Growth conditions

The assay was developed in a zenithal plastic type greenhouse of the Instituto de Investigaciones Agropecuarias y Forestales of the Universidad Michoacana de San Nicolás de Hidalgo (19° 45’ 95’’ N, 101° 09’ 16’’ W, 1900 m) under light, temperature, and environmental humidity conditions.

Arbuscular mycorrhizal fungus inoculant

Five native mycorrhizal consortia and a commercial inoculant made by INIFAP^® (Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, MX) were assessed. The consortia came from soils with Agave cupreata plantations of the State of Michoacán, Mexico and were named according to their collection site: Las Campesinas (LC), Carlos Rojas (CR), Paso Ancho (PA), El Limón (EL), Cerro del Metate (CM). The inoculants were previously propagated in tramp pots in the greenhouse for 12 months; each consortia contained from four to six species, of which the most abundant of each mycorrhizal consortia were the following: CM: Glomus glomerulatum, PA: Acaulospora delicata, CR and EL: Glomus deserticola, LC: Acaulospora scrobiculata (^{Trinidad-Cruz et al., 2017a}). The commercial INIFAP inoculant was previously used in other experiments where its viability, colonization, and growth promotion was assessed in other plant species.

Plant material

Commercial guava seeds were disinfected with a commercial chlorine solution at 3% (active chlorine at 6%) for 10 min and then washed with distilled water thrice for 5 min. Subsequently, they were placed in plastic germinator trays with sterilized sand under light and environmental conditions. The seeds and substrate were kept hydrated all the time. From the 28 days after sowing, the seeds were considered germinated when they showed a homogenous size (in average, 2 cm high, four leaves, and 9 mm of stem diameter), and transplanted to greenhouse bags.

Inoculation

The guava plants were transplanted in black polyethylene bags with 2.5 kg of sand and soil (1:1 v/v) sterilized with growth medium. The seedlings were placed in a cavity made in the center of the bag; at the moment of transplanting, inoculation of the different mycorrhizal consortia was performed directly in the radicle system. The number of spores of each AMF consortium was quantified per gram, and the necessary grams were taken to inoculate 80 spores, approximately; for the control group without AMF (w/AMF) treatment, 10 g of sterile sand per treatment were inoculated. Once inoculation was performed, the seedlings remained for 125 days in the greenhouse and were irrigated with demineralized water at field capacity as required. Seven treatments (five native consortia, the commercial INIFAP inoculant and one w/AMF) were generated; each treatment was replicated seven times, and the experimental unit was one guava plant. The factorial arrangement was a completely randomized block design.

Plant growth, quality, and mycorrhization variables

A destructive sampling was performed at 125 days after transplant; plant height was measured manually with a flexometer; the stem diameter with a Digital Vernier Caliper; leaf area with digital planimetry (LI‑COR, LI-3100, Lincoln, NE, U.S.A.); and the fresh biomass of each organ was weighed with an analytical balance (Mettler Toledo AT200, Columbus, OH, U.S.A.); subsequently, the samples were stove-dried at 60 °C for four days to determine dry weight. The relationship DAPB / DRB (dry aerial part biomass and dry root biomass) was determined as plant quality variable to know the balance between the plant transpiration and absorption surfaces (^{Prieto et al., 2009}), robust index related to the plant photosynthetic capacity (^{Rueda-Sánchez et al., 2013}), and Dickinson’s index, which expresses the equilibrium of biomass and robustness distribution, avoiding the selection of disproportionate plants and discarding those of lower height but with greater vigor (^{Dickson et al., 1960}). With respect to the microbiological variables, the mycorrhizal colonization percentage was determined by ^{Phillips and Hayman’s (1970)} technique, subsequently determining the mycorrhizal colonization percentage with the methods described by ^{Mcgonigle et al. (1990)}. For spore quantification in the substrate, the technique proposed by ^{Gerdemann and Nicolson (1963)} and ^{Brundrett et al. (1996)} was used.

Statistical analysis

A one-way analysis of variance (ANOVA) and Tukey’s multiple range test were performed to the data, both with a value of P ≤ 0.05. The percentage values were transformed (ArcSen n%/100) for homogenization of variance (^{Gomez and Gomez, 1984}). The statistical software Statgraphics ver. XV (^{Statgraphics, 2005}) was used for data processing.

Guava plant growth

The statistical analysis showed significant statistical differences (P ≤ 0.05) among the different treatments in all the growth variables assessed (Table 1). At 125 days after inoculation, the guava plants with the INIFAP inoculant and those w/AMF obtained the lowest growth values in all the cases and resulted statistically equal between them.

On the other hand, the greatest mortality after transplant and inoculation was recorded in the plants with the treatments w/AMF and those with the INIFAP inoculant with 43 and 58%, respectively. In that respect, ^{Estrada-Luna et al. (2000)} and ^{Machineski et al. (2018)} mentioned that the AMF increased stress resistance in fruit trees during transplant and acclimation because of the advantages that the symbiotic association promoted by improving hydric relationships, nutrient acquisition, and plant growth, among others.

Likewise, the plants that survived in the treatments w/AMF and those with the INIFAP inoculant did not produce leaves during the time the experiment lasted (Table 1). The fact of finding the greatest mortality and the least plant growth in plants w/AMF or with the INIFAP inoculant might have been due to their disadvantage with respect to the mycorrhized plants; in the same manner, the effect that the colonization may have on guava plant survival was notorious in those inoculated with the CM consortium, which had a survival of 85% with only 3% of colonization, and their growth was greater than that reached by the plants w/AMF and those with the INIFAP inoculant.

On the other hand, the greatest guava plant growth obtained from seeds and inoculated with different species and the arbuscular mycorrhizal fungus combination has been reported previously (^{Estrada-Luna et al., 2000}; ^{Schiavo and Martins, 2002}; ^{da Silva-Campos et al., 2013}; ^{Mohandas et al., 2013}; ^{Das et al., 2017}). In general, this study found an increase in guava plants propagated by seeds when a mycorrhizal consortium was applied. The effectiveness of some of this native consortia has been proven in maize, chili, and bean (^{Reyes-Tena et al., 2015}, ²⁰¹⁶), Agave inaequidens (^{Quiñones-Aguilar et al., 2016}), and Agave cupreata (^{Trinidad-Cruz et al., 2017b}). This promotion effectiveness may have been caused by the diversity of the AMF species that contain inoculants; with this respect, ^{Gavito and Varela (1995)} and ^{Bashan et al. (2000)} mentioned that the inoculants constituted by more than one AMF species showed a greater benefit in their hosts. Furthermore, native mycorrhizal consortia are also known to be more effective than those constituted by exotic species or only one species (^{Bashan et al., 2000}; ^{Ortas and Ustuner, 2014}). Another possible cause of greater growth promotion in mycorrhizal consortia could have been an effect of the propagation method (tramp pot) when rhizosphere soil propagates; besides the AMF, this soil had microorganism diversity, mainly bacteria associated to free-life spores that have the capacity of promoting plant growth. Some studies have reported a greater AMF effectiveness when they were applied jointly with other microorganisms (^{Panneerselvam et al., 2012}; ^{Mohandas et al., 2013}). Similarly, other studies have reported the presence of actinomycetes, such as Streptmyces fradiae, S. avermitilis, S. cinnamonensis, S. canus and Leifsonia poae (^{Mohandas et al., 2013}) and some pseudomonas species, such as Pseudomonas putida, P. aeruginosa, Brevibacillus sp. and Bacillus subtilis (^{Panneerselvam et al., 2012}), colonizing AMF spores of the guava plant rhizosphere. Guava plant growth promotion was differentiated and only significant in the plants inoculated with the LC, PA, and EL consortia, with respect to those w/AMF or with the INIFAP inoculant. The difference found among the consortia was likely due to the species diversity of each one of them, which could indicate differential effectiveness (^{Costa et al., 2001}; ^{Robles-Martínez et al., 2013}). The inoculated plants with the LC and EL consortia reached the greatest values in the variables assessed. The increments reached by these plants with respect to those w/AMF were 9, 4.4, 332 in average and more than 145 times for plant height, stem diameter, total dry biomass, and leaf area, respectively. Similar increments in growth variables have been reported in guava by the inoculation effect with AMF (Estrada-Luna et al., 2000; ^{da Silva-Campos et al., 2013}; Das et al., 2017). On the other hand, despite recording an increase in size in guava plants inoculated with the CR, PA, and CM consortia, the result was not significant with respect to those w/AMF or with the INIFAP inoculant (Table 1). Mycorrhization of guava plants with some AMF species has not promoted growth; ^{da Silva-Campos et al. (2013)} did not find differences in leaf number and dry biomass of the aerial part of guava plants when they were inoculated with Acaulospora lonbgula and Gigaspora albida.

Guava plant quality parameters

With respect to the quality attributes for broad-leaf trees (^{Rueda-Sánchez et al., 2013}), which are considered growth variables, an increase was found when guava plants were inoculated with any AMF consortia (Table 2).

The plants w/AMF or with the INIFAP inoculant showed the lowest quality, according to the DAPB/DRB relationship and Dickinson index. According to the DAPB/DRB relationship, two types of plant quality were obtained, low (DAPB/DRB > 2.0) for plants without AMF or with the INIFAP inoculant and high (DAPB/DRB < 2.0), when they were inoculated with any AMF consortium, which had a bearing in greater plant survival at the moment of transplanting to the field or in drought conditions (^{Prieto et al., 2009}). For the robustness index, no difference was found in plant quality between the plants inoculated with AMF and those w/AMF, which according to this index, the plants showed high quality (< 6.0). With respect to Dickinson’s Index, two types of plant quality were obtained: low (< 0.2), for guava plants w/AMF and with the INIFAP inoculant and CM; average (0.2 - 0.4), for the plants inoculated with CR and PA; and high (≥ 0.5), for plants inoculated with EL and LC (Table 2). According to plant quality parameters, those inoculated with AMF were the ones with the best size and vigor, which would be benefitted at the moment of transplanting to the field (^{Ortas and Ustuner, 2014}). This greater vigor in plants may have been caused by an increase in the photosynthetic activity that plants show when they are mycorrhized. In this particular case, ^{Estrada-Luna et al. (2000)} reported that guava plants propagated by seeds and inoculated with a mixture of Glomus diaphanum. Glomus albidum and Lomus claroides increased their net stomatal conductance and photosynthesis compared to those without mycorrhizal.

Mycorrhizal colonization percentage

Guava has been mentioned to be a highly mycotrophic plant with a mycorrhizal dependence of more than 100% (^{Estrada-Luna et al., 2000}). These authors reported colonization values of 94% when a mixture of Glomus diaphanum, G. albidum and G. claroides was used. On the other hand, ^{da Silva-Campos et al. (2013)} reported percentages of 32, 48, and 62% when guavas were inoculated with G. etunicatum, Gigasora albida, and Acaulospora longula, respectively. ^{Panneerselvam et al. (2012)} reported 65% of colonization in guavas inoculated with Glomus mosseae. This study found that guava plants with the INIFAP inoculant did not show colonization (Figure 1), which could have generated the high mortality and stunted growth found in these plants. Several causes might have taken place if the INIFAP inoculant did not function, for example, spore viability and growth medium, among others. The EL consortium reached the greatest colonization (90%) although it resulted significantly similar to that obtained with the consortia PA, LC, CR. In average, a colonization of 70% was recorded among the four consortia, which was 23 times more than that obtained with the CM consortium that only colonized 3%. The low colonization for this consortium could have been due to species diversity that this inoculant contained, indicating a possible plant selectivity by the AMF symbiont (^{Costa et al., 2001}; ^{Barea and Azcón-Aguilar, 2013}).

w/AMF = without arbuscular mycorrhizal fungus; PA = Paso Ancho; LC = Las Campesinas; CR = Carlos Rojas; EL = El Limón; CM = Cerro del Metate. Lines on bars indicated standard error. Different letters on bars indicate significant differences (P ≤ 0.05).

Figure 1: Mycorrhizal colonization in guava plant roots with different AMF, at 125 days of establishment.  

Spore production

Spore production in the environment varied significantly (P ≤ 0.05) depending on the AMF inoculant (Figure 2). The greatest spore production was recorded in the EL consortium, producing more than 40 spores/g of dry substrate, which was statistically different to the rest of the inoculants. In average the production in this consortium was five times more than that produced by the rest of the inoculants, followed by LC and CR inoculants where they produced 15 spores/g in average; the least spore production was recorded for the INIFAP, PA, and CM inoculants where they produced 3 spores/g of substrate in average. On the other hand, ^{Panneerselvam et al. (2012)} reported a production of 14.7 spores g^-1 of dry substrate when the guava plants were inoculated only with Glomus mosseae. The spore production with the EL and CM consortia was related to the colonization percentage and plant growth, which indicated a certain selectivity by the species contained in the respective inoculants. In the case where a greater spore production was observed, it could have been due to a greater red mycelial production by these AMF that increased spore production (^{Azcón and Barea, 2015}).

w/AMF = without arbuscular mycorrhizal fungus; PA = Paso Ancho; LC = Las Campesinas; CR = Carlos Rojas; EL = El Limón; CM = Cerro del Metate. Lines on bars indicate standard error. Different letters on bars indicate significant differences (P ≤ 0.05).

Figure 2: Spore production in the substrate of guava plants with different arbuscular mycorrhizal fungi (AMF) at 125 days of establishment.