Phenazines are Involved in Biocontrol of Pythium myriotylum on Cocoyam by Pseudomonas aeruginosa PNA1

Root rot of cocoyam (Xanthosoma sagittifolium) caused by Pythium myriotylum is the most devastating disease of this important tropical tuber crop with yield reductions of up to 90%. Bioassays were conducted in vitro and in sterile volcanic soil artificially infested with Pythium myriotylum, isolate CRPm, to test whether Pseudomonas aeruginosa PNA1 can control the cocoyam root rot disease. P. aeruginosa PNA1 (wild type) produces phenazine-1-carboxylic acid and phenazine-1-carboxamide (oxychlororaphin), while its tryptophan auxotrophic mutant FM13 is phenazine negative and secretes anthranilate in vitro. PNA1 and FM13 have previously been shown to control Pythium debaryanum and Pythium splendens on lettuce and bean. PNA1 and FM13 significantly inhibited growth of P. myriotylum in dual cultures, while their supernatants highly reduced mycelial dry weight in potato dextrose broth. However, in the presence of tissue culture derived cocoyam plantlets, only strain PNA1 strongly reduced root rot disease severity. Soil experiments involving strain PNA1 in comparison to phenazine-deficient mutants suggested that the biocontrol activity of PNA1 against P. myriotylum may involve phenazines. Phenazine involvement was further strengthened by the fact that FM13 fed with exogenous tryptophan (so that phenazine production is restored) significantly reduced disease severity on cocoyam. The efficiency of PNA1 to control P. myriotylum on cocoyam was significantly improved when the strain and the pathogen were allowed to interact for 24 h prior to transplanting cocoyam plantlets, while doubling the inoculum density of the pathogen negatively affected its efficiency.     

Characterization of resistance mechanisms activated by Trichoderma harzianum T39 and benzothiadiazole to downy mildew in different grapevine cultivars

Downy mildew, caused by Plasmopara viticola, is one of the most destructive diseases of grapevine and is controlled with intense application of chemical fungicides. Treatment with Trichoderma harzianum T39 (T39) or benzothiadiazole‐7‐carbothioic acid S‐methyl ester (BTH) has been previously shown to activate grapevine resistance to downy mildew and reduce disease symptoms in the Pinot noir cultivar. However, enhancement of plant resistance can be affected by several factors, including plant genotype. In order to further extend the use of resistance inducers against downy mildew, the physiological and molecular properties of T39‐ and BTH‐activated resistance in different cultivars of table and wine grapes were characterized under greenhouse conditions. T39 treatment reduced downy mildew symptoms, but the degree of efficacy differed significantly among grapevine cultivars. However, efficacy of BTH‐activated resistance was consistently high in the different cultivars. Expression profiles of defence‐related genes differed among cultivars in response to resistance inducers and to pathogen inoculation. T39 treatment enhanced the expression of defence‐related genes in the responsive cultivars, before and after P. viticola inoculation. A positive correlation between the efficacy of T39 and the expression level of defence‐related genes was found in Primitivo and Pinot noir plants, while different genes or more complex processes were probably activated in Sugraone and Negroamaro. The data reported here suggest that the use of a responsive cultivar is particularly important to maximize the efficacy of resistance inducers and new natural inducers should be explored for the less responsive cultivars.     

Induction of systemic resistance to<Emphasis Type="Italic">Colletotrichum lindemuthianum</Emphasis> in bean by a benzothiadiazole derivative and rhizobacteria

Plants have developed mechanisms to resist secondary infection upon inoculation with a necrotizing pathogen, chemical treatment as well as treatment with some non-pathogenic microorganisms such as rhizosphere bacteria. This phenomenon has been variously described as induced systemic resistance (ISR) or systemic acquired resistance. In the present study, the chemical benzo(1,2,3)thiadiazole-7-carbothioic acid-S-methyl ester (BTH, acibenzolar-S-methyl), and the rhizobacteriaPseudomonas aeruginosa KMPCH andP. fluorescens WCS417 were tested for their ability to induce resistance toColletotrichum lindemuthianum in susceptible and moderately resistant bean plants (Phaseolus vulgaris L.). BTH induced local and systemic resistance when bean leaves were immersed in 10⁻³ to 10⁻⁷ M BTH 3 days before the challenge inoculation. At a high concentration (10⁻³ M), BTH induced resistance of the same order as resistance induced by the pathogenC. lindemuthianum, although at this high concentration BTH appeared to be phytotoxic. Soil and seed treatment with 1 mg kg⁻¹ BTH protected beans against anthracnose. BTH-mediated induced resistance was effective in susceptible and moderately resistant plants.P. aeruginosa KMPCH induced resistance in bean againstC. lindemuthianum only in a moderately resistant interaction. KMPCH-567, a salicylic acid mutant of KMPCH, failed to induce resistance, indicating that salicylic acid is important for KMPCH to induce resistance in the bean—C. lindemuthianum system.P.fluorescens WCS417 could induce resistance toC. lindemuthianum in a susceptible and in moderately resistant interactions. http://www.phytoparasitica.org posting Jan. 16, 2002.     

Benzothiadiazole and BABA improve resistance to <Emphasis Type="Italic">Uromyces pisi</Emphasis> (Pers.) Wint. in <Emphasis Type="Italic">Pisum sativum</Emphasis> L. with an enhancement of enzymatic activities and total phenolic content

Benzothiadiazole (BTH) and DL-β-aminobutyric acid (BABA) induced systemic resistance was investigated in susceptible and resistant pea genotypes against Uromyces pisi. Resistance was characterized by reduced infection frequency mainly due to decreases in appressorium formation, stomatal penetration, growth of infection hyphae and haustorium formation. Changes in β-1,3-glucanase, chitinase, phenylalanine ammonia-lyase and peroxidase activities and in total phenolics content, demonstrate that U. pisi resistance is induced by BTH and BABA treatments at early and late stages of the fungal infection process, but that the chemicals operate via different mechanisms. In fact, our study showed that BTH treatment primed the activity of pathogenesis related-proteins such as β-1,3-glucanase, chitinase and peroxidase in both susceptible and resistant genotypes. On the other hand, BABA treatment did not increase the enzymatic activities in the studied genotypes, but significantly increased their total phenolic contents.     

Silicon amendment improves wheat defence against Fusarium graminearum and complements the control by fungicide of Fusarium head blight

A three-year field experiment with two wheat cultivars evaluated the effect of soil-applied silicon (Si), with and without fungicide spraying, on Fusarium head blight (FHB) control. Silicon treatment alone reduced FHB severity and the percentage of damaged wheat kernels, regardless of the cultivar. The best disease control was obtained for the cultivar with moderate disease resistance (MR), supplied with silicon and treated with fungicide during flowering. Silicon treatment alone promoted an increase in deoxynivalenol (DON) concentration in the disease-susceptible cultivar; however, in the MR cultivar, silicon amendment associated with fungicide treatment led to a reduction in DON concentration. Greenhouse experiments evaluated the effect of silicon combined with different timings of fungicide application on wheat defences against Fusarium graminearum. Plants supplied with silicon had a longer pathogen incubation period, lower FHB severity and lower DON concentration when compared to plants without silicon. In addition, silicon-supplied plants had higher soluble phenolic content and altered antioxidant enzyme activities (SOD, CAT, POX and PPO) that favoured early accumulation of hydrogen peroxide when compared to plants without silicon. Greater control of FHB and lower DON concentration in plants treated with silicon and fungicide before inoculation and up to 1 day after inoculation was associated with increased levels of defence-associated metabolites. Silicon contributed to the reduction of FHB and DON concentration in wheat, especially for the MR cultivar and, when combined with fungicide spraying, both MR and disease-susceptible cultivars had enhanced performances upon silicon amendment.     

The yam nematode (Scutellonema bradys), a potential threat to potato (Solanum tuberosum) production in West Africa

Potato (Solanum tuberosum) production in Africa is rapidly expanding and becoming increasingly important. As its geographical production range broadens, so does its potential to host new pests and diseases. Following the discovery that potato can be affected by Scutellonema bradys, further studies were undertaken to assess its potential pathogenicity on potato under screenhouse and field conditions, and on marketed tubers. Potato plants inoculated with S. bradys produced tubers with substantial cracking and evident tuber rot, compared with tubers from uninoculated plants. Symptoms of nematode infection on tubers included a scaly appearance, surface cracking as well as deeper tissue cracks, distortions, and darkened surface patches. In most cases these patches were related to sub‐surface rot. Nematodes were recovered from the soil, roots and tubers of inoculated plants. Eight weeks after inoculation, the reproduction factor of the nematode was greatest (2·0) at the lowest inoculation rate assessed (1000 nematodes per 2·5‐L pot) and least (0·4) at the highest inoculation rate (5000 nematodes per pot). In the screenhouse, potato tuber weights were low and mostly unaffected by nematode inoculation rate, except at 5000 nematodes per pot. In the field, non‐inoculated plants yielded over nine times more tubers than plants inoculated with 2000 S. bradys. Low densities of S. bradys were also recovered from 10 of 15 (67%) samples collected from market stalls, indicating field infection. This study confirms that potato can host and be damaged by S. bradys, raising its prospect as a likely significant biotic constraint to the crop.     

Plant age and ambient temperature: significant drivers for powdery mildew (Erysiphe cruciferarum) epidemics on oilseed rape (Brassica napus)

Powdery mildew (Erysiphe cruciferarum) is an important disease in oilseed rape crops worldwide, but of sporadic importance in most southern Australian crops. Six Brassica napus cultivars were exposed to E. cruciferarum simultaneously in four plant age cohorts. First symptoms of powdery mildew appeared 9 days after inoculation (dai) on the oldest plants [42 days after seeding (das)], but 44 dai in the youngest plants that were exposed to inoculum from sowing, although final disease severity did not differ with the plant age at exposure. The maximum level of pod peduncle infestation was unaffected by plant age (P = 0.37) or cultivar (P = 0.28). The effect of temperature was also investigated. The development of disease on plants was slower and final severity reduced at a day/night temperature 14/10 °C compared with 22/17 °C. In vitro, maximum growth of germ tubes from conidia of E. cruciferarum was at 15–20 °C and survival of conidia reduced by temperatures >30 °C. The results explain the sporadic nature of powdery mildew outbreaks in winter‐grown oilseed rape in Australia, where slow rates of infection occur when seasonal colder prevailing winter conditions coincide with the presence of younger plants, together curtailing rapid disease development until temperatures increase in late winter/early spring. These results explain why epidemics are most severe in the two warmer cropping regions, viz. the northern agricultural region of Western Australia and New South Wales. This study suggests that with increases in winter temperatures under future climate scenarios, earlier and more severe powdery mildew outbreaks in Australia will be favoured.     

A high-throughput method for early screening of coffee (<Emphasis Type="Italic">Coffea</Emphasis> spp.) genotypes for resistance to root-knot nematodes (<Emphasis Type="Italic">Meloidogyne</Emphasis> spp.)

Root-knot nematodes (Meloidogyne spp.) threaten the livelihood of millions of farmers producing coffee worldwide. The use of resistant plants either as cultivars or rootstocks appears to be the single most effective method of control. A screening method was developed to evaluate large populations of plants for resistance to root-knot nematodes. Two coffee cultivars, one susceptible and the other resistant to Meloidogyne paranaensis, were grown under controlled conditions in two substrates: a commercial sieved potting compost and an inert substrate containing sand with a water-absorbent synthetic polymer. Plant growth and development and nematode multiplication were compared for two inoculation dates (2 and 8 weeks after planting) and two evaluation dates (eight and 13 weeks after inoculation). Root growth, but not nematode multiplication, was influenced by the choice of substrate. Evaluation of the differences in root weight and nematode numbers between the different cultivars, substrates and dates of inoculation suggested that an optimal condition could be defined. The best discrimination between susceptible and resistant plants was found in the experiment where inoculation occurred at 2 weeks after planting and evaluation occurred at 8 weeks after inoculation. Because the total duration of this experiment was only 3 months, high-throughput evaluation was possible, opening up new possibilities for screening large germplasm collections and studying the genetic control of root-knot nematode resistance in coffee.     

Novel methodologies in screening and selecting olive varieties and root-stocks for resistance to <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

An innovative inoculation process, involving the drilling of a trunk hole in 3 year-old olive trees and injecting a dense conidial suspension of Verticillium dahliae, was developed to study differentiation in foliar symptom expression between olive cultivars tolerant or susceptible to the pathogen. It was demonstrated that V. dahliae conidia could be translocated and colonize the xylem at the same distance above and below the point of trunk injection in both cultivars. However, the pathogen could be subsequently isolated at statistically significant percentages in susceptible cv. Amphissis compared to the tolerant cv. Kalamon, indicating operation of resistance mechanisms in the vascular phase of the disease. Consequently symptom development in the susceptible cultivar was at least sixfold more intensive compared to the tolerant cultivar, 6–11 months after trunk inoculation. Perennial olive orchard experiments, aimed at selecting Verticillium-resistant root-stocks, were conducted by applying the novel method in 2–3 year-old root-stock suckers of Amphissis olive trees and in the tolerant cvs Lianolia of Corfu and Koroneiki. It was indicated that potentially resistant root-stocks could be obtained following the trunk drilling technique. Resistance differentiation between cvs Amphissis and Kalamon was further verified through root inoculation by various V. dahliae microsclerotial concentrations and demonstrated that the trunk drilling inoculation procedure is equally efficient in resistance evaluation of olives to Verticillium wilt. The trunk inoculation procedure could be useful in selecting and screening root-stocks for resistance to V. dahliae and other vascular pathogens and could elucidate resistance mechanisms in woody plants against vascular wilt diseases.     

Susceptibility of wild carrot (Daucus carota ssp. carota) to Sclerotinia sclerotiorum

Sclerotinia soft rot, caused by Sclerotinia sclerotiorum, is a severe disease of cultivated carrots (Daucus carota ssp. sativus) in storage. It is not known whether Sclerotinia soft rot also affects wild carrots (D. carota ssp. carota), which hybridise and exchange genes, among them resistance genes, with the cultivated carrot. We investigated the susceptibility of wild carrots to S. sclerotiorum isolates from cultivated carrot under controlled and outdoor conditions. Inoculated roots from both wild and cultivated plants produced sclerotia and soft rot in a growth chamber test. Two isolates differed significantly in the ability to produce lesions and sclerotia on roots of both wild carrots and cv. Bolero. Flowering stems of wild carrots produced dry, pale lesions after inoculation with the pathogen, and above-ground plant weight was significantly reduced 4 weeks after inoculation in a greenhouse test. Wild and cultivar rosette plants died earlier and fewer plants survived when inoculated with the pathogen under outdoor test conditions. Cultivar plants died earlier than wild plants, but survived as frequently. Plants inoculated in the crown died earlier and at a lower frequency than plants inoculated on leaves. Wild carrots may thus serve as a host of S. sclerotiorum and thus eventually benefit from any uptake of resistance genes, among them transgenes, via introgression from cultivated carrots.     

Spongospora subterranea root infection assessed in two potato cultivars differing in susceptibility to tuber powdery scab

Infection by Spongospora subterranea of roots of two potato (Solanum tuberosum) cultivars, either very resistant or very susceptible to powdery scab on their tubers, was studied in a glasshouse experiment. Plants grown in sand/nutrient solution culture were inoculated with S. subterranea sporosori 2 weeks after planting. Plant parameters, the intensity of zoosporangium infection in roots, numbers of Spongospora root galls and amounts of Spongospora DNA in roots, measured using quantitative PCR (qPCR), were assessed at sequential harvests. Inoculation with S. subterranea reduced water use (56 days after planting) by 26% in the tuber resistant cultivar compared with uninoculated plants, and by 60% in the susceptible cultivar. Inoculation did not affect growth of the resistant cultivar, nor shoot mass of the susceptible cultivar, but caused a 38% reduction in root mass of the susceptible cultivar. The intensities of zoosporangium development in both cultivars were similar. The susceptible cultivar had approximately four times more Spongospora root galls g^?1 root mass than the resistant cultivar. Quantitative PCR detected S. subterranea DNA in roots 1 week after inoculation, and indicated a twofold greater amount of pathogen DNA in roots of the susceptible than the resistant cultivar. This study suggests that the S. subterranea zoosporangium stage in host roots is affected differently by host resistance factors than the sporosorus (root gall and tuber scab) stages. The study has also demonstrated the usefulness of qPCR for sensitive and consistent detection of S. subterranea across the duration of potato root infection.     

Using metabolic profiling to assess plant-pathogen interactions: an example using rice (<Emphasis Type="Italic">Oryza sativa)</Emphasis> and the blast pathogen <Emphasis Type="Italic">Magnaporthe grisea</Emphasis>

A metabolomics based approach has been used to study the infection of the Hwacheong rice cultivar (Oryza sativa L. cv. Hwacheong) with compatible (KJ201) and incompatible (KJ401) strains of the rice blast fungal pathogen Magnaporthe grisea. The metabolic response of the rice plants to each strain was assessed 0, 6, 12, 24, 36, and 48 h post inoculation. Nuclear Magnetic Resonance (NMR) spectroscopy and Gas and Liquid Chromatography Tandem Mass spectrometry (GC/LC-MS/MS) were used to study both aqueous and organic phase metabolites, collectively resulting in the identification of 93 compounds. Clear metabolic profiles were observed at each time point but there were no significant differences in the metabolic response elicited by each pathogen strain until 24 h post inoculation. The largest change was found to be in alanine, which was ~30% (±9%) higher in the leaves from the compatible, compared to the resistant, plants. Together with several other metabolites (malate, glutamine, proline, cinnamate and an unknown sugar) alanine exhibited a good correlation between time of fungal penetration into the leaf and the divergence of metabolite profiles in each interaction. The results indicate both that a wide range of metabolites can be identified in rice leaves and that metabolomics has potential for the study of biochemical changes in plant-pathogen interactions.     

Comparison of the development <Emphasis Type="Italic">in planta</Emphasis> of a pyrrolnitrin-resistant mutant of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> and its sensitive wild-type parent isolate

Botrytis cinerea is able to build-up resistance to pyrrolnitrin, an antibiotic produced by diverse biocontrol agents, possibly compromising the durability of this method of disease control. The development of two near-isogenic lines of B. cinerea differing in their level of resistance to pyrrolnitrin was compared in tomato plants and on PDA medium. In tomato plants, significant differences in the percentage of infected petioles 1 day after inoculation and in symptom progression on petioles and stems were observed between the resistant mutant and the sensitive wild-type parent, suggesting a difference in their level of aggressiveness. Cytohistological investigations revealed that conidia of both near-isogenic lines germinated 6 h after inoculation and mycelium developed within petiole tissues 12 h after inoculation. However, while the wild-type parent isolate spread throughout the petiole and rapidly invaded the stem tissues via the leaf-abscission zone 72 h after inoculation, the pyrrolnitrin-resistant mutant failed to extend beyond petiole tissues to invade the stem. Moreover, 72 h after inoculation, the mycelial development of the pyrrolnitrin-resistant mutant was accompanied by abnormal glycogen accumulation and chlamydospore-like cell formation. In contrast, wild-type parent mycelium was normally structured with intensive colonization of stem tissues. Additionally, on PDA medium the mycelium of the pyrrolnitrin-resistant mutant was less vigorous than the wild-type isolate. These results suggest that the acquisition of pyrrolnitrin-resistance in B. cinerea is accompanied by changes in mycelial structure and reduction in mycelial growth, leading to a noticeable loss of aggressiveness on tomato plants.     

Assessment of recent outbreaks of <Emphasis Type="Italic">Dickeya</Emphasis> sp. (syn. <Emphasis Type="Italic">Erwinia chrysanthemi</Emphasis>) slow wilt in potato crops in Israel

Suspected Dickeya sp. strains were obtained from potato plants and tubers collected from commercial plots. The disease was observed on crops of various cultivars grown from seed tubers imported from the Netherlands during the spring seasons of 2004–2006, with disease incidence of 2–30% (10% in average). In addition to typical wilting symptoms on the foliage, in cases of severe infection, progeny tubers were rotten in the soil. Six strains were characterised by biochemical, serological and PCR-amplification. All tests verified the strains as Dickeya sp. The rep-PCR and the biochemical assays showed that the strains isolated from blackleg diseased plants in Israel were very similar, if not identical to strains isolated from Dutch seed potatoes, suggesting that the infection in Israel originated from the Dutch seed. The strains were distantly related to D. dianthicola strains, typically found in potatoes in Western Europe, and were similar to biovar 3 D. dadanti or D. zeae. This is the first time that the presence of biovar 3 strains in potato in the Netherlands is described. One of the strains was used for pathogenicity assays on potato cvs Nicola and Mondial. Symptoms appeared 2 to 3 days after stem inoculation, and 7 to 10 days after soil inoculation. The control plants treated with water, or plants inoculated with Pectobacterium carotovorum, did not develop any symptoms with either method of inoculation. The identity of Dickeya sp. and P. carotovorum re-isolated from inoculated plants was confirmed by PCR and ELISA.     

Programmed cell death pathways induced by early plant‐virus infection are determined by isolate virulence and stage of infection

Programmed cell death (PCD) pathways caused by Turnip mosaic virus (TuMV) infection before symptom appearance were studied by light microscopy and electrolyte leakage following sap inoculation of Brassica carinata (Ethiopian mustard) TZ‐SMN‐44‐6 plants. Leaf responses to inoculation with avirulent (TuMV‐avir) and virulent (TuMV‐vir) isolates, and mock‐inoculation, were compared at 2, 20 and 52 h after inoculation (hai). The phenotypes induced were localized resistance (TuMV‐avir) and systemic susceptibility (TuMV‐vir). No visible TuMV symptoms were recorded in any inoculated plants during the 2–52 hai sampling period, but appeared as chlorotic spots in inoculated leaves at 5 days after inoculation. With TuMV‐vir alone, they were followed by systemic infection (mosaic). Dead cell number, deformation, percentage area and percentage integrated intensity, and conductivity of electrolyte leakage data, were analysed to examine their possible roles in stimulating cell death pathways. At 2 hai, dead cell number and percentage area were significantly greater for TuMV‐avir than TuMV‐vir infection or mock‐inoculation. Overall, isolate TuMV‐vir caused significantly greater cell deformation than TuMV‐avir, whereas wounding by mock‐inoculation had negligible effects. By 52 hai, isolate TuMV‐avir caused significantly greater electrolyte leakage than isolate TuMV‐vir or mock‐inoculation. This suggests both isolates triggered morphological changes consistent with apoptotic‐like PCD and necrosis‐like PCD that depended upon isolate virulence and stage of infection, respectively. These findings highlight how quantification of dead cell deformation and electrolyte leakage offer a new understanding of compatible and incompatible plant responses to early virus infection in plants.     

Histopathological aspects of mango resistance to the infection process of Ceratocystis fimbriata

Mango wilt, caused by Ceratocystis fimbriata, is one of the most important diseases affecting mango yields in Brazil. Information regarding the infection process of C. fimbriata in the stem tissues of mango from different cultivars and the basis of host resistance to the pathogen is rare in the literature. Thus, the objective of the study was to investigate how infection by two isolates of C. fimbriata can be affected by mango cultivar‐specific mechanisms of resistance. Disease progress on the inoculated stem tissues of the mango cultivars was evaluated and stem sections were obtained from the site of inoculation and prepared for histopathological observations using light microscopy. The factors mango cultivars and C. fimbriata isolates and their interaction were significant for all measures of disease development. Plants from the cultivars Espada, Haden and Palmer inoculated with isolates of C. fimbriata were more susceptible, whereas plants from the cultivars Tommy and Ubá were moderately resistant and resistant, respectively. Histopathologically, fungal isolates apparently massively colonized the stem tissues of plants from the susceptible cultivars Espada, Haden and Palmer, starting from the collenchyma and moving in the direction of the cortical parenchyma, xylem vessels and pith parenchyma. By contrast, on stem tissues of plants from the resistant cultivars Tommy Atkins and Ubá, most of the cells reacted to C. fimbriata infection by accumulating amorphous material. The results from the present study strongly indicated the importance of phenolic compounds for mango cultivar resistance against infection by Brazilian C. fimbriata isolates.     

Influence of temperature on infection and establishment of ‘Candidatus Liberibacter americanus’ and ‘Candidatus Liberibacter asiaticus’ in citrus plants

The objectives of this work were (i) to determine the influence of temperature on infection of citrus by ‘Candidatus Liberibacter asiaticus’ and ‘Candidatus Liberibacter americanus’, the two bacterial species associated with citrus huanglongbing (HLB) in Brazil, and (ii) to determine the influence of temperature on citrus colonization by ‘Ca. L. asiaticus’, which has taken over from ‘Ca. L. americanus’ as the predominant species in Brazil since 2008. Two experiments were carried out with graft‐inoculated Valencia oranges on Rangpur lime rootstocks. Immediately after inoculation the plants were maintained for 423 days in growth chambers under the following night/day temperature conditions: 17/22, 22/27 or 27/32°C, with a dark/light photoperiod of 8/16 h. Infection and colonization of plants were determined using quantitative PCR (qPCR). ‘Candidatus Liberibacter americanus’ did not infect the plants maintained at 27/32°C; however, infection by ‘Ca. L. asiaticus’ occurred at all studied temperatures. Two months after inoculation, ‘Ca. L. asiaticus’ was distributed throughout the inoculated plants, with mean C_t values in the range of 30–31 for leaves and 25–28 for roots. Over time, ‘Ca. L. asiaticus’ reached the highest titres in mature leaves (mean C_t value = 26·7) of citrus plants maintained at 22/27°C. ‘Candidatus Liberibacter asiaticus’ colonization of citrus plants was negatively affected by the daily temperature regime of 27/32°C (mean C_t value in mature leaves = 33·6).     

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.     

Spread of potato virus Y in potato cultivars (Solanum tuberosum L.) with different levels of sensitivity

The aim of this work was to correlate the appearance of the symptoms, multiplication and spread of virus after mechanical inoculation of potato (Solanum tuberosum L.) cultivars showing different levels of susceptibility and sensitivity to Potato virus Y^NTN (PVY^NTN). The potato cultivars used were the resistant cultivar Sante and susceptible cultivars Igor, Pentland squire and Désirée. The spread of the virus PVY^NTN in infected plants was monitored using different methods: DAS-ELISA, tissue printing, immuno-serological electron microscopy and real-time PCR. In all three susceptible cultivars, the virus was detected in the inoculated leaves 4–5 days after inoculation. From there virus spread rapidly, first into the stem, then more or less simultaneously to the upper leaves and roots. Real-time PCR was shown to be very sensitive and enabled viral RNA to be detected in non-inoculated leaves of susceptible cultivar Igor earlier than other methods. Therefore, for exact studies of plant–virus interaction, a combination of methods which detect viruses on the basis of their different properties (coat protein, morphology or RNA) should be used to monitor the spread of viruses.