Natural infection potential and efficacy of the entomopathogenic fungus Beauveria bassiana against Orosanga japonica (Melichar)

In this study, potential of the entomopathogenic fungi (EPF) isolates from dead samples of Orosanga japonica (Melichar) (Hemiptera: Ricaniidae) was evaluated. Infected specimens with fungi results confirmed that the samples were Beauveria bassiana after morphological and molecular identification. Amplicons produced two haplotypes (h = 2), which included one polymorphic site after sequence and named isolates 1 and 2. Two haplotypes were rooted with MK229193.1, MG345084.1 B. bassiana Genbank samples. Two-year survey results of the EPF effects revealed the highest mortality and natural infection rates in the field. Natural mortality rates varied between 80.35% (Fındıklı-August) and 94.8% (Çayeli-September) in 2018 and between 79.82% (Alipaşa-August) and 97.75% (Fındıklı- September) in 2019. For nymphs, the lowest LT50 value was found at 2.92 days for isolate 1 and 2.56 days for isolate 2, with a concentration of 1 × 106 conidia/ml, using the leaf dipping method. For adults, the lowest LT50 value was found at 3.02 days for isolate 1 and 3.15 days for isolate 2, with a concentration of 1 × 106 conidia/ml, using the direct spraying method. Nymph LT50 values were found a little bit lower than adults. Direct spraying methods results gave a high LT50 value for nymph in contrast to adult. Although the LT50 ratios gave high/low degree in different isolates, methods and life stages, non-significant differences were found between each other’s (p > 0.05). In general, B. bassiana natural infection rates were found high in August and September during the 2 years. Efficacy of the two isolates, which derived from naturally infected O. japonica specimens, increased with concentration. The two isolates can potentially be used for O. japonica integrated management, as a fungal biocontrol agent, but their toxicological effects on beneficial insects, such as honeybees, will need to be determined.


Background
The Eastern Black Sea region in Turkey is characterized by a mild and rainy climate, favoring the survival of numerous insect pest species from tropical areas. This region has limited agricultural areas, and the most important agricultural products are tea, hazelnut, and kiwi. Insecticide usage is limited because of the precipitation regime and the "zero chemical" perspective of the General Directorate of Tea Enterprises in Turkey.
Orosanga japonica (Melichar) (Hemiptera: Ricaniidae) is an invasive species in the western Palearctic region and widespread in the Black Sea area countries (Georgia, Ukraine, Russia, Turkey, and Iran) (Demir, 2018 andMozaffarian, 2018). Rising population levels of O. japonica have become a considerable problem in the Eastern Black Sea region in Turkey, since the species was first detected in 2006 (Demir, 2009). O. japonica is a pest insect in tropical regions and recently, it was reported on chestnut in Abhazia, orchards in Iran as well as tea and kiwifruit in Turkey (Mozaffarian, 2018). Adults and nymphs feed on plant sap, and adults lay their eggs on a wide range of crop plants such as grape, kiwi, tea, beans, corn, cabbage, pepper, and eggplant (Gokturk and Aksu, 2014). Synthetic insecticides-based pest control is an important measure, but they also linked to adverse effects such as environmental contamination and insecticide resistance. Additionally, toxic and detrimental effects on non-target organisms are a problem in many areas (Dinesh et al. 2015). Although different types of biological control agents and mechanical control methods against the adult and nymph stages of O. japonica have been investigated (Ak et al. 2013 andGokturk et al. 2018), more advanced studies are needed.
For insect control, the family Clavicapitaceae constitutes a favorable alternative in this regard (Sung et al. 2007), such as entomopathogenic fungi (EPF), which are distributed throughout many diverse ecological habitats (White et al. 2003). Beauveria bassiana is a well-known EPF species and has a worldwide distribution (Ownley et al. 2008). It belongs to the order Hypocrales (Ascomycota) and infects numerous insect species (Zimmermann, 2007). Several studies have investigated its effectiveness as a biological control agent in agricultural and forestry areas (Inanli et al. 2012 andGuven et al. 2015). B. bassiana can infect more than 70 insect pests, with a potential to control these species (Roy et al. 2006 andVan Lenteren, 2012).
The present study aimed to identify the EPF species that caused high rates of O. japonica mortality in the field, to determine natural infection rates under field conditions, and to confirm their efficacy against O. japonica under laboratory conditions.

Isolation of entomopathogenic fungi from naturally infected samples
Infected O. japonica specimens were collected from the field in 2018 around two locations in Rize city by hand picking. The specimens were placed in falcon tubes and transferred to the laboratory. Specimens collected on kiwifruit in Rize City Center and Fındıklı, whole collection points were fruit garden and situated around tea gardens area. Dead O. japonica specimens, infected with fungi, were transferred on potato dextrose agar (PDA) medium and incubated for 15-17 days at 25 ± 2°C in the dark and their spores were collected ) and transferred to falcon tubes containing 50 ml of Tween 80 solution (0.1% v/v). The conidia concentrations of the stock solution were determined via direct counting on a Thoma slide under a light microscope. Test conidia concentrations contained 1 × 10 4 , 1 × 10 5 , 1 × 10 6 conidia/ ml for the bioassay. Each fungal set received a code (Rize City Center islolate 1, Fındıklı isolate 2), and the collection area was noted (location, elevation, host plant, date). Isolates stored under sub-culture in PDA and subcultured every months according to the Oliveira et al. (2011) in the RTEU Vector Ecology and Control Laboratory in Rize Turkey.

Determination of fungal isolates
EPF were identified morphologically and molecularly. Morphological species distinction was made according to Humber (1997). Morphologically identified species were confirmed molecularly for exact determination.
DNA isolation, PCR, and analysis of the sequence data The DNA isolation was performed, using the Gene JET genomic DNA Extraction Kit (Thermo Scientific®), according to the manufacturer's instructions. Isolated DNA amounts were measured Qbit and diluted for final PCR volume (50 μl). The ITS4-5 regions were amplified according to White et al. (1990). Each PCR reaction was performed, using T100 ™ Thermal Cycler-Bio-Rad ® ; the PCR mix contained 100 ng of genomic DNA, 1 x buffer, 2.5 mM MgCl2, 250 mM each dNTP, 100 nM each primer (ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATCG-3′)) and 1 unit of Biolabs® Taq polymerase. Amplification conditions were as follows: 3 min at 95°C for initial denaturation, 35 cycles of 60 s at 95°C for denaturation, 30 s at an annealing temperature of 53°C, 90 s at 72°C for extension and 72°C for 5 min for final extension. Amplicons were visualized using 1.5% agarose gel and sequenced at Macrogen Inc. (Amsterdam). The sequences were edited using the BIOEDIT program, version 7.0.5.2 (Hall, 1999) and aligned in CLUSTAL W (Thompson et al. 1994). The numbers of haplotypes (h) and polymorphic sites as well as nucleotide diversity (π) were calculated using DNAsp version 5.0 (Rozas et al. 2017). The haplotypes identified in the present study were also compared to those available in GenBank samples. The neighbor-joining tree was generated by 1000 bootstrap replications according to the Mega 7 software (Kumar et al. 2016 . Whole collection points are fruit garden and situated around tea gardens area. Collection was performed with electric aspirator during 5 min and collected samples were placed in the net cages. Collected samples were transferred to the laboratory and placed in previously sterilized rearing cages. Samples were kept at 25 ± 2°C, 65 ± 10% RH and a photoperiod regime of 12:12 h light/dark in the insectarium. Fresh plant branches (blackberry) were placed into the cages for feeding. Fresh plant branches were washed by distilled water to remove soil and other pollutants, then washed with 85% ethyl alcohol solution and treated with distilled water to prevent infection of adults. Samples were kept for 120 h in the insectarium to acclimate to the environment and to avoid naturally infected specimens.

Survey of natural Orosanga japonica infections with Beauveria bassiana
In August and September 2018, wild O. japonica populations on host plants were surveyed for natural infection rates of B. bassiana. Surveys were performed monthly between August and September with an electric aspirator. The area had been checked monthly before the survey, but no infected samples were found. Counts were made over a period of 5 min by randomly selecting 3 trees (same species) infected and the O. japonica individuals were seen. Specimens were collected with and without visual sign with infection (dead and alive) during 5 min. Death/alive specimen ratio was evaluated as the sum of 3 plant collections. Collected death and alive individuals were placed in separate plastic boxes (34 × 22 × 12 cm) furnished with screen. Each individual was examined under a stereomicroscope for signs of infection by B. bassiana. Alive specimens were washed in a 0.5% sodium hypochlorite solution with 0.01% Triton X-100 after kiling with ethly acetate for avoiding any cuticular contamination. Then, they were dissected and rinsed twice with sterilized water then placed onto PDA and incubated at 25 ± 2°C for 2 weeks. The presence of fungal infection was evaluated visually. Infection rates were evaluated on all collected (dead, alive) samples.

Efficacy of Beauveria bassiana against Orosanga japonica in the laboratory
Laboratory assays were to evaluate the efficacy against O. japonica with 3 concentrations of B. bassiana. The conidia ratios for efficacy experiments were arranged at 1 × 10 4 , 1 × 10 5 , 1 × 10 6 conidia/ml in the suspensions (Tween 80 solution (0.1% v/v)). The direct effect of B. bassiana on nymphs and adults O. japonica was evaluated by leaf dipping and direct spray methods for leaves with slight modifications (Inanli et al. 2012 andGuven et al. 2015).

Leaf dipping bioassay
Field blackberry branches with leaves were collected and used. Collected 10 cm branches with leaves (4-5) were selected. Branches were dipped in the conidia suspension (Tween 80 solution (0.1% v/v)) for 5 s and then kept at room temperature for 15-20 min for drying. A piece of cotton, moistened in 5 ml distilled water, was placed on the bottom of the glass jar. Branches with leaves were placed on top of the cotton to prevent drying. Then, 20 acclimatized individuals (nymphs and adults separately) were placed in the glass jars for testing. Tween 80 solution (0.1% v/v) was used as a control with the same procedure.

Direct spray bioassay
First, cotton moistened with 5 ml distilled water was placed on the bottom of the sterilized glass jar. Leaves were placed with the branches on top of the cotton to prevent drying. Then, 20 acclimatized individuals (nymphs and adults separately) were placed in the glass jars for testing. Sprays were performed with 1 ml of conidia suspension at a time in the glass jar (each concentration included the same amount of conidia) with hand sprayer. Sprayers washed with 1/10 diluted chromic acid and sterilized with 95% ethyl alcohol between tests. Tween 80 solution (0.1% v/v) was used as a control with the same procedure. The system was maintained at 25 ± 2°C, with a 12:12 light:dark cycle, over 9 days. Dead specimens were determined 3 times over those 9 days. Whole specimens were washed by sterilized water, placed onto potato dextrose agar (PDA) plates and incubated for 2 weeks at 25 ± 2°C in the dark. Colonies were determined microscopically. The presence of fungal infection was evaluated visually. Infection rates were evaluated on all (dead, alive) samples after testing.

Data analysis
Mortality rates were evaluated via Probit analysis (LT 50 ). Differences between groups were analyzed via one-way ANOVA, and Tukey's test was applied for the evaluation between doses with p < 0.05 significance level, using the IBM® SPSS® statistics program version 22.

Results and discussion
Two different fungal isolates were visually determined according to Humber (1997) as B. bassiana. The ITS4-5 region amplicons showed similar agarose gel electrophoresis images (Fig. 1). Sequence results were almost similar for 12 B. bassiana isolates. Amplicons produced 2 haplotypes (h = 2), which included one polymorphic site; nucleotide diversity was π = 0.5333. Isolates gene sequences submitted to Genbank with MT350119  (Fig. 2). Isolation and identification of the local pathogens are essential to possible manage pest species in an ecologically sustainable manner. Ribosomal RNA genes are conserved to different degrees and highly divergent spacer area. Internal transcribed spacer (ITS) sequence results have been widely used in fungal separation (Driver et al. 2000 andCai et al. 2013). The two haplotypes were identified by one polymorphic site. Rehner and Buckley (2005) reported that the two unrelated and morphologically indistinguishable clades from different B. bassiana isolates by using ITS1-5.8-ITS2 and EF-1a sequences. Sevim (2010)    genetic variation by different genetic markers. Even though many authors reported the B. bassiana populations have low genetic diversity (Gaitan et al. 2002), Beauveria isolates showed differences for the virulence (Cruz et al. 2006). Some studies also reported that the genetic homogenity for B. bassiana isolates from a small geographical regions/areas (Muro et al. 2005 andFernandes et al. 2006). Mar et al. (2012) reported that 99% similarity between the two B. bassiana isolates from different sites and hosts in Northern Thailand. Obtained B. bassiana isolates originating from different points but from the same host. Similarly, the sequences showed a high similarity and one mutational point.

Natural infection of Orosanga japonica with Beauveria bassiana
The insect species O. japonica is widely distributed in the Eastern Black Sea area and has also been found in the western part of the Black Sea (Demir, 2018 and Arslangundogdu and Hizal 2019). Obtained results also showed that from 2018 to 2019, the population of this species increased. In the 2018 and 2019 active seasons, O. japonica adult specimens, naturally infected with B. bassiana, were collected from 4 different locations in Rize City (Table 1). Big epizootic situation has not detected yet. Naturally infected specimens are shown in Fig. 3. In 2018, 2325 specimens were collected during the field study with 5 min/human collection from 4 different collection points. Mortality rates (NDS%) ranged between 80.35% (Fındıklı-August) and 94.8% (Cayeli-September). Actual infection rates (AIS%) showed similarity and were higher than mortality rates. Mortality and actual infection rates were higher in September than in August. In August, the mortality rate in Rize City (88.64%) was higher than in other areas. The lowest mortality rate was found in Fındıklı (80.35%). In September 2018, the highest mortality rate was found in Çayeli (94.8). A correlation between mortality rates and infected specimen number was observed.
In 2019, 3192 specimens were collected. Mortality rates ranged between 79.82% (Alipasa-August) and 97.75% (Fındıklı-September). In August, Fındıklı showed the highest mortality rate (97.4%). The lowest mortality rate (79.82%) and actual infection rate (81.35%) were found in Alipaşa. In September, the highest mortality rate was found for Fındıklı (97.75%), similar to August, with a correlation with the actual infection rate. The lowest mortality rate was found for Rize City (95.62%) and the lowest actual infection rate for Çayeli (98.54%). In general, more specimens were collected in August than in September, although the mortality and actual infection rates were higher in September than in August. In 2019, more specimens were collected than in 2018 (Table 1).
B. bassiana occurred in various natural soils and is not related to a certain region (Bing and Xing 2008). This species has been isolated from several insect hosts in nature from different regions, including Turkey (Padmavathi et al. 2003).

Efficacy of B. bassiana against O. japonica in the laboratory
The results of the efficacy of the 2 different isolates of B. bassiana with the 2 different application methods against O. japonica nymphs are presented in Table 2. For isolate 1, leaf dipping method LT 50 results varied between 6.634 (1 × 10 4 ) and 2.92 days (1 × 10 6 ). Actual infection results showed correlation and were slightly higher than the mortality rates. For isolate 2, leaf dipping method LT 50 results varied between 7.23 (1 × 10 4 ) and 2.56 days (1 × 10 6 ). The most efficient results were found via applying the leaf dipping method at the concentration of 1 × 10 6 conidia/ml concentration, and the LT 50 time decreased to almost 2.56 days for isolate 2 at a 1 × 10 6 conidia/ml concentration.
The results regarding the efficacy of the two different isolates of B. bassiana with two different methods against O. japonica adults are presented in Table 3. Similarity with the nymphs' results and consistency between the 2 isolates were noticed. The most efficient results were found via direct spraying at a concentration of 1 × 10 6 conidia/ml, and the LT 50 time decreased to almost 3.02 days for isolate 1 at a concentration of 1 × 10 6 conidia/ml.
In general, direct spraying mortality showed a higher degree than the leaf dipping method, but without a significant difference (p > 0.05) (F = 0.79, significance = 0.37). The application of isolate 2 resulted in a slightly higher LT 50 value than isolate 1 for the same concentration (p > 0.05) (F = 0.09 significance = 0.762). Although adult LT 50 results were generally higher than those for nymphs, non-significant differences (p > 0.05) (F = 0.196, significance = 0.66) between nymphs and adults were found. The ANOVA results for concentration effects showed significant differences between concentrations (F = 0.196 significance = 0.000). Tukey's test results showed differences (p < 0.05) between the first (1 × 10 4 conidia/ml) and the other two concentrations (significance between 1st and 2nd dose = 0.02; significance between first (1 × 10 4 conidia/ml) and third (1 × 10 6 conidia/ml) concentration = 0.000). The second (1 × 10 5 conidia/ml) and third (1 × 10 6 conidia/ml) concentrations were non-significantly different (significance = 0.08), but close to the p < 0.05 level. Three different concentrations against nymphs and adults were tested, of which the concentration of 1 × 10 6 conidia/ml was the most effective. The highest efficacy of local B. bassiana isolates against two life stages of O. japonica was reported. The LT 50 values decreased with increasing dosage. Numerous studies have investigated the effectiveness of EPF in biological pest control, but studies using O. japonica are limited (Ak et al. 2013 andGokturk et al. 2018). Guclu et al. (2010) reported that nymphs were more sensitive than adults against the EPF Lecanicillium muscarium, and Lecanicillium muscarium. The LT 50 values ranged from 2.34 to 3.90 days, with a single concentration (1 × 10 7 conidia/ml) against nymphs. Ak et al. (2014) also reported that the Conidiobolus coronatus strain Cc3 had potential in the O. japonica IPM program. LT 50 value variation was from 3.19 to 3.66 days with three different C. coronatus isolates in a  fixed 1 × 10 7 conidia/ml concentration against nymphs. Obtained results varied from 2.92 to 3.026 days with B. bassiana against nymphs and found that the best concentration was 1 × 10 6 conidia/ml. This variation and concentration efficiency may explain with EPF species and their virulence. Eken et al. (2006) reported that LT 50 value variation from 4.6 to 4.4 days with 1 × 10 7 and 1 × 10 8 conidia/ml for B. bassiana against Saperda populnea. Tuncer et al. (2018) showed that the 4.64 and 8.53 days LT 50 value against Curculio nucum adults with one B. Bassiana isolate and commercially product in a fixed 1 × 10 8 conidia/ml concentration. Obtained results showed the variations around the 3-8 days LT 50 values testing 3 different concentrations. These results implied that the from 1 × 10 6 to 1 × 10 8 concentrations were the best scale for the possible usage of B. bassiana isolates as a bio control agent. Göktürk et al. (2018) reported bio-pesticide efficiency against O. japonica and found a low efficacy for B. bassiana against nymphs and adults (under 20%). In contrast, obtained results showed a high efficacy against O. japonica nymphs and adults with the two natural B. bassiana isolates and a high rate of natural infection. These differences may explain preserving conditions after isolation from the field or usage of different test method.
Our isolates preserved sub-culturing condition according to the Oliveira et al. (2011) who reported that the subculturing was the best method for B. bassiana preserving. Gokturk et al. (2018) preserved the isolated samples at -86°C before the identification and did not give information about the keep condition before and after tests.
Several EPF showed different degrees of efficacy against this pest species, but further detailed studies are necessary to develop environmentally friendly and economically viable biocontrol options. The species B. bassiana may be a good control agent for this pest species, but area conditions and toxicity against other arthropods are important when considering commercially usage of it. The Eastern Black Sea area is an important honeybee production area for Turkey and has economically important honey products. There are some reports of the negative effects of B. bassiana on the honeybee nest mate recognition ability and longevity (Potrich et al. 2018 andCappa et al. 2019). The EFSA (European Food Safety Authority) has also released a review of the effects of B. bassiana on different animal, soil, and plant species, including the bee brood and nest mate recognition (EFSA, 2015). Against this context, more detailed studies on different isolates, persistence, and toxicological effects on non-target organisms are needed.

Conclusion
EPFs have become an important component for pest control. B. bassiana is one of the most studied and wellknown EPFs. Obtained results revealed positive potentials against the nymphs and adults of O. japonica under field and laboratory conditions. B. bassiana could be a good component for the area wide insect pest management options for O. japonica but further detailed studies related to the non-target organisms' effects are still needed.