Location: Emerging Pests and Pathogens Research
2016 Annual Report
Objectives
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.
Approach
To address the need for new biological control agents, this project maintains the ARS Collection of Entomopathogenic Fungi (ARSEF), one of three official ARS microbial germplasm repositories (http://www.ars.usda.gov/Main/docs.htm?docid=12125). ARSEF is the world’s largest, most diverse, and most comprehensive culture collection of fungal pathogens of insects and other invertebrates, and provides services and conducts a mission unduplicated elsewhere within ARS. New species and strains of agriculturally important fungi are discovered, described and accessioned into ARSEF, as well as incorporating isolates from orphaned culture collections dealing with entomopathogenic fungi. The collection includes fungi from more than 100 countries, and serves many hundreds of clients throughout the world who contribute and receive isolates. ARSEF has the world’s best facilities to culture and preserve fungi from insects, arachnids, and other invertebrates, and the curator is an active collaborator and co-author on publications from throughout the world dealing with a very wide range of topics involving fungal entomopathogens. We conduct research on the systematics, genetic diversity, and biology of these fungi.
Progress Report
ARS Collection of Entomopathogenic Fungal Cultures (ARSEF): As of July 15, 2016, ARSEF comprises 13051 isolates from 715 fungal taxa (up from 704 in 2015) from 2439 locations worldwide (up from 2399 in 2015), and 1308 host taxa or other non-insect substrates. A Brazilian ‘Science Without Borders’ project is funding research on the diversity and activity of fungal pathogens of dipteran insects (mainly mosquitoes, blackflies, and other fly-like insects with immature stages in aquatic habitats) that transmit animal, including human, pathogens. We collected additional isolates of the oomycete pathogen Leptolegnia chapmanii that has excellent known capacities to kill larvae of Aedes aegypti (the principal vector of Zika virus and many other significant pathogens). New fungi previously encountered under this program for the first time in South America have included several isolates of another significant mosquito-pathogenic oomycete genus, Lagenidium (or, possibly, of the related genus Aphanomyces; positive identifications are awaiting genomic sequencing confirmation), and of an unidentified (possibly new) species of the blastocladiomycotan genus Coelomomyces. Metarhizium anisopliae reduced Aedes aegypti egg viability, and negatively affected all other life stages of this important mosquito. The ovicidal activity of this fungus is not due to direct infections of the eggs but may be due to either smothering of the eggs by fungal growth or by secretion of biologically active compounds toxic to the eggs. Collaboration with scientists at the Michigan State University on the entomopathogenic oomycetes classified in Lagenidium indicated that L. giganteum (a well-recognized mosquito pathogen) is phylogenetically resolvable into at least two species, one of which is being described as new, and has raised significant issues about the validity of other genera of the order Lagenidiales (Peronosporomycetes). The importance of the discovery of all of these rapidly effective mosquito pathogens under this project has been underscored by the now acute global health concerns about A. aegypti and its ability to transmit Zika virus. An entomophthoralean pathogen identified as Conidiobolus macrosporus was found to cause significant mortality of adult houseflies that are economically significant pests in poultry houses worldwide. This was the first demonstration of any Conidiobolus species causing such significant mortality (up to 100% within three days after exposure).
CHEMICAL BIOLOGY OF ENTOMOPATHOGENS: The use of insect-pathogenic fungi as biopesticides provides environmentally friendly and cheaper alternatives to chemical insecticides, but they tend to be less efficacious in most agriculture settings and some produce animal toxins that compromise their safe release as commercial products. Our work has focused on improving commercially important broad-spectrum insect-pathogenic biocontrol fungi through a better understanding of the genetic mechanisms that regulate their success as pathogens of insects. We have produced a large panel of genetically modified mutants of Metarhizium robertsii (MR) in which genes that are important to virulence and are required for the production of small molecules including toxins have been inactivated. We have now produced a mutant lacking a master enzyme required for the synthesis of the vast majority of small molecule metabolites and determined that its virulence toward insect hosts was severely reduced relative to the parent strain indicating that one or more of these metabolites plays a key role in pathogenicity.
Pathogens and their hosts in most disease systems engage in a battle for iron that has driven the evolution of small molecule metabolites called siderophores that are produced by many microbes including insect pathogenic fungi and are highly effective at scavenging and retaining iron. Our experiments comparing the expression of genes of iron-supplied and iron-deprived cultures of MR revealed that iron-restriction vigorously stimulates production of several different types of genes related to iron acquisition. These included two genes that we have now shown to be responsible for adding the sugar groups that are a unique chemical feature of MR siderophores that we characterized previously. This was accomplished by genetically engineering MR to lack one or both of the genes responsible for adding the sugar groups. In addition, a gene coding for a polyketide synthase enzyme (PKS) with a presumed small molecule end product also was upregulated under iron-deprivation conditions. We successfully produced a mutant strain of MR in which this gene was disrupted. The significance of these unique chemical signatures to the performance of the fungus as a biopesticide can now be assayed using these genetically engineered mutant strains.
Genomic comparisons showed that the entire suite of genes that is upregulated in MR in response to iron starvation, most importantly the genes that chemically modify the siderophores with sugar groups, is present in a few other fungi including Beauveria bassiana (BB), another important cosmopolitan insect pathogenic biocontrol fungus. Guided by this discovery, we recently purified and structurally characterized a large family of novel iron-scavenging molecules that differ from those produced by MR but also contain modified sugar groups.
We have also chemically purified and have preliminary chemical structural data for yet another group of novel siderophores from the fungus Hirstuella citriformis which occurs in nature as a pathogen of the Asian citrus psyllid (ACP), the vector that spreads the bacterium, Candidatus Liberibacter asiaticus (CLas), associated with citrus greening disease (aka Huanglongbing or HLB). Study of these compounds advances our knowledge of how these biocontrol fungi acquire and manage iron and how these traits contribute to the ability of these fungi to successfully infect and kill insects injurious to agriculture, and in the case of ACP, how they may affect the microbial community associated with HLB, and the vector's ability to transmit the CLas.
VECTOR BIOLOGY: We are developing novel HLB management tools that block insect vector transmission of the bacterial pathogen. Control of the vector is the most effective way of preventing the spread of citrus greening disease. The citrus greening bacterium, CLas, must spread throughout the body of the psyllid vector relying on highly tuned interactions between psyllid and bacterial protein molecules. We are using a revolutionary technology called Protein Interaction Reporter (PIR) technology to characterize in vivo protein interactions that regulate transmission of CLas by psyllids and develop inhibitors of these interactions as vector control agents. We used mass spectrometry-based proteomics to discover proteins in the psyllid associated with acquisition and transmission of the citrus greening pathogen. Developmental factors play a central role in CLas transmission by the psyllid – while both nymphs and adults are able to acquire the pathogen, only insects that have acquired CLas as nymphs are able to effectively transmit the pathogen. The proteome changes reflect this developmental difference in CLas acquisition. The ACP is host to the bacterial endosymbionts ‘Candidatus Carsonella rudii’, ‘Candidatus Profftella armatura’, and Wolbachia. These symbionts are required by the ACP for metabolic and possibly defensive functions, and interactions between the psyllid and its endosymbionts represent promising targets for vector control. Proteomics, qPCR, and fluorescent microscopy are being applied to elucidate the role of the psyllid endosymbionts in CLas transmission. Our research program is also working on the fundamental and applied biology of aphids and circulative plant viruses, including the Potato leafroll virus (PLRV). The RTP is the PLRV minor capsid protein. This protein functions both as a structural component of the virion and as a non-structural protein that operates in an unknown manner to retain virus in phloem (vascular) tissues of the plant. Knowing what host proteins interact with proteins on the virus particle or the soluble RTP protein can identify potential targets to exploit in plants that may prevent or reduce virus infection. By quantifying differences in the protein interaction networks, we identified four distinct classes of PLRV-plant interactions: those plant and non-structural viral proteins interacting with assembled coat protein; plant proteins in complex with both coat protein and RTP; plant proteins in complex with the RTP; and plant proteins that had higher affinity for virions lacking the RTP. To better understand how the various proteins interact we used PIR. This work is the first report of a host-pathogen protein interaction network that provides topological features of the protein-protein interactions. PLRV particles have hot spots of protein interaction and multifunctional surface features, revealing how these plant viruses maximize their use of binding interfaces. These data can also be used to map structural features of proteins that are difficult to crystallize or study using X-ray technologies. Demonstrating and mapping the direct interactions between host and virus proteins during infection has been a major challenge for the field of virology and these results illuminate the functional diversity of the PLRV-host protein interaction network and demonstrate the usefulness of PIR technology for precision mapping of functional host-pathogen protein interaction topologies.
Accomplishments
1. Significant activity of a fungal pathogen against unembryonated eggs of Aedes aegypti mosquitoes proven. Metarhizium anisopliae s.l. is well known to be effective against adults and larvae of Aedes aegypti, and has also been shown to suppress the eclosion of the eggs of this mosquito. Studies on exposures to this fungus of unembryonated (newly deposited) eggs of A. aegypti have shown that even this very earliest stage of the host can be negatively affected by the presence of this fungus, but it appears that this suppression is not due to any ability of the fungus to penetrate and to grow inside the eggs. The implications of this study suggest that applications of M. anisopliae conidia to the intermittently wetted habitats in which A. aegypti usually lays its eggs may help to reduce the populations even without any direct applications of the fungus to the target host.
2. Expanded knowledge about species of Metarhizium present in Brazil. Work with two Brazilian isolates of Metarhizium–one that was described from a cockroach, M. blattodeae, collected as part of the Science Without Borders project, and another that has been used for a large number of studies by the laboratory that is home to this project over a course of several years that has been identified as M. anisopliae sensu lato but that is now understood through genomic data on this isolate to be a new, unidentified species–were both shown in studies of the protein profiles on the surfaces of their spores as shown with MALDI-TOF mass spectrometry to differ distinctly from M. anisopliae sensu stricto based on genomic sequence data. This result further validates the utility of MALDI-TOF for species identifications published in 2014 jointly between ARSEF and Brazilian scientists for recognizing new taxa within this important genus of entomopathogens.
3. Clarification of systematics of mosquito-pathogenic oomycetes in genus Lagenidium. Collaboration between ARSEF and scientists at Michigan State University on the genome-based identifications of mosquito-pathogenic species of the oomycete genus Lagenidium confirm that L. giganteum (which has become fairly well known as a mosquito pathogen) is a heterogeneous assemblage of at least two species (one of which is being described as new), and that both of these Lagenidium species have host spectra that include, for one species, human and environmentally derived isolates, and for the newly recognized segregate, a series of isolates affecting nematodes in Taiwan.
4. Long confusion about identification of a major fungal pathogen of scales resolved. After many years of study in Brazil, a major fungal pathogen affecting citrus orthezia scales, Praelongorthezia praelonga, has been definitively identified as Colletotrichum nymphaeae based on genomic and morphological/developmental characteristics. The identification of this fungus has been problematic and in question before 2005. This particular fungal genus from the ascomycete order Glomerellales has been known to have some capacities to be pathogenic for insects, but the few species involved are only infrequently (and poorly) known from insect hosts amid a genus whose species are primarily plant-associated. C. nymphaeae has been demonstrated to be a very effective pathogen for helping to control populations of this serious scale insect pest.
5. Detailed analysis of secondary metabolite genes from insect-pathogenic Metarhizium biocontrol fungi completed. The fungal genus Metarhizium comprises greater than 30 insect-pathogenic species that infect a wide range of insect species, worldwide, including many injurious agricultural pests. Thus, this group of fungi is important because of its potential as a source of natural biocontrol agents of insects and several species of Metarhizium have been commercialized as safe pesticides and are now important components of integrated pest management programs. Metarhizium fungi produce an exceptionally large number of unique small molecules, aka secondary metabolites. In light of the key role played by these small molecules in pathogenic processes an ARS researcher in Ithaca, New York in collaboration with a scientist from Cornell University conducted a detailed analysis of the genomes of nine sequenced Metarhizium species to identify and compare the genes that control small molecule metabolite biosynthesis. This study opens the door to functional analyses of these genes using modern genetic engineering techniques that can inactivate specific biosynthetic pathways and allow us to understand how these contribute to the ability of this fungus to successfully infect and kill insects and, ultimately, how they impact the success of these fungi as biocontrol agents of pests of agriculture.
6. Drechemeria coniospora genome sequence completed and toxins characterized. Although annual crop losses to plant-parasitic nematodes are estimated at a staggering $157 billion worldwide, options for nematode pest management are very limited due to environmental safety concerns. This situation warrants further research to discover effective but environmentally responsible alternatives to replace legislatively withdrawn nematicides. Nematode-pathogenic fungi exploited as biocontrol agents, may be part of the answer when applied in the context of integrated pest management systems. Thus, understanding the mechanisms governing the interactions between these fungi and their nematode hosts is a key issue for crop protection. Drechmeria coniospora (DC) is a highly specialized fungal pathogen that attacks plant-pathogenic nematodes and lives out most of its life cycle in the host. It is critical to deepen our understanding of the basic biology of this fungus to progress toward its use for biological control of economically injurious nematodes. Thus, in collaboration with scientists at Cornell University, the University of Arizona, and the Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, ARS scientists at Ithaca, New York contributed to the sequencing of the DC genome from ARSEF fungi, identified genes responsible for the biosynthesis of potent antifungal peptide toxins involved in the interaction with the nematode host, and chemically characterized these toxins, which may have agro-economic applications. Analysis of the sequenced genome and comparisons with other fungal pathogens reveal that although DC has reduced the number of genes related to the ability to subsist on soil detritus (i.e. dead plant and animal material) it shares most of the genes that are responsible for successfully parasitizing its host, most notably genes coding for proteins required for attachment to and penetration of the host.
None.
Review Publications
Pinheiro, P., Kliot, A., Ghanim, M., Cilia, M. 2015. Is there a role for symbiotic bacteria in plant virus transmission? Current Opinion in Insect Science. 8:69-78.
Ramsey, J.S., Johnson, R., Hoki, J., Kruse, A., Mahoney, J., Hilf, M.E., Hunter, W.B., Hall, D.G., Shroeder, F., Maccoss, M., Cilia, M. 2015. Metabolic interplay between the Asian citrus psyllid and its Profftella symbiont: An Achilles’ heel of the citrus greening insect vector. PLoS One. DOI: 10.1371/journal.pone.0140826.
Montalva, C., Collier, K., Fernandes, E., Lopes, R., Inglis, P., Luz, C., Humber, R.A. 2016. A natural fungal infection of a sylvatic cockroach with Metarhizium blattodeae sp. nov., a member of the M. flavoviride species complex. Fungal Biology. 120:655-665.
Montalva, C., Collier, K., Luz, C., Humber, R.A. 2016. Pandora bullata (Entomophthorales) affecting calliphorid flies in Central Brazil. ACTA TROPICA. 158:177-180.
Montalva, C., Dos Santos, K., Collier, K., Fernandes, E., Luz, C., Humber, R.A. 2016. First report of Leptolegnia chapmanii (Peronosporomycetes: Saprolegniales) affecting mosquitoes in Central Brazil. Journal of Invertebrate Pathology. 136:109-116.
Duarte, R.T., Goncalves, K.C., Espinosa, D., Moreira, L.F., De Bortoli, S.A., Humber, R.A., Polanczyk, R.A. 2016. Entomopathogenic fungi as biological control agents of diamondback moth (Lepidoptera: Plutellidae)and compatibility with chemical insecticides. Journal of Economic Entomology. 109(2): 594-601.
Deblasio, S.L., Chavez, J., Alexander, M., Ramsey, J.S., Eng, J., Mahoney, J., Gray, S.M., Bruce, J., Cilia, M. 2015. Visualization of host-polerovirus interaction topologies using Protein Interaction Reporter technology. Journal of Virology. 90:1973-1987.
Rosen, R., Kanakala, S., Kliot, A., Pakkianathan, B., Abu Farich, B., Santana-Magal, N., Elimelech, M., Kontsedalov, S., Lebedev, G., Cilia, M., Ghanim, M. 2015. Persistent, circulative transmission of begomoviruses by whitefly vectors. Current Opinion in Virology. 15:1-8.
Hong, I., Kang, P., Kim, K., Nam, S., Lee, M., Choi, Y., Kim, N., Kim, K., Lee, K., Humber, R.A. 2011. Fruit body formation on silkworm by Cordyceps militaris. Mycobiology. 38:128-132.
Mascarin, G.M., Molina Guarin, J.H., Pauli, G., Humber, R.A., Delalibera, Jr., I. 2016. Seasonal prevalence of the insect pathogenic fungus Colletotrichum nymphaeae in Brazilian citrus groves under different chemical pesticide regimes. Fungal Ecology. 22:43-51.
Humber, R.A. 2016. Book Chapter Title: Entomophthoromycota: a new overview of some of the oldest terrestrial fungi. In Biology of Microfungi (D. Li, ed). Cham: Springer International Publishing Switzerland. p. 127-145.
Montalva, C., Luz, C., Humber, R.A. 2016. First report on the entomopathogenic genus Neozygites (Entomophthoromycota) and Neozygites osornensis on aphids in Brazil. Neotropical Entomology. 45:227-230.
Donzelli, B.G., Krasnoff, S. 2016. Molecular genetics of secondary chemistry in Metarhizium fungi. Advances in Genetics. 94:365-436.
Zhang, L., Zhou, Z., Guo, Q., Fokkens, L., Miskei, M., Pocsi, I., Zhang, W., Chen, M., Wang, L., Sun, Y., Donzelli, B., Gibson, D.M., Nelson, D.R., Luo, J., Rep, M., Hang, L., Yang, S., Wang, J., Krasnoff, S., Xu, Y., Molnar, I., Lin, M. 2016. Insights into adaptations to a near-obligate nematode endoparasitic lifestyle from the finished genome of Drechmeria coniospora. Scientific Reports. DOI: 10.1038/srep23122.
