Spatial Distribution of Venturia inaequalis Airborne Ascospores in Orchards

ABSTRACT Apple scab (Venturia inaequalis) causes important economic losses in many apple production areas of the world. The disease is controlled by numerous fungicide applications regardless of the presence of ascospores in the orchard. Airborne ascospore concentration (AAC) can be measured in real time to time fungicide applications. However, the level of heterogeneity of the AAC in commercial orchards was unknown. Consequently, the spatial distribution of V. inaequalis ascospores was studied in a commercial apple orchard of 0.43 ha. The potential ascospore dose (PAD) and AAC were measured in 40 quadrats each of 108 m(2). In each quadrat, the AAC was monitored during the major rain events in spring 1999 and 2000 using spore samplers. The variance-to-mean ratio for the PAD and for most of the AAC sampling dates was >1, indicating an aggregated pattern of distribution. None of the frequency distributions of the most important ascospore ejection events followed the Poisson probability distribution, indicating that the pattern of distribution was not random. For all events, AAC had an aggregated pattern of distribution as suggested by the negative binomial distribution. The PAD followed neither the Poisson nor the negative binomial distribution. Geostatistical analyses confirmed the aggregated pattern of distribution. The cultivars had an effect on the PAD and AAC distribution pattern, but both PAD and AAC were not uniformly distributed within a block of the same cultivar. Therefore, the number, location, and height of samplers required to estimate AAC in orchards need to be investigated before using information on AAC for decision making.     

A model simulating deposition of Venturia inaequalis ascospores on apple trees*

Based on existing physical theories and models, a dynamic model estimating the concentration of Venturia inaequalis ascospores in the orchard air and their deposition on apple leaves was elaborated. The model produces two main outputs: number of ascospores deposited per leaf and proportion of ascospores discharged from pseudothecia deposited onto the leaves. The model has a relatively simple structure, and computations are based on few algorithms, which are implemented on an electronic data sheet of common use. Nevertheless, it preserves the accuracy of more complex physical models reasonably well. The model includes the effect of meteorological conditions and horticultural characteristics, and thus provides information for each type of orchard. Few input variables are required: wind speed and rainfall rate can be measured in standard meteorological stations; horticultural characteristics of the orchard can be determined for each type of orchard. The model produces conservative estimates of ascospore deposition, because it assumes a complete retention of the spores deposited by rainfall and does not consider either deposition on stems and flowers or the spatial distribution of plant surfaces. After further validation under orchard conditions, the model will be used to obtain better estimates of scab infection risk in current scab control strategies.     

The relative importance of conidia and ascospores as primary inoculum of Venturia inaequalis in a southeast England orchard

Apple scab, caused by Venturia inaequalis, can lead to large losses of marketable fruit if left uncontrolled. The disease appears in orchards during spring as lesions on leaves. These primary lesions are caused by spores released at bud burst from overwintering sources; these spores can be sexually produced ascospores from the leaf litter or asexual conidia from mycelium in wood scab or within buds. The relative importance of conidia and ascospores as primary inoculum were investigated in an orchard in southeast England, UK. Potted trees not previously exposed to apple scab were placed next to (c. 1 m) orchard trees to trap air‐dispersed ascospores. Number and position of scab lesions were assessed on the leaves of shoots from both the potted trees (infection by airborne ascospores) and neighbouring orchard trees (infection by both ascospores and splash‐dispersed, overwintered conidia). The distribution and population similarity of scab lesions were compared in the two tree types by molecular analysis and through modelling of scab incidence and count data. Molecular analysis was inconclusive. Statistical modelling of results suggested that conidia may have contributed approximately 20–50% of the primary inoculum in early spring within this orchard: incidence was estimated to be reduced by 20% on potted trees, and lesion number by 50%. These results indicate that, although conidia are still a minority contributor to primary inoculum, their contribution in this orchard is sufficient to require current management to be reviewed. This might also be true of other orchards with a similar climate.     

渭北旱塬苹果黑星病的初侵染来源

 苹果黑星病是世界各苹果产区的重要病害之一,也是我国渭北旱塬苹果产区的新病害。近5年调查研究证明,渭北旱塬苹果落叶上分生孢子数量随时间推移呈急剧下降趋势,在春季苹果芽鳞萌动之前,分生孢子呈无色透明薄片状,均已失去存活力,芽鳞内外检测到分生孢子的数量很少,且均已丧失萌发力。果园中孢子捕捉结果表明,子囊孢子出现在先,分生孢子在后,两者出现时间相差15d以上,且捕捉到分生孢子的日期是在黑星病发病以后。对洛川县、白水县、长武县、永寿县、泾川县和平凉市等地苹果黑星病普查中未发现发病枝条,以菌丝体在枝条上越冬的可能性极小。综合以上试验结果,初步确定子囊孢子是渭北旱塬苹果黑星病的初侵来染,分生孢子不能越冬。     

Spatial heterogeneity,incidence-incidence and incidence-lesion density relationship of apple scab (<Emphasis Type="Italic">Venturia inaequalis</Emphasis>) in managed orchards

The spatial pattern of apple scab was characterized using 10 years of disease incidence and lesion density data collected in managed orchards located in Quebec, Canada. Distributional analyses indicated that scab incidence was better characterized by the beta-binomial than the binomial distribution in 53 and 65% of the data sets at the leaf and shoot scales, respectively. Median values of the beta-binomial parameter θ, a measure of small-scale aggregation, were near 0 (0.003 and 0.028) at both sampling scales, indicating that disease incidence was close to being randomly distributed (low degree of aggregation). For lesion density, the negative binomial distribution fitted the data better than the Poisson distribution in 86% of the data sets at the leaf scale. The median value of the index of dispersion k was 0.068, indicating that aggregation was present. For all apple scab measurements, the power law models provided a good fit to the data. The estimated slope and intercept parameters were significantly greater than 1 and 0, respectively, suggesting that spatial heterogeneity changed systematically with disease incidence. Results of a covariance analysis showed that spatial heterogeneity of scab incidence at both scales and lesion density was not dependent upon shoot type but that spatial heterogeneity of scab incidence and lesion density at the leaf scale was influenced by the sampling period. A hierarchical analysis showed that scab incidence at the tree scale increased as a saturation-type curve with respect to incidence at the leaf or shoot scales. A similar relationship was observed for incidences at the shoot and leaf scales. An effective sample size model based on the binary power law parameters (Madden and Hughes, Phytopathology 89:770–781, 1999) gave the best fit to the leaf and shoot data, respectively. The incidence-lesion density relationship at both scales was well described by a complementary log-log (CLL) and log transformation model ( R_adj² = 0.97 and R_adj² = 0.94 ) \left( {R_{{adj}}^2 = 0.97\,and\,R_{{adj}}^2 = 0.94} \right) , however, the models tended to underestimate lesion density. The information of the spatial relations of apple scab within and between hierarchical scales acquired from this study can be used in developing and evaluating practical disease management strategies and to improve apple scab assessments for fungicide or cultivar susceptibility trials.     

Ascospores of the Japanese pear scab fungus (<Emphasis Type="Italic">Venturia nashicola</Emphasis> Tanaka &; Yamamoto) are discharged during the day

We investigated the diurnal pattern of ascospore discharge of the Japanese pear scab fungus (Venturia nashicola Tanaka & Yamamoto) in an orchard. Ascospores of V. nashicola were mainly discharged during the day. Most ascospores were discharged from 7:00 to 19:00: 99.6% in 2001, 99.3% in 2002, and 93.8% in 2005. Because the ascospores were discharged only when the fallen diseased leaves were wet from precipitation, the wetness of these leaves is probably imperative for spore discharge. Ascospore discharge began immediately after precipitation in the daytime. When it rained at night, however, ascospore discharge did not begin until the following morning and never began immediately after precipitation. We also investigated other meteorological factors. When fallen diseased leaves were wet, the percentage of ascospore discharge was positively correlated with the amount of solar radiation and atmospheric temperature and negatively correlated with relative humidity. Ascospore discharge was interrupted by a decrease in solar radiation and atmospheric temperature and by increased relative humidity at night. This report is the first that V. nashicola discharges ascospores primarily during the day.     

A‐scab (Apple‐scab), a simulation model for estimating risk of Venturia inaequalis primary infections*

A‐scab (Apple‐scab) is a dynamic simulation model for Venturia inaequalis primary infections on apple. It simulates development of pseudothecia, ascospore maturation, discharge, deposition and infection during the season based on hourly data of air temperature, rainfall, relative humidity and leaf wetness. A‐scab produces a risk index for each infection period and forecasts the probable periods of symptoms appearance. The model was validated under different epidemiological conditions: its outputs were successfully compared with daily spore counts and actual onset and severity of the disease under orchard conditions, and neither corrections nor calibrations have been necessary to adapt the model to different apple‐growing areas. Compared to other existing models, A‐scab: (i) combines information from literature and data acquired from specific experiments; (ii) is completely ‘open’ because both model structure and algorithms have been published and are easily accessible; (iii) is not written with a specific computer language but it works on simple‐to‐use electronic sheets. For these reasons the model can be easily implemented in the computerized systems used by warning services.     

苹果黑星病在辽宁省的分布及其发生规律

苹果黑星病是辽宁省植物检疫对象。目前主要分布于小苹果栽植区及大、小苹果混栽区,已接近辽宁南部的大苹果主产区。经自然感病和人工接种证明,大、小苹果上的黑星病菌可以交互侵染,目前一些主栽的大苹果品种均能感染此病。辽宁地区苹果黑星病的初次侵染来源是落地越冬病叶上于翌春产生的子囊孢子。分生孢子不能越冬成活。苹果枝条及芽鳞不带菌。黑星病菌的两种孢子随气流传播,使病害逐步扩大蔓延。经过6年的观察,苹果黑星病发生始期早晚及发病轻重,与早春和夏季的降水量多少成正相关。田间药剂防治试验结果,以托布津、乙磷铝、多菌灵、特克多等药剂防治效果良好。     

Detection and quantification of airborne inoculum of Hymenoscyphus pseudoalbidus using real‐time PCR assays

A method based on real‐time polymerase chain reaction (PCR) and the use of rotating‐arm spore traps was developed for quantifying airborne Hymenoscyphus pseudoalbidus ascospores. The method was sensitive and reproducible, and the collection efficiency was 10% of the spores present in the air. The temporal ascospore dispersal pattern was studied over 3 years by collecting spores every 15 days for a 24 h air‐sampling period during the ash‐growing season. The highest production was detected from the end of June to the beginning of September. The overall ascospore production did not differ significantly among stands within a specific year but there were differences from year to year. There was a positive correlation between air temperature and the number of ascospores trapped, with most of the positive samples being observed at temperatures above 12°C. The vertical profile of ascospore dispersal showed a strong decrease in ascospore density within a height of 3 m, regardless of date of collection. An analysis of the spore traps installed at increasing distances from an infected stand showed that most of the ascospores were deposited downwind within 50 m of the stand. These data are discussed in context of the epidemiology of the disease.     

Effects of environmental factors on discharge and germination of ascospores of Venturia nashicola

Experiments were conducted under controlled environmental conditions to study the effects of temperature, duration of wetness, relative humidity (RH) and light on the discharge and germination of ascospores of Venturia nashicola , the causal agent of pear scab in China. Discharge of ascospores from pseudothecia required free water or 100% RH. A period of soaking in water as short as 10 s was sufficient to initiate the discharge of ascospores. Temperatures from 10 to 30°C did not significantly affect the temporal trend of ascospore discharge. A greater proportion of ascospores was discharged under light than in the dark. However, a period of light as short as 10 min, either during the initial wetting of pseudothecia or interrupting the darkness, was sufficient to reduce the inhibitory effect of darkness on ascospore discharge. Ascospores were discharged within 10 min after pseudothecia were wetted and most ascospores ( c. 80%) were discharged within the first hour. The temporal pattern of ascospore discharge could be well described by a logistic model, which estimated that 50% of ascospores were discharged within half an hour of wetting. Ascospores germinated over a wide range of temperatures from 5 to 30°C, with an optimum at c . 20°C. Temporal dynamics of ascospore germination at six temperatures (5, 10, 15, 20, 25 and 30°C) were satisfactorily described by logistic models.     

Environmental Influences on the Release of Ophiosphaerella agrostis Ascospores Under Controlled and Field Conditions

ABSTRACT Ophiosphaerella agrostis, the causal agent of dead spot of creeping bentgrass (Agrostis stolonifera), can produce prodigious numbers of pseudothecia and ascospores throughout the summer. The environmental conditions and seasonal timings associated with O. agrostis ascospore release are unknown. The objectives of this research were to (i) determine the influence of light and relative humidity on ascospore release in a controlled environment, (ii) document the seasonal and daily discharge patterns of ascospores in the field, and (iii) elucidate environmental conditions that promote ascospore release under field conditions. In a growth chamber, a sharp decrease (100 to approximately 50%; 25 degrees C) in relative humidity resulted in a rapid (1- to 3-h) discharge of ascospores, regardless of whether pseudothecia were incubated in constant light or dark. In the field, daily ascospore release increased between 1900 and 2300 h and again between 0700 and 1000 h local time. The release of ascospores occurred primarily during the early morning hours when relative humidity was decreasing and the canopy began to dry, or during evening hours when relative humidity was low and dew began to form. Few ascospores were released between 1100 and 1800 h when the bentgrass canopy was dry. The release of ascospores also was triggered by precipitation. Of the ascospores collected during precipitation events, 87% occurred within 10 h of the beginning of each event.     

Effect of Fall Application of Fungal Antagonists on Spring Ascospore Production of the Apple Scab Pathogen, Venturia inaequalis

ABSTRACT The influences of Microsphaeropsis sp., M. arundinis, Ophiostoma sp., Diplodia sp., and Trichoderma sp., all antagonists of Venturia inaequalis, on ascospore production were evaluated under natural conditions and compared with urea and Athelia bombacina, a known antagonist. In the autumn, the fungi were applied to leaf disks artificially inoculated with V. inaequalis and to scabbed apple (Malus domestica) leaves incubated under controlled and natural conditions. In addition, large-scale trials were conducted with Microsphaeropsis sp. applied either as a foliar postharvest spray or as a ground application at 90% leaf fall. All fungal isolates, except Ophiostoma sp., were recovered from the leaf material that overwintered in the orchard. All treatments, except those with Ophiostoma sp., resulted in a significant reduction in V. inaequalis ascospore production on the leaf disks incubated under controlled conditions or in the orchard. In 1997, leaves with apple scab lesions treated with urea or Microsphaeropsis sp. produced significantly fewer ascospores of V. inaequalis than did nontreated leaves, with a reduction of 73.0 and 76.3%, respectively. In 1998, leaves treated with Microsphaeropsis sp., urea, Trichoderma sp., A. bombacina, and M. arundinis reduced ascospore production by 84.3, 96.6, 75.2, 96.6, and 52.2%, respectively. Based on all tests combined, the most efficient isolate was Microsphaeropsis sp. Postharvest applications of Microsphaeropsis sp. reduced the total amount of airborne ascospores trapped by 70.7 and 79.8% as compared with the nontreated plots in 1997 and 1998, respectively. Microsphaeropsis sp. provided a significant and consistent reduction in ascospore production in all tests.     

Estimating the dynamics of airborne ascospores of Venturia inaequalis*

A system was elaborated to estimate the dynamics of primary inoculum of Venturia inaequalis in apple orchards. It separates the primary inoculum season into five periods with different risks: absent (ascospores not yet mature); potential (ascospores mature but not yet ready to be discharged); actual (ascospores can be discharged when favourable conditions occur); present (ascospores are airborne); exhausted (all ascospores have been ejected). These periods were determined by two mathematical models, which use meteorological parameters as driving variables. The first model estimates the development stage of the overwintering pseudothecia and then determines when the first pseudothecia contain pigmented and mature ascospores. A threshold of mature ascospores inside pseudothecia defines when the ascospores become ready for discharge. The second model estimates the proportion of the season's ascospores that are airborne on each discharging event, using temperature and leaf wetness, expressed as the degrees accumulated daily in the hours when leaves are wet. Estimates of absent and potential risk were verified by collecting data on the first ascospore discharge in the period 1991/1998 at Bologna and Modena (northern Italy), and they were always found to be accurate. To verify the estimates of actual, present and exhausted risk, the model outputs were compared with data collected by spore samplers at Modena and Bologna in 1997 and 1998: they were sufficiently accurate because the greatest part of the records from the spore sampler fell inside the confidence limits of the model.     

Ascospore Release and Infection of Apple Leaves by Conidia and Ascospores of Venturia inaequalis at Low Temperatures

ABSTRACT Mills' infection period table describes the number of hours of continuous leaf wetness required at temperatures from 6 to 25 degrees C for infection of apple leaves by ascospores of Venturia inaequalis and reports that conidia require approximately two-thirds the duration of leaf wetness required by ascospores at any given temperature. Mills' table also provides a general guideline that more than 2 days of wetting is required for leaf infection by ascospores below 6 degrees C. Although the table is widely used, infection times shorter than those in the table have been reported in lab and field studies. In 1989 a published revision of the table eliminated a potential source of error, the delay of ascospore release until dawn when rain begins at night, and shortened the times reported by Mills for ascospore infection by 3 h at all temperatures. Data to support the infection times below 6 degrees C were lacking, however. Our objective was to quantify the effects of low temperatures on ascospore discharge, ascospore infection, and infection by conidia. In two of three experiments at 1 degrees C, the initial release of ascospores occurred after 131 and 153 min. In the third experiment at 1 degrees C, no ascospores were detected during the first 6 h. The mean time required to exceed a cumulative catch of 1% was 143 min at 2 degrees C, 67 min at 4 degrees C, 56 min at 6 degrees C, and 40 min at 8 degrees C. At 4, 6, and 8 degrees C, the mean times required to exceed a cumulative catch of 5% were 103, 84, and 53 min, respectively. Infection of potted apple trees by ascospores at 2, 4, 6, and 8 degrees C required 35, 28, 18, and 13 h, respectively; substantially shorter times than previously were reported. In parallel inoculations of potted apple trees, conidia required approximately the same periods of leaf wetness as ascospores at temperatures from 2 to 8 degrees C, rather than the shorter times reported by Mills or the longer times reported in the revision of the Mills table. We propose the following revisions to infection period tables: (i) shorter minimum infection times for ascospores and conidia at or below 8 degrees C, and (ii) because both ascospores and conidia are often present simultaneously during the season of ascospore production and the required minimum infection times appear to be similar for both spore types, the adoption of a uniform set of criteria for ascosporic and conidial infection based on times required for infection by ascospores to be applied during the period prior to the exhaustion of the ascospore supply. Further revisions of infection times for ascospores may be warranted in view of the delay of ascospore discharge and the reduction of airborne ascospore doses at temperatures at or below 2 degrees C.     

Viability and release of Neonectria ditissima ascospores on apple fruit in Brazil

During European canker monitoring in an apple experimental orchard, 14 mummified fruit (two and three trees with 10 and four positive records in 2018 and 2019, respectively) showed perithecia. Perithecium production on apple fruit, confirmation of pathogenicity of Neonectria ditissima isolated from mummified fruit, and ascospore release from fruit tissues has rarely been reported, and their role in the epidemiology of European canker has been largely overlooked. Thus, the objectives of our study were to (a) prove the presence of both conidia and ascospores of N. ditissima in mummified fruit in an experimental field, confirming pathogenesis in different apple cultivars, and (b) monitor production of the two types of inoculum in infected apple fruit over time. Canker incidence in this orchard was 47% of trees with symptoms in 2018 and 48% in 2019. Molecular and morphological tests confirmed that the fungus detected in the mummified apple fruit was N. ditissima. Apple fruit with sporodochia and perithecia washed immediately after collection from the orchard showed conidia but no ascospores of N. ditissima. However, after 4 days’ incubation, perithecia on mummified fruit showed many ascospore cirri. Koch's postulates were fulfilled on apple plants and mature fruit. Fruit inoculated with N. ditissima released spores for over a year under Brazilian field conditions. The release of both spore types peaked in May (Brazilian leaf fall) and October (spring); release of conidia also peaked in February (early harvest). These results support our hypothesis that fruit can serve as primary inoculum for European canker in Brazilian apple orchards.     

Selection and orchard testing of antagonists suppressing conidial production by the apple scab pathogen <Emphasis Type="Italic">Venturia inaequalis</Emphasis>

Apple scab caused by Venturia inaequalis is a major disease in apple production. Epidemics in spring are initiated by ascospores produced on overwintering leaves whereas epidemics during summer are driven by conidia produced on apple leaves by biotrophic mycelium. Fungal colonisers of sporulating colonies of V. inaequalis were isolated and their potential to reduce the production of conidia of V. inaequalis was evaluated on apple seedlings under controlled conditions. The four most effective isolates of the 63 screened isolates were tested subsequently under Dutch orchard conditions in 2006. Repeated applications of conidial suspensions of Cladosporium cladosporioides H39 resulted in an average reduction of conidial production by V. inaequalis of approximately 40%. In 2007, applications of conidial suspensions of C. cladosporioides H39 reduced conidial production by V. inaequalis by 69% on August 6 and by 51% on August 16, but no effect was found on August 20. However, viability of available conidia of C. cladosporioides H39 was low at the end of the experiment. Epiphytic and endophytic colonisation by Cladosporium spp. of leaves treated during the experiment with C. cladosporioides H39 was significantly higher than on control leaves sampled 6 weeks after the last application. It is concluded that C. cladosporioides H39 has promising potential as a biological control agent for apple scab control. More information is needed on the effect of C. cladosporioides H39 on apple scab epidemics as well as on mass production, formulation and shelf life of conidia of the antagonist.     

Effect of Timing of Application of the Biological Control Agent Microsphaeropsis ochracea on the Production and Ejection Pattern of Ascospores by Venturia inaequalis

ABSTRACT Field and in vitro trials were conducted to establish the influence of the biological control agent Microsphaeropsis ochracea on the ejection pattern of ascospores by Venturia inaequalis and on apple scab development, and to establish the best timing of application. The ejection pattern of ascospores was similar on leaves sprayed with M. ochracea and on untreated leaves. Fall application of M. ochracea combined with a delayed-fungicide program was evaluated in orchards with intermediate and high scab risk. For both orchards, it was possible to delay the first three and two infection periods in 1998 and 1999, respectively, without causing significant increase or unacceptable leaf and fruit scab incidence. To evaluate the best timing of application, sterile leaf disks were inoculated with V. inaequalis and then with M. ochracea 0, 2, 4, 6, 8, 10, 12, 14, and 16 weeks later. After incubation under optimal conditions for pseudothecia development, the number of ascospores was counted. Similarly, M. ochracea was sprayed on scabbed leaves on seven occasions from August to November 1999 and 2000. Leaves were overwintered on the orchard floor and ascospore production was evaluated the following spring. Ascospore production was reduced by 97 to 100% on leaf disks inoculated with M. ochracea less than 6 weeks after inoculation with V. inaequalis, but ascospore production increased with increasing period of time when M. ochracea was applied 8 to 16 weeks after the inoculation with V. inaequalis. In the orchard, the greatest reduction in production of ascospores (94 to 96% in 2000 and 99% in 2001) occurred on leaves sprayed with M. ochracea in August. The production of ascospores was reduced by 61 to 84% in 2000 and 93% in 2001 on leaves sprayed with M. ochracea in September, reduced by 64 to 86% in 2000 and 74 to 89% in 2001 on leaves sprayed in October, and reduced by 54 and 67% in 2000 and 2001, respectively, on leaves sprayed in November. It was concluded that M. ochracea should be applied in August or September and that ascospore maturation models and delayed-fungicide program could be used in orchards treated with this biological control agent.     

Evaluating predictors of apple scab with receiver operating characteristic curve analysis

ABSTRACT Apple scab (Venturia inaequalis) is a perennial threat to apple production in temperate climates throughout the world. In the eastern United States, apple scab is managed almost exclusively through the regular application of fungicides. Management of the primary phase of disease is focused on preventing infection by ascospores. Management of secondary cycles of infection is largely dependent on how well primary infections were controlled. In this study, we used receiver operating characteristic curve analysis to evaluate how well mid-season assessments of the incidence of apple scab on cluster leaves, clusters (i.e., the whorl of cluster leaves), or immature fruit can serve as predictors of apple scab on harvested fruit (harvest scab) and whether these mid-season assessments of scab could be used reliably to manage scab under various damage thresholds. Results showed that assessment of scab on immature fruit was superior at predicting harvest scab than were assessments made on clusters or cluster leaves at all damage thresholds evaluated. A management action threshold of 7% scab incidence on immature fruit was identified by Youden's index as the optimal action threshold to prevent harvest scab incidence from exceeding 5%. Action thresholds could be higher or lower than 7% when economic assumptions were factored in to the decision process. The utility of such a predictor is discussed.     

Germination of <Emphasis Type="Italic">Gibberella zeae</Emphasis> ascospores as affected by age of spores after discharge and environmental factors

The effects of age of ascospores (0–18 days after discharge), photon flux density (0–494 mol m^–2 s^–1 PAR), temperature (4–30 °C), frost (–15 °C for 30 min), relative humidity (RH; 0–100%), pH (2.5–6.5) and dryness (0 and 53% RH for up to 40 min) on the germination of the ascospores of the mycotoxin-producing fungus Gibberella zeae (anamorph Fusarium graminearum) were studied. Freshly discharged ascospores germinated within 4 h at 20 °C and 100% RH. The rate of germination and the percentage of viable ascospores decreased over time after the spores were discharged from perithecia. The time course of ascospore germination was not significantly affected by photon flux density. The period of time required to obtain 50% germinated ascospores at 100% RH was 26.90 h at 4 °C, 10.40 h at 14 °C, 3.44 h at 20 °C and 3.31 h at 30 °C. There was no significant effect of frost on the percentage of viable ascospores. A small percentage (6.6 ± 3.8%) of the ascospores germinated at 53% RH. At RH 84% and 20 °C almost 100% of the freshly discharged ascospores germinated. The time course of ascospore germination was affected by pH. The maximum rate of ascospore germination was estimated to be at pH 3.76. Ascospores lost their ability to germinate following exposure to 0% RH almost instantaneously. No germinating spores were detected after an incubation period of 1 min at 0% RH. Incubating the ascospores at 53% RH decreased the percentage of viable spores from 93 to 6% within 10 min. The data demonstrate that age of spores, relative humidity, temperature and pH, but not photon flux density, are key factors in germination of G. zeae ascospores.     

A method to monitor airborne Venturia inaequalis ascospores using volumetric spore traps and quantitative PCR

Apple scab caused by the fungus Venturia inaequalis can result in significant crop losses if not managed effectively. Sanitation as part of an integrated management strategy aims to significantly reduce primary inoculum to lower the disease pressure. This study evaluates the possibility of molecular detection and quantification of ascospore discharge and the use of this method to test for efficacy of orchard sanitation treatments. A method to detect and quantify airborne ascospores was developed using volumetric spore traps (VSTs). V. inaequalis specific primers were tested on daily VST samples from two orchard sections (leaf litter removed compared to leaf litter left) during spring. A molecular method to detect and quantify ascospores was tested by amplifying genomic regions of the mitochondrial CYP51A1 gene, and the ITS region using SYBR® green. Timing of ascospore discharge was compared to predicted infection risk and a degree day model using weather data. The average spore detection limit was estimated to be at levels of 1 pg μl^?1 DNA (approximately 37 ascospores) per daily spore trap reading using CYP51A1 primers. Using the CYP51A1 primer pair, primary inoculum was estimated to be 51 % lower in the orchard sections where leaves had been removed, indicating that this method could be used to evaluate the efficacy of alternative control strategies such as leaf removal to reduce potential ascospore dose. This is the first report of combining VSTs and quantitative PCR to monitor airborne V. inaequalis ascospores.