Histological Characterization of Resistance to Different Root-Knot Nematode Species Related to Phenolics Accumulation in Capsicum annuum

ABSTRACT In the pepper Capsicum annuum CM334, which is used by breeders as a source of resistance to Phytophthora spp. and potyviruses, a resistance gene entirely suppresses reproduction of the root-knot nematode (Meloidogyne spp.). The current study compared the histological responses of this resistant line and a susceptible cultivar to infection with the three most damaging root-knot nematodes: M. arenaria, M. incognita, or M. javanica. Resistance of CM334 to root-knot nematodes was associated with unidentified factors that limited nematode penetration and with post-penetration biochemical responses, including the hypersensitive response, which apparently blocked nematode migration and thereby prevented juvenile development and reproduction. High-performance liquid chromatography analysis suggested that phenolic compounds, especially chlorogenic acid, may be involved in CM334 resistance. The response to infection in the resistant line varied with root-knot nematode species and was correlated with nematode behavior and pathogenicity in the susceptible cultivar: nematode species that quickly reached the vascular cylinder and initiated feeding sites in the susceptible cultivar were quickly recognized in CM334 and stopped in the epidermis or cortex. After comparing our data with those from other resistant pepper lines, we suggest that timing of the resistance response and the mechanism of resistance vary with plant genotype, resistance gene, and root-knot nematode species.     

拮抗细菌对番茄植株抗灰霉病的诱导

通过拮抗细菌多粘类芽孢杆菌W3、Y2和地衣芽孢杆菌W1 0诱导接种和灰霉病菌挑战接种试验 ,明确了 3菌株培养液和去菌液对番茄植株抗灰霉病的诱导作用。 3株拮抗细菌的诱导抗病效果为 2 3 .4%～ 64.5% ,其中W3诱导作用最强。W 3培养液及其去菌液处理后 5d ,诱导效果达最大值 ,且 1 2d后仍有诱抗作用。在 1 0 2 ～ 1 0 1 0 cfu/ml范围内 ,拮抗细菌的诱导活性随浓度增加而增强 ,以≥ 1 0 8cfu/ml的效果最好。拮抗细菌处理叶上部各叶片间诱抗效果无明显差异。     

Induced Systemic Resistance by Fluorescent Pseudomonas spp

ABSTRACT Fluorescent Pseudomonas spp. have been studied for decades for their plant growth-promoting effects through effective suppression of soilborne plant diseases. The modes of action that play a role in disease suppression by these bacteria include siderophore-mediated competition for iron, antibiosis, production of lytic enzymes, and induced systemic resistance (ISR). The involvement of ISR is typically studied in systems in which the Pseudomonas bacteria and the pathogen are inoculated and remain spatially separated on the plant, e.g., the bacteria on the root and the pathogen on the leaf, or by use of split root systems. Since no direct interactions are possible between the two populations, suppression of disease development has to be plant-mediated. In this review, bacterial traits involved in Pseudomonas-mediated ISR will be discussed.     

植物激活蛋白对番茄抗病性的诱导作用

用2μg.ml-1植物激活蛋白处理番茄植株,测定了番茄叶片过氧化物酶、过氧化氢酶活性和过氧化氢的含量。处理番茄4d后,过氧化物酶活性比对照增加117.14%;10min过氧化氢酶活性即迅速上升为对照的2.5倍,12h其活性显著低于对照;过氧化氢含量随过氧化氢酶活性的降低逐渐增加,在36h达最大值134.67μmol.g-1,比对照提高27.5%。通过半定量RT-PCR方法测定了参与蜡质合成基因Cer1表达水平,结果表明,处理番茄2d后Cer1表达量约为对照的2倍。植物激活蛋白处理番茄植株,对灰霉病的防治效果21d时达71.30%。     

Biological Control of the Root-Knot Nematode Meloidogyne javanica by Trichoderma harzianum

ABSTRACT The fungal biocontrol agent, Trichoderma harzianum, was evaluated for its potential to control the root-knot nematode Meloidogyne javanica. In greenhouse experiments, root galling was reduced and top fresh weight increased in nematode-infected tomatoes following soil pretreatment with Trichoderma peat-bran preparations. The use of a proteinase Prb1-transformed line (P-2) that contains multiple copies of this gene improved biocontrol activity in the greenhouse experiments compared with the nontransformed wild-type strain (WT). All the Trichoderma strains showed the ability to colonize M. javanica-separated eggs and second-stage juveniles (J2) in sterile in vitro assays, whereas P-2 also penetrated the egg masses. This protease-transformed line presented the same nematicidal and overall proteolytic activity as the WT in in vitro tests in which concentrated soil extracts from Trichoderma-treated soils immobilized the infective J2. However, the J2 immobilization and proteolytic activities of both P-2 and the WT were higher than those obtained with strain T-203. Characterization of the activity of all Trichoderma strains soil extracts on J2 showed that it was heat resistant and restricted to the low-molecular-weight fraction (less than 3 kDa). It is suggested that improved proteolytic activity of the antagonist may be important for the biological control of the nematodes.     

Role of Salicylic Acid in Systemic Resistance Induced by Pseudomonas spp. Against Pythium aphanidermatum in Cucumber Roots

Pseudomonas corrugata strain 13 and P. aureofaciens strain 63-28, applied to roots, induced systemic resistance against Pythium aphanidermatum in cucumber roots. Salicylic acid (SA) from bacterial culture or plant tissues was quantified by high performance liquid chromatography. Both strains produced SA in King's B broth and also induced cucumber root to accumulate endogenous SA one day after bacterial inoculation. Using a split root system, more SA accumulated in roots treated with bacteria than in distant roots on the opposite side of the root system in the first two days, but this difference disappeared after 3–4 days. SA levels were significantly higher in plants treated with bacteria compared to the split control, from one to five days after bacterization. SA did not inhibit mycelial growth of Pythium aphanidermatum at 100–200µgml^–1 in vitro, but higher levels inhibited mycelial growth. Zoospore germination increased at concentrations of 10–500µgml^–1, but decreased at 1000µgml^–1 compared to lower concentrations. Exogenously applied SA failed to induce local or systemic resistance against a challenge infection by the pathogen in planta. The results of this study show that exogenous applied SA does not induce systemic resistance to cucumber root rot caused by P. aphanidermatum, but endogenous SA accumulation in cucumber roots may be involved in induced systemic resistance.     

内生细菌TF28诱导番茄抗病分子机制研究

为了解内生解淀粉芽胞杆菌Bacillus amyloliquefaciens TF28诱导番茄抗病分子机制,本文采用相对定量RT-PCR方法研究内生细菌TF28对番茄抗病信号传递途径中促分裂原活化蛋白激酶基因MAPK和转录因子WRKY基因表达的影响,采用双向电泳、质谱检测和生物信息学等蛋白质组学技术研究接种菌株TF28后番茄叶片差异蛋白的表达情况。结果表明接种菌株TF28后,番茄叶片中的基因MAPK和WRKY上调表达,基因WRKY表达增幅高于基因MAPK,2个基因均在接种后2 d表达量最高。从接种内生细菌2 d后的番茄叶片中分离到373个蛋白点,与对照相比,有23个蛋白点特异表达,18个蛋白点上调表达,53个蛋白点下调表达,280个蛋白表达量与对照相当,对14个特异表达蛋白进行质谱分析和蛋白数据库检索,9个与抗病相关,3个与品质相关,2个与生长调控相关。     

Induced Systemic Resistance and Promotion of Plant Growth by Bacillus spp

ABSTRACT Elicitation of induced systemic resistance (ISR) by plant-associated bacteria was initially demonstrated using Pseudomonas spp. and other gram-negative bacteria. Several reviews have summarized various aspects of the large volume of literature on Pseudomonas spp. as elicitors of ISR. Fewer published accounts of ISR by Bacillus spp. are available, and we review this literature for the first time. Published results are summarized showing that specific strains of the species B. amyloliquefaciens, B. subtilis, B. pasteurii, B. cereus, B. pumilus, B. mycoides, and B. sphaericus elicit significant reductions in the incidence or severity of various diseases on a diversity of hosts. Elicitation of ISR by these strains has been demonstrated in greenhouse or field trials on tomato, bell pepper, muskmelon, watermelon, sugar beet, tobacco, Arabidopsis sp., cucumber, loblolly pine, and two tropical crops (long cayenne pepper and green kuang futsoi). Protection resulting from ISR elicited by Bacillus spp. has been reported against leaf-spotting fungal and bacterial pathogens, systemic viruses, a crown-rotting fungal pathogen, root-knot nematodes, and a stem-blight fungal pathogen as well as damping-off, blue mold, and late blight diseases. Reductions in populations of three insect vectors have also been noted in the field: striped and spotted cucumber beetles that transmit cucurbit wilt disease and the silver leaf whitefly that transmits Tomato mottle virus. In most cases, Bacillus spp. that elicit ISR also elicit plant growth promotion. Studies on mechanisms indicate that elicitation of ISR by Bacillus spp. is associated with ultrastructural changes in plants during pathogen attack and with cytochemical alterations. Investigations into the signal transduction pathways of elicited plants suggest that Bacillus spp. activate some of the same pathways as Pseudomonas spp. and some additional pathways. For example, ISR elicited by several strains of Bacillus spp. is independent of salicylic acid but dependent on jasmonic acid, ethylene, and the regulatory gene NPR1-results that are in agreement with the model for ISR elicited by Pseudomonas spp. However, in other cases, ISR elicited by Bacillus spp. is dependent on salicylic acid and independent of jasmonic acid and NPR1. In addition, while ISR by Pseudomonas spp. does not lead to accumulation of the defense gene PR1 in plants, in some cases, ISR by Bacillus spp. does. Based on the strains and results summarized in this review, two products for commercial agriculture have been developed, one aimed mainly at plant growth promotion for transplanted vegetables and one, which has received registration from the U.S. Environmental Protection Agency, for disease protection on soybean.     

Pine Wilt Disease Caused by the Pine Wood Nematode: The Induced Resistance of Pine Trees by the Avirulent Isolates of Nematode

Since the beginning of the 20th century, pine trees in Japan have been seriously damaged by the pine wilt disease. This disease is caused by the pine wood nematode, Bursaphelenchus xylophilus, which is transmitted by the Japanese pine sawyer, Monochamus alternatus. The control of disease depends to a large extent on chemicals, but the public is now demanding environmentally friendly control methods. The virulence of B. xylophilus varies very widely. Pre-inoculation of young pine trees in a nursery with avirulent B. xylophilus has induced systemic resistance of trees against a subsequent inoculation with virulent B. xylophilus. This induced resistance was considered a hopeful means for developing a biological control for the disease. The induced resistance by the avirulent nematodes was also expressed in mature pine trees in a forest where the disease was naturally epidemic. However, the effects of induced resistance were not satisfactory for practical biological control. Since the inoculation with higher concentrations of the avirulent B. xylophilus induced the resistance more effectively, the pre-inoculation method will need to be improved to develop the biological control. The induced resistance of pine trees by avirulent B. xylophilus should be one of the candidate biological control methods against pine wilt disease. This induced resistance also provides an experimental system to clarify physiological interactions between the nematodes and pine trees.     

Implication of Systemic Induced Resistance in the Suppression of Fusarium Wilt of Tomato by Pseudomonas fluorescens WCS417r and by Nonpathogenic Fusarium oxysporum Fo47

Fluorescent pseudomonads and nonpathogenic Fusarium oxysporum have been shown to suppress fusarium wilts. This suppression has been related to both microbial antagonism and induced resistance.The aim of the present study was to assess the relative importance of systemic induced resistance in the suppression of fusarium wilt of tomato in commercial-like conditions by a reference strain of each type of microorganism (P. fluorescens WCS417r and nonpathogenic F. oxysporum Fo47). The spatial separation of the pathogen and the biocontrol strains excluded any possible microbial antagonism and implicated the involvement of the systemic induced resistance; whereas the absence of any separation between these microorganisms allowed the expression of both mechanisms. Since systemic induced resistance has often been associated with the synthesis of PR-proteins, their accumulation in tomato plants inoculated with WCS417r or with Fo47 was determined.The analysis of the results indicates that the suppression of fusarium wilt by P. fluorescens WCS417r was ascribed to systemic induced resistance without any detection of the PR-proteins tested (PR-1 and chitinases). In contrast, the suppression achieved by nonpathogenic F. oxysporum Fo47 appeared to be mainly ascribed to microbial antagonism but also to a lesser extent to systemic induced resistance. This induced resistance could be related to the accumulation of PR-1 and chitinases.The possible relationship between the ability of Fo47 to suppress fusarium wilt more efficiently than WCS417r and its ability to show both mechanisms is discussed.     

Salicylic Acid Induces Resistance to Alternaria solani in Hydroponically Grown Tomato

ABSTRACT Alternaria solani is the causal agent of early blight disease in tomato and is responsible for significant economic losses sustained by tomato producers each year. Because salicylic acid (SA) is an important signal molecule that plays a critical role in plant defense against pathogen invasion, we investigated if the exogenous application of SA would activate systemic acquired resistance (SAR) against A. solani in tomato leaves. The addition of 200 muM SA to the root system significantly increased the endogenous SA content of leaves. Free SA levels increased 65-fold over basal levels to 5.85 mug g(-1) fresh weight (FW) after 48 h. This level of SA had no visible phytotoxic effects. Total SA content (free SA + SA-glucose conjugate) increased to 108 mug g(-1) FW after 48 h. Concomitant with elevated SA levels, expression of the tomato pathogenesis-related (PR)-1B gene was strongly induced within 24 h of the addition of 200 muM SA. PR-1B expression was still evident after 48 h; however, PR-1B induction was not observed in plants not receiving SA treatment. Challenge inoculation of SA-treated tomato plants using conidia of A. solani resulted in 83% fewer lesions per leaf and a 77% reduction in blighted leaf area as compared with control plants not receiving SA. Our data indicate that root feeding 200 muM SA to tomato plants can (i) significantly elevate foliar SA levels, (ii) induce PR-1B gene expression, and (iii) activate SAR that is effective against A. solani.     

Heritability Of Rhizobacteria-mediated Induced Systemic Resistance And Basal Resistance In Arabidopsis

Selected strains of non-pathogenic rhizobacteria have the ability to trigger an induced systemic resistance (ISR) response in plants. In Arabidopsis, rhizobacteria-mediated ISR has been extensively studied, using Pseudomonas fluorescens WCS417r as the inducing agent and P. syringae pv. tomato DC3000 (Pst) as the challenging pathogen. To investigate how far expression of ISR depends on the level of basal resistance, 10 different Arabidopsis ecotypes were screened for their potential to express WCS417r-mediated ISR and basal resistance against Pst. Two Arabidopsis ecotypes, RLD and Wassilewskija (Ws), were found to be blocked in their ability to express ISR. This ISR-noninducible phenotype correlated with a relatively low level of basal resistance against Pst. Genetic analysis of crosses between the ISR-inducible ecotypes Columbia (Col) and Landsberg erecta (Ler), on the one hand, and the non-inducible ecotypes RLD and Ws, on the other hand, revealed that ISR inducibility and basal resistance against Pst were inherited as monogenic dominant traits that are genetically linked. Neither ISR inducibility, nor basal resistance against Pst was complemented in the F₁ progeny of a cross between RLD and Ws, indicating that both ecotypes are affected in the same locus. This locus, designated ISR1, was mapped between markers Ein3 and GL1 on chromosome III. Interestingly, ecotypes RLD and Ws also failed to express ISR against the oomycetous pathogen Peronospora parasitica, but they were not affected in their level of basal resistance against this pathogen. Thus, the ISR1 locus controls the expression of ISR against different pathogens but basal resistance only against Pst and not against P. parasitica. Like ecotypes RLD and Ws, ethylene-insensitive mutants showed the isr1 phenotype in that they were unable to express WCS417r-mediated ISR and show enhanced susceptibility to Pst infection. Analysis of ethylene responsiveness of RLD and Ws revealed that both ecotypes exhibit reduced sensitivity to ethylene. Therefore, it is proposed that the Arabidopsis ISR1 locus encodes a component of the ethylene-response pathway that plays an important role in ethylene-dependent resistance mechanisms.     

Expression of the Arabidopsis MCM Gene PROLIFERA During Root-Knot and Cyst Nematode Infection

ABSTRACT Expression of the Arabidopsis thaliana gene PROLIFERA (PRL) was examined during development of root-knot and cyst nematode feeding sites. These obligate plant parasites establish specialized feeding structures in roots that allow them to withdraw nutrients from the host. In the process of establishing feeding sites, nematodes alter cell cycle regulation. PRL is normally expressed specifically in dividing cells at all stages of plant development and was used here as a marker for cell division. PRL expression, reported from a PRL::GUS fusion protein, was detected in nematode feeding sites of both root-knot and cyst nematodes from the earliest stages of infection in both giant cells and syncytia. However, unlike other cell cycle genes, expression of PRL was detected only occasionally in cells surrounding the feeding sites. PRL::GUS activity persisted until late in the infection cycle, past the time when other cell cycle genes are expressed. These data indicate that some aspects of the PRL expression pattern during nematode infection differ from that of other cell cycle genes.     

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.     

Priming as a Mechanism in Induced Systemic Resistance of Plants

Induced systemic resistance is a plant defence state that is associated with an enhanced ability – the so-called priming – to resist pathogen attack by stronger activation of cellular defence responses. So far, however, priming has not been widely appreciated when studying induced plant disease resistance. During the past several years, it has been demonstrated that pre-treatment of cultured parsley cells with inducers of systemic resistance, salicylic acid or a benzothiadiazole, leads to the direct activation of a set of defence-related genes and also primes the cells for stronger elicitation of another set of defence genes including those encoding phenylalanine ammonia-lyase. From these results, it was concluded that the resistance inducers have at least a dual role in plant defence-gene activation. When elucidating whether priming plays a role in induced systemic resistance of Arabidopsis, pre-treating plants with benzothiadiazole was found to augment the subsequent activation of phenylalanine ammonia-lyase genes by Pseudomonas infection, wounding and osmotic stress and also to enhance wound/osmotic stress-induced callose production. The augmentation of phenylalanine ammonia-lyase gene activation or/and callose deposition was not seen in the Arabidopsis non-expresser of pathogenesis-related genes1 mutant which is compromised in induced resistance, while it was present, without benzothiadiazole pre-treatment, in the constitutive expresser of pr genes1 and 5 mutants in which induced resistance is constitutive. Together these studies point to priming as an important cellular mechanism in induced systemic resistance of plants which requires the intact non-expresser of pathogenesis-related genes1 gene.     

Concepts and Direction of Induced Systemic Resistance in Plants and its Application

Resistance to plant disease is often specific and metabolites and receptors contributing to this specificity may have specific structures. However, simple, structurally-unrelated compounds induce systemic resistance in unrelated plants to diverse pathogens including fungi, bacteria and viruses. Both resistance and induced systemic resistance (ISR) are associated with the rapid accumulation of the same structurally unrelated putative defense compounds that have diverse functions. It has been suggested that cultivar (race)-specific resistance is initiated by the specific interaction of a pathogen product (or pathogen induced product) and a plant receptor. However, restricted infection by pathogens can result in ISR and many different compounds can cause ISR. It is thus evident that there are both specific and non-specific routes to the master switch for ISR and there may be more than one master switch. Are reactive oxygen species and free radicals regulating the master switch(es) via both routes? It is also evident there are many switches, other than the master switch. Adding to the complexity of resistance and ISR are the observations that different compounds and pathways may mediate different biochemical resistances. Activation of one of the pathways may antagonize or enhance the activation or effectiveness of another. The review will address these complexities and questions and propose directions of research which require high priority. Factors which encourage and suppress the application of ISR in agriculture will also be addressed.     

低钾逆境下根结线虫感染所诱导的番茄根巨型细胞的组织病理学反应

 Comparison of histopathological response and quantitative measurement of giant cell(GC) induced by Meloidogyne javanica in tomato root were studied under potassium-deficient(0.2 mmol/L K⁺) and replete conditions(control,6.0 mmol/L K⁺).K⁺-deficient stress did not impede the formation and maintenance of GC.The mean number of GC per feeding site as well as the mean diameter of GC did not differ between the treatments.However,the thickness of cell wall including components resulted from the accumulated polysaccharide and the length of cell-wall ingrowth increased 5-25 d after inoculation in K⁺-deficient as compared with K⁺-replete conditions.An increase of cell-wall ingrowth suggested a kind of compensational response to the potassium stress.