Reflects the coat protein variability of apple mosaic virus host preference?

Apple mosaic virus (ApMV) is a widespread ssRNA virus which infects diverse species of Rosales. The phylogenetic analysis of complete capsid protein gene of the largest set of ApMV isolates discriminated two main clusters of isolates: one cluster correlates with Maloideae hosts and Trebouxia lichen algae hosts; a second with hop, Prunus, and other woody tree hosts. No correlation was found between clusters and geographic origin of virus isolates, and positive selection hypothesis in distinct hosts was not confirmed: in all virus populations, purifying selection had occurred. GGT→AAT substitution resulted in Gly→Asn change inside the zinc-finger motif in the capsid protein was revealed specific for discrimination of the clusters and we hypothesise that could influence the host preference.

First Report of Polymyxa graminis f. sp. temperata, a Vector of Soilborne Cereal Viruses in the Czech Republic.

Polymyxa graminis L. is an eukaryotic, obligate, biotrophic parasite of plant roots (1), which belongs to a poorly studied, discrete, taxonomic unit informally called the 'plasmodiophorids'. P. graminis has the ability to acquire and transmit a range of soilborne viruses that belong to at least three separate genera and can cause economically significant diseases in cereal crops. For example, the winter barley disease caused by Barley yellow mosaic virus (BaYMV) and/or Barley mild mosaic virus (BaMMV) is widespread in Europe, Japan, and China; yield losses of >50% may occur when susceptible barley cultivars are grown on severely infested soils (2). Monitoring for P. graminis was started in the Czech Republic in May 2008. Fifty-six soil samples were collected from different localities of cereal production (wheat and barley) in the Královéhradecký Region (eastern Czech Republic). Soil from each sample was placed in five replicate pots (12 × 12 cm) in a greenhouse at 22 to 25°C. Seeds of barley cv. Florian were sown into the soil (10 seeds per pot). Negative control soil (noninfested soils from the Czech Republic) and positive control soil (known P. graminis-infested soil from Germany) were also planted to barley in five replicate pots. After 90 days, plants were collected and the roots were washed thoroughly in sterilized water and examined with a light microscope without staining. The fungus was identified as P. graminis on the basis of morphology of resting spores (cystosori) and sporangia and the size of individual cystosori (4 to 5 µm in diameter) according to Thouvenel et al. (3). Cystosori of P. graminis were observed in the roots of plants grown in 20 of the 56 soil samples, especially the samples from Ceské Mezirici. The presence of P. graminis in the roots of plants grown in the soil samples and the positive control sample versus the absence of the vector in roots of plants in the negative control soil was verified by PCR assay with DNA extracts and the Psp1 and Psp2rev primers according to Legrève et al. (Page 40 in: Proceedings of the Fifth Symposium of the International Working Group on Plant Viruses with Fungal Vectors, 2003). The PCR assay included denaturation at 94°C for 2 min, then 35 cycles including denaturation of 30 s at 94°C, annealing at 60°C for 1 min, and elongation at 72°C for 35 s. A final elongation was completed at 72°C for 7 min. To characterize the P. graminis isolates, the amplified PCR product (a DNA fragment of 472 bp) was sequenced and blasted for each of the samples that tested positive. These sequences were aligned with a known sequence (GenBank Accession No. AM259276) for P. graminis. The sequences from P. graminis on barley were 100% homologous to the published sequence of P. graminis f. sp. temperata. To our knowledge, this is the first report of P. graminis f. sp. temperata in the Czech Republic. References: (1) G. A. Ledingham. Can. J. Res. 17:50, 1939. (2) R. T. Plumb et al. Plant Pathol. 35:314, 1986. (3) J. C. Thouvenel et al. Plant Dis. 64:957, 1980.

First Report of Peach latent mosaic viroid Infecting Peach in Egypt.

Peach latent mosaic viroid (PLMVd) is a widespread pathogen of stone fruit trees in some European and Mediterranean countries and also in North America. To access the presence of the viroid in Egypt, a survey was conducted that covered five commercial peach orchards in the El Khatatba Region in Al Minufiya Governorate. During 2003 and 2004, 73 peach trees (cv. Florida grafted on Nemagard rootstock) were visually inspected and sampled. No symptoms characteristic of PLMVd infection, such as mosaic, delayed growth, or fruit suture cracking, were observed. All samples were tested for the presence of PLMVd using dot-blot hybridization and reverse transcription (RT)-PCR. Aliquots (5 µl) of total nucleic acids extracted from approximately 2 mg of leaf tissue were spotted onto positively charged nylon membranes and hybridized under stringent conditions with a digoxigenin-labeled riboprobe (2). The extracts (1 µl) also were used in RT-PCR as described (1). Only 1 of the 73 peach trees was positive for PLMVd using these detection techniques. The RT-PCR product was of the size expected for PLVMd and was cloned and sequenced. The 339 nucleotide sequence was deposited in GenBank as Accession No. DQ839564. The sequence of this Egyptian PLMVd isolate was 94% identical to the reference PLMVd variant (GenBank Accession No. M83545) and most closely (95%) related to Canadian isolate variant 16 (GenBank Accession No. AJ550911). Such a low incidence compared with other countries may be because the survey was restricted to a limited number of samples, conducted on newly reclaimed lands where no sources of infection existed before, and material with relatively low PLMVd incidence might have been used for planting. Although the incidence of PLMVd was low in this survey, the occurrence represents a threat to the stone fruit tree industry in this country and regular screening of PLMVd in certification programs is suggested. To our knowledge, this is the first report of PLMVd on peach in Egypt. References: (1) S. Loreti et al. EPPO Bull. 29:433, 1999. (2) A. M. Shamloul et al. Acta Hortic. 386:522, 1995.

Detection of beet yellows virus by RT-PCR and immunocapture RT-PCR in Tetragonia expansa and Beta vulgaris.

Two sensitive methods, RT-PCR with phenol-extracted RNA or Triton X-100-released RNA and immunocapture RT-PCR (IR-RT-PCR) were used for the detection of Beet yellows virus (BYV) in young and old leaves of Tetragonia expansa and sugar beet (Beta vulgaris) and in sugar beet roots. Four oligonucleotide primer pairs proved suitable for the detection of BYV. The release of BYV RNA with Triton X-100 was shown to be a very effective and easy as compared to isolation of total RNA by phenol extraction with the same or higher sensitivity of subsequent PCR. Using the Triton X-100 release of RNA and IC-RT-PCR the sensitivity of detection was so high that pg amounts of BYV RNA occurring in dilutions up to 10(-6) of saps from young Tetragonia and sugar beet leaves could be detected.

First Report of Peach latent mosaic viroid and Hop stunt viroid Infecting Peach Trees in the Czech Republic.

Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd) are known to naturally infect stone fruits, but their contemporary presence in peach trees has been reported only recently (3). During a field validation of detection methods developed for sanitary screening of propagation material, PLMVd and HSVd, alone or in mixed infections, were detected in peach trees grown in the trial orchard of the Czech University of Agriculture in Prague. Leaf samples were collected in September 2002 from symptomless trees of peach cultivars imported from the United States (cvs. Sunhaven, Redhaven, Fairhaven, Cresthaven, Dixired, Halehaven, and NJC 103), Slovakia (cv. Luna), and a tree of Chinese wild peach, Prunus davidiana, and analyzed by reverse transcription-polymerase chain reaction (RT-PCR). PLMVd cDNA was amplified as previously reported (2) or by using two sets of primer pairs designed to amplify partial cDNAs, one reverse primer R: GTTTCTACGG CGGTACCTGA, complementary to the nucleotide positions 204 to 223 and forward primers F1: CGTATCTCAACGCCTCATCA, homologous to the positions 109 to 128, and F2: CTGCAGTTCCCGCTAGAAAG, homologous to the positions 15 to 34 of PLMVd reference sequence (2). The two pairs using the R sequence produced the expected size PCR products of 115 and 209 bp, respectively. RT-PCR for HSVd detection was performed as reported (1). The same total RNA preparations were also analyzed by molecular hybridization with nonisotopic riboprobes specific for each viroid. With minor exceptions, both methods gave similar results. Of 66 tested trees, 5 were infected with PLMVd, 46 were infected with PLMVd and HSVd, and 15 were free of both viroids. Viroid free plants included cvs. Luna, Cresthaven, Dixired, and Halehaven and the species P. davidiana. The high number of infections by both viroids was unexpected because mixed infections are generally rare (3). Most likely, mixed infections occurred during field manipulations and propagation of infected materials. To our knowledge, this is the first report of PLMVd in the Czech Republic. Although further investigations are needed to ascertain the spread of stone fruit viroids in the Czech Republic, our results also report an unusually high incidence of mixed infections of peach trees in this country. These results stress the need for a certification program to help control the spread of stone fruit viroids in the Czech Republic. References: (1) K. Amari et al. J. Gen. Virol. 82:953, 2001. (2) A. M. Shamloul et al. Acta Hort. 386:522, 1995. (3) M. Tessitori et al. Plant Dis. 86:329, 2001.

First Report of the Potato Cyst Nematode (Globodera pallida) in the Czech Republic.

Potato cyst nematode, Globodera pallida, was detected in several soil samples collected from various areas of the Czech Republic. Globodera rostochiensis pathotype Ro1 is known to be widespread in the Czech Republic. G. pallida was reported from the neighboring countries of Austria, Germany, and Poland, and also was suspected to be present in the Czech Republic, but it has never been unambiguously proved (1). Recently, nematode isolates have been recovered that multiply readily on Ro1 resistant potato cultivars. These isolates were identified on the basis of three tests: (i) microscopic observations of cyst vulval area morphology and stylets of second-stage larvae; (ii) enzyme-linked immunosorbent assay using a commercial kit; and (iii) a polymerase chain reaction method (2). A complete series of G. pallida and G. rostochiensis pathotypes from Scotland and Germany served as controls. Results were identical for all three methods used. One sample contained G. pallida only, five samples contained mixtures of G. rostochiensis and G. pallida, and one sample contained G. rostochiensis only. The origin of G. pallida contamination is unknown. Strict quarantine measures have been taken to prevent G. pallida from spreading into neighboring areas. To our knowledge, this is the first evidence of the occurrence of G. pallida in the Czech Republic. References: (1) J. Potocek et al. EPPO Bull. 21:81, 1991. (2) M. Zouhar et al. Plant Prot. Sci. 36:81, 2000.

Occurrence of the Root-Knot Nematode Meloidogyne hapla in the Czech Republic.

Meloidogyne hapla was detected in several commercial vegetable fields in the Province of Central Bohemia in the Czech Republic. Crops grown in the area of infestation include leek, onion, cauliflower, early potatoes, and carrot. During the past 2 to 3 years, growers have observed typical symptoms of Meloidogyne infection as stunting and galls on roots of carrot. Identification of the causal organism was based on microscopic observation of the perineal patterns of females obtained from root galls and polymerase chain reaction (1,2). Known isolates of M. chitwoodi, M. arenaria, M. javanica, M. incognita, and M. fallax from M. Phillips (SCRI, Dundee) and C. Zijlstra (PRI, Wageningen), and M. hapla from M. Liskova (PI, Kosice) were used as controls. M. hapla was identified in galled roots using both methods. Several fields with carrots were inspected in the area during vegetation in 2000 and 2001. In most fields, only isolated and limited outbreaks occurred, but recently, we observed a widespread and fairly homogeneous occurrence of root knot attributed to M. hapla in a 2-ha field. This nematode may become a more widespread and damaging pest, especially of carrot, in the Czech Republic. Other than carrot, vegetable crops grown in the area and tested in the greenhouse and field in soil highly infested by our isolates of M. hapla had no visible symptoms of infection. Therefore, exclusion of carrot cultivation or a longer crop rotation is recommended to minimize economic losses to growers. To our knowledge, this is the first report of the occurrence of M. hapla in the Czech Republic. References: (1) T. S. Harris et al. J. Nematol. 22:518, 1990. (2) C. Zijlstra et al. Genetics 85:1231, 1995.