Physiological and molecular characterization of Metarhizium isolates and their acaricidal activity against Tetranychus urticae Koch (Trombidiformes: Tetranychidae)

The two-spotted spider mite, Tetranychus urticae Koch (Trombidiformes: Tetranychidae), is one of the most damaging mites in agriculture. Due to the concern for the intensive use of synthetic acaricides, entomopathogenic fungi represents a feasible alternative to T. urticae management. In the present study, 7 isolates of Metarhizium were characterized physiological and molecularly (based on the ITS1-5.8s-ITS2 rDNA) and evaluated for their acaricidal activity [mortality, mean and 90 lethal concentration (LC50: LC90) and mean and 90 lethal time (LT50: LT90)] against T. urticae under laboratory conditions. Sequencing of the ITS1-5.8s-ITS2 rDNA region indicated that the 7 isolates belong to M. anisopliae. The isolates Ma114 (3.7 ± 0.006 mm day−1), Ma109 (3.5 ± 0.009 mm day−1) and Ma106 (3.5 ± 0.006 mm day−1) had the highest radial growth rate and Ma114 (92.2 ± 0.86%) and Ma108 (94.4 ± 1.07%) had the highest germination percentage. All isolates were pathogenic to T. urticae, causing mortality that ranged from 45.3 to 85.3%. The LC50 and LC90 were 1.2 and 2.8, 1.1 and 2.5, and 1.2 and 2.8 × 108 conidia mL−1 for isolates Ma110, Ma109 and Ma106, respectively, while the LT50 and LT90 were 7.7 and 16.5, and 7.2 and 16.1 days for isolates M110 and Ma109, respectively. The isolates Ma110 and Ma109 of M. anisopliae were moderately pathogenic and virulent against T. urticae.


Background
The two-spotted spider mite (Tetranychus urticae Koch) is one of the most damaging pests in agriculture. Damage by T. urticae is typical in leaves, but at high population density inflorescences and fruits are also affected. Infested plants undergo a decrease in photosynthetic rate, plant growth and fruit production (Landeros et al. 2013).
To control T. urticae, synthetic acaricides have been intensively used, but even though this strategy has alleviated the problem to some extent, issues associated with the continuous use of these chemicals include a decrease in population of beneficial arthropods, increased risk for human health and selection of acaricide resistant populations (Shin et al. 2017).
The use of biological control agents such as entomopathogenic fungi (EPF) represents an alternative to reduce the dependence of synthetic acaricides to control of phytophagous mites. Among EPF, Metarhizium spp. (Ascomycota: Clavicipitaceae) are considered Page 2 of 7 Cua Basulto et al. Egyptian Journal of Biological Pest Control (2022) 32:30 an effective biological control agent against a wide range of phytophagous mites (Souza et al. 2014). In laboratory assays, Metarhizium anisopliae has shown high activity against T. urticae with mortalities range from 80 to 100%, when mites were exposed to 1.0 × 10 7 to 1.0 × 10 8 conidia mL −1 (Dogan et al. 2017). In the field and greenhouse conditions, M. anisopliae at concentration of 1 × 10 8 conidia mL −1 had also showed efficacy to reduce the population of T. urticae (Bugeme et al. 2014a). Also, Metarhizium has been proven to cause mortality on insects from different orders including: Hemiptera, Heteroptera, Coleoptera, Lepidoptera, Thysanoptera, Orthoptera, Diptera and Isoptera (Carolino et al. 2014). In addition, it has already used in commercial formulation of bioinsecticides for pest insects of foliage, root and stored grain (Kepler and Rehner 2013). Due to the importance of the biological control of twospotted spider mite, the objective of this study was to characterize physiological and molecularly fungal isolates of Metarhizium and evaluate their acaricidal activity against the T. urticae.

Fungal isolates
Seven isolates of Metarhizium were obtained from the Laboratory of Biological Control of the Faculty of Biological and Agricultural Sciences at the University of Colima in Mexico. The isolates were obtained from soil samples in the states of Colima and Jalisco, Mexico (Table 1). The fungi were grown on Sabouraud dextrose agar (SDA, MCD ® Lab, Mexico) in Petri dishes under laboratory conditions 25 ± 1 °C. To obtain the spore suspension, the conidia were harvested from the surface of a fungal colony (12 days old) by scrapping using 10 mL of sterile distilled water plus Tween 80 (0.05%), and then, it was filtered with a sterile gauze to avoid the mycelium and to recover only the conidia (Chan-Cupul et al. 2010). The conidia suspension was used for the bioassays.

Molecular identification of Metarhizium spp.
Genomic DNA extraction was performed from monosporic cultures in GPY culture medium, according to the method developed in the GeMBio laboratory (Tapia-Tussell et al. 2006). The ITS1-5.8s-ITS2 region of the rDNA was amplified using the primers ITS1 (5′ TCC GTA GGT GAA CCT GCG G 3′) and ITS4 (5′TCC TCC GCT TAT TGA TAT GC 3′) (White et al. 1990), at a final volume of 50 µL, which they contained 25 ng of genomic DNA, 0.20 mM of each dNTP (Invitrogen), 1.5 mM of MgCl 2 , 1 µM of primers and 1 U of Taq (Kumar et al. 2018).

Physiological characterization
The radial growth and conidial germination were used as parameters of physiological characterization of the Metarhizium isolates (Permandi et al. 2020). To evaluate the radial growth rate (RGR), 5 μL of a conidial suspension of 1 × 10 6 conidia mL −1 of each isolate was inoculated individually in the center of a Petri dish with SDA. The conidia suspension was obtained from an 11-dayold colony in a Petri dish, 10 mL of sterile distilled water with Tween (0.05%) was deposited on the surface of the colony and scraped with a sterile spatula, and conidia were recovered by filtration in a falcon tube (50 mL) with sterile gauze. The conidia concentration was counted and adjusted in a Neubauer chamber. Petri dishes were incubated at 25 ± 1 °C and photoperiod of 16: 8 h light: darkness. The diameter of the growing colony was measured daily for 10 days using a Vernier, with the colony diameter values were calculate the radial growth rate (mm d −1 ) (Chan-Cupul et al. 2010). A Petri dish served as a replicate and 10 replicates per fungal isolate were used. The evaluation of conidial germination of each isolate was evaluated using the same Petri dishes from the RGR test. Ten microliters of 1 × 10 6 conidia mL −1 were deposited on SDA Petri dishes. The plates were incubated at 25 °C. Conidia germination was recorded each 12 h by 3 times (36 h) in a microscope (40×), 100 conidia for each plate were counted. Germinated conidia were considered when the germ tube has the same length than the conidia Table 1 Source and origin of the Metarhizium isolates

Acaricidal activity of fungal isolates
A stock culture of T. urticae was established on common bean plants (Phaseolus vulgaris L.) into an entomological cage under greenhouse conditions (minimum temperature) = 24.0 °C, maximum temp = 38.5 °C, average temp = 31.4 °C, minimum relative humidity (RH) = 43.15%, maximum RH = 73.97%, average RH = 58.5% and 12:12 h of light: darkness photoperiod). For bioassays, fungal spore suspensions were obtained by 15-day-old colonies grown in SDA as previously described by Ayala-Zermeño et al. (2015). To test the pathogenicity of the fungal isolates, an initial assessment was conducted using a spore concentration of 1 × 10 7 conidia mL −1 . Then, for the most active fungal isolates, a range of spore concentrations (1 × 10 7 , 1 × 10 6 , 1 × 10 5 and 1 × 10 4 conidia mL −1 ) was evaluated as described by Jeyarani et al. (2011). Spore suspensions were sprayed until runoff onto both surfaces of P. vulgaris leaves using hand sprayer. The leaves were air-dried for 20 min under the laminar flow cabinet, and then placed on wet cotton wool in Petri dishes. Adult mites (20 individuals per Petri dish) were then placed onto the treated leaves. Control leaves were sprayed with sterile distilled water containing 0.05% Tween 80. Petri dishes were placed in an incubator at 25 ± 1 °C, 75 ± 5% RH, and photoperiod of 16:8 h light: darkness. Mortalities were recorded daily for 12 days. Dead mites were transferred and kept for 15 days in Petri dishes lined with moist filter paper to observe mycosis (Eken and Hayat 2009). Each Petri dish served as a replicate, 8 replicates per fungal isolate were used.

Data analysis
All experiments were set in a completed randomized design. Data subjected to analyses of variance were checked for normality and homoscedasticity. Significant differences (P < 0.05) among means were determined by the Tukey test. Mean and ninety lethal times (LT 50 and LT 90 ) and mean and 90 lethal concentrations (LC 50 and LC 90 ) were calculated using Probit analysis. All analyses were performed in the Statistical Package for Statgraphics ® .

Molecular identification
Sequencing of the ITS1-5.8s-ITS2 rDNA gene showed that the 7 isolates had homology with M. anisopliae (Metschn.) Sorokin 1883, 6 isolates with identity of 99.8-100% and the MA91 isolate of 99.2%, identity were obtained from BLAST algorithm NCBI database (Zhang et al. 2000). Phylogenetic analysis showed that isolates MA97, MA106, MA114 and MA109 were very close to each other, while isolates MA103 and MA110 shared the same clade, and isolate MA91 shared a clade with another isolated of M. anisopliae (Fig. 1).

Discussion
In order to find a more safety method to control the twospotted spider mite, this study evaluated the acaricidal activity of Metarhizium isolates against T. urticae. In addition, Metarhizium isolates were molecularly physiologically characterized. Phylogenetic analysis showed variability among strains, isolates Ma103 and Ma110 were grouped in the same clade, while isolate Ma91 was more distant and shared the clade with M. anisopliae     Fig. 2 Mean (± standard error) mortality of Tetranychus urticae adults exposed to 1 × 10 7 conidia mL −1 of seven isolates of Metarhizium anisopliae under laboratory conditions. Bars with different letters are significantly different (Tukey test, n = 5, p < 0.05) Page 5 of 7 Cua Basulto et al. Egyptian Journal of Biological Pest Control (2022) 32:30 The physiological characterization of Metarhizium isolates indicated significant differences among isolates in radial growth and conidial germination. This result could be due by the genetic diversity of Metarhizium or at their biochemical activity, because fungal growth can be stimulated by the culture media (Gandarilla-Pacheco et al. 2012). In this regard, the present results are similar to those reported by Dimni et al. (2004) in M. anisopliae isolates, which achieved a mycelial growth from 2.3 to 3.2 mm day −1 . In contrast, Nussenbaum et al. (2013) reported higher values for radial growth rate for Metarhizium isolates from 4.0 to 5.3 mm day −1 .
Fungal growth rate is an important characteristic to select isolates for biological control programs, because it is expected that isolates with the highest growth rate would also have high acaricidal effects. Talaei-Hassanloui et al. (2007) suggested that there was a positive association between fungal radial growth and fungal virulence in EPF. Another important predictor of the acaricidal effect of a fungus is the conidial germination rate. In the present study, this variable ranged from 62.6 to 94.4% at 24 h post-inoculation. In another studies, Bugeme et al. (2009) observed in M. anisopliae 86-96% of conidial germination at 24 post-inoculation. Likewise, Onsongo et al. (2019) found similar results, and observed that the highest rate of conidial germination was achieved at temperatures of 25-30 °C. The rate of conidial germination is considered an important indicator of fungal virulence, based on the assumption that a spore on the insect cuticle that germinates rapidly would also have higher probability to penetrate and initiate the infection process in the host insect (Andersen et al. 2006); in M. anisopliae a positive relationship between fungal virulence and rate of conidial germination has been well documented (Ummidi et al. 2013). However, this asseveration is not a general rule, because in the present study M. anisopliae Ma114 achieved the highest RGR and conidia germination, but the isolate was not the most virulent.
All fungal isolates evaluated in this work caused significant mortality against T. urticae adults. Similar to other studies, the mortality caused by the fungal isolates ranged from 45.4 to 83.5% using 1 × 10 7 conidia mL −1 . For example, Bugeme et al. (2014b) found 65 to 100% mortality using a spore concentration of 1 × 10 7 conidia mL −1 . However, Chandler et al. (2005) observed that M. anisopliae isolates caused no more than 43% of mortality of T. urticae using 1 × 10 7 conidia mL −1 .
In the present study, the calculated LT 50 ranged from 7.2 to 13.2 days using a conidial suspension of 1 × 10 7 conidia mL −1 . The lowest values for LT 50 were observed for the isolates Ma110 (7.7 days) and Ma 109 (7.2 days). These values were relatively high compared to those reported in other studies, where calculated LT 50 for Metarhizium spp. was within 2.2-4.0 days (Castro et al. 2018). Regarding to the LT 90 , Bugeme et al. (2009) reported values from 3.1 (ICIPE48) to 11.7 (ICIPE97)  By other hand, the calculated LC 50 for the most pathogenic isolates (Ma110, Ma109 and Ma106) ranged from 1.1 × 10 8 to 1.2 × 10 8 conidia mL −1 . These values are similar to those reported by Hassan et al. (2017), who documented LC 50 of 9.3 × 10 7 and 4.57 × 10 8 conidia mL −1 for M. anisopliae isolates on T. urticae. In contrast, the LC 50 values in the present study were relatively higher than the LC 50 values of 2.0 × 10 5 to 5.0 × 10 5 conidia mL −1 reported in previous studies by Elhakim et al. (2020). Regarding the LC 90 , the isolates Ma110, Ma109 and Ma106 values that ranged from 2.5 to 2.8 × 10 8 conidia mL −1 ) were lower than those (LC 90 , 2.24 to 2.85 × 10 10 conidia mL −1 ) reported by Elhakim et al. (2020). Taken both, the LT 50 and LC 50 as indicator of the fungal virulence, obtained fungal isolates may be considered moderately virulent. It is important to note that the virulence of EPF not only depends on intrinsic characteristics of the fungal isolates, but also on the concentration and frequency of the applications, as well as on the environmental conditions (Oyku et al. 2017).

Conclusions
Metarhizium anisopliae isolate Ma114 achieved the highest germination percentage and the isolates Ma114, Ma109 and Ma106 reached the highest mycelial growth rate. According to the sequencing of the ITS1-5.8s-ITS2 region (rDNA), the all studied isolates were identified as Metarhizium anisopliae (Metschn.) Sorokin 1883. The most pathogenic isolates were the M. anisopliae Ma110 and Ma106 that caused 83.46 and 80.76% mortality of T. urticae. According to the LT 50 , the most virulent M. anisopliae isolates against T. urticae were the Ma110 (7.7 days) and Ma109 (7.2 days). The calculated LC 50 for the isolates Ma110 and Ma109 were 1.2 and 1.1 × 10 8 conidia mL −1 , with nonsignificant difference between them.