Biological Control 50 (2009) 164171

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Biological Control
journal homepage: www.elsevier.com/locate/ybcon

Screening of plant epiphytic yeasts for biocontrol of bacterial fruit blotch

(Acidovorax avenae subsp. citrulli) of hami melon
Xiaodong Wang a,b,c, Guoqing Li a,b,*, Daohong Jiang a,b, Hung-Chang Huang d
a

The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, No. 1 of the Lion Mountain Street, Hong Shan District,
Wuhan, Hubei Province 430070, China
b
The Key Laboratory of Plant Pathology of Hubei Province, Huazhong Agricultural University, Wuhan 430070, China
c
Department of Plant Protection, Shihezi University, Shihezi, Xinjiang 832003, China
d
Agriculture and Agri-Food Canada, Research Centre, Lethbridge, Alta., Canada T1J 4B1

a r t i c l e

i n f o

Article history:
Received 1 December 2008
Accepted 23 March 2009
Available online 31 March 2009
Keywords:
Hami melon
Cucumis melo var. saccharinus
Bacterial fruit blotch
Acidovorax avenae subsp. citrulli
Pichia anomala
0732-1
Biocontrol

a b s t r a c t
Bacterial fruit blotch (BFB) caused by Acidovorax avenae subsp. citrulli (Aac) is a serious disease of hami
melon (Cucumis melo var. saccharinus) in Northern China. A study was conducted to screen plant epiphytic yeasts for use as biocontrol agents of BFB. Results showed that 24 out of 463 yeast strains isolated
from leaves or owers of plants collected from three provinces in China were antibiotic against Aac on
agar medium and eight antagonistic yeast strains including strain 0732-1 formed inhibition zones larger
than 18 mm in diameter. Spray application of strain 0732-1 isolated from watermelon grown in Xinjiang
was effective in reducing incidence and severity of disease caused by Aac on leaves of hami melon. Treatment of hami melon seeds with cell-free cultural ltrates of the yeast strain 0732-1 resulted in a significant reduction in severity of seedling blight caused by seedborne Aac, and the efcacy was not
signicantly different (P > 0.05) from that of chemical seed treatments including streptomycin sulfate
(0.1%, w/v) and hydrochloric acid (2%, v/v). Based on morphological and physiological characteristics
and analysis of the DNA sequence of the internal transcribed spacer of ribosomal DNA, the yeast strain
0732-1 was identied as Pichia anomala Kurtzman. This study suggests that the yeast strain 0732-1 is
an agent with potential for biocontrol of BFB of hami melon caused by Aac.
2009 Elsevier Inc. All rights reserved.

1. Introduction
Bacterial fruit blotch (BFB) caused by Acidovorax avenae subsp.
citrulli (Schaad et al.) Willems et al. (formerly Pseudomonas pseudoalcaligenes subsp. citrulli) (Willems et al., 1992) is a serious disease of cucurbits including hami melon (Cucumis melo L. var.
saccharinus Naud.). The pathogen also causes BFB on watermelon
(Citrullus lanatus L.) in many countries including the USA (Hopkins,
1989; Latin and Rane, 1990; Somodi et al., 1991; Jacobs et al., 1992;
Black et al., 1994; Hamm et al., 1997; Langston et al., 1999),
Australia (Martin and Horlock, 2002), Brazil (Silveira et al., 2003),
Canada (Walcott et al., 2004), China (Zhang et al., 1998; Zhao
et al., 2001), Israel (Burdman et al., 2005), Japan (Shirakawa
et al., 2000), Mariana Islands (Wall et al., 1990), Thailand (Walcott
et al., 2004) and Turkey (Demir, 1996). Besides fruit blotch,
A. avenae subsp. citrulli (Aac) also causes leaf blight, seedling blight
and/or blossom rot of cucurbitaceous plants. Annual yield loss of
* Corresponding author. Address: The State Key Laboratory of Agricultural
Microbiology, Huazhong Agricultural University, No. 1 of the Lion Mountain Street,
Hong Shan District, Wuhan, Hubei Province 430070, China.
E-mail address: guoqingli@mail.hzau.edu.cn (G. Li).
1049-9644/$ - see front matter 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.biocontrol.2009.03.009

watermelon caused by BFB in USA reached 550%, depending on

the stage of infection and environmental conditions (Latin and
Hopkins, 1995; Lessl et al., 2007). In China, BFB was reported on
watermelon in 1998 in Hainan province (Zhang et al., 1998) and
on hami melon in 2001 in Xinjiang Uygur Autonomous Region
(Zhao et al., 2001), also resulting in heavy losses to these two crops.
Since BFB is a seedborne disease (Latin and Hopkins, 1995; Walcott et al., 2003), control strategies for this disease include preventive measures such as use of pathogen-free seeds and seedlings,
seed fermentation and seed treatment with peroxyacetic acid,
hydrochloric acid or copper-containing bactericides (Zhao et al.,
2003; Hopkins et al., 2003; Feng et al., 2007). Although preventive
control measures could provide some efcacy against BFB (Fessehaie and Walcott, 2005), seed fermentation and seed treatment
with chemicals sometimes could have negative impacts on seed
quality, including reduction of seed germination and seedling
growth (Hopkins et al., 2003; Zhao et al., 2003). Moreover, public
concerns over the use of chemical pesticides suggest a need to utilize other environmentally safe measures for control of BFB on
watermelon or hami melon.
Biological control is considered an ecologically sound and environmentally friendly approach for management of plant diseases

X. Wang et al. / Biological Control 50 (2009) 164171

including BFB on cucurbits (Fravel, 2005). Fessehaie and Walcott

(2005) reported that seed treatment with either Acidovorax avenae
subsp. avenae AAA99-2 or Pseudomonas uorescens A506 was effective in reducing incidence of BFB on seedlings of watermelon under
growth chamber conditions. They also found that treatment of Aaccontaminated blossoms of watermelon with each of these two
antagonists signicantly lowered the percentage of watermelon
seeds infested with Aac under greenhouse conditions (Fessehaie
and Walcott, 2005). Other antagonistic microorganisms such as
Bacillus spp. (Santos et al., 2006) and Streptomyces spp. (Yaeram
et al., 2006), and unidentied endophytic bacteria isolated from
hami melon (Lu and Luo, 2004) were reported as potential biocontrol agents of BFB. However, information on control of Aac on
cucurbits by yeasts remains unavailable.
Yeasts are single-cell fungi, which are widely distributed in soil
and on the surface of leaves, stems, ower petals, fruits and seeds
of plants (Wilson and Wisniewski, 1989). Numerous studies indicated that some yeast species or isolates are ideal biocontrol agents,
as they are natural plant epiphytic colonizers, nonpathogenic to
plants and human beings in most cases and can rapidly proliferate
(Wilson and Wisniewski, 1989). Commercial yeast products such
as Aspire based on Candida oleophila Montrocher (Ecogen Inc., Langhorn, PA, USA), YieldPlus based on Cryptococcus albidus (Saito) Skinner (Anchor Yeast, Cape Town, South Africa), have been developed
for control of postharvest diseases of fruits and vegetables caused
by a wide range of phytopathogenic fungi, including Botrytis cinerea,
Colletotrichum spp., Penicillium spp. and Rhizopus spp. (Fravel, 2005).
The objectives of this study were (i) to screen yeast strains
antagonistic to Aac; (ii) to evaluate the potential of the selected
yeast strain 0732-1 for control of Aac on leaves and seedlings of
hami melon; and (iii) to characterize the identity of the yeast strain
0732-1.

2. Materials and methods

2.1. Bacterial strain, media and preparation of bacterial cultures
Strain XJ05-1 of A. avenae subsp. citrulli (Aac) used in this study
was isolated from a diseased plant of hami melon grown in Chang
Ji of Xinjiang Uygur Autonomous Region, China. Cultural media
used in this study included Kings medium B (KB), Kings medium
B agar (KBA), potato dextrose agar (PDA), potato dextrose broth
(PDB) and soybean sprouts extract broth (SSEB). KB was prepared
according to the description by King et al. (1956), whereas KBA
was a modied KB containing 20 g of agar per liter. KB and KBA
were used for culturing Aac and KBA was also used for testing
the antibiotic activity of yeast strains against Aac. PDA and PDB
were made of fresh potato according to the procedure described
by Fang (1998) and used for isolating and culturing yeasts. SSEB
contained the same ingredients as PDB except for replacement of
peeled potato with fresh soybean sprouts.
Aac cultures were prepared by adding an aliquot of 100 ll of a
bacterial suspension of Aac (1  109 cfu/ml) into 50 ml of KB medium in an Erlenmeyer ask (150 ml). The culture was incubated at
28 C for 2 days and used for in vitro and in vivo screening of yeast
strains.
2.2. Isolation of yeast strains and preparation of yeast cultures
Yeast strains were isolated from leaves or owers of cucumber
(Cucumis sativus L.), celery (Apium graveloens L.), coriander (Coriandrum sativum L.) and lettuce (Lactuca sativa L.) grown in Wuhan of
Hubei Province, China, from leaves of watermelon (Citrullus lanatus
L.), soybean (Glycine max L.) and apricot (Prunus armenica L.) grown
in Shi He Zi of Xinjiang, and from leaves of tea plants (Camellia sin-

165

ensis Kuntze) grown in Xin Yang of Henan province, China. Each

plant sample was cut into pieces of ca 1  1 cm (width  length),
and 10 g of the sample was added to 100 ml of sterilized distilled
water (SDW) in a 250 ml Erlenmeyer ask. After shaking
(150 rpm) on a rotary shaker (Model HQL150B, the Instrument
Company of the Chinese Academy of Sciences, Wuhan, China) for
30 min, the mixture in each ask was ltered through a single-layered cheesecloth. The resulting ltrate was designated as 101 and
was serially diluted up to 102, 103, 104 and 105 with SDW. Aliquots of each diluted suspension were pipetted onto PDA medium
amended with 0.1% (w/v) streptomycin sulfate in a Petri dish (9 cm
diameter), 100 ll per dish, and the suspension drop in each dish
was spread evenly using a ame-sterilized glass rod. The dishes
were incubated at 28 C for 2 days and yeast colonies on the medium were individually identied by appearance, color and smell.
Yeast cells of selected colonies were transferred onto PDA slants
in glass tubes (18  2 cm, length  diameter) using a sterilized
inoculation loop. The stock cultures were labelled, incubated at
28 C for 4 days and stored at 4 C until use.
For preparation of yeast cultures, 100 ll of a cell suspension of
each yeast strain (1  108 cells/ml) was inoculated into 100 ml of
PDB in an Erlenmeyer ask (250 ml) and the cultures were incubated at 28 C for 2 days. The concentration of yeast cells in each
yeast culture was determined using a haemocytometer and a compound light microscope. The two-day-old yeast cultures were used
for in vitro and in vivo screenings of yeast strains.
2.3. Antibiosis of yeast strains against Aac (in vitro screening)
Yeast strains were tested for antibiosis against Aac using the
method described by Arun et al. (2007) with modications. An aliquot of 500 ll of a 2-day-old liquid culture of Aac grown in KB medium (1  109 cfu/ml) was pipetted onto 120 ml of KBA medium in a
Petri dish (18 cm diameter) and spread evenly on the surface of the
agar medium. A sterilized cork borer (6 mm diameter) was used to
remove agar pieces and create 20 wells in each dish, at 3 cm between
wells, and used for testing 19 yeast strains. An aliquot of 100 ll of a 2day-old culture (28 C) of a yeast strain was pipetted into a well.
Wells lled with PDB at 100 ll/well were used as control. For each
dish, there were 19 wells for 19 yeast strains and one well for the
control. There were three replicates for each yeast strain. The cultures were incubated at 28 C for 2 days and the presence of a clear
zone surrounding a well in each dish was recorded.
To conrm the production of antibacterial substances by the
yeast strain 0732-1, an aliquot of 60 ll of a cell suspension
(1  108 cells/ml) of this strain was inoculated in a 150 ml ask
containing 60 ml of PDB and a total of 27 asks were inoculated.
The asks were mounted on the shaker (Model HQL150B, the
Instrument Company of the Chinese Academy of Sciences, Wuhan,
China) and incubated at 28 C and 150 rpm. Three asks of the
yeast cultures were randomly removed from the shaker at 12 h
intervals for a period of 96 h. The yeast suspension in each ask
was centrifuged for 15 min at 4 C and 12,000 rpm. The supernatant was pipetted out and lter-sterilized. The cell-free cultural ltrate was tested for pH using a pH meter (Model pHS-3C, Shanghai
Hongyi Instrument Company, Ltd., Shanghai, China) and for inhibition of Aac growth on Aac-containing KBA plates (9 cm diameter)
using the method described previously. The precipitate (yeast
cells) was dried at 60 C overnight and weighed.
2.4. Suppression of Aac infection of hami melon leaves by antagonistic
yeasts (in vivo screening)
Seeds of hami melon (C. melo var. saccharinus cultivar 86-1)
were sown in autoclaved loess-clay soil in plastic pots
(10  15 cm, height  diameter), 10 seeds/pot. The pots were kept

166

X. Wang et al. / Biological Control 50 (2009) 164171

in a greenhouse (3035 C and 85100% relative air humidity) and

plants were watered as required. After emergence, the seedlings
were thinned to 6 seedlings per pot. At the 2-true-leaf stage, the
seedlings in each pot were sprayed with 10 ml of PDB cultures
(15  108 cells/ml) of each selected yeast strain. The hami melon
seedlings sprayed with SDW was used as controls. There were
three pots (replicates) of hami melon seedlings for each selected
yeast strain and six pots of hami melon seedlings for the control
treatments. The hami melon seedlings pre-treated with yeasts for
24 h were then inoculated with Aac by spraying the bacterial bacterial suspension (1  108 cfu/ml) of Aac on these seedlings at
10 ml per pot. Three pots of hami melon seedlings pre-treated with
SDW were sprayed with the bacterial suspension of Aac as the positive control and the other three pots of hami melon seedlings
pre-treated with SDW were sprayed with SDW as the negative control. All the hami melon seedlings were incubated in the greenhouse for 3 days and then examined for incidence and severity of
disease on leaves. Disease incidence was dened as percentage of
diseased leaves in total leaves examined in each pot. Disease severity of each leaf was rated using a scale of 04, where 0 represented
healthy, and 1, 2, 3 and 4 represented diseased with lesion area
<15%, 1650%, 5180% and >80%, respectively. Disease index (DI)
of hami melon leaf blight for each pot was calculated by the following formula (Fang, 1998):

,
4
4
X
X
DI 100 Li  i 4
Li
i0

i0

where i is the leaf blight severity rating, and Li is the number of

hami melon leaves in each pot corresponding to the disease severity
rating i. The experiment was repeated three times.
2.5. Suppression of seed transmission of Aac by cultural ltrates of the
yeast strain 0732-1
Seeds of hami melon (cv 861) were surface-sterilized with 2%
sodium hypochlorite (v/v) for 15 min and rinsed twice in SDW.
After air-drying for 2 days, seeds were soaked in 100 ml of an

Aac suspension (1  106 cfu/ml) for 30 min and ltered through

double-layered cheesecloth to remove the excess bacterial suspension. The seeds were then air-dried and divided into four lots, 72
seeds/lot. The four seed lots were soaked in the cell-free cultural
ltrate of the yeast strain 0732-1 (pH3.4), 2% (v/v) HCl (Shennong
Chemical Reagent Co. Ltd., Yi Chang, China), 0.1% (w/v) streptomycin sulfate (The Northern China Pharmaceutical Company Ltd., Shi
Jia Zhuang, China) or SDW (control), respectively. The seeds of the
four treatments were separately ltered, air-dried and individually
sown in autoclaved soil in plastic pots (10  15 cm, height  diameter) at 8 seeds/pot and 9 pots/treatment with three pots of each
treatment being treated as one replicate. The pots were kept under
outdoor conditions (3033 C) with treatments being arranged in a
randomized complete block design. Ten days later, seedlings in
each pot were examined for blight symptoms on cotyledons, true
leaves and stems. The experiment was repeated once.
2.6. Identication of the yeast strain 0732-1
The yeast strain 0732-1 was identied by morphological and
physiological features and molecular feature of the internal transcribed spacer (ITS) of the ribosomal gene. The cultures on PDA
at 28 C for 2 days were used for observation of colony morphology
(size, color and texture), and the cultures in SSEB at 28 C and
150 rpm for 2 days were used for observation of morphology and
proliferation of yeast cells, whereas the cultures on autoclaved carrot slices at 20 C for 10 days were used for observation of ascospores (Barnett et al., 1983; Kurtzman and Fell, 1998; Liang and Chi,
2002). A minimum of 50 cells or ascospores were measured for size
of cells or ascospores of the yeast strain 0732-1. Physiological characteristics of the yeast strain 0732-1 were determined by detecting
its ability to ferment sugars under semi-anaerobic conditions; and
to assimilate a variety of carbon compounds as the major carbon
sources or to assimilate a variety of nitrogen compounds as the
major nitrogen sources under aerobic conditions (Barnett et al.,
1983). The sugars used in the fermentation test included D-glucose,
D-galactose, maltose, sucrose and lactose. The carbon compounds
used in the assimilation test included D-glucose, D-galactose, maltose, lactose, D-xylose, starch D-mannose, inositol, L-arabinose, amri-

Fig. 1. A histogram showing inhibition of growth of Acidovorax avenae subsp. citrulli (Aac) by different yeast strains on Kings medium B agar (28 C, 2 days). Inoculum of
yeasts was from cultures in potato dextrose broth and Aac from cultures in Kings medium B. Vertical bars represent the standard errors of means (n = 3). Bars headed by the
same letters are not signicantly different (P > 0.05) according to Duncans Multiple Range Test.

X. Wang et al. / Biological Control 50 (2009) 164171

ta, glycerol, ethanol, methanol and sorbitol. The nitrogen compounds used in the assimilation test included L-lysine, creatine,
ammonium sulfate and potassium nitrate. Additional tests such
as production of acids, esters and starch, and growth at 42 C in
the presence of high concentration of D-glucose (50 or 60%, w/v)
were included (Barnett et al., 1983). Each test was repeated three
times.
For analysis of the ITS sequence, genomic DNA was extracted
from cells of the yeast strain 0732-1 collected from 2-day-old
PDA cultures using the procedure described by Heras-Vazquez
et al. (2003). Polymerase chain reaction (PCR) based method was
used to amplify the ITS region (ITS15.8S rDNAITS2) with primers
ITS1 (50 -TCCGTAGGTGAACCTGCGG-30 ) and ITS4 (50 -TCCTCCGCTTA
TTGATATGC-30 ) (White et al., 1990). Amplication was performed
in a PTC-100TM Peltier Thermal Cycler (Hercules, CA, USA) programmed as follows: the initial denaturation at 95 C for 5 min, followed by 35 cycles each beginning with denaturation at 94 C for
30 s, annealing at 52 C for 30 s and ending with DNA synthesis
at 72 C for 1 min, and the nal extension at 72 C for 5 min. The
amplied DNA fragment was puried from agarose gel after electrophoresis using the Fermentas DNA Extraction Kit (Jingmei Biotech Co., Ltd., Shengzhen, China), ligated into the pMD18-T vector
(TaKaRa Biotechnology Co., Ltd., Dalian, China) and transformed
into competent cells of Escherichia coli JM109. Positive E. coli clones
grown on LuriaBertani agar medium containing ampicillin
(50 lg/ml) were selected, individually tested for the size of the
DNA insert by the PCR method and three E. coli clones harboring
the plasmid containing the expected DNA insert were sent to SinoGenoMax Co. Ltd. (Beijing, China) for sequencing on the ABI
PRISM 37796 automated sequencer with the universal primer
M13. The sequence were aligned using the CLUSTAL W program
and compared with all available sequences in GenBank database
through internet (http://www.ncbi.nlm.nih.gov) using Basic Local
Alignment Search Tool (BLAST) (Altschul et al., 1990).

167

ery (Hubei), coriander (Hubei), apricot (Xinjiang), cucumber (Hubei), lettuce (Hubei) and tea plants (Henan), respectively. In
contrast, the remaining 439 yeast strains were not antibiotic
against Aac, as no clear zones surrounding agar wells containing
PDB cultures of these yeast strains appeared after incubation at
28 C for 2 days.

2.7. Data analyses

Data on diameter of inhibition zones, disease incidence and disease index for the antagonistic yeast strains used in this study were
analyzed using analysis of variance (ANOVA) in SAS software (SAS
Institute, Cary, NC, USA, Version 8.0, 1999). Data on the disease
incidence (percentage) were arcsin-transformed to angular data
prior to ANOVA. After each analysis, means were individually
back-transformed to numerical values. Means for different yeast
strains or different treatments in each experiment or trial were
separated using Duncans Multiple Range Test at P = 0.05 level.

3. Results
3.1. Isolation and screening of antagonistic yeast strains (in vitro
screening)
A total of 463 yeast strains were isolated from 283 plant samples collected from three provinces (Henan, Hubei and Xinjiang)
in China. Results of in vitro screening showed that 24 of the 463
yeast strains tested on KBA medium were antibiotic against A. avenae subsp. citrulli (Aac), as indicated by the appearance of clear
zones surrounding agar wells containing PDB cultures of these
yeast strains after incubation at 28 C for 2 days. The average diameter of clear zones varied with yeast strains, ranging from 8.0 mm
for strain 074101-2 to 19.7 mm for strain 0732-1 (Fig. 1). There
was no signicant difference (P > 0.05) in the average diameter of
clear zones among yeast strains 0732-1, 074111-5, 074112-1,
074102-2, 07457-1, 703030, 19-1 and xin12-6. These yeast strains
were isolated from watermelon (Xinjiang), soybean (Xinjiang), cel-

Fig. 2. Antibacterial activity of the yeast strain 0732-1 against Acidovorax avenae
subsp. citrulli (Aac). A, Inhibition of Aac growth by cell-free cultural ltrates of the
yeast strain 0732-1 on Kings medium B agar (28 C, 2 days) showing inhibition
zone (right well), whereas sterile distilled water (left well) did not form any
inhibition zones. (BD) Time-course of the yeast biomass (B), ambient pH (C) and
the antibacterial activity of the liquid cultures of the yeast strain 0732-1 against Aac
(D). Vertical bars represent the standard errors of means (n = 3).

168

X. Wang et al. / Biological Control 50 (2009) 164171

Fig. 3. Effect of spray-treatment of leaves of hami melon at the seedling stage with
cultures of the yeast strain 0732-1 (A) or sterile distilled water (B) on severity of leaf
blight caused by Acidovorax avenae subsp. citrulli.

Inhibition of Aac growth by the cell-free cultural ltrate of the

yeast strain 0732-1 was observed (Fig. 2A). After incubation in
PDB at 28 C for 2496 h, strain 0732-1 grew and the biomass
reached 2.02.9 mg/ml (Fig. 2B) and the pH of the liquid culture
declined from the initial of 6.2 to the nal of 3.24.4 (Fig. 2C).
The average diameter of inhibition zones caused by the cell-free
cultural ltrate of strain 0732-1 was consistently increased from
4.0 mm after incubation for 24 h to 19.4 mm after incubation for
96 h in three replicates (Fig. 2D).
3.2. Effect of antagonistic yeasts on Aac infection of leaves of hami
melon (in vivo screening)
Eleven antagonistic strains of yeasts were evaluated for control
of bacterial fruit blotch leaf symptoms on hami melon. Results
showed that while the hami melon leaves in the negative control
(SDW alone) were healthy, the hami melon leaves in the positive

Fig. 4. Effect of seed treatment with the cultural ltrates of the yeast strain 0732-1
(0732-1), streptomycin sulfate (Strep) or hydrochloric acid (HCl) on seedling blight
of hami melon caused by Acidovorax avenae subsp. citrulli (Aac). (A) Seedlings of
10 days after inoculation showing severe seedling blight in the treatment of Aac
alone (Aac), but slight seedling blight in the treatments of the yeast strain 0732-1
(0732-1), streptomycin sulfate (Strep) and hydrochloric acid (HCl). (B) A histogram
showing difference in disease incidence of seedling blights of hami melon for these
four treatments. Vertical bars represent the standard errors (n = 3).

control (Aac) were severely diseased in all the three tests (Fig. 3
and Table 1). The yeast strain 0732-1 showed consistent suppression of leaf blight symptoms of hami melon caused by Aac with
reduction of disease incidence to 43.148.8% and disease index to
13.119.1, compared to the disease incidence of 75.390.0% and
the disease index of 40.075.3 for the Aac treatment (Fig. 3 and Table 1). In contrast, the yeast strains 079-1, 074102-2, 074112-1 and

Table 1
Efcacy of different yeast strains in suppression of leaf blight of hami melon caused by Acidovorax avenae subsp. citrulli (Aac).
Yeast strain

CK11
CK22
0732-1
07488-1
07463-4
074111-5
xin12-6
0714-1
19-1
074112-1
079-1
074102-2
07114-1
1
2
3
4

Test 1

Test 2

Test 3

Disease incidence (%)

Disease index (0100)

Disease incidence (%)

Disease index (0100)

Disease incidence (%)

Disease index (0100)

0.0c3
90.0a
48.8b
51.5b
54.4b
55.0b
57.1b
57.6b
58.5b
69.0b
81.1a
82.0a
90.0a

0.0e3
40.0a
14.2d
17.0cd
16.4cd
25.0b
19.2cd
17.5cd
20.8bcd
37.5a
37.5a
30.8ab
30.0ab

0.0e3
90.0a
46.4d
64.4b
82.6a
ND4
54.8bcd
52.0cd
59.0bc
ND
54.8bcd
90.0a
90.0a

0.0f3
73.2a
19.1e
26.2de
72.6a
ND
46.4bc
39.4cd
51.4bc
ND
34.5cde
60.0ab
46.7bc

0.0d3
82.0a
43.1c
54.8bc
ND
ND
50.8bc
58.5b
62.2b
ND
50.0bc
ND
ND

0.0d3
75.3a
13.3cd
34.5b
ND
ND
28.3b
33.3b
30.6b
ND
25.8bc
ND
ND

CK1, negative control without inoculation of Aac or yeast cultures.

CK2, positive control with inoculation of Aac alone.
Means followed by the same letters within each column are not signicantly different (P > 0.05) according to Duncans Multiple Range Test.
ND, not determined.

X. Wang et al. / Biological Control 50 (2009) 164171

169

Fig. 5. Pichia anomala strain 0732-1 showing formation of colonies (A) on potato dextrose agar at 28 C for 2 days and pseudo-hyphae at the colony margin (B), formation of
yeast cells and small budding propagules in soybean sprout extract broth at 28 C for 2 days (C) and formation of hat-shaped ascospores (arrowheads) on autoclaved carrot
slices at 20 C for 10 days (D).

07114-1 were ineffective or slightly effective for suppression of

leaf blight of hami melon caused by Aac (Table 1). The remaining
six yeast strains (07488-1, 07463-4, 074111-5, 0714-1, 19-1 and
xin12-6) were intermediate regarding efcacy of reducing disease
incidence or disease index on leaves of hami melon (Table 1).
3.3. Suppression of Aac seedling blight of hami melon by the yeast
strain 0732-1
Treatment of hami melon seeds with Aac alone resulted in
development of seedling blights with severe symptoms on cotyledons and leaves (Fig. 4A). The disease incidence was high, 97.6% in
the rst trial and 83.3% in the second trial (Fig. 4B). In contrast,
treatment of hami melon seeds with the cultural ltrates of the
yeast strain 0732-1, streptomycin sulfate or HCl resulted in development of seedling blights with mild symptoms on cotyledons and
leaves (Fig. 4A). The disease incidence was low, 2.8, 0 and 2.6% in
the rst trial and 7.0, 8.8 and 3.7% in the second trial, for the treatments of strain 0732-1, streptomycin sulfate and HCl, respectively
(Fig. 4B).
3.4. Identication of the yeast strain 0732-1
The yeast strain 0732-1 was identied as Pichia anomala (Hansen) Kurtzman on the basis of morphological and physiological
characteristics and analysis of the ITS sequence. Colonies of strain
0732-1 on PDA for 2 days were circular in shape, 0.61.1 cm in
diameter, smooth surface, milky white in color (Fig. 5A) and with
pseudohyphae around the colony margin (Fig. 5B). Cells of the
yeast strain 0732-1 were oval in shape, 57  35 lm in size
(length  width) and multiplied by budding (Fig. 5C). Ascospores
of strain 0732-1 were hat-shaped and 3.54.2  1.21.8 lm in size
(Fig. 5D). These morphological characteristics of strain 0732-1
matched the descriptions for P. anomala (Barnett et al., 1983).

Results of the physiological tests showed that under

semi-anaerobic conditions, the yeast strain 0732-1 could ferment
D-glucose, D-galactose, maltose and sucrose, but could not ferment
lactose (Table 2). Under aerobic conditions, it could assimilate Dglucose, D-galactose, maltose, lactose, D-xylose, starch, D-mannose,
D-arabinose, mannitol, glycerol, ethanol and sorbitol, but could not
assimilate methanol, lactose and inositol, as the major source carbon; and could assimilate L-lysine, KNO3 and (NH4)2SO4, but could
not assimilate creatine, as the major source nitrogen (Table 2).
Meanwhile, cultures of the yeast strain 0732-1 at 42 C showed
production of acids and esters, but not starch (Table 2). The physiological characteristics of the yeast strain 0732-1 were also similar
to those for P. anomala described by Barnett et al. (1983).
The ITS and the anking regions of the rDNA of the yeast strain
0732-1 comprised 617 base pairs. Without the anking regions,
the sequence of ITS (ITS1+5.8S rDNA+ITS2) is composed of only
529 base pairs. This DNA sequence was submitted to GenBank under the Accession No. EU380207. BLAST analysis showed that the
ITS sequence of the yeast strain 0732-1 was identical to that for
Pichia anomala strains WM 828, MTCC 237, MCCL 13, MCCL 1569,
MCCL 209/2K and FY-102, which were assigned with GenBank
Accession Nos. DQ249195, AY231606, AY231609, AY231610,
AY231611 and AY270936, respectively.
4. Discussion
Numerous studies indicate that some yeast species, including
Pichia anomala, are effective agents for control of post-harvest diseases caused by fungal pathogens in crops such as fruits, vegetables (Wilson and Wisniewski, 1989; Fravel, 2005; Lassois et al.,
2008) and cereal grains (Petersson and Schnrer, 1995, 1998; Petersson et al., 1999; Druvefors et al., 2002). However, information
on control of plant bacterial diseases by yeasts is rare. This study
demonstrates the effectiveness of P. anomala strain 0732-1 as a

170

X. Wang et al. / Biological Control 50 (2009) 164171

Table 2
Comparison of physiological characteristics between the yeast strain 0732-1 and
Pichia anomala (Pa).
Test

0732-1

Pa1

Fermentation
D-Glucose
D-Galactose
Maltose
Sucrose
Lactose

+
+
+
+


+
V
V
+


Assimilation
Carbon source
D-Glucose
D-Galactose
Maltose
Lactose
D-Xylose
Starch
D-Mannose
Inositol
L-Arabinose
Mannitol
Glycerol
Ethanol
Methanol
Sorbitol

+
+
+

+
+
+

+
+
+
+

+

+
V
+

V
+
NL
NL

+
+
+

+

Nitrogen source
L-Lysine
Creatine
KNO3
(NH4)2SO4

+

+
+

+

+
+

Additional tests
Starch production
Grew at 42 C
In 50% D-Glucose
In 60% D-Glucose
Acids production (PDB)
Ester production


+
+
+
+
+


NL
V
V
NL
+

1
Physiological characteristics of Pichia anomala (Pa) were listed in Barnett et al.
(1983). +,  and V represent positive reaction, negative reaction and variable
reaction, respectively. NL represents the characteristics not listed in Barnett et al.
(1983).

biocontrol agent for control of leaf blight and seedling blight of

hami melon caused by A. avenae subsp. citrulli. Since A. avenae
subsp. citrulli is an important bacterial pathogen of cucurbitaceous
crops, including hami melon. Further investigation is warranted on
efcacy and practicality of P. anomala strain 0732-1 in control of
bacterial fruit blotch of hami melon under eld conditions.
Previous studies showed that P. anomala could suppress mycelial growth or sporulation of Penicillium spp. and Aspergillus spp. in
cereal grains stored under oxygen-limited conditions possibly
through competition (Petersson and Schnrer, 1995, 1998; Petersson et al., 1999; Druvefors et al., 2002). Friel et al. (2007) reported
that production of b-1,3-glucanase by P. anomala strain K played an
important role in suppression of Botrytis cinerea, the causal agent of
grey mould diseases. Meanwhile, production of killer toxins by P.
anomala for suppression of the human pathogen Candida albicans
(Mathews et al., 1998) and spoilage yeasts Dekkera and Brettanomyces (Comitini et al., 2004) was also reported. The present study
showed that P. anomala strain 0732-1 could produce anti-bacterial
substances (ABS), which are effective for suppression of Aac
growth on KBA medium. Production of ABS by the yeast strain
0732-1 might be responsible for control of seedling blight of hami
melon caused by Aac, as treatment of hami melon seeds with cellfree cultural ltrates of strain 0732-1 signicantly reduced incidence and severity of this disease.
Production of acidic compounds by P. anomala strain 0732-1 was
evidenced by lowering of the pH value in cultures observed in this
study. Pusey et al. (2008) reported that acidic ambient pH signi-

cantly enhanced the antibiotic activity and biocontrol efcacy of

Pantoea agglomerans strain E328 against Erwinia amylovora, the causal agent of re blight of pear and apple. This phenomenon might occur in the interaction between P. anomala strain 0732-1 and Aac on
hami melon. Additional studies are required to characterize the nature of acidic compounds produced by P. anomala strain 0732-1 and
the role of the acidic compounds in biocontrol of Aac.
Acknowledgements
This research was funded by the Natural Science Foundation of
China (Grant No. 30570079). We thank Mr. M.D. Wu of Huazhong
Agricultural University for help of graphic preparations.
References
Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local
alignment search tool. Journal of Molecular Biology 215, 403410.
Arun, C., Pintip, R., Bhinyo, P., 2007. Screening and identication of yeasts strains
from fruits and vegetables: potential for biological control of postharvest chilli
anthracnose (Colletotrichum capsici). Biological Control 42, 326335.
Black, M.C., Isakeit, T., Barnes, L.W., Kucharek, T.A., Hoover, R.J., Hodge, N.C., 1994.
First report of bacterial fruit blotch of watermelon in Texas. Plant Disease 78,
831.
Burdman, S., Kopelowitz, J., Kritzman, G., Kots, N., 2005. Molecular, physiological,
and host-range characterization of Acidovorax avenae subsp. citrulli isolates
from watermelon and melon in Israel. Plant Disease 89, 13391347.
Barnett, J.A., Payne, R.W., Yarrow, D., 1983. Yeasts: characteristics and
identication. Cambridge University Press. pp. 306307.
Comitini, F., Ingeniis De, J., Pepe, L., Mannazzu, I., Ciani, M., 2004. Pichia anomala and
Kluyveromyces wickerhamii killer toxins as new tools against Dekkera/
Brettanomyces spoilage yeasts. FEMS Microbiology Letters 238, 235240.
Demir, G., 1996. A new bacterial disease of watermelon in Turkey: bacterial fruit
blotch of watermelon (Acidovorax avenae subsp. citrulli (Schaad et al.) Willems
et al.. Journal of Turkey Phytopathology 28, 4349.
Druvefors, U., Jonsson, N., Boysen, M.E., Schnrer, J., 2002. Efcacy of the biocontrol
yeast Pichia anomala during long-term storage of moist feed grain under
different oxygen and carbon dioxide regimens. FEMS Yeast Research 2, 389
394.
Feng, J.J., Chen, K.J., Kim, J.Y., Xu, Y., Zhou, X.Y., Li, J.Q., 2007. Effect of seed priming
on seedling growth and disinfection to Acidovorax avenae subsp. citrulli in
triploid watermelon seeds. Acta Phytopathologica Sinica 37, 528534.
Fessehaie, A., Walcott, R.R., 2005. Biological control to protect watermelon blossoms
and seed from infection by Acidovorax avenae subsp. citrulli. Phytopathology 95,
413419.
Fang, Z.D., 1998. Research Methodology for Plant Diseases (third ed.). Chinese
Agriculture Press, Beijing. pp. 812.
Fravel, D.R., 2005. Commercialization and implementation of biocontrol. Annual
Review of Phytopathology 43, 337359.
Friel, D., Pessoa, N.M.G., Vandenbol, M., Jijakli, M.H., 2007. Separate and combined
disruptions of two exo-b-1, 3-glucanase genes decrease the efciency of Pichia
anomala (strain K) biocontrol against Botrytis cinerea on apple. Molecular
Plant-Microbe Interaction 20, 371379.
Hamm, P.B., Spink, D.S., Clough, G.H., Mohan, K.S., 1997. First report of bacterial fruit
blotch of watermelon in Oregon. Plant Disease 81, 113.
Heras-Vazquez, F.J.L., Mingorance-Cazorla, L., Clemente-Jimenez, J.M., RodriguezVic, F., 2003. Identication of yeast species from orange fruit and juice by RFLP
and sequence analysis of the 5.8S rRNA gene and the two internal transcribed
spacers. FEMS Yeast Research 3, 13.
Hopkins, D.L., 1989. Bacterial fruit blotch of watermelon: a new disease in the
eastern USA. Cucurtaceae 89, 7475.
Hopkins, D.L., Thompson, C.M., Hilgren, J., Lovic, B., 2003. Wet seed treatment with
peroxyacetic acid for the control of bacterial fruit blotch and other seedborne
diseases of watermelon. Plant Disease 87, 14951499.
Jacobs, J.L., Damicone, J.P., McCraw, B.D., 1992. First report of bacterial fruit blotch of
watermelon in Oklahoma. Plant Disease 76, 1185.
King, E.O., Ward, M.K., Raney, D.E., 1956. Two simple media for the demonstration
of pyocyanin and uorescin. Journal of Laboratory and Clinical Medicine 44,
301307.
Kurtzman, C.P., Fell, J.W., 1998. The Yeasts: Taxonomic Study (fourth ed.). Elsevier
Science, Amsterdam.
Langston Jr, D.B., Walcott, R.R., Gitaitis, R.D., Sanders, F.H., 1999. First report of a
fruit rot of pumpkin caused by Acidovorax avenae subsp. citrulli in Georgia. Plant
Disease 83, 100.
Lassois, L., de Bellaire, L., Jijakli, M.H., 2008. Biological control of crown rot of
bananas with Pichia anomala strain K and Candida oleophila strain O. Biological
Control 45, 410418.
Latin, R., Rane, K., 1990. Bacterial fruit blotch of watermelon in Indiana. Plant
Disease 74, 331.
Latin, R., Hopkins, D.L., 1995. Bacterial fruit blotch of watermelon: the hypothetical
exam question becomes reality. Plant Disease 79, 761765.

X. Wang et al. / Biological Control 50 (2009) 164171

Lessl, J.T., Fessehaie, A., Walcott, R.R., 2007. Colonization of female watermelon
blossoms by Acidovorax avenae ssp. citrulli and the relationship between
blossom inoculum dosage and seed infestation. Journal of Phytopathology 155,
114121.
Liang, Q.F., Chi, Z.M., 2002. Study on screening of one strain of yeast with biological
control effect and its physiological and biochemical characters. Journal of
Shangdong Institute of Light Industry 16, 6670.
Lu, Y., Luo, M., 2004. Isolation of endophytic bacteria from hami melon and
screening of antagonistic bacteria. Journal of Shihezi University (Natural
Science Edition) 22 (Suppl.), 104109.
Martin, H.L., Horlock, C.M., 2002. First report of Acidovorax avenae subsp. citrulli as a
pathogen of gramma in Australia. Plant Disease 86, 1406.
Mathews, H.L., Conti, S., Witek-Janusek, L., Polonelli, L., 1998. Effect of Pichia
anomala killer toxin on Candida albicans. Medical Mycology 36, 199204.
Petersson, S., Schnrer, J., 1995. Biocontrol of mould in high-moisture wheat stored
under airtight conditions by Pichia anomala, Pichia guilliermondii and
Saccharomyces cerevisiae. Applied and Environmental Microbiology 61, 1027
1032.
Petersson, S., Schnrer, J., 1998. Pichia anomala as a biocontrol agent of Penicillium
roqueforti in high-moisture wheat, rye, barley, and oats stored under airtight
conditions. Canadian Journal of Microbiology 44, 471476.
Petersson, S., Jonsson, N., Schnrer, J., 1999. Pichia anomala as a biocontrol agent
during storage of high-moisture feed grain under airtight conditions.
Postharvest Biology and Technology 15, 175184.
Pusey, P.L., Stockwell, V.O., Rudell, D.R., 2008. Antibiosis and acidication by
Pantoea agglomerans strain E328 may contribute to suppression of Erwinia
amylovora. Phytopathology 98, 11361143.
Santos, E.R., Gouveia, E.R., Mariano, R.L.R., Souto-Maior, A.M., 2006. Biocontrol of
bacterial fruit blotch of melon by bioactive compounds produced by Bacillus
spp.. Summa Phytopathologica 32, 376378.
Somodi, G.C., Jones, J.B., Hopkins, D.L., Stall, R.E., Kucharek, T.A., Hodge, N.C.,
Watterson, J.C., 1991. Occurrence of a bacterial watermelon fruit blotch in
Florida. Plant Disease 75, 10531056.
Shirakawa, T., Kikuchi, S., Kato, T., Abiko, K., Kaiwa, A., 2000. Occurrence of
watermelon bacterial fruit blotch in Japan. Annals of the Phytopathological
Society of Japan 66, 223231.

171

Silveira, E.B., Mariano, R.L.R., Michereff, S.J., 2003.Variabilidade de isolados de

Acidovorax avenae subsp. citrulli provenientes de melo produzido no estado do
Rio Grande do Norte. Summa Phytopathologica 29, 285261.
Wall, G.C., Santos, V.M., Cruz, F.J., Nelson, D.A., Cabrea, I., 1990. Outbreak of
watermelon fruit blotch in the Mariana Islands. Plant Disease 74, 80.
Walcott, R.R., Gitaitis, R.D., Castro, A.C., 2003. Role of blossoms in watermelon
seed infestation by Acidovorax avenae subsp. citrulli. Phytopathology 93, 528
534.
Walcott, R.R., Fessehaie, A., Castro, A.C., 2004. Differences in pathogenicity between
two genetically distinct groups of Acidovorax avenae subsp. citrulli on cucurbit
hosts. Journal of Phytopathology 152, 277285.
White, T.J., Bruns, T., Lee, S., Taylor, J., 1990. PCR protocols: a guide to methods and
applications. In: Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J. (Eds.),
Amplication and Direct Sequencing of Fungal Ribosomal RNA genes for
Phylogenetics. Academic Press, San Diego, pp. 315322.
Willems, A., Goar, M., Thielemans, S., Gills, M., Kersters, K., Deley, J., 1992. Transfer
of several phytopathogenic Pseudomonas species to Acidovorax as Acidovorax
avenae subsp. citrulli, Acidovorax avenae subsp. cattleyae and Acidovorax konjaci.
International Journal of Systematic Bacteriology 42, 107119.
Wilson, C.L., Wisniewski, M.E., 1989. Biological control of postharvest diseases of
fruits and vegetables: an emerging technology. Annual Review of
Phytopathology 27, 428441.
Yaeram, C., Thummabenjapone, P., Pachinburavan, A., 2006. Suitable medium for
increasing biomass of antagonistic Streptomyces spp. against bacterial fruit
blotch disease of watermelon. Khon Kaen Agriculture 34, 1219.
Zhang, R.Y., Tan, Z.Q., Wen, Y.T., Zhang, H.M., Jiang, R.M., 1998. Description and
identication of the causal organism of bacterial fruit blotch of watermelon.
Chinese Journal of Tropical Crops 19, 7076 (in Chinese with an English
abstract).
Zhao, T.C., Sun, F.Z., Wang, J.R., Xie, Y.J., 2003. Control of bacterial fruit blotch of
hami melon by seed treatment with bactericides. Plant Protection 29, 5052 (in
Chinese with an English abstract).
Zhao, T., Sun, F., Wang, B., Hui, W., 2001. Pathogen identication of hami melon
bacterial fruit blotch. Acta Phytopathologica Sinica 31, 357364 (in Chinese
with English abstract).