Association of citrus psorosis B symptoms with a sequence variant of the Citrus psorosis virus RNA 2

Citrus psorosis virus (CPsV), the type species of genus Ophiovirus, is the presumed causal agent of a bark scaling disease in citrus plants. CPsV virions are kinked filaments composed of three negative‐strand RNA molecules and a ～48‐kDa coat protein. The virus induces two different syndromes: psorosis A (PsA), characterized by limited bark scaling lesions in the trunk and main limbs, and a more aggressive form of the disease called psorosis B (PsB) with rampant bark lesions affecting even thin branches and chlorotic blotches in old leaves. In the greenhouse, the PsA and PsB syndromes can be induced by graft inoculating healthy citrus seedlings with non‐lesion or with lesion bark inoculum from PsA‐affected field trees. PsA‐ and PsB‐inducing CPsV sub‐isolates obtained by this procedure from the same tree showed identical single‐strand conformation polymorphism (SSCP) profiles in homologous segments of the RNAs 1 and 3, whereas segments of the RNA 2 enabled discrimination between PsA‐ and PsB‐associated sequence variants. SSCP analysis of the RNA 2 population present in different tissues of psorosis‐infected plants showed that: (i) PsA‐inducing isolates contain PsB‐associated sequence variants at low frequency, (ii) the PsB‐associated sequence variant is predominant in blistered twigs and gummy pustules affecting old leaves, characteristic of PsB isolates, and (iii) the PsB‐associated sequence variant accumulates preferentially in bark lesions of the trunk and limbs. SSCP analysis of the RNA 2 population also enabled monitoring of interference between PsA‐ and PsB‐associated variants in plants co‐inoculated with both psorosis types.     

Molecular analysis suggests that recent <Emphasis Type="Italic">Citrus tristeza virus</Emphasis> outbreaks in Italy were originated by at least two independent introductions

Citrus tristeza virus (CTV) is the causal agent of the most important virus disease of citrus. Numerous CTV isolates differing in biological and molecular characteristics have been reported worldwide. Recently, CTV was detected in Italy in several citrus crops from three separate areas: (1) Cassibile, province of Syracuse; (2) Massafra, province of Taranto; and (3) Belpasso, province of Catania. CTV isolates from Massafra and Cassibile were mild, whereas isolates from Belpasso induced severe symptoms. To study the genetic variation of CTV populations of these areas, 150 samples per area were examined by single strand conformation polymorphism (SSCP) and nucleotide sequence analysis of CTV gene p20. All isolates from the same area showed the same SSCP pattern whereas for each area a different SSCP pattern was obtained. The Massafra and the Cassibile isolates had a nucleotide identity higher than 99% with a mild isolate from Spain and about 92% with the Belpasso isolates, which were similar (identity higher than 99%) to severe isolates from California and Japan. These results suggest at least two independent introductions of CTV in Italy, probably by import of CTV-infected budwoods. Within each area, the virus population was homogeneous suggesting diffusion of CTV by aphid transmission. The GenBank accession numbers of the sequences reported in this paper are: AY262000, AY263360 and AY263361 corresponding to gene p20 of CTV isolates from Massafra, Cassibile and Belpasso (Italy), respectively.     

Infection of potato by Rhizoctonia solani: effect of anastomosis group

Glasshouse and field experiments showed that the pathogenicity and disease type on potato varied between different anastomosis groups (AGs) of Rhizoctonia solani. For example, severe stem and stolon disease developed in plants inoculated with a single isolate of AG3PT and AG5. Severe root disease was observed with single isolates of AG8 and to a lesser extent AG3PT, but rarely with single isolates of the other AGs tested. In both field and glasshouse experiments the AG2‐1 isolate (X81) produced only small lesions (<5 mm). However, this was not representative of two other AG2‐1 isolates. When AG2‐1 isolates of the three different rDNA IGS1 types were tested in a glasshouse trial, one caused more severe stem and stolon infection than AG3PT. In the field experiment, the yield of tubers, by weight, was significantly less (P < 0·05) in all inoculated plants than for uninoculated (control) plants. Yield losses were greatest and tuber numbers smallest in plots inoculated with an AG8 isolate, suggesting that root infection is important in determining quantitative yield loss. The incidence of black scurf was greatest in the progeny tubers in plots inoculated with AG3PT (83·9%), whereas only very small amounts of black scurf developed on tubers from plants infected with AG2‐1 (510 bp) or AG5 isolates. This is supported by laboratory tests, where isolates of AG3PT produced significantly more sclerotia on potato dextrose agar than isolates of AGs 2‐1, 4, 5 and 8.     

Variation in the pathogenicity of two Turnip mosaic virus isolates in wild UK Brassica rapa provenances

Brassica rapa can be infected with Turnip mosaic virus (TuMV) as a result of manual inoculation or aphid transmission, but infected plants have not been found in the field. In this study, B. rapa plants grown from seed collected from two field sites in southern England were mechanically inoculated with one of two distinct isolates (pathotypes) of TuMV under glasshouse conditions. These had either been isolated from Brassica oleracea growing wild in Wales, (GBR 83, pathotype 3) or Dorset (GBR 98, pathotype 1). Use of ELISA as an index of infection in manually inoculated B. rapa showed that although seed provenance had a small effect on the proportion of plants infected, the biggest factor was the virus isolate. Both virus isolates infected both lines of B. rapa , but invaded at different rates, although both resulted in easily discernible symptoms. The severity of symptoms was not related to amounts of virus in the infected plants. A significantly greater proportion of plants were infected with GBR 83 at 45 days post-inoculation (d.p.i.) than GBR 98. but GBR 98 caused significantly more severe and obvious symptoms as well as greater mortality at 119 d.p.i., in plants from both sites than GBR 83.     

The effects of co‐infection by different Potato virus Y (PVY) isolates on virus concentration in solanaceous hosts and efficiency of transmission

The influence of co‐infection on concentration and accumulation of genetically different isolates of Potato virus Y (PVY) in potato and tobacco plants and the efficiency of transmission by Myzus persicae of PVY isolates from doubly versus singly infected plants were evaluated. The vector ability to simultaneously transmit two virus isolates was examined. Eight PVY isolates represented three strain groups: PVY^O (pathotype and serotype O), PVY^NW (pathotype N and serotype O), and PVY^NTN (pathotype and serotype N). Different diagnostic methods, including DAS‐ELISA, multiplex RT‐PCR, aphid transmission tests and bioassays, were applied to detect the presence of PVY isolates in source and assay plants. Significant reductions in concentrations of certain PVY isolates during co‐infection with other isolates were found both in potato and tobacco plants. The observed effects were both isolate‐ and host‐dependent in form. The highest rates of virus transmission by single aphids were recorded with PVY^NTN isolates, and the lowest ones with PVY^O isolates. Individual aphids of M. persicae were able to simultaneously transmit two PVY isolates. The frequency of transmission was generally low, but it reached as high as 20% for one of the isolate combinations. The findings presented in the work provide proof for antagonistic within‐plant interactions between isolates of PVY, with some implications of these interactions for virus transmission by aphid vectors. Consequently, this research contributes to a better understanding of the epidemiology of the disease caused by PVY.     

Occurrence of the Andean strain of potato virus S in imported potato material and its effects on potato cultivars

Infection with potato virus S Andean (PVS^A) and ordinary (PVS^o) strains was found in potato breeder's selection No. 8163-511 imported from West Germany; the two PVS strains were differentiated by their reactions on Chenopodium quinoa Tests on potato leaf samples using enzyme-linked immunosorbent assay followed by inoculation to C quinoa were subsequently used to detect PVS^A and PVS^o in a large-scale survey of imported and domestic potato material. PVS^A was detected in breeders' selections and cultivars imported from the Netherlands and West Germany, but not in domestic certified seed potato stocks or farmers' once-grown stocks. PVS^o was found in both imported and domestic certified stocks, but infection was commoner in the imported ones. When plants of C. quinoa, C. amaranticolor, C. murale and Nicotiana debneyi were inoculated with four isolates of PVS^A, one induced mild symptoms while the reactions of the others ranged from moderate to severe. When plants of different potato cultivars were inoculated with three isolates, the plants were mostly infected without symptoms. However, when tubers from some were grown on, the progeny plants of most of the different combinations of cultivar and isolate of PVS^A developed one or more of the following symptoms: vein deepening, rugosity, interveinal chlorosis, premature senescence and early loss of lower leaves.     

Pepino mosaic virus isolates and differential symptomatology in tomato

Based on a survey conducted in commercial tomato production in Belgium in 2006, four Pepino mosaic virus (PepMV) isolates that differed in symptom expression in the crop of origin were selected for greenhouse trials. The selected isolates were inoculated onto tomato plants grown in four separate plastic tunnels. PepMV symptom development was assessed regularly and extensive sampling followed by ELISA analyses, genotyping and sequencing was performed to study viral presence and variation in PepMV sequences throughout the trial period. Two isolates (EU-mild and CH2-mild) that were selected based on mild symptom expression in the crop of origin caused only mild symptoms in the trial, while two other isolates (CH2-aggressive and EU + CH2) that were selected for severe symptom display, caused considerably more severe symptoms. Sequence homology between CH2-mild and CH2-aggressive was as high as 99·4%. Results of this study show that differential symptom expression can, at least partially, be attributed to the PepMV isolate, which may be related to minor differences at the nucleotide level between isolates.     

Identification of a defective molecule derived from DNA-A of the bipartite begomovirus of East African cassava mosaic virus

Geminivirus defective interfering DNAs arise spontaneously in mechanically inoculated test plants, and have previously been found with DNA-B of the bipartite cassava mosaic geminiviruses, but not DNA-A. Reported here for the first time is the cloning and characterization of a naturally occurring truncated form of cassava mosaic geminivirus DNA-A, which at 1525 nt is around half the expected full size. Sequence analysis has shown it to be a defective (df) form of East African cassava mosaic virus (EACMV) DNA-A that has retained its cis elements essential for replication by the helper virus, and it has been termed df DNA-A 15. Phylogenetic comparisons placed the df DNA-A 15 molecule close to mild and severe isolates of EACMV-UG2. Biolistic inoculation of Nicotiana benthamiana with infectious df DNA-A 15 clone and East African cassava mosaic Cameroon virus (EACMCV) resulted in symptom amelioration as compared with EACMCV singly inoculated plants, and there was an accumulation of df DNA-A 15 in systemically infected leaves. In addition, the level of EACMV DNA-B accumulation was reduced in the coinoculated plants compared with those inoculated with EACMCV alone. PCR and sequence analysis confirmed the helper virus as EACMV.     

Competition between metalaxyl-sensitive and metalaxyl-resistant isolates of Phytophthora infestans in the absence of metalaxyl

Three metalaxyl-sensitive (MS, wild type) and three metalaxyl-resistant (MR) field isolates of Phytophthora infestans were compared, in the absence of metalaxyl, for non-competitive and competitive fitness on potato leaf and tuber tissues. When inoculated singly onto intact plants MR isolates produced larger lesions in leaflets than MS isolates, but no significant differences were recorded in infection frequency or sporulation capacity. When mixtures of MS and MR isolates were inoculated onto intact plants, all MR isolates exhibited a strong competitive ability: their proportion increased in the sporangial populations from 10 to 100% after eight to 10 sporulation cycles. In contrast, when mixtures were inoculated onto detached leaflets or tuber slices in a moisture-saturated atmosphere, only MR2 was a strong competitor; MR1 was a weak and MR3 a moderate competitor. The results showed that in intact plants all MR isolates were able to compete successfully with their MS partner isolates, because of the larger lesions they produced and the unlimited availability of host tissue. However, in detached tissues only MR2 was able to compete successfully with MS2, because of its higher infection and sporulation capabilities compared to MS2. The results explain the severe MR-induced late blight epidemics in potato crops in Israel.     

Separation and interference of strains from a citrus tristeza virus isolate evidenced by biological activity and double-stranded RNA (dsRNA) analysis

Separation of strains of citrus tristeza virus (CTV), differentiated by their double-stranded RNA (dsRNA) profiles, was obtained by graft-inoculating citron plants from a Mexican lime that had been recently aphid- or graft-inoculated with a mild CTV isolate (T-385). Up to 24 sub-isolates with differing dsRNA profiles were obtained from the aphid-inoculated lime. Some of these sub-isolates induced stronger symptoms in several citrus species than the original T-385 isolate. One sub-isolate, T-385-33, was mild in Mexican lime, but induced stem pitting on sweet orange. Inoculation of this isolate on Mexican lime, sour orange and Eureka lemon induced mild or no symptoms when inoculum was taken from citron, but very severe symptoms when the inoculum was from sweet orange. Mexican lime and sweet orange plants co-inoculated with T-385-33 from sweet orange in combination with the other 23 sub-isolates showed mild symptoms. The results obtained suggest that there is natural cross-protection among sub-isolates in the original T-385 isolate.     

Analysis of natural populations and possible natural reassortment of Cucumber mosaic virus

Plants naturally infected with Cucumber mosaic virus (CMV) were collected and analyzed by electrophoresis of the replicative form of dsRNA and by Northern blot hybridization using CMV RNA-specific probes. Some of the CMV-infected plants, especially winter crops, contained two kinds of RNA 1 segments or RNA 2 segments (or both), suggesting that mixed infections of CMV occurred naturally. Single-aphid-transmitted isolates (SATIs) from the field isolate containing two RNA 1 segments were grouped into three types by the electrophoretic mobility of RNA 1 (i.e., those containing one slow segment, those containing one fast segment, and those containing both). Furthermore, SATIs and single-lesion isolates, generated from the plants inoculated with a mixture of two CMV isolates that could be differentiated by their electrophoretic dsRNA profiles, were analyzed by dsRNA, indicating that nonparental progenies were observed. These results suggested that genetic reassortment of CMV RNA may occur in nature and that this is an important mechanism in CMV evolution.     

Variability among turnip mosaic potyvirus isolates

ABSTRACT Eight turnip mosaic potyvirus (TuMV) isolates from the Campania region of Italy were characterized. Experiments based on host range and symptomatology indicated that the isolates were biologically different. In addition, the isolates, with the exception of ITA1 and ITA3, were distinguished from each other by using a combination of monoclonal antibodies recognizing the coat protein. Single-strand conformation polymorphism (SSCP) analysis of the coat protein gene revealed that each isolate produced a specific SSCP profile, except for isolates ITA1 and ITA3. This study indicates that (i) even in a small geographical region, there is a great deal of variation in TuMV isolates; (ii) the use of a set of four differential hosts does not always specify the same pathotype in different environments; (iii) the TuMV isolates with the same pathotype on Brassica napus test lines can still differ in host range, symptoms, serology, and SSCP; and (iv) there was perfect correlation between the panel of antibodies and SSCP in differentiating among the isolates; ITA1 and ITA3 were indistinguishable by either assay.     

Assessing the virulence of four bark beetle-associated bluestain fungi using Norway spruce seedlings

The virulence of two isolates of each of four different bark beetle-associated bluestain fungi was evaluated by wound-inoculating 2- and 4-year-old Norway spruce seedlings. One isolate of Ceratocystis polonica killed 40% of the 2-year-old plants and 20% of the 4-year-old plants, whereas the other fungi ( Ophiostoma piceae , Ophiostoma sp., an unidentified fungus with dark sterile mycelium) and the control treatments (inoculation with sterile agar and unwounded plants) did not kill any plants during the 11-week incubation period. Only C. polonica and the Ophiostoma sp. caused any bluestain of the sapwood. The two C. polonica isolates caused significantly deeper bluestain penetration into the sapwood and longer necrotic lesions on the sapwood surface than all other isolates. The symptoms caused by the other fungi were similar to those on the sterile inoculated control plants. The virulence of the fungal isolates tested in this study agrees largely with results from a previous mass inoculation study using the same isolates inoculated into 40-year-old Norway spruce trees. Thus, inoculation of seedlings seems to be a reliable, inexpensive and convenient bioassay for determining the virulence of bark beetle-associated bluestain fungi.     

Systemic growth of Leptosphaeria maculans from cotyledons to hypocotyls in oilseed rape: influence of number of infection sites, competitive growth and host polygenic resistance

The influence of competitive effects between two isolates, of the number of infection sites on cotyledons and of host polygenic resistance on the systemic growth of Leptosphaeria maculans , the cause of phoma stem canker in oilseed rape ( Brassica napus ), were investigated. Controlled-condition experiments were conducted with two oilseed rape doubled haploid lines, one susceptible and the other with a high level of polygenic resistance, inoculated via wounded cotyledons with conidial suspensions obtained from two isolates. Expression of cankers in plants was enhanced by exposing inoculated plants to low temperature (6°C) followed by warm temperature (20°C). The fungus was detected by PCR amplifications of three minisatellite markers in all stems with visible canker symptoms and also in the stems of 14 of the 59 plants without visible cankers on the hypocotyls. Disease severity increased with the number of infection sites on cotyledons: in one of the three replicate experiments, the mean external necrosis length on the hypocotyl ranged from 6·47 to 35·3 mm for one and eight infections sites on cotyledons, respectively. The probability of an isolate reaching the hypocotyl from inoculated cotyledons decreased with increasing competing inoculum load on cotyledons: for instance, for isolate A290v it decreased from 1 when inoculated alone to 0·28 when coinoculated with six drops of competing isolate P27d. Polygenic resistance significantly reduced disease incidence and severity. For instance, in one of the three replicate experiments, disease incidence ranged from more than 74% in susceptible plants to 16% in resistant ones, while mean external necrosis length was up to 35·3 and 6·5 mm on susceptible and on resistant plants, respectively. This study offers new possibilities for assessing levels of polygenic resistance to stem canker in B. napus and studying the aggressiveness of L. maculans isolates.     

Biological diversity of Rhizoctonia solani (AG-3) in a northern potato-cultivation environment in Finland

A total of 119 isolates of Rhizoctonia were collected from stem canker lesions, stolon and root lesions, hymenia on stems, or from black scurf on tubers of potato plants ( Solanum tuberosum ) in Finland (latitudes 60–67°N). All isolates except three belonged to anastomosis group 3 (AG-3) of R. solani , as determined by phylogenetic analysis of the internal transcribed spacer sequences (ITS1 and ITS2) of ribosomal RNA (rRNA) genes. Sensitivity of the 119 isolates to the fungicide flutolanil was tested in vitro (EC₅₀ values 0·14–0·75  µ g active ingredient mL⁻¹). The isolates also varied considerably in growth rate (5·1–14·8 mm day⁻¹). The severity of disease caused by 99 isolates was determined based on the proportion of potato sprouts affected by lesions, discoloration or death, which was c . 1–60%. Only two isolates that were able to cause severe symptoms showed particularly low sensitivity to the fungicide and rapid growth rate. One isolate each of anastomosis groups AG-2-1 and AG-5 and an unknown, binucleate Rhizoctonia sp. were detected. The AG-5 isolate and the binucleate isolate caused mild symptoms on potato sprouts, whereas the AG-2-1 isolate was not pathogenic. Taken together, AG-3 of R. solani was the predominant causal agent of the stem canker and black scurf diseases of potato in Finland and showed considerable variability in disease severity, fungicide sensitivity and growth rate in vitro .     

Host Effect on the Molecular and Biological Properties of a Citrus exocortis viroid Isolate from Vicia faba

ABSTRACT Citrus exocortis viroid (CEVd) is the casual agent of citrus exocortis disease, and has been found in naturally infected citrus and noncitrus hosts. Field isolates of CEVd may infect susceptible hosts as a complex of genetically related sequence variants (haplotypes). In the present work, a CEVd isolate recovered from a symptomless broad bean plant was characterized as a heterogeneous population with a nucleotide diversity of 0.026, which did not contain a predominant haplotype. When nucleic acid extracts of this infected broad bean were used to inoculate tomato, the plants displayed symptoms and the CEVd population was more homogeneous, with a nucleotide diversity of 0.007. However, when nucleic acid extracts from this tomato isolate were back inoculated to new broad bean plants, this isolate did not revert to the original population, because it showed low nucleotide diversity (0.001) and induced symptoms in the broad bean plants. Symptomless broad bean plants may act as reservoirs of highly heterogeneous populations of CEVd variants, providing an excellent inoculum source in terms of its potential to infect a broad range of putative hosts. The epidemiological implications are discussed.     

Field occurrence of vinclozolin resistance in Monilinia fructicola

Strains of Monilinia fructicola resistant to vinclozolin were isolated from fruit affected by brown rot from an orchard where the fungicide had been used over four seasons. Resistant isolates were pathogenic to peach fruit, and resistance of one isolate was confirmed following dipping of inoculated fruit in fungicide suspensions. In culture, one of four resistant isolates was identical in colony morphology and sporulation to sensitive isolates from different geographic areas. The other resistant isolates produced dark mycelium on PDA and were slower growing. Vinclozolin-resistant isolates were resistant in vitro to two other dicarboximide fungicides, iprodione and procymidone.