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Article

Characterisation of the Pathogenicity of Beauveria sp. and Metarhizium sp. Fungi Against the Fall Armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae)

by
Nonthakorn (Beatrice) Apirajkamol
1,2,
Bishwo Mainali
1,
Phillip Warren Taylor
1,
Thomas Kieran Walsh
1,2 and
Wee Tek Tay
1,2,*
1
Applied BioSciences, Macquarie University, Sydney, NSW 2109, Australia
2
Black Mountain Laboratories, Commonwealth Scientific and Industrial Research Organisation, Canberra, ACT 2601, Australia
*
Author to whom correspondence should be addressed.
Agriculture 2025, 15(2), 170; https://doi.org/10.3390/agriculture15020170
Submission received: 20 November 2024 / Revised: 20 December 2024 / Accepted: 9 January 2025 / Published: 14 January 2025
(This article belongs to the Section Crop Protection, Diseases, Pests and Weeds)
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Abstract

:
Previously, we assessed the pathogenicity of eleven endemic entomopathogenic fungi (EPF), including six Beauveria isolates, four Metarhizium isolates, and one M. pingshaense, against the agricultural pest Spodoptera frugiperda (fall armyworm, FAW). We found that four Beauveria and one Metarhizium isolates were effective, with Beauveria isolates B-0571 and B-1311 exhibiting high mortality within 24 h post-spore application. This study aimed to identify and characterise the entomopathogenesis mechanisms of these isolates as potential FAW biocontrol agents. All Beauveria isolates were determined as B. bassiana, the Metarhizium isolates as two M. robertsii, one M. majus, and an unknown Metarhizium species. Despite the high mortality from B-0571 and B-1311 isolates, scanning electron microscopy showed no fungal spore germination on dead larvae 24 h after spore application. Four insecticide compound gene clusters, i.e., bassianolide, beauvericin, beauveriolide, and oosporein, were identified and characterised in all B. bassiana isolates. These compounds are hypothesised to contribute to the high early mortality rates in FAWs. Identifying and characterising gene clusters encoding these insecticide compounds in B-0571 and B-1311 will contribute to a better understanding of the entomopathogenicity of these isolates that will be vital to developing these EPF isolates as sustainable alternative FAW biocontrol agents.

1. Introduction

The fall armyworm (Spodoptera frugiperda, FAW) is an invasive noctuid moth (Lepidoptera) that was first reported in Australia early in 2020 [1]. The FAW is highly polyphagous [2,3,4], causing significant damage to economically important crops through larval feeding on leaves and fruit [5]. Consequently, the introduction of the FAW poses a considerable risk to Australia’s agriculture, including sorghum, wheat, cotton, sugarcane, and various vegetables [6,7,8]. To reduce reliance on broad-spectrum synthetic insecticides, there is substantial interest in potential biocontrol options as part of an integrated pest management (IPM) strategy to help mitigate damage from the FAW.
Entomopathogenic fungi (EPF) represent a group of fungi that infect and kill insect hosts and are valuable resources for natural pest control [9,10]. EPF are ubiquitous in nature, and more than 18,000 species have been reported to exhibit entomopathogenesis [11]. Species in the order Hypocreales, including the genera Beauveria and Metarhizium, are particularly well-recognised for their biocontrol potential and applications in sustainable agricultural practices [12]. This is due to the simplicity of their culture media and growth conditions, as well as their effectiveness [13]. The general mode of action of EPF in the order Hypocreales starts with infection of insects topically through the cuticle [14]. Once inside, the fungi proliferate throughout the insect’s body, absorbing nutrients required for their growth, which leads to the death of the insect host [15,16,17]. The EPF then emerges from the host’s carcass, sporulates, and the cycle repeats [17]. Incorporating EPF as part of IPM in agriculture could foster sustainability and serve as an alternative to chemical insecticides, addressing the challenges of pesticide resistance, insecticide residues on produce, and environmental contamination.
In addition to the ability to parasitise host species, the production of insecticidal bioactive compounds could also be a crucial factor in the virulence of EPF. A wide range of insecticide compounds is known to be produced by fungi in the genus Beauveria, including beauvericin [18], bassiacridin [19], bassianolide [20], dipicolinic acid [21], beauveriolide [22], and more [18,23,24]. Some of these compounds, including beauvericin [18,23], bassiacridin, and bassianolide [20], have shown efficacy against the FAW. For example, beauvericin could reduce the SF-9 (FAW, cell line) cell population by 50% within 48 h, even when exposed to a low dosage (2.5 ± 0.5 μM) [23]. In addition, other metabolites, i.e., oosporein, can suppress the immunity of insects [21,25,26,27], which assists other metabolites or the EPF itself to synergistically take over the insect host’s body.
In our previous study [28], eleven entomopathogenic fungal isolates were tested against FAWs at various life stages. Two Beauveria isolates, namely B-0571 and B-1311, exhibited high efficacy, causing 73.96  ±  7.85% and 62.08  ±  3.67% mortality in 3rd instar FAW larvae within 24 h [28]. Given the rapidity of larval mortality, it was hypothesised that the mode of action of these isolates might be attributed to the production of insecticidal compounds rather than direct parasitism. Therefore, the objective of this study is to test this hypothesis and to identify and characterise the compounds produced by these two highly virulent Beauveria isolates, B-0571 and B-1311, that are lethal to the FAW.

2. Materials and Methods

2.1. Fungal Isolates

Eleven isolates of entomopathogenic fungi were chosen from the CSIRO fungal collection for their efficacy against the FAW [28]. This includes six isolates of Beauveria species (B-0016, B-0077, B-0079, B-0571, B-0698, and B-1311), four isolates in the genus Metarhizium (M-0121-0123 and M-0999), and one isolate of M. pingshaense (M-1000, JBCAUT000000000). Fungal host species, geographic origins, and collection dates have been previously reported [28].
Fungal isolates were revived and maintained in Sabouraud dextrose agar media with 1% yeast extract (SDAY; pH 5.6) and incubated at 28 ± 1 °C under dark conditions. For further investigation, once fungi have fully sporulated, spores were collected following the protocol as described in [28].

2.2. Genomic DNA Extraction and Whole Genome Sequencing

High molecular weight genomic DNA of fungal isolates was extracted using a protocol from [29]. The quality of extracted DNA was quantified using a Qubit 2.0 fluorometer (Life Technologies Corporation, Carlsbad, CA, USA), gel electrophoresis, and NanoDrop® (Thermo ScientificTM, Waltham, MA, USA). The extracted DNA was submitted to Genomics WA, Western Australia, for library preparation and long-read sequencing using PacBio HiFi Sequel® ll sequencer (Menlo Park, CA, USA) with SMRTBell technology.

2.3. Bioinformatics Analysis

PacBio HiFi reads were de novo assembled using Canu assembler with default parameter [30] https://github.com/marbl/canu (accessed on 28 March 2024). The quality of assembly was analysed through BUSCO (Benchmarking Universal Single-Copy Orthologs) [31] https://busco.ezlab.org/ (accessed on 30 April 2024) with hypocreales_odb10 (the order of Beauveria and Metarhizium genera) dataset and QUAST (QUality ASsessment Tool) [32] https://github.com/ablab/quast (accessed on 4 October 2024).

2.4. Species Identification and Phylogeny

The genera of the fungal isolates were previously identified through morphology and internal transcribed spacer (ITS) sequencing [28]. However, a sole ITS region has insufficient resolution power to identify species of fungi from the genera Beauveria and Metarhizium [28,33,34]. Therefore, a Multi Locus Sequence Typing (MLST) approach was carried out to aid the identification process [33,34].
To identify the species of Beauveria isolates, the sequences of four DNA markers, i.e., B locus nuclear intergenic region (Bloc), the RNA polymerase II largest (RPB1) and second largest (RPB2) subunits, and translation elongation factor (TEF), were used for MLST analysis. For the Metarhizium isolates, seven DNA markers, i.e., DNA lyase (APN2), beta tubulin (BTUB), RPB1a, RPB1b, RPB2a, RPB2b, and TEF, were used. The marker sequences were acquired from the whole genome sequence. The reference sequences were obtained from [33] for Beauveria and [34] for Metarhizium and NCBI databases.
Sequences of twenty reference Beauveria isolates (six species, Table S1) and twenty-two taxa of ten species within Metarhizium were used (Table S2). The marker sequences were aligned using MUSCLE with default parameters through Geneious Prime 2023.2.1 (Muscle 5.1 with algorithm: PPP). Maximum likelihood phylogenetic placements of concatenated sequences were constructed through the IQ-TREE web server http://iqtree.cibiv.univie.ac.at/ (accessed on 5 April 2024) with an auto selection for the optimal substitute model and 1000 ultrafast bootstrap replications [35] to estimate node confidence. The phylogenetic trees were modified via iTOL v.6 <https://itol.embl.de/> (accessed on 5 April 2024) [36].

2.5. Insect Samples and Insect Bioassay

The virulence of the two highly virulent fungal isolates B-0571 and B-1311 was tested on a lab-maintained FAW colony, originally established from 30 field-collected pupae collected from a field station owned by the University of Queensland (Rex Road, Walkamin, QLD, Australia) [37].
The insect bioassay was conducted following the protocol in [28]. 32 3rd instar FAW larvae were treated with 0.1% Tween 80® solutions as control samples and spore suspension of B-0571 or B-1311 at spore concentration ≥ 107 conidia/mL as fungal-treated samples. Three caterpillars were collected from day 1 to day 7 (24 h following treatment = day 1) and preserved in 3:1 ethanol-acetic acid. The samples were stored at room temperature (21 ± 1 °C) for further investigation.

2.6. Microscopic Analysis

The two most highly virulent fungal candidates, B-0571 and B-1311, were microscopically assessed. Subsequently, the exterior morphology of the samples was visualised at the CSIRO Black Mountain microImaging Centre.
For investigations of external morphology, scanning electron microscopy (SEM) was used. Samples were critical point dried (Autosamdri®-931, Tousimis, Rockville, MD, USA) and mounted on aluminium stubs with double-sided adhesive carbon tabs (IA0201, ProSciTech, Kirwan, QLD, Australia). The samples were then imaged through an SEM (ZEISS EVO LS 15, ZEISS Microscopy (Oberkochen, Germany)) with 10 kV accelerating voltage, 10 Pa vacuum, and a backscattered electron detector.
For internal morphology examination, samples were rehydrated through a series of decreasing ethanol solutions (70, 50, and 25%) each for 15 min. The samples were then cleared using 10% (w/v) potassium hydroxide (KOH) at 85 °C for 5–15 min, with the treatment time varied based on sample size. The treatments ended when samples had optical clarity while also maintaining structural integrity. The samples were then gently washed three times in distilled water, each time for 10 min. Subsequently, the samples were stained by soaking in 20 μg/mL of wheat germ agglutinin conjugated with tetramethylrhodamine isothiocyanate-dextran (WGA-TRITC, W849, Thermo Fisher Scientific) at room temperature for 30 min with occasional gentle agitation. The samples were washed twice in distilled water and were mounted using glass slides and coverslips. Widefield images were obtained using an optical microscope (ZEISS AxioImager Z1, ZEISS Microscopy) equipped with white and fluorescence light emitting diode illumination (Colibri 7), ZEISS Axiocam 712 colour charge-coupled device camera (ZEISS Microscopy) and a plan-apochromat 10 × NA = 0.3 and 20 × NA = 0.5 objectives. ZEISS filter sets 43 HE (DsRed, excitation BP550/25; beam splitter FT 570 HE; emission BP605/70 HE) and 02 (DAPI, excitation 365; beam splitter 395; emission LP 420) filters were used to visualise WGA-TRITC and background tissue autofluorescence, respectively. Image capture and post-acquisition image processing were carried out using ZEN blue v3.2 (ZEISS Microscopy).

2.7. Biosynthesis Gene Cluster Prediction

Biosynthesis gene clusters (BGCs) of fungal isolates were predicted from their genome via AntiSMASH–fungal version 7.0.1 (Antibiotics & Secondary Metabolite Analysis Shell [38]), available at <https://fungismash.secondarymetabolites.org> (accessed on 13 October 2023). The detective strictness of the prediction was set to ‘relaxed’, and all extra features (e.g., KnownClusterBlast and MIBiG cluster comparison) were activated. Consequently, the results were manually assessed to verify the accuracy of the prediction.

3. Results

3.1. Species Identification

The genomes of ten isolates were assembled, with the average sizes ranging from 37 to 39 Mb for Beauveria isolates and 42 to 45 Mb for Metarhizium isolates, both showing BUSCO completions of 96.9–97% and 96.7–97.5%, respectively (Table S3). An exception was M-0999, whose genome was estimated to be larger at 101 Mb and exhibited a high number of duplications, with 88.5% duplicated BUSCOs. Consequently, it is believed that M-0999 possesses a diploid genome with heterozygosity.
To identify the species of six Beauveria isolates, a phylogenetic tree was constructed using MLST of four commonly used DNA markers. The analysis showed that these Beauveria isolates are closely related to eight reference sequences of B. bassiana, as they were grouped in the same clade. This grouping demonstrated high node confidence levels of 100%, which is highlighted in Figure 1 with a red box.
The phylogenetic placement established from seven DNA markers was used to help identify species of four Metarhizium isolates. The analysis clustered M-0121 and M-0122 with the four M. robertsii reference sequences with strong bootstrap support (99%, Figure 2, indicated in red box). The M-0123 isolate was placed as a sister branch to M. guizhouense but with only 63% node support (Figure 2, highlighted in yellow). Finally, for M-0999, it was clustered with three isolates of M. majus with 100 node confidence (Figure 2, in the blue box).

3.2. Microscope Analysis

3.2.1. External Morphology Analysis

To better understand the pathogenicity of highly virulent B. bassiana isolates towards the FAW, the external morphology of control and fungus-treated samples was observed. The results suggest that no spore germination occurred within 24 h post-treatment. While there were no fungal spores detected in the control samples (Figure 3a,b), a substantial number of fungal spores were visible on the surface of the fungus-treated samples (Figure 3c–e). The spores were mostly found on the bodies of insects (thorax and abdomen), with fewer on the heads. The fungal spores were located around the spiracles (external respiratory pores of the caterpillars, Figure 3f); however, they did not appear to obstruct the spiracles. In addition, the fungal spores appear to be much smaller than insect spiracles.
Seven days following the fungal treatments, the bodies of the insects were covered in hyphae, which could be seen easily by the naked eye. The germination of spores and signs of parasitism were evident throughout the whole insect (Figure 3f–h). On day 7 following the treatment, control samples developed into 5th–6th instar caterpillars, displaying a noticeable size difference when compared to the treated samples. Therefore, the control for the 7-day fungus-treated samples was collected from the first 24 h post-treatment (Figure 3a,b). For the fungus-treated samples (Figure 3f–h), signs of fungal germination and parasitism were detected throughout the bodies of insects, including the head. However, the hyphae appeared to be located mostly on the bodies.

3.2.2. Internal Morphology Analysis

To locate signs of fungal germination and parasitisation, the internal morphology of treated third instar caterpillars was examined using an optical microscope. The control sample is shown in Figure 4 (Figure 4a, before clearing; Figure 4b–d, after clearing), while the fungal-treated sample is shown in Figure 4e. Spots in the digestive tract of 24 h fungal-treated samples (Figure 4e) were observed, but they did not appear in the control samples. These spots were suspected to be fungal spores, although the image resolution was not sufficiently high enough to enable better confirmation or to determine if early spore germination processes had commenced. Hence, control and fungus-treated samples were stained with WGS-TRITC to assist with confirmation.
The WGS-TRITC fluorescent dye stained both the insect cuticle and fungal spore cell walls in control (Figure 5a,b) and fungus-treated samples (Figure 5c,d). Distinct spots appeared in the digestive tracts of fungus-treated caterpillars (Figure 5c,d) but were absent in controls (Figure 5a,b). As such, these spots are believed to be fungal spores. However, no signs of germination were detected in the 24 h fungus-treated samples (Figure 5d).

3.3. Biosynthesis Gene Clusters Prediction

There are 43–51 biosynthesis gene clusters (BGCs) recognised in six B. bassiana isolates and 56–111 in five Metarhizium isolates. Although many remain unknown, 13–16 of the BGCs from B. bassiana and 24–42 from Metarhizium spp. share similarities with reference clusters from the Minimum Information about a Biosynthesis Gene cluster (MIBiG) database. The clusters that share more than 50% similarity with the reference clusters from the MIBiG database are listed in Table S4.

3.3.1. Beauveria bassiana

Among the known BGCs found in tested B. bassiana isolates, there is evidence suggesting that some possess insecticide properties. This includes bassianolide, beauvericin, and beauveriolide. Although oosporein does not exhibit insecticidal properties, it has been shown to suppress the immune response in insects [25], which is also discussed further below.

Bassianolide

A bassianolide gene cluster was found in all tested B. bassiana isolates, with their entire cluster sharing a similarity of 53–66% to a reference gene cluster (BGC0000312 from B. bassiana, Table 1). All these clusters contained a core biosynthesis gene for bassianolide production (bsls), with the B. bassiana isolates exhibiting 85–97% similarity to the reference bsls.

Beauvericin

All tested B. bassiana isolates also possess one beauvericin gene cluster, with the clusters exhibiting 70–90% similarity to the MIBiG-reported cluster (BGC0000313 from B. bassiana, Table 1). The core biosynthesis gene, beauvericin nonribosomal cyclodepsipeptide synthetase (Beas), is found in all B. bassiana isolates, showing 88–97% similarity.

Beauveriolide B/C/D

The gene cluster for beauveriolide B/C/D was recognised in all tested B. bassiana isolates, the whole cluster exhibiting similarities ranging from 66% to 83% compared to a reference cluster (BGC0002203 from B. bassiana, Table 1). The beauveriolide B/C/D gene cluster contained two core biosynthesis genes, BesA and BesB. These two genes were detected in all B. bassiana isolates. The BesA of three isolates including B-0077, B-0079, and B-1311, however, appears to be inserted by an unknown sequence (132 nucleotides (nt), Figure 6). The similarities of the nrps in B. bassiana isolates to the reported gene ranges from 81% to 97%, while it is 95 to 96% for the BesB gene.

Oosporein

All tested B. bassiana isolates possessed an oosporein gene cluster. While not identical, they all shared 85% similarity across the entire cluster compared to the reference cluster (BGC0001720 from B. bassiana, Table 1). For the core biosynthesis gene, they shared 89–92% similarity compared to the reference gene. However, B-0077 and B-1311 had an incomplete transport gene, and it remains to be investigated whether this would impact oosporein production.

3.3.2. Metarhizium Species

Among the fungal isolates, four distinct species of Metarhizium were identified (Figure 2; see also [28]), with each exhibiting considerable variance in their BGCs. In this study, our focus was on one specific isolate, M-0121 (M. robertsii), because of the highest efficacy demonstrated against the FAW when compared to other tested Metarhizium isolates [28]. Two insecticide compound gene clusters, i.e., destruxin and enniatin, were identified in M-0121 and will be further discussed below.

Destruxin

A destruxin (Dtx) gene cluster is found only in the M-0121 genome, with the whole cluster sharing 76% similarity to the reported cluster (BGC0000337 from M. robertsii). BGC0000337 has two core biosynthesis genes, i.e., nrps GliP-like and DtxS1. However, only DtxS1 is found in M-0121 with 98% similarity (Figure 7). In addition, the core biosynthesis genes DtxS2, DtxS3, and DtxS4 are also missing in the M-0121 isolate.

Enniatin

A reported enniatin gene cluster (BGC0000342 from Fusarium equiseti) contains only one gene: esyn1. Although esyn1 is found in four isolates of Metarhizium, including M-0121, M-0122, M-0999, and M-1000, esyn1 in these isolates contains less than half the length of the reported esyn1 (Figure 8). Moreover, the percentage of similarity on their core biosynthesis gene is only 51–52%. Therefore, enniatin gene clusters in Metarhizium isolates are incomplete, and it is hypothesised that they might not be able to produce enniatin.

4. Discussion

4.1. Species Identification

We inferred the six Beauveria isolates (i.e., B-0016, B-0077, B-0079, B-0571, B-0698, and B-1311) to likely represent different isolates of B. bassiana based on MLST analysis.
The phylogenetic results suggest that there are four species within tested Metarhizium isolates. M-0121 and M-0122 are grouped with various isolates of M. robertsii, which suggests that these two isolates are also likely to be M. robertsii. The species of M-0123, however, remained uncertain since M-0123 is the only sample currently found to be basal to M. guizhouense but divergent from M. indigotica. More sequences (which currently are not available) from other closely related species will be required to better define the species status of the M-0123 isolate. Finally, the isolate M-0999 is likely to be M. majus, as it grouped with three other isolates of M. majus.

4.2. Microscope Analysis

The external and internal morphology of dead B-0571 or B-1311-treated samples indicates that no fungal germination occurred within 24 h post-fungal treatment. While many fungal spores were detected on the surface of caterpillars, they did not appear to obstruct insect spiracles. Hence, the cause of insect death is unlikely to be due to fungal parasitisation or hypoxia. We hypothesise that these fungal candidates produce bioactive compounds with insecticidal properties harmful to FAW larvae. In addition, optical white and fluorescence light microscopy was used to observe the internal morphology of caterpillars. Spores from B-0571 and B-1311 were observed in the digestive tracts of caterpillars, suggesting that these insecticidal bioactive compounds may require oral ingestion to be effective. This hypothesis is supported by the lack of effectiveness of B-0571 and B-1311 against pupae and adults in the first 24 h [28], as these later instar stages have lower to no consumption of food.

4.3. Biosynthesis Gene Cluster Prediction

To better understand the genomic basis of high virulence observed in the previously tested fungal isolates, including the production of compounds harmful toward FAWs, their BGCs were annotated and characterised. Our findings indicated that four insecticidal BGCs were recognised in all tested B. bassiana isolates. This includes bassianolide, beauvericin, beauveriolide B/C/D, and oosporein. At least one of these compounds is speculated to be expressed in high-virulence isolates (i.e., B-0571 and B-1311). In the case of B-0077, B-0079, and B-1311, the besA gene within the beauveriolide B/C/D gene cluster was shown to be disrupted. This cluster contained two main biosynthetic core genes, namely besA and besB. The research by Yin et al. [39] indicated that these genes were likely responsible for producing different analogues of beauveriolide. In addition, there are no studies comparing the virulence of ΔbesA mutants to the wild type. Therefore, the effects of besA disruption on B-0077, B-0078, and B-1311, such as whether these isolates still express functional BesA, and how the absence of BesA might affect the virulence of these isolates, remained to be investigated. Furthermore, the function of more than half of BGCs predicted in B. bassiana remained unidentified. The remaining possibility is that some of these could encode compounds with insecticide properties.
For M-0121, the isolate with the highest virulence among tested Metarhizium isolates, two insecticide compound gene clusters were detected: Dtx and enniatin. However, the enniatin gene clusters appeared to be incomplete, and they were therefore hypothesised to either not produce enniatin or possibly produce unknown compounds instead. The Dtx gene cluster appeared to be different from the reference gene cluster, missing one of the core genes and many additional biosynthesis genes. The work of Wang and colleagues [40] suggests that the absence of DtxS2, DtxS3, and DtxS4, which encode cytochrome P450, aldo/keto reductase, and decarboxylase enzymes, respectively, affects Dtx production by reducing the ability to convert Dtx B to Dtx A, C, D, and E, and significantly reduced its virulence against insects. However, the effect of the missing nrps GliP-like gene is still unknown. Therefore, the production of Dtx B in M-0121 remains unknown. In addition, as with the B. bassiana isolates, there may be unidentified insecticide compounds produced by M-0121. Alternatively, the virulence of M-0121 might not be contingent on insecticidal compounds but rather on another factor yet to be determined.
Herein, six Beauveria species and four Metarhizium species were identified. All Beauveria isolates were identified as B. bassiana. Among the Metarhizium isolates, two were identified as M. robertsii, one as M. majus, and one as an unknown species. We analysed the entomopathogenicity of two highly effective B. bassiana isolates (i.e., B-0571 and B-1311) using microscopy, with results showing no germination of fungi in the dead, fungus-treated FAW caterpillars after 24 h. The absence of fungal germination indicated that the virulence of the fungal candidates likely depended on factors other than parasitisation. These factors may include the production of compounds with insecticidal properties. Annotation and characterisation of the predicted secondary metabolite gene clusters in these fungal isolates identified four insecticidal compound gene clusters in all B. bassiana isolates. At least one of these compounds was hypothesised to contribute to the virulence of B-0571 and B-1311. Further research, such as transcriptomic, metabolomic, or proteomic studies, will be necessary to confirm this hypothesis and to better understand the virulence mechanisms of these fungal isolates.
Apart from isolates M-0121 and M-0122, the other Metarhizium isolates were different species and may exhibit varying levels of virulence toward FAWs. Additionally, an insecticide gene cluster (i.e., Dtx) was found only in M-0121, which exhibited the highest efficacy among the Metarhizium isolates. The expression of the Dtx gene cluster is therefore hypothesised to underpin the superior efficacy of M-0121 compared to the other Metarhizium isolates.

5. Conclusions

The discovery and characterisation of two highly virulent EPFs targeting FAWs opens promising new avenues for the development of sustainable pest management tools. A better understanding of their entomopathogenic properties allows these isolates to be applied directly, or their derived toxins can be utilised as biocontrol agents. These can be employed as standalone treatments or integrated with existing pest management approaches. This advancement holds the potential to provide more effective and sustainable solutions to mitigate the impact of FAWs on crops, ultimately leading to improved crop productivity and quality.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agriculture15020170/s1. Table S1: Information on Beauveria species, including their origin, host, and accession numbers, used in this study; Table S2: Strains used in the Metarhizium phylogenetic analysis, including geography, host, and GenBank numbers; Table S3: Summary of genome assembly of the fungal isolates; Table S4: The biosynthesis gene clusters of the fungal candidates and their similarity to MIBiG reference clusters.

Author Contributions

Conceptualisation, N.A., W.T.T., T.K.W., B.M. and P.W.T.; Methodology N.A., W.T.T. and T.K.W.; Software, N.A.; Validation, W.T.T.; Formal analysis, N.A.; Investigation, N.A.; Resources, T.K.W. and W.T.T.; Data Curation, N.A. and T.K.W.; Writing—Original Draft Preparation, N.A.; Writing—Review and Editing, W.T.T., B.M., P.W.T. and T.K.W.; Visualisation, N.A.; Supervision, W.T.T., B.M., P.W.T. and T.K.W.; Project Administration, N.A. and W.T.T.; Funding Acquisition, T.K.W. All authors have read and agreed to the published version of the manuscript.

Funding

The fungal genomes were sequenced with support from the CSIRO Applied Genomics Initiative (awarded to T.K.W.). This project was part of an N.A.’s PhD funded by MQ through the International Macquarie University Research Excellence Scholarship (iMQRES). The project was completed with support from the CSIRO Health and Biosecurity and Environment. The full waiver of the article processing charge for this article was awarded to W.T.T. by Agriculture (Voucher: cf1c2c48cfb2a338).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The genomes of fungal isolates are available at NCBI under BioProject (PRJNA1153139). Bioinformatics scripts for Section 2.3 are available on request to the first author.

Acknowledgments

Leon Court (CSIRO) helped with sample submissions for sequencing. Cecile Gueidan (CSIRO) provided guidance on phylogenetic analysis. Timothy Hogarty (CSIRO) and Ray Yang (Murdoch University/CSIRO) helped take care of the insects. Phil Hands and Vivien Rolland (BMIC and CSIRO) provided discussions on experiment design and assisted with microscopy analysis.

Conflicts of Interest

Authors declare no conflicts of interest.

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Figure 1. Maximum likelihood phylogenetic analysis of six Beauveria isolates, including B-0016, B-0077, B-0079, B-0571, B-0698, and B-1311 (in bold). All six Beauveria isolates were clustered with eight B. bassiana reference sequences with high node confidence (100%).
Figure 1. Maximum likelihood phylogenetic analysis of six Beauveria isolates, including B-0016, B-0077, B-0079, B-0571, B-0698, and B-1311 (in bold). All six Beauveria isolates were clustered with eight B. bassiana reference sequences with high node confidence (100%).
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Figure 2. Maximum Phylogeny placements of four Metarhizium isolates (M-0121, M-0122, M-0123, and M-0999). Both M-0121 and M-0122 isolates were clustered with four M. robertsii reference sequences (indicated in the red box, 99% bootstrap value). The M-0123 isolate was placed as a solitary branch (highlighted in yellow), sharing 63% node confidence with the M. guizhouense sister clade. The M-0999 isolate was shown to group with three reference sequences of M. majus with 100% node support (shown in the blue box).
Figure 2. Maximum Phylogeny placements of four Metarhizium isolates (M-0121, M-0122, M-0123, and M-0999). Both M-0121 and M-0122 isolates were clustered with four M. robertsii reference sequences (indicated in the red box, 99% bootstrap value). The M-0123 isolate was placed as a solitary branch (highlighted in yellow), sharing 63% node confidence with the M. guizhouense sister clade. The M-0999 isolate was shown to group with three reference sequences of M. majus with 100% node support (shown in the blue box).
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Figure 3. External morphology of control (a,b) and fungus-treated (c–h) third instar FAWs. The living control samples (0.1% (v/v) Tween 80® solution) and the dead fungus-treated samples (B-0571 or B-1311 at spore concentration ≥ 107 conidia/mL) were collected from day 1 (24 h post-treatment, a–e) to day 7 (g–h). The samples were preserved in 3:1 ethanol-acetic acid, and the surface morphology of the insect body (a,c,f), head (b,d,g), spiracles (e), and close-up surface (h) was visualised using an SEM with 96–1.65 K× magnification. The red arrows indicate the hyphae/germination of B. bassiana spores.
Figure 3. External morphology of control (a,b) and fungus-treated (c–h) third instar FAWs. The living control samples (0.1% (v/v) Tween 80® solution) and the dead fungus-treated samples (B-0571 or B-1311 at spore concentration ≥ 107 conidia/mL) were collected from day 1 (24 h post-treatment, a–e) to day 7 (g–h). The samples were preserved in 3:1 ethanol-acetic acid, and the surface morphology of the insect body (a,c,f), head (b,d,g), spiracles (e), and close-up surface (h) was visualised using an SEM with 96–1.65 K× magnification. The red arrows indicate the hyphae/germination of B. bassiana spores.
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Figure 4. The internal morphology of control (a–d) and fungus-treated (e) third instar FAW larvae. The larvae were treated with 0.1% (v/v) Tween 80® solution for the control samples, or in a spore suspension of B-0571 or B-1311 isolates (spore concentration ≥ 107 conidia/mL) as fungal-treated samples. The samples were clarified with 10% KOH at 85 °C for 10–15 min. The internal morphology of the samples was captured before (a) and after (b–e) the clearing process with an optical microscope. The scale bar in figure (c) indicates 0.64 mm. The photos were captured with an optical microscope using white light. The suspect fungal spores in treated samples were indicated by a red arrow (e).
Figure 4. The internal morphology of control (a–d) and fungus-treated (e) third instar FAW larvae. The larvae were treated with 0.1% (v/v) Tween 80® solution for the control samples, or in a spore suspension of B-0571 or B-1311 isolates (spore concentration ≥ 107 conidia/mL) as fungal-treated samples. The samples were clarified with 10% KOH at 85 °C for 10–15 min. The internal morphology of the samples was captured before (a) and after (b–e) the clearing process with an optical microscope. The scale bar in figure (c) indicates 0.64 mm. The photos were captured with an optical microscope using white light. The suspect fungal spores in treated samples were indicated by a red arrow (e).
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Figure 5. Fluorescent images of WGA-TRITC staining of the control (a,b) and fungal-treated (c,d) third instar FAW larvae. Living third instar FAWs treated with 0.1% (v/v) Tween 80® solution (control samples) and dead caterpillars treated with spore suspension of B-0571 or B-1311 isolates (≥107 conidia/mL) were collected 24 h following the treatment (N = 3). The samples were clarified with 10% KOH and stained with WGA-TRITC. The fluorescence signal was captured with an optical microscope using ZEISS DsRed and DAPI filter sets. The stained spots of the fungal cell wall were indicated by white arrows in (c,d).
Figure 5. Fluorescent images of WGA-TRITC staining of the control (a,b) and fungal-treated (c,d) third instar FAW larvae. Living third instar FAWs treated with 0.1% (v/v) Tween 80® solution (control samples) and dead caterpillars treated with spore suspension of B-0571 or B-1311 isolates (≥107 conidia/mL) were collected 24 h following the treatment (N = 3). The samples were clarified with 10% KOH and stained with WGA-TRITC. The fluorescence signal was captured with an optical microscope using ZEISS DsRed and DAPI filter sets. The stained spots of the fungal cell wall were indicated by white arrows in (c,d).
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Figure 6. Comparison of the beauveriolide B/C/D gene clusters identified in the B. bassiana isolates (B-0016, B-0077, B-0079, B-0571, B-0698, and B-1311) against the reference beauveriolide gene cluster from MIBiG (BGC0002203 from B. bassiana). The beauveriolide B/C/D gene clusters are predicted from the genomes of B. bassiana isolates. The core biosynthesis genes are in red; additional biosynthesis genes are coloured in pink, and non-specific genes are shaded in grey. Source: modified from figure generated by antiSMASH 7.0.1.
Figure 6. Comparison of the beauveriolide B/C/D gene clusters identified in the B. bassiana isolates (B-0016, B-0077, B-0079, B-0571, B-0698, and B-1311) against the reference beauveriolide gene cluster from MIBiG (BGC0002203 from B. bassiana). The beauveriolide B/C/D gene clusters are predicted from the genomes of B. bassiana isolates. The core biosynthesis genes are in red; additional biosynthesis genes are coloured in pink, and non-specific genes are shaded in grey. Source: modified from figure generated by antiSMASH 7.0.1.
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Figure 7. The gene clusters for destruxin predicted from the genome of M-0121 and the MIBiG reference cluster BGC0000337 from M. robertsii are compared. The reference cluster was identified to have all the components required to produce destruxin (complete cluster). Additional synthesis genes are indicated in pink, core biosynthesis genes in red, and genes related to transportation in blue. Source: modified from figure generated by antiSMASH 7.0.1.
Figure 7. The gene clusters for destruxin predicted from the genome of M-0121 and the MIBiG reference cluster BGC0000337 from M. robertsii are compared. The reference cluster was identified to have all the components required to produce destruxin (complete cluster). Additional synthesis genes are indicated in pink, core biosynthesis genes in red, and genes related to transportation in blue. Source: modified from figure generated by antiSMASH 7.0.1.
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Figure 8. Comparison of the enniatin gene clusters predicted from genomes of four Metarhizium spp. (i.e., M-0121, M-0122, M-0999, and M-1000) with the Fusarium equiseti reference cluster BGC0000342 from MIBiG. Blue indicates genes related to transportation, pink denotes additional biosynthesis genes, and red represents core biosynthesis genes. Source: modified from figure generated by antiSMASH 7.0.1.
Figure 8. Comparison of the enniatin gene clusters predicted from genomes of four Metarhizium spp. (i.e., M-0121, M-0122, M-0999, and M-1000) with the Fusarium equiseti reference cluster BGC0000342 from MIBiG. Blue indicates genes related to transportation, pink denotes additional biosynthesis genes, and red represents core biosynthesis genes. Source: modified from figure generated by antiSMASH 7.0.1.
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Table 1. Insecticide compound gene clusters predicted in the fungal isolates.
Table 1. Insecticide compound gene clusters predicted in the fungal isolates.
Biosynthesis Gene ClustersCSIRO IsolatesOverall Similarity (%)Core Biosynthesis Gene Similarity (%)
123
Beauveria bassiana
BassianolideB-00165385
B-00776097
B-00795397
B-05716697
B-06986088
B-13115397
BeauvericinB-00167097
B-00778093
B-00797093
B-05717088
B-06987097
B-13118093
Beauveriolide B/C/DB-00166681 96
B-007783917195
B-007966978696
B-05718385 95
B-06986681 96
B-131183977196
OosporeinB-00168589
B-00778591
B-00798589
B-05718592
B-06988589
B-13118591
Metarhizium species
DestruxinM-012176-98-
EnniatinM-012110052
M-012210052
M-099910051
M-100010052
The bold characters indicate the genera of fungal isolates.
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Apirajkamol, N.; Mainali, B.; Taylor, P.W.; Walsh, T.K.; Tay, W.T. Characterisation of the Pathogenicity of Beauveria sp. and Metarhizium sp. Fungi Against the Fall Armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae). Agriculture 2025, 15, 170. https://doi.org/10.3390/agriculture15020170

AMA Style

Apirajkamol N, Mainali B, Taylor PW, Walsh TK, Tay WT. Characterisation of the Pathogenicity of Beauveria sp. and Metarhizium sp. Fungi Against the Fall Armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae). Agriculture. 2025; 15(2):170. https://doi.org/10.3390/agriculture15020170

Chicago/Turabian Style

Apirajkamol, Nonthakorn (Beatrice), Bishwo Mainali, Phillip Warren Taylor, Thomas Kieran Walsh, and Wee Tek Tay. 2025. "Characterisation of the Pathogenicity of Beauveria sp. and Metarhizium sp. Fungi Against the Fall Armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae)" Agriculture 15, no. 2: 170. https://doi.org/10.3390/agriculture15020170

APA Style

Apirajkamol, N., Mainali, B., Taylor, P. W., Walsh, T. K., & Tay, W. T. (2025). Characterisation of the Pathogenicity of Beauveria sp. and Metarhizium sp. Fungi Against the Fall Armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae). Agriculture, 15(2), 170. https://doi.org/10.3390/agriculture15020170

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