TY - JOUR
AU - Ruiz-Montoya, Lorena
AB - Abstract The knowledge of the diversity and genetic structure of pest insects under management contributes to the improvement of control strategies. An experiment was run to investigate whether the addition of the fungus Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Cordycipitaceae) (BB) and compost to soil affects the presence and genetic diversity of adults and larvae of Phyllophaga obsoleta Blanch (Coleoptera: Melolonthinae) larvae in maize crops. We collected adults in and used mating pairs under four treatments (BB, compost, soil, blank). Genetic diversity and structure were determined through five allo/iso-enzymatic loci. Beauveria bassiana affected the presence and mortality of P. obsoleta in the laboratory but not under field conditions. The genetic diversity of P. obsoleta ranged from moderate to high (Ho = 0.26–0.31), with a low genetic differentiation among localities or treatments (Phi < 0.05), indicating high levels of gene flow. Our results showed a weak effect of B. bassiana on P. obsoleta in the field. Still, our laboratory observations suggest that the fungus may be a suitable alternative for biological control. Graphical Abstract Open in new tabDownload slide Graphical Abstract Open in new tabDownload slide This research carried out to evaluate how the addition of Beauveria bassiana and compost to corn crops affects the presence and survival of Phyllophaga obsoleta. biological control, isoenzymes, Mexico, pest insect genetics, Phyllophaga The main factors associated with low maize production include high input costs (Jurado et al. 2013) to avoid losses caused by soil pests (Maag et al. 2015). Melolonthid larvae (Hexapoda, Coleoptera, Scarabaeoidea, Melolonthinae), also known as the complex as the white-grub complex (Zitlalpopoca-Hernandez et al. 2017), are the most common pests. These can cause overall economic losses estimated at $135 million US dollars per year in all crops in Latin America, including maize in Mexico (Marín and Bujanos 2008, Solis et al. 2016). In the state of Chiapas, Mexico, agriculture is carried out under precarious conditions (Maldonado-López et al. 2017). Controlling soil pests such as the white-grub demands the application of fertilizers and insecticides that increase production costs and thus reduce profits per hectare (Ramírez-Salinas and Castro-Ramírez 2000, Álvarez and Anzueto 2004). For these reasons, it is necessary to find methods for soil pest control that maintain soil fertility and the health condition of local inhabitants (Bernardino-Hernández et al. 2016). Root-feeding pests, as the white-grub complex, have been controlled using entomopathogenic organisms (e.g., Guzmán-Franco et al. 2012, Lopes et al. 2013). Among these, Beauveria bassiana (Balsamo) Vuillemin (an entomopathogenic fungus) is recognized as an effective strategy for pest management, because it is highly virulent for pest insects but involves no risk for human health (Gandarilla-Pacheco et al. 2013, Chávez et al. 2014, Solis et al. 2016). Another strategy has been to supply nutrients to crop plants to stimulate tolerance to pests. Compost supplementation improves soil quality (Guo et al. 2016) and promotes maize growth (Lima et al. 2004, Manirakiza and Şeker 2020), which likely compensate for damage from root-feeding pests while fostering the development of entomopathogenic organisms (Velázquez et al. 2006). Additionally, compost has also been considered a food source for the white-grub complex (Ramírez-Salinas and Castro-Ramírez 2000, Vallejo et al. 2007) and might serve as a potential distractor to reduce the pressure exerted on the roots of cultivated plants. It is well known that insects rapidly develop resistance to insecticides (Onstad et al. 2001, Roderik 1996), and the same could happen with other control systems. Population genetic diversity can help to understand the adaptative potential and dispersal capacity of populations (Szalanski et al. 1999, Zhou et al. 2002, Zhang et al. 2018, Petzold-Maxwell et al. 2012). Thus, studying genetic diversity in pest insect populations is essential to improve management strategies and avoid resistance to control methods (Alphey et al. 2009, Sumerford et al. 2013). Management of cultivated plants and their pests can have a different impact on the population dynamics of pest insects and modify their genetic diversity (Fig. 1). Management targeting the crop plant, including fertilization, may not influence the population size of the pest insects (N), and hence its genetic structure would not be affected. By contrast, management targeting the pest insect implies a drastic reduction of its population size, likely involving the selective reduction of alleles and genotypes, at least in the time of fungus application (t1). Understanding how the management of crops and their pest insects affect the genetic diversity of latter—aspect little studied in the field—can help to increase the harvest and identify environmentally friendly methods for pest insect control. Fig. 1. Open in new tabDownload slide A conceptual overview of the potential relationship between the application of compost and an entomopathogenic fungus (Beauveria bassiana) on the genetic diversity of Phyllophaga obsoleta. The management targeting the plant does not affect insect pest, while the management targeting the pest insect reduce their population size (N), at least at the time of fungus application (t1). Fig. 1. Open in new tabDownload slide A conceptual overview of the potential relationship between the application of compost and an entomopathogenic fungus (Beauveria bassiana) on the genetic diversity of Phyllophaga obsoleta. The management targeting the plant does not affect insect pest, while the management targeting the pest insect reduce their population size (N), at least at the time of fungus application (t1). The genetic diversity levels of any of the species in the white-grub complex remain unknown. Phyllophaga obsoleta Blanck is a white group that is a common rhizophagous pest of maize crops in Chiapas, with a widespread distribution in America (Ramírez-Salinas et al. 2011, Romero-López 2012). The addition of the fungus B. bassiana and compost may be useful methods for managing P. obsoleta Blanch (Nájera-Rincón et al. 2005, Scorza-Dagert and Cova 2006), impacting on both survival and genetic diversity. We carried out this study to provide evidence about the effectiveness of the use of compost and an entomopathogenic fungus on the survival of P. obsoleta larvae, and how this combined management of the crop and its pest insect affects the genetic diversity of the latter in the field. The specific objectives were (1) record the genetic diversity of P. obsoleta in the study area, (2) identify changes in the genetic structure of larvae under various treatments, compared to parental adults, and (3) determine the effect of B. bassiana and compost on the survival and genetic diversity of P. obsoleta larvae. We expected a high genetic diversity in both adults and larvae and a poor structure of their wild (untreated) populations due to a high genetic flow across populations. Also, we expected to observe low genetic diversity and survival in the sample of P. obsoleta larvae infected with B. bassiana, and higher genetic diversity and survival in samples of larvae in compost-enriched soil. Materials and Methods Study Area The study was carried out in two municipalities of Chiapas where larvae of Phyllophaga spp. cause serious productivity issues in maize crops cultivated for self-consumption (Ramírez-Salinas and Castro-Ramírez 2000, García et al. 2006). Amatenango del Valle (hereafter Amatenango) in Los Altos (Highlands) region, and Venustiano Carranza, adjacent to Amatenango toward the Central Depression slope. Amatenango is located between coordinates 16°32′N and 92°26′W; the local climate is temperate with summer rains, mean annual precipitation of 1366.5 mm, and mean annual temperature of 16.8°C. The dominant vegetation is pine-oak forest alternating with large areas of maize crops in the lowlands. Venustiano Carranza is located at 16°21′′N and 92°34′′W; the climate is warm subhumid with summer rains and mean annual precipitation of 616 mm; and mean annual temperature of 22°C, the dominant vegetation is low tropical forest (García 1973). Species Studied Phyllophaga obsoleta is a beetle of the Melolonthinae subfamily distributed from the southern United States to Venezuela (Morón 2006). It thrives in humid environments between 800 and 2500 meters above sea level and is common in various types of vegetation (Morón 2001, 2006, 2010). The adults measure 16–24 mm long by 6–8 mm wide on average and display a bright reddish- or yellowish-brown color (Morón 2006). Larvae are grayish-white or off-white, with a yellowish-brown head, strong jaws, and three pairs of well-developed legs (Ramírez-Salinas and Castro-Ramírez 2000). Newly hatched larvae feed on organic matter, undergo accelerated growth, and upon reaching the third instar stage, feed voraciously on roots of both crop and surrounding plants (Vallejo et al. 2007). The eggs, larvae, and pupae dwell exclusively in soil, while adults emerge above-ground in the afternoons after the first rains to feed and reproduce, dying after a few days (Vallejo et al. 2007). The sex ratio of this species in the study area is 2:1 (Cruz-López et al. 2001), and females lay an average of 42 eggs (Ramírez-Salinas and Castro-Ramírez 2000). Field Experiments One experimental plot was established in one maize crop field in each of three localities in Chiapas (Mexico): San Nicolás and Aljó, in Amatenango, and Los Olivos, in Venustiano Carranza (Fig. 2). Sixty seeding points were set in each plot and were randomly assigned to one of the following treatments until 15 seeding points (replicates) were completed per treatment per plot: (1) enrichment with compost, (2) application of the fungus Beauveria bassiana (BB), (3) a control treatment free from competitors and predators, hereafter called ‘soil’, and (4) a blank treatment where competitors or predators were not controlled (see below). Experimental plots measured 14 m × 4 m, delimited by a 1 m strip. Each seeding point was isolated by 0.5 cm × 0.5 cm-mesh steel cylinders measuring 30 cm high and 50 cm in diameter. Each cylinder had an inner lining of dark plastic pierced with a needle to allow water filtration. Cylinders were buried on each seeding point and filled in with soil from each plot (locality), representing a pot. The soil for the plot was sieved, and any animal individuals were removed, as these could bias the outcome of treatments. Fig. 2. Open in new tabDownload slide Location of experimental plots and capture and collection sites of Phyllophaga obsoleta in Chiapas, Mexico. Fig. 2. Open in new tabDownload slide Location of experimental plots and capture and collection sites of Phyllophaga obsoleta in Chiapas, Mexico. Four maize seeds were planted in each pot. Except for blank plots, all pots were covered with a 1 m-high mesh to prevent the entrance of other rhizophagous and predator insects. To inoculate each treatment with P. obsoleta larvae, adults were collected at the Amatenango town square park, given the high abundance of beetles attracted by incandescent lights. Two P. obsoleta male-female couples were placed in each pot of the treatments described below. Treatments Compost (C) The soil was enriched with organic matter using 2 kg of compost, expecting to result in higher maize tolerance to insect attacks. The compost used was processed from organic kitchen waste, stubble, and manure in equal proportion. Larvae used in this treatment were not intentionally inoculated with the fungus, and their general appearance showed no signs of the presence of fungi. Beauveria bassiana (BB) We use the entomopathogenic fungus BB from the strain 5APr R3 (native strain 5 infecting adult Phyllophaga ravida Reseeding 4) produced in the Bioassay Laboratory at ECOSUR (Polanco Mendoza 2008). Phyllophaga obsoleta mating couples were inoculated with BB following the technique described by Cruz-López (1999), i.e., placing the insects in a jar containing fungal spores and shaking gently to promote the attachment of spores to beetles. The jar contained 2 kg of dried conidia (1.88 × 107 conidia/g) of B. bassiana in rice as substrate. This procedure is intended to pass the fungus from parental adults to beetle larvae, aiming to increase larval mortality and reduce maize seedling damage. To obtain data on the effect of BB under laboratory conditions, 30 P. obsoleta couples collected from the different localities (i.e., Amatenango, Aljó, San Nicolás, Los Olivos) were transported to the bioassay laboratory at El Colegio de la Frontera Sur (San Cristobal campus, Chiapas) in 500 ml plastic containers filled with soil from the collection sites. Ten of these 30 couples were randomly selected for inoculation with the fungus. Containers with mating couples were reviewed at 4-day intervals to record mortality and the number of eggs laid. Any eggs detected were counted and placed in moist soil to promote the development of the first larval instar. Soil This treatment consisted of compost-free soil containing P. obsoleta mating couples free from fungal infections. The entry of competitors and predators was prevented by the protective mesh, as in the other treatments. This treatment was included as an experimental control in addition to the blank treatment. We expected to observe the natural development of P. obsoleta larvae. Blank No treatment was applied. Unlike the treatments mentioned above, neither P. obsoleta mating couples were placed in these pots nor was the entry of predators or competitors controlled. The purpose was to allow the normal oviposition of any species in the beetle complex. Six months after the start of each treatment, the soil at each planting site was thoroughly searched for developed larvae. The larvae were counted; then, to determine whether there was an association between the number of larvae and treatment we carry out a Chi-square (χ 2) per locality (Sokal and Rholf 1995). Genetic Diversity of Adults and Larvae Thirty adults from Amatenango, Aljó, Los Olivos, San Nicolás, and all larvae from all planting sites in the three experimental plots were collected (Aljó, Los Olivos, San Nicolás) to analyze their genetic diversity. Samples were stored at −70°C until analysis. Fifteen enzymes were tested in adults (females and males) and larvae in cellulose acetate, according to Hebert and Beaton (1993). The activity and legible staining pattern were observed for three enzymes: Glutamate-Oxaloacetate Transaminase (2.6.1.1, GOT), Malic Enzyme (1.1.1.40, ME), and Esterase (3.1.1.1, EST), in cellulose acetate. ME and EST were run in Tris-Maleate buffer pH 7.8 for 60 and 40 min, respectively. GOT was revealed in phosphates pH 7 for 60 min. Enzymes were extracted by macerating a whole organism with 200 μl of the extraction buffer using 3/4 of YO buffer (Yeh and O’Malley 1980) and 1/4 of VEG II buffer (Cheliak and Pitel 1984). Conventional genetic diversity estimators were obtained, such as the average number of alleles, observed heterozygosity, and expected heterozygosity under the Hardy Weinberg Equilibrium (HWE) by treatment and locality for P. obsoleta larvae and adults using the software GenAlEx v.6.4 (Peakall and Smouse 2006). The genetic structure by treatment and locality was assessed for larvae and adults separately using a Molecular Variance Analysis (AMOVA); and the fixation indices equivalent to Wright’s F statistics (Excoffier et al. 1992), were estimated using the software GenAlex v.6.4 (Peakall and Smouse 2006) with 999 iterations. Besides, Bayesian cluster analysis was performed for larvae and adults separately, and to compare larvae versus sample from Amatenango, i.e., the place of origin of the parental generation of larvae. These analyses were run using Structure v. 2.3. (Pritchard et al. 2000). The clustering method is based on a multilocus genotype model that infers two or more hypothetical ancestral populations (K); individuals are probabilistically assigned to one or more populations depending on the combination of genotypes. The admixture model was used, with independent allelic frequencies. To estimate K, the program was run with a burn-in length of 5,000 iterations and 10,000 MCMCs (Markov chain Monte Carlo) with 15 replicates (to calculate mean and variance to be used to obtain optimal K), without additional information on the population. Then, we manually determined the optimal value of K following the second-order difference ΔK method of Evanno et al. (2005). For larvae, we set a maximum value of K = 8; for adults, K = 4; and to compare larvae versus adults, K = 2. Results Larvae Under Field Conditions A total of 78 larvae were found in the Los Olivos experimental plot: nine larvae in compost, 32 in soil, and 37 in BB. Twenty larvae were recovered in Aljó: 13 in compost, three in soil, and four in BB. Only 11 larvae were found in San Nicolás: six in compost and five in soil. The χ 2 test of the data obtained in each locality revealed that the number of larvae in Aljó and San Nicolás was independent of the treatment, contrary to the findings in Los Olivos (χ 2 = 11.73; P = 0.008). Larvae Under Laboratory Conditions A total of 69 eggs were recovered from eight of 30 mating couples. Of these eggs, 12 (17.93%) came from two couples inoculated with BB, with none developing to the larval stage. From the other 57 eggs (from six couples not inoculated with BB), 22 larvae were produced within 15 d. Each female laid several batches of eggs, a large one (10 to 20 eggs) and several small ones (one to three eggs laid every two days). Eggs from BB-infected mating couples survived for seven days, while the offspring of the uninoculated couples survived for 15 d (from egg to the first-stage larva). Males inoculated with BB died within five days, while females survived for ten days. Uninoculated females and males survived for 21 d and uninoculated for 14 d. Genetic Diversity Larvae The total sample of larvae showed three alleles for the locus EST and two for ME-1, ME-2, GOT-1, and GOT-2, with frequencies that differed between treatments (Fig. 3); the exception was the locus GOT-2, which was virtually monomorphic across treatments and localities (Fig. 3E). EST Allele 3 (the fastest or with the highest migration rate) was more frequent in Aljó in all treatments and less frequent in Los Olivos and San Nicolás relative to Aljó; it was not recorded in the compost treatment in San Nicolás (Fig. 3A). ME-1 Allele 2 (fast) showed the highest frequency across treatments and localities and was the only allele found in the Los Olivos compost treatment (Fig. 3B). The two ME-2 alleles showed almost the same frequency in all treatments and localities (Fig. 3C). Loci GOT-1 and GOT-2 included two alleles, with allele 2 (the fastest) showing a markedly higher frequency (Fig. 3D). Fig. 3. Open in new tabDownload slide Allele frequencies of Phyllophaga obsoleta larvae based on five isoenzymatic loci for three treatments in three localities of Chiapas, Mexico. C, Compost; BB, Beauveria bassiana; Compost: pots with soil enriched with organic matter and mating couples of P. obsoleta, covered only for a few days at the start of the experiment; BB: pots with mating couples of P. obsoleta inoculated with the fungus B. bassiana and covered at the start of the experiment; Soil: pots with compost-free soil and P. obsoleta free from B. bassiana and covered. EST: a locus for Esterase; ME-1: locus 1 for Malic Enzyme; ME-2: locus 2 for Malic Enzyme; GOT-1: locus 1 for Glutamate-Oxaloacetate Transaminase; GOT-2: locus 2 for Glutamate-Oxaloacetate Transaminase. Fig. 3. Open in new tabDownload slide Allele frequencies of Phyllophaga obsoleta larvae based on five isoenzymatic loci for three treatments in three localities of Chiapas, Mexico. C, Compost; BB, Beauveria bassiana; Compost: pots with soil enriched with organic matter and mating couples of P. obsoleta, covered only for a few days at the start of the experiment; BB: pots with mating couples of P. obsoleta inoculated with the fungus B. bassiana and covered at the start of the experiment; Soil: pots with compost-free soil and P. obsoleta free from B. bassiana and covered. EST: a locus for Esterase; ME-1: locus 1 for Malic Enzyme; ME-2: locus 2 for Malic Enzyme; GOT-1: locus 1 for Glutamate-Oxaloacetate Transaminase; GOT-2: locus 2 for Glutamate-Oxaloacetate Transaminase. The genotypic frequencies of three loci, namely EST, ME-1, and ME-2, were found to deviate from the expected HWE in the compost treatment. The locus EST was unbalanced in Aljó (χ 2 = 8.21, P = 0.04); for its part, the locus ME-1 was also unbalanced in Aljó (χ 2 = 13.0, P < 0.001), Los Olivos (χ 2 = 9.0, P = 0.003), and San Nicolás (χ 2 = 6, P = 0.014). The locus EM-2 was outside the HWE in Aljó (χ 2 = 7.0, P = 0.008) and Los Olivos (χ 2 = 9.0, P = 0.003). The soil treatment showed two unbalanced loci: ME-2 in Los Olivos (χ 2 = 30.0, P < 0.001) and ME-1 in San Nicolás (χ 2 = 26.24, P < 0.001). For its part, the BB treatment showed only a single unbalanced locus, namely ME-2 in Los Olivos (χ 2 = 31.0, P < 0.001). The average number of alleles per treatment ranged between 1.6 and 2 alleles, with an average of effective alleles between 1.4 and 1.7 alleles (Table 1). The observed heterozygosity varied between 0.267 and 0.320 in larvae samples per treatment and locality, while He ranged from 0.234 to 0.406, being generally higher than Ho (Table 1). Polymorphism ranged between 40% and 100%; these extreme values were observed in the compost and BB treatments in Los Olivos (Table 1). Table 1. Genetic diversity was estimated for samples of Phyllophaga obsoleta larvae from three maize crop treatments in three localities of Chiapas, Mexico. BB: Beauveria bassiana; SD: standard error; N: sample size; Na: mean number of alleles; Ne: mean number of effective alleles (1/(Σp2); F: Fixation index (1-Ho/He); Ho: observed heterozygosity; He: expected heterozygosity; P%: percent polymorphism Locality . Treatment . N . Na . Ne . Ho . He . F . P% . Aljó Compost 13 2.0 1.728 0.277 0.358 0.140 80 SE 0 0.316 0.293 0.159 0.121 0.327 BB 4 2.0 1.489 0.300 0.336 0.007 80 SE 0 0.316 0.156 0.146 0.096 0.309 Soil 3 1.6 1.437 0.267 0.293 −0.067 60 SE 0 0.245 0.204 0.194 0.128 0.450 Los Olivos Compost 8.8 1.6 1.502 0.300 0.234 −0.416 40 SE 0.2 0.40 0.318 0.200 0.145 0.370 BB 35 2.2 1.586 0.320 0.277 0.216 100 SE 0 0.20 0.321 0.199 0.123 0.365 Soil 30 1.8 1.647 0.320 0.283 0.035 60 SE 0 0.374 0.370 0.203 0.135 0.433 San Nicolás Compost 6 1.8 1.698 0.267 0.406 0.314 80 SE 0 0.2 0.178 0.172 0.102 0.376 Soil 5 2.0 1.649 0.320 0.356 0.064 80 SE 0 0.316 0.290 0.162 0.121 0.310 Average 13.10 1.88 1.59 0.30 0.32 0.08 70 SE 1.86 0.10 0.09 0.06 0.04 0.11 100 Locality . Treatment . N . Na . Ne . Ho . He . F . P% . Aljó Compost 13 2.0 1.728 0.277 0.358 0.140 80 SE 0 0.316 0.293 0.159 0.121 0.327 BB 4 2.0 1.489 0.300 0.336 0.007 80 SE 0 0.316 0.156 0.146 0.096 0.309 Soil 3 1.6 1.437 0.267 0.293 −0.067 60 SE 0 0.245 0.204 0.194 0.128 0.450 Los Olivos Compost 8.8 1.6 1.502 0.300 0.234 −0.416 40 SE 0.2 0.40 0.318 0.200 0.145 0.370 BB 35 2.2 1.586 0.320 0.277 0.216 100 SE 0 0.20 0.321 0.199 0.123 0.365 Soil 30 1.8 1.647 0.320 0.283 0.035 60 SE 0 0.374 0.370 0.203 0.135 0.433 San Nicolás Compost 6 1.8 1.698 0.267 0.406 0.314 80 SE 0 0.2 0.178 0.172 0.102 0.376 Soil 5 2.0 1.649 0.320 0.356 0.064 80 SE 0 0.316 0.290 0.162 0.121 0.310 Average 13.10 1.88 1.59 0.30 0.32 0.08 70 SE 1.86 0.10 0.09 0.06 0.04 0.11 100 Open in new tab Table 1. Genetic diversity was estimated for samples of Phyllophaga obsoleta larvae from three maize crop treatments in three localities of Chiapas, Mexico. BB: Beauveria bassiana; SD: standard error; N: sample size; Na: mean number of alleles; Ne: mean number of effective alleles (1/(Σp2); F: Fixation index (1-Ho/He); Ho: observed heterozygosity; He: expected heterozygosity; P%: percent polymorphism Locality . Treatment . N . Na . Ne . Ho . He . F . P% . Aljó Compost 13 2.0 1.728 0.277 0.358 0.140 80 SE 0 0.316 0.293 0.159 0.121 0.327 BB 4 2.0 1.489 0.300 0.336 0.007 80 SE 0 0.316 0.156 0.146 0.096 0.309 Soil 3 1.6 1.437 0.267 0.293 −0.067 60 SE 0 0.245 0.204 0.194 0.128 0.450 Los Olivos Compost 8.8 1.6 1.502 0.300 0.234 −0.416 40 SE 0.2 0.40 0.318 0.200 0.145 0.370 BB 35 2.2 1.586 0.320 0.277 0.216 100 SE 0 0.20 0.321 0.199 0.123 0.365 Soil 30 1.8 1.647 0.320 0.283 0.035 60 SE 0 0.374 0.370 0.203 0.135 0.433 San Nicolás Compost 6 1.8 1.698 0.267 0.406 0.314 80 SE 0 0.2 0.178 0.172 0.102 0.376 Soil 5 2.0 1.649 0.320 0.356 0.064 80 SE 0 0.316 0.290 0.162 0.121 0.310 Average 13.10 1.88 1.59 0.30 0.32 0.08 70 SE 1.86 0.10 0.09 0.06 0.04 0.11 100 Locality . Treatment . N . Na . Ne . Ho . He . F . P% . Aljó Compost 13 2.0 1.728 0.277 0.358 0.140 80 SE 0 0.316 0.293 0.159 0.121 0.327 BB 4 2.0 1.489 0.300 0.336 0.007 80 SE 0 0.316 0.156 0.146 0.096 0.309 Soil 3 1.6 1.437 0.267 0.293 −0.067 60 SE 0 0.245 0.204 0.194 0.128 0.450 Los Olivos Compost 8.8 1.6 1.502 0.300 0.234 −0.416 40 SE 0.2 0.40 0.318 0.200 0.145 0.370 BB 35 2.2 1.586 0.320 0.277 0.216 100 SE 0 0.20 0.321 0.199 0.123 0.365 Soil 30 1.8 1.647 0.320 0.283 0.035 60 SE 0 0.374 0.370 0.203 0.135 0.433 San Nicolás Compost 6 1.8 1.698 0.267 0.406 0.314 80 SE 0 0.2 0.178 0.172 0.102 0.376 Soil 5 2.0 1.649 0.320 0.356 0.064 80 SE 0 0.316 0.290 0.162 0.121 0.310 Average 13.10 1.88 1.59 0.30 0.32 0.08 70 SE 1.86 0.10 0.09 0.06 0.04 0.11 100 Open in new tab The Analysis of Molecular Variance indicated that the greatest genetic variation occurred within individuals (estimated variance 92%), and the rest among samples of larvae per treatment and locality (8%) (PhiPT = 0.076, P = 0.006), i.e., a low but significant genetic structure between larvae samples. According to an AMOVA pairwise comparison, significant genetic differences were found between the compost treatment in Aljó and San Nicolás versus the compost, BB, and soil treatments in Los Olivos (Table 2). Differences were also found between the compost in San Nicolás and the BB and soil treatment in Los Olivos (Table 2). Table 2. PhiPT values below the diagonal from AMOVA pairwise comparisons between samples of Phyllophaga obsoleta larvae recovered from treatments per locality in Chiapas, México. P-values are above the diagonal. BB: Beauveria bassiana treatment. Figures in bold indicate statistically significant differences . . Locality . Locality . Treatment . Aljó . Los Olivos . San Nicolás . . . Compost . BB . Soil . Compost . BB . Soil . Compost . Soil . Aljó Compost 0.389 0.462 0.054 0.043 0.038 0.122 0.429 BB 0 0.407 0.159 0.187 0.073 0.157 0.499 Soil 0 0 0.074 0.189 0.061 0.062 0.581 Los Olivos Compost 0.129 0.120 0.225 0.101 0.143 0.010 0.360 BB 0.081 0.064 0.094 0.058 0.113 0.002 0.372 Soil 0.088 0.163 0.215 0.044 0.031 0.020 0.373 San Nicolás Compost 0.096 0.149 0.218 0.229 0.297 0.166 0.352 Soil 0 0 0 0 0 0 0.026 . . Locality . Locality . Treatment . Aljó . Los Olivos . San Nicolás . . . Compost . BB . Soil . Compost . BB . Soil . Compost . Soil . Aljó Compost 0.389 0.462 0.054 0.043 0.038 0.122 0.429 BB 0 0.407 0.159 0.187 0.073 0.157 0.499 Soil 0 0 0.074 0.189 0.061 0.062 0.581 Los Olivos Compost 0.129 0.120 0.225 0.101 0.143 0.010 0.360 BB 0.081 0.064 0.094 0.058 0.113 0.002 0.372 Soil 0.088 0.163 0.215 0.044 0.031 0.020 0.373 San Nicolás Compost 0.096 0.149 0.218 0.229 0.297 0.166 0.352 Soil 0 0 0 0 0 0 0.026 Open in new tab Table 2. PhiPT values below the diagonal from AMOVA pairwise comparisons between samples of Phyllophaga obsoleta larvae recovered from treatments per locality in Chiapas, México. P-values are above the diagonal. BB: Beauveria bassiana treatment. Figures in bold indicate statistically significant differences . . Locality . Locality . Treatment . Aljó . Los Olivos . San Nicolás . . . Compost . BB . Soil . Compost . BB . Soil . Compost . Soil . Aljó Compost 0.389 0.462 0.054 0.043 0.038 0.122 0.429 BB 0 0.407 0.159 0.187 0.073 0.157 0.499 Soil 0 0 0.074 0.189 0.061 0.062 0.581 Los Olivos Compost 0.129 0.120 0.225 0.101 0.143 0.010 0.360 BB 0.081 0.064 0.094 0.058 0.113 0.002 0.372 Soil 0.088 0.163 0.215 0.044 0.031 0.020 0.373 San Nicolás Compost 0.096 0.149 0.218 0.229 0.297 0.166 0.352 Soil 0 0 0 0 0 0 0.026 . . Locality . Locality . Treatment . Aljó . Los Olivos . San Nicolás . . . Compost . BB . Soil . Compost . BB . Soil . Compost . Soil . Aljó Compost 0.389 0.462 0.054 0.043 0.038 0.122 0.429 BB 0 0.407 0.159 0.187 0.073 0.157 0.499 Soil 0 0 0.074 0.189 0.061 0.062 0.581 Los Olivos Compost 0.129 0.120 0.225 0.101 0.143 0.010 0.360 BB 0.081 0.064 0.094 0.058 0.113 0.002 0.372 Soil 0.088 0.163 0.215 0.044 0.031 0.020 0.373 San Nicolás Compost 0.096 0.149 0.218 0.229 0.297 0.166 0.352 Soil 0 0 0 0 0 0 0.026 Open in new tab Adults A total of 242 adults were analyzed. No genetic differences were found between females and males, so the results reported correspond to both sexes pooled. Three alleles were detected in the loci EST and GOT-1, with the predominance of one of these alleles (intermediate movement) (Fig. 4A, D). For their part, loci ME-1, ME-2, and GOT-2 showed two alleles with a frequency close to 1.0 of the most mobile allele in loci ME-1 and GOT-2 (Fig. 4B, E), and a similar frequency in ME-2 (Fig. 4C). Fig. 4. Open in new tabDownload slide Allele frequencies of Phyllophaga obsoleta adults based on five isoenzymatic loci for three treatments in four localities of Chiapas, Mexico. Amatenango: Amatenango del Valle; C: Compost; BB: Beauveria bassiana. Fig. 4. Open in new tabDownload slide Allele frequencies of Phyllophaga obsoleta adults based on five isoenzymatic loci for three treatments in four localities of Chiapas, Mexico. Amatenango: Amatenango del Valle; C: Compost; BB: Beauveria bassiana. Allele richness in adult samples was similar across localities (Na = 2.4), except for Amatenango, where we found an average of 2.2 alleles (Table 3). The average effective number of alleles was also similar across localities (Ne from 1.884 to 1.936) (Table 3). The observed heterozygosity, Ho, was slightly variable across localities, from 0.348 to 0.400. The expected heterozygosity by the Hardy–Weinberg Equilibrium was slightly higher than Ho for two out of three samples. A 100% polymorphism was recorded for three out of four samples (Table 3). Almost all loci deviated from the Hardy–Weinberg Equilibrium, except for EST and GOT-1 in San Nicolás (Table 4). The AMOVA showed no genetic differences across localities (PhiPT = 0.009, P = 0.093, d.f. = 3), with an overall genetic variation of 1%. The pairwise comparisons (AMOVA) detected slight genetic differences only between San Nicolás and Amatenango (PhiPT = 0.026, P = 0.025) (Table 5). Table 3. Genetic diversity in samples of Phyllophaga obsoleta adults from four localities in Chiapas, Mexico, based on five enzymatic loci. SD: standard error; N: sample size; Na: mean number of alleles; Ne: mean number of effective alleles (1/(Σp2); F: Fixation index (1-Ho/He); Ho: observed heterozygosity; He: expected heterozygosity; P%: percent polymorphism, F: Fixation index (1-Ho/He) Locality . N . Na . Ne . Ho . He . F . P% . Amatenango del Valle 78.0 2.200 1.936 0.400 0.381 0.092 80 SE 0.894 0.374 0.385 0.176 0.136 0.320 Aljó 59.2 2.400 1.981 0.348 0.384 0.288 100 SE 0.583 0.245 0.419 0.155 0.140 0.281 Los Olivos 53.0 2.400 1.884 0.385 0.380 0.301 100 SE 0.775 0.245 0.349 0.185 0.128 0.366 San Nicolás 57.8 2.400 1.855 0.357 0.373 0.351 100 SE 0.80 0.245 0.341 0.156 0.127 0.306 Total 62.0 2.350 1.914 0.373 0.379 0.267 95 SE 2.212 0.131 0.172 0.077 0.061 0.148 Locality . N . Na . Ne . Ho . He . F . P% . Amatenango del Valle 78.0 2.200 1.936 0.400 0.381 0.092 80 SE 0.894 0.374 0.385 0.176 0.136 0.320 Aljó 59.2 2.400 1.981 0.348 0.384 0.288 100 SE 0.583 0.245 0.419 0.155 0.140 0.281 Los Olivos 53.0 2.400 1.884 0.385 0.380 0.301 100 SE 0.775 0.245 0.349 0.185 0.128 0.366 San Nicolás 57.8 2.400 1.855 0.357 0.373 0.351 100 SE 0.80 0.245 0.341 0.156 0.127 0.306 Total 62.0 2.350 1.914 0.373 0.379 0.267 95 SE 2.212 0.131 0.172 0.077 0.061 0.148 Open in new tab Table 3. Genetic diversity in samples of Phyllophaga obsoleta adults from four localities in Chiapas, Mexico, based on five enzymatic loci. SD: standard error; N: sample size; Na: mean number of alleles; Ne: mean number of effective alleles (1/(Σp2); F: Fixation index (1-Ho/He); Ho: observed heterozygosity; He: expected heterozygosity; P%: percent polymorphism, F: Fixation index (1-Ho/He) Locality . N . Na . Ne . Ho . He . F . P% . Amatenango del Valle 78.0 2.200 1.936 0.400 0.381 0.092 80 SE 0.894 0.374 0.385 0.176 0.136 0.320 Aljó 59.2 2.400 1.981 0.348 0.384 0.288 100 SE 0.583 0.245 0.419 0.155 0.140 0.281 Los Olivos 53.0 2.400 1.884 0.385 0.380 0.301 100 SE 0.775 0.245 0.349 0.185 0.128 0.366 San Nicolás 57.8 2.400 1.855 0.357 0.373 0.351 100 SE 0.80 0.245 0.341 0.156 0.127 0.306 Total 62.0 2.350 1.914 0.373 0.379 0.267 95 SE 2.212 0.131 0.172 0.077 0.061 0.148 Locality . N . Na . Ne . Ho . He . F . P% . Amatenango del Valle 78.0 2.200 1.936 0.400 0.381 0.092 80 SE 0.894 0.374 0.385 0.176 0.136 0.320 Aljó 59.2 2.400 1.981 0.348 0.384 0.288 100 SE 0.583 0.245 0.419 0.155 0.140 0.281 Los Olivos 53.0 2.400 1.884 0.385 0.380 0.301 100 SE 0.775 0.245 0.349 0.185 0.128 0.366 San Nicolás 57.8 2.400 1.855 0.357 0.373 0.351 100 SE 0.80 0.245 0.341 0.156 0.127 0.306 Total 62.0 2.350 1.914 0.373 0.379 0.267 95 SE 2.212 0.131 0.172 0.077 0.061 0.148 Open in new tab Table 4. χ 2 tests to assess the Hardy–Weinberg Equilibrium on samples of Phyllophaga obsoleta adults from four localities of Chiapas, México. Amatenango: Amatenango del Valle; d.f.: degrees of freedom; p: P-value Locality . Locus . d.f. . χ 2 . p . Amatenango EST 3 13.889 0.004 GOT-1 3 13.389 0.004 GOT-2 1 58.317 0.000 EM-1 Monomorphic EM-2 1 58.432 0.000 Aljó EST 3 13.931 0.003 GOT-1 3 14.036 0.003 GOT-2 1 13.983 0.000 EM-1 1 59.000 0.000 EM-2 1 30.149 0.000 Los Olivos EST 3 9.386 0.025 GOT-1 3 9.386 0.025 GOT-2 1 54.000 0.000 EM-1 1 53.000 0.000 EM-2 1 50.000 0.000 San Nicolás EST 3 4.535 0.209 GOT-1 3 4.535 0.209 GOT-2 1 57.000 0.000 EM-1 1 59.000 0.000 EM-2 1 22.812 0.000 Locality . Locus . d.f. . χ 2 . p . Amatenango EST 3 13.889 0.004 GOT-1 3 13.389 0.004 GOT-2 1 58.317 0.000 EM-1 Monomorphic EM-2 1 58.432 0.000 Aljó EST 3 13.931 0.003 GOT-1 3 14.036 0.003 GOT-2 1 13.983 0.000 EM-1 1 59.000 0.000 EM-2 1 30.149 0.000 Los Olivos EST 3 9.386 0.025 GOT-1 3 9.386 0.025 GOT-2 1 54.000 0.000 EM-1 1 53.000 0.000 EM-2 1 50.000 0.000 San Nicolás EST 3 4.535 0.209 GOT-1 3 4.535 0.209 GOT-2 1 57.000 0.000 EM-1 1 59.000 0.000 EM-2 1 22.812 0.000 Open in new tab Table 4. χ 2 tests to assess the Hardy–Weinberg Equilibrium on samples of Phyllophaga obsoleta adults from four localities of Chiapas, México. Amatenango: Amatenango del Valle; d.f.: degrees of freedom; p: P-value Locality . Locus . d.f. . χ 2 . p . Amatenango EST 3 13.889 0.004 GOT-1 3 13.389 0.004 GOT-2 1 58.317 0.000 EM-1 Monomorphic EM-2 1 58.432 0.000 Aljó EST 3 13.931 0.003 GOT-1 3 14.036 0.003 GOT-2 1 13.983 0.000 EM-1 1 59.000 0.000 EM-2 1 30.149 0.000 Los Olivos EST 3 9.386 0.025 GOT-1 3 9.386 0.025 GOT-2 1 54.000 0.000 EM-1 1 53.000 0.000 EM-2 1 50.000 0.000 San Nicolás EST 3 4.535 0.209 GOT-1 3 4.535 0.209 GOT-2 1 57.000 0.000 EM-1 1 59.000 0.000 EM-2 1 22.812 0.000 Locality . Locus . d.f. . χ 2 . p . Amatenango EST 3 13.889 0.004 GOT-1 3 13.389 0.004 GOT-2 1 58.317 0.000 EM-1 Monomorphic EM-2 1 58.432 0.000 Aljó EST 3 13.931 0.003 GOT-1 3 14.036 0.003 GOT-2 1 13.983 0.000 EM-1 1 59.000 0.000 EM-2 1 30.149 0.000 Los Olivos EST 3 9.386 0.025 GOT-1 3 9.386 0.025 GOT-2 1 54.000 0.000 EM-1 1 53.000 0.000 EM-2 1 50.000 0.000 San Nicolás EST 3 4.535 0.209 GOT-1 3 4.535 0.209 GOT-2 1 57.000 0.000 EM-1 1 59.000 0.000 EM-2 1 22.812 0.000 Open in new tab Table 5. PhiPT-values below the diagonal from AMOVA pairwise comparisons between samples of Phyllophaga obsoleta adults of four localities from Chiapas, México. Amatenango: Amatenango del Valle; P-values are above the diagonal Locality . Amatenango . Aljó . Los Olivos . San Nicolás . Amatenango 0.204 0.285 0.025 Aljó 0.006 0.295 0.258 Los Olivos 0.003 0.004 0.006 San Nicolás 0.026 0.004 0.247 Locality . Amatenango . Aljó . Los Olivos . San Nicolás . Amatenango 0.204 0.285 0.025 Aljó 0.006 0.295 0.258 Los Olivos 0.003 0.004 0.006 San Nicolás 0.026 0.004 0.247 Open in new tab Table 5. PhiPT-values below the diagonal from AMOVA pairwise comparisons between samples of Phyllophaga obsoleta adults of four localities from Chiapas, México. Amatenango: Amatenango del Valle; P-values are above the diagonal Locality . Amatenango . Aljó . Los Olivos . San Nicolás . Amatenango 0.204 0.285 0.025 Aljó 0.006 0.295 0.258 Los Olivos 0.003 0.004 0.006 San Nicolás 0.026 0.004 0.247 Locality . Amatenango . Aljó . Los Olivos . San Nicolás . Amatenango 0.204 0.285 0.025 Aljó 0.006 0.295 0.258 Los Olivos 0.003 0.004 0.006 San Nicolás 0.026 0.004 0.247 Open in new tab The cluster analysis using Structure was consistent with the AMOVA; no genetic structure was observed in adults since the two ancestral groups inferred were similarly allocated across adult sample sets (Fig. 5A–D). In larvae, cluster 2 predominated in all localities, with no clear structure among treatment within localities (Fig. 6). A difference was observed between Amatenango adults and the total sample of larvae: adults showed a composition consistent with the hypothetical cluster 1, while the ancestral cluster 2 prevailed in larvae (Fig. 7A, B). Fig. 5. Open in new tabDownload slide Genetic cluster analysis of Phyllophaga obsoleta adults implemented in Structure v. 2.3 (Pritchard et al. 2000). Each bar represents an individual with an ancestry ratio for an optimal K (hypothetic clusters) value equal to 2. Fig. 5. Open in new tabDownload slide Genetic cluster analysis of Phyllophaga obsoleta adults implemented in Structure v. 2.3 (Pritchard et al. 2000). Each bar represents an individual with an ancestry ratio for an optimal K (hypothetic clusters) value equal to 2. Fig. 6. Open in new tabDownload slide Genetic cluster analysis of Phyllophaga obsoleta larvae implemented in Structure v. 2.3 (Pritchard et al. 2000). Each bar represents an individual with an ancestry ratio for optimal K value (hypothetic clusters) equal to 2. The treatment and location where larvae were recovered are indicated. Compost: pots with soil enriched with organic matter and mating couples of P. obsoleta, and covered only for a few days at the start of the experiment; BB: pots with mating couples of P. obsoleta inoculated with the fungus Beauveria bassiana and covered at the start of the experiment; Soil: pots with compost-free soil and P. obsoleta free from B. bassiana and covered. Fig. 6. Open in new tabDownload slide Genetic cluster analysis of Phyllophaga obsoleta larvae implemented in Structure v. 2.3 (Pritchard et al. 2000). Each bar represents an individual with an ancestry ratio for optimal K value (hypothetic clusters) equal to 2. The treatment and location where larvae were recovered are indicated. Compost: pots with soil enriched with organic matter and mating couples of P. obsoleta, and covered only for a few days at the start of the experiment; BB: pots with mating couples of P. obsoleta inoculated with the fungus Beauveria bassiana and covered at the start of the experiment; Soil: pots with compost-free soil and P. obsoleta free from B. bassiana and covered. Fig. 7. Open in new tabDownload slide Genetic cluster analysis of Phyllophaga obsoleta adults from Amatenango del Valle (Amatenango) using the software Structure v. 2.3 (Pritchard et al. 2000). (A), the locality of the parental generation of larvae, and (B) larvae. Each bar represents an individual with an ancestry ratio for K (hypothetic clusters) equal to 2. The treatment and locality where larvae were recovered are indicated. Compost: pots with soil enriched with organic matter and mating couples of P. obsoleta and covered only for a few days at the start of the experiment; BB: pots with mating couples of P. obsoleta inoculated with the fungus Beauveria bassiana and covered at the start of the experiment; Soil: pots with compost-free soil and P. obsoleta free from B. bassiana and covered. Fig. 7. Open in new tabDownload slide Genetic cluster analysis of Phyllophaga obsoleta adults from Amatenango del Valle (Amatenango) using the software Structure v. 2.3 (Pritchard et al. 2000). (A), the locality of the parental generation of larvae, and (B) larvae. Each bar represents an individual with an ancestry ratio for K (hypothetic clusters) equal to 2. The treatment and locality where larvae were recovered are indicated. Compost: pots with soil enriched with organic matter and mating couples of P. obsoleta and covered only for a few days at the start of the experiment; BB: pots with mating couples of P. obsoleta inoculated with the fungus Beauveria bassiana and covered at the start of the experiment; Soil: pots with compost-free soil and P. obsoleta free from B. bassiana and covered. Discussion The results obtained in this work suggest a minor effect of the inoculation with B. bassiana or compost enrichment on the presence of P. obsoleta under field conditions. However, the results of the trial under laboratory conditions indicate that BB can be used as a control on eggs and adults of P. obsoleta. As regards genetic diversity, the findings show moderate diversity in larvae and high diversity in adults, indicating a high adaptive potential in adults. A low but significant genetic structure was found in larvae of compost samples, suggesting a minor effect of these treatments. Larvae Under Field Conditions The number of larvae recovered from the study sites was highly heterogeneous, both between localities and treatments. This may be due to several factors that were not considered at the beginning of the study, such as 1) infertile mating couples or no mating, 2) egg viability, or 3) random larval mortality. The factors that induce mating in Melolonthinae are still unknown (Wenninger and Averill 2006), and this may be a relevant topic for managing P. obsoleta. Another possibility is that the local environmental conditions may have influenced the number of larvae obtained. The higher number of larvae observed in Los Olivos was probably due to more rainy days in this locality, which probably favored larvae survival. Soil moisture is one of the most important characteristics affecting the survival and abundance of rhizophagous insects (Régnière et al. 1981, Vallejo et al. 2007). King (1994) noted that living roots and slightly acidic soils appear to be essential for the survival P. obsoleta during its soil life, and that females show a marked preference for laying eggs on humus-rich soil and in areas covered by grasses. Los Olivos had a pH of 7.2 and 8.4% organic matter, conditions that may have contributed to a favorable environment for larvae. Our results indicate the importance of conducting further research on the characteristics of the microhabitat in the field to improve the effectiveness of entomopathogenic fungi against P. obsoleta. The measurement of microenvironmental variation is an aspect that should be included in future research, as it likely influences the survival and reproduction of P. obsoleta. Larvae Under Laboratory Conditions Although larvae abundance was not consistently lower in the BB treatment in the field, we recorded a smaller number of larvae from adults inoculated with the fungus under laboratory conditions. Besides, the life expectancy of these adults dropped by 52%, consistent with the rate (62.5% at day 8) recorded by Chávez et al. (2014). The effectiveness of B. bassiana may have dropped in the field since the peak contamination of larvae with BB was recorded toward the end of the crop cycle (October-November), when larvae have already damaged crops (Ramírez-Salinas and Castro-Ramírez 2000). Thus, any positive effect of BB as pest control would be evident until the next agricultural cycle, as contaminated larvae do not reach the adult stage. This indicates that a longer period may be required for its effect to become evident (Cabrera-Mora et al. 2019). Our laboratory results and the low number of larvae recorded in the BB treatment in Aljó, as well as their absence in San Nicolás, suggest that using B. bassiana may be a suitable biological control of P. obsoleta in maize fields. This is especially relevant for low-income farmers, whose crops are mainly for self-consumption (Bernardino-Hernández et al. 2016, Maldonado-López et al. 2017). We recommend conducting further field experiments with follow-up over at least three annual cycles, inoculating B. bassiana directly into soil once the emergence of P. obsoleta adults is observed and when evidence of the presence of larvae is obtained (Solis et al. 2016). To note, females survived longer than males when both were inoculated with B. bassiana, suggesting that P. obsoleta may develop differential resistance to B. bassiana between sexes. Females lay eggs 11 d after mating (Ramírez-Salinas and Castro-Ramírez 1998, 2000); even females inoculated with B. bassiana can produce offspring, thus lowering the effectiveness of B. bassiana. Genetic Diversity of Larvae Samples of P. obsoleta larvae recovered from experimental plots had moderate-to-high diversity levels, with low differentiation between the eight experimental units where larvae were recorded (treatment by locality). The initial expectation was to observe a lower genetic diversity (He) in the BB treatment compared to soil and compost, assuming that B. bassiana is a selective factor so that only those genotypes that are resistant to this factor survive, hence reducing the total genetic diversity (Butlin 2010). The selective death of P. obsoleta larvae, if any, necessarily leads to the genetic differentiation between the BB treatment and all other treatments. Within localities, no significant differences were observed between treatments, suggesting that the application of compost or BB produced a random effect on the genetic diversity of larvae in the observation sites. This random effect could be attributed to the small sample size in most treatments. In the BB treatment in Aljó, the sample size was small (n = 4), which undoubtedly introduces a bias in any of the genetic diversity parameters affected by sample size. The pairwise comparison analysis revealed that larvae from the compost treatment in Aljó and San Nicolás differed only slightly, although significantly, versus larvae from any of the treatments in Los Olivos, which is reflected in the cluster analysis as the dominance of the hypothetic cluster 1 in Los Olivos. This finding suggests that the effects of treatments were mediated by the local environmental conditions, indicating the need to improve the experimental design to show, with a greater statistical power, the selective effects of BB and the promotion of resistance of maize plants through field composting. Additionally, further studies should involve a greater number of loci and molecular markers that are expressed similarly in adults and larvae. The present study showed three alleles of the locus GOT in larvae and two in adults, which is expected for enzymes (Richardson et al. 1986). Our results suggest the need to consider ecological and genetic aspects for the development of larval control strategies with long-term efficiency by considering an evolutionary genetic perspective in the management of P. obsoleta. Genetic Diversity of Adults As expected, the genetic diversity observed in adult populations of P. obsoleta was generally high, probably because it supports large populations (Cruz-López et al. 2001). Also, the flight capacity of adults (Morón 2001) contributes to the genetic flow between localities, thus contributing to increase the diversity of alleles in the populations involved. Across samples, polymorphism showed higher values versus those observed in other coleopteran species. For example, González-Rodríguez et al. (2000) found 23.3% polymorphism in Acanthoscelides obvelatus Bridwell (bean weevil, bean pest) in Mexico using alloenzymes, while our results showed a mean polymorphism of 60%, similar to that of populations of A. obtectus Say (bean weevil, a pest of beans and other legumes; 72.2%), and Chrysomela tremulae Fabricius (defoliating beetle; 71%) obtained with enzymes (Génissel et al. 2000). In addition, mean heterozygosity was higher in populations of P. obsoleta (HO = 0.28) relative to populations of A. obvelatus (0.09) (González-Rodríguez et al. 2000). The heterozygosity observed in P. obsoleta adults, although high, is lower than the levels observed in other pest species; for example, the defoliating insect of eucalyptus crops, Chrysophtharta agricola Chapuis, with H = 0.50 (Nahrung and Allen 2003) and the western conifer seed bug, Leptoglossus occidentalis Heidemann, with H = 0 0.45–0.73 (Lesieur et al. 2014). However, our values were close to the figure reported for invertebrates in general (H = 0.30; Falconer and Mackay 2001) and for pest species such as A. obtectus and Chrysomela tremulae Fabricius (H = 0.26) (Génissel et al. 2000, González-Rodriguez et al. 2000). The genotypic frequency of most loci was skewed relative to the values expected under HWE, showing that populations of P. obsoleta undergo at least one of the following processes: migration, mutation, natural selection, nonrandom mating, and gene drift (Hartl and Clark 1997). It should be stressed that although we observed a deviation from HWE, no genetic differentiation between localities was detected. The low differentiation found suggests, in principle, a high genetic flow between localities (Whitlock and McCauley 1999). However, there is probably some barrier between San Nicolás and Amatenango and Los Olivos, since a low but significant differentiation was detected between these localities. Potential barriers that can be suggested included the urban area of Amatenango itself, as the public lighting of the urban area may restrain the displacement from one site to another. The migration of adults probably contributes to the genetic flow and the homogenization of populations, as long as natural selection exerts no negative effect on migrating individuals (Núñez-Farfán and Eguiarte 1999, Frankham et al. 2004). Our results suggest that the adult population is genetically homogeneous across the region comprising the four localities, strengthened by genetic flow and that the potential local selection is of lower intensity than flow; the combined effect of these two factors would lead to high local and regional diversity. The cluster analysis evidenced the lack of genetic structure in the set of adult samples. Clusters 1 and 2 show a similar composition across all localities. The high reproductive potential and the sustained high genetic diversity support the resistance of P. obsoleta to temporal and spatial environmental changes, with the potential to withstand chemical, biological, and even mechanical control measures (e.g., mass capture of adults). Interestingly, a likely change in the genetic composition of larvae relative to adults was observed in the cluster analysis. This change may have resulted from genetic drift produced by the random transmission of alleles across generations, as well as from a possible effect of the local environmental conditions on both adults and larvae. We observed that cluster 2 was the genetic group that prevailed in larvae recovered in Aljó, whereas cluster 1 was the dominant genetic group in larvae from San Nicolás was cluster 1. To note, the sample size was small in both San Nicolás and Aljó, which may have contributed to the clustering pattern observed. To note, a possible random effect that may also affect the genetic diversity in the larval samples is the fact of having collected the adults in Amatenango and subdividing them into groups to be placed in the treatments. The subdivision randomly reduces the genetic diversity of the next generation. Conclusion The results of this investigation offer valuable advances on the genetic knowledge of P. obsoleta, a major pest insect, and alternative potential control methods in the field. However, our findings are insufficient to conclude that B. bassiana eliminates P. obsoleta larvae in the field. Nonetheless, we consider that P. obsoleta management with B. bassiana is likely a useful strategy to control this pest and, therefore, mitigate damages to maize crops intended for self-consumption. The variation in the number of larvae per treatment underlines the need to conduct further research on the factors involved in mating and survival, which should be considered in future experimental designs. This research detected the high evolutionary potential and resistance of P. obsoleta, given by the levels of heterozygosity and polymorphism. Future studies on population genetics should include a larger sample size to analyze the genetic structure under different treatments, which will contribute to supporting the implementation of effective pest management actions. Acknowledgments We thank Fundación Produce Chiapas, A.C. (FPCH/011/03), for the financial support granted to carry out this research. To José Luis Navarrete Heredia, Aaron Rodríguez Contreras, Gustavo Moya Raygoza and Marcelino Vázquez García for the revision to a preliminary version. To Eduardo Velásquez-Cruz and Concepción Ramírez-Salinas for their assistance in field work. To Edgar Alberto Aguirre for his assistance in the construction of the location map. María Elena Sánchez-Salazar translated the manuscript into English and Jean Benoit Pinard revised. To Tania Bautista Gutiérrez for design and elaboration of graphical abstract (tania.bautista.editora@gmail.com). References Cited Alphey , N. , M. B. Bonsall, and L. Alphey. 2009 . Combining pest control and resistance management: synergy of engineered insects with Bt crops . J. Econ. Entomol . 102 : 717 – 732 . Google Scholar Crossref Search ADS PubMed WorldCat Álvarez , S. J. D. , and M. M. de J. Anzueto. 2004 . Actividad microbiana del suelo bajo diferentes sistemas de producción de maíz en los Altos de Chiapas, México . Agrociencia 38 : 13 – 22 . Google Scholar OpenURL Placeholder Text WorldCat Bernardino-Hernández , H. U. , R. Mariaca-Méndez, A. Nazar-Beutelspacher, J. D. Álvarez-Solis, A. Torres-Dosal, and C. Herrera-Portugal. 2016 . Factores socieconómicos y tecnológicos en el uso de agroquímicos en tres sistemas agrícolas en Los Altos de Chiapas Mexico . Interciencia 41 : 382 – 392 . Google Scholar OpenURL Placeholder Text WorldCat Butlin , R. K . 2010 . Population genomics and speciation . Genetica 138 : 409 – 418 . Google Scholar Crossref Search ADS PubMed WorldCat Cabrera-Mora , J. A. , A. W. Guzmán-Franco, M. T. Santillán-Galicia, and F. Tamayo-Mejía. 2019 . Niche separation of species of entomopathogenic fungi within the genera Metarhizium and Beauveria in different cropping systems in Mexico . Fungal Ecol . 39 : 349 – 355 . Google Scholar Crossref Search ADS WorldCat Chávez , I. E. , S. Rodríguez-Navarro, L. C. Sánchez-Pérez, A. H. Partida, and J. E. Barranco-Florido. 2014 . Actividad insecticida in vitro de extracto crudo de Beauveria bassiana (Bálsamo) Vueillemin sobre larvas de Phyllophaga spp (Harris) . Rev. Prot. Veg . 29 : 226 – 230 . Google Scholar OpenURL Placeholder Text WorldCat Cheliak , W. M. , and J. A. Pitel. 1984 . Techniques for starch gel electrophoresis of enzymes from forest tree species. Petawawa National Forestry Institute Information Report P1-X-42 . Canadian Forestry Service. Agriculture , Chalk River, Ontario, Can . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Cruz-López , J. A . 1999 . Thesis or dissertation alternativas de manejo de ‘Gallina Ciega’ (Coleoptera: Melolonthidae) en maíz en Amatenango del Valle, Chiapas, Universidad de Ciencias y Artes del Estado de Chiapas, Escuela de Biología . Tuxtla Gutierrez, Chiapas, México . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Cruz-López , J. A. , A. E. Castro-Ramírez, C. Ramírez-Salinas, and B. Gómez-Gómez. 2001 . Supresión manual de adultos de Phyllophaga ssp. y Anomala spp. en maíz en México . M. I. P . 59 : 41 – 47 . Google Scholar OpenURL Placeholder Text WorldCat Evanno , G. , S. Regnaut, and J. Goudet. 2005 . Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study . Mol. Ecol . 14 : 2611 – 2620 . Google Scholar Crossref Search ADS PubMed WorldCat Excoffier , L. , P. E. Smouse, and J. M. Quattro. 1992 . Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data . Genetics 131 : 479 – 491 . Google Scholar Crossref Search ADS PubMed WorldCat Falconer , D. S. , and T. F. C. Mackay. 2001 . Introduction to quantitative genetics . Prentice Hall , Harlow, Eng . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Frankham , R. , J. D. Balloy, and K. H. McInnes. 2004 . A primer of conservation genetics . University of Cambridge , Cambridge, Eng . Google Scholar Crossref Search ADS Google Preview WorldCat COPAC Gandarilla-Pacheco , F. L. , L. J. Galán-Wong, K. Arévalo-Niño, M. Elías-Santos, and I. Quintero-Zapata. 2013 . Evaluación de aislados nativos mexicanos de Beauveria bassiana (Bals.) Vuill. (Hypocreales: Cordycipitaceae) provenientes de zonas citrícolas para su producción masiva en cultivo sumergido y bifásico . Agrociencia 47 : 255 – 266 . Google Scholar OpenURL Placeholder Text WorldCat García , E . 1973 . Modificaciones al sistema de clasificación climática de Köpen (para adaptarlo a las condiciones de la República Mexicana) . Instituto de Geografía, Universidad Nacional Autónoma de México , Ciudad de México, Mex . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC García , L. O. , A. E. Castro-Ramírez, A. G. Flores-Ricárdez, and C. Ramírez-Salinas. 2006 . Evaluación del daño a las raíces de leguminosas y solanáceas por ‘gallina ciega’ (Coleoptera: Melolonthidae), pp. 135 – 146 . In A. E. Castro-Ramírez, M. A. Morón, and A. Aragón (eds.), Diversidad, importancia y manejo de escarabajos edafícolas, El Colegio de la Frontera Sur, Fundación Produce Chiapas . A.C. y Benemérita Universidad Autónoma de Puebla , Ciudad de México, Mex . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Génissel , A. , F. Viard, and D. Bourguet. 2000 . Population genetics of Chrysomela tremulae: a first step towards management of transgenic Bacillus thuringiensis poplars Populus tremula x.P. tremuloides . Hereditas 133 : 85 – 93 . Google Scholar Crossref Search ADS PubMed WorldCat González-Rodríguez , A. , B. Bemrey, A. Castañeda, and K. Oyama. 2000 . Population genetic structure of Acanthoscelides obtectatus and A. obvelatus (Coleoptera: Bruchidae) from wild and cultivated Phaseolus sp. (Leguminosae) . Ann. Entomol. Soc. Am . 93 : 1100 – 1107 . Google Scholar Crossref Search ADS WorldCat Guo , L. , G. Wu, Y. Li, C. Li, W. Liu, J. Meng, H. Liu, X. Yu, and G. Jiang. 2016 . Effects of cattle manure compost combined with chemical fertilizer on topsoil organic matter, bulk density and earthworm activity in a wheat-maize rotation system in Easter China . Soil Till. Res . 156 : 140 – 147 . Google Scholar Crossref Search ADS WorldCat Guzmán-Franco , A. , J. Hernández-López, J. N. Enríque-Vara, R. Alatorre-Rosas, F. Tamayo-Mejía, and L. D. Ortega-Arenas. 2012 . Susceptibility of Phyllophaga polyphylla and Anomala cincta larva to Beauveria bassiana and Metarhizium anisopliae isolates, and the interaction with soil properties . BioControl 57 : 553 – 563 . Google Scholar Crossref Search ADS WorldCat Hartl , D. L. , and A. G. Clark. 1997 . Principles of population genetics . Sinauer Associates., Sunderland , Massachusetts, USA . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Hebert , P. D. N. , and M. J. Beaton. 1993 . Methodologies for allozyme analysis using cellulose acetate electrophoresis. Technical Manual of Cellulose Acetate Electroporesis . Helena Laboratories , TX, USA . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Jurado , A. J. , G. H. Aranda, J. N. Callejas, M. F. I. Ortega. 2013 . Situación económica de la producción de maíz en condiciones de riego en el estado de Chihuahua . Rev. Mex. Agronegocio 33 : 504 – 512 . Google Scholar OpenURL Placeholder Text WorldCat King , A. B. S . 1994 . Biología e identificación de (Phyllophaga) de importancia económica en América Central, pp. 33 – 43 . In J. P. Shannon and M. Carballo (eds.), Seminario-Taller Centroamericano Sobre la Biología y Control de Phyllophaga sp . CATIE , Turrialba, CR . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Lesieur , V. , B. Courtial, A. Roques, and M. A. Auger-Rozenberg. 2014 . Isolation and characterization of 11 polymorphic microsatellite markers in the highly invasive Western conifer seed bug, Leptoglossus occidentalis (Heteroptera, Coreidae) . Conserv. Genet. Resour . 6 : 617 – 619 . Google Scholar Crossref Search ADS WorldCat Lima , J. S. , J. E. G. De Queiroz, and H. B. Freitas. 2004 . Efecto del compost de residuos urbanos seleccionado y no seleccionado sobre el crecimiento inicial del maíz . Recur. Conserv. Reciclaje 42 : 309 – 315 . Google Scholar Crossref Search ADS WorldCat Lopes , R. B. , D. A. Souza, C. M. Oliveira, and M. Faria. 2013 . Genetic diversity and pathogenicity of Metarhizium spp. associated with the white grub Phyllophaga capillata (Blanchard) (Coleoptera: Melolonthidae) in a soybean field . Neotrop. Entomol . 42 : 436 – 438 . Google Scholar Crossref Search ADS PubMed WorldCat Maag , D. , M. Erb, J. S. Bernal, J. L. Wolfender, T. C. Turlings, and G. Glauser. 2015 . Maize domestication and anti-herbivore defences: leaf-specific dynamics during early ontogeny of maize and its wild ancestors . PLoS One 10 : e0135722 . Google Scholar Crossref Search ADS PubMed WorldCat Maldonado-López , L. G. , R. Mariaca-Méndez, A. Nazar Beutelspacher, P. Rosset, and U. L. E. Contreras-Cortés. 2017 . Women: clay and corn. Peasant women and subsistence strategies in Amatenango del Valle, Chiapas . Rev. Geog. Agr . 59 : 55 – 85 . Google Scholar OpenURL Placeholder Text WorldCat Manirakiza , N. , and C. Şeker. 2020 . Effects of compost and biochar amendments on soil fertility and crop growth in a calcareous soil . J. Plant Nutr . 43 : 3002 – 3019 . Google Scholar Crossref Search ADS WorldCat Marín , J. A. , and M. R. Bujanos. 2008 . Especies del complejo ‘gallina ciega’ del género Phyllophaga en Guanajuato, México . Agric. Tec. Mex . 34 : 349 – 355 . Google Scholar OpenURL Placeholder Text WorldCat Morón , M. A . 2001 . Larvas de escarabajos del suelo en México (Coleoptera: Melolonthidae) . Acta. Zool. Mex . 1 : 111 – 130 . Google Scholar OpenURL Placeholder Text WorldCat Morón , M. A . 2006 . Revisión de las especies de Phyllophaga (Phytalus) grupos obsoleta y pallida (Coleoptera: Melolonthidae: Melolonthinae) . Folia Entomol. Mex . 45 : 1 – 104 . Google Scholar OpenURL Placeholder Text WorldCat Morón , M. A . 2010 . Diversidad y distribución del complejo «gallina ciega» (Coleoptera: Scarabaeoidea), pp. 41 – 64 . In L. A. Rodríguez-del Bosque and M. A. Morón (eds.), Plagas del suelo . Mundi-Prensa , Ciudad de México, Mex . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Nahrung , F. H. , and G. R. Allen. 2003 . Geographical variation, population structure and gene flow between population of Chrysophtharta agricola (Coleoptera: Crhysomelidae), a pest of Australian eucalypt plantations . Bull. Entomol. Res . 93 : 137 – 144 . Google Scholar Crossref Search ADS PubMed WorldCat Nájera-Rincón , M. B. , M. M. García, R. L. Crocker, V. Hernández-Velázquez, and L. A. Rodríguez del Bosque. 2005 . Virulencia de Beauveria bassiana y Metarhizium anisopliae, nativos del occidente de México, contra larvas de tercer estadio de Phyllophaga crinita (Coleoptera: Melolonthidae) bajo condiciones de laboratorio . Fitosanidad 9 : 33 – 36 . Google Scholar OpenURL Placeholder Text WorldCat Núñez-Farfán , J. , and L. E. Eguiarte(Comp). 1999 . Evolución biológica . Universidad Nacional Autónoma de México, Facultad de Ciencias: Comisión Nacional para el Conocimiento y Uso de la Biodiversidad , Mex . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Onstad , D. W. , J. L. Spencer, C. A. Guse, E. Levine, and S. A. Isard. 2001 . Modeling evolution of behavioral resistance by an insect to crop rotation . Entomol. Exp. Appl . 100 : 195 – 201 . Google Scholar Crossref Search ADS WorldCat Peakall , R. , and P. E. Smouse. 2006 . GenAlEx v5: genetic analysis in excel. Populations genetic software for teaching and research . Bioinformatics 28 : 2537 – 2539 . Google Scholar Crossref Search ADS WorldCat Petzold-Maxwell , J. L. , X. Cibils-Stewart, B. W. French, and A. J. Gassmann. 2012 . Adaptation by western corn rootworm (Coeloptera: Chrysomelidae) to Bt Maize: Inheritance, fitness costs, and feeding preference . J. Econ. Entomol . 105 : 1407 – 1418 . Google Scholar Crossref Search ADS PubMed WorldCat Polanco Mendoza , J. C . 2008 . Patogenicidad de aislamientos nativos de hongos entomopatógenos sobre el complejo ‘Gallina ciega’ (Coleoptera: Melolonthidae) de Los Altos de Chiapas, México . Bachelor Thesis, Universidad de Ciencias y Artes de Chiapas , Tuxtla Gutiérrez, Chiapas . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Pritchard , J. K. , M. Stephens, and P. Donnelly. 2000 . Inference of population structure using multilocus genotype data . Genetics 155 : 945 – 959 . Google Scholar Crossref Search ADS PubMed WorldCat Ramírez-Salinas , C. and A. E. Castro-Ramírez. 1998 . Estudio morfológico del estado larval de seis especies de Phyllophaga (Coleoptera: Melolonthidae) en la región Altos de Chiapas, México, pp. 37 – 50 . In M. A. Morón and A. Aragón (eds.), Avances en el estudio de la diversidad, importancia y manejo de los coleópteros edaficotas americanos . Publicación especial de la Benemérita Universidad Autónoma de Puebla y la Sociedad Mexicana de Entomología , A. C. Puebla, Mex . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Ramírez-Salinas , C. and A. E. Castro-Ramírez. 2000 . El complejo ‘Gallina Ciega’ (Coleoptera: Melolonthidae) en el cultivo de maíz, en el Madronal, Municipio de Amatenango del Valle Chiapas, México . Acta Zool. Mex . 79 : 17 – 41 . Google Scholar OpenURL Placeholder Text WorldCat Ramírez-Salinas , C. , M. A. Morón, and A. E. Castro-Ramírez. 2011 . Descripciones de los estados inmaduros de cuatro especies de Phyllophaga, Paranomala macrodactylus (Coleoptera: Melolonthidae) de Los Altos de Chiapas, México . Acta Zool. Mex . 27 : 527 – 545 . Google Scholar OpenURL Placeholder Text WorldCat Régnière , J. , R. L. Rabb, and R. E. Stinner. 1981 . Popillia japonica: effect of soil moisture and texture on survival and development of eggs and first instar grubs . Environ. Entomol . 10 : 654 – 660 . Google Scholar Crossref Search ADS WorldCat Richardson , B. J. , P. R. Baverstock, N. Adams. 1986 . Allozyme electrophoresis. A handbook for animal systematics and populations studies . Academic Press , London, Eng . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Roderik , G. K . 1996 . Geographic structure of insect population: gene, flow, phylogeography, and their uses . Annu. Rev. Entomol . 41 : 325 – 352 . Google Scholar Crossref Search ADS PubMed WorldCat Romero-López , A. A . 2012 . Uso de feromonas sexuales para el conocimiento y manejo de los ‘ensambles gallina ciega’ . Interciencia 37 : 559 – 564 . Google Scholar OpenURL Placeholder Text WorldCat Scorza-Dagert , J. V. , and L. J. Cova. 2006 . Acción patógena de una cepa venezolana de Beauveria bassiana para Musca domestica (Diptera: Muscidae) . Bol. Malariol. y Sal. Amb . 46 : 119 – 130 . Google Scholar OpenURL Placeholder Text WorldCat Sokal , R. R. , F. J. Rholf. 1995 . Biometry . Third edition. W. H. Freeman and Company , New York, USA . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Solis , P. O. , G. A. Castillo, C. G. Peña, G. A. Alvear, M. M. M. Serrano, R. R. Suárez, and V. V. M. Hernández. 2016 . Pathogenicity, virulence and the interaction of Metarhizium anisopliae and Beauveria bassiana against Phyllophaga vetula (Colepotera: Melolonthidae) . J. Pure Appl. Microbiol . 10 : 2607 – 2612 . Google Scholar OpenURL Placeholder Text WorldCat Sumerford , D. V. , G. P. Head, A. Shelton, J. Greenplate, and W. Moar. 2013 . Field-evolved resistance: assessing the problem and ways to move forward . J. Econ. Entomol . 106 : 1525 – 1534 . Google Scholar Crossref Search ADS PubMed WorldCat Szalanski , A. L. , R. L. Roehrdanz, D. B. Taylor, and L. Chandler. 1999 . Genetic variation in geographical populations of western and Mexican corn rootworm . Insect Mol. Biol . 8 : 519 – 525 . Google Scholar Crossref Search ADS PubMed WorldCat Vallejo , F. , M. A. Morón, and S. Orduz. 2007 . Biología de Phyllophaga obsoleta Blanchard (Coleoptera: Melolonthidae), especie rizófaga del complejo ‘Chisa’ de Colombia . Bol. Cient. Mus. His. Nat . 11 : 188 – 204 . Google Scholar OpenURL Placeholder Text WorldCat Velázquez , C. E. J. , A. E. Castro-Ramírez, and C. Ramírez-Salinas. 2006 . Manejo agroecológico de ‘gallina ciega’ (Coleoptera: Melolonthidae ), pp. 209 – 220 . In A. E. Castro-Ramírez, M. A. Morón and A. Aragón (eds.), Diversidad, Importancia y Manejo . Publicación especial de El Colegio de la Frontera Sur, Fundación Produce Chiapas, A.C. and Benemérita Universidad Autónoma de Puebla , Puebla, Mex . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Wenninger , E. J. , and A. L. Averill. 2006 . Effects of delayed mating on reproductive output of female oriental beetle Anomala orientalis (Coleoptera: Scarabaeidae) . Agr. Forest Entomol . 8 : 221 – 231 . Google Scholar Crossref Search ADS WorldCat Whitlock , M. C. , and D. E. McCauley. 1999 . Indirect measures of gene flow and migration: FST not equal to 1/(4Nm + 1) . Heredity (Edinb) . 82 ( Pt 2 ): 117 – 125 . Google Scholar PubMed OpenURL Placeholder Text WorldCat Yeh , F. C. H. , and D. O’Malley. 1980 . Enzyme variations in natural populations of Douglas-fir, Pseudotsuga menziesii (Mirb.) Franco, from British Columbia. 1. Genetic variation patterns in coastal populations . Silvae Genet . 29 : 83 – 92 . Google Scholar OpenURL Placeholder Text WorldCat Zhang , L. , W. Cai, J. Luo, S. Zhang, W. Li, C. Wang, L. Lv, and J. Cui. 2018 . Population genetic structure and expansion patterns of the cotton pest Adelphocoris fasciaticollis . J. Pest Sci . 91 : 539 – 550 . Google Scholar Crossref Search ADS WorldCat Zhou , X. , M. E. Scharf, S. Parimi, L. J. Meinke, R. J. Wright, L. D. Chandler, and B. D. Siegfried. 2002 . Diagnostic assays based on esterase-mediated resistance mechanisms in western corn rootworms (Coleoptera: Chrysomelidae) . J. Econ. Entomol . 95 : 1261 – 1266 . Google Scholar Crossref Search ADS PubMed WorldCat Zitlalpopoca-Hernandez , G. , M. B. Najera-Rincon, E. del Val, A. Alarcon, J. Jackson, and J. Larsen. 2017 . Multitrophic interactions between maize mycorrhyzas, the root feeding insect Phyllophaga vetula and the entomopathogenic fungus Beauveria bassiana . Appl. Soil Ecol . 115 : 38 – 43 . Google Scholar Crossref Search ADS WorldCat © The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - How Does the Application of Beauveria bassiana and Compost on Corn Crops Affect the Survival and Genetic Diversity of Phyllophaga obsoleta (Coleoptera: Melolonthinae)?
JF - Environmental Entomology
DO - 10.1093/ee/nvab054
DA - 2021-06-24
UR - https://www.deepdyve.com/lp/oxford-university-press/how-does-the-application-of-beauveria-bassiana-and-compost-on-corn-kdinnRMVFs
SP - 1
EP - 1
VL - Advance Article
IS - 
DP - DeepDyve
ER -