The Effect of Exposure to Decreasing Relative Humidity on the Viability of Phytophthora ramorum sporangia

Sporangia of three isolates of Phytophthora ramorum representing three different clonal lineages were subjected to relative humidity (RH) levels between 80 and 100% for exposure periods ranging from 1 to 24 h at 20°C in darkness. Plastic containers (21.5 × 14.5 × 5 cm) were used as humidity chambers with 130 ml of glycerine solution added to each container. Glycerine concentrations corresponded to 100, 95, 90, 85 and 80% RH based on refractive index measurements. Sporangia suspensions were pipeted onto nitrile mesh squares (1.5 × 1.5 cm, 15 micron pore size) which were placed in the humidity chambers and incubated at 20°C in darkness. Following exposure periods of 1, 2, 4, 8, 12 and 24 h, mesh squares were inverted onto Petri dishes of selective medium and sporangia germination assessed after 24 and 48 h. At 100% RH, we observed a mean value of 88% germination after 1 h exposure declining to 18% germination following 24 h incubation. At 95% RH, a steeper decline in germination was noted, with means ranging from 79% at 1 h to less than 1% at 24 h exposure. At 90% RH, no germination was noted after 8 or more h exposure, and values were 57%, 22% and 3% germination for the 1, 2 and 4 h exposures, respectively. Germination was only observed at 1 h exposure for both the 85% RH treatment (52% germination) and the 80% RH treatment (38% germination). The three isolates responded similarly over the range of RH values tested. The germination response of P. ramorum sporangia to RH values between 80% and 100% was comparable to that reported for other Phytophthora species. Knowledge of conditions that affect P. ramorum sporangia germination can shed light on pathogenesis and epidemic potential and lead to improved control recommendations.     

Inoculum Density Relationships for Infection of Some Eastern US Forest Species by Phytophthora ramorum

Our objectives were to establish inoculum density relationships between P. ramorum and selected hosts using detached leaf and whole‐plant inoculations. Young plants and detached leaves of Quercus prinus (Chestnut oak), Q. rubra (Northern red oak), Acer rubrum (red maple), Kalmia latifolia (mountain laurel) and Rhododendron ‘Cunningham's White’ were dip‐inoculated with varying numbers of P. ramorum sporangia, and the total number of diseased and healthy leaves recorded following incubation at 20°C and 100% relative humidity. Calibration threshold estimates for obtaining 50% infected leaves based on linear analysis ranged from 36 to 750 sporangia/ml for the five hosts. Half‐life (LD50) estimates (the number of spores for which the per cent of diseased leaves reaches 50% of its total) from asymptotic regression analysis ranged from 94 to 319 sporangia/ml. Statistically significant differences (P = 0.0076) were observed among hosts in per cent infection in response to increased inoculum density. Inoculum threshold estimates based on studies with detached leaves were comparable to those obtained using whole plants. The results provide estimates of inoculum levels necessary to cause disease on these five P. ramorum hosts and will be useful in disease prediction and for development of pest risk assessments.     

The biology of Peronospora viciae on pea: laboratory experiments on the effects of temperature, relative humidity and light on the production, germination and infectivity of sporangia

Germination of Peronospora viciae sporangia washed off infected leaves varied from 20% to 60%. Sporangia shaken off in the dry state gave 11–19% germination. Most sporangia lost viability within 3 days after being shed, though a few survived at least 5 days. Infected leaves could produce sporangia up to 6 weeks after infection, and sporulating lesions carried viable sporangia for 3 weeks. Sporangia germinated over the range 1–24 °C, with an optimum between 4 and 8 °C. Light and no effct. The temperature limits for infection were the same as for germination, but with an optimum between 12 and 20 °C. A minimum leaf-wetness period of 4h was required, and was independent of temperature over the range 4–24 °C. Maximum infectivity occurred after 6h leaf wetness at temperatures between 8 and 20 °C. Infection occurred equally in continuous light or in darkness. After an incubation period of 6–10 days sporangia were produced on infected leaves at temperatures between 4 and 24 °C, with an optimum of 12–20 °C. Exposure to temperatures of 20–24 °C for 10 days reduced subsequent sporulation. Sporangia produced at suboptimal temperatures were larger, and at 20 °C. smaller, than those produce at 12–16 °C. Viability was also reduced. No sporangia were produced in continuous light, or at relative humidities below 91%. For maximum sporulaiton an r.h. of 100% was required, following a lower r.h. during incubation. Oospores wre commonly formed in sporulating lesions, and also where conditons limited or prevented sporulation. The results are discussed briefly in relaiton to disease development under field conditions.     

Enhanced Recovery of Phytophthora ramorum from Soil Following 30 Days of Storage at 4°C

Chlamydospores of Phytophthora ramorum were used to infest field soil at densities ranging from 0.2 to 42 chlamydospores/cm³ soil. Recovery was determined by baiting with rhododendron leaf discs and dilution plating at time 0 and after 30 days of storage at 4°C, as recommended by USDA‐APHIS. Baiting was slightly more sensitive than dilution plating in recovering P. ramorum immediately following infestation of soil and allowed detection from samples infested with as little as 0.2 chlamydospores/cm³ compared with 1 chlamydospore/cm³ for dilution plating. After 30 days of infested soil storage at 4°C, P. ramorum was detected at significantly (P = 0.05) higher levels than at time 0 with both recovery methods. The results indicate that storage of P. ramorum‐infested soil at 4°C may allow for pathogen activity, such as sporangia production, which may enhance recovery from soil.     

The effect of salinity on the survival,growth, sporulation and infection of Phytophthora ramorum

Phytophthora ramorum has been found in waterways outside infested nurseries, but little is known about its behavior in water. This study examined the effect of salinity on survival, growth, sporulation, and infection. P. ramorum survival and growth was negatively correlated with salt concentration (range of 0–45 g l⁻¹), but showed a level of tolerance even at 45 g l⁻¹. No sporangia were observed in cultures with higher than 20 g l⁻¹ of salt and zoospores were not released from sporangia above 14 g l⁻¹. Water sources with different salinity were used to understand the environment where P. ramorum can survive and infect host material. Water from natural bodies and water amended with different salt concentrations were added to P. ramorum-infested sand and baited with rhododendron leaf disks. Infection decreased with increasing salt concentration and increased with higher initial concentration of P. ramorum. This research helps to better understand the effects of water quality on survival and infectivity of P. ramorum, expanding the potential survey range.     

New Insights in the Life Cycle and Epidemics of Phytophthora porri on Leek

White tip, caused by Phytophthora porri, is a devastating disease in the autumn and winter production of leek (Allium porrum) in Europe. This study investigated the disease cycle of P. porri in laboratory and field conditions. Oospores readily germinated in the presence of non‐sterile soil extract at any temperature between 4 and 22°C, with the formation of sporangia which released zoospores. The zoospores survived at least 7 weeks in water at a temperature range of 0 till 24°C. Microscopic examinations revealed that zoospores encysted and germinated on the leek leaf surface and hyphae entered the leaf directly through stomata or by penetrating via appressoria. Oospores were formed in the leaves within 6 days, while sporangia were not produced. By monitoring disease progress in fields with a different cropping history of leek, it could be deduced that P. porri survives in soil for up to 4 years. Disease progress during three consecutive years was correlated with average daily rainfall in the infection period. Disease incidence on leek was reduced when rain splash was excluded by growing the plants in an open hoop greenhouse. Based on these findings, we propose a disease cycle for P. porri in which oospores germinate in puddles, and zoospores reach the leaves by rain splash and survive in water in the leaf axils, from where they infect the plant by direct penetration or via stomata. When conditions become unfavourable, oospores are produced in the leaves which again reach the soil when leaves decay. Secondary spread of the disease by sporangia does not seem to be important.     

The Influence of Temperature on Phytophthora infestans (Mont.) de Bary

The influence of temperature on the growth rate, sporulation density and zoospore release of Phytophthora infestans, cultivated on rye agar, has been studied. Temperature significantly influenced all the features of the fungus mentioned above. The highest yield of sporangia per 1 cm² of aerial mycelium occurred at 24°C while the highest percentage of sporangia releasing zoospores was observed when the fungus was grown at 15 °C. When considering the size of the fungal colony the highest production of sporangia was obtained at 20°C. It was concluded that the temperature at which the fungus was cultured predetermined the way it germinated.     

Effects of temperature on germination and hyphal growth from ascospores of A-group and B-group Leptosphaeria maculans (phoma stem canker of oilseed rape)

Ascospores of both A‐group and B‐group Leptosphaeria maculans germinated at temperatures from 5–20°C on distilled water agar or detached oilseed rape leaves. After 2 h of incubation on water agar, some A‐group ascospores had germinated at 10–20°C and some B‐group ascospores had germinated at 5–20°C. The percentages of both A‐group and B‐group ascospores that had germinated after 24 h of incubation increased with increasing temperature from 5–20°C. The observed time (Vo₅₀) which elapsed from inoculation until 50% of the spores had germinated was shorter for B‐group than for A‐group ascospores. Germ tube length increased with increasing temperature from 5–20°C for both ascospore groups. Germ tubes from B‐group ascospores were longer than germ tubes from A‐group ascospores at all temperatures tested, but the mean diameter of germ tubes from A‐group ascospores (1.8 μm) was greater than that of those from B‐group ascospores (1.2μm) at 15°C and 20°C. The average number of germ tubes produced from A‐group ascospores (3.8) was greater than that from B‐group ascospores (3.1) after 24 h of incubation at 20°C, on both water agar and leaf surfaces. Germ tubes originated predominantly from interstitial cells or terminal cells of A‐group or B‐group ascospores, respectively, on both water agar and leaf surfaces. Hyphae from A‐group ascospores grew tortuously with extensive branching, whilst those from B‐group ascospores were predominantly long and straight with little branching, whether the ascospores were produced from oilseed rape debris or from crosses between single ascospore isolates, and whether ascospores were germinating on water agar or leaf surfaces.     

Prevailing sea surface temperatures inhibit summer reproduction of the kelp Laminaria digitata at Helgoland (North Sea)

The impact of abiotic factors on kelp sporophyte reproduction has rarely been investigated. Laminaria digitata (Hudson) J.V. Lamouroux is one of the few summer fertile Laminaria species worldwide and reproduction is subjected to relatively high water temperatures. We investigated the impact of prevailing summer temperatures (~18°C in August) on the induction of sporangia, meiospore release, and germination at the island of Helgoland (North Sea). At Helgoland, fertile sporophytes are found between April and December with a maximum in late summer. While released meiospore numbers were constant between June and October, germination rates decreased significantly in summer. Short‐term exposure of mature sori to 17°C–22°C induced a significantly higher meiospore release indicating enhancement of sporulation by elevated temperatures. Induction of sporangia on vegetative blade disks was not possible at 20°C, and fertility was only 20% at 18°C–19°C, but it was 100% in cool temperatures of 1°C–10°C. It was shown for the first time in a kelp species that “sporogenesis” is the life‐cycle process with the narrowest temperature window compared to growth or survival of the sporophyte or reproduction, growth, and survival of the gametophyte. We incorporated several parameters (induction time, fertile area, and relative fertility) into a “Reproductive efficiency index.” This indicates that sporogenesis of L. digitata is a cold‐adapted process with an optimum at (5)–10°C. The results show that the population at Helgoland is at its reproduction limit despite the existence of other geographically more southerly located populations.     

Effects of Inoculum Density and Wounding on Stem Infection of Three Eastern US Forest Species by Phytophthora ramorum

Seedlings of three Eastern US forest species Quercus rubra (northern red oak), Quercus prinus (chestnut oak) and Acer rubrum (red maple) were inoculated by applying Phytophthora ramorum sporangia to stems at different inoculum densities with and without wounding. Disease occurred in all treatments involving wounds, and no disease was observed in unwounded treatments. Younger seedlings (2–3 years old) did not differ significantly from older seedlings (5–6 years old) in disease incidence, but older seedlings sustained smaller lesions compared with younger seedlings. For both old and young seedlings, disease on wounded stems was observed down to the lowest sporangia concentration utilized (500 sporangia/ml for old seedlings and 100 sporangia/ml for young seedlings). The results show that in the presence of wounding, even very low sporangia concentrations can result in disease, and further suggest that wounding caused by insects and other factors may play an important role in P. ramorum epidemiology in forest environments.     

Effect of Variation of Sporulation Time and Temperature on Thermostability of Bacillus cereus Spores

The optimum temperature for sporulation of a strain of Bacillus cereus was estimated at 30°–35°C, where the maximum yield of spores was obtained between 18 and 24 hours’ incubation. Sporulation was more rapid, but less extensive at 40°C and did not occur at all at 45°C. The heat resistance of the spores increased with the sporulation temperature from 20° to 40°C. The spores appear to be more susceptible to heat destruction in the early stage of spore production than after further incubation.     

High-level expression and characterization of a highly thermostable chitosanase from <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The sequence of an endo-chitosanase gene (CSN) from Aspergillus fumigatus was optimized based on the preferred codons of Pichia pastoris and synthesized in vitro through overlapping PCR (CSN-P). The gene was cloned into a yeast expression vector, pHBM905A, and secretorily expressed in Pichia pastoris GS115. The yield of CSN-P reached ~3 mg/ml with a high-density fermentation in a 14 l fermenter and the enzyme activity was ~25,000 U/ml. The enzyme had half-lives of 2.5 h at 80°C, 1 h at 90°C and 32 min at 100°C. It retained 70% activity after incubation with 10 M urea at room temperature for 30 min. This enzyme was used for a large-scale preparation of oligosaccharides: 3 g enzyme converted 200 kg chitosan into oligosaccharides in 24 h at 60°C.     

Viability of Rhodococcus equi and Parascaris equorum Eggs Exposed to High Temperatures

There is great concern about the potential pathogen contamination of horse manure compost spread in the same fields horses graze in. To ensure that pathogen destruction occurs, temperatures need to be sufficiently high during composting. Here, we investigated the survival rate of two marker organisms, Rhodococcus equi and Parascaris equorum eggs, exposed to temperatures potentially encountered during horse manure composting. Our results show that the time required to achieve a 1 log10 reduction in R. equi population (D-value) are 17.1 h (±1.47) at 45°C, 8.6 h (±0.28) at 50°C, 2.9 h (±0.04) at 55°C and 0.7 h (±0.04) at 60°C. For P. equorum eggs we show that at 45 and 50°C, 2 log10 reduction of viability is reached between 8 and 24 h of incubation and that it takes less than 2 h at 55 and 60°C to achieve a viability reduction of 2 log10. These results are useful for identifying composting conditions that will reduce the risk of environmental contamination by R. equi and P. equorum eggs.     

Climatic factors influencing spore production in Alternaria brassicae and Alternaria brassicicola

Sporulation in A. brassicae and A. brassicicola on naturally-infected leaf discs of oilseed rape and cabbage required humidities equal to or higher than 91.5% and 87% r.h. respectively. The optimum temperatures for sporulation were 18–24°C for A. brassicae and 20–30°C for A. brassicicola at which temperatures both fungi produced spores in 12–14 h. Above 24°C sporulation in A. brassicae was inhibited. At sub-optimal temperatures sporulation times for A. brassicicola were significantly longer than for A. brassicae with the differences increasing with decrease in temperature. Interrupting a 16-h wet period at 20°C with a period of 2 h at 70% or 80% r.h. did not affect sporulation in either fungus but a dry interruption of 3–4 h inhibited sporulation in both. Exposure of both fungi to alternating wet (18 h at 100% r.h., 20°C) and dry periods (6 or 30 h at 5565% r.h., 20°C) did not affect the concentration of spores produced in each wet period. Sporulation times were not affected by either the host type of the age of the host tissue. White light (136 W/m²) inhibited sporulation in A. brassicae with the degree of inhibition increasing with increasing light intensity. The effect of light on sporulation in A. brassicicola was not tested.     

Freezing tolerance of <Emphasis Type="Italic">Porphyra yezoensis</Emphasis> (Bangiales,Rhodophyta) gametophyte assessed by chlorophyll fluorescence

In January and February 2010, heavy sea ice formed along the coast of the Bohai Sea and the northern Yellow Sea, China. Intertidal organisms were subjected to serious freezing stress. In this study, we investigated the freezing tolerance of the upper intertidal economic seaweed Porphyra yezoensis. The maximum photochemical efficiency of PS II (F _v/F _m) in undehydrated thalli remained high after 24 h at −2°C and that in dehydrated thalli decreased in a proportion to thallial water loss. F _v/F _m dropped sharply after 24 h at −20°C, regardless of absolute cellular water content (AWC). The F _v/F _m in frozen thalli recovered rapidly at 0–20°C. A wide range of water loss in the thalli enhanced their tolerance to freezing. F _v/F _m values in undehydrated thalli dropped sharply after 3 d at −2°C or 10 d at −20°C while those in dehydrated thalli (20–53% AWCs) remained at high levels after 9 d at −2°C or 30 d at −20°C. These results indicate that P. yezoensis has high freezing tolerance by means of dehydration during the ebb tide and rapid recovery of F _v/F _m from freezing. A strategy of P. yezoensis industry to avoid heavy loss during freezing season is discussed based on these findings.     

Temperature‐dependent development of the European larch bark beetle,Ips cembrae

The temperature‐dependent development of the European larch bark beetle, Ips cembrae, was studied under long‐day conditions L:D 16:8 at three temperature regimes, 15°C, 20°C and 25°C, using the sandwich plate method. By observing the individual developmental progress, we calculated the developmental times and lower developmental thresholds of one entire generation and various ontogenetic stages. The mean developmental time of one generation was about 120, 64 and 37 days at 15°C, 20°C and 25°C, respectively. The egg stage comprised about 9% of the total development or about 16% of the pre‐imaginal development. The larval stages took about 39% of the entire and about 66% of the pre‐imaginal development. The pupal stage needed about 11% of the total or about 18% of the pre‐imaginal development. The lower developmental threshold for one generation was 11.2°C. The egg stage had the highest lower developmental threshold of 12.0°C, the pupa the lowest of 9.8°C and the total larval stages showed a value of 11.2°C. The thermal requirements for I. cembrae have never been studied in detail before. The results will be a valuable contribution for monitoring and risk assessment models to estimate the beetle's phenology and its potential impacts on forest ecosystems under changing climate conditions.     

Impact of increasing temperatures and exposure duration on egg hatch in the hemlock looper,Lambdina fiscellaria

As the growing season is expected to begin earlier under climate change, insects should initiate reproduction several days or weeks earlier than they used to. In eastern Canada, hemlock looper (HL) Lambdina fiscellaria (Guenée) (Lepidoptera: Geometridae) females generally oviposit in September, with eggs entering an obligatory diapause quickly after their deposition. We therefore simulated an early start of the HL reproduction cycle of 2, 4, 6, or 8 weeks to examine the extent to which freshly laid eggs from two populations (island and mainland) can withstand exposure to four temperature conditions (15, 20, 25 °C, or fluctuating temperature in an outdoor insectary), with all treatments ending on 1 September 2007. On this date, half the eggs from each population were immediately incubated at 15 °C, while the rest were stored in an outdoor insectary until their incubation at 15 °C the following spring. In a separate experiment, the effect of temperature on pre‐diapause duration was determined from the number of days required for eggs to change colour after oviposition. The pre‐diapause phase was completed faster as temperature increased. Regardless of incubation date and population, percent hatch decreased significantly after 6‐8 weeks of exposure to 25 °C or in the outdoor insectary. Under most treatments, the odds of dying as pharate larvae increased with exposure duration. When eggs were incubated at 15 °C immediately after treatment, time to hatch and diapause duration remained constant over treatments, except at 25 °C when they both decreased. After 8 weeks of exposure to 15 or 20 °C, eggs transferred outdoors were more likely to hatch precociously than those exposed to 25 °C or insectary conditions. Globally, mortality seemed greater among eggs stored outdoors than among those kept indoors. Most eggs that survived the winter hatched synchronously after incubation in spring. Overall, larger eggs from the island population survived better than smaller eggs from the mainland population.     

Enhanced inhibition of Aspergillus niger on sedge (Lepironia articulata) treated with heat‐cured lime oil

Aims

This study aimed to examine heat curing effect (30–100°C) on antifungal activities of lime oil and its components (limonene, p‐cymene, β‐pinene and α‐pinene) at concentrations ranging from 100 to 300 μl ml^?1 against Aspergillus niger in microbiological medium and to optimize heat curing of lime oil for efficient mould control on sedge (Lepironia articulata).

Methods and Results

Broth dilution method was employed to determine lime oil minimum inhibitory concentration, which was at 90 μl ml^?1 with heat curing at 70°C. Limonene, a main component of lime oil, was an agent responsible for temperature dependencies of lime oil activities observed. Response surface methodology was used to construct the mathematical model describing a time period of zero mould growth on sedge as functions of heat curing temperature and lime oil concentration. Heat curing of 90 μl ml^?1 lime oil at 70°C extended a period of zero mould growth on sedge to 18 weeks under moist conditions.

Conclusions

Heat curing at 70°C best enhanced antifungal activity of lime oil against A. niger both in medium and on sedge.

Significance and Impact of the Study

Heat curing of lime oil has potential to be used to enhance the antifungal safety of sedge products.     

Responses of Aquatic Fungal Communities on Leaf Litter to Temperature‐Change Events

Increases of extreme weather events are predicted to occur with ongoing climate change, but impacts to freshwaters have rarely been examined. We assessed the effects of temperature on leaf‐litter associated fungi by exposing leaves colonized in a stream to 18 °C (control), 25 °C, or 18 °C after freezing. Treatments altered fungal dominance on leaves; Lunulospora curvula sporulation was stimulated by increased temperature and stopped by the freeze‐thaw treatment. Fungal biomass and diversity decreased at 18 °C after freezing, but not at 25 °C. Leaf decomposition was retarded by the freeze‐thaw treatment (k = –0.024 day^–1) and stimulated at 25 °C (k = –0.069 day^–1). Results suggest that occasional freezing may constrain fungal diversity and their ecological functions, while warming appears to accelerate plant‐litter decomposition in streams. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

A rapid effect of applied brassinolide on abscisic acid concentrations in <Emphasis Type="Italic">Brassica napus</Emphasis> leaf tissue subjected to short-term heat stress

Three-week old canola (Brassica napus L.) seedlings grown at 20/16°C (day/night) were subjected to short-term (4 and 8 h) heat stress (45°C) or maintained at a normal temperature of 20°C. Half of the plants under each treatment received a 10⁻⁶ M solution of brassinolide (BL) 1 h prior to beginning the temperature treatments. The concentration (ng/g dry weight) of endogenous abscisic acid (ABA) was subsequently determined in young leaves via the stable isotope dilution method. Applied BL had no effect on endogenous ABA for plants maintained at normal temperatures. However, ABA concentration was significantly elevated by heat stress alone and doubled by heat stress + BL. These results suggest that the well-known enhancement of tolerance to high temperature stress that can be obtained by BL or 24-epi-BL applications may be caused by a brassinosteroid-induced elevation in endogenous ABA concentration.