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.     

A Large-Scale Survey of Races of Leptosphaeria maculans Occurring on Oilseed Rape in France

Nine avirulence genes (AvrLm1–AvrLm9) were identified in Leptosphaeria maculans, the causal agent of stem canker of oilseed rape (OSR), combinations of which could theoretically generate up to 512 different races of the fungus. L. maculans displays a high evolutionary potential to adapt to novel resistance genes as illustrated by the Rlm1 breakdown in France, where virulent populations became prevalent within three growing seasons. An improved knowledge of the race structure of the fungal population is therefore needed to ensure a better use of available major resistance genes. The objective of this study was to characterise the L. maculans population structure in France using a large-scale, rationalised sample of isolates. Experimental fields, planted with “trap plants” harbouring no major resistance gene, were sown at 20 locations. Single-pycnidium isolates were collected from leaf lesions that developed in early autumn and 1797 isolates were genotyped at Avr loci. The frequency of AvrLm6 and AvrLm7 was higher than 99%, whereas avrLm2 and avrLm9 alleles were fixed in the population. AvrLm1, AvrLm4, AvrLm5 and AvrLm8 were polymorphic. AvrLm3 isolates were detected at a very low frequency (less than 1%). Only 11 races were identified in France, with one race prevalent, namely Av5-6-7-(8) (i.e. virulent on Rlm1, Rlm2, Rlm3, Rlm4 and Rlm9), representing around 65% of the population. Disparities between the locations sampled were evident at all scales analysed. Some virulent races, such as those harbouring avrLm5, were present before the introduction of the corresponding resistance gene in the commercial OSR crop.     

World-Wide Importance of Phoma Stem Canker (Leptosphaeria maculans and L. biglobosa) on Oilseed Rape (Brassica napus)

Phoma stem canker is an internationally important disease of oilseed rape (Brassica napus, canola, rapeseed), causing serious losses in Europe, Australia and North America. UK losses of €56M per season are estimated using national disease survey data and a yield loss formula. Phoma stem canker pathogen populations comprise two main species, Leptosphaeria maculans, associated with damaging stem base cankers, and Leptosphaeria biglobosa, often associated with less damaging upper stem lesions. Both major gene and quantitative trait loci mediated resistance to L. maculans have been identified in B. napus, but little is known about resistance to L. biglobosa. Leptosphaeria maculans, which has spread into areas in North America and eastern Europe where only L. biglobosa was previously identified, now poses a threat to large areas of oilseed rape production in Asia. Epidemics are initiated by air-borne ascospores; major gene resistance to initial infection by L. maculans operates in the leaf lamina of B. napus. It is not clear whether the quantitative trait loci involved in the resistance to the pathogen that can be assessed only at the end of the season operate in the leaf petioles or stems. In countries where serious phoma stem canker epidemics occur, a minimum standard for resistance to L. maculans is included in national systems for registration of cultivars. This review provides a background to a series of papers on improving strategies for managing B. napus resistance to L. maculans, which is a model system for studying genetic interactions between hemi-biotrophic pathogens and their hosts.     

Dissemination of Information About Management Strategies and Changes in Farming Practices for the Exploitation of Resistance to Leptosphaeria maculans (Phoma Stem Canker) in Oilseed Rape Cultivars

The management of phoma stem canker (blackleg disease, caused by Leptosphaeria maculans) is an integral component of oilseed rape production. In this paper, we discuss the information about management strategies that is disseminated in Europe and Australia. New cultivars have been introduced with improved resistance to disease, but sometimes this resistance has been overcome as new races of the pathogen have emerged. When cultivars with single major gene resistance have been introduced into areas with high inoculum concentrations, significant economic damage has been caused by new races of L. maculans within 2–3 years. Quantitative or polygenic resistance has also been used successfully against stem canker and offers more durable disease resistance if plant breeders and farmers deploy this resistance more effectively. Strategies to improve the durability of resistance need to be developed and tested in practice. New information on the occurrence of virulence and avirulence genes in populations of Leptosphaeria maculans and modelling of the durability of resistance provide opportunities for plant breeders, specialist technical organisations, cooperatives, advisory services and farmers to collaborate and better exploit cultivar resistance. Changing economic and environmental factors influence cropping practices and, if to be considered successful, management strategies must show clear financial benefits. Technology transfer will need to address all aspects of managing stem canker and other diseases of oilseed rape and using effective written, verbal and electronic methods of communication.     

Frequency of Avirulence Alleles in Field Populations of Leptosphaeria maculans in Europe

This paper describes the first large-scale Europe-wide survey of avirulence alleles and races of Leptosphaeria maculans. Isolates were collected from the spring rape cultivar Drakkar, with no known genes for resistance against L. maculans, at six experimental sites across the main oilseed rape growing regions of Europe, including the UK, Germany, Sweden and Poland. Additionally in Poland isolates were collected from cv. Darmor, which has resistance gene, Rlm9. In total, 603 isolates were collected during autumn in 2002 (287 isolates from Germany and the UK) and 2003 (316 isolates from Poland and Sweden). The identity of alleles at eight avirulence loci was determined for these isolates. No isolates had the virulence allele avrLm6 and three virulence alleles (avrLm2, avrLm3 and avrLm9) were present in all isolates. The isolates were polymorphic for AvrLm1, AvrLm4, AvrLm5 and AvrLm7 alleles, with virulence alleles at AvrLm1 and AvrLm4 loci and avirulence alleles at AvrLm7 and AvrLm5 loci predominant in populations. Virulent avrLm7 isolates were found at only one site in Sweden. Approximately 90% of all isolates belonged to one of two races (combinations of avirulence alleles), Av5-6-7 (77% of isolates) or Av6-7 (12%). Eight races were identified, with four races at frequencies less than 1%. The study suggested that Rlm6 and Rlm7 are still effective sources of resistance against L. maculans in oilseed rape in Europe. The results are comparable to those of a similar survey done in France in autumn 2000 and 2001.     

Improved Resistance Management for Durable Disease Control: A Case Study of Phoma Stem Canker of Oilseed Rape (Brassica napus)

Specific resistance loci in plants are generally very efficient in controlling development of pathogen populations. However, because of the strong selection pressure exerted, these resistances are often not durable. The probability of a resistance breakdown in a pathosystem depends on the evolutionary potential of the pathogen which is affected by: (i) the type of resistance (monogenic and/or polygenic), (ii) the type of reproduction of the pathogen (sexual and/or asexual), (iii) the capacity of the pathogen for dispersal, (iv) the resistance deployment strategy (pyramiding of specific resistances, mixture of cultivars, spatio-temporal alternation), (v) the size of the pathogen population, which is affected by control methods and environmental conditions. We propose the concept of Integrated Avirulence Management (IAM) to enhance the durability of specific resistances. IAM involves a strategy to limit the selection pressure exerted on pathogen populations and, at the same time, reduce the size of pathogen populations by combining cultural, physical, biological or chemical methods of control. Several breakdowns of resistance specific to Leptosphaeria maculans, the causal agent of phoma stem canker have occurred in Europe and in Australia. This review paper examines control methods to limit the size of L. maculans populations and discusses how this limitation of population size can enhance the durability of specific resistances. It proposes pathways for the development of a spatially explicit model to define IAM strategies. Simulation results are presented to demonstrate the potential uses of such a model for the oilseed rape/L. maculans pathosystem.     

A 10-year Survey of Populations of Leptosphaeria maculans in France Indicates a Rapid Adaptation Towards the Rlm1 Resistance Gene of Oilseed Rape

Leptosphaeria maculans, the cause of stem canker of oilseed rape (OSR), exhibits gene-for-gene interactions with its host plant. The race structure of L. maculans was assessed on the basis of the analysis of 1011 isolates collected in France between 1990 and 2000, with regards to three AVR genes, AvrLm1, AvrLm2 and AvrLm4. The effect of selection pressure, due to large-scale cropping of Rlm1 cultivars, on the evolution of races of the fungus was also evaluated. The results revealed a scarcity or complete absence of isolates harbouring AvrLm2, whereas isolates harbouring AvrLm4 were present at a variable level, that was as high as 17.2–31.2% depending on the sample year and location. When obtained from rlm1 cultivars, isolates harbouring AvrLm1 always represented more than 83% of the populations until the 1997–1998 growing season. As a consequence, the Rlm1 cultivars had been highly efficient at controlling the disease and were grown on an estimated 43.7% of the total French acreage in OSR in 1998–1999. However, the increased commercial success of Rlm1 cultivars was paralleled by a decrease in the proportion of isolates harbouring AvrLm1 in 1997–1998 and 1998–1999. This resulted in less than 13% of isolates harbouring AvrLm1 in populations being collected from rlm1 cultivars in 1999 and 2000, and contributed to the loss of efficiency of the Rlm1 resistance in the field. The present study is an illustration of one round of a `boom and bust' cycle that occurred for a pathosystem where it has never been reported before. These data and the high evolutionary potential of L. maculans are fully supportive of one pathogen species with a high risk of breaking down resistance genes in OSR and suggest that the development of integrated strategies aiming at maximising the durability of novel resistance is now a priority for this pathosystem.     

Major gene resistance in Brassica napus (oilseed rape) is overcome by changes in virulence of populations of Leptosphaeria maculans in France and Australia

Resistance of Brassica napus (oilseed rape, canola) conferred by three different major resistance genes has been overcome by changes in virulence of Leptosphaeria maculans populations in France and Australia. In South Australia where B. napus cultivars with major gene resistance derived from Brassica rapa ssp. sylvestris were grown extensively, resistance was rendered ineffective within 3 years of commercial release of the cultivar. Disease severity was higher on cultivars with sylvestris-derived resistance than cultivars with polygenic resistance. This Australian situation is compared to that in France, where resistance conferred by the Rlm1 gene was overcome nation-wide in 5 years under commercial cropping practices, and also where a source of resistance introgressed into B. napus from B. juncea was rendered inefficient in 3 years in experimental field plots near Rennes.     

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.     

Cytological responses in the hypersensitive reaction in cotyledon and stem tissues of Brassica napus after infection by Leptosphaeria maculans

A hypersensitive response (HR) occurs after infection of cotyledon, leaf and stem of Brassica napus cv. Surpass 400 by an avirulent strain of Leptosphaeria maculans. The cotyledon or the sixth true-leaf stages of plants were inoculated with pycnidiospores of L. maculans (strain UWA P11). For the first time in this specific pathosystem and its HR, we report condensation of cytoplasm, shrinkage in cell size and nuclear DNA fragmentation in cotyledon tissues, and in stem tissues, shrinkage and condensation of the cytoplasm, chromatin fragmentation and lobing of the nucleus.     

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.     

Study of Resistance to Orobanche ramosa in Host (Oilseed Rape and Carrot) and Non-host (Maize) Plants

Orobanche ramosa is a parasitic Angiosperm responsible for severe yield losses in several economical crops. It is a serious threat in oilseed rape in France and Morocco and is appearing in carrot crops in Morocco. In this study, several varieties of oilseed rape and carrot were screened in order to identify resistant cultivars and to characterize the resistance mechanisms involved. All the 15 oilseed rape varieties tested were susceptible. In carrot, the varieties 'Colmar à coeur rouge' and 'Nantaise demi-longue' were susceptible, whereas 'Palaiseau' and 'Buror' were resistant. In the susceptible 'Colmar à coeur rouge' carrot no defence reactions were found and the development of the parasite inhibited carrot tap root formation. In the resistant carrot varieties, the parasite germinated, became attached to the host root but became necrotic before emergence. In 'Buror' carrot, formation of a mechanical barrier was associated with the restriction to the cortex of the parasite. In maize cv. 'Vigni', a non-host of O. ramosa, thickening of xylem vessels, cell divisions in the central cylinder and formation of an encapsulation layer were observed in association with restricted development of Orobanche haustoria.     

不同覆盖处理对甘肃中部地区甘蓝型冬油菜越冬率及产量的影响

试验研究了地膜覆盖、土覆盖、农家肥覆盖处理对甘肃中部地区甘蓝型冬油菜的越冬率、生育期、产量的影响。试验结果表明,覆盖处理较露地栽培可缩短总生育期2~11 d,各生育时期也相应缩短。其中地膜覆盖效果最好,可缩短生育期11 d左右,农家肥覆盖与土覆盖处理分别可缩短生育期2 d与5 d。地膜覆盖、土覆盖、农家肥覆盖越冬率分别为95.45%、81.25%、80.77%,露地栽培越冬率为66.67%。覆盖处理较露地栽培的越冬率增加14.1~28.78个百分点。覆盖处理的株高、主花序长度、单株角果数、千粒重、单株产量以及角粒数等性状优于露地栽培。覆盖处理可提高产量,其中地膜覆盖产量达到5 358.3 kg/hm2,土覆盖为3 854.25 kg/hm2,农家肥覆盖产量为3 704.25 kg/hm2,分别较对照增产50.94%、8.57%和4.34%。覆盖对提高土壤温度有良好影响,无论地膜覆盖、土覆盖还是农家肥覆盖,土壤温度均高于露地栽培的处理。根据覆盖栽培越冬保苗效果、增产效果以及农事作业的可操作性,建议甘肃中部地区甘蓝型冬油菜栽培以地膜覆盖和土覆盖为主要覆盖方式。     

甘肃临夏高寒区油菜种植气候区划与产量预测气候模型

采用数理统计方法,分析了临夏地区油菜气候生态条件以及气候条件对油菜产量及其构成因素的影响.结果表明:影响该地区油菜生长发育的主要气候要素为降水、气温和日照;根据高寒地区油菜生长季节对不同气候要素的依赖程度,对高寒地区油菜种植进行气候区划,并根据气候区划分析了双低油菜不同生长阶段所需气候条件;把双低油菜从播种到成熟划分为五个生育期,建立各生育期动态气候预测模式,实现了双低油菜产量气象预测.     

Systemic resistance to bacterial leaf speck pathogen in Arabidopsis thaliana induced by the culture filtrate of a plant growth-promoting fungus (PGPF) Phoma sp. GS8-1

The culture filtrate (CF) from the plant growth-promoting fungus Phoma sp. GS8-1 was found to induce systemic resistance in Arabidopsis thaliana against the bacterial leaf speck pathogen Pseudomonas syringae pv. tomato DC3000 (Pst), and the underlying mechanism was studied. Roots of A. thaliana were treated with CF from GS8-1, and plants expressed a clear resistance to subsequent Pst infection; disease severity was reduced, and proliferation of pathogen was suppressed. Various mutants of A. thaliana were used to test whether the CF induced resistance through one of the known signaling pathways: salicylic acid (SA), jasmonic acid (JA), and ethylene (ET). The CF was fully protective against Pst in Arabidopsis mutants jar1 and ein2 similar to wild-type plants. However, its efficacy was reduced in plants containing transgene NahG. Examination of systemic gene expression revealed that CF modulates the expression of SA-inducible PR-1, PR-2 and PR-5 genes, the JA/ET-inducible ChitB gene, and the ET-inducible Hel gene. Moreover, the expression of these genes was further enhanced upon subsequent stimulation after attack by Pst. Our data suggest that in addition to a partial requirement for SA, the signals JA and ET may also play a role in defense signaling in Arabidopsis.     

粘虫脂动激素基因MsepAKH1的克隆与成虫期表达模式分析

为明确脂动激素基因AKH在粘虫Mythimna separata(Walker)能源代谢过程中的作用,采用RT-PCR和RACE技术克隆得到粘虫脂动激素基因MsepAKH1的cDNA全长,并通过实时荧光定量PCR技术测定该基因在成虫期的组织与发育阶段表达模式。结果表明,粘虫脂动激素基因MsepAKH1的cDNA序列全长为449 bp,GenBank登录号为KP979739.1,开放阅读框为207 bp,编码68个氨基酸,具有昆虫脂动激素基因家族典型的结构特征;预测该基因编码的MsepAKH1蛋白分子量为7.61 kD,等电点为8.46,MsepAKH1的氨基酸序列与烟草天蛾Manduca sexta、二化螟Chilo suppressalis、小菜蛾Plutella xylostella、棉铃虫Helicoverpa armigera、家蚕Bombyx mori的AKH氨基酸序列同源性较高。MsepAKH1基因在粘虫初羽化雌蛾不同组织间的表达量差异显著,主要在头部和飞行肌内表达,分别约为同日龄雌蛾脂肪体内表达量的15.4倍与8.1倍,在脂肪体、卵巢与中肠内的表达量显著降低。对不同日龄雌蛾头部和飞行肌内的表达量检测发现,在7日龄雌蛾头部和3日龄雌蛾飞行肌内的表达量分别显著高于其它日龄雌蛾相同组织中的表达量,分别约为初羽化雌蛾相同组织表达量的2.4倍与1.6倍。表明MsepAKH1基因的表达因粘虫雌蛾组织与日龄间的不同而呈现动态变化。     

Colonization of cabbage by the western black flea beetle (Phyllotreta pusilla) as affected by mulch and time of day

A 3-year study was conducted in Colorado (USA) to evaluate the response of the western black flea beetle (WBFB),Phyllotreta pusilla Horn (Coleoptera: Chrysomelidae), to different colored mulches and to determine if there were diurnal differences in the effect of mulch on their colonization on plants. The plants surrounded by aluminum mulch had the highest number of WBFB compared with the rest of the treatments in 1999 and 2000, but not in 2002; the black mulch had the lowest number of WBFB in 3 years of sampling. The highest number of WBFB were observed in the late day (4 p.m.) counting in 1999, whereas none of the treatments resulted in significant differences among early, mid, and late day counts in 2000 and in 2002. http://www.phytoparasitica.org posting May 20, 2005.     

重庆市萝卜芜菁花叶病毒的检测与序列分析

为明确重庆市萝卜感染芜菁花叶病毒(Turnip mosaic virus,TuMV)的情况,在重庆市巴南区、九龙坡区、北碚区等14个区县共采集了146份萝卜病毒病样品,采用酶联免疫吸附试验法(enzymelinked immunosorbent assay,ELISA)对病样进行TuMV检测,并通过PCR、克隆和测序等技术获得13个TuMV分离物CP核苷酸序列,利用软件分析重庆市TuMV CP基因序列的相似性,利用系统进化树分析CP基因遗传变异与系统进化。结果表明,共有105份样品的TuMV检测为阳性,检出率为71.9%;所得13个分离物CP基因均为864 bp,测序的13个分离物CP核苷酸相似性为89.1%~99.2%,与国内已报道的TuMV各分离物CP核苷酸相似性在88.3%~99.4%之间;所得的13个分离物均分布在basal-BR和world-B组中,在Asian-BR和basal-B组中均无分布,进一步分析发现有9个分离物属于word-B组,仅4个分离物属于basal-BR组。研究表明,TuMV在重庆市萝卜各种植区普遍发生,且word-B组为重庆市萝卜的优势毒源。     

东北地区亚洲玉米螟不同寄主植物种群线粒体基因遗传多样性

为了解不同寄主植物对亚洲玉米螟Ostrinia furnacalis(Guenée)寄主专化性及遗传多样性的影响,以东北地区取食4种寄主植物的亚洲玉米螟种群为研究对象,选取其线粒体COⅠ和COⅡ基因作为分子标记,通过序列比对分析,研究了亚洲玉米螟种群间的遗传多样性、基因流水平及分子变异。结果显示,亚洲玉米螟线粒体基因具有较丰富的遗传多态性;COⅠ和COⅡ基因总寄主植物种群Fu’s Fs检验结果分别为-1.82和-2.04,种群历史呈扩张趋势,基因流Nm分别为6.38和3.24,说明各种群间基因交流水平较高;遗传变异主要来自于种群内部;亚洲玉米螟的单倍型在BI树和NJ树上的分布拓扑结构与寄主植物间无对应关系。研究表明,取食不同寄主植物的亚洲玉米螟种群间的遗传多态性较高,基因交流频繁,且尚未发生遗传分化。