Developmental regulated mechanisms affect the ability of a fungal pathogen to infect and colonize tobacco leaves.

During tobacco development, a transition state from susceptibility to resistance to fungal pathogen infection is observed. Leaves acquire resistance to Phytophthora parasistica when the plant becomes committed to flowering. The ability to develop resistance does not imply pathogen-induced defence responses as for the onset of systemic acquired resistance (SAR). Throughout flowering growth, fungal establishment is restrained at two levels. The first level is the control of infection effectiveness. Using the salicylic acid non-accumulating NahG plants, we demonstrate that this control does not require salicylic acid accumulation. The intercellular fluids (IFs) from tobacco leaves committed to flowering exhibit a cytotoxic activity on fungal zoospore cells based on in vitro germination assays. Its accumulation is correlated to the control of infection effectiveness that occurs during flowering growth. The expression of this activity appears to constitute a developmental regulated mechanism that inhibits early steps of fungal pathogen installation. A second level of fungal growth control is the restriction of fungal hyphae expansion. In contrast to infection initiation, fungal hyphae spreading appears to be restricted by similar mechanisms induced during SAR as it is attested by the requirement of salicylic acid accumulation and by the correlating apoplastic accumulation of PR1 proteins. These results provide evidence for the activation of a set of at least two regulatory pathways during flowering growth. This activation leads to the induction of mechanisms which control fungal development by affecting the ability of the fungus to both infect and colonise plant tissues.

The effect of benzoic acid derivatives on Nicotiana tabacum growth in relation to PR-b1 production.

Using HPLC quantification, we have shown that benzoic acid derivatives stimulate PR-b1 protein synthesis in the leaf discs of Nicotiana tabacum Xanthi nc. The stimulation of PR-b protein synthesis during treatment with several benzoic acid derivatives is described for the first time in the root system of in vitro grown Nicotiana tabacum plantlets. In healthy in vitro grown plantlets the PR-b1 concentration is similar in roots and leaves (200 ng per gram of fresh material). During chemical treatment, however, the PR-b1 concentration increases to a lesser extent in roots than in leaves (10-fold higher in treated roots and 100-fold higher in treated leaves). Benzoic acid derivatives also have a detrimental effect on the growth of in vitro plantlets, which may be related to the accumulation of PR-b proteins.

Survey of Prunus necrotic ringspot virus in Rose and Its Variability in Rose and Prunus spp.

ABSTRACT A survey for viruses in rose propagated in Europe resulted in detection of only Prunus necrotic ringspot virus (PNRSV) among seven viruses screened. Four percent of cut-flower roses from different sources were infected with PNRSV. Progression of the disease under greenhouse conditions was very slow, which should make this virus easy to eradicate through sanitary selection. Comparison of the partial coat protein gene sequences for three representative rose isolates indicated that they do not form a distinct phylogenetic group and show close relations to Prunus spp. isolates. However, a comparison of the reactivity of monoclonal antibodies raised against these isolates showed that the most prevalent PNRSV serotype in rose was different from the most prevalent serotype in Prunus spp. All of the 27 rose isolates tested infected P. persica seedlings, whereas three of the four PNRSV isolates tested from Prunus spp. were poorly infectious in Rosa indica plants. These data suggest adaptation of PNRSV isolates from Prunus spp., but not from rose, to their host plants. The test methodologies developed here to evaluate PNRSV pathogenicity in Prunus spp. and rose could also help to screen for resistant genotypes.

Enzyme-Linked Immunosorbent Assay Testing of Shoots Grown In Vitro and the Use of Immunocapture-Reverse Transcription-Polymerase Chain Reaction Improve the Detection of Prunus necrotic ringspot virus in Rose.

We developed and evaluated two different methods to improve the detection of the most prevalent virus of rose in Europe, Prunus necrotic ring-spot virus (PNRSV). Immunocapture-reverse transcription-polymerase chain reaction was estimated to be about 100 times more sensitive than double-antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) and showed an equivalent specificity. Based on the observation that PNRSV multiplies actively in young growing tissues (axillary shoots and cuttings), an in vitro culture method allowing rapid (about 15 days) and homogeneous development of dormant axillary buds with high virus titers was standardized. ELISA tests of these young shoots showed, in some cases, a 10(4) to 10(5) increase in sensitivity in comparison to adjacent leaf tissues from the rose mother plants. Between 21 and 98% (depending on the season) more samples were identified as positive by using ELISA on samples from shoot tips grown in vitro rather than on leaves collected directly from the PNRSV-infected mother plants. This simple method of growing shoot tips in vitro improved the confidence in the detection of PNRSV and eliminated problems in sampling appropriate tissues.

Diversity of Virulence in Phytophthora parasitica on Tobacco, as Reflected by Nuclear RFLPs.

ABSTRACT A worldwide collection of P. parasitica isolates was investigated for the ability to infect tobacco and tomato, as related to elicitin production. Elicitin was produced by all nontobacco isolates, and nonproducing strains all were isolated from tobacco. In addition, producing strains were isolated from tobacco and coexisted with nonproducing (TE ) strains. Elicitin production generally was associated with low virulence on tobacco and frequent pathogenicity on tomato, whereas TE isolates generally were highly virulent and specialized to tobacco. Analysis of both mitochondrial and nuclear DNA restriction fragment length polymorphisms indicated, for the first time, that black shank isolates can be distinguished from other P. parasitica isolates on the basis of genetic criteria. Our results suggest that severe black shank is caused by a limited number of TE strains that have been disseminated by clonal evolution. Mutations in the TE phenotype seem to have arisen independently in several genetic backgrounds and distinct geographic areas. The fortuitous absence of elicitin production has precluded population replacements in areas of intensive tobacco cultivation. Thus, monitoring the loss of elicitin production in developing tobacco areas should be considered in disease management.

First Report of Cucumber mosaic virus in Teucrium fruticans.

Teucrium fruticans (shrubby germander), family Lamiaceae, is a hardy shrub. Being drought tolerant, it is widespread in the Mediterranean area. Because it is readily propagated through cuttings, it is also planted in hedges. In 1997 and 2000, respectively, yellow chlorotic areas were observed on the foliage of T. fruticans in Saint Jean Cap Ferrat (France) and San Remo (Italy). These symptoms were distinct from those produced by a rust that frequently affects T. fruticans in these areas. Viruses from both locations were identified as Cucumber mosaic virus (CMV) based on the following: (i) symptoms after mechanical inoculation of Nicotiana tabacum cv. Xanthi nc, N. tabacum cv. Samsum, Chenopodium quinoa, C. amaranticolor, Vigna unguiculata cv. Black, and Cucumis sativus cv. Poinsett; (ii) the morphology of particles observed in electron microscopy of uranyl acetate stained leaf dips from tobacco; and (iii) positive result from leaves of diseased T. fruticans and mechanically inoculated host plants cited above based on enzyme-linked immunosorbent assay (ELISA) using CMV antisera. On tobacco cv. Xanthi nc, the French (F) and Italian (I) isolates first induced essentially necrotic rings on the inoculated leaves followed by the same systemic symptoms as described above. The two isolates were cloned from local lesions after two successive inoculations in V. unguiculata cv. Black, multiplied in tobacco, purified with the citrate-chloroform method, and stabilized with formaldehyde (1). The serotype determination was made by double immunodiffusion in agar gel with the CMV-D and CMV-To strains and homologous antisera (1,2). The formation of spurs and antigen-antibody lines indicated that both isolates belonged to the ToRS serotype (1). Thirty plants of T. fruticans cv. Azureum, first tested negative for CMV using ELISA, were mechanically inoculated with the F isolate (25 plants) and the CMV-D strain (five plants) and cultivated in a hydroponic system. Three months later, plants inoculated with the F isolate were positive for CMV using ELISA and displayed clear symptoms with chlorotic spots, which were sometimes ring-shaped. As plants mature, symptoms tend to disappear on young shoots. For the CMV-D strain, three plants of five were ELISA positive, but did not show any typical symptoms. This report demonstrates the infection of T. fruticans by CMV and the symptom induction by some CMV isolates. In September 2002, two CMV isolates were collected from T. fruticans in public gardens in Menton (France) and Genoa (Italy). These new isolates have the same characteristics as those described in this report. References: (1) J. C. Devergne and L. Cardin. Ann. Phytopathol. 7:225, 1975. (2) M. H. V. van Regenmortel. Adv. Virus Res. 12:207, 1966.

RNase activity prevents the growth of a fungal pathogen in tobacco leaves and increases upon induction of systemic acquired resistance with elicitin.

The hypersensitive response and systemic acquired resistance (SAR) can be induced in tobacco (Nicotiana tabacum L.) plants by cryptogein, an elicitin secreted by Phytophthora cryptogea. Stem application of cryptogein leads to the establishment of acquired resistance to subsequent leaf infection with Phytophthora parasitica var nicotianae, the agent of the tobacco black shank disease. We have studied early events that occur after the infection and show here that a tobacco gene encoding the extracellular S-like RNase NE is expressed in response to inoculation with the pathogenic fungus. Upon induction of SAR with cryptogein, the accumulation of NE transcripts coincided with a rapid induction of RNase activity and with the increase in the activity of at least two different extracellular RNases. Moreover, exogenous application of RNase activity in the extracellular space of leaves led to a reduction of the fungus development by up to 90%, independently of any cryptogein treatment and in the absence of apparent necrosis. These results indicate that the up-regulation of apoplastic RNase activity after inoculation could contribute to the control of fungal invasion in plants induced to SAR with cryptogein.

Pathogen-induced elicitin production in transgenic tobacco generates a hypersensitive response and nonspecific disease resistance.

The rapid and effective activation of disease resistance responses is essential for plant defense against pathogen attack. These responses are initiated when pathogen-derived molecules (elicitors) are recognized by the host. We have developed a strategy for creating novel disease resistance traits whereby transgenic plants respond to infection by a virulent pathogen with the production of an elicitor. To this end, we generated transgenic tobacco plants harboring a fusion between the pathogen-inducible tobacco hsr 203J gene promoter and a Phytophthora cryptogea gene encoding the highly active elicitor cryptogein. Under noninduced conditions, the transgene was silent, and no cryptogein could be detected in the transgenic plants. In contrast, infection by the virulent fungus P. parasitica var nicotianae stimulated cryptogein production that coincided with the fast induction of several defense genes at and around the infection sites. Induced elicitor production resulted in a localized necrosis that resembled a P. cryptogea-induced hypersensitive response and that restricted further growth of the pathogen. The transgenic plants displayed enhanced resistance to fungal pathogens that were unrelated to Phytophthora species, such as Thielaviopsis basicola, Erysiphe cichoracearum, and Botrytis cinerea. Thus, broad-spectrum disease resistance of a plant can be generated without the constitutive synthesis of a transgene product.