Introduction

The interaction between the components of a microbial community in agricultural systems may manifest or not any morphological or physiological attribute of anthropocentric interest of the host plant. In some annuals and perennials found in the different components increase performance by inoculating fungi and bacteria together (^{Aguirre-Medina et al, 2012}). These benefits have been tried in other crops in order to identify synergism between some associations of soil microorganisms and growing interest. Some of the benefits on plant growth are expressed with the combination of associative bacteria and mycorrhizal fungi.

The interaction between rhizobacteria promoting plant growth and fungi endomycorrhizal, can be selective and dependent bacteria and fungus involved (^{Azcon, 2000}). The positive interaction in the growth and yield of some crops has encountered inoculating of Rhizophagus intraradices, Azospirillum brasilense and/or Rhizobium etli (^{Aguirre Medina, 2006}), along with increases in the nitrogen content (N) and phosphorus (P) in plant tissue and grain (^{Aguirre-Medina et al, 2012}). The pseudomonas are an important group of microorganisms that can promote the growth of plants and protect them from pathogens (^{Unno et al, 2005}).

For the production of chili is of great importance the overall evaluation of the use of bio-fertilizers based fungi-rhizobacteria that have been evaluated in other cultures and have proven effective action on plants. Given this background, this study aimed to study agricultural biological effectiveness of mycorrhiza-rhizobacterias co-inoculation on the growth and yield of chile.

Materials and methods

Results and discussion

In the Table 1 shows the growth of C. annuum L. plant with the treatments applied. The plant height was statistically different (p≤ 0.05) for all three samples. In the first two sampling the plant height increased by inoculating seeds with a microorganism compared to the inoculation of two or all three microorganisms. The maximum height was 63 cm and recorded in plants inoculated with A. brasilense. With Azospirillum is favored the plant growth by the increase in the radical development of the host plant by producing hormones and nitrogen fixation (^{Bashan and Bashan-2010}).

Table 1 Comparisons of average plant height, leaf number and stem diameter of Capsicum annuum L. bofertilized with different microorganisms in andosol-mollic of Soconusco, Chiapas soil. 

CV= coeficiente de variación (%). 'Valores con diferente letra dentro de cada factor y columna son estadísticamente diferentes (p ≤0.05).

The inoculated with microorganisms three plants showed about half of the height compared to A. brasilense.^{Alonso and Galán (2006)} cite in tomato increase in plant height with A. brasilense.^{Karthikeyan et al. (2009)} recorded increase in plant height to inoculate the seeds of Catharanthus roseus (L.) G. Don. two bacteria, A. brasilense and P. fluorescens and ^{Neetu et al. (2012)} cite tallest plants to inoculate Linum usitatissimum L. with P. fluorescens + G. mosseae. Meanwhile ^{Reyes-Ramírez et al. (2014)} reported increased height habanero pepper plants (Capsicum chinense Jacq.) after 90 days of being inoculated with Pseudomonas spp. compared with plants inoculated with A. brasilense and the witness.

In some cultures the response to inoculation is higher with a single microorganism and may be related to the increased demand for carbon colonization more symbionts (^{Sylvia, 2005}). In annual crops such as corn and beans, we found differential response to the simple inoculation and co-inoculation of more than one microorganism (^{Aguirre-Medina, 2006}), the previous answer seems to be influenced by the ability of colonization of microorganisms, by interaction with the ground and naturalized microorganisms present in the soil.

Stem diameter also showed statistically significant difference (p≤ 0.05) between treatments during the first two samples. At 28 days after transplanting (ddt), the largest stem thickness was found with inoculation A. brasilense and P. fluorescens. At 56 ddt, stem diameter was very similar among treatments, but the statistical differences favored inoculated with P. fluorescens treatment. Results contrary presented ^{Reyes-Ramírez et al. (2014)} in habanero chile (Capsicum chinense Jacq.) Who found no differences between treatments for stem diameter at the first sampling dates, but from 90 ddt and was higher with inoculation of Pseudomonas spp. and compared to the control plants inoculated with A. brasilense.

In other perennial crops it has consigned the same effect but with G. intraradices (^{Chattopadhyay et al., 2006}; ^{Aguirre-Medina et al., 2007} and ²⁰¹¹⁾, and with Gigaspora margarita in plants of C. canephora (^{Tristáo et al., 2006}). The number of sheets presents contrasting response between treatments at 28 ddt and individually inoculated A. brasilense was statistically different (p≤ 0.05) the rest ofthe treatments. In the following samples, the number of leaves was similar between treatments, including the witness, and the least amount of leaves was presented with the inoculation of the three microorganisms. In this regard there is evidence that the combination of more than one microorganisms not necessarily induce an additive or synergistic effect on the host plant (^{Trabelsi and Mhamdi, 2013}), as in our investigation.

Sampling in two and three the number of leaves of plants inoculated with a microorganism increased and was very similar between the two microorganisms coinoculaciones. It is likely that this time has been improved plant-microbe relationship. Between plants and bacteria there is also a complex and dynamic language for colonization, as their ability to use root exudates as carbon sources and their ability to interact and collaborate with other microorganisms in the rhizosphere (^{Holguin, 2008)}. In perennial greenhouse crops, plant microorganisms response thereofis expressed about 90 days after inoculation, as in Tabebuia donnell-smithii Rose (^{Aguirre-Medina et al., 2014}), in Cedrela odorata L. (^{Aguirre-Medina et al., 2014}) and Robusta coffea Coffea canephora (Pierre) ex Froehner (^{Ibarra-Puon et al, 2004}).

For the control treatment, the response is due to the presence of other microorganisms associated naturalized in their root system that favored its development. In this research, only counting the radical colonization was performed by the endomycorrhizal fungus and in the case of the witness, the initial radical colonization was 72% and finally 27%. Very similar values to those found in the treatments where was applied R. intraradices. These facts suggest the contrasting functionality coinoculaciones interacting with plants (^{Jáderlund et al, 2008}).

Dry weight of leaf blade, stem and root. The greatest accumulation of dry biomass of the leaf in plants inoculated was presented with A. brasilense, P. fluorescens and the combination R. intraradices + A. brasilense at 28 ddt, R. intraradices and A. brasilense inoculated separately 56 ddt, and at the end of the evaluation at 84 ddt, all treatments outperformed the treatment inoculated with the three microorganisms (Table 2). ^{Reyes-Ramírez et al. (2014)} found no difference in the leaf biomass habanero chile (Capsicum chinense Jacq.) inoculated with Pseudomonas spp., R. irregularis or A. brasilense. In perennial crops such as Leucaena leucocephala (Lam.) De Wit and Theobroma cacao L. coinoculated with Glomus-Rhizobium and Azospirillum microorganisms respectively, increases occur in these same yield components (^{Aguirre-Medina et al., 2007}).

Table 2 Dry weight of leaf blade, stem and root in plants Capsicum annnum L. biofertilizers with different microorganisms in soil andosol-mollic in Soconusco, Chiapas. 

CV= coeficiente de variación (%). * ± error estándar. *Valores con diferente letra dentro de cada factor y columna son estadísticamente diferentes (p≤ 0.05).

The slow initial growth in the treatments inoculated with two microorganisms, is likely related to the increased demand for photosynthates required by them during the establishment phase of symbiosis and consequently decreases its initial growth. In Tabebuia donnell-smithii Rose to evaluate various collections of endomycorrhizal fungi from different regions of Southeast Mexico, he found that induction in the growth of yield components was expressed at different times (^{Aguirre-Medina et al., 2014}).

The dry weight of main stem showed higher biomass in treatments inoculated with P. fluorescens at 28 and 84 ddt and symbiosis R. intraradices + A. brasilense at 56 ddt compared with inoculation of the three organisms and were statistically higher (p≤ 0.05) than the other treatments. With Pseudomonas spp. stem biomass habanero chile (C. chinense Jacq.) increased but not with the combination of endomycorrhizal fungus (^{Reyes-Ramírez et al., 2014}). In Coffea canephora (Pierre) ex Froehner, stem biomass also increased with inoculation separately from the same microorganisms (^{Aguirre-Medina et al., 2011}).

Most biomass accumulated in the root system was recorded with P. fluorescens and was statistically different (p≤ 0.005) to the other treatments and coincides with that reported by ^{Reyes-Ramírez et al. (2014)} in C. chinense Jacq. During the next sampling, this response was also presented with A. brasilense. The greatest growth in the root system may be related to the increased growth of some substances, the result of the symbiosis between the plant and microorganisms. A. brasilense and P. fluorescens promote root development by producing indole acetic acid (^{Patten and Glick, 2002}; ^{Hungria et al, 2004}; ^{Ahmad et al, 2006}; ^{Neetu et al, 2012}) amending root morphology and increase biomass.

Similarly, A. brasilense can induce greater root development in annual plants are inoculated togetherAzospirillum+Glomus in Phaseolus vulgaris L. and Zea mays L. (^{Dobbelaere et al., 2003}). P. fluorescens can stimulate proliferation of roots and has been linked to the promotion of plant growth (^{Gamalero et al., 2004}) and the possible removal of pathogenic microorganisms in the soil (^{Neetu et al., 2012}). The above results indicate that dry matter accumulation in the plant components of Capsicum annuum L., applied microorganism varies over time. At the beginning of the evaluation, the treatments inoculated with A. brasilense, P. fluorescens and double symbiosis R. intraradices+A. brasilense, they accumulated the highest amount of dry matter in different yield components, and this time most mycorrhizal colonization was reached in the combined treatment, which was higher than 90%.

With the individual record of the weight of the fruits were classified into three categories. Of total average fruit per plant, which weighed up to ten g represented 13.7% of total production. 41% of fruits weighed between five and 10 g and less than 5 g, 45.5% of them (Figure 1).

Figure 1 Number and fresh weight of fruits of Capsicum annuum L. biofertilizers with different microorganisms alone or combined in a soil Andosol mollic of Chiapas. The values are averages of five repetitions. 

The fruits with greater than 10 g weight was two on average almost in all treatments, except treatment symbiosis R. intraradices, P. fluorescens and the three microorganisms.

Weight in the range of five to ten g, the ratio between treatments was very similar, i.e., there were six to seven fruits. The greatest variation occurred in the fruits that weighed less than 5 g. In this regard, with the double symbiosis R. intraradices+ A. brasilense as many fruits of less than 5 g size was found, and represented approximately 50% of this surplus. This suggests the importance of inoculating a microorganism chili plants to encourage fruiting. If you enter more than one organism, it is likely that the demand for photosynthates by them, reduce the possibility that they are transported to the flowering and fruit set. It has been shown that P. fluorescens in the rhizosphere increased biomass and crop productivity (^{Sood, 2003}), as in the case of habanero (^{Reyes-Ramírez et al, 2014}).

Colonization radical. The root colonization of plants at 28 ddt, was high in all treatments where microorganisms (Figure 2) were applied. On average, the range of colonization was the order of 60% to 96%. In the case of control without application of microorganisms initial radical colonization was high, 72%, probably because endomycorrhizal fungi present in the substrate and it is common in species that have adapted to the environmental conditions of the region.

Figure 2 Radical colonization of Capsicum annuum L. biofertilizers with different microorganisms alone or combined in a soil adosol mollic of Chiapas. The values are averages of five replicates ± standard error for each sample. 

It was expected that the inoculation of Azospirillum and Pseudomonas bacteria favor increased root colonization, as has happened in other plants (^{Aguirre-Medina, 2006}); however, in the second and third sampling values of root colonization, when applied alone microorganisms, it was 60 to 68% and increased 80 to 86% from when included R. intraradices.

The lowest percentage of mycorrhizal colonization in treatments inoculated with bacteria due to the colonization of endomycorrhizal fungi present in the substrate. When R. intraradices+P. fluorescens, were applied together, also the percentage of colonization was low, on the order of 66% and with the two bacteria together, it was 29%. This indicates that not all microorganisms have the ability to associate to induce greater benefits to the host plant.

In the final evaluation, the percentages of root colonization by mycorrhizal fungi was 76% with R. intraradices, 69% with A. brasilense, 64% with P P. fluorescens and 73% with double symbiosis R. intraradices + A. brasilense and was these treatments where the greatest effect on increasing plant biomass, the thicker the stem diameter, number of leaves, flowers and fruit found. In the other co-inoculation treatments two microorganisms, root colonization was less than 53 and 29% for R. intraradices + P. fluorescens and A. brasilense + P. fluorescens and none of the evaluated variables showed significant or higher values others, not statistically significant differences.

The effects of microorganisms in plant development of plants have been documented (^{Barea et al., 2002}) and in other cases, the application of selected mycorrhizal fungi have favored the performance of various crops, when applied alone, or in combination with bacteria, such as P. fluorescens, which stimulates mycorrhizal colonization tomato and increases production (^{Gamalero et al., 2004}).

Conclusions

The morphological and physiological components chili plant present contrasting initial and final changes in interaction with various microorganisms. Individual inoculation of microorganisms Pseudomonas fluorescens, Azospirillum brasilense and co-inoculation of R. intraradices +A. brasilense increased the number of fruits. The co-inoculation of R. intraradices+P. fluorescens and individual inoculation of A. brasilense induced larger fruits