Brown apical necrosis of walnut in Turkey - Cost 873

The two years of experience on
etiology of apical necrosis on
walnut in Turkey
Hatice ÖZAKTAN
Senem AKAT Lalehan YOLAGELDİ
Department of Plant protection, Faculty of Agriculture, University of
Ege, 35100 Bornova, İzmir / TURKEY
hatice.ozaktan@ege.edu.tr

Brown apical necrosis of walnut
in Turkey
• SYMPTOMS: externally
Brown to dark brown patches
originated at the stigmatic end of
nuts

Brown apical necrosis of walnut
in Turkey
• SYMPTOMS: internally
• Symptoms on fruits remaining the tree were a brown to dark-brown patch
originating exclusively at the blossom end and a brown to blackish rot of
inner tissues

Brown apical necrosis of walnut in Turkey
Rot of internal tissues is variable.
Internal tissues become necrotic
and blackish
Fruits dropped to the soil
Fruits remined in the tree
Necrosis of stigma and style can spread
to internal tissues and reaches the kernel
Fruits dropped to the soil

External and internal symptoms
and severity of BAN disease
sometimes do not correlate the
incidence of symptoms
HOWARD ON THE SOIL
VINA SYMPTOMATIC

AIM OF THIS RESEARCH
• In 2009 and 2010, this research was
carried out in a walnut orchard
located in Marmara Region of Turkey
to substantiate the etiology of BAN.
• symptom evolution and disease
progress,
• the microorganisms associated with
brown apical necrozis (BAN),
• Pathogenicity of isolated
microorganisms, and
• cultivar susceptibility have been
monitored in a walnut orchard from
May to August

Susceptibility of walnut cultivars to brown apical necrosis depending
MARMARA SEA
on causal agents
According to results of 2009 year studies, we selected susceptible walnut cvs.
to BAN for monitoring the etiology of the disease in the orchard

1.Monitoring the simptom development in survey area
depending on the vegetation period
SAMPLING DATES:
• From May to August 2010 as
twice in a month
• 10 fruits per date and each
cultivar in tree
WALNUT CULTIVARS:
• Hartley
• Rendede
• Vina
• Howard
• Bilecik (Local veriety)
WALNUT ORCHARD:
• The experimental orchard in
Yalova, Marmara Region of Turkey

Vina Symptomatic
26 .05.2010
Rendede Symptomatic

11.06.2010
Hartley Symptomatic

25 June 2010
Fruits dropped to the soil
The disease started to cause a
premature walnut fruit drop
and a yield reduction at the
mid of June 2010

12.07.2010

27.07.2010
Severe fruit drops was
observed on 27 June,2010

11.08.2010
• Late infections (August) of walnut
fruits after the shell hardening satage
localized only epicarp and mesocarp
tissues
• Fruit drops was more severe and
apical necrosis extended to all internal
tissues

Disease incidence (%)
80
BAN incidence in survey area depending on the sampling dates
70
60
50
40
30
20
10
0
26.05.2010 11 June 26 June 12 July 29 July 11 August
Hartley Vina Howard Rendede Bilecik
Sampling dates

2. microorganisms associated with brown apical necrosis
EXOCARP
ENDOCARP
SEED
SAMPLES FOR ISOLATION
OF CAUSAL AGENTS:
• 3 fruits per date and
cultivar
Symptomatic
fruits in tree
Symptomatic
fruits dropped to
the soils
Non symptomatic
fruits
Samples of walnut fruits affected from BAN were taken to isolate the
microorganisms associated to the disease on each tissue

Isolation Techniques :
a
b
If the bacteria produced yellow colonies on
YDC medium, we restriked them on TMM
medium for observation prespitation zone on
TMM medium. Identification of
microorganism was based on morphological
traits and growth on the media and
pathogenicity on tomato leaflets and nuts.
Piece of tissues were break into pieces with bistoury and we cultured on YDC,TMM
medium for bacterial development.(a)
Piece of tissues were cultured on PDA with streptomicin for fungal microorganisms.(b)

Isolation frquency (%)
the isolated microorganisms associated with brown
apical necrozis from May to August 2010
100
90
80
70
60
50
40
30
20
10
0
26.May 11 June 26 June 12 July 29 July
Xaj Alternaria Fusarium

Isolated microorganisms
90
80
70
60
50
40
30
20
10
0
Isolated microorganisms depending on the different sampling dates
May June July
Months
X.a.j Alternaria sp. Fusarium sp.

Microorganims isolated from simptomless
walnut fruits
• X.a.pv juglandis isolated
from simptomless fruits in
2009 and 2010 years at the
rate of 5% and 14%,
respectively.
cv. Vina simptomless
cv.Vina simptomless
cv.Rendede simptomless

Microorganims isolated from walnut fruits dropped on
the soil
Cv .Rendede on the soil
3%
2009
20%
12%
32%
16%
13%
2010
Xaj Fusarium Alternaria
Xaj Fusarium Alternaria

Pathogenicity tests on detached walnut fruits
of isolated microorganisms from BAN simptoms
• Inoculations on detached immature fruits (GF+30, cv. Hartley)
• Walnut fruits were inoculated by placing a mycelial disc (2-3mm) on the
stigmatic end for fungal pathogens.

Pathogenicity tests of isolated microorganisms from
BAN simptoms on walnut fruits
• Bacterial suspensions were infiltrated into stigmatic end of fruits as
30µl per each nut
• The incubation lasted 14 days at 24 0 C, RH over 90%.
• Microorganisms 5 replicates, 2 fruit/replicate
• external and internal infection was evaluated by 0-4 scale

Pathogenicity tests of isolated microorganisms from
BAN simptoms on walnut fruits
Tested
Isolates
Disease severity of tested strains on walnut fruit tissue
%0 0-5% 6-25% 26-50% 51-88%
X. arboricola
pv. Juglandis
(33 strains)
Alternaria
sp. (7 strains)
Fusarium sp.
(9 strains)
Other Fungi
(11 strains)
6 1 6 12 8
6 1 - - -
4 - 5
11 - - - -
* BAN symptoms on inoculation points of fruits were evaluated by 0-4 scale

Xaj inoculation on immature nuts
CONTROL (-)

Pathogenicity test
results of Fungal strains
on immature nuts
Fusarium spp.
Alternaria spp
Fungal organisms isolated from BAN symptoms were inoculated on the nuts,
but, they couldn’t produce typical BAN symptoms

Brown Apical Necrosis incidence (%)
Brown Apical Necrosis Incidence
(%)
Susceptibility of walnut cultivars to brown apical
necrosis depending on causal agents
60
50
2009
40
30
20
10
0
Howard Rendede Hartley Vina Bilecik
Walnut Cultivars
X.a.j A.alternata Fusarium
2010
90
80
70
60
50
40
30
20
10
0
Hartley Vina Howard Rendede Bilecik
Walnut cultivars
X.a.j
Alternaria sp.
Fusarium sp.

IN CONCLUSION:
• Infections could be observed in walnut fruits from fruit set
to harvest, but they are more intense at early fruit
development stages, when cause major fruit drop.
• Initial infections were observed in exocarp tissues at the
end of May in Turkey.
• Symptoms were more visible in the first half of June than
May, and the incidence of BAN increased during the
growing season, with full expression in the first and
second half of July
• Infections in young fruits observed in July affected
epicarp tissues and reached kernel whereas late
infections (August) of walnut fruit localized only epicarp
tissues.
• The extent of external and internal lesions of the style
were not correlated on symptomatic fruit.
• Premature fruit drops on the soil observed from
25.06.2010 to 11.08.2010

In Conclusion
• All fruits inoculated with X. arboricola pv. juglandis produced
typical external and internal symptoms of BAN, similar to
those observed with natural infections within fifteen days after
inoculation. Fusarium spp. caused only limited external
necroses of the style on the inoculated fruit. Alternaria spp
couldn’t produce typical symptoms after artificial inoculation
on detached nuts.
• Fusarium spp. and Alternaria spp. also seem to be involved in
apical necrosis of walnut causing secondary infections or
growing as saprophyte on bacterial infected tissues, enhancing
the disease symptoms and severity.
• Most commercially cultivars were susceptible to apical
necrosis. “Hartley”, “Bilecik”, “Vina”, “Rendede”, “Howard”,
and “Serr” were highly susceptible to apical necrosis.
• According to these results, Xaj can be considered as the main
causal organism of apical necrosis of walnut whereas Fusarium
spp. can occasionally be involved in the disease, interacting
with bacterial infections. The presence of Alternaria spp. on
necrotic tissues may be related more to the opportunistic
colonization of dead tissues previously infected by the
bacteria.

THANK YOU FOR YOUR ATTENTION

BAN
BACTERIAL
BLIGHT OF
WALNUT
CAUSED BY
XAJ