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High-throughput gene-expression quantification of grapevine defense responses in the field using microfluidic dynamic arrays

View Article: PubMed Central - PubMed

ABSTRACT

Background: The fight against grapevine diseases due to biotrophic pathogens usually requires the massive use of chemical fungicides with harmful environmental effects. An alternative strategy could be the use of compounds able to stimulate plant immune responses which significantly limit the development of pathogens in laboratory conditions. However, the efficiency of this strategy in natura is still insufficient to be included in pest management programs. To understand and to improve the mode of action of plant defense stimulators in the field, it is essential to develop reliable tools that describe the resistance status of the plant upon treatment.

Results: We have developed a pioneering tool (“NeoViGen96” chip) based on a microfluidic dynamic array platform allowing the expression profiling of 85 defense-related grapevine genes in 90 cDNA preparations in a 4 h single run. Two defense inducers, benzothiadiazole (BTH) and fosetyl-aluminum (FOS), have been tested in natura using the “NeoViGen96” chip as well as their efficacy against downy mildew.

Results: BTH-induced grapevine resistance is accompanied by the induction of PR protein genes (PR1, PR2 and PR3), genes coding key enzymes in the phenylpropanoid pathway (PAL and STS), a GST gene coding an enzyme involved in the redox status and an ACC gene involved in the ethylene pathway.

Results: FOS, a phosphonate known to possess a toxic activity against pathogens and an inducing effect on defense genes provided a better grapevine protection than BTH. Its mode of action was probably strictly due to its fungicide effect at high concentrations because treatment did not induce significant change in the expression level of selected defense-related genes.

Conclusions: The NeoViGen96” chip assesses the effectiveness of plant defense inducers on grapevine in vineyard with an excellent reproducibility. A single run with this system (4 h and 1,500 €), corresponds to 180 qPCR plates with conventional Q-PCR assays (Stragene system, 270 h and 9,000 €) thus a throughput 60–70 times higher and 6 times cheaper. Grapevine responses after BTH elicitation in the vineyard were similar to those obtained in laboratory conditions, whereas our results suggest that the protective effect of FOS against downy mildew in the vineyard was only due to its fungicide activity since no activity on plant defense genes was observed. This tool provides better understanding of how the grapevine replies to elicitation in its natural environment and how the elicitor potential can be used to reduce chemical fungicide inputs.

Electronic supplementary material: The online version of this article (doi:10.1186/s12864-016-3304-z) contains supplementary material, which is available to authorized users.

No MeSH data available.


Efficacy of potential defense inducers on leaves against grapevine downy mildew (P. viticola). Tests were carried out on a randomized block design with 4 blocks and 3 grapevine plants per block of Cabernet Sauvignon. Three modalities were studied: untreated, treated every week with 1Kg Ha-1 of active ingredient of BTH (Acibenzolar-S-methyl 50%, Bion® 50WG, Syngenta) and treated with 2.5 Kg Ha-1 of active ingredient of fosetyl aluminum (Fosetyl-Al 80%, Aliette®Flash, Bayer). Treatments were carried out between 3rd May and 19th July 2011 (12 treatments, T1 to T12 and red arrows) and with artificial inoculation performed on 19th May 2011 (green arrow). Disease severity was assessed 5 times between 9th June 2011 (after 5 treatments and 3 weeks after artificial inoculation) and 28th July by assessing the extent of attack on 30 leaves per block during the season. Leaves were sampled throughout the season: before any treatment to check the homogeneity of the parcel (S1), 48 h after the second treatment (S2), 48 h after the third treatment and just before artificial inoculation (S3), 48 h after artificial inoculation (S4), then later, 48 h after the seventh treatment (S5)
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Fig5: Efficacy of potential defense inducers on leaves against grapevine downy mildew (P. viticola). Tests were carried out on a randomized block design with 4 blocks and 3 grapevine plants per block of Cabernet Sauvignon. Three modalities were studied: untreated, treated every week with 1Kg Ha-1 of active ingredient of BTH (Acibenzolar-S-methyl 50%, Bion® 50WG, Syngenta) and treated with 2.5 Kg Ha-1 of active ingredient of fosetyl aluminum (Fosetyl-Al 80%, Aliette®Flash, Bayer). Treatments were carried out between 3rd May and 19th July 2011 (12 treatments, T1 to T12 and red arrows) and with artificial inoculation performed on 19th May 2011 (green arrow). Disease severity was assessed 5 times between 9th June 2011 (after 5 treatments and 3 weeks after artificial inoculation) and 28th July by assessing the extent of attack on 30 leaves per block during the season. Leaves were sampled throughout the season: before any treatment to check the homogeneity of the parcel (S1), 48 h after the second treatment (S2), 48 h after the third treatment and just before artificial inoculation (S3), 48 h after artificial inoculation (S4), then later, 48 h after the seventh treatment (S5)

Mentions: The effect of BTH and FOS on downy mildew was evaluated. The mode of action of BTH is only through stimulation of plant defenses [17, 27], while that of FOS is more complex with direct and indirect effects [9]. Previous studies showed that this complex mode depends on the dose applied by soil drenching [33], with an indirect effect at low dose (<10 mM) and a direct effect at high dose (>50 mM). In our study, FOS was applied on grapevine foliage at the authorized dose (2.5 kG Ha−1 corresponding to 7.05 mM) for which a direct action has already been shown leading to an inhibition of 87.5% of downy mildew spores germination at a dose 5 times lower (1.13 mM) [27]. Figure 5 shows that treatment of grapevine leaves with BTH and FOS in field conditions led to a significant reduction in downy mildew symptoms compared to untreated control leaves, with a better efficiency of FOS. The severity of grapevine downy mildew (Plasmopara viticola) in FOS and BTH-treated blocks at the end of 28th July were 85 and 70% lower than on untreated blocks, respectively (5.2% ± 1.6 and 12.4% ± 2.5 of downy mildew severity respectively compared to 39.3% ± 2.8 on untreated control). Area Under Disease Progress Curves (AUDPC) [48], which summarize repeated data such as the change in intensity of an epidemic as a unique value (AUDPC), were 55 and 45% lower than in untreated controls, respectively (Fig. 6).Fig. 5


High-throughput gene-expression quantification of grapevine defense responses in the field using microfluidic dynamic arrays
Efficacy of potential defense inducers on leaves against grapevine downy mildew (P. viticola). Tests were carried out on a randomized block design with 4 blocks and 3 grapevine plants per block of Cabernet Sauvignon. Three modalities were studied: untreated, treated every week with 1Kg Ha-1 of active ingredient of BTH (Acibenzolar-S-methyl 50%, Bion® 50WG, Syngenta) and treated with 2.5 Kg Ha-1 of active ingredient of fosetyl aluminum (Fosetyl-Al 80%, Aliette®Flash, Bayer). Treatments were carried out between 3rd May and 19th July 2011 (12 treatments, T1 to T12 and red arrows) and with artificial inoculation performed on 19th May 2011 (green arrow). Disease severity was assessed 5 times between 9th June 2011 (after 5 treatments and 3 weeks after artificial inoculation) and 28th July by assessing the extent of attack on 30 leaves per block during the season. Leaves were sampled throughout the season: before any treatment to check the homogeneity of the parcel (S1), 48 h after the second treatment (S2), 48 h after the third treatment and just before artificial inoculation (S3), 48 h after artificial inoculation (S4), then later, 48 h after the seventh treatment (S5)
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5120521&req=5

Fig5: Efficacy of potential defense inducers on leaves against grapevine downy mildew (P. viticola). Tests were carried out on a randomized block design with 4 blocks and 3 grapevine plants per block of Cabernet Sauvignon. Three modalities were studied: untreated, treated every week with 1Kg Ha-1 of active ingredient of BTH (Acibenzolar-S-methyl 50%, Bion® 50WG, Syngenta) and treated with 2.5 Kg Ha-1 of active ingredient of fosetyl aluminum (Fosetyl-Al 80%, Aliette®Flash, Bayer). Treatments were carried out between 3rd May and 19th July 2011 (12 treatments, T1 to T12 and red arrows) and with artificial inoculation performed on 19th May 2011 (green arrow). Disease severity was assessed 5 times between 9th June 2011 (after 5 treatments and 3 weeks after artificial inoculation) and 28th July by assessing the extent of attack on 30 leaves per block during the season. Leaves were sampled throughout the season: before any treatment to check the homogeneity of the parcel (S1), 48 h after the second treatment (S2), 48 h after the third treatment and just before artificial inoculation (S3), 48 h after artificial inoculation (S4), then later, 48 h after the seventh treatment (S5)
Mentions: The effect of BTH and FOS on downy mildew was evaluated. The mode of action of BTH is only through stimulation of plant defenses [17, 27], while that of FOS is more complex with direct and indirect effects [9]. Previous studies showed that this complex mode depends on the dose applied by soil drenching [33], with an indirect effect at low dose (<10 mM) and a direct effect at high dose (>50 mM). In our study, FOS was applied on grapevine foliage at the authorized dose (2.5 kG Ha−1 corresponding to 7.05 mM) for which a direct action has already been shown leading to an inhibition of 87.5% of downy mildew spores germination at a dose 5 times lower (1.13 mM) [27]. Figure 5 shows that treatment of grapevine leaves with BTH and FOS in field conditions led to a significant reduction in downy mildew symptoms compared to untreated control leaves, with a better efficiency of FOS. The severity of grapevine downy mildew (Plasmopara viticola) in FOS and BTH-treated blocks at the end of 28th July were 85 and 70% lower than on untreated blocks, respectively (5.2% ± 1.6 and 12.4% ± 2.5 of downy mildew severity respectively compared to 39.3% ± 2.8 on untreated control). Area Under Disease Progress Curves (AUDPC) [48], which summarize repeated data such as the change in intensity of an epidemic as a unique value (AUDPC), were 55 and 45% lower than in untreated controls, respectively (Fig. 6).Fig. 5

View Article: PubMed Central - PubMed

ABSTRACT

Background: The fight against grapevine diseases due to biotrophic pathogens usually requires the massive use of chemical fungicides with harmful environmental effects. An alternative strategy could be the use of compounds able to stimulate plant immune responses which significantly limit the development of pathogens in laboratory conditions. However, the efficiency of this strategy in natura is still insufficient to be included in pest management programs. To understand and to improve the mode of action of plant defense stimulators in the field, it is essential to develop reliable tools that describe the resistance status of the plant upon treatment.

Results: We have developed a pioneering tool (&ldquo;NeoViGen96&rdquo; chip) based on a microfluidic dynamic array platform allowing the expression profiling of 85 defense-related grapevine genes in 90 cDNA preparations in a 4&nbsp;h single run. Two defense inducers, benzothiadiazole (BTH) and fosetyl-aluminum (FOS), have been tested in natura using the &ldquo;NeoViGen96&rdquo; chip as well as their efficacy against downy mildew.

Results: BTH-induced grapevine resistance is accompanied by the induction of PR protein genes (PR1, PR2 and PR3), genes coding key enzymes in the phenylpropanoid pathway (PAL and STS), a GST gene coding an enzyme involved in the redox status and an ACC gene involved in the ethylene pathway.

Results: FOS, a phosphonate known to possess a toxic activity against pathogens and an inducing effect on defense genes provided a better grapevine protection than BTH. Its mode of action was probably strictly due to its fungicide effect at high concentrations because treatment did not induce significant change in the expression level of selected defense-related genes.

Conclusions: The NeoViGen96&rdquo; chip assesses the effectiveness of plant defense inducers on grapevine in vineyard with an excellent reproducibility. A single run with this system (4&nbsp;h and 1,500 &euro;), corresponds to 180 qPCR plates with conventional Q-PCR assays (Stragene system, 270&nbsp;h and 9,000 &euro;) thus a throughput 60&ndash;70 times higher and 6 times cheaper. Grapevine responses after BTH elicitation in the vineyard were similar to those obtained in laboratory conditions, whereas our results suggest that the protective effect of FOS against downy mildew in the vineyard was only due to its fungicide activity since no activity on plant defense genes was observed. This tool provides better understanding of how the grapevine replies to elicitation in its natural environment and how the elicitor potential can be used to reduce chemical fungicide inputs.

Electronic supplementary material: The online version of this article (doi:10.1186/s12864-016-3304-z) contains supplementary material, which is available to authorized users.

No MeSH data available.