Studies on variability of mungbean yellow mosaic virus isolates of mungbean and ecofriendly ...

212 Pages·2017·1.76 MB·English

Studies on variability of mungbean yellow
mosaic virus isolates of mungbean and
ecofriendly management of disease
A
THESIS
SUBMITTED TO THE
ANAND AGRICULTURAL UNIVERSITY
IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE AWARD OF THE DEGREE
OF
DOCTOR OF PHILOSOPHY
(AGRICULTURE)
IN
PLANT PATHOLOGY
BY
PRIYA JOHN
M.Sc. (Agri.)
DEPARTMENT OF PLANT PATHOLOGY
B.A. COLLEGE OF AGRICULTURE
ANAND AGRICULTURAL UNIVERSITY
ANAND-388 110
2005
Regn. No. 04-04853-2001
Studies on variability of mungbean yellow mosaic virus isolates of
mungbean and ecofriendly management of disease
Name of student Major Advisor
Priya John Dr. Ashok Mishra
DEPARTMENT OF PLANT PATHOLOGY
B.A. COLLEGE OF AGRICULTURE
ANAND AGRICULTURAL UNIVERSITY
ANAND-388 110
ABSTRACT
Amidst the din of biodiversity within begomoviruses, a quantum leap in the
occurrence of yellow mosaic disease (YMD) in pulses was realized. Gujarat is not an
exception to this plague, especially Mungbean yellow mosaic virus (MYMV)
crippling Vigna radiata, have attracted considerable attention. Vigna belongs to the
family: Leguminosae; subfamily: Papilionoidae; tribe: Phaseoleae; subtribe:
Phaseolinae. Mungbean repletes excellent nutritional qualities. Inquisitive about the
natural variability of MYMV, the study was divided into two parts, the first part
included the collection and characterization of natural variants of MYMV from Anand
followed by the second part which comprised DNA hybridization based investigations
of the biodiversity among MYMV isolates from different areas of Gujarat.
Concomitant repertoire of specific variants showing symptoms of yellow mosaic, was
carried out from diverse areas of Gujarat including Anand, Navsari, Ahmedabad,
Sardarkrushinagar, Himmatnagar and Rajkot. Nomenclature of Anand isolates
understudy was done according to their original host and place of collection. So the
mungbean isolates from Anand were named as mb-AND1, mb-AND2, mb-AND3
and mb-AND4. In a case-by-case basis evaluation of Anand isolates, mb-AND1 was
commendably selected for major studies. Isolate mb-AND1 has the potential to infect
Abstract
mungbean cultivars (K 851, GM-2-12-24 and GM-02-01), French bean, mothbean,
dolichos, soybean and cowpea but not blackgram and pigeonpea by whitefly-mediated
transmission, which makes it distantly distinct from other, reported MYMV from
India. Transmission by graft and not by sap, distant it from MYMV of Thailand and
West Malaysia. The induction of yellow mosaic symptoms, changes in micronutrient
level and reduction in yield are complementary mode effects of MYMV including
mb-AND1.
Polymerase chain reaction (PCR) was carried out by using Mungbean yellow
mosaic India virus (MYMIV) primers. Amplification was seen only with DNA
extracted from mungbean, soybean and cowpea and the amplicon product of 1.8 kb
was obtained in the case of mungbean (mb-AND3), soybean (mb-AND1) and cowpea
(mb-AND1 and mb-AND2) samples, that represents left half of DNA A with AC abut
and Rep C2 primers. A 750 bp amplicon representing right half of DNA A was
obtained with CPV1 and AC abut primers in mungbean (mb-AND4) samples. PCR
amplification with abutting primers could not be achieved at all. Consequently, the
viral replicative forms, identified to represent double stranded super coiled form by
analogy with the profile of other begomoviruses, were obtained in mb-AND1
(mungbean), mb-AND1 (cowpea) and mb-AND1 (mothbean) by cesium chloride
(CsCl ) density gradient centrifugation and confirmed by Southern hybridization.
2
Radiolabelled probes prepared from PCR amplified DNA A fragment coding for coat
protein (CP) of MYMIV were used. mb-AND1 (cowpea) responded appreciably as
the concentration of viral replicative forms were predominantly high. Cowpea
(mb-AND1) fractions were pooled together and was taken for cloning in pUC 18
DNA at Kpn I and Bam HI loci followed by digestion with Bgl I. Southern
ii
Abstract
hybridization using probe of CP and movement protein (MP) gene of MYMIV,
restriction analysis and PCR amplification revealed clones pCGK 25 and pCGB 31
representing DNA A and DNA B. Sequencing of these clones was carried out in an
automated DNA sequencer (Microsynth GmbH, Sequencing Group, Schutzenstrasse
15 PostFach, CH-9436 Balgach, Switzerland; [email protected] & ABI
Prism, Perkin Elmer and University of Delhi, South campus, New Delhi) and it was
designated in the light of guidelines comprehended by the study group on
Geminiviridae, International Committee on Taxonomy of Viruses (ICTV) as
Mungbean yellow mosaic India virus-cowpea (Gujarat) {MYMIV-Cp (Guj)} which
harnesses more than 89 % identity with MYMIV. These sequences are available in
nucleotide sequence data libraries-DNA Data Bank of Japan (DDJB), European
Molecular Biology Laboratory (EMBL) and GenBank databases, USA, under
accession numbers AY937195 (DNA A) and AY937196 (DNA B) respectively. The
genome structure of MYMIV-Cp (Guj) embodies two virion sense genes, AV2 and
AV1 in DNA A and one BV1 in DNA B and complementary sense genes are AC1,
AC2, AC3 and AC4 in DNA A and BC1 in DNA B. Comparative nucleotide sequence
analysis evidenced the alignment of MYMIV-Cp (Guj) within the MYMIV cluster.
Koch’s postulates of the cloned DNA components of mb-AND1 (cowpea)
were proved by sprouted seed method of agroinoculation on mungean cv. K 851. The
explored host range was confined to mungbean (GM-2-12-24 and Delhi local), French
bean and blackgram, but no symptom induction was seen in mungbean (GM-9907,
GM-9908, GM-9922 and GM-02-01), cowpea and Nicotiana benthamiana. DNA A
alone of MYMIV-Cp (Guj) did not impassion any symptom on all the tested plant
species. Pseudorecombination of the MYMIV-Cp (Guj) DNA A and MYMIV-
iii
Abstract
Cp (Del) DNA B elucidated the importance of DNA B in symptom development by
agroinoculation as the resistance of above-mentioned mungbean cultivars was
overcome by the artificial recombination.
Lack of variability was found between the full length
[mb-AND1=MYMIV-Cp (Guj)] and the viral fragments cloned and sequenced from
the Anand isolates of MYMV (mb-AND2, mb-AND3 and mb-AND4), which
indicated that isolates from Anand may be considered as a single population of
MYMIV-Cp (Guj). The accession numbers for cowpea (mb-AND2) 1.8 kb (L),
mungbean (mb-AND3) 1.8 (L) and mungbean (mb-AND4) 750 (R) are AY937197,
AY937198 and AY937199 respectively.
However, differentiation of natural variants of MYMV isolates of mungbean
in Gujarat could be detected by employing MYMIV-Cp (Guj) as probe in DNA-DNA
hybridization and they could be classified into three groups (i) Strong hybridization to
DNA A and B {mungbean (Anand), soybean (Navsari and Rajkot)}; (ii) Weak
hybridization to DNA A and B {mungbean (Himmatnagar, Navsari and Ahmedabad),
cowpea (Ahmedabad)}; (iii) Weak hybridization to DNA A alone {mungbean
(Sardarkrushinagar), dolichos (Anand)}. It clearly revealed that the MYMV isolate of
mungbean from Sardarkrushinagar is having genomic variation in DNA B component
as compared to MYMIV-Cp (Guj).
Satellite DNA β (1.3 kb) was found to be associated with begomovirus
infected leguminous and non-leguminous hosts (dot blot positive with DNA A probe
of Indian cassava mosaic virus) in Anand, whose role is yet to be understood.
The botanicals possessing antiviral principles, Clerodendrum inerme and
Boerhaavia diffusa inhibited yellow mosaic disease induced by isolate mb-AND1
iv
Abstract
upto 93.33 % (co-inoculation) and 86.67 % (pre-inoculation) respectively, implicating
the role of botanicals as an effective and efficient ecofriendly measure to manage this
vexed problem. Maximum disease inhibition (60.00 %) was shown in aureofungin
(50 ppm) treated plants in the co-inoculation strategy.
v
Acknowledgements
Prior to all concerns, I take this delightful moment of
my life to quote eternal glory and honour to my Heavenly
Father “Daddy Jesus” who said “Is any thing too hard for
the Lord?” (Gen. 18:14).
I greatly acknowledge Dr. Ashok Mishra, Principal &
Dean, College of Agriculture, Junagadh Agricultural
University, Junagadh with deepest sense of gratitude, who
as an ideal Guide, budded the enthusiasm in taking up
Plant Virology as major subject and helped me to look into
the mysteries of plant viruses. Also influenced enormously
to learn molecular techniques. Sir always buoyed me to
work hard which erased all the signs of fatigue. Strategic
designing of the present investigation is obviously the
insight of my major advisor which had brought a paradigm
shift in me in understanding plant pathogens.
I was blessed with the revelation that the real joy of
doing things can be found in one’s life, that was my
meeting, sharing and spending the time with a humble,
brilliant mind with full of immense affection-Dr. V.G.
Malathi, Principal Scientist, Advanced Center for Plant
Virology, IARI, whose inspiration, generosity in sharing her
valuable time and knowledge, who has selflessly enriched
me as an elder sister and as a good teacher, also who
opened up the secrets in the beauty of “Indian
Geminiviruses”, words are inadequate to incorporate the
love showered beyond measure by madam; I unveil with
pride privilege my sincere regards, admiration and respect
to Dr. V.G. Malathi madam.
I am thankful to Dr. Y.S. Ahlawat for giving me
permission to carryout the committed molecular work in
ACPV, New Delhi. Also I am grateful to Prof. M.C.
Varshneya, VC, AAU allowing me to complete my work
under Dr Ashok Mishra.
Dr. R. Bhatnagar, my minor Guide, whose unspoken
support took me to unsophisticated conversations with
childlike enthusiasm in enjoying the field of biochemistry,
is greatly acknowledged.
Acknowledgement
I am thankful to committee members, Dr. G.B. Valand
(Professor & Head, Dept. of Plant Pathology, BACA) and Dr.
N.P. Patel for their valuable suggestions in appraisal of the
thesis.
My sincere gratitude and regards are due to cheerful,
amicable and energetic teachers of Dept. of Plant
Pathology, BACA who helped me in establishing myself as
a part of Plant Pathology family in Anand, with sufficient
passion for one’s chosen field of study.
The cheerful company provided by Shilpa, Sharda,
Kuldeep and Hema is at forefront. My thanks to Mitesh and
Ramji who helped me in carrying out whitefly rearing and
other accessory works. My gratitude to Khadu bhai for his
help.
My sincere thanks to all the inmates of ACPV, New
Delhi, Dr. Usha, Padma, Ahmad, Perisamy, Raja, Sourab,
Sumiya, Sarita, Shanika and Tamilvannan who helped me
directly or indirectly is greatly acknowledged. Words are
less to say the sincere thanks to Radhu, Betsy and Sujatha
who helped me feel at home during my stay in Madakini
hostel. Also remembrances of Manju and Jaini will remain
with me. My thanks to Sivalingam for his intellectual
fervor, clarity of thought and passion for perfection in
teaching me the molecular techniques, rightly called as
Guru.
My deepest gratitude to my loving parents (both
sides) for their unfailing, emotional support with their
sacrificial love during studies, research and writing,
because I seek in them that attitude towards life which I
loved, admired and wanted to come out in this work.
It is greatly appreciated for the cooperation,
patience, continuous encouragement and generous
sharing granted to me by my loving sister-Mammachy and
Aby chachan; Talitha and Kareena, which can never be
forgotten. I also give my heartfelt thanks for the affection
given by my loving chachan and chechi; Allen and little
Abyn. Words are few to express my sincere love to
younger brother “Sonchidiya” for his benevolent presence
during vacation and other times making me prepare
always for a fresh start.
ii
Acknowledgement
My personal triumphs and tribulations in achieving
me to self-sufficiency to the committed work is not just
possible without my constant companion-my loving
husband Naveen as an unsparing critic for his tough
comments, scientific thinking accompanied, all
compromises and affection is sincerely expressed with
heartfelt gratitude.

Anand Priya
John
Date: July 2005

iii
CONTENTS
CHAPTER TITLE PAGE
NO. NO.
I INTRODUCTION 1 – 8
II REVIEW OF LITERATURE 9 – 37
III MATERIALS AND METHODS 38 – 71
IV RESULTS 72 – 118
V DISCUSSION 119 – 143
VI SUMMARY AND CONCLUSION 144 – 149
REFERENCES i – xxxi
APPENDICES I - XV