Zoospore Concentration Research Articles

Nitrate content in roots of pepper seedlings exposed to Phytophthora capsici

Phytophthora capsici causes root rot, a deadly plant disease. Resistance to diseases is produced by the activation of many defense substances, so knowledge of this natural defense mechanism allows the development of new strategies for disease control. In this study, the response of nitrate (NO3-), which is effective in plant growth and development, in different pepper genotypes exposed to pathogen infection was investigated. For this, resistant and sensitive pepper genotypes were exposed to 102, 103, and 104 zoospore/mL of P. capsici-22 strain and changes in NO3- content were determined from root samples taken on the 2nd, 4th and 6th days after infection. All zoospore concentrations resulted in an overall increase in NO3- content in roots of CM-334 on all days. In KM-181 and SD-8 genotypes, the highest NO3- content was determined on the 6th day of 103 zoospore/mL application. In SD-8 and KM-181 genotypes, a significant decrease in the amount of NO3- was determined on the 4th and 6th days of treatment of 104 zoospore/mL. In these genotypes, a decrease in the amount of NO3- was found with the increase in infection time at high zoospore concentration. When the three pepper genotypes were compared, the highest NO3- content was determined in the resistant CM-334 genotype, which was exposed to 104 zoospore/mL on the 6th day following the infection. In this study, changes in the amount of NO3- in resistant and susceptible pepper genotypes indicated that NO3- may be effective in plant defense against P. capsici-22.

Pathogenicity and infectivity of Phytophthora ramorum vary depending on host species, infected plant part, inoculum potential, pathogen genotype, and temperature

AbstractA total of 25 ornamental plant species representing 10 families were inoculated using three genotypes, each representing one of the genetic lineages NA1, NA2, and EU1 of the pathogen Phytophthora ramorum. Leaves were inoculated using suspensions with two zoospore concentrations and exposure at three temperatures, while stems were inoculated using agar plugs colonized by mycelia. Susceptibility was determined by measuring either the success of pathogen reisolation or lesion length caused by the pathogen. Infectivity was determined by counting sporangia in washes of inoculated leaves or stems. Results from all three pathogen genotypes combined were used to rank each of the 25 plant species for susceptibility and infectivity, while pooled results per genotype from all 25 hosts combined were employed for a preliminary comparison of pathogenicity and infectivity among genotypes. Statistical analyses showed that leaf results were affected by the concentration of zoospores, temperature, plant host, pathogen genotype, and by the interaction between host and pathogen genotype. Stem results were mostly affected by host and by the interaction between host and pathogen genotype. Hosts ranked differently when looking at the various parameters, and differences in rankings were also significant when comparing stem and leaf results. Differences were identified among the 25 hosts and the three pathogen genotypes for all parameters: results can be used for decision‐making regarding regulations or selection of plants to be grown where infestations by P. ramorum are an issue.

Evaluating type III polyketide synthase (PKS) and terpene synthase (TPS) expression in incompatible interactions of Zingiber zerumbet to Pythium myriotylum Drechsler

Pythium species are aggressive soil-borne necrotrophic oomycetes causing soft-rot disease of ginger. Disease severity indices determined following infection with P. myriotylum Drechsler in ginger cultivar, Zingiber officinale cv. Varada and a wild congener, Z. zerumbet at varying zoospore concentrations (104–1012 spores/ml) revealed high disease severity (100%) in ginger cultivar whereas Z. zerumbet displayed resistance. Absence of positive correlation between Z. zerumbet resistance and polyphenolic content indicates role of polyketides and zerumbone in preventing pathogen ingress, as reported earlier. Towards elucidating this, Z. zerumbet specific polyketide synthase (PKS) and terpene synthase (TPS) gene sequences designated ZzTPS and ZzPKS respectively were characterised. Phylogenetic analysis clustered ZzTPS with TPS-b sub-family and ZzPKS with non-chalcone forming PKS. ZzTPS and ZzPKS showed biphasic expression with first at 6 hours post infection (hpi) and then at 8 hpi, indicative of rapid induction followed by reinforcement to sustain resistance mechanisms in the wild taxon.

Bullfrog farms release virulent zoospores of the frog-killing fungus into the natural environment

Bullfrog farming and trade practices are well-established, globally distributed, and economically valuable, but pose risks for biodiversity conservation. Besides their negative impacts on native amphibian populations as an invasive species, bullfrogs play a key role in spreading the frog-killing fungus Batrachochytrium dendrobatidis (Bd) in the natural environment. Bullfrogs are tolerant to Bd, meaning that they can carry high infection loads without developing chytridiomycosis. To test the potential of bullfrog farms as reservoirs for diverse and virulent chytrid genotypes, we quantified Bd presence, prevalence and infection loads across approximately 1,500 farmed bullfrogs and in the water that is released from farms into the environment. We also described Bd genotypic diversity within frog farms by isolating Bd from dozens of infected tadpoles. We observed individuals infected with Bd in all sampled farms, with high prevalence (reaching 100%) and high infection loads (average 71,029 zoospore genomic equivalents). Average outflow water volume from farms was high (60,000 L/day), with Bd zoospore concentration reaching approximately 50 million zoospores/L. Because virulent pathogen strains are often selected when growing in tolerant hosts, we experimentally tested whether Bd genotypes isolated from bullfrogs are more virulent in native anuran hosts compared to genotypes isolated from native host species. We genotyped 36 Bd isolates from two genetic lineages and found that Bd genotypes cultured from bullfrogs showed similar virulence in native toads when compared to genotypes isolated from native hosts. Our results indicate that bullfrog farms can harbor high Bd genotypic diversity and virulence and may be contributing to the spread of virulent genotypes in the natural environment. We highlight the urgent need to implement Bd monitoring and mitigation strategies in bullfrog farms to aid in the conservation of native amphibians.

Characterization of Pythium chondricola associated with red rot disease of Pyropia yezoensis (Ueda) (Bangiales, Rhodophyta) from Lianyungang, China

Pyropia yezoensis (formerly Porphyra yezoensis) is an economically important red alga that is cultured extensively in China. The red rot disease occurs commonly during Pyropia cultivation, causing serious economic losses. An incidence of red rot disease was found in a P. yezoensis farm from mid-November to mid-December 2015 at Lianyungang, Jiangsu Province, China. Histopathological examination revealed that the naturally infected thalli were infected apparently by a pathogen, leading to red rot symptoms. The causative agent was isolated and identified as the oomycete Pythium chondricola by morphological analysis and sequence analysis of the internal transcribed spacer and cytochrome oxidase subunit 1 (cox1). In artificial infection experiments on the P. yezoensis blades, the P. chondricola isolate was able to cause the same characteristic histopathology seen in natural infections. P. chondricola grew well at a wide range of temperatures in the range 8–31°C, salinities at 0–15 and pH 5–9. In an orthogonal test used to determine the effects of environmental factors (temperature, salinity, and zoospore concentration) on infection, the data revealed that temperature was the most important factor to affect red rot disease development, with the optimal conditions for disease expansion being 20°C, 35 salinity, and a zoospore concentration of 106 zoospores/mL. The results obtained from the present study prompted us to set up a comprehensive epidemiological study on Pyropia, which will provide support to maintain the healthy development of the Pyropia industry in China.

Effects of temperature, zoospore concentration, infection period, and fruit maturity on Phytophthora palmivora infection of figs

White powdery rot in figs caused by Phytophthora palmivora is an important disease resulting in severe fruit rot, but is not currently effectively controlled in Japan due to a lack of understanding of its epidemiology. Therefore, the effects of temperature, zoospore concentration, infection period, and fruit maturity on infection of figs were examined by inoculating the fruit with a suspension of P. palmivora zoospores. The zoospores germinated at temperatures from 5 to 35 °C, with the optimum temperature range being 20–35 °C. Germ tube length in zoospore cysts was greatest at 20–30 °C. The disease developed in green figs at temperatures from 20 to 30 °C. Figs inoculated with as few as 10 zoospores per fruit developed severe symptoms at the optimum temperature (25 °C). The minimum infection period required for infection was 2 h at 20–28 °C. All of the figs developed symptoms within an 8 h infection period at 25 or 28 °C, and with a 6 h infection period at 25 °C. All fruit at different stages of development (immature fruit, yellow fruit, and mature fruit) developed symptoms. These results indicate that P. palmivora is capable of infecting figs over a wide range of temperatures, within a short infection period, at a low concentration of zoospores, and at any stage of development. These data could be used to construct forecasting models and develop effective control systems for white powdery rot.

Embryonated chicken eggs: An experimental model for Pythium insidiosum infection.

Pythiosis is a severe disease caused by Pythium insidiosum. Currently, the research on the treatment of pythiosis uses rabbits as an experimental infection model. To reduce the use of animals in scientific experimentation, alternative models are increasingly necessary options. The objective of this study was to establish a new experimental infection model for pythiosis using embryonated chicken eggs. First, we tested the inoculation of 4 zoospore concentrations into the egg allantoic cavity at 3 embryonic days. We observed that increased zoospore concentration causes a decrease in survival time, and at a later embryonic day (the 14th) of infection, embryos showed delayed mortality. To confirm the reproducibility of the model, we chose the 14th embryonic day for the inoculation of 50 zoospores/egg, and the experiment was repeated twice. Mortality began with 30% embryos 48hours after inoculation, and 95% embryos died within 72hours. There was no mortality in the uninfected control group. The infection was confirmed by culture, PCR and histopathology. Immunohistochemistry confirmed the presence of hyphae in blood vessels in the umbilical cords in 95% of embryos and only 1 liver (5%). Our results suggest that embryonated eggs can be a very useful alternative infection model to study pythiosis.

Consistency of Published Results on the Pathogen Batrachochytrium dendrobatidis in Madagascar: Formal Comment on Kolby et al. Rapid Response to Evaluate the Presence of Amphibian Chytrid Fungus (Batrachochytrium dendrobatidis) and Ranavirus in Wild Amphibian Populations in Madagascar.

Consistency of Published Results on the Pathogen Batrachochytrium dendrobatidis in Madagascar: Formal Comment on Kolby et al. Rapid Response to Evaluate the Presence of Amphibian Chytrid Fungus (Batrachochytrium dendrobatidis) and Ranavirus in Wild Amphibian Populations in Madagascar.

Development of quantification bioassays for live propagules of Phytophthora sojae in soil

To estimate inoculum density of Phytophthora sojae in natural soils to predict disease risk and to study the ecology of P. sojae in the soil, we developed two bioassays to quantify zoospores and another for oospores of P. sojae in soil based on inoculation of Mollisol (the most conducive soil type for this disease according to our disease survey) with a known density of zoospores or oospores of enhanced green fluorescent protein (EGFP)-labeled P. sojae Eps597-3 (pathotype 1a3c7). A linear regression equation, \( y_{ 1} = 1.0 9 2 7x_{ 1} + 7. 3 6 5 2 \) (Eq. 1), and an index regression equation, \( {\text{y}}_{ 2} \, \;{ = }\; 1 8 6 2. 9 {\text{e}}^{{0.00 9 5 {\text{x}}_{ 2} }} \) (Eq. 2), were established to quantify the zoospores in the soil, where x1 is the concentration of zoospores counted directly using a light microscope and y1 is the concentration of zoospores added to a soil suspension, x2 is the number of zoospores at the margin of 18 leaf discs, and y2 is the number of zoospores added to 10 mL of a soil suspension. Another linear regression equation, \( y_{ 3} = 3. 3 8 3 6x_{ 3} + 20 7.0 5 \) (Eq. 3), was determined to quantify the oospores in the soil, where x3 is the number of zoosporangia at the margin of 18 leaf discs, and y3 is the number of oospores added to 10 mL of a soil suspension. These three equations provided high reproducibility and accuracy. To verify their applicability, two other isolates with different pathotypes (3a3b678 and 1a1d23a3c578) and two other soil types (Albic luvisols and Aquic cambosols) were tested. Pathotype and soil type did not markedly affect the parameters in the three equations, suggesting that these three equations are accurate enough to serve as universal quantification bioassays of live zoospores and oospores of P. sojae isolates of any pathotype in any soil. Equations (1) and (2) can be used only to estimate zoospores, Eq. (2) for zoospores, and Eq. (3) for oospores of P. sojae in natural or inoculated soil. This method can be finished within 2–3 h for Eq. (1), 4–5 h for Eq. (2), and 7–10 days for Eq. (3).

Differential efficiency among DNA extraction methods influences detection of the amphibian pathogen Batrachochytrium dendrobatidis

Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is responsible for massive declines and extinctions of amphibians worldwide. The most common method for detecting Bd is quantitative polymerase chain reaction (qPCR). qPCR is a highly sensitive detection technique, but its ability to determine the presence and accurately quantify the amount of Bd is also contingent on the efficiency of the DNA extraction method used prior to PCR. Using qPCR, we compared the extraction efficiency of 3 different extraction methods commonly used for Bd detection across a range of zoospore quantities: PrepMan Ultra Reagent, Qiagen DNeasy Blood and Tissue Kit, and Mobio PowerSoil DNA Isolation Kit. We show that not all extraction methods led to successful detection of Bd for the low zoospore quantities and that there was variation in the estimated zoospore equivalents among the methods, which demonstrates that these methods have different extraction efficiencies. These results highlight the importance of considering the extraction method when comparing across studies. The Qiagen DNeasy kit had the highest efficiency. We also show that replicated estimates of less than 1 zoospore can result from known zoospore concentrations; therefore, such results should be considered when obtained from field data. Additionally, we discuss the implications of our findings for interpreting previous studies and for conducting future Bd surveys. It is imperative to use the most efficient DNA extraction method in tandem with the highly sensitive qPCR technique in order to accurately diagnose the presence of Bd as well as other pathogens.

Carbon Dioxide as a Potential Water Disinfestant for Phytophthora Disease Risk Mitigation.

The spread of Phytophthora spp. through irrigation systems and natural waterways can have a significant impact on plant health and requires mitigation. Pressurized carbon dioxide (CO2) can inactivate Phytophthora nicotianae zoospores but its effectiveness at low pressure and on other species was unknown. This study evaluated the effect of injected CO2 at 63 to 4,000 ppm in irrigation water on zoospore survival of four Phytophthora spp. and infectivity of P. nicotianae zoospores. Zoospore survival of P. nicotianae, P. tropicalis, and P. pini was reduced by over 90% at 4,000 ppm and was reduced by 40% at 125 to 2,000 ppm after a 2-h exposure. Survival of P. megasperma was less affected by injected CO2, with a reduction of 37.1% at ≤4,000 ppm. CO2 treatments at 4,000 ppm for 30 or 120 min of water infested with P. nicotianae at zoospore concentrations of 1,000 and 5,000 ml-1 reduced disease incidence of annual vinca (Catharanthus roseus) by 92 and 75%. Comparable efficacy was observed in the CO2 treatment at 2,000 ppm. The CO2 treatments at <2,000 ppm also significantly reduced disease caused by water infested at 1,000 zoospores ml-1. These results indicate that CO2 may have potential as a safe and effective water disinfestant for Phytophthora spp.

Infectivity and Inoculum Production of Phytophthora ramorum on Roots of Eastern United States Oak Species.

Little is known about colonization of roots of trees by Phytophthora ramorum. We examined zoospore concentration and exposure time needed to infect six Quercus (oak) species and the inoculum produced from their roots. Sprouted acorns, exposed to zoospores (3,000/ml) for different times and transplanted to potting soil, were susceptible to infection within 1 h of exposure but root weights were not impacted after 4 weeks (P = 0.952). Roots of Quercus prinus seedlings, inoculated with sporangia, had 0.6 to 3.2% colonization of the total root mass after 5 months. Neither root lesions nor obvious root sloughing were observed. Inoculum threshold levels were tested by exposing radicles to varying zoospore concentrations for 24 h. Results showed that radicle infection occurred even at 1 zoospore/ml. To test inoculum production, roots were inoculated with sporangia and transplanted into pots. Periodically, samples of runoff were collected and plated on selective medium. Afterward, root segments were plated to calculate percent colonization. After 16 and 35 days, root colonization and inoculum production from oak was lower than that of Viburnum tinus, a positive control. This study shows that P. ramorum is able to infect sprouted oak acorns and produce secondary inoculum, which may be important epidemiologically.

Defence responses in leaves of resistant and susceptible pepper ( Capsicum annuum L.) cultivars infected with different inoculum concentrations of Phytophthora capsici Leon

Three pepper cultivars (PM-702: resistant, Demre-8 and KM-hot: susceptible) with different resistances to Phytophthora capsici-22 ( P. capsici-22) were inoculated with different concentrations of zoospores to analyze the time course of phenylalanine ammonia lyase (PAL) activity, malondialdehyde (MDA), hydrogen peroxide (H 2O 2), proline and total protein. Samples were collected from the leaves of three pepper cultivars on the 2nd, 4th, and 6th days after infection; important differences were observed in PAL activity, MDA amount, H 2O 2, proline, and total protein with respect to zoospore concentration, infection time, and type of pepper cultivars. Activity of phenylalanine ammonia-lyase (PAL), the first enzyme in the phenylpropanoid biosynthetic pathway, was increased in inoculated leaves of all three cultivars. The increase in production of PAL upon challenge was higher in the infected plants compared to the non-infected (control) plants. An early induction of PAL was observed in the leaves of all peppers infected with P. capsici within two days, making it one of the detected earlier responses to infection. Moreover, pathogen stress increased the rate of lipid peroxidation (which is indicated by increasing MDA content). Production of reactive oxygen species, H 2O 2, and lipid peroxidation increased in pepper leaves by increasing time of infection and inoculum concentration. The findings suggested that PM-702 pepper cultivar was inherently better protected against lipid peroxidation under P. capsici stress than KM-hot and Demre-8. In addition, it was observed that proline content in the leaves of infected susceptible cultivars increased with time, but highest proline and total protein in non-infected (control) leaves were recorded in resistant cultivar.

Histological, physiological and molecular investigations of Fagus sylvatica seedlings infected with Phytophthora citricola

The aim of the work was to shed light into histological, physiological and molecular changes of Fagus sylvatica seedlings infected with the root pathogen Phytophthora citricola with the final goal to distinguish between local and systemic responses. Real-time quantitative PCR analysis proved that P. citricola was able to grow from infected roots into hypocotyl and epicotyl tissue of F. sylvatica seedlings. Light microscopy showed many collapsed parenchyma cells of the cortex without being penetrated by the pathogen. Hyphae were mainly growing intracellular in parenchyma and xylem tissue. Transmission electron microscopy displayed disintegration of xylem vessels and of parenchyma cells. Inhibition of water uptake of infected beech seedlings was positively correlated with the concentration of zoospores used in the experiment. In addition, a split root experiment indicated that invertases were possibly involved locally and systemically in the conversion of sucrose of P. citricola infected roots. During the growth of the pathogen in roots, a transient expression of the 1-aminocyclopropane-1-carboxylic acid (ACC)-oxidase gene was quantified in leaves which was detected in parallel with the first peak of a biphasic ethylene outburst. Additionally a systemic upregulation of aquaporin transcripts was mainly detected in leaves of beech seedlings infected with P. citricola.

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

AbstractIn 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 × 103P. 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 × 104P. 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.

Infective Potential of Sporangia and Zoospores of Phytophthora ramorum.

Phytophthora species produce sporangia that either germinate directly or release zoospores, depending upon environmental conditions. Previous Phytophthora spp. inoculation trials have used both sporangia and zoospores as the inoculum type. However, it is unknown what impact propagule type has on disease. Rhododendron leaf disks were inoculated with P. ramorum zoospores (75, 500, or 2,400 per disk), sporangia (75 per disk), or sporangia plus trifluoperazine hydrochloride (TFP) (75 per disk), a chemical that inhibits zoospore formation. Combining results from two different isolates, the highest concentration of zoospores (2,400 per disk) induced a significantly higher percentage of necrotic leaf disk area (96.6%) than sporangia (87.6%) and 500 zoospores per disk (88.7%). The sporangia plus TFP treatment had the lowest necrosis at 47.5%. Rooted rhododendron cuttings had a higher percentage of necrotic leaves per plant when inoculated with zoospores (3,000 or 50,000 per ml) or cysts (50,000 per ml) than with sporangia (3,000 per ml) with or without TFP. The percentage of necrotic leaf area was significantly higher when cysts or zoospores were inoculated at 50,000 per ml than sporangia without TFP and zoospores at 3,000 per ml. All treatments were significantly higher in the percentage of necrotic leaf area than the leaves treated with sporangia plus TFP. This demonstrates that the full inoculum potential may not be achieved when sporangia are used as the inoculum propagule.

Defence Responses of Calli and Seeds of Hevea brasiliensis to Zoospores and the Elicitin of Phytophthora palmivora

AbstractThe defence responses of calli and seeds of two cultivars (resistant; BPM‐24 and susceptible; RRIM600) of the rubber tree, Hevea brasiliensis, against zoospores and elicitin purified from its pathogen, Phytophthora palmivora, were investigated. Both zoospores and elicitin induced the biosynthesis of the phytoalexin, scopoletin (Scp), in Hevea calli ranging from 5 × 105 to 4.5 × 106 zoospores/ml and 0.5 to 2 μg elicitin/g fresh weight of calli. At the highest concentration of zoospores (4.5 × 106 zoospores/ml) or elicitin (2 μg/g fresh weight of calli), the rate of Scp production was fastest but then it rapidly decreased and produced lower peak value than detected at the optimum concentration. The decline of Scp level at the highest zoospore/elicitin concentration was correlated to the amount of cell death measured by Evans Blue method. Peroxidase (POD) activities in Hevea calli were also measured using the optimum level of zoospores or elicitin. Induction of Scp preceded the production of Scp POD and o‐dianisidine POD then followed by the guaiacol POD. The Scp and POD accumulations were approximately two to three times higher in the resistance cultivar than those in the susceptible one. As the responses of the calli to elicitin were faster than those to the zoospores, it demonstrates that zoospores require more time to act on the host cells. The pattern of Scp and POD activities monitored in elicitin‐treated Hevea seeds was similar to that of Hevea calli after treating with zoospores or elicitin. Therefore, the callus cultures could be used as a tool for studying other defence mechanisms in H. brasiliensis. The achieved knowledge will be applied to enhance resistance and led to the protection of Hevea young seedlings from the pathogen in the plantation.

First Report of Crown and Root Rot Caused by Phytophthora capsici on Hydroponically Grown Cucumbers in Norway.

In December 2004, symptoms of root and crown rot were observed on cucumbers (Cucumis sativus L.) in a greenhouse in Norway. Cucumbers were the only crop of the greenhouse that used rockwool as a growing substrate in a hydroponical system. The first symptoms were detected in propagation material. One week after planting, symptoms of root and crown rot were observed and approximately 10% of the plants died. Later, losses of 50% in some greenhouses were observed. A yield reduction as much as 65% was estimated in the winter period (January and February). The two main cucumber cultivars planted were Armada and Lopez. In February 2005, Phytophthora capsici (Leonian) (1) was isolated on potato dextrose agar from a sample of cv. Lopez. The isolate produced deciduous, papillate sporangia (occasionally with two or three papilla) and pedicels that were sometimes longer than the sporangia. Sequencing of amplicons of the internal transcribed spacer region (ITS) rDNA and of the mitochondrial cytochrome c oxidase subunit 1 (Cox1) gene (2) confirmed the identification. Three isolates collected through 2005 from the same greenhouse were crossed with tester strains of P. cryptogea. Formation of oogonia and amphigynous antheridia was always observed in crosses with mating type A2; thus, all isolates were the A1 mating type. All three isolates grew well at 35°C and did not produce chlamydospores. A pathogenicity test was performed with one isolate of P. capsici. Four plants of cucumber cvs. Indira and Jessica were grown in a growth chamber at 24°C. Plants at the two-leaf stage were drenched with 20 ml of a zoospore suspension of 106 zoospores per ml per plant. After 18 days, all plants of both cultivars developed symptoms of crown rot or wilted and died. P. capsici was reisolated from inoculated plants of both cultivars. The pathogenicity test was repeated in the same way, but in a greenhouse with temperatures that ranged between 18 and 29°C. In addition, four plants of both cultivars at the four-leaf stage were inoculated with a suspension of 105 zoospores per ml. After 1 week, all plants developed crown rot or were irreversibly wilted, independently of the plant age or the zoospore concentration. To our knowledge, this is the first report of P. capsici in Norway.

Susceptibility of New Zealand flora toPhytophthora ramorumand pathogen sporulation potential: an approach based on the precautionary principle

Phytophthora ramorum, the cause of sudden oak death in the westernUSAand a damaging pathogen in Europe, is a biosecurity threat of unknown magnitude to New Zealand and Australasia because of its presence in traded ornamental plants. Knowledge of potential hosts acting as carriers and of symptoms caused by the pathogen on such hosts will strengthen precautionary quarantine regulations to prevent inadvertent introductions of P. ramorum into the region. Also, the identification of potential hosts will permit determination of areas at risk within countries that do not have P. ramorum. Susceptibility of New Zealand plants, including 17 endemic and three commercial species (Eucalyptus globulus, Pinus radiata and Acacia melanoxylon), as well as two known Rhododendron cultivar hosts, was determined by analysing the size of lesions on inoculated excised leaves and branches, while infectivity was determined by counting sporangia produced on leaves. In order to identify extremely susceptible hosts, seven species were inoculated using three concentrations of zoospores ranging from low (1×102 zoospores/mL) to high (5×103 zoospores/mL). In branch inoculations, P. radiata and Nothofagus fusca were as susceptible as the Rhododendron cultivars. Pseudopanax arboreus, Fuchsia excorticata and one Rhododendron cultivar were equally susceptible in leaf inoculations. However, F. excorticata was the only species with 100% infected leaves, high foliar sporulation and was highly susceptible at all three zoospore concentrations. Leptospermum scoparium was the only asymptomatic foliar host that had high reisolations of the pathogen. F. excorticata, P. radiata, N. fusca, P. arboreus and L. scoparium should be added to the potential host list for P. ramorum and monitored for symptoms and sporulation in gardens and nurseries in theUSAand Europe. As part of a precautionary strategy, these species are suitable candidates for targeted surveillance programs in high-risk incursion areas of New Zealand. Furthermore, the sympatry of foliar hosts with high infectivity and of highly susceptible stem hosts was identified: these areas may be at risk for the development of a forest epidemic.

Effects of temperature, pH and salinity on the infection of Leptolegnia chapmanii Seymour (Peronosporomycetes) in mosquito larvae

The effects of temperature, pH, and NaCl concentrations on the infectivity of zoospores of Leptolegnia chapmanii (Argentine isolate) were determined for Aedes aegypti and Culex pipiens under laboratory conditions. Zoospores of L. chapmanii were infectious at temperatures between 10 and 35 °C but not at 5 or 40 °C. At the permissive temperatures, mortality rates in young instars were much higher than in older instars and larvae of Ae. aegypti were more susceptible to L. chapmanii than larvae of Cx. pipiens. At 25 °C, Ae. aegypti larvae challenged with L. chapmanii zoospores resulted in 100% infection at pH levels ranging from 4 to 10. Larvae of Cx. pipiens exposed to similar pH and zoospore concentrations resulted in increasing mortality rates from 62% to 99% at pH 4 to 7, respectively, and then decreased to 71% at pH 10. Aedes aegypti larvae exposed to L. chapmanii zoospores in NaCl concentrations ranging from 0 to 7 parts per thousand (ppt) at 25 °C resulted in 100% mortality while mortality rates for Cx. pipiens decreases from 96% in distilled water to 31.5% in water with 6 ppt NaCl. Control Cx. pipiens larvae died when exposed at a NaCl concentration of 7 ppt. Vegetative growth of L. chapmanii was negatively affected by NaCl concentrations. These results have demonstrated that the Argentinean isolate of L. chapmanii tolerated a wide range of temperatures, pH, and salinity, suggesting that it has the potential to adapt to a wide variety of mosquito habitats.