Aedia leucomelas (Lepidoptera: Noctuidae)–pathogenic Entomophaga aulicae (Zygomycetes: Entomophthorales) in sweet potato fields

Journal of Asia-Pacific Entomology 19 (2016) 71–79

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Aedia leucomelas (Lepidoptera: Noctuidae)–pathogenic Entomophaga aulicae (Zygomycetes: Entomophthorales) in sweet potato fields Seon-Wu Choi a, Ju-Rak Lim a, Hyung-Cheol Moon a, Ju-Hee Kim a, Young-Ju Song a, Yu-Shin Nai b,c,⁎, Jae Su Kim c,⁎ a b c

Jeollabukdo Agricultural Research & Extension Services, Iksan 54591, Republic of Korea Department of Biotechnology and Animal Science, National Ilan University, Ilan, Taiwan Department of Agricultural Biology, College of Agriculture & Life Sciences, Chonbuk National University, Jeonju 561-756, Republic of Korea

a r t i c l e

i n f o

Article history: Received 29 August 2015 Accepted 13 November 2015 Available online 27 November 2015 Keywords: Entomopathogenic fungi Ecology Entomophaga aulicae Aedia leucomelas Sweet potato

a b s t r a c t Entomopathogenic fungi have high potential in pest management; however, little attention was given to phylum Zygomycota (order Entomophthorales). Herein we investigated the ecology of Entomophaga aulicae at three sweet potato fields (Iksan, Wanju, and Gimje) in Korea and further characterized its biological features, finally suggesting the possible factors inducing the occurrence of the fungal pathogen, which can be effectively used as a biological control agent. In 2002, E. aulicae were primarily observed in Aedia leucomelas (sweet potato leaf worm) larval populations (average infection rate = 41.3%). Further investigation at Iksan and Gimje in 2004 and 2005 showed that this fungal epizootic mostly occurred during the autumn season and the occurrence was related to precipitation. From the laboratory characterization, the fastest mycelial growth and the highest number of satellite colonies were observed at 20–24 °C on Sabouraud dextrose agar with 1% yeast extract medium (SDAY). E. aulicae produced large amounts of protoplasts within this temperature range; the production was significantly enhanced when the medium contained fetal bovine serum (FBS). This work provided useful information about E. aulicae and suggests that this isolate can be an effective biological control agent against the serious sweet potato leaf worm. © 2015 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved.

Introduction Knowledge concerning entomopathogenic fungi is important to understand the ecological aspects of insects (Steinhaus, 1949). Experts on these fungi have recognized that they are significantly involved in the control of insect populations. However, in the pest management schemes of agroecosystems, biological control agents, such as predators and parasitoids, are used primarily, instead of microorganisms such as those in the kingdom Fungi. Efforts are being given to the successful industrialization of entomopathogenic fungi. Hamm (1984) suggested that deep insights about the relationships among pathogens, target pests, and host plants should be understood prior to the application of the fungal control agents. A variety of research and development has been conducted on fungal insecticides, which might lead to an increase in the scale of the fungal insecticide market. Entomopathogenic fungi are categorized into two groups of orders, Hypocreales and Entomophthorales. Consideration has been given to ⁎ Corresponding authors at: Department of Agricultural Biology, College of Agriculture & Life Sciences, Chonbuk National University, Jeonju 561-756, Republic of Korea. Tel.: +82 63 270 2525; fax: +82 63 270 2531. E-mail addresses: [email protected] (Y.-S. Nai), [email protected] (J.S. Kim).

members of the Hypocreales order, such as Beauveria, Metarhizium, Lecanicillium, and Isaria because of their high virulence and broad spectrum. In contrast, little interest has been given to the Entomophthorales although this order includes many genera having pathogenicity against agriculturally serious pests (Hajek, 1999). Additionally, although many studies focus on the morphological and cultural characterization of the genera of the Hypocreales (e.g. Beauveria and Metarhizium), few studies examined genera of the Entomophthorales even though these fungi (e.g. Entomophthora planchoniana, Neozygites fresenii, Pandora neoaphidis, Zoophthora phalloides, Conidiobolus obscurus) infect some serious agricultural pests in South of Korea (Yoon et al., 1998a,b,c,d, 1999a,b). In 2002, we found that sweet potato leaf worms (Aedia leucomelas) were frequently infected by Entomophaga aulicae (Zygomycetes: Entomophthorales) in sweet potato (Ipomoea batatas) fields during a routine survey of natural enemies. Generally, E. aulicae infects lepidopteran insects in Europe, Asia, and North America (Hajek et al., 1996). This fungal species has been reported in the soybean pest, green clover worm (Plathypena scabra) (Kalkar and Carner, 2005), and as cooccurring with nucleopolyhedrovirus in populations of the forest pest, white marked tussock moth (Orgyia leucostigma) (van Frankenhuyzen et al., 2002). Additionally, in 1976, E. aulicae was isolated from A. leucomelas in sweet potatoes Japan (Hajek et al., 1996).

http://dx.doi.org/10.1016/j.aspen.2015.11.008 1226-8615/© 2015 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved.

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Larvae of A. leucomelas cause serious damage to sweet potato leaves resulting in poor crop production. Damage from the pest in Korean sweet potato fields has been cumulative since 1990. Each year, 3–4 generations are produced. The population of A. leucomelas significantly increases from late August and reaches a peak in early to middle September (Lee et al., 2003). Chemical pesticides (e.g. methoxyfenozide and etofenprox) have been applied to control the pests in Korean sweet potato fields. In this work, we collected sweet potato leaf worms that were infected by E. aulicae in three regions (Iksan, Wanju, and Gimje) of Korean fields from 2002 to 2004. The ecological aspects affecting occurrence of the pest and fungal infection were investigated in two different fields (Gimje and Iksan) and included measurements of abiotic environmental factors from 2004 to 2005. To better understand the biological characteristics of E. aulicae, it was isolated from the cadavers in the fields and the morphology and cultural features were further studied. Our data help to elucidate the relationships among E. aulicae, sweet potato leaf worms, and the cropping environment so that this fungal control agent may become an effective biopesticide in sweet potato fields. Materials and methods Sampling Entomophaga aulicae-infected sweet potato leaf worms (Aedia leucomelas) were first collected in October 2002, in the sweet potato fields of Iksan, Wanju, and Gimje areas, where 10 sampling sites were determined in each area. Each sampling site had 5–10 replicates of 1 m2 size. Additional information about the sampling sites (e.g. size of cultivated area, % leaf damage by A. leucomelas, and numbers of A. leucomelas larvae) is shown in Table 1. The E. aulicae-mediated infection rates were estimated by calculating the numbers of infected larvae per total numbers of collected sweet potato leaf worms. The host rage of E. aulicae in lepidopteran larvae was investigated in sweet potato fields

in Iksan, Wanju, and Gimje during the autumn seasons from 2002 to 2004. Relationship among E. aulicae, A. leucomelas, and environmental features Occurrence of A. leucomelas and E. aulicae in fields was investigated from 2004 to 2005. For 2 years, one area of Iksan and one area of Gimje were assigned for monitoring. Monitoring involved the close inspection of collections from 4 spots per area and each spot was 1 m2 in size. The numbers of A. leucomelas and E. aulicae-infected dead larvae were counted from August to October in both years. Metrological records of rainfall, relative humidity (RH), and average temperature in the monitoring areas in 2004 and 2005 were provided by Jeonju National Weather Forecast Service, Korea. Characterization of E. aulicae M32 Infected sweet potato leaf worms collected from Gimje were store at 4 °C for fungal morphological observation and molecular identification. Fresh cadavers of E. aulicae-infected larvae were placed on glass slides in moist Petri dishes to collect single discharged conidium on surface of Sabouraud dextrose agar with 1% yeast extract (SDAY) agar and then cultured at 20 °C in darkness. The isolated fungi were observed under a stereoscopic microscope (Nikon Eclipse 80i), stained with acetoorcein, and then observed under an optical microscope (Nikon SMZ1000). The cellulose and chitin cell wall of resting spores were stained with 20 μl of 0.1% calcofluor-white, 5 μl of 0.85% sodium chloride, and 5 μl of PTU (50 μg/ml) and then observed under a fluorescence microscope (Olympus Ix70). For scanning electron microscopy (SEM), fungal samples were prefixed in 2.5% glutaraldehyde for 2 hours at 4 °C and washed with phosphate buffer (pH 7.0) three times for 20 minutes each time. The prefixed samples were further fixed in 2% osmium tetroxide (OsO4) at 4 °C for 2 hours, followed by washing with phosphate buffer (pH 7.0)

Table 1 Occurrence of E. aulicae-infected sweet potato leaf worm (A. leucomelas) in three locations (Iksan, Wanju, and Gimje) of sweet potato fields in Jeollabuk-Do, Korea, October of 2002. Locations

Iksan city

Wanju county

Gimje city

Altitude (m) 15 7 39 38 19 18 19 21 27 20 28 33 35 33 33 32 31 36 28 39 31 33 29 27 17 34 31 33 31 37

Geographic coordinates E

N

35°56′24.23″ 35°56′24.23″ 36°00′49.23″ 36°00′41.21″ 36°01′29.64″ 36°01′40.28″ 36°01′50.43″ 36°01′55.15″ 36°01′20.02″ 36°02′18.08″ 35°49′58.58″ 35°49′53.96″ 35°49′39.99″ 35°49′32.95″ 35°49′29.50″ 35°52′07.86″ 35°52′28.25″ 35°51′45.52″ 35°52′40.92″ 35°51′35.29″ 35°48′53.09 35°48′55.93″ 35°48′51.17″ 35°48′55.17″ 35°48′44.86″ 35°49′00.00″ 35°48′57.86″ 35°48′37.14″ 35°48′36.25″ 35°48′29.74″

126°59′35.87″ 126°59′34.43″ 127°01′23.46″ 127°01′26.41″ 126°58′19.11″ 126°58′06.68″ 126°56′30.05″ 126°56′32.57″ 126°58′57.10″ 126°56′22.59″ 126°59′32.48″ 126°59′42.38″ 126°59′39.74″ 127°00′00.12″ 126°59′36.11″ 127°09′53.02″ 127°09′33.18″ 127°10′36.71″ 127°09′21.77″ 127°10′53.30″ 126°59′26.34″ 126°59′17.24″ 126°58′54.41″ 126°58′17.28″ 126°55′19.37″ 126°59′50.29″ 126°59′49.19″ 127°00′09.99″ 127°00′20.51″ 127°00′22.22″

Average cultivation size (m2)

Leaf damage rate by moth larvae (%)

A. leucomelas larva (No./m2)

Infected larvae by E. aulicae (No./m2)

Average infected rate (%)

5000

43

5.4

2.2

41

3600

47

10.0

4.9

49

4200

68

8.5

3.6

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and finally dehydrated with ethanol series (50, 70, 80, 90, 95, and 100%). Dehydrated samples were subjected to gold coating then observed and photographed with an SEM (JSM-5410LV, Jeol). The fungal isolate was also molecularly identified based on the sequencing of the 18S-ITS1 region, which was amplified using PCR with primers p1f2 (5′-ACC TGG TTG ATC CTG CCA GTA G-3′) and p1r (5′-CCG AGA GAT CCA TCG TC-3′); the method was performed in accordance with the Entomopathogenic Fungal Identification manual (Humber, 1998). PCR products were sequenced commercially (MACRO GEN, Korea) and analyzed with NCBI BLAST (Wheeler et al., 2003). Insect rearing Sweet potato leaf worms were collected from Iksan field and reared in laboratory conditions. All insect rearing equipment was sterilized using 2% sodium hypochloride. Insects were kept at 25 ± 2 °C, 70 ± 10% RH, with a photoperiod of 16:8 (L:D), and fed with sweet potato leaves supplemented with an artificial diet. After mating, female adults were placed in a rearing cage (35 × 35 × 50 cm3) containing sweet potato leaves for laying eggs. Cultivation of E. aulicae M32 The E. aulicae strain (M32) was isolated from the A. leucomelas larval cadavers in Gimje and cultured on Sabouraud dextrose agar with 1% yeast extract (SDAY) medium at 20 °C in darkness for 20 days. Protoplasts from the culture were sub-cultured in Grace's Insect Medium (Sigma–Aldrich) supplemented with 5% fetal bovine serum (FBS) using a 125 ml neck tissue culture flask at 20 °C for 2 days. Hyphal bodies and protoplasts were kept at −70 °C in a 15% glycerol for the bioassays and growth tests. Bioassay Virulence of E. aulicae M32 against sweet potato leaf worms was assayed in laboratory conditions using an injection method as described below. Protoplasts of E. aulicae were harvested from the liquid culture and adjusted to 2 × 105 protoplast/ml. Abdominal legs of five- and six-instar A. leucomelas larvae were injected with 5 μl of the protoplast suspension (1 × 103 protoplast). Injection of Grace culture medium and non-injection larvae served as controls. The control and the injected larvae were placed on a Petri dish (Ø10 × 4 cm) with one moisturized filter paper. Each treatment had 20 larvae per replicate and it was replicated three times. All larvae were kept at 25 ± 2 °C, 70 ± 10% RH, with a photoperiod of 16:8 (L:D), and fed with sweet potato leaves supplemented with an artificial diet. Lethal times of 50% (LT50) and 90% (LT90) were calculated by using a probit analysis of the program R (Version 3.0.3). Growth of E. aulicae M32 in solid and liquid cultures Solid culture The growth of E. aulicae M32 in several media: 2% water agar (WA), potato dextrose agar (PDA), SDAY, and sabouraud dextrose agar with egg yolk and low fat milk (EYSDA) were compared. One agar block (2 mm in diameter) from a satellite colony was transferred to the center of each media and cultured at 20 °C. Mycelial growth, spore discharge, and satellite mycelium formation were observed 10 days after the cultures. Secondly, to investigate the relationship between cultural temperature and hyphal growth, 2 mm diameter satellite colonies were transferred to the center of SDAY medium and cultured at 16, 20, 24, and 28 °C. Mycelial growth and the numbers of satellite colonies were observed at 4, 6, 8, 10, 12, 14, 16 days after the inoculation. Lastly, the discharging distances of satellite colonies were investigated as described below. E. aulicae M32 was cultured on WA medium at 12, 16, 20, 24, 28, 32, and 36 °C. At each temperature, one agar block (4 mm

73

in diameter) of EYSDA culture plate was transferred to the WA Petri dish (Ø10 × 4 cm) and covered with a lid. The distance of discharging was measured 4 days after the culture. This experiment was replicated three times. Liquid culture The influence of FBS, temperature, and agitation speed on the production of protoplasts was investigated in liquid culture conditions, which were conducted in darkness at 16, 20, 24, and 28 °C. FBS was added to the Grace's Insect Medium (Sigma–Aldrich) with 2.5, 5.0, and 7.5% (v/v). Additionally, the effect of agitation speed on the production of protoplasts was investigated and compared at the following conditions: non-agitation, 50 and 100 rpm in a shaking incubator (Rotary shaker, Jeio Tech. SI-600). The initial protoplast concentration was adjusted to 1 × 103 propagules/ml; protoplasts were counted using a hemocytometer. This experiment was replicated three times. Data analysis Data were analyzed using a general linear model (GLM) after the normal distribution test, and it was followed by Tukey's honest significant difference (HSD) test for multiple comparisons. All analyses were conducted using R program 3.1.2 (2014) (The R Foundtion for Statistical Computing) at the 0.05 (α) level of significance. Results Occurrence of E. aulicae in sweet potato fields In three regions of sweet potato fields in 2002, the average population of A. leucomelas was 5.4–10 larvae/m2 and caused 43–68% leaf damage. In the sweet potato leaf worm populations, ~34–49% of populations were infected by E. aulicae (Table 1). During the 2002 to 2004 field surveys in the three sweet potato fields, several pests were observed in autumn (e.g. Aedia leucomelas, Agrius convolvuli, Acanthoplusia agnate, and Spodoptera litura) but only A. leucomelas was severely infected by E. aulicae (Table 2). Infected A. leucomelas larvae were covered with white conidia and external conidiophores and became symptomatically flaccid (Fig. 1). Interaction of E. aulicae, A. leucomelas, and environmental features In the field surveys of two sweet potato fields during 2004 to 2005, E. aulicae-infected A. leucomelas larvae were mostly found in the autumn season (August to October) (Figs. 2 and 3). Additionally, Table 2 E. aulicae-infected insect host species in three locations (Iksan, Wanju, and Gimje) of sweet potato fields in Jeollabuk-Do, Korea, 2002–2004. Locations

Infected host species

No. of fields where E. aulicaeinfected insect host was founda

Time period

Occurrence rangeb

Iksan

Aediae leucomelas Agrius convolvuli Acanthoplusia agnata Spodoptera litura A. leucomelas Ag. convolvuli Ac. agnata S. litura Unidentified species A. leucomelas Ag. convolvuli Ac. agnata S. litura

10 1 2 2 10 1 1 2 1 10 3 4 1

2002−2004 2003−2004 2003−2004 2003−2004 2002−2004 2004 2004 2004 2004 2002−2004 2002−2004 2002−2004 2002−2004

++ + + + ++ + + + + ++ + + +

Wanju

Gimje

a b

Sweet potato fields in Iksan, Wanju, and Gimje were investigated. ++: Moderate; +: slight.

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Fig. 1. Aedia leucomelas larvae infected by Entomophaga aulicae: (a) healthy larva, (b) early stage of summit disease syndrome, and (c) dead larva with white conidia of Entomophaga aulicae.

the outbreak of E. aulicae was significantly related to environmental abiotic factors, especially precipitation. Importantly, the population of A. leucomelas larvae dramatically decreased following the increase in E. aulicae infection rate. In 2004, A. leucomelas larvae occurred in the Iksan field from August to October; the highest E. aulicae infection rate (~25%) was observed in late September. A similar pattern of pest occurrence was found in the Gimje field in 2004; two high-infection peaks were observed: late August (~40%) and middle October (~35.7%) (Fig. 2). A delayed occurrence of A. leucomelas larvae was found in both Iksan and Gimje fields in 2005 (Fig. 3); pest occurrence was first observed in the beginning of September and reached its highest population in the middle of September. The

E. aulicae infection rate within the population of A. leucomelas larvae increased to ca. 66% in October and to ca. 100% by the end of October into November 2005. Through an examination of the metrological records of rainfall and RH along with E. aulicae infection rate in the two monitoring areas, it was apparent that the infection rate of the entomopathogenic fungus was significantly related to local precipitation (Figs. 2 and 3). In the figures, relatively large amounts of precipitation were recorded in August and September, followed by high humidity conditions, and this phenomenon was more pronounced in 2005. Survey data from the two sweet potato fields in 2005 show that E. aulicae infected A. leucomelas larvae primarily from October to

Fig. 2. Occurrence of A. leucomelas and E. aulicae in sweet potato fields in Iksan and Gimje, Korea, 2004. (a) Iksan, (b) Gimje, and (c) climate record of the areas.

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Fig. 3. Occurrence of A. leucomelas and E. aulicae in sweet potato fields in Iksan and Gimje, Korea, 2005. (a) Iksan, (b) Gimje, and (c) climate record of the areas.

November; this occurrence was significantly related to the environmental abiotic factors of precipitation (Fig. 3). Although A. leucomelas larvae were observed in late August, E. aulicae-infected larvae were not found during this period. Instead of E. aulicae-infected larvae, Nomuraea rileyiinfected larvae were found during this period of late August (data not shown). Morphological characterization and virulence of E. aulicae M32 The isolated E. aulicae M32 strain has similar morphology to previously reported isolates (Hamm, 1980; Humber, 1984; Kalkar and Carner, 2005). Details on the morphology of mycelia, hypha, and the conidia that discharged from external conidiophores on larva cadavers, were observed and described (Table 3 and Fig. 4). Conidia of E. aulicae M32 attached to the larval cuticle, followed by conidial sporulation and growth of appressoria (Figs. 4a and b). Unbranched conidiophores were observed on the larval surface and conidia were formed directly on top of a single conidiophore (Fig. 4c). Primary conidia were pyriform to ovoid in shape with a papilla apex; they were contained in one globule (Fig. 4d). Conidia stained with

aceto-orcein showed multiple nuclei in one conidium (Fig. 4e). The average size of conidia was 34.0 ± 1.69 × 24.4 ± 1.45 μm (n = 100). Resting spores of E. aulicae M32 in S. litura larvae were stained with calcofluor-white and observed under light and fluorescence microscopes. The resting spores were spherical in shape (Figs. 4f–h). The hyphae of E. aulicae M32 were 8.5 ± 1.54 μm in width (Fig. 4i) and showed yellowish and white mycelial growth on SDAY medium. The PCR result revealed that the 18S sequence of this fungal (GenBank No.: KT952324) isolate was 99% identical to the previously reported isolate (GenBank No.: U35394). From the bioassay data, E. aulicae M32 showed high virulence against A. leucomelas larvae 5 days post-injection in laboratory conditions. The LT50 and LT90 values were 4.3 ± 0.03 and 4.9 ± 0.05 days, respectively (additionally LT95 = 5.03 ± 0.71 days) (Table 4). Growth of E. aulicae M32 in solid and liquid cultures Cultural characterization of E. aulicae M32 showed that EYSDA medium was superior to the other WA and PDA media in the mycelial growth and satellite mycelia formation (Table 5). The fastest mycelial growth

Table 3 Morphological characteristics of E. aulicae isolate M32 on SDAY medium compared to the previously reported isolates. Characteristics

Odor Mycelial density Mycelial color on the SDAY Hyphal width (μm) Nucleus Conidiophores Primary conidia size Length × width (μm) Primary conidia shape

References Hamm (1980)

Humber (1984)

Kalkar and Carner (2005)

E. aulicae M32 (This study)

– – – – – – 30–42.4 × 22.9–30.4a

No – – – Multinuclei No branched –

– – – – Multinuclei No branched 28 ± 3 × 19.5 ± 1.9

No Thick mycelia Yellowish white 8.5 ± 1.54 Multinuclei No branched 34.0 ± 1.69 × 24.4 ± 1.45

Pyriform to broadly ovoid

Pyriform to ovoid

Pyriform to obovate

Pyriform to ovoid

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Fig. 4. Morphological characteristics of E. aulicae. (a) E. aulicae on larval surface (×80); (b) germination of conidia on larval surface (×750); (c) conidiophores on larval surface (×750); (d) conidium (×200); (e) conidium stained with aceto-orcein (×200); (f) resting spore (×200); (g) resting spore attached to hyphal body (×200); (h) resting spore from hyphal body (×200); and (i) hyphal bodies in a hemocoel (×200).

and the highest number of satellite colonies were observed at 20–24 °C on SDAY solid medium, but these excellent cultural features were not observed at 16 °C, even in dramatic decrease at 28 °C (Table 6). E. aulicae M32 totally produced 75 and 46 satellite colonies per plate at 20 °C and 24 °C conditions, respectively; in each temperature, the average colony size was less than 1 mm in diameter (Fig. 5a). However, any significant satellite colonies were not observed at 16 °C and 28 °C conditions. The discharging distance of E. aulicae M32 conidia gradually increased at the temperature range of 12 °C (17 mm) to 24 °C (28.8 mm), but decrease of the discharging distance was observed at 28 °C (Fig. 5b). In liquid cultures, the production of E. aulicae protoplasts was significantly enhanced when the SDAY medium was supplemented with 2.5– 5% FBS (Fig. 6). The highest production of E. aulicae protoplasts was observed at 20 °C in medium supplemented with 5% FBS. The second highest production occurred at 24 °C in the same medium supplemented with 2.5–5% FBS.

(and vice versa), isolates, cultural features, and occurrences in field conditions should be studied. Occurrence of E. aulicae was mostly observed in sweet potato fields, compared to other crops in Korea. E. aulicae was frequently isolated in A. leucomelas larva; its insect host range was narrow in the sweet potato fields. There are more than 10 species in the genus Entomophaga. In the past, there were some synonymous species in the fungal group; E. aulicae has been reported as the same species of E. maimaiga, now it has been re-classified (Walsh, 1996). Morphologically, significant differences in the size of conidia, resting spores, and the number of nuclei are observed between E. aulicae and E. maimaiga, but other characteristics are highly similar to each other (Macleod and Mullerko, 1973; Keller, 2007). Recently, the different host spectrum led to re-classification of the two species. E. maimaiga is pathogenic against only gypsy moth (Lymantria dispar) (Hajek et al., 1990a; Hajek and Humber, 1991; Bidochka et al., 1995). In contrast, E. aulicae has been isolated from several species of lepidopteran insects, and it was isolated from A. leucomelas in Japan in 1976 (Hajek, 1999). According to our field

Discussion

Table 5 Mycelial growth, spore discharge, and the formation of satellite colonies of E. aulicae M32 on several solid culture media.

Entomopathogenic fungi are very sensitive to abiotic environmental factors during pathogenesis, making understanding these factors an important background for the successful application of this fungal group. In order to characterize responses of the host insects to fungi Table 4 Virulence of E. aulicae protoplasts against 5–6th instar of A. leucomelas larvae. Larval stage 5-6 stage larval stage a b

Injection concentration 103 protoplast ml-1

LT50a (days)

LT90b (days)

Medium

Mycelial growth (mm)

Spore discharge⁎

Satellite mycelium formation§

WAa PDAb SDYAc EYSDAd

0 3.2 ± 0.17 8.5 ± 0.26 9.0 ± 0.51

++ ++ +++ +++

− _ ++ +++

a

Water agar. Potatp dextrose agar. Saubouraud dextrose agar with 1% yeast extract. d Saubouraud dextrose agar with egg yolk and low fat milk. ⁎ ++: moderate; +++: severe. § −: no satellite mycelium formation; +: slight; ++: moderate; +++: severe. b c

4.3 ± 0.03

LT50: Median lethal time, lethal time to 50% mortality. LT90: Lethal time to 90% mortality.

4.9 ± 0.05

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Table 6 Mycelial growth of E. aulicae M32 on SDAY solid medium at different temperature conditions.§ Temp. (°C)

16 20 24 28 §

Mycelial growth (mm); days after inoculation 4

6

8

10

12

14

16

2.0 ± 0.82 3.5 ± 0.58 3.0 ± 1.53 2.1 ± 1.73

3.7 ± 0.96 4.7 ± 0.58 5.0 ± 0.58 2.5 ± 0.58

5.2 ± 1.89 6.5 ± 0.58 6.7 ± 1.00 2.7 ± 0.58

6.7 ± 2.36 9.0 ± 1.15 11.2 ± 2.02 8.0 ± 0.58

8.1 ± 2.17 10.2 ± 2.18 11.2 ± 2.02 8.0 ± 0.58

8.5 ± 1.73 14.5 ± 1.73 14.0 ± 1.17 8.0 ± 2.89

11.5 ± 3.32 19.5 ± 4.04 19.0 ± 4.00 8.0 ± 5.03

Means with the same lower case letters in the same column are not significantly different according to the Tukey’s HSD (p N 0.05).

survey data, E. aulicae was mostly isolated from A. leucomelas larvae. However, E. aulicae is now divided into three sub-groups (Hajek, 1999), so E. aulicae from other host insects may have unknown genetic information. It is worth noting that during this survey, we also found Zoophthora radicans (Family: Entomophthoraceae) in diamondback moth, rice leaf roller and aphids, consequently revealing that fungal species of the family Entomophthoraceae have a broad spectrum. Virulence varied among the isolates of Z. radicans, which is similar to E. aulicae; thus, a variety of pathotypes may exist in Z. radicans (Pell et al., 2001). In addition, N. rileyi, which was categorized into Hypocreales, was also frequently isolated in our field survey conditions. In the sweet potato fields, E. aulicae was rarely isolated during the summer time. In combination with the in vitro cultural characterization results, it could be determined that this fungus is sensitive to the temperature; the optimal growth temperature was 20–24 °C. This result clarifies why this fungus was not observed during the hot temperature season. Being different from Hypocreales, Entomophthorales strongly discharge conidia from conidiophores, which enables this fungal group to grow faster than that of Hypocreales. Moreover, the germination and sporulation of Entomophaga maimaiga in the laboratory occurred between 2 and 25 °C with maximal rates between 20 and 25 °C (Hajek et al., 1990b). Domestic reports on the Entomophthorales have focused on their morphology, with little information provided concerning cultural and ecological features (Yoon et al., 1998a,b,c,d, 1999a,b). Now, it is possible to research this fungal group through in vitro cultivation. Samson et al. (1988) suggested that, compared to other fungal groups, entomopathogenic fungi had different optimal temperatures for growth as follows: Hypocreales (N25 °C) and Entomophthorales (b25 °C), a suggestion supported by our data. In this study, for the liquid culture of E. aulicae, FBS was added to the medium, enabling this fungus to produce large numbers of protoplasts. The optimal temperature for protoplast production was 20–24 °C, but shaking of the liquid culture reduced the protoplast yield. At 20 °C, E. aulicae can infect host insects, followed by the production of protoplasts in the body. However, when the environmental temperature increases to 28 °C (or in hosts with higher body temperatures), protoplast production by E. aulicae can be inhibited, thus delaying the death of host insects.

The range of temperature affecting biological development depends on the species and ecological habitats involved. Abiotic factors, particularly temperature, play an important role in the pathogenesis of entomopathogenic microorganisms. For example, small amounts of temperature change can impact host resistance levels, resurgence of host insect populations, and virulence levels of pathogens (Thomas and Blanford, 2003a). Additionally, it has been suggested that the activities of natural enemies and internally cooperative organisms in host insects can be expected at temperatures over the upper and under the lower host insect temperature limitations (Thomas and Blanford, 2003b). The temperature range for growth of entomopathogenic fungi overlaps with their insect hosts, thus simple studies on the relationship between fungal growth and abiotic temperature can be used to assume fungal virulence levels according to environmental conditions. When host insects are reared in high-temperature conditions, they can show resistance to the fungal infection. In the cases of Trichoplusia ni (Behnke and Paschke, 1966), Anticarsia gemmatalis (Boucias et al., 1984), and Heliothis zea, N. rileyi was applied to the insects at 30 °C but little pathogenesis was observed (Gardner, 1985). Although entomopathogenic fungi can grow in high-temperature conditions, insects usually have an effective immune system range and control the infection of fungal pathogens at those temperatures. Gardner (1985) described that at a high temperature, insect hosts acquire immunization factors and consequently inhibit fungal growth, followed by resistance to the fungal infection. However, in this work, the growth rate of the isolated E. aulicae dramatically decreased at high-temperature conditions (28 °C). Based on our field survey data and metrological records in the monitoring areas during 2004 and 2005, A. leucomelas larvae occurred in high numbers from September to October in both survey fields. During this season, the average temperature was ca. 20–25 °C. For the purpose of biological control of this pest, isolated E. aulicae can be effectively used in the sweet potato fields at the temperatures preferred by the host insect. In the 2002 survey, many sweet potato fields (~ 3000 m2) were a monoculture. However, less consideration was given to the sweet potatoes compared to the other crops (such as cabbage, pepper, cucumber, etc.) because of its lower market value. The use of chemical pesticides in large areas of sweet potato fields is not a very economic strategy;

Fig. 5. Cultural characteristics of E. aulicae M32 isolate on SDAY medium after 16 days of culture. (a) Satellite colony number- and size-distribution at different temperatures and (b) the discharging distance of satellite colonies at different temperatures. Bars with the same lower case letters are not significantly different according to Tukey's HSD (p N 0.05).

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Fig. 6. Growth curves of E. aulicae protoplast cultured at 16, 20, 24, and 28 °C in SDAY medium supplemented with 0%, 2.5%, 5%, and 7.5% FBS, respectively.

it increases the production cost and reduces net income. Pesticides could also negatively affect the maintenance of the beneficial fungal pathogens and natural enemies. In sweet potato fields, pesticides such as insecticides and fungicides can be alternatively applied in practice; thus, the interaction between pesticides and entomopathogenic fungi should be carefully investigated to effectively inform integrated pest management. Based on the temperature and virulence studies, and the economic considerations as mentioned above, an integrated pest management system should be emphasized and established. Entomopathogenic fungi produce asexual spores (conidia) attached on the host insect cuticle, where they germinate and penetrate into the cuticle. After entering the cuticle by enzymatic degradation and mechanical penetration with the release of secondary metabolites, it produces hyphal bodies and protoplasts in the insect hemocoel. Entomopathogenic fungi parasite the insects until the available nutrients of the hosts are consumed. Sporulated conidia and hyphae move to other population for pathogenesis or enter a dormant stage and wait for favorable conditions (Kaya and Tanada, 1993). E. aulicae protoplasts lack of ß (1, 3)-glucan and chitin, which can be recognized by the host immune system, so the fungus could escape host defense mechanisms when in protoplast form (Hajek, 1999). In this work, resting spores and protoplasts were found in A. leucomelas and S. litura; we assumed that this was due to different hosts' responses to infection with E. aulicae. Further studies on the mode of action of E. aulicae should be conducted to comprehensively understand biological aspects of this fungus and its interactions with host insects. Considerations can be given to the spectrum of the fungus, including investigation of beneficial insects for efficient application and the negative fungal effect to crops. In spite of the several advantages of the E. aulicae M32 shown in this work, this isolate showed slow pathogenesis against A. leucomelas unless applied at 16–28 °C under high humidity conditions. Further study to solve these problems should be conducted in near the future to establish successful pest control. Similar to Hypocreales, Entomophthorales have great potential in pest management. This group has been used successfully for a long time as biological control agents in some areas (Hajek, 1999; Shah and Pell, 2003). To industrialize E. aulicae in practice, understanding population genetics and insect pathology are the basic studies to be done (Pell et al., 2001). In conclusion, the entomopathogenic fungal pathogen E. aulicae dominantly colonized in the larval populations of A. leucomelas in sweet potato fields from August to October as an effective biological control agent, and precipitation, humidity, and moderate temperatures induced its occurrence. Cultural characterizations indicated that the fastest mycelial growth and the highest number of satellite colonies were observed at 20–24 °C on SDAY medium. The E. aulicae isolate M32 was characterized in regard to morphology, cultural features, and

ecological occurrence. It has a high potential to be used in the successful integrated pest management. Acknowledgments We appreciate Dr. R. A. Humber from United States Department of Agriculture (USDA) and Dr. D. J. Lim from Rural Development Administration (RDA), Republic of Korea, for their scientific advises about our works. This study was supported by the Site Joint Cooperating Agricultural Research-Promoting Project, RDA, Republic of Korea and Advanced Production Technology Development Program, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea. References Behnke, C.N., Paschke, J.D., 1966. Spicaria rileyi (Farlow) charles and Aspergillus flavus Link, entomogenous fungi of the cabbage looper, Trichoplusia ni (Hübner), in Indiana and Wisconsin. J. Invertebr. Pathol. 8, 103–108. Bidochka, M.J., Walsh, S.R., Ramos, M.E., Leger, R.J., Silver, J.C., Roberts, D.W., 1995. Pathotypes in the Entomophaga grylli species complex of grasshopper pathogens differentiated with random amplification of polymorphic DNA and cloned-DNA probes. Appl. Environ. Microbiol. 61, 556–560. Boucias, D., Bradford, D.L., Barfield, C.S., 1984. Susceptibility of the velvetbean caterpillar and soybean looper (Lepidoptera: Noctuidae) to Nomuraea rileyi: effects of pathotype, dosage, temperature, and host age. J. Econ. Entomol. 77, 247–253. Gardner, W.A., 1985. Effects of temperature on the susceptibility of Heliothis zea larvae to Nomuraea rileyi. J. Invertebr. Pathol. 46, 348–349. Hajek, A.E., 1999. Pathology and epizootiology of Entomophaga maimaiga infections in forest Lepidoptera. Microbiol. Mol. Biol. Rev. 63, 814–835 (table of contents). Hajek, A.E., Humber, R.A., 1991. Sympatric occurrence of two Entomophaga aulicae (Zygomycetes: Entomophthorales) complex species attacking forest lepidoptera. J. Invertebr. Pathol. 58, 373–380. Hajek, A.E., Carruthers, R.I., Soper, R.S., 1990a. Temperature and moisture relations of sporulation and germination by Entomophaga maimaiga (Zygomycetes, Entomophthoraceae), a fungal pathogen of Lymantria dispar (Lepidoptera, Lymantriidae). Environ. Entomol. 19, 85–90. Hajek, A.E., Humber, R.A., Elkinton, J.S., May, B., Walsh, S.R., Silver, J.C., 1990b. Allozyme and restriction fragment length polymorphism analyses confirm Entomophaga maimaiga responsible for 1989 epizootics in North American gypsy moth populations. Proc. Natl. Acad. Sci. U. S. A. 87, 6979–6982. Hajek, A.E., Elkinton, J.S., Witcosky, J.J., 1996. Introduction and spread of the fungal pathogen Entomophaga maimaiga (Zygomycetes: Entomophthorales) along the leading edge of gypsy moth (Lepidoptera: Lymantriidae) spread. Environ. Entomol. 25, 1235–1247. Hamm, J.J., 1980. Epizootics of Entomophthora aulicae in lepidopterous pests of sorghum. J. Invertebr. Pathol. 36, 60–63. Hamm, J.J., 1984. Invertebrate pathology and biological-control. J. Georgia Entomol. Soc. 19, 6–13. Humber, R.A., 1984. The identity of entomophaga species (Entomophthorales, Entomophthoraceae) attacking Lepidoptera. Mycotaxon 21, 265–272. Humber, R.A., 1998. Entomopathogenic fungal identification. APS/ESA workshop. Cornell Univ. 26. Kalkar, O.Z., Carner, G.R., 2005. Characterization of the fungal pathogen, Entomophaga aulicae (Zygomycetes: Entomophthorales) in larval populations of the green cloverworm, Plathypena scabra (Lepidoptera: Noctuidae). Turk. J. Biol. 29, 243–248. Kaya, H.K., Tanada, Y., 1993. Insect Pathology. Academic Press, Inc, San Diego, CA, p. 666. Keller, S., 2007. Arthropod-pathogenic Entomophthorales from Switzerland. III. First additions. Sydowia 59, 75–113.

S.-W. Choi et al. / Journal of Asia-Pacific Entomology 19 (2016) 71–79 Lee, G.H., Paik, C.H., Kim, D.H., Choi, M.Y., Na, S.Y., Kim, S.S., 2003. Morphological characteristics, developmental periol, seasonal occurrence, and sweetpotato consumption of Aedia leuconelas (L.) (Lepidoptera:Noctuidae). Korean J. Appl. Entomol. 42, 1–7. Macleod, D.M., Mullerko, E., 1973. Entomogenous fungi—Entomophthora species with pear-shaped to almost spherical Conidia (Entomophthorales–Entomophthoraceae). Mycologia 65, 823–893. Pell, J.K., Eilenberg, J., Hajek, A.E., Steinkraus, D.C., 2001. Biology, ecology and pest management potential of entomophthorales. In: Butt, T.M., Jackson, C., Magan, N. (Eds.), Fungi as Biocontrol Agents. CABI, pp. 71–153. Samson, R.A., Evans, H.C., Latgé, J.-P., 1988. Atlas of Entomopathogenic Fungi. SpringerVerlag, Berlin. Shah, P.A., Pell, J.K., 2003. Entomopathogenic fungi as biological control agents. Appl. Microbiol. Biotechnol. 61, 413–423. Steinhaus, E.A., 1949. Principles of Insect Pathology. 1st ed. McGraw-Hill Book Co., New York. Thomas, M.B., Blanford, S., 2003a. Thermal biology in insect-parasite interactions. Trends Ecol. Evol. 18, 455-350. Thomas, M.B., Blanford, S., 2003b. Thermal biology in insect-parasite interactions. Trends Ecol. Evol. 18, 344–350. van Frankenhuyzen, K., Ebling, P.M., Thurston, G.S., Lucarotti, C.J., Royama, T., Guscott, R., Georgeson, E., Silver, J., 2002. Incidence and impact of Entomophaga aulicae (Zygomycetes: Entomophthorales) and a nucleopolyhedrovirus in an outbreak of the whitemarked tussock moth (Lepidoptera: Lymatriidae). Can. Entomol. 134, 825–845. Walsh, S., 1996. Development of molecular markers for the detection and differentiation of Entomophaga strains pathogenic for insects (PhD Dissertation) University of Toronto, Toronto, Ontario, Canada, p. 383.

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Wheeler, D.L., Church, D.M., Federhen, S., Lash, A.E., Madden, T.L., Pontius, J.U., Schuler, G.D., Schriml, L.M., Sequeira, E., Tatusova, T.A., Wagner, L., 2003. Database resources of the National Center for Biotechnology. Nucleic Acids Res. 31, 28–33. Yoon, C.S., Sung, G.H., Lee, G.H., Park, H.S., Lee, J.-O., 1998a. Entomophthora planchoniana Cornu (Zygomycetes: Entomophthoraceae), the first observed pathogen of the green peach aphid Myzus persicae in Korea. Korean J. Mycol. 26, 403–406. Yoon, C.S., Yun, T.Y., Lee, G.H., Yoo, J.K., 1998b. First record of the entomopathogenic fungus Zoophthora radicans on the green peach aphid Myzus persicae in Korea. Korean J. Mycol. 26, 399–402. Yoon, C.S., Sung, G.H., Choi, B.R., Yoo, J.K., Lee, J.O., 1998c. The aphid-attacking fungus Pandora neoaphidis; the first observation and its host range in Korea. Korean J. Mycol. 26, 407–410. Yoon, C.S., Sung, G.H., Lee, S.H., Yun, T.Y., Lee, J.O., 1998d. Zoophthora phalloides Batko (Zygomycetes: Entomophthoraceae), a fungal parasite of the aphid Dactynotus species in Korea. Korean J. Mycol. 26, 413–415. Yoon, C.S., Lee, M.H., Lee, S.H., Yoo, J.K., Lee, G.H., 1999a. First record of the entomopathogenic fungus Neozygites fresenii on the aphid in Korea. Korean J. Mycol. 27, 66–67. Yoon, C.S., Sung, G.H., Park, H.S., Yoo, J.K., Lee, J.O., 1999b. Two entomopathogenic Conidiobolus species first observed on the aphids in Korea. Korean J. Mycol. 27, 63–65.