Complete genome sequences of two isolates of spiraea yellow leafspot virus (genus Badnavirus) from Spiraea x bumalda 'Anthony Waterer'.

The complete genome sequences of two isolates of spiraea yellow leafspot virus (SYLSV) were determined. Spiraea (Spiraea x bumalda) 'Anthony Waterer' plants showing virus-like symptoms including yellow spotting and leaf deformation were used for sequencing. The viral genome of SYLSV-MN (Minnesota) and SYLSV-MD (Maryland) is 8,017bp in length. The sequences share 95% identity at the nucleotide level. Both isolates have the same genome organization containing three open reading frames (ORFs), with ORF3 being the largest, encoding a putative polyprotein of 232 kDa with conserved domains including a zinc finger, pepsin-like aspartate protease, reverse transcriptase (RT), and RNase H. Pairwise comparisons between members of the genus Badnavirus showed that gooseberry vein banding associated virus GB1 (HQ852248) and rubus yellow net virus isolate Baumforth's Seedling A (KM078034) were the closest related virus sequences to SYLSV, sharing 73% identity at the nucleotide level. Bacilliform virions with dimensions of 150 nm × 30 nm were observed in virus preparations from symptomatic, but not asymptomatic, plants.

Genomic characterization of cycad leaf necrosis virus, the first badnavirus identified in a gymnosperm.

A previously undescribed badnavirus was isolated from Zamia fischeri showing symptoms of chlorosis, necrosis, and ringspot. The virus has bacilliform virions 30 nm in diameter and averaging 120 nm in length. The viral genome is 9227 bp in length and contains three open reading frames characteristic of members of the genus Badnavirus. The largest open reading frame (ORF3) encodes a putative polyprotein, with predicted domains including zinc finger, aspartic protease, reverse transcriptase (RT) and RNase H. The virus is tentatively named "cycad leaf necrosis virus" (CLNV). Within the genus Badnavirus, CLNV was most closely related to sugarcane bacilliform Guadeloupe D virus (FJ439817), sharing 69% identity at the nucleotide level in the RT + RNase H region. This virus is the first badnavirus reported to infect cycads, and it has the largest genome among the currently characterized badnaviruses.

Complete genome sequence of aglaonema bacilliform virus (ABV).

Aglaonema bacilliform virus (ABV), a member of the genus Badnavirus in the family Caulimoviridae, is associated with leaf deformation and chlorosis in Aglaonema modestum. The complete genome sequence of a Minnesota isolate of ABV was determined. The ABV genome is 7,178 bp in length and similar in size and organization to those of the members of the genus Badnavirus, containing three open reading frames (ORFs) with the potential to encode three proteins of 14.92, 13.33 and 207.95 kDa, respectively. The last ORF (ORF3) encodes a putative polyprotein with conserved domains, including zinc finger, aspartic protease, reverse transcriptase (RT) and RNase H domains, in that order. Phylogenetic analysis using the amino acid sequence of the ORF3 polyprotein showed that ABV clusters with several isolates of taro bacilliform CH virus (TaBCHV). Pairwise alignment using the highly conserved RT/RNase H region reveals that ABV has the highest level of identity (71%) at the nucleotide level to a Hawaiian isolate of TaBCHV.

Complete genome sequence of a previously undescribed badnavirus occurring in Polyscias fruticosa L. (Ming aralia).

A previously undescribed badnavirus was identified in plants of Polyscias fruticosa (Ming aralia) showing symptoms of mild mosaic and leaf senescence. Characteristic bacilliform virions of the Polyscias badnavirus averaging 30 × 120 nm in size were observed by transmission electron microscopy in partially purified leaf tissue extracts from symptomatic but not asymptomatic plants collected in the USA and Nigeria. The isolate from the USA was complete sequenced. The genome is 7592 bp in length and contains three open reading frames with an arrangement similar to that of other members of the genus Badnavirus. The largest open reading frame (ORF3) encodes a putative polyprotein, with predicted domains including zinc finger, aspartic protease, reverse transcriptase (RT) and RNase H, in that order. The USA and Nigeria isolates of the virus had a high level (98%) of nucleotide sequence identity in the RT+RNase H region. Within the genus Badnavirus, these viruses were most closely related to schefflera ringspot virus (SRV), sharing 63% identity at the nucleotide level. Based on the ICTV species demarcation criteria for the genus Badnavirus (more than 20% nucleotide sequence divergence in the RT+RNase H region), the Polyscias virus is proposed to be a new member of the genus, and the name polyscias mosaic virus (PoMV) is proposed. The complete genome sequence was deposited in the NCBI GenBank database under accession no. MH475918.

Identification of Tobacco streak virus in Cranberry and the Association of TSV with Berry Scarring.

Cranberry plants bearing disfigured, scarred fruit were reported by growers in the major cranberry-growing region of central Wisconsin in July 2012. Plants bearing scarred fruit have since been observed in Massachusetts and New Jersey. Three complementary methods provided evidence of Tobacco streak virus (TSV) in symptomatic plants: (i) leaves and scarred berries tested positive for TSV by double-antibody sandwich enzyme-linked immunosorbent assay; (ii) quasi-isometric particles approximately 33 nm in diameter were extracted from leaves of symptomatic plants and visualized using transmission electron microscopy; and (iii) coat protein gene sequence analysis revealed 94 to 99% nucleotide similarity with reference TSV sequences. In newer cultivars, 99% of uprights with scarred berries tested positive for TSV. In older cultivars, 31% of uprights with scarred berries tested positive for TSV and the remaining 69% of uprights with scarred berries tested positive for Blueberry shock virus. TSV overwintered in cranberry plants, and leaves, pollen, and fruit tested positive for TSV the year following symptom occurrence. Attempts to inoculate cranberry using infected pollen or sap as inoculum failed, but several herbaceous hosts tested TSV positive following mechanical inoculation. Phylogenetic analysis of the coat protein gene of 26 TSV isolates from various cultivars of cranberry in Wisconsin, New Jersey, and Massachusetts revealed diversity. This work provides information that will be useful in understanding the epidemiology of TSV in cranberry and in the development of management strategies.

First Report of Tobacco etch virus Infection in Coleus in the United States.

Virus-like disease symptoms consisting of foliar and veinal necrosis similar to those caused by Coleus vein necrosis virus (CVNV) (2) were observed in plants of coleus (Coleus blume Benth.) 'Rustic Orange' obtained from retail greenhouse outlets in Missouri and Minnesota. Flexuous, filamentous, 750 to 770 nm virus-like particles (vlps) were observed by transmission electron microscopy in negatively stained partially purified leaf tissue extracts from symptomatic 'Rustic Orange' leaf tissue. No other virus-like particles were observed and none were detected in extracts from asymptomatic leaves. These vlps were longer than those of CVNV (640 nm) (2) and were not detected by immunosorbent electron microscopy (ISEM) using antibodies to CVNV (2). Degenerate potyvirus primers PNIbF1 (5'GGBAAYAATAGTGGNCAACC3') and PCPR1 (5'GGGGAGGTGCCGTTCTCDATRCACCA3') (1) and total RNA extracted from 'Rustic Orange' leaf tissue with a Qiagen RNeasy Kit were used for reverse transcription-PCR with Ready-To-Go RT-PCR Beads (GE Healthcare). A 950-bp amplicon was obtained from total RNA from diseased but not from healthy leaf tissue. The nucleotide sequence of the amplicon (GenBank Accession No. GQ268818) had levels of identity to published Tobacco etch virus (TEV) sequences comprising portions of the nuclear inclusion body (NIb) and coat protein (CP) gene regions ranging from 89% (L38714) to 93% (M15239, M11458). The identity of the virus occurring in 'Rustic Orange' was further confirmed by ISEM. Virions were trapped and decorated by antibodies to TEV (ATCC PVAS 32). Systemically infected leaf tissue from Datura stramonium in which the coleus TEV isolate was propagated was used to mechanically inoculate Carborundum-dusted leaves of virus-free test plants of 'Rustic Orange' (Park Seed, Greenwood, SC). Inoculated plants developed foliar necrosis symptoms similar to those observed originally, and the presence of TEV was confirmed by ISEM and RT-PCR and nucleotide sequence analysis as described above. To our knowledge, this is the first report of a disease of coleus caused by TEV. Many of approximately 30 'Rustic Orange' plants in one nursery in Minnesota showed similar necrotic foliar symptoms and randomly selected plants tested positive for TEV by ISEM. This suggests that TEV infection in this variety may be spread by vegetative propagation from infected stock plants. References: (1) Y.-C. Hsu et al. J. Virol. Methods 128:54. 2005. (2) D. S. Mollov et al. Plant Dis. 91:754. 2007.

First Report of Tobacco rattle virus in Sedum in Minnesota.

Sedums (Sedum spp.; Crassulaceae) are perennial landscape plants that are grown widely because they are drought tolerant and winter hardy. Plants of Sedum 'Matrona' showing faint foliar ringspot symptoms were collected at a nursery retail outlet in St. Paul, MN in July 2008 and tested for possible viral infection by transmission electron microscopic (TEM) examination of negatively stained, partially purified leaf tissue extracts (1). The only virus-like particles observed were rigid, rod-shaped particles similar to those of Tobacco rattle virus (TRV) and other tobraviruses. A random sample of 100 measurements showed particles 20 nm in diameter with two modal lengths of 115 nm and 175 nm. These virus-like particles were confirmed to be those of TRV by immunosorbent electron microscopy (1) using antiserum to TRV (ATCC PVAS 75) and by reverse transcription (RT)-PCR using total RNA extracted with the RNeasy Kit (Qiagen, Valencia, CA) and primers that yield a 462-bp amplicon from TRV RNA 1 (4). An amplicon of the expected size was obtained by RT-PCR and its nucleotide sequence (GenBank Accession No. GQ268817) had 95 to 99% identity to published TRV sequences (AAW13192 and AAB48382). Two additional amplicons generated by RT-PCR from separate plants were identical in size and nucleotide sequence to the first. On the basis of virion morphology, serological relatedness, and sequence identity, the virus associated with mild ringspot symptoms in sedum was identified as an isolate of TRV. To our knowledge, this represents the first report of TRV incidence in sedum. Although Arabis mosaic virus is the only other virus reported to occur in sedum (2), we have observed numerous, flexuous filamentous 750 to 800 nm virus-like particles in partially purified extracts of a range of sedums showing mild mosaic and/or vein-clearing symptoms in Minnesota. Similar virus-like particles were not observed by TEM in partially purified extracts from TRV-infected 'Matrona' plants, suggesting that they did not contribute to the symptoms observed. We have reported previously (3) the occurrence of TRV in a variety of widely grown perennial ornamentals that provide potential sources of inoculum for spread of this virus by nematode vectors (Trichodorus and Paratrichodorus spp.) that occur commonly in garden soil, and Sedum is now added to the list of potential TRV reservoir plants. References: (1) Y. S. Ahlawat et al. Plant Dis. 80:590, 1996. (2) A. Gera et al. Acta Hortic. 722:175, 2006. (3) B. E. Lockhart et al. Plant Dis. 79:1249, 1995. (4) D. J. Robinson. J. Virol. Methods 40:57, 1992.

First Report of Alternanthera mosaic virus Infection in Angelonia in the United States.

Stunting, chlorosis, and light yellow mottling resembling symptoms of nutrient deficiency were observed in angelonia (Angelonia angustifolia) in commercial production in New York. Numerous, filamentous particles 520 to 540 nm long and spherical virus particles 30 nm in diameter were observed by transmission electron microscopy (TEM) in negatively stained partially purified extracts of symptomatic Angelonia leaf tissue. Two viruses, the filamentous potexvirus Alternanthera mosaic virus (AltMV) and the spherical carmovirus Angelonia flower break virus (AnFBV) were subsequently identified on the basis of nucleotide sequence analysis of amplicons generated by reverse transcription (RT)-PCR using total RNA isolated from infected leaf tissue. A 584-bp portion of the replicase-encoding region of the AltMV genome was obtained with the degenerate primers Potex 2RC (5'-AGC ATR GNN SCR TCY TG-3') and Potex 5 (5'-CAY CAR CAR GCM AAR GAT GA-3') (3). Forward (AnFBV CP 1F-5'-AGC CTG GCA ATC TGC GTA CTG ATA-3') and reverse (AnFBV CP 1R-5'-AAT ACC GCC CTC CTG TTT GGA AGT-3') primers based on the published AnFBV genomic sequence (GenBank Accession No. NC_007733) were used to amplify a portion of the viral coat protein (CP) gene. The nucleotide sequence of the amplicon generated using the potexvirus-specific primers (GenBank Accession No. EU679362) was 99% identical to the published AltMV (GenBank Accession No. NC_007731) sequence and the nucleotide sequence of the amplicon obtained using the AnFBV CP primers was 99% identical to the published AnFBV genomic sequence (GenBank Accession No. EU679363). AnFBV occurs widely in angelonia (1) and AltMV has been identified in phlox (2). These data confirm the presence of AltMV and AnFBV in diseased angelonia plants showing stunting and nutrient deficiency-like symptoms and substantiates, to our knowledge, this first report of AltMV in angelonia in the United States. References: (1) S. Adkins et al. Phytopathology 96:460, 2006. (2) J. Hammond et al. Arch. Virol. 151:477, 2006. (3) R. A. A. van der Vlugt and M. Berendeson. Eur. J. Plant Pathol. 108:367, 2002.

Molecular characterization of banana streak acuminata Vietnam virus isolated from Musa acuminata siamea (banana cultivar).

An isolate of banana streak virus (BSV) that does not also occur as an integrant in the Musa balbisiana genome was sought in order to investigate the biological role of BSV in the evolution of either the Musa genome or of the virus itself. We isolated BSV virions from a Musa acuminata siamea accession from Vietnam and sequenced the entire viral genome. The molecular organization is similar to that described for other BSV but slightly larger (7801 bp vs. 1611-7568 bp), and ORF I has a non-conventional start codon. This genome was sufficiently different to propose it as a member of a distinct species named Banana streak virus strain acuminata Vietnam (BSAcVNV).

Banana streak virus Identified for the First Time in Peru in Cavendish Banana (Musa AAA).

In October of 2005, a field survey was done in the province of Piura in northern Peru to determine the cause of a disease known locally as "mosaico" that was affecting organic Cavendish banana (Musa AAA) grown for the export market. Disease symptoms consisted of pronounced chlorotic and necrotic lesions on leaves of affected plants. Twenty-four farms were visited, and at each location, 10 randomly selected plants at flowering stage were evaluated for disease incidence and severity. Plants showing virus-like symptoms were observed in 18 of the 24 locations (75%). Fifty-two banana leaf samples, 27 from plants showing virus-like symptoms and 25 from asymptomatic plants, were tested for the presence of Banana streak virus (BSV), Cucumber mosaic virus (CMV), and Banana mild mosaic virus (BanMMV) by immunosorbent electron microscopy (ISEM) using partially purified leaf tissue extracts (2).The same extracts were also tested by immunocapture PCR (IC-PCR) for presence of BSV and specific BSV isolates (BSV-OL, BSV-GF, BSV-IM, and BSV-CAV) using badnavirus-specific degenerate primers and BSV isolate-specific primers, respectively (1). Seventeen of 27 leaf samples showing virus-like symptoms (63%) tested positive for BSV by ISEM and IC-PCR using badnavirus, but not isolate-specific, primers. The symptoms on the 10 samples that tested negative were not typical of BSV infection. One asymptomatic leaf sample (4%) also tested positive for BSV. To validate the PCR results, the nucleotide sequence of the amplicon from a plant showing the most prevalent foliar symptom type was determined. This sequence (GenBank Accession No. DQ674317) had ≤86% homology to the corresponding ORF III polyprotein region of BSV and other badnaviruses. Neither CMV nor BanMMV was detected in any of the 52 samples tested. From these results, it was concluded that "mosaico" disease of organic Cavendish bananas in northern Peru is associated frequently with BSV infection and that there is a high incidence of BSV infection in this area. To our knowledge, this is the first report of BSV occurrence in Peru. It was both surprising and interesting that neither BSV-OL nor BSV-GF, the two BSV isolates found most commonly in banana (Musa AAA) and plantain (Musa AAB) in South and Central America (B. E. L. Lockhart, unpublished), was detected in Cavendish banana in northern Peru. Failure to detect BSV-OL and BSV-GF suggests that field infection may be due to vertical transmission by clonal propagation rather than to horizontal transmission from local plantain and that control of "mosaico" disease could therefore be achieved by use of virus-free planting material. References: (1) A. D. W. Geering et al. Phytopathology 90:921, 2000. (2) B. E. L. Lockhart et al. Phytopathology 82:921, 1992.

Occurrence of Arabis mosaic virus in Hostas in the United States.

Hostas (Hosta spp.) are one of the most widely grown and economically important landscape perennials in the nursery industry in North America. Several viruses including Hosta virus X (HVX), Tobacco rattle virus (TRV), Tobacco ringspot virus (ToRSV), Tomato ringspot virus (TomRSV), Impatiens necrotic spot virus (INSV), and Tomato spotted wilt virus (TSWV) are known to occur in hostas (4). This report confirms the occurrence of an additional virus, Arabis mosaic virus (ArMV), in hostas in North America. This virus was first identified during the summer of 2004 in Hosta fortunei 'Sharmon' in several garden centers in Minneapolis and St. Paul, MN. Entire lots of this variety, numbering several dozen plants, showed symptoms consisting of blanching of the foliage similar to those caused by ToRSV and TomRSV infection (4). Symptoms persisted throughout the growing season. Virus-like particles, 28 to 30 nm in diameter, were observed by electron microscopy in partially purified extracts of symptomatic leaf tissue following fixation with 5% glutaraldehyde and negative staining with 2% sodium phosphotungstate, pH 7.0. Particles had an angular outline and some were penetrated by stain. No other virus-like particles were observed in these extracts. The particles were identified as those of ArMV. Identification was made using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and immunosorbent electron microscopy (ISEM) with antiserum to ArMV (PVAS-587) obtained from the American Type Culture Collection, Manassas, VA. In the spring and summer of 2005, ArMV was again identified as described above in 'Sharmon', H. undulata 'Albomarginata' samples from Minnesota, Michigan, and Nebraska, and H. 'Marion Bachman' and H. 'Touch of Class' from two wholesale nurseries in Minnesota. Symptoms in these hosta cultivars were similar to those observed in 'Sharmon' and were accompanied by stunting and leaf deformation. A portion of the coat protein (CP) gene of the ArMV isolate from 'Sharmon', designated ArMV-H, was amplified using reverse transcription-polymerase chain reaction (RT-PCR) with ArMV-specific CP primers (3) and total RNA extracted with a RNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA). Amplicons of the expected size (220 bp) were cloned and five clones were sequenced. Nucleotide sequence identities of the ArMV-H CP sequence to corresponding ArMV databank entries varied from 94 to 88% (Genbank Accession Nos. AY017339 and D10086 and X55460 and X81815, respectively). Interestingly, the hosta ArMV isolate was not transmitted by mechanical inoculation to diagnostically susceptible indicator plants (cucumber, tobacco, and petunia) (2) or to hosta (H. undulata 'Albormarginata', H. 'Honeybells', and H. 'Royal Standard'). Testing by using ELISA and ISEM showed that 'Sharmon' source plants contained high levels of ArMV antigen and virions, and a high percentage of virions were not penetrated by negative stain, indicating that they were not empty (i.e., devoid of RNA). It appears that ArMV-H may be transmitted only vertically, (i.e., clonal propagation) and this raises some interesting questions about the molecular basis of this anomaly. An isolate of ArMV from hops was similarly reported to have a very restricted host range (1) suggesting a possibility of a common mechanism of host range restriction. References: (1) K. R. Bock. Ann. Appl. Biol. 57:431, 1966. (2) A. A. Brunt et al. Viruses of Plants. CAB Internacional Mycological Institute, Wallingford, UK, 1995. (3) P. Kominek et al. Acta Virol. 47:199, 2003. (4) B. E. L. Lockhart and S. Currier. Acta Hortic. 432:62, 1996.

Characterisation of Banana streak Mysore virus and evidence that its DNA is integrated in the B genome of cultivated Musa.

We have sequenced the complete genome of an isolate of Banana streak virus from banana cv. 'Mysore' and show that it is sufficiently different from a previously characterised isolate from cv. 'Obino l'Ewai' to warrant recognition as a distinct species, for which the name Banana streak Mysore virus (BSMysV) is proposed. The structure of the BSMysV genome was typical of badnaviruses in general, although ORF I had a non-conventional start codon. Evidence that at least part of the BSMysV genome is integrated in the B genome of cultivated Musa is presented and transmissibility by the mealybug Planococcus citri also demonstrated.

Three Previously Unrecorded Viral Diseases of Astilbe, Fuschia, and Thermopsis Species in Minnesota.

Interest in virus diseases of perennial ornamentals has been increasing because of their increasing monetary value, because wholesale producers perceive an advantage in marketing disease-free stock, and because widespread international movement of these plants carries the risk of introduction of exotic viruses. In an ongoing study to identify and document viral diseases of perennial ornamentals used in the United States commercial horticultural industry, three virus-like diseases of astilbe (Astilbe chinensis), fuschia (Fuschia cv. Gartenmeister) and false lupine (Thermopsis caroliniana) occurring in Minnesota were investigated. Symptomatic plants were selected from lots in commercial greenhouses and garden centers in several locations in Minnesota. Astilbe with systemic chlorosis was found to be infected with Tobacco ringspot virus (TRSV). Fuschia with leaf mottling and leaf deformation was found to be infected with Cucumber mosaic virus (CMV), and false lupine with mosaic and leaf deformation symptoms was found to be infected with Bean yellow mosaic virus (BYMV). Identification of the three viruses was based on: 1) virion presence and morphology in partially purified leaf extracts using electron microscopy (EM) and immunosorbent electron microscopy (1); 2) enzyme-linked immunosorbent assay using crude leaf extracts; and 3) biological properties, including symptoms produced in indicator plants. Antisera to BYMV (ATCC PVAS-368), CMV (ATCC PVAS-30), and TRSV (ATCC PVAS-157) were obtained from the American Type Culture Collection, Manassas, VA. No other virus-like particles were observed with EM in partially purified leaf extracts of the three plants, no virus-like particles were observed in similar preparations from asymptomatic plants, and indicator plant tests did not indicate the presence of any other mechanically transmissible viruses. The TRSV isolate from astilbe and the BYMV isolate from false lupine produced typical symptoms on indicator plants susceptible to known isolates of these two viruses. The CMV isolate from fuschia was similar to previously described isolates of CMV (2) in most respects and was readily transmitted in a nonpersistent manner by Myzus persicae, but was unusual in that it did not infect Nicotiania benthamiana, N. glutinosa, and tomato, which are normally highly susceptible to infection by CMV. The identity of the fuschia CMV isolate was further confirmed by reverse transcription-polymerase chain reaction (RT-PCR) amplification with CMV-specific oligonucleotide primers (3). The PCR product was of the predicted size (500 bp) and was cleaved by restriction digestion with EcoRI, suggesting that the fuschia virus is a Type II CMV isolate (3). To my knowledge, this is the first report of TRSV infection in astilbe, CMV infection in fuschia, and of a viral disease of false lupine. References: (1) Y. C. Ahlawat et al. Plant Dis. 80:590, 1996. (2) A. A. Brunt et al. Viruses of Plants. CAB International, Wallingford, UK, 1995. (3) S. Wylie et al. Aust. J. Agric. Res. 44:41, 1993.

Sequencing, Improved Detection, and a Novel Form of Kalanchoë top-spotting virus.

Virions of Kalanchoë top-spotting virus (KTSV) were purified from infected leaf tissue of Kalanchoë blossfeldiana using a procedure that prevented loss of virus in the initial extraction step. The double-stranded DNA viral genome was cloned and sequenced. The KTSV genome was 7,591 bp in size and contained three open reading frames capable of encoding proteins of 21, 14, and 223 kDa, respectively. The size and organization of the KTSV genome were similar to those of other mealybug-transmitted badnaviruses. Several oligonucleotide primer pairs, based on the KTSV genomic sequence, were used to efficiently detect the virus in plants, thereby removing a major constraint to reliable screening of kalanchoë propagating stock and breeding lines for KTSV infection. Two KTSV sequences, one symptom-inducing and the other not, were identified and differentiated by polymerase chain reaction (PCR) amplification and digestion of the resulting amplicon with restriction endonucleases. Preliminary results from graft-transmission tests and PCR indexing suggest that the nonsymptomatic form of KTSV may represent an integrated viral element. The occurrence of such integrated pararetroviral elements poses practical problems for routine PCR indexing of breeding and propagating stock, and also raises the possibility of symptomatic episomal infections arising from these viral integrants.

Identification of genetic markers linked to banana streak disease expression in inter-specific Musa hybrids.

Recently-introduced inter-specific Musa hybrids, bred for improved yield and resistance to diseases, have been found to be widely infected with banana streak virus (BSV), the causal agent of banana streak disease (BSD). One hypothesis suggests: (1) that BSD occurrence in these inter-specific hybrids results from activation of BSV-Ol endogenous pararetrovirus sequences (EPRV) integrated into the Musa genome rather than from external sources of infection, and (2) that the process of genetic hybridisation may be one factor involved in triggering episomal expression of the BSV integrants. In order to test this hypothesis we carried out a genetic analysis of BSD incidence in a F1 triploid ( Musa AAB) population produced by inter-specific hybridisation between virus and disease-free diploid Musa balbisiana (BB) and tetraploid Musa acuminata (AAAA) parents. Half of the F1 progeny of this cross expressed BSV particles. Using PCR amplification to determine the presence or absence of BSV-Ol EPRVs, it was determined that this endogenous sequence was specific to the M. babisiana genome and occurred in a homozygous state. Using bulk segregant analysis, ten AFLP markers co-segregating with the absence and/or presence of BSV infection were identified in the M. balbisiana genome, but were absent from the M. acuminata genome. Seven of these markers segregated with the presence of a BSV particle and three with the absence of BSV particles. Analysis of the segregation of these markers using a test-cross configuration allowed the construction of a genetic map of the linkage group containing the locus associated with BSV infection in the F1 hybrid population. These data indicate that a genetic mechanism is involved in BSV appearance, and suggest that a monogenic allelic system confers the role of carrier to the M. balbisiana parent.

Report of Sugarcane yellow leaf virus in Ecuador, Guatemala, and Nicaragua.

In 1998, sugarcane plants with symptoms similar to yellow leaf syndrome were observed in Ecuador, Guatemala, and Nicaragua. These plants showed yellowing of the central portion of the third to sixth leaves on the abaxial surface from the youngest expanding spindle leaf. Intense yellowing and necrosis of the leaf tip and the central portion of the leaf blade near the midrib occurred in severe cases. A tissue blot immunoassay was used to detect Sugarcane yellow leaf virus (SCYLV) in the midrib of the top visible dewlap leaf (2) using an antiserum specific to a Florida isolate of SCYLV (1). Since the virus can be detected in asymptomatic plants, leaf samples were collected from both symptomatic and asymptomatic plants. Symptom expression was most intense in plants at maturity that were under stress. Cut ends of leaf samples were imprinted on nitrocellulose membranes in the country of origin, and control samples of healthy and SCYLV-infected leaves were imprinted in Florida on each membrane prior to serological processing. The results from the following locations and cultivars, and the ratio of SCYLV-positive samples over the total samples is indicated: Milagro, Ecuador, PR 70-2085 (11/24) and PR 76-3385 (48/63) in 1999; Escuintla, Guatemala, CP 57-603 (1/10), CP 73-1547 (0/10), CP 72-2086 (120/308), PR 75-2002 (8/11), PR 78-294 (10/10), and PR 87-2080 (13/13) in both 2000 and 2001; Tipitapa, Nicaragua, L 68-40 (21/70) in 1998; and Chinandega, Nicaragua, CP 72-2086 (30/30) and CP 74-2005 (13/45) in 2000. CP 72-2086 is a major commercial cultivar in Central American countries and was infected in both Guatemala and Nicaragua. SCYLV was detected in 9 of 10 cultivars sampled. An exception was noticed in CP 73-1547 in Guatemala where none of the 10 plants tested were infected; however this cultivar has a high incidence of SCYLV in Florida. Only 1 of 10 samples of CP 57-603 was SCYLV positive in Guatemala; however, this cultivar has a low incidence of infection in Florida and is considered more resistant than the other CP cultivars sampled. To our knowledge, this is the first report SCYLV in Ecuador, Guatemala, and Nicaragua. References: (1) S. M. Scagliusi and B. E. L. Lockhart. Phytopathology 90:120, 2000. (2) S. Schenck et al. Sugar Cane 4:5, 1997.

Analysis of the distribution and structure of integrated Banana streak virus DNA in a range of Musa cultivars.

Summary Banana streak virus strain OL (BSV-OL) commonly infects new Musa hybrids, and this infection is thought to arise de novo from integrated virus sequences present in the nuclear genome of the plant. Integrated DNA (Musa6+8 sequence) containing the whole genome of the virus has previously been cloned from cv. Obino l'Ewai (Musa AAB group), a parent of many of the hybrids. Using a Southern blot hybridization assay, we have examined the distribution and structure of integrated BSV-OL sequences in a range of Musa cultivars. For cv. Obino l'Ewai, almost every restriction fragment hybridizing to BSV-OL was predicted from the Musa6+8 sequence, suggesting that this is the predominant type of BSV-OL integrant in the genome. Furthermore, since only two junction fragments of Musa/BSV sequence were detected, and the Musa6+8 sequence is believed to be integrated as multiple copies in a tandem array, then the internal Musa spacer sequences must be highly conserved. Similarly sized restriction fragments were detected in four BB group cultivars, but not in six AA or AAA group cultivars, suggesting that the BSV-OL sequences are linked to the B-genome of Musa. We also provide evidence that cv. Williams (Musa AAA group) contains a distinct badnavirus integrant that is closely related to the 'dead' virus integrant previously characterized from Calcutta 4 (Musa acuminata ssp. burmannicoides). Our results suggest that the virus integrant from cv. Williams is linked to the A-genome, and the complexity of the hybridization patterns suggest multiple sites of integration and/or variation in sequence and structure of the integrants.

Evidence that the proliferation stage of micropropagation procedure is determinant in the expression of banana streak virus integrated into the genome of the FHIA 21 hybrid (Musa AAAB).

Banana streak virus (BSV) is causing increasing concern in almost every producing area of banana and plantain (Musa spp.) worldwide. This situation appeared partially linked to some breeding lines and micropropagated hybrids. A complete BSV sequence integrated into the genome of a triploid plantain has been recently characterised and it has been hypothesised that it could give rise to infectious virus via recombination. In this study, we evaluated the effect of a routine micropropagation procedure on the expression of BSV in the FHIA 21 tetraploid hybrid. The widespread presence of integrated sequences and the absence of episomal BSV in thirty FHIA 21 "mother plants" selected for micropropagation were first confirmed by specific PCR and IC-PCR tests. The proliferation stage of the procedure, characterised by an intensive production of neoformed buds, appeared determinant in BSV expression whereas the rooting and acclimatisation stages had little or no effect. The duration in culture and the way of subdividing the clumps of proliferation influenced greatly the percentage of episomal BSV infections, reaching 58% of infected micropropagated lines after six in vitro subcultures. These data suggest that the expression of episomal BSV observed during the in vitro procedure is correlated with the presence of an integrated form.

First Report of Sugarcane yellow leaf virus (ScYLV) in Costa Rica.

In Costa Rica, sugarcane plants with symptoms similar to those described for yellow leaf syndrome (YLS) (1,2) were first observed in 1994 in research plots of imported material in the midland areas of San Carlos and Turrialba. Recently, the same symptoms have been observed in commercial plantations in Turrialba. Symptomatic plants were characterized by yellowing of the leaves and central veins, the yellowing being more intense near the leaf tips. In severe cases, veins became reddish, and necrosis developed along the leaf edges, beginning at the leaf tip and extending to the base of the leaf. Growth of stems and roots was also reduced in infected plants. Minipurifications of six plants of four different varieties were examined by immunospecific electron microscopy (ISEM) using polyclonal antibodies (1). They were: one symptomatic plant each of the varieties H782313 and H608521; two symptomatic plants of H657052, and one asymptomatic plant each of H608521 and H827318. Isometric particles of approximately 28 nm were observed in the asymptomatic H827318 plant and in all symptomatic plants, with the exception of one plant of H657052. The size and morphology of the particles was similar to those reported for Sugarcane yellow leaf virus (ScYLV) (2). The presence of ScYLV was verified by indirect enzyme-linked immunosorbent assay (ELISA) using polyclonal antibodies (1). Twenty-two of 24 symptomatic plants and five of 13 asymptomatic plants were positive for ScYLV. These findings confirm the association of ScYLV with the yellows syndrome of sugarcane observed in Costa Rica. However, as was also reported by Scaglusi and Lockhart (1), ScYLV was not detected in several symptomatic plants, and research is continuing to determine whether other pathogens are associated with this syndrome in Costa Rica. References: (1) S. Scagliusi and B. E. L. Lockhart. Phytopathology 90:120, 2000; (2) J. Vega et al. Plant Dis. 81:21, 1997.

Detection of Sugarcane yellow leaf virus in Quarantine and Production of Virus-free Sugarcane by Apical Meristem Culture.

Sugarcane yellow leaf virus (SCYLV) was detected for the first time in 1996 in the Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD) sugarcane quarantine at Montpellier by reverse transcription-polymerase chain reaction (RT-PCR) in varieties from Brazil, Florida, Mauritius, and Réunion. Between 1997 and 2000, the virus was found by RT-PCR and/or tissue-blot immunoassay (TBIA) in additional varieties from Barbados, Cuba, Guadeloupe, Indonesia, Malaysia, Philippines, Puerto Rico, and Taiwan, suggesting a worldwide distribution of the pathogen. An excellent correlation was observed between results obtained for the two diagnostic techniques. However, even though only a few false negative results were obtained by either technique, both are now used to detect SCYLV in CIRAD's sugarcane quarantine in Montpellier. The pathogen was detected by TBIA or RT-PCR in all leaves of sugarcane foliage, but the highest percentage of infected vascular bundles was found in the top leaves. The long hot water treatment (soaking of cuttings in water at 25°C for 2 days and then at 50°C for 3 h) was ineffective in eliminating SCYLV from infected plants. Sugarcane varieties from various origins were grown in vitro by apical bud culture and apical meristem culture, and the latter proved to be the most effective method for producing SCYLV-free plants.