Early stages of septoria tritici blotch epidemics of winter wheat: build‐up,overseasoning, and release of primary inoculum

Septoria tritici blotch (STB), caused by Mycosphaerella graminicola, is the most prevalent disease of wheat worldwide. Primary inoculum and the early stages of STB epidemics are still not fully understood and deserve attention for improving management strategies. The inoculum build‐up and overseasoning involves various fungal structures (ascospores, pycnidiospores, mycelium) and plant material (wheat seeds, stubble and debris; wheat volunteers; other grasses). Their respective importance is assessed in this review. Among the mechanisms involved in the early stages of epidemics and in the year‐to‐year disease transmission, infection by ascospores wind‐dispersed from either distant or local infected wheat debris is the most significant. Nevertheless, infection by pycnidiospores splash‐dispersed either from neighbouring wheat debris or from senescent basal leaves has also been inferred from indirect evidence. Mycosphaerella graminicola has rarely been isolated from seeds so that infected seed, although suspected as a source of primary inoculum for a long time, is considered as an epidemiologically anecdotal source. Mycosphaerella graminicola can infect a few grasses other than wheat but the function of these grasses as alternative hosts in natural conditions remains unclear. Additionally, wheat volunteers are suspected to be sources of STB inoculum for new crops. This body of evidence is summarized in a spatio‐temporal representation of a STB epidemic aimed at highlighting the nature, sources and release of inoculum in the early stages of the epidemic.     

Is the onset of septoria tritici blotch epidemics related to the local pool of ascospores?

To elucidate the early epidemic stages of septoria tritici blotch, especially the relationship between the onset of epidemics, the local availability of primary inoculum, and the presence of wheat debris, the early disease dynamics and airborne concentration in Zymoseptoria tritici ascospores were concomitantly assessed at a small spatiotemporal scale and over two years, using spore traps coupled with a qPCR assay. One plot, with the crop debris left, provided a local source of primary inoculum, while the other plot, without debris, lacked any. According to the assay's limits of detection, daily spore trap samples were classified as not detectable or not quantifiable, detectable, and quantifiable. The proportions of samples assigned to the different classes and numbers of spores in samples classified as quantifiable were significantly different between years, time periods (from November to March) and spore trap location (field with or without debris). The effect of year on the airborne ascospore concentration was high: 22 daily peaks with more than 230 ascospores m^?3 day^?1 were identified in the autumn of 2012/13, but none in the autumn of 2011/12. The local presence of wheat debris had no obvious effect on the amount of airborne ascospores or on the earliness of the two epidemics, especially in the year with high inoculum pressure (2012/13). These results suggest that the amount of primary airborne inoculum available in a wheat crop is not a limiting factor for the onset of an epidemic.     

Comparative pathogenicity of sexual and asexual spores of Zymoseptoria tritici (septoria tritici blotch) on wheat leaves

Zymoseptoria tritici ascospores and pycnidiospores are considered the main forms of primary and secondary inoculum, respectively, in septoria tritici blotch epidemics. The pathogenicity of the two types of spores of the same genotypic origin were compared through a two‐stage inoculation procedure in controlled conditions. Adult wheat leaves were inoculated with ascospores collected from field sources, yielding 119 lesions; pycnidiospores collected from 12 lesions resulting from these ascospore infections were then used for inoculation. Lesion development was assessed for 5 weeks; latent period, lesion size, and pycnidium density were estimated for different isolates. The latent period was calculated as the maximum likely time elapsed between inoculation and either the appearance of the majority of the sporulating lesions (leaf scale) or the appearance of the first pycnidia (lesion scale). The latent period was significantly longer (c. 60 degree‐days, i.e. 3–4 days) after infection with ascospores than with pycnidiospores. No difference was established for lesion size and density of pycnidia. A comparison with other ascomycete fungi suggested that the difference in latent period might be related to the volume of spores and their ability to cause infection. Fungal growth before the appearance of lesions may be slower after inoculation with an ascospore than with a pycnidiospore. The mean latent period during the very beginning of epidemics, when first lesions are mainly caused by ascospores, may be longer than during spring, when secondary infections are caused by pycnidiospores. Disease models would be improved if these differences were considered.     

Airborne ascospores ofDidymella rabiei as a major primary inoculum for Ascochyta blight epidemics in chickpea crops in southern Spain

The incidence and severity of Ascochyta blight in potted chickpea trap plants exposed for 1-wk periods near infested chickpea debris in Córdoba, Spain, or in chickpea trap crops at least 100 m from infested chickpea debris in several locations in southern Spain were correlated with pseudothecial maturity and ascospore production ofDidymella rabiei from nearby chickpea debris. The period of ascospore availability varied from January to May and depended on rain and maturity of pseudothecia. The airborne concentration of ascospores ofD. rabiei was also monitored in 1988. Ascospores were trapped mostly from the beginning of January to late February; this period coincided with that of maturity of pseudothecia on the chickpea debris. Most ascospores were trapped on rainy days during daylight and 70% were trapped between 12.00 and 18.00 h. Autumn-winter sowings of chickpea were exposed longer to ascospore inoculum than the more traditional spring sowings because the autumn-winter sowings were exposed to the entire period of ascospore production on infested chickpea debris lying on the soil surface.     

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.     

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.     

The use of ascospores of the dieback fungus Hymenoscyphus fraxineus for infection assays reveals a significant period of biotrophic interaction in penetrated ash cells

Ascospores, discharged naturally from apothecia growing on rachis debris, were used as inoculum to examine the invasion of ash tissues by Hymenoscyphus fraxineus in order to understand the critical, but poorly understood, early interactions between host and pathogen. Methods were developed to collect ascospores for controlled infection assays on detached leaves, petioles and stem internode tissues. Light microscopy, using plasmolytic techniques, allowed the invasion of living plant cells to be observed. Ascospores were readily available from late May to September. On the plant surface, most spores differentiated directly to form appressoria without germ‐tube growth. Direct penetration was followed by a significant period of biotrophic fungal growth before lesions developed. Following the formation of a vesicle‐like structure after penetration, bulbous and elongated intracellular hyphae were produced in living plant cells. The use of ascospore inoculum, rather than mycelia, will allow natural and rapid screening of ash genotypes for resistance to the devastating dieback disease. The identification of the biotrophic phase of infection suggests that host range is controlled by effector‐triggered immunity.     

Environmental Factors Affecting the Release and Dispersal of Ascospores of Mycosphaerella citri

ABSTRACT Greasy spot, caused by Mycosphaerella citri, produces a leaf spot disease affecting all citrus species in Florida and the Caribbean Basin. M. citri produces pseudothecia and ascospores, which are considered the principal source of inoculum, in decomposing leaves on the grove floor. In studies using a computer-controlled environmental chamber, a single rain event triggered release of most mature ascospores beginning 30 to 60 min after the rain event. Additional rain events did not bring about further release. High relative humidity without rain triggered release of low numbers of ascospores, but vibration and red/infrared irradiation had little or no effect on ascospore release. After three to four cycles of wetting and drying of leaves, all pseudothecia had matured and released their ascospores. In the field, ascospores were detectable starting about 2 h after the beginning of a rain or irrigation and most ascospores were released within 16 h. Ascospore release was greatest following rain events and somewhat less following irrigations, and low numbers of ascospores were detectable on days without precipitation. Ascospore numbers declined linearly with horizontal distance from the source and as a function of the logarithm of ascospore numbers with vertical distance. Low numbers of ascospores were detected 7.5 m above the ground and 90 m downwind from the grove. Ascospore release can be advanced by irrigating frequently during dry, nonconducive conditions to stimulate ascospore release when environmental conditions are unfavorable for infection, but the eventual effects on disease severity are uncertain.     

Environmental Factors Affecting Pseudothecial Development and Ascospore Production of Mycosphaerella citri, the Cause of Citrus Greasy Spot

ABSTRACT Mycosphaerella citri, the cause of citrus greasy spot, produces pseudothecia and ascospores in decomposing leaf litter on the grove floor. In laboratory studies, the effect of wetting and drying and temperature on the formation, maturation, and production of pseudothecia and ascospores was evaluated on mature, detached grapefruit leaves. Production of pseudothecia was most rapid when leaves were soaked five times per week for 2 h per day, but pseudothecial density and total ascospore production were greatest when leaves were soaked three times per week for 2 h per day. In duration of wetting studies, 3 h per day, 3 days per week brought about the most rapid production, but 10 to 30 min per day resulted in production of the most pseudothecia and ascospores. Pseudothecia and ascospore production were greatest at 28 degrees C and declined rapidly at lower and higher temperatures. Maturation of pseudothecia was slow at 20 and 24 degrees C, but production was high at 24 degrees C; at 32 degrees C, pseudothecia matured rapidly, but degenerated quickly. No mature pseudothecia were produced on leaves maintained continuously under wet conditions. In field studies, leaves were placed on the grove floor monthly from April 2000 to September 2001. Pseudothecia production was rapid during the summer rainy season from June to September. Pseudothecia produced on leaves placed in the grove from October to May developed and matured more slowly but were produced in much larger numbers than in summer. The number of days to first pseudothecial initials, 50% maturation, first discharge of ascospores, leaf decomposition, as well as pseudothecial density and incidence, were negatively related to average temperature. Total ascospore production was unrelated to temperature.     

Germination and invasion by ascospores and pycnidiospores of <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> on spring-type <Emphasis Type="Italic">Brassica napus</Emphasis> canola varieties with varying susceptibility to blackleg

The infection processes of ascospores and pycnidiospores of Leptosphaeria maculans were studied on cotyledons of six cultivars of spring-type Brassica napus: one with resistance controlled by a single dominant gene (cv. Surpass 400), three with polygenic resistance (cvs. Dunkeld, Grouse, and Outback), and two susceptible cultivars (Westar and Q2). On all cultivars, ascospore germination, penetration, and development of symptoms on cotyledons were much earlier than that with pycnidiospores. At 2h after inoculation ascospores began to germinate, by 4h about 50% had germinated, and by 6–8h 85%–90% had germinated. In contrast, pycnidiospores began to germinate 1 day after inoculation (dai) and reached only 50% germination by 3 dai. Ascospores began germinating from terminal cells and then later from the interstitial cells. Pycnidiospores germinated predominantly from one end and sometimes from both ends. Germ tubes from ascospores penetrated stomata as early as 4h after inoculation, whereas those from pycnidiospores penetrated at 2 dai. Symptom development with ascospores was 2 days earlier than that with pycnidiospores. Symptoms on Surpass 400 were evident as early as 3–5 dai with ascospores and 5–7 dai with pycnidiospores. However, on other cultivars, symptoms were not evident until 10 dai with ascospores and 12 dai with pycnidiospores. This report is the first on differences in the infection processes by the two spore types. Ascospore and pycnidiospore attachment, germination, and penetration did not differ between resistant and susceptible cultivars, but there were major differences after penetration. Under high humidity, 80%–90% of stomata of susceptible Westar and Q2 had aerial hyphae emerging from stomatal pores. However, fewer stomata (5%–10%) had aerial hyphae on Surpass 400 by 10 dai with ascospores and 12 dai with pycnidiospores, but even these were usually poorly developed. Host differences in spring-type B. napus in relation to production of aerial hyphae have not previously been reported. In Surpass 400, rapid necrosis of guard cells occurred within a few hours of penetration by either type of spore, and subsequently one or a few cells immediately adjacent to the penetration site died. This necrosis then spread to the cells around the penetration site to form a hypersensitive response (in the form of a small, dark lesion) to both ascospores and pycnidiospores. This is the first detailed report on interactions between spring-type B. napus and L. maculans in relation to single dominant gene-based resistance. Neither the cultivars with polygenic resistance nor the susceptible cultivars had such a response.     

Phenology of Didymella rabiei Development on Chickpea Debris Under Field Conditions in Spain

ABSTRACT The development of Didymella rabiei on debris of naturally infected chickpea was investigated in four chickpea-growing areas with different climatic conditions in Spain during 1987 to 1992. D. rabiei extensively colonized chickpea debris and formed pseudothecia and pycnidia. Differentiation of pseudothecial initials occurred regularly across experimental locations by November, 1 month after placement of debris on the soil. Ascospore maturation occurred mainly from late January to late March, depending on location and year. Maximum ascospore discharge from sampled debris pieces placed under suitable environmental conditions occurred 2 to 4 weeks after ascospore maturation, after which ascospore release decreased sharply. Pseudothecia were exhausted, due to ascospore discharge, by the beginning of summer. New asci did not develop in empty pseudothecia and no pseudothecia formed in tissues after the first season. Ascospore maturation and liberation in cooler locations were more uniform and occurred later compared to maturation in warmer locations. Also, production of asci and ascospores per pseudothecium was much higher in cooler than in warmer locations. A similar relationship was found for density of pseudothecia and pycnidia and conidia production per pycnidium. The percentage of mature pseudothecia increased according to the logistic model, with the cumulative number of Celsius degree days calculated by computing the mean of the maximum and minimum daily air temperatures on rainy days from the date of debris placement on the soil. There were significant differences among model parameter estimates between cooler and warmer locations, but minor differences were found among parameters for locations with similar environmental conditions. There was an inverse linear relationship between the average temperature during the period of pseudothecia maturation and the number of asci produced per pseudothecium.     

Dynamics of the ascospore dispersal of Stagonosporopsis citrulli,a causal agent of gummy stem blight of cucurbits

Sexually reproduced, airborne ascospores of Stagonosporopsis citrulli may play a role in its dispersal. S. citrulli causes gummy stem blight (GSB), one of the most important foliar diseases of cucurbits. Four studies were conducted with S. citrulli to investigate for how long ascospores are released and how far they can be dispersed from a source field. In the first study, colonized watermelon debris was sampled during three seasons and samples were assayed for ascospore release. Ascospores were detected 292, 313, and 306 days after inoculation of the source. In the second study, the active release of ascospores from pseudothecia in a Petri dish was monitored for 7 days. The release of ascospores decreased by ≤90% from 1 day after the start of the assay until 7 days after. In the third study, trap plant assays were conducted to measure the dispersal gradient of ascospores up to 366 m from the source. Generally, frequency of pathogen recovery from trap plants decreased with increasing distance from the source. The ascospore dispersal data fitted the exponential model better than the power law model. In the final study, dispersal experiments were conducted under controlled conditions. The incidence of GSB decreased with increasing distance, up to 55 m, from the source. It was concluded that ascospores of S. citrulli can serve as primary inoculum for epidemics and could easily spread among fields. Debris from cucurbit crops can be the source of ascospores for up to 10 months and should be cleared expeditiously.     

Differences Between Ascospores and Conidia of Didymella rabiei in Spore Germination and Infection of Chickpea

ABSTRACT Studies were performed to compare the germination and infection of ascospores and conidia of Didymella rabiei under different temperature and moisture conditions. Germination of ascospores and conidia on cover glasses coated with water agar began after 2 h, with maximum germination (>95%) occurring in 6 h at 20 degrees C. No germination occurred at 0 and 35 degrees C. Ascospores germinated more rapidly than conidia at all temperatures. Germination declined rapidly as the water potential varied from 0 to -4 MPa, although some germination occurred at -6 MPa at 20 and 25 degrees C. Ascospores germinated over a wider range of water potentials than conidia and their germ tubes were longer than those of conidia at most water potentials and temperatures. The optimum temperature for infection and disease development by both ascospores and conidia was around 20 degrees C. Disease severity was higher when ascospores were discharged directly onto plant surfaces from naturally infested chickpea debris compared with aqueous suspensions of ascospores and conidia sprayed onto plants Disease severity increased as the length of the wetness period increased. When dry periods of 6 to 48 h occurred immediately after inoculation, disease severity decreased, except for the shorter periods which had the opposite effect. Disease severity was higher with ascospore inoculum when no dry periods occurred after inoculation.     

The Occurrence of Mycosphaerella graminicola and its Anamorph Septoria tritici in Winter Wheat during the Growing Season

The disease septoria tritici blotch of wheat is initiated by ascospores of the teleomorph Mycosphaerella graminicola or pycnidiospores of the anamorph Septoria tritici. We report for the first time the presence of the teleomorph, M. graminicola, in Denmark. With the objective of elucidating the importance of the teleomorph for the development of septoria tritici blotch, data on the occurrence of fruit bodies of the anamorph (pycnidia) and the teleomorph (pseudothecia) stages were collected over three growing seasons. Pseudothecia were present in the springs, however, high numbers of pseudothecia compared to pycnidia were not observed until July, too late to influence the epidemic. On an individual leaf layer, pycnidia were observed well before pseudothecia. As the leaves aged, progressively higher proportions of fruit bodies were observed to be pseudothecia. The period from the appearance of pycnidia to detection of pseudothecia was estimated as 29–53 days. At harvest, high proportions of sporulating fruit bodies in the crop were pseudothecia, suggesting that the primary source of inoculum for new emerging wheat crops in autumn is likely to be ascospores.     

Relationship between ascochyta blight on field pea (Pisum sativum) and spore release patterns of Didymella pinodes and other causal agents of ascochyta blight

Ascochyta blight of field pea, caused by Didymella pinodes, Phoma medicaginis var. pinodella, Phoma koolunga and Didymella pisi, is controlled through manipulating sowing dates to avoid ascospores of D. pinodes, and by field selection and foliar fungicides. This study investigated the relationship between number of ascospores of D. pinodes at sowing and disease intensity at crop maturity. Field pea stubble infested with ascochyta blight from one site was exposed to ambient conditions at two sites, repeated in 2 years. Three batches of stubble with varying degrees of infection were exposed at one site, repeated in 3 years. Every 2 weeks, stubble samples were retrieved, wetted and placed in a wind tunnel and up to 2500 ascospores g^?1 h^?1 were released. Secondary inoculum, monitored using seedling field peas as trap plants in canopies arising from three sowing dates and external to field pea canopies, was greatest in early sown crops. A model was developed to calculate the effective number of ascospores using predictions from G1 blackspot manager (Salam et al., 2011b; Australasian Plant Pathology, 40 , 621–31), distance from infested stubble (Salam et al., 2011a; Australasian Plant Pathology, 40 , 640–7) and winter rainfall. Maximum disease intensity was predicted based on the calculated number of effective ascospores, soilborne inoculum and spring rainfall over two seasons. Predictions were validated in the third season with data from field trials and commercial crops. A threshold amount of ascospores of D. pinodes, 294 g^?1 stubble h^?1, was identified, above which disease did not increase. Below this threshold there was a linear relationship between ascospore number and maximum disease intensity.     

Effects of temperature,water regime and irrigation system on the release of ascospores of Mycosphaerella nawae,causal agent of circular leaf spot of persimmon

Experiments were conducted under controlled conditions to quantify the effects of temperature, water regime and irrigation system on the release of Mycosphaerella nawae ascospores from leaf litter in Spanish persimmon orchards. The effect of temperature on ascospore release was best described by a Gompertz model. The end of the lag phase of ascospore release occurred at 9·75°C, and the end of the exponential phase at 15·75°C. Few ascospores were discharged from dry leaves wetted with 0·1 or 0·5 mm water, but significant amounts were recovered with 1–50 mm water. About half of the total ascospores were released after three wetting and drying cycles, but 32 cycles were necessary for a complete discharge. No significant difference in ascospore release was detected when the leaf litter was wetted by flood and drip irrigation. However, considering the proportion of soil area wetted in both systems, inoculum release was significantly reduced by drip irrigation. The potential of drip irrigation as a cultural control measure should be investigated.     

Biology and control of Polystigma ochraceum, the cause of almond red leaf blotch

Polystigma ochraceum is a major leaf pathogen of almond in Fars Province of Iran. Over a 4-year study period it was found that ascospore discharge began at flowering and continued for 4–5 weeks. The maximum discharge occurred at petal fall. The incubation period was estimated to be 4–5 weeks under experimental conditions. Although the mature ascospores could produce short germ tubes in distilled water or water agar, the fungus could not be cultured or grown on conventional media from either ascospores, pycnidiospores or stromatic tissues under laboratory conditions.
Of several systemic and non-systemic fungicides evaluated under field conditions, triforine at 100–400 μ/ml was most effective. Other fungicides which significantly reduced leaf infection were, in order of efficacy, copper oxychloride (2000 μg/ml), copper hydroxide (2000 mUg/ml), Bordeaux mixture (10 000 μg/ ml) and mancozeb (2000 μg/ml). Carbendazim and thiophanate methyl (500 μg/ml) increased the level of infection. One application of the fungicide at petal fall and then two at 14-day intervals were found to be effective in reducing the disease.     

Factors Affecting the Release of Ascospores of Anisogramma anomala

ABSTRACT Relationships between environmental factors and release of ascospores of Anisogramma anomala, the causal agent of eastern filbert blight, were examined in four European hazelnut (Corylus avellana) orchards during a 2-year period. In each orchard, Burkhard volumetric spore traps and automated weather-monitoring equipment were deployed for 12-week periods beginning at budbreak, when hazelnut becomes susceptible to infection. Ascospores of A. anomala were released when stromata on the surface of hazelnut branches were wet from rain but not from dew. Release of ascospores ceased after branch surfaces dried. The duration of free moisture on branch surfaces regulated the initiation and rate of ascospore release, but no significant effects of temperature, relative humidity, wind, or light on ascospore release were apparent. Most (>90%) ascospores were captured during precipitation events that exceeded 20 h in duration, which represented about 10% of the total precipitation events each season. Quantitative relationships between the hourly capture of A. anomala ascospores and hours since the beginning of a precipitation event were developed. With the onset of precipitation, the hourly rate of ascospore capture increased until the fifth hour of rain, remained relatively constant between the fifth and twelfth hours, and then declined gradually. During the 12-week spore-trapping periods, the likelihood and rates of ascospore release associated with precipitation were highest at budbreak and then declined through April and May until early June, when the reserve of ascospores in the perithecia was depleted. Large numbers of ascospores were captured in the volumetric spore traps, indicating that ascospores may be commonly dispersed long distances on air currents as well as locally by splash dispersal within the canopy, as reported previously. The results indicate that monitoring seasonal precipitation patterns may be useful for estimating the quantity and temporal distribution of airborne inoculum during the period that the host is susceptible to infection.     

Early stages of infection of rapeseed petals and leaves by Sclerotinia sclerotiorum revealed by scanning electron microscopy

The primary ascospore inoculum of Sclerotinia sclerotiorum initially infects rapeseed (Brassica napus var oleifera) via petals. Infected petals fall onto leaf surfaces, resulting in infection of those organs. A scanning electron microscopy (SEM) study of this process was undertaken to elucidate the host-parasite relationship and to determine the best plant organ for detection by serology of early field infection as an aid to disease forecasting and cost-effective disease control. The behaviour of ascospores deposited on young petals and on leaves was compared. Ascospores were deposited by inverting a mature apothecium above either a leaf disc, a young petal or young petal placed on a leaf surface. Spore germination, host penetration and colonization were examined by SEM. On young petals, the following steps in pathogenesis were observed: ascospore adhesion and germination, penetration of the host from short germ tubes and collapse of epidermal cells. Petals were then covered with extensive mycelium. From these sites, the mycelium invaded leaf tissues and infection proceeded. In contrast, ascospores landing directly on leaf surfaces failed to germinate. The role of petals as sites of pre-election in the aetiology of the disease is discussed in relation to the published literature.     

Real-time PCR quantification and spatio-temporal distribution of airborne inoculum of Mycosphaerella graminicola in Belgium

Two kinds of propagules play a role in Mycosphaerella graminicola dissemination: splash-dispersed pycnidiospores and airborne sexual ascospores. A method based on real-time polymerase chain reaction (PCR) assay and using Burkard spore traps was developed to quantify M. graminicola airborne inoculum. The method was tested for its reliability and applied in a spore trap network over a 2-year period in order to investigate the spatio-temporal distribution of airborne inoculum in Belgium. At four experimental sites, airborne inoculum was detected in both years. A seasonal distribution was observed, with the highest mean daily quantities (up to 351.0 cDNA) trapped in July and with clusters detected from September to April. The first year of trapping, a mean daily quantity of 15.7 cDNA of M. graminicola airborne inoculum was also detected in the air above a building in a city where the spatio-temporal distribution showed a similar pattern to that in the field. Mean daily quantities of up to 60.7 cDNA of airborne inoculum were measured during the cereal stem elongation and flowering stages, suggesting that it contributes to the infection of upper leaves later in the season. Most detection, however, tended to occur between flowering and harvest, suggesting significant production of pseudothecia during that period. Variations in mean daily quantities from 1.0 to 48.2 cDNA were observed between sites and between years in the patterns of airborne inoculum. After stem elongation, the quantities detected at a site were positively correlated with the disease pressure in the field. Quantities trapped at beginning of the growing season were also well correlated with the disease level the previous year. Multiple regressions revealed that some factors partly explain the daily variations of airborne inoculum.