Nano-Metals Forming Bacteria in Egypt. II. Efficacy towards Some Bacterial Soft Rot/Blackleg Genera ex vivo and Bio-Control in vivo as Eco-Friendly Therapeutic against Dickeya Solani

The antibacterial activity of Cu, Fe, Co, and ZnNPs which were formed by Enterococcus thailandicus, Pseudomonas putida, Marinobacter hydrocarbonoclasticus, and P. geniculate sequentially was tested against some soft rot/blackleg genera. The effects of NPs were recorded on bacterial DNA, proteins, and carbohydrates concentration of Pectobacterium carotovorum, Enterobacter cloacae (soft rot), and Dickeya solani (soft rot/blackleg). Treated cells showed degradation in the DNA isolated, and a decrease in proteins, and carbohydrates concentration compared with untreated cells. The treated cells using SEM showed collapsed, and small pits in the cell wall. Internal changes using TEM showed penetration of NPs inside the tested bacterial cells, the appearance of periplasmic space, formation vacuoles, and condensation of cytoplasm. Disease severity ex vivo of tuber infected with tested genera demonstrated that NPs treatment didn't show any rotted tissue compared with untreated. FeNPs were tested to control soft rot/blackleg disease caused by D. solani in comparison with copper pesticide. Present data recorded an increase in shoot and root length, in addition to, increase in dry and fresh weight, compared with either infected or healthy plants. In studying the ability of treated potato (Solanum tuberosum) seedlings with NPs to uptake and accumulate FeNPs from the soil, ICP-OES recorded a small increase in Fe content of treated plants compared with untreated. FeNPs can be used to control soft rot/blackleg disease caused by D. solani instead of copper pesticide and can be considered as a new and alternative approach to traditional disease management methods, and also increase the nutritional value of the plants

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.

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.

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.

Epidemiology of Blackleg Disease in Ethiopia

Blackleg is an infectious bacterial disease of cattle and rarely of other ruminants. This bacteria is caused by Clostridium chauvoei which is an anaerobic, gram positive, motile, rod-shaped bacillus bacterium and persists in the soil as resistant spores. Blackleg is an acute or subacute but chronic disease may occur. It occurs most frequently in animals 6-24 months of age and the disease mainly affects none vaccinated as well as animals in good nutritional condition. It produces persistent spores when conditions are not ideal and spores are highly resistant to environmental factors and disinfectants. Infected ruminants do not directly transmit the disease to other animals. The bacteria enter the body through the alimentary mucosa after ingestion of contaminated feed. Secretion of cytolytic toxins that cause necrosis of vascular endothelia .The toxins are absorbed into the animal’s bloodstream which makes the animal acutely sick and causes rapid death. Economic importance due to blackleg is loss of animals, milk production and draft oxen, and cost for treatment and vaccination. Fatality rate of blackleg in fully susceptible populations approaches 100%. Clinical Signs include lethargy anorexia, reluctance to move lameness and recumbence. When superficial muscles are affected, swelling and crepitus are evident. Cattle found dead of blackleg are lying on the side with the affected hind limb stands out stiffly, bloating and putrefaction occur quickly and bloodstained from exudates, nostrils and anus. The disease can be diagnosed using laboratory diagnosis, Immune Fluorescent, Cell Culture and PCR. Control and prevention relies mainly on vaccination.

Constitutive expression of transcription factor SirZ blocks pathogenicity in Leptosphaeria maculans independently of sirodesmin production

Sirodesmin, the major secondary metabolite produced by the plant pathogenic fungus Leptosphaeria maculans in vitro, has been linked to disease on Brassica species since the 1970s, and yet its role has remained ambiguous. Re-examination of gene expression data revealed that all previously described genes and two newly identified genes within the sir gene cluster in the genome are down-regulated during the crucial early establishment stages of blackleg disease on Brassica napus. To test if this is a strategy employed by the fungus to avoid damage to and then detection by the host plant during the L. maculans asymptomatic biotrophic phase, sirodesmin was produced constitutively by overexpressing the sirZ gene encoding the transcription factor that coordinates the regulation of the other genes in the sir cluster. The sirZ over-expression strains had a major reduction in pathogenicity. Mutation of the over-expression construct restored pathogenicity. However, mutation of two genes, sirP and sirG, required for specific steps in the sirodesmin biosynthesis pathway, in the sirZ over-expression background resulted in strains that were unable to synthesize sirodesmin, yet were still non-pathogenic. Elucidating the basis for this pathogenicity defect or finding ways to overexpress sirZ during disease may provide new strategies for the control of blackleg disease.

Validating the Strategic Deployment of Blackleg Resistance Gene Groups in Commercial Canola Fields on the Canadian Prairies

Blackleg, caused by the fungal pathogen Leptosphaeria maculans, is a serious threat to canola (Brassica napus L.) production in western Canada. Crop scouting and extended crop rotation, along with the use of effective genetic resistance, have been key management practices available to mitigate the impact of the disease. In recent years, new pathogen races have reduced the effectiveness of some of the resistant cultivars deployed. Strategic deployment and rotation of major resistance (R) genes in cultivars have been used in France and Australia to help increase the longevity of blackleg resistance. Canada also introduced a grouping system in 2017 to identify blackleg R genes in canola cultivars. The main objective of this study was to examine and validate the concept of R gene deployment through monitoring the avirulence (Avr) profile of L. maculans population and disease levels in commercial canola fields within the Canadian prairies. Blackleg disease incidence and severity was collected from 146 cultivars from 53 sites across Manitoba, Saskatchewan, and Alberta in 2018 and 2019, and the results varied significantly between gene groups, which is likely influenced by the pathogen population. Isolates collected from spring and fall stubble residues were examined for the presence of Avr alleles AvrLm1, AvrLm2, AvrLm3, AvrLm4, AvrLm5, AvrLm6, AvrLm7, AvrLm9, AvrLm10, AvrLm11, AvrLepR1, AvrLepR2, AvrLep3, and AvrLmS using a set of differential host genotypes carrying known resistance genes or PCR-based markers. The Simpson’s evenness index was very low, due to two dominant L. maculans races (AvrLm2-4-5-6-7-10-11 and AvrLm2-5-6-7-10-11) representing 49% of the population, but diversity of the population was high from the 35 L. maculans races isolated in Manitoba. AvrLm6 and AvrLm11 were found in all 254 L. maculans isolates collected in Manitoba. Knowledge of the blackleg disease levels in relation to the R genes deployed, along with the L. maculans Avr profile, helps to measure the effectiveness of genetic resistance.

Genetic Analysis of a Horizontal Resistance Locus BLMR2 in Brassica napus

Leptosphaeria maculans causes blackleg disease in Brassica napus. The blackleg disease is mainly controlled by resistance genes in B. napus. Previous studies have shown that the blackleg resistant BLMR2 locus that conferred horizontal resistance under field conditions, is located on chromosome A10 of B. napus. The purpose of this study is to fine map this locus and hence identify a candidate gene underlying horizontal resistance. The spectrum of resistance to L. maculans isolates of the resistance locus BLMR2 was analyzed using near isogenic lines, resistant, and susceptible cultivars. The results showed that this locus was horizontally resistant to all isolates tested. Sequence characterized amplified regions (SCAR), simple sequence repeats (SSR), and single nucleotide polymorphism (SNP) markers were developed in the chromosome region of BLMR2 and a fine genetic map was constructed. Two molecular markers narrowed BLMR2 in a 53.37 kb region where six genes were annotated. Among the six annotated genes, BnaA10g11280D/BnaA10g11290D encoding a cytochrome P450 protein were predicted as the candidate of BLMR2. Based on the profiling of pathogen induced transcriptome, three expressed genes in the six annotated genes were identified while only cytochrome P450 showed upregulation. The candidate corresponds to the gene involved in the indole glucosinolate biosynthesis pathway and plant basal defense in Arabidopsis thaliana. The molecular markers identified in this study will allow the quick incorporation of the BLMR2 allele in rapeseed cultivars to enhance blackleg resistance.

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

The metabolites produced in elicited leaves of the wild crucifer <i>Neslia paniculata</i> (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 <i>in planta</i> 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. <i>N. paniculata</i> is potentially useful to generate hybrid plants resistant to blackleg disease caused by <i>Leptosphaeria maculans.</i>