Seasonal patterns of viruses, bacteria and dissolved organic carbon in a riverine wetland

SUMMARY 1. Viral and bacterial abundances were studied in relation to environmental attributes over an annual period, for both planktonic and attached (sediment, aquatic macrophyte and submerged wood) habitats, in a riverine wetland.
2. Annual mean abundance of planktonic viruses ranged from 2.3 × 10⁵−3.8 × 10⁵ particles mL⁻¹ and varied according to sampling site. Significant seasonal patterns in viral abundance were evident and appeared to be linked to variations in bacterial abundance, dissolved organic carbon and inorganic nutrients.
3. Annual mean abundance of viruses associated with surfaces ranged from 1.3 × 10⁶ particles cm⁻² on aquatic macrophytes to 1.1 × 10⁷ particles cm⁻² on wood and also showed seasonal patterns. The difference in viral dynamics among the different sites emphasizes the importance of considering habitat diversity within wetlands when examining microbial communities.     

Spatiotemporal distribution of microbial communities in a coastal, sandy aquifer system (Doñana, SW Spain)

The aquifer system of Doñana (SW Spain) represents the most important freshwater source in the Doñana Natural Area. Its spatiotemporal dynamics favours the hydrological connection between surface and subsurface ecosystems, and promotes matter fluxes among the different terrestrial and aquatic systems present here. This aquifer has been intensively studied from a hydrogeological point of view but little is known from an ecological perspective. In order to understand the ecological roles played by microbial communities in this system, we conducted a long-term seasonal study of bacterial abundance, cell biomass, bacterial biomass and functional activities over a 2-year period. Bacterial abundance ranged between 2.11 ± 1.79 × 10⁵ and 8.58 ± 6.99 × 10⁷ bacteria mL⁻¹ groundwater, average cell biomass was estimated to be 77.01 ± 31.56 fgC and bacterial biomass varied between 8.99 ± 4.10 × 10⁻² and 5.65 ± 0.70 µgC mL⁻¹. Iron-related bacteria showed the highest activities among the functional groups studied. Moreover, among the variables that usually control spatial distributions of microbial communities in aquifer systems, depth did not have a relevant effect on this aquifer, at least in the range of depths studied, but grain size, probably due to its direct effects on hydrogeological parameters, such as permeability or porosity, appeared to exert moderate control, principally in terms of bacterial abundance. Finally, significant seasonal differences in the means of these microbiological variables were also observed; temperature seems to be the main factor controlling the temporal distribution of microbial communities in this aquifer system.     

Farm-scale trials to compare the entomopathogenic fungus Beauveria bassiana with pirimiphos methyl + deltamethrin and essential oil of lemon grass for protection of stored cowpea against Callosobruchus maculatus (Coleoptera: Bruchidae)

In trials conducted in Benin, conidia of Beauveria bassiana were evaluated as a control method against the cowpea weevil, Callosobruchus maculatus , in stored cowpea. In the first trial using a high artificial infestation of C.   maculatus in 8-kg batches of cowpea in jerry cans under typical conditions, concentrations of 1 × 10⁹ and 1 × 10¹¹ conidia kg⁻¹ were compared with lemon grass oil at 2 mL kg⁻¹ and the synthetic pesticide mixture of 1.5% pirimiphos methyl + 0.05% deltamethrin at 0.5 g kg⁻¹. After 2 months of storage, seed losses (SD) were 20.63 (5.3)% in the untreated control, 8.04 (3.2)% in the low-dose B.   bassiana group, 3.12 (1.3)% in the high-dose B.   bassiana group, 2.52 (0.4)% in the lemon grass oil group and 0% in the pirimiphos methyl + deltamethrin group. In a second trial with natural infestations in 50-kg batches of cowpea in 200-L drums, treatment with B.   bassiana 1 × 10¹¹ conidia kg⁻¹ was compared with pirimiphos methyl + deltamethrin at 0.5 g kg⁻¹. After 6 months of storage on six farms, losses reached 30.76 (1.5)% in the control, 1.28 (0.2)% in the pirimiphos methyl + deltamethrin group and 3.69 (0.6)% in the B.   bassiana group.     

Effect of starvation time on the prey capture behaviour, functional response and population growth of Asplanchna sieboldi (Rotifera)

1. We examined the effect of different periods of prior starvation(from 30 min to 16 h) on the prey capture behaviour, and functional and numerical responses of the predatory rotifer Asplanchna sieboldi using the rotifer Brachionus calyciflorus as prey.
2. Feeding activity (i.e. encounter, attack, capture and ingestion) by Asplanchna increased significantly with increasing prey densities from 2 to 16 mL⁻⁻¹ and with increasing prior starvation periods from 0.5 to 8 h.
3.  Asplanchna sieboldi showed a type II functional response at all the prior starvation periods tested. The asymptotic prey density was highest after 8 h of starvation.
4. The instantaneous population growth rate of A. sieboldi ranged from 0.089 ± 0.044 (when starved for 8 h in every 24 h and at a prey density of 2 individuals mL⁻⁻¹ for the other 16 h) to 1.015 ± 0.142 in the control (no starvation and at a prey density of 16 individuals mL⁻⁻¹). The effect of starvation time on the numerical response was evident only at the higher prey density.     

Automated concentration and recovery of micro-organisms from drinking water using dead-end ultrafiltration

Aims:  Concentration of pathogens diluted in large volumes of water is necessary for their detection. An automated concentration system placed online in drinking water distribution systems would facilitate detection and mitigate the risk to public health.
Methods and Results:  A prototype concentrator based on dead-end hollow fibre ultrafiltration was used to concentrate Bacillus atrophaeus spores directly from tap water. Backflush was used to recover accumulated particulates for analysis. In field tests conducted on a water utility distribution system, 3·2 × 10⁴–1·4 × 10⁶ CFU ml⁻¹ (6·1 × 10⁶–3·0 × 10⁸ CFU) were recovered from the filter when 2·9 × 10⁷–1·0 × 10⁹ CFU were spiked into the system. Per cent recovery ranged from 21% to 68% for flow volumes of 15–21 l. Tests using spore influent levels <10 CFU l⁻¹ (spike < 1000 CFU) yielded 23–40% recovery for volumes >100 l.
Conclusions:  B. atrophaeus spores at levels <10 CFU l⁻¹ were concentrated directly from tap water using an automated dead-end hollow-fibre ultrafiltration system.
Significance and Impact of the Study:  The prototype concentrator represents a critical step towards an autonomous system that could be installed in drinking water distribution lines or other critical water lines to facilitate monitoring. Recovered samples can be analysed using standard or rapid biosensor methods.     

Real-time quantification of Pseudomonas fluorescens cell removal from glass surfaces due to bacteriophage varphiS1 application

Aims:  To study the efficacy of the lytic phage φS1 in eliminating Pseudomonas fluorescens in the early stage of biofilm formation, using an in situ and real time methodology for cell quantification.
Methods and Results:  Cell adhesion and phage infection studies were carried out in a parallel plate flow chamber under laminar conditions. Cells were allowed to adhere until reaching 1·7–1·8 × 10⁶ cells cm⁻² and phage infection was performed with two different phage concentrations (2 × 10⁹ PFU ml⁻¹ and 1 × 10¹⁰ PFU ml⁻¹). Phage concentration clearly affects the speed of infection. The less concentrated phage solution promoted a three times slower rate of cell removal but did not affect the overall percentage of cell removal. In fact, after a longer infection period the less concentrated phage solution reached the same 93% cell removal value.
Conclusions:  Phages are efficient in the eradication of bacterial cells at the early stage of biofilm formation and their presence at the surface did not allow bacterial recolonization of the surface.
Significance and Impact of the Study:  To date, no published studies have been made concerning in situ and real time quantification of cell removal from surfaces due to phage action.     

Bacterivory by the ciliate Euplotes in different states of hunger

Abstract: The feeding of the marine ciliate Euplotes mutabilis was studied using bacteria ( Vibrio natriegens ) doubly labelled with ³H-thymidine and ¹⁴C-leucine. In the presence of abundant bacteria (30 × 10⁶ bacteria ml⁻¹), an average Euplotes cell (initially without food vacuoles) with a protein content of 12 ng consumed 16 × 10³ bacteria in the first hour and 27 × 10³ bacteria over four hours, accumulating about 60% of the bacterial protein into ciliate macromolecules. Euplotes which had been starved or under-fed to reduce cell protein biomass to 7 or 9 ng consumed significantly fewer bacteria, but the gross growth efficiency for protein did not change. The rate of consumption of bacteria by large Euplotes of protein content 15 ng was initially less than that of 12 ng cells, and it decreased markedly before the end of a 4-hour experiment. Recently divided cells ingested bacteria rapidly, but showed a reduced gross growth efficiency of about 40%. At low bacterial concentrations (6 × 10⁶ bacteria ml⁻¹) the rates of ingestion were markedly reduced to between     and     of maximal levels; the smallest cells could not sustain feeding activity at the low prey concentration and gross growth efficiency fell from 43 to 20% during a 4-hour experiment. The strategy adopted by Euplotes in response to local fluctuations in food supply involves rapid consumption with high growth efficiency in times of plenty, but slow shrinkage without cell division to survive in times of shortage.     

Effects of triploidy on rainbow trout myogenesis in vitro

To examine the effects of triploidy on rainbow trout myogenesis in vitro , mononuclear cells were liberated enzymatically from the lateralis muscles of diploid and triploid trout. The muscle of diploids yielded 1 × 10⁶± 1 × 10⁵ (± s.e.m. ) mononuclear cells g⁻¹ muscle compared to 0.7 × 10⁶± 8 × 10⁴ cells g⁻¹ from triploids ( P <0.01). The plating efficiencies of diploid and triploid mononuclear cells on Matrigel™ following 18 h of culture in Leibovitz's L-15 + 10% foetal bovine serum were not significantly different, 35.0 ± 3.5% and 33.0 ± 2.9%, respectively. For most time points examined, the proportion of nuclei in multinucleated cells and the proportion of nuclei in myosin positive cells were not significantly different for diploid and triploid trout. Taken together, these data suggest that diploid and triploid myogenic cells will differentiate similarly when compared under identical, in vitro conditions.     

Source of cell injected is a critical factors for short and long engraftment in xeno-transplantation

Abstract. This study aims to investigate engraftment of human cord blood and foetal bone marrow stem cells after in utero transplantation via the intracoelomic route in the sheep. Here, we performed transplantation in 14 single and 1 twin sheep foetuses at 40–47 days of development, using a novel schedule for injection. (i) Single injection of CD34⁺ human cord blood stem cells via the coelomic route (from 10 to 50 × 10⁴) in seven single foetuses. (ii) Single injection of CD34⁺ foetal bone marrow stem cells via the intracoelomic route with further numbers of cells (20 × 10⁵ and 8 × 10⁵, respectively) in three single and in one twin foetuses. (iii) Double fractioned injection (20–30 × 10⁶) via the coelomic route and 20 × 10⁶ postnatally, intravenously, shortly after birth of CD3-depleted cord blood stem cells in four single foetuses. In the first group, three single foetuses showed human/sheep chimaerism at 1, 8 and 14 months after birth. In the second group, the twin foetuses showed human/sheep chimaerism at 1 month after birth. In the third group, only two out of four single foetuses that underwent transplantation showed chimaerism at 1 month. While foetal bone marrow stem cells showed good short-term engraftment (1 month after birth), cord blood stem cells were able to persist longer in the ovine recipients (at 1, 8 and 14 months after birth).     

Growth and volatile compound production by Brettanomyces/Dekkera bruxellensis in red wine

Aims:  Brettanomyces / Dekkera bruxellensis is a particularly troublesome wine spoilage yeast. This work was aimed at characterizing its behaviour in terms of growth and volatile compound production in red wine.
Methods and Results:  Sterile red wines were inoculated with 5 × 10³ viable cells ml⁻¹ of three B. bruxellensis strains and growth and volatile phenol production were followed for 1 month by means of plate counts and gas chromatography-mass spectrometry (GC-MS) respectively. Maximum population levels generally attained 10⁶–10⁷ colony forming units (CFU) ml⁻¹ and volatile phenol concentrations ranged from 500 to 4000 μg l⁻¹. Brettanomyces bruxellensis multiplication was also accompanied by the production of organic acids (from C₂ to C₁₀), short chain acid ethyl-esters and the 'mousy off-flavour' component 2-acetyl-tetrahydropyridine.
Conclusions:  Different kinds of 'Brett character' characterized by distinct metabolic and sensory profiles can arise in wine depending on the contaminating strain, wine pH and sugar content and the winemaking stage at which contamination occurs.
Significance and Impact of the Study:  We identified new chemical markers that indicate wine defects caused by B. bruxellensis. Further insight was provided into the role of some environmental conditions in promoting wine spoilage.     

Differing effects of suspended sediments on the performance of native and exotic Daphnia

1. Although large cladocerans are usually uncommon in large rivers, Daphnia lumholtzi (an exotic species in North America) is widespread and occasionally abundant. We collected zooplankton on the Illinois River (Illinois, U.S.A.) in 1995 and 1996 and found that the peak density of D. lumholtzi (25 L⁻¹) typically exceeded that of all native species combined. Maximum density occurred during warm periods (up to 27 °C) when concentrations of inorganic suspended sediments were high (>50 mg L⁻¹).
2. Using a life table experiment, we examined the effects of variation in suspended sediment (0 and 80 mg L⁻¹) and food (10⁴ and 10⁵ Ankistrodesmus cells mL⁻¹) on fitness of D. lumholtzi and the native Daphnia parvula. Daphnia lumholtzi had greater survivorship than D. parvula in most treatments and higher life-time fertility than D. parvula in all treatments. Both species achieved their fastest intrinsic rates of growth in treatments with high food, but their responses to suspended solids differed. The growth rate of D. lumholtzi in high food was slightly increased by higher turbidity, whereas that of D. parvula was depressed.
3. Results suggest that the ability of D. lumholtzi to tolerate suspended solids is an important factor contributing to its success in invading North American rivers.     

Growth and development of larval northern cricket frogs (Acris crepitans) in relation to phytoplankton abundance

SUMMARY. 1. Tadpoles of Acris crepitans were collected from two ponds in central Illinois, Depth, water temperature, distance from shore and water samples were taken at each sample site. The potential for food limitation and non-random habitat selection were examined.
2. Algal densities from water samples averaged over 1.0× 10⁶ cells ml⁻¹ in Scott's pond hut only 4.6×10³ cells ml⁻¹ in Vic's pond.
3. Guts of tadpoles from Scott's pond contained more algal cells than did guts of tadpoles from Vic's pond. Tadpoles from Scott's pond were consistently larger and more advanced in development than tadpoles collected from Vic's pond at the same lime. Therefore, tadpoles of Scott's pond were able to utilize the higher algal densities whereas tadpoles of Vic's pond may be food-limited, causing reduced growth and development.
4. Canonical discriminant analysis was used to test whether sites with different numbers of tadpoles could be distinguished based on environmental variables. Canonical function I was primarily a measure of water depth at a sample site. This function was significant in Scott's pond only. Sites containing one or more tadpoles were not easily distinguished from each other, but were found in consistently shallower water than sites where no tadpoles were found.     

Permeability of the Chara cell membrane for ethylene glycol, glycerol, meso-erythritol, xylitol and mannitol

The permeability of internodal cells of Chara australis R. Brown for polyol molecules was determined by using a turgor balance to measure the increase in the osmotic pressure of an internodal cell incubated in artificial pond water containing one of the polyol compounds tested. The permeabilities for ethylene glycol, glycerol, meso -erythritol, xylitol and mannitol were (4. 39 ± 0. 52) × 10⁻⁹, (1. 49 ± 0. 40) × 10¹⁰, (4. 92 ± 0. 27) × 10⁻¹⁰ (9. 9 ± 3. 4) × 10¹¹ and (7. 6 ± 4. 8) × 10⁻¹² m s⁻³, respectively. The permeability for glycerol was slightly smaller than that for meso -erythritol, whose molecular weight is larger than that of glycerol in this homologous series: but the reason for this is not clear.     

Attempts to control cabbage root flies Delia radicum L. and Delia floralis (Fall.) (Dipt., Anthomyiidae) with entomopathogenic fungi: laboratory and greenhouse tests

One laboratory and three greenhouse experiments were conducted to study the pathogenicity and efficacy of Finnish isolates of entomopathogenic hyphemycetous fungi against cabbage root flies. In Petri dishes, exposure to 1.5 × 10¹⁰ spores of Metarhizium anisopliae per dish caused 40–50% mortality of undifferentiated second- and third-stage larvae of Delia floralis , and 1 × 10⁷ spores per dish caused 40–50% mortality of Delia radicum larvae. In one greenhouse test, 1 × 10⁸ and 1 × 10⁹ spores of M. anisopliae and Paecilomyces fumosoroseus reduced the root damage of head cabbage by 20–70% compared with untreated controls, although this was not accompanied by significant reductions in the number of pupae. Only M. anisopliae consistently grew out of larvae and pupae of D. floralis during incubation that followed their recovery from the soil at the end of an experiment testing different formulations of M. anisopliae and Beauveria bassiana , but the frequency of the latent infections of the pest by M. anisopliae was not associated with reduced severity of damage to seedlings of head cabbage.     

Stimulation of biomethanation by Clostridium sp. PXYL1 in coculture with a Methanosarcina strain PMET1 at psychrophilic temperatures

Aim:  Bioaugumentation of low temperature biogas production was attempted by addition of cold-adapted Clostridium and a methanogen.
Methods and Results:  A psychrotrophic xylanolytic acetogenic strain Clostridium sp. PXYL1 growing optimally at 20°C and pH 5·3 and a Methanosarcina strain, PMET1, growing optimally on acetate and producing methane at 15°C were isolated from a cattle manure digester. Anaerobic conversion of xylose at 15°C with the coculture of the two strains was performed, and batch culture methane production characteristics indicated that methanogenesis occurred via acetate through 'acetoclastic' pathway. Stimulation studies were also undertaken to evaluate the effect of exogenous addition of the coculture on biogas yields at 15°C. Addition of 3 ml of PXYL1 at the rate of 12 × 10² CFU ml⁻¹ increased the biogas 1·7-fold (33 l per kg cowdung) when compared to control (19·3 l per kg cowdung) as well as increased the volatile fatty acid (VFA) levels to 3210 mg l⁻¹ when compared to 1140 mg l⁻¹ in controls. Exogenous of addition of 10 ml PMET1 inoculum at the rate of 6·8 ± 10² CFU ml⁻¹ in addition to PXYL1 served to further improve the biogas yields to 46 l kg⁻¹ as well as significantly brought down the VFA levels to 1350 mg l⁻¹.
Conclusions:  Our results suggest that the rate-limiting methanogenic step at low temperatures could be overcome and that biogas yields improved by manipulating the population of the acetoclastic methanogens.
Significance and Impact of the Study:  Stimulation of biomethanation at low temperature by coculture.     

Evaluation of an entomopathogenic fungus, Paecilomyces fumosoroseus (Wize) Brown and Smith (Deuteromycota: Hyphomycetes) obtained from Formosan subterranean termites (Isop., Rhinotermitidae)

Abstract:  An isolate of Paecilomyces fumosoroseus was obtained from Coptotermes formosanus collected in Hong Kong, and a commercially available isolate of Metarhizium anisopliae , were both tested against C. formosanus shipped live from China. Survivorship of termites treated with a suspension of 5 × 10⁵ M. anisopliae conidia/ml and kept alone declined more rapidly than for those treated at the same concentration of P. fumosoroseus conidia. At a 5 × 10⁶ conidia/ml concentration, no significant differences in terms of termite survivorship were observed between the two fungal species. However, among termites kept in groups of 10 after treatment, those sprayed with P. fumosoroseus conidia at either 5 × 10⁵ or 5 × 10⁶ conidia/ml had significantly lower survivorship than those sprayed with M. anisopliae conidia. All the cadavers of termites treated with P. fumosoroseus and kept alone sporulated and among grouped termites 29% of the cadavers sporulated. By comparison, 53% of the cadavers of termites treated with M. anisopliae and kept alone sporulated, and only 4% of the cadavers of treated termites kept in groups sporulated.     

Lysogens and free viruses in fresh, brackish, and marine waters: a Bayesian analysis

A yearlong study was conducted to determine factors that affect the abundance and distribution of lysogens and free viruses at fresh-, brackish-, and saltwater stations in Newport Bay, CA. The viral and bacterial abundance were highest in the freshwater (average 1.1 × 10⁸ and 1.1 × 10⁷ mL⁻¹, respectively) and lowest in the marine water (average 0.4 × 10⁸ and 0.5 × 10⁷ mL⁻¹, respectively). Bacterial and viral counts were also several times higher during the summer than in winter. Approximately, 35% of the 141 samples were inducible in the presence of mitomycin C. The highest percentage of inducible lysogens was observed in marine waters (42%), while the lowest percentage was observed in the warmer freshwater (23%). A statistical model for the joint occurrence of lysogens and free viruses was formulated and estimated using Bayesian techniques to understand the key environmental determinants of viruses and lysogens. Our results support the existence of significant heterogeneity between the saltwater and freshwater sites. A parsimonious model that combines the two saltwater sites performs best among the specifications that were considered. Bacteria and water temperature were significant determinants of virus counts, whereas lysogen relationships are unclear. Importantly, conditional on the covariates, viruses and lysogen fractions exhibit robust negative correlation.     

Mg2+-free buffer elevates transformation efficiency of Vibrio parahaemolyticus by electroporation

Aims:  The ability to transform Vibrio spp. is limited by the extracellular nuclease that their cells secrete. The reported transformation efficiency of this organism is 10²–10⁵ transformants per microgram DNA. We tried different buffers and conditions, aiming to elevate its transformation efficiency.
Methods and Results:  MgCl₂ and sucrose are often included in the washing and/or electroporation buffers to stabilize the cell membrane. However, Mg²⁺ is required for production and activity of the extracellular nuclease. A simple electroporation buffer lacking Mg²⁺ was found to increase transformation efficiency dramatically, to levels 50-fold more than the buffers containing Mg²⁺. To maintain the stability of the cell membranes, Mg²⁺ was replaced with high concentrations of sucrose, from 272 to 408 mmol l⁻¹. With the new buffers, the transformation efficiency of Vibrio parahaemolyticus was increased to 2·2 × 10⁶ transformants per microgram DNA.
Conclusions:  Mg²⁺ in the buffer adversely affected transformation of V. parahaemolyticus by electroporation. The cell membranes of vibrio can be stabilized by high concentration of sucrose when Mg²⁺ is absent.
Significance and Impact of the Study:  A greater transformation efficiency can facilitate the genetic analysis of an organism and its pathogenicity. Buffers lacking Mg²⁺ can be used for other nuclease-producing organisms.     

Effect of Verticillium lecanii on biological characteristics and life table of Serangium japonicum (Coleoptera: Coccinellidae), a predator of whiteflies under laboratory conditions

Effects of entomopathogenic fungus Verticillium lecanii on biological characteristics and life table of Serangium japonicum , a predator of whiteflies against five different conidial concentrations (1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, and 1×10⁸ conidia/mL) were studied under laboratory conditions. The developmental periods for all immature stages (from eggs, 1st, 2nd, 3rd, 4th instar nymph and pupae up to emergence) among the treatments were significantly different when compared to that of control, and the longest development period was observed as treated with 1×10⁸ spore/mL. However, no significant difference on the percent survival of all immature stages was observed among the treatments and control. Also, there were no significantly different effects of V. lecanii on mean generation time, intrinsic rate, the finite rate of increase and longevity of S. japonicum among the treatments and control.     

Molecular Detection of Phytophthora cryptogea on Calendula officinalis and Gerbera jamesonii Artificially Inoculated with Zoospores

In this work, a protocol for zoospores production of Phytophthora cryptogea , an economically important plant pathogen was optimized. Five different concentrations of zoospores (5 × 10⁵, 5 × 10⁴, 5 × 10³, 5 × 10², 5 × 10¹ zoospores/ml) from four different isolates of P. cryptogea (Maria 1, Maria 2, S3 1-A, Amazzone) were used as inoculum on pot marigold ( Calendula officinalis ) and gerbera ( Gerbera jamesonii ) plants. Maria 1 was the most virulent isolate both on pot marigold and gerbera plants according to disease severity. A rapid and sensitive pathogen DNA extraction protocol suitable for large quantities of plant samples was adopted. Conventional polymerase chain reaction (PCR) was able to detect the pathogen in artificially inoculated symptomless pot marigold (collected day 12) and gerbera plants (day 8) after pathogen inoculation, with the suspension of 5 × 10⁵, 5 × 10⁴, 5 × 10³ P. cryptogea  zoospores/ml. Real-time PCR showed the possibility to detect the pathogen in artificially inoculated symptomless pot marigold (collected day 8) and gerbera plants (day 4) after pathogen inoculation, with the suspension of 5 × 10⁵, 5 × 10⁴ P. cryptogea  zoospores/ml. The first symptoms appeared on pot marigold plants 14 days after pathogen inoculation and on gerbera plants 10 days after inoculation. Real-time PCR showed the possibility to detect the pathogen 4 days before conventional PCR and 6 days before the appearance of disease symptoms both on pot marigold and gerbera plants.