Journal
Journal of Applied Horticulture, 19(3): 249-252, 2017
Appl
Inhibition of Indian citrus ringspot virus from kinnow by
phytoproteins treatment in budding
Vikas Bishnoi* Anshul Goyal1 and H.N. Verma1
Department of Agriculture, Faculty of Sciences, Jagannath University-303901, Jaipur, Rajasthan, India.
1
Jaipur National University, Jaipur-302025, Rajasthan, India. *E-mail: drvikasbishnoi@gmail.com
Abstract
This paper reports the elimination of ICRSV (Indian citrus ringspot virus) from Kinnow when aqueous extract of different antiviral
phytoproteins were used during budding. Present study also reports the effect of these phytoproteins on the bud survival rate, growth and
proliferation of newly budded Kinnow plants. The phytoproteins were obtained from air dried roots of Boerhaavia diffusa and leaves
of Clerodendrum aculeatum. Budsticks from ICRSV infected mother plants (Indexed with RT-PCR) were soaked with phytoproteins
for different time periods (1, 2 and 3 h) before taking buds and ‘T’ budded on one year healthy rough lemon (Citrus jambhiri) plants.
Both the phytoproteins were found effective in all the parameters as compared to control. Phytoproteins from C. aculeatum (43.33
%) were found more prominent in respect of elimination of virus than B. diffusa (28.57 %), however B. diffusa was more effective in
promotion of growth and proliferation of buds than the C. aculeatum. Plants were considered virus-free when they showed absence of
ICRSV in both DAC-ELISA and RT-PCR tests. The study promised towards production of ICRSV free Kinnow plant material through
regular cost effective budding method of Kinnow with little modification.
Key words: Kinnow, phytoprotein, budding, ICRSV.
Introduction
Citrus fruits appeared on globe 30 million years ago. It is a longlived perennial crop and is grown in more than 100 countries
across the world (Saunt, 1990). Kinnow mandarin is a hybrid
between the King Sweet Orange (C. nobilis Loureiro) and Willow
Leaf Mandarin (C. deliciosa Tenore) developed in 1915 by H.B.
Frost (Frost and Krug, 1942). Kinnow is propagated vegetatively
by budding. Kinnow bud is grafted on one year old rootstock.
In India, more than 80 percent citrus plants are being raised on
rough lemon (Citrus jambhiri). The attentiveness of farmers in
acceptance of Kinnow cultivation in India is increasing day by
day due to appropriate agro-climatic condition, higher crop yield
and demand in international market. The productivity of the citrus
fruits in India is comparatively low due to many biotic stresses
of which particularly some viruses, virus-like pathogens play a
very significant role.
A ringspot disease (caused by Indian Citrus Ringspot Virus) of
citrus was first described by Wallace and Drake (1968) and can
be easily recognized in affected trees by conspicuous rings on
mature leaves which may be several per leaf. The incidence of
ringspot disease was observed up to 100 percent in most of the
Kinnow mandarin orchards in North Indian subcontinent. The
yield loss (number of fruits) in 7 year old Kinnow trees was from
20.54-98.38 percent (Byadgi and Ahlawat, 1995). The health of
the affected trees deteriorated year after year and finally they
collapsed therefore the investigation was aimed to eliminate
virus through the main source of transmission because except
bud transmission, no other mode of natural spread of the disease
could be established so far (Awasthi et al., 1984).
Scientists have tried to explore the use of different antiviral
compounds to obtain virus-free plants from infected cultivars.
Several workers have suggested a number of plants having
antiviral properties (Singh and Verma, 1981; Khan et al., 1990;
Watanabe et al., 1997; Pun et al., 1999; An et al., 2001; Narwal
et al., 2001).
Antiviral properties of Boerhaavia diffusa and Clerodendrum
aculeatum were reported for controlling plant viral diseases in
vivo (Verma and Awasthi,1979; Awasthi et al., 1984; Verma et al.,
1984; Verma et al., 1985; Verma et al., 1996; Verma et al., 1999;
Srivastava et al., 2004; Singh et al., 2004; Awasthi and Verma,
2006). These studies were done under in vivo conditions where
the extract was applied to the leaves of infected plants and the
decrease in virus concentration/virus infection was observed, but
there seems to be no information about the use of plant extracts
in budding.
The present study was planned to eliminate ICRSV from Kinnow
by using aqueous extracts of B. diffusa roots and C. aculeatum
leaves for the treatment of scion (bud) material before budding.
In the present study antiviral phytoproteins were not only studied
for elimination of ICRSV but also on bud survival, growth and
proliferation of Kinnow when they coupled with budding.
Material and methods
The roots of B. diffusa were collected from completely developed
plants and the leaves of C. aculeatum were collected from the
plants growing as hedge. The extraction of phytoproteins from
these plant parts was carried out separately, essentially according
to Verma et al. (1980). In brief, the roots of B. diffusa were washed
with distilled water, cut into small pieces, air dried under room
temperature protected from the direct sunlight and ground to fine
powder in the grinder. The root powder was then filtered with
flour sieve and the root powder (200 g) was then mixed with one
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250
Inhibition of ICRSV from Kinnow by phytotherapy in budding
litre of distilled water and shaken overnight on a shaker at 4oC.
It was then filtered through two folds of muslin cloth and filtrate
so obtained was centrifuged at 5000 rpm for 15 min to obtain
clear supernatant. Ammonium sulfate [60% (w/v)] was added
to the supernatant with continuous stirring and left overnight
at 4oC. Thereafter the mixture was centrifuged at 5000 rpm for
15 min and the supernatant was discarded. The precipitate was
retained and suspended in small amount of distilled water and then
dialyzed to obtain total protein fraction. After that, this solution
was filtered with Vacuum filter of pore size 0.22 µM. It was used
as aqueous extract. Essentially a similar protocol was adopted for
obtaining phytoproteins from the leaves of C. aculeatum.
For treatment, budsticks were taken from 10 year old ICRSV
infected mother plants of Kinnow. The mother plants were indexed
with RT-PCR (Prabha and Baranwal, 2011) for the presence of
ICRSV. Round shaped branches with dormant buds were collected
from the canopy of the low seeded Kinnow plants. Leaves were
removed from these budsticks with the help of scissor. These
budsticks were cut into 8-9 cm pieces and washed with running
tap water for 30 min and then with distilled water 2-3 times.
Budsticks were then soaked in aqueous extracts of phytoproteins
for 1, 2 and 3 h time period, individually. After treatment,
buds were taken from these treated budstics with the help of
fine sterilized (by NaOCl) knife. After treatment, buds were
‘T’ budded on rootstock of one year old, healthy and standard
length plants of C. jambhiri (rough lemon). Spraying of same
phytoprotein was done after every 2 weeks interval. The budding
was done with each type of phytochemical in randomized block
design. Each treatment consists of 24 plants for three different time
periods (1, 2 and 3 h). Plants were also treated with distilled water
for each time period and served as control.
After 1 year of budding, the survived plants were tested through
DAC-ELISA for the presence of ICRSV. Further confirmation
was done by RT-PCR (Prabha and Baranwal, 2011). Virus-free
plants were multiplied and maintained in the polyhouse. Data
acquired from the experiments was statistically analyzed with
MATLAB 13 for Windows 7 Ultimate version for linear model/
general factorial. To evaluate homogenous subsets for various
treatments, post hoc test/Tukey’s honesty significant difference
(HSD) at a level of significance of α=0.05 was applied.
Fig. 1. Showing comparative effect of treatment of 3 h of phytoproteins
on budding growth after 4 months of budding.
Distilled water as control. Leaves of Clerodendrum aculeatum. Roots
of Boerhaavia diffusa
Results and discussion
The data regarding the effect of different treatments of
phytoproteins extracted from roots of B. diffusa and leaves of C.
aculeatum on growth and proliferation of survived buds are given
in Table 1. The data were recorded after three and six months of
the budding.
Both the phytoproteins were found to produce beneficial effect
on the growth of shoots as well as their proliferation. However,
the phytoprotein extracted from B. diffusa were more efficacious
in both of these aspects than those from C. aculeatum. The
growth and proliferation of shoots progressively improved with
the increase of time of treatment (1, 2 and 3 h). In all the cases
of phytoprotein treatment, the growth and proliferation of the
buds were remarkably better as compared to the control. 3 h
treatment of both the phytoproteins had significant effect on
growth as well as proliferation of Kinnow plants followed by 2
h treatment (Fig. 1).
The role of phytoproteins was neither purely nutritional nor
hormonal. The Kinnow plants nurtured in the presence of
phytoproteins did require compulsory nutritional supplements
Table 1. Effect of treatments of phytoproteins extracted from roots of B. diffusa and leaves from C. aculeatum on growth and proliferation on budding
of Kinnow. Distilled water was also used as control for the same periods of time.
Phytoproteins Duration of Length of main shoot after 3 months
After 6 months of budding
treatment
of budding (cm)
Length of main shoot (cm)
Number of proliferated shoots
1h
Control
4.89±0.0524a
19.31±0.4306a
4.92±0.1244a
B. diffusa
5.25±0.0265abc
21.72±0.1162ab
6.92±0.1132ab
C. aculeatum
5.58±0.0416
20.93±0.1396
5.25±0.0265abc
5.00±0.0521a
19.28±0.1475a
5.28±0.0902a
6.05±0.0404
ab
25.76±0.4419
8.34±0.0754abc
5.88±0.0361ac
24.08±0.5129c
7.21±0.0666abc
a
4.99±0.0467
a
19.17±0.4104
5.18±0.1065a
ab
6.88±0.0786
ab
30.72±0.6302
9.91±0.1271ab
Control
abc
2h
B. diffusa
ab
C. aculeatum
Control
B. diffusa
3h
c
6.70±0.1040ac
28.82±0.4449c
9.17±0.1212c
C. aculeatum
Data shown is mean±SEM of three treatments; each experiment consisted of 24 plants. Means followed by the same letter are significantly different
from each other (general factorial/Tukey’s HSD at α= 0.05).
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Inhibition of ICRSV from Kinnow by phytotherapy in budding
251
Table 2. Effect of treatments of phytoproteins extracted from roots of B. diffusa and leaves from C. aculeatum on percent survival of buds and
elimination of ICRSV in Kinnow. Distilled water was used as control for the same period of time to study the effect of soaking on buds.
Phytoproteins
Duration of
treatment
Percent of survival buds after
two months of budding
Percent virus elimination
DAC-ELISA
RT-PCR
Control
67.89±1.2200a
0.00
0.00
B. diffusa
1h
70.66±2.4105b
0.00
0.00
C. aculeatum
69.11±1.2200c
5.88
0.00
Control
54.17±2.4047a
0.00
0.00
B. diffusa
2h
56.94±1.3867b
17.94
7.69
C. aculeatum
55.56±1.3867ac
26.82
17.07
Control
38.89±1.3900a
0.00
0.00
B. diffusa
3h
44.44±1.3867ab
46.42
28.57
C. aculeatum
41.67±2.4047ac
63.33
43.33
Data shown is mean±SEM of three treatments; each treatment consisted of 24 plants. Means followed by the same letter are significantly different
from each other (general factorial/Tukey’s HSD at α= 0.05).
and also needed hormonal treatments. Thus, the phytoproteins
did not substitute for growth hormones. However, at the same
time the phytoproteins showed their carry-forward effect on
growth and proliferation which is closely similar to action of
growth hormones.
The carry-forward effects of phytoproteins observed in the present
investigation in respect of growth and proliferation of shoot is
equivalent to resistance to viral infection when buds were treated
with aqueous extracts of both the phytoproteins. The plants raised
from budding coupled with phytotherapy were free from ICRSV
(Table 2).
Considering the factors of percent surviving budding success
rate and percent virus elimination, the best antiviral effect
(43.33% virus elimination) was observed with C. aculeatum at 3
h treatment with 41.67% budding success followed by B. diffusa
(28.57% virus elimination) at 3 h treatment with 44.44% budding
success rate. When treatment time of phytoproteins was increased,
percentage of virus elimination was also increased but the percent
budding success rate was decreased. One of the main causes of the
spread of the disease is the use of infected budwood. When the
budsticks were treated with phytoproteins, the budsticks became
to a large extant free from virus.
The percent success rate of bud survival was different for each
treatment with different soaking durations. When duration of
soaking of buds prior to budding was increased, the percent
success rate of surviving buds was decreased, even if soaked
in distilled water. Soaking of budsticks for 3 h in different
phytoproteins (even in distilled water) had most adverse effect
on bud survival followed by 2 h and 1 h. The percent survival
rate was comparatively better in buds treated with phytoproteins
(B. diffusa) as compared to distilled water. The less survival
after prolonged soaking may be because the buds of Kinnow are
highly susceptible for moisture. Soaking in certain phytoproteins
however was beneficial for budding success.
The roots of Boerhaavia diffusa and leaves of Clerodendrum
aculeatum have been shown to contain potent endogenous virus
inhibitory proteins called BD-SRIP and CA-SRIP respectively
(Verma et al., 1996; Verma and Baranwal, 1999). These
phytoproteins confer strong systemic resistance in several plants
against a number of plant viruses (Verma et al., 1984; Verma et al.,
1996; Verma et al., 1999; Srivastava et al., 2004). Phytoproteins
isolated from B. diffusa and C. aculeatum are highly stable and
could be easily purified (Verma et al., 1996).
Effect of these phytoproteins on virus resistance capability and
vegetative growth of cultured tissue was also seen by Verma et
al. (1999), Gupta (1999) and Thakare et al. (2015). Phytoproteins
from C. aculeatum, B. diffusa and many other plant spp.
have useful role to play in enhancing growth as well as virus
elimination in certain plants.
In this way it may be possible to produce virus-free plants without
resorting to preying somatic hybridization or transgenosis. In any
case, such studies provide ample scope for further investigations
which can be utilized for improvement of plant species to increase
the useful products from them, be their fruit yield or active
principles.
Since, ICRSV has no vector for transmission, phytoproteins
coupled with budding can work for management of the disease
as a better option. During this investigation, therefore the
ICRSV-free plant material of the Kinnow has been developed.
The study promised towards production of ICRSV free Kinnow
plant material through regular cost effective production method
of Kinnow plant.
Fig. 2. Agarose gel electrophoresis of Kinnow plants raised from budding
employing treatments for various time periods to budsticks. Lane M
is 1kb DNA ladder, Lane 1 and 2 are positive and negative controls,
respectively; lane 3 to 10, correspond to the Kinnow plant raised at 3 h
treatment of both the phytoproteins, respectively. Lane 3 to 7, where no
amplification was observed, was showing the absence of ICRSV. Lane 8
to 10, where amplification found, was showing the presence of ICRSV.
In conclusion, phytoproteins extracted from B. diffusa roots and
C. aculeatum leaves were incorporated with budding of Kinnow.
Shoots from infected Kinnow plants were treated with aquous
extracts of both phytoproteins and “T” budded on one year old
C. jambhiri plants. Phytoproteins were not only found effective
to eliminate ICRSV in in vivo generated Kinnow plants but also
they enhanced the growth and proliferation of newly budded
Kinnow plants.
Journal of Applied Horticulture (www.horticultureresearch.net)
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Inhibition of ICRSV from Kinnow by phytotherapy in budding
Acnowledgement
The authors are thankful to Dr. V. K. Baranwal, Principal Scientist
and Incharge, The Virology unit, Division of Plant Pathology,
Indian Agriculture Research Institute, Pusa, New Delhi for
helping in indexing of virus through RT-PCR.
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Received: June, 2017; Revised: September, 2017; Accepted: September, 2017
Journal of Applied Horticulture (www.horticultureresearch.net)