009_COST(Ozaktan)_S53_COL 15-06-2012 10:47 Pagina 53
Journal of Plant Pathology (2012), 94 (1, Supplement), S1.53-S1.56
BIOLOGICAL CONTROL OF BACTERIAL BLIGHT OF WALNUT
BY ANTAGONISTIC BACTERIA
H. Ozaktan1, M. Erdal2, A. Akkopru1 and E. Aslan3
1 University of Ege Faculty of Agriculture, Department of Plant Protection 35100 Bornova-Izmir, Turkey
2 Ministryof Agriculture and Rural Affairs, Agricultural Quarantine Directorate, 35100 Izmir, Turkey
3 University of Ege Faculty of Engineering, Department of Bioengineering 35100 Bornova-Izmir, Turkey
SUMMARY
The goal of this study was to determine whether bac-
terial antagonists could be used to control Xanthomonas
arboricola pv. juglandis (Xaj), the causal agent of bacteri-
al blight of walnut. A total of 35 potentially bacterial an-
tagonists, 29 of which were isolated as epiphytes from
the phylloplane of healthy walnut trees, were screened
for their in vitro biological control activity against Xaj.
Pantoea vegans strain C9/1 was also tested as reference
biological control agent. Eighteen strains, that inhibited
the growth of Xaj between 3.0 mm to 13.0 mm on TSA
medium, were selected and further tested for their abili-
ty to control bacterial blight on immature nuts. Approx-
imately 60% of the antagonistic strains, significantly re-
duced bacterial blight by 41% to 77% as compared to
the positive control. Bacterial antagonistic strains,
which effectively inhibited the development of Xaj on
immature nuts were selected for further testing on wal-
nut seedlings. Antagonistic strains were sprayed in a
suspension of 109 CFU ml1 on the leaves which were
subsequently inoculated with suspension of Xaj strain
W7/1 (108 CFU ml-1), 24 h after the antagonist treat-
ments. According to the in vivo biocontrol tests on wal-
nut seedlings (cv. Chandler), 4 putative antagonists sig-
nificantly reduced the symptoms on walnut leaves
(41% to 82%) compared with the positive conttol.
Strains WH77/1, WH68 and WH48/1A were the most
effective in the seedling test, yielding 55%, 42%, and
82% disease reduction, respectively, in two consecutive
years. These strains were tentatively identified as
Pseudomonas fluorescens on the basis of biochemical
and physiological tests. P. vagans strain C9/1 agent, re-
duced the severity of bacterial blight on the leaves by
71%. The results obtained both nut and seedling tests
agreed, except for P. fluorescens strain WH55/4 which
was less effective in inhibiting symptom development in
walnut leaves than in the fruits. Prohexadione-Ca (Pro-
Ca) treatments of walnut leaves reduced by 66% blight
symptoms.
Corresponding author: H. Ozaktan
Fax: +90.232.3744848
E-mail: hatice.ozaktan@ege.edu.tr
Edizioni ETS Pisa, 2012 S1.53
Key words: prohexadione-Ca, biocontol, Pantoea va-
gans, Peudomonas fluorescens, Xanthomonas aboricola
pv. juglandis.
Turkey is the third biggest walnut producer in the
world, after the USA and China with an average yearly
production of ca. 171,000 tons (www.faostat.fao.org).
Bacterial blight of Persian (English) walnut (Juglans re-
gia), caused by Xanthomonas arboricola pv. juglandis
(Xaj,) is a disease of economic importance in all walnut-
producing areas, the severity of its outbreaks depending
to a great extent on spring weather conditions (Vauterin
et al., 2000). The disease is currently an important limit-
ing factor in many walnut orchards (Frutos, 2010). Bac-
terial blight of walnut caused by Xaj has been very de-
structive in Turkey due to frost injury and rainy weather
conditions in the Marmara region during spring 2007
(Ozaktan et al., 2007). New walnut tissues are suscepti-
ble to infection and necrosis can occur in catkins, fe-
male flowers, leaves, fruits, and green shoots. Early in-
fected nutlets develop dark lesions on the hull and most
of them drop off the tree, whereas mature nuts, even
when symptomatic, may remain on the tree. Host sus-
ceptibility differs not only among cultivars (Mulrean
and Schroth, 1982), but also among plant organs within
the same cultivar (Aleta et al., 2001). Copper-based
compounds have been the only means of control for
more than 40 years, but their efficacy is often limited
(Gardan et al., 1993). Copper-resistant strains of Xaj
have been isolated from walnut orchards where inten-
sive copper spraying was used for disease control (Gar-
dan et al., 1993). Applications of intensive copper treat-
ments over many years in commercial orchards in USA
have resulted in the accumulation of copper in soils,
with subsequent environmental pollution. Excess cop-
per in the soil can alter the plants ability to metabolize
nitrogen. When frequently used in walnut orchards, the
resulting high levels of copper can have undesired ef-
fects on the soil, disturb tree performance, and predis-
pose fruits to necrosis (Radix et al., 1998). Biological
control of walnut blight pathogen may be an alternative
to chemical control.
The purpose of this study was to evaluate the efficacy
009_COST(Ozaktan)_S53_COL 15-06-2012 10:47 Pagina 54
S1.54 Biological control of X. aboricola pv. juglandis Journal of Plant Pathology (2012), 94 (1, Supplement), S1.53-S1.56
of bacterial antagonists for walnut blight control via
testing their effect on immature nuts and walnut
seedlings. Xaj W7/1, a very virulent strain isolated from
diseased buds of walnut trees at Izmir in 2007 was used
in all experiments. Before inoculation the bacterium
was grown on Kings B medium at 24C for 48 h, and
was then suspended in sterile distilled water to a final
concentration of 108 CFU ml1 (spectrophotometrically
determined at 600 nm).
A total of 35 bacterial antagonists were screened for
their in vitro and in vivo biocontrol activity against Xaj,
29 of which were isolated as epiphytes from the phyllo-
plane of healthy walnut trees. The others had been pre-
viously isolated from beans, cucurbits, and olive trees
and found promising when tested in different host-
pathogen combinations in Turkey. P. vagans strain C9/1
was used as reference biocontrol agent. Tests for an-
tibiosis between antagonistic bacteria and Xaj were con-
ducted in TSA (tryptic soy agar) plates. One hundred
l of a 108 CFU/ml suspension of Xaj strain W7/1
grown for 2 days on TSA medium was spread over the
agar surface and let to dry. Three sterile assay disks (5
mm diameter) were placed on the agar and a suspension
of each candidate antagonistic bacteria (109 CFU ml-1,
10 l per disk) was deposited on each disk. Plates were
incubated at 24C for 48 h and examined for the ap-
pearance of growth inhibition zones whose size (mm)
was recorded (Jetiyanon and Kloepper, 2002). There
were four replications (or agar plates) per antagonistic
bacterium. Tests were performed in duplicate.
Symptomless immature nuts (cv. Hartley) were col-
lected 30-40 days after setting (Gf) and used for testing
the biocontrol activity of bacterial antagonists. De-
tached fruits were surface-disinfected with alcohol
(70%), rinsed with sterile distilled water (SDW) and
dried before inoculation. Nuts were submitted to infil-
tration by injecting antagonistic bacteria (30 l) and
then Xaj on marked areas under controlled environ-
mental conditions. Four inoculations per nut were made
and eight replicates of 2 nuts per cultivar were tested.
SDW was injected into the immature nuts as control
treatment. Xaj strain W7/1 was used as positive control
in nuts where no antagonistic bacteria were infiltrated
whereas P. vagans strain C9/1 was used as reference bio-
control agent. The incubation lasted 5 to 7 days at 24C
and over 90% relative humidity (RH). Bacterial blight
incidence on immature walnut fruits was evaluated us-
ing an empirical scale from 0 to 4 where: 0 = no symp-
toms; 1 = very small superficial spots at the inoculation
point; 2 = less then 2 mm wide superficial spots on the
inoculation point; 3 = blackening of the inoculation
point of nut 2 to 5 mm in diameter, extending in the in-
ner tissues by 1 mm; 4 = blackening around the inocula-
tion point more than 5 mm in diameter, extending in
the inner tissues by 2 to 4 mm (Aleta et al., 2001). A to-
tally randomized experiment design was used for the as-
say, which was repeated twice.
A set of one-year-old potted walnut seedlings of the
Xaj-susceptible cv. Chandler, was used for evaluating
the antagonists activity by spraying with bacterial sus-
pensions (109 CFU ml-1). P. vagans strain C9/1 was again
used as reference biocontrol strain. Another set of
seedlings was sprayed with copper-oxychloride +
maneb (0.2%,37.5%+20% a.i. per 100 litres of water)
and Prohexadione Ca [(Regalis, BASF), 125 g a.i. per
100 litres of water]. Water-treated seedlings and strain
Xaj W7/1 were included as negative and positive con-
trol, respectively. Each treatment was applied to six wal-
nut seedlings in completely randomized blocks. The
seedlings were covered with plastic bags and kept in an
air conditioned room at 24C and 90-95% RH.
Seedlings were evaluated for Xaj damage to the leaves,
depending on symptom development, 28 to 30 days af-
ter inoculation. Disease symptoms on the leaves were
assessed evaluated using an empirical scale from 0 to 4
where: 0 = no symptom; 1 = yellowish spots without
necrosis; 2 = no more than 3 necrotic spots; 3 = more
than 10 necrotic spots; 4 = more than 50% of the leaf
area with extensive necrosis and blight. The in vivo ex-
periments were repeated twice yielding similar results.
Hence, one representative trial of each experiment is re-
ported. Statistical analyses of variance were conducted
using the SPSS version 15.0, and means were compared
using the Duncan test (P=0.05). Identification of prom-
ising bacterial antagonists at the species level was car-
ried out by biochemical and physiological tests. The
presence of oxidase, fluorescent pigment production,
gelatinase, arginine dihydrolase, levan sucrose, reduc-
tion of nitrate, pectinolytic activity on potato slices and
hypersensitive reaction on tobacco leaves were ascer-
tained according to Lelliott et al. (1966). Utilization of
trehalose, L (+) tartrate, and L(-) tryptophan was tested
using basal medium [(NH4)H2PO4 1 g, KCl 0.2 g,
MgSO4 7H2O 0.2 g, agar 3 g, bromothymol blue 0.08
g, distilled water 1,000 ml, pH 7.2]. All these tests were
carried out to differentiate species and biovars accord-
ing to Stanier et al. (1970) and Palleroni (1984). The
ability of bacterial strains to assimilate 49 carbohy-
drates, 49 organic acids, and 49 amino acids was evalu-
ated using API50CH, API50AO, and API50A strips ac-
cording to manufacturers instructions (Biomerieux,
France).
Of the 35 bacterial strains tested, 18 inhibited Xaj
with zones ranging from 3 to 13 mm and were used for
immature nut tests. Eight of these strains significantly
reduced fruit symptoms (73 to 88%) compared with the
positive control (Fig. 1, 2). Strains WH48/1A,
WH39/1A, and WH68 were the most effective (88%,
84% and 86% reduction, respectively). By contrast,
strains WH43/1B, WH84/2, and WH42/1a were totally
ineffective in protecting immature nuts from Xaj infec-
tions (Fig. 1). P. vagans strain C9/1 reduced blight by
009_COST(Ozaktan)_S53_COL 15-06-2012 10:47 Pagina 55

Journal of Plant Pathology (2012), 94 (1, Supplement), S1.53-S1.56 Ozaktan et al. S1.55

Fig. 1. Biocontrol activity of tested bacterial antagonists against Xanthomonas arboricola pv.
juglandis on immature nut test.

40%, although it did not produce any inhibition zones
against Xaj. Strains WH48/1A and WH68 and that ef-
fectively inhibited Xaj on immature nuts plus a couple
of other efficient inhibitors (WH77/1, WH55/4) were
selected for seedling inoculation. Three out of the 4
tested antagonists and the two chemicals tested, signifi-
cantly reduced symptom development on the walnut
leaves compared with the positive control. In particular,
strain WH48/1A was the most effective (82% blight re-
duction) followed by strain WH77/1 (55%). P. vagans
C9/1 reduced blight by 71% and Prohexadione Ca by
63%, a value comparable to that afforded by copper-
based compound estimated at 57% reduction (Table 1).
Walnut blight is currently controlled by multiple ap-
plications of copper from bud break until the end of
August (Gardan et al., 1993; Radix et al., 1998). Alter-
native control chemicals are few, and even more toxic,
while no effective plant resistance has been identified.
Potential alternatives including bacterial antagonists
and other bactericidal products (Serenade), and sys-
temic acquired host resistance (SAR) compounds may
be proposed as control measures for bacterial blight of
walnut. With no proven alternatives for walnut blight
control, investigating the potential of an antagonistic
bacteria-based biocontrol is warranted. Streptomyces ly-
dicus strain WYEC 108 yielded a reduction of 85% of
walnut bacterial blight (www.epa.gov/oppbppd1/
biopesticides). Bacteriophages of Xaj showed the ability
of lysing all New Zealand isolates of the pathogen tested
(Jenkins et al., 2010).
Our tests have now shown that some strains of P. fluo-
rescens were promising Xaj biocontrol agents, in accord
with the suggestion that antagonistic bacteria isolated
from healthy walnut plants may be better adapted to this
species and provide better control of bacterial blight than
organisms isolated from other plant species. This re-
search showed also that the detached nut assay is a quick
and reliable method for screening promising Xaj biocon-

Fig. 2. P. fluorescens strains WH48/1A, WH39/1A, and WH68 (from left to the right) were the most effective for inhibition of Xaj
on immature nuts (left), compared to positive control (Xanthomonas arboricola pv. juglandis) (middle) and treated with sterile
water (right).
009_COST(Ozaktan)_S53_COL 15-06-2012 10:47 Pagina 56
S1.56 Biological control of X. aboricola pv. juglandis Journal of Plant Pathology (2012), 94 (1, Supplement), S1.53-S1.56
Table 1. Effect of some bacterial antagonist strains and chemicals for suppression of bacterial blight caused by
Xaj on walnut seedlings (cv. Chandler).
Treatments
P. fluorescens strain WH 77/1
P. fluorescens strain WH 68
P. fluorescens strain WH 55/4
P. vagans strain C 9/1
P. fluorescens strain WH 48/1 A
Prohexadione Ca (125 g a.i./100L water)
Copper-oxychloride+maneb (0.2%,37,5%+20% a.i./100l water)
Pathogen alone treatment
Non-treated Control
* Mean values for six replications of 2 walnut seedlings in each replicate block.
** Percentage of reduction in bacterial blight compared to walnut seedlings treated with water alone.
*** Means within a column followed by the same letter are not significantly different according to Duncans Multiple
Range Test at P = 0.05.
trol agents. Additional studies are nevertheless needed
for developing a suitable scale up production and appli-
cation to walnut orchards, in the hope to produce a fur-
ther tool for the management of walnut blight.
ACKNOWLEDGEMENTS
This research was supported by the Scientific and
Technical Research Council of Turkey (research project
TUBITAK#TOVAG 106O825) and Ege University Sci-
ence#Technology Research and Application Center
(EBILTEM). We thank the COST#873 Action for sup-
porting this short communication.
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