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.     

Biochemical defense mechanisms induced in winter oilseed rape seedlings with different susceptibility to infection with Leptosphaeria maculans

The activity of key biochemical defense mechanisms in seedlings of winter oilseed rape cultivars, differing in tolerance to infection with Leptosphaeria maculans, was investigated. The studied winter oilseed rape cultivars differed in their biochemical defense potential. In the resistant cultivar, a significant increase in the activity of chitinase and β-1,3-glucanase was observed that can be regarded as a symptom of a systemic defense reaction. In addition, the resistant cultivar had a more efficient antioxidant system, that is, higher activity of specific SOD isoforms and higher level of low-molecular antioxidants. Only the resistant cultivar had three Fe-SOD sub-isoforms. A correlation between the content of cell wall-bound phenolics and hydrogen peroxide in the resistant cultivar may indicate that an important method of H₂O₂ neutralization was its participation in the process of phenolics incorporation into the cell wall. Elevated content of cell wall-bound phenolics significantly enhances the cell wall integrity, which then becomes a more effective firewall against L. maculans. Moreover, high utilization of soluble sugars during synthesis of the phenolic compounds in the pathogenesis was demonstrated in the resistant cultivar. We conclude that the key factors contributing to a greater tolerance to L. maculans were an effective antioxidant system and higher activity of PR proteins, i.e., chitinase and β-1,3-glucanase.     

Latent infection of winter oilseed rape by Leptosphaeria maculans

Plants of oilseed rape, cultivars Primer and Jet Neuf, were grown in a glasshouse and inoculated at G.S. 2.4–2.7 with pycnidiospores or ascospores of Leptosphaeria maculans. The plants were kept for a further 2–4 weeks at 14°C and then transferred, together with uninoculated plants, to a polythene tunnel in winter. The majority of stems of inoculated plants did not have macroscopic symptoms of L. maculans infection 6 weeks after inoculation. Examination of whole mounts of peripheral tissue and transverse sections of fixed and embedded portions of these stems revealed intercellular septate fungal hyphae, often deep in non-necrotic cortical tissue, in symptomless inoculated plants but not in uninoculated plants. L. maculans was recovered following surface sterilization of adjacent portions of the same stems. When symptomless inoculated plants were transferred to a glasshouse at 18–20°C, cankers soon developed. The significance of these latent mycelial infections to canker development in the field is discussed.     

Screening for resistance to canker (Leptosphaeria maculans) in winter oilseed rape (Brassica napus ssp. oleifera)

A test involving infection of seedlings in a glasshouse was evaluated for potential use in screening breeding material by comparing the results with those of field tests. The two tests correlated well for some resistant selections, particularly those closely related to Jet Neuf, and for very susceptible selections. However, two small groups of selections were resistant in the field but consistently susceptible in the glasshouse. Inoculation of mature plants in the glasshouse resulted in similar discrepancies and therefore gave no advantage over the seedling test. These discrepancies limit the usefulness of glasshouse techniques for routine resistance screening. A very high correlation was found overall between internal and external canker symptoms of the crown in nine field trials. Significant differences were found, however, between selections and cultivars in the relative severity of these two symptoms. Upper stem canker and crown canker were found to be very poorly correlated for individual plants, but highly correlated for eultivar means. The significance of these observations is discussed with respect to scoring canker in the field.     

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.     

The full life cycle of Leptosphaeria maculans completed on inoculated oilseed rape incubated under controlled conditions

Because epidemics of successive cropping seasons are not independent, epidemiological studies need to encompass the processes occurring during the transmission of epidemics from one season to the next. With Leptosphaeria maculans, infected stubble allows carry‐over of the fungus. Generation experiments using recurrent selection on field plots are a useful means of comparing the effects of selection pressures. However, the full life cycle of the fungus, from plant infection to the next generation of ascospores, has not yet been achieved under controlled conditions. Studies were undertaken to achieve an experimental set‐up with sexual reproduction under controlled conditions. Cankered oilseed rape stems were produced under controlled conditions, after inoculation with a mixture of 12 isolates across both mating types. Stems were cut longitudinally and attached to styropore plates. Stem halves were incubated outside or in climate chambers regularly soaked in tap water to ensure maturation. Incubation was stopped when mature pseudothecia were observed. In all three independent experiments, more stem halves had pseudothecia when incubated under controlled conditions (30–100%) than incubated outside (0–80%). To the authors’ knowledge, this is the first study achieving the full life cycle of the fungus under controlled conditions, from infection of the plant to mature pseudothecia. This opens up the prospect of running experiments year‐round to better understand inoculum production, to compare fungal fitness, or to run generation experiments with exotic pathogen populations.     

First record of ascomata of Leptosphaeria maculans on winter-grown oilseed rape in Denmark

The ascomata of Leptosphaeria maculans were observed for the first time in Denmark in 1991. Morphological data and cultural characteristics were consistent with previously published data.     

The establishment of systemic infection in leaves of oilseed rape by Leptosphaeria maculans

In a series of growth room experiments in which leaves of Brassica napus var. oleifera were inoculated with ascospores or pycnidiospores of Leptosphaeria maculans successful infections progressed through three consecutive phases. Initial establishment in the mesophyll was succeeded by a phase of intercellular exploration, when hyphal proliferation was highly variable and host cell necrosis always ensued, and then by a systemic phase when hyphae were consistently sparse. Host cells associated with the hyphal front were capable of autofluorescence, accumulation of vital stains and plasmolysis, indicating that they were viable and that the pathogen was biotrophic throughout this sequence. During either of the first two phases permanent fungistatic containment, involving the formation of vesicles by disintegration of the hyphae, often occurred. Localization at the first phase was symptomless; at the second it was signified by a lesion with a clearly defined margin.
There was a negative correlation between biotrophic potential and necrotrophic potential of three pathogenic isolates, on both the moderately susceptible cultivar Primor and the resistant cultivar Jet Neuf. As leaves aged, a progressively larger proportion of infections failed to become systemic. With increasing inoculum load, symptomless localization of infection diminished, the phase of necrosis extended, and the probability of irreversible systemic development increased.     

A systemic pathway in the infection of oilseed rape plants by Leptosphaeria maculans

In growth room regimes arranged to simulate field conditions which coincide with natural infection of oilseed rape by Leptosphaeria maculans , leaf inoculation resulted in systemic infection. After colonizing intercellular spaces in the spongy mesophyll of the lamina, the fungus reached a vascular strand and spread down the petiole mainly in xylem vessels or between cells of the xylem parenchyma and cortex, eventually invading and killing cells of the stem cortex and causing the stem canker symptom. The intercellular systemic phase of growth, which was biotrophic and virtually sytnptomless, occurred under a wide range of temperatures.     

Quantitative resistance to symptomless growth of Leptosphaeria maculans (phoma stem canker) in Brassica napus (oilseed rape)

Quantitative resistance to Leptosphaeria maculans in Brassica napus was investigated in field and controlled environments using cultivars Darmor (with quantitative resistance) and Eurol (without quantitative resistance). In field experiments, numbers of phoma leaf spot lesions in autumn/winter and severity of stem canker the following summer were assessed in three growing seasons. There were no differences between Darmor and Eurol in number of leaf lesions in autumn/winter. However, stem cankers were less severe on Darmor than Eurol at harvest the following summer. In controlled-environment experiments, development of leaf lesions at different temperatures (5–25°C) and wetness durations (12–72 h) was investigated using ascospore inoculum; symptomless growth of L. maculans along leaf petioles towards the stem was quantified using quantitative PCR and visualized using GFP-expressing L. maculans ; growth of L. maculans within stem tissues was investigated using GFP-expressing L. maculans . There were more leaf lesions on Darmor than Eurol, although there was no difference between Darmor and Eurol in L. maculans incubation period. There were no differences between Darmor and Eurol in either distance grown by L. maculans along leaf petioles towards the stem or quantity of L. maculans DNA in leaf petioles, but L. maculans colonized stem tissues less extensively on Darmor than Eurol. It was concluded that quantitative resistance to L. maculans operates during colonization of B. napus stems by the pathogen.     

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.     

Entwicklung einer Herbstprognose für den Erreger der Wurzelhals- und Stängelfaule Phoma lingam (Teleomorph: Leptosphaeria maculans)

Based on experimental data of oilseed rape monitoring in Schleswig-Holstein during the last seven years and actually established monitoring at four sites in Germany, a forecasting system for a determined autumn application against the important rape pathogen root collar (Phoma lingam; teleomorph: Leptosphaeria maculans) was developed. Early primary infections by ascospores in autumn are the elicitors of severe yield-reducing root collar epidemics, which depend on weather conditions, especially rainfall, leaf wetness and precipitation. Based on measured data and on trial weather records, a model was developed to predict the daily infection risk. Threshold values for a determined application were calculated based on disease incidence monitoring values. Applications predetermined from the calculated “daily infection value” reduced pathogen populations in autumn in comparison to the untreated control. Effects of the autumn treatments on root collar could be recorded; however, a significant yield effect occurred at one site only. Weather conditions over winter until vegetation start in the following year influence root collar severely. Due to this conclusion it is difficult to evaluate prosperous autumn treatments.     

Splash dispersal of Leptosphaeria maculans pycnidiospores and the spread of blackleg on oilseed rape

The fungus Leptosphaeria maculans causes blackleg (phoma stem canker), one of the most serious diseases of oilseed rape. The role of pycnidiospores produced during asexual reproduction is poorly documented and limits the understanding of the pathogen's population dynamics. The objectives of this study were to assess rain-splash dispersal of pycnidiospores of L. maculans from phoma leaf spots, and transmission of the disease from oilseed rape stubble carrying pycnidia. The work was conducted in still air with either a drop generator or a rain simulator. The impact of simulated incident drops on phoma leaf spots resulted in the dispersal of L. maculans pycnidiospores within splash droplets. Ninety per cent of the spores were collected within 14 cm of the source and a few were regularly observed up to 40 cm. Pycnidiospores produced on oilseed rape stubble and dispersed by simulated rain infected oilseed rape trap plants in a spatial pattern that matched the spatial dispersal of the pycnidiospores. In the field, rain-splash dispersal of pycnidiospores could increase the pathogen population and may enhance sexual reproduction by facilitating the mating of initially spatially separated isolates of opposite mating type.     

Epidemiology and management of Leptosphaeria maculans (phoma stem canker) on oilseed rape in Australia, Canada and Europe

Phoma stem canker (blackleg), caused by Leptosphaeria maculans , is an important disease on oilseed rape (canola, rapeseed, Brassica napus , Brassica juncea , Brassica rapa ) causing seedling death, lodging or early senescence in Australia, Canada and Europe, but not in China. The two forms of L. maculans (A group and B group) that occur on oilseed rape are now considered to be separate species. The epidemiology and severity of phoma stem canker differs between continents due to differences in the pathogen population structure, oilseed rape species and cultivars grown, climate and agricultural practices. Epidemics are most severe in Australia, where only the A group occurs, and can be damaging in Canada and western Europe, where both A and B groups occur, although their proportions vary within regions and throughout the year. Epidemics are slight in China, where the A group has not been found. Dry climates (Australia, western Canada) lengthen the persistence of infected debris and may synchronize the release of airborne ascospores (after rain) with seedling emergence. L. maculans spreads from cotyledon and leaf infections down petioles to reach the stem, with infections on cotyledons and leaves early in the season producing the most damaging stem cankers at the stem base (crown). Development of both crown cankers and phoma stem lesions higher up stems is most rapid in regions with high temperatures from flowering to harvest, such as Australia and Canada. Breeding for resistance (genetic, disease escape or tolerance), stubble management, crop rotation and fungicide seed treatments are important strategies for control of phoma stem canker in all areas. Fungicide spray treatments are justified only in regions such as western Europe where high yields are obtained, and accurate forecasts of epidemic severity are needed to optimize their use.     

Differentiating A and B groups of Leptosphaeria maculans, causal agent of stem canker (blackleg) of oilseed rape

Stem canker or blackleg of brassicas, caused by Leptosphaeria maculans , is one of the most damaging diseases of winter oilseed rape in the UK. Airborne ascospores, released in autumn and winter, initiate leaf infections which may lead to colonization of the petiole and, later in the season, formation of stem lesions and cankers. Although isolates of the pathogen differ in ability to cause damaging stem cankers, this is not readily apparent from leaf spotting or stem lesion symptoms. However, several cultural, biochemical and genetic characteristics appear to be associated with the ability to form damaging stem cankers and isolates can be assigned to one of two groups, termed A and B, on the basis of differences in these characteristics. To investigate the relationship between leaf spotting symptoms and subsequent stem canker formation, and to improve understanding of the epidemiology of this pathogen, it is desirable to differentiate between the stem canker forming A group and the less damaging B group of L. maculans . Characterization of isolate type is also important in seed testing and crop breeding programs, particularly in countries such as Canada and Poland where the A type is not ubiquitous. This article reviews methods, including plant assays, assessments of growth characteristics in vitro , isozyme analyses, secondary metabolite profiling, serology, and nucleic acid analyses, that can be used to differentiate the A and B groups.     

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.