Detection of The Causal Agent of Leaf Mosaic of Jute

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
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
2
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
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Prof. Dr. M. Ashrafuzzaman Supervisor
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Prof. Dr. Md. Ayub Ali Co-supervisor
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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 cosupervisor 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 cooperation 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 cooperation 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
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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 Tbudding) 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.
CONTENTS
CHAPTER
TITLE
PAGE
ACKNOWLEDGEMENTS ABSTRACT CONTENTS LIST OF FIGURES LIST OF PLATES LIST OF TABLES CHAPTER 1 INTRODUCTION CHAPTER 2 REVIEW OF LITERATURE
iv v vi x xi xiii 1 5
2.1 Symptoms 2.2 The causal agent and mode of inheritance 2.3 Transmission of the causal agent 2.4 Incidence of leaf mosaic of jute and its effect on yield
5 6 7 9 11 12 15 15 15 15 16
2.5 Observation of inclusion body by light microscopy 2.6 Molecular detection
CHAPTER 3 MATERIALS AND METHODS
3.1 Place and time 3.2 Collection of seeds 3.3 Study of leaf mosaic of jute on the growing plants
3.4 Seed to seedling transmission of the causal agent of leaf mosaic of jute by cassette holder method
6
CHAPTER
TITLE
PAGE
3.5
Seed to plant to seed transmission of the causal agent of leaf mosaic of jute
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3.6 Graft transmission of the causal agent of leaf mosaic of jute 3.6.1 Peg grafting 3.6.2 Veneer grafting 3.6.3 Gooti (approach) grafting 3.6.4 T- budding 3.6.5 Root grafting 3.7 Vector Transmission of the causal agent of leaf mosaic of jute
17 18 18 19 19 19 20
3.7.1 Vector population 3.7.2 Acquisition Feeding Period (AFP) 3.7.3 3.7.4 3.8 Inoculation Feeding Period (IFP) Persistence of causal agent in the vector Study of inclusion bodies of the causal agent of leaf mosaic of jute under light microscope 3.9 Detection of the causal agent of leaf mosaic of jute by molecular techniques 3.9.1 Collection of leaf samples
7
20 20 21 21
24 24
24
CHAPTER
TITLE
PAGE
3.9.2
Preparation of DNA samples
25 25 26 29 32 32 33 33 33
3.9.3 Protocol for preparation of 1% agarose gel 3.9.4 Amplification of DNA by PCR 3.9.5 Selection and design of primer 3.9.6 Electrophoresis, gel staining and documentation 3.9.7 Observation of DNA Bands
CHAPTER 4 RESULTS
4.1 4.2
Study of leaf mosaic of jute on growing plants Seed to seedling transmission of the causal agent of leaf mosaic of jute by cassette holder method
4.3
Seed to plant to seed transmission of the causal agent of leaf mosaic of jute Graft transmission of the causal agent of leaf mosaic of jute
39
4.4
42 42 42 43 43 43
4.4.1 Peg grafting 4.4.2 Veneer grafting 4.4.3 Gooti or Approach grafting 4.4.4 T-budding 4.4.5 Root grafting 4.5 Vector transmission of the causal agent of leaf mosaic of jute
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CHAPTER
TITLE
PAGE
4.5.1 Vector population 4.5.2 Acquisition Feeding Period (AFP) 4.5.3 Inoculation Feeding Period (IFP) 4.5.4 Persistence of causal agent in the vector 4.6 Study of inclusion body of the causal agent of leaf mosaic of jute under light microscope 4.7 Detection of the causal agent of leaf mosaic of jute by molecular techniques
CHAPTER 5 DISCUSSION CHAPTER 6 SUMMARY AND CONCLUSION REFERENCES APPENDICES
55 55 55 56 64
68
70 75 77 86
LIST OF FIGURES
FIGURE
TITLE
PAGE
1 2 3 4 5
Diagrammatic representation of the PCR cycle Status of percentage of germination and seedling with chlorotic spots as grown in aluminum tray Status of percentage of germination and seedling with chlorotic spots as grown in cassette holder Status of mosaic infection at different days in two cultivars of jute Relationship between number of whiteflies and transmission of the jute mosaic causal agent at 24 hours of AFP and IFP Relationship between number of whiteflies and transmission of the jute mosaic causal agent at 48 hours of AFP and IFP Relationship between Aquision Feeding Period (AFP) and transmission of jute leaf mosaic causal agent Relationship between Inoculation Feeding Period (IFP) and transmission of jute leaf mosaic causal agent Relationship between days (persistence) and plants infected after acqusition
28 37 38 41
61
61
62
62
63
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LIST OF PLATES
PLATE
TITLE
PAGE
1 2 3 4
Whitefly Inoculation of healthy jute seedling by vector (whitefly) Management of inoculated plant in insect proof net-cage Seedling with yellow dot spot on cotyledon (Grown on sand in aluminium tray)
22 22 23
35
Seedling with yellow dot spot on cotyledon growing cassette holders 35 36 36 47 48 49 50 51 52 53 successful 54
Symptoms of leaf mosaic of jute at seedling stage (20 days old)
7 8 9
Symptoms of leaf mosaic of jute on 60 days old plant Peg grafting between D-154 D-154 at the initial stage Peg grafting between D-154 D-154 after successful transmission
10 11
Veneer grafting between D-154 D-154 at initial stage Veneer grafting between D-154 D-154 after successful transmission
12 13
Approach grafting between CVL-1 CVL-1 at initial stage Approach grafting between CVL-1 CVL-1 after successful transmission
14 15
T-budding between CVL-1 CVL-1 at initial stage T-budding transmission between CVL-1CVL-1 after
PLATE
TITLE
11
PAGE
16
Distribution of inclusion bodies in the nucleus (100X magnification)
65
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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
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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
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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 2 3 4 5 6 7 8 9 10 11 12 13
Sequences of primers used in the study Percentage seedling with mosaic symptom with in two tests Seed to plant transmission of the causal agent of leaf mosaic of jute transmission of jute leaf mosaic in seeds collected from infected plants (1st season) grown in 2nd season Transmission efficiency of jute leaf mosaic causal agent by peg grafting Transmission efficiency of jute leaf mosaic causal agent by veneer grafting Transmission efficiency of jute leaf mosaic causal agent by approach grafting Transmission efficiency of jute leaf mosaic causal agent by T- budding Transmission efficiency of jute leaf mosaic causal agent by root grafting Effect of number of viruliferous insects on the transmission of causal agent of leaf mosaic jute Effect of AFP on transmission of leaf mosaic causal agent Effect of IFP on transmission of the causal agent of jute mosaic Persistence (days) of the causal agent of leaf mosaic of jute in the vector insect
29 34 40 40 44 44 45 45 46 57 58 59 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
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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.
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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 begomovirusspecific degenerate primers and suggested further study to confirm the association of virus with yellow mosaic disease of jute.
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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).
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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.
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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.
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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 preflowering 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.
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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).
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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
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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
26
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.
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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, CC45, 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.
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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
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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
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Severity was assessed based on scale: 0 4
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
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.
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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
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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)

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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.
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Plate 1: Whitefly
Plate 2: Inoculation of healthy jute seedling by vector (whitefly)
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Plate 3: Management of inoculated plant in insect proof net-cage
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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 2methoxy 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
37
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 l 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 65C for 10 minutes in water bath (WB-2400, Taiwan) and then centrifuged for 10 minutes at 10,000g.The supernatant fluid (approximately 250l) was transferred to a clean microfuge tube and 50l 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 200l 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 300l of distilled water. Finally the DNA samples were stored in a refrigerator at -20C. 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 (15152 cm in size) that was placed on a level table and the appropriate
38
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 9498 C for 2030 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 5065 C for 2040 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 primertemplate hybrid and began DNA synthesis.
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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 7580 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.
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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.
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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

Nucleotide sequence 5-GCATATGCAGGCCCACATYGTCTTYCCNGT-3 5-AATACTGCAGGGCTTYCTRTACATRGG-3
Primer * PAL1v1978 PAR1c496
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.
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Preparation of working solution of DNA sample Before PCR, DNA concentrations were adjusted to 25ng/l using following formula: V1 S1 = V2 S2 Where, V1 = final volume of DNA solution (l) S1 = final DNA concentration (ng/l) V2 = initial volume of DNA solution (l) S2 = initial DNA concentration (ng/l) Therefore, V1 = V2 S2 / S1 Reaction mix preparation to perform Polymerase Chain Reaction (PCR) A 10l PCR reaction mix contained the following reagents: Reagents 10X Ampli Taq polymerase buffer 5 M primer PAL1v1978 (+) 5 M primer PAR1c496 (-) 1.5 mM dNTP Ampli Taq DNA polymerase sample DNA extract Sterile NPW Total Quantity (l) 1.0 1.25 1.25 1.0 0.2 4.0 1.3 10
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Amplification buffer, 10X, pH 8.3: Tris HCl KCl MgCl2 BSA .05M .05M .07M .02% (w/v)
Taq DNA polymerase: Tris HCl Na2-EDTA DTT Glycerol .05 1mM 1mM 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.
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Thermal profile DNA amplification was performed in a thermal cycler (Master Cycler Gradient, Eppendorf, Germany). The thermal cycle was as follows 95 C for 3 minutes 95 C for 50 seconds 55 C for 50 seconds 72 C for 1 minutes 72 C for 10 minutes : Denaturation : 35 cycles : Annealing : Elongation or extension : Final step/complete extension
After completion of cycling program, reactions were held at 4 C
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 (0.1g/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.
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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 (BJC7370), 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).
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Table 2: Percentage seedling with mosaic symptom with in two tests

Variety Aluminum tray Germination (%) V1 (CVE-3) V2 (D-154) V3 (BJC-7370) V4 (CVL-1) V5 (BJC-43) V6 (O-795) V7 (CC-45) 74 82 55 65 69 56 62 Symptom bearing seedling (%) 6 8 3 4 5 1 4 Cassette holder Germination (%) 75 83 60 65 70 50 62 Symptom bearing seedling (%) 5 6 2 3 3 1 3
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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
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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

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Germination (%)
90
Symptom bearing seedling (%)
80
70
60
50
40
30
20
10
0 V1 V2 V3 V4 V5 V6 V7
Variety
Fig 2: Status of percentage of germination and seedling with chlorotic spots as grown in aluminum tray
50
90
Germination (%)
80
Symptom bearing seedling (%)
70
60
50
40
30
20
10
0 V1 V2 V3 V4 V5 V6 V7
Variety
Fig 3: Status of percentage of germination and seedling with chlorotic spots as grown in cassette holder
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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.
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Table 3: Seed to plant transmission of the causal agent of leaf mosaic of jute
Name of variety D-154 CVL-1 Mosaic plant (%) Mosaic leaf (%) No. of pods/plant No. of seeds/pod
Diseased Healthy Diseased Healthy 1.6 0.9 39.5 27.0 37 33 46 49 21 19 30 25
Table 4: transmission of jute leaf mosaic in seeds collected from infected plants (1st season) grown in 2nd season
Name of variety D-154 CVL-1
% germination
Mosaic plant (%) 30 DAS 60 DAS 56.43 38.69
Mosaic leaf (%)*
73.5 59.75
28.55 26
90.25 83.14
* 60 days after sowing
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Fig 4: Status of mosaic infection at different days in two cultivars of jute
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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, Tbudding 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-1CVE-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-1O-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%) combination CVL-1O-795.
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was found in the host
4.4.3 Gooti or Approach grafting In case of gooti (approach) grafting initial symptoms developed within 34 weeks and the full syndrome within 12 months. The variety CVL-1 gave highest percentage of successful grafts (90%) followed by grafting between host combination D-154CVL-1(80%). The highest percentage of successful transmission (85.71%) was observed in the host combination (D-154CVE-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-1CVE-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-1O-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.
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Table 5: Transmission efficiency of jute leaf mosaic causal agent by peg grafting
Variety Stock
D-154 CVL-1 CVE-3 CVE-3 CVL-1
Scion
D-154 CVE-3 CVE-3 CVL-1 O-795
Grafts Successful Successful attempted grafts transmission

5 5 5 5 5 5 3 4 3 1 4 2 3 1 0
Successful grafts (%)

100 60 80 60 20
Successful Severity Transmission (%)

80 66.67 75 33.33 0 3.12 2.68 3.54 1.98 0
Table 6: Transmission efficiency of jute leaf mosaic causal agent by Veneer grafting
Variety
Stock D-154 CVL-1 CVE-3 CVE-3 CVL-1 Scion D-154 CVE-3 CVL-1 CVE-3 O-795
Grafts attempted
6 5 5 6 4
Successful grafts
5 4 4 4 2
Successful transmission
4 3 2 4 1

83.33 80 60 66.67 50
Successful Transmission (%)

80 75 50 100 50
Severity
2.78 3.31 1.79 3.10 0.98
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Table 7: Transmission efficiency of jute leaf mosaic causal agent by approach grafting
Variety
Grafts
Successful grafts
Severity
Healthy Infected attempted
CVL-1 D-154 D-154 O-795
CVL-1 CVE-3 CVL-1 CVL-1
10 10 10 10
9 7 8 3
7 6 6 1
90 70 80 30
77.77 85.71 75 33.33
2.56 1.98 3.54 3.12
Table 8: Transmission efficiency of jute leaf mosaic causal agent by Tbudding

Variety
Budding attempted
5 6 5 6 5
Successful buds
4 4 2 5 0
3 3 2 3 0
Successful buds (%)

60 66.67 40 83.33 0

75 75 100 60 0
Severity
Stock
CVL-1 CVL-1 CVE-3 CVE-3 CVL-1
Scion
CVL-1 CVE-3 CVL-1 CVE-3 O-795
3.18 2.87 3.01 2.67 0
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Table 9: Transmission efficiency of jute leaf mosaic causal agent by root grafting
Variety Grafts attempted 5 5 D-154 5 5 5 Successful grafts 3 3 2 2 4 No. successful transmission 2 1 0 0 1 28.57 Successful transmission (%)
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Plate 8: Peg grafting between D-154 D-154 at the initial stage
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Plate 9: Peg grafting between D-154 D-154 after successful transmission.
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Plate 10: Veneer grafting between D-154 D-154 at initial stage
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Plate 11: Veneer grafting between D-154 D-154 after successful transmission
63
Plate 12: Approach grafting between CVL-1 CVL-1 at initial stage
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Plate 13: Approach grafting between CVL-1 CVL-1 after successful transmission
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Plate 14: T-budding between CVL-1 CVL-1 at initial stage
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Plate 15: T-budding between CVL-1 CVL-1 after successful transmission
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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 corelation 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,
68
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= 11.57x27.66, 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 BJC7370, which started declining after the fifth day (Table 13).
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Table 10: Effect of number of viruliferous insects on the transmission of causal agent of leaf mosaic jute
No. of insects/plant No. of inoculated plants 1 3 5 10 15 20 20 20 20 20 No. of infected plants 0 6 9 14 19 Transmission (%) 0 30 45 70 95 No. of inoculated plants 20 20 20 20 20 No. of infected plants 3 6 9 15 20 Transmission (%) 15 30 45 75 100 24 hours AFP*, IFP** 48 hours AFP, IFP
*AFP= Acquisition Feeding Period **IFP= Inoculation Feeding Period
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Table 11: Effect of AFP on transmission of the causal agent of jute mosaic
Acquisition Feeding Period (AFP) 1 minute 5 minutes 10 minutes 15 minutes 30 minutes 1 hour 3 hours 5 hours 24 hours 48 hours
No. of inoculated plants 20 20 20 20 20 20 20 20 20 20
No. of infected plants 0 0 0 0 2 6 8 11 16 18
Transmission (%)
0 0 0 0 10 30 40 55 80 90
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Table 12: Effect of IFP on transmission of the causal agent of jute mosaic
Inoculation Feeding Period (IFP)
1 minute 5 minutes 10 minutes 15 minutes 30 minutes 1 hour 3 hours 5 hours 24 hours 48 hours No of inoculated plants No of infected plants Transmission (%)
20 20 20 20 20 20 20 20 20 20
0 0 0 1 3 8 11 12 17 19
0 0 0 10 15 40 55 60 85 95
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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 CVL-1 CVE-3 D-154 BJC-7370 5 4 5 3 2 5 5 5 5 3 5 5 5 5 4 5 5 5 5 5 4 3 5 5 6 2 2 3 3 7 2 1 2 2 8 1 1 1 1 9 1 0 1 1 10 0 0 1 1 11 0 0 0 0 12 0 0 0 0 13 0 0 0 0 14 0 0 0 0
Five plants were used for each test
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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
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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
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Fig. 9: Relationship between days after acqusition (persistence) and plants infected
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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.
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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)
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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)
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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)
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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).
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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).
82
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
83
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
84
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).
85
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
86
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.
87
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.
88
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.
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REFERENCES
Ahmed, M. 1978. A whitefly vectored yellow mosaic jute. Food and Agricultural Organization (FAO). Plant Pathology Bulletin. 26(4): 169-171. Ahmed, Q. A. 1968. Diseases of jute in East Pakistan. Jute and Jute Fabrics Pakistan. 7(8): 147-151. Ahmed, Q. A.; Bishwas A. C.; Farukuzzaman A. K. M.; Kabir M. Q. and Ahmed N. 1980. Leaf mosaic disease of jute. Jute and Jute Fab. Bangladesh. Pp 913. Alam, M.1998. Effectiveness of three insecticides for the control of the spiraling whitefly of guava. Bangladesh journal of Entomology. 8: 53-58. Anonymous, 1953. Indian Central Jute Committee (ICJC) report. Jute Bulletin. 15: 545. Anonymous, 1959. Pakistan Central Jute Committee (PCJC) report. Jute Bulletin. 38: 356. Anonymous, 1985. Annual Report. Plant Pathology Division. Bangladesh Jute Research Institute (BJRI), Dhaka. Pp-381. Atzmon, G.; Oss, H. V. and Czosnek. H. 1998. PCR amplification of tomato yellow leaf curl virus (TYLCV) DNA from squashes of plants and whitefly vectors: Application to the study of TYLCV acquisition and transmission. European Journal of Plant Pathology. 104: 189194. Azad, A.K. and Wahab, M.A. 1984. Survey of leaf mosaic in Corchorus capsularis jute. Jute and Jute fabrics Bangladesh. 5-7. Banerjee, K.C. 1924. Chlorosis. Bengal Agricultural Journal. 4: 118. Banglapedia, 2006. http://www.banglapedia.org/httpdocs/HT/J_0135.HTM
90
Bawden, F.C. 1963. Plant virus and Virus Diseases, Waetham, Mass, USA. 3rd Edition. Page: 89-91. BBS, 2011. Statistical Year Book of Bangladesh. 22nd Edition, Statistics Division. Ministry of Planning, Govt. of the Peoples Rep. of Bangladesh, Dhaka. Page: 97-98, 317. Bist, N. S. and Mathur, R. S. 1964. Occurrence of two strains of jute mosaic virus in the Uttar Pradash. Current Science. 14: 434. Biswas, A. C. 1982. Studies of the leaf yellow mosaic disease and development of methods for screening jute against fungal pathogens. A Research Report submitted to the Department of Plant Pathology and Crop Physiology. Luisiana State Unv. USA. 15: 63-66. Biswas, A. C.; Asaduzzaman, M.; Sultana. K. and Taher M. A. 1989. Effect of leaf mosaic disease on loss of yield and quality of jute fibre. Bangladesh Journal of Jute Fibre Research, 14: 43-46. Biswas, A. C.; Sultana, K.; Begum, H. A.; Iqbal. S. and Farukuzzaman, A.K.M. 1992. Diseases of Kenaf (Hibiscus canabinas) and Mesta (Hibiscus sabdariffa) recorded in Bangladesh. Bangladesh Journal of Jute Fibre Research, 17: 103-109. Candresse, T.; Hammond, R. W. and Hadidi, A. 1998a. Detection and identifi cation of plant viruses and viroids using polymerase chain reaction (PCR). Pages 399416 in control of plant virus.diseases, edited by A. Hadidi, R.K. Khetarpal, and K. Koganezawa. APS Press, St. Paul, MN. Capoor, S. P. and Ahmad, A. U. 1975. Yellow vein mosaic disease of field pumpkin and its relationship with the vector, Bemisia tabaci. Indian Phytopathology, 28: 241-246.
91
Cohen, S. P.; Duffas, J. E.; Larsen, R. C.; Liu, H. Y. and Flock, R. A. (1983). Purification, Serology and Vector relationship of squash leaf curl virus-a whitefly transmitted Gemini virus, Phytopathology 73: 1669-1673. Conti, M. A. 1996. Whiteflies (Bemisia tabaci) as vector of plant viruses. Review of Plant Pathology. 75(2): 107. Cristie, R. G. 1977. Rapid staining procedures for differentiating plant virus inclusions in epidermal strips. Virology 31: 298-271. Cristie, R. G. and Edwardson, J. R. 1967. Light and Electron microscopy of plant virus inclusions. Florida Agricultural Experiment Station. Monogr. Ser. Pp- 9. Cristie, R. G.; Ko, N. J.; Falk, B. W.; Hiebert, E.; Lastra, R. and Kim, K. S. 1986. Light microscopy of geminivirus-induced inclusion nuclear inclusion bodies. Phytopathology 76: 124-126. Das, S.; Khokon, M. A. R.; Haque, M. M. and Ashrafuzzaman, M. 2001. Jute leaf mosaic and its effects on jute production. Pakistan Journal of Biological Science. 4(12): 1500-1502. de Mejia, M. V. G.; Hiebert, E. and Purcifull, D. E. 1985. Isolation and partial characterization of amorphous cytoplasmic inclusions associated with infection caused by two potyvirus. Virology. 142: 24-33 Dempsey, J. M. 1975. Fibre crops. University press. Florida. USA. Pp. 147. Deng, D.; Mcgrath, P. F.; Robinson, D. J. and Harrison, B. D. 1994. Detection and differentiation of whitefly-transmitted geminiviruses in plants and vector insects by the polymerase reaction with degenerate primers. Ann. Applied Biol., 125: 327-336.
92
Dennis, B. B. and Bajet, N. B. 2007. Molecular Detection of WhiteflyTransmissible Geminiviruses (Family Geminiviridae, Genus Begomovirus) in the Philippines. Philippine Journal of Science.136 (2): 87-101. Dijkstra, J.and Jager. C. P. 1998. Practical Plant Virology, Protocol and practice, Springe Laboratory Manual. New York. Pp- 384. Esau, K. and Magyarosy, A. C. 1979. Nuclear abnormalities and
cytoplasmicinclusions in Amsinkia infected with curly tor virus. J. Ultrastruc. Res. 66: 11-21. Finlow, R. S. 1917. Annual Reports of the Department of Agriculture. Bengal "chlorosis. Bengal Agricultural Journal. 4: 118. Finlow, R.S. 1939. The production of jute. Journal Text. Institute. 30: 352-373. Ghosh, R.; Paul, S.; Das, S.; Palit, P.; Acharyya, S.; Das, A.; Mir, J. I.; Ghosh, S. K. and Roy, A. 2008. Molecular evidence for existence of a New
World begomovirus associated with yellow mosaic disease of Corchorus capsularis in IndiaAustralasian Plant Disease Notes. 3: 59-62. Ghosh, T. and Basak, M. 1951. Chlorosis of jute. Science and Culture, 17 (6): 262-264. Ghosh, T. and Basak, M. 1961. Transmission of leaf mosaic (chloroosis) in jute. Jute Bulletin, 1-5. Gilbertson, R. L.; Rojas, M. R.; Russell, D. and Maxwell, D. P.1991. The use of asymmetric polymerase chain reaction and DNA sequencing to determine genetic variability among isolates of bean golden mosaic geminiviruses in the Dominican Republic. J. Gen. Virol. 74: 23-31. Goodman, R. M. 1981. Geminiviruses: the single-stranded DNA viruses of plants. Pages 879-910 in: Handbook of Plant Virus Infection. E. Kurstak, ed. North Holland/ Elsevier, Amsterdam. 943pp.
93
Hadidi, A.; Levy, L. and Podleckis. E.V. 1995 Polymerase chain reaction technology in plant pathology. Pages 167187 in Molecular methods in plant pathology, edited by R.P. Singh and U.S. Singh. CRC Press, Boca Raton, Florida, USA. Haque, M.; Ahmed and Ashrafuzzaman, M. 1998. Survey on the incidence of leaf mosaic of jute in different agroecological zones of Bangladesh. Annual report of Bangladesh Jute Research Institute, Plant Pathology Division. Pp.169-171 Harender, R.; Bhardwaj, M. L.; Sharma I. M. and Sharma, N.K. 1993. Performance of commercial okra (Hibiscus exculentus) varieties inrelation to disease and insect pests. Indian Journal of Agricultural Science. 63(11): 747-748 Henson, J. M. and French, R. 1993. The polymerase chain reaction and plant disease diagnosis. Annu. Rev. Phytopathol. 31: 81109 Hiebert, E.; Purcifull, D. E. and Christie, R. G. 1984. Purification and immunological analysis of plant virul inclusion bodies. Pages 225-2801 in: Methods in plant Virology. Vol. VIII. K. Maramorosch and H. Koprowski, eds. Academic Press, New York. Innis, M. A.; Gelfand, D. H.; Sninksy, J. J. and Ehite, T. J. 1990. PCR protocols a guide to methods and application. Academic Press. New York. Pp-482. Islam, K. M. M. 1993. Severity and sources of jute mosaic virus on white jute. Jute & Jute febrics Bangladesh. 19: 1-12. Islam, M. M. and Rahman, M. M. 2008. Hand book on agricultural technologies of Jute, Kenaf and Mesta crops. Bangladesh Jute Research Institute (BJRI). Dhaka. Pp-02.
94
Kim, K. S.; Shock, T. L. and Goodman, R. M. 1978. Infection of phaseolus vulgaris by bean golden mosaic virus: ultrastructural aspects. Virology 89: 22-23. Lange, L. 1980. Virology report, Danish Govt. Inst. of Seed Pathology for the Developing countries, 78 Ryvangs Alle, DK 2900, Hellerup. Denmark. Lastra, R., and Gil, F.1981. ultrstructural host cell changes associated with tomato yello mosaic. Phytopathology 71: 236-241. Li, R.; Salih, S. and Hurtt, S. 2004. Detection of geminiviruses in sweetpotato by polymerase chain reaction. Plant Dis. 88:1347-1351. Mahesh, Y.S.; Shankarappa, K.S.; Rangaswamy, K.T.; Prameela, H.A. and D.S.A. Narayana, D.S.A. 2010. Detection and characterisation of a begomovirus associated with leaf curl disease of ornamental croton (Codiaeum variegatum). J. Hort. Sci. Biotechnol., 85: 101-105. Maruthi, M. N.; Rekha, A. R.; Govindappa, M. R.; Colvin, J. and Muniyappa, V. 2006. A distinct begomovirus causes Indian dolichos yellow mosaic disease. Plant Pathol., 55: 290-290. Maruthi, M.N.; Rekha, A.R. and Muniyappa, V. 2007. Pumpkin yellow vein mosaic disease is caused by two distinct begomoviruses: Complete viral sequences and comparative transmission by an indigenous Bemisia tabaci and the introduced B- biotype. EPPO Bull., 37: 412-419. Mathews, R. E. F. 1982. Classifiation and nomenclature of viruses. Intervirology 17: 4-99. Mitra, D. K.; Ahmed, Q. A. and Prakash, N. 1984. Electron microscopy of the virus causing leaf mosaic of jute in leaf exudates. Science and culture. 50: 127-128.
95
Mullis, K. 1990. The unusual origin of the polymerase chain reaction. Scientific American. 262 (4): 5665. Muniyappa, V.; Maruthi, M. N.; Babitha, C. R.; Colvin, J.; Briddon, R.W. and Rangaswamy, K.T. 2003. Characterisation of pumpkin yellow vein mosaic virus from India. Ann. Applied Biol., 142: 323-331. Muniyappa, V.; Venkatesh, H. M.; Ramappa, H.K.; Kulkarni, R. S. and Zeidan, M. 2000. Tomato leaf curl virus from Bangalore (ToLCV-Ban4): Sequence comparison with Indian ToLCV isolates, detection in plants and insects and vector relationships. Arch. Virol., 145: 1583-1598. Naidu, R.A.; Hughesa, J. A. and Shoyinka, S. A. 2001. Future of plant virus disease research in sub-Saharan African agriculture. International Institute of Tropical Agriculture. Nigeria 261-276 Narayana, D.S.A.; Rangaswamy, K.T.; Shankarappa, K.S.; Maruthi, M.N. and Reddy, C.N.L. 2007. Distinct begomovirus closely related to cassava mosaic viruses cause Indian jatropha mosaic disease. Int. J. Virol., 3: 1-11. Rabindran, R.; Samy, K. P.; Subbalaksmi, B; Jeyarajan, R. and Doraisamy, S. 1988. Phyllody a new disease of jute (cotchorus olitorius L.) Review of Plant Pathology, 69 (6): 383. Raghupathy, N. 1989. Studies on the yellow mosaic disease of soybean (Glycine max). M. Sc. thesis, Namil Nadu Agricultural University, Coimbatore. Pp91. Raghupathy, N. 1995. Studies on the leaf curl virus disease of tomato (Lycopersicon esculentum). PhD. thesis, Namil Nadu Agricultural University, Coimbatore. Pp-189.
96
Rahman, M. M. 2010. Policy & prospect of jute & allied fibers with special reference to Bangladesh, Ministry of Textiles & Jute. Govt. of the People's Republic of Bangladesh. Bangladesh Secretariat. Dhaka. Pp-2. Raj, S. K.; Snehi, S. K.; Kumar, S.; Khan, M. S. and Pathre, U. 2008. First molecular identification of a begomovirus in India that is closely related to Cassava mosaic virus and causes mosaic and stunting of Jatropha curcas L. Australasian Plant Disease Notes 3(1): 6972. Robertson, N. L.; French, R. and Gray, S. M. 1991. Use of group specific primers and the polymerase chain reaction for the detection and identification of luteoviruses. J. Gen. Virol. 72: 1473-1477. Rojas, M. R.; Gilbertson, R. L.; Russell, D. R. and Maxwell, D. P. 1993. Use of degenerate primers in the polymerase chain reaction to detect whitefly transmitted geminiviruses. Plant Dis. 77: 340-347. Saha. R. 2001. Study on the transmission of jute leaf mosaic causal agent. M.S thesis. Department of Plant Pathology, Bangladesh Agricultural university, Mymensingh-2202. Saiki, R. K.; Gelfand, D. H.; Stoffel, S.; Scharf, S. J; Higuchi, R; Horn, G.T.; Mullis, K. B. and Erlich, H. A. 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science; 239(4839):487-91. Sastry, K. S. and Singh. S. J. 1973. Field evaluation of insecticides for the control of whitefly in relation to the incidence of YVM of okra. Indian Phytopathology. 26(1): 129-138. Schneider, H. 1959. The anatomy of tristeza-virus-infected citrus. In Wallace, J.M., (ed.), Citrus virus diseases, Univ. California. Div. Agri. Sci. Berkeley. Pp : 73-84.
97
Sharma, A.; Shankarappa, K. S.; Rangaswamy, K. T.; Dubey, R. K. and Maruthi, M. N. 2009. First record of duranta leaf curl, a begomovirus associated disease in India. Ind. J. Virol., 20: 88-89. Subramanian, K. S. 1979. Studies on yellow mosaic disease of lablab niger madivals (Dolichos lablab). PhD thesis, Namil Nadu Agricultural University, Coimbatore. Pp-102. Sultana, K.; Akter, N.; Begum, H. A. and Taher, A. 1995. Transmission of jute (Corchorus capsularies) leaf mosaic disease by infected pollen. Bangladesh journal of Jute fibre Research. 20: 85-87. Thongmeearkon, P.; Honda, Y.; Satio. Y. and Syamananda, R. 1981. Nuclear Ultrstructural changes and aggregation og virus like particles in mungbean yellow mosaic disease. Phytopathology 71: 41-44. Verma, P. M.; Rao, G. G. and Capoor, S. P. 1966. Yellow mosaic of Corchorus trilocalaris. Science and culture, 32:466 Zaman, M. A. and Albrechtsen. 1999. Studies on the seed borne leaf mosaic of jute. Danish Govt. Inst. of Seed Pathology for the Developing Countries. 78, Ryvangs Alle, DK-2900 Hellerup, Copenhagen, Denmark.
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Appendix I: Reagents used for DNA extraction Extraction buffer: pH: 8 100 mM Tris-HCl 100M EDTA (Ethylene di-ammine tetra acetic acid) 2.5M NH4Cl 500l isopropanol 70% Ethanol 100-300l 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 l (.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 l 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
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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
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DETECTION OF THE CAUSAL AGENT OF LEAF MOSAIC OF JUTE

MASTER OF SCIENCE (MS) IN PLANT PATHOLOGY

DEPARTMENT OF PLANT PATHOLOGY BANGLADESH AGRICULTURAL UNIVERSITY MYMENSINGH