Diversity and Global Distribution of Whitefly-Transmitted Geminiviruses of Cotton
J. K. Brown, Plant Sciences Department
Geminivirus diseases of cotton are on the rise, worldwide, yet few have been studied in
adequate detail to permit the implementation of rational approaches to disease control.
The rising costs of managing the whitefly vector, coupled with substantial losses caused by
geminivirus-incited diseases now hinder cotton production by requiring inputs that are beyond
economic feasibility. The need for geminivirus disease resistant cultivars in diverse cotton
producting areas and against different viral genotypes presents a new challenge. To meet this
need, information about the identity, distribution, and relevant biotic characteristics of
cotton-infecting geminiviruses is needed. This project addresses this problem through the
molecular analysis of the genomes of cotton-infecting geminivirus from cotton throughout the
world. Here, sequence similarities of the coat protein gene and of the non-coding IR/CR
involved in regulating virus replication and transcription were examined by comparative
sequence analysis to achieve virus identification. This is the first effort to determine
virus identity and to map the distribution of geminiviruses on a global basis.
The outcome of this effort will be a data base containing biotic and molecular information
that will permit rapid and accurate geminivirus identification, and the selection of relevant
viral species for development of cotton cultivars with disease resistance to the geminiviruses
specific to individual production areas.
Until quite recently, whitefly-transmitted (WFT) geminiviruses were known primarily to
infect non-cultivated plants in dry subtropical and tropical climates. The increasingly wider
distribution and higher population levels of the whitefly vector in agroecosystems are directly
implicated in the development of new geminivirus epidemics in cotton-vegetable agroecosystems
throughout the world. This is due to the increased use of pesticides to control primary insect
and vector pests, and to the cultivation of high yielding cotton cultivars that are not
tolerant or resistant to geminivirus pathogens.
Whitefly-transmitted geminivirus-incited diseases result in reduced growth and significant
reductions in yield and quality of cotton. Virus infection at seedling or subsequent, early
developmental stages typically results in little to no production in most widely grown
vegetable and cotton cultivars. To date, there are few geminivirus-resistant crop varieties
available, though much effort is in progress to control geminivirus diseases through
development of resistant varieties that are suitable to specific locations and market needs.
Only a few geminiviruses of cotton have been studied to date. Cotton leaf crumple disease
was first reported in the irrigated desert southwest in the 1950's (Brown, 1992). This disease
of cotton was shown to be caused by a whitefly-transmitted geminivirus, CLCV. CLCV is not
usually economically limiting to cotton production in the US because in most years, although
virs is widely prevalent, infection occurs late in the season,doing minimal damage (Brown and
Nelson, 1984; 1987; Brown et al., 1987; Butler et al., 1986). During the 1950 to the 1980's,
declining cotton production in Central America and the Caribbean Basin was attributed to
whitefly-transmitted geminviruses (J. Bird, per comm). In the Eastern Hemisphere, cotton
production has experienced increasing threats from diseases caused by locally occurring
geminiviruses that are transmitted by the indigenous B. tabaci populations (Brown, 1992; 1998).
Among the additional countries reporting geminivirus diseases of cotton are Cameroon, India,
Malawi, Mali, South Africa, Sudan, and Pakistan (J. K. Brown, 1998, APS Cotton Compendium, in
press) though for the most part, these viruses are generally poorly studied. In Pakistan, at
least one whitefly-transmitted geminivirus has been described from cotton and is thought to be
a primary causal agent in the 1993-1995 epidemic (Mansoor et al., 1993).
Members of the Bemisia tabaci (Genn.) complex, among which are the tobacco, cotton, or
sweet potato whitefly, and B. argentifolli, or the B biotype of B. tabaci, are the only known
whitefly vectors of subgroup III geminiviruses (Brown, 1996). B. tabaci is readily capable of
establishing to extreme population levels, particularly in crops grown under irrigated, arid
conditions in both field and greenhouse systems. In addition, this whitefly has the potential
to colonize a wide range of dicotyledonous species, among which are primarily vegetable and
fiber species of great importance to worldwide agricultural production efforts. Recent
studies indicate that there are numerous populations of B. tabaci that vary somewhat in their
capacity to develop high population densities and cause direct feeding damage, in the extent
of their host ranges, and in the efficacy with which they can transmit geminiviruses (Bedford
et al., 1994; Brown and Bird, 1995; Brown et al., 1995a,b,c). The establishment of the B
biotype in cotton-vegetable agroecosystems in the sunbelt states of the US and throughout
Latin America during 1986-1996, is considered the primary factor driving force behind the
emergence of geminiviruses in cotton-vegetable agroecosystems.
Many distinct virus species are though to infect cotton on a worldwide basis, though very
little specific information is available. Consequently, there is much to be learned about the
identity, the distribution, and the specific threats that these emerging geminiviruses pose to
cotton production. Most problematic in devising measures to control these new diseases is the
paucity of knowledge about the biotic and molecular characteristics of the most problematic
viruses and their molecular epidemiology. For example, most viruses are as yet unidentified
and remain unstudied. As a result, information about their distribution, host range, and
virus-vector relationships are not available. This report describes the results of a two
year study of the molecular sequences of taxonomically key regions of the genomes of
cotton-infecting geminivirus in an initial effort toward identifying and mapping
whitefly-transmitted geminiviruses of cotton on a global basis.
Methods and Materials
Total nucleic acid extraction, PCR, and DNA sequencing
Nucleic acids were extracted from leaves using the method of Doyle and Doyle, 1990 with minor modifications. Leaves were ground
to powder while frozen in liquid nitrogen and immediately suspended in warm (60°C)
cetyltrimethylammonium bromide (CTAB) buffer. Homogenates were extracted with an equal amount
of chloroform:isoamyl alcohol (24:1), phases were separated by centrifugation at 9,000 x g for
10 min at 4°C, and supernatants were precipitated overnight with 2/3 vol isopropyl alcohol.
Nucleic acids were collected by centrifugation at 9,000 x g for 10 min at 4°C, washed with 1
vol wash buffer containing 76% ethanol and 0.2 M sodium acetate, and collected by
microcentrifugation at 4°C. Pellets were air- or vacuum-dried and resuspended (1:1 wt:vol) in
Tris-EDTA (TE) buffer, pH 8.0.
The CR of the DNA A component and the coat protein gene (AV1) were amplified using
polymerase chain reaction (PCR) [Saiki, 1988] and two different pairs of degenerate primers
(Idris and Brown, 1998). PCR primers were synthesized at the Biotechnology Facility,
University of AZ, Tucson. PCR was carried out as described (Idris and Brown, 1998; Wyatt and
Brown, 1996). PCR products of the expected sizes were cloned into the plasmid vector
(pCRŽ 2.1) using the TA Cloning Kit (Invitrogen, San Diego, CA) per manufacturer's
instructions. Fragments containing putative viral sequences were identified by size
approximation in miniprep screening, and clones were confirmed positive for viral inserts
using the respective PCR primers used for initial amplification. Viral DNA sequences were
obtained for a minimum of three clones each by automated sequencing at the Molecular Genetics
Facility, University of Georgia (Athens, GA) using an automated Applied Biosystems 373A
(version 2.1.1) Sequencer. Clones were sequenced in both directions using the same primers
employed to obtain PCR products or universal primers on the vector.
Reference viral CR and AV1 sequences used are of representative geminiviruses and were
obtained by truncating full length sequences obtained from GenBank. Cotton geminivirus
sequences were assembled with the aid of SeqEdit in the DNASTAR software package (DNASTAR,
Madison, WI). AV1 and CR sequences were aligned using the Clustal multiple sequence alignment
program 'MegAlign' (DNASTAR) which aligns using a distance matrix method with stepwise,
cummulative comparative clustering, generating an arithmetic mean for all subsequent pairwise
comparisons. Clustal calculates the distances between all pair-wise character comparisons,
and makes no a priori assumptions about evolutionary histories. Reconstructed trees were
analyzed by maximum parsimony (MP) with Phylogenetic Analysis Using Parsimony (PAUP) program,
version 3.1.1 [Swofford, 1993 #240]. A single most parsimonious tree(s) was sought using a
heuristic search method with stepwise addition of sequences and the tree-bisection-reconnection
(TBR) random branch-swapping options [Swofford, 1993]. Bootstrap values were calculated for
major nodes using the 50% majority rule to place confidence limits on the tree exhibiting the
most parsimonious reconstruction. Geminivirus sequences used in comparative sequence analyses
were obtained from GenBank.
The results of a preliminary analysis of viral common region sequences by maximum parsimony
(PAUP) (Figure 1) and mean calculated pairwise distances expressed
as percent similarities are shown, along with iterations identified in the common region of
select cotton isolates (Table 1;
Table 2). Also presented are results of maximum parsimony
analysis of the coat protein gene (Figure 2.)
and corresponding mean % similarities calculated from the pairwise mean distance matrix
(Table 3) for select geminivirus isolates from cotton.
Distance and phylogenetic analyses of the coat protein gene of these isolates indicate that
they have an distinct origin in either an Old or a New World site. For example, from these
analyses it can clearly be determined that cotton leaf curl virus-Pak 1 (CLCuV-Pak1) is
distinct from all other geminiviruses found thus far in either locale, and is an Old World
virus species, quite distinct from the well-studied cotton leaf crumple virus of the
southwestern US and Mexico desert.
Among the New World isolates examined here, cotton leaf crumple virus (CLCV), and possibly,
strains thereof, was documented as the sole geminivirus species in Arizona (CLCVAZCgr94 &95),
California (CLCVCalif 94&95), and in Caborca and the Mexicali Valley of Sonora, Mexico
(MexCaborcaCYM, MexicaliVLCr 17&18), all three sites in which the disease has been previously
documented. In addition, a close relative or strain of CLCV was documented for the first time
in Guatemala (Guat57cot94). Also, at least one and possibly two apparently distinct and as yet
undescribed geminiviruses of cotton were found in Texas (CotV9Tx61, Txcotgrmos, TxcotLCr,
TxMontAl and TxcotLCr & CotV7Tx) and another in Guatemala (Guat2cot).
Further studies will now be required to substantiate these preliminary findings, including
the complete DNA sequence from infectious clones and examination of the potential for trans
activation of replication and movement functions between the components of different isolates.
Additional geminivirus isolates from the Dominican Republic (1992), Brazil (1997-98), Sudan
(1997), India (1998), and numerous other cotton growing regions of the world are presently
under investigation using these approaches.
Recent advances in the application of molecular biological methods to the
characterization of geminiviruses have facilitated the investigation of cotton
infecting geminiviruses. New approaches involve the application of polymerase
chain reaction (PCR) and universal subgroup III that amplify the middle or core
region of the coat protein gene (Wyatt and Brown, 1996), or geminivirus-specific
primers to direct the amplification of key regions of the virus genome (Idris and
Brown, 1998). Partial genome sequences of relevant genes or genomic regions are
useful for establishing virus identity and relationships between viruses in the
absence of a complete genomic sequence, the latter, a time-consuming and arduous
task. Used in conjunction with biotic information about the isolate, and when
compared for multiple viruses, coat gene and LIR/CR sequences provide important
clues about relationships between whitefly-transmitted geminiviruses, as based
upon the specific targeted or marker sites within the viral genome.
The geminivirus coat protein gene (AV1) and large viral intergenic/common
region (LIR/CR) sequences have been shown to be useful in establishing the
relative identity of geminiviruses (Brown et al., 1998, in preparation). The
coat protein gene is informative because it contains sequences that are both
highly conserved and regions that are variable to such an extent that
phylogenetic inferences can be correlated to biotic and geographic
characteristics (Padidam et al., 1995). The coat contains sequences that are
conserved to function in the formation of the characteristic 'geminate' coat
protein that encapsidates the ssDNA genome, plays a role in virus movement in
the plant (Pooma et al., 1996), and is required for vector-mediated transmission
The large intergenic region is considered to be an informative sequence of the
geminivirus genome because it contains viral regulatory sequences essential for
the disease cycle (Eagle and Hanley-Bowdoin, 1997) and for potentially
interacting with other geminiviruses when they occur in a mixture in the same
host, a possible means by which additional genotypes can emerge, possibly with
distinct biological properties (Arguello-Astorga et al., 1994). Specifically,
these sequences are important for predicting the likelihood of cross-replication,
or pseudo-recombination, between compatible, and therefore, closely related
isolates (in question), and is conserved at key sites to perform essential
functions in the disease cycle.
Phylogenetic analysis of geminiviruses based upon the coat protein or LIR/CR
sequences position the most closely related viruses within the same 'cluster' or
group, whereas, those that are not as closely related are placed on different
branches with their closest sequence relatives. Inclusion of the leafhopper/
planthopper relatives within the Geminiviridae in the analysis reveals clear
separation of whitefly-transmitted viruses from subgroup I and II viruses.
Within the whitefly subgroup, the viruses are clearly further separated by
geography of origin (Eastern or Western Hemisphere), and at times by a further
sub-geographic separation (Brown, 1996; Brown and Wyatt, 1995; 1996; Padidam
et al., 1995).
Using this approach, the introduction of a geminivirus from one geographic
world region into another can be readily detected. In line with predictive
approach for the rational selection of relevant isolates for resistance screening
or as sources of viral genes toward pathogen-derived resistance, we are examining
the prospects of applying phylogenetic relationships to development of disease
resistant germplasm with well-defined breadths of virus resistance. In this
approach, it is possible to make predictions about the genotype and numbers of
closely or distantly related geminiviruses in germplasm from breeding programs
and in plants protected by virus-derived resistance. Though the approaches
differ in possible resistance mechanisms, both hope to achieve resistance to as
many viruses and strains as possible to accomodate for extant virus pathogens
and those that may emerge from extant relatives in the future.
We hypothesize that germplasm with resistance to a geminivirus from the same
or an adjacent geographic region may also afford protection against other closely
related viruses, and provide less or little protection against more divergent
viruses from distant geographic regions, the viruses having evolved either under
more or less the same conditions and/or possibly from a biogeographically related
common ancestor. Consequently, germplasm or transgenic plants expressing the
gene of a particular geminivirus genotype will be most likely be effective
against most closely related viruses as opposed to those evolving in
biogeographic isolation. Clearly, this hypothesis cannot claim a priori
knowledge about any particular mechanism operating in a resistant genotype, or
whether there is a traceable evolutionary basis for a mechanism that is
necessarily congruent with geminivirus pathogen evolution. None the less, the
broad theoretical and practical utilities of this diagnostic and predictive tool
can not be underestimated.
The initial goal of this effort is to determine the identity and map the
geographic distribution of the most prevalent geminiviruses of cotton on a global
basis. Second, viral sequence data will be used to establish the phylogenetic
relationships between cotton-infecting geminiviruses in relation to well-studied
geminiviruses, using the analogous sequences as reference sequences having a
biotic and geographic basis. Third, particular regulatory sequences will be
evaluated to predict if interactions may be possible between viruses if present
in mixed infections, a result that also lends insights to relationships at the
virus strain versus species level.
The long range goal is to catalog and map whitefly-transmitted geminiviruses
of cotton on a global basis, and a establish a rational means for the selection
of relevant viruses and/or strains toward developing customized geminivirus
disease resistant cotton varieties. The most important virus species will
then be selected according to the criteria of association with substantial
disease losses and a widespread distribution in cotton. These viruses will be
subjected to molecular cloning to obtain a full length infectious virus clones
and their complete DNA sequences, thus achieving for the first time, the
isolation of these viruses in 'pure culture', reproduction of disease symptoms
after inoculating cotton with the infectious clones, and the end result being,
characterized geminiviruses of cotton for disease resistance efforts.
- Bedford, I.D., P.G. Markham, J.K. Brown and R.C. Rosell. 1994.
Geminivirus trnsmission and biological characterization of whitefly (Bemisia tabaci)
biotypes from different world regions. Ann. appl. Biol. 125: 311-325.
- Brown, J. K. 1994. Current Status of Bemisia tabaci Genn. as a pest and vector in world agroecosystems. FAO Plant Prot. Bull. 42: 3-32.
- Brown, J. K. 1996. Chapter 5 in: Molecular Biology and Epidemiology of Subgroup III, Geminiviridae. Plant-Microbe Interactions Review Series, G. Stacey and N. Keen, eds (Chapman and Hall) pp 125-195.
- Brown, J. K., D. Frohlich, and R. Rosell, 1995a. The Sweetpotato/Silverleaf
Whiteflies: Biotypes of Bemisia tabaci Genn., or a Species Complex? Ann. Rev. Entomology 40: 511-534.
- Brown, J. K., S. Coats, I . D. Bedford, P. G. Markham, J. Bird, and D. R.
Frohlich. 1995c. Characterization and distribution of esterase electromorphs
in the whitefly, Bemisia tabaci (Genn.) (Homoptera:Aleyrodidae). Biochemical Genetics: 33: 205-214.
- Brown, J.K. 1992. Virus Diseases of Cotton, pages 275-330 in: Cotton Diseases. R. J. Hillocks, ed. Commonwealth Agricultural Bureaux International, Oxon, United Kingdom. 415 pp.
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- Brown, J.K. and Nelson, M.R. 1984. Geminate particles associated with cotton leaf crumple disease in Arizona. Phytopathology 74:987-990.
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- Brown, J.K., Mihail, J.D., and Nelson, M.R. 1987. The effects of cotton leaf crumple virus on cotton inoculated at different growth stages. Plant Dis. 71:699-703.
- Eagle, P. A. and L. Hanley-Bowdoin. 1997. cis Elements that contribute to geminivirus transcriptional regulation and the efficiency of DNA replication. Journal of Virology 71: 6947-6955.
- Heyraud, F., S. Schumacher, J. Laufs, S. Schaefer, J.Schell, and B. Gronenborn. 1995. Determination of the origin cleavage and joining domain of geminivirus rep proteins. 1995. Nucleic Acids Res. 23: 910-916.
- Idris, A. M. and J. K. Brown. 1998. Sinaloa tomato leaf curl virus: biological and molecular evidence for a new subgroup III geminivirus. Phytopathology (in press).
- Lazarowitz, S. G., L. C. Wu, S. G.Rogers, and J. S. Elmer. 1992. Sequence-specific interaction with the viral AL1 protein identifies a geminivirus DNA replication origin. 1992. Plant Cell 4:799-809.
- Padidam, M., R. N. Beachy, and C. F. Fauquet. 1995. Classification and identification of geminiviruses using sequence comparisons. J. Gen. Virol. 76: 249-263.
- Swofford, D. 1993. PAUP: Phylogenetic Analysis Using Parsimony, version 3.1.1. Washington D.C.: Smithsonian Institution.
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