Isolation, Identification, and Preserval of Filamentous Fungi Found at Soils of Viotá, Cundinamarca

Objectives: This study focuses on the isolation, identification, and conservation of strains of filamentous fungi recovered from soils of Viotá, Cundinamarca; and the subsequent conformation of a strain collection. Methods: An adaptation of the sampling method proposed by Arias and Piñeros was carried out. Identification was carried out by macroscopic, microscopic, and DNA sequencing. Conservation techniques were evaluated based on the index of effectiveness of the behavior of viability, purity, and morphological stability. An ANOVA TUKEY test was performed using both an alpha value of α = 0.05 with 95% confidence to assess significant differences. Results: Of the 125 samples analyzed, 165 isolates were recovered, and nine species were identified: Trichoderma spirale 15.53%; Trichoderma ovalisporum 13.66%; Hypocrea nigricans 13.04%; Penicillium implicatum 13.04%; Penicillium citrinum 11.80%; Fusarium incarnatum 11.18%; Fusarium verticiloides 9.94%; Cunninghamella elegans 6.83%, and Aspergillus cf. versicolor 4.97% that had the lowest number of isolates. Aspergillus, Penicillium, Trichoderma, Fusarium, and Cunninghamela are among the most common genera isolated soils. The most efficient conservation method was cryopreservation, with an efficiency rate of 100%. However, although the cultivation technique had an efficiency percentage of 70%, it is considered that it can be used in collections with limited financial support or as a backup preservation method. Ángel, in 2006 supports the use of serial cultivation for periods of time such as those handled in this study (15 days). Application/Improvements: The results obtained will contribute to the knowledge of the diversity of this group and its biotic potentials; for the generation of strategies that promote the development of this region.


Method of Sampling
Five samples were taken between the months of October 2016 and May 2017 in which the diagonal sampling pattern [5] was applied, consisting of a zig-zag displacement in 5 m 2 quadrants and obtaining 22.5 cm 3 of corner soil and the center of the quadrant with a hole, as shown in Figure 1. The samples were transported in hermetic bags in a refrigerated container to the INBIBO Biology Research laboratory at Universidad El Bosque for analysis.

Laboratory Phase
In the laboratory, the samples were cleaned by extracting the waste material and then sieving (2 mm pore) to determine pH, moisture percentage, and isolation in selective agars.
The pH of the samples was measured potentiometrically by mixing 20 g of soil in distilled water at a ratio of 1:1.25.
To determine the percentage of moisture, a gravimetric method was used, which consists of taking 10 g of sample to dry in an oven at 110 °C for 24 hours. The moisture percentage was obtained by dividing the difference of the wet soil mass minus the dry mass over the dry mass, multiplied by 100 [5].

Isolation of Strains
A modification was made to the strain isolation method referenced by Arias and Piñeros [5]. A solution of 100 ml of pre-enrichment of 0.005 mg/ml of biochemical chloramphenicol, Tween 80 at 5% and 1% of peptone and 10 g of the soil sampled was used. Subsequently, they were kept in constant agitation at 150 rpm for 48 hours. They were then subjected to static incubation at 28 °C for 7 days. Subsequently, triplicate isolation was performed in PDA, incubated at 28 ° C for 7 days.

Identification of Isolates
The identification was carried out by macroscopic observation of aspects such as color and texture on both the reverse and the front of the petri dish. For microscopic identification, lactophenol blue staining was used using the adhesive tape printing technique; taxonomic keys. In addition, DNA and PCR extraction tests were carried out to amplify the ITS regions of the isolated species for subsequent sequencing to corroborate what was found.

Conservation Methods
For the conservation of the strains, inoculums from the samples supplemented in the chloramphenicol solution were used. They were kept in constant agitation at 150 rpm during a period of 5 days to achieve an inoculum concentration of 3 × 10 8 spores/ml. This concentration was confirmed by the Neubauer chamber count.
From these inoculums, tests were performed to evaluate the two conservation methods; inclined tube cultivation with PDA and Sabouraud agar (SAB), and cryopreservation. In order to verify the results obtained, the effectiveness of each conservation method was determined by calculating the percentage of successful trials in the tests carried out, multiplying it by 100, and thus choosing the appropriate method to form the filamentous fungi collection.

Serial Cultivation Method
For the serial cultivation, the cultivation was made in PDA and SAB simultaneously, from inoculates of each species with a concentration of 3 × 10 8 spores/ml, in quadruplicate in an inclined tube to avoid the drying of the cultivation medium. These were stored at room temperature for 7 days without completely sealing the tubes as aerobic organisms. Once the 7 days are fulfilled, the samples are transferred to refrigeration at 4 °C to delay the aging of the strains and to postpone the period between the reseeding to 15 days, adapting the method used by Angel in the evaluation of conservation techniques for filamentous fungi and yeasts [6] for a total of 6 repetitions.

Cryopreservation Method
For cryopreservation, the microorganisms were preserved in cryogenic vials in liquid nitrogen, from cultivation that was at the beginning of the stationary phase of growth of the strain in the inoculum, using 10% glycerol v/v as cryoprotector to avoid cell damage and decrease the freezing point allowing a quick freezing [7][8]. This has as premises to use cells that were in the stationary phase of growth; to maintain a short time during the freezing and thawing of the vials t its last one to a temperature of 37 °C. After one month, the criterion was evaluated by recovering three of the five cryogenic vials of each species in PDA and SAB, in triplicate, incubated at 28 °C for five days [6].

Evaluation Criteria
The parameters established were viability: formation of typical colonies in the cultivation medium [9]; purity: only typical colonies of evaluated isolation are recovered [6]; and morphological stability: the strains maintain the macroscopic and microscopic characteristics [6] corresponding to those defined during their isolation and identification.

Conformation of the Cepary
Finally, in order to deposit the strains, an admission format and technical sheets were designed with the pertinent information of the isolated and identified species, together with a protocol for the entry of material and the maintenance of the strains. An Arduino DTH111 (Arduino) sensor was also programmed and installed to control the temperature and relative humidity conditions of the strain collection.

Statistical Analyses
For statistical analyses, once the data on the number of isolates vs. sampling times are established, as the assumptions of homogeneity of variances and normality are fulfilled, an ANOVA test is performed in conjunction with a TUKEY test using in both an alpha value of α = 0.05 with 95% confidence to evaluate significant differences. In the conservation process, the percentage of effectiveness of each conservation method was evaluated for each species and for all of them, understanding the effectiveness as the capacity of each method to present the expected results of maintaining viability, morphological stability, and purity.

Characteristics of Filamentous Fungal Species
The isolated strains were identified considering their microscopic and macroscopic morphological characteristics. Each of these characteristics is contrasted with other characteristics reported by different authors in the tables: • Aspergillus cf. versicolor: Figure 3a: the colonies of slow growth, small, that are initially of whitish color and from the fifth day to acquire a very abundant yellow pigmentation and an incrustants aspect, although when the sowing in dilution its growth can occupy all the means. The conidia are small and abundant up to 2 microns and the conidiophores are long and very hyaline, as seen in the table the description of the species made by the authors is similar in comparison to the description of Arias and Piñeros in Czapeck agar [5] (Table 1).

• Cunninghamela elegans (Lendn. 1905):
Description, Figure 3b: the colonies show a rapid growth in PDA, in the first 48 h the colonies occupy three quarters of the box with a diameter of 7 cm, after 72 h they can occupy the entire box. Its color is initially white, and the passage of time acquires the color gray storm. Mycelium without septa. The sporangiophores have a variable size; they are usually abundant, with a width of between 2.7 and 3 μm in the basal part and up to 7 μm in the apical part. The sporangioles are globose or pyriform, slightly abundant grayish or measured between 2 and 4 μm. As can be seen in Table 2 for the authors' species descriptions compared to the Lendn strain description in PDA [11].

• Fusarium incarnatum:
Description, Figure 3c: Slow growing colonies, 1 cm at 7 days in PDA, initially white, flocky, cream color on the inverse, with salmon pigmentation as the vine ages. With hyaline hyphae, long partitions and thin conidiophores, with polyphialideslides, although also with simple phialides, the macroconidia have 3 septa, are straight and elongated, and can measure up to 9 μm, the description of the species, made in this text agrees with the description of Carrillo in PDA [12] in Table 3.

• Fusarium verticillioides:
Description, Figure 3d: Colonies of fast growth, cottony, of 3 cm of diameter to the first 48 h, of 4.5 cm to the first 72 h, with abundant aerial mycelium. Coloration initially white, then violet. Pigment violet agar. Cream back. Conidiophores densely branched, septa rarely evident. Macroconidia are abundant, almost always between 7 and 10 µm, predominance of globose chlamydiapores. As can be seen in Table 4 for the description of the species made by the authors, which agrees with the description of Arias and Piñeros in PDA [5].  Description, Figure 4c: After the first 96 h it already occupies the middle completely in PDA. Its color is bright white with a dusty blue-green center. After one week, it has a green disc on the outermost part, with rigid and whitish pustules. With a cream-colored back, the yellow agar is slightly pigmented. It has cylindrical phialides with globular conidia of smooth walls. The description of the species by the authors corresponds to the description of Kamala and his collaborators [13] in Table 5.   smooth walls. As can be seen in Table 6, the description of this species made by the authors of this text agrees with the description made by Arias and Piñeros in Czapeck agar [5].
• Penicillium implicatum (Biourge, 1923): Description, Figure 4b: Small colonies of slow growth in PDA, velvety, initially whitish later with greenish center and white and thick irregular edges, produce pigmentation in the agar from the first 24 h. The conidia are short and tend to be biverticillated, although it is common to observe monoverticillates, with long metulas, one per branch, to which up to 8 cylindrical phialides can be attached, the conidia are globose with thick walls and roughness, as observed in Table 7 for the description of the species by the authors in accordance with the description of Arias and Piñeros in Czapeck agar [5].  • Trichoderma ovalisporum: Description, Figure 4d: Rapidly growing colonies, initially white and flocky, but which quickly (48 h) acquire a greenish powder coating, presenting rigid pustules. The conidiophores are long, with phyalides paired in the shape of a flask, produce abundant green globular conidia with thin walls that tend to accumulate. As can be seen in In Czapek agar, colonies grow slowly after 7 days and the diameter becomes 0.5 cm. Green color with wide white and irregular border.
Czapek agar colonies grow slowly and reach a diameter of 1.5 cm in 7 days. Light green to bluish green, yellow back. Colony round, no exudation.
2 μm wide and up to 60 μm long, smooth walls and hyalines.
a Description of Arias and Piñeros in Czapeck agar.

Growth and development in agar.
In slow-growing, reducedgrowing czapeck agar colonies less than 3 cm in diameter, accumulation of spores in the centre, slightly greenish in colour, inverse cream in colour.
The colonies grow very little and slowly reach 1.5 to 2 cm in diameter in 7 days. The thin white margin produces abundant spores of light and dark green color, powdery texture, exudation of limited transparent color. Back cream color with coffee center. Conidiophores.
Globular conidia with smooth and thick walls or with slight roughness. Table 8, the description of the authors' species matches the description of Kamala and his collaborators in PDA agar [13].
• Trichoderma spirale: Description, Figure 4e: Cotton colonies, slow growth, initially whitish, from the ninth day shows a change from color to light green. Agar pigmented yellow, white on the back with the inside cream to dark brown. As seen in the description in Table 9 made for this species

Fungus Behavior in Relation to pH
Additionally, to understand the behavior of these species, the pH and average moisture percentage were recorded in Table 10, followed by the number of isolates for each sampling period shown in Figure 5. Initially, for the months of October 2016 and March 2017, an average of humidity was evidenced in relation to the other sampling periods. In April 2017, neutral pH values were recorded.
Similarly, in the number of isolates for each species per sampling period ( Figure 5), you can see how many isolates were recorded on the dates of the sampling events, generally for the species of the genus Trichoderma recorded between four and five isolates, for the genus Fusarium between two and five isolates, for Penicillium between two and six,  Cunninghamela and Aspergillus between one and four isolates on the five dates the soil samples were collected.
On the table of average values for pH and humidity percentage (Table 10) for the March sampling event in which the pH is 6.31, slightly acidic, and the average percentage of humidity is 34.12% up to 10 isolates were recorded, while for the next time when the pH is slightly neutral and the humidity is higher, 45.85% did not register any. For Cunninghamella elegans, the highest number of isolates was on March and none for April. Otherwise, for Aspergillus cf. versicolor for the month of March there were no isolations, for the next date, which corresponds to the month of April, it had the highest number of isolations, corresponding to three. The number of isolations registered in relation to the sampling times statistically does not present significant differences with a significance of P = 0.391. Aspergillus, Fusarium, and Trichoderma present a high tolerance so they can grow in a variety of conditions and can be easily isolated in different environments [4], in the case of Cunninghamella they can grow and sporulate quickly and easily in environments that are rich in carbohydrates and in soils [14].

Serial Cultivation
The nine isolated and identified species were preserved in La Universidad El Bosqueceparium collection. Seven of the nine species were conserved under the serial cultivation method in an inclined tube and all species in cryopreservation, the results show the relevance in the use of each method according to the requirements for the maintenance of each species.
Fusarium incarnatum and Aspergillus cf. versicolor could only be recovered by purifying the vine; in the case of Fusarium incarnatum, it was only possible to preserve it in petri dishes with PDA and by cryopreservation. In the case of Aspergillus cf. versicolor, after recovering the vine, it was possible to continue preserving it through serial cultivation in both PDA and Sabouraud agar, in addition to cryopreservation.
Of these nine species analyzed, only seven had an efficiency percentage higher than 70.83% under this method, viability higher than 79%, purity higher than 83% and for the tubes without contamination showed a morphological stability higher than 77%, both in PDA and in Sabouraud agar (see Figure 6a). The contamination generated in some of the tubes for Cunninghamella elegans and Penicillium implicatum were yeasts of the genus Rhodotorula.
Fusarium incarnatum showed contamination after 15 days, in both agars, in all tubes with another fungus of the genus Penicillium, 15 days later it was no longer viable. In contrast, Aspergillus cf. versicolor lost purity in the second trial and viability in the third trial, with the effectiveness of this method in these two species being only 41.67% and 16.67%, respectively, in PDA (Figure 6b).
It can be seen that, in relation to purity, there is only a difference of 0.46% between the agars (Figure 6a), being slightly greater when singing the PDA. Morphological stability is greater when the growth in PDA to preserve it by subcultivation in periodic series, with a difference of 1.39% using Sabouraud agar. On the contrary, for viability, a difference of 0.46% was greater in Sabouraud agar than in PDA.
For the first trial, Cunninghamella elegans, showed incomplete growth in the PDA, the remaining species grew completely in most of the tubes. As for Sabouraud agar, Fusarium incarnatum was viable only in two tubes, but was not pure, and Trichoderma spirale grew in three out of four tubes, the rest of the strains were viable and pure in most of the tubes.
For the second trial, the only morphotype in PDA not showing viability was Fusarium incarnatum, all other morphotypes showed growth, by Aspergillus cf. versicolor a purity of 50% was obtained. Similarly, in Sabouraud agar, Fusarium incarnatum showed no viability, exclusively Cunninghamela elegans and Trichoderma spirale, showed no growth in a single tube and Aspergillus cf. Versicolor showed contamination in all tubes. In contrast, during the third trial, Trichoderma ovalisporum was viable, pure, and morphologically stable in 25%, unlike the rest of the strains in PDA, excluding Aspergillus cf. versicolor and Fusriumincarnatum, which were no longer viable in PDA in the first case and for both agars in the second. Penicillium implicatum was viable in 75%, Cunninghamela elegans, showed contamination in one of the tubes, Aspergillus cf. versicolor grew in two tubes but was contaminated, and the other strains were optimally met with the three criteria mentioned in the Sabouraud agar.
As for the fourth trial, in both agars the optimum purity, viability, and morphological stability were observed for all strains, excluding Aspergillus cf. versicolor which was viable at 25%, but without purity or morphological stability and Fusarium incarnatum, without viability, purity, or morphological stability.
In the fifth trial, viability, purity, and morphological stability were optimal in both agars for the strains, even after recovery and purification, Aspergillus cf. versicolor presented 50% purity in Sabouraud and PDA, unlike Fusarium incarnatum.
The last trial maintained the viability, purity, and morphological stability of all strains in both agars, with the exception of Penicillium implicatum in Sabouraud agar.

Viability Percentage
The viability of Aspergillus cf. versicolor, Penicillium implicatum, and Fusarium incarnatum was compromised by using this method on Sabouraud agar compared with other species (Figure 7a).

Percentage of Purity
The purity of Trichoderma ovalisporum in PDA, Penicillium implicatum and Cunninghamela elegans in Sabouraud agar was good but not optimal, since at some point in the procedure they presented contamination of the medium with yeasts of the genus Saccharomyces and Rhodotorula, fungi of the genus Penicillium and actinobacteria of the genus Streptomyces, so the percentage of purity and efficacy of these species was affected (Figure 7b).

Percentage of Morphological Stability
The percentage of morphological stability for the species remained above 80% in both agars, however, there were certain particularities such as Trichoderma ovalisporum, which their morphological stability in PDA did not exceed 71% (Figure 7c). In addition, as mentioned above, Aspergillus cf. versicolor and Fusarium incarnatum in any of the agars did not exceed 50%.

Cryopreservation
This conservation method was carried out with 100% efficacy for all strains, starting in September and performing an analysis of purity, viability, and morphological stability, one month later. Indian Journal of Science and Technology Vol 13(08), DOI: 10.17485/ijst/2020/v13i08/146345, February 2020 According to the statistical analysis, it is possible to appreciate that the sampling dates did not have a significant incidence on the variation of the isolates obtained from the soil samples.
In order to guarantee the cepary, temperature and humidity must be measured constantly. For cryopreservation with liquid nitrogen, the tank should be filled once every three months, which ensures that the average temperature inside is kept below −70 °C, in addition, it is necessary to keep the strains in culture media in hermetic boxes, covered individually with parafilm at an average temperature of 4 ° to 8 °C.
The most efficient conservation method was cryopreservation, with an efficacy percentage of 100%, maintaining optimal viability, morphological stability, and purity, although the subculture in periodic series had an efficacy higher than 90%, especially for Trichoderma ovalisporum, in agar Sabouraud, Cunninghamela elegans, Penicillium citrinum and Penicillium implicatum in PDA, finally, the efficacy of Hipocrea nigricans was the same in both agars. The best culture medium that maintained morphological stability and purity for longer was PDA, Sabouraud agar was better to maintain viability; however, the difference between the two agars was 1.39% effective.
These results were used to establish the protocol for the conservation of filamentous fungal strains of La Universidad El Bosqueceparium.