The plants of the Agave genus represent a valuable non-timber forest resource for soil recovery, particularly in arid and semi-arid zones. The leaves (pencas) of agaves are covered with a cuticular layer that performs relevant functions, such as: gas exchange, control of temperature changes, as a barrier against pathogens, among others (^{Tafolla-Arellano et al., 2013}). The cuticular layer that covers the leaves of Agave salmiana Otto ex Salm-Dyck provides highly appreciated characteristics in order to cook some traditional-food dishes; it is made up of epidermal cells, cutin, polysaccharides, waxes and stomata (^{Reina-Pinto and Yephremov, 2009}; ^{Staiger et al., 2019}; ^{Pérez-España et al., 2019}). Stomata are formed by two guard or occlusive cells, which give rise to a pore called an ostiole. Guard cells control the opening and closing of ostioles in response to hormonal and environmental signals to regulate gas exchange (^{Zeng and He, 2010}; ^{Biswapriya et al., 2015}). Agaves have stomata on the abaxial and adaxial face of the leaves (^{Hernández-Valencia et al., 2003}) that open at night and remain closed during the day to prevent excessive loss of liquids in the plant (^{Lee, 2010}).

Phytopathogenic fungi use different mechanisms to colonize plants (^{Gudesblat et al., 2009}) such as: adhesion of spores to the cuticular layer, formation of appressoria and adhesive, invasive and infective structures, development of a high pressure of turgidity in the invaded cells (^{Mendgen et al., 2016}) and degradation of the cell wall and the cuticular layer through the secretion of enzymes. Other phytopathogens have the ability to modulate the opening or closing of stomata through the release of chemical compounds (^{Staples and Macko, 1980}; ^{Lee et al., 1999}; ^{Bury et al., 2013}).

At present, A. salmiana plants show a disease known as smallpox or “negrilla”; caused by the Asterina mexicana Ellis & Everh. fungus (Ellis y Everhart, 1900; ^{Cesaveg, 2008}), of which there is little information. This disease causes dark spots on both sides of the leaves and can lead to necrosis and dryness. The objective of this work was to describe the importance of the cuticular layer during the colonization of the fungus that causes the negrilla in Agave salmiana Otto ex Salm-Dyck ssp. salmiana.

The cuticular layers were obtained from leaves of agave plantations in Pachuca de Soto, Apan, Cardonal (state of Hidalgo) and Calpulalpan (state of Tlaxcala) municipalities, from February to June 2019. Three leaves which carried symptoms of the disease were collected per ~8-year-old plant (according to the experience of the producers) by municipality. From the adaxial face of each penca, a sample was taken from the apical, middle and base sites (Figure 1a). Cuticular layer samples were removed from the collection site and kept in a 2 % glycerol solution until they were analyzed.

Figure 1 Cuticular layer of Agave salmiana Otto ex Salm-Dyck ssp. salmiana, (a) Extraction of the cuticular layer in three areas of the adaxial face: 1) distal area, 2) middle area and 3) proximal area; (b) Adverse of stomata, guard cells and epidermal cells; (c) appressoria observed in black spot (4X), (d) dehydrated penca due to invasion of the bold and (e) appressoria obstructing the stomata (SEM). 

Stomatal index (SI) and stomatal density (SD) were determined with an optical microscope (Zeigen, ZB-7300). Cuticles were stained with methylene blue for better definition. The SI was calculated according to what was recommended by ^{Sosa et al. (2014)} from the following formula:

Where:

NE = Number of stomata per observation field

CE = Number of epidermic cells in the same observation field

SD was determined by calculating the number of stomata found in 19.635 mm-2, equivalent to the field observed (40X) in a 2.5 mm radius. The SI and SD results were analyzed with ANOVA and the significant differences (P < 0.05) between the groups with the Bonferroni test (GraphPad Prism Software version 5.00 for Windows, GraphPad Software Inc.).

For the analysis of the microstructure, the cuticular layers were dehydrated in a vacuum oven (Shel-Lab, Mod. 1407) at 40 °C and 45 kPa. Subsequently, they were fixed with carbon tape to an aluminum slide, covered with gold-palladium and observed with a scanning electron microscope (SEM; Model JEOL JMS-5600LV).

The distribution of the stomata in the cuticular layer of the abaxial face showed homogeneity in the analyzed areas. In the micrographs of the cuticular layer of A. salmiana ssp. salmiana with a 121 ± 2.8 mmascomata thickness, the presence of waxes, epidermal cells and stomata was recognized. The average thickness recorded for the cuticle of A. tequilana F. A. C. Weber is 8.67 μm for the abaxial face (^{Hernández-Valencia et al., 2003}).

SD showed homogeneity in the analyzed areas. The tetracytic-type stomata, the size of the ostioles of 57.9 mm ± 5 long and 23.75 mm ± 1.25 wide, and the isodiametric polygonal epidermal cells (Figure 1b) are similar to those reported by ^{Vargas-Rodríguez et al. (2017)} and by ^{Chávez-Güitrón et al. (2019)}. The difference in the size of the ostioles is attributed to variations in the thickness and distribution of the cuticular wax, to the species and to the environmental conditions in which the plants grow (^{García, 2007}; ^{Bernardino-Nicanor et al., 2012}).

Macroscopically, bold disease is seen as gray spots that turn black over time (Figure 1d). Inside the stain, the existence of ascomata with hyphae specialized in the invasion of plant tissue is identified (Figure 1c) that accumulate in the Agave leaf and obstruct the stomata; in addition, other hyphae adhered to the cuticular layer are observed (Figure 1e).

SD (22.67 to 27.67 stomata mm-2) did not show significant differences between the areas of the adaxial face of the analyzed leaves or between municipalities (Figure 2a); the values (mm-2) were similar to those recognized for other Agave species, such as: A. mapisaga Trel in L. H. Bailey (20-50 stomata; ^{Nobel, 1994}), A. deserti Engelm. (34 stomata; ^{Gentry and Sauck, 1978}) and A. atrovirens Karw. Ex Salm-Dyck (29-30 stomata; ^{Bernardino-Nicanor et al., 2012}); but they differ from A. promontorii Trel. (18 stomata) and A. sisalana Perrine (11 stomata; ^{Neto and Martins, 2012}).

Densidad estomática = Stomatal density; Índice estomático = Stomatal index; Apical = Apical; Media = Middle; Basal =Base.

Figure 2 Index and stomatal density in the cuticular layer of Agave salmiana Otto ex Salm-Dyck ssp. salmiana in different municipalities. 

SI for A. salmiana ssp. salmiana is 10.61-14.15 (Figure 2b) while for A. tequilana it is between 5.72 and 6.68 stomata/epidermal cells (^{Hernández-Valencia et al., 2003}). Both parameters (IE and SD) are influenced by some environmental and nutritional conditions (^{Wilkinson, 1979}; ^{Roth et al., 1986}).

In the paradermal and transverse sections, it was noted that the cuticular layer acts as a mechanical barrier and prevents the passage of the reproductive structures of the pathogenic fungus into the interior of the Agave leave. With the SEM micrographs, it was detected that the hyphae and appressoria of the phytopathogen only appear on the obverse side of the cuticular layer since they could not be identified on the reverse side. In addition, it was observed that the appressoria are located mainly in the stomata, which suggests that the mechanism of invasion of this fungus consists of drying the leaves through the modulation of the stomata, by preventing them from completing the cycle (day-night) in a normal way for affected plants.

SEM micrographs show the importance of the cuticular layer as a mechanical barrier by preventing hyphae from entering the innermost layers of the maguey leaf. Therefore, the infection is restricted to the obverse side of the cuticular layer.

On the other hand, SD coincides with the records for other species of the Agave genus. However, this is the first report on the importance of the cuticular layer of Agave salmiana ssp. salmiana during its interaction with the phytopathogenic fungus responsible for the bold disease. Complementary studies are necessary to propose alternatives to prevent or control fungal invasion.