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A THESIS

FOR THE DEGREE OF MASTER OF SCIENCE

Juvenile hormone antagonistic activity of actinobacteria and their

insecticidal activity against Plutella xylostella

방선균의 곤충 유약호르몬 길항제 탐색 및

배추좀나방 살충활성 검정

By

Jun Young Kim

Major in Entomology

Department of Agricultural Biotechnology

Seoul National University

February, 2020





UNDER THE DIRECTION OF ADVISER YEON HO JE

SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF

SEOUL NATIONAL UNIVERSITY

By

Jun Young Kim

Major in Entomology



February, 2020

APPROVED AS A QUALIFIED THESIS OF Jun Young Kim

FOR THE DEGREE OF MASTER OF SCIENCE

BY THE COMMITTEE MEMBERS

CHAIRMAN Joon-Ho Lee _________________________

VICE CHAIRMAN Yeon Ho Je _________________________

MEMBER Jun-Hyung Tak _________________________

II



Major in Entomology



February 2020

Jun Young Kim

ABSTRACT

Insect growth regulators (IGRs) are promising alternatives to conventional chemical

insecticides because they are specific to target insects and low toxic to non-target organisms.

Actinobacteria produce a wide range of secondary metabolites with insecticidal and insect

growth regulatory activities, and therefore, these bacteria could be potential sources of

novel IGR compounds. In this study, to identify novel IGR compounds from actinobacteria,

culture media of 1,904 actinobacteria isolated from Korean soil samples were investigated

for their juvenile hormone (JH)-based IGR activities. Whereas there were no actinobacteria

III

isolates with JH agonist (JHA) activity, 25 isolates showed high JH antagonist (JHAN)

activity. Among them, 4 isolates exhibited high level of insecticidal activity against 3rd

instar larvae of Plutella xylostella. Two isolates IMBL-1412 and IMBL-1823 showing

relatively higher insecticidal activities were identified as Streptomyces lactacystinicus

based on culture characteristics on various ISP media and nucleotide sequence of 16S rRNA

gene. Although the two isolates showed 100% of similarity in 16S rRNA nucleotide

sequence, nucleotide sequence of another marker, recA gene, was different each other,

demonstrating that these two isolates could belong to different subspecies. When the culture

media of these two isolates were extracted sequentially using hexane, ethyl acetate and

butyl alcohol, ethyl acetate fractions of both isolates showed high JHAN and insecticidal

activities against P. xylostella larvae at a concentration of 100 ppm. These results suggested

that secondary metabolites of actinobacteria could be efficiently applied as novel IGR

insecticides.

Key words: Actinobacteria, Streptomyces lactacystinicus, insect growth regulator, Juvenile

hormone antagonist, Plutella xylostella

Student Number; 2018-20499

IV

TABLE OF CONTENTS

ABSTRACT ........................................................................................................... II

TABLE OF CONTENTS ................................................................................... IV

LIST OF TABLES .............................................................................................. VI

LIST OF FIGURES ........................................................................................... VII

INTRODUCTION ................................................................................................. 9

LITERATURE REVIEW ................................................................................... 12

1. Actinobacteria ..................................................................................... 12

2. Juvenile hormone ............................................................................... 14

3. Insect growth regulator (IGR) .......................................................... 16

METERIAL AND METHODS ........................................................................... 20

1. Insects .................................................................................................. 20

2. Actinobacteria ..................................................................................... 20

3. Yeast two-hybrid β-galactosidase assay ........................................... 21

4. Yeast growth inhibition tests ............................................................. 22

5. Insect bioassay .................................................................................... 22

6. Taxanomic characteristics of the selected actinobacteria ............... 23

V

7. Isolation of JHAN compounds from the selected actinobacteria ... 23

RESULTS ............................................................................................................. 26

1. Screening of actinobacteria for IGR activity ...................................... 26

2. Insecticidal activity of actinobacteria with JHAN activity ............. 29

3. Concentraiton-dependent activities of the selected actinobacteria

strains .................................................................................................. 35

4. Taxonomic identification of the selected actinobacteria strains .... 36

5. Activities of the selected actinobacteria culture extracts ................ 43

DISCUSSION ....................................................................................................... 48

LITERATURES CITED ..................................................................................... 50

ABSTRACT IN KOREAN .................................................................................. 62

VI

LIST OF TABLES

Table 1. Cultural characteristics of the IMBL-1412 and IMBL-1823 strains................. 40

VII

LIST OF FIGURES

Figure 1. Schematic diagram for extraction of actinobacteria culture media. ................ 25

Figure 2. Screening of actinobacteria culture media for their IGR activites. ................. 27

Figure 3. JHAN activity of actinobacteria strains. ......................................................... 28

Figure 4. Insecticidal activity of actinobacteria strains against P. xylostella. ................. 30

Figure 5. Assay method-dependent insecticidal activities of actinobacteria strains against

P. xylostella. ................................................................................................. 31

Figure 6. Time-course insecticidal activities of actinobacteria strains against P. xylostella.

..................................................................................................................... 32

Figure 7. Insecticidal activity of actinobacteria strains against A. albopictus. ............... 33

Figure 8. Insecticidal activity of actinobacteria strains against B. tabaci. ...................... 34

Figure 9. Concentration-dependent JHAN activities of actinobacteria strains. .............. 36

Figure 10. Anti-yeast activity tests of actinobacteria strains with JHAN activities. ...... 37

Figure 11. Concentration-dependent insecticidal activities of actinobacteria strains

against P. xylostella. ..................................................................................... 38

Figure 12. Phylogenetic relationship of the IMBL-1412 and IMBL-1823 strains based on

their nucleotide sequence of 16S rRNA gene. ............................................. 41


their nucleotide sequence of recA gene. ...................................................... 42

Figure 14. Concentration-dependent JHAN activities of the strain IMBL-1412 culture

extracts. ........................................................................................................ 44

VIII


extracts. ........................................................................................................ 45

Figure 16. Concentration-dependent insecticidal activity of the strain IMBL-1412 culture

extracts against P. xylostella. ....................................................................... 46


extracts against P. xylostella. ....................................................................... 47

9

INTRODUCTION

Insects not only cause severe economic damage to agricultural products but also act as

vectors of human diseases by carrying various pathogens (Boyer, Zhang, and Lempérière

2012; Hill et al. 2005). Chemical insecticides have been commonly used to minimize the

losses to agriculture and to protect human health. However, due to their toxicity to the

environment and the development of insect resistance, the demands for environmentally

benign insecticides are on the rise.

Actinobacteria are filamentous gram-positive bacteria with a high G+C content and

widely distributed in various terrestrial and aquatic environments (Sharma, Dangi, and

Choudhary 2014). Actinobacteria represent one of the most studied classes of bacteria

because they produce a broad spectrum of biologically active compounds (Mahajan and

Balachandran 2014; Solecka et al. 2012). Approximately 10,000 bioactive secondary

metabolites are reported to be produced by actinobacteria, which represents 45% of all

bioactive microbial metabolites discovered (Jackson et al. 2018).

Actinobacteria have also been identified as potential biological pest control agents

(Omura 2011; Montesinos 2003). It has been reported that secondary metabolites derived

from actinobacteria show insecticidal activities against a variety of insect pests belonging

to Lepidoptera and Diptera (Vijayabharathi et al. 2014; Ababutain, Abdul Aziz, and AL-

Meshhen 2012; Karthik et al. 2011; El-Khawagh, Hamadah, and El-Sheikh 2011;

Dhanasekaran et al. 2010). Furthermore, recent studies have shown that actinobacteria

produce secondary metabolites with insect growth regulatory activities (Samri et al. 2017;

Kaur et al. 2014; Arasu et al. 2013).

10

Insect growth regulators (IGRs) are promising alternatives to conventional chemical

insecticides because of their selectivity and relatively low toxicity (Pener and Dhadialla

2012; Dhadialla, Retnakaran, and Smagghe 2009). Depending on their mode of action,

commercially available IGRs have been divided into three classes: juvenile hormone (JH)

agonists, ecdysone agonists and chitin synthesis inhibitors (Dhadialla, Retnakaran, and

Smagghe 2009). As JH is insect specific hormone and regulates development, reproduction,

diapause and many other aspects of insect physiology, JH-based IGRs, including JH

agonists (JHAs) and antagonists (JHANs), fatally disrupt the endocrine system, which

causes abnormal development and larval death (S.-H. Lee et al. 2015; Pener and Dhadialla

2012).

It has been reported that methoprene-tolerant (Met) functions as the JH receptor (Jindra,

Palli, and Riddiford 2013; Charles et al. 2011). Met is a member of a protein family known

as basic-helix-loop-helix (bHLH)-Per-Arnt-Sim (PAS) transcription factors, which have

significant roles in JH activity and downstream transcriptional activation. This family of

transcription factors must dimerize to regulate transcription (Kewley, Whitelaw, and

Chapman-Smith 2004). Met of Aedes aegypti dimerizes with other bHLH-PAS

transcription factors, such as Ftz-F1-interacting steroid receptor coactivator (FISC) or

Cycle (CYC), in a JH-dependent manner (Shin et al. 2012; M. Li, Mead, and Zhu 2011).

Previously, this JH-mediated interaction of Met and its binding partners have been

replicated in vitro using yeast cells transformed with the Met and FISC/CYC genes of A.

aegypti (S.-H. Lee et al. 2015). Using this in vitro yeast two-hybrid β-galactosidase ligand

binding assay, various compounds with JHA or JHAN activity have been identified from

plant essential oils and chemical libraries (S.-H. Lee, Lim, et al. 2018; S.-H. Lee, Ha, et al.

11

2018; S.H. Lee, Choi, et al. 2018).

In this study, it was assumed that actinobacteria could be another source of natural JHAs

and JHANs because they produce a variety of secondary metabolites with insecticidal and

insect growth regulatory activities. In order to explore novel IGR compounds from

actinobacteria, culture media of 1,904 actinobacteria isolates were screened for their

JHA/JHAN activities, and insecticidal activities of culture media with high JHAN activity

were evaluated.

12

LITERATURE REVIEW

1. Actinobacteria

Actinobacteria are Gram-positive filamentous bacteria with a high guanine-plus-

cytosine (GC) content in their genomes. They grow by a combination of tip extension and

branching of the hyphae. This is what gave them their name, which derives from the Greek

words for ray (aktis or aktin) and fungi (mukes). Traditionally, actinobacteria were

considered transitional forms between fungi and bacteria. Indeed, like filamentous fungi,

many Actinobacteria produce a mycelium, and many of these mycelial actinobacteria

reproduce by sporulation. However, the comparison to fungi is only superficial: like all

bacteria, actinobacteria’ cells are thin with a chromosome that is organized in a prokaryotic

nucleoid and a peptidoglycan cell wall; furthermore, the cells are susceptible to

antibacterial agents. Physiologically and ecologically, most Actinobacteria are aerobic, but

there are exceptions. Further, they can be heterotrophic or chemoautotrophic, but most are

chemoheterotrophic and able to use a wide variety of nutritional sources, including various

complex polysaccharides (Zimmermann 1990; Lechevalier and Lechevalier 1965).

Actinobacteria may be inhabitants of soil or aquatic environments (e.g., Streptomyces,

Micromonospora, Rhodococcus, and Salinispora species), plant symbionts (e.g., Frankia

spp.), plant or animal pathogens (e.g., Corynebacterium, Mycobacterium, or Nocardia

species), or gastrointestinal commensals (e.g., Bifidobacterium spp.).

Actinobacteria are of great importance in the field of biotechnology, as producers of a

plethora of bioactive secondary metabolites with extensive industrial, medical, and

13

agricultural applications. In particular, Actinobacteria produce the majority of the naturally

occurring antibiotics. The first antibiotics discovered in Actinobacteria were actinomycin

from a culture of Streptomyces antibioticus in 1940 (Waksman and Woodruff 1940),

streptothricin from Streptomyces lavendulaein 1942 (Waksman and Woodruff 1942), and

streptomycin from Streptomyces griseus in 1944 (Schatz and Waksman 1944), all of which

were discovered by Waksman and colleagues. Streptomycetes have been the major source

of clinical antibiotics and are responsible for over 80% of all antibiotics of actinobacterial

origin (Ilic et al. 2007). That actinomycin, streptomycin, and streptothricin were the first to

be found is not surprising, as these molecules occur at much higher frequencies than many

other antibiotics. The producing capacity of individual actinobacteria can also vary

enormously. Some Streptomyces species produce a single antibiotic, while others produce

a range of different compounds and compound classes. Besides antibiotics, Actinobacteria

also produce a wide variety of other secondary metabolites with activity as herbicides

(Tindall et al. 2006), antifungals, antitumor or immunosuppressant drugs, and anthelmintic

agents (Běhal 2000). Examples are given below.

Macrotetrolides are active against mites, insects (Jizba et al. 1991; Sagawa et al. 1972;

Oishi et al. 1970), coccidia (Sakamoto et al. 1978), and helminths, and they also show

immunosuppressive effects (Shichi, Tanouchi, and Kamada 1989). They are produced by a

variety of Streptomyces species. However, with regard to the composition of the

macrotetrolide complex, only S. aureus S-3466 (Ando et al. 1971), which produces a

mixture of tetranactin (the most active member of the compound group) with dinactin and

trinactin (Ando et al. 1971; Oishi et al. 1970), has been utilized for commercial purposes.

Tetranactin, a cyclic antibiotic produced by Streptomyces aureus with a molecular structure

14

related to cyclosporine, is used as emulsion against carmine mites of fruits and tea. A true

success story in terms of anthelmintics is ivermectin (Ōmura and Crump 2014), which is a

dehydro derivative of avermectin produced by Streptomyces avermitilis. After its

appearance in the late 1970s, ivermectin was the world’s first endectocide, which at the

time was a completely novel class of antiparasitic agents, with strong and broad-spectrum

activity against both internal and external nematodes and arthropods. Recently, the Nobel

Prize for Physiology or Medicine 2015 was awarded to Satoshi Omura and William C.

Campbell for their discovery of avermectin, jointly with Youyou Tu for the discovery of

the antimalarial drug artemisinin.

2. Juvenile hormone

It has been almost two centuries since studies relating juvenile hormone (JH) were

started. Müller described specific organs in the cockroach which were renamed as the

corpora allata (CA) in 1899. However, until then, the CA was described as sympathetic

ganglia concerned with the innervation of the digestive system. Although Police (1910)

suggested that the CA is endocrine organs concerned with nervous function, it had remained

to be proved that. In 1934, Wigglesworth (1934) began historical studies on insect JH,

making efficient use of surgical techniques. He assumed at first that the CA is the source

of the molting hormone, an “inhibitory factor” which prevents the first four larval stages

from molting directly into adults in Rhodnius. In 1936, he showed that the CA is the source

of the inhibitory hormone that prevents metamorphosis in young larvae and that the CA

from young larvae when implanted into fifth instars caused them to undergo a

15

supernumerary molt (V.B. Wigglesworth 1936). Wigglesworth concluded that the

concentration of the inhibitory hormone from CA determines the extent of metamorphosis

at the next molt. Then, there have been many studies on JH function. The modern era of JH

research began with the critical finding by Carroll Williams (1956) that he discovered a

natural repository for juvenile hormone, “golden oil”, and JH was extracted and diluted in

peanut oil or mineral oil to conduct hundreds or even thousands of experiments from male

Hyalophora cecropia. About 10 years after, the structure of JH was identified using gas

chromatographic analysis. Röller and colleague (Röller et al. 1967) identified the first

juvenile hormone from lipid extracts of the wild silk moth, H. cecropia. This JH, methyl

(2E, 6E, 10-cis) -10,11-epoxy-7-ethyl-3,11-dimethyl-2,6-tridecadienoate, was termed

cecropia JH or C18 JH in older literature, but it is now recognized as JH I. Meyer (Meyer

et al. 1968) identified a minor component that is called JH II, which differed from JH I by

a methyl group at C7 in the H. cecropia extracts. A third JH homologue, JH III, methyl 10,

11–epoxy–farnesoate, was identified from media in which the CA of the tobacco hornworm,

Manduca sexta, had been contained (Judy et al. 1973). JH III differs from the other

homologues in that all three branches of the carbon skeleton, at C3, C7, and C11, are methyl

groups. JH III appears to be the most common homologue among the species studied

(Schooley et al. 1984). The trihomosesquiterpenoids JH 0 and its isomer 4–methyl JH I

(iso–JH 0) were identified in M. sexta eggs (Bergot, Schooley, and De Kort 1981), but

nothing is currently known of their functions. JH III bisepoxy (JHB3) was identified from

in vitro cultures of larval ring glands of Drosophila melanogaster (Richard, Applebaum,

and Gilbert 1989). These historical studies formed the basis of many JH-related studies

today. JHs are a group of acyclic sesquiterpenoids that secreted from endocrine glands

16

called CA. Main role of JHs were first recognized and described by Wigglesworth in the

blood sucking bug, Rhodnius prolixus (V.B. Wigglesworth 1934). JHs roles mainly in

various physiological functions including molting, metamorphosis, reproduction,

polyphenism, caste differentiation, and various physiological functions in insects

(Hartfelder and Emlen 2012; Raikhel, Brown, and Belles 2005; Nijhout 1998; Riddiford

1994). Although JHs are very important for insect physiology, their regulatory mechanisms

have remained elusive (Riddiford 2008).

3. Insect growth regulator (IGR)

Carol Williams (1967) suggested the use of insects own hormone to pest control, and he

termed as “third-generation insecticides”. Schneiderman (1972) used the term insect

growth regulators (IGRs) that regulate insect growth and development. Now IGRs are

termed as chemicals that interfere with insect specific development, normal growth and

reproduction. The first IGRs used for pest control were JH mimics or JH agonist. And chitin

synthesis inhibitors and ecdysteroid agonists have been added later.

These insecticides possess relatively low environmental toxicity, such as low toxicity to

off-target like man, wildlife, and environment. Furthermore, IGRs have high specificity

cause effect against only targeted specific taxa (Pener and Dhadialla 2012).

Juvenile hormone agonists (JHAs)

JH regulates molting, metamorphosis and reproduction in insects. Due to its importance

JH has long been considered as novel pesticides (Cusson, Sen, and Shinoda 2013). The first

JH active compounds were sesquiterpenoid farnesol and farnesal (V. Wigglesworth 1961).

17

Later these compounds were announced as JH precursors and chemical structures of JHs

were elucidated. But chemical properties of natural JHs are unstable and have vulnerable

sites for degradation caused by lights, water, and temperature to use them into pest

management (Sláma 1999; Judy et al. 1973; Meyer et al. 1968; Röller et al. 1967).

The first botanical JH agonist “The paper factor” containing Canadian balsam fir was

first identified by Slama and Willians (Sláma and Williams 1966b). Pyrrhocoris apterus

reared on the paper towels made from Canadian balsam fir cause abnormal development

like metamorphosis failure and became nymphal-adult intermediate creatures or extra

instar nymphs. Also eggs from adults that normally developed showed reduced hatch rate.

Eventually, it was discovered that the balsam fir containing juvabione acts as a JHA (Slama

1971; Sláma and Williams 1966a).

After the discovery of juvabione, numerous plant derived sesquiterpenoids were

screened for their JHA activities (W.S. Bowers and Bodenstein 1971). Despite extensive

endeavors, however, only few JHAs have been identified as of now (W.S. Bowers 2012).

But large number of synthetic sesquiterpenoid JHA was revealed and Zoecon Corporation

registered hydroprene and methoprene (isopropyl 11-methoxy 3,7,11 trimethyldodeca-2,4-

dienoate), which became first JHA commercialized IGR insecticide (HENRICK 1982).

They have been successfully used against mosquitoes, ants and flies and they are still

favored as the least toxic, environmentally safe insecticides.

In 1981, Hoffmann-LaRoche laboratories reported that juvenoid containing 4-

phenoxyphenyl group shows high JH activity. The most active molecule in 4-

phenoxyphenyl series was fenoxycarb (Masner et al. 1981). As the one of the most

successful JHA, the pyriproxyfen has been commercialized in 1986. It is also fenoxycarb

18

derivatives in which side chain has been replaced by pyridyl structure (HATAKOSHI,

AGUI, and NAKAYAMA 1986).

Juvenile hormone antagonists (JHANs)

Since JHA discovered, inspired thoughts that the reverse principle, anti-juvenile

hormone agent could be explored to complement the use of JHA (Staal 1986). And it could

offer more attractive method of control because accelerate metamorphosis would shorten

the larval lifetime (Quistad et al. 1981).

Fluoromevalonate (FMev), tetrahydro-4-fluoromethyl-4-hydroxy-2H-pyran-2-one, was

previously known for its hypocholesteremic activity in mammalian systems, showed anti

JH activity in Lepidoptera. FMev induced precocious metamorphosis in several

Lepidoptera larvae. And later, it was discovered that FMev acts as a reversible inhibitor in

JH biosynthesis (Quistad et al. 1981). Imidazole caused precocious metamorphosis in

Bombyx mori. Later, substituted imidazoles act as methyl farnesoate inhibitor in JH

synthesis (Unnithan et al. 1995; ASANO, KUWANO, and ETO 1986).

Bowers discovered prococene 1 and prococene 2 that showed anti JH activity in the

extract of Ageratum houstonianum (W.S. Bowers 1977; W. Bowers 1976). These

compounds induce precocious metamorphosis, inhibition of vitellogenic development in

oocytes. These compounds were shown allactocidal activity by forming highly reactive

epoxides in the CA (Hamnett et al. 1981; W.S. Bowers 1981; Barovsky and Brooker 1980;

W.S. Bowers 1977).

Recent studies have identified JHANs from plants Lindera erythrocarpa and Solidago

serotine. These compounds were found by yeast two-hybrid system and their JHAN activity

19

and insecticidal activity to Aedes aegypti larvae were characterized. Also, topical

application of these compounds caused a retardation of follicle development in female

mosquito ovaries. The discovery of JHANs, along with plant derived JHAs like juvabione,

indicates that plants produce IGRs, and that they use these substances as a part of their

defense system against herbivores (S.-H. Lee et al. 2015).

20

METERIAL AND METHODS

1. Insects

The diamondback moth Plutella xylostella (L.) (Lepidoptera: Plutellidae) was reared on

rape sprouts and maintained at 25°C and 70% relative humidity with a 16 h light/8 h dark

cycles. Aedes albopictus (Skuse) (Diptera: Culicidae) was maintained in breeding

chambers at 28°C and 70% relative humidity with a 12 h light/12 h dark cycles in aged tap

water. Larvae were fed on a diet of TetraMin fish flakes, and adults were reared using 10%

sucrose solution. Bemisia tabaci was reared on solanum melongena in plastic cages under

a 16 h light /8 h dark cycles at 25°C and 70% relative humidity.

2. Actinobacteria

A total of 1,904 actinobacteria were isolated from soil samples collected from various

sites in Korea. One gram of soil samples was serially diluted with distilled water and

homogenized by vortexing for 10 min. An aliquot of the soil suspension was plated on

humic acid-vitamin agar (1 g humic acid, 0.5 g Na2HPO4, 1.71 g KCl, 0.05 g MgSO4·7H2O,

0.01 g FeSO4·7H2O, 1 g CaCl2, B vitamins (0.5 mg each of thiamine-HCl, riboflavin,

niacin, pyridoxine, Ca-pantothenate, inositol and p-aminobenzoic acid, and 0.25 mg of

biotin), 15 g agar and 1 L water, pH 7.2) and incubated at 28°C for 7 days. Selected isolates

were maintained as a spore suspension in glycerol (20%, v/v) at -80°C. All 1,904 isolates

were fermented in a 500 ml baffled Erlenmeyer flask containing 100 ml of M3 culture

media (1 g soytone, 1 g glucose, 2 g soluble starch, 0.3 g CaCO3, 0.02 g FeSO4· 7H2O),

21

which were incubated on a rotary shaker (150 rpm) at 30°C for 7 days. After fermentation,

the culture media were harvested by centrifugation at 10,000 rpm for 15 min at 4°C.

3. Yeast two-hybrid β-galactosidasea ssay

The Y-187 yeast cells transformed with A. aegypti Met-FISC were incubated at 30°C in

DDO (SD -Leu/-Trp) media until OD600 values reached 0.3-0.4. After harvest, the cells

were suspended in the fresh media at a concentration of 2.0×106 cells / ml and 100 μl of

the cells was distributed in 96-well plates. To estimate JHA activity, 1 μl of each

actinobacteria culture media was added into each well, and the cells were incubated for 3

h and subjected to the β-galactosidase assays using the yeast β-galactosidase assay kit

(Thermo Scientific, USA). A positive control treated with 0.033 ppm of pyriproxyfen and

a negative control treated with 1 μl of M3 culture media was placed in each tested plate.

The assay reaction mixtures in the 96-well plates were incubated at 30°C for 5 h, and the

OD420 was measured using an iMarkTM microplate reader (BIO-RAD, USA). The obtained

OD420 values were converted to an arbitrary unit representing JHA activity.

JHA activity = OD420 of sample

OD420 of pyriproxyfen (0.033 ppm)

For JHAN activity, 100 μl of yeast cells (2.0×106 cells / ml) distributed in 96-well plates

was treated with 0.033 ppm of pyriproxyfen and 1 μl of each actinobacteria culture media.

A negative control treated with 0.033 ppm of pyriproxyfen and 1 μl of M3 culture media

was placed in each tested plate. The cells were incubated for a further 3 h and subjected to

the β-galactosidase assays as described above. The obtained OD420 values were converted

to an arbitrary unit representing JHAN activity.

22

JHAN activity = OD420 of pyriproxyfen (0.033 ppm) - OD420 of treated sample

OD420 of pyriproxyfen (0.033 ppm)

4. Yeast growth inhibition tests

The transformed Y187 yeast cells with A. aegypti Met-FISC were incubated at 30°C in

DDO (SD -Leu/-Trp) media until OD600 values reached 0.3-0.4. After harvesting, the cells

were suspended in the fresh media at a concentration of 2.0×106 cells / ml, and 200 μl of

the cells was treated with 2 μl of each actinobacteria culture media in 96 well plates. The

treated cells were incubated at 30°C with shaking, and the OD600 of each sample was

measured every 3 h for 1 day.

5. Insect bioassay

Insecticidal activities of actinobacteria culture media against P. xylostella larvae were

evaluated using two separate assay methods, Cabbage leaf dipping and larval immersion.

For Cabbage leaf dipping assay, thirty 3rd instars were fed on Chinese cabbage leaf disc

(60 mm diameter) soaked in serially diluted culture media or various concentrations (1, 10,

50, and 100 μg/ml) of culture extracts. In case of larval immersion, thirty 3rd instars were

soaked in undiluted culture media for 30 sec and the treated larvae were provided Chinese

cabbage leaf discs (60 cm diameter). Thirty A. albopictus 3rd instars were treated with

actinobacteria culture media by adding 2.5 m1 of culture media in 2.5 ml tap water with

food mixtures. Thirty adults of B. tabaci were fed on Solanum melongena leaf disc (15 mm

diameter) treated with undiluted culture media. The number of dead larvae was checked at

24 h intervals and calculated after treatment for 5 days. All experiments were performed in

23

triplicate.

6. Taxanomic characteristics of the selected actinobacteria

The cultural characteristics of the strains IMBL-1412 and IMBL-1823 were determined

on various International Streptomyces Project (ISP) media. The colony color was evaluated

using the Inter-Society Color Council-National Bureau of Standards (ISCC-NBS) color

chart (Kelly and Judd 1965). PCR amplification and nucleotide sequence analysis of the

16S rRNA and recA genes of the strains was performed as previously reported (Meyers

2015; W.-J. Li et al. 2007). The 16S rRNA sequences from closely related actinobacteria

were retrieved from the National Center for Biotechnology Information

(http://www.ncbi.nlm.nih.gov/GenBank/index.html) and aligned using the CLUSTAL X

program (Thompson et al. 1997). Molecular phylogeny of the selected strains for 16S rRNA

and recA, respectively, was inferred using the neighbor-joining method under the Jones-

Taylor-Thornton (JTT) matrix-based model incorporated in the MEGA7 software (Jones,

Taylor, and Thornton 1992).

7. Isolation of JHAN compounds from the selected actinobacteria

To separate JHAN compounds from the selected strains, the culture media of IMBL-

1412 and IMBL-1823 was sequentially extracted with equivalent volumes of n-hexane,

ethyl acetate and n-butanol (Fig.1). These extracts were concentrated in a rotary vacuum

and redissolved in dimethyl sulfoxide (DMSO) for activity tests. The JHAN and

24

insecticidal activities of the extracted substances were determined as described above.

25

Figure 1. Schematic diagram for extraction of actinobacteria culture media.

26

RESULTS

1. Screening of actinobacteria for IGR activites

To isolate novel compounds with JHA or JHAN activity, culture media of 1,904

actinobacteria strains were tested using in vitro yeast two-hybrid β-galactosidase assay (Fig.

2). Among 1,904 actinobacteria strains, there were no culture media showing JHA activity.

In contrast, culture media of 25 actinobacteria strains highly interfered with the binding of

A. aegypti Met-FISC, suggesting that these actinobacteria strains produce secondary

metabolites with relatively high JHAN activity (Fig. 3).

27

Figure 2. Screening of actinobacteria culture media for their IGR activities.

28

Figure 3. JHAN activity of actinobacteria strains. To estimate JHAN activity, 0.033 ppm of pyriproxyfen and 1 μl of each undiluted

culture media were applied to yeast two-hybrid β-galactosidase assay.

29

2. Insecticidal activities of actinobacteria strains with JHAN activity

To evaluate insecticidal activities of actinobacteria with JHAN activity, 3rd instar larvae

of P. xylostella and A. albopictus and adult of B. tabaci were treated with culture media of

each actinobacteria strains, respectively. Among the 25 actinobacteria strains, culture media

from strains IMBL-0719, IMBL-1412, IMBL-1752 and IMBL-1823 showed high levels of

insecticidal activities against P. xylostella 3rd instar larvae, with mortalities greater than

80% (Fig. 4).

To investigate insecticidal activities of these 4 strains further, culture media of them were

applied to 3rd instar larvae of P. xylostella using cabbage leaf dipping and larval immersion

method, respectively. In all 4 strains tested, larval mortalities from cabbage leaf dipping

assay were higher than those from larval immersion assay, demonstrating that JHAN

compounds from these actinobacteria strains have both oral and topical toxicities (Fig. 5).

Also, mortalities of P. xylostella larvae treated with these culture media were gradually

increased with elapsed time (Fig. 6).

The insecticidal spectrum of actinobacteria was further investigated against A. albopictus

larvae and B. tabaci adults (Fig. 7 and 8). All 4 culture media of actinobacteria showing

high insecticidal activities against P. xylostella larvae exhibited very low insecticidal

activities against both A. albopictus and B. tabaci, with mortalities smaller than 20%,

demonstrating that JHAN compounds from these actinobacteria strains are highly specific

to P. xylostella larvae.

30

Figure 4. Insecticidal activity of actinobacteria strains against P. xylostella. Third instar larvae of P. xylostella were treated with

undiluted culture media of each actinobacteria strain and the mortality was calculated at 5 days after treatment.

31

Figure 5. Assay method-dependent insecticidal activities of actinobacteria strains against P. xylostella. Third instar larvae of P.

xylostella were treated with undiluted culture media of each actinobacteria strain using cabbage leaf dipping method (Left) and

larval immersion method (Right), and the mortality was calculated at 5 days after treatment.

32

Figure 6. Time-course insecticidal activities of actinobacteria strains against P. xylostella. Third instar larvae of P. xylostella were

treated with undiluted culture media of each actinobacteria strain using cabbage leaf dipping method (Left) and larval immersion

method (Right), and the mortality was calculated every 24 h for 5 days after treatment.

33

Figure 7. Insecticidal activity of actinobacteria strains against A. albopictus. Third instar

larvae of A. albopictus were treated with 50% of each actinobacteria culture media and the

mortality was calculated at 5 days after treatment.

34

Figure 8. Insecticidal activity of actinobacteria strains against B. tabaci. B. tabaci adults

were treated with undiluted culture media of each actinobacteria strain and the mortality

was calculated at 5 days after treatment.

35

3. Concentration-dependent activities of the selected actinobacteria strains

The IMBL-0719, IMBL-1412, IMBL-1752 and IMBL-1832 strains showing high

insecticidal activities against P. xylostella were subjected to a concentration-dependent β-

galactosidase assay to test the effects of increasing concentration on their JHAN activities.

The absorbance of these actinobacteria culture media increased with regards to increasing

concentration in the JHAN test (Fig. 9), while they showed no JHA activity even at high

concentrations (data not shown).

Yeast growth inhibition tests were conducted to investigate the possibility of false signals

originating from the anti-yeast activities of actinobacteria (Fig. 10). The addition of

actinobacteria culture media resulted in the normal growth of Y187 yeast cells transformed

with Met and FISC in non-selective double dropout minimal (DDO, -Leu/-Trp) media,

indicating that culture media of these actinobacteria strains directly disrupt the JH receptor

complex and exhibit JHAN activity.

In addition, insecticidal activities of these actinobacteria strains against P. xylostella

larvae were increased with increasing concentration of culture media (Fig. 11). Among 4

strains, the IMBL-1412 and IMBL-1823 strains showed superior insecticidal activities with

mortalities greater than 60% at a concentration of 2-fold dilution, and were selected for

further studies.

36

Figure 9. Concentration-dependent JHAN activities of actinobacteria strains. To estimate

the JHAN activity, 0.033 μg/ml of pyriproxyfen and serially diluted culture media of each

strain were applied to a yeast two-hybrid β-galactosidase assay.

37

Figure 10. Anti-yeast activity tests of actinobacteria strains with JHAN activities. Culture

media of each strain were tested for their anti-yeast activity to investigate whether the

reduced β-galactosidase activity was resulted from JHAN activity or yeast toxicity.

38

Figure 11. Concentration-dependent insecticidal activities of actinobacteria strains against

P. xylostella. Third instar larvae of P. xylostella were treated with serially diluted culture

media of each actinobacteria strain using cabbage leaf dipping method and the mortality

was calculated at 5 days after treatment.

39

4. Taxonomic identification of the selected actinobacteria strains

The cultural and physiological characteristics of the IMBL-1412 and IMBL-1823 strains

observed at 14 days after growing on ISP media (Table 1). Aerial mycelium produced by

the selected strains on all media were grey in color. Whereas substrate mycelium produced

by the selected strains on ISP2 and ISP5 were brown in color, those on ISP4 were dark

brown in color, suggesting that these strains belong to the genus Streptomyces.

For further phylogenetic profiling of these strains, the nucleotide sequence of their 16S

rRNA gene was compared with those of representative Streptomyces strains. A

phylogenetic tree constructed using the neighbor-joining method showed that both strains

were most closely related to Streptomyces lactacystinicus OM-6519 with 99.5% 16S rRNA

nucleotide sequence similarity (Fig. 12). Although nucleotide sequence of 16S rRNA were

identical, these two strains showed differences in nucleotide sequence of another marker

gene, recA, suggesting that they belong to different subspecies each other (Fig. 13).

40

Table 1. Cultural characteristics of the IMBL-1412 and IMBL-1823 strains.

41


their nucleotide sequence of 16S rRNA gene. The nucleotide sequences of 16S rRNA gene

from the genus Streptomyces were compared by the neighbor-joining method. Numbers at

each branch node indicate the bootstrap percentage of 1,000 replications.

42


their nucleotide sequence of recA gene. The nucleotide sequences of recA gene from the

genus Streptomyces were compared by the neighbor-joining method. Numbers at each

branch node indicate the bootstrap percentage of 1,000 replications.

43

5. Activities of the selected actinobacteria culture extracts

To purify JHAN compounds from the IMBL-1412 and IMBL-1823 strains, culture media

of these strains were sequentially extracted with n-hexane, ethyl acetate and n-butanol,

respectively. Whereas non-polar n-hexane and high-polar n-butanol fractions showed very

low level of JHAN activity, ethyl acetate fraction of both strains displayed high JHAN

activity at a concentration of 100 ppm (Fig. 14 and 15). Also, these fractions exhibited

increasing JHAN activities with increasing concentrations, demonstrating that compounds

extracted with ethyl acetate interfere with the pyriproxyfen-mediated binding of A. aegypti

Met-FISC in a concentration-dependent manner.

In addition, insecticidal activities of the ethyl acetate fraction of IMBL-1412 and IMBL-

1823 strains showing high JHAN activity were investigated against P. xylostella. Ethyl

acetate fraction of both strains showed insecticidal activities in a concentration-dependent

manner and the highest activity at a concentration of 100 ppm, with mortalities of 100%

(Fig. 16 and 17).

44


extracts. To estimate the JHAN activity, 0.033 μg/ml of pyriproxyfen and corresponding

concentrations (5, 10, 50 and 100 ppm) of each fraction were applied to a yeast two-hybrid

β-galactosidase assay.

45


extracts. To estimate the JHAN activity, 0.033 μg/ml of pyriproxyfen and corresponding

concentrations (5, 10, 50 and 100 ppm) of each fraction were applied to a yeast two-hybrid

β-galactosidase assay.

46


extracts against P. xylostella. Third instar larvae of P. xylostella were treated with

corresponding concentrations (1, 10, 50 and 100 ppm) of each fraction using cabbage leaf

dipping method and the mortality was calculated at 5 days after treatment.

47


extracts against P. xylostella. Third instar larvae of P. xylostella were treated with

corresponding concentrations (1, 10, 50 and 100 ppm) of each fraction using cabbage leaf

dipping method and the mortality was calculated at 5 days after treatment.

48

DISCUSSION

During the last several decades, studies searching for eco-friendly insecticidal

compounds have been extensively carried out to overcome the undesirable effects caused

by synthetic insecticides such as toxicity to the environment and the development of

resistant pests. Many natural products originating from plants and microorganisms have

been successfully utilized as environmentally benign alternatives to synthetic insecticides

(Cantrell, Dayan, and Duke 2012). Specifically, actinobacteria have been regarded as

plentiful sources for natural products as they have been reported to produce various

secondary metabolites with a diverse range of biological activities, including insecticidal,

antifeedant, and insect growth inhibitory activities (Vijayabharathi et al. 2014; Arasu et al.

2013; Omura 2011). In this study, among 1,904 actinobacteria isolated from Korean soil

samples, culture media of 25 strains showed high JHAN activities, with the culture media

of IMBL-1412 and IMBL-1823 strains causing 100% mortality against P. xylostella larvae,

demonstrating that these strains might produce novel JHAN compounds with high

insecticidal activities. Several endophytic actinobacteria have been reported to play a role

as beneficial symbionts with plants, and diverse bioactive metabolites from symbiotic

actinobacteria could have been utilized by plants to endure environmental stresses, such as

plant diseases and insect pests, over the course of plant and actinobacteria coevolution

(Kinkel et al. 2012; Qin et al. 2011). Thus, potential JHAN compounds derived from

endophytic actinobacteria might be adopted as effective defense mechanisms for symbiotic

plants because IGRs are insect-specific, and insects may have difficulty acquiring

49

resistance to these IGRs (W.S. Bowers 2012). These results provided novel insights into

the interactions between actinobacteria, plants, and insects.

Botanical precocenes isolated from Ageratum houstonianum and their synthetic

analogues have also been reported to show JHAN activities (Banerjee et al. 2008). They

cause precocious metamorphosis in several insect species by inducing irreversible

degeneration of the corpora allata and inhibiting JH production (Azambuja and Garcia

1991). However, precocenes have not been commercialized as insecticides because of their

low insecticidal activities and potential carcinogenic effects in mammals (Alzogaray and

Zerba 2017). Because the JHAN compounds from actinobacteria identified in this study

interrupt the formation of the JH receptor complex by competing with JH for binding to

Met, their mode of action as JHANs is different from that of precocenes. Additionally, the

high insecticidal activities of these compounds suggested that actinobacteria might be

plentiful sources of novel IGR compounds, which could be exploited for the development

of biopesticides.

50

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62

ABSTRACT IN KOREAN



서울대학교

농생명공학부 곤충학전공

김준영

초 록

곤충생장조절제 (insect growth regulator: IGR)은 화학 살충제에 비해 상대

적으로 높은 특이성과 환경에 대해 낮은 독성 등의 장점을 가지기 때문에 화

학 농약의 대안으로 떠오르고 있다. 그램 양성에 속하는 토양 세균인 방선균

은 배양 과정 중에 살충 및 곤충 생장 조절 활성을 가지는 다양한 2차 대사

산물을 생산하는 것으로 알려져 있어서 새로운 IGR 활성 물질의 잠재적인

source로 활용될 수 있다. 본 연구에서는 방선균으로부터 새로운 IGR 물질을

탐색하기 위하여, 국내토양으로부터 분리된 1,904개 방선균 균주 배양액의

IGR 활성을 평가하였다. 그 결과, 25개의 균주가 0.4 이상의 높은 JHAN

63

(juvenile hormone antagonist) 활성을 보였다. 우수한 JHAN 활성을 보인 방

선균 균주 중, 4개의 균주 배양액이 배추좀나방 3령 유충에 대하여 우수한 살

충 활성을 보여주었다. 그 중, 상대적으로 높은 JHAN 및 살충 활성을 보인

IMBL-1412와 IMBL-1823 균주는 ISP 배지 상에서의 배양 생리학적 특성과

16S rRNA 유전자 염기서열을 이용한 분자생물학적 동정을 통하여

Streptomyces lactacystinicus 균주인 것으로 나타났다. 또한, 이 두 균주는

recA 유전자 염기서열에서 차이를 보임으로써 Streptomyces lactacystinicus

에 속하는 서로 다른 subspecies임을 확인할 수 있었다. IMBL-1412와

IMBL-1823 균주가 생산하는 JHAN 활성 물질을 분리하기 위하여, 두 균주의

배양액을 다양한 용매를 이용하여 순차적으로 추출하고 각각의 추출물에 대하

여 IGR 및 살충 활성을 조사하였다. 그 결과, ethyl acetate 추출물이 높은

JHAN 활성과 배추좀나방 유충에 대한 살충 활성을 가지는 것을 확인할 수

있었다. 이러한 결과는 방선균 유래의 2차 대사산물이 새로운 IGR 살충제로

유용하게 이용될 수 있음을 시사하였다.

Key words : 방선균, Streptomyces lactacystinicus, 곤충생장조절제, 유충

호르몬 길항제, 배추좀나방

학번 : 2018-20499

INTRODUCTION LITERATURE REVIEW1. Actinobacteria2. Juvenile hormone3. Insect growth regulator (IGR)

METERIAL AND METHODS1. Insects2. Actinobacteria3. Yeast two-hybrid β-galactosidase assay4. Yeast growth inhibition tests5. Insect bioassay6. Taxanomic characteristics of the selected actinobacteria7. Isolation of JHAN compounds from the selected actinobacteria

RESULTS1. Screening of actinobacteria for IGR activity2. Insecticidal activity of actinobacteria with JHAN activity3. Concentraiton-dependent activities of the selected actinobacteria strains4. Taxonomic identification of the selected actinobacteria strains5. Activities of the selected actinobacteria culture extracts

DISCUSSIONLITERATURES CITEDABSTRACT IN KOREAN

3INTRODUCTION 9LITERATURE REVIEW 12 1. Actinobacteria 12 2. Juvenile hormone 14 3. Insect growth regulator (IGR) 16METERIAL AND METHODS 20 1. Insects 20 2. Actinobacteria 20 3. Yeast two-hybrid β-galactosidase assay 21 4. Yeast growth inhibition tests 22 5. Insect bioassay 22 6. Taxanomic characteristics of the selected actinobacteria 23 7. Isolation of JHAN compounds from the selected actinobacteria 23RESULTS 26 1. Screening of actinobacteria for IGR activity 26 2. Insecticidal activity of actinobacteria with JHAN activity 29 3. Concentraiton-dependent activities of the selected actinobacteria strains 35 4. Taxonomic identification of the selected actinobacteria strains 39 5. Activities of the selected actinobacteria culture extracts 43DISCUSSION 48LITERATURES CITED 50ABSTRACT IN KOREAN 62

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