Candida krusei and Kloeckera apis inhibit the causal agent of pineapple fusariosis, Fusarium guttiforme.

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Candida krusei and Kloeckera apis inhibit the causal agent of

pineapple fusariosis, Fusarium guttiforme

Adriana M. N. KORRES

a

, David S. BUSS

b

, Jose A. VENTURA

c

, Patricia M. B. FERNANDES

b,

*

a_{Instituto Federal de Educac¸~ao, Ci^encia e Tecnologia do Espırito Santo (IFES), Av. Vitoria, 1729, Vitoria, ES, Brazil}

b_{Nucleo de Biotecnologia, Universidade Federal do Espırito Santo (UFES), Av. Marechal Campos, 1440, Maruipe, Vitoria, ES, Brazil} c_{Instituto Capixaba de Pesquisa, Assist^encia Tecnica e Extens~ao Rural (INCAPER), Rua Afonso Sarlo 160, Vitoria, ES, Brazil}

a r t i c l e i n f o

Article history: Received 7 April 2011 Received in revised form 31 August 2011

Accepted 7 September 2011 Available online 29 September 2011 Corresponding Editor: Stephen W. Peterson Keywords: Antagonist yeasts Biocontrol Microbial interactions

a b s t r a c t

Studies based on microbial ecology and antagonistic interactions play an important role in the development of new alternative strategies in controlling plant pathogens and are relevant to further biotechnological applications. Antagonistic interactions between the yeasts Candida krusei and Kloeckera apis isolated from rotten pineapple fruits, and two isolates of the patho-genic filamentous fungus Fusarium guttiforme (Syn.: Fusarium subglutinans f. sp. ananas) resis-tant and susceptible to fungicide benzimidazole were studied in broth culture, and on plate assays. The yeasts significantly reduced Fusarium conidial germination after 24 h of cocultiva-tion in broth culture, and also mycelial growth on plate assays. Slide coculture appeared to show attachment of yeasts to the hyphal surface and also slight morphological abnormalities caused by C. krusei. Filtrates of cocultures of fungi and yeasts inhibited fungal growth, but fil-trates of the yeast cultures alone did not, suggesting that the antagonistic action of the yeasts is inducible.

The F. guttiforme isolate sensitive to benzimidazole was most affected by both yeasts in pine-apple juice, reaching a maximum of 36.5 % germ tube inhibition. This isolate was also in-hibited by yeasts in mycocinogenic plate assay. These results demonstrated that C. krusei and K. apis are effective in inhibiting F. guttiforme growth and that the mode of action is as-sociated with hyperparasitism and mycocinogenic activity.

Introduction

Brazil is a tropical country with great potential for the export of fruit to many regions of the world. In this context, pineapple is commercially very significant and Brazil is the number one world producer with almost 2 500 000 ton y1(FAO 2009). How-ever, plant and fruit diseases are major problems in field and postharvest conditions and fusariosis, caused by the fungus Fusarium guttiforme (Syn.: Fusarium subglutinans f. sp. ananas) is a constant concern in crops, leading to 30e40 % losses of fruit and 15e20 % losses of propagative material (Ventura et al.

2009). This fungus infects pineapple plants and fruits with the characteristic symptoms being morphological changes in dis-eased plants and exudation of gum from infected fruits (Ventura & Zambolim 2002). Chemical control is recommended, but constant use of systemic benzimidazole fungicides has se-lected resistant isolates of the pathogen (Ventura et al. 1994; Santos et al. 2002).

In natural environments microbial interactions exert both beneficial and harmful effects on pathogen populations and some microorganisms act as natural biocontrol agents (Saligkarias et al. 2002; Hiltunen et al. 2009). Studies of microbial

* Corresponding author. Tel.:þ55 27 3335 7348.

E-mail addresses:akorres@gmail.com,dsbuss@gmail.com,ventura@incaper.es.gov.br,patricia.fernandes@pq.cnpq.br j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / f u n b i o

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ecology and antagonistic interactions between microorganisms may lead to the development of new alternative strategies for controlling plant pathogens (Rosa et al. 2010).

Yeasts are an important component of the microbiota of the aerial part of the plants. Because they are adapted to these niches, they can colonize and compete for nutrients and space (El-Tarabily & Sivasithamparam 2006). Many authors have re-ported the use of yeasts as biocontrols of phytopathogenic fil-amentous fungi (Montesinos et al. 2002; Reyes et al. 2004; Coelho et al. 2007). Thus, efforts to elucidate antagonistic in-teractions between microorganisms in order to further biolog-ical control of phytopathogens are important.

Candida krusei and Kloeckera apis are yeasts associated with plants and animals, and have been isolated from insect legs, a common means of dispersal. Kloeckera apis in particular is associated with pineapples (Robbs et al. 1989). The study of these yeasts associated with plants has increased with respect to their potential as fungal biocontrols (McLaughlin & Wilson 1992; Hua et al. 1999).

This study aimed to investigate the antagonistic interaction between the yeasts C. krusei and K. apis, isolated from rotten pineapple fruit, and two isolates of F. guttiforme (Syn.: F. subglu-tinans f. sp. ananas), resistant and susceptible to benzimidazole.

Materials and methods

Microorganisms

Two yeasts (Candida krusei BT0701, and Kloeckera apis BT0703) were isolated from rotten pineapple fruits and identified by phenotypic characteristics and by partial sequence of the 26S rDNA D1/D2 region. Cultures were maintained on Yeast Extract Peptone Dextrose (YEPD) (20 % glucose; 10 % yeast ex-tract; 20 % peptone, 20 % agar) at 4C. Biochemical character-ization was performed using the method ofYarrow (1998)and conducted in Yeast Nitrogen Base media (YNB, Difco). As-similation of carbon sources in replica plating was performed with 7 d culture grown in 2 ml 0.1 % YNB glucose. Growth at 36 and 40C (in a water bath) was evaluated and also the ability of yeasts to grow in media with 0.01 and 0.1 % cycloheximide and in 10 and 15 % NaCl. Newly isolated yeasts were also tested as starch producers. Chitinase production was assessed using the method ofSantos et al. (2004), where chitin was used as sole source of carbon and energy, using the same procedure as in the standard tests used for identification.

Colony aspect in plates was observed after 7, 14, and 21 d of cultivation at 28C (Carvalho 2007). Cellular morphology was observed by light microscopy (Leica, model DMLS, Leica Micro-systems, Wetzlar, Germany) of fresh slide preparations and by slide culture in Cornmeal Agar (Barnet et al. 1990; Yarrow 1998). Genetic sequence identification was performed as de-scribed bySampaio et al. (2001)andAlmeida (2005)by partial amplification of the D1/D2 26S rDNA region sequence using primers NL1 (50GCA TAT CAA TAA GCG GAG GAA AAG 30) and NL4 (50GGT CCG TGT TTC AAG ACG G 30) (Sampaio et al. 2001; Almeida 2005). Samples were sequenced in an automatic DNA sequencer (ABI PRISM model 377, Applied Biosystems). Se-quences were compared to type strain seSe-quences deposited in

GenBank (http://www.ncbi.nlm.nih.gov/) using BLAST N (Altschul et al. 1997).

Isolates of filamentous fungus Fusarium guttiforme used in this study were obtained from the Plant Pathology Laboratory of INCAPER (Instituto Capixaba de Pesquisa, Assist^encia Tecnica e Extens~ao Rural, Brazil) and were isolated from pine-apple fruit with symptoms of fusariosis. Two isolates were studied, one resistant to benzimidazole, E-261 fungicide resis-tant (Fr) and one susceptible, E-203 fungicide sensitive (Fs). The cultures were maintained on slants of Potato Dextrose Agar (PDA, Oxoid Unipath Ltd., Basingstoke, Hampshire, UK) at 4C. For all experiments, an isolate of C. krusei ATCC 6258 was used as a comparison parameter. Kloeckera apis does not sur-vive long time storage at low temperature and therefore there is no available collection strain.

Mycelial growth inhibition

Yeasts were cultivated on solid YEPD for 24 h at 28C and sus-pended in sterile distilled water to 105_{cells ml}1_{. Fusarium}

gut-tiforme isolates were cultivated on PDA for 7e10 d at 28_{C and}

suspended in sterile distilled water to 105conidia ml1. Experiments were performed by streaking 10ml of the F. guttiforme conidia suspension on the centre of the plate and streaking the same volume of the yeast suspension 3 cm on either side (Ahrendts & Carrillo 2004). Plates were incu-bated at 28C for 7e10 d. The area of the fungal colony was measured and compared to control. The experiment was arranged in a randomized complete design with three replica-tions and performed twice. Positive and negative controls used 0.1 % tebuconazole fungicide (Folicur, Bayer, S~ao Paulo, Brazil) and sterile distilled water, respectively.

Inhibition efficacy (IE) was determined as follows: IEð%Þ ¼ ½1 ðAFY=AFCÞ 100

where AFY¼ area of F. guttiforme culture with yeasts, AFC¼ area of F. guttiforme in control.

Broth coculture

Experiments in broth coculture were conducted by addition of 104_{conidia ml}1_{and 10}5_{yeast cells ml}1_{suspension to tubes}

containing 3 ml of sterile pineapple juice or YEPD broth (Coelho et al. 2007). Pineapple juice was prepared by blending peeled pineapple fruit without addition of water and filtering twice in cheesecloth. Both YEPD broth and pineapple juice were sterilized in autoclave at 121C for 15 min. For the control, broth alone was used. Tubes were incubated at 28C in static condition. Spore germination was evaluated after 24 h and phal width measured after 120 h. Conidial germination and hy-phal width were measured for 40 randomly selected hyphae for each repetition and the mean value was calculated for each treatment (Coelho et al. 2007). Another assay was conducted us-ing yeast culture filtrate. The yeasts were cultivated in static condition in YEPD broth for 24 h, after which 1.5 ml of the cul-ture was centrifuged (14 750g 5 min1) and the supernatant fil-tered in a 0.22mm pore filter (Millipore). The filtered supernatant of each yeast culture was evaluated in terms of its ability to inhibit the growth of the filamentous fungus cul-tures in broth YEPD and pineapple juice as described above for

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the presence of yeasts (modified fromCoelho et al. 2007). Hyphal measurements and images were recorded using a light micro-scope (Leica model DMLS optical micromicro-scopee Leica Microsys-tems, Wetzlar, Germany). In all cases, the pH of YEPD broth was adjusted to 4.5 to be similar to pineapple juice. Mycelial growth inhibition was also tested with addition of coculture fil-trate of each yeast with each Fusarium guttiforme isolate. All ex-periments were arranged in a completely randomized design with three replications and conducted twice.

IE in all treatments was calculated as follows: IEð%Þ ¼ ½1 ðFGY=FGCÞ 100

where FGY is F. guttiforme growth with yeasts (germ tube length or hyphae width) or filtrate and FGC is F. guttiforme growth in control media without yeasts (germ tube length or hyphae width).

Plate diffusion assay

Three different filtrate materials were obtained as follows: (1) fil-trate of yeast culture: yeasts were cultivated separately in YEPD broth for 24 h/28C in static condition, and the culture filtered in a 0.22mm pore filter (Millipore); (2) filtrate of coculture of each yeast with each Fusarium guttiforme isolate: each yeast was cultivated together with each F. guttiforme isolate in YEPD broth in static condition for 48 h/28C; (3) filtrate of culture of yeast and sonicated material obtained from fungi growth: each F. guttiforme isolate was cultivated in potato dextrose broth (48 h/28C) and sonicated for 30 min. Yeasts were cultivated (28C/48 h) in tubes with 5 ml of YEPD broth with the addition of the sonicated material from each fungi culture. Yeast cul-tures were centrifuged (14 750g 5 min1) and filtered in a 0.22mm pore filter (Millipore). The three filtrates were evalu-ated for F. guttiforme inhibition by plate diffusion assays in PDA. The conidia suspension of each F. guttiforme culture was prepared as reported above and 100ml of this suspension was spread in each PDA plate. A volume of 100ml of the filtrate of each treatment was added to a well of 1 cm diameter made with a sterile tip in the centre of each plate. Plates were incu-bated at 28C for 7 d and observed daily for inhibition zone. Pos-itive and negative controls were performed with 0.1 % tebuconazole fungicide (Folicur Bayer, S~ao Paulo, Brazil) and sterile distilled water, respectively.

Slide coculture

Slide coculture was performed by inoculating each yeast spe-cies and each Fusarium guttiforme isolates in combinations in the centre of a slide covered with solid YEPD. A volume of 10ml of a suspension of 105_{cells ml}1_{of each microorganism}

was inoculated in the centre of the slide. Inoculated media was covered with a sterile coverslip and maintained in a humid chamber at 28C for 7 d, when the culture was observed under light microscope (Leica model DMLS optical microscope e Leica Microsystems, Wetzlar, Germany).

Mycocinogenic activity of the yeast isolates

Mycocinogenic activity was evaluated in triplicate by cultivat-ing the isolates in Yeast Malt (YM) media (0.3 % yeast extract;

0.3 % malt extract; 0.5 % peptone; 1 % glucose; 2 % agar) with 0.003 % methylene blue, pH 4.2 with 0.01 M citrate buffer an in-dicator of the presence of killer toxins (Somers & Bevan 1969; Vital et al. 2002). Killer toxin activity was defined as a region of blue coloured cells, or by a clear zone of inhibition bounded by blue coloured cells, surrounding the wells. A solid medium was used as the medium state is very important, since myco-cins are more stable in solid than in liquid media, because ag-itation can cause their inactivation (Vital et al. 2002).

Both Fusarium guttiforme isolates were tested for their sensi-tivity to mycocins that might be produced by pineapple isolated yeasts and Candida krusei ATCC 6258. A suspension of 1 ml of 105conidia ml1was spread on the surface of the assay me-dium and the potential killer toxin producing yeasts (grown on agar slants for 48 h/28C) were inoculated in the centre of the plates. Plates were incubated for 5 d at 28C and observed daily (Vital et al. 2002; Vadkertiova & Slavikova 2007). Candida albicans (12A) was used as sensitive control (Carreiro et al. 2002). Yeasts were considered as mycocinogenic when a clear zone with no growth, or an adjacent blue zone indicating cellu-lar death of the sensitive isolate was produced (Vital et al. 2002).

Statistical analysis

All experiments were conducted at least twice and in tripli-cate. Statistical analysis was performed by ANOVA using Mul-tistat software by comparing the mean values by Tukey test with 5 % probability (Shane 1989).

Results

Chitinase production

Neither Candida krusei BT0701 nor Kloeckera apis BT0703 were able to grow in media in which the sole carbon source was chi-tin, suggesting that they could not produce chitinase under these circumstances.

Mycelial growth inhibition

Significant mycelial growth inhibition of both isolates of Fusa-rium guttiforme, E-261 (Fr) and E-203 (Fs), occurred in streak ex-periments conducted in solid medium (Fig 1). Growth inhibition caused by Candida krusei BT0701 (75.8 % inhibition of Fr and 75.3 % inhibition of Fs) was greater than growth inhi-bition caused by Kloeckera apis BT0703 (41.4 % inhiinhi-bition of Fr and 47.9 % inhibition of Fs), even though collection strain C. kru-sei ATCC 6258 did not inhibit growth of any F. guttiforme isolates. There was no statistically significant difference (P< 0.05) in growth inhibition between the fungal isolates for each yeast treatment.

Germ tube elongation inhibition

Yeasts demonstrated considerable antagonistic activity on Fusarium isolates in YEPD broth and pineapple juice by inhibi-tion of germ tube elongainhibi-tion (Table 1). Inhibition of Fusarium guttiforme E-203 (Fs) was consistently higher than E-261 (Fr) by both Candida krusei BT0701 and Kloeckera apis BT0703, and

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in both media. In contrast to the mycelial growth inhibition shown on solid medium there was little difference in inhibi-tion between K. apis BT0703 and C. krusei BT0701 except that inhibition of E-261 (Fr) was higher by K. apis BT0703 than C. krusei BT0701 in pineapple juice. The collection strain C. kru-sei ATCC 6258 caused inhibition, but at a lower level than the two yeasts isolated from pineapple.

Filtrate supernatant of cultures of C. krusei BT0701 and K. apis BT0703 did not inhibit germ tube growth in broth cul-ture (Table 2). In fact growth was stimulated in each case. In contrast, data showed germ tube elongation was inhibited in broth culture when fungi were cultured with coculture filtrate of yeasts and fungi (Table 3), the exception being inhibition by C. krusei BT0701 in YEPD.

Plate diffusion assays with different filtrate material (filtrate of individual culture of yeasts, coculture of yeasts and F. gutti-forme isolates, and filtrate of yeast culture with sonicated mate-rial from F. guttiforme isolates culture) did not show any inhibitory effect on fungi growth after 7 d.

Physical interaction of yeasts and fungus

In order to observe physical interaction of fungi and yeasts, slide coculture assays were performed. Slide coculture of each yeast

with the fungal isolates showed that the yeasts were able to ad-here to the hyphal surface and that Candida krusei BT0701 and C. krusei ATCC 6258 caused abnormalities on hyphae, making them narrower (Fig 2A and B). Adhesion of Kloeckera apis BT0703 seemed not to affect the hyphae surface (Fig 2C). Some hyphae of both fungi isolates seemed exceptionally swollen, large, and vacuolated in the presence of yeasts (Fig 3).

Mycocinogenic activity of yeasts

The toxicity profile of yeasts isolated from rotten pineapple fruit was determined for the interaction with Fusarium gutti-forme cultures on medium indicative of killer toxins (Table 4). Both yeasts were mycocinogenic to the benzimidazole sensi-tive F. guttiforme isolate E-203 (Fs) by killer toxin production, but no inhibition was observed of the benzimidazole resistant isolate F. guttiforme E-261 (Fr).

The collection strain Candida krusei ATCC 6258 was weakly toxic to F. guttiforme E-203 (Fs), but not to E-261 (Fr).

Discussion

To determine the effect of two yeasts isolated from pineapple on the pineapple pathogen Fusarium guttiforme we coincubated combinations of the yeasts and two F. guttiforme isolates, and also exposed F. guttiforme to filtrate of yeast cultures and yeast/fungus cocultures. We measured fungal germination and growth, and physical contact between Fusarium hyphae and the yeasts.

Significant mycelial growth inhibition of both fungi isolates [E-261 (Fr) and E-203 (Fs)] by Candida krusei BT0701 and Kloeckera apis BT0703 on plate media suggests synthesis and secretion of suppressive substances into the medium and also the diffusion of them on the medium surface when in presence of the fungi. Such results are often attributed to synthesis of antifungal dif-fusible and volatile metabolites, although there are very few re-ports about the nature of these substances (El-Tarabily & Sivasithamparam 2006). Many features must be considered, such as the different potential of the inhibitory compounds, some presenting higher inhibitory efficacy on growth of the tar-get microorganism, which may be more or less sensitive to each compound. In addition, media pH and constitution, and inoculum concentration are also important (Ostrosky et al. 2008; Sales 2009). By using specific media (Vital et al. 2002) we Fig 1e Inhibition (%) of mycelial growth of F. guttiforme

(Syn.: F. subglutinans f. sp. ananas) isolate E-261 (Fr), and isolate E-203 (Fs), by C. krusei BT0701 and K. apis BT0703 isolated from pineapple fruit and C. krusei ATCC 6258.

Table 1e Percentage inhibition of germ tube length of F. guttiforme isolates E-261 (Fr) and E-203 (Fs) in broth culture (YEPD

and pineapple juice) with yeasts C. krusei BT0701, K. apis BT0703, and C. krusei ATCC 6258. Values are mean ± standard

deviation of the germ tube measures (mm).

Test pathogens

% Inhibition by yeasts

C. krusei BT0701 K. apis BT0703 C. krusei ATCC 6258

YEPD Pineapple juice YEPD Pineapple juice YEPD Pineapple juice

E-261 (Fr) 19.6 2.6 17.9 2.5 22.4 2.5 25.6 2.3 6.6 2.5 7.1 2.4

E-203 (Fs) 31.8 3.0 36.1 2.4 31.9 2.9 36.5 2.8 19.0 2.2 þ3.0a_2.5

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determined that the inhibitory factors produced included a mycocin.

The F. guttiforme isolates showed different reactions to killer toxin from yeasts, as they also reacted differently to benzimidazole fungicide. On media specific for killer toxins, E-203 (Fs) was inhibited whilst E-261 (Fr) was not. The main mode of action of benzimidazole fungicide is related to inhibi-tion of mechanisms of mitosis and cell division by disrupting tubulin biosynthesis and, consequently, the mitotic fuse (Zambolim et al. 2007). Some killer toxins also have the poten-tial to inhibit the cell cycle (Marquina et al. 2002) and so differ-ences in resistance to both benzimidazole and yeast mycocins may be due to the same target site. Alternatively, benzimid-azole resistance in fungi is frequently the result of over ex-pression of ATP binding cassette (ABC) membrane pumps (Andrade et al. 2000;Buss & Callaghan 2008) which typically have low substrate specificity and so conceivably provide re-sistance to killer proteins and other inhibitory factors as well. Inhibition of fungal growth in broth was only found with a fil-trate of yeast and fungus together, not with a filfil-trate of yeast alone, suggesting that the presence of fungi is necessary to in-duce the production of yeast inhibitory factors. Again there was a difference between the two F. guttiforme isolates E-261 (Fr) and E-203 (Fs), suggesting a difference in resistance to inhib-itory factors or possibly a difference in rate of, or capacity for, in-duction. This raises the possibility that differences in the toxicity of yeasts towards F. guttiforme isolate E-261 (Fr) and E-203 (Fs) may also be due to differences in inductive capacity of the fungal isolates. The focus of our study was the possible antagonistic effects of yeasts on Fusarium. However, we cannot exclude the possibility that Fusarium in the presence of yeasts produced substances antagonistic to the yeasts (Boeira et al. 2000), and that these substances in the filtrate affected Fusarium hyphal growth (Boeira et al. 2000). However, as Fusarium isolates were only tested with a filtrate from the same isolate, ie. Fr vs Fr and Fs vs Fs, we can exclude cross-isolate effects.

In addition, the ability of both C. krusei BT0701 and K. apis BT0703 to inhibit the filamentous fungi isolates Fr and Fs may be related to cellecell interaction. Both C. krusei BT0701 and K. apis BT0703 appeared to adhere to the hyphae surface (Fig 2A and B). Attachment of yeasts to pathogen hyphae has been described as a factor related to competition for nutrients, hyphae lysis, and disruption of filamentous fungi (Chaurasia et al. 2005).Ganter & Starmer (1992)referred to this type of in-teraction as interference competition.

Although both yeasts appeared able to adhere to the fungal surface, only C. krusei BT0701 led to abnormalities (Fig 2A and B). Induction of morphological changes in hyphae could be con-sidered as a mechanism of antagonistic interaction. It is notice-able that this result is complementary to work published by Marquina et al. (2002)which demonstrated that the mode of ac-tion of killer toxins from C. krusei is associated with cell mem-brane damage, which can explain the abnormalities on the hyphae surface. These morphological changes may be due to micro-lysis of hyphal segments, attributed to synthesis of en-zymes such as glucanase, chitinase, and others (Janisiewicz & Korsten 2002). However, the yeasts studied here demonstrated negative for chitin degradation in biochemical characterization performed during identification possibly indicating that these enzymes have been induced by the presence of the fungus. Why the killer toxin(s) of K. apis did not cause abnormalities is unclear.

Although the yeasts C. krusei BT0701 and K. apis BT0703 were both capable of inhibiting fungal growth and both produced mycocins, there was considerable difference in effectiveness in different situations. Filtrate of a coculture of K. apis BT0703 and fungus was more effective than a coculture of C. krusei in inhibiting F. guttiforme growth, and in fact C. krusei BT0701 cocul-ture filtrate was entirely ineffective when supplied to F. gutti-forme growing in YEPD media (we discuss differences in medium below). Although both C. krusei BT0701 and K. apis BT0703 were isolated from pineapple plants, K. apis is more

Table 2e Percentage inhibition of germ tube length of F. guttiforme isolates E-261 (Fr) and E-203 (Fs) in broth culture

(YEPD and pineapple juice) with filtrate of yeast culture (C. krusei BT0701, K. apis BT0703, and C. krusei ATCC 6258).

Values are mean ± standard deviation of the germ tube measures (mm).

Test pathogens % Inhibition by yeast filtrate

E-261 (Fr) 11.8 2.8 1.6 1.1 20.9 2.9 20.8 2.6 7.8 2.7 16.8 2.4

E-203 (Fs) 8.6 2.4 24.0 3.0 6.6 2.5 5.1 2.7 11.7 2.5 0.3 2.2

Table 3e Percentage stimulus/inhibition of germ tube length of F. guttiforme isolates E-261 (Fr) and E-203 (Fs) in broth

culture (YEPD and pineapple juice) with filtrate of coculture of yeast (C. krusei BT0701, K. apis BT0703, and C. krusei ATCC

6258) and fungi. Values are mean ± standard deviation of the germ tube measures (mm). Positive values indicate stimulus of

germ tube elongation, negative values indicate inhibition.

Test pathogens % Inhibition by yeast filtrate

E-261 (Fr) 1.3 2.5 7.0 2.3 7.8 1.8 10.1 2.7 9.5 2.1 4.3 2.2

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commonly found associated with pineapple plants (Robbs et al. 1989) in nature and so could be expected to compete better with F. guttiforme, which is a specialist of pineapple. The mechanisms of competition appear to include cellular exudates. However, inhibition of F. guttiforme was greater by C. krusei BT0701 than K. apis BT0703 when cells were present. This is possibly related to the greater cellecell interaction found with C. krusei BT0701 and F. guttiforme, which caused considerable abnormalities in the hyphae. This is similar to the results found byMcLaughlin

& Wilson (1992)who found that cell-free filtrate of a culture of Kloeckera apiculata did not reduce decay in grapes caused by Rhi-zopus stolonifer, but the presence of cells of K. apiculata signifi-cantly reduced disease in grape, peach, and apple caused by R. stolonifer, Botrytis cinerea, and Penicillium digitatum.

The collection yeast C. krusei ATCC 6258 showed low myco-cinogenic activity on F. guttiforme isolate E-203 (Fs) and no ac-tivity on F. guttiforme isolate E-261 (Fr), in contrast to C. krusei BT0701 and K. apis BT0703. Both these yeasts were isolated from rotten pineapple fruit, which may indicate a greater nat-ural competitive ability in pineapple fruit or juice. This is in agreement with the literature since the frequency of mycoci-nogenic strains isolated from natural environments is greater than that found among isolates from culture collections, and also, most reports of mycocinogenic yeasts in natural environ-ments refer to habitats like fruits (Vital et al. 2002). In addition, the collection isolate C. krusei ATCC 6258 was not able to in-hibit E-203 (Fs) isolate growth in pineapple juice, although it had a significant inhibitory effect in YEPD broth. This is simi-lar to the findings ofReyes et al. (2004), that inhibition of Cha-lara paradoxa by Pichia guilliermondii was higher on Glycerol Yeast Extract Agar (GYEA) media than in pineapple juice. Pine-apple juice is a relatively poor medium with, for example, little nitrogen (Gonc¸alves & Carvalho 2007) whilst YEPD is a rela-tively rich artificial medium and nutrient deprivation may ex-plain lack of impact of C. krusei ATCC 6258 in pineapple juice. This study suggests that the mode of action of these yeast species on F. guttiforme growth is complex and involves a num-ber of processes. A diffusible mycocin is induced in yeast by the presence of filamentous fungi, with the potential to inhibit fila-mentous fungal growth. There is also a suggestion of interfer-ence competition through physical contact (Ganter & Starmer

Fig 3e F. guttiforme in culture: (A) with C. krusei BT0701 showing hyphal swelling and vacuolation; (B) without C. krusei BT0701. Bar[ 10 mm.

Table 4e Mycocinogenic activity of yeasts C. krusei

BT0701 and K. apis BT0703, and C. krusei ATCC 6258 on isolates of F. guttiforme E-261 (Fr) and E-203 (Fs). 0: no

inhibition;D: weak inhibition; DD: medium inhibition.

Yeast E-261 (Fr) E-203 (Fs)

C. krusei BT0701 0 þþ

K. apis BT0703 0 þþ

C. krusei ATCC 6258 0 þ

Fig 2e Contact of yeasts to hyphae surface of F. guttiforme (Syn.: F. subglutinans f. sp. ananas). (A and B) abnormalities (arrows) on hyphae by C. krusei BT0701 (A) and C. krusei ATCC 6258 (B). (C) K. apis BT0703 attached to hyphae surface. Bar[ 10 mm.

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1992) though this is a hypothesis at this stage. The characteriza-tion performed above suggests that if chitinase is involved in this process then production is induced by the presence of fungi, rather than with only chitin alone. In addition, there is a further process of abnormality formation, only found with some yeasts which might be a result of mycocins. This has yet to be determined.

Yeasts are good candidates for biological control. Sapro-phytic yeasts are commonly observed on leaves and fruits, and some of them compete with plant pathogenic filamentous fungi for space and nutrients leading to plant protection (Saligkarias et al. 2002; Reyes et al. 2004). Competitive interac-tions have been shown to include hyperparasitism as well as the secretion of various agents such as wall degrading enzymes and killer toxins (Saligkarias et al. 2002; Janisiewicz & Korsten 2002; Ahrendts & Carrillo 2004; Santos et al. 2009; Cabral et al. 2009). Our results indicate that the interaction between the yeasts C. krusei BT0701 and K. apis BT0703 isolated from pineap-ple, and F. guttiforme, the etiological agent of pineapple fusario-sis, can be described as antagonistic, with hyperparasitism, mycocin production, and nutrient competition proposed as modes of actions. This is supported by reports of similar spe-cies in the literature such as the inhibition of Aspergillus flavus by C. krusei (Hua et al. 1999), inhibition of R. stolonifer by K. apicu-lata and Candida guilliermondii (McLaughlin & Wilson 1992), and inhibition of Fusarium oxysporum by Candida steatolytica (El-Mehalawy 2004). Both yeasts studied grow successfully on pineapple in field conditions and are antagonistic to a common pineapple pathogen and thus would appear to be worth further study as biological control agents.

Acknowledgements

This work was financially supported by the National Council of Scientific and Technological Development (CNPq, Brazil), the Research and Projects Financing (FINEP), and The State of Espırito Santo Science and Technology Foundation (FAPES). We are grateful to Professors A. N. Hagler, Universidade Fed-eral do Rio de Janeiro, and F. C. Pagnocca, Universidade Estad-ual Paulista, Rio Claro, SP, for helping in yeast identification and for providing yeast isolates for comparison experiments.

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