Entomophthoralean and hypocrealean fungal pathogens of the sugarcane aphid, Melanaphis sacchari (Hemiptera: Aphididae), on sorghum in Georgia.

The sugarcane aphid, Melanaphis sacchari, is a widely distributed insect that attacks grasses in different genera including Miscanthus, Saccharum, and Sorghum. The invasive aphid superclone was first discovered in the U.S. attacking grain sorghum in Texas in 2013. Since then, it has been found in at least 25 states including Georgia. We conducted a survey of naturally occurring fungal pathogens of sugarcane aphids on five farms in Georgia, and identified a hypocrealean fungus, Akanthomyces dipterigenus, and two entomophthoralean fungi, Neoconidiobolus spp. From 2018 to 2020, fungal activity differed across farms but at one farm both major fungal species, A. dipterigenus and N. thromboides, were found each of the 3 years infecting sugarcane aphids, attacking adults, both alatae and apterae, and nymphs.

Cryptic diversity and virulence of Beauveria bassiana recovered from Lycorma delicatula (spotted lanternfly) in eastern Pennsylvania.

The entomopathogenic fungus Beauveria bassiana is cosmopolitan and known to infect a variety of sap-sucking pests like aphids, mealybugs, and scales in the order of Hemiptera. In Fall 2017, spotted lanternfly (SLF) adults killed by the fungal entomopathogen B. bassiana were found in Berks County, Pennsylvania. In 2018-2020 we collected SLF and nearby non-target insects killed by Beauveria spp. from 18 field sites in southeastern Pennsylvania. We identified 159 Beauveria isolates from SLF and six isolates from non-targets. Five isolates of B. bassiana and one isolate of B. brongniartii were identified from the non-targets. Based on sequence data from the nuclear B locus (Bloc) intergenic region, all the isolates from SLF were identified as B. bassiana, but there were 20 different strains within this species, grouped into two clades. Three B. bassiana strains (A, B, and L) were found in most field sites and were the most prevalent. Representative isolates for these three strains were used in laboratory bioassays and were compared to a commercial B. bassiana strain (GHA). Strain B was inferior to A, L, and GHA against nymphs; strains A and L had greater efficacy than B and GHA against adults. We also quantified conidial production on SLF cadavers. This paper discusses the diversity of these B. bassiana strains in SLF populations and implications for biological control of this abundant invasive.

Discovery of two hypocrealean fungi infecting spotted lanternflies, Lycorma delicatula: Metarhizium pemphigi and a novel species, Ophiocordyceps delicatula.

In the eastern United States, populations of the invasive spotted lanternfly, Lycorma delicatula, can be infected by native fungal entomopathogens, including Batkoa major and Beauveria bassiana. In some areas of southeastern Pennsylvania, localized population collapses have been observed in L. delicatula populations to be caused by these pathogens. Two additional fungal pathogens were discovered infecting L. delicatula at low levels, and these were identified as Metarhizium pemphigi and Ophiocordyceps delicatula, a new species that has not been previously described. Therefore, four species of native entomopathogenic fungi have now been documented infecting this abundant, invasive planthopper that is spreading in the United States.

Identification and Virulence of Cordyceps javanica Strain wf GA17 Isolated From a Natural Fungal Population in Sweetpotato Whiteflies (Hemiptera: Aleyrodidae).

A new strain of the entomopathogenic fungus, identified as Cordyceps javanica (Frieder. & Bally) Kepler, B. Shrestha & Spatafora (Hypocreales: Cordycipitaceae) wf GA17, was found naturally infecting the sweetpotato whitefly, Bemisia tabaci (Gennadius) MEAM1 in southern Georgia, US, in September 2017. The fungus was tested for pathogenicity and virulence in comparison with commercially available entomopathogenic fungal strains against several insect species in the laboratory. In specific, it was compared with Cordyceps fumosorosea (Wize) Kepler, B. Shrestha & Spatafora (Hypocreales: Cordycipitaceae) Apopka 97, Beauveria bassiana (Bals.-Criv.) Vuill. (Hypocreales: Cordycipitaceae) strain GHA, and Metarhizium brunneum Petch (Hypocreales: Clavicipitaceae) strain F52 for virulence against B. tabaci (4th instars) and cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae) (4th instars with or without wingbuds), on leaf-discs, and against last instars of pecan weevil Curculio caryae (Horn) (Coleoptera: Curculionidae) and citrus root weevil Diaprepes abbreviatus (L.) (Coleoptera: Curculionidae) in soil cups. Against B. tabaci, C. javanica exhibited higher mortality and mycosis development at 5 d post inoculation than other fungi. In assays against A. gossypii with and without wingbuds, C. javanica and C. fumosorosea had the highest mortality and mycosis levels and B. bassiana had the lowest; nymphs with wingbuds were more susceptible to some fungal infection than those without. Against C. caryae, B. bassiana was more effective than other fungi. For D. abbreviatus, B. bassiana also caused the highest mortality while M. brunneum had the lowest, with Cordyceps spp. being intermediate. Overall, the findings suggest high potential of the new strain, C. javanica wf GA17, for managing whiteflies and aphids, while it was not as effective as B. bassiana against the curculionids.

Genetic diversity among naturally-occurring strains of Beauveria bassiana associated with the introduced coffee berry borer, Hypothenemus hampei, (Coleoptera: Curculionidae) on Hawai'i Island.

The coffee berry borer (CBB), Hypothenemus hampei, is considered the most important insect pest of coffee worldwide. CBB was discovered on Hawai'i Island in 2010 and soon thereafter on the islands of O'ahu (2014) and Maui (2016). As part of an areawide effort to manage CBB in Hawai'i, we conducted a survey of naturally-occurring Beauveria associated with the beetle to complement field efficacy studies of the commercial B. bassiana strain GHA. Sampling of CBB from coffee farms or unmanaged sites in various districts on the islands of Hawai'i and O'ahu, and also from Puerto Rico, resulted in >1800 Beauveria isolates. These were initially characterized using colony morphology to differentiate strain GHA, registered for use in Hawai'i, from indigenous congenerics. A total of 114 isolates representative of these indigenous morphotypes were selected for further characterization. Sequencing of the intergenic regions B locus and EFutr identified all as Beauveria bassiana sensu stricto. Sixteen haplotypes were observed, with one more common haplotype present in 12 of 16 sites sampled on Hawai'i Island. This B locus-EFutr haplotype, designated Bb1, was the only haplotype observed in 2016 epizootics on two high-elevation coffee farms on Hawai'i Island with no history of GHA application. Many of the haplotypes showed genetic similarity to those collected from CBB from other countries, including Brazil, Columbia, Nicaragua, and Kenya, but a few were identical to those from other insect species collected in Hawai'i before 2010. This diversity suggests a mixed lineage among B. bassiana strains associated with CBB in the three Hawaiian islands.

A pair of native fungal pathogens drives decline of a new invasive herbivore.

Two North American fungal pathogens caused a coepizootic leading to localized collapse of an outbreak population of the newly invasive planthopper pest, the spotted lanternfly (Lycorma delicatula), in the eastern United States. The pathogens partitioned the habitat, with the majority of L. delicatula on tree trunks killed by Batkoa major, while cadavers of L. delicatula killed by Beauveria bassiana were usually on the ground. The future will show whether these pathogens will be drivers in boom-bust cycles or will result in recurrent low population densities of this new invasive species.

Sleeping Beauties: Horizontal Transmission via Resting Spores of Species in the Entomophthoromycotina.

Many of the almost 300 species of arthropod-pathogenic fungi in the Entomophthoromycotina (Zoopagomycota) are known for being quite host-specific and are able to cause epizootics. Most species produce two main types of spores, conidia and resting spores. Here, we present a review of the epizootiology of species of Entomophthoromycotina, focusing on their resting spores, and how this stage leads to horizontal transmission and persistence. Cadavers in which resting spores are produced can often be found in different locations than cadavers of the same host producing conidia. Resting spores generally are dormant directly after production and require specific conditions for germination. Fungal reproduction resulting from infections initiated by Entomophaga maimaiga resting spores can differ from reproduction resulting from conidial infections, although we do not know how commonly this occurs. Reservoirs of resting spores can germinate for variable lengths of time, including up to several months, providing primary infections to initiate secondary cycling based on conidial infections, and not all resting spores germinate every year. Molecular methods have been developed to improve environmental quantification of resting spores, which can exist at high titers after epizootics. Ecological studies of biological communities have demonstrated that this source of these spores providing primary inoculum in the environment can decrease not only because of germination, but also because of the activity of mycopathogens.

Starvation and Imidacloprid Exposure Influence Immune Response by Anoplophora glabripennis (Coleoptera: Cerambycidae) to a Fungal Pathogen.

In several insect systems, fungal entomopathogens synergize with neonicotinoid insecticides which results in accelerated host death. Using the Asian longhorned beetle, Anoplophora glabripennis (Motschulsky), an invasive woodborer inadvertently introduced into North America and Europe, we investigated potential mechanisms in the synergy between the entomopathogenic fungus Metarhizium brunneum Petch and the insecticide imidacloprid. A potential mechanism underlying this synergy could be imidacloprid's ability to prevent feeding shortly after administration. We investigated whether starvation would have an impact similar to imidacloprid exposure on the mortality of fungal-inoculated beetles. Using real-time PCR to quantify fungal load in inoculated beetles, we determined how starvation and pesticide exposure impacted beetles' ability to tolerate or resist a fungal infection. The effect of starvation and pesticide exposure on the encapsulation and melanization immune responses of the beetles was also quantified. Starvation had a similar impact on the survival of M. brunneum-inoculated beetles compared to imidacloprid exposure. The synergy, however, was not completely due to starvation, as imidacloprid reduced the beetles' melanotic encapsulation response and capsule area, while starvation did not significantly reduce these immune responses. Our results suggest that there are multiple interacting mechanisms involved in the synergy between M. brunneum and imidacloprid.

First report of Leptolegnia chapmanii (Peronosporomycetes: Saprolegniales) affecting mosquitoes in central Brazil.

Numerous isolates of an oomycete 'fungus', Leptolegnia chapmanii, are reported from Brazil for the first time. This aquatic pathogen was baited with Aedes aegypti sentinel larvae from stagnant, temporary bodies of water in selected locations under secondary tropical forest in and near the central Brazilian city of Goiânia and from more distant sites in the western and northern regions of the state of Goiás. Isolates were identified based on their morphological and developmental characters, comparative sequence data for the ITS and TEF loci, as well as their rapid activity against A. aegypti larvae. Taxonomic issues affecting the application of the name L. chapmanii and its typification are rectified. This study contributes to a better understanding of the presence and distribution of this oomycete in Brazil, its sequence-based identification, and of its potential as a biological agent against mosquito vectors.

Three genera in the Ceratocystidaceae are the respective symbionts of three independent lineages of ambrosia beetles with large, complex mycangia.

The genus Ambrosiella accommodates species of Ceratocystidaceae (Microascales) that are obligate, mutualistic symbionts of ambrosia beetles, but the genus appears to be polyphyletic and more diverse than previously recognized. In addition to Ambrosiella xylebori, Ambrosiella hartigii, Ambrosiella beaveri, and Ambrosiella roeperi, three new species of Ambrosiella are described from the ambrosia beetle tribe Xyleborini: Ambrosiella nakashimae sp. nov. from Xylosandrus amputatus, Ambrosiella batrae sp. nov. from Anisandrus sayi, and Ambrosiella grosmanniae sp. nov. from Xylosandrus germanus. The genus Meredithiella gen. nov. is created for symbionts of the tribe Corthylini, based on Meredithiella norrisii sp. nov. from Corthylus punctatissimus. The genus Phialophoropsis is resurrected to accommodate associates of the Xyloterini, including Phialophoropsis trypodendri from Trypodendron scabricollis and Phialophoropsis ferruginea comb. nov. from Trypodendron lineatum. Each of the ten named species was distinguished by ITS rDNA barcoding and morphology, and the ITS rDNA sequences of four other putative species were obtained with Ceratocystidaceae-specific primers and template DNA extracted from beetles or galleries. These results support the hypothesis that each ambrosia beetle species with large, complex mycangia carries its own fungal symbiont. Conidiophore morphology and phylogenetic analyses using 18S (SSU) rDNA and TEF1α DNA sequences suggest that these three fungal genera within the Ceratocystidaceae independently adapted to symbiosis with the three respective beetle tribes. In turn, the beetle genera with large, complex mycangia appear to have evolved from other genera in their respective tribes that have smaller, less selective mycangia and are associated with Raffaelea spp. (Ophiostomatales).

Multilocus genotyping of Amylostereum spp. associated with Sirex noctilio and other woodwasps from Europe reveal clonal lineage introduced to the US.

Sirex noctilio is a woodwasp of Eurasian origin that was inadvertently introduced to the southern hemisphere in the 1900s and to North America over a decade ago. Its larvae bore in Pinus spp. and can cause significant mortality in pine plantations. S noctilio is associated with a symbiotic white rot fungus, Amylostereum areolatum, which females inject into trees when they oviposit and which is required for survival of developing larvae. We compared the genetic diversity of A. areolatum isolated from S. noctilio and other woodwasps collected from Europe and from northeastern North America to determine the origin of introduction(s) into the United States. Multilocus genotyping of nuclear ribosomal regions and protein coding genes revealed two widespread multilocus genotypes (MLGs) among the European samples, one of which is present in the US. The other two MLGs associated with S. noctilio in the US represented unique haplotypes. These latter two haplotypes were likely from unrepresented source populations, and together with the introduced widespread haplotype reveal multiple A. areolatum MLGs introduced by S. noctilio and indicate possible multiple S. noctilio introductions to North America from Europe. Our results also showed a lack of fidelity between woodwasp hosts and Amylostereum species.

Detection of presumptive mycoparasites associated with Entomophaga maimaiga resting spores in forest soils.

The fungal pathogen Entomophaga maimaiga can provide high levels of control of the gypsy moth, Lymantria dispar, an important forest defoliator. This fungus persists in the soil as resting spores and occurs naturally throughout many areas where gypsy moth is established. Studies on the spatial dynamics of gypsy moth population have shown high variability in infection levels, and one possible biological factor could be the variable persistence of E. maimaiga resting spores in the soil due to attacks by mycoparasites. We surveyed presumptive mycoparasites associated with parasitized E. maimaiga resting spores using baiting and molecular techniques and identified an ascomycete (Pochonia sp.) and oomycetes (Pythium spp.).

Transstadial transmission of Pythium in Bradysia impatiens and lack of adult vectoring capacity.

Fungus gnats have been shown to transmit a variety of plant-pathogenic fungi that produce aerial dispersal stages. However, few studies have examined potential interactions between fungus gnats and oomycetes, including Pythium spp. A series of laboratory experiments were conducted to determine whether fungus gnat adults are vectors of several common greenhouse Pythium spp., including Pythium aphanidermatum, P. irregulare, and P. ultimum. An additional objective was to determine whether P. aphanidermatum can be maintained transstadially in the gut of a fungus gnat larva through the pupal stadium to be transmitted by the subsequent adult. Adult fungus gnats did not pick up infectious Pythium propagules from diseased plants and transmit them to healthy plants in any experiment. Species-specific primers and a probe for real-time polymerase chain reaction were developed to detect the presence of P. aphanidermatum DNA in fungus gnat tissue samples. P. aphanidermatum DNA was detectable in the larval and pupal stages; however, none was detected in adult fungus gnats. These results are in agreement with previous studies that have suggested that adult fungus gnats are unlikely vectors of Pythium spp.

Detection and quantification of Entomophaga maimaiga resting spores in forest soil using real-time PCR.

Environmental sampling to monitor entomopathogen titre in forest soil, a known reservoir of insect pathogens such as fungi and viruses, is important in the evaluation of conditions that could trigger epizootics and in the development of strategies for insect pest management. Molecular or PCR-based analysis of environmental samples provides a sensitive method for strain- or species-based detection, and real-time PCR, in particular, allows quantification of the organism of interest. In this study we developed a DNA extraction method and a real-time PCR assay for detection and quantification of Entomophaga maimaiga (Zygomycetes: Entomophthorales), a fungal pathogen of the gypsy moth, in the organic layer of forest soil. DNA from fungal resting spores (azygospores) in soil was extracted using a detergent and bead mill homogenization treatment followed by purification of the crude DNA extract using Sephadex-polyvinylpolypyrrolidone microcolumns. The purification step eliminated most of the environmental contaminants commonly co-extracted with genomic DNA from soil samples but detection assays still required the addition of bovine serum albumin to relieve PCR inhibition. The real-time PCR assay used primers and probe based on sequence analysis of the nuclear ribosomal ITS region of several E. maimaiga and two E. aulicae strains. Comparison of threshold cycle values from different soil samples spiked with E. maimaiga DNA showed that soil background DNA and remaining co-extracted contaminants are critical factors determining detection sensitivity. Based on our results from comparisons of resting spore titres among different forest soils, estimates were best for organic soils with comparatively high densities of resting spores.