Hypovirulence and Double-Stranded RNA in Sclerotinia homoeocarpa

ABSTRACT One hundred and thirty-two isolates of Sclerotinia homoeocarpa, the causal agent of dollar spot of turfgrass, were evaluated for virulence on swards and detached leaves of creeping bentgrass and for the presence of double-stranded RNA (dsRNA). In at least four isolates, the hypovirulent phenotype was associated with the presence of specific segments of dsRNA. In addition, these hypovirulent isolates often grew slowly on potato dextrose agar (PDA), formed thin colonies with atypical colony margins, and failed to produce typical black stroma. The hypovirulent phenotype and dsRNA were transmitted from hypovirulent isolate Sh12B to virulent isolate Sh48B, and the converted isolate was hypovirulent and contained dsRNA. The hypovirulent phenotype and dsRNA also were transmitted to at least four other isolates of the pathogen, including the fungicide-resistant, dsRNA isolate KY-7. Converted isolates of KY-7 developed the hypovirulent phenotype, grew on fungicide-amended medium, and contained dsRNA. Subcultures of hypovirulent isolate Sh12B that did not contain dsRNA were obtained through curative treatment using cycloheximide-containing medium and heat. Cured subcultures grew faster on PDA, had more typical colony morphologies, were more virulent on bentgrass leaves, and did not contain dsRNA. No cured subcultures were obtained from hypovirulent isolate Sh09B. Isolates regenerated from protoplasts of hypovirulent isolate Sh12B were not cured, remained hypovirulent, and contained dsRNA. Transmission of hypovirulence and dsRNA in S. homoeocarpa has potential as a novel approach to the management of dollar spot of turfgrass.     

Hypovirulence detected in Brazilian isolates of Cryphonectria cubensis

A single 3 kb segment of double-stranded (dsRNA) was present in three of 30 Brazilian isolates of Cryphonectria cubensis . These dsRNA-containing isolates showed morphological characteristics suggestive of hypovirulence and were significantly less virulent than dsRNA-free isolates. One isolate, however, with morphological characteristics suggestive of hypovirulence, showed reduced virulence, but was free from dsRNA. Conversion of virulent isolates with normal morphology to a morphology associated with hypovirulence was achieved by pairing hypovirulent and virulent isolates of the same vegetative compatibility group (VCG). This suggests that dsRNA can be transmitted to isolates of the same vegetative compatibility group by hyphal anastomosis. Converted isolates exhibited the same hypovirulence-associated traits as those of the original dsRNA-containing hypovirulent isolates. These studies suggest that a single 3 kb segment of dsRNA alters both morphology and virulence by conferring hypovirulence on the pathogen; the first such report for Brazilian isolates of C. cubensis .     

Elevated mitochondrial alternative oxidase activity in dsRNA-free, hypovirulent isolates of Cryphonectria parasitica

In Cryphonectria parasitica, the causal agent of chestnut blight, hypovirulence is commonly associated with the presence of cytoplasmically transmissible, double-stranded RNA (dsRNA). Recently, hypovirulent isolates of C. parasitica that do not have detectable levels of dsRNA have been obtained from healing cankers on chestnut trees. In these isolates, most of the respiratory activity is cyanide-resistant and salicylhydroxamate-sensitive, indicating that the mitochondrial alternative oxidase was induced. In contrast, the respiration of virulent isolates from the same trees and most of the dsRNA-containing hypovirulent isolates from a variety of geographical locations was cyanide sensitive. In all of these isolates, the alternative oxidase could be induced by growing them in the presence of chloramphenicol, an inhibitor of mitochondrial protein synthesis. The attenuated virulence and altered respiratory phenotypes could be transferred from the dsRNA-free hypovirulent isolates to virulent strains through hyphal anastomosis. Some of the dsRNA-free hypovirulent isolates grew more slowly than virulent strains. These results indicate that, in Cryphonectria, cytoplasmically transmitted hypovirulence may have more than one cause, and that mitochondrial mutations sometimes may be involved in the generation of this phenotype.     

Hypovirulence and Double-Stranded RNA in Botrytis cinerea

ABSTRACT Twenty-one strains of Botrytis cinerea isolated from 13 species of plants grown in China were compared for pathogenicity on Brassica napus, mycelial growth on potato dextrose agar, and presence of double-stranded (ds)RNA. The results showed that the strain CanBc-1 was severely debilitated in pathogenicity and mycelial growth, compared with the 20 virulent strains. A dsRNA of approximately 3.0 kb in length was detected in CanBc-1 and 4 hypovirulent single-conidium (SC) isolates of CanBc-1, but was not detected in the 20 virulent strains of B. cinerea and 4 virulent SC isolates of CanBc-1. Results of the horizontal transmission experiment showed that the hypovirulent trait of CanBc-1 was transmissible and the 3.0-kb dsRNA was involved in the transmission of hypovirulence. Analysis of a 920-bp cDNA sequence generated from the 3.0-kb dsRNA of CanBc-1 indicated that the dsRNA element was a mycovirus, designated as B. cinerea debilitation-related virus (BcDRV). Further analyses showed that BcDRV is closely related to Ophiostoma mitovirus 3b infecting O. novo-ulmi, the causal agent of Dutch elm disease. Mitochondria and cytoplasm in hyphal cells of CanBc-1 became degenerated, compared with the virulent isolate CanBc-1c-66 of B cinerea. This is the first report on the occurrence of Mitovirus-associated hypovirulence in B. cinerea.     

A Satellite RNA of Ophiostoma novo-ulmi Mitovirus 3a in Hypovirulent Isolates of Sclerotinia homoeocarpa

ABSTRACT Two genetically distinct double-stranded RNA (dsRNA) elements were identified in hypovirulent isolates of Sclerotinia homoeocarpa, the causal agent of dollar spot of turfgrass. The large dsRNA (L-dsRNA) was consistently present in all hypovirulent isolates, whereas the small dsRNA (S-dsRNA) was found only in some hypovirulent isolates. Virulence comparisons revealed that there was no significant difference between isolates containing one or both dsRNAs. Therefore, the L-dsRNA appears to be the genetic determinant of hypovirulence, while the S-dsRNA is not essential for hypovirulence in S. homoeocarpa. The L-dsRNA in hypovirulent isolate Sh12B of S. homoeocarpa was previously characterized as a fungal mitochondrial virus and designated Ophiostoma novo-ulmi mitovirus 3a-Sh12B (OnuMV3a-Sh12B) because it was conspecific with O. novo-ulmi mitovirus 3a-Ld from O. novo-ulmi, the causal agent of Dutch elm disease. In the present study, the nucleotide sequences of the S-dsRNAs (738 to 767 nucleotides) in hypovirulent isolates Sh12B, Sh279B, and Sh286B were determined. Nucleotide sequence analysis indicated that the S-dsRNA was not derived from the OnuMV3a dsRNA and it could not encode an RNA-dependent RNA polymerase. These results are consistent with biological data that the S-dsRNA was always associated with the L-dsRNA and was never found independently. Therefore, the S-dsRNA can be regarded as a satellite RNA of OnuMV3a in S. homoeocarpa. Northern blotting analysis indicated that nucleic acid extracts from isolate Sh12B of S. homoeocarpa contained more single (+) stranded RNA than dsRNA for this satellite RNA. The 5'- and 3'-terminal sequences of the positive strand of the S-dsRNA each could be folded into a stem-loop structure and the terminal 21 nucleotides were complementary to each other, potentially forming a panhandle structure.     

Aphid Transmission Alters the Genomic and Defective RNA Populations of Citrus tristeza virus Isolates

ABSTRACT A total of 14 Spanish isolates of Citrus tristeza virus (CTV) and 1 isolate from Japan were transmitted by Aphis gossypii, and the subisolates obtained were compared with the source isolates for symptom expression and double-stranded RNA (dsRNA) pattern. Of the 14 Spanish isolates, 9 showed altered dsRNA patterns after aphid transmission but only minor variations in the intensity of symptoms induced on Mexican lime. Northern blot hybridization with complementary DNA (cDNA) probes corresponding to both the 5' and the 3' termini of the CTV genomic RNA (gRNA) showed that the dsRNA bands that could be used to discriminate between the dsRNA pattern of the source and the aphid-transmitted isolates were the replicative forms of defective RNAs (D-RNAs). Conversely, the Japanese isolate and two subisolates obtained from it by aphid transmission had the same dsRNA pattern, but one of the subisolates induced milder symptoms in several hosts. Dot-blot hybridization with cDNA probes representing several regions of the gRNA showed that most of the aphid-transmitted isolates differed from the corresponding source isolate by their hybridization pattern. Our results indicate that aphid transmission often sorts the populations of gRNA variants and D-RNAs present in CTV isolates.     

Isolation and identification of hypovirulent Rhizoctonia spp. from soil

Two-hundred and forty-eight isolates of Rhizoctonia spp, were obtained from 13 locations in Gifu Prefecture in Japan using the plant debris particles isolation, colonization of bait tissue, and soil-clump plating methods. Of the isolates, 143 were binucleate Rhizoctonia spp., 60 were R. solani and 45 were R. zeae. Three isolates of R. solani and 54 of binucleate Rhizoctonia spp, were hypovirulent on radish, whilst all isolates of R. zeae were highly virulent, Hypovirulent strains were isolated most frequently by the plant debris particles isolation method, Hypovirulent isolates of R. solani belonged to anastomosis group 4, whilst the hypovirulent binucleate Rhizoctonia isolates belonged to AG A, AG Ba, AG G, and AG O.
Thirty-two isolates of Rhizoctotria spp, selected for hypovirulence on radish were tested on cucumber in vitro. Only five binucleate Rhizoctonia isolates and one R. solani isolate were hypovirulent on both species, and these isolates were also hypovirulent on seven other crop species. Cucumber showed wide variation in disease susceptibility to different isolates but hypovirulent isolates exhibited a consistent reaction on five different host cultivars, Pathogenicity tests using cucumber grown in soil also showed consistent reactions with isolates selected either for hypovirulence or virulence. The results support the use of cucumber in bioassays for identifying hypovirulent isolates of binucleate Rhizoctonia spp.     

核盘菌中dsRNA种类及其与致病力的关系

 对源于我国黑龙江省佳木斯市同一块茄子田的14个核盘菌(Sclerotinia sclerotiorum)菌株及其中1个菌株Ep-1PNA5的2个衍生菌株的致病力和菌丝中的双链RNA(dsRNA)进行了分析。在马铃薯琼脂培养基(PDA)上对5个弱致病型核盘菌菌株(Ep-1PD、Ep-1PI、Ep-1PL、Ep-1PM、Ep-1PN)异常性状(菌丝生长慢,且异常分枝)的传染特性进行了测定。结果表明:在离体油菜叶片上,16个供试核盘菌菌株中,7个属于强致病类型,7个属于弱致病类型,2个属于中等致病类型。从10个核盘菌菌株的菌丝中检测到dsRNA因子,并可分成3类dsRNA电泳谱型。第一类dsRNA谱型只含有7.4kb大小的dsRNA分子,3个强致病型核盘菌菌株属于这种谱型;第二类dsRNA谱型含有2种dsRNA分子,大小分别为6.4kb和7.4kb。6个弱致病型菌株属于这种谱型;第三类dsRNA谱型只含有6.4kb大小的dsRNA分子。1个弱致病类型菌株属于这种谱型。从另外6个核盘菌菌株的菌丝中没有检测到任何dsRNA因子,其中4个菌株属于强致病型菌株,2个菌株属于中等致病型。可见,6.4kb大小的dsRNA因子与核盘菌弱致病性状密切相关。5个弱致病型核盘菌菌株(Ep-1PD、Ep-1PI、Ep-1PL、Ep-1PM、Ep-1PN)的异常性状可以通过菌丝接触传染给一些强致病型核盘菌菌株,使其菌丝生长变慢及分枝异常。结果还表明:这些弱致病型核盘菌异常性状的传染具有菌株特异性。     

Potential for Biocontrol of Monosporascus Root Rot/vine Decline Under Greenhouse Conditions using Hypovirulent Isolates of Monosporascus cannonballus

Monosporascus root rot/vine decline (MRR/VD) causes root necrosis and severe stunting of muskmelon and watermelon plants in several countries around the world. MRR/VD is caused by the soilborne ascomycete fungus, Monosporascus cannonballus. Currently, there are few options available for control of MRR/VD. This research describes experiments to test the possibility of using naturally occurring M. cannonballus isolates containing double-stranded RNA (dsRNA) for the biological control of MRR/VD. These isolates often develop a degenerate phenotype characterized by slow growth and reduced ascospore production. In addition, these degenerate isolates are hypovirulent on muskmelon. Plants co-inoculated with a hypovirulent, dsRNA⁺ isolate (Tx93-449⁺) and a virulent, dsRNA^- isolate (Az90-33^-) at an inoculum ratio of 10:1 (hypovirulent:virulent) were indistinguishable from the uninoculated plants in greenhouse pathogenicity trials. In vitro infection assays using fluorescence microscopy on aniline-stained muskmelon roots suggested that although the hypovirulent dsRNA⁺ isolate Tx93-449⁺ penetrated and partially colonized roots of the seedlings, it was not as efficient in colonizing the roots as the virulent, dsRNA^- isolate Az90-33^-. While more extensive experiments are needed, these data suggest that hypovirulent dsRNA⁺ isolates of M. cannonballus have potential for development as biological control agents to reduce disease pressure associated with MRR/VD.     

Transmission of double-stranded RNA and a disease factor in Ophiostoma ulmi

A diseased isolate of Ophiostoma ulmi was found to contain 10 segments of double-stranded RNA (dsRNA) with molecular weights ranging from 2.40 x 10⁶ to 0.23 x 10⁶. In contrast seven other healthy isolates in the same vegetative compatibility group contained either no dsRNA or up to four dsRNA segments. Transmission of the disease to healthy isolates by hyphal anastomosis was accompanied by-transmission of the 10 dsRNA segments. In a genetic cross in which the diseased isolate acted as the female parent single-ascospore progeny were healthy and either contained no dsRNA or only one segment of dsRNA. When elm trees were inoculated with diseased isolates, subsequent reisolations were healthy and retained only two to seven of the dsRNA segments or were diseased and retained all 10 dsRNA segments. Following conidiogenesis a diseased isolate gave rise to single-conidial progeny which were either slow growing and diseased, like their parent, with all 10 dsRNA segments, or faster growing like healthy isolates. Some of the faster growing conidial isolates retained only two to seven of the dsRNA segments and were disease-free. However a majority of the faster growing conidial isolates retained all 10 dsRN A segments and were shown to carry the disease in a latent form. The possibility that the disease of O. ulmi is conferred by specific segments of dsRNA and the potential of d-factors for the control of Dutch elm disease are discussed.     

Suppression of Dollar Spot by Hypovirulent Isolates of Sclerotinia homoeocarpa

ABSTRACT Selected hypovirulent isolates of Sclerotinia homoeocarpa were evaluated for efficacy in suppressing dollar spot of turfgrass under growth room and field conditions. Under growth room conditions, hypovirulent isolates Sh12B, Sh09B, or Sh08D of S. homoeocarpa caused 3.4 to 30.4% diseased turf in comparison to virulent isolates Sh48B and Sh14D, which caused 80.2 to 90.2% disease. In treatments that received both virulent and hypovirulent isolates, only hypovirulent isolate Sh12B significantly reduced disease as compared with the control with virulent isolates alone. In a field experiment in 1993 on swards of creeping bentgrass artificially inoculated with a virulent isolate of the pathogen, all treatments containing hypovirulent isolate Sh12B applied as a mycelial suspension, granular mix, or alginate pellets developed significantly less disease (6.3 to 20.8% diseased turf) compared with their respective formulation controls (23.8 to 31.2%). Suppression of dollar spot by treatment with mycelial suspensions of isolate Sh12B was evident up to 45 days postinoculation, and disease suppression was still significant 1 year after application when compared with the water control. Applications of hypovirulent isolate Sh09B did not reduce dollar spot in any treatments. Significant suppression of dollar spot by isolate Sh12B was also observed in the experiment conducted in 1994. In addition, suppression of dollar spot by hypovirulent isolate Sh12B was evaluated on swards with naturally occurring inoculum during 1994. Treatments with a mycelial suspension and alginate pellets of hypovirulent isolate Sh12B significantly reduced dollar spot compared with their respective formulation controls. With few exceptions, there was no statistical difference between treatments with hypovirulent isolate Sh12B and the fungicide chlorothalonil (Daconil 2787). Multiple applications of the hypovirulent isolate did not result in greater suppression of dollar spot as compared with a single application. The results indicate that hypovirulence has potential as an effective strategy for the management of dollar spot.     

Protection of Citrus Rootstocks Against Phytophthora spp. with a Hypovirulent Isolate of Phytophthora nicotianae

ABSTRACT Phytophthora root rot of citrus in Florida is caused by Phytophthora nicotianae and P. palmivora. A naturally occurring isolate of P. nicotianae (Pn117) was characterized as hypovirulent on citrus roots. Pn117 infected and colonized fibrous roots, but caused significantly less disease than the virulent isolates P. nicotianae Pn198 and P. palmivora Pp99. Coincident inoculation of rootstock seedlings of Cleopatra mandarin (Citrus reticulata) or Swingle citrumelo (C. paradisi x Poncirus trifoliata) with the hypovirulent Pn117 and the virulent isolates Pn198 and Pp99 did not reduce the severity of disease caused by the virulent Phytophthora spp. When either rootstock was inoculated with the hypovirulent Pn117 for 3 days prior to inoculation with virulent isolates, preinoculated seedlings had significantly less disease and greater root weight compared with seedlings inoculated with the virulent isolates alone. Recovery of the different colony types of Phytophthora spp. from roots of sweet orange (C. sinensis) or Swingle citrumelo was evaluated on semiselective medium after sequential inoculations with the hypovirulent Pn117 and virulent Pp99. Pn117 was isolated from roots at the same level as the Pp99 at 3 days post inoculation. Preinoculation of Pn117 for 3 days followed by inoculation with Pp99 resulted in greater recovery of the hypovirulent isolate and lower recovery of the virulent compared with coincident inoculation.     

Attenuation of Virulence in Sclerotinia homoeocarpa during Storage is Associated with Latent Infection by Ophiostoma mitovirus 3a

The hypovirulence-associated mitovirus, Ophiostoma mitovirus 3a (OMV3a), has been shown to be widespread in eastern Canadian populations of Sclerotinia homoeocarpa in the form of latent infection. Latent infection by OMV3a was not associated with an apparent phenotype and did not significantly reduce the growth and virulence of the pathogen. In the present study, we found that isolates of S. homoeocarpa latently infected by OMV3a can change to hypovirulent isolates after storage at 4 °C, and that this attenuation of virulence was associated with increased concentration of the OMV3a virus. Recurrent observations revealed that up to 29.8% of latently infected isolates changed to hypovirulent isolates after 21 months of storage. Transmission of OMV3a dsRNA from latently infected isolates to virus-free isolates resulted in latent infection of the recipient isolates, indicating that latent infection by OMV3a was not associated with genetic differences in the fungal host. The RNA genomes of the OMV3a virus in an isogenic pair of latently infected and hypovirulent isolates were sequenced and compared. Each of the two RNAs contained an open reading frame of 726 amino acids with conserved motifs typical of RNA-dependent RNA polymerase (RdRp). The OMV3a RNA sequences in these two isolates share 95.1% nucleotide and 94.6% amino acid sequence identities. The development of hypovirulence from latent infection by OMV3a virus may provide new strategies to improve the biological control efficacy of hypovirulence in dollar spot management.     

Transmission of tomato spotted wilt tospovirus by Thripstabaci populations originating from leek

The transmission of tomato spotted wilt tospovirus (TSWV) by Thrips tabaci collected from leek was studied using the petunia local-lesion leaf-disc assay. After an acquisition-access period of 72 h given to newborn larvae up to 8 h old, the efficiency of transmission by adults was determined in three inoculation-access periods of 48 h. This efficiency varied for six T. tabaci populations from 0.7 to 11.6% in experiments using the Greek TSWV isolate GR-04. Males were more efficient transmitters than females (19 out of 176 versus five out of 494). Frankliniella occidentalis transmitted the same virus with a higher efficiency (34.8%). The transmission rate differed also among TSWV isolates, as shown in tests with four T. tabaci using two isolates. The virus was more efficiently acquired from infected leaf material of Datura stramonium than from that of Emilia sonchifolia . Plants of the latter species were more susceptible than Nicotiana tabacum in thrips transmission tests.     

Characterization of hypovirulent isolates of the chestnut blight fungus, Cryphonectria parasitica from the Marmara and Black Sea regions of Turkey

Chestnut blight caused by the introduced fungus Cryphonectria parasitica has been responsible for the decline of Castanea sativa in Turkey since the 1960s. In this study, 72 C. parasitica isolates were recovered from the Marmara and Black Sea regions of Turkey showing white or cream-coloured culture morphology and were subjected to various tests to determine if they were infected by Cryphonectria hypovirus 1 (CHV-1). The vast majority of the isolates (69 out of 72) were vc type EU-1. Both mating types were found among a subsample of the isolates. The hypovirus was detected in 55 isolates by dsRNA extraction and/or virus specific RT-PCR on total RNA extracts. All but one isolates showed no or only weak phenol oxidase activity on agar medium containing tannic acid, typical of CHV-1 infected isolates. Through sequencing of a specific region of the hypovirus genome, we found that 24 hypovirus isolates belonged to the CHV-1 subtype I and six to the CHV-1 subtype F2. The distribution of the two CHV-1 subtypes in Turkey showed a clear geographic pattern. CHV-1 subtype I was only detected in the Marmara and western Black Sea region, whereas subtype F2 was restricted to the eastern part of the Black Sea region. The effectiveness of 23 hypovirulent isolates was tested against a virulent isolate on 2–3 years old chestnut sprouts. Ten hypovirulent isolates, all infected by CHV-1 subtype I, prevented canker development by more than 80 % suggesting that they might be suitable for biological control of chestnut blight in Turkey.     

Double-stranded RNA: distribution and analysis among isolates of Rhizoctonia solani AG-2 to -13

The occurrence, distribution, and genetic relatedness of double-stranded RNA (dsRNA) components from 36 isolates of Rhizoctonia solani belonging to nine anastomosis groups (AGs) were studied using electrophoretic analysis and RNA–RNA blot hybridization. DsRNA was consistently detected in all 36 isolates. The size of the dsRNA components varied considerably, ranging from 0·74 to 23 kb. Two thirds of isolates possessed different size dsRNA components. Only two of the isolates had small size dsRNA between 0·5 and 1·0 kb. The biotin-labelled dsRNA probes provided the sensitivity and specificity required to study genetic relationships of dsRNA. As little as 10 pg of 'hybridizing' dsRNA could be detected using biotin-labelled total dsRNA with no detectable nonspecific-hybridization. Results from several dot-spot as well as RNA–RNA gel hybridization experiments revealed considerable sequence heterogeneity among dsRNA components within each isolate or isolates from the same AG.     

Hypovirulence-Associated Double-Stranded RNA from Sclerotinia homoeocarpa Is Conspecific with Ophiostoma novo-ulmi Mitovirus 3a-Ld

ABSTRACT The nucleotide sequence of the hypovirulence-associated double-stranded RNA (dsRNA) in hypovirulent isolate Sh12B of Sclerotinia homoeocarpa, the causal agent of dollar spot of turf grass, was determined. This large dsRNA (L-dsRNA) is 2,632 bp long and is A and U rich (61.0% A+U residues). One strand of this dsRNA contains an open reading frame (ORF) with the potential to encode a protein of 720 amino acids. This ORF contains 12 UGA codons, predicted to encode tryptophan in ascomycete mitochondria, and has a codon bias typical of mitochondrial genes, which is consistent with a mitochondrial localization of this dsRNA. The amino acid sequence contains conserved motifs typical of RNA-dependent RNA polymerases (RdRps). Sequence analyses of the nucleotide and RdRp-like protein revealed that the L-dsRNA is homologous with previously characterized mitochondrial viruses and dsRNAs from other phytopathogenic fungi, and shares 92.4% nucleotide and 95.1% amino acid sequence identities with the Ophiostoma novo-ulmi mitovirus 3a-Ld from Ophiostoma novo-ulmi, the causal agent of Dutch elm disease. The results indicate that these two dsRNAs are conspecific. This is the first report that a hypovirulence-associated dsRNA virus naturally occurs in two taxonomically distinct fungi, and indicates that horizontal transmission of this dsRNA virus may have occurred between these fungi.     

Variation for isozymes and double-stranded RNA among isolates of Puccinia striiformis and two other cereal rusts

Gel electrophoresis was used to examine the variation in isozymes and dsRNA within and between the rust species Puccinia striiformis, P. recondita and P. hordei. No differences in isozyme phenotype were found among 29 diverse isolates of the wheat-attacking form of P. striiformis (WYR). Smaller numbers of isolates of the barley-attacking form (BYR) of P. recondita and of P. hordei showed similar intra-group uniformity. There were major differences in isozyme phenotypes between the three species, while WYR and BYR differed for two of 12 enzymes. Double-stranded RNA was detected in each species and in all 26 isolates examined. For WYR and BYR, all isolates within each group had the same or a similar phenotype. In contrast, each isolate of P. recondita and P. hordei had a unique phenotype. There were differences in dsRNA phenotypes both between the three species and between WYR and BYR.
The uniformity of these rust populations and species for isozyme phenotype is contrasted with their variability in pathogenicity and with the variability in isozymes encountered in higher organisms. Uniformity may result from a feature of the biology of the rust species and populations examined, or from the relative homogeneity of the environment of biotrophic pathogens. Consistent differences in isozyme and dsRNA phenotype between the WYR and BYR isolates of P. striiformis indicate that these are two discrete populations, supporting the view that they should be recognized as f.sp. tritici and f.sp. hordei , respectively.     

Variation in transmission of two BYDV-MAV isolates by multiple clones of Rhopalosiphum padi L.

Vector efficiency of up to 17 Rhopalosiphum padi L. clones originating from Europe, North America and North Africa, was evaluated by transmitting two isolates of the serotype MAV for which this species is normally an inefficient vector. When test plants were inoculated by batches of 3 aphids, both isolates were rather well transmitted by one clone (Rp5), isolate MAV2 was poorly transmitted by all other clones tested and isolate MAV11 was not transmitted by eight clones and poorly transmitted by two clones. When eight aphids were used by test plants, all clones transmitted both isolates. The epidemiological consequences of MAV transmission by some R. padi clones are discussed, as well as the interest of these clones for studying aphid-derived components of luteovirus transmission.     

Two paths of sequence divergence in the citrus tristeza virus complex

ABSTRACT Comparison of a sampling of complementary DNA (cDNA) sequences from the Florida citrus tristeza virus (CTV) isolates T3 and T30 to the sequence of the genome of the Israeli isolate VT showed a relatively consistent or symmetrical distribution of nucleotide sequence identity in both the 5' and 3' regions of the 19.2-kb genome. In contrast, comparison of these sequences to the sequence of isolate T36 showed a dramatic decrease in sequence identity in the 5' proximal 11 kb of the genome. A cDNA probe derived from this region of the T36 genome hybridized to double-stranded RNA (dsRNA) of only 3 of 10 different Florida CTV isolates. In contrast, analogous probes from T3 and T30 hybridized differentially to the seven isolates not selected by the T36 probe. Primers designed from cDNA sequence for polymerase chain reaction (PCR) selectively amplified these 10 isolates, allowing them to be classified as similar to T3, T30, or T36. In contrast, individual cDNA probes derived from the 3' terminal open reading frames of the T3, T30, and T36 genomes all hybridized to dsRNA from all Florida CTV isolates tested, and PCR primers designed from the T36 capsid protein gene sequence amplified successfully from all isolates. Based on these data, we propose the creation of two groups of CTV, exemplified by the VT and T36 isolates, respectively. Isolates in the VT group, which include isolates VT, T3, and T30, have genomic sequence divergence that is relatively constant in proportion and distribution throughout the genome, and candidate isolates for that group could be considered strains of the same virus. The T36 group is differentiated from the VT group by the highly divergent 5' genomic sequence. This 5' region of the CTV genome, thus, can serve as a measure of the extent of sequence divergence and can be used to define new groups and group members in the CTV complex.