Evaluation of new strain (AAD16) of Beauveria bassiana recovered from Japanese rhinoceros beetle: Effects on three coleopteran insects

A strain (AAD16) of the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin was isolated from field-collected Japanese rhinoceros beetle, Allomyrina dichotoma (L.) (Coleoptera: Scarabaeidae). Its virulence was compared with another strain (ARP14) recovered from a cadaver of Riptortus pedestris (F.) (Hemiptera: Alydidae) focusing on its effect on three coleopteran, i.e., Tenebrio molitor L., A. dichotoma, and Monochamus alternatus Hope. The LT50 value of T. molitor for two larval sizes, i.e., 16–18 and 22–24 mm, was 15.3 and 19.4% lower for strain AAD16 compared to strain ARP14, respectively. Furthermore, after 8 and 10 days of exposure, the mycosis rate of strain AAD16 was 1.3 and 1.2 times higher than that of strain ARP14 in the 16–18 and 22–24 mm larval sizes, respectively. The LT50 for M. alternatus larvae was 23.2% lower on strain AAD16 than on strain ARP14. In addition, the LT50 for M. alternatus adults was 47.1% lower for strain AAD16 compared to control. The mycosis rate of strain AAD16 on M. alternatus larvae was 1.8 higher than that of strain ARP14 after 120 hours of exposure. The strain AAD16 also showed higher larval mortality (90%) for A. dichotoma compared to strain ARP14 (45.0%) at 28 days after exposure. These results suggest that B. bassiana AAD16 can be a potential biological control agent against coleopteran pests.


Introduction
Biological control is an important component of Integrated Pest Management due to its high efficiency and minimal negative effects on the environment [1,2].Predators, parasitoids, and entomopathogens including fungi, bacteria, viruses, and nematodes have potential for use as biological pest control agents [3].Entomopathogenic fungi (EPF) are effective biocontrol agents of several destructive pest species [4,5].Several species of entomopathogenic fungi have been used for pest control, including Beauveria bassiana (Balsamo), Metarhizium anisopliae (Metschnikoff), Nomuraea rileyi (Farlow), Verticillium lecanii (Zimmerman), and Paecilomyces fumosoroseus (Wize) [6].Nonetheless, only a small number of entomopathogenic fungal species have been effectively marketed as biopesticides, and there is a need to continue developing better species.Among these species, B. bassiana is a candidate for the biological control of many agricultural pests, including species of Lepidoptera, Coleoptera, and Diptera [7,8].B. bassiana is a ubiquitous, mitosporic fungus that is pathogenic on insects and has a broad host range [9].It is relatively widely used as a microbial control agent for agricultural and forest insect pests.Populations of B. bassiana comprise both genetically and phenotypically diverse isolates [9].Different isolates, which often show wide variation in their virulence to different insects, can easily be obtained from the environment using simple media for their isolation [10].However, it is well known that entomopathogenic fungi are more virulent on their native host species than on other species [11], although the mortality rate is dependent on not only the fungal strain's host-specificity but also other parameters such as the host-pathogen interactions and the prevailing environmental conditions [12].The high genetic diversity of B. bassiana strains creates potential for microbial control of insect pests and has allowed researchers to continue to develop new mycoinsecticides based on this fungus [13].
Several B. bassiana isolates have been assessed and found to be promising biological control agents for coleopteran insects [14][15][16].In this study, we report a new strain of B. bassiana, designated AAD16, and then we determine the relative pathogenicity of the new isolate (AAD16) against three coleopteran insects, i.e., Tenebrio molitor L. (Coleoptera: Tenibrionidae), Monochamus alternatus Hope (Coleoptera: Cerambycidae), and Allomyrina dichotoma (L.) (Coleoptera: Scarabaeidae), compared to isolate B. bassiana ARP14 in a laboratory bioassay.B. bassiana ARP14 is effective against different hemipteran and lepidopteran insects [17][18][19].The information on the virulence and epizootic conditions of B. bassiana that we present here would be helpful in the development of a new EPF strain for use against coleopteran pests.

Source of entomopathogenic fungi
B. bassiana AAD16 was isolated from an adult Japanese rhinoceros beetle, A. dichotoma collected in Andong, Republic of Korea in 2016 (36.550909, 128.802944).B. bassiana ARP14 was recovered from Riptortus pedestris (F.) (Hemiptera: Alydidae) in our laboratory.An adult R. pedestris infected with B. bassiana was collected from a soybean field in Songcheon, Andong, Republic of Korea in 2014 [17].

Isolation and mass production of the pathogen B. bassiana
The infected insect has been preserved in a sterilized falcon tube in a freezer.The fungus was isolated and cultured in Sabouraud Dextrose Agar (SDA) media (Difco™, Maryland, USA) for 14 days.After isolating the fungus from the host, a single colony was removed and cultured, and a pure culture of the B. bassiana isolate was produced after two cycles of plating.The purified fungal culture was replated using the loop streak dilution method.A single colony of the fungus was isolated and transferred after 72 hours and then grown for 14 days in SDA media.
The Japanese pine sawyer (M.alternatus) and Japanese rhinoceros beetle (A.dichotoma) were obtained as larvae from a commercial insect rearing company (OsangKinsect Co., Ltd., Guri, Republic of Korea).Larvae of M. alternatus and A. dichotoma were provided with an artificial diet maintained in a growth chamber at 24.8 ± 0.0˚C and 53.5 ± 0.9% RH for M. alternatus and 24.9 ± 0.0˚C and 52.3 ± 0.8% RH for A. dichotoma with a photoperiod of 16:8 hour (Light:Dark).

Morphological identification of B. bassiana strains
The cultured B. bassiana colonies were transferred onto slides with PVA mounting medium (PVA MTNG, BioQuip Products, Gladwick Street, CA, USA) and incubated at 25˚C for 48 hours.The slides were observed under an optical microscope (DM500, Leica, Wetzlar, Germany) with 50× and 100× magnification.The morphology of the fungal pathogen's synnema was studied under scanning electron microscopy (VEGA II LMU, Tescan Orsay Holding, Brno-Kohoutovice, Czech Republic) according to the taxonomic description of Rehner et al. [21].

Molecular identification of B. bassiana strains
Genomic DNA (gDNA) was extracted using a commercial kit (BioFact, Daejeon, Republic of Korea).An internal transcribed spacer (ITS) region was amplified using ITS forward primer (5' TCCGTAGGTGAACTTGCGG-3') and ITS reverse primer (5' TCCTCCGCTTATTGA TATGC-3') as reported by Schoch et al. [22].For PCR amplification of the ITS region, the extracted gDNA was used as a template, with 35 cycles under the conditions followed: 1 minute at 94˚C for denaturation, 1 minute at 46˚C for annealing, and 1 minute at 72˚C for extension, and the resulting product was then sequenced by SolGent Co. (Daejeon, Republic of Korea).
The obtained nucleotide sequence of the AAD16 strain (Accession No. MN481507.1)was analyzed using the BlastN program of the National Center for Biotechnology Information (NCBI, www.ncbi.nlm.nih.gov).The evolutionary relationship was inferred using a Neighbor-Joining phylogenetic tree with MEGA6.06 [23].Bootstrap values on the branches were estimated with 1,000 replications.

Preparation of conidial suspensions
B. bassiana AAD16 and B. bassiana ARP14 (Accession No. MG952537.1)were grown under dark conditions at 24.9 ± 0.0˚C and 48.7 ± 0.5% RH for 14 days.Conidial suspensions of the two strains were prepared by scraping the surface of the fungal culture and placing the material obtained into a 20 mL liquid scintillation vial (240804, Wheaton, Millville, NJ) containing autoclaved Triton X-100 (0.1%) solution (Duksan Pure Chemicals Co. Ltd., Ansan, Republic of Korea).To separate the conidial clumps, the suspension was stirred for 2-5 minutes with a Vortex mixer.

Topical bioassay on mealworm (Tenebrio molitor)
The virulences of the two strains of B. bassiana (AAD16 and ARP14) at a concentration of 1×10 8 conidia/ml were evaluated using two larval sizes (16-18 mm and 22-24 mm) of mealworms (T.molitor).Larvae of T. molitor were treated with 4 μl of fungal solution on the dorsal part of abdomen using regular plunger-type syringes and then placed in Petri dishes (60 mm diameter × 15 mm height) and held at 24.7 ± 0.0˚C, 99.2 ± 0.1% RH, and 16:8 hour (Light: Dark) photoperiod in the incubator.Triton X-100 ddH 2 O (0.1%) used as a control.There was one larva in each Petri dish.Bioassays were conducted with 40 larvae in the 16-18 mm size group and 20 larvae in the 22-24 mm size group (1 larva / replication).Mortality of larva was observed at 24 hours intervals from the exposure until death.

Topical bioassay on long horned beetle (Monochamus alternatus)
The virulences of B. bassiana strains AAD16 and ARP14 at a concentration of 1×10 8 conidia/ ml were evaluated on the larvae and adults of M. alternatus.Fourteen day-old larvae and seven day-old adults of M. alternatus were used in this experiment.Larvae and adults of M. alternatus were treated with 4 μl of fungal solution applied on the dorsal part of abdomen using regular plunger-type syringes and then placed in Petri dishes (60 mm diameter × 15mm height) and held at 25.1 ± 0.0˚C, 94.4 ± 0.2% RH, and 16:8 hour (Light:Dark) photoperiod in the incubator.Triton X-100 ddH 2 O (0.1%) was used as a control.In the bioassay, one Petri dish contained a single larva or a single adult.A total of 33 larvae and 8 adults per treatment were used in the bioassays (1 insect / replication).Mortality of larva was observed at 24 hours intervals from the exposure until the death.

Topical bioassay on Japanese rhinoceros beetle (Allomyrina dichotoma)
The virulences of B. bassiana strains AAD16 and ARP14 strains at a concentration of 1×10 8 conidia/ml were evaluated on larva (larval body size, 7-10 cm) of A. dichotoma.Larva of A. dichotoma was treated with 50 μl of fungal solution on the dorsal part of abdomen and placed in a breeding dish (100 mm diameter × 40 mm height) and held at 24.9 ± 0.01˚C, 96.8 ± 0.3% RH, and 16:8 hour (Light:Dark) photoperiod in the incubator.Triton X-100 ddH 2 O (0.1%) was used as a control.A total of 20 larvae of A. dichotoma were used in the bioassay (1 larva / replication).Mortality of larva was observed at 24 hours intervals from the exposure until 32 days.

Statistical analysis
Insect mortality was corrected using Abbott's formula [25].Mortality data for T. molitor and M. alternatus were subjected to log-probit regression analysis to calculate lethal median time (LT 50 ) using SAS 9.4 software [26].For A. dichotoma, survival curves and median lethal times (LT 50 ) was estimated using Kaplan-Meier survival analysis because mortality did not reach 100% and compared using the Log-rank test at the 95% level using MedCalc software [27].Mycosis developmental rates and corrected mortality rates were analyzed using a Chi-square test with a post-hoc multiple comparison test analogous to Tukey's test [28].

Morphological identification of B. bassiana strains
For strain AAD16, the short, globose-shaped clusters of conidiogenous cells were grouped in ball-shaped structures (Fig 1A and 1B), and the conidia terminated in a rachis with a narrow apical extension (Fig 1C).The zig-zag extension of the elongated rachis formed globose to subglobose shaped conidia, which are designated as B. bassiana.

Molecular identification of B. bassiana strains
The ITS region that we amplified had a sequence that showed high similarity (> 99%) with known ITS sequences of several B. bassiana strains (Table 1).To evaluate the relationship of the strain with other fungal strains, a phylogenetic tree was constructed using the ITS sequences (Fig 2).This strain AAD16 is designated as B. bassiana type clade (Fig 2).

Topical bioassay on mealworm (Tenebrio molitor) larvae
Significant differences in mealworm mortality were found between the two fungal strains tested within each of the two mealworm larval sizes (16-18 mm and 22-24 mm) (Fig 3).B.  bassiana strain AAD16 caused higher mortality in both sizes of mealworms compared to the B. bassiana strain ARP14.

Topical bioassay on Japanese rhinoceros beetle (Allomyrina dichotoma)
The virulence of B. bassiana AAD16 strain was also higher on the larvae of A. dichotoma compared to B. bassiana ARP14, with a LT 50 of 17.7 days for AAD16 (Table 4).However, the corresponding lethal median time (LT 50 ) could not be obtained for B. bassiana ARP14 because mortality lower than 50% did not occur after 30 days.Survival analysis A. dichotoma exposed to entomopathogenic fungi showed significant differences among treatments (χ 2 = 16.36,df = 2, P < 0.001).After 30 days of exposure, the survival rate of A. dichotoma larvae treated with B. bassiana AAD16 was 5.0%, whereas for larvae treated with the B. bassiana ARP14 strain, the survival rate was 55.0% (Fig 7).

Discussion
The new entomopathogenic fungal isolate collected from A. dichotoma was identified as B. bassiana and designated as strain AAD16 based on morphology [29] and intraspecies and interspecies divergence rate with different Beauveria species and strains [30].Entomopathogenic fungi are important pathogens of many arthropod pests such as aphids, leafhoppers, whiteflies, stink bugs, lepidopterans, and coleopterans [31][32][33][34][35][36][37][38].The efficacy of the entomopathogenic fungus varies from strain to strain [39], so we evaluated two strains of B. bassiana (ARP14 and AAD16) against three coleopteran insects, i.e., T. molitor, M. alternatus, and A. dichotoma.Tenebrio molitor is known for its susceptibility to entomopathogenic fungal infection and is a suitable test organism for assessing fungal virulence [40].Strain AAD16 was more virulence than the ARP14 strain to T. molitor larvae when applied topically.In the study by Rodrı ´guez-Go ´mez et al. [41] who reported similar virulence levels for two B. bassiana isolates, i.e., the wild type and its mutant type, against the larvae of T. molitor, the LT 50 value of mutant type isolate was 5.7 days, which was longer than LT 50 value of 4.2 days that we found in our study for large larvae (22-24 mm).In another study by Praprotnik et al. [40], among five B. bassiana isolates, B. bassiana 2121 showed higher virulence to larvae of T. molitor.
For the longhorned beetle M. alternatus, the B. bassiana AAD16 strain killed the host faster, causing 100% mortality by day 5 after exposure while our other treatment, B. bassiana ARP14, caused only 69% mortality in the same time period.Sone et al. [42] reported that exposing adult M. alternatus to non-woven fabric strips containing B. bassiana spores at 1.4×10 8 conidia/cm 2 caused 97% mortality in 14 days.Other strains of B. bassiana, such as F-263 and ERL836, have also been shown to be effective against M. alternatus [15,43,44].In another  study [45], application of B. bassiana F-263 at 5.5×10 6 conidia/individual of on 10 day-old beetles killed 50% of M. alternatus, while younger beetles that were only 4 days old, suffered 50% mortality from a lower dose (1.9×10 6 conidia/individual) in a somewhat longer time period (by 14 days) when conidial mixtures were applied to the tarsi of CO 2 -anesthetized adults with a fine hairbrush.However, in our experiment where higher concentration of fungus was applied, B. bassiana AAD16 kills beetles faster, causing 50% adult mortality in this species within only 8 days.
For our third test species (A.dichotoma, the beetle from which the AAB16 strain was recovered), we found that the virulence of the AAD16 strain was higher than that for the other strain ARP14.Allomyrina dichotoma larvae exposed to B. bassiana AAD16 had a low (5.0%) survival rate after 30 days of exposure, compared to a substantial (55.0%) survival rate at the same time point if treated with the other strain, B. bassiana ARP14.Nevertheless, B. bassiana ARP14 is highly virulent against R. pedestris, the host from which it was originally isolated [17].Similarly, another B. bassiana isolate (F-263), collected from a cadaver of a larva of M. alternatus, was most virulent against its original host [43,46].In Japan and China, isolates of B. bassiana have been successfully applied for the control of M. alternatus using inoculative methods of fungal bands and baited traps, respectively [47] and so, having the most virulent strain available is important in achieving good control with these programs against M. alternatus.
The basis for the above discussed intra-specific variation in virulence in this group of pathogens could be any of several modes of action [35] known in these fungi, including mechanical damage resulting from tissue invasion, depletion of nutrient resources and toxicosis, and production of toxins in host insect body [48].The toxins Beauvericin, Bassianolide, Isarolides, and Beauverolides have been isolated from B. bassiana infected hosts [49,50], but the capacity to produce toxic compounds varies depending on the strain of B. bassiana [39].
The mortality rate of insect hosts caused by entomopathogenic fungus could vary depending on the fungal strain, host-pathogen interaction, fungal specificity for the host, and other factors [12].Strains are known to often be more virulent on their natal host species than on novel species [11].As B. bassiana AAD16 strain was isolated from A. dichotoma while B. bassiana ARP14 strain from R. pedestris, B. bassiana AAD16 showed higher virulence to the three coleopteran insects we tested than did ARP14.This finding may indicate that B. bassiana AAD16 is probably more specific to coleopteran insects.Thus, we conclude that B. bassiana AAD16 has the potential to be used as microbial control agent against coleopteran insects.

Table 4 . Calculation of LT 50 values for A. dichotoma.
& Mortality did not occur above 50% LT 50 value followed by the same letters is not significantly different among the treatment based on 95% C.I. https://doi.org/10.1371/journal.pone.0296094.t004