Introduction

The rhizosphere may be defined as that portion the soil zone that surrounds and is influenced by the roots of plants (Paul and Clark 1996). The adoption of different land use management can have negative or positive effects on the rhizosphere soil microbiota (Barrios 2007, Notaro et al. 2008). The number of Colony Forming Units (CFUs) of bacteria, fungi, and actinobacteria in the rhizosphere may largely determine microsite occupancy, depending of the distances from the root surface (Kent and Triplett 2002).

The major cause of changes in forest use in Villavicencio is land clearing for monoculture, mainly rice monoculture, than according to Delgado et al. (2009) influenced soil microbiota. Several research have examined the effects of soil management practices on number of CFUs of rhizosphere soil microbiota in different land use type (Balota et al. 2003, Bausenwein et al. 2008, Jangid et al. 2008, Ceja-Navarro et al. 2010, Prevost-Boure et al. 2011); monoculture is one of the most significant anthropogenic activities that alter soil biological properties and processes (Brussaard et al. 2007, Jangid et al. 2008) due to that conventional tillage can affect above ground residues content (Alvear et al. 2005, Roger-Estrade et al. 2010, Mathew et al. 2012).

Agroforestry systems appear to be a good alternative as land use patterns for increase soil microbiota in the humid tropics, due to high litter production and increases in soil organic matter, the large root system of trees in agroforestry systems potentially accumulates nutrients from a large volume of soil, whereas fallen litter concentrates nutrients near the soil surface (Reis et al. 2007, Notaro et al. 2008). The return of residue to the field in agroforestry systems is a useful cultural practice to improve both soil fertility and soil organic carbohydrate storage ensuring the survival of soil microorganisms (Barreto et al. 2008, Notaro et al. 2008, Paudel et al. 2012).

Consequently, fungi, bacteria and actinobacteria are the most sensitive and rapid indicators of soil perturbations due to land use changes. In this sense, a quantitative description of soil microbial population has great interest as a potential tool for soil quality evaluation (Brussaard et al. 2007).

We hypothesized that soil management practices in different land use patterns would result in changes on rhizosphere soil microbiota, that can provide important information for sustainable production systems in tropical regions of Colombia.

In this context, the aims of this research was to estimate the effect of different land use patterns: Secondary forest (T1), rice monoculture (T2), pineapple monoculture (T3) and agroforestry system of Theobroma cacao associated with Acacia mangium (T4), with different soil management factors and at different distances from the root surface on number of Colony Forming Units (CFUs) of soil rhizosphere fungi, bacteria and actinobacteria in an Oxisol of Villavicencio, Orinoquia, Colombia East.

Materials and methods

The study site is located between the coordinate 4°04’47’’ N and 73°35’17’’W. The experiment was conducted on an Oxisol in a humid tropics zone of Villavicencio, Orinoquia, Colombia East, specifically in Barcelona farm, Unillanos University (Figure 1). The temperature is from 28 to 30ºC with long dry season (Igac 2004) and average annual rainfall of 3,663 mm. Average altitude of 525 m. a.s.l. Relative humidity of 65 %.

The work was developed in an unrestricted random design. The experimental design consisted of four treatments of land use patterns (Figure 2): Secondary forest system (T1): This area contained one ha, this system had been maintained for nearly 10 years and although there had been no management, high influence of above ground residue input. Rice monoculture (T2): This area contained 0.5 ha, with more than 5 years grown, conventional tillage was conducted by disking (0–20 cm) after harvest, followed by chiselplowing (0–20 cm), a second disking, and ridging,

reduced inputs from above-ground residues. Pineapple monoculture (T3): This area corresponds to a small area of investigation, which contained < 0.5 ha, with 1 year grown. Conventional tillage, reduced inputs from above ground residues. Agroforestry system of Theobroma cacao associated with Acacia mangium (T4): This area contained one ha, with 5 years grown, the area was managed with reduced tillage, and it was made only loosening the floor to make holes for planting of the trees, with high influence of above ground residue input.

All systems exhibit the same agroecological conditions (Figure 2).

Three soil samples from each study system were collected in dry season along a transect at regular intervals. Soil sampling was done up to a depth of 20 cm and were collected from five different points (distances from the root surface), 0-10 cm (D1), 10- 20 cm (D2), 20-30 cm (D3), 30-40 cm (D4), 40-50 cm (D5). A composite sample for each depth was made by thoroughly mixing three samples in a given site and triplicate of subsamples were considered for further analysis.

A total of 60 samples (4 systems × 5 distances from the root surface x 3 replicates) were analysed.

The samples underwent serial dilution technique and pour plate method to detect the number of CFUs of bacteria, fungi and actinobacteria (Alexander 1982). Ten grams of soil were shaken with 90 ml of distilled water. From this suspension, the serial solution was prepared. An amount of 0.5 ml of the serially diluted solution was plated on agar by the pour plate technique (Alexander 1982). Three main types of agar were used. Nutrient agar (NA), potato dextrose agar (PDA) and starch ammonia (SA) medium were used for enumeration of bacteria, fungi and actinobacteria respectively. The results are expressed as Colonial Forming Units CFUs g^-1, for fungi x 10³, bacteria x 10⁶, actinobacteria x 104 (Alexander 1982).

The plates for fungus and actinobacteria are incubated at 25–30ºC, for bacteria at 37ºC, and the populations of bacteria, fungi and actinobacteria were observed at 2, 5 and 7 days after inoculation, respectively.

Was used analysis of variance and significant differences were analyzed using multiple comparison of means of LSD by test of Fischer ranges (P ≤ 0.05) applying InfoStat statistical software 2010 version (Di Rienzo et al. 2010).

Results and discussion

The influence of land use patterns and distances from the root surface on the total soil fungi, bacteria and actinobacteria counts are presented in Figure 3 and Table 1. Figure 3 and Table 1 showed that population of bacteria, fungi and actinobacteria were decreased as distances from the root surface increased in all land use patterns. Figure 3 also showed that soil microbial population in the rice monoculture T2 and pineapple monoculture T3 were negatively affected. Following the description of the results by type of soil microorganism investigated.

Land use types: 1T1= secondary forest; 2T2=rice monoculture; 3T3= pineapple monoculture and 4T4= agroforestry system of Theobroma cacao associated with Acacia mangium. Distances from the root surface: ⁵D1: 0-10 cm, ⁶D2: 10-20 cm, ⁷D3: 20-30 cm, ⁸D4: 30-40 cm, ⁹D5: 40-50 cm of each land use types. In column, mean followed by common letters do no differ significantly at 5% level by LSD.

Fungal population

High rates of fungal number of CFUs are related to secondary forest T1 at 0-10 cm from the root surface D1 (49.23×10³ CFU g^-1 soil), compared to pineapple monoculture T3 at 40-50 cm from the root surface D5 (0.61 ×10³ CFU g-1 soil) (P≤0.05) (Table 1, Figure 3). Fungal population are relatively more abundant in forest than agricultural soils as monocultures (Lauber et al. 2013), because carbon products secreted by trees roots, which are preferential nutrients and energy for fungal metabolism.

Secondary forest T1 at 10-20 cm from the root surface D2 is associated with moderate increases of the CFUs of fungi (32.1 ×10³ CFU g^-1 soil), which was equal to secondary forest T1 at 20-30 cm from the root surface D3 (28.3 ×10³ CFU g^-1 soil), indicating that there is a range of best action of fungal populations up to 30 cm (Table 1, Figure 3). The reduction in fungal population may be explained by secondary forest T1at 30-40 cm from the root surface D4 at 40-50 cm from the root surface D5 (13.5, 13.8 ×10³ CFU g^-1 soil) (P≥0.05) (Table 1, Figure 3). The stimulatory effect on microorganisms is known as the “rhizosphere effect” as indicated by the interaction of soil and rhizosphere microbes and their ratio (Paul and Clark 1996).

This indicates that secondary forest T1, affected positively fungal population in the area closest to the roots directly by generating inputs of organic matter above and belowground and indirectly by the physical effects of shading, soil protection and uptake of water and nutrients by roots (Smith 2008, Lauber et al. 2013, Voříšková and Baldrian 2013). Forest from natural regeneration may show higher resistance to microclimatic site conditions, which can also contribute to their growth and litter yields, which favors increases in soil microbiota (Notaro et al. 2008). The microbial community in forest soil is dominated by fungi. Fungi are more efficient than bacteria, in terms of decomposing organic compounds (Sylvia et al. 2005).

Rice monoculture T2 and pineapple monoculture T3 exhibiting both conventional tillage and low inputs from residues, as the distances increased of D1 to D5 from the root surface had detrimental effects on fungal number of the CFUs accounting 9.7, 8.7, 5.7, 4.2, 3.9 ×10³ CFU g^-1 soil and 4.2, 2.2, 1.0, 0.8 and 0.6 ×10³ CFU g^-1 soil respectively (P≥0.05) (Table 1, Figure 3).

In a study of an Oxisol of the Eastern Plains of Colombia, a leguminous cover with C. Ochroleuca in rice and corn crops increased by 86 % the greater diversity of fungi, mainly Aspergillus, Cladosporium, Trichoderma and Penicillium (Delgado et al. 2009), which have been reported as phosphate solubilizing microorganisms (Perez et al. 2005), of great importance in phosphorus deficient soils in tropical zones.

Low increment of fungal number of CUFs for pineapple monoculture T3 at 40-50 cm from the root surface D5 are due to that monoculture cropping systems and intensive soil management can markedly affect the activity of soil microorganisms and their diversity (Balota et al. 2003), around 89 % decline in microbial diversity index is observed from the distance closest to the rhizosphere to the most distanced (Kent and Triplett 2002).

Contrary to what is stated, other authors say that plant roots do not alter and enhance the total count of the CFUs of fungi in the rhizosphere, in contrast to their effects on bacteria (Whipps 2001, Kent and Triplett 2002).

The chemical and physical nature of the root zone is quite different from the soil away from the root zone, being that fungi population are more likely to regulate decomposition of surface-applied residues in no-till soils in the area adjacent to the root (Paul and Clark 1996, Beare 1997).

The rhizosphere is also a metabolically busier and more competitive environment than the surrounding soil, also known as bulk soil. Unlike the soil associated with the rhizosphere, bulk soil is not penetrated by plant roots and typically have lower microbial communities within it (Kent and Triplett 2002), corroborating this result.

The management of agroforestry system of Theobroma cacao associated with Acacia mangium T4 may provide advantages for soil microbiota, due to that all distances from the root surface, which steadily increased from D5 to D1 from the root surface accounting for 16.6, 17.3, 18.3, 19.6 and 19.6 ×10³ CFU g^-1 soil (Table 1, Figure 3), was significant similar that the number de CFUs of fungi in secondary forest T1 at D4 and D5 distances from the root surface (13.5, 13.8 ×10³ CFU g^-1) (P>0.05), respectively (Table 1, Figure 3). According to Wagg et al. (2014), the function and structure of agroforestry systems may have a similar biological performance that secondary forest. Similarly, Beare (1997) states that this behavior is characteristic of microbial populations in both mature ecosystems and those in process of succession.

Acacia mangium is a leguminous plant that can to be use associated with Theobroma cacao in tropical zones, since it contributes a great amount of litter to the soil, most of the nitrogen is recycled into the soil (Beare 1997).

It is found that surface residues in agroforestry systems retain moisture, dampen temperature fluctuations, and provide a continuous substrate that promotes fungal growth (Beare 1997); trees roots in agroforestry systems exude amino acids and sugars that serve as a food source for microorganisms, since fungi population are poor degraders of cellulose and other plant wall material (Pii et al. 2016), and this is the reason for the high increments of CFUs of fungi.

Bacterial population

A greater rhizosphere effect was observed in bacteria than actinobacteria and fungi in all land use patterns (Figure 3), due to those bacteria known as plant growth promoting rhizobacteria (PGPR) colonize roots very efficiently (Kent and Triplett 2002, Sylvia et al. 2005).

The population of bacteria ranged from 286.33 to 4.99 ×10⁶ CFU g^-1 soil corresponding to secondary forest T1 at 0-10 cm from the root surface D1 and pineapple monoculture T3 at 40-50 cm from the root surface D5, respectively (P≤0.05) (Table 1, Figure 3). This last treatment showed similar amount to the average of the values reported by Muhammed et al. (2015) for humid tropical climate in Indian for an okra monoculture at fruiting (6.3 ×10⁶ CFU g^-1 soil).

Higher count of bacteria was observed in secondary forest T1 and all the distances from the root surface compared to others treatments (p≤0.05), the number of CFUs of bacteria no reflected a positive response of the increases of the distances of D1 to D5 from the root surface accounting for 286.3, 231.3, 209.3, 171.0 and 164.6 ×10⁶ CFU g^-1 soil respectively (Table 1, Figure 3).

The bacterial population cover about 4-10 % of the total root area occurring profusely on the root hair region and rarely in the root tips. There is predominance of amino acids and growth factors required by bacteria, are readily provided by the root exudates in the region of rhizosphere (Kent and Triplett 2002). The most common genera of soil bacterias are: Pseudomonas, Arthrobacter, Agrobacterium, Alcaligenes, Azotobacter, Mycobacterium, Flavobacter, Cellulomonas, Micrococcus, and others have been reported to be either abundant or sparse in the rhizosphere (Whipps 2001).

According to Whipps (2001), the aerobic bacteria are relatively less in the rhizosphere because of the reduced oxygen levels due to root respiration.

Other authors as Kent and Triplett (2002) argue that in addition to the respiration of the roots themselves, the carbon-rich environment of the rhizosphere promotes high levels of respiration by other macro and microorganisms, to a far greater extent than what is occurring in the bulk soil.

In contrast, secondary forest T1 at 30-40 cm from the root surface D4 and at 40-50 cm from the root surface D5 (171.0 and 164.6 ×10⁶ CFU g^-1 soil) was equal that agroforestry system T4 at 0-10 cm from the root surface (155.3 ×10⁶ CFU g^-1) and at 40-50 cm from the root surface D5 (133.6 ×10⁶ CFU g^-1 soil) (Table 1, Figure 3).

According Da C Jesus et al. (2009) agroforestry communities are related to intermediate values of the soil biology attributes and crop and young secondary forest communities do not constitute well-defined groups.

The values of counts of bacterial UCFs in rice monoculture T2 at all distances are slightly below average for secondary forests (T1) accounting for 61.6, 56.0, 48.6, 45.6 and 37.0 ×10⁶ CFU g^-1 soil, being statistically differences (P≤0.05) (Table 1, Figure 3) and above average for pineapple monoculture (T3) at all distances, but with no statistically significant differences between rice monoculture T2 at 40-50 cm from the root surface D5 and pineapple monoculture T3 at 10-20 cm from the root surface D5 (P≥0.05) (Table 1, Figure 3) (P≤0.05).

According to Beare (1997) plant residue is distributed throughout the plough layer in fields managed with conventional tillage, these soils are dominated by bacteria and their predators such as bacterial-feeding nematodes and are considered to be in an early stage of succession, may possibly explain this response obtained. Gopalakrishnan et al. (2009) also reported the inhibitory effect of root exudates on bacteria.

Bacterial populations have their lowest value when pineapple monoculture T3 at 40-50 cm distance maximal from the root surface D5 accounting for 4.9 ×10⁶ CFU g^-1 soil (Table 1, Figure 3). On the other hand, pineapple monoculture (T3) at all distances from the root surface presented the least count of CFUs of bacteria with no statistically significant differences between these (P≥0.05) responsible for 13.0, 9.3, 8.0, 7.1 and 4.9 ×10⁶ CFU g^-1 soil (Table 1, Figure 3).

The magnitude of these responses indicates the need to intensify the studies involving monoculture management and its relation with soil microbiota bioactivity and mechanisms of interaction with root surface.

Actinobacteria population

The population of actinobacteria ranged from 97.33 ×10⁴ CFU g^-1 soil to 3.00 ×10⁴ soil CFU g^-1 corresponding to secondary forest T1 at 0-10 cm from the root surface and pineapple crop T3 at 40- 50 cm from the root surface D5 revealed statistically significant differences (P≤0.05) (Table 1, Figure 3).

Higher actinobacteria population was recorded for T1 at 0-10 cm from the root surface D1 (97.3 ×104 CFU g-1 soil) followed by T4 at D1 (94.0 ×10⁴ CFU g^-1 soil) and T1 at D2 (78.0 ×10⁴ CFU g^-1 soil), in turn this treatment equaled to T4 at D3 (69.6 ×10⁴ CFU g^-1 soil), being that secondary forest T1 and agroforestry system T4 by effect of all distances from the root surface showed similarity in actinobacteria population (P≥0.05) (Table 1, Figure 3).

Soil community composition with agroforestry systems resembles that of natural ecosystem soils more closely than in the case with annual crops (Brussaard et al. 2007). Among different microbial population, the bacterial population was recorded maximum (64 %), followed by actinobacteria (23 %) and fungi (13 %) in agroforestry systems (Radhakrishnan and Varadharajan 2016).

The populations of soil microbiota found in the Leucaena plantation were generally similar to the Atlantic forest and higher than in the Corymbia plantation (Reis et al. 2007).

Stimulation of actinobacteria in the rhizosphere has not been studied in much detail so far. It is generally understood that the actinobacteria are less stimulated in the rhizosphere than bacteria. However, when antagonistic actinobacteria increase in number they suppress bacteria (Wagg et al. 2014).

The actinobacteria population was reduced drastically by effect of rice monoculture T2 and pineapple monoculture T3 at all the distances from the root surface accounting for 28.3, 22.6, 18.6, 13.6, 11.3 and 12.0, 8.0, 7.6, 6.3, 3.0 ×10⁴ CFU g^-1 soil respectively, revealed no statistically significant differences between these (P≥0.05) (Table 1, Figure 3). The root endosphere microbiota results from gradual community shifts including enrichment and depletion processes from the surrounding soil microbiota (Sylvia et al. 2005; Barreto et al. 2008).

As rice and pineapple monocultures and soil management practices influenced soil actinobacteria populations at all distances from the root surface, it is important to propitiate the effect of soil microbiota enrichment process begin acting at a distance in the rhizosphere, continues at the rhizoplane, and is largely driven by root exudation, through efficient practices of soil management such as direct seeding and crop rotation. Difference in microbial population is a reflection of many factors such as nutrient and oxygen levels, temperature and availability of minerals (Sylvia et al. 2005).

Conclusions

The positive relation of fungi, bacteria and actinobacteria organisms with secondary forest (T1) at all distances from the root surface, is important for the establishment of the natural vegetation in tropical zones of Villavicencio, East Colombia.

The negative impact of rice monoculture (T2) and pineapple monoculture (T3) at all distances from the root surface on Oxisol microbiota disturbance, suggest that it should be taken in consideration for more sustainable soil management factors that contribute great amount of soil organic matter in monocultures systems.

The soil microbiota comparison in land use patterns and distances from the root surface revealed that agroforestry system of Teobroma cacao associated with Acacia mangium (T4) have potential to increase fungi, bacteria and actinobacteria in a tropical Oxisol of Villavicencio.

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