Journal of Phytopathology and Pest Management 6(1): 11-23, 2019
pISSN:2356-8577 eISSN: 2356-6507
Journal homepage: http://ppmj.net/
∗
Corresponding author:
Ahmed A. Elsisi,
E-mail: ahmed.elsisi@fagr.bu.edu.eg
11
Copyright © 2019
Bacterial blight disease caused by
Pseudomonas
cichorii
on chrysanthemum in Egypt
Ahmed A. Elsisi*
Plant Pathology Department, Faculty of Agriculture, Benha University, Moshtohor, Egypt
Abstract
Keywords: bacterial blight, Pseudomonas cichorii, Dendranthema grandiflorum, PCR, identification, host range.
12
1. Introduction
The cut flower industry is expanding
worldwide and Egypt. Chrysanthemum
(
Dendranthema grandiflorum
) is a
perennial herb grown well in Egypt. It is
highly attractive and charming short-day
plant, which behaves both as an annual as
well as perennial flowering herb (Arora,
1990). There are about 160 species of
chrysanthemum. The inflorescences
which call a flower head are greatly
required in markets because of its
beautiful shape and longevity in vases.
The chrysanthemum plants are flowering
only under short day conditions. The
importance role of chrysanthemum is
grown both as potted plants and as cut
flowers during the fall months when the
other flowers are scarce to supply the
flower markets. They are the best keeping
flowers for home use, and one of the most
adaptable to design work.
Chrysanthemum widely used in two types
namely standard "Art Queen and White
Zambia" (Elnemr, 2018).
Chrysan-
themum is vegetative propagated
flowering plant and is affected by many
diseases caused by fungi like vascular
wilt diseases caused by
Fusarium
oxysporum
f.sp.
chrysanthemi
and
Verticillium dahlia
(Dreistadt, 2001)
and
bacterial diseases severely reducing the
crop yields were bud blight
(
Pseudomonas syringae
)
,
bacterial wilt
(
Erwinia chrysanthemi
)
(Vegh
et.al.
,
2014).
The major bacterial diseases
severely reducing the crop yields stem
necrosis (
Pseudomonas cichorii
)
according to
(Shamala & Janardhana,
2018).
Bacterial blight caused by
Pseudomonas species affects a broad
range of ornamentals and horticultural
crops including Gerbera, Tulip, Cabbage,
Coriander
etc.
(Wehlburg, 1963)
.
A
sudden appearance of bacterial blight
disease in Egypt is of serious concern for
Chrysanthemum growers as it directly
affects the quality of cut flowers. Stead
et
al.
(2013)
reported that
Pseudomonas
cichorii
have a wide host range affecting
high economic importance plants from
different family including tomato, lettuce
and watermelon. The pathogen was
isolated in Florida on chrysanthemum,
celery, geranium, and hibiscus recently
on
Stevia rubidian
(Strayer
et al.
,
2012)
and
Duranta erecta
(Gumtow
et al
,
.
2013).
The bacterium is widely
distributed in worldwide which recent
reports from Belgium (Cottyn
et al.
2009),
Greece (Trantas
et al.
,
2013),
Italy
(Scortichini
et al.
,
2002),
Turkey (Aysan
et al.
,
2003),
Tanzania (Testen
et al.
,
2015).
In recent years, many new marker
techniques have been developed in line
with the rapid growth of genomic
research. Start Codon Targeted (SCoT)
polymorphisms are dominant and
reproducible markers that are based on
the short-conserved region in plant genes
surrounding the ATG translation start (or
initiation) codon and use a single 18-mer
primer in the polymerase chain reaction
(PCR) assays and higher annealing
temperature (50°C) (Collard & Mackill
,
2009).
SCoT markers are generally
reproducible, and it is suggested that
primer length and annealing temperature
are not the sole factors determining
reproducibility. SCoT markers have been
successfully used to assess genetic
diversity and structure, identify cultivars,
and for quantitative trait loci (QTL)
mapping and DNA fingerprinting in
different species (Caho
et al.
,
2014;
Amirmoradi
et al
., 2012).
The level of
genetic diversity and its molecular
variation of the bacterial blight disease
caused by
Pseudomonas cichorii,
three
isolations from five geographic regions in
Egypt. The purposes of this study were:
(a) to assess the genetic diversity and
phylogenetic relationship among three
Pseudomonas cichorii
isolates and (b) to
examine the effectiveness of the SCoT
13
markers in
Pseudomonas cichorii
genetic
diversity study. These results could
facilitate
Pseudomonas cichorii
isolation,
conservation and future microbial
Identification. In this paper, we reported
the pathogenic and genetic diversity
found, using traditional and Start Codon
Targeted (Scot) technique, among the
different bacterial blight pathogen
isolates obtained from Chrysanthemum
orchards collected from different
geographical areas of Egypt.
2. Materials and methods
2.1 Sampling and isolation of causal
organism
Diseased samples of Chrysanthemum
i.e.
flowers, leaves, stems and buds with
typical bacterial blight symptoms were
collected from various locations of
Qalubia, Menoufia and Giza
governorates, Egypt, in the autumn of
2016 season. Collected samples of
different chrysanthemum varieties were
transferred to the laboratory in plastic
bags, kept in a refrigerator at 7°C, where
each sample was kept alone for further
studies. Isolation and identification
procedures carried out in Plant Pathology
Department, Faculty of Agriculture,
Benha University, Moshtohor, Egypt.
The infected leaves collected were
washed with tap water and then air dried.
Bacterial lesions were then cut and
surface disinfected by first dipping in
95% ethanol for 5 sec., then sodium
hypochlorite solution 1.25% for 20 sec.
followed by two successive rinses in
sterile water. Each lesion was
homogenized in 2.0 ml sterilized distilled
water. The homogenate was then streaked
onto nutrient agar medium. Five colonies
from each lesion were selected and
retained randomly. A loopful of the
resulting suspension was streaked on the
surface of nutrient agar (NA) and King’s
B media. These plates were incubated at
28 ± 2°C for 2-3 days. Observations
were daily recorded and any emerged
colony was picked up and transferred to
nutrient glucose agar slant medium for
maintenance till use in subsequent tests.
All picked colonies were purified using
the single colony technique (Fahy &
Persley, 1983).
2.2 Pathogenicity test and
hypersensitivity reaction
Bacterial isolates were grown at yeast
dextrose carbonate agar (YDC) medium
and were scraped with a sterile pin.
Chrysanthemum seedlings were
inoculated with bacterial inoculum (10
8
cfu/ ml) as foliar spray until it run off.
Other plants were untreated as control
treatment. The inoculated seedlings were
kept moist under greenhouse conditions
until appearance of symptoms (Shamala
& Janardhana, 2018).
Hypersensitivity
test was carried out on Seedlings of
tobacco (
Nicotiana tabaccum
) which
were inoculated with infiltrating bacterial
suspension (10
8
cfu/ mL) using a fine
syringe into the intercellular spaces of
the lower side of the leaves.
Characteristic HR symptoms were
recorded at 48 h. In order to fulfill
Koch's postulates, disease symptoms
were observed and the pathogen re-
isolated in culture medium.
2.3 Identification of isolated bacteria
2.3.1 Using the traditional techniques
Identification of the bacterial isolates
14
were conducted on the basis of their
morphological, cultural and physiological
characteristics according to schemes
suggested by Fahy and Persley (1983),
Krieg and Holt (1984) and Lelliott and
Stead (1987).
2.3.2 Molecular identification using
SCoT- PCR technique
2.3.2.1 Bacterial DNA Extraction
DNA was extracted from the three
Pseudomonas cichorii
(Wl-1, Pf-4 and
Rs-9) isolates using DNeasy Tissue Mini
Kit (QIAGEN). The concentration of
DNA was then determined based on a
comparison of the DNA samples with
standard lambda DNA on 1% (w/v)
agarose gel, after which it was adjusted to
5 ng/µl.
2.3.2.2 SCoT-PCR amplification and
detection
The identification techniques were done
in Molecular biology Laboratory,
Agricultural Research Park, Faculty of
Agriculture, Benha University, Qalubia,
Egypt. Six SCoT primers were randomly
selected (Table 1). SCoT-PCR reaction
volume was 25 µL, containing 1.5 µL of
template DNA (25 ng/µL), 1.0 µL primer
at 10 µM, 2.0 µL dNTPs at 10 µM, 0.125
µL Taq DNA polymerase at 5 U/µL, 2.5
µL I0X PCR buffer, and 17.875 µL
ddH2O. SCoT-PCR was performed on an
TProfessional PCR mechanics (Biometra,
Germany). Initial denaturation was
carried out at 94 °C for 5 min, followed
by 35 cycles of 94°C for 1 min, 50°C for
1 min, 72°C for 2 min, and final
extension at 72°C for 5 min. The
amplification products were separated in
1.5% agarose gels containing 0.5 µg/mL
of ethidium bromide through
electrophoresis in IX TBE buffer
solution at 5 V/cm and photographed by
gel documentation system, Fair Reader -
Gel Doc.55+ with Image Lab Software
with A 100 bp DNA plus ladder was
used as a molecular size standard (El-
Shaer
et al.
,
2014).
Table 1: The sequences of the six used SCoT primers.
Primer name
Sequence (5'-3')
SCoT-1
CGACATGGCGACCACGC
SCoT-2
ACCATGGCTACCACCGGC
SCoT-3
CGACATGGCGACCCACA
SCoT-4
ACCATGGCTACCACCGCA
SCoT-5
CAATGGCTACCACTAGCG
SCoT-6
CAATGGCTACCACTACAG
2.3.2.3 Data Analysis
Similarity coefficients were calculated
according to dice matrix (Rohlf, 1993;
Nei & Li, 1979).
Parents were grouped
by cluster analysis with the similarity
matrix and unweighted pair group
method based on arithmetic mean
(UPGMA).
2.4 Host range of
Pseudomonas
cichorii
bacterium
In this trial, bacterial isolates of
P.
cichorii
were tested for their pathogenic
reactions on different host plants from
different plant families. The isolates were
tested for pathogenic reactions on eight
different cultivars from four different
plant families
i.e.,
Chrysanthemum cv.
White Zambla (
Chrysanthemum
grandiflorum
) and Lettuce cv. Balady
(
Lactuca saliva
) from family
Asteraceae,
cabbage cv. Sabeany
(Brassica
oleracea),
from family
Brassicaceae,
Mint cv. Balady (
Mentha viridis
),
from
family
Lamiaceae
and pepper cv.
California wander
(Capsicum annuum),
15
tomato cv. super strain b (
Solanum
lycopersicum
), Datura cv. Balady
(
Datura metel
),
eggplant cv. Balady long
white (
Solanum melongena
) from family
Solanaceae
under greenhouse conditions
by injecting 0.2 ml of the previously
prepared bacterial suspension (10
8
cfu/ml) in the tip of growing transplants
using a fine hypodermic syringe. All
seedlings were covered with plastic sheet
for 24 hrs. Disease symptoms were
recorded after 15 days post inoculation
(Marques
et al.
,
2016).
3. Results
3.1 Disease symptoms
The bacterial blight disease occurs on
leaves, stem, buds and flower. The most
conspicuous symptoms in infected leaves
appear as water-soaked spots then
become brown and dry that are often
bordered or ringed by yellowing tissue.
Moreover, the symptoms on stem appear
darkened areas often at the base of buds.
In contrast, the disease appears on
flowers in blast
symptoms
.
3.2 Sampling and isolation of causal
organism
Data in Table (2) showed that ten
bacterial isolates were isolated from
different parts of Chrysanthemum plants
which were collected from different
localities of Egypt. Four different isolates
from Qualubia governorate (Moshtohor
and El-kanater El-khairia) were isolated
from white and purple variety (leaf, bud,
stem and flower) which designated as
Wl-1, Wb-2, Ps-3 and Pf-4. while,
two
isolates from Menoufia governorate
(Shebeen El-kom) were isolated from
white variety (stem and flower) which
coded as Ws-5 and Wf-6. In this respect,
four different isolates from Giza
governorate (Kirdasah and Elmariotia)
were isolated from yellow and red
variety (leaf, bud, stem and flower)
which coded as Yl-7, Yb-8, Rs-9 and Rf-
10.
Table 2: Source of bacterial isolates which isolated from chrysanthemum plants which
collected from different governorates, during growing season 2016.
Governorate
Locality
Variety
Infected sample
Qualubia
Moshtohor
White
leaf
bud
El kanater El khairia
Purple
stem
flower
Menoufia
Shebeen El-kom
White
stem
flower
Giza
Kirdasah
Yellow
leaf
bud
Elmariotia
Red
stem
flower
3.3 Pathogenicity test and
hypersensitivity reaction
In this experiment, ten bacterial isolates
were examined for their reaction on
different hosts tobacco and
chrysanthemum (white, yellow, red and
purple, CVs) plants. In this respect, data
in Table (3) reveal that Pf-4 and Rs-9
isolates produced HR on tobacco and
pathogenic to Chrysanthemum (white,
yellow, red and purple, CVs) plants.
16
Table 3: Hypersensitive reaction and virulence of tested bacterial isolates
on differential hosts.
Code of
isolate
Reaction
Tobacco
Chrysanthemum
HR
White
Yellow
Red
Purple
Wl-1
++
++
++
++
++
Wb-2
-
-
-
-
-
Ps-3
-
-
-
-
-
Pf-4
+
+
+
+
+
Ws-5
-
-
-
-
-
Wf-6
+
-
-
-
-
Yl-7
-
-
-
-
-
Yb-8
+
-
-
-
-
Rs-9
+
+
+
+
+
Rf-10
-
-
-
-
-
Meanwhile, Wl-1 isolate was highly
virulence on tobacco and highly
pathogenic to all Chrysanthemum
varieties used. Wf-6 and Yb-8 isolates
were pathogenic only on tobacco plants.
Meantime, Wb-2, Ps-3, Ws-5, Yl-7 and
Rf-10 isolates were not pathogenic on all
tested hosts. Symptoms on the tested
hosts were recorded clearly on tobacco
seedlings, appeared as water-soaking of
inoculated tissue with 48 h. then dryness,
light-brown localized necrosis with 3
days. Meantime, on chrysanthemum
plants, appeared as brown spots in first
on inoculated leaves then become blight,
small dark lesion at stem and flower blast
on all varieties.
3.4 Identification of isolated bacteria
3.4.1 Morphological, physiological and
biochemical identification
Data in Table (4)
showed that the four
isolates
i.e.
Wl-1, Pf-4, Yb-8 and Rs-9
gave negative reaction with gram
reaction but, Wf-6 isolate gave positive
reaction in the same test. All five isolated
bacterial gave positive reaction in KOH
(3%) and catalase activity. On the other
hand, these isolates gave negative
reaction with production of indole and
growth at 41°C test. Meantime, they
gave negative reaction with starch
hydrolysis and gelatin liquefaction
except isolate Wf-6 gave positive
reaction on those tests. Meanwhile, the
three isolates
i.e.
Wl-1, Pf-4 and Rs-9
gave different reaction on methyl red test
(M.R.) and production of H
2
S.
Meantime, the two isolates Wf-6 and
Yb-8 gave negative reaction with the
same tests. The isolates
i.e.
Wl-1, Pf-4,
Yb-8 and Rs-9 gave negative reaction
with urease production but, Wf-6 isolate
gave positive reaction in the same test.
Meantime, these isolates Wl-1, Wf-6, Pf-
4, and Rs-9 gave positive reaction with
nitrate reduction and oxidase reaction.
The isolate Yb-8 gave negative reaction
in the same test. The isolate Wf-6 gave
negative reaction with levan production
test while, the four isolates gave positive
reaction in this test. Finally, the
aforementioned tests and their result
revealed that the three isolates
i.e.
Wl-1,
Pf-4 and Rs-9 could be identified as
Pseudomonas cichorii,
while, the
isolates
i.e.
Yb-8 could be identified as
Pseudomonas fluorescens
but the isolate
Wf-6 was identified as
Bacillus subtilis.
17
Table 4: Identification of bacterial isolates which isolated from Chrysanthemum plants.
Test
Reaction
Wl-1
Wf-6
Pf-4
Yb-8
Rs-9
Gram reaction
-
+
-
-
-
KOH 3%
+
+
+
+
+
Starch hydrolysis
-
+
-
-
-
Gelatin Liquefaction
-
+
-
-
-
Catalase activity
+
+
+
+
+
Methyl red test
d
*
-
d
-
d
Production of H
2
S
d
-
d
-
d
Production of indole
-
-
-
-
-
Urease production
-
+
-
-
-
Nitrate reduction
+
+
+
-
+
Growth at 41°C
-
-
-
-
-
Levan production
+
-
+
+
+
Oxidase reaction
+
+
+
-
+
Bacterial species
P. cichorii
B. subtilis
P. cichorii
P. fluorescens
P. cichorii
*
d = different reaction
3.4.2 Molecular identification of the
bacterial blight disease
Amplification of bacterial blight
(
Pseudomonas cichorii
)
DNA from three
bacterial isolates were done with Six
SCoT primers of 115 amplified
fragments, 115 were polymorphic
(19.17%) / primers and the percentage of
polymorphism ranged from 100%in all
Scot primers; the fragment sizes were
108bp to 1202bp (Table 5 and Figure 1).
The number of bands which obtained by
each primer was 28 with Scot- I primer,
26 with the Scot-2 primer, 13 with Scot-
3 primer, 12 with both Scot-4 primer and
24 with Scot-5 primer and12 with Scot-6.
The Scot-I primer had the higher
polymorphism bands; the fragment sizes
were 108bp to 1202bp but Scot- 4 and
Scot-6 primers gave the lowest
polymorphism bands; the fragment sizes
were 146bp to 920 bp.
3.4.3 Jaccard's similarity coefficient
The UPGMA cluster analysis based on
pairwise genetic similarity coefficient
revealed that the similarity between the
three-bacterial blight bacterial isolates
ranged from 0.848 between Rs-9 and Pf-
4 to 0.889 between Pf-4 and Wl-1; the
average of similarity among genotypes
was 0.867 (Table 6 and Figure 2). The
dendrogram constructed from SCoT
analysis of
Pseudomonas cichorii
isolates collected from geographically
diverse zones of Egypt separated the
three isolates into two major SCoT
groups (Figure 2).
Table 5: Characteristics of Scot marker banding profiles of Pseudomonas cichorii isolates.
Scot
Primer
Total
number
of bands
Monomorphic
bands
Specific
bands
Polymorphic
bands
% of
polymorphis
m
Molecular
weight (bp)
Scot-1
28
0.0
0.0
28
100
108-1202
Scot-2
26
0.0
0.0
26
100
120-787
Scot-3
13
0.0
0.0
13
100
282-999
Scot-4
12
0.0
0.0
12
100
217-768
Scot-5
24
0.0
0.0
24
100
126-998
Scot-6
12
0.0
0.0
12
100
146-920
Total
115
0.0
0.0
115
100
--
Average
19.17
0.0
0.0
19.17
100
--
18
Figure 1: SCoT marker profiles for the three Pseudomonas cichorii isolates Wl-1, Pf-4
and Rs-9 with (a): Scot-1, 2 and 3 primers;(b): Scot-4 ,5 and 6 primers and M: 100 bp
DNA ladder (Lane 1).
Table 6: Similarity Matrix computed with Dice coefficient.
Isolates
Wl-1
Pf-4
Rs-9
Wl-1
1
0.889
0.865
Pf-4
1
0.848
Rs-9
1
Figure 2: Dendrogram generated using Un-Weighted Pair Group Method with arithmetic average
(UPGMA) analysis showing relationships among different Pseudomonas cichorii isolates (Wl-1, Pf-4
and Rs-9) from Egyptian, using SCOT.
The first group was formed by
(
Wl-1
and
Pf-4); the second group only one isolate
(Rs-9) of this group. Finally, found the
different within groups, the various
bacterial strains diverged slight from
each other.
3.5 Host range of
Pseudomonas
cichorii
bacterium
From the previous experiments it was
clearly shown that three isolates of the
identification bacteria isolated from
chrysanthemum parts could be identified
as
Pseudomonas cichorii i.e.
Wl-1, Pf-4
and Rs-9. But, Wl-1 isolate was highly
virulence on pathogenic of all
chrysanthemum varieties. Results in
(Table 7)
clearly showed virulence of
Pseudomonas cichorii
isolate (Wl-1) on
eight different host plants from four
different plant families. It is clear from
the obtained results that Chrysanthemum
(
Chrysanthemum grandiflorum
)
cv.
White Zambla and Lettuce (
Lactuca
saliva
) cv. Balady from family
Asteraceae were highly susceptible one
19
among the eight tested hosts to infection
with the tested isolate (Wl-1) isolate with
injection method of inoculation at 15
days post inoculation. Meanwhile,
cabbage (
Brassica oleracea
)
cv.
Sabeany, from family
Brassicaceae,
Mint (
Mentha viridis
)
cv. Balady, from
family
Lamiaceae
and pepper (
Capsicum
annuum
)
cv. California wander, tomato
(
Solanum lycopersicum
)
cv. super strain
b, Datura (
Datura metel
)
cv. Balady,
eggplant (
Solanum melongena
) cv.
Balady long white from family
Solanaceae
were infected with the tested
isolate (Wl-1) isolate at 15-day
inoculation.
Table 7: Host range reaction and virulence of Pseudomonas cichorii isolate (Wl-1) on differential hosts
from different plant families.
Common name
Scientific name
Family
Reaction
Chrysanthemum
Chrysanthemum grandiflorum cv. White Zambla
Asteraceae
++
Lettuce
Lactuca saliva cv. Balady
Asteraceae
++
Cabbage
Brassica oleracea cv. Sabeany
Brassicaceae
+
Mint
Mentha viridis cv. Balady
Lamiaceae
+
Pepper
Capsicum annuum cv. California wander
Solanaceae
+
Tomato
Solanum lycopersicum cv. super strain b
Solanaceae
+
Datura
Datura metel cv. Balady
Solanaceae
+
Eggplant
Solanum melongena cv. Balady
Solanaceae
+
The results clearly reveal that (Wl-1)
isolate was able to infect all tested
different host plants from different plant
families.
4. Discussion
Chrysanthemum is one of the most
important commercial floriculture crops
of Egypt. The chrysanthemum is
subjected to attack by a number of
disease (Cavallini
et al.,
1992).
Chrysanthemum is vegetative propagated
flowering plant and is affected by many
diseases caused by fungi and bacteria.
(Shamala & Janardhana, 2018).
Major
bacterial blight disease caused by
Pseudomonas cichorii
has become a
serious problem in many parts of the
world (Timilsina
et al.,
2017).
Shamala
and Janardhana (2018)
found that the
bacterial blight symptoms include dark
brown spots and blotches on leaves
extending beyond plant leaves with
water-soaked lesions on stems,
darkening and death of buds and stem
terminals. Affected plants showed brown
to black decay at the base. As for
sampling and isolation of the disease, the
bacterial disease occurs on flowers,
stems, buds and leaves. Leaf infections
appear as water-soaked spots then
become brown and dry. Also, ten
bacterial isolates were isolated from
different parts of Chrysanthemum which
collected from different localities of
Egypt. In this respect, the bacterial
isolates designated as Wl-1 and Wb-2
were isolated from leaf and bud
respectively of chrysanthemum white
variety in Qualubia (Moshtohor).
Meanwhile, the isolates coded as Ps-3
and Pf-4 were isolated from stems and
flower of purple variety respectively in
the same governorate (El-kanater El-
20
khairia). On the other hand, the bacterial
isolates coded as Ws-5 and Wf-6 were
isolated from stems and flower of white
variety Menoufia governorate (Shebeen
El-kom). while, the isolates coded as Yl-
7 and Yb-8 were isolated from leaf and
buds of yellow variety respectively in
Giza governorate (Kirdasah). The isolate
Rs-9 and Rf-10 were isolated from stems
and flower of red variety from the same
governorate (Elmariotia). Isolation from
Chrysanthemum parts are in harmony
with those obtained by (Janse, 1981) who
identified a bacterium, as
Pseudomonas
cichorii,
isolated from Chrysanthemum
plants and Jokes
et al
. (1983) described a
similar disease, also caused by
P.
cichorii
, on field grown chrysanthemum
in USA. Moreover, Bolick (1960) and
Mcfadden (1961)
found that
P. cichorii
had only been the cause of bud blight
and leaf spot of chrysanthemum.
Horita
(1993) found that bacterial disease
characterized by
Pseudomonas cichorii
incited Chrysanthemum plants made
visible symptoms in infected leaves
appear as marginal legion in japan.
Isolates were pathogenic to
chrysanthemum by spray inoculation.
The causal organism produced a
fluorescent pigment on King's B medium
and was arginine dihydrolase negative,
gelatin liquefaction negative and oxidase
positive, which identified it as
P.
cichorii
. In relation to the identification
of isolated bacteria using traditional
techniques according to their
morphological, biochemical and physio-
logyical characteristics, these traditional
tests showed that these isolates could be
of three isolates
i.e.
Wl-1, Pf-4 and Rs-9
could be identified as
Pseudomonas
cichorii,
while, the isolates i.e. Yb-8
could be identified as
Pseudomonas
fluorescens
but the isolate Wf-6 was
identified as
Bacillus subtilis
. SCoT
markers have been successfully used to
assess genetic diversity and structure,
identify cultivars, and for quantitative
trait loci (QTL) mapping and DNA
fingerprinting in different species (Caho
et al
., 2014). Stead
et al.
(2013) reported
that
Pseudomonas cichorii
have a wide
host range of high economic importance
including tomato, lettuce and
watermelon. Moreover, Marques
et al
.
(2016) found that isolates of
Pseudomonas cichorii
were pathogenic
to the other 24 plants in artificial
inoculations. This bacterium has a wide
distribution and range of hosts, and this
report is important for cataloging plant-
pathogenic bacteria. Timilsina
et al
.
(2017) reported that the strains were
pathogenic on tomato and were also
pathogenic on lettuce. Based on isolation
and identification tests, these results
show that the cause of bacterial blight of
chrysanthemum belongs to
P. cichorii
isolates. In addition, isolates were
pathogenic for eight plants from
different plant families by artificial
inoculations. This bacterium has a wide
host range and it is important to list the
plant pathogenic bacteria, which can
become epidemic in whole Egypt under
special circumstances.
References
Amirmoradi B, Talebi R, Karami E, 2012.
Comparison of genetic variation and
differentiation among annual Cicer
species using start codon targeted
21
(SCoT) polymorphism, DAMD-PCR,
and ISSR markers. Plant Systematics and
Evolution 298: 1679–1688.
Arora JS, 1990. Introductory Ornamental
Horticulture. Kalyani Publishers, New
Delhi, India, 48 pp.
Aysan Y, Sabin S, Ulke G, Sabin F, 2003.
Bacterial rot of lettuce caused by
Pseudomonas cichorii in Turkey. Plant
Pathology 52(6): 782–782.
Bolick JH, 1960. Bacterial bud blight of
chrysanthemum. Proceedings of the
Florida State Horticultural Society 73:
346–351.
Caho S, Ferreira L, Carvalho A, Martins-
Lopes P, Martin A, Lima-Brito JE, 2014.
Potential of Start Codon Targeted
(SCoT) markers for DNA fingerprinting
of newly synthesized tritordeum and
their respective parents. Journal of
Applied Genetics 55: 307–312.
Cavallini G, D'Ercole N, Passerini G, 1992.
Altemaria and Fusarium: two adverse
diseases of chrysanthemum
(Chrysanthemum indicum). Informatore
Agrario 48: 31–48.
Collard BC, Mackill DJ, 2009. Start Codon
Targeted (SCoT) polymorphism: A
simple, novel DNA marker technique for
generating gene-targeted markers in
plants. Plant Molecular Biology Reporter
27: 86–93.
Cottyn B, Heylen K, Heyrman J,
Vanhouteghem K, Pauwelyn E, Bleyaert
P, Baerenbergh JV, Hofte M, De Vos P,
Maes M, 2009. Pseudomonas cichorii as
the causal agent of midrib rot, an
emerging disease of greenhouse-grown
butterhead lettuce in Flanders.
Systematic and Applied Microbiology
22: 211–225.
Dreistadt SH, 2001. Integrated pest
management for floriculture and
nurseries. Publication 3402, Division of
Agriculture and Natural Resources,
University of California, USA.
Elnemr AM, 2018. Physiological studies on
Dendranthema grandiflorum plant.
M.Sc. Thesis, Faculty of Agricultural,
Benha University, Egypt, 224 pp.
EL-Shaer HF, Attallah AG, EL-Namouly SD,
2014. Genetic Diversity Based on SCoT
and ISSR Markers in Agrobacterium
isolated from Egyptian soil. Research
Journal of Pharmaceutical, Biological
and Chemical Sciences 5(4): 1605–
1616.
Fahy PC, Persley GJ, 1983. Plant Bacterial
Disease, a diagnostic guide. Academic
press, London, England, 377 pp.
Gumtow RL, Khan AA, Bocsanczy AM,
Yuen, JM, Palmateer AJ, Norman DJ,
2013. First Report of a Leaf Spot
Disease of Golden Dewdrop (Duranta
erecta) Caused by Pseudomonas cichorii
and a Xanthomonas Species in Florida.
Plant Disease 97(6): 836–836.
Horita H, 1993. Occurrence of bacterial leaf
spot disease of chrysanthemum incited
by pseudomonas cichorii (swingle)
Stapp in Hokkaido. Annual Report of
the Society of Plant Protection of North
Japan 44: 71–73.
Janse JD, 1981. Pseudonionas cichorii in
gerbera. Verslagen en Mededelingen van
de Plantenziektenkundige Dienst 157:
28.
Jokes JB, Engelhard AW, Raiu BC, 1983.
Outbreak of a stem necrosis on
chrysanthemum incited by
Pseudonionas cichorii in Florida. Plant
Disease 67: 431–433.
22
Krieg NR, Holt JR, 1984. Bergey’s Manual
of Systematic Bacteriology. Baltimore,
MD : Williams & Wilkins, 38–46 pp.
Lelliott RA, Stead DE, 1987. Methods for the
diagnosis of bacterial diseases of plants.
Blackwell Scientific Publications,
Oxford, London 216 pp.
Marques E, Borges R, Uesugi C, 2016.
Identification and pathogenicity of
Pseudomonas cichorii associated with a
bacterial blight of gerbera in the Federal
District. Horticultura Brasileira 34: 244–
248.
Mcfadden LA, 1961. A bacterial leaf spot of
florists' chrysanthemums,
Chrjsunthemum morifolium. Plant
Disease Reporter 45: 16–19.
Nei M, Li W, 1979. Mathematical model for
studying genetic variation in terms of
restriction endonucleases. Proceedings of
the National Academy of Sciences of the
United States of America76: 5269–5273.
Rohlf FJ, 1993. NTSYS-pc: Numeric
taxonomy and multivariate analysis
system, Exeter Software, East Setauket,
New York, USA.
Scortichini M, Marchesi U, Rossi M,
Diprospero P, 2002. Bacteria associated
with hazelnut (Corylus avellana L.)
decline are of two groups: Pseudomonas
avellanae and strains resembling P.
syringae pv. syringae. Applied and
Environmental Microbiology 68(2):
476–484.
Shamala G, Janardhana G, 2018. Isolation
and identification of pseudomonas
putida from bacterial leaf blight disease
of chrysanthemum and its management
using bactericides and bio-agents in vitro
and in vivo in karnataka (India).
International Journal of Pharmaceutical,
Chemical and Biological Sciences 8(1):
125–133.
Stead DE, Simpkins SA, Weller SA,
Hennessy J, Aspin A, Stanford H, Smith
NC, Elphinstone JG, 2013.
Classification and identification of plant
pathogenic Pseudomonas species by
REP-PCR derived genetic fingerprints.
In: Pseudomonas syringae and related
pathogens. Springer, Dordrecht,
Holland, 411–420 pp.
Strayer A, Garcia-Maruniak A, Sun X,
Schubert T, Sutton B, 2012. First report
of Pseudomonas cichorii causing leaf
spot of stevia detected in Florida. Plant
Disease 96(11): 1690–1690.
Testen AL, Nahson J, Mamiro DP, Miller
SA, 2015. First report of tomato pith
necrosis caused by Pseudomonas
cichorii in Tanzania. Plant Disease
99(7):1035–1035.
Timilsina S, Adkison H, Testen AL,
Newberry EA, Miller SA, Paret ML,
Minsavage GV, Goss EM, Jones JB,
Vallad GE, 2017. A Novel phylogroup
of Pseudomonas cichorii identified
following an unusual disease outbreak
on tomato. Phytopathology 107: 1298–
1304.
Trantas EA, Sarris P F, Mpalantinaki EE,
Pentari MG, Ververidis FN, Goumas
DE, 2013. A new genomovar of
Pseudomonas cichorii, a causal agent of
tomato pith necrosis. European Journal
of Plant Pathology 137(3): 477–493.
Vegh A, Nemethy ZS, Salamon P, Mandoki
Zand Palkovics L, 2014. First report of
bacterial wilt on Chrysanthemum caused
by Dickeya chrysanthemi (syn. Erwinia
chrysanthemi) in Hungary. Plant Disease
98(7): 988.
23
Wehlburg C, 1963. A bacterial spot of
cabbage caused by Pseudomonas
cichorii. Florida Agricultural Experiment
Station Journal Series 1724: 119–122.