BIOFERTILIZERS AND BIOCONTROLLERS AS AN ALTERNATIVE TO THE USE OF CHEMICAL FERTILIZERS AND FUNGICIDES IN THE PROPAGATION OF YERBA MATE BY MINI-CUTTINGS

– The production of yerba mate seedlings through seeds has several limitations, which can be overcome by ex vitro vegetative propagation techniques such as the mini-cuttings, in which it is usually necessary to use synthetic chemical fertilizers and fungicides. However, there is a tendency towards sustainable agriculture, using biofertilizers (growth-promoting bacteria) and biocontrollers ( Trichoderma sp.). Therefore, the objectives of this work were to evaluate the eﬀ ect of biofertilizers on the production of mini-cuttings from yerba mate mini-stumps; as well as the eﬀ ect, of biocontrollers on survival and rooting capacity of mini-cuttings. Strains of Bacillus sp . and Trichoderma asperelloides of yerba mate were used under two radiation conditions. There was a positive relationship between the availability of radiation and the production of mini-cuttings and the rooting capacity. All the mini-stumps sprouted regardless of treatments. The largest production of viable mini-cuttings occurred in a situation of high radiation and fertilization; while the treatments with growth-promoting bacteria and high radiation had intermediate values. The mini-cuttings inoculated with Trichoderma asperelloides had higher rooting percentage, greater number and length of roots than the mini-cuttings treated with fungicide. Therefore, we demonstrated that the use of chemical products can be replaced by biological ones and achieves acceptable yields. que uso de químicos pode ser substituído por produtos biológicos e esse fato atingem alcança rendimentos aceitáveis.


INTRODUCTION
The Ilex genus (Aquifoliaceae) is distributed around the world, particularly in temperate, tropical and subtropical regions. One of the most important species, within the genus, is Ilex paraguariensis Saint Hilaire, known as yerba mate, typical of northeastern Argentina, southern Brazil and eastern Paraguay. This species constitutes one of the main crops in the province of Misiones, with approximately 165,327 hectares cultivated (INYM, 2016).
Nowadays, there is a tendency towards sustainable agriculture, avoiding the indiscriminate use of chemical products. In this sense, the use of biological products that enhance growth of crops in the area is one of the most promising alternatives (Alarcón and Ferrera-Cerrato, 2012). Biofertilizers and biocontrollers are products obtained from microorganisms found in the soil that can improve the physiological response of plants. In addition, it will contribute to microorganism biodiversity conservation, and a more stable production in the long term (Adesemoye et al., 2009;Grageda-Cabrera et al., 2012;Santos et al., 2012).
Among these microorganisms are plant growth promoting bacteria (PGPR), which can stimulate the growth and development of agricultural crops due to their activity as biofertilizers (Çakmakçi and Tingir, 2001;Glick, 2012). In the rhizosphere and roots of yerba mate a wide diversity of bacteria has been found with the capacity to promote plant growth (Bergottini et al., 2015;Bergottini et al., 2017). On the other hand, among the most widely studied species, as biocontrollers, are those of the genus Trichoderma due to their effi ciency, reproductive capacity, ecological plasticity, stimulating eff ect on crops and their action as inducer of systemic resistance to diff erent pathogens (Vega, 2001;Woo et al., 2014). They have antagonistic action against a wide range of phytopathogenic fungi, such as: Fusarium oxysporum, Fusarium roseum, Botrytis cinerea, Rhizoctonia solani, Sclerotium sp., Sclerotinia sp., Pythium sp., Phytophthora sp., Alternaria sp., among others (Montealegre et al., 2014;Woo et al., 2014). Although there are some examples of the use of PGPR bacteria (Teixeira et al., 2007;Peralta et al., 2012) and of Trichoderma sp. (Zaldúa and Sanfuentes, 2010), in the production of mini-cuttings for diff erent Eucalyptus sp., there is no precedent for the use of these microorganisms in Ilex paraguariensis production.
The combination of modern vegetative propagation techniques associated with the application of biofertilizers and biocontrollers would allow us to obtain homogeneous plant material in a friendly environment. Therefore, the objectives of this work were to evaluate the eff ect of biofertilizers on the production of mini-cuttings from yerba mate mini-stumps; as well as the eff ect of biocontrollers on survival and rooting capacity of mini-cuttings.

MATERIALS AND METHODS
2.1. Biofertilizer eff ect on sprout capacity and minicuttings production from young yerba mate ministumps (Experiment 1).
This experiment was carried out with 6-monthold yerba mate seedlings, grown in 1-liter pots with composted pine bark substrate (110 g of substrate per pot). The mini-stumps production and growth was developed according to Rocha et al. (2019). The nutrient source treatments were applied two weeks after the detopping of the seedlings as follow: PGPR (inoculation with the selected combination of PGPR, 3 applications of 5 ml each, every 15 days of a suspension of 1.5x108 CFU / ml) and Fertilization (F, slow release fertilizer application to the substrate, Plantacote® Plus 6M, 3 kg / m 3 ). A combination of native bacteria strains belonging to Bacillus genus with plant growth promoting capacity isolated from yerba mate roots from the Province of Misiones and available in the internal strain repository of the Misiones Biotechnology Institute (UNaM) were used. At the same time, two radiation conditions were evaluated: low radiation (L, 25% of photosynthetically active radiation (PAR)) and high radiation (H, 50% of PAR). The measurements of PAR were made with a Ceptometer (Cavadevices) during the day and several days during the experiment for a correct characterization inside the greenhouse, over the mini-stumps, and at the same time directly to the sun. Three measurements were made at 70-day intervals (September, December and February) after decapitation, where mini-cuttings were harvested. The variables measured were sprout capacity, shoots number and number of viable mini-cuttings (diameter greater than 2mm, 4 to 6 cm long and at least two internodes) per mini-stumps. Therefore, this experiment consists of three factors: Evaluation date (September, December or February), nutrient source (F or PGPR) and radiation (L or H).
The rooting capacity assessment was carried out with mini-cuttings obtained from experiment 1, for the September (experiment 2.1) and February (experiment 2.2) harvests. The same treatments of experiment 1 were maintained (Nutrient source and Radiation). Though, in this case, fungal control factor with 2 levels were added. The fi rst was the application of commercial fungicide (Z, Zineb) and the second was the inoculation with Trichoderma asperelloides IBM193 (T). This strain was isolated from roots of yerba mate plants in the Province of Misiones and is available in the internal strain repository of Misiones Biotechnology Institute (UNaM). This strain was selected given its biological control abilities. Three inoculations of IBM193 of 5 ml each were performed every 15 days, from a suspension of 1.5x106 spores / ml. Rooting was performed under controlled conditions of humidity and temperature in greenhouse with micro-irrigation. After 60 days of installation, the following variables were evaluated: survival and rooting percentage (alive mini-cuttings with roots of at least 5 mm in length); number of roots/ mini-cuttings and maximum root length/mini-cuttings (mm). These experiments consisted of three factors: nutrient source (F or PGPR), radiation (L or H) and fungal control (T or Z).

Statistical analysis.
The experiments were carried out in a completely randomized design, with a minimum of 10 replications per treatment, the experimental unit was the mini-stump or the mini-cutting. In experiment 1, ANOVA was performed considering evaluation date (September, December or February), nutrient source (F or PGPR) and radiation (L or H) as factors. Complete interactions between the three factors were analyzed. If any interaction was signifi cant (more than two levels), means were compared by Tukey test (p< 0.05). In experiments 2.1 and 2.2, ANOVA was performed considering nutrient source (F or PGPR), radiation (L or H) and fungal control (T or Z) as factors. The same post hoc comparison of means was done, if the interaction was signifi cant. Presumptions of independency, normality and variance homogeneity were proven for the variables (regrowth percentage, shoots number, viable mini-cuttings number, survival percentage, rooting percentage, roots number and roots maximum length).

RESULTS
Regardless of the treatments, all the mini-stumps sprouted (data non show). The number of shoots/ mini-stumps, showed interaction between evaluation date and source of nutrients; September showed no signifi cant diff erences between fertilizers or PGPR treatment, with 2.55 and 2.95 shoots/mini-stumps respectively. However, on the following dates, fertilizer treatment showed higher number of shoots, 3.95 shoots/mini-stumps for F and 3.25 shoots/ministumps for PGPR treatments in December; and 4.35 shoots/mini-stumps for F and 3.50 shoots/ministumps for PGPR treatments in February (Table 1, Figure 1  and radiation was observed; shoots production was always higher at high radiation intensities. However, differences in number of shoots/ministumps grown at high radiation and low radiation increased depending on the evaluation dates; the difference between low and high radiation treatments were 0.61, 0.67 and 1.69 in September, December and February respectively (Table 1, Figure 1.A).

.A). Interaction between evaluation date
Viable mini-cuttings production/mini-stumps was modified by triple interaction (D, N and R). The lowest production of viable mini-cuttings/ mini-stumps, both with low and high radiation, was observed in February with PGPR treatment, and in December with low radiation; not exceeding 2 viable mini-cuttings/ministumps. While the highest production was achieved, with fertilizer and high radiation treatments, in December and February, with 6 or more viable mini-cuttings/ mini-stumps. The rest of the treatments presented intermediate values between 3 and 4 viable minicuttings/mini-stumps (Table 1, Figure 1.B).
Mini-cuttings survival rate for experiment 2.1 varied depending on radiation and the type of fungal control. The higher the intensity of radiation in the mini-stumps, higher the survival rate, 70% and 50% for high and low radiation respectively; and higher survival rate in fungicide-treated minicuttings compared to inoculated ones, 72% and 47% respectively (Table 2, Figure 2.A). However, for experiment 2.2, mini-cuttings survival rate was only modifi ed by intensity of radiation. Mini-cuttings obtained from mini-stumps growing with high radiation presented the highest values, more than 96% compared to 70% for low radiation. There were no interactions between factors for this variable in both experiments (Table 2, Figure 2.B).
Rooting percentage for experiment 2.1 was not modifi ed by any of the factors and there were no interactions between them (Table 2, Figure 2.C). However, in experiment 2.2 there was an eff ect of nutrient source and radiation. Mini-cuttings from mini-stumps inoculated with PGPR presented a value of 67% while for mini-cuttings obtained from fertilized mini-stumps was 41%. Regarding radiation, mini-cuttings from mini-stumps growing with high radiation had a value of 79%, while those from ministumps growing in low radiation were 30% (Table 2, Figure 2.D).
Roots number/mini-cutting variations, in experiment 2.1, were observed according to the fungal control method. Mini-cuttings inoculated with Trichoderma asperelloides IBM193 presented higher number of roots than those that received fungicide application, 7.8 and 4.7 respectively (Table 3, Figure  2.E). In experiment 2.2, roots number was modifi ed by the conditions of the mini-stumps, source of nutrient and intensity of radiation. Mini-cuttings from mini-stumps growing with high radiation had greater roots number compared to mini-cuttings obtained from mini-stumps with low radiation, 3.9 and 2.0 respectively. Also, mini-cuttings from mini-stumps inoculated with PGPR had greater number of roots than those from fertilized mini-stumps, 3.9 and 2.0 respectively (Table 3, Figure 2.F). There were no interactions between factors for this variable in both experiments (Table 3).
Maximum root length in experiment 2.1 was modifi ed by intensity of radiation and by type of fungal control. Mini-cuttings inoculated with Trichoderma sp. presented a value of 1.9 mm while mini-cuttings with fungicide application approximately 1.0 mm (Table 3, Figure 2.G). In experiment 2.2 there was only eff ect of radiation intensity. Mini-cuttings from mini-stumps growing at greater intensity showed higher values of root length than those from lower radiation, 1.2 mm and 0.5 mm respectively (Table 3, Figure 2.H). There were no interactions between factors for this variable in both experiments (Table 3).

DISCUSSION
Yerba mate vegetative propagation is a technique that will help to mitigate problems regarding sexual propagation. Nevertheless, it is still necessary to adjust protocols for its application on a commercial scale, taking into account certain limitations such as environmental management and nutrition (Wendling et al., 2007). However, its technical viability has been demonstrated, with an average production of 291 minicuttings/m2 of garden, 95% survival of mini-stumps and 85% of mini-cuttings rooting capacity (Wendling et al., 2007). In our experiment, all yerba mate ministumps sprouted, in accordance with results observed for this species in similar systems in Brazil (Wendling et al., 2010;Pimentel et al., 2019).
Production of viable mini-cuttings per I. paraguariensis mini-stumps varies depending on the mini-garden system adopted (Wendling et al., 2010). For our system, viable mini-cuttings production varied depending on date of collection, source of nutrients and radiation level, with interaction between three factors. The highest production was observed in December and February for fertilization and high radiation treatments with more than 6 viable minicuttings/mini-stumps; while the lowest production was between 1-2 viable mini-cuttings for treatments inoculated with PGPR and low radiation (Table 1, Figure 1.B). The production values obtained for fertilization and high radiation treatments are similar to those obtained in Colombo (Brazil), although in that case there were 6 collections dates (Wendling et al., 2010).  Fertilization aff ects production of mini-cuttings, greater nitrogen availability is associated with greater production (Martínez-Alonso et al., 2012). Although composted pine bark is widely used as substrate in forest nurseries in Misiones province, due to its abundance and low price, it is characterized by high cation exchange capacity and mainly low nitrogen concentration, making fertilization necessary (Jerez, 2007). The traditional way is the application of chemical fertilizers; however, use of PGPR bacteria is possible. One of the physiological mechanisms that might explain the positive interaction between PGPR and plants is the fact that microorganisms improve the nutritional status of plants. The nutrients directly involved, could be explained by nitrogen fi xation, iron chelation by microorganism releasing substances that made it available to the roots (Pii et al., 2015), and the release of organic acids that solubilize phosphorus (Know et al., 2011). However, the positive eff ect of PGPR may also be due to the release of auxins and other hormones (Ramos et al., 2003) that stimulate the proliferation of fi ne roots, and consequently the ability to absorb nutrients present in the soil (Domínguez-Nuñez et al., 2012). In experiment 1, inoculated ministumps produced low or intermediate values of viable mini-cuttings compared to chemical fertilization. The mechanisms previously described, in the present work, could only be related to nitrogen fi xation and hormone production, due to the substrate (composted pine bark) used for the mini-stumps production.
The other factor analyzed in experiment 1 was the intensity of radiation received by mini-stumps. Yerba mate is a plant that grows under canopy in natural conditions (Eibl et al., 2000), where intensity of radiation is regulated by the upper canopy. Therefore, under direct sunlight, the excess of radiation, being unable to dissipate it, causes photoinhibition of photosystem II and the consequent loss of growth and yield (Nishiyama and Murata, 2014). The greatest development of leaves and accumulation of biomass in yerba mate plants occurs when radiation is 50% of direct radiation, being less under full sun or very low radiation intensities (Sansberro et al., 2002;Sansberro et al., 2004). Our results agree with this idea, the highest values of production of viable mini-cuttings were produced in high intensity radiation treatments, which in our case corresponded to 50% of direct solar radiation (Table 1, Figure 1.B).
At the same time, radiation intensity was also the main factor that infl uenced the performance of the mini-cuttings; higher survival rates, rooting percentages, roots number and roots length were achieved in high radiation treatments (Table 2 and 3, Figure 2). In Corylus avellanda mini-cuttings, survival and rooting are positively aff ected by availability of radiation, whereas higher the available radiation, greater the concentration of carbon reserves in stems (Tombesi et al., 2015).
Optimal conditions for mini-cuttings growth are characterized by high relative humidity and high temperature (Zaldúa and Sanfuentes, 2010); conditions that also favor diseases development, making necessary to control or prevent such development. When we compared use of chemical fungicide and inoculation with Trichoderma asperelloides IBM193, diff erences in mini-cuttings survival were slightly higher in fungicide treatment in experiment 2.1. Meanwhile in experiment 2.2, there were no diff erences between treatments, only higher percentage of rooting was observed (Table 2, Figure  2). We do not strictly measure diseases incidence, but higher mini-cuttings survival could be related to a better phytosanitary status. On the other hand, inoculation of IBM193 strain has a positive eff ect on mini-cuttings rooting (number and length of roots). Inoculation of mini-cuttings of Eucalyptus globulus with strains of Trichoderma sp. and Clonostachys sp. reduced infection levels of Botritys cinerea and at the same time, increased rooting percentage (Zaldúa and Sanfuentes, 2010). In mini-cuttings of Passifl ora edulis it was demonstrated that the application of Trichoderma sp. increased root production and that this increase was strongly infl uenced by the inoculation form (Pereira, 2012). This radical stimulation is possibly due to the ability of Trichoderma sp. to produce auxins or their precursors (López-Bucio et al., 2015). The application of PGPR could enhance rooting in addition to its proven capacity to stimulate plant growth, also associated with the production of auxins (Teixeira et al., 2007).

CONCLUSIONS
We have studied yerba mate vegetative propagation by mini-stumps and mini-cuttings propagation system, under conditions of conventional use of fertilizers and fungicides; and under the paradigm of a more sustainable and environmentally friendly production, using biological products (PGPR and Trichoderma sp.); and two radiation conditions. There is a positive relationship between availability of radiation and production of viable minicuttings as well as rooting capacity.
The highest production of viable mini-cuttings was achieved in fertilization treatments compared to inoculated with PGPR ones. However, treatments inoculated with PGPR give acceptable yields.
In the comparison of fungicide and inoculation with Trichoderma sp. treatments, there were no diff erences, or they were minimal. Diff erences on rooting percentage, roots number and roots length/ mini-cutting were greater in inoculated plants.
Therefore, it is demonstrated that the use of chemical products can be replaced by biological ones and achieve acceptable yields.