Mealybug Population Dynamics: A Comparative Analysis of Sampling Methods for Saccharicoccus sacchari and Heliococcus summervillei in Sugarcane (Saccharum sp. Hybrids).

This research is focused on a comparative field-based study of the population dynamics and sampling methods of two mealybug species, Saccharicoccus sacchari (Cockerell, 1895) (Hemiptera: Coccomorpha, Pseudococcidae) and Heliococcus summervillei (Brookes, 1978) (Hemiptera: Coccomorpha, Pseudococcidae), in sugarcane (Saccharum sp. hybrids) (f. Poaceae) over consecutive growing seasons. The research monitored and compared the above- and belowground populations and seasonal abundance of these two mealybug species in sugarcane fields in Far North Queensland, with non-destructive sampling techniques of yellow sticky traps, pan traps, and stem traps, and destructive sampling of the whole leaf and whole plant. The results indicated that S. sacchari (n = 29,137) was more abundant and detected throughout the growing season, with population peaks in the mid-season, while H. summervillei (n = 2706) showed peaks of the early-season activity. S. sacchari is primarily located on sugarcane stems and roots, compared to H. summervillei, which is located on leaves and roots. The whole-leaf collection and stem trap were the most effective sampling techniques for quantification of H. summervillei and S. sacchari, respectively. This study enhanced the understanding of S. sacchari and the first-ever record of H. summervillei on sugarcane in Australia and will contribute to the development of more effective pest management strategies.

Resporulation of Calcium Alginate Encapsulated Metarhizium anisopliae on Metham®-Fumigated Soil and Infectivity on Larvae of Tenebrio molitor.

Metarhizium anisopliae infects and kills a large range of insects and is a promising biocontrol agent to manage soil insects, such as wireworm in sweetpotato. The presence of other soil microbes, which exhibit competitive fungistasis, may inhibit the establishment of M. anisopliae in soil. Microbially depleted soil, for example, sterilized soil, has been shown to improve the resporulation of the fungus from nutrient-fortified M. anisopliae. Prior to planting, sweetpotato plant beds can be disinfected with fumigants, such as Metham®, to control soil-borne pests and weeds. Metham® is a broad-spectrum soil microbial suppressant; however, its effect on Metarhizium spp. is unclear. In the research presented here, fungal resporulation was examined in Metham®-fumigated soil and the infectivity of the resulting granule sporulation was evaluated on mealworm, as a proxy for wireworm. The fungal granules grown on different soil treatments (fumigated, field and pasteurized soil) resporulated profusely (for example, 4.14 × 107 (±2.17 × 106) conidia per granule on fumigated soil), but the resporulation was not significantly different among the three soil treatments. However, the conidial germination of the resporulated granules on fumigated soil was >80%, which was significantly higher than those on pasteurized soil or field soil. The resporulated fungal granules were highly infective, causing 100% insect mortality 9 days after the inoculation, regardless of soil treatments. The results from this research show that the fungal granules applied to soils could be an infective inoculant in sweetpotato fields in conjunction with soil fumigation. Additional field studies are required to validate these results and to demonstrate integration with current farming practices.

Transmission of Metarhizium anisopliae and Beauveria bassiana to adults of Kuschelorhynchus macadamiae (Coleoptera: Curculionidae) from infected adults and conidiated cadavers.

Kuschelorhynchus macadamiae is a major pest of macadamias in Australia, causing yield losses of up to 15%. Our previous studies have shown the weevil is susceptible to Beauveria bassiana and Metarhizium anisopliae. The aim of this study was to investigate horizontal transmission of both fungal species to healthy weevils from both infected adults and weevil cadavers. In a confined environment the mortality of healthy adults caused by the transmission of conidia from live fungus-infected adults was < 50%. Under similar experimental conditions, the mortality of healthy adults reached 100% when exposed to conidiated cadavers. However, when conidiated cadavers were used in more spacious environments (insect cages), the mortality of adults was < 80%. Using scanning electron microscopy, it was observed that all healthy adults had conidia attached to all external parts of the body. This suggests that although the conidia were readily transferred to the adults, the lower mortality in the larger insect cages could be the result of an unfavourable environmental factor such as low humidity. The presence of conidia attached to all the adults indicated that they did not show any discriminatory behaviour such as avoidance of conidiated cadavers infected by these two fungal species. The results from this study show that there is potential for enhanced control of adult K. macadamiae via transmission from either fungus-infected adults or conidiated cadavers and this could strengthen sustainable pest management in macadamias.

Integration of Entomopathogenic Fungi into IPM Programs: Studies Involving Weevils (Coleoptera: Curculionoidea) Affecting Horticultural Crops.

Weevils are significant pests of horticultural crops and are largely managed with insecticides. In response to concerns about negative impacts of synthetic insecticides on humans and the environment, entomopathogenic fungi (EPF) have been developed as an alternative method of control, and as such appear to be "ready-made" components of integrated pest management (IPM) programs. As the success of pest control requires a thorough knowledge of the biology of the pests, this review summarises our current knowledge of weevil biology on nut trees, fruit crops, plant storage roots, and palm trees. In addition, three groups of life cycles are defined based on weevil developmental habitats, and together with information from studies of EPF activity on these groups, we discuss the tactics for integrating EPF into IPM programs. Finally, we highlight the gaps in the research required to optimise the performance of EPF and provide recommendations for the improvement of EPF efficacy for the management of key weevils of horticultural crops.

Response of the macadamia seed weevil Kuschelorhynchus macadamiae (Coleoptera: Curculionidae) to Metarhizium anisopliae and Beauveria bassiana in laboratory bioassays.

Macadamia seed weevil, Kuschelorhynchus macadamiae Jennings and Oberprieler, is a major pest of macadamia in eastern Australia, causing yield losses of up to 15%. Current control methods involve two applications of acephate per season but more recently have moved to a single application of indoxacarb, combined with the collection and destruction of fallen nuts that contain developing larvae. As a first step towards reducing the dependence of the industry on synthetic insecticides, we tested six isolates of M. anisopliae, six isolates of B. bassiana and one commercial B. bassiana product (Velifer® biological insecticide) against adult macadamia seed weevil under laboratory conditions. All isolates were pathogenic against adult weevils with M. anisopliae accession ECS1/BRIP 70272 and B. bassiana accession B27/BRIP 70267 causing 97.5% and 92.5% mortality 12 days after being treated at 1 × 107 conidia/mL. Isolates ECS1/BRIP 70272 and B27/BRIP 70267 had the shortest LT50 values of 5.13 days and 5.37 days respectively. The median lethal concentrations (LC50) for ECS1/BRIP 70272 and B27/BRIP 70267 were 1.48 × 105 and 1.65 × 105 conidia/mL respectively. Results of this study indicate that M. anisopliae accession ECS1/BRIP 70272 and B. bassiana accession B27/BRIP 70267 have considerable potential for K. macadamiae control, and should be developed into biological insecticides for integration into macadamia pest management programs.

Occurrence and diversity of entomopathogenic fungi (Beauveria spp. and Metarhizium spp.) in Australian vineyard soils.

Entomopathogenic Ascomycetes: Hypocreales fungi occur worldwide in the soil; however, the abundance and distribution of these fungi in a vineyard environment is unknown. A survey of Australian vineyards was carried out in order to isolate and identify entomopathogenic fungi. A total of 240 soil samples were taken from eight vineyards in two states (New South Wales and Victoria). Insect baiting (using Tenebrio molitor) and soil dilution methods were used to isolate Beauveria spp. and Metarhizium spp. from all soil samples. Of the 240 soil samples, 60% contained either Beauveria spp. (26%) or Metarhizium spp. (33%). Species of Beauveria and Metarhizium were identified by sequencing the B locus nuclear intergenic region (Bloc) and elongation factor-1 alpha (EFT1) regions, respectively. Three Beauveria species (B. bassiana, B. australis and B. pseudobassiana) and six Metarhizium species (M. guizhouense, M. robertsii, M. brunneum, M. flavoviride var. pemphigi, M. pingshaense and M. majus) were identified. A new sister clade made up of six isolates was identified within B. australis. Two potentially new phylogenetic species (six isolates each) were found within the B. bassiana clade. This study revealed a diverse community of entomopathogenic fungi in sampled Australian vineyard soils.

Sweetpotato weevil, Cylas formicarius (Fab.) (Coleoptera: Brentidae) avoids its host plant when a virulent Metarhizium anisopliae isolate is present.

Metarhizium anisopliae has a wide range of coleopteran hosts, including weevils. Some susceptible insects are known to modify their behavior to prevent infection, typically detecting virulent strains by olfaction, and avoiding physical contact with sources of infection. Laboratory olfactometer assays were conducted on the sweetpotato weevil Cylas formicarius to test the hypothesis that insects would avoid a more virulent strain of M. anisopliae when presented with a strain of low virulence or an untreated control. When adult weevils were allowed to choose between paired test arenas containing sweetpotato roots and M. anisopliae isolates on agar cores, weevils avoided arenas with the highly virulent isolate QS155, showing a preference for either roots with uninoculated agar cores or cores with the low virulence isolate QS002-3. When roots or whole sweetpotato plants were inoculated with M. anisopliae, the preferences of weevils remained broadly similar; weevils were repelled by the highly virulent isolate QS155 when tested against either QS002-3 or uninoculated roots and plants, however weevils were not repelled by the low virulence isolate QS002-3 tested against uninoculated controls. When single-sex groups of weevils were tested separately in the olfactometer using uninoculated whole plants and plants treated with isolate QS155, males and females responded similarly and statistically identical preferences were found for the untreated plants. When weevils were released singly at different times of the day the response time for males was significantly shorter in the afternoon compared to the morning. Males were always significantly faster to respond to olfactory stimuli than females. Understanding factors that may lead to avoidance of virulent M. anisopliae strains by C. formicarius will be an essential part of developing an 'attract-and-infect' strategy for the management of C. formicarius.

Coconut Lethal Yellowing Diseases: A Phytoplasma Threat to Palms of Global Economic and Social Significance.

The recent discovery of Bogia coconut syndrome in Papua New Guinea (PNG) is the first report of a lethal yellowing disease (LYD) in Oceania. Numerous outbreaks of LYDs of coconut have been recorded in the Caribbean and Africa since the late Nineteenth century and have caused the death of millions of palms across several continents during the Twentieth century. Despite the severity of economic losses, it was only in the 1970s that the causes of LYDs were identified as phytoplasmas, a group of insect-transmitted bacteria associated with diseases in many other economically important crop species. Since the development of polymerase chain reaction (PCR) technology, knowledge of LYDs epidemiology, ecology and vectors has grown rapidly. There is no economically viable treatment for LYDs and vector-based management is hampered by the fact that vectors have been positively identified in very few cases despite many attempted transmission trials. Some varieties and hybrids of coconut palm are known to be less susceptible to LYD but none are completely resistant. Optimal and current management of LYD is through strict quarantine, prompt detection and destruction of symptomatic palms, and replanting with less susceptible varieties or crop species. Advances in technology such as loop mediated isothermal amplification (LAMP) for detection and tracking of phytoplasma DNA in plants and insects, remote sensing for identifying symptomatic palms, and the advent of clustered regularly interspaced short palindromic repeats (CRISPR)-based tools for gene editing and plant breeding are likely to allow rapid progress in taxonomy as well as understanding and managing LYD phytoplasma pathosystems.

Determining putative vectors of the Bogia Coconut Syndrome phytoplasma using loop-mediated isothermal amplification of single-insect feeding media.

Phytoplasmas are insect vectored mollicutes responsible for disease in many economically important crops. Determining which insect species are vectors of a given phytoplasma is important for managing disease but is methodologically challenging because disease-free plants need to be exposed to large numbers of insects, often over many months. A relatively new method to detect likely transmission involves molecular testing for phytoplasma DNA in sucrose solution that insects have fed upon. In this study we combined this feeding medium method with a loop-mediated isothermal amplification (LAMP) assay to study 627 insect specimens of 11 Hemiptera taxa sampled from sites in Papua New Guinea affected by Bogia coconut syndrome (BCS). The LAMP assay detected phytoplasma DNA from the feeding solution and head tissue of insects from six taxa belonging to four families: Derbidae, Lophopidae, Flatidae and Ricaniidae. Two other taxa yielded positives only from the heads and the remainder tested negative. These results demonstrate the utility of combining single-insect feeding medium tests with LAMP assays to identify putative vectors that can be the subject of transmission tests and to better understand phytoplasma pathosystems.

The genome sequence of the biocontrol fungus Metarhizium anisopliae and comparative genomics of Metarhizium species.

BACKGROUND: Metarhizium anisopliae is an important fungal biocontrol agent of insect pests of agricultural crops. Genomics can aid the successful commercialization of biopesticides by identification of key genes differentiating closely related species, selection of virulent microbial isolates which are amenable to industrial scale production and formulation and through the reduction of phenotypic variability. The genome of Metarhizium isolate ARSEF23 was recently published as a model for M. anisopliae, however phylogenetic analysis has since re-classified this isolate as M. robertsii. We present a new annotated genome sequence of M. anisopliae (isolate Ma69) and whole genome comparison to M. robertsii (ARSEF23) and M. acridum (CQMa 102). RESULTS: Whole genome analysis of M. anisopliae indicates significant macrosynteny with M. robertsii but with some large genomic inversions. In comparison to M. acridum, the genome of M. anisopliae shares lower sequence homology. While alignments overall are co-linear, the genome of M. acridum is not contiguous enough to conclusively observe macrosynteny. Mating type gene analysis revealed both MAT1-1 and MAT1-2 genes present in M. anisopliae suggesting putative homothallism, despite having no known teleomorph, in contrast with the putatively heterothallic M. acridum isolate CQMa 102 (MAT1-2) and M. robertsii isolate ARSEF23 (altered MAT1-1). Repetitive DNA and RIP analysis revealed M. acridum to have twice the repetitive content of the other two species and M. anisopliae to be five times more RIP affected than M. robertsii. We also present an initial bioinformatic survey of candidate pathogenicity genes in M. anisopliae. CONCLUSIONS: The annotated genome of M. anisopliae is an important resource for the identification of virulence genes specific to M. anisopliae and development of species- and strain- specific assays. New insight into the possibility of homothallism and RIP affectedness has important implications for the development of M. anisopliae as a biopesticide as it may indicate the potential for greater inherent diversity in this species than the other species. This could present opportunities to select isolates with unique combinations of pathogenicity factors, or it may point to instability in the species, a negative attribute in a biopesticide.