SciELO - Scientific Electronic Library Online

vol.34 número1Identificación y actividad antagónica in vitro de aislados de Bacillus spp. y Trichoderma spp. contra hongos fitopatógenos comunesEtiología de la marchitez y pudrición basal de Jatropha curcas en Arriaga, Chiapas, México índice de autoresíndice de materiabúsqueda de artículos
Home Pagelista alfabética de revistas  

Servicios Personalizados




Links relacionados

  • No hay artículos similaresSimilares en SciELO


Revista mexicana de fitopatología

versión On-line ISSN 2007-8080versión impresa ISSN 0185-3309

Rev. mex. fitopatol vol.34 no.1 Texcoco  2016 

Phytopathological notes

In vitro toxicity of tropical mexican micromycetes on infective juveniles of Meloidogyne incognita

M. Gamboa Angulo1 

J.A. Moreno Escobar1 

E. Herrera Parra2 

J. Pérez Cruz3 

J. Cristóbal Alejo3  * 

G. Heredia Abarca4 

1Unidad de Biotecnología, Centro de Investigación Científica de Yucatán; Mérida, Yucatán, México

2Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, INIFAP; Mocochá, Yucatán, México

3Instituto Tecnológico de Conkal, Conkal, Yucatán, México

4Red de Biodiversidad y Sistemática, Instituto de Ecología A.C., Xalapa, Veracruz, México


Culture filtrates and mycelia extracts (methanol and ethyl acetate) from nine selected Mexican tropical fungal strains were tested against second stage juveniles (J2) of Meloidogyne incognita, in vitro conditions. The micromycetes Acremonium kiliense TA31, Aspergillus sp. 2XA5, Gliocladium sp. MR41, Selenosporella sp. MR26, Stagonospora sp. TA34, and four unidentified strains (TA13, 2TA6, 2TA7, and 2XA7) were cultured on Czapeck-Dox medium for 14 days and mycelial mat was separated by filtration for metabolites extraction. Aspergillus sp. 2XA5 and Selenosporella sp. MR26 showed the highest nematotoxic activity both in culture filtrates (100 % mortality) and methanol extracts (> 90 % mortality). The lowest EC50 (0.08 mg mL-1) was exhibited by the methanol extract of the unidentified strain 2XA7. The results obtained indicate that tropical mycobiota have potential as biological control agents of plant parasitic nematodes.

Key words: Aspergillus sp.; nematotoxic; Micromycetes; root-knot nematodes; Selenosporella sp


Filtrados de cultivos y extractos de micelio (acetato de etilo y metanol) de nueve cepas fúngicas Mexicanas tropicales se evaluaron en condiciones in vitro contra juveniles de segundo estadio (J2) de Meloidogyne incognita. Los micromicetos Acremonium kiliense TA31, Aspergillus sp. 2XA5, Gliocladium sp. MR41, Selenosporella sp. MR26, Stagonospora sp. TA34 y cuatro cepas no identificadas (TA13, 2TA6, 2TA7 y 2XA7) se cultivaron en medio Czapeck-Dox durante 14 días y la masa micelial se separó por filtración para la extracción de metabolitos. Aspergillus sp. 2XA5 y Selenosporella sp. MR26 mostraron la mayor actividad nematóxica, tanto en filtrados de cultivo (100% de mortalidad) como en extractos metanólicos (> 90% de mortalidad). La CE50 más baja (0.08 mg mL-1) se obtuvo con el extracto de metanol de la cepa 2XA7 no identificada. Los resultados obtenidos indican que la micobiota tropical tiene potencial como agentes de control biológico de nematodos fitopatógenos.

Palabras clave: Aspergillus sp.; nematóxico; Micromycetes; nematodos agalladores; Selenosporella sp.

Meloidogyne incognita is one of the most important root-knot nematode in Mexican agriculture. In Yucatan affects several important crops such as habanero pepper (Capsicum chinense Jacq.), aloe (Aloe vera L.), and tomato (Solanum lycopersicum L.), among many others crops (Herrera-Parra et al., 2011). A number of studies have reported that the metabolic production of some fungi could be used as part of management strategy (Xalxo et al., 2013; Bhattacharjee and Dey 2014). These metabolites include caryospomycins (Dong et al., 2007), chitinases, glucanases, peroxidases, viridine, gliotoxin, Trichodermine (Szabo et al., 2013) among others.

However, there is highly necessary development more investigations on fungal potential in this topic (Hernández-Carlos and Gamboa-Angulo, 2011). In this sense, our natural products research group has focused on screening potential regional sources of eco-friendly compounds to be developed as natural nematicides to control this important nematode. As results, the Selenosporella sp. MR26 strain has shown to have good effectivity against M. incognita (Reyes-Estebanez et al., 2011). However, in this study the nematotoxic effect detected was low (2 %), when strains were grown in fermented rice.

Also, is well documented that microbial biosynthesis of metabolites are in dependence on the substrate and conditions of culture (Shinya et al., 2008; Regaieg et al., 2010). Then, to continue the screening of our fungal collections, nine active strains were choosen (Acremonium kiliense TA31, Aspergillus sp. 2XA5, Gliocladium sp. MR41, Selenosporella sp. MR26, Stagonospora sp. TA34, and four unidentified strains: TA13, 2TA6, 2TA7, and 2XA7). These strains were grown in Czapeck Dox, a defined liquid medium (Gamboa-Angulo et al., 2012; Reyes-Estebanez et al., 2011).

Therefore, the aim of this study was to test in vitro culture filtrates, and mycelia organic extracts (methanol and ethyl acetate) against second juvenile stage (J2) of M. incognita, and to measure the median effective concentration of the most active samples.

Materials and methods

Fungal material

Fungal strains (Table 1) from the culture collection of the biotechnology unit of the Scientific Research Center of Yucatán, C.A., were originally isolated from leaves and water samples collected in Tabasco, Veracruz, and Yucatan, Mexico (Reyes-Estébanez et al., 2011; Gamboa-Angulo et al., 2012).

Table 1 Effect of fungal culture filtrate (1 mL) and mycelium methanol extracts (0.3 mg mL-1) from micromycetes strains against J2 of Meloidogyne incognita

Means with the same letter(s) in the column are not significantly different at P = 0.05 according to Tukey's studentized range test. Positive control: Vydate (Oxamyl 1 mL L-1). Blank: Czapeck-Dox medium/ organic extract.

Negative control: water. Nt= no tested.

1: Yucatán 2:Veracruz 3:Tabasco. A:water B: leaves.

Culture conditions and metabolites fungal extraction

All strains were cultured in potato dextrose agar (PDA) media at room temperature (25 ± 2 °C, 16/8 hours light/darkness) for one week, to obtain a suspension of hyphal fragments-spore suspension (Reyes-Estebanez et al., 2011). One mL of the suspension was transferred to Czapek-Dox liquid medium (200 mL) contained in Roux bottles, with three replicates per isolate. These cultures were incubated for 2 weeks at room temperature, in stationary conditions. Then the culture y s were filtered through filter paper (Whatman(r) No. 42), to separate the mycelial mat and the culture filtrate. Mycelial mat was lyophilized (Labconco), ground to powder using a glass mortar, and the respective extract was obtained using ethyl acetate (3×, 100 mL for 24 h, each time), followed by methanol (3×, 100 mL for 24 h, each time) to obtain non-polar and polar compounds, respectively. Solvent was eliminated under reduced pressure until dryness to get organic crude extracts which were stored at 4 °C in the dark. Culture filtrates, ethyl acetate extracts (from mycelia and filtrate), as well as methanol extracts were tested for nematotoxic effects (Reyes-Estébanez et al., 2008).

Nematotoxic assay

The nematode inoculum was prepared as previously described (Cristóbal-Alejo et al., 2006). All fungal extracts (300 mg mL-1) were dissolved in 0.25 % tween 20, and culture filtrates (1 mL) were sterilized by filtration through 0.45 µm filter (Millipore) before use in bioassays (Cristóbal-Alejo et al., 2006; Candelero-de la Cruz et al., 2015). Initial assessments were conducted with freshly hatched J2 (10 each replicate) which were placed in suspension, and incubated at room temperature in special glass dishes for J2 mortality studies. Vydate (Oxamyl, 1 mL L-1 of water) was used as positive control and negative control included distilled water and a blank (Czapeck-Dox medium extracts). Each extract was replicated four times and maintained under laboratory conditions using a completely randomized design. After 24 and 48 h, under a stereoscopic microscopic, the J2 were touched with a needle and those that did not respond were classified as immobile. All J2 were counted and classified as mobile and immobile. After 72 h, death of J2 was confirmed by the transfer of immobile J2 to distilled water and further examination (24 h).

Dose-inhibitory response curves using a dilution series (0, 50, 100, 200, 300, 400 and 500 mg mL1) were prepared for three of the most active extract in culture filtrates and extracts (Aspergillus sp. 2XA5, Selenosporella sp. MR26, and unidentified 2XA7). In the case of culture filtrates, these were diluted to 50 and 25 % (v:v).

Results were reported as percentage of J2 M. incognita mortality. For the analyses of variance data were arcosin transformed [y = arcosin (sqrt (y/100))] and significant differences among treatments were detected by Tukey´s test (P=0.05) (Steel and Torrie, 1988). Effective concentrations (EC50 and EC95) were obtained by transforming to "Probit" and ten-base logarithms the calculated percent mortality of data from the second assay (Throne et al., 1995).

Results and discussion

Nematotoxic activity of culture filtrates and mycelium methanol extracts on M. incognita is summarized in Table 1. Ethyl acetate extracts did not show nematotoxic effect (<50 %). The results showed that the extracts from six strains (67 %) immobilize J2 M. incognita in at least one of their filtrate or mycelial extract tested. Five culture filtrates at undiluted concentration of Aspergillus sp. 2XA5, Selenosporella sp. MR26, Stagonospora sp. TA34, and two unidentified strains (TA13 and 2TA7), together two methanol extracts (Aspergillus sp. 2XA5 and unidentified 2XA7) displayed good effect (85-100 % mortality) on J2. Moreover, there was observed that Aspergillus sp. 2XA5, followed by Selenosporella sp. MR26 displayed the most significant nematotoxic effect, in both, their culture filtrate and methanol mycelia extracts.

The toxicity of the filtrates and methanol extract obtained from liquid culture medium from Selenosporella sp. MR26 was higher as compare to the one shown by extracts derived from solid medium (Reyes-Estebanez et al., 2011).

When the juveniles were exposed to three different concentrations of the Aspergillus sp. 2XA5, Selenosporella sp. MR26 and unidentified 2XA7 active filtrates (Figure 1), there was a concentration-dependent effect. The number of immobile J2 was higher at 50 % concentration and almost all nematodes were paralyzed with a mean value of 75.8 % mortality at 48 h. The highest toxicity was observed with all undiluted filtrates from the active isolates. Similar results were reported for culture filtrate from Nigrospora sp., cultivated on Czapeck medium, when tested against juveniles of M. incognita (Amin, 2013) The nematicidal action of culture filtrates can be attributed to the production of toxic metabolites or enzymes, as in F. solani, where the action was a result of fungal toxins and unused sugar and salt residues present in the culture filtrate (Jain et al., 2008; Regaieg et al., 2010). On the other hand, enzymes could play a key role in fungal infection processes (Segers et al., 1994). Extracellular hydrolytic enzymes such as lipases, chitinases and proteases are considered to be virulence determinants of entomopathogenic fungi and they are involved in complex processes leading to host cuticle penetration and cell digestion (Regaieg et al., 2010).

Figure 1 Nematotoxic effect of fungal culture filtrates at different dilutions (0, 25, 50, 100 % v/v) against J2 of Meloidogyne incognita at 48 h. 

The results of nematotoxic assays from culture filtrates and mycelia methanol extracts against M. incognita showed to be effective with only five polar extracts (Table 1). The best effect was produced by Aspergillus sp. 2XA5 and the unidentified strain 2XA7 (100 % mortality), followed by Selenosporella sp. MR26 (90 % mortality) at 48 h of exposure. Other strains such as Stagonospora sp. TA34 and the unidentified strain 2TA7 were less active than those mentioned above with 47 % nematode mortality at 24 h and up to 80 % mortality at 48 h. This nematotoxic effect was particularly interesting since the fungal strains could be causing nematode immobility as a result of intracellular (endotoxins) and extracellular metabolites (exotoxins). The nematotoxic effect of the unidentified fungus 2XA7 only occurred in the methanol extract and not in filtrate, indicating that the toxic substance is not secreted by fungal hyphae (endotoxin).

According to Table 1, mycelia methanol extracts with the highest nematotoxic activity obtained from Aspergillus sp. 2XA5, Selenosporella sp. MR26, and the unidentified strain 2XA7 were chosen for estimation of EC50 and EC95 (Table 2). The most potent fungal extracts was the unidentified strain 2XA7 with a median effective dose of 0.08 mg mL-1, followed by Aspergillus sp. 2XA5 and Selenosporella sp. MR26 (EC50 values of 0.20 and 0.26 mg mL-1, respectively). These values are good in comparison with other fungal compounds isolated from Paecilomyces lilacinus 6029 (LC values of 3.03 mg mL1) and Verticilllium chlamydosporum (500 mg L-1) (Khambay et al., 2000; Sharma et al., 2014). Furthermore, it was interesting to detect that Selenosporella sp. MR26 produces non polar nematotoxic metabolites when grown on solid fermented rice media (EC50 values of 0.91 mg mL-1) and polar in Czapeck-Dox liquid medium (Reyes-Estebanez et al., 2011). Actually, the isolation and identification of the metabolites of Selenosporella sp. MR26 are in progress.

Table 2 Effective concentrations (EC50 and EC95) of methanol extracts of those strains with the highest nematotoxic activity against J2 of Meloidogyne incognita at 48 h. 

On other hand, Aspergillus is an extensively known genus with a highly prolific production of active metabolites, several species have shown nematotoxic properties such as A. awamori and A. niger against M. incognita, and A. quadrilineatus against M. javanica (Bath and Wani, 2012). From A. niger were brevianamide A, itaconitin, canadensic and mycophenolic acids, and A. quadrilineatus produces flavoskyrin, dehydrocanadensolide and α-Collatolic acid (Siddiqui and Futai, 2009; Akhtar and Panwar, 2013).


The experimental data obtained indicate that Aspergillus sp. 2XA5 and Selenosporella sp. MR26 are the most promissory fungi herein detected to control J2 of M. incognita. Both fungi produce great nematotoxic effect in their culture filtrates and methanol mycelia extracts which indicate polar nature of the metabolites responsible of the activity. Furthermore investigations are required to identify and characterize the molecules responsible for the activity of the potential candidates detected in this study. It will also be necessary to carry out greenhouse trials to tests these nematotoxic metabolites, and finally verify the environmental safety of their use.


The authors thank Irma L. Medina-Baizabal, Manuela Reyes, Narcedalia Gamboa, and Sergio Pérez for their valuable technical assistance. This research was supported by CONACyT (Projects No. 47549 and 2009/CB131256), and by Undergraduate Student Fellowship to Jaime Perez Cruz.

Literature cited

Akhtar MS, and Panwar J. 2013. Efficacy of root-associated fungi and PGPR on the growth of Pisum sativum (cv. Arkil) and reproduction of the root-knot nematode Meloidogyne incognita. Journal of Basic Microbiology 53:318-326. ]

Amin N. 2013. Investigation of culture filtrate of endophytic fungi Nigrospora sp. isolate RS 10 in different concentrations towards root-knot nematode Meloidogyne spp. Indian journal of Science and Technology 6:5177-5181. ]

Bhat MY, and Wani AH. 2012. Bio-activity of fungal culture filtrates against root-knot nematode egg hatch and juvenile motility and their effects on growth of mung bean (Vigna radiata L. Wilczek) infected with the root-knot nematode, Meloidogyne incognita. Archives of Phytopathology and Plant Protection 45:1059-1069. ]

Bhattacharjee R, and Dey U. 2014. An overview of fungal and bacterial biopesticides to control plant pathogens/diseases. African Journal of Microbiology Research 17:1749-1762. ]

Candelero-De la Cruz J, Cristóbal-Alejo JRA, Reyes-Ramírez A, Tun-Suárez JM, Gamboa-Angulo MM, and Ruíz-Sánchez E. 2015. Trichoderma spp. promotoras del crecimiento en plántulas de Capsicum chinense Jacq. y antagónicas contra Meloidogyne incognita. Phyton International Journal of Experimental Botany 84:113-119. Disponible en línea: ]

Cristóbal-Alejo J, Tun-Suárez JM, Moguel-Catzin S, Marbán-Mendoza N, Medina-Baizabal IL, Simá-Polanco P, Peraza-Sánchez SR, andGamboa-Angulo MM. 2006. In vitro sensitivity of Meloidogyne incognita to extracts from native yucatecan plants. Nematropica 36:89-97. Disponible en línea: ]

Dong J, Zhu YA, and Song HB. 2007. Nematicidal resorcylides from the aquatic fungus Caryospora callicarpa YMF1.01026. Journal of Chemical Ecology 33:1115-1126. ]

Gamboa-Angulo M, De la Rosa-García SC, Heredia-Abarca G, Medina-Baizabal IL. 2012. Antimicrobial screening of tropical microfungi isolated from sinkholes located in the Yucatan peninsula, Mexico. African Journal of Microbiology Research 6:2305-2312. ]

Hernández-Carlos B, Gamboa-Angulo MM. 2011. Metabolites from freshwater aquatic microalgae and fungi as potential natural pesticides. Phytochemistry Reviews 10:261-286. ]

Herrera-Parra E, Cristóbal-Alejo J, Tun-Suárez JM, Góngora-Jiménez JA, y Lomas-Barrie CT. 2011. Nematofauna nociva (Meloidogyne spp.) en cultivos hortícolas tropicales: Distribución y perspectivas de manejo en Yucatán. In: Gamboa AM, Rojas HR (eds.). Recursos genéticos microbianos en la zona Golfo-Sureste de México. Vol 1. México: Subnargem, Sagarpa. Morevalladolid S. de R.L. de C.V., 138-150. ]

Jain A, Mohan J, Singh M, and Goswami BK. 2008. Potentiality of different isolates of wilt fungus Fusarium oxysporum collected from rhizosphere of tomato against root-knot nematode Meloidogyne incognita. Journal of Environmental Science and Health B 43:686-691. ]

Khambay BPS, Bourne JM, Cameron S, Kerry BR, and Zaki MJ. 2000. A nematicidal metabolite from Verticillium chlamydosporium. Pest Management Science 56:1098-1099.<1098::AID-PS244>3.0.CO;2-HLinks ]

Regaieg H, Ciancio A, Raouani NH, Grasso G, and Rosso L. 2010. Effects of culture filtrates from the nematophagous fungus Verticillium leptobactrum on viability of the root-knot nematode Meloidogyne incognita. World Journal of Microbiology and Biotechnology 26:2285-2289. ]

Reyes-Estebanez M, Heredia-Abarca G, y Gamboa-Angulo MM. 2008. Perfil biológico de hongos anamórficos del sureste de México. Revista Mexicana de Micología 28:49−56. Disponible en línea 2009/10/RMM_2009_28_049-056.pdfLinks ]

Reyes-Estebanez M, Herrera-Parra E, Cristóbal-Alejo J, Heredia-Abarca G, Canto-Canché B, Medina-Barizabal BI, and Gamboa-Angulo M. 2011. Antimicrobial and nematicidal screening of anamorphic fungi isolated from plant debris of tropical areas in Mexico. African Journal of Microbiology Research 5:1083-1089. ]

Segers R, Butt TM, Kerry BR, and Peberdy JF. 1994. The nematophagous fungus Verticillium chlamydosporium produces a chymoelastase-like protease which hydrolyses host nematode proteins in situ. Microbiology 140:2715-2723. ]

Sharma A, Sharma S, and Dalela M. 2014. Nematicidal activity of Paecilomyces lilacinus 6029 cultured on Karanja cake medium. Microbial Pathogenesis 75:16-20. ]

Shinya R, Aiuchi D, Kushida A, Tani M, Kuramochi K, and Koike M. 2008. Effects of fungal culture filtrates of Verticillium lecanii (Lecanicillium spp.) hybrid strains on Heterodera glycines eggs and juveniles. Journal of Invertebrate Pathology 97:291-297. ]

Siddiqui ZA, and Futai K. 2009. Biocontrol of Meloidogyne incognita on tomato using antagonistic fungi, plant-growth-promoting rhizobacteria and cattle manure. Pest Management Science 65:943-948. ]

Steel RDG, and Torrie JH. 1988. Bioestadística. Principios y procedimientos. Segunda edición. McGraw-Hill. DF, México. 662 p. [ Links ]

Szabó M, Urbán P, Virányi F, Kredics L, and Fekete C. 2013. Comparative gene expression profiles of Trichoderma harzianum proteases during in vitro nematodes egg-parasitism. Biological Control 67: 337-346. ]

Throne JE, Weaver DK, and Baker JE. 1995. Probit analysis: Assessing goodness-of-fit based on block transformation and residuals. Journal of Economic Entomology 88:1513-1516. ]

Xalxo PC, Karkur D, and Poddar A.N. 2013. Rhizospheric fungal association of root knot nematode infested cucurbits: in vitro assessment of their nematicidal potential. Research Journal of Microbiology 2: 81-91. ]

Received: July 06, 2015; Accepted: December 10, 2015

Autor de correspondencia:

Creative Commons License This is an open-access article distributed under the terms of the Creative Commons Attribution License