ABSTRACT

Stem rot in rapeseed, caused by Sclerotinia sclerotiorum, is an important cause of yield loss in China and Europe. The European Union has funded a joint project, which started in August 1997, between two Chinese and two European partners to develop strategies for integrated control of Sclerotinia stem rot of rapeseed using biotechnological and epidemiological approaches. The aims of the project are to develop new, more efficient biotechnology-based methods to strengthen Chinese and European resistance-breeding programmes, and identify improvements to existing cultivation systems that will reduce the chances of breakdown in new resistances. Three parallel approaches have been adopted: identification of novel sources of resistance or tolerance to stem rot and the development of molecular markers for resistance; the study of biochemical systems which contribute to resistance; and the study of the epidemiology of the pathogen. The structure of the project and the approaches used are described and progress in each area highlighted including: the identification of potential sources of tolerance in Chinese germplasm; the identification of QLT’s for reststance; comparison of inoculation methods; observation of local and systemic changes in biochemical constituents in response to infection; and effects of sowing date on potential disease.

INTRODUCTION

Rapeseed is an important oil crop in China (about 6M ha grown annually) and in Europe (about 3M ha grown annually). Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, is the most important disease in rapeseed crops in China, causing an average national yield loss of about 20%. The disease can also cause significant yield losses in Europe (up to 50% on severely infected crops). The disease can be controlled by the timely application of fungicides, however this is not often an option for Chinese growers. Alternative control strategies include the use of disease resistant varieties combined with agronomic practices which use knowledge of the epidemiology of the pathogen to minimise the impact of the disease. No commercial varieties possess good resistance to Sclerotinia stem rot, but, there are promising sources of resistance, expressed as disease tolerance or avoidance, which are being used to develop hybrid rapeseed varieties. However, it is difficult to use these sources in breeding programmes because disease tolerance and disease avoidance are both likely to be polygenically controlled and their genetics are not well understood. All stages of the development of the disease in rapeseed crops are greatly influenced by environmental factors, but the interactions between environment, pathogen and crops are not clearly understood either in China or in Europe. Likewise, little is known of the influence that agronomic practices can have on crop environments and disease development.

The European Union has funded a three year project (started August 1997) with the objective of reducing the impact of Sclerotinia stem rot in China and Europe. It is a collaboration between two Chinese and two European partners: the Anhui Academy of Agricultural Sciences (AAAS), Institute of Oil Crops Research (IOCR), IACR-Rothamsted (IACR) and Rustica Prograin Génétique (RPG). The project has adopted an integrated approach which aims not only to develop new, more efficient, biotechnology based methods, base on the use of molecular markers, to strengthen Chinese and European resistance-breeding programmes, but also to identify improvements to existing cultivation systems that will reduce the effects of stem rot epidemics. This paper outlines the structure and methodologies adopted in this project and highlights progress during its first 18 months. Details of some of the work are presented in other papers in these proceedings.

PROJECT OBJECTIVES AND STRUCTURE

The main objectives of the project are: to develop improved method for screening for resistance to stem rot; to identify new sources of resistance in Chinese germplasm; to identify molecular markers associated with resistance; to identify constitutive and inducible biochemical systems that contribute to resistance; to identify cultural practices that discourage stem rot development; and to devise strategies for the integrated control of stem rot that combine improved rapeseed varieties and better cultural practices. These objectives are being approached by dividing the work into five interrelated areas, with each partners responsible for work in several of the areas:

1 (IACR, RPG, AAAS): Comparison of methods for screening for resistance to S. sclerotiorum.

2 (AAAS, IOCR, RPG): Identification of new sources of resistance to S. sclerotiorum and evaluation and incorporation of resistance into rapeseed:.

3 (RPG, AAAS, IOCR): The genetic study of resistance the development of molecular markers for resistance to S. sclerotiorum.

4 (IACR, IOCR): Biochemistry of resistance of rapeseed to S. sclerotiorum:

5 (IACR, AAAS): Epidemiology of S. sclerotiorum and cultural control of disease.

There is also an element of training built into the co-operation between the four partners. This consists of four 12 month training visits, two from each Chinese partner to both European partners. The Chinese trainees visiting RPG are being trained in molecular marker technology while one trainee from AAAS has received training in epidemiological methods and one from IOCR has been trained in the biochemical methods. During the training visits the visitors also contribute directly to the research programme.

APPROACHES AND PROGRESS

Comparison of methods for screening for resistance

Much of the work in this project requires that germplasm be screened for resistance to S. sclerotiorum. Thus, to choose a common method for screening for resistance, four methods were evaluated against a set of Chinese and European lines. The methods were: (a) inoculation of plants during flowering by placing a small mycelium coated wooden peg (a wooden match-stick) into a pre-drilled hole in the stem about 30cm from the plant base and measuring lesion length at regular intervals after inoculation; (b) placing mycelial plugs about one centimetre in diameter on leaves of young plants, and enclosing the leaves in a plastic bag to maintain high humidity, and assessing lesion growth regular intervals; (c) placing mycelial plugs on detached leaves and covering with a wet cotton pad; (d) inserting 5mm long wooden wedges inoculated with mycelium into the vein of broken rosette leaves and assessing the length of lesions after 10 days. Some differences in the ranking of genotypes were found depending on the resistance evaluation method used. However method a evaluated tolerance on the stem while the other methods tested tolerance on the leaf, suggesting that different mechanisms of resistance could exist within the lines. Method a had a smaller variability between sites and because of its ease of use, both in the field and greenhouse, it was decided that this method should be use to screen for resistance for the rest of the project.

Identification of new sources of resistance

Field trials at IOCR using natural infection to screen 100 Chinese germplasm sources for resistance have identified ten lines that appear to be tolerant. However, none of these lines are double low (low erucic acid and low glucosinolates), but hybrids incorporating, disease tolerance, double low characteristics and high yields are being developed at IOCR. At AAAS a hybrid variety, C022, has been developed from a genetic male sterile line (9012A) combined with an inbred line from a tolerant variety already released in China. This is a double low line and it is more resistant than other available double low varieties.

The genetic study of resistance the development of molecular markers

Diellel crosses of 6 genotypes (one European: Cheyenne,one Korean: Norin 9; and 4 Chinese: CH712, CH713, L88, Y16) have been done at RPG, AAAS and IOCR. Plants of the 36 crosses will be screened for resistance to S. sclerotiorum and the results analysed using conventional genetic methods.

One of the main objective of this project is to find QLT’s for resistance. To achieve this the work has been divided into three main parts: production of progenies from susceptible and tolerant lines; evaluation of the resistance of these progenies; and identification and characterisation of molecular markers corresponding to resistance genes.

Crosses of one tolerant (Norin 9) and one susceptible (Cheyenne) winter type and one tolerant (Wuhan 6) and 13 spring elite European lines, have been done. F3 seeds of these crosses have been produced and the plants will be screened for resistance.

About two hundred F2 plants derived from the winter type cross and about two hundred of F2 plants derived from the spring type cross have been grown in the field at two RPG sites: Mondonville (south of France) and Boisseaux (north of France). Young leaf tissue has been sampled, for genomic DNA production, from each F2 individual of these crosses.

Three different PCR-based markers, SSR (single sequence repeats), STS (sequence tagged site) and EST (expressed sequence tag), are being developed. These markers are being screened against a set of 8 oilseed rape genotypes and validated on a collection of 24 genotypes to reveal polymorphism. One hundred and twenty STS have been produced and analysed. About 30% present polymorphism on the collection of 24 genotypes. An additional 30 STSs have been developed, using the sequencing data of 30 rapeseed DNA fragments, and 15 were polymorphic for at least one of the two crosses. Forty nine ESTs and 30 SSRs have been identified from published gene sequences or gene sequence database analysis. Sixteen ESTs gave polymorphisms between Norin 9 and Cheyenne, while, 7 SSTs were polymorphic for the same cross. Further polymorphic markers are being sought. Molecular mapping work will begin as soon as sufficient polymorphic markers have been developed (probably July 1999).

Biochemistry of resistance

This area of the project is investigating the biochemistry of resistance or tolerance to infection of rapeseed by S. sclerotiorum, with the aim of identifying pathogenesis-related protein or enzyme systems. This information will contribute to the development of resistance gene markers by using peptide sequence analysis of proteins expressed in resistance to design DNA probes which can be evaluated as resistance markers.

Responses in leaf glucosinolates to infection by S. sclerotiorum have been measured in young oilseed rape plants of 6 cultivars: 2 European (Bienvenue, single low, Capricorn, winter double low) and 4 Chinese (IOCR lines 014, 016, 020 and 024). In the experiments leaf glucosinolate concentrations were measured at different times after inoculation with mycelial plugs. The experiments measured the local response, that is in the inoculated leaf, and systemic response, that is in non-inoculated leaves. Bienvenue, a cultivar with good disease resistance, showed significant changes in glucosinolate levels both locally and systematically, especially in young leaves. In contrast the other European cultivar, Capricorn, a susceptible cultivar, only had significant increases locally. Two of the Chinese lines used (014 and 020) were tolerant to the disease and two (016 and 024) were susceptible. Post inoculation glucosinolate levels increased in one of the tolerant lines (014) both locally and systematically, especially in young leaves, but there was little response in the other tolerant line. In both the susceptible lines local glucosinolate levels increase, but, levels decrease in non infected leaves. The results suggest that the response of the glucosinolate system to infection may be related to the levels of disease resistance or susceptibility.

Measurements of changes in enzyme activity in response to infection have also been done. The responses of four cultivars (Zhongyou 821, Bienvenue, Starlight, and Norin 9) have been studied. Experiments were mainly done on two enzyme systems: Phenylalanine Ammonia-Lyase (PAL) and peroxidases (POX). There were no significant differences in local and systemic changes in PAL in all lines. However, there were significant differences in local and systemic changes in POX between tolerant and susceptible lines. There was a 10 fold increase in POX in local (inoculated) areas of leaf tissue three days after inoculation. In the cultivar Zhongyou 821 there was about a 70 fold increase in POX in non inoculated leaves (systemic increase) about 7 days after inoculation. In the spring type cultivar Starlight there were smaller increases in both local and systemic changes in POX

Epidemiology of S. sclerotiorum and cultural control of disease

This part of the project is aimed at developing improved cultural practices which reduce infection by developing better understanding of the epidemiology of the disease. The work covers detailed epidemiology studies in the field and under controlled environments and field studies of the effects of cultural practices such as sowing date and sowing density.

S. sclerotiorum infection of rapeseed is primarily through petals infected by airborne ascospores deposited on leaves and leaf nodes. The infected petals provide a substrate for ascospores growth and allow infection. Little is known of the patterns of petal deposition on leaves or on the effects of environmental factors on their duration on plant parts or on infection processes. The results of the epidemiological studies in the project are reported in detail elsewhere in these Proceedings, however, the main findings can be summarised as follows. The rate of petal fall and number of petals on leaves follow the pattern of flowering but delayed by about 4-5 days. Petal retention on leaves depends on height above ground with retention on lower leaves tending to be shorter than on upper leaves. At all levels many petals do not stick for more than 2 days but a few may be retained for more that 15 days. Ascospores germinate on petals within a few hours of deposition providing there is enough water available and petals containing germinated ascospores can infect plants for at least 24h. The effectiveness of infection may depend on the ascospore load on the leaf up to about 100 ascospores per leaf, above this number infection appears to become independent of ascospore load. Provided that humidity is high (>95%), free water may not be necessary for infection, but it may be more efficient when water is present. Petal age may influence the effectiveness of infection with “old” petals being more effective than “young” petals. Between 24 and 48h of continuous high humidity (c100% RH) are needed to initiate lesion formation from ascospores on petals placed on leaves.

Field studies of the effect of cultural practices are being done in China (AAAS). The effect of sowing date and sowing density on disease development in two Chinese double low hybrid varieties, one tolerant (5C21) and one susceptible (Huaza No. 3) have been studied in multifactorial trials. The resistant variety had about half the disease incidence of the susceptible variety in all treatments. The initial results suggest that: variety is important; the earlier the sowing date the more the severe disease; there may be little influence of plant density, but this may have been due to wet weather conditions. A long term rotation/regression combination trial with 5 factors and 5 levels per factor is also being done at AAAS. After one year it is difficult to be confident in the results, but they suggest that the use of balanced fertiliser is important and the earlier the sowing date the more serious the disease.

The effects of cultural practices under Chinese conditions are also being studied by surveying farmer’s fields. The first year’s results are similar to the field experiments and suggest that early sowing dates correspond to high disease incidence and that unbalanced fertiliser "encourages" disease.

CONCLUSIONS

This project is an attempt to integrate two approaches to the management and control of Sclerotinia stem rot in rapeseed crops: breeding for resistance and the use of cultural practices to reduce the effects of disease. The development of disease resistant, or at least tolerant, cultivars offers the best solution in terms of both economic returns and environmental effects (reduction in the use of agrochemical). However, the effective lifetime of new cultivars will depend on the disease pressure to which it is exposed, especially with a pathogen such as S. sclerotiorum which has a wide host range and reproduces sexually. Understanding the interaction between the host pathogen and environment may allow the development of cultural practices which reduce or inhibit disease epidemic development and so lessen disease pressure on new cultivars, thus extending their “useful life”. It is hoped that the results of the work will lead not only to the identification of new sources of resistance and more efficient methods for germplasm selection in breeding programmes but more effective systems for rapeseed cultivation for use in China and in Europe.

ACKNOWLEDGEMENTS

This work is supported by a grant from the European Union, contract number ERBIC18CT970173. IACR-Rothamsted is suppored by a grant in air from the UK Biotechnology and Biological Sciences Research Council.