Leptosphaeria maculans Isolates Reveal Their Allele Frequency in Western Canada.

Blackleg, caused by Leptosphaeria maculans, is a major disease of canola in Canada, Australia, and Europe. For effective deployment of resistant varieties and disease management, it is crucial to understand the population structure of L. maculans. In this study, we analyzed L. maculans isolates from commercial fields in western Canada from 2014 to 2016 for the presence and frequency of avirulence (Avr) genes. A total of 1,584 isolates were examined for the presence of Avr genes AvrLm1, AvrLm2, AvrLm3, AvrLm4, AvrLm6, AvrLm7, AvrLm9, AvrLepR1, AvrLepR2, and AvrLmS via a set of differential host genotypes carrying known resistance genes and a PCR assay. Several Avr genes showed a higher frequency in the pathogen population, such as AvrLm6 and AvrLm7, which were present in >90% of isolates, whereas AvrLm3, AvrLm9, and AvrLepR2 showed frequencies of <10%. A total of 189 races (different combinations of Avr genes) were detected, with Avr-2-4-6-7-S, Avr-1-4-6-7, and Avr-2-4-6-7 as the three predominant races. When the effect of crop rotation was assessed, only a 3-year rotation showed a significantly higher frequency of AvrLm2 relative to shorter rotations. This study provides the information for producers to select effective canola varieties for blackleg management and for breeders to deploy new R genes in disease resistance breeding in western Canada.

LmCBP1, a secreted chitin-binding protein, is required for the pathogenicity of Leptosphaeria maculans on Brassica napus.

Leptosphaeria maculans is the causal agent of blackleg disease on Brassica napus. Determining the underlying functions of genes required for pathogenesis is essential for understanding the infection process. A chitin-binding protein (LmCBP1) was discovered as a pathogenicity factor for the infection of B. napus by L. maculans through gene knockout using the CRISPR-Cas9 system. Chitin-binding activity was demonstrated through a chitin-protein binding assay. A secreted signal peptide was detected using a yeast secreted-signal peptide trap assay. An increased expression level during the infection stage was also observed, suggesting that LmCBP1 is a secreted protein. The knockout mutants showed decreased infection on B. napus, with reduced pathogenicity on ten cultivars with/without diverse R genes. The mutants were more sensitive to H2O2 compared to wild type L. maculans isolate JN3. This study provides evidence of the virulence of a novel chitin-binding protein LmCBP1 on B. napus through mutants created via the CRISPR-Cas9 system.

Author Correction: Comparison of DNA-, PMA-, and RNA-based 16S rRNA Illumina sequencing for detection of live bacteria in water.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Comparison of DNA-, PMA-, and RNA-based 16S rRNA Illumina sequencing for detection of live bacteria in water.

The limitation of 16S rRNA gene sequencing (DNA-based) for microbial community analyses in water is the inability to differentiate live (dormant cells as well as growing or non-growing metabolically active cells) and dead cells, which can lead to false positive results in the absence of live microbes. Propidium-monoazide (PMA) has been used to selectively remove DNA from dead cells during downstream sequencing process. In comparison, 16S rRNA sequencing (RNA-based) can target live microbial cells in water as both dormant and metabolically active cells produce rRNA. The objective of this study was to compare the efficiency and sensitivity of DNA-based, PMA-based and RNA-based 16S rRNA Illumina sequencing methodologies for live bacteria detection in water samples experimentally spiked with different combination of bacteria (2 gram-negative and 2 gram-positive/acid fast species either all live, all dead, or combinations of live and dead species) or obtained from different sources (First Nation community drinking water; city of Winnipeg tap water; water from Red River, Manitoba, Canada). The RNA-based method, while was superior for detection of live bacterial cells still identified a number of 16S rRNA targets in samples spiked with dead cells. In environmental water samples, the DNA- and PMA-based approaches perhaps overestimated the richness of microbial community compared to RNA-based method. Our results suggest that the RNA-based sequencing was superior to DNA- and PMA-based methods in detecting live bacterial cells in water.

The biocontrol agent Pseudomonas chlororaphis PA23 primes Brassica napus defenses through distinct gene networks.

BACKGROUND: The biological control agent Pseudomonas chlororaphis PA23 is capable of protecting Brassica napus (canola) from the necrotrophic fungus Sclerotinia sclerotiorum via direct antagonism. While we have elucidated bacterial genes and gene products responsible biocontrol, little is known about how the host plant responds to bacterial priming on the leaf surface, including global changes in gene activity in the presence and absence of S. sclerotiorum. RESULTS: Application of PA23 to the aerial surfaces of canola plants reduced the number of S. sclerotiorum lesion-forming petals by 91.1%. RNA sequencing of the host pathogen interface showed that pretreatment with PA23 reduced the number of genes upregulated in response to S. sclerotiorum by 16-fold. By itself, PA23 activated unique defense networks indicative of defense priming. Genes encoding MAMP-triggered immunity receptors detecting flagellin and peptidoglycan were downregulated in PA23 only-treated plants, consistent with post-stimulus desensitization. Downstream, we observed reactive oxygen species (ROS) production involving low levels of H2O2 and overexpression of genes associated with glycerol-3-phosphate (G3P)-mediated systemic acquired resistance (SAR). Leaf chloroplasts exhibited increased thylakoid membrane structures and chlorophyll content, while lipid metabolic processes were upregulated. CONCLUSION: In addition to directly antagonizing S. sclerotiorum, PA23 primes the plant defense response through induction of unique local and systemic defense networks. This study provides novel insight into the effects of biocontrol agents applied to the plant phyllosphere. Understanding these interactions will aid in the development of biocontrol systems as an alternative to chemical pesticides for protection of important crop systems.

Construction and characterization of a cDNA library from wheat infected with Fusarium graminearum Fg 2.

Total RNA from wheat spikes infected with F. graminearum Fg2 was extracted and the mRNA was purified. Switching Mechanism at 5' end of the RNA Transcript (SMART) technique and CDS Ill/3' primer were used for first-strand cDNA synthesis using reverse transcriptase by RT-PCR. Primer extension polymerase chain reaction was used to construct the double-strand cDNA that was digested by proteinase K, then by Sfi I and fractionated. cDNAs longer than 0.5 kb were collected and ligated to λTriplEx2 vector followed λ phage packaging reaction and library amplification. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. One hundred and sixty five plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. A high quality cDNA library from wheat spikes that have been infected by F. graminearum was successfully constructed.

Developing rainfall- and temperature-based models to describe infection of canola under field conditions caused by pycnidiospores of Leptosphaeria maculans.

Blackleg, also known as Phoma stem canker, caused by Leptosphaeria maculans (Phoma lingam), is one of the most serious diseases of canola worldwide. In this study, the mean disease severity (Ds) and incidence (Di) of canola cv. Westar plants infected at the cotyledon, three-leaf, and six-leaf stages by pycnidiospores of L. maculans were monitored in the greenhouse after infection of the plants under field conditions in two successive years and associated with meteorological data during infection time. Pearson's correlation coefficient showed that total rainfall per week (R) was significantly correlated to Ds on plants infected at the cotyledon, three-leaf, and six-leaf stages, and average maximum temperature per week (Tmax) only showed significant correlation with plants infected at the cotyledon and six-leaf stages. These results also indicated that there is correlation between Di and R for plants infected at all three growth stages. A nonlinear model was developed to evaluate the combined effects of R and Tmax on Ds. The best model comprised monomolecular function and beta probability density function for plants infected at the above three growth stages. Parameters, including maximum potential for Ds at a given rainfall (d(max)), rate of changes with respect to rainfall (k), constant of integration (B), maximum potential for Ds with respect to Tmax (e), rate of increase with increasing Tmax to optimum (n), and rate of decrease as Tmax increased and passed the optimum Tmax (p), were estimated for plants infected at the above three growth stages. The effect of plant growth stage was characterized by differences in the upper limit parameter a. This parameter was greater for the plants infected at the cotyledon stage than for plants infected at the other two stages. The estimate of parameter k was the same for the plants infected at the cotyledon and three-leaf stages. This parameter was much lower for the plants infected at the six-leaf stage compared with two other stages. The logistic model could describe the disease incidence with respect to R slightly better than the other two models in the plants infected at all three growth stages. Based on the model, upper-limit estimate (d(max)) was approximately 100, 94.4, and 88.8% in the plants infected at cotyledon, three-leaf, and six-leaf stages, respectively. Di increased until rainfall reached approximately 18, 10, and 13 mm/week and became constant in the plants at cotyledon, three-leaf, and six-leaf stages, respectively. Effects of plant growth stage on the rate of change with respect to R (parameter k) were lower in the plants infected at cotyledon than at the other two stages. The accuracy of the nonlinear models suggests that they could be used to develop a comprehensive model to evaluate epidemics of blackleg based on pycnidiospores as sources of inoculum. However, additional years of data collection should improve model fit and evaluation of introduced models and contribute to the development of a more robust predictive model.

Effect of timing of application and population dynamics on the degree of biological control of Sclerotinia sclerotiorum by bacterial antagonists.

Antagonistic Pseudomonas spp. (DF-41 and PA-23) were evaluated for inhibition of germination of ascospores, and for the effect of timing of application and its effect on biological control of Sclerotinia sclerotiorum (Lib.) de Bary, causal agent of stem rot of canola. Population dynamics were also assessed. In all studies, a petal inoculation technique was used. Significant inhibition (P < 0.05) of germination of ascospores was observed at both log 4 and log 8 cfu (colony forming units) ml(-1) of bacterial populations. In the population study, the pathogen had no significant effect (P < 0.05) on bacterial populations; however, a significant (P < 0.05) increase in bacterial populations was observed after 24 h and a decrease occurred between 96 and 120 h. Significant differences in disease severity (P < 0.05) were found with respect to timing of ascospore applications in the control treatments (ascospores only). One isolate completely suppressed disease when co-applied with ascospores, while only minor suppression occurred when applied 24 or 48 h after. Results from all studies indicate PA-23 and DF-41 to be effective biocontrol agents against S. sclerotiorum of canola and to have practical implications for biological control of this disease by bacteria in the field.