Endophytic bacteria isolated from wild jojoba [<Emphasis Type="Italic">Simmondsia chinensis</Emphasis> L. (Schneider)] roots improve in vitro propagation

Endophytic bacteria promote plant growth, reduce stress caused by biotic and abiotic factors, and can trigger active defense reactions in plants. This study aimed to evaluate enzyme activity of in vitro jojoba (Simmondsia chinensis) plants inoculated with endophytic bacteria. In vitro shoots of female and male plants were inoculated with strains of Azospirillum brasilense (Cd), Methylobacterium aminovorans (JRR11), Rhodococcus pyridinivorans (JRR22) or co-inoculated with a mixture of JRR11?+?JRR22. A total of 10 treatments were performed to evaluate shoot and root length; changes in key enzymes involved in plant defense (superoxide dismutase, catalase, peroxidase, ascorbate peroxidase and phenylalanine ammonia lyase) after post-inoculation (45 days). All endophytic bacteria strains used promoted plant growth and rhizogenesis. Differences were found in enzyme activity between female and male plants. The plants inoculated with JRR22 strain, showed the highest enzyme activity suggesting an induced systemic response and a potential increase in plant resistance to pathogen attack.     

丛枝菌根真菌与高羊茅协同修复镉污染土壤

本研究采用温室盆栽试验,利用丛枝菌根（AM）真菌摩西管柄囊霉Funneliformis mosseae进行接种试验,研究了在Cd胁迫下（0、5、15和30mg/kg）接种AM真菌对高羊茅Festuca elata ‘Crossfire II’的生物量、防御酶活性、磷和镉（Cd）含量的影响。结果表明,随着Cd浓度的增加,高羊茅的菌根侵染率和菌根相对依赖性有所增加。接种AM真菌改善了磷从植株根系向地上部的转运,有助于植株在地上部积累更多的磷。此外,AM真菌和Cd胁迫对高羊茅植株抗氧化酶活性都有显著影响,在镉胁迫下,与未接种植株相比,接种AM真菌显著提高了植株的过氧化氢酶活性,而显著降低了植株的丙二醛含量。与未接种植株相比,接种摩西管柄囊霉显著提高了寄主植物对Cd的富集能力,有利于重金属在根部的积累,同时降低了地上部的Cd含量。本研究表明,高羊茅-丛枝菌根共生体在Cd污染土壤的修复中具有潜在应用价值。     

Changes in specific activities of peroxidase, chitinase and phenylalanine ammonia-lyase and phenolic content in cucumber leaves inoculated with Podosphaera fusca, the causal agent of powdery mildew

In this investigation, the effects of powdery mildew disease [caused by Podosphaera fusca (syn. Sphaerotheca fuliginea)] on specific activities of several defense-related enzymes and phenolic content were studied in cucumber leaves. Spore suspension of the fungus was sprayed on cucumber (cv. Super Dominus) plants in greenhouse and leaves from both inoculated and non-inoculated control plants were sampled at 0, 24, 48, 72 and 144 hours after inoculation (HAI). Spore germination and tissue colonization of P. fusca were microscopically studied on the inoculated surface of leaf samples. Further, Phenolic content (PHE) and specific activities of peroxidase (POX), chitinase (CHI) and phenylalanine ammonia-lyase (PAL) were spectrophotometrically measured in leaf extracts. Time-course of disease progress on the leaf surface showed that maximum spore germination occurred within 24 HAI and host penetration and disease development process began during 24-48 HAI. Evaluation of enzyme activities showed that POX specific activity in inoculated plants significantly increased at 72 HAI onwards and reached 2.5 times of that of control at 144 HAI. CHI specific activity showed a transient reduction in inoculated plants between 48-72 HAI and thereafter increased significantly in relation to control. PAL specific activity in inoculated plants was not significantly different from that of control. PHE in inoculated plants showed a significant increase compared to control at 48 HAI and thereafter. Comparison of time-course of disease progress with changes in enzyme activities indicated that POX activity had an increasing trend during disease progress whereas CHI activity showed a transient decrease at the early stages and then increased during the later stages of infection: PAL activity did not show any changes during the infection and PHE increased at the early stages of infection process and remained constant at rest of the time.     

<Emphasis Type="Italic">Piriformospora indica</Emphasis> and <Emphasis Type="Italic">Sebacina vermifera</Emphasis> increase growth performance at the expense of herbivore resistance in <Emphasis Type="Italic">Nicotiana attenuata</Emphasis>

A Sebacinales species was recovered from a clone library made from a pooled rhizosphere sample of Nicotiana attenuata plants from 14 native populations. Axenic cultures of the related species, Piriformospora indica and Sebacina vermifera, were used to examine their effects on plant performance. Inoculation of N. attenuata seeds with either fungus species stimulated seed germination and increased growth and stalk elongation. S. vermifera inoculated plants flowered earlier, produced more flowers and matured more seed capsules than did non-inoculated plants. Jasmonate treatment during rosette-stage growth, which slows growth and elicits herbivore resistance traits, erased differences in vegetative, but not reproductive performance resulting from S. vermifera inoculation. Total nitrogen and phosphorous contents did not differ between inoculated and control plants, suggesting that the performance benefits of fungal inoculation did not result from improvements in nutritional status. Since the expression of trypsin proteinase inhibitors (TPI), defensive proteins which confer resistance to attack from Manduca sexta larvae, incur significant growth and fitness costs for the plant, we examined the effect of S. vermifera inoculation on herbivore resistance and TPI activity. After 10 days of feeding on S. vermifera-inoculated plants, larval mass was 46% higher and TPI activity was 48% lower than that on non-inoculated plants. These results suggest that Sebacina spp. may interfere with defense signaling and allow plants to increase growth rates at the expense of herbivore resistance mediated by TPIs.     

Characterization of a mitogen-activated protein kinase gene from cucumber required for trichoderma-conferred plant resistance

The fungal biocontrol agent Trichoderma asperellum has been recently shown to induce systemic resistance in plants through a mechanism that employs jasmonic acid and ethylene signal transduction pathways. Mitogen-activated protein kinase (MAPK) proteins have been implicated in the signal transduction of a wide variety of plant stress responses. Here we report the identification and characterization of a Trichoderma-induced MAPK (TIPK) gene function in cucumber (Cucumis sativus). Similar to its homologs, wound-induced protein kinase, MPK3, and MPK3a, TIPK is also induced by wounding. Normally, preinoculation of roots with Trichoderma activates plant defense mechanisms, which result in resistance to the leaf pathogen Pseudomonas syringae pv lachrymans. We used a unique attenuated virus vector, Zucchini yellow mosaic virus (ZYMV-AGII), to overexpress TIPK protein and antisense (AS) RNA. Plants overexpressing TIPK were more resistant to pathogenic bacterial attack than control plants, even in the absence of Trichoderma preinoculation. On the other hand, plants expressing TIPK-AS revealed increased sensitivity to pathogen attack. Moreover, Trichoderma preinoculation could not protect these AS plants against subsequent pathogen attack. We therefore demonstrate that Trichoderma exerts its protective effect on plants through activation of the TIPK gene, a MAPK that is involved in signal transduction pathways of defense responses.     

Ectopic expression of an Arabidopsis calmodulin-like domain protein kinase-enhanced NADPH oxidase activity and oxidative burst in tomato protoplasts

Among plant defense responses to pathogen attack, the release of active oxygen species (AOS), termed the oxidative burst, may affect the attacking pathogen and the host plant cells at the infection site, thereby limiting the spread of the pathogen. Plasma membrane-associated NADPH oxidase represents a key enzyme in mediating the oxidative burst. The mechanisms of NADPH oxidase activation, however, remains unclear. Ectopic expression of AK1-6H, an Arabidopsis calmodulin-like domain protein kinase (CDPK) in tomato protoplasts enhanced plasma membrane-associated NADPH oxidase activity. Arabidopsis protein phosphatase 2A abolished this enhancement, whereas Arabidopsis dual-specificity protein tyrosine phosphatase 1 or maize protein phosphatase 1 had no effect tMEK2MUT, a constitutively activated, mitogen-activated protein kinase kinase from tomato, did not enhance NADPH oxidase activity when overexpressed. In a cell-free system, AK1-6H moderately stimulated the NADPH oxidase activity on plasma membrane. AK1-6H, but not tMEK2MUT, also enhanced production of AOS in intact protoplasts. Our results show that ectopic expression of a heterologous CDPK can enhance NADPH oxidase activity and stimulate an oxidative burst in tomato protoplasts.     

Down-regulation of free riboflavin content induces hydrogen peroxide and a pathogen defense in Arabidopsis

Riboflavin mediates many bioprocesses associated with the generation of hydrogen peroxide (H?O?), a cellular signal that regulates defense responses in plants. Although plants can synthesize riboflavin, the levels vary widely in different organs and during different stages of development, indicating that changes in riboflavin levels may have physiological effects. Here, we show that changing riboflavin content affects H?O? accumulation and a pathogen defense in Arabidopsis thaliana. Leaf content of free riboflavin was modulated by ectopic expression of the turtle gene encoding riboflavin-binding protein (RfBP). The RfBP-expressing Arabidopsis thaliana (REAT) plants produced the RfBP protein that possessed riboflavin-binding activity. Compared with the wild-type plant, several tested REAT lines had >70% less flavins of free form. This change accompanied an elevation in the level of H?O? and an enhancement of plant resistance to a bacterial pathogen. All the observed REAT characters were eliminated due to RfBP silencing (RfBPi) under REAT background. When an H?O? scavenger was applied, H?O? level declined in all the plants, and REAT no longer exhibited the phenotype of resistance enhancement. However, treatment with an NADPH oxidase inhibitor diminished H?O? content and pathogen defense in wild-type and RfBPi but not in REAT. Our results suggest that the intrinsic down-regulation of free flavins is responsible for NADPH oxidase-independent H?O? accumulation and the pathogen defense.     

Brassinosteroid-Mediated Stress Responses

Brassinosteroids (BRs) are a group of naturally occurring plant steroidal compounds with wide-ranging biological activity that offer the unique possibility of increasing crop yields through both changing plant metabolism and protecting plants from environmental stresses. In recent years, genetic and biochemical studies have established an essential role for BRs in plant development, and on this basis BRs have been given the stature of a phytohormone. A remarkable feature of BRs is their potential to increase resistance in plants to a wide spectrum of stresses, such as low and high temperatures, drought, high salt, and pathogen attack. Despite this, only a few studies aimed at understanding the mechanism by which BRs promote stress resistance have been undertaken. Studies of the BR signaling pathway and BR gene-regulating properties indicate that there is cross-talk between BRs and other hormones, including those with established roles in plant defense responses such as abscisic acid, jasmonic acid, and ethylene. Recent studies aimed at understanding how BRs modulate stress responses suggest that complex molecular changes underlie BR-induced stress tolerance in plants. Analyses of these changes should generate exciting results in the future and clarify whether the ability of BRs to increase plant resistance to a range of stresses lies in the complex interactions of BRs with other hormones. Future studies should also elucidate if BRI1, an essential component of the BR receptor, directly participates in stress response signaling through interactions with ligands and proteins involved in plant defense responses.     

Chemical defense,mycorrhizal colonization and growth responses in <Emphasis Type="Italic">Plantago lanceolata</Emphasis> L.

Allelochemicals defend plants against herbivore and pathogen attack aboveground and belowground. Whether such plant defenses incur ecological costs by reducing benefits from plant mutualistic symbionts is largely unknown. We explored a potential trade-off between inherent plant chemical defense and belowground mutualism with arbuscular mycorrhizal fungi (AMF) in Plantago lanceolata L., using plant genotypes from lines selected for low and high constitutive levels of the iridoid glycosides (IG) aucubin and catalpol. As selection was based on IG concentrations in leaves, we first examined whether IG concentrations covaried in roots. Root and leaf IG concentrations were strongly positively correlated among genotypes, indicating genetic interdependence of leaf and root defense. We then found that root AMF arbuscule colonization was negatively correlated with root aucubin concentration. This negative correlation was observed both in plants grown with monocultures of Glomus intraradices and in plants colonized from whole-field soil inoculum. Overall, AMF did not affect total biomass of plants; an enhancement of initial shoot biomass was offset by a lower root biomass and reduced regrowth after defoliation. Although the precise effects of AMF on plant biomass varied among genotypes, plants with high IG levels and low AMF arbuscule colonization in roots did not produce less biomass than plants with low IG and high AMF arbuscule colonization. Therefore, although an apparent trade-off was observed between high root chemical defense and AMF arbuscule colonization, this did not negatively affect the growth responses of the plants to AMF. Interestingly, AMF induced an increase in root aucubin concentration in the high root IG genotype of P. lanceolata. We conclude that AMF does not necessarily stimulate plant growth, that direct plant defense by secondary metabolites does not necessarily reduce potential benefits from AMF, and that AMF can enhance concentrations of root chemical defenses, but that these responses are plant genotype-dependent.     

Silencing NOA1 elevates herbivory-induced jasmonic acid accumulation and compromises most of the carbon-based defense metabolites in Nicotiana attenuata(F)

Nitric oxide-associated protein 1 (NOA1) is involved in various abiotic stress responses and is required for plant resistance to pathogen infections. However, the role of NOA1 in plant-herbivore interactions has not been explored. We created NOA1-silenced Nicotiana attenuata plants (irNaNOA1). Compared with wild-type (WT) plants, irNaNOA1 plants had highly decreased photosynthesis rates. We further examined various traits important for plant defense against its specialist herbivore Manduca sexta by treating WT and irNaNOA1 plants with mechanical wounding and M. sexta oral secretions (OS). NOA1-silenced plants showed elevated levels of herbivory-induced jasmonic acid (JA), but decreased JA-isoleucine conjugate (JA-Ile) levels. The decreased JA-Ile levels did not result from compromised JAR (jasmonic acid resistant) activity in irNOA1 plants. Moreover, nitrogen-rich defensive compounds, nicotine and trypsin proteinase inhibitors, did not differ between WT and irNaNOA1 plants. In contrast, concentrations of most carbon-based defensive compounds were lower in these plants than in WT plants, although the levels of chlorogenic acid were not changed. Therefore, silencing NOA1 alters the allocation of carbon resources within the phenylpropanoid pathway. These data suggest the involvement of NOA1 in N. attenuata's defense against M. sexta attack, and highlight its role in photosynthesis, and biosynthesis of jasmonates and secondary metabolites.     

Plant-mediated interactions between insects and a fungal plant pathogen and the role of plant chemical responses to infection

Diverse organisms simultaneously exploit plants in nature, but most studies do not examine multiple types of exploiters like phytophagous insects and fungal, bacterial, and viral plant pathogens. This study examined patterns of induction of antipathogenic peroxidase enzymes and phenolics after infection by the cucurbit scab fungus, Cladosporium cucumerinum, and then determined if induction mediated ecological effects on Colletotrichum orbiculare, another fungal pathogen, and two insect herbivores, spotted cucumber beetles, and melon aphids. Peroxidase induction occurred in inoculated, `local,' symptom-bearing leaves 3 days after inoculation, and in `systemic,' symptom-free leaves on the same plants 1 day later. Phenolics were elevated in systemic but not in local leaves 3 days after inoculation. Detached systemic leaves from plants inoculated with C. cucumerinum developed significantly fewer and smaller lesions after challenge with C. orbiculare. Spotted cucumber beetles did not show consistently significant preferences for infected versus control leaf disks in comparisons using local or systemic leaves, but trends differed significantly between leaf positions. In no-choice tests, beetles removed more leaf area from local but not from systemic infected leaves compared to control leaves, and melon aphid reproduction was enhanced on local infected leaves. In the field, cucumber beetle and melon aphid densities did not differ between infected and control plants. Antipathogenic plant chemical responses did not predict reduced herbivory by insects. Other changes in metabolism may explain the positive direction and spatially dependent nature of plant-mediated interactions between pathogens and insects in this system. Received: 28 September 1997 / Accepted: 9 February 1998     

Enhancement of in vitro growth and resistance to gray mould of Vitis vinifera co-cultured with plant growth-promoting rhizobacteria

The potential of a plant growth-promoting rhizobacterium, Pseudomonas sp. (strain PsJN), to stimulate the growth and enhancement of the resistance of grapevine (Vitis vinifera L.) transplants to gray mould caused by Botrytis cinerea has been investigated. In vitro inoculation of grapevine plantlets induced a significant plant growth promotion which made them more hardy and vigorous when compared to non-inoculated plantlets. This ability increased upon transplanting. When grown together with B. cinerea, the causal agent of gray mould, significant differences of aggressiveness were observed between the inoculated and non-inoculated plants. The presence of bacteria was accompanied by an induction of plant resistance to the pathogen. The beneficial effect from this plant-microbe association is being postulated.     

The effects of wilt symptom development and peroxidase induction on interactions between vascular wilt bacteria and cucumber beetles

Infection of cucumber (Cucumis sativus L.) with the bacterial pathogen Erwinia tracheiphila E. F. Smith causes vascular wilt disease in leaves, which may alter the suitability of the host plant for insects and other pathogens. In this study, densities of spotted (Diabrotica undecimpunctata howardi Barber) and striped (Acalymma vittata (Fab.) cucumber beetles (Coleoptera: Chrysomelidae) were higher on wilted leaves of mature and seedling field plants inoculated with E. tracheiphila. Bacterial infection or feeding by D. undecimpunctata howardii beetles increased total peroxidase enzyme activity in inoculated or infested leaves of greenhouse seedlings, but only beetle feeding induced higher activities in untreated systemic leaves on the same plants. Neither bacterial infection nor beetle infestation led to the development of systemic acquired resistance (SAR) to the fungal pathogen Colletotrichum orbiculare (Berk & Mont.) Arx. Susceptibility to this fungus was greater on E. tracheiphila-infected plants than on controls. The positive association between leaf wilt symptom development and beetle occurrence thus occurs in the presence of an oxidative but not anti-pathogenic response induced by both the insect and the pathogen.     

Molecular communications between plant heat shock responses and disease resistance

As sessile, plants are continuously exposed to potential dangers including various abiotic stresses and pathogen attack. Although most studies focus on plant responses under an ideal condition to a specific stimulus, plants in nature must cope with a variety of stimuli at the same time. This indicates that it is critical for plants to fine-control distinct signaling pathways temporally and spatially for simultaneous and effective responses to various stresses. Global warming is currently a big issue threatening the future of humans. Reponses to high temperature affect many physiological processes in plants including growth and disease resistance, resulting in decrease of crop yield. Although plant heat stress and defense responses share important mediators such as calcium ions and heat shock proteins, it is thought that high temperature generally suppresses plant immunity. We therefore specifically discuss on interactions between plant heat and defense responses in this review hopefully for an integrated understanding of these responses in plants.     

Diverse RNA viruses elicit the expression of common sets of genes in susceptible Arabidopsis thaliana plants

Systemic infections of plants by viruses require that viruses modify host cells in order to facilitate infections. These modifications include induction of host factors required for replication, propagation and movement, and suppression of host defense responses, which are likely to be associated with changes in host gene expression. Past studies of the effects of viral infection on gene expression in susceptible hosts have been limited to only a handful of genes. To gain broader insight into the responses elicited by viruses in susceptible hosts, high-density oligonucleotide probe microarray technology was used. Arabidopsis leaves were either mock inoculated or inoculated with cucumber mosaic cucumovirus, oil seed rape tobamovirus, turnip vein clearing tobamovirus, potato virus X potexvirus, or turnip mosaic potyvirus. Inoculated leaves were collected at 1, 2, 4, and 5 days after inoculation, total RNA was isolated, and samples were hybridized to Arabidopsis GeneChip microarrays (Affymetrix). Microarray hybridization revealed co-ordinated changes in gene expression in response to infection by diverse viruses. These changes include virus-general and virus-specific alterations in the expression of genes associated with distinct defense or stress responses. Analyses of the promoters of these genes further suggest that diverse RNA viruses elicit common responses in susceptible plant hosts through signaling pathways that have not been previously characterized.     

Accumulation of gentisic acid as associated with systemic infections but not with the hypersensitive response in plant-pathogen interactions

In the present work we have studied the accumulation of gentisic acid (2,5-dihydroxybenzoic acid, a metabolic derivative of salicylic acid, SA) in the plant-pathogen systems, Cucumis sativus and Gynura aurantiaca, infected with either prunus necrotic ringspot virus (PNRSV) or the exocortis viroid (CEVd), respectively. Both pathogens produced systemic infections and accumulated large amounts of the intermediary signal molecule gentisic acid as ascertained by electrospray ionization mass spectrometry (ESI-MS) coupled on line with high performance liquid chromatography (HPLC). The compound was found mostly in a conjugated (β-glucoside) form. Gentisic acid has also been found to accumulate (although at lower levels) in cucumber inoculated with low doses of Pseudomonas syringae pv. tomato, producing a nonnecrotic reaction. In contrast, when cucumber was inoculated with high doses of this pathogen, a hypersensitive reaction occurred, but no gentisic-acid signal was induced. This is consistent with our results supporting the idea that gentisic-acid signaling may be restricted to nonnecrotizing reactions of the host plant (Bellés et al. in Mol Plant-Microbe Interact 12:227–235, 1999). In cucumber and Gynura plants, the activity of gentisic acid as inducing signal was different to that of SA, thus confirming the data found for tomato. Exogenously supplied gentisic acid was able to induce peroxidase activity in both Gynura and cucumber plants in a similar way as SA or pathogens. However, gentisic-acid treatments strongly induced polyphenol oxidase activity in cucumber, whereas pathogen infection or SA treatment resulted in a lower induction of this enzyme. Nevertheless, gentisic acid did not induce other defensive proteins which are induced by SA in these plants. This indicates that gentisic acid could act as an additional signal to SA for the activation of plant defenses in cucumber and Gynura plants.     

Intercellular 27 kD protein is a chitinase induced by water stress or Pseudoperonospora cubensis in cucumber leaves]

Cucumber seedlings were drought-stressed or inoculated with Pseudoperonospora cubensis. After 3 or 6 d the intercellular fluids of treated cucumber leaves were extracted and analyzed. Protein contents increased after pathogen inoculation and a 27-kD protein was found in intercellular fluids (Figs.1, 7). Both 27 kD proteins were purified from the intercellular fluids of cucumber leaves after drought stress or pathogen inoculation by SDS-PAGE and electro-elution protocol respectively (Fig.2, 3). Purified proteins from drought-stressed and P. cubensis infected seedlings were analyzed by MALDI-TOF MS and their peptide mass fingerprinting (PMF) results were obtained (Figs.4, 5). The PMF results were compared with protein database using the software Profound. The results show that the 27 kD proteins from seedlings after drought stress and after P. cubensis infection were the same protein, i.e. an acidic chitinase (Tables 1, 2; Fig.6). The activities of chitinase in the intercellular fluids of cucumber leaves after pathogen inoculation and in those drought stress were also analyzed. Results showed that both treatments induced the increase in chitinase activity (Fig.8), which indicated that chitinase may be involved in the protection of cucumber plant against both pathogen attack and water stress.     

The effects of <Emphasis Type="Italic">β</Emphasis>-amino-butyric acid on resistance of cucumber against root-knot nematode, <Emphasis Type="Italic">Meloidogyne javanica</Emphasis>

In this study, the effects of β-amino-butyric acid (BABA) on root-knot nematode(Meloidogyne javanica) infection of cucumber and accumulation of total phenolic compounds, hydrogen peroxide and activity of some enzymes related to plant defense mechanisms, i.e., guaiacol peroxidase (GPOX), polyphenol oxidase (PPO), catalase (CAT) in cucumber roots infected with nematode were investigated. Results of this study show that treating the cucumber seedlings with the above elicitor significantly reduces the nematode infection level (the nematode galls, number of egg masses per plant and number of eggs per individual egg mass) compared to control. Additionally, treatment of cucumber roots by BABA and BABA + nematode, significantly increased peroxidase, polyphenol oxidase and catalase activities in root tissues, 1 day after nematode inoculation in comparison to nematode inoculated plants as control and sterile water-treated plants. Enzyme activities reached to a maximum level at 4, 4 and 3 days after nematode inoculation, respectively. Additionally, the amount of H₂O₂, a product of oxidative stress, was significantly increased in the BABA and BABA + nematode treatments in comparison to control. Such increases have occurred in two phases and maximum levels of it were observed at 5 days after inoculation. Inoculation of cucumber plants by BABA also significantly increased accumulation of total phenol in comparison to control and maximum level of it was observed at 7 days after nematode inoculation. The results suggest that the inhibitory effect of BABA on the root-knot nematode (M. javanica) may be related to its ability to enhance defense responses in the cucumber roots.