Study area, plant material and crop management

The experiments were carried out in the months from March to July in a greenhouse belonging to the Universidad Autonoma Agraria Antonio Narro, located in Saltillo, Coahuila, Mexico, with geographical coordinates of latitude 25° 27’ N 101° 02’ W and an altitude of 1610 meters.

The Swiss chard seedlings Beta vulgaris var. Forhook Giant (seeds purchased from ITSCO Agro, SAPI de CV) was carried out on polystyrene trays with 200 cavities, in which one seed per cavity was sown with peat moss substrate. Treatments are described in Table 1; a total of 800 seeds of Swiss chard were used (Beta vulgaris var. Forhook Giant). After 29 days the seedlings were transplanted to 3 kg pots with a substrate containing soil, peat moss, and perlite (1: 1: 1) added at this time the Steiner solution and compost tea were applied once a week (CT, CTIF, IF, CTCi and CiIF) depending on each case as described in Table 1 and were irrigated with tap water twice a week. In the cases of the C and Ci treatments, only irrigation twice a week was done during the entire experiment.

Treatments

A total of seven treatments and twenty-one repetitions with seven samplings were established for the present work as described in Table 1. Every sampling consisted in withdrawing 3 (of the 21) replicates of each treatment. The samplings were carried out every ten days after transplanting.

Co-inoculation

The disinfection and inoculation of seeds were carried out as described by Pérez-García (2017¹), all seeds were previously disinfected (using Tween 20, ethanol, sodium hypochlorite, sodium thiosulfate), and half of the seeds (400) were co-inoculated with Azospirillum brasilence and Glomus intraradices (using gum arabic as adherent). The sown co-inoculated seeds were used to form the seedling for the CTIF, CTCi, and Ci (Table 1) treatments. Co-inoculation was only carried out during this stage.

Compost tea

Compost tea was prepared using mature compost (5 months of composting) constituted by vegetal and animal waste, as well as cow manure (80:20). The compost tea was prepared by adding 1 kg of compost per 20 liters of water. The compost tea was brewed for 7 days with aeration.

Inorganic fertilization

Inorganic fertilization was made with Steiner nutrient solution (Steiner, 1984²), carried out in both cases, in co-inoculated and non-inoculated seeds. The 25% nutrient solution was applied once a week during the first 45 days after transplantation and at 50% for the remaining trial.

Pest and disease management

Two commercial products were used for pest and disease control. The first commercial product is Bralic^® (Allium spp. 12.5%), and the second commercial product was DiPel DF (Bacillus thuringiensis var. Kurstaki).

Bralic attacks the nervous system of insects with sulfur substances called allomones and is usually applied to eliminate Trips spp., Liriomyza sp., and Bemisia tabaci. The product DiPel DF is made of different toxins that target insect larvae, and it is mainly used to eliminate caterpillars of lepidopterous.

Analytical methods

All samples (for all treatments) were separated into roots, stems, and leaves. Previous to measuring, the roots were cleansed to remove the substrate. The roots and stems were measured with a Vernier caliper and the leaf area was determined with a leaf area meter (LI-COR Model LI3100C). After measuring, the roots, stems and leaves were sampled and dried (65 °C for 72 h) in the drying oven (TERLAB model TE-H70DM) and subsequently weighed in an analytical balance (US SOLID model USS-DBS15-3) to determine the root dry weight, stem dry weight and leaf dry weight. The biomass partitioning coefficient for root (BPC_R), stem (BPC_S), and leaf (BPC_L) were calculated according to the following equations:

In the case of the last samplings, the whole plant was dried in a dry oven at 65 °C for 72 h, and subsequently, the plants were subjected to a grinding process in a mortar for further determinations of macro and micronutrients. Macro and micronutrients K, Ca, Mg, Fe, Cu, Zn, Mn, and B were determined with an atomic emission spectrometer ICP Optical Varian 725-ES), and the total was determined with the semi-micro Kjeldahl method as described by ^{Bremner (1965)}. For each treatment, only one analysis was carried out.

Experimental design and statistical analysis

A complete randomized block experimental design with 21 replicates for each of the seven treatments was applied. Every sampling consisted in withdrawing 3 (of the 21) replicates of each treatment. An analysis of variance (ANOVA) was conducted along with a Tukey test (P ≥ 0.05) using the InfoStat software 2020 (^{Di Rienzo et al., 2020}).

Emergence

The emergence of the Swiss chard was observed on the fourth day after seedling, achieving an 88% after 14 days for seeds with and without co-inoculation, it is noteworthy that seedlings from seeds that were co-inoculated had an emergence rate 25% higher, than those without co-inoculation. The results obtained in the present work showed that co-inoculation had a beneficial effect on the emergence of Swiss chard plants. Different authors have reported similar results in the use of biofertilizers, such as ^{Delshadi, Ebrahimi and Shirmohammadi (2017)}, who reported that the use of inoculation or co-inoculation with Azotobacter vinelandii, Pantoea agglomerans,and Pseudomonas putida in Onobrychis sativa L seeds, showed increased germination compared to the seeds that were not inoculated or co-inoculated. Another report on the use of co-inoculation by ^{Zeffa et al. (2019)}, reported an increase in the concentration of indole acetic acid (IAI) in seeds of different maize genotypes that were inoculated with Azospirillum brasilense, also mentioning that this contributed to improved also the germination percentage and growth in the plants.

Dry weight

Swiss chard plant root dry weight results showed that there was no significant difference in samples 1, 3, 4, and 5 in all treatments assessed, meanwhile, in samples 2, 6, and 7 in the case of CTIF and CT treatments, the root dry weight was greater than the other treatments (Table 2). In 6 out of 7 samples of plants treated with CTIF, the root dry weight results were the highest, followed by 5 out of 7 samples for the plants treated with CT. The use of compost tea in tomato plants has been previously reported, mentioning that plants under this treatment have produced three times more root dry weight, and, additionally, a suppressive effect over Fusarium oxysporum has also been observed (^{Morales-Corts, Pérez and Gómez, 2018}), ^{González-Solano, Rodríguez, Trejo, García and Sánchez (2013)}, reported that the use of vermicompost tea in lettuce, basil, and coriander led to an increase in dry biomass weight in similar amounts as when using the Steiner solution. The use of compost tea (grape marc) with no aeration in pepper seedlings produced an increase in root dry weight (^{Marín et al., 2014}). ^{Ingham (2005)} mentioned that the increase in the root size is attributed to the nutrients and growth-promoting substances that are present in the vermicompost or compost teas. In the present work, the highest values for root length were obtained in plants with Ci treatment (Table 2). In five of the seven samplings, the Ci treatment showed the greatest results in root length (statistically significant difference). This can be attributed to the fact that mycorrhiza (Glomus intraradices) is known to increase the root exploration volume due to the mycelium being an extension of the roots (^{Ardakani et al., 2011}). Additionally, the rhizobacteria Azospirillum, a vegetable plant growth promoter (present in the commercial product AzoFert^®) increases the root length in tomato seedlings (^{Terry, Núñez, Pino and Medina, 2001}). The greater dry matter weight in the stems was obtained in the plants with CTFI treatment (in 5 out of 7 samples), meanwhile, the lowest values were obtained in the case Ci treatment (Table 4).

It is worth mentioning that the IF and CTIF treatments increased the stem length of fresh plants (Table 3). In five of the seven samplings, the IF and CTIF treatments obtained higher values for root length (Table 4). ^{González-Solano et al. (2013)}, reported that using vermicompost tea rendered similar values of stem length (dry weight) in basil when using the Steiner solution, and higher values were obtained in comparison to the Steiner solution when using vermicompost tea in lettuce and coriander plants. ^{Marín et al. (2014)}, reported that in pepper seedlings that were treated with compost tea (from grape marc without aeration), the dry stem weight was increased. In the case of leaf dry weight for 3 out of 7 samples (samples 3, 6, and 7, Table 3) there was no significant difference in all treatments. The highest values for dry leaf weight were obtained in the cases of plants treated with IF, CT, and CTIF (in 5 out of 7 samples). The highest number of leaves per plant was obtained in the cases of plants treated with IF, followed by those treated with CTIF (data not shown). ^{González-Solano et al. (2013)}, also mentioned that the use of vermicompost tea led to an increase in the leave dry weight of basil, coriander, and lettuce, being the last two crops with even higher values compared to plants treated with the Steiner solution. ^{Haggag, Merwad, Shahin, Hoballah and Mahdy (2014)}, reported that the use of compost tea alone or combined with chemical fertilizer increases the values of leaves dry weight and leaves per plant. ^{Marín et al. (2014)}, mentioned that the leaf dry weight values increased (pepper seedlings) when using vermicompost tea.

The use of compost or vermicompost tea with and without the addition of nutrients has been reported to increase the values for dry weight in plants. ^{Siddiqui et al. (2011)}, mentioned that the use of compost tea and NPK fertilizers in a ratio of 1:1 increased the dry matter obtained in Centella asiatica. ^{Moncayo-Luján, Álvarez, González, Salas and Chávez (2015)}, mentioned that the use of vermicompost and compost tea increased the dry matter obtained in basil plants. ^{Salas-Pérez, Borroel, Ramírez and Moncayo (2018)}, reported that the use of compost tea and compost tea with ascorbic acid increased 18% the dry matter obtained in a hydroponic green fodder crop. Additionally, it can be mentioned that fresh weight is also increased with the use of compost tea (this value was not determined in the present work), as described by ^{Kim et al. (2015)}, in lettuce plants that were treated with compost and vermicompost teas.

Leaf area

From an agroindustrial perspective, the most important is to promote organ growth, in the case of Swiss chard plants, when big, green, and healthy leaves are desired (directly proportional to leaf area). The Swiss chard plants that were treated with IF and CTIF obtained the greatest leaf area as shown in Table 5. Similar results as those obtained in the present work are described by ^{Marín et al. (2014)}, where the pepper seedlings that were treated with compost tea from grape marc showed an increase in the leaf area. A leaf area increase in coriander plants was also reported by ^{González-Solano et al. (2013)} when using vermicompost tea compared to Steiner solution.

Biomass partitioning coefficient

The biomass partitioning coefficient provides an insight into how biomass is distributed among its different organs, and these calculations are of great importance when new organs appear in plants, such as tubers, bulbs, fruits, and so forth (^{Di Benedetto and Tognetti, 2016}). The results obtained in the present work showed that in the first sampling, the photosynthates were mainly used for the formation of biomass in roots and leaves, and to a lesser extent, stems (Table 6). In the following samplings, it can be observed that the photosynthates were used to form leaves, followed by the formation of the stem, and to a lesser extent, roots. This is advantageous in the case of Swiss chard plants, in order to have leaves continue their size increase, while the stem must grow thicker.

Nutrient analysis in leaves?

Ten micro-and macronutrients were determined, N (Nitrogen), P (Phosphorus), K (Potassium), Ca (Calcium), Mg (Magnesium), Fe (Iron), Cu (Copper), Zn (Zinc), Mn (Manganese), and B (Boron), (Table 7). Results obtained showed that plants that were subject to the CT treatment presented a higher concentration of Ca, Fe, Zn, and B (9630, 213.5, 25.6 and 54 mg kg^-1 respectively). The Ci treatment led to an increase in N, Ca, Zn, Mn, and B (28 600, 8700, 19.3, 39.7 and 55 mg kg^-1 respectively); the IF treatment increased the Ca, Fe, Cu, Zn, and B (10 400, 217, 8, 25.1, and 52.3 mg kg^-1 respectively) content in plants. Results also showed that when these treatments were combined in some cases, the nutrient content increased; however, in other cases, the nutrient content declined. The CTIF treatment led to an increase in N, Ca, Fe, Cu, Zn, Mn, and B (26 800, 10 700, 246, 8.3, 31.5, 39.7, and 60.5 mg kg^-1 respectively) content; the CTCi treatment increased the N, K, Ca, Zn, Mn, and B (30 000, 2600, 9000, 15.5, 41.9, and 54.1 mg kg^-1 respectively) content; meanwhile the CiIF treatment increased the Ca, Zn and B (8600, 19.4, and 52.3 mg kg^-1 respectively). The CTIF was the treatment that led to an increase in seven of the ten nutrients that were determined in the present work. The plants that presented a higher N content were Ci, CTCi, and CTIF, ^{Terry et al. (2001)} and ^{Ardakani et al. (2011)}, reported similar results. Meanwhile, the plants with the CiIF and IF treatments presented lower N values. ^{Velasco-Velasco, Ferrera and Almaraz (2001)}, reported that N content increased by 30% in plants when Azospirillum, Glomus, and vermicompost treatments were applied, and a 100% increase in N was observed when a combination of vermicompost+Glomus and vermicompost+Glomus+Azospirillum were applied; additionally, these combinations also led to an increase of a 100% in P content. However, in the present work, the Ci treatment did not increase the P content as expected, attributing this to Glomus being a phosphorus solubilizing microorganism (^{Ardakani et al. 2011}).