TITLE: FruitRescue! - Prunus phenotyping
AUTHORS: Mathieu Brisson, Amandine Cornille

[DESCRIPTION]
The FruitRescue project aims to create a pipeline to predict fruit trees' genomic offset. To confirm the pipeline predictions, tree fitness parameters are measured in crop and wild tree orchards along a longitudinal gradient. Fitness is assessed through measurements of trunk diameter and the Nitrogen Balance Index (NBI), as well as monitoring phenological parameters such as bud break, flowering time, fruit set, and crop load.
This protocol is meant for Apricot and Peach (Prunus species) phenotyping.

[BEFORE_START]
Emails concerning the progress of the project should be sent to Mathieu Brisson cc Amandine Cornille.

Mathieu Brisson
mathieu.brisson@inrae.fr
+33 (0) 6 41 55 24 32
-
Amandine Cornille
amandine.cornille@nyu.edu
 


[GUIDELINES]
Orchard Management
In each orchard, standard cultural management practices should be followed as typically implemented on the site. One exception is to be noted : to prevent influencing bitter pit, calcium spraying is not applied.
Each year, a summary of these treatments will be sent to Mathieu Brisson and Amandine Cornille.
During winter a pruning might be required between November and December.
In June, thinning by hand might be required. A maximum of 2 fruit per cluster should remain.

Data management
Throughout the years, you will collect a large amount of data. To ensure good quality and readability of the data when opening spreadsheets on your computer, it is crucial to be vigilant. 
Here are some important guidelines concerning the data export step:
File Type: Always check the file type and favor .tsv or .csv for single sheet documents.
Date Formatting: Format the sheet's cells as text for dates in ISO 8601 (YYYY-MM-DD) format to facilitate reading across multiple computers and software.
Raw Data Backup: Always make a copy of the raw data file before opening it.
Cell and Decimal Separators: Check the cell and decimal separators before opening the file. For example, Dualex ".csv" output files contain data separated by semicolons, and opening the file carelessly could lead to the removal of the decimal separator.
Data Integrity: It is better to reference the original file in another sheet rather than writing directly inside it.
Data Structure: It is easier to understand the data when it is represented as a list rather than a map. Fill the given template with the data you have. If you have trouble filling it properly, leave it blank and contact the team who can provide you help.
After the data acquisition and eventual formatting, you will need to send it by mail to Mathieu Brisson, cc Amandine Cornille.

[STEPS]
SECTION: Trunk diameter
1. The trunk diameter is an indicator of the tree vigor (Waring 1987). Genetic information as well as environmental cues including abiotic and biotic stresses can modify the source-sink relationships and therefore modify the trunk growth during the year. Tree adaptation to its location can thus be assessed through this measurement.

SECTION: Dualex
2. 1 month after the end flowering, in general from mid-April to the end of May. The Dualex measures Chlorophyll and Flavonoid content and enables the calculation of the Nitrogen Balance Index (NBI). These elements are used to assess the tree photosynthetic activity and nitrogen nutrition (Cerovic et al. 2012).
SECTION: Trunk diameter
1.1. For grafted trees, a mark is placed 50cm above the grafting point with marking spray. For ungrafted trees, the mark is placed 50cm from the ground.
SECTION: Trunk diameter
1.2. After cold hardening, during winter, and 1 month after flowering using a digital caliper, measure the trunk diameter on the uppermost part of the white mark :
make a first measurement then a second at 90°.

 

 

SECTION: Phenology
3. Bud break / Bud burst
After cold hardening, buds become dormant. Entering in endodormancy, buds are in a stage of physiological inhibition of bursting (Lang, 1987). Only heat accumulation can lift this stage of growth inhibition, even in favorable conditions, switching the buds to their ecodormant stage. During the ecodormancy, buds are physiologically capable of sensing their environment but the bud break is repressed until conditions are favorable (Fadon et al. 2020). The lifting of the ecodormancy encompassing the bud swelling and burst stage is therefore a fitness trait or an indicator of local adaptation (Anderson et al. 2012).
SECTION: Dualex
2.2. There are some points of vigilence when taking measurements with the Dualex :
ID your Dualex if you use more than one.
Write down Line and Number of each tree associated with the group given by the Dualex (and eventually Dualex ID).
Write down the reason of a missing measurement (not enough leaves, tree death...)
Always look at the Dualex screen after each measurements to check on the integrity of the data (no star (*) should appear on the screen).


 

SECTION: Dualex
2.1. 1 month after the end flowering, in general from the end of may to the end of july. The Dualex measures Chlorophyll and Flavonoid content and enables the calculation of the Nitrogen Balance Index (NBI). These elements are used to assess the tree photosynthetic activity and nitrogen nutrition (Cerovic et al. 2012).
SECTION: Phenology
3.1. In February, the bud burst usually occurs for apple trees. Measure trees at stage 2 and 3 of the BBCH scale: note the date when the trees reach 10% of buds at these stages. 
Head twice a week to the orchard and record the stage of all trees.


 

SECTION: Fruit
7. Fruits play a crucial role in the maturation, protection and the dispersal of the seeds and thus the effective reproduction of the mother plant. Fruit pigmentation can be a protection against stresses (Espley and Jaakola 2023) or a signal perceived by animals feeding on them in order to disseminate the seeds (Steyn, 2008). Furthermore the fruit is also a major sink thus reflecting resources allocation and overall tree fitness.
SECTION: Phenology
4. Flowering and fruit set 
Flowering is a critical step for reproduction success. Its timing and its intensity has long term effect on multiple life history trait (Pallas et al. 2018). Genetical, physiological and environmental factors are thus controlling this complex set of traits that can have a long term effect on the survival and reproduction  of trees . Flowering Time intertwined in this complex set of characters is therefore a critical indicator of tree fitness in regard of climate change (Anderson et al. 2012). 
SECTION: Phenology
4.1. Flowering Time
From February to March, Apricot and Peach trees start flowering. Measure trees at stage 59 to 61 of the BBCH  scale (Baglioni E2/F1) when 10% of the flower have bloomed.
Head twice a week to the orchard and record the stage of all trees.
SECTION: Phenology
5. Flowering intensity
Flowering intensity or floribundity will be recorded as the number of bloomed flowers over the maximum number of possible flowers (%). The maximum number of possible flowers is set when all the buds have flowered. The trees are graded when they reach 50% of Flowering and it can be then re-evaluated at 100%. The most important thing is not to look at the flower number but to see if the flower distribution on the tree is according to the number of buds.

 


SECTION: Phenology
6. The fruit set rate
When pollination is successful, flowers will develop into swollen, immature and very small green fruits but when unsuccessful, the flowers that did not receive enough compatible pollen will not develop into a fruit and look like small green stars. 
The number of fruits are counted 2 to 3 weeks after flowering (=mean fruit number by clusters after abscission). This fruit set is the quantitative measurement of the proportion of flowers developing into fruits.
This will allow to calculate the percentage of fruit set. This is the percentage of flowers that become fruit.
% fruit set per tree = (# fruit / # Flowering buds)* 100
SECTION: Phenology
6.1. Crop Load
Environmental and Internal cues can also lead to fruit drop after pollination, diminishing the total fruit set in the weeks following pollination and establishing the tree's crop load.
Estimation of the crop load is done after the June fruit drop but before hand thinning. It is expressed in percent of present fruits compared to the maximum possible number of fruits. The tree express its maximum possible number of fruits when bearing fruits on each buds. We use the following scale :

1 = 0% of fruit set meaning 0 fruits on the majority of the tree's cluster
2 = around 25% of the fruit set.
3 = around 50% of the fruit set.
4 = More than 50% of the fruit set without any thinning.
5 = High production with reduced thinning.
6 = High production with heavy thinning
SECTION: Fruit
7.1. Fruit sample weight
When the fruits are ripe, harvest a sample of 51 fruits (a cellular crate) and weight the sample. 
SECTION: Fruit
7.3. Relative surface of red color cover on the background
Estimation of the % of surface red blushes above the background color, given by a scale from 0 to 5, mean by tree. 
0%: absence of red blush,
1- 1 to 20% of the fruit covered by red blush,
3- 40 to 60% of the fruit covered by red blush,
5- 80 to 100% of the fruit covered by red blush,
SECTION: Fruit
7.2. Grade the size variability among the sample.
0-  The sample is more homogeneous (more than 3-4 fruits seem to have a similar size)
1- The sample is more heterogeneous