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Advances in Environmental Biology, 5(1): 209-215, 2011
ISSN 1995-0756
This is a refereed journal and all articles are professionally screened and reviewed
ORIGINAL ARTICLE
Systemic Resistance Induced in Potato Plants Against Potato Virus Y Common Strain
(PVYo) by Plant Extracts in Iraq
Rakib A. Al-Ani, Mustafa A. Adhab, Sabir N.H. Diwan
Plant Protection Department, College of Agriculture, University of Baghdad, IRAQ.
Rakib A. Al-Ani, Mustafa A. Adhab, Sabir N.H. Diwan: Systemic Resistance Induced in Potato Plants
Against Potato Virus Y Common Strain (PVYo) by Plant Extracts in Iraq
ABSTRACT
Potato virus Y is one of the most important viruses affecting potato wherever grown in Iraq. The virus
is transmitted by sap and by more than 50 species of aphids, in non–persistant manner. The present study was
conducted to evaluate the efficacy of frog fruit (Lippia nodiflora L.), Datura (Datura metel L.), and Thuja
(Thuja orientalis L.) extracts for inducing systemic resistance against Potato virus Y common strain (PVYO)
in potato plants. Air dried of aerial parts powdered was extracted with 70% ethanol (1:3) weight: volume, then
concentrated to consistence syrup in water bath at 42<C. The alcoholic extract was used as tuber-dipping before
cultivation or foliage spraying after germination. The PVYO multiplication in the plants was followed
serologically by Enzyme-linked immunosorbent assay (ELISA). Results showed that dipping the tubers in the
extracts, at 1, 2, 3 g/L for 24 hrs., or spraying the foliage by the extracts at the same concentrations exhibited
a protection period to the plants against PVYO infection up to one month in the presence of virus source with
the vector Myzus persicae Sulz. Means of ELISA readings at 405 nm for samples from treated plants were
found to be 0.11 and 0.125 for L. nodiflora extract, 0.13 and 0.126 for D. metel extract, 0.13 and 0.12 for T.
orientalis extract for the three concentrations and the two methods of application respectively, compared with
2.52 for untreated plants. No significant differences between the concentrations or between the methods of
application on virus multiplication were observed.
Key words: Systemic resistance, PVYO, medicinal plant extracts, control.
Introduction
The potato (Solanum tuberosum L., Solanaceae)
is considered among the most nutritive in the world
and in Iraq. Potato plants are susceptible to many
viruses that caused heavy losses in the yield of both
quality and quantity [23,30,34]. Potato virus Y (PVY)
the type member of the genus potyvirus, belong to
the largest plant virus family Potyviridae [12,31] one
of the most wide distributed and the most important
viruses infected potato and some other species in the
family Solanaceae [12,16,32]. PVY is occurred
worldwide wherever potatoes are grown [11].
PVY is easily transmitted by sap and is
transmitted by more than 50 species of aphids in
non-persistant mode [14,23]. Green peach aphid
Myzus persicae Sulz. was found to be the most
efficient vector to PVY [18,23]. The aphid can
acquire the virus from infected plants in less than 60
seconds and transmit in to a healthy plant in less
than 60 seconds [40].
Several strain groups of PVY were identified on
the basis of symptoms in naturally infected potatoes
S. tuberosum, Nicotiana tabacum, and Physalis
floridana. The common (ordinary) strain PVYO
induce mild to severe mosaic often associated with
crinkling, yellowing, leaf necrosis and leaf drop on
potato, vein clearing and mottle in tobacco. The
tobacco veinal necrosis group (PVYN) produces
milder mottle than PVYO on potato and severe
Corresponding Author
Mustafa A. Adhab, Plant Protection Department, College of Agriculture, University of Baghdad,
IRAQ.
Email: [email protected]
Adv. Environ. Biol., 5(1): 209-215, 2011
necrosis on leaf veins and petiols in N. tabaccum.
This strain (PVYN) was identified in previous study
in our laboratory in Iraq [6]. The PVYC isolates
induce stipple streak symptoms on some potato
cultivars and is not transmissible by aphids [12,33].
The objective of this study is to characterize the
common strain of PVY (PVYO) prevailing among
isolates collected from different potato production
regions in Abu-Ghraib – Iraq on the basis of
serology and bioassay, and to evaluate the possibility
of inducing systemic resistance in potato plants
against this strain by some medicinal plants extracts.
Materials and Methods
Virus isolates:
potato leaves from plants showing mottling, leaf
necrosis lesions, rugosity, yellowing, and stunting
suspected to be of Potato virus Y (PVY) infection
were collected from potato fields in Abu-Ghraib area
(West of Baghdad). The virus was identified
serologically by polyclonal antibodies to PVY
provided by Bioreba Company (Switzerland) as
immunostrip. The isolate that gives positive reaction
were purified by single-spot on Chenopodium
amaranticolor [5], and propagated in Nicotiana
tabacum cv. Samsun plants.
Virus strain characterization: leaves surfaces of
test plants consist of Chenopodium amaranticolor, N.
tabacum cv. Samsun, Capsicum annum, Datura
stramonium, Lycopersicon esculentum, Physalis
floridana and Solanum tuberosum were slightly
dusted with carborandum 600 mesh and mechanically
inoculated by sap from systemically PVY-infected N.
tabacum cv. Samsun. The sap was prepared by
grinding leaves showing virus symptoms in 0.01 M
phosphate buffer containing 0.2%
Diethyldithiocarbamate (DIECA) pH 7.2 at 1g/4ml.
Virus transmission by vector:
Apterous Myzus persicae were collected from
Apricot, and placed on PVY-infected N. tabacum cv.
Samsun for 15 min. to acquire the virus. Viruliferous
aphids were then transferred to healthy plants (10
aphids/plant) for 30 min. for inoculation. Also,
healthy plants were exposed to non-viruliferous
aphids as control. The plants were maintained in an
insect-free glasshouse at 25±2 <C for 4 weeks.
Virus purification:
Leaves from N. tabacum cv. Samsun showing
virus symptoms (100g) were grounded in a warring
blender with 0.5 M phosphate buffer (Na2HPO4) pH
7.0 containing 1% 2-mercaptoethanol 1:1 (g/ml). The
extract was passed through 2 layers of cheese cloth,
210
and n-butanol was added to the filtrate at 7%. The
mixture was agitated for one hour at 4<C and
centrifuged at 5000 rpm for 45 minutes in cooling
centrifuge.
Polyethylene glycol (PEG) 6000 MW and NaCl
were added at 4% and 2% respectively to the
supernatant with agitation. The solution was
maintained at 4<C for 24 hours and submitted to a
centrifugation at 5000 rpm for 45 min. The pellet
was solved in 1 ml of 0.01M phosphate buffer pH
7.2 and centrifuged at 5000 rpm for 20 min. The
supernatant was loaded on the surface of sucrose
gradient 10-50% with reverse gradient of Ammonium
sulfate 50-10% in gradient tubes. The tubes were
kept at 4<C for 24 hours and centrifuged at 5000 rpm
in swinging rotor for 60 min in cooling centrifuge.
Virus bands were collected from the gradient and
dialyzed against 0.01 M phosphate buffer pH 7.2 for
24 hour with agitation.
Serological identification
ELISA:
Leaves of infected potato plants were grounded
in phosphate buffer saline (PBS) pH 7.0 at 1 g/10
ml. The homogenate was filtered through double
layer of cheese cloth followed by a centrifugation at
5000 rpm for 5 min. and tested by DAS-ELISA
using polyclonal antibodies as described by Clark and
Adams [10]. The absorbance of ELISA reactions
were read at 405 nm one hour after adding the
substrate (P-nitrophenol phosphate) in ELISA-reader.
Sample from healthy plants were treated in the same
way and used as control. Absorbance values twice
the average of uninfected plants was considered
positive.
Immunostrip test:
Polyclonal antibodies to PVY provided by
Bioreba Co. (Switzerland) as immunostrip were used.
Leaves samples of 0.15 g from plants showing virus
symptoms were homogenized in plastic sacs
containing extraction buffer by pestle. The end of the
strip was dipped in the extract to 1 cm for 15
min[1]. An extract from healthy plants was treated in
the same way as control.
Induced systemic resistance against PVYO:
Aerial parts of medicinal plants, Thuja orientalis,
Datura metel, and Lippia nodiflora were collected
from the gardens of the College of Agriculture,
University of Baghdad, IRAQ. Samples of air dried
were ground with a mortar and pestle and 100 g of
the powder obtained was added to 300 ml of ethanol
80%, in a 1000 ml flask. The mixture was agitated
for 24 h at room temperature and passed through
Adv. Environ. Biol., 5(1): 209-215, 2011
filter paper (whatman 2) in Buchner funnel with
vacuum. The filtrate was concentrated at 42 EC in a
water bath and stored at -20 EC. Three
concentrations of each extract, 1, 2, 3 g/L in distilled
water amended with 0.1% of tween-20 were used.
Potato tubers were soaked in each concentration for
24 h and sown in plastic pots (25 * 18 * 18 cm)
containing soil mix and the plants were inoculated by
PVYO at 3 leaves stage. Also, potato plants generated
from healthy tubers were sprayed by the extracts and
inoculated by PVYO after 48 h of application. The
virus in the plants was serologically detected by
ELISA.
Results and Discussion
Virus detection:
Symptoms:
Leaf mottling often associated with chlorotic
spots appeared on the lower leaves of potato plants
mechanically inoculated by sap from leaves of N.
tabacum cv. Samsun exhibited virus symptoms or
transmitted to potato plants by aphids Myzus
persicae. These symptoms were developed to
necrosis on the upper leaves began by the main veins
then to secondary ones and to leaf petiol led finally
to death and leaf drop (fig. 1). Similar results were
previously obtained by many workers on PVY
[9,22,25].
Immunostrip test:
A band specific to PVY appeared on the strip
containing polyclonal antibodies to PVY treated with
extract from potato plants showing virus-symptoms
(fig. 2). This band is absent on the strip treated with
extract from healthy plants which indicates that the
virus under identification is PVY[1].
211
Das-elisa:
The isolates showing symptoms similar to those
exhibited by the ordinary strain were reacted
positively with monoclonal antibodies specific to
PVYO purchased from sigma chemical company
USA. The absorbance of ELISA reactions at 405 nm
were found to be 3.11, 3.04, and 2.07 for extracts
from N. tabacum cv. Samsun, Physalis floridana, and
S. tuberosum showing virus symptoms respectively
compared with 0.059 for extract from healthy plants.
The PVY-strain under identification in this study is
considered as PVY-common strain (PVYO), because
it induced typical mild mosaic on the leaves of N.
tabacum cv. Samsun without any indication of veinal
necrosis, while PVYN cause severe veinal necrosis on
this plant [13], and reacted positively with
monoclonal antibodies PVYO. It was found that the
present strain is easily transmissible by aphids M.
persicae in non persistant manner, while PVYC is not
transmissible by aphids [12,33].
Induced systemic resistance against PVYO:
Lippia nodiflora extract:
Results obtained showed that the concentrations
1, 2, 3 g/l of Lippia extract were highly effective in
inducing systemic resistance in potato plants against
PVYO infection. The means of absorbance values of
ELISA reactions at 405 nm were found to be 0.11
for extracts from plants generated from tubers treated
with the extract, compared with 2.55 for extract from
PVY-infected plants (control). Similar results were
obtained from plants sprayed by the extracts with
absorbance values 0.125 compared with 2.55 for the
control (virus infected plants) (Table 1). It was
shown in previous work that the alcoholic extract of
Lippia contain phenolic compounds; flavonoids and
terpenoids such Lippacin acts as antioxidants [8,29].
Datura metel extract:
Virus strain characterization:
Symptoms:
The virus induced systemic mild mottling on N.
tabacum cv. Samsun, Capsicum annum, and
Lycopersicon esculentum mechanically inoculated by
sap from PVY-infected N. tabacum cv. Samsun
leaves. Chlorotic local lesions were developed on the
inoculated leaves of Ch. amaranticolor, vein clearing
associated with mottle symptoms induced on the
leaves of Physalis floridana upon inoculation by
extract from virus infected N. tabacum cv. Samsun
(fig. 3). No symptoms were observed on sap
inoculated Datura stramonium and no virus was
detected from this plant. Similar results were
previously reported concerning PVYO strain
[9,11,18,25].
It has been found that the alcoholic extract of D.
metel induced systemic resistance in treated potato
plants. No virus was detected in the treated plants for
a period up to one month of PVY inoculation. The
means of ELISA readings at 405 nm were 0.13 for
samples from plants generated from treated tubers,
0.126 for samples from plants sprayed by the extracts
compared with 2.53 for the control (Table 1). It has
been reported that D. metel contain alkaloids, tannins,
flavonoids, and carbohydrates. The medical effect of
this plant is often attributed to its content
scopolamine (an alkaloid) [38].
Thuja orientalis extract:
The treatment of potato tubers with the alcoholic
Adv. Environ. Biol., 5(1): 209-215, 2011
extract of Thuja at 1, 2, 3 g/L induced systemic
resistance in the plant against PVYO for the same
period mentioned for Lippia and Datura extract. The
means of absorbance values at 405 nm were 0.13 for
samples from plants generated from treated tubers,
0.12 for samples from plants sprayed by the extracts
compared with 2.52 for the control (Table 1). The
effect of Thuja extracts may be due to the presence
of tannins as shown by previous study in our
laboratory [2,4].
The results obtained showed that the three
extracts from Lippia, Datura, and Thuja, used in this
study induced resistance against PVY. High
significant differences were found in ELISA readings
between the plants treated and non-treated as control.
No significant differences were observed between the
extracts or methods of application. These results
indicate that the use of substances derived from
higher plant as biological control against viruses
appears to be promising.
212
Discussion:
The symptoms manifested on, potato, Nicotiana
tabacum cv. Samsun, serological characteristic and
transmissibility by Myzus persicae, indicates that the
current isolate of PVY represent the ordinary strain
(PVYO). This isolate was found to be more prevalent
in all the fields submitted to the study, and causing
heavy losses in potato yields. The prevalence of
PVYO can be attributed to PVY asymptomatic hosts,
identified in previous studies in our department [20],
which may acts as virus reservoir to hasten virus
spread by the aphids in the same field and to those
in the vicinity.
Fig. 1: Symptoms of necrosis on blades (A), leaf dropping and death (B) on PVY-infected plants, compared
with healthy plant (C).
Fig. 2: Polyclonal antibodies to PVY on immunostrip, reacted with extracts from potato plant showing
virus-symptoms (arrow) indicates the presence of PVY in the extract (A), compared with a healthy
plant (control) (B).
Fig. 3: Symptoms of vein clearing and mild mottling on N. tabacum cv. Samsun (A), chlorotic local lesions
on Ch. amaranticolor, (B) and mild mottling on Capsicum annum (C) sap inoculated by PVY.
Adv. Environ. Biol., 5(1): 209-215, 2011
213
Table 1: Elisa readings of samples from virus-infected plants generated from infected tubers and treated with extracts with two
application methods
Date
Lippia nodiflora
Datura metel
Thuja oreintalis
--------------------------------------------------- ------------------------------------------------ -----------------------------------------------Tuber dipped Foliage spray Control Tuber dipped Foliage spray Control Tuber dipped Foliage spray Control
26 March
0.9
0.11
2.56
0.12
0.13
2.53
0.11
0.11
2.48
29 March
0.11
0.12
2.57
0.13
0.12
2.55
0.13
0.11
2.51
1 April
0.12
0.13
2.53
0.13
0.13
2.54
0.14
0.11
2.53
3 April
0.12
0.13
2.56
0.13
0.12
2.56
0.13
0.12
2.55
6 April
0.10
0.13
2.55
0.12
0.12
2.54
0.14
0.12
2.53
9 April
0.10
0.12
2.55
0.13
0.14
2.53
0.13
0.12
2.55
12 April
0.11
0.12
2.56
0.12
0.12
2.53
0.13
0.12
2.52
15 April
0.11
0.13
2.57
0.13
0.14
2.51
0.12
0.12
2.51
18 April
0.12
0.13
2.55
0.13
0.13
2.53
0.12
0.12
2.53
21 April
0.11
0.13
2.56
0.14
0.13
2.55
0.14
0.12
2.55
24 April
0.11
0.13
2.55
0.13
0.11
2.53
0.15
0.12
2.51
26 April
0.12
0.12
2.56
0.13
0.13
2.53
0.12
0.13
2.51
Mean
0.11
0.125
2.55
0.13
0.126
2.53
0.13
0.12
2.52
Each value in the table represents the mean of 3 replicates.
The values in the table represent the absorbance at 405 nm.
Several strategies have been adopted to manage
the virus diseases on potato through using
insecticides without success especially for viruses
transmitted by insects in non-persistant manner [30].
Recently the efforts were directed toward searching
for substances more effective including chemicals,
biological or natural plants products that can induce
resistance in the treated plants against viruses [21].
This idea came from fact that when a plant infected
by a pathogen, the reaction of the plant toward this
pathogen is developing systemic resistance to
subsequent infection [19,26].
These observation indicates that plants possess
inducible defense system against pathogen attack, and
this system can be triggered prior infection by certain
elements may be biotic or abiotic [28,35,37]. The
induce resistance render the plant resist to broad
spectrum of pathogens included virus, bacteria, and
fungi [26,39].
This study was focused on the use of extracts
from some medicinal plants (Lippia, Datura, Thuja)
in attempt to induce resistance against PVYO. The
idea came from indication that these plants contain
phenolic and alkaloids, substance had inhibition
effects towards different pathogens [1,28,7,39]. These
substances in addition to proteinacious compound
present in the extracts may acts directly on the virus
or indirectly through inducing host-resistance genes
leading to produce proteins have the capacity to
inhibit virus multiplication (antiviral proteins). The
results obtained reveal effectively the capacity of
these substances to induce systemic resistance in
potato plants against PVY as was shown by DASELISA using polyclonal antibodies to PVYO. This
resistance may enhance and activate by subsequent
application of the extracts.
Several previous studies reported that extracts
from, Phytolacca americana, Mirabilis jalaba,
Eucalyptus camalduliensis, Glycyrriza glabra,
Euphorbia tinctoria, Chenopodium spp., and Datura
spp. contains substance can induce systemic
resistance in treated plants [3,7,15,36].
Some other studies found that the onset of
induced resistance correlates with the accumulation of
Salisylic acid (SA) and pathogenesis related proteins
(PR-Proteins) [27]. Exogenous application of SA
found to induce SAR and activates the same set of
PR-Proteins genes in several plant species [24,17].
This mechanism is not excluded in this study and
suggests that these proteins may play an essential
role in the defense capacity of induced tissue. The
use of substances derived from plants as biological
elements to induce systemic resistance in the plants
against virus appear to be promising in virus
management strategy.
Conclusion:
The ordinary strain of Potato virus Y (PVYO)
was found to be the most prevailing in potato fields
in Iraq. The alcoholic extracts from Lppia nodiflora,
Datura metel, and Thuja orientalis exhibited
protection period to potato plants against PVYO
infection up to 30 days in the presence of virus
source with the vector Myzus persicae.
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