Micología Aplicada International ISSN: 1534-2581 [email protected] Colegio de Postgraduados México

Munguía-Pérez, R.; Díaz-Cabrera, E.; Martínez-Montiel, N.; Muñoz-Rojas, J.; Martínez-Contreras, R. Fungal diversity in soil samples from a Mexican region with endemic dermatomycoses Micología Aplicada International, vol. 23, núm. 1, enero, 2011, pp. 11-19 Colegio de Postgraduados , México

Available in: http://www.redalyc.org/articulo.oa?id=68516112002

How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative Micologia Aplicada InternFungalation diversityal, 23(1), in 2011, soil pp. 11-1911 © 2011, Berkeley, CA, U.S.A. www.micaplint.com

Fungal diversity in soil samples from a Mexican region with endemic dermatomycoses

R. Munguía-Pérez, E. Díaz-Cabrera, N. Martínez-Montiel, J. Muñoz-Rojas and R. Martínez-Contreras

Centro de Investigaciones Microbiológicas, Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, Edificio 103J, Ciudad Universitaria, Puebla 72570, Puebla, México.

Accepted for publication December 17, 2010

ABSTRACT

Forty soil samples were collected from four rural communities in the Municipality of (Puebla, ), a region with endemic dermatomycoses. Classical and molecular approaches allowed the identification of 30 different species, including several agents of superficial, subcutaneous and opportunistic mycoses. The most prevalent pathogenic agents identified by micro-morphological characteristics were: Trichophyton mentagrophytes (12.5%), T. rubrum (7.5%), and Aspergillus flavus (7.5%). A lower number of isolates was obtained in soils having acidic pH (5.19). Fungal diversity of Ascomycetes was also found in the studied area by sequence analysis of the ITS1-5.8S-ITS2 rDNA region. Our results showed a high prevalence of pathogenic and potential agents of mycoses, as well as the importance of molecular tools to identify microbial populations in soil.

Key words: Diversity, fungi, mycosis, phylogeny, soil.

Corresponding author: R. Martínez-Contreras, e-mail: [email protected] . Tel.: +52 (222) 2295500, ext. 2529. Fax: +52 (222) 2295650. Micol. Apl. Int., 23(1), 2011, pp. 11-19 12 R. Munguía-Pérez et al.

INTRODUCTION Mexico). Different fungal species were identified in soil samples by both classical The soil constitutes the main reservoir of and molecular approaches, including po- fungi and potentially pathogenic species tentially pathogen and opportunistic fungi are widespread worldwide. The vast ma- for the inhabitants of the region studied. jority of fungi producing diseases in hu- mans and animals exist freely in nature as soil saprophytes or plant pathogens, and MATERIALS AND METHODS gain entrance into the body through abra- sion, implantation or inhalation. The distri- Region of study. Huauchinango lies in the bution of these fungi depends on different Northern Mountains of the State of Puebla, factors, including physico-chemical char- central Mexico (20o 03’ 00’’N, 98o 08’ 00’’ acteristics of the soil, the association with W). The area comprises 160.72 km2, includ- plants and, of course, human and/or animal ing 44 different communities with warm to presence. Mycotic infections are continu- mild humid weather all year around (Fig. ously reported everywhere and some of 1). This Municipality has mainly pine and them result into extremely contagious cas- pine-oak forest associations. Typical soil es. Studies on the isolation of fungi from is loamy-sand and organic-rich (andosol). soil have been investigated throughout the The local economy is based on flower world using different techniques8. growing and agriculture, including the pro- Endemic mycoses remain a major pub- duction of coffee, fruits, and woods for ex- lic health problem in Mexico10. Most port. About one third (33.6%) of the popu- cases originate in rural areas, particularly lation is rural of a medium social level. in groups of low socio-economic level. Soil sample collection. A total of 40 soil Unfortunately, little is known about the samples were collected between July and most common species present in endemic August (2008) from different locations in areas. The Municipality of Huauchinango the Municipality of Huauchinango. The (Puebla, Mexico) has been considered an communities were Ahuacatlán (20o 08’ N, endemic area for some dermatomycoses 98o 01’ W), Huilacapixtla (20o 13’ N, 98o according to previous studies. There has 05’ W), Nigromante (20o 07’ N, 98o 06’ been a high frequency of superficial -my W), and Tenexalco (20o 12’ N, 97o 59’ W). cosis in the rural population from northern All samples were collected randomly from Puebla. In this region, eighty six cases of locations related to daily activities of the mycosis were detected in 110 patients, and people, and they were associated either 18 strains of dermatophytes were isolated, with corn, bougainvillea, rosebush or wild mainly Trichophyton rubrum and T. menta- plants. About 100 g of soil were collected, grophytes. Non-dermatophyte filamentous for each sample, and taken to the labora- fungi and yeast strains were also isolated, tory for characterization. most of them belonging to Candida spp., Soil physico-chemical characterization. but surprisingly none to C. albicans 15,16. In General characteristics were recorded for this work, we determined fungal popula- each soil sample, including pH, soil tex- tions of clinical interest isolated from soil ture, electric conductivity and presence of samples of different locations within the organic matter, nitrogen, soluble cations Municipality of Huauchinango (Puebla, and available micronutrients. Soil pH in a

Micol. Apl. Int., 23(1), 2011, pp. 11-19 Fungal diversity in soil 13

Fig. 1. Location map of Huauchinango (State of Puebla, Mexico) showing different communities studied.

1:1 (soil:water) suspension was measured mum period of 4 weeks, and fungal growth using a digital pH meter14. For this pur- was observed under a stereoscopic binocu- pose, 10 g of soil were shaken in double lar microscope. Samples were stained with distilled water (10 ml), and the mixture cotton blue and observed at 40x to describe was allowed to stand for about 30 min. colony morphology. Strains were identified General methods were used to charac- by their morphological and physiological terize each sample. Organic matter was characteristics, such as size, shape, color, determined by measuring total organic texture and consistency according to pro- carbon present in soil according to the pro- cedures previously described2,13. cedure described originally by Walkley DNA extraction, PCR and sequenc- and Black19. Quantitative determination of ing. Humic acids present in soil were nitrogen was measured by the micro-Kjel- eliminated, and total DNA was extract- dahl method5. Soil texture was determined ed using the ZR Soil Microbe DNA Kit by the Bouyoucos Hydrometer Method4. (Zymo Research Corp., U.S.A.), ac- Conductivity was measured using an EC cording to the manufacturer’s instruc- Meter18. Micronutrients present in soil tions. PCR amplification was carried were extracted using the chelating proper- out using the following primers: ITS1F ties of DTPA12. (5’-CTTGGTCATTTAGAGGAAGT- Analysis of isolates. One gram of sieved AA-3’)6; and ITS4A (5’-CGCCGTTACT- soil was added to 9 ml of sterile water. GGGGCAATCCCTG-3’)11. These primers Serial dilutions (1:10 factor) were used to have been used previously to amplify the inoculate Sabouraud dextrose agar contain- ITS region between the 18S and 28S rRNA ing chloramphenicol (100 µg/ml). Cultures fungal genes, showing specificity for were examined regularly during a maxi- Ascomycetes6,20. Primers were purchased

Micol. Apl. Int., 23(1), 2011, pp. 11-19 14 R. Munguía-Pérez et al. from IDT Technologies Inc. (U.S.A.), BLAST search of the sequences was per- and PCR amplification was performed as formed using the NCBI/Gene bank data- previously described11 in a MultiGene II base (www.ncbi.nlm.nih.gov/genbank), Thermal Cycler (Labnet International, Inc., and compared with deposited sequences for U.S.A.). The PCR protocol consisted of an identification. Sequences were aligned us- initial denaturation step at 94 C for 2.5 min, ing CLUSTALW2 (EMBL-EBI), exported followed by 35 cycles of 15 s denaturation to Bioedit 7.0 (Ibis Biosciences), and the at 94 C, 30 s annealing at 62 C, and 90 s phylogenetic tree was constructed using elongation at 72 C. The PCR amplification MEGA 4. was terminated incubating for 10 min at 72 C. Successful PCR amplification was confirmed visualizing the products (7 µL) RESULTS AND DISCUSSION by electrophoresis on a 1% agarose/TBE gel, and subsequent staining in ethidium Physico-chemical characteristics of the bromide for 10 min. Single bands corre- soil. Quantitative and qualitative indices sponding to different PCR products were of fungal occurrence change according to purified from gel using the QIAEX II Gel soil characteristics. For example, soil tex- extraction kit (QIAGEN, U.S.A.). Pure ture affects how well nutrients and water DNA was sequenced in both orientations are retained affecting all microorganisms. using the Perkin Elmer/Applied Biosystems In this study, the soil texture was predomi- Fluorescence-based Sequencer Model 3730 nantly loamy-sandy (Table 1), except for from a DNA Sequencing Facility (Institute Huilacapixtla where soil was sandy-loam. of Biotechnology, UNAM, Mexico). As water drains from sandy soils, it often

Table 1. Physico-chemical characteristics of different soil samples studied.

Variable Community Ahuacatlán Huilacapixtla Nigromante Tenexalco (n= 10) (n= 10) (n= 10) (n= 10) pH 6.48 ± 0.026 5.19 ± 0.127 6.82 ± 0.09 6.01 ± 1.043 Texture Loamy-sand Sandy-loam Loamy-sand Loamy-sand Organic matter (%) 6.25 ± 0.03 3.52 ± 0.05 6.62 ± 0.1 4.01 ± 0.13 Total nitrogen (mg kg-1) 0.308 ± 0.0012 0.217 ± 0.0021 0.294 ± 0.0012 0.266 ± 0.006 Conductivity (µS) 77.4 ± 2.81 34.4 ± 0.63 52.4 ± 1.73 83.4 ± 1.19 Na+ (mg kg-1) 6.3 ± 0.001 1.8 ± 0.0153 1.1 ± 0.001 4 ± 0.0058 K+ (mg kg-1) 2.7 ± 1.292 2.7 ± 0.462 1.3 ± 0.176 1.3 ± 1.149 Fe++ (mg kg-1) 5 ± 0.0252 3.5 ± 0.1249 1 ± 0.0954 3.3 ± 0.3764 Cu++ (mg kg-1) < 0.2 < 0.2 < 0.2 < 0.2 Zn++ (mg kg-1) < 0.5 < 0.5 < 0.5 < 0.5

Micol. Apl. Int., 23(1), 2011, pp. 11-19 Fungal diversity in soil 15

Table 2. Fungal distribution in different soil samples examined. Fungal species were identified on the basis of morphology.

Municipality Ahuacatlán Huilacapixtla Nigromante Tenexalco Total F (%)

Altitude (m) 1720 1747 1780 1300 Number of samples examined 10 10 10 10 40 Number of isolates 7 3 7 13 30 Distribution (%) 75 Trichophyton mentagrophytes 1 - - 4 5 12.5 T. rubrum - - - 3 3 7.5 T. tonsurans 1 - - - 1 2.5 Microsporum canis - - - 1 1 2.5 Phialophora verrucosa - - - 1 1 2.5 Cladosporium carrionii - 1 - - 1 2.5 Aspergillus flavus 2 - 1 - 3 7.5 A. fumigatus 2 - - - 2 5 Paecilomyces sp. - 1 5 - 6 15 Scopulariopsis sp. 1 1 - - 2 5 Humicola sp. - - - 1 1 2.5 Ulocladium sp - - 1 1 2 5 Fusarium sp. - - - 1 1 2.5 Epicoccum sp. - - - 1 1 2.5

F= Frequency.

carries nutrients diminishing the organic tral, ranging from 5.19 to 6.82 (Table 1), a matter available, and affecting fungal sur- condition that favors fungal development. vival. In accordance to this, the soil from All other parameters measured varied in Huilacapixtla showed the lowest values the different communities studied, show- for conductivity, organic matter, and few- ing mainly low content of nitrogen and mi- er isolates were recovered from this soil cronutrients. (Table 2). A pH level of the soil ranging Classical identification of fungal - spe from 6.3-6.8 is preferred by most soil mi- cies. Fungi isolated are shown in Table 2. croorganisms, including some fungi; how- A total of 14 different species were pheno- ever, some species have shown a broader tipically identified, including 13 agents of tolerance and tend to multiply at lower opportunistic, subcutaneous or superficial or higher pH values8. In the communities mycoses. The recovered agents for super- studied here, pH values were close to neu- ficial mycoses correspond mainly to the

Micol. Apl. Int., 23(1), 2011, pp. 11-19 16 R. Munguía-Pérez et al. genus Trichophyton, including T. menta- end of the 28S gene and the intermedi- grophytes, T. rubrum, and T. tonsurans. ate region which includes ITS1, the 5.8S Microsporum canis was also found. In gene and ITS2. Using this approach, six- terms of distribution, these species were teen operational taxonomic units (OTUs) isolated from different locations, and have corresponding to sequences reported for also previously been reported as abun- different characterized clones and un- dant16. Scopulariopsis sp., Fusarium sp., cultured fungi were identified Table ( 3). and Paecilomyces sp. were also identi- Interestingly, several sequences ampli- fied. The recovery of agents associated to fied were highly similar to fungi reported subcutaneous mycoses showed that dif- as mycological agents, including several ferent species were present in each loca- species of Fusarium, Colletotrichum cap- tion, including Phialophora verrucosa sici (Sydow) E. J. Butler & Bisby, and and Cladosporium carrionii. This finding Chaetomiaceae. In this regard, many spe- suggested a possible relationship between cies belonging to the genus Fusarium have location and the predominance for a giv- been associated to keratitis and ocular in- en species. Several species have recently fections3,7, and isolated from patients pre- been suggested as the origin of opportu- senting different types of mycoses16. These nistic mycoses, particularly in immuno- studies show not only the clinical relevance compromised patients17. Our work showed of Fusarium as a prevalent agent of several that many opportunistic fungi were pres- mycoses, but they also show the necessity ent in different soil samples analyzed, in- to identify fungal species for each clinical cluding Aspergillus flavus, A. fumigatus, case. Our data showed that opportunistic Fusarium sp., and Scopulariopsis sp. At fungi are widespread in the area studied, least one of these species was present in and they may be the origin of several clini- each community. Some saprophytic fungi cal cases developed in the endemic region. widely distributed in soil or in associa- This finding becomes relevant consider- tion with plants, like Humicola sp. and ing the increase in the number of severe- Paecilomyces sp., were identified as well ly immunocompromised patients, as well being the latter considered as an agent as those communities facing the growing for superficial mycosis16. In terms of fre- problem of opportunistic fungal infections9. quency, Paecilomyces sp. (15%) predomi- Treatment of these infections is long and nated, followed by Trichophyton mentag- difficult so the identification of the fungal rophytes (12.5%), T. rubrum, T. tonsurans, agent becomes important, involving un- Microsporum canis, Phialophora verruco- derstanding of their epidemiology in order sa, Cladosporium carrionii, Fusarium sp., to develop effective diagnostic and preven- and Epicoccum sp. (2.5%) [Table 2]. Some tive strategies. The spread of opportunistic of these species have previously been re- fungi in the environment occurs predomi- covered from patients of northern Puebla, nantly via the air. Although transmission including T. rubrum, T. mentagrophytes, T. by air is probably the most important route tonsurans, Fusarium sp., Scopulariopsis of infection, inhabitants of the endemic re- sp. and Paecilomyces sp.16. gion studied here are in close contact with Molecular identification of fungal - spe soil, suggesting that other possible routes cies. The PCR products obtained com- of transmission might also exist that need prised the 3’ end of the 18S gene, the 5’ further studies.

Micol. Apl. Int., 23(1), 2011, pp. 11-19 Fungal diversity in soil 17

Table 3. Operational taxonomic units (OTUs) identified by molecular techniques in this study.

C Isolate GenBank Sequence Closest homolog GenBank Query E-value Maximal accession lenght accession coverage identity number (bp) number (%) (%)

A UAP7728 HQ871907 616 Retroconis fusiformis (Reddy & EU040239 97 0.0 95 Bilgrami) de Hoog & Batenburg- van der Vegte UAP7766 HQ871920 163 Colletotrichum capsici (Sydow) EU056738 41 1e-20 95 E.J. Butler & Bisby UAP7767 HQ871921 117 Fusarium sp. EU791919 46 3e-14 94 UAP7770 HQ871922 359 Uncultured Chaetomiaceae GU055740 97 8e-145 93 UAP7771 HQ871923 234 Ascomycota sp. GQ922579 88 6e-47 82 UAP7727* HQ871927 178 - - - - - H UAP7747 HQ871909 241 Fusarium chlamydosporum GU457416 73 1e-26 80 Wollenw. & Reinking UAP7748 HQ871910 234 Gibberella moniliformis Wineland EF158026 73 2e-24 79 UAP7752 HQ871911 243 Uncultured Trichocladium GU055745 99 5e-105 95 UAP7753 HQ871912 244 Chaetomidium leptoderma FJ666353 98 2e-89 91 UAP7754 HQ871913 401 Uncultured Trichocladium GU055745 96 0.0 99 UAP7756 HQ871914 240 Stachybotrys chartarum GU945205 95 2e-85 91 (Ehrenberg) S. Hughes UAP7720* HQ871925 286 - - - - - UAP7722* HQ871926 258 - - - - - UAP7755* HQ871928 273 - - - - - N UAP7759 HQ871915 228 Uncultured Tarzetta FJ197021 30 3e-23 97 UAP7760 HQ871916 348 Pezizomycetes sp. GQ153164 80 2e-105 91 UAP7761 HQ871917 323 Fusarium incarnatum Berkeley EF158029 99 1e-158 99 & Ravenel UAP7763 HQ871918 245 Chaetomidium leptoderma FJ666353 97 1e-55 81 UAP7764 HQ871919 320 Chaetomidium leptoderma FJ666353 83 1e-92 87 T UAP7729 HQ871908 594 Podospora austroamericana GQ922535 56 4e-40 76 UAP7773 HQ871924 202 Fusarium sp. EF453092 92 1e-35 79 UAP9771 HQ871929 241 Uncultured Trichocladium GU055745 96 1e-91 93 UAP9773 HQ871930 360 Uncultured Lophiostomataceae DQ974708 97 5e-72 81

* Isolates did not show high similarity with sequences deposited at the GenBank database. C= Community. A= Ahuacatlán. H= Huilacapixtla. N= Nigromante. T= Tenexalco.

Fungal diversity in Huauchinango. Twenty sequences corresponding to dif- Very little is known about the diversity of ferent Ascomycota were analyzed, and fungi occurring in Mexican soils, which their closest homologs are shown in Table are associated with dermatomycoses. 3. Species identified in this study are in

Micol. Apl. Int., 23(1), 2011, pp. 11-19 18 R. Munguía-Pérez et al. agreement with classes reported elsewhere. techniques for describing whole microbial The most prevalent class corresponded to populations. Sordariomycetes, which comprises differ- In summary, this study represents a ent species of the genus Fusarium. combined approach to assess the diversity Classical versus molecular identifica- of fungi associated to dermatomycoses in an tion of fungi in soil. Numerous studies on endemic region by classical and molecular fungal diversity in natural habitats have techniques. The sequence data were used shown that the identification of fungi is for phylogenetic analysis, allowing the often very complex due to species rich- identification of hard-to-recover fungi. ness and high micromorphological simi- The high fungal diversity from Mexican larities7. Furthermore, these organisms soils found in this study should be further exhibit multiple life-cycle types and often examined. they cannot be cultured using standard techniques. In this regard, molecular ap- proaches involving the analysis of complex ACKNOWLEDGEMENTS community DNA are widely recognized as having potential for overcoming these This work was partially funded by PROMEP and 1 Programa de Consolidación CONACYT. Authors would limitations , as species that cannot be cul- also like to thank Dolores Castañeda Antonio for her tivated are characterized and identified. In technical advice. this study, 18S rRNA and 5.8S rRNA gene sequences and internal transcribed spacer (ITS) analyses were performed to assess LITERATURE CITED fungal diversity. Several species of hard- to-recover fungi were identified, includ- 1. Anderson, I. C. and J. W. G. Cairney. 2004. Diversity ing Stachybotrys chartarum (Ehrenberg) and ecology of soil fungal communities: increased understanding through the ap- S. Hughes, Retroconis fusiformis (Reddy plication of molecular techniques. Environ. & Bilgrami) de Hoog & Batenburg-van Microbiol. 6: 769-779. der Vegte, Tarzetta and other saprophyt- 2. Arenas, R. 2008. Micología Médica Ilustrada. Mc ic organisms (Table 3). Additionally, we Graw-Hill, Mexico. 423 pp. 3. Azor, M., J. Gené, J. Cano, P. Manikandan, N. found sequences that might correspond to Venkatapathy and J. Guarro. 2009. Less- new fungal species, which have not yet frequent Fusarium species of clinical in- been described (UAP7720, UAP7722, terest: correlation between morphological and molecular identification and antifun- UAP7727, UAP7755; Table 3). These gal susceptibility. J. Clin. Microbiol. 47: four sequences did not show any match 1463-1468. when compared to the GenBank database, 4. Bouyoucos G. J. 1962. Hydrometer method im- suggesting possible undescribed species proved for making particle-size analysis of soils. Agron. J. 43: 434-438. because there was only about 30% ho- 5. Bremner, J. M. and C. S. Mulvaney. 1982. Nitrogen mology with closest fungi. For example, total. Pp. 595-624. In: Methods of Soil the sequence corresponding to the sample Analysis. Ed. A. L. Page, R. H. Miller UAP7727 was similar to that reported for and D. R. Keeney. Agronomy Monograph 9, part 2: Chemical and Microbiological Colletotrichum coccodes. Although se- Properties, 2nd. ed. Am. Soc. Agron., quencing more loci for further character- Madison. ization is needed, the finding of these four 6. Gardes, M. and T. D. Bruns. 1993. ITS primers with enhanced specificity for Basidiomycetes - sequences shows the value of molecular application to the identification of mycor-

Micol. Apl. Int., 23(1), 2011, pp. 11-19 Fungal diversity in soil 19

rhizae and rusts. Molecular Ecology 2: Eds. A. L. Page, R. H. Miller and D. R. 113-118. Keeney. Agronomy Monograph 9, part 2: 7. Greif, M. D., A. M. Stchigel, A. N. Miller and S. M. Chemical and Microbiological Properties, Huhndorf. 2009. A re-evaluation of genus 2nd. ed. Am. Soc. Agron., Madison. Chaetomidium based on molecular and 15. Méndez-Tovar, L. J., A. Anides-Fonseca, A. morphological characters. Mycologia 101: Vázquez-Hernández, M. Galindo- 554-564. González, M. Díaz-Madrid, A. Berdón- 8. Jain, N. and M. Sharma. 2011. Distribution of Castro, P. Manzano-Gayosso, B. Millán- dermatophytes and other related fungi in Chiu, F. Hernández-Hernández and R. Jaipur city, with particular reference to soil López-Martínez. 2006. Micosis observadas pH. Mycoses 54: 52-58. en cinco comunidades mexicanas con alto 9. Karkowska-Kuleta, J., M. Rapala-Kozik and A. grado de marginación. Gac. Med. Mex. Kozik. 2009. Fungi pathogenic to humans: 142: 381-386. molecular bases of virulence of Candida 16. Méndez-Tovar, L. J., A. Lemini-López, F. Hernández- albicans, Cryptococcus neoformans and Hernández, F. Manzano-Gayosso, R. Aspergillus fumigatus. Acta Biochim. Pol. Blancas-Espinosa and R. López-Martínez. 56: 211-224. 2003. Frecuencia de micosis en tres comu- 10. Laniado-Laborín, R. 2007. Coccidioidomycosis and nidades de la sierra norte de Puebla. Gac. other endemic mycoses in Mexico. Rev. Med. Mex. 139: 118-122. Iberoam. Micol. 24: 249-258. 17. Pfaller, M. A. and D. J. Diekema. 2010. Epidemiology 11. Larena, I., O. Salazar, V. González, M. C. Julián and of invasive mycoses in North America. V. Rubio. 1999. Design of a primer for ri- Crit. Rev. Microbiol. 36: 1-53. bosomal DNA internal transcribed spacer 18. Richards, L. A. 1969. Diagnosis and Improvement with enhanced specificity for Ascomycetes. of Saline and Alkali Soils. USDA Agric. J. Biotech. 75: 187-194. Handbook 60. Washington, D.C. 631 pp. 12. Lindsay, W. L. and W. A. Norvell. 1978. Development 19. Walkley, A. and I. A. Black. 1934. An examination of a DTPA soil test for zinc, iron, manga- of the Degtjareff method for determining nese, and copper. Soil Sci. Soc. Am. J. 42: soil organic matter, and a proposed modifi- 421-428. cation of the chromic acid titration method. 13. López, R., L. J. Méndez Tovar, F. Hernández Soil Sci. 37: 29-38. Hernández and R. Castañón Olivares. 1995. 20. Weber, S. D., A. Hofmann, M. Pilhofer, G. Wanner, Micología Médica, Procedimientos para R. Agerer, W. Ludwig, K. H. Schleifer and el Diagnóstico de Laboratorio. Editorial J. Fried. 2009. The diversity of fungi in Trillas, México, .D.F. 192 pp. aerobic sewage granules assessed by 18S 14. McLean, E. O. 1982. Soil pH and lime requirement. rRNA gene and ITS sequence analyses. Pp. 199-224. In: Methods of Soil Analysis. FEMS Microbiol. Ecol. 68: 246-254.

Micol. Apl. Int., 23(1), 2011, pp. 11-19