Effect of synthetic fertilization dose on the diameter increase, height and mortality of Cinchona officinalis L. (Rubiaceae)

Abstract Cinchona officinalis, is a South American tree species, commonly used for medicine, and is currently threatened by agricultural expansion and cattle ranching. The objective was to determine the effect of chemical fertilization on the nursery growth to increase growth potential and survival of C. officinalis. A completely randomized design with six treatments and three replicates was used; 20 C. officinalis plants were used per replicate. Two months after transplanting the C. officinalis seedlings to the polyethylene bags, inorganic fertilizer (YaraMila® HYDRAN) was applied. Monthly evaluations were carried out and the number of dead plants, plant height, diameter and number of leaves were recorded. The highest mortality rate was recorded when fertilizer was applied (73%) while in the non-fertilized treatment mortality reached 36%. Regarding the increase in height, diameter and number of leaves in all cases, the best results were obtained in the fertilized treatments, exceeding by 85, 70 and 17% (respectively) those obtained in the treatment to which fertilization was not applied. This study shows the effects that the application of fertilizers to C. officinalis plants at the nursery level can have on growth and mortality variables, the results suggest the use of this product for a sustainable and large-scale production of this species taking into consideration the appropriate dosage.


Introduction
Cinchona officinalis is a forest species of medicinal importance, as it was the only cure for malaria for more than three centuries (Cóndor et al. 2009;Bharadwaj et al. 2018) and its alkaloids are considered to be the most influential tree bark-derived medicine in human history (Prendergast and Dolley 2001).
Cinchona officinalis needs specific conditions to ensure its growth, and the distribution range is limited (Armijos-González and Pérez-Ruiz 2016).The species faces different negative conditions that hinder the location of C. officinalis populations in natural forests (Buddenhagen et al. 2004).The first problem is regarding its natural habitat, since it has been affected by migratory farming and agriculture (Huamán et al. 2019;Fernandez et al. 2022).Second problem, is the low survival rate of seedlings in nursery (Fernandez et al. 2021) and finally the limited natural regeneration due to its low germination capacity influenced by factors such as seed quality, humidity, temperature and microbial activity (De-la-Cruz et al. 2007;Santos et al. 2010;Valdiviezo et al. 2018).These conditions have driven the prioritization of the conservation and recovery of C. officinalis in Peru (Albán-Castillo et al. 2020).
To ensure the success of afforestation and reforestation plans from the establishment of C. officinalis seedlings grown in forest nurseries, it is necessary to standardize protocols for obtaining seedlings suitable for the edaphoclimatic conditions of the area to be restored (Massone et al. 2018).Among the parameters to consider are management factors, fertilization (chemical or organic fertilization) is an activity that can significantly influence plant growth, health and productivity, as well as increase plant tolerance to many biotic and abiotic stresses (Souri and Hatamian 2019;Serri et al. 2021).
During the search for scientific information, no report was found in which the response of C. officinalis to the application of inorganic fertilizers had been studied, so this research is pioneering and generating a range of opportunities to continue developing research in this area, in addition, the results of this research will allow optimizing the management of fertilization of C. officinalis at the nursery level and ensure the quality of seedlings to be used in reforestation programs (Oliet et al. 2005).In this context, the study sought to determine the effect of the application of inorganic fertilizer application on some growth parameters and mortality of C. officinalis at the nursery level.

Study area
The research was carried out in the La Cascarilla locality (5° 40′ 21.12″ S and 78° 53′ 55.65″ W), province of Jaén, Peru, which is located at 1810 m.In this area the annual precipitation is 1730 mm, the minimum temperature is 13.0 °C and the maximum is 20.5 °C (Fernandez et al. 2021;Fernandez et al. 2022).

Plant material
C. officinalis seedlings were obtained from seeds.These were collected from a single population, from the locality of San Luis (6°22′ 6.68″ S and 79°3′ 29.50″ E) at 2489 m altitude.We collected 0.5 kg of mature capsules (brown to dark brown in color), packed in cloth bags and transported them to the community of La Cascarilla, where they were stored in the shade.After 20 days, seeds without visible cracks, fungi and/or nematodes were selected for use in the study (Fernandez-Zarate et al. 2022).For germination, an subirrigation chamber described by (Fernandez et al. 2021) was used, seeds were sown in forest substrate (Fernandez-Zarate et al. 2022).C. officinalis seedlings were transplanted into 269.25 cm 3 polyethylene cylindrical bags (7 cm diameter and 7 cm high).

Soil characterization
The physical and chemical properties of the substrate used for the transplanting of C. officinalis were determined before applying the fertilizer and at the end of the trial.The methods used were: For texture: hydrometer method, for electrical conductivity (EC) was determined in aqueous extract soil: water (1:1), organic matter (OM): method established by Walkley and Black, nitrogen (N): Kjeldahl method, phosphorus (P): Olsen method, cation exchange capacity (CEC) and exchangeable cations: saturation with ammonium acetate, all analyses were determined at the Soil and Water Research Laboratory of the National University Toribio Rodriguez de Mendoza.

Experimental design
A completely randomized design with six treatments and three replicates per treatment was used; 20 C. officinalis plants per replicate and 360 plants were used throughout the trial.Two months after transplanting C. officinalis to the polyethylene bags, inorganic fertilizer (YaraMila® HYDRAN) was applied and then followed up monthly.This fertilizer is composed of NPK and micronutrients; it is also one of the most widely used fertilizers in Peru.The nutritional composition of which is shown in Table 1.

Measurements
To determine the effect of fertilization on nursery growth of C. officinalis, monthly evaluations were conducted for six months to record the number of dead plants, plant height (from the top of the polyethylene bag to the apex of the plants), diameter (measured with a digital vernier at substrate level).

Data analysis
Data were processed in StatGraphics Centurion XVI software (StatPoint Technologies Inc, Warrenton, VA, USA).The assumptions of normality (Shapiro Wilk) and homogeneity of variances (Levene's test) were checked.Then, an analysis of variance (ANOVA) was performed for each response variable and the mean values were compared through Tukey's test (α = <0.05), in addition, a correlation test was performed in the R programming environment between the growth variables evaluated and the chemical properties of the substrate at the end of the trial.

Results
Thirty days after applying the fertilizer to the C. officinalis plants, the first records of mortality were observed in treatments T4, T5 and T6, whose trend is sustained throughout the trial, reaching percentages of mortality of 64, 73 and 54% respectively, it can also be seen that treatment T2 reports its first mortality data 60 days after fertilizer application, reaching 73% mortality at the end of the trial (Figure 1A).Finally, the treatment to which no fertilizer was applied reported the lowest mortality rate (36%), which suggests that the application of chemical fertilizers on C. officinalis has a negative impact on plant survival at the nursery level.
It can be seen that in T5 there is a decrease in the number of leaves until day 90, thereafter a linear  increase is observed until the end of the trial, with treatments T2, T3 and T4, an increase in the number of leaves after fertilization of C. officinalis plants until day 60, thereafter there was a decrease in the number of leaves; T1 shows a slight tendency to decrease the number of leaves.In general, T5 reached 17% more leaves with respect to the number of leaves recorded at the beginning of the trial, followed by T2 with 4%, and in treatments T1, T3, T4 and T6 a decrease of -14, -21, -3 and -10%, respectively, is reported (Figure 1B).
For the parameters of diameter increment (Figure 1C) and height increment (Figure 1D), similar behaviors were observed, the highest values were observed in treatment T5, followed by T3 and T2.In both parameters, the lowest increases were recorded in treatment T1.When comparing the results of T5 with those of T1, it was observed that the values obtained in treatment T5 for diameter and height were 70 and 85% higher than the results obtained in treatment T1.These results show the effect of fertilization on the growth of C. officinalis plants at nursery level.
Table 2 shows the results of the multifactorial ANOVA obtained by considering the growth parameters (mortality, height, diameter and number of leaves) as dependent variables and fertilization dose and sampling time as factors.With a significance level of 0.05, the dose and sampling time have significant effects on the diameter and height increment.Of the four parameters analyzed, only for height increment the interaction between dose and time had significant effects.
Table 3 shows the statistical differences among the six treatments evaluated.It is evident that there are no differences between T2 and T5 for the mortality parameter, but there are statistical differences between these two treatments and the other four treatments.Regarding the increase in height, T5 is the one that has registered the highest value, despite this, there are no differences with treatments T4, T3 and T2; however, there are differences between T5 (higher increase in height) and T1 (lower increase in height).Regarding the increase in diameter, T5 is the treatment that obtained the best results, with significant differences between T5 and T1; similar behavior is observed in the increase in the number of leaves.

Discussion
Several investigations were developed to evaluate the effect of fertilization on the growth of forest species, however, most of these investigations developed in tropical areas focused on introduced species of commercial interest (Alvarado and Raigosa 2012).This research, to the best of our knowledge, is pioneering in analyzing the response to chemical fertilization in C. officinalis, a species of medicinal, civic and highly threatened importance (Huamán et al. 2019;Fernandez et al. 2022), with potential commercial interest and for ecological recovery programs.
The increase in the growth of C. officinalis plants is relatively slow, as reported by (Fernandez-Zarate et al. 2022).However, the results obtained in this research show a significantly higher growth in the fertilized treatments with respect to the treatment that was not fertilized and regardless of the fertilization dose, diameter and height have shown linear increases until the end of the trial (six months after fertilization).In general, fertilization of C. officinalis seedlings at nursery level showed positive results, contributing to the increase in diameter, height and number of leaves.These positive effects could contribute to revegetation in soils with competing vegetation, as has been reported for other species and site conditions (Oliet et al. 2005;Salifu et al. 2009).The significant increase in the growth parameters evaluated could be attributed, although it was not measured in this study, to the reserve of nutrients that usually accumulate in the stems and roots of fertilized plants (Hu et al. 2015;Pokharel and Chang 2016).
The mortality rate of C. officinalis plants varied with the treatments; in general, fertilization contributed to an increase in seedling mortality, reaching 73% of dead  plants compared to 36% mortality when no fertilizer was applied; this is the average mortality rate at the nursery level for this species (Fernandez et al. 2021).This high percentage of mortality would be associated with the mineral composition of the product used in the study, since an imbalance in nutrients usually has a negative effect on plant survival rate when there is an excess of any of them (Grzyb et al. 2013;Christophe et al. 2019).
If the nutritional distribution of the fertilizer used in this trial is observed, it is evident that it has a high nitrogen content (19%), which although it can have positive effects on the number of leaves on the plant (Onyango 2002), the excess of this nutrient could lead to an increase in the mortality rate since it tends to generate yellowing of the leaves and trigger their dehiscence and subsequent death of the plants (Westerveld et al. 2003;Perner et al. 2007).
Regarding potassium, it is associated with photosynthesis (Sardans and Peñuelas 2015), enzymatic activity and protein synthesis (Armengaud et al. 2009).It is also known that, if adequate potassium fertilization is performed, it generates protection to plants from biotic and abiotic stresses (Wang et al. 2013;Sardans and Peñuelas 2015) and with respect to magnesium, if the dose is small, it could trigger growth inhibition and tissue aging (Verbruggen and Hermans 2013), since there are stomatal limitations and low CO 2 assimilation and, with respect to phosphorus, its excess could have a defoliating effect leading to plant death (Novoa et al. 2018), this can be observed in Figure 2, since there is a high positive correlation between mortality and phosphorus content in the soil.

Conclusion
The application of inorganic fertilizer to C. officinalis plants was beneficial for the parameters of diameter increase, height and number of leaves; however, the same results were not obtained with the mortality rate, which was higher in those treatments in which fertilizers were applied.Since this is the first research that reports on the behavior of C. officinalis in response to fertilization, it is suggested to follow this line of research and analyze the response of C. officinalis to each nutrient separately, which could probably better explain the behavior observed in the trial.In spite of this, the results show promise in the application of fertilizers in nurseries to produce C. officinalis plants in order to help the recovery programs of the species.

Figure 2 .
Figure 2. correlation matrix between growth variables (M = mortality, h = height, di = diameter) of C. officinalis plants obtained at the end of the trial versus the chemical properties of the substrate (n = nitrogen, P = phosphorus, K = potassium, Mg = magnesium, ca = calcium, oM = organic matter, cec = cation exchange capacity, ec = electrical conductivity) analyzed at the end of the trial.

Table 1 .
Fertilizer dose distribution according to trial treatment.

Table 2 .
Multifactorial anoVa with growth parameters as dependent variables.

Table 3 .
differences between the means of the 6 treatments.

Table 4 .
chemical properties of the substrate used as transplanting medium of C. officinalis in each treatment.