Martin J. Barbetti

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.

Analysis of Leptosphaeria maculans Race Structure in a Worldwide Collection of Isolates

ABSTRACT Leptosphaeria maculans, the causal agent of stem canker of oilseed rape, develops gene-for-gene interactions with its hosts. To date, eight L. maculans avirulence (Avr) genes, AvrLm1 to AvrLm8, have been genetically characterized. An additional Avr gene, AvrLm9, that interacts with the resistance gene Rlm9, was genetically characterized here following in vitro crosses of the pathogen. A worldwide collection of 63 isolates, including the International Blackleg of Crucifers Network collection, was genotyped at these nine Avr loci. In a first step, isolates were classified into pathogenicity groups (PGs) using two published differential sets. This analysis revealed geographical disparities as regards the proportion of each PG. Genotyping of isolates at all Avr loci confirmed the disparities between continents, in terms of Avr allele frequencies, particularly for AvrLm2, AvrLm3, AvrLm7, AvrLm8, and AvrLm9, or in terms of race structure, diversity, and complexity. Twenty-six distinct races were identified in the collection. A larger number of races (n = 18) was found in Australia than in Europe (n = 8). Mean number of virulence alleles per isolate was also higher in Australia (5.11 virulence alleles) than in Europe (4.33) and Canada (3.46). Due to the diversity of populations of L. maculans evidenced here at the race level, a new, open terminology is proposed for L. maculans race designation, indicating all Avr loci for which the isolate is avirulent.

Screening techniques and sources of resistance to foliar diseases caused by major necrotrophic fungi in grain legumes

SummaryNecrotrophic pathogens of the cool season food legumes (pea, lentil, chickpea, faba bean and lupin) cause wide spread disease and severe crop losses throughout the world. Environmental conditions play an important role in the development and spread of these diseases. Form of inoculum, inoculum concentration and physiological plant growth stage all affect the degree of infection and the amount of crop loss. Measures to control these diseases have relied on identification of resistant germplasm and development of resistant varieties through screening in the field and in controlled environments. Procedures for screening and scoring germplasm and breeding lines for resistance have lacked uniformity among the various programs worldwide. However, this review highlights the most consistent screening and scoring procedures that are simple to use and provide reliable results. Sources of resistance to the major necrotrophic fungi are summarized for each of the cool season food legumes. Marker-assisted selection is underway for Ascochyta blight of pea, lentil and chickpea, and Phomopsis blight of lupin. Other measures such as fungicidal control and cultural control are also reviewed. The emerging genomic information on the model legume, Medicago truncatula, which has various degrees of genetic synteny with the cool season food legumes, has promise for identification of closely linked markers for resistance genes and possibly for eventual map-based cloning of resistance genes. Durable resistance to the necrotrophic pathogens is a common goal of cool season food legume breeders.

Breakdown of a Brassica rapa subsp. sylvestris Single Dominant Blackleg Resistance Gene in B. napus Rapeseed by Leptosphaeria maculans Field Isolates in Australia

Blackleg, caused by Leptosphaeria maculans, is a major disease of oilseed rape (Brassica napus) grown in Canada, Europe, and Australia. Cv. Surpass 400 was released in Australia in 2000 as the most resistant cultivar to L. maculans. It carries a single dominant resistance gene from B. rapa subsp. sylvestris. This cultivar usually shows a hypersensitive response to L. maculans characterized by small, dark brown lesions that are necrotic, localized, and without pycnidia on cotyledons, leaves, and stems. However, in 2001 on a Western Australian experimental farm, a small proportion of the lesions on the lower stem and crown region of cv. Surpass 400 were typical of those observed in susceptible cultivars, which were brown, necrotic lesions with a darker margin, but they contained fewer pycnidia. Forty seedlings of cv. Surpass 400 and susceptible cv. Westar were inoculated with pycnidiospore suspensions (106/ml) of each of 18 isolates taken from lesions on cv. Surpass 400. All 18 isolates caused collapse of cotyledons of susceptible cv. Westar. Four of these isolates caused large cotyledon lesions with some pycnidia on cv. Surpass 400. Three of these four isolates were subsequently inoculated onto 60 seedlings per isolate, at each of the four cotyledon lobes of each seedling of the two cultivars. Inoculated plants were assessed for disease severity on cotyledons and transplanted to the field 14 days after inoculation. The cotyledons of inoculated cv. Surpass 400 showed characteristic large, necrotic lesions with pycnidia, while the cotyledons of cv. Westar had collapsed and contained a mass of pycnidia. Blackleg disease severity in the crown region of the stem was assessed at 2 weeks before harvest. Fifty-four percent of the cv. Surpass 400 transplanted inoculated plants subsequently developed susceptible symptoms of crown cankers on stems. These symptoms were deep, girdling, brown lesions on the plant crowns with some pycnidia. One hundred percent of cv. Westar plants were infected and dead at this stage. This confirmed the ability of these field isolates to overcome the single dominant resistance gene present in cv. Surpass 400. To our knowledge, this is the first report of breakdown of a single dominant B. rapa subsp. sylvestris gene based resistance to blackleg in oilseed rape in the field.

Blackleg sporacle: A model for predicting onset of pseudothecia maturity and seasonal ascospore showers in relation to blackleg of canola

ABSTRACT A simple model has been developed to predict the onset of pseudothecia maturity and seasonal ascospore showers in relation to blackleg disease in canola, caused by the fungus Leptosphaeria maculans. The model considers a combination of two weather factors, daily mean temperature and daily total rainfall, to drive progress of maturity of pseudothecia on the infested canola stubble left from past crops. Each day is categorized as suitable or not suitable for progress of the maturation process. The onset of pseudothecia maturity occurs when approximately 43 suitable days have occurred. Following the onset of maturity, ascospore showers are triggered when daily rainfall exceeds a threshold. The model satisfactorily predicted the timing of the onset of pseudothecia maturity when tested with 3 years of field observations at four locations in Western Australia, which characteristically has a Mediterranean climate. The model also agreed reasonably well with the daily pattern of ascospore release observed in two locations. Sensitivity analysis was performed to show the relative importance of the parameters that describe the onset of pseudothecia maturity.

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.

Expression of field resistance under Western Australian conditions to Sclerotinia sclerotiorum in Chinese and Australian Brassica napus and Brassica juncea germplasm and its relation with stem diameter

Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, has become one of the most serious disease problems in oilseed rape-growing areas in Australia. Sources of resistance to this disease have been sought worldwide. In this study, germplasm comprising 42 Brassica napus and 12 Brassica juncea accessions from China and Australia, was screened for resistance to Sclerotinia stem rot under Western Australian field conditions. Resistance was confirmed in some germplasm from China and new sources of resistance were identified in germplasm from Australia. Furthermore, our study found that the severity of stem lesions was related to stem diameter and percentage of the host plants that were dead. It was evident that both stem lesion length and percentage of plant death were at the lowest level when the stem diameter was approximately 10 mm. Smaller or greater stem diameter resulted both in increased stem lesion length and plant death. Stem diameter may be a useful parameter in breeding cultivars of oilseed Brassicas with Sclerotinia resistance.

New sources of resistance to Sclerotinia stem rot caused by Sclerotinia sclerotiorum in Chinese and Australian Brassica napus and B.juncea germplasm screened under Western Australian conditions

Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, is a serious problem in oilseed rape in China and Australia. Locating effective sources of host resistance offers the best long-term prospects for improved management of this disease. For these reasons, 93 genotypes of Brassica napus and B. juncea from China and Australia were screened in the field for resistance to S. sclerotiorum under semi-field conditions in Western Australia using a stem inoculation test. There were significant differences (P < 0.001) among genotypes in relation to the severity of the disease measured as stem lesion length. Amongthe B. napus genotypes, ZY006 showed outstanding resistance, with a mean stem lesion length of <0.45 cm, a level of resistance far superior to that ever identified previously in Australia for B. napus or B. juncea. B. napus genotypes 06-6-3792 and ZY004 from China and RT108 from Australia showed very high levels of resistance, with mean stem lesion lengths <3 cm. The most resistant B. juncea genotypes were JM06018 and JM06006 from Australia and B. juncea 2 from China with mean stem lesion lengths of ≤4.8 cm. The outstanding resistance to S. sclerotiorum identified in B. napus ZY006provides the first such level of resistance available for oilseed Brassica breeding programs in Australia. Australasian Plant Pathology Society

Germination and invasion by ascospores and pycnidiospores of Leptosphaeria maculans on spring-type Brassica napus canola varieties with varying susceptibility to blackleg

The infection processes of ascospores and pycnidiospores of Leptosphaeria maculans were studied on cotyledons of six cultivars of spring-type Brassica napus: one with resistance controlled by a single dominant gene (cv. Surpass 400), three with polygenic resistance (cvs. Dunkeld, Grouse, and Outback), and two susceptible cultivars (Westar and Q2). On all cultivars, ascospore germination, penetration, and development of symptoms on cotyledons were much earlier than that with pycnidiospores. At 2 h after inoculation ascospores began to germinate, by 4 h about 50% had germinated, and by 6–8 h 85%–90% had germinated. In contrast, pycnidiospores began to germinate 1 day after inoculation (dai) and reached only 50% germination by 3 dai. Ascospores began germinating from terminal cells and then later from the interstitial cells. Pycnidiospores germinated predominantly from one end and sometimes from both ends. Germ tubes from ascospores penetrated stomata as early as 4 h after inoculation, whereas those from pycnidiospores penetrated at 2 dai. Symptom development with ascospores was 2 days earlier than that with pycnidiospores. Symptoms on Surpass 400 were evident as early as 3–5 dai with ascospores and 5–7 dai with pycnidiospores. However, on other cultivars, symptoms were not evident until 10 dai with ascospores and 12 dai with pycnidiospores. This report is the first on differences in the infection processes by the two spore types. Ascospore and pycnidiospore attachment, germination, and penetration did not differ between resistant and susceptible cultivars, but there were major differences after penetration. Under high humidity, 80%–90% of stomata of susceptible Westar and Q2 had aerial hyphae emerging from stomatal pores. However, fewer stomata (5%–10%) had aerial hyphae on Surpass 400 by 10 dai with ascospores and 12 dai with pycnidiospores, but even these were usually poorly developed. Host differences in spring-type B. napus in relation to production of aerial hyphae have not previously been reported. In Surpass 400, rapid necrosis of guard cells occurred within a few hours of penetration by either type of spore, and subsequently one or a few cells immediately adjacent to the penetration site died. This necrosis then spread to the cells around the penetration site to form a hypersensitive response (in the form of a small, dark lesion) to both ascospores and pycnidiospores. This is the first detailed report on interactions between spring-type B. napus and L. maculans in relation to single dominant gene-based resistance. Neither the cultivars with polygenic resistance nor the susceptible cultivars had such a response.

Pathogenicity of morphologically different isolates of Sclerotinia sclerotiorum with Brassica napus and B. juncea genotypes

Sclerotinia stem rot caused by Sclerotinia sclerotiorum is a serious threat to oilseed production in Australia. Eight isolates of S. sclerotiorum were collected from Mount Barker and Walkway regions of Western Australia in 2004. Comparisons of colony characteristics on potato dextrose agar (PDA) as well as pathogenicity studies of these isolates were conducted on selected genotypes of Brassica napus and B. juncea. Three darkly-pigmented isolates (WW-1, WW-2 and WW-4) were identified and this is the first report of the occurrence of such isolates in Australia. There was, however, no correlation between pigmentation or colony diameter on PDA with the pathogenicity of different isolates of this pathogen as measured by diameter of cotyledon lesion on the host genotypes. Significant differences were observed between different isolates (P ≤ 0.001) in two separate experiments in relation to pathogenicity. Differences were also observed between the different Brassica genotypes (P ≤ 0.001) in their responses to different isolates of S. sclerotiorum and there was also a significant host × pathogen interaction (P ≤ 0.001) in both experiments. Responses between the two experiments were significantly correlated in relation to diameter of cotyledon lesions caused by selected isolates (r = 0.79; P < 0.001, n = 48). Responses of some genotypes (e.g., cv. Charlton) were relatively consistent irrespective of the isolates of the pathogen tested, whereas highly variable responses were observed in some other genotypes (e.g., Zhongyou-ang No. 4, Purler) against the same isolates. Results indicate that, ideally, more than one S. sclerotiorum isolate should be included in any screening programme to identify host resistance. Unique genotypes which show relatively consistent resistant reactions (e.g., cv. Charlton) across different isolates are the best for commercial exploitation of this resistance in oilseed Brassica breeding programmes.