Soil sampling

In December 2012, were taken soil samples of 1 kg in a natural area (not cultivated) and soil for agricultural use. The sample was taken from the rhizosphere of cultivated tomatillo (rendidora variety) and wild tomatillo in the profile of 0-30 cm deep. In two locations of Arteaga, Coahuila, Mexico 25° 20' 56.35" north latitude - 100° 40' 45.84" west longitude and 25° 23' 23.63' north latitude - 100° 37' 33.97 west longitude respectively. Note that tomatillo has been grown for 10 consecutive years and regarding tomatillo from the natural system it has remained the same time without cultural practices.

Physicochemical analysis of soil

Two soil analysis were performed at the Universidad Autonoma Agraria Antonio Narro (25° 23' north latitude - 101° 02' west longitude) in the soil department, in order to determine the physicochemical conditions where tomatillo and wheat plants developed, organic matter was determined using the method of Walkley and Black ^{Bornemisza (1979)}, nitrogen (N) with Kjeldahl ^{Bremner (1960)}, phosphorus (P) ^{Olsen (1954)}, potassium (K) (atomic absorption spectrometry), pH (soil: water 2: 4) and texture (^{Bouyoucos, 1936}).

Monosporic culture

The spores from arbuscular mycorrhizal fungi were extracted from the soil samples through wet sieving (sieve. 50, 325 and 400µm) and decanting ^{Gerdemann et al. (1963)} followed by a gradient of 2M sucrose (^{Furlan et al., 1980}; ^{Horn et al., 1992}). A morphological clustering was performed considering shape, color and size (^{INVAM, 2014}). The obtained morphotypes were inoculated in Triticum aestivum L. (1 spore per plant / 10 reps) seedlings. The monosporic culture was established in pots of 1 liter capacity, and as substrate sterile sand, also to avoid contamination the pots were covered with plastic. The plants were kept in a greenhouse at an average temperature of 40 °C, ^{Steiner et al., (1973)} nutrient solution was applied, modified in phosphorus 20%, pH 7.5 - 8, watering daily. Soil samplings were performed monthly to evaluate the propagation of the inoculated spore with different morphotypes; in the fourth month, one wheat seed was placed per pot for a second crop cycle in order for the spores to multiply and establish symbiosis. In addition, the plants were subjected to water stress, completely suspending the irrigation in the last two months in order to accelerate this symbiosis.

Root colonization (Triticum aestivum L.)

In order to determine the degree of root colonization, an analysis was carried out in the eighth month of each of pot by morphotype. To assess successful symbiosis from morphotypes of arbuscular mycorrhizal fungi on wheat were clarified and stained with trypan blue wheat root fragments of 1cm length from the different morphotypes following technique Clarification-staining roots and estimating root colonization by mycorrhizal -arbuscular fungi (^{Walker et al., 2005}). Finally, the root fragments were mounted vertically on slides (3 slides per plant / 50 observations) adding a drop of fresh acidified glycerol for observation under microscope to estimate colonization (^{McGonigle et al., 1990}).

Morphological analysis

The morphological characteristics of spores were described from spores mounted in a mixture (1: 1) reagent PVLG and Melzer (^{Brundrett et al., 1994}). For color and shape was taken as reference the international collection of HMA (^{INVAM, 2014}). The pictures were taken in light microscope (Axio Scope A1, Carl Zeiss Microscopy GmbH, Gottingen, Germany) with integrated camera Axio Cam Icc 1 10X objective, measurements were performed using the software Axion Vision Release 4.8.2.

Statistical analysis

For data processing Statistical Analisys Systems (SAS) 2013 was used. An analysis of variance (ANOVA) and mean comparison Tukey (≤ 0.05) was conducted.

Physicochemical analysis of soil

Table 1 shows in detail the results of physicochemical analysis where tomatillo plants were grown under agricultural systems (TC) and natural system (TS). It is noted that tomatillo plants of both systems grew in alkaline soils, soil texture clay and sandy clay loam respectively; organic matter content (MO), nitrogen (N), phosphorus (P) and potassium is higher in soil for agricultural use (TC) compared to a natural soil system (TS). Long-term studies have shown that fertilization cause significant impact on arbuscular mycorrhizal fungi communities both in species diversity and its functioning (^{Thomson et al., 1992}; ^{Kahiluoto et al., 2009}).

Table 1 Physicochemical characteristics of soil where plants tomatillo developed under two different systems. 

* TC= tomatillo cultivado; TS= tomatillo silvestre.

Characteristics from arbuscular mycorrhizal fungi differ, because studies have shown that some species form more than one spore morphotype ^{Stockinger et al. (2009)}; however, the current characterization performed in this study, indicates the types of arbuscular mycorrhizal fungi present in soil and their abundance. This likewise allows isolation to study more deeply the body belongs.

Morphological analysis

The morphological diversity of spores from arbuscular mycorrhizal fungi that were associated to tomatillo rhizosphere from wild soil (TS) was higher compared to cultivated tomatillo (TC) (Figure 1). It has been shown in several studies that agricultural systems are exposed to intense microbial selection pressures due to short cycles and crop management, therefore sporulation of arbuscular mycorrhizal fungi is affected by agriculture, being the opposite in natural systems ^{Burdon and Thrall (2008)}; ^{Oehl et al. (2003)}, which is consistent with the results obtained in this study.

Figure 1 Morphological diversity of arbuscular mycorrhizal fungi associated with rhizosphere of wild tomatillo (TS) and tomatillo cultivated (TC) (objective 3 X). 

According to the morphological characteristics of the spores associated to the rhizosphere of cultivated tomatillo (rendidora variety) nine morphotypes (Table 2) were grouped.

Table 2 Morphological characteristics of spores of arbuscular mycorrhizal fungi associated to rhizosphere grown tomatillo (Physalis ixocarpa cv. Rendidora). 

*0-20-60-0= red- brown, 0-50-60-0=reddish-orange, 20-80-100-0= red- brown (^{INVAM, 2014}). DE= diámetro ecuatorial; DM= Diámetro meridional. Promedios con la misma letra indican diferencia no significativa (Tukey´s 0.05).

Means comparison test Tukey (≤ 0.05) indicates that the C7 and C4 morphotypes have greater equatorial and meridional diameter with 202 µm - 190 µm; 184 µm - 188μm respectively; while morphotype C6 has smaller diameter 100 µm - 117 µm, certain spore colors are 0-50-60-0 (reddish-orange), 20-80-100-0 (red- brown) and 0- 20-60-0 (red- brown) being this the most frequently observed, most morphotypes showed a globular - subglobular shape.

Table 3 shows the grouping of 12 morphotypes associated with rhizosphere of wild tomatillo according to the morphological characteristics of the spore. Means comparison test Tukey (≤ 0.05) indicates that morphotype S9 presents higher equatorial and meridional diameter with 249 μm and 238 μm- while morphotype S7 presents diameters of 95 μm - 104 μm considered as morphotype with the smallest size; determined spore colors are: 20-80-100-0 (red-brown), 0-1090-5 (orange), 0-60-100-10 (orange brown) and 0-50-60-0 (reddish-orange) observed more frequently at 0-60-100-10 (orange brown), the shape of all morphotypes is globular or subglobular.

Table 3 Morphological arbuscular mycorrhizal fungi spores associated with rhizosphere of wild tomatillo Features. 

*20-80-100-0=red-brown, 0-10-90-5=orange, 0-60-100-10=orange brown, 0-10-60-0=pale orange-yellow, 0-50-60-0=reddish-orange, 0-20-60-0=red brown (^{INVAM, 2014}). Promedios con la misma letra indican diferencia no significativa (Tukey p ≤ 0.05).

In this study it was determined that there is greater morphological diversity of arbuscular mycorrhizal fungi spores associated with the rhizosphere of wild tomatillo with 12 morphotypes compared to nine morphotypes of cultivated tomatillo, it is important to note that tomatillo plants from the two systems have been grown on this site during 10 years. There have been recent publications ^{Oehl et al. (2009)} showing that in cultivated land with crop rotation and presence of weeds or perennial grasses (clover) were more rich in species of arbuscular mycorrhizal fungi, contrary to that happening in crops consecutively planted (monoculture) and even richer than natural grasslands, so it is clear that the results of morphological diversity of spores of arbuscular mycorrhizal fungi are lower in cultivated tomatillo because it is an intensive agricultural system, therefore the presence of other hosts (weeds) is zero or minimum for very short periods that there is no opportunity for more mycorrhizal morphotypes to express and vice versa in the natural wild tomatillo system.

Long-term studies have shown the impact of fertilization on arbuscular mycorrhizal fungi communities, reporting negative impacts on the composition and functionality of the different species (^{Thomson et al., 1992}; ^{Kahiluoto et al., 2009}). It has been found that the addition of nitrogen fertilizers can alter the functional diversity of arbuscular mycorrhizae, besides, performing high nitrogen fertilization in soils rich in phosphorus decrease the richness and diversity of the communities of arbuscular mycorrhizal fungi (^{Egerton and Warburton et al., 2007}). From the latter can be understood that wild tomatillo had higher spore diversity of mycorrhizal due to nitrogen limitations presenting 0.16% rated according to the physicochemical analysis as mid-soil compared to cultivated tomatillo (rendidora variety) with 0.22% considered moderately rich.

Organic matter and pH are parameters that influence on the composition of arbuscular mycorrhizal fungi; studies made in Colombia found high diversity and abundance of arbuscular mycorrhizal fungi in acid soils (^{Seralde and Ramirez, 2004}).The real relationship whether strong or weak (^{Hijri et al., 2006}) between soil properties and distribution of arbuscular mycorrhizal fungi species under field conditions is little known (^{Carvalho et al., 2003}).

Morphological analysis of mycorrhizal isolated in wheat (Triticum aestivum L.)

The resulting spore from isolates have a globular- sub globular shape, the equatorial diameter (DE) of isolates from wild and cultivated system range from 108 μm - 170 μm and meridional diameter (DM) from 112 μm- 196 μm, the thickness of its spore wall (EPE) is 7 μm - 12.8 μm, the spore colors of the different isolates were: 0-60-100-10 (orange-brown), 0-10-60-0 (pale orange-yellow), 0-10-90-5 (orange), 0-0-40-0 (pale yellow), 0-50-60-0 (reddish-orange), 0-20-60-0 (red-brown), the number of layers of the spore is 1 on its majority except morphotype S1 which had two spore layers, all morphotypes has gallbladder and arbuscule (Table 4).

Table 4 Morphological characteristics of isolates of arbuscular mycorrhizal fungi associated with rhizosphere of wheat (Triticum aestivum L.). 

*0-50-60-0= reddish-orange, 0-20-60-0= red-brown, 0-10-90-5= orange, 0-60-100-10= orange- brown, 0-10-60-0= pale orange-yellow, 0-040-0= pale yellow (^{INVAM, 2014}). Promedios con la misma letra indican diferencia no significativa (Tukey p≤ 0.05).

According to statistical analysis in equatorial diameter the S9 isolate is significantly different with a mean 183a according to the mean group (Tukey p≤ 0.05) followed by S10 with a mean of 170ba; regarding to the meridional diameter isolated S4 showed significant difference wotj a mean of 196a followed by S10 with a mean of 173ba. Isolate S7 showed the lowest diameter both equatorial and meridional averaging 108d-112e respectively. The thickness of the spore wall according to statistical analysis and means comparison test (Tukey p≤ 0.05) isolate S4 and S10 are not statically different but numerically their diameter differs being larger S10 with an average of 12.8a followed by S4 with 12.6a, isolate S7 shows less thickness in spore wall averaging 7c.

It is appreciated that morphological characteristics from isolates differ from its natural system both cultivated and wild, as previously mentioned some species form more than one spore morphotype ^{Stockinger et al. (2009)}. Recently ^{Ehinger et al. (2009)} studied individuals genetically different of G. intraradices isolated from the same field and found that isolates showed different growth strategies depending on the host and different levels of phosphorus, this means that the availability of phosphate affects the genotype of a forming arbuscular micorrhizal fungi isolated through multiple generations.

Similar dynamics have been found by ^{Oliveira et al. (2010)} working with isolates of Glomus geosporum during a year in two soils with different pH (acid-alkaline), these studies have proven that lineages resulting from arbuscular mycorrhizal fungi share a third of their genetic markers which means that when grown under the same conditions, mycelium density and spores density differ significantly. Situation that could be different in morphological characters from isolates of native mycorrhizal since its natural environment was modified subjecting them to Steiner nutrient solution modified to 20% in phosphorus and pH of 7.5- 8 and in substrate sand type under controlled conditions.

Inoculum potential of isolated mycorrhizal on wheat (Triticum aestivum L.)

From the 21 morphotypes that were obtained from wild system and 12 from cultivated, not all established successfull simbiosis in wheat plants (Figure 2). Of which 10 morphotypes belong to the wild system (S1, S2, S3, S4, S5, S6, S7, S9, S10 and S11) and 2 (C2 and C5) to cultivated system.

Figure 2 Colonization of mycorrhizal fungi isolated in root wheat (Triticum aestivum L.) 8 months after inoculation. 

According to statistical analysis, isolate S1 is significantly different with 57.81 according to grouping mean (Tukey p≤ 0.05); followed by C2 with an average of 47.2ba, isolate S11 was who set lower symbiosis in wheat root with an average of 27.16c. Isolates C1, C2, C3, C4, C6, C7, C8 and C9 from the cultivated system and S8, S12 from the wild system did not establish symbiosis in wheat plants so they are not considered as potential inoculum in this research. In this study it can be said that shape, diameter and wall thickness characteristics are not indicators of mycorrhizal fungi with inoculum potential. It has been shown that there is a strict specificity between host and arbuscular mycorrhizal fungi, the result of the interactions dependent on its host and environmental conditions (^{Walder et al., 2012}; ^{Smith and Smith, 2015}). Wheat is a crop that does not depend heavily on the association with arbuscular mycorrhizal fungi to obtain nutrients, but there are studies showing that wheat plants benefit in establishing symbiosis (^{Manske, 1990}; ^{Tawaraya, 2003}).

Figure 3 shows morphological characteristics from isolates that established successful symbiosis in wheat (Triticum aestivum L.), which are considered as potential inoculum.

Figure 3 Morphological characters isolated arbuscular mycorrhizal fungi inoculum potential in wheat. S1A, S1B, S1C, S2A, S2B, S2C, S3A, S3B, S3C,S4A, S4B, S4C, S5A, S5B, S5C, S6A, S6B, S6C, S7A, S7B, S7C, S9A, S9B, S9C, S10A, S10B, S10C, S11A, S11B and S11C are wild morphotypes system; C2A, C2B, C2C and C5a, C5B, C5C are cultivated morphotypes of the system where (A) spore complete; (B) fractional spore; and (C) in root colonization respectively.