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
Figure 12. Phylogenetic relationship of the IMBL-1412 and IMBL-1823 strains based on 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
Figure 13. Phylogenetic relationship of the IMBL-1412 and IMBL-1823 strains based on 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 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
Figure 14. Concentration-dependent JHAN activities of the strain IMBL-1412 culture 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
Figure 15. Concentration-dependent JHAN activities of the strain IMBL-1823 culture 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
Figure 16. Concentration-dependent insecticidal activity of the strain IMBL-1412 culture 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
Figure 17. Concentration-dependent insecticidal activity of the strain IMBL-1823 culture 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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