Implication of peroxynitrite in defence responses of potato to Phytophthora infestans

In this study peroxynitrite (ONOO^?) is proposed as an important player in defence responses during the interaction of potato (Solanum tuberosum) and the oomycete pathogen Phytophthora infestans. The potato–avr P. infestans model system exhibited a transient programme of boosted ONOO^? formation correlated in time with the burst of nitric oxide (NO) and superoxide during the first 6 h post‐inoculation (hpi). The early ONOO^? over‐accumulation was not accompanied by TPx gene expression. In contrast, the compatible interaction revealed a 24 h delay of ONOO^? formation; however, an enhanced level of NO and superoxide correlated with TPx up‐regulation was recorded within the earlier stages of pathogen infection. Peroxynitrite over‐accumulation in the susceptible potato coincided with an enhanced level of protein tyrosine nitration starting from 24 hpi. Surprisingly, the nitroproteome profile of the resistant potato did not show any visible difference after inoculation, apart from one band containing subtilisin‐like protease‐like proteins, which appeared 48 h after pathogen attack. An additional pharmacological approach showed that treatment of the susceptible genotype with ONOO^? followed by inoculation with P. infestans contributed to slowing down of the colonization of host tissues by the pathogen via a faster and stronger up‐regulation of the key defence markers, including the PR‐1 gene. Taken together, the results obtained indicate that a precise control of emitted NO and superoxide in cooperation with thioredoxin‐dependent redox sensors in sites of pathogen ingress could generate a sufficient threshold of ONOO^?, triggering defence responses.     

Callose and β‐1,3‐glucanase inhibit Phytophthora cinnamomi in a resistant avocado rootstock

Phytophthora root rot (PRR) of avocado, caused by Phytophthora cinnamomi, is a significant threat to sustainable production wherever the crop is grown. Resistant rootstocks in combination with phosphite applications are the most effective options for managing this disease. Recently, the mechanisms underpinning PRR resistance have been investigated by the avocado community. Here, biochemical assays and confocal and scanning electron microscopy were used to investigate early defence responses in PRR resistant and ‐susceptible avocado rootstocks. Zoospore germination and subsequent hyphal growth for the pathogen were significantly inhibited on the surface of resistant avocado roots. When penetration occurred in the resistant R0.06 rootstock, callose was deposited in the epidermal cells, parenchyma and cortex of roots. In addition, β‐1,3‐glucanase was released early (6 h post‐inoculation, hpi) in response to the pathogen, followed by a significant increase in catalase by 24 hpi. In contrast, susceptible R0.12 roots responded only with the deposition of lignin and phenolic compounds incapable of impeding pathogen colonization. In this study, PRR resistance was attributed to a timely multilayered response to infection by P. cinnamomi.     

Factors affecting the activity of ethephon in stimulating seed germination of Striga hermonthica (Del.) Benth.

Ethephon (2-chloroethylphosphonic acid), which generates ethylene, stimulated the germination of pre-conditioned seeds of Striga hermonthica when it was added to the alkaline Gezira clay soil at concentrations of 2.5 to 30 mg kg^?1. As little as 5 min contact with treated soil was enough to stimulate germination. Ethephon in soil did not cause germination of unconditioned seeds for periods of up to 12 days and also had an adverse effect on seed germination when such seeds were given a second ethephon exposure after a storage period which was adequate, in untreated soil, to give the necessary pre-conditioning. Ethephon activity persisted in air-dry soil but declined over a 14 day period in moist soil. In the field ethephon at 0.6 to 4.8 kg ha^?1 decreased the number of Striga shoots and increased sorghum height and flowering.     

Expression of defence‐related genes in stems and leaves of resistant and susceptible field pea (Pisum sativum) during infection by Phoma koolunga

The differential expression of 13 defence‐related genes during Phoma koolunga infection of stems and leaves of susceptible versus resistant field pea (Pisum sativum) was determined using qRT‐PCR. Expression, in terms of relative mRNA level ratios, of genes encoding ferredoxin NADP oxidoreductase, 6a‐hydroxymaackiain methyltransferase (hmm6), chalcone synthase (PSCHS3) and ascorbate peroxidase in leaves and stems differed during 6–72 hours post‐inoculation (hpi) and reflected known host resistance levels in leaves versus stems. In comparison to the susceptible genotype, at 24, 48 and 72 hpi, two genes, hmm6 (122.43‐, 206.99‐ and 32.25‐fold, respectively) and PSCHS3 (175.00‐, 250.13‐ and 216.24‐fold, respectively), were strongly up‐regulated in leaves of the resistant genotype, highlighting that resistance against P. koolunga in field pea is governed by the early synthesis of pisatin. At 24 hpi, leaves infected by P. koolunga showed clear differences in expression of target genes. For example, the gene encoding a precursor of the defensin ‘disease resistance response protein 39’ was substantially down‐regulated in leaves of both the susceptible and the resistant genotypes inoculated with P. koolunga. This contrasts with other studies on another pea black spot pathogen, Didymella pinodes, where this same gene is strongly up‐regulated in leaves of resistant and susceptible genotypes. The current study provides the first understanding of defence‐related genes involved in the resistance against P. koolunga, opening novel avenues to engineer new field pea cultivars with improved leaf and stem black spot disease resistance as the basis for developing more effective and sustainable management strategies.     

十一种杀菌剂防治马铃薯晚疫病的适宜施用时期

为了明确当前市场上具有代表性的11种防治马铃薯晚疫病杀菌剂的适宜施用时期,采用人工接种马铃薯叶片的方法,测定了其中5种药剂在接种晚疫病菌前10 d内不同时间施用的预防效果和其中7种药剂在接种后24 h内不同时间施用的治疗效果。结果显示:在保证良好防治效果的前提下,药剂在病原菌接种前保护性施用的适宜时期比在接种后治疗性施用的适宜时期要长。在测试浓度下,接种前5种供试药剂保持100%防治效果的适宜施用时期为接种前3~10 d;而接种后7种供试药剂保持相同防效的适宜施用时期仅为接种后6~12 h,在生产实际中难以操作。研究结果说明,在田间马铃薯晚疫病的化学防治中,为了取得良好防治效果,每次用药均应在病菌侵入之前采用保护性施用,尽量避免在病菌侵入之后进行治疗性施用。     

Fusarium wilt‐resistant lines of Brazil banana (Musa spp., AAA) obtained by EMS‐induced mutation in a micro‐cross‐section cultural system

This study combined the micro‐cross‐section cultural system with in vitro mutagenesis induced by ethyl methanesulphonate (EMS) to screen for fusarium wilt‐resistant lines of Brazil banana (Musa spp., AAA). The results indicated that the optimum EMS concentration and duration for the treatment of micro‐cross‐sections cut from the pseudostem of tissue‐cultured plantlet were 300 mm and 60 min, respectively. Under the optimal treatment, an average of 2·2 regenerated shoots were produced from each explant. One hundred regenerated plantlets were used for screening for fusarium wilt‐resistant lines by the early screening technique. The initial disease symptom – yellowing in lower leaves of susceptible plantlets – was observed 2 weeks after inoculation. After 2 months, only six plants survived – the putative fusarium wilt‐resistant lines. The fusarium wilt pathogen Fusarium oxysporum f. sp. cubense race 4, was identified in the preliminary test field by a SCAR marker technique. Of the six putative resistant lines, five survived the preliminary field test. The regenerated plantlets from these five fusarium wilt‐resistant lines were subjected to early screening again, where they showed markedly reduced disease incidences compared with regenerated plantlets of Brazil banana (control). It was concluded that EMS‐induced mutation of banana through the micro‐cross‐section cultural system is potentially useful for banana improvement.     

Formation of highly branched hyphae by Colletotrichum acutatum within the fruit cuticles of Capsicum spp.

This study showed that Colletotrichum acutatum penetrates the cuticle layer of Capsicum spp. fruits by forming a previously uncharacterized structure from appressoria. This unusual structure was localized in the cuticle layer. The structure, formed within 24 h post‐inoculation (hpi), was a highly branched, well‐differentiated hypha which penetrated the epidermal cell at 72 hpi. The novel structure, with abnormally thick walls (about 250 nm), often formed multiple branches in the affected chilli pepper. This dendroid structure, probably required for penetration, was formed exclusively in the cuticle layer of chilli pepper fruits and was not found when C. acutatum was inoculated onto pepper petals, mango leaves, or fruits of tomato and aubergine. Colletotrichum acutatum produced similar dendroid structures within resistant chilli pepper fruits, but eventually these structures turned dark brown and no further infection in the epidermal cells occurred, implicating the presence of inhibitors of the formation and development of the dendroid penetration structure in the resistant line.     

Spatiotemporal patterns of oxidative burst and micronecrosis in resistance of wheat to brown rust infection

The accumulation of H₂O₂ (oxidative burst) and the progress of pathogen development were studied in compatible and incompatible wheat‐brown rust interactions. The accumulation of H₂O₂ was detected in 98·7% of guard cells with appressoria 8 h post inoculation (hpi). The reaction in both susceptible and resistant plants declined 2–3 days post inoculation (dpi). The second phase of the oxidative burst was observed in the mesophyll and/or epidermis. In susceptible plants it began 4–5 dpi and was detected only in the epidermis. In resistant plants the response was observed in the mesophyll. In moderately resistant plants it was induced 1–3 dpi, and the percentage of infection units reached 80–90% 8 dpi. This corresponded with severe necrotic symptoms. In highly resistant plants, the oxidative burst was short and transient. The percentage of infection units with H₂O₂ accumulation reached its highest level (60–70%) 2 dpi, and decreased thereafter. Four days later, the low percentage and weak DAB staining indicated very low H₂O₂ accumulation. The localization and the time‐course changes of the oxidative burst correlated with the profiles of the micronecrotic response, haustorium mother cell formation and pathogen development termination. An early and localized induction of oxidative burst followed by its rapid quenching correlated with high resistance and almost no disease symptoms. The possible correlation of the oxidative burst and pathogen development patterns with the level and durability of resistance conferred by Lr genes are discussed.     

Effects of Verticillium dahliae on tomato root morphology considering plant growth response and defence

The soilborne pathogen Verticillium dahliae invades its host via the root, and spreads systemically throughout the plant. Although a functional root system of appropriate size is essential for water and nutrient uptake, to date, effects of pathogens on root morphology have not been frequently investigated. Therefore, this study aims to improve knowledge of how V. dahliae infection impairs root morphological characteristics of tomato, considering plant growth and physiological responses, particularly those involved in defence in roots and leaves over a growing period of up to 28 days post‐inoculation. Verticillium dahliae infection suppressed the growth of both shoot and root. Diseased plants developed a smaller leaf area, and exhibited a reduction in the rate of photosynthesis and stomatal conductance. An early response to pathogen invasion in the host root was the up‐regulation of several defence‐related genes, such as proteinase inhibitor II (Pin2), β‐1,3‐glucanase A (GluA) and two pathogenesis‐related genes (PR‐1a, PR‐1b). However, this response did not prevent colonization of the roots by the pathogen. Although a high variability in pathogen density was found within the root system, a significant increase of both the specific root length and surface area was observed in response to pathogen invasion; these traits correlated with water use efficiency. Morphological changes of the root may represent an adaptive response evolved to sustain the supply of both water and nutrients in the presence of the pathogen.     

Infection process of <Emphasis Type="Italic">Pseudocercospora musae</Emphasis> on banana leaf

Yellow Sigatoka that is caused by Pseudocercospora musae is an important banana disease. The aim of this study was to elucidate the infection process of P. musae in banana leaves by scanning electron microscopy. Leaf samples were inoculated on the abaxial surface with P. musae and then analysed at 12, 24, 36, 48, 72, 96, 120, 144, and 168 h post inoculation (hpi) and at 36 and 50 days post inoculation (dpi). The conidia were found to be germinated between 24 and 36 hpi and penetrated through the stomata between 96 and 120 hpi, or more generally from 144 hpi. P. musae colonized the spongy parenchyma at 36 dpi and the palisade parenchyma at 50 dpi. Sporulation occurred at 50 dpi on the adaxial surface of leaves through the emergence of conidia on conidiophores through the stomata. Considering the importance of yellow Sigatoka in banana production, our results provide a better understanding of the life cycle of the fungus for treatment processes.     

<Emphasis Type="Italic">Trichoderma harzianum</Emphasis> elicits defence response genes in roots of potato plantlets challenged by <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

Biological control of Rhizoctonia solani with Trichoderma harzianum has been demonstrated in several studies. However, none have reported the dynamics of expression of defence response genes. Here we investigated the expression of these genes in potato roots challenged by R. solani in the presence/absence of T. harzianum Rifai MUCL 29707. Analysis of gene expression revealed an induction of PR1 at 168 h post-inoculation (hpi) and PAL at 96 hpi in the plants inoculated with T. harzianum Rifai MUCL 29707, an induction of PR1, PR2 and PAL at 48 hpi in the plants inoculated with R. solani and an induction of Lox at 24 hpi and PR1, PR2, PAL and GST1 at 72 hpi in the plants inoculated with both organisms. These results suggest that in the presence of T. harzianum Rifai MUCL 29707, the expression of Lox and GST1 genes are primed in potato plantlets infected with R. solani at an early stage of infection. Mycothèque de l’Université catholique de Louvain of S. Cranenbrouck's affiliation is part of the Belgian Coordinated Collections of Micro-organisms (BCCM).     

Histology of waxflower (Chamelaucium spp.) flower infection by Botrytis cinerea

Botrytis cinerea infects waxflower (Chamelaucium spp.) flowers and can induce them to abscise from their petioles before disease becomes evident. Botrytis cinerea infection of flowers was studied on two waxflower cultivars by light and electron microscopy. Pot‐grown waxflower flowers were harvested, inoculated with aqueous suspensions of B. cinerea conidia, incubated at 20–22°C and >95% RH and examined within 96 h post‐inoculation (hpi). Conidial germination on petals started 4 hpi, penetration via germ tube tips was 6 hpi and protoappressoria were formed 8 hpi. Germination on petals approximately doubled every 4–6 h to 18 hpi. Conidial germination was ca. 50% at 22–24 hpi. Botrytis cinerea infected most waxflower flower organs, including petals, anthers and filaments, stigma and hypanthium, within 24 hpi. Hyaline and lobate appressoria were observed 36 hpi. Infection cushions on stamen bases were formed 36 hpi by saprophytic hyphae that originated from anthers. This infection process can give rise to tan‐coloured symptoms typical of botrytis disease that radiate from this part of the flower. Subcuticular hyphae were present at high density near stamen bases and evidently resulted from multiple penetrations from single infection cushions. The subcuticular hyphae grew within the hypanthium and towards the centre of the floral tube. When flower abscission occurred, floral tube tissues close to the abscission zone remained uninfected. This observation infers possible transmission of a signal (e.g. ethylene) upon B. cinerea infection. Thus, B. cinerea causes flower abscission apparently as a defence response.     

Induced resistance to Verticillium longisporum in Brassica napus by β‐aminobutyric acid

A preinoculative soil drench application of 0·5 mm β‐aminobutyric acid (BABA) significantly inhibited colonization of oilseed rape (Brassica napus, susceptible cultivar Falcon) by Verticillium longisporum and also prevented plant stunting caused by the pathogen. To better understand the defence responses induced by BABA, the presence of occlusions in the plant hypocotyl, levels of salicylic acid (SA) and hydrogen peroxide (H₂O₂), phenylalanine ammonia lyase (PAL) activity and expression of PR‐1 and PDF1.2 genes were examined. Transverse sections through the hypocotyl region of BABA‐treated plants showed clear vessels surrounded by phenol‐storing cells, in contrast to numerous obstructed vessels in water‐treated plants, in response to the pathogen. A significant increase in SA levels was observed in the hypocotyls of both water‐ and BABA‐treated plants in response to the pathogen; however, SA levels were unrelated to disease resistance. The level of H₂O₂ decreased in both treatments in response to the pathogen. A significant increase in PAL activity was observed in hypocotyl tissues of BABA‐treated plants. The expression patterns of PR‐1 and PDF1.2 were similar in the two treatments in response to the pathogen, indicating no involvement of these genes in resistance. The results indicate a similar organ specificity of the plant hypocotyl for chemically induced internal resistance as for genotype‐related resistance, two phenomena which, however, are based on contrasting cytological responses in the vascular tissues. Nonetheless, evidence is provided that the activity of the phenylpropanoid pathway plays a crucial role in both types of resistance.     

Comparison of root and foliar applications of potassium silicate in potentiating post‐infection defences of melon against powdery mildew

The application of silicon to the roots or leaves reduces the severity of powdery mildew (Podosphaera xanthii) in melon but the latter treatment is less effective. This study compared key biochemical defence responses of melon triggered by P. xanthii after root or foliar treatment with potassium silicate (PS). Treatments consisted of pathogen‐inoculated or mock‐inoculated plants supplied with PS via roots or foliarly, as well as a non‐treated control. The activity of defence enzymes and the concentration of phenolic compounds, lignin and malondialdehyde were determined from leaf samples at different time points after inoculation. Pathogen‐inoculated plants irrigated with PS showed both an accumulation of silicon and primed defence responses in leaves that were not observed in pathogen‐inoculated plants either sprayed with PS or not treated. These responses included the anticipated activity of peroxidase and accumulation of soluble phenols, the activation of chitinase and repression of catalase, and the stronger activation of superoxide dismutase, peroxidase and β‐1,3‐glucanase. Moreover, the lignin concentration increased in response to inoculation, whereas the malondialdehyde concentration decreased. For the foliar treatment, however, only an increase in lignin deposition was observed compared with the control plants. The results show that silicon strongly plays an active role in modulating the defence responses of melon against P. xanthii when supplied to the roots as opposed to the foliage.     

Expression of biotic stress response genes to Phytophthora infestans inoculation in White Lady,a potato cultivar with race-specific resistance to late blight

Expression changes of biotic stress response genes were analyzed during a 65 h period post inoculation with Phytophthora infestans in potato cultivar White Lady that possesses race-specific resistance to this pathogen. All analyzed respiratory burst oxidase homologs, the PR proteins, the serine-, cysteine- and aspartic protease inhibitors, as well as the Rpi-bt1 gene homolog were up-regulated in the biotrophic phase. The R1 and R2 gene homologs showed up-regulation only at 65 hpi, and interestingly, the R3a gene showed only a very slight expressional increase. It is concluded, that beside the constitutively expressed R genes a number of non-specific stress response genes contribute to the successful resistance response in race-specific defense.