Ubiquity of Insect-Derived Nitrogen Transfer to Plants by Endophytic Insect-Pathogenic Fungi: an Additional Branch of the Soil Nitrogen Cycle

ABSTRACTThe study of symbiotic nitrogen transfer in soil has largely focused on nitrogen-fixing bacteria. Vascular plants can lose a substantial amount of their nitrogen through insect herbivory. Previously, we showed that plants were able to reacquire nitrogen from insects through a partnership with the endophytic, insect-pathogenic fungusMetarhizium robertsii. That is, the endophytic capability and insect pathogenicity ofM. robertsiiare coupled so that the fungus acts as a conduit to provide insect-derived nitrogen to plant hosts. Here, we assess the ubiquity of this nitrogen transfer in fiveMetarhiziumspecies representing those with broad (M. robertsii,M. brunneum, andM. guizhouense) and narrower insect host ranges (M. acridumandM. flavoviride), as well as the insect-pathogenic fungiBeauveria bassianaandLecanicillium lecanii. Insects were injected with15N-labeled nitrogen, and we tracked the incorporation of15N into two dicots, haricot bean (Phaseolus vulgaris) and soybean (Glycine max), and two monocots, switchgrass (Panicum virgatum) and wheat (Triticum aestivum), in the presence of these fungi in soil microcosms. AllMetarhiziumspecies andB. bassianabut notL. lecaniishowed the capacity to transfer nitrogen to plants, although to various degrees. Endophytic association by these fungi increased overall plant productivity. We also showed that in the field, where microbial competition is potentially high,M. robertsiiwas able to transfer insect-derived nitrogen to plants.Metarhiziumspp. andB. bassianahave a worldwide distribution with high soil abundance and may play an important role in the ecological cycling of insect nitrogen back to plant communities.

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MrSkn7 Controls Sporulation, Cell Wall Integrity, Autolysis, and Virulence in Metarhizium robertsii

Eukaryotic Cell ◽

10.1128/ec.00266-14 ◽

2015 ◽

Vol 14 (4) ◽

pp. 396-405 ◽

Cited By ~ 17

Author(s):

Yanfang Shang ◽

Peilin Chen ◽

Yixiong Chen ◽

Yuzhen Lu ◽

Chengshu Wang

Keyword(s):

Cell Wall ◽

Target Genes ◽

Response Regulator ◽

Pathogenic Fungus ◽

Functional Divergence ◽

Pathogenic Fungi ◽

Wild Type ◽

Metarhizium Robertsii ◽

Content Type ◽

Cell Autolysis

ABSTRACT Two-component signaling pathways generally include sensor histidine kinases and response regulators. We identified an ortholog of the response regulator protein Skn7 in the insect-pathogenic fungus Metarhizium robertsii , which we named MrSkn7. Gene deletion assays and functional characterizations indicated that MrSkn7 functions as a transcription factor. The MrSkn7 null mutant of M. robertsii lost the ability to sporulate and had defects in cell wall biosynthesis but was not sensitive to oxidative and osmotic stresses compared to the wild type. However, the mutant was able to produce spores under salt stress. Insect bioassays using these spores showed that the virulence of the mutant was significantly impaired compared to that of the wild type due to the failures to form the infection structure appressorium and evade host immunity. In particular, deletion of MrSkn7 triggered cell autolysis with typical features such as cell vacuolization, downregulation of repressor genes, and upregulation of autolysis-related genes such as extracellular chitinases and proteases. Promoter binding assays confirmed that MrSkn7 could directly or indirectly control different putative target genes. Taken together, the results of this study help us understand the functional divergence of Skn7 orthologs as well as the mechanisms underlying the development and control of virulence in insect-pathogenic fungi.

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The Intermediates in Branched-Chain Amino Acid Biosynthesis Are Indispensable for Conidial Germination of the Insect-Pathogenic Fungus Metarhizium robertsii

Applied and Environmental Microbiology ◽

10.1128/aem.01682-20 ◽

2020 ◽

Vol 86 (20) ◽

Author(s):

Feifei Luo ◽

Hongxia Zhou ◽

Xue Zhou ◽

Xiangyun Xie ◽

You Li ◽

...

Keyword(s):

Amino Acid ◽

Pathogenic Fungus ◽

Pathogenic Fungi ◽

Conidial Germination ◽

Keto Acid ◽

Metarhizium Robertsii ◽

Content Type ◽

Branched Chain Amino Acid ◽

Key Enzyme ◽

Branched Chain

ABSTRACT Metarhizium spp. are well-known biocontrol agents used worldwide to control different insect pests. Keto-acid reductoisomerase (ILVC) is a key enzyme for branched-chain amino acid (BCAA) biosynthesis, and it regulates many physiological activities. However, its functions in insect-pathogenic fungi are poorly understood. In this work, we identified MrilvC in M. robertsii and dissected its roles in fungal growth, conidiation, germination, destruxin biosynthesis, environmental stress response, and insecticidal virulence. BCAA metabolism affects conidial yields and germination. However, BCAAs cannot recover the conidial germination of an MrilvC-deficient strain. Further feeding assays with intermediates showed that some conidia of the ΔMrilvC mutant start to germinate. Therefore, it is the germination defect that causes the complete failures of conidial penetration and pathogenicity in the ΔMrilvC mutant. In conclusion, we found intermediates in BCAA biosynthesis are indispensable for Metarhizium robertsii conidial germination. This study will advance our understanding of the fungal germination mechanism. IMPORTANCE Branched-chain amino acid (BCAA) metabolism plays a significant role in many biological activities beyond protein synthesis. Spore germination initiates the first stage of vegetative growth, which is critical for the virulence of pathogenic fungi. In this study, we demonstrated that the keto-acid reductoisomerase MrILVC, a key enzyme for BCAA biosynthesis, from the insect-pathogenic fungus Metarhizium robertsii is associated with conidial germination and fungal pathogenicity. Surprisingly, the germination of the ΔMrilvC mutant was restored when supplemented with the intermediates of BCAA metabolism rather than three BCAAs. The result was significantly different from that of plant-pathogenic fungi. Therefore, this report highlights that the intermediates in BCAA biosynthesis are indispensable for conidial germination of M. robertsii.

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Three sympatrically occurring species of Metarhizium show plant rhizosphere specificity

Microbiology ◽

10.1099/mic.0.051102-0 ◽

2011 ◽

Vol 157 (10) ◽

pp. 2904-2911 ◽

Cited By ~ 88

Author(s):

Michael Wyrebek ◽

Cristina Huber ◽

Ramanpreet Kaur Sasan ◽

Michael J. Bidochka

Keyword(s):

Tree Species ◽

Acer Saccharum ◽

Panicum Virgatum ◽

Sugar Maple ◽

Root Exudate ◽

Pathogenic Fungus ◽

Plant Roots ◽

Metarhizium Robertsii ◽

Grass Roots

Here we tested the hypothesis that species of the soil-inhabiting insect-pathogenic fungus Metarhizium are not randomly distributed in soils but show plant-rhizosphere-specific associations. We isolated Metarhizium from plant roots at two sites in Ontario, Canada, sequenced the 5′ EF-1α gene to discern Metarhizium species, and developed an RFLP test for rapid species identification. Results indicated a non-random association of three Metarhizium species (Metarhizium robertsii, Metarhizium brunneum and Metarhizium guizhouense) with the rhizosphere of certain types of plant species (identified to species and categorized as grasses, wildflowers, shrubs and trees). M. robertsii was the only species that was found associated with grass roots, suggesting a possible exclusion of M. brunneum and M. guizhouense. Supporting this, in vitro experiments showed that M. robertsii conidia germinated significantly better in Panicum virgatum (switchgrass) root exudate than did M. brunneum or M. guizhouense. M. guizhouense and M. brunneum only associated with wildflower rhizosphere when co-occurring with M. robertsii. With the exception of these co-occurrences, M. guizhouense was found to associate exclusively with the rhizosphere of tree species, predominantly Acer saccharum (sugar maple), while M. brunneum was found to associate exclusively with the rhizosphere of shrubs and trees. These associations demonstrate that different species of Metarhizium associate with specific plant types.

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Insect herbivory facilitates the establishment of an invasive plant pathogen

ISME Communications ◽

10.1038/s43705-021-00004-4 ◽

2021 ◽

Vol 1 (1) ◽

Author(s):

Martin M. Gossner ◽

Ludwig Beenken ◽

Kirstin Arend ◽

Dominik Begerow ◽

Derek Peršoh

Keyword(s):

Invasive Plant ◽

Pathogenic Fungus ◽

Feeding Activity ◽

Insect Herbivory ◽

Forest Health ◽

Mechanical Damage ◽

Pathogenic Fungi ◽

Fungal Colonization ◽

Feeding Damage ◽

The Impact

AbstractPlants can be severely affected by insect herbivores and phytopathogenic fungi, but interactions between these plant antagonists are poorly understood. We analysed the impact of feeding damage by the abundant herbivore Orchestes fagi on infection rates of beech (Fagus sylvatica) leaves with Petrakia liobae, an invasive plant pathogenic fungus. The fungus was not detected in hibernating beetles, indicating that O. fagi does not serve as vector for P. liobae, at least not between growing seasons. Abundance of the fungus in beech leaves increased with feeding damage of the beetle and this relationship was stronger for sun-exposed than for shaded leaves. A laboratory experiment revealed sun-exposed leaves to have thicker cell walls and to be more resistant to pathogen infection than shaded leaves. Mechanical damage significantly increased frequency and size of necroses in the sun, but not in shade leaves. Our findings indicate that feeding damage of adult beetles provides entry ports for fungal colonization by removal of physical barriers and thus promotes infection success by pathogenic fungi. Feeding activity by larvae probably provides additional nutrient sources or eases access to substrates for the necrotrophic fungus. Our study exemplifies that invasive pathogens may benefit from herbivore activity, which may challenge forest health in light of climate change.

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Glycerol-3-Phosphate Acyltransferase Contributes to Triacylglycerol Biosynthesis, Lipid Droplet Formation, and Host Invasion in Metarhizium robertsii

Applied and Environmental Microbiology ◽

10.1128/aem.02905-13 ◽

2013 ◽

Vol 79 (24) ◽

pp. 7646-7653 ◽

Cited By ~ 38

Author(s):

Qiang Gao ◽

Yanfang Shang ◽

Wei Huang ◽

Chengshu Wang

Keyword(s):

Pathogenic Fungus ◽

Single Copy ◽

Fungal Genome ◽

Model Organisms ◽

Glycerol Phosphate ◽

Fungal Virulence ◽

Metarhizium Robertsii ◽

Content Type ◽

Rate Limiting Step

ABSTRACTEnzymes involved in the triacylglycerol (TAG) biosynthesis have been well studied in the model organisms of yeasts and animals. Among these, the isoforms of glycerol-3-phosphate acyltransferase (GPAT) redundantly catalyze the first and rate-limiting step in glycerolipid synthesis. Here, we report the functions of mrGAT, a GPAT ortholog, in an insect-pathogenic fungus,Metarhizium robertsii. Unlike in yeasts and animals, a single copy of the mrGAT gene is present in the fungal genome and the gene deletion mutant is viable. Compared to the wild type and the gene-rescued mutant, the ΔmrGATmutant demonstrated reduced abilities to produce conidia and synthesize TAG, glycerol, and total lipids. More importantly, we found that mrGAT is localized to the endoplasmic reticulum and directly linked to the formation of lipid droplets (LDs) in fungal cells. Insect bioassay results showed thatmrGATis required for full fungal virulence by aiding fungal penetration of host cuticles. Data from this study not only advance our understanding of GPAT functions in fungi but also suggest that filamentous fungi such asM. robertsiican serve as a good model to elucidate the role of the glycerol phosphate pathway in fungal physiology, particularly to determine the mechanistic connection of GPAT to LD formation.

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A novel cascade allows Metarhizium robertsii to distinguish cuticle and hemocoel microenvironments during infection of insects

PLoS Biology ◽

10.1371/journal.pbio.3001360 ◽

2021 ◽

Vol 19 (8) ◽

pp. e3001360

Author(s):

Xing Zhang ◽

Yamin Meng ◽

Yizhou Huang ◽

Dan Zhang ◽

Weiguo Fang

Keyword(s):

Transcription Factor ◽

Histone Deacetylase ◽

Pathogenic Fungus ◽

Pathogenic Fungi ◽

Insect Cuticle ◽

Metarhizium Robertsii ◽

Fungal Pathogenicity ◽

Histone 3 ◽

Underlying Mechanisms ◽

Epigenetic Repression

Pathogenic fungi precisely respond to dynamic microenvironments during infection, but the underlying mechanisms are not well understood. The insect pathogenic fungus Metarhizium robertsii is a representative fungus in which to study broad themes of fungal pathogenicity as it resembles some major plant and mammalian pathogenic fungi in its pathogenesis. Here we report on a novel cascade that regulates response of M. robertsii to 2 distinct microenvironments during its pathogenesis. On the insect cuticle, the transcription factor COH2 activates expression of cuticle penetration genes. In the hemocoel, the protein COH1 is expressed due to the reduction in epigenetic repression conferred by the histone deacetylase HDAC1 and the histone 3 acetyltransferase HAT1. COH1 interacts with COH2 to reduce COH2 stability, and this down-regulates cuticle penetration genes and up-regulates genes for hemocoel colonization. Our work significantly advances the insights into fungal pathogenicity in insects.

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Availability of carbon and nitrogen in soil affects Metarhizium robertsii root colonization and transfer of insect-derived nitrogen

FEMS Microbiology Ecology ◽

10.1093/femsec/fiz144 ◽

2019 ◽

Vol 95 (10) ◽

Author(s):

Larissa Barelli ◽

Scott W Behie ◽

Michael J Bidochka

Keyword(s):

Ammonium Nitrate ◽

Root Colonization ◽

Pathogenic Fungus ◽

Broth Culture ◽

Nitrogen Transfer ◽

Symbiotic Association ◽

Metarhizium Robertsii ◽

Carbon And Nitrogen ◽

Ecological Importance ◽

Fungal Gene Expression

ABSTRACT The endophytic, insect pathogenic fungus, Metarhizium, exchanges insect-derived nitrogen for photosynthate as part of a symbiotic association similar to well-known mycorrhizal relationships. However, little is known about this nitrogen transfer in soils where there is an abundance of nitrogen and/or carbon. Here, we applied D-glucose and ammonium nitrate to soil to examine the effect on root colonization and transfer of labelled nitrogen (15N) from an insect (injected with 15N-ammonium sulfate) to Metarhizium robertsii, into leaves of the common bean, Phaseolus vulgaris, over the course of 28 days. Application of exogenous carbon and/or nitrogen to soils significantly reduced detectable 15N in plant leaves. Metarhizium root colonization, quantified with real-time PCR, revealed colonization persisted under all conditions but was significantly greater on roots in soil supplemented with glucose and significantly lower in soil supplemented with ammonium nitrate. Fungal gene expression analysis revealed differential expression of sugar and nitrogen transporters (mrt, st3, nrr1, nit1, mep2) when Metarhizium was grown in pure broth culture or in co-culture with plant roots under various carbon and nitrogen conditions. The observation that Metarhizium maintained root colonization in the absence of nitrogen transfer, and without evidence of plant harm, is intriguing and indicates additional benefits with ecological importance.

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Divalent Metal Cations Potentiate the Predatory Capacity of Amoeba forCryptococcus neoformans

Applied and Environmental Microbiology ◽

10.1128/aem.01717-17 ◽

2017 ◽

Vol 84 (3) ◽

Cited By ~ 13

Author(s):

Man Shun Fu ◽

Arturo Casadevall

Keyword(s):

Contaminated Soils ◽

Divalent Cations ◽

Pathogenic Fungus ◽

Acanthamoeba Castellanii ◽

Pathogenic Fungi ◽

Important Variable ◽

Yeast Cells ◽

Experimental Conditions ◽

Content Type ◽

Practical Applications

ABSTRACTAmong the best-studied interactions between soil phagocytic predators and a human-pathogenic fungus is that ofAcanthamoeba castellaniiandCryptococcus neoformans. The experimental conditions used in amoeba-fungus confrontation assays can have major effects on whether the fungus or the protozoan is ascendant in the interaction. In the presence of Mg2+and Ca2+in phosphate-buffered saline (PBS),C. neoformanswas consistently killed when incubated withA. castellanii.A. castellaniisurvived better in the presence of Mg2+and Ca2+, even when incubated withC. neoformans. In the absence of Mg2+and Ca2+,C. neoformanssurvived when incubated withA. castellanii, and the percentage of dead amoebae was higher than when incubated without yeast cells. These results show that the presence of Mg2+and Ca2+can make a decisive contribution toward tilting the outcome of the interaction in favor of the amoeba. Of the two metals, Mg2+had a stronger effect than Ca2+. The cations enhancedA. castellaniiactivity againstC. neoformansvia enhanced phagocytosis, which is the major mechanism by which amoebae kill fungal cells. We found no evidence that amoebae use extracellular killing mechanisms in their interactions withC. neoformans. In summary, the presence of Mg2+and Ca2+enhanced the cell adhesion on the surfaces and the motility of the amoeba, thus increasing the chance for contact withC. neoformansand the frequency of phagocytosis. Our findings imply that the divalent cation concentration in soils could be an important variable for whether amoebae can controlC. neoformansin the environment.IMPORTANCEThe grazing of soil organisms by phagocytic predators such as amoebae is thought to select for traits that enable some of them to acquire the capacity for virulence in animals. Consequently, knowledge about the interactions between amoebae and soil microbes, such as pathogenic fungi, is important for understanding how virulence can emerge. We show that the interaction between an amoeba and the pathogenic fungusC. neoformansis influenced by the presence in the assay of magnesium and calcium, which potentiate amoebae. The results may also have practical applications, since enriching soils with divalent cations may reduceC. neoformansnumbers in contaminated soils.

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Virulence of the insect-pathogenic fungi Metarhizium spp. to Mormon crickets, Anabrus simplex (Orthoptera: Tettigoniidae)

Bulletin of Entomological Research ◽

10.1017/s0007485321000663 ◽

2021 ◽

pp. 1-8

Author(s):

Drauzio E. N. Rangel ◽

Helen G. Bignayan ◽

Hernani G. Golez ◽

Chad A. Keyser ◽

Edward W. Evans ◽

...

Keyword(s):

Pathogenic Fungi ◽

Field Application ◽

Control Measures ◽

Metarhizium Robertsii ◽

Metarhizium Acridum ◽

Threatened And Endangered Species ◽

Insect Pathogenic Fungi ◽

The Usa ◽

Mormon Cricket ◽

Anabrus Simplex

Abstract The Mormon cricket (MC), Anabrus simplex Haldeman, 1852 (Orthoptera: Tettigoniidae), has a long and negative history with agriculture in Utah and other western states of the USA. Most A. simplex populations migrate in large groups, and their feeding can cause significant damage to forage plants and cultivated crops. Chemical pesticides are often applied, but some settings (e.g. habitats of threatened and endangered species) call for non-chemical control measures. Studies in Africa, South America, and Australia have assessed certain isolates of Metarhizium acridum as very promising pathogens for Orthoptera: Acrididae (locust) biocontrol. In the current study, two isolates of Metarhizium robertsii, one isolate of Metarhizium brunneum, one isolate of Metarhizium guizhouense, and three isolates of M. acridum were tested for infectivity to MC nymphs and adults of either sex. Based on the speed of mortality, M. robertsii (ARSEF 23 and ARSEF 2575) and M. brunneum (ARSEF 7711) were the most virulent to instars 2 to 5 MC nymphs. M. guizhouense (ARSEF 7847) from Arizona was intermediate and the M. acridum isolates (ARSEF 324, 3341, and 3609) were the slowest killers. ARSEF 2575 was also the most virulent to instar 6 and 7 nymphs and adults of MC. All of the isolates at the conidial concentration of 1 × 107 conidia ml−1 induced approximately 100% mortality by 6 days post application of fungal conidia. In conclusion, isolates ARSEF 23, ARSEF 2575, and ARSEF 7711 acted most rapidly to kill MC under laboratory conditions. The M. acridum isolates, however, have much higher tolerance to heat and UV-B radiation, which may be critical to their successful use in field application.

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Iron Metabolism, Pseudohypha Production, and Biofilm Formation through a Multicopper Oxidase in the Human-Pathogenic Fungus Candida parapsilosis

mSphere ◽

10.1128/msphere.00227-20 ◽

2020 ◽

Vol 5 (3) ◽

Author(s):

Tanmoy Chakraborty ◽

Zsófia Tóth ◽

Renáta Tóth ◽

Csaba Vágvölgyi ◽

Attila Gácser

Keyword(s):

Iron Metabolism ◽

Biofilm Formation ◽

Iron Uptake ◽

Candida Parapsilosis ◽

Iron Acquisition ◽

Pathogenic Fungus ◽

Pathogenic Fungi ◽

Multicopper Oxidase ◽

Immunocompromised Patients ◽

Content Type

ABSTRACT Among all the essential micronutrients, iron plays an important role in mammalian biology. It is also essential for pathogens infecting mammalian hosts, including bacteria, fungi, and protozoans. As the availability of accessible iron is limited within the mammalian host, several human-pathogenic fungal pathogens, such as Candida albicans, Cryptococcus neoformans, Candida glabrata, and Aspergillus fumigatus, have developed various iron uptake mechanisms. Although Candida parapsilosis is the second or third most common non-albicans Candida species associated with systemic and superficial Candida infections in immunocompromised patients, the mechanisms of iron uptake and homoeostasis remain unknown in this fungus. In the current report, we show that a homologue of the multicopper oxidase gene FET3 is present in the genome of C. parapsilosis (CPAR2_603600) and plays a significant role in iron acquisition. We found that homozygous deletion mutants of CPAR2_603600 showed defects under low-iron conditions and were also sensitive to various stressors. Our results also revealed that the levels of pseudohypha formation and biofilm formation were reduced in the null mutants compared to the wild type. This phenotypic defect could be partially rescued by supplementation with excess iron in the growth medium. The expression levels of the orthologues of various iron metabolism-related genes were also altered in the mutants compared to the parental strain. In conclusion, our report describes the role of CPAR2_603600 in iron homoeostasis maintenance as well as morphology and biofilm formation regulation in this pathogenic fungus. IMPORTANCE C. parapsilosis is the second or third most common opportunistic human-pathogenic Candida species, being responsible for severe fungal infections among immunocompromised patients, especially low-birth-weight infants (0 to 2 years of age). Among the major virulence factors that pathogenic fungi possess is the ability to compete with the host for essential micronutrients, including iron. Accessible iron is required for the maintenance of several metabolic processes. In order to obtain accessible iron from the host, pathogenic fungi have developed several iron acquisition and metabolic mechanisms. Although C. parapsilosis is a frequent cause of invasive candidiasis, little is known about what iron metabolic processes this fungus possesses that could contribute to the species’ virulent behavior. In this study, we identified the multicopper oxidase FET3 gene that regulates iron homeostasis maintenance and also plays important roles in the morphology of the fungus as well as in biofilm formation, two additional factors in fungal virulence.

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