detection of the causal agent of leaf mosaic of jute
DESCRIPTION
A Master thesis submitted to the Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh By K. M. Golam Dastogeer2012TRANSCRIPT
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DETECTION OF THE CAUSAL AGENT OF LEAF MOSAIC OF JUTE
A Thesis By
K. M. GOLAM DASTOGEER
Examination Roll No. 10 Ag. P. Path. JD 05M Registration No. 32160
Session: 2005-06 Semester: July- December, 2011
MASTER OF SCIENCE (MS) IN
PLANT PATHOLOGY
DEPARTMENT OF PLANT PATHOLOGY BANGLADESH AGRICULTURAL UNIVERSITY
MYMENSINGH
NOVEMBER 2011
2
DETECTION OF THE CAUSAL AGENT OF LEAF MOSAIC OF JUTE
A Thesis By
Examination Roll No. 10 Ag. P. Path. JD 05M Registration No. 32160
Session: 2005-06 Semester: July- December, 2011
Submitted to the Department of Plant Pathology Bangladesh Agricultural University, Mymensingh
In partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE (MS) IN
PLANT PATHOLOGY
DEPARTMENT OF PLANT PATHOLOGY BANGLADESH AGRICULTURAL UNIVERSITY
MYMENSINGH
NOVEMBER 2011
3
DETECTION OF THE CAUSAL AGENT OF LEAF MOSAIC OF JUTE
A Thesis By
Examination Roll No. 10 Ag. P. Path. JD 05M
Registration No. 32160 Session: 2005-06
Semester: July- December, 2011
Approved as to style and content by
.........................................................................
Prof. Dr. M. Ashrafuzzaman Supervisor
........................................................................
Prof. Dr. Md. Ayub Ali Co-supervisor
.......................................................................... Dr. Md. Rashidul Islam
Chairman, Examination Committee and
Head, Department of Plant Pathology Bangladesh Agricultural University
Mymensingh
NOVEMBER 2011
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ACKNOWLEDGEMENTS
At first the author likes to express his extreme and humble gratitude and endless praises to Almighty Allah, the omnipresent, omnipotent and omniscient whose blessing has enabled the author in successful planning, materialization and fulfillment of the research work.
The author deems it a pride to express the deepest sense of gratitude, sincere appreciation, immense indebtedness and best regards to his reverent research supervisor, Professor M. Ashrafuzzaman, Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh for his scholastic and dynamic guidance, constant inspiration, cordial assistance, affectionate feeling, sympathetic supervision, valuable suggestions and comments and continuous active help during the entire tenure of the research work and preparation of the manuscript.
The author is ever grateful and immensely indebted to his honorable teacher and research co-supervisor Professor Dr. Md. Ayub Ali, Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh for his constructive suggestions, steady encouragement and incisive criticism during the study period and in the preparation of this thesis.
The author would like to humbly express gratitude and high regards to his respected teacher Professor Dr. Md. Rashidul Islam, Head of the Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh for his all out support and encouragement during the reach work.
The author is deeply indebted and grateful to his all respected teachers of the Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh for their valuable instructions and kind help during the entire period of the study.
The author will always remember the sympathy and sincere co-operation that he has received during the research work from Zinnat Ara, Scientific Officer, Department of Plant Pathology, Bangladesh Agricultural Research Institute, Gazipur.
The author desires to express his cordial thanks to all the staff of Seed Pathology Centre, Bangladesh Agricultural University, Mymensingh and Plant Pathology Laboratory, Bangladesh Agricultural Research Institute (BARI), Gazipur for their assistance and co-operation during the period of research work.
The author express heartfelt gratitude to his beloved parents, sister and brothers for their blessings, logistic supports, best wishes and sacrifices during his entire period of student life.
The author happily and emotionally remembers his grandparents and relatives for their inspiration, encouragement and blessing for higher study.
The author is pleased to extend his gratefulness to Shawpon, Mokshed. Muzammel, Mamata, Samsia, Monju, Nuru Bhai, Ismail and all other friends and well wishers for their helpful co-operation and good wishes.
In fine, the author would like to thank the Bangladesh Agricultural University Research System (BAURES), Bangladesh Agricultural University, Mymensingh for the financial support which enabled the author to proceed with the research work this far. The Author The Author The Author The Author
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ABSTRACT
Several experiments were conducted in the glass house, net house and in the laboratory
to check the transmission pathways and to identify the causal agent of leaf mosaic of
jute. Seed to plant transmission was studied in aluminium tray and in cassette holders.
Again seed to plant to seed transmission study was conducted in successive two seasons.
In the second year seeds collected from the infected plants only were sown. In graft
transmission study five grafting techniques (viz. peg, veneer, gooti, root grafting and T-
budding) were employed. Vector transmission was studied under insect proof cage.
Again, infected leaves were subjected to study under light microscope to observe the
inclusion body. In molecular detection polymerase chain reaction (PCR) was employed
using begomovirus specific primes in nucleic acid preparation from mosaic infected jute
leaf to confirm the causal agent. It was observed that the cultivar D-154 showed the
highest percentage of seed to plant transmission of the causal agent in both aluminium
tray and cassette holders. In seed to plant to seed transmission it was observed that seeds
obtained from the infected plants gave higher percentage of infected plants in the
succeeding year than those obtained from healthy ones. In the graft transmission study it
was noted that the causal agent was readily transmitted through all grafting techniques
attempted. Graft transmission was more successful when hosts of same cultivar were
used as both scion and stock. In the vector transmission study results obtained indicated
that the causal agent was transmitted persistently by whitefly (Bemisia tabaci). The
results showed that at least 3 and 1 whiteflies were required to transmit the causal agent
when both AFP and IFP were 24hr and 48hr respectively. The minimum AFP and IFP
were 30 minutes and 15 minutes respectively. The persistence of causal agent inside the
vector was at best 10 days. Under light microscope large, blue-violet, prominent nuclear
inclusion bodies were readily detected from infected leaf tissues which are indicative of
geminivirus infection. This is probably the first study of this kind in mosaic infected jute
leaf. In molecular detection the primers amplified 1.2 kb of the DNA fragment. The
results obtained in present study conclude that the causal agent of leaf mosaic of jute is
transmitted through seed, grafts and vector whitefly and microscopic and molecular
study confirm that begomoviruses are responsible for the leaf mosaic in jute.
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CONTENTS
CHAPTER TITLE PAGE
ACKNOWLEDGEMENTS iv
ABSTRACT v
CONTENTS vi
LIST OF FIGURES x
LIST OF PLATES xi
LIST OF TABLES xiii
CHAPTER 1 INTRODUCTION 1
CHAPTER 2 REVIEW OF LITERATURE 5
2.1 Symptoms 5
2.2 The causal agent and mode of inheritance 6
2.3 Transmission of the causal agent 7
2.4 Incidence of leaf mosaic of jute and its effect on yield 9
2.5 Observation of inclusion body by light microscopy 11
2.6 Molecular detection 12
CHAPTER 3 MATERIALS AND METHODS 15
3.1 Place and time 15
3.2 Collection of seeds 15
3.3 Study of leaf mosaic of jute on the growing plants 15
3.4 Seed to seedling transmission of the causal agent of
leaf mosaic of jute by cassette holder method 16
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CHAPTER TITLE PAGE
3.5 Seed to plant to seed transmission of the causal agent of leaf mosaic of jute
17
3.6 Graft transmission of the causal agent of leaf
mosaic of jute 17
3.6.1 Peg grafting 18
3.6.2 Veneer grafting 18
3.6.3 Gooti (approach) grafting 19
3.6.4 T- budding 19
3.6.5 Root grafting 19
3.7 Vector Transmission of the causal agent of leaf
mosaic of jute 20
3.7.1 Vector population 20
3.7.2 Acquisition Feeding Period (AFP) 20
3.7.3 Inoculation Feeding Period (IFP) 21
3.7.4 Persistence of causal agent in the vector 21
3.8 Study of inclusion bodies of the causal agent of
leaf mosaic of jute under light microscope
24
3.9 Detection of the causal agent of leaf mosaic of jute
by molecular techniques 24
3.9.1 Collection of leaf samples 24
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CHAPTER TITLE PAGE
3.9.2 Preparation of DNA samples 25
3.9.3 Protocol for preparation of 1% agarose gel 25
3.9.4 Amplification of DNA by PCR 26
3.9.5 Selection and design of primer 29
3.9.6 Electrophoresis, gel staining and documentation 32
3.9.7 Observation of DNA Bands 32
CHAPTER 4 RESULTS 33
4.1 Study of leaf mosaic of jute on growing plants 33
4.2 Seed to seedling transmission of the causal agent of
leaf mosaic of jute by cassette holder method
33
4.3 Seed to plant to seed transmission of the causal agent of leaf mosaic of jute
39
4.4 Graft transmission of the causal agent of leaf mosaic of jute 42
4.4.1 Peg grafting 42
4.4.2 Veneer grafting 42 4.4.3 Gooti or Approach grafting 43
4.4.4 T-budding 43
4.4.5 Root grafting 43
4.5 Vector transmission of the causal agent of leaf mosaic of jute
55
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CHAPTER TITLE PAGE
4.5.1 Vector population
55
4.5.2 Acquisition Feeding Period (AFP)
55
4.5.3 Inoculation Feeding Period (IFP)
55
4.5.4 Persistence of causal agent in the vector
56
4.6 Study of inclusion body of the causal agent of
leaf mosaic of jute under light microscope
64
4.7 Detection of the causal agent of leaf mosaic of jute
by molecular techniques
68
CHAPTER 5 DISCUSSION 70
CHAPTER 6 SUMMARY AND CONCLUSION 75
REFERENCES 77
APPENDICES 86
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LIST OF FIGURES
FIGURE TITLE PAGE
1 Diagrammatic representation of the PCR cycle 28
2 Status of percentage of germination and seedling with chlorotic spots as grown in aluminum tray 37
3 Status of percentage of germination and seedling with chlorotic spots as grown in cassette holder 38
4 Status of mosaic infection at different days in two cultivars of jute 41
5 Relationship between number of whiteflies and transmission of the jute mosaic causal agent at 24 hours of AFP and IFP
61
6 Relationship between number of whiteflies and transmission of the jute mosaic causal agent at 48 hours of AFP and IFP
61
7 Relationship between Aquision Feeding Period (AFP) and transmission of jute leaf mosaic causal agent
62
8 Relationship between Inoculation Feeding Period (IFP) and transmission of jute leaf mosaic causal agent
62
9 Relationship between days after acqusition (persistence) and plants infected 63
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LIST OF PLATES
PLATE TITLE PAGE
1 Whitefly 22
2 Inoculation of healthy jute seedling by vector (whitefly) 22
3 Management of inoculated plant in insect proof net-cage 23
4 Seedling with yellow dot spot on cotyledon (Grown on sand in
aluminium tray)
35
5 Seedling with yellow dot spot on cotyledon growing cassette
holders
35
6 Symptoms of leaf mosaic of jute at seedling stage
(20 days old) 36
7 Symptoms of leaf mosaic of jute on 60 days old plant 36
8 Peg grafting between D-154 × D-154 at the initial stage 47
9 Peg grafting between D-154 × D-154 after successful
transmission 48
10 Veneer grafting between D-154 × D-154 at initial stage 49
11 Veneer grafting between D-154 × D-154 after successful
transmission 50
12 Approach grafting between CVL-1 ×CVL-1 at initial stage 51
13 Approach grafting between CVL-1 ×CVL-1 after successful
transmission 52
14 T-budding between CVL-1 ×CVL-1 at initial stage 53
15 T-budding between CVL-1×CVL-1 after successful
transmission 54
PLATE TITLE PAGE
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16 Distribution of inclusion bodies in the nucleus (100X
magnification) 65
17 Nuclear inclusion body in the mosaic infected leaf of jute (cv.
CVE-3) (1000X magnification) 65
18 Nuclear inclusion body in the mosaic infected leaf of jute (cv.
D-154) (1000X magnification) 66
19 Nuclear inclusion body in the mosaic infected leaf of jute (cv.
CVL-1) (1000X magnification 66
20 Healthy leaf sample (cv. CVL-1) stained with azure-A showing
no inclusion body (1000X magnification) 67
21 Healthy leaf sample (cv. CVE-3) stained with azure-A showing
no inclusion body (1000X magnification)
67
22 Agarose gel electrophoresis illustrating begomovirus-specific
PCR products obtained using the primers PAL1v 1978 and
PAR1c 496
69
LIST OF TABLES
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TABLE TITLE PAGE
1 Sequences of primers used in the study 29
2 Percentage seedling with mosaic symptom with in two tests 34
3 Seed to plant transmission of the causal agent of leaf mosaic of jute 40
4 transmission of jute leaf mosaic in seeds collected from infected plants (1st season) grown in 2nd season 40
5 Transmission efficiency of jute leaf mosaic causal agent by peg grafting 44
6 Transmission efficiency of jute leaf mosaic causal agent by veneer grafting 44
7 Transmission efficiency of jute leaf mosaic causal agent by approach grafting 45
8 Transmission efficiency of jute leaf mosaic causal agent by T- budding 45
9 Transmission efficiency of jute leaf mosaic causal agent by root grafting 46
10 Effect of number of viruliferous insects on the transmission of causal agent of leaf mosaic jute 57
11 Effect of AFP on transmission of leaf mosaic causal agent 58
12 Effect of IFP on transmission of the causal agent of jute mosaic 59
13 Persistence (days) of the causal agent of leaf mosaic of jute in the vector insect 60
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INTRODUCTION
Jute (Corchorus capsularis L. and Corchorus olitorius L.) is an important fibre
crop of Bangladesh. The word jute is coined from the word “jhuta or jota”, an
Orrisan (Indian) word. The use of “Jutta potta” cloth was mentioned in the Bible
(Banglapedia, 2006). Jute is being grown in Bangladesh for more than one
hundred years. It is biodegradable and does not create any adverse effect on the
ecosystem. It maintains an excellent harmony with the ecosystem by balancing
the nutritional status of soil. Bangladeshi jute produces the good quality fibre due
to favourable climate and soil condition. Jute is one of the mainstays of
Bangladesh economy. It accounts for about 6 per cent of the foreign currency
earning from export. Among the jute growing countries of the world, Bangladesh
ranks second in respect of production (Islam and Rahman, 2008).In 2010-2011,
8.40 million bales of jutes were produced in the country from 1.75 million acres
of land. Bangladesh earned foreign currency worth about 5378.28 crore taka from
exporting 0.30 million tones of raw jute and 0.79 million tones of jute goods in
the year 2009-2010 (BBS, 2011). Still today Bangladesh is the largest supplier of
jute and jute goods in the international markets. Bangladesh meets nearly 95% of
world raw jute demand and about 60% of jute goods demand. About 35 million
people (25% of the total population) of Bangladesh are directly or indirectly
dependent on jute cultivation and manufacturing, trading of jute and jute goods
(Rahman, 2010).
Jute fibres have versatile uses for making hessians, blankets, sacks, gunny bags,
carpets, furnishing fabrics, mats, ropes and packaging materials. It is also used for
the production of different types of domestic products. Besides the use of jute
fibres, jute sticks and root stamps are traditionally being used as house
construction materials and fuels in the rural areas. Jute sticks are also being
used in producing compressed sheets of hardboard after processing in the mills
which replaces building materials where wood is needed. These are water
resistance and fire proof with longevity as good as timber. The young green
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leaves of jute contain minerals and proteins, which are edible and are popular as
leafy vegetable. Now a days attempt is being made to popularize the jute plants
for making pulp in paper industries.
Jute plants suffer from different diseases among them leaf mosaic has been
reported to be the most damaging one. This disease was first reported by Finlow
in 1917. The leaf mosaic of jute has wide spread occurrence in the major jute
growing countries of the world, namely Bangladesh, Burma, India (Ghosh and
Basak, 1951), Nepal and Pakistan (Dempsy, 1975). Leaf mosaic of jute has been
considered to be one of the most important limiting factors of jute cultivation in
India and some other jute growing countries (Harender et al., 1993) The disease
is characterized by symptoms such as small yellow flakes on the lamina during
the initial infection stage which gradually increases in size to form green and
chlorotic intermingled patches producing a yellow mosaic appearance. The
incidence of the disease has been found to be around 50% on some of the leading
C. capsularis cultivars. It was also observed from the survey that infection
reduces plant height to the extent of 20% and thus adversely affects the yield of
the fiber (Ghosh et al., 2008).
The disease has been reported to be transmitted through grafts, seed and pollen
(Ghosh and Basak, 1951; Saha, 2001). Whitefly transmission of the disease has
also been reported (Verma et al. (1966); Ahmed (1978) and Ahmed et al. (1980).
Severe infestation of whitefly may result in defoliation of jute and it causes
reduction of yield. The secretion of wax and honeydew of the insects significantly
reduces the photosynthetic area of the plant (Alam, 1998).
Plants raised from seed collected from symptom bearing plants have been
reported to be showing up to 81% seed transmission (Ghosh and Basak, 1951).
Seed transmission of the causal agent of jute leaf mosaic disease of C. capsularis
cv. D-154 was studied. The F1 generation of the reciprocal crosses between
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healthy and infected plants of D-154 produced 22 to 23% hybrid progenies with
typical leaf mosaic symptom against up to 70% progenies with symptoms in
reciprocal crosses when the pollens from infected plants pollinated 70%
progenies with mosaic symptom. Hybridization between infected female and
infected male showed 94% infected progenies (Sultana et al., 1995). The
pathogen was successfully transmitted by vectors in plants of C. capsularies
(Ghosh and Basak, 1951). The causal agent was not transmitted mechanically to
the plants of C. capsularies (Lange, 1980; Biswas, 1982; Saha, 2001).
There is a lot of mystification about the precise identity of the causal agent of jute
leaf mosaic or chlorosis of jute. Many, however, believe that the causal agent is a
virus (Ghosh and Basak, 1951, 1961; Mitra et al., 1984, Ghosh et al., 2008). It
has also been anticipated that the causal agent could be mycoplasma or rickettsia
(Rabindran et al., 1988; Biswas, 1982; Biswas et al., 1992). Another thought is
that it could be a genetic disorder in the capsularis cultivars of the jute.
Zaman and Albrechtsen (1999) endeavored to identify the pathogen and tried to
extract virus particles through partial purification and ultracentrifugation. The
attempt was not successful. They also attempted the procedure described by Mitra
et al., (1984) but could not locate virus particles. Double stranded RNA
extraction followed by electrophoresis protocols also failed to produce doubtless
indication that the causal agent is a RNA virus. Ghosh et al., (2008) reported that
the yellow mosaic of jute is associated with a bipartite begomovirus and revealed
its molecular evidence by using begomovirus-specific degenerate primers and
suggested further study to confirm the association of virus with yellow mosaic
disease of jute.
It is evident from the above mentioned literature that there is no systematic
research on the causal agent of the leaf mosaic of jute. Most of the studies were
done in abroad. Confirmation of the causal agent of leaf mosaic of jute is now a
17
national demand to formulate control measures against the disease. Therefore the
study was undertaken with the following objectives:
1. To identify the causal agent of leaf mosaic of jute by light microscopic
study and molecular techniques.
2. To elucidate the mode of transmission of causal agent of leaf mosaic of
jute.
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REVIEW OF LITERATURE
Leaf mosaic of jute is considered as one of the most serious diseases of jute that
has got profound effect in reducing yield and quality of jute. For climatic reasons,
jute is cultivated mostly in Bangladesh, some parts of India and a few other
countries of Asia and Africa. As jute is not grown worldwide, whatever research
work has been done on jute and its diseases is concentrated mostly in Bangladesh
and India. Amongst the jute disease leaf mosaic has been the least studied.
However, the literatures related to the study are summarized in this chapter under
different subheadings.
2.1 Symptoms
Finlow (1917) first observed the yellow and light green patches on the leaf of jute
leading to variegated appearance and was termed as ‘Chlorosis’ to describe the
phenomenon. Since then the term ‘Chlorosis’ has been using by the researchers
dealing with this problem of jute. Afterwards, Finlow (1939) reviewing the works
on jute, referred to ‘Chlorosis’ as a ‘morphological imperfection’ and suggested
concentrated attention to study this phenomenon.
Ghosh and Basak (1951) described that the symptoms sometimes remain latent in
the early stage and are revealed afterwards. In case of severe attack, the infected
plants remained stunted and ultimately died. Those which survived till flowering
usually failed to produce pods and most of the pods, if at all formed, failed to
develop seeds. The author preferred to name the disease as “leaf mosaic”.
Lange (1980) observed that symptom might start to appear with yellow spots on
cotyledons following severe mosaic on all the leaves or on the 3rd or 4th true
leaves or later.
Rabindran et al. (1988) observed that in the infected plants, leaves were crinkled,
leathery and the top of the plant become needle like floral parts become
deformed. Internodes become shortened and the branches become proliferated.
19
Ghosh et al. (2008) stated that the disease was characterized by symptoms such as
small yellow flakes on the lamina during the initial infection stage which
gradually increase in size to form green and chlorotic intermingled patches
producing a yellow mosaic appearance
2.2 The causal agent and mode of inheritance
Banerjee (1924) said that chlorosis in jute was possibly due to virus and not a genetic
disorder. Crossing between chlorotic and non chlorotic plants indicated that the
mode of inheritance was not mendelian.
Bist and Mathur (1964) opined that the occurrence of two types of leaf mosaic
indicated that they might have been caused by two strains of the same virus.
Biswas (1982) followed all conventional methods of extraction purification and
mechanical transmission to detect the causal agent of leaf yellow disease. But he
found no virus particles under the electron microscope. He suggested that the
causal agent might be something other than viral in nature. Observing the
agent's seed-borne nature and the presence of Rickettsia (RLO) in infected
flower buds and infected seeds he assumed that the organism might be the
causal agent of the disease.
Mitra et al., (1984) reported the detection of virus particles by electron
microscope from mosaic infected jute leaves following modified leaf-dip
method. The virus particles were characteristic in shape and size. The particles
were spherical in shape and measured about 23 nm in diameter.
Ghosh et al., (2008) reported that the yellow mosaic of jute is associated with a
bipartite begomovirus and revealed its molecular evidence by using begomovirus-
specific degenerate primers and suggested further study to confirm the association
of virus with yellow mosaic disease of jute.
20
2.3 Transmission of the causal agent
Bawden (1963) cited known cases of transmission of pathogenic virus through
the seed of the infected plant or by seeds of plants of different species.
Ghosh and Basak (1951) reported that the disease is graft and seed transmissible,
although they failed to induce chlorosis by sap inoculation. They noted the
sporadic occurrence of chlorosis (leaf mosaic) in the field. The population raised
from seeds of affected plants, showed as high as 81.09% incidence.
Anonymous (1953) observed some branches may have leaf mosaic symptoms but
the others may remain green and healthy. They collected seeds separately from
the chlorotic and non- chlorotic branches and sown in the following year. They
obtained higher percentage of chlorotic individuals from seeds of chlorotic branch
than from the non-chlorotic branch of the same plant. Seeds collected from the
chlorotic branch gave 13.2% while seeds of other branch gave only 1.07%
chlorotic progenies.
Graft transmission of leaf mosaic was successful in case of Corchorus capsularis
plants but unsuccessful in Corchorus olitorius (Anonymous, 1959)
Ghosh and Basak (1961) observed that the progenies of a chlorotic jute plant
almost segregated into two groups, one showing chlorotic symptoms and the
other apparently normal and green. The ratio of chlorotic to non chlorotic
progeny was never the same for any two selections. They proved that the causal
agent was seed transmissible.
Pollen and embryo sac transmission of the causal agent was made by crossing
between chlorotic and non chlorotic individuals. The presence of chlorotic plants
in the F1 from a cross between non chlorotic male parents indicated that the pollen
can carry the agent. They claimed to have found additional evidences to indicate
that the disease was due to a virus that the embryo sac can carry the virus (Ghosh
and Basak, 1961).
21
Verma et al. (1966) recorded the whitefly (Bemicia tabci) transmission of the
disease from affected plants to healthy plants. They were successful to transmit
the causal agent of leaf mosaic of jute from infected plants to healthy plants by
insect vector, whitefly within insect proof cage. Mosaic symptom began to appear
on leaves after 23 days of release of viruliferous insects.
Surveys in the Punjab revealed that C. capsularis was seriously affected by a
yellow mosaic disease. The disease exhibited viral characteristics. It was
transmitted by grafting and whitefly (Bemisia tabaci), but not by mechanical
means, dodder, soil or nematodes. There was no transovarial transmission by
whitefly (Ahmed, 1978).
Sultana et al. (1995) studied the seed transmission of the causal agent of leaf mosaic
of jute in C. capsularis (cv. D-154) by doing reciprocal crosses between healthy and
infected plants. They obtained 22 to 23% hybrid progenies with leaf mosaic
symptoms in F1 generation. Hybridization between the infected female and infected
male parents showed 94% infected progenies.
Conti (1996) reported that whitefly vectors mostly infect plants in the
Chenopodiaceae, Compositae, Cruciferae, Cucurbitaceae and Solanaceae, causing
symptoms such as chlorotic spots, yellowing or chlorosis, and thickening, brittleness
and downward curling of leaves. Transmission has been obtained by grating but not
by sap or aphid inoculation, or by contact or through seed. The whitefly vectors can
transmit with acquisition and inoculation feedings of at least 5 min but the
transmission rate increases as both feeding periods are increased.
Das et al. (2001) conducted that growing on test by seeds. The test cultivars were
grown in net house in two successive years. Seed collected from infected plants were
used in the second year. Significantly higher percentage of seed transmission was
recorded.
22
2.4 Incidence of leaf mosaic of jute and its effect on yield
Anonymous (1959) reported 18%-46% less fiber yield from leaf mosaic affected
plants than that obtained from healthy plants depending upon the percentage of
affected plants and severity of symptom.
Ahmed (1968) found the incidence of leaf mosaic affected plants in various
percentages in jute fields of different districts of Bangladesh. Incidence of 100%
affected plants was reported from Rangpur in 1966 while 59.3% was reported from
Bogra in 1963.
Sastry and Sigh (1973) stated that when the plant are infected within 20 days of
planting the loss may be up to 92% while infection at 35 and 50 days old crop
result in 74% and 20% loss respectively.
Ahmed et al. (1980) conducted an experiment in Dhaka for screening 24
varieties of C. capsularis against leaf mosaic disease. Plants of the test varieties
were raised in lines. In between two test lines, leaf mosaic affected plants were
grown from seeds collected from recurrent selection of affected plants. First
record, taken after one month of sowing, showed from 2.9% to 73.3% infected
plants in presence of whitefly population. Within 10 weeks, all the test lines had
81 % to 96% infected plants. Plant height and base diameter appeared more in
healthy plants than those of primarily and secondarily affected populations.
Azad and Wahhab (1984) surveyed the occurrence of leaf mosaic of jute in
Corchorus capsularis. They reported least percent (3.8%) of leaf mosaic in
breeder seed. Most severely affected variety was CC-45 having 79.8% leaf
mosaic plants. Occurrence of leaf mosaic was higher in all varieties raised from
foundation seeds except for variety. CC-45 had 39.6% chlorosis infection with
plant raised from foundation seeds. CVL-1 and D-154 were less affected by
leaf mosaic of jute. The variety CC-45 was severely affected.
23
Survey on the occurrence of diseases of jute in different locations was conducted
by plant pathology Division of BJRI during 1985. At Kishoreganj, leaf mosaic
disease was found in local varieties, 70-80%. Trishal and Ishoreganj area, 10-85%
leaf mosaic affected plants were observed in plants raised from local varieties
(Anonymous, 1985).
Biswas et al. (1989) reported that the infected plants raised from infected seeds
gave 16.8 to 65.9% less fibre yield. Healthy plants infected secondarily at pre-
flowering stage showed 11.4% less fibre. With the increase of percentage of
infected plants, green weight and plant height were reduced. Leaf mosaic
infected plants had lower percentage of cellulose (46.02); lignin (12.0), pectin
(1.82) and protein (1.87) indicating weaker strength of fiber.
Islam (1993) observed that the mosaic or chlorotic symptom of white jute
resulted fiber loss. The sources of seed other than BJRI, viz. BADC, local
market farmers were more infected.
Haque et al. (1998) reported the highest percent of leaf mosaic expressing plants
in variety CC-45 (26.01%) at Kishorganj with and the lowest percent at Rangpur
in variety CVL-1 (5.54%). They also found plant height, base diameter, plant
height and leaf weight was lower in leaf mosaic infected plants than those of
healthy plants.
Ghosh et al. (2008) conducted a survey on the disease within different jute
growing regions in India over the 4 years. The results of this survey indicated that
the incidence of the disease has increased from nearly 20% to above 40% in They
reported that the incidence of the disease was around50% on some of the leading
C. capsularis cultivars such as JRC-7447 and JRC-212. It was also observed from
the survey that infection reduces plant height to the extent of 20% and thus
adversely affects the yield of the fibre.
24
2.5 Observation of inclusion body by light microscopy
Inlusion bodies are typical of virus infection. Their presence in disesaed plant is
of diagonstic value as they are characteristic of virus groups and the type of
inclusion depends solely on the the virus and not on the host plant. Inclusion
bodies may contain virus particle, viral genome coded protein, modified cellular
materials or mixtures of these constituents in various proportions. Depending on
their composition, the inclusions may be crystalline, paracrystalline or
noncrystalline (amorphous). The may occur in the nucleus (nuclear inclusion) or
in the cytoplasm (cytoplasmic inclusions). Some inclusion may be seen with the
light microscope, othe can only be made visible with electron microscope
(Dijkstra and Jager,1998).
Nucler changes such as segregated nucleoli, fibrillar bodies, and virus particle
aggregates in cells of the vascular region are cytopathic effects constantly
associated with the ultracture of geminivirus infections (Esau and Magyarosy,
1979, Goodman, 1981; Kim et.al., 1987; Mathews, 1982; thongmeearkon et.al.,
1981). There is only few reported geminivirus light microscopy of geminivirus
infections (Lastra and Gil, 1981). Light microscopy on virul infection has proved
to be very useful in viral disease diagnosis, in the selection of tissue fo ultractural
studies, for monitoring host tissue for virus infections, and for monitoring viral
inclusion purification (Cristie, 1977; de Mejia et.al., 1985). The large field of
view, the selective strain, and the speed ease of tissue preparation and
examination are of the advantages of light microscopy over electron microscopy
in studying viral infections (Cristie et.al.,1986).
25
2.6 Molecular detection
PCR (polymerase chain reaction)
Polymerase chain reaction ('''PCR''') is a Molecular Biology technique for
enzymatically replicating DNA without using a living Organism , such as '' E.
Coli '' or Yeast . The technique allows a small amount of the DNA molecule to be
amplified many times, in an exponential manner. The polymerase chain reaction
(PCR) is a scientific technique in molecular biology to amplify a single or a few
copies of a piece of DNA across several orders of magnitude, generating
thousands to millions of copies of a particular DNA sequence. Developed in 1983
by Kary Mullis (Mullis, 1990)), PCR is now a common and often indispensable
technique used in medical and biological research labs for a variety of
applications. The polymerase chain reaction (PCR) has been used as the new
standard for detecting a wide variety of templates across a range of scientific
disciplines, including virology. The method employs a pair of synthetic
oligonucleotides or primers, each hybridizing to one strand of a double stranded
DNA target, with the pair spanning a region that will exponentially reproduced.
The hybridized primer acts as a substrate for a DNA polymerase, which creates a
complementary strand via sequential addition of de-oxynucleotides. The
polymerase chain reaction (PCR) is an extremely sensitive and specific technique
(Innis et.al., 1990, Saiki et al., 1988) for the detection and identification of plant
pathogens, and it can be used to investigate precise questions about the
composition of plant pathogen populations and the genetic diversity of plant
viruses (Gilbertson, et al., 1991, Robertson, et. al., 1991). PCR is an in-vitro
method for amplifying target nucleic acid sequences. The speed, specificity,
sensitivity, and versatility of PCR made it suitable in many areas of research in
biology. Since PCR has the power to amplify the target nucleic acid present at an
extremely low level and form a complex mixture of heterologous sequences, it
has become an attractive technique for the diagnosis of plant virus diseases
(Henson et.al., 1993; Hadidi et al., 1995; Candresse et al. 1998a). This procedure
26
is applicable directly to DNA plant viruses caulimo, gemini, and badnaviruses
(Naidu et al., 2001).
Atzmon et al. (1998) reported that the DNA of tomato yellow leaf curl virus
(TYLCV), a geminivirus transmitted by the whitefly Bemisia tabaci, was
amplified from squashes of infected tomato plants and of viruliferous vectors
using the polymerase chain reaction (PCR). The reaction products were subjected
to gel electrophoresis, blotted and hybridized with a radio-labeled virus specific
DNA probe. TYLCV DNA was amplified from squashes of leaves, roots, and
stem of infected tomato and from individual viruliferous whiteflies. DNA
fragments were amplified using the primers V61, C473; V781, C1256; V1769
and C2120. The products of the reaction were collected, subjected to
electrophoresis, blotted and hybridized with the virus specific DNA probe to
confirm the identity of the amplified viral DNA fragment. They reported that a
410 bp DNA fragment was amplified from tissue squashes of viruliferous insect
and of infected plant.
Li et al., (2004) observed that geminivirus infection of sweet potato (Ipomoea
spp.). A protocol of polymerase chain reaction (PCR) was developed for the
detection of geminiviruses in a variety of sweet potato. PCR assays using three
primer pairs detected nine uncharacterized isolates of the geminiviruses in sweet
potato from Asia and America. However, the best PCR result was obtained with
degenerate primers SPG1/SPG2, which detected a Taiwan isolate of Sweet potato
leaf curl virus (SPLCV-Taiwan) in a sample.
Dennis and Bajet (2007) detected geminiviruses by the polymerase chain reaction
(PCR) in total nucleic acid preparations of tomato and squash leaf samples from
different areas in the USA. Begomovirus DNA fragments were detected by PCR
using 3 sets of degenerate primers that amplify different regions of the genomic A
DNA component of begomoviruses. Some samples produced bands in all 3
primer sets, some in only 2 of the 3 sets of primers, while some produced PCR
27
fragments in only 1 of the 3 primer pairs, which suggests variation in the virus
DNA sequence.
Ghosh et al. (2008) used the begomovirus-specific primers to amplify the
corresponding genomic fragments of the causal agent of leaf mosaic of jute. The
primers PAL1v1978 and PAR1c496 amplified the expected 1.2-kb segment of
DNA-A from all the 12 samples tested. Gel-eluted amplicons (eight amplicons
from glasshouse samples and two amplicons from field samples) were cloned into
the pJET1 positive selection vector using the Gene JET_ PCR Cloning Kit and
competent Escherichia coli cells (strain DH5-a) were transformed following
standard molecular biology procedures. Sequencing of a representative clone
from each of the 10 amplicons revealed that all the inserted fragments were 1263
nucleotides in length and identical in sequence except for two clones in which
only two nucleotides were found to vary. These sequence variations may be due
PCR or sequencing error. As the segment of sequence they reported shared the
highest sequence identity with Corchorus golden mosaic virus, they concluded
that yellow mosaic disease of C. capsularis is associated with a begomovirus.
Raj et al. (2008) carried out polymerase chain reaction (PCR) using begomovirus
genus specific primers PALIv 1978 and PARIc 496 to confirm the association of
a begomovirus in naturally mosaic infected J. curcas leaf tissues. PCR products
were analysed by electrophoresis in 1.2% agarose gels. As expected, bands of
1.1kb was consistently amplified which confirmed the association of a
begomovirus with the mosaic disease of J. curcas. They also reported from the
study that Jatropha mosaic virus possessed highest identities and closest
relationships with Indian and Sri Lankan cassava mosaic virus isolates.
28
MATERIALS AND METHODS
3.1 Place and time
The experiments were carried out in the glass house of Seed Pathology Centre,
Net house of the Department of Plant Pathology, Bangladesh Agricultural
University, Mymensingh and Plant Pathology Laboratory of Bangladesh
Agriculture Research Institute (BARI), Gazipur, during the period January- 2010
– November 2011.
3.2 Collection of seeds
Seeds of seven varieties of jute namely D-154, CVL-1, CVE-3, BJC-2142, CC-
45, O-795, BJC-7370 were collected from the Plant Breeding Division of
Bangladesh Jute Research Institute (BJRI), Dhaka.
3.5 Study of leaf mosaic of jute on the growing plants
A number of 100 seeds of each seven varieties were taken at random from the
working samples and sown in 20 perforated polythene bags. The polythene bags
were placed on trays and watered regularly. The first reading of symptoms which
develop on the cotyledonous leaves and counting was done during 10 days after
sowing. Numbers of seedlings with yellow dot marks on the cotyledons were
counted. Such seedlings individually transferred to earthen pot for further
observations.
29
3.4 Seed to seedling transmission of the causal agent of leaf mosaic of jute by cassette holder method
Seeds of each sample (7 varieties) were tested. Twenty five seeds of each sample
were used. Percent germination and percent seed borne infection was evaluated
using in cassette holders. In this method 2- folds blotting paper strips were put in
the compartments of a photographic slide cassette holder. Two seed, taken at
random, were taken in between each paper folding. The loaded cassette holder
was placed in a suitable tray containing tap water. The cassette with trays was
then placed in screen house at room temperature or normal light. Number of seeds
sprouted was counted after 10 days of placing the seeds in the cassette holders.
The seedlings were observed for 20 days. Individual leaf was observed for mosaic
or chlorosis symptom on the growing seedlings.
Number of infected seedlings Seedling with mosaic symptoms (%) =-------------------------------------------×100
Total number of seedlings
Number of leaves with mosaic symptom Leaf with mosaic symptom (%) =----------------------------------------------------×100
Total number of leaves
30
3.5 Seed to plant to seed transmission of the causal agent of leaf mosaic of
jute
Seed to plant to seed transmission of leaf mosaic of jute was studied in the glass
house of Seed Pathology Centre under insect proof condition. Two varieties
namely D-154 and CVL-1 were used in this experiment. Two lots of seeds were
used in each variety. Four hundred seeds of each variety were sown in 16 pots
with 25 seeds/ pot. Symptoms bearing seedlings were taken out carefully and
transplanted in new pots. Mosaic affected plants were tagged with numbering.
Seeds were collected from those plants for next season sowing. In the successive
season 400 seeds of each variety collected from mosaic affected plants were sown
in pots with 25 seeds in each pot. Data were recorded on the basis of symptom
expression.
3.6 Graft transmission of the causal agent of leaf mosaic of jute
Several grafting techniques were performed viz. Gooti (approach) grafting, peg
grafting, veneer grafting, T- budding and root grafting in the Net-house of Plant
Pathology Department. To prevent the whitefly (Bemisia tabaci) infestation the
entire experimental area was periodically sprayed with insecticide namely Rogor
@ 0.2%. Appropriate techniques of each grafting were followed.
Number of plants successfully grafted Successful grafts (%) = ----------------------------------------------------- x 100%
Total number of plants tested Number of plants infected Successful transmission (%) = ------------------------------------------------ x 100%
Number of plants successfully grafted
31
Severity was assessed based on scale: 0– 4
3.6.1 Peg grafting
Two test plants one of them was healthy and another diseased was taken
preferably of same age which was 40 days after sowing. The top portion of
healthy plant was taken and defoliated leaving the apical bud only and scion was
prepared by making vertical inward cut tangentially at its base from two sides
forming a peg like structure. The length of scion was about 10-12cm. For
preparation of stock plant the top portion of an infected plant was removed and a
downward cut of about 2-3 cm was made at the middle of the stock. The sharp
peg of the scion was inserted into the stock plant carefully to best fit having no
gape between them. The stems thus joined together were tightly fastened with a
parafilm strip and then with cotton thread for proper tightening. The peg grafting
was also made between infected scion and healthy stock. The grafts were
observed carefully for any kind of symptom in the grafts.
3.6.2 Veneer grafting
Here a sharp peg was made at the scion following the same procedure as
described in section 3.5.2. The stock was prepared by giving a downward and
inward incision at one side of stem at 10-12 cm below its top. The sharp peg of
the scion was inserted into the stock carefully. The stems thus joined together
were tightly fastened with a parafilm strip and then with cotton thread for proper
tightening.
0= No symptom
1= ≤25% of the leaf area infected
2= 26-50% of the leaf area infected
3= 51-75% of the leaf area infected
4= ≥ 76% of the leaf area infected
32
3.6.3 Gooty (approach) grafting
Two test plants one of them healthy and diseased, preferably of same height, was
taken. Their pots were brought as close as possible. A piece of vertical bark was
removed from each stem at the same height of the two plants. The bark removed
area of both the stems was joined at attached faces of surface tied together with a
parafilm strip and then wrapped with thread. After two weeks of grafting stems of
the plants the shoot of infected was cut at above the point of joint of the stems.
3.6.4 T- budding
A scion was prepared with a bud from a healthy young plant with a sterile scalpel.
A plant with typical mosaic symptom was selected At an axil of a selected
diseased stalk plant, two incisions, one across the bark of the stem and the other
longitudinally at a 900 angle to the former incision, was given in a way that it
resembles the letter “T”. The scion was then placed carefully in the pocket of the
bark “T” facing the growing point outward. The structure was wrapped with
parafilm strip and tied with thread. The grafts were maintained and observed for
symptoms developed in the shoot growing from the bud.
3.6.5 Root grafting
One hundred seeds of the D-154 cultivar were sown on sand in polythene bags.
The polythene bags were punctured at the base and kept in trays. The trays were
watered regularly. After 20 days of sowing 10 seedlings with mosaic symptoms
on leaves were selected. Ten healthy seedlings without mosaic symptoms were
also selected. The seedlings were uprooted from the sand. A vascular connection
between root of a healthy and a diseased plant was attempted. Little injury on root
system (tap root) was made. The roots of healthy and diseased plants were tied
together carefully with cotton thread. Then couple of seedlings was transferred in
pot soil. The pots were maintained in insect proof net for symptom development.
Individual pair was observed regularly and recorded.
3.7 Vector Transmission of the causal agent of leaf mosaic of jute
33
3.7.1Vector population
The vector whitefly (Bemisia tabaci Genn.) was collected from guava plant.
Infested guava leaves were collected and dislodged the insects inside a net box.
The collected whiteflies were then reared on healthy tobacco (Nicotiana tabacum)
plant in insect proof wooden cages. The adult whiteflies that emerged from
nymphs grown on the tobacco were used for transmission. Non-viruliferous
adults of whiteflies were confined to symptom bearing jute plants (cv. D-154) for
24 or 48 hr acquisition feeding period (AFP). Insects either singly or in groups of
3, 5, 10 and 20 per plant were transferred to healthy jute seedlings (20 plants /
treatment) kept in insect proof cage. Insects were given 24 hr or 48 hr IFP.
The results were examined visually and calculated as percentage.
Number of infected plants Transmission (%) = ---------------------------------------- × 100 Number of inoculated plants
3.7.2 Acquisition Feeding Period (AFP)
AFP was determined by allowing the adults of whitefly (B. tabaci) to feed on
mosaic infected jute plants (cv. D-154) for 1, 5, 10, 15, 30 min, 1, 3, 5, 24 and
48hours. After virus acquisition, the viruliferous whiteflies was released onto 30
healthy seedlings (five seedlings per pot) contained in a net-cage to allow
inoculation feeding for 48 hours of Inoculation Feeding Period (IFP) and were
sprayed with 0.2% dimethoate (Rogor). Twenty plants and 20 insects/ per plants
were used. Percentage of infection was calculated from plants showing mosaic
symptoms after 30 days of inoculation feeding.
3.7.3 Inoculation Feeding Period (IFP)
34
A set 20 non-viruliferous adults of whiteflies were transferred into transparent
plastic bottles containing jute plants showing typical yellow mosaic symptom and
allowed to feed for 48 hours of AFP. After virus acquisition, each set of
viruliferous whiteflies was released onto 30 healthy seedlings (five seedlings per
pot) contained in a net-cage to allow inoculation feeding for 1, 5, 10, 15, 30 min,
1, 3, 5, 24 and 48 hours. After the respective time period of feeding the seedlings
were sprayed with 0.2% dimethoate (Rogor) to kill the vectors. The same
numbers of healthy plants were also inoculated with non-viruliferous whitefly as
controls in each replication. Symptoms of infection were recorded at seven days
intervals for 30 days.
3.7.4 Persistence of causal agent in the vector
A group of adult non-viruliferous B. tabaci was caged for 48 hours of acquisition
feeding on jute plants showing mosaic symptoms. The infected plants were then
removed from the cage. The viruliferous whiteflies were transferred to healthy
jute plants of four cultivars. A number of twenty whiteflies were transferred to
each variety daily and allowed to feed 48 hours for inoculation feeding. The
plants were sprayed 0.2% dimethoate (Rogor) after 48 hours of each transfer and
monitored for disease symptom development.
35
Plate 1: Whitefly
Plate 2: Inoculation of healthy jute seedling by vector (whitefly)
36
Plate 3: Management of inoculated plant in insect proof net-cage
37
3.8 Study of inclusion bodies of the causal agent of leaf mosaic of jute under
light microscope
Young growing tips from mosaic symptom bearing plant and healthy plant leaf
tissue were collected. The tissue were prepared for staining in Azure- A (Cristie
and Edwardson, 1967) by abrading with sand paper (600mess) to remove the
cuticle so that the stain could penetrate into the mesophyll and vascular cell
(Hiebert et.al., 1984). The abraded tissue were placed in 2-methoxyethanol for
15-30minutes in order to remove the chlorophyll and then in 0.1% azure-A stain
for 15-30minutes. The tissues were washed sequentially in 95% ethanol and 2-
methoxy ethyl acetate for 15-30minutes each to remove the stain. The tissue were
then blotted dry mounted in a drop of Euparal on a glass slide, and cover slip
before viewing in a light microscope (Cristie et.al.,1986: Hiebert et.al.,
1984).The specimen ware then examined under the microscope at magnification
ranging 100x to 1000x.The type, color and the location inclusion body were then
described.
3.9 Detection of the causal agent of leaf mosaic of jute by molecular technique
Molecular techniques based on hybridization or amplification, and especially on
PCR, have been developed for the most important plant pathogenic viruses. Their
main advantages are specificity and rapidity. Specificity is directly related both to
the design of the primers or probes and to the amplification.
3.9.1 Collection of leaf samples
Both leaves with typical mosaic symptoms of four jute cultivars viz. CVL-1,
CVE-3, D-154, CC-45 and healthy leaves of one cultivar (D-154) were collected
from plants grown in the field when they were 50 days old. Symptom bearing
leaves of one cultivar (D-154) were also collected after successful insect
38
transmission grown under insect proof net. Collected leaves were dried in the
laboratory at normal room temperature. The samples were stored at room
temperature until use for DNA extraction.
3.9.2 Preparation of DNA samples
DNA from each leaf sample was extracted from young typical symptom bearing
and healthy leaves following the protocol as described by Rojas et al. (1993).
Briefly, approximately 25mg leaf tissue were taken in mortar and ground with
pestle in 300 µl extraction buffer solution and taken in 1.5 microfuge tube. The
ground samples were vortexed (Vortex-Mixture: VM-2000, Taiwan) for 20
seconds for proper mixing. The samples were incubated at 65ºC for 10 minutes in
water bath (WB-2400, Taiwan) and then centrifuged for 10 minutes at
10,000g.The supernatant fluid (approximately 250µl) was transferred to a clean
microfuge tube and 50µl isopropanol was added.Then the tubes were vortexed
and centifuged for 10 minutes at 10,000g and supernatant fluids were removed.
The pellete were washed with 200µl of 70% ethanol and centrifuged for 3
minutes at 10,000g. The supernatant was discarded completely without disturbing
the DNA pellete and dried for 5 minutes in a Speed Vac-drier. The pellete were
re-suspended in 300µl of distilled water. Finally the DNA samples were stored in
a refrigerator at -20ºC.
3.9.3 Protocol for preparation of 1% agarose gel (50 mL)
An amount 1.2 g agarose powder was weighed and taken into 500 ml Erlenmeyer
flask. Then 150 ml of (0.5X TBE) buffer was added into the flask. The flask was
heated in microwave oven with occasional swirling for generating uniform
suspension until no agarose powder was seen and the agarose solution become
transparent. Then the agarose solution cooled to 50oC (flask cool enough to
comfortably hold with bare hand). Then the gel was poured onto the gel casting
tray (15×15×2 cm in size) that was placed on a level table and the appropriate
39
comb was inserted. Melted agarose allowed for solidifying on the bench for 20
min.
3.9.4 Amplification of DNA by PCR
The polymerase chain reaction (PCR) was employed for the amplification of
large number of copies of DNA.
The cycling reactions:
There were three major steps in PCR, which were repeated for 30 or 40 cycles,
done on an automated cycler, which heated and cooled the tubes with the reaction
mixture in a very short time (Fig. 2).
Denaturation: The first regular cycling event was done by heating the reaction to
94–98 °C for 20–30 seconds. It melts template by disrupting the hydrogen bonds
between complementary bases, yielding single-stranded DNA molecules. All the
enzymatic reaction was stopped (for example the extension from a previous
cycle).
Annealing: The primers were jiggling around caused by the Brownian movement
Ionic bonds were constantly formed and broken between the single stranded
primer and the single stranded template. The more stable bonds retained a little
bit long periods (the primer that fit exactly) and on that little piece of double
stranded DNA (template and primer); the polymerase started to attach and started
copying the template. Once there were a few bases built in, the ionic bond was
strong between the template and the primer, that it did not break anymore. The
reaction temperature was lowered to 50–65 °C for 20–40 seconds allowing
annealing of the primers to the single-stranded DNA template. Typically the
annealing temperature is about 3-50 C below the Tm of the primers used. Stable
DNA-DNA hydrogen bonds were only formed when the primer sequence very
closely matches the template sequence. The polymerase bound to the primer-
template hybrid and began DNA synthesis.
40
Extension/elongation: The primers where there were few bases built in, already
had a stronger ionic attraction to the template than the forces breaking these
attractions. The primers that were on positions with no exact match get loose
again (because of the higher temperature) and did not give an extension of the
fragment. The bases (complementary to the template) were coupled to primer on
the 3’ side. The temperature at this step dependent on the DNA polymerase used;
Taq polymerase had its optimum activity temperature at 75–80 °C, and a
temperature of 72 °C was used with this enzyme. At this step the DNA
polymerase synthesizes a new DNA strand complementary to the DNA template
strand by adding dNTPs that were complementary to the template in 5' to 3'
direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl
group at the end of the nascent (extending) DNA strand. The extension time was
dependent both on the DNA polymerase used and on the length of the DNA
fragment to be amplified. As a thumb rule, at the optimum temperature, the DNA
polymerase would polymerize a thousand bases per minute. Under optimum
conditions, the amount of DNA target was doubled, leading to exponential
(geometric) amplification of the specific DNA fragment.
41
Fig 1: Diagrammatic representation of the PCR cycle. (1) Denaturing at 94–96 °C. (2) Annealing at ~65 °C (3) Elongation at 72 °C. Four cycles are shown here. Blue lines = DNA template, Red arrows=Primers, Light green circles= DNA polymerase, Green lines=DNA products.
42
3.9.5 Selection and design of primer
To perform PCR reaction begomovirus-specific degenerate primers (Rojas et al.
1993) were used to amplify the corresponding genomic fragments of the virus.
The degenerate primer was a mixture of molecules in which the nucleotides at
one or more defined position varied by design. The degenerate number for primer
was the product of all the numbers that designated how many nucleotides might
occur at each position in that primer. Degenerate primers for whitefly transmitted
geminiviruses were designed to anneal to highly conserved nucleotide sequence
regions of the open reading frames (ORFs) or the common region of DNA.
Primer PAL1v1978 was designed to anneal to the complementary sense strand of
the replicative form AL1 sequence encoding the derived amino acid sequence
ThrGlyLysTh-rMet TrpAla, which was a conserved, putative NTP-binding site
present in viral replication associated proteins. Primer PAR1c496 was designed to
anneal to the viral sense strand of the AR1 ORF sequence encoding for the
conserved, derived amino acid sequence ProMetTyrArg LysProArg, which was
located near the amino terminus of the coat protein.
Table 1. Sequences of primers used in the study
Primer *
Nucleotide sequence
PAL1v1978
5’-GCATATGCAGGCCCACATYGTCTTYCCNGT-3’
PAR1c496
5’-AATACTGCAGGGCTTYCTRTACATRGG-3’
* Primer nomenclature was coded as follows: P = primer; AR1 = open reading frame (OFR) for AR1, AL1 = ORF for AL1; v = viral sense primer (anneals to complementary sense strand of the replicative form and gives viral sense sequence) or c = complementary sense primer (anneals to viral sense strand of the replicative form and gave complementary sequence). Nucleotides at degenerate positions were represented by a single letter of IUPAC ambiguity code: Y = C, T; R = A, G.
43
Preparation of working solution of DNA sample
Before PCR, DNA concentrations were adjusted to 25ng/µl using following
formula:
V1 × S1 = V2 × S2
Where, V1 = final volume of DNA solution (µl)
S1 = final DNA concentration (ng/µl)
V2 = initial volume of DNA solution (µl)
S2 = initial DNA concentration (ng/µl)
Therefore, V1 = V2 × S2 / S1
Reaction mix preparation to perform Polymerase Chain Reaction (PCR)
A 10µl PCR reaction mix contained the following reagents:
Reagents Quantity (µl)
• 10X Ampli Taq polymerase buffer 1.0
• 5 µM primer PAL1v1978 (+) 1.25
• 5 µM primer PAR1c496 (-) 1.25
• 1.5 mM dNTP 1.0
• Ampli Taq DNA polymerase 0.2
• sample DNA extract 4.0
• Sterile NPW 1.3
• Total 10
44
Amplification buffer, 10X, pH 8.3:
• Tris HCl .05M
• KCl .05M
• MgCl2 .07M
• BSA .02% (w/v)
Taq DNA polymerase:
• Tris HCl .05
• Na2-EDTA 1mM
• DTT 1mM
• Glycerol 50% (v/v)
During the experiment, PCR buffer, dNTPs, and primer solution were thawed
from frozen stocks, mixed by vortexing and placed on ice. DNA samples were
also thawed out and mixed gently. The primers were pipetted first into PCR tubes
compatible with the thermocycler used (0.2 ml). For each DNA sample being
tested, a pre-mix was then prepared in the following order: buffer, dNTPs, DNA
template and sterile distilled water. Taq DNA polymerase enzyme was then added
to the pre-mix. The pre-mix was then mixed well and aliquoted into the tubes
containing primers. The tubes were then sealed and placed in thermo-cycler and
the cycling was started immediately.
45
Thermal profile
DNA amplification was performed in a thermal cycler (Master Cycler Gradient,
Eppendorf, Germany).
The thermal cycle was as follows
• 95 ºC for 3 minutes : Denaturation
• 95 ºC for 50 seconds : 35 cycles
• 55 ºC for 50 seconds : Annealing
• 72 ºC for 1 minutes : Elongation or extension
• 72 ºC for 10 minutes : Final step/complete extension
• After completion of cycling program, reactions were held at 4 ºC
3.9.6 Electrophoresis, gel staining and documentation
The amplified products were separated electrophoretically on 1% agarose gel.
The gel was prepared using 3.75g agarose powder (Genei, India) mixed with 250
ml 0.5× TBE buffer. The electrophoresis was done at 120 V for 90 min. The gel
was stained with ethidium bromide (0.1µg/ml) solution after electrophoresis for
15 min at room temperature. Thereafter the gel was removed from the ethidium
bromide. DNA ladder set (1 Kb, MBI Fermentas, and Germany) was included as
sized molecular marker. DNA from healthy plants and double distilled water were
used as experimental controls.
3.9.7 Observation of DNA Bands
The gel was placed under UV illuminator inside of a gel documentation system.
DNA bands were observed, focused and the photograph was saved as a file and
printed out.
46
RESULTS
4.1 Study of leaf mosaic of jute on growing plants
The results of present study are presented in Table 2. Seed samples belonging to
cultivar V2 (D-154) showed the highest seed germination (82%) as well as seed to
plant transmission (8%) of the causal agent. The lowest seed germination (55%)
was observed in V3 (BJC-7370) and the lowest seed to plant transmission (1%)
was observed in the cultivar V6 (O-795). The cultivars V1 (CVE-3), V3 (BJC-
7370), V4 (CVL-1), V5 (BJC-43) and V7 showed almost similar seed to plant
transmission. After transferring to the pots the symptom was observed till seed
formation. At the seedling stage the symptoms appeared as small yellow flakes on
the leaf lamina which gradually increases and observed as yellow and light green
patches giving variegated appearances (Plate 6). As plant grows symptoms
became severe and infected leaf showed yellowing and became crinkled and
leathery (Plate 7).
4.2 Seed to seedling transmission of the causal agent of leaf mosaic of jute by cassette holder method The V2 (D-154) expressed the highest mosaic symptom in number of seedlings
about 6%, cassette holder trial. The cultivar V6 (O-795) expressed the lowest
(1%) of symptom bearing seedlings. V1 expressed 5% seed to plant transmission.
V4, V5 and V7 expressed symptom in 3% seedlings. The range of seed
germination was 50-83% among the jute cultivars. V2 (D-154) showed the highest
germination (Table 2).
47
Table 2: Percentage seedling with mosaic symptom with in two tests
Variety Aluminum tray Cassette holder
Germination (%)
Symptom bearing seedling (%)
Germination (%)
Symptom bearing seedling (%)
V1 (CVE-3) 74 6 75 5
V2 (D-154) 82 8 83 6
V3 (BJC-7370) 55 3 60 2
V4 (CVL-1) 65 4 65 3
V5 (BJC-43) 69 5 70 3
V6 (O-795) 56 1 50 1
V7 (CC-45) 62 4 62 3
48
Plate 4: Seedling with yellow dot spot on cotyledon (Grown on sand in
aluminium tray)
Plate 5: Seedling with yellow dot spot on cotyledon growing in cassette
holders
49
Plate 6: Symptoms of leaf mosaic of jute at seedling stage (20 days old)
Plate 7: Symptoms of leaf mosaic of jute on 60 days old plant
50
0
10
20
30
40
50
60
70
80
90
V1 V2 V3 V4 V5 V6 V7
Variety
Germination (%)
Symptom bearing seedling (%)
Fig 2: Status of percentage of germination and seedling with chlorotic spots
as grown in aluminum tray
51
0
10
20
30
40
50
60
70
80
90
V1 V2 V3 V4 V5 V6 V7
Variety
Germination (%)
Symptom bearing seedling (%)
Fig 3: Status of percentage of germination and seedling with chlorotic spots as grown in cassette holder
52
4.3 Seed to plant to seed transmission of the causal agent of leaf mosaic of
jute
The transmission of leaf mosaic disease of jute from seed to plant to seed was
studied in two consecutive seasons. Results obtained from the study are given in
Table-3 and Table-4. Mosaic symptom appeared on the plants of both varieties
(D-154 and CVL-1) just after 30 days of seeds sowing. In the first season D-154
variety had 1.6% of leaf mosaic plants with 39.5% of mosaic leaf in mosaic
plants, whereas CVL-1 variety had 0.9% of mosaic plants with 27% of mosaic
leaves in each plant on an average. In both cases number of pods and number of
seeds per pod were more in healthy plants than that of diseased one.
In the following season seeds collected from the infected plants were sown. At
this time also first mosaic symptom prominently appeared on the plants after
more or less a month of sowing. At the second season, the incidence of mosaic
symptoms was recorded at two stages of plant growth, 1st t at 30 days of sowing
and 2nd at 60 days of sowing. At the first time D-154 and CVL-1 had 28.55%
and 26% of leaf mosaic incidence that increased at the second date of record
taking which was 56.43% and 38.69% for D-154 and CVL-1 respectively.
Percent mosaic leaf in mosaic affected plants were 90.25% and 83.12% for D-154
and CVL-1 variety respectively. This data was recorded at the 60 day age of
plant.
53
Table 3: Seed to plant transmission of the causal agent of leaf mosaic of
jute
Name of
variety
Mosaic plant (%)
Mosaic leaf (%)
No. of pods/plant No. of seeds/pod
Diseased Healthy Diseased Healthy
D-154 1.6 39.5 37 46 21 30
CVL-1 0.9 27.0 33 49 19 25
Table 4: transmission of jute leaf mosaic in seeds collected from infected
plants (1st season) grown in 2nd season
Name of
variety
% germination Mosaic plant (%) Mosaic leaf (%)*
30 DAS 60 DAS
D-154 73.5 28.55 56.43 90.25
CVL-1 59.75 26 38.69 83.14
* 60 days after sowing
54
Fig 4: Status of mosaic infection at different days in two cultivars of jute
55
4.4 Graft transmission of the causal agent of leaf mosaic of jute
The result of graft transmission of the causal agent of leaf mosaic of jute are
presented in Table 5-9.The causal agent of the jute leaf mosaic syndrome was
found to be fairly graft transmissible. However, the transmissibility dependent on
the host combination and the grafting technique employed. In almost all cases
transmission was highly successful when stalk and scion were both belonging to
C. capsularis cv. D-154.
Several grafting techniques such as gooti (approach), vineer, peg gafting, T-
budding and root grafting were employed in this study. The results are discussed
below:
4.4.1 Peg grafting
In case of peg grafting the variety D-154 gave the highest percentage of
successful grafts (100%) as well as successful transmission (80%) in the same
host combination (D-154 × D-154) followed by variety CVE-3 when same host
combination. The host combination CVL-1×CVE-3 established medium number
of successful grafts (4) as well as successful transmission (4). Lowest percentage
of successful grafts (20%) was found in the host combination CVL-1×O-795 and
no transmission was found in that combination.
4.4.2 Veneer grafting
In case of veneer grafting the variety D-154 gave the highest percentage of
successful grafts (83.33%) in the same host combination followed by host
combination (CVL-1× CVE-3). Highest percentage of successful transmission
(100%) was recorded in the variety CVE-3 followed by in the variety D-154
(80%) both in the same host combination. The lowest percentage of successful
grafts (50%) as well as successful transmission (50%) was found in the host
combination CVL-1×O-795.
56
4.4.3 Gooti or Approach grafting
In case of gooti (approach) grafting initial symptoms developed within 3–4 weeks
and the full syndrome within 1–2 months. The variety CVL-1 gave highest
percentage of successful grafts (90%) followed by grafting between host
combination D-154×CVL-1(80%). The highest percentage of successful
transmission (85.71%) was observed in the host combination (D-154×CVE-3)
followed by in CVL-1 in the same host combination. Lowest percentage of
successful grafts (30%) as well as successful transmission (85.71%) was found
when C. olitorius cv. O-795 was grafted to CVL-1.
4.4.4 T-budding
In case of T-budding the variety CVE-1 gave highest percentage of successful
buds (83.33%) in the same host combination followed by in the host combination
CVL-1×CVE-3. Highest percentage successful transmission (100%) was recorded
in the host combination (CVE-3× CVL-1) followed by the cultivar CVL-1 in the
same host cultivar as well as in combination with cultivar CVE-3. No bud was
found successful between CVL-1×O-795.
4.4.5 Root grafting
The results of root graft transmission are sown in Table 8. Out of 25 such grafts
attempted 14 grafts took hold and successful transmission occurred in 4 grafts. In
the other pairs root grafts did not held and as a result no transmission apparent
after 30 days. These plants were found separate at the root area when sand on
which they were placed was removed.
57
Table 5: Transmission efficiency of jute leaf mosaic causal agent by peg grafting Variety Grafts
attempted Successful
grafts Successful
transmission Successful grafts (%)
Successful Transmission
(%)
Severity
Stock Scion
D-154 D-154 5 5 4 100 80 3.12
CVL-1 CVE-3 5 3 2 60 66.67 2.68
CVE-3 CVE-3 5 4 3 80 75 3.54
CVE-3 CVL-1 5 3 1 60 33.33 1.98
CVL-1 O-795 5 1 0 20 0 0
Table 6: Transmission efficiency of jute leaf mosaic causal agent by Veneer grafting Variety Grafts
attempted
Successful
grafts
Successful
transmission
Successful
grafts (%) Successful
Transmission (%) Severity
Stock Scion
D-154 D-154 6 5 4 83.33 80 2.78
CVL-1 CVE-3 5 4 3 80 75 3.31
CVE-3 CVL-1 5 4 2 60 50 1.79
CVE-3 CVE-3 6 4 4 66.67 100 3.10
CVL-1 O-795 4 2 1 50 50 0.98
58
Table 7: Transmission efficiency of jute leaf mosaic causal agent by approach grafting Variety Grafts
attempted
Successful
grafts
Successful
transmission
Successful
grafts (%) Successful
Transmission (%) Severity
Healthy Infected
CVL-1 CVL-1 10 9 7 90 77.77 2.56
D-154 CVE-3 10 7 6 70 85.71 1.98
D-154 CVL-1 10 8 6 80 75 3.54
O-795 CVL-1 10 3 1 30 33.33 3.12
Table 8: Transmission efficiency of jute leaf mosaic causal agent by T- budding Variety Budding
attempted
Successful
buds
Successful
transmission
Successful
buds (%) Successful
Transmission (%) Severity
Stock Scion
CVL-1 CVL-1 5 4 3 60 75 3.18
CVL-1 CVE-3 6 4 3 66.67 75 2.87
CVE-3 CVL-1 5 2 2 40 100 3.01
CVE-3 CVE-3 6 5 3 83.33 60 2.67
CVL-1 O-795 5 0 0 0 0 0
59
Table 9: Transmission efficiency of jute leaf mosaic causal agent by root grafting
Variety Grafts attempted
Successful grafts
No. successful transmission
Successful transmission
(%)
D-154
5 3 2
28.57
5 3 1
5 2 0
5 2 0
5 4 1
60
Plate 8: Peg grafting between D-154 × D-154 at the initial stage
61
Plate 9: Peg grafting between D-154 × D-154 after successful transmission.
62
Plate 10: Veneer grafting between D-154 × D-154 at initial stage
63
Plate 11: Veneer grafting between D-154 × D-154 after successful transmission
64
Plate 12: Approach grafting between CVL-1 ×CVL-1 at initial stage
65
Plate 13: Approach grafting between CVL-1 ×CVL-1 after successful transmission
66
Plate 14: T-budding between CVL-1 ×CVL-1 at initial stage
67
Plate 15: T-budding between CVL-1 ×CVL-1 after successful transmission
68
4.5 Vector transmission of the causal agent of leaf mosaic of jute
4.5.1 Vector population
The result of transmission efficiency of jute mosaic causal agent by different
number of whitefly is shown in the Table 10. It was observed that even one
individual whitefly was capable of transmitting the virus. When 3, 5 and 10
viruliferous whiteflies plant-1 were released; the disease transmission was 20, 30
and 70 percent, respectively. It was found that 15 whiteflies could transmit
the causal agent to a range of hundred percent transmissions. The findings
of the study showed that a maximum of 15 viruliferous whiteflies
required for effective transmission of the causal agent. A positive co-
relation was found between number of whitefly and percentage of transmission of
jute leaf mosaic causal agent. Regression analysis produced regression equation
y= 6.273x+5.341, r=0.975 and y= 6.040x+11.92, r=0.996 when both AFP and
IFP were 24 hours and 48 hours respectively (Fig 5-6).
4.5.2 Acquisition Feeding Period (AFP)
The results of the present study are presented in Table 11. It was found that the
whitefly required a minimum period of 30 minutes acquisition feeding period to
acquire the causal agent for transmission. But the vector required 8 hr acquisition
feeding period for successful transmission of the causal agent into jute plants,
where 100% plants were found to show the disease symptoms. There found a
positive co-relation between acquisition feeding period and percentage of
transmission of jute leaf mosaic causal agent. Regression analysis produced a
regression equation: y= 10.81x-29, r=0.944 (Fig 7).
4.5.3 Inoculation Feeding Period (IFP)
The results of the present study are presented in Table 12. It was found that at
least 30 minutes of inoculation feeding period were required to transmit the
causal agent, though the percentage of transmission was 10. However,
69
100per cent transmission was recorded when 5 hrs of inoculation
feeding period was given to whitefly. There exists a positive co-relation between
inoculation feeding period and percentage of transmission of jute leaf mosaic
causal agent. Regression analysis produced a regression equation: y= 11.57x-
27.66, r=0.966 (Fig 8).
4.4.4 Persistence of causal agent in the vector
The results of the persistence study are presented in Table 13. After a 24-h
AFP, the whiteflies retained the ability to transmit the virus for up to 10 days for
the D-154 and BJC- 7370 cultivars. However, there was a gradual decline in the
number of infected plants after the fourth day, except for the D-154 and BJC-
7370, which started declining after the fifth day (Table 13).
70
Table 10: Effect of number of viruliferous insects on the transmission of causal agent of leaf mosaic jute
No. of
insects/plant
24 hours AFP*, IFP** 48 hours AFP, IFP
No. of inoculated
plants
No. of infected plants
Transmission (%)
No. of inoculated
plants
No. of infected plants
Transmission (%)
1 20 0 0 20 3 15
3 20 6 30 20 6 30
5 20 9 45 20 9 45
10 20 14 70 20 15 75
15 20 19 95 20 20 100
*AFP= Acquisition Feeding Period **IFP= Inoculation Feeding Period
71
Table 11: Effect of AFP on transmission of the causal agent of jute mosaic
Acquisition Feeding
Period (AFP)
No. of inoculated
plants
No. of infected
plants
Transmission (%)
1 minute 20 0 0
5 minutes 20 0 0
10 minutes 20 0 0
15 minutes 20 0 0
30 minutes 20 2 10
1 hour 20 6 30
3 hours 20 8 40
5 hours 20 11 55
24 hours 20 16 80
48 hours 20 18 90
72
Table 12: Effect of IFP on transmission of the causal agent of jute mosaic
Inoculation Feeding
Period (IFP)
No of inoculated
plants
No of infected
plants
Transmission (%)
1 minute 20 0 0
5 minutes 20 0 0
10 minutes 20 0 0
15 minutes 20 1 10
30 minutes 20 3 15
1 hour 20 8 40
3 hours 20 11 55
5 hours 20 12 60
24 hours 20 17 85
48 hours 20 19 95
73
Table 13: Persistence (days) of causal agent of jute leaf mosaic in the vector insects (Bemisia tabaci)
Variety No. of test plant infected out of five
Days after Acquisition Feeding
1 2 3 4 5 6 7 8 9 10 11 12 13 14
CVL-1 5 5 5 5 4 2 2 1 1 0 0 0 0 0
CVE-3 4 5 5 5 3 2 1 1 0 0 0 0 0 0
D-154 5 5 5 5 5 3 2 1 1 1 0 0 0 0
BJC-7370 3 5 5 5 5 3 2 1 1 1 0 0 0 0
Five plants were used for each test
74
Fig 5: Relationship between number of whiteflies and transmission of the jute mosaic causal agent at 24 hours of AFP and IFP
Fig 6: Relationship between number of whiteflies and transmission of the jute mosaic causal agent at 48 hours of AFP and IFP
75
Fig 7: Relationship between Aquision Feeding Period (AFP) and transmission of jute leaf mosaic causal agent
Fig 8: Relationship between Inoculation Feeding Period (IFP) and transmission of jute leaf mosaic causal agent
76
Fig. 9: Relationship between days after acqusition (persistence) and plants infected
77
4.6 Study of inclusion body of the causal agent of leaf mosaic of jute under light microscope The tissue of a leaf with mosaic symptom was stained with Azure-A to
investigate the inclusion bodies incited by virus. Inclusion bodies were observed
in the nucleus of the host cell. The inclusion bodies were observed as large, black
or blue-violet structure in the nucleus of the cells. Inclusion bodies were visible in
the phloem parenchyma cells of the mosaic infected leaves. Typical nuclear
inclusion bodies were prominent in the nucleus of the cells of young expanding
leaves during early stages of symptom development. Inclusion bodies in the
nucleus of expanded mature tissues of the leaves with mosaic symptom were
minimal. Nuclei in which numerous inclusion bodies were visible become
hypertrophied. The distribution of the nuclear inclusion bodies under light
microscope at 100X is shown Plate 16. The inclusion bodies were not uniformly
distributed throughout the vascular system of leaf. The resolution of the stained
inclusion bodies under the light microscope was confirmed at a higher
magnification of selected area (Plate 17-19). The tissue of healthy plants was free
from any kind of inclusion body (Plate 20-21). Inclusion bodies appeared similar
in the leaf samples of the plants of all cultivar of infected jute. In some cases,
quantitative differences in inclusions between the cultivars were noted. Inclusions
sometimes were observed in parenchyma cells immediately outside the phloem.
78
Plate 16: Distribution of inclusion bodies in the nucleus (100X magnification)
Plate 17: Nuclear inclusion body in the mosaic infected leaf of jute (cv. CVE-3) (1000X magnification)
79
Plate 18: Nuclear inclusion body in the mosaic infected leaf of jute (cv. D-154) (1000X magnification)
Plate 19: Nuclear inclusion body in the mosaic infected leaf of jute (cv. CVL-1) (1000X magnification)
80
Plate 20: Healthy leaf sample (cv. CVL-1) stained with azure-A showing no inclusion body (1000X magnification)
Plate 21: Healthy leaf sample (cv. CVE-3) stained with azure-A showing no inclusion body (1000X magnification)
81
4.7 Detection of the causal agent of leaf mosaic of jute by molecular techniques
Molecular based detection of begomovirus has been reported by Rojas et. al.
(1993). In this study jute leaf mosaic causal agent belongs to the same group of
virus as reported by Ghosh et al.(2008). A total of seven samples were collected
all but one of which were from infected plants and rest one was from healthy
plant. DNA of each sample was extracted following method as described by
Rojas et. al.(1993). DNA fragment of Approximately 1.2 kb amplified by PCR
using primers PAL1v1978 and PARI c 496 was observed on 1% agarose gels for
the sample corresponding to infected plants, while no amplification products were
obtained from nucleic acids extracted from healthy plants and distilled water
control (Plate 22).
82
Plate 22: Agarose gel electrophoresis illustrating begomovirus-specific PCR
products obtained using the primers PAL1v 1978 and PAR1c 496. Lanes: 1--4:
field infected jute leaf samples; Lanes 5--6: whitefly inoculated samples; lane: 7
healthy jute leaf sample and lane 8: distilled water control; M: DNA 1 kb DNA
ladder (Fermentas, Germany).
83
DISCUSSION
The main objective of the study was to detect and identify the causal agent of leaf
mosaic of jute and to know its mode of transmission. Transmission through seeds,
grafts and vector were studied. Light microscopy and molecular techniques were
employed to identify the causal agent. Seven seed samples were tested and found
to be affected by jute leaf mosaic. Though the seeds of cultivars varied in
successfully transmitting the causal agent to seedlings and to the plant, symptom
expression differed among varieties. Mosaic appeared on very young seedlings as
diffused chlorotic spots on the cotyledons (Plate 1 and 2). Similar symptoms were
also observed by many scientists (Lange, 1980; Zaman and Albrechtsen, 1999;
Saha 2001). In this experiment such 10 days old symptom bearing seedlings were
transferred to separate pots. In an average 96% of these seedlings develop into
plants having pronounced leaf mosaic symptoms on their true leaves. A few
apparently healthy also produced plant expressing pronounced symptoms. This
indicated that chlorotic spots on the cotyledons can be considered as a positive
sign of seed transmitted infection, whereas apparently healthy looking seedlings
do not indicate freedom from seed-borne infection. This may have indicated that
in these cases the seed borne infections were latent and were expressed at the later
growth stage.
Symptoms appeared on the first true leaf, or on the third or fourth true leaf or on
later leaves as the seedlings were allowed to grow. The symptom bearing true
leaves were crinkled, leathery and sometimes, at the top of the plant, somewhat
needle-like. The floral organs were more of less deformed. Internodes were
shortened and branches proliferated.
By studying the seed to plant to seed transmission of jute leaf mosaic causal agent
it has been found that seeds obtained from the infected plant gave higher
percentage of infected plants in the following year(Table-3 and Table-4) which is
59.29% and 38.69% for D-154 and CVL-1 respectively. The result obtained
confirms the findings of Ghosh and Basak (1951). From the table 3, it is evident
84
that D-154 variety is more susceptible to the disease than CVL-1 variety in terms
of incidence of the disease and its severity. But pod formation was more in the
diseased plant of D-154 variety than that of CVL-1 (Table-3). Whereas the
healthy plants of CVL-1 variety produced more pods than that of D-154 variety.
It proves that though pod production is affected by the disease in case of variety
but the diseased plant of D-154 showed more tolerance to the disease after getting
affected where the performance of healthy plants of CVL-1 variety were better in
terms of pod production . From the table-3 it is seen that the number of seeds/pod
were affected by the disease in case of both variety. But diseased plant as well as
the healthy plants of D-154 produced more seeds/pod than that of CVL-1 variety.
All these depend on variety performance and comparative varietal reaction to the
disease. From the table-4 it is found that after 25 days of recording 1st data
number of mosaic plants increased in case of both varieties. For D-154 variety it
was 30% and 12% for CVL-1.This indicates that the symptom remained masked
in some plants which appeared later that might have revealed viral nature of the
causal.
In this piece of research work experiments were set for graft transmission.
Different grafting techniques were employed to achieve the shoot grafting and
transmission of the pathogen through grafting. Root grafting was also done as the
causal agent was supposed to present in the root system. Transmission of the leaf
mosaic pathogen from diseased (symptom bearing) host to healthy host was
found to be quite frequent and easy when grafts were successful using shoot as
both stalk and scion (Plate 8-15). This kind of graft transmission was also
confirmed by many workers ( Bist and Mathur, 1964; Ahmed, 1978; Conti, 1996;
Saha, 2001).
Root to root grafting was attempted in this research work. This approach of
grafting was found to be effective in transmitting the causal agent from infected
plant to healthy plants .This type of grafting was supported by the previous work
(Saha, 2001). This successful root to root grafting clearly indicated that the jute
85
leaf mosaic causal agent also present in the root system of the jute plant. This has
got an extra significance for extraction of DNA/RNA from root for molecular
characterization of the causal agent as the nucleic acid extraction from jute leaf is
quite tricky and difficult due to having mucilaginous substances in the leaf of jute
plant, whereas, root does not have this problem.
The causal agent was successfully transmitted to healthy jute plants using
whitefly vector B. tabaci. Healthy jute seedlings inoculated with viruliferous
whiteflies developed symptoms similar to those of naturally infected plants in the
field. Studies on the vector transmission of the leaf mosaic causal agent revealed
that even a single whitefly was capable of transmitting the disease although 15
whiteflies are required to cause 95-100% transmission. There found a positive
correlation between number of whitefly and transmission of the causal agent. The
results were comparable to that of other begomovirus like Tomato Leaf Curl
Virus (Muniyappa et al., 2000) and Pumpkin Yellow Vein Mosaic Virus
(Muniyappa et al., 2003; Maruthi et al., 2007) which required 5-15 adult
viruliferous whiteflies to get 100% transmission. Earlier Capoor and Ahmad
(1975) noticed a maximum infection 77.3% with 20 whiteflies. Subramanian
(1979) reported that 15 whiteflies were required to cause cent percent
transmission of yellow mosaic virus in Lablab niger. Cohen et.al. (1983) found
that five whiteflies caused 100 percent infection in squash leaf curl virus infected
squash plant. Raghupathy (1989) reported that 15 to 20 whiteflies were required
to cause effective transmission of yellow mosaic disease of urdbean and soybean,
respectively. However, three whiteflies were sufficient to secure hundred percent
transmission of TYLCV in tomato (Raghupathy, 1995).
The minimum acquisition feeding period required for the vector (B. tabaci) for
successful transmission of the causal agent of the jute leaf mosaic was found to be
30 minutes though the percentage of infection increased with the increase in the
acquisition feeding period (AFP). This has been supported by (Ghosh et al.
2008).
86
In the present study the minimum inoculation feeding period (IFP) required by
the whitefly for successful transmission of the virus was 30 minutes although the
percentage of infection increased with the increase in the inoculation feeding
period. The findings of the present study are in accordance with the finding of
Ghosh et al. (2008).
Light microscopic techniques was tried to observe the inclusions associated with
the presumed viral infection of the mosaic affected jute leaf. The result gave the
excellent indication of the association of virus with the mosaic infected jute leaf.
In this piece of work light microscopy of azure-A stained tissue readily resolved
the aggregation of particles. Conspicuous nuclear inclusions were observed in the
mosaic infected jute leaf which is the diagnostic character of geminivirus
infection as described by Cristie et. al. (1986). The microscopy techniques
described have several significant advantages over other procedures. Perhaps,
most importantly, the infection can be detected within minutes, whereas even the
relatively rapid serology procedures described in the companion paper normally
require at least 24 hours. These microscopy procedures also provide physical
information about the location of the causal agent within the host. This may not
be critical for practical, routine indexing but is important for many research
applications. The detection of stained inclusions by light microscopy requires no
antiserum and only simple laboratory equipment. Slides can even be prepared
from freehand sections and examined in the field with a portable microscope.
Probably this is the first experiment of this kind with mosaic infected jute plant.
Similar inclusions were observed earlier by Schneider (1959) and were
interpreted as viral inclusions. Similar result was obtained by Kim et al. (1979)
from bean leaf tissue infected by bean golden mosaic virus and Lastra and Gil
(1981) from tomato leaf infected with tomato yellow leaf curl geminivirus.
Polymerase chain reaction (PCR) was employed to amplify the presumed
begomovirus infection of the infected jute leaf after extraction of DNA. The
begomovirus specific primer pairs PAL1v 1986 and PARc 496 amplify the
87
expected 1.2 kb of the DNA extracted from symptom bearing leaf both from field
grown and insect transmitted plant. On the contrary no amplification was
observed in the DNA obtained from healthy jute leaf and water control. This
outstanding result strongly suggests that mosaic infected plant is associated with a
begomovirus. This result is in accordance with the result reported by Ghosh et
al.(2008). These primers in have been used extensively for the identification of
begomoviruses in a wide range of crop plants and their vector B. tabaci
previously (Deng et al., 1994; Maruthi et al., 2006; Narayana et al., 2007;
Sharma et al., 2009; Mahesh et al., 2010). However, this result is in opposition
with the result obtained by Zaman Albrechtsen, (1999) who tried extract virus
particle by ultracentrifugation and partial purification and failed to locate the
causal agent. Probably they failed, due to they presumed that the causal as a RNA
virus. But a present finding suggests that the causal agent of leaf mosaic of jute to
be DNA containing begomovirus.
88
SUMMERY AND CONCLUSION
Growing on (seedling symptom) test was conducted in aluminum trays and in
cassette holders to observe the germination and seed to seedling transmission of
jute leaf mosaic disease of seven Corchorus capsularies cultivars. Higher
percentage of germination was observed in aluminum trays than that of cassette
holders though seedling symptoms on cotyledonous leaves were better expressed
in the cassette holders. Cultivar D-154 showed the highest percentage of
transmission of the disease causal agent.
Seed to plant to seed transmission study were conducted in successive to seasons.
In the second year seeds collected from the infected plants only were sown. It was
observed that seeds obtained from the infected plants gave higher percentage of
infected plants in the succeeding year than the percentage of infected plants
expressing seed-borne transmission of mosaic causal agent in the previous year.
Grafting techniques were employed to know the transmissibility of the causal
agent through graft joint. Transmissibility of the leaf mosaic causal agent from
diseased host to healthy host was found to be quite frequent and easy when the
grafts made were successful using shoot as both stock and scion. Graft
transmission was more successful when hosts of same cultivar were used.
Root to root graft transmission clearly indicated that the disease inciting agent is
present in the root system of an infected jute plant. This successful experiment
promised that the root system of infected jute plants can be used as the source of
the causal agent for extraction procedure.
Studies on the vector transmission of the leaf mosaic causal agent revealed that
the causal agent was successfully transmitted to healthy jute plants using whitefly
vector B. tabaci. Healthy jute seedlings inoculated with viruliferous whiteflies
developed symptoms similar to those of naturally infected plants in the field.
There exists a positive correlation between number of whitefly and transmission
of the causal agent.
89
Light microscopic technique was employed to locate the inclusions associated
with the presumed viral infection of the mosaic affected jute leaf. The result gave
the excellent indication of the association of virus with the mosaic infected jute
leaf. Conspicuous nuclear inclusions were observed in the mosaic infected jute
leaf which is the diagnostic character of geminivirus infection.
In the polymerase chain reaction (PCR) detection of the causal agent of leaf
mosaic of jute the begomovirus specific primers amplify the expected 1.2 kb of
the DNA extracted from symptom bearing jute leaf. In contrast, no amplification
was found in the DNA obtained from healthy jute leaf and water control using the
same primers. This extra-ordinary finding suggests that mosaic infected plant
may be associated with a virus.
Based on symptom observations, transmission studies of the virus through seed,
grafts and vector (B. tabaci) and observation of nuclear inclusion bodies under
light microscope and PCR detection of the begomovirus-specific DNA products
from the infected plants, it is concluded that the leaf mosaic of jute disease is
caused by a virus belonging to begomovirus group. The disease is seed and graft
transmissible and can be transmitted in the field by vector insect whitefly
(Bemisia tabaci).
Further studies can be undertaken to find out the whole genome sequence of the
causal agent of the leaf mosaic of jute by employing advanced molecular
techniques.
90
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Appendix I: Reagents used for DNA extraction
• Extraction buffer: pH: 8
⋅ 100 mM Tris-HCl
⋅ 100µM EDTA (Ethylene di-ammine tetra acetic acid)
⋅ 2.5M NH4Cl
• 500µl isopropanol
• 70% Ethanol
• 100-300µl distilled water
Appendix II: Reagent used for preparation of Agarose gel
• Agarose powder (Bangalore Genei, India)
• 5X TBE Buffer (pH 8.3); Composition(for 1L):
• Tris: 54g (Bio Basic Inc., Canada)
• Boric Acid 27.5g (Bio Basic Inc., Canada)
• EDTA: 20 µl (.05M, pH 8.0) (Bio Basic Inc., Canada)
• Ethidium Bromide (SRL, India)
Appendix III: Preparation of 5X TBE (1L):
• At first 54g Tris base was taken in 800 mL ddH2O.
• The mixture was stirred for some time.
• An amount of 27g boric acid was added.
• Stirring was done for several minutes.
• An amount of 20 µl EDTA (.05M, pH 8.0) was added.
Appendix IV: Materials and Chemicals used for inclusion body study under light microscope
♦ Materials
• Plant materials (leaves)
• Razor blade
100
• Tweezers
• Watch glass
• Dropping bottles
• Deionised water
• Cover slip
• Light microscope with oil emersion objectives
• Hot plate with temperature regulation
• Immersion oil
♦ Chemicals and solutions
• 0.1% azure-A stain (Eastman Kodak Co.)
• 2-methoxyethanol (Eastman Kodak Co.)
• 2-methoxy ethyl acetate (Eastman Kodak Co.)
• 95% ethanol
• Euparal (embedding materials) (GBI [Labs] Ltd., Manchester,
England)
• Deionised water