Origin of arbuscular mycorrhizal fungi

The AMF were isolated from soil from the lowland deciduous forest of southeastern Mexico, in the Cuxtal ecological reserve, with locations of 20°52’ 07.44’’ N and 89°36’ 51.64’’ W. The soil type is Leptosol with depths varying from 0 to 25 cm (^{Díaz-Garrido et al., 2005}). The climate is warm sub-humid with rain in summer and winter. The average annual precipitation is 900 mm with an average annual temperature of 27.5 °C (^{García, 1973}). The dominant vegetation is tropical deciduous forest (^{Flores & Espejel, 1994}).

Propagation, extraction and identification of arbuscular mycorrhizal fungi

To identify the AMF, the collected soil was sifted and mixed with sterile sand (1:1 v/v) and placed in 10 kg pots for propagation. Sorghum (Sorghum bicolor L.) seeds were then planted as a trap crop (^{Sieverding, 1991}). The pots were kept in greenhouses at an average of 36 ± 2 °C and relative humidity of 64 %, and were irrigated at field capacity. After 12 weeks and with the purpose of stimulating the sporulation of the AMF, irrigation was suspended. In week 16, spore extraction was carried out, according to the method of wet sifting and decanting and centrifugation (^{Gerdermann & Nicholson, 1963}), where 100 g of soil from each propagation pot were processed and homogenized with Tween 20 (0.05 %) in water, the solution was filtered through a series of sieves with 600, 425, 90 and 25 μm mesh opening, and from the retained fractions, spores were extracted and deposited in 1.5 mL vials provided with sterile distilled water.

The identification was made by comparing and contrasting the morphological characteristics of the spores, with genus- and species-specific descriptors, available on the website of the International Culture Collection of Arbuscular and Vesicular-Arbuscular Mycorrhizal Fungi (^{West Virginia University, 2020}), the taxonomy of Glomeromycota (^{Schenck & Perez, 1990}; ^{Schüßler & Walker, 2010}). Spores were separated by species and deposited in vials. According to morphological characteristics three species were identified: Funneliformis geosporum C. Walker & A. Schüßler, Claroideoglomus claroideum C. Walker A. and Glomus ambisporum C. Walker & A. Schüßler. For the conservation of the spores, they were disinfected by removing the sterile water from the vials and adding Tween 20 at 0.05 %, they were centrifuged at 500 rpm for one minute, then the supernatant was removed and 2 % Chloramine T was added. The water was centrifuged at 500 rpm for 15 minutes, both procedures were repeated twice. Finally, the supernatant was removed and a mixture of gentamicin (100 ppm) and streptomycin (200 ppm) was added to each vial and kept at 4 °C until inoculation.

Evaluation of arbuscular mycorrhizal fires in the initial growth of sweet pepper (C. annuum).

Five treatments were evaluated, three constituted by each species of AMF: T1 = F. geosporum, T2 = C. claroideum and T3 = G. ambisporum, with the incorporation of 50 % of chemical fertilization, two control treatments: T4 = 100 % chemical fertilization (Polyfeed^®, 17-17-17, Haifa, Mexico, 2 g L^-1 of water) without AMF, applied twice a week, from 10 days after sowing (das) and T5 = one control (only water, without AMF and without fertilization).

Before the establishment of the experiment, soil was sterilized by means of steam dragging for three days for 1 hour at 90 °C, and ten germination trays of 72 cavities were filled. In six of these, 30 spores of each AMF were deposited in each cavity, the remaining four served as controls, without AMF inoculation. In the trays were planted sweet pepper seeds (C. annuum) type Criollo previously disinfected with sodium hypochlorite 1 % followed by two washings with sterile distilled water. The trays were covered until the germination of the seeds and were kept in the greenhouse at an average of 28 ± 2 °C, with an average relative humidity of 64 % and an average light intensity of 400 lux with the application of irrigation at field capacity.

After 47 das the growth variables were estimated at 30 seedlings: seedling height, dry air biomass, fresh and dry root weight. The percentage of mycorrhizal colonization was estimated in five plants per treatment. The roots were washed with running water and deposited in cassettes for tissue, then they were passed through KOH at 10 %, H₂0₂ at 30 %, HCL at 30 %, with heating and rinsing with running water. Finally, trypan blue was added and they were left to rest for 24 hours. After this time, trypan blue was removed and the roots were rinsed with running water (^{Phillips & Hayman, 1970}).

Later, permanent preparations were made, 10 sections of roots were taken and mounted on an object holder, with the help of brown paper they were pressed to soften them, then two drops of polyvinyl alcohol and an object cover were added. The samples were labeled and placed in the drying oven at 47 °C for 72 hours. Finally, the structures of the AMF (mycelium, spores, vesicles and coils) were observed in an optical microscope and the percentage of colonization was quantified (^{McGonigle et al., 1990}).

Obtaining J₂ from M. incognita

Commercial plantations in production of sweet pepper (C. annuum) parasitized by M. incognita were sampled in the municipality of Tixpehual, Yucatan, located 20°58’40”N and 89°26’30”W at an altitude of 8 meters above sea level. The gilled roots were deposited in paper bags and kept at 6 °C in refrigeration for 24 hours. They were washed with running water and egg masses were extracted with syringes under the stereoscopic microscope (Leica M80, USA). They were disinfected with 1 % sodium hypochlorite for one minute and washed with sterile distilled water until the disinfectant was removed. The eggs were incubated in a microbiological culture oven at 25 ± 1 °C for three days until the J₂ hatched. Then, they were concentrated in a 500 mL flask, 1 mL was taken and counted to calibrate the inoculum. Adult females were extracted from the gilled roots to identify the species by morpho-taxonomic characters (^{Ayuob, 1980}).

Control bioassay of M. incognita in greenhouse

In order to estimate the biocontrol of M. incognita with AMF, plastic pots of 2 kg capacity were filled with sterile soil. Before transplanting, a hole of 3 cm in diameter and 5 cm deep was made and 1 mL of water was inoculated containing 1,000 larvated eggs and 300 J₂ of M. incognita, then a 47-day-old sweet chili bell pepper (C. annuum) seedling inoculated with the AMF was transplanted, as indicated in the first experiment and coming from this one.

In this experiment there was an adjustment of the nutrition of the plants from the inoculation of the nematode. This consisted in a fertilization with chemical balance 2:1:1, with the sources: urea (Magro^®, 46-0000, Fertinova, Mexico), potassium nitrate (Ultrasol^®, 1200-46, SQM, Mexico) and monoammonium phosphate (MAP^®, 12-61-00, Greenhow S. A. de C. V. Mexico), applied twice a week.

Five treatments were also evaluated in this experiment: three constituted the AMF species: T1 = F. geosporum, T2 = C. claroideum and T3 = G. ambisporum, two control treatments: T4 = a 24 % oxamyl nematicide in a dose of 1 mL L^-1 of water (Vydate^®, Dupont, Mexico), applied to the soil at the time of transplant, with nematodes and without AMF and T5 = a control, with nematodes and without AMF. Each treatment was constituted by 15 plants that constituted the repetitions, distributed in greenhouse conditions, indicated in the first experiment.

The treatments were evaluated 50 days after inoculation with M. incognita, and the following variables were considered as biocontrol variables of the nematode: the galling index estimated with a scale of radical damage for root-knot nematodes (^{Taylor & Sasser, 1983}), the number of eggs and females per gram of root (^{Herrera-Parra et al., 2014}). As growth variables were measured: plant height, number of flower buds per plant, dry aerial biomass per plant, fresh root weight, root length and finally by volumetric displacement, root volume. As variables associated with AMF, the percentage of colonization in the roots of five plants per treatment was estimated according to the techniques of ^{Phillips and Hayman (1970)} and ^{McGonigle et al. (1990)} indicated in the first experiment.

Response of sweet pepper (C. annuum) seedlings to inoculation with arbuscular mycorrhizal fungi

AMF promoted significant growth (p ≤ 0.01) of C. annuum seedlings at 47 das compared to control seedlings. Treatments involving C. claroideum and G. ambisporum increased the height of the seedlings by up to 20.50 % relative to those inoculated with F. geosporum and 100 % chemical fertilization, and by up to 73 % relative to those of the control (Table 1).

A significant effect (p ≤ 0.01) of dry aerial biomass production was obtained with G. ambisporum with gains of 97 % in relation to the control seedlings, in addition it surpassed the 100 % chemical fertilization and F. geosporum with which up to 0.32 ± 0.03 g of dry aerial biomass was registered. Also, C. claroideum improved this response variable as did G. ambisporum and 100 % chemical fertilization (Table 1).

Root growth was significantly increased (p ≤ 0.01) with C. claroideum and G. ambisporum and significantly increased fresh (90 %) and dry (98 %) root biomass, relative to the control. The averages of fresh and dry root biomass with 100 % chemical fertilization were equal to those caused by F. geosporum and lower than those of C. claroideum and G. ambisporum (Table 1). In the cultivation of C. annuum, a preliminary growth phase under protected conditions is required to obtain seedlings with desirable characteristics for establishment in production systems (^{Macías-Rodríguez et al., 2013}). At this stage, inoculation with C. claroideum and G. ambisporum is an alternative to improve seedling growth (height, aerial and root biomass), even with better characteristics than those obtained with 100 % chemical fertilization. These effects have been observed in cultivars of C. annuum Ancho with Glomus spp + Gigaspora sp. and the incorporation of medium-dose fertilization (P 22 µg mL^-1) where higher dry biomass was estimated (^{Alonso-Contreras, 2013}). In cultivars such as Jalapeño, De árbol and Serrano with the inoculation Rhizophagus intraradices (formerly Glomus intraradices) and Glomus zac-19 improved the emission of leaves, stem diameters and aerial biomass (^{González-Mendoza et al., 2015}). In C. frutescens they favored initial growth with F. mosseae and Racocetra fulgida (formerly Scutellospora fulgida) in combination with 50 % of bovine manure (^{Jiménez et al., 2017}). Other reports indicate that AMF significantly increase photosynthetic activity, chlorophyll content, and assimilation of water and immobile nutrients from the soil (^{Alvarado-Carrillo et al., 2014}; ^{Lehman & Rilling, 2015}), reduce biotic and abiotic stress (^{Khalid et al., 2016}; ^{Tchabi et al., 2019}), which favors the growth of the initial phase of crops as observed in this study.

Of the three AMF species, F. geosporum was the least favorable to seedling growth. This response may have been modulated by compatibility between AMF and the host and no functional mutualistic symbiosis was established (^{Jin et al., 2017}; ^{Bazghaleh et al., 2018}), which does not show a positive effect on plant biomass production (^{Rosheim, 2016}; ^{Avio et al., 2017}). The results indicated that AMF with medium fertilization doses favored the growth of C. annuum seedlings and that the incorporation of these microorganisms can be a complementary alternative for seedling production, which reduces the use of chemical fertilizers and damage to the environment.

The AMF species: F. geosporum, C. claroideum and G. ambisporum colonized the roots of C. annuum seedlings, with significant difference (p ≤ 0.01). In particular, G. ambisporum had greater colonization (100 ± 0.00 %), followed by F. geosporum (98.66 ± 0.95 %) and C. claroideum (96.97 ± 1.73 %). Seedlings with 100 % chemical fertilization and those of the control had less than 10 % root colonization (Table 1). The estimated colonization percentages in the seedlings inoculated with these fungi suggest that in this growth stage they allow the colonization of their roots, which may be a strategy to increase growth success in early stages of the crop, where better P translocation, photosynthetic rate and dry biomass production have been reported (^{Alonso-Contreras et al., 2013}; ^{Nana et al., 2015}).

Effect of arbuscular mycorrhizal fungi on M. incognita

In the control plants inoculated with M. incognita, without arbuscular mycorrhizal fungi and without the application of nematicide, the symptoms of the disease were observed: chlorosis, decrease in growth and galls formation. In these plants there were higher index of galls (51.60 ± 37.24), number of eggs (408.30 ± 282.54) and females (60.80 ± 41.97) (p ≤ 0.01) (Table 2).

The galling index was significantly reduced (p ≤ 0.01) with the inoculation of F. geosporum and G. ambisporum, which reduced damages by 79.65 % in relation to the control plants (Table 2). This potential has been reported with R. intraradices (formerly G. intraradices) against Nacobbus aberrans (^{Lax et al., 2011}), with Rhizophagus intraradices, Funneliformis mosseae against Rotylenchulus reniformis (^{Herrera-Parra et al., 2014}), with G. bagyarajii, G. macrocarpum against M. incognita (^{Raghavendra et al., 2016}), with Gigaspora albida, Claroideoglomus etunicatum and Acaulospora morrowiae (formerly Acaulospora longula) against M. arenaria (^{Da Silva et al., 2017}), which evidences the effectiveness as microbial biocontrol agents.

C. claroideum and the nematicide treatment, although statistically not different from the control, showed a tendency to reduce the galling index by up to 38 %. The effect with C. claroideum was related to its ability to improve root length and branching, which allowed new spaces available for nematode feeding and new infections, which caused more damage and up to 87 % of egg production and 51 % of female formation, in relation to the plants treated with nematicide. This treatment did not prevent the penetration of J₂, but significantly reduced the reproduction of the nematode with 44.40 ± 3.20 eggs and 18.40 ± 1.89 females, and its effect to reduce the damage was the same as with F. geosporum and G. ambisporum, which limited the production of eggs by 89 % and the formation of females by 69.73 %, in relation to the control plants (Table 2).

The ability of AMF to reduce damage and reproduction of endoparasitic nematodes (^{Lax et al., 2011}; ^{Herrera-Parra et al., 2014}; ^{Tchabi et al., 2019}), is associated with competition among organisms for carbon sources, spaces and sites of infection, since these are organisms with the same physiological needs and struggle to establish themselves in the same place where they obtain resources for their feeding (^{Vos et al., 2014}; ^{Schouteden et al., 2015}). It is also related to the production of radical exudates, such as phenols, flavonoids and organic acids (^{Hage-Ahmed et al., 2013}); which disorient the J₂ of the nematode and prevent its penetration and allow functional symbiosis between plants and AMF, allowing the activation of the host defense system (^{Cervantes-Gámez et al., 2015}). This action prepares the plant and allows a defense response also against foliar pathogens (^{Pérez-Ortega et al., 2015}, ^{Reyes-Tena et al., 2016}).

Effect of AMF on C. annuum growth

The decrease in root damage and reduction of nematode reproduction by AMF favored plant growth. In the control plants the growth was significantly lower (p ≤ 0.01) than in the plants treated with AMF and nematicide.

The plants with higher height were the ones inoculated with F. geosporum and C. claroideum, these treatments presented the same effect as nematicide and registered plants 75 % higher than control ones (Table 3). Similar reports showed height increases of C. annuum cv plants. Cacho de Cabra with the inoculation of C. claroideum (^{Castillo et al., 2009}) and in the Jalapeño, Serrano and De árbol cultivars, with the inoculation of Glomus spp. and R. intraradices significantly favored the growth in height, in relation to the control plants (^{González-Mendoza et al., 2015}). The functional interaction between AMF and plants increases the production of gibberellins and cytokines (^{Alonso-Contreras et al., 2013}; ^{Pereira et al., 2016}), reduces abiotic stress and nematode damage (^{Baum et al., 2015}). Factors that favor the growth of plants still parasitized by these phyto-parasites.

The highest number of flower buds was presented in the plants inoculated with the AMF and the nematicide, on average they ranged from 8 ± 0.78 to 11 ± 0.87, the control plants did not emit flower buds (Table 3). This response was associated to the capacity that the AMF have to translocate nutrients such as N, P, K, Ca, Cu, Mg, Zn, Fe (^{Nana et al., 2015}; ^{He et al., 2017}) which favors better nutrition, growth and emission of flower buds, e.g. studies with G. caledonium and R. intraradices in C. annuum promoted greater emission of flowers, associated with better contents of P and Zn (^{Ortas et al., 2011}). In species such as Heliconia stricta, inoculation of Rhizophagus intraradices and Zac 29 strain increased the number of buds and flowers, improved nutritional status and photosynthetic activity (^{Uc-Ku et al., 2019}).

A significant increase was registered (p ≤ 0.01) of dry aerial biomass with the nematicide (2.69 g±0.45) and F. geosporum (2.20 g ± 0.30), with the rest of the AMF, increases higher than the control were registered up to 95 % (Table 3). This response has been observed in crops inoculated with AMF (^{González-Mendoza et al., 2015}; ^{Zayed et al., 2017}; ^{Uc-Ku et al., 2019}) in which changes in photosynthetic activity are reported to favor CO₂ fixation, capture efficiency and P translocation, which is reflected in greater growth, foliar expansion and biomass production.

With C. claroideum and G. ambisporum the plants had 21 % longer roots than those treated with the nematicide and 75 % than those of the control. The roots with the highest fresh weight (p ≤ 0.01) were achieved with the nematicide (5.86 ± 1.80) and with F. geosporum (5.03 ± 1.53). With the rest of the AMF it increased up to 88 % in relation to the control plants. The largest volume of roots was estimated with the nematicide (8.70 ± 1.56 cm³) and a significant increase (95 %) in root size with the incorporation of the AMF in relation to those of the control (Table 3). The highest percentage of mycorrhizal colonization was estimated with G. ambisporum (68.83 %). With C. claroideum and F. geosporum the roots had 42.98 %. The plants treated with the nematicide and those of the control presented a 6.36 % of colonization, which could be due to spores introduced by air currents.

The results obtained in this study showed that with native species of AMF as a biological resource, it can be used to reduce the biotic stress induced by root pathogens such as M. incognita and to favor the growth of C. annuum in the study conditions evaluated.