Reclassification of aster yellows group phytoplasmas in Korea

Aster yellows group phytoplasmas were reclassified by analysis of the 16S rRNA gene sequence, their phylogeny and the presence of interoperon heterogeneity. Nine phytoplasmas were classified into subgroups 16SrI-B and 16SrI-D using the 16S rRNA gene sequence. Then, based on the presence of interoperon heterogeneity, subgroup 16SrI-B phytoplasmas were differentiated into three subunits as 16SrI-B(a): mulberry dwarf, sumac witches’ broom and porcelain vine witches’ broom; 16SrI-B(b): angustata ash witches’ broom and Japanese spurge yellows; and 16SrI-B(c): onion yellow dwarf, water dropwort witches’ broom and hare’s ear yellow dwarf phytoplasma.     

Weed control in conventional and herbicide tolerant winter oilseed rape (Brassica napus) grown in rotations with winter cereals in the UK

Two winter oilseed rape (Brassica napus) cultivars, tolerant to glyphosate and glufosinate, were compared with a conventional cultivar at three sites over 4 years, in 3‐year crop rotations in the UK. The winter oilseed rape was grown in Years 1 and 4, with winter cereals, which received uniform herbicide treatments, in the intervening years. The second winter oilseed rape treatments were applied to randomised sub‐plots of the original plots. Weed densities were recorded in autumn and spring and weed biomass was measured in summer. At most sites, there was only one application of glufosinate or glyphosate, whereas two products were often used on the conventional variety. The timing of glyphosate and glufosinate application was, on average, 34 days later than that of the conventional broad‐leaved weed control treatments. Overall weed control, across all sites and years, was not statistically different between the conventional, glyphosate and glufosinate treatments. However, glyphosate achieved higher control of individual weed species more frequently than the other treatments. Glufosinate and the conventional treatments were similar in performance. The treatments in Year 1 sometimes affected weed populations in the subsequent cereal crops and, in rare instances, those in the rape in Year 4. Carry‐over effects were small after most treatments. In general, weed survival was greater in the oilseed rape crops, irrespective of the treatment, than it was in the intervening cereal crops.     

Detection and differentiation of grapevine yellows phytoplasmas belonging to the elm yellows group and to the stolbur subgroup by PCR amplification of non-ribosomal DNA

Primer pairs were designed from a cloned DNA probe of a strain of flavescence dorée (FD) phytoplasma and from a cloned DNA probe of a strain of stolbur phytoplasma. Among an array of reference phytoplasma strains maintained in periwinkle, pair FD9f/r amplified a 1.3 kb DNA fragment only with phytoplasma strains of elm yellows (EY) group, i.e. two strains of FD and two strains of EY. Tru9I restriction analysis of the fragment amplified by FD9f/r revealed a diversity among EY-group phytoplasmas. The FD strains differed from the strains isolated from elm. The profile of the phytoplasmas infecting the grapevine samples from Catalonia and most of the samples from Northern Italy were identical to that of a FD strain. Three other profiles were detected in grapevine from Palatinate, in Germany.The two primer pairs derived from a stolbur strain, STOL4f/r and STOL11f2/r1, specifically amplified a 1.7 kb and a 0.9 kb DNA fragment, respectively, with all strains in the stolbur subgroup. However, the pair STOL4f/r did not recognise strain MOL. Both pairs allowed to detect phytoplasmas in diseased grapevines from France, Italy, Spain and Israel. Attempts to differentiate between phytoplasmas in the stolbur subgroup by restriction analyses failed. The pairs FD9f/r and STOL11f2/r1 could be used in the same reaction (multiplex PCR) to detect EY-group phytoplasmas, stolbur-subgroup phytoplasmas or both phytoplasmas simultaneously when template DNAs were mixed.     

Electron microscopy and molecular identification of phytoplasmas associated with strawberry green petals in the Czech Republic

The presence of phytoplasma inFragaria ananassa x Duch cv Senga Sengana showing strawberry green petals symptoms was observed by electron microscopy of phloem tissue. No phytoplasmas were found in asymptomatic strawberry plants used as controls. Nucleic acids extracted from these plants were used in nested-PCR assays with primers amplifying 16S rRNA sequences specifie for phytoplasmas. Bands of 1.2 kb were obtained and the subsequent nested-PCR with specific primers and RFLP analyses allowed to classify the detected phytoplasmas in the aster yellows group (16SrI). They belonged to the subgroup I-C of which type strain is clover phyllody phytoplasma.     

Predicting the risk of weed infestation in winter oilseed rape crops

Chemical weed control before crop and weed emergence is a systematic practice in winter oilseed rape crops in France. It would be profitable both for farmers and the environment to predict the level of weed infestation early on in the growing season and to control weeds only when necessary using post‐emergence weed control. The objective of this paper was to develop and evaluate simple models to predict weed biomass in oilseed rape crops. The model input variables were related to weed population characteristics and farmers’ practices. The models can be used to classify oilseed rape plots into two categories: plots with a level of weed infestation above a threshold or those with level of weed infestation below a threshold. A data set including 3 years of experiments, conducted across several regions in France, was used to estimate the parameters and to evaluate the models. High values of sensitivity and specificity were obtained when weed biomass was predicted as a function of sowing date, type of soil tillage, soil mineral nitrogen, crop density, weed density at emergence, and main characteristics of the most abundant weed species. Model performance strongly decreased when input variables related to the weed population were not taken into account. The best models correctly classified 90% of the plots with high weed infestation and 64% of the plots with low weed infestation.     

Screening for resistance to canker (Leptosphaeria maculans) in winter oilseed rape (Brassica napus ssp. oleifera)

A test involving infection of seedlings in a glasshouse was evaluated for potential use in screening breeding material by comparing the results with those of field tests. The two tests correlated well for some resistant selections, particularly those closely related to Jet Neuf, and for very susceptible selections. However, two small groups of selections were resistant in the field but consistently susceptible in the glasshouse. Inoculation of mature plants in the glasshouse resulted in similar discrepancies and therefore gave no advantage over the seedling test. These discrepancies limit the usefulness of glasshouse techniques for routine resistance screening. A very high correlation was found overall between internal and external canker symptoms of the crown in nine field trials. Significant differences were found, however, between selections and cultivars in the relative severity of these two symptoms. Upper stem canker and crown canker were found to be very poorly correlated for individual plants, but highly correlated for eultivar means. The significance of these observations is discussed with respect to scoring canker in the field.     

Pathogenic variability of Plasmopara halstedii infecting sunflower in the Czech Republic

Plasmopara halstedii was isolated from diseased sunflowers collected from eight locations in the Czech Republic from 2007 to 2014. Races of the pathogen were determined based on 84 isolates collected during the study. In total, eight races of P. halstedii were detected using a set of nine sunflower differential lines. Races 700, 704, 705, 710, 714 and 715 were proven by soil drench inoculation, and two additional races (730 and 770) proposed by the previously applied leaf disc inoculation method. Race 700 was the most dominant in the Czech P. halstedii populations, with race 710 being the second most frequent. Races 704 and 714 were found over three seasons, while other races were recorded only in one growing season (race 730 in 2010, and the new races 705 and 715 in 2014). A comprehensive study was further conducted for isolates collected in 2013–14 using an extended differential set consisting of 15 sunflower lines. According to the latter methodology which marks races with five‐digit virulence codes, races 70060, 70471, 70571, 71060, 71461 and 71571 were recorded. The growing complexity of P. halstedii pathogenicity exhibited by the ability to infect higher numbers of differential genotypes and resulting in determination of the new pathogen races (virulence profiles) 70571, 71461 and 71571 is alarming. Although the limited number of isolates studied cannot characterize the entire pathogen diversity in the Czech Republic, the trend towards more diverse virulence in P. halstedii populations is clearly demonstrated by the new records of races 704, 705, 714 and 715, all capable of overcoming the resistance gene Pl₆.     

Biochemical defense mechanisms induced in winter oilseed rape seedlings with different susceptibility to infection with Leptosphaeria maculans

The activity of key biochemical defense mechanisms in seedlings of winter oilseed rape cultivars, differing in tolerance to infection with Leptosphaeria maculans, was investigated. The studied winter oilseed rape cultivars differed in their biochemical defense potential. In the resistant cultivar, a significant increase in the activity of chitinase and β-1,3-glucanase was observed that can be regarded as a symptom of a systemic defense reaction. In addition, the resistant cultivar had a more efficient antioxidant system, that is, higher activity of specific SOD isoforms and higher level of low-molecular antioxidants. Only the resistant cultivar had three Fe-SOD sub-isoforms. A correlation between the content of cell wall-bound phenolics and hydrogen peroxide in the resistant cultivar may indicate that an important method of H₂O₂ neutralization was its participation in the process of phenolics incorporation into the cell wall. Elevated content of cell wall-bound phenolics significantly enhances the cell wall integrity, which then becomes a more effective firewall against L. maculans. Moreover, high utilization of soluble sugars during synthesis of the phenolic compounds in the pathogenesis was demonstrated in the resistant cultivar. We conclude that the key factors contributing to a greater tolerance to L. maculans were an effective antioxidant system and higher activity of PR proteins, i.e., chitinase and β-1,3-glucanase.     

Note: A comparison of molecular diagnostic procedures for the detection of aster yellows phytoplasmas (16Sr-I) in leafhopper vectors

Different molecular procedures were compared for the detection of aster yellows phytoplasmas (AYP) in the leafhopper vectorsMacrosteles quadripunctulatus (Kirschbaum),Euscelidius variegatus (Kirschbaum) andEuscelis incisus (Kirschbaum). Polymerase chain reaction (PCR) with universal and group-specific primers designed on the 16S-rDNA sequence was most sensitive in nested assays. A dot-blot procedure with an oligoprobe designed on the 16S-rDNA was less sensitive and consistent to detect phytoplasmas in total insect DNA, but consistently detected amplicons from direct PCR. The dot-blot assay with a probe based on a phytoplasma plasmid sequence detected AYP in most vector specimens and did not react with DNAs from leafhoppers infected by flavescence dorée and psyllids infected by apple proliferation phytoplasmas. This last assay is almost devoid of contamination risks, faster and cheaper compared to PCR, therefore it has to be preferred for field-scale analysis of leafhopper populations. http://www.phytoparasitica.org posting Feb. 24, 2004.     

Winter oilseed rape with high levels of resistance to Pyrenopeziza brassicae derived from wild Brassica species

Oilseed rape cultivars possess inadequate levels of resistance to light leaf spot disease, caused by the ascomycete Pyrenopeziza brassicae Sutton & Rawlinson. High levels of resistance to this disease were found within wild accessions of Brassica oleracea and B. rapa . This resistance was introgressed into agronomically acceptable winter oilseed rape breeding lines. Seedling resistance was determined by two genes. One of these, derived from B. rapa and positioned on linkage group N1, resulted in no apparent symptoms following infection, while the other, derived from B. oleracea and positioned on N16, resulted in black necrotic flecks and a reduced amount of sporulation compared with standard cultivars. Several agronomically acceptable double haploid lines were developed which expressed very high levels of adult plant resistance.     

Geostatistical analysis of the distribution of Leptosphaeria species causing phoma stem canker on winter oilseed rape (Brassica napus) in England

In June/July 2001, 2002, 2003 and 2006, regional variation in distribution of the pathogens Leptosphaeria maculans and L. biglobosa that are causally associated with phoma stem canker was surveyed on winter oilseed rape crops in England. In 2001–2003, when isolates from basal cankers were visually identified as L. maculans or L. biglobosa based on cultural morphological characteristics, 70% were L. maculans and 30% L. biglobosa . In 2001, 2002, 2003 and 2006, when amounts of DNA of each species in basal cankers were determined by quantitative PCR, the abundance of L. maculans DNA was greater than that of L. biglobosa DNA in 77% of samples. When regional differences in amounts of L. maculans and L. biglobosa DNA were mapped geostatistically, quantities of L. maculans DNA were greater in cankers from southern England and those of L. biglobosa DNA were greater in northern England. A comparison with geostatistically mapped predictions made using a weather-based model describing stages in development of phoma stem canker epidemics suggested that these differences in Leptosphaeria populations may have been a consequence of differences in temperature after onset of leaf spotting between northern and southern England. Both PCR and morphological evidence suggested that the abundance of L. maculans in England has increased since the last surveys in the 1980s. Implications of these surveys for control of phoma stem canker are discussed.     

Pathotypes of <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis> causing damage to oilseed rape in the Czech Republic and Poland

Winter oilseed rape (Brassica napus) is an important crop in the Czech Republic and Poland. Clubroot disease caused by the pathogen Plasmodiophora brassicae is a serious and still-growing problem for oilseed rape growers in both countries. The aim of this study was to evaluate the pathotype composition of P. brassicae populations from the Czech Republic and Poland, according to the three evaluation systems, and to determine soil inoculum loads for representative fields via traditional end-point PCR as well as quantitative PCR analysis. There were considerable differences between the populations of P. brassicae from both countries, and the number of pathotypes varied depending on the evaluation system and the threshold used to distinguish susceptible vs. resistant plant reactions. This is the first study comparing the effect of different thresholds. Using an index of disease (ID) of 25 % to distinguish susceptible vs. resistant reactions, there was a total of seven pathotypes identified based on the differentials of Williams, five with the system of Somé et al., and 18 with the European Clubroot Differential (ECD) set. However, based on a threshold of 50 %, there were nine pathotypes according to the evaluation system by Williams, four based on the differentials of Somé et al., and 15 with the ECD set. Changing of the thresholds led to the reclassification of some pathotypes. Several pathotypes were common in both countries. High amounts of pathogen DNA were found in many of the field soils analysed by quantitative PCR. There was a weak correlation between soil pH and infestation of P. brassicae for the Polish soils.     

Visual and PCR assessment of light leaf spot (Pyrenopeziza brassicae) on winter oilseed rape (Brassica napus) cultivars

Methods to assess light leaf spot ( Pyrenopeziza brassicae ) on winter oilseed rape cultivars were compared in laboratory, controlled-environment and field experiments. In controlled-environment experiments with seedling leaves inoculated at GS 1,4, the greatest differences in percentage area affected by P. brassicae sporulation were observed with inoculum concentrations of 4 × 10³ or 4 × 10⁴ spores mL⁻¹, rather than 4 × 10² or 4 × 10⁵ spores mL⁻¹, but older leaves had begun to senesce before assessment, particularly where they were severely affected by P. brassicae . In winter oilseed rape field experiments, a severe light leaf spot epidemic developed in 2002/03 (inoculated, September/October rainfall 127·2 mm) but not in 2003/04 (uninoculated, September/October rainfall 40·7 mm). In-plot assessments discriminated between cultivars best in February/March in 2003 and June in 2004, but sometimes failed to detect plots with many infected plants (e.g. March/April 2004). Ranking of cultivar resistance differed between seedling experiments done under controlled-environment conditions and field experiments. The sensitivity of detection of P. brassicae DNA extracted from culture was greater using the PCR primer pair PbITSF/PbITSR than using primers Pb1/Pb2. P. brassicae was detected by PCR (PbITS primers) in leaves from controlled-environment experiments immediately and up to 14 days after inoculation, and in leaves sampled from field experiments 2 months before detection by visual assessment.     

Early development of light leaf spot (Pyrenopeziza brassicae) on winter oilseed rape (Brassica napus) in relation to temperature and leaf wetness.

In controlled environment experiments to study early development of light leaf spot, lesions developed with leaf wetness durations of 16 to 48 h after inoculation of oilseed rape with conidial suspensions of Pyrenopeziza brassicae at 12 or 18°C, but not with leaf wetness durations of 0 to 13h. The incubation period was 21 to 22 days at 12°C and 14 to 18 days at 18°C for leaf wetness durations of 16 to 48 h. The latent period was 21 to 23 days at 12°C and 18 to 19 days at 18°C, and the total number of lesions increased with increasing leaf wetness duration at both temperatures. In field experiments, light leaf spot always developed on oilseed rape with a leaf wetness duration of 48 h after inoculation in both 1990/1991 and 1991/1992, but the percentage leaf area affected was less on plants placed in an oilseed rape crop than on those placed in a glasshouse. Plants moved to an oilseed rape crop immediately after inoculation nearly always developed light leaf spot symptoms when they were inoculated between 19 October 1990 and 1 March 1991 or between 27 September 1991 and 14 February 1992, but plants inoculated between 31 August and 16 October 1990 or on 20 September 1991, when estimated leaf wetness duration was less than 16 h for several days after they were placed in crops, did not develop symptoms. The latent period of light leaf spot on plants transferred to the oilseed rape crop was 15 to 40 days, and there was an approximately linear relationship between 1 (latent period) and mean temperature during this period. The accumulated temperature during the latent period ranged from c. 150 to 250 day-degrees. The severity of lesions on these plants increased with increasing temperature from 5 to 15°C.     

Induction of defense mechanisms in seedlings of oilseed winter rape inoculated with Phoma lingam (Leptosphaeria maculans)

The aim of this study was to find the biochemical markers of blackleg, evoked by the fungus Leptosphaeria maculans in seedlings of oilseed winter rape. Analyses of antioxidant enzymes activity and reactive oxygen species detection were performed 24, 48 and 72 h after inoculation. Moreover, measurements of the total pool of phenolic compounds, trans-cinnamic acid and ferulic acid were carried out. The results proved the virulence of Ph L5 isolate towards the rape seedlings. An increase in total phenolic content, including trans-cinnamic acid, was observed 24 h after inoculation. High activity of phenylalanine ammonia-lyase is probably another symptom of the defense mechanisms, despite the fact that it was not accompanied by elevated phenolics level, which were probably rapidly exhausted during Phoma lingam inoculation. A drop in phenolics content was balanced by higher activity of SOD, CAT and non-specific peroxidases. Enhanced activity of these enzymes was a response to the reactive oxygen species overproduction. ROS accumulation in indirectly inoculated first true leaves after inoculation may suggest a systemic defense response.     

Biofumigation with Brassica plants and its effect on the inoculum potential of Fusarium yellows of Brassica crops

The use of Brassica crops as green manure in the so-called biofumigation treatment has been successfully exploited for the management of soilborne pathogens and is gaining interest particularly in the case of less intensive agricultural systems. A study was undertaken to investigate possible negative side-effects of biofumigation in order to prevent possible damage caused by wilt pathogens able to attack both plants used for biofumigation as well as agricultural crops. To do so, firstly the response of different Brassicas, including some used in biofumigation, to the formae speciales of Fusarium oxysporum known for being pathogenic on Brassica crops was evaluated. Secondly, the effect of green manure treatments on yield, quality of crops, and inoculum densities, infection and survival of Fusarium oxysporum f. sp. conglutinans and F. oxysporum f. sp. raphani was evaluated. In the second part of the work, four Brassica crops, selected for their response (susceptibility or resistance) to F. oxysporum f. sp. conglutinans and to F. oxysporum f. sp. raphani were evaluated in order to determine their response to the two pathogens during subsequent crops grown in soil where plants were incorporated as green manure into the soil at the end of each cycle. Moreover, the dynamics of the populations of F. oxysporum f. sp. conglutinans and F. oxysporum f. sp. raphani in the soil after several biofumigation cycles was studied. Many of the Brassica crops used for biofumigation tested were susceptible to F. oxysporum f. sp. conglutinans and or to F. oxysporum f. sp. raphani. Green manure treatment, carried out by growing nine cycles of biocidal plants, with a short crop cycle of 30–35 days, did not reduce Fusarium wilts on susceptible Brassica hosts. The population of the pathogen was partially increased as a result of the incorporation of tissues of the susceptible plants. When Brassica crops grown were resistant to the two F. oxysporum pathogens used for soil infestation, green manure simulation did inhibit both pathogens, thus confirming its biocidal activity. The results obtained under our experimental conditions show that biofumigation treatment is not applicable for soil disinfestation on crops susceptible to the same formae speciales of F. oxysporum affecting Brassica species used for biofumigation. Brassica crops resistant to Fusarium yellows should be grown where biofumigation is applied. Moreover, alternation of crops used for biofumigation should be encouraged.