Journal of Basic Microbiology 2009, 49, 441–451 441

© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Research Paper

Diversity of endophytic yeasts from sweet orange and their localization by scanning electron microscopy

Cláudia Santos Gai1, Paulo Teixeira Lacava1, Walter Maccheroni Jr.2, Chirlei Glienke3, Welington Luiz Araújo1, Thomas Albert Miller4 and João Lúcio Azevedo1

1 Departamento de Genética, Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de São Paulo, Piracicaba, SP, Brazil

2 Canavialis, Campinas, SP, Brazil 3 Departamento de Genética, Universidade Federal do Paraná, Curitiba, PR, Brazil 4 Department of Entomology, University of California Riverside, Riverside, CA, USA

Endophytes are microorganisms that colonize plant tissues internally without causing harm to

the host. Despite the increasing number of studies on sweet orange pathogens and endophytes,

yeast has not been described as a sweet orange endophyte. In the present study, endophytic

yeasts were isolated from sweet orange plants and identified by sequencing of internal

transcribed spacer (ITS) rRNA. Plants sampled from four different sites in the state of São

Paulo, Brazil exhibited different levels of CVC (citrus variegated chlorosis) development. Three

citrus endophytic yeasts (CEYs), chosen as representative examples of the isolates observed,

were identified as Rhodotorula mucilaginosa, Pichia guilliermondii and Cryptococcus flavescens. These

strains were inoculated into axenic Citrus sinensis seedlings. After 45 days, endophytes were re-

isolated in populations ranging from 106 to 109 CFU/g of plant tissue, but, in spite of the high

concentrations of yeast cells, no disease symptoms were observed. Colonized plant material

was examined by scanning electron microscopy (SEM), and yeast cells were found mainly in the

stomata and xylem of plants, reinforcing their endophytic nature. P. guilliermondii was isolated

primarily from plants colonized by the causal agent of CVC, Xylella fastidiosa. The supernatant

from a culture of P. guilliermondii increased the in vitro growth of X. fastidiosa, suggesting that

the yeast could assist in the establishment of this pathogen in its host plant and, therefore,

contribute to the development of disease symptoms.

Keywords: Pichia guilliermondii / Rhodotorula mucilaginosa / Cryptococcus flavescens / Xylella fastidiosa / Citrus variegated chlorosis

Received: October 16, 2008; accepted: May 04, 2009

DOI 10.1002/jobm.200800328

Introduction*

Endophytes are defined as microorganisms isolated

from surface-sterilized plant tissues that do not cause

any damage to the host plant [1, 2]. In 2000, Azevedo

et al. [3] defined endophytes as microorganisms that

inhabit the inner tissues of plants without causing

damage to the host or developing external structures;

this definition excludes microorganisms such as my- Correspondence: Dr. P. T. Lacava, Departamento de Genética, Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de São Paulo, Piracicaba, SP, 13400-970, Brazil E-mail: ptlacava@esalq.usp.br Phone: 55-19-342-94251 Fax: 55-19- 3433-6706

corrhizal fungi and plant-nodulating bacteria. Endo-

phytic microorganisms can colonize an ecological niche

similar to that of phytopathogens, which may make

them useful as biocontrol agents [4]. Indeed, previous

work has suggested that endophytic microorganisms

have the potential to control pathogens [5–10], insects

[3, 11] and nematodes [4]. In some cases, endophytes

can also accelerate seed emergence, help plant estab-

lishment under adverse conditions [12] and increase

plant growth and development [13–16].

Compared with many reports dealing with endo-

phytic filamentous fungi and bacteria, there are only a

few reports in the literature on the isolation, localiza-

tion or diversity of endophytic yeasts. Sporobolomyces,

442 C. S. Gai et al. Journal of Basic Microbiology 2009, 49, 441–451

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Rhodotorula, Debaryomyces and Cryptococcus were recently

reported as apple endophytes [17]. Cryptococcus laurentii

was isolated from Eucalyptus camaldulensis, and Da Costa

et al. [18] have proposed an endophytic relationship

between yeast and host plants. Rhodotorula pinicola sp.

nov. has been isolated from the xylem of surface-

sterilized pine (Pinus tabulaeformis) twigs [19], and others

have described the isolation of yeasts from black

spruce (Picea mariana) [20], Musa acuminata [21], wheat

[22] and Sequoia sempervirens [23]. Candida and Rhodoto-

rula mucilaginosa were isolated from samples of sponta-

neously fermented orange fruit and juice [24], suggest-

ing that these species may also colonize sweet orange

plants.

The role of yeast in the biology of sweet orange

plants is poorly described in the literature. Therefore,

the aims of this work were (i) to isolate endophytic

yeasts from sweet orange plants; (ii) to identify the

predominant isolates by internal transcribed spacer

(ITS) sequencing; (iii) to evaluate the endophytic status

of yeasts by re-inoculation into sweet orange seedlings;

(iv) to localize yeast cells in plant tissues by scanning

electron microscopy (SEM); (v) to elucidate a possible

relationship between the endophytic yeast population

and the presence of Xylella fastidiosa, the causal agent of

citrus variegated chlorosis (CVC); and (vi) to evaluate

the influence of yeast supernatants on X. fastidiosa

growth in vitro.

Materials and methods

Yeast isolation The diversity of endophytic yeasts associated with

sweet orange (Citrus sinensis) plants was assessed in

samples of leaves collected from four different sweet

orange-growing areas of the Brazilian state of São

Paulo: Catanduva, Colina, Elisiário and Novais. In order

to evaluate a possible interaction between endophytic

yeasts and the sweet orange pathogen X. fastidiosa, the

plants selected for sampling exhibited a range of CVC

status: uninfected plants lacking the pathogen, CVC-

infected plants showing symptoms and CVC-asympto-

matic plants (plants with no symptoms of the disease

but colonized by the pathogen). Tangerine plants

(C. reticulata), known to be naturally resistant to X. fas-

tidiosa, were also sampled. A random sampling from

plants representing each condition consisted of 80

leaves from 20 different trees, 5 from each growing

area. After surface disinfection [25], each leaf was cut

into fragments (4–6 mm), which were placed onto

complete medium (CM), as described by Pontecorvo

et al. (1953) [26], supplemented with tetracycline antibi-

otic (100 μg/ml). After 3–7 d of incubation at 28 °C, the

number of pieces showing yeast growth was counted.

Yeast strains isolated from leaf fragments were sub-

cultured and transferred onto 2% malt extract agar for

later identification. Isolates were divided into three

morphologic groups, white (A), beige (B) and pink (C),

and the frequency of isolation was calculated as the

number of samples showing yeast growth divided by

the total number of fragments [27].

DNA extraction and ITS amplification DNA extraction of yeast isolates was performed by us-

ing the Wizard total DNA extraction kit (Promega,

Madison, WI, USA).

PCR amplification was carried out in a 50 μl final

volume, containing 5 ng DNA template, 0.2 mM of

primers ITS1 (Sigma, Genosys, USA) (5′-TCC GTA GGT

GAACCT GCG G-3′) and ITS4 (Sigma, Genosys, USA) (5′-

TCC TCC GCT TAT TGA TAT GC-3′), 3.7 mM MgCl2 and

0.4 U of Taq DNA polymerase (Invitrogen, Carlsbad, CA,

USA) in 1X PCR buffer. The amplification profile was as

follows: 5 minutes initial denaturation at 94 °C, 30 cy-

cles of 30 sec at 94 °C, 30 sec at 55 °C and 30 sec at

72 °C, followed by a final step at 72 °C for 7 min. PCR

products were analyzed in 1% agarose gels stained with

ethidium bromide. Negative controls for PCR reactions,

containing distilled water instead of DNA, were used in

all experiments.

Sequence identification and sequence similarity Twenty-four isolates were chosen as representatives of

the three morphological groups and were identified by

ITS sequencing [28, 29], followed by BLAST analysis.

Isolates analyzed were referred to as citrus endophytic

yeasts (CEYs).

The resulting sequences were aligned using Mega 3.1

software [30]. Phylogenetic trees were calculated based

on the method of Jukes and Cantor [31], and neigh-

bor-joining [32] and bootstrap analyses were perform-

ed with 1000 repetitions. Phytophthora citricola was

used as an out group. The nucleotide sequences ob-

tained in this study have been submitted to GenBank

(http://www.ncbi.nlm.nih.gov/BLAST/) and were assign-

ed accession numbers AY700120 to AY700144.

Plant material Axenic seedlings were obtained from surface-disinfect-

ed seeds (seeds treated with 70% ethanol for 20 min,

sodium hypochlorite solution [2% available Cl–] for

30 min, 70% ethanol for 30 sec and finally washed with

sterile distilled water), followed by germination on MS

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media [33]. Seedlings were cultured under a photope-

riod of 12 h of light at 25 °C for 60 d.

Endophytic colonization The endophytic status of one isolate of each morpho-

logical group (A, B and C) was confirmed using sweet

orange plants. The strains were inoculated into sweet

orange seedlings by immersing the roots, after cutting

off the tip ends, for one hour in a suspension of the

yeast being tested (107 CFU/ml in 0.8% NaCl). The inocu-

lated seedlings were transferred onto MS medium

without glucose to prevent yeast from growing on the

medium. Control seedlings were inoculated with saline

solution (0.8% NaCl). Seedlings were kept under a pho-

toperiod of 12 h of light at 25 °C for 30 d.

For re-isolation assays, seedlings were surface disin-

fected using ethanol and hypochlorite as previously

described, 45 d after inoculation. Plant tissues (roots,

stems and leaves) were separated, cut into pieces,

weighed and macerated in 1 ml of 0.8% NaCl. Dilutions

were plated onto CM. Incubations were carried out at

28 °C for 2 d to allow yeast growth. The number of

CFUs per g of fresh tissue was determined. For each

strain, 25 seedlings were analyzed.

Scanning electron microscopy Plant material for scanning electron microscopy was

treated according to the method described by Rodri-

guez and Wetzstein [34]. Briefly, 45 days after inocula-

tion, seedlings were cut into small pieces, which were

fixed in a cacodylic acid (2 M) and glutaraldehyde (8%)

solution overnight, washed twice in cacodylic acid solu-

tion (1 M) and dehydrated in a sequence of ethanol

solutions (30%, 45%, 60%, 75%, 85%, 90% and 100%)

for 20 min each. The final wash in 100% ethanol was

repeated three times. Material was critical-point dried,

fixed on stubs and coated with gold. Scanning electron

micrographs were taken on a model DSM-960 (Carl

Zeiss, Oberkochen, Germany) at 10 kV, 15 mm focal

length, at magnifications from 1000 to 5000 ×.

In vitro competition assay Endophytic yeasts were grown in 50 ml of YEPD (2%

peptone, 1% yeast extract, 2% glucose, pH 6.8) at 28 °C

overnight. Yeast cultures were filtered with 0.2 mm

diameter filters (Millipore, USA), and the filtrates

were used to test the ability to control X. fastidiosa

growth in vitro [6]. A starter culture of X. fastidiosa strain

9a5c [35] was grown in PW [36] and centrifuged to

reach a concentration of 106 CFU/ml of media. Tubes

with final volumes of 5 ml of PW were prepared, and

each tube was inoculated with 10% (v/v) X. fastidiosa

culture and yeast supernatant in one of the following

quantities: 0.02%, 0.2%, 1%, 2% or 10% (v/v). Addition-

ally, 10% (v/v) of YEPD media was added to some tubes

as a control to check for the influence of the yeast me-

dia on the growth of X. fastidiosa. The experiment was

done in triplicate. The optical density at 500 nm was

measured after 20 d of incubation at 28 °C. Results

show average values of two independent biological

assays.

Data analysis Statistical analysis (Tukey’s test, with P < 0.01 consid-

ered to be significant) was performed using SAS 8

(www.sas.com) software.

Figure 1. Frequency of colonization of citrus leaf samples by endophytic yeasts isolated from field plants of different disease conditions (mean ± standard error (SE); data from 4 different sites, 5 leaves per site per plant condition). Group A is composed mainly of Pichia, Candida and A. pullulans, group B of Cryptococcus spp. and group C of Rhodotorula spp.

444 C. S. Gai et al. Journal of Basic Microbiology 2009, 49, 441–451

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Figure 2. Phylogenetic trees comparing sequences obtained from yeasts isolated in the present study (citrus endophytic yeast [CEY] numbered isolates) to sequences from GenBank (with accession numbers). The tree was constructed based on the rRNA ITS fragment sequence. Bootstrap analysis was performed with 1000 repetitions, and bootstrap values are shown next to branches.

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Results

Isolation of yeast from the leaves of sweet orange plants The endophytic yeast community isolated from sweet

orange plants included Pichia guilliermondii, Candida

parapsilosis and Aureobasidium pullulans for group A,

Cryptococcus flavescens and C. laurentii for group B and

Rhodotorula mucilaginosa and R. dairenensis for group C.

The isolation frequencies (IFs) were different for

uninfected, CVC-symptomatic and CVC-asymptomatic

sweet orange plants and tangerine (C. reticulata) plants

(Fig. 1). Plants lacking X. fastidiosa (uninfected and tan-

gerine groups) contained higher concentrations of

endophytic yeasts, on average. Yeasts were isolated

from 80.9% and 85.6% of the leaf pieces from unin-

fected and tangerine plants, as compared to 34.8% and

50% of the leaf pieces from CVC-symptomatic and CVC

asymptomatic plants, respectively.

Group B (Cryptococcus spp.) yeasts were prevalent in

uninfected sweet orange and tangerine plants, but were

replaced by group A (Pichia spp.) yeasts in the presence

of X. fastidiosa.

Yeasts from group C (Rhodotorula spp.) were isolated

from every plant tested in the treatments, and the per-

centage of group C yeast in the total population did not

vary according to plant condition. (Fig. 1).

Variability of endophytic yeasts by ITS sequencing From the total collection of endophytic yeasts, twenty-

four strains representing the three morphological

groups (A, B and C) were chosen to be genotyped by ITS

sequencing. The sequences were clustered with ITS

sequences from GenBank, and the resulting dendro-

gram (Fig. 2) showed that some of them clustered with

identified yeasts with high bootstrap values. Isolates

represented diverse genera, and fell into the same three

groups that were determined according to morphology,

group A (Candida, Pichia and Aureobasidium), group B

(mainly Cryptococcus spp.) and group C (Rhodotorula spp.).

Colonization and distribution of endophytic yeasts in sweet orange seedlings Three endophytic isolates were chosen for this assay,

according to group and the host plant from which they

were most frequently isolated. CEY 22 (Rhodotorula muci-

laginosa) was isolated from an uninfected sweet orange

plant, CEY 24 (Pichia guilliermondii) was isolated from a

CVC-symptomatic plant, and CEY 21 (Cryptococcus flaves-

cens) was isolated from an uninfected plant. Yeasts were

introduced into axenic sweet orange seedlings, and the

populations of yeast colonizing the plants ranged from

Figure 3. Population density of endophytic yeasts colonizing C. sinensis seedlings. Each bar represents the log CFU/g of fresh plant tissue. Capital letters indicate comparisons between parts of the plant within each treatment. Small letters indicate comparisons between different treatment groups using the same plant part. Means with the same letter are not significantly different (i.e., P > 0.01 by Tukey’s test).

106 to 109 CFU g–1 of wet, healthy tissue. The highest

density was observed for CEY 24 (P. guilliermondii) in

roots, while the lowest density was observed for CEY 22

(R. mucilaginosa) in stems. Isolates CEY 22 and CEY 24

were more frequently found in roots than in stems; in

contrast, CEY 21 (C. flavescens) was more frequently

found in stems than in roots or leaves (Fig. 3). No other

microorganisms were observed during isolation, indi-

cating that no contamination had occurred during the

incubation period.

Scanning electron microscopy The inoculated isolates were observed mainly in and

around the stomata of host plants (Fig. 4). The surface

of leaves was also analyzed, but no yeast was found

colonizing leaves epiphytically. Moreover, yeast cells

were also found residing in the lumen of xylem vessels

and in intercellular spaces (Fig. 5). In Fig. 5, black ar-

rows indicate the yeast cells, and white arrows indicate

the spiral structures of xylem vessels. Xylem vessels

were completely full of CEY 21 (C. flavescens) yeast cells

(Fig. 5A). CEY 24 (P. guilliermondii) was also found colo-

nizing the interior of the xylem lumen, with cells fixed

on xylem walls (Fig. 5B). The yeast isolate CEY 22

(R. mucilaginosa) was found colonizing stem tissue in a

lower concentration than the other isolates evaluated

(Fig. 5C). The black arrow points to the lone yeast cell

found in a xylem lumen. Interestingly, the endophytic

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Figure 4. Scanning electron micrographs of yeast cells on culture media were taken to aid in identification of yeast in plant tissues (A) CEY 22 (Rhodotorula mucilaginosa), scale bar = 2.5 mm. (B) Leaf stomata of citrus seedlings inoculated with CEY 22 (R. mucilaginosa), shown in a transverse cut with a yeast cell inside (arrow; scale bar = 3.0 mm). (C) CEY 22 (R. mucilaginosa) and (D) CEY 24 (Pichia guilliermondii) colonizing the surface and interior of leaf stomata (arrows). Scale bar = 3 mm.

isolate CEY 24 was observed colonizing intracellular

spaces of plant tissues, close to xylem vessels (Fig. 5D).

In control plants, inoculated with saline solution, no

yeast was detected by isolation or SEM (Fig. 5E and F).

Competition assay Group A was isolated predominantly from plants colo-

nized by X. fastidiosa (CVC-symptomatic and CVC-

asymptomatic sweet orange plants; Fig. 1), so a repre-

sentative yeast strain from this group, CEY 24

(P. guilliermondii) was tested to determine whether it has

any influence on the in vitro growth of X. fastidiosa.

Group B was predominantly found in uninfected sweet

orange and tangerine plants, and the isolate CEY 21

(Cryptococcus flavescens) was also included in the competi-

tion in vitro test, as this isolate could be acting as a bio-

control agent to protect plants from the colonization by

X. fastidiosa. Competition assays (Fig. 6) of the two iso-

lates showed that the addition of 10% cell-free filtrate

of P. guilliermondii resulted in 40% growth enhancement

of X. fastidiosa. On the other hand, cell-free filtrates

from the yeast C. flavescens had the same effect on

pathogen growth as the control treatments.

Discussion

In the present study, endophytic yeasts were isolated

from surface-sterilized sweet orange plants, and a more

diverse range of endophytic species was isolated as

compared to reports from other host plant species [20,

22]. The collection of isolates was formed mostly of

Candida sp., Pichia sp., Rhodotorula spp., Sporobolomyces sp.,

Aureobasidium sp., Cryptococcus spp. and Phialophora sp.,

which belong to the basidiomycota and ascomycota

phyla. When comparing the four sites of sampling, no

significant difference in the endophytic yeasts associ-

ated with sweet orange trees is noticeable; however,

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Figure 5. Scanning electron micrographs of endophytic yeast cells (black arrows) colonizing xylem vessels (white arrows indicate the spiral structure of xylem vessels) of citrus stems. (A) CEY 21 (Cryptococcus flavescens; scale bar = 3.0 mm), (B) CEY 24 (Pichia guilliermondii; scale bar = 2.0 mm), (C) CEY 22 (Rhodotorula mucilaginosa; scale bar = 3.0 mm). (D) CEY 24 (Pichia guilliermondii) found intracellularly colonizing the vessels of a citrus stem (scale bar = 3.0 mm). (E) and (F) scanning electron microscopy of vessels of citrus stems of control plants. Note that xylem vessels (indicated by white arrows) are empty (scale bar = 2.0 mm).

clear differences in the population with respect to the

development of CVC can be seen. The presence of the

endophytic yeast population appears to have a signifi-

cant effect on the plant-pathogen interaction.

The population of each representative isolate re-

inoculated into axenic sweet orange seedlings reached

high numbers (106 to 109 CFU/g tissue), which could

result from the lack of competition inside the host

plant. Despite this fact, the plants did not exhibit overt

signs of injury during the period of the experiment,

confirming the endophytic condition of these yeasts

inside sweet orange plants.

Colonization of vascular plant tissue has been widely

reported for bacteria [44, 45, 46], but not for yeasts. In

the present study, endophytic yeasts were observed

inside the stomata and xylem vessels of sweet orange

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Figure 6. Effects of cell-free filtrates of the citrus endophytic yeasts Pichia guilliermondii (CEY 24) and Cryptococcus flavescens (CEY 21) on the growth of X. fastidiosa. Bars represent the absorbance (OD 500 nm) of X. fastidiosa cultures after 20 days of cultivation. As controls, PW represents the bacterial growth without the addition of yeast culture supernatants, and 10% YEPD represents the treatment in which only yeast media was added. A different quantity of yeast filtrate was added to each treatment, to a final concentration of 0.02%, 0.2%, 1%, 2% or 10% (v/v). An asterisk indicates that the treatment significantly differed from control (P < 0.01 by Tukey’s test).

seedlings (Figs. 4 and 5). The presence of these yeasts in

the stomata, after root inoculation, may suggest that

yeast can colonize plants and translocate from roots to

leaves through the vasculature. After endophytic colo-

nization, these microorganisms could also colonize the

plant surface, as some endophytic bacteria do [47]. Be-

sides colonizing xylem vessels, P. guilliermondii (CEY 24)

also colonized plant intracellular spaces (Fig. 5D). This

may not be as common as intercellular colonization,

but it might be functionally important [4]. Bacteria

have been shown to intracellularly colonize grasses [46],

wheat [48], sugarcane [49] and cotton [38]. However, no

yeast has been described in this niche until this study.

The plant-associated habitat is a dynamic environ-

ment in which many factors affect the structure and

species composition of the microbial communities that

colonize roots, stems, branches and leaves [25]. It has

previously been shown that endophytic communities

vary spatially within a plant [37] and almost certainly

interact with other endophytic and pathogenic organ-

isms as well as the host plant itself [6, 38]. We observed

that the predominance of some colonizing yeast genera

varied according to the plant’s disease condition. Pichia,

Candida and Aureobasidium (group A) were isolated

mainly from plants colonized by X. fastidiosa (CVC-

symptomatic and CVC-asymptomatic sweet orange

plants). On the other hand, group B (Cryptococcus spp.)

yeasts were better established in plants not affected by

CVC (uninfected sweet orange and tangerine plants).

Lacava et al. [6] have highlighted the relationships

among bacterial populations and suggested that CVC

symptoms in sweet orange plants could be a result of

the population balance among the endophytic bacteria

Methylobacterium spp., Curtobacterium flaccumfaciens and

X. fastidiosa. This study contributes further to the un-

derstanding of the complex environment involved in

the development of CVC in sweet orange trees. The

endophytic population occupying the same niche as

X. fastidiosa is composed of bacteria, yeasts and fungi,

and the physiological status of the host plant and envi-

ronmental conditions also contribute to disease estab-

lishment.

An indication of the importance of the presence of

yeasts in plants and their capacity to interact with the

endophytic community is found in their potential to

produce metabolites that could play a role in the bio-

logical control of phytopathogenic microorganisms or

in inducing systemic resistance [19, 39, 40–43].

Our results showed that C. flavescens culture super-

natant did not influence the in vitro growth of X. fas-

tidiosa (Fig. 6). Perhaps the capacity of Cryptococcus to

control other pathogens is due to the competition for

space and/or nutrients in the environment instead of

the production of a biocontrol compound. In contrast,

P. guilliermondii, which was isolated more often from

plants contaminated with X. fastidiosa (CVC-sympto-

matic and CVC-asymptomatic plants) than from plants

that were not infected, appeared to produce and secrete

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a factor (or factors) that enhanced the in vitro growth of

X. fastidiosa cultures (Fig. 6).

Our study may be the first to describe a diverse endo-

phytic yeast population isolated from sweet orange

plants. The presence of endophytic yeasts in axenic

seedlings did not affect the fitness of the plants, even in

high concentrations, confirming the endophytic state

of these yeasts in sweet orange plants.

CVC is a very complex disease, and a considerable

amount of research has been done to try to understand

its development and to explain the presence of CVC-

resistant plants surrounded by symptomatic plants in

infected groves formed by clones of sweet orange trees.

The endophytic microbial population could contribute

to whether or not the disease develops in an individual

tree. However, this is a complex scenario in which each

component contributes to the overall outcome. Yeasts

are often neglected when studies of microbial popula-

tions are carried out, and this is the first work that

discusses the importance of the presence of endophytic

yeasts in sweet orange plants, showing that they may

play an important role in disease development.

Although the results shown suggest that the pres-

ence of endophytic yeasts in the plant environment

could influence the development of the phytopathogen

X. fastidiosa and consequent development of CVC symp-

toms, it is known that adult plants are physiologically

different from seedlings and that the presence of other

microorganisms in the plants changes the population

dynamics. For these reasons, additional experiments

using adult greenhouse plants should be performed to

support and confirm the results from our in vitro ex-

periments.

Acknowledgements

We thank Dr. Elliot W. Kitajima (NAP/MEA, ESALQ/USP,

Piracicaba, SP, Brazil) for access to scanning electron

microscope facilities. This work was supported by a

grant from the FAPESP (Proc. No. 06/55494-4). Also, we

thank CNPq/RHAE for providing a fellowship to C.S.G.

References

[1] Petrini, O., 1991. Fungal endophytes of tree leaves. In: Andrews, J.H., Hirano S.S., (eds.), Microbial Ecology of the Leaves. Springer-Verlag, New York, pp. 179–197.

[2] Hallmann, J., Quadt-Hallmann, A., Mahaffee, W.F. and Kloepper, J.W., 1997. Bacterial endophytes in agricultural crops. Can. J. Microbiol., 43, 895–914.

[3] Azevedo, J.L., Maccheroni, W. Jr, Pereira, J.O. and Araújo, W.L., 2000. Endophytic microorganisms: a review on in-sect control and recent advances on tropical plants. EJB: Electronic Journal of Biotechnology [Online]. 15 April 2000, 3: [cited 05 May 2000]. Available on the Web: http://www.ejb.org/content/vol3/issue1/full/3/4. ISSN: 0717-3458.

[4] Hallmann, J., Quadt-Hallmann, A., Rodríguez-Kábana, R. and Kloepper, J.W., 1998. Interactions between Meloido- gyne incognita and endophytic bacteria in cotton and cu-cumber. Soil Biol. Biochem., 30, 925–937.

[5] Duijff, B.J., Gianinazzi-Pearsonand, V. and Lemanceau, P., 1997. Involvement of the outer membrane lipopolysac-charides in the endophytic colonization of tomato roots by biocontrol Pseudomonas fluorescens strain WCS417r. New Phytol., 135, 325–334.

[6] Lacava, P.T., Araújo, W.L., Marcon, J., Maccheroni, W. Jr. and Azevedo, J.L., 2004. Interaction between endophytic bacteria from citrus plants and the phytopathogenic bac-teria Xylella fastidiosa, causal agent of citrus variegated chlorosis. Lett. Appl. Microbiol., 39, 55–59.

[7] Sharma, V.K. and Nowak, J., 1998. Enhancement of Verti-cillium wilt resistance in tomato transplants by in vitro co-culture of seedlings with a plant growth promoting rhi-zobacterium (Pseudomonas sp. strain PsJN). Can. J. Micro-biol., 44, 528–536.

[8] Sturz, A.V. and Matheson, B.G., 1996. Populations of endophytic bacteria which influence host-resistance to Erwinia-induced bacterial soft rot in potato tubers. Plant and Soil, 184, 265–271.

[9] Sturz, A.V., Christie, B.R. and Matheson, B.G., 1998. Asso-ciation of bacterial endophyte populations from red clo-ver and potato crops with potential for beneficial allelo-pathy. Can. J. Microbiol., 44, 162–167.

[10] Lacava, P.T., Li, W., Araújo, W.L., Azevedo, J.L. and Har-tung, J.S., 2007. The endophyte Curtobacterium flaccumfa-ciens reduces symptoms caused by Xylella fastidiosa in Ca-tharanthus roseus. J. Microbiol., 45, 388–393.

[11] Petrini, L.E., Petrini, O. and Laflamme, G., 1989. Recovery of endophytes of Abiens balsamea from needles and galls of Paradiplosis tumifex. Phytoprotection, 70, 97–103.

[12] Chanway, C.P., 1997. Inoculation of tree roots with plant growth promoting soil bacteria: An emerging technology for reforestation. Forest Science, 43, 99–112.

[13] Bent, E. and Chanway, C.P., 1998. The growth-promoting effects of a bacterial endophyte on lodgepole pine are par-tially inhibited by the presence of other rhizobacteria. Can. J. Microbiol., 44, 980–988.

[14] Lazarovits, G. and Nowak, J., 1997. Rhizobacteria for im-provement of plant growth and establishment. Hort-science, 32, 188–192.

[15] Nassar A.H., El-Tarabily, K.A. and Sivasithamparam, K., 2005. Promotion of plant growth by an auxin-producing isolate of the yeast Williopsis saturnus endophytic in maize (Zea mays L.) roots. Biology and Fertility of Soils, 42, 97–108.

[16] Pillay, V.J. and Nowak, J., 1997. Inoculum density, tem-perature and genotype effects on in vitro growth promo-tion and epiphytic and endophytic colonization of tomato (Lycopersicum esculentum L.) seedlings inoculated with a

450 C. S. Gai et al. Journal of Basic Microbiology 2009, 49, 441–451

© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

pseudomonads bacterium. Can. J. Microbiol., 43, 354–361.

[17] Camatti-Sartori, V., Silva-Ribeiro, R.T., Valdebento-San-hueza, R.M., Pagnocca, F.C., Echeverrigaray, S. and Azeve-do, J.L., 2005. Endophytic yeasts and filamentous fungi associated with southern brasilian apple (Malus domestica) orchards subjected to conventional, integrated or organic cultivation. J. Basic Microbiol., 45, 397–402.

[18] Da Costa, E.F., Lazera, M.S., Wanke, B. and Gompertz, O.F., 1997. Association between Cryptococcus laurentii and Eucalyptus camaldulensis. Revista de Microbiologia, 28, 239–244.

[19] Zhao, J.H., Bai, F.Y., Guo, L.D. and Jia, J.H., 2002. Rhodoto-rula pinicola sp nov., a basidiomycetous yeast species iso-lated from xylem of pine twigs. FEMS Yeast Res., 2, 159–163.

[20] Johnson, J.A. and Whitney, N.J., 1992. Isolation of fungal endophytes from black spruce (Picea mariana) dormant buds and needles from New Brunswick, Canada. Can. J. Bot., 70, 1754–1757.

[21] Cao, L.X., You, J.L. and Zhou, S.N., 2002. Endophytic fungi from Musa acuminata leaves and roost in South China. W. J. Microbiol. Biotech., 18, 169–171.

[22] Gindrat, D. and Pezet, R., 1994. Le paraquat, un outil pour la revelation rapide d’infections fongiques latentes et de champignons endophytes. J. Phytopathol., 141, 86–98.

[23] Middelhoven, W.J., 2003. The yeast flora of the coast redwood, Sequoia sempervirens. Folia Microbiol., 48, 361–362.

[24] Heraz-Vazquez, F.J.L., Mingorance-Cazorla, L., Clemente-Jimenes, J.M. and Rodriguez-Vico, F., 2003. Identification of yeast species from orange fruit and juice by RFLP and sequence analysis of 5.8S rRNA gene and the two internal transcribed spacers. FEMS Yeast Research, 3, 3–9.

[25] Araújo, W.L., Maccheroni, W. Jr., Aguilar-Vildoso, C.I., Barroso, P.A.V., Saridakis, H.O. and Azevedo, J.L., 2001. Variability and interactions between endophytic bacteria and fungi isolated from leaf tissues of citrus rootstocks. Canad. J. Microbiol., 47, 229–236.

[26] Pontecorvo, G., Roper, J.A., Hemmons, L.H., McDonald, K.D. and Bufton, W.J., 1953. The genetics of Aspergillus ni-dulans. Advances in Genetics, 5, 141–238.

[27] Araújo, W.L., Marcon, J., Maccheroni, W. Jr, van Elsas, J.D., van Vuurde, J.W.L. and Azevedo, J.L, 2002. Diversity of endophytic bacterial population and their interaction with Xylella fastidiosa in citrus plants. Appl. Environm. Microbiol., 68, 4906–4914.

[28] Chen, Y.C., Eisner, J.D., Kattar, M.M., Rassoulian-Barrett, S.L., Lafe, K., Yarfitz, S.L., Limaye, A.P. and Cookson, B.T., 2000. Identification of medically important yeasts using PCR-based detection of DNA sequence polymorphism in the internal transcribed spacer 2 region of the rRNA ge-nes. J. Clinic Microbiol., 38, 2302–2310.

[29] White, T.J., Bruns, T., Lee, S. and Taylor, J., 1990. Amplifi-cation and direct sequencing of fungal ribosomal RNA genes for phylogenetics, p. 315–322. In: Innis, M.A., Gefland, D.H., Sninsky, J.J., White, T.J. PCR Protocols: a Guide to Methods and Applications. Academic Press, Inc., New York, N.Y.

[30] Kumar S., Tamura, K. and Nei, M., 2004. MEGA3: Integrat-ed software for Molecular Evolutionary Genetics Analysis and sequence alignment. Briefings in Bioinformatics, 5, 150–163.

[31] Jukes, T.H. and Cantor, C.R., 1969. Evolution of protein molecules. In: Mammalian Protein Metabolism (Munro, H.N., ed.). New York, USA: Academic Press, pp. 21–132.

[32] Saitou, N. and Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol., 4, 406–425.

[33] Murashige, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant., 15, 473–497.

[34] Rodriguez, A.P.M. and Wetzstein, H.Y., 1998. A morpho-logical and histological comparison of the initiation and development of pecan (Carya illinoinensis) somatic embryo-genic cultures induced with naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid. Protoplasma, 204, 71–83.

[35] Simpson, A.J.G., Reinach, F.C., Arruda, P. and Abreu, F.A. et al., 2000. The genome sequence of the plant pathogen Xylella fastidiosa. Nature. 406, 151–157.

[36] Davis, M.J., French, W.J. and Schaad, N.W., 1981. Axenic culture of the bacteria associated with phony disease of peach and plum leaf scald. Curr. Microbiol., 6, 309–314.

[37] Fisher, P.J., Petrini, O. and Scott, H.M.L., 1992. The distri-bution of some fungal and bacterial endophytes in maize (Zea mays L.). New Phytol., 122, 299–305.

[38] Quadt-Hallmann, A., Hallmann, J. and Kloepper, J.W., 1997. Bacterial endophytes in cotton: location and inter-action with other plant associated bacteria. Can. J. Micro-biol., 43, 254–259.

[39] Middelhoven, W.J., 1997. Identity and biodegradative abilities of yeasts isolated from plants growing in an arid climate. Antonie Van Leeuwenhoek Internationl. J. Gen. Mol. Microbiol., 72, 81–89.

[40] Schisler, D.A., Kurtzman, C.P., Bothast, R.J. and Slininger, P.J., 1995. Evaluation of yeasts for biological-control of fu-sarium dry rot of potatoes. Am. Potato J., 72, 339–353.

[41] Chand-Goyal, T. and Spotts, R.A., 1996. Post harvest bio-logical control of blue mold of apple and brown rot of sweet cherry by natural saprophytic yeasts alone or in combination with low doses of fungicides. Biological Control, 6, 253–259.

[42] Lil, B.Q., Tian, S.P., 2006. Effects of trehalose on stress tolerance and biocontrol efficacy of Cryptococcus laurentii. J. Appl. Microbiol., 100, 854–861.

[43] Qin G.Z., Tian, S.P., Xu, Y., Chan, Z.L. and Li, B.Q., 2006. Combination of antagonistic yeasts with two food addi-tives for control of brown rot caused by Monilinia fructicola on sweet cherry fruit. J. Appl. Microbiol., 100, 508–515.

[44] Gardner, J.M., Feldman, A.W. and Zablotowicz, M., 1982. Identity and behavior of xylem-residing bacteria in rough lemon roots of Florida citrus trees. Appl. Environ. Micro-biol., 43, 1335–1342.

[45] Bell, C.R., Dickie, G.A., Harvey, W.L.G. and Chan, J.W.Y.F., 1995. Endophytic bacteria in grapevine. Can. J. Microbiol., 41, 46–53.

[46] Hurek T., Reinhold-Hurek, B., Van Montagu, M. and Kel-lemberger, E., 1994. Root colonizing and systemic spread-

Journal of Basic Microbiology 2009, 49, 441–451 Endophytic yeasts from citrus plants 451

© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

ing of Azoarcus sp. strain BH72 in grasses. J. Bacteriol., 176, 1913–1923.

[47] Kuklinsky-Sobral, J., Araujo, W.L., Mendes, R., Geraldi, I.O., Pizzirani-Kleiner, A.A. and Azevedo, J.L., 2004. Isola-tion and characterization of soybean-associated bacteria and their potential for plant growth promotion. Env. Mi-crobiol., 6, 1244–1251.

[48] Gantar, M., Kerby, N.W. and Rowell, P., 1991. Coloniza-tion of wheat (Triticum vulgare) by N2-fixing cyanobacteria. II. An ultrastructural study. New Phytol., 118, 485–492.

[49] James, E.K., Reis, V.M., Olivares, F.L., Baldani, J.I. and Döbereiner, J., 1994. Infection of sugarcane by the nitro-gen-fixing bacterium Acetobacter diazotrophicus. J. Exp. Bot., 45, 757–766.

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