This protocol presents an optimized detached-leaf bioassay system for evaluating the effectiveness of entomopathogenic fungi (EPF) against the mustard aphid (Lipaphis erysimi (Kalt.)), a parthenogenetic insect. The method outlines the data collection process during Petri dish experiments, enabling researchers to consistently measure the virulence of EPF against mustard aphids and other parthenogenetic insects.
The mustard aphid (L. erysimi) is a pest that infests various cruciferous crops and transmits plant viruses. To achieve eco-friendly pest management, entomopathogenic fungi (EPF) are potential microbial control agents for controlling this pest. Therefore, virulence screening of EPF isolates under Petri dish conditions is necessary before field application. However, the mustard aphid is a parthenogenetic insect, making it difficult to record data during Petri dish experiments. A modified system for detached-leaf bioassays was developed to address this issue, using a micro-sprayer to inoculate conidia onto aphids and prevent drowning by facilitating air-drying after spore suspension. The system maintained high relative humidity throughout the observation period, and the leaf disc remained fresh for over ten days, allowing parthenogenetic reproduction of the aphids. To prevent offspring buildup, a process of daily removal using a painting brush was implemented. This protocol demonstrates a stable system for evaluating the virulence of EPF isolates against mustard aphids or other aphids, enabling the selection of potential isolates for aphid control.
The mustard aphid (L. erysimi) is a notorious pest that infests a variety of cruciferous crops, causing significant economic losses1. While several systematic insecticides have been recommended to combat aphid infestations, the frequent use of these insecticides raises concerns about pesticide resistance2,3. Therefore, in terms of eco-friendly pest management, entomopathogenic fungi (EPF) could serve as a suitable alternative control strategy. EPF is an insect pathogen with the ability to infect hosts by penetrating their cuticles, making it a potent agent for controlling aphids and other plant-sucking insects4. Furthermore, EPF has proven to be a feasible and sustainable pest management technique, offering benefits such as plant pathogen antagonism and plant growth promotion5.
EPF can be obtained through insect-soil baiting or isolated from insect cadavers in the field6,7. However, before further use of fungal isolates, pathogenicity screening is necessary. Several studies have been conducted on the effectiveness of EPF against aphids, which are significant crop pests that can cause severe damage8,9. Mustard aphids, among various species of aphids, have been tested for susceptibility to several strains of Beauveria spp., Metarhizium spp., Lecanicillium spp., Paecilomyces spp., and even Alternaria, which is primarily known as a saprophytic and plant pathogenic fungus but has shown some lethal effects against mustard aphids10,11,12.
To evaluate the effectiveness of EPF against aphids under laboratory conditions, bioassays can be divided into two main parts: the inoculation chamber and fungal inoculation. The current protocol describes the construction of an inoculation chamber, where aphids can be maintained using various methods such as an excised leaf with a petiole wrapped in moist cotton, an excised leaf disc with a Petri dish lined with damped filter paper, direct maintenance on pot plants, or an excised leaf disc embedded in water agar within a Petri dish or container10,11,13. Common methods for fungal inoculation include conidia spraying, aphid immersion into a conidia suspension, leaf dipping into a conidia suspension, and plant endophyte inoculation11,14,15,16. While various inoculation methods exist, the bioassays should simulate field application conditions. For example, in the case of the leaf dipped method12,17, the efficiency of EPF can be evaluated, but since the aphids infest the fungus-loaded leaves, the dorsal side of the aphid, which is a preferential penetration site, does not usually get exposed to the fungus.
To evaluate the aphidicidal effect of EPF under laboratory conditions, this protocol suggests using the detached-leaf method described by Yokomi and Gottwald18 with some modifications, followed by conidia inoculation using a micro-sprayer. This method maintains approximately 100% humidity in the bioassay chamber for at least seven days without requiring additional replenishment of water18,19. Additionally, confining aphids to one surface ensures their exposure to conidia spraying and facilitates observations20. However, aphids may become stuck in the exposed agar surface while moving within the inoculation chamber. Furthermore, recording data in the Petri dish experiment with mustard aphids, which are parthenogenetic insects, can be challenging due to their rapid development and reproduction. It is difficult to distinguish between inoculated adults and their progeny without removal. The details of how to proceed with this step are seldom mentioned, and some inconsistent factors, such as leaf consumption area, need to be optimized.
This protocol demonstrates a stable system for screening the virulence of EPF isolates against mustard aphids, enabling the selection of potential isolates against various aphid species from an extensive EPF library. Field-collected aphids can be identified, and a sufficient laboratory population of mustard aphids can be established to evaluate the aphidicidal effect of various fungal isolates using an easy and feasible methodology with consistent outcomes. Aphids have developed multiple evolutionary mechanisms in response to intense and repeated anthropogenic pressures in agroecosystems, posing challenges to food security9. Therefore, this described method could be extended to evaluate potential EPF isolates against various aphid species.
NOTE: The complete flowchart is shown in Figure 1.
1. Mustard aphid collection and maintenance
2. Molecular identification of mustard aphid
NOTE: To confirm the species of field-collected mustard aphid, molecular identification was performed using two molecular markers: the sequence characterized amplified region (SCAR) based A05Le designed by Lu et al.21, and mustard aphid cytochrome oxidase subunit 1 (COI) region of the mustard aphid.
3. Preparation of Entomopathogenic fungi
NOTE: The EPF used in this study is listed in Table 1.
4. Virulence screening against mustard aphid
5. Bioassay of selected EPF isolates
NOTE: EPF isolates that showed high virulence, which was selected from step 4, were subjected to a bioassay against mustard aphids using four concentrations of conidia suspensions (ranging from 104 to 107 conidia/mL).
6. Statistical analysis
The presented flowchart illustrates the stable condition of the mustard aphids from field collection to virulence screening. The maintenance of aphids from field collection ensured a stable increase in aphid colonies with an adequate food supply. The field-collected aphids were confirmed as mustard aphids through the use of molecular markers, including PCR amplicon size and LeCO1 sequencing. The virulence screening, conducted using the detached-leaf method, revealed a consistent survival rate for mustard aphids, with the control group exhibiting an 85% survival rate (Figure 4).
During the virulence screening, Cc-NCHU-213 demonstrated the fastest aphid-killing ability, resulting in 50% and 90% mortalities at 3 days and 4.5 days post-inoculation (d.p.i.), respectively (Figure 4). However, varying aphid-killing abilities were observed among the five B. bassiana isolates. Bb-NCHU-141, -143, and -153 exhibited slow aphid-killing abilities, with only 5% mortality at 3 d.p.i., even when excluding the effects of 0.03% Tween 80 spraying or other lethal factors (Figure 4). Hence, the corrected mortality formula was employed to normalize the control mortality. Most EPF isolates against mustard aphids exhibited corrected mortality rates higher than 70% within 5 d.p.i., except for Pl-NCHU-152 (Table 3). Among these EPF isolates, Bb-NCHU-286 demonstrated the highest mortality rate of 100% (Table 3). Additionally, EPF mycosis was observed on cadavers of mustard aphids infected with Metarhizium spp., Beauveria spp., Purpureocillium lilacinum, and Cordyceps cateniannulata during the virulence screening, indicating the effectiveness of this system (Figure 5).
Based on the results of the virulence screening, two EPF isolates, namely Mb-NCHU-197 and Cc-NCHU-213, exhibiting rapid insect-killing activity (40% and 50% mortality at 3 d.p.i., respectively), were selected for bioassay against mustard aphids. The results demonstrated significantly different corrected mortalities for Mb-NCHU-197 and Cc-NCHU-213 at 3 and 4 d.p.i. with an inoculation of 107 conidia/mL (Figure 6). In the LT50 assay, the treatment with 107 conidia/mL of Cc-NCHU-213 exhibited a significantly shorter duration compared to other treatments (Table 4). Furthermore, the LC50 value of Cc-NCHU-213 (9.32 × 104) was lower than that of Mb-NCHU-213 (2.30 × 105), indicating that Cc-NCHU-213 possesses greater virulence against mustard aphids (Table 5).
Figure 1: Experimental flowchart for screening EPF virulence against mustard aphids. (A) Establishment of a mustard aphid-rearing system. (B) Preparation of EPF. (C) Fungal inoculation. (D) Statistical analysis. Please click here to view a larger version of this figure.
Figure 2: Electrophoresis of mustard aphid genomic DNA amplified with A05Le and Le CO1 primer sets. Electrophoresis was performed on a 1% agarose gel. M = 100 bp DNA ladder; bp = base pairs.The red asterisk indicates the target bend of PCR amplification. Please click here to view a larger version of this figure.
Figure 3: Differences between apterous fourth-instar nymph and adult mustard aphids. (A) Fourth-instar nymph. (B) Adult. The tibiae of the hind legs of the fourth-instar nymph are whitish (marked with red arrow). Newly emerged aphids were removed by fine camel brush during virulence tests. Scale bar = 1 mm. Please click here to view a larger version of this figure.
Figure 4: Mortality heat map of 13 EPF isolates against mustard aphids through the detached-leaf method. Please click here to view a larger version of this figure.
Figure 5: Observation of fungal mycosis of 13 EPF isolates. Mepe-NCHU-2 = Metarhizium pemphigi; Mp-NCHU-11 = Metarhizium pinghaense; Mb = Metarhizium baoshanense;Cc = Cordyceps cateniannulata; Ba = Beauveria australis;Bb = Beauveriabassiana; Pl = Purpureocillium lilacinum. Scale bar = 1 mm. Please click here to view a larger version of this figure.
Figure 6: Corrected mortality of fungal isolates Mb-NCHU-197 and Cc-NCHU-213 against mustard aphid. The error bars represent the standard deviation (SD). The mortalities of Mb-NCHU-197 and Cc-NCHU-213 at the same time point with the same inoculated concentration were compared using independent t-test, and mortalities marked with an asterisk were found to be significantly different (p < 0.05). Please click here to view a larger version of this figure.
Isolate | Species | Host or source* | Location |
Mp-NCHU-2 | Metarhizium pemphigi | soil | Yilan |
Ml-NCHU-9 | Metarhizium lepidiotae | soil | Yilan |
Mp-NCHU-11 | Metarhizium pinghaense | soil | Yilan |
Ba-NCHU-113 | Beauveria australis | soil | Taichung |
Bb-NCHU-141 | Beauveria bassiana | Hypothenemus hampei | Chiayi |
Bb-NCHU-143 | Beauveria bassiana | Hypothenemus hampei | Chiayi |
Pl-NCHU-152 | Purpureocillium lilacinum | Tessaratoma papillosa | Chiayi |
Bb-NCHU-153 | Beauveria bassiana | Rhynchophorus ferrugineus | Chunghua |
Bb-NCHU-157 | Beauveria bassiana | Rhynchophorus ferrugineus | Chunghua |
Mb-NCHU-196 | Metarhizium baoshanense | soil | Taichung |
Mb-NCHU-197 | Metarhizium baoshanense | soil | Taichung |
Cc-NCHU-213 | Cordyceps cateniannulata | soil | Taichung |
Bb-NCHU-286 | Beauveria bassiana | Cerambycidae | Taichung |
Table 1: EPF isolates used in this study.
Primer name | Sequence (5' – 3') | Product size (bp) | Reference | |
A05Le | F-GGGTCTTGGATGGTGTGGTG | 953 | Lu et al. [21] | |
R-AGGGGTCTTGTCGCCATTTT | ||||
LeCO1 | F-CTTTTCCCATGATCAATTTT | 593 | This study | |
R-ACGTAGTGGAAATGAGCAAC |
Table 2: Primer pairs used for molecular identification of mustard aphids.
Isolate | Species | Corrected mortality (%) |
Mp-NCHU-2 | Metarhizium pemphigi | 76.47 |
Ml-NCHU-9 | Metarhizium lepidiotae | 82.35 |
Mp-NCHU-11 | Metarhizium pinghaense | 76.47 |
Ba-NCHU-113 | Beauveria australis | 82.35 |
Bb-NCHU-141 | Beauveria bassiana | 88.24 |
Bb-NCHU-143 | Beauveria bassiana | 88.24 |
Pl-NCHU-152 | Purpureocillium lilacinum | 35.29 |
Bb-NCHU-153 | Beauveria bassiana | 82.35 |
Bb-NCHU-157 | Beauveria bassiana | 88.24 |
Mb-NCHU-196 | Metarhizium baoshanense | 76.47 |
Mb-NCHU-197 | Metarhizium baoshanense | 88.24 |
Cc-NCHU-213 | Cordyceps cateniannulata | 94.12 |
Bb-NCHU-286 | Beauveria bassiana | 100.00 |
Table 3: Corrected mortality rates of 13 EPF isolates against mustard aphids at 5 days post inoculation (d.p.i.).
Isolate | Species | Conc.(conidia/mL) | N* | LT50 (days)† | 95% confidence limits | Slope (SE) | X2 (df)‡ |
Mb-NCHU-197 | Metarhizium baoshanense | 104 | 60 | 3.816 a | 3.61–4.05 | 0.60 (0.05) | 15.64 (28) |
105 | 60 | 3.112 bd | 2.95–3.28 | 0.72 (0.05) | 14.61 (28) | ||
106 | 60 | 2.908 b | 2.76–3.06 | 0.85 (0.06) | 15.04 (28) | ||
107 | 60 | 2.549 c | 2.40–2.69 | 0.90 (0.06) | 24.31 (28) | ||
Cc-NCHU-213 | Cordyceps cateniannulata | 104 | 60 | 3.948 a | 3.71–4.23 | 0.53 (0.05) | 8.81 (28) |
105 | 60 | 3.237 d | 3.07–3.41 | 0.72 (0.05) | 22.86 (28) | ||
106 | 60 | 2.414 c | 2.28–2.54 | 1.08 (0.07) | 28.30 (28) | ||
107 | 60 | 2.132 e | 2.02–2.25 | 1.41 (0.10) | 28.96 (28) | ||
*Number of insects observed. | |||||||
†LT50 values marked with different letters were considered significantly different as their 95% confidence limits did not overlap. | |||||||
‡X2, Chi-square value in Pearson's goodness-of-fit test; df, degrees of freedom |
Table 4: LT50 values of fungal isolates Mb-NCHU-197 and Cc-NCHU-213 against mustard aphids under different conidia concentrations. *Number of insects observed. †LT50 values marked with different letters were considered significantly different as their 95% confidence limits did not overlap. ‡X2, Chi-square value in Pearson's goodness-of-fit test; df, degrees of freedom.
Isolate | Species | N* | LC50 (conidia/mL)† | 95% confidence limits | Slpoe (SE) | X2 (df)‡ |
Mb-NCHU-197 | Metarhizium baoshanense | 240 | 2.30 × 105 a | 8.63 × 104–5.70 × 105 | 0.43 (0.08) | 10.14 (10) |
Cc-NCHU-213 | Cordyceps cateniannulata | 240 | 9.32 × 104 a | 4.97 × 104–1.62 × 105 | 0.76 (0.09) | 4.33 (10) |
*Number of insects observed. | ||||||
†LC50 values marked with different letters were considered significantly different as their 95% confidence limits did not overlap. | ||||||
‡X2, Chi-square value in Pearson's goodness-of-fit test; df, degrees of freedom |
Table 5: LC50 values of fungal isolates Mb-NCHU-197, and Cc-NCHU-213 against mustard aphids. *Number of insects observed. †LC50 values marked with different letters were considered significantly different as their 95% confidence limits did not overlap. ‡X2, Chi-square value in Pearson's goodness-of-fit test; df, degrees of freedom.
Crucifers, a group of vegetables, are frequently infested by multiple aphid species, including mustard aphid (L. erysimi) and cabbage aphid (Brevicoryne brassicae)26. Both species have been reported in Taiwan27, and it is possible for them to coexist at the collection site. To distinguish closely related aphid species, this study employed a molecular identification technique using a multiplex primer set21. By designing a molecular marker from the mustard aphid COI gene fragment, we successfully identified the mustard aphid, thus confirming the reliability of the molecular marker A05Le for this purpose21.
The observations revealed that distinguishing between apterous fourth-instar nymphs and adult mustard aphids based solely on their size or morphology is challenging without experience. However, a crucial distinguishing feature is the tibia of the hind legs, which appears white in fourth-instar nymphs but not in adults (Figure 3). Previous studies on L. attenuatum, B. bassiana against cotton aphids, and M. brunneum against green peach aphids have demonstrated that molting can be a strategy to avoid infection28,29,30. Therefore, misidentifying aphid stages can lead to errors in virulence assessment, as molting can affect the effectiveness of EPF28,29,31. To address this issue and ensure greater accuracy and precision in virulence evaluation, exclude aphids with whitish portions on their hind leg tibiae that have not reached the adult stage unless intentionally using aphid nymphs. These aphids do not fully mature and may undergo molting, which could allow them to escape the infection process of EPF. Additionally, a time course of observation and data recording every 12 h is recommended. The 12 h interval is preferable for demonstrating differences between multiple promising isolates with similar aphid-killing abilities and facilitates the precise calculation of LT50. However, the time interval can be adjusted based on the different life cycles of other insect species or determined through a small-scale pretest.
Although the detached-leaf method may leave minimal honeydew on the leaf disc, most of the honeydew adheres to the Petri dish cover since the leaf disc is in close proximity. It may be wiped or washed away during the observation period. However, honeydew is undoubtedly present in the practical environment of crop cultivation when aphids invade32. The honeydew present in the inoculation chamber may somewhat simulate the situation when EPF is inoculated into aphids in the field. Aphid cuticles mostly contain honeydew, which serves as a source of nutrients for the fungus and may stimulate the germination of EPF16. Thus, the combination of EPF application and honeydew production by aphids can be advantageous.
To confirm that aphid death is due to infection, cadavers are typically transferred from the EPF inoculation chamber to damp filter paper or a culture medium for observing fungal outgrowth11,14,33,34. However, in this study, water agar was used in the inoculation chamber to maintain high humidity, allowing the aphid cadavers to remain on the leaves without needing to be moved for fungal outgrowth observation. Although the detached-leaf method provides information on the aphidicidal effect of EPF isolates, it still has limitations. The water agar used to maintain the leaf disc condition can cause aphids to become stuck while moving in the inoculation chamber. To address this issue, the percentage of water agar was increased to 3%, providing a firm enough surface for Russian wheat aphids (Diuraphis noxia) to walk on20. In this experiment, mustard aphids were able to move comfortably on water agar surfaces with concentrations ranging from 1.5% to 3%. However, as the observation period progressed, a few aphids became stuck upside down with their legs upward and had to be rescued back to the leaf disc. This may be due to the differences in aphid size or mobility, indicating that increasing the agar concentration alone may not solve the problem. Therefore, ensure that the leaf disc fully covers the agar surface by using a leaf that fits more snugly to the Petri dish size, which can help alleviate this issue. In comparison to the detached-leaf method described by Yokomi and Gottwald18, this study has improved upon one aspect by using a leaf size that is approximately a good fit for the Petri dish. This allows for relative quantification of food consumption and minimizes the exposed agar surface. Additionally, the leaf needs to be "embedded" in the semi-solidified water agar, whereas in the original reference, it was described as "placed" on the water agar18. Embedding the leaf disc into the water agar reduces the likelihood of aphids getting stuck to the agar surface and facilitates observation, as the aphids are confined to one side.
This protocol provides detailed instructions on how to proceed with the detached-leaf method and EPF inoculation, along with some modifications that have facilitated operations, observations, and consistent results. It also establishes a standardized method for selecting EPF isolates against plant-sucking pests under laboratory conditions. Furthermore, conducting a standard check with known effective or commercially available products can be performed for the future commercialization of EPF isolates.
The authors have nothing to disclose.
This research was supported by 109-2313-B-005 -048 -MY3 from the Ministry of Science and Technology (MOST).
10 μL Inoculating Loop | NEST Scientific | 718201 | |
100 bp DNA Ladder III | Geneaid | DL007 | |
2x SuperRed PCR Master Mix | Biotools | TE-SR01 | |
50 mL centrifuge tube | Bioman Scientific | ET5050-12 | |
6 cm Petri dish | Alpha Plus Scientific | 16021 | |
6 mm insect aspirator | MegaView Science | BA6001 | |
70 mm filter paper NO.1 | Toyo Roshi Kaisha | ||
70% ethanol | |||
9 cm Petri dish | Alpha Plus Scientific | 16001 | |
Agar | Bioman Scientific | AGR001.1 | Microbiology grade |
Agarose | Bioman Scientific | PB1200 | |
BioGreen Safe DNA Gel Buffer | Bioman Scientific | SDB001T | |
Chromas | Technelysium | ||
GeneDoc | |||
GenepHlow Gel/PCR Kit | Geneaid | DFH300 | https://www.geneaid.com/data/files/1605861013102532959.pdf |
Gene-Spin Genomic DNA Isolation Kit | Protech Technology | PT-GD112-V3 | http://www.protech-bio.com/UserFiles/file/Gene-Spin%20Genomic%20DNA%20Kit.pdf |
Hemocytometer | Paul Marienfeld | 640030 | |
Komatsuna leaves (Brassica rapa var. perviridis) | Tai Cheng Farm | 1-010-300410 | |
Microsprayer | |||
MiniAmp Thermal Cycler | Thermo Fisher Scientific | A37834 | |
Mustard aphid (Lipaphis erysimi) | |||
Painting brush | Tian Cheng brush company | 4716608400352 | |
Parafilm M | Bemis | PM-996 | |
Pellet pestle | Bioman Scientific | GT100R | |
Sabouraud Dextrose Broth | HiMedia | MH033-500G | |
SPSS Statistics | IBM | ||
TAE buffer 50x | Bioman Scientific | TAE501000 | |
Tween 80 | PanReac AppliChem | 142050.1661 |