Qixing Zhou

Molecular characterization of a serine protease Pro1 from Plasmodiophora brassicae that stimulates resting spore germination

Clubroot, caused by Plasmodiophora brassicae, is one of the most serious diseases of cultivated cruciferous crops in the world. However, the basis for pathogenicity in P. brassicae is not well understood. In this study, a serine protease gene (PRO1) was cloned from P. brassicae and its molecular characteristics were investigated. Southern analysis and specific polymerase chain reaction (PCR) amplification indicated that PRO1 is a single-copy gene present in a broad range of P. brassicae pathotypes. Northern analysis revealed that the expression of PRO1 was induced during plant infection, and that the quantity of transcript fluctuated according to the stage of pathogenesis. Amino acid sequence analysis suggested that the encoded protein (Pro1) belongs to the S28 family of proteases, with a predicted signal peptide and a theoretical molecular mass of 49.4 kDa. The open reading frame (ORF) of PRO1 was transferred into Pichia pastoris and Pro1 was heterologously produced. Pro1 showed proteolytic activity on skimmed milk and N-succinyl-Ala-Ala-Phe-7-amido-4-methylcoumarin, and the activity could be inhibited by serine protease inhibitors and the chelating agent ethylenediaminetetraacetic acid. The optimal temperature of Pro1 was 25 degrees C, and it exhibited high activity at pH 6.0-6.4. These values coincide with the temperature and pH conditions favourable for P. brassicae resting spore germination in the field. When Pro1 was used to treat canola root exudates, it enhanced the stimulating effect of the root exudates on P. brassicae resting spore germination, indicating that Pro1 may play a role during clubroot pathogenesis by stimulating resting spore germination through its proteolytic activity.

An inexpensive method for extraction of genomic DNA from fungal mycelia

Abstract A rapid and efficient protocol for the extraction of genomic DNA from plant pathogenic fungi was developed. Key features of the protocol include the SDS-assisted lysis of fungal mycelium with inclusion of a glass bead to help break hyphal walls, followed by isopropanol precipitation of the DNA. The protocol was used to extract genomic DNA from a collection of 26 fungal species, representing many important plant pathogens. Yield of DNA ranged from 2.1–4.9 μg per 20 mg of mycelium or 0.4–0.6 μg per 20 mg of spores. The DNA was of sufficient purity to be digested by restriction enzymes, to serve as a template in the PCR-amplification of genomic fragments as large as 4.9 kb, and to be used in dot-blot hybridization for the detection of multiple- and single-copy genes.

Pathogenicity and genetic diversity of Rhizoctonia solani isolates from lupin and other crops in Alberta, Canada

Narrow-leaved lupin (Lupinus angustifolius) is a potentially important crop in Canada. During the summers of 2003, 2004, and 2007, a total of 78 isolates of Rhizoctonia solani were isolates from diseased lupin plants and various other crops in Alberta, Canada. When the isolates were assigned to anastomosis groups (AGs) according to anastomosis behaviour, 33 (42.3%) of the isolates were identified as AG-4, 21 (26.9%) were AG-2-1, 20 (25.6%) were AG-2-2, and 4 isolates were not determined. Isolates belonging to AG-4 produced typical symptoms of stem rot and root rot on lupin seedlings and showed greater virulence compared with AG-2-1 and AG-2-2 isolates. The genetic variability among these isolates was evaluated by phylogenetic analysis based on the ribosomal DNA internal transcribed spacer (ITS) sequences and random amplified polymorphic DNA (RAPD) markers. For the ITS sequence analysis, a neighbour-joining tree, which was constructed using the PAUP program, clustered the R. solani isolates into three groups (groups I–III). Based on the phylogenetic analysis, ITS sequences were unsuitable for distinguishing the AG-2-1 and AG-2-2 isolates of R. solani but were valuable for identifying the AG-4 isolates. For the RAPD analysis, 62 polymorphic RAPD-phenotypes were identified, indicating a high level of diversity among the isolates. Based on analysis with the POPGENE program, all the isolates were clustered into five groups (groups I–V). This grouping was not closely associated with location, AG, or virulence.

Genetic Diversity and Aggressiveness of Fusarium spp. Isolated from Canola in Alberta, Canada

Canola (Brassica napus) is one of the most economically important oilseed crops in Canada. Fusarium seedling blight is a root disease with the potential to cause severe yield reductions in canola. Fusarium spp. are commonly isolated root pathogens from fields in Alberta. Fusarium infection can also cause root rot in adult plants. In this study, 128 isolates identified as Fusarium spp. were recovered from field soils in central Alberta and from the roots of diseased canola plants with typical Fusarium seedling blight symptoms. Six species of Fusarium were identified, with Fusarium acuminatum as the predominant species (57 of 128 isolates, 44.5%). Phylogenetic analyses based on the translation elongation factor 1-α and the internal transcribed spacer sequence data were used for evaluation of genetic variations, and also used for Fusarium spp. identification in combination with morphological characteristics and polymerase chain reaction-based analyses. Based on disease ratings in pathogenicity tests, six isolates of F. avenaceum showed high aggressiveness on canola. Also, the aggressiveness varied within all Fusarium spp. No correlation was observed between aggressiveness and the geographic origin of the isolates.

Blackleg (Leptosphaeria maculans) Severity and Yield Loss in Canola in Alberta, Canada.

Blackleg, caused by Leptosphaeria maculans, is an important disease of oilseed rape (Brassica napus L.) in Canada and throughout the world. Severe epidemics of blackleg can result in significant yield losses. Understanding disease-yield relationships is a prerequisite for measuring the agronomic efficacy and economic benefits of control methods. Field experiments were conducted in 2013, 2014, and 2015 to determine the relationship between blackleg disease severity and yield in a susceptible cultivar and in moderately resistant to resistant canola hybrids. Disease severity was lower, and seed yield was 120%–128% greater, in the moderately resistant to resistant hybrids compared with the susceptible cultivar. Regression analysis showed that pod number and seed yield declined linearly as blackleg severity increased. Seed yield per plant decreased by 1.8 g for each unit increase in disease severity, corresponding to a decline in yield of 17.2% for each unit increase in disease severity. Pyraclostrobin fungicide reduced disease severity in all site-years and increased yield. These results show that the reduction of blackleg in canola crops substantially improves yields.

Effects of Fusarium avenaceum and Rhizoctonia solani on growth of soybean in saline soils

Abstract: Soybean (Glycine max) acreage on the Canadian Prairies has increased rapidly in recent years. Production has expanded into semiarid regions where irrigation and drainage problems often result in the accumulation of salts in the soil. Fusarium avenaceum and Rhizoctonia solani are the two dominant pathogens in the disease complex that cause root rot and seedling blight of legume crops on the Canadian Prairies. The effects of F. avenaceum or R. solani in combination with soil salinity on soybean root rot were evaluated under greenhouse and mini-plot conditions. As expected, inoculation with F. avenaceum or R. solani consistently reduced seedling emergence and increased root rot severity in soybean. At high soil electrical conductivity values and inoculum densities, seedling emergence decreased and root rot severity increased in soybean in both trials with F. avenaceum and R. solani. Twenty short-season soybean cultivars that were well suited for production in Alberta were evaluated for their reactions to inoculation with F. avenaceum or R. solani in a saline soil (21.1 dS m-1). High seedling emergence was observed for cultivars 900Y61, P002T04R, 900Y01, TH27005RR, P001T34R, and 900Y81 in the non-inoculated control, for P002T04R and 900Y61 in the F. avenaceum treatment, and for 900Y61, 900Y81, and 900Y71 in the R. solani treatment. Root rot severity was low for cultivars NSC Portage and 900Y61 in the non-inoculated control and P002T004R in the F. avenaceum treatment. The cultivar 900Y61 also consistently had lower disease severity over the trials in the mini-plot test.

Suppression of clubroot by dazomet fumigant

Abstract: Clubroot, caused by Plasmodiophora brassicae, is an important disease in canola (Brassica napus) and other crucifers. Experiments were conducted to examine the effectiveness of the prill formulation of the soil fumigant dazomet (tetrahydro-3,5-dimethyl-2H-1,3,5-thiadiazine-2-thione, trade name Basamid) on infection by P. brassicae, clubroot severity, and the growth and yield of canola. In greenhouse studies, seedling emergence and plant height increased while infection (both primary and secondary) and clubroot severity decreased, with increasing rates of a dazomet pretreatment. Under field conditions, seedling emergence, gall mass, and seed yield were reduced, especially at high rates of dazomet application (e.g., 0.4–0.8 t a.i. ha-1). A study conducted under controlled conditions indicated that an inadequate interval between the dazomet treatment and seeding was the underlying cause of the phytotoxic effect on canola in the field experiments. Further field studies showed that covering the soil with construction-grade plastic after dazomet application increased its efficacy, reducing gall weight and clubroot severity and increasing seed yield. Fumigation with dazomet also increased emergence, plant survival, and plant biomass in P. brassicae-infested soils that were inoculated with the soil-borne fungal pathogens Fusarium avenaceum, Pythium ultimum, and Rhizoctonia solani. These results suggest that dazomet is effective against both clubroot and seedling blight in canola.

First report of Verticillium dahliae Kleb. causing wilt symptoms in canola (Brassica napus L.) in North America

Abstract In Canada, Verticillium wilt of canola (Brassica napus L.), caused by Verticillium longisporum, was first confirmed in Manitoba in 2014. Verticillium dahliae, however, has not been reported to cause this disease. In 2016, a field survey in Alberta revealed canola plants with vascular wilt symptoms. Symptomatic plants were collected and infected stem tissue was cultured on agar medium, with one isolate designated A1-SS05 identified as a putative Verticillium spp. based on its colony characteristics. The colony was off-white in colour with a felt-like surface. On potato dextrose agar, the underside of the colony was dark only in the central area with radiating ridges. On Howell’s medium, only the test isolate exhibited polyphenol oxidase activity. The isolate formed conidiophores with 4–5 verticillate phialides. Mean conidial length was 5.96 µm (range of 4.65–6.64 µm) and mean width was 2.37 µm (range of 1.71–2.79 µm). The isolate also produced irregularly elongated chain-like microsclerotia of various sizes. Based on these criteria, isolate A1-SS05 was tentatively identified as V. dahliae. Sequencing of PCR products amplified with the primer sets ITS5/4 and VeruniF2/VeruniR3 revealed 99 and 100% identity, respectively, with V. dahliae sequences in GenBank, confirming the identity of this isolate. The isolate was pathogenic on the canola cultivar ‘Westar’, causing wilt symptoms, stunting, and leaf yellowing and senescence. Microscopic analysis of infected vascular tissues revealed irregular shaped microsclerotia, as were observed in pure culture. This is the first report of V. dahliae causing Verticillium wilt of canola in North America.