Beauveria bassiana is a cosmopolitan entomopathogenic fungus that can infect over 1000 insect species. During growth inside the host, B. bassiana transitions from hyphal to yeast-like unicellular growth as blastospores. Blastospores are well suited as an active ingredient in biopesticides due to their ease of production by liquid fermentation. Herein, we investigated the impact of hyperosmotic growth environments mediated by ionic and non-ionic osmolytes on two strains of B. bassiana (ESALQ1432 and GHA) relevant to growth morphology, blastospore production, desiccation tolerance, and insecticidal activity. Polyethylene glycol (PEG200) increased osmotic pressure in submerged cultures leading to decreased blastospore size but higher blastospore yields for one strain. Morphologically, decreased blastospore size was linked to increased osmotic pressure. However, smaller blastospores from PEG200 supplemented cultures after air-drying exhibited delayed germination. Ionic osmolytes (NaCl and KCl) generated the same osmotic pressure (2.5–2.7 MPa) as 20 • Osmotic pressure plays a critical role in submerged fermentation of B. bassiana. • Ionic/non-ionic osmolytes greatly impact blastospore morphology, fitness, and yield. • Desiccation tolerance and bioefficacy of blastospores are affected by the osmolyte.

When left unmanaged, the invasive aquatic weed hydrilla [Hydrilla verticillata (L. f.) Royle; Hydrocharitaceae] causes serious environmental and economic impacts by choking out native plants and impeding flood control, navigation, and recreation. In Florida, millions of dollars are spent annually to control infestations of hydrilla, primarily with herbicides. However, during the past 15 yr, some hydrilla populations developed resistance to two of the most used aquatic herbicides. Since 2010, we have been testing a novel integrated weed management (IWM) system for hydrilla control that integrates selective insect herbivory by the hydrilla tip miner Cricotopus lebetis Sublette (Diptera: Chironomidae) with a disease-causing fungal pathogen [Mycoleptodiscus terrestris (Gerd.) Ostaz.] (Mt), and the reduced-risk aquatic herbicide imazamox. Over a period of 2 yr, field testing of the two biological control agents and imazamox was performed in four limnocorrals (1 m diam. by 1 m depth) installed in man-made ponds naturally infested with hydrilla to determine the most effective combination for use in hydrilla management. Establishment of the biological control agents and hydrilla damage were measured by collecting apical meristem samples and harvesting the hydrilla to count turions and calculate biomass. Cricotopus lebetis and Mt together and the two biological control agents in combination with imazamox caused the most damage to hydrilla, both in terms of short-term damage to apical meristems and long-term damage through reduced turion production and biomass. Overall, these findings indicate that a combination of different biological and chemical tactics can be used for integrated management of hydrilla.

If there is anything we can learn from the media, it is the frequency and severity of cyber-attacks is increasing and there are not enough qualified people to combat the risk organizations are facing. Current estimates say there are 3.5 million available cybersecurity related jobs globally and there has been a 350% growth in cybersecurity jobs since 2013 (Group, 2020). The Idaho Cyber Research Project (ICRP) is focused on finding an implementing solution to the problems in the workforce development pipeline. Our team consists of Cohort 2 of the ICRP, we are tasked with solving issues faced by organizations hiring new cyber personnel. To provide solutions to these issues we focused our research on four components of workforce availability and competency: resume and transcript analysis, apprenticeships, cyber incident response plan development, and adversarial mindset training. From this research we have produced the following focus areas and subsequent steps for each component of workforce capability: transcript and knowledge skills abilities (KSA) focused analysis, cybersecurity apprenticeships programs, the value of an adversarial mindset, and a guide to setting up cyber incident response plans for underprepared organizations. These solutions can be further developed and implemented to reduce the gap in workforce demand and talent.

The commercial use of the entomopathogenic fungi Metarhizium spp. in biopesticides has gained more interest since the discovery that several species of this genus are able to colonize roots. In general, commercial products with Metarhizium are formulated based on conidia for insect pest control. The process of mass production, harvesting, and formulation of infective conidia can be detrimental for conidial viability. Entomopathogenic fungi such as Metarhizium spp. are able to produce high concentrations of resistant structures, known as microsclerotia, when grown in liquid media. Microsclerotia are desiccation tolerant, with excellent storage stability, and are capable of producing high quantities of infective conidia after rehydration. The aim of this study was to evaluate microsclerotia production by different isolates of Metarhizium spp. and determine the effect of microsclerotia coated onto maize seeds on plant growth in the presence of soil-borne pathogen Fusarium graminearum . On average, ~1 × 10 5 microsclerotia/mL were produced by selected isolates of M. anisopliae (A1080 and F672) and Metarhizium robertsii (F447). Microsclerotia were formulated as granules with diatomaceous earth and used for seed coating, after which propagules produced around 5 × 10 6 CFU/g of seeds. In the presence of the plant pathogen, maize plants grown from untreated seeds had the lowest growth, while plants treated with the Metarhizium microsclerotia had significantly greater growth than the control plants. Hyphae were observed growing on and in root tissues in all the Metarhizium spp. treatments but not in samples from control plants. Metarhizium hyphal penetration points' on roots were observed 1 month after sowing, indicating the fungi were colonizing roots as endophytes. The results obtained indicate that microsclerotia can be coated onto seeds, providing plant protection against soil plant pathogens and a method to establish Metarhizium in the ecto- and endo-rhizosphere of maize roots, allowing the persistence of this biocontrol agent.

The Mexican INIFAP-Hir-2 Hirsutella citriformis strain was cultured in six liquid media at different C:N ratios. Glucose and hydrolysed amino acids were used as C and N sources, respectively. During fermentation, low-cost nitrogen sources were used to obtain an improved biomass production, and these cultures were then air-dried, vacuum packed, and evaluated for germination. Cultures containing mycelia were inoculated on different clays to observe spore formation. The highest biomass yield (19.47 g/L) was obtained from the medium supplemented with 36 g/L glucose and hydrolysed casamino acids added at a C:N ratio of 10:1. Furthermore, upon the use of low-cost nitrogen source, the highest dry biomass weights were obtained when cottonseed flour (51.30 g/L) and soybean meal (48.40 g/L) were used. This showed a significant difference with respect to the media supplemented with casamino acids, yeast extract or corn steep powder. Synnemata production on clays was observed only for those cultures grown on soybean meal. Germination and conidia production were evaluated on 8 different clays and diatomaceous earth (HyfloA (R)). Here, hydrous aluminosilicate (HAS) and montmorillonite (MONT) clay showed a higher percentage of germination, whereas Attapulgite (GA) granulometry -325 y Verge montmorillonite S-140 showed lower germination percentages. Twelve days post inoculation of liquid cultures on various clays, Attapulgite 16/30, HAS 24/48 and HyfloA (R) showed a higher production of conidia where values reached more than 1 x 10(7) conidia/g.

The invertebrate pests most commonly affecting New Zealand's pastoral-based production in 'average' years cause losses of between $1.7B and $2.3B p.a. of which up to $0.9B occur on sheep and beef farms and $1.4B on dairy farms. The native scarab grass grub is the most costly pest causing losses of $140-380 M on dairy farms and $75-205 M on sheep and beef farms annually. The exotic scarab, black beetle, although only affecting approximately 1 M ha, costs dairy farmers up to $223 M and sheep and beef farmers up to $19 M annually. Porina cause losses up to $84 M and $88 M respectively. Pasture nematodes are estimated to cost up to $274 M p.a. for dairy farmers and $326 M p.a. for sheep and beef farmers. Two exotic pests, Argentine stem weevil (ASW) and clover root weevil (CRW) are causing damage estimated at up to $200 M p.a. and $235 M p.a. respectively in dairy and sheep and beef pastures. While CRW is subject to successful biological control management it still causes considerable losses. Lesser pests also contribute to lost production, particularly as they often coexist with more major pests. However, their economic cost to New Zealand is difficult to calculate due to the variable nature of infestations on both temporal and spatial scales. At farm and paddock level, it is abundantly clear that substantial savings could be made if pest management is achieved. It is equally clear that in many instances the tools to do so are limited but if developed would contribute substantially to farm profitability.

The palladium-catalyzed decarboxylative asymmetric allylic alkylation (DAAA) is comprehensively reviewed, from its original development to recent advances in terms of substrate scope, reactivity, regio- and enantioselectivity. The current understanding of the mechanistic details, based on a combination of experimental and computational studies, is described. Selected examples of the application of this asymmetric synthetic methodology in natural product synthesis are given. The exploration of Pd-catalyzed decarboxylative asymmetric catalysis, in particular, the related interceptive decarboxylative asymmetric allylic alkylation, has become a focus in recent years and this is also summarized.

Autodissemination and foliar sprays of PFR-97 (Certis Inc., Columbia, MD) microbial insecticide, a blastospore formulation of Isaria fumosorosea Wize (Hypocreales: Cordycipitaceae), were evaluated for control of Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), on residential citrus. Seasonal trials on dooryard trees in South Texas evaluated: 1) pathogenicity of I. fumosorosea (Ifr) spores on autodisseminators (dispensers) deployed up to 3 wk on grapefruit trees; 2) psyllid control on several citrus species by dispensers and sprays; 3) infection range of the dispenser. Decline in spore pathogenicity over time was similar among dispensers during fall, winter, or spring and decreased by 30% after 1 d, 59% after 7 d, 81% after 14 d, and 100% after 21 d. Dispensers or sprays were equally effective for psyllid control on heavily infested lime trees from fall to spring and reduced mean reproduction (cumulative eggs) by 90% and mean attack intensity (cumulative psyllid-days) of adults by 76% and nymphs by 82%. Dispensers or sprays were also equally effective for psyllid control on lightly infested lime trees from spring to mid-summer and on orange or grapefruit trees from fall to winter. Very light infestations on grapefruit trees from spring to mid-summer were not significantly reduced by dispensers or sprays. Psyllid control was not improved by combining dispensers and sprays. Adult psyllids infected by Ifr were recovered in trees located 3-4 m away from trees with dispensers but not at greater distances. PFR-97 dispensers could be a treatment option for D. citri in settings where chemical control is problematic.

AimsNitrogen is a critical element in industrial fermentation media. This study investigated the influence of various nitrogen sources on blastospore production, desiccation tolerance and storage stability using two strains of the cosmopolitan insect-pathogenic fungus Beauveria bassiana. Methods and ResultsComplex organic sources of nitrogen such as soy flour, autolysed yeast and cottonseed flour induced great numbers of blastospores after 2-3days of fermentation, which also survived drying and remained viable (32-56% survival) after 9months storage at 4 degrees C, although variations were found between strains. Nitrogen availability in the form of free amino acids directly influenced blastospore production and resistance to desiccation. Increasing glucose and nitrogen concentrations up to 120 and 30gl(-1), respectively, did not improve blastospore production but enhanced desiccation tolerance. Cell viability after drying and upon fast-rehydration was increased when 25g acid-hydrolysed casein per litre was supplemented in the liquid culture medium. ConclusionsThese findings indicate that low-cost complex nitrogen compounds are suitable to enhance yeast-like growth by B. bassiana with good desiccation tolerance and therefore support its further scale-up production as a mycoinsecticide. Significance and Impact of the StudyNitrogen is the most expensive nutrient in liquid media composition, but this study underscores the feasibility of using low-cost nitrogen compounds composed mainly of agro-industrial by-products for rapid production of desiccation-tolerant B. bassiana blastospores by liquid culture fermentation.

This study compared the insecticidal activity of liquid culture-produced blastospores and solid substrate-produced aerial conidia of Beauveria bassiana GHA and Isaria fumosorosea ARSEF3581 strains against Diaphorina citri adults. Insects exposed to 10(7) propagules/ml in a spray residue contact leaf bioassay died within 6 days at 25 degrees C, with no significant differences between fungal treatments. At higher concentrations (10(8) propagules/ml), I.fumosorosea conidia killed psyllids faster compared to its blastospore formulation, i.e. 4 versus 5 days, respectively. In greenhouse tests, the same treatments applied to infested citrus plants (2x10(6) spores/ml) all significantly reduced the number of nymphs compared with the untreated controls over 3 weeks; however, only I.fumosorosea blastospores significantly reduced the number of F1 adult psyllids when compared with controls. Similar results were observed in the follow-up greenhouse test, where I.fumosorosea blastospores were the most effective treatment overall, reducing D.citri populations by about 60% after 21 days; by contrast, imidacloprid killed almost 100% of psyllids within a week in both tests. Fewer psyllids exhibited mycosis in the greenhouse (i.e. approximate to 20 versus87% in the laboratory). This is the first report comparing both conidial and blastospore formulations of B.bassiana and I.fumosorosea for the control of a psyllid pest. Field testing is required to determine how successful different spore formulations might be under various environmental conditions.

We have developed the first catalytic asymmetric preparation of sterically hindered tertiary -aryl oxindoles via enantioselective palladium-catalyzed decarboxylative protonation of the corresponding -aryl--amido allyl esters. The reaction occurs under very mild conditions and in short reaction times, providing excellent yields and promising enantioselectivities (ee's up to 78%). We have also performed an experimental investigation of the reaction mechanism and employed theoretical calculations to understand the nature of the enantioselectivity-determining step.

The efficacy of microsclerotia of Metarhizium brunneum (Petch) (Hypocreales: Clavicipitaceae) strain F52 (ARSEF 7711) was tested using samples which had been exposed on forest trees, allowing time for conidia to be produced. An aqueous slurry of microsclerotial granules (61.3% of dry mass), a straw mulch hydroseeding product (29.4%), xanthan gum as a tackifier (5.4%), and water-absorbing hydrogel (3.9%), was sprayed on small wood-veneer samples attached to tree trunks within the Ohio USDA quarantine zone for eradication of Asian longhorned beetles, Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae). Samples were collected biweekly June to August, and adult female beetles in a quarantine laboratory were exposed for 2 days to individual samples after each collection. Median survival time (ST50) after exposure to samples which had been outdoors four weeks was 9.5 days (95% CI 8.5-10.5); the beetles died faster than with samples from 2 or 6-10 weeks, but slower than using positive control microsclerotial samples (ST50 6.5 days). Conidial density of field-collected samples peaked at 4 weeks, while the sprayed material gradually weathered to 33% of initial dry mass by 10 weeks. A separate field experiment in New York State compared three formulations: the hydrogel-hydromulch mixture, straw hydromulch without hydrogel, or hydrogel alone. Results were highly weather-dependent, but median beetle survival in the August bioassays was between 8.5-9.5 days for all three formulations. Overall, the hydrogel addition did not significantly improve efficacy, either during dry or rainy periods, and hydrogel alone (without hydromulch) appeared to be an additional viable option.

Microsclerotia of the entomopathogenic fungus (Metarhizium brunneum Petch strain F52; Hypocreales: Clavicipitaceae) were sprayed in a hydromulch formulation onto wood samples to test persistence and biological control efficacy when exposed on forest and orchard trees. The density of viable conidia and retention of hydromulch were not significantly affected by the tackifier in the formulation (psyllium, xanthan, or a mixture of both). Forest samples had a maximum conidial density and more viable conidia (7.2 × 105 conidia cm−2) than orchard samples (1.4 × 105 conidia cm−2). Increased rainfall, temperature and the time that samples were outdoors were critical factors for conidial density. Asian longhorned beetles [Anoplophora glabripennis (Motschulsky); Coleoptera: Cerambycidae] exposed to orchard samples in laboratory bioassays did not have significantly less mortality compared to forest samples, indicating that hydromulch may be feasible in open sunny conditions, if sufficient moisture is present.

The highly enantioselective synthesis of sterically hindered α-allyl-α-aryl oxindoles possessing an all-carbon quaternary stereocenter at the oxindole 3-position has been developed. The key step in the synthetic route employed was a novel one-pot, two-step synthesis of α-aryl-β-amido allyl ester substituted oxindoles in good yields of 41-75% (13 examples) by interception of an unstable allyl ester intermediate through reaction with aryllead triacetate reagents. Pd-Catalyzed decarboxylative asymmetric allylic alkylation (DAAA) was optimized with 2,4,6-trimethoxyphenyl as the aryl-containing substrate. A screen of chiral P,N- and P,P-based ligands showed that the ANDEN-phenyl Trost ligand was the most effective, affording the corresponding α-allyl-α-aryl oxindole product in 96% yield and 99% ee. A substrate scope of a further 12 α-aryl-β-amido allyl ester substituted oxindoles showed that products containing bulky di-ortho-methoxy substituted arenes and naphthyl groups were formed in very high ee's (94-98%), whereas those lacking this substitution pattern were formed in more moderate levels of enantioselectivities (56-63% ee). Surprisingly, the 2,6-dimethylphenyl-substituted substrate afforded the O-allylated product in contrast to the expected C-allylated product. A crystal structure was obtained of the 2,4,6-trimethoxyphenyl-substituted α-allyl-α-aryl oxindole product which enabled us to identify the absolute stereochemistry of the quaternary stereocenter formed. A plausible explanation to rationalise the sense of enantioselection observed in these DAAA transformations is also proposed.

The current study evaluates the potential of using high resolution DNA melting assays to discriminate species in the genus Isaria. The study utilizes a previously identified 103 base pair PCR amplicon, which was reported to be selective for Isaria fumosorosea. Our study finds the amplicon selective for Isaria javanica and Isaria poprawskii when assayed against all members of the genus. In addition, the high resolution melting profile of this amplicon can be used to discriminate between I. javanica, I. poprawskii and a 1:1 mixture of the two species. The practical application of this technique was confirmed using a bioassay on whitefly nymphs (Bemisia tabaci biotype B) inoculated with I. javanica, I. poprawskii or a 1:1 mixture of the two species. This assay provides a simple assay to identify these two species of entomopathogenic fungi.

Fungal microsclerotia ("small" sclerotia) are compact hyphal aggregates, typically 50-600 μm in diameter, that are formed under unfavorable nutritional and/or environmental conditions. These structures are often melanized and desiccated to some degree containing endogenous nutritional reserves for use when favorable conditions return. Many fungi, mostly plant pathogens, produce microsclerotia as a survival structure. Liquid culture methods have been developed for producing microsclerotia of the Ascomycota Metarhizium spp, Colletotrichum truncatum, Mycoleptodiscus terrestris, and Trichoderma spp. While these fungi have varying culture conditions that optimize microsclerotia production, all share common nutritional and environmental requirements for microsclerotia formation. Described are the general liquid culture techniques, media components, and harvesting and drying methods necessary to produce stable microsclerotial granules of these fungi.

Microsclerotia (MS) of Metarhizium brunneum strain F52 (Hypocreales: Clavicipitaceae) were processed as granules using three carriers: kaolin clay, microcrystalline cellulose (MCC), or diatomaceous earth (DE). In a series of experiments aimed at comparing viable conidial production, each MS carrier type was hydrated using a decreasing range of water activities on glycerol or polyethylene glycol (PEG200)-amended media. Conidial density and percent germination of conidia declined significantly as water activity was lowered. All three carrier types produced >2-6 x 10(9) viable conidia/gram at higher water activities (a(w) >0.987) but were still capable of producing 2.9 x 10(7) +/- 1.6 x 10(6) conidia/g (30% glycerol a(w) = 0.883) and 1.9 x 10(7) +/- 6.5 x 10(6) conidia/g (30% PEG200 a(w) = 0.924) at the lowest tested water activities. MS carrier types were sprayed onto 0.4 m length logs with or without hydromulch formulation. The median survival times of Asian longhorned beetles, Anoplophora glabripennis (Coleoptera: Cerambycidae), exposed by climbing double the length of the logs of hydromulch-treated bark, were 16.5-20.5 d while beetles exposed to logs without hydromulch formulation had median survival times of 22-25.5 d. Overall, experiments showed that there were few biologically significant differences between the MS carrier types.

The yeast form (blastospore) of the dimorphic insect-pathogenic fungus Beauveria bassiana can be rapidly produced using liquid fermentation methods but is generally unable to survive rapid dehydration processes or storage under non-refrigerated conditions. In this study, we evaluated the influence of two convective drying methods, various modified atmosphere packaging systems, and storage temperatures on the desiccation tolerance, storage stability, and virulence of blastospores of B. bassiana ESALQ 1432. All blastospore formulations were dried to <5 % water content equivalent to aw < 0.3. The viability of B. bassiana blastospores after air drying and spray drying was greater than 80 %. Vacuum-packaged blastospores remained viable longer when stored at 4 °C compared with 28 °C with virtually no loss in viability over 9 months regardless the drying method. When both oxygen and moisture scavengers were added to sealed packages of dried blastospore formulations stored at 28 °C, viability was significantly prolonged for both air- and spray-dried blastospores. The addition of ascorbic acid during spray drying did not improve desiccation tolerance but enhanced cell stability (∼twofold higher half-life) when stored at 28 °C. After storage for 4 months at 28 °C, air-dried blastospores produced a lower LC80 and resulted in higher mortality to whitefly nymphs (Bemisia tabaci) when compared with spray-dried blastospores. These studies identified key storage conditions (low aw and oxygen availability) that improved blastospore storage stability at 28 °C and will facilitate the commercial development of blastospores-based bioinsecticides.