Phoma stem canker disease on oilseed rape (Brassica napus) in China is caused by Leptosphaeria biglobosa ‘brassicae’

This document is the Accepted Manuscript version of the following article: Ze Liu, Akinwunmi O. Latunde-Dada, Avice M. Hall, Bruce D. L. Fitt, ‘Phoma stem canker disease on oilseed rape (Brassica napus) in China is caused by Leptosphaeria biglobosa ‘brassicae’’, European Journal of Plant Pathology, Vol. 140(4): 841-857, December 2014. The final publication is available at Springer via: http://dx.doi.org/10.1007/s10658-014-0513-7 © Koninklijke Nederlandse Planteziektenkundige Vereniging 2014Phoma stem canker of oilseed rape (Brassica napus) is a globally important disease that is caused by the sibling ascomycete species Leptosphaeria maculans and L. biglobosa. Sixty fungal isolates obtained from oilseed rape stems with phoma stem canker disease symptoms collected from four provinces in China in 1999, 2005 and 2006 were all identified as Leptosphaeria biglobosa, not L. maculans, by PCR diagnostics based on species-specific primers. There were no differences in cultural characteristics (e.g. pigmentation and in vitro growth) between these L. biglobosa isolates from China and those of 37 proven L. biglobosa isolates from Europe or Canada. In studies using amplified fragment length polymorphism (AFLP) markers, Chinese L. biglobosa populations were genetically more similar to European L. biglobosa populations than to the more diverse Canadian L. biglobosa populations. Sequencing of gene fragments of β-tubulin, actin and the internal transcribed spacer (ITS) region of rDNA from L. biglobosa isolates from China, Europe, Australia and Canada showed a closer taxonomic similarity of Chinese L. biglobosa to the European L. biglobosa ‘brassicae’ than to Canadian L. biglobosa ‘canadensis’ or to the Australian L. biglobosa ‘occiaustralensis’ or ‘australensis’ subclades. These results suggest that the Chinese L. biglobosa population in this study is in the same subclade as European L. biglobosa ‘brassicae’ populationsPeer reviewe

Detection and quantification of Leptosphaeria maculans in the leaf petiole of Brassica napus

In controlled environment experiments winter oilseed rape (Brassica napus) cultivars Canberra and Courage did not differ significantly in leaf lesion size and systemic growth in petioles, measured after inoculation of leaves with conidia of Leptosphaeria maculans isolate ME24 (transformed to express green fluorescent protein). In field experiments (2003/2004) quantification of L. maculans within petioles of six doubled haploid (DH) lines of oilseed rape derived from Canberra x Courage showed differences in the number of colony forming units obtained from maceration of petiole parts. Differences between lines did not relate to differences in stem canker severity at harves

OREGIN collection of oilseed rape fungal pathogen isolates managed by a relational database accessible to stakeholders via the Internet

One aim of the Defra-funded Oilseed Rape Genetic Improvement Network (OREGIN) is to establish, characterise and catalogue a collection of Leptosphaeria maculans (phoma stem canker) and Pyrenopeziza brassicae (light leaf spot) isolates from around the world and to determine genetic diversity in the world-wide populations of these pathogens. Five hundred and twenty isolates of L. maculans and 253 isolates of P. brassicae have been assembled at Rothamsted Research from sources representative of the world-wide distribution of phoma stem canker and light leaf spot. Information on these isolates (including geographical origin, host species and cultivar of origin, pathogenicity group, mating type and other properties such as culture medium, stock type [slope culture/ glycerol spore suspension stocks]) is now being collected and saved to a Rothamsted Research MySQL open source relational database server. This is being made accessible to the research community and stakeholders via the OREGIN website (www.oregin.info). Once characterised and described, the isolates will be available and the website will generate the appropriate Material Transfer Agreement (MTA) forms. The OREGIN website is being expanded to include access to information relating to other public domain plant resources being made available via OREGIN. This includes information about the Brassica napus Diversity Fixed Foundation Set (BnDFFS) and reference genetic mapping populations that are held in secure storage at Warwick HRI

Leptosphaeria maculans avirulence management for durable control of phoma stem canker of oilseed rape

Non peer reviewe

Efficient qPCR estimation and discrimination of airborne inoculum of Leptosphaeria maculans and L. biglobosa, the causal organisms of phoma leaf spotting and stem canker of oilseed rape : qPCR detection of Leptosphaeria airborne inoculum

© 2023 Society of Chemical Industry. This is the accepted manuscript version of an article which has been published in final form at https://doi.org/10.1002/ps.7800BACKGROUND: Detection of the inoculum of phytopathogens greatly assists in the management of diseases, but is difficult for pathogens with airborne fungal propagules. Here, we present experiments to determine the abundance and distribution frequencies of the ascospores of Leptosphaeria (Plenodomus) species that were collected on the tapes of volumetric Hirst-type traps near oilseed rape fields in Poznan, Poland and Harpenden, UK. Fungal detection and species discrimination were achieved using a SYBR-Green quantitative polymerase chain reaction (qPCR) with two different pairs of primers previously reported to differentiate Leptosphaeria maculans (Plenodomus lingam) or L. biglobosa (P. biglobosus). RESULTS: Detection was successful even at fewer than five spores per m 3 of air. The primer pairs differed in the correlation coefficients obtained between DNA yields and the daily abundance of ascospores that were quantified by microscopy on duplicate halves of the spore trap tapes. Important differences in the specificity and sensitivity of the published SYBR-Green assays were also found, indicating that the Liu primers did not detect L. biglobosa subclade ‘canadensis’, whereas the Mahuku primers detected L. biglobosa subclade ‘canadensis’ and also the closely related Plenodomus dezfulensis. CONCLUSIONS: Comparisons confirmed that application of qPCR assays to spore trap samples can be used for the early detection, discrimination and quantification of aerially dispersed L. maculans and L. biglobosa propagules before leaf spot symptoms are visible in winter oilseed rape fields. The specificity of the primers must be taken into consideration because the final result will greatly depend on the local population of the pathogen.Peer reviewe

Quantitative PCR analysis of abundance of airborne propagules of Leptosphaeria species in air samples from different regions of Poland

When airborne propagules of Leptosphaeria maculans and L. biglobosa were collected in Poland at three ecologically different sites from 1 September to 30 November in 2004 to 2008, using a Hirst-type seven-day volumetric spore trap, there were fluctuations in timing of ascospore release and diverse ratios between airborne propagules of both species depending on season, field location and weather conditions. The detection was done using the microscope as well as quantitative PCR with species-specific primers targeted against fragments of β-tubulin genes and quantified with a dual-labelled fluorescent probe approach. This detection chemistry is described for the first time for L. maculans and L. biglobosa. Its advantage over the previous ITS-based SYBR-Green chemistry resides in improved sensitivity and the virtual absence of false positives in the detection of these fungi. There were significant, positive correlations between data obtained using visual assessment of ascospore numbers and DNA concentrations that were measured by qPCR. Climatic differences between the oilseed rape growing regions could have significantly affected the biological processes of pseudothecial maturation and ascospore development of the pathogens. The data suggest that regular rain events of intermediate intensity recorded in the Maritime region favoured the maturation of the pathogen more than the drier weather recorded in the Silesia or Pomerania regions. It was observed that the number of rainy days was of greater importance than the cumulative rainfall to obtain the generative sporulation of the pathogen. Accurate detection of airborne inoculum of pathogenic Leptosphaeria spp. facilitates improved targeting of disease management decisions for oilseed rape protection against phoma stem canker and stem necrosis diseases.Peer reviewe

Comparison of Leptosphaeria biglobosa-induced and chemically induced systemic resistance to L-maculans in Brassica napus

Brassica napus (cv. Madrigal) seedlings pre-treated with ascospores of Leptosphaeria biglobosa or foliar sprays of either acibenzolar-S-methyl (ASM) or menadione sodium bisulphite (MSB) were challenge inoculated with L. maculans ascospores and assessed for phoma leaf spot development and tissue morphology and gene expression responses to infection. Rates of increase in phoma leaf spot area 8-21 d after challenge inoculation were significantly greater on water pre-treated plants than on plants pre-treated with L. biglobosa, ASM or MSB on both pre-treated leaves (local effect) and younger leaves without pre-treatment (systemic effect). Ninety-six h after challenge inoculation, the invasive hyphae of L. maculans were encircled by rings of necrotic mesophyll cells on leaves pre-treated with L. biglobosa, ASM or MSB but not those pre-treated with water. Quantification of transcript levels of genes commonly used as markers of the major defence signalling pathways (PDF1.2, PR-1, NPR1, APX, CHB4) 0-96 h after L. maculans challenge inoculation showed expression patterns indicating preferential activation of the jasmonate/ethylene pathway and involved induction of NPR1 locally and systemically in leaves of plants pre-treated with L. biglobosa ascospores.Peer reviewe

Geographic variation in severity of phoma stem canker and Leptosphaeria maculans/L. biglobosa populations on UK winter oilseed rape (Brassica napus)

“The original publication is available at www.springerlink.com” Copyright SpringerPhoma stem canker, caused by Leptosphaeria maculans and L. biglobosa, is the most important disease of oilseed rape in Europe. Differences between L. maculans and L. biglobosa in their life-cycles enable the two species to co-exist on oilseed rape crops over a cropping season. This review considers the factors affecting geographic variation in the severity of phoma stem canker epidemics and in the structure of the population of the pathogens in the UK, where the most severe epidemics occur in the south of England and cankers do not develop in Scotland. It is concluded that this variation is directly related to differences in climate, since weather-based models show that stem canker severity increases with increasing winter/spring temperature and temperatures are greater in the south of the UK. It may also be related to differences in pathogen populations, since surveys showed that the proportion of the more damaging L. maculans in stem cankers was greatest in southern England, with most L. biglobosa in northern England. Regional variation in agronomic practices such as cultivar choice and fungicide use may also indirectly influence phoma stem canker severity. Differences in cultivar choice result in differences in L. maculans race structure, which may influence the severity of epidemics. Differences in fungicide use may also influence pathogen populations, since L. maculans and L. biglobosa differ in their sensitivities to different azole fungicides. These factors are discussed in relation to strategies for sustainable production of oilseed rape by adaptation to threats posed by climate change.Peer reviewe