Artículos

 

Genetic characterisation of Metarhizium anisopliae (Metchnikoff) Sorokin isolates from sugarcane fields and their pathogenicity against Aeneolamia postica (Walker) (Hemiptera: Cercopidae)

 

Caracterización genética de aislados de Metarhizium anisopliae (Metchnikoff) Sorokin de cañaverales y su patogenicidad contra Aeneolamia postica (Walker) (Hemiptera: Cercopidae)

 

A Bautista-Galvez¹*, JF Barrera², E Payró de la Cruz³, S Salgado-García⁴, J Gómez-Ruiz², JF Gomez-Leyva⁵

 

¹ Universidad Tecnológica del Usumacinta, Libramiento Glorieta, Carretera E. Zapata-Tenosique Km. 1. Colonia Las Lomas C.P. 86901 Emiliano Zapata, Tabasco, México. Correo Electrónico: arelybautistagalvez@yahoo.com.mx

² El Colegio de la Frontera Sur, Carretera Antiguo Aeropuerto km 2.5, Tapachula, 30700, Chiapas, México

³ Instituto Tecnológico de la Zona Olmeca, Tabasco, México

⁴ Colegio de Postgraduados, Campus Tabasco, Tabasco, México

⁵ Instituto Tecnológico de Tlajomulco, Jal. Km 10 Carretera a San Miguel Cuyutlán. Tlajomulco de Zúñiga, Jalisco 45640 México

 

Recibido: 1 de julio de 2012
Aceptado: 12 de julio de 2012

 

Abstract

A commercial strain of Metarhizium anisopliae (Metchnikoff) Sorokin was used by farmers in 2005 to control the sugarcane froghopper Aeneolamia postica (Walker) in Los Ríos, Tabasco, Mexico. The persistence and the effect of this introduced isolate on the genetic diversity and pathogenicity of the native fungus population are unknown. The purpose of this study was to characterise the genetic diversity and pathogenicity of M. anisopliae isolates collected from sugarcane fields in the region. The Galleria baiting method was used to sample the fungus from the soil, whereas the insect netting method was used to capture adults infected by A. postica. Samples were collected from a total of 33 sugarcane fields. Ten isolates of M. anisopliae were obtained, three in 2007 and seven in 2008; seven isolates were collected near the Usumacinta river. Random amplified polymorphic DNA markers and amplified ribosomal DNA restriction analysis of the 10 isolates revealed the presence of seven subgroups. Two of the isolates obtained in 2008 were genetically similar to the commercial strain used in 2005. At least two genotypes of M. anisopliae were found in this region. No clear relationship between genotype similarity and isolate pathogenicity against A. postica was recorded. Some of the native M. anisopliae isolates are promising candidates as agents of biological control against A. postica.

Key words: Sugarcane, entomopathogenic fungi, biological control, molecular characterisation, pathogenicity.

 

Resumen

En 2005, una cepa comercial de Metarhizium anisopliae (Metchnikoff) Sorokin fue aplicada por agricultores contra la mosca pinta Aeneolamia postica (Walker) en Los Ríos, Tabasco, México. Se desconoce la persistencia y el efecto de este aislado introducido sobre la diversidad genética y patogenicidad de las poblaciones nativas del hongo. El objetivo de este estudio fue caracterizar la diversidad genética y patogenicidad de aislados de M. anisopliae colectados en cañaverales de esta región. La técnica del insecto trampa con larvas de Galleria se usó para colectar el hongo del suelo, mientras que una red entomológica se usó para capturar adultos infectados de A. postica. Las muestras se colectaron en un total de 33 cañaverales. En total, se obtuvieron 10 aislados de M. anisopliae, tres en 2007 y siete en 2008; siete de los aislados se colectaron cerca del Río Usumacinta. Marcadores del ADN polimórfico amplificado al azar y análisis de restricción de ADN ribosomal amplificado de los diez aislados, revelaron siete subgrupos. Dos de los aislados obtenidos en 2008 fueron genéticamente similares a la cepa comercial usada en 2005. Al menos dos genotipos de M. anisopliae se encontraron distribuidos en esta región. No hubo una clara relación entre la similitud genética y la patogenicidad de los aislados contra A. postica. Algunos de los aislados nativos de M. anisopliae son candidatos promisorios como agentes de control biológico contra A. postica.

Palabras clave: Caña de azúcar, hongos entomopatógenos, control biológico, caracterización molecular, patogenicidad.

 

Introduction

Sugarcane (Saccharum officinarum L.) production is currently the most important agroindustry in Mexico. The cultivation of sugarcane involves 812 000 ha distributed over 15 states, and 57 sugar factories. With an average yield of 69 ton ha⁻¹, Mexico is one of the 10 main producers of sugarcane in the world and, with a per capita consumption of 50 kg year⁻¹, it is the second greatest sugar consumer. Approximately three million Mexicans depend on this economic activity, which also generates an important amount of foreign currency (Salgado et al. 2005). Approximately 28 000 ha of sugarcane are grown in the regions of La Chontalpa and Los Ríos in the state of Tabasco. Mean sugarcane yield in Tabasco is 60 ton ha⁻¹ (Salgado et al. 2010). Sugarcane production is limited by the froghopper Aeneolamia postica (Walker) (Hemiptera: Cercopidae), the most damaging insect pest present over extensive areas of the Gulf of Mexico and Pacific Ocean coastal plains. This pest reduces sugarcane yield up to 60 %. It is controlled mainly by pesticides, which increases production costs substantially (Bautista-Gálvez & Gonzalez-Cortes 2005). As the froghopper has become resistant to many insecticides, sugarcane producers in Tabasco have searched for alternative control methods. One such alternative is Metarhizium anisopliae (Metchnikoff) Sorokin (Moniliales: Moniliaceae), an entomopathogenic fungus that is widely used in the biological control of insect pests (Zimmermann 2007).

In 2005, some farmers applied a commercial strain of M. anisopliae as part of an integrated pest management strategy against A. postica in the sugarcane fields of Los Ríos, Tabasco (Bautista-Gálvez & Gonzalez-Cortes 2005). To this day, there is no information on whether the strain has managed to survive in the region's sugarcane fields. Some studies have indicated that M. anisopliae is a highly genetically diverse species (Riba et al. 1985; Fegan et al. 1993; Becerra et al. 2007; Zimmermann 2007), however the diversity of the native populations of this fungus in the Los Ríos region, their pathogenicity against A. postica and their potential as biological control agents are similarly unknown. The present study was carried out in order to increase understanding of these issues and to characterise the genetic diversity and pathogenicity of Metarhizium isolates collected from the main sugarcane growing areas in the Los Rios region of Tabasco, Mexico. Polymerase chain reaction (PCR)-based methods, such as the random amplified polymorphic DNA (RAPD) markers and the internal transcribed spacer (ITS-rDNA) sequence analysis, were used to determine the genomic variability of this fungus. These methods have been applied in studies of genomic variation of M. anisopliae (Cobb & Clarkson 1993; Fegan et al. 1993; Bridge et al. 1997; Driver et al. 2000; Castrillo et al. 2003; Entz et al. 2005; Becerra et al. 2007; Freed et al. 2011).

 

Materials and methods

Study Area

The study took place in the sugarcane fields of the Los Rios region, municipality of Tenosique, Tabasco, Mexico. Sampling was carried out in the fields that supply the AZSUREMEX S.A. de C.V. sugar refinery, that is located on the La Palma highway, Tenosique, at 17°25' N, 91°24' W, and 60 m altitude, with a cultivated area of 4,210 ha (Bautista-Gálvez & Gonzalez-Cortes 2005).

Fungal sampling

Samples were collected from the fields of 33 farmers (two fields with and 31 fields without the use of M. anisopliae to control A. postica), representing 5.1 % of the sugarcane producers and 4.7 % of the area cultivated with sugarcane in the Los Ríos region. On average, each farmer had 6 ha of sugarcane crops. The area is predominantly characterised by a Cambisol soil type. Soil samples were taken to obtain A. postica adults. Five sampling points were located in each field, one at each corner and another at the centre. Soil samples were collected down to a depth of 10 cm within the rhizosphere, as described by Almeida et al. (1997). Each soil sample consisted of five 0.5 kg soil sub-samples of the upper 10 cm of soil, close to the crop's rhizosphere, with a distance of 5 cm between each sample. The samples were placed in plastic bags and transported to the laboratory in insulated boxes at 4 °C. Samples of A. postica adults were collected near the base of the sugarcane stems using an insect net. The captured insects were placed in Petri dishes (100 × 15 mm) and transported to the laboratory. All A. postica adults were placed in a sterile Petri dish containing a moistened filter paper and sealed with Parafilm to promote fungus sporulation (Goettel & Inglis 1997). The samples were collected between September and December of 2007 and 2008.

Fungal isolation

The Galleria mellonella L. (Lepidoptera: Pyralidae) larvae baiting method was used to isolate M. anisopliae from the soil (Vanninen 1997). The soil samples, each containing five sub-samples per field, were sieved through a 2 mm metallic mesh sieve, after which 300 g of soil were moistened and placed in a 500 mL plastic cup. Five larvae were then placed in each cup. The cups were sealed with sellotape and incubated for seven days at 25 °C, with a photoperiod of L:D of 16:8 h (Bedding & Akurst 1975; Doberski & Tribe 1980). The larvae were examined after incubation. The moribund insects were separated from the soil and disinfected in a 0.025 % sodium hypochlorite (NaOCl) solution for one minute, followed by 70 % alcohol for five seconds. They were then rinsed three times in sterile distilled water, and the excess water was removed using tissue paper (Inglis et al. 2008). The larvae were then placed in a 100 × 15 mm Petri dish with a double layer of damp filter paper and subjected to the aforementioned incubation conditions to favour fungus development and sporulation (Hatting et al. 1999). Mycosed insects were observed under a stereoscopic microscope to detect the body areas with the greatest sporulation. The procedures mentioned above for G. mellonella mycosed larvae were followed in order to isolate entomopathogenic fungi from the field-collected samples of A. postica adults. In total, 33 soil samples and 165 A. postica adults were processed. The isolated fungi were identified following Tulloch's (1976) taxonomic keys.

Fungal genomic DNA purification

Monosporic cultures were used as inoculi and were grown on potato dextrose agar (PDA) (Lilly & Barnett 1951). Fungal inoculi were incubated and agitated for three days, at 27 °C, in a potato dextrose broth, and the mycelium was collected on a sterile filter. The mycelium was ground with liquid nitrogen to a fine dust, and transferred to a sterile 1.5 mL plastic microcentrifuge tube. Each sample was mixed with lysis buffer (containing Tris HCL 50 mM, pH 7.0; EDTA 50 mM; SDS 3 %; and 1-2 β-mercaptoethanol 1 %), incubated at 65 °C for 1 h, extracted with phenol:chloroform and precipitated with isopropanol (Lee & Taylor 1990). The resulting DNA was analysed on 0.8 % agarose gel in 1x SB buffer (sodium borate 10 mM), stained with ethidium bromide and observed under ultraviolet light (UV). DNA samples were stored at 4°C (Gómez-Leyva et al. 2008).

PCR amplification of the rDNA ITS region and restriction analysis (ARDRA)

The Internal Transcribed Spacer (ITS) region of the rDNA of each sample was amplified by PCR using the following reaction mixture: 1x buffer, pH 8.5, ITS1 and ITS4 primers at 20 pmol each (White et al. 1990), 0.2 mM dNTPs, 2.0 mM of MgCl₂, 2.5 U of Taq polymerase DNA and 100 pmol DNA, and the following thermocycler conditions: 3 min at 94 °C followed by 30 cycles of: 1 min at 94 °C, 1 min at 55 °C, 1 min at 72 °C and a final extension of 7 min at 72 °C. The amplified products were extracted after electrophoresis on agarose 1.2 % gel using a DNA extraction kit (Promega, Madison, WI). The PCR products were ligated in the TOPO TA vector (Invitrogen) and cloned in Escherichia coli Top10. The recombinant TOPO vector was sequenced in both directions and the results were compared with GenBank records using the DNASTAR Lasergene software (DNASTAR Inc. 2009). In total, 14 isolates were sequenced dideoxi sequencing, Sanger method.

In the case of the amplified ribosomal DNA restriction analysis, the ITS-PCR products were digested using Hae II according to the manufacturer's instructions (this enzyme was obtained from Promega, Madison, WI). The restriction fragments were separated on 1.4 % agarose gel in 1x SB buffer. Ethidium bromide gel staining was applied, and the DNA fragments were observed under UV light and photographed. The molecular size of the fragments was estimated by comparison with a 100 bp DNA ladder (Entz et al. 2005; Inglis et al. 2008).

The ARDRA technique was applied to a group of 19 fungus samples consisting of 10 M. anisopliae isolates obtained from the sampled sugarcane fields and the following nine reference samples: one commercial strain of M. anisopliae from the "Tiemelonlà Nich Klum" laboratory, four M. anisopliae isolates (M370, M371, M372 and M374) characterised and provided by the Centro Nacional de Referencia de Control Biológico (CNRCB), Tecomán, Colima, Mexico, two Beauveria bassiana (Balsamo) Vuillemin isolates, and two unidentified fungus isolates obtained from the sugarcane fields sampled in this study.

RAPD-PCR analysis

Fungal isolates were characterised by RAPD analysis (Williams et al. 1990). The OPB07 (GGT-GACGCAG) and OPB09 (TGGGGGACTC) Series B Operon primers were used (Operon Technologies Inc., Alameda, CA) (Cobb & Clarkson 1994; Guerrero et al. 2000; Kendall & Rygiewicz 2005). The reaction mixture consisted of 0.2 mM dNTPs, 10 µM of primer, 2.5 U of Taq polymerase DNA (Pro-mega, Madison, WI) and 100 ng of DNA. Polymerase Chain Reaction (PCR) amplifications were carried out in a MJ Research thermocycler programmed as follows: 2 min at 94°C followed by 40 cycles of 1 min at 94 °C, 1 min at 36 °C, 90 s at 72 °C, and then 7 min at 72 °C and 4 °C until removed. The amplified fragments were separated by horizontal gel electrophoresis in 1.2 % agarose (Ultrapure, Gibco BRL, Gaithersburg, MD). The gels were stained with ethidium bromide and photographed on a UV Cole Palmer 97500 transilluminator.

The RAPD technique was applied to a group of 10 M. anisopliae samples that included the first nine isolates obtained from the sugarcane fields and the L0909 commercial strain provided by the "Tiemelonlà Nich Klum" laboratory (Palenque, Chiapas, Mexico), which was used as the reference isolate.

Estimation of fungal genetic diversity

The genetic diversity of the 17 fungus samples was analysed through the polymorphism expressed by the primers. A 0,1 matrix, referring to the absence or presence of bands in each isolate, was generated from the RAPD amplifications and ARDRA results. Variations in band intensity were not considered as differences. The matrix was prepared using the Neighbour-Joining coefficient. Dendrograms were generated by a UPGMA analysis and NTSYS 2.0 (Numeral Taxonomy and Multivariate Analysis System) programme (Rohlf 1993).

Fungal pathogenicity

Netting was used to collect A. postica adults in the field between the hours of 06:00 to 09:00. The captured insects were transported to the laboratory in 500 mL bottles with perforated tops to allow ventilation. They were fed cane leaves to favour survival during transportation. Once in the laboratory, the insects fed on the Chontalpo-variety grass Brachiaria decumbens Stapf during the experiment. Pathogenicity was compared for nine M. anisopliae isolates collected in the sugarcane fields, two isolates provided by the CNRCB and the standard stock provided by the "Tiemelonlà Nich Klum" laboratory. The control consisted of a sterile distilled water solution of Inex with a coadjuvant (Inex, 2 µlL⁻¹). The insects were treated with a 1 × 10⁸ conidia mL⁻¹ solution of each isolate. The conidia concentration was determined using a hemocytometer (Goettel & Inglis 1997). The adult insects were submerged in the fungal solution contained in a Petri dish for 1 minute using a soft paintbrush. Each experimental unit consisted of a 100 × 15 mm box with 10 A. postica insects. Four replicates were used. Insect mortality was recorded 24 h after starting the experiment. Each dead insect was placed in a 100 × 15 mm Petri dish with a double layer of damp filter paper and incubated at 25 ± 1 °C to favour fungus development and sporulation and to corroborate the inoculated species. The mortality percentage was calculated by relating the number of dead insects to the total number of insects at the start of the experiment. The bioassays were repeated when the control mortality was ≥ 20 % (Alves et al. 1998). An analysis of variance and a Tukey multiple Benchmark test of averages (p < 0.01) were used to analyse the data. Before running the variance analysis, the percentage data were normalised by arcsine transformation. The analyses were carried out using the Minitab ® Statistical Software (Minitab Inc. 2007).

Relationship between fungal genetic diversity, fungal pathogenicity and geographic distance

The Spearman's rank correlation test (Minitab Inc. 2007) was used to analyse the relationships among the genetic diversity of M. anisopliae isolates (Jaccard coefficient), their pathogenicity against A. postica (mortality percentage) and the geographic distance between the sites where the isolates were collected (Figure 1).

 

Results

Fungal isolates collected in the sampled sugarcane fields

Fourteen fungal isolates were collected in the sugarcane fields in this study (Table 1; Figure 1). Three were collected in 2007 and 11 in 2008, representing 9.1 % and 33.3 % of the sampled sites, respectively. In total, 10 isolates were morphologically identified as M. anisopliae (Tulloch 1976), two as B. bassiana (Humbert 1997), and two were not identified. In 2008, two M. anisopliae isolates were obtained from A. postica adults that were collected in sugarcane fields where the reference isolate of M. anisopliae (L0909) was sprayed in 2005. The other eight M. anisopliae isolates were obtained with the Galleria baiting method in sugarcane fields without M. anisopliae application. Most of the isolates (seven) were collected in the western area of the Los Ríos region where M. anisopliae has not been used against A. postica, due to a historically low pest population.

PCR amplification of the rDNA ITS region and restriction analysis (ARDRA)

Six hundred to 800 bp of DNA fragments were obtained from the PCR amplification of the rDNA ITS1-ITS4 region for the analysed fungal isolates (Figure 2a). The size of the DNA fragments of the M. anisopliae isolates collected in sugarcane fields was very similar to that recorded for the M. anisopliae reference strain L0909. All the fungal isolates gave rise to a 650 bp product, with the exception of the isolate YR0812 for which the amplicon was 800 bp in length and the isolate JC0816 with a 700 bp amplicon.

The restriction rDNA of the products following treatment with Hae II (Figure 2b) resulted in the generation of 200 to 700 bp fragments. The restriction profile of the M371 and M372 isolates differed from that of other isolates, possibly as the result of an incomplete digestion. These results were not analysed in this study.

A comparison of the obtained rDNA ITS of the fungal isolates with respect to the GenBank (NCBI) database indicated that isolates MM0801, AD0702, AD0803, CD0804, FC0805, FC0706, AS0807, GB0808, L0909, BC0710, SF0811, M370 and M374 corresponded to M. anisopliae and isolates AR0814 and JC0816 were from B. bassiana.

In the dendrogram generated from the restriction analysis (Figure 3), two neighbouring clades grouped together the reference strain of M. anisopliae (L0909) and the 10 isolates of this fungus that were collected in the sugarcane fields. One clone was observed in each clade, the first clone with isolates MM0801 and AD0702, and the second with isolate GB0808 and the L0909 reference strain. This last clone was genetically similar to isolate AS0807. The dendrogram indicated a genetic similarity between isolates CD0804 and FC0805. Four isolates of M. anisopliae collected in the sugarcane fields, two from each clade, formed solitary subgroups. Two isolates of M. anisopliae var. anisopliae provided by the CNRCB formed a separate subgroup. Both B. bassiana isolates (AR0814 and JC0816) were classified in the same subgroup. Finally, differences were observed between the unidentified isolates YR0812 and ES0813, and both also differed from the remainder of the fungal isolates.

RAPD-PCR analysis

The RAPD amplification of the DNA of M. anisopliae isolates 1 to 10 from Table 1 with OPB07 and OPB09 and its dendrogram is presented in figures 4 and 5. Genetic variation was observed amongst the isolates, with polymorphic bands ranging between 250 and 2500 bp (Figure 4). The dendrogram formed three subgroups with two isolates each and four subgroups with only one isolate each (Figure 5). Isolate FC0805 was genetically similar to the reference isolate L0909. One of the subgroups was made up of two isolates (CD0804 and AS0807) which were collected in two sugarcane fields that were located nearby (1.8 km). However, in another subgroup, the isolates (MM0801 and AD0702) were collected in two sugarcane fields that were separated by a distance of at least 20 km (Figure 1).

Fungal pathogenicity

Mortality of M. anisopliae isolates on A. postica adults ranged from 5 % to 55 % (Figure 6). There were significant differences regarding A. postica mortality of isolates AD0702 (55.0 ± 8.66 %), AD0803 (52.5 ± 6.29 %), MM0801 (50.0 ± 18.26 %), CD0804 (45.0 ± 2.89 %), BC0710 (42.5 ± 8.54 %) and SF0811 (27.5 ± 6.29 %), versus the control (0 %) (F = 5.74; df = 12, 39; p < 0.001). In this group, only the isolate MM0801 was obtained from A. postica adults. The others were collected using the Galleria baiting method.

Relationship between fungal genetic diversity, fungal pathogenicity and geographic distance

No significant association was detected between the genetic diversity of M. anisopliae isolates (Jaccard coefficient) and the pathogenicity against A. postica (mortality percentage) (S = 18318.84; P = 0.1729). Similarly, no significant associations were detected between genetic diversity and geographic distance between sites where isolates were collected (S = 14700.15; P = 0.8367), or between pathogenicity and geographic distance (S = 17366.10; P = 0.3453).

 

Discussion

Fourteen fungus isolates were collected from sugarcane fields in the Los Rios region of Tabasco, Mexico (Table 1), distributed from east to west across the study area (Figure 1). As stated by Salgado et al. (2010), the sampled east-west strip is wetter than the northern and southern areas of the region. Ten of the isolates were identified as M. anisopliae considering their morphological characteristics and comparing their genetic sequences with the GenBank database. The number of positive collections of M. anisopliae was lower in this study in comparison with those reported by other authors. For example, compared with the study by Inglis et al. (2008) that was carried out in British Columbia, Canada, our 10 isolates represent a lower percentage of positive cases of this fungus from the total number of analysed samples (33 % vs 57 %). This percentage is even lower considering that three isolates were collected in 2007 and seven in 2008. These differences may be explained by the environmental conditions that predominate in both studies (temperate region vs tropical region), and also because Inglis et al. (2008) sampled a greater number of habitats, and sampling was restricted to rural sugarcane growing areas in our study.

Eight of the M. anisopliae isolates were obtained with the Galleria baiting method and two by netting A. postica adults, suggesting that this fungus is generally easier to collect from the soil, possibly because it is more abundant there. The ability of M. anisopliae to survive in soil is well recognised (Vanninen et al. 2000; Bidochka et al. 2001; Sallam et al. 2007; St. Leger 2008).

Regarding isolate geography, the two isolates obtained from A. postica were collected in the eastern area of the Los Rios region (Figure 1). Historically, sugarcane fields in this area have suffered more damage from this pest given the close proximity to pastures, and some sugarcane farmers have used M. anisopliae to control A. postica (Bautista-Gálvez & Gonzalez-Cortes 2005). It is worth mentioning that the soil in the sugarcane fields is even wetter in the western area of the region, where seven M. anisopliae isolates were collected (Figure 1), as this area tends to flood occasionally when the Usumacinta river overflows. The higher frequency of M. anisopliae in this area supports reports of increased fungus survival in wet soils (Zimmermann 2007). The presence of B. bassiana in the sugarcane fields was lower than that of M. anisopliae, with only two isolates, one obtained with the Galleria method and the other by capturing A. postica adults. The lower presence of B. bassiana was possibly because this fungus has not been applied to control other pests like the sugarcane borer Diatraea saccharalis (F.) in the Los Ríos region.

From the genetic point of view, the isolates were separated into two large groups or clades, one grouping six isolates in two subgroups of two and two subgroups of one each, and the other grouping four isolates in one subgroup of two and two subgroups of one each. These groups demonstrate the existence of genetic heterogeneity among the M. anisopliae isolates collected in the study area, in agreement with the observations of other authors that have reported a high level of genetic diversity (Riba et al. 1985; Becerra et al. 2007). The genetic analysis suggests that two of the M. anisopliae isolates obtained in 2008, AS0807 isolated from Galleria larvae and GB0808 isolated from A. postica adults, collected in the western and eastern areas of the Los Rios region respectively, and separated by a distance of approximately 20 km (Figure 1), are similar to the L0909 reference isolate that was used by some producers in 2005 to control A. postica. This suggests two points. First, that commercial M. anisopliae products can persist in sugarcane fields for at least three years after application, in agreement with experiments carried out by Milner et al. (2003) who reported a persistence of 3.5 years for M. anisopliae in a sugarcane crop in Australia. Second, from its origin in commercial applications, a fungus can disperse great distances from the original treated site, possibly aided by farmers' practices like the shared use of tractors to plough the soil and of lorries to transport sugarcane from fields to sugar refineries. Indeed, Bidochka et al. (2001) suggested that certain agricultural practices may favour the dispersion of M. anisopliae. Taking into consideration our results, it may be stated that the L0909 isolate has spread throughout the Los Rios region from a few sprayings, presenting the possibility of recombination between introduced and native strains of the species, as well as the eventual impact that this phenomenon could have on the genetic diversity of M. anisopliae in the region and its pathogenicity towards A. postica. Although the methods used in this study did not allow detection of recombination between fungal isolates, recombination events have been detected using isoenzyme techniques (Bidochka et al. 2001).

Genetic analyses also confirmed the existence of a native genotype of M. anisopliae that may be distributed throughout the whole region, represented by the isolates MM0801 (to the east) and AD0702 (to the west), collected in sugarcane fields a fair distance apart (ca 20 km), that were genetically identical. In contrast, a genotype that was only found in the western area of the region was represented by isolates AD0804 and FC0805. These findings suggest the existence of different adaptations to environmental conditions among the native isolates. In this respect, Bidochka et al. (2001) pointed out that the habitat can affect the genetic population of M. anisopliae as, in the absence of host insects, the fungus possesses the ability to survive in the soil where it is exposed to factors such as organic matter, pesticides, desiccation, solar radiation and fluctuations in temperature.

The genetic analyses of two isolates typified as M. anisopliae var. anisopliae, collected in sugarcane fields, one in Oaxaca, Mexico (M370) and the other in Veracruz, Mexico (M374), both provided by the CNRCB, indicated that the isolates collected in the Los Ríos region of Tabasco do not belong to this variety of Metarhizium, as the CNRCB isolates formed a genetically similar group that was different from our isolates (Figure 3).

The pathogenicity towards A. postica of the M. anisopliae isolates collected in the Los Rios region, expressed as a percentage of adult mortality, varied from very low (5 %) to intermediate (55 %). Six of the isolates caused significantly greater A. postica mortality than the control. These may therefore be promising candidates for the second stage of the selection process consisting of virulence tests involving lethal concentrations and mean lethal time assays. It is clear that pathogenicity studies for the selection of M. anisopliae isolates are a prerequisite in a biological control programme for A. postica. In contrast with other similar studies on the genetic diversity of M. anisopliae, our study explored the relationship between pathogenicity and the degree of genetic similarity and geographical distance between isolates. According to our results, these variables are not related. In Chile, Becerra et al. (2007) found no association between the genetic diversity of M. anisopliae and the geographical origin. At least in the Los Rios region, there are no factors or environmental conditions that favour the spatial clustering of isolates with similar genetic and/or pathogenic characteristics. Furthermore, it is not possible to forecast isolate pathogenicity with respect to their degree of genetic similarity. Nevertheless, this study made it possible to identify several native M. anisopliae isolates that merit more detailed research due to their pathogenicity against A. postica and their widespread distribution throughout the Los Ríos region, particularly the MM0801 and AD0702 clonal isolates.

RAPD markers and ITS-rDNA sequence analyses were applied to determine the genetic diversity of M. anisopliae isolates, as has been previously done in studies of this fungus (Cobb & Clarkson 1993; Fegan et al. 1993; Bridge et al. 1997; Castrillo et al. 2003; Driver et al. 2000; Entz et al. 2005; Becerra et al. 2007; Freed et al. 2011). In order to determine the phylogenetic relationships within the M. anisopliae isolates from the Los Ríos region, studies that employ a multigene phylogenetic approach using near-complete sequences from nuclear encoded EF-1α, RPB1, RPB2 and β-tubulin gene regions will be necessary, as Bischoff et al. (2009) recently proposed.

In conclusion, a high diversity of M. anisopliae genotypes exists, with a wide range of pathogenicity characteristics against A. postica, in the sugarcane fields of Los Ríos, Tabasco, Mexico, particularly in the wetter areas of the region. It was demonstrated that the reference isolate L0909, commercially used for the control of this pest, may persist three years after its application in the sugarcane fields of the region, and may be found at sites 20 km from where it was originally applied. Finally, some of the native M. anisopliae isolates collected in the Los Rios region presented characteristics that make them promising candidates as agents of biological control against A. postica.

 

Acknowledgements

The authors thank the Fondos Mixtos CONACyT-Tabasco State Government (Project TAB-2007-C09-74101) for financial support, Javier Valle-Mora for statistical support and Trevor Williams for helpful comments to the manuscript. We especially thank the sugarcane producers of the Los Rios region of Tabasco for facilitating access to their sugarcane fields.

 

Cited literature

Almeida JE, Alves MSB, Pereira RM (1997) Selection of Beauveria spp. isolates for control of the termite Heterotermes tenuis. Journal of Applied Entomology 121: 539-543.         [ Links ]

Alves SB, Almeida JE, Minor Jr A, Alves L (1998) Técnicas de laboratorio, pp. 637-710. In Alves SB (ed) Controle Microbiano de Insetos. Fundação de Estudos Agrários Luiz de Queiroz, Piracicaba, Sao Paulo, Brazil.         [ Links ]

Bautista-Galvéz A, González-Cortes N (2005) Metarhizium anisopliae en mosca pinta en caña de azúcar. Universidad y Ciencia 41: 37-40.         [ Links ]

Becerra VV, Paredes MC, Meriño CR, Iglesias AF, Franco JD (2007) Intraspecific differentiation of Chilean isolates of the entomopathogenic fungi Metarhizium anisopliae var. anisopliae as revealed by RAPD, SSR and ITS markers. Genetics and Molecular Biology 30: 89-99.         [ Links ]

Bedding RA, Akurst RJ (1975) A simple technique for the detection of insect parasitic rhabditid nematodes in soil. Nematologica 21: 109-116.         [ Links ]

Bidochka MJ, Kamp MAA, Vender LMT, Dekoning J, Croos ANJ (2001) Habitat association in two genetic groups of the insect pathogenic fungus Metarhizium anisopliae: Uncovering cryptic species? Applied and Environmental Microbiology 67: 1335-1342.         [ Links ]

Bischoff FJ, Rehner SA, Humber RA (2009) A multilocus phylogeny of the Metarhizium anisopliae lineage. Mycologia 101: 512-530.         [ Links ]

Bridge DP, Prior C, Sagbohan J, Lomer J, Carey M, Buddie A (1997) Molecular characterization of isolate of Metarhizium from locusts and grasshoppers. Biodiversity and Conservation 6: 177-189.         [ Links ]

Castrillo AL, Vandenberg DJ, Wraight PS (2003) Strain-specific detection of introduced Beauveria bassiana in agricultural fields by use of sequence-characterized amplified region markers. Journal of Invertebrate Pathology 82: 75-83.         [ Links ]

Cobb BD, Clarkson JM (1993) Detection of molecular variation in the insect pathogenic fungus Metarhizium using RAPD-PCR. Microbiology 112: 319-324.         [ Links ]

Cobb BD, Clarkson JM (1994) A simple procedure for optimizing the polymerase chain reaction (PCR) using modified Taguchi methods. Nucleic Acids Research 22: 3801-3805.         [ Links ]

Driver F, Milner RJ, Trueman JWH (2000) A taxonomic revision of Metarhizium based on a phylogenetic analysis of rDNA sequence data. Mycological Research 104: 134-150.         [ Links ]

Doberski JW, Tribe HT (1980) Isolation of entomogenous fungi elm bark and soil with reference to ecology of Beauveria bassiana and Metarhizium anisopliae. Transactions of the British Mycological Society 74: 95-100.         [ Links ]

DNASTAR Inc. (2009) Getting Started with DNASTAR® Lasergene® User's guide: Statistical software. Eleventh Ed., v. 8.1. Printed in Madison, WI. 209 p.         [ Links ]

Entz CS, Johson LD, Kawchuk ML (2005) Development of a PCR-based diagnostic assay for the specific detection of the entomopathogenic fungus Metarhizium anisopliae var. acridum. Mycological Research 109: 1302-1312.         [ Links ]

Fegan M, Manners JM, Maclean DJ, Irwin JAG, Samuels KDZ, Holdom DG, Li DP (1993) Random amplified polymorphic markers reveal high degree of genetic diversity in the entomopathogenic fungus Metarhizium anisopliae var. anisopliae. Journal of General Microbiology 139: 2075-2081.         [ Links ]

Freed S, Liang JF, Xiang RS (2011) Determination of genetic variability among the isolates of Metarhizium anisopliae var. anisopliae from different geographical origins. World Journal of Microbiology and Biotechnology 27: 359-370.         [ Links ]

Goettel SM, Inglis DG (1997) Fungi: Hyphomycetes, pp. 213-244. In Lacey AL (ed), Manual of Techniques in Insect Pathology. Academic Press. San Diego.         [ Links ]

Gómez-Leyva JF, Lara-Reyna J, Hernández-Cuevas LV, Martínez-Soriano JP (2008) Specific polymerase chain reaction-based assay for the identification of the arbuscular mycorrhizal fungus Glomus intraradices. Journal of Biological Sciences 8: 563-569.         [ Links ]

Guerrero JC, Mera KM, Carrillo LR (2000) Discriminación de cepas nativas chilenas del hongo entomopatógeno Metarhizium anisopliae var. anisopliae con marcadores moleculares RAPD. Agro Sur 28: 81-91.         [ Links ]

Hatting LJ, Humber RA, Poprawski TJ, Miller RM (1999) A survey of fungal pathogens of aphids from South Africa, with special reference to cereal aphids. Biological Control 16: 1-12.         [ Links ]

Humbert AR (1997) Fungi: Identification, pp. 153-185. In Lacey AL (ed), Manual of Techniques in Insect Pathology. Academic Press. San Diego.         [ Links ]

Inglis DG, Duke GM, Goettel MS, Kabaluk JT (2008) Genetic diversity of Metarhizium anisopliae var. anisopliae in southwestern British Columbia. Journal of Invertebrate Pathology 98: 101-113.         [ Links ]

Kendall JM, Rygiewicz PT (2005) Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts. BMC Microbiology 5: 1-11.         [ Links ]

Lee SB, Taylor JW (1990) Isolation of DNA from fungal mycelia and single spores, pp. 282-287. In Innis M, Gelfand D, Sninsky J, White T (eds) PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego.         [ Links ]

Lilly VG, Barnett HL (1951) Physiology of the Fungi. McGraw-Hill, NY. 464 p.         [ Links ]

Milner JR, Samson P, Morton R (2003) Persistence of conidia of Metarhizium anisopliae in sugarcane fields: Effect of isolate and formulation on persistence over 3.5 years. Biocontrol Science and Technology 13: 507-516.         [ Links ]

Minitab Inc. (2007) User's guide: Statistical software, v. 15. State College, PA. Minitab Inc.         [ Links ]

Riba G, Azevedo LJ, Messias C, Dalsiveira DW, Tuveson R (1985) Studies of the inheritance of virulence in the entomopathogenic fungus Metarhizium anisopliae. Journal of Invertebrate Pathology 46: 20-25.         [ Links ]

Rohlf FJ (1993) NTSYS-pc Numerical Taxonomy and Multivariate Analysis System. Version 1.8. Applied Biostatistics Inc. Exeter Software Setauket. NY.         [ Links ]

Sallam NM, Mcavoy AC, Samson RP, Bull JJ (2007) Soil sampling for Metarhizium anisopliae spores in Queensland sugarcane fields. BioControl 52: 491-505.         [ Links ]

Salgado GS, Nuñez ER, Palma LDJ, Lagunes ELC, Ortiz GCF, Ascencio JMR (2005) Bases para generar un programa sustentable de fertilización en un ingenio de Tabasco, México. Interciencia 30: 395-403.         [ Links ]

Salgado GS, Palma LDJ, Zavala CJ, Lagunes ELC, Ortiz GCF, Castelan EM, Guerrero PA, Aranda IEM, Moreno CE, Rincón RJA (2010) Lotificación del campo cañero: Una metodología para iniciar la agricultura de precisión en ingenios de México. Interciencia 35: 183-190.         [ Links ]

St Leger JR (2008) Studies on adaptations of Metarhizium anisopliae to life in the soil. Journal of Invertebrate Pathology 98: 271-276.         [ Links ]

Tulloch M (1976) The genus Metarhizium. Transactions of the British Mycological Society 66: 407-411.         [ Links ]

Vanninen I (1997) Distribution and occurrence of four entomopathogenic fungi in Finland: Effect of geographical location, habitat and soil type. Mycological Research 91: 93-101.         [ Links ]

Vanninen I, Tyni JJ, Hokkanen H (2000) Persistence of augmented Metarhizium anisopliae and Beauveria bassiana in Finnish agricultural soils. BioControl 45: 201-222.         [ Links ]

White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, pp. 315-322. In Innis M, Gelfand D, Sninsky J, White T (eds) PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego.         [ Links ]

Williams J, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18: 6531-6535.         [ Links ]

Zimmermann G (2007) Review on safety of the entomopathogenic fungus Metarhizium anisopliae. Biocontrol Science and Technology 17: 879-920.         [ Links ]