油菜茎基溃疡病菌的实时荧光PCR检测

 根据油菜茎基溃疡病菌Leptosphaeria maculans与其近似种ITS序列的差异,设计了检测L. maculans的引物Lmb3/R2和探针Probe-M,建立了L. maculans的实时荧光PCR检测方法。试验结果表明,来自加拿大、澳大利亚和乌克兰等国的22株L. maculans菌株都能得到阳性扩增,而供试的30株L. biglobosa菌株和6株其他菌株以及空白对照没有荧光信号的增加。该检测方法的灵敏度达到4 pg菌丝DNA,整个检测过程控制在4 h内,其快速、特异和灵敏的特点可以满足进境油菜籽样品的快速初检以及病菌分离物的快速鉴定。     

Detection of airborne inoculum of Leptosphaeria maculans and Pyrenopeziza brassicae in oilseed rape crops by polymerase chain reaction (PCR) assays

The potential use of DNA-based methods for detecting airborne inoculum of Leptosphaeria maculans and Pyrenopeziza brassicae , both damaging pathogens of oilseed rape, was investigated. A method for purifying DNA from spores collected using Hirst-type spore samplers and detecting it using polymerase chain reaction (PCR) assays is described. For both pathogens, the sensitivities of the DNA assays were similar for spore-trap samples and pure spore suspensions. As few as 10 spores of L. maculans or P. brassicae could be detected by PCR and spores of both species could be detected against a background of spores of six other species. The method successfully detected spores of P. brassicae collected using spore traps in oilseed rape crops that were infected with P. brassicae. Leptosphaeria maculans spores were detected using spore traps on open ground close to L. maculans -infected oilseed rape stems. The potential use of PCR detection of airborne inoculum in forecasting the diseases caused by these pathogens is discussed.     

Detection and quantification of airborne inoculum of Sclerotinia sclerotiorum using quantitative PCR

This paper reports the development of a new specific diagnostic technique to accurately quantify airborne inoculum of Sclerotinia sclerotiorum and discusses its potential use in disease-forecasting schemes, using examples of three contrasting epidemic seasons: 2007, when there was a severe epidemic of sclerotinia stem rot (SSR) in England and high numbers of airborne ascospores were trapped at Rothamsted, and, in contrast, 2003 and 2004, when the incidence of SSR in England was low and low numbers of airborne ascospores were trapped at Rothamsted. DNA was extracted from wax-coated plastic tapes, such as those used in Burkard (Hirst-type) spore traps and rotating-arm traps. A SYBR-green quantitative PCR (qPCR) method produced a linear relationship between ascospore numbers and S. sclerotiorum DNA (mean 0·35 pg DNA per spore) and was able to detect DNA representing as few as two ascospores. The technique was insensitive to DNA of the host plant, Brassica napus , and other plant pathogens, including Sclerotinia minor , S. trifoliorum and Botrytis cinerea , and common airborne fungal genera such as Cladosporium and Penicillium . There was no relationship between rainfall and numbers of airborne ascospores of S. sclerotiorum present at Rothamsted during the period of infection in the severe SSR season (2007).     

进境澳大利亚油菜籽中茎基溃疡病菌的检测

 41 fungal isolates with similar morphological characteristics to Leptosphaeria maculans were obtained by the deep-freezing filter paper method from 2100 seeds of Brassica napus imported from Australia.The isolate 8129-5 showed a slower growth on PDA at 20℃with growth rate of 2.8 mm/day.The colonies on PDA at 20℃ had an irregular or regular margin with white or grayish white compact aerial mycelium.No diffusible pigment was produced on PDA at 31℃ or in liquid Czapek-Dox media at 20℃.PCR detection showed that the isolate 8129-5 could be amplified by L.maculans-specific primers LmacF/LmacR and got expected product of 331 bp.The sequence analysis revealed that the ITS sequence of isolate 8129-5 had 99.8% identity with L.maculans.Pathogenicity of the isolate 8129-5 was confirmed on cotyledons of rape seed by artificial inoculation compared with typical symptom of L.maculans.Based on the morphological characteristics, PCR detection and the result of pathogenicity test, the isolate 8129-5 was identified as L.maculans.     

Effect of rotation of canola (Brassica napus) cultivars with different complements of blackleg resistance genes on disease severity

Blackleg disease (phoma stem canker) caused by the fungus Leptosphaeria maculans is a major disease of canola (oilseed rape, Brassica napus) worldwide. Canola plants in pots were exposed to blackleg‐infested stubble of canola with different complements of resistance genes and then assessed for disease. Plant mortality was reduced when plants were exposed to stubble from a cultivar with a different complement of resistance genes compared to stubble of a cultivar with the same resistance gene. These findings were consistent with 7 years of field surveys, which showed that changes in selection pressure as a result of extensive sowing of cultivars with major‐gene resistance, termed ‘sylvestris resistance’, dramatically influenced the frequency of virulent isolates in the population towards particular resistance genes, and therefore disease severity. All these data were supported by PCR‐genotyping surveys of fungal populations whereby the frequency of virulence alleles of avirulence genes AvrLm1 and AvrLm4 changed significantly depending on the resistance gene present in the cultivar from which the isolates were cultured. This is the first example of a study showing that sowing of canola cultivars with different complements of resistance genes in subsequent years, i.e. rotation of resistance genes, minimizes disease pressure by manipulating fungal populations. This approach provides a valuable disease management strategy for canola growers and is likely to be applicable to other plant diseases.     

Survival of Leptosphaeria maculans and associated mycobiota on oilseed rape stubble buried in soil

Leptosphaeria maculans , the causal agent of phoma stem canker on oilseed rape, is an important pathogen in oilseed rape growing regions of the world, including Australia. Survival of L. maculans and associated mycobiota on oilseed rape stubble buried for 13 months in field soil and in sandy soil was studied under South Australian environmental conditions. Stubble weight decreased significantly by the end of the burial period, more so in field (53·7%) than in sandy soil (22%). Pseudothecia did not develop on stubble buried in field soil and few formed when buried in sandy soil. Moist incubation of stubble following retrieval from both media generated pseudothecia; however, pseudothecial development ceased on stubble that had been buried for 10 and 12 months in field and sandy soil, respectively. In total, 20 and 36 genera of fungi were isolated from stubble before and after burial, respectively. Alternaria spp., L. maculans and Stemphylium botryosum were isolated from 81·7, 70 and 60% of stubble pieces before burial, respectively. Isolation frequency of these species decreased significantly throughout the burial period in both media. Conversely, isolation frequency of Stachybotrys chartarum , Fusarium spp. and Trichoderma spp., having pre-burial frequencies of 26·7, 16·7 and 2·5%, respectively, increased over the burial period regardless of soil type. These findings suggest that inoculum production of L. maculans decreases with the increasing burial duration in field soil over 10 months, before ceasing, and this may be due to associated mycobiota.     

Effects of Severity and Timing of Stem Canker (Leptosphaeria maculans) Symptoms on Yield of Winter Oilseed Rape (Brassica napus) in the UK

The relationships between yield loss and incidence (% plants with stems affected) or severity (mean stem score, 0–4 scale) of stem canker in winter oilseed rape were analysed using data from experiments at Rothamsted in 1991/92, Withington in 1992/93, Boxworth in 1993/94 and Rothamsted in 1997/98. Critical point models and area under disease progress curve (AUDPC) models were better than multiple point models for describing relationships between yield (tha^–1) and incidence or severity of stem canker for the four experiments. Since yield is influenced by many factors other than disease, % yield loss was calculated and critical point models and AUDPC models relating % yield loss to stem canker were constructed. The critical point models for % yield loss on stem canker incidence for three of the four experiments were similar, but differed from that for Rothamsted in 1991/92. There were also no differences between models of % yield loss on AUDPC of both incidence and severity for these three experiments. Therefore, general models of % yield loss (L) against AUDPC of incidence (X) or severity (S) of stem canker from growth stages 4.8 to 6.4 were derived from the combined data sets for the three experiments: L=–0.76+0.0075X (R²=35%, p<0.001), L=0.26+0.53S (R²=37%, p<0.001). The relationships between % yield loss and % plants with different stem canker severity scores at different growth stages were also analysed; the greatest yield losses were generally associated with the largest severity scores, for plants assessed at the same crop growth stage, and were also associated with the early development of stem lesions. Further analyses showed that % yield loss was related to incidence or severity of both basal stem cankers and upper stem lesions in experiments at Boxworth in 1993/94 and at Rothamsted in 1997/98.     

Effects of cultivar resistance and fungicide application on stem canker of oilseed rape (Brassica napus) and potential interseasonal transmission of Leptosphaeria spp. inoculum

Phoma stem canker is a damaging disease of oilseed rape (Brassica napus) that causes annual yield losses to UK oilseed rape growers worth approximately £100 million, despite the use of fungicides. In the UK, oilseed rape is sown in August/September and harvested in the following July. The disease epidemics are initiated by ascospores released from Leptosphaeria spp. pseudothecia (ascocarps) on stem stubble in the autumn/winter. Control of this disease is reliant on the use of cultivars with “field resistance” and azole fungicides. This study investigated the effects of cultivar resistance and application of the fungicide prothioconazole on the severity of stem canker before harvest and the subsequent production of pseudothecia on the infected stubble under natural conditions in the 2017/2018, 2018/2019, and 2019/2020 cropping seasons. The application of prothioconazole and cultivar resistance decreased the severity of phoma stem canker before harvest, and the subsequent production of Leptosphaeria spp. pseudothecia on stubble in terms of pseudothecial density. Results showed that stems with less severe stem cankers produced fewer mature pseudothecia of Leptosphaeria spp. on the infected stubble. This investigation suggests that the most sustainable and effective integrated control strategy for phoma stem canker in seasons with low quantities of inoculum is to use cultivars with medium or good field resistance and apply only one spray of prothioconazole when required.     

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.     

Effectiveness of Rlm7 resistance against Leptosphaeria maculans (phoma stem canker) in UK winter oilseed rape cultivars

The Rlm7 gene in Brassica napus is an important source of resistance for control of phoma stem canker on oilseed rape caused by the fungus Leptosphaeria maculans. This study shows the first report of L. maculans isolates virulent against Rlm7 in the UK. Leptosphaeria maculans isolates virulent against Rlm7 represented 3% of the pathogen population when cultivars with the Rlm7 gene represented 5% of the UK oilseed rape area in 2012/13. However, the Rlm7 gene has been widely used since then, representing >15% of the UK oilseed rape area in 2015/16. Winter oilseed rape field experiments included cultivars with the Rlm7 gene, with the Rlm4 gene or without Rlm genes and took place at five sites in the UK over four cropping seasons. An increase in phoma leaf spotting severity on Rlm7 cultivars in successive seasons was observed. Major resistance genes played a role in preventing severe phoma leaf spotting at the beginning of the cropping season and, in addition, quantitative resistance (QR) in the cultivars examined made an important contribution to control of phoma stem canker development at the end of the cropping season. Deployment of the Rlm7 resistance gene against L. maculans in cultivars with QR in combination with sustainable disease management practices will prolong the use of this gene for effective control of phoma stem canker epidemics.     

Monitoring of plant and airborne inoculum of Sclerotinia sclerotiorum in spring oilseed rape using real‐time PCR

Sclerotinia stem rot of spring oilseed rape (Brassica napus) is caused by Sclerotinia sclerotiorum. In Sweden, the disease leads to severe crop damage that varies from year to year. A real‐time PCR assay was developed and used to determine the incidence of S. sclerotiorum DNA on petals and leaves of spring oilseed rape as well as in air samples, with the aim of finding tools to improve precision in disease risk assessment. Five field experiments were conducted from 2008 to 2010 to detect and study pathogen development. Assessments of stem rot showed significant differences between experimental sites. The real‐time PCR assay proved fast and sensitive and the relationship between percentage of infected petals determined using a conventional agar test and the PCR assay was linear (R² > 0·76). There were significant differences in S. sclerotiorum incidence at different stages of flowering. The incidence of S. sclerotiorum DNA on the leaves varied (0–100%), with significantly higher incidence on leaves at lower levels. In one field experiment, S. sclerotiorum DNA was not detected on petals during flowering, whereas the pathogen was detected on leaves, with a corresponding stem rot incidence of 7%. The amount of S. sclerotiorum DNA in sampled air revealed that spore release did not coincide with flowering on that experimental site. Thus, using a real‐time PCR assay to determine the incidence of S. sclerotiorum on oilseed rape leaves, rather than on petals, could potentially improve disease risk assessment.     

Methods for Sampling and Assessment in Relation to the Spatial Pattern of Phoma Stem Canker (Leptosphaeria maculans) in Oilseed Rape

A sound assessment of phoma stem canker symptoms is needed to develop epidemiological, agronomical and physiological studies on the pathosystem. A specific analysis was therefore carried out to: (i) compare four methods of crown canker assessment; (ii) test the among and within assessor repeatability of one of the methods compared; (iii) characterise the spatial pattern of the disease; and (iv) define the sample size required to achieve a given level of disease assessment precision. The methods compared examined the symptoms with different procedures and graded the plants observed into six severity classes. A disease index (DI) summarised the severity distribution observed. Examination of crown cross-sections was the most precise method for assessing crown cankers. The method was repeatable, though an 'assessor effect' was apparent. The disease generally had a random pattern although significant spatial correlations were detected for four out of the fifteen plots studied at the scales examined. A relationship between the coefficient of variation of the DI and the sample size was established, evaluated with experimental field data and exemplified for typical severity distributions.     

Spatiotemporal variation in airborne sporangia of Phytophthora infestans: characterization and initiatives towards improving potato late blight risk estimation

This study investigated the value of using real‐time monitoring of Phytophthora infestans airborne inoculum as a complement to decision support systems (DSS). The experiment was conducted during the 2010, 2011 and 2012 potato production seasons in two locations in New Brunswick, Canada. Airborne sporangia concentrations (ASC) of P. infestans were monitored using 16 rotating‐arm spore samplers placed 3 m above the ground. The first cases of late blight (2010 and 2011) were detected 6–7 days after the first ASC peak, and all samplers captured their first sporangia within the same week (at 3‐ and 9‐day periods). The cumulative ASC curve and the risk curves from two DSS (PLANT‐Plus and Pameseb Late Blight) had the same shape but different magnitudes. In both locations, the negative binomial distribution fitted the data better than the Poisson distribution, which is indicative of heterogeneity, and based on Taylor's power law, the heterogeneity increased with increasing ASC. Therefore, the present results suggest that spore‐sampling network devices may be a suitable approach for early detection of incoming inoculum and, when combined with DSS, represent a potential aid for targeting the optimal time to apply a disease‐control product. In this context, cumulative ASC can be a counterweight to the DSS risk estimate: a high risk combined with significant ASC will trigger fungicide spraying. Moreover, spore sampling can be used to assess the efficiency of management strategies by means of examining the area under the inoculum progress curve.     

Detection and quantification of Ilyonectria spp. associated with black‐foot disease of grapevine in nursery soils using multiplex nested PCR and quantitative PCR

Three nursery fields and three rootstock mother fields from commercial nurseries located in Comunidad Valenciana region (central‐eastern Spain) were surveyed in July 2011 to detect the presence and to quantify Ilyonectria spp. in the soil. In each field, ten soil samples were taken randomly with a soil probe at a depth of 10–30 cm, and 10–20 cm from the base of the plant. Three replicate subsamples (10 g each) were taken from each soil sample. DNA was extracted and a multiplex nested PCR with species‐specific primer pairs (Mac1/MaPa2, Lir1/Lir2 and Pau1/MaPa2) was used to identify the species present. Among the 180 soil DNA samples analysed, Ilyonectria spp. were detected in 172 of them. Ilyonectria macrodidyma complex was the most frequently detected, being identified in 141 samples from all the fields evaluated. However, I. liriodendri was detected in only 16 samples, but was present in all open‐root field nurseries and in two rootstock mother fields. In addition, quantitative PCR (qPCR) assays were done to assess the levels of I. liriodendri and I. macrodidyma‐complex DNA in the soil samples. Detection of Ilyonectria spp. DNA using qPCR correlated with the fields found positive with the nested multiplex PCR. DNA concentrations of Ilyonectria spp. ranged from 0·004 to 1904·8 pg μL^?1. In general, samples from rootstock mother fields showed the highest DNA concentrations. The ability to detect and quantify Ilyonectria spp. genomic DNA in soil samples from nursery fields and rootstock mother fields confirms soils from both field types as important inoculum sources for black‐foot pathogens.