Suppressive effect of abscisic acid on systemic acquired resistance in tobacco plants

Recent studies have indicated that the phytohormone abscisic acid (ABA), induced in response to a variety of environmental stresses, plays an important role in modulating diverse plant–pathogen interactions. In Arabidopsis thaliana, we previously clarified that ABA suppressed the induction of systemic acquired resistance (SAR), a plant defense system induced by pathogen infection through salicylic acid (SA) accumulation. We investigated the generality of this suppressive effect by ABA on SAR using tobacco plants. For SAR induction, we used 1,2-benzisothiazole-3(2H)-one 1,1-dioxide (BIT) and benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) that activate upstream and downstream of SA in the SAR signaling pathway, respectively. Wild-type tobacco plants treated with BIT or BTH exhibited enhanced disease resistance against Tobacco mosaic virus (TMV) and tobacco wildfire bacterium, Pseudomonas syringae pv. tabaci (Pst), however, which was suppressed by pretreatment of plants with ABA. Pretreatment with ABA also suppressed the expression of SAR-marker genes by BIT and BTH, indicating that ABA suppressed the induction of SAR. ABA suppressed BTH-induced disease resistance and pathogenesis-related (PR) gene expression in NahG-transgenic plants that are unable to accumulate SA. The accumulation of SA in wild-type plants after BIT treatment was also suppressed by pretreatment with ABA. These data suggest that ABA suppresses both upstream and downstream of SA in the SAR signaling pathway in tobacco.     

Expression of the tobacco β-1,3-glucanase gene, PR-2d, following induction of SAR with Peronospora tabacina

Systemic acquired resistance (SAR) is induced following inoculation of Peronospora tabacina sporangia into the stems of Nicotiana tabacum plants highly susceptible to the pathogen. Previous results have shown that accumulation of acidic β-1,3-glucanases (PR-2's) following induction of SAR by P. tabacina may contribute to resistance to P. tabacina. We showed that up-regulation of the PR-2 gene, PR-2d, following stem inoculation with P. tabacina, is associated with SAR. Studies using plants transformed with GUS constructs containing the full length promoter from PR-2d or promoter deletions, provided evidence that a previously characterized regulatory element that is involved in response to salicylic acid (SA), may be involved in regulation of PR-2d following induction of SAR with P. tabacina. This work provides evidence that regulation of PR-2 genes during P. tabacina-induced SAR may be similar to regulation of these genes during infection of N-gene tobacco by TMV or following exogenous application of SA, and provides further support for the role of SA in regulation of genes during P. tabacina-induced SAR.     

Contribution of both lignin content and sinapyl monomer to disease resistance in tobacco

As a major component of the cell wall, lignin has been suggested to play an important role in the plant defence response to various pathogens. However, how lignin is involved in plant pathogen interaction is still unclear. Here, a series of transgenic tobacco lines were cultivated with a range of differences in lignin content and composition. Evaluation of pathogen resistance in these plants indicated that lower total lignin content aggravated the severity of tobacco black shank and bacterial wilt diseases, while increased sinapyl lignin (S) alleviated the disease symptoms. The regression analysis indicated both lignin content and S lignin were positively correlated with disease resistance. These two factors had additive effects, exhibiting stronger correlation with disease resistance when they were combined. Neither guaiacyl lignin (G) nor S/G ratio showed close correlation with disease resistance. The expression of pathogenesis‐related protein genes PR2 and PR3 was induced after pathogen inoculation. However, the up‐regulation of PR2 and PR3 was not associated with a disease resistance‐induced increase in lignin content. These data collectively suggest that both total lignin content and S lignin are main factors that contribute to the basic defence response in tobacco.     

Plant responses to plant growth-promoting rhizobacteria

Non-pathogenic soilborne microorganisms can promote plant growth, as well as suppress diseases. Plant growth promotion is taken to result from improved nutrient acquisition or hormonal stimulation. Disease suppression can occur through microbial antagonism or induction of resistance in the plant. Several rhizobacterial strains have been shown to act as plant growth-promoting bacteria through both stimulation of growth and induced systemic resistance (ISR), but it is not clear in how far both mechanisms are connected. Induced resistance is manifested as a reduction of the number of diseased plants or in disease severity upon subsequent infection by a pathogen. Such reduced disease susceptibility can be local or systemic, result from developmental or environmental factors and depend on multiple mechanisms. The spectrum of diseases to which PGPR-elicited ISR confers enhanced resistance overlaps partly with that of pathogen-induced systemic acquired resistance (SAR). Both ISR and SAR represent a state of enhanced basal resistance of the plant that depends on the signalling compounds jasmonic acid and salicylic acid, respectively, and pathogens are differentially sensitive to the resistances activated by each of these signalling pathways. Root-colonizing Pseudomonas bacteria have been shown to alter plant gene expression in roots and leaves to different extents, indicative of recognition of one or more bacterial determinants by specific plant receptors. Conversely, plants can alter root exudation and secrete compounds that interfere with quorum sensing (QS) regulation in the bacteria. Such two-way signalling resembles the interaction of root-nodulating Rhizobia with legumes and between mycorrhizal fungi and roots of the majority of plant species. Although ISR-eliciting rhizobacteria can induce typical early defence-related responses in cell suspensions, in plants they do not necessarily activate defence-related gene expression. Instead, they appear to act through priming of effective resistance mechanisms, as reflected by earlier and stronger defence reactions once infection occurs.     

Induced systemic resistance in Trichoderma harzianum T39 biocontrol of Botrytis cinerea

Biocontrol of Botrytis cinerea with Trichoderma spp. is generally believed to result from direct interaction of the biocontrol agent with the pathogen or from a Trichoderma-induced change in environmental conditions that affects B. cinerea development. In this work we provide arguments for the participation of induced plant defence in T. harzianum T39 control of B. cinerea. In tomato, lettuce, pepper, bean and tobacco, T. harzianum T39 application at sites spatially separated from the B. cinerea inoculation resulted in a 25–100%percnt; reduction of grey mould symptoms, caused by a delay or suppression of spreading lesion formation. Given the spatial separation of both micro-organisms, this effect was attributed to the induction of systemic resistance by T. harzianum T39. The observation that in bean the effect of T. harzianum T39 was similar to that of the rhizobacterium Pseudomonas aeruginosa KMPCH, a reference strain for the induction of systemic resistance, confirmed this hypothesis. Since B. cinerea control on tobacco leaves sprayed with T. harzianum T39 was similar to the control on leaves from T. harzianum T39 soil-treated plants, induction of plant defence might also participate in biocontrol on treated leaves.     

Ethylene signalling is essential for the resistance of Nicotiana attenuata against Alternaria alternata and phytoalexin scopoletin biosynthesis

The phytohormone ethylene plays an important role in plant defence responses to pathogen attack. When infected by the necrotrophic fungal pathogen Alternaria alternata (tobacco pathotype), which causes severe diseases in Nicotiana species, the wild tobacco plant Nicotiana attenuata accumulates a high amount of the jasmonate (JA)‐dependent phytoalexin scopoletin to defend itself against this fungal pathogen. However, it is still not known whether ethylene signalling is also involved in scopoletin biosynthesis and the resistance of N. attenuata. After infection, ethylene biosynthetic genes were highly elicited. Furthermore, plants strongly impaired in ethylene biosynthesis or perception had dramatically decreased scopoletin levels, and these plants became more susceptible to the fungus, while A. alternata‐elicited JA levels were increased, indicating that the decreased defence responses were not due to lower JA levels. Thus, it is concluded that after infection, ethylene signalling is activated together with JA signalling in N. attenuata plants and this subsequently regulates scopoletin biosynthesis and plant resistance.     

Biochemical responses associated with common bean defence against Sclerotinia sclerotiorum

The aim of this study was to investigate changes in defence compounds of common bean cultivars with different levels of resistance to the fungus Sclerotinia sclerotiorum and determine the relation of the compounds to pathogen tolerance. The lines were inoculated with the pathogen and assessed for enzymatic and non-enzymatic parameters related to plant defence: peroxidases (POX), polyphenol oxidases (PPO), catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX), total soluble phenol and lignin contents. Stem tissue samples were collected from two regions of the plant for biochemical analyses. Stem tissue samples were collected from two regions of the plant for biochemical analyses. In the position one, 5 cm of the stem was collected from the region with necrosis caused by the pathogen, and in the position two, 5 cm of the stem was collected from the end of the position one at the times of 12, 24, 48, 72, 96 and 120 h after inoculation (HAI). Greater lignin and total soluble phenol contents and greater induction of POX and SOD activity in inoculated plants in the region near the inoculation (position one) indicate local activation with later signalling for activation of defence mechanisms in other regions of the plant. The genotype with a greater level of resistance was superior to the susceptible one in regard to lignin production and the activities of POX, APX and SOD defence enzymes. These results suggest that a combination of these defence responses in common bean may contribute to greater plant resistance to the pathogen and that these enzymes have potential use in selection of common bean genotypes.     

Acquired resistance triggered by elicitins in tobacco and other plants

Elicitins are a family of proteins excreted byPhytophthora spp. They exhibit high sequence homology but large net charge differences. They induce necrosis in tobacco plants which then become resistant to the tobacco pathogenPhytophthora parasitica var.nicotianae. In stem-treated plants, resistance was not restricted to the site of elicitin application, but could be demonstrated by petiole inoculation at all levels on the stem. Resistance was already maximum after two days and lasted for at least two weeks. It was effective not only towardsP. p. var.nicotianae infection, but also against the unrelated pathogenSclerotinia sclerotiorum. In contrast to dichloroisonicotinic acid, an artificial inducer of systemic acquired resistance, which was increasingly effective with doses ranging from 0.25 to 5mole per plant, the basic elicitin cryptogein exhibited a threshold effect, inducing near total resistance and extensive leaf necrosis above 0.1 nmole per plant. Between 1 and 5 nmole, acidic elicitins (capsicein and parasiticein) protected tobacco plants with hardly any necrotic symptom. Elicitins exhibited similar effects in various tobacco cultivars andNicotiana species, although with quantitative differences, but induced neither necrosis nor protection in other SolanaceÆ (tomato, petunia and pepper). Among 24 additional species tested belonging to 18 botanical families, only some BrassicaceÆ, noticeably rape, exhibited symptoms in response to elicitins, in a cultivar-specific manner. Elicitins appear to be natural specific triggers for systemic acquired resistance and provide a tool for unraveling the mechanisms leading to its establishment.Abbreviations AR acquired resistance - HR hypersensitive response - INA 2,6-dichloroisonicotinic acid - Ppn Phytophthora parasitica var.nicotianae - SAR systemic acquired resistance     

Harpin-elicited hypersensitive cell death and pathogen resistance require the NDR1 and EDS1 genes

Plants sprayed with harpin, a bacterial protein that induces hypersensitive cell death (HCD), develop systemic acquired resistance (SAR) without macroscopic necrosis. HCD sometimes accompanies the development of resistance conferred by resistance (R) genes. In Arabidopsis, some R genes require one or both of the signalling components NDR1 and EDS1 for function. This study addresses whether HCD, NDR1 and EDS1 are required for induction of SAR by harpin. When Arabidopsis and tobacco leaves were sprayed with harpin, microscopic hypersensitive response (micro-HR) lesions developed. Systemic expression of PR genes and the development of resistance were accompanied by micro-HR, except in the ndr1-1 mutant, in which harpin induced micro-HR without the development of resistance or expression of the PR-1 gene. Cell death and resistance did not occur following treatment with harpin in plants that could not accumulate salicylic acid. Harpin also failed to induce resistance in Arabidopsis eds1-1 mutants. Therefore, harpin-induced resistance seems to develop concomitantly with cell death and resistance requires NDR1 and EDS1.     

Enhancement of natural disease resistance in potatoes by chemicals

The mechanism involved in systemic acquired resistance (SAR) can be non-specifically induced in susceptible plants. In response to pathogens, plants' natural defence mechanisms include the production of lignin and phytoalexins and the induction of plant enzymes. The aim of this research was to study the induction of SAR mediated by the chemical activator DL-3-aminobutyric acid (BABA) and the fungicide fosetyl-aluminium in potato cultivars with different levels of resistance against Phytophthora infestans (Mont) de Bary. To study the chemical induction of the resistance, the foliage of several potato cultivars was sprayed with BABA, fosetyl-aluminium or water (as a control treatment). After 3 days the foliage was inoculated with P. infestans. Seven days after inoculation, development of disease symptoms in the foliage was assessed. In postharvest tuber samples, evidence for enhancement of the defence response was evaluated by measuring the protein content of several hydrolytic enzymes as well as the phenol and phytoalexin content. The highest level of protection against late blight was observed when the chemicals were applied at early stages of crop development. An increase in resistance to late blight was also detected in tubers after harvest. There was also an increase in the protein level of beta-1,3-glucanase and aspartic protease as well as in the phenol and phytoalexin content of potato tuber discs obtained from postharvest tubers of treated plants. Thus the protective effect seemed to persist throughout the whole crop cycle. This treatment may offer the possibility of controlling both foliage and tuber blight and could have a major impact in reducing over-winter survival of P. infestans in tubers.     

Colonization of <Emphasis Type="Italic">Arabidopsis</Emphasis> roots by <Emphasis Type="Italic">Trichoderma atroviride</Emphasis> promotes growth and enhances systemic disease resistance through jasmonic acid/ethylene and salicylic acid pathways

Trichoderma spp. are common soil fungi used as biocontrol agents due to their capacity to produce antibiotics, induce systemic resistance in plants and parasitize phytopathogenic fungi of major agricultural importance. The present study investigated whether colonization of Arabidopsis thaliana seedlings by Trichoderma atroviride affected plant growth and development. Here it is shown that T. atroviride promotes growth in Arabidopsis. Moreover, T. atroviride produced indole compounds in liquid cultures. These results suggest that indoleacetic acid-related indoles (IAA-related indoles) produced by T. atroviride may have a stimulatory effect on plant growth. In addition, whether colonization of Arabidopsis roots by T. atroviride can induce systemic protection against foliar pathogens was tested. Arabidopsis roots inoculation with T. atroviride provided systemic protection to the leaves inoculated with bacterial and fungal pathogens. To investigate the possible pathway involved in the systemic resistance induced by T. atroviride, the expression profile of salicylic acid, jasmonic acid/ethylene, oxidative burst and camalexin related genes was assessed in Arabidopsis. T. atroviride induced an overlapped expression of defence-related genes of SA and JA/ET pathways, and of the gene involved in the synthesis of the antimicrobial phytoalexin, camalexin, both locally and systemically. This is the first report where colonization of Arabidopsis roots by T. atroviride induces the expression of SA and JA/ET pathways simultaneously to confer resistance against hemibiotrophic and necrotrophic phytopathogens. The beneficial effects induced by the inoculation of Arabidopsis roots with T. atroviride and the induction of the plant defence system suggest a molecular dialogue between these organisms.     

Induction of systemic resistance byPseudomonas fluorescens in radish cultivars differing in susceptibility to fusarium wilt,using a novel bioassay

Pseudomonas fluorescens-mediated induction of systemic resistance in radish against fusarium wilt (Fusarium oxysporum f. sp.raphani) was studied in a newly developed bioassay using a rockwool system. In this bioassay the pathogen and bacterium were confirmed to be confined to spatially separate locations on the plant root, throughout the experiment. Pathogen inoculum obtained by mixing peat with microconidia and subsequent incubation for four days at 22 °C, yielded a better percentage of diseased plants than a microconidial suspension drench, an injection of a microconidial suspension into the hypocotyl, or a talcum inoculum.Pseudomonas fluorescens strain WCS374 applied in talcum or peat, but not as a suspension drench, induced systemic resistance. A minimal initial bacterial inoculum density of 10⁵ CFU WCS374 root^–1 was required to significantly reduce the percentage diseased plants. At least one day was necessary between bacterization of strain WCS374 in talcum on the root tips and inoculation of the pathogen in peat on the root base, for an optimal induction of systemic resistance. Strain WCS374 induced systemic resistance in six radish cultivars differing in their susceptibility toF. oxysporum f. sp.raphani. Significant suppression of disease by bacterial treatments was generally observed when disease incidence in the control treatment, depending on pathogen inoculum density, ranged between approximately 40 to 80%. Strains WCS374 and WCS417 ofPseudomonas fluorescens induced systemic resistance against fusarium wilt, whereasP. putida WCS358 did not. This suggests that the induction of systemic resistance byPseudomonas spp. is dependent on strain-specific traits.Abbreviations CFU colony forming units - IFC immunofluorescence colony-staining - ISR induced systemic resistance - PBS phosphate buffered saline - SAR systemic acquired resistance     

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 .     

Functional imaging of biophoton responses of plants to fungal infection

Each living cell of a plant produces photons in certain conditions. Under normal physiological conditions, cell photon emission is stationary and minimal. Disturbance in the oxidative homeostasis by biotic stress is manifested by increased ‘biophoton’ production. Such biophoton responses of plants may be used as an integral indicator of the degree of oxidative homeostasis misbalance. Our results demonstrate that biophoton generation has been much higher in a resistant potato variety than in a susceptible one till 10 h after Phytophthora infestans inoculation. In contrast, ultra-weak luminescence from detached susceptible potato and moderately resistant pelargonium leaves increased from 1–4 to 4–5 days after inoculation with Phytophthora infestans or Botrytis cinerea, respectively. Pre-treatment of susceptible potato leaves with a defence inducer, arachidonic acid, resulted in a transient burst of light in response to P. infestans lasting for 30–45 h post inoculation (hpi). This study presents the potential adaptation of functional imaging of ultra-weak luminescence to monitor time-dependent free radical processes during disease development and its application to draw conclusions on plant resistance to pathogens of different lifestyle. Moreover, it has been shown that imaging of temporal biophoton generation from potato leaves treated with arachidonic acid might be a helpful marker in mapping oxidative changes leading to systemic acquired resistance (SAR).     

壳寡糖诱导对烟草体内TMV-CP基因表达的抑制作用

 采用实时荧光定量PCR方法, 研究了壳寡糖诱导对烟草体内TMV-CP基因表达量的影响。结果表明, 壳寡糖诱导后烟草表达了对TMV侵染的系统获得抗病性, 植株显症推迟4~7 d, 症状减轻, 平均严重度比对照降低了82.9%。同时烟草叶片中CP基因的表达量显著降低。壳寡糖诱导处理的植株在接种病毒时(0 h), CP基因的表达量(拷贝数/2μL)为0.918, 接种后168 h增加到167.730。而在不诱导对照植株内, CP基因的表达量在0 h为1.218, 接种后168 h猛增到648.623。换言之, 不处理对照的表达量在此期间增长了532.53倍, 而壳寡糖诱导处理植株中仅增长了182.71倍。试验证明壳寡糖诱导的系统获得抗病性强烈抑制了烟草体内TMV-CP基因的表达。     

Control of plant diseases by natural products: Allicin from garlic as a case study

This review aims to increase awareness of the potential for developing plant protection strategies based on natural products. Selected examples of commercial successes are given and recent data from our own laboratory using allicin from garlic are presented. The volatile antimicrobial substance allicin (diallylthiosulphinate) is produced in garlic when the tissues are damaged and the substrate alliin (S-allyl-l-cysteine sulphoxide) mixes with the enzyme alliin-lyase (E.C.4.4.1.4). Allicin is readily membrane-permeable and undergoes thiol-disulphide exchange reactions with free thiol groups in proteins. It is thought that these properties are the basis of its antimicrobial action. We tested the effectiveness of garlic juice against a range of plant pathogenic bacteria, fungi and oomycetes in vitro. Allicin effectively controlled seed-borne Alternaria spp. in carrot, Phytophthora leaf blight of tomato and tuber blight of potato as well as Magnaporthe on rice and downy mildew of Arabidopsis. In Arabidopsis the reduction in disease was apparently due to a direct action against the pathogen since no accumulation of salicylic acid (a marker for systemic acquired resistance, SAR) was observed after treatment with garlic extract. We see a potential for developing preparations from garlic for use in organic farming, e.g. for reducing the pathogen inoculum potential in planting material such as seeds and tubers. We have tested various encapsulation formulations in comparison to direct treatment. Note added in proof: The complete chemical synthesis of azadirachtin has now been achieved. Nature (2007) 448: 630–631     

Defence Responses Against TNV Infection Induced by Galactoglucomannan-derived Oligosaccharides in Cucumber Cells

Galactoglucomannan-derived oligosaccharides (GGMOs) showing biological activity in growth, morphogenesis and cell viability were tested in a host pathogen interaction. As a model system, cucumber (Cucumis sativus L. cv. Laura) reacting hypersensitively to tobacco necrosis virus (TNV) was used. The defence reactions were dependent on the degree of polymerisation and concentration of oligosaccharides, as well as on the time of application of virus to plant cotyledons. Disease symptoms were inhibited by 60–75%. The average number of lesions per cotyledon was significantly decreased when oligosaccharides were used simultaneously or 24h prior to virus inoculation. Significant changes in peroxidase, beta-glucanase and chitinase activities accompanied the defence reaction. It can be concluded that oligosaccharides derived from spruce galactoglucomannan induce non-specific resistance to local viral infection in plants. GGMOs probably act as inhibitors of the virus infection, rather than inhibitors of direct virus multiplication.