Introduction

The collection and commercialization of non-timber forest resources is one of the main economic activities in the arid and semi-arid zones of northeastern Mexico (^{Castillo et al., 2015}). However, the inadequate management of the exploited species, as well as clandestine collection, have had effects on the diversity, the area of distribution and the abundance of these species, to the extent of leading them to different levels of risk of extinction (^{Durán and Núñez, 2015}). Among the threatened species in Mexico is Agave victoriae-reginae T. Moore (Asparagaceae) (^{Tropicos, 2019}), known as “noa” or “queen’s agave”, a perennial species, endemic to the Chihuahuan Desert, distributed in northern Mexico, in the states of Coahuila, Durango and Nuevo León (^{Durán and Núñez, 2015}).

From its peculiar beauty, this species has been the focus of attention by collectors for ornamental use (^{González et al., 2011}). This situation has led this taxon to be included in the Official Mexican Standard NOM-059-SEMARNAT-2010 under the category of "endangered species" (^{Semarnat, 2010}), as well as in Appendix II of CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora) (^{CITES, 2017}). Therefore, the assisted reproduction of A. victoriae-reginae seedlings and the subsequent reforestation in their habitat constitutes an alternative to mitigate the loss of natural populations.

Germination is the most crucial stage of the plant cycle, as it depends on a set of environmental conditions and resources such as soil and nutrients, which limit or promote the establishment of the seedling on a substrate, and on a broader scale, the regeneration of a plant population (^{Donohue et al., 2005}). Therefore, understanding the germination process is key to improving nursery production (^{Barnett and Varela, 2004}) and thus promoting the conservation of forest populations.

Maturity, viability (period during which seeds retain their germination capacity) and dormancy (inability to germinate under optimal environmental conditions and resources) are intrinsic factors on which seed germination depends, as well as extrinsic factors such as temperature, substrate, light intensity and humidity (^{Khurana and Singh, 2001}; ^{Doria, 2010}). The use of pregerminative treatments allows to accelerate and homogenize germination, among which are mechanical, thermal and chemical scarification, dehydration, imbibition, and the use of growth regulators (^{Cubillos et al., 2011}; ^{Hernández et al., 2017}).

There is evidence that certain microorganisms have the ability to stimulate seedling germination and growth (^{Cubillos et al., 2011}). On the one hand, the use of fungi such as Trichoderma spp. reduces the mechanical resistance of the testa in the seeds and facilitates the breaking of dormancy (^{Delgado-Sánchez et al., 2013}). On the other hand, the use of bacteria such as Bacillus spp. allows the solubilization of nutrients such as phosphates, which improves the nutrition of the embryos (^{Cabra et al., 2017}).

In general, the use of pregerminative treatments in seeds of Agave species has been limited and has focused on evaluating the effect of environmental conditions such as temperature in eight species of this genus, including Agave lechuguilla Torr. and Agave cupreata Trel. et Berger (^{Ramírez et al., 2012}), the origin of the seed in Agave potatorum Zucc. (^{Rangel et al., 2015}), or the substrate in A. victoriae-reginae (^{Sánchez et al., 2017}). Furthermore, in this genus, the use of microorganisms as germination facilitators has not been evaluated. Therefore, the objective of this study was to evaluate the effect of pregerminative treatments with Trichoderma spp. and Bacillus spp. on the percentage of germination of seeds of A. victoriae-reginae. The hypothesis of this research is that the use of Trichoderma spp. and Bacillus spp. as pregerminative treatments, the GP can be increased compared to sowing without these microorganisms.

Materials and Methods

Results

The analysis of covariance showed that the treatment and the time had a significant influence on the germination of the seeds (Table 1). the Trichoderma and Bacillus consortium (P <0.001) treatment determined a final PG statistically higher than control, with an increase in germination of 14.8 % and 17.1 %, respectively. The germination time after sowing also influenced GP (P <0.001), as during the first days a notable increase in germination was observed, to follow afterwards a stabilization trend after the 12^th day of sowing.

Germination of A. victoriae seeds began on the 5^th day after sowing, with 29.6 %, 19.6 % and 12.6 % for the seeds treated with Trichoderma spp., Bacillus spp. and control, respectively. Between the 8^th and 12^th day, an exponential increase in germination was observed, and later, the increase in germination began a relative stabilization period until the end of 26 days, when the GP reached 85 % for seeds treated with Trichoderma spp., 86.7 % with Bacillus spp. and 74 % with the control treatment (tap water) (Figure 1).

Figure 1 Percentage of germination of seeds of Agave victoriae-reginae T. Moore according to the treatment with respect to time. 

The type of treatment did not influence the germination time (P> 0.345), since the three treatments followed similar temporal trajectories, but there was significant evidence that the use of microorganisms accelerated the GP in the first days, for example, on the 5^th day, the GP with Trichoderma spp. was 29.7 %, a figure higher than that registered with the control on the 10^th day (28 %). Also in the last days of germination, the control reached a GP of 74 % at 26 days, while with microorganisms this value was exceeded at 17 days (79.3 % with Trichoderma spp. and 77 % with Bacillus spp.). During the first 12 days, there were significant differences between the three treatments. After this period, the GPs with the microorganisms were similar, but they did differ in contrast to the control.

Discussion

The total GP of the three treatments were higher than those calculated for Agave mapisaga Trel. (70 %) and A. angustifolia Haw. subsp. tequilana (F.A.C. Weber), with 28 % germination (^{Ramírez et al., 2016}), in which no pre-germination treatments with microorganisms were used. Also, the seeds of A. victoriae-reginae were viable after one year of being collected. Since metabolic processes such as perspiration and metabolisms that cause natural aging occur in stored seeds, and, consequently, viability tends to decrease over time (^{Moncaleano et al., 2013}), it is possible that if A. victoriae-reginae seeds are sown during the year of collection, they might achieve higher GPs. However, the results of this study suggest that it is possible to store the seeds for at least one year, and have acceptable GPs.

In addition, under the environmental conditions during the experiment, it can be stated that seeds did not show dormancy, characteristic of other Agave species from arid areas, such as Agave lechuguilla Torr. (^{Freeman et al., 1977}) and Agave parryi Engelm. var. parryi (^{Freeman, 1975}). The absence of dormancy in the evaluated seeds in this study is similar to that known for seeds stored for two years of Agave salmiana Otto ex Salm-Dyck, which had lower dormancy than those sown in the same year of collection; such differences are attributable to the effect of the storage temperature (^{Peña et al., 2006}).

The germination start time was similar to that reported in another study with A. victoriae-reginae (^{Sánchez et al., 2017}), which varied between 3 and 4 days. The time after sowing significantly influenced the GP in which significant differences were identified between the three treatments before 12 days, expressed in a period of acceleration of germination; meanwhile, in the second half of the evaluated time there were no differences between Bacillus spp. and Trichoderma spp., but they were found with the control. This behavior is similar to that of other Agave species, with a fast-growth period followed by a saturation of the germination curve (^{Ramírez et al., 2012}; ^{Ramírez et al., 2016}).

The increase in GP during the first days of the experiment could be attributed to the fact that the largest seeds could have been the first to germinate, as they were genotypically the most vigorous (^{Sánchez et al., 2011}). Another factor could be the high moisture due to the covered container used, which favored the ideal humidity conditions to promote germination (^{Castillo et al., 2014}).

The response of A. victoriae-reginae to the microorganisms added as pregerminative treatments was satisfactory, indicating that the use of Trichoderma spp. and Bacillus spp. can improve the nursery production of this species. These results coincide with those recorded in Opuntia streptacantha Lem, another plant from semi-arid areas, in which fungi have the ability to dissolve the testa, without affecting the endocarp, which facilitates the emergence of the embryo (^{Delgado-Sánchez et al., 2010}). In addition, fungi and bacteria release radical exudates, among which are sugars, mucilage, organic acids and amino acids, which can provide nutrients for the embryos, and even promote the subsequent growth of seedlings (^{Ahmad et al., 2008}; ^{Gómez et al., 2013}).

In addition, some of the biocomposites produced by these inoculants, such as antimicrobial substances or that stimulate the seedling's immune system, inhibit the development of phytopathogens, which represents an advantage for new seedlings (^{Guillén et al., 2006}). Therefore, the management of Trichoderma spp. and Bacillus spp., widely used in agriculture (^{Moreno et al., 2018}), is highly recommended to promote the germination of A. victoriae-reginae seeds, since they increase the GP.

In forest plantations with coniferous species, the incorporation of microorganisms such as mycorrhizae has also shown increases in seedling survival (^{Gómez et al., 2013}). This confirms the great role that microorganisms play during the first stages of the life cycle of plant species such as pine trees and also agaves, through biotic interactions such as mutualism (^{Sieber, 2007}). For this reason, it is of vital importance to introduce this type of microorganisms in the production of forest species in nurseries (^{Ortega et al., 2004}).

Because the seeds of A. victoriae-reginae respond favorably to the use of Trichoderma spp. and Bacillus spp., the nursery production of this species is feasible, which means a great opportunity for the recovery of the populations of this endangered species. In subsequent studies, it will be important to evaluate the effect of other environmental conditions, such as humidity, temperature and light, on the germination of the seeds of this species and others at risk.

Conclusions

The germination of seeds of A. victoriae-reginae responded favorably to treatment with Trichoderma spp. and Bacillus spp., increasing the PG. These characteristics represent a great opportunity for the assisted reproduction of this species at risk and the recovery of natural populations in the long term.