A comparison of the biofouling potential of field-collected and laboratory-cultured Ulva.

The marine algae Ulva spp. are commonly used as model biofouling organisms. As biofouling studies are primarily conducted using field-collected specimens, factors including species identity, seasonal availability, and physiological status can hinder the replicability of the results. To address these limitations, a protocol was developed for the on-demand laboratory culture and release of Ulva zoospores. The biofouling potential of laboratory-cultured and field-collected Ulva blades was compared using a waterjet. No significant differences were found between field and laboratory-cultured samples in either spore adhesion (before waterjet) or the proportion of spores retained after waterjet exposure. However, there was significant variability within each session type in pre- and post-waterjet exposures, indicating that spore adhesion and retention levels vary significantly among trial runs. In addition, all our laboratory cultures were Ulva Clade C (LPP complex). In contrast, our field samples contained a mix of Ulva Clade C, U. compressa clade I, and U. flexuosa Clade D. This protocol for on-demand production of Ulva spores can improve biofouling research approaches, enables comparison of results across laboratories and regions, and accelerate the development of anti-biofouling strategies.

Biocontrol Mechanisms of Three Plant Essential Oils Against Phytophthora infestans Causing Potato Late Blight.

Late blight, caused by the notorious pathogen Phytophthora infestans, poses a significant threat to potato (Solanum tuberosum) crops worldwide, impacting their quality as well as yield. Here, we aimed to investigate the potential use of cinnamaldehyde, carvacrol, and eugenol as control agents against P. infestans and to elucidate their underlying mechanisms of action. To determine the pathogen-inhibiting concentrations of these three plant essential oils (PEOs), a comprehensive evaluation of their effects using gradient dilution, mycelial growth rate, and spore germination methods was carried out. Cinnamaldehyde, carvacrol, and eugenol were capable of significantly inhibiting P. infestans by hindering its mycelial radial growth, zoospore release, and sporangium germination; the median effective inhibitory concentration of the three PEOs was 23.87, 8.66, and 89.65 µl/liter, respectively. Scanning electron microscopy revealed that PEOs caused the irreversible deformation of P. infestans, resulting in hyphal shrinkage, distortion, and breakage. Moreover, propidium iodide staining and extracellular conductivity measurements demonstrated that all three PEOs significantly impaired the integrity and permeability of the pathogen's cell membrane in a time- and dose-dependent manner. In vivo experiments confirmed the dose-dependent efficacy of PEOs in reducing the lesion diameter of potato late blight. Altogether, these findings provide valuable insight into the antifungal mechanisms of PEOs vis-à-vis late blight-causing P. infestans. By utilizing the inherent capabilities of these natural compounds, we could effectively limit the harmful impacts of late blight on potato crops, thereby enhancing agricultural practices and ensuring the resilience of global potato food production.

An improved method to study Phytophthora cinnamomi Rands zoospores interactions with host.

Phytophthora cinnamomi Rands is a highly prevalent phytopathogen worldwide, ranking among the top ten in terms of distribution. It inflicts crown rot, canker, and root rot on numerous plant species, significantly impacting the biodiversity of both flora and fauna within affected environments. With a host range spanning over 5,000 species, including important plants like Quercus suber, Quercus ilex, Castanea sativa, and commercially significant crops such as avocado (Persea americana), maize (Zea mays), and tomato (Solanum lycopersicum), Phytophthora cinnamomi poses a substantial threat to agriculture and ecosystems. The efficient dissemination of the oomycete relies on its short-lived asexually motile zoospores, which depend on water currents to infect host roots. However, managing these zoospores in the laboratory has long been challenging due to the complexity of the life cycle. Current protocols involve intricate procedures, including alternating cycles of growth, drought, and flooding. Unfortunately, these artificial conditions often result in a rapid decline in virulence, necessitating additional steps to maintain infectivity during cultivation. In our research, we sought to address this challenge by investigating zoospore survival under various conditions. Our goal was to develop a stable stock of zoospores that is both easily deployable and highly infective. Through direct freezing in liquid nitrogen, we have successfully preserved their virulence. This breakthrough eliminates the need for repeated culture transfers, simplifying the process of plant inoculation. Moreover, it enables more comprehensive studies of Phytophthora cinnamomi and its interactions with host plants.

Differential temperature sensitivity of haploid and diploid spores of two red intertidal algae from the Magellan Strait.

Spores have crucial importance in the establishment and development of seaweed populations. When the spore release matches with the low tidal period, they experience an extreme variation in the environmental conditions including the temperature. In this study, we assess the photosynthetic responses and growth of haploid (tetraspores) and diploid (carpospores) spores of two Gigartinales species (Mazzaella laminarioides and Iridaea cordata) from sub-Antarctic populations when exposed to an increasing temperature. In the laboratory, freshly released spores were exposed to a temperature gradient (7 [control], 10, 15, and 20 °C) recreating the temperature increase experienced by these spores during typical spring tides. Germination and further growth of spores previously exposed to temperature treatments were assessed. Carpospores and tetraspores exhibited variation in their photosynthetic response (measured as effective quantum yield; ΦPSII) to temperature increase. In Mazzaella laminarioides, only carpospores exhibited a reduction in ΦPSII (by 7-24% at 15-20 °C), while both types of spores of Iridaea cordata were sensitive to temperature increase (12-24% of ΦPSII reduction at 10-20 °C). Spores previously exposed to temperature treatments and maintained at 7 °C and low PAR germinated and developed in germlings. In general, germlings originated from carpospores pre-treated at high temperatures showed higher growth rates. The different responses to temperature increase exhibited by haploid and diploid propagules of both species highlight their ecophysiological capacity to face high-temperature variation ensuring successful recruitment survival.

An adventurous journey toward and away from fern apomixis: Insights from genome size and spore abortion patterns.

PREMISE: Apomixis in ferns is relatively common and obligatory. Sterile hybrids may restore fertility via apomixis at a cost of long-term genetic stagnation. In this study, we outlined apomixis as a possible temporary phase leading to sexuality and analyzed factors relating to transitioning to and away from apomixis, such as unreduced and reduced spore formation in apomict and apo-sex hybrid ferns. METHODS: We analyzed the genome size of 15 fern species or hybrids ("taxa") via flow cytometry. The number of reduced and unreduced gametophytes was established as a proxy for viable spore formation of either type. We also calculated the spore abortion ratio (sign of reduced spores) in several taxa, including the apo-sex hybrid Dryopteris × critica and its 16 apomictically formed offspring. RESULTS: Four of 15 sampled taxa yielded offspring variable in genome size. Specifically, each variable taxon formed one viable reduced plant among 12-451 sampled gametophytes per taxon. Thus, haploid spore formation in the studied apomicts was very rare but possible. Spore abortion analyses indicated gradually decreasing abortion (haploid spore formation) over time. In Dryopteris × critica, abortion decreased from 93.8% to mean 89.5% in one generation. CONCLUSIONS: Our results support apomixis as a transitionary phase toward sexuality. Newly formed apomicts hybridize with sexual relatives and continue to form haploid spores early on. Thus, they may get the genomic content necessary for regular meiosis and restore sexuality. If the missing relative goes extinct, the lineage gets locked into apomixis as may be the case with the Dryopteris affinis complex.

The effect of sedimentation on spore settlement and recruitment of the endemic Arctic kelp, Laminaria solidungula (Phaeophyceae).

Environmental changes associated with rapid climate change in the Arctic, such as the increased rates of sedimentation from climatic or anthropogenic sources, can enhance the impact of abiotic stressors on coastal ecosystems. High sedimentation rates can be detrimental to nearshore kelp abundance and distribution, possibly due to increased mortality at the spore settlement stage. Spore settlement and viability of the Arctic kelp Laminaria solidungula were examined through a series of lab-based sedimentation experiments. Spores were exposed to increasing sediment loads in three experimental designs simulating different sedimentation scenarios: sediment deposition above settled spores, settlement of spores on sediment-covered substrate, and simultaneous suspension of spores and sediments during settlement. Spore settlement was recorded upon completion of each experiment, and gametophyte abundance was assessed following a growth period with sediments removed to examine short-term spore viability via a gametophyte-to-settled-spore ratio. In all three types of sediment exposure, the addition of sediments caused a 30%-40% reduction in spore settlement relative to a no-sediment control. Spore settlement decreased significantly between the low and high sediment treatments when spores were settled onto sediment-covered substrates. In all experiments, increasing amounts of sediment had no significant effect on spore viability, indicating that spores that had settled under different short-term sediment conditions were viable. Our results indicate that depending on spore-sediment interaction type, higher rates of sedimentation resulting from increased sediment loading could affect L. solidungula spore settlement success with potential impacts on the long-term persistence of a diverse and productive benthic habitat.

Non-chlorophyllous and crypto-chlorophyllous fern spores differ in their mobilisation of fatty acids during priming.

During fern spore germination, lipid hydrolysis primarily provides the energy to activate their metabolism. In this research, fatty acids (linoleic, oleic, palmitic and stearic) were quantified in the spores exposed or not to priming (hydration-dehydration treatments). Five fern species were investigated, two from xerophilous shrubland and three from a cloud forest. We hypothesised that during the priming hydration phase, the fatty acids profile would change in concentration, depending on the spore type (non-chlorophyllous and crypto-chlorophyllous). The fatty acid concentration was determined by gas chromatograph-mass spectrometer. Chlorophyll in spores was vizualised by epifluorescence microscopy and quantified by high-resolution liquid chromatography with a DAD-UV/Vis detector. Considering all five species and all the treatments, the oleic acid was the most catabolised. After priming, we identified two patterns in the fatty acid metabolism: (1) in non-chlorophyllous species, oleic, palmitic, and linoleic acids were catabolised during imbibition and (2) in crypto-chlorophyllous species, these fatty acids increased in concentration. These patterns suggest that crypto-chlorophyllous spores with homoiochlorophylly (chlorophyll retained after drying) might not require the assembly of new photosynthetic apparatus during dark imbibition. Thus, these spores might require less energy from pre-existing lipids and less fatty acids as 'building blocks' for cell membranes than non-chlorophyllous spores, which require de novo synthesis and structuring of the photosynthetic apparatus.

The relationship between chlorophyllous spores and mycorrhizal associations in ferns: evidence from an evolutionary approach.

PREMISE: Approximately 14% of all fern species have physiologically active chlorophyllous spores that are much more short-lived than the more common and dormant achlorophyllous spores. Most chlorophyllous-spored species (70%) are epiphytes and account for almost 37% of all epiphytic ferns. Chlorophyllous-spored ferns are also overrepresented among fern species in habitats with waterlogged soils, of which nearly 60% have chlorophyllous spores. Ferns in these disparate habitat types also have a low incidence of mycorrhizal associations. We therefore hypothesized that autotrophic chlorophyllous spores represent an adaptation of ferns to habitats with scarce mycorrhizal associations. METHODS: We evaluated the coevolution of chlorophyllous spores and mycorrhizal associations in ferns and their relation to habitat type using phylogenetic comparative methods. RESULTS: Although we did not find support for the coevolution of spore type and mycorrhizal associations, we did find that chlorophyllous spores and the absence of mycorrhizal associations have coevolved with epiphytic and waterlogged habitats. Transition rates to epiphytic and waterlogged habitats were significantly higher in species with chlorophyllous spores compared to achlorophyllous lineages. CONCLUSIONS: Spore type and mycorrhizal associations appear to play important roles in the radiation of ferns into different habitat types. Future work should focus on clarifying the functional significance of these associations.

Fern fronds that move like pine cones: humidity-driven motion of fertile leaflets governs the timing of spore dispersal in a widespread fern species.

BACKGROUND AND AIMS: The sensitive fern, Onoclea sensibilis, is a widespread species in eastern North America and has an atypical timing of spore dispersal among temperate ferns. During early summer, this dimorphic species produces heavily modified spore-bearing fronds with leaflets tightly enveloping their sporangia and spores. These fronds senesce and persist above ground as dead mature structures until the following early spring when the leaflets finally open and spores are dispersed. While this timing of spore dispersal has been observed for over 120 years, the structural mechanisms underpinning this phenology have remained elusive. METHODS: Based on field observations, growth chamber manipulations and scanning electron microscopy, the mechanisms underlying this distinctive timing of spore dispersal in the sensitive fern were investigated. KEY RESULTS: I show that fertile leaflets of the sensitive fern move in direct response to changes in humidity, exhibiting structural and functional parallels with multicellular hygromorphic structures in seed plants, such as pine cones. These parallels include differences in cellulose microfibril orientation in cells on the abaxial and adaxial sides of the leaflet. The dynamics of this hygroscopic movement concomitant with regular abscission zones along the pinnules and coordinated senescence lead to the specific timing of early spring spore dispersal in the sensitive fern. CONCLUSIONS: While hygroscopic movement is common in seed-free plants, it mostly occurs in small structures that are either one or a few cells in size, such as the leptosporangium. Given its multicellular structure and integration across many cells and tissues, the movement and construction of the sensitive fern pinnules are more similar to structures in seed plants. The evolution of this complex trait in the sensitive fern efficiently regulates the timing of spore release, leading to early spring dispersal. This phenology likely gives gametophytes and subsequent sporophytes an advantage with early germination and growth.

C-coordinated O-carboxymethyl chitosan Cu(II) complex exerts antifungal activity by disrupting the cell membrane integrity of Phytophthora capsici Leonian.

Damage to the cell membrane is an effective method to prevent drug resistance in plant fungal diseases. Here, we proposed a negative remodeling model of the cell membrane structure induced by the C-coordinated O-carboxymethyl chitosan Cu (II) complex (O-CSLn-Cu). FITC-labeled O-CSLn-Cu (FITC-O-CSLn-Cu) was first synthesized via a nucleophilic substitution reaction and confirmed by FT-IR. FITC-labeled O-CSLn-Cu could pass through the fungal cell membrane, as detected by confocal laser scanning microscopy (CLSM) coupled with fluorescein isothiocyanate (FITC)-fluorescence. O-CSLn-Cu treatment led to apparent morphological changes in the membranes of P. capsici Leonian and giant unilamellar vesicles (GUVs) by transmission electron microscopy (TEM). Then, we performed component analysis of the cell membrane from the P. capsici Leonian affected by O-CSLn-Cu with a particular interest in membrane physicochemical properties. Many unsaturated fatty acids (UFAs) and key enzymes promoting UFA synthesis of the cell membrane were downregulated. Similarly, a large number of membrane proteins responsible for substance transport and biochemical reactions were downregulated. Furthermore, O-CSLn-Cu treatments increased plasma membrane permeability with significant leakage of intercellular electrolytes, soluble proteins and sugars, and lipid peroxidation with decreasing membrane fluidity. Finally, aquaporin 10 was proven to be a potential molecular target sensitive to antimicrobial agents according to composition analysis of membrane structure and immunohistochemistry.

Prebiotics-Encapsulated Probiotic Spores Regulate Gut Microbiota and Suppress Colon Cancer.

While microbial-based therapy has been considered as an effective strategy for treating diseases such as colon cancer, its safety remains the biggest challenge. Here, probiotics and prebiotics, which possess ideal biocompatibility and are extensively used as additives in food and pharmaceutical products, are combined to construct a safe microbiota-modulating material. Through the host-guest chemistry between commercial Clostridium butyricum and chemically modified prebiotic dextran, prebiotics-encapsulated probiotic spores (spores-dex) are prepared. It is found that spores-dex can specifically enrich in colon cancers after oral administration. In the lesion, dextran is fermented by C. butyricum, and thereby produces anti-cancer short-chain fatty acids (SCFAs). Additionally, spores-dex regulate the gut microbiota, augment the abundance of SCFA-producing bacteria (e.g., Eubacterium and Roseburia), and markedly increase the overall richness of microbiota. In subcutaneous and orthotopic tumor models, drug-loaded spores-dex inhibit tumor growth up to 89% and 65%, respectively. Importantly, no obvious adverse effect is found. The work sheds light on the possibility of using a highly safe strategy to regulate gut microbiota, and provides a promising avenue for treating various gastrointestinal diseases.

Microbe Profile: Bacillus subtilis: model organism for cellular development, and industrial workhorse.

Bacillus subtilis is the best studied model organism of the Gram-positive lineage. It is naturally transformable and has an extremely powerful genetic toolbox. It is fast growing and easy to cultivate. It is an important industrial organism, being proficient at secreting proteins and making small fine chemicals, as well as acting as a plant growth promoter. It has been an important model system for studying biofilms. Finally, it makes endospores, which have provided an exceptionally fruitful system for studying various central problems of cellular development, including the generation of asymmetry, cell fate determination and morphogenesis.

Cold climate adaptation is a plausible cause for evolution of multicellular sporulation in Dictyostelia.

Unicellular protozoa that encyst individually upon starvation evolved at least eight times into organisms that instead form multicellular fruiting bodies with spores. The Dictyostelia are the largest and most complex group of such organisms. They can be subdivided into 4 major groups, with many species in groups 1-3 having additionally retained encystment. To understand fitness differences between spores and cysts, we measured long-term survival of spores and cysts under climate-mimicking conditions, investigated spore and cyst ultrastructure, and related fitness characteristics to species ecology. We found that spores and cysts survived 22 °C equally well, but that spores survived wet and dry frost better than cysts, with group 4 spores being most resilient. Spore walls consist of three layers and those of cysts of maximally two, while spores were also more compacted than cysts, with group 4 spores being the most compacted. Group 4 species were frequently isolated from arctic and alpine zones, which was rarely the case for group 1-3 species. We inferred a fossil-calibrated phylogeny of Dictyostelia, which showed that its two major branches diverged 0.52 billion years ago, following several global glaciations. Our results suggest that Dictyostelium multicellular sporulation was a likely adaptation to a cold climate.

Effects of Kudoa septempunctata infections in a human intestinal epithelial model (Caco-2): a DNA microarray study.

Kudoa septempunctata, a myxosporean parasite infecting the trunk muscles of olive flounder (Paralichthys olivaceus), is reported to cause food poisoning in humans. The molecular mechanisms underlying the toxicity of K. septempunctata spores remain largely unknown. In the present study, we examine the molecular basis of such toxicity using DNA microarray analysis of K. septempunctata-inoculated human colon adenocarcinoma cells (Caco-2). We observed that the transepithelial resistance of the K. septempunctata-inoculated Caco-2 cell monolayers decreased markedly. DNA microarray analysis revealed that the mRNA expression profiles of control and inoculated cells clearly differed. Inflammatory and bacteria-related pathways, such as interleukin-8 (IL-8) production and MAPK/NF-kappa B pathway, were enriched. The concentrations of IL-8 and serotonin (5-HT) were higher in inoculated cells than in controls. K. septempunctata invasion damages the human intestinal epithelium, causing increased production of IL-8 and 5-HT, which likely results in the vomiting associated with K. septempunctata invasion.Abbreviations: AP-1: activator protein 1; DAVID: Database for Annotation, Visualization and Integrated Discovery; ENS: enteric nervous system; FARMS: Factor Analysis for Robust Microarray Summarization; FDR: false discovery rate; GO: Gene Ontology; 5-HT: 5-hydroxytryptamine; IL-8: Interleukin-8; KEGG: Kyoto Encyclopedia of Genes and Genomes; K. septempunctata: Kudoa septempunctata; NF-kappa B: nuclear factor-kappa B; TJ: tight junction; TER: transepithelial electrical resistance.

Small RNA inhibits infection by downy mildew pathogen Hyaloperonospora arabidopsidis.

Gene silencing exists in eukaryotic organisms as a conserved regulation of the gene expression mechanism. In general, small RNAs (sRNAs) are produced within the eukaryotic cells and incorporated into an RNA-induced silencing complex (RISC) within cells. However, exogenous sRNAs, once delivered into cells, can also silence target genes via the same RISC. Here, we explored this concept by targeting the Cellulose synthase A3 (CesA3) gene of Hyaloperonospora arabidopsidis (Hpa), the downy mildew pathogen of Arabidopsis thaliana. Hpa spore suspensions were mixed with sense or antisense sRNAs and inoculated onto susceptible Arabidopsis seedlings. While sense sRNAs had no obvious effect on Hpa pathogenicity, antisense sRNAs inhibited spore germination and hence infection. Such inhibition of infection was not race-specific, but dependent on the length and capping of sRNAs. Inhibition of infection by double stranded sRNA was more efficient than that observed with antisense sRNA. Thus, exogenous sRNA targeting conserved CesA3 could suppress Hpa infection in Arabidopsis, indicating the potential of this simple and efficient sRNA-based approach for deciphering gene functions in obligate biotrophic pathogens as well as for R-gene independent control of diseases in plants.

Effects of inter-organism interactions in biofouling on microtopographic surfaces.

An extended model of the surface energetic attachment (SEA) model is introduced to study the fouling of marine organisms on microtopographic surfaces, taking into account the excluded volume interaction and the attraction between the organisms. It is shown that the excluded volume interaction leads to changes in the site-typed attachment probabilities which increase with the average spore density on the surface. As a result of these changes, the spore density map is flattened under very high density fouling. The attractive interaction on the other hand leads to aggregation of spores and the average aggregate size increased with the strength of attraction. The model can be mapped to a specific experiment to determine the attachment energy parameters. In contrast to various prior empirical approaches, the extended SEA model is rigorous from the statistical mechanics viewpoint, thus it provides a reliable tool for studying complex attachment behaviors of microorganisms on topographic surfaces.

Immobilization of planktonic algal spores by inkjet printing.

The algal cell immobilization is a commonly used technique for treatment of waste water, production of useful metabolites and management of stock culture. However, control over the size of immobilized droplets, the population of microbes, and production rate in current techniques need to be improved. Here, we use drop-on-demand inkjet printing to immobilize spores of the alga Ecklonia cava within alginate microparticles for the first time. Microparticles with immobilized spores were generated by printing alginate-spore suspensions into a calcium chloride solution. We demonstrate that the inkjet technique can control the number of spores in an ejected droplet in the range of 0.23 to 1.87 by varying spore densities in bioink. After the printing-based spore encapsulation, we observe initial sprouting and continuous growth of thallus until 45 days of culture. Our study suggest that inkjet printing has a great potential to immobilize algae, and that the ability to control the number of encapsulated spores and their microenvironments can facilitate research into microscopic interactions of encapsulated spores.

Fitness of an allopolyploid rupicolous fern compared with its diploid progenitors: from sporogenesis to sporophyte formation.

PREMISE: When two populations of related cytotypes grow in sympatry, the rarer cytotype tends to be excluded due to a frequency-dependent mating disadvantage. Evolutionary models predict that polyploids, which are typically the rarer cytotype upon first formation, should have higher relative fitness and/or higher selfing rates to establish and then coexist with diploid parents. METHODS: Performance during early recruitment was compared among three co-occurring rupicolous fern species: the allotetraploid Cheilanthes tinaei and its diploid ancestors, C. hispanica and C. maderensis. In culture experiments, fresh spores and samples of soil spore banks were tested for variation among cytotypes in germination, survival, fecundity, and mating system of gametophytes. RESULTS: Compared with its diploid parents, C. tinaei fresh spores had higher abortion percentages, lower dispersal ability as a result of its larger spores, and similar vigor at germination. For gametophytes from soil spore banks, C. tinaei had high survival similar to C. maderensis, but its sex expression resembled that of C. hispanica, with a high proportion of males. Patterns of sporophyte formation by females and bisexuals indicate that the polyploid does not have an increased gametophytic selfing rate. Gametophytes were larger in C. tinaei, but its reproductive success (sporophyte formation) was intermediate relative to diploids. CONCLUSIONS: Our results show no evidence of higher selfing or fitness advantage of the allopolyploid over both diploid parents at any stage of early recruitment. These two unexpected findings suggest that further factors, such as niche differentiation, play a more important role in cytotype coexistence.

Establishment and application of CRISPR interference to affect sporulation, hydrogen peroxide detoxification, and mannitol catabolism in the methylotrophic thermophile Bacillus methanolicus.

Bacillus methanolicus is a thermophilic, Gram-positive, rod-shaped bacterium. It is a facultative methylotroph which can use carbon and energy sources including mannitol and the one-carbon (C1) and non-food substrate methanol for growth and overproduction of amino acids, which makes it a promising candidate for biotechnological applications. Despite a growing tool box for gene cloning and expression, tools for targeted chromosomal gene knockouts and gene repression are still missing for this organism. Here, the CRISPRi-dCas9 technique for gene repression was established in B. methanolicus MGA3. Significantly reduced spore formation on the one hand and increased biofilm formation on the other hand could be demonstrated when the stage zero sporulation protein A gene spo0A was targeted. Furthermore, when the mannitol-1-phosphate 5-dehydrogenase gene mtlD was targeted by CRISPRi, mtlD RNA levels, and MtlD specific activities in crude extracts were decreased to about 50 % which resulted in reduced biomass formation from mannitol. As a third target, the catalase gene katA was chosen. Upon targeting katA by CRISPRi, catalase activity was decreased to about 25 % as shown in H2O2 drop assays and by determination of specific catalase activity in crude extracts. Our results support the predicted functions of Spo0A in sporulation and biofilm formation, of MtlD for mannitol catabolism, and of catalase in hydrogen peroxide dismutation. Thus, CRISPR interference as developed here serves as basis for the functional characterization of B. methanolicus physiology as well as for its application in biotechnology.

The oospore stage of Plasmopara obducens, impatiens downy mildew.

Impatiens downy mildew is caused by Plasmopara obducens, a pathogen known in the United States for over a hundred years, but newly attacking ornamental Impatiens walleriana in production and in the landscape. Little is known about the life cycle of P. obducens; thus, in this study an attempt was made to determine whether the pathogen is homothallic or heterothallic. Fourteen single-sporangium isolates and three single-zoospore isolates were used in single and dual inoculations of stem tissue to see whether the pathogen was homothallic or heterothallic; all isolates tested were able to produce oospores when inoculated singly, suggesting homothally. Cold treatment at 0 C for at least 1 mo induced oospores to germinate and produce primary sporangia. Inoculation of plant tissue with germinating oospores resulted in infection. Other incubation temperatures (-10, 10, and 20 C) did not induce germination, but fluctuating temperatures (between -10 and 0 C, or 0 and 10 C) induced some germination. Spores incubated at -10 C had significantly thicker walls than spores incubated at other temperatures. Evidence suggests that oospores can serve as an overwintering stage.