Plant biotic agent phenotyping platform and process of phenotyping

Abstract

The present invention relates to a plant biotic agent phenotyping platform comprising a container hermetically sealed with a lid delimiting an internal space divided into two spaces: -a lower internal space which comprises at least one temperature control means immersed in a temperature transfer liquid; and -an upper internal space, the surface of the temperature transfer liquid being the border between the lower internal space and the upper internal space; the lid comprising at least one orifice which is adapted to fit a pot and at least one pot whose bottom part is contained in the upper internal space and which is adapted to receive plant seeds and soil parasite and to enable development of such soil parasites and plants. The invention also related to the use of this platform for screening plant resistance or tolerance to soil parasite.

Claims

1. A plant biotic agent phenotyping platform comprising a container hermetically sealed with a lid delimiting an internal space divided into two spaces: a lower internal space which comprises at least one thermostat immersed in a temperature transfer liquid; and an upper internal space, the surface of the temperature transfer liquid being the border between the lower internal space and the upper internal space; the lid comprising at least one orifice which is adapted to fit a pot and at least one pot whose bottom part is contained in the upper internal space and which is adapted to receive plant seed and soil parasites, and to enable growth of the plant and development of such soil parasites, wherein the pot has such a size that when it is placed within the orifice, the pot is not in contact with the temperature transfer liquid; wherein the pot comprises a plant seed sowed on a substrate; wherein the pot further comprises at least one soil parasite; and wherein the platform is configured to determine a resistance or a tolerance between the plant and the soil parasite.

2. The plant biotic agent phenotyping platform of claim 1, further comprising a decontaminator.

3. The plant biotic agent phenotyping platform of claim 2, wherein the decontaminator further comprises a chlorinated water.

4. The plant biotic agent phenotyping platform according to claim 1 further comprising a drip system delivering a controlled and equal amount of water independently to each pots.

5. The plant biotic agent phenotyping platform according to claim 1, wherein the soil parasite is broomrape and the plant is sunflower or rapeseed.

6. The plant biotic agent phenotyping platform according to claim 1, wherein the temperature transfer liquid is water.

7. The plant biotic agent phenotyping platform according to claim 1, wherein the container also comprises an insulating material.

8. The plant biotic agent phenotyping platform according to claim 1, wherein the container is 1 to 3 m of length and 50 cm to 1 m of width.

9. The plant biotic agent phenotyping platform according to claim 1, comprising from 50 to 500 orifices and pots.

10. A kit comprising the phenotyping platform according to claim 1, and further comprising at least one element selected from the group consisting of an irrigator, pot lighting, and a decontaminator.

11. A phenotyping greenhouse comprising at least one phenotyping platform according to claim 1, further comprising at least one element selected from the group consisting of an irrigator, pot lighting, and a decontaminator and/or a thermostat for controlling temperature and/or an instrument for controlling hygrometry of the greenhouse.

12. A method of identification of plant resistance or tolerance to a root parasite or interaction with a symbiotic organism comprising: sowing a seed plant on a substrate containing a standard quantity of soil parasite in at least one pot of a phenotyping platform, wherein the phenotyping platform is the phenotyping platform according to claim 1: cultivating the plant; recovering the plants after a predetermined duration of culture; and noting the roots and/or aerial damage on the plant.

13. The method according to claim 12 for the identification of plant resistance or tolerance to an Orobanche parasite comprising: sowing the seed of the plant on a substrate containing a standard quantity of Orobanche in at least one pot of the phenotyping platform of claim 1, cultivating the plant; recovering the roots of the plant; counting the number of nodules created by the parasite on the plant roots; and determining whether the plant is resistant or tolerant to the Orobanche parasite.

14. The method according to claim 13 further comprising the recovery of DNA and/or RNA of Orobanche for genotyping.

15. The method according to claim 12 further comprising the genotyping of the plants having shown resistance or higher tolerance and the use of this plant for breeding new varieties.

16. A method of identification of soil parasite population aggressiveness to a plant comprising: sowing seeds plant on a substrate containing different quantity of soil parasite from different origin in at least one pot of a phenotyping platform according to claim 1; cultivating the plant; recovering the plants after a predetermined duration of culture; and noting the roots and/or aerial damage on the plant.

17. The method according to claim 16 for the identification of Orobanche population aggressiveness to a plant comprising: sowing Orobanche plants or seeds plants on a substrate containing a standard quantity in at least one pot of the phenotyping platform of claim 1, cultivating the plant; recovering the roots of the plant; counting the number of nodules created by the parasite on the plant roots; and determining whether the Orobanche is aggressive to the plant.

18. A method of identification of plant resistance or tolerance to drought comprising: sowing a seed plant on a substrate with a standard moisture in at least one pot of a phenotyping platform comprising: a container hermetically sealed with a lid delimiting an internal space divided into two spaces, a lower internal space which comprises at least one thermostat immersed in a temperature transfer liquid; and an upper internal space, the surface of the temperature transfer liquid being the border between the lower internal space and the upper internal space; the lid comprising at least one orifice which is adapted to fit a pot and at least one pot whose bottom part is contained in the upper internal space and which is adapted to receive plant seed and to enable growth of the plant; wherein the pot has such a size that when it is placed within the orifice, the pot is not in contact with the temperature transfer liquid; wherein the pot comprises a plant seed sowed on a substrate; and wherein the platform is configured to determine the resistance or tolerance to drought; cultivating the plant, according to controlled drought condition; recovering the plants after a predetermined duration of culture; and noting the roots growth and architecture on the plant.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention will better be defined based on the following drawings.

(2) FIG. 1: represents a platform (1) according to the invention comprising a container (2) hermetically closed by a lid (5) comprising two pots (7) and delimiting an internal space (3) into which are place an insulating mean (6) and temperature control means (4) immersed in a temperature transfer liquid.

(3) FIG. 2: represents a platform (1) according to the invention comprising a container (2) hermetically closed by a lid (5) comprising eleven pots (7) and delimiting an internal space (3) into which are place temperature control means (4) immersed in a temperature transfer liquid.

(4) FIG. 3: represents an assembly of 5 platforms (I to V) according to the invention, each platform comprising 72 orifices (12 lines (9) (1 to 12) and 6 columns (8) (A to F)).

(5) FIG. 4: represents an irrigation system according to the invention, the arrows representing the flow of water.

(6) FIGS. 5 to 7: represents respectively the distribution (histograms) of the frequency of data obtained with the method of the invention for sunflower and orobanche race F (two screening (FIGS. 5 and 6) and field data (FIG. 7).

(7) FIGS. 8 to 10: represents respectively the correlation (scatter plots with pearson r value) of the results obtained in screening 1 and 2 (FIG. 8), of the results obtained in screening 1 and field (FIG. 9) and of the results obtained in screening 2 and field (FIG. 10).

EXAMPLES OF THE INVENTION

(8) The below tests were carried out in a greenhouse comprises a platform composed of an aluminum container (2500?800?200 mm) and a lid (thickness 6 mm) composed of a polyethylene sheet of 5.6 mm thick covered in its two faces with an aluminum sheet of 0.21 mm.

(9) The lid enables to hermetically close the container and is maintained thanks to spacer riveted. The lid comprises 360 orifices of diameter 4.5 or 5.5 cm (12 lines of 30 orifices) and pots. The pots have a diameter of 4.5 or 5.5 cm to fit the orifice's diameter and are 11 cm length.

(10) The pots present are perforated and a cotton balls is placed in the hole. The pots are then filed with a substrate comprising blonde sphagnum peat and NPK fertilizer.

(11) An aqua napping is placed at the bottom of the container. A heating cable (whose heating power is 300 W/m.sup.2) is placed on the aqua napping and 4 cm of water (temperature transfer liquid) is added (the heating cable is totally immersed in water).

(12) The 30 lines of orifices are irrigated by 5 independent irrigation unit, each irrigation unit is adapted to irrigate 6 lines.

(13) Each pot can be identified by number of the irrigation unit 1 to 5, a letter A to E to address the position of the pot on the irrigation unit, and a range 1 to 12 to address the position of the plot on the table.

Example 1

Homogeneous and Controlled Temperature Around the Pot

(14) The heating cable is set in order to have a temperature comprised between 27.5 and 28.5 in the container. After 14 hours of running the temperature was measured into the pots. The results are shown in the table below:

(15) TABLE-US-00001 I A B C D E F II A B C D E F III A B C D E F 1 29 28.9 28.8 29 1 28.6 28.6 28.4 28.4 28.3 28.4 1 28.3 28.2 28 28.1 28.1 28.1 2 28.9 28.7 28.5 28.7 28.8 2 28.6 28.6 28.6 28.5 28.3 28.3 2 28 28 28.1 28.3 28.3 28.2 3 29 28.9 28.8 28.5 28.5 28.5 3 28.5 28.5 28.4 28.3 28.3 28.2 3 28 28.1 28.2 28 28 28.1 4 29 28.6 28.6 28.6 28.6 28.6 4 28.6 28.6 28.3 28.3 27.8 28.1 4 27.8 28 28 28.1 28.1 28.2 5 29 28.9 28.7 28.7 28.6 28.7 5 28.4 28.4 28.3 28 27.9 28 5 28 28 27.9 27.9 28 28 6 29 28.8 28.7 28.5 28.6 28.5 6 28.6 28.5 28.3 28 28 27.8 6 27.8 27.5 27.7 27.7 27.7 27.8 7 29 28.8 28.7 28.5 28.5 7 28.4 28.2 28 28 27.9 27.7 7 27.8 27.8 27.7 27.6 27.7 27.8 8 29 28.8 28.6 28.6 28.6 28.5 8 28.6 28.4 28.3 28 27.7 27.7 8 27.5 27.6 27.6 27.6 27.6 27.5 9 29 29 28.8 28.8 28.6 28.5 9 28.6 28.6 28 27.9 27.5 27.6 9 27.5 27.5 27.4 27.4 27.5 27.5 10 29 28.8 28.7 28.5 28.3 28.4 10 28.3 28.2 28.1 28.9 27.7 27.5 10 27.2 27.3 27.2 27.4 27.4 27.5 11 29 29 28.9 28.8 28.5 28.4 11 28.1 27.9 27.7 27.6 27.5 27.5 11 27.4 27.2 27.1 27.1 27.1 27 12 28.7 28.8 28.5 28.3 28.3 28.4 12 28.1 28.2 28.1 28 27.7 27.6 12 27.2 27 27 27.4 27.4 27.4 IV A B C D E F V A B C D E F 1 28.1 28.1 28 27.9 28 27.9 1 28 28.1 28 28 28 28 2 28.1 28.2 28.2 28.1 28.1 28 2 28.2 28.2 28.1 27.9 28.1 27.9 3 28.3 28.2 28.2 28.2 28.2 28.3 3 28.2 28.1 28.2 28.2 28.2 28.1 4 28 28.1 28 28.2 28.2 28.2 4 28.3 28.3 28.3 28.2 28.9 28.1 5 28.1 28.1 28.1 28.1 28.2 28.2 5 28.3 28.4 28.4 28.4 28.4 28.4 6 27.6 27.9 28 28 28 28.2 6 28.4 28.4 28.5 28.6 28.6 28.5 7 27.9 27.9 28 28.1 28.2 28.1 7 28.4 28.4 28.4 28.2 28.5 28.5 8 27.7 27.7 27.6 27.8 27.8 28 8 28.2 28.2 28.3 28.3 28.4 28.4 9 27.6 27.9 27.6 27.7 28 27.9 9 28.2 28.3 28.3 28.4 28.4 28.5 10 27.3 27.4 27.4 27.4 27.5 27.6 10 27.9 28 28.1 28.1 28.2 28.3 11 27.1 27.2 27.3 27.4 27.6 27.6 11 28.2 28.3 28.4 28.4 28.4 28.4 12 27.4 27.5 27.5 27.5 27.5 27.5 12 27.6 27.8 28 28 28.2 28.1

(16) The temperatures varied between 27 and 29.5. The temperature is considered homogeneous. This example clearly shows that the platform according to the invention enables to have controlled and homogeneous temperature conditions around each pot.

Example 2

Determination of Resistance or Tolerance of Sunflower Races to Orobanche

(17) The greenhouse and the 360 pots platform disclosed above are used. The pots not used are hermetically closed.

(18) An equal quantity of Orobanche seeds of race F (Spain) are placed in each pot with a seed of sunflower. 300 different sunflower genotype are placed on the platform and studied, six to twelve repeats are studied by genotype and this experiment has been done twice (screening 1 and 2 respectively FIGS. 5 and 6).

(19) The heating cable is set in order to have a temperature comprised between 27.5 and 28.5 in the container. The temperature inside the pots is checked once a week and is regulated thanks to a thermostat if needed.

(20) The platform is located in a greenhouse where plants are cultivated under Sodium vapor lights (400 W), at a temperature of 18 to 22? C. and a hygrometry comprised between 40 and 60%. The pots are watered once a week during 50 seconds with a drop wise irrigation system.

(21) Five weeks after sowing the heating cable, the irrigation system and the lights are shut down. The roots of each plant are carefully washed in separate container comprising water. The numbers of nodules on the roots are count.

(22) The results are shown on the histograms of FIGS. 5 and 6. The frequency distribution of results of the first and second screening is shown in FIGS. 5 and 6 respectively. The average numbers of nodules per plants for one genotype varied between 0 and 20 and are represented by the histogram (axe x) for each line of sunflower, the axe y represents the frequency of sunflower genotypes having the considered average number of nodules.

(23) The comparison of results from screening 1 and 2 are shown in FIG. 8. Each genotype studied in this experiment corresponds to a point. The projection of the point in the Y-axis gives the number of nodules obtained for the first screening and the projection of the point in the X-axis gives the number of nodules obtained for the second screening.

(24) This graph allows calculating the Pearson correlation coefficient with the following formula:

(25) $r=\frac{{?}_{\mathrm{xy}}}{{?}_x{?}_y}$

(26) ?.sub.xy is the covariance between variable x and y

(27) ?.sub.x is the standard deviation of variable x

(28) ?.sub.y is the standard deviation of variable y

(29) The Pearson correlation coefficient calculated between screening 1 and screening 2 is 0.7. The results show what, the method of the invention and the use of the platform according to the invention are repeatable.

Example 3

Correlation between Data Obtained in Fields

(30) The correlation used the data obtained from the two screening of example 2. The same 300 sunflower genotypes were phenotyped in field in order to address the reliability of the test and its ability to predict field behavior. One line per genotype with around 20 plants was sown in Spain field, supposed to contain Orobanche seeds form race F. At ad hoc plant stage, the number of plants with at least 1 emerged Orobanche is scored per line. Based on this scoring the percentage of plants on the line with at least on emerged Orobanche is computed.

(31) FIG. 7 gives the frequency distribution of the percentage of plants with at least on emerged Orobanche per sunflower genotype based on Spain field experiment.

(32) FIGS. 9 and 10 display the correlation between respectively screening 1 and 2 and field scoring, produced for 300 sunflower genotypes with Orobanche seeds from race F (Spain origin), according to the present invention and data set obtain in field on the same set of sunflower genotype in Spain, known to contain race F Orobanche seeds.

(33) The comparison of results from screening 1 and the field scoring and from screening 2 and the field scoring are shown respectively in FIGS. 9 and 10. Each genotype is represented by a point. The projection of the point in the Y-axis gives the number of average nodules per plant tested during the first or second screening (respectively FIGS. 9 and 10 and the projection of the point in the X-axis gives the percentage of plants with at least one emerged orobanche during the field screening).

(34) These graphs allow calculating the Pearson correlation coefficient which is from 0.41 for the first screening and 0.38 for the second screening.

(35) Therefore, the method of the invention has been demonstrated to be an accurate predictive tool for field trial (r=0.4), and can be used a prescreening tool in breeding program. The results show that the use of the platform according to the invention enables to determine the resistance or tolerance to a plant with respect of a soil parasite.

Example 4

Destruction of Trash and Cleaning of the Platform

(36) Water contained in the container (water initially added in the container and water evacuated by the pots (evacuation of excessive amount of water caused by the irrigation by the hole in the bottom of each pot) and water resulting from the washing of the platform are heated at 80? C. during one hour in order to eliminate Orobanche and sunflower seeds.

(37) The solid trash (especially the plants and the substrate) are placed in autoclavable bag comprising two layers. The autoclavable bag is placed in another autoclavable bag and then put into a dump to which a vapor generator is connected. The vapor generator is switched. After treatment the resulting trash are disposed of with household waste.

(38) The elements of the platform (container, lid, heating material) are washed by soaking in Javel water.