Microbiological material

Phytophthora capsici strain (PC): the strain CH11 provided by the Plant Pathology Laboratory of the IIAF- UMSNH was used. This strain was isolated from diseased plants wilting from a culture of pepper in the State of Michoacan, Mexico. The infectivity of the CH11 strain previously revived in plants serrano pepper, placing discs mycelium in the root system of plants causing infection and development of necrotic lesions, where he was re- isolated and purified in culture medium potato dextrose agar (PDA).

Inocula HMA: seven mycorrhizal consortia from soils with plantations of Agave cupreata the State of Michoacan, which were given a nomenclature according to the site which were used were extracted: El Huizachal (EH), Las Campesinas (LC), Rancho Carlos Rojas (RCR), Paso Ancho (PA), El limón (EL), Cerro del Metate (CM), Barranca de las Nueces (BN). The inocula previously were propagated for 12 months in pots in the greenhouse trap. Consortia were composed of different species of HMA, finding a total of 40 species in 13 genera, 9 families and 3 orders (^{Rincon-Enriquez et al., 2012}; ^{Trinidad-Cruz, 2014}).

Vegetal material

Pepper varieties: the pepper varieties used in this work were: serrano pepper (cultivar Tampiqueña) and poblano pepper (cultivar San Luis). The seeds were sown in aluminum trays with wells containing 100 sterile river sand (120 °C for three h, three consecutive days). The plants were transplanted when they were four true leaves approximately 35 days after planting.

Establishment of the experiment

HMA and inoculation of substrate: the substrate containing the pots consisted of a mixture of clay loam soil with sand in the ratio 1:1 v/v sterilized (120 °C for three hours three consecutive days). The plants were transplanted to pots with 500 g substrate, then inoculated into the root system with 80 spores HMA contained in a given amount of sand as carrier material. Technique wet sieving and decanting of ^{Gerdemann and Nicholson (1963)} was used for extraction, identification and standardization of the number of spores of each inoculum in combination with the flotation technique sugar sucrose gradient (Walker, 1997). For counting a stereomicroscope it was used (VELAB).

Inoculation of PC: at 75 days after inoculation with HMA (DDI), at which time the plants had higher mycorrhizal colonization 50%, were inoculated with a zoospore suspension of Phytophthora capsici adjusted to a concentration of 1 x 10⁴ zoospores ml^-1 (^{Ristaino, 1990}), using 1 mL per plant 500 g^-1 substrate (20 zoospores g^-1 substrate).

Response variables

Growth variables: to determine the effect of mycorrhizae on growth of pepper plants at 56 DDI, plant height (AP) was measured starting from the stem base to the apex of the last sheet (ruler), stem diameter (DT) storey (digital vernier), and the number of sheets (NH). A 95 DDI was terminated the experiment and proceeded to record total fresh biomass (root, stem, leaves and fruit) with an analytical balance (Mettler Toledo AT200).

Micorrización variables: the 56 DDI substrate samples and roots of each treatment were obtained to quantify the production of spores of HMA and mycorrhizal root colonization. Evaluating the number of spores it was made by wet sieving technique and decanting (^{Gerdemann and Nicholson, 1963}) in combination with sugar flotation technique sucrose gradient (Walker, 1997). For counting a stereomicroscope (VELAB) it was used. Mycorrhizal colonization was determined by the technique described by Hayman and ^{Phillips (1970)} and using trypan blue was used to determine the percentage of colonization by the method described ^{McGonigle et al. (1990)}.

Disease severity: the 20 days after inoculation of PC (95 DDI), was terminated the experiment and the level of damage was determined on the ground by recording the symptomatology of the disease according to the scale of virulence wilt in pepper plants described by ^{Quinto-Alvarez (2014)}. This scale includes six zero values: 0= healthy plant; 1= plant with leaves partially collapsed; 2= plant with leaves collapsed and some detached, slightly inclined stem; 3= plant with leaves and chlorotic collapsed, with some detached stem slightly inclined; 4= plants with leaves collapsed, most of them detached, collapsed stems and necrosis at the base; 5= plants completely collapsed leaves, almost all detached, collapsed and presence of necrosis from the base to the middle of the stalk stalks; and 6= plants completely collapsed leaves detached most part, collapsed and presence of necrosis in more than 50% of the stem stems.

Experimental design and statistical analysis

Two experiments (one for each variety of pepper) under an experimental design of randomized complete block with four replications were made. In each experiment nine treatments for seven consortia HMA native rhizosphere Agave, a positive control which consisted of an inoculum of commercial HMA made from Rhizophagus intraradices and a negative control without mycorrhizal (S/HMA) were evaluated. The experimental units (UE) consisted of a potted plant chili with their respective treatment. A data obtained from the growth variables and mycorrhizae, was applied an analysis of variance and to determine statistical differences between treatments a comparison test of Tukey (p≤ 0.05) was performed. For fresh air biomass variable, data were transformed with the exponential function to meet the normality and homoscedasticity of variances. For the scale of virulence of the disease, non-parametric analysis with the Kruskal-Wallis and confidence interval for the median with the Dunn test was performed (p≤ 0.05). The Pearson correlation coefficients were calculated (p≤ 0.05) between mycorrhizal colonization number of spores of HMA and complete with the level of virulence of the disease fresh biomass. Statistical analyzes were performed with StatGraphics Centurion program (^{Statgraphics, 2005}).

The HMA as plant growth promoters in pepper

Analysis of variance showed significant differences (p≤ 0.05) for all variables in serrano pepper growth (Table 1). In AP (p= 0.0206) inoculum CM showed the highest values with 23.0 cm; DT (p= 0.012) with 4.05 mm and NH (p= 0.0003) with 33.5 leaves. In all of these variables CM proved to be statistically different from control without HMA where the lowest values were recorded. For poblano pepper, no significant differences in DT (p= 0.71) and NH (p= 0.63), only to AP, where the inoculum LC obtained 18.03 cm were found. These results agree with those reported by ^{Alonso-Contreras et al. (2013)}, where different mycorrhizal consortia isolated from the rhizosphere of apple (Malus domestica B.) had a positive effect in promoting stem diameter and leaf area of C. annuum plants.

Table 1 Growth of chili effect of different inocula of arbuscular mycorrhizal fungi (HMA) under greenhouse conditions at 56 days after transplantation. 

^{Diaz-Franco et al. (2013)}, in an experiment in pepper (C. annuum) under greenhouse conditions they found that the mycorrhizae with Rhizophagus intraradices promoted growth by increasing fruit weight by 30% compared to non-mycorrhizal plants. In another study, when evaluating mycorrhizal inoculants in tomato plants (Lycopersicon esculentum M.) a significant increase in height and dry biomass compared to non-mycorrhizal plants was reported (^{Fernandez et al., 2006}).

This reflects that the HMA is a biological resource of importance in organic-biological fertilization of plants of economic interest, which could even be an alternative to other types of management and chemical fertilization (^{Armenta-Bojorquez et al., 2010}).

For variables mycorrhizal (Table 2) statistical differences in mycorrhizal colonization (p= 0.00) and spore density (p= 0.00) in both varieties of chili at 56 days after inoculation (DDI) met the HMA . Not mycorrhizal colonization was observed in the negative control, the inoculates recorded higher mycorrhizal colonization of roots in serrano pepper were BN and PA with 84.93% and 83.43%, Rhizophagus intraradices showed only 33.6% of colonization, the rest of inoculates were statistically equal the CM inoculum promoted further growth in serrano pepper recorded only 44.96% of mycorrhizal colonization, therefore it is not a very infective inoculum but effective in promoting plant growth. The concepts of infectivity, defined as the ability to colonize the root and effectiveness, ability to promote plant growth, are not related and are known to exist HMA inocula that colonize the root lesser percentage (15-40%) and have an excellent effect on plant nutrition and growth (^{Tapia-Gone et al., 2010}).

Table 2 Mycorrhization and spore density of arbuscular mycorrhizal fungi (HMA) in pepper plants under greenhouse conditions at 56 days after transplantation. 

The high percentage of mycorrhizal colonization found in this study is similar to that shown by ^{Lucas-Santoyo (2011)} who reported a 92.92% colonization up in plants guajillo (C. annuum L.) inoculated with mycorrhizal different consortia to 150 days after transplantation; also ^{Tanwar et al. (2013)} report a high percentage of colonization in the root (93.98%) in pepper, however this was even greater in the presence of Pseudomonas fluorescens (97.62%) which was reflected in increased growth. The high percentages of colonization found in the present study suggest that these varieties of pepper are highly micotróficas and not self-regulate the degree of colonization in their roots.

Regarding the density of spores of HMA analysis of variance showed statistically significant differences (p≤ 0.05). In serrano pepper where all treatments were inoculated HMA recorded statistically similar values between them however were higher than the negative control. In poblano pepper with R. intraradices control showed the lowest density of spores (130 in 100 g of dry soil), the highest spore density was recorded in the inoculum EH with 406.4 in 100 g of dry soil that was statistically equal to LC, PA and BN. correlation coefficients of Pearson significant between mycorrhizal colonization and number of spores in poblano (r= 0.5469) and serrano (r= 0.7475) were found, this is consistent with that reported in the literature, which indicates that high densities of spores foster greater mycorrhizal colonization (^{Vazquez et al., 2010}). However, both variables are not always necessarily dependent and depending on the species or plant variety and species of HMA used (^{Mendoza et al., 2002}).

The spore density is a variable that indicates the abundance of propagules of HMA that could compete against plant pathogens, providing for space and nutrients directly to the plant biosecurity (^{Azcon-Aguilar and Barea, 1996}). In this case, CM inoculum was the best in reducing the severity of disease caused by PC (Figures 1 and 2) but not the densest produced spores. This might suggest that the sickest plants being in a situation of greater biotic stress, could have induced greater sporulation of the HMA. This effect is related to survival mechanisms of HMA, which may be able to increase the amount spores being threatened carbon supplies the plant. Some authors suggest that some species of Glomus are strategists "r"; i.e. increase reproduction with a larger number of spores in the presence of a limiting survival factor (^{Ijdo et al., 2010}).

Figure 1 Severity of wilting caused by PC plants serrano pepper (A) and poblano (B) inoculated with HMA under greenhouse conditions at 95 days after transplantation. El Huizachal (EH); Las Campesinas (LC); Rancho Carlos Rojas (RCR); Paso Ancho (PA); El Limón (EL); Cerro del Metate (CM); Barranca de las Nueces (BN); without HMA (S/HMA). Different letters indicate statistically significant differences according to Dunn's test (p≤0.05). 

Figure 2 Viewing the severity of wilt caused by Phytophthora capsici in plants serrano pepper (1) and poblano pepper (2) to 95 DDI under greenhouse conditions. Cerro del Metate (level 1: a and d); without HMA (level 3: b; Level 4 = f); The Huizachal (level 5= c); Paso Ancho (level 3= e). 

This coincides with that reported by ^{Schalamuk et al. (2014)}, where on wheat plants (Triticum sp.) That were severely affected by septoria caused by Mycosphaerella graminicola the spore density was higher than in plants selectively with lower disease damage.

Bioprotector effect of HMA against Phytophthora capsici L. (PC) in pepper plants

After inoculation of PC, all plants showed symptoms of the disease, however the degree of severity of the inoculated mycorrhizal consortium depended. The nonparametric Kruskal-Wallis statistical analysis showed significant differences in serrano pepper (statistic= 15.6042, value- p= 0.0484086) and poblano (statistic= 15.6959, value- p= 0.0469459) for the scale of virulence. In both varieties of pepper all inoculates HMA except bioprotector CM did not exert effect against wilting plants meeting the same level of disease than the negative control (Figure 1).

According to the scale of virulence, plants inoculated with CM they found a severity index of one, indicating partially collapsed leafy plants with erect stem (Figure 2). This shows that the mycorrhizal consortium CM is able to bioproteger chili plants against Phytophthora capsici given the reduction in the severity of wilting. This is consistent with that reported by ^{Ozgonen and Erkilic (2007)} who by inoculating spores of Glomus etunicatum, Gl. fasciculatum and Gigaspora margarita in pepper plants, found a decrease in disease caused by PC up to 91.7% under greenhouse conditions and 57.2% under field conditions. On the other hand, plants inoculated with CM had the highest fresh biomass radical, which could give them a larger area of exploration for nutrients and water, which could favor their nutritional status, development and protection against Phytophthora capsici. This is consistent with that reported by ^{Gomez-Dorantes et al. (2008)}, who found a larger size of the root in tomato plants inoculated with HMA, which showed less damage by Phytophthora capsici compared to non-mycorrhizal plants which showed a significant loss of root biomass (p≤ 0.05).

The synthesis of phytoalexins is documented as a plant response to attack by pathogens which are involved jasmonic acid and ethylene that promote systemic resistance can be induced by HMA (^{Carreon-Abud et al., 2008}). Meanwhile ^{Gómez-Dorantes et al. (2008)} attributed the reduced susceptibility to PC in tomato to improve the nutritional status of plants and competition for carbon compounds in the root. Considering these results it is possible that the bioprotector effect found in this study by the HMA, reflected in a lower severity of illness, it is due among other things to the induction of biochemical signaling to give defense responses prompted by the activity of plant hormones such as salicylic and jasmonic acid (^{Kapoor, 2008}).

This bioprotective effect has been reported in Tomato plants where inoculation with G. mosseae induced systemic response against Phytophthora parasitica in a root system divided, where resistance was associated with the accumulation of phenolic compounds, thickening of the cell wall and proteins related pathogenesis (PR-1) (^{Cordier et al., 1998}). Tomato systemic response against P. parasitica was also reported by ^{Pozo et al. (1999)} where hydrolytic enzymes isoforms possibly related to this response were found. The protection PC via protein synthesis PR peroxidase activity has been reported in pepper plants inoculated with Trichoderma harzianum, where colonization by the antagonist induces systemic resistance that can trigger the mechanism of hypersensitive defense (^{Ezziyyani et al., 2005}).

Regarding the air fresh and radical biomass PC infected plants at 95 days after transplantation significant differences (p≤ 0.05) between treatments (Table 3) were found. In serrano pepper CM inoculum biomass promoted as fresh air with 15.4 g was statistically superior to the negative control (S/HMA) which registered 8.26 g. For poblano pepper trend was the same, the plants inoculated with CM showed the air fresh biomass of 7.95 g and statistically different treatments without HMA (2.83 g). In the radical fresh biomass HMA different consortiums including CM, they failed to promote root growth since in both varieties of pepper were statistically equal to the negative control without HMA.

Table 3 Fresh aerial and radical biomass in plants and poblano pepper serrano inoculated with different arbuscular mycorrhizal fungi (AMF) and infected with Phytophthora capsici at 95 days after transplantation under greenhouse conditions. 

However, increased air fresh biomass by the CM consortium plants serrano pepper poblano was reflected in the lowest level of severity of the disease who had plants of this treatment (Figure 2), which was corroborated by making a correlation analysis between the total fresh biomass and the level of severity of wilting in plants of both varieties where a significant negative correlation in both cases (-0.47 and -0.37) was found. Finally, it is important to study the biological effectiveness in promoting growth and biocontrol of plant pathogens by consortia of HMA isolated from soil A. cupreata because it allows to know the associated species and their potential biotechnological application in this and other plant species of economic importance such as chili.