Identification by suppression subtractive hybridization and expression analysis of Arabidopsis thaliana putative defence genes during Orobanche ramosa infection

A suppression subtractive hybridization strategy was used to identify genes that were induced 2 h after infection of Arabidopsis thaliana by broomrape (Orobanche ramosa) seedlings. Among these genes, the expression of twelve was analysed from the first hour to the seventh day of this compatible interaction by cDNA blot analyses. The twelve genes showed a transient expression, which occurred in seven cases as early as the first or second hour of interaction and ended 2 or 3 h later. Most of the proteins encoded by these genes are already known to be involved in different A. thaliana response pathways to pathogen attack. The first two genes to be induced (Rc kinase and ACaM5) encoded a receptor and a calmodulin, and could be involved in signal transduction. The two following genes encoding a sucrose carrier (SUC1) and a protein with a pectin methylesterase inhibitor domain were found to be overexpressed; their roles are consistent with plant defence emergence. The gst1, gst11 and peptide methionine sulfoxide reductase genes, which were turned on early, are known to play a role in the detoxification of reactive oxygen species. The accumulation of mRNAs for lox1, a latex allergen and a myrosinase binding protein could be related to a jasmonate dependent pathway, while genes for a class III peroxidase and a caffeoyl-CoA 3-O-methyltransferase, both likely to be involved in cell wall reinforcement, were also upregulated.     

In vitro culture of Orobanche ramosa

Orobanche spp. (broomrapes) are holoparasites that subsist on the roots of many important crops and can considerably reduce yield. The control of Orobanche spp. includes physical, chemical and biological methods. Interactions between parasitic angiosperms and their hosts first occur at the level of parasite seed germination. The seeds of all Orobanchaceae germinate in soil under natural conditions only in response to specific chemical exudates from the host plant. This study describes the influence of different plant growth regulators and host plant root exudates on germination and development of calli from Orobanche seeds in vitro . The effect of indole-3-acetic acid, gibberellic acid and kinetin on the germination of Orobanche seeds varied with concentration. These plant growth regulators also affected the period of germination and the structure of calli and protrusions. An in vitro system for the collection of tobacco root exudates was established. Compounds released from the host roots of three different tobacco cultivars were found to provoke high levels of germination of the Orobanche seeds without any period of pre-conditioning. This study developed methods for the investigation of host–parasite interactions and the effect of germination stimulants in Orobanche spp.     

The effect of salt stress on Arabidopsis thaliana and Phelipanche ramosa interaction

Salinity and Orobanche or Phelipanche spp. infection are important crop stress factors in agricultural areas. In this study, we investigated the effect of salt stress on Phelipanche ramosa seed germination and its attachment onto Arabidopsis thaliana roots. We also evaluated the effect of both stresses on the expression of genes regulated by abiotic and biotic stresses. According to our results, high concentration of NaCl delayed P. ramosa seed germination in the presence of a strigolactone analogue (GR24). A similar pattern was observed in the presence of A. thaliana plants. Furthermore, we found that salt‐treated A. thaliana seedlings were more sensitive to P. ramosa attachment compared with the untreated plants, indicating that there was a positive correlation between salt sensitivity and the ability of P. ramosa to infect A. thaliana plants. At the molecular level, a synergystic effect of both salt and P. ramosa stresses was observed on the cold‐regulated (COR) gene expression profile of treated A. thaliana seedlings. Our data clarify the interaction between parasitic plants and their hosts under abiotic stress conditions.     

Possible involvement of gibberellins and ethylene in Orobanche ramosa germination

Summary Six inhibitors acting at different stages of gibberellin biosynthesis, applied during conditioning of Orobanche ramosa seeds, reduced subsequent germination in the presence of GR₂₄ (a strigol analogue). Ethylene seems to be involved in the induction of germination of conditioned seeds by GR₂₄, as inhibitors of its synthesis or action, applied to conditioned seeds, also strongly reduced induction of germination by GR₂₄. Exogenous ethylene did not induce germination of conditioned seeds, but 2-chloroethylphosphonic acid was able to do so. When inhibitors of gibberellin biosynthesis were applied to conditioned seeds in the presence of GR₂₄, they inhibited germination. These same inhibitors also strongly inhibited germination of conditioned Striga hermonthica seeds in response to GR₂₄; this inhibitory effect was reversed by the addition of 2-chloroethylphosphonic acid. The effect of these inhibitors on S. hermonthica , in which ethylene is a necessary mediator of germination induction by GR₂₄, strongly suggests that ethylene synthesis is also required for the induction of O. ramosa seed germination by GR₂₄. These growth regulators, which inhibit the two steps of germination in O. ramosa , could be useful for the development of methods for early season control of this parasite.     

Pathogenicity of branched broomrape (Orobanche ramosa) populations on tobacco cultivars

Parasitic weed species of the genus Orobanche are a serious threat for the production of several crops in Europe, Africa and Asia. In contrast to other broomrape species of agronomic importance, O. ramosa (branched broomrape) has a broad host range and in Europe particularly affects hemp, tobacco, tomato and, in recent times, oilseed rape. Two separate sets of experiments investigated the effect of two populations of O. ramosa on nine tobacco cultivars grown in Europe and belonging to the three major tobacco types: Virgin (flue-cured), Burley (light air-cured) and dark air-cured under standardized glasshouse conditions. The two broomrape populations were discriminated by means of polymorphic DNA fragments obtained by PCR of the intersimple sequence repeat regions (ISSRs). The Orobanche populations exhibited different levels of pathogenicity but all various tobacco cultivars were susceptible. Dark air-cured tobacco cultivars were the least susceptible to both broomrape populations. Virgin and Burley tobacco cultivars were more susceptible to one population of O. ramosa .     

Methyl jasmonate and methyl salicylate, but not cis-jasmone, evoke defenses against infection of Arabidopsis thaliana by Orobanche aegyptiaca

The mechanism by which some plant species develop resistance to the root parasite, broomrape ( Orobanche aegyptiaca ), is still not clear. Resistance to other pathogens can be induced by methyl jasmonate and systemic acquired resistance can be induced by treatment with salicylic acid, while cis -jasmone can act as a signaling molecule in plant–insect interactions. The three compounds studied, methyl jasmonate, cis -jasmone, and methyl salicylate, were applied to Arabidopsis thaliana seedlings that were then transferred to Nunc cell culture plates and exposed to the germinating seeds of O. aegyptiaca . The number of infections of the roots of single seedlings of A. thaliana was then quantified. Exposure for 24 h to very low concentrations of methyl jasmonate or methyl salicylate, which were then removed, effectively induced resistance to infection of A. thaliana by O. aegyptiaca , reducing attachment and tubercle formation by 90%. cis -Jasmone was far less effective in inducing a similar resistance to infection. These results support the view that methyl jasmonate can induce almost full resistance to infection by broomrape. The fact that such resistance is not observed under normal conditions of infection supports the idea that the root parasite does not evoke the full defensive response in the host plant.     

A novel strain of Fusarium oxysporum from Germany and its potential for biocontrol of Orobanche ramosa

A newly isolated Fusarium oxysporum strain was investigated for its biocontrol potential against the root parasitic weed Orobanche ramosa (branched broomrape). The fungus was found to affect all developmental stages of the parasite. Orobanche seed germination was reduced by 40% in the presence of fungal conidia in vitro . The number of underground tubercles and shoots of the weed was reduced by the fungus by 55% compared with the control and 92% of tubercles were recorded as diseased in root chambers. In pot experiments, soil application of a granular formulation of the fungus resulted in a reduction of number and dry matter of Orobanche shoots by more than 90%. Spraying of a conidial suspension on aboveground Orobanche shoots caused the death of 75% of them within 2 weeks. Data from initial host-range experiments indicate that the isolate is very host-specific, not even attacking shoots of other Orobanche species. Because of these promising results, we conclude that the fungal isolate should be investigated under field conditions and be compared with other Fusarium isolates proposed for biocontrol of O. ramosa .     

Possible peroxidase functions in the interaction between the parasitic plant, Orobanche aegyptiaca, and its host, Arabidopsis thaliana

The mechanism by which the flowering holoparasitic plant, Orobanche aegyptiaca , infects its host without evoking a defence mechanism is still poorly understood. In this work, we studied several mechanisms used by phytopathogenic fungi. We focussed on the possible role of peroxidases during O. aegyptiaca penetration into the roots of Arabidopsis thaliana . A convenient experimental system for studying the interaction under sterile conditions was developed. The formation of extracellular reactive oxygen species (ROS) was detected at the interaction site before, during, and after the parasite penetrated into the host. These extracellular ROS probably originated from the parasite. However, no intracellular ROS could be detected at the site of the interaction. Peroxidase activity was observed mainly at the apex of the root of the parasite and in the adventitious roots of the tubercle. Benzhydroxamic acid, a peroxidase inhibitor, was used to probe the possible role of peroxidase in the infection process. Peroxidase activity was observed in the root apex and adventitious roots of O. aegyptiaca, but no evidence was found for its participation in the actual infection process. Peroxidase activity was also found in the later stages of the interaction between the host and the parasite. We propose that peroxidases could have a role in generating extracellular ROS for loosening the cell wall of the host in order to facilitate penetration. Alternatively, the ROS could act in facilitating the root elongation of the parasite.     

Herbicides for control of broomrape (Orobanche ramosa L.) in tobacco (Nicotiana tabacum L.)

In field tests conducted on oriental and flue-cured tobacco, six herbicides were evaluated for their effect on broomrape (Orobanche ramosa L.) control and on tobacco growth, yield and chemical composition. Imazaquin+pendi-methalin (1:6) at 0.09+0.55 kg a.i. ha^?1 soil in corporated before transplanting controlled broomrape effectively only in 1990 and signifi cantly reduced tobacco yield. Glyphosate (as Roundup 360 ga.e.1^?1 and Armada (90 ga.e.l^?1) and sulfosate, each at 0.2+0.3 kg a.e. ha^?1 and imazaquinat 0.07+0.1 kg a.i. ha^?1 foliar applied at 40 days and 60 days after transplanting gave excellent control of broomrape, with no adverse effect on tobacco growth or yield; however, the 9% formulation of glyphosate was damaging. Maleic hydrazide gave moderate to good (up to 80%) control of broomrape. Reducing sugars in flue-cured tobacco and nicotine in both oriental and flue-cured tobacco were higher where a her bicide was used. Des herbicides pour lutter contre Orobanche ramosa L. dans le tabac (Nicotiana tabacum L). Dans des expérimentations au champ conduites sur du tabac oriental et ‘flue cured’, six herbicides ont étéévalués pour leur effet sur Orobanche ramosa L. ainsi que sur la croissance, le rendement et la composition chimique du tabac. Le mélange imazaquin+pendiméthalin (1:6 à 0.09+0.55 kg ha^?1, incorporé avant le repiquage, n'a permis de détruire l'orobanche qu'en 1990 et réduisait de manière significative le rendement du tabac. Le glyphosate (Roundup, 360 m.a. 1^?1 et Armada, 90 g m.a. l^?1) et le sulfosate chacun aux doses 0.2+0.3 kg m.a. ha^?1 et l'imazaquin 0.07+0.1 kg m.a. ha^?1 appliqués en post-levée 40 et 60 jours après le repiquage donnaient d'excellents résultats contre l'orobanche, sans effet sur la croissance du tabac et sur son rendement, si l'on excepte la formulation de giyphosate 9%. L'hydrazide maléique donnait des résultats moyens à bons (jusqu'à 80% d'efficacité). Les teneurs en sucres réducteurs chez le tabac ‘flue cured’ et en nicotine chez les tabacs ‘flue cured’ et oriental étaient plus élevées lorsqu'un herbicide avait été utilisé. Herbizide zur Bekämpfung von Orobanche ramosa L. in Tabak (Nicotiana tabacum L.) In Freilandversuchen wurden in Orient- und Vir-gin-Tabak 6 Herbizide hinsichtlich der Bekämpfung der Sommerwurz Orobanche ramosa sowie des Wachstums, des Ertrags und der chemischen Zusammensetzung des Tabaks geprüft. Mit einer Mischung von Imazaquin mit Pendimethalin zu 0,09+0,55 kg AS ha^?1, vor dem Pflanzen in den Boden eingearbeitet, konnte die Sommerwurz nur 1990 wirkungsvoll bekämpft werden, aber der Tabakertrag wurde signifikant reduziert. Glyphosat (als Roundup [360 g AS 1^?1] und als Armada [90 g AS 1^?1) mit Sulfosat zu 0,2+0,3 kg AS ha^?1 und Imazaquin zu 0,07 sowie 0,1 kg AS ha^?1, 40 bzw. 60 Tage nach dem Pflanzen angewandt, wirkten hervorragend, ohne Wuchs und Ertrag des Tabaks zu beeinträchtigen, ausgenommen bei der 9% igen Glyphosatformulierung. Mit Maleinhydazid ließ sich die Sommerwurz befriedigend bis gut (bis zu 80 %) bekämpfen. Der Gehalt an reduzierenden Zuckern im Virgin-Tabak und Nikotin in beiden Tabaken war erhöht, wenn ein Herbizid angewandt worden war.     

Study of Resistance to Orobanche ramosa in Host (Oilseed Rape and Carrot) and Non-host (Maize) Plants

Orobanche ramosa is a parasitic Angiosperm responsible for severe yield losses in several economical crops. It is a serious threat in oilseed rape in France and Morocco and is appearing in carrot crops in Morocco. In this study, several varieties of oilseed rape and carrot were screened in order to identify resistant cultivars and to characterize the resistance mechanisms involved. All the 15 oilseed rape varieties tested were susceptible. In carrot, the varieties 'Colmar à coeur rouge' and 'Nantaise demi-longue' were susceptible, whereas 'Palaiseau' and 'Buror' were resistant. In the susceptible 'Colmar à coeur rouge' carrot no defence reactions were found and the development of the parasite inhibited carrot tap root formation. In the resistant carrot varieties, the parasite germinated, became attached to the host root but became necrotic before emergence. In 'Buror' carrot, formation of a mechanical barrier was associated with the restriction to the cortex of the parasite. In maize cv. 'Vigni', a non-host of O. ramosa, thickening of xylem vessels, cell divisions in the central cylinder and formation of an encapsulation layer were observed in association with restricted development of Orobanche haustoria.     

A study of assimilation and translocation in Cuscuta hyalina Heyne ex Roth., Orobanche ramosa L. and Striga hermonthica Benth.

Quantitative determinations of chlorophyll showed that Cuscuta hyalina and Striga hermonthica possess chlorophyll. No trace of chlorophyll was found in Orobanche ramosa. Autoradiographic evidence showed that both radiocarbon and radiophosphorous moved from host to parasite in alt three species. Photosynthesis occurs in S. hermonthica when the parasite is isolated from its host. The adventitious roots of S. hermonthica and O. ramosa appear to be functional in absorbing a proportion of the required inorganic substances and water. S. hermonthica is a hemiparasite, O. ramosa is a holoparasite but C. hyalina is best described as a partial parasite.     

Natural history of Arabidopsis thaliana and oomycete symbioses

Molecular ecology of plant–microbe interactions has immediate significance for filling a gap in knowledge between the laboratory discipline of molecular biology and the largely theoretical discipline of evolutionary ecology. Somewhere in between lies conservation biology, aimed at protection of habitats and the diversity of species housed within them. A seemingly insignificant wildflower called Arabidopsis thaliana has an important contribution to make in this endeavour. It has already transformed botanical research with deepening understanding of molecular processes within the species and across the Plant Kingdom; and has begun to revolutionize plant breeding by providing an invaluable catalogue of gene sequences that can be used to design the most precise molecular markers attainable for marker-assisted selection of valued traits. This review describes how A. thaliana and two of its natural biotrophic parasites could be seminal as a model for exploring the biogeography and molecular ecology of plant–microbe interactions, and specifically, for testing hypotheses proposed from the geographic mosaic theory of co-evolution.     

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.     

Resistance and Susceptibility of Arabidopsis thaliana to Bacterial Wilt Caused by Ralstonia solanacearum

ABSTRACT Tomato bacterial wilt caused by Ralstonia solanacearum is a model system for studying plant-bacterial interactions, because it is genetically one of the best characterized plant diseases. We demonstrate here that four different strains of R. solanacearum, two from radishes (Rd4 and Rd15) and two from tomato (Ps21 and Ps95), can infect 27 different ecotypes of Arabidopsis thaliana, causing different responses. All ecotypes tested were highly susceptible to strain Rd15, which caused symptoms similar to those observed in tomato plants. For example, leaf drooping and discoloration developed just 3 days after inoculation, and plants completely wilted within 1 week. Strains Rd4 and Ps95 were less infectious than Rd15. With these two strains, a variety of disease responses were observed among different ecotypes at 2 weeks after inoculation; both susceptible and resistant ecotypes of A. thaliana were identified. Ps21 was the least infectious of the four strains and caused almost no symptoms in any of the ecotypes of Arabidopsis tested. Direct bacterial isolation and plant skeleton hybridization analysis from infected plants indicated that bacterial colonization was correlated with the severity of symptoms. Growth of bacteria was limited to the infection site in resistant plants, whereas the bacteria spread throughout susceptible plants by 1 week after inoculation.     

Ustilago maydis Infection of the Nonnatural Host Arabidopsis thaliana

ABSTRACT The experimental infection of Arabidopsis thaliana by the maize phytopathogenic hemibasidiomycete Ustilago maydis under axenic conditions is described. When plantlets were inoculated with mixtures of compatible haploids, the fungus was able to grow on the plant surface of inoculated seedlings in the form of white mycelium and invade the tissues, probably penetrating through stomata; however, it did not form teliospores. Symptoms of disease were increased anthocyanin formation, development of chlorosis, increased formation of secondary roots, induction of malformations in the leaves and petioles, induction of tissue necrosis, and stunting. In several cases, death of the invaded plants occurred. Interestingly, inoculation of single U. maydis haploid strains produced similar symptoms in Arabidopsis plantlets. In contrast, several mutants avirulent to maize also were avirulent or less virulent than wildtype strains on Arabidopsis. Collectively, the reported data suggest that the U. maydis-Arabidopsis pathosystem may constitute a useful experimental model for the analysis of some aspects of the virulence factors of the fungus. With the study of nonhost responses and their comparison to those occurring during maize infection, we will be able to elucidate some obscure aspects of U. maydis pathogenicity in the future.     

Arabidopsis thaliana resistance to insects, mediated by an earthworm-produced organic soil amendment

BACKGROUND: Vermicompost is an organic soil amendment produced by earthworm digestion of organic waste. Studies show that plants grown in soil amended with vermicompost grow faster, are more productive and are less susceptible to a number of arthropod pests. In light of these studies, the present study was designed to determine the type of insect resistance (antixenosis or antibiosis) present in plants grown in vermicompost‐amended potting soil. Additionally, the potential role of microarthropods, entomopathogenic organisms and non‐pathogenic microbial flora found in vermicompost on insect resistance induction was investigated. RESULTS: Findings show that vermicompost from two different sources (Raleigh, North Carolina, and Portland, Oregon) were both effective in causing Arabidopsis plants to be resistant to the generalist herbivore Helicoverpa zea (Boddie). However, while the Raleigh (Ral) vermicompost plant resistance was expressed as both non‐preference (antixenosis) and milder (lower weight and slower development) toxic effect (antibiosis) resistance, Oregon (OSC) vermicompost plant resistance was expressed as acute antibiosis, resulting in lower weights and higher mortality rates. CONCLUSION: Vermicompost causes plants to have non‐preference (antixenosis) and toxic (antibiosis) effects on insects. This resistance affects insect development and survival on plants grown in vermicompost‐amended soil. Microarthropods and entomopathogens do not appear to have a role in the resistance, but it is likely that resistance is due to interactions between the microbial communities in vermicompost with plant roots, as is evident from vermicompost sterilization assays conducted in this study. Copyright © 2010 Society of Chemical Industry     

Assessment of resistance pathways induced in Arabidopsis thaliana by hypovirulent Rhizoctonia spp. isolates

Certain hypovirulent Rhizoctonia isolates effectively protect plants against well-known important pathogens among Rhizoctonia isolates as well as against other pathogens. The modes of action involved in this protection include resistance induced in plants by colonization with hypovirulent Rhizoctonia isolates. The qualifications of hypovirulent isolates (efficient protection, rapid growth, effective colonization of the plants, and easy application in the field) provide a significant potential for the development of a commercial microbial preparation for application as biological control agents. Understanding of the modes of action involved in protection is important for improving the various aspects of development and application of such preparations. The hypothesis of the present study is that resistance pathways such as systemic acquired resistance (SAR), induced systemic resistance (ISR), and phytoalexins are induced in plants colonized by the protective hypovirulent Rhizoctonia isolates and are involved in the protection of these plants against pathogenic Rhizoctonia. Changes in protection levels of Arabidopsis thaliana mutants defective in defense-related genes (npr1-1, npr1-2, ndr1-1, npr1-2/ndr1-1, cim6, wrky70.1, snc1, and pbs3-1) and colonized with the hypovirulent Rhizoctonia isolates compared with that of the wild type (wt) plants colonized with the same isolates confirmed the involvement of induced resistance in the protection of the plants against pathogenic Rhizoctonia spp., although protection levels of mutants constantly expressing SAR genes (snc1 and cim6) were lower than that of wt plants. Plant colonization by hypovirulent Rhizoctonia isolates induced elevated expression levels of the following genes: PR5 (SAR), PDF1.2, LOX2, LOX1, CORI3 (ISR), and PAD3 (phytoalexin production), which indicated that all of these pathways were induced in the hypovirulent-colonized plants. When SAR or ISR were induced separately in plants after application of the chemical inducers Bion and methyl jasmonate, respectively, only ISR activation resulted in a higher protection level against the pathogen, although the protection was minor. In conclusion, plant colonization with the protective hypovirulent Rhizoctonia isolates significantly induced genes involved in the SAR, ISR, and phytoalexin production pathways. In the studied system, SAR probably did not play a major role in the mode of protection against pathogenic Rhizoctonia spp.; however, it may play a more significant role in protection against other pathogens.     

CEP peptide induces susceptibility of Arabidopsis thaliana to non-adapted pathogens

CEP peptide was synthesized and tested for induction of disease susceptibility using Arabidopsis Col-0. When Colletotrichum tropicale was used as a non-adapted fungal pathogen, the conidia germinated to form hyphal-like structures, which successfully penetrated epidermis, eventually causing disease symptoms. In such case, PEN2-, but not PEN3-dependent resistance was likely suppressed by CEP peptide. Similarly, the CEP peptide-mediated disease susceptibility was also effective to a non-adapted bacterial pathogen. Notably, such induced susceptibility was also evident on Arabidopsis mutants lacking the previously identified receptors, suggesting that the CEP peptide modulates Arabidopsis immunity through an unidentified receptor(s).