A comparison of Peronospora parasitica (Downy mildew) isolates from Arabidopsis thaliana and Brassica oleracea using amplified fragment length polymorphism and internal transcribed spacer 1 sequence analyses

Amplified fragment length polymorphism (AFLP) fingerprints and internal transcribed spacer 1 (ITS1) sequences from 27 Peronospora parasitica isolates (collected from Arabidopsis thaliana or Brassica oleracea), 5 Albugo candida isolates (from the same hosts and from Capsella bursa-pastoris), and 1 Bremia lactucae isolate (from Lactuca sativa) were compared. The AFLP analysis divided the isolates into five groups that correlated with taxonomic species and, in most cases, with host origin. The only exception was a group consisting of A. candida isolates from both B. oleracea and C. bursa-pastoris. ITS1 sequence analysis divided the isolates into the same five groups, demonstrated the divergence between P. parasitica isolates from A. thaliana and B. oleracea, and, using previously published ITS1 sequences, clearly showed the relationship between A. candida isolates from different hosts.     

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.     

Basic compatibility of Albugo candida in Arabidopsis thaliana and Brassica juncea causes broad-spectrum suppression of innate immunity

A biotrophic parasite often depends on an intrinsic ability to suppress host defenses in a manner that will enable it to infect and successfully colonize a susceptible host. If the suppressed defenses otherwise would have been effective against alternative pathogens, it follows that primary infection by the "suppressive" biotroph potentially could enhance susceptibility of the host to secondary infection by avirulent pathogens. This phenomenon previously has been attributed to true fungi such as rust (basidiomycete) and powdery mildew (ascomycete) pathogens. In our study, we observed broad-spectrum suppression of host defense by the oomycete Albugo candida (white blister rust) in the wild crucifer Arabidopsis thaliana and a domesticated relative, Brassica juncea. A. candida subsp. arabidopsis suppressed the "runaway cell death" phenotype of the lesion mimic mutant lsd1 in Arabidopsis thaliana in a sustained manner even after subsequent inoculation with avirulent Hyaloperonospora arabidopsis (Arabidopsis thaliana downy mildew). In sequential inoculation experiments, we show that preinfection by virulent Albugo candida can suppress disease resistance in cotyledons to several downy mildew pathogens, including contrasting examples of genotype resistance to H. arabidopsis in Arabidopsis thaliana that differ in the R protein and modes of defense signaling used to confer the resistance; genotype specific resistance in B. juncea to H. parasitica (Brassica downy mildew; isolates derived from B. juncea); species level (nonhost) resistance in both crucifers to Bremia lactucae (lettuce downy mildew) and an isolate of the H. parasitica race derived from Brassica oleracea; and nonhost resistance in B. juncea to H. arabidopsis. Broad-spectrum powdery mildew resistance conferred by RPW8 also was suppressed in Arabidopsis thaliana to two morphotypes of Erysiphe spp. following pre-infection with A. candida subsp. arabidopsis.     

Characterization of eds1, a mutation in Arabidopsis suppressing resistance to Peronospora parasitica specified by several different RPP genes.

The interaction between Arabidopsis and the biotrophic oomycete Peronospora parasitica (downy mildew) provides an attractive model pathosystem to identify molecular components of the host that are required for genotype-specific recognition of the parasite. These components are the so-called RPP genes (for resistance to P. parasitica). Mutational analysis of the ecotype Wassilewskija (Ws-0) revealed an RPP-nonspecific locus called EDS1 (for enhanced disease susceptibility) that is required for the function of RPP genes on chromosomes 3 (RPP1/RPP14 and RPP10) and 4 (RPP12). Genetic analyses demonstrated that the eds1 mutation is recessive and is not a defective allele of any known RPP gene, mapping to the bottom arm of chromosome 3 (approximately 13 centimorgans below RPP1/RPP14). Phenotypically, the Ws-eds1 mutant seedlings supported heavy sporulation by P. parasitica isolates that are each diagnostic for one of the RPP genes in wild-type Ws-0; none of the isolates is capable of sporulating on wild-type Ws-0. Ws-eds1 seedlings exhibited enhanced susceptibility to some P. parasitica isolates when compared with a compatible wild-type ecotype, Columbia, and the eds1 parental ecotype, Ws-0. This was observed as earlier initiation of sporulation and elevated production of conidiosporangia. Surprisingly, cotyledons of Ws-eds1 also supported low sporulation by five isolates of P. parasitica from Brassica oleracea. These isolates were unable to sporulate on > 100 ecotypes of Arabidopsis, including wild-type Ws-0. An isolate of Albugo candida (white blister) from B. oleracea also sporulated on Ws-eds1, but the mutant exhibited no alteration in phenotype when inoculated with several oomycete isolates from other host species. The bacterial resistance gene RPM1, conferring specific recognition of the avirulence gene avrB from Pseudomonas syringae pv glycinea, was not compromised in Ws-eds1 plants. The mutant also retained full responsiveness to the chemical inducer of systemic acquired resistance, 2,6-dichloroisonicotinic acid; Ws-eds1 seedlings treated with 2,6-dichloroisonicotinic acid became resistant to the Ws-0-compatible and Ws-0-incompatible P. parasitica isolates Emwa1 and Noco2, respectively. In summary, the EDS1 gene appears to be a necessary component of the resistance response specified by several RPP genes and is likely to function upstream from the convergence of disease resistance pathways in Arabidopsis.     

Systemic spread of downy mildew in basil plants and detection of the pathogen in seed and plant samples

Downy mildew on sweet basil (Ocimum basilicum L.) occurs worldwide. Contaminated seeds are considered as the primary inoculum source. So far no strategy to control the disease is available. Hence, the use of pathogen-free seeds is the only alternative to prevent disease outbreaks. Therefore, a rapid diagnostic method for seed testing is urgently needed. The sensitivity of a specific PCR method for direct detection of the downy mildew pathogen Peronospora belbahrii on basil samples, particularly on seeds, was evaluated. The applied PCR method proved to be very sensitive for direct detection of the pathogen on seeds and plant samples. The PCR detection limit of P. belbahrii in artificially infested seeds corresponded to the DNA amount of a single spore per seed. Additionally, the systemic spread of the pathogen from naturally infected seeds was investigated. The experiments showed that outgrowing basil plants were latently infected with the downy mildew pathogen, and the infection continued within the plant. Contaminated seeds were harvested from symptomless latently infected plants. These results support the implementation of PCR-based detection in a seed certification scheme and the necessity to control the pathogen on seeds. The PCR method can also be used for evaluation of pathogen control on seeds based on detection of the pathogen in outgrowing plants.     

Phenotypic characterization and molecular mapping of the Arabidopsis thaliana locus RPP5, determining disease resistance to Peronospora parasitica

Peronospora parasitica causes downy mildew on crucifers. An isolate of P. parasitica (denoted NoCO2) was identified that infected Arabidopsis plants of the land race Columbia (Col-0) but not plants of land race Landsberg erecta (La- er ). Segregation analysis of F₂ plants derived from a La- er x Col-0 cross established that the resistance was inherited as a single locus, denoted RPP5 . Macroscopic and microscopic examinations of inoculated La- er and Col-0 cotyledons showed that restriction of fungal growth in La- er was accompanied by massive callose accumulation and death of plant cells in direct contact with points of attempted fungal penetration. La- er x Col-0 F₁ plants exhibited an intermediate resistance response in all aspects of fungal development, indicating that RPP5 is semi-dominant in its action. F₈ recombinant inbred lines generated between La- er and Col-0 were used to map RPP5 to a narrow interval (<1.1 cM) on chromosome 4, utilizing existing restriction fragment length polymorphic (RFLP) markers and newly generated random amplified polymorphic DNA (RAPD) markers. The data provide a basis for the isolation of the RPP5 locus by positional cloning as a first step towards understanding recognitional specificity in plant-pathogen interactions at a molecular level.     

Genetic and physical mapping of the RPP13 locus, in Arabidopsis, responsible for specific recognition of several Peronospora parasitica (downy mildew) isolates.

Fifteen isolates of the biotrophic oomycete Peronospora parasitica (downy mildew) were obtained from a population of Arabidopsis thaliana plants that established naturally in a garden the previous year. They exhibited phenotypic variation in a set of 12 Arabidopsis accessions that suggested that the parasite population consisted of at least six pathotypes. One isolate, Maks9, elicited an interaction phenotype of flecking necrosis and no sporulation (FN) in the Arabidopsis accession Nd-1, and more extensive pitting necrosis with no sporulation (PN) in the accession Ws-2. RPP13 was designated as the locus for a single dominant resistance gene associated with the resistance in Nd-1 and mapped to an interval of approximately 60 kb on a bacterial artificial chromosome (BAC) contig on the lower arm of chromosome 3. This locus is approximately 6 cM telomeric to RPP1, which was previously described as the locus for the PN interaction with five Peronospora isolates, including resistance to Maks9 in Ws-2. New Peronospora isolates were obtained from four other geographically distinct populations of P. parasitica. Four isolates were characterized that elicited an FN phenotype in Nd-1 and mapped resistance to the RPP13 locus. This suggests that the RPP13 locus contains either a single gene capable of multiple isolate recognition or a group of tightly linked genes. Further analysis suggests that the RPP11 gene in the accession Rld-0 may be allelic to RPP13 but results in a different recognition capability.     

Occurrence of downy mildews on ornamental plants and their control by chemical compounds

The downy mildew on Coreopsis grandiflora caused by Plasmopara halstedii was observed during summer, mainly in July and August. Symptoms of the disease were first seen on external leaves and progressively spread to inner parts of plant rosette. On Alyssum saxatile downy mildew symptoms induced by Peronospora parasitica were observed during whole vegetation period with the strongest expression in early spring and late summer. Amistar 250 SC (25% azoxystrobine), Mildex 711,9 WG (66.7% phosethyl aluminium + 4.4% fenamidone), Previcur Energy 840 SL (530 g/l propamocarb + 310 g/l phosetyl aluminium) and Tanos 50 WG (25% cymoxanil + 25% famoxate) were used for pathogens control. In the protection of Coreopsis grandiflora against P. halstedii all tested compounds, applied curatively, decreased sporulation of the pathogen. On treaded plants at least 4-time less leaves were diseased. In the control of P. parasitica on Alyssum saxatile, the smallest number of swallowed structures on leaves was noticed on plants treated with azoxystrobine at conc. 250 microg/cm3.     

Re-consideration of Peronospora farinosa infecting Spinacia oleracea as distinct species, Peronospora effusa

Downy mildew is probably the most widespread and potentially destructive global disease of spinach (Spinacia oleracea). The causal agent of downy mildew disease on various plants of Chenopodiaceae, including spinach, is regarded as a single species, Peronospora farinosa. In the present study, the ITS rDNA sequence and morphological data demonstrated that P. farinosa from S. oleracea is distinct from downy mildew of other chenopodiaceous hosts. Fifty-eight spinach specimens were collected or loaned from 17 countries of Asia, Europe, Oceania, North and South America, which all formed a distinct monophyletic group. No intercontinental genetic variation of the ITS rDNA within Peronospora accessions causing spinach downy mildew disease was found. Phylogenetic trees supported recognition of Peronospora from spinach as a separate species. Microscopic examination also revealed morphological differences between Peronospora specimens from Spinacia and P. farinosa s. lat. specimens from Atriplex, Bassia, Beta, and Chenopodium. Consequently, the name Peronospora effusa should be reinstated for the downy mildew fungus found on spinach. Here, a specimen of the original collections of Peronospora effusa is designated as lectotype.     

Arabidopsis is susceptible to infection by a downy mildew fungus.

A population of Arabidopsis thaliana growing locally in a suburb of Zürich called Weiningen was observed to be infected with downy mildew. Plants were collected and the progress of infection was investigated in artificial inoculations in the laboratory. The plants proved to be highly susceptible, and pronounced intercellular mycelial growth, haustoria formation, conidiophore production, and sporulation of the causal organism Peronospora parasitica were all observed. The formation of oogonia, antheridia, and oospores also occurred. In contrast, Arabidopsis strain RLD was resistant to infection and none of the above structures was formed. The fungus was localized very soon after penetration of RLD leaf cells, which responded with a typical hypersensitive reaction. The differential interaction of an isolate of P. parasitica with two strains of Arabidopsis opens up the possibility of cloning resistance determinants from a host that is very amenable to genetic and molecular analysis.     

Arabidopsis SGT1b is required for defense signaling conferred by several downy mildew resistance genes

We describe the identification of a mutant in the Arabidopsis accession Columbia (Col-0) that exhibits enhanced downy mildew (edm1) susceptibility to several Peronospora parasitica isolates, including the RPP7-diagnostic isolate Hiks1. The mutation was mapped to chromosome IV and characterized physically as a 35-kb deletion spanning seven genes. One of these genes complemented the mutant to full wild-type resistance against all of the Peronospora isolates tested. This gene (AtSGT1b) encodes a predicted protein of 39.8 kD and is an Arabidopsis ortholog of yeast SGT1, which was described originally as a key regulatory protein in centromere function and ubiquitin-mediated proteolysis. AtSGT1b contains three tetratricopeptide repeats at the N terminus followed by a bipartite chord-containing SGT domain and an SGT-specific domain at the C terminus. We discuss the role of AtSGT1b in disease resistance and its possible involvement in ubiquitin-mediated proteolysis in plants.     

Morphological and Molecular Characterization of the Causal Agent of Downy Mildew on Quinoa (<Emphasis Type="Italic">Chenopodium quinoa</Emphasis>)

Downy mildew is an economically important and widespread disease in quinoa (Chenopodium quinoa) growing areas. Although in many studies Peronospora farinosa is most commonly regarded as the causal agent of the disease, identification and classification of the pathogen remain still uncertain due to its taxonomic confusion. Thirty-six Peronospora isolates from quinoa with different geographic origins including Argentina, Bolivia, Denmark, Ecuador, and Peru were morphologically and molecularly compared with Peronospora species from other Chenopodium species. The morphology of three herbarium specimens was similar to that of P. variabilis, which originated from C. album, characterized by flexuous to curved ultimate branchlets and pedicellated conidia. Phylogenetic analysis based on ITS rDNA sequences also placed the quinoa pathogen within the same clade as P. variabilis. Within the ITS rDNA sequences of the quinoa pathogens, two base substitutions were found, which separated the majority of the Danish isolates from isolates from South America, but no sequence difference was found among the isolates from different cultivars of quinoa. The present results indicate that the pathogen responsible for the quinoa downy mildew is identical to Peronospora variabilis and that it should not be lumped with P. farinosa as claimed previously by most studies.     

Analysis of expressed sequence tags derived from pea leaves infected by Peronospora viciae f. sp. pisi

A cDNA library was constructed from field pea leaves infected by the downy mildew pathogen, Peronospora viciae f. sp. pisi, using a suppression subtractive hybridisation approach. The library consists of 399 expressed sequence tags, from which 207 unisequences were obtained after sequence assembly. Of the unisequences, six were shown to be of Peronospora viciae f. sp. pisi origin. The remaining unisequences were subjected to gene ontology analysis and their functions were predicted in silico. Eleven of these unisequences (representing 24 clones) shared significant sequence similarities with Arabidopsis genes known to be involved in downy mildew resistance, including the well‐characterised genes RPP5, RPP6 and RPP27. Expression analysis of five selected unisequences by real‐time PCR indicated that all five were up‐regulated during downy mildew pathogenesis, suggesting a significant role for these genes in the host response to downy mildew infection.     

Arabidopsis downy mildew resistance gene RPP27 encodes a receptor-like protein similar to CLAVATA2 and tomato Cf-9

The Arabidopsis Ler-RPP27 gene confers AtSgt1b-independent resistance to downy mildew (Peronospora parasitica) isolate Hiks1. The RPP27 locus was mapped to a four-bacterial artificial chromosome interval on chromosome 1 from genetic analysis of a cross between the enhanced susceptibility mutant Col-edm1 (Col-sgt1) and Landsberg erecta (Ler-0). A Cf-like candidate gene in this interval was PCR amplified from Ler-0 and transformed into mutant Col-rpp7.1 plants. Homozygous transgenic lines conferred resistance to Hiks1 and at least four Ler-0 avirulent/Columbia-0 (Col-0) virulent isolates of downy mildew pathogen. A full-length RPP27 cDNA was isolated, and analysis of the deduced amino acid sequences showed that the gene encodes a receptor-like protein (RLP) with a distinct domain structure, composed of a signal peptide followed by extracellular Leu-rich repeats, a membrane spanning region, and a short cytoplasmic carboxyl domain. RPP27 is the first RLP-encoding gene to be implicated in disease resistance in Arabidopsis, enabling the deployment of Arabidopsis techniques to investigate the mechanisms of RLP function. Homology searches of the Arabidopsis genome, using the RPP27, Cf-9, and Cf-2 protein sequences as a starting point, identify 59 RLPs, including the already known CLAVATA2 and TOO MANY MOUTHS genes. A combination of sequence and phylogenetic analysis of these predicted RLPs reveals conserved structural features of the family.     

PCR amplification of species-specific DNA sequences can distinguish among Phytophthora species.

We used PCR to differentiate species in the genus Phytophthora, which contains a group of devastating plant pathogenic fungi. We focused on Phytophthora parasitica, a species that can infect solanaceous plants such as tomato, and on Phytophthora citrophthora, which is primarily a citrus pathogen. Oligonucleotide primers were derived from sequences of a 1,300-bp P. parasitica-specific DNA segment and of an 800-bp P. citrophthora-specific segment. Under optimal conditions, the primers developed for P. parasitica specifically amplified a 1,000-bp sequence of DNA from isolates of P. parasitica. Primers for P. citrophthora similarly and specifically amplified a 650-bp sequence of DNA from isolates of P. citrophthora. Detectable amplification of these specific DNA sequences required picogram quantities of chromosomal DNA. Neither pair of primers amplified these sequences with DNAs from other species of Phytophthora or from the related genus Pythium. DNAs from P. parasitica and P. citrophthora growing in infected tomato stem tissue were amplified as distinctly as DNAs from axenic cultures of each fungal species. This is the first report on PCR-driven amplification with Phytophthora species-specific primers.     

Nucleotide polymorphisms associated with Internal Transcribed Spacer (ITS) regions of ocular isolates of Aspergillus flavus

PURPOSE: To analyse the genetic similarity among ocular isolates of Aspergillus flavus by Polymerase chain reaction based Restriction Fragment Length Polymorphism (PCR-RFLP) and DNA sequencing. MATERIALS AND METHODS: Seven ocular isolates of A. flavus from 5 patients (3 from paient 1, and four isolates from patients no. 2, 3, 4, and 5 respectively) consisting of 2 Aqueous Humor (AH), 2 Vitreous fluid (VF), 1 eviscerated material, 1 corneal button were included in the study. The three specimens from 1 were one each of AH, VF and corneal button. The fungal isolates were amplified using primers targeting ITS region and the amplicons were subjected to PCR-RFLP using Hae-III enzyme and DNA sequencing to analyse the genetic similarity. RESULTS: A. flavus isolates yielded a specific product of 595 bp after amplification. All the seven A. flavus isolates showed similar pattern of digestion with Hae-III . However, DNA sequencing of ITS amplicons revealed 97.7% genetic similarity and 2.3% dissimilarity with nucleotide polymorphisms -- single, double and multiple pertaining to inversion, substitution, insertion and deletion in comparison with that of standard strain of A. flavus ATCC 16883 [Accession Number ]. A. flavus isolated from AH, VF and corneal button from the same patient showed similar nucleotide polymorphisms as against other isolates which exhibited distinct polymorphisms. This pattern of nucleotide polymorphisms in A. flavus isolates is novel and first time reported in literature to the best of our knowledge. CONCLUSION: DNA sequencing proves to be a useful molecular tool in screening for nucleotide polymorphisms among fungal isolates.     

Use of polymerase chain reaction to detect the soft rot pathogen,Pythium myriotylum,in infected ginger rhizomes

AIMS: The aims are to establish a polymerase chain reaction (PCR)-based method for detecting Pythium myriotylum in the rhizome of ginger and diagnosing ginger soft rot and screening health seed ginger. METHODS AND RESULTS: A booster PCR method was established for detection of P. myriotylum using a specific primer selected from rDNA ITS1 region coupled with universal primer ITS2. It successfully applied to the detection of P. myriotylum in naturally infected ginger rhizomes but not from DNA of ginger rhizomes collected from field without target fungus. CONCLUSIONS: A specific method for detecting P. myriotylum was achieved. SIGNIFICANCE AND IMPACT OF THE STUDY: The new PCR method has allowed us to monitor ginger for the presence of P. myriotylum as a way of disease diagnosis or healthy seed ginger examination.     

The maintenance of extreme amino acid diversity at the disease resistance gene, RPP13, in Arabidopsis thaliana

We have used the naturally occurring plant-parasite system of Arabidopsis thaliana and its common parasite Peronospora parasitica (downy mildew) to study the evolution of resistance specificity in the host population. DNA sequence of the resistance gene, RPP13, from 24 accessions, including 20 from the United Kingdom, revealed amino acid sequence diversity higher than that of any protein coding gene reported so far in A. thaliana. A significant excess of amino acid polymorphism segregating within this species is localized within the leucine-rich repeat (LRR) domain of RPP13. These results indicate that single alleles of the gene have not swept through the population, but instead, a diverse collection of alleles have been maintained. Transgenic complementation experiments demonstrate functional differences among alleles in their resistance to various pathogen isolates, suggesting that the extreme amino acid polymorphism in RPP13 is maintained through continual reciprocal selection between host and pathogen.     

大白菜霜霉病菌寄生霜霉孢子囊的保藏方法

通过离体子叶冷冻法、离体叶片冷冻法和10%二甲基亚砜+5%脱脂乳冷冻法等3种方法来保藏大白菜霜霉病菌寄生霜霉,并采用离体子叶接种法测定孢子囊的致病力,以筛选出一种较好的保藏方法。结果表明,保藏6个月后,离体子叶冷冻法和离体叶片冷冻法保藏的孢子囊的萌发率、发病率和病情指数均较高;而10%二甲基亚砜+5%脱脂乳冷冻法保藏6个月后仅有2.16%的孢子囊能够萌发,菌株致病力丧失,发病率和病情指数均为0。保藏12个月后,离体子叶冷冻法保藏效果最好,孢子囊萌发率达到62.22%,发病率为90%,病情指数为48.89;离体叶片冷冻法保藏的孢子囊的致病力较弱;10%二甲基亚砜+5%脱脂乳冷冻法保藏12个月的孢子囊全部失活。此外,采用离体子叶冷冻法保藏12个月的32株寄生霜霉复壮成功率达到了93.75%。离体子叶冷冻法适用于寄生霜霉的保藏,其孢子囊可长时间保持较高的萌发率。