Abundance of Beauveria spp. and Metarhizium spp. in maize and banana agroecosystems in central Cuba Received for publication: February 4, 2022. Accepted for publication: April 22, 2022. Doi: 10.15446/agron.colomb.v40n1.100886 1 Instituto de Investigaciones Agropecuarias y Forestales, Universidad Michoacana de San Nicolás de Hidalgo, Tarímbaro, Michoacán (México). 2 Departamento de Agronomía, Facultad de Ciencias Agropecuarias, Universidad Central “Marta Abreu” de Las Villas, Santa Clara (Cuba). 3 Instituto de Investigaciones Fundamentales en Agricultura Tropical “Alejandro de Humboldt”, OSDE Grupo Agrícola, Santiago de Las Vegas, La Habana (Cuba). 4 Departamento de Biología, Facultad de Ciencias Agropecuarias, Universidad Central “Marta Abreu” de Las Villas, Santa Clara (Cuba). 5 Centro de Investigaciones Agropecuarias, Facultad de Ciencias Agropecuarias, Universidad Central “Marta Abreu” de Las Villas, Santa Clara (Cuba). * Corresponding author: yordanys.ramos@umich.mx Agronomía Colombiana 40(1), 141-146, 2022 ABSTRACT RESUMEN Entomopathogenic fungi are an ecological alternative for the control of agricultural pests. These fungi live in organic matter in the soil and can cause natural epizootics in many arthropods associated with the rhizosphere. The aim of this study was to evaluate the abundance of Beauveria and Metarhizium spp. in maize and banana agroecosystems in central Cuba. Selective medium and insect baiting methods were used to isolate the entomopathogenic fungi from the soil. Metarhizium spp. were significantly more abundant than Beauveria spp. in both types of fields of agroecosystems. The abundance of Metarhizium spp. was higher in Sagua la Grande than in Santa Clara and Camajuaní municipalities. The insect bait method resulted as the most successful way to isolate entomopathogenic fungi from soil. These results show the composition of the entomo- pathogenic fungi in different agroecosystems, and they are an advance in the understanding of their ecology. Los hongos entomopatógenos son una alternativa ecológica para el control de plagas agrícolas. Estos hongos viven en la materia orgánica contenida en el suelo y pueden causar epizoo- tias naturales a muchos artrópodos asociados a la rizosfera. El objetivo de este estudio fue evaluar la abundancia de Beauveria y Metarhizium spp. dentro de los agroecosistemas de maíz y banano en el centro de Cuba. Se utilizaron los métodos de medio selectivo e insecto cebo para aislar los hongos entomopa- tógenos del suelo. Metarhizium spp. fue significativamente más abundante que Beauveria spp. en ambos agroecosistemas. La abundancia de Metarhizium spp. fue mayor en Sagua la Grande que en los municipios de Santa Clara y Camajuaní. Además, el método de insecto cebo constituye el más apropiado para aislar hongos entomopatógenos. Estos resultados muestran la composición de los hongos entomopatógenos en diferentes agroecosistemas y constituyen un avance en la comprensión de su ecología. Key words: entomopathogenic fungi, fungal diversity, insect bait method, selective medium. Palabras clave: hongos entomopatógenos, diversidad fúngica, método del insecto cebo, medio selectivo. Abundance of Beauveria spp. and Metarhizium spp. in maize and banana agroecosystems in central Cuba Abundancia de Beauveria spp. y Metarhizium spp. en agroecosistemas de maíz y banano en el centro de Cuba Yordanys Ramos1, 2*, Alberto Daniel Taibo2, Andy Luis Alvarez Vega2, Chabeli Abreu Lemes2, Rafael Felipe Castañeda-Ruiz3, and Orelvis Portal4, 5 Introduction Entomopathogenic fungi constitute an important biotic component in the natural regulation of arthropod popula- tions (Meyling & Eilenberg, 2007). Beauveria spp. have been found in several ecosystems worldwide including forest, seminatural habitats, and agricultural fields (Clifton et al., 2015). In contrast, Metarhizium spp. are more abundant in temperate regions, but not in colder regions (Steinwender et al., 2015). These entomopathogenic fungi show potential as microbial control agents against different agricultural pests, and they can be artificially reproduced. Among the attributes of these fungi, we can mention a high mortality of the targeted pest population, high genetic diversity across a wide number of strains, infection of multiple life stages, penetration through the integument, and capacity for both horizontal and vertical transmission (Destefano et al., 2004; Jaronski, 2014). The environmental and ecological variations within eco- systems have become a major factor influencing the biocon- trol effects of Beauveria and Metarhizium species. A more detailed understanding of environmental and ecological SCIENTIFIC NOTE https://doi.org/10.15446/agron.colomb.v40n1.100886 mailto:yordanys.ramos@umich.mx 142 Agron. Colomb. 40(1) 2022 interactions, especially the distributions of these fungi in different ecological areas, is needed to improve consis- tency in the control capacity of these fungi. In this sense, particular stages in the life cycle of Beauveria spp. and Metarhizium spp., including their persistence and dispersal in the environment, are unresolved in Cuba. The aim of this study was to evaluate the abundance of Beauveria spp. and Metarhizium spp. in maize (Zea mays L.) and banana (Musa paradisiaca L.) fields or agroecosystems. These are the most important crops in Cuba. Materials and methods Field sampling Field samplings were conducted from April to July 2018 in three maize (Zea mays (L.), cv. ‘Jibara’) and three banana (Musa paradisiaca (L.), cv. ‘Grande Naine’) fields located in three municipalities in Villa Clara province, Cuba. The selected municipalities were Camajuaní (22°28’4” N, 79°43’26” W), Santa Clara (22°24’49” N, 79°57’58” W) and Sagua la Grande (22°48’24” N, 80°4’32” W), where five collection points spaced 20 m apart were selected in each of the maize and banana fields. Two soil samples of 500 g each were collected with a garden spade around selected points to a depth of about 15 cm after removal of surface litter. The garden spade was disinfected with 70% ethanol between every collection to avoid contamination (Klingen et al., 2002). The soil samples from each point were placed into polyethylene bags and transferred to the Microbiology Laboratory at the Universidad Central “Marta Abreu” de Las Villas. Collected soil samples were thoroughly homo- genized by hand and stored at 4°C until processing. Isolation methods The selective medium and the insect bait methods were used to isolate entomopathogenic fungi from soil samples. The first method was used through serial dilutions of soil in a culture medium, and the insect bait method employed the use of Galleria mellonella L. (Lepidoptera: Pyralidae) larvae. Galleria mellonella larvae were used for this purpose due to their high susceptibility to many fungal pathogens and because they are commercially reproduced in the Entomophagous and Entomopathogenic Reproduction Center in Cuba. A growth selective medium for Beauveria spp. and Metarhizium spp. was formulated using saboraud dextrose agar (SDA) (BioCen, Cuba) according to Meyling and Eilenberg (2007). The SDA culture medium was mixed with 1 mg L-1 (w/v) of thiabendazole, 0.05% streptomycin sulfate, and 250 mg L-1 (w/v) of chloramphenicol to avoid bacterial and some saprophytic fungi. One g of each soil sample was placed in 20 ml of sterile distilled water with 0.01% Tween 80® in a 40 ml f lat bottom glass tube. The tubes were mixed by vortexing for 1 min, and 100 µl of the soil solution was serially diluted to 10-3 conidia/ml and then inoculated into Petri dishes (9 cm diameter) with the selective medium described above. The Petri dishes with the soil dilution were incubated at 25 ± 1ºC, and 75% relative humidity (RH) in the dark, for the emergence of fungal colonies. There were four replicates for each sample. The insect bait method was conducted with the use of G. mellonella larvae. Soil samples (500 g) were placed in glass containers (500 ml) and five healthy 5-week-old G. mellonella larvae, obtained from the Entomophagous and Entomopathogenic Reproduction Center in Santa Clara, Cuba, were added. To prevent cocoon production and fur- ther webbing, G. mellonella larvae were conditioned before they were added by immersion of the larvae in water at 56ºC for 15 sec, followed by the pouring of cold water at 4ºC for 30 sec. Finally, the immobile larvae were placed on paper towels until they regained their movement (Woodring & Kaya, 1988). Containers were covered with lids perforated with 15 holes for aeration and placed at 25ºC, 90% RH in the dark. No food was provided for the larvae. Containers were inverted every day to ensure that the larvae remained exposed to the soil. They were checked every two days for mortality until all larvae were dead. All cadavers were rinsed with distilled water and transferred to a moist cham- ber in Petri dishes (9 cm diameter) with moistened filter paper to stimulate fungal growth. A total of 150 larvae were used and the evaluations lasted 24 d. When larvae showed external fungal growth, the fungi were isolated on SDA chloramphenicol (250 mg L-1 (w/v)) and incubated at 25 ± 1ºC and 90% RH in the dark. Colony colors were treated according to Kornerup and Wanscher (1984). Entomopathogenic fungi identification Entomopathogenic fungal isolates obtained from the soil were mounted on standard microscope slides (7.5 x 2.5 cm) and then mixed with a drop of lactophenol. Glass coverlips (2.5 x 5.0 cm and 0.16 cm thick) were then attached to the slide and sealed with resin. Fungal isolates were morpho- logically identified under a compound microscope (Motic, USA, 400x magnification) according to morphological characteristics described by Humber (2012) for each fun- gal species. The fungal isolates were kept in refrigeration at 4ºC in tubes with SDA in the culture collections at the Departamento de Agronomía, Universidad Central “Marta Abreu” de Las Villas and the Instituto de Investigaciones Fundamentales en Agricultura Tropical ”Alejandro de 143Ramos, Taibo, Alvarez Vega, Abreu Lemes, Castañeda-Ruiz, and Portal: Abundance of Beauveria spp. and Metarhizium spp. in maize and banana agroecosystems in central Cuba Humboldt” (WDCM 853). Abundance was determined through the number of samples in which Beauveria and Metarhizium were found. Statistical analysis Analysis of variance (ANOVA) was applied to evaluate differences in frequencies of entomopathogenic fungi in maize and banana fields as well as to compare the effective- ness of the isolation methods. Means of entomopathogenic fungi were separated using Fisher's least significant diffe- rence (LSD) test. ANOVA were run using STATGRAPHICS Plus 5.1 (Manugistics Inc.) with significance level of 0.05. Results and discussion A total of 151 fungal isolates were obtained from the dif- ferent maize and banana fields with both selective medium and insect bait methods. The identified entomopathogenic fungi are described below: Beauveria spp. Colonies on SDA attaining 50 mm in 7 d at 25ºC, cot- tony at center, radially sulcate to filamentous toward the filiform margin, white (Fig. 1). Reverse colonies were reddish at the center and yellow around the periphery. Mycelium superficial and immersed. Hyphae septate, branched, hyaline, smooth, 1-2 µm wide. Conidiogenous cells polyblastic, lageniform, integrated or discrete, in- determinate ampulliform to subcylindrical at the base, geniculate, sympodial extended forming a rachis, with several distinct or inconspicuous denticles at the coni- diogenous loci, arise from aerial hyphae. Conidia solitary, acropleurogenous, globose, unicellular, smooth-walled, hyaline, dry with 3.1 µm of diameter. FIGURE 1. Beauveria sp. obtained from maize and banana fields. A) Colony of Beauveria sp. on SDA culture medium 7 d after inoculation at 25ºC. B) Spore balls representing dense clusters of large numbers of conidiogenous cells and conidia. C) Conidium formed successively on each denticle. A B C Spore balls 144 Agron. Colomb. 40(1) 2022 olivaceous 6-8 × 1.5-2 µm, accumulating in a columnar, dark olivaceous masses. A total of 36 and 52 Metarhizium spp. isolates were obtained from maize and banana fields, respectively. This fungal spe- cies was significantly (F=15.30; df=1; P=0.0001) more abun- dant than Beauveria spp., which were represented by 25 and 38 isolates in both the maize and banana fields. Beauveria spp. and Metarhizium spp. were the most frequently found entomopathogenic fungi in Mexican agroecosystems, and Beauveria bassiana, Beauveria pseudobassiana and Metarhizium robertsii were widely distributed (Pérez- González et al., 2014). Our results were in accordance with the results obtained by Korosi et al. (2019) who obtained more Metarhizium spp. (33%) than Beauveria spp. (26) in Australian vineyard soils. The abundance and diversity of entomopathogenic fungi have not been reported in maize and banana fields in Cuba before and, thus, constitutes a new record for the country. Beauveria spp. isolates obtained from Santa Clara (10), Camajuaní (10) and Sagua la Grande (13) municipalities did not show significant differences (P>0.05) in abundance. However, Metarhizium spp. isolates in Sagua la Grande (19) were higher in number of infected larvae (F=10.18; df=2; P=0.0001) than in Santa Clara (13) and Camajuaní (12) (Tab. 1). TABLE 1. Abundance (number of infected larvae) of Beauveria spp. and Metarhizium spp. obtained from maize and banana fields in three muni- cipalities in Villa Clara, Cuba. Location Entomopathogenic fungi Beauveria spp. (mean ± SE) Metarhizium spp. (mean ± SE) Santa Clara 10 ± 0.77 ab 13 ± 1.88 b Camajuaní 10 ± 0.94 ab 12 ± 1.61 b Sagua la Grande 13 ± 1.86 a 19 ±1.74 a Different letters in the same column indicate significant differences in the abundance of Beauveria spp. and Metarhizium spp. isolates according to the Fisher’s test (P<0.05). These results can be supported by the fact that Metarhi- zium is reported to be more abundant than other ento- mopathogenic fungi in cultivated fields (Tkaczuk et al., 2014). In contrast, Pérez-González et al. (2014) obtained 112 Beauveria spp. and 9 Metarhizium spp. isolates from the soil of 11 locations of Guanajuato State, Mexico. These results demonstrated that the abundance and dis- tribution of entomopathogenic fungi is still unclear, and more studies are needed to clarify this aspect. However, the abundance of Metarhizium spp. over Beauveria spp. in banana and maize fields in Cuba could be explained FIGURE 2. Metarhizium spp. obtained from maize and banana fields. A) Colony of Metarhizium spp. on SDA culture medium 7 d after inocu- lation at 25ºC. B) Branched conidiophore. C) Conidial chains. A B C Metarhizium spp. Colonies on SDA attaining 80 mm in 7 d at 25ºC, cottony to f loccose at center, curled toward the slightly filiform margin that is colored white, with several sporodochial conidiomata, green or olivaceous (Fig. 2). Reverse was brownish. Mycelium was superf icial and immersed. Hyphae were septate, branched, hyaline, smooth, 1-2 µm wide. Conidiomata were sporodochial, columnar, scattered or conf luent, green, olivaceous to olivaceous brown. Co- nidiophores were macronematous, septate, penicillate or irregularly branched, hyaline, smooth, forming a compact cluster or clumps in the sporodochial conidiomata. Coni- diogenous cells were monophialidic, cylindrical, discrete, determinate, smooth, hyaline. Conidia were basocatenu- late, cylindrical, truncated at the ends, unicellular, pale 145Ramos, Taibo, Alvarez Vega, Abreu Lemes, Castañeda-Ruiz, and Portal: Abundance of Beauveria spp. and Metarhizium spp. in maize and banana agroecosystems in central Cuba through the hypothesis that the association of Metarhi- zium spp. with insect host species has a tropical origin. In addition, Metarhizium comprises an assemblage of cryptic species, many of which traverse large geographical barriers (Bidochka & Small, 2005). Biotic (interaction with other species) and abiotic factors (mainly temperature) are considered primary determinants of abundance and population genetic structure of Metarhi- zium (McGuire & Northfield, 2020). According to these data we infer that the tropical conditions of Cuba allowed a greater abundance of Metarhizium spp. in banana and maize fields compared with Beauveria spp. The mean of Beauveria spp. (22) and Metarhizium spp. (30) isolates recovered with the insect baiting method were higher (F=25.12; df=1; P=0.0018) than those obtained with the selective medium (10 Beauveria spp. and 15 Metarhi- zium spp. isolates) (Tab. 2). TABLE 2. Abundance of Beauveria spp. and Metarhizium spp. obtained by selective medium and insect bait methods. Isolation method Entomopathogenic fungi Beauveria spp. (mean ± SE) Metarhizium spp. (mean ± SE) Selective medium 10 ± 0.70 b 15 ± 0.60 b Insect bait 22 ± 0.99 a 30 ± 1.35 a The selective medium indicates the number of colonies per Petri dish, while insect bait shows the number of infected larvae. Different letters in the same column indicate significant differ- ences in the abundance of Beauveria and Metarhizium isolates obtained with selective medium and insect bait methods according to the Fisher’s test (P<0.05). Different results have been obtained about methods of isolating entomopathogenic fungi in the same soil sample (Hernández-Domínguez et al., 2016). Our results demon- strated that the insect bait method is better for obtaining entomopathogenic fungi. The selective medium is targeted at particular fungal species, while insect baiting could de- tect a larger number of species (Keller et al., 2003). However, Tkaczuk et al. (2014) did not find difference in Metarhizium spp. from organic fields using the insect baiting and selec- tive medium methods. The possible explanation for this result is focused on the insect bait method. The absence of water within the plastic boxes could limit the growth of the entomopathogenic fungi. In a similar study conducted by Ramos et al. (2017) the authors used sterile water to moisten the soil before introducing it to the plastic boxes. Water contents in the soil helps to maintain a high relative humidity which in turn helps the growth of the entomo- pathogenic fungi (Lazzarini et al., 2006; Jaronski, 2009). Conclusion According to our results, the entomopathogenic fungi Metarhizium spp. were signif icantly more abundant than Beauveria spp. in both maize and banana plots. The abundance of Metarhizium spp. in Sagua la Grande was higher than in Santa Clara and Camajuaní. The insect bait method resulted in the most appropriate method to isolate entomopathogenic fungi from soil. These results contribute to a better understanding of hypocrealean fungi ecology and their composition in both maize and banana fields in central Cuba. Acknowledgments We thank the Universidad Central “Marta Abreu” de Las Villas for funding this study. Further, Entomophagous and Entomopathogenic Reproduction Center in Santa Clara, Cuba, is acknowledged as the supplier of the Galleria mellonella larvae. Conflict of interest statement The authors declare that there is no conf lict of interest regarding the publication of this article. Author’s contributions YR and OP designed the experiment. YR, ADT, CA and AA conducted the experiment. YR performed the statistical analysis. ADT, CA, ALA and RCR wrote the initial draft. YR, RCR and OP wrote the final version of the manuscript. All authors have reviewed the manuscript. Literature cited Bidochka, M. J., & Small, C. L. (2005). Phylogeography of Metarhi- zium, an insect pathogenic fungus. In F. E. Vega, & M. Black- well (Eds.), Insect-fungal associations: ecology and evolution (pp. 75–118). Oxford University Press. Clifton, E. H., Jaronski, S. T., Hodgson, E. W., & Gassmann, A. J. (2015). Abundance of soil-borne entomopathogenic fungi in organic and conventional fields in the Midwestern USA with an emphasis on the effect of herbicides and fungicides on fungal persistence. PLoS ONE, 10(7), Article e0133613. https://doi. org/10.1371/journal.pone.0133613 Hernández-Domínguez, C., Cerroblanco-Baxcay, M. L., Alvarado- Aragón, L. U., Hernández-López, G., & Guzmán-Franco, A. W. (2016). Comparison of the relative efficacy of an insect baiting method and selective media for diversity studies of Metarhi- zium species in the soil. Biocontrol Science and Technology, 26(5), 707–717. https://doi.org/10.1080/09583157.2016.1152458 Humber, R. A. (2012) Identification of entomopathogenic fungi. In L. Lacey (Ed.), Manual of techniques in invertebrate pathology (pp. 151–187). Academic Press. https://doi.org/10.1371/journal.pone.0133613 https://doi.org/10.1371/journal.pone.0133613 https://doi.org/10.1080/09583157.2016.1152458 146 Agron. Colomb. 40(1) 2022 Jaronski, S. T. (2009). Ecological factors in the inundative use of fungal entomopathogens. BioControl, 55, 159–185. https://doi. org/10.1007/s10526-009-9248-3 Jaronski, S. T. (2014). Mass production of entomopathogenic fungi: State of the art. In J. Morales-Ramos (Ed.), Mass production of beneficial organisms (pp. 357–413). Academy Press. https:// doi.org/10.1016/B978-0-12-391453-8.00011-X Keller, S., Kessler, P., & Schweizer, C. (2003). Distribution of the in- sect pathogenic soil fungi in Switzerland with special reference to Beauveria brongniartii and Metarhizium anisopliae. Bio- Control, 48, 307–319. https://doi.org/10.1023/A:1023646207455 Klingen, I., Eilenberg, J., & Meadow, R. (2002). Effects of farm- ing system, field margins and bait insect on the occurrence of insect pathogenic fungi in soils. Agriculture, Ecosystem & Environment, 91(1–3), 191–198. https://doi.org/10.1016/ S0167-8809(01)00227-4 Kornerup, A., & Wanscher, J. H. (1984). Methuen handbook of colour (2nd ed.). Hastings House Daytrips Publishers. Korosi, G. A., Wilson, B. A. L., Powell, K. S., Ash, G. J., Reineke, A., & Savocchia, S. (2019). Occurrence and diversity of entomo- pathogenic fungi (Beauveria spp. and Metarhizium spp.) in Australian vineyard soils. Journal of Invertebrate Pathology, 164, 69–77. https://doi.org/10.1016/j.jip.2019.05.002 Lazzarini, G. M. J., Rocha, L. F. N., & Luz, C. (2006). Impact of moisture on in vitro germination of Metarhizium anisopliae and Beauveria bassiana and their activity on Triatoma in- festans. Mycological Research, 110(4), 485–492. https://doi. org/10.1016/j.mycres.2005.12.001 McGuire, A. V., & Northfield, T. D. (2020). Tropical occurrence and agricultural importance of Beauveria bassiana and Metarhizium anisopliae. Frontiers in Sustainable Food Systems, 4, Article 6. https://doi.org/10.3389/fsufs.2020.00006 Meyling, N. V., & Eilenberg, J. (2007). Ecology of the entomopatho- genic fungi Beauveria bassiana and Metarhizium anisopliae in temperate agroecosystems: potential for conservation bio- logical control. Biological Control, 43(2), 145–155. https://doi. org/10.1016/j.biocontrol.2007.07.007 Pérez-González, V. H., Guzmán-Franco, A. W., Altatorre-Rosas, R., Hernández-López, J., Hernández-López, A., Carrillo-Benítez, M. G., & Baverstock, J. (2014). Specific diversity on the ento- mopathogenic fungi Beauveria and Metarhizium in Mexican agricultural soils. Journal of Invertebrate Pathology, 119, 54–61. https://doi.org/10.1016/j.jip.2014.04.004 Ramos, Y., Portal, O., Lysøe, E., Meyling, N. V., & Klingen, I. (2017). Diversity and abundance of Beauveria bassiana in soils, stink bugs and plants tissues of common bean from organic and conventional fields. Journal of Invertebrate Pathology, 150, 114–120. https://doi.org/10.1016/j.jip.2017.10.003 Steinwender, B. M., Enkerli, J., Widmer, F., Eilenberg, J., Kristensen, H. L., Bidochka, M. J., & Meyling, N. V. (2015). Root isolations of Metarhizium spp. from crops ref lect diversity in the soil and indicate no plant specificity. Journal of Invertebrate Pathology, 132, 142–148. https://doi.org/10.1016/j.jip.2015.09.007 Tkaczuk, C., Król, A., Majchrowska-Safaryan, A., & Nicewicz, Ł. (2014). The occurrence of entomopathogenic fungi in soils from fields cultivated in a conventional and organic system. Journal of Ecological Engineering, 15(4), 137–144. https://doi. org/10.12911/22998993.1125468 Woodring, J. L., & Kaya, H. K. (1988). Steinermematid and Heter- orhabditid nematodes: A handbook of biology and techniques. Arkansas Agricultural Experimental Station. https://doi.org/10.1007/s10526-009-9248-3 https://doi.org/10.1007/s10526-009-9248-3 https://doi.org/10.1016/B978-0-12-391453-8.00011-X https://doi.org/10.1016/B978-0-12-391453-8.00011-X https://doi.org/10.1023/A:1023646207455 https://doi.org/10.1016/S0167-8809(01)00227-4 https://doi.org/10.1016/S0167-8809(01)00227-4 https://doi.org/10.1016/j.jip.2019.05.002 https://doi.org/10.1016/j.mycres.2005.12.001 https://doi.org/10.1016/j.mycres.2005.12.001 https://doi.org/10.3389/fsufs.2020.00006 http://dx.doi.org/10.1016/j.biocontrol.2007.07.007 https://doi.org/10.1016/j.jip.2014.04.004 https://doi.org/10.1016/j.jip.2017.10.003 https://doi.org/10.1016/j.jip.2015.09.007 https://doi.org/10.12911/22998993.1125468 https://doi.org/10.12911/22998993.1125468