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.

Warm and Wet Autumns Favour Yield Losses of Oilseed Rape Caused by Phoma Stem Canker

Winter oilseed rape (Brassica napus L.) is the main source of domestic oil in central and northern Europe, bringing profits to farmers, but the plants are often damaged by stem canker, caused by two fungal species belonging to the genus Leptosphaeria. Due to environmental concerns, the benefits of fungicide applications must outweigh disadvantages. The aim of this work was to determine the effect of stem canker on seed yield and its quality and find out the best timing of fungicide application. The multi-year field experiments were done at two sites in south-west Poland, where the disease is regarded as a serious problem. The fungicide treatments with the azole-containing preparation followed the same scheme each year; a single application was made at one-week intervals, starting in late September through mid-November for a total of eight treatments. Seed yield, oil and protein content, mass of thousand seeds as well as indole-and alkenyl-glucosinolate contents in seeds were statistically unrelated with the incidence and severity of phoma leaf spotting and stem canker symptoms. The significant decrease of the seed yield was observed in three (site × year combinations) of eight, in which phoma leaf spotting and stem canker were severe. Yield loss was noted only in years with warm and wet autumns, when cumulative mean temperatures between BBCH14 and BBCH19 plant growth stages exceeded 60 °C and precipitation in this period exceeded 110 mm of rain. Under these conditions, fungicide treatments were highly effective when they were done between BBCH15–BBC16 growth stages (5–6 true leaves).

Mycovirus-Induced Hypervirulence of Leptosphaeria biglobosa Enhances Systemic Acquired Resistance to Leptosphaeria maculans in Brassica napus

Phoma stem canker (blackleg) is one of the most important diseases of winter oilseed rape (Brassica napus) worldwide and is caused by a complex that comprises at least two species: Leptosphaeria maculans and L. biglobosa. Screening a panel of field Leptosphaeria isolates from B. napus for the presence of mycoviruses revealed the presence of a novel double-stranded RNA quadrivirus in L. biglobosa and no viruses in L. maculans. Following elimination of the mycovirus, virus-infected and virus-free isogenic lines of L. biglobosa were created. A direct comparison of the growth and virulence of these isogenic lines illustrated that virus infection caused hypervirulence and resulted in induced systemic resistance toward L. maculans in B. napus following lower leaf preinoculation with the virus-infected isolate. Analysis of the plant transcriptome suggests that the presence of the virus leads to subtle alterations in metabolism and plant defenses. For instance, transcripts involved in carbohydrate and amino acid metabolism are enriched in plants treated with the virus-infected isolate, while pathogenesis-related proteins, chitinases and WRKY transcription factors are differentially expressed. These results illustrate the potential for deliberate inoculation of plants with hypervirulent L. biglobosa to decrease the severity of Phoma stem canker later in the growing season. [Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

The Harmfulness of Phoma Stem Canker, Sclerotinia Stem Rot, and Phytoplasma on Winter Oilseed Rape with Regard to Czech Breeding Programs

New genotypes of winter oilseed rape bred under the Czech breeding program were subjected to performance tests within the framework of inter-institutional pre-tests. At four monitored localities in the Czech Republic, field trials were carried out on 50 genotypes in the years 2015 to 2017. In these trials, evaluations were made on infestation by the fungal pathogens Leptosphaeria maculans, Leptosphaeria biglobosa, and Sclerotinia sclerotiorum, as well as the level of infestation caused by phytoplasmas. The evaluation of new oilseed rape genotypes confirmed a medium to high level of resistance to phoma stem canker and Sclerotinia stem rot compared to the resistant reference varieties. The most severe infestations caused by Leptosphaeria spp. were observed in Chlumec nad Cidlinou in 2015 (mean severity score 22.38%) and in Opava in 2016 (mean severity score 20.72%). The same trend was observed for infestations caused by S. sclerotiorum, where the most damage to trial plots were observed at the same localities, in 2015 in Chlumec nad Cidlinou (mean severity score 36.85%) and in 2016 in Opava (mean severity score 18.84%). PCR determination of Leptosphaeria spp. showed that 98% of sampled leaves in autumn season showing symptoms were infected by L. maculans, while the remaining 2% were identified as bearing L. biglobosa. Molecular detection of infected parts of oilseed rape plants with symptoms of phyllody confirmed in all cases the presence of phytoplasma isolates from the 16SrI Aster Yellows group.

Careful deployment of oilseed rape crops with Rlm6 resistance gene against L. maculans is recommended to prevent the loss of efficacy of this resistance gene in French condiment mustard

Breeding varieties for increased disease resistance is a major means to control epidemics. However, the deployment of resistance genes through space and time drives the genetic composition of the pathogen population, with predictable changes in pathotype frequencies. In France, Leptosphaeria maculans causes disease on Brassica napus oilseed rape crops but not on B. juncea condiment mustard. Prior to the deployment of winter B. napus varieties with Rlm6 resistance gene introduced from B. juncea, the aim of our study was to investigate if this deployment could impact disease control in condiment mustard. We assessed the presence of resistance genes against phoma stem canker in a set of current French B. juncea varieties and breeding lines. Rlm6 was detected in all the 12 condiment mustard varieties. Rlm5 was also detected in 8 varieties. No additional resistance genes were detected with the set of isolates used. Because frequency of isolates virulent on Rlm6 is very low, these results indicate that Rlm6 gene is a major component of disease control in the French B. juncea mustards tested. Using Rlm6 in oilseed rape varieties will very likely induce an increase in frequency of Rlm6 virulent isolates. This raises the acute concern of a wise deployment of oilseed rape around the condiment mustard growing area. Scientific knowledge on adaptation dynamics, spatial segregation of crops and cooperation between actors is currently available in order to mitigate the risk and advert negative consequences of the introduction of Rlm6 resistance gene in oilseed rape varieties.

Analysis of Leptosphaeria species complex causing phoma leaf spot and stem canker of winter oilseed rape (Brassica napus) in the Czech Republic

In the Czech Republic, increases in the area sown with oilseed rape (Brassica napus L.) and shifts to intensive crop farming systems have contributed to increased incidence and harmfulness of phoma stem canker. This study comprised a large-scale survey of the occurrence of two closely related causal agents of the disease, Leptosphaeria maculans and L. biglobosa, in oilseed rape tissues and the country-wide distribution of each fungal species. In the 2007–11 growing seasons, 1132 leaves with phoma leaf spot symptoms were sampled; from those, 977 L. maculans-type and 477 L. biglobosa-type leaf spots were sampled and analysed by species-specific PCR without pathogen isolation. There were 1159 leaf spots confirmed as infected by Leptosphaeria spp., with 65% of 907 L. maculans-type leaf spots infected by L. maculans only and 35% co-infected by both species; and with 88% of 252 L. biglobosa-type leaf spots infected by L. biglobosa only and 12% co-infected by both species. Furthermore, 217 monopycnidial isolates were collected from selected leaf spots and identified based on pigment production during solid- and liquid-media culture and PCR assay. Most (82%) isolates originating from L. maculans-type leaf lesions were L. maculans, and most (69%) isolates collected from L. biglobosa-type leaf lesions were L. biglobosa. Co-infection by both species was found in both L. maculans-type and L. biglobosa-type leaf lesions. In 2007–12, 708 stems with phoma stem canker symptoms and 2635 plant tissues from upper stem, stem base, root collar and taproot of each stem were sampled for PCR; symptoms on the four parts of each stem were assessed before taking tissue samples. There were 1495 plant tissues confirmed as infected by Leptosphaeria spp., with the proportion of plant tissue in which only L. biglobosa DNA (62%) was amplified greater than that with only L. maculans DNA (11%) or with both L. maculans and L. biglobosa DNA (27%). Although both species were detected in leaf samples in autumn, L. biglobosa was the more frequently detected species in stem samples in summer, suggesting that L. biglobosa is the more successful in colonising oilseed rape tissues in later growth stages of the plant in the Czech Republic.