Intraspecific and within-isolate sequence variation in the ITS rRNA gene region of Pythium mercuriale sp. nov. (Pythiaceae)

Sixteen Pythium isolates from diverse hosts and locations, which showed similarities in their morphology and sequences of the internal transcribed spacer (ITS) region of their rRNA gene, were investigated. As opposed to the generally accepted view, within single isolates ITS sequence variations were consistently found mostly as part of a tract of identical bases (A-T) within ITS1, and of GT or GTTT repeats within the ITS2 sequence. Thirty-one different ITS sequences obtained from 39 cloned ITS products from the 16 isolates showed high sequence and length polymorphisms within and between isolates. However, in a phylogenetic analysis, they formed a cluster distinct from those of other Pythium species. Additional sequencing of two nuclear genes (elongation factor 1 alpha and beta-tubulin) and one mitochondrial gene (nadh1) revealed high levels of heterozygosity as well as polymorphism within and between isolates, with some isolates possessing two or more alleles for each of the nuclear genes. In contrast to the observed variation in the ITS and other gene areas, all isolates were phenotypically similar. Pythium mercuriale sp. nov. (Pythiaceae) is characterized by forming thin-walled chlamydospores, subglobose to obovoid, papillate sporangia proliferating internally and smooth-walled oogonia surrounded by multiple antheridia. Maximum likelihood phylogenetic analyses based on both ITS and beta-tubulin sequence data place P. mercuriale in a clade between Pythium and Phytophthora.     

拟南芥与大豆疫霉菌的非寄主互作及一个感病突变体的遗传分析

非寄主抗病性是一种普遍的自然现象, 该文通过建立拟南芥-大豆疫霉菌(Arabidopsis thaliana-Phytophthora sojae)非寄主互作系统, 筛选对大豆疫霉菌感病的拟南芥突变体, 为研究植物对卵菌的非寄主抗病性遗传机制奠定基础。以大豆疫霉菌游动孢子接种拟南芥T-DNA插入突变体离体叶片, 从代表12 000个独立转化株系的40 000株T₃代T-DNA插入拟南芥突变体中获得一系列对大豆疫霉菌感病的突变体。其中突变体581-51感病性状表现稳定, 离体叶片接菌后3天内出现明显的水渍状病斑, 4–5天后产生大量卵孢子和/或孢子囊。细胞学观察发现有典型的吸器形成。Southern杂交和遗传分析结果表明, 581-51突变体含有4个T-DNA插入事件, 其感病性状可能由隐性单基因控制。     

Sodium,Potassium-ATPases in Algae and Oomycetes

We have investigated the presence of K⁺-transporting ATPases that belong to the phylogenetic group of animal Na⁺,K⁺-ATPases in the Pythium aphanidermatum Stramenopile oomycete, the Porphyra yezoensis red alga, and the Udotea petiolata green alga, by molecular cloning and expression in heterologous systems. PCR amplification and search in EST databases allowed one gene to be identified in each species that could encode ATPases of this type. Phylogenetic analysis of the sequences of these ATPases revealed that they cluster with ATPases of animal origin, and that the algal ATPases are closer to animal ATPases than the oomycete ATPase is. The P. yezoensis and P. aphanidermatum ATPases were functionally expressed in Saccharomyces cerevisiae and Escherichia coli alkali cation transport mutants. The aforementioned cloning and complementary searches in silicio for H⁺- and Na⁺,K⁺-ATPases revealed a great diversity of strategies for plasma membrane energization in eukaryotic cells different from typical animal, plant, and fungal cells.     

Effects of eukaryotic pathogens (Chytridiomycota and Oomycota) on marine benthic diatom communities in the Solthörn tidal flat (southern North Sea,Germany)

The presence of eukaryotic parasites (i.e. chytridiomycetes and oomycetes) infecting benthic marine diatoms was revealed by a reconnaissance survey in the Solthörn tidal flat (southern North Sea, Germany) followed by 5 months of regular monitoring in order to assess the impact of these zoosporic fungal pathogens on microphytobenthic diatom communities. Additionally, variation of environmental factors such as sediment composition and nutrient concentrations were monitored. Pre-treatment of sediment samples using short ultrasound pulses and gradient centrifugation, in combination with CalcoFluor White, were used for the visualization of both pathogen groups. The highest prevalence of infected benthic diatoms was observed in late September (6.3% of the total benthic diatom community), correlating with the highest abundance of benthic diatoms recorded during the survey (6.5 ± 1.3 × 10⁴ cells cm^–2). Most infections were caused by chytrids (up to 99.8%) and, only in a few cases, by oomycetes. The analysis of individual host abundances/infection prevalence showed in most cases a decline in cell numbers of the host species with increasing numbers of the eukaryotic parasite. Several shifts in the diatom community composition were observed. Statistical analysis revealed that the abundance of the benthic diatom hosts and their parasites was related to seasonal variation in temperature, irradiance and nutrient availability, particularly of dissolved inorganic nitrogen.     

Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR/Cas9

Phytophthora sojae is an oomycete pathogen of soybean. As a result of its economic importance, P. sojae has become a model for the study of oomycete genetics, physiology and pathology. The lack of efficient techniques for targeted mutagenesis and gene replacement have long hampered genetic studies of pathogenicity in Phytophthora species. Here, we describe a CRISPR/Cas9 system enabling rapid and efficient genome editing in P. sojae. Using the RXLR effector gene Avr4/6 as a target, we observed that, in the absence of a homologous template, the repair of Cas9‐induced DNA double‐strand breaks (DSBs) in P. sojae was mediated by non‐homologous end‐joining (NHEJ), primarily resulting in short indels. Most mutants were homozygous, presumably as a result of gene conversion triggered by Cas9‐mediated cleavage of non‐mutant alleles. When donor DNA was present, homology‐directed repair (HDR) was observed, which resulted in the replacement of Avr4/6 with the NPT II gene. By testing the specific virulence of several NHEJ mutants and HDR‐mediated gene replacements in soybean, we have validated the contribution of Avr4/6 to recognition by soybean R gene loci, Rps4 and Rps6, but also uncovered additional contributions to resistance by these two loci. Our results establish a powerful tool for the study of functional genomics in Phytophthora, which provides new avenues for better control of this pathogen.     

Paenibacillus polymyxa antagonizes oomycete plant pathogens Phytophthora palmivora and Pythium aphanidermatum

Aim:  To find sustainable alternatives to the application of synthetic chemicals for oomycete pathogen suppression.
Methods and Results:  Here, we present experiments on an Arabidopsis thaliana model system in which we studied the antagonistic properties of rhizobacterium Paenibacillus polymyxa strains towards the oomycete plant pathogens Phytophthora palmivora and Pythium aphanidermatum . We carried out studies on agar plates, in liquid media and in soil. Our results indicate that P. polymyxa strains significantly reduced P. aphanidermatum and P. palmivora colonization in liquid assays. Most plants that had been treated with P. polymyxa survived the P. aphanidermatum inoculations in soil assays.
Conclusions:  The antagonistic abilities of both systems correlated well with mycoidal substance production and not with the production of antagonistic substances from the biocontrol bacteria.
Significance and Impact of the Study:  Our experiments highlight the need to take biofilm formation and niche exclusion mechanisms into consideration for biocontrol assays performed under natural conditions.     

Approaches to functional genomics in filamentous fungi

The study of gene function in filamentous fungi is a field of research that has made great advances in very recent years. A number of transformation and gene manipulation strategies have been developed and applied to a diverse and rapidly expanding list of economically important filamentous fungi and oomycetes. With the significant number of fungal genomes now sequenced or being sequenced, functional genomics promises to uncover a great deal of new information in coming years. This review discusses recent advances that have been made in examining gene function in filamentous fungi and describes the advantages and limitations of the different approaches.     

The C-terminal half of Phytophthora infestans RXLR effector AVR3a is sufficient to trigger R3a-mediated hypersensitivity and suppress INF1-induced cell death in Nicotiana benthamiana

The RXLR cytoplasmic effector AVR3a of Phytophthora infestans confers avirulence on potato plants carrying the R3a gene. Two alleles of Avr3a encode secreted proteins that differ in only three amino acid residues, two of which are in the mature protein. Avirulent isolates carry the Avr3a allele, which encodes AVR3aKI (containing amino acids C19, K80 and I103), whereas virulent isolates express only the virulence allele avr3a, encoding AVR3aEM (S19, E80 and M103). Only the AVR3aKI protein is recognized inside the plant cytoplasm where it triggers R3a-mediated hypersensitivity. Similar to other oomycete avirulence proteins, AVR3aKI carries a signal peptide followed by a conserved motif centered on the consensus RXLR sequence that is functionally similar to a host cell-targeting signal of malaria parasites. The interaction between Avr3a and R3a can be reconstructed by their transient co-expression in Nicotiana benthamiana. We exploited the N. benthamiana experimental system to further characterize the Avr3a-R3a interaction. R3a activation by AVR3aKI is dependent on the ubiquitin ligase-associated protein SGT1 and heat-shock protein HSP90. The AVR3aKI and AVR3aEM proteins are equally stable in planta, suggesting that the difference in R3a-mediated death cannot be attributed to AVR3aEM protein instability. AVR3aKI is able to suppress cell death induced by the elicitin INF1 of P. infestans, suggesting a possible virulence function for this protein. Structure-function experiments indicated that the 75-amino acid C-terminal half of AVR3aKI, which excludes the RXLR region, is sufficient for avirulence and suppression functions, consistent with the view that the N-terminal region of AVR3aKI and other RXLR effectors is involved in secretion and targeting but is not required for effector activity. We also found that both polymorphic amino acids, K80 and I103, of mature AVR3a contribute to the effector functions.     

The genus Pythium in Taiwan, China (1) — a synoptic review

The genus Pythium, with slightly over 280 described species, has been classified traditionally with other filamentous, coenocytic, sporangia-producing fungi as “Phycomyetes”. However, with recent advances in chemical, ultrastructural and molecular studies, Pythium spp. are now considered as “fungus-like organisms” or “pseudo-fungi” and are placed in the Kingdom Chromista or Kingdom Straminopila, distinct from the true fungi of the Kingdom Fungi or Kingdom Mycota. They are widely distributed throughout the world as soil saprophytes or plant pathogens. Because of the warm moist maritime climate, Taiwan, China, is especially rich in Pythium species. To date, 48 species of Pythium have been reported from Taiwan, China, with the dominant species being Py. vexans, Py. spinosum, Py. splendens, Py. aphanidermatum, Py. dissotocum and Py. acanthicum. There is no definite geographical distribution of Pythium spp. in Taiwan, China. Twenty nine species of Pythium have proven to be plant pathogens attacking a wide variety of woody and herbaceous plants primarily causing pre- and post-emergence seedling damping-off, root rot, stem rot and rotting of fruits, tubers and ginger rhizomes, resulting in serious economic losses. The most important plant pathogenic species include Py. aphanidermatum and Py. Myriotylum, which are most active during the hot and wet summer months; whereas Py. splendens, Py. spinosum, Py. ultimum and Py. irregulare cause the greatest damage in the cool winter. Most Pythium spp. are non-specific pathogens, infecting mainly juvenile or succulent tissues. This review attempts to assess the taxonomic position of the genus Pythium and provide details of the historical development of the study of Pythium as pathogens in Taiwan, China, causing diseases of sugarcane, trees, vegetables, fruits, specialty crops and flowering plants, as well as measures to control these diseases. Of special note is the introduction of the S-H mixture which, when used as soil amendment, effectively controls many soil-borne Pythium diseases during the early stages of plant growth. The diversity of Pythium species in Taiwan, China, is discussed in comparison with the situation in the mainland of China and suggestions are made to fully utilize Pythium spp. as agents for biological control, in industry and medicine.     

Clonal expansion of the Belgian Phytophthora ramorum populations based on new microsatellite markers

Co-existence of both mating types A1 and A2 within the EU1 lineage of Phytophthora ramorum has only been observed in Belgium, which begs the question whether sexual reproduction is occurring. A collection of 411 Belgian P. ramorum isolates was established during a 7-year survey. Our main objectives were genetic characterization of this population to test for sexual reproduction, determination of population structure, evolution and spread, and evaluation of the effectiveness and impact of control measures. Novel, polymorphic simple sequence repeat (SSR) markers were developed after screening 149 candidate loci. Eighty isolates of P. ramorum, broadly representing the Belgian population, were analyzed using four previously described and three newly identified polymorphic microsatellite loci as well as amplified fragment length polymorphisms. SSR analysis was most informative and was used to screen the entire Belgian population. Thirty multilocus genotypes were identified, but 68% of the isolates belonged to the main genotype EU1MG1. Although accumulated mutation events were detected, the overall level of genetic diversity within the Belgian isolates of P. ramorum appears to be limited, indicating a relatively recent clonal expansion. Based on our SSR analysis there is no evidence of sexual recombination in the Belgian population of P. ramorum . Metalaxyl use decreased the genetic diversity of P. ramorum until 2005, when the majority of the isolates had become resistant. Most genotypes were site-specific and despite systematic removal of symptomatic and neighbouring plants, some genotypes were detected over a period of several years at a single site, sometimes discontinuously, indicating (latent) survival of the pathogen at those sites.