Life and death: the destiny of Phytophthora sojae determined by a receptor-like kinase.

Leucine-rich repeat receptor-like kinases (LRR-RLKs) are well known to act in plant growth, development, and defense responses. Plant LRR-RLKs locate on cell surface to sense and initiate responsive signals to a variety of extracellular stimuli, such as microbe-associated molecular patterns (MAMPs) released from microorganisms. LRR-RLKs are also present in microbes and function in microbial growth and development, but their roles in communicating with hosts are largely unknown. A recent study published in Nature Communications uncovered that a microbial LRR-RLK, PsRLK6, is required for oospore development in the sexual reproduction of Phytophthora sojae, an oomycete pathogen that causes root and stem rot in soybean. Meanwhile, PsRLK6 is recognized as a novel type of MAMP by an unknown plant LRR receptor-like protein and triggers immune responses in soybean, tomato, and Nicotiana benthamiana. The findings reveal dual roles of a pathogen LRR-RLK in determining both life through sexual reproduction and death through triggering plant immunity.

Inoculum dose-disease response relationships for the pea root rot pathogen, Aphanomyces euteiches, are dependent on soil type and other pathogens.

The oomycete pathogen, Aphanomyces euteiches, was implicated for the first time in pea and lentil root rot in Saskatchewan and Alberta in 2012 and 2013. Subsequent surveys from 2014 to 2017 revealed that Aphanomyces root rot (ARR) was widespread across the Canadian prairies. The absence of effective chemical, biological, and cultural controls and lack of genetic resistance leave only one management option: avoidance. The objectives of this study were to relate oospore levels in autoclaved and non-autoclaved soils to ARR severity across soil types from the vast prairie landscape and to determine the relationship of measured DNA quantity of A. euteiches using droplet digital PCR or quantitative PCR to the initial oospore inoculum dose in soils. These objectives support a future end goal of creating a rapid assessment method capable of categorizing root rot risk in field soil samples to aid producers with pulse crop field selection decisions. The ARR severity to oospore dose relationship was statistically significantly affected by the soil type and location from which soils were collected and did not show a linear relationship. For most soil types, ARR did not develop at oospore levels below 100/g soil, but severity rose above this level, confirming a threshold level of 100 oospores/g soil for disease development. For most soil types, ARR severity was significantly higher in non-autoclaved compared to autoclaved treatments, demonstrating the role that other pathogens play in increasing disease severity. There was a significant linear relationship between DNA concentrations measured in soil and oospore inoculum concentration, although the strength of the relationship was better for some soil types, and in some soil types, DNA measurement results underestimated the number of oospores. This research is important for developing a root rot risk assessment system for the Canadian prairies based on soil inoculum quantification, following field validation of soil quantification and relationship to root rot disease severity.

Plastid DNA sequences and oospore characters of some European taxa of Tolypella section Tolypella (Characeae) identify five clusters, including one new cryptic Tolypella taxon from Sardinia, but they do not coincide with current morphological descriptions.

In Europe, the genus Tolypella (Characeae) comprises four to eight Tolypella taxa in sections Rothia and Tolypella that have been distinguished by vegetative morphology and gametangial characters such as antheridial size and oospore wall ornamentation. However, morphological differentiation is difficult in some cases due to overlapping and variable vegetative features, which in many cases are difficult to observe clearly. To clarify the taxonomic status of the five European taxa of Tolypella in section Tolypella, sequence data of the plastid genes atpB, rbcL and psbC for Tolypella glomerata (Desv.) Leonh., Tolypella hispanica Allen, Tolypella nidifica (O.F. Müll.) A. Braun, Tolypella normaniana (Nordst.) Nordst. and Tolypella salina Cor. were combined with data on oospore morphology, including oospore wall ornamentation. Gene sequence data identified five distinct clusters, but they were not consistent with the morphologically identified five taxa. T. glomerata consisted of some of the samples morphologically identified as T. glomerata and seven samples of T. normaniana, while the remaining T. glomerata samples clustered with specimens of unclear affiliation (Tolypella sp.). We identified two clusters of T. hispanica within the European material: cluster T. hispanica I consisted of samples from various locations, whereas the second cluster (T. hispanica II) consisted of samples of T. hispanica from Sardinia Island. The remaining cluster consisted of all the specimens that had been determined as T. salina or T. nidifica in addition to two specimens of T. normaniana. Oospore morphology was most clearly distinguishable for T. glomerata. Oospore characteristics for all other taxa were not as informative but showed some geographical and/or environmentally influenced differences, especially for T. nidifica and T. salina. Our results suggest the need to further check the different taxonomy of Tolypella sect. Tolypella in which specimens normally identified as T. glomerata might be two different taxa, T. glomerata and an unidentified taxon; T. nidifica and T. salina are not separate taxa; T. normaniana is a diminutive variant of two different Tolypella taxa; and T. hispanica comprises two different taxa, one from the Mediterranean island Sardinia.

Signal and regulatory mechanisms involved in spore development of Phytophthora and Peronophythora.

Oomycetes cause hundreds of destructive plant diseases, threatening agricultural production and food security. These fungus-like eukaryotes show multiple sporulation pattern including the production of sporangium, zoospore, chlamydospore and oospore, which are critical for their survival, dispersal and infection on hosts. Recently, genomic and genetic technologies have greatly promoted the study of molecular mechanism of sporulation in the genus Phytophthora and Peronophythora. In this paper, we characterize the types of asexual and sexual spores and review latest progress of these two genera. We summarize the genes encoding G protein, mitogen-activated protein kinase (MAPK) cascade, transcription factors, RNA-binding protein, autophagy-related proteins and so on, which function in the processes of sporangium production and cleavage, zoospore behaviors and oospore formation. Meanwhile, various molecular, chemical and electrical stimuli in zoospore behaviors are also discussed. Finally, with the molecular mechanism of sporulation in Phytophthora and Peronophythora is gradually being revealed, we propose some thoughts for the further research and provide the alternative strategy for plant protection against phytopathogenic oomycetes.

Field Occurrence and Overwintering of Oospores of Pseudoperonospora cubensis in the Southeastern United States.

In the United States, the cucurbit downy mildew pathogen, Pseudoperonospora cubensis, has been shown to form oospores under laboratory conditions, but there are no reports on the formation of oospores in naturally infected cucurbit plants in the field. This study investigated the occurrence of oospores in naturally infected leaves from cucurbit fields in North Carolina and South Carolina from 2018 to 2020. Oospore viability and survival was also determined outdoors during the winter in North Carolina during this study period using soil containing leaves infested with oospores. About 5% of 1,658 naturally infected cucumber and cantaloupe leaves sampled during the study had oospores, with a mean density of 585 oospores per cm2 of infected leaf tissue. Absolute oospore viability, as assessed using the plasmolysis method, declined linearly (slope = -0.27; P < 0.0001) over the 6-month exposure period from 67.8% in November to 19.3% in May. Other variables being equal, the decrease in oospore viability was significantly affected by soil temperature (b = -0.03 to -0.05; P < 0.0001) and number of rainy days (b = 21.6 to 40.46; P < 0.05), while the effects of soil moisture on oospore viability were less clear. About 20% of the oospores exposed to outdoor conditions at the end the study period were putatively viable and deemed potentially infective. However, these putatively viable oospores failed to germinate or initiate disease when inoculated onto cucumber or cantaloupe leaves. These results indicate that oospores might require some unrecognized stimuli or physiological factors to initiate germination and infection. Nonetheless, viability of oospores at the end of the winter season suggests that once exposed to the right conditions that stimulate germination, these oospores could potentially serve as a primary inoculum source in the southeastern United States where winter temperatures are cold enough to kill cucurbits plants.

A C2H2 Zinc Finger Protein PlCZF1 Is Necessary for Oospore Development and Virulence in Peronophythora litchii.

C2H2 zinc finger is one of the most common motifs found in the transcription factors (TFs) in eukaryotes organisms, which have a broad range of functions, such as regulation of growth and development, stress tolerance and pathogenicity. Here, PlCZF1 was identified to encode a C2H2 zinc finger in the litchi downy blight pathogen Peronophythora litchii. PlCZF1 is conserved in P. litchii and Phytophthora species. In P. litchii, PlCZF1 is highly expressed in sexual developmental and early infection stages. We generated Δplczf1 mutants using the CRISPR/Cas9 method. Compared with the wild type, the Δplczf1 mutants showed no significant difference in vegetative growth and asexual reproduction, but were defective in oospore development and virulence. Further experiments revealed that the transcription of PlM90, PlLLP and three laccase encoding genes were down-regulated in the Δplczf1 mutant. Our results demonstrated that PlCZF1 is a vital regulator for sexual development and pathogenesis in P. litchii.

A Real-Time PCR Assay for the Quantification of Plasmopara viticola Oospores in Grapevine Leaves.

Grapevine downy mildew caused by Plasmopara viticola is one of the most important diseases in vineyards. Oospores that overwinter in the leaf litter above the soil are the sole source of inoculum for primary infections of P. viticola; in addition to triggering the first infections in the season, the oospores in leaf litter also contribute to disease development during the season. In the current study, a quantitative polymerase chain reaction (qPCR) method that was previously developed to detect P. viticola DNA in fresh grapevine leaves was assessed for its ability to quantify P. viticola oospores in diseased, senescent grapevine leaves. The qPCR assay was specific to P. viticola and sensitive to decreasing amounts of both genomic DNA and numbers of P. viticola oospores used to generate qPCR standard curves. When the qPCR assay was compared to microscope counts of oospores in leaves with different levels of P. viticola infestation, a strong linear relationship (R2 = 0.70) was obtained between the numbers of P. viticola oospores per gram of leaves as determined by qPCR vs. microscopic observation. Unlike microscopic observation, the qPCR assay was able to detect significant differences between leaf samples with a low level of oospore infestation (25% infested leaves and 75% non-infested leaves) vs. samples without infestation, and this ability was not influenced by the weight of the leaf sample. The results indicate that the qPCR method is sensitive and provides reliable estimates of the number of P. viticola oospores in grapevine leaves. Additional research is needed to determine whether the qPCR method is useful for quantifying P. viticola oospores in grapevine leaf litter.

Sporangiospore Viability and Oospore Production in the Spinach Downy Mildew Pathogen, Peronospora effusa.

Downy mildew of spinach, caused by the obligate pathogen Peronospora effusa, remains the most important constraint in the major spinach production areas in the United States. This disease can potentially be initiated by asexual sporangiospores via "green bridges", sexually derived oospores from seed or soil, or dormant mycelium. However, the relative importance of the various types of primary inoculum is not well known. The ability of P. effusa sporangiospores to withstand abiotic stress, such as desiccation, and remain viable during short- and long-distance dispersal and the ability of oospores to germinate and infect seedlings remain unclear. Thus, the primary objectives of this research were to evaluate the impact of desiccation on sporangiospore survival and infection efficiency and examine occurrence, production, and germination of oospores. Results indicate that desiccation significantly reduces sporangiospore viability as well as infection potential. Leaf wetness duration of 4 h was needed for disease establishment by spinach downy mildew sporangiospores. Oospores were observed in leaves of numerous commercial spinach cultivars grown in California in 2018 and Arizona in 2019. Frequency of occurrence varied between the two states-years. The presence of opposite mating types in spinach production areas in the United States was demonstrated by pairing isolates in controlled crosses and producing oospores on detached leaves as well as intact plants. Information from the study of variables that affect sporangiospore viability and oospore production will help in improving our understanding of the epidemiology of this important pathogen, which has implications for management of spinach downy mildew.

Identification of differentially activated pathways in Phytophthora sojae at the mycelial, cyst, and oospore stages by TMT-based quantitative proteomics analysis.

Phytophthora sojae is a widely distributed, destructive oomycete plant pathogen that has been developed as a model for oomycete biology. Given the important but limited reports on the comparison of the sexual and asexual stages in Phytophthora, we performed a large-scale quantitative proteomics study on two key asexual life stages of P. sojae-the mycelium and cyst-as well as on the oospore, which is a key sexual stage. Over 29,631 peptides from 4688 proteins were analyzed. Briefly, 445 proteins, 624 proteins, and 579 proteins were defined as differentially quantified proteins in cyst vs mycelium, oospore vs cyst, and oospore vs mycelium comparisons, respectively (|log2 fold change| > 1 and P < 0.05). Compared to the mycelium and oospore, fatty acid and nitrogen metabolism were specifically induced in cysts. In oospores, the up-regulated proteins focused on RNA transport and protein processing in endoplasmic reticulum, indicating translation, folding, and the secretion of core cellular or stage-specific proteins active in oospores, which might be used for oospore germination. The data presented expand our knowledge of pathways specifically linked to asexual and sexual stages of this pathogen. BIOLOGICAL SIGNIFICANCE: The sexual spores (oospores) in oomycetes have thick cell walls and can survive in the soil for years, thus providing a primary source and allowing the reinfection of their host plant in subsequent growing seasons. However, the proteomic study on oospores remains very limited as they are generally considered to be dormant. In the present study, we successfully isolated oospores, and performed a large-scale comparative quantitative proteomics study on this key sexual stage and two representative asexual stages of P. sojae. The results provide an improved understanding of P. sojae biology and suggest potential metabolic targets for disease control at the three different developmental stages in oomycetes.

Danos na soja causada por míldio / Damage to soybean caused by downy mildew

RESUMO: O míldio da soja é disseminado em todo mundo. Entretanto, o dano que ele causa não tem sido estudado no Brasil. O objetivo deste trabalho foi avaliar quais componentes de rendimento da soja são afetados por essa doença e determinar o coeficiente de dano. Dois experimentos foram conduzidos, um em Castro (2006/07) e outro em Ponta Grossa (2007/08), no Estado do Paraná, Brasil. O delineamento experimental foi de blocos ao acaso com (i) seis tratamentos e cinco repetições para os experimentos de campo, safras 2006/07, e (ii) oito tratamentos e quatro repetições para 2007/08. Foram aplicados fosfito de potássio (750g ia ha-1), propamocarb + fenamidona (900g ia ha-1) e mancozeb (2,400g ia ha-1). Em todos os tratamentos, a severidade máxima da infecção pelo míldio ocorreu no estádio R5.3 e a porcentagem de severidade variou entre 0 e 43%. O gradiente da doença foi obtido apenas na segunda safra. Equações de danos foram geradas para o peso de grãos e produtividade. O número de grãos por vagem e o número de vagens por planta não foram afetados pelo míldio mas o peso de 1000 sementes por planta foi reduzido linearmente com o aumento da severidade do míldio.

ABSTRACT: Downy mildew is widespread throughout the world. However, the damage that it causes has not been studied in Brazil yet. The objective of this work was to evaluate which components of soybean yield are affected by downy mildew and to determine the coefficient of damage. Two field experiments were conducted in Castro (2006/07) and Ponta Grossa (2007/08) in the state of Parana, Brazil. The experimental design consisted of completely randomized blocks of (i) six treatments and five replications for the 2006/07 and (ii) eight treatments and four replications for the 2007/08 field experiments. Potassium phosphite (750g a.i. ha-1), propamocarb + fenamidone (900g a.i. ha-1) and mancozeb (2400g a.i. ha-1) were applied. In all of the treatments, the maximum severity of downy mildew infection occurred at growth stage R5.3, and the percentage of severity ranged between 0 and 43%. The disease gradient was obtained only on the second season. Equations of damage were generated for the yield and grain weight. The number of grains per pod and the number of pods per plant were not affected by downy mildew but, the weight of 1000 seeds per plant was reduced linearly with increasing of mildew severity.

The importin α subunit PsIMPA1 mediates the oxidative stress response and is required for the pathogenicity of Phytophthora sojae.

The sensing of extracellular signals and their transduction into an appropriate response are crucial for the survival and virulence of plant pathogens. Eukaryotic plant pathogens must overcome the obstacles posed by nuclear membranes to manipulate gene expression to adapt to the host challenge. A highly sophisticated mechanism is the use of importins to transport proteins into the nucleus. In this study, we identified a conserved importin α gene, PsIMPA1, in Phytophthora sojae that was differentially expressed during the life cycle of this soybean pathogen. PsIMPA1 expression was lowest in zoospores and cysts but relatively consistent during the other life cycle stages, except for a slight increase at 6h post infection. Silenced mutants Psimpa1 had a decreased growth rate, an aberrant mycelial morphology, and a severely impaired ability to form oospores and sporangia. In addition, the Psimpa1 mutants exhibited reduced pathogenicity compared to the wild type. 3,3-Diaminobenzidine (DAB) staining and in vitro hydrogen peroxide tolerance assays showed that the scavenging of reactive oxygen species by these mutants was significantly impaired. Taken together, these results indicate that PsIMPA1 regulates multiple processes during the life cycle of P. sojae.

Oospore dimensions and morphology in North American Tolypella (Charophyceae, Charophyta).

Characteristics of the oospores have been used to delimit sections and, in some cases, species in the genus Tolypella A. Braun. To test the utility of oospore characters for identifying North American species of Tolypella, we investigated oospores from field-collected and herbarium specimens. Oospore dimensions (length, width, and length to width ratio) and morphology (color, ridge number and shape, wall ornamentation, and basal impression number) were measured. Oospore dimensions were statistically analyzed and oospore morphology was studied with light and scanning electron microscopy. Statistical analyses showed significant differences in length, width, and length to width ratios among most Tolypella species and populations but there was considerable overlap, which suggested that species identification based on oospore measurements alone is not wholly reliable. In addition, oospore morphology was not unique for every species.

PsMPK7, a stress-associated mitogen-activated protein kinase (MAPK) in Phytophthora sojae, is required for stress tolerance, reactive oxygenated species detoxification, cyst germination, sexual reproduction and infection of soybean.

The sensing of stress signals and their transduction into appropriate responses are crucial for the adaptation, survival and infection of phytopathogenic fungi and oomycetes. Amongst evolutionarily conserved pathways, mitogen-activated protein kinase (MAPK) cascades function as key signal transducers that use phosphorylation to convey information. In this study, we identified a gene, designated PsMPK7, one of 14 predicted genes encoding MAPKs in Phytophthora sojae. PsMPK7 was highly transcribed in each tested stage, but was up-regulated in the zoospore, cyst and cyst germination stages. Silencing of PsMPK7 affected the growth of germinated cysts, oospore production and the pathogenicity of soybean. PsMPK7 transcription was induced by stresses from sorbitol, NaCl and hydrogen peroxide. Transformants in which PsMPK7 expression was silenced (PsMPK7-silenced) were significantly more sensitive to osmotic and oxidative stress. Aniline blue and diaminobenzidine staining revealed that the silenced lines did not suppress the host reactive oxygen species (ROS) burst, indicating that either the inoculated plants activated stronger defence responses to the transformants and/or the PsMPK7-silenced transformants failed to overcome plant defences. In addition, extracellular secretion of laccase decreased in the silenced lines. Overall, our results indicate that the PsMPK7 gene encodes a stress-associated MAPK in P. sojae that is important not only for responses to various stresses, but also for ROS detoxification, cyst germination, sexual oospore production and infection of soybean.

Phylogeny of North American Tolypella (Charophyceae, Charophyta) based on plastid DNA sequences with a description of Tolypella ramosissima sp. nov.

Characeae (Charophyceae, Charophyta) contains two tribes with six genera: tribe Chareae with four genera and tribe Nitelleae, which includes Tolypella and Nitella. This paper uses molecular and morphological data to elucidate the phylogeny of Tolypella species in North America. In the most comprehensive taxonomic treatment of Characeae, 16 Tolypella species worldwide were subsumed into two species, T. intricata and T. nidifica, in two sections, Rothia and Tolypella respectively. It was further suggested that Tolypella might be a derived group within Nitella. In this investigation into species diversity and relationships in North American Tolypella, sequence data from the plastid genes atpB, psbC, and rbcL were assembled for a broad range of charophycean and land plant taxa. Molecular data were used in conjunction with morphology to test monophyly of the genus and species within it. Phylogenetic analyses of the sequence data showed that Characeae is monophyletic but that Nitelleae is paraphyletic with Tolypella sister to a monophyletic Nitella + Chareae. The results also supported the monophyly of Tolypella and the sections Rothia and Tolypella. Morphologically defined species were supported as clades with little or no DNA sequence differences. In addition, molecular data revealed several lineages and a new species (T. ramosissima sp. nov.), which suggests greater species diversity in Tolypella than previously recognized.

Oospores of Pustula helianthicola in sunflower seeds and their role in the epidemiology of white blister rust.

White blister rust (WBR) of sunflower caused by Pustula helianthicola is an important and often underestimated disease in many countries of the world. The epidemiology of the pathogen is not yet fully understood; particularly the role of oospores in primary infection and long distance dispersal. We analysed WBR severity in sunflower under natural conditions and found disease incidence of 97-99 % in fields where infected sunflower had first been observed ca. 8 yr ago. Besides the typical blisters of mitotic sporangia on leaves, large amounts of oospores were observed on the involucral bracts. Inoculation of sunflower seedlings with oospores from these bracts resulted in disease incidence of ca. 30 %, thus confirming their infectivity without a period of dormancy. Bracts of infected flower heads from the field were checked for oospores using a binocular microscope and seeds were checked by light microscopy. Oospores were found in all of the bracts and in up to 28 % of the achenes. Light microscopy revealed that oospores developed in the thin-walled, crushed parenchymatic cells of the inner layer and in the parenchymatic rays of the fibrous layer of the pericarp. Dried seeds were grown in soil to assess the occurrence of seed borne infection. Within 3 wk, up to 58 % of seedlings showed typical WBR pustules on cotyledons. Asymptomatic infections were confirmed in phenotypically healthy plants by using a PCR-based diagnostic test for P. helianthicola. The results showed the importance of oospores of P. helianthicola as the primary inoculum for WBR development in sunflower, and pointed to the potential role of contaminated seeds in the long distance transmission of the pathogen.

Oospore Wall Ornamentation in the Genus Tolypella (Charales, Charophyceae).

The ultrastructural features of oospore wall ornamentation in the genus Tolypella were examined using scanning electron microscopy (SEM). The taxonomic relationships among several species were discussed on the basis of oospore ultrastructure and measurements. In the case of T. glomerata and T. nidifica, our results support the status of separate species. Close relationships and transitional forms may exist between T. nidifica and T. normaniana, and not only in oospore wall ornamentation. Oospores of T. hispanica exhibited the same distinct type of reticulate oospore wall as previously reported, but our results do not support the recognition of T. hispanica as a separate species. Ultrastructure of the oospore walls of T. prolifera and T. intricata was almost identical, suggesting that these species are closely related. We therefore reject previous suggestions that morphological characteristics of oospores as observed in SEM are sufficient for identification of individual species. Although significant differences were found among oospores in individual species of Tolypella, large variation among populations, and among individuals belonging to the same population, caused substantial overlap among species.

A new species of Albugo parasitic to Arabidopsis thaliana reveals new evolutionary patterns in white blister rusts (Albuginaceae).

The obligate biotrophic lineages of the white blister rusts (Albuginales, Oomycota) are of ancient origin compared to the rather recently evolved downy mildews, and sophisticated mechanisms of biotrophy and a high degree of adaptation diversity are to be expected in these organisms. Speciation in the biotrophic Oomycetes is usually thought to be the consequence of host adaptation or geographic isolation. Here we report the presence of two distinct species of Albugo on the model plant Arabidopsis thaliana, Albugo candida and Albugo laibachii, the latter being formally described in this manuscript. Both species may occupy the same host within the same environment, but are nevertheless phylogenetically distinct, as inferred from analyses of both mitochondrial and nuclear DNA sequences. Different ways of adapting to their host physiology might constitute an important factor of their different niches. Evidence for this can be gained from the completely different host range of the two pathogens. While Albugo candida is a generalist species, consisting of several physiological varieties, which is able to parasitize a great variety of Brassicaceae, Albugo laibachii has not been found on any host other than Arabidopsis thaliana. Therefore, Albugo laibachii belongs to a group of highly specialised species, like the other known specialist species in Albugo s.s., Albugo koreana, Albugo lepidii and Albugo voglmayrii. The comparative investigation of the effector genes and host targets in the generalist and the specialist species may constitute a model system for elucidating the fundamental processes involved in plant pathogen co-adaptation and speciation.