Arbuscular mycorrhizal fungi might alleviate aluminium toxicity in banana plants

Under defined laboratory conditions it was shown that two glucosinolate-containing plant species, Tropaeolum majus and Carica papaya , were colonized by arbuscular mycorrhizal (AM) fungi, whereas it was not possible to detect AM fungal structures in other glucosinolate-containing plants (including several Brassicaceae). Benzylglucosinolate was present in all of the T. majus cultivars and in C. papaya it was the major glucosinolate. 2-Phenylethylglucosinolate was found in most of the non-host plants tested. Its absence in the AM host plants indicates a possible role for the isothiocyanate produced from its myrosinase-catalysed hydrolysis as a general AM inhibitory factor in non-host plants. The results suggest that some of the indole glucosinolates might also be involved in preventing AM formation in some of the species. In all plants tested, both AM hosts and non-hosts, the glucosinolate pattern was altered after inoculation with one of three different AM fungi ( Glomus mosseae , Glomus intraradices and Gigaspora rosea ), indicating signals between AM fungi and plants even before root colonization. The glucosinolate induction was not specifically dependent on the AM fungus. A time-course study in T. majus showed that glucosinolate induction was present during all stages of mycorrhizal colonization.     

Isolation of a Microsomal Enzyme System Involved in Glucosinolate Biosynthesis from Seedlings of Tropaeolum majus L

An in vitro system that converts phenylalanine to phenylacetaldoxime in the biosynthesis of the glucosinolate glucotropaeolin has been established in seedlings of Tropaeolum majus L. exposed to the combined treatment of jasmonic acid, ethanol, and light. The treatment resulted in a 9-fold induction, compared with untreated, dark-grown seedlings, of de novo biosynthesis measured as incorporation of radioactively labeled phenylalanine into glucotropaeolin. Formation of the inhibitory degradation product benzylisothiocyanate during tissue homogenization was prevented by inactivation of the thioglucosidase myrosinase by addition of 100 mM ascorbic acid to the isolation buffer. This allowed the isolation of a biosynthetically active microsomal preparation from the induced T. majus plant material. The enzyme, which catalyzes the conversion of phenylalanine to the corresponding oxime, was sensitive to cytochrome P450 inhibitors, indicating the involvement of a cytochrome P450 in the biosynthetic pathway. It has previously been shown that the oxime-producing enzyme in the biosynthesis of p-hydroxybenzylglucosinolate in Sinapis alba L. is dependent on cytochrome P450, whereas the oxime-producing enzymes in Brassica species have been suggested to be flavin monooxygenases or peroxidase-type enzymes. The result with T. majus provides additional experimental documentation for a similarity between the enzymes converting amino acids into the corresponding oximes in the biosynthesis of glucosinolates and cyanogenic glucosides.     

Plasmodiophora brassicae: a review of an emerging pathogen of the Canadian canola (Brassica napus) crop

Plasmodiophora brassicae causes clubroot disease in cruciferous plants, and is an emerging threat to Canadian canola (Brassica napus) production. This review focuses on recent studies into the pathogenic diversity of P. brassicae populations, mechanisms of pathogenesis and resistance, and the development of diagnostic tests for pathogen detection and quantification. TAXONOMY: Plasmodiophora brassicae is a soil-borne, obligate parasite within the class Phytomyxea (plasmodiophorids) of the protist supergroup Rhizaria. DISEASE SYMPTOMS: Clubroot development is characterized by the formation of club-shaped galls on the roots of affected plants. Above-ground symptoms include wilting, stunting, yellowing and premature senescence. DISEASE CYCLE: Plasmodiophora brassicae first infects the root hairs, producing motile zoospores that invade the cortical tissue. Secondary plasmodia form within the root cortex and, by triggering the expression of genes involved in the production of auxins, cytokinins and other plant growth regulators, divert a substantial proportion of plant resources into hypertrophic growth of the root tissues, resulting in the formation of galls. The secondary plasmodia are cleaved into millions of resting spores and the root galls quickly disintegrate, releasing long-lived resting spores into the soil. A serine protease, PRO1, has been shown to trigger resting spore germination. PHYSIOLOGICAL SPECIALIZATION: Physiological specialization occurs in populations of P. brassicae, and various host differential sets, consisting of different collections of Brassica genotypes, are used to distinguish among pathotypes of the parasite. DETECTION AND QUANTIFICATION: As P. brassicae cannot be cultured, bioassays with bait plants were traditionally used to detect the pathogen in the soil. More recent innovations for the detection and quantification of P. brassicae include the use of antibodies, quantitative polymerase chain reaction (qPCR) and qPCR in conjunction with signature fatty acid analysis, all of which are more sensitive than bioassays. RESISTANCE IN CANOLA: Clubroot-resistant canola hybrids, recently introduced into the Canadian market, represent an important new tool for clubroot management in this crop. Genetic resistance must be carefully managed, however, as it has been quickly overcome in other regions. At least three resistance genes and one or two quantitative trait loci are involved in conferring resistance to P. brassicae. Root hair infection still occurs in resistant cultivars, but secondary plasmodia often remain immature and unable to produce resting spores. Fewer cell wall breakages occur in resistant hosts, and spread of the plasmodium through cortical tissue is restricted. More information on the genetics of clubroot resistance in canola is needed to ensure more effective resistance stewardship. USEFUL WEBSITES: http://www.canolacouncil.org/clubroot/resources.aspx, http://tu-dresden.de/die_tu_dresden/fakultaeten/fakultaet_mathematik_und_naturwissenschaften/fachrichtung_biologie/botanik/pflanzenphysiologie/clubroot, http://www.ohio.edu/people/braselto/plasmos/     

Yield reduction in Brassica napus, B. rapa, B. juncea, and Sinapis alba caused by flea beetle (Phyllotreta cruciferae (Goeze) (Coleoptera: Chrysomelidae)) infestation in northern Idaho

Phyllotreta cruciferae is an important insect pest of spring-planted Brassica crops, especially during the seedling stage. To determine the effect of early season P. cruciferae infestation on seed yield, 10 genotypes from each of two canola species (Brassica napus L. and Brassica rapa L.) and two mustard species (Brassica juncea L. and Sinapis alba L.) were grown in 2 yr under three different P. cruciferae treatments: (1) no insecticide control; (2) foliar applications of endosulfan; and (3) carbofuran with seed at planting plus foliar application of carbaryl. Averaged over 10 genotypes, B. rapa showed most visible P. cruciferae injury and showed greatest yield reduction without insecticide application. Mustard species (S. alba and B. juncea) showed least visible injury and higher yield without insecticide compared with canola species (B. napus and B. rapa). Indeed, average seed yield of S. alba without insecticide was higher than either B. napus or B. rapa with most effective P. cruciferae control. Significant variation occurred within each species. A number of lines from B. napus, B. juncea, anid S. alba showed less feeding injury and yield reduction as a result of P. cruciferae infestation compared with other lines from the same species examined, thus having potential genetic background for developing resistant cultivars.     

Identification and quantification of three active auxins in different tissues of Tropaeolum majus

Indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and phenylacetic acid (PAA) were identified as endogenous compounds with auxin activity in nasturtium ( Tropaeolum majus L.) by full scan gas chromatography-mass spectrometry. The endogenous concentrations of the three auxins were measured by GC-selected ion monitoring-MS and isotope dilution analysis using stable labelled isotopes. PAA was present at concentrations about 10- to 100-fold lower than IAA, whereas IBA was found to be in the same concentration range as IAA. Free IAA was highest in roots followed by young leaves. IBA was also highest in the roots, and relatively high concentrations were found in young leaves and flowers. The distribution of PAA was quite different from that found for IBA. No PAA could be detected in young leaves and flowers, and in all other tissues studied the concentrations were well below those of the other two auxin compounds. The presence of a nitrilase gene family and nitrilase activity in extracts from T. majus suggests that PAA might be synthesized by the nitrilase pathway using benzylglucosinolate as precursor.     

Identification of expressed genes during infection of Chinese cabbage (Brassica rapa subsp. pekinensis) by Plasmodiophora brassicae

Plasmodiophora brassicae is an obligate, biotrophic pathogen causing the club-root disease of crucifers. Despite its importance as a plant pathogen, little is known about P. brassicae at the molecular level as most of its life cycle takes place inside the plant host, and axenic culturing is impossible. Discovery of genes expressed during infection and gene organization are the first steps toward a better understanding of the pathogen-host interaction. Here, suppression subtractive hybridization was used to search for the P. brassicae genes expressed during plant infection. One-hundred and forty ESTs were found of which 49% proved to be P. brassicae genes. Ten novel P. brassicae genes were identified, and the genomic sequences surrounding four of the ESTs were acquired using genome walking. Alignment of the ESTs and the genomic DNA sequences confirmed that P. brassicae genes are intron rich and that the introns are small. These results show that it is possible to discover new P. brassicae genes from a mixed pool of both plant and pathogen cDNA. The results also revealed that some of the P. brassicae genes expressed in Chinese cabbage (Brassica rapa subsp. pekinensis) were identical to the genes expressed in the infection of Arabidopsis plants, indicating that these genes play an important role in P. brassicae infection.     

Infection of Chinese cabbage by Plasmodiophora brassicae leads to a stimulation of plant growth: impacts on cell wall metabolism and hormone balance

The importance of plant hormones in clubroot infection has long been recognized. The morphological changes, such as cell division and cell elongation leading to gall formation are triggered in the early stages of infection. We analysed cell expansion by localizing Xyloglucan endoTransglucosylase/Hydrolase (XTH)-action and screened the endogenous concentrations of several classes of phytohormones by mass spectrometry in the early stages of Plasmodiophora brassicae infection in Chinese cabbage (Brassica rapa spp. pekinensis). Infected plants showed a general transient growth promotion early in infection. Furthermore a clear XTH action was visible in the epidermal layer of infected roots. Complex changes in the endogenous phytohormone profile were observed. Initially infection resulted in an increased total auxin pool. The auxin increase, together with an increased XTH action, results in wall loosening and consequently cell expansion. When the first secondary plasmodia are formed, thirteen days after infection (DAI), can be considered a switch point in phytohormone metabolism. Twenty-one DAI the plasmodia might act as a plant hormone sink resulting in a reduction in the active cytokinin pool and a lower indole-3-acetic acid content in the infected plants.     

Synthesis of Benzylglucosinolate in Tropaeolum majus L. (Isothiocyanates as Potent Enzyme Inhibitors)

Benzylglucosinolate accumulates in mature plants of Tropaeolum majus L. The biosynthetic capacity for synthesis of benzylglucosinolate and the total content of benzylglucosinolate have been investigated during plant development and in different tissues. The content increased from 5 mg of benzylglucosinolate in the fresh seed to between 200 and 400 mg in the adult plant, depending on size. The biosynthetic capacity was measured using L-[U-14C]phenylalanine as precursor. Incorporation levels of approximately 30% were obtained with green leaves, whereas the incorporation levels obtained with other tissues were in the range of 0 to 5%. Leaves were the primary site of benzylglucosinolate synthesis. The high amounts of benzylglucosinolate accumulated in other tissues (e.g. developing seeds) reflected transport of benzylglucosinolate from the leaves. The initial steps in the biosynthesis of glucosinolates and cyanogenic glycosides are thought to be similar and to be localized on microsomal membranes. However, a microsomal system prepared from T. majus was biosynthetically inactive. Inclusion of T. majus plant material during preparation of sorghum microsomes also inhibited their activity. Benzylisothiocyanate, generated by degradation of benzylglucosinolate during the homogenization procedure, strongly inhibited the sorghum enzyme system, and its presence may thus explain why the isolated T. majus microsomal system is inactive.     

The infected root-hair count for estimating the activity of Plasmodiophora brassicae Woron. in the soil

A method is described for estimating the relative numbers of Plasmodiophora brassicae spores germinating in different soils, by counting infected root hairs. Cabbage seedlings are grown for 1 week at 25C. in glass tumblers of the infected soils; after washing out, the tap roots are stained in 1% aceto-carmine and a count is made of the number of root hairs containing zoosporangia of P. brassicae , along 2 cm. of root. It is shown that by this method it is possible to study, for example, the action of different bases in inhibiting root-hair infection; the main inhibiting factor was found to be soil alkalinity, however produced. Other factors were found to influence infection in lesser degree; thus the number of infected root hairs was reduced in soils receiving N /10 sodium and potassium chlorides in place of distilled water. Root-hair infection was also inhibited by low soil-moisture content.     

Resistance of some cultivated Brassicaceae to infestations by Plutella xylostella (Lepidoptera: Plutellidae)

Selecting insect-resistant plant varieties is a key component of integrated management programs of oligophagous pests such as diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), but rigorous research on important life history parameters of P. xylostella in relation to host plant resistance is rare. We evaluated six conventional brassicaceous species, namely, Brassica napus L. 'Q2', B. rapa L., B. juncea (L.) Czern., B. carinata L., B. oleracea L., and Sinapis alba L., and two herbicide-tolerant cultivars, namely, B. napus 'Liberty' and B. napus 'Conquest' for their resistance against P. xylostella. Brassicaceae species and cultivars varied considerably in their susceptibilities as hosts for P. xylostella. Sinapis alba and B. rapa plants were highly preferred by ovipositing females and trichome density on adaxial and abaxial leaf surfaces had nonsignificant effects on P. xylostella oviposition. Larval survival was similar on the genotypes we tested, but host plants significantly affected larval and pupal developmental time, herbivory, pupal weight, silk weight, adult body weight, forewing area and longevity (without food) of both male and female P. xylostella. Larval and pupal development of females was fastest on B. juncea and S. alba, respectively. Specimens reared on B. napus Liberty and B. oleracea, respectively, produced the lightest female and male pupae. Defoliation by both female and male larvae was highest on B. rapa, whereas least herbivory occurred on S. alba. Females reared on S. alba were heavier and lived longer in the absence of food than their counterparts raised on other tested host plants. Brassica oleracea could not compensate for larval feeding to the level of the other species we evaluated. B. napus Conquest, B. napus Q2, B. carinata, B. rapa, and S. alba produced, respectively, 1.6-, 1.8-, 1.8-, 3.9-, and 5.5-fold heavier root systems when infested than their uninfested counterparts, suggesting that these species were better able to tolerate P. xylostella infestations.     

Efficacy of dried seed powder of some plant species as soil amendment against Meloidogyne incognita (Tylenchida: Meloidogynidae) on tomato

The nematicidal activity of dried ground seeds of Ammi majus, Matricaria chamomilla, Ricinus communis, Brassica alba, B. oleracea, Peganum harmala, Solanum nigrum, Raphanus sativus and Eucalyptus sp. was assessed against the root-knot nematode, Meloidogyne incognita, infecting tomato in a glasshouse. The powdered seeds of the tested plants were incorporated into the soil at the rate of 5 g/kg and their nematicidal activity was compared with that of the synthetic nematicide carbofuran at the rate of 0.01 g a.i./kg. The effects of the treatments on the growth of tomato were also examined. The populations of M. incognita in the soil and root galling of tomato were significantly suppressed by the powdered seeds of all the plant species tested, with the greatest reduction occurring in soil amended with M. chamomilla, followed by soil treated with powdered seeds of A. majus, S. nigrum, R. communis and Eucalyptus sp. The efficacy of B. oleracea, B. alba, M. chamomilla and R. communis in reducing the number of J₂ in the soil was similar to that of carbofuran. All amendments, except powdered seeds of M. chamomilla and A. majus significantly increased shoot length compared to the untreated inoculated plants. Shoot weight was significantly increased in soil amended with powdered seeds of B. oleracea, B. alba, R. communis, P. harmala and S. nigrum, but not in soil amended with the other seed powders when compared with untreated inoculated soil. Significant increases in root length occurred in pots amended with seed powder of B. alba, R. communis and Eucalyptus and in root weight for P. harmala. None of the tested dried seeds was phytotoxic to tomato plants at the applied rate.     

Quantitative trait loci for glucosinolate accumulation in Brassica rapa leaves

Glucosinolates and their breakdown products have been recognized for their effects on plant defense, human health, flavor and taste of cruciferous vegetables. Despite this importance, little is known about the regulation of the biosynthesis and degradation in Brassica rapa. Here, the identification of quantitative trait loci (QTL) for glucosinolate accumulation in B. rapa leaves in two novel segregating double haploid (DH) populations is reported: DH38, derived from a cross between yellow sarson R500 and pak choi variety HK Naibaicai; and DH30, from a cross between yellow sarson R500 and Kairyou Hakata, a Japanese vegetable turnip variety. An integrated map of 1068 cM with 10 linkage groups, assigned to the international agreed nomenclature, is developed based on the two individual DH maps with the common parent using amplified fragment length polymorphism (AFLP) and single sequence repeat (SSR) markers. Eight different glucosinolate compounds were detected in parents and F(1)s of the DH populations and found to segregate quantitatively in the DH populations. QTL analysis identified 16 loci controlling aliphatic glucosinolate accumulation, three loci controlling total indolic glucosinolate concentration and three loci regulating aromatic glucosinolate concentrations. Both comparative genomic analyses based on Arabidopsis-Brassica rapa synteny and mapping of candidate orthologous genes in B. rapa allowed the selection of genes involved in the glucosinolate biosynthesis pathway that may account for the identified QTL.     

Broccoli and turnip plants display contrasting responses to belowground induction by Delia radicum infestation and phytohormone applications

Induced responses to insect herbivory are a common phenomenon in the plant kingdom. So far, induced responses have mostly investigated in aerial plant parts. Recently it was found that root herbivore may also elicit both local and systemic responses affecting aboveground herbivores and their natural enemies. Using broccoli (Brassica oleracea subsp. italica L.) and turnip (Brassica rapa subsp. rapa L.), two cultivated brassicaceaous plants differing in their chemistry and morphology, we analysed the local and systemic induced responses triggered by Delia radicum L. damage, JA and SA application. We also assessed whether the root induction treatments affected D. radicum larval performance. Both D. radicum damage and JA induced changes in glucosinolate and sugar content as well as affected D. radicum performance, while SA application did not. Despite the uniform chemical responses, the effect on larval performance on broccoli and turnip plants was very different. On broccoli, JA root treatment reduced herbivore performance, whereas in turnips the same treatment enhanced it. JA- and D. radicum-induced responses followed similar patterns, which suggests that the JA signalling pathway is involved in root-induced responses to larval feeding. Glucosinolate induction cannot fully explain the differences found in the performance of D. radicum on the different species. Changes in other resistance factors might significantly contribute to the induced resistance in these brassicaceaeous species as well.     

A DIGESTIVE LIPASE OF Pieris brassicae L. (LEPIDOPTERA: PIERIDAE): PURIFICATION, CHARACTERIZATION, AND HOST PLANTS EFFECTS

The properties of a digestive lipase from the larval midgut of Pieris brassicae were studied by performing biochemical purification, characterization, effect of host plants, and extracted inhibitors. The purification process revealed a lipase with a purification fold of 42, recovery of 18.12%, molecular weight mass of 72.3 kDa, optimal pH at 11, and optimal temperature at 30°C, as well as stability at the optimal temperature for 12 h. The purified enzyme was inhibited by the ions Na(+) , Mn(+) , Fe(2+) , and Cu(2+) and the inhibitors SDS, EDTA, TTHA, and mercaptoethanol. Ca(2+) and Mg(2+) increased activity of the purified lipase, but urea, PMSF, EGTA, and DTC had no effect on enzymatic activity. Feeding of larvae on three host plants, Trepaeolus majus, Brassica olearcea var. alba, and B. olearcea var. rubra revealed the highest lipase activity on T. majus, but the two varieties of B. olearcea significantly decreased lipase activity. Extraction of a crude inhibitor from two varieties of B. olearcea demonstrated that the crude inhibitor inhibited the purified lipase up to 75%. The inhibitor changed the kinetic parameters of the enzyme by elevating the K(m) , as in competitive inhibition. The data suggest a possible role for plant lipase inhibitors in host plant resistance.     

Metabolomic analysis of methyl jasmonate treated Brassica rapa leaves by 2-dimensional NMR spectroscopy

The metabolomic analysis of Brassica rapa leaves treated with methyl jasmonate was performed using 2-dimensional J-resolved NMR spectroscopy combined with multivariate data analysis. The principal component analysis of the J-resolved NMR spectra showed discrimination between control and methyl jasmonate treated plants by principal components 1 and 2. While the level of glucose, sucrose and amino acids showed a decrease after methyl jasmonate treatment, hydroxycinnamates and glucosinolate were highly increased. Methyl jasmonate treatment resulted in a long-term accumulation of indole glucosinolate and indole-3-acetic acid, lasting up to 14 days after treatment. Malate conjugated hydroxycinnamates also exhibited an increase until 14 days after methyl jasmonate treatment, these compounds might play an important role in plant defence responses mediated by methyl jasmonate.     

Mapping QTLs for root morphological traits inBrassica rapa L. based on AFLP and RAPD markers

Root growth and thickening plays a key role in the final productivity and even the quality of storage roots in root crops. This study was conducted to identify and map quantitative trait loci (QTLs) affecting root morphological traits in Brassica rapa by using molecular markers. An F2 population was developed from a cross between Chinese cabbage (Brassica rapa ssp. chinensis) and turnip (B. rapa ssp. rapifera), which differed greatly in root characters. A genetic map covering 1837.1 cM, with 192 marker loci and 11 linkage groups, was constructed by using this F2 population. The F3 families derived from F2 plants were grown in the field and evaluated for taproot traits (thickness, length, and weight). QTL analysis via simple interval mapping detected 18 QTLs for the 3 root traits, including 7 QTLs for taproot thickness, 5 QTLs for taproot length, and 6 QTLs for taproot weight. Individually, the QTLs accounted for 8.4-27.4% of the phenotypic variation. The 2 major QTLs, qTRT4b for taproot thickness and qTRW4 for taproot weight, explained 27.4% and 24.8% of the total phenotypic variance, respectively. The QTLs for root traits, firstly detected in Brassica crops, may provide a basis for marker-assisted selection to improve productivity in root-crop breeding.     

Intracellular localization of sucrose-phosphate phosphatase in storage cells

The intracellular location of sucrose-phosphate phosphatase (SPP; EC 3.1, 3.24) was investigated by comparing the ratios of SPP to vacuolar and cytosolic markers in protoplasts and vacuoles from storage tissues of red beet ( Beta vulgaris ) and turnip ( Brassica rapa L. var. rapa ) hypocotyl, and carrot ( Daucus carota ) root. In all three instances, SPP activity was found not to be associated with vacuolar markers ß-acetyl-glucosaminidase and α-mannosidase, but proportionally associated with the cytosolic marker alcohol dehydrogenase. It was determined that in storage cells of red beet and turnip hypocotyl, as well as in carrot root, SPP is located in the cytoplasm. Discrepancies with earlier reports indicating SPP of vacuolar origin are discussed.     

Plasmodiophora brassicae-induced Cell Death and Medium Alkalization in Clubroot-resistant Cultured Roots of Brassica rapa

Plasmodiophora brassicae causes clubroot in the turnip, Brassica rapa L. We used organ cultures of adventitious roots from B. rapa seedlings to investigate the initial response of resistant and susceptible cultivars to P. brassicae infection. Primary plasmodia of P. brassicae were observed in root hairs of both susceptible and resistant cultured roots. On the other hand, secondary plasmodia were able to proliferate only in the susceptible root culture but not in the resistant one. Root cultures from the susceptible cultivar all developed clubroot 4 weeks after treatment with 10⁴, 10⁵ or 10⁶ spores/ml, but roots from the resistant cultivar did not develop clubroot under the same conditions. Cell death, as measured by Evans blue and TTC dye methods, was observed in cultured roots from the resistant cultivar but did not occur in roots from the susceptible cultivar after exposure to P. brassicae spores. Cell death was inhibited almost completely by EGTA and verapamil but not by the calmodulin antagonist W7. These results suggest the involvement of Ca²⁺ in P. brassicae‐induced cell death. Alkalization of the root culture medium of the resistant cultivar was observed 2 days after treatment with P. brassicae spores but was not observed in root culture medium from the susceptible strain. We conclude that our root culture system must be a useful tool for further studies of the molecular mechanism of clubroot resistance.     

Randomly amplified polymorphic DNA (RAPD) profiling of Plasmodiophora brassicae

M. MÖLLER AND R. HARLING. 1996. A technique is described for the preparation of DNA suitable for use in RAPD analysis from pure, sterile resting spores of Plasmodiophora brassicae . Using this technique, random 10-base pair primers were applied to P. brassicae DNA from three single spore isolates. The resulting profiles were compared with the race classification based on inoculation of the European Clubroot Differential (ECD) series of Brassica hosts. Out of 40 primers tested, 23 gave amplification products, three gave isolate-specific profiles and one a profile which corresponded with the ECD race classification of the isolates. RAPD profiling can provide a faster means of race classification in P. brassicae .