Coryphodema tristis (Drury) (Lepidoptera: Cossidae) is native to
Neither insecticides nor natural enemies are currently in use or registered for the cossid moth. More effective and sustainable control systems should thus be developed, as for example the recent pheromone determination (Bouwer et al., 2015) . This study evaluates twelve Metarhizium anisopliae (Metschnikoff) Sorokin (Hypocreales: Clavicipitaceae) isolates aggressiveness and their mycelial growth, sporulation rate, virulence genes presence, and insecticide-compatibility. The genetic variability of the assayed population was also determined. A selected isolate, based on the indicated parameters results, was tested in simulated field conditions.
2. Material and Methods
Initially, infectivity assays were performed. Infested E. nitens logs were obtained at Lothair area in
Table 1. Source, colony growth, sporulation rate and virulence genes presence among the tested twelve Metarhizium anisopliae isolates.
aEx-type isolate. Means with the same letter are not significantly different (P > 0.05), by ANOVA followed by Tukey test. Isolates were incubated on PDA plates at 20˚C for 15 days and 16:8 h L:D. + Positive amplicons.
2.2. Compatibility with Deltamethrin
To evaluate M. anisopliae isolates sensitivity to deltamethrin (Decis 1.5% w/v; Aventis), potato dextrose agar (PDA) media were amended with deltamethrin at 0, 0.0125, 0.025, or 0.05 µg/ml of active ingredient. Six replicates of each concentration were used for each isolate. Five millimeter diameter mycelial plugs taken from the edge of a two weeks-old colony of each isolate were placed onto PDA plates amended with each concentration. After the plates were incubated at 20˚C for 7 days in the dark, occupied areas were measured. Besides, eight control, eight deltamethrin-treated, eight ARSEF2518-treated, and eight mixed-treated E. nitens logs (20 cm long) were placed on emerging cages. Logs were respectively sprayed with 400 ml of sdH2O (negative control), sterile aqueous solution of Decis (0.002%, v/v), sterile aqueous solution of a conidial suspension of 109 conidia/ml (1%, v/v), and a mixed treatment solution. Ten C. tristis larvae were added to each log. Every week during one month, larvae mortality percentage was measured on two logs of each type and compared by using ANOVA using SPSS Statistics (version 19).
2.3. Mycelial Growth, Sporulation Rate, and Virulence Genes Detection
To study mycelial growth, six replicate two weeks-old mycelial plugs (0.5 cm diam.) were placed face down at the centre of 60-mm dish containing 10 ml of PDA. After 15 days at 20˚C and 16:8 h L:D, occupied areas were traced and measured. Sporulation rate was estimated by collecting conidia with 4 ml of 0.01% Tween 80 at 200 rpm for 30 min at 22˚C.
As regards some virulence genes detection, PCR amplification of Pr1a protease gene was performed by nested PCR with outer primers METPR1 (5’-CACTCTTCTCCCAGCCGTTC-3’), METPR4 (5’-GTAGCTCAACTTCTGCACTC-3’), and inner primers METPR2 (5’-AGGTAGGCAGCCAGACCGGC-3’) and METPR5 (5’-TGCCACTATTGGCCGGCGCG-3’). The template DNA was amplified in a 50 μL PCR reaction volume, consisting of 5 μL of 10X Reaction Buffer, 5 μL of MgCl2 (25 mM), 5 μL of dNTPs (10 mM), 1 μL of each primer (10 μM), 1 μL of DNA solution and 0.5 μL of Super-Therm Taq polymerase. PCR reactions were performed on a GeneAmp PCR System 9700 (Applied Biosystems) with an initial denaturation step of 4 min at 95˚C. This step was followed by 40 cycles of denaturation at 95˚C (60 s), annealing at 58˚C (60 s) and elongation at 72˚C (2 min). A final extension was conducted for 8 min at 72˚C. The second PCR used 1 μl of the first PCR as template. Products were visualized under UV on a 1% agarose gel stained with Gelred (Biotium), run in a Wide Mini-Sub Electrophoresis System at 90V, 400 mA for 30 min and digitalized in a ChemiDoc Gel Documentation System (BioRad). Pr1b protease gene was amplified using the same PCR and conditions but with primers Pr1B1, Pr1B2, and inner primers Pr1B3 and Pr1B4 (Wang et al., 2002) .
2.4. Screening of RAPD Markers
To determine the genetic variability of the tested isolates, a total of twenty-one 10-mer random primers (Inqaba Biotech) were screened (Table 2). PCRs were carried out in volumes of 50 μl containing 5 μL of 10X Reaction Buffer, 3 μL of MgCl2 (25 mM), 4 μL of dNTPs (10 mM), 2.5 μL of primer (10 μM), 1.5 μL of DNA solution and 0.5 μL of Super-Therm Taq polymerase. Conditions were 94˚C for 4 min, followed by 35 cycles at 94˚C (60 s), 47˚C (60 s), 72˚C (2 min), and then 72˚C for 7 min. Electrophoresis was as before but through 2% agarose gels for 90 min. A genetic similarity matrix was calculated using the method of simple matching coefficient. The matrix was used to perform hierarchical cluster analysis based on the un-weighted pair-group method using arithmetic averages using GelQuest and ClusterVis (Sequentix). One diagnostic marker was recovered using the QIAquick gel extraction kit (Qiagen), sub-cloned into the pGEM-T easy cloning vector (Promega), and both stranded sequenced.
Table 2. Twenty-one decamer random primers used for the screening of RAPDs.
Obtained results indicate considerable variation in aggressiveness among isolates, being isolates number 6, 3, 11 and 5 pathogenic whereas the rest of the isolates were not pathogenic. Isolate number 6 (ARSEF2518) presented the highest cumulative mortality value (Figure 1(a)). Mean lethal dose values could not be calculated through probit analysis because pathogenic isolates were only effective at the highest assayed conidia concentration.
Among the twelve isolates, isolates number 2, 3, 6, 8, 9, 10 and 11 growth was not significantly reduced by the tested deltamethrin concentrations (Figure 1(b)). Deltamethrin treatment killed all the larvae after 7 days. Treatment with ARSEF2518 fungus and mixed treatments respectively reached the same mortality percentage after 28 and 21 days (Figure 1(c)).
Neither mycelial growth (Pearson correlation coefficient (P) = 0.516, F = 0.086) nor sporulation rate (P = 0.360, F = 0.250) data were correlated with cumulative mortality values. Tested virulence genes were present among all the isolates (Table 1).
(a) (b) (c)
Figure 1. (a) Mortality of Coryphodema tristis larvae after inoculation with Metarhizium anisopliae isolates (curves correspond with four aggressive isolates at the highest tested conidia concentration 109 conidia/ml). (b) Colonized area of twelve M. anisopliae isolates grown for one week at 20˚C on potato dextrose agar (PDA) media amended with deltamethrin (Decis 1.5% w/v; Aventis) at 0, 0.0125, 0.025, or 0.05 µg of active ingredient/ml. (c) Larvae mortality percentage when infesting E. nitens logs (20 cm long) respectively sprayed with four treatments. See experimental details. Means with the same letter are not significantly different (P > 0.05), by ANOVA followed by Tukey test. Isolates details given in Table 1.
Aggressive isolates grouped together in the topology of the 21 decamer primers (Table 2)-based consensus tree (Figure 2). Cluster analysis of the banding patterns revealed two tightly clustered groups among the M. anisopliae isolates. Group 1 consisted of six isolates with a mean similarity of 88.6%, and group 2 consisted of six isolates with a mean similarity of 89%. Group 1 and group 2
Figure 2. Dendrogram (a) generated from genetic similarity coefficients (b) obtained from presence or absence of DNA bands in twelve Metarhizium anisopliae isolates and based on the unweighted pair-group method using arithmetic averages (UPGMA). Matrix calculated from bands yielded by twenty-one primers. Isolates details given in Table 1.
isolates diverged at the 12% similarity level. One primer with reproducible amplified band of 760 bp unique to the aggressive isolates group was selected.
This is the first study investigating the aggressiveness of M. anisopliae to C. tristis. There was considerable variation in aggressiveness among isolates, presenting isolate number 6 (ARSEF2518) the highest cumulative mortality value. It is interesting to note that some Coleoptera or Homoptera-derived isolates showed more aggressiveness than some isolates from Lepidoptera, supporting that the original host is not a reliable indicator of higher virulence in some entomopathogenic fungi (Varela & Morales, 1996; Devi et al., 2008; Romón et al., 2017) . Entomopathogenic fungi and deltamethrin have been used in integrated management research to control ticks (Bahiense et al., 2008) and Triatoma infestans (Forlani et al., 2013) , with similar mortality rates among individual and mixed treatments and with no negative effect on certain isolates of the fungus after its combination with deltamethrin at the usual or higher doses than those applied in the field. As expected, considering previous studies, no synergistic effect was found in the combined treatment probably because entomopathogenic fungi present high detoxification abilities (Forlani et al., 2013) .
Random amplified polymorphic DNA (RAPD) markers are polymorphic DNA sequences that are amplified using PCR with decamer primers. This procedure is useful, simple, fast, requires only trace amounts of template DNA, and it does not require prior genome information. RAPD-PCR has been used in a number of surveys of M. anisopliae. In general, these studies have shown that the species consists of numerous genotypes and is considered very diverse. Correlations of distinctive groups of isolates defined by host (Bridge et al., 1997) , geographical range (Leal et al., 1994) , morphology (Bidochka et al., 1994) and/or pathogenicity (Fegan et al., 1993) have been demonstrated previously. In the present study, the nucleotide sequence of the cloned diagnostic RAPD-marker fragment from OPA-08 has been deposited in the GenBank database under accession numbers KJ543536-KJ543539, and it presents 96% homology with a protein mRNA from Metarhizium robertsii related with host adaptation (Hu et al., 2014) . Biocontrol agent development requires isolation in pure culture of the microorganism, its identification, efficacy laboratory trials, pilot trials under field conditions, implementation of formulation and biosafety studies. The phase of screening for proper isolates is very time-consuming and expensive and there is a need for faster and easier methods. The development of fingerprinting of higher aggressiveness could be valuable. Our results suggest that the isolate ARSEF2518 appears to have the best potential among the tested isolates for future evaluation and development as fungal biocontrol agent to C. tristis. However, further laboratory and field experiments are needed. More studies should be directed to determine effects of different formulations and storage on conidial viability, and to test its ability and the other aggressive isolates in controlling C. tristis populations in the field.
We acknowledge the University of Pretoria, the National Research Foundation of South Africa (NRF), the Department of Education, Universities and Research of Basque Government, as well as the members of Tree Protection Co-operative Program (TPCP), specially to Mr. Marc Bouwer, and the NRF/DST Centre of Excellence in Tree Health Biotechnology (CTHB) of Forestry and Agricultural Biotechnology Institute (FABI), for financial and logistical support.
 Bahiense, T. C., Fernandes, E. K. K., Angelo, I. C., Perinotto, W. M. S., & Bittencourt, V. R. E. P. (2008). Performance of Metarhizium anisopliae and Its Combination with Deltamethrin against a Pyrethroid-Resistant Strain of Boophilus microplus in a Stall Test. Animal Biodiversity and Emerging Diseases, 1149, 242-245.
 Bidochka, M. J., McDonald, M. A., St. Leger, R. J., & Roberts, D. W. (1994). Differentiation of Species and Strains of Entomopathogenic Fungi by Random Amplification of Polymorphic DNA (RAPD). Current Genetics, 25, 107-113.
 Boreham, G. R. (2004). A Survey of Cossid Moth Attack in Eucalyptus nitens on the Mpumalanga Highveld of South Africa. Southern African Forestry Journal, 206, 23-26.
 Bouwer, M. C., Slippers, B., Degefu, D., Wingfield, M. J., Lawson, S., & Rohwer, E. R. (2015). Identification of the Sex Pheromone of the Tree Infesting Cossid Moth Coryphodema tristis (Lepidoptera: Cossidae). PLoS ONE, 10, e0118575.
 Bridge, P. D., Prior, C., Sagbohan, J., Lomer, C. J., Carey, M., & Buddie, A. (1997). Molecular Characterization of Isolates of Metarhizium from Locusts and Grasshoppers. Biodiversity and Conservation, 6, 177-189.
 Devi, K. U., Padmavathi, J., Maheswara Rao, C. U., Khan, A. A. P., & Mohan, M. C. (2008). A Study of Host Specificity in the Entomopathogenic Fungus Beauveria bassiana (Hypocreales, Clavicipitaceae). Biocontrol Science and Technology, 18, 975-989.
 Fegan, M., Manners, J. M., Maclean, D. J., Irwin, J. A. G., Samuels, K. D. Z., Holdom, D. G., et al. (1993). Random Amplified Polymorphic DNA Markers Reveal a High Degree of Genetic Diversity in the Entomopathogenic Fungus Metarhizium anisopliae var. anisopliae. Journal of General Microbiology, 139, 2075-2081.
 Forlani, L., Juárez, M. P., Lavarías, S., & Pedrini, N. (2013). Toxicological and Biochemical Response of the Entomopathogenic Fungus Beauveria bassiana after Exposure to Deltamethrin. Pest Management Science, 70, 751-756.
 Hu, X., Xiao, G., Zheng, P., Shang, Y., Su, Y., Zhang, X., et al. (2014). Trajectory and Genomic Determinants of Fungal-Pathogen Speciation and Host Adaptation. Proceedings of National Academy of Science USA, 111, 16796-16801.
 Leal, S. C. M., Bertioli, D. J., Butt, T. M., & Peberdy, J. (1994). Characterization of Isolates of the Entomopathogenic Fungus Metarhizium anisopliae by RAPD-PCR. Mycological Research, 98, 1077-1081.
 Romón, P., Hatting, H., Goldarazena, A., & Iturrondobeitia, J. C. (2017). Variation in Virulence of Beauveria bassiana and B. pseudobassiana to the Pine Weevil Pissodes nemorensis in Relation to Mycelium Characteristics and Virulence Genes. Fungal Biology, 121, 189-197.
 Varela, A., & Morales, E. (1996). Characterization of Some Beauveria bassiana Isolates and Their Virulence toward the Coffee Berry Borer Hypothenemus hampei. Journal of Invertebrate Pathology, 67, 147-152.
 Wang, C., Typas, M. A., & Butt, T. M. (2002). Detection and Characterization of pr1 Virulent Gene Deficiencies in the Insect Pathogenic Fungus Metarhizium anisopliae. FEMS Microbiology Letters, 10575, 1-5.