Antiphytoviral Activity of the Plectranthus tenuiflorus on Some Important Viruses

Abstract

Crude extract of Plectranthus tenuiflorus crude extract was tested as an antiphytoviral agent against different plant viruses like Tobacco Necrosis Virus (TNV), Tobacco Mosaic Virus (TMV), and Tomato Spotted Wilt Virus (TSWV). When it was applied onto Phaseolus vulgaris, Datura stramonium, and Chenopodium amaranticolor as pre-inoculation spray (in vivo), it reduced the infectivity of above viruses by 90.6, 85.8 and 77.7%, respectively. However, when the extract was mixed with the virus inoculum (in vitro), it inhibited the local lesion development by 100% after one hour of mixing for TNV, and three hours for both TMV and TSWV. Effect of P. tenuiflorus on the systemic TMV infection was also studied. It delayed the onset of the disease development from 4 to 5 days, although it had less apparent effect on the virus accumulation. Studying the effect of temperature and rate of dilution on the antiphytoviral activity revealed that the thermostable property of this extract as well as remaining of its antiphytoviral activity up to 10 -2 dilution rate. In order to investigate the bioactive constituent of the P. tenuiflorus. Crude extract, its proteins, carbohydrates, and storage oil were separated and tested as an antiphytoviral agent. The results indicated that the antiviral activity was not mainly attributed to these constituents.

Full text

INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY
1560–8530/2004/06–5–844–849
http://www.ijab.org
Antiphytoviral Activity of the Plectranthus tenuiflorus on Some
Important Viruses
B.A. OTHMAN AND S.A. SHOMAN1†
Department of Microbiology, Faculty of Agriculture, Ain Shams University, Cairo–Egypt
†Department of Microbiology, Faculty of Science, Ain Shams University, Cairo–Egypt
1Corresponding author’s e-mail: saharshoman@hotmail.com
ABSTRACT
Crude extract of Plectranthus tenuiflorus crude extract was tested as an antiphytoviral agent against different plant viruses like
Tobacco Necrosis Virus (TNV), Tobacco Mosaic Virus (TMV), and Tomato Spotted Wilt Virus (TSWV). When it was
applied onto Phaseolus vulgaris, Datura stramonium, and Chenopodium amaranticolor as pre-inoculation spray (in vivo), it
reduced the infectivity of above viruses by 90.6, 85.8 and 77.7%, respectively. However, when the extract was mixed with the
virus inoculum (in vitro), it inhibited the local lesion development by 100% after one hour of mixing for TNV, and three hours
for both TMV and TSWV. Effect of P. tenuiflorus on the systemic TMV infection was also studied. It delayed the onset of the
disease development from 4 to 5 days, although it had less apparent effect on the virus accumulation. Studying the effect of
temperature and rate of dilution on the antiphytoviral activity revealed that the thermostable property of this extract as well as
remaining of its antiphytoviral activity up to 10-2 dilution rate. In order to investigate the bioactive constituent of the P.
tenuiflorus. Crude extract, its proteins, carbohydrates, and storage oil were separated and tested as an antiphytoviral agent. The
results indicated that the antiviral activity was not mainly attributed to these constituents.
Key Words: Antiviral; P. sp; Plant virus
INTRODUCTION
The plant family Lamiaceae (mint family) represents a
valuable pool of plant species, which contain biologically
active molecules (Harley & Reynolds, 1992). The genus P.
consists of about 350 species distributed from Africa
through to Asia and Australia (Godd, 1985).
Many P. species are plants of economic and medicinal
interest: Plectranthus hereroensis (Batista et al., 1995), P.
elegans (Dellar et al., 1996), and P. grandidentatus
(Teixeira et al., 1997). It has been reported that the
medicinal value of these P. species attributed to the
antibacterial, antifungal, and antiviral activities. These
activities mainly depend on the phytochemical constituent
characteristic of this genus (Abdel-Mogib et al., 2002).
Concerning the phytopathogenic viruses, various
substances of natural and synthetic origins have been used
for control of virus infection. However none of them
possessed a satisfactory selective action, which could
enables them to be used as a therapy of plant viral diseases
(Allam et al., 1979; Verma & Baranwel, 1983; Barakat,
1988; Hansen, 1989; Takanami et al., 1990; Othman et al.,
1991; Meyer et al., 1995; Yordanova et al., 1996;
Eldougdoug, 1997; Shoman, 2002).
P. tenuiflorus is a small, downy, very leafy herb, with
stems of about 60 cm long, found in Abha, Saudia Arabia
(Collenette, 1985). Its medicinal value has been reported by
Abulfatih (1987). This work was undertaken to study the
effectiveness of P. tenuiflorus extract on different plant
virus infections as an antiphytoviral agent. To our
knowledge, this is the first study to address this topic in
Egypt.
MATERIALS AND METHODS
Source of viruses. The viruses used in this study were
Tobacco Necrosis Virus (TNV-D), Tobacco Mosaic Virus
(TMV-8) and Tomato Spotted Wilt Virus (TSWV) isolates.
The viruses were provided by Dept. of Microbiology,
Faculty of Science, Ain Shams Univ. and Dept. of Agric.
Microbiology, Faculty of Agriculture, Ain Shams Univ.,
Cairo, Egypt.
The viral inocula were prepared from infected frozen
leaves of Phaseolus vulgaris, Nicotiana tabacum, var white
barley and Chenopodium amaranticolor previously infected
with TNV, TMV and TSWV, respectively.
Five grams of leaf material was ground with 50 mL of
distilled water in a mortar. The extract was strained and the
filtrate made up to 200 mL with distilled water. This
dilution of sap extract was found to give a suitable number
of discrete local lesions on test plants.
Source of P. tenuiflorus. Part of the stem of plant (about 15
cm) was obtained from Saudia Arabia and planted in 10 cm
pots, kept in the glass green house at 25±5oC. Parts of the
new branches were taken and subcultured to obtain a large
number of the plant material.
Preparation of the antiphytoviral. To obtain the crude
extract, 10 g of the small, downy, herbaceous leaves of P.

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tenuiflorus were ground completely in porcelain mortar with
20 mL of distilled water then clarified
Plant materials. Seeds of Datura stramonium,
Chenopodium amaranticolor and Nicotiana tabacum, var
white barley were sown in trays, maintained in a glass house
at 25±5oC and watered as required. When the seedlings
were large enough, they were planted individually into 10-
cm plastic pots. They were selected for testing when they
had 4-6 true leaves. Seeds of Phaseolus vulgaris were
potted directly in the plastic pots, plants were selected when
they had the full expanded primary (cotyledonary) leaves.
Care was taken to ensure that the experimental plants were
as uniform in size as possible.
Assay of the antiphytoviral activity
In vitro. To investigate the effect of the antiphytoviral agent
on the viruses, 1.0 mL of each virus suspension was mixed
with 1.0 mL of the P. tenuiflorus. crude extract. The mixture
was left for 0, 1, 2, and 3 h at room temperature. The
samples (mixtures of both viruses & antiphytoviral agent)
were inoculated mechanically by dipping the forefinger in
inoculum and rubbing once over the upper surface of the
local lesion host leaves (Phaseolus vulgaris for TNV,
Datura stramonium for TMV, & Chenopdium
amaranticolor for TSWV), using carborandum as abrasive
material. The number of local lesions on the inoculated
leaves was counted 5-6 days after inoculation. The
antiphytoviral inhibitory effect on the infectivity of viruses
was assayed using the following equation: % of
inhibition=number of local lesions on control-number of
local lesions on treated / number of local lesions on control
X 100. For control, distilled water was used instead of the
antiphytoviral agent. Four to six plants of each host were
used for each treatment.
In vivo. This experiment was carried out by applying
different dilutions (10-1, 10-2, & 10-3) of antiphytoviral
extract to the local lesion hosts (Phaseolus vulgaris, Datura
stramonium, and Chenopdium amaranticolor) for 2
successive days prior to TNV, TMV, and TSWV
inoculation respectively. Nicotiana tabacum, var white
barley was used also as a systemic host for assay of the
antiphytoviral activity of P. tenuiflorus. With systemic
infection.
ELISA. TMV was serologically detected in its systemic
hosts using ELISA. The tests were done as described by
Clark and Adams (1977). After two weeks of TMV
inoculation the sap of the upper leaves of both P. tenuiflorus
extract-treated and untreated tobacco plants were prepared
by homogenizing leaf tissues in 0.02 M phosphate buffer
saline containing 0.05% tween 20 and 2%
polyvinylpyrrolidine.
TMV antiserum was obtained by the repeated
injections of 50 μg of purified virus preparations (provided
from Dept. of Agric. Microbiology, Faculty of Agriculture,
Ain Shams University) into Newzland healthy rabbits. The
purified preparation of virus was measured with
spectrophotometer (230-320 nm) as a quick and convenient
method confirm on the purity of the virus (Donbrow, 1967).
The doses were applied at weakly intervals. The TMV
suspension was emulsified with incomplete then complete
adjuvants, finally antiserum was collected and partially
purified by using saturated ammonium sulphate solution.
In all tests microplate ELISA plates were used with
coating TMV-antiserum at 10 μg/mL and antibody-enzyme
conjugate at 1:1000. The enzyme substrate paranitrophenyl
phosphate at concentration 0.6 mg/mL was allowed to act
for 30 min at room temperature. The reaction was then
stopped by adding 50 μL of 3N NaOH and the absorbance
was measured at 405 nm with a spectrophotometer.
Effect of dilution and heating on the antiphytoviral
agent. The crude extracts of P. tenuiflorus. was serially
diluted with distilled water to 10-1, 10-2 and 10-3. For heat
treatment, test tubes each containing 2 mL of the crude
extract were heated in water bath for 10 min at 40, 50, 60,
70, 80, 90 and 100oC and cooled immediately with tap
water. Each treatment of dilutions and heating was assayed
for the antiphytoviral activity.
Extraction of P. tenuiflorus Proteins. The total soluble
proteins of P. tenuiflorus leaves were extracted as described
by Parent and Asselin (1984). Protein concentration was
estimated by using Sigma diagnostic kit and bovine serum
albumin (BSA) as standard. To investigate the possible role
of the P. tenuiflorus proteins as antiphytoviral, soluble
solutions of different protein concentrations were prepared
and applied to the host plant for two successive days before
TNV inoculation.
Extraction of P. tenuiflorus Carbohydrates. The total
water-soluble carbohydrates of P. tenuiflorus were extracted
according to the method of Cerning and Guilbot (1973). The
Quantitative determination of the total soluble
carbohydrates was carried out by using Anthron reagent.
The developing colour was measured in a colourimeter at
620 nm (Umbreit et al., 1969). Different concentrations of
these extractable carbohydrates were prepared and tested as
antiphytoviral agents as previously described.
Extraction of P. tenuiflorus Oil. Extraction of storage oil
and determination of its concentration in the dry leaf tissues
of P. tenuiflorus was carried out according to AOAC (1990)
using petrolium ether as a solvent (40-60 BP) for 16 h by
Soxhelt apparatus.
Soluble solution of oil in distilled water was prepared
for testing the antiphytoviral effect. To overcome the
insolubility of the oil in water, appropriate amount of oil
was mixed with 0.1 mL of tween 80 containing 0.05 mL of
ethanol and subsequently made up to 100 mL with distilled
water. Different concentrations of the oil were prepared.
The control one was water/ ethanol/tween 80.
Statistical analysis. The significance of difference between
mean value for treatment and control was estimated
statistically using one tailed student T-test.

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846
RESULTS
Effect of P. tenuiflorus crude extract on the TNV, TMV,
and TSWV infectivity
In vitro assay. Three RNA viruses (TNV, TMV, and
TSWV) were selected from different virus groups. In vitro
tests showed that the infectivity of each virus was affected
by treating with crude extract of P. tenuiflorus. When the
extract and the virus were mixed and inoculated directly (at
zero time) into their local lesion hosts, the percentage of
viral inhibition was 54.5% for TNV infection, while with
TSWV it was 2.5% (Table І). Treatment of TMV revealed
that there was no inhibition detected at this time (Table І).
Once the virus particles were incubated for longer time with
the P. tenuiflorus extract, the inhibitory effect was rapidly
increased. The reduction of local lesions reached to 100%
after one hour of mixing with TNV and three hours for both
TMV and TSWV (Table І).
In vivo assay. Results with TNV showed that the P.
tenuiflorus extract (antiphytoviral) induced resistance
against infection by this virus, where the mean number of
local lesions reduced by 90.6% (Table П). In both TMV and
TSWV infections, the resistance also developed in their
treated hosts. The degree of this induced resistance was
varied according to the plant-virus system used (85.8% for
TMV and 77.7% for TSWV infections). The positive
correlation was statistically significant between the P.
tenuiflorus extract and the induced resistance when the
different dilutions of the antiphytoviral were applied (10-1,
10-2 & 10-3) and the percentage of inhibition declined with
these serial dilutions (Table II).
Effect of P. tenuiflorus crude extract on the TMV
infectivity in its systemic host. Since the previous
observation indicated that P. crude extract had a major
inhibitory effect on the hypersensitive viral infection, one
might expect that it would have similar effect on the
systemic disease symptoms. Surprisingly, this proved not to
be the case. Tobacco plants sprayed with crude extract of P.
tenuiflorus for 2 days prior to TMV inoculation consistently
showed a delay in the onset of systemic symptoms
compared with untreated plants. Faint necrotic spots were
developed on TMV-inoculated leaves in 3-4 days for treated
or untreated plants. The typical mosaic symptoms were first
evident on the new foliage of untreated plants 8 to 10 days.
However, the average time of appearance systemic
symptoms was delayed by 4 to 5 days, especially for plants
treated with high concentration of P. tenuiflorus extract.
Detection of TMV in systemic hosts by ELISA. The
specificity of TMV-antiserum was preliminary confirmed
with two-fold serial dilutions of purified viral samples using
ELISA. The results revealed the fidelity of TMV-antisera
used when the average absorption at A405 nm decreased with
increase in viral dilutions (Fig. 1).
Detection of TMV particles in the treated tissues of
tobacco plants was examined using these specific-tested
antisera. It was showed that TMV particles accumulated in
the leaves of both P. tenuiflorus extract –treated and
untreated plants where the average of absorbance at 405 nm
was slightly changed from 0.285 to 0.308 OD, respectively.
However, there was a delay in the appearance of systemic
symptoms TMV accumulation weakly was affected as
compared with control even with using serial dilutions of P.
tenuiflorus extract (Table III).
Effect of dilutions and heat treatments on the inhibitory
activity of the P. tenuiflorus crude extract. The dilution
rate of the P. tenuiflorus crude extract (antiphytoviral)
exhibited important role in the viral inhibition. The
percentage of inhibition was 96.2, 82.4 and 24.1% when the
Table І. Effect of P. tenuiflorus crude extract on TNV, TMV, and TSWV infectivity (in vitro assay)
TNV TMV TSWV Hours post mixing with
Plectranthus extract Mean of L.
L±SEM.
% of inhibition Mean of L.
L±SEM
% of inhibition Mean of L.
L±SEM
% of inhibition
Control 33.0±6.8 40.2±4.0 11.8±2.4
Zero time 14.6±2.2** 54.5 45.5±4.9 -13.1 11.5±2.1 2.5
One hour _ 100 20.5±5.1** 49.0 10.3±1.7 12.7
Two hours _ 100 1.6±0.4 ** 96.0 2.4±0.9** 79.6
Three hours _ 100 _ 100 _ 100
Mean of L.L= the mean number of local lesions; SEM=Standard Error Mean; **=High Significance reduction in disease compared with control
Table II. Effect of P. tenuiflorus crude extract on the induced resistance of the host plant against TNV, TMV, and
TSWV (in vivo assay)
TNV TMV TSWV Dilution of applying
Plectranthus extract Mean of L.
L±SEM
% of inhibition Mean of L.
L±SEM
% of inhibition Mean of L.
L±SEM
% of inhibition
Control 21.5±5.0 40.9±6.6 12.6±3.0
10-1 1.6±0.4** 90.6 5.8±1.1 85.8 2.8±0.3** 77.7
10-2 5.6±0.9** 71.7 15.5±2.0** 62.1 7.6±1.6** 39.6
10-3 12.2±2.0** 42.8 26.8±4.9 * 34.4 9.2±1.9* 26.9
**, *=High Significance, Significance reduction in disease compared with control

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virus treated with the serial dilutions of the antiphytoviral
10-1, 10-2, and 10-3, respectively (Table IV).
The effect of heat on antiphytoviral activity of the
extract of P. tenuiflorus was examined at different degrees
of temperature. The results in table (V) show that the
inhibitory antiphytoviral activity of P. tenuiflorus was
slightly affected by heating. The extract boiled for 10
minutes at 100oC still reduced the number of local lesions
by 65.4% whereas the percentage of inhibition was 85.8,
83.9, 79.2, and 71.1 at room temperature, 40, 60, and 80oC,
respectively.
Effect of certain P. tenuiflorus phytochemical
constituents on viral infectivity
Effect of P. tenuiflorus proteins. It was found that each
100 g of P. tenuiflorus leaf tissues contained 0.93 mg of
soluble proteins. Various concentrations (0.1, 0.01 and
0.001 mg) of these proteins were prepared and for their
inhibitory effect against TNV. The results in Table (VI)
indicated that these proteins had no inhibitory effect on
TNV infectivity, rather it caused enhancement in its
infectivity that was independent on the protein
concentrations.
Effect of P. tenuiflorus carbohydrates. About 1.5 g of
carbohydrates was obtained form 100 g of P. tenuiflorus
leaf tissues. The infectivity of TNV for producing local
lesions on the host plant was slightly decreased by 14.7% at
5.0 g of carbohydrates. Surprisingly, this low inhibitory
effect was moderately increased to 50% with decrease in
carbohydrates concentration at 0.05 g (Table VI).
Effect of P. tenuiflorus oils. Presence of high oil content in
P. tenuiflorus. (9.08 g/100 g tissues) encouraged us for
testing its inhibitory effect as antiphytoviral agent. The
results in Table VI showed that there are significant
difference between the number of lesions on control and
oil–treated plants, 51.6% inhibition relative to control..
Moreover, there was a trend towards more lesions as the oil
concentration decreased (Table VI).
DISCUSSION
The use of natural resources from plant species in the
treatment of plant viral diseases is an extensively explored
area. P. tenuiflorus was proven to have a powerful
inhibitory microbial activity against several bacterial and
fungal pathogens (Dellar et al., 1996) and viruses affecting
vertebrates (Batista et al., 1995). P. tenuiflorus also showed
to possess inhibitory effects against phytopathogenic
viruses. The previous reports support our observation that P.
tenuiflorus effectively inhibited infection by a wide range of
viruses (TNV, TMV, and TSWV) from groups with
different genome components and replication strategies.
The mechanisms of P. tenuiflorus antiviral activity
could be attributed to either inactivation of viral particles or
inhibition of virus through inducing resistance response in
the host (Zinnen & Fulton, 1986; Yalpani et al., 1993). In
the present investigation, P. tenuiflorus as an antiphytoviral
showed both mechanisms through in vitro and in vivo assays
toward the three tested viruses. The possibility of other
mechanism for the antiviral action enable it to be more
potent inhibitory agent may be there.
Our results also indicated that the antiphytoviral
activity in case of the local infection was higher than in
systemic infection. This may be attributed to several
possibilities; the virus replication might faster than the
inducing resistance in treated plants (Fraser, 1979), and the
antiphytoviral might have an effect on the virus movement
Table III. Analysis of TMV using ELISA in both
treated tobacco with serial dilutions of P. ten. crude
extract and untreated (control) plants. The numbers are
the average A405nm of 4 replicates
Treatment Average absorbance at 405 nm±SEM
Control 0.308±3.3x10-2
Plectranthus extract at 10-1 0.285±2.2x10-2 NS
Plectranthus extract at 10-2 0.311±8.8x10-3 NS
Plectranthus extract at 10-3 0.297±2.4x10-2 NS
Table IV. Effect of dilutions on the viral inhibitory
activity of P. tenuiflorus crude extract
Dilutions Mean no. of L.L ±SEM % of inhibition
Control 41.6±4.8
10-1 1.6±0.2** 96.2
10-2 7.3±1.6** 82.4
10-3 31.6±6.7* 24.1
Table V. Effect of temperature on the viral inhibitory
activity of P. tenuiflorus crude extract
Temperature Mean no. of L.L. ±SEM % of inhibition
Control 48.6±7.4
Room temp. 6.9±0.9** 85.8
40Co 7.8±1.7** 83.9
60Co 10.1±3.4** 79.2
80Co 14.0±2.2** 71.1
100Co 16.8±4.1** 65.4
**=High Significance reduction in disease compared with control
Table VI. Effect of P. tenuiflorus phytochemical
constituents as antiphytoviral against TNV
Phytochemical Concentration /mg
Mean of L.L
±SEM
% of
inhibition
Proteins
0
0.1
0.01
0.001
51.6±7.1
69.6±9.9
56.6±6.3
79.3±5.0
-34.8
-9.6
-47.8
Carbohydrates 0
5.0
0.5
0.05
40.8±4.5
34.8±6.1
19.2±4.4**
20.4±4.9**
14.7
52.9
50.0
Oil 0
18.0
1.8
0.18
49.6±5.6
24.0±4.5**
32.0±6.6*
30.8±6.8*
51.6
35.4
37.9
**, *=High Significance, Significance reduction in disease compared with
control; NS= non significant

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848
more than virus multiplication thereby delaying the onset of
systemic symptoms without significant effect on the virus
accumulation. Similar assumption has been reached by
Naylor et al. (1998).
The antiphytoviral of P. tenuiflorus extract was found
to be thermostable and was active up to dilution of 1:100. A
possible explanation for these unique properties lies in its
active component which is a stable complex in the P.
tenuiflorus extract. It might also be the occurrence of this
substance in the crude extract which exhibits the antiviral
activity and their concentration decreased with increase of
dilution rate.
Several reports indicated that proteins may have
antiviral agents (Smookler, 1971; Irvin et al., 1980; Mousa,
1986; Schonfelder et al., 1992, Verma et al., 1996; Shoman
et al., 2002). The results of this study showed the weak
antiviral activity of P. tenuiflorus proteins and the lower
content of it in the P. tenuiflorus tissues. This suggests the
involvement of other phytochemical constituents of P.
tenuiflorus in this viral inhibitory activity. These might be
the diterpenoids, essential oils or phenolic compounds
(Abdel Mogib et al., 2002) which is consistent with the role
of diterpens of different Plectranthus sp. as antimicrobial
agents (Teixeira et al., 1997). This is supported by the fact
that different extracts of many plants were tested for
antiviral activity and it was explained due to their content of
diterpenoids and essential oils (Remero et al., 1989; Bishop,
1995).
In conclusion, the study provided preliminary
information that the application of the P. tenuiflorus extract
(antiphytoviral) was a potential candidate for the protection
of plants in the field or greenhouses. In particular, it is
characterized by the broad spectrum effectiveness, stability,
and comparative safety for the environment.
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Fig. 1 Histogram representing the serological relationship between the serial dilutions of purified TMV and OD read
by ELISA reader corresponding to the homologous TMV and TMV–antibody complex that result in a color change
0
0.1
0.2
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0.5
0.6
1 0.5 0.25 0.125 0.0625 0.0312
TMV- dilutions
OD at 405 nm

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(Received 20 May 2004; Accepted 16 July 2004)