Substrate preparation and biological material

Compost was provided by the Government of Mexico DF (GDF) and the bio-fertilizers (A. brasilense and G. intraradices) by the trading house Biosustenta. The compost is made in Texcoco with trash collection from DF and product of the pruning of gardens, no contain carries animal manure and reported a neutral pH (7.31), an organic matter content of 27.36% and humidity very low (4.08%). The soil used presented a slightly alkaline pH (7.65), an organic matter content of 2.88% and very low humidity percentage (1.84%). The compost and soil were autoclaved at 121 °C for 1 h. To sterilize the seeds from Zea mays was determined as described ^{Wenny and Dumroese 1987}. After this treatment, they were impregnated with a non-toxic adherent based on carboxymethyl cellulose. To finish, we mixed G. intraradices (333 g) and A. brasilense (117 g) and apply this mixture on the seeds.

Treatments

The treatments used in this study are CN (soil and seed) T1 (soil, compost and seed), T2 (Soil, compost sterile and seed), T3 (soil, composte, seed and Biofertilizer), T4 (soil, compost sterile, seed and biofertilizer), T5 (soil, compost, seed sterile and biofertilizer) and T6 (soil sterile, compost sterile, seed sterile and biofertilizer.

Determining the compost physicochemical properties

The organic matter content of C, P and N was determined as described ^{González-Chávez et al. (2009)}. The pH was determined in water and CaCl₂ in a ratio 1:2.5 of extractsolution a potentiometer 420ª was used for the measurements (Orion Research, Beverly, MA).

Determining human pathogens in the compost and heavy metals in the compost

To characterize the microbiology of the compost, we did serial dilutions which were inoculated in specific media for bacteria considered potentially pathogenic for the human (Mac Conkey, Salt and mannitol and Salmonella-Shigella). To determine the heavy metals in the compost, the procedure was done in the Laboratory of Geochemistry, belonging to the Academic Unit of Earth Sciencies, located in Taxco el Viejo, Guerrero. Metals were analyzed by ICP-AES (^{Talavera et al., 2006}).

Isolation of A. brasilense and observation of G. Intraradices from maize roots with compost

We did some tests as the glucose fermentation and Gram´s method to identify the microorganism of the roots (^{Baca et al., 2010}). To observe mycorrhizal roots by G. intraradices, we used the technique by ^{Phillips and Haymann (1970)}. Additionally, we counted spores in the rhizosphere as described by ^{Gerdemann and Nicholson (1963)}.

Physicochemical analysis of the compost

One of the goals of this work was to determine the physicochemical properties of the compost. The analysis reported that the compost has a neutral pH (7.31), an average content of organic matter (27.36%) and low humidity (4.08%). The amount of N, K, Ca, Mg, Fe, P, and Mn is average (^{Álvarez-Solís et al., 2010}, ^{Pérez et al., 2010}). The electrical conductivity registered a high value and a sandy texture (NTC-5167 2004). Regarding the compost, the three main elements considered for the growth of the microorganism (N, P y K), are present below the levels reported by ^{Álvarez-Solís et al. (2010)}, N= 0.095%, P= 0.65% and K= 0.83%. These results show that in those composts where trash collection from DF and vegetable waste are included, there is a decrease in the content of those nutrients (^{Castillo et al., 2000}). The organic matter content (OMC), considered an important source of nutrients (^{Pérez et al., 2010}), was 27.36%, which is a good amount for compost. The OMC in the compost has a positive effect on the soil fertility, besides improving its physical, chemical and biological characteristics. However, the percent of OM in the used compost showed a lower value compared to that reported by ^{Zapata et al. (2005)}, in a range from 46 to 56%, who used compost based on local muds and pine bark residues.

Determining human pathogens in the compost

The microbiological analysis of the compost reported the presence of Staphylococcus aureus and Pseudomonas aeruginosa. We confirmed this result with the coagulase test for S. aureus and a biochemical set for P. aeruginosa. The results obtained in the microbiological analysis of the compost (Table 1) showed the existence of S. aureus, although the growth was not significant to affect the health of those manipulating the compost (NMX-F-310-1978). Several authors have reported the growth of this bacterium in samples of vermicompost, suggesting that its components have a fecal animal origin (^{Ogefere et al., 2010}) nonetheless, the compost used in this study does not have these origins, no carries animal manure.

The growth of P. Aeruginosa was observed due to the capacity of this bacterium to grow at high temperatures which makes it resistant to the auto-sterilization process undergone by the compost. The auto-sterilization consists in a temperature rise because of the metabolic activity of the microorganisms present in the compost (^{Muñoz, 2005}). ^{Pérez et al. (2010)} showed that the manure compost has higher densities of the genus Pseudomonas (420 x 10³ CFU g^-1), which has a high adaptation capacity.

Determining heavy metals in the compost

The analysis of heavy metals in the compost is shown in Table 2. We compared three international norms, considering the amounts of heavy metals found in the compost falling inside the maximum permissive limits. Despite the presence of heavy metals in the analyzed compost, the concentrations of As, Cd, Cr, Cu, Ni and Pb are below the maximum permissive levels in the NTC-5167 2004, Norm 503 of EPA and the EPA in 2006. These concentrations suggest that the compost was made using a big variety of waste, which normally is not subject to an appropriate selection and residue separation. Even more, some authors report the use of waste water to maintain the humidity during the compost process, being one of the ways to incorporate heavy metals to the compost. Likewise, heavy metals as Pb, As, Cd, Cu, Zn, Ni and Hg, could have been added in a continuous manner to the soils through agricultural and/or industrial activities, and incineration (^{Khan, 2006}).

Isolation of A. brasilense in the roots and observation of G. intraradices in the maize rhizosphere

We used the method reported by ^{Baca et al. (2010)} to obtain pure cultures A. brasilense, here we observed that the concentration of heavy metals from compost used in this work did not affect the native or external microbiology that could exist in the compost and the soil since the analysis showed positive results to microbial growth and to the isolation of A. brasilense. The observation of how the fungus colonized the roots was performed at the end of the experiment. We used two techniques, the first one was to isolate the fungus from the roots, being able to observe a high amount of vesicles, spores and hyphae (Figure 1a), mainly belonging to the genus Glomus. The second technique was the one described by ^{Gerdemann and Nicholson, (1963)}, where we counted and analyzed the rhizosphere (Figure 1b). In this case, we were able to identify more than one sample of the genus Glomus and particularly G. intraradices in the treatments where we added bio-fertilizers (T3 and T6 mainly).

Treatment 6. Image A) spores (sp); B) hyphae (hf) and spores (sp); C) spores (sp); D) vesicles (vs) and spores (sp); E) spores (sp) and F) spores (sp) and vesicle (vs). Usual taxonomy shown by the spores belonging to G. intraradices.

Figure 1 Morphology (a) and spores of G. intraradices (b) observed in the maize roots and the rhizosphere by the technique of ^{Phillips and Haymann, 1970}.