Further observations on variation of lettuce mosaic virus in relation to lettuce (Lactuca sativa), and a discussion of resistance terminology

Two potyvirus isolates from endive, originating from southern France (Ls252) and from the Netherlands (Ls265), that were highly and poorly pathogenic on lettuce, respectively, were compared with a common isolate (Ls1) of lettuce mosaic virus (LMV) and with two highly deviant Greek isolates fromHelminthia (Picris) echioides (Gr4) and endive (Gr5), earlier recognized as LMV. The isolates could not be distinguished by particle morphology and serology, and were all identified as LMV. Leaf curling, plant stunting and necrosis were more characteristic of the virus than mosaic. The isolates studied varied considerably on differential host species and a range of lettce cultivars including pathotype differentials of Pink et al. [1992b]. Ls1 and Ls265 reacte largely as pathotype II, including the common strain of the virus, but Ls265 was least pathogenic on lettuce. Ls252 fitted pathotype IV and was very similar to LMV-E (the Spanish strain). The Greek isolates were very similar to each other in causing very severe symptoms on some non-lettuce hosts and a number of lettuce cultuvars. In lettuce variectal reaction Gr4 resembled pathotype I, but Gr5 severely affected Salinas 88, resistant to pathotypes I, II and III, and it appears to be a novel pathotype. Genetic interaction between lettuce and LMV is not following a simple yes-or-no pattern, and it is not a mere matter of resistance versus susceptibility. Adoption of a more realistic resistance terminology is proposed. None of the lettuce cultivars tested was resistant to the most pathogenic isolate Ls252, but resistance to it was found in 2 out of 12 wildLactuca species tested (Lactuca perennis andL. tatarica) while the symptomless plants ofL. perennis clearly reacted in ELISA.     

Evidence for Lettuce big‐vein associated virus as the causal agent of a syndrome of necrotic rings and spots in lettuce

Lettuce big‐vein associated virus (LBVaV, genus Varicosavirus) was shown to be responsible for characteristic necrotic symptoms observed in combination with big‐vein symptoms in lettuce breeding lines when tested for their susceptibility to lettuce big‐vein disease (BVD) using viruliferous Olpidium virulentus spores in a nutrient film technique (NFT) system. Lettuce plants showing BVD are generally infected by two viruses: Mirafiori lettuce big‐vein virus (MiLBVV, genus Ophiovirus) and LBVaV. New mechanical inoculation methods were developed to separate the two viruses from each other and to transfer both viruses to indicator plants and lettuce. After mechanical inoculation onto lettuce plants MiLBVV induced vein‐band chlorosis, which is the characteristic symptom of BVD. LBVaV caused a syndrome of necrotic spots and rings which was also observed earlier in lettuce plants inoculated in the NFT system, resembling symptoms described for lettuce ring necrosis disease (RND). This observation is in contrast with the idea that LBVaV only causes latent infections in lettuce. De novo next‐generation sequencing demonstrated that LBVaV was the only pathogen present in a mechanically inoculated lettuce plant with symptoms, providing evidence that LBVaV was the causal agent of the observed necrotic syndrome and thus fulfilling Koch’s postulates for this virus. The necrotic syndrome caused by LBVaV in lettuce is referred to as LBVaV‐associated necrosis (LAN).     

Identification and characterization of a potyvirus causing chlorotic spots on <Emphasis Type="Italic">Phalaenopsis</Emphasis> orchids

A putative virus-induced disease showing chlorotic spots on leaves of Phalaenopsis orchids was observed in central Taiwan. A virus culture, phalaenopsis isolate 7-2, was isolated from a diseased Phalaenopsis orchid and established in Chenopodium quinoa and Nicotiana benthamiana. The virus reacted with the monoclonal antibody (POTY) against the potyvirus group. Potyvirus-like long flexuous filament particles around 12–15 × 750–800 nm were observed in the crude sap and purified virus preparations, and pinwheel inclusion bodies were observed in the infected cells. The conserved region of the viral RNA was amplified using the degenerate primers for the potyviruses and sequence analysis of the virus isolate 7-2 showed 56.6–63.1% nucleotide and 44.8–65.1% amino acid identities with those of Bean yellow mosaic virus (BYMV), Beet mosaic virus (BtMV), Turnip mosaic virus (TuMV) and Bean common mosaic virus (BCMV). The coat protein (CP) gene of isolate 7-2 was amplified, sequenced and found to have 280 amino acids. A homology search in GenBank indicated that the virus is a potyvirus but no highly homologous sequence was found. The virus was designated as Phalaenopsis chlorotic spot virus (PhCSV) in early 2006. Subsequently, a potyvirus, named Basella rugose mosaic virus isolated from malabar spinach was reported in December 2006. It was found to share 96.8% amino acid identity with the CP of PhCSV. Back-inoculation with the isolated virus was conducted to confirm that PhCSV is the causal agent of chlorotic spot disease of Phalaenopsis orchids in Taiwan. This is the first report of a potyvirus causing a disease on Phalaenopsis orchids.     

Evaluation and QTL mapping of resistance to powdery mildew in lettuce

Lettuce (Lactuca sativa) is the major leafy vegetable that is susceptible to powdery mildew disease under greenhouse and field conditions. Quantitative trait loci (QTLs) for resistance to powdery mildew under greenhouse conditions were mapped in an interspecific population derived from a cross between susceptible L. sativa cultivar Salinas and the highly susceptible L. serriola accession UC96US23. Four significant QTLs were detected on linkage groups LG 1 (pm‐1.1), LG 2 (pm‐2.1 and pm‐2.2) and LG 7 (pm‐7.1), each explaining between 35 to 42% of the phenotypic variation. The four QTLs are not located in the documented hotspots of lettuce resistance genes. Alleles for the disease resistance at the four QTLs originated from both parents (two from each), demonstrating that even highly susceptible accessions may provide alleles for resistance to powdery mildew. These QTLs appeared to operate during limited periods of time. Results of the field trials with F_2:3 and F_3:4 families derived from a Soraya (moderately resistant) × Salinas cross demonstrated effective transfer of resistance to powdery mildew in this material. An integrated rating approach was used to estimate relative levels of resistance in 80 cultivars and accessions tested in a total of 23 field and greenhouse experiments. Generally, very low resistance was observed in crisphead‐type lettuces, while the highest relative resistance was recorded in leaf and butterhead types. Comparison of two disease assessment methods (percentage rating and the area under the disease progress steps, AUDPS) for detection of QTLs shows that the two approaches complement each other.     

Evaluation of triticale accessions for resistance to wheat bacterial leaf streak caused by Xanthomonas translucens pv. undulosa

The bacterium Xanthomonas translucens pv. undulosa (Xtu) causes bacterial leaf streak (BLS) on wheat and other small grains. Several triticale accessions were reported to possess high levels of resistance to wheat Xtu strains. In this study, a worldwide collection of triticale accessions as well as the major North Dakota hard red spring and durum wheat cultivars were evaluated for reaction to two local Xtu strains. All wheat cultivars showed a susceptible reaction but a wide range of reactions was observed among triticale accessions. Out of the 502 accessions tested, 45 and 10 accessions were resistant to the two virulent strains BLS‐LB10 and BLS‐P3, respectively, with five accessions, PI 428736, PI 428854, PI 428913, PI 542545 and PI 587229, being highly resistant to both strains. Statistical analysis showed significant difference among the accessions, strains, and the accession by strain interaction (P < 0.001). Bacterial population growth in resistant triticale was significantly slower than that in susceptible triticale. Molecular cytogenetic characterization in four representative triticale accessions confirmed the hexaploid level of the species and the presence of 12 or 14 rye chromosomes. The triticale accessions identified are valuable materials for developing wheat germplasm with high levels of BLS resistance.     

Differential interactions between strains of Rhizorhapis,Sphingobium, Sphingopyxis or Rhizorhabdus and accessions of Lactuca spp. with respect to severity of corky root disease

Over 100 isolates of Rhizorhapis suberifaciens, Sphingobium (Sb.) sp., Sb. mellinum, Sb. xanthum, Rhizorhabdus sp., and Sphingopyxis sp. (Sphingomonodaceae) were tested for pathogenicity on lettuce (Lactuca sativa) cultivars Salinas and Green Lakes, susceptible and resistant, respectively, or their resistant descendent breeding line (B.L.) 440‐8, to R. suberifaciens type strain CA1^T. Rhizorhabdus sp. CA15 and NL2, R. suberifaciens CA3, and Sphingopyxis CA32 were equally virulent to Green Lakes or B.L. 440‐8 and Salinas. Over 40 accessions from four Lactuca species were tested for resistance to R. suberifaciens CA1^T/CA3 or Rhizorhabdus sp. CA15/NL2. All lettuce accessions with resistance to CA1^T were susceptible to isolates CA15, NL2 and/or CA3. None of the Lactuca lines were highly resistant to all four isolates. There was a significant differential interaction between eight Lactuca lines and ten isolates of Rhizorhapis and related genera with respect to corky root severity. Three strains of isolates were distinguished: (i) isolates with a similar virulence pattern as R. suberifaciens CA1^T, (ii) isolates with a virulence pattern similar to that of R. suberifaciens CA3 and Sphingopyxis sp. CA32, and (iii) isolates of Rhizorhabdus being moderately aggressive to all Lactuca lines. Thus, strains belonging to several genera can cause similar symptoms (a rare phenomenon) but have different virulence patterns on Lactuca species and cultivars.     

Detection of four calla potyviruses by multiplex RT-PCR using nad5 mRNA as an internal control

Dasheen mosaic virus (DsMV), Turnip mosaic virus (TuMV), Konjac mosaic virus (KoMV) and Zantedeschia mild mosaic virus (ZaMMV) are important potyviruses previously identified in calla lily plants in Taiwan. In order to save time and cost of virus detection, a multiplex RT-PCR assay was developed for these calla potyviruses. Specific primers for each virus were designed based on the sequences of 3′ terminal region of respective viruses. To prevent false negative results, a primer pair specific to plant mitochondrial nad5 mRNA was used to produce a 185-bp fragment as an internal control of RT-PCR. The specificities of primers were confirmed by means of simplex and multiplex PCR assays. Optimal primer concentration ratio was identified by multiplex PCR assay. Total RNAs purified from virus-infected plants were used directly or mixed in different combinations, and then tested by multiplex RT-PCR. The result indicated that the expected RT-PCR products could be specifically amplified and identified on the basis of their molecular sizes. The detection sensitivity of multiplex RT-PCR was 25–625 times higher than that of indirect-ELISA (I-ELISA) depending on the virus. When applied to field surveys, multiplex RT-PCR could detect more single as well as mixed infection samples than I-ELISA. Accordingly, our multiplex RT-PCR assay provides a simple, rapid and reliable method for multiple potyvirus detection in calla lily.     

Production of discernible disease phenotypes in Canna by five plant viruses belonging to the genera Potyvirus,Cucumovirus and Badnavirus

Cannas are tropical and subtropical flowering perennial plants. The genus contains many species but most commercially grown cultivars are interspecific hybrids selected for their attractive foliage and flowers. Canna production is so lucrative that there are farmers and nurseries dedicated solely to its production. The specific issue that the canna industry faces is virus diseases. In this study, rhizomes of 24 canna cultivars were gathered and diagnostics conducted to detect Bean yellow mosaic virus (BYMV, Potyvirus), Canna yellow mottle virus (CaYMV, Badnavirus), Canna yellow streak virus (CaYSV, Potyvirus), Cucumber mosaic virus (CMV, Cucumovirus) and Tomato aspermy virus (TAV, Cucumovirus). Visual assessment of disease symptoms and diagnostic tests were carried out to identify the prevalent diseases and describe the symptoms that are associated with virus infection. BYMV, CaYMV and CaYSV caused severe mosaic and necrosis either in the leaf lamina or veins of infected leaves. Potyvirus infection suppressed red colouration in the foliage of some varieties. CaYMV and CaYSV often appeared in the same plant, suggesting they might represent a viral complex. CMV and TAV were rarely seen in these populations. Interestingly, CaYMV but not CaYSV could be mechanically inoculated to Phaseolus vulgaris plants.     

Phylogenetic investigations in the downy mildew genus <Emphasis Type="Italic">Bremia</Emphasis> reveal several distinct lineages and a species with a presumably exceptional wide host range

Bremia lactucae is one of the most devastating and widespread pathogens in lettuce production worldwide. Despite its economical importance, uncertainty prevails about the species delimitation in the genus Bremia. Commonly, Bremia is considered to be monotypic, containing only Bremia lactucae, while taxonomists have described additional species, and molecular phylogenetic studies have shown significant sequence divergence between accessions from different hosts. Here, we report that several previously described species are genetically highly distinct from Bremia lactucae parasitic to Lactuca sativa. These include Bremia lapsanae, Bremia sonchicola, and Bremia taraxaci. In addition to these host-specific species, a plurivorous species is revealed, which infects hosts from three different tribes in the Asteraceae subfamilies Asteroideae and Carduoideae. The broad host range of clade 1 is exceptional for downy mildews and only paralleled by Pseudoperonospora cubensis, which infects a broad range of Cucurbitaceae. The taxonomic status of Bremia cirsii and of Bremia centaureae remains unresolved, as the accessions from Cirsium and Centaurea, respectively, did not form a monophylum but were partly contained in the plurivorous clade 1. Bremia lactucae was found to be restricted to Lactuca sativa and Lactuca serriola. Thus, it can be assumed that Bremia infections on weeds apart from Lactuca species do not pose a significant risk for lettuce production. However, it is unlikely that breeding resistance genes from Lactuca serriola into Lactuca sativa will result in durable resistance of lettuce to downy mildew disease, because the current study provides additional evidence that Bremia accessions from both hosts form a population continuum.     

Differentiation of Bean pod mottle virus isolates based upon host symptoms

Accessions from Glycine, Phaseolus, and Vigna genera were screened for their reactions to different subgroups of isolates of Bean pod mottle virus (BPMV) in order to establish a differential host system. Screening results indicated that the BPMV isolates differed in pathogenic aggressiveness but not in virulence. No major resistance genes were found in soybean (Glycine max) or G. soja since all screened accessions showed mosaic or necrotic symptoms to BPMV inoculation. However, these accessions expressed differences in severity of symptoms when challenged by various BPMV isolates. The inoculation of G. tomentella accessions did not result in mosaic symptoms, and some accessions did not support systemic infection of some of the isolates. Resistance, presented as a hypersensitive reaction, was observed in some of Phaseolus and Vigna genotypes, and resistant response or susceptibility was stable to all the isolates used in the screening. In conclusion, the selected G. soja genotypes PI 407019, PI 464889A, and PI 464928, and ‘Amsoy 71’ soybean may help to separate severe (reassortant) from mild isolates of BPMV based upon their phenotypic reactions.     

Resistance to Sclerotinia sclerotiorum in wild Brassica species and the importance of Sclerotinia subarctica as a Brassica pathogen

Brassica crops are of global importance, with oilseed rape (Brassica napus) accounting for 13% of edible oil production. All Brassica species are susceptible to sclerotinia stem rot caused by Sclerotinia sclerotiorum, a generalist fungal pathogen causing disease in over 400 plant species. Generally, sources of plant resistance result in partial control of the pathogen although some studies have identified wild Brassica species that are highly resistant. The related pathogen S. subarctica has also been reported on Brassica but its aggressiveness in relation to S. sclerotiorum is unknown. In this study, detached leaf and petiole assays were used to identify new sources of resistance to S. sclerotiorum within a wild Brassica ‘C genome’ diversity set. High‐level resistance was observed in B. incana and B. cretica in petiole assays, whilst wild B. oleracea and B. incana lines were the most resistant in leaf assays. A B. bourgeai line showed both partial petiole and leaf resistance. Although there was no correlation between the two assays, resistance in the detached petiole assay was correlated with stem resistance in mature plants. When tested on commercial cultivars of B. napus, B. oleracea and B. rapa, selected isolates of S. subarctica exhibited aggressiveness comparable to S. sclerotiorum indicating it can be a significant pathogen of Brassica. This is the first study to identify B. cretica as a source of resistance to S. sclerotiorum and to report resistance in other wild Brassica species to a UK isolate, hence providing resources for breeding of resistant cultivars suitable for Europe.     

Response of UK winter wheat cultivars to <Emphasis Type="Italic">Soil-borne cereal mosaic</Emphasis> and <Emphasis Type="Italic">Wheat spindle streak mosaic viruses</Emphasis> across Europe.

Twenty-one UK winter wheat cultivars were grown over three seasons at sites with natural inoculum sources of Soil-borne cereal mosaic virus (SBCMV) and Wheat spindle streak mosaic virus (WSSMV) located in France, Italy and the UK. Plants were assessed visually for virus symptoms and leaf extracts were tested for the presence of each virus using enzyme-linked immunosorbent assays (ELISA). Cultivars showing little or no foliar symptoms and low levels of virus in leaf tissue were classified as resistant to each virus. All the trials were taken to harvest and agronomic data collected. At the most heavily infected sites, severe symptoms of SBCMV were observed in all UK cultivars except Aardvark, Charger, Claire, Cockpit, Hereward and Xi 19. The latter cultivars exhibited either light or no symptoms and little or no SBCMV infection in leaves. In fields with WSSMV, the virus failed to develop in Italy, but was detected in the leaves of all the susceptible control cultivars at a site in France. However, no UK cultivar tested positive for WSSMV. Multi-site analysis indicated that the presence of WSSMV did not increase the impact of SBCMV on the height, thousand-grain weight or yield of UK cultivars. The wheat cultivars on test gave a similar response to SBCMV across three European countries. Possible sources of SBCMV resistance are discussed.     

Serotypic variation in turnip mosaic virus

A panel of 30 monoclonal antibodies (MAbs) was produced against four isolates of turnip mosaic virus (TuMV). The panel was tested in plate-trapped antigen ELISA tests against 41 TuMV isolates (with different host and geographical origins and of differing pathotypes). The antibodies were also tested against four other potyviruses (bean common mosaic virus, bean common mosaic necrosis virus, lettuce mosaic virus and zucchini yellow mosaic virus). The reactions were assessed quantitatively (using multivariate analysis) and qualitatively (using the standard deviation obtained against healthy leaf material). The MAbs recognized 16–17 TuMV epitopes that were not present in the other potyviruses and a further two potyvirus epitopes. The isolates were grouped into three serotypes. Only one isolate did not fit this grouping. The classification of seven isolates in coat protein amino acid sequence homology groups correlated with serotypes. There was no correlation between serotype and pathotype, or between reactions to individual MAbs and single lines. There was therefore no evidence that the epitopes recognized by the MAbs are elicitors for the resistance genes present in the Brassica napus lines. However, the sensitivity and specificity of the MAbs will be useful for both routine detection of TuMV and fundamental studies on plant–virus interactions.     

Unterschiedliche Formen der Resistenz gegen bodenbürtige Viren des Weizens

In Germany the furovirus Soil-borne cereal mosaic virus (SBCMV) and the bymovirus Wheat spindle streak mosaic virus (WSSMV) occur often together particularly in several rye production areas. Soil-borne wheat mosaic virus (SBWMV), a wheat infecting furovirus, has so far been found only in one field near Heidelberg. Each of these viruses is transmitted by Polymyxa graminis. The cultivation of resistant varieties is the only promising measure to prevent yield losses caused by soil-borne viruses. Resistance of wheat against the bymovirus WSSMV is comparable to the immunity of barley to the bymoviruses Barley yellow mosaic virus and Barley mild mosaic virus. In case of immunity no virus multiplication is observed in resistant cultivars. In contrast, all wheat cultivars are hosts of the furoviruses. All cultivars – including the resistant ones – can be infected following mechanical inoculation with SBWMV and SBCMV. Resistance to furoviruses is based on reduced levels of virus multiplication in roots and on inhibition of virus movement from roots to leaves. Because of the inhibited virus movement from roots to aerial parts of plants this type of resistance is referred to as translocation resistance. In spite of the different resistance mechanisms the absence of virus symptoms on the leaves is a common selection criterion for both immunity and translocation resistance. Therefore, the symptom free development of plants on uniformly infested fields is the best criterion for selecting wheat lines with resistance to soil-borne viruses. The limited suitability of other selection methods is discussed.     

Characterizations of Carrot thin leaf virus based on host reactions and complete genomic sequences

Host range of a cilantro isolate of Carrot thin leaf virus (CTLV-Cs) was determined to include 15 plant species. The virus was also transmitted to nine of 11 tested apiaceous species by aphids. Complete genomic sequences of CTLV-Cs and a carrot isolate of CTLV (CTLV-Dc) were determined to be 9,491 and 9,490 nucleotides, respectively, excluding the 3′-poly(A) tail. Sequence analyses showed that the two isolates shared 98 % identities at both genomic and polyprotein sequence levels. Their genomic organization is typical of potyviruses, and their polyproteins contain conserved motifs found in members of the genus Potyvirus. Pairwise comparisons show that the virus shares 52.0–59.6 % identities to those of other members in the genus Potyvirus at genomic sequence level. Phylogenetic analyses confirm that CTLV is a distinct potyvirus most closely related to Konjac mosaic virus. Both biological and molecular results also showed that the two CTLV isolates were very similar although they were isolated from different hosts at different times.     

Survey of viruses infecting open‐field pepper crops in Côte d'Ivoire and diversity of Pepper veinal mottle virus and Cucumber mosaic virus

The prevalence of viruses in pepper crops grown in open fields in the different agro‐ecological zones (AEZs) of Côte d'Ivoire was surveyed. Pepper veinal mottle virus (PVMV; genus Potyvirus) and Cucumber mosaic virus (CMV; genus Cucumovirus) were the most frequent viruses among those surveyed, while tobamoviruses (genus Tobamovirus) were detected at low frequency. PVMV showed a high heterogeneity across AEZs, which may be related to climatic, ecological or agronomical conditions, whereas CMV was more homogeneously distributed. The molecular diversity of CMV and PVMV were analysed from partial genome sequences. Despite the low number of CMV isolates characterized, two molecular groups were revealed, one corresponding to subgroup IA and the other to reassortants between subgroups IA and IB. RNAs 1 and 3 of the reassortants clustered with the IB subgroup of CMV isolates, whereas their RNA 2 clustered with the IA subgroup. Importantly, RNA 1 of CMV isolates of the IB subgroup has been shown to be responsible for adaptation to pepper resistance. The diversity of PVMV in the VPg‐ and coat protein‐coding regions revealed multiple clades. The central part of the VPg showed a high level of amino acid diversity and evidence of positive selection, which may be a signature of adaptation to plant recessive resistance. As a consequence, for efficient deployment of resistant pepper cultivars, it would be desirable to examine the occurrence of virulent isolates in the CMV or PVMV populations in Côte d'Ivoire and to follow their evolution as the resistance becomes more widely deployed.     

Behaviour of Solanum spp. on inoculation with different isolates of Fusarium oxysporum f. sp. melongenae*

Twelve wild Solanum accessions were tested in a glasshouse at the seedling stage for resistance to Fusarium oxysporum f. sp. melongenae, the causal agent of fusarium wilt of aubergine. Four isolates of the fungus (three Turkish and one Italian) were used. Solanum incanum and S. linneanum were highly susceptible, whereas S. sisymbrifolium, S. torvum and S. aethiopicum Gilo group (one accession) were resistant. In Solanum aethiopicum Aculeatum (two accessions), S. aethiopicum Gilo, S. viarum and S. macrocarpon there were both resistant and susceptible individuals. The sources of resistance found in these wild Solanum spp. could be conveniently used to breed aubergine cultivars resistant to fusarium wilt.