Advances in understanding the Leptosphaeria maculans - Brassica pathosystem and their impact on disease management

Figures & data

Table 1. Canola/rapeseed (Brassica napus) production (million metric tonnes), area sown (million hectares) and yield (tonnes/hectare) over the past five years for specific countries.

Country/Region	Production (mmt)^1					Area (mha)^1					Yield (t/ha)^1
	2013/14	2014/15	2015/16	2016/17	2017/18	2013/14	2014/15	2015/16	2016/17	2017/18	2013/14	2014/15	2015/16	2016/17	2017/18
Australia	3.8	3.5	3.0	4.4	3.8	2.7	2.5	2.3	2.8	2.7	1.4	1.8	1.3	1.2	1.4
Canada	18.6	16.4	18.4	19.7	21.3	9.3	8.3	8.4	8.4	8.2	2.3	2.4	2.2	2.0	2.3
China	11.6	10.0	8.0	5.7	5.0	3.1	3.8	5.2	6.2	6.8	1.6	1.5	1.6	1.6	1.7
France	4.4	5.5	5.3	4.7	5.2	1.4	1.5	1.5	1.5	1.4	3.7	3.1	3.5	3.7	3.0
Germany	5.8	6.1	4.9	4.6	4.3	1.3	1.3	1.3	1.4	1.5	3.3	3.5	3.8	4.3	3.9
UK	2.1	2.5	2.5	1.8	2.1	0.6	0.6	0.7	0.7	0.7	3.7	3.1	3.9	3.6	3.0
World^2	71.7	70.4	68.7	69.4	74.0

¹Production figures for individual countries were provided by the Australian Oilseeds federation.

²World oilseed production figures sourced from the United States Department of Agriculture.

Fig. 1 (Colour online) Interaction of Leptosphaeria maculans avirulence genes and Brassica resistance genes. (a) Fifteen resistance genes have been identified; two of which have been cloned (green). Eight of the corresponding avirulence genes have been cloned. Of these, AvrLm1-L3, AvrLm4-7 and AvrLm5-9 have dual specificity, recognised by two different resistance genes. The AvrLm10-Rlm10 interaction involves two avirulence genes (AvrLm10A/AvrLm10B). (b) The AvrLm4-7 gene is epistatic over AvrLm3 and AvrLm9 (represented by red lines in (a)). When the avirulent allele of AvrLm7 is present, isolates will be virulent towards Rlm3 (b) or Rlm9, regardless of the genotype at the AvrLm3 and AvrLm9 locus, respectively.

Table 2. Avirulence genes of Leptosphaeria maculans, mechanisms for virulence and technologies used to monitor their frequencies.

Avirulence gene	Major mechanisms for virulence	Minor mechanisms for virulence	Molecular techniques for monitoring allele frequency in field populations	References
AvrLm1-L3^a	Deletion of entire gene	RIP	Standard PCR, KASP, qPCR	Gout et al. (2006); Gout et al. (2007); Van de Wouw et al. (2010b); Larkan et al. (2013); Kaczmarek et al. (2014); Simes (2018)
AvrLm2	AA sub (E^146 – Q)	RIP, small deletion	RFLP, KASP	Van de Wouw et al. (2010a); Ghanbarnia et al. (2015); Simes (2018)
AvrLm3	AA sub (I^58-H, G^131-R)	N/A	KASP	Plissonneau et al. (2016); Plissonneau et al. (2017b); Simes (2018)
AvrLm4-7^a	AvrLm4 – AA sub (G^120-R)^c AvrLm7 – Deletion or RIP	AvrLm7, AA sub	RFLP, KASP, pyrosequencing, high resolution mapping, ARMS-PCR	Parlange et al. (2009); Daverdin et al. (2012); Van de Wouw and Howlett (2012); Carpezat et al. (2014); Plissonneau et al. (2017a); Simes (2018); Zou et al. (2018)
AvrLm5-9^a	AvrLm5 – AA sub (R^29-Stop) AvrLm9 – AA sub (K^55-R)	N/A	RFLP, KASP	Van de Wouw et al. (2014b); Ghanbarnia et al. (2018); Simes (2018)
AvrLm6	Deletion	RIP, AA sub	Standard PCR, KASP, qPCR	Fudal et al. (2009); Van de Wouw et al. (2010a); Van de Wouw et al. (2010b); Kaczmarek et al. (2014)
AvrLm10A/AvrLm10B^b	Deletion^d	N/A	N/A	Petit-Houdent et al. (2019)
AvrLm11	Deletion	N/A	Standard PCR, KASP	Balesdent et al. (2013)

Abbreviations: AA sub = amino acid substitution; N/A = not applicable; RIP = Repeat-induced point mutation; RFLP = Restriction fragment length polymorphism; KASP = Kompetitive allele specific PCR; qPCR = quantitative PCR; ARMS-PCR = tetra-primer amplification refractory mutation system-PCR

^aAvrLm1-L3 recognises both Rlm1 and LepR3. AvrLm4-7 recognises both Rlm4 and Rlm7 and AvrLm5-9 recognises both Rlm5 and Rlm9.

^bTwo avirulence genes, AvrLm10A and AvrLm10B, are required for AvrLm10-Rlm10 – mediated resistance.

^cIf an isolate is virulent towards Rlm7, it will also be virulent towards Rlm4. Therefore, RIP and deletion mutations can also confer virulence towards Rlm4.

^dPresence/absence polymorphism has been reported in the avirulent and virulent parents used for crossing to identify the genes, however no population surveys have been performed to determine whether deletion is responsible for virulence in field populations.

Table 3. Classification of resistance genes of Australian and Canadian commercial cultivars of Brassica napus.

Resistance gene	Australian Resistance group	Canadian Resistance group
Rlm1/LepR3	Group A	Group A
Rlm2		Group B
Rlm3	Group C	Group C
Rlm4	Group B	Group E1
Rlm5	Group G
Rlm6	Group F
Rlm7	Group H	Group E2
Rlm9		Group F
RlmS	Group S	Group G
LepR1	Group D	Group D
LepR2		Group H

Fig. 2 Changes in avirulence allele frequencies for AvrLm1 (a), AvrLm4 (b) and AvrLm6 (c) of Leptosphaeria maculans isolates collected from cultivars with different resistance genes in Australia. The frequency of the avirulence alleles differs depending on genotype of the cultivars from which the isolates were collected. The mating-type (d) allele was used as a control as the presence of resistance genes does not alter the frequency of the two mating types in fungal populations.

Gout L, Fudal I, Kuhn ML, Blaise F, Eckert M, Cattolico L, Balesdent MH, Rouxel T. 2006. Lost in the middle of nowhere: the AvrLm1 avirulence gene of the Dothideomycete Leptosphaeria maculans. Mol Microbiol. 60:67–80.

 PubMed Web of Science ®Google Scholar

Gout L, Kuhn ML, Vincenot L, Bernard-Samain S, Cattolico L, Barbetti M, Moreno-Rico O, Balesdent MH, Rouxel T. 2007. Genome structure impacts molecular evolution at the AvrLm1 avirulence locus of the plant pathogen Leptosphaeria maculans. Environ Microbiol. 9:2978–2992.

 PubMed Web of Science ®Google Scholar

Van de Wouw AP, Stonard JF, Howlett BJ, West JS, Fitt BD, Atkins SD. 2010b. Determining frequencies of avirulent alleles in airborne Leptosphaeria maculans inoculum using quantitative PCR. Plant Pathol. 59:809–818.

 Web of Science ®Google Scholar

Larkan NJ, Lydiate DJ, Parkin IA, Nelson MN, Epp DJ, Cowling WA, Rimmer SR, Borhan MH. 2013. The Brassica napus blackleg resistance gene LepR3 encodes a receptor-like protein triggered by the Leptosphaeria maculans effector AVRLM1. New Phytol. 197:595–605.

 PubMed Web of Science ®Google Scholar

Kaczmarek J, Latunde-Dada AO, Irzykowski W, Cools H, Stonard JF, Brachaczek A, Jedryczka M. 2014. Molecular screening for avirulence alleles AvrLm1 and AvrLm6 in airborne inoculum of Leptosphaeria maculans and winter oilseed rape (Brassica napus) plants from Poland and the UK. J Appl Gen. 55:529–539.

 PubMed Web of Science ®Google Scholar

Simes J. 2018. [accessed 2018 Nov]. https://www.producer.com/2018/04/new-lab-test-helps-farmers-protect-against-blackleg/.

 Google Scholar

Van de Wouw AP, Cozijnsen AJ, Hane JK, Brunner PC, McDonald BA, Oliver RP, Howlett BJ. 2010a. Evolution of linked avirulence effectors in Leptosphaeria maculans is affected by genomic environment and exposure to resistance genes in host plants. PLoS Pathog. 6:e1001180.

 PubMed Web of Science ®Google Scholar

Ghanbarnia K, Fudal I, Larkan NJ, Links MG, Balesdent M, Profotova B, Fernando DWG, Rouxel T, Borhan H. 2015. Rapid identification of the Leptosphaeria maculans avirulence gene AvrLm2 using an intraspecific comparative genomics approach. Mol Plant Pathol. 16:699–709.

 PubMed Web of Science ®Google Scholar

Plissonneau C, Daverdin G, Ollivier B, Blaise F, Degrave A, Fudal I, Rouxel T, Balesdent M. 2016. A game of hide and seek between avirulence genes AvrLm4-7 and AvrLm3 in Leptosphaeria maculans. New Phytol. 209:1613–1624.

 PubMed Web of Science ®Google Scholar

Plissonneau C, Rouxel T, Chevre AM, Van de Wouw AP, Balesdent MH. 2017b. One gene-one name: the AvrLmJ1 avirulence gene of Leptosphaeria maculans is AvrLm5. Mol Plant Pathol. 19:1012–1016.

 PubMed Web of Science ®Google Scholar

Parlange F, Daverdin G, Fudal I, Kuhn ML, Balesdent MH, Blaise F, Grezes-Besset B, Rouxel T. 2009. Leptosphaeria maculans avirulence gene AvrLm4-7 confers a dual recognition specificity by the Rlm4 and Rlm7 resistance genes of oilseed rape, and circumvents Rlm4-mediated recognition through a single amino acid change. Mol Microbiol. 71:851–863.

 PubMed Web of Science ®Google Scholar

Daverdin G, Rouxel T, Gout L, Aubertot JN, Fudal I, Meyer M, Parlange F, Carpezat J, Balesdent MH. 2012. Genome structure and reproductive behaviour influence the evolutionary potential of a fungal phytopathogen. PLoS Pathog. 8:e1003020.

 PubMed Web of Science ®Google Scholar

Van de Wouw AP, Howlett BJ. 2012. Estimating frequencies of virulent isolates in field populations of a plant pathogenic fungus, Leptosphaeria maculans, using high-throughput pyrosequencing. J Appl Microbiol. 113:1145–1153.

 PubMed Web of Science ®Google Scholar

Carpezat J, Bothorel S, Daverdin G, Balesdent MH, Leflon M. 2014. Use of high resolution melting analysis to genotype the avirulence AvrLm4-7 gene of Leptoaphaeria maculans, a fungal pathogen of Brassica napus. Ann Appl Biol. 164:430–444.

 Web of Science ®Google Scholar

Plissonneau C, Blaise F, Ollivier B, Leflon M, Carpezat J, Rouxel T, Balesdent MH. 2017a. Unusual evolutionary mechanisms to escape effector-triggered immunity in the fungal phytopathogen Leptosphaeria maculans. Mol Ecol. 26:2183–2198.

 PubMed Web of Science ®Google Scholar

Zou Z, Liu F, Fernando DWG. 2018. Rapid detection of Leptosphaeria maculans avirulence gene AvrLm4-7 conferring the avirulence/virulence specificity on Brassica napus using a tetra-primer ARMS-PCR. Eur J Plant Pathol. 152:515–520.

 Web of Science ®Google Scholar

Van de Wouw AP, Lowe RGT, Elliott CE, Dubois DJ, Howlett BJ. 2014b. An avirulence gene, AvrLmJ1, from the blackleg fungus, Leptosphaeria maculans, confers avirulence to Brassica juncea cultivars. Mol Plant Pathol. 15:523–530.

 PubMed Web of Science ®Google Scholar

Ghanbarnia K, Ma L, Larkan NJ, Haddadi P, Fernando DWG, Borhan MH. 2018. Leptosphaeria maculans AvrLm9: a new player in the game of hide and seek with AvrLm4-7. Mol Plant Pathol. 19:1754–1764.

 PubMed Web of Science ®Google Scholar

Fudal I, Ross S, Brun H, Besnard AL, Ermel M, Kuhn ML, Balesdent MH, Rouxel T. 2009. Repeat-induced point mutation (RIP) as an alternative mechanism of evolution toward virulence in Leptosphaeria maculans. Mol Plant Microbe Interact. 22:932–941.

 PubMed Web of Science ®Google Scholar

Petit-Houdent Y, Degrave A, Meyer M, Blaise F, Ollivier B, Marais CL, Jauneau A, Audran C, Rivas S, Veneault-Fourrey C, et al. 2019. A two genes-for-one gene interaction between Leptosphearia maculans and Brassica napus. New Phytol. 223:397–411. doi:10.1111/nph.15762

 Web of Science ®Google Scholar

Balesdent MH, Fudal I, Ollivier B, Bally P, Grandaubert J, Eber F, Chevre AM, Leflon M, Rouxel T. 2013. The dispensable chromosome of Leptosphaeria maculans shelters an effector gene conferring avirulence towards Brassica rapa. New Phytol. 198:887–898.

 PubMed Web of Science ®Google Scholar