvest diseases of citrus [2, 17] and temperate fruit [9, 19] . In several previous publications this isolate was referred to as Debaryomyces hansenii (Zopf) Lodder Et Kreger-Van Rij ; however, more detailed characterization of this isolate has changed the classification to P . guilliermondii [10] . Abbreviations used in text : PDA, potato dextrose agar : LTSEM, low temperature scanning electron microscopy . 0885-5765/91/100245+14 S03 .00/0

C 1991 Academic Press Limited Mi'P 39



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'1 o evaluate the use of this vcast as a biocontrol agent, an understanding of the antagonistic interaction bctween the yeast and postharvest pathogens is needed . Although nutrient competition has been suggested as the principle mode of antagonism [21 . attachment of the yeast to pathogen hyphae and extensive production of an extracellular matrix have been observed [19 ] . These factors may also play a key role by either enhancing nutrient competition or by some other undetermined mechanism . Attachment mechanisms are generally recognized to play a major role in cell-cel] interactions in fungi and various cell surface receptors : carbohydrate binding proteins) have been identified in prokaryotic and eukaryotic cells [7] . The role of lectins in attachment has been elucidated in other biological control systems utilizing Trichoderma harzianum [3] and Enterobacter cloacae 112, 20 ] . Jansson et al . [51 also demonstrated that attachment of an endoparasitic fungus . .tleria coniospora, to nematodes was affected by the larval stage of the nematodes and that sialic acids located on the nematode cuticle were critical for recognition by the endoparasite . The principal object of the present study was to examine the nature of , the attachment of the yeast to hyphae of the postharvest pathogen of apple . Bolrytis cinerea Pers . : Fr . It was of interest to determine if the attachment was due to a specific lectintype binding or was of a more general, non-specific nature involving the extracellular matrix . Furthermore, we wanted to investigate whether or not attachment played a direct role in antagonism, by facilitating enzymatic hydrolysis of hyphal cell walls by P . guilliermondii .

MATERIALS AND METHODS Botrylis cinerea cultures were obtained from apples and maintained on potato dextrose agar PDA) . P. guilliermondii (isolate 87 ; was obtained from a lemon surface as previously described [17] . D . hansenii (isolate 117, ATCC No . 36239) was obtained from the American Type Culture Collection, Rockville, Maryland, U .S .A . Yeast isolates were maintained on silica gel and grown in a yeast medium in culture flasks for 24-48 h before experimental use, as previously described [9] .

Fungal and yeast interaction The pathogen (1 x 10' conidia ml -' 1 was plated on either acidic PDA, apple juice agar or apple slices (cv . Golden Delicious) overlayed with cellophane (Spectra/Por, Spectrum Medical Industries, Inc ., Los Angeles, California .) . After 24 h, 40 µl of a yeast isolate (1 x 10' cfu ml") was placed at the margin of fungal growth . At 4, 24 or 48 h intervals, the cellophane portion where fungal and yeast interaction could be observed was removed from the agar plate or apple slice, washed with a stream of distilled water for 30-60 s and viewed with a light microscope or SEM . To characterize the attachment mechanism, several compounds were applied to the yeast and pathogen to test if a disruption of attachment would occur . Cellophane with the pathogen alone was removed and placed in a 45 mm Petri dish . The yeast with the specified compound was applied to the fungus and allowed to incubate for 4 h . 'The compounds or physical treatments used are listed in Tables 1 and 2 . After incubation,



Pichia guilliermondii attachment to Botrytis cinerea

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TABLE I

Attachment of Pichia guilliermondii (isolate 87) to Botrytis cinerea in the presence of carious salts . sugars and chemical agents Treatments Yeast isolate 87 ;control` CaCl2 (1 ° , MnCl s ! 1 MgCI 2 (1 ° Protease (2 mg ml ') Protease 2 mg ml - ', boiled Trypsin (2 mg Trypsin ( 1 in g ml - ') Trypsin (0 . 5 mg ml -' Trypsin (2 mg ml ', boiled] Laminarasc (2 mg ml - ' Tween 20 :1 SDS (0 . 01 "„) f-mercaptoethanol (0 . 01 n-glucose ) °,,) 2-Deoxyglucose (5" ) Methyl-n-glucose (5°-1,l n-galactose (5 °-,,) n-mannose (5' ) ( ,% Trehalose 5 ° Sorbose (5 ~, Raffinose Sodium azide P),) )

Attachment after 4 h ° +

+

+ + + + +++ + + + 4+ + +

All reagents were prepared w/v, except where indicated . ') ++, High level of attachment ; +, attachment : +-, attachment but sparse ; -, no attachment . a

the fungal hyphae were observed with a light microscope for any attachment . Attachment was rated as + or -, where - indicates no attachment and + or + -1indicates increasing levels of attachment .

Enzyme activity To test whether isolate 117 or 87 exhibited fi-(l-3) glucanase activity, the isolates were grown on several carbon substrates consisting of l ° o (w/v) carbon source in a minimal salt medium (Lilly-Barnett) or on cell walls of various fruit rotting fungi . Carbon substrates utilized were chitin, fructose, D-mannose, D-glucosamine, sucrose, Dgalactose and cellobiose . Hyphal cell walls were obtained from shake cultures of malt extract broth inoculated with spores of Botrytis cinerea, Monilinia fructicola (G . Wint .) honey, and Penicillium expansum Link . Cultures of Rhizopus stolonifer (Ehrenb . : Fr .) Vuill were grown in potato dextrose broth . Cell walls were prepared as described below . The yeast isolates were grown in shake culture for 48 h (stationary phase) . The individual cultures were then centrifuged (10000g) and the supernatant dialysed for 18 h against three changes of deionized water in a 4 1 container . Samples were then lyophilized and reconstituted with 2 ml of 0 . 05 M acetate buffer, pH 5 . 0 . A 200 gl sample was added to ts?



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M . Wisniewski _=lttachnrent

I Ast.E 2 ;isolate 87) to Botrytis cinerea after pretreatment organism with protein degrading enzymes and chemical agents or after boilin, ;

of Pichia guillicrmondii

Treatment°

et al .

either

Attachment after 4 h"

1' . guilliermondn (isolate 871 Control no treatment) Boiled (I h ; Boiled 10 r n : Protease 2 mg ml Ti `-psin i 2 mg ml` 0 . 1 N NaOH 0-l N HCl

++

B . cinerea Control ;no treatment', Boiled I h ; Boiled ; 10 m ) Protease 2 mg ml -r l Try psin 2 mg ml - r 0-1 N NaOH 0-1 N HCI

'All treatments were followed by a wash in deionized water prior to co-culturing . "++, High level of attachment : +, attachment ; +-, attachment but sparse ; -, no attachment .

800 µl of 1 % (w/v) laminarin (No . L-9634, Sigma, St Louis, Missouri) and 0 . 02 (w/v) sodium azide . The enzyme-substrate mixture was incubated for 2 or 24 h at 37 ° C and tested for total reducing sugars using the dinitrosalicylic acid reagent [ii] . Glucose was used as a standard . The amount of reducing groups was measured Spec trophotometrically at 500 nm . Each experiment contained three replicates . Background levels of reducing sugars were determined with a time 0 supernatant extract from each treatment . The time 0 extract was added to the laminarin enzyme substrate just prior to boiling at 100 ° C for 5 min . Boiling the supernatant prior to incubation with laminarin gave similar results . These experiments were repeated twice . In the initial carbon substrate experiments (Table 3) results are reported as total activity . Later experiments conducted with hyphal cell walls are reported on an equal protein basis . Protein content in reconstituted samples of supernatant was determined with a Bio-Rad protein assay (Bio-Rad, Richmond, California) . Preparation offungal cell walls Botrytis cinerea, M. fructicola, P . expansum

and R . stolonifer were grown in 500 ml of nutrient media (as described above) for 4 days at 20 ° C . The fungal material was then collected on cheese cloth, washed several times with distilled water and homogenized with a Tissuemizer homogenizer for 2 min . These homogenates were stored at -20 ° C overnight . The samples were then thawed and again homogenized as above to break up the pellet . Samples were centrifuged for 2 min at 1625 r min' . The fungal material was transferred to a Braun homogenizer flask and 20 g of 1 mm diameter glass beads were



Pichia guilliermondii attachment to Botrytis cinerea

24 9

TABLE 3

The effect of various carbon substrates on the activity of extracellular /1-(1-3)-glucanase produced by Pichia guilliermondii (isolate 87) and Debaryomyces hansenii (isolate 117) grown on a minimal medium fl-Glucanase activity'`

Carbon source Chitin Fructose D-mannose n-glucosamine Sucrose D-galactose Cellobiose

Isolate 117 87 117 87 117 87 117 87 117 87 117 87 117 87

(µmol reducing sugars released per 1) 2 li 24 h ND ND ND 0 . 55+ _ 0 .02 ND 0. 84+ _ 0 .04 ND ND ND 0. 30+0 . 16 0. 16±0 . 12 1 . 02+0 . 02 0. 25+0 . 11 ND

ND ND 0 . 62+0. 04 2 .85+_ 0. 09 ° 1 .09+0. 04 2 . 91+0 .05 ° ND 0 . 56+0 . 07 1 .06+ _ 0 .07 3 . 04+0 .08 0 . 51±0 .02 3 . 02+ _ 0 .04 0 . 27+ _ 0 .09 1 . 37+0 .07

'All measurements of l3-(1-3) glucanase activity were determined using laminarin as a substrate . Numbers represent mean+ SE of three replications . All experiments were repeated twice. ND = not determined . ° Significantly different utilizing t-test, P < 0 .05 . Comparisons are between isolates at a given time on a specific substrate .

added . After 2 min of CO 2 cooled homogenization, the samples were stored for 10-15 min at 4 ° C to allow the glass beads to settle . The supernatant was then removed and sonicated with a probe type sonicator for 15 min and centrifuged for 2 min at 1625 r min' . The supernatant was discarded and the pellet resuspended in water . The samples were subjected to sonication and centrifugation as above for a total of six times . The final pellet of fungal wall fragments was frozen at -80 ° C and lyophilized for 2 days . These preparations were used to replace the carbon source in a minimal salt medium . Low temperature scanning electron microscopy (LTSE,W) The majority of experiments were conducted with hyphae of B . cinerea incubated for 24 or 48 h with the yeasts as described above . In some experiments, attachment of P. guilliermondii to hyphae of Penicillium expansum was also examined . P. expansum cultures were maintained as previously described [9] and handled in a manner identical to B . cinerea . Attachment to cotton or glass fibres was also examined . Small sections of cellophane (6 mm 2 ) in areas of fungal-yeast interaction were removed from the surface of the PDA, rinsed thoroughly with a stream of distilled water and mounted on an aluminium stub using silver paint as an adhesive . To enhance the removal of the yeast, some samples were rinsed with either 1% Tween 20 (v/v) or 2% CaC1 2 (w/v) .



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Additionally, some samples were briefly sonicated . An Oxford C"l'-1500 Cavo Preparation System and Cold Stage (Oxford, England ; mounted on a Cambridge ,5120 SEM (Cambridge, Englandl were used to prepare and examine the specimens . Samples were quenched in liquid N ..,-shush, transferred to the cold stage, and etched at -80 ° C for 3-5 min . The samples were then removed from the specimen chamber . coated with gold/palladium at -160 ° C and subsequently transferred hack to the cold stage and kept at - 165 to - 175 ° C . Specimens were viewed at .5-10 kV .

Transmission electron microscopy ( TE~1I ) Samples of B . cinerea hyphae incubated with the yeast for 48 h were examined . Small samples of material (1 mm 2 ) were placed in 3 °-0 (v/v) gluteraldehyde in 25 mm sodium phosphate buffer (pH 6 . 8) at 4 ° C and postfixed in 2 °-0 osmium tetroxide (w/v) at 4 °C . Samples were dehydrated in a graded ethanol series, followed by propylene oxide and embedded in epoxy resin . The material was sectioned with a diamond knife, mounted on copper grids and stained with uranyl acetate and lead citrate . Grids were examined at 75 kV, using a Hitachi H-600 TEM .

RESULTS Ultrastructure LTSEM observations of 24 h co-cultures of P. guilliermondii/B . cinerea indicated that the yeast cells were tenaciously attached to the hyphae and to each other despite extensive rinsing of samples with distilled water or 1 1',, Tween from a wash bottle during sample preparation (Fig . 1) . In contrast, co-culturing ofB . cinerea with an isolate ofD . hansenii (117), selected for use as a comparison because of its inability to exhibit effective biological control of B . cinerea, indicated only a loose association of the yeast with the fungus (Fig . 2) . D . hansenii (117) was also more easily detached from the hyphae by rinsing of samples with distilled water or 1 % Tween 20 . Closer examination of attachment of P. guilliermondii to B . cinerea indicated that, in many instances, individual yeast cells gave the appearance of having sunk into the hyphal cell wall, resulting in a concave appearance of the hyphal surface under the attached yeast cell (Figs 3 and 4) . Some samples were rinsed with 2",, CaCl2 or sonicated prior to sample preparation to enhance dislodgment of P . guilliermondii . These treatments were mildly effective in dislodging the yeast from some areas of the hyphae . When areas of hyphae were observed where yeast cells presumably had either become detached on their own or dislodged during sample preparation, irregularities in wall conformation were observed (Figs 5-8) . Discrete areas of hyphal cell walls appeared concave, giving the hyphal strand a general appearance of being pitted . In some areas, the pitting was quite extensive (Figs 7 and 8) . Further observations were made of sectioned material. The fixation protocol gave adequate preservation of hyphal ultrastructure, but little internal structure of the yeast cells was visible ; although plasmolysis or extraction of yeast protoplasm was not observed . In co-cultures of D . hansenii/B . cinerea, hyphal ultrastructure was normal in appearance and did not exhibit evidence of stress, despite the presence of numerous

Pichia guilliermondii attachment to Botrytis cinerea

FIG, 1 . Fastidious attachment of yeast cells (Pichia guilliermondii` to hyphae !H) of Botrvtis cinerea after extensive rinsing with water or 1 ° o Tween . Bar = 20 pm . FIG, 2 . Lack of attachment of yeast cells Debaryomvcec hansenii ; to hyphae (H) of Botr_rtis cinerea . Bar = 20 µm . FIGS 3 and 4 . Embedding of Pichia guilliermondii into by phal wall of Boirvtis cinerea . Note concave appearance of hyphal wall (arrows ; . Bars = 25 pm . i

251



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u,s 5 8 Dislodgment of yeast cells iPichia guilliermondii' from hyphae of Bo I?lli~ rinrrrar . Aoir pitting present on hyphae arrows, v, here yeast ccIts have been presumably dislodged . Pitting li 8 _ and 4µm appeared quite extensive at bunts Fig,= fi R' . liars = 25 prn Fig .

yeast cells (Fig . 9 . The protoplasm was rich in glycogen and a full complement of' fungal organelles was visible . In contrast, hyphae that had been co-cultured with P . guilltermondt were often convoluted in appearance and appeared moribund (Fig . 10i . It also appeared that in many hyphae the wall had undergone some swelling and partial degradation (Figs 1 1 and 12) . Although many areas of the wall appeared to have a concave appearance, it

Pichia guilliermondii attachment to Botrytis cinerea

Fin . 9 . Co-culture of Debaryornyces hansenii (isolate 117' and Botryt+ . inerea . Note healthy appearance of hyphae (H) despite presence of yeast . Hyphal cells were rich in glycogen and a full complement of organelles was visible . Bar = 5 pm . . 10 . Co-culture of Pichia guilliermondii (lisolate 87j and Bat win cinerea . Hyphae (H) appeared Fin moribund when grown in the presence of the yeast and man\ hyphal strands appeared to have been lysed . Bar = 5 mm . Fins 1 1 and 12 . Ultrastructure of hyphae of Botryti .s cinerea from eu-cultures of Pichia guilliermondii and B. cinerea . Pitting and partial degradation of the walls was observed on numerous hyphae arrows) .. Swelling of the wall was also visible . Although the ultrastructure of the hyphae was difficult to discern the hyphal cells did not appear to be plasmolysed except in cases where the cells had been lysed . Bars = 1 pm ;Fig . I1' and 0. 5 pin (Fig . 12' .

253



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was not clear if this related to the pitting observed with LTSEM or was a more general phenomenon of cell wall degradation . In areas where P. guilliermondii appeared to be in direct contact with hyphae of B . cinerea, yeast cells appeared to be lying within a depression of the hyphal cell wall . I t was difficult to discern where the hyphal wall ended and the extracellular matrix of the yeast began (Fig . 13) . In contrast, yeast cells of' D . hansenii were only superficially attached to the hyphal cell wall via an extracellular matrix (Fig . 1I . 'phis was in agreement with observations using LTSEM . Similar hyphal-yeast cell interactions were observed in co-cultures of P . guilliermondii/Penicillium expansum . Examination of samples using LTSEM indicated a similar pitting appearance in the hyphae of P . expansum after 24 h or co-culture . The pitting, however, was more extensive and in some areas distinct holes in the hyphal cell walls were observed (Figs 15 and 16) . In contrast to P . guilliermondii/B . cinerea cocultures, yeast cells were less firmly attached in P . guilliermondii/P. expansum co-cultures and yeast cells were more readily removed by washing with distilled water or I "1'ween 20 . Effect of carious salts, sugars and chemical treatments on attachment Various agents that have been reported to block attachment or agglutination in fungi or bacteria were utilized in co-culture and pretreatment experiments to try and block attachment of P . guilliermondii (87) to hyphae of B . cinerea . Similar results were obtained from all growth media (PDA, apple juice agar and apple slices) . The results of coculture experiments, where both P . guilliermondii and B . cinerea were incubated together in the presence of the test compound, indicated that all the salt solutions tested (CaCl2, MgC1 2 and MnC1 2 ) and the protein degrading enzymes (protease and trypsin) blocked attachment (Table 1) . Enzymes that had been boiled prior to use, however, were ineffective . Most of the sugars tested and also laminarase were unsuccessful in preventing attachment of P. guilliermondii to hyphae of B . cinerea but two exceptions were 2-deoxyglucose and raffinose . Soaps (SDS and Tween 20) and f-mercaptoethanol were mildly effective in blocking attachment . Inhibiting respiration with sodium azide also blocked attachment . The results of experiments where either P. guilliermondii or B . cinerea were treated before co-culture are reported in Table 2 . Pretreatment of B . cinerea with protease (2 mg ml -r ), trypsin (2 mg ml - '), 0 . 1 N NaOH and 0 . 1 N HCl followed by washing the hyphae twice in deionized water also deterred attachment . Ati'hen the same experiments were conducted with P . guilliermondii, protease and NaOH did not completely block attachment . Boiling the yeast for 1 h and then applying it to nonboiled B . cinerea hyphae blocked attachment . The inverse experiment conducted with B . cinerea hyphae being boiled and applying non-boiled P. guilliermondii also resulted in no attachment . Boiling the yeast for 10 min only partially blocked attachment, whereas boiling of the fungus for 10 min again prevented attachment . In further experiments to characterize the specificity= of attachment of P . guilliermondii, it was observed that yeast cells were unable to attach to either cotton or glass fibres . Enzyme activittr Preliminary experiments detected /3-(I-3) glucanase activity in several supernatant culture extracts of P . guilliermondii and D . hansenii grown on different carbon sources

Pichia guilliermondii attachment to Botrytis cinerea

Ftc . 13 . Pichia guilliermondii yeast cell (Y) in contact with hypha (H) of Botrytis cinerea . Note partial degradation of hyphal wall . It was difficult to determine where the hyphal wall ended and the extracellular material of the yeast began . Bar = 0. 2 pm . Fm . 14 . Deharyomyces hansenii yeast cell (Y) in contact with hypha Fl of Botrvtts cinerea . Notc superficial attachment of the yeast cell and integrity of the hyphal cell wall . Bar = 0. 5 pm . Fins 15 and 16 . Co-culture of Pichia guilliermondii and Penicillium expansion after dislodgement of the yeast . Note the pitted appearance of the hyphae ;straight arrows' . Actual holes in the hyphal wall were sometimes observed (curved arrows ' . Bars = 4 pm .

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M . Wisniewski et al.

256

'Fable 3) . Extracellular culture extracts of P . guilliermondii exhibited at least two- to five-fold greater fi-)1-3 glucanase activity than D . hansenii culture extracts depending on the carbon source . Greatest differences were present when fructose, o-glucosamine . D-galactose or cellobiosee were used as a carbon source . Cell walls of different fruitrotting pathogenic fungi were used in later experiments . P . guilliermondii culture extracts had significantly higher fl-(1-3) glucanase activity than D . hansenii culture extracts on all fungal cell walls tested (Table 4) . 'The highest fl-(1--31 glucanase activity

FABLE 4 The effect

of hyphal

cell walls of various fungi used as a carbon substrate on the activity of extracellular ~i-

1-3)-glucanase produced b_y

Pichia guilliermondii

(isolate 87' and

Debarvomvces hansenii

!isolate 117 ; groan on a minimal medium

f-Glucanasc activity``

Carbon source .tlonilinia Rhizopus Penicillium

Botrytu

Isolate 117 87 117 87 117 87 117 87

(µmol reducing sugars released per log protein 2h 24 h 0 . 15+0 . 15 0 .93+0 . 08" 1 .01+0. 21 1 . 15+0.08 1 .00+0. 10 8 . 15+1 . 26" 1 . 58±0. 40 1'49+0 . 19

181+0. 66 4 .46+0. 11" 1 . 72±0. 26 3 . 62+ _ 0. 09" 5 . 80+ _ 0. 92 16. 78+0. 21 ° 1 . 86±0. 18 3 . 68+0. 30"

'All measurements of l3-(1-3) glucanase activity were determined using laminarin as a substrate . Numbers represent mean ± SE of three replications . All experiments were repeated twice . 'Significantly different utilizing t-test, 1' < 0. 05 . Comparisons are between isolates at a given time on a specific substrate .

of both yeasts grown on fungal cell walls was detected with Penicillium . Enzyme activity and differences between the two yeasts were more readily detected after 24 h than after a 2 h incubation period with laminarin as a substrate .

DISCUSSION Attachment of yeast and other fungal cells to host organisms has been shown to play a major role in their biological activity [1, 6, 7] . The isolate of P . guilliermondii investigated in this study appears to have a strong attachment mechanism that is blocked most readily when yeast cells or fungal hyphae are exposed to compounds that effect protein integrity or when respiration is inhibited 'Tables 1 and 2) . Nelson et al . [12] showed that Enterobacter cloacae attachment could be negated if sugars were applied . They concluded that a lectin-type binding was functioning in attachment . A lectin was also concluded to play a major role in the agglutination of spores of Trichoderma harzianum by culture supernatants of Sclerotium roll ii and Rhizocionia solani [3] . This agglutination was blocked both by sugars and trypsin . In our study, 5'), ) 2deoxyglucose and raffinose were the only sugars that blocked attachment . 'This does



Pichia guilliermondii attachment to Botrytis cinerea

257

not rule out the possibility of blocking attachment with the use of other sugars that were not tested . The yeast was also unable to attach to cotton or glass fibres . Cell surface proteins on P . guilliermondii and B . cinerea both appeared to play an important role in attachment of the yeast to the fungal pathogen . When trypsin or protease were present in the culture medium, attachment was blocked . Pretreatment of B . cinerea with protease, trypsin, 0 . 1 N NaOH and 0. 1 N HCI inhibited attachment of P. guilliermondii . Pretreatment of P. guilliermondii with trypsin or 0 . 1 N HCl also inhibited attachment, but pretreatment with 0 . 1 N NaOH or protease only partially blocked attachment . In general, the inhibition of attachment by protein degrading enzymes, NaOH, and HCl indicates the involvement of proteins or glycoproteins in recognition of carbohydrates at the cell surface [8] . The yeast proteins that are necessary for attachment, however, appear to be insensitive to the mild alkaline wash and protease . Salt solutions either denatured the proteins or, more likely, removed them from the cell surface . Denaturing agents, such as SDS and /3-rnercaptoethanol, also inhibited attachment . Collectively, the data indicate that a lectin or other type of agglutinin may be involved in binding P. guilliermondii to other fungi . The blocking of attachment by raffinose (Table 1) indicates that a sugar with a similar isomeric configuration may be important in attachment . Toda et al . [15] have studied in detail the ability of L-fucose to react with an antibody that blocks cell recognition and adhesion in

Polysphond_ylium

pallidum . Further experiments indicated that the isolates of P . guilliermondii and D . hansenu produce /3-(1-3) glucanase when grown on various carbon substrates or cell walls of several fungal pathogens . The effective antagonist, P . guilliermondii (isolate 87i, however, produced higher levels of this cell wall hydrolase (Tables 3 and 4) . In addition, the fastidious attachment of P . guilliermondii to the fungal cell wall would enhance the effectiveness of any hydrolase secreted by the yeast to the extracellular matrix . Chitinases and fl-glucanases have been observed in yeast [1] and are instrumental in the budding process . Filamentous fungal hydrolases have also been shown to degrade pathogen cell walls [3] . Notario [13] demonstrated fl-(1-3) glucanase production by Candida albicans . Mild acid treatment of the yeast decreased /3-(1-3) glucanase activity indicating that the /3(1-3) glucanase activity was associated with the cell wall (extracytoplasmic) portion of the yeast . Fleet & Phaff [4] also found fl-(1-3) glucanase tightly bound to yeast cell walls and this enzyme has been found in cell-free yeast extracts [14 ] and culture fluids of the yeast C. albicans [16] . The anamorph of P . guilliermondii is in the same genus as C. albicans [10] . The production of #-(1-3) glucanase may also enhance the ability of P . guilliermondii to adhere to other fungi as is hypothesized for C . albicans [13] and may be the reason for the observed degradation of fungal hyphae observed in our study . Droby et al . [2] have suggested that nutrient competition plays a major role in the biocontrol activity of P . guilliermondii . Our results indicate that the yeast is firmly attached to hyphae of B . cinerea and that this attachment in conjunction with the production of /3-(l-3) glucanase results in a partial degradation of the hyphal cell wall . The cell wall degradation was evidenced by a pitted appearance, visible with both LTSEM and TEM . In contrast, the ineffective isolate of D . hansenii (117) showed only a superficial ability to attach to hyphae of B . cinerea and exhibited less /3-(1-3'.



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glucanase activity when grown on several carbon substrates . Therefore . the eflicacA of

P . guilliermondi appears to be dependent not only on its ability to colonize a wound site and compete for nutrients [21, but may also depend oil its ability to attach firmly to hyphae of' the pathogen and exhibit high levels of /3-f 1--3) glucanase activity .

'Phis research was supported in part by grant No . US--1378-87 from

BARD,

The

United States-Israel Binational Agricultural Research Development Fund .

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20 .

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