Shoot and root phenological relationships in hybrid walnut growing in a Mediterranean alley cropping system

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Test 1: previously scanned and measured root systems

To allow for a fully comprehensive comparison of data between scanning, manual and automated methods, we tested each method on previously scanned and measured root system (n = 35), and likewise on a measuring tape placed in different positions (Fig. 3) of known dimensions in a rhizotron (50 x 50 cm). The scanned root systems were measured using four methods: flatbed scanner, handheld scanner, smartphone scanners and the time-lapse camera. Data were imported to the SmartRoot software.

Test 2. Measurements in rhizotrons using scanners and manual tracing

We performed measurements at Le Beil, Madic, in the Cantal region, France (45°22’7.95″N, 2°28’1.46″E) (see section on study site for more details). We started the observations in October 2014 and fine root growth was measured every month from April to June 2015 using four methods: flatbed scanner, handheld scanner, smartphone scanner (iphone4) and the manual tracing (n= 25). Walnut fine roots are quite thick and so roots ≤ 4 mm in diameter were classed as fine roots.

Test3. Measurements in rhizotrons using a time-lapse camera

A third set of measurements was performed at Cormont, in the Pas de Calais region, France (50°33’27.87″N, 1°44’3.08″E), (see section on study site for more details). Root growth was monitored in six rhizotrons twice a day from May to September. We focused our study on 21 roots growing over a period of 10 days for un easier understanding and comparison of results.

Image analysis

Once images of root growth had been acquired, we conducted analyses of images using the semi-automated SmartRoot software (Lobet et al 2011). SmartRoot is an operating system independent freeware based on ImageJ and uses cross-platform standards (RSML, SQL, and Java) for communication with data analysis softwares (Lobet et al 2011, Mathieu et al 2015).
Before analyzing roots with SmartRoot, when necessary, images need “stitching” together (e.g. with Adobe Photoshop CS3 software), if several have been taken (when the rhizotron surface area was greater than the field of the scanner). In our case, we transformed all images to 8 bit gray scale and then inverted them using ImageJ software so that roots were darker than the background of the image. The length and diameter of each root produced during one interval time (i.e. one month) were calculated for each rhizotron. Before analyzing a new sequence of images, SmartRoot provides the user with an icon to import the traces of the same roots from the previous image data file to superimpose them on this new image, which helps root elongation. This preceding image also helps determine whether the root is live (usually cream in color) or in a phase of senescence (shriveled, transparent or turning black) (Anderson et al 2003, Germon et al 2016, Graefe et al 2008, Huck & Taylor 1982, Tierney & Fahey 2002). We declared a root dead when it became completely black in color.

Study site

We measured fine root growth in situ in two agroforests. One was located at Le Beil, Madic, in the Cantal region, France (45°22’7.95″N, 2°28’1.46″E) at an elevation of 530 m, hereafter termed ‘continental’ climate. The agroforest comprised three transplanted tree species: hybrid walnut (Juglans major (MJ209) × Juglans regia L.), cherry (Prunus avium L.), sycamore maple (Acer pseudoplatanus L.) at 12 m x 8 m tree spacing and intercropped with permanent pasture (ovine or bovine pasture). All national guidelines and legislation were complied with when using these cultivars. Mean diameter at breast height (DBH) of all walnut trees at the site was 0.20 ± 0.02 m and mean height was 12.09 ± 1.30 m. Data are means ± standard error.
All trees were planted in 1994 at a density of 100 trees ha-1. Hybrid walnut at this study site starts leafing in early May and shedding in mid-November. The climate is continental with a mean annual temperature of 9.95°C and a mean annual rainfall of 1174 mm (Météo France). The soil is silty and not deep, attaining an average maximum depth to bedrock of approximately 110 cm, on a 5° – 10° slope.
The second agroforest was located at Cormont, in the Pas de Calais region, France (50°33’27.87″N, 1°44’3.08″E), hereafter termed ‘oceanic’ climate. The site is at an altitude of, 40m. The climate is oceanic, with a mean annual temperature of 11°C and a mean annual rainfall of 777.9 mm (Météo France). Tree species comprised hybrid walnut (Juglans nigra × regia L.) and Maple (Acer laurinum L.) at 13 x 7.5 m tree spacing intercropped permanent pasture (ovine pasture). All trees were planted in 1999. The soil is silty clay and < 2.5 m deep. The site is next to La Dordonne River. Mean DBH of walnut trees at the site was 0.30 ± 0.03 m and mean height was 14.75 ± 3.50 m. Hybrid walnut at this site starts leafing in early May and shedding in mid-November.

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Rhizotron installation

In the continental agroforest (Madic), we dug eight (1 m x 1 m x 1 m) trenches by hand in three rows of trees. Each trench was at a distance of 2 m from the nearest tree stem. Eight rhizotrons, or root windows (50 cm long x 50 cm wide x 0.5 cm thick), were installed. In the oceanic agroforest (Cormont), soil was deep (4 m) and comprised four (2 m long x 1 m wide x 2 m depth) trenches in one row of trees placed at 2 m from the nearest tree stem. One rhizotron was installed on two opposing faces of the trench (n = 8 rhizotrons in total). All rhizotrons were placed vertically at an angle of 15° from the face of the profile. This angle will permit the roots to grow downwards due to positive geotropism (Huck & Taylor 1982, Mao et al 2013). Where the rhizotrons were to be placed on the trench, we gently removed the soil to make a flat surface and cut all roots on the profile with secateurs. The soil removed during the digging of the trenches was kept aside, and then sieved through a 5 mm size sieve and air-dried for several hours. The sieved and air-dried soil was then poured into the space between the window and the soil profile and slowly compacted using a wooden plank. Each rhizotron was covered with foil backed felt insulation and black plastic sheeting to protect roots from light and temperature variations. All trenches were then covered with wooden boards and corrugated plastic to avoid damage from passing animals and to prevent direct rainfall and sunlight onto the rhizotrons. In the first three months after installation, no root growth was recorded to avoid over estimations of root growth (Strand et al 2008).

Root indicator calculation

We used the following method to estimate root elongation rate:
individual root growth was evaluated by calculating the difference between the root length at t -1 and at t. To determine the daily root elongation rate (RER), the mean of all individual root lengths produced between time t and t -1 was divided by the duration of the corresponding period (Germon et al 2016). According to the literature, the characterization of dead roots is not obvious, particularly behind a transparent window (Tierney & Fahey 2002). We considered root as live when it had a cream color and dead when it had turned black with no growth between two or more successive sessions until the last observation date occurred (Germon et al 2016).
A Shapiro-Wilk test was performed before each test to ensure if the investigated indicator followed a normal or non-normal distribution. Homogeneity of variances was checked. For data not normally distributed, analyses were followed by a Kruskal-Wallis Test for each factor. A post-hoc analysis between root diameters was performed using the Nemenyi test of Kruskal Wallis at p<0.05 to determine which levels of the independent variable differ from every other level. All analyses were performed using R software, Version 2.15.3 (R Development Core Team 2013) at a significance level of <0.05.

Table of contents :

Chapter I: General Introduction
1. What are fine roots?
2. Why study fine roots?
3. Why Agroforestry?
4. Root methodological problems
5. What drives fine root dynamics?
6. The role of deep fine roots
7. Are above and below ground phenology in sync?
8. General hypotheses and objectives
9. General approach and study sites
10. Chapter arrangement
11. References
Chapter II: An evaluation of inexpensive methods for root image acquisition when using rhizotrons
Abstract
1. Background
2. Materials and methods
3. Results
4. Discussion
5. Conclusion
6. Abbreviations
7. General informations
8. References
Chapter III: Above and belowground phenological relationships in hybrid walnut growing in agroforests along a climatic gradient
Abstract
1. Introduction
2. Materials and methods
3. Results
4. Discussion
5. Conclusion
6. Abbreviations
7. Figures
8. Appendix
9. References
Chapter IV: Shoot and root phenological relationships in hybrid walnut growing in a Mediterranean alley cropping system
Abstract
1. Introduction
2. Materials and methods
3. Results
4. Discussion
5. Conclusion
6. Figures
7. Appendix
8. References
Chapter V: General discussion
1 Root methodological problems
2 Root growth and mortality drivers
3 Shoot and root relationships
4 Root survivorship
5 Deep fine root phenology
General conclusion and perspectives
References
Résumé: Objectifs, résultats, conclusions générales
Les hypothèses principales:
Les objectifs:
Les approches générales et les sites d’étude
Les résultats généreux
Conclusion générale et perspectives

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