Efficacy of Beauveria bassiana and Metarhizium brunneum isolates against the pine processionary moth, Thaumetopoea wilkinsoni Tams, 1926 (Lepidoptera: Notodontidae)

The pine processionary moth Thaumetopoea wilkinsoni Tams, 1926 (Lepidoptera: Notodontidae), causes severe skin reactions to animals and humans. The insect also destroys pine ecosystems by feeding on pine leaves at its larval stage. Instead of chemical control, eco-friendly biological control methods should be preferred to combat this species. The purpose of this study was to evaluate the efficacy of Beauveria bassiana isolate (GOPT-331) and Metarhizium brunneum isolates (ORP-13 and ORP-18) against the second and fourth larval instars of T. wilkinsoni under laboratory conditions. T. wilkinsoni eggs were collected from pine trees at Ondokuz Mayıs University in Samsun, Turkey, in 2021, and the second and fourth larval instars were used for the experiment. Two milliliters of a spray of the three fungal isolates was applied to the larvae for each concentration (1 × 105–1 × 108 conidia ml−1). The mortality rates of GOPT-331, ORP-13, and ORP-18 were changed between 91.1 and 100% for the second-instar larvae and between 86.6 and 97.7% for the fourth-instar larvae at 1 × 108 conidia ml−1. The ORP-13 isolate showed the lowest LC50 values. In conclusion, it is suggested that all the three isolates were virulent to T. wilkinsoni and can be used for biological control of this species. The promising results from the study were obtained from trials conducted under controlled laboratory conditions, and it will be critical to explore the potential of these promising entomopathogenic fungi in field conditions.

Page 2 of 7 Yanar et al. Egyptian Journal of Biological Pest Control (2023) 33:32 Background Forest ecosystems are essential as they provide habitat for various living species, prevent soil erosion, play a crucial role in the water cycle, sequester carbon, and help mitigate the effects of global warming (Ramachandra et al. 2018). The genus Pinus contains the most economically valuable species of forest trees worldwide, and these species are distributed all over a wide geographical area (Wang et al. 2019). They contribute significantly to the economy because they are used in various industries such as paper and furniture. Additionally, almost all parts of the Pinus species, particularly the needles, are used in traditional medicine. Because of all these features, Pinus species are essential economically, ecologically, and medically.
Pinus species are distributed almost worldwide, including Turkey. One extremely harmful insect threatens this important species: the pine processionary moth Thaumetopoea wilkinsoni Tams (Lepidoptera: Notodontidae). This insect is distributed in the Mediterranean Basin and Turkey and can cause up to 100% leaf loss by eating pine leaves at the larval stage (Kanat et al. 2005). Defoliated trees become susceptible to secondary pests such as bark beetles and pine lice (Cebeci et al. 2010), making them more vulnerable. This insect destroys pine forests, particularly young ones. Additionally, this insect has urticating setae at the larval stage and causes severe allergic reactions in humans and domestic pets (İpekdal et al. 2016). For all these reasons, it is inevitable to control this pest.
Mechanical, biotechnical, chemical, and biological methods have been used to control the pine processionary moth (Erkan 2018). Given the adverse effects of chemical control on the ecosystem and the difficulty of mechanical control, biological control methods should be used to control this species. The use of entomopathogenic microorganisms as biological control agents is critical for controlling harmful species because they can target a specific pest to be eco-friendly. Entomopathogenic fungi (EPF) are one group of the organisms used in this context, and they are effective in pest control (Mantzoukas and Eliopoulos 2020). EPFs Beauveria bassiana (Bals.-Criv.) Vuill (Hypocreales: Cordycipitaceae) and Metarhizium brunneum Petch (Hypocreales: Clavicipitaceae) are widely used against many insect species. Several studies (Martínez et al. 2021) have shown the effects of these pathogenic fungi on various insects. Various studies have investigated the efficacy of different Beauveria and Metarhizium strains in developing safe biological control strategies for the pine processionary moth (Topkara et al. 2022).
To control this species, it is essential to reduce the destruction caused by this insect in forest ecosystems, protect pine populations, and reduce the threat to human and animal health. Most of the studies on the pine processionary moth were conducted with T. pityocampa Denis & Schiffermüller (Lepidoptera: Thaumetopoeidae), while studies on its sister T. wilkinsoni are less limited. Therefore, in this study, the efficacy of EPFs B. bassiana (GOPT-331) and M. brunneum  isolates against the second and fourth larval instars of T. wilkinsoni was evaluated under laboratory conditions.

Sampling
Thaumetopoea wilkinsoni eggs were collected from the needles of Pinus sylvestris L. (Pinales: Pinaceae) at Ondokuz Mayıs University Kurupelit Campus in Samsun, Turkey (N 41°22′26.5116″ E 36°13′17.6340″), in 2021, and brought to the laboratory. The eggs were disinfected for about 7 min with 10% sodium hypochlorite and then washed and rinsed with distilled water for about 7 min. The disinfected eggs were placed in an air-conditioning room at 24 °C, 70 ± 5% RH, a 16:8-h light/dark period. The second and fourth larval instars of T. wilkinsoni were used in the study.

Fungal cultures
The EPF M. brunneum (ORP-13 and ORP-18) and B. bassiana (GOPT-331) isolates, isolated from soil samples collected from Tokat and Ordu provinces, were identified by Dr. Yusuf YANAR at Tokat Gaziosmanpasa University, Agricultural Faculty, Department of Plant Protection, Tokat/Turkey, and tested in the study. DNA extractions of fungi were performed to identify the isolates. ITS4 (5′-TCC TCC GCT TAT TGA TAT TGC-3′) and ITS5 (5′-GGA AGT AAA AGT CGT AAC AAGG-3′) primers were used for genomic DNA amplification. The isolates were diagnosed using sequence analysis and recorded at the GenBank database (Table 1). The isolates had already been tested for pathogenicity on Galleria mellonella L. (Lepidoptera: Pyralidae) and Spodoptera littoralis Boisd (Lepidoptera: Noctuidae), which were considered virulent (Şahin and Yanar 2021). They were grown on potato dextrose agar (PDA; Merck Ltd., Darmstadt, Germany) medium in an incubator at 24 ± 2 °C and a 16-h photoperiod for 15-30 days.

Preparation of conidial suspensions
The fungi were subcultured by conidial transfer to the PDA plates to produce inoculum for experiments. After getting sporulation, fungal conidia were harvested by scraping with a scalpel. The conidial suspension was prepared by adding 10 ml of sterile distilled water containing 0.02% Tween 80. The conidial suspension was vortexed for 1-2 min and filtered through four layers of sterile cheesecloths to remove mycelial fragments. The resulting spore suspensions were adjusted to concentrations of 1 × 10 5 -1 × 10 8 conidia ml −1 using a hemocytometer. The viability of conidia was determined by applying 0.1 ml of the suspension to the PDA plates. A sterile microscope coverslip was placed on each plate and incubated at 27 °C and 65 ± 5% under a 16:8-h light/dark photoperiod. Viability of conidia was determined by spreading a drop of conidial suspensions PDA and scored for germination after 24 h at 25 ± 2 °C. Conidia with germ tubes equal or greater than the width were considered to have germinated. The viability was > 90% for all isolates.

Experimental setup
The newly hatched larvae were fed on P. sylvestris leaves that had been disinfected with 50% ethanol and then washed with distilled water. The second and fourth larval instars of T. wilkinsoni were placed in plastic containers. Sterilized P. sylvestris leaves were given to the larvae in the control group. P. sylvestris leaves were contaminated with various concentrations (1 × 10 5 , 1 × 10 6 , 1 × 10 7 , and 1 × 10 8 conidia ml −1 ) of B. bassiana (GOPT-331) and M. brunneum (ORP-13 and ORP-18) isolates at 2 ml per concentration, and the leaves were given to both the second and fourth larval instars for the infected groups. Fifteen larvae were placed in each group to determine the survival rates, and each experiment was repeated three times. A total of 585 larvae, 45 in each group, were used for each concentration to be applied to the second-instar larvae. Likewise, 585 larvae (45 in each group) were used for each concentration to be applied to the fourth-instar larvae. In this experiment, the larvae were observed for 25 days.

Statistical analysis
Efficacy of three different fungal isolates (GOPT-331, ORP-13, and ORP-18) against the second and fourth larval instars of T. wilkinsoni was compared to the control groups. The log-rank test was used to compare the fungal isolates and the control group. The Cox regression analysis was used to compare the mortality risk of larvae exposed to three isolates. In addition, the lethal concentrations (LC 50 ) were determined by the Probit analysis. SPSS version 21.0 software (IBM Corp., Armonk, NY, USA) was used for these tests.
In the larvae treated with the ORP-13 isolate, 51.1% mortality rate was recorded at 1 × 10 5 conidia ml −1 concentration, while 100% mortality rate occurred at 1 × 10 8 conidia ml −1 concentration. The mortality rates of the fourth-instar larvae of T. wilkinsoni, exposed to various fungal isolates, are shown in Fig. 2. Mortality rate of the control larvae was 4.4%. The mortality rates of larvae exposed to the GOPT-331 Fig. 1 Mortality rates of the second larval instar of Thaumetopoea wilkinsoni exposed to different fungal isolates isolate were 17.7, 33.3, 57.7, and 88.8% at 1 × 10 5 , 1 × 10 6 , 1 × 10 7 , and 1 × 10 8 conidia ml −1 concentrations, respectively. The mortality rates of larvae treated with the ORP-18 isolate were 31.1, 40, 66.6, and 86.6% at 1 × 10 5 , 1 × 10 6 , 1 × 10 7 , and 1 × 10 8 conidia ml −1 concentrations, respectively, whereas the mortality rates of larvae treated with the ORP-13 isolate were recorded as 44.4, 57.7, 75.5, and 97.7% at 1 × 10 5 , 1 × 10 6 , 1 × 10 7 , and 1 × 10 8 conidia ml −1 concentrations, respectively. The Cox regression analysis results of the second-and fourth-instar larvae of T. wilkinsoni are shown in Table 2. The risk of death of larvae exposed to the GOPT-331 isolate increased 21 times, while that exposed to the ORP-13 isolate increased 27 times against the second-instar larvae. The risk of death of larvae exposed to the GOPT-331 isolate increased 13 times, while that exposed to the ORP-13 isolate increased 24 times for the fourth-instar larvae. The risk of death of larvae exposed to the ORP-18 isolate increased 16 times against the fourth-instar larvae, while it increased 22 times against the second-instar larvae.
Log-rank (Mantel-Cox) test results of the secondand fourth-instar larvae of T. wilkinsoni are shown in Table 3. Mortality rates were high at 1 × 10 7 and 1 × 10 8 conidia ml −1 concentrations. Log-rank (Mantel-Cox) test for these groups was calculated. Logrank (Mantel-Cox) test revealed that the control group   Table 3 Log-rank (Mantel-Cox) test results of the second and fourth larval instars of Thaumetopoea wilkinsoni exposed to 1 × 10 7 and 1 × 10 8 conidia ml −1 concentrations for each isolate of GOPT-301, ORP-18, and ORP-13 was statistically different (p < 0.01) than other groups exposed to GOPT-301, ORP-18, and ORP-13 isolates for the second-and fourth-instar larvae. Means and medians for survival time results of the second and fourth larval instars of T. wilkinsoni larvae exposed to 1 × 10 7 and 1 × 10 8 conidia ml −1 concentrations of  isolates are shown in Table 4. For both second and fourth larval instars, the longest were found in the control groups, while the shortest survival times were in the groups exposed to the ORP-13 isolate. There was non-statistically significant difference between the groups in terms of median survival times for the second-instar larvae (p > 0.05). For the fourth-instar larvae, there was a significant difference between the groups in terms of median survival times (p < 0.05), but there was non-statistically significant difference among GOPT-331 and ORP-18 (p > 0.05).

Discussion
EPF is one of the effective applications that can be used as alternative to chemical insecticide due to its potential as a biocontrol agent. Since the EPFs M. brunneum and B. bassiana are the most commonly used fungi against pests, the efficacy of B. bassiana (GOPT-331) and M. brunneum (ORP-13 and ORP-18) isolates against the second and fourth larval instars of T. wilkinsoni was evaluated in this study.
Several studies have proven that EPF showed promising results against the pine processionary moth.  Table 4 Means and medians for survival time results of the second and fourth larval instars of Thaumetopoea wilkinsoni exposed to 1 × 10 7 and 1 × 10 8 conidia ml −1 concentrations for each isolate of     et al. Egyptian Journal of Biological Pest Control (2023) 33:32 moth have been shown to be pathogenic to this insect (Sönmez et al. 2017). Gök et al. (2018) demonstrated that the B. bassiana LD2016 strain applied to the pine processionary moth caused 100% mortality on the 5th day, while the B. bassiana BMAUMM6-4 strain caused this mortality rate on the 3rd day. Er et al. (2007) investigated the efficacy of 13 different EPF isolates, including Beauveria and Metarhizium, against the fourth-instar larvae of T. pityocampa under laboratory conditions and revealed that the tested isolates caused 16-100% mortality rates. Topkara et al. (2022) investigated the efficacy of various B. bassiana isolates (TR-SM-10, TR-SM-11, TR-SM-2, TR-SK-1, and TR-D-1) against the fourth-instar larvae of T. wilkinsoni under laboratory conditions and noted that all isolates caused 100% mortality at a concentration of 1 × 10 8 conidia ml −1 . In this study, the mortality rates of the second-instar larvae of T. wilkinsoni increased with increasing conidial concentrations of all three isolates. The mortality rates of the larvae exposed to the highest concentration (1 × 10 8 conidia ml −1 ) of all three isolates were extremely high. This situation indicates that the isolates are highly virulent to T. wilkinsoni larvae. The mortality rates of the fourth-instar larvae of T. wilkinsoni also increased with increasing conidial concentrations. The larvae exposed to the highest concentration (1 × 10 8 conidia ml −1 ) of all three isolates had the highest mortality rates but lower than those of the second-instar larvae. This situation suggests that instar in age decreases the virulence of the fungal isolates. The findings of this study are consistent with the result of Sönmez et al. (2017), who found the fourthinstar larvae of T. pityocampa to be less sensitive than the earlier instars. Furthermore, Sönmez et al. (2017) investigated the efficacy of Metarhizium and Beauveria isolates against the first-to the fourth-instar larvae of the pine processionary moth using concentrations ranging from 1 × 10 5 to 1 × 10 8 conidia ml −1 . At the end of the study, it was shown that while the virulence of the three isolates varied slightly, they all caused 100% mortality in all larval stages at the highest concentration.
The highest LC 50 values were found in the larvae exposed to the GOPT-331 isolate for the second and fourth larval instars, while the larvae exposed to the ORP-13 isolate had the lowest LC 50 values. Since a low LC 50 value means that the applied fungal isolate was effective even in small amounts, the ORP-13 isolate was the most effective as compared to all isolates. Also, the LC 50 values for the fourth-instar larvae were higher than those for the second ones. This situation indicates that as the larval instar age increases, so will the concentration of the isolate to be applied.

Conclusion
This study aimed to control T. wilkinsoni, which is highly harmful to pine forests, humans, and animals. With high humidity and low average temperatures, the Black Sea Region, where this species is common, has a significant potential for using EPFs. In this study, B. bassiana (GOPT-331) and M. brunneum (ORP-13 and ORP-18) isolates were found to be virulent for T. wilkinsoni larvae at the highest concentration (1 × 10 8 conidia ml −1 ), with ORP-13 being the most virulent isolate. Additionally, the ORP-13 isolate had the lowest LC 50 values for both second and fourth larval instars of T. wilkinsoni. It will be critical to explore the potential of these promising EPFs in field conditions.

EPF
Entomopathogenic fungi PDA Potato dextrose agar