Pathogenicity of Phaeoacremonium Species on Grapevines

Several Phaeoacremonium species have been recently described to include some species involved in disease of decline of woody plants and others associated with human infections. Thirteen species are currently reported on grapevines and they are suspected to be involved in Esca and Petri disease. The pathogenic character of new defined species is still unknown and, therefore, pathogenicity studies were conducted in this work. The pathogenicity of the following species was studied on grapevine seedlings and cuttings: Phaeoacremonium aleophilum , P. angustius , P. inflatipes , P. krajdenii , P. mortoniae , P. parasiticum , P. scolyti , P. venezuelense , P. viticola , and Phaeomoniella chlamydospora included as positive control. Two-month-old grapevine seedlings of Vitis vinifera cv. Malvar and cv. Airen were inoculated by watering 10 individual pots with a spore suspension (10⁷ spores/ml) of each Phaeoacremonium species. All inoculated seedlings showed typical symptoms of a vascular disease 2 months after inoculation. Grapevine cuttings of Vitis vinifera cv. Monastrell were vacuum-inoculated with a spore suspension (10⁸ spores/ml) and individually planted. Plants rated after 5 months showed that all Phaeoacremonium species and P. chlamydospora (used as positive control) caused a significant vascular discoloration, while only Phaeomoniella chlamydospora , Phaoeacremonium mortoniae and P. aleophilum caused a significant root weight reduction compared with a non-inoculated control. Phaoeacremonium parasiticum , P. angustius , P. inflatipes and P. venezuelense caused significant foliar symptoms that included interveinal chlorosis and stunted leaves.     

Real-time PCR detection of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum

Phaeomoniella chlamydospora and Phaeoacremonium aleophilum are the two main fungal causal agents of Petri disease and esca. Both diseases cause significant economic losses to viticulturalists. Since no curative control measures are known, proactive defensive measures must be taken. An important aspect of current research is the development of sensitive and time-saving protocols for the detection and identification of these pathogens. Real-time PCR based on the amplification of specific sequences is now being used for the identification and quantification of many infective agents. The present work reports real-time PCR protocols for identification of P. chlamydospora and P. aleophilum. Specificity was demonstrated against purified DNA from 60 P. chlamydospora isolates or 61 P. aleophilum isolates, and no amplification was obtained with 54 nontarget DNAs. The limits of detection (i.e., DNA detectable in 95% of reactions) were around 100 fg for P. chlamydospora and 50 fg for P. aleophilum. Detection was specific and sensitive for P. chlamydospora and P. aleophilum. Spores of P. chlamydospora and P. aleophilum were detected without the need for DNA purification. The established protocols detected these fungi in wood samples after DNA purification. P. chlamydospora was detectable without DNA purification and isolation in 67% of reactions. The detection of these pathogens in wood samples has great potential for use in pathogen-free certification schemes.     

Togninia (Calosphaeriales) is confirmed as teleomorph of Phaeoacremonium by means of morphology, sexual compatibility and DNA phylogeny

Petri disease, or black goo, is a serious disease of vines in most areas where grapevines are cultivated. The predominant associated fungus is Phaeomoniella chlamydospora (Chaetothyriales). Several species of Phaeoacremonium (Pm.) also are associated, of which Pm. aleophilum is the most common. Although no teleomorph is known for Phaeoacremonium, the genus Togninia previously has been linked to phaeoacremonium-like anamorphs. To investigate the possible anamorph-teleomorph connection of Phaeoacremonium to Togninia, anamorphs of Togninia minima, T. fraxinopennsylvanica and T. novae-zealandiae morphologically were compared with Pm. aleophilum and some representative cultures were mated in all combinations. Although no interspecies mating proved fertile, matings between isolates of Pm. aleophilum produced a Togninia teleomorph within 3-4 weeks. Certain field isolates of Pm. aleophilum commonly produced the teleomorph, demonstrating that both mating types can occur in the same vine and thus also explaining the genetic diversity observed for this fungus in some vineyards. To elucidate the phylogenetic relationships among these taxa, isolates were subjected to sequence analysis of the nuclear ribosomal internal transcribed spacers (ITS1, ITS2) and the 5.8S rRNA gene, as well as portions of the translation elongation factor 1 alpha (EF-1α) gene. The generic placement of teleomorphs within Togninia (Calosphaeriales) further was confirmed via phylogenetic analyses of 18S small subunit (SSU) DNA. From these sequences, morphological and mating data, we conclude that T. minima is the teleomorph of Pm. aleophilum, and that it has a biallelic heterothallic mating system. An epitype and mating type tester strains also are designated for T. minima.     

Effect of hot-water treatments in vitro on conidial germination and mycelial growth of grapevine trunk pathogens

In this study, the sensitivity of Cadophora luteo-olivacea, Cylindrocarpon liriodendri, Cn. macrodidymum and eight species of the genus Phaeoacremonium to hot-water treatments (HWTs) in vitro was evaluated. Conidial suspensions and plugs of agar with mycelia were placed in Eppendorf vials and incubated for 30, 45 or 60 min in a hot-water bath at 41, 42, 43, 44, 45, 46, 47, 48 or 49°C for Cylindrocarpon spp. and at 49, 50, 51, 52, 53, 54 or 55°C for Ca. luteo-olivacea and Phaeoacremonium spp. In general, conidial germination and the colony growth rate of all pathogens decreased with increased temperature and time combinations. Cylindrocarpon spp. were more sensitive than Ca. luteo-olivacea and Phaeoacremonium spp. to HWT temperatures. Conidial germination of Ca. luteo-olivacea was inhibited by treatments above 51°C–30 min, while treatments up to 54°C–60 min were necessary to inhibit the mycelial growth. For Cylindrocarpon spp., conidial germination was inhibited by treatments above 45°C–45 min, while treatments above 48°C–45 min were necessary to inhibit the mycelial growth. Regarding Phaeoacremonium spp., treatments up to 54°C–60 min were necessary to completely inhibit both conidial germination and mycelial growth. These results suggest that current HWT protocols at 50°C for 30 min may be sufficient to control Cylindrocarpon spp. However, it would be necessary to develop HWT using higher temperatures to reduce the incidence of Ca. luteo-olivacea and Phaeoacremonium spp. infections.     

PCR-based strategy to detect and identify species of Phaeoacremonium causing grapevine diseases

Species of Phaeoacremonium (especially Phaeoacremonium aleophilum) are associated with two severe diseases in grapevines, Petri disease in young plants and Esca disease in adult plants. Phaeoacremonium species grow slowly on culture medium, and it is difficult to identify these species on the basis of morphological characteristics. Primers Pm1 and Pm2 were designed in the ribosomal DNA internal transcribed spacer (ITS) regions ITS1 and ITS2, respectively. They yielded a single amplicon of 415 bp for nine species of Phaeoacremonium that may occur in grapevines. A nested PCR (using general fungal primers ITS1F/ITS4 in the primary reaction) was developed to detect Phaeoacremonium directly in grapevine wood. Molecular detection was more sensitive than the traditional method of culturing in growth medium was. Identification of Phaeoacremonium species was achieved by digesting the PCR-amplified fragment with the restriction enzymes BssKI, EcoO109I, and HhaI. It was possible to distinguish these species by their restriction fragment length polymorphism patterns, except for Phaeoacremonium viticola and Phaeoacremonium angustius, which had 100% similarity in their ITS region sequences. A species-specific PCR amplification of the partial beta-tubulin gene using the primer pair Pbr4_1/T1 and Pbr8/T1 was necessary to differentiate P. angustius from P. viticola, respectively. An easy and fast protocol was developed to detect and identify species of Phaeoacremonium in a few hours. Primers defined here can be used in a plant nursery sanitation program to produce plants free of Phaeoacremonium spp. Use of healthy grapevine plants in new plantations is the most effective measure to manage Petri disease.     

Development of microsatellite markers for the grapevine fungal pathogen Phaeomoniella chlamydospora

Twenty polymorphic microsatellite markers from microsatellite-enriched genomic DNA of the grapevine fungal pathogen, Phaeomoniella chlamydospora, were developed and characterized. The markers were used to genotype isolates from Australia and from Europe/Eurasia. The number of alleles per locus ranged from two to 11. Gene diversity per locus ranged from 0.08 to 0.63 among Australian isolates, and from 0.2 to 0.77 among isolates from Europe/Eurasia, demonstrating the suitability of these markers for population genetic studies of P. chlamydospora. Eighteen of the 20 markers also amplified a product in the closely related Phaeoacremonium aleophilum.     

Effect of temperature and water activity on in vitro germination of Monilinia spp.

Aims:  This study evaluated the effect of temperature (0–38°C) and water activity ( a _w: 0·87–0·99) on the lag phase prior to germination and the percentage of germination over time for Monilinia laxa , Monilinia fructicola and Monilinia fructigena .
Methods and Results:  More than 80% of viable conidia germinated at 25°C and 0·99 a _w within 2 h for M. fructicola and M. fructigena and 4 h for M. laxa . There was no germination at 38°C, and all three Monilinia spp. germinated at 0°C. At the lowest a _w (0·87), none of the Monilinia spp. was able to germinate at any of the incubation temperatures studied. Whereas at 0·90 a _w, conidia were only able to germinate at 15, 25 and 30°C for the three species studied, except for M. fructicola at 15°C. In contrast, at 0·95, 0·97 and 0·99 a _w, germination occurred at all studied temperatures less 38°C. Generally, the lag phase was longer at low levels of a _w (0·90–095), and differences were more evident as temperatures were far from the optimum (0–5°C).
Conclusions:  Germination and lag phase period were markedly influenced by temperature and a _w, and in general when conditions of temperature and a _w were suboptimal, the lag phase was longer and the percentage of germination was lower.
Significance and Impact of the Study:  Knowledge of the germination requirements of this fungus is important in order to understand their behaviour in natural situations and to provide baseline data required for the construction of new prediction models. Our study might be used to develop a predictive model to understand and control the disease caused by Monilinia spp.     

Characterization of Metarhizium species and varieties based on molecular analysis, heat tolerance and cold activity

Aims:  The genetic relationships and conidial tolerances to high and low temperatures were determined for isolates of several Metarhizium species and varieties.
Methods and Results:  Molecular-based techniques [AFLP and rDNA (ITS1, ITS2 and 5·8S) gene sequencing] were used to characterize morphologically identified Metarhizium spp. isolates from a wide range of sources. Conidial suspensions of isolates were exposed to wet heat (45 ± 0·2°C) and plated on potato dextrose agar plus yeast extract (PDAY) medium. After 8-h exposure, the isolates divided clearly into two groups: (i) all isolates of Metarhizium anisopliae var. anisopliae ( Ma-an ) and Metarhizium from the flavoviride complex ( Mf ) had virtually zero conidial relative germination (RG), (ii) Metarhizium anisopliae var. acridum ( Ma-ac ) isolates demonstrated high heat tolerance ( c . 70–100% RG). Conidial suspensions also were plated on PDAY and incubated at 5°C for 15 days, during which time RGs for Ma-an and Ma-ac isolates were virtually zero, whereas the two Mf were highly cold active (100% RG).
Conclusions:  Heat and cold exposures can be used as rapid tools to tentatively identify some important Metarhizium species and varieties.
Significance and Impact of the Study:  Identification of Metarhizium spp. currently relies primarily on DNA-based methods; we suggest a simple temperature-based screen to quickly obtain tentative identification of isolates as to species or species complexes.     

Effects of environmental factors on virulence of the entomopathogenic fungus, Nomuraea rileyi, against the corn earworm, Helicoverpa armigera (Lep., Noctuidae)

The effects of environmental factors on infection of the entomopathogenic fungus, Nomuraea rileyi , isolated from the corn earworm, Helicoverpa armigera , in Taiwan, to its host insect were studied in the laboratory. The fungus caused higher larval mortality at 20°C than at 30°C when 5 × 10⁶ conidia/ml were sprayed on the fourth instar. However, mortality of the fifth instar injected with 1 × 10³ conidia/larva was not significantly different when the inoculated larvae were incubated from 15 to 30°C. The fungal development in inoculated larvae was best at 20 and 25°C after shifting from 20°C to either lower or higher temperatures. The germination rate was higher at 20 and 25°C than at 30 or 35°C. Conidial germination was better on the wash-off of insect cuticle than on Sabouraud maltose agar with yeast extract. Sporulation on chill-dried cadavers was maximal at 95 or 100% relative humidity than at lower levels of relative humidity. The time required for sporulation was 2 days less at 100% than at 95% relative humidity. Although photoperiod did not affect fifth instar mortality caused by N. rileyi , the median lethal time (LT₅₀) values were shorter upon incubating under light than in darkness. Incubation of infected cadavers under 12 or 24 h light resulted in 20-fold more conidial production than under full darkness. Therefore, illumination is necessary for development of this isolate on insect cadavers.     

Occurrence of Fungal Pathogens Associated with Grapevine Nurseries and the Decline of Young Vines in Spain

The occurrence of fungal grapevine trunk pathogens associated with grapevine nurseries and the decline of young vines in Spain was determined in extensive surveys conducted in nurseries and vineyards with young plants. The presence of Phaeomoniella chlamydospora, Phaeoacremonium aleophilum, Cylindrocarpon spp., Botryosphaeria obtusa and Botryosphaeria spp. was detected in all the surveys that were carried out. This study provides evidence for the presence of these pathogens in Spanish grapevine nurseries. Cylindrocarpon spp., B. obtusa and Botryosphaeria spp., were isolated very early in the planting material production process, nevertheless, P. aleophilum and P. chlamydospora were not detected until the rootstock–scion combinations were planted in the field to develop shoots and roots during summer. The occurrence of trunk disease pathogens in symptomatic young vineyards was also high, suggesting that infected plant material might have been used. Phaeomoniella chlamydospora and P. aleophilum were the main species associated with the decline of young vines, followed by Botryosphaeria spp., Cylindrocarpon spp. and B. obtusa. This research confirmed the importance of fungal grapevine trunk pathogens associated with the decline of young vineyards in Spain and suggested the connection between the presence of these fungi in the nurseries and the disease in the fields.     

Phaeoacremonium spp. and Botryosphaeriaceae spp. Associated with Date Palm (Phoenix dactylifera L.) Decline in Iran

During spring and summer of 2011, a survey was undertaken on some palm groves in the Kerman province (south‐eastern Iran) to determine the fungal pathogens associated with date palm (Phoenix dactylifera L.) decline diseases. Samples were taken from date palm trees showing yellowing, wilting and dieback symptoms. Isolations were made from symptomatic tissues on malt extract agar (MEA) supplemented with 100 mg/l streptomycin sulphate (MEAS). Two species of Phaeoacremonium, Phaeoacremonium aleophilum and Pm. parasiticum, and two species of Botryosphaeriaceae, Botryosphaeria dothidea andDiplodia mutila, were isolated from affected trees and identified on the basis of morphological, cultural and molecular characteristics. Pathogenicity tests were performed on date palm (4‐year‐old potted plants) under greenhouse conditions. Based on the pathogenicity tests, Pm. aleophilum was the most virulent and caused the longest lesions. This is the first report of Pm. aleophilum and B. dothidea and their pathogenicity on date palm tree.     

Formation and Germination of Septoria tritici Secondary Conidia as Affected by Environmental Factors

The effects of medium, isolate, temperature, light and pH on the formation and germination of Septoria tritici secondary conidia were tested. Of the six media tested, the malt–yeast extract agar was the best and generated 1.82 × 10⁹ conidia/plate. The ten isolates tested showed different ability of conidia production. Darkness significantly reduced conidial formation and enhanced the transition of intermediates. The conidial germination and germ tube growth was strongly inhibited at 30°C. The suitable pH for conidial budding in malt–yeast broth (MYB) was between 5 and 9. At pH 2, 10 and 11, almost no new conidia were formed. The number of conidia reached 1.27 × 10⁸ conidia/ml after 7 days in MYB, significantly more than that in potato dextrose broth, wheat leaf extract and H₂O.     

PCR-Based Strategy To Detect and Identify Species of Phaeoacremonium Causing Grapevine Diseases

Species of Phaeoacremonium (especially Phaeoacremonium aleophilum) are associated with two severe diseases in grapevines, Petri disease in young plants and Esca disease in adult plants. Phaeoacremonium species grow slowly on culture medium, and it is difficult to identify these species on the basis of morphological characteristics. Primers Pm1 and Pm2 were designed in the ribosomal DNA internal transcribed spacer (ITS) regions ITS1 and ITS2, respectively. They yielded a single amplicon of 415 bp for nine species of Phaeoacremonium that may occur in grapevines. A nested PCR (using general fungal primers ITS1F/ITS4 in the primary reaction) was developed to detect Phaeoacremonium directly in grapevine wood. Molecular detection was more sensitive than the traditional method of culturing in growth medium was. Identification of Phaeoacremonium species was achieved by digesting the PCR-amplified fragment with the restriction enzymes BssKI, EcoO109I, and HhaI. It was possible to distinguish these species by their restriction fragment length polymorphism patterns, except for Phaeoacremonium viticola and Phaeoacremonium angustius, which had 100% similarity in their ITS region sequences. A species-specific PCR amplification of the partial β-tubulin gene using the primer pair Pbr4_1/T1 and Pbr8/T1 was necessary to differentiate P. angustius from P. viticola, respectively. An easy and fast protocol was developed to detect and identify species of Phaeoacremonium in a few hours. Primers defined here can be used in a plant nursery sanitation program to produce plants free of Phaeoacremonium spp. Use of healthy grapevine plants in new plantations is the most effective measure to manage Petri disease.     

Seed germination ecology of Portulaca oleracea L.: an important weed of rice and upland crops

Portulaca oleracea , a C₄ species, is reported to be a serious weed in 45 crops in 81 countries. Experiments were conducted in the laboratory, the screenhouse and the field to determine the influence of environmental factors on seed germination and seedling emergence of P. oleracea . In the laboratory, germination in the dark was low and was not influenced by the tested temperatures (35/25°C, 30/20°C and 25/15°C alternating day/night temperatures). In the light/dark regime, however, germination was lower at 25/15°C and 35/25°C than at 30/20°C (70%, 75% and 81% germination, respectively). In conditions of 106 mM sodium chloride or −0.34 MPa osmotic potential, seeds germinated to only 50% of maximum germination of the control. Germination was not influenced by buffered pH solutions ranging from 5 to 9. In the screenhouse, germination was greatest for seeds placed on the soil surface, but emergence declined with increasing seed burial depth in soil; no seedlings emerged from the depth of 2 cm. Seedling emergence and seedling dry matter were markedly reduced by the addition of rice residue to the soil surface at rates equivalent to 4 to 6 t ha⁻¹. In the field, seedling emergence of P. oleracea was greater under zero till (ZT) (17–20%) than under minimum tillage (6–10%), a likely reflection of low seed burial and exposure of seeds to light with a ZT system. This study identifies some of the factors enabling P. oleracea to be a widespread weed in the humid tropics, and the information could contribute to improved control strategies.     

Detection of black-foot and Petri disease pathogens in soils of grapevine nurseries and vineyards using bait plants

Background and aims

Little information is currently available regarding the number of species of black-foot and Petri disease pathogens present in soil and their capacity to infect grapevine roots and reach the xylem vessels.

Methods

Seedlings of grapevine rootstock 41-B, and cvs. Bobal and Palomino were planted both in pots containing soil samples collected from commercial vineyards and in nursery fields. Roots and xylem vessels were later analyzed for fungal isolation.

Results

Black-foot pathogens: Ilyonectria alcacerensis, I. macrodidyma, I. novozelandica and I. torresensis were frequently isolated from roots of seedlings grown in all soils evaluated, whereas Petri disease pathogens: Cadophora luteo-olivacea, Phaeoacremonium aleophilum, Pm. parasiticum and Phaeomoniella chlamydospora were only isolated from xylem vessels of seedlings grown in nursery soils, with a low incidence. Ilyonectria alcacerensis, I. novozelandica and I. torresensis were isolated for the first time from grapevines in Spain, and Pm. parasiticum and Ca. luteo-olivacea were detected for the first time in nursery soils.

Conclusions

Our results confirm nursery and vineyard soils as an important inoculum source for black-foot pathogens and demonstrate the presence of several Petri disease pathogens in nursery soils.     

Morphological dormancy in seeds of the autumn-germinating shrub Lonicera caerulea var. emphyllocalyx (Caprifoliaceae)

To better understand the germination ecophysiology of the genus Lonicera , the dormancy class, temperature requirements for embryo growth and radicle emergence and phenology of seedling emergence were determined for Lonicera caerulea var. emphyllocalyx . At maturity, seeds have an underdeveloped embryo (approximately 28% of the length of full-grown embryos). Embryos in fresh seeds grew to full length at 15, 20, 20/10 and 25/15°C within 3 weeks, but failed to grow at ≤ 10°C and at 30°C. Radicles emerged from 86–100% of freshly matured seeds in light at 15, 20, 20/10 and 25/15°C within 28 days, but failed to emerge at 10°C. Radicles emerged equally well in a 12 h photoperiod and in continuous darkness at 25/15°C. Rapid embryo growth and germination over a range of conditions indicate that seeds of this taxon have morphological dormancy (MD); this is the first report of MD in a species of Lonicera . Seeds are dispersed in summer, at which time high temperatures promote embryo growth. Embryos grow to the critical length for germination in approximately 1 month; the peak of seedling emergence occurs in early autumn. Radicles emerged within 2 months from 98% of seeds buried at soil depths of 2 cm and 10 cm in the field in August in Sapporo, Japan; thus, seeds have no potential to form a persistent soil seed bank. However, seeds sown too late in autumn for embryos to grow remained viable and germinated the following summer when temperatures were high enough to promote embryo growth.     

The temperature response of photosynthesis in tobacco with reduced amounts of Rubisco

The reasons for the decline in net CO₂ assimilation ( A ) above its thermal optimum are controversial. We tested the hypothesis that increasing the ratio of Rubisco activase to Rubisco catalytic site concentration would increase the activation state of Rubisco at high temperatures. We measured photosynthetic gas exchange, in vivo electron transport ( J ) and the activation state of Rubisco between 15 and 45 °C, at 38 and 76 Pa ambient CO₂, in wild-type (WT) and anti- rbc S tobacco. The Rubisco content of the anti- rbc S lines was 30% (S7-1) or 6% (S7-2) of WT, but activase levels were the same in the three genotypes. Anti- rbc S plants had lower A than WT at all temperatures, but had a similar thermal optimum for photosynthesis as WT at both CO₂ levels. In WT plants, Rubisco was fully activated at 32 °C, but the activation state declined to 64% at 42 °C. By contrast, the activation state of Rubisco was above 90% in the S7-1 line, between 15 and 42 °C. Both A and J declined about 20% from T _opt to the highest measurement temperatures in WT and the S7-1 line, but this was fully reversed after a 20 min recovery at 35 °C. At 76 Pa CO₂, predicted rates of RuBP regeneration-limited photosynthesis corresponded with measured A in WT tobacco at all temperatures, and in S7-1 tobacco above 40 °C. Our observations are consistent with the hypothesis that the high temperature decline in A in the WT is because of an RuBP regeneration limitation, rather than the capacity of Rubisco activase to maintain high Rubisco activation state.     

Temperature conditions control embryo growth and seed germination of Corydalis solida (L.) Clairv., a temperate forest spring geophyte

Spring is often the most suitable period for seedling establishment of temperate woodland species. Different physiological mechanisms resulting in spring emergence have evolved in seeds of such plants. The aim of this study was to determine the requirements for breaking dormancy and for seed germination of the European perennial spring geophyte Corydalis solida (Fumariaceae). Ripe seeds of C. solida contain an underdeveloped embryo, consisting of no more than a clump of cells. As a consequence, the embryo has to differentiate and grow to a critical length before germination can occur. In nature, seeds are dispersed in spring, while growth of the embryo starts in the autumn and continues in winter. Germination starts in late winter, immediately after embryo growth is completed, resulting in seedling emergence in the following spring. Experiments in controlled conditions showed that temperature is the main factor controlling dormancy and germination. Incubation at autumn temperatures (15/6 °C; 20/10 °C) for at least 8 weeks is required to initiate embryo growth, while a transfer to 5 °C is needed for completion of embryo growth and germination. Growth of the embryo of C. solida occurs at different temperatures over an extended period, a feature typical of temperate forest herbs. Our results indicate that the dormancy mechanism in seeds of C. solida is very similar to mechanisms in other Corydalis species studied thus far, suggesting that stasis in the dormancy trait has occurred.     

Seed dormancy and germination behaviour in two Euphrasia species (Orobanchaceae) occurring in the Swiss Alps

The optimal time for germination of a seed depends on environmental conditions of its habitat, the life cycle of the germinating plant, and the conditions for successful establishment, growing and reproduction. We studied the germination behaviour of the alpine annual Euphrasia minima and an alpine ecotype of E. salisburgensis in a lowland garden experiment. Seeds of both species and their hybrids germinated at constant (5 °C) and at varying temperatures (3–10 °C), and never before spring after seed ripening. Germination was spread over 3 years, which suggests that the seeds formed a persistent seed bank. The two species together with E. minima and E. minima hybrids differed significantly in the germination rate in the first and second spring.  © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 156 , 649–656.     

Competitive ability of the antagonists Ulocladium atrum and Gliocladium roseum at temperatures favourable for Botrytis spp. development

Ulocladium atrum and Gliocladium roseum are fungal antagonists capable of suppressing sporulation of Botrytis spp. on dead plant parts. The effect of temperature (3 to 36 °C) on antagonist conidial germination and mycelial growth was assessed on agar. In addition conidial germination of U. atrum was measured on dead lily leaves. The optimum temperature of both antagonists for both conidial germination and mycelial growth was between 27 and 30 °C. U. atrum was less affected by lower temperatures than G. roseum. At optimum temperature, 50% of conidia of U. atrum and G. roseum germinated within 2.6 and 10.0 hrs, respectively. At low sub-optimal temperatures (6 °C), 50% of conidia germinated within 18 and 96 hours, respectively.In bioassays on dead onion leaves, U. atrum suppressed sporulation of B. cinerea and B. aclada at all temperatures tested (6 to 24 °C) by more than 85%. On dead cyclamen leaves, G. roseum was more efficient than U. atrum at 21 and 24 °C but, in contrast to U. atrum, showed no antagonistic activity at temperatures below 21 °C. On dead hydrangea leaves, U. atrum significantly reduced sporulation of B. cinerea at temperatures as low as 3 and 1 °C. Under Dutch growing conditions, the mean air temperature during leaf wetness periods in onion and lily fields was 15 °C with temperatures only occasionally above 20 °C. In greenhouse crops of cyclamen, the mean temperature during high humidity periods was 17 °C. It is therefore concluded that U. atrum is better adapted than G. roseum to temperatures which occur in the field, in greenhouse crops such as cyclamen, or during cold storage of plant stocks.