Epidemiology of Leptosphaeria maculans in relation to forecasting stem canker severity on winter oilseed rape in the UK

In the UK, ascospores of Leptosphaeria maculans first infect leaves of oilseed rape in the autumn to cause phoma leaf spots, from which the fungus can grow to cause stem cankers in the spring. Yield losses due to early senescence and lodging result if the stem cankers become severe before harvest. The risk of severe stem canker epidemics needs to be forecast in the autumn when the pathogen is still in the leaves, since early infections cause the greatest yield losses and fungicides have limited curative activity. Currently, the most effective way to forecast severe stem canker is to monitor the onset of phoma leaf spotting in winter oilseed rape crops, although this does not allow much time in which to apply a fungicide. Early warnings of risks of severe stem canker epidemics could be provided at the beginning of the season through regional forecasts based on disease survey and weather data, with options for input of crop-specific information and for updating forecasts during the winter. The accuracy of such forecasts could be improved by including factors relating to the maturation of ascospores in pseudothecia, the release of ascospores and the occurrence of infection conditions, as they affect the onset, intensity and duration of the phoma leaf spotting phase. Accurate forecasting of severe stem canker epidemics can improve disease control and optimise fungicide use.     

Factors affecting development of phoma canker (Leptosphaeria maculans) on stems of winter oilseed rape (Brassica napus) in southern England

In winter oilseed rape experiments at Rothamsted in 1997/98 (cvs Lipton and Capitol), 1998/99 (cv. Apex) and 1999/2000 (cvs Apex, Lipton and Capitol), development of crown canker and phoma stem lesions in spring was related to development of phoma leaf spot in the previous autumn/winter. There were differences in thermal time (degree‐days) from the first appearance of phoma leaf spot (autumn) to the first appearance of crown canker (spring) between cultivars (cvs Lipton and Capitol, 1220–1240; cv. Apex, 1120–1140 degree‐days) but not between growing seasons. In 1998/99 and 1999/2000, fungicide (November) treatment delayed the start of crown canker development in the spring but did not affect the rate of increase in severity. In 1997/98, fungicide treatments did not delay the appearance of crown canker but decreased the rate of increase in crown canker severity. In all three seasons, fungicide treatments generally decreased the proportions of plants at harvest with crown canker severity scores 3 or 4 and increased the proportions with scores 0 or 1. There were differences between seasons in the distributions of crown canker severity scores at harvest. The severity of both crown canker and phoma stem lesions increased linearly with accumulated degree‐days in plots with or without fungicide treatment in 1997/98 (cv. Lipton), 1998/99 (cv. Apex) and 1999/2000 (cv. Apex). Regressions showed that severity of crown canker at harvest in July was related to severity in the spring in 1997/98 (early June, cv. Lipton), 1998/99 and 1999/2000 (April, cv. Apex).     

Association mapping of quantitative resistance for <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Stem canker caused by the fungus Leptosphaeria maculans is a major disease of Brassica napus. Quantitative resistance factors appear to be important components for effective and durable control of this pathogen. Quantitative trait loci (QTL) for stem canker resistance have previously been identified in the Darmor variety. However, before these QTL can be used in marker-assisted selection (MAS) to breed resistant varieties, they must be validated in a wide range of genetic backgrounds. We used an association mapping approach to confirm the markers located within the QTL previously identified in Darmor and establish their usefulness in MAS. For this, we characterized the molecular diversity of an oilseed rape collection of 128 lines showing a large spectrum of responses to infection by L. maculans, using 72 pairs of primers for simple sequence repeat and other markers. We used different association mapping models which either do or do not take into account the population structure and/or family relatedness. In all, 61 marker alleles were found to be associated with resistance to stem canker. Some of these markers were associated with previously identified QTL, which confirms their usefulness in MAS. Markers located in regions not harbouring previously identified QTL were also associated with resistance, suggesting that new QTL or allelic variants are present in the collection. All of these markers associated with stem canker resistance will help identify accessions carrying desirable alleles and facilitate QTL introgression.     

Effects of inoculum concentration, leaf age and wetness period on the development of dark leaf and pod spot (Alternaria brassicae) on oilseed rape (Brassica napus)

Experiments were done under controlled environment and glasshouse conditions to study the effects of inoculum concentration, leaf age and wetness period on the development of dark leaf and pod spot (Alternaria brussicae) on oilseed rape (Brassica napus). On leaves of potted oilseed rape plants (cv. Bienvenu) inoculated with A. brassicae conidial suspensions, the severity (number of lesions cm^-2) of dark leaf spot increased as inoculum concentration increased from 80 to 660 spores ml^-1and as leaf age increased from 4 to 14 days. On pods on detached racemes of spring oilseed rape (cv. Starlight), the incidence of dark pod spot (% of pods diseased) increased as inoculum concentration increased from 80 to 10⁴spores ml^-1. Increasing inoculum concentration above 10⁴spores ml^-1did not increase the incidence but did increase the severity of dark pod spot. A minimum wetness period of 4 h was needed for infection of oilseed rape leaves (cv. Envol) by A. brussicue at 18°C and disease severity increased with increasing wetness period up to 12 h. The length of dry interruptions after 3–8 h of initial wetness affected the severity of dark leaf spot. A second wetness period increased the severity of dark leaf spot if the dry interruption was ≤ 6 h and if the first wetness period was ≤ 8 h. The incubation period of A. brassicae decreased from 3.5 to 2.5 days as inoculum concentration increased from 80 to 660 spores ml^-on leaves (cv. Bienvenu) at 17–25°C and from 3.8 to 1.0 day as inoculum concentration increased from 80 to ≥2 ≥ 10³spores ml^-1on pods (cv. Starlight) at 18°C.     

The roles of ascospores and conidia of Pyrenopeziza brassicae in light leaf spot epidemics on winter oilseed rape (Brassica napus) in the UK

Ascospores of Pyrenopeziza brassicae were produced in apothecia (cup‐shaped ascomata) on oilseed rape debris. The conidia, which were morphologically identical to the ascospores, were produced in acervular conidiomata was greater than for lesions caused by ascospores. In June 2000, on the ground under a crop with light on the surface of living oilseed rape tissues. Ascospores were more infective than conidia on oilseed rape leaves. The proportion of lesions caused by conidia located on leaf veins leaf spot, numbers of petioles with apothecia decreased with increasing distance into the crop from the edge of pathways. Air‐borne ascospores of P. brassicae were first collected above debris of oilseed rape affected with light leaf spot on 5 October 1998 and 18 September 1999,12 or 23 days, respectively, after the debris had been exposed outdoors. P. brassicae conidia were first observed on leaves of winter oilseed rape on 6 January 1999 and 15 February 2000, respectively, after plots had been inoculated with debris in November 1998 and October 1999. In 1991/92, numbers of ascospores above a naturally infected crop were small from January to April and increased in June and July. P. brassicae conidia were first observed in February and the percentage plants with leaves, stems or pods with light leaf spot increased greatly in May and June. In 1992/93, in a crop inoculated with debris, numbers of airborne ascospores were small from October to January and increased from April to June. P. brassicae conidia were first observed on leaves in late November and light leaf spot was seen on stems and pods in March and June 1993, respectively.     

Relationships between phoma leaf spot and development of stem canker (Leptosphaeria maculans) on winter oilseed rape (Brassica napus) in southern England

Models were constructed to describe the relationships between incidence of phoma leaf spot at different growth stages in autumn/winter or early spring and incidence of stem canker (basal canker or stem lesions) in summer on winter oilseed rape in southern England. Model 1, describing the phoma leaf spot/basal canker relationship, was y₁=β₀+β₁x₁+β₂(x₂ ‐ x₁) if x₂ > x₁, and y₁=β₀+β₁x₁ if x₂≤x₁, in which y₁ was the incidence (% plants affected) of basal canker at harvest, x₁ was the maximum incidence of phoma leaf spot during the period from sowing to growth stage (G.S.) 1,6‐1,7 (about 100 days after sowing) and x₂ was the maximum incidence of phoma leaf spot between G.S. 1,7 and G.S. 2,0 (start of stem extension). Model 2, describing the phoma leaf spot/stem lesion relationship, was y₂=α₀+α₁x₃+α₂x₄, in which y₂ was the incidence of stem lesions at harvest, x₃ was the incidence of phoma leaf spot at G.S. 3,3–3,5 (flower buds visible) and x₄ was the incidence of phoma leaf spot at G.S. 4,5–5,5 (flower buds opening). Data from field experiments with four winter oilseed rape cultivars at Boxworth or Rothamsted in the 1992/93, 1993/94, 1996/97, 1997/98 or 1998/99 seasons were used to test the models. The values of R² for the regression equations testing model 1 for the phoma leaf spot/basal canker relationship were 0.75, 0.93, 0.91 and 0.89 for cvs Apex, Bristol, Capitol and Envol, respectively. The values of R² for the regression equations testing model 2 for the phoma leaf spot/stem lesion relationship were 0.58, 0.57, 0.54 and 0.71 for cvs Apex, Bristol, Capitol and Envol, respectively. The phoma leaf spot/basal canker relationship (model 1) could also be fitted to the combined data set for all four cultivars (R²= 0.65), whereas the phoma leaf spot/stem lesion relationship (model 2) could not to be fitted to the combined data set for the four cultivars. The relationships between incidence and severity of stem canker were examined and the values of R² for the regressions of severity on incidence were 0.91 for basal canker and 0.89 for stem lesions.     

Effects of previous cropping and fungicide timing on the development of light leaf spot (Pyrenopeziza brassicae), seed yield and quality of winter oilseed rape (Brassica napus)

Light leaf spot, caused by Pyrenopeziza brassicae, was assessed regularly on double-low cultivars of winter oilseed rape during field experiments at Rothamsted in 1990-91 and 1991-92. Previous cropping and fungicide applications differed; seed yield and seed quality were measured at harvest. In each season, both the initial incidence of light leaf spot and the rate of disease increase were greater in oilseed rape crops sown after rape than those sown after cereals. The incidence of diseases caused by Phoma lingam or Alternaria spp. was also greater in second oilseed rape crops. In 1991-92 there was 42% less rainfall between September and March than in 1990-91, and much less light leaf spot developed. However, P. lingam and Alternaria spp. were more common. Only fungicide application schedules including an autumn spray decreased the incidence of light leaf spot on leaves, stems and pods, as indicated by decreased areas under the disease progress curves (AUDPC) and slower rates of disease increase. Summer sprays decreased incidence and severity of light leaf spot on pods only. In 1990-91, all fungicide treatments which included an autumn spray increased seed and oil yields of cv. Capricorn but only the treatment which included autumn, spring and summer sprays increased yields of cv. Falcon. No treatment increased the yields of cv. Capricorn or cv. Falcon in 1991-92. Fungicide applications decreased glucosinolate concentrations in the seed from a crop of cv. Cobra severely infected by P. brassicae in 1990-91, but did not increase yield.     

Effects of temperature, cultivar and isolate on the incubation period of white leaf spot (Mycosphaerella capsellae) on oilseed rape {Brassica napus)

When leaves of oilseed rape (cv. Cobra) were inoculated with conidial suspensions of Mycosphaerella capsellae (white leaf spot) and incubated in controlled environments, the lag period from inoculation to the appearance of the first lesions decreased, and the total number of lesions produced increased, as temperature increased from 5^oC to 20^oC, although differences between 15^oC and 20^oC were small. With incubation period estimated as the time from inoculation until 5%, 50% or 95% of the lesions were produced, there was a linear relationship between l/(incubation period in days) and temperature over the range 5^oC to 20^oC, from which values at intermediate temperatures could be estimated. Summed mean daily temperatures from inoculation to the production of 5% of the lesions were estimated as 115–130 degree-days in the controlled environment experiments at 5^oC to 20^oC. When pods or leaves of plants in oilseed rape crops (cv. Cobra or cv. Libravo) were inoculated with conidial suspensions of M. capsellae on five occasions from January to October, with variable temperatures during the incubation period, degree-days until the first appearance of lesions were in the range 115–230. The numbers of white leaf spot lesions cm^-2 which developed on inoculated leaves differed greatly between nine oilseed rape cultivars, with most on cv. Tapidor and fewest on cv. Libravo, but the incubation period differed little between cultivars. Similarly, the number of lesions which developed differed between four M. capsellae isolates from different regions but the incubation period did not.     

Survival of Alternaria brassicae and Alternaria brassicicola on crop debris of oilseed rape and cabbage

Alternaria brassicae and A. brassicicola lesions present on infected leaves of oilseed rape and cabbage placed outdoors on soil produced viable spores for as long as leaf tissues remained intact. For oilseed rape this was up to 8 wk and for cabbage up to 12 wk. On leaves exposed in November and January spore concentrations decreased with time but on leaves exposed between April and June spore concentrations increased up to 9-fold in the first 4–6 wk and then declined. On stem sections of seed plants of oilseed rape and cabbage similarly placed on the soil, the fungi produced viable spores for up to 23 wk with spore concentrations increasing up to 11-fold in the first 6–8 wk after harvest. These results indicate that infected debris of brassica crops remaining on the ground after harvest may provide a source of dark leaf spot infection which may be implicated in the spread of the disease within and between crops.     

Methods for assessment of light leaf spot (Pyrenopeziza brassicae) on winter oilseed rape (Brassica napus) in the UK

Light leaf spot (Pyrenopeziza brassicae) was assessed as % plants with light leaf spot, % leaves with light leaf spot or % leaf area with light leaf spot in winter oilseed rape field experiments done at different sites (Rothamsted, Hertfordshire; Boxworth, Cambridgeshire; near Aberdeen, Scotland), with different cultivars (e.g. Bristol and Capitol), different fungicide treatments, on plants sampled at different dates. Regression analyses on data from these experiments showed that there were consistently good relationships between % leaves with light leaf spot and % plants with light leaf spot for plants sampled during the autumn and winter, until the % plants with light leaf spot approached 100%. The slopes and positions of regression lines were sometimes affected by cultivar, fungicide treatment or sampling date, but not by site. The relationship between % leaf area with light leaf spot (square root-transformed) and % leaves with light leaf spot was less consistent than that between % leaves with light leaf spot and % plants with light leaf spot and was sometimes affected by cultivar, fungicide treatment or sampling date but not by site. The relationship between % leaf area with light leaf spot (square root-transformed) and % plants with light leaf spot was also inconsistent and was sometimes affected by cultivar, fungicide treatment, sampling date and site.     

A Comparison of the Disease Reactions of Stems and Detached Leaves of Soil and in vitro Grown Plants and Regenerants of Oilseed Rape to Leptosphaeria maculans and Protocols for Selection for Novel Disease Resistance

Protocols for selecting plant tissues of winter oilseed rape (Brassica napus subsp. oleifera) with resistance to Leptosphaeria maculans by either stem or leaf inoculation of both soil and in vitro grown plant material are described. The stem inoculation procedure gave good correlation (r = 0. 92) between the 50 day stem disease scores of eight out of nine cultivars of soil grown winter oilseed rape inoculated with isolate 41A4 of L. maculans and the N. A. B. esistance ratings or resistance data from field trials. The exception was the cultivar Liradonna. Inoculation of stems of five cultivars with isolates 41A4, 433 and 478 indicated a range of isolate virulence 478 > 41A4 > 433. This was the inverse of that observed in leaf inoculations. Application of the stem inoculation procedure to in vitro shoot cultures allowed differentiation of resistant and susceptible cultivars, including the cultivar Liradonna, after 20 days incubation at 20°C. The protocol was also applicable to plantlets regenerated from thin cell layer explants grown in vitro. Inoculations with isolate 433 allowed the differentiation of resistant, intermediately resistant and susceptible leaf material of soil grown plants, when leaf discs from young leaves were incubated on water agar supplemented with BAP (1 × 10^?5 M) at 25°C for 10 days. Intermediately resistant leaves were resistant after 10 days and susceptible after 15 days of incubation. Leaves of shoot cultures grown in vitro were more susceptible than the corresponding soil grown material. However, inoculation of old leaves with isolate 41A4 (an isolate of less virulence on leaves than 433) distinguished the cultivars after 15 days of incubation. These protocols allow the accurate assessment of resistance to L. maculans at the stem or leaf level and are of use in traditional as well as in vitro selection programmes.     

Crystal structure of the effector AvrLm4–7 of Leptosphaeria maculans reveals insights into its translocation into plant cells and recognition by resistance proteins

The avirulence gene AvrLm4–7 of Leptosphaeria maculans, the causal agent of stem canker in Brassica napus (oilseed rape), confers a dual specificity of recognition by two resistance genes (Rlm4 and Rlm7) and is strongly involved in fungal fitness. In order to elucidate the biological function of AvrLm4–7 and understand the specificity of recognition by Rlm4 and Rlm7, the AvrLm4–7 protein was produced in Pichia pastoris and its crystal structure was determined. It revealed the presence of four disulfide bridges, but no close structural analogs could be identified. A short stretch of amino acids in the C terminus of the protein, (R/N)(Y/F)(R/S)E(F/W), was well‐conserved among AvrLm4–7 homologs. Loss of recognition of AvrLm4–7 by Rlm4 is caused by the mutation of a single glycine to an arginine residue located in a loop of the protein. Loss of recognition by Rlm7 is governed by more complex mutational patterns, including gene loss or drastic modifications of the protein structure. Three point mutations altered residues in the well‐conserved C–terminal motif or close to the glycine involved in Rlm4‐mediated recognition, resulting in the loss of Rlm7‐mediated recognition. Transient expression in Nicotiana benthamiana (tobacco) and particle bombardment experiments on leaves from oilseed rape suggested that AvrLm4–7 interacts with its cognate R proteins inside the plant cell, and can be translocated into plant cells in the absence of the pathogen. Translocation of AvrLm4–7 into oilseed rape leaves is likely to require the (R/N)(Y/F)(R/S)E(F/W) motif as well as an RAWG motif located in a nearby loop that together form a positively charged region.     

Effects of experimental warming on fungal disease progress in oilseed rape

Global warming will influence the growth and development of both crops and pathogens. The aims of this study were to investigate potential effects of future warming on oilseed rape growth and the epidemiology of the three economically important pathogens Verticillium longisporum, Sclerotinia sclerotiorum, and Leptosphaeria maculans (anamorph: Phoma lingam). We utilized climate chambers and a soil warming facility, where treatments represented regional warming scenarios for Lower Saxony, Germany, by 2050 and 2100, and compared results of both approaches on a thermal time scale by calculating degree‐days (dd) from day of sowing, December 1st and March 1st until sampling, the latter correlating best with disease progress. Regression analysis showed that plant growth and growth stages in spring responded almost linearly to increasing thermal time until 1000–1500 dd. Colonization of plant tissue by V. longisporum showed an exponential increase when exceeding 1300–1500 dd and reaching plant growth stage BBCH 74/75 (pod development). V. longisporum colonization of plants may be advanced, potentially leading to higher inoculum densities after harvest and increased economic importance of this pathogen under future warming. Sclerotia germination of S. sclerotiorum reached its maximum at 600–900 dd. Advance of these critical degree‐days may lead to earlier apothecia production, potentially advancing the infection window, whereas the future importance of S. sclerotiorum may remain constant. Severity of phoma crown canker increased linearly with increasing thermal time, but showed also large variation in response to the warming scenarios, suggesting that factors such as canopy microclimate in fall or leaf shedding over winter may play a bigger role for L. maculans infection and disease severity than higher soil temperatures. Thermal time was a suitable tool to combine and integrate data on biological responses to soil and air temperature increases from climate chamber and field experiments.     

Analyses of air samples for ascospores ofLeptosphaeria maculans andL.biglobosa by light microscopy and molecular techniques

Spores of many fungal pathogens are dispersed by wind. Detection of these airborne inocula is important in forecasting both the onset and the risk of epiphytotics. Species-specific primers targeted at the internal transcribed spacer (ITS) region ofLeptosphaeria maculans andL. biglobosa — the causal organisms of phoma stem canker and stem lesions ofBrassica spp., including oilseed rape — were used to detect DNA extracted from particles deposited on tapes obtained from a spore trap operated in Rarwino (northwest Poland) from September to November in 2004 and 2006. The quantities of DNA assessed by traditional end-point PCR and quantitative real-time PCR were compared to microscopic counts of airborne ascospores. Results of this study showed that fluctuations in timing of ascospore release corresponded to the dynamics of combined concentrations of DNA fromL. maculans andL. biglobosa, with significant positive correlations between ascospore number and DNA yield. Thus the utilization of PCR-based molecular diagnostic techniques enabled the detection, identification, and accurate quantification of airborne inoculum at the species level. Moreover, real-time PCR was more sensitive than traditional PCR, especially in years with low ascospore numbers.     

Effects of light leaf spot (Pyrenopeziza brassicae) on yield of winter oilseed rape (Brassica napus)

The relationship between development of light leaf spot and yield loss in winter oilseed rape was analysed, initially using data from three experiments at sites near Aberdeen in Scotland in the seasons 1991/92, 1992/93 and 1993/94, respectively. Over the three seasons, single-point models relating yield to light leaf spot incidence (% plants with leaves with light leaf spot) at GS 3.3 (flower buds visible) generally accounted for more of the variance than single-point models at earlier or later growth stages. Only in 1992/93, when a severe light leaf spot epidemic developed on leaves early in the season, did the single-point model for disease severity on leaves at GS 3.5/4.0 account for more of the variance than that for disease incidence at GS 3.3. In 1991/92 and 1992/3, when reasonably severe epidemics developed on stems, the single-point model for light leaf spot incidence (stems) at GS 6.3 accounted for as much of the variance. Two-point (disease severity at GS 3.3 and GS 4.0) and AUDPC models (disease incidence/severity) accounted for more of the variance than the single-point model based on disease incidence at GS 3.3 in 1992/93 but not in the other two seasons. Therefore, a simple model using the light leaf spot incidence at GS 3.3 (x) as the explanatory variable was selected as a predictive model to estimate % yield loss (y_r): y_r= 0.32x– 0.57. This model fitted all three data sets from Scotland, When data sets from Rothamsted, Rosemaund and Thurloxton in England were used to test it, this single-point predictive model generally fitted the data well, except when yield loss was clearly not related to occurrence of light leaf spot. However, the regression lines relating observed yield loss to light leaf spot incidence at GS 3.3 often had smaller slopes than the line produce, by the model based on Scottish data.     

Studies on taphrina maculans butler,inciting leaf spot of turmeric (curcuma longa L.). I. Isolation of the pathogen

Summary T. maculans inciting leaf spot of turmeric is difficult to be isolated into a pure culture. The best stage of the spot development on turmeric which yielded successful isolations was either the initial or the middle stage expressing yellowish necrotic areas. The successful course of development of asci, ascospore and conidia has been worked out. By streaking ascospore and conidial suspensions, the spot bearing portions of leaves on the inner side of the lid thus placing them in hanging position above the plates poured with cleared and acidified potato dextrose agar or turmeric leaf decoction agar or by streaking sterile water washings from the chocolate brown spots on these media, it was possible to isolate successfully the pathogenic culture ofT. maculans at 20° C.Condensed from the thesis submitted by the senior author to the University of Poona for M. Sc. (Agri.) under the guidance of the junior author. Respectively Assistant Professor of Plant Pathology, College of Agri. Poona and Wheat Rust Mycologist, Mahabaleshwar, India.     

Defoliation of Brassica napus increases severity of blackleg caused by Leptosphaeria maculans: implications for dual-purpose cropping

Canola (Brassica napus) crops for grazing and grain (dual-purpose) production provide an economic break-crop alternative for dual-purpose cereals in Australian mixed farming systems. Infection by Leptosphaeria maculans is the most prevalent disease in Australian canola crops with airborne inoculum released throughout the autumn and winter when crops are grazed. Glasshouse and field experiments were conducted to investigate the effect of mechanical defoliation (simulated grazing) on disease severity at plant maturity. In glasshouse experiments, stem canker severity increased from 4% to 24% in severely defoliated plants, but light defoliation had no effect compared with undefoliated control plants. Disease severity was increased with defoliation in all field experiments. Defoliation increased crown canker severity from 22.6% to 39.3% at Wagga Wagga and from 3.0% to 7.1% at Canberra and lodging from 9.6% to 11.9% at Naracoorte in the same set of cultivars assessed at each site. The increase in disease severity with defoliation was less in canola lines with moderate to high levels of stem canker resistance. Plants defoliated before stem elongation tended to develop less disease than those defoliated during the reproductive phase of plant growth. These findings suggest that the impact of grazing on L. maculans infection of canola crops can be minimised by sowing cultivars with a high level of stem canker resistance and grazing during the vegetative stage of plant growth prior to stem elongation. Further research is required to determine whether these management strategies are applicable in canola crops defoliated by grazing animals.