Plasmodiophora brassicae resting spore dynamics in clubroot resistant canola (Brassica napus) cropping systems

The soilborne pathogen Plasmodiophora brassicae, causal agent of clubroot of canola (Brassica napus), is difficult to manage due to the longevity of its resting spores, ability to produce large amounts of inoculum, and the lack of effective fungicides. The cropping of clubroot resistant (CR) canola cultivars is one of the few effective strategies for clubroot management. This study evaluated the impact of the cultivation of CR canola on P. brassicae resting spore concentrations in commercial cropping systems in Alberta, Canada. Soil was sampled pre-seeding and post-harvest at multiple georeferenced locations within 17 P. brassicae-infested fields over periods of up to 4 years in length. Resting spore concentrations were measured by quantitative PCR analysis, with a subset of samples also evaluated in greenhouse bioassays with a susceptible host. The cultivation of CR canola in soil with quantifiable levels of P. brassicae DNA resulted in increased inoculum loads. There was a notable lag in the release of inoculum after harvest, and quantifiable P. brassicae inoculum peaked in the year following cultivation of CR canola. Rotations that included a ≥2-year break from P. brassicae hosts resulted in significant declines in soil resting spore concentrations. A strong positive relationship was found between the bioassays and qPCR-based estimates of soil infestation. Results suggest that CR canola should not be used to reduce soil inoculum loads, and crop rotations in P. brassicae infested fields should include breaks of at least 2 years away from B. napus, otherwise the risk of selecting for virulent pathotypes may increase.     

Effect of temperature on primary infection by Plasmodiophora brassicae and initiation of clubroot symptoms

A study was conducted to assess the effect of temperature on infection and development of Plasmodiophora brassicae in root hairs of Shanghai pak choi (Brassica rapa subsp. chinensis) and on initiation of clubroot symptoms. Ten‐day‐old seedlings were grown in liquid‐sand culture, inoculated with resting spores and maintained in growth cabinets at 10, 15, 20, 25 and 30°C. Seedlings were harvested at 2‐day intervals, starting 2 days after inoculation (DAI) and continuing until swelling of the tap root was observed (maximum 28 days). Roots were assessed for root hair infection (RHI), stage of development of infection (primary plasmodia, zoosporangia, release of zoospores, secondary plasmodia), symptom development, and for clubroot severity at 24 DAI. Temperature affected every stage of clubroot development; RHI was highest and visual symptoms initiated earliest at 25°C, intermediate at 20 and 30°C, and lowest and latest at 15 and 10°C. Root hair infection was observed at every temperature, but clubroot symptoms developed only above 15°C. A substantial delay in the development of the pathogen was observed at 10 and 15°C. No symptoms were observed at 28 DAI in plants grown at 10°C. Swelling of the tap root was visible at 28 DAI in plants at 15°C, 14 DAI at 20 and 30°C, and 10 DAI at 25°C. These results support and explain the observation in companion studies that cool temperatures result in slower development of clubroot symptoms in brassica crops.     

Effect of susceptible and resistant canola plants on Plasmodiophora brassicae resting spore populations in the soil

Clubroot, caused by Plasmodiophora brassicae, has become a serious threat to canola (Brassica napus) production in western Canada. Experiments were conducted to assess the effect of growing resistant and susceptible canola genotypes on P. brassicae soil resting spore populations under greenhouse, mini‐plot and field conditions. One crop of susceptible canola contributed 1·4 × 10⁸ spores mL^?1 soil in mini‐plot experiments, and 1 × 10¹⁰ spores g^?1 gall under field conditions. Repeated cropping of susceptible canola resulted in greater gall mass compared to resistant canola lines. It also resulted in reduced plant height, increased clubroot severity in susceptible canola, and increased numbers of resting spores in the soil mix.     

Potential biological control of clubroot on canola and crucifer vegetable crops

Clubroot caused by Plasmodiophora brassicae is an emerging threat to canola (Brassica napus) production in western Canada, and a serious disease on crucifer vegetable crops in eastern Canada. In this study, seven biological control agents and two fungicides were evaluated as soil drenches or seed treatments for control of clubroot. Under growth cabinet conditions, a soil‐drench application of formulated biocontrol agents Bacillus subtilis and Gliocladium catenulatum reduced clubroot severity by more than 80% relative to pathogen‐inoculated controls on a highly susceptible canola cultivar. This efficacy was similar to that of the fungicides fluazinam and cyazofamid. Under high disease pressure in greenhouse conditions, the biocontrol agents were less effective than the fungicides. Additionally, all of the treatments delivered as a seed coating were less effective than the soil drench. In field trials conducted in 2009, different treatments consisting of a commercial formulation of B. subtilis, G. catenulatum, fluazinam or cyazofamid were applied as an in‐furrow drench at 500 L ha^?1 water volume to one susceptible and one resistant cultivar at two sites seeded to canola in Alberta and one site of Chinese cabbage in Ontario. There was no substantial impact on the susceptible canola cultivar, but all of the treatments reduced clubroot on the susceptible cultivar of Chinese cabbage, lowering disease severity by 54–84%. There was a period of 4 weeks without rain after the canola was seeded, which likely contributed to the low treatment efficacy on canola. Under growth cabinet conditions, fluazinam and B. subtilis products became substantially less effective after 2 weeks in a dry soil, but cyazofamid retained its efficacy for at least 4 weeks.     

A molecular marker for the specific detection of new pathotype 5‐like strains of Plasmodiophora brassicae in canola

Clubroot of crucifers, caused by Plasmodiophora brassicae, is managed in canola (Brassica napus) by the deployment of resistant cultivars. Recently, however, new strains of P. brassicae have been detected in Alberta, Canada, that can overcome this resistance. Some of these strains are classified as pathotype 5 on the differential system of Williams, but are distinguished by their ability to overcome host resistance. In order to expedite the identification of these new pathotype 5‐like strains, three primer sets were developed based on the 18S‐ITS region of the pathogen. With primers P5XF3 and P5XR3, a 127 bp product was amplified from all new pathotype 5‐like strains following optimized PCR analysis. A TaqMan probe‐based quantitative assay was also developed. These protocols could be used to detect as little as 0.5 pg P. brassicae DNA, and as few as 10⁴ mL^?1 pathogen resting spores; infection of host tissues could be detected as soon as 4 days after inoculation. The PCR and qPCR assays described in this study represent useful tools for the rapid and reliable diagnosis and quantification of new pathotype 5‐like strains of P. brassicae.     

Virulence and inoculum density‐dependent interactions between clubroot resistant canola (Brassica napus) and Plasmodiophora brassicae

To mitigate the impact and dissemination of clubroot in western Canada, canola (Brassica napus) producers have relied on clubroot resistance traits. However, in 2013 and 2014, new strains of the clubroot pathogen, Plasmodiophora brassicae, emerged that are virulent on most clubroot‐resistant (CR) canola genotypes. Novel strains of the pathogen were inoculated onto two susceptible canola cultivars, one resistant line and six CR cultivars. Although all cultivars/lines showed a susceptible response to inoculation with the new strains of P. brassicae, the severity of disease reaction, root hair infection rates and the amount of P. brassicae DNA present in each canola genotype varied depending on the strain. In addition, the effect of inoculum density on disease severity and gall formation was recorded for one of these new strains on a universally susceptible Chinese cabbage cultivar and one susceptible and 10 resistant canola genotypes. Although root galls were observed at an inoculum density of 10³ spores per mL of soil, clear differentiation of susceptible and resistant reactions among canola cultivars/lines was not observed until the inoculum density reached 10⁵ spores mL^?1. At a spore density of 10⁶ spores mL^?1 and above, all cultivars/lines developed susceptible reactions, although there was some differentiation in the degree of reaction. This study shows the potential to develop a unique disease profile for emergent clubroot pathotypes and shows a useful range of spore densities at which to study new P. brassicae strains.     

Efficacy of Vapam fumigant against clubroot (Plasmodiophora brassicae) of canola

Clubroot, caused by Plasmodiophora brassicae, has become a serious threat to canola (Brassica napus) production in western Canada. Experiments were conducted under greenhouse and field conditions to assess the effect of Vapam fumigant (dithiocarbamate; sodium N‐methyldithiocarbamate) on primary and secondary infection by P. brassicae, clubroot severity, and growth parameters in canola. Preliminary trials showed a 12–16‐fold reduction in primary and secondary infection and clubroot severity at all of the Vapam application rates (0·4–1·6 mL L^?1 soil) assessed. Vapam was also found to be effective in reducing clubroot severity and improving seed yield of canola under field conditions. Application of Vapam at soil moisture levels in the range of 10–30% (v:v) had a large effect on both disease severity and infection rates and plant growth parameters. The results suggest that Vapam can effectively reduce clubroot severity and may be useful for the treatment of transplant propagation beds in brassica vegetable production, and for the containment of small, localized clubroot infestations in commercial canola crops.     

Growth and movement of secondary plasmodia of Plasmodiophora brassicae in turnip suspension-culture cells

Growth of secondary plasmodia of the clubroot pathogen Plasmodiophora brassicae was studied in dual culture of P. brassicae and turnip suspension cells. Suspension culture of P. brassicae -infected turnip cells was achieved by using P. brassicae -infected callus in Murashige and Skoog medium supplemented with 0·1 mg 2,4-D L⁻¹ and 0·02 mg kinetin L⁻¹. The shape of secondary plasmodia in suspension cells was spherical-to-subspherical. A few young plasmodia divided and became numerous spherical, small plasmodia which eventually formed a plasmodial cluster. The plasmodia fused and became vegetative plasmodia. Infected cells were significantly larger than noninfected cells. Secondary plasmodia moved within transformed turnip suspension host cells by cytoplasmic streaming of the host cells. Secondary plasmodia divided in synchrony with the transformed turnip cells.     

Suppression of clubroot disease under neutral pH caused by inhibition of spore germination of Plasmodiophora brassicae in the rhizosphere

To elucidate the mechanism of clubroot suppression under neutral soil pH, a highly reproducible germination assay system under soil culture conditions was designed based on the hypothesis that germinated spores of Plasmodiophora brassicae could be identified by the absence of a nucleus (i.e. having released a zoospore to infect a root hair of the host plant). Brassica rapa var. perviridis seedlings were inoculated with a spore suspension of P. brassicae at a rate of 2·0 × 10⁶ spores g⁻¹ soil and grown in a growth chamber for 7 days. The spores were recovered from rhizosphere and non-rhizosphere soils and stained with both Fluorescent Brightener 28 (cell-wall-specific) and SYTO 82 orange fluorescent nucleic-acid stain (nucleus-specific stain). Total numbers of spores were counted under UV-excitation, and spores with a nucleus that fluoresced orange under G-excitation were counted. The significant increase in the percentage of spores without a nucleus (germinated spores) in the rhizosphere after 7 days' cultivation and the correlation with root-hair infections validated the assay system. Applications of calcium-rich compost or calcium carbonate to neutralize the soil significantly reduced the percentage of germinated spores in the rhizosphere, as well as the number of root-hair infections. The present study provides direct evidence that the inhibition of spore germination is the primary cause of disease suppression under neutral soil pH.     

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.     

Influence of cultivar resistance and inoculum density on root hair infection of canola (Brassica napus) by Plasmodiophora brassicae

The impact of cultivar resistance and inoculum density on the incidence of primary infection of canola root hairs by Plasmodiophora brassicae, the causal agent of clubroot, was assessed by microscopy. The incidence of root hair infection in both a resistant and a susceptible cultivar increased with increasing inoculum density, but was two‐ to threefold higher in the susceptible cultivar; the relationship between root hair infection and inoculum density was also substantially stronger and more consistent in the susceptible cultivar. In the susceptible cultivar, the root hair infection rate peaked between 6 and 8 days after sowing and then declined. In the resistant cultivar, it increased over the 14‐day duration of each study. It appears that examination of root hair infection by microscopy in a bait crop of susceptible canola could serve as a useful tool for estimating P. brassicae inoculum levels in soil. In a separate trial, the relationship between inoculum density and clubroot severity, plant growth parameters, and seed yield was assessed under greenhouse conditions. Inoculum density in the susceptible genotype was strongly and positively correlated with clubroot severity and negatively correlated with plant height and seed yield. In addition, a single cropping cycle of the susceptible cultivar contributed significantly higher levels of resting spores to the soil in a greenhouse test than did a cycle of the resistant cultivar, as assessed by quantitative PCR and microscope analysis.     

Assessment of the impact of resistant and susceptible canola on Plasmodiophora brassicae inoculum potential

The impact on clubroot severity of growing susceptible canola or mixtures of resistant and susceptible canola genotypes was examined. Bioassays revealed greater clubroot severity and incidence, and reduced plant height, where 100% of a susceptible cultivar had been grown. A higher proportion of susceptible plants within a resistant canola crop increased root hair and secondary infections. Regression analysis of root hair infection and the amount of Plasmodiophora brassicae DNA (as determined by quantitative PCR) revealed strong linear relationships between the two parameters. The linear relationships between root hair infection and P. brassicae DNA were stronger for the resistant cultivar than for the susceptible cultivar when regression analysis was conducted by cultivar over the sampling dates. In conclusion, the cropping of a resistant cultivar reduced clubroot severity, while the presence of susceptible volunteer canola increased inoculum potential. Quantitative PCR was a reliable tool for the quantification of root hair infection.     

不同杀菌剂对油菜根肿病的防效及对油菜产量损失的影响

本研究使用5种杀菌剂和1种微肥于油菜苗期2~3片真叶期进行灌根处理,以传统的石灰处理方法比较,评价不同处理对油菜根肿病的防治效果和对油菜产量损失的影响。5种杀菌剂处理、微肥以及石灰处理均对油菜根肿病有较好的防效,其中50%多菌灵可湿性粉剂防效达90.82%;从产量损失方面比较,10%氰霜唑悬浮剂、50%多菌灵可湿性粉剂,75%百菌清可湿性粉剂和60%硫磺·敌磺钠可湿性粉剂处理可显著减少根肿病引起的产量损失;50%氟啶胺悬浮剂处理较对照增产不显著,而且对油菜生长有抑制作用;油菜根肿病的病情指数与油菜产量呈显著的负相关性,相关系数r=-0.796 2,油菜产量(y)与病情指数(x)的回归方程为y=-32.65x+2 372。     

In‐field distribution of Plasmodiophora brassicae measured using quantitative real‐time PCR

A protocol using real‐time polymerase chain reaction (PCR) for the direct detection and quantification of Plasmodiophora brassicae in soil samples was developed and used on naturally and artificially infested soil samples containing different concentrations of P. brassicae. Species‐specific primers and a TaqMan fluorogenic probe were designed to amplify a small region of P. brassicae ribosomal DNA. Total genomic DNA was extracted and purified from soil samples using commercial kits. The amount of pathogen DNA was quantified using a standard curve generated by including reactions containing different amounts of a plasmid carrying the P. brassicae target sequence. The PCR assay was optimized to give high amplification efficiency and three to four copies of the target DNA sequence were detected. Regression analysis showed that the standard curve was linear over at least six orders of magnitude (R² > 0·99) and that the amplification efficiency was >92%. The detection limit in soil samples corresponded to 500 resting spores g^?1 soil. The intersample reproducibility was similar to, or higher than, that of assays for other pathogens quantified in soil samples. Bait plants were used to validate the real‐time PCR assay. The protocol developed was used to investigate the spatial distribution of P. brassicae DNA in different fields and a significant difference was found between in‐field sampling points. The reproducibility of soil sampling was evaluated and showed no significant differences for samples with low levels of inoculum, whereas at higher levels differences occurred. Indicator kriging was used for mapping the probability of detecting P. brassicae within a 2‐ha area of a field. A threshold level of 5 fg plasmid DNA g^?1 soil, corresponding to approximately 3 × 10³P. brassicae resting spores g^?1 soil, is suggested for growing resistant cultivars. The results provide a robust and reliable technique for predicting the risk of disease development and for assessing the distribution of disease within fields.     

Variation in pathotypes and virulence of Plasmodiophora brassicae populations in Germany

Between 2012 and 2015, 49 new clubroot‐infested fields were identified in 12 German federal states. Clubroot disease incidence varied within these fields from 22% to 92%. Field information revealed that in 85% of fields, oilseed rape was grown in rotation once every 2 or 3 years. Frequency of OSR in the rotation was significantly correlated with the incidence and prevalence of clubroot disease. The disease was detected in fields with soil pH ranging from 5.1 to 8.3, and a significant negative correlation was found between soil pH and the disease incidence of infested fields. Furthermore, more cases of disease and severe incidences were observed in sandy loam and loamy sand as compared with other soil types. Pathotype classification of the 49 Plasmodiophora brassicae populations was conducted on two differential sets, the European Clubroot Differential set and the set of Somé (1996). Additionally, the degree of virulence of the collected isolates was analysed on the clubroot‐resistant oilseed rape cv. Mendel. The results showed variation in pathotype distribution in different regions in Germany. The majority of isolates according to Somé were pathotypes 1 and 3, respectively, with pathotypes 2 and 5 in the minority. Detailed classification according to Buczacki showed the dominance of 16/31/31, 16/14/30 and 16/14/31 populations among 20 distinct virulence patterns of collected isolates. From all populations tested for virulence on cv. Mendel, 15 isolates were found to be moderately or highly virulent. These virulent populations were not restricted to a small geographical area in the country.     

Effect of temperature on cortical infection by Plasmodiophora brassicae and clubroot severity

A study was conducted to assess the effect of temperature on infection and development of Plasmodiophora brassicae in the root cortex of Shanghai pak choy (Brassica rapa subsp. chinensis) and on subsequent clubroot severity. Ten-day-old seedlings were grown individually, inoculated with resting spores, and maintained in growth cabinets at 10, 15, 20, 25, and 30?C. Seedlings were harvested at 2-day intervals, starting 8 days after inoculation (DAI) and continuing until 42 DAI. Roots were assessed at 4-day intervals for the incidence of cortical infection and stage of infection (young plasmodia, mature plasmodia, and resting spores), at 2-day intervals for symptom development and clubroot severity, and at 8-day intervals for the number of spores per gram of gall. Temperature affected every stage of clubroot development. Cortical infection was highest and symptoms were observed earliest at 25?C, intermediate at 20 and 30?C, and lowest and latest at 15?C. No cortical infection or symptoms were observed at 42 DAI in plants grown at 10?C. A substantial delay in the development of the pathogen was observed at 15?C. Resting spores were first observed at 38 DAI in plants at 15?C, 26 DAI at 20 and 30?C, and 22 DAI at 25?C. The yield of resting spores from galls was higher in galls that developed at 20 to 30?C than those that developed at 15?C over 42 days of assessment. These results support the observation in companion studies that cool temperatures result in slower development of clubroot symptoms in brassica crops, and demonstrate that the temperature has a consistent pattern of effect throughout the life cycle of the pathogen.     

Direct evidence of surface infestation of seeds and tubers by Plasmodiophora brassicae and quantification of spore loads

Using quantitative PCR, DNA of Plasmodiophora brassicae, the causal agent of clubroot, was detected and quantified on canola, pea and wheat seeds, as well as on potato tubers, all harvested from clubroot‐infested fields in Alberta, Canada. Quantifiable levels of infestation were found on seven of the 46 samples analysed, and ranged from <1·0 × 10³ to 3·4 × 10⁴ resting spores per 10 g seeds; the vast majority (80–100%) of resting spores on these samples were viable, as determined by Evan’s blue vital staining. However, the levels of infestation found were generally lower than that required to cause consistent clubroot symptoms in greenhouse plant bioassays. While the occurrence of P. brassicae resting spores on seeds and tubers harvested from clubroot‐infested fields suggests that seedborne dissemination of this pathogen is possible, practices such as commercial seed cleaning may be sufficient to effectively mitigate this risk.     

Reducing the build‐up of Plasmodiophora brassicae inoculum by early management of oilseed rape volunteers

Clubroot of oilseed rape (OSR), caused by Plasmodiophora brassicae, is a disease of increasing economic importance worldwide. Previous studies indicated that OSR volunteers, Brassica crops and weeds play a critical role in the predisposition of the disease. To determine the effect of timing of foliar application of the herbicide glyphosate or mechanical destruction of OSR volunteers in reduction of clubroot severity and resting spore production, a series of studies was conducted under controlled conditions with a susceptible OSR cultivar and an isolate of P. brassicae. Plants were inoculated by injecting a spore suspension beside the root hairs at growth stage 11–12 (BBCH scale) and were terminated at 7 (early) or 21 (late) days post‐inoculation (dpi). Under controlled conditions, the first symptoms on roots were observed as early as 7 dpi. The early application of glyphosate as well as early mechanical destruction resulted in significant (P ≤ 0.05) reduction in the development of clubroot symptoms, root fresh weight and the number of resting spores?g root. Furthermore, the effect of volunteer management on clubroot severity in the succeeding OSR was studied by inoculating plants with the resting spores obtained from treated clubbed roots. Inoculated OSR exhibited root clubs similar to the initial symptoms after 35 dpi. Plants that were inoculated with spore suspension from early treated roots resulted in significant reductions in clubroot incidence and severity. Conversely, plants inoculated with the spore suspension from the late treated roots displayed levels of clubroot similar to the plants inoculated with the spore solutions of positive controls.     

土壤pH对芸薹根肿菌侵染及病害发生的影响

通过设置不同pH梯度,研究土壤pH对根肿菌侵染及病害发生的影响。结果表明:土壤酸性时病菌侵染速度快,碱性时慢,而强酸性和碱性土壤条件则抑制孢子萌发;pH为6.0时最有利于根肿菌休眠孢子萌发,萌发率最高,为53.96%;碱性条件可使初级原生质团变形凝结成球状,不能正常分裂或延迟形成游动孢子囊,从而不利于根肿菌侵染。白菜发病率与病情指数随pH升高,呈先上升后下降趋势。其中,pH为5.0时,发病率和病情指数最高,pH 7.0~8.0时发病轻。因此,适宜的偏酸性环境条件下,通过作用于病菌休眠孢子萌发和侵染,提高病害危害程度,而中性或碱性条件干扰该过程并降低病害发生。     

Reduction of clubroot (Plasmodiophora brassicae) formation in Arabidopsis thaliana after treatment with prohexadione‐calcium,an inhibitor of oxoglutaric acid‐dependent dioxygenases

Recently, flavonoids were shown to modulate the outcome of clubroot development in Arabidopsis thaliana after infection with the obligate biotrophic pathogen Plasmodiophora brassicae. Therefore, the development of clubroot disease was investigated in Arabidopsis after treatment with prohexadione‐calcium (ProCa), an inhibitor of ascorbic acid/2‐oxoglutaric acid‐dependent dioxygenases such as flavanone‐3‐hydroxylase. The treatment resulted in a reduction of the flavonols quercetin and kaempferol in clubroots, whereas the precursor naringenin highly accumulated. The root system of ProCa‐treated plants was better developed although galls were still visible. Thus, ProCa treatment resulted in reduced gall size. Flavonoids are thought to inhibit polar auxin transport by modulating auxin efflux carriers. It was investigated whether the auxin response might change as a consequence of the accumulation of naringenin in ProCa‐treated plants. In the areas of gall development an auxin response was indicated by the auxin‐responsive promoter DR5 coupled to the reporter β‐glucuronidase (GUS), whereas very little staining was found in healthy root parts. No differences in GUS activity were found between P. brassicae‐infected and ProCa‐treated plants, and plants only infected with P. brassicae, indicating that the effect of ProCa treatment on clubroot reduction is not via changes in auxin responses. As ProCa is also an inhibitor of late steps in gibberellin biosynthesis, a specific gibberellin biosynthesis inhibitor, chlormequatchloride (CCC), was tested on club development. However, CCC did not reduce disease symptoms, indicating that the observed reduced gall development was not because of gibberellin biosynthesis inhibition by ProCa.