CHLOROTIC SPOTS ON Clerodendrum , A DISEASE CAUSED BY A NUCLEAR TYPE OF Brevipalpus ( ACARI : TENUIPALPIDAE ) TRANSMITTED VIRUS

Chlorotic spots have been observed in plants of Clerodendrum x speciosum growing in residential gardens and parks in Piracicaba, SP, Brazil. Thin sections of diseased tissues revealed characteristic cytopathic effects of the nuclear type of the Brevipalpus (Acari: Tenuipalpidae) mitetransmitted viruses (BTrV). Brevipalpus mites, identified as B. phoenicis, infesting symptomatic C. x speciosum plants transmitted the pathogen to healthy C. x speciosum and to C. thomsonae, Gomphrena globosa, Hibiscus cannabinus, H. coccineus, H. schizopetalus, Salvia leucantha, Spathiphyllum wallasi and Tetragonia expansa causing chlorotic spots on their leaves. Mechanical inoculation using leaf extracts from infected C. x speciosum resulted in chlorotic spots on inoculated C. x speciosum, Chenopodium quinoa, C. amaranticolor, G. globosa, H. cannabinus, H. coccineus and T. expansa leaves. C. amaranticolor and C. quinoa kept at 28 30°C became systemically infected. The same cytopathic effects caused by the nuclear type of BTrV were seen in tissues from all infected test plants by electron microscopy. The virus was purified from systemically infected leaves of C. amaranticolor and C. quinoa. A polyclonal antiserum obtained from an immunized rabbit presented a strong reaction with the homologous antigen in ELISA tests. The results suggest that this chlorotic spot disease of C. x speciosum is caused by a new species of the nuclear type of BTrV, tentatively named Clerodendrum chlorotic spot virus (ClCSV).


INTRODUCTION
The genus Clerodendrum comprises about 400 species, most of which grow in warm temperate or tropical climates.The name is derived from the Greek words "Clerodendrum" for chance (klero) and tree (dendrum) and refers back to the original species name "fortunate".Many publications refer to this genus as Clerodendron but Clerodendrum is the official spelling.Originally considered as a member of the family Verbenaceae, the genus Clerodendrum is now considered as a member of the family Lamiaceae based on cladistic analysis of the chloroplast DNA and internal transcriber spacer sequences (Steane et al., 2004).Clerodendrum plants appear as trees, shrubs and scrabbles and are commonly used as ornamentals (KVL, 2006).Clerodendrum thomsonae Baulf.(bleeding heart), C. x speciosum Tiej.et Bin.(glorybower, Java glory vine, heart vine, Pagoda flower) and C. splendens G. Don.(flaming glorybower) are among the most cultivated in home gardens and parks in Brazil and elsewhere, covering fences and walls (Lorenzi & Souza, 2001) (Figure 1, A-C).There are few reports of viral diseases affecting plants of this genus.A yellow mosaic of C. inerme, caused by a begomovirus was reported in India (John et al., 2006) and a vein clearing of C. x speciosum, associated with an unidentified rhabdovirus, was found in Brazil (Schuta et al., 1997).
Plants of C. x speciosum exhibiting chlorotic and necrotic spots on their leaves (Figure 1, G) were found in a residential garden at Piracicaba, SP, Brazil, infested by mites identified as Brevipalpus phoenicis (Geijskes) (Figure 1 D, F).Electron microscopic examination of the tissue sections of these spots revealed cytopathic effects of the nuclear type of the Brevipalpus mite transmitted viruses (BTrV) (Kitajima et al., 2003).Similar symptoms, always associated with Brevipalpus mite infestation, were also found in C. thomsonae and C. splendens (Figure 1 H, I) in Piracicaba and other cities of the states of São Paulo, Santa Catarina, Amazonas and Distrito Federal.In C. thomsonae, brownish spots were also observed on flower petals (Figure 1, J) when infested by Brevipalpus (Figure 1, E).
Brevipalpus (Acari: Tenuipalpidae) mites transmit an ever increasing list of plant viruses among them some of economical importance such as Citrus leprosiscytoplasmic type (CiLV-C), Orchid fleck (OFV), Cof-      1 and 2).Three to five test plants of each species were mechanically inoculated with the extract.Inoculation was made by rubbing the sap on the leaves dusted with carborundum.  1 and 2) were surveyed for symptoms of chlorotic/necrotic lesions on the leaves.When leaves with suspected lesions were found, samples were collected and processed for electron microscopy analysis.

Survey on possible natural infection by
Electron microscopy -Small pieces of the chlorotic spots on the leaves (and in one case from a flower petal of C. thomsonae) were fixed in a modified Karnovsky solution (2% paraformaldehyde, 2.5% glutaraldehyde in 0.05 M cacodylate buffer, pH 7.2) for 1 -2 h, washed with buffer and post-fixed in 1% osmium tetroxide in the same buffer for 1 h, dehydrated in a graded series of acetone, infiltrated and embedded in the low viscosity Spurr epoxy resin (Maunsbach & Afezelius, 1999).Blocks were sectioned in a Leica UT ultramicrotome equipped with a diamond knife.The sections were collected on copper grids, stained with 3% uranyl acetate and Reynold's lead citrate, and examined in a Zeiss EM 900 transmission electron microscope.Virus purification -Fresh leaves of C. amaranticolor and C. quinoa exhibiting symptoms of systemic infection were used for virus purification, based on the protocol used for CoRSV purification (Boari et al., 2004), which is the procedure used to purify OFV (Chang et al., 1976) with small modifications.Infected leaf tissues frozen in liquid nitrogen were ground in 0.1 M phosphate buffer, pH 7.0, containing 0.01 M sodium diethyl ditiocarbamide, 0.1% ascorbic acid and 5% Triton X-100.After clarification by low speed centrifugation, the suspension was centrifuged on top of a 20% sucrose cushion (150 min /40,000 g).The pellet was resuspended in 0.1 M phosphate buffer, pH 7.0, and submitted to a cycle of differential centrifugation and then centrifuged in a sucrose gradient (10 -40%) for 90 min at 40,000 g.One ml fraction were collected and analyzed by spectrophotometry, and the fraction with the peak of nucleic acid absorbance was separated and centrifuged for 150 min at 40,000 g.The pellet was resuspended and stored for antiserum production.A sample was examined in the electron microscope by negative staining with 1% uranyl acetate.
Antiserum production and ELISA -Four aliquots of 250 μL of a purified preparation (ca.20 μg mL -1 ), mixed with an equal volume of incomplete Freund´s adjuvant, were injected intramuscularly in a rabbit at weekly intervals.One week after the last injection, the serum was collected and stored at -20°C (Van Regenmortel, 1982).This antiserum was tested in Plate trapped antigen -Enzyme linked immunosorbent assay (PTA-ELISA) (Mowat & Dawson, 1987) against purified virus and extracts from infected Clerodendrum.Antisera against OFV (Kondo et al., 1995) and CoRSV (Boari et al., 2004), and the respective antigens were also included in the test to analyze possible serological relationships.Appropriate healthy controls were included in homologous and heterologous reactions.In some few cases, the chlorotic spots became greenish when the leaves became senescent (Figure 2 A, C, G, H, I).Necrotic stem lesions were only no-ticed in H. cannabinus.In one instance, the white corolla of C. thomsonae growing in a residential garden presented brownish spots (Figure 1 J). 1 D, E, F) infesting C. x speciosum, C. splendens and C. thomsonae exhibiting chlorotic lesions were identified as B. phoenicis based on external morphological characteristics (Welbourn et al., 2003).When mites collected from symptomatic C. x speciosum plants were transferred to healthy ones, chlorotic spots became visible four to five weeks later.Electron microscopic examination confirmed the presence of cytopathic effects similar to those found in the source plant.After this initial experiment, similar mite transmission tests were performed on plants of 39 species (Tables 1 and 2).   1 and 2).In all of them the characteristic cytopathic effects of the nuclear type of BTrV were observed.

Mite transmission tests -Mites (Figure
Electron microscopy -Cell alterations characterized by an electron lucent inclusion (viroplasm) in the nucleus and the presence of short rod shaped particles (ca.40 nm × 100 -110 nm) in the nucleus or cytoplasm were observed in the tissues of all symptomatic plants, obtained by natural or experimental (mite or mechanical) transmission of the virus (Figure 3 A-D).In the nucleus, these rodlike particles could appear scattered within viroplasm or nucleoplasm and sometimes arranged side-by-side forming lamellar aggregates (Figure 3 B).Also, they commonly appear either in the nucleus or in the cytoplasm arranged perpendicularly onto membranous system of the nuclear envelope or endoplasmic reticulum (Figure 3 C).This arrangement is reminiscent of early phases of the budding process during morphogenesis of most of the membrane bound viruses, which ends up with the complete envelopment of the nucleocapsid by the membrane.However, with the nuclear type of BTrV (Kitajima et al., 2003) this budding process appears to be incomplete in most cases.In some instances the endoplasmic reticulum cisternae form a tubular array with the particles arranged in a radial configuration inside, producing a figure referred to as "spoke wheel" (Figure 3 C, D).These cell changes are characteristic of the so-called nuclear type of BTrV, and served as evidence that these plants are infected by ClCSV.
Virus purification -The protocol used for purification of OFV and CoRSV was successfully used to purify ClCSV from extracts of systemically infected leaves of C. amaranticolor and C. quinoa.Examination of the fraction which had a UV absorption for nucleic acid contained a large amount of short bullet shaped particles 40 nm wide and 100 -110 nm long, with cross striation of 5 nm, essentially similar to those observed in purified preparations of OFV and CoRSV (Figure 3 E).Mechanical inoculation of these preparations caused local lesions in C. quinoa.Transmission electron microscopy of these lesions indicated the presence of typical cytopathic effects caused by a nuclear type of BTrV, thus confirming that the purified preparations were infectious.
PTA-ELISA -The purified preparations of ClCSV strongly reacted with the produced homologous antiserum in PTA-ELISA.Antisera against OFV and CoRSV only reacted weakly with purified ClCSV (data not shown), suggesting that it may differ from OFV and CoRSV, but may share some common antigens or epitopes.

DISCUSSION
The chlorotic spots occurring naturally in at least three Clerodendrum species (C.x speciosum, C. thomsonae, C. splendens) are caused by a nuclear type of BTrV, which is named Clerodendrum chlorotic spot virus (ClCSV).The virus is naturally transmitted by mites and by mechanical inoculation to several other plant species.Nineteen of 87 plants growing near ClCSV-infected C. x speciosum (Table 1) showed localized infection with cytopathic effects characteristic of the nuclear type of BTrV.They were possibly infected by B. phoenicis that acquired the ClCSV from infected C. x speciosum plants.Few isolated C. splendens plants were also found to be infected by ClCSV.Experimental mite transmission tests resulted in the infection of some of these plants (G. globosa, H. cannabinus, H. coccineus, S. leucantha, S. wallasi).Infection of H. cannabinus, H. coccineus, S. leucantha and Dendrobium sp., could be demonstrated by mechanical inoculation on C. amaranticolor and C. quinoa.With 14 species of these plants, mechanical recovery of the virus was unsuccessful possibly due to inhibitors present in the extracts.Further studies are required to confirm infection of these plants by immunological or molecular tools to detect ClCSV.
Two orchid species (Dendrobium sp. and Oncidium sp.) growing amidst ClCSV infected C. x speciosum presented leaf lesions undistinguishable from those caused by OFV.However, extracts from the lesions did not react with OFV antiserum, and no amplification was obtained by RT-PCR using primers specific for OFV (data not shown).It is likely that these orchid species were infected by an isolate of ClCSV but further tests are required to confirm this hypothesis.
As it happens with two other nuclear type BTrV, respectively OFV (Kondo et al., 1995) and CoRSV (Boari et al., 2003), mechanical infection of  Based on symptoms and electron microscopy examination of the infected tissues, the causal agent of some reported diseases like Malvaviscus ringspot (Kitajima et al., 2003), Soursop (Annona muricata) ringspot (Bitancourt, 1955;Kitajima et al., 2003) and Hibiscus chlorotic spot (Kitajima & Rodrigues, 2001) may be caused by ClCSV.Similar symptoms were observed for these plants growing near to ClCSV-infected C. x speciosum as above cited, and presumed to be naturally inoculated by the mite vector.This possibility will be evaluated using immunological and molecular techniques.
Many hosts, which appeared not to be infected by ClCSV in this study like Citrus sinensis (Rodrigues et al., 2003a), Pittosporum tobira (Rodrigues et al., 2003b), Piper nigrum (Yamashita et al., 2004), Allamanda schottii, Hedera canariensis, Bidens pilosa and Mussaenda erythrophylla (Rodrigues et al., 2005) were reported to be infected by the nuclear type of BTrV.Now that immunological and molecular tools are available for three of the nuclear types of BTrV, respectively ClCSV, CoRSV and OFV, it will be possible to assess whether or not other diseases for wich cytopathology indicates the presence of the nuclear type of BTrV represent infection by isolates of one of these viruses or a different virus.
So far, except for OFV, known BTrV are restricted to the American continent (Kitajima et al., 2003;2006a) although Brevipalpus species that serve as vector are dispersed worldwide in tropical and subtropical regions (Childers et al., 2003b).The worldwide occurrence of OFV is explainable by the intense exchange of living plants which certainly resulted in the introduction of the virus and the vector, even in temperate regions.The absence of BTrVs in other parts of the world, however, is circumstantial and based on observations made only in parts of South Africa, Australia and few countries in Southeast Asia (J.C.V. Rodrigues and E.W. Kitajima, unpublished results) and a wider survey is required to confirm these observations.ClCSV has been found naturally infecting Clerodendrum species in the states of São Paulo, Santa Catarina, Amazonas and Distrito Federal indicating a wide dispersion in Brazil and is likely to be present in other parts of the American continent.
Examination of thin sections of the tissues from the lesions of experimental or naturally ClCSV-infected plants appeared similar to those reported for other nuclear types of BTrV as OFV and CoRSV (Kitajima et al., 2003;Kondo et al., 2003;Chagas et al., 2003).Typical electron lucent viroplasm in the nucleus and short rod shaped particles were seen mostly in mesophyl parenchyma cells, and rarely in vascular parenchyma.The rod-like particles were demonstrated to be the virions by immunogold labeling in OFV-infected cells (Kitajima et al., 2001) and by analogy, those present in ClCSV-infected cells may represent ClCSV particles, though experimental demonstration is still required.These particles were commonly naked, without surrounding membrane, and quite often were perpendicularly arranged to the nuclear envelope or endoplasmic reticulum membranes, as shown for other nuclear type of BTrV, a process suggesting that the envelopment of the particles is not completed after an initial start.In very rare instances, membrane bounded particles could individually be seen within endoplasmic reticulum elements, possibly after a successful budding process.A possible explanation might be that glycoprotein of most nuclear types of BTrV have some defect by which the budding process can not be completed and results in the accumulation of naked nucleocapsids.Similar observations were described for Tomato spotted wilt virus (Resende et al., 1991).
The same pattern of cell alterations observed in plant tissues was observed also in gland cells of B. phoenicis collected from ClCSV-infected plants (Kitajima et al., 2006a).This fact strongly suggests that the virus multiplies in the mite, thus B. phoenicis/ClCSV relationship is of the circulative/propagative type.This is in agreement with the conclusions reached in the transmission of OFV by B. californicus (Kondo et al., 2003).
Because systemically infected tissues contain more virus than in local lesions, as noticed by electron microscopy, this tissue was used to purify ClCSV.This was achieved using the protocol developed for OFV (Chang et al., 1976;Boari et al., 2004), and the purified particles looked essentially similar to those of OFV and CoRSV.The antiserum raised in rabbit against purified ClCSV was specific.Comparative PTA-ELISA using antibodies against OFV, CoRSV and ClCSV, and the respective homologous antigens, revealed strong homologous and faint heterologous reactions (data not shown).These results indicate that they are different viruses, but share some common epitopes.This is considered supplementary evidence that though belonging to the same genus, these three viruses are of different species.OFV genome has been completely sequenced and revealed to be negative sense ssRNA.Its genome is divided in two pieces of about 6 kb with gene organization similar to that of Rhabdoviruses.A new genus Dichorhabdovirus has been proposed for OFV (Kondo et al., 2006), of which CoRSV and ClCSV will be possible members.As soon as more informa- tion about ClCSV genome emerges, a better understanding of the phylogenetic relationship among these viruses is expected.Also, the development of molecular and immunological tools to detect ClCSV will help to confirm the cases of its natural transmission as well as the establishment of its natural host range, which is much wider than that of CoRSV and OFV.

Figure 1 -
Figure 1 -A-C.Inflorescences of : A. Clerodendrum x speciosum.B. C. thomsonae.C. C. splendens.D. Part of a leaf of C. x speciosum heavily infested by B. phoenicis.E. The same mite infesting a petal of the flower of C. thomsonae.F. An adult female of B. phoenicis on the leaf of C. x speciosum.G-I.Leaf symptoms caused by Clerodendrum chlorotic spot virus (ClCSV) in Clerodendrum species.G. Chlorotic spots with necrotic centers in C. x speciosum.H. Chlorotic spots in C. thomsonae.I. Chlorotic spots, some of them with necrotic center, in C. splendens.J. Brownish spots in the petal of C. thomsonae flower.K-N.Local lesions and systemic infection by ClCSV in mechanically inoculated Chenopodium quinoa and C. amaranticolor.Local lesions (K) and systemic symptoms (L) on the leaves of C. amaranticolor.Systemic infection (M) and local lesions (N) on the leaves of inoculated C. quinoa.

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Symptoms caused by natural or experimental (mite or mechanical inoculation) transmission of ClCSV remained restricted to the inoculated leaves of susceptible hosts.The symptoms consisted usually of chlorotic spots (Figure 1 H, I, K, N; Figure 2 A, B, C, F, I, J, L, N, O), occasionally with a necrotic center (Figure 1 G, 2 E, H).Some hosts developed brownish (Figure 2 I, K) or necrotic spots (Figure 2 D, M).

Figure 2 -
Figure 2 -A-O.Leaf symptoms on several plant species experimentally (mite or mechanical) or naturally infected by Clerodendrum chlorotic spot virus (ClCSV).A. Chlorotic spots on green leaves and green spots in senescent leaves of mite infected Salvia leucantha.B. Chlorotic spots or rings on the leaves naturally infected Cestrum nocturnum.C. Ringspot on a leaf of naturally infected Commelina sp.D. Necrotic ringspot on a leaf of naturally infected Oncidum sp.E. Chlorotic spots on a leaf of naturally infected Hibiscus schizopetalus.F. Chlorotic spots on a leaf of mite infected Spathiphyllum wallasi.G. Green spots on senescent leaves of naturally infected Thunbergia erecta.H. Chlorotic spots on a green leaf and green spots on senescent leaves of naturally infected Malvaviscus arboreus.I. Brownish and chlorotic spots on green leaves and green spots on a senescent leaf of Hibiscus cannabinus mechanically infected.J. Chlorotic spots on mite-inoculated leaves of Hibiscus coccineus.K. Large brownish spots on the leaves of naturally infected Annona muricata.L. Chlorotic spots on the leaf of naturally infected Hydrocotyle leucocephala.M. Necrotic spots on the leaves of mechanically inoculated Gomphrena globosa.N. Chlorotic spots on a leaf of naturally infected Diffenbachia sp.O. Chlorotic spots on a leaf of mechanically infected Tetragonia expansa.
and C. quinoa with ClCSV resulted in local chlorotic spots and if the plants are maintained at high temperatures (28 -30ºC), systemic infection occurs in the form of chlorotic spots or vein clearing.The systemic infection in hosts as C. amaranticolor and C. quinoa may be a common fea-ture for most of the nuclear type of BTrV and deserves attention to understand why temperature may affect the mechanisms that tend to restric the infection.In other susceptible plant species, however, systemic infection was not yet observed under similar conditions.

Figure 3 -
Figure 3 -A-D.Transmission electron micrographs of thin sections of Clerodendrum chlorotic spot virus (ClCSV)-infected leaf tissues: A. A nucleus in a leaf parenchyma cell of Clerodendrum x speciosum.A large viroplasm (Vp) and a mass of presusmed rodlike virions are visible.B. Large parallel aggregate of presumed rodlike particles near to the viroplasm (Vp).A group of particles can also be seen arranged perpendicularly onto the inside of the nuclear envelope in a leaf parenchyma cell of Chenopodium quinoa mechanically infected.C. Part of the cytoplasm of a leaf parenchyma cell in a chlorotic spot of Hibiscus cannabinus experimentally infected by Brevipalpus phoenicis.Rod-shaped, presumed virions, appear arranged perpendicularly onto the endoplasmic reticulum elements.A spoke wheel configuration is visible (*).D. Detail of a spoke wheel configuration in a leaf parenchyma cell of a chlorotic spot of naturally infected Malvaviscus arboreus.E. Purified preparation of ClCSV obtained from systemically infected Chenopodium amaranticolor and C. quinoa negatively stained with uranyl acetate.A round end and cross striations are clearly visible on the rod shaped virions of ClCSV.

Table 1 -
List of plants tested for susceptibility to Clerodendrum chlorotic spot virus (ClCSV) by mite, mechanical and natural transmission and respective symptoms.
1 Brevipalpus phoenicis.2Extractsfromlocallesions of Chenopodium quinoa or C. amaranticolor used as inoculum.3Plantsgrowing next to a group of Clerodendrum x speciosum plants naturally infected by Clerodendrum chlorotic spot virus (ClCSV) and infested by B. phoenicis. - no reaction.nt 5 -not tested.+ 6 Local chlorotic lesions.Infection by ClCSV demonstrated by electron microscopic examination of the lesions and mechanical inoculation of C. quinoa and C.amaranticolor.nf 7 -Plants not found near ClCSV -infected C.x splendens or C. thomsonae.++ 8 Local chlorotic lesions followed by systemic infection (vein clearing and chlorotic spots) in uninoculated leaves when plants were kept at 28 -30 o C for 2 weeks. 9Natural infection in an isolated plant.

Table 2 -
Other plant species tested negative for mite, mechanical and natural transmission of the Clerodendrum chlorotic spot virus.