Journal of Phytopathology and Pest Management 7(1): 64-78, 2020
pISSN:2356-8577 eISSN: 2356-6507
Journal homepage: http://ppmj.net/
∗
Corresponding author:
Naglaa M. Balabel,
E-mail: naglaa.balabel@arc.sci.eg
64
Copyright © 2020
Detection of
Ralstonia solanacearum
phylotype II,
sequevar 1 in seasonal weed plants associated with
potato cultivations in Egypt
Naglaa M. Balabel
1,2*
1
Bacterial Disease Research Department, Plant Pathology Research Institute, Agricultural
Research Center, Giza, Egypt
2
Potato Brown Rot Project, Ministry of Agriculture and Land Reclamation, Dokki, Egypt
Abstract
Keywords: potato bacterial wilt, immunofluorescence antibody stain, real-time PCR, phylotype analysis, seasonal weed flora.
65
1. Introduction
Potato Bacterial wilt (syn. brown rot)
caused by
Ralstonia solanacearum
phylotype II (sequevar 1) (previously
Pseudomonas solanacearum
) is one of
the most serious plant diseases. It was
possibly first reported in Egypt by Briton-
Jones (1925) in El-Gemmeiza farm, El-
Gharbeya governorate based on
symptomology only. Although the first
introduction of the disease is not well
documented, it has been assumed to
coincide with the mass importation of
potatoes at the time of Mohamed Ali
Pasha (1805 ac), the Ottoman Viceroy
also, the French invasion of Egypt by
Napoleon Bonaparte (1798 ac) might be
another possible mean of introduction
from Europe (Farag, 2000). Potato brown
rot is being described as a quarantine
disease that means no tolerance is
allowed (zero tolerance) for any
consignment, during export (EFSA Panel
on Plant Health, 2019; Anonymous,
2000).
Ralstonia solanacearum
invades
intercellular elements of roots where it
multiplies before invading xylem vessels
and producing exopolysaccharide (EPS),
leading to wilt of the infected plant. The
vascular element classified the pathogen
as causing wilt of potato plants and
rotting of tubers and may be described as
sore eye or jammy eye (visual
symptoms), however it may survive
latently in the plants without causing any
symptoms (Prior et al., 1998). The
pathogen has a wide range of
economically important crops including
different weeds. It is literally described as
vascular disease for more than 450 plant
species belonging to 54 different
botanical families (Allen, 2005). Many
infected plant species do not show
symptoms after infection and therefore
escape attention as hosts, enabling the
pathogen to persist though rotations with
non-host crops. Granada and Sequeira
(1983) reported that the bacterium of
R.
solanacearum
can infect the roots of
different plants considered as non-hosts
but it does not survive for long periods in
vegetation-free soil. The survival of this
bacterium can be by infecting susceptible
plants or by colonizing the rhizospheres
of non-host plants. The bacterium
survived for only few weeks in
artificially inoculated soils (Balabel
et al.,
2005; Granada & Sequeira, 1983).
However, under field conditions the
pathogen survived in the absence of
potato for long periods (Pradhanang,
1998a; 1998b). Previous studies have
shown that
R. solanacearum
could be
infect solanaceous and certain non
solanaceous weeds such as
Solanum
dulcamara
,
Tropaeolum majus
(Olsson,
1976a;
1976b),
S. cinereum
(Graham &
Lloyd, 1978),
Portulaca oleracea
,
Tagetes
sp.,
Ipomoea
sp. (Zambrano,
1990),
Urtica dioica
(Van-Elsas, et al.,
2001),
Solanum nigrum
and
Rumex
sp.
(Balabel et al., 2005). All these weeds
may not show wilt symptoms and, thus,
were not previously considered as hosts.
This study was initiated to identify
different weeds that could act as an
occasional natural host to
R.
solanacearum
race 3 to explain extended
natural survival of
R. solanacearum
and
to explore the role of weeds in the
epidemiology of
R. solanacearum
phylotype II, sequevar 1 (race 3 biovar
2).
2. Materials and methods
2.1 Sampling area unit(s)
All weed flora raised in potato fields, at
locations in concern 70 days of planting
potato in winter and summer plantation
were gently uprooted and subject to
botanical identification as well as
R.
solanacearum
detection. Sampling was
66
made in term of uproding all weeds
developed per one square meter (Kirat
area) and three replicated areas per each
feddan (Acre) were considered.
2.2 Survey and sample collection
Samples of different weeds developing
seasonally in different old traditional
potato villages such as Digwa, El-
Saadany, Al- Rifai, Kafr -Yaqoub, Abu
Sawyer and Talia at Al-Kalubeiah, El-
Behira, El-Giza, El-Gharbeya, El-
Ismailia and El-Menofiya governorates
respectively, were
collected
during 2017-
2018 and 2018-2019 growing season and
were identified according to Zaki (1991).
2.3 Isolation of
R. solanacearum
from
weeds
Isolation of
R. solanacearum
was carried
out from crown area of the weed stems,
as described by Pradhanang et al. (2000).
Stems were washed thoroughly with tap
water and surface disinfected by flaming.
Thin sections were made under aseptic
conditions and macerated in 1 ml sterile
phosphate buffer (0.01 M) in small sterile
plastic bags then allowed to stand for 30
min. The supernatant was plated
(0.1ml/plate) on modified Semi Selective
Medium of South Africa (SMSA)
(Elphinstone et al., 1996). Incubation was
made at 28°C for 72 hour. A single
typical phenotype colony (reddish,
irregular and fluidal) from each sample
was selected for further work. All isolates
were maintained for long-term duration
as a suspensions in sterile tap water and
were revived by plating on tetrazolium
chloride (TTC) medium (Kelman, 1954),
when desired. A total of 75
R.
solanacearum
isolates taken from various
25 plant species and 6 governorates were
selected as a representative population.
These subcultures were used for
identification and DNA extraction.
2.4 Identification of
R
.
solanacearum
isolates
2.4.1 Immunofluorescence antibody
stain (IFAS) test
Typical colonies of the selected isolates
were selected and propagated on nutrient
agar medium for 48 hours, colony-
morphology determination was
confirmed by a serological test (IFAS),
immunofluorescent antibody staining
(Janse, 1988). The polyclonal antibodies
(cat. No. 07356) manufactured by Lowe
Biochemica GmbH, Germany and
produced in goats against
R.
solanacearum
, race 3 biotype II, was
used while, the anti-rabbit anti-goat
(RAG/Ig (H+L) (FITC) antiserum (cat.
No. 07200) manufactured by Nordic
Immunological Laboratories, Nether-
lands was used as a conjugate.
2.4.2 Real-Time PCR (Taq-Man) assay
Identification of
R. solanacearum
via
qPCR was performed on the selected
isolates according to Weller et al. (2000)
by using the apparatus of Applied
Biosystems 7500. The reaction mixture
consisted of 12.5 µl of master mix, 1 µl
of primer forward, 1 µl of primer reverse,
1 µl of probe and 7 µl of water and 2.5 µl
of nucleic acid extract. The following
program conditions was used: (1) 2 min.
at 50 C°, (2) 10 min. at 95 C°, (3)
followed by 40 two-step cycles of 10 sec
at 95 C° then 1 min. at 60 C°. The
sequence of primers and probe used is
67
shown in Table (1) and were provided by
OPRON, USA. Every run included
controls, DNA extraction (69/20)
provided by Potato Brown Rot Project as
a positive control and sterile pure water
instead of bacterial suspension as a
negative control.
2.5 Differentiation of
R. solanacearum
isolates into biovar(s) and race(s)
2.5.1 Biovar determination
Selected isolates (75) were checked for
the ability to oxidize three disaccharides
(cellobiose, lactose, maltose) and three
hexose alcohols (dulcitol, mannitol,
sorbitol) to determine biovar of
R.
solanacearum
as described by Hayward
(1964).
2.5.2 Race determination
Analyses of selected isolates were
performed by using the Opina primers
759/760 as internal markers specific for
the
R. solanacearum
strains and a set of
four phylotype - specific forward primers
with a unique and conserved reverse
primer targeted in the 16S-23S
Intergentic Spacer region (Opina et al
.,
1997) that allows discrimination between
different races. Information of the
primers are presented in Table (2). The
reaction mixture prepared by adding:
12.5 μl of ready master mix, 1 μl from
each primer, 7.5 μl of water and 2 μl of
nucleic acid extract. The following
cycling program was used in a thermal
cycler (Biometra T personal): (1) 96°C
for 5 min. (2) then cycled through 30
cycles of 94°C for 15s, 59°C for 30s and
72°C for 30s, (3) followed by a final
extension period of 10 min. at 72°C. 13
μl aliquot of each amplified PCR
products was subjected to electrophoresis
on 2 % (w/v) agarose gels, stained with
ethidium bromide (0.5% μgL-1) and
imaged (Sagar et al
.,
2014).
Table 1: Characteristics of primers and Taq-Man probe used to detect R. solanacearum by Real-time PCR.
Primer or probe
Sequence(5'→3')
Length
Dye
RS-I-F
GCA TGC CTT ACA CAT GCA AGTC
22
RS-II-R
GGC ACG TTC CGA TGT ATT ACT CA
23
RS-P
AGC TTG CTA CCT GCC GGC GAG TG
23
FAM
Table 2: Bases sequence(s) of used primers and their length(s) for phylotype analysis of R.
solanacearum by Multiplex- PCR.
Primer
Sequence (5'→3')
Length
759
GTC GCC GTC AAC TCA CTT TCC
21
760
GTC GCC GTC AGC AAT GCG GAA TCG
24
Nmult:21:1F
CGT TGA TGA GGC GCG CAA TTT
21
Nmult:21:2F
AAG TTA TGG ACG GTG GAA GTC
21
Nmult:23:AF
ATT ACS AGA GCA ATC GAA AGA TT
23
Nmult:22:InF
ATT GCC AAG ACG AGA GAA GTA
21
Nmult:22:RR
TCG CTT GAC CCT ATA ACG AGT A
22
2.6 Pathogenic potential of selected
isolates
Selected isolates (75) were tested for
pathogenic potential(s) via inoculation
into young tomato (
Solanum
lycopersicum
cv. Pinto) seedlings with a
suspension of a 48 hour nutrient agar
culture, 10
6
cells ml
-
1 in sterile water
(Janse, 1988). Injection was made at the
68
leaf axis by a needle laden with the
bacterial growth of the pathogen. Control
treatments were prepared by applying
few drops of sterile water instead of
bacteria. The inoculated plants were
covered with polyethylene bags for one
day, kept at 30°C, then bags were
removed and pots were irrigated as
required and examined for wilting
symptoms after bench incubation.
3. Results
3.1 Isolation of
R. solanacearum
from
different weeds
Using SMSA medium, seventy five
isolates were selected by the
characteristics previously reported. They
selected from different villages (21
isolates from El-Saadany, 15 isolates
from Al- Rifai, 15 isolates from Kafr-
Yaqoub, 8 isolates from
Abu Sawyer, 8
isolates from
Digwa and 8 isolates from
Talia). Selected colonies were irregular,
reddish, and fluidal white with red center
(Figure 1). Out of (1609) samples
collected from different weeds only
(272) were found infected, the rates of
successful isolation from these weeds
were generally low, and account for
16.9%. Also, Table (3) shows that, the
highest infection percentage of weed
plants were shown from El-Gharbeya
and Al-Kalubeiah governorates (44.7 and
31.7 % respectively) followed by El-
Behira governorate (16.1%), whereas the
lowest percentage (11.5%) was observed
in El- Menofiya governorate. On the
other hand, both El-Giza and El-Ismailia
governorates showed almost similar
percentage of infected weeds (12.7 and
13.9% respectively).
Figure 1: Typical colony with milky white and fluidal central
blood red color of R. solanacearum on modified Semi
Selective Medium of South Africa.
69
Table 3: Detection of R. solanacearum in naturally growing weed species during two growing seasons (2017-2018
and 2018-2019) in plots with potato crop in the six governorates of Egypt.
Governorate (Village)
Total no. of
tested plants
Number of
healthy plants
Frequency of detection of R.
solanacearum in stem base
Infection (%)*
Al-Kalubeiah (Digwa )
63
43
20/63
31.7
El-Behira (El-Saadany)
762
639
123/762
16.1
El-Giza (Al-Rifai)
332
290
42/332
12.7
El-Gharbeya (Kafr-Yaqoub)
94
52
42/94
44.7
El-Ismailia (Abu Sawyer)
158
136
22/158
13.9
El- Menofiya (Talia)
200
177
23/200
11.5
Total
1609
1337
272
*
Data in Table (4) showed that these
weeds were belonged to twenty five
species affiliated to thirteen families
marking the potato fields. The results
obtained indicate that, according to the
(272) number of infected weeds may be
divided into three groups according to
their predominance. The first group
included species with large numerical
numbers such as:
Chenopodium album
L.,
Cichorium pamilum
,
Malva parviflora
L.,
Dactyloctenium aegyptium
L.,
Cynodon dactylon
L.,
Amaranthus
ascendens
Lois
and Portulaca oleracea
L
.
The second group included the
infected weeds in medium numbers as:
Brassica nigra
L.,
Convolvulus arvensis
,
Polypogon monspeliensis
,
Cyperus
rotundus
,
Rumex dentatus
and
Beta
vulgaris
. The third group included
species of poorly dominated weeds such:
Amaranthus cruentus
L.,
Arachis
hypogaea
,
Chenopodium mural
L.,
Centaurea calcitrapa
L.,
Cyperus
difformis
L.,
Conyza aegyptiaca
L.,
Dicanthium annulatum
,
Medicago
polymorpha
L.,
Sonchus oleraceus
L.,
Solanum nigrum
L.,
Sisymbrium irio
L.
and
Urtica urens
L.
The results revealed
that, the winter annual weeds were the
most affected weeds followed by the
summer annual weeds while the
perennial and biennial weeds included
almost the same number of infested
weeds 4 and 3, respectively.
3.2 Identification and characterization
of
R. solanacearum
isolates
3.2.1 Immunofluorescence antibody
stain (IFAS) test
Immunofluorescence antibody stain
(IFAS) test was carried out on selected
colonies to confirm identity. The cells
showed short rod morphology stained
evenly as bright green fluorescent
(Figure 2).
3.2.2 Real-Time PCR (Taq-Man) assay
Real-time PCR is a sensitive test for
detection of low concentrations of
R.
solanacearum
and is being considered a
confirmatory test in the detection work.
The RS primers and probe were
employed to detect all biovars and races
of
R. solanacearum
. Positive results were
noticed with all tested isolates indicating
that the 75 isolates were
R.
solanacearum.
70
Table 4: Common, scientific names and families for different weeds collected randomly from different governorates.
Group*
No. of positive
plants
Growing season
Family
Scientific name
Common name
Governorate (Village)
3
5
Annual winter
Asteraceae
Sonchus oleraceus L.
Annual sowthistle
Al-Kalubeiah( (Digwa)
3
4
Annual summer
Asteraceae
Conyza aegyptiaca L.
Fleabane
3
2
Perennial
Poaceae
Dicanthium annulatum
Forssk
3
2
Annual winter
Brassicaceae
Sisymbrium irio L.
London rocket
3
7
Annual summer
Fabaceae
Arachis hypogaea
Peanut
1
20
Annual winter
Amaranthaceae
Amaranthus ascendens Lois
Livid amaranth
El-Behira (El-Saadany)
1
34
Annual winter
Chenopodiaceae
Chenopodium album L.
Common lambsquarters
3
4
Annual winter
Chenopodiaceae
Chenopodium mural L.
Goosefoot
3
3
Annual summer
globe
Cyperaceae
Cyperus difformis L.
Small flower umbrella plant
1
25
Annual winter
Malvaceae
Malva parviflora L.
Cheese weed (Little Mallow)
3
2
Annual winter
Fabaceae
Medicago polymorpha L.
Burclover
2
12
Annual summer
Poaceae
Polypogon monspeliensis L.
Rabbitfoot grass
2
10
Perennial
Cyperaceae
Cyperus rotundus L.
Purple nutsedge
3
3
Annual summer
Solanaceae
Solanum nigrum L.
Black nightshade
2
10
Annual or biennial
Amaranthaceae
Beta vulgaris L.
Wild beet
1
21
Annual summer
Poaceae
Dactyloctenium aegyptium L.
Crowfoot grass
El-Giza (Al-Rifai)
1
21
Perennial
Poaceae
Cynodon dactylon L.
Bermuda grass
1
25
Annual winter
Asteraceae
Cichorium pamilum
Chicory
El-Gharbeya (Kafr-Yaqoub)
1
17
Annual summer
Portulacaceae
Portulaca oleracea L.
Common purslane
2
10
Annual winter
Polygonaceae
Rumex dentatus L.
Dock
El-Ismailia (Abu Sawyer)
3
2
Biennial
Asteraceae
Centaurea calcitrapa L.
Purple starthistle
2
10
Annual herb
Brassicaceae
Brassica nigra L.
Black mustard
3
3
Annual winter
Amaranthaceae
Amaranthus cruentus L.
Pigweed
El- Menofiya (Talia)
2
11
Perennial
Convolvulaceae
Convolvulus arvensis L.
Field bindweed
3
9
Annual winter
Urticaceae
Urtica urens L.
Burning nettle
*The isolates were classified according to their predominance into three groups: 1 = greater than or equal 15, 2 = 10 to 14, 3 = 1 to 9.
Figure 2: Cell morphology of R. solanacearum in the serological immunofluorescent
antibody staining (IFAS) test.
3.3 Differentiation of
R. solanacearum
isolates into biovar(s) and race(s)
3.3.1 Biovar determination
The biovars determination was based on
the ability of isolates to produce acids
from hexose and alcohol sugars. The
studied Seventy five isolates were able
to produce acids from lactose, maltose,
and cellibiose. All isolates, however,
were unable to produce acids from
sorbitol, mannitol, and dulcitol denoting
that, these seventy five isolates were
assigned to biovar 2 which is the only
race in Egypt described as race 3,
biovar II, in previous work (Table 5).
71
Table 5: Biovar determination of the selected R. solanacearum isolates from different villages in Egypt.
Governorate
(Village)
Isolate's
number
Utilization of (Acid without gas)
Maltose
Lactose
Cellobiose
Mannitol
Sorbitol
Dulcitol
El-Behira
(El-Saadany)
1
+
+
+
-
-
-
2
+
+
+
-
-
-
3
+
+
+
-
-
-
4
+
+
+
-
-
-
5
+
+
+
-
-
-
6
+
+
+
-
-
-
7
+
+
+
-
-
-
8
+
+
+
-
-
-
9
+
+
+
-
-
-
10
+
+
+
-
-
-
11
+
+
+
-
-
-
12
+
+
+
-
-
-
13
+
+
+
-
-
-
14
+
+
+
-
-
-
15
+
+
+
-
-
-
16
+
+
+
-
-
-
17
+
+
+
-
-
-
18
+
+
+
-
-
-
19
+
+
+
-
-
-
20
+
+
+
-
-
-
21
+
+
+
-
-
-
El-Giza
(Al-Rifai)
22
+
+
+
-
-
-
23
+
+
+
-
-
-
24
+
+
+
-
-
-
25
+
+
+
-
-
-
26
+
+
+
-
-
-
27
+
+
+
-
-
-
28
+
+
+
-
-
-
29
+
+
+
-
-
-
30
+
+
+
-
-
-
31
+
+
+
-
-
-
32
+
+
+
-
-
-
33
+
+
+
-
-
-
34
+
+
+
-
-
-
35
+
+
+
-
-
-
36
+
+
+
-
-
-
El-Gharbeya
(Kafr-Yaqoub)
37
+
+
+
-
-
-
38
+
+
+
-
-
-
39
+
+
+
-
-
-
40
+
+
+
-
-
-
41
+
+
+
-
-
-
42
+
+
+
-
-
-
43
+
+
+
-
-
-
44
+
+
+
-
-
-
45
+
+
+
-
-
-
46
+
+
+
-
-
-
47
+
+
+
-
-
-
48
+
+
+
-
-
-
49
+
+
+
-
-
-
50
+
+
+
-
-
-
51
+
+
+
-
-
-
El-Ismailia
(Abu Sawyer )
52
+
+
+
-
-
-
53
+
+
+
-
-
-
54
+
+
+
-
-
-
55
+
+
+
-
-
-
56
+
+
+
-
-
-
57
+
+
+
-
-
-
58
+
+
+
-
-
-
59
+
+
+
-
-
-
Al-Kalubeiah
(Digwa)
60
+
+
+
-
-
-
61
+
+
+
-
-
-
62
+
+
+
-
-
-
63
+
+
+
-
-
-
64
+
+
+
-
-
-
65
+
+
+
-
-
-
66
+
+
+
-
-
-
67
+
+
+
-
-
-
El-Menofiya
(Talia)
68
+
+
+
-
-
-
69
+
+
+
-
-
-
70
+
+
+
-
-
-
71
+
+
+
-
-
-
72
+
+
+
-
-
-
73
+
+
+
-
-
-
74
+
+
+
-
-
-
75
+
+
+
-
-
-
72
3.3.2 Race determination
According to the results of the Pmx-
PCR, all selected isolates
belonged to the
phylotype II sequevar I, as 372- bp
amplicon was produced in their reactions
(Figure 3).
Figure 3: PCR amplification products (280 and 372 bp) to detect R solanacearum in weeds from different locations using
pmx primers (Opina et al., 1997). Lane NC, negative control; lane PC, Positive control; lane 1-75, PCR amplicons
derived from 75 separate DNA extracts isolated from different weeds.
3.3.3 Pathogenic potential of
representative isolates
The pathogenic potential of randomly
selected isolates (75 isolates) was tested
for producing wilt to tomato seedlings, 3
days after stem inoculation under
greenhouse conditions. The results
showed that all tested isolates from
different locations were able to wilt
tomato seedlings (Figure 4).
Figure 4: Pathogenicity test of R. solanacearum isolates from different locations sources on tomato plants.
73
4. Discussion
Early detection of latent infection with
bacterial wilt caused by
R. solanacearum
may be playing an important role to
decrease the risk of crop loss. Several
detection methods have been developed
for
R. solanacearum
such as direct
plating on modified (SMSA) medium
,
enzyme-linked immunosorbent assay
(ELISA), IFAS, PCR-based methods and
bioassay in tomato seedlings (Weller et
al., 2000; Elphinstone et al., 1996) and
phylotype assignment (Sagar et al.,
2014).
All selected isolates were
collected from the different weeds in this
study were
identified as
R. solanacearum
using isolation on modified SMSA
medium, IFAS test, also real-time PCR
assay. On the other hand, the results of
biovar determination indicating that all
the tested isolates belong to biovar 2 or
the so called a member of potato race 3.
Moreover, phylotype specific multiplex
(Pmx)- PCR revealed that all seventy
five isolates of
R. solanacearum
belonged to phylotype II as a 372-bp
amplicon was observed for all the tested
isolates after electrophoresis (Agarose
gel 2% w/v). These results indicating
that, the race 3, biovar 2 (phylotype II,
sequevar I) is dominant in Egypt, the
same results were observed by other
searchers (Hanafy et al., 2018; Hassan, et
al., 2017; Mikhail et al., 2017). In this
study,
t
he pathogenicity tests showed
that all the tested isolates were virulent to
tomato plants using stem puncture
inoculation. The presence of
R.
solanacearum
can be detected by SMSA
medium at 10
3
CFU /ml (Mikhail et al.,
2016). This concentration was adequate
for the population analysis of this
bacterium in the weed samples. Also,
IFAS test is rapid and inexpensive
method but lack in sensitivity besides
giving false positive results due to cross-
reactions with other bacteria (Balabel,
2014). Other methods were able to detect
a lower concentration of the bacterium in
low densities. Methods such as real time-
PCR may be very successful in detecting
the bacterium's presence, but
preparation, time and money would be
needed for this technique. Although PCR
as a sensitive and precise detection tool
has great potential, it has not been
widely used for field samples.
Meanwhile, PCR detection results were
recorded from plant and soil samples
(Elphinstone & Stanford, 1998).
Inhibition of the enzymatic PCR reaction
by different compounds present in plant
and soil samples may be attributable to
inconsistent findings (Wilson, 1997;
Picard et al., 1992). Also, one of the
disadvantages of the PCR method is to
obtain false negative results in some
cases due to the presence of some
inhibitors (Farag & Balabel, 2014; Farag
et al
.,
2010). In this study, randomized
weed samples belong to (13) families
with (23) genera and (25) species were
collected from different potato fields
during two successive growing seasons
(2017-2018 and 2018-2019) of winter
and summer plantation. Seventy five
isolates were selected from these weeds
and identified as
R. solanacearum
. These
results indicate the important role of
weeds in overwintering and extended
survival of the pathogen in soil. In this
point several researchers have clarified
the role of the plant weeds. Tusiime et
74
al
.
(1998) reported that there are a large
number of latently infected non-
solanaceous weeds in highland Uganda
such as
Amaranthus
spp.,
Bidens pilosa
,
Galinsoga perviflora
,
Oxalis latifolia
,
Spergula arvensis
,
Rumex abyssinicum
,
Tagetes minuta
, and
Stellaria sennii
.
Dittapongpitch and Surat (2003) found
that weed samples belong to (13)
families including (17) genera and (18)
species were infected with
R.
solanacearum,
among
these families:
Amaranthaceae, Asteraceae, Chenopodi-
aceae, Cyperaceae, Portulacaceae and
Solanaceae. Also, Hamad et al. (2016)
revealed that,
Portulaca olracea
,
Solanum nigrum
,
Rumex dentatus
,
Chenopodium album, Brassica kaber
and
Beta vulgaris
are considered as hosts for
R. solanacearum.
These results are
consistent with the results of this study.
Many studies have suggested the
relationship between the presence of
weeds and the survival of bacterium is
due to infecting susceptible plants or by
colonizing the rhizospheres of non-host
plants. The pathogen able to colonize the
root systems of non-host plants including
many weeds, without causing any visible
symptoms. In this case weed hosts can
act as "Sheltered sites" and this way is
considered as one of the ways for the
survival of bacteria in the absence of the
suitable host (Graham et al., 1979) so,
controlling potato bacterial wilt is
difficult (Hayward, 1986). Latent
infection can play an important role in
spreading disease. The bacterium can
survive for a long time in soils
(Tomlinson et al., 2011), infested surface
irrigation water and infected weeds
(Tomlinson et al., 2009). From these
sources the bacterium can spread from
infested to healthy fields by soil transfer
on machinery, and surface runoff water
after irrigation or rainfall. Also, infected
semi-aquatic weeds may spread the
pathogen through releasing bacterium
from roots into irrigation waters (Hong
et al., 2008; Elphinstone et al., 1998).
There are a large number of weeds that
are considered an alternative host for
bacteria in areas planted with potatoes,
and as a result, the rate of bacterial
growth is slow, which leads to these
weeds being a constant source of
infection (Pradhanang et al., 2000). So,
control of weed hosts and volunteer
plants may be the most important ways
in control of
R. solanacearum.
It is
interesting to note that, there is little
information in Egypt that has looked at
the species of weeds that
R.
solanacearum
can enter in the absence
of a suitable host. Most of the weeds
reported as alternative hosts of the
pathogen including the following
species:
Brassica nigra
,
Beta vulgaris
,
Chenopodium album
,
Portulaca
oleracea
,
Rumex dentatus
and
Solanum
nigrum.
However, in this study many
species of weeds were reported. So, it is
very important to continue surveys to
determine new natural hosts and the role
they play in disease spread whereas,
weeds can act as host reservoirs of
infection. Therefore, elimination of weed
hosts could be part of an integrated
management strategy for the control of
potato bacterial wilt. In order to prevent
this source of inoculum, potato growers
need to take these findings into account
and use management strategies. Further
investigations would be needed to carry
75
out the presence of
R. solanacearum
in
weeds, water, and soil and with the
potato disease incidence to verify the
importance of various inoculum sources.
Acknowledgements
I would like to express
acknowledgements to all the team of Pest
Free Areas (PFA) in potato brown rot
project, Egypt for their assistance in
collecting weed samples from different
areas.
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