Parasitica Isolates Research Articles

Diversity of vegetative compatibility types, distribution of mating types and occurrence of hypovirulence of Cryphonectria parasitica in chestnut stands in NW Spain

An extensive survey on chestnut stands in NW Spain was carried out to study the population biology of Cryphonectria parasitica in order to design future biological control programmes of chestnut blight based on the field introduction of native hypovirulent strains. Five hundred and thirty-nine Cryphonectria parasitica isolates, belonging to five populations (A Coruña, Lugo, Ourense, Pontevedra and León), were obtained. The diversity of vegetative compatibility was very low. Six vc types were found, each population showing only from 2 to 4 vc types. Two vc types were the known European testers EU1 and EU66, and the other four were undescribed vc types that were named E1, E2, E3 and E4. E1 was the dominant vc type in Lugo and Ourense populations, and EU1 in Pontevedra and León. Hypovirulent isolates (a total of 15) were only detected in the León population, all but one being compatible with the vc type EU1. Hypovirulent strains belonged to the French subtype F1 of the Cryphonectria hypovirus-1 (CHV1). The study of the sexual compatibility in the León population showed that the idiomorph MAT-1 was present in most isolates (97%). In León, the simple structure of the population (reproduction mainly asexual and very low vc diversity) and the presence of hypovirulent strains compatible with the dominant vc type are factors that favour the application of biological control of chestnut blight in the near future.

A role for a menthone reductase in resistance against microbial pathogens in plants.

Plants elaborate a vast array of enzymes that synthesize defensive secondary metabolites in response to pathogen attack. Here, we isolated the pathogen-responsive CaMNR1 [menthone: (+)-(3S)-neomenthol reductase] gene, a member of the short-chain dehydrogenase/reductase (SDR) superfamily, from pepper (Capsicum annuum) plants. Gas chromatography-mass spectrometry analysis revealed that purified CaMNR1 and its ortholog AtSDR1 from Arabidopsis (Arabidopsis thaliana) catalyze a menthone reduction with reduced nicotinamide adenine dinucleotide phosphate as a cofactor to produce neomenthol with antimicrobial activity. CaMNR1 and AtSDR1 also possess a significant catalytic activity for neomenthol oxidation. We examined the cellular function of the CaMNR1 gene by virus-induced gene silencing and ectopic overexpression in pepper and Arabidopsis plants, respectively. CaMNR1-silenced pepper plants were significantly more susceptible to Xanthomonas campestris pv vesicatoria and Colletotrichum coccodes infection and expressed lower levels of salicylic acid-responsive CaBPR1 and CaPR10 and jasmonic acid-responsive CaDEF1. CaMNR1-overexpressing Arabidopsis plants exhibited enhanced resistance to the hemibiotrophic pathogen Pseudomonas syringae pv tomato DC3000 and the biotrophic pathogen Hyaloperonospora parasitica isolate Noco2, accompanied by the induction of AtPR1 and AtPDF1.2. In contrast, mutation in the CaMNR1 ortholog AtSDR1 significantly enhanced susceptibility to both pathogens. Together, these results indicate that the novel menthone reductase gene CaMNR1 and its ortholog AtSDR1 positively regulate plant defenses against a broad spectrum of pathogens.

Characterization ofCryphonectria parasitica isolates collected from Aydın province in Turkey

The major chestnut-growing areas in Aydin Province, which yield nearly 35% of Turkey’s production, were examined for the presence of chestnut blight and bark samples were collected from the canker formations. Generally virulent type cankers were observed. Of 31 villages, 22 were found to be contaminated with the disease. A total of 97Cryphonectria parasitica (Murrill) M.E. Barr. isolates were characterized for the presence of potential hypovirulence by looking at cultural characteristics on media and virulence in cv. ‘Golden Delicious’ apple fruit. The isolates were placed into four groups based on pigmentation and abundance of pycnidia formation. Isolates produced lesions of various sizes on apple fruits but lesion size was not correlated with cultural characteristics typical to hypovirulent isolates. Among 97 isolates, ten possessing the characteristics associated with hypovirulence were assayed for the presence of dsRNA. Among these isolates only one, coded M1-3, was found to contain dsRNA. No dsRNA was detected in the single conidium isolates derived from M1-3. All of the isolates were screened for vegetative compatibility and two European vc types, EU-1 and EU-12, were detected. This low vc-type diversity in the region may indicate a high potential for spread of transmissible hypovirulence.

Importance ofCryphonectria parasiticastromata production and intermediate-pigmented isolates in spread ofCryphonectria hypovirus 1on grafted American chestnut trees

Summary Large, surviving American chestnut trees, grafted in 1980, were inoculated in 1982 and 1983 with four ‘white’ European (French and Italian) hypovirulent strains of Cryphonectria parasitica infected with Cryphonectria hypovirus 1 (CHV1). Spread of Italian CHV1-Euro7, indicated by nucleotide sequence analysis of CHV1 isolates, and a high level of blight control, occurred on these trees for over 20 years. However, the means by which CHV1 spreads and the possible role of stromata production in that spread are unknown. In this study, 249 C. parasitica isolates were recovered from stromata excised from natural cankers on the grafted trees and plated on an agar medium; 5.2% of the stromata yielded white phenotype isolates, 9.2% yielded intermediate-pigmented isolates (30–70% pigmentation) and the remainder were normal-pigmented isolates. For comparison, cankers artificially established on blight-free, forest-clear-cut American chestnut trees, following inoculation with three Italian white hypovirulent strains, were evaluated in a similar manner. Of 241 C. parasitica isolates recovered from stromata, 66.4% had a white colony phenotype, 19.1% had an intermediate-pigmented phenotype and the remainder were normal-pigmented isolates. For single conidia collected from stromata and plated, nearly equal frequencies of only white and intermediate-pigmented colony phenotypes were obtained. Following dsRNA extraction and electrophoresis, 21 of 33 intermediate-pigmented isolates were positive for CHV1. Some normal pigmented isolates also were positive for CHV1. Single-sporing a CHV1-positve, normal-pigmented, natural-canker, stroma isolate (Str 1) from the grafts resulted in several deeply red-orange pigmented (JR) isolates as well as some white isolates. The dsRNA in the JR isolate was extracted and cDNAs made by reverse transcriptase-polymerase chain reaction (RT-PCR) for part of a region (p29) in ORF A. Nucleotide sequencing indicated 100% identity to CHV1 present in the inoculated Italian white strain, Ep 47. The results indicate that stromata production on the grafted trees may contribute to CHV1 spread, and the presence of CHV1 in intermediate-pigmented isolates and some normal pigmented isolates indicates these isolates, often overlooked, may be important in CHV1 spread and the high level of blight control on the grafted trees.

Arabidopsis DMR6 encodes a putative 2OG‐Fe(II) oxygenase that is defense‐associated but required for susceptibility to downy mildew

The Arabidopsis mutant downy mildew resistant 6 (dmr6) carries a recessive mutation that results in the loss of susceptibility to Hyaloperonospora parasitica. Here we describe the map-based cloning of DMR6 (At5g24530), which was found to encode a 2-oxoglutarate (2OG)-Fe(II) oxygenase of unknown function. DMR6 transcription is locally induced during infections with both compatible and incompatible H. parasitica isolates. High DMR6 transcript levels were also observed in constitutive defense mutants and after treatment with salicylic acid analog BTH, suggesting that DMR6 has a role during plant defense. Expression analysis of dmr6 mutants, using DNA microarrays and quantitative PCR, showed the enhanced expression of a subset of defense-associated genes, including DMR6 itself, suggesting dmr6-mediated resistance results from the activation of plant defense responses. Alternatively, resistance could be caused by the accumulation of a toxic DMR6 substrate, or by the absence of a DMR6 metabolic product that is required for H. parasitica infection.

Pathogens on wild radish,Raphanus raphanistrum(Brassicaceae), in south-western Australia – implications for biological control

Biological control options for wild radish (Raphanus raphanistrum L.) were explored through a pathogen survey in south-western Australia, inoculation studies and a literature review. Twelve fungal species were isolated from diseased wild radish, including an undescribed species of Mycosphaerella and Hyaloperonospora parasitica, which have not been previously recorded on wild radish in Western Australia. H. parasitica was the most damaging and widespread pathogen of wild radish. Phylogenetic analysis based on the internal transcribed spacer regions of the rRNA genes showed that the H. parasitica isolates from wild radish are genetically distinct from the isolates found on Brassica species including canola. This finding opens up the possibility of developing a conservation or augmentative approach to the biological control of wild radish using H. parasitica. The possibility of using other pathogens in a biocontrol strategy is remote because of likely nontarget effects on canola. Leptosphaeria biglobosa and L. maculans were widespread pathogens of wild radish suggesting this weed may provide an inoculum source for blackleg disease on canola (Brassica napus) by providing a bridge to disease along roadsides and paddocks throughout grain-growing areas.

The Downy Mildew Effector Proteins ATR1 and ATR13 Promote Disease Susceptibility in Arabidopsis thaliana

The downy mildew (Hyaloperonospora parasitica) effector proteins ATR1 and ATR13 trigger RPP1-Nd/WsB- and RPP13-Nd-dependent resistance, respectively, in Arabidopsis thaliana. To better understand the functions of these effectors during compatible and incompatible interactions of H. parasitica isolates on Arabidopsis accessions, we developed a novel delivery system using Pseudomonas syringae type III secretion via fusions of ATRs to the N terminus of the P. syringae effector protein, AvrRPS4. ATR1 and ATR13 both triggered the hypersensitive response (HR) and resistance to bacterial pathogens in Arabidopsis carrying RPP1-Nd/WsB or RPP13-Nd, respectively, when delivered from P. syringae pv tomato (Pst) DC3000. In addition, multiple alleles of ATR1 and ATR13 confer enhanced virulence to Pst DC3000 on susceptible Arabidopsis accessions. We conclude that ATR1 and ATR13 positively contribute to pathogen virulence inside host cells. Two ATR13 alleles suppressed bacterial PAMP (for Pathogen-Associated Molecular Patterns)-triggered callose deposition in susceptible Arabidopsis when delivered by DC3000 DeltaCEL mutants. Furthermore, expression of another allele of ATR13 in plant cells suppressed PAMP-triggered reactive oxygen species production in addition to callose deposition. Intriguingly, although Wassilewskija (Ws-0) is highly susceptible to H. parasitica isolate Emco5, ATR13Emco5 when delivered by Pst DC3000 triggered localized immunity, including HR, on Ws-0. We suggest that an additional H. parasitica Emco5 effector might suppress ATR13-triggered immunity.

Species-Specific Primers for Eutypella parasitica, the Causal Agent of Eutypella Canker of Maple.

Eutypella parasitica was recently reported in Europe for the first time, and this study reports the molecular evaluation of the internal transcribed spacer (ITS)1/5.8S/ITS2 regions of 68 isolates of the fungus obtained in pure culture with polymerase chain reaction restriction fragment length polymorphism (RFLP). The RFLP patterns of all isolates proved identical and the restriction profiles served to differentiate E. parasitica from Eutypa lata, another pathogenic member of the family Diatrypaceae. Low intraspecific variability was detected in the sequenced ITS1/5.8S/ITS2 regions of eight Eutypella parasitica isolates originating from different hosts and geographical locations. Based on this ITS region, EpR/F primers specific to E. parasitica were constructed and tested with a wide range of fungal isolates. The EpR/F primer pair successfully amplified the expected fragment size of 341 bp from isolates of E. parasitica and also directly from infected maple wood shavings. The RFLP patterns and species-specific primers represent a step toward routine, large-scale, and rapid molecular diagnostics and identification of E. parasitica.

Cryphonectria parasitica in Portugal: diversity of vegetative compatibility types, mating types, and occurrence of hypovirulence

Summary Cryphonectria parasitica, the causal agent of chestnut blight, has been responsible for the decline in chestnut in Portugal for the last two decades. In order to improve understanding of C. parasitica diversity, a total of 617 isolates from all affected chestnut‐growing areas in continental Portugal, Madeira and the Azores archipelagos were studied. Nine vegetative compatibility (vc) types were identified among the isolates. EU‐11 was the most widespread vc type comprising 80.2% of the isolates, followed by EU‐12 (7.1%) and EU‐66 (6.6%). Two of the Portuguese vc types could not be assigned to a known European vc type. The diversity of vc types was low in the Portuguese populations of C. parasitica, but comparable with other areas where C. parasitica was introduced recently. The frequent occurrence of perithecia and both mating types of C. parasitica indicates that sexual reproduction of the chestnut blight fungus is common in Portugal. One C. parasitica isolate from Trás‐os‐Montes showed a white culture morphology and contained dsRNA, indicating the presence of hypovirulence in this area.

Biologische Bekämpfung des Kastanienrindenkrebses auf der Alpennordseite der Schweiz|Biological control of chestnut blight north of the Swiss Alps

Since 1986 several isolated stands of chestnut (Castanea sativa) located north of the Swiss Alps have been infected with the chestnut blight fungus (Cryphonectria parasitica). At all sites (1–3), the diversity of the vegetative compatibility types was low. To control the disease, the hypovirus CHV1 was introduced at seventeen sites in five cantons. In total, 571 cankers were treated with local C. parasitica isolates containing CHV1. Re-inspection of the cankers one to two years after treatment demonstrated that the percentage of active cankers was significantly reduced in three cantons. Re-isolations of C. parasitica showed that the hypovirus persisted in 33% to 75% of the treated cankers and was disseminated to new cankers at a low rate. The difficulties and the potential of hypovirus treatment of small chestnut stands are discussed.

The rules of engagement

The rules of engagement

Detection of ‘long-haired’ Saprolegnia ( S. parasitica) isolates using monoclonal antibodies

The ability of five monoclonal antibodies (Mabs) raised against a pathogenic Saprolegnia parasitica isolate from brown trout to detect and differentiate between isolates with bundles of long hairs ( S. parasitica) and other Saprolegnia species was determined by means of an indirect immunofluorescence assay. Four of the Mabs used recognized some of the long-haired S. parasitica isolates but also cross-reacted with other Saprolegnia species without bundles of hairs and with Achlya sp. The other Mab (named 18A6) was able to differentiate between the asexual and most of the sexual isolates in the group of long-haired S. parasitica isolates, but did not recognize Achlya sp. or the Saprolegnia species without bundles of hairs, with the exception of S. hypogyna. These results indicate that isolates with bundles of long hairs are closely related with other members of genus Saprolegnia and share several antigens. However, Mab 18A6 seems to recognize an epitope that is expressed mainly in the asexual isolates in the long-haired S. parasitica isolates.

Diversity of viruses in Cryphonectria parasitica and C. nitschkei in Japan and China, and partial characterization of a new chrysovirus species

We surveyed native populations of the chestnut blight fungus, Cryphonectria parasitica, in Japan and China, and C. nitschkei, a sympatric species on chestnut trees in Japan, to learn more about the diversity of hypoviruses and other double-stranded (ds) RNA viruses. In a sample of 472 isolates of C. parasitica and 45 isolates of C. nitschkei from six prefectures in Japan, we found 27 containing one or more dsRNAs. Twelve isolates of C. parasitica and two isolates of C. nitschkei were infected with Cryphonectria hypovirus 1 (CHV-1); four of these 12 C. parasitica isolates also contained other dsRNAs that did not hybridize to CHV-1. In China, only one of 85 C. parasitica isolates was CHV-1-infected; no dsRNAs were detected in the other isolates from China. No other known hypoviruses were found in this study. However, we found two previously undescribed dsRNAs in Japan approximately 9 kb in size that did not hybridize to each other or to any known dsRNAs from C. parasitica. We also found three additional groups of dsRNAs, one of which represents the genome of a new member of the virus family Chrysoviridae and was found only in C. nitschkei; the other two dsRNAs were found previously in isolates of C. parasitica from Japan or China. The most significant result of this survey is the discovery of novel dsRNAs that can be characterized in future research.

EDM2 is required for RPP7‐dependent disease resistance in Arabidopsis and affects RPP7 transcript levels

Specific disease resistance of Arabidopsis thaliana against the Hyaloperonospora parasitica isolate Hiks1 (HpHiks1) is mediated by RPP7. Although this disease resistance gene encodes a typical nucleotide binding site leucine-rich repeat (NB-LRR) disease resistance protein, its function is independent of the defense hormone salicylic acid and most known genes required for plant immune responses. We identified EDM2 (enhanced downy mildew 2) in a genetic screen for RPP7 suppressors. Mutations of EDM2 phenocopy RPP7 mutations, but do not affect other tested disease resistance genes. We isolated EDM2 by map-based cloning. The predicted EDM2 protein is structurally unrelated to previously identified components of the plant immune system, bears typical features of transcriptional regulators, including plant homeodomain (PHD)-finger-like domains, and defines a plant-specific protein family. In edm2 mutants both constitutive and HpHiks1-induced RPP7 transcript levels are reduced, suggesting that EDM2 is either a direct or an indirect regulator of RPP7 expression. Microarray analyses defined a set of defense-associated genes, the expression of which is suppressed during successful HpHiks1 colonization of either rpp7 or edm2 plants. This transcriptional phenotype is counteracted by an EDM2/RPP7-dependent mechanism.

The Arabidopsis thaliana TIR‐NB‐LRR R‐protein, RPP1A; protein localization and constitutive activation of defence by truncated alleles in tobacco and Arabidopsis

Specific recognition of Hyaloperonospora parasitica isolate Cala2 by Arabidopsis thaliana Ws-0 is mediated by the resistance gene RPP1A. Transient expression of different truncations of RPP1A in tobacco leaves revealed that its TIR-NB-ARC portion is sufficient to induce an elicitor-independent cell death. In stable transgenic lines of Arabidopsis, overexpression of the RPP1A TIR-NB-ARC domains (E12) using the 35S promoter leads to broad-spectrum resistance to virulent strains of H. parasitica and Pseudomonas syringae DC3000. The TIR-NB-ARC-mediated constitutive immunity is due to activation of the salicylic acid-dependent resistance pathway and is relieved by either a mutation in EDS1 or the presence of the salicylate hydroxylase gene, NahG. Growth of 35S::E12 plants is reduced, a phenotype observed in many constitutively resistant mutants. RPP1A carries a hydrophobic peptide at its N-terminus that directs the RPP1A protein into membranes, though it may not be the sole determinant mediating membrane association of RPP1A. Two-phase partitioning and sucrose density gradient sedimentation established that RPP1A resides in the endoplasmic reticulum and/or Golgi apparatus.

Genetic Analysis of Developmentally Regulated Resistance to Downy Mildew (Hyaloperonospora parasitica) in Arabidopsis thaliana

Although developmentally regulated disease resistance has been observed in a variety of plant-pathogen interactions, the molecular basis of this phenomenon is not well understood. Arabidopsis thaliana ecotype Columbia-0 (Col-0) expresses a developmentally regulated resistance to Hyaloperonospora parasitica isolate Emco5. Col-0 seedlings support profuse mycelial growth and asexual spore formation in the cotyledons. In contrast, Emco5 growth and reproduction is dramatically (but not completely) restricted in the first set of true leaves. Subsequent leaves exhibit progresssively increased resistance. This adult resistance is strongly suppressed by expression of the salicylic acid-degrading transgene NahG and by loss-of-function mutations in the defense-response regulators PAD4, NDR1, RAR1, PBS3, and NPR1. In contrast to Col-0, the Wassilewskija-0 (Ws-0) ecotype supports profuse growth of Emco5 at all stages of development. Gene-dosage experiments and segregation patterns indicate that adult susceptibility in Ws-0 is incomepletely dominant to adult resistance in Col-0. Genetic mapping in a Col x Ws F2 population revealed a major locus on the bottom arm of chromosome 5, which we named RPP31. Analysis of T-DNA insertion lines indicated that the Columbia allele of RPP8, though tightly linked to RPP31, is not necessary for adult resistance.

Sources of resistance to downy mildew (Peronospora parasitica (Pers. (ex Fr.) Fr.) in Sicilian germplasm of cauliflower and broccoli

Downy mildew (Peronospora parasitica (Pers. ex Fr.) Fr.) is a serious disease of brassicas in several countries. Seedlings are very susceptible to this pathogen and crops require frequent fungicide treatments to reach a good marketable yield. The use of resistant cultivars can be the most economical, reliable and environmental friendly method for managing this disease. In this work 32 Sicilian landraces and 16 commercial cultivars of cauliflower and broccoli (B. oleracea) were screened for downy mildew resistance at the cotyledon stage using one P. parasitica strain from Portugal and one from Sicily (Italy). Seven-day old seedlings were inoculated by deposing a droplet of a spore suspension on the cotyledons, incubated under controlled environment and scored 7 days later using a seven-class scale of interaction phenotype (IP), which took into consideration host response and pathogen sporulation. There were no differences in virulence between the two P. parasitica isolates. Accessions ranged from very susceptible to highly resistant to downy mildew showing a variable number of resistant individuals per accession. Forty accessions were very susceptible to downy mildew and are of no interest as sources of resistance, since most of the seedlings were scored in the most susceptible IP classes. Seven accessions had intermediate resistance and included individuals that expressed some degree of resistance. Accession Cv 90 (‘Cavolfiore Torino’) and Br 63 (‘Sparaceddu’) showed the majority of seedlings in the resistant IP classes and may constitute valuable sources of resistance to downy mildew to be used in breeding programs.

Susceptibility of Gisela 5 and Maxma 14 cherry rootstocks to four Phytophthora species

Susceptibility of Gisela 5 and Maxma 14 cherry rootstocks to Phytophthora cactorum, P. citrophthora, P. citricola and P. parasitica was evaluated. All Phytophthora species were pathogenic to both of the cherry rootstocks tested. The level of susceptibility, however, varied according to Phytophthora species. P. citrophthora and P. parasitica isolates were the most virulent. P. citricola showed moderately virulence. The least virulence was shown from the P. cactorum isolates. Both rootstocks showed similar susceptibility.

Screening and evaluation of resistance to downy mildew (Peronospora parasitica) and clubroot (Plasmodiophora brassicae) in genetic resources of Brassica oleracea

52 entries including landraces, old cultivars and wild accessions of B. oleracea and closely related Brassica species were screened for resistance against downy mildew and clubroot. Several accessions resistant to downy mildew and a few to clubroot were found. Genetic inheritance of the resistance in downy mildew was investigated by screening F1 and BC1F1 offspring from three resistant landrace accessions crossed with both a resistant and a susceptible father. The seedling resistance against downy mildew was found to be inherited recessively. This is a bit surprising as earlier papers mostly report of inheritance controlled by a single dominant gene. Previous screenings of B. oleracea resistance against downy mildew at the cotyledon stage have been done with P. parasitica isolated from B. oleracea as the original host plant. The recessive nature of the cotyledon resistance found in this screening might be due to the fact that the P. parasitica isolate was collected from B. napus fields. The clubroot seedling resistance was found to be controlled by recessive inheritance after screening the F1 offspring, this in agreement with earlier results/reports.

Situation of damage caused by Cryphonectria parasitica to forest stands and orchards of Castanea sativa by 2001 in Slovakia

In the present paper the knowledge of evaluation of the health condition of Castanea sativa (Mill.) in forest stands was summarised. It was found out that the fungus Cryphonectria parasitica (Murr.) Barr damaged the coppices until 60 years. Degree of damage (I<sub>hc</sub>) in the evaluated localities ranged from 1.11 to 3.50. Between 1992 and 2001 the biological protection in orchards by the method of hypovirulent converted strains from France was used. This method had not been used in forest stands until then. The efficiency of canker treatment ranged from 38.9% to 64.9%. On the basis of genetic variability of C. parasitica isolates they were classified into 4 vegetative-compatible (v-c) groups.