Rawiwan Chotiphan

Laurent Vaysse

Regis Lacote

Eric Gohet

Philippe Thaler

Kannika Sajjaphan

Celine Bottier

Christine Char

Siriluck Liengprayoon

Dr Frederic Gay
email: fgay@cirad.fr

Can fertilization be a driver of rubber plantation intensification?

Keywords: Natural rubber, fertilization, ethylene stimulation, yield, latex metabolism, latex properties

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Natural rubber (NR) extracted from rubber trees (Hevea brasiliensis) plays a crucial role in the socio-economic development of many tropical countries. NR is an indispensable raw material for several manufacturing industries, particularly the tyre sector which absorbs 70% of the total supply. During the last 20 years, high demand on the world market has led to the expansion of rubber plantations, particularly in South East Asia [START_REF] Fox | Expansion of rubber (Hevea brasiliensis) in Mainland Southeast Asia: what are the prospects for smallholders?[END_REF]. Expansion raises questions about their environmental impacts as it triggers forest clearance [START_REF] Guardiola-Claramonte | Hydrologic effects of the expansion of rubber (Hevea brasiliensis) in a tropical catchment[END_REF][START_REF] Hauser | Environmental and socio-economic impacts of rubber cultivation in the Mekong region: challenges for sustainable land use[END_REF]2015;[START_REF] Hughes | Understanding the drivers of Southeast Asian biodiversity loss[END_REF][START_REF] Warren-Thomas | Protecting tropical forests from the rapid expansion of rubber using carbon payments[END_REF] and about the socioeconomic impact when rubber trees are planted on agricultural land where they compete with food crops [START_REF] Fox | Expansion of rubber (Hevea brasiliensis) in Mainland Southeast Asia: what are the prospects for smallholders?[END_REF][START_REF] Chambon | Can the cocoa cycle model explain the continuous growth of the rubber (Hevea brasiliensis) sector for more than a century in Thailand?[END_REF]. One way to limit such impacts is to increase productivity per planted area by intensifying agricultural practices.

In most crops, intensification relies mainly on nutrient management and the use of fertilizers [START_REF] Cassman | Ecological intensification of cereal production systems: Yield potential, soil quality, and precision agriculture[END_REF][START_REF] Witt | The need to increase profitability in oil palm plantations: matching crop and nutrient management principles with evolving strategies[END_REF]. However, rubber tree is a crop where the harvested component, latex, is not a vegetative or reproductive organ whose biomass is directly linked to primary production. Natural rubber (NR) obtained from the latex of the trees is mainly comprised of cis, 1-4, polyisoprene, a secondary metabolite very rich in carbon (see review by [START_REF] Vaysse | Natural Rubber[END_REF].

Latex does not naturally exude from the trees and only tapped trees need to regenerate latex through a tapping-induced metabolism. Hence, improving tapping techniques has been the main driver of rubber plantation intensification along with the release of modern high-yielding clones.

In particular, stimulation of latex metabolism with ethylene-based stimulant has made it possible to maintain or even increase the yield while reducing tapping frequency [START_REF] Eschbach | Avantages de la stimulation à l'Ethrel en association avec une réduction de l'intensité de saignée en Côte d'Ivoire[END_REF][START_REF] Gohet | Clone, croissance et stimulation, facteurs de la production de latex[END_REF][START_REF] Thanh | Long-term Effect of Tapping and Stimulation Frequency on Yield Performance of Rubber Clone GT1[END_REF][START_REF] Zhu | Ethylene stimulation of latex production in Hevea brasiliensis[END_REF][START_REF] Lacote | Longterm effect of ethylene stimulation on the yield of rubber trees is linked to latex cell biochemistry[END_REF]. [START_REF] Lacote | Longterm effect of ethylene stimulation on the yield of rubber trees is linked to latex cell biochemistry[END_REF] found the maximum increase in the yield of stimulated trees ranged from +30% to +78%, compared to that of unstimulated trees, depending on the clone. Conversely, the effect of fertilization on latex yield are still unclear, and this research topic has been almost ignored for the last 40 years, as most references in the literature date from the 1970s in Malaysia [START_REF] Sivanadyan | Nutrition of Hevea brasiliensis in relation to Ethrel stimulation[END_REF][START_REF] Pushparajah | Recent developments in the nutrition of Hevea in west Malaysia[END_REF]Yogaratnam and Weerasuriya 1984) or in Côte d'Ivoire [START_REF] Compagnon | Progression de l'usage des fertilisants et plus particulièrement de la fumure potassique en rapport avec la productivité de l'hévéa[END_REF][START_REF] Du Plessix | Effet de la fumure potassique sur les hévéas francs de pied cultivés sur sables tertiaires en savane de Basse Côte d'Ivoire[END_REF]. Since the 1980s, the maximum reported increase in yield due to fertilization has been 26% [START_REF] Onuwaje | Yield response of rubber (Hevea brasiliensis) to fertilizers on an Ultisol in Nigeria[END_REF]) but most other reports showed no effects below 20% or no effects at all [START_REF] Murbach | Adubação npk e produção de borracha seca pela seringueira (Hevea brasiliensis)[END_REF][START_REF] Virgens Filho | Efeito da calagem e adubação da seringueira no estado nutricional e produção de borracha seca[END_REF][START_REF] Gohet | Sustainable rubber production through good latex harvesting practices: An update on mature rubber fertilization effects on latex cell biochemistry and rubber yield potential[END_REF].

For several authors, a mature rubber plantation is a forest-like ecosystem, which is "selfsustainable" or in a "steady-state" with respect to the nutrient requirements of the trees [START_REF] Krishnakumar | Nutrition of Hevea[END_REF]Sivanadiyan et al., 1995; further developed by [START_REF] George | Natural rubber plantation: a nutritionally self-sustaining ecosystem[END_REF]. Hence, the management of fertilization of a rubber plantation is different than that of annual crops. In particular, high nutrient returns to the soil through leaf fall, self-pruning and fine root turnover need to be taken into account [START_REF] Samarappuli | Economics and efficiency of fertilizer utilization in mature rubber[END_REF]. The recycling of these nutrients is hypothesized to be enough to support the yield and the functioning of the trees thanks to the low nutrient exports through latex harvesting, which is rather low compared to the tree biomass (4-5 kg/tree/year) and contains only small amounts of mineral nutrients [START_REF] Bolton | The manuring and cultivation of Hevea brasiliensis[END_REF][START_REF] Samarappuli | Economics and efficiency of fertilizer utilization in mature rubber[END_REF][START_REF] Murbach | Nutrient cycling in a RRIM 600 clone rubber plantation[END_REF]. According to these authors, annual exports of nitrogen range from 5 to 12 kg.ha -1 .year -1 while recycling of N through litter falls amount to more than 20 kg.ha -1 .year -1 [START_REF] Murbach | Nutrient cycling in a RRIM 600 clone rubber plantation[END_REF]. These figures suggest that rubber tree only needs a small amount of fertilizer to support latex production and to enable a satisfactory girth increment during the mature stage. Based on experiments by [START_REF] Sivanadyan | Manuring of mature hevea: recent evidences and a possible new outlook[END_REF], [START_REF] Watson | Nutrition[END_REF] reported that fertilization only needed to start four years after the beginning of tapping, and that N application would be sufficient if the plantation was well managed during the immature phase (e.g. with both leguminous cover and regular fertilizer applications). If required P, K and Mg fertilizers could be applied at 3 to 5year intervals.

Fertilization can also change the mineral composition of the latex with possible detrimental effects on quality. For instance, divalent cations such as Mg 2+ are known to be detrimental to latex stability, and industrial production of latex concentrate generally includes Mg 2+ precipitation through phosphate addition prior to centrifugation. [START_REF] Collier | Effect of fertilizer applications on latex properties[END_REF] observed that nitrogen fertilization increased the Mg 2+ content of field latex while decreasing dry matter of the latex and increasing nitrogen content of air-dried sheet rubber. The higher Mg 2+ content reduced the mechanical stability of concentrated latex. Conversely, applying potassium or phosphate fertilizers may counteract the effect of Mg 2+ on latex. A long-term experiment carried out by the Rubber Research Institute of Malaysia (cited in [START_REF] Watson | Nutrition[END_REF]) revealed a positive effect of K fertilizers on latex stability. The decrease in Mg content after K fertilizer is applied leads to antagonism of root uptake between the two cations. A study by [START_REF] Philpott | Stability and mineral composition of Hevea latex[END_REF] demonstrated the beneficial role of combined K and P fertilizers in latex stability, which reduced the Mg/P ratio in the latex. However, in Thailand, the world's biggest producer of NR, the amount of fertilizer recommended by R&D institutions is high and, in practice, farmers sometimes apply even more than the recommended rate of NPK fertilizers in tapped plantations [START_REF] Chambon | What determine smallholders' fertilization practices during the mature period of rubber plantations in Thailand?[END_REF]. A recent socioeconomic survey showed that farmers believe such practices are beneficial [START_REF] Chambon | What determine smallholders' fertilization practices during the mature period of rubber plantations in Thailand?[END_REF].

However, this assumption is not supported by field data and such practices may not only be a waste of money for the farmers, but a source of environmental pollution if the nutrients are not effectively taken up by the trees. Such contradictions may arise from the indirect effects of NPK fertilization on latex yield through the improvement of the nutrient status of the whole tree, resulting in long-term benefits. As farmers' benefits rely on good management to maintain a balance between rubber production and plant growth, it is also important to understand how regular fertilization affects tree growth. Another possible explanation for these contradictions? is that Thai rubber farmers have adapted their fertilization practices to the very intensive tapping frequencies they use [START_REF] Chambon | A Comparative Analysis of Smallholders Tapping Practices in Four Rubber Producing Regions of Thailand[END_REF]. According to [START_REF] Gohet | Sustainable rubber production through good latex harvesting practices: An update on mature rubber fertilization effects on latex cell biochemistry and rubber yield potential[END_REF], potential gain from fertilization can only be achieved through the intensification of tapping practices. As tapping drives the demand for latex regeneration, it is not surprising that no effect of fertilization can be demonstrated if the same tapping system is applied in fertilized and unfertilized plots. However, interactions between latex harvesting technologies and fertilization of tapped rubber trees are poorly scientifically documented, particularly the use of ethylene stimulation to enhance latex yield, although, [START_REF] Sivanadyan | Nutrition of Hevea brasiliensis in relation to Ethrel stimulation[END_REF] reported a positive interaction between fertilization and ethylene stimulation, which was partly confirmed by [START_REF] Gohet | Sustainable rubber production through good latex harvesting practices: An update on mature rubber fertilization effects on latex cell biochemistry and rubber yield potential[END_REF].

In this context, the main objective of our work was to re-assess the possible effects of fertilizers combined with ethylene stimulation on latex yield in rubber plantations in Thailand. Our starting hypothesis was that there is a positive interaction between fertilization and stimulation, as the higher metabolic demand induced by stimulation would require additional nutrient resources. To this end, we analysed yield data from a three-year experiment with four fertilizer treatments applied to stimulated and unstimulated trees. To better understand the effect of fertilization and stimulation on yield, we collected data on the latex metabolism using the latex diagnosis approach [START_REF] Jacob | Yield limiting factors, latex physiological parameters, latex diagnosis, and clonal typology[END_REF][START_REF] D'auzac | The regulation of cis-polyisoprene production (natural rubber) from Hevea brasiliensis[END_REF]). The underlying assumption was that fertilization would have an effect on latex metabolism before having a statistically noticeable effect on yield. We also measured certain latex properties to assess possible negative effects of fertilization on yield and on the quality of the latex.

Material and methods

Site description

The three-year experiment was carried out from May 2014 to April 2017 in a traditional rubber growing area at the Sithiporn Kridakorn Research Station (latitude 10 ๐ 59'13.35"N, longitude 99 ๐ 29'22.41"E) of Kasetsart University, Prachuap Khirikhan province, Thailand (Fig. 1). The research station is located at the limit between two main climatic regions of Thailand: one with an equatorial climate, which extends southward from Chumphon province to the border with Malaysia, and the other with a tropical monsoon climate with long rainy season, which covers several provinces scattered in the northern, central and the east-central parts of the country.

Weather data collected at the station from 2000 to 2010 showed average annual rainfall of 1985 mm (s.e. 383 mm) with 106 rainy days (s.e. 11 days) (Table 1). The rainiest months are October and November with more than 250 mm on average. The driest month is January with 64 mm on average. These climatic conditions are perfect for rubber tree cultivation with high latex yield potential [START_REF] Gohet | Sustainable rubber production through good latex harvesting practices: An update on mature rubber fertilization effects on latex cell biochemistry and rubber yield potential[END_REF]. Total rainfall in the two first years of the experiment (2014-15 and 2015-16) was respectively 20% and 27% lower than in the 2000-2010 reference period, and 22% higher in the third year (Table 1). The number of rainy days was higher than the reference period in all three years with respectively, 112, 109 and 129 days of rain. The soil of the experimental plot, classified as Rhodic Kandiudults, is deep with a sandy-loam texture. The topsoil (0-30 cm) has the following average characteristics: 7.7% clay; 71.4% sand; 20.9% loam; pH4.9 (1:1 water); 0.25% C and 0.032% N (Elemental Analysis); CEC 2.1 meq/100g (pH7 ammonium-acetate); available P 12 ppm and available K 32 ppm (ICP-AES).

Figure 1 and Table 1 to be inserted here

Experimental design

The experimental plot was a ca. 9 ha rubber plantation set up in 2007 using the Hevea brasiliensis RRIM 600 clone, which has medium to high metabolic activity and is used in 90% of rubber plantations in Thailand [START_REF] Delarue | La Thaïlande : premier exportateur de caoutchouc naturel grâce à ses agriculteurs familiaux[END_REF]. The plot previously contained a coconut plantation. From planting to the beginning of the experiment in May 2014, the plantation was regularly weeded but not fertilized. The trees were planted with a spacing of 8 m between tree lines and 2.5 m between trees resulting in an average density of 500 trees.ha -1 . Tapping the tree bark to harvest latex started at the beginning of the experiment in May 2014. Only the trees with a trunk girth of 47 cm measured 100 cm above the ground were opened on this occasion. The first tapping cut was made 150 cm from the ground by removing a thin layer of bark along a downward half spiral on the tree trunk. The trees were then tapped every two days without any tapping rest in a week until the trees completely shed their leaves in February 2015. Tapping stopped during the refoliation period and resumed in May 2015 for another 10-month period using the same tapping system. This tapping system is noted S/2 d2 7d7 10m(MAY-FEB)/12 with respect to the revised international notation for latex harvesting technology [START_REF] Vijayakumar | Revised international notation for latex harvest technology[END_REF]. We did not use rain guards to make it possible to tap the trees on rainy days as it is not a common practice in Thailand. Hence, a substantial number of tapping days were loss every year because 90% of the rainy days occurred during the tapping period from May to February. In the end, trees were tapped 106, 115 and 106 days in the first, second and third year, respectively, out of the 150 tapping days expected with the d2 tapping frequency (i.e. 23% to 30% loss of tapping days).

The trial was set up as a split plot design in randomized complete blocks with four fertilizer treatments in the main plots, two tapping treatments in the subplots and four replications (blocks).

Elementary plots contained 108 trees and covered an area of 2,160 m². Each plot was separated from adjacent plots or plantation edges by two lines of tree (16 m) or six trees (15 m) (Fig. 1). The four fertilization treatments were (T1) no fertilization, (T2) 75/45/100 g.tree -1 .year -1 of N,P (as P2O5) and K (as K2O) in one application in June in each year, (T3) 180/80/170 g.tree -1 .year -1 of N,P and K in two applications in June and October in each year, and (T4), 306/136/289 g.tree - 1 .year -1 of N,P and K also in two applications (Table 2). Treatment T2 corresponded to the fertilization programme recommended by CIRAD in rubber estates [START_REF] Gohet | Sustainable rubber production through good latex harvesting practices: An update on mature rubber fertilization effects on latex cell biochemistry and rubber yield potential[END_REF], while T3 corresponded to the current recommendation in Thailand [START_REF] Chambon | What determine smallholders' fertilization practices during the mature period of rubber plantations in Thailand?[END_REF]. Treatment T4 was based on the highest rate of fertilization found in a survey of fertilization practices used by rubber growers in Thailand [START_REF] Chambon | What determine smallholders' fertilization practices during the mature period of rubber plantations in Thailand?[END_REF]. The two tapping treatments differed in stimulation, no stimulation (NS) or four stimulations per year, i.e. in June, July, August and September (ST). Stimulation was done by applying ca. 0.8 gram.tree -1 of a ready to use paste containing 2.5% of ethephon on 1 cm of the tapping panel located above the tapping cut.

Stimulation is not usually applied when trees are tapped every two days (d2) but only when the tapping frequency is reduced (d3 and more). Our stimulated treatments thus represented a very intensive system in which we expected a clearer effect of fertilization according to the hypothesis that the potential gain from fertilization can only be achieved through intensified tapping.

The experiment included three tapping seasons hereafter referred to as "years" (first year or year 1 = May 2014-April 2015; second year or year 2 = May 2015-April 2016; third year or year 3 = May 2016-April 2017). In May 2015 and May 2016, trees that had reached 47cm of trunk girth measured 100 cm above the ground in the previous cropping seasons were opened. On average, 57%, 66% and 70% of the 54 trees in each subplot were tapped in 2014-15, 2015-16 and 2016-17, respectively. There was no statistical difference in the number of tapped trees between blocks and treatments (Table 1). We did not observed any significant rate of tapping panel dryness as well.

Measurements and data processing

Latex and rubber yield

On each tapping day, tapping started at 8 P.M. and latex was collected separately from each subplot between 5 and 8 A.M. The fresh latex was weighed immediately, and a 200 mL sub-sample was mixed with 400 mL of water to determine the dry rubber content (DRC) using the Metrolac method [START_REF] Smith | Use of hydrometers to estimate dry rubber content of latex[END_REF]. DRC measurements with Metrolac were calibrated against the standard NF ISO126:

2005 twice a year (see 'latex and rubber quality' sub-section). This calibration indicated that the Metrolac method underestimated DRC by 17%. Rubber yield per subplot was obtained by multiplying fresh latex weight by calibrated DRC values. We then calculated the rubber yield per tree in g.tree -1 by dividing the rubber yield per subplot by the number of tapped trees in the subplot.

In May at the end of each tapping season, the number of tapped trees was updated to take the newly opened trees and the trees affected by tapping panel dryness into account. At the same time, we measured the trunk girth of all the trees in the experimental plots 1.7 m above the ground.

Physiological status of the latex cells (latex diagnosis).

Latex diagnosis (LD) is a method used to assess the impact of tapping on the physiological status of the latex cells [START_REF] Jacob | Yield limiting factors, latex physiological parameters, latex diagnosis, and clonal typology[END_REF][START_REF] D'auzac | The regulation of cis-polyisoprene production (natural rubber) from Hevea brasiliensis[END_REF]. According to [START_REF] Jacob | Yield limiting factors, latex physiological parameters, latex diagnosis, and clonal typology[END_REF], the biochemical ability of latex cells to produce rubber is based on their sucrose (SUC), inorganic phosphorus (Pi) and thiol (RSH) contents. SUC reflects the balance between sucrose consumption by the latex cells for latex biosynthesis, and the transfer of sucrose from the apoplast to the latex cells. Pi indicates the intensity of metabolic activity in the latex cells and RSH indicates the efficiency of scavengers in counteracting oxidative stress. We applied the LD method as developed by CIRAD [START_REF] Jacob | Métabolisme de la cellule laticifère et production chez l'Hevea brasiliensis[END_REF][START_REF] Jacob | Le diagnostic latex[END_REF]. The latex diagnosis was performed each year in September or October during the most regular and high yielding period. In each subplot, a composite sample was obtained from ten randomly selected tapped trees and ten drops of latex were collected from each tree. The LD parameters were measured using the Ashwell anthrone method (1957) for SUC, the Taussky and Shorr method (1953) for Pi, and the Boyne and Ellman method (1972) for RSH.

Sucrose, thiol and inorganic phosphorus contents were expressed in millimoles per litre of latex (mmol.1 -1 ).

Latex and rubber quality

Two sampling campaigns were carried out each year in October and June to collect latex and dry rubber samples for quality analysis. The campaigns were systematically planned around 15 days after fertilizer application. As it was not possible to obtain representative unsmoked sheet due to drying problems following the first sampling campaign in October 2014, supplementary sampling was conducted in January 2015. Several indicators of the technological properties of either latex or rubber were measured: dry rubber content (DRC), mechanical stability (MST) of the fresh latex; initial plasticity (P0), the plasticity retention index (PRI) and its component P30 (SMR bulletin N°7 1992 -part B.8), and the ash content of rubber processed as unsmoked sheet (USS). Mineral content (N, P, K, Ca and Mg) was also analyzed in both latex films and rubber USS. Figure 2 summarizes the processing of the latex samples collected in each plot and the methods used to measure those indicators. Latex samples were collected between 4 am and 6 am in half the 32 subplots on a given tapping day, and in the other half on the following tapping day (normally 48 h later, if no rain was falling). However, MST was measured in all the subplots on the two collection days. We used the average of those two measurements as the MST value for one subplot. The standard NF ISO35:2006 method was used with the following adaptations: the sample was made of 80 mL of fresh latex instead of 80 mL of concentrated latex. The latex was not heated, and all measurements were made within two hours after collection. The rest of the latex sample was divided into three sub-samples used separately for fresh latex and rubber analysis, and DRC measurement. As mentioned above, the standard NF ISO126: 2005 was applied for the DRC with the following adaptations: the sample comprised 2 mL of fresh latex instead of 10 mL of concentrated latex. The volume and concentration of added acid were 15 mL of 2% acetic acid instead of 25-35 mL of 5% acetic acid. Mineral and ash contents of the latex were measured on latex films. The latex films were made as follows: within the two hours after latex collection, 50 mL of latex were spread on a flat square recipient (22.5 x 22.5 x 3.75 cm) and were placed in a ventilated oven at 70 °C until no more white spots were visible on the surface of the sample (approx. duration 24 h). The resulting latex films were rolled and stored in plastic bags containing desiccant (5 g silica gel) prior to analysis. The mineral content expressed versus dry film weight were converted into a fresh latex reference by multiplying by the estimated total solid content of latex (TSC). The TSC was estimated by dividing measured DRC by 0.9. This numerical conversion value of the DRC/TSC ratio is regularly used in the field. It was checked experimentally in two consecutive sampling campaigns by measuring both DRC and TSC on the whole set of samples ( 2x 32), and the average ratio obtained was 0.90 (SD 0.03). Unsmoked sheets (USS) were made following the recommendations of the Rubber Research Institute of Thailand. First, latex was coagulated by mixing 3 L of latex with 2 L of water and 300 mL of formic acid solution 1.56% (94% diluted 60x). After 45 min, the coagulum was manually pressed, then passed 1-2 time(s) through a crusher (final thickness 10 mm), 3-4 times through a flat hand mangle and twice through a ribbed hand mangle (final thickness 2-3 mm). Finally, the USS were hung up outside in the shade to dry for one to two weeks (turned daily). The nitrogen content of both latex films and USS rubber was determined with a CHN Determinator (LECO CHN628) on a 100 mg sub-sample.

Total Mg, Ca, P and K content of both latex films and USS rubber were determined with a ICP-AES (ICP Agilent 720 AES) on ashes from a 2 g sub-sample placed in a furnace at 500 °C for two hours.

Figure 2 to be inserted here

Statistical analysis

A three-way ANOVA was performed on data collected each year to test the effect of blocks (4 modalities, n=8), fertilization (4 modalities, n= 8) and stimulation treatments (2 modalities, n=16), and their respective interactions on rubber yield (in g.tree-1 .year-1 ), LD parameters (SUC, Pi, RSH), latex and rubber quality parameters of the two sampling campaigns. Additionally, an ANOVA was performed on cumulative rubber yield (g.tree -1 ) at the end of the third cropping season. Tukey's HSD test was used to compare the means at the 5% probability level. The relationship between LD parameters and yield was tested every year with a multiple linear regression. Statistical analyses were performed with Xlstat software (2018.6 version, Addinsoft, Paris, France).

Results and Discussion

Our study is the first assessment of the interactions between latex harvesting technologies (stimulation) and fertilization on yield of tapped rubber trees in Thailand, the world's largest producer of natural rubber. We found only one publication in English reporting on a fertilizer trial in a mature rubber plantation in Thailand [START_REF] Mak | Effect of fertilizer and irrigation on yield and quality of rubber (Hevea brasiliensis) grown in Chanthaburi province of Thailand[END_REF]. However, this study only compared different formulae of fertilizers applied at the same dose. We also investigated the influence of stimulation and fertilization on latex physiology and latex quality parameters for the first time.

Indeed, the literature on the effect of fertilization on natural rubber production is scarce and not very recent [START_REF] Gohet | Sustainable rubber production through good latex harvesting practices: An update on mature rubber fertilization effects on latex cell biochemistry and rubber yield potential[END_REF]. According to [START_REF] Gohet | Sustainable rubber production through good latex harvesting practices: An update on mature rubber fertilization effects on latex cell biochemistry and rubber yield potential[END_REF] and [START_REF] Bolton | The manuring and cultivation of Hevea brasiliensis[END_REF], one reason for the lack of studies on this topic is the technical constraints to the implementation of fertilizer trials. This kind of trial requires a large homogeneous plantation area covering several hectares, which is difficult to find in rubber smallholdings.

The average yield of our experimental plot (all treatments) was high with ca. 5.8 kg. tree -1 . year -1 in the first year and more than 7.2 kg. tree -1 . year -1 in the second and third year (Table 3). Annual rubber yield per tree recorded for the control treatment without fertilization nor stimulation (T1NS) ranged between 5.3 and 6.5 kg.tree -1 .year -1 , which is in the upper range of yield data reported in rubber production trials in Thailand using the same tapping system on the clone RRIM600 [START_REF] Mak | Effect of fertilizer and irrigation on yield and quality of rubber (Hevea brasiliensis) grown in Chanthaburi province of Thailand[END_REF][START_REF] Chantuma | Distribution patterns of latex sucrose content and concurrent metabolic activity at the trunk level with different tapping systems and in latex production bark of Hevea brasiliensis[END_REF][START_REF] Chantuma | An innovative tapping system, the double cut alternative, to improve the yield of Hevea brasiliensis in Thai rubber plantations[END_REF][START_REF] Chantuma | Effects of Different Tapping Rest Periods during Wintering and Summer Months on Dry Rubber Yield of Hevea brasiliensis in Thailand[END_REF][START_REF] Sdoodee | First tests of "Double Cut Alternative" rubber tapping system in Southern Thailand[END_REF][START_REF] Sainoi | Low frequency tapping systems applied to young-tapped trees of Hevea brasiliensis (Willd. ex A. Juss.) Müll. Arg. in Southern Thailand[END_REF]. In general, the good performances of the plantation was explained by the high tapping frequency (d2) despite the loss of tapping days. Yet, the number of tapping days was similar to that used by rubber smallholders in Thailand [START_REF] Chambon | A Comparative Analysis of Smallholders Tapping Practices in Four Rubber Producing Regions of Thailand[END_REF]. Our results suggest that the low soil fertility (low organic matter and low N, P and K contents) was not particularly limiting. The depth of the soil and the absence of any obstacle to root growth may have compensated for the low mineral content. Roots were observed at a depth of more than 2 m (data not shown), in agreement with the results of [START_REF] Maeght | Seasonal Patterns of Fine Root Production and Turnover in a Mature Rubber Tree (Hevea brasiliensis Müll. Arg.) Stand-Differentiation with Soil Depth and Implications for Soil Carbon Stocks[END_REF]. Moreover, the absence of significant tapping panel dryness (TPD) on tapped trees at the end of the third year suggests that the trees were not over-exploited. This observation was confirmed by the results of the latex diagnosis (see sub-section 3.2). The good performances of the unfertilized and unstimulated plots (T1NS, control treatment), along with the absence of major block effects on yield (Table 3), suggest that our experimental field was well managed and was located in a favourable environment. We can therefore go further with the analysis of the treatment effects on rubber yield (section 3.1), latex metabolism indicators (section 3.2) and latex properties (section 3.3).

Effects of stimulation and fertilization on rubber yield (Table 3)

Our results show a positive effect of fertilization on yield from the first year, but the effect was only statistically significant (p=0.023) in the third year. This year, we observed that yield increased with the dose of fertilizers: +8% than the yield of T1 for T2, +10% for T3 and +13% for T4 in the third year. After three years, the cumulative yield of T3 and T4 treatments was 8% higher than T1. This effect was only significant at the 10% level (p=0.071). Stimulation by applying ethephon on the tapping cut four time a year had a strongly significant positive effect (p<0.0001) on rubber yield the three years of the experiment. The strongest effect of stimulation was observed in the third year with a 20% increase in yield, whereas in the first two years, the effect was similar (+12-13%). After three years of tapping, the cumulative yield of stimulated trees was 15% higher than that of unstimulated trees. It is noteworthy that the yield of stimulated and fertilized treatments (ST or T2, T3, T4) increased the third year compared to the second year, while it decreased for control treatments (NS or T1). We did not find significant interactions between fertilization and stimulation treatments.

The maximum effect of fertilization on rubber yield (+13%) we observed was similar to that reported by [START_REF] Gohet | Sustainable rubber production through good latex harvesting practices: An update on mature rubber fertilization effects on latex cell biochemistry and rubber yield potential[END_REF] and [START_REF] Murbach | Adubação npk e produção de borracha seca pela seringueira (Hevea brasiliensis)[END_REF], two of the most recent works on the effect of fertilization on modern high-yielding varieties with intensive tapping systems. Yield increases of +20% and more were obtained in works conducted before the 1990s in less intensive rubber cultivation systems [START_REF] Sivanadyan | Nutrition of Hevea brasiliensis in relation to Ethrel stimulation[END_REF][START_REF] Pushparajah | Recent developments in the nutrition of Hevea in west Malaysia[END_REF]Yogaratnam et al., 1984;[START_REF] Onuwaje | Yield response of rubber (Hevea brasiliensis) to fertilizers on an Ultisol in Nigeria[END_REF]. However, in our specific conditions, the response to fertilization may have been restricted because the yield of the unfertilized control was already high, despite the apparent low soil fertility. Increasing yield through external inputs is of course harder when the base line yield is already high. In other words, there were probably no major nutrient limitations in the rubber plot before the beginning of the experiment. Nevertheless, the response of rubber yield to fertilizer addition was quite rapid. We observed a positive but not significant effect from the first year that became significant at p<0.05 in the third year. The effect on cumulative yield over the three years of the experiment was significant at the 10% probability threshold. According to [START_REF] Bolton | The manuring and cultivation of Hevea brasiliensis[END_REF], we could have expected a slower response of yield to fertilizer. Bolton argued that additional fertilizer would first affect tree growth before increasing yield because of the low level of nutrients exported by latex harvesting compared to the total amount of nutrients required for tree growth. However, we observed no effect of the fertilization treatments on trunk radial growth (Table 3).

As expected, stimulation with an ethylene based product had the strongest effect on yield. Unlike fertilization, stimulation significantly increased rubber yield since the first year of the experiment.

The effect of stimulation we observed with 4 ethephon applications per year (+15% in cumulated yield over the three-year experiment) was much lower than the effects reported by [START_REF] Lacote | Longterm effect of ethylene stimulation on the yield of rubber trees is linked to latex cell biochemistry[END_REF] that ranged from +29% to +85%. This result can be explained by the more intensive tapping frequency in our study (d2, meaning tapping every other day) than in the study of [START_REF] Lacote | Longterm effect of ethylene stimulation on the yield of rubber trees is linked to latex cell biochemistry[END_REF], which was with a d4 (tapping once in 4 days). Although stimulation is not recommended with d2 tapping frequency, we choose to include a stimulated d2 tapping system in our experiment to test the hypothesis that fertilization can have a bigger effect on yield when trees are stimulated because of their increased nutrient requirements, or that only an increase in tapping intensity can exploit the higher yield potential enabled by fertilization [START_REF] Sivanadyan | Nutrition of Hevea brasiliensis in relation to Ethrel stimulation[END_REF][START_REF] Gohet | Sustainable rubber production through good latex harvesting practices: An update on mature rubber fertilization effects on latex cell biochemistry and rubber yield potential[END_REF]. Our results do not support this hypothesis as we did not find any significant interaction between the stimulation and the fertilization treatments. In other words, yield response to fertilizer application was similar with and without stimulation. In particular, we observed that fertilization contributed, as stimulation did, to maintain or even increase rubber yield the third year while yield of the control (unfertilized) treatment decreased compared to the second year.

Altogether, these results demonstrate that fertilization, independently of the use of ethylene stimulation, can increase the yield of rubber plantation under high tapping frequency system (tapping once every 2 or 3 days) such as those used by smallholders in Thailand. As ethylene stimulation is not recommended with these tapping systems, improving the nutrition of the trees can thereby be a main driver of rubber plantation intensification. However, the maximum effect, and the most significant, of fertilization in our experiment was obtain with the highest dose of fertilizer (T4 treatment, Eq. to 306/136/289 g.tree -1 .y -1 , Table 2). This treatment corresponds to the highest dose of inorganic fertilizers reported by rubber smallholders in Thailand [START_REF] Chambon | What determine smallholders' fertilization practices during the mature period of rubber plantations in Thailand?[END_REF]). This dose is 1.7 times higher than the standard fertilizer recommendation in Thailand and 4 times higher than the CIRAD recommendation for industrial estates. [START_REF] Chambon | What determine smallholders' fertilization practices during the mature period of rubber plantations in Thailand?[END_REF] calculated that this dose of fertilizer with a 10% increase in yield would only be profitable if the price of rubber is more than USD4.kg -1 , while the price had rarely exceeded USD 2.kg -1 over the last 10 years. Additionally, the environmental impacts, i.e. the risks of nutrient leaching to groundwater and N20 emissions to the atmosphere, with such a high fertilization rate must be high [START_REF] Zhou | The effects of nitrogen fertilization on N2O emissions from a rubber plantation[END_REF].

In the next sub-section, we analysed the effect of fertilization and stimulation on the indicators of latex metabolism we measured. We collected these data in order to better understand the effect of fertilization on rubber yield. The underlying assumption was that fertilization would have an effect on latex metabolism before having a statistically noticeable effect on yield in relation to better latex regeneration.

Table 3 to be inserted here

Effects of stimulation and fertilization on latex metabolism

Latex diagnosis (LD) is a method used to assess the impact of tapping on the physiological status of the latex cells [START_REF] Jacob | Yield limiting factors, latex physiological parameters, latex diagnosis, and clonal typology[END_REF][START_REF] D'auzac | The regulation of cis-polyisoprene production (natural rubber) from Hevea brasiliensis[END_REF]. Both fertilization and stimulation treatments had significant effects on the LD indicators, but the effects were different among the years and among the indicators. Inorganic phosphorus content (Pi) was the indicator most affected by the treatments. Pi increased with both stimulation and fertilization (Table 4). These effects were statistically significant in both the second and third year but not in the first year. Fertilization had a stronger effect on Pi than stimulation with an average increase of 16% increase with high levels of fertilization (T3 and T4) compared to control plots (T1) in 2015-2016, and of about 30% in 2016-2017. Low fertilization (T2) had little or no effect. There was also a slight interaction between stimulation and fertilization treatments in the second and third year (significant in the third year). Without stimulation, Pi increased with the dose of fertilizers but, with stimulation, only T3 had a significantly higher Pi than control (T1). This means that the effect of fertilization on Pi was stronger in non-stimulated trees. The sucrose content of the latex (SUC) decreased with stimulation the three years and with fertilization on years 2 and 3 (Table 4). These effects were only significant for stimulation on year 1 and 2. Indeed, we observed a decrease in the effect of stimulation on SUC over time (-38% in 2014-2015, -18% in 2015-2016 and -12% in 2016-2017).

There was no consistent effect of either stimulation or fertilization on thiol content (RSH).

Stimulation had a strong negative effect on RSH in the first tapping season (-24%), but no effect in the second and third years. Fertilization had a positive effect on RSH in general but with no clear significant trend linked with the dose of fertilizer. The high variability of this parameter (up to 21% within the same treatment) partly explains the lack of statistical significance. For a better understanding of the effect of latex diagnosis parameters on yield, we performed multiple linear regressions between the three latex diagnosis parameters and the latex yield expressed in g.tree-1.year-1 in each year. The results in Table 5 show that the p-value of the model decreased from the first to the third cropping season. This means that the fitness of the multiple linear regression to predict yield improved year after year. In 2014-2015, the three latex parameters explained only 30% of the yield variation whereas they explained nearly 60% in 2016-2017. SUC had a significant effect on yield in the second and the third year (p-value<0.05). Pi had a significant effect on yield only in the third year but this effect was stronger than for SUC.

Our starting hypothesis was that stimulation and fertilization would have a positive effect on latex yield through improved latex regeneration. The latex diagnosis (LD) indicators confirmed this hypothesis. Taken together, the results of the LD showed that latex metabolism was well activated after the first year of tapping and was well balanced in the two following years. In agreement with [START_REF] Jacob | Yield limiting factors, latex physiological parameters, latex diagnosis, and clonal typology[END_REF], the increase in sucrose content (SUC) along with a decrease in inorganic phosphorus (Pi) and thiol (RSH) content we observed in the third year suggest that the risk of overexploitation of the trees was low. Similarly, the negative correlation between SUC and yield, and the positive correlation between Pi and yield revealed by the multiple linear regression between LD indicators and rubber yield are consistent with the results of previous studies showing that SUC decreases and Pi increases with an increase in tapping intensity (Tupy, 1985;[START_REF] Gohet | Clone, croissance et stimulation, facteurs de la production de latex[END_REF][START_REF] Jacob | Les mécanismes biologiques de la production de caoutchouc par Hevea brasiliensis[END_REF]. In particular, these correlations and their underlying mechanisms have been well documented in the case of increases in yield obtained by ethylene stimulation [START_REF] Amalou | Ethrel (Ethylene Releaser)-Induced Increases in the Adenylate Pool and Transtonoplast ΔpH within Hevea Latex Cells[END_REF][START_REF] Dusotoit-Coucaud | Sucrose importation into laticifers of Hevea brasiliensis, in relation to ethylene stimulation of latex production[END_REF][START_REF] Lacote | Longterm effect of ethylene stimulation on the yield of rubber trees is linked to latex cell biochemistry[END_REF].

In addition, our results provide original insights into the combined effect of stimulation and fertilization on latex metabolism and yield. The main effect of fertilization on latex metabolism was on Pi. The maximum effect of fertilization on Pi was even stronger than that of stimulation after the first year. In the third year, both treatments had a highly significant positive impact on Pi with +33.5% for T4 compared to T1, and +24.7% for stimulated trees compared to unstimulated ones. The multiple linear regression between yield and LD indicators showed that Pi became the main explanatory factor in the third year. Positive correlation between rubber yield and Pi is wellknown [START_REF] Amalou | Ethrel (Ethylene Releaser)-Induced Increases in the Adenylate Pool and Transtonoplast ΔpH within Hevea Latex Cells[END_REF]. The metabolism of rubber particles involves active biochemical mechanisms which require a lot of energy and then fast cycling of ATP and NADPH, releasing free Pi into the cell cytoplasm. The positive effect of stimulation on Pi is in line with the results of [START_REF] Lacote | Longterm effect of ethylene stimulation on the yield of rubber trees is linked to latex cell biochemistry[END_REF]. [START_REF] Gohet | Sustainable rubber production through good latex harvesting practices: An update on mature rubber fertilization effects on latex cell biochemistry and rubber yield potential[END_REF] also reported a +20% significant increase in Pi in response to a combination of intensive stimulation and a high dose of fertilizer. However, the effect of fertilization on Pi in our study was stronger and was significant in both the stimulated and unstimulated treatments. Moreover, the effect of fertilization on Pi was stronger on unstimulated trees.

As expected, stimulation had a significant negative effect on SUC [START_REF] Lacote | Longterm effect of ethylene stimulation on the yield of rubber trees is linked to latex cell biochemistry[END_REF]. The decrease in SUC content in stimulated trees can be explained by activation of rubber biosynthesis, which consumes more sucrose (Tupy and Primot,1976). Surprisingly, the positive effect of fertilization on yield did not result in a significant reduction in SUC with the dose of fertiliser.

However, this result does not mean that fertilization had no effect on SUC. On the contrary, as a decrease in SUC was expected, this could mean that fertilization enhanced the supply of SUC to latex cells to cope with the increased latex productivity. The positive, but not significant, effect of fertilization on SUC observed in the first year is consistent with this hypothesis. This hypothesis is also in line with the results of [START_REF] Gohet | Sustainable rubber production through good latex harvesting practices: An update on mature rubber fertilization effects on latex cell biochemistry and rubber yield potential[END_REF] who observed a significant increase in SUC in response to high dose of fertilizer combined with intensive stimulation. In their study, SUC was low (<5 mMol) even in the control treatment. In our study, SUC levels were rather high in all treatments particularly in the third year [START_REF] Chantuma | Distribution patterns of latex sucrose content and concurrent metabolic activity at the trunk level with different tapping systems and in latex production bark of Hevea brasiliensis[END_REF]. Thus, another possible explanation for the absence of response of SUC to fertilization is that sucrose was not limiting for rubber production in our experimental conditions. This would explain the high yields obtained with the control treatment right from the onset of the experiment. This would also explain the increase in SUC in the third year in all treatments and the decline in the effect of stimulation on SUC over time (-38% in 2014-2015 with p<0.0001, -18% in 2015-2016 with p=0.001 and -12.3% in 2016-2017 with p>0.05).

Our analysis demonstrated that fertilization had a positive impact on latex metabolism by 1) increasing inorganic phosphorus (Pi) content and hence the energy available to regenerate rubber particles, 2) supporting the supply of sucrose (SUC) in the latex despite the increased output of latex. These results suggest that the effect of fertilization on yield was directly related to the effect of fertilization on latex metabolism, likely through an effect on the enzymatic activity of the latex cells. Our analysis showed that SUC and Pi were the LD parameters the most correlated with yield and that they are good predictors of the latex yield in response to fertilization.

Next, we presented the results on the effect of the treatments on latex and rubber properties. As we observed significant effects on LD indicators, we could wonder whether these properties, which are linked to the quality of latex and rubber, were also affected by the experimental treatments.

Table 4 and 5 to be inserted within this sub-section

Effect of stimulation and fertilization on latex and rubber properties.

We quantified six indicators of the technological properties of latex and rubber: the dry rubber content (DRC) and the mechanical stability (MST) of the fresh latex; the initial plasticity (P0), the plasticity retention index (PRI) and its component P30, and the ash content of rubber processed as unsmoked sheet (USS) (Table 6). Simultaneously, we analysed the mineral content (N, P, K, Ca, Mg) of fresh latex and dry rubber (Table 7).

Over the three-year period, we observed a decrease in MST (from 61 to 50 s.), PRI (from 104 to 91) and rubber USS ash content (from 0.33 to 0.27%) and an increase in P0 (from 34 to 42). We found no significant effects of fertilization on the technological properties of the latex and USS rubber. Stimulation had a significant negative effect on DRC (p=0.001, ST/NS=-3.50%) and a significant positive effect on PRI (p=0.004, ST/NS=+3.10%) in the third year only. Although not significant, stimulation and fertilization tended to destabilize the latex in the two first cropping years (-5.9% and -12.4% MST with stimulation; -12.6% and -15.6% MST for T4 treatment).

Over the three-year period, Mg and N contents in both fresh latex and USS rubber tended to increase whereas P, K and Ca contents decreased. The only significant effect of fertilization on mineral contents was on the N content of fresh latex in the third year (+6.3% on average, p=0.046).

The significance of this difference was not conserved in USS rubber, even though USS rubber from T4 contained 4.5% more nitrogen than USS rubber from T1. The P content of latex also tended to increase with fertilization during the third cropping year, but the effect was not statistically significant (T4/T1 +13.9%, p=0.122). Several effects of stimulation on mineral contents were observed. N, P, K and Mg contents of USS rubber from trees growing in the stimulated plot were higher, and this effect became significant in the third cropping year.

Logically, ash content of USS rubber from stimulated plots was up to 5% higher than that of nonstimulated ones (Table 6).

To our knowledge, this study is the first to provide a full set of data on latex and rubber properties, including mineral contents and several indicators of technological properties such as MST or PRI, in response to stimulation and fertilization treatments. The results revealed no significant effects of fertilization on the technological properties of either fresh latex or USS rubber. However, although not significant, the reduced stability of the latex in the fertilized treatments compared to controls observed in the first two years is in accordance with the findings of [START_REF] Collier | Effect of fertilizer applications on latex properties[END_REF]. The only significant effect of fertilization on latex mineral content and properties, an increase in N content, was observed the third year. Although not significant, latex P content showed the same tendency. These results are in agreement with data on rubber yield and latex metabolism and support the conclusion that fertilization significantly modified the latex properties three years after the beginning of the experiment. Longer-run observations will be necessary to confirm this conclusion.

Significant effects of stimulation on latex properties also appeared in the third year. Increased mineral contents (except Ca) with stimulation are consistent with reports in the literature [START_REF] Jacob | Yield limiting factors, latex physiological parameters, latex diagnosis, and clonal typology[END_REF]. It is noteworthy that stimulation affected the mineral contents of USS rubber and not those of fresh latex. This can be attributed to a concomitant decrease in dry rubber content (DRC), which is a well-documented effect of stimulation [START_REF] Coupé | Physico-chemical and biochemical mechanisms of hormonal (ethylene) stimulation[END_REF]. This phenomenon was particularly clear in the October measurements, which were preceded by four applications of stimulant (data not shown). USS rubber is indeed the dry matter of latex from which the hydrophilic matter is removed during USS processing (from 8% to 10% of total dry matter). In fact, our measurements showed that significant amounts of minerals were lost during this process. [START_REF] Vaysse | Effect of fertilization and stimulation of Hevea brasiliensis trees on mineral compositions and properties of produced latex and rubber[END_REF] calculated that about 33% of the nitrogen contained in latex was lost during USS processing. Losses of K (ca. 90% of initial content in latex), Mg (ca. 80%) and P (ca. 60%) were even greater. Only Ca content was preserved. This finding highlights the complex relationships between the mineral composition of fresh latex and dry rubber, and their possible effects on rubber quality. However, in our study, the observed differences in the mineral content of USS rubber had no direct effect on the technological properties of USS rubber. In particular, the increase in ash content of USS rubber induced by stimulation led to a content that was less than half the maximum accepted by the Technically Specified Rubber standard (0.6% for TSR5 grade, ISO 2000:2003, TSR standard is used as reference as no ash content standard is available for sheet rubber). Similarly, stimulation had significant effects on P0 and PRI but technologically speaking, the averaged differences (less than 2 points "P0" and less than 3 points "PRI" respectively, see supplementary Table 4) are not significant [START_REF] Bateman | Significance of PRI in raw and vulcanized Natural Rubber[END_REF].

Finally, the absence of an effect of both fertilization and stimulation treatments on the technological properties of NR is a positive result. Indeed, it proved that, while having a positive effect on production, none of the treatment had a detrimental effect on quality.

Table 6 and 7 to be inserted here

Conclusion and perspectives

Our study is the first experimental work to investigate the effects of increased doses of mineral fertilizers combined with stimulation of trees on the yield, latex metabolism and technological properties of latex and dry rubber in a rubber plantation in Thailand. Our results show a positive and significant effect of fertilization on yield three years after the beginning of the experiment.

The effect of fertilization was similar with and without ethylene stimulation. The hypothesis that the effect of fertilization on latex yield would only be higher under more intensive tapping was thus not confirmed. In practice, the effect of fertilization and stimulation were additive. We also demonstrate that the effect of fertilization on yield was linked to a direct effect on latex metabolism mainly through an increase in inorganic phosphorus content (Pi). We observed no significant effect of the fertilizer treatments on latex and rubber properties. Therefore, increasing latex yield by fertilization combined with intensive tapping did not have any identifiable risks on the technological properties of rubber. Thus, our study suggest that fertilization can be an efficient driver of the intensification of rubber plantation. In order to validate this result on the long-term, our study is continuing. In addition to direct effects on yield and the latex regeneration processes, we are analysing the effect of fertilization on the growth and the nutritional status of the trees. We are also collecting data in order to quantify the actual mineral balance of the different fertilization treatments and to assess their respective environmental impacts through nutrient leaching or N2O emissions. Similarly, we are studying the feasibility of recycling the water used for USS processing which contains large amounts of minerals. Finally, the profitability of the fertilization practices must be assessed with respect to the expected effect on yield and the price of natural rubber.

Altogether, these new data will contribute to a multi-criteria evaluation of fertilization practices in rubber plantation that can help farmers to decide whether or not using fertilizers in their rubber plantations.

Acknowledgments

The authors would like to thank the Faculty of Agriculture of Kasetsart University, Khampaeng Sean campus, for granting the access to the Sithiporn Kridakorn Research Station; the staff of Sithiporn Kridakorn Research Station for their contribution to the fieldwork; Mr. Jirasak Chehsoh for latex diagnosis sampling and analysis; Ms. Supawan Sathornluck and the staff of the Laboratory of Biochemistry and Technology of Natural Rubber of Kasetsart University for their participation in the latex and rubber quality sampling campaign and analysis; Jerome Sainte-Beuve and Frederic Bonfils from CIRAD, UMR IATE, for their support and valuable advice on the design and the interpretation of quality measurements; and Marie Tella and Gael Senecal from US Analyses of CIRAD for their efforts to adapt mineral analysis to latex and USS rubber samples. This work was financially supported by Yara International ASA and the "Towards the improvement of rubber tree productivity" project of the Thai International Cooperation Agency in the framework of the Hevea Research Platform in Partnership (HRPP)

Author contributions

Project coordination: F. Gay, K. Sajjaphan Conceptualization of the experiment: F. Gay, L. Vaysse, R. Lacote, E. Gohet, , P. Thaler Table 3. Average annual and cumulative dry rubber yield (g.tree -1 ), and average trunk girth (cm) at the end of the third year in each treatment (fertilization x stimulation). Letters in brackets show significant differences among the calculated means (Tukey's test, p<0.05). The rows "Stimulation effect", "Fertilization effect", "Block effect" and "Fert.xStim effect" give the results of the ANOVA of the main treatment, sub-treatment, block and interaction effect. p-values in bold are statistically significant effects. Percentages represent the ratio of the yield of a given treatment to the control (ST to NS for stimulation treatment, T2, T3 or T4 to T1 for fertilization treatment).

Treatment

Rubber yield 2014-15 (g.tree -1 .year -1 ) Rubber yield 2015-16 (g.tree -1 .year -1 )

Rubber yield 2016-17 (g.tree -1 .year


Clément Dondé

Muriel Senn

Julien Eche

Lassaad Kallel

Mohamed Saoud

Jérôme Brunelin

® psychoeducation program for schizophrenia improves knowledge about, but not insight into, the illness

Keywords: Schizophrenia, Psychoeducation, Insight, Knowledge, Cognition

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Schizophrenia (SZ) is a serious mental illness. Its prevalence approaches 1% internationally. Besides the traditional symptoms of delusions, disorganized behavior/thinking and reduced ability to function, patients with SZ have profound cognitive deficits that mostly hamper their integration into the community. These deficits may involve neurocognitive processes (e.g., attention, executive functions, learning), social skills, and cognitive insight, i.e. the capacity to gain an accurate and deep understanding of something or someone. Most individuals diagnosed with schizophrenia (SZ) SZ individuals have little to no insight regarding the presence of their illness [START_REF] Amador | Psychopathologic domains and insight in schizophrenia[END_REF][START_REF] Mccormack | Lack of insight in psychosis: theoretical concepts and clinical aspects[END_REF].

In other words, SZ is associated with impaired ability to label own psychotic experiences as pathological, being aware of the illness course and risk of relapse, and acknowledge the interest of validated treatment [START_REF] Dam | Insight in schizophrenia: a review[END_REF][START_REF] David | Insight and psychotic illness. Cross-sectional and longitudinal associations[END_REF]. Lack of insight in these individuals ultimately leads to poor treatment adherence, more relapses and longer hospitalizations [START_REF] Schwartz | Does insight affect long-term impatient treatment outcome in chronic schizophrenia?[END_REF]. Poor insight is also correlated with lower global functioning [START_REF] Parellada | Trait and state attributes of insight in first episodes of early-onset schizophrenia and other psychoses: a 2-year longitudinal study[END_REF], altered quality of life [START_REF] Mohamed | Cross-sectional and longitudinal relationships between insight and attitudes toward medication and clinical outcomes in chronic schizophrenia[END_REF][START_REF] Pu | Right Frontotemporal Cortex Mediates the Relationship between Cognitive Insight and Subjective Quality of Life in Patients with Schizophrenia[END_REF] and directly associates with worsened outcome [START_REF] Lincoln | Effectiveness of psychoeducation for relapse, symptoms, knowledge, adherence and functioning in psychotic disorders: a metaanalysis[END_REF]. As a result, when working patients with SZ, improving insight into the illness is an important objective since currently available therapeutic approaches such as antipsychotic drugs have only a limited influence on insight capacities [START_REF] Phahladira | Changes in insight over the first 24months of treatment in schizophrenia spectrum disorders[END_REF].

During the last decade, numerous psychoeducational approaches have been developed and are now considered as state-of-the-art interventions for SZ [START_REF] Galletly | Royal Australian and New Zealand College of Psychiatrists clinical practice guidelines for the management of schizophrenia and related disorders[END_REF][START_REF] Lehman | Practice guideline for the treatment of patients with schizophrenia, second edition[END_REF]. Psychoeducation consists in delivering stabilized patients with accurate knowledge about their illness and treatment, thereby improving symptoms, adherence and prognosis (Xia et al., 2011). The programs are preferentially based on an interactive group model with a definite number of structured sessions. In such model, a healthcare provider, oftentimes with the assistance of other professionals, videos or demonstrations, takes on the role of an educator to help patients become better cognizant and informed about their condition. To foster learning and "real-life" application of new knowledge, psychoeducation often involves skill learning and positive reinforcement throughout the group. Meta-analyses covering almost 50 studies have reported better adherence, fewer relapse, as well as improvements of clinical symptoms and global functioning after psychoeducation (Xia et al., 2011;Zhao et al., 2015).

Seminal viewpoint of [START_REF] Amador | Poor insight in schizophrenia[END_REF] has long suggested that poor insight of patients could stem from, among others potential deficits, a lack of general knowledge about the illness [START_REF] Amador | Poor insight in schizophrenia[END_REF]. More recent studies have supported this assumption by demonstrating significant associations between levels of illness-related knowledge and insight in SZ individuals [START_REF] Alenius | Knowledge and insight in relation to functional remission in patients with long-term psychotic disorders[END_REF][START_REF] Chan | Medication adherence, knowledge about psychosis, and insight among patients with a schizophreniaspectrum disorder[END_REF]. Studies investigating the effectiveness of the core psychoeducational component (i.e., conveying new knowledge) revealed significant improvements of illness knowledge questionnaires scores after psychoeducation [START_REF] Jahn | Neurocognitive prediction of illness knowledge after psychoeducation in schizophrenia: results from the Munich COGPIP study[END_REF][START_REF] Lincoln | Effectiveness of psychoeducation for relapse, symptoms, knowledge, adherence and functioning in psychotic disorders: a metaanalysis[END_REF]. Regarding insight, recent metaanalyses reported moderate, yet non-significant, effect sizes at post-test [START_REF] Pijnenborg | Changes in insight during treatment for psychotic disorders: a meta-analysis[END_REF]Xia et al., 2011;Zhao et al., 2015). However, psychoeducation studies involved heterogeneous programs in term of number of session and content. Only one study has shown a positive impact of psychoeducation on both insight and knowledge levels in a same group of patients (von Maffei et al., 2015). Hence, the effect of psychoeducation programs on these related outcomes needs to be explored further.

In France, The P.A.C.T. ® (Psychose, Aider, Comprendre, Traiter) program has been developed to provide French-speaking SZ patients with a validated psychoeducational approach in their treatment regiment [START_REF] Bayle | Dire la maladie, vivre avec son traitement, vivre avec la maladie[END_REF][START_REF] Salomé | Un programme d'information sur la maladie et ses traitements destiné aux patients atteints de schizophrénie : P.A.C.T.®[END_REF]. Although this program showed promising results on symptoms and illness-related knowledge [START_REF] Dumas | Programme PACT : évaluer l'apport psychoéducatif[END_REF], no studies have yet investigated its concomitant impact on insight.

The primary objective of this study was Here, we propose to compare levels of knowledge and insight before and after the psychoeducational P.A.C.T. ® program to investigate how this approach affects both outcomes in a same group of SZ subjects, along with clinical symptoms potential improvement. We hypothesized that extending knowledge through psychoeducation will enable patients to learn and internalize relevant information about the illness and, in turn, develop better insight into their condition. As a secondary objective, we sought to explore whether the P.A.C.T. ® program improves the clinical symptoms of SZ. We believed that our study can could provide useful evidence about the potential benefits of psychoeducational approaches in SZ and allow for future refinement of the psychoeducation P.A.C.T. ® program.

METHODS

Participants

In this open label study, 30 patients diagnosed with SZ or schizo-affective disorder without a history of substance dependency according to the Diagnostic and Statistical manual of Mental Disorders (DSM-IV) criteria were recruited at a tertiary care facility (Centre Hospitalier Le Vinatier University Adult Psychiatry unit, Bron, France). All patients were interviewed with the French version of the Mini-International Neuropsychiatric Interview (MINI) to assess the presence of clinical symptoms and diagnose of SZ. The mean age was 30.5 ± standard deviation 1.3 years old, sex ratio 7F/ 23M and illness duration 7.8 ± 1.1 years.

All participants had never been involved in a psychoeducation program before the study.

All assessments were performed at before and after the P.A.C.T. ® program by trained clinicians, in accordance with relevant guidelines and regulations.

P.A.C.T.® psychoeducational program

The P.A.C.T. ® program was divided into 16 weekly sessions consisting of watching a short thematic video followed by supportive group therapy focusing on problem-solving skills for approximately 90 min. After each session, participants were given printed material summarizing the session's content. The program was divided into three modules (~5 sessions each) placing emphasis on (i) what is actually like to live with SZ, (ii) vulnerability and relapse, and (iii) progress toward recovery. Each session was conducted by two nurses and one psychiatrist from the psychiatry unit team. A patient stakeholder participated in some sessions. The study was completed across 4 groups involving 6-8 patients each.

Evaluations

Knowledge: Knowledge about SZ was measured with the self-questionnaire "What do I know?" developed for this study, and tailored to the psychoeducational content of the P.A.C.T. ® program. Its 30 questions are related to those topics discussed in the psychoeducation groups, concerning clinical symptoms, medication, vulnerability and recovery. Each question includes 3 true/false items (e.g., "auditory hallucinations is a symptom of SZ", "antipsychotic medication can help me decreasing the voices"). Total correct responses were used for analysis, with higher scores indicating higher knowledge.

Insight: insight levels were measured using a French version of the Scale to Assess Unawareness of Mental Disorder (SUMD) [START_REF] Raffard | French adaptation and validation of the scale to assess unawareness of mental disorder[END_REF]. This eleven-item semistructured interview evaluates global awareness of having a mental disorder, the social consequences of the disorder, the effects achieved from antipsychotic medication, and specific insight into clinical symptoms and their attribution to the psychiatric disorder.

Dimensions of insight were rated in present time on a five-point Likert scale ranging from 1 (aware) to 5 (unaware). Total SUMD score was used for analysis, with higher scores indicating lower awareness.

Symptoms: All patients were interviewed with The Positive and Negative Syndrome Scale (PANSS) [START_REF] Kay | The positive and negative syndrome scale (PANSS) for schizophrenia[END_REF] that assesses the presence of SZ clinical dimensions (positive symptoms, general psychopathology, negative symptoms) rated from 1 (absence) to 7 (extreme).

Analyses

Normality of the distributions was investigated with Shapiro-Wilkinson tests.

Between-time comparisons were performed with paired t-tests and Mann-Whitney z-tests for normal and non-normal continuous outcomes, respectively. Effect of potential confounds (age, sex ratio and illness duration) were assessed using covariance analyses. Cohen's d effect sizes (ES) were computed to quantify between-groups significant differences, with 0.20 = small, 0.50 = medium and 0.80 = large ES. Relationships between measures were conducted with correlation tests. Significance threshold was set at p < .05. All analyses were performed using JASP software version 0.9.1. (https://jasp-stats.org).

RESULTS

A significant improvement of knowledge scores ("What do I know?" questionnaire) was observed after the psychoeducation program with medium effect size (z(2,29)=3.44; p<0.001; d=0.77; Fig. 1A). By contrast, the analysis reported no effect of psychoeducation on insight (SUMD) scores (z(2,28)=0.18; p=0.86; d=0.07; Fig. 1B). Clinical symptoms indexed by the PANSS total score were significantly improved after treatment (t(2,28)= -3.53 ; p=0.001; d=0.66; Fig. 1C). When considering PANSS subscales, only the general psychopathology dimension was significantly improved (PANSS positive: z(2,27)= -0.97, p=0.33; PANSS negative: z(2,27)= -0.58, p=0.56; PANSS general psychopathology: t(2,28)= -3.02, p=0.0005).

Pre-to-post improvement of Knowledge was still significant when age, gender and illness duration were entered in a covariance analysis (p=0.03), but symptoms improvement lost significance. The relationship between knowledge and PANSS total improvements (measured as the difference post-pre) was not significant (Spearman's r = 0.30, p=0.15).

DISCUSSION

The main objective of this study was to investigate the impact of the P.A.C.T. ® psychoeducation program on knowledge about and insight into SZ in a sample of patients with SZ. In contrast to our primary hypothesis (i.e., concomitant improvement of both illnessrelated knowledge and insight after psychoeducation), the P.A.C.T. ® program did not have a significant impact on insight as assessed by the SUMD. By contrast, we observed a significant improvement of learnt knowledge and overall symptomatology after the psychoeducational intervention, which reproduces a previous investigation of the P.A.C.T. ® program in SZ groups [START_REF] Dumas | Programme PACT : évaluer l'apport psychoéducatif[END_REF]. This differential impact means that the P.A.C.T. ® procedure did not allow patients to manipulate and appropriate new knowledge in a sufficient manner to gain better insight. A potential explanation could be related to the alteration of semantic knowledge described in SZ, a phenomenon where patients present with impaired ability to use new salient semantic knowledge appropriately [START_REF] Doughty | Semantic memory impairment in schizophrenia--deficit in storage or access of knowledge?[END_REF]. Another study demonstrated that self-rating levels of insight (often referred as "cognitive insight") are not correlated with clinical ratings of insight as measured by the SUMD (Tranulis et al., 2008). Therefore, it is possible that subjective aspects of insight in our sample differentiated from the SUMD outcome and may have improved the same manner as subjective levels of knowledge evaluated by the "What do I know" self-questionnaire. This hypothesis remains to be explored by further studies including both self-and clinician-evaluations of insight. We initially posit that providing patients with relevant information about their condition would improve levels of insight, which implies that poor insight is related or even induced by a lack of knowledge regarding the illness. However, it is likely that knowledge-focused psychoeducation alone was not sufficient to overcome other mechanisms that potentially concurred to poor insight in our sample of SZ subjects. For instance, Amador et al., suggested that lack of insight might also be a consequence of shared neural alterations with anosognosia, that is, fronto-parietal impairments [START_REF] Amador | Awareness of illness in schizophrenia[END_REF]. This assumption is supported by lower neuropsychological performance of frontal executive functioning [START_REF] Laroi | Unawareness of illness in chronic schizophrenia and its relationship to structural brain measures and neuropsychological tests[END_REF][START_REF] Raffard | Insight and executive functioning in schizophrenia: a multidimensional approach[END_REF] and altered neuroimaging measures of frontal lobes [START_REF] Orfei | The neuroanatomical correlates of cognitive insight in schizophrenia[END_REF][START_REF] Sapara | Prefrontal cortex and insight in schizophrenia: a volumetric MRI study[END_REF] that were demonstrated strongly associated with poor insight in patients with SZ.

The absence of significant impact of the P.A.C.T.® program on insight in our group corroborates recent meta-analyses that have demonstrated moderate but non-significant improvement of insight measures after psychoeducation [START_REF] Pijnenborg | Changes in insight during treatment for psychotic disorders: a meta-analysis[END_REF]Xia et al., 2011;Zhao et al., 2015). This highlights a need for complementary approaches that specifically target cognitive processes that are relevant for insight. For instance, brief intervention focused on self-reflection improvement [START_REF] Pijnenborg | REFLEX, a social-cognitive group treatment to improve insight in schizophrenia: study protocol of a multi-center RCT[END_REF]metacognitive training (de Jong et al., 2019b) have shown promising results on insight impairments of SZ subjects. Ideally, core components of such approaches could be embedded to the P.A.C.T.® program. More broadly, the program may complement other cognitive interventions in SZ that efficiently target social skills deficits and internalized stigma [START_REF] Li | Community-based comprehensive intervention for people with schizophrenia in Guangzhou, China: Effects on clinical symptoms, social functioning, internalized stigma and discrimination[END_REF][START_REF] Thonse | Social cognition intervention in schizophrenia: Description of the training of affect recognition program -Indian version[END_REF], especially since a lack of insight can hamper the effect of such intervention (de Jong et al., 2019a).

Several limitations to the present study should be acknowledged. First, conversely to prior studies investigating knowledge gain after psychoeducation [START_REF] Jahn | Neurocognitive prediction of illness knowledge after psychoeducation in schizophrenia: results from the Munich COGPIP study[END_REF][START_REF] Lincoln | Effectiveness of psychoeducation for relapse, symptoms, knowledge, adherence and functioning in psychotic disorders: a metaanalysis[END_REF], the questionnaire "What do I know" was not a validated assessment. Second, no measures were performed at further endpoints, which prevent us to draw conclusions about the potential long-term impacts of the P.A.C.T.® program on knowledge, insight and symptoms. Third, no control group was included in the study, which makes it impossible to rule out a placebo effect. Furthermore, we cannot know whether knowledge and symptoms improvement were due to the psychoeducational program or to other factors, such as spontaneous improvement, treatments changes, other concurrent therapies or lifestyle interventions.

In sum, although the P.A.C.T.® program is a promising tool for improving symptoms and illness-related knowledge in patients with SZ, its use is not sufficient to significantly improve levels of insight. Future investigations are warranted to define how to effectively target and assess levels of insight in this specific population. Before After

FIGURE CAPTION

Figure 1 :

 1 Figure 1: Outcomes at pre-(before) and post-(after) psychoeducation assessments.