Studies of infection withxanthomonas campestris pv.vesicatoria,causal agent of bacterial scab of pepper in Israel

Xanthomonas campestris pv. vesicatoria, the causal agent of bacterial scab of pepper, was isolated in several regions in Israel. When artificial inoculation was practiced, pathogen growth was enhanced by high temperatures (30-36°C), and an inoculum concentration of 10^p6 colony-forming units (CFU) per ml was optimal for symptoms to develop on plants. Pre-inoculation treatments such as wounding the leaves by rubbing them with carborundum powder or spraying them with diluted wax solvents, markedly increased disease severity, but were not essential. Pre-inoculation conditioning at two different relative humidity levels (R.H. = 100% or R.H. < 40%) did not affect disease severity. Young leaves were more severely affected following infection than older leaves. Disease severity was similar with several isolates ofX. campestris pv. vesicatoria.     

Infection processes of Ustilaginoidea virens during artificial inoculation of rice panicles

Ustilaginoidea virens is a ubiquitous plant pathogen that causes rice false smut disease, one of the most destructive diseases of rice (Oryza sativa L.) production. However, data concerning the effect of inoculation on disease development and the infection process of this pathogen are not comprehensive. In this study, the developmental processes of U. virens in rice panicles were characterized using an enhanced green fluorescent protein (EGFP) labelled strain. A mixture of hyphae and conidia of U. virens was used to inoculate rice panicles by leaf sheath injection during the booting stage of rice plants grown in a greenhouse. The panicles were assessed to determine the relationship between artificial inoculation and disease occurrence. Increasing volumes of inocula (0.2, 0.5, 1, and 2 ml of a mixture of hyphae-fragment and 2?×?10⁶ conidia/ml suspension) caused more severe infections, and small differences were also observed for the different inoculation sites at the base, apex and mid-point of rice panicles. The optimum inoculation condition was 1–2 ml inoculum injected into the mid-point of rice panicles. Spikelet samples were collected as the disease progressed and observed by confocal laser scanning microscopy and scanning electron microscopy. The images collected showed that the primary site of U. virens colonization was at the base of the filaments with the inner spikelets becoming infected by hyphae at 24 h post inoculation (hpi). The accumulation of hyphae reached its highest level at 168 hpi, before the rice heading stage, as the infection extended upward from basal filaments to the anther apex, and then enclosed all the floral organs to produce a velvety smut ball.     

Studies on the colonization of axenically grown tomato plants by a GFP-tagged strain of Clavibacter michiganensis subsp. michiganensis

In this study, colonization and disease development of axenically-grown tomato plants by Clavibacter michiganensis subsp. michiganensis (Cmm), the causative agent of bacterial wilt and canker, was investigated. For this, a spontaneous rifampicin resistant strain of Cmm was tagged with a marker that expressed a green fluorescent protein (GFP) in a stable way and which possessed a similar virulence to the parental strain. In vitro plants were drop-inoculated at the stem base and the population dynamics was determined by dilution pour-plating in a selective medium. At 3 h after inoculation, Cmm was already present in low densities in roots, stems and leaves. At 16 dpi, Cmm was found throughout the entire plant in high densities of ca. 10¹⁰ cfu g^?1. Symptoms developed in the in vitro plants typical for Cmm, such as canker, wilting and growth reduction. The presence of Cmm in vascular and parenchymatic tissue of in vitro tomato plants was confirmed by epifluorescence stereo- and confocal laser scanning microscopy. This study showed that in vitro tomato plants can be effectively used for detailed studies on interactions between Cmm and its host, in particular if a GFP-tagged strain of the pathogen is used.     

Role of earhead bug (leptocorisa acuta) feeding on sheath rot disease caused bysarocladium oryzae inoryza sativa in India

A study was conducted to determine the role of the earhead bug (Leptocorisa acuta Thumb. [Heteroptera: Alydidae]) in the manifestation of sheath rot disease caused bySarocladium oryzae (Sawada) W. Gams & D. Hawksw. [syn.Acrocylindrium oryzae (Sawada)], with the aim of formulating effective control measures against the disease. In greenhouse studies, rice plants (Oryza sativa, cv. IR 20) exhibited typical sheath rot within 12 days whenS. oryzae spore suspensions (5 x 10⁷ conidia/ml) were sprayed on earhead bug-infested plants (5 bugs/tiller). In a comparison of various inoculation methods, swabbing a spore suspension (using a camel?s hair brash) on the sheath surface of earhead bug-infested plants, and inserting a single-grain culture inside the sheath of infested plants, produced the greatest disease severity within 18 days of inoculation. The results demonstrated (a) the role of the earhead bug in sheath rot disease manifestation and (b) that rice genotypes can be screened effectively and rapidly in the greenhouse for resistance to the disease, using the described inoculation techniques.     

Possibilities of biological and chemical control of Verticillium wilt in pepper

In pathogen populations in Serbia, the incidence, pathogenic and morphological characters ofVerticillium spp. were studied. Biological and chemical control ofVerticillium was investigated in pepper plants (Capsicum annuum L. cv. ‘Soroksari’) with the biofungicide Polyversum^® (Pythium oligandrum) and the conventional fungicides benomyl and propamocarb-hydrochloride. On the basis of macroscopic and microscopic characters of the isolates originating from eight localities in Serbia, it was established that they apparently belong to the speciesVerticillum dahliae. The isolates differed in their pathogenic characters. However, all of them caused marked wilting symptoms on pepper plants 40 days after inoculation, conducted when there were more than nine fully developed leaves on the primary stem. The fungicides were applied either before or after inoculation. Benomyl was the most efficient fungicide in wilt control (88.2% when applied after inoculation and 94.6% when applied before inoculation). Polyversum proved more efficient (66.6%) when applied before rather than after inoculation. Propamocarb-hydrochloride provided sufficient Verticillium wilt control; its efficacy and that of Polyversum were similar, and less efficient than benomyl, but still significantly different from the disease control.     

Metabolic profiling in the roots of coffee plants exposed to the coffee root-knot nematode, Meloidogyne exigua

To understand the effect of nematode Meloidogyne exigua infestation on coffee plants, resistant and susceptible coffee seedlings were inoculated with second-stage juveniles of M. exigua, and root metabolites were studied for four time intervals at 0, 24, 48 and 96 h. During this important period for parasite establishment, the concentrations of phenols, carbohydrates, amino acids and alkaloids in the roots were measured, and hydrogen nuclear magnetic resonance spectra of the root extracts were used to identify and quantify other metabolites. One of the most striking changes was the concentration of fumaric acid on resistant plants, which varied from 59 μg g(of root)^?1 to 138 μg g(of root)^?1 during the first 24 h of the nematode inoculation. The same level of variation was observed much later (96 h) in susceptible plants. Similarly, formic and quinic acid concentrations increased more rapidly in the resistant plants compared to the susceptibles. Sucrose concentrations increased to 370 % in the first 48 h in the resistant plants but showed no significant variation in the susceptible plant. Besides, the concentration of alkaloids was much higher at 24 and 48 h in the susceptibles compared to the resistant plants. These results suggest that the higher production of sucrose as well as formic, fumaric and quinic acids, and the lower production of alkaloids by the resistant cultivar in the first 48 h after the nematode inoculation are associated with the resistance of coffee plants to M. exigua.     

Risks for infection of strawberry plants with an aerosolized inoculum of <Emphasis Type="Italic">Xanthomonas fragariae</Emphasis>

Xanthomonas fragariae is the causative agent of angular leaf spot of strawberry, a quarantine organism in plant propagation material in the European Union. Field experiments were conducted to assess the risks for infection of strawberry plants through dispersal of an aerosolized inoculum. In practice, pathogen aerosols can be formed during mowing of an infected crop or by water splashing on symptomatic plants during overhead irrigation or rain. In our experiments, aerosols were generated by spraying suspensions of X. fragariae with a density of 10⁸ cfu ml^?1 or water under pressure vertically up into the air. In strawberry plants (cv Elsanta) placed at 1.3, 5 and 10 m distance downwind from the spray boom, infections were found, as evidenced with a combination of dilution–plating and molecular techniques, but more frequently in plants wetted prior to inoculation than in plants kept dry. A logarithmic decrease in infection incidence was found with the distance to the inoculum source. Symptomatic plants were found up to 5 m distance from the inoculum source. No infected plants were found in plants placed 4 m upwind or treated with water. In glasshouse studies, it was shown that under conditions favorable for disease development, spray-inoculation of strawberry plants with estimated densities of X. fragariae as low as 2000 cfu per plant were able to cause symptoms both in cv Elsanta and cv Sonata. Results indicate that there is a considerable risk on infections of strawberry plants exposed to aerosolized inoculum.     

A method of inoculating barley seedlings with Ramularia collo‐cygni

A method of inoculating seedlings with the fungus Ramularia collo-cygni, the causal agent of ramularia leaf spot (RLS), an increasingly important problem in barley in Europe and elsewhere, is described. Symptoms of RLS similar to those found in the field were reproduced on seedlings and the fungus was re-isolated from them, fulfilling the third and fourth of Koch's postulates. The method was similar to one used for the related fungus, Mycosphaerella graminicola (anamorph Septoria tritici), a pathogen of wheat. Briefly, plants were sprayed with a suspension of R. collo-cygni mycelium fragments, incubated at 15°C, first in darkness for 48 h then in a 16-h-light/8-h-dark cycle. Disease levels reached saturation when plants were sprayed to runoff with a suspension of 480 cm² of mycelium, scraped from the entire surface of 7·5 Petri dishes (9 cm diameter) and sieved, in 50 mL water. Growth of seedlings in high light intensity (900 µmol m⁻² s⁻¹, 16-h daylength) before inoculation increased disease symptoms, but reduced disease when applied after inoculation. In contrast to M. graminicola, near-ultraviolet light after inoculation reduced symptom development. It is proposed that for the full development of RLS, plants should be grown in a stressful environment before inoculation. Nine barley lines were assessed for their resistance to RLS as seedlings and a subset were tested in field trials with natural infection by R. collo-cygni. There was cultivar-by-isolate interaction in the amount of RLS symptoms on seedlings. RLS levels on adult plants in field plots were correlated with RLS scores on seedlings formed by one isolate but not the other.     

Ulvan effect on conidial germination and appressoria formation of Colletotrichum gloeosporioides

Ulvan is an algal polysaccharide known for its ability to induce resistance to plant diseases such as the Glomerella leaf spot of apple caused by Colletotrichum gloeosporiodes. This study was aimed at investigating microscopically, in tests in vitro and in vivo, whether ulvan interferes in the development of pre-infective structures of C. gloeosporioides. Conidial germination and appressoria formation were monitored hourly on agar and cellophane, and at 48 h on water- and ulvan-treated susceptible as well as resistant apple leaves. Amendment of agar with ulvan (10 mg ml^?1) enhanced the germination and resulted in longer germ tubes at 7 h of incubation. On cellophane it significantly delayed appressoria formation up to 8 h, but later after 14 h increased the number of appressoria per conidium. Spraying of susceptible leaves with ulvan 6 days before inoculation decreased disease severity by 50%. This was associated with inhibition of appressoria formation and stimulus in growth of germ tubes, without interfering with conidial germination, when compared with both water-treated control and resistant plants. Appressorium formation occurred preferentially on anticlinal walls of epidermal cells and its location was not influenced by host resistance or by ulvan treatment. This study suggests a new mode of action for ulvan interfering with appressorium formation that could protect apple plants against C. gloeosporioides infection.     

In vitro andin vivo evaluation of fungicides for control ofStemphylium botryosum F.lactucae on lettuce leaves

Maneb was the most efficient chemical in inhibiting germination ofStemphylium botryosum f. lactucae sporesin vitro. At 0.1 ppm it inhibited germination of all spores in the original population. Growth on PDA was markedly inhibited by 100 ppm daconil or maneb, withca. 90-95% spore mortality. The inhibitory effects of chemicals at sublethal doses on the rate of colony growth resulted both in retarded radial growth and in prolonged incubation period in culture. Maneb at 100 ppm and daconil at 10⁴ ppm had an inhibitory effect on inoculated leaf discs only when applied immediately after inoculation, whereas sodium dimethyldithiocarbamate (SDMC) inhibited fungal development at 10⁴ ppm both immediately after inoculation and 24 h later. SDMC kept the tissue very fresh during the experimental period. Fungal development in inoculated lettuce heads was prevented by 10³ ppm maneb or SDMC when applied before inoculation, but not when applied 24 h after inoculation, although inin vitro tests germinating spores were more sensitive to maneb than non-germinating ones.     

内生放线菌A-1对苹果果实轮纹病的防效及防御性酶活性的影响

为揭示内生放线菌A-1对苹果果实轮纹病的防效及防病机制,采用平板法和喷雾处理刺伤接种法,测定了其对苹果轮纹病菌的抑制作用及果实内防御性酶活性的影响。结果表明：菌株A-1能显著抑制轮纹病菌菌丝生长,10⁴ CFU/mL菌悬液的抑制率达90%以上;喷施10⁷ CFU/mL A-1菌悬液后,间隔12 h以上接种的各处理,3 d和7 d时防效分别为91.79%~95.67%和77.41%~94.00%,均与对照农药苯醚甲环唑相当。喷施10⁷ CFU/mL A-1菌悬液后接种或不接种轮纹病菌的处理,果实内过氧化物酶、过氧化氢酶、多酚氧化酶和苯丙氨酸解氨酶活性显著升高,且均高于只接种轮纹病菌的处理;喷施A-1菌悬液后接种病原菌的处理酶活性增加最显著,其峰值是对照的2.30~11.00倍。表明内生放线菌A-1可通过产生拮抗物质、提高寄主防御性酶活性等机制实现对苹果轮纹病的有效防治。     

Accumulation of phytoalexins in susceptible and resistant near-isogenic lines of tomato infected with Verticillium albo-atrum or Fusarium oxysporum f.sp. lycopersici

The time course of accumulation of two phytoalexins, the terpenoid rishitin and the polyacetylene cis-tetradeca-6-ene-1,3-diyne-5,8-diol, was determined in near-isogenic susceptible and resistant tomato lines inoculated with either Verticillium albo-atrum or Fusarium oxysporum f.sp. lycopersici.Cultivars containing the Ve gene for verticillium wilt resistance accumulated phytoalexins at a rate similar to that in susceptible plants following stem inoculation with V. albo-atrum. Higher amounts of phytoalexins were isolated from susceptible than from resistant plants at 11 days after inoculation. Inoculum concentrations of 10⁵, 10⁶, 10⁷ and 10⁸ conidia ml⁻¹ had no differential effect on phytoalexin accumulation at 3 days after inoculation. Also, no differences were observed between fungal growth in susceptible and resistant cultivars during that period.A cultivar containing the I-1 gene for fusarium wilt resistance contained more rishitin than did susceptible plants at 2 and 3 days after inoculation with 10⁷ conidia of F. oxysporum f.sp. lycopersici ml⁻¹, but at 7 and 11 days after inoculation more rishitin had accumulated in the susceptible plants.No difference was observed between the rate of accumulation of phytoalexin in stem segments from resistant and susceptible plants inoculated by vacuum-infiltration.To estimate the concentration of phytoalexins in the xylem fluid, sap was expressed from vascular tissue and amounts of phytoalexins were determined in the sap and in the expressed tissue. Less than 5% of the phytoalexins present in stem segments was recovered from the sap, indicating that their concentration in the xylem fluid may be relatively low.The role of phytoalexins in resistance to verticillium and fusarium wilt is discussed.     

Direct antifungal activity of tiadinil,a systemic acquired resistance inducer,and thymol formulations on <Emphasis Type="Italic">Stagonosporopsis citrulli</Emphasis> and control of watermelon gummy stem blight

This study evaluated the direct antifungal activity of tiadinil [N-(3-chloro-4-methylphenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide], a systemic acquired resistance (SAR) inducer and two formulations of thymol (thymol I and thymol II) against Stagonosporopsis citrulli, the causal agent of gummy stem blight (GSB) disease of watermelon. Tiadinil, thymol I and thymol II completely inhibited the mycelial growth of S. citrulli in vitro at ≥?100 ppm. Conidial germination and germ tube elongation were completely inhibited by tiadinil at ≥?2000 ppm and by thymol-based formulations at ≥?100 ppm. A single foliar application of tiadinil at ≥?10 ppm or a single application of thymol I and II at ≥?1 ppm, 48 h before or after pathogen inoculation, significantly reduced disease severity of watermelon seedlings inoculated with 10⁵/ml conidial suspension of S. citrulli, compared to respective nontreated controls. Plants treated with foliar application of tiadinil at ≥?1000 ppm before pathogen inoculation had significantly lower disease severity than plants that received an equivalent drench application. The efficacy of foliar application of tiadinil was affected by concentration and frequency of application. The study suggests direct antifungal activity of tiadinil, indicating a new mode of action of tiadinil against GSB disease of watermelon. The study also demonstrated direct antifungal action of thymol, a formulated active compound of essential oils, against S. citrulli and GSB disease of watermelon.     

Avirulent strain of Colletotrichum induces a systemic resistance in strawberry

Strawberry plants exposed to an avirulent isolate of Colletotrichum fragariae acquired strong resistance against a virulent strain of C. acutatum. Biochemical, morphological and molecular markers indicated that the strong defence response was associated with an oxidative burst and a transient accumulation of salicylic acid (SA). A maximum accumulation of H₂O₂ and O₂ ^? was observed 8 h after inoculation (hai), callose was detected 48 hai, and a peak of SA was observed 48 hai. Biochemical and phytopathogenic analyses carried out in non-treated tissues revealed that the defence response was systemic and remained fully active 60 days after the first inoculation. Experiments also showed that the resistance acquired by mother plants after the inoculation with the avirulent isolate could be passed to daughter plants through runners. Further characterization of the induced systemic resistance showed that the resistance was not only effective against a virulent strain of C. acutatum but also against Botrytis cinerea.     

Inhibition of Tomato Root Growth in Culture by aVerticillium Dahliae Phytotoxin

To assess the relationship betweenVerticilliun dahliae toxin and the stunting effect in V.dahliae-infected plants, an assay was developed based on the inhibition of elongation of cultured tomato roots. The toxin inhibited growth of roots and development of secondary roots in disease-susceptible plants, tomato cv. ’Hosen-Eilon’(veve) and eggplant, whereas root growth was enhanced in tomato cv. ’VF-134’, which carries theVe gene for resistance, and in a non-host plant, onion. The toxin also inhibited the incorporation of [¹⁴C]-glutamic acid and [¹⁴C]-uridine into TCA-precipitable material in susceptible but not in resistant tomato roots. Cells from toxin-treated susceptible roots were smaller than those from resistant roots, suggesting that the toxin impeded cell growth rather than cell division. Since toxin-induced stunting cannot be detected using leaf bioassays, we suggest that the root inhibition assay can be used as a reliable tool for Verticillium disease tolerance screening studies with theV. dahliae toxin as probe.     

The occurrence of rust disease,and biochemical and physiological responses on <Emphasis Type="Italic">Apocynum venetum</Emphasis> plants grown at four soil water contents,following inoculation with <Emphasis Type="Italic">Melampsora apocyni</Emphasis>

We studied the development of rust disease, and biochemical and physiological responses, on Apocynum venetum plants inoculation with Melampsora apocyni that were growing in a greenhouse at four relative soil water contents. The soil conditions were 25% (severe drought), 50% (mild drought), 75% (optimal) and 100% (waterlogging) relative soil water content. Plants exposed to drought and waterlogging stress had a lower number of open stomata before inoculation, corresponding with the disease index on the 10th day after inoculation being lower than that of the optimal soil water condition. Inoculated plants exposed to severe and mild drought stress had a gradually enhanced resistance to the rust disease from the 10th day after inoculation, corresponding with the enhanced activity of polyphenol oxidase and phenylalanine ammonialyase. For the inoculated plants exposed to severe drought stress, hydrogen peroxide always remained at the highest level for any treatment, and they had a rapidly enhanced activity of peroxidase, two factors that were associated with suppression of disease development. A. venetum plants exposed to double stress of waterlogging and disease had a high activity of peroxidase that not only removed reactive oxygen to prevent or reduce cell injury but also enhanced resistance to the rust disease. In addition, a rapidly enhanced activity of phenylalanine ammonialyase in the waterlogging condition from the 25th day after inoculation was also associated with an enhanced resistance to the rust disease. Drought and waterlogging stress had a negative effect on the leaf photosystem, and in particular, there was a significant decrease in the net photosynthetic rate with an increase in the duration and degree of drought stress, and this lead to a statistically significant decrease in the weight of aboveground tissue compared with that of plants under optimal soil water condition (P < 0.05). Inoculating with M. apocyni had a slight effect on photosynthesis of plants during early disease development, but the physiological function of diseased leaves under the drought stress was damaged more seriously than that of non-inoculated plants in later disease development, leading to a large reduction in the net photosynthetic rate. However, this reduction did not cause a statistically significant (P > 0.05) decrease in the weight of aboveground tissue compared with that of non-inoculated plants under drought stress.     

Cross protection provides evidence for race-specific avirulence factors inFusarium oxysporum

Simultaneous inoculation with races 1 and 2 of the vascular wilt pathogenFusarium oxysporumf.sp.lycopersiciprovided a high level of protection against race 2 in three tomato cultivars carrying resistance geneI, which confers resistance to race 1 but not race 2. However, simultaneous inoculation did not provide any protection in cultivars lacking this gene. Protection resulted in reduction and delay of wilt symptoms. Similarly, avirulent races ofF. oxysporumf.sp.melonisprotected muskmelon plants against virulent races of the sameforma specialis.A ratio 10:1 between spore concentrations of inducer and challenger organism gave the highest cross protection, but ratio 0.1:1 still provided significant disease reduction. Cross protection was also obtained when inoculation with the inducer organism was performed 6 or 12 h before inoculation with the challenger organism. Autoclaved spores of the inducer did not have any protective effect, indicating that living propagules were required to initiate protection. The results suggest the presence of a gene-for-gene interaction betweenF. oxysporumf.sp.lycopersici-tomato andF. oxysporumf.sp.melonis-muskmelon, in which cross protection against a virulent race is mediated by recognition of a specific elicitor from the avirulent race by the plant resistance gene product and by subsequent induction of the plant defense reaction.     

Development of an effective screening method for partial resistance to Alternaria brassicicola (dark leaf spot) in Brassica rapa

In order to develop a method to measure resistance to Alternaria brassicicola (cause of dark leaf spot disease) in Brassica rapa, the effects of inoculum concentration, leaf stage, leaf age and incubation temperature of inoculation on infection were studied under controlled conditions using several B. rapa genotypes. Three inoculation methods (cotyledon, detached leaf and seedling inoculation) were evaluated for this purpose. The detached leaf inoculation test was the most suitable for screening B. rapa genotypes because clear symptoms were observed on the leaves in less than 24 h, and there was a significant positive correlation between the results from the detached leaf inoculation test and the seedling inoculation test, an established method considered to yield reliable results. In addition, it was very easy to screen plants for resistance on a large scale and to maintain standard physical conditions using detached leaves. For successful infection, inoculum concentration should be adjusted to 5 × 10⁴ conidia ml⁻¹, and incubation temperature should be between 20 °C and 25 °C. The 3rd/4th true leaves from 30 day-old plants were optimal for inoculation. In a screening test using 52 cultivars of B. rapa, the detached leaf test effectively discriminated between various levels of partial resistance among cultivars. As a result, we identified two cultivars, viz Saori and Edononatsu, as highly resistant and five cultivars, viz Tokinashi Taisai, Yajima Kabu, Purara, Norin-F₁-Bekana and Tateiwa Kabu, as having borderline resistance.     

Studies into primary and secondary infection processes by Plasmodiophora brassicae on canola

The early stages of infection of canola roots by the clubroot pathogen Plasmodiophora brassicae were investigated. Inoculation with 1 × 10⁵ resting spores mL^?1 resulted in primary (root hair) infection at 12 h after inoculation (hai). Secondary (cortical) infection began to be observed at 72 hai. When inoculated onto plants at a concentration of 1 × 10⁴ mL^?1, secondary zoospores produced primary infections similar to those obtained with resting spores at a concentration of 1 × 10⁵ mL^?1. Secondary zoospores caused secondary infections earlier than resting spores. When the plants were inoculated with 1 × 10⁷ resting spores mL^?1, 2 days after being challenged with 1 × 10⁴ or 1 × 10⁵ resting spores mL^?1, secondary infections were observed on the very next day, which was earlier than the secondary infections resulting from inoculation with 1 × 10⁷ resting spores mL^?1 alone and more severe than those produced by inoculation with 1 × 10⁴ or 1 × 10⁵ resting spores mL^?1 alone. Compared with the single inoculations, secondary infections on plants that had received both inoculations remained at higher levels throughout a 7‐day time course. These data indicate that primary zoospores can directly cause secondary infection when the host is under primary infection, helping to understand the relationship and relative importance of the two infection stages of P. brassicae.