Notes
病原菌上澄み製剤の殺アリマキ活性に及ぼすイソトリデシルアルコールのエトキシル化の効果
2013 年 38 巻 2 号 p. 85-87
詳細
2013 年 38 巻 2 号 p. 85-87
Insect-killing fungi have high potential for controlling pests with piercing and sucking mouth parts. Several species from the genera Beauveria, Metarhizium, Isaria, and Lecanicillium have been registered in the United States and commercialized.1) These products constitute a small percentage of the total insecticide market,2) although insect killing fungi are still among the most practical biological control agents.
Commercialized entomopathogenic fungal products are normally based on conidia and have limitations when applied on phyllospheres. They are slower to kill target pests than chemical insecticides.3) Some pests have a short life cycle and may have a chance to escape any conidia attached to their cuticles before they germinate and penetrate into their bodies. Another limitation is that their insecticidal activities are severely affected by environmental abiotic factors during pathogenesis.4) The germination of conidia and their enzymatic activations are influenced by humidity, temperature, and sunlight.
The direct application of liquid culture broth or spent culture medium (hereafter referred to as supernatant) can enhance the fungus-induced mortality of Isaria fumosorosea or Metarhizium flavoviride with little susceptibility to the abiotic factors.5–7) The fungal culture broth contains various pathogenesis-related components such as blastospores, mycelium, and metabolites with different functions in insect-killing. Enzymes such as chitinase [EC 3.2.1.14], Pr1- [EC 3.4.21], and Pr2- [EC 3.4.21] proteases, among the metabolites, have been reported to play an important role in penetrating insect cuticles.8,9) They have been studied intensively to improve the insecticidal activity of fungal isolates and to determine the influence of these enzymes on virulence.10–12)
When considering fungal supernatants for pest control, incorporation of an appropriate surfactant is required to allow the supernatant to wet all of the hydrophobic surfaces of the insect cuticle. Various non-ionic and non-toxic surfactants have been used previously, such as polyoxyethylene sorbitan monooleate (Tween 80), -sorbitan monolaurate (Tween 20), -isooctylphenyl ether (Triton X-100), and polysiloxane polyether (Silwet L-77).13) Recently, in the application of the supernatant of Beauveria bassiana (Balsamo) Vuillemin SFB-205 (KCCM 10892P),14) we found that ethoxylated (3 mol) isotridecyl alcohol (TDE-3) as a surfactant was more effective than Tween 80 for controlling the cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae)15) and could even be used in an oil-based formulation.16)
The present work discusses the role that the ethoxylation of TDE-n (n=the number of ethoxy groups) plays in the control of cotton aphid adults when the SFB-205 supernatant was applied with the surfactant. This non-ionic surfactant might allow the supernatant effectively to bind to the hydrophobic surface of aphid cuticles in an ethoxylation-dependent manner, followed by destruction of the cuticle structures, in a similar manner to that of some non-ionic surfactants. Ethoxylation could be involved in the control efficacy of the fungal supernatant.
B. bassiana SFB-205, deposited at the Korean Culture Center of Microorganisms (KCCM; www.kccm.or.kr), was stored at −80°C. SFB-205 was isolated from a soil sample in a Korean agricultural field in 2005. The isolate has high virulence against cotton aphid (A. gossypii Glover), green peach aphid (Myzus persicae Sulzer), and the two-spotted spider mite (Tetranychus urticae Koch).17) It was propagated on a Sabouraud dextrose agar medium supplemented with 0.5% (w/v) yeast extract (SDAY, pH 6.0) in Petri dishes in darkness at 25±1°C for 14 days.18)
2. Production of supernatantA liquid culture at pH 6 was produced in a soluble starch-yeast extract with a glycerol (SYG) medium as follows: (/L) 10 g soluble starch (Gibco), 5 g yeast extract (Gibco), and 5 mL glycerol (Fisher Scientific). A 1-mL aliquot of conidial suspension (1×106 conidia/mL) was added to 200 mL culture broth. The inoculated broth was incubated on a rotary shaker at 150 rpm at 27±1°C for 3 days. The supernatant was separated from the culture broth by centrifugation at 16000×g at 4°C for 10 min and subsequently filtered through a 0.2-µm syringe filter.
3. Aphicidal activityThe aphicidal activity of the supernatant+TDE-n against cotton aphid adults was determined using a leaf-dipping method under laboratory conditions.19) The supernatant solution (10 mL/L, pH 6) was prepared with TDE-3, TDE-5, and TDE-7 (all from Hannong Chemicals Inc., Republic of Korea (www.hannong.co.kr) and >99.5% of purity) at 0.1 mL/L. Three red hot pepper leaf discs (variety: Kwangmyung), infested with 150–170 adults per disc, were dipped into 100 mL of the diluted SFB-205 supernatant+TDE-n solution in a 500 mL glass beaker for 10 sec. Solutions of TDE-n alone and supernatant served as controls. Two days after the treatment, the number of live and dead cotton aphids per leaf disc was counted to assess mortality. The experiment was repeated twice using different batches of samples on different days.
4. Data analysisData on the percentage of cotton aphid population were analyzed by a general linear model (GLM), followed by Tukey’s honestly significant differences (HSD). All analyses were conducted using SPSS ver. 17.0 (SPSS Inc., IL, USA) at the 0.05 (α) level.
SFB-205 supernatant+TDE-n treatments reduced the cotton aphid population in an ethoxylation-dependent manner (F7,48=1174.5, p<0.001) (Table 1). The less ethoxylated TDE-n+supernatant treatments had higher mortality. Dead cotton aphids turned dark-brown 2 days after the treatment, whereas the living populations in the non-treated control and individual TDE-n and supernatant treatments remained green. Proteolytic enzymes, chitinases and lipases, which are known to be major extracellular cuticule-degradation enzymes in entomopathogenic fungi,20) might be involved in the biological performance of the supernatant in aphid control. This enzymatic degradation was possibly enhanced by the less ethoxylated TDE-n as a cuticle destroyer. At each ethoxylation level, a synergistic interaction between the supernatant and TDE-n was evident, and it was analyzed using the Webb method.21) No foliar damage, i.e., phytotoxicity, was observed in any treatment.
| Treatment | Dosage (mL/L) | Percentage of cotton aphid population (A) (mean±SE)* (Abbott control efficacy)** | Estimation of synergy (Webb method) | |
|---|---|---|---|---|
| Predicted value*** (B) | Determination**** | |||
| Supernatant | 10 | 103.2±9.3 (4.3) d | ||
| Supernatant+TDE-3 | 10+0.1 | 12.3±3.4 (88.6) a | 115.9 (=103.2×112.3/100) | synergistic |
| Supernatant+TDE-5 | 10+0.1 | 31.2±7.5 (71.0) b | 110.2 (=103.2×106.8/100) | synergistic |
| Supernatant+TDE-7 | 10+0.1 | 58.9±9.8 (45.3) c | 111.4 (=103.2×107.9/100) | synergistic |
| TDE-3 | 0.1 | 112.3±5.9 (0.0) d | ||
| TDE-5 | 0.1 | 106.8±2.8 (1.0) d | ||
| TDE-7 | 0.1 | 107.9±0.6 (0.0) d | ||
| Water | — | 107.8±2.1 d | ||
Means followed by the same lower case letter in the column do not differ according to the Tukey’s HSD test (p<0.05).*: (the number of aphid per plant at 2 days post-application/the number of aphid per plant before the application)×100%. **: ((% aphid population in control−% aphid population in a treatment)/(% aphid population in control))×100%. ***: (% aphid population in the supernatant alone×% aphid population in the corresponding TDE-n treatment alone)/100. ****: if the observed % aphid population (A) was less than 70% of the predicted value (B), then it was determined synergistic.
In a preliminary test before the bioassay, wetting of cotton aphid adults was examined by dropping the adults on the supernatant+TDE-n solutions used for the bioassay. Cotton aphids were more easily wetted in the less ethoxylated TDE-n+supernatant solution. The wetting activity of TDE-n seemed to be maintained even when the surfactant was mixed with the fungal supernatant, just like other non-ionic surfactants. Cotton aphid adults in the supernatant without TDE-n and in water took a longer time to become wet. Cotton aphids that had become wet and had sunk in the supernatant+TDE-n solution changed from light-green to dark-brown, while the controls, with TDE-n alone, did not change color. The color changes could have resulted from the possible penetration of the TDE-n solutions into aphid cuticles, followed by the supernatant-mediated enzymatic degradation; however, this still needs to be clarified.
The ethoxylation-dependent wetting and aphicidal activity might be explained by the hydrophile-lipophile balance (HLB) values of TDE-n. A low HLB value indicates that TDE-n is more lipophilic. The HLB values of TDE-n are 9.0 (TDE-3), 10.5 (TDE-5), and 12.0 (TDE-7).22) The less ethoxylated TDE-n has a lower HLB value and more easily reduces the surface tension of hydrophobic (lipophilic) cement and wax layers, which are the outer parts of insect cuticles. This suggestion is based on the hydrophobic natures of cotton aphid cuticles, in particular, their specialized structures and lipid composition for the maintenance of hydrophobicity.23) Cotton aphids have a smooth surface without any grids and lattices to mechanically reduce their hydrophobic natures, and their cuticles are mainly composed of lipophilic triacylglycerols and free fatty acids.
In terms of the mode of action, the less ethoxylated TDE-n might allow the fungal supernatant to bind more easily to the aphid epicuticles (the outer parts of cuticles) and at the same time destroy the cuticles, including many proteins,24–26) which possibly results in faster enzymatic degradation of the epicuticles, particularly by the Pr1 and Pr2 proteases in the supernatant.27) Once the fungal supernatant degrades the epicuticles, degradation of procuticles (exo- and endo-cuticles) under the epicuticles would easily follow. Particularly, in the endo-cuticles, where chitin–protein matrixes are found, TDE-n probably disrupts the proteins in the chitin-protein matrixes or suppresses the function of quinone compounds, which are responsible for the solidification of cuticle protein structures (sclerotinization).28) TDE-n-mediated disruption of proteins could cause the chitin fibers to be more susceptible to attack by chitinolytic enzymes, followed by the acceleration of the degradation of chitins. The entire process of degradation might be affected by the levels of ethoxylation.
In conclusion, these results suggest that the B. bassiana (SFB-205) supernatant with less ethoxylated isotridecyl alcohol (TDE) had higher insecticidal activity against cotton aphid adults. Incorporation of TDE to the supernatant increased the potency of the fungal supernatant in an ethoxylation-dependent manner. This finding represents a practical approach to effectively control agriculturally harmful insects using an entomopathogenic fungal supernatant or spores (conidia). Future studies will be required to clarify the mode of action of the fungal supernatant under the differently ethoxylated TDE-n.
We are grateful to Dr. Bong-Jin Chung (Dongbu Hannong Co., Republic of Korea) for his comments and passionate assistance. This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ009147)” Rural Development Administration, Republic of Korea. This work is also supported by research funds of Chonbuk National University in 2012.
