Species Diversity of Entomopathogenic Fungi Infecting the Sugarcane Aphid Melanaphis sacchari : A Recently Introduced Pest in Mexico

The sugarcane aphid, Melanaphis sacchari, is a key pest that affects sorghum in Mexico. During 2014 to 2016, in South of Tamaulipas sites and Bajio region in Guanajuato, the populations of this aphid were infected by different species of Hypocrealean fungi. Based in the morphometric identification and molecular characterization, the species associated with sugarcane aphid in South of Tamaulipas were Lecanicillium longisporum, Beauveria bassiana and Isaria javanica. In this region, the higher infection levels were caused by L. longisporum, mortality range from 30.0% to 50.0%. The presence of I. javanica and B. bassiana represented less than 26.0% and 10.0%, respectively. In Guanajuato, the species found corresponded to L. longisporum and B. bassiana. The infection levels of both species in sugarcane aphid populations in Guanajuato sites were less than 1.00%. The natural occurrence of entomopathogenic fungi on sugarcane aphid populations was associated with climactic factors such as temperature and relative humidity and development of infections was possibly affected by abiotic factors such as crop phenological stage and applications of chemical insecticides realized by farmers for control of this aphid. Further studies on the ecology and physiology of these fungi and trials to determine virulence and persistence in M. sacchari populations are needed. This is the first report on natural presence of L. longisporum, B. bassiana and I. javanica causing disease on Melanaphis sacchari in Guanajuato and Tamaulipas, Mexico. How to cite this paper: Zambrano-Gutiérrez, J., Alatorre-Rosas, R., Carrillo-Benítez, M.G., Lomelí-Flores, J.R., Guzmán-Plazola, R.A., Azuara-Domínguez, A. and Terán-Vargas, A.P. (2019) Species Diversity of Entomopathogenic Fungi Infecting the Sugarcane Aphid Melanaphis sacchari: A Recently Introduced Pest in Mexico. Advances in Microbiology, 9, 38-55. https://doi.org/10.4236/aim.2019.91004 Received: December 12, 2018 Accepted: January 11, 2019 Published: January 14, 2019 Copyright © 2019 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access DOI: 10.4236/aim.2019.91004 Jan. 14, 2019 38 Advances in Microbiology J. Zambrano-Gutiérrez et al.


Introduction
The sugarcane aphid (SCA) Melanaphis sacchari (Zehntner) (Hemiptera: Aphididae) is an invasive species and the most important insect pest of Sorghum spp.
in Mexico and the United States of America [1] [2] [3].This aphid can infest sorghum in all its stages of development and cause production losses of 20 to 100% [1] [2] [4].The foliar application of chemical insecticides such as imidacloprid, spirotetramat, thiamethoxam, sulfoxaflor, and fluopyradifurone is themain strategy to control SCA [1] [2] [5].However, the inadequate management of these chemicals in the field can induce the development of resistance in M. sacchari populations.This situation has been documented in other Aphididae species with some of the insecticides used for the control of SCA [6] [7] [8].
Thus, other strategies such as sorghum genotypes tolerant to M. sacchari have been evaluated [9] [10] [11] [12].Predators, parasitoids, and species of entomopathogenic fungi (EPF) such as Lecanicillium lecanii (Zimm.)Zare and Gams and L. longisporum (Petch) Zare and Gams can offer a sustainable control M. sacchari populations [2] [13] [14] [15].EPF is the most important organism for the biological control of aphids; however their infection on insect populations can be regulated by climatic conditions such as temperature, relative humidity, rain, and wind [16] [17] [18].Worldwide, L. longisporum and L. lecanii have been reported causing epizooties in SCA populations [15] [19].Other genera such as Beauveria, Isaria and several species of Entomophthorales can infect other species of aphids [16] [20].Given the recent introduction of this pest in Mexico, it is important to look for species of fungi that are infecting this pest in the most important sorghum producing areas, in order to look for (in future works) candidates for their microbial control.Thus, we identified the species of EPF which were infecting the sugarcane aphid in some regions of Tamaulipas and Guanajuato, in first states infested by M. sacchari in Mexico.

Study Area
The study was carried out on agricultural fields of commercial sorghum in different phenological stages planted in Tamaulipas and Guanajuato.For sampling in Tamaulipas, populations of the sugarcane aphid were monitored in December 2014 as well as February, March, and May 2015 in sorghum crops of the autumn-winter cycle located in the municipalities of Aldama, Altamira, and Mante.Samples in Guanajuato were collected during the spring to summer of 2016 in the municipalities of Irapuato, Jaral del Progreso, and Salvatierra (Table 1

Sampling of Aphid Populations
Within each location, five sampling points were established (5 × 5 m at each point).There were four points in the corners and one in the middle of each plot.
At each point, one sample of 10 sorghum leaves was randomly selected and the number of fungal infected and healthy aphids was recorded.Due to the difficulty of identifying fungal pathogens in the field, infected aphids were collected in plastic containers with an airtight lid (15 cm wide by 7 cm deep).These containers were labeled and then kept at 10˚C for 72 hours until processing.Growth stage of the crop was recorded on each sampling occasion using the growth stages for sorghum proposed by Vanderlip and Reeves [21].

Weather Data
The local temperature, precipitation, and relative humidity (RH) data before sampling were obtained from the local weather station of the National Institute of Forestry, Agriculture and Livestock (INIFAP) (Table 4).

Isolation of Entomopathogenic Fungi Infecting the Sugarcane Aphid
The EPF Hypocreales were isolated using the rain conidia descending method proposed by Papierok and Hajek [22] for Entomopthoralean fungi with some modifications.The mycosed aphids were adhered individually with a double-sided adhesive tape to the lid of a 60-mm-diameter Petri dish with 10 mL of Papa Dextrose Agar (PDA: BD Bioxon, Becton Dickinson of Mexico S. A. de C. V., Cuautitlán Izcalli, state of Mexico).The dishes were incubated at 25˚C ± 2˚C in the dark inside a bioclimatic chamber (Thermo Fisher Scientific, Model 3721. Marietta, OH, USA) until colonies developed.Each isolate was purified as monosporic culture and incubated for 15 days at the conditions described above.

Morphological Characterization
For morphological studies of EPF genus, isolates were grown on PDA at 25˚C ± 2˚C for 15 days.Common saprophytic species were excluded from this research.
These included species from the genera Penicillium, Cladosporium, Mucor, and Aspergillus.For detailed morphological comparisons, the characteristics of the colony were described along with the size and shape of the conidia and conidiogenous cells.To determine the shape and size of conidia and conidiogenous cells, microcultures were grown on PDA incubated at 25˚C ± 2˚C in the dark for

Amplification and Sequencing
For the phylogenetic placement of the genus of each isolate, the ITS1/5.8SrDNA/ITS2 region was amplified using the Polymerase chain reaction (PCR) [24].PCR conditions consisted of an initial denaturation at 94˚C for 5 minutes followed by 35  For species identification were amplified the 18S rDNA region and Bt2 of the β-tubulin gen in the isolates of Lecanicillium [25].The mitochondrial intergenic region atp9-nad3 and Bloc in the isolates of Beauveria [26] [27].The small subunit ribosomal ribonucleic acid (SSU rRNA) was amplified in the isolates of Isaria [28].The amplification of each region was realized using the primers indi- cated in Table 2.
The PCR mixture for 18S rDNA region and Bt2 of the β-tubulin were performed with 50 ng of DNA genomic, 1× PCR reaction buffer, 1.5 mM MgCl 2 , 0.2 mM dNTP's mix (QIAGEN GmbH, Hilden, Germany), 0.6 mM of each primer, and 1 U Taq DNA Polymerase.For both regions, the amplification conditions included an initial denaturation at 94˚C for 5 minutes; 35 cycles of denaturation at 94˚C for 60 seconds, annealing at 55˚C for 60 seconds, and extension at 72˚C for 60 seconds with a final extension at 72˚C for 10 minutes.
The mitochondrial intergenic region atp9-nad3 was amplified using 50  The mixture reaction for SSU region was performed using the same mixture reaction described for amplification of ITS region for Isaria spp.isolates.Cycling conditions for the SSU rRNA region consisted of denaturation at 94˚C for 2 minutes followed for 30 cycles of denaturation at 94˚C for 60 seconds, annealing at 50˚C for 60 seconds, and extension at 70˚C for 3 minutes; the final extension was at 70˚C for 2 minutes.PCR reactions were performed at a final volume of 25 μl using a MyCycler TM thermocycler (BIO-RAD Laboratories Inc. Hercules, California, USA).All amplification products were examined by electrophoresis in 1.5% agarose gel stained with ethidium bromide.Bands were visualized under UV light in a transluminator (Infinity-1000 WL/26 MX, Vilber Lourmat®, Marne la Vallée, France).Samples were sent to Macrogen Inc. (Seoul, Korea) for sequencing with the primers used in the initial amplification.

DNA Sequences Analysis
For obtained the consensus sequences of amplified regions in each isolate, nucleotide sequences were edited and assembled with BioEdit v7.0.5 [29].First, the consensus sequences of ITS region were compared with sequences from the GenBank database of the National Center for Biotechnology Information (NCBI) by Basic Local alignment search Tool (BLAST).For species identification, similar 18S rDNA, Bt2 of the β-tubulin gen, mitochondrial intergenic region atp9-nad3, Bloc, and SSU rRNA sequences were selected of the NCBI and the European Bioinformatics Institute (EMBL-EBI).The select sequences of da-tabases and the amplified in the isolates were aligned with Mega 7.0 using Clustal W [30]. Phylogenetic analysis of aligned sequences was performed by Maximum Likelihood method (MLE), and the dendogram for each region was generated with a statistical analysis by Bootstrap [31] based on 1000 replications.

Morphological Identification
The size of conidia and conidiogenous cells showed high variation between isolates of each genus (Table 3).Six isolates (CP-LlonPAS) were considered into the genus Lecanicillium; these had verticillated conidiogenous cells with a mucilaginous head and macro and micro ellipsoidal conidia with rounded ends.
Colonies of the CP-BbPAS isolates had white mycelium with a powdery aspect and abundant sporulation.The micro cultures had short globose to flask-sharped conidiogenous cells that were clustered or whorled.The rachis was elongated and long in shape of zigzag after each conidium.The detached conidiogenous cells had a succession of global to round and densely packed conidia with average dimensions of 1.97 to 2.30 × 1.65 to 2.02 μm (long × wide).The size of conidiogenous cells was of 3.84 to 3.94 × 1.32 to 1.49 μm (long × wide).The morphological characteristics and the dimensions of the conidia and conidiogenous cells were similar to B. bassiana (Blas.)Vuill [33] [34] [35].
The CP-IjaPASisolates were characterized by his powdery appearance and white to gray coloration that changed to pink during sporulation.The shape of conidia was elongated with dimensions of 2.61 to 5.24 × 1.35 to 2.22 μm (long × wide) and placed formed long chains.Conidiogenous cells have a globose basal portion with a narrow and long neck that connects with the conidia.These structures varying in size ranged of 3.63 to 8.37 × 1.17 to 2.55 μm (long × wide).
The macro and microscopic characteristics of those isolates are similar to that described for the genus Isaria.The dimensions of conidia and conidiogenous cells are similar to descriptions reported by others authors for I. javanica (Frieder.& Bally) [36] [37].

Molecular Analyses
BLAST analysis showed that sequences ITS1/5.8SrDNA/ITS2 of the isolates CP-LlonPAS presented high similarity (94.0% to 99.0%) with some sequences corresponding to Lecanicillium species deposited in the GenBank.Phylogenetic tree for the sequences of the region Bt2 of β-tubulin gen showed that the CP-LlonPAS isolates were grouped within L. longisporum supported 80.0% bootstrap values (Figure 1(a)).Based in the phylogenetic analysis of 18S rDNA region, the six isolates CP-LlonPAS were grouped with L. longisporum DOI: 10.4236/aim.2019.91004The results of the BLAST analysis showed that sequences ITS1/5.8SrDNA/ITS2 of the isolates CP-Ija1PAS and CP-Ija2PAS presented between 97.0% to 99.0% of similarity with several species of genus Isaria.Phylogenetic tree indicated that both isolates were grouped within I. javanica supported by bootstrap value of 100.0%(Figure 3).

Natural Incidence of Mycosed Aphids
The highest aphid mortality was caused by L. longisporum in the Tamaulipas sites, where the infection levels were 30.0% to 50.0%, while in Guanajuato this specie represented less than 1.00%.B. bassiana infected SCA populations in ranges of 6.0% to 10.0% in Tamaulipas sites; while this pathogen in Guanajuato  showed low levels of infection.The presence of I. javanica was specific for Mante and Aldama, Tamaulipas; with infection range from 19.5% to 26.0% (Figure 4).

Climatic Conditions in the Sampled Sites
In Guanajuato, the infections for L. longisporum occurred after eight days with a mean value of RH of 68.0% to 88.0% and medium temperature of 19˚C (Table 4).The presence of L. longisporum in the Tamaulipas sites was preceded by a period of RH higher than 87.0% and temperature range of 15˚C to 24˚C (Table 4).The natural presence of B. bassiana in Guanajuato and Tamaulipas was DOI: 10.4236/aim.2019.91004observed when the RH was from 68.0% to 81.0% with a mean temperature of 13˚C to 30˚C in the days before detection.The presence of aphids infected by I. javanica in Tamaulipas was remarkable when the temperature was 18˚C to 30˚C and RH from 69.0% to 77.0%.

Discussion
The search for native species of EPF is important for implementing a microbial DOI: 10.4236/aim.2019.91004control strategy of the SCA.However, to establish a successful strategy it is important to know the exact identity of the EPF collected [37] [38].Based on the morphological study (size and shape of the conidia and conidiogenous cells and characteristics of the colonies), the EPF isolates obtained in the Bajío region and southern Tamaulipas were grouped into three genera Lecanicillium (six isolates: CP-LlonPAS), Beauveria (four isolates: CP-BbPAS) and Isaria (two isolates: CP-IjaPAS).This classification was validated with a BLAST analysis that confirmed that the sequences ITS1/5.8SrDNA/ITS2 of the CP-LlonPAS, CP-BbPAS and CP-IjaPAS isolates showed high similarity with the homologous sequences of Lecanicillium, Beauveria and Isaria species deposited in the GenBank.For the morphological identification of the species, the dimensions of the conidia and the conidiogenous cells were determined.The dimensions of these structures in the isolates CP-LlonPAS, CP-BbPAS and CP-IjaPAS were similar to those documented by other authors for L. longisporum, B. bassiana and I. javanica or I. fumosorosea, respectively.However, morphological characteristics and classical taxonomic studies often do not provide sufficient evidence for species differentiation within each genus of EPF [39] [40] [41].
The identification of CP-LlonPAS isolates was confirmed with phylogenetic analysis of the regions 18S rDNA and Bt2 of the β-tubulin gen.The combination of morphometric studies and phylogenetic analyses of the amplified regions confirm that the six CP-LlonPAS isolates belong to L. longisporum.This specie was previously reported as a natural regulator of SCA populations in Tecomán, Colima and on others aphids species in different regions worldwide [15] [18] [42].Molecular identification of species within the Beauveria genus may be possible by sequencing of the mitochondrial intergenic region nad3-atp9 and Bloc [26] [27] [41].The four CP-BbPAS isolates were identified as B. bassiana based in the combination of morphometric characteristics and molecular analysis of intergenic mitochondrial region nad3-atp9 and Bloc.The genus Beauveria is a common pathogen among several insect species of agricultural importance [34] including SCA.Based in the morphological studies, the isolates CP-IjaPAS belong to I. javanica or I. fumosorosea.BLAST analysis indicated that the isolates CP-IjaPAS presented high similarity with some sequences corresponding to I. javanica or I. fumosorosea.However, phylogenetic tree constructed with SSU rRNA region sequences grouped both isolates within I. javanica.
EPF natural infections on insect populations are regulated by biotic and abiotic factors, such as temperature, relative humidity, rain, wind, and cultural practices [16] [43] [44].The climate in Tamaulipas is usually warm, dry, and temperate [45], the municipalities in which sampling was performed are located in the southern region near the Gulf of Mexico.This zone is characterized by tropical weather (Aw) with an annual average temperature highest to 18˚C and a high percentage of humidity coming from the sea [45] [46].In Guanajuato state, the aphid collections were from Celaya and Jaral del Progreso; these municipalities are part of the Bajio region that has a humid and temperate climate (Cw) with an average annual rainfall of 600 to 800 mm that L. longisporum causes infection in an average temperature range of 20˚C to 25˚C with a relative humidity greater than 70.0% in populations of M. sacchari, Brevicoryne brassicae (L.) and Myzus persicae (Sulzer) [15] [18].Similar conditions were observed before the detection of aphids with mycosis in the sampling sites of Guanajuato and Tamaulipas.However, in Guanajuato infection levels were lower than 1.0%.A similar situation was observed with the incidence of B. bassiana in Guanajuato and Los Aztecas, Tamaulipas; in these sites RH was less than 70.0%.Conidia of B. bassiana require a relative humidity greater than 95.0% to germinate [44].The highest incidence of B. bassiana was observed in Cuahutemoc, Tamaulipas, where the relative humidity was higher than 80.0%, while that in the sites with low presence of infected aphids by B. bassiana the RH was less than 70.0%.The presence of aphids infected by I. javanica in Tamaulipas was remarkable when the temperature was between 15˚C to 33˚C and the RH was 65.0% to 82.0%.Some species of Isaria have thermotolerance, for example, I. javanica can develop at 35˚C [47].The CP-IjaPAS isolates, collected in Tamau- lipas, might have this characteristic because they were collected in areas where the maximum temperature fluctuated from 30˚C to 35˚C.
Others factors that may be related to the low levels of infection observed in Guanajuato are cultural practices and agrochemicals applications.The use of chemical fertilizers and insecticides affect the survival of EFP [16] [17].Generally, when aphid population increases, producers apply insecticides by reducing the population of SCA.These control actions can prevent the propagation of the EFP species associated with this aphid, since low density can affect the potential of transmission and development of epizootics [16] [48].Also, in some sites the detection of infected aphids was carried out during the physiological maturity stage of the crops when the presence of SCA was observed in isolated colonies on the leaves.
Adoption of these species for control of SCA will rely on achieving efficacy, cost reduction, and an ability to broaden the range of pest species that may be targeted.Detailed knowledge of fungal ecology is needed to better understand their role in nature and limitations in biological control.Testing under field conditions is required to identify effects of biotic and abiotic factors and their interactions on efficacy, persistence, and potential limitations to the use of these biocontrol agents in certain crops or locations.There are great opportunities to use these fungi in classical and conservation biological control approaches that can improve environmental stability, efficacy and the cost effectiveness.Temperature is one of the most important abiotic factors affecting the biology and ecology of entomopathogenic fungi [49] [50].Therefore, the estimation of the in vitro growth profiles of entomopathogenic fungi is important in order to gain more information towards the understanding of the abiotic factors affecting specific diversity and distribution of fungal species.

Conclusion
The diversity and presence of EF associated with SCA was highest in Tamauli-pas, and wherein the species L. longisporum, B. bassiana and I. javanica were isolated.In Guanajuato, only the species of L. longisporum and B. bassiana were detected.B. bassiana and I. javanica represent the first record of other native species associated with M. sacchari.A more detailed study of the ecology of L. longisporum, B. bassiana and I. javanica within this agroecosystem is needed to analyze their possible role in the insect population dynamics.

Figure 3 .
Figure 3. Phylogenetic trees for isolates of Isaria javanica: CP-IjaPAS and CP-Ija2PAS derived by Maximum Likelihood method with sequences of SSU region.Bootstrap values based on 1000 replications.The analysis involved 10 nucleotide sequences of SSU region (NS1-FS2).

Table 1 .
Geographic location and phenological stage of the crops of sorghum with presence of entomopathogenic fungi associated with sugarcane aphid in Tamaulipas and Guanajuato.
). DOI: 10.4236/aim.2019.91004N = North latitude.W = West longitude.† Isolates obtained from naturally infected individuals of SCA.¶ It was determined at the time of sampling in field based in the established by Vanderlip and Reeves [21].The samplings were carried on: *Commercials hybrids or varieties and **Soca of sorghum.

2.4. Genetic Characterization 2.4.1. DNA Extraction
ferred to sterile 20 mL vials and lyophilized for 48 h using a freeze dryer (Free-Zone 4.5, Labconco Coporation.Kansas City, Missouri, USA).For the DNA extraction, 0.2 g of lyophilized mycelium was deposited in 1.5 mL Eppendorf tube with 1 mL of liquid nitrogen and then was macerated using a No. 14 knitting DOI: 10.4236/aim.2019.91004hook (Fabrics Parisina S. A. de C. V. Texcoco, State of Mexico, Mexico).Total genomic DNA was extracted with the DNeasy® PlantMinikit following the manufacturer's instructions with some modifications [23].DNA concentration was quantified using a spectrophotometer NanoDrop TM 2000/2000 C (Thermo Fisher Scientific, Inc. Waltham, MA, USA), and the samples were stored at −20˚C until used.
cycles of denaturation at 94˚C for 60 seconds, annealing at 55˚C for 60 seconds, and extension at 72˚C for 90 seconds; the final extension was at 72˚C for 7 minutes.
For Lecanicillium sp.isolates, the PCR mix for ITS region contained 30 ng of genomic DNA, 1× PCR reaction buffer, 3.0 mM MgCl 2 , 0.6 mM dNTPs mix (QIAGEN GmbH, Hilden, Germany), 0.6 mM of each primer, and 2.5 U Taq DNA Polymerase (BIOLASE TM ).The mixture reaction for ITS region of Beauveria sp. was performed with 20 ng of genomic DNA, 1× PCR reaction buffer, 2.0 mM MgCl 2 , 0.2 mM dNTP's mix, 0.4 mM of each primer, and 2 U Taq DNA Polymerase.The PCR reaction mixture for Isaria sp.consisted of 80 ng of genomic DNA, 1× PCR reaction buffer, 2.0 mM MgCl 2 , 1.0 mM dNTP's mix, 1.0 mM of each primer, and 2.5 U Taq DNA Polymerase.

Table 2 .
Primers used for PCR ¶ amplification of regions of genomic DNA of entomopathogenic fungi associated with the sugarcane aphid.
turation at 95˚C for 30 seconds, annealing at 55˚C for 30 seconds, extension at 72˚C for 60 seconds, and a final extension at 72˚C for 7 minutes.The PCR reaction for Bloc region was performed with 50 ng of genomic DNA, 1× PCR reaction DOI: 10.4236/aim.2019.91004buffer, 2.0 mM MgCl 2 , 0.2 mM dNTPs mix, 0.6 mM of each primer, and 1 U Taq DNA Polymerase.The amplification conditions for this region consisted of an initial denaturation at 94˚C for 2 minutes; 40 cycles of denaturation at 94˚C for 30 seconds, annealing at 56˚C for 30 seconds, and extension at 72˚C for 60 seconds; and a final extension at 72˚C for 15 minutes.

Table 3 .
Conidial and conidiogenous cells measurements (in μm ± SE) of L. longisporum, B. bassiana and I. javanica isolates of sugarcane aphid in Tamaulipas and Guanajuato, †Mean for length and wide of conidia and conidiogenous cells (n = 100).¶ Minimum and maximum values observed for each data set.**Obtained from micro cultures development in PDA and incubated at 25˚C ± 2˚C in darkness for 10 to 15 days.‡ Dimensions reported by other authors and used as comparison data with those obtained in the present study.SE = Standard error.NA = Data no available.

Table 4 .
Meteorological recorded in the sites with presence of infected sugarcane aphid by entomopathogenic fungi in Guanajuato and Tamaulipas, México.