Wee Tek Tay’s research while affiliated with State Library of New South Wales and other places

Bacillus thuringiensis ( Bt ) endotoxins are often considered environmentally friendly pest control tools. However, the development of resistance to Bt toxins and emergence of exotic pests necessitate the characterisation of new Bt isolates. This study aims to identify and characterise novel Bt toxins and bioactive compounds that may be utilised to mitigate the impact of Spodoptera frugiperda (fall armyworm, FAW), a polyphagous agricultural pest species that has recently established populations in over 80 countries including Australia. Eight Bt isolates were used in bioassays to ascertain toxicity to FAW neonates. Six Bt isolates (Bt_01‐02 and Bt_05‐08) exhibited potential insecticidal activities. Three isolates including Bt_01 and Bt_07‐08 caused 100% mortality, while Bt_02, Bt_05, and Bt_06 induced 71.27 ± 21.17, 98.44 ± 2.21 and 92.19 ± 11.05% mortality, respectively. Genome analysis was conducted to characterise the toxins and secondary metabolite gene clusters of each isolate. Four isolates (Bt_01, Bt_06, Bt_07, Bt_08) contained the Cry1Na‐partial and Cry1I toxins, while Bt_05 contained Cry2A, Cry1H and Cry1‐like amino acid sequences. In addition, the gene cluster for zwittermicin A, a crystal toxin enhancer, was present in all isolates. However, no known toxins or insecticidal compounds were identified in Bt_02 despite inducing high mortality. The pathogenicity of Bt_02 was also tested against two Australian native pest species: Helicoverpa armigera conferta and H. punctigera . This includes both the susceptible and Cry1Ac‐resistant (Hp9‐3784) lines of H. punctigera . Bt_02 caused 74.88 ± 19.82% mortality in H. armigera , 95.65 ± 6.15% mortality in H. punctigera and 90.91 ± 12.86% mortality in Hp9‐3784. This suggests that Bt_02 may possess unknown toxins or bioactive compounds responsible for its effectiveness against three species of lepidopteran pests including those that exhibited Cry1Ac resistance.

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.

The entomopathogenic fungus Metarhizium pingshaense infects diverse insect host species. We present an annotated draft genome of M. pingshaense (Commonwealth Scientific and Industrial Research Organisation [CSIRO] strain M-1000) isolated from a Spodoptera species individual, thereby contributing to future research of M. pingshaense as a potential biological control agent.

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 exhibited high mortality within 24 hours post spore application. This study aimed to identify and characterise 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 hours 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 FAW. Identifying and characterising of gene clusters encoding these insecticide compounds in B-0571 and B-1311 will contribute to better understanding the entomopathogenicity of these isolates that will be vital to developing these EPF isolates as sustainable alternative FAW biocontrol agents.

BACKGROUND Helicoverpa armigera is a highly polyphagous species that causes huge losses to agricultural and horticultural crops worldwide. In the cotton industry, H. armigera, including the Australian subspecies Helicoverpa armigera conferta, is largely managed using genetically modified crops that express insecticidal toxins, such as Cry1Ac. Resistance to Cry1 proteins occurs and, in some cases, is mediated by changes to HaCad1, a gene that encodes the midgut protein cadherin. Around the world, numerous resistance‐associated polymorphisms have been identified in the HaCad1 gene of H. armigera, but Cry1Ac resistance is rare in the Australian subspecies. We used CRISPR/Cas9 to disrupt the cadherin gene in H. armigera conferta and characterised the resulting phenotype with bioassays and transcriptomics. RESULTS Compared to the parental strain, the newly generated HaCad1 knockout strain is 44‐fold and 16‐fold more resistant to Cry1Ac and Cry1A.105, respectively, while wild‐type and knockout insects were equally insensitive to Cry1F. CONCLUSION The disruption of the HaCad1 gene causes Cry1Ac resistance in Australian H. armigera conferta. However, Cry1Ac resistance remains rare in Australian field populations suggesting that Australia's approach to pest management in cotton has prevented widespread Cry1Ac resistance. © 2024 CSIRO. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

A partial mitochondrial DNA Cytochrome Oxidase subunit I (mtCOI) gene haplotype variant of the coconut rhinoceros beetle (CRB) Oryctes rhinoceros, classed as ‘CRB-G (clade I)’, has been the focus of much research since 2007, with reports of invasions into new Pacific Island locations (e.g., Guam, Hawaii, Solomons Islands). For numerous invasive species, inference of invasion biology via whole genome is superior to assessments via the partial mtCOI gene. Here, we explore CRB draft mitochondrial genomes (mitogenomes) from historical and recent collections, with assessment focused on individuals associated within the CRB-G (clade I) classification. We found that all Guam CRB individuals possessed the same mitogenome across all 13 protein-coding genes and differed from individuals collected elsewhere, including ‘non-Guam’ individuals designated as CRB-G (clade I) by partial mtCOI assessment. Two alternative ATP6 and COIII partial gene primer sets were developed to enable distinction between CRB individuals from Guam that classed within the CRB-G (clade I) haplotype grouping and CRB-G (Clade I) individuals collected elsewhere. Phylogenetic analyses based on concatenated ATP6–COIII genes showed that only Guam CRB-G (clade I) individuals clustered together, and therefore Guam was not the source of the CRB that invaded the other locations in the Pacific assessed in this study. The use of the mtCOI and/or mtCOIII genes for initial molecular diagnosis of CRB remained crucial, and assessment of more native CRB populations will further advance our ability to identify the provenance of CRB invasions being reported within the Pacific and elsewhere.

Background: Bacillus thuringiensis (Bt) insecticidal proteins, including Cry proteins and vegetative insecticidal proteins (Vips), are extensively utilized in transgenic crops due to their efficacy and safety. The fall armyworm, Spodoptera frugiperda, has evolved practical resistance to Cry1Fa, yet no practical resistance to Vip3Aa has been documented. However, both laboratory selection and field screen studies indicate a high potential for this pest to evolve resistance to Vip3Aa, making it crucial to evaluate potential resistance genes. HaVipR1 has recently been identified as a key determinant of Vip3Aa resistance in the cotton bollworm, Helicoverpa armigera. This study investigated whether the HaVipR1-homologous gene in S. frugiperda (SfVipR1) is similarly involved in Vip3Aa resistance. Results: We employed CRISPR/Cas9 technology to generate a homozygous knockout strain of SfVipR1. In comparison with the parent susceptible YJ-19 strain, the knockout strain (Sfru-KO) exhibited high-level resistance to Vip3Aa (>1875-fold) but showed no resistance to Cry1Fa. This acquired resistance to Vip3Aa is autosomal, recessive, and genetically linked to the deletion mutation in SfVipR1 within the Sfru-KO strain of S. frugiperda. Conclusion: Disruption of SfVipR1 results in high-level resistance to Vip3Aa, highlighting SfVipR1 has a critical role in Vip3Aa toxicity in S. frugiperda, despite the exact mechanism remaining unclear. Early detection of SfVipR1 mutant alleles in the field is essential for developing adaptive resistance management strategies against S. frugiperda.

The global reliance on Bacillus thuringiensis (Bt) proteins for controlling lepidopteran pests in cotton, corn, and soybean crops underscores the critical need to understand resistance mechanisms. Vip3Aa, one of the most widely deployed and currently effective Bt proteins in genetically modified crops, plays a pivotal role in pest management. This study identifies the molecular basis of Vip3Aa resistance in Australian Helicoverpa armigera through genetic crosses, and integrated genomic and transcriptomic analyses. We identified a previously uncharacterized gene, LOC110373801 (designated HaVipR1 ), as a crucial determinant of Vip3Aa resistance in two field-derived resistant lines. Functional validation using CRISPR-Cas9 knockout in susceptible lines confirmed the gene’s role in conferring resistance. Despite extensive laboratory selection of Vip3Aa-resistant colonies in Lepidoptera, the biochemical mechanisms underlying resistance have remained elusive. Our research demonstrates that HaVipR1 -mediated resistance operates independently of known resistance genes, including midgut-specific chitin synthase and the transcription factor SfMyb. The identification of HaVipR1 offers further insights into the Vip3Aa mechanism of action. This discovery is vital for devising strategies to counteract resistance and sustain the efficacy of Bt crops. Future research should focus on elucidating the biochemical pathways involving HaVipR1 and investigating its interactions with other resistance mechanisms. Our findings underscore the utility of analysing field-derived resistant lines in providing biologically relevant insights and stress the necessity for comprehensive management strategies to maintain agricultural productivity. Significance Statement This is the first identification of a specific gene in Helicoverpa armigera which mediates resistance to the Bacillus thuringiensis (Bt) protein Vip3Aa. We identify that this gene is disrupted in two different ways in separate field-derived resistant lines, one being a large transposable element insertion in the first intron of the HaVipR1 gene, confirmed using long-read sequencing. Disruption of this gene using CRISPR-Cas9 knockout in a susceptible H. armigera line confers Vip3A resistance. The identification of a specific gene is important for molecular monitoring and management of H. armigera as well as other global pests of concern like Spodoptera frugiperda . These findings also offer insights for researchers aiming to understand how Vip3Aa functions, as the action pathway remains unclear.

Multiple Epiphyas species inhabit southwestern Western Australia, including Light Brown Apple Moth (LBAM) Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), a globally significant, polyphagous pest. This study evaluated the efficacy and specificity of lures designed for 3 Epiphyas species: E. postvittana, Epiphyas pulla (Turner), and the undescribed Epiphyas sp. (1) (Common). Additionally, the study sought to determine the presence and distribution of Epiphyas species in 3 significant apple-growing localities. Trapping, together with partial sequencing of the mitochondrial COI gene, found LBAM to be restricted to the Perth Hills and E. pulla, to apple orchards near Manjimup and Pemberton. This geographic disjunction remains unexplained. Epiphyas sp. (1) was not recorded despite using a specifically designed lure. The E. pulla and LBAM traps demonstrated superior efficacy in capturing their target species, while the catch in Epiphyas sp. (1) traps did not significantly differ between the 2. Both E. pulla and LBAM exhibited peak abundance from late spring to the end of summer (October–February), with variations in timing and peak catch of male moths across species, locations, and years. Surveys conducted in April during the harvest period (February–May), when moth traps caught an average of 1–1.8 moths/trap/week, found no Epiphyas larvae or damage on 140,400 mature apples or on 26,000 leaves. While E. pulla and LBAM traps effectively monitor their target moths, genetic identification of trap catch would be necessary if they co-occurred. Encouragingly, the results indicate that both species become relatively rare as harvest season approaches, and neither inflicts significant damage to mature apples under existing management.

A partial mitochondrial DNA Cytochrome Oxidase subunit I (mtCOI) gene haplotype variant of the coconut rhinoceros beetle (CRB) Oryctes rhinoceros classed as ‘CRB-G (clade I)’ has been the focus of much research since 2007 with reports of invasions into new Pacific Island locations (e.g., Guam, Hawaii, Solomons Islands). For numerous invasive species, inference of invasion biology via whole genome is superior to assessments via the partial mtCOI gene. Here, we explore CRB draft mitochondrial genomes (mitogenomes) from historical and recent collections, with assessment focused on individuals associated within the CRB-G (clade I) classification. We found that all Guam CRB individuals possessed the same mitogenome across all 13 protein coding genes and differed from individuals collected elsewhere, including ‘non-Guam’ individuals designated as CRB-G (clade I) by partial mtCOI assessment. Two alternative ATP6 and COIII partial gene primer sets were developed to enable distinction between CRB (clade I) that invaded Guam and CRB-G (Clade I) individuals collected elsewhere. Phylogenetic analyses based on concatenated ATP6-COIII genes showed that only Guam CRB-G (clade I) individuals clustered together, and therefore Guam was not the source of the CRB that invaded the other locations in the Pacific assessed in this study. The use of the mtCOI and/or mtCOIII genes for initial molecular diagnosis of CRB remained crucial, and assessment of more native CRB populations will further advance our ability to identify the provenance of CRB invasions being reported within the Pacific and elsewhere