Mechanisms of resistance in Brassica carinata,B. napus and B. juncea to Pseudocercosporella capsellae

Studies were undertaken to compare susceptible and resistant host responses to Pseudocercosporella capsellae in cotyledons of Brassica carinata, B. juncea and B. napus in order to define the mechanisms of resistance in these three species. On both resistant and susceptible hosts, hyphal penetration was always through stomatal openings and without infection pegs or appressoria. On resistant B. carinata ATC94129P, up to 72% of spores disintegrated and, generally, germination (<22%) and germ tube lengths (<25 μm) were comparatively low. Resistant B. napus Hyola 42 had the lowest germination (8%) and susceptible B. carinata UWA#012 had the highest (51%). On resistant B. carinata ATC94129P, germ tube extension was impeded across 24–60 h post‐inoculation (hpi) and percentage stomatal penetration lower (4%) at 60 hpi compared with susceptible B. carinata UWA#012 (26%). Stomatal densities (stomata/14 757 μm²) on resistant B. juncea Dune (2·12) and B. napus Hyola 42 (1·62) were lower than for susceptible B. juncea Vardan (2·40) and B. napus Trilogy (2·03). Resistant B. carinata ATC94129P had greater stomatal density (1·89) than susceptible B. carinata UWA#012 (1·58). Overall, B. juncea had greater stomatal density (2·26) compared with B. napus (1·83) and B. carinata (1·74). In resistant B. carinata ATC94129P, P. capsellae induced 28% stomata to close, while in susceptible B. carinata UWA#012 no such closure was induced. Epicuticular wax crystalloids were present only on resistant B. carinata ATC94129P and probably also contribute towards resistance.     

Potential loss of clubroot resistance genes from donor parent Brassica rapa subsp. rapifera (ECD 04) during doubled haploid production

Clubroot resistance derived from the oilseed rape/canola Brassica napus ‘Mendel’ has been overcome in some fields in Alberta, Canada, by the emergence of ‘new’ strains of the protist Plasmodiophora brassicae. Resistance to the pathogen was assessed in 112 doubled haploid (DH) lines, derived from B. rapa subsp. rapifera (European clubroot differential (ECD) 04). The lines were evaluated against five single‐spore isolates representing the ‘old’ pathotypes 2, 3, 5, 6 and 8, and 15 field populations representing new strains of P. brassicae. The disease severity index (ID%) data revealed that none of the DH lines were resistant or moderately resistant to the new pathotype 5X (field populations L‐G1, L‐G2, L‐G3) and D‐G3, while 3–42% were resistant or moderately resistant to the other 11 new strains. Using the mean ID induced by the old pathotype 3 (approx. 13.5%) as the baseline, clubroot severity increased by 300–600% when inoculated with the new pathotypes. A significant finding of this study was the fact that ECD 04 showed absolute resistance to all of the old and new P. brassicae strains while the B. napus ‘Mendel’, although resistant to all of the old pathotypes, was resistant to only about 50% of the new strains. Similarly, all of the selected clubroot‐resistant commercial canola cultivars evaluated in this study were susceptible to 87% of the new P. brassicae strains. The molecular data revealed that the breakdown of clubroot resistance in Mendel and the canola cultivars was in part due to the non‐inheritance of the Crr1 gene on the A08 chromosome from ECD 04.     

Phenotypic and phylogenetic studies associated with the crucifer white leaf spot pathogen,Pseudocercosporella capsellae,in Western Australia

Pseudocercosporella capsellae (white leaf spot disease) is an important disease on crucifers. Fifty‐four single‐conidial isolates collected from Brassica juncea (Indian mustard), B. napus (oilseed rape), B. rapa (turnip), and Raphanus raphanistrum (wild radish) across Western Australia were investigated for differences in pathogenicity and virulence using cotyledon screening tests, genetic differences using internal transcribed spacer (ITS) sequencing and phylogenetic analysis, and growth rates on potato dextrose, V8 juice and malt extract agars. All isolates from the four crucifer hosts were pathogenic on the three test species: B. juncea, B. napus and R. raphanistrum, but showed differences in levels of virulence. Overall, isolates from B. juncea, B. napus and B. rapa showed greatest virulence on B. juncea, least on R. raphanistrum and intermediate virulence on B. napus. Isolates from R. raphanistrum showed greatest virulence on B. juncea, least on B. napus and intermediate virulence on R. raphanistrum. Growth and production of a purple‐pink pigment indicative of cercosporin was greatest on malt extract agar and cercosporin production on V8 juice agar was positively correlated with virulence of isolates on B. juncea and B. napus. ITS sequencing and phylogenetic analysis showed that isolates collected from B. napus, B. juncea and B. rapa, in general and with few exceptions, had a high degree of genetic similarity. In contrast, isolates from R. raphanistrum were clearly differentiated from isolate groups collected from Brassica hosts. Pseudocercosporella capsellae reference isolates from other countries generally grouped into a single separate cluster, highlighting the genetic distinctiveness of Western Australian isolates.     

Effects of R gene‐mediated resistance in Brassica napus (oilseed rape) on asexual and sexual sporulation of Pyrenopeziza brassicae (light leaf spot)

The phenotype of the R gene‐mediated resistance derived from oilseed rape (Brassica napus) cv. Imola against the light leaf spot plant pathogen, Pyrenopeziza brassicae, was characterized. Using a doubled haploid B. napus mapping population that segregated for resistance against P. brassicae, development of visual symptoms was characterized and symptomless growth was followed using quantitative PCR and scanning electron microscopy on leaves of resistant/susceptible lines inoculated with suspensions of P. brassicae conidia. Initially, in controlled‐environment experiments, growth of P. brassicae was unaffected; then from 8 days post‐inoculation (dpi) some epidermal cells collapsed (‘black flecking’) in green living tissue of cv. Imola and from 13 to 36 dpi there was no increase in the amount of P. brassicae DNA and no asexual sporulation (acervuli/pustules). By contrast, during this period there was a 300‐fold increase in P. brassicae DNA and extensive asexual sporulation in leaves of the susceptible cv. Apex. However, when leaf tissue senesced, the amount of P. brassicae DNA increased rapidly in the resistant but not in the susceptible cultivar and sexual sporulation (apothecia) was abundant on senescent tissues of both. These results were consistent with observations from both controlled condition and field experiments with lines from the mapping population that segregated for this resistance. Analysis of results of both controlled‐environment and field experiments suggested that the resistance was mediated by a single R gene located on chromosome A1.     

Resistance to a highly aggressive isolate of Sclerotinia sclerotiorum in a Brassica napus diversity set

Sclerotinia stem rot (SSR) of oilseed rape (OSR, Brassica napus), caused by Sclerotinia sclerotiorum, is a serious problem in the UK and worldwide. As fungicide‐based control approaches are not always reliable, identifying host resistance is a desirable and sustainable approach to disease management. This research initially examined the aggressiveness of 18 Sclerotinia isolates (17 S. sclerotiorum, one S. subarctica) on cultivated representatives of B. rapa, B. oleracea and B. napus using a young plant test. Significant differences were observed between isolates and susceptibility of the brassica crop types, with B. rapa being the most susceptible. Sclerotinia sclerotiorum isolates from crop hosts were more aggressive than those from wild buttercup (Ranunculus acris). Sclerotinia sclerotiorum isolates P7 (pea) and DG4 (buttercup), identified as ‘aggressive’ and ‘weakly aggressive’, respectively, were used to screen 96 B. napus lines for SSR resistance in a young plant test. A subset of 20 lines was further evaluated using the same test and also in a stem inoculation test on flowering plants. A high level of SSR resistance was observed for five lines and, although there was some variability between tests, one winter OSR (line 3, Czech Republic) and one rape kale (line 83, UK) demonstrated consistent resistance. Additionally, one swede (line 69, Norway) showed an outstanding level of resistance in the stem test. Resistant lines also had fewer sclerotia forming in stems. New pre‐breeding material for the production of SSR resistant OSR cultivars relevant to conditions in the UK and Europe has therefore been identified.     

Comparative microscopic and molecular analysis of Thatcher near‐isogenic lines with wheat leaf rust resistance genes Lr2a,Lr3, LrB or Lr9 upon challenge with different Puccinia triticina races

Thatcher near‐isogenic lines (NILs) of wheat carrying resistance gene Lr2a, Lr3, LrB or Lr9 were inoculated with Puccinia triticina races of virulence phenotype BBBD, MBDS, SBDG and FBDJ. Puccinia triticina infection structures were analysed under the fluorescence microscope over a course of 14 days after inoculation (dai). The relative proportion of P. triticina and wheat genomic DNA in infected leaves was estimated with a semiquantitative multiplex PCR analysis using P. triticina‐ and wheat‐specific primers. The occurrence of a hypersensitive response (HR), cellular lignification and callose deposition in inoculated plants was investigated microscopically. In interactions producing highly resistant infection type (IT) ‘0;’, a maximum of two haustorial mother cells per infection site were produced, and there was no increase in the proportion of P.  triticina genomic DNA in infected leaves, indicating the absence of P. triticina growth. In comparison, sizes of P. triticina colonies increased gradually in interactions producing moderately resistant IT ‘1’ and ‘2’, with the highest proportion of P. triticina genomic DNA found in leaves sampled at 14 dai. In interactions producing susceptible IT ‘3–4’, the highest proportion of P. triticina genomic DNA was found in leaves sampled at 10 dai (45·5–51·5%). HR and cellular lignification were induced in interactions producing IT ‘0;’ and ‘1’ at 1 dai but they were not observed in interactions producing IT ‘2’ until 2 dai. No HR or cellular lignification were induced in interactions producing susceptible IT ‘3–4’. Furthermore, a strong deposition of callose was induced in Lr9 + BBBD and Lr9 + FBDJ (IT ‘0;’), whereas this defence response was not induced in resistant or susceptible interactions involving Lr2a, Lr3 or LrB, indicating that Lr9 mediated resistance was different from that conditioned by Lr2a, Lr3 or LrB.     

Temperature adaptation in isolates of Sclerotinia sclerotiorum affects their ability to infect Brassica carinata

Sclerotinia stem rot (Sclerotinia sclerotiorum) is a serious disease in oilseed Brassica crops worldwide. In this study, temperature adaptation in isolates of S. sclerotiorum collected from differing climatic zones is reported for the first time on any crop. Sclerotinia sclerotiorum isolates from oilseed rape (Brassica napus) crops in warmer northern agricultural regions of Western Australia (WW3, UWA 7S3) differed in their reaction to temperature from those from cooler southern regions (MBRS‐1, UWA 10S2) in virulence on Brassica carinata, growth on agar, and oxalic acid production. Increasing temperature from 22/18°C (day/night) to 28/24°C increased lesion diameter on cotyledons of B. carinataBC054113 more than tenfold for warmer region isolates, but did not affect lesion size for cooler region isolates. Mean lesion length averaged across two B. carinata genotypes (resistant and susceptible) fell from 4·6 to 2·4 mm for MBRS‐1 when temperature increased from 25/21°C to 28/24°C but rose for WW3 (2·35 and 3·21 mm, respectively). WW3, usually designated as low in virulence, caused as much disease on stems at 28/24°C as MBRS‐1, historically designated as highly virulent. Isolates collected from cooler areas grew better at low temperatures on agar. While all grew on potato dextrose agar between 5 and 30°C, with maximum growth at 20–25°C, growth was severely restricted above 32°C, and only UWA 7S3 grew at 35°C. Oxalate production increased as temperature increased from 10 to 25°C for isolates MBRS‐1, WW3 and UWA 7S3, but declined from a maximum level of 101 mg g^?1 mycelium at 20°C to 24 mg g^?1 mycelium at 25°C for UWA 10S2.     

High level resistance to Pseudocercosporella capsellae offers new opportunities to deploy host resistance to effectively manage white leaf spot disease across major cruciferous crops

Field and controlled environment studies were undertaken to define the range and extent of available host resistances to Pseudocercosporella capsellae (white leaf spot) across diverse oilseed, forage and vegetable crucifers, including some wild and/or weedy species, and also within and/or derived from Brassica carinata. In each experiment, there was a wide range in host response from high resistance to high susceptibility as assessed by four disease parameters, viz. in the field for: (i) Area Under Disease Progress Curve (AUDPC) for percent leaves diseased with values ranging from 0 to 375.5; (ii) Percent Leaf Collapse Index (%LCI) for leaf collapse due to disease with values ranging from 0 to 23.0; and (iii), Percent Pod Area Disease Index (%PADI) for pod area affected with values ranging from 0 to 52.1; and (iv) under controlled environmental conditions for Percent Cotyledon Disease Index (%CDI) for cotyledon lesion size with values ranging from 0 to 27.5. At the Crawley field site, B. carinata ATC 94129 was the most resistant genotype with AUDPC?=?1.2, followed by Crambe abyssinica (AUDPC 8.7), Eruca sativa Eruc-01 (AUDPC 19.3) and E. vesicaria Yellow rocket (AUDPC 19.4). B. carinata ATC 94129 and B. oleracea var. capitata had the least leaf collapse, with %LCI?=?0.2. At the Shenton Park field site, 21 genotypes of B. carinata and B. oleracea var. acephala Tuscan kale showed total resistance, all with AUDPC values of 0. Of the B. napus genotypes carrying one or more B. carinata B genome introgressions, genotypes NC8 (AUDPC 23.0) and NC9-1 (AUDPC 26.2) were the most resistant. Genotypes as assessed on these disease criteria as having high level resistance generally showed no pod infection; in contrast to %PADI values up to 52 on the most susceptible genotypes. Under controlled environmental conditions, the most resistant genotype was B. carinata ATC 94129 with %CDI values of 0 and 0.2, respectively, across two experiments, along with B. napus genotypes Zhongyou 821 and Hyola 42, with a %CDI value of 0 in one of the two experiments. There was a high degree of correlation both within individual experiments across the different disease parameters and also between field and controlled environment experiments. Within both B. napus and B. juncea genotypes tested, the most resistant genotypes were from China, the most susceptible from India, with those from Australia intermediate.     

Pathotypes and phylogenetic variation determine downy mildew epidemics in Brassica spp. in Australia

Isolates of Hyaloperonospora brassicae inoculated onto cotyledons of 28 diverse Brassicaceae genotypes, 13 from Brassica napus, two from B. juncea, five from B. oleracea, two from Eruca vesicaria, and one each from B. nigra, B. carinata, B. rapa, Crambe abyssinica, Raphanus sativus and R. raphanistrum, showed significant effects (P ≤ 0.001) of isolate, host and their interaction. Host responses ranged from no visible symptom or a hypersensitive response, to systemic spread and abundant pathogen sporulation. Isolates were generally most virulent on their host of origin. Using an octal classification, six host genotypes were identified as suitable host differentials to characterize pathotypes of H. brassicae and distinguished eight distinct pathotypes. There were fewer, but more virulent, pathotypes in 2015–2016 isolates than 2006–2008 pathogen populations, probably explaining the increase in severity of canola downy mildew over the past decade. Phylogenetic relationships determined across 20 H. brassicae isolates collected in 2006–2008 and 88 isolates collected in 2015–2016 showed seven distinct clades, with 70% of 2006–2008 isolates distributed within clade I (bootstrap value (BVs) of 100%) and the remaining 30% in clade V (BVs 83.3%). This is the first study to define phylogenetic relationships of H. brassicae isolates in Australia, setting a benchmark for understanding current and future genetic shifts within pathogen populations; it is also the first to use octal classification to characterize pathotypes of H. brassicae, providing a novel basis for standardizing phenotypic characterization and monitoring of pathotypes on B. napus and some crucifer species in Australia.     

Host suitability of Solanum torvum cultivars to Meloidogyne incognita and M. javanica and population dynamics

Several experiments were carried out to assess the performance of commercial Solanum torvum cultivars against the root knot nematodes Meloidogyne incognita and M. javanica in Spain. The response of S. torvum rootstock cultivars Brutus, Espina, Salutamu and Torpedo against M. incognita and Mi-1.2 (a)virulent M. javanica isolates was determined in pot experiments, and of ‘Brutus’ to an N-virulent isolate of M. incognita, compared with that of the eggplant S. melongena ‘Cristal’. The relationship between the initial and final population densities of M. javanica on ungrafted and grafted ‘Cristal’ onto the S. torvum ‘Brutus’ was assessed, together with the effect on dry shoot biomass. Finally, the population growth rate and the resistance level of the four S. torvum cultivars against M. incognita was assessed under plastic greenhouse conditions in two cropping seasons. All S. torvum rootstocks responded as resistant to the M. incognita isolates and from highly resistant to susceptible against M. javanica isolates. The maximum multiplication rates of M. javanica on the ungrafted or grafted eggplant were 270 and 49, respectively, and the equilibrium densities were 1318 and 2056 eggs and J2 per 100 cm³ soil, respectively. The tolerance of the ungrafted eggplant was 10.9 J2 per 100 cm³ soil, and the minimum relative dry shoot biomass was 0.76. The population growth rate of M. incognita on eggplant cv. Cristal differed from that of the S. torvum cultivars in both cropping seasons. These results suggest that S. torvum is a valuable rootstock for managing the two Meloidogyne species irrespective of the (a)virulence status.     

Colonization of lettuce cultivars and rotation crops by Fusarium oxysporum f. sp. lactucae,the cause of fusarium wilt of lettuce

The severity of fusarium wilt is affected by inoculum density in soil, which is expected to decline during intervals when a non‐susceptible crop is grown. However, the anticipated benefits of crop rotation may not be realized if the pathogen can colonize and produce inoculum on a resistant cultivar or rotation crop. The present study documented colonization of roots of broccoli, cauliflower and spinach by Fusarium oxysporum f. sp. lactucae, the cause of fusarium wilt of lettuce. The frequency of infection was significantly lower on all three rotation crops than on a susceptible lettuce cultivar, and the pathogen was restricted to the cortex of roots of broccoli. However, F. oxysporum f. sp. lactucae was isolated from the root vascular stele of 7·4% of cauliflower plants and 50% of spinach plants that were sampled, indicating a greater potential for colonization and production of inoculum on these crops. The pathogen was also recovered from the root vascular stele of five fusarium wilt‐resistant lettuce cultivars. Thus, disease‐resistant plants may support growth of the pathogen and thereby contribute to an increase in soil inoculum density. Cultivars that were indistinguishable based on above‐ground symptoms, differed significantly in the extent to which they were colonized by F. oxysporum f. sp. lactucae. Less extensively colonized cultivars may prove to be superior sources of resistance to fusarium wilt for use in breeding programmes.     

Virulence of ‘Dickeya solani’ and Dickeya dianthicola biovar‐1 and ‐7 strains on potato (Solanum tuberosum)

Studies were conducted to explain the relative success of ‘Dickeya solani’, a genetic clade of Dickeya biovar 3 and a blackleg‐causing organism that, after recent introduction, has spread rapidly in seed potato production in Europe to the extent that it is now more frequently detected than D. dianthicola. In vitro experiments showed that both species were motile, had comparable siderophore production and pectinolytic activity, and that there was no antagonism between them when growing. Both ‘D. solani’ and biovar 1 and biovar 7 of D. dianthicola rotted tuber tissue when inoculated at a low density of 10³ CFU mL^?1. In an agar overlay assay, D. dianthicola was susceptible to 80% of saprophytic bacteria isolated from tuber extracts, whereas ‘D. solani’ was susceptible to only 31%, suggesting that ‘D. solani’ could be a stronger competitor in the potato ecosystem. In greenhouse experiments at high temperatures (28°C), roots were more rapidly colonized by ‘D. solani’ than by biovar 1 or 7 of D. dianthicola and at 30 days after inoculation higher densities of ‘D. solani’ were found in stolons and progeny tubers. In co‐inoculated plants, fluorescent protein (GFP or DsRed)‐tagged ‘D. solani’ outcompeted D. dianthicola in plants grown from vacuum‐infiltrated tubers. In 3 years of field studies in the Netherlands with D. dianthicola and ‘D. solani’, disease incidence varied greatly annually and with strain. In summary, ‘D. solani’ possesses features which allow more efficient plant colonization than D. dianthicola at high temperatures. In temperate climates, however, tuber infections with ‘D. solani’ will not necessarily result in a higher disease incidence than infections with D. dianthicola, but latent seed infection could be more prevalent.     

Temperature and plant age drive downy mildew disease epidemics on oilseed <Emphasis Type="Italic">Brassica napus</Emphasis> and <Emphasis Type="Italic">B. juncea</Emphasis>

Studies were undertaken on the effects of temperature (14/10 °C and 22/17 °C day/night) and plant age (15, 23, 31 and 40 day-old-plants) on the severity of downy mildew (Hyaloperonospora parasitica) on oilseed Brassica cultivars (temperature: Brassica juncea Montara, B. napus Atomic, ATR-Hyden, Hyola 432, Hyola 450 TT, Thunder TT; plant age: B. juncea Dune, B. napus Surpass 402 and Hyola 450 TT). For temperature studies, there were significant (P?<?0.001) effects of temperature, cultivar, and cultivar x temperature interaction. On cotyledons of susceptible cultivars (B. napus Hyola 450 TT and Thunder TT), plants were symptomatic at 22/17 °C by 48 h post inoculation (hpi) and with abundant sporulation evident by 72 hpi, and with all cotyledons of B. napus Thunder TT collapsed by 7 days post inoculation (dpi). However, at 14/10 °C, there were no symptoms on the same cultivars until 5 dpi, and sporulation only observed at 7 dpi. Percent disease index values (DI%) at 22/17 °C of B. juncea Montara and B. napus ATR-Hyden, Hyola 432, Atomic, Hyola 450 TT and Thunder TT were 4.5, 49.0, 51.4, 65.8, 86.3 and 96.0, respectively, with all except B. juncea Montara having significantly lower (P?<?0.001) disease at 14/10 °C with DI% values of 2.8, 30.4, 27.9, 31.1, 44.4 and 76.4, respectively. For plant age studies, there were significant (P?<?0.001) effects of plant age, cultivar, and cultivar x plant age interaction. DI% was significantly higher at 15 compared to 40 day-old-plants (dop) across all cultivars. B. juncea Dune showed greatest resistance, particularly on 40 dop, with DI% values of 25.8, 24.6, 22.9 and 7.5, for 15, 23, 31 and 40 dop, respectively. B. napus Surpass 402 showed high susceptibility on cotyledons of 15 dop but moderate resistance on leaves of other ages, with DI% values of 59.0, 31.2, 27.1 and 26.2 for 15, 23, 31 and 40 dop, respectively. B. napus Hyola 450 TT showed very high susceptibility at the cotyledon stage on 15 dop, but some resistance on 23 dop and more so on 31 and 40 dop, with DI% values of 84.0, 41.2, 35.4 and 32.9 for 15, 23, 31 and 40 dop, respectively. Together, these findings explain for the first time why development of downy mildew epidemics on susceptible cultivars occurs early in the growing season when warmer seasonal temperatures in autumn coincide with presence of seedlings; in contrast to later in the growing season on less susceptible older plants coinciding with cooler and less favourable winter temperatures. Increasing maximum and minimum temperatures associated with climate change have likely fostered the increased severity of downy mildew over the past 15 years.     

In‐field classification of herbicide‐resistant Papaver rhoeas and Stellaria media using an imaging sensor of the maximum quantum efficiency of photosystem II

Reliable in‐season and in‐field tools for rapidly quantifying herbicide efficacy in dicotyledonous weeds are missing. In this study, the maximum quantum efficiency of photosystem II (F_v/F_m) of susceptible and resistant Papaver rhoeas and Stellaria media populations in response to treatments with acetolactate synthase (ALS) inhibitors were examined. Seedlings (4–6 leafs) were transplanted into the field immediately after the application of the ALS inhibitors florasulam, metsulfuron‐methyl and tribenuron‐methyl. The F_v/F_m values were assessed 1–7, 9 and 14 days after treatment (DAT). Based on the F_v/F_m values of all fluorescing pixels in the images of herbicide‐treated plants, discriminant maximum‐likelihood classifiers were created. Based on this classifier, an independent set of images were classified into ‘susceptible’ or ‘resistant’ plants. The classifiers’ accuracy, false‐positive rate and false‐negative rate were calculated. The F_v/F_m values of sensitive P. rhoeas and S. media plants decreased within 3 DAT by 28–43%. The F_v/F_m values of the resistant plants of both species were 20% higher than those of the sensitive plants in all herbicide treatments. The classifier separated sensitive and resistant plants 3 DAT with accuracies of 62–100%. False‐positive and false‐negative classifications decreased with increasing DAT. We conclude that by the assessment of the F_v/F_m value in combination with the classification sensitive and resistant P. rhoeas and S. media populations could be separated 3 DAT. This technique can help to select effective control methods and speed up the monitoring process of susceptible and resistant weeds.     

Simple sequence repeat analysis of genetic diversity among Acetyl‐CoA carboxylase inhibitor‐resistant and ‐susceptible Echinochloa crus‐galli and E. oryzicola populations in Korea

Echinochloa species are amongst the most problematic weeds in rice fields of Korea. The steady reliance on the Acetyl‐CoA carboxylase (ACCase) and acetolactate synthase inhibiting herbicides for control of these weeds has led to resistance to these herbicides. The objective of this study was to assess the genetic diversity among populations of ACCase inhibitor‐resistant and ‐susceptible Echinochloa crus‐galli and E. oryzicola in Korea, to better understand their population structure and possible origins of resistance. Seven simple sequence repeat markers were applied to assess the genetic diversity between resistant and susceptible E. crus‐galli and E. oryzicola from 12 populations in Korea. Genetic diversity was slightly higher in the resistant group. The Unweighted Pair Group Method using Arithmetic algorithm (UPGMA) dendrogram generated two distinct clades. One clade consisted of Echinochloa spp. from three populations, i.e. Anmyeondo, Gimje 4 and Gongju, which are resistant and differentiated from the susceptible populations, and the other clade contained the rest of the populations. Structure modelling supported two clades of UPGMA clustering. Based on these data, we can infer that some resistant populations are greatly differentiated, whereas other resistant biotypes are still building up resistance in rice fields in Korea. Resistance traits will be fixed and continue to spread over time without proper control measures.     

Transmission and detection of toria [<Emphasis Type="Italic">Brassica rapa</Emphasis> L. subsp. <Emphasis Type="Italic">dichotoma</Emphasis> (Roxb.)] phyllody phytoplasma and identification of a potential vector

Phyllody disease associated with 16SrIX phytoplasma was observed in the range of 4.1–11% in 10 different lines of toria [Brassica rapa L. subsp. dichotoma (Roxb.)] in experimental fields of the Indian Agricultural Research Institute, New Delhi, India during 2008 and 2009. The toria phyllody (TP) phytoplasma was detected in all the symptomatic and 13.3% of asymptomatic toria plants by nested PCR. The phytoplasma was detected in midrib, flower part, siliquae, stem, and root of infected plants as well as seeds. TP was transmitted by grafting and by dodder to toria and nine other rapeseed/mustard species as confirmed by nested PCR. However, symptoms of phytoplasma infection were induced only in toria, yellow sarson [Brassica rapa L. subsp. trilocularis (Roxb.)], brown sarson [Brassica rapa L. subsp. sarson (Prain)], rapeseed (B. napus subsp. oleifera), and rocket or taramira (Eruca sativa) but not in mustard (B. juncea), black mustard (B. nigra), Ethiopian mustard (B. carinata), B. tournefortii and white mustard (Sinapis alba). Transmission of TP phytoplasma to periwinkle (Catharanthus roseus) was successful only through dodder, but no transmission to tomato (Lycopersicon esculentum) or brinjal (Solanum melongena) was found. TP phytoplasma was detected in Laodelpax striatellus, an abundant planthopper in toria fields, which indicates that this planthopper may be a potential vector for TP phytoplasma.     

Pathogenic and non‐pathogenic Agrobacterium tumefaciens,A. rhizogenes and A. vitis strains form biofilms on abiotic as well as on root surfaces

Pathogenic and non‐pathogenic Agrobacterium tumefaciens, A. rhizogenes and A. vitis strains growing in minimal liquid medium adhered to different abiotic surfaces, forming biofilms at initial stages of development. Agrobacterium tumefaciens and A. vitis strains were able to attach to both polystyrene and polypropylene materials, whereas the A. rhizogenes strains only bound to polystyrene surfaces. Strains of the three species were also able to form biofilms on borosilicate coverslips. It is concluded that their ability to adhere to and form nascent biofilms on abiotic surfaces is dependent on the Agrobacterium species (biovar), surface material and growth conditions. Furthermore, tumorigenic A. tumefaciens and A. vitis strains, and the biological control agent A. rhizogenes strain K84, bound tightly to and formed complex biofilms on the surface of tomato root tips ex planta. More importantly, in planta assays confirmed that all three Agrobacterium spp. strains efficiently colonized tomato seedlings and also formed biofilms on roots. These complex structures, as revealed by scanning electron microscopy, were composed of numerous bacterial cells arranged in different ways: either dense and continuous carpets, large aggregates embedded in extra‐cellular material or globular mushrooms traversed internally by channels. Confocal laser scanning microscopy, using GFP‐marked derivative strains, corroborated the presence of live, three‐dimensional and thick green fluorescent structures attached to plant material. This study illustrates that besides A. tumefaciens, strains of the species A. rhizogenes and A. vitis are also able to build biofilms on abiotic as well as on root surfaces.     

Identification of Brassica accessions resistant to ‘old’ and ‘new’ pathotypes of Plasmodiophora brassicae from Canada

Genetic resistance is the main tool used to manage clubroot of canola (Brassica napus) in Canada. However, the emergence of new virulent strains of the clubroot pathogen, Plasmodiophora brassicae, has complicated canola breeding efforts. In this study, 386 Brassica accessions were screened against five single-spore isolates (represented by pathotypes 2F, 3H, 5I, 6M and 8N on the Canadian Clubroot Differential Set) and 17 field isolates (represented by 12 unique pathotypes: 2B, 3A, 3D, 3O, 5C, 5G, 5K, 5L, 5X, 8E, 8J and 8P) of P. brassicae to identify resistance sources effective against these strains. The results showed that one B. rapa accession (CDCNFG-046, mean index of disease (ID) = 3.3%) and two B. nigra accessions (CDCNFG-263, mean ID = 3.1%; and CDCNFG-262, mean ID = 4.7%) possessed excellent resistance to all 22 of the isolates evaluated. Fifty other accessions showed differential clubroot reactions (resistant, moderately resistant or susceptible), including 27 (one B. napus, two B. rapa, four B. oleracea and 20 B. nigra) accessions that were each resistant to 8–21 P. brassicae isolates, but developed mean IDs in the range of 5.3–29.6%. The remaining 23 accessions (two B. napus, one B. rapa, five B. oleracea and 15 B. nigra) were each resistant to 3–13 isolates, but developed mean IDs in the range of 30.3–47.0%. The three accessions that showed absolute resistance and the 50 accessions that showed differential clubroot reactions could be used to breed for resistance to the new P. brassicae strains.     

Development of a real‐time PCR method for the detection of the dagger nematodes Xiphinema index,X. diversicaudatum,X. vuittenezi and X. italiae,and for the quantification of X. index numbers

The ectoparasitic dagger nematodes Xiphinema index and Xiphinema diversicaudatum, often at low numbers in the soil, are vectors of grapevine nepoviruses, which cause huge agronomical problems for the vineyard industry. This study reports a method, based on real‐time PCR, for the specific detection of these species and of the closely related non‐vector species Xiphinema vuittenezi and Xiphinema italiae. Specific primers and TaqMan probes were designed from the ribosomal DNA internal transcribed spacer 1 (ITS1), enabling the specific detection of single individuals of each of the X. index, X. diversicaudatum, X. italiae and X. vuittenezi species whatever the nematode population. The specificity of detection and absence of false positive reaction were confirmed in samples of each species mixed with the three other Xiphinema species or mixed with nematodes representative from other genera (non‐plant‐parasitic Dorylaimida, Longidorus sp., Meloidogyne spp., Globodera spp. and Pratylenchus sp.). The method was shown to be valid for the relative quantification of X. index numbers through its use, from crude nematode extracts of soil samples, in a greenhouse assay of grapevine accessions ranging from highly susceptible to resistant. As an alternative to time‐consuming microscopic identification and counting, this real‐time PCR method will provide a fast, sensitive and reliable diagnostic and relative quantification technique for X. index nematodes extracted from fields or controlled conditions.