Sporocarp collection

Boletus luridellus species were collected from various localities of the states of Nuevo León and Tamaulipas and examined for the herbarium; their morphological and ecological data and their color in fresh state were recorded (^{Maerz and Paul, 1930}). The microscopic analysis of the material was conducted using a Primo Star Carl Zeiss compound microscope with an ocular micrometer, KOH (5 %) and Melzer’s solution. The identification of the material was based on the studies by ^{Singer (1986)}, ^{García (1999)}, and ^{Bessette et al. (2000}; ²⁰¹⁶).

In vitro culture

The strains were isolated in a laminar flux bell; small pieces of tissue of the context of young sporomes were removed using fine sterile tweezers and opened longitudinally with a sterile scalpel. They were placed on several lines on Melin-Nokrans solid culture medium with a pH of 6.5 in Petri dishes (^{Garza et al., 2014}). The dishes were incubated at 25 ^oC in the darkness.

Having isolated the four strains in a pure culture, an experiment was conducted in order to measure the radial growth on a daily basis, during one month, in vitro in the Melin-Norkrans culture media and Malt Extract Agar. Ten replications were utilized for each strain and culture medium (a total of 80); pH was adjusted to 6.5, and growth was measured in orthogonal lines marked at the base of the Petri dishes.

A variance analysis (ANOVA) and a mean comparison test (p≤0.05) were made using the SPSS statistical software package, 2.0 version, in order to assess the significant differences in the relationship between the media and the strains.

Production of mycorrhizal inoculum

The four strains were used individually in the mycorrhization of oak seedlings; inoclulant was produced from each of these, in 500 mL wide-mouth jars, with vermiculite and peat moss (proportional volume 4:1), moisturized (to 70 %) with a modified Melin-Norkrans liquid culture medium (^{Garza et al., 2014}). The jars with the substrate were sterilized at 120 ^oC during 40 minutes; they were then left to cool and were later individually inoculated with fresh mycelium obtained from the Petri dishes with Melin-Norkrans solid medium.

The jars were incubated during five weeks at 25 ^oC, in the dark, and shaken on a weekly basis in order to accelerate the colonization process of the substrate.

Germination of Quercus fusiformis seeds

The acorns were obtained from trees previously established in the gardens of the Facultad de Ciencia Forestales de la Universidad Autónoma de Nuevo León, UANL (School of Forest Science of the Autonomous University of Nuevo León). They were superficially sterilized with H₂O₂ at 30 % during 15 minutes; they were subsequently washed with sterile distilled water in three 15-minute cycles in order to eliminate the excess peroxide. The germination was carried out in Petri dishes, in water with 1.6 % agar, placed in the dark in a Shellab^TM LI20 incubator at 25 ^oC during one week.

Inoculant substrate mix

The inoculant substrate in each strain consisted of a mixture of 50 % inoculant washed with sterile distilled water and 50 % sterile vermicompost soil. The sterile acorns which germinated during one week were placed upside down, half buried in the substrate in groups of 10 in each 250 mL styrofoam container cup, adding up to a total of 100 cups and 1000 acorns. 25 container cups (250 seedlings) were inoculated with each strain; the bottom of each cup was pierced at four points using a sterile scalpel in order to favor drainage. The cups were then placed in a nursery during six months (November to April) at an average temperature of 26 ^oC and relative humidity of 60 %.

Mycorrhization percentage

The percentage of mycorrhizae in the root system of the plants was determined through random sampling of 10 container cups, i.e. 100 seedlings per strain (adding up to a total of 400). The complete root systems were drawn from the cups and washed in the laboratory with distilled water in order to eliminate all residues of soil particles. The tips of the roots with mycorrhizae were counted using a Zeiss Stemi 2000C stereomicroscope. Their morphology was observed under a Primo Star Carl Zeiss microscope; fine cross-cuts were made in order to observe the Hartig network and measure the fungal mantle (^{Brundrett et al., 1996}).

Taxonomic description

Boletus luridellus (Murr.) Murrill Mycologia 30:525. 1938.

Synonymy

Ceriomyces luridellus Murr. Mycologia 30: 523.1938.

Ceriomyces subsensibilis Murr. Mycologia 31: 111. 1939.

Boletus subsensibilis Murr., Mycologia 31: 112. 1939.

Convex pileus with a 36 to 105 mm diameter and a smooth, viscid to partly tomentous surface of an orange to reddish brown (6E8 - 7E8 K. & W.) or yellowish brown hue (5D7 - E7) (^{Maerz and Paul, 1930}), and with slightly appendiculate margins.

Subattached hymenophore with 5 - 12 mm long tubes, of an olive yellow color, stained blue when exposed; angular, with pores of a 0.3 - 0.7 mm diameter, of a 3C8 yellow color, stained blue when exposed.

75 - 110 × 13 - 29 mm subcylindrical stipe with an acute base and a strongly reticulated surface, except in the lower third, where it is smooth or slightly pruinose, of a 4B6 K. & B. yellow color (^{Maerz and Paul, 1930}) in its upper part, and reddish purple, reddish brown or copper red at its base; the reticulation is concolored with the surface of the stipe; it stains blue rapidly when touched.

Context 1.2-2.0 cm thick, yellow, stains blue rapidly when exposed, with a slightly acidic or sweet fruity odor and taste, according to the ripeness of the specimens.

The KOH on the pileus reacts producing a reddish brown color, with an orange hue in the context and surface of the stipe, and with orange brown pores. The NH₄OH on the pileus produces a dark brown color, with a somewhat ochre-like color, dark olive brown pores and orange to olive-colored stipe.

Spores sized (11.2-) 12 - 15 (-19) × 4 - 5.6 (-6.4) μm, ellipsoid to fusoid, smooth, some with a subacute apex or inversely attached; some with a thick wall of up to 1 - 1.2 mµ, of an olive brown color in KOH, and orange brown in Melzer’s solution. Olive brown spore print.

Basidia sized 20 - 32 × 8 - 12.8 μm, hyaline in KOH, and with a granular content in Melzer’s solution. Pleurocystidia sized 26 - 48 × 8 - 10 μm, fusoid-ventricose, with medium-sized necks and a subacute apex, hyaline or yellowish in KOH and in Melzer’s solution.

Cheilocystidia sized 32 - 40 × 7.2 - 9 μm, fusoid to somewhat ventricose, with a subacute apex, of a pale yellowish hue in KOH and in Melzer’s solution.

Bilateral trama of the tubes, with a middle layer of somewhat gelatinized hyphae and a lateral layer of tubular hyphae, both with hyaline cells in KOH and in Melzer’s solution, and with a 3.5 - 6 μm diameter.

Epicutis formed by a trichoderm of interwoven hyphae, with terminal elements of cylindrical, claviform or cystidioid hyphae, most of them with a rounded apex, sized 36 - 77 × 6.4 - 11.2 μm, with a few short, intermingled subglobous to claviform elements, sized 26 - 32 × 10.4 - 13 μm, hyaline or of a pale yellow color in KOH and orange in Melzer’s solution.

Stipe surface formed by a layer of cystidioid subglobous cells, some of them of a sphaerocyst type, claviform or fusoid-ventricose, hyaline or of a pale yellow color in KOH and in Melzer’s solution, and sized 20 - 36 × 5.6 - 12.8 μm. Some tetrasterigmate caulobasidia, sized 20 - 26.5 × 7.2 - 12 μm and hyaline to pale yellow in KOH and Melzer’s solution, are also present.

Habitat

Solitary to subrgregarious in Quercus forests, particularly beneath Quercus fusiformis, Q. polymorpha and Q. canbyi, at altitudes of 300 to 1 600 m (Figure 1).

Scale 10mµ.

a= Spores; b = Basidia; c = Cystidia; d = Pleurocystidia; e = Cheilocystidia.

Figure 1 Microscopy of Boletus luridellus (Murr.) Murrill. 

Geographic distribution

The species is distributed from North Carolina and Florida to southern Texas. In Mexico, it is known in Nuevo León and Tamaulipas, and possibly in other northeastern states as well (Figure 2).

Figure 2 Known distribution of Boletus luridellus (Murr.) Murrill in the states of Nuevo León and Tamaulipas, Mexico. 

Edibility

Boletus luridellus is edible, according to ^{Metzler and Metzler (1992)} and ^{Bessette et al. (2016)}. There is no history of consumption of this or other Boletaceae species of the northeastern region of Mexico.

Examined material

Nuevo León: Linares municipality, Universidad Autónoma de Nuevo León, Facultad de Ciencias Forestales. 15-X-2011, 10-X-2012; 12-X-2014; 15-10-2016; 9-X-2017. Garza 3900, 4025, 4189, 4230, 4600(CFNL). Nuevo León: Santiago municipality, El Ranchito, 14-IX-1979, García 59 (UNL, ITCV) El Cercado, 10-X-1984, A. Moreno s/n (ITCV), Puerto Genovevo, 14-X-1979, García 63 and Marmolejo 30 (UNL, ITCV). Tamaulipas: Victoria municipality, Cañón de La Libertad, 14-IX-1991, García 7405 (ITCV); the same municipality, km 155 of the Tula - Cd. Victoria road, Altas Cumbres ejido, 15-IX-1991, García 7433; Las Mulas ejido, 13-IX-2008, García 17,468 (ITCV). Casas municipality, km 80 of the Cd. Victoria-Soto La Marina road, 1-VII-1993, García 8530,1-X-1993; García 8751, 6-X-1993; García 8796, 27-VI-1994; García 8949 and 8951, 24-V-1997; García 10,580 and 10,586 (all at the Instituto Tecnológico de Ciudad Victoria (Technological Institute of Ciudad Victoria), ITCV. Victoria municipality, 30-VIII-1998, García 11050 and 11053 (ITCV); road from Huizachal to Puerto Arrazolo, 29-VIII-1998, García 11002 (ITCV). Victoria municipality, Cañón del Novillo, 21-X-1992, García 8280(ITCV); Jaumave municipality, near Rancho La Reja, 8-IX-1994, García and Martínez Ávalos 9028-D.

In vitro growth

Multiple mean test for differences among strains

The results of the ANOVA show that there are significant differences in the effect of the strain*medium interaction with a model F value F= 482.78, and significance values below (P<0.05.) with F=81.05 for the effect of the strain*medium interaction. Tukey’s test evidenced that the treatment of strain 4 with the Melin-Norkrans medium was the best, as it exhibited the greatest growth of the Boletus luridellus colony (Figure 3). Table 1 shows the results of the analysis of the test of between-subjects effects.

Scale 2 cm.

Figure 3 Pure in vitro culture of Boletus luridellus (Murr.) Murrill in Melin-Norkrans medium at 15 days. 

The results for growth in a pure in vitro culture prove that the four strains grow better in the Melin medium than in Malt Extract. The monthly average growth of strains 4, 1, 3 and 2 in the Melin-Norkrans medium was 3.9 cm, while the average monthly growth observed in Malt Extract was 2.8 cm. Strain 4 had a higher growth in both culture media, and strains 2 and 3 registered the lowest values in the Melin-Norkrans medium and in Malt Extract, respectively. The growth rate differed significantly between the four strains and in the two culture media utilized (Figure 4).

Figure 4 Average growth (cm) in vitro of the four Boletus luridellus (Murr.) Murrill strains. 

Description of the strains

Mycelium growth in vitro was observed to be somewhat shiny, of a brown color and releasing a dark brown pigment around the colony, while the agar adopts a pale ochre hue in the Melin-Norkrans medium (Figure 3). The colony has a semicircular growth, mainly on the surface on top of the agar, although it also exhibits this growth into the medium; however, the mycelium does not penetrate all the way to the base of the dish. The superficial mycelium in the colony is compact and tight at the center of the colony, and lax or loose in its most aerial part.

The colony has a light ochre color at its margins, while its center is darker, with dark brown droplets; the agar takes on a light ochre hue at the edges of the colony. The hyphae have a diameter of 2 - 3.5 μm; they are hyaline in a 10 % KOH solution, and yellow in Melzer’s reagent; they are smooth and branched, septated, with sparse fibulae; the walls of the hyphae are thin, barely 1μm thick.

Production of fruits and ectomycorrhizae

Sporomes were produced in 75 % of the containers. The mycorrhizae are shiny and brown, monopodial and branched; they exhibit a well developed fungal mantle of a brown color and a thickness of 120 μm; the mantle has compact hyphae with no emerging cells and is dextrinoid in Melzer’s reagent. Development of the Hartig network was observed.

Colonization

The average percentage of mycorrhization in the root system was 75-100 %. Formation of mycorrhizae in 25 % of the tips of the root and the presence of a scarcely developed fungal mantle were observed (figures 5 and 6).

Scale 1 mm

Figure 5 Branched mycorrhizae and mycelium of Boletus luridellus (Murr.) Murrill and Quercus fusiformis Small.  

Figure 6 Production of Boletus luridellus(Murr.) Murrill sporomes in Quercus fusiformis Small seedlings inoculated in a nursery at six months of the inoculation. 

Boletus luridellus grows by associating with Quercus virginiana, Q. fusiformis and Quercus polymorpha in Nuevo León and Tamaulipas. it is likely that it may be associated to other species of oaks of the northeast region of the country. In the United States of America, it occurs in North Carolina, southern Florida and western Texas (^{Bessette et al., 2016}).

The oak species mentioned above have been planted in many urban and suburban areas in Nuevo León and Tamaulipas and they also exist in commercial nurseries in the citrus producing area of the state of Nuevo León.

This macromycete is mycorrhizal, and its presence in Texas has been cited by ^{Metzler and Metzler (1992)} and by ^{Bessette et al. (2016)}, according to whom it is edible and has an exquisite flavor. Boletus luridellus does not involve any toxicity issues, unlike the Scleroderma texense Berk. S. Pers. strain, which grows in gardens, squares, and recreational centers in urban and suburban areas of Nuevo León, where various species of oaks have been planted.

The fungal material collected in Nuevo León has a slightly acidic taste in young specimens, and a somewhat sweet taste in mature specimens. It is edible, and only young individuals that are free of insect larvae are recommended for consumption. Abundant sporomes weighing 1.5 to 2 kg per tree are produced in inoculated trees growing in gardens. The inoculation procedures would have positive repercussions in order to reforest with preinoculated seedlings that may later produce edible sporomes of the selected species.

Cistus ladanifer L. (gum rockrose or labdanum) seedlings are inoculated in vitro in Spain; these belong to the family Cystaceae and are obtained from the culture of tissues with Boletus edulis, of which experimental plantations have already been made quite successfully in terms of the production of sporomes (^{Agueda et al., 2006}; ^{Mediavilla et al., 2016}).

Boletus luridellus was registered for the first time in Mexico by ^{García (1999)}, and this study documents the first record for Nuevo León.

There are no previous differences for the species in regard to their culture. The obtained strains produce a colony of dark brown mycelia and dye the agar dark brown beneath the colony in both media; these results are similar to those obtained for Boletus luridus Schaeff. (^{Iotti et al., 2005}). This dyeing characteristic also occurs in other macromycete cultures obtained by the first author, such as Suillus lakei (Murr.) A.H. Sm. & Thiers and Suillus granulatus (L.) Russell or Exsudoporus frostii (J.L. Russell) Vizzini, Simonini & Gelardi, besides other species like Pisolithus arhizus (Scop) Rasuchert. ^{Díaz et al. (2009)} studied certain species of Boletaceae in vitro and succeeded in producing small primordia, although they did not conduct experiments at a nursery to produce sporomes. ^{Pantidou (1961}, ¹⁹⁶²) obtained them thus in vitro from the saprobe fungus Phlebopus sulphureus (Quél.) Singer and from P. lignicola (Kallenb.) M. M. Moser ex Groves. ^{Chung et al. (2010)} inoculated Pinus radiata D. Don seedlings with Boletus edulis Bull. and B. pinicola (Vittad.). In Chile, Vassilkov, C. R. determined that the mycorrhizae persisted in the field after 24 months, and that these seedlings had better growth responses than those that were not inoculated.