Nitric oxide is involved in chitosan‐induced systemic resistance in pearl millet against downy mildew disease

BACKGROUND: The nature and durability of resistance offered by chitosan and the involvement of nitric oxide (NO) in chitosan‐induced defence reactions in pearl millet against downy mildew disease were investigated. RESULTS: It had previously been reported that chitosan seed priming protected pearl millet plants against downy mildew disease. Further elucidation of the mechanism of resistance showed that chitosan seed priming protects the plants systemically. A minimum 4 day time gap is required between the chitosan treatment and pathogen inoculation for maximum resistance development, and it was found to be durable. Chitosan seed priming elevated NO accumulation in pearl millet seedlings, beginning from 2 h post‐inoculation, and it was found to be involved in the activation of early defence reactions such as hypersensitive reaction, callose deposition and PR‐1 protein expression. Pretreatment with NO scavenger C‐PTIO and nitric oxide synthase (NOS) inhibitor L‐NAME before pathogen inoculation reduced the disease‐protecting ability of chitosan, and defence reactions were also downregulated, which indicated a possible role for NO in chitosan‐induced resistance. CONCLUSION: Protection offered by chitosan against pearl millet downy mildew disease is systemic in nature and durable. Chitosan‐induced resistance is activated via NO signalling, as defence reactions induced by chitosan were downregulated under NO deficient conditions. Copyright © 2009 Society of Chemical Industry     

Basal tomato defences to Botrytis cinerea include abscisic acid-dependent callose formation

In Arabidopsis, abscisic acid (ABA) application can induce resistance by priming for callose deposition; this resistance is impaired in ABA-deficient and -insensitive mutants. In tomato, ABA-deficiency causes resistance to Botrytis cinerea. Here, we show that callose deposition after B. cinerea inoculation is weaker in the ABA-deficient sitiens tomato mutant compared to the wild type (WT). Inhibition of callose synthesis did not affect resistance in sitiens, but caused additional susceptibility in WT. These findings indicate that callose deposition is not part of sitiens defence responses that are effective in blocking B. cinerea and suggest that callose deposition only contributes to WT basal resistance. Furthermore, also in tomato callose formation is at least in part ABA-dependent. However, it seems that in contrast to Arabidopsis, basal ABA levels in tomato are sufficiently high to prime for callose deposition.     

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 disease resistance and pathogen defence responses in Asparagus officinalis inoculated with nonpathogenic strains of Fusarium oxysporum

The ability of nonpathogenic isolates of Fusarium oxysporum (np Fo ) to induce systemic resistance and defence responses against subsequent challenge with a pathogenic strain of F. oxysporum f. sp. asparagi ( Foa ) was examined in Asparagus officinalis . In a split-root experiment, roots inoculated with np Fo exhibited a hypersensitive response and those subsequently inoculated with Foa displayed resistance. Induction of systemic resistance in np Fo -treated plants led to significantly fewer necrotic lesions ( P  = 0·05) and reduced Foa disease severity compared with plants not treated with np Fo . In hyphal-sandwich root inoculation experiments, activities of peroxidase and phenylalanine ammonia-lyase and lignin content were higher in np Fo -treated plants and increased more rapidly than in np Fo -untreated plants after Foa inoculation. Antifungal activity (inhibition of fungal spore germination and germ-tube growth) from exudates of roots inoculated with Foa were observed for np Fo -treated plants but not for np Fo -untreated plants. Thus, isolates of np Fo may function as inducers of systemic acquired resistance (SAR) and defence responses against Foa invasion in A. officinalis .     

Expression patterns of defence genes and antioxidant defence responses in a rice variety that is resistant to leaf blast but susceptible to neck blast

The rice stage resistance variety Jiajing3768 is resistant to leaf blast but susceptible to neck blast. This variety was used to analyze the expression patterns of defence genes and antioxidant defence responses in the leaves at the seedling stage (LSS) and necks at the preliminary heading stage (NPHS), after inoculation with Magnaporthe oryzae. We found that defence genes PR1b (pathogenesis-related class 1b), PR4, PR10a, JIOsPR10 (jasmonic acid induced rice PR 10), Gns1 (1,3; 1,4-β-glucanase), Cht-1 (chitinase), and LOX (lipoxygenase) may play roles in the stage resistance in Jiajing3768; PAL (phenylalanine ammonia-lyase) and CHS (chalcone synthase) could participate in defending Jiajing3768 against neck blast but not leaf blast. The antioxidant enzymes superoxide dismutase, peroxidase, catalase, glutathione reductase, and malondialdehyde coordinately participate in the stage resistance to blast in Jiajing3768, and that oxidative damage is less in the LSS than in the NPHS.     

Peanut priming induced by biocontrol agents

Selected strains of rhizobacteria induce systemic resistance in plant (ISR), enhancing the capacity to mobilize infection-induced cellular defense responses (priming). Bacillus sp. CHEP5 and Pseudomonas sp BREN6 strains reduced root and stem wilt disease severity caused by Sclerotium rolfsii in Arachis hypogaea L. Strains inoculation increased the activity of phenylalanine ammonia-lyase and peroxidase, after pathogen-challenge, indicating priming. CHEP5 primes plants to produce more ethylene upon stimulation with ACC. A. hypogaea L is an ISR-positive plant and BREN6 and CHEP5 strains enhance the plant’s defense capacity by priming for potentiated activity of defense proteins and ACC-converting capacity.     

毁灭炭疽菌Colletotrichum destructivum诱抗蛋白对烟草抗病性的诱导

为明确毁灭炭疽菌Colletotrichum destructivum诱抗蛋白诱导烟草的抗病性及其作用,采用喷雾、摩擦接种方法及RT-PCR技术研究了诱抗蛋白的预防保护作用,以及烟草悬浮细胞经诱导后过氧化物酶(POD)、多酚氧化酶(PPO)、苯丙氨酸解氨酶(PAL)活性及脯氨酸(Pro)含量和病程相关基因表达的变化。结果表明,接种3、5和7 d后,该诱抗蛋白对烟草炭疽病的诱抗效果分别为58.00%、48.99%和49.65%,对烟草白粉病的诱抗效果分别为83.26%、80.76%和78.60%,并可以抑制烟草普通花叶病毒的复制及在寄主体内的扩增;经诱抗蛋白处理后,烟草悬浮细胞POD、PPO、PAL活性及Pro含量明显提高;诱抗蛋白能够诱导烟草病程相关蛋白基因PR-1a、PR-1b以及抗病信号传导途径关键基因NPR1的表达。表明毁灭炭疽菌诱抗蛋白可诱导烟草产生抗病性,可能与烟草悬浮细胞中POD、PAL、PPO的活性及Pro的含量提高以及相关病程基因表达有关。     

Rhizobacteria-mediated Induced Systemic Resistance: Triggering, Signalling and Expression

Selected strains of rhizosphere bacteria reduce disease by activating a resistance mechanism in the plant named rhizobacteria-mediated induced systemic resistance (ISR). Rhizobacteria-mediated ISR resembles pathogen-induced systemic acquired resistance (SAR) in that both types of induced resistance render uninfected plant parts more resistant towards a broad spectrum of plant pathogens. Some rhizobacteria trigger the salicylic acid (SA)-dependent SAR pathway by producing SA at the root surface. In other cases, rhizobacteria trigger a different signalling pathway that does not require SA. The existence of a SA-independent ISR pathway has been demonstrated in Arabidopsis thaliana. In contrast to pathogen-induced SAR, ISR induced by Pseudomonas fluorescens WCS417r is independent of SA accumulation and pathogenesis-related (PR) gene activation but, instead, requires responsiveness to the plant hormones jasmonic acid (JA) and ethylene. Mutant analyses showed that ISR follows a novel signalling pathway in which components from the JA and ethylene response are successively engaged to trigger a defensive state that, like SAR, is controlled by the regulatory factor NPR1. Interestingly, simultaneous activation of both the JA/ethylene-dependent ISR pathway and the SA-dependent SAR pathway results in an enhanced level of protection. Thus combining both types of induced resistance provides an attractive tool for the improvement of disease control. This review focuses on the current status of our research on triggering, signalling, and expression of rhizobacteria-mediated ISR in Arabidopsis.     

A PAL1 Gene Promoter–Green Fluorescent Protein Reporter System to Analyse Defence Responses in Live Cells of Arabidopsis thaliana

Arabidopsis thaliana ecotype Columbia-0 was transformed with a green fluorescent protein (GFP) gene under control of a phenylalanine ammonia-lyase (PAL) promoter. PAL is a key enzyme of the phenylpropanoid pathway and is induced to high levels during plant stress. Constitutive expression of PAL1 promoter-controlled GFP occurred in vascular tissues within stems, leaves and roots and in developing flowers. PAL1 promoter–GFP expression was examined in leaves of transgenic plants subjected to an abiotic elicitor, mechanical wounding or to inoculation with the pathogens Pseudomonas syringae pv. tomato or Peronospora parasitica. Wounding of leaves and treatment with an abiotic elicitor and compatible interactions produced low to moderate levels of GFP. However, in incompatible interactions there were high levels of GFP produced. In incompatible interactions, the intensity of GFP fluorescence was similar to that produced in transgenic plants expressing GFP driven by the CaMV promoter. The bright green fluorescence produced in live cells and tissues was readily visualised using conventional fluorescence microscopy and was quantified using spectroflourometry. This is the first report of the use of GFP as a reporter of defence gene activation against pathogens. It has several advantages over other reporter genes including real time analysis of gene expression and visualisation of defence gene activation in a non-invasive manner.     

Activation of defence responses to Phytophthora infestans in potato by BABA

Late blight caused by Phytophthora infestans is one of the most devastating diseases of the potato crop. Resistance breeding and current fungicides are unable to control the rapidly evolving P. infestans and new control strategies are urgently needed. This study examined mechanisms of dl ‐β‐aminobutyric acid (BABA)‐induced resistance (IR) in the potato–P. infestans system. Leaves from two cultivars that differ in their degree of resistance, Bintje and Ovatio, were analysed after foliar treatment with BABA. Rapid activation of various defence responses and a significant reduction in P. infestans growth were observed in leaves treated with BABA. In the more resistant cultivar, Ovatio, the activation was both faster and stronger than in Bintje. Microscopic analysis of leaves treated with BABA revealed induction of small hypersensitive response (HR)‐like lesions surrounded by callose, as well as production of hydrogen peroxide (H₂O₂). Molecular and chemical analyses revealed soluble phenols such as arbutin and chlorogenic acid and activation of PR‐1. These results show a direct activation of defence responses in potato, rather than priming as reported for other plant species. They also show that the efficiency of BABA‐IR differs between cultivars, which highlights the importance of taking all aspects into consideration when establishing new methods for disease management.     

Potential role for salicylic acid in induced resistance of asparagus roots to Fusarium oxysporum f.sp. asparagi

Pre-inoculation of asparagus ( Asparagus officinalis ) roots with selected nonpathogenic isolates of Fusarium oxysporum (np Fo ) has previously been shown to induce systemic resistance against infection by F. oxysporum f.sp. asparagi ( Foa ) through activation of plant-defence mechanisms. To elucidate the putative np Fo -mediated defence pathways, the effect of salicylic acid (SA) was examined in a split-root system of asparagus where one half of the seedling root system was drenched with SA and the activation of defence responses was measured subsequently on the remaining roots. SA-treated plants exhibited enhanced systemic resistance, with a significant reduction in disease severity of the roots inoculated with Foa , compared with untreated plants. SA activated peroxidase and phenylalanine ammonia-lyase, as well as lignification, upon Foa attack, in a manner similar to that observed with np Fo pretreatment. In addition, application of diphenyleneiodonium, an SA biosynthesis inhibitor, led to failure of np Fo to induce lignin deposition and systemic resistance. Treatment of fungal spores with SA did not affect germination and growth of either np Fo or Foa in in vitro antifungal assays. Production of SA at the site of np Fo infection may be involved in the induction of Foa resistance in asparagus roots.     

Effects of foliar- and root-applied silicon on the enhancement of induced resistance to powdery mildew in Cucumis sativus

Two cucumber ( Cucumis sativus ) cultivars differing in their resistance to powdery mildew, Ningfeng No. 3 (susceptible) and Jinchun No. 4 (resistant), were used to study the effects of foliar- and root-applied silicon on resistance to infection by Podosphaera xanthii (syn. Sphaerotheca fuliginea ) and the production of pathogenesis-related proteins (PRs). The results indicated that inoculation with P. xanthii significantly suppressed subsequent infection by powdery mildew compared with noninoculation, regardless of Si application. Root-applied Si significantly suppressed powdery mildew, the disease index being lower in Si-supplied than in Si-deprived plants, regardless of inoculation treatment. The resistant cultivar had a more constant lower disease index than the susceptible cultivar, irrespective of inoculation or Si treatment. Moreover, with root-applied Si, activities of PRs (for example peroxidase, polyphenoloxidase and chitinase) were significantly enhanced in inoculated lower leaves or noninoculated upper leaves in inoculated plants of both cultivars. Root-applied Si significantly decreased the activity of phenylalanine ammonia-lyase in inoculated leaves, but increased it in noninoculated upper leaves. However, Si treatment failed to change significantly the activity of PRs in plants without fungal attack. Compared to the control (no Si), foliar-applied Si had no effects either on the suppression of subsequent infection by P. xanthii or on the activity of PRs, irrespective of inoculation. Based on the findings in this study and previous reports, it was concluded that foliar-applied Si can effectively control infections by P. xanthii only via the physical barrier of Si deposited on leaf surfaces, and/or osmotic effect of the silicate applied, but cannot enhance systemic acquired resistance induced by inoculation, while continuously root-applied Si can enhance defence resistance in response to infection by P. xanthii in cucumber.     

Signal interactions in pathogen and insect attack: systemic plant-mediated interactions between pathogens and herbivores of the tomato,Lycopersicon esculentum

Plant-mediated interactions (i.e., induced resistance) between plant pathogens and insect herbivores were investigated using several pests of the cultivated tomato,Lycopersicon esculentum. Single leaflets of tomato leaves were injured by allowing a third-instarHelicoverpa zealarva to feed on the leaflets or by inoculating the leaflets withPseudomonas syringaepv.tomato(the causal agent of bacterial speck in tomato;Pst) or withPhytophthora infestans(the causal agent of late blight). Leaflets on separate plants were sprayed with benzothiadiazole, a chemical inducer of resistance toPst. The effects of these treatments on the resistance of uninoculated or undamaged leaflets to bothPstandH. zeawere then assessed after appropriate periods of time. The levels or activities of several defense-related proteins were determined in parallel. Infection of leaflets byPstdecreased the suitability of uninoculated leaflets of the same leaf for bothH. zeaand forPst. Similarly, feeding byH. zeacaused leaf-systemic increases in resistance to bothH. zeaandPst. Infection of leaflets byP. infestans, in contrast, had no effect on resistance of leaflets toH. zea. Treatment of leaves with benzothiadiazole induced resistance toPstbut improved suitability of leaflets forH. zea. Feeding byH. zeacaused the systemic accumulation of proteinase inhibitor mRNA and the systemic induction of polyphenol oxidase activity; in contrast, treatment with benzothiadiazole and inoculation withP. infestanscaused the systemic accumulation of pathogenesis-related protein mRNA and the systemic induction of peroxidase activity. Inoculation of leaflets withPstcaused the leaf-systemic accumulation of both pathogenesis-related protein and proteinase inhibitor mRNA and the systemic induction of both peroxidase and polyphenol oxidase activity. These results provide clear evidence for reciprocal induced resistance involving certain pathogens and arthropod herbivores of tomato. In addition, these results provide several insights into the integration and coordination of the induced defenses of tomato against multiple pests and suggest that the expression of resistance against some pests may compromise resistance to others.     

Involvement of kaempferol in the defence response of virus infected Arabidopsis thaliana

The roles of several phenolic compounds in plant defence response have been extensively studied, yet little is known about the role of flavonoids in plant-virus interaction. Quantitative and qualitative changes of selected phenolics in Arabidopsis thaliana induced by Cucumber mosaic virus containing satellite RNA (CMVsat) infection were analysed accompanied by plant hormone, chalcone synthase and pathogenesis-related gene expression analysis. Lower leaves of infected plants had a lower concentration of total phenolics compared to control plants. The concentration of kaempferol in upper leaves of all infected plants was significantly lower compared to control plants, while the expression of the chalcone synthase gene in those leaves was in most cases upregulated. All infected plants had a higher concentration of indole-3-acetic acid in lower leaves, which was accompanied with a lower concentration of kaempferol in upper leaves. Our research demonstrates a correlation between kaempferol and indole-3-acetic acid in response to CMVsat infection in Arabidopsis. We demonstrated two different metabolic patterns in infected plants suggesting the activation of two different defence responses. We also propose kaempferol to be an important part of the auxin-dependent defence response which limits systemic movement of CMVsat and that this defence response is activated prior to the well-known salicylic acid dependent defence response. Further research on kaempferol and its role in Arabidopsis-CMVsat interaction will improve our understanding on the role of flavonoids in plant defence.     

Differential necrotic lesion formation in soybean cultivars in response to soybean mosaic virus

In this study, we examined the necrosis phenotype on leaves of two cultivars of soybean (ZheA8901 and Nannong1138-2) that show varying level of resistance to soybean mosaic virus (SMV). The necrotic symptoms seen on inoculated and systemic leaves of soybean cultivar ZheA8901 were reminiscent of programmed cell death (PCD). The cell death phenotypes were evaluated using the TUNEL method, quantification of hydrogen peroxide (H₂O₂) and salicylic acid, callose production, as well as by monitoring expression of defence genes GmPR-1 and GmNPR1. Our results show that SMV inoculation induced PCD on ZheA8901 is associated with rapid increase in H₂O₂, increased SA and callose accumulation and higher defence gene expression.     

棉疫病菌90kD胞外蛋白激发子诱导烟草过敏性反应的研究

 就棉疫病菌90 kD胞外蛋白激发子诱导烟草过敏反应(HR)过程中细胞死亡和防卫反应酶系活性变化及病程相关蛋白PR5的诱导进行研究。结果是,以10 nmol/L激发子溶液注射处理W38烟草叶片,HR枯斑周围5 mm宽组织在UV光下呈现蓝色荧光,对处理部位进行Evans blue染色测定结果是至20 h处理部位细胞全部死亡;激发子可诱导烟草防卫反应中苯丙氨酸解氨酶(PAL)的活性提高;可快速诱导PR5基因的转录。上述结果表明90 kD蛋白激发子可诱发烟草的细胞死亡、苯丙烷代谢和PR基因的表达等多条信号途径。     

The Relationship Between Pathogen-induced Systemic Resistance (ISR) and Multigenic (horizontal) Resistance in Plants

Plants have developed mechanisms to successfully co-exist in the presence of pathogenic organisms. Some interactions between plants and pathogens are based on recognition of specific elicitor molecules from avirulent pathogen races (avr gene products), which is described in the gene-for-gene resistance theory. Another type of resistance, multigenic (horizontal) resistance, is a less well-studied phenomenon that depends upon multiple genes in the plant host. All plants possess resistance mechamisms which can be induced upon pre-treatment of plants with a variety of organisms or compounds. This general phenomenon is known as induced systemic resistance (ISR). At least in some plant species, ISR depends on the timely accumulation of multiple gene products, such as hydrolytic enzymes, peroxidases or other gene products related to plant defences. The pre-treatment of plants with an inducing organism or compound appears to incite the plant to mount an effective defense response upon subsequent encounters with pathogens, converting what would have been a compatible interaction to an incompatible one. Our studies in three plant–pathogen systems clearly document that multigenic-resistant plants constitutively express specific isozymes of hydrolytic enzymes that release cell wall elicitors, which in turn may activate other defense mechanisms. ISR induces constitutive accumulation of these and other gene products prior to challenge. ISR is known to function against multiple organisms, and there is no specificity observed in the accumulation patterns of defense-related gene products when ISR is induced. It is therefore hypothesized that the constitutive accumulation of specific isozymes of hydrolytic enzymes, or other defense related gene products, is an integral part of both multigenic resistance and the phenomenon of ISR. Further, plants in which ISR has been activated appear to move from a latent resistance state to one in which a multigenic, non-specific form of resistance is active.