Metabolic Capacity Differentiates Plenodomus lingam from P. biglobosus Subclade ‘brassicae’, the Causal Agents of Phoma Leaf Spotting and Stem Canker of Oilseed Rape (Brassica napus) in Agricultural Ecosystems

In contrast to the long-lasting taxonomic classification of Plenodomus lingam and P. biglobosus as one species, formerly termed Leptosphaeria maculans, both species form separate monophyletic groups, comprising sub-classes, differing considerably with epidemiology towards Brassicaceae plants. Considering the great differences between P. lingam and P. biglobosus, we hypothesized their metabolic capacities vary to a great extent. The experiment was done using the FF microplates (Biolog Inc., Hayward, CA, USA) containing 95 carbon sources and tetrazolium dye. The fungi P. lingam and P. biglobosus subclade ‘brassicae’ (3 isolates per group) were cultured on PDA medium for 6 weeks at 20 °C and then fungal spores were used as inoculum of microplates. The test was carried out in triplicate. We have demonstrated that substrate richness, calculated as the number of utilized substrates (measured at λ490 nm), and the number of substrates allowing effective growth of the isolates (λ750 nm), showed significant differences among tested species. The most efficient isolate of P. lingam utilized 36 carbon sources, whereas P. biglobosus utilized 60 substrates. Among them, 25–29 carbon sources for P. lingam and 34–48 substrates for P. biglobosus were efficiently used, allowing their growth. Cluster analysis based on Senath criteria divided P. biglobosus into two groups and P. lingam isolates formed one group (33% similarity). We deduce the similarities between the tested species help them coexist on the same host plant and the differences greatly contribute to their different lifestyles, with P. biglobosus being less specialized and P. lingam coevolving more strictly with the host plant.

Regulation of effector gene expression as concerted waves in Leptosphaeria maculans: a two-players game

During infection, plant pathogenic fungi secrete a set of molecules collectively known as effectors, involved in overcoming the host immune system and in disease establishment. Effector genes are concertedly expressed as waves all along plant pathogenic fungi lifecycle. However, little is known about how coordinated expression of effector genes is regulated. Since many effector genes are located in repeat-rich regions, the role of chromatin remodeling in the regulation of effector expression was recently investigated. In Leptosphaeria maculans, causing stem canker of oilseed rape, we established that the repressive histone modification H3K9me3 (trimethylation of Lysine 9 of Histone H3), deposited by the histone methyltransferase KMT1, was involved in the regulation of expression of genes highly expressed during infection, including effectors. Nevertheless, inactivation of KMT1 did not induce expression of these genes at the same level as observed during infection of oilseed rape, suggesting that a second regulator, such as a transcription factor (TF), might be involved. Pf2, a TF belonging to the Zn2Cys6 fungal specific TF family, was described in several Dothideomycete species as essential for pathogenicity and effector gene expression. We identified the orthologue of Pf2 in L. maculans, LmPf2, and investigated the role of LmPf2 together with KMT1, by inactivating and over-expressing LmPf2 in a wild type (WT) strain and a ∆kmt1 mutant. Functional analyses of the corresponding transformants highlighted an essential role of LmPf2 in the establishment of pathogenesis. Transcriptomic analyses during axenic growth showed that LmPf2 is involved in the control of effector gene expression. We observed an enhanced effect of the over-expression of LmPf2 on effector gene expression in a ∆kmt1 background, suggesting an antagonist role between KMT1 and LmPf2.

Brassica napus genes Rlm4 and Rlm7, conferring resistance to Leptosphaeria maculans, are alleles of the Rlm9 wall-associated kinase-like resistance locus

Brassica napus (canola/rapeseed) race specific resistance genes against blackleg disease, caused by the ascomycete fungus Leptosphaeria maculans, have been commonly used in canola breeding. To date, LepR3, Rlm2 and Rlm9 R genes against L. maculans have been cloned from B. napus. LepR3 and Rlm2 are Receptor Like Proteins (RLP) and the recently reported Rlm9 is a Wall Associated Kinase-Like (WAKL) protein. Rlm9 located on chromosome A07 is closely linked with Rlm3, Rlm4, RLm7 genes. Recognition of AvrLm5-9 and AvrLm3 by their corresponding Rlm9 and Rlm3 proteins is masked in the presence of AvrLm4-7. Here we report cloning of Rlm4 and Rlm7 by generating genome sequence of the doubled haploid (DH) B. napus cv Topas DH16516 introgression lines Topas-Rlm4 and Topas-Rlm7. Candidate Rlm4 and Rlm7 genes were identified form the genome sequence and gene structures were determined by mapping RNA-sequence reads, generated from infected cotyledon tissues, to the genome of Topas-Rlm4 and Topas-Rlm7. Rlm4 and Rlm7 genomic constructs with their native promoters were transferred into the blackleg susceptible B. napus cv Westar. Complementation of resistance response in the transgenic Westar-Rlm4 and Westar-Rlm7 that were inoculated with L. maculans transgenic isolates 2367-AvrRlm4-7 or 2367-AvrLm7 confirmed the function of Rlm4 and Rlm7 genes. Wild type L. maculans isolate 2367 that does not contain AvrLm4-7 or AvrLm7, and transgenic 2367-AvrLm3 and 2367-AvrLm5-9 did not induce resistance proving the specificity of Rlm4 and Rlm7 response. Rlm4 and Rlm7 alleles are also allelic to Rlm9. Rlm4 and Rlm7 genes encode WAKL proteins. Comparison of highly-homologous sequences of Rlm4 and Rlm7 with each other and with the sequence of additional alleles identified a limited number of point mutation located within the predicted extracellular receptor domains.

Detection of the Phoma pathogens Plenodomus biglobosus subclades ‘brassicae’ and ‘canadensis’ on wasabi, and ‘canadensis’ in Europe

Phoma stem canker / blackleg is an internationally important disease of Brassicas including B. napus (oilseed rape, OSR), caused by multiple genetic subclades of the fungi Plenodomus lingam (formerly Leptosphaeria maculans) and P. biglobosus (L. biglobosa). In Spring 2021, Phoma-like disease symptoms were observed on leaves and stems of Eutrema japonicum (wasabi) crops at three UK sites (Northern Ireland, Southern England and the West Midlands). Fungal isolation from wasabi leaf spots yielded colonies with two distinct phenotypes on potato dextrose agar (PDA). Isolates from the Northern Ireland and Southern England sites had white colonies with abundant pink cirri that were confirmed (based on ITS rDNA, beta tubulin and actin sequences) as P. biglobosus subclade ‘canadensis’ (Pbc). Those from the West Midlands site, however, had yellow pigmented colonies and were confirmed by sequencing as P. biglobosus subclade ‘brassicae’ (Pbb). Greenhouse pathogenicity testing showed that Pbb and Pbc wasabi isolates were pathogenic not only to this host but also OSR, B. oleracea (cabbage), and B. rapa (pak choi). Re-isolation of the fungi was attempted and confirmed from lesions that developed on inoculated OSR and wasabi, thus completing Koch’s postulates. These findings represent new discoveries for both Pbb and Pbc on wasabi, plus for Pbc in Europe. The crop health implications of these results are briefly considered.

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

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.

Development of Plant–Fungal Endophyte Associations to Suppress Phoma Stem Canker in Brassica

Endophytic microorganisms are found within the tissues of many plants species, with some conferring several benefits to the host plant including resistance to plant diseases. In this study, two putative endophytic fungi that were previously isolated from wild seeds of Brassica, identified as Beauveria bassiana and Pseudogymnoascus pannorum, were inoculated into cultivars of three Brassica species—Brassica napus, Br. rapa and Br. oleracea. Both fungal endophytes were reisolated from above- and below-ground tissues of inoculated plants at four different plant-growth stages, including cotyledon, one-leaf, two-leaf, and four-leaf stages. None of the plants colonised by these fungi exhibited any obvious disease symptoms, indicating the formation of novel mutualistic associations. These novel plant–endophyte associations formed between Brassica plants and Be. bassiana significantly inhibited phoma stem canker, a devastating disease of Brassica crops worldwide, caused by the fungal pathogen Leptosphaeria maculans. The novel association formed with P. pannorum significantly suppressed the amount of disease caused by L. maculans in one out of two experiments. Although biological control is not a new strategy, endophytic fungi with both antiinsect and antifungal activity are a highly conceivable, sustainable option to manage pests and diseases of economically important crops.

Growth Promotion of Rapeseed (Brassica napus L.) and Blackleg Disease (Leptosphaeria maculans) Suppression Mediated by Endophytic Bacteria

Rapeseed is an important oil crop strongly dependent on high agrochemical inputs. Some pathogens, including Leptosphaeria maculans, cause blackleg disease and can drastically decrease yields. Microbial inoculants seem to be a promising solution to these problems. However, a selection of potent bacterial strains able to improve growth and/or suppress disease is needed. Endophytic bacteria (n = 38) isolated from rapeseed plants with exceptionally good growth were screened for plant growth promoting (PGP) traits and L. maculans antifungal activity. A majority of isolates (35) showed the ability to produce siderophores, 17 isolates solubilized phosphate, and 28 isolates inhibited the growth of L. maculans. The six most promising isolates belonging to Bacillus genera were characterized in detail and compared to two previously published PGP strains. Plant growth measured as total weight and root length of rapeseed seedlings was stimulated by all isolates in comparison to control. The best isolate, 1L6, preliminary identified as Bacillus pumilus showed the highest phosphate solubilization, IAA and HCN production, and growth promotion of plants. Isolates with high antifungal activity in screening showed good potential to suppress disease on plants, with 87% reduction of lesions caused by L. maculans. These strains are good candidates to be explored under field use either solely or in combination.