Lone Buchwaldt

Patterns of differential gene expression in Brassica napus cultivars infected with Sclerotinia sclerotiorum

SUMMARY The fungal pathogen Sclerotinia sclerotiorum infects a broad range of dicotyledonous plant species and causes stem rot in Brassica napus. To elucidate the mechanisms underlying the defence response, the patterns of gene expression in the partially resistant B. napus cultivar ZhongYou 821 (ZY821) and the susceptible cultivar Westar were studied using a B. napus oligonucleotide microarray. Although maximum differential gene expression was observed at 48 h post-inoculation (hpi) in both cultivars, increased transcript levels were detected in cv. ZY821 at the earlier stages of infection (6-12 hpi) for many genes, including those encoding defence-associated proteins, such as chitinases, glucanases, osmotins and lectins, as well as genes encoding transcription factors belonging to the zinc finger, WRKY, APETALA2 (AP2) and MYB classes. In both cultivars, genes encoding enzymes involved in jasmonic acid, ethylene and auxin synthesis were induced, as were those for gibberellin degradation. In addition, changes in the expression of genes encoding enzymes involved in carbohydrate and energy metabolism appeared to be directed towards shuttling carbon reserves to the tricarboxylic acid cycle and generating reactive oxygen species. Transcripts from genes encoding enzymes involved in glucosinolate and phenylpropanoid biosynthesis were highly elevated in both cultivars, suggesting that secondary metabolites are also components of the response to S. sclerotiorum in B. napus.

Sources of Resistance to Anthracnose (Colletotrichum truncatum) in Wild Lens Species

Lentil anthracnose (Colletotrichum truncatum (Schwein.) Andrus et W.D. Moore is a potential threat in many lentil (Lens culinaris Medik.) production regions of North America. In the lentil germplasm maintained in Germany and North America, 16 lines were reported to have resistance to race Ct1, but none has resistance reported to race Ct0. The objective of this study was to examine accessions of wild Lens species for their resistance to races Ct1 and Ct0 of lentil anthracnose. Five hundred and seventy-four wild accessions of six species and control lines were screened in two replications under both field and greenhouse conditions using a 1–9 scoring scale (1, highly resistant; 2–3, resistant; 4–5, moderately resistant; 6–7, susceptible; and 8–9, highly susceptible). Indianhead and PI 320937 were resistant while Eston and Pardina were susceptible to race Ct1 as expected. However, none of the check lines were resistant to race Ct0. Among the six Lens wild species tested, accessions of Lens ervoides (Brign.) Grande had the highest level of resistance, 3–5 to race Ct1 and Ct0 followed by L. lamottei Czefr. in the field and greenhouse. Lens orientalis (Boiss.), L. odemensis L., L. nigricans (M. Bieb.) Godron and L. tomentosus L. were highly susceptible, 8–9 to race Ct0 in the greenhouse. The highest frequency of resistance, especially in L. ervoides (Brign.) Grande, was found in accessions originating from Syria and Turkey. The usefulness of these L. ervoides (Brign.) Grande accessions as sources of resistance to the more virulent race of anthracnose in a lentil breeding program is discussed.

Brassica napus possesses an expanded set of polygalacturonase inhibitor protein genes that are differentially regulated in response to Sclerotinia sclerotiorum infection, wounding and defense hormone treatment

Most plants encode a limited set of polygalacturonase inhibitor (PGIP) genes that may be involved in aspects of plant development, but more importantly in the inactivation of polygalacturonases (PG) secreted by pathogens. Previously, we characterized two Brassica napus PGIP genes, BnPgip1 and BnPgip2, which were differentially expressed in response to pathogen infection and wounding. Here we report that the B. napus genome encodes a set of at least 16 PGIP genes that are similar to BnPgip1 or BnPgip2. This is the largest Pgip gene family reported to date. Comparison of the BnPGIPs revealed several sites within the xxLxLxx region of leucine rich repeats that form β-sheets along the interacting face of the PGIP that are hypervariable and represent good candidates for generating PGIP diversity. Characterization of the regulatory regions and RT-PCR studies with gene-specific primers revealed that individual genes were differentially responsive to pathogen infection, mechanical wounding and signaling molecules. Many of the BnPgip genes responded to infection by the necrotic pathogen, Sclerotinia sclerotiorum; however, these genes were also induced either by jasmonic acid, wounding and salicylic acid or some combination thereof. The large number of PGIPs and the differential manner in which they are regulated likely ensures that B. napus can respond to attack from a broad spectrum of pathogens and pests.

Foliar fungicides to manage ascochyta blight [Ascochyta rabiei] of chickpea in Canada

Ascochyta blight, caused by Ascochyta rabiei, is a major constraint to chickpea production in Saskatchewan. Foliar fungicides were evaluated at various rates and timings for managing blight epidemics over 18 station years from 1998 to 2000. Dry weather in 1998 resulted in low disease pressure, and fungicide application had no effect on blight severity and yield. In 1999 and 2000, wet conditions favored the development of epidemics. Under high disease pressure, a single fungicide application often reduced disease severity, but had no effect on yield. Two applications (early + mid flowering) of chlorothalonil (Bravo® 500) at 1 kg active ingredients (a.i.)/ha, two applications of azoxystrobin (Quadris®) at 125 g a.i./ha, or chlorothalonil + azoxystrobin reduced ascochyta blight and increased yield. An alternative formulation of chlorothalonil, Bravo Ultrex®, was less effective than Bravo 500, but more effective than mancozeb (Dithane®). Fungicide application had a substantial impact on seed yield; at one site, yield in the untreated control was less than 5% of the best fungicide treatment. Regression analysis showed a strong relationship between disease severity and seed yield. With high disease pressure in 2000, the incidence of seed-borne A. rabiei was 30–;48%. Applying azoxystrobin at early + mid flowering reduced it to 7–9%. Fungicide application had no impact on other seed-borne pathogens such as Botrytis cinerea, Sclerotinia sclerotiorum, and Fusarium spp. These results indicate that fungicide application can complement partial resistance to reduce blight severity and increase seed yield and quality in chickpea.

Host specificity of Colletotrichum truncatum from lentil

Isolates of Colletotrichum truncatum from lentil (Lens culinaris) and soybean (Glycine max) were shown to have different host ranges and patterns of latent infection. A smaller number of studies on an isolate from scentless chamomile (Matricaria perforata) indicated that this isolate likely differs from either lentil or soybean isolates. In growth-cabinet trials, lentil isolates were pathogenic on lentil, faba bean (Vicia faba), field pea (Pisum sativum), narrow-leaf vetch (Vicia americana), and chickpea (Cicer arietinum). Latent infection by lentil isolates was observed only in scentless chamomile, and no symptoms were observed on alfalfa (Medicago sativa), dry bean (Phaseolus vulgaris), or lupin (Lupinus albus). Isolates from scentless chamomile had a short latent period and produced abundant disease on scentless chamomile and limited symptoms on lentil, faba bean, field pea, and chickpea. In contrast, soybean isolates had an extended latent period and produced visible lesions on soybean, chickpea, lupin, and dry bean, but not on faba bean, field pea, or narrow-leaf vetch. Latent infection by soybean isolates was identified in inoculated plants of all the plants species in the trial. Field trials confirmed that C. truncatum from lentil causes disease symptoms on faba bean and field pea (but not chickpea) under field conditions. There were no substantive differences in the pattern of pathogenicity or disease severity between the races of C. truncatum from lentil (Ct0 and Ct1) on the plant species in the study. Narrow-leaf vetch was generally susceptible to C. truncatum from lentil

Expression and regulation of Sclerotinia sclerotiorum necrosis and ethylene-inducing peptides (NEPs).

Successful host colonization by necrotrophic plant pathogens requires the induction of plant cell death to provide the nutrients needed for infection establishment and progression. We have cloned two genes encoding necrosis and ethylene-inducing peptides from Sclerotinia sclerotiorum, which we named SsNep1 and SsNep2. The peptides encoded by these genes induce necrosis when expressed transiently in tobacco leaves. SsNep1 is expressed at a very low level relative to SsNep2 during infection. The expression of SsNep2 was induced by contact with solid surfaces and occurred in both the necrotic zone and at the leading margin of the infection. SsNep2 expression was dependent on calcium and cyclic adenosine monophosphate signalling, as compounds affecting these pathways reduced or abolished SsNep2 expression coincident with a partial or total loss of virulence.

Differential expression of duplicated peroxidase genes in the allotetraploid Brassica napus

Gene redundancy due to polyploidization provides a selective advantage for plant adaptation. We examined the expression patterns of two peroxidase genes (BnPOX1 and BnPOX2) in the natural allotetraploid Brassica napus and the model diploid progenitors Brassica rapa (Br) and Brassica oleracea (Bo) in response to the fungal pathogen Sclerotinia sclerotiorum. We demonstrated that the Bo homeolog of BnPOX1 was up-regulated after infection, while both BnPOX2 homeologs were down-regulated. A bias toward reciprocal expression of the homeologs of BnPOX1 in different organs in the natural allotetraploid of B. napus was also observed. These results suggest that subfunctionalization of the duplicated BnPOX genes after B. napus polyploidization as well as subneofunctionalization of the homeologs in response to this specific biotic stress has occurred. Retention of expression patterns in the diploid progenitors and the natural allotetraploid in some organs indicates that the function of peroxidase genes has been conserved during evolution.

IGS Minisatellites Useful for Race Differentiation in Colletotrichum lentis and a Likely Site of Small RNA Synthesis Affecting Pathogenicity.

Colletotrichum lentis is a fungal pathogen of lentil in Canada but rarely reported elsewhere. Two races, Ct0 and Ct1, have been identified using differential lines. Our objective was to develop a PCR-probe differentiating these races. Sequences of the translation elongation factor 1α (tef1α), RNA polymerase II subunit B2 (rpb2), ATP citrate lyase subunit A (acla), and internal transcribed spacer (ITS) regions were monomorphic, while the intergenic spacer (IGS) region showed length polymorphisms at two minisatellites of 23 and 39 nucleotides (nt). A PCR-probe (39F/R) amplifying the 39 nt minisatellite was developed which subsequently revealed 1–5 minisatellites with 1–12 repeats in C. lentis. The probe differentiated race Ct1 isolates having 7, 9 or 7+9 repeats from race Ct0 having primarily 2 or 4 repeats, occasionally 5, 6, or 8, but never 7 or 9 repeats. These isolates were collected between 1991 and 1999. In a 2012 survey isolates with 2 and 4 repeats increased from 34% to 67%, while isolated with 7 or 9 repeats decreased from 40 to 4%, likely because Ct1 resistant lentil varieties had been grown. The 39 nt repeat was identified in C. gloeosporioides, C. trifolii, Ascochyta lentis, Sclerotinia sclerotiorum and Botrytis cinerea. Thus, the 39F/R PCR probe is not species specific, but can differentiate isolates based on repeat number. The 23 nt minisatellite in C. lentis exists as three length variants with ten sequence variations differentiating race Ct0 having 14 or 19 repeats from race Ct1 having 17 repeats, except for one isolate. RNA-translation of 23 nt repeats forms hairpins and has the appropriate length to suggest that IGS could be a site of small RNA synthesis, a hypothesis that warrants further investigation. Small RNA from fungal plant pathogens able to silence genes either in the host or pathogen thereby aiding infection have been reported.

Plant Gene Resources of Canada and the Canadian plant germplasm system

Plant Gene Resources of Canada is part of Agriculture and Agri-Food Canada and has the mandate to acquire, maintain, and characterize plant germplasm and microorganisms for preserving biodiversity and supporting development of economically important crops. About 34 staff members are located at 12 sites across Canada. The Canadian Collection of Fungal Cultures is located in Ottawa, Ontario, and the Virus Collection, in Summerland, British Columbia. Seed-propagated crops are maintained at the Saskatoon Research Centre, Saskatoon, Saskatchewan, and encompass 113 000 accessions representing 850 plant species. Fruit trees and small fruits are propagated at Harrow, Ontario, while potatoes are maintained in tissue culture at Fredericton, New Brunswick. These latter three locations are responsible for most plant germplasm entering and leaving Canada. Staff members with expertise in breeding, entomology, plant pathology, or other crop-specific research are located at specific research centres (nodes); some of these locations were selected because of high natural levels of certain diseases or specific growing conditions. The production of seed stocks for winter cereals takes place in Lethbridge, Alberta, and in Delhi, Ontario, while screening for resistance takes place in Lacombe, Alberta, for barley scald [Rhynchosporium secalis], in Charlottetown, Prince Edward Island, for barley net blotch [Pyrenophora teres], in Brandon, Manitoba, for fusarium head blight [Fusarium graminearum] of barley, and in Winnipeg, Manitoba, for crown rust [Puccinia coronata f. sp. avenae] and stem rust [Puccinia graminis f. sp. avenae] of oat. Morphological and agronomical data are usually collected during regeneration of accessions of the gene bank seed stock. Information is publicly available in a searchable database, GRIN-CA (Germplasm Resources Information Network, Canada), and accessions can be ordered online, free of charge, by plant breeders, scientists, and others who can demonstrate a valid use. Molecular characterization of genetic diversity within plant species has most recently been undertaken for oat and flax. Plant pathology research is being conducted to help solve important production problems, such as anthracnose [Colletotrichum truncatum] and ascochyta blight [Ascochyta lentis] on lentil, ascochyta blight on chickpea [A. rabiei], and sclerotinia stem rot [Sclerotinia sclerotiorum] on Brassica napus.

Lentil anthracnose: epidemiology, fungicide decision support system, resistance and pathogen races

Abstract Colletotrichum lentis causes anthracnose of lentil (Lens culinaris) in Canada that results in defoliation, stem girdling and severe yield losses, but the disease is rarely reported elsewhere. The pathogen survives as microsclerotia on lentil debris for up to 3 years when buried in the soil, but loses viability on the soil surface. Windborne debris spreads the pathogen to neighbouring fields, while seedborne infection is less important. Foliar fungicides were registered, and a fungicide decision support system was developed which assessed disease risk with 85% accuracy. Around 2300 L. culinaris accessions from 50 countries were screened for resistance. Congruently, two races – Ct1 and Ct0 – were identified on differential lentil lines. Resistant lines were generated by cycles of inoculation and selfing of single resistant plants which resulted in the following three accessions resistant to both races: VIR2633 (Georgia), VIR2058 and VIR2076 (Czech Republic), while six and 49 lines had resistance to Ct0 and Ct1, respectively. Ct1 resistance is controlled by recessive and dominant genes crt1 and CtR3 in variety ‘Indianhead’, ctr2 and CtR5 in accession PI345629 and CtR4 in PI320937. Molecular markers linked to Ct1 resistance were identified on linkage group six, close to Ascochyta lentis resistance, and were used to combine resistance to both pathogens in breeding lines. Two repeat rich regions in the intergenic spacer (IGS) of ribosomal DNA can be used to differentiate the two C. lentis races. Utilizing length polymorphisms in a 39 nucleotide repeat region showed the races were equally frequent among isolates collected between 1991 and 1999, while 95% belonged to race Ct0 in 2010, likely because lentil varieties are susceptible to race Ct0, but around one-third of the varieties had Ct1 resistance.