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First report of airborne begomovirus infection in Melia azedarach (Pride of

India), an ornamental tree in India.

Article  in  Aerobiologia · October 2014

DOI: 10. 1007/s10453-013-9319-x

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Aerobiologia
DOI 10.1007/s10453-013-9319-x

BRIEF COMMUNICATION

First report of airborne begomovirus infection

in Melia azedarach (Pride of India), an ornamental
tree in India
Avinash Marwal • Anurag Kumar Sahu •

R. K. Gaur

Received: 8 May 2013 / Accepted: 3 October 2013

Ó Springer Science+Business Media Dordrecht 2013

Abstract The begomovirus infection in plants has
been widely reported throughout the world. The chief
carrier of this virus is the whitefly. All of the reports,
however, concern plants that grow at a stumpy height
from the ground; moreover, the whitefly transmits the
begomovirus infection to plants at this low height only
by residing under their leaves. To date, there has been
no record of the begomovirus infection in trees as the
prevalence of the whitefly at tree level is unlikely. For
this reason, this study focuses on and presents the first
report of airborne begomovirus infection in an
ornamental tree—the Melia azedarach (or Pride of
India) found on the Indian subcontinent.
Keywords Begomovirus  Airborne 
Ornamental tree  Melia azedarach
1 Introduction
Apart from curtovirus, mastrevirus, and topocuvirus, the
begomovirus represents one of the largest genera of the
Geminiviridae family (Fauquet et al. 2005). The
whitefly (Bemisia tabaci) is the chief vector for
begomovirus infections in ornamentals, weeds, and
crop plants (Boulton 2003), and it is especially prevalent
in its favorite environment—the tropical and subtropical
regions of the world. The begomovirus is the only genus
of Geminiviridae that has a bipartite genome with virus
genes resident on two circular ssDNA molecules (DNA-
A and DNA-B) (Marwal et al. 2013a).
Throughout the life, we are encircled by an
indistinguishable stratum of atmosphere. We know
that the atmosphere contains copious microscopic
particles such as pollens, allergens, dust particles, and
species of microorganisms ranging from bacteria to
fungi and viruses. And the viruses are considered the
foremost environmental danger to humans, animals,
and plants. Until now, very little has been known about
atmospheric virology. For example, an airborne
begomovirus was recently identified and lucratively
characterized (Whon et al. 2012). In this report, we
identify a begomovirus infection in an ornamental
tree—the Melia azedarach—that is transmitted aeri-
ally without assistance from the whitefly.

2 Materials and methods

Avinash Marwal and Anurag Kumar Sahu have contributed
equally to this work.
2.1 Virus sources
A. Marwal  A. K. Sahu  R. K. Gaur (&)
Department of Science, Faculty of Arts, Science and
Over the past few decades, there has been an
Commerce, Mody Institute of Technology and Science,
Lakshmangarh, Sikar 332311, Rajasthan, India increasing interest in geminiviruses, especially
e-mail: gaurrajarshi@gmail.com begomoviruses, as many of the diseases they cause

123

have now reached epidemic proportions (Brown and
Czosnek 2002). Ornamental plants serve as an alter-
native host for the begomovirus in gardens, and they
may facilitate begomovirus transmission to crop
plants, thus enhancing the host range of this virus in
different regions of the India. Keeping this in mind, we
surveyed different gardens during 2011. M. azedarach
trees were found with leaf curling and crinkled leaves
disease—symptoms typical of a begomovirus infec-
tion. To investigate their possible begomovirus infec-
tion, we collected infected M. azedarach tree samples
(designated as isolate DB) from a location at latitude:
30N 200 84.40 and longitude: 74E 990 24.8500 (the
Army Cantonment, Bhatinda, Punjab province, India).
2.2 Extraction of total DNA
The leaf samples were cleaned, cut, rolled in a piece of
tissue paper, and stored at -20 °C until DNA isolation.
To obtain the molecular characterization, total DNA was
extracted from leaves of infected tree as well as from
leaves of healthy trees using the cetyltrimethylammo-
nium bromide (CTAB) method (Manen et al. 2005).
2.3 Identification of begomovirus components
by PCR
PCR was performed using a pair of degenerate primers
specific to the coat protein region of the begomovirus.
The forward primer sequence was GGRTTDGARG
CATGHGTACATG (AC 1048), and the reverse
primer sequence was GCCYATRTAYAGRAAGCC-
MAG (AV 494). The primers had been validated and
used previously in screening begomovirus varieties.
Nearly 40 begomoviruses had been screened earlier
such as the Croton yellow vein mosaic virus
(HQ631429), Croton yellow vein mosaic Hisar virus
(JN000701), Datura leaf curl virus (JN000702), Vinca
yellow vein virus (JQ693139), and Cotton leaf curl
virus (JQ693143) (Marwal et al. 2012).
A typical PCR contained about 100 ng DNA
template, Taq 109 buffers (10 mmol/L Tris–HCl,
pH 8.8; 50 mmol/L KCl) 25 mmol/L MgCl2,
200 lmol/L of each dNTPs, 2 units of Taq DNA
polymerase, nuclease free water, and 10 pmol/L of
each primer. The PCR thermal profile was pre-PCR by
denaturation at 94 °C for 120 s followed by 35 cycles
of denaturing at 94 °C for 60 s, annealing at 55 °C for
60 s, extension at 72 °C for 60 s, and a final extension
123
Aerobiologia
at 72 °C for 5 min. After amplification, 4 ll aliquot
from each sample was electrophoresed in a 1 %
agarose gel and visualized by staining with ethidium
bromide and UV illumination (Marwal et al. 2013b).
To detect any DNA-B component in the diseased
trees, the primer pair PCRc1 and PBLlv2040 was used
(Rojas et al. 1993; Bela-ong and Bajet 2007) as the pair
displays the same PCR conditions and reaction as that
in begomovirus DNA-A. To test whether a satellite
molecule was associated with the isolate GD, a
universal primer pair specific to alphasatellite and
betasatellite was also used to amplify the putative DNA
(Briddon et al. 2002; Bull et al. 2003). The PCR for
alphasatellite and betasatellite was the same as for
DNA-A, which we mentioned above. The PCR thermal
profile was pre-PCR by denaturation at 94 °C for 120 s
followed by 35 cycles of denaturing at 94 °C for 60 s,
annealing at 68 °C for 60 s, extension at 72 °C for
60 s, and a final extension at 72 °C for 5 min.
2.4 Cloning and sequencing
The amplified PCR product was purified and cloned
into the Promega pGEM-T vector system following
the manufacturer’s instructions. The clones were
sequenced and the details were submitted to NCBI.
BLAST analysis was performed to reveal their sim-
ilarity to other begomovirus sequences in the database.
2.5 Koch postulate and Southern hybridization
Moreover, to confirm Koch’s postulate for the virus
and assess the infectivity and phenotype symptoms of
the variants, infected clones (three construct) of virus
variants were constructed. The constructs were mobi-
lized into Agrobacterium tumefaciens by the freeze–
thaw method, which were agroinoculated 2–3 times
with the Hamilton syringe in N. benthimiana at 4–6 leaf
stage. For each construct, 15 plants were tested
individually. The presence of a begomovirus was
further confirmed by Southern blot hybridization using
the Papaya leaf curl virus as a general probe for
begomoviruses (Kon et al. 2003).
3 Results
The beautiful tree M. azedarach, commonly known as
Pride of India or the Chinaberry tree, belongs to the
Aerobiologia
Fig. 1 Symptoms such as leaf curling and crinkled leaves
exhibited by Melia azedarach (Pride of India) from which the
begomovirus DNA-A and DNA-B were isolated
Meliaceae family. The tree grows to a height of 15 m
and is used for timber in the Indian subcontinent
(Langeland and Burks 2005). It has an esthetic beauty
seen in its twin-colored leaves and beautifully pat-
terned purple flowers. It is widely cultivated as an
ornamental tree to line gardens.
During our 2011 survey of begomovirus infections,
leaf curling and crinkled leaves, diseases typical of
begomovirus, were observed in M. azedarach trees
(Fig. 1). Considerable time and expense are required
to obtain complete genome sequences. However, full
length coat protein gene sequences are accepted by the
International Committee on the Taxonomy of Viruses
(ICTV) for provisional classification of begomovirus-
es when complete genome sequences are unavailable
(Mayo and Pringle 1998).
Positive PCR confirmed the begomovirus infection
in the isolate DB of M. azedarach. The PCR product of
a complete coat protein region of DNA-A having an
expected size (*550) was obtained from infected
trees along with the DNA-B component (*450). They
were suitably cloned into the pGEM-T vector and
sequenced, the sequence having accession numbers
JQ693137 (Melia leaf curl virus) and KC206080
(Melia leaf curl virus segment DNA-B), respectively.
We did not find any satellite molecules associated with
the begomovirus.
In nucleotide alignments, the begomovirus DNA-A
revealed the highest nucleotide sequence identities—92
to 97 % with Papaya leaf crumple virus (HE580236),
Papaya leaf crumple virus (HM140369), Vinca yellow
vein virus (JQ693139), Rose leaf curl virus (GQ4783
42), and Pedilanthus leaf curl virus (HM134231), and it
had a lower sequence identity of 87–89 % that matched
with the Tomato leaf curl virus (GU732204) and Cotton
leaf curl Pakistan virus (HF549184). In contrast, the
begomovirus DNA-B had the highest nucleotide
sequence identities (90–95 %) with Tomato leaf curl
New Delhi virus DNA-B component (X89653), and
Bhendi yellow vein mosaic virus DNA-B component
(HQ586007), and it showed a lower sequence identity of
83–88 % with Gossypium punctatum mild leaf curl
virus DNA-B component (FJ218489), Tomato leaf curl
New Delhi virus DNA-B component (GU112087), and
Bhendi yellow vein mosaic virus DNA-B component
(HQ586005).
The positive PCR showed the presence of begomo-
virus. For further confirmation of begomovirus infec-
tivity, Southern hybridization was performed. In the
Southern hybridization technique, all samples from
symptomatic plants were hybridized with a probe,
whereas samples extracted from non-symptomatic
plants did not show positive results. Hybridization of
the Papaya leaf curl virus probe with the DNA
fragment on the filter membrane further indicated that
this fragment contained a DNA sequence comple-
mentary to the probe. This strong indicator showed
that the virus in M. azedarach had some homology
with the Papaya leaf curl virus.
Moreover, to identify the parental begomovirus
responsible for infection in M. azedarach, various
samples of begomovirus-infected plants were collected
and analyzed that had been growing on the ground
within a 1 km range of the infected trees. The samples
included an ornamental plant, Vinca rosea (Marwal
et al. 2013a), crop species such as radish (Raphanus
sativus), chili (Capsicum annuum), tomato (Solanum
lycopersicum), luffa (Luffa cylindrica), and spinach
(Spinacia oleracea). Their DNA was extracted, fol-
lowed by PCR, cloning, and sequencing using the same
protocol described above for M. azedarach.
The obtained sequences were deposited in NCBI
and have the following accession numbers JQ693139
(Vinca yellow vein virus), JN998450 (Raphanus
sativus leaf curl Bhatinda virus), JN000700 (Chili
leaf curl Bhatinda virus), JN009664 (Tomato leaf curl
Bhatinda virus), JN998451 (Luffa cylindrica yellow
mosaic Bhatinda virus), and JN998453 (Spinacia
oleracea leaf curl Bhatinda virus). Sequence analysis
(Zhang et al. 2000) showed that the virus in M.
123

Table 1 Depicting the results of Koch’s postulates
S. no. Construct No of Percentage
test plants of infection
1 1 15 100
2 2 15 100
3 3 15 100
azedarach had a 97 % identity with the Vinca yellow
vein virus isolated from Vinca rosea growing at
ground level at a distance of 30 m from the M.
azedarach tree, which was located at latitude: 30N 200
87.5300 and longitude: 74E 990 06.1600 .
A test was performed to conclusively show that the
virus was transmitted through the air. The whitefly
(B. tabaci) is the chief vector for begomovirus
transmission in plants. To identify the route of
begomovirus infection in M. azedarach and to find
whiteflies in M. azedarach trees, daily continuous
observation during early morning hours was performed
for a four-month period. During the course of this
investigation, the leaf curling symptoms continued
spreading to the healthy trees, even though the
prevalence of whiteflies at tree height (6–7 m) under
the surface of infected leaves was not observed.
Therefore, one might readily conclude that the aerial
route of begomovirus infection in these M. azedarach
trees is not attributable to whiteflies.
To show that trees can be infected in the absence of
the whitefly vector, control conditions provided in a
greenhouse permitted the exposure route of the virus to
also be studied during the experiments using N. benth-
imiana. The construct developed to test Koch’s postu-
lates caused typical symptoms when inoculated into N.
benthimiana (Wege et al. 2000). For each construct,
about 15 N. benthimiana plants grown in an insect-free
greenhouse were taken (Table 1). These control plants
developed the same symptoms as were observed in M.
azedarach. Further, these infected plants along with
healthy plants were subjected to whiteflies. In the
presence of whitefly, the infections were successfully
transmissible into healthy plants from infected plants
within a few days. But, during the course of the absence
of whiteflies, the transmission of begomovirus infections
to healthy N. benthimiana was negative in the first
3 weeks. But surprisingly, the symptoms started to
develop at the end of the fourth week on healthy plants
growing in proximity to the infected plants. The
123
Aerobiologia
Observed symptoms
Leaf curling, crinkled leaves, stunted growth, and leaf distortion
Leaf curling and stunted growth
Leaf curling, crinkled leaves, and stunted growth
authenticity of the infectivity assay was confirmed by
using begomovirus coat protein primers in PCR.
4 Discussion
Whitefly-mediated viral transmission has long been
considered the major route of infection in plants
(Mansoor et al. 2003). Until now, if there is a
begomovirus infection in plant, whiteflies are com-
monly seen under the leaf surface of the infected plant
in the early morning hours (Markham et al. 1994).
Notwithstanding, we found whiteflies present under
the leaf surface of infected plants growing on the
ground, but they were totally absent in M. azedarach at
heights (6–7 m) above the ground. The biotic and
abiotic factors that might be involved in maintaining
the viability of the begomovirus during air transmis-
sion could be (1) soil contaminated with the litter of
infected dead leaves (Shevchenkoa et al. 2007) blow
off by wind or infected pollen grains, or (2) aerial
transmission of infected pollen grains by wind, or by
honey bees or butterflies probing for nectar (Singh
et al. 2010). (3) Birds and small animals like squirrels
could also transmit the begomovirus while searching
for food from plant to plant or by contaminating the
healthy M. azedarach trees through infected feces. (4)
Yet another important factor for possible transmission
of the infection could be either trichomes (Waigmann
and Zambryski 1995), which are fine outgrowths on
the surface of plants, or guttation fluid containing the
begomovirus (Traoré et al. 2008). These means of
transmission have been studied for many viruses but
have not yet been studied for the begomovirus
infection. Therefore, we will consider these possibil-
ities with regard to the begomovirus using air mon-
itoring within a greenhouse as the basis of our future
research to identify begomovirus infection carriers
other than its chief carrier, the whitefly.
Aerobiologia
5 Conclusions
Thus, identification of transmission of the begomovi-
rus by other than whiteflies represents a new possibil-
ity, and as such, the begomovirus poses a serious threat
to economically important ornamental and crop plants.
Therefore, there is a need for a more comprehensive
study that focuses on amplification of the complete
genome of the virus and the recombination analysis
that has put a light on its origin. Moreover, future
studies should focus on identifying additional routes of
begomovirus infection to assess the contribution each
makes to crop loss and with a view to devising control
strategies. This will form the basis of our future
investigations. The possible association of a begomo-
virus with Melia azedarach (the Pride of India) had
not been investigated previously other than by white-
fly transmission. To the best of our knowledge, this is
the first report from the Indian subcontinent of the
begomovirus associated with leaf curl disease in an
ornamental tree Melia azedarach through aerial
transmission.
Acknowledgments The authors would like to acknowledge a
vote of thanks to Department of Biotechnology (DBT Project No.
BT/PR13129/GBD/27/197/2009) and Department of Science
and Technology (DST Project No SR/FT/LS-042/2009), India,
for their financial support.
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