Visualization of grapevine root colonization by the Saharan soil isolate Saccharothrix algeriensis NRRL B-24137 using DOPE-FISH microscopy

Background and aim

There is currently a gap of knowledge regarding whether some beneficial bacteria isolated from desert soils can colonize epi- and endophytically plants of temperate regions. In this study, the early steps of the colonization process of one of these bacteria, Saccharothrix algeriensis NRRL B-24137, was studied on grapevine roots to determine if this beneficial strain can colonize a non-natural host plant. An improved method of fluorescence in situ hybridization (FISH), the double labeling of oligonucleotide probes (DOPE)-FISH technique was used to visualize the colonization behavior of such bacteria as well as to determine if the method could be used to track microbes on and inside plants.

Methods

A probe specific to Saccharothrix spp. was firstly designed. Visualization of the colonization behavior of S. algeriensis NRRL B-24137 on and inside roots of grapevine plants was then carried out with DOPE-FISH microscopy.

Results

The results showed that 10 days after inoculation, the strain could colonize the root hair zone, root elongation zone, as well as root emergence sites by establishing different forms of bacterial structures as revealed by the DOPE-FISH technique. Further observations showed that the strain could be also endophytic inside the endorhiza of grapevine plants.

Conclusions

Taking into account the natural niches of this beneficial strain, this study exemplifies that, in spite of its isolation from desert soil, the strain can establish populations as well as subpopulations on and inside grapevine plants and that the DOPE-FISH tool can allow to detect it.     

The 4-phosphopantetheinyl transferase of Trichoderma virens plays a role in plant protection against Botrytis cinerea through volatile organic compound emission

Aims

This work was conducted to examine the effects of volatile organic compounds (VOCs) from Trichoderma virens and the 4-phosphopantetheinyl transferase 1 (TvPPT1) mutant in growth promotion and induction of defense responses of Arabidopsis thaliana seedlings using a co-cultivation system in vitro.

Methods

The contribution of VOCs to plant development and immunity was assessed by comparing the effectiveness of WT and Δppt1 mutant strains of T. virens in the formation of lateral roots and protection conferred against Botrytis cinerea. VOCs released by T. virens and Δppt1 mutant were compared by gas chromatography–mass spectrometry.

Results

Plants exposed to volatiles from WT T. virens showed 2-fold increase in fresh weight when compared to axenically-grown seedlings, which correlated with increased root branching and enhanced expression of the jasmonic acid-responsive marker pLox2:uidA as well as accumulation of jasmonic acid and hydrogen peroxide. T. virens produced a series of hydrocarbon terpenes, including the sesquiterpenes β-caryophyllene, (?)-β-elemene, germacrene D, τ-cadinene, δ-cadinene, α-amorphene, and τ-selinene and the monoterpenes β-myrcene, trans-β-ocimene, and cis-β-ocimene that were absent in TvPPT1 mutant.

Conclusions

Our results indicate that T. virens VOCs elicit both development and defense programs and that PPT1 plays an important role in biosynthesis of terpenes and plant protection against B. cinerea.     

Trichoderma-induced plant immunity likely involves both hormonal- and camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistance against necrotrophic fungus Botrytis cinerea

Filamentous fungi belonging to the genus Trichoderma have long been recognized as agents for the biocontrol of plant diseases. In this work, we investigated the mechanisms involved in the defense responses of Arabidopsis thaliana seedlings elicited by co-culture with Trichoderma virens and Trichoderma atroviride. Interaction of plant roots with fungal mycelium induced growth and defense responses, indicating that both processes are not inherently antagonist. Expression studies of the pathogenesis-related reporter markers pPr1a:uidA and pLox2:uidA in response to T. virens or T. atroviride provided evidence that the defense signaling pathway activated by these fungi involves salicylic acid (SA) and/or jasmonic acid (JA) depending on the amount of conidia inoculated. Moreover, we found that Arabidopsis seedlings colonized by Trichoderma accumulated hydrogen peroxide and camalexin in leaves. When grown under axenic conditions, T. virens produced indole-3-carboxaldehyde (ICAld) a tryptophan-derived compound with activity in plant development. In Arabidopsis seedlings whose roots are in contact with T. virens or T. atroviride, and challenged with Botrytis cinerea in leaves, disease severity was significantly reduced compared with axenically grown seedlings. Our results indicate that the defense responses elicited by Trichoderma in Arabidopsis are complex and involve the canonical defense hormones SA and JA as well as camalexin, which may be important factors in boosting plant immunity.Key words: Arabidopsis, Trichoderma, phytostimulation, defense responses, jasmonic acid, salicylic acid, camalexin     

Differential inducible defense mechanisms against bacterial speck pathogen in Arabidopsis thaliana by plant-growth-promoting-fungus Penicillium sp. GP16-2 and its cell free filtrate

Although a wealth of information is available regarding resistance induced by plant growth-promoting rhizobacteria (PGPR), not much is known about plant growth-promoting fungi (PGPF). Hence, the goal of the present research was to provide more information on this matter. In Arabidopsis thaliana L., root colonizing PGPF Penicillium sp. GP16-2 or its cell free filtrate (CF) elicited an induced systemic resistance (ISR) against infection by Pseudomonas syringae pv. tomato DC3000 (Pst), leading to a restriction of pathogen growth and disease development. We demonstrate that signal transduction leading to GP16-2-mediated ISR requires responsiveness to JA and ET in a NPR1-dependent manner, while CF-mediated ISR shows dispensability of SA, JA, ET and NPR1-dependent signaling (at least individually). In addition, root colonization by GP16-2 is not associated with a direct effect on expression of known defense-related genes, but potentiates the activation of JA/ET-inducible ChitB, which only becomes apparent after infection by Pst. However, CF-mediated ISR was partly associated with the direct activation of marker genes responsive to both SA and JA/ET signaling pathways and partly associated with priming, leading to activation of JA-/ET-inducible ChitB and Hel genes. These suggest that CF may contain one or more elicitors that induce resistance by way where at least SA, JA and ET may play a role in defense signaling in Arabidopsis. Therefore, defense gene changes and underlying signaling pathways induced by Penicillium sp. GP16-2 root colonization and its CF application are not the same and only partially overlap.     

Endogenous jasmonic and salicylic acids levels in the Cd-hyperaccumulator Noccaea (Thlaspi) praecox exposed to fungal infection and/or mechanical stress

Key message

Sensitivity to Erysiphe in Noccaea praecox with low metal supply is related to the failure in enhancing SA. Cadmium protects against fungal-infection by direct toxicity and/or enhanced fungal-induced JA signaling.

Abstract

Metal-based defense against biotic stress is an attractive hypothesis on evolutionary advantages of plant metal hyperaccumulation. Metals may compensate for a defect in biotic stress signaling in hyperaccumulators (metal-therapy) by either or both direct toxicity to pathogens and by metal-induced alternative signaling pathways. Jasmonic acid (JA) and salicylic acid (SA) are well-established components of stress signaling pathways. However, few studies evaluate the influence of metals on endogenous concentrations of these defense-related hormones. Even less data are available for metal hyperaccumulators. To further test the metal-therapy hypothesis we analyzed endogenous SA and JA concentrations in Noccaea praecox, a cadmium (Cd) hyperaccumulator. Plants treated or not with Cd, were exposed to mechanical wounding, expected to enhance JA signaling, and/or to infection by biotrophic fungus Erysiphe cruciferarum for triggering SA. JA and SA were analyzed in leaf extracts using LC–ESI(?)–MS/MS. Plants without Cd were more susceptible to fungal attack than plants receiving Cd. Cadmium alone tended to increase leaf SA but not JA. Either or both fungal attack and mechanical wounding decreased SA levels and enhanced JA in the Cd-rich leaves of plants exposed to Cd. High leaf Cd in N. praecox seems to hamper biotic-stress-induced SA, while triggering JA signaling in response to fungal attack and wounding. To the best of our knowledge, this is the first report on the endogenous JA and SA levels in a Cd-hyperaccumulator exposed to different biotic and abiotic stresses. Our results support the view of a defect in SA stress signaling in Cd hyperaccumulating N. praecox.     

The role of phytohormones in basal resistance and Trichoderma-induced systemic resistance to Botrytis cinerea in Arabidopsis thaliana

Thirty-six phytohormone-affected mutants of Arabidopsis thaliana (L.) Heynh. and their parental ecotypes were tested for resistance/susceptibility to Botrytis cinerea Pers.; Fr. and ability to develop Trichoderma-mediated induced systemic resistance (ISR). Ecotype Colombia-0 (Col-0) was relatively resistant to B. cinerea, and Trichoderma harzianum Rifai T39 application at sites spatially separated (roots) from the B. cinerea inoculation (leaves) resulted in reduction of grey mold symptoms. Ecotypes Wassilewskija-4, Nossen-0 and Landsberg-0 had low levels of basal resistance to B. cinerea and were unable to express ISR. Mutants derived from ISR-non-inducible ecotypes displayed ISR-non-inducible phenotypes, whereas the ISR inducibility of mutants derived from the ISR-inducible genotype Col-0 varied according to the type of mutant. Thus, salicylic acid (SA)-impaired mutants derived from Col-0 were ISR-inducible, while ethylene/jasmonic acid (ethylene/JA)-impaired mutants of the same origin were ISR-non-inducible. SA-impaired mutants retained basal level of resistance to B. cinerea, while most ethylene/JA-impaired mutants were highly susceptible. Abscisic acid- and gibberellin-impaired mutants were highly susceptible to B. cinerea and showed ISR-non-inducible phenotypes irrespective of their lines of origin. Auxin-resistant mutants derived from Col-0 were ISR-inducible; mutant originating from Landsberg-0 and mutants which were resistant to both auxin and ethylene were ISR-non-inducible. Most of the arabidopsis genotypes which were unable to express Trichoderma-mediated ISR against B. cinerea exhibited enhanced susceptibility to this pathogen. T. harzianum treatments enhanced the growth of arabidopsis plants regardless of genotype or ISR inducibility.     

Arabidopsis Brassinosteroid-overproducing gulliver3-D/dwarf4-D mutants exhibit altered responses to Jasmonic acid and pathogen

Key message

Arabidopsis gulliver3 - D/dwarf4 - D displays growth-promoting phenotypes due to activation tagging of a key brassinosteroid biosynthetic gene DWARF4. In gul3-D/dwf4-D , the Jasmonate and Salicylate signaling pathways were relatively activated and suppressed, respectively.

Abstract

Energy allocation between growth and defense is elegantly balanced to achieve optimal development in plants. Brassinosteroids (BRs), steroidal hormones essential for plant growth, are regulated by other plant hormones, including auxin and jasmonates (JA); auxin stimulates the expression of a key brassinosteroid (BR) biosynthetic gene, DWARF4 (DWF4), whereas JA represses it. To better understand the interaction mechanisms between growth and defense, we isolated a fast-growing mutant, gulliver3-D (gul3-D), that resulted from the activation tagging of DWF4, and examined the response of this mutant to defense signals, including JA, Pseudomonas syringae pv. tomato (Pst DC3000) infection, and wounding. The degree of root growth inhibition following MeJA treatment was significantly decreased in gul3-1D/dwf4-5D relative to the wild type, suggesting that JA signaling is partially desensitized in gul3-1D. Quantitative RT-PCR analysis of the genes involved in JA and salicylic acid (SA) responses, including MYC2, PDF1.2, CORI3, PR1, and PR2, revealed that JA signaling was preferentially activated in gul3-1D, whereas SA signaling was suppressed. As a result, gul3-1D was more susceptible to a biotrophic pathogen, Pst DC3000. Based on our results, we propose a model in which BR and JA cooperate to balance energy allocation between growth and defense responses. In ambient conditions, BRs promote plant growth; however, when stresses trigger JA signaling, JA compromises BR signaling by downregulating DWF4 expression.     

Differential effectiveness of salicylate-dependent and jasmonate/ethylene-dependent induced resistance in Arabidopsis

Salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are each involved in the regulation of basal resistance against different pathogens. These three signals play important roles in induced resistance as well. SA is a key regulator of pathogen-induced systemic acquired resistance (SAR), whereas JA and ET are required for rhizobacteria-mediated induced systemic resistance (ISR). Both types of induced resistance are effective against a broad spectrum of pathogens. In this study, we compared the spectrum of effectiveness of SAR and ISR using an oomycete, a fungal, a bacterial, and a viral pathogen. In noninduced Arabidopsis plants, these pathogens are primarily resisted through either SA-dependent basal resistance (Peronospora parasitica and Turnip crinkle virus [TCV]), JA/ET-dependent basal resistance responses (Alternaria brassicicola), or a combination of SA-, JA-, and ET-dependent defenses (Xanthomonas campestris pv. armoraciae). Activation of ISR resulted in a significant level of protection against A. brassicicola, whereas SAR was ineffective against this pathogen. Conversely, activation of SAR resulted in a high level of protection against P. parasitica and TCV, whereas ISR conferred only weak and no protection against P. parasitica and TCV, respectively. Induction of SAR and ISR was equally effective against X. campestris pv. armoraciae. These results indicate that SAR is effective against pathogens that in noninduced plants are resisted through SA-dependent defenses, whereas ISR is effective against pathogens that in noninduced plants are resisted through JA/ET-dependent defenses. This suggests that SAR and ISR constitute a reinforcement of extant SA- or JA/ET-dependent basal defense responses, respectively.     

Priming for JA-dependent defenses using hexanoic acid is an effective mechanism to protect Arabidopsis against B. cinerea

Soil drench treatments with hexanoic acid can effectively protect Arabidopsis plants against Botrytis cinerea through a mechanism based on a stronger and faster accumulation of JA-dependent defenses.Plants impaired in ethylene, salicylic acid, abscisic acid or glutathion pathways showed intact protection by hexanoic acid upon B. cinerea infection. Accordingly, no significant changes in the SA marker gene PR-1 in either the SA or ABA hormone balance were observed in the infected and treated plants. In contrast, the JA signaling pathway showed dramatic changes after hexanoic acid treatment, mainly when the pathogen was present. The impaired JA mutants, jin1-2 and jar1, were unable to display hexanoic acid priming against the necrotroph. In addition, hexanoic acid-treated plants infected with B. cinerea showed priming in the expression of the PDF1.2, PR-4 and VSP1 genes implicated in the JA pathways. Moreover, JA and OPDA levels were primed at early stages by hexanoic acid. Treatments also stimulated increased callose accumulation in response to the pathogen. Although callose accumulation has proved an effective IR mechanism against B. cinerea, it is apparently not essential to express hexanoic acid-induced resistance (HxAc-IR) because the mutant pmr4.1 (callose synthesis defective mutant) is protected by treatment.We recently described how hexanoic acid treatments can protect tomato plants against B. cinerea by stimulating ABA-dependent callose deposition and by priming OPDA and JA-Ile production. We clearly demonstrate here that Hx-IR is a dependent plant species, since this acid protects Arabidopsis plants against the same necrotroph by priming JA-dependent defenses without enhancing callose accumulation.     

Involvement of ethylene signaling in Azospirillum sp. B510-induced disease resistance in rice

A bacterial endophyte Azospirillum sp. B510 induces systemic disease resistance in the host without accompanying defense-related gene expression. To elucidate molecular mechanism of this induced systemic resistance (ISR), involvement of ethylene (ET) was examined using OsEIN2-knockdown mutant rice. Rice blast inoculation assay and gene expression analysis indicated that ET signaling is required for endophyte-mediated ISR in rice.

Abbreviations: ACC: 1-aminocyclopropane-1-carboxylic acid; EIN2: ethylene-insensitive protein 2; ET: ethylene; ISR: induced systemic resistance; JA: jasmonic acid; RNAi: RNA interference; SA: salicylic acid; SAR: systemic acquired resistance     

Organic hydroponics induces systemic resistance against the air-borne pathogen,Botrytis cinerea (gray mould)

Here, we propose that organic hydroponics trigger induced systemic resistance (ISR) in lettuce against air-borne Botrytis cinerea, which causes gray mold. We compared effects of organic and chemical hydroponics, assessed presence of ISR elicitors in the hydroponic nutrient solution, and investigated molecular mechanism of ISR. Organic hydroponics significantly reduced gray mold lesions in lettuce (cultivated hydroponically) and cucumber (cultivated in soil and foliar sprayed with nutrient solution). The 1-aminocyclopropane-1-carboxylic acid synthase gene in lettuce and lipoxygenase and ethylene receptor-related gene in cucumber showed heightened expression, suggesting that the jasmonic acid/ethylene (JA/ET)-signaling pathway was involved in ISR for both crops. Low salicylic acid β-glucoside levels confirmed role of the ISR signaling pathway. ISR in both lettuce and cucumbers indicated that elicitors in organic hydroponics were nonhost-specific and that the JA/ET pathway was activated without microbe–root interaction. Thus, organic hydroponics can be an effective method for both soil-borne and air-borne disease control.     

Systemic Resistance Induced by Volatile Organic Compounds Emitted by Plant Growth-Promoting Fungi in Arabidopsis thaliana

Volatile organic compounds (VOC) were extracted and identified from plant growth-promoting fungi (PGPF), Phoma sp., Cladosporium sp. and Ampelomyces sp., using gas chromatography–mass spectrometry (GC-MS). Among the three VOC extracted, two VOC blends (emitted from Ampelomyces sp. and Cladosporium sp.) significantly reduced disease severity in Arabidopsis plants against Pseudomonas syringae pv. tomato DC3000 (Pst). Subsequently, m-cresol and methyl benzoate (MeBA) were identified as major active volatile compounds from Ampelomyces sp. and Cladosporium sp., respectively, and found to elicit induced systemic resistance (ISR) against the pathogen. Molecular signaling for disease suppression by the VOC were investigated by treating different mutants and transgenic Arabidopsis plants impaired in salicylic acid (SA) or Jasmonic acid (JA)/ethylene (ET) signaling pathways with m-cresol and MeBA followed by challenge inoculation with Pst. Results show that the level of protection was significantly lower when JA/ET-impaired mutants were treated with MeBA, and in SA-, and JA/ET-disrupted mutants after m-cresol treatment, indicating the involvement of these signal transduction pathways in the ISR primed by the volatiles. Analysis of defense-related genes by real-time qRT-PCR showed that both the SA-and JA-signaling pathways combine in the m-cresol signaling of ISR, whereas MeBA is mainly involved in the JA-signaling pathway with partial recruitment of SA-signals. The ET-signaling pathway was not employed in ISR by the volatiles. Therefore, this study identified two novel volatile components capable of eliciting ISR that may be promising candidates in biological control strategy to protect plants from diseases.