Comparison of non-subjective relative fungal biomass measurements to quantify the Leptosphaeria maculans—Brassica napus interaction

Abstract Background Blackleg disease, caused by the fungal pathogen Leptosphaeria maculans, is a serious threat to canola (Brassica napus) production worldwide. Quantitative resistance to this disease is a highly desirable trait but is difficult to precisely phenotype. Visual scores can be subjective and are prone to assessor bias. Methods to assess variation in quantitative resistance more accurately were developed based on quantifying in planta fungal biomass, including the Wheat Germ Agglutinin Chitin Assay (WAC), qPCR and ddPCR assays. Results Disease assays were conducted by inoculating a range of canola cultivars with L. maculans isolates in glasshouse experiments and assessing fungal biomass in cotyledons, petioles and stem tissue harvested at different timepoints post-inoculation. PCR and WAC assay results were well correlated, repeatable across experiments and host tissues, and able to differentiate fungal biomass in different host-isolate treatments. In addition, the ddPCR assay was shown to differentiate between L. maculans isolates. Conclusions The ddPCR assay is more sensitive in detecting pathogens and more adaptable to high-throughput methods by using robotic systems than the WAC assay. Overall, these methods proved accurate and non-subjective, providing alternatives to visual assessments to quantify the L. maculans-B. napus interaction in all plant tissues throughout the progression of the disease in seedlings and mature plants and have potential for fine-scale blackleg resistance phenotyping in canola.

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Recent Findings Unravel Genes and Genetic Factors Underlying Leptosphaeria maculans Resistance in Brassica napus and Its Relatives

International Journal of Molecular Sciences ◽

10.3390/ijms22010313 ◽

2020 ◽

Vol 22 (1) ◽

pp. 313

Author(s):

Aldrin Y. Cantila ◽

Nur Shuhadah Mohd Saad ◽

Junrey C. Amas ◽

David Edwards ◽

Jacqueline Batley

Keyword(s):

Brassica Napus ◽

Genetic Factors ◽

Quantitative Resistance ◽

Genetic Resistance ◽

Leptosphaeria Maculans ◽

Gene Interaction ◽

R Gene ◽

R Genes ◽

Blackleg Resistance ◽

Avr Gene

Among the Brassica oilseeds, canola (Brassica napus) is the most economically significant globally. However, its production can be limited by blackleg disease, caused by the fungal pathogen Lepstosphaeria maculans. The deployment of resistance genes has been implemented as one of the key strategies to manage the disease. Genetic resistance against blackleg comes in two forms: qualitative resistance, controlled by a single, major resistance gene (R gene), and quantitative resistance (QR), controlled by numerous, small effect loci. R-gene-mediated blackleg resistance has been extensively studied, wherein several genomic regions harbouring R genes against L. maculans have been identified and three of these genes were cloned. These studies advance our understanding of the mechanism of R gene and pathogen avirulence (Avr) gene interaction. Notably, these studies revealed a more complex interaction than originally thought. Advances in genomics help unravel these complexities, providing insights into the genes and genetic factors towards improving blackleg resistance. Here, we aim to discuss the existing R-gene-mediated resistance, make a summary of candidate R genes against the disease, and emphasise the role of players involved in the pathogenicity and resistance. The comprehensive result will allow breeders to improve resistance to L. maculans, thereby increasing yield.

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Assessing Quantitative Resistance against Leptosphaeria maculans (Phoma Stem Canker) in Brassica napus (Oilseed Rape) in Young Plants

PLoS ONE ◽

10.1371/journal.pone.0084924 ◽

2014 ◽

Vol 9 (1) ◽

pp. e84924 ◽

Cited By ~ 23

Author(s):

Yong-Ju Huang ◽

Aiming Qi ◽

Graham J. King ◽

Bruce D. L. Fitt

Keyword(s):

Brassica Napus ◽

Oilseed Rape ◽

Quantitative Resistance ◽

Leptosphaeria Maculans ◽

Stem Canker ◽

Phoma Stem Canker

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Limitations of transcriptome-based prediction of pathogenicity genes in the plant pathogen Leptosphaeria maculans

FEMS Microbiology Letters ◽

10.1093/femsle/fnz080 ◽

2019 ◽

Vol 366 (7) ◽

Cited By ~ 2

Author(s):

Andrew S Urquhart ◽

Alexander Idnurm

Keyword(s):

Gene Expression ◽

Brassica Napus ◽

Leptosphaeria Maculans ◽

Simple Expression ◽

Gene Transcript ◽

Blackleg Disease ◽

Fungal Pathogenicity ◽

Transcript Levels ◽

Oilseed Crops ◽

High Gene

ABSTRACT Identification of pathogenicity determinants in Leptosphaeria maculans, a major cause of disease of oilseed crops, has been a focus of research for many years. A wealth of gene expression information from RNA sequencing promises to illuminate the mechanisms by which the fungus is able to cause blackleg disease. However, to date, no studies have tested the hypothesis that high gene transcript levels during infection correlate with importance to disease progression. In this study, we use CRISPR-Cas9 to disrupt 11 genes that are highly expressed during the early stages of disease and show that none of these genes are crucial for fungal pathogenicity on Brassica napus. This finding suggests that in order to understand the pathogenicity of this fungus more sophisticated techniques than simple expression analysis will need to be employed.

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Genome-Wide Identification and Analysis of VQ Motif-containing Gene Family in Brassica napus and Functional Characterization of BnMKS1 in Response to Leptosphaeria maculans

Phytopathology ◽

10.1094/phyto-04-20-0134-r ◽

2020 ◽

Cited By ~ 1

Author(s):

Zhongwei Zou ◽

Fei Liu ◽

Shuanglong Huang ◽

DILANTHA GERARD FERNANDO

Keyword(s):

Brassica Napus ◽

Gene Family ◽

Functional Characterization ◽

Leptosphaeria Maculans ◽

Defense Responses ◽

Marker Genes ◽

Blackleg Disease ◽

Genome Wide ◽

A Genome

Proteins containing Valine-glutamine (VQ) motifs play important roles in plant growth and development, as well as in defense responses to both abiotic and biotic stresses. Blackleg disease, which is caused by Leptosphaeria maculans, is the most important disease in canola (Brassica napus L.) worldwide. H; however, the identification of B. napus VQs and their functions in response to blackleg disease have not yet been reported. In this study, we conducted a genome genome-wide identification and characterization of the VQ gene family in B. napus, including chromosome location, phylogenetic relations, gene structure, motif domain, synteny analysis, and cis-elements categorization of their promoter regions. To understand B. napus VQ gene function in response to blackleg disease, we overexpressed BnVQ7 (BnaA01g36880D, also known as the mitogen-activated protein kinase4 substrate1 (MKS1) gene) in a blackleg-susceptible canola variety Westar. Overexpression The overexpression of BnMKS1 in canola did not improve its resistance to blackleg disease at the seedling stage. H; however, transgenic canola plants overexpressing BnMKS1 displayed an enhanced resistance to L. maculans infection at the adult plant stage. Expression levels of downstream and defense marker genes in cotyledons increased significantly at the necrotrophic stage of L. maculans infection in the overexpression line of BnMKS1, suggesting that the SA salicylic acid (SA)- and jasmonic acid (JA )-mediated signaling pathways were both involved in the defense responses. Together, these results suggest that BnMKS1 might play an important role in the defense against L. maculans.

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Zinc suppresses sirodesmin PL toxicity and protects Brassica napus plants against the blackleg disease caused by Leptosphaeria maculans

Plant Science ◽

10.1016/0168-9452(90)90155-h ◽

1990 ◽

Vol 68 (1) ◽

pp. 77-86 ◽

Cited By ~ 9

Author(s):

Thierry Rouxel ◽

Albert Kollmann ◽

Jean-Francois Bousquet

Keyword(s):

Brassica Napus ◽

Leptosphaeria Maculans ◽

Blackleg Disease

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Genome Wide Identification of the Immunophilin Gene Family in Leptosphaeria maculans: A Causal Agent of Blackleg Disease in Oilseed Rape (Brassica napus)

OMICS A Journal of Integrative Biology ◽

10.1089/omi.2014.0081 ◽

2014 ◽

Vol 18 (10) ◽

pp. 645-657 ◽

Cited By ~ 12

Author(s):

Khushwant Singh ◽

Miloslav Zouhar ◽

Jana Mazakova ◽

Pavel Rysanek

Keyword(s):

Brassica Napus ◽

Gene Family ◽

Oilseed Rape ◽

Leptosphaeria Maculans ◽

Causal Agent ◽

Blackleg Disease ◽

Genome Wide

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

Plant Disease ◽

10.1094/pdis.2003.87.6.752a ◽

2003 ◽

Vol 87 (6) ◽

pp. 752-752 ◽

Cited By ~ 85

Author(s):

H. Li ◽

K. Sivasithamparam ◽

M. J. Barbetti

Keyword(s):

Brassica Napus ◽

Resistance Gene ◽

Disease Severity ◽

Brassica Rapa ◽

Oilseed Rape ◽

Leptosphaeria Maculans ◽

Blackleg Disease ◽

Field Isolates ◽

Western Australian ◽

Lower Stem

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.

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Phytoalexins and Signalling Metabolites Produced in the Wild Crucifer Neslia Paniculata: Camalexins and Arabidopsides

10.26434/chemrxiv.14166866 ◽

2021 ◽

Author(s):

M. Soledade C. Pedras ◽

Mahla Alavi ◽

Abbas Abdoli

Keyword(s):

Plant Species ◽

Leptosphaeria Maculans ◽

Blackleg Disease ◽

In Planta ◽

First Report ◽

Signalling Molecules ◽

The Third ◽

Hybrid Plants ◽

In The Wild ◽

Number Of Plant Species

The metabolites produced in elicited leaves of the wild crucifer Neslia paniculata (L.) Desv. were investigated in abiotically stressed plants. Herein the phytoalexins camalexin, 7-methoxycamalexin and 6,7-dimethoxycamalexin together with the signalling metabolites arabidopsides A and D are reported. This is the first report of occurrence of 7-methoxy and 6,7-dimethoxycamalexins in planta and the third example of synchronized biosynthesis of phytoalexins and galacto-oxylipins. It is suggested that arabidopsides and structurally similar metabolites functioning as signalling molecules are likely to occur in a much larger number of plant species than originally hypothesized. N. paniculata is potentially useful to generate hybrid plants resistant to blackleg disease caused by Leptosphaeria maculans.

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