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VIROLOGICAL STUDIES ON SOME POTYVIRUSES OF PEPPER
IN EGYPT
By
MOHAMMAD AL-SAYED HAFEZ B.Sc. Agric. (Plant Pathology), 1985
Fac. Agric., Zagazig Univ. M.Sc. (Plant Pathology), 1993
Fac. Agric., Moshtohor Zagazig University
THESIS Submitted in Partial Fulfillment of the
Requirements for the Degree of
DOCTOR OF PHILOSOPHY
IN
PLANT PATHOLOGY
Fungus and Plant Pathology Branch
Agricultural Botany Department
Faculty of Agriculture
Zagazig University
Benha Branch
1999
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دراسات فريولوجية على بعض فريوسات واي البطاطس اخلاصة بالفلفل يف مصر
ـافــظـد السيد حـحممـ 5891( أمراض نبات)بكالوريوس العلوم السراعية
كلية السراعة بالسقازيق
5881( أمراض نبات)ماجستري العلوم السراعية
كلية السراعة مبشتهر
سقازيقجامعة ال
أمراض نبات
قسم الهبات السراعي
كلية السراعة مبشتهر
فرع بهها/ جامعة السقازيق
9111
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ACKNOWLEDGMENT
Firstly, Ultimate Thanks to “Allah”
The author wishes to express his deepest thanks and in recognition
to Prof. Dr. Nawal A. Eisa, Professor of Plant Pathology, Faculty of
Agriculture, Moshtohor, Zagazig University for suggesting the research
problem, planning of this work, valuable discussion during the tenure of
investigation and for her kind help in the preparation of this manuscript,
valuable advises and always encouragement.
This work is present to Soul of late Prof. Dr. Abo El-Yazid Emam
Badr, for effective scientific supervision, guidance, generous personal
and help during first stage of this work.
Greatest thanks are extended to Dr. Mohammad A. Abo El-Nasr,
Professor of Virology, Faculty of Agriculture, Ain-Shams University, for
enthusiastic encouragement and continuous support during this study,
reviewed the final work, and adding his beautiful magic touch on this
study.
It is pleasure to acknowledge Dr. Taymour M. Nasr El-Deen,
Professor of Virology, Agriculture Genetic Engineering Research
Institute (AGERI), Giza for providing the facilities of ELISA for check
the existence and severity of the isolated virus in this work, and
constructive criticism and guidance during this study.
Sincere thanks are extended to all the staff members of Agric.
Botany Dept., Fac. Agric. Moshtohor, Zagazig University for being
ready to help me when needed.
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CONTENTS
Page
INTRODUCTION 1
REVIEW OF LITERATURE 5
MATERIALS AND METHODS 38
EXPERIMENTAL RESULTS 54
1- Isolation and Symptomatology 54
2- Host range of the tested virus isolate 57
3- Physical Properties 62
A- Thermal inactivation point (TIP) 62
B- Dilution end point (DEP) 62
C- Longevity in vitro (LIV) 62
4- Mode of Transmission 64
A- Aphid transmission 64
B- Seed transmission 64
5- Virus purification 64
6- Electron Microscopy 64
7- Serological Studies 69
8-Antiviral activity of some selected healthy medicinal plant extracts on
the incidence of virus infection under the greenhouse conditions 73
9-Response of some pepper cultivars to infection with the isolated virus
under greenhouse conditions 79
10-Determination of distribution and severity of natural infection with
the seemed like tested virus in some Governorates 82
11- Determination of Capsiacin and Vitamin C in the natural infected
pepper plants 85
DISCUSSION and CONCLUSION 90
SUMMARY 111
REFERENCES 117
ARABIC SUMMARY
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INTRODUCTION
Capsicum terminology is confusing. Pepper, chili, chile, chilli,
aji, paprika, and Capsicum are used interchangeably for plants in
the genus Capsicum. Capsicum investigators use chile, pepper, or
aji as vernacular terms. Chile pepper has come to mean pungent
chile cultivars. However, chile means pepper (Capsicum) whether
or not the fruits are pungent. Bell pepper, generally, refers to non-
pungent blocky chile types.
The five domesticated Capsicum spp. are C. annuum, C.
frutescens, C. chinense, C. baccatum, and C. pubescens have close
wild relatives with which they cross readily, producing viable and
fertile hybrids. The wild relatives have not been evaluated
extensively but may contain useful sources of resistance to viruses,
bacteria, and fungi.
Capsicum is a small shrub of the Solanaceae, or nightshade
family. It is perennial in suitable climatic conditions, living for a
decade or more in tropical Central America, but is more often
cultivated as an annual. Pepper has been cultivated for thousands of
years. A handful of seeds found in a South America caves were
estimated to be 7000 years old. This makes chile one of the oldest
domesticated crops. (Bosland, 1992).
There are many different cultivars, forms, and uses of
Capsicum.
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Another group of popular chiles is the ornamental ones.
Ornamental chiles have all the colors of the rainbow, often
displaying pods in four or five colors on the plant at the same time.
Pepper are very important vegetables worldwide. They
provide spice and color to foods, while providing essential vitamins
and minerals. In Many poor households, peppers provide the variety
needed to enhance intake of otherwise bland diets. Peppers likewise
are good sources of income to small producers in many developing
countries. (Green & Kim, 1991).
A green, New Mexican (such as Anaheium, Chimayo, Dixon
and Velarde) chile pod contains three times the vitamin C of a
Valencia orange and provides the minimum daily requirement. As
green pod turn red, vitamin A content increases until they contain
twice the vitamin A of carrot. (Bosland, 1992).
Chile heat (pungency) is a complex of seven closely related
alkaloids or capsaicinoids. They often are called capsaicin, for the
most prevalent one. The capsaicinoids are produced in glands on the
placenta. While, seeds are not the source of pungency or “heat”,
they occasionally absorb capsaicin because of their proximity to the
placenta. Capsaicin is a powerful and stable alkaloid that can be
detected by human taste buds in solution of one part per million. A
more objective measure of the amount of capsaicinoids present in
chiles is measured by high-performance liquid chromatography
(HPLC).
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From an economic point of view, pepper yield is often low and
variable. Virus diseases are an important factor contributing to low
yields and reduced fruit quality.
Symptoms of virus infection vary greatly in expression and
severity, and include mild mottle, mosaic, veinbanding, ringspots,
various types of necrosis, leaf discoloration, deformation and
blistering and severe stunting of the whole plant. Leaves, stems and
flowers, as well as fruits, may be affected (Green & Kim, 1991).
Some 35 viruses have been reported to infect peppers
(Capsicum spp.). Of these, more than half are transmitted by aphids.
The other viruses are transmitted by nematodes, thrips, leafhoppers,
whiteflies, beetles and fungi. Several are transmitted by contact
and/or through the soil by mechanisms not yet understood. Most
pepper viruses are distributed worldwide with the exception of chili
veinal mottle virus, pepper severe mosaic virus, pepper veinal
mottle virus, pepper mild mosaic virus and pepper mottle virus.
These have been reported only in certain geographic areas. Virus-
infected peppers generally exhibit a variety of symptoms, the most
common of which are mosaic, mottle, necrosis and leaf distortion.
Many of these viruses, including ten potyviruses, cause
considerable yield losses (Green & Kim, 1991).
The present study aims to isolate and identify some
potyviruses, which seriously infected pepper plants. Regarding, the
massive application of pesticides has created serious problems such
as the build-up of pest resistance, the upsetting of natural balance,
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and acute and chronic hazards to man and animals. It is therefore
necessary to complement out reliance on synthetic pesticides with
less hazardous, safe, and biodegradable substances. Recently, the
search for naturally occurring antiviral, antifeedants and insecticides
against plant viruses and their vector pests of field crops has been
intensified. So, the present work, also, aims to established an
effective means to produce virus-free peppers by using natural
antiviral, antifeedant and insecticide substances extracted from
medicinal higher plants. Symptomatology and host range, physical
properties, mode of transmission, serological studies and electron
microscope were used for identification the isolated virus.
Determination of the distribution and incidence of some
pepper-associated potyviruses in Northern Egyptian governorates
was carried out and results were recorded.
Determination of both Capsaicinoids and Vitamin C in viral-
infected and healthy pepper fruits and recorded the relationship
between the differences in their amount and virus infection.
Response of some hot and sweet pepper cultivars under
greenhouse conditions to virus infection by the isolated virus was
studied.
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REVIEW OF LITERATURE
The present work concerning virological studies on some
potyviruses infecting pepper plants in Egypt. So, these reviews
interesting with the some previously virological studies of
potyviruses group may be infect pepper plants.
Peppers (Capsicum spp.) originated in Mexico, Southern Peru
and Bolivia (Greenleaf, 1986). They are now grown worldwide
under various environmental and climatic conditions, covering an
area of nearly one million hectares (Martelli & Quacquarelli,
1983).
A general overview of some 45 pepper viruses - their
morphological characteristics, vector transmission, geographic
distribution, the disease syndrome, diagnosis and control - is
reported by Green & Kim (1991).
From an economic point of view, pepper yield is often low
and variable. Virus diseases are an important factor contributing to
low yields and reduced fruit quality (Alonso et al., 1989). One
hundred percent losses of marketable fruit have been reported
(Marte & Wetter, 1986), and in some areas infection with viruses
has rendered the growing of peppers uneconomical, causing whole
fields to be abandoned prior to harvest (Greenleaf, 1986).
The Potyviridae now contains 198 viruses. The Potyvirus
genus is by far the largest of the five genera with a current
membership of 180 viruses (82 definite and 98 possible members),
although there is considerable uncertainly about its precise size.
Potyviruses have flexuous filamentous particles measured between
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650–900 nm long, including single-stranded RNA, particles contain
c. 90% protein and 5% nucleic acid, and mostly monopartite genera.
(Shukla et al., 1994).
Richter et al. (1995) reported that, the family Potyviridae
comprises the largest and economically most important group of
plant viruses which has recently been subdivided into the three
ICTV-approved genera Potyvirus, Rymovirus and Bymovirus as
well as the tentative genus Ipomovirus. This subdivision into genera
is largely based upon molecular characteristics but also on
properties such as taxonomic position of the vector and host range.
The genus Potyvirus comprising the aphid-transmitted potyviruses
is very large, consisting of more than 150 definite and possible
members.
Potyviruses were found to cause severe infections wherever
Capsicum was grown. Disease incidence and yield loss in most
areas surveyed were estimated at 40 – 100% (Agranovsky, 1993).
Isolation and identification of some pepper associated
potyviruses could be performed using several techniques, including
host range, distinguish symptoms, mode of transmission,
cytological diagnostic inclusions, light and electron microscopy
examinations, purification, and serological diagnostic protocols as
follows:
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1- Host Range and Symptomatology:
Matthews (1993) recorded that symptoms on plants in the
field may be unreliable because: (1) several viruses may cause
similar symptoms in the same crop; (2) a single virus may cause
highly variable symptoms, depending on virus strain; (3) a mixture
of viruses, or virus strains, or the presence of a satellite RNA may
greatly affect disease expression; (4) different crop cultivars may be
affected differently by the same virus; and (5) different soil and
weather conditions may alter disease expression.
For these reasons many searches have been made for
experimental host plants, which under reasonably standardized
conditions, usually in the glasshouse, will give consistent and
distinguishing disease symptoms with particular viruses. Such
plants are known as indicator species. However, even in a
glasshouse, disease symptoms can be greatly influenced by various
factors, (e.g., temperature, day length, light intensity, age of plants,
virus strains, plant cultivars, and accessions). Furthermore, it is
known that the accessions or varieties of the same species may give
different responses to the same virus. Thus, symptomatology in
itself is usually not sufficient for the exact identification of viruses.
(Matthews, 1993).
Virus identification should never be based on symptoms alone
because symptoms vary with the strain of the virus, the host
cultivar, the age of the host, environmental conditions and possible
coinfection with other viruses. Furthermore, different viruses may
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cause similar symptoms, and insect damage, particularly by thrips
and mites, may mimic virus symptoms. Certain herbicides, such as
2,4-D, and growth hormones may also cause reactions in the plant,
which resemble virus symptoms. Exact identification of pepper
viruses should be based on differential host plant tests, confirmed
by serological tests or vice versa, and if possible supplemented by
electron microscopic characterization of the virus particle and virus-
induced inclusions and by vector transmission tests. (Green &
Kim, 1991).
Shukla et al., (1994) recorded that, symptomatology has
played a significant role in early attempts to identify potyviruses.
Potyviruses induce an array of symptoms in their natural and
experimental hosts. These symptoms may include mosaic, strip,
mottling, vein-clearing, vein-banding, ringspots, necrotic or
chlorotic lesions, flower breaking, necrosis, stunting, wilting, etc.,
and a combination of these. Some potyviruses induce characteristic
symptoms in some of their hosts and can be identified on the basis
of these symptoms.
Many potyviruses have very restricted natural and
experimental host ranges which are often confined to a few species
within one genus or a few closely related genera. Some potyviruses
have moderately wide host ranges but comparatively few infect an
extensive range of plant species. Although some viruses of the
Potyviridae usually infect their hosts symptomlessly, most induce
conspicuous symptoms either permanently or sporadically in their
hosts; many cause stunting and yield losses.
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Potyviruses infecting dicotyledonous species often induce
chlorotic vein-banding, mosaic mottling, necrosis and/or distortion
of leaves; some also induce color “breaking” of flowers, a few
cause the production of discolored and smaller seeds, and others
severe distortion and discoloration of fruits.
Pepper associated potyviruses have narrow or intermediate
host range including few members belonging families:
Amaranthaceae, Chenopodiaceae, Leguminosae, and Solanaceae;
and species susceptible to many viruses are Tetragonia expansa,
Gomphrena globosa, Chenopodium amaranticolor, C. murale, Ch.
quinoa, Phaseolus vulgaris, Nicotiana clevelandii, N. benthamiana,
N. tabacum and Petunia hybrida.
2- Mode of Transmission:
Mechanical transmission:
Most pepper viruses are difficult to transmit from pepper to
other hosts because of inhibitors in the pepper plant thought to be
phenolic substances. This problem can be overcome by using
additives such as sodium bisulfite or sodium diethyldithiocarbamate in
the inoculum. The following buffer has been recommended for routine
inoculations of pepper viruses: 0.03 M disodium phosphate, pH 7.0,
amended with 0.5% sodium bisulfite, 0.5% sodium diethyldithio-
carbamate, 0.5% caffeine and 20 mg/ml activated charcoal. Passing
the plant extract through a column of Sephadex G or of Controlled
Pore Glass (CPG10) has also been found useful in reducing the
inhibitory effect of pepper plant extracts. (Green & Kim, 1991).
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Insect Transmission:
Most of the pepper-infecting potyviruses also infect other
solanaceous crops such as tomato and potato. They are transmitted
in a nonpersistent (stylet-borne) manner by aphids. The green peach
aphid (Myzus persicae) is considered to be the single most
important vector, although several other aphid species such as Aphis
gossypii, Macrosiphum solanifolli, M. pisi and A. spiraecola are
also known to transmit these viruses (Raccah et al. 1985).
Seed Transmission:
A number of potyviruses are seed-borne and infected seed-
lots constitute a serious hazard to the health of the crop. This danger
is particularly acute because young plants are usually much more
susceptible to infection than are older plants, and early infections
result in much more severe disease and loss of crop. Seed-borne
infections are especially important as the primary introduction focus
with viruses such as bean common mosaic, pea seed-borne mosaic,
soybean mosaic and lettuce mosaic. There were no evident that
pepper associated potyviruses can be transmitted through pepper
seeds. (Hollings & Brunt, 1981a).
3- Purification of Potyviruses:
Polson, (1993) recorded that, the leaves of tobacco plants
infected with PVYo (300 g) were ground in the presence of the
following stabilizing and reducing agents: citric acid – 200 nM
Na2HPO4 buffer pH 7.4, 10 mM EDTA, 20 nM 2-mercaptoethanol
and 1% Na-diethyl-dithio-carbamate. The volume of the mixture
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was 100 ml. After mincing the leaves, the pulp was ultracentrifuged
in the virus extraction rotor. The rotor was designed for extracting
and clarifying the plant extract in a single operation. Four hundred-
ml pulps were spun at 15000 g for 30 min yielding a clarified
extract of 360 ml. The virus was centrifuged in the thin layer rotor
at 15000 g for 60 min. Because the volume of infected extract was
too large for a single centrifugation it was necessary to limit the
batch volume treated in one operation to 120 ml. After the first
batch was freed of virus, the supernatant fluid was collected. A
second batch of 120 ml was next introduced into the rotor bowl
through the valve in the lid. After centrifugation the supernatant
was removed gently and the virus in the remaining extract was
concentrated.
4- Serological diagnostic protocols:
Most of the serological techniques which have been used in
the diagnosis of other plant and animal viruses have also been
applied to potyviruses (Koenig, 1988). The following techniques
appear to be most useful for the detection and identification of
potyviruses.
Agglutination tests:
In these tests either antibodies or antigens are attached to
larger particles such as bentonite, barium sulphate, latex, bacterial
cells or red blood cells. A positive serological reaction is indicated
by the formation of precipitates or clumps. The procedure increases
the sensitivity of detection by many fold over that of unbound
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reactant since the clumps formed by an attached reactant are much
more readily detected than the small precipitates formed when
nonattached reactant is used. When antibodies are attached to larger
carrier particles the tests permit the detection of viruses at much
lower concentration. The detection limit with these tests ranges
from 5 to 20 ng virus ml-1
(Shukla et al., 1994).
Precipitin test:
This technique, performed in liquid medium, has been one of
the earliest serological techniques used to study antigenic
relationships of plant viruses including potyviruses. It involves
mixing equal volumes of diluted antisera and solutions containing
homologous or heterologous virus isolates on a slide, a Petri dish or
in a tube. Positive serological reaction is indicated by the
appearance of floccular precipitates with filamentous plant viruses.
The precipitin test can be used to establish close and distant
serological relationships between strains and viruses on the basis of
average serological differentiation indices (SDI) of reciprocal tests.
SDI is the serological cross-reactivity between two viruses
expressed as the number of two-fold dilution steps separating
homologous and heterologous titres. (Shukla et al., 1994).
Precipitin tests are straightforward, quick, require simple
equipment and can be used to screen large number of samples. They
were routinely used to screen millions of samples of seed potatoes
each year for filamentous plant viruses, including potyviruses, in
many countries before the development of more sensitive
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serological techniques. However, the technique suffers from its low
sensitivity and the fact that an unbalanced concentration ratio of
reactants may lead to inhibition of precipitate formation. This
necessitates the testing of a number of dilutions of antiserum and
virus solutions (Koenig, 1988).
Sodium dodecyl sulfate (SDS)-double diffusion test:
The double diffusion test in agar or agarose without SDS is
one of the most widely used techniques for identification of
isometric viruses because of its simplicity, economical use of
reagents, and the fact that it provides a visible demonstration of
relationship between antigens (Van Regenmortel, 1982). However,
filamentous viruses with lengths above 500 nm diffuse poorly in
agar gel. Therefore, for successful immunodiffusion particles of
such viruses must be fragmented or disrupted by sonication or the
addition of disrupting chemicals into the gel. When SDS was shown
to be very effective for the immunodiffusion studies of PVY and
TEV potyviruses, the SDS-immunodiffusion test became widely
used to study the antigenic relationships of many potyviruses
(Koenig, 1988). However, it is the most insensitive serological
technique known and gives the lowest antiserum titre. For example,
an antiserum to PVY had a homologous titre of 1/2056 when tested
by the tube precipitin test but only 1/128 in the immunodiffusion
test using sonicated viral preparations (Moghal & Francki, 1976).
The SDS-immunodiffusion test has also been used to
investigate antigenic relationships of nonstructural proteins
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pinwheel, nuclear and amorphous inclusions of potyviruses.
(Mowat et al., 1989).
Enzyme-linked immunosorbent assay (ELISA):
The sensitivity of antigen-antibody reactions can be increased
many fold by attaching a label to the antibody that can be detected
in minute quantities. This has led to the development of different
ELISA procedures in which enzyme-linked antibodies are used for
the detection of antigen-antibody complexes on solid supports such
as polystyrene or polyvinylchloride microtitre plates (Van
Regenmortel, 1982). Presently ELISA procedures are among the
most sensitive serological techniques available for the diagnosis of
viruses. ELISA techniques are also the most widely used since they
are particularly suited to large-scale screening of samples. Among
the different ELISA procedures described, indirect-ELISA and
direct double antibody sandwich (DAS)-ELISA are perhaps the
most useful for the diagnosis of plant viruses including potyviruses.
The indirect-ELISA procedure commonly used involves
coating microtitre plate wells with virus suspension, additioning
virus-specific antibody, binding enzyme-labeled globulin-specific
antibodies from a different animal species and additioning enzyme
substrate for colorimetric visualization of the bound enzyme
conjugate (Van Regenmortel, 1982). This procedure is known to
detect a much broader range of relationships than DAS-ELISA.
Thus, it is well suited to the detection of distantly related strains of a
virus and to study serological relationships among distinct viruses.
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(Koenig, 1988). The broad-specificity of the indirect-ELISA
procedure is due to the fact that virus particles directly adsorbed to
microtitre plates are denatured to some extent, thus exposing the
internal coat protein epitopes which are generally conserved among
members of a plant virus group including potyviruses (Shukla et
al., 1989).
The standard DAS-ELISA procedure involves coating of
microtitre plate wells with specific antibody, the addition of virus
suspension, substrate for colorimetric revelation of bound enzyme
conjugate (Clark & Adams, 1977). DAS-ELISA is extremely
specific; enzyme conjugate prepared with antibodies against one
virus often does not react with closely related strains. This extreme
specificity of the DAS-ELISA is probably due to a reduction in
antibody avidity following conjugation with enzyme.
Variations in solid phase material, extraction medium,
conjugate buffer detection system, incubation time and temperature
and blocking agent have been used to improve the level of detection
of different potyviruses by ELISA procedures. (Shukla et al.,
1994).
Chemical analysis of pepper plants:
Chemical composition of sweet and hot pepper have been
conducted during different several studies especially when facilities
and techniques were more developed. High performance liquid
chromatography (HPLC) is one of very effective method, which
used widely for detection, determination isolation and identification
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several components as analysis tool.
Capsicum (sweet or hot) fruits was analyzed for their
constituents and analysis resulted that fresh mature (ripening stage,
about 50 days from transplanting) fruits consists of carbohydrates
(50.5%), protein (12%), ascorbic acid (1550 mg/100g), capsaicin
(200 mg/100g), carotenoids, (65 mg/100g), free amino acids (0.9g/
100g), in additional to minerals (N, P, K, Ca, Fe, Mn, Zn, Cu, Pb,
Cd, Ni, Cr) with different concentrations. These contents were
altered with different pepper cultivars (El-Saeid, 1995 and
Soohyun et al., 1997).
Vitamin C (Ascorbic acid) content:
In the extremely studies including 21 plant viruses (3 of them
were infect Capsicum annuum, i.e. CMV, PVY and TEV),
Pennazio et al., (1996) reported that, the relationships between L-
ascorbic acid (vitamin C) and localized or systemic virus infections
in higher plants have so far been little investigated and poorly
known. Experimental evidence indicates that the vitamin plays a
protective (antinecrotic) role during the hypersensitive reaction, but
its mechanism is quite unknown.
The results concerning the vitamin content in systemically
infected plants are not univocal because it has been described to
increase, decrease or remain unaltered. It must be pointed out that
papers devoted to this subject are really very few and often the
results they report concern only the plant leaves and not other
organs, which are consumed by man.
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Capsaicinoids content:
The pungent principle of chili pepper fruits is complex of
seven closely related alkaloids or capsaicinoids. They often are
called capsaicin, for the most prevalent one. The capsaicinoids are
produced in glands on the placenta. While seeds are not the source
of pungency or “heat”, they occasionally absorb capsaicin because
of their proximity to the placenta. Capsaicin is a powerful and stable
alkaloid that can be detected by human taste buds in solutions of
one part per million. A more objective measure of the amount of
capsaicinoids present in chiles is measured by high-performance
liquid chromatography (Bosland, 1992).
Some investigators observed that there is relationship between
high content of Capsaicinoids in the hot pepper fruits and their
tolerance or resistance to virus infection. For instance, Awasthi &
Singh (1975) reported that, both ascorbic acid and capsaicin content
were decrease in the of fresh Capsicum fruit samples infected with
CMV, and observed that this was more pronounced in the
susceptible varieties than in the tolerant one. They also, suggested
that pungency might be correlated with resistance.
Tewari (1990) attempts, through hybridization programs
depending on heritability, to developed high capsaicin content
chillies. Capsicum annuum (Pusa Jwala cv.) was produced with
high content of capsaicin and resistance to virus infection.
Hundal et al.,(1995) released a new Capsicum variety, its
fruits are suitable for salad, pickle and dry powder and are also a
good source of ascorbic acid, capsaicin and oleoresin. The variety is
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tolerant of mosaic virus and resistant to leafcurl virus.
Berke (1997) screened 20 pepper varieties for productivity
characteristics, including fruit capsaicinoid content, and for
resistance to some important fungal, bacterial and viral diseases. He
found that, some screening varieties, with high content of
capsaicinoid, were resistance for chilli veinal mottle and potato Y
potyviruses.
Recently, in Japan Kobata et al. (1998) isolated two novel
capsaicinoid-like compounds, named capsiate and dihydrocapsiate
from fruits of Capsicum cv. CH-19 Sweet (selected from a pungent
Thai cultivar, CH-19). Their structures were determined from
spectral data. Kobata et al. (1999), also in Japan, isolated
nordihydrocapsiate, (a new capsinoid) from the fruits of a
nonpungent pepper, Capsicum annuum.
Control of viral diseases:
Potyviruses are very largely a problem of outdoor cultivation
even an unscreened glasshouse offers a surprising degree or
protection against the introduction and subsequent epidemic spread.
As with many other viruses, control depends upon planting healthy
material and preventing the introduction of virus from outside
sources. The production and distribution of virus-free planting
material has formed the basis for effective control measures in a
wide variety of vegetatively propagated crops. Where existing
stocks still free from virus cannot be found, the techniques of
thermotherapy and meristem-tip culture have been successfully
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applied, and foundation clones established free from potyviruses
and other pathogens. (Hollings & Brunt, 1981a).
Virus inhibitory activity of extracts from different higher plants:
Cheesin et al. (1995) recorded that a number of healthy
plants, especially herbaceous species, have been reported to contain
virus inhibitory substances. Duggar and Armstrong reported for the
first time in 1925 that the crude extract of pokeweed (Phytolacca
decandra L.) markedly inhibited the activity of tobacco mosaic
virus. Several have appeared which have listed the different plants
showing virus inhibitory activities.
The realization that plants contain virus inhibitory substances
was first made when it was found that certain sap-transmissible
viruses could not be transmitted from a few host plants to other hosts.
Obviously, the presence of certain inhibitory substances in such
extracts interfered with virus transmission. Thus, the first evidence
that plants contain inhibitory substances came from virus-infected
plants and subsequently the occurrence of virus inhibitory substances
was noticed in a number of healthy plants, belonging to different
families of Angiosperms, such as Amaranthaceae, Caryophyllaceae,
Chenopodiaceae, Nyctaginaceae, Phytolaccaceae, Solanaceae, and
Verbenaceae.
Healthy extracts from plants, when incubated with the virus
inoculum and then inoculated on the leaves of susceptible plants,
either decreased the production of local lesions or, in systemically
reacting hosts, delayed symptom production, or the symptoms
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produced were mild or totally suppressed. Such inhibitor-containing
plants posed great difficulty in mechanical transmission of plant
viruses. Although the effect of these inhibitors may appear more or
less the same, there is no indication that all these plant extracts
contain the same type of inhibitor or than the mechanism is the
same in all cases.
A number of plants have been shown to prevent the
establishment of plant viruses in host plants. More than 180 plants
from different taxonomic families of angiosperm have been
reported to possess potent inhibitors of plant virus infection.
(Baranwal & Verma, 1993).
Some biological compounds may inactivate in vitro the
infectivity of plant viruses by interacting with their chemical
structure. The compounds, defined as exogenous inhibitors of virus
infection, do not induce resistance by affecting plant cell
metabolism. Three classes of compounds are considered, including
phenolic compounds, embracing a wide range of substances, such
as phenolic acid and derivatives, quinones, flavonoids and tannins;
lytic enzymes, such as ribonucleases and proteases; and virus-
specific antibodies. The mechanisms of inactivation differ for
different compounds and some of them are discussed. It is
concluded that investigations on the in vitro chemical inactivation
of plant viruses can be considered a useful preliminary step to
understand inactivation mechanisms occurring during the early
events of in vitro infection leading to plant immunity and possibly
to control viral diseases by biosynthesis of inactivating compounds
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21
in biomanipulated plants (Pennazio & Roggero, 1996).
Suriachandraselvan & Narayanasamy (1987) tested
extracts from disease-free leaves of 22 non-host species against
potato Y virus in inoculated chilli [Capsicum] plants. Inhibition of
infection ranged from 18.5 to 71.0%, with extracts from Basella
rubra [B. alba] being most effective. They are suggested that
resistant plants may contain protein antiviral compounds.
Nagaraju et al. (1997) investigated the effect of exogenously
applied plant products on pepper vein banding virus transmission,
multiplication and symptoms production in bell pepper (Capsicum
annuum L.). They tested two neem based products, neemark (5%)
and neem seed kernel extract (4%), and leaf extracts from 7 plant
species (Azadirachta indica, Pongamia glabra [P. pinnata],
Ocimum sanctum, Vinca rosea [Catharanthus roseus], Phyllanthus
niruri, Tagetes erecta and spinach) against the transmission of
pepper vein banding virus on bell pepper (Capsicum) under
glasshouse conditions. They found that, spinach was inhibitory,
resulting in only 25% transmission compared with 86.6% in the
inoculated control. Ocimum sanctum had no inhibitory effect on the
virus recording 100% transmission.
Singh et al. (1985) observed that when leaf extracts from 50
plant species (27 families), 42 showed inhibitory activity against
both the mild and severe strains of arhar (pigeonpea) mosaic virus
(AMV). Extracts from Capsicum annuum and Datura stramonium
prevented infection of the cv. Sharda when applied before
inoculation.
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22
Properties of some Pepper Associated Potyviruses:
Ten potyviruses have been reported to infect peppers: potato
virus Y (PVY) (Arteaga et al., 1997), tobacco etch virus (TEV)
(Ariyaratne et al., 1996), pepper mottle virus (PeMV) (Escudero,
1996), pepper mild mosaic virus (Ladera et al., 1982), pepper
veinal mottle virus (PVMV) (Atiri, 1992), chili veinal mottle virus
(CVMV) (Duriat, 1996), pepper severe mosaic virus (PeSMV)
(Feldman & Gracia, 1977), Peru tomato virus (PTV) (Fernandez-
Northcote & Fulton 1980), pepper vein banding virus (PVBV)
(Ravi et al., 1997) and chilli vien-banding mottle virus (CVbMV)
(Siriwong et al., 1995). Not all of these agents have been well
characterized and their status as separate viruses seems to need
further confirmation, e.g., pepper mild mosaic, pepper severe
mosaic, Peru tomato virus, pepper vein banding virus and chilli
vien-banding mottle virus.
Properties of most important potyviruses, which caused
economically serious viral diseases in all pepper fields allover the
world, could be summarized as follows:
1-Potato virus Y (PVY):
Potato virus Y (PVY) is the most common potyvirus infecting
pepper. It occurs worldwide although it appears to be more
important in warmer areas (Mills & Abdul-Magid 1987). Disease
incidence may be as high as 100% in some areas, resulting in
considerable crop loss (Sharma et al. 1989).
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23
Many investigators isolated PVY from Capsicum annuum
showing mosaic (Erkan, 1986; Thakur et al., 1988; Davino et al.,
1989; and El-Sanusi et al, 1991).
PVY induced mosaic with typical dark vein-banding on fully
expanded or mottling and crinkling on apical leaves, stunting, leaf
abscission, distortion and chlorotic spots, without symptoms on
fruits. Chlorotic local lesions induced on Chenopodium
amaranticolor, Ch. quinoa and Solanum demissum. (Erkan, 1986).
Thermal inactivation point of infectious sap with PVY was
65-70°C (Khatri & Sekhon, 1974). Dilution end point was 10
-3 –
10-4
(Erkan, 1986). Longevity in vitro at room temperature was 72
h. (Khatri & Sekhon, 1974); 7-8 days (Erkan, 1986) and 12 days
(El-Sanusi et al., 1991).
PVY is easily transmitted by sap inoculation (Thakur et al.,
1988). Myzus persicae and Aphis gossypii were found to be PVY-
vectors (Gowda & Reddy, 1989). PVY is not known to be seed-
transmitted in pepper (Raccah et al., 1985).
PVY has flexuous thread-like (filamentous) particles ranging
from 700-800 x 12 nm. (Thakur et al., 1988).
Erkan (1986) used agar-gel diffusion test for serological
reaction and found that antigen of PVY did not react with antisera
against Potato virus X (PVX), Cucumber mosaic virus (CMV) and
Tobacco mosaic virus (TMV), but gave a strong positive reaction
with PVY-antiserum with titre of 1/8.
Sherwood et al. (1988) used the protein-A Sandwich enzyme-
linked immunosorbent assay (PAS-ELISA) and agar-gel double-
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24
diffusion tests to study serological reaction of PVY isolate from
Bahamian hot chile, and found that there was no significant reaction
with antisera against tobacco etch virus (TEV) and pepper mottle
virus (PMV), whereas, reacted with PVY-anti serum raised against
the Wisconsin isolate of PVY.
El-Sanusi et al. (1991) used agar-gel double-diffusion test to
identifier a strain of PVY in Libya, and stated that, there were
serological relationship between antigen of this strain and antisera
of PVY and pepper mottle virus (PMV), but not to tobacco etch
virus (TEV).
2-Pepper Mottle Virus (PMV or PeMV):
Pepper mottle virus is one of the Potyviruses, which caused
mosaic disease of pepper cultivars as reported by Nelson &
Wheeler (1978) in USA and Chandrasrikul & Patrakosol (1986)
in Thailand.
PeMV induced systemic mottling on certain Solanaceous
species such as: Capsicum annuum L., Lycopersicon esculentum
MilL, Nicotiana hybrid, N. tabacum "Havana-425" and " Turkish-
NN", Physalis floridana and Solanum sp. (night shade) (Purcifull et
al., 1975).
Hill (1984) recorded that, thermal inactivation point was 50-
60°C, longevity in vitro was a few days at 20
°C, and concentration
in sap 5-35 mgl-1
.
PeMV was transmitted mechanically to test plants (Nelson &
Wheeler, 1978). PMV was transmitted with Myzus persicae
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25
(Zitter, 1975).
Flexuous, rod-shaped particles were found in crude extracts
from tobacco leaves infected with PeMV measured between 729-
745nm. long with the modal class at 737nm. The virus preparations
were immunogenic and the PeMV antisera gave strong reactions
with PeMV in gels containing SDS, but they did not react with sap
from healthy plants. PeMV antiserum may be specific for PeMV
and did not react with antigens of some viruses infect pepper, e.g.,
TEV, PVY and pepper veinal mottle viruses. PeMV antiserum also
did not react with lettuce mosaic, bidens mottle and turnip mosaic
viruses. PeMV antiserum could be reacted weakly with PVY and
pepper veinal mottle viruses. All antisera tested gave positive
reactions with homologous antigens. Purcifull et al. (1975)
PeMV is serologically distinct from PVY and TEV (Nelson &
Wheeler, 1978).
3- Pepper Severe Mosaic virus (PSMV):
Pepper severe mosaic potyvirus (PSMV), the suggested name
for our virus isolate in this study, was isolated for the first time from
pepper crops (C. annuum L. cv. Calahorra) in San Juan and
Mendoza provinces, Argentina, during 1970 caused severe mosaic
and necrotic streak and/or spots on stems and fruits, consequently,
its responsible for important crop losses (Feldman & Gracia,
1977).
In the subsequently study, during 1982, Feldman & Gracia
(1985), isolated pepper severe mosaic potyvirus (PSMV) from
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26
pepper crops (Capsicum annuum L. cvs. Hungarian Yellow Sweet),
caused serious pepper losses in Catamarca province, Argentina.
The findings of theses studies could be summarized as
follows:
PSMV causes a severe disease in peppers with strong mottle,
necrotic spots on the stems, necrotic streaks on the leaves and leaf
abscission. Serious yield losses occur as a result of infection with
this virus. Systemic infection (mosaic, vein clearing, leaf
deformation, sometimes necrosis and defoliation) induced on
Capsicum annuum L. cvs. Calahorra, California Wonder and Volo
V, and C. frutescens L. cvs. New Carolina (hot pepper) and
Tabasco. Local lesions developed on inoculated leaves of
Chenopodium amaranticolor and Ch. quinoa. Local lesions
followed by systemic infection induced on Nicandra physaloides,
Nicotiana rustica, and N tabacum cv."White Burley" and N.
debneyi. Systemic symptoms (mosaic, vein clearing, leaf
deformation, sometimes necrotic and defoliation) developed on
Capsicum annuum cv. "Calatauco" Datura metel, N. clevelandi4 N
glutinosa, N sylvestris, N. tabacum cvs. ' 'Samsun; Turkish &
Xanthi-nc", Petunia hybrida, Physalisfioridana, P. peruviana,
Solanum nigrum. Tomatoes, Lycopersicum esculentum cvs.
"Plantense & Roma" and L. pimpinelhfolium, showed no symptoms
but recovery tests were positive.
Stability of PSMV in sap of Nicotiana tabacum cv. "White
Burley" was tested and found that, thermal inactivation point was
85-90 °C; dilution end point was 10
-3 – 10
-4 and longevity in vitro
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27
was 32-64 days.
PSMV was easily transmitted by sap inoculation while was
not transmitted by Myzus persicae Sluzer in San Juan, Argentina.
Other strain of PSMV was transmitted by Myzus persicae in the
non-persistent manner, in Catamarca, Argentina, where 10 C.
annuum cv. Calatauco were infected from 20 plants.
In infected epidermal and hair cells, PSMV and its variants
induce numerous amorphous cytoplasmic inclusions visible by light
microscopy.
Flexuous-rod particles measured 761x13nm. in sap of PSMV-
infected plants examined by electron microscope.
The virus is related to, but serologically distinct from other
potyviruses such as PVY, TEV, PeMV and PVMV.
PSMV was serologically distinct, but distantly related to
potato virus Y (PVY) and tobacco etch virus (TEV). Moreover,
neither PSMV antigen nor its antiserum could react with either
antisera or antigens of pepper mottle virus (PMV) and turnip mosaic
virus (TurMV). Also, PSMV antiserum failed to react with pepper
veinal mottle virus (PVMV).
Capsicum annuum L. cv. "Calahorra" were more susceptible
to infection with PSMV also, California Wonder and Yolo Y, C.
frutescens L. cvs. "New Carolina Hot" pepper and Tabasco were
infected. Whereas, Yolo Wonder, Early Calwonder, Keystone,
Resistant Giant, Gigantic de Nag, Sweet Banana, Saltiness, Ambato
and Nora pepper cultivars were not naturally infected with PSMV.
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4- Pepper Veinal Mottle virus (PVMV):
Pepper veinal mottle virus (PVMV) has been reported in
several countries where disease incidences as high as 100% have
frequently been observed (Atiri, 1992 and Agranovsky, 1993).
Leaves of PVMV-infected plants commonly develop
chlorosis of the veins followed by systemic interveinal chlorosis.
Mottle, vein chlorosis and small-distorted leaves also occur. Leaf
abscission and fruit distortions have also been reported (Brunt &
Kenten, 1971).
PVMV had an unusually narrow experimental host range
(Agranovsky, 1993).
The host range of the PVMV was mainly restricted to the
Solanaceae, although, symptoms were also produced on three
species of both Amaranthaceae and Chenopodiaceae, in addition
one sp. of Cucurbitaceae (Atiri, 1986).
Allan et al. (1975) found that thermal inactivation point was
65°C, dilution end point and longevity in vitro was 3 days at 20-
25°C using sap extracted from infected pepper leaves.
PVMV was easily transmitted mechanically to several pepper
cultivars (Allan et al., 1975).
Alegbejo (1986) found that PVMV was transmitted by 7
species of Aphids (Myzus persicae; Hysteroneura setariae, Aphis
gossypii, A. fabae, A. cracivora, Rhopalosiphum sp. and R. maidis).
Of these, M. persicae was the most efficient.
Agranovsky (1993) found that pepper veinal mottle potyvirus
had filamentous particles c. 700 –750 nm in length.
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29
There were serological relationships between PVMV and both
Onion yellow dwarf virus and Colombian Datura virus. (De Wijs,
1973).
The virus is serologically unrelated to TEV, PVY, PeMV and
other potyviruses (Brunt & Kenten, 1972).
5- Tobacco Etch Virus (TEV):
Incidences of nearly 100% at harvest time have been reported.
Yield reductions due to TEV can be as high as 70% (Koenning &
McClure, 1981).
Many investigators isolated TEV from different plant species
(showing mosaic symptoms) of which pepper (C. annuum L.) in
different countries such as: Hawaii (Milbrath & Cook, 1971),
Turkey (Palloix et al., 1994), Cuba (Depestre et al., 1993), India
(Bidari & Reddy, 1991), Northeastern Georgia, USA (Padgett et
al., 1990) and Southern California, USA (Ariyaratne et al., 1996).
The virus causes chlorotic mottle and necrosis of pepper
(Purcifull & Hiebert, 1982). Veinbanding along the whole length
of the veins is another typical symptom (Zitter et al. 1984).
Wilting, often followed by death, of C. frutescens Tabasco is a
useful diagnostic symptom to distinguish the virus from other
potyviruses, most of which generally cause only mottling on this
host (Nelson & Wheeler 1978). Capsicum frutescens Greenleaf
Tabasco is, however, immune to TMV (Purcifull et al., 1975).
The ability of TEV to infect D. stramonium systemically
distinguishes it from PVY, to which D. stramonium is immune
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30
(Nelson & Wheeler, 1978).
Agrios (1988) recorded that, TEV infected pepper leaves
showed mottling, mosaic and distortion; pepper fruit were distorted,
and the entire plant may be stunted.
Padgett et al. (1990) observed that, bell pepper (C. annuum)
"Yolo Wonder B", showed severe (mosaic, leaf curling, stunting) 3-
5 days after TEV-inoculation. But, Tambel-2 cultivar and Asgrow
hybrid (both have moderate resistance to TEV were developed
moderate mosaic and little or no stunting 2-3 weeks after
inoculation.
Chenopodium amaranticolor was local lesion host only when
tobacco was used as the source of inoculation (Nelson & Wheeler,
1978).
Local lesions followed by systemic infection were developed
on Nicotiana rustica, N. sylvestris, Petunia hybrida and Physalls
floridana (Laird et al., 1964).
Severely systemic infection including (vein clearing, mosaic,
mottling, curling, chiorosis, vein banding, and malformations)
developed on Datura metel, D. stramonium, Lycopersicon
esculentum, N. benthamiana, N. glutinosa, N. tabacum cvs. "White
Burley" & "Xanthi" (Zitter & Tsai, 1981).
Thermal inactivation point was 53-55°C for a severe strain of
TEV and 54°C for a mild strain, but it was generally ranged from
55-59°C for mild or severe TEV-strains in three different separate
studies. Dilution end point was ranging between 1:5000 to 1: 104.
The virus was not inactivated after 12 days and its longevity in vitro
Page 36
31
is comparable to the 13 days at 25°C for a severe TEV strain (Laird
et al., 1964).
Tobacco etch virus (TEV) was easily transmitted
mechanically (Zitter & Tsai, 1981).
The virus is transmitted by more than 10 species of Aphids in
the nonpersistent manner (Agrios, 1988).
Puga & Perez (1986) reported that, Myzus persicae acquired
TEV from Capsicum annuum in less than 60 seconds and
transmitted it efficiently within 30 seconds.
TEV is a potyvirus, which has a flexuous filamentous
particles 705 um length (Laird et al., 1964) and 730x12nm
(Agrios, 1988).
Some serological studies revealed relationship between TEV
and hen bane mosaic and not to either cowpea aphid-borne mosaic,
clover yellow vein (Hollings & Brunt, 1981a), pepper severe
mosaic (Feldman & Gracia, 1977), pepper mottle (Nelson &
Wheeler, 1978) or potato virus Y (Sherwood et al., 1988).
6- Pepper Mild Mosaic Virus (PMMV):
Ho et al. (1982) identified a strain of pepper mild mosaic
virus, which caused mosaic disease of pepper in the suburb of
Guangzhou, China.
Debrot et al. (1983) studied a new virus disease of peppers
(Capsicum) occurring in Aragua State, Venezuela, and was found to
be caused by a Potyvirus (for which the name pepper mild mosaic
virus was proposed) which was transmitted in the laboratory in a
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32
non-persistent manner by Myzus persicae. The virus was also
isolated from the field weed Physails angulata.
The virus does not react with antisera to PVY, PeMV, PVMV
and TEV in SDS agar gel immunodiffusion tests.
7- Chili Veinal Mottle Virus (CVMV)
Chili veinal mottle virus (CVMV) is the most important virus
of C. annuum and C. frutescens in Malaysia.
Symptoms include dark green mottle, reduced leaf size and
distortion, and fewer and smaller fruits (Abu Kassim 1986). Yield
reductions of more than 50% have been reported when the crop
became infected at an early growth stage (Ong et al., 1980).
8- Peru tomato virus
Peru tomato virus (PTV) has been reported in Peru as the
causal agent of a pepper disease causing mottle, necrotic leaf spots,
crinkling of the leaves and epinasty (Fernandez-Northcote &
Fulton 1980).
Fribourg, (1979) reported that, the host range is restricted to
species of the Solanaceae and Chenopodiaceae. Lycopersicon
pimpinellifolium, Nicotiana occidentalis, N. glutinosa, and
Chenopodium amaranticolor were useful indicator species. The
weeds Nicandra physaloides, Physalis peruviana and Solanum
nigrum are natural hosts.
Pepper (Capsicum annuum) reacted to PTV with severe mosaic,
C. frutescens „Tabasco‟ with leaf epinasty, and systemic necrotic
spots, and C. pubescens with leaf epinasty and mild mosaic.
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33
PTV can be differentiated from other potyviruses by circular
chlorotic spots and chlorotic rings on N. debneyi. The virus
produces local lesions on C. amaranticolor and Ch. quinoa and
causes necrosis and death of L. pimpinelhfohum.
Myzus persicae readily transmitted PTV from infected N.
occidentalis source plants to healthy N. occidentalis in short probes.
For example, in one trial with single aphids given an acquisition
period of 30 sec and an inoculation access time of 15 min, three of
10 plants became infected. No symptoms were observed in seedling
grown from seeds obtained from systemic infected pepper plants.
In infective N. occidentalis sap, the thermal inactivation point
was 50-55°C, dilution end point 10-4
– 10-5
, and longevity in vitro at
room temperature (18 – 26°C) 4 – 5 days. Infectivity also was
maintained for at least one year in N. occidentalis leaves desiccated
over silica gel and stored at 2°C. Expressed sap from infected plants
contained long flexuous particles. When 40 individual particles
from infective N. occidentalis sap were measured they ranged from
740 to 800 nm in length with a mean of 775 nm.
Peru tomato virus-C antiserum reacted with partially purified
antigens of seven different potyviruses indicating that PTV-C is
related to all of them. Reciprocal tests, however, showed that this
relationship was only one-sided with potato virus A (PVA), pepper
veinal mottle virus (PVMV), and tobacco etch virus (TEV). Taking
into consideration the homologous and heterologous titers the results
suggest that PTV is more closely related to potato virus Y (PVY) and
tobacco vein mottling virus (TVMV) than to any of the others.
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9- Pepper Vein Banding Virus (PVBV):
This virus has been isolated in Cuba (Lopez-Cardet &
Blanco, 1972), China (Ho et al., 1982); India (Ravi et al., 1997).
This virus produced mosaic symptoms on inoculated
Capsicum (Bidari & Reddy, 1991), vein banding, rat tailing leaves,
mosaic mottling, leaf distortion and reduction in leaf size. In host
range studies, 56 species or cultivars belonging to five families
(Solanaceae, Chenopodiaceae, Cruciferaceae and Leguminaceae),
were tested for infectivity by PVBV. Susceptibility to PVBV was
confined to the family Solanaceae. (Ravi et al., 1997).
In activation occurred on heating at 72-73°C. Dilution end
point was 1:1100. At room temperature sap remained infective for
75 hrs, but not 4 days. The virus was transmitted mechanically and
by Myzus persicae (Lopez-Cardet & Blanco, 1972).
Pepper vein banding virus was transmitted by Myzus persicae
and Aphis gossypii at 90 and 100%, respectively (Gowda & Reddy,
1989).
Electron microscopy revealed flexuous rod-shaped particles
(900 nm length) in the purified preparations. The coat protein (CP)
molecular weight was 35000, which is similar to members of
Potyvirus group. Using SDS-PAGE, and direct antigen coating-
(DAC)-ELISA in cross-reactivity studies, PVBV is closer
serologically to tobacco etch virus (TEV), peanut mottle virus
(PeMoV) and peanut stripe virus (PStV) antisera, but failed to react
with sorghum potyvirus antiserum. (Ravi et al., 1997).
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35
10- Chilli Vein-Banding Mottle Virus (CVbMV):
Siriwong et al., (1995) isolated, in Thailand, a virus from
infected chilli spur pepper (Capsicum annuum cv. Acuminatum),
which showed systemic dark green mottling that was mainly
confined to the veins of chilli pepper leaves and also caused leaf
distortion, was initially isolated in 1989 and was first tentatively
identified as chilli veinal mottle virus (CVMV). In the new study in
1995, according to typical symptoms, which occur on pepper
species, chilli vein-banding mottle virus (CVbMV) is the newly
proposed name.
CVbMV induced vein-clearing within 5 days after inoculation
on the upper leaves of pepper (Capsicum annuum and C.
frutescens). This was soon followed by mottling, dark green
spotting or patching and green banding along the vein. The newly
infected leaves were generally smaller in size and showed varying
degrees of distortion in established infections. The infected plants
were stunted, showed dark green streaks on the stem and branches,
and most of their flowers dropped before bearing fruits. The few
fruits that were produced were usually small, mottled and/or
distorted.
The host range of this virus was restricted to the Solanaceae.
Of the 48 plant species or cultivars from 11 families that were
mechanically inoculated with this virus, 19 species or cultivars were
infected. Systemic infections occurred only in solanaceous species.
Symptoms observed 5 – 10 days after inoculation.
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36
The symptoms on tobacco differed slightly depending on the
species or cultivars. N. glutinosa exhibited small chlorotic spots on
inoculated leaves at 5 days after inoculation, followed by vein-
banding, mottling and leaf distortion. N. tabacum cvs. “Xanthi,
Xanthi-nc, Samsun, and hybrid Xanthi x Samsun generally
exhibited larger chlorotic spots on inoculated leaves, followed by
mottling and\or mosaic or ringspots on the upper leaves, whereas
cv. Sanderae displayed severe small chlorotic spots and leaf
distortion. N. tabacum cv. “White Burley” is considered to be a
useful system is lesions host for CVbMV, since it showed chlorotic
spots which developed further to form necrotic lesions. On tomato
CVbMV induced a mild mottle but showed severe mosaic and leaf
distortion on Petunia hybrida and Physalis floridana.
CVbMV did not infect test plants belonging to the following
families: Aizoaceae, Amaranthaceae, Chenopodiaceae, Compositae,
Convolvulaceae, Cruciferae, Cucurbitaceae, Gramineae, Labiatae
and Leguminosae. A diagnostic non-susceptible species for
CVbMV was Chenopodium amaranticolor.
Flexuous particles were consistently observed in leaf-dip
preparations of CVbMV-infected C. annuum, N. glutinosa and P.
hybrida. The particle length ranged from 680 to 847 nm, with a
mean length of 765 nm and a mean width of 13 nm.
The virus preparations were immunogenic and the CVbMV
antisera reacted strongly with homologous antigens, giving a
specific precipitin band in agarose gels containing SDS with both
crude extracts from infected leaves and virus preparations, but they
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37
did not react with crude extracts from healthy plants. The CVbMV
failed to react with antisera against potyviruses which commonly
infect peppers, including PepMoV, PVY, PVY-O, PVMV, TEV and
other potyviruses which naturally infect cucurbits and legumes,
PRSV and PStV, respectively. All of the antisera tested gave
positive reactions with homologous antigens.
Similarly, crude extracts from freshly ground PepMoV, PVY-
O and PVY-T infected tissues gave no precipitin bands with
CVbMV antiserum during SDS-immunodiffusion tests. Using the
Western blotting technique, the intensity of the immuno-precipitin
signals using antiserum against the CVMV from Malaysia was
almost as strong as that of CVbMV homologous antiserum, while
very weak signals were observed in the case of TEV, PVY and
PVMV antisera, and no visible signal was observed that CVbMV is
closely related to CVMV from Malaysia and distantly related to
TEV, PVMV and PVY, but unrelated to PepMoV.
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38
MATERIALS AND METHODS
1- Source of Virus Isolate:
In this study, thirty samples (ten for each governorate) as
naturally infected young leaves showed similar systemic virus
infection my be due to poytviruses, were collected from different
locations (pepper fields) of Qalubia, Menofyia, and Sharkia
governorates. These samples were divided into three groups.
Infectious sap was obtained by grinding infected leaves in the
presence of 0.02 M phosphate buffer of pH 7.0, with mortar and
pestle. Extracted sap was strained through two layers of
cheesecloth. The obtained crude sap from each group was
separately used for mechanical inoculation of 600 mesh-dusted
carborundum-leaves of Chenopodium quinoa Willd., C.
amaranticolor Coste & Ryn., D. stramonium Jimson, Solanum
tuberosum L. hybrid A6 leaves as diagnostic host plants. Rubbing
of tested plants with the forefinger dipped in inoculum then rinsed
with tap water made inoculation. All inoculations were performed
in an insect-proof greenhouse maintained at approximately 30°C
and examined for external symptoms (within 20 days).
Chlorotic local lesions were observed on C. quinoa (9 days
after inoculation). Single local lesions (showed the same color, the
same pattern, the same size and on the same leaf) were cut out from
inoculated leaves and macerated with few drops of buffer on a glass
slide and inoculated onto the aforementioned diagnostic host as
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39
described by Basu & Giri (1992). Resultant extracts from the
reformed local lesions on C. quinoa, were used to inoculate pepper
(Capsicum annuum L.) cv. "California Wonder" plants which
served as the source of virus inoculum throughout this study.
2- Host Range and Symptomatology:
Twenty-five species and cultivars belonging to 4
dicotyledonous families (Amaranthaceae: Amaranthus ascendens
Lois. Gomphrena globosa L.; Chenopodiaceae: Beta vulgaris L.
cv. Raspoly, Chenopodium quinoa Willd., C. amaranticolor Coste
& Ryn.; Leguminosae: Phaseolus vulgaris L. cv. Pinto, Vigna
unguiculata L. cv. Blackeye and Solanaceae: Capsicum annuum L.
cvs. Balady hot, Balady sweet, Yolo Wonder; C. frutescens, cv.
Tabasco; Datura metel L.; D. stramonium L.; Lycopersicum
esculentum Mill.; Nicotiana clevelandii Gray; N. debneyi Domin.;
N. glutinosa L.; N. rustica L.; N. tabacum L., cvs. Samsun, White
Burley, Xanthi; Petunia hybrida Vilm.; Physalis fioridana Ryd.;
Solanum nigrum L.; Solanum tuberosum L. cv. Hybrid A6) were
used in host range studies. The all test plants were grown from seed
in a mixed soil (clay: peat : sand 1:1:1 v/v/v), fertilized weekly
(crystalon 20N: 20P : 20K) and regularly irrigated. The virus
inoculum was prepared by grinding virus infected leaves in 0.01 M
phosphate buffer, pH 7.0 containing 0.2% 2-mercaptoethanol
(PBM) at 10-1
dilution. The inoculum was rubbed with muslin cloth
pad to test plants dusted with 600-mesh carborundum and observed
for development of symptoms for 4 weeks. Inoculated and
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40
subsequently developed leaves were back inoculated to C. quinoa to
confirm the virus infection under greenhouse conditions.
Five test plants sown in each clay pots (30 cm in diameter)
were mechanically inoculated at the cotyledonary or four- to eight-
leaf stage (according to the species) and were kept in insect-proof
greenhouse. Plants without subsequent symptoms were inoculated
again after 15-20 days, uninoculated plants were included as
controls.
Seeds of the tested plants used in this study were obtained
kindly from Agricultural Research Center, Ministry of Agriculture,
Cairo, Egypt and from Agronomy and Horticulture Departments,
Faculty of Agriculture, Moshtohor, Zagazig Univ. "Benha Branch".
3- Physical Properties:
Crude sap from systemically infected leaves of pepper
(Capsicum annuum, L.) cv. "California Wonder", was used to study
the thermal inactivation point (TIP), dilution end point (DEP) and
longevity in vitro (LIV) according to the method described by Fox
(1993). C. quinoa was used as an indicator plant.
A- Thermal Inactivation Point (TIP):
To determine thermal inactivation point of virus isolate, two
ml of the infected sap pipetted separately into each specimen tube.
The tubes were heated for 10 minutes in a thermostatically
controlled water-bath at the required temperature, i.e. 55, 60, 65, 70,
75, 80, 85, 90, and 95°C. The tubes were then immediately cooled
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41
by dipping in cold water. One tube of each infected sap was left
without heating for comparison. Treated and untreated saps were
used to inoculate five leaves of C. quinoa, which were previously
dusted with 600-mesh carborundum. The experiment was repeated
three times and local lesion numbers were recorded.
B- Dilution End Point (DEP):
Infectious sap of pepper cv."California Wonder" leaves was
diluted with distilled water. Several dilutions up to 10-7
were
prepared. Each particular dilution was mechanically inoculated on
five leaves of C. quinoa plants. The experiment was repeated three
times and average numbers of local lesions were estimated.
C- Longevity In Vitro (LIV):
To determine the in vitro stability of the virus isolate, infected
sap extracted from pepper "California Wonder" cv. leaves was
placed in sterilized small tubes (without any additives). The tubes
were plugged and kept at room temperature (25°C). Infectivity of
virus isolate was determined up to 45 days through inoculation on
C. quinoa. Numbers of local lesions were determined.
4- Mode of Transmission:
A- Aphid transmission:
Among several aphids collected from different pepper fields,
the green peach Myzus persicae Sluzer, was the most frequency.
The colony was virus-free by serial transmission on healthy pepper
plants and then re-examined on different solanceous hosts for non-
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42
viruliferous assurance. Non-viruliferous Myzus persicae were
reared on pea plants (Pisum sativus). In transmission studies, aphids
were starved for 2 h and then given a 2-min acquisition feeding
period on viral-infected leaves of Capsicum annuum plant, then
transferred to 5 healthy pepper seedlings (five aphids per seedling)
for inoculation, feeding period of 24 h.
For the control, the same procedure was used, but virus-free
aphids where feeding for acquisition on healthy pepper plants.
The inoculated seedlings were then sprayed with 0.75% (v/v)
insecticide [Pirimor, from ICI Agrochemicals Co.]. Symptoms and
percentage of transmission were recorded within 4 weeks after
inoculation.
B- Seed transmission:
In seed transmission, 50 healthy pepper (cv. California
Wonder) seedlings were mechanically inoculated with virus isolate,
in the same time, an equal number of seedlings were mechanically
inoculated with phosphate buffer and kept as control. Healthy
controls and virus-infected seedlings were left in the green house till
harvesting. Seeds were obtained separately from completely mature
fruits were used for seed transmission test. Three hundred seeds of
each were sown in clay pots (30 cm diameter) containing steam
sterilized soil and received regularly the same cultural practices till
harvesting (five seeds per pot) and kept in an insect-proof greenhouse.
Seedlings were examined at regular intervals for one month to
detect the development of any external symptoms.
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43
5- Virus Purification:
The isolated virus was purified partially by the following
procedure. Systemically infected leaves of Nicotiana tabacum L.
type White Burley, harvested 25 days after inoculation, were
homogenized with 0.1 M potassium phosphate buffer, pH 7.0,
containing 0.3% 2-mercaptoethanol (1:1 w/v) in a Waring Blendor.
The homogenized extract was strained through two layers of
cheesecloth and clarified by adding 4% ethanol plus 4% CCl4,
followed by slow-speed centrifugation. The extract was stayed
overnight, and the slow-speed centrifugation was repeated, if
necessary. The virus was precipitated from the clarified juice by
dissolving 0.5 M NaCl + 6% (w/v) of polyethylene glycol mol. wt.
6000, incubating the solution for 30 min and collecting the
precipitated virus by low-speed centrifugation. The pellets were
resuspended in the original buffer (1/10 initial volume) and
subjected to one or two cycles of differential centrifugation with
suspension of the high-speed pellets in the same buffer. High-speed
centrifugations were made for 45 min at 40000 rpm. Low-speed
centrifugations were made in a Sorvall refrigerated centrifuge for 20
min at 7000 rpm in the SS-34 rotor.
Sucrose density-gradient centrifugation has been widely used
for further purification of partially purified preparations of many
potyviruses. With sedimentation coefficients of approx. 155 S, most
potyviruses are well separated from host constituents by this
method, although there may be very considerable loss when the
virus-containing zone is then centrifuged to pellet the virus.
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44
Separation of the virus from host contaminants has also been
achieved by permeation chromatography for example, with pepper
veinal mottle using controlled-pore glass bead columns. In this case,
the virus eluted immediately after the void volume, followed by the
host material.
Fractions collected at the end of the rotor were monitored for
absorbance at 280 and 260 nm using spectrophotometer to check the
presence and concentration of virus.
Purified isolated virus tested biologically using Chenopodium
quinoa by using concentrated and diluted (10-1
) purified virus
suspension for mechanical inoculation.
6- Electron Microscopy:
To study the morphology and length of the isolated virus
particles, appropriate amount of highly purified preparation was
micropipette onto formvar-carbon coated grids, then allows drying.
With special forceps, the grids was inverted put onto drop of the
negative staining of 2% uranyl acetate, pH 4.0 for 5 sec. Staining
dried prepared grids were examined by “Philips 400-T” electron
microscope in Specialized Hospital, Ain-Shams University, Cairo,
Egypt.
7- Serological Studies:
Antiserum preparation:
Specific antiserum against the isolated virus was produced by
using the purified virus preparation. Two healthy white New
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45
Zealand rabbits were injected with purified virus emulsified 1 : 1
(v/v) in Freund‟s complete adjuvant. Four injections (500 g of the
virus for first intramuscular injection, and 250 g for three
subsequent injections) were used at weekly intervals. Animal was
bled two times after 21 and 27 days from last injection. After
removal from the rabbit, the blood is allowed to clot overnight at
(37°C) and the serum is carefully separated from the clot. The
serum is then centrifuged at low speed 3,000 g for 5 min. to remove
any remaining corpuscles, and the resulting supernatant stored.
Stored specific antiserum against the isolated virus was subjected to
serological tests. The binding of antibodies in the serum to the
antigen was assayed with goat anti-rabbit immunoglobulin (IgG)
conjugated to alkaline phosphatase to use for ELISA tests.
Stored antiserum against the isolated virus was tested
serologically against the antisera of four potyviruses, which infected
pepper plants, such as: Pepper severe mosaic potyvirus (PSMV),
kindly obtained from Argentina (J. M. Feldman); Pepper veinal
mottle virus (PVMV), Nigeria (G.I. Atiri); Potato Y virus (PYV),
Germany (Hans L. Weidemann) and Tobacco etch virus (TEV),
Finland (J.P.T. Valkonen). In addition to four Tobamoviruses
antisera previously obtained from Holland (E.Z.Maat) were used in
the gel diffusion test. Serological studies used in this work were:
A- Precipitation test:
Drops of the partially purified antigens of the isolated virus
suspension was tested serologically using precipitation procedure
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46
(Matthews, 1993). Purified virus obtained was diluted with saline
solution (NaCl, 0.85%) in tube (7 x 10 mm) in serial two-fold
dilutions ranging from 1/2 to 1/1024. Three replicates were made
for each dilution. An equal volume of antiserum at constant dilution
was added to drop of the virus solution in the glass plate. Drops
were mixed gently using sterilized needle, and density of the bulky
flocculent precipitates at different dilutions was recorded.
B- Ouchterlony gel Immunodiffusion test:
In this test the antibody-antigen reaction is carried out in a gel
agarose plates. The reactants are allowed to diffuse through the gel
and combine.
Medium consists of 0.8-g agarose, 0.85 g NaCl and 0.5 g SDS
were dissolved in 100-ml phosphate buffer. Bring to the boil and
allow cooling to approximately 50°C then 0.02-g sodium azide
(NaN3) was added. Medium maintains in water bath to prevent
setting. Pipette about 15 ml of media onto new clean plastic Petri
dishes (10 cm ). Allow medium to solidify, standing dishes on a
level surface.
The pattern in the medium plates consisted of 6 peripheral
wells (5 mm each) around a central well (6 mm ), with 5-mm
distance between the closest edges of the center and peripheral
wells. Both antibody (antiserum) and antigen (partial purified
virus), were diluted with phosphate buffer at the same dilutions.
Appropriate amounts of each were pipette into wells in the
prescribed pattern, and arrangement was recorded.
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Plates were incubated in the humid chamber at room
temperature for 1 week. Precipitin resulting bands (spurs) were
photographed without staining and results were recorded.
C- ELISA test:
Double Antibody Sandwich Enzyme-Linked Immunosorbent
Assay (DAS-ELISA) was performed for diagnostic purposes in this
work as follows:
1-Coating microtiter (ELISA) plates:-
a. Dilute 100 l IgG in 100 ml coating buffer.
b. Add 200 l of this mixture to each well.
c. Incubate for 3 hours at 37°C.
d. Wash the plate 3 times with washing buffer (PBST).
2- Add 200 l aliquots of the test sample (1g fresh infected
tissue/10 ml of extraction buffer or sample buffer).
3- Incubate overnight at 4°C.
4- Wash the plate 3 times with washing buffer (PBST).
5- Dilute 100 l conjugated IgG in 100 ml conjugated buffer
and add 200 l to each well.
6- Incubate at 37°C for 3 hours.
7- Wash the plate 3 times with washing buffer (PBST).
8- Prepare substrate solution immediately before use by adding
p-nitrophenyl-phosphate at 0.75 mg/ml to the substrate
buffer, add 200 l to each well.
9- Incubate at room temperature for one hour to observe reaction
(development of different intensities of yellow colour).
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10- Stop reaction by adding 50 l of 3 M NaOH to each well.
11- Assess results by:
a. Visual observation
b. Measurement of absorbance at 405 nm.
8- Antiviral Activity of Some Selected Healthy Medicinal Plant
Extracts on the Incidence of Virus Infection under
Greenhouse Conditions:
To study the inhibitory activities (as antiviral agents), the
leaves, flowers or fruits of 12 selected medicinal plant [belonging to
10 families and containing different active integrated compounds]
were used. Extracts of these plants were prepared by triturating 20
g of plant parts in 200 ml distilled water, then squeezed through
double layer muslin cloth and filtrates were centrifuged for 20 min
at 3000 rpm. Efficiency of crude sap of medicinal plants in the
resistance of the isolated virus infection was determined as the
inhibition of local lesion formation on the indicator plant.
Preparation of inoculum:
The isolated virus inoculum was prepared by triturating 1 g of
young systemically infected pepper leaves cv. “California Wonder”
in sterilized mortar by adding 10 ml of sterile distilled water. The
extract was filtered through muslin cloth then centrifuged for 20
min at 3000 rpm. The supernatant was used as inoculum.
Four leaves of 3 test seedling plants (Nicotiana debenyi as
local lesion host) were treated with crude sap of medicinal plants
and inoculated with virus inoculum. Eight and hundred N. debenyi
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seedlings were divided into three groups and treated with sap of
medicinal plants as follows:
a)- One day prior to the inoculation with the virus (pre-inoculation
application).
b)- One day after inoculation with the virus (post-inoculation
application).
c)- Crude sap mixed with virus inoculum and immediately
inoculation (mixed application).
Equal number of N. debenyi seedlings plants were sprayed
with distilled water and inoculated as mentioned with virus
inoculum and kept to serve as control. Appropriately amount of
distilled water was mixed with equal amount of virus inoculum for
the mixed application.
Treated plants were kept under insect proof house and
observed for the appearance of local lesion formation.
Inhibitory effect of the tested medicinal plants were
determined by using the following equation:
Relative inhibition % = 100A
B - A
where:
A = Total number of local lesion on untreated plants
(Untreated with medicinal plants crude sap, but with distilled
water + virus inoculation).
B = Total number of local lesion on treated plants
(Treated with medicinal plants crude sap + virus inoculation).
Percentages of the inhibitory effect were transformed to the
arcsine. The arcsine percentages of the inhibitory effect of
medicinal plant extracts were subjected to the proper analysis of
variance (Clarke & Kempson, 1997).
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9- Response of some pepper cultivars to infection with the
isolated virus under greenhouse conditions:
A greenhouse-pot experiment was conducted to determine the
response of some commercial pepper cultivars to mechanical
inoculation with the isolated viral strain under test. It was carried
out under greenhouse conditions at Fac. Agric., at Moshtohor.
Eight pepper cultivars (three sweet peppers and three hot peppers,
all obtained from the Egyptian Agricultural Organization, Ministry
of Agriculture, Cairo Egypt, while seeds of peppers Serrano very
sweet cultivar and Yellow Banana very hot cultivar were obtained
from Preservation of Germplasm Laboratory, Horticulture
Department, Faculty of Agriculture, Moshtohor, by Dr. Ahmad R.
Aggour) used. The experiment was conducted in a randomized
complete block design comprised of four blocks each contains 40
identical replicates (5 pots/cultivar). Pepper seedlings of each
cultivar were randomly transplanted within each block (4
seedlings/pot or replicate). All test plants were grown from seed in a
mixed soil (clay: peat : sand 1:1:1 v/v/v), fertilized weekly
(crystalon 20N: 20P : 20K) and regularly irrigated. Four-leaf stage
pepper seedlings were mechanically inoculated. The plants were
observed and the systemically infected plants were counted until
consistent numbers were reached. The percentages of infection of
each cultivar per block were calculated according to the following
equation:
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% of infection = 100ckplants/blo inoculated of No. Total
block plants infected lysystemical No.of
Percentages of infection were transformed to the arcsine. The
arcsine percentages of virus infection were subjected to the proper
analysis of variance (Clarke & Kempson, 1997).
10- Determination of the distribution and severity of natural
infection with the seemed like tested virus in some
Governorates:
During summer season (July-September), 1997 screening for
the potyvirus isolate infection incidence undertaken in some
Governorates in the Northern of Egypt (e.g., El-Behera, Dakahlia,
Domiat, Gharbia, Giza, Ismailia, Kafr El-Sheikh, Menofyia,
Qalubia, and Sharkia). Upper Egypt don‟t included in this screening
because there is not enough cultivated Capsicum plants. Young
leaves and fruits of pepper plants (commercial lines and other
Capsicum species and varieties, sweet and hot) naturally-infected
with typical potyvirus symptoms from different fields were
collected and investigated for the virus isolate. Three samples from
ten different locations for each governorate were taken. Double
Antibody Sandwich Enzyme-Linked Immunosorbent Assay (DAS-
ELISA) technique in indirect mode used for further indexing and
identification of the concerned virus in crude sap extracted from
infected leaves of the diseased pepper plants were performed at
Agricultural Genetic Engineering, Research Institute (AGERI),
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Giza, Egypt. Whereas, vitamin C (ascorbic acid), and capsaicin
were determined in the infected and healthy collected fruits.
Results of the screening according to natural infection
severity were compared with pepper productivity (ton/feddan)
during summer, 1997, which recorded in the yearbook of the
Economic Institute, Ministry of Agriculture, Dokky-Giza.
11- Determination of Capsiacin and Vitamin C in the natural
infected pepper plants:
The influence of potyvirus infection on changes in vitamin C and
the pungent alkaloid “capsaicin” content were determined in naturally
infected pepper plants.
A- Ascorbic acid (vitamin C):
Vitamin C (ascorbic acid) content was determined in both
sweet and hot green mature naturally infected fruit samples.
Ascorbic acid determination using 2,6-dichlorophenol indophenol
dye titration methods of Ali and Phillippo (1996).
B- Capsaicinoids:
Capsaicin content was extracted and estimated with liquid
chromatographic method (HPLC), using the extract of the fruit
samples in warm EtOH in a reflux condenser and injected onto LC
column, then compared with the NVN-standard, 99% which
available as synthetic capsaicin (from Segma Chemical Co., USA)
according to Parrish (1996) as follows:
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- Calculation:
Capsaicinoids contain 3 major compounds: nordihydro-
capsaicin (N), capsaicin (C), and dihydrocapsaicin (D). Calculate
Capsaicinoids as sum of these compounds [N+C+D; in Scoville
heat units (SHU); 1 g total Capsaicinoids/g = ca 15 SHU], as
follow:
- UV detection.
- Ground peppers and chili pepper:
N = (PN/PS) x (CS/WT) x (200/0.98) x 9300
C = (PC/PS) x (CS/WT) x (200/0.89) x 16100
D = (PD/PS) x (CS/WT) x (200/0.93) x 16100
where: PN, PC and PD = average peak areas of nordihydrocapsaicin,
capsaicin, and dihydrocapsaicin, respectively, from duplicate
injections; PS = average peak area of appropriate standard
solution; CS = concentration of standard solution (mg/mL); WT
= weight of test sample (g).
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EXPERIMENTAL RESULTS
1- Isolation and Symptomatology:
The isolated virus was isolated for the first time from pepper
(Capsicum annuum L. Balady) crops, during this study, in Qalubia,
Menofyia, and Sharkia governorates. The virus causes severe
mosaic whose main field symptoms are characterized by necrotic
spots on the stems, fruits and leaves, followed by premature foliar
abscission. The leaves were developing subsequently in the
defoliated plants, and especially the top leaves, show severe mosaic
(Fig., 1).
The crude sap of the virus isolate used in this study was
obtained from naturally infected pepper plants showing severe
mosaic symptoms and grown in different areas at Qalubia,
Menofyia, and Sharkia Governorates. The infectious sap was
mechanically inoculated on 600 mesh-dusted carborundum
Chenopodium amaranticolor Coste & Ryn. and C. quinoa, Datura
stramonium Jimson, Solanum tuberosum L. “hybrid A6” as
diagnostic host plants. Inoculated plants were kept in an insect-
proof greenhouse. Chlorotic local lesions were observed on C.
amaranticolor and C. quinoa (20 and 9 days of inoculation,
respectively). Single lesions were cut out and macerated on a glass-
slide and inoculated onto the former diagnostic hosts. Extracts
obtained from the reformed local lesions were used to inoculate
Capsicum annuum L. “California Wonder”, which served as the
source of virus inoculum throughout this study.
Symptoms induced by the virus isolate in experimentally
inoculated pepper plants (C. annuum L. cv. “California Wonder”)
maintained in a greenhouse were similar to those expressed by filed
plants (Fig. 2).
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Fig. (1): Naturally infected leaves and fruits of pepper (Capsicum
annuum L.) cv. Balady hot, used as initial source of the
virus isolate.
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Fig. (2): Symptoms induced by isolated virus in experimentally
inoculated pepper leaves (C. annuum L. cv. “California
Wonder”).
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2- Host range of the tested virus isolate:
The host range of isolated virus was shown in Table (1). Of
the 25 plant species or cultivars belonged 4 families were
mechanically inoculated with this virus, 18 species or cultivars
(belonging 3 families, i.e., Amaranthaceae, Chenopodiaceae, and
Solanaceae) were infected under greenhouse conditions.
Five test plants per pot (30 cm in diameter) were inoculated
mechanically at the cotyledonary or four- to eight-leaf stage
(according to the species) and were kept in an insect-proof
glasshouse. Plants without subsequent symptoms were inoculated
again after 15-20 days. Uninoculated plants were included as
controls.
Virus symptoms were observed for a long period at regular
intervals. In order to check symptomless plants and the virus in
plants with symptoms, back inoculations were made on
Chenopodium quinoa.
Susceptibility of the tested hosts differed in their symptoms
according to the species and cultivars as follows:
Necrotic or chlorotic local lesions without systemic infection
appeared on Amaranthus ascendens Lois. Chenopodium
amaranticolor Coste & Ryn., and C. quinoa WilId. (Fig., 3, A, B, C
& D).
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Whereas, Nicotiana debneyi Domin; N. rustica L., and N.
tabaccum “White Burley” were reacted with local lesions followed
by systemic infection.
On the other hand, systemic symptoms only (according to
host plant species or cultivars) were appeared on Capsicum annuum
L. cvs. “Balady Hot, Balady Sweet, California Wonder”, C.
frutescens “Tabasco” Datura metel L., N. glutinosa L., N.
clevelandii Gray, N. tabacum “Samsun and Xanthi-nc” Petunia
hybrida Vilm., Physalis floridana Rydi. and Solanum nigrum L.
(Fig., 3 E, F, G & H).
On the contrary, hosts showing no symptoms with negative
back inoculation tests were: Gomphrena globosa L. (Amaranthaceae),
Beta vulgaris L. cv. “Raspoly”, (Chenopodiaceae), Phaseolus
vulgaris L. cv. “Pinto”, Vigna unguiculata (L.) Waip. cv. “Blackeye”
(Leguminosae), Lycopersicum esculentum Mill, Datura stramonium
L. and Solanum tuberosum L. cv. hybrid A6 (Solanaceae).
Clear necrotic local lesions without systemic infection
appeared on Amaranthus ascendens Lois this first record for this
host may serve later as indicator or differential host plant.
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Table (1): The host range of the isolated virus. Results of the
inoculations onto selected indicator plants.
Families Indicator species Symptoms
Amaranthaceae Amaranthus ascendens Lois.
Gomphrena globosa L.
NLL NS
Chenopodiaceae Beta vulgaris L. cv. Raspoly
Chenopodium quinoa Willd.
C. amaranticolor Coste & Ryn.
NS CLL CLL
Leguminosae Phaseolus vulgaris L. cv. Pinto NS Vigna unguiculata L. cv. Blackeye NS
Solanaceae: Capsicum annuum L.
cv. Balady hot
VC, MM, LD, SG cv. Balady sweet VC, SM, LD, SG cv. California Wonder
C. frutescens cv. Tabasco SM, VB, LD, SG MM
Datura metel L. MM, LA D. stramonium L. NS Lycopersicum esculentum Mill. NS Nicotiana clevelandii Gray VC, SM, SG N. debneyi Domin. NLL/SM N. glutinosa L. M, LD N. rustica L. CLL/SM N. tabacum L.
cv. Samsun
cv. White Burley
cv. Xanthi
VC, M CLL/SM VC, M
Petunia hybrida Vilm.
Physalis fioridana Ryd.
MM, LD MM
Solanum nigrum L. M Solanum tuberosum L. cv.Hybrid A6 NS
MM=Mild mosaic, SM=Severe mosaic, LD=Leaf distortion, CLL=Chlorotic local
lesions, NLL=Necrotic local lesions, SG=Stunting growth, LA=Leaf abscission,
LD= Leaf deformation, VC=Vein clearing, and NS=No symptoms
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Fig. (3): Host plants reacted with local lesion symptoms as
follows:
A = Chlorotic local lesions on Chenopodium amaranticolor.
B = Chlorotic local lesions on Chenopodium quinoa.
C = Necrotic local lesions on Amaranthus ascendens
D = Necrotic lesions followed by mosaic on Nicotiana debneyi.
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Fig. (3): Host plants reacted with systemic symptoms as follows:
E = Vein clearing on Nicotiana tabacum “White Burley”.
F = Mosaic on N. tabacum cv. Samsun.
G = Mosaic on Petunia hybrida.
H = Mild mosaic and distortion on Physalis fioridana.
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3- Physical Properties:
The results of the in vitro virus property tests are shown in
Table (2) as follows:
A- Thermal inactivation point (TIP):
Infectious sap extracted from Capsicum annuum L.
“California Wonder” as a source of the virus, was used to determine
thermal inactivation point of the present virus isolate. Inoculum was
heated to 55, 60, 65, 70, 75, 80, 85, 90 and 95°C for 10 minutes.
Treated and untreated sap was tested on leaves of C. quinoa as an
indicator plant.
The obtained results were cleared that, the virus isolate was
inactivated at 80°C but not at 75°C after 10 min.
B- Dilution end point (DIP):
Several dilutions up to 10-9
was prepared from infectious sap
of C. annuum L. “California Wonder” leaves. Each dilution was
separately inoculated on leaves of C. quinoa as local lesion an
indicator plant.
Results of trials showed that, the infectivity of the present
virus isolate was preserved at dilution between 10-6
and 10-7
.
C- Longevity in vitro (LIV):
In regard to effect of storing at room temperature (25°C) on
the infectivity of the virus isolate in crude sap extracted from C.
annuum L. cv. “California Wonder”, was determined. Obtained data
indicated that, the present virus isolate kept its infectivity for a
period between 28-35 days on C. quinoa plants.
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4- Mode of Transmission:
A-Aphid transmission:
The isolated virus was readily transmitted by Myzus persicae
Sulzer, from infected pepper (C. annuum L. „California Wonder‟)
source plants to healthy C. quinoa in a non-persistent manner.
B- Seed transmission:
Seeds were harvested from seriously infected pepper plants
(C. annuum L., „California Wonder‟) with the isolated virus. No
symptoms were observed in the obtained seedlings. To check for
the presence of the isolated virus, inoculum were obtained from
infected C. annuum L. cv. „California Wonder‟ seedlings and
inoculated to C. quinoa plants. No symptoms were observed on the
tested plant.
5- Virus purification:
The final pellet, during highly purified preparations of the
isolated virus, was resuspended in a minimum volume of the
extraction buffer and was used for further studies.
The infectivity of the purified isolated virus was tested
biologically using Chenopodium quinoa by using concentrated and
diluted (10-1
) suspensions. Clear chlorotic local lesions were
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observed 7-days after inoculation with highly purified isolated
virus, either concentrated or diluted.
Fractions collected at the end of the rotor were monitored for
absorbance at 280 and 260 nm using spectrophotometer to check the
presence and concentration of virus. The results showed that, the
ratio of A min/max A 260/280 was 1.23. While, the ratio of A
max/min A 280/260 was 1.11 with average 1.17 (Fig., 4). This mean
that high yield of purified homologous virus particles was obtained
6- Electron Microscopy:
Electron microscopic examination of the purified preparation
of the isolated virus, negatively staining with uranyl acetate (2%)
pH 4, showed the presence of filamentous flexuous virus particles.
(Fig., 5).
Results of the estimation of the particle length and width
showed that obtained particles measured averaged 760 nm length
and 15 nm width (Fig., 6).
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Fig. (4): Ultraviolet spectrum of the purified preparation of the
isolated virus obtained from systemically infected
pepper (Capsicum annuum L. cv. California Wonder)
plants.
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Fig. (5): Electron micrograph of the purified preparation of the
isolated virus from systemically infected pepper
(Capsicum annuum cv. California Wonder) plants, after
staining with 2% uranyl acetate, pH 4.0 (X 81420).
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Fig. (6): Average particle length distribution of purified virus
preparation from systemically infected pepper
(Capsicum annuum cv. California Wonder) plants,
stained with 2% uranyl acetate, pH 4.0
0
5
10
15
20
25
598 650 702 741 780 806 910
Particle length (nm)
Nu
mb
er
of
Part
icle
s
760
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7- Serological Studies:
A- Precipitation test:
Tube precipitin test carried out to determine serological
affinity of the isolated virus. Antisera of some Potyviruses, i.e.,
pepper severe mosaic (PSMV) from Argentina, pepper veinal
mottle virus (PVMV) from Nigeria, potato virus Y (PVY) from
Germany, and tobacco etch virus (TEV) from Finland kindly
obtained and used for serologically identification tests. Results
indicated that, specific antiserum of the isolated virus strongly
reacted with clear density precipitin with its antigen. Antigens of
the isolated virus also reacted, with the same strong, with the
antiserum of the PSMV (from Argentina). Reaction, less strong,
was observed between isolated virus antigen and antisera of both
PVY (Germany) and TEV (Finland). No precipitin observed
between antigen of isolated virus and antiserum of PVMV
(Nigeria).
B- Ouchterlony gel Immunodiffusion test:
In this test the antibody-antigen reaction is carried out in a gel
agarose plates. The reactants are allowed to diffuse through the gel
and combine.
Results illustrated in Fig. (7) clearly showed that, precipitin
bands between antigen of the isolated virus (partially purified
Page 75
70
virus), and specific antiserum, produced against isolated virus, were
sharp and homologous without any cross in the ended, meaning
that, there were strong serological relationship between both. The
same properties between isolated virus antigens and antibodies of
PSMV imported from Argentina. Meanwhile, diffused and weak
were induced between isolated virus antigen and antibodies of both
PVY and TEV imported from Germany and Finland, respectively.
On the other hand, No reactions observed between isolated virus
antigen and antibodies of PVMV imported from Nigeria, or five
Tobamoviruses imported from Holland.
Serological relationship confirmed between isolated virus (V)
antigen and antisera of isolated virus (S), PSMV (P) PVY (Y), and
TEV (H). No reaction observed between isolated virus antigen and
antisera of PVMV (1), and Five Tobamoviruses [PMMV-P11 (2),
PMMV-SL (3), ToMV (4), TMV-WU1 (5) and PepMV-3 (6)].
Central wells contained isolated virus antigen (V). No materials
were added in (0) peripheral wells.
Obtained results indicated that, the isolated virus was related
serologically to Potyviruses group. This may lead, to concede the
isolated virus as a member of the plant Potyviruses.
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71
Table (3): Serological tube precipitation reactions of isolated virus
against specific antisera for PSMV, PVY, TEV, and
PVMV.
Antigen
Dilutions
Antibodies (specific antisera) of
Isolated
virus
PSMV PVY TEV PVMV
1 : 2 ++++ ++++ +++ +++ ---
1 : 4 ++++ ++++ +++ +++ ---
1 : 8 ++++ +++ ++ ++ ---
1 : 16 +++ +++ ++ ++ ---
1 : 32 +++ +++ ++ ++ ---
1 : 64 +++ ++ + + ---
1 : 128 +++ ++ + ± ---
1 : 256 ++ ++ ± --- ---
1 : 512 ++ + --- --- ---
1 : 1024 + ± --- --- ---
Control
(saline
solution)
--- --- --- --- ---
-- = No reaction; ± = Rarely visible precipitation; + = Slight precipitation; ++ = Moderate
precipitation; +++ = Heavy precipitation and ++++ = Very heavy precipitation.
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72
Fig. (7): Gel Immunodiffusion SDS-test showing the serologically
relationships between antigen of the isolated virus (V)
and antisera against of isolated virus (S), PSMV (P)
PVY (Y), and TEV (H). No materials were added in (0)
peripheral wells. Five Tobamoviruses (2, 3, 4, 5 & 6).
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73
8- Antiviral activity of some selected healthy medicinal plant
extracts on the incidence of virus infection under
greenhouse conditions:
Leaves, flowers and fruits of 12 selected medicinal plant (Fig.
8) (belonging 10 families and containing different active integrated
compounds) extracts were used to study the inhibitory activities (as
antiviral agents), on systemic virus infection. Inhibition effect of
tested plants was determined as the percentage of local lesion
number on infected N. debenyi plants out of 12 healthy inoculated
seedlings.
Four leaves of 3 test seedling plants (Nicotiana debenyi as
local lesion host) were treated with crude sap of medicinal plants
and inoculated with virus inoculum. One hundred and eight N.
debenyi seedlings were divided into three groups and treated with
sap of medicinal plants as follows:
a)- One day prior to the inoculation with the virus (pre-
inoculation application).
b)- One day after inoculation with the virus (post-inoculation
application).
c)- Crude sap mixed with virus inoculum and immediate
inoculation (mixed application).
Equal number of N. debenyi seedlings plants were sprayed
with distilled water and inoculated as mentioned with virus
inoculum and kept to serve as control. Appropriately amount of
distilled water was mixed with equal amount of virus inoculum for
the mixed application.
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74
Figure (8): Properties of selected medicinal plants used for antiviral
activity against the isolated virus infection.
Pic
ture
Family: Acanthaceae Apocynaceae
S. N.: Adhatoda vasica Vinca rosea
C. N.: Malabar nut tree Vinca, Periwinkle
A. I.: Alkaloids, volatile oils Alkaloids (Vincristine)
U. P.: Leaves, Roots All plants
Pic
ture
Family: Chenopodiaceae Euphorbiaceae
S. N.: Chenopodium anaranticolor Acalypha fruticosa
C. N.: Worm seed Copper leaf
A. I.: Alkaloids (Ascaridole) Pigments (Hisbidine)
U. P.: Leaves All plants
Pic
ture
Family: Euphorbiaceae Euphorbiaceae
S. N.: Euphorbia peplus Euphorbia pulcherrima
C. N.: Wild purslane Easter flower
A. I.: Toxic (Phorbol) Flavonoids (Kaempferol)
U. P.: All plants All plants
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75
Figure (8): Continued
Pic
ture
Family: Geraniaceae Labiatae
S. N.: Pelargonium zonale Salvia officinalis
C. N.: Horse-shoe geranium Common sauge
A. I.: Essential oil (Geraniol) Terpene hydroxide
U. P.: All plants Leaves
Pic
ture
Family: Malvaceae Meliaceae
S. N.: Hibiscus rosa sinensis Azadirachta indica
C. N.: Shoe flower Margosa tree
A. I.: Glycosides (Saponins) Limonoid (Azadirachtin)
U. P.: Leaves, Flowers Leaves, Fruits, Cortix
Pic
ture
Family: Solanaceae Verbenaceae
S. N.: Datura metel Lantana camara
C. N.: Downy thorn apple Lantana
A. I.: Alkaloids (Hyoscyamine) Triterpenes
U. P.: Leaves All plants
S.N.= Scientific Name, C.N.= Common Name, A.I.= Active ingredients, and U.P.=
Used Parts.
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76
Treated plants were kept under insect proof house and
observed for the appearance of local lesion formation.
Percentages of the inhibitory effect were transformed to the
arcsine. The arcsine percentages of the inhibitory effect of
medicinal plant extracts were subjected to the proper analysis of
variance.
Data presented in Table (4) and Fig. (9), showed that leaves
extract of Chenopodium amaranticolor gave the superior antiviral
activity either applied pre- (99.3%), mixed (98.9%) or post-
inoculation (97.1%) with isolated virus. Anterior petals extract of
Hibiscus rosa sinensis was second with the same applications
(98.3% pre-, 97.7% mixed & 94.5% post-inoculation with isolated
virus). Strong inhibitory effect was induced by the leaf extract of
Vinca rosea (95.3%, 94.7% & 90.0% for pre-, mixed & post-
inoculation with isolated virus).
Tip and expanded leaves extract of Pelargonium zonale,
Lantana camara, Euphorbia pulcherrima, Datura metel,
Azadirachta indica, and Adhatoda vasica gave antiviral activity as
descending manner, while applied pre-, mixed or post-inoculation
with isolated virus (88.4, 86.7, 79.2%; 64.2, 62.1, 58.3%; 54.9,
51.1, 49.9%; 54.2, 50.6, 48.9%; 41.6, 39.9, 34.4% and 27.6, 20.1,
17.8%, respectively).
Extract of expanded leaves of Acalypha fruticosa showed the
lowest inhibition effect while applied as mentioned before (1.7, 0.9
and 0.8%, respectively).
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77
Generally, pre-inoculation applications gave the best
protection against infection with isolated virus. Mixed application
was the second, while post-inoculation applications were less
effective in the same trend.
Table (4): Effect of the extract of some medicinal plants (as
antiviral agents) on the inhibition of isolated virus
infection (percent of inhibition).
Test plants Part used
Relative inhibition (%) Pre-
treatment
Mixed-
treatment
Post-
treatment
Acalypha fruticosa Forsk Leaves 1.7 0.9 0.8
Adhatoda vasica Ness Leaves 27.6 20.1 17.8
Azadirachta indica A. Juss Fruits 41.6 39.9 34.4
Chenopodium amaranticolor
Coste & Reyn. Leaves 99.3 98.9 97.1
Datura metel L. Leaves 54.2 50.6 48.9
Euphorbia peplus L. Leaves 19.2 13.6 11.5
Euphorbia pulcherrima Willd. Leaves 54.9 51.1 49.9
Hibiscus rosa sinensis L. Flowers 98.3 97.7 94.5
Lantana camara L. Leaves 64.2 62.1 58.3
Pelargonium zonale L‟Hérit Leaves 88.4 86.7 79.2
Salvia officinalis L. Leaves 49.8 46.4 36.9
Vinca rosea L. Leaves 95.3 94.7 90.0
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Fig. (9): The inhibition percentages of virus-infection by isolated
virus as affected by the extract of some medicinal plants
(as antiviral agents).
0
20
40
60
80
100
120
Acaly
ph
a f
ruti
co
sa
Ad
hato
da v
asic
a
Azad
irach
ta in
dic
a
Ch
. A
mara
nti
co
lor
Datu
ra m
ete
l
Eu
ph
orb
ia p
ep
lus
Eu
ph
orb
ia p
ulc
herr
ima
Hib
iscu
s r
osa s
inen
sis
Lan
tan
a c
am
ara
Pela
rgo
niu
m z
on
ale
Salv
ia o
ffic
inalis
Vin
ca r
osea
Tested medicinal plants
% o
f in
fecti
on
in
hib
itio
nPre-treatmentMixed-treatmentPost-treatment
Page 84
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9- Response of some pepper cultivars to infection with the
isolated virus under greenhouse conditions:
A pot experiment arranged in a randomized complete block
design was conducted under semi-controlled conditions in a glass-
house to determine the response of some pepper cultivars to
mechanical inoculation with the isolated virus strain under test.
Eight pepper cultivars, species and hybrids (most of them usually
used for local production under protected cultivation in Egypt). The
percentages of infection of each cultivar were calculated,
transformed to arcsine then subjected to the proper analysis of
variance.
Data obtained were recorded in Table ( 5 ) and Fig. (10). The
results showed that, all tested pepper cultivars were susceptible, in
different categories, to mechanical inoculation with the present
virus isolate. Statistical analysis revealed that there were highly
significant differences between cultivars.
Yellow Banana cv. (Sweet pepper) among tested cultivars
was more susceptible to infected with isolated virus (96.25%), then
California Wonder (93.75%), Marconi (85.00%) and Gedion
(58.75%). On the other hand, Serrano cv. (Hot pepper) showed
lowest rate of infection with isolated virus (6.25%), then Cayenne
Large (13.75%), Anheium (15.00%), Pical (22.50%).
Generally it could be concluded that, sweet pepper cultivars
are more susceptible to artificial infection with the isolate tested
virus than hot ones.
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81
Fig. (10): Response of some tested pepper cultivars to infection
with the isolated virus under greenhouse conditions.
0
20
40
60
80
100
120
Anhei
um
Cay
enne
Large
Pical
Serra
no
Cal
iforn
ia W
onder
Ged
eon
Mar
coni
Yello
w B
anan
a
Pepper Cultivars
% o
f in
fecti
on
Page 87
82
10- Determination of the distribution and severity of natural
infection with the seemed like tested virus in some
Governorates:
Screening for distribution and severity of natural infection
with the tested isolated virus was carried out during summer season
(July-September), 1997 including some Northern Egyptian
Governorates (e.g., El-Behera, Dakahlia, Domiat, Gharbia, Giza,
Ismailia, Kafr El-Sheikh, Menofyia, Qalubia, and Sharkia). Upper
Egypt governorates are not included in this screening because there
is not enough cultivated Capsicum plants. Young leaves and fruits
of pepper plants (commercial lines and other Capsicum species and
varieties, sweet and hot) naturally-infected with typical potyvirus
symptoms from different fields were collected and investigated for
the virus isolate. Three samples from different ten locations for each
governorate were taken.
Data including pepper cultivated area (feddan), production
quantity (ton) and productivity (ton/feddan) during 1997 for the ten
screened governorates were obtained from the Agriculture
Economic Reports (1997), Ministry of Agriculture, Cairo, Egypt.
Double Antibody Sandwich Enzyme-Linked Immunosorbent
Assay (DAS-ELISA) technique in indirect mode used for further
indexing and identification of the concerned virus in crude sap
extracted from infected leaves of the diseased pepper plants were
performed at Agricultural Genetic Engineering, Research Institute
(AGERI), Giza, Egypt.
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83
DAS-ELISA detection obtained results could be correlated
with those data to study the effect of virus distribution in the
Northern Egyptian Governorates on the productivity of pepper crop.
Results recorded in the Table ( 6 ) revealed that there were
reduction in the productivity of pepper crop in some surveyed
governorates may be due to severity of isolated virus infection in
these governorates. Meanwhile, there were no relation between
severity infection and productivity in the other governorates.
Although El-Behera governorate gave the highest quantity of
pepper fruiting (42999 ton), but reduction in its productivity (5.64
ton/feddan) was noticed and may be due to severity of isolated virus
infection. On the contrary, the productivity of Kafr El-Sheikh and
Dakahlia governorates was best (7.16 and 6.99 ton/feddan), with no
detection to the isolated virus.
Qalubia governorate gave the highest productivity in the
Egypt (10.93 ton/feddan), but showed low level of isolated virus
infection. In this trend, Gharbia and Giza governorates were came
in the next (8.5 and 7.69 ton/feddan).
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84
Table ( 6 ): Results of survey for PSMV in some Northern Egyptian
provinces using DAS-ELISA test compared with the
pepper productivity (ton/feddan) during summer
season (1997).
Governorates 1997 (summer season)
ELISA results Ton Feddan Productivity (ton/feddan)
El-Behera ++++ 42999 7627 5.64
Dakahlia -- 930 133 6.99
Domiat -- 268 57 4.70
Gharbia + 2091 246 8.50
Giza ++ 20254 2633 7.69
Ismailia ++++ 13511 2125 6.36
Kafr El-Sheikh -- 1390 194 7.16
Menofyia +++ 2588 295 8.77
Qalubia + 8562 783 10.93
Sharkia +++ 22495 3492 6.44
Total of Egypt 240034 37833 6.34
++++ = Severe infection
+++ = High infection
++ = Moderate infection
+ = Low infection
-- = No infection
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85
11- Determination of Capsiacin and Vitamin C in the natural
infected pepper plants:
Vitamin C (ascorbic acid) content was determined in both sweet
and hot green mature fruit samples naturally virus-infected and healthy
fruit samples (mg/100g fresh-weight) collected from field in the
surveyed governorates using 2,6-dichlorophenol indophenol dye titration
method.
Capsaicinoids (pungent alkaloids) content, as the major compound
of them “capsaicin”, was determined in hot green mature naturally virus-
infected fruit samples (mg/100g dry-weight) collected from field in the
surveyed governorates using liquid chromatographic method.
Obtained resulted clearly showed that, there were the inverse
correlation between virus-infection and both ascorbic acid and
capsaicin content, i.e. there were markedly to slight reduction in the
content of ascorbic acid and capsaicin observed in the samples
according to severity to slight infection.
Slight significant reduction observed in the ascorbic acid
content of hot or sweet fruit samples collected from infected plant.
But, non-significant recorded in the ascorbic acid content of hot or
sweet fruit samples collected from healthy ones (Table, 7) and Fig.
(11).
Capsaicin content showed significant reduction in the hot fruit
samples collected from infected plants. However, capsaicin content
were significantly high in fruit samples collected from healthy ones
(Table, 8) and Fig. (12).
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86
Table (7): Vitamin C content (mg/100 g fresh weight base) in the
healthy and naturally infected hot and sweet pepper
fruit samples collected from different ten Northern
Egyptian Governorates.
Governorates Hot peppers Sweet peppers
Healthy Infected Healthy Infected
El-Behera 1221* 802.0* 1475 890.1
Dakahlia 1234 932.4 1532 1007.4
Domiat 1051 781.2 1356 985.6
Gharbia 1164 756.3 1485 891.3
Giza 1321 801.8 1652 811.5
Ismailia 1335 851.6 1541 586.7
Kafr El-Sheikh 1033 764.3 1530 1128.3
Menofyia 1021 771.1 1428 472.0
Qalubia 1258 791.1 1489 655.4
Sharkia 1135 799.5 1387 460.3
L.S.D. at 0.05 N.S. 71.897 N.S. 57.521
L.S.D. at 0.01 N.S. 95.500 N.S. 76.410
*Mean of 10 replicates as samples collected from 10 different locations for each governorates. N.S. = Non significant.
Page 93
88
Table ( 8 ): Capsaicin content (mg/100 g dry weight base) in the
healthy and naturally infected hot pepper fruit samples
collected from different ten Northern Egyptian
Governorates.
Governorates Hot peppers
Healthy Infected
El-Behera 112* 84.7*
Dakahlia 250 202.0
Domiat 223 186.8
Gharbia 130 83.3
Giza 135 105.1
Ismailia 115 96.6
Kafr El-Sheikh 165 145.4
Menofyia 120 79.5
Qalubia 127 101.5
Sharkia 136 101.7
L.S.D. at 0.05 3.131 10.183
L.S.D. at 0.01 4.294 13.530
*Mean of 10 replicates as samples collected from 10 different locations for each governorates.
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89
Fig. (12): Capsaicin content (mg/100 g dry weight base) in the
healthy and naturally infected hot pepper fruit samples
collected from different ten Northern Egyptian
Governorates.
0
50
100
150
200
250
300
El-
Be
he
ra
Da
ka
hlia
Do
mia
t
Gh
arb
ia
Giz
a
Ism
ailia
Ka
fr E
l-S
he
ikh
Me
no
fyia
Qa
lub
ia
Sh
ark
ia
Governorates
Ca
ps
aic
in c
on
ten
t (m
g/1
00
g d
ry w
eig
ht)
Healthy Infected
Page 95
90
DISCUSSION
1- Isolation and Symptomatology:
Seriously naturally virus-infected pepper plants appeared
recently through pepper (Capsicum annuum L. cv. Balady) crops at
Qalubia, Menofyia and Sharkia governorates. This studies aims to
isolate and identify the pathogen which causing an important yield
losses in peppers. Beside the severe mosaic symptoms, the isolated
virus causes a vein-clearing, defoliation on the infected top leaves;
necrotic spots on the stems, fruits and leaves, followed by
premature foliar abscission.
Pepper severe mosaic potyvirus (PSMV), the suggested name
for our virus isolate in this study, was isolated for the first time from
pepper crops (C. annuum L. cv. Calahorra) in San Juan and
Mendoza provinces, Argentina, during 1970 caused severe mosaic
and necrotic streak and/or spots on stems and fruits, consequently,
its responsible for important crop losses (Feldman & Gracia,
1977).
In the subsequently study, during 1982, Feldman & Gracia
(1985), isolated pepper severe mosaic potyvirus (PSMV) from
pepper crops (Capsicum annuum L. cvs. Hungarian Yellow Sweet),
caused serious pepper losses in Catamarca province, Argentina.
Page 96
91
New additional characteristics or studies concerning pepper
severe mosaic potyvirus (PSMV) weren‟t reviewed all over the
world, up till now.
The crude sap of the virus isolate used in this study was
obtained from naturally infected pepper plants showing severe
mosaic symptoms and grown in different areas at Qalubia and
Sharkia governorates. Homologous single chlorotic local lesions
observed on Chenopodium quinoa (9 days after inoculation), were
cut out and macerated on a glass-slide and inoculated onto the
Capsicum annuum L. “California Wonder”, as a propagative host,
which served as the source of virus inoculum throughout this study.
These results are in harmony with those obtained, with other
potyviruses, by some investigators (Makkouk & Gumpf, 1976;
Güldür, et al., 1994 and D’Aquino et al., 1995).
2- Host range of the tested isolated virus:
The host range of the isolated virus was found to be restricted
mainly in some Solanaceae plants, but it could be infecting some
plant species and cultivars belonged to 3 families, i.e.,
Amaranthaceae, Chenopodiaceae, and Solanaceae.
Chlorotic local lesions without systemic infection were
appeared on Chenopodium amaranticolor Coste & Ryn.; C. quinoa
WilId., mechanically inoculated with isolated virus. Whereas,
Nicotiana debneyi Domin. (necrotic local lesions), N. rustica L.,
and N. tabaccum “White Burley” (chlorotic local lesions) were
Page 97
92
reacted with local lesions followed by systemic infection.
Amaranthus ascendens Lois, belonging the family
Amaranthaceae, may be the first record as necrotic local lesion host
could be used, latter, as indicator or differential host, where its
reacted with clearly small necrotic local lesions.
Systemic infection only with differentiated external symptoms
according to host plant species or cultivars was appeared on
Capsicum annuum L. cvs. “Balady Hot, Balady Sweet, California
Wonder”, C. frutescens “Tabasco” Datura metel L., N. glutinosa L.,
N. clevelandii Gray, N. tabacum “Samsun and Xanthi-nc” Petunia
hybrida Vilm., Physalis floridana Rydi. and Solanum nigrum L.
Hosts showed no symptoms with negative back inoculation
tests include Gomphrena globosa L. (Amaranthaceae), Beta
vulgaris L. cv. “Raspoly”, (Chenopodiaceae), Phaseolus vulgaris L.
cv. “Pinto”, Vigna unguiculata (L.) Waip. cv. “Blackeye”
(Leguminosae), Lycopersicum esculentum Mill, Datura stramonium
L. and Solanum tuberosum L. cv. hybrid A6 (Solanaceae).
The symptoms expressed by the isolated virus in the various
solanaceous and other species of indicator plants used in this study
were similar, somewhat, to those reported previously on PSMV by
Feldman and Gracia, (1977, 1985).
There were some differences between host plants response
against infection by the isolated virus in the current study (in Egypt)
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93
and other studies (in Argentina). Walkey (1991) recorded that,
these differences may be due to various factors influence the
development and severity of symptoms in virus infected plants.
Among the most important are those related to the genetical
composition of the host plant and the virus, the age of the host and
existing environmental conditions prior to, and after infection.
3- Physical Properties:
The results of the in vitro virus property tests are as follows:
A- Thermal inactivation point (TIP):
The obtained results were cleared that the virus isolate was
inactivated at 80°C but not at 85°C after 10 min.
B- Dilution end point (DEP):
Results of trials showed that, the infectivity of the present
virus isolate was preserved at dilution between 10-6
and 10-7
.
C- Longevity in vitro (LIV):
Obtained data indicated that, the present virus isolate kept its
infectivity for a period between 28-35 days (at 20-25°).
The data of physical properties of the isolated virus were far
from other potyviruses member recorded as pathogen infected
pepper plants all over the world. However, these results are in
agreement, only somewhat, with those obtained by Feldman &
Gracia (1977), who found that TIP, DEP and LIV for PSMV were
32-64 days, 85-90°C and 10-6
-10-7
, respectively. The differences in
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94
the results between this study and the findings recorded by
Feldman & Gracia (1977), may be due to the differences in the
virus strain, test plant species, and environmental conditions.
4- Mode of Transmission:
A- Aphid transmission:
Isolated virus was easily transmitted by Myzus persicae
Sulzer, from virus-infected pepper (C. annuum L. „California
Wonder‟) to healthy C. quinoa in the non-persistent manner.
From several studies, all over the world, could be noticed that
the green peach aphid, Myzus persicae Sulzer, non-persistently
transmissible all potyviruses which infect pepper plants with
different percentages (Zitter, 1975; Fribourg, 1979; Debrot et al.,
1983; Feldman & Gracia, 1985; Puga & Perez, 1986; Gowda &
Reddy, 1989 and Agranovsky, 1993).
B- Seed transmission:
Regarding seed transmission of the present virus isolate.
Obtained data clearly stated that, the isolated virus did not transmit
through seeds of pepper.
Seed-transmission or seed-borne potyviruses were not
confirmed yet in some previously studies concerning pepper
associated potyviruses (Fribourg, 1979; Raccah et al., 1985;
Agranovsky, 1993 and Sharma et al., 1993).
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95
5- Virus purification:
Partial and highly purified preparations of the isolated virus
could be obtained using two cycles of differential centrifugation.
The infectivity of the purified isolated virus was tested
biologically using Chenopodium quinoa by using concentrated and
diluted (10-1
) suspensions. Clear chlorotic local lesions were
observed 7-days after inoculation with highly purified isolated
virus, either concentrated or diluted.
Fractions collected at the end of the rotor were monitored for
absorbance at 280 and 260 nm using spectrophotometer to check the
presence and concentration of virus. The results showed that, the
ratio of A 260/280 nm was 1.23. While, the ratio of A max/min was
1.11 and the ratio of A 280/260 was 0.86. This mean that high yield
of purified homologous virus particles was obtained.
These results came in line with those of Hollings and Brunt,
(1981a), who reported that, some potyviruses are easily purified and
high yields (up to 20 mg/kg leaf tissues) are readily obtained.
6- Electron Microscopy:
Electron microscopic examination of the purified preparation
of the isolated virus, negatively staining with uranyl acetate (2%)
pH 4, showed the presence of filamentous flexuous virus particles.
Obtained particles measured averaged 760 nm length and 15 nm
width.
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96
Particles of isolated virus had a properties similar to those
stated by many investigator such as: Shukla et al.,(1994) recorded
that, potyviruses members have flexuous filamentous particles
measured between 650 – 900 nm. Feldman and Gracia (1977)
observed flexuous-rod particles measured 761 x 13 nm in sap of
PSMV-infected pepper plants examined by electron microscope, in
Argentina. Erkan (1986) isolated PVY particles measured 710-760
nm from pepper in Turkey. Agranovsky (1993) found that, pepper
veinal mottle potyvirus had filamentous particles 700 – 750 nm in
length.
7- Serological Studies:
A- Precipitation test:
Precipitation test performed between antigen of isolated virus
and its antibodies. Also, tested antibodies (antisera) of some
Potyviruses, i.e., pepper severe mosaic (PSMV) from Argentina,
pepper veinal mottle virus (PVMV), Nigeria, potato virus Y (PVY),
Germany, and tobacco etch virus (TEV), Finland kindly obtained
and used for serologically identification tests. Results indicated that,
specific antiserum prepared against the isolated virus, strongly
reacted, with its antigen. Antigens of the isolated virus also reacted,
with the same strong, with the antiserum of the PSMV. Reaction,
less strong, was observed between isolated virus antigen and
antisera of both PVY and TEV. No precipitin observed between
antigen of isolated virus and antiserum of PVMV.
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B- Ouchterlony gel Immunodiffusion test:
Results showed that, precipitin bands (spurs) between antigen
of the isolated virus (partially purified virus), and specific
antiserum, produced against isolated virus, were sharp and
homologous without any cross in the ended, meaning that, there
were strong serological relationship between both. The same
properties of the spur were confirmed between isolated virus
antigens and antibodies of PSMV. Meanwhile, diffused and weak
spurs were induced between isolated virus antigen and antibodies of
both PVY and TEV. On the other hand, No reactions (spurs)
observed between isolated virus antigen and antibodies of PVMV,
or five Tobamoviruses. Characteristics of the obtained spurs
according to Matthews (1993), means that, there were closely
serologically relationship between the isolated virus and antiserum
against pepper severe mosaic potyvirus (PSMV) obtained from
Argentina (J.M. Feldman). Obtained results indicated that, the
isolated virus, surely, related serologically to Potyviruses group, but
not Tobamovirus. This may lead, after the finishing of the study, to
record the isolated virus as a member of the plant Potyviruses.
Obtained results concerning, symptomatology, host range,
physical properties, mode of transmission, electron microscopy and
serological studies, showed that, isolated virus characteristics,
somewhat, identical with those recorded by Feldman and Gracia,
(1977 & 1985), so its suggested that, the isolated virus called
pepper severe mosaic potyvirus (PSMV). These first records to this
virus isolate in Egypt.
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8- Antiviral activity of some selected healthy medicinal
plant extracts on the incidence of virus infection
under the greenhouse conditions:
In this study, the inhibitory activity of the tested plant extracts
against PSMV local lesion production on N. debenyi plants did not
vary significantly with the different three times of spray
applications. Pre-inoculation application of plant extracts was better
than post-inoculation application. Baranwal & Verma (1993)
suggested that, the inhibitors, as a result of 24 hours pre-inoculation
application, might have acted in initial establishment phase of virus
infection.
Leaves extract of Chenopodium amaranticolor, petals extract
of Hibiscus rosa sinensis, leaves extract of Vinca rosea, gave the
superior antiviral activity against of PSMV infection in the all three
times of applications.
Tip and expanded leaves extract of the other tested plants,
except of Acalypha fruticosa which showed the lowest inhibition
effect, gave antiviral activity as descending manner.
Further studies concerning antiviral activity of the extracts of
the tested and other medicinal plants against pepper associated
potyviruses, especially pepper severe mosaic potyvirus (PSMV), are
needed.
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Lantana camara L. aqueous leaf extract, in this study, gave
64.2, 62.1 and 58.3% inhibitory effect when applied pre-, mixed
and post-inoculation of PSMV, respectively. On the other hand, for
example, ethanol (96%) extracts of the Lantana camara L. leaves,
in the other study, gave toxic effect (80.3±5.4%) towards the green
peach aphid Myzus persicae, the main vector for the most pepper
viruses (Stein & Klingaui, 1990). This might be open a new wide
scope to more useful from some plant extracts as both antiviral and
insecticidal materials.
9- Response of some pepper cultivars to infection with the
isolated virus under greenhouse conditions:
Eight pepper cultivars, species and hybrids (most of them
usually used for local production under protected cultivation in
Egypt), were tested for response to infected by PSMV under
greenhouse conditions.
Serrano cv. (hot pepper) and Gedeon cv. (sweet pepper)
showed highly resistance against infection with PSMV than other
tested peppers. On the contrary, Pical cv. (hot pepper) and Yellow
Banana cv. (sweet pepper) were more susceptible to infect by
PSMV.
The results showed that, sweet pepper cultivars are more
susceptible to artificial infection with the tested virus isolate than
hot ones.
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Many investigators, through several different studies,
observed that there are some factors related with resistance property
for viral diseases for instance:
Resistance to viral infection is an inheritance property
subjected to Mendel‟s low and depends on some genes responsible
for resistance. The resistance genes may be functioned by partially
inhibiting virus multiplication (Soh et al., 1977).
Chemical content may also associated with the plant
resistance to viral infection. Increase in sugar content may be
induced as response to virus-infection, then sugars play an
important role in the resistance to virus infection. Also, the increase
in phenolics in some pepper cultivars might be associated with
resistance (Thakur et al., 1986). Šubíková et al. (1994) suggested
that there is a possible role of the cell wall polysaccharides or their
oligosaccharide fragments in the mechanism of induced resistance
against viruses.
High content in the vitamin C (ascorbic acid) may be play a
role in the resistance to viral diseases (Liu et al., 1992 and Hundal
et al., 1995). On the other hand, correlated between high content of
pungency compounds (capsaicinoids), especially capsaicin, in
addition to oleoresin, in some pepper cultivars or hybrids and
resistance to virus infection had been reported by Awasthi & Singh
(1975); Tewari (1990); Liu et al., (1992); Hundal et al. (1995)
and Berke (1997).
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On the contrary, Walkey (1991) recorded that, the effect of
host plant nutrition upon virus symptoms may be quite variable, but
in general, nutritional conditions that favour plant growth, also
favour increased host susceptibility to virus infection. High nitrogen
levels for instance, have been reported to increase the susceptibility
of marrow seedlings to virus infection by CMV.
High temperature may affect a host plant‟s ability to resist
virus infection by arrested virus replication and inactivated a
reversible resistance mechanism in the host cells. Also, high light
intensities produce „hard‟ plants, which are less susceptible to virus
infection than plants grown under low light intensities (Walkey,
1991).
10- Determination of the distribution and severity of natural
infection with the seemed like tested virus in some
Governorates:
Naturally virus-infected pepper leaf samples, collected from
different locations of ten Northern Egyptian Governorates were
subjected to detection of virus using direct system (double-antibody
sandwich) of enzyme-linked immunosorbent assay (DAS-ELISA),
utilizing PSMV antiserum prepared in our laboratory. Antiserum for
PSMV was kindly supplied by Dr. J.M. Feldman, Mendoza,
Argentina. The ELISA reader as the degree of disease severity
estimated incidence of virus disease.
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Results of 1997 survey indicated the following regional
distribution of the studied virus (listed according to their importance):
El-Behera, Ismailia, Menofyia, Sharkia, Giza, Gharbia and Qalubia. But
not detected in the Dakahlia, Domiat and Kafr El-Sheikh governorates.
Regarding to correlation between the pepper productivity and virus
infection with PSMV, this study clearly showed that the reduction in the
pepper productivity of El-Behera and Ismailia governorates might due to
severity of virus infection of both.
This survey demonstrates the widespread occurrence and high
incidence of the studied virus. This result is in agreement of that
previously obtained by Gracia et al. (1990). Also, Escudero
(1996), in Puerto Rico, showed that potyviruses could be reduced
pepper yields by as much as 90% during survey of viruses affecting
pepper plants.
11- Determination of Capsiacin and Vitamin C in the natural
infected pepper plants:
Pepper content of vitamin C (ascorbic acid) altered with pepper
species, variety, cultivar and hybrids. Many workers recorded some
peppers showed high content of ascorbic acid, for example, Anaheium is
Mexican type contains three times the vitamin C of a Valencia orange and
provides the minimum daily requirement (Bosland, 1992). Punjab Surkh, a
new high yielding variety of pepper also a good source of ascorbic acid
(Hundal et al., 1995).
Pepper content of ascorbic acid is affected by different factors. For
instance, in the sweet or hot pepper, ascorbic acid content was increased
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gradually with the increasing of pepper fruit ripening age, then being to
decrease after colouring stage and may be trend to decrease significantly
during storage (Audisio et al., 1995; El-Saeid, 1995; Ishikawa et al.,
1997 and Simonne et al., 1997). Vitamin C content were higher for
peppers fertilized by Ca(NO3)2, than in those grown with (NH4)2SO4, as
a nitrogen source (Kitis & Aktas, 1997).
Regarding the correlation between pepper content of ascorbic acid
and virus infection. Awasthi & Singh (1975) observed that CMV
infection caused a decrease in the ascorbic acid content of fresh
Capsicum fruit samples, especially susceptible varieties than in the
tolerant one. They also added that, the reduction of ascorbic acid content
might be associated with the increase in ascorbic acid oxidation activity.
Liu et al. (1992) recorded that the pepper resistance to infection with
TMV and CMV correlated positively with the increase in the pepper
vitamin C content.
In the present study, the changes of vitamin C content as affected
by virus infection under natural infection conditions showed slight
correlation. This result was harmony with those recorded by Pennazio et
al. (1996) who studied the correlation between L-ascorbic acid content
and virus infection (by 19 different plant viruses belonged various
groups) in higher plans (including peppers). They reported that,
experimental evidence indicates that the vitamin plays a protective
(antinecrotic) role during the hypersensitive reaction, but its mechanism
is quite unknown. The results concerning the vitamin content in
systemically infected plants are not univocal because it has been
described to increase, decrease or remain unaltered.
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Vitamin C (ascorbic acid) content was determined in both sweet
and hot green mature naturally potyvirus-infected fruit samples.
Ascorbic acid determination using 2,6-dichlorophenol indophenol dye
titration method. Capsaicin content (mg/100g dry-weight) was
extracted and estimated with liquid chromatographic method.
Regarding the pungent alkaloid “capsaicin” content changes
as affected by virus infection (mg/100g fresh-weight and dry-
weight, respectively) were determined, in this work, in the fruit
samples collected (infected and healthy) from field under natural
virus-infection in the aforementioned governorates.
Many investigators observed that pepper contents of
capsaicinoids (the pungency) are correlated with tolerance or
resistance to virus infection by many viruses. So, hot peppers showed
different degrees of resistance to virus diseases were recorded and/or
developed (Awasthi & Singh, 1975; Tewari, 1990; Tewari,
1991a,b; Liu et al., 1992; Hundal et al., 1995; Berke, 1997).
The pungency of Capsicum spp. is due to capsaicinoids,
comprising capsaicin, dihydrocapsaicin, nordihydrocapsaicin,
homocapsaicin and homodihydrocapsaicin. The first three, the
major compounds in Capsicum spp., give rapid pungent sensation in
the back of palate and throat while the others tend to produce
prolonged pungent sensation of low intensity in the mid-mouth and
mid-palate regions (Sudhakar et al., 1995).
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Capsaicinoids, i.e. acid amide derivatives of vanillylamine and
C9 to C11 branching chain fatty acids. Capsaicin [N-(4-hydroxy-3-
methoxybenzyl)-nontrans-6-enamide] is the main component of most
Capsicum spp. The aromatic skeleton of capsaicinoids is
biosynthetically derived from phenylalanine and the branched fatty
acid moiety from valine (Salgado-Garciglia & Ochoa-Alejo, 1990).
Ishikawa, et al., (1998) studied the contents of capsaicinoids
and their phenolic intermediates (trans-cinnamic acid, trans-
coumaric acid, trans-caffeic acid, trans-ferulic acid and
vanillylamine). They noticed that, capsaicinoids accumulated
primarily in the placenta of fruits (average of 33 and 38 mol/g of
capsaicin and dihydrocapsaicin, respectively). Aproximately 58 and
49% of vanillylamine and phenolic intermediates, respectively,
accumulated in the placenta. Their distribution was not correlated
with the production of capsaicinoids.
Capsaicinoids are synthesized through the cinnamic acid
pathway; their degradation is thought to be aided by the action of
peroxidases. The content of capsaicinoids (particularly capsaicin
and dihydrocapsaicin) increased continuously and reached a peak
45-50 days after fruit set in some pepper varieties, and then
declined. Peroxidase activity increased at the time when the
concentrations of capsaicinoids started to decrease. There was an
inverse relationship between the evolution of capsaicinoids and
peroxidase activity that might indicate that this enzyme is involved
in capsaicinoid degradation (Contreras-Padilla & Yahia, 1998).
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Peroxidase is an enzyme that catalyses the oxidation of a large
number of aromatic structures at the expense of H2O2. Two main
groups of isoperoxidases have been distinguished, acidic and basic.
Peroxidases have recently been considered to play a role in the
metabolism of alkaloids. In hot peppers, like other plant alkaloids,
capsaicinoids accumulate and later undergo a rapid turnover and
degradation during fruit development. While considerable progress
has been made on the biosynthesis of capsaicinoids, the
enzymology of the last steps in capsaicinoid metabolism and
degradation is still incomplete. Basic peroxidase may be directly
related to capsaicinoid metabolism since both capsaicin,
dihydrocapsaicin and their phenolic precursors are easily oxidized
by this enzyme (Pomar et al., 1997).
Peroxidase specific enzyme activity increased throughout the
growing season as chiles ripened. The highest level of activity was
detected 68-75 days after ripening. In chile, the increase in
peroxidase activity correlates with enhancements of other
physiological parameters including an increase in ethylene
production, increase in fruit colour change from green to red
(chlorophyll loss), decrease in respiration, and cuticle thickening.
Peroxidases have been correlated in plants with disease resistance,
wound healing, lignification, phenol polymerization, suberization,
protection against H2O2 and other oxidants, drought tolerance,
chlorophyll degradation and senescence. Alternate forms of chile
fruit peroxidase vary during fruit ripening, are stable at elevated
temperature, and demonstrate broad pH optima during the ripening
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stages. Recently, pepper peroxidases have been implicated in the
oxidation of dihydrocapsaicins of pepper, therefore influencing
pungency (Biles et al., 1997).
As described above, capsaicinoids and their phenolic
intermediates, which synthesized through the cinnamic acid
pathway and the inverse relationship between the evolution of
capsaicinoids and peroxidase activity, might partially inhibiting
virus multiplication.
Recently, fortunately, in Japan Kobata et al. (1998) & (1999)
isolated three novel capsaicinoid-like substances called capsiate,
dihydrocapsiate and nor dihydrocapsiate from the fruits of a non-
pungent cultivar, CH-19 Sweet of pepper (Capsicum annuum L.). This
discovery might be open a glimpse hope to obtain good quality of
sweet peppers free from, or less infected by, viral diseases.
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CONCLUSIONS
Seriously naturally virus-infected pepper plants recently
appeared through pepper (Capsicum annuum L. cv. Balady) crops,
at Qalubia, Menofyia and Sharkia governorates, causing an
economic important yield losses. This studies aims to isolate and
identify the virus(s) which cause this problem, establish the
distribution and incidence of the isolated virus, and testing some
natural antiviral substances to prevent or reduce the severity of
virus-infection in pepper crops. Determination of Vitamin C and
Capsaicinoids (pungency matter) in the fruits of pepper plant
(cultivated in the different governorates fields) healthy, or naturally
virus-infected and correlated the content of both with naturally
virus-infection. Relationship between virus-infection incidence and
productivity of pepper was recorded.
A new potyvirus was isolated from naturally infected pepper
fields (Capsicum annuum L. cv. Balady) at Qalubia, Menofyia and
Sharkia governorates. Beside the severe mosaic symptoms, this
virus causes a vein-clearing, defoliation on the infected top leaves;
necrotic spots on the stems, fruits and leaves, followed by
premature foliar abscission. So, pepper severe mosaic potyvirus
(PSMV) was suggested for it.
The host range of pepper severe mosaic virus (PSMV) was
found to be restricted mainly in some Solanaceae plants, but it
could be infecting some plant species and cultivars belonging 3
families, i.e., Amaranthaceae, Chenopodiaceae, and Solanaceae.
Amaranthus ascendens Lois, belonged the family
Amaranthaceae, reacted with clearly small necrotic local lesions,
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may be the first record and could be used as diagnostic host.
The isolated virus was easily transmissible by green peach
aphid, Myzus persecae Sulz. and by mechanical means, but not
through pepper seeds.
High temperature (between 80 - 85°C) inhibited the
pathogenicity of the isolated virus. Infective of isolated virus was
lost at the dilution between 10-6
to 10-7
, but tested virus preserved in
vitro between 28-35 days.
Severity of virus infection symptoms might be altered
according the differences in the pepper cultivars and associated
inheritability factors, season of cultivation, fertilization, the
distribution of virus-vector insects, and other environmental
conditions. This means, the careful by available the optimum of
these factors might be reduced the severity of virus infection,
consequently, enhanced the productivity and total yield of pepper.
Production of new pepper hybrids showed natural tolerant or
resistant to virus infection was the most effective means to obtain
good quality and quantity of peppers.
Cultivation of Serrano cv. (hot pepper) and Gedeon cv. (sweet
pepper) are recommended for their highly resistance to virus
infection. Also, Anheium cv. (Mexican hot pepper) showed
considerable resistance with high content of vitamin C.
Using the aqueous leaves extracts of Chenopodium
amaranticolor Coste & Reyn., Vinca rosea L., Pelargonium zonale
L‟Hérit and the aqueous flowers extracts Hibiscus rosa sinensis L.,
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showed the most effective antiviral activity agents among 12
medicinal tested plant.
Also, the aqueous leaves extracts of Lantana camara L.,
Euphorbia pulcherrima Willd. and Datura metel L., showed
considerable role in the reduction of virus infection, beside their
role in the reduction of virus-vector population in the treated plants.
Further studies regarding the using of natural extracts of some
medicinal plants against pepper associated potyviruses, and the
studying the relation between these extracts as antiviral agents and
their considerable role in the reduction virus-vector insect
populations in the pepper field, are needed.
Regarding to the relation between both vitamin C and
capsaicinoids contents, either in the fruits of healthy or virus-
naturally infected pepper plants, and the incidence of virus
infection. There are conflict results in regard to increase or decrease
of both as resulting to virus infection. This may be due to many
factors including: time of cultivation and when infection cause,
plant species, soil type, fertilization, environmental conditions,
interaction between more than on pathogen in the same plant, …
etc. This relation would be more clearly after more specific works.
Generally, pepper severe mosaic potyvirus (PSMV), a new
potyvirus occurring and severity incidence in some pepper
productive Egyptian governorates may cause the considerable
reduction in the pepper yield.
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SUMMARY
The present work was carried out at Laboratory, Greenhouse,
and Farm of Faculty of Agriculture, Moshtohor, Zagazig
University, during 1996/97 – 1997/98 seasons, to isolate and
identified some pepper-associated potyviruses, which caused
serious reduction in the pepper crops during the last seasons. This
work also aims to establish an effective means to produce virus-free
peppers by using natural antiviral substances, and survey
occurrence and incidence of the isolated virus(es).
A new potyvirus, suggesting called pepper severe mosaic
potyvirus (PSMV), was isolated from pepper crops (Capsicum
annuum L. Balady) in Qalubia, Menofyia and Sharkia governorates.
The distinguishing symptoms of this virus isolate were severe
mosaic on top leaves; necrotic spots on the stems, fruits and leaves;
followed by premature foliar abscission. Infected plants, generally,
showed severe stunting.
Isolated virus was identified using sympomatology, host
range, mode transmission, physical properties, and serological tests.
The host range of pepper severe mosaic virus (PSMV) were
18 species or cultivars (belonging 3 families, i.e., Amaranthaceae,
Chenopodiaceae, and Solanaceae), during pot-trial under
greenhouse conditions.
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Only necrotic local lesions followed by no systemic infection
were appeared on Amaranthus ascendens Lois. Meanwhile,
chlorotic ones appeared on mechanically inoculated leaves of
Chenopodium amaranticolor Coste & Ryn., and C. quinoa WilId.
Necrotic local lesions followed by systemic infection
produced on Nicotiana debneyi Domin. While, chlorotic ones
followed by systemic infection appeared on N. rustica L., and N.
tabaccum “White Burley”.
Systemic symptoms only and differentiated according to host
plant species or cultivars was appeared on Capsicum annuum L.
cvs. “Balady Hot, Balady Sweet, California Wonder”, C. frutescens
“Tabasco” Datura metel L., N. glutinosa L., N. clevelandii Gray, N.
tabacum “Samsun and Xanthi-nc” Petunia hybrida Vilm., Physalis
floridana Rydi. and Solanum nigrum L.
Physical properties of the isolated virus recorded as follows:
Thermal inactivation point (TIP), was 80°C but not at 85°C
after 10 min., dilution end point (DIP), was between 10-6
and 10-7
,
and longevity in vitro (LIV), was between 28-35 days.
Tested transmission means demonstrated that, the isolated
virus was easily transmitted by mechanical inoculation and by green
peach aphid, Myzus persicae Sulz. as non-persistent manner. But
non-transmissible though pepper seeds.
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Particles of the isolated virus, negatively stained with uranyl
acetate (2%), were filamentous fexuous and had a 760 nm length
and 15 nm width when examined with electron microscope.
Serological studies using tube precipitin, double diffusion test
and directs system (double-antibody sandwich) of enzyme-linked
immunosorbent assay (DAS-ELISA) tests were performed. Results
demonstrated that there is a clearly positive reaction between
antiserum against isolated virus (PSMV) and antisera against some
other potyviruses such as PSMV, PVY and TEV, but not with
PVMV, which imported kindly from Argentina, Germany, Finland
and Nigeria, respectively.
This result clearly showed that, serologically, the isolated
virus is a member of Potyvirus group.
Aqueous extracts of 12 plant species showing potent antiviral
activity belong to 10 families and containing different active
integrated compounds used in the pot-trial.
Extracts of Chenopodium amaranticolor Coste &
Reyn.(leaves), Hibiscus rosa sinensis L.(flowers), Vinca rosea
L.(leaves), and Pelargonium zonale L‟Hérit (leaves), showing
marked inhibitory effect, especially if sprayed 24h before
inoculation with the tested virus.
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Leaves extracts of Lantana camara L., Euphorbia
pulcherrima Willd., Datura metel L., Salvia officinalis L., and fruit
extract of Azadirachta indica A. Juss came in the next in this trend.
Slight inhibitory effect induced using leaves extracts of both
Adhatoda vasica Ness and Euphorbia peplus L. While, no effect
showed using leaves extract of Acalypha fruticosa Forsk.
There were no significant differences between sprayed the
tested extracts pre-, mixed or post-inoculation with the virus isolate.
Response of some pepper cultivars, species and hybrids (most
frequently cultivated in the greenhouse) against virus infection were
studied using pot experiment.
Serrano (hot pepper), and Gedeon (sweet pepper) gave the
superior resistant against infection by isolated virus. Came in the
next Cayenne Large, Anheium and Pical (hot pepper); Marconi,
California Wonder and Yellow Banana, respectively.
Generally, hot pepper showing more resistance to virus
infection than sweet peppers.
Screening for existence pepper severe mosaic virus (PSMV)
including some Northern Egyptian Governorates (e.g., El-Behera,
Dakahlia, Domiat, Gharbia, Giza, Ismailia, Kafr El-Sheikh,
Menofyia, Qalubia, and Sharkia) was carried out during summer
season (July-September), 1997.
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Young leaves and fruits of pepper plants (commercial
Capsicum sweet and hot lines, species or varieties) naturally-
infected with typical potyvirus symptoms from different fields were
collected and investigated for the virus isolate. Crude sap from
infected leaves subjected to detection tests using double antibody
sandwich Enzyme-Linked Immunosorbent Assay (DAS-ELISA)
technique in indirect system at Agricultural Genetic Engineering,
Research Institute (AGERI), Giza, Egypt. Meanwhile, fruit samples
were subjected to vitamin C and capsaicinoids determination.
Seven of ten screened Egyptian governorates (El-Behera,
Ismailia, Menofyia, Sharkia, Giza, Gharbia and Qalubia, arranged
descendingly according to severity infection) showed incidence of
the isolated virus with different severity. Results clearly established
when compared with the productivity of these governorates from
pepper (recorded in the yearbook of Agriculture Ministry), where
found consecutive correlation between severity infection and
reduction in the productivity. Isolated virus not detected in the other
three screening Egyptian governorates (Dakahlia, Domiat and Kafr
El-Sheikh).
Fruit samples collected from infected and healthy pepper
plants during the screening were subjected to chemical analyses for
their content of both vitamin C (ascorbic acid) and capsaicin.
Correlation between virus infection and fruit pepper content of
vitamin C and capsaicin were estimated.
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Results showed marked reduction in the content of both
vitamin C and capsaicin in the virus infected pepper plants. Positive
correlation between pepper content of pungency „capsaicin‟ and
resistance to virus infection was naturally found during the survey.
Occurrence of the isolated virus during this study, suggested
called pepper severe mosaic potyvirus (PSMV), was the first record
in Egypt
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REFERENCES
Abu Kassim, A. B. (1986): Virus disease of horticultural crops in
Malaysia. pp.3-6 In: FFTC Book Series. No.33:3-6. Food
and Fertilizer Technology Center for the Asian and Pacific
Region, Taipei, Taiwan. 193 pp.
Agranovsky, A.A. (1993): Virus diseases of pepper (Capsicum
annuum L.) in Ethiopia. J. of Phytopathology, 138(2):89-
97.
Agrios, G.N. (1988): Plant Pathology, 3rd
Edt Academic Press Inc.
(London) Ltd. 703 pp.
Alegbejo, M.D. (1986): Aphid transmission of pepper veinal
mottle virus. Samaru J. of Agric. Res., 4:71-77. [c.f. Rev.
of Appi. Ento.,A, 75(10):5561].
Ali, M. Sher and Phillippo, E.T. (1996): Simultaneus
determination of ascorbic, dehydroascorbic, isoascorbic
and dehydroisoascorbic acids in meat-based food products
by liquid chromatography with postcolumn fluorescence
detection: A method extension. AOAC International,
79(3): 803-808.
Allan, O.L.; Gilmer, R.M.; Wilson, G.F. and Shoyinka, S.A.
Page 123
118
(1975): An unusual new virus, possibly of the potyvirus
group, from pepper in Nigeria. Phytopathology, 65:1329-
1332.
Alonso, E.; Garcia-Luque, L.; Avila-Rincon, M.J.; Wicke, B.;
Serra, M.T. and Diaz-Ruiz, J.R. (1989): A
tobamoviruses causing heavy losses in protected pepper
crops in Spian. J. Phytopathology, 125:67-76.
Ariyaratne, I.; Hobbs, H.A.; Valverde, R.A.; Black, L.L. and
Dufresne, D.J. (1996): Resistance of Capsicum spp.
genotypes to tobacco etch potyvirus isolates from the
Western Hemisphere. Plant Disease, 80(11): 1257-1261.
Arteaga, M.L.; Andres, M.A. and Ortega, R.G. (1997): New potato
virus Y pathotype in pepper. Capsicum & Eggplant
Newsletter, No. 16, 85-86. [c.f. Rev. Plant Path., 77(7):5814].
Atiri, G.I. (1986): A disease of fluted pumpkin (Telfairia
occidentalis Hook. F.) caused by a yellow vein-clearing
strain of pepper veinal mottle virus in Nigeria. J. of Plant
Protec. in the Tropic, 3:105-110. [c.f. Rev. Plant Path,
66(10):4545].
Atiri, G.I. (1992): Progress of pepper veinal mottle disease in
Capsicum peppers. Crop Protection, 11(3): 255-259.
Page 124
119
Audisio, M.; Dante, D.; Cicco, A. de. and Siciliano, M. (1995):
The vitamin C content in peppers (Capsicum annuum) of
the Rubra and Golden King cultivars in relation to degree
of ripeness and type of storage. Rivista di Scienza
dell‟Alimentazione, 24 (4): 543-547. [c.f. FSTA, 28(6):
230].
Awasthi, D.N. and Singh, B.P. (1975): Influence of cucumber
mosaic virus on ascorbic acid and capsaicin content from the
fruits of tolerant and susceptible varieties of chilli. Indian
Phytopathology, 28(2): 272-274.
Baranwal, V.K. and Verma, H.N. (1993): Virus inhibitory
activity of leaf extracts from different taxonomic groups of
higher plants. Indian Phytopathology, 64(4): 402-403.
Basu, A.N. and Giri, B.K. (1992): The essentials of viruses,
vectros and plant disease. Wiley Eastern Ltd.,
Berke, T. (1997): Current status of the International Chilli Pepper
Nursery. Capsicum & Eggplant Newsletter, No.16: 80-81.
Bidari, V.B. and Reddy, H.R. (1991): Incidence of chilli mosaic
and its distribution in the commercial fields of Karnataka.
J. of Plantation Crops, 19: 21-25. [c.f. Rev. Plant Path.,
7(8): 4895].
Page 125
120
Biles, C.L.; Kuehn, G.D.; Wall, M.M.; Bruton, B.D. and Wann,
V. (1997): Characterization of chile pepper fruit
peroxidases during ripening. Plant Physiol. Biochem.,
35(4): 273-280.
Bosland, P.W. (1992): Chiles: A Diverse Crop. HortTechnology,
2(1): 7-10.
Brunt, A.A. and Kenten, R.H. (1971): Pepper veinal mottle virus
- a new member of the potato virus Y group from peppers
(Capsicum annuum L. and C. frutescens L.) in Ghana.
Ann. Appl. Biol. 69:235-243.
Brunt, A.A., and Kenten, R.H. (1972): Pepper veinal mottle
virus. CMI/AAB Descriptions of Plant Viruses No.104.
Commonwealth Mycological Institute. Kew, England. 4 p.
Chandrasrikul, A. and Patrakosol, P. (1986): Viruses disease of
horticultural crops in Thailand. Taipei, Taiwan; Food and
Fertilizer Technology Center for Asian and Pacific
Region, (1986):7-11. [c.f. Rev. Plant Path., 66(7):2752].
Page 126
121
Cheesin, M.; DeBorde, D. and Zipf, A.E. (1995): Antivarl
proteins in higher plants. CRC Press Inc.,
Clark, M.F. and Adams, A.N. (1977): Characteristics of the
microplate method of enzyme-linked immunosorbent
assay for the detection of plant viruses. J. of Gen.
Virology, 34, 475-483.
Clarke, G.M. and Kempson, R.E. (1997): Introduction to the
design and analysis of experiments. Arnold, a Member of
the Holder Headline Group, 1st Edt., London, UK.
Contreras-Padilla, M. and Yahia, E.M. (1998): Changes in
capsaicinoids during development, maturation, and
senescence of chile peppers and relation with peroxidase
activity. J. of Agric. and Food Chemistry, 46(6): 2075-
2079. [c.f. Hort. Abstr., 68(10): 8665].
D’Aquino, L.; Dalmay, T.; Burgyán, J.; Ragozzino, A. and
Scala, F. (1995): Host range and sequence analysis of an
isolate of potato virus Y induced veinal necrosis in pepper.
Plant Disease, 79(10):1046-1050.
Davino, M.; Areddia, R.; Polizzi, G. and Grimaldi, V. (1989):
Observations on pitting in pepper fruit in Sicily. Difesa
delle Piante, 12:65-73.[c.f. Rev. Plant Path., 70(10):6939].
Page 127
122
De Wijs, J.J. (1973): Pepper veinal mottle virus in Ivory Coast.
Neth. J. P1. Path., 79(5):189-193. [c.f. Rev. Plant Path.,
53:2018].
Debrot, E.A.; Lastra, P. and Ladera, P. (1983): Detection of a
new potyvirus attacking sweet pepper (Capsicum annuum
L.) in Venezuela. Agronomia Tropical, 30(1.6):85-96.
[c.f. Rev. Plant Path.,63:5704].
Depestre, T.; Palloix, A.; Camino, V. and Selassie, K.G. (1993):
Identification of virus isolates and of tobacco etch virus
(TEV) pathotypes infecting green pepper in Caujeri valley
(Guantanamo, Cuba): Capsicum & Eggplant Newsletter,
No. 12, 73-73. [c.f. Rev. Plant Path.,73(5):2981].
Duriat, A.S. (1996): Management of pepper viruses in Indonesia:
problems and progress. Indonesian Agric. Res. &
Development J., 18(3): 45-50. [c.f. Rev. Plant Path.,76
(10):8140].
El-Saeid, H.M. (1995): Chemical composition of sweet
and hot pepper fruits grown under plastic house
conditions. Egypt. J. Hort., 22 (1): 11-18.
El-Sanusi, O.; Shagrun, M. and Khalil, J. (1991): Isolation and
identification of potato virus Y from pepper plants in
Page 128
123
Libya. Arab Journal of Plant Protection, 9(1):47-51.
Erkan, S. (1986): Potato virus Y on pepper, in Turkey. Phytopath.
Medit., 25:149-150.
Escudero, J. (1996): Survey of viruses affecting pepper
(Capsicum annuum L.) in southern Puerto Rico, J. of
Agric. of the Univ. of Puerto Rico, 80(1/2): 77-80. [c.f.
Rev. Plant Path.,76(5):3909].
Feldman, J.M. and Gracia, O. (1977): Pepper severe mosaic
virus, a new potyvirus from pepper in Argentina.
Phytopathologische Zeitschrift 89(2): 146-160.
Feldman, J.M. and Gracia, O. (1985): Pepper severe mosaic
virus in Catamarca. IDIA, No.433/436,75-81. (in Spanish )
Fernandez-Northcote, F.N., and Fulton, W.R. (1980): Detection
and characterization of Peru tomato virus strains infecting
pepper and tomato in Peru. Phytopathology 70:315-320.
Fox, R.T.V. (1993): Principles of diagnostic techniques in plant
pathology. CAB International Publ., UK.
Fribourg, C.E. (1979): Host plant reactions, some properties, and
serology of Peru tomato virus. Phytopathology, 69(5):
441-445.
Page 129
124
Gowda, K.T.P. and Reddy, H. R. (1989): Aphid-transmitted
viruses infecting chilli. Current Research-University of
agricultural Sciences, 18(5):71-72. [c.f. Rev. Plant Path.,
69(4):1968].
Gracia, O.; Iglesias, V.A.; Garcia Lampasona, S.C. and J.M.
Feldman (1990): Distribucion e incidencia de algunos
virus de pimiento en Argentina. Revista de Investigaciones
Agropecuarias (RIA), XXII (1): 226 – 233. (in Spanish,
English Abstr.)
Green, S.K. and Kim, J.S. (1991): Characteristics and control of
viruses infecting peppers: a literature review. Asian
Vegetable Research and Development Center, AVRDC
Publication, No. 91-339, Technical Bulletin, No. 18, 60
pp.
Greenleaf, W.H. (1986): Pepper breeding. p.67-134 In: Breeding
Vegetable Crops. M. J. Bassett, ed. AVI Publishing Co.
Inc., Westport, Connecticut. 584 p.
Güldür, M.E.; Özaslan, M.; Baloğlu, S. and Yilmaz, M.A.
(1994): Pepper mild mottle virus in pepper in Türkiye. 9th
Congress of the Mediterranean Phytopathological Union-
Kusadasi-Aydin-Türkiye, 1994. pp. 465-467.
Hill, S.A. (1984): Methods in Plant Virology. 1st Edt., Alden Press,
Page 130
125
Oxford, Great Britain, 167pp.
Ho, S.C.; Faan, H.C.; Gao, C.W. and Lou, S.H. (1982):
Identification of the causal viruses of the mosaic disease
of pepper in the suburb of Guangzhou. J. of the South
China Agric., College, 3(3): 73-86. [c.f. Rev. Plant
Path.,63: 4202].
Hollings, M. and Brunt, A.A. (1981a): “Potyviruses”. In
Handbook of Plant Viruses infections and comparative
diagnosis. ed. E. Kurstack, Amsterdam, Elsevier/North-
Holland, Biomedical Press, p.731-807.
Hundal, J.S.; Khurana, D.S. and Kaur, S. (1995): Punjab Surkh
– a new high yielding variety of chilli. J. of Res., Punjab
Agric. Univ., 32(2): 240. [c.f. Hort. Abstr., 68(1): 439].
Ishikawa, K.; Janos, T.; Sakamoto, S. and Nunomura, O.
(1998): The contents of capsaicinoids and their phenolic
intermediates in the various tissues of the plants of
Capsicum annuum L. Capsicum & Eggplant Newsletter,
No. 17, 22-25. [c.f. Hort. Abstr., 68(11): 9613].
Ishikawa, K.; Nunomura, O.; Nakamura, H.; Matsufuji, H. and
Takeda, M. (1997): High ascorbic acid contents in the
fruits of a deep-green cultivar of Capsicum annuum
throughout the fruit development. Capsicum & Eggplant
Page 131
126
Newsletter, No. 16: 52-55. [c.f. Hort. Abstr., 68(6): 5044].
Khatri, H.L. and Sekhon, L.S. (1974): Studies on a virus causing
mosaic disease of chilli. Indian J. of Mycology and Plant
Pathology, 4(2):121-125. [c.f. Rev. Plant Path., 55(9):
4412].
Kitis, M and Aktas, M (1997): The effect of manganese
fertilization and nitrogen form on the content of vitamin C
in pepper grown by NFT. Turkish J. of Agric. and
Forestry, 21(1): 23-28. [c.f. FSTA, 29(5): 155].
Kobata, K., Sutoh, K.; Todo, T.; Yazawa, S.; Iwai, K. and
Watanabe, T. (1999): Nordihydrocapsiate, a new
capsinoid from the fruits of a nonpungent pepper,
Capsicum annuum. J. of Natural Products, 62(2): 335-336.
Kobata, K., Todo, T.; Yazawa, S.; Iwai, K. and Watanabe, T.
(1998): Novel capsaicinoid-like substances, capsiate and
dihydrocapsiate, from the fruit of a nonpungent cultivar,
CH-19 Sweet, of pepper (Capsicum annuum L.): J. of
Agric. and Food Chemistry, 46(5): 1695-1696.
Koenig, R. (1988): Serology and immunochemistry. In: Milne,
R.G. (ed.), The Plant Viruses: The Filamentous Plant
Viruses, Volume 4. Plenum Press, New York, pp. 111-
158.
Page 132
127
Koenning, S. R., and McClure, M. A. (1981): Interaction of two
potyviruses and Meloidogyne incognita in chili pepper.
Phytopathology, 71:404-408.
Ladera, P.; Lastra, R. and Debrot, E.A. (1982): Purification and
partial Characterization of potyvirus infecting peppers in
Venezuela. Phytopathol, Z. 104(2):97-103. [c.f. Rev. Plant
Path., 62(1):520].
Laird, E.F.; Desjardins, P.R. and Dickson, R.C. (1964):
Tobacco etch virus and potato virus Y from pepper in
southern California. Plant Disease Reporter, 48(10):772-
776.
Liu, J.; Yang, Y. and Zou, X. (1992): Effects of the content of
dry matter, vitamin C, and capsaicin on the resistance to
TMV, CMV and anthracnose in Capsicum annuum.
HortScience, 27(6): 628-629.
Lopez-Cardet, Y. and Blanco, N. (1972): Green vein-banding of
pepper. Revista de Agric., Cuba, 5(2):30-36. [c.f. Rev.
Plant Path.,54:1083].
Makkouk, K.M., and Gumpf, D.J. (1976): Characterization of
potato virus Y strains isolated from pepper.
Phytopathology, 66:576-581.
Page 133
128
Marte, M., and Wetter, C. (1986): Occurrence of pepper mild
mottle virus in pepper cultivars from Italy and Spain. J.
Plant Dis. and Protection 93(1):3743.
Martelli, G. P., and Quacquarelli, A. (1983): The present status
of tomato and pepper viruses. Acta Hort. 127:39-64.
Matthews, R.E.F. (1993): Diagnosis of Plant Virus Diseases.
CRC Press, pp.406
Milbrath, G.M. and Cook, A.A. (1971): Virus diseases of pepper
(Capsicum sp.) in Hawaii. Plant Dis. Reptr., 55:783-785.
Mills, P.R., and Abdul-Magid, A.G.M. (1987): Infection of
Capsicum frutescens with potato virus Y and tobacco etch
virus in the Sudan. Plant Disease, 71:557. (Abstr.)
Moghal, S.M. and Francki, R.I.P. (1976): Towards a system for
the identification and classification of potyviruses. II.
Virus particle length, symptomatology and cytopathology
of six distinct viruses. Virology, 112, 210-216.
Mowat, W.P.; Dawson, S. and Duncan, G.H. (1989): Production
of antiserum to non-structural potyviral protein and its use
to detect narcissus yellow stripe and other potyviruses. J.
of Virological Methods, 25:199-210.
Page 134
129
Nagaraju, N.; Reddy, H.R. and Ravi, K.S. (1997): Effect of
exogenously applied plant products on pepper vein
banding virus transmission, multiplication and symptom
production in bell pepper (Capsicum annuum L.): Indian J.
of Virology, 13(2): 161-163.
Nelson, M.R. and Wheeler, R.E. (1978): Biological and
serological characterization and separation of potyviruses
that infect peppers. Phytopathology, 68:979-984.
Ong, C.A.; Varghese, O. and Poh, T.W. (1980): The effect of
chilli veinal mottle virus on yield of chilli (Capsicum
annuum L.): Malaysian Agriculture Research and
Development Institute (MARDI) Res. Bull. 8(1):74-79.
Padgett, G.B.; Nutter, F.W.Jr.; Kuhn, C.W. and All, J.N.
(1990): Quantification of disease resistance that reduces
the rate of tobacco etch virus epidemics in bell pepper.
Phytopathology, 80(5):451-455.
Palloix, A.; Abak, K.; Gognalons, P.Daubeze, A.M., Güldür,
M.E.; Memouchi, G. and Gebre-Selassie, K. (1994): Virus
diseases infecting pepper crops in Türkiye. 9th Congress of
the Mediterranean Phytopathological Union-Kusadasi-
Aydin-Türkiye, 1994. pp. 469-472.
Page 135
130
Parrish, M. (1996): Liquid chromotographic method for
determining capsaicinoids in Capsicums and their
extractives: Collaborative study. J. of AOAC
International, 79(3): 738-745.
Pennazio, S. and Roggero, P. (1996): Biological compounds
inactivating the infectivity of plant viruses in vitro. Difesa
delle Piante, 19(1): 1-13. [c.f. Rev. Path., 76(6): 4417].
Pennazio, S.; Roggero P. and Conti, M. (1996): Acido ascorbico
ed infezione virale nelle piante superiori. Informatore
Fitopatologico, 12:3-6. (in Italian, English Abstract):
Polson, A. (1993): Virus separation and purification methods.
Marcel Dekker Inc. ISBN 0-8247-9149-5, pp.291.
Pomar, F.; Bernal, M.A.; Jose-Diaz and Merino, F. (1997):
Purification, characterization and kinetic properties of
pepper fruit acidic peroxidase. Phytochemistry, 46(8):
1313-1317.
Puga, R.F. and Pérez, G.F. (1986): Acquisition from and
transmission to Capsicum of tobacco etch virus by Myzus
persicae. Ciencias de la Agricultura, No.27: 55-60. [c.f.
Rev. Plant Path., 67(4): 2190].
Purcifull, D.E. and Hiebert, F. (1982): Tobacco etch virus.
Page 136
131
CMI/AAB Descriptions of Plant Viruses No.258.
Commonwealth Mycological Institute, Kew, England. 4
pp.
Purcifull, D.E.; Zitter, T.A. and Hiebert, E. (1975):
Morphology, host range, and serological relationships of
pepper mottle virus. Phytopathology, 65: 559-562.
Raccah, B.; Gal-On, A. and Eastop, V. F. (1985): The role of
flying aphid vectors in the transmission of cucumber
mosaic virus and potato virus Y to peppers in Israel. Ann.
Appl. Biol. 106:451-460.
Ravi, K.S.; Joseph, J.; Nagaraju, N.; Krishna Prasad, S.;
Reddy, H.R. and Savithri, H.S. (1997): Characterization
of a pepper vein banding virus from chili pepper in India.
Plant Disease, 81(6): 673-676.
Richter, J.; Rabenstein, F.; Proll, E. and Vetten, H.J. (1995):
Use of cross-reactive antibodies to detect members of the
Potyviridae. J. Phytopathology, 143, 459-464
Salgado-Garciglia, R. and Ochoa-Alejo, N. (1990): Increased
capsaicin content in PFP-resistant cells of chili pepper
(Capsicum annuum L.): Plant Cell Reports, 8: 617-620.
Sharma, O.P.; Sharma, P.P. and Chowfla, S.C. (1989):
Page 137
132
Inheritance of resistance to potato virus Y in garden
pepper (Capsicum annuum L): Euphytica, 42(1-2):31-33.
[c.f. Rev. Plant Path., 68(10): 4612].
Sharma, P.N.; Chowfla, S.C.; Garg, I.D. and Paul Khurana,
S.M. (1993): Properties of the viruses associated with
mosaic disease complex of bell pepper. Indian Phytopath.,
46(4):347-353.
Sherwood, J.L.; Reddick, B.B. and Conway, K.E. (1988):
Reactions of Bahamian hot chile to single and double
infections with tobacco mosaic virus and potato virus Y.
Plant Disease, 72(1):14-16.
Shukla, D.D.; Ford, R.E.; Tosic, M. and Ward, C.W. (1989):
Possible members of the potyvirus group transmitted by
mites or whiteflies share epitopes with aphid transmitted
definitive members of the group. Archives of Virology,
105, 143-151.
Shukla, D.D.; Ward, C.W. and Brunt, A.A. (1994): The
Potyviridae, CAB International, Wallingford, Oxon OX10
8DE, UK, 516 pp.
Simonne, A.H.; Simonne, E.H.; Eitenmiller, R.R.; Mills, H.A.
and Green, N.R. (1997): Ascorbic acid and provitamin A
contents in unusually colored bell peppers (Capsicum
Page 138
133
annuum L.): J. of Food Composition and Analysis, 10 (4):
299-311. [c.f. Hort. Abstr., 68(9): 7789].
Singh, R.; Mall, T.P. and Singh, R.R. (1985): Inhibitory activity
of leaf extracts on the infectivity of arhar (pigeonpea)
mosaic virus. International Pigeonpea Newsletter, 4: 38-
40.
Siriwong, P.; Kittipakorn, K. and Ikegami, M. (1995):
Characterization of chilli vein-banding mottle virus
isolated from pepper in Thailand. Plant Pathology, 44:
718-727.
Soh, A.C.; Yap, J.C. and Graham, K.M. (1977): Inheritance of
resistance to pepper veinal mottle virus in chilli.
Phytopathology, 67(1): 115-117.
Soohyun, K.; Younghwan, K.; Zeewon, L.; Byungdong, K. and
Kwonsoo, H. (1997): Analysis of chemical constituents in
fruits of red pepper (Capsicum annuum L. cv. Bugang): J.
of the Korean Soc. for Hort. Sci., 38(4): 384-390. [c.f.
Hort. Abstr., 68(1):447].
Stein, U. and Klingaui, F. (1990): Insecticidal effect of plant
extracts from tropical and subtropical species. Traditional
methods are good as long as they are effective. J. Appl.
Ent., 112:160-166.
Page 139
134
Šubíková, V.; Slováková, L. and Farkaš, V. (1994): Inhibition of
tobacco necrosis virus infection by xyloglucan fragments.
Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz,
101(2): 128-131. [c. f. Rev. Plant Path.,74(2):981].
Sudhakar Johnson; Ravishankar, G.A. and Dhanaraj, S.
(1995): Pungency threshold of capsaicin produced by in
vitro culture of placental tissues of Capsicum frutescenes
Mill. Food Biotechnology, 9(3):167-173.
Suriachandraselvan, N. and Narayanasamy, P. (1987):
Inhibition of potato virus Y infection on chilli by plant
extracts. Madras Agric. J., 74(3): 154-156. [c. f. Rev. Plant
Path.,68(6):2429].
Tewari, V.P. (1990): Development of high capsaicin chillies
(Capsicum annuum L.) and their implications for the
manufacture of export products. J. of Plantation Crops,
18(1): 1-13.
Tewari, V.P. (1991a): A multipurpose perennial chilli “Pusa
Sadabahar”. Indian Hort., 35(4): 29-31. [c. f Plant
Breeding Abst.,62:1642].
Tewari, V.P. (1991b): Chilli „Pusa Jwala‟: chiefly for industry and
export. Indian Horticulture, 35(4): 24-27.
Page 140
135
Thakur, P.D.; Chowfia, S.C. and Khurana, S.M.P. (1988):
Natural occurrence of an atypical strain of potato virus Y
on bell pepper in Himachal Pradesh. Indain J. of
Virology,4(1-2): 91-96. [c. f. Rev. Plant Path.,69:4558].
Thakur, P.D.; Chowfla, S.C.; Bhardwaj, S.S. and Dohroo, N.P.
(1986): Changes in phenolic and sugar contents in leaves
of mosaic susceptible and resistant cultivars of pepper
(Capsicum annuum L.): Himachal J. of Agric. Res., 12 (1):
60 [c.f. Rev. Pl. Path., 68(6): 2430].
Van Regenmortel, M.H.V. (1982): Serology and
immunochemistry of plant viruses. Academic Press, Imc.,
New York, pp.302.
Walkey, D.G.A. (1991): Applied Plant Virology. Chapman and
Hall, 2nd
Edt. ISBN 0412357402, 338pp.
Zitter, T.A. and Tsai, J.H. (1981): Viruses infecting tomato in
southern Florida. Plant Disease, 65(10): 787-791.
Zitter, T.A. (1975): Transmission of pepper mottle virus from
susceptible and resistant pepper cultivars. Phytopathology,
65:110-114.
Page 141
136
Zitter, T.A.; Florini, D. and Provvidenti, R. (1984): Virus
diseases of pepper. Vegetable Crops Fact Sheet. pp.730,
Cornell University, USA
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