MPPOL

Short Report
Acknowledgements
References

Molecular Plant Pathology On-Line [http://www.bspp.org.uk/mppol/1998/0929rybicki]

Novel Grass and Wheat Strains of Maize Streak Virus Detected by DNA Amplification and Sequencing

Edward P Rybicki1, Susan Dennis, Grant Napier, and Fiona L Hughes

1ed@molbiol.uct.ac.za

Department of Microbiology, University of Cape Town, PB Rondebosch, 7701, South Africa



Corresponding
author


Edward P Rybicki, Department of Microbiology, University of Cape Town, PB Rondebosch, 7701, South Africa. Tel: +27-21-650-3265; Fax: +27-21-689-7573; Email: ed@molbiol.uct.ac.za



ABSTRACT


The investigation of genetic diversity among maize, grass and wheat isolates of maize streak virus (MSV) and other Mastreviruses (family: Geminiviridae)   is described. The products of a degenerate primer polymerase chain reaction (PCR) DNA amplification from a variety of virus isolates were cloned and sequenced, and the +200 bp sequences aligned and compared for phylogenetic analysis. MSV isolates could be grouped into three distinct strain classes, one of which comprised closely-related isolates from maize, another closely-related viruses from wheat and grasses, and the third a single distinct grass type. The sequences represent a significant expansion of our knowledge of MSV diversity.




SHORT REPORT


Maize streak virus (MSV) is the type member of genus Mastrevirus, family Geminiviridae: it is an obligately leafhopper-transmitted (Cicadellidae: Cicadulina spp.) virus with a single-strand, circular (ssc) DNA genome and geminate virions ( Briddon and Markham, 1995; van Regenmortel et al., 1997). The virus occurs only in Africa and the neighbouring Indian Ocean islands of Mauritius, La RĂ©union, and Madagascar, where it can cause serious crop losses ( Rybicki and Pietersen, 1998).

In recent times, all viruses causing "streak disease" in grasses in Africa were included as strains of an all-encompassing MSV: these included viruses of maize as well as of grasses and sugarcane   (eg: Storey and McClean, 1930; McClean, 1947 Pinner et al. 1988; Dekker et al., 1988). Recently, however, sequencing and other nucleic acid characterisation techniques such as restriction mapping, polymerase chain reaction (PCR) amplification and sequencing of viral DNA, have allowed delineation of a number of distinct species. These include maize isolates of MSV, Panicum streak virus (PanSV), and sugarcane streak viruses (SSV) ( Howell, 1984; ( Mullineaux et al., 1984; Lazarowitz, 1988; Rybicki and Hughes, 1990; Hughes et al., 1991, 1992, 1993; Briddon et al., 1992 and 1994; Peterschmitt et al., 1996).   This group of viruses has been referrred to as the "African streak virus group" ( Hughes et al., 1992).  This work has shown that maize isolates of MSV are very closely related to one another - to 96% or greater total sequence similarity ( Briddon et al., 1994; Peterschmitt et al., 1996) - with distinct species of "African streak viruses" being separated by up to 40% difference in nucleotide sequence.

Although many workers have studied non-maize isolates of presumptive MSV, very few have characterised the viruses by any other means than host range/symptomatology and perhaps serology (eg: McClean, 1947; Pinner et al., 1988; von Wechmar and Hughes, 1990; Mesfin et al., 1992). Thus, although there was a broad recognition that viruses found in grasses generally do not cause severe disease in maize, there was no good idea of how similar these viruses were to maize isolates. This leads plant breeders, among others, to assume that the MSV found in maize overwinters in grasses and/or crops such as wheat, which may overlap in growing seasons.

We have previously addressed the question of MSV genetic variability by means of restriction mapping of whole genomic clones of virus DNA (Clarke et al., 1989; Hughes et al., 1992); however, although there were good indications that grass and other non-maize MSV isolates were more diverse than maize isolates, this could not be satisfactorily quantified. Briddon et al. (1994) used PCR amplification and sequencing of the coat protein gene to explore diversity of MSVs from around Africa; however, they found no more than 4% sequence variation among isolates.

We have continued our study of variation among isolates of MSV by means of PCR amplification of viral DNA isolated from maize, grasses and wheat; DNA sequencing of the products, and phylogenetic analysis. We used a highly degenerate set of primers already known to amplify a range of viruses from MSV to PanSV and SSV, and potentially capable of amplifying the very distantly related wheat dwarf and chloris striate mosaic mastreviruses (WDV and CSMV; Rybicki and Hughes, 1990).

Viruses, DNA samples and sequences used in this work are detailed in Table 1.  Local viruses were maintained as described by Hughes et al. (1992); samples of Australasian viruses were supplied as whole-plant DNA extracts by R Briddon, Dept Virus Research, John Innes Centre.

Total DNA extractions from infected plants were done for local plants as described by Hughes et al. (1992), and for Australasian plants as described by Briddon et al. (1994).

PCR amplifications were performed as described by Rybicki and Hughes (1990), using the pair of degenerate 17-mer oligonucleotide primers described therein. These amplify a +250-bp section of the C2 ORF of Mastreviruses. All products were electrophoresed in 2-3% TBE-containing agarose gels and stained for photography using 50 ng/ml ethidium bromide.

Two variants of Sanger dideoxy sequencing were used: these were direct sequencing of PCR products by the method of Bachmann et al. (1990), and sequencing of products cloned into pUC or pBLUESCRIPT vectors by the T-tailing method of Marchuk et al. (1990). In the latter case, at least two clones were completely sequenced for each product. Kits from US Biochemicals were used for all sequencing, with 35S-labelled dATP.

Sequences were analysed using DNAMAN ver. 3.0 (Lynnon BioSoft, Quebec, Canada) sequence manipulation and multiple alignment and TreeView dendrogram production software (Page, 1996) for PC. Tree descriptions were generated using the neighbour-joining algorithm in DNAMAN.

DNA sequences from the following viruses / isolates were successfully amplified by PCR (see also Table1):

CSMV-C, MSV-CT, MSV-Dig, MSV-Kom, MSV-RSE, MSV-Set, MSV-Tas, MSV-VW, MSV-WES and MSV-VM (results not shown).

Although products of about the right size were amplified from PanSV-Kar and PasSMV-BC DNA preparations, sequenced clones were of unidentified amplimers which were not virus related (not shown). No product could be amplified from any other DNA sample. Nucleotide sequences have been deposited in Genbank (see Table 1).



Figure 1


Figure 2


Figure 3


Aligned sequences are shown in Figure 1, with sequences from WDV, CSMV, Miscanthus streak virus (MiSV), PanSV-Kenya strain and sequenced MSVs included for sake of comparison. Sequences were truncated to exclude primers, and to allow comparison of MSV-WES, which was sequenced directly from the PCR product. The alignments were used to produce a relationship dendrogram, shown in Figure 2. Pairwise sequence similarities of MSVs only are shown in Figure 3.

It is obvious from Figure 3 that although all MSV isolates group together when compared to SSVs, PanSV, etc., there is a clear division of MSVs into what appears to be three distinct groupings: namely,

  • closely-related isolates which infect maize (termed the "maize type"),
  • closely-related isolates which infect wheat or grasses (termed the "wheat/grass type"),
  • and MSV-Set from a Setaria sp. as a unique grouping.

Bootstrap values of 100% indicate each grouping is "robust". The only other DNA amplified in this study from outside the MSV group - that of CSMV-C - is most closely related to the completely-sequenced CSMV (99%, 3 nucleotide exchanges), as expected from a serological study (Pinnerb et al., 1992). It is not clear why other DNAs did not amplify, as the primers were designed to handle CSMV and WDV amplification; however, with PanSV-Kar at least, the nucleotide sequence around one of the oligonucleotide priming sites does not agree with the consensus previously determined ( Rybicki and Hughes, 1990; Schnippenkoetter, 1998).

Novel findings in this work are the most different isolates of MSV yet characterised: MSV-Set, -Dig, -Tas, -VW and -WES all differ from maize MSVs by 9-15% in this sequence, whereas MSV-Reu (previously the most distinct; Briddon et al., 1994; Peterschmitt et al., 1996) differs by only 3-4% (see Figure 3). Moreover, the wheat/grass grouping of MSVs -Dig, -Tas, -VW and -WES are as similar to one another (98-100%) as are maize isolates (96-100%): this was an unexpected finding, as the wheat viruses were isolated from plants grown in winter wheat areas separated by up to 1000 km (eg: Free State and Western Cape), and the grass viruses come from areas as far apart as the Western Cape and Natal (1500 km).  The similarity of the maize-type viruses is less striking given previous evidence (eg: Briddon et al., 1994); however, two of the most similar isolates tested here (MSVs -Kom and -RSE) came from locations separated by over 2000 km.  By contrast, two viruses from the same location but from different hosts - MSVs VM and VW, from maize and wheat grown in consecutive seasons in the Vaalharts irrigation scheme in Mpumalanga Province, South Africa (MB von Wechmar, personal communication) - were strikingly dissimilar (91% identity)

The wheat/grass type viruses and the maize types are biologically different in that the former all cause mild symptoms in maize, whereas the maize types all cause severe symptoms ( Schnippenkoetter, 1998; Martin and Rybicki, 1998): note that MSV-VM and MSV-VW were switched in the latter reference. The new discoveries possibly complicate the presumed epidemiology of maize MSVs, in that the conventional wisdom was that maize MSV overwinters in wheat and/or in grasses, which should result in wheat viruses looking like the maize types.  However, no  maize-type MSVs were amplified out of three wheat and two grass samples, and no wheat/grass-types from maize.  While it is tempting to speculate further on epidemiology, the small sample size studied here precludes any firm conclusions.  The results obtained here do indicate, however, that more attention should be paid to determining the true over-wintering hosts of especially the maize types of MSV.

We have previously shown that comparisons of the short sequence amplified in this work yield effectively the same quantitative relationship shown by comparison of the whole sequence or parts thereof ( Rybicki, 1994; Rybicki and Hughes, 1990; Palmer and Rybicki, 1998); thus, we are confident that the results shown here are indicative of the relationships of whole virus genomes. Moreover, restriction map comparisons of whole genomes ( Hughes et al., 1992), and longer sequence comparisons from some of the same viruses show exactly the same overall relationships (EP Rybicki, W Schnippenkoetter, M Fyvie, J Willment, D Martin, unpublished).  Thus, while it is possible that recombination between isolates or even strain groups could complicate this picture, there is as yet no evidence that this has happened with MSV outside of the laboratory. 

Our characterisation of these novel viruses considerably expands our conception of the potential genetic diversity of MSV-like viruses, as well as indicating that their epidemiology may be more complicated than was hitherto believed, and has resulted in a number of projects aimed at elucidating the diversity here as well as in other parts of Africa. Determination of the different groups of viruses as strains rather than as species depends on other work on trans-replication between strains: the fact that viruses from different strain groups (eg: MSV-Set and MSV-Kom) can trans-replicate each other as well as form viable recombinants in the laboratory ( Schnippenkoetter, 1998) is a pointer to the necessity for doing further work on the natural diversity of these viruses.



ACKNOWLEDGE
-MENTS


EPR acknowledges the help of Prof Barbara von Wechmar in obtaining and maintaining virus isolates, of several generations of students for maintaining the research, and of the Foundation for Research Development for financial support.



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