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ARS Home » Northeast Area » Ithaca, New York » Robert W. Holley Center for Agriculture & Health » Emerging Pests and Pathogens Research » Research » Research Project #430048

Research Project: Management and Biology of Arthropod Pests and Arthropod-borne Plant Pathogens

Location: Emerging Pests and Pathogens Research

2017 Annual Report

Objective 1. Using and developing genetic resources and associated information of the ARS Collection of Entomopathogenic Fungal Cultures (ARSEF), conserve, characterize (including taxonomic revision), and exchange insect pathogenic fungi such as Beauveria, Metarhizium, and Hirsutella species complexes to facilitate use of these fungi as biocontrol agents of key arthropod pests and disease vectors. Subobjective 1.1. Continue the curation, operation, and expansion of the ARSEF culture collection and associated information resources. Subobjective 1.2. Improve methods to isolate, culture, and preserve fungal entomopathogens. Subobjective 1.3. Conduct research on the systematics, taxonomy, and organismal biology of these fungi. Objective 2. Identify genetic, environmental and behavioral mechanisms that regulate circulative transmission of insect-borne plant pathogens. Subobjective 2.1. Identification of pathogen, host, and vector components that regulate uptake and transmission of pathogens by sap-sucking insects. Subobjective 2.2. Functional analysis of genes, proteins and metabolites involved in circulative plant pathogen transmission. Objective 3. Explore the utility of novel interdiction molecules that could interfere with plant pathogen acquisition and transmission. Subobjective 3.1. Continue efforts to define the chemistry of fungal secondary metabolites and characterize their effects on phloem-feeding insects, their endosymbionts, and on plant pathogen transmission. Subobjective 3.2. Develop RNA aptamers that bind to transmission related compounds and test their ability to interfere with pathogen acquisition and transmission.

Control of arthropods that transmit pathogens is arguably one of the biggest challenges to human health and agriculture. Many serious plant and animal pathogens are dependent upon arthropod vectors for transmission between hosts. Nearly all arthropod-transmitted animal pathogens are internalized and circulate in their insect vectors, while plant pathogens are divided between those that circulate in their vectors and those that are carried on the cuticle linings of mouthparts and foreguts. The mechanisms of circulative transmission are only beginning to be dissected, but already commonalities among transmission of both circulative plant and animal pathogens have been discovered. Our experimental systems offer innovative approaches to manage circulative-transmitted plant pathogens that have been recalcitrant to the development of host resistance and for which the economic and environmental costs of vector control has been prohibitive, unsustainable and/or ineffective. Scientists' incomplete understanding of interactions among insect vectors, plant pathogens and plant hosts limits the development of new tools to block or interfere with pathogen transmission by insects in the field. We address this problem by attempting to discover genes and products that mediate the associations among insect vectors, circulative plant pathogens and plant hosts. The new technologies and knowledge are expected to be extended and applied to the study of other circulative pathogens and will greatly impact growers, industry stakeholders, and other research communities. The project will also focus on maintaining the extensive ARS Collection of Entomopathogenic Fungal Cultures (ARSEF). ARSEF contains 12,500 isolates representing 700 fungal taxa from 1,300 hosts and 2,400 locations worldwide, and will be managed to ensure ongoing accession, preservation, identification, and distribution of fungal isolates for development and deployment as biocontrol agents and for research purposes. The ARSEF collection also plays a central role in revising taxonomies of fungi using the state-of-the-art systematic methods.

Progress Report
Objective 1: The ARS Collection of Entomopathogenic Fungal Cultures (ARSEF) remains actively involved with distributing fungal isolates to a national and global clientele to sustain a range of research and development activities. A total of 635 isolates were shipped in response to a total of 75 orders since July 2016. Since that time, 166 isolates were processed for cryopreservations, and 172 isolates have been freeze-dried for later distribution in response to incoming requests. ARSEF lost one technician at the end of 2015 and hired a microbiologist who is being trained in all aspects of maintaining ARSEF and serving its clientele. The user interface for the ARSEF database will be updated to reflect current demands, an iterative, 1-2 year process between the curators and ARS computational biologists. The Science Without Borders project terminated after three active years of research to discover central Brazilian biodiversity of fungal pathogens affecting mosquitoes, flies, and other related host insects that transmit serious diseases of humans and animals; to isolate and to identify the cultures obtained from these collections, to preserve these isolates at the Institute of Tropical Pathology and Public Health, Federal University of Goiás; Goiânia, Brazil, to train students in mycology and pathobiology, and to verify the pathogenicity of these fungal isolates against the mosquito, Aedes aegypti. Objective 2: To identify mechanisms that regulate circulative transmission of insect-born plant pathogens, we characterized plant proteins that interact with aphid-borne poleroviruses to facilitate plant infection. Our results showed that the targeting of chloroplasts and vesicle transport pathways negatively regulate virus infection. In collaboration with university cooperators, we solved the crystal structure of a Potato leafroll virus (PLRV) virion protein, the first ever for this family of viruses. The structure provides a wealth of information on the function of this protein in aphid transmission. Additional data on the structure of the related Turnip yellows virus enabled homology modeling and prediction of sites involved in aphid transmission and virion assembly. Characterization of the function of these host-virus protein interactions and structures is ongoing and may lead to the development of novel sources of virus resistance in cereal crops and potato. We identified 8 aphid proteins that interact with PLRV. The function of these virus-interacting aphid proteins in virus acquisition, virus transmission and aphid biology are currently being probed using a variety of microscopic, cellular and biochemical approaches. In a serendipitous discovery, PLRV transmission efficiency was significantly reduced when a clonal lineage of the green peach aphid, Myzus persicae, was reared on turnip as compared with the weed physalis, and this was a transient effect caused by a host-switch response inducing the expression of the enzyme cathepsin B in the aphid gut. Chemical inhibition of cathepsin B restored the ability of aphids reared on turnip to transmit PLRV in a dose-dependent manner, showing that the increased activity of gut enzymes can be modulated to control virus transmission by aphids. In a second serendipitous discovery, we found that M. persicae densovirus titer is increased in PLRV viruliferous aphids as compared to non-viruliferous aphids and that this effect is not seen when aphids are viruliferous for Potato virus Y, a non-circulative virus. We showed that the effect on densovirus titer in the aphid is regulated by the PLRV silencing suppressor protein, P0. As insect viruses are being developed by ARS scientists and others as biocontrol agents, understanding the interaction between P0 and other viral silencing suppressors of plant pathogens and insect vectors will be crucial for the deployment of a vector control strategy targeting hemipteran pests that relies on insect-infecting viruses. We have systematically performed mass spectrometry-based quantitative proteomics for discovery of proteins in the Asian citrus psyllid (ACP) associated with acquisition and transmission of the citrus greening pathogen (‘Candidatus Liberibacter asiaticus”, CLas) in whole insects and a variety of insect tissues. Collaboration with scientists at the Boyce Thompson Institute in Ithaca, New York focused on additional sequencing and annotation of the ACP genome which resulted in an improved ACP protein database for data analysis. We are studying the role of a number of ACP proteins in CLas transmission, including hemocyanin. Hemocyanin is a copper binding protein with a distinct blue color, and is one of the most highly expressed proteins in CLas-exposed ACP. ACP populations contain different color morphs (blue, yellow, gray), and western blot analysis revealed that blue insects have higher amounts of hemocyanin than non-blue insects from the same population, and elemental metal analysis revealed that blue ACP have significantly more copper than non-blue ACP. Blue ACP have a lower CLas titer than non-blue ACP, and are less effective at CLas transmission. Silencing hemocyanin gene expression leads to a reduction of CLas titer in ACP. DsRNA sequences have been designed against additional ACP targets, and will be delivered using microinjection and artificial diet protocols that have been optimized in our lab. We received citrus trees from a cooperator at the University of Florida in Gainesville, Florida, which have been infected with citrus tristeza virus (CTV) constructs designed to silence these ACP genes. Proteome analysis has also pinpointed a role for Wolbachia in the gut of the ACP in CLas acquisition. We found that the bacteria Wolbachia and CLas occupy similar but non-overlapping regions of insect organs. We modified a published method to extract and purify diaphorin, a small molecule produced by the ACP symbiont Profftella, which resulted in double the published yield per insect. This afforded pure material to probe the biological activity of diaphorin in a range of experiments, including structure determination and detection in insect samples. We detected diaphorin-like compounds in an artificial sucrose-solution diet fed upon by infected ACP. Thus, diaphorin compounds must be present in ACP saliva, and are likely transferred to the host-plant tissue with unknown effects on the plant immune response. Profftella occurs only in ACP and in all ACP, and diaphorin production is changed in response to CLas acquisition. This highly specific relationship of Profftella to ACP is seen as a key point of leverage in our effort to combat citrus greening. We found that CLas induces nuclear fragmentation in adult ACP gut tissues, but not nymphal ACP gut tissues. The nuclear fragmentation is indicative of apoptosis, or programmed cell death, and we are now trying to understand how nymph insects are resistant to this effect of CLas. This could be critical to understanding why ACP must acquire CLas as nymphs to transmit the bacteria as adults. In collaboration with ARS scientists in Fort Pierce, Florida, we tested for natural variation in CLas transmission ability between different populations of the ACP collected from various locations in Florida. A highly significant difference (P<0.0001) was found among the 15 isofemale lines with respect to mean acquisition rates (percentage of psyllids that tested positive for CLas) and mean titer of CLas in infected psyllids. Molecular and genetic analysis of these lines is critical for the citrus industry. Objective 3: Significant progress was made on isolation and structural characterization of siderophores, iron scavenging molecules, and related compounds from fungi in ARSEF. Isolation and characterization of siderophores and related compounds from Beauveria bassiana continued and we have now have total of 15 compounds of which nine are new to science. We also continued to structurally characterize novel siderophores in a complex mixture of compounds from seven strains of Hirsutella biocontrol fungi all of which were isolated from field-collected ACP. We confirmed the structure of the primary and secondary component of the siderophore mixture from a strain of H. citriformis, studied the variability of the siderophore blends and acquired spectral data for preliminary characterization of several minor components that reveal variation among the strains in the composition of the siderophore mixtures. In addition we found that mutant strains of another biocontrol fungus, Metarhizium robertsii, accumulate much larger quantities of siderophore analogs than are produced by the naturally occurring strain. We leveraged this discovery to build a supply of these rare compounds for testing. Progress was made in developing artificial diet bioassays to measure the effects of isolated fungal compounds on phloem-feeding insects, their endosymbiont titers and on plant pathogen transmission. We also employed an in vitro disk-diffusion assay to test for antibacterial effects of siderophores on a panel of gram-negative and -positive bacteria (including Liberibacter crescens, a close relative to CLas that is culturable) that serve as surrogates for the unculturable endosymbionts of ACP as well as CLas, the results of which will be described in more detail in an invention disclosure that is in preparation. Under objective 3.2, we analyzed ACP gut transcriptome and stylet sheath and gut proteomes of healthy and CLas + insects to identify targets for the development of interdiction molecules that block CLas transmission. Targets have been selected from these analyses and will be expressed and purified in year 2. The transcriptome analysis revealed the presence of about 900 long non-coding RNAs (lncRNAs) and several were among the most highly expressed RNAs detected in gut tissue. We are now exploring how these lncRNAs may also be targeted for interdiction and functional analysis.

1. The first collections from Brazil of any verified species of the fungal genus Coelomomyces was made and determined to represent a new, undescribed species. A 3-year Science Without Borders project involving an ARS researchers from Ithaca, New York, with collaborators from the Institute for Tropical Pathology and Public Health (Federal University of Goiás, in Goiânia, Brazil) sought to explore the central Brazilian biodiversity of fungal pathogens affecting mosquitoes and other disease-bearing dipteran insects. During the closing months of this project (which ended on 1 March 2017), two collections separated in time by several months were made from one area in a small biological preserve of a new fungal pathogen, Coelomomyces santabrancae, from mosquito larvae of an unidentified Onirion species. The diseased larvae were found in open, hollow sections of bamboo stems (artificial treeholes) hung on vegetation at the short of a river. This fungal genus, whose swimming spores have a single posterior flagellum, are well known pathogens (in the diploid portion of their life history) of mosquito larvae. The haploid phase of the life history and produces sexual gametes affects other aquatic arthropods that are not insects. These Brazilian collections represent a new species distinguished by the identification of its mosquito host and the morphology of its thick-walled resistant sporangia that allow the fungus to survive periods of dryness or seasonal absences of susceptible hosts.

2. The systematics of some unusual Brazilian collections of mosquito-pathogenic watermolds is being clarified and expanded. This work is a joint effort of an ARS scientist in Ithaca, New York, and scientists at Michigan State University, and seeks to clarify the systematics of some of the mosquito-pathogen found during the Brazilian Science Without Borders project. This work is being prepared for publication in a pair of manuscripts that will report on the diversity and systematics of these watermolds; the second manuscript will involve these same ARS and Michigan State University scientists as well as Brazilian partners in the Science Without Borders project. The identification of the pathogens being treated in this second manuscript appear to be of a species well known from diverse locations around the world where it affects mammalian hosts, but the Brazilian collections are the first recognition of this species from any invertebrate host as well as the first collections of this species from Brazil.

3. Over several decades now, a series of collections of cranefly species (Diptera: Tipulidae) that produce on a single generation in each year have yielded the thick-walled resting spores of fungi that clearly belonged in the order Entomophthorales, but because only these overwintering spore forms were produced, could not be classified among the majority of genera in this fungal order whose species are defined by their production of short-lived, thin-walled spore forms. DNA sequences were used to link these thick-walled resting spores for the first time to the genus Zoophthora, and resulted in the description of a new species and a transfer to this genus from another genus based on only resting spores.

4. It is widely recognized that the identifications and revisions of organismal taxonomy needs to be based now on genomic rather than on traditional criteria and systematics practices, but it is necessary to be much more careful about the use of genomic data than has been widely understood. A keynote address by an ARS scientist from Ithaca, New York, at the 2016 meeting of the Korean Society for Applied Entomology addressed several key issues involving the use of fungal pathogens of insects as practical biological control agents and offered a series of highly pertinent warnings about difficulties in the use of genomic databases as tools to identify organisms, and the general lack of recognition that identifications or taxonomic revisions should be based only sequences from nomenclatural type materials or from taxonomically verified sources when trying to make gene-based identifications. The use of sequences ascribed to inaccurately or unverifiable identified organisms for identifications or taxonomic revisions introduces unnecessary–and possibly significant–errors into the literature that are difficult to impossible to correct later. Many of these same warning were also repeated later at a conference in China on Cordyceps species and their relatives (these fungi are among the most numerous, taxonomically complex, and more biologically important of all fungal pathogens affecting insects).

5. A long-standing mystery about which of two competing explanations for how a highly unusual form of forcible discharge of some fungal spores is accomplished was resolved. The spores of nearly all fungi in the order Entomophthorales are forcibly discharged to a substantial distance from their infected invertebrate hosts, and several differing modes of spore discharge are known to occur. It was long believed that the spores of Entomophthora muscae were discharged by a rocket-like mechanism in which the contents of the cell forming the spore are forced through the apex of that cell and carry the spore on a droplet of the contents of that cell. Much more recently, it was proposed that the prominent droplet of ‘goo’ these discharged spores was not the contents of the spore-producing cell but was derived from a rapid gelatinization of the outer wall of on the spore. Following biological background information and procedural suggestions from an ARS scientist in Ithaca, New York, a graduate student at the University of California Berkeley completed a high-speed video study that unambiguously confirmed that the discharge of E. muscae spores is rocket-like and propelled on the contents of the cells that produce them.

6. Master genes controlling small molecule production are critical for efficacy of an insect pathogenic fungus as an insect biocontrol agent. Metarhizium fungi infect a wide range of agricultural pest insects. Thus, these fungi have potential as natural biocontrol agents of insects and several species of Metarhizium have been marketed as environmentally friendly biopesticides. Metarhizium fungi produce a large number of unique small molecule metabolites, also known as secondary metabolites. Some of these molecules may impact the safety and efficacy of the fungus as a biocontrol agent and a biopesticide. To evaluate the role played by these molecules in pathogenic processes ARS scientists at Ithaca, New York collaborated with Cornell University scientists to analyze the M. robertsii genome to identify several master genes that control the production of secondary metabolites. The genome sequence information will enable ARS scientists to develop and commercialize biopesticides from M. robertsii.

7. The genetic basis of biosynthesis of swainsonine, a forage-contaminating mycotoxin, was characterized using genetically engineered mutants. Swainsonine is a small molecule mycotoxin produced by a broad range of fungi including endophytes of plants. Swainsonine constitutes a forage-contamination hazard that can have severe negative impacts on livestock. Candidate swainsonine biosynthesis genes were identified by genomic screening of swainsonine-producing fungi, which include the insect biocontrol fungus Metarhizium robertsii. Genetic engineering techniques were used to produce mutants of M. robertsii in which the swainsonine biosynthetic pathway was inactivated. Chemical analysis confirmed that these mutants failed to produce swainsonine. Bioassays also showed that mutants lacking swainsonine were slightly more virulent to insects than the natural strain. This research demonstrated that the safety of a genetic engineered mutant of an insect biocontrol agent could be improved by abolishing the production of a hazardous mycotoxin. The identification of swainsonine biosynthesis genes will lead to more efficient genome-based strategies for discovering unknown fungal/plant sources of swainsonine contamination in forage crops.

8. Proteomic analysis of Candidatus Liberibacter-Asian citrus psyllid interactions identified unique, pathogen-induced changes in protein expression and protein interactions in the insect vector. During acquisition, the citrus greening bacterial pathogen, Candidatus Liberibacter asiaticus (CLas) interacts with tissues of the Asian citrus psyllid vector (Diaphorina citri), but the impact on the vector's molecular biology was unknown. Using state of the art quantitative mass spectrometry analysis, insect proteins differentially expressed between CLas(+) and CLas(-) D. citri included defense and immunity proteins, proteins involved in energy storage and utilization, and proteins associated with microbial invasion of host cells. Protein interaction networks between CLas and D. citri were identified and quantified during infection and insect development. The work produced conceptually novel information about the metabolic interdependence among the insect vector, its endosymbionts, and the citrus greening pathogen. These data reveal novel opportunities for control of this disease for the citrus industry. These findings led to the ARS scientist becoming a team leader (co-PI) for a $10 million dollar USDA SCRI grant to build upon these studies to develop new molecules that interfere with acquisition and transmission of the citrus greening pathogen and a PI on a second, $2 million dollar USDA SCRI grant focused on the development of a new biosensor for detecting CLas positive insects. The findings also enabled ARS scientists to extend two California Citrus Research Board-funded projects in new directions.

9. Interactions between ACP and the endosymbionts Profftella were identified as a novel target for citrus greening interdiction strategies. In collaboration with scientists from the University of Washington, ARS scientists in Ithaca, New York found that proteins involved in polyketide biosynthesis by the psyllid endosymbiont Profftella were up-regulated in CLas(+) Asian citrus psyllid (ACP) insects. A novel polyketide in the psyllid was also discovered, and the ratio of two polyketides was drastically altered in CLas(+) insects. Profftella occurs only in ACP and in all ACP. This highly specific relationship of Profftella to ACP is seen as a key point of leverage in our effort to find highly target-specific strategies to control ACP and combat HLB. ARS scientists modified a published method to extract and purify the polyketide, called diaphorin, from ACP that resulted in double the published yield per insect. This afforded pure material to probe the biological activity of diaphorin in a range of experimental systems. Purified diaphorin also allowed us to crystallize the compound and obtain a three-dimensional structure of the molecule, which is critical for understanding the biochemical basis for its biological activity and provides insights into how to chemically modify the molecule to exploit it for control of ACP and, ultimately HLB disease. In addition we structurally characterized two related compounds that co-extract from ACP along with diaphorin. One of these is downregulated in CLas-infected ACP and the other is up-regulated. The latter was also detected artificial sucrose-solution diet fed upon by infected ACP therefore must be present in ACP saliva, is likely transferred to the host-plant tissue and may have effects on the plant's immune response to ACP feeding and/or CLas that can reveal unknown control points in CLas transmission that can be exploited for control.

10. Microscopy analysis of the gut of the Asian citrus psyllid vector revealed that Candidatus Liberibacter asiaticus (CLas) induces apoptosis, or programmed cell death. The gut is the first interface between the vector and the pathogen, and thus an important interface for the development of interdiction strategies to block the transmission of the citrus greening pathogen. An ARS scientist in Ithaca, New York, discovered that CLas induces gut cells to commit suicide, that is, to initiate a process called programmed cell death (apoptosis). This finding is critically important to our understanding of pathogen transmission by this insect vector. Research by ARS scientists and others have shown that for adult insects to efficiently transmit the citrus greening pathogen, the pathogen must be acquired during the nymphal stage of development. The apoptosis phenotype is not induced in nymphs. The finding of CLas-induced apoptosis in the adult gut may explain why adult insects must acquire the pathogen during the nymphal stage. Moreover, ARS scientists in Ithaca, New York showed that nymphs do not respond as dramatically to the pathogen as do adult insects at the molecular level. Taken together, these findings could be leveraged to develop new, targeted molecular strategies to block nymphs specifically from acquiring the pathogen.

11. Development of new proteomic methods aids in sensitive protein analysis and identification in insect vectors that spread plant diseases. Until recently, no proteomic methods were systematically tested for their suitability for studying aphids, psyllids and other insect vectors of plant pathogens. The success of any proteomics experiment depends on the initial protein extraction method that is used. There are a few properties of aphids, as with all insects, that makes protein extraction technically challenging. systematically comparing protein extraction methods, ARS scientists in Ithaca, New York determined the most efficient workflow for proteomics sample preparation in aphids, a method that has since been applied to study psyllids and whiteflies. Moreover, ARS scientists in Ithaca, New York improved the mass spectrometry data analysis pipeline for psyllids by contributing to the annotation of the Asian citrus psyllid genome. These studies provided critical insights into how to optimize proteomics experiments in recalcitrant, complex systems such as aphid and psyllid vectors. The method enabled an ARS scientist in Ithaca, New York to obtain a collaborative grant with a cooperator at Columbia University and ARS scientists at Fort Pierce, Florida, to develop a biosensor for detecting psyllids harboring the citrus greening bacterium.

12. ARS scientists in Ithaca, New York reported the first example where an insect vector modulates its virus transmission efficiency according to interactions with its host plant. During experiments, they unexpectedly observed that a clonal lineage of the aphid, Myzus persicae, reared on different host plants showed a dramatic difference in its ability to transmit the Polerovirus, Potato leafroll virus (PLRV). PLRV is a circulative plant virus that is exclusively transmitted by aphids in nature. It infects potato and causes net necrosis in some cultivars, leading to economic damage of the infected crop. Aphids reared on turnip transmitted PLRV less well as compared to conspecifics on Physalis floridana. Using quantitative proteomics, ARS scientists showed that cathepsin B is the major protein differentially expressed in M. persicae reared on turnips vs. P. floridana. Inhibiting cathepsin B activity in turnip-reared aphids increased virus transmission efficiency and inhibiting cathepsin B activity in P. floridana-reared aphids reduced virus transmission efficiency. These data show that cathepsin B is the first aphid protein target whose activity can be modulated for regulation of virus transmission - data that pose a tremendous opportunity for the development of novel plant and animal virus transmission control strategies.

Review Publications
Torres-Acosta, R., Humber, R.A., Sanchez-Pena, S. 2016. Zoophthora radicans (Entomophthorales), fungal pathogen of Bagrada hilaris and Bactericera cockerelli (Hemiptera: Pentatomidae & Triozidae) in Mexico: Prevalence, bioassays, & environmental influences on conidial morphology. Journal of Invertebrate Pathology. doi:10.1016/j.jip.2016.07.017.
Montalva, C., Silva, J.J., Buchter, S., Fernandes, E.K., Luz, C., Humber, R.A. 2016. Conidiobolus macrosporus (Entomophthorales), a mosquito pathogen in Central Brazil. Journal of Invertebrate Pathology. 139:102-108.
Humber, R.A. 2016. Seeking stability for research and applied uses of entomopathogenic fungi as biological control agents. Journal of Asia-Pacific Entomology. 19:1019-1025.
Chavez, J.D., Eng, J.K., Cilia, M., Rivera, K., Zhong, X., Wu, X., Schweppe, D., Allen, T., Khurgel, M., Kumar, A., Lampropoulos, A., Larsson, M., Maity, S., Morozov, Y., Pathmasiri, W., Perez-Neut, M., Pineyro-Ruiz, C., Polina, E., Post, S., Rider, M., Tokmina-Roszyk, D., Parrine Sant'Ana, D., Bruce, J.E. 2016. A general method for targeted quantitative cross-linking mass spectrometry. PLoS One. 11(12):e0167547.
Donzelli, B.G., Turgeon, B., Gibson, D.M., Krasnoff, S. 2016. Disruptions of the genes involved in lysine biosynthesis, iron acquisition, and secondary metabolisms affect virulence and fitness in Metarhizium robertsii. Fungal Genetics and Biology. DOI: 10.1016/j.fgb.2016.11.005.
Montalva, C., Batra, M., Rojas, E., Arismendi, N., Rodrigues, J., Valenzuela, E., Humber, R.A. 2017. Characterization and virulence of Chilean Lecanicillium (Hypocreales: Cordycipitaceae) isolates on Cinara cupressi (Hemiptera: Aphididae). Biocontrol. doi:10.1007/s10526-017-9817-9.
Rodrigues, J., Luz, C., Humber, R.A. 2017. New insights into the in vitro development and virulence of Culicinomyces spp. as fungal pathogens of Aedes aegypti. Journal of Invertebrate Pathology. doi:10.1016/j.jip.2017.03.012.
Alexander, M., Cilia, M. 2017. A molecular tug-of-war: Global plant proteome changes during viral infection. Current Plant Biology. 5:13-24.
Scorsetti, A., Jensen, A., Lopez Lastra, C., Humber, R.A. 2011. First report of Pandora neoaphidis resting spore formation in vivo in aphid hosts under field conditions. Fungal Biology. doi:10.1016/j.funbio.2011.11.002.
Luangsa-Ard, J., Mongkolsamrit, S., Thanakitpipattana, D., Khonsanit, A., Tasanathai, K., Noisripoom, W., Humber, R.A. 2017. Clavicipitaceous entomopathogens: New species of Metarhizium and a new genus Nigelia. Mycological Progress. 16(4):369-391.
Pinheiro, P., Ghanim, M., Rebelo, A., Santos, R., Orsburn, B.C., Gray, S.M., Cilia, M. 2016. Host plants indirectly influence plant virus transmission by altering gut cysteine protease activity of aphid vectors. Molecular and Cellular Proteomics. DOI: 10.1074/mcp.M116.063495.
Deblasio, S.L., Johnson, R., Maccoss, M., Gray, S.M., Cilia, M. 2016. Model system-guided protein interaction mapping for virus isolated from phloem tissue. Journal of General Virology. 15:4601-4611.