Transcriptional induction of two phosphate transporter 1 genes and enhanced root branching in grape plants inoculated with Funneliformis mosseae.

We investigated the effects of the arbuscular mycorrhizal fungus (AMF) Funneliformis mosseae on the growth and root architecture of plantlets of the grape rootstock 41B MGt under hydroponic conditions, and analyzed the concomitant expression of putative mycorrhizal-specific phosphate transporter 1 (PHT1) genes. In vitro propagated plantlets were acclimatized to ex vitro culture before AMF inoculation and grown under low phosphate (Pi) nutrition conditions during 6 weeks. Grape roots could be efficiently colonized by F. mosseae in this culture system, as shown by high mycorrhization frequency and intensity. The presence of many arbuscules in the cortex was coupled with high-level expression of two PHT1 genes in grape roots. These two very similar genes, named VvPht1-1 and VvPht1-2, present P1BS and MYCS regulatory motifs in their promoter, consistent with a specific role in the mycorrhizal pathway of Pi uptake. Although AMF inoculation significantly increased shoot growth, no effect on root biomass was observed. However, inoculated grapes exhibited an enhanced branched root system compared with non-inoculated controls, with a twofold higher number of tips and a higher proportion of fine roots usually involved in nutrient uptake from the soil. Taken together, these results suggest that root colonization by F. mosseae improved grape growth by favoring the uptake of Pi from the substrate via VvPht1-1 and VvPht1-2 high-level expression.

Use of a RT-qPCR Method to Estimate Mycorrhization Intensity and Symbiosis Vitality in Grapevine Plants Inoculated with Rhizophagus irregularis.

Assessing the mycorrhization level in plant roots is essential to study the effect of arbuscular mycorrhizal fungi (AMF) on plant physiological responses. Common methods used to quantify the mycorrhization of roots are based on microscopic visualization of stained fungal structures within the cortical cells. While this method is readily accessible, it remains time-consuming and does not allow checking of the symbiosis vitality. The aim of this work is thus to develop an efficient method for assessing the intensity and vitality of mycorrhiza associated with grapevine through gene expression analyses by RT-qPCR. To this end, grapevine plants were inoculated with the AMF Rhizophagus irregularis (Ri). The relationship between mycorrhization level, assessed by microscopy, and expression of several fungus and grapevine genes involved in the symbiosis was investigated. In AMF-inoculated plants, transcript amounts of fungal constitutively-expressed genes Ri18S, RiTEF1α and RiαTub were significantly correlated to mycorrhization intensity, particularly Ri18S. Grapevine (VvPht1.1 and VvPht1.2) and AMF (GintPT, Ri14-3-3 and RiCRN1) genes, known to be specifically expressed during the mycorrhizal process, were significantly correlated to arbuscular level in the whole root system determined by microscopy. The best correlations were obtained with GintPT on the fungal side and VvPht1.2 on the plant side. Despite some minor discrepancies between microscopic and molecular techniques, the monitoring of Ri18S, GintPT and VvPht1.2 gene expression could be a rapid, robust and reliable method to evaluate the level of mycorrhization and to assess the vitality of AMF. It appears particularly useful to identify AMF-inoculated plants with very low colonization level, or with non-active fungal structures. Moreover, it can be implemented simultaneously with the expression analysis of other genes of interest, saving time compared to microscopic analyses.

Characterization of natural hybrids of Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum.

Nine yeast strains were isolated from spontaneous fermentations in the Alsace area of France, during the 1997, 1998 and 1999 grape harvests. Strains were characterized by pulsed-field gel electrophoresis, PCR-restriction fragment length polymorphism (RFLP) of the MET2 gene, delta-PCR, and microsatellite patterns. Karyotypes and MET2 fragments of the nine strains corresponded to mixed chromosomal bands and restriction patterns for both Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum. They also responded positively to amplification with microsatellite primers specific to both species and were demonstrated to be diploid. However, meiosis led to absolute nonviability of their spores on complete medium. All the results demonstrated that the nine yeast strains isolated were S. cerevisiaexS. bayanus var. uvarum diploid hybrids. Moreover, microsatellite DNA analysis identified strains isolated in the same cellar as potential parents belonging to S. bayanus var. uvarum and S. cerevisiae.

Evolution of the population of Saccharomyces cerevisiae from grape to wine in a spontaneous fermentation.

To determine the grape or winery origin of the Saccharomyces cerevisiae involved in spontaneous fermentation, musts were collected at different stages of wine-making process and fermented. First, grapes were collected in two different vineyards and crushed at the laboratory. Second, musts were collected after crushing and clarification in the cellar. Third, musts collected in the cellar were sterilized and inoculated with tartar deposit collected in the vats. The fourth fermentation was in the cellar. For the two vineyards, two hundred of S. cerevisiae clones were isolated for each of the four fermentations, driving to a library of 1600 clones. All the library was analysed by inter-delta PCR with a basic set of primers and about 20% of the library was further analysed by inter-delta PCR with an improved set of primers. Six, and more than 30 different PCR patterns were obtained from basic- and improved-PCR analysis, respectively. The amounts of each family were analysed at the different stages of wine making. Our study demonstrates that the two vineyards present different S. cerevisiae populations. Moreover the S. cerevisiae strains involved in spontaneous fermentation in the cellar originate partly from the vineyard and partly from the winery, in amounts varying with the must.