Myrcianthes Cisplatensis seedling obtention - Approximately, 200 M. cisplatensis seeds were collected from the natural population site (32°29'16.1" S 60°50' 57.5" O) and were germinated under controlled conditions in plastic trays with blotting paper (25-27 °C, constant humidity with reverse osmosis water, photoperiod of 12 h) (Eynard; Calviño; Ashworth, 2017). Seeds germinated within 6 - 10 days; individual seedlings were transplanted to pots of 100 mL filled with perlite, peat and compost in a proportion of 5:3:2, respectively. M. cisplatensis were grown under semi-controlled conditions (controlled irrigation, without control on photoperiod and temperature) for 11 months; the seedlings were watered to field capacity regularly.

ii) Compost elaboration

The compost used for the experiment was produced at the Vivero Forestal Agroecológico from the Facultad de Ciencias Agrarias, Universidad Nacional de Rosario, Argentina. Organic wastes, predominantly pumpkin and potato peel, lettuce, onion and tomato, from the university cafeteria were mixed with Populus sp. and Ligustrum lucidumdry leaves at a 1:1 proportion and were disposed of as a compost pile over the soil. Organic wastes and leaves were weekly incorporated into the pile, at the same 1:1 proportion. The compost was watered frequently and the pile was periodically turned over during 4 months in order to avoid anoxic conditions. Humidity was checked weekly with the hand squeeze test (Román; Martínez; Pantoja, 2013). Organoleptic properties (color, structure, odor, moisture and color of the suspension) and the maturity of the compost were assessed  by La Enmienda (Table 1). The maturity of the compost was determined by the quantification of the trophic microbiologic network (Inghman and Slaughter, 2004). Briefly, compost samples are dissolved 1:5 in water and 25 fields of water drops are observed under a light microscope. The different metabolic links are quantified following the protocol developed by Dra. Elaine Ingham (Inghman and Slaughter, 2004). The trophic microbiologic network analysis established that the compost was almost mature. Compost C:N relation was 11% (HSE soluciones, Argentina, with S.A.M.L.A. method) .

Table 1. Organoleptic properties and trophic biological network of compost. One hundred grams of compost was sampled and send for further analysis to La Enmienda, were the analysis are made following the protocol developed by Dra. Elaine Inghman (Inghman and Slaughter, 2004). Data is expressed as media and standard error of 25 field observations.

Source: La Enmienda, 2022.

iii) Compost extract preparation

A compost extract was prepared in order to evaluate the effect of compost on AMF symbiosis with M. cisplatensis seedlings. The compost extract was prepared from a compound compost sample by an incubation of 10 minutes of 500 gr of compost (inside a mesh, 400-500 µm pore diameter, La Enmienda) in 20 lts of reverse osmosis water. Mineral (Table 2) and potentially toxic elements (Table 3) in compost extract were quantified by ICP-MS (Perkin-Elmer, CCT Rosario) together with total Nitrogen determination (HSE soluciones, with Kjeldahl method).

For the evaluation of the effect of compost extract on AMF symbiosis, a 1:500,000 dilution was made with reverse osmosis water. In order to distinguish between the effect of microorganisms and the effect of biologically active molecules, the dilutions were filtered with a nanopore filter (0.45 µm pore diameter). Dilution selection was made considering previous results of the research group (de la Torre et al., 2022).

Table 2: Element composition of Compost Extract (CE). Compost Extract element composition was quantified by ICP-MS. Total amount (mg) of each element present in the 50 ml of the 1:500,000 dilution used at the experiment is expressed together with the concentration and total amount of each element present in the low P Hoagland solution used as the nutritive solution during the experiment.

Source: Fernández, 2022.

Table 3: Potentially Toxic Element composition of Compost Extract (CE). Compost Extract Potentially Toxic Element (PTE) composition was quantified by ICP-MS. Total amount (mg) of each PTE present in the 50 ml of the 1:500,000 dilution used at the experiment is expressed.

Source: Fernández, 2022.

iv) Experimental design

Eleven-month M. cisplatensis seedlings were transplanted to 1 lt pots previously filled with a mixture of autoclaved (121 °C, 30 minutes) sand : perlite (4:1 proportion) up to 50% of its volume. A fine layer of the TC mixture (100 mL) was added over the autoclaved substrate. Then, pots were filled to complete its volume with the same mixture of substrate. Previous to the transplant, M. cisplatensis seedlings height was measured so that each treatment contained a representative group of seedlings. The treatments were: Inoculation with 100 mL of the TC (AMF); AMF + 1:500,000 filtrate compost extract (AMF+CEF dil); AMF + 1:500,000 compost extract (AMF+CE dil) and AMF with autoclaved TC (no AMF). Each treatment consisted of 5 replicates. The TC used for the AMF treatments was a mixture of TC1, TC2 and TC3; a mixture of TC4, TC5 and TC6 was autoclaved and used for the no AMF control. The compost extract treatment consisted in one irrigation with 50 mL of each dilution, 3 days after the transplant. Plants were grown for 6 months. They were regularly irrigated with 100 mL of low phosphorous Hoagland solution (Gil-Cardeza et al., 2021).

v) Determinations and analysis

Myrcianthes cisplatensis growth-related -  Seedlings height (from the base of the pot to the apical meristem) was measured at 30, 60, 90 and 150 days. At the end of the experiment, plants were harvested, shoots were separated from the roots and fresh weight was measured. Then, the tissues were dried at 80 °C for 96 h and dry weight was determined. Both determinations were made with an analytical balance (0.001 gr).

Arbuscular mycorrhizal symbiosis - A representative portion of dry roots were placed in Falcon tubes and re-hydrated for 48 h in deionized water (Gil-Cardeza et al., 2021). The roots were then incubated at 70 °C in a water bath for 30 min in 50 mL KOH 10% w/V (Cicarelli, pro-analysis), KOH was discarded and fresh 50 ml were added and roots were incubated for another 30 min at 70°C. The KOH was removed and roots were rinsed thoroughly with tap water and rinsed for 5 min with HCl 1% w/V (Cicarelli, pro-analysis (36.5-38% w/w)). Roots were further stained in 25 mL of cotton blue 0.05% w/V (Biopack, suitable microscopy) containing HCl (0.05% w/V) and glycerin (50% w/V; Biopack, pro-analysis (99.5% w/w)) and incubated at 90 °C in a water bath for 20 min. The roots were finally rinsed and stored in acid glycerin before observation (Phillips and Hayman, 1970).

For observation, twenty root fragments of ~10 mm length were mounted on microscope slides and examined under a compound microscope at 20-40x magnifications. The intensity of mycorrhizal association (%I) was calculated as follows: (v + 5w + 30x + 70y + 95z)/(v+w+x+y+z), where v, w, x, y , z are the number of root fragments containing an increasing proportion (i.e. v: <1%, w: 1-10%, x: 11-50%, y: 51-90%,z:  > 90%) of AMF structures (Dodd et al., 2001).

Statistical analysis - All analyses were conducted using INFOSTAT (Di Renzo et al., 2016). Differences between averages of Intensity of the mycorrhizal association (%I) and fresh and dry weight were analyzed with One-way ANOVA (p ≤ 0.05). Multiple comparisons between medias were made with the Tukey post test (p ≤ 0.05). Intensity of the mycorrhizal association didn't assume the assumptions for homoscedasticity so data were transformed with arcsine √(x+1). Height curves were analyzed with the repeated measures test (p ≤ 0.05).

Compost extract effect on AMS and growth of M. cisplatensis seedlings - All M. cisplatensis seedlings grown in presence of viable AMF established the symbiosis (Fig. 1). The highest mycorrhizal intensity values (%I) were observed in M. cisplatensis roots irrigated with the 1:500,000 compost extract dilution, independently of the filtration through the nanopore filter: 31% no filtration and 26% filtrated (Fig. 1 AMF+CEdil and AMF+CEFdil, respectively). Mycorrhizal intensity in M. cisplatensis control AMF roots was 6%. No AMF structures were observed in no-AMF control.  

Figure 1: Mycorrhizal intensity (%I) in Myrcinathes cisplatensis roots. Eleven months old M. cisplatensis seedlings were transplanted to 1lt pots filled with autoclaved substrate with (AMF) or without (no AMF, autoclaved TC) arbuscular mycorrhizal fungi. Three days after the transplant pots were irrigated with 50 mL of a dilution of compost extract previously filtered or not filtered through a nanopore filter (1:500,000 dilution without filtration: AMF + CEdil; 1:500,000 dilution filtered AMF + CEFdil). Data are expressed as means ± SEM (N = 4). Values with the same lower-case letters in a graph do not differ at P ≤ 0.05 (one-way ANOVA; multiple-comparisons were made by a multiple range Tukey test).

These findings show a stimulatory effect on AMS of a 1:500,000 compost extract dilution. Among the variables that could influence the symbiosis are minerals, microorganisms and hormones, growth factors and/or other metabolic products of microbial activity developed in the metabolism of the compost (Parniske, 2008; Koskey et al., 2022). The experimental design carried out, in the sense of the dilution chosen and the selection of a single application of the compost extract at the beginning of the experiment, reduced the possibility of an influence on AMS of the minerals present in the compost extract. This is in accordance with the mineral concentrations determined in the compost extract and the amounts administered by Hoagland's solution, where it was observed that the amount of minerals provided by the extract is lower than those provided by the solution (Table 2).

Even though it was not the principal aim of the work, height (Fig. 2) and weight (dry and fresh) were determined on M. cisplatensis seedlings, so as to gather more knowledge regarding the effects of the treatments. M. cisplatensis seedlings that grown in presence of AMF and that were irrigated with the 1:500,000 compost extract dilution, independently of the filtration treatment, showed the highest values of height at the end of the experiment (Fig. 2, ○ and ●, (36 ± 3) and (35 ± 3) cm, respectively). No AMF control seedlings heights (Fig. 2, □, (31± 2) cm) did not differed statistically from heights of AMF controls seedlings (Fig. 2, ▪, (29 ± 2) cm). These findings are in accordance with the stimulation on AMS observed in M. cisplatensis roots that grown in the pots irrigated with the compost extract. Since it was not the principal aim of the work, no control without AMF and compost extract irrigation was made, so it is not possible to conclude a cause-effect relation between AMS stimulation and the higher heights for observed in the compost extraction treatments. Nevertheless, it is possible to affirm that the positive effect on height was independent of the presence of microorganisms in the compost extract.

Regarding fresh and dry weights, no differences were observed between treatments when fresh and dry weight of shoots and roots were analyzed (data not shown). Mean fresh weights (gr) were in the range of (4.3 ± 0.5) - (7 ± 1) and (5.4 ± 0.6) - (7.8 ± 1) in roots and shoots, respectively. Mean dry weights (gr) were in the range of (0.9 ± 0.2) - (1.5 ± 0.2) and (2.5 ± 0.3 - 3.5 ± 0.6) in roots and shoots, respectively.

The methodological approach used in this study (the production of soil enriched with AMF indigenous populations and the use of compost extract to promote AMS) could be applicable in agroecological transitions that incorporates trees in the agroecosystem design. Planting young trees that are already in symbiotic association with AMF populations isolated from the field where trees will be transplanted can help the trees to overcome stress during the transplant (Rivillas; Calle; Angel. 2019). Moreover, it could promote the restoration of soil life since AMF mycelium has the potential to interconnect with the pre-existing CMN and thus help to stabilize AMF mycelium that could become AMS inoculum for future crops (Giovannetti; Avio; Sbrana, 2015). In addition, the promotion of AMS in agroecosystems has also the potential to stimulate carbon sequestration into the soil, thus contributing to the mitigation of climate change (Moriën et al., 2017).

As a possible strategy to develop a sustainable agriculture, plenty of efforts are currently put into the development of biological products that contain isolated species of microorganisms or a mixture of them. Overall, our results demonstrate that another strategy can be undertaken, a strategy that stimulates the growth of the indigenous microorganisms populations, in this case of AMF, and that the symbiosis can be stimulated with extremely diluted compost extracts solutions possibly due to the presence of soluble biomolecules. Moreover, the techniques used in the study (i.e. compost and trap culture of indigenous AMF) are not expensive and could be implemented by native trees plant nurseries and/or family farmers. Particularly, we demonstrated that a 1:500,000 dilution of a compost extract stimulated AMS in M. cisplatensis seedlings.

ACKNOWLEDGMENTS

We thank Sebastián Ferlini, María Victoria Flores and Mariano Craviotto for their guidance and companion during the recollection of the soil used to cultivate AMF; Ing. Agr. David Balabán for helping with the irrigation of the plants; Lic. Paula Frassón for her guidance during the early steps of this work and Dra. Ayelén Pagani for the determination of minerals and toxic elements.

This work was supported by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Project PUE 22920160100043) and by the Universidad de Rosario, (Project PID 80020180100057UR), Argentina.

Copyright (©) 2025 - Antonella Fernández, Lourdes Gil-Cardeza

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