Nonthakorn ( Beatrice) Apirajkamol

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.

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.

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.

A modified extraction protocol is required to extract high quantity and quality DNA from fungal spores. We optimised DNA extraction protocols to obtain a sufficient amount of high molecular weight DNA from fungal spores for long read sequencing i.e., PacBio HiFi. Note: If required, the DNA extraction protocol can be scaled up to achieved the desired amount of genomic DNA

A modified extraction protocol is required to extract high quantity and quality DNA from fungal spores. We optimised DNA extraction protocols to obtain a sufficient amount of high molecular weight DNA from fungal spores for long read sequencing i.e., PacBio HiFi. Note: If required, the DNA extraction protocol can be scaled up to achieved the desired amount of genomic DNA

The development of effective pest management strategies for Spodoptera frugiperda is a high priority for crop protection across its invasive ranges. Here, we examined six Beauveria and five Metarhizium fungal isolates against this pest. Two Beauveria isolates (B-0571, B-1311) induced high mortality toward 3rd and 6th instar caterpillars and adults. For B-0571 mortality was 82.81 ± 5.75%, 61.46 ± 6.83%, and 93.75 ± 3.61%, and 73.72 ± 2.51%, 71.88 ± 5.41%, and 97.92 ± 2.08% for B-1311, with deaths in caterpillars largely occurring under 24 h (3rd instar control 0.74 ± 0.33%, B-0571 73.96 ± 7.85% and B-1311 62.08 ± 3.67%; 6th instar control 0%, B-0571 66.67% ± 11.02% and B-1311 62.5% ± 9.55%). Infection from both Beauveria isolates fully prevented reproduction in surviving S. frugiperda females. In contrast, all five Metarhizium isolates tested and the remaining four Beauveria isolates exhibited lower virulence. The discovery of two highly virulent Beauveria fungal isolates to S. frugiperda opens avenues to develop novel biological control tools against this highly invasive pest.

Since 2016, the fall armyworm (Spodoptera frugiperda, J.E. Smith) has spread from the Americas to invade many regions of the world, including Australia in early 2020. The development of effective pest management strategies for S. frugiperda is a high priority for crop protection. In the invasive range, the degree to which endemic biological control agents can constrain S. frugiperda remains sparsely investigated. This study examines 11 isolates of fungi including six Beauveria isolates and five Metarhizium isolates from Oceania for their potential as S. frugiperda biocontrol. Two Beauveria isolates (B-0571, B-1311) exhibited high virulence toward S. frugiperda caterpillars and adults. The overall mortalities over seven days in 3rd and 6th instar caterpillars and moths (respectively) for B-0571 was 82.81 ± 5.75, 61.46 ± 6.83, and 93.75 ± 3.61, and for B-1311 was 73.72 ± 2.51, 71.88 ± 5.41, and 97.92 ± 2.08%. The majority of deaths in the caterpillars occurred within the first 24 hours (3rd instar control 0.74 ± 0.33%, B-0571 73.96 ± 7.85 and B-1311 62.08 ± 3.67%; 6th instar control 0%, B-0571 66.67 ± 11.02% and B-1311 62.5 ± 9.55%). Infection from both isolates fully prevented reproduction in surviving S. frugiperda females. The two isolates, however, cause a significantly lower mortality in a native noctuidae species: Helicoverpa armigera (3rd instar B-0571 44.79 ± 4.54% and B-1311 19.80 ± 7.51%). The discovery of two fungal isolates with high virulence to S. frugiperda caterpillars and adults opens new avenues for the development of biological control tools for this invasive moth pest. Such tools may also provide additional options for control of native pest lepidoptera, and reduce selection pressure for resistance to Bt and synthetic insecticides.