Trading nitrogen for carbon: Nitrogen and carbon translocation in a

plant/fungal (Metarhizium spp.) symbiosis

Scott William Behie, BSc (Hons)

A thesis submitted to the Centre of Biotechnology

in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

Faculty of Mathematics and Science,

Brock University -St. Catharines, Ontario

© Scott W. Behie 2015

 
Thesis Outline

The overall hypothesis that was being tested in this thesis was that root colonization

by endophytic fungi is predicated on reciprocal nutrient exchange between symbiotic

partners. It was predicted that Metarhizium would be able to provide the plants with insect-

derived nitrogen in exchange for photosynthetically derived carbon.

The endophytic, insect pathogenic fungi, that I are explored is Metarhizium.

Metarhizium spp. are traditionally known as insect pathogens and research has focused on

their utility as biological control agents. Here I connected the dual life styles of this fungus

by showing that Metarhizium can provide insect-derived nitrogen to its plant host.

In Chapter 1, I explore the literature that deals with fungal nutrient transfer to plants,

as well as the many ways plants are able to utilize insects as a nitrogen source. The ability of

numerous fungal species to transfer nitrogen to plants through symbiotic association as well

as the overall ability of plants to exploit insect nitrogen suggested that the dual life cycle of

Metarhizium, as both an insect pathogen and plant root colonizer, is coupled to provide plant

roots with insect-derived nitrogen.

Chapter 2 is an experimental chapter where I test the ability of Metarhizium to

transfer insect-derived nitrogen to both monocots and dicots. Here I found that Metarhizium

was able to infect insect larvae, harvest nitrogen, and transfer insect-derived nitrogen to its

plant host.

Chapter 3 is an experimental chapter where I was able to expand on the nitrogen

transfer capabilities of Metarhizium by testing 5 species of Metarhizium with 4 different

 
types of plants. All species of Metarhizium were able to transfer insect-derived nitrogen to

their plant hosts, although at varying efficiencies. I also tested other species of endophytic,

insect pathogenic fungi (Beauveria bassiana, Lecanicillium lecanii) for their ability to

transfer insect-derived nitrogen to haricot beans and switchgrass. It was found that B.

bassiana was capable of acting as an insect nitrogen vector while L. lecanii was not. I also

tested the ability of Metarhizium to transfer insect-derived nitrogen in field settings where

microbial competition for plant root space is potentially high. It was found that Metarhizium

is able to cycle insect nitrogen to its plant host in the field.

Chapter 4 is an experimental chapter where I test the reciprocity between

Metarhizium and its plant host. Here I found that Metarhizium is provided with

photosynthetically fixed carbon from its plant host.

Chapter 5 reviews potential evolutionary strategies that have led to the emergence of

plant colonizing insect pathogens. Through phylogenetic analysis, I show that Metarhizium

species are more closely related to plant colonizing fungi, as opposed to pure insect

pathogens.

Chapter 6 consists of concluding remarks as well as a review dedicated to the future

direction of this research, and potential downstream industrial applications.

 
Abstract

While nitrogen is critical for all plants, they are unable to utilize organically bound nitrogen in

soils. Therefore, the majority of plants obtain useable nitrogen through nitrogen fixing bacteria

and the microbial decomposition of organic matter. In the majority of cases, symbiotic

microorganisms directly furnish plant roots with inorganic forms of nitrogen. More than 80% of

all land plants form intimate symbiotic relationships with root colonizing fungi. These common

plant/fungal interactions have been defined largely through nutrient exchange, where the plant

receives limiting soil nutrients, such as nitrogen, in exchange for plant derived carbon. Fungal

endophytes are common plant colonizers. A number of these fungal species have a dual life cycle,

meaning that they are not solely plant colonizers, but also saprophytes, insect pathogens, or plant

pathogens. By using 15N labeled, Metarhizium infected, wax moth larvae (Galleria mellonella) in

soil microcosms, I demonstrated that the common endophytic, insect pathogenic fungi

Metarhizium spp. are able to infect living soil borne insects, and subsequently colonize plant

roots and furnish ts plant host with useable, insect-derived nitrogen. In addition, I showed that

another ecologically important, endophytic, insect pathogenic fungi, Beauveria bassiana, is able

to transfer insect-derived nitrogen to its plant host. I demonstrated that these relationships

between various plant species and endophytic, insect pathogenic fungi help to improve overall

plant health. By using 13C-labeled CO2, added to airtight plant growth chambers, coupled with

nuclear magnetic resosnance spectroscopy, I was able to track the movement of carbon from the

atmosphere, into the plant, and finally into the root colonized fungal biomass. This indicates that

Metarhizium exists in a symbiotic partnership with plants, where insect nitrogen is exchanged for

plant carbon. Overall these studies provide the first evidence of nutrient exchange between an

insect pathogenic fungus and plants, a relationship that has potentially useful implications on

plant primary production, soil health, and overall ecosystem stability.

 
Acknowledgements

First, I would like to thank my supervisor Dr. Michael Bidochka. Dr. Bidochka has been an

incredible mentor, teacher, and friend over the past 5 years. He was always there to guide me

through my experiments and gave me advice and help when I needed it, not only about

research, but about life as well. I am a better scientist and a better-rounded person because of

my time spent in his lab, and with him. What I have learned from Dr. Bidochka will stay with

me always, and I will be forever grateful to him for the opportunity he gave me.

Secondly, I would like to thank Dr. Paul Zelisko, who was instrumental in the completion of

this research. Dr. Zelisko’s door has always been open to me, something I made great use of,

and am very thankful for.

I would like to thank my other incredible committee members Dr. Liette Vasseur and Dr.

Alan Castle. Both of these professors provided valuable insights into my research and

allowed me to sit with them to ask questions about areas of my research in which they are

experts. They made themselves available to me whenever I needed, and helped me see my

research from different perspectives, I am grateful to them both for the time they put in on

this committee.

I would like to thank my external examiners, Dr. Jonathan Newman and Dr. Brian Roy for

their valuable insight into this thesis, and for making themselves available to be at my

defense, it is greatly appreciated.

I wish to thank Larissa Barelli, Israel Padilla, Irina Semetchoukova, Camila Moreira, Soumya

Moonjely, Ramanpreet Sasan, and all of my lab mates that I have worked with in the past 5

 
years. The friendship and support provided to me by these amazing young scientists will

always be remembered.

I would like to express my gratitude and love to my entire family, especially my father, Dr.

Stewart Behie, my two sisters, Dr. Alison Behie, and Jennifer Ratzlaff, and my brother in law,

Robert Ratzlaff. They have been nothing but supportive during my entire time at Brock

University, I would not have been able to succeed without their love and encouragement.

Last, but not least, I would like to thank my wife Kristin Schaven. Kristin has been incredibly

supportive of me during my time as a graduate student. She has stuck by me during all the

highs and lows, was there with encouragement, support, and has always believed in me. I am

a better person because of my relationship with Kristin, and would not be where I am without

this amazing woman by my side.

“In nature's infinite book of secrecy

A little I can read.”
-Soothsayer
-Antony and Cleopatra (1.2.13)

 
Table of Contents  

Chapter 1: Introduction and Literature Review .................................................................................1

Insects as a Nitrogen Source for Plants………………………………………….. ……………....4

1.1 Abstract ……………………………………………………………………………………….5

1.2 Introduction ……………………………………………………………………………...5

1.3 Nitrogen in the Soil – Why is it a Deficit? …………………………………………………..7

1.4 Insects as a Nitrogen Reservoir ……………………………………………………………9

1.5 Carnivorous Plants…………………………………………. ………………………………..11

1.6 Insect Cadavers and Insect Frass: Potential Involvement of Mycorrhizal Fungi…….......12

1.7 Endophytic Insect Pathogenic Fungi ………………………………………………….15

1.8 Evolutionary Implication of Nitrogen Transfer by Fungal Symbionts ……………...18

1.9 Conclusions ……………………………………………………………………………...19

Nutrient transfer in plant-fungal symbioses …………………………………………………..19

1.10 Abstract ……………………………………………………………………………...21

1.11 Introduction ……………………………………………………………………………...21

1.12 Nitrogen transfer from fungal symbiont to plant hosts ………………………………..24

1.13 Transfer of phosphate from fungus to plant …………………………………………29

1.14 Non-limiting soil nutrients ……………………………………………………………32

1.15 Reciprocal exchange: trading carbon for nutrients ………………………………..33

1.16 Applications ……………………………………………………………………………...35

1.17 Conclusions ……………………………………………………………………………...36

1.18 Acknowledgements …………………………………………………………………….37

Chapter 2: Endophytic-insect parasitic fungi translocate nitrogen directly from insects to plants38

2.1 Abstract ……………………………………………………………………………………….39

2.2 Introduction ……………………………………………………………………………...40

2.3 Materials and Methods …………………………………………………………………….41

 
 2.3.1 Seed Sterilization and Plating ..............................................................................................42

2.3.2 G. mellonella Injection .........................................................................................................43

2.3.3 G. mellonella Infection .........................................................................................................43

2.3.4 Soil Sampling and Plating....................................................................................................43

2.3.5 MMN Treatment ...................................................................................................................44

2.3.6 Microcosm setup ..................................................................................................................44

2.4 Results and Discussion …………………………………………………………………….45

2.5 Acknowledgements …………………………………………………………………….48

Chapter 3: An additional branch of the soil nitrogen cycle: Ubiquity of insect-derived nitrogen

transfer to plants by endophytic insect pathogenic fungi ................................................................. 49

3.1 Abstract ……………………………………………………………………………………….49

3.2 Introduction ……………………………………………………………………………...50

3.3 Materials and Methods …………………………………………………………………….52

3.3.1 Sources of fungi and plant material .....................................................................................52

3.3.2 Seed Sterilization and Plating ..............................................................................................53

3.3.3 Galleria mellonella Injection and Infection .........................................................................54

3.3.4 Soil Sampling and Plating....................................................................................................54

3.3.5 Microcosm setup ..................................................................................................................55

3.3.6 Preparation of samples for 15N Analysis..............................................................................55

3.3.7 Plant Growth Measurements ...............................................................................................56

3.3.8 Confocal Microscopy of GFP fungi/plant root associations ...............................................56

3.3.9 Field Experiment ..................................................................................................................57

3.3.10 Calculation of insect nitrogen available to plants through EIPF ......................................57

3.4 Results ……………………………………………………………………………………….58

3.4.1 Nitrogen transfer to plants by Metarhizium species ............................................................58

3.4.2 Beauveria bassiana and Lecanicillium lecanii: Insect-derived nitrogen transfer to switchgrass

(Panicum virgatum) and haricot bean (Phaseolus vulgaris) ........................................................62

 
 3.4.3 Soil Samples .........................................................................................................................64

3.4.4 GFP confocal imaging .........................................................................................................64

3.4.5 Nitrogen transfer from Metarhizium to grass roots under natural conditions ....................66

3.4.6 Plant Growth ........................................................................................................................67

3.5 Discussion ……………………………………………………………………………...70

3.6 Conclusion ……………………………………………………………………………...73

Chapter 4: Trading insect nitrogen for photosynthate- Carbon translocation from plant to

endophytic, insect pathogenic, fungus ................................................................................................75

4.1 Abstract ……………………………………………………………………………………….76

4.2 Introduction ……………………………………………………………………………...76

4.3 Materials and Methods …………………………………………………………………….78

4.3.1 Fungal Culture sand Plant Material ....................................................................................78

4.3.2 Seed sterilization and plating ...............................................................................................78

4.3.3 Microcosm set up and plant growth .....................................................................................79

4.3.4 Assimilation chamber set up and CO2 addition ...................................................................80

4.3.5 Sequential Extraction of soluble sugars and N-acetylglucosamine .....................................80

4.3.6 Nuclear magnetic resonance spectroscopy ..........................................................................80

4.3.7 Soil analysis and plating ......................................................................................................81

4.3.8 Fungus colonized root staining ............................................................................................81

4.3.9 Root colonization assay .......................................................................................................82

4.4 Results and Discussion……………………………………………...………………………..96

Chapter 5: Nutrient transfer to plants by phylogenetically diverse fungi suggests convergent

evolutionary strategies in rhizospheric symbionts ............................................................................93

5.1 Introduction …………………………………………………………………………….94

5.2 Nutrient transfer in AM and endophytic fungi ………………………………………..95

5.3 Convergent evolution in nutrient transfer in AM and endophytic fungi …………….97

 
 5.4 Materials and Methods …………………………………………………………………...116

5.5 Conclusions ………………………………………………………………………………..116

Chapter 6: Conclusions and Perspectives ........................................................................................118

6.1 Nitrogen transfer by EIPF …………………………………………………………..105

6.2 Reciprocal exchange: Carbon transfer from plant to Metarhizium ……………………...106

6.3 Evolution of EIPF …………………………………………………………………...109

Potential agricultural benefits through biotechnological manipulation of plant fungal associations

……………………………………………………………………………………….……...110

6.4 Introduction …………………………………………………………………………….111

6.5 Could Biotechnology Be Used To Facilitate Beneficial Fungal-Crop Symbioses? …...112

6.6 Agricultural Sustainability in Drought or Saline Conditions Through Fungal

Symbiosis…………………………………………………………………………………………115

6.7 Nutrient Exchange in Plant-Fungal Symbiosis ………………………………………..115

6.8 Potential of Endophytic Fungi To Protect Crops Against Insect Pests …………….117

6.9 Conclusion …………………………………………………………………………….117

References ...........................................................................................................................................119

Appendix A .........................................................................................................................................137

Appendix B .........................................................................................................................................158

 
List of Figures

Figure 1 Representation of 3 major routes by which plants are able to obtain insect bound
nitrogen ……………………………………………………………………………….......................17
Figure 2 Plant–fungal root symbioses ……………………………………………………....…….29

Figure 3 Percentage of plant nitrogen derived from waxmoth larvae by an endophytic, insect-
pathogenic fungus …………………………………………………….…………………..………...46
Figure 4 Representation of the transfer of the insect-derived nitrogen to plants through an
association with endophytic, insect parasitic Metarhizium …………………………...……..….48
15
Figure 5 Percentage of plant nitrogen derived from N-injected waxmoth larvae by five
species of the endophytic, insect pathogenic fungus, Metarhizium (robertsii, acridum,
guizhouense, brunneum, flavoviridae)………………………...…………………………...……...61
15
Figure 6 Percentage of plant nitrogen derived from
N-injected waxmoth larvae by the
endophytic, insect pathogenic fungus Beauveria bassiana and Lecanicillium
lecanii………………………………….……………………………………………………..……....63
Figure 7 Time course, after insect infection of fungal propagules found per gram of
soil……………………………………………………………………...……………………......…....65
Figure 8 Confocal micrographs obtained after plants had grown in soil microcosms with
waxmoth larvae and Metarhizium-GFP for 7 days ….…………………………………..….…...66
15
Figure 9 Percentage of plant nitrogen derived from
N-injected waxmoth larvae by the
endophytic insect pathogenic fungi M. robertsii under field
conditions………….………………………………………………………….……………………...68
Figure 10 Plant growth measurements taken every four days for 16 days ….............................69
13
Figure 11 Relative % C in Metarhizium and plant derived sugars..……………...…………...85
13
Figure 12 Relative percent C in fungal produced sugars as a function of plant root weight
………………………………………………………………………………………………...………87
13
Figure 13 Ratio of trehalose to sucrose in C labeled plants colonized by
Metarhizium……………………………………………………………...……………...…………...89
Figure 14 Internal cross section of M. robertsii colonized haricot bean plant roots stained with
Chlorazol black ……………………………………………………………………………...….…106
Figure 15 Phylogenetic positioning of 4 major fungal phyla with examples of plant symbionts,
ability of these symbionts to transfer nutrients, insect pathogens and plant pathogens
………………………………………………………………………..………………………….……91

Figure 16 Representation of the flow of nutrients between Metarhizium and its plant
host………………………………………………………………………….……………....108

 
Figure 17 Potential plant benefits from symbiotic fungal gene manipulations or plant gene
manipulations ………………………………………………………………..…………......114
Supplementary Figure 1 Microcosm design and experimental setup ….……………..…….....137

Supplementary Figure 2 Galleria melonella larvae infected with Metarhizium robertsii and
Aspergillus flavus ………………………………………………………..…………………….......138
Supplementary Figure 3 Time course, after insect infection of M. robertsii (close circles) and
A. flavus (open circles) propagules found within 0.5 cm from plant roots at a depth of 2 cm
……………………………………………………………………………………………………….139
13
Supplementary Figure 4 C CO2 microcosm design and experimental setup……………...140
13
Supplementary Figure 5 C NMR spectra of both trehalose and N-acetylglucosamine (1, 7,
14, and 21 days) obtained from Metarhizium colonized plant roots or Metarhizium colonized
soil grown in 13C or natural CO2 environment, as well as control
treatments…………………………………………………………………...………………...….....141
Supplementary Figure 6 NMR spectra of soluble sugars extracted from plants grown without
13
C CO2………………………………………………………...………………………………..…..142

 
List of Abbreviations

AM – arbuscular mycorrhizal fungi

BCA – biological control agent

CFU – colony forming unit

EIPF - endophytic, insect pathogenic fungi

EM – ecotmycorrhizal fungi

GlcNAc – N-acetylglucosamine

ITS – internal transcribed spacer

JA – jasmonic acid

MMN - modified Melin-Norkrans

MY – million years

NMR – nuclear magnetic resonance

PAM – peri arbuscular membrane

ppm – parts per million

PDA – potato dextrose agar

rRNA - ribosomal ribonucleic acid

SA – salicylic acid

Spp. – species

UV – ulra violet

AMT – ammonium transporter

NRT – nitrate transporter

NL – nonlimiting soil nutrients

MtSULTR – Medicago truncatula sulfate transporter

SE – standard error

GFP – green flourescent protein

mrt – Metarhizium raffinose transporter

 
 Chapter 1: Introduction and Literature Review
 

1  
 The majority of terrestrial plants form symbioses with soil fungi (Gaude et al. 2012).

Well-studied plant/fungal symbioses (mycorrhizal fungi) are typically defined through

nutrient transfer; where root colonized fungal species provide plants with limited soil

nutrients such as nitrogen (Parniske 2008). Non-saprophytic, plant-colonizing fungi, however,

are at a disadvantage with respect to carbon acquisition, and must use their plant hosts as a

carbon source (Tiunov & Scheu 2004). The fungus and plant are engaged in a reciprocal

relationship where the plant receives critical soil nutrients in exchange for plant-derived

carbon (Parniske 2008). These symbioses are over 400 million years old, have helped shape

the land distribution of plants, and are critical to ecosystem health (Bonfante & Genre 2010).

Insects have high nitrogen content (protein and chitin), and therefore represent a

potentially important nitrogen reservoir for plants. The deposition of insect nitrogen into soil

systems through frass and cadavers has been shown to be useful towards plant nitrogen

acquisition (Fielding et al. 2012). Some plants have evolved unique mechanisms by which to

obtain insect bound nitrogen. Carnivorous plants, for example, are able to survive in low

nitrogen environments by exploiting the nitrogen present in insects through direct ingestion

(Brewer 2001). Thus, in a number of ways, plants are able to access insect-bound nitrogen.

Metarhizium spp. are soil borne insect pathogenic fungi, capable of infecting over 200

species of insects, and have been used extensively in agriculture as biological control agents

(Roberts & Hajek 1992). This insect pathogen is ubiquitous in soil, and has been isolated

from diverse ecosystems such as arctic tundra, temperate soils, and tropical environments

(Hajek & St. Leger 1994). Metarhizium has recently been shown to be a plant endophyte, and

is therefore capable of internal colonization of plant tissue (Sasan & Bidochka 2012).

Through this plant colonization, Metarhizium is able to increase lateral root hair growth,
2  
overall plant size, and mitigate soil stress to plants (Sasan & Bidochka 2012, Khan et al.

2012). These two distinct life styles of Metarhizium, combined with the plants need to obtain

soil limited nitrogen, suggests that the insect pathogenesis and root colonizing abilities of

Metarhizium are coupled to provide nutrients to the plant host, in exchange for carbon.

I hypothesized that, like mycorrhizal fungi, the Metarhizium/plant partnership is

predicated on nutrient exchange. It was predicted that Metarhizium spp. in the soil would be

able to harvest nitrogen from the insects they infect, and provide this nitrogen to the plant in

exchange for carbon.

These ideas will be explored in greater detail in the following two reviews. The first

discusses plant utilization of insects as a nitrogen source, and the second is a comprehensive

review of nutrient transfer in plant-fungal symbioses.

   

3  
 Insects as a Nitrogen Source for Plants

Published as: Behie SW, & Bidochka MJ. Insects as a nitrogen source for plants.
Insects 2013 4, 413-424.
Behie wrote the manuscript with additions, edits and guidance from Bidochka.

4  
1.1 Abstract
 
Many plants have evolved adaptations in order to survive in low nitrogen environments. One

of the best-known adaptations is that of plant symbiosis with nitrogen-fixing bacteria; this is

the major route by which nitrogen is incorporated into plant biomass. A portion of this plant-

associated nitrogen is then lost to insects through herbivory and insects represent a nitrogen

reservoir that is generally overlooked in nitrogen cycles. In this review I show three

specialized plant adaptations that allow for the recovery of insect nitrogen; that is, plants

gaining nitrogen from insects. First, I show specialized adaptations by carnivorous plants in

low nitrogen habitats. Insect carnivorous plants such as pitcher plants and sundews

(Nepenthaceae/Sarraceniaceae spp. and Drosera spp., respectively) are able to obtain

substantial amounts of nitrogen from the insects they capture. Secondly, numerous plants

form associations with mycorrhizal fungi that can provide soluble nitrogen from the soil,

some of which may be insect-derived nitrogen, obtained from decaying insects or insect frass.

Finally, a specialized group of endophytic, insect-pathogenic fungi (EIPF) provide host

plants with insect-derived nitrogen. These soil-inhabiting fungi form a remarkable symbiosis

with certain plant species. They can infect a wide range of insect hosts and also form

endophytic associations in which they transfer insect-derived nitrogen to the plant. Root

colonizing fungi are found in disparate fungal phylogenetic lineages, indicating possible

convergent evolutionary strategies between taxa, evolution potentially driven by access to

carbon-containing root exudates.

5  
1.2 Introduction
 
Nitrogen exists in the soil in numerous forms, and these forms can be rapidly

converted from inorganic to organic and vice versa (Schulten & Schnitzer 1998). It is a

limiting resource for primary producers and this lack of useable nitrogen is due, in large part,

to the inability of plants to directly utilize atmospheric nitrogen and their subsequent reliance

on microbial nitrogen fixation. As a result, many plant species (e.g. legumes) form symbiotic

associations with microorganisms that can fix atmospheric nitrogen that is then made

accessible to the plant (Cocking 2003). Free-living nitrogen-fixing bacteria (e.g. Azotobacter)

also contribute to the nitrogen flux in the soil, as they are able to release useable nitrogen

(nitrate or ammonia) into the soil. Despite the abundance of microbial nitrogen fixation,

nitrogen is still a limiting resource, particularly for many plants without nitrogen-fixing

symbionts (Mary et al. 1996). However, numerous plant adaptations, and novel plant

microbe symbioses, have evolved specifically for the acquisition of nitrogen (Behie et al.

2012, Ellison & Gotelli 2001).

Soil bacteria and fungal symbionts, such as Rhizobia spp. and Glomus spp., are able

to provide plants with substantial amounts of inorganic nitrogen per year in natural settings,

as well as in commercial agricultural fields (Cocking 2003, Sprent 1986, Johansson 2004).

These plant symbionts are able to convert N2 into inorganic nitrogen (e.g. Rhizobia) or

convert organic nitrogen in the soil to a form that is transferred to plants (e.g. Glomus).

Organic nitrogen is present in soil ecosystems bound within leaf litter and other plant detritus,

as well as in insect cadavers, insect frass and living insects (Koller et al. 2013). This organic

nitrogen is then made available to plants through microbial denitrification. Nitrogen can also

be made available for plants through atmospheric fixation of nitrogen when lightning strikes
6  
convert N2 into NO and NO2 that is subsequently deposited in soils through precipitation;

however, this type of nitrogen fixation is inconsistent over time and space (Liaw 1990).

Despite the many methods by which nitrogen is converted to an inorganic form that

can be used by plants, leaching of nitrogen from the soil due to erosion or poor farming

practices, biological processes such as denitrification and microbial competition, all act to

remove and limit soil nitrogen levels (Blossey & Hunt-Joshi 2003, Post et al. 1985, Parson et

al. 1991, Goulding 1998).

Many insects are primary herbivores that strip nitrogen from plants; the nitrogen

amassed in insects can comprise a substantial amount of the total nitrogen flux in the

ecosystem (Whittaker 2001). The current paradigm of the nitrogen cycle represents the flow

of nitrogen within an entire ecosystem, yet there are few models that consider the reservoir of

nitrogen in insects. On an ecosystem level, insects can be an essential nitrogen source for

plants and should not be overlooked. When viewed on a per plant basis, insects potentially

become increasingly important as a source of nitrogen. In this review I discuss how the

nitrogen reservoir present within insects is an integral part of the soil nitrogen cycle, and the

three major adaptations by which insects can be exploited by plants for their nitrogen.

1.3 Nitrogen in the Soil – Why is it a Deficit?

 
The majority of soil ecosystems are nitrogen-limited (Wardle 1992) and nitrogen,

along with phosphorus, are the nutrients most likely to limit plant productivity in terrestrial

environments (Vitousek & Howarth 1991, Elser et al. 2007). Primary producers require more

nitrogen and phosphorus than other nutrients, and nitrogen is energetically expensive to

acquire and utilize (Gutschick 1981). Nitrogen has proven to be critical in the overall health

7  
of plants, and the introduction of nitrogen into forested areas, grasslands, or tundra has been

shown to increase overall plant productivity (Miller 1981, Agren 1983, Huenneke et al. 1990,

Shaver et al. 1980). Plant photosynthate production is strongly limited by N availability and

plants require substantial amounts of N (2–5% N content by dry weight, compared with 0.3–

0.5% phosphorus content), and well over 50% of leaf N is devoted to photosynthesis (Field

& Mooney 1986). Therefore, the acquisition of soil nitrogen by plants is directly related to

their ability to fix carbon via photosynthesis.

One of the main reasons for the nitrogen deficit in soils is its mobility. When

compared to other important soil nutrients, such as phosphorous, nitrogen is more readily lost

through leaching to aquatic systems, and is more sensitive to microbial processes, such as

denitrification, that remove nitrogen from the soil (Vitousek & Howarth 1991). Leaching of

nitrogen from natural ecosystems can increase as a result of environmental disturbances, such

as insect defoliation (Swank et al. 1981). For example, increased deforestation by canker

worms has been linked to an increase in nitrate levels in forest streams (Swank et al. 1981).

Over 70% of the total nitrogen inputs into terrestrial ecosystems can be lost due to natural

disturbance and microbial processes (Vitousek & Howarth 1991).

Another major reason for the nitrogen deficit is that some nitrogen is immobilized

within decaying plant material. Plant litter is high in nitrogen-containing polymers such as

lignin, which is recalcitrant to decomposition, and as such, plant detritus contains large

quantities of immobilized nitrogen (Melillo et al. 1982). In this way, organic nitrogen can

collect in large quantities in soil ecosystems, but remains unavailable for plant use (Melilo et

al. 1989). Organically bound nitrogen within decaying plant and animal material, and soil

8  
organic matter can account for as much as 90% of the nitrogen in surface soils (Kelley &

Stevenson 1995).

While it is clear that there are a number of reasons to explain why nitrogen is limiting

in soil ecosystems, there remains an inconsistency concerning this lack of nitrogen. If large

nitrogen deficits persist in soil ecosystems, how are primary producers able to acquire the

nitrogen required for sustained growth and development? Potentially, there are other nitrogen

sources that are unaccounted for by the current models of nitrogen cycling; that is, nitrogen

sources such as insects.

1.4 Insects as a Nitrogen Reservoir

 
In this review, I specifically focus on insects as a source of nitrogen for plants. Insects

represent a potential nitrogen loss to plants. Insect herbivory causes the loss of plant material

and, therefore, a loss of critical nitrogen. Below ground insect herbivory, specifically, has

been found to represent a possible significant loss of plant nitrogen, nitrogen that cannot be

replaced through atmospheric or bacterial fixation alone (Ayers 2007). In this regard, insects

can be viewed as nitrogen thieves, whereby herbivorous insects incorporate plant nitrogen.

Plants are composed mostly of carbohydrates, and the nitrogen content in actively growing

plants typically ranges from 3–7% by weight (Mattson 1980); as such, the loss of any plant

material can be devastating to overall plant health.

Despite its potentially damaging effect, below ground insect herbivory has been

largely ignored in models of the nitrogen cycle. A reason for this may be that nitrogen

cycling is viewed primarily on a whole ecosystem level, which ignores the potential nitrogen

9  
deficit that exists at the plant level. A large number of studies have focused on plant

predation by above ground insect herbivores; however, root-feeding insects can also cause

potentially damaging effects to plants (Maron 1998). Below ground herbivory represents a

loss of nitrogen from the plants roots, not only in the plant material that has been fed upon,

but also through increased levels of nitrogen in the root exudate (Blossey & Hunt-Joshi 2003)

nitrogen that can then be taken up by competing plants in the soil. On an ecosystem level this

does not represent a loss of nitrogen; however, there is a specific loss for the individual plant

(Sprent, 1986). The loss of plant material due to insect herbivory of plant roots represents a

potentially lethal loss of nitrogen to the plant, and total plant biomass can be reduced by as

much as 70% (Murray et al. 1996).

Insects contain approximately 10% nitrogen by weight, primarily in protein and chitin

(Fagan et al. 2002); in a square hectare of temperate ecosystem soil there is approximately 15

g of insect nitrogen below ground and approximately 100-200 g of insect-bound nitrogen

above ground (van Emden 1989). This amount is not insignificant, particularly when

compared to microbially fixed nitrogen in the soil whereby microbes can fix approximately

100 g of nitrogen per hectare per year (Kummerow et al. 1978).

This below ground loss of nitrogen can however partially be counterbalanced through

the incorporation of insect-derived nitrogen back into plants. Plants are able to accomplish

this through: (1) direct ingestion of insects; (2) indirect transfer of insect-derived nitrogen (i.e.

decomposing insect cadavers or insect frass) through bacterial or fungal ammonification and

nitrification, or transfer of this nitrogen through symbiotic mycorrhizal fungi; or (3) a direct

transfer of insect-derived nitrogen through a partnership with endophytic insect pathogenic

fungi.
10  
1.5 Carnivorous Plants
 
There are over 600 known species of carnivorous plants, the majority of which thrive

in bogs, marshes and rocky outcrops, or other nitrogen-poor areas where sunlight and water

are abundant (Brewer 2001). These plants have evolved the ability to trap and digest insects,

which are an excellent source of nitrogen as they contain around 10% nitrogen by mass

(Fagan et al. 2002). Carnivorous plants are able to obtain between 10 and 80% of their total

nitrogen from insects, depending on environment conditions and type of trap employed

(Ellison & Gotelli 2001).

Found both on land and in fresh water, pitcher plants (Nepenthacea spp.,

Sarraceniaceae spp.), flypaper traps (Droseraceae, Lentibulariaceae), snap traps

(Droseraceae), combination traps (Aldrovanda) (Ellison & Gotelli 2001), bladder traps

(Utricularia) and lobster pot traps (Genlisea) (Müller et al. 2006) have evolved to exploit

insect nitrogen. These plants use sophisticated traps and lures in order to capture insect prey,

such as emitting specific odors or employing displays visible only in the UV spectrum

(Moran et al. 2012, Jaffi et al. 1995). Insects are then enzymatically digested and insect-

derived nutrients are metabolized (Adamec 1997). A number of carnivorous plants, notably

Sarracenia and Drosera, are able to alter their consumption of insects in response to soil

nitrogen fluctuations (Ellison & Gotelli 2002). Insects remain, however, a critical source of

nitrogen for all carnivorous plants.

The most famous of these prey capture mechanisms are the snap traps found in Venus

flytraps (Dionaea muscipula) and waterwheel plants (Aldrovanda vesiculosa). These traps

consist of two lobed leaves that are able to close around insect prey when the plant senses

11  
movement of one of its trigger hairs (Volkov et al. 2008). One of the most remarkable

aspects of the snap trap is that it employs simple memory to ensure it does not close on

precipitation or debris (Volkov et al. 2009).

Pitcher plants (Nepenthaceae, Sarraceniaceaer) are able to attract foraging, flying or

crawling insects to their liquid-filled pits; once inside the pit, the insect is enzymatically

digested (Jaffe et al. 1992). The lip of the pit fall trap is coated with a slippery mucous-like

substance secreted by the plant; when an insect lands there, it is unable to escape (Gaume &

Forterre e1185). There has been some disagreement over whether nectar bribes or visual cues

play the key role in attracting insect prey (Joel 1988); however, the end result is that plants

are able to acquire nitrogen from trapped insects. Carnivorous plants are able to thrive in

areas of extremely low nitrogen, a fact that speaks to the enormous potential of insects as a

source of nitrogen.

1.6 Insect Cadavers and Insect Frass: Potential Involvement of Mycorrhizal Fungi
1.6.1 Insect Cadavers/Insect Frass
 
Insect cadavers present in the soil represent a potentially large reservoir of organic

nitrogen (Fielding 2012). Decaying insects are more easily broken down in soil than other

organic material such as leaf litter, and the nitrogen in insect cadavers is therefore more

readily available to plants (Hunter 2001). Insect frass also contains a large amount of organic

nitrogen, in the form of uric acid, that can be converted to inorganic forms, such as ammonia

and nitrate (Frost & Hunter 2007), and there have been a number of studies that have focused

on frass deposition by foraging insects such as termites and gypsy moths (Zaady et al. 2003,

Lovett 1995). It has been shown that, in certain forest ecosystems, insect cadavers and frass
12  
deposition can represent up to 70% of the nitrogen returning to the soil when insect

populations are large (Hollinger 1986). Insect frass may also provide a source of nutrients for

microbial decomposers in the soil, which accelerates the decomposition of natural leaf litter

and insect cadavers, increasing the amount of inorganic nitrogen released into the soil

(Hollinger 1986). In soil ecosystems, this organic nitrogen is converted to ammonia by a

number of ammonifying bacteria such as Bacillus, Pseudomonas and Streptomyces, after

which bacteria such as Nitrobacter and Nitrosomonas are then able to oxidize ammonia into

nitrate (Antonie 1981). Soil fungi also play a key role in the decomposition of insect

cadavers and release of organic nitrogen. In the case of insect cadavers and insect frass,

extracellular fungal hydrolytic enzymes, such as proteases and chitinases, are capable of

breaking down organic nitrogen and converting it into a form useable by plants (Sánchez

2009).

1.6.2 Potential Role of Myccorhizal Fungi in Insect Nitrogen Transfer

Root-associated fungi have long been studied for the benefits they provide through

their symbiotic relationship with plants. A particular focus has been placed on the ubiquitous

mycorrhizal fungi that form an internal association with plant roots. These symbiotic fungi

are able to increase the overall fitness of their plant partners through an increase in the uptake

of critical nutrients, such as nitrogen (George 1995). These fungi provide nutrients by

effectively increasing the surface area of the plant roots and by mobilizing nitrogen

(Marschner & Dell 1994). In return, the plant provides the fungus with plant-synthesized

carbohydrates, typically in the form of monosaccharides (Govindarajulu et al. 2005).

13  
 Mycorrhizal fungi are able to scavenge important soil nutrients, some of which are

transferred to plants during internal root colonization (Smith & Read 1997). The nutrients

that are scavenged can be from any decaying organic matter; however, some mycorrhizal

fungi transfer nitrogen from insect cadavers to their plant host. For example, the

ectomycorrhizal basidiomycete, Laccaria bicolor, is found to harvest nitrogen bound in

insect cadavers and subsequently furnish plant roots with this scavenged nitrogen

(Klironomos & Hart 2001). Several mycorrhizal species are decomposers of organic detritus

in the soil (Talbot et al. 2008). These fungi are able to actively decompose organic matter,

and transfer useable inorganic nitrogen to plant roots. In this case, the mycorrhizae are able

directly to break down insect cadavers or frass in order to obtain nitrogen that can be

exchanged within the plant.

The exchange of nitrogen between mycorrhizal fungi and plant roots is mediated

internally by the formation of a nutrient transfer interface. Embedded within this interface are

transport proteins that facilitate the movement of phosphorous, nitrogen and carbon between

the two symbionts (Harrison 1999). This has been observed most prominently in arbuscule

mycorrhizal fungi when promoter GUS (β-glucuronidase) fusion has been used to identify

the location and expression of fungal and plant transporters within arbuscule-containing cells

(Gaude et al. 2012). These include ammonium and nitrate transporters, such as gmAMT4 and

MtGim, respectively (Kobae et al. 2010, Wulf et al. 2003), as well as the fungal phosphate

transporters GmosPT and GvPT and the sugar transporter Mtst1 (Benedetto et al. 2005,

Harrison 1996).

The reciprocal nature of this symbiotic partnership has been elucidated by tracking

the translocation of nitrogen, phosphorous and sugars at the plant/fungal interface (Pearson &
14  
Jakobsen 1993, Govindarajulu et al. 2005). When the plant is provided with high levels of

carbon, there is a greater transfer of nutrients from the fungus to the plant, indicating that the

movement of carbon, from the plant to the fungus, is an essential component of nutrient

transfer in plant/fungal symbioses (Hobbie & Hobbie 2006). This partnership increases plant

productivity and overall plant health. Plants that are able to form complex and symbiotic

relationships with soil fungi are stronger competitors with respect to gaining access to soil

nutrients such as nitrogen.

1.7 Endophytic Insect Pathogenic Fungi

 
Endophytic fungi are a class of plant symbionts that form intercellular root

associations, similar to those formed by the mycorrhizae. Unlike mycorrhizal fungi, however,

it was considered that endophytes did not develop a specific nutrient transfer interface once

inside the plant; as such, it was initially thought that endophytic fungi were not able to

provide nutrients to plant roots (Newsham 2011). Subsequently, it was found that

Heteroconium chaetospira, a dark septate endophyte, was able to transfer nitrogen to the

roots of cabbage plants (Usuki & Narisawa 2007). H. chaetospira is a saprobe that transfers

nutrients to plants which have been acquired from decaying organic matter (Hashiba &

Narisawa 2005). Several other examples of nutrient transfer by endophytic fungi are known

(Porras-Alfaro & Bayman 2011). Recently a new class of endophytic insect pathogenic fungi

(EIPF) has been elucidated, which is able to transfer nitrogen obtained from the insects that

they infect directly to their plant symbionts (Behie et al. 2012).

Metarhizium and Beauveria are examples of insect pathogenic fungi that have

recently been discovered to have endophytic capabilities (Sasan & Bidochka 2012, Akello et

15  
al. 2007). Historically, research on these fungi focused on their insect pathogenicity and

their use as biological control agents. These fungi are broad range insect pathogens and are

able to infect over 200 insect species (Roberts & Hajek 1992). Why would insect pathogenic

fungi possess the ability to colonize plant roots? Work done on Metarhizium robertsii has

shown that this EIPF is able to acquire nitrogen from the insects it infects and, through

endophytic association, transfers the insect-derived nitrogen to plants (Behie et al. 2012).

This finding represents a remarkable adaptation by which plants are able to utilize insects in

the soil as a nitrogen source, albeit through a fungal partner.

Root feeding insects represent a substantial loss of nitrogen to the plant through leaf

and root material as well as through root exudates (Blossey & Hunt-Joshi 2003). This

nitrogen loss, if not balanced, may have detrimental effects on plant growth and development

(Goulding et al. 1998). The partnership between an EIPF and a plant can allow plant

reacquisition of nitrogen previously lost through below ground insect herbivory.

16  
 A B C

   

Ammonification  &
nitrification

Figure 1. Representation of 3 major routes by which plants are able to obtain insect bound
nitrogen. A) Carnivorous plants capture and digest insects and acquire insect derived
nitrogen. Shown is a depiction of a pitcher plant where insects are captured in a pitfall trap
containing fluid and are subsequently digested. B) Organic nitrogen within insects can be
made available to plants from insect cadavers and from insect frass. Insect-derived nitrogen
is microbially converted by ammonification and nitrification and made directly available to
plants or, translocated to plants through a symbiotic association with mycorrhizal fungi
(shown in yellow). C) Plants are able to acquire insect derived nitrogen through a symbiotic
association with EIPF. EIPF (shown as purple) infect and kill insects in the soil and insect-
derived nitrogen is translocated directly to plants.

17  
1.8 Evolutionary Implication of Nitrogen Transfer by Fungal Symbionts
 
The majority of land plants are able to form intimate associations with soil fungi,

either mycorrhizae or endophytes. What is intriguing about these associations is that these

fungi are from disparate phylogenetic lineages (Arnold & Lutzoni 2007, Porras-Alfaro et al.

2008). This suggests that these fungi have undergone convergent evolution with respect to

plant symbiosis. The intimacy of this association was potentially driven by fungal acquisition

of plant-fixed carbon. Plants can fix carbon dioxide and are a carbon reservoir, a valuable

commodity to fungi (Behie et al. 2013). Plants would allow carbon exchange to a partner that

could provide nutrients. Nitrogen is a valuable commodity to plants and symbionts that

provide nitrogen may have access to plant carbon. In this review, we considered insects as a

nitrogen reservoir and plant adaptations that allow access to insect nitrogen. Several

examples of fungal symbionts that provide nitrogen to plants were outlined, and the

implication is that the plant host may be driving the evolution of endophytism.

The traditional paradigm concerning the population structure of insect pathogenic

fungi has focused on the insect host as the driving force. However, the fact that some

pathogenic fungi are endophytes (Beauveria and Metarhizium) suggests that plant host

symbiosis may be a major evolutionary influence (Behie et al. 2013). Insect pathogenic

fungi that are incapable of forming plant associations would be more likely to evolve insect

host specificity and, in this case, fungal evolution would be driven most influentially by the

range of insect hosts. This type of specificity would provide an advantage for these insect

pathogenic fungi under certain conditions. However, EIPF have a broad insect host range,

which would be advantageous to the plant as well as to the fungus, i.e., a larger insect

nitrogen reservoir to use in trading for carbon. This implies that the plant host is driving the
18  
evolution of the broad range EIPF, since it would be beneficial for the plant to associate with

fungal partners that are able to exploit a broad range of insects.

1.9 Conclusions
 
Nitrogen cycling in the soil has largely ignored insects as a nitrogen reservoir. Many

species of plants have evolved adaptations by which to exploit nitrogen present in insects.

Carnivorous plants survive, almost exclusively, from nitrogen found in insects (Plummer et

al. 1964). While carnivorous plants are one of the best known examples of a plant adaptation

in order to access insect nitrogen, plants that are incapable of enzymatic/mechanical

digestion have developed novel fungal partnerships in order to exploit insects as a nitrogen

source. By partnering with mycorrhizal fungi, plants can gain access to nitrogen, of which

one form is derived from decomposing insects and insect frass. Another, more active

symbiosis for the acquisition of insect nitrogen is through EIPF, such as Metarhizium or

Beauveria. EIPF are able to infect live insects and translocate insect-derived nitrogen to their

plant partners, potentially in exchange for plant-synthesized carbon, as has been found for

mycorrhizal fungi. These associations have theoretically evolved to satisfy fungal acquisition

of carbon, and have driven EIPF to a broad insect host range that provides plants with the

largest potential source of nitrogen. These symbioses have potentially driven the evolution of

soil fungi (e.g. mycorrhizal and EIPF) in a manner that allows non-carnivorous plants to

reacquire the nitrogen lost through insect herbivory.

19  
 Nutrient Transfer in Plant-Fungal Symbioses

Published as: Behie SW, & Bidochka MJ. Nutrient transfer in plant-fungal symbioses.
Trends in Plant Science 2014 19, 734-740.

Behie wrote the manuscript with additions, edits, and guidance from Bidochka.

20  
1.10 Abstract
 
Almost all plant species form symbioses with soil fungi, and nutrient transfer to

plants is largely mediated through this partnership. The majority of research into fungal

nutrient transfer to plants has largely focused on limiting soil nutrients, such as nitrogen and

phosphorus, transferred to plants by mycorrhizal fungi. Innovative research into fungal-

mediated nutrient transfer has also revealed that certain fungal endophytes are able to transfer

nitrogen to their plant hosts. Here, I focus on recent discoveries (since 2008) of genes and

their encoded transporters involved in the movement of soil nutrients between symbionts.

These discoveries could lead to applications in agricultural and horticultural settings and in

the development of model fungal systems that could further elucidate the role of fungi in

these symbioses.

1.11 Introduction
 
Over 90% of all plant species form symbioses with soil fungi. These partnerships

were initiated over 450 million years ago, and this early origin suggests that beneficial

fungal/plant interactions played an important role in the global colonization of land plants

(Bonfante & Genre 2010). Benefits imparted to the plant include increased mineral nutrient

absorption, water use efficiency, disease resistance, and may facilitate plant-to-plant

communication (Parniske 2008, Azcon-Aguilar & Barea 1997). Of particular interest is the

transfer of soil nutrients mediated by the fungal symbiont to the plant host. Colonization of

plant roots by fungi increases the surface area from which plants can scavenge nutrients.

Thus, root-colonizing fungi facilitate the absorption of critical and often limiting soil
21  
nutrients, which results in increased photosynthetic ability, enhanced growth, productivity,

and overall health of the plant (Clark & Zeto 2000)

It is estimated that 5,000 fungal species are capable of colonizing roots and

subsequently furnishing plants with soil nutrients (Peterson et al. 2004). The two major

classifications of beneficial plant-associating fungi are mycorrhizal fungi and endophytic

fungi. One distinguishing characteristic between mycorrhizal and endophytic fungi is their

ability to survive in the absence of their plant hosts, that is, the majority of mycorrhizal fungi

are obligate biotrophs (Bonfante & Genre 2010). Endophytes, however, do not necessarily

require internal plant colonization for survival, and many of these fungi have multiple life

stages as decomposers of organic detritus, plant pathogens, or pathogens of soil insects

(Fukasawa et al. 2009, Slippers & Wingfield 2007, Sasan & Bidochka 2012)

There are seven classes of mycorrhizal fungi including ectomycorrhizal,

endomycorrhizal (arbuscular mycorrhizal), ectendomycorrhizal, ericoid, arbutoid,

monotropoid, and orchid (Peterson et al. 2004). While little is known about the specific

evolutionary origins of these symbioses, currently more than 2,000 species of mycorrhizal

fungi are able to colonize the roots of over 300,000 species of plants in ecosystems globally

(Klironomos 2000). The majority of mycorrhizal fungi can be classified as either

ectomycorrhizal (EM) or arbuscular mycorrhizal (AM) (Saikkonen 2007). EM fungi

typically fall within the Ascomycetes or Basidiomycetes and occur, predominantly, in

forested areas where they colonize roots of trees including tropical species

(Dipterocarpaceae), pine trees (Pinaceae), and beech (Fagaceae) (Smith & Read 1997). These

fungi initially form symbioses with lateral roots and create a mycelial sheath that envelops

the root tip. The fungus is then able to penetrate the plant root creating a hartig net, a
22  
complex network of mycelia surrounding the epidermal and outer cortical cells (Smith &

Read 1997).

AM fungi are obligate biotrophic symbionts and all belong to the phylum

Glomeromycota (Helgason & Fitter 2009). AM fungi differ from EM fungi in that the

hyphae penetrate cells of the inner root cortex, forming specialized branched structures called

arbuscules (Jayot et al. 2007). Arbuscules create a specific nutrient transfer interface that is

embedded with a number of plant and fungal transporters, facilitating nutrient transfer

between the symbionts (Genre et al. 2005, Genre et al. 2005). AM fungi, however, do not

enter the protoplast of the plant cell; instead the plant cell wall accommodates the entering

arbuscule, forming the new and distinct peri-arbuscular membrane (PAM) (Genre et al.

2005).

Endophytes can be defined as endosymbiotic and asymptomatic colonizers of plants.

Endophytes have been found to associate with almost all plant species, and some are capable

of internal migration within plant tissue, moving to inhabit foliage, stems, and bark, as well

as roots (Gazis & Cheverri 2010, Porras-Alfaro & Bayman 2011). Root-associating

endophytic fungi share numerous similarities with their mycorrhizal counterparts, however,

the majority of endophytes are not obligate biotrophs and live, at least part of their life cycle,

outside of the root (Rodriguez et al. 2009). The mechanism of plant colonization, and the

nature of the symbiosis, however, is poorly understood when compared to mycorrhizal fungi

(Saikkonen 2007). Specifically, their ability to transfer nutrients to plant hosts is a relatively

recent discovery, and the mechanisms of this transfer are unknown.

Nutrient transfer is common among the majority of plant/fungal symbioses; however,

23  
the specific mechanisms and gene products involved in nutrient transfer of many of these

fungi remain to be elucidated. Despite the importance of nutrient transfer to plants by

mycorrhizal fungi, there is a paucity of information with respect to the fungal genes involved,

and the majority of genetic information obtained has been inferred from a plant perspective.

Mycorrhizal fungi are difficult to grow and difficult to manipulate using traditional

transformation technology. Therefore, research has focused on plant molecular biology,

using model plants such as Medicago that can be genetically modified (Genre et al. 2008,

Doidy et al. 2012, Javot et al. 2011). However, recent discoveries on the plant-colonizing

ability of a number of endophytic fungi have provided the opportunity to genetically analyze

the fungal role in this symbiosis, as these fungi can be stably transformed (Mukherjee et al.

2010, Fernanda 2012, Fang et al. 2004). It is established that fungal symbionts are able to

transfer limiting soil nutrients, including phosphorus, nitrogen, and sulfur to their plant hosts,

and the transfer is reciprocated with plant-derived carbohydrates to the fungus (Kiers 2011).

There are several good reviews on mycorrhizal fungi, however, here we will focus the

information available on nutrient transfer in mycorrhizal and endophytic fungi since 2008.

We will specifically focus on recent elucidation of novel symbiotic pathways that relate to

widespread plant/fungal nutrient exchange.

24  
1.12 Nitrogen Transfer from Fungal Symbiont to Plant Hosts
 
In almost all ecosystems, including agricultural conditions, nitrogen availability limits

primary productivity, and nitrogen cycling and storage is highly variable (Boring 1988). The

rate of photosynthesis can be directly linked to nitrogen availability, and plants require

relatively large quantities of nitrogen compared to phosphorus (Yoneyama 2007). Over 50%

of leaf nitrogen is devoted to photosynthesis (Field & Mooney 1986), thus, nitrogen

availability is integral for carbon fixation.

There are two routes by which atmospheric nitrogen is deposited into terrestrial

ecosystems, atmospheric deposition and nitrogen fixation (Boring et al. 1988). In the

atmosphere, several forms of nitrogen exist, such as N2 (the most abundant, comprising 78%

of the atmosphere), NO, NO3-, and NH3 gas (Boring et al. 1988). While some of these forms

can be readily incorporated into terrestrial ecosystems, N2 requires fixation by soil and/or

plant symbiotic bacteria (Steppe 1996). Once fixed in terrestrial systems, nitrogen may be

bound in organic matter and must subsequently be mineralized in order to release inorganic

nitrogenous compounds that can be utilized by plants (Van Der Heijden et al. 2007).

Nitrogen conversion is largely mediated by fungal decomposers in the soil (Hodge & Fitter

2010), and a number of soil fungi are able to exploit the energy stored in the redox potential

of organic nitrogen (Sinsabaugh 2010). For example, mycorrhizal fungi are able to excrete

exoenzymes that break down organic polymers and subsequently capture the nitrogen-

containing monomers, thus, providing a direct path from organically bound nitrogen in the

soil to the plant (Schimel & Bennet 2004). That is, mycorrhizal fungi provide the host plant

access to nitrogenous reserves previously inaccessible in the soil.

25  
 Typically, nitrogen bound in organic matter is present as peptides, proteins, and free

amino acids, which can be directly absorbed by fungi (Schimel & Bennet 2004). Both AM

and EM fungi release peptidases and proteases into the soil that cleave organically bound

nitrogen and subsequently absorb the nitrogenous monomers (Bonfante & Genre 2010,

Nygren et al. 2007). Inorganic nitrogen in the soil is preferentially taken up by fungi as

nitrates (NO3-) or ammonium (NH4+), and it has been theorized that this nitrogen, once

absorbed by the extraradical mycelia, is converted into the amino acid arginine for transport

into the intraradical mycelia (Ngwene et al. 2013). Once internal migration is complete, the

arginine is broken down through the urease cycle into ammonium for transport into the plant.

This hypothesized pathway has been supported by work that showed that during fungal

association, arginine in host plant roots is increased three-fold, and was the most abundant

free amino acid (Tian et al. 2010). This pathway would indicate preferential absorption of

NH4+ by fungal hyphae, however, this is not always the case. Cowpea plants furnished with

NO3- showed a greater root/shoot ratio than that of cowpeas grown with NH4+, indicating that

the movement of nitrate within the plant is faster than that of ammonium, and that the

transfer of NO3- may rely on independent pathways within the fungus (Ngwene et al. 2013).

Preference for the form of inorganic nitrogen absorbed by the hyphae may also relate directly

to soil type. In the majority of soils, nitrate is more mobile than ammonium; this effect is

exacerbated in clay-based soils since clay minerals adsorb NH4+ due to its positive charge

(Mian et al. 2009). Thus preference for a specific nitrogen type may be a result of soils in

which the symbiosis occurs.

While the transport of nitrogen in the majority of plant/fungal symbioses seems to

focus heavily on nitrates and ammonium, the specifics of the transfer vary between fungal

26  
species (Fig. 2). For example, AM fungi construct a specific nutrient transfer interface with

the host plant, the aforementioned PAM, and ammonium transporters in Glomus intraradices

have been characterized within this membrane (Perez-Tienda et al. 2011). Specifically, the

ammonium transporters GintAMT1 and GintAMT2 are expressed by mycorrhizal fungi

colonizing cortical cells (Perez-Tienda 2011). Other Glomus species have also been studied

for their nitrogen transfer ability, and a Glomus mosseae ammonium transporter

(GmAMT4.1) has been identified during arbuscule development inside soybean roots (Kobae

et al. 2010). In several of the endomycorhizal symbioses, a distinct up regulation of the AMT

family of ammonium transporters has been observed. In ectomycorrhizal fungal-colonized

roots, fungal nitrate transporters have been found. In both Laccaria bicolor and Hebeloma

cylindrosporum, high-affinity nitrate transporters in the NRT family are expressed during

plant colonization (Kemppainen & Pardo 2013, Jargeat et al. 2003).

Endophytic fungi are also capable of transferring nitrogen to their plant hosts. The

dark septate endophyte, Heteroconium chaetospira, was able to transfer nitrogen obtained

from decaying organic matter in the soil to the roots of cabbage (Usuki & Narisawa 2007).

Other endophytes have been found to increase the nitrogen uptake efficiency in young plants

in nitrogen-depleted soils (Upson et al. 2009). Nitrogen can also be cycled into plants

through endophytic, insect-pathogenic fungi (EIPF). This unique group of root-colonizing

fungi is able to transfer nitrogen harvested from insect hosts into different plant species

(Behie et al. 2012, Behie & Bidochka 2014). Specifically, species of the insect pathogens

Metarhizium and Beauveria are able to infect soil insects and subsequently transfer insect-

derived nitrogen to plants (Behie et al. 2012, Behie & Bidochka 2014). The specific form of

nitrogen transfer is unknown. However, it is postulated that nitrogen is converted to

27  
ammonium within the intraradical mycelia and transferred into plant roots in this form, much

like the theorized pathways in the AM and EM/plant symbiosis (Behie et al. 2013).

A reciprocal upregulation of nitrogen transporters has also been observed in plants

that accommodate fungal translocation of nitrogen. Plant ammonium transporters are up-

regulated in arbuscule-containing cells. For example, in sorghum the expression of plant

ammonium transporters SbAMT3;1 and SbAMT4 were induced only in arbuscule-containing

cells (Koegel et al. 2013). Similarly, nitrate transporters were expressed in arbusculated cells

of Medicago truncatula (Gaude et al. 2012). Specifically, the low-affinity nitrate

transporters, medtr7g116510.1, medtr4g136330.1, mtr.27765.1.s1, and medtr4g159100.1, all

had increased expression in cortical cells colonized by AM fungi (Gaude et al 2012). This

coordinated and specific expression of both plant and fungal ammonium and nitrate

transporters in mycorrhizal-colonized cortical cells suggests the critical importance of fungal

nitrogen transfer in plants.

28  
Figure 2. Plant–fungal root symbioses. Plant genes (depicted in green) and fungal genes
(depicted in black) involved in nitrogen (N), phosphorus (P), and other nonlimiting soil
nutrient (NL) transport in arbuscular mycorrhizal fungal (Perez-Tienda et al. 2011, Kobae et
al. 2010, Harrison & Van Buuren 1995, Maldonado-Mendoza et al. 2001, Casieri et al. 2012,
Cappellazzo 2008, Allen & Shachar-Hill 2009), endophytic fungal (Yadav et al. 2010,
Zuccaro et al. 2011), and ectomycorrhizal fungal (Tatry et al. 2009, Garcia et al. 2014,
Kemppainen et al. 2009, Mansouri-Bauly et al. 2006) symbioses.

29  
1.13 Transfer of Phosphate from Fungus to Plant
 
Phosphorous may also be limiting in some ecosystems; however, unlike nitrogen,

atmospheric levels of phosphate do not play an important role in the cycling of phosphate to

plants (Ruttenberg 2003). The majority of phosphorus is bound in rocks, minerals, and large

oceanic deposits (Ruttenberg 2003). Geological processes such as weathering release

phosphorus as phosphate ions into the soil where it can be absorbed by plant roots

(Ruttenberg 2003).

The most abundant form of phosphate in soils is orthophosphate (PO43-) and in this

form it can be absorbed directly by plant roots or by roots colonized by mycorrhizal fungi

(Vasquez et al. 2000). PO43- is then rapidly translocated to fungal structures inside the root,

such as intercellular hyphae or highly branched arbuscules, for transport directly into root

cortical cells (Smith & Smith 2011). The transfer of PO43- is a stepwise process that involves

the conversion of PO43- to a polyphosphate molecule for movement through the mycelia; the

polyphosphate is then converted back into an inorganic phosphate molecule for transport into

the plant (Smith & Smith 2011). Mycorrhizal fungi secrete enzymes such as

phosphomonoesterases and phosphatases that are able to hydrolyze organic P compounds in

soil, such as phenolphthalein diphosphate, providing plant hosts with phosphorous typically

unavailable to non-mycorrhizal roots (Plassard & Bell 2010).

Initial studies on fungal nutrient transfer focused on the ability of Glomus spp. to

transfer phosphate to their plant partners. It is now apparent that a number of mycorrhizal

fungi play a key role in the cycling of soil P (Smith et al. 2011) and a number of transporters

involved in the process have been elucidated. For example, microarray analysis of G.

30  
intraradices and an associated plant (M. truncatula) has shown a coordinated up-regulation

of fungal and plant phosphate transporters, specifically GmosPT and GvPT in the fungus and

MtPt4 in the plant (Harrison & Van Buuren 1995, Maldonado-Mendoza et al. 2001, Bendetto

et al. 2005). These transporters are specific to AM symbiosis and are localized in the PAM

(Bendetto et al. 2005). In addition, various transporters critical to the cycling of phosphate

have been reported in EM fungi. In the model organisms Laccaria bicolor and Hebeloma

cylindrosporum, there is increased expression of the phosphate transporters HcPT2 and LbPT

during plant root association (Tatry et al. 2009). Expression is further increased when soil

phosphorous levels are low, indicating the importance of these transporters in plant

phosphate nutrition (Tatry et al. 2009).

Significant up-regulation of high-affinity phosphate transporters has also been

reported in a number of mycorrhizal plants, including legumes and woody plant species

(Loth-Pereda et al. 2011). For example, proteins in the Pht1 family are highly expressed

during ectomycorrhizal symbiosis, and knockdown experiments have shown the importance

of these transporters in phosphate transfer between the EM Laccaria bicolor and poplar trees

(Loth-Pereda et al 2011).

Endophytes are also capable of phosphorous transfer to plants, however, there is a

paucity of information on this process. Piriformospora indica, a basidomycetous root

endophyte, was shown to increase the phosphate uptake of its host plant, specifically through

the increased expression of the phosphate transporter PiPT (Yadav et al. 2010).

31  
1.14 Non-Limiting Soil Nutrients
 
Not all nutrients transferred from symbiotic fungi to plants are limited in soils; and

both AM and EM are able to transfer mineral nutrients such as sulfur, cesium, zinc,

potassium, and copper (Sieh et al. 2013, Gyuricza et al. 2010, Cartmill et al. 2008). These

minerals enter soil systems in a manner similar to phosphorous; however, large quantities are

typically not required by plants nor are they particularly mobile in soil, and as such are

relatively abundant in the majority of ecosystems (Nord & Lynch 2009).

In AM fungi, the transfer of non-limiting and limiting soil nutrients is linked such that

the overall efficiency of total mineral transfer is dependent on the relative abundance of

limited nutrients. For example, examination of the plant genetics underlying fungal nutrient

transfer revealed that during mycorrhizal association several Medicago sulfate transporters

(MtSULTRs) were upregulated, indicating that root colonization by fungi increased the rate

of plant sulfur absorption (Casieri et al. 2012). However, the efficiency of sulfur uptake in

plant roots was directly tied to phosphate availability, and there was an increase in sulfur

transfer only when soil phosphate content was low (Sieh et al. 2013). A similar relationship

was observed between potassium and phosphate in ectomycorryhizal nutrient transfer. The

transfer of potassium to cluster pine trees (Pinus pinaster) was significantly increased when

colonized by the ectomycorrhizal fungi Hebeloma cylindrosporum (Garcia et al. 2014).

However, expression of the fungal potassium transporter HcTrk1 was correlated with a

decrease in the phosphate transporter HcPT1 (Garcia et al. 2014).

There is little information available on the ability of endophytes to transfer non-

limiting soil nutrients to their plant hosts. Historically, endophytes were viewed as

32  
asymptomatic plant colonizers with no nutrient transfer capabilities; however, we now know

that is not the case (Usuki & Narisawa 2007, Upson et al. 2009, Behie et al. 2012, Behie &

Bidochka 2014). Future research on fungal endophytes should focus on the extent of

endophyte-mediated nutrient cycling as well as overall ecological impact.

1.15 Reciprocal Exchange: Trading Carbon for Nutrients

 
In soil ecosystems, readily available carbon is limited particularly with respect to

fungi that are incapable of breaking down complex organic compounds. Plants fix carbon and

offer easily metabolizable carbohydrates to fungi that have evolved to colonize plant roots

and offer, in exchange, limiting nutrients to the plant (Smith et al. 2009).

This reciprocity has been observed in several mycorrhizal fungi. Glomus intraradices

transfers phosphorus to the roots of wild carrot (Daucus carota) and barrel clover (Medicago

truncatula) in exchange for carbon (Maldonado-Mendoza et al. 2001). Here it was found that

an efflux of carbon from the plant that was available for the fungus resulted in an increase in

phosphate transfer to the plant (Maldonado-Mendoza et al. 2001). Conversely, a decrease in

available carbon slowed the overall rate of phosphorous transfer (Maldonado-Mendoza et al.

2001). These results are significant since they not only indicate a close symbiotic relationship

between the plant and the fungus, but also that the fungus and plant are capable of sensing

their immediate environments and actively participating in the reciprocal exchange of

nutrients.

33  
 A similar relationship was observed with nitrogen transfer, where nitrogen is

provided to plants in exchange for photosynthetically fixed carbon with G. intraradices and

wild carrot (St. Arnaud et al. 1996). Here again, nitrogen transfer to the plant was reliant on

plant carbon provided to the fungus (St. Arnaud et al. 1996). Furthermore, it was observed

that G. intraradices would not transfer nitrogen until plant sugars had initially been

transferred to the fungus (St. Arnaud et al. 1996). Thus, reciprocal nutrient exchange

stabilizes the cooperative relationship between the symbiotic partners.

The mechanisms that control carbon transport into intraradical mycelia have yet to be

elucidated; however, plant sugar transporters and enzymes play a key role in the movement

of plant-derived carbohydrates into the fungus. In Glomus spp., a high-affinity

monosaccharide transporter, MST2 has been characterized (Bucking & Shachar-Hill 2005).

The expression of this transporter correlates with expression patterns of the mycorrhizal

phosphate transporter, PT4, indicating that reception of carbon into the fungal symbiont is

directly related to phosphate transfer efficiency (Bucking & Shachar-Hill 2005). Plant sugar

transporters also show elevated expression during mycorrhizal colonization. In the model

mycorrhizal association between M. truncatula and Glomus intraradices, there was increased

expression of MtSucS1, a plant sucrose synthase gene, surrounding internal hyphae and

arbuscules (Doidy et al. 2012). Furthermore, the expression of a family of M. truncatula

sucrose transporters, MtSUTs, increased in mycorrhizal-colonized roots (Doidy et al. 2012).

There is little information available on nutrient reciprocity in endophytic fungi.

However, it has been shown that carbon exchange may be required for EIPF root association.

A raffinose transporter (Mrt) was found to be crucial for rhizospheric competence in

Metarhizium and a gene knockout led to poor growth in soybean root exudate (Fang & St.
34  
Leger 2010). Metarhizium also produces an extracellular invertase which, when knocked out,

led to poor growth in root exudate but improved the root-colonizing ability of the fungus

(Liao et al. 2013). They hypothesized that the invertase knockout caused a reduction in

carbon catabolite repression resulting in increased production of plant cell wall-degrading

depolymerases that subsequently allowed for root colonization.

Several non-limiting soil nutrients are transferred to plants through fungal

intermediaries. Despite the lack of knowledge surrounding the transfer of these nutrients, it

has been seen that, with respect to certain micronutrients, transfer is dependent on plant

reciprocity (Casieri et al. 2010, Cairney 2012). The question of plant carbon reciprocity to

fungal endophytes in exchange for soil nutrients has yet to be elucidated.

1.16 Applications
 
Although arbuscular mycorrhizal, ectomycorrhizal, and endophytic fungi are

ubiquitous in soils, specific applications of these fungi in agricultural fields, with respect to

increasing plant productivity and survival, remain intangible. The inability to culture

mycorrhizal fungi effectively in the laboratory is a major impediment to producing

mycorrhizal fungi on an industrial scale for commercial applications, and large-scale

culturing on plants is a cumbersome process that yields inconsistent results (Ceballos et al.

2013). Recently, some of these inconsistencies have been alleviated through novel culture

techniques (Ceballos et al. 2013) and, advances in the study of mycorrhizal genetics may

lead to the creation of easily manipulated laboratory strains for study as well as for industrial

applications.
35  
 Regardless of laboratory difficulties, there is great potential for the industrial

application of mycorrhizal fungi. In agricultural and horticultural settings, mycorrhizal fungi

have been shown to improve water-uptake efficiency, nutrient acquisition, and the survival

rate of young crops (Parniske 2008). By increasing the presence of mycorrhizal fungi in

industrial soils, there should be further increases in positive, fungal-conferred benefits to

plants.

EIPF have been manufactured on an industrial scale for the treatment of insect pests

(Kassa 2008). Metarhizium and Beauveria are of particular interest because they infect a

large range of insects and have nitrogen transfer capabilities (Javelle et al. 2003,

Kemppainen & Pardo 2013, Roberts & Hajek 1992, Behie et al. 2014). These fungi live in a

wide range of hosts, their genomes have been sequenced, and they are amenable to traditional

molecular genetic techniques, such as gene knockouts. As such, these fungi may provide

insight into the roles of fungal genes in reciprocal nutrient exchanges, particularly if there are

homologous transfer genes in mycorrhizal fungi.

1.17 Conclusions
 
Despite the integral roles plant-associating fungi play in the cycling of soil nutrients,

they remain relatively enigmatic from a research perspective. Although several studies have

been conducted on the nutrient transfer capabilities of mycorrhizal fungi, a combination of

their inability to be effectively cultured and their recalcitrance to traditional genetic

manipulation, leaves a paucity of information on these fungi (Parniske 2004). Endophytes

have the potential to be an important model for the study of plant root interactions. In
36  
contrast to the mycorrhizal fungi, a number of endophytes can be easily cultured in the lab,

and stable transformation systems are available (Fang et al. 2004, Sebastienes et al. 2012).

Through the use of novel methodology and EIPF as a model, there is the potential to

characterize genes involved in fungal nutrient transfer. The application of optimized root-

colonizing fungi into agricultural and horticultural settings could increase fertilizer use

efficiency, limit run-off contamination, and decrease the total energy inputs into

agroecosystems.

1.18 Acknowledgements
This work was supported by a Natural Sciences and Engineering Research Council of

Canada Discovery grant to M.J.B. We thank E.K. Bidochka for artwork in Figure 1.

37  
 Chapter 2: Endophytic-Insect Parasitic Fungi Translocate
Nitrogen Directly from Insects to Plants

Published as: Behie SW, Zelisko PM, & Bidochka MJ. Science 2012 336, 1576-1577.

Behie developed experimentation under supervision of Bidochka and Zelisko. Behie performed all
experimentation and analyses. Behie wrote the manuscript with additions, edits, and guidance from
Bidochka and Zelisko.

38  
2.1 Abstract
 
Most plants obtain nitrogen through nitrogen-fixing bacteria and microbial decomposition of

plant and animal material. Most vascular plants are able to form close symbiotic associations

with endophytic fungi. Metarhizium is a common plant endophyte found in a large number of

ecosystems. This abundant soil fungus is also a pathogen to a large number of insects, a

source of nitrogen. It is possible that endophytic capability and insect pathogenicity of

Metarhizium are coupled to provide an active method of nitrogen transfer to plant hosts via

fungal mycelia. In this study we used soil microcosms to test the ability of M. robertsii to

translocate insect-derived nitrogen to plants. Insects were injected with 15N-labelled nitrogen,

and we tracked the incorporation of 15N into amino acids in two plant species, haricot bean

(Phaseolus vulgaris) and switchgrass (Panicum virgatum), in the presence of M. robertsii.

These findings are evidence that active nitrogen acquisition by plants in this tripartite

interaction may play a larger role in soil nitrogen cycling than previously thought.

39  
2.2 Introduction
 
Nitrogen gas, although comprising 78% of the atmosphere, is unavailable to plants as

a source of nitrogen unless it is fixed by microbial symbionts (e.g. Rhizobium) or free-living

(e.g. Azotobacter) microbes (Cocking 2003). In many natural, as well as agricultural, settings

nitrogen is the limiting nutrient for plant growth. The current model of the soil nitrogen cycle

relies heavily on nitrogen fixing bacteria to furnish plants with useable nitrogen (some is

fixed by lightning strikes) (Galloway 1998). However, there are some examples where plants

have evolved mechanisms to scavenge nitrogen from insects. Carnivorous plants are able to

obtain significant amounts of nitrogen from insects they ingest. Pitcher plants (families

Nepenthaceae and Sarraceniaceae) trap insects in a deep cavity filled with liquid and insect-

derived nitrogen can comprise up to 70% of the plant nitrogen content (Schulze et al.

2001). There is an example of a fungus-mediated transfer of insect-derived nitrogen to plants.

The ectomycorrhizal fungus, Laccaria bicolor transfers nitrogen from soil dwelling

collembola to white pine (Pinus strobus) whose roots it colonizes (Klironomos & Hart 2001).

The ability of L. bicolor to transfer insect-derived nitrogen is specific to white pine

and generally L. bicolor associates with roots of pine and spruce in temperate forests (Richter

& Brun 1993, Buschena et al. 1992). However, it suggests that a more general example of

insect-derived nitrogen transfer via fungal mycelia to plants may exist. Metarhizium spp. are

ubiquitous soil dwelling insect pathogenic fungi that are found in a variety of ecosystems

worldwide (St. Leger 1993), occur in soils up to 106 propagules per gram (Milner 1991), and

can infect over 200 species of insects (Roberts & Hajek 1992). Insects contribute substantial

amounts of nitrogen to soil. There is approximately 0.4-4 g (by weight) of available insect

nitrogen per square meter of habitat (Supp. Calc. 1)

40  
 During a routine survey of plant root symbionts we found that Metarhizium spp.

formed endophytic associations with many plant species (Wyrebek et al. 2011, Sasan &

Bidochka 2011). Endophytes live internally within the plant and the host plant may benefit

from the interaction (Saikkonen et al. 1998). Here, we hypothesized that Metarhizium can

parasitize and kill a soil born insect and then transfer the insect-derived nitrogen to plants via

fungal mycelia and endophytic association.

We used 15N -labeled waxmoth (Galleria mellonella) larvae as a model prey insect

and used this model in the experimental design to measure Metarhizium-mediated

translocation of 15N to the foliage of haricot bean (Phaseolus vulgaris) or switchgrass

(Panicum virgatum).

2.3 Materials and Methods

 
2.3.1 Materials
 
Fungal cultures – Metarhizium robertsii strain Ma2575, M. acridum strain 7486 and

Beauveria bassiana strain 252 were obtained from the U. S. Department of Agricultural

Research Service Collection of Entomopathogenic Fungal Cultures, Ithaca, New York. M.

flavoviridae rf08A, M. brunneum strain 43a-2i, M. guizhouense strain b77-ai and,

Lecanicillium lecanii strain 313 were isolated from the field and cultured in lab. Aspergillus

flavus strain 6982 was obtained from University of Alberta Microfungus Collection and

Herbarium, Edmonton, Alberta. M. robertsii-GFP, M. guizhouese-GFP, B. bassiana-GFP,

and L. lecanii-GFP were used for confocal micrographs, the construction of the GFP

41  
expressing plasmids, as well as transformation of Metarhizium species have been previously

described (Fang et al. 2006). Stock cultures were grown at 27OC in potato dextrose agar

(PDA; Difco laboratories, Mississauga, Ontario, Canada).

Plant Material – Phaseolus vulgaris (Haricot Bean) and Panicum virgatum (switchgrass)

seeds were obtained from OSC seeds, Waterloo, Ontario, Canada. Triticum aestivum (winter

wheat) seeds were obtained from sprout master, Elmvale, Ontario, Canada. Glycine max (soy

bean) seeds were obtained from Agriculture Canada Research Station, London, Ontario,

Canada.

 
2.3.2 Seed Sterilization and Plating
 
Seeds were surface sterilized before use in order to prevent any unwanted fungal or

bacterial growth. Seeds were immersed in sterile distilled water for 30 min in a 50 mL

capped plastic tube. They were then washed in a 4% sodium hypochlorite solution three

times, 10 min for each wash. The seeds were rinsed with sterile distilled water in between

each wash. The fluid was drained and seeds were washed with sterile distilled water. The

seeds were then placed in 15% hydrogen peroxide for 30 min and then washed three times

with sterile distilled water. The seeds were then kept overnight at -5°C to allow for

synchronization of growth, then plated on water agar, in sterile conditions, and kept at 25°C

for a photoperiod of 16 h a day for a minimum of 10 days in order to obtain seedlings, which

were further used in the study.

42  
2.3.3 G. mellonella Injection
 
The Galleria mellonella larvae were injected with a series of increasing

concentrations of an ammonium sulfate solution in order to determine the maximum volume

and concentration of injection the insects were able to take while surviving for 48 h

subsequent to injection. The larvae were injected with a sterilized syringe through the rear

proleg, with 10 µl of a 5 % 15N ammonium sulfate solution and kept for 48 h to allow healing.

After 48 h the larvae were infected with M. robertsii or Aspergillus flavus.

2.3.4 G. mellonella Infection

 
The Galleria mellonella were infected with conidia 48 h after 15N injection by placing

the larva on a 10 day old conidiating culture of M. robertsii or A. flavus. The plate was

agitated for 2 min allowing the fungus to sufficiently adhere to the insects.

2.3.5 Soil Sampling and Plating

 
Soil was taken from pots containing microcosms with G. mellonella larvae and either

M. robertsii or A. flavus and plant seedlings. Soil was sampled 0.5 cm from the plant roots at

a depth of 2 cm. One gram of soil was sampled from 5 separate plants every two days.

Metarhizium soil samples were plated on Metarhizium selective Potato Dextrose Agar that

contained 9.75 g potato dextrose agar, 0.125 g cylcohexamide, 0.05 g chloramphenicol,

0.125 g 65% dodine, and 0.0025 g crystal violet (all weights per 250 mL). Aspergillus

samples were plated on Aspergillus selective media that contained 5 g yeast extract, 2.5 g

43  
peptone, 0.025 g chloramphenicol, 0.125 g ferric ammonium citrate, 250 µL 0.2% dichloran

in ethanol and 3.75 g agar (all weights per 250 mL). Samples were spread with a plate

spreader and kept at 27°C for 5 days. After 15 days of growth, colony forming units (CFU)

were counted.

2.3.6 MMN Treatment

 
Plants were watered once a week with 50 mL of 50% modified melin-norkins (MMN)

solution. These solutions contained 0.05 g CaCl2, 0.025 g NaCl, 0.05 g KH2PO4, 0.5 g

(NH4)2PO4, 0.15 g MgSO4•7H20, 1 mg FeCl3•6H20, 5 g glucose monohydrate, 10 mL trace

elements solution (all weights per 1 L). The trace elements solution contained 3.728 g KCl,

1.546 g H3BO3, 0.845 G MnSO4•H20, 0.05 g ZnS04•7H20, 0.0125 g CuS04, 0.05 g

(NH4)6Mo7O24•4H20 (all weights are per 1 L).

2.3.7 Microcosm setup

 
Microcosms were created by cutting a circle in the lid of a Petri dish approximately 7

cm in diameter and subsequently covering the lid with 30 micron mesh. The mesh was

adhered to the Petri dish lid with silicone adhesive. The pots used were plastic garden pots 20

cm tall. The microcosms were sterilized by UV light for 2.5 hours prior to use. Once

sterilized the Petri dishes were filled with sterile soil and five fungal infected, 15N injected

larvae and sealed with parafilm. The Petri dishes were then placed in the pot and covered

with soil completing the microcosm. The soil was filled in the pot 3 cm from the top and a

44  
seedling was planted into the soil (Supp. Fig. 1). Only the fungi were able to penetrate the

mesh and move across both compartments of the microcosm.

2.3.8 Plant material preperation for 15N Analysis.

 
Plant tissue was dried at 60°C until all moisture was removed. It was then crushed

into a fine powder with mortor and pestle. The comminuted plant material was encapsulated

in 4 mm tin cups and analyzed for 15N content through NOI-5-emissions spectrophotometry

2.4 Results and Discussion

 
15
N labeled waxmoth larvae were added to microcosms in which the roots of the

plants were separated from each insect using a 30 µm mesh (Supp. Fig. 1). The insects were

infected by Metarhizium 48 h after 15N injection, placed into the microcosm, and the amount

of 15N transfer to plant tissues was determined during a one-month period. After 14 days, in

the presence of Metarhizium, insect-derived nitrogen comprised 28% and 32% of the

nitrogen content in haricot bean and switchgrass, respectively, and was statistically greater

than 15N incorporation in the presence of uninfected 15N -labelled waxmoth larvae (Fig.3,

ANOVA, p< 0.01). After 28 days insect-derived nitrogen comprised 48% and 12% of

switchgrass and bean nitrogen content in the presence of Metarhizium, and again, this was

statistically greater 15N incorporation than in the presence of uninfected 15N -labelled

waxmoth larvae (Fig. 3; factorial ANOVA, p<0.05). Similar results were observed when the

plant seeds were first inoculated with conidia of Metarhizium

45  
 Haricot Bean Switchgrass
45 A D

40

35

30

25

20
Insect-derived N in leaves (%)

15

10

-5
1 7 14 28 1 7 14 28
10 B E

-5
1 7 14 28 1 7 14 28
C
10 F

-5
1 7 14 28 1 7 14 28
Time (days)

Figure 3. Percentage of plant nitrogen derived from waxmoth larvae by an endophytic,

insect-pathogenic fungus. Two plant species were used, haricot bean (Phaseolus vulgaris:
left panel) and switchgrass (Panicum virgatum: right panel). Results obtained with (A & D)
Metarhizium robertsii (B & E) Aspergillus flavus; and (C & F) without fungus. Nitrogen
source: closed circles, waxmoth larvae; open circles, no waxmoth larvae. The amount of
insect-derived nitrogen in the plant issues was determined using a NOI-5 emissions
spectrophotometer. Nitrogen content was calculated as 100 X % 15N in seedling leaves.
Results were analyzed using ANOVA. Error bars represent 1 S.E. N= 6. Results are the
means of three separate trials done in duplicate.
46  
and subsequently formed a root endophytic association. We therefore concluded that the

hyphae infected the insect and transferred nitrogen back to the plant. Plants grown in a soil

microcosm containing a non-endophytic insect pathogen (Aspergillus flavus strain 6982)

(Scully and Bidochka 2005) in the presence of 15N -labeled waxmoth larvae contained less

than 10% insect-derived nitrogen, which was not statistically different to plants grown in the

presence of uninfected 15N -labelled waxmoth larvae (Fig. 3; factorial ANOVA, p>0.05). In

this experiment, insects were infected by M. robertsii or A. flavus within 2 days of placement

into the microcosm. Insects were alive when placed into the microcosm but within two days

of inoculation the larvae were dead. Six days after inoculation insects were mummified with

fungal conidia, this was not observed in control experiments (Supp. Fig. 2). Fungal

propagules of both species were found within 0.5 cm of the plant roots within 6 days (Supp.

Fig. 3).

We concluded in our experiments that plants derived a significant proportion of

nitrogen from soil insects through their endophytic associations with the insect pathogen

Metarhizium spp. Possibly M. robertsii provides nitrogen to the plant in exchange for carbon.

A plant carbon transporter, Mrt (Metarhizium raffinose transporter), has been reported for

Metarhizium and is required for successful root colonization (Fang and St. Leger 2010).

Symbiotic association of Metarhizium with plants may aid in plant survival in nitrogen-

limited soils as vectors for acquiring nitrogen from insects.

47  
Figure 4. Representation of the transfer of the insect-derived nitrogen to plants through an
association with endophytic, insect parasitic Metarhizium. Metarhizium infects and kills a
soil born insect. From the dead, parasitized, insect, fungal mycelia then associate
endophytically with plant roots, through which nitrogen translocation occurs. The reverse
may also occur, that is, endophytically-associated Metarhizium could parasitize and kill and
insect.

2.5 Acknowledgements
This research was conducted with the assistance of an NSERC Discovery Grant to M.J.B.
We thank Maia K. Bidochka for Figure 4.

48  
 Chapter 3: An Additional Branch of the Soil Nitrogen Cycle:
Ubiquity of Insect-Derived Nitrogen Transfer to Plants by
Endophytic Insect Pathogenic Fungi

Published as: Behie SW, & Bidochka MJ. An additional branch of the soil nitrogen cycle:
Ubiquity of insect-derived nitrogen transfer to plants by endophytic insect pathogenic fungi.
Applied Environmental Microbiology 2014 80, 1553-1560.
Behie developed experimentation under supervision of Bidochka. Behie performed all experimentation
and analyses. Behie wrote the manuscript with additions, edits, and guidance from Bidochka.

49  
3.1 Abstract
 
Symbiotic nitrogen transfer in soil has largely focused on nitrogen fixing bacteria. Vascular

plants can lose a substantial amount of this nitrogen through insect herbivory. Previously we

have shown that plants were able to re-acquire nitrogen from insects through a partnership

with the endophytic, insect pathogenic fungus Metarhizium robertsii. That is, the endophytic

capability and insect pathogenicity of M. robertsii are coupled so that the fungus acts as a

conduit to provide insect-derived nitrogen to plant hosts. Here we assessed the ubiquity of

this nitrogen transfer in five Metarhizium species representing those with broad (M. robertsii,

M. brunneum and M. guizhouense) and narrower insect host ranges (M. acridum and M.

flavoviride) as well as the insect pathogenic fungi Beauveria bassiana and Lecanicillium

lecanii. Insects were injected with 15N-labeled nitrogen, and we tracked the incorporation of
15
N into dicots, haricot bean (Phaseolus vulgaris) soybean (Glycine max) and monocots,

switchgrass (Panicum virgatum) and wheat (Triticum aestivum), in the presence of these

fungi in soil microcosms. All Metarhizium species and B. bassiana, but not L. lecanii,

showed the capacity to transfer nitrogen to plants, although to varying degrees. Endophytic

association by these fungi increased overall plant productivity. We also showed that M.

robertsii was able to transfer insect derived nitrogen to plants in the field, where microbial

competition is potentially high. Metarhizium spp. and B. bassiana have a worldwide

distribution, ranging from arctic to tropical habitats, with high soil abundance, and may play

an important role in the ecological cycling of insect nitrogen back to plant communities.

50  
3.2 Introduction
 
Despite its atmospheric abundance, nitrogen gas (N2) is not directly available as a

source of nitrogen to plants. Free living or symbiotic soil microbes fix N2 and produce

nitrogen-containing compounds that are directly utilized by plants (Cocking 2003).

Traditionally, the paradigm of symbiotic nitrogen transfer to plants has focused on nitrogen

fixing bacteria such as Rhizobium. However, once this nitrogen is fixed and transferred to

the plant, it can be lost primarily through microbial mineralization of decaying vegetation or

through insect herbivory. With respect to mineralized nitrogen, more than 90% of land

plants are able to form symbiotic relationships with soil fungi and it has been demonstrated

that a number of these fungi are able to transfer this nitrogen to plants (Malloch et al. 1980).

Mycorrhizal fungi, specifically from the phylum Glomeromycota, have been shown to

provide plants with nitrogen obtained from the soil in exchange for plant-derived carbon

(Fellbaum et al. 2012).

Up to 31% of plant nitrogen in an ecosystem can be lost to insects through herbivory

(Lovett et al. 2002). Recently, we illustrated a strategy whereby plants can reacquire nitrogen

previously lost to herbivorous insects through an association with an endophytic, insect

pathogenic fungus (EIPF) (Behie et al. 2012). The EIPF, Metarhizium robertsii, transferred

insect-derived nitrogen directly to plants. This strategy suggests the potential that other

endophytic, insect pathogenic fungal species, such as species of Beauveria and Lecanicillium,

are also able to transfer nitrogen from infected insects to plants.

Here we tested the potential ubiquity of insect-derived nitrogen transfer to four plant

species (representing monocots and dicots) via five species in the genus Metarhizium

51  
representing broad range insect pathogens (M. robertsii, M. guizhouense, M. brunneum) and

narrow range insect pathogens (M. flavoviridae and M. acridum) as well as Beauveria

bassiana and Lecanicillium lecanii. These fungi are found worldwide, from arctic to tropical

ecosystems (Hajek & St. Leger 1994), occur up to 106 propagules per gram of soil (Milner

1991), and the broad range insect pathogens can infect up to 200 different species of insects

(Roberts & Hajek 1992). We measured insect-derived nitrogen transfer by these fungi in

soybean (Glycine max), haricot bean (Phaseolus vulgaris), switchgrass (Panicum virgatum),

and wheat (Triticum aestivum). We also tested insect-derived nitrogen transfer to plants by M.

robertsii under field conditions. The results suggested that all Metarhizium species as well as

B. bassiana were able to transfer significant amounts of insect derived nitrogen to plants.

Endophytic association by these fungi increased overall plant productivity. Also, under

natural settings, where competition is potentially high, Metarhizium is an effective conduit

for the transfer of insect derived nitrogen to plants.

3.3 Materials and Methods

 
3.3.1 Sources of fungi and plant material
 
Fungal cultures – Metarhizium robertsii strain Ma2575, M. acridum strain 7486 and

Beauveria bassiana strain 252 were obtained from the U. S. Department of Agricultural

Research Service Collection of Entomopathogenic Fungal Cultures, Ithaca, New York. M.

flavoviridae rf08A, M. brunneum strain 43a-2i, M. guizhouense strain b77-ai and,

Lecanicillium lecanii strain 313 were isolated from the field and cultured in lab. Aspergillus

flavus strain 6982 was obtained from University of Alberta Microfungus Collection and

52  
Herbarium, Edmonton, Alberta. M. robertsii-GFP, M. guizhouese-GFP, B. bassiana-GFP,

and L. lecanii-GFP were used for confocal micrographs, the construction of the GFP

expressing plasmids, as well as transformation of Metarhizium species have been previously

described (Fang et al. 2006). Stock cultures were grown at 27OC in potato dextrose agar

(PDA; Difco laboratories, Mississauga, Ontario, Canada.

Plant Material – Phaseolus vulgaris (Haricot Bean, cultivar: soldier) and Panicum virgatum

(switchgrass, cultivar: blackwell) seeds were obtained from OSC seeds, Waterloo, Ontario,

Canada. Triticum aestivum (winter wheat, cultivar: accipiter) seeds were obtained from

sprout master, Elmvale, Ontario, Canada. Glycine max (soy bean, cultivar: harosoy) seeds

were obtained from Agriculture Canada Research Station, London, Ontario, Canada.

3.3.2 Seed Sterilization and Plating

 
Seeds were surface sterilized before use in order to prevent any unwanted fungal or bacterial

growth. Seeds were immersed in sterile distilled water for 30 min in a 50 mL capped, plastic

tube and subsequently immersed in a 4% sodium hypochlorite solution three times for five

minutes. After each sodium hypochlorite wash, seeds were rinsed with sterile distilled water.

Seeds were then placed in 15% hydrogen peroxide for ten minutes and subsequently washed

three times with sterile distilled water to remove all hydrogen peroxide. Seeds were kept

overnight at 4°C to allow for synchronization of growth, then plated on water agar and kept

at 25°C for a photoperiod of 16 hours a day for a minimum of 7 days in order to obtain

seedlings.

53  
3.3.3 Galleria mellonella Injection and Infection
 
15
G. mellonella larvae were injected through the rear proleg with 10 µL of a 5% N

ammonium sulfate solution using a sterile syringe. After 48 h, live larvae were infected on a

10-day-old conidiating culture of Metarhizium spp., B. bassiana, L. lecanii or A. flavus and

agitated for 2 min.

3.3.4 Soil Sampling and Plating

 
Soil was taken from microcosms with G. mellonella larvae infected with Metarhizium

robertsii, B. bassiana, L. lecanii, or A. flavus and plant seedlings. Soil was sampled 0.5 cm

from the plant roots at a depth of 2 cm. One gram of soil was sampled from 5 separate plants

every 2 days. Soil samples were plated on selective PDA that contained 9.75 g potato

dextrose agar, 0.125 g cyclohexamide, 0.05 g chloramphenicol, 0.125 g 65% Dodine, and

0.0025 g crystal violet (all weights per 250 mL). Samples were spread with a plate spreader

and kept at 27°C for 10 days. After 10 days of growth colony forming units (CFU) were

counted.

54  
3.3.5 Microcosm setup
 
A 7 cm diameter hole was cut in the lid of a Petri dish and covered with a 30 micron mesh

and adhered with silicon glue. Plants were grown in plastic garden pots (height 20 cm,

diameter 15 cm). The garden pots and the modified Petri dishes were sterilized with UV light

for 2.5 hours prior to use. Once sterilized the Petri dishes were filled with sterile soil and five

fungal infected, 15N injected, larvae and sealed inside the Petri dish with parafilm. The Petri

dishes were then placed in the pot and covered with soil completing the microcosm. The pots

were filled with soil 3 cm from the top and a seedling was planted into the soil. Only the

fungi were able to penetrate the mesh and move across both compartments of the microcosm.

Plants were watered daily with sterile, distilled water and once a week with 50 mL of 50%

MMN solution [(0.05 g CaCl2, 0.025 g NaCl, 0.05 g KH2PO4, 0.5 g (NH4)2PO4, 0.15 g

MgSO4•7H20, 1 mg FeCl3•6H20, 5 g glucose monohydrate, 10 mL trace elements solution

(3.728 g KCl, 1.546 g H3BO3, 0.845 g MnSO4•H2O, 0.05 g ZnSO4•7H2O, 0.0125 G CuSO4,

0.05 g (NH4)6Mo7O24•4H2O per 1 L) per 1 L].

3.3.6 Preparation of samples for 15N Analysis.

 
Plant tissue was removed at seven day intervals and dried at 60°C until all moisture was

removed. Plant material was then crushed into a fine powder using a mortar and pestle. The

comminuted plant material was encapsulated in 4 mm by 4 mm tin cups and analyzed for 15N

content by NOI-5-emissions spectrophotometer.

55  
3.3.7 Plant Growth Measurements
 
Plants were grown under previously described experimental conditions. In plants grown with

the fungus alone, conidia from 10 day old cultures were used to inoculate the soil within the

Petri dish section of soil microcosms. In insect only treatments, insects were placed in the

Petri dish section of microcosms with no fungus. After four days, and every 4 days

subsequent for 16 days, plants were harvested (N=6), washed with sterile distilled water to

remove soil, dried at 60 degrees for 24-48 h, and sectioned for weighing. Plants roots and

leaves were removed from the whole plant with a sterile scalpel. Plant growth parameters

measured were leaf, root and whole plant weights of haricot bean (Phaseolus vulgaris), shoot

and whole plant weights of switchgrass (Panicum virgatum) and wheat (Triticum aestivum).

3.3.8 Confocal Microscopy of GFP fungi/plant root associations

 
The associations between the various fungi and plant roots were visualized microscopically.

Roots were observed using confocal microscopy to confirm association by root colonizing by

EIPF. Plants were grown under previously described experimental conditions. Whole roots

were harvested at 7 days and washed with sterile, distilled water to partially remove

particulate soil. Roots were then cut into 5 cm sections and placed on glass slides for

visualization.

Plant tissue was examined using a Leica DMIRE2 inverted display confocal microscope

utilizing an argon–krypton laser operated at excitation wavelengths of 480 ± 10 nm and 518

nm.

56  
3.3.9 Field Experiment
 

The experimental plot (ca. 100 m2) was on an old field (near St. Catharines, ON, Canada)

dominated by three grass species, timothy (Phaleum pretense), smooth bromegrass (Bromus

inermis), and orchard grass (Dactylis spp.). Within the field site, the modified Petri dishes,

with 30 or 1 micron meshes were buried approximately 15 cm below the surface. Treatments

in the modified Petri dishes were: 15N insect + M. robertsii , 30 µm mesh (experimental), 15N

insect + M. robertsii, 1 micron mesh, 15N insect, 1 µm mesh, 15N insect, 30 µm mesh,

micron mesh. A sample (ca. 50 gm) of grass was harvested at 1, 7, 14, and 28 days, collected

from distances of 5, 10, and 30 cm from the center of the buried Petri dish.

3.3.10 Calculation of insect nitrogen available to plants through EIPF

 
The estimated amount of atmospheric nitrogen that is fixed in an ecosystem varies between

10-160 kg/ha/yr (Boring et al. 1988). Approximately 31% of this fixed, plant incorporated,

nitrogen is then lost to insect herbivores (Lovett et al. 2002). Therefore the amount of plant

nitrogen lost through insect herbivory varies between 3.1kg/ha/yr (10kg/ha/yr x 0.31) to

49.6kg/ha/yr (160kg/ha/yr x 0.31). We estimated that approximately 30% of soil insects may

become infected by EIPF (Bidochka et al. 1998) however this number could be as high as

66%. Therefore, the amount of insect derived nitrogen potentially provided to plants by EIPF

varies between 0.93kg/ha/yr (3.1kg/ha/yr x 0.30) and 14.88kg/ha/yr (49.6kg/ha/yr x 0.30).

We then calculated that the percentage of the total fixed nitrogen provided to plants

through EIPF as 9.3%. That is 0.93kg/ha/yr/insect derived nitrogen provided to plants

57  
through EIPF divided by 10kg/ha/yr average atmospheric fixed nitrogen. Similarly,

14.88kg/ha/yr insect derived nitrogen provided to plants through EIPF divided by

160kg/ha/yr average atmospheric fixed nitrogen in an ecosystem is also 9.3%.

3.4 Results
 
3.4.1 Nitrogen transfer to plants by Metarhizium species
 
Soy Bean (Glycine max)

After 14 days of growth in soil microcosms containing Metarhizium infected, 15N

injected wax moth larvae, insect derived nitrogen levels in soybean ranged from 9% (M.

robertsii) to 55% (M. acridum ) (Fig. 5). At 28 days, M. flavoviridae had transferred the

highest levels of insect derived nitrogen to soybean roots with insect derived nitrogen

constituting 60% of the total plant nitrogen content (Fig. 5). With the exception of soybean

grown in the presence of M. robertsii infected 15N injected larvae, soybean plants with

Metarhizium showed significantly greater levels of 15N incorporation when compared to

soybean grown with 15N-injected but uninfected wax moth larvae at 14 and 28 days

(ANOVA, p<0.05).

15
N incorporation into soybean plants under experimental conditions was also

compared to soybean harvested from microcosms that contained the opportunistic insect

pathogenic, yet non-endophytic fungus, Aspergillus flavus. Again, with the exception of

soybean grown in microcosms with M. robertsii infected 15N-injected insects, all

58  
experimental plants analyzed had significantly higher levels of 15N incorporation (ANOVA,

p<0.05).

Haricot bean (Phaseolus vulgaris)

All species of Metarhizium were able to transfer significant amounts of insect derived

nitrogen to haricot beans at 14 and 28 days. Plants grown in the presence of M. guizhouense -

infected, 15N injected, wax moth larvae were found to contain the highest levels of insect-

derived nitrogen while M. robertsii was found to have the lowest levels of 15N incorporation

(Fig. 5). However, all plants grown in soil microcosms containing 15N labeled, Metarhizium

infected larvae had significantly higher levels of 15N incorporation when compared to plants

grown in the presence of uninfected, 15N injected, insects or with A. flavus (ANOVA,

p<0.05).

Switchgrass (Panicum virgatum)

After 14 and 28 days, all species of Metarhizium were able to transfer significant

levels of insect derived nitrogen to switchgrass. Plants grown in microcosms containing M.

guizhouense had the highest 15N content (48% and 62% of total plant nitrogen at days 14 and

28, respectively) (Fig. 5). At 14 and 28 days all switchgrass grown in the presence of

Metarhizium and 15N injected insects had significantly higher levels of 15N content when

compared to plants grown in the presence of uninfected 15N injected insect or A. flavus

(ANOVA, p<0.05).

59  
Wheat (Triticum aestivum)

At 14 days, all species of Metarhizium transferred significant levels of insect derived

nitrogen to wheat. Plants grown in microcosms with 15N-injected insects infected with M.

robertsii showed the highest level of 15N incorporation (82% of total nitrogen). Plants grown

with M. brunneum showed the lowest levels of 15N incorporation (61%) (Fig. 5).

At 28 days, 88% of total wheat nitrogen was insect derived when grown with 15N

insects and M. flavoviridae while M. acridum had the lowest levels of 15N incorporation

(58%) (Fig. 5). Wheat grown in the presence of 15N injected insects alone or with 15N

injected, A. flavus infected insects had significantly less 15N incorporation when compared to

all experimental treatments (ANOVA, p<0.05).

60  
Figure 5. Percentage of plant nitrogen derived from 15N-injected waxmoth larvae by five
species of the endophytic, insect pathogenic fungus, Metarhizium (robertsii, acridum,
guizhouense, brunneum, flavoviridae). Four plant species were used, wheat (Triticum
aestivum), switchgrass (Panicum virgatum), soybean (Glycine max), and haricot bean
(Phaseolus vulgaris). Results are means of three separate trials done in duplicate. Solid
circles represent treatments with waxmoth larvae, open circles are without. Insect derived
nitrogen in leaves was determined by NOI-5 emission spectrophotometer. Results were
analysed using ANOVA. Standard deviations not shown are less than 1% of the mean.

61  
3.4.2 Beauveria bassiana and Lecanicillium lecanii: Insect-derived nitrogen transfer to
switchgrass (Panicum virgatum) and haricot bean (Phaseolus vulgaris)
 
After 28 days in the presence of B. bassiana and 15N injected insects, insect derived

nitrogen content in switchgrass and haricot bean was significantly higher when compared to

plants in the presence of uninfected, 15N injected, wax moth larvae alone (ANOVA, p<0.05)

(Fig. 6). Plants grown with A. flavus did not contain more than 9% 15N, which was

significantly lower than plants grown under experimental conditions (ANOVA, p<0.05).

At 7, 14, and 28 days switchgrass and haricot bean plants grown in the presence of L.

lecanii infected, 15N injected insects, did not have significantly higher 15N content than plants

grown in the presence of 15N injected insects alone or plants grown in microcosms containing

A. flavus infected, 15N injected wax moth larvae (ANOVA, p<0.05) (Fig. 6).

62  
Figure 6. Percentage of plant nitrogen derived from 15N-injected waxmoth larvae by the
endophytic, insect pathogenic fungus Beauveria bassiana and Lecanicillium lecanii. Two
plant species were used, switchgrass (Panicum virgatum), and haricot bean (Phaseolus
vulgaris). Results are the means of three separate trials done in duplicate. Solid circles
represent treatments with waxmoth larvae, open circles are without. Insect derived nitrogen
in leaves was determined by NOI-5 emission spectrophotometer. Results were analyzed
using ANOVA. Standard deviations not shown are less than 1% of the mean.

63  
3.4.3 Soil Samples
 
Soil samples (1 g of soil) from microcosms containing Metarhizium, Beauveria,

Lecanicillium, and Aspergillus-infected insects were taken at 0, 2, 4, 6, and 8 days at a depth

of 2 cm and 0.5 cm distance from the plant root. Samples were then plated on selective media

and colony-forming units (CFU) per gram of soil were counted. Metarhizium and Beauveria

were found within 0.5 cm of the plant root at 4 days with an average of 15 and 3.5 CFU/g

respectively. L. lecanii and A. flavus were found within 0.5 cm of the plant root at 6 days

with an average of 13 and 29 CFU/gram of soil, respectively (Fig. 7) (all results are the mean

of five separate trials).

3.4.4 GFP confocal imaging

M. guizhouense, M. robertsii, B. bassiana, and L. lecanii hyphae were observed on

root surfaces of haricot bean (Phaseolus vulgaris) at 7 days (Fig. 8). These images, as well as

the soil samples, indicated that after one week, M. guizhouense and M. robertsii were able to

migrate to the plant and form root associations more efficiently than either B. bassiana or L.

lecanii.

64  
Figure 7. Time course, after insect infection of fungal propagules found per gram of soil.
Soil was sampled within 0.5 cm from plant roots at a depth of 2. Soil was plated on selective
media and colony-forming units were counted. A) Metarhizium species CFU/gram of soil.
Open circles represent M. brunneum, black circles represent M. robertsii, grey circles
represent M. flavoviridae, black squares represent M. acridum, and open squares represent M.
guizhouense. B) Beauveria bassiana and Lecanicillium lecanii CFU/gram of soil. Open
circles represent B. bassiana, closed circles black circles represent L. lecanii. Standard
deviations of the mean are shown. N=5 for each time point.

65  
Figure 8. Confocal micrographs obtained after plants had grown in soil microcosms with
waxmoth larvae and Metarhizium-GFP for 7 days. Panels are GFP images of EIPF
associating with plant roots at 7 days, 400x magnification. GFP micrographs are overlaid
over bright field images. Bars represent 10 µm A) Metarhizium guizhouense B) Metarhizium
robertsii C) Beauveria bassiana D) Lecanicillium lecanii

66  
3.4.5 Nitrogen Transfer from Metarhizium to Grass Roots under Natural Conditions
 
In field experiments, M. robertsii transferred insect derived nitrogen to three grass

species, timothy (Phleum pratense), smooth bromegrass (Bromus inermis), and orchard grass

(Dactylis spp.). There was a progressive decline in the amount of insect-derived nitrogen

incorporated by these grasses harvested further away from the soil-implanted source of the

Metarhizium infected-15N injected insects. After 28 days, the 15N content in these grass

species was 26.2%, 13.74% and 1%, at 5, 10 and 30 cm from the source of the Metarhizium

infected-15N injected insects, respectively (Fig. 9). Controls included insect only as well as

Metarhizium infected, 15N injected insects, contained in a 1 µm mesh separated from plants.

The 15N content in field grass samples at 28 days, 5 and 10 cm away from the Metarhizium

infected-15N injected insect source, were significantly greater than all control treatments

harvested at the same time point at the same distances (ANOVA, p<0.05).

3.4.6 Plant Growth

 
Plant growth parameters were taken every 4 days in order to ascertain the overall

health of the plant under varying experimental conditions. Bean plants grown in the presence

of M. robertsii and infected insect larvae had larger average leaf weight, root weight, and

overall plant weight than plants grown with the fungus or the insect alone (ANOVA, p<0.05)

(Fig. 10). In switchgrass and wheat grown in the presence of Metarhizium species and insect

larvae, root weight and whole plant weight were greater than in plants grown with either the

fungus or the insect alone (ANOVA, p<0.05) (Fig. 10) (results are the mean of five separate

trials).

67  
Figure 9. Percentage of plant nitrogen derived from 15N-injected waxmoth larvae by the
endophytic insect pathogenic fungi M. robertsii under field conditions. Three dominant grass
species were present in the field site, timothy (Phleum pratense), smooth bromegrass
(Bromus inermis), and orchard grass (Dactylis spp.). Open circles represents 15N insect + M.
robertsii – 30 micron mesh, black squares represents 15N insect + M. robertsii - 1 micron
mesh, closed circles represents 15N insect – 1 micron mesh, grey circles represents 15N insect
– 30 micron mesh, and the white squares represents grass only controls. Results are means of
two trials done in duplicate. Standard deviations are less than one percent of the mean.

68  
 Figure 10. Plant growth measurements taken every four days for 16 days. 3 plant species were used
in order to ascertain overall plant health under 3 separate growth conditions. Closed circles represent
plants grown in the presence of Metarhizium infected insects, open circles represent plants grown in
the presence of Mertarhizium only, and grey circles are measurements taken from plants grown in the
presence of insects alone. A) Whole plant weights of haricot bean B) Whole plant weights of
switchgrass C) Whole plant weights of wheat D) root weight of haricot bean E) root weight of
switchgrass F) root weight of wheat.

69  
3.5 Discussion
 
Here we found that five species of Metarhizium, as well as Beauveria bassiana, were

able to endophytically associate with, and transfer insect derived nitrogen to, four plant

species representing monocots as well as dicots. B. bassiana shares a number of

characteristics with Metarhizium, including a wide insect host range, wide habitat

distribution from the arctic to the tropics (Humber 1992, Samson et al. 1988), and shown

here, the ability to endophytically associate with several plant species, as well as the ability

to transfer insect-derived nitrogen to plants. This overlooked branch of the soil nitrogen cycle

represents a potentially large transfer of insect-derived nitrogen to plants in a number of

ecosystems globally. We calculated that there is between ca. 0.93 and 14.88 kg/hectare/year

of insect derived nitrogen provided to plants through EIPF, or approximately 9.3% of the

total fixed nitrogen, in a given ecosystem (Supp. Calc. 1).

The five Metarhizium species studied here have varying degrees of insect host

specificity. For example M. robertsii has a broad insect host range and can infect over 200

species of insects, whereas M. acridum is specifically virulent to acridids such as

grasshoppers and locusts (Wang & St. Leger 2005). Despite the varying insect host range

within the genera of Metarhizium tested here, the strains tested were all able to transfer

insect-derived nitrogen to plants using Galleria mellonella larva as the host insect (this larvae

is readily infected by all fungal species tested). These results were intriguing and suggest that

insect host ranges of the Metarhizium tested and plant host ranges are not linked. Similarly,

other endophytic fungi do not display plant host preferences and broad plant range

colonization is a common feature in the arbuscule mycorrizal (AM) (Klironomos et al. 2000).
70  
There are ca. 150 species of AM fungi, and ca. 2000 species of mycorrhizal fungi overall,

that are capable of colonizing 300,000 plant species (Klironomos 2000). This low fungal

species to plant host species ratio suggests that most plant associated fungi do not have

fastidious preferences with respect to plant host (Klironomos et al. 2000). These plant

symbioses, however, show a commonality with respect to the overall positive effect on plant

productivity and nutrient uptake (Klironomos et al. 2000).

The variability of insect-derived nitrogen transfer by EIPF to plants found in our

study suggests that there may be other ecophysiological criteria with respect to efficient

endophytic association and subsequent nitrogen transfer. For example, we did not find

significant levels of insect derived nitrogen transfer between M. robertsii and soybean.

Potentially, M. robertsii requires specific environmental indicators from soybean in order to

transfer nitrogen. Previous work done with M. robertsii and soybean suggests that this may

be the case. Successful soybean root colonization by M. robertsii alleviated salt induced

oxidative stress in plants soils (Khan et al. 2012). While this is a specific example of EIPF

mitigating stress to plant partners, it indicates a potential for this unique group of fungi to aid

in plant growth, development, and sustainability in a number of fluctuating soil conditions.

For example, in soils with high concentrations of heavy metals such as nickel, chromium, or

cobalt, endophytes are able to increase the heavy metal tolerance of plants prone to

hyperaccumulation of metals from the soil (Hegg et al. 1990).

Our study found that the EIPF, L. lecanii was unable to transfer insect derived

nitrogen to either monocots or dicots. The inability of L. lecanii to transfer insect derived

nitrogen to plants despite its ability to infect insects and form endophytic associations was

surprising since L. lecanii is similar to Metarhizium and Beauveria with respect to insect host
71  
range and endophytic competence (Krauss et al. 2004, Gurulingappa et al. 2011). We can

only speculate as to the reason for this. Perhaps there may be differences in the pathways by

which L. lecanii metabolizes insect derived nitrogen. Metabolic pathways of nutrient

assimilation can vary between species of insect pathogenic fungi (Marzful 1997). With

respect to nitrogen, we postulate that insect-derived nitrogen may be directly metabolized by

the fungus and not necessarily converted to inorganic nitrogen that can be transferred to plant

roots. If this is indeed the case, the symbiotic relationship that exists between L. lecanii and

its plant partners may rely on the transfer of other important soil nutrients such as phosphorus,

a relationship that has been observed in some species of mycorrhizal fungi (George et al.

1995).

We observed an increase in plant health and productivity such that plants grown in

the presence of an EIPF infected insect were larger than plants grown in the presence of EIPF

without insect which was larger than plants grown without a fungal partner. Plant

photosynthate production is positively correlated with N availability. Plants require

substantial amounts of N (2–5% N content by dry weight, compared with 0.3–0.5%

phosphorus content), and well over 50% of leaf N is devoted to photosynthesis (Field &

Mooney 1986). Therefore the acquisition of nitrogen by plants is directly related to their

ability to fix carbon via photosynthesis. EIPF can act as a conduit for insect-derived nitrogen

availability to plants. Several species of endophytic fungi have been shown to provide plants

with nitrogen. Heteroconium chaetospira, a dark septate endophyte, was able to transfer

nitrogen to the roots of cabbage plants (Usuki & Narisawa 2007). It was also found that the

roots of Ranunculus adoneus, colonized by endophytic fungi, had increased nitrogen

72  
acquisition early in the growing season resulting in increased plant survival rate (Mullen et al.

1998).

Our field study suggested that nitrogen transfer from M. robertsii to plants occurs at

high levels in natural environments. Root colonizing fungi show strong competition for

available root space, and this competition has been predicated on carbon availability from the

plant (Pearson et al. 1993). However, even where competition in the rhizosphere is

potentially high, Metarhizium was able to effectively transfer insect derived nitrogen to

plants. This suggests that the plants in our field experiments accommodated colonization by

Metarhizium. Metarhizium has also been found in soils at relatively high concentrations

when compared to other root colonizing microbes (Bruck 2004).

Metarhizium and Beauveria are ubiquitous endophytic soil fungi isolated from

habitats as diverse as the arctic tundra, temperate soil, and tropical environments (Hajek & St.

Leger 1994). The worldwide distribution of these fungi and broad plant host ranges, as well

as their ability to infect a broad range of insect species, and their ability to contribute to the

nitrogen flux in plants, suggests insect-derived nitrogen transfer to plants mediated by these

fungi is a common feature in soil environments worldwide and is an overlooked branch of

the global soil nitrogen cycle.

3.6 Conclusion
 
In most ecosystems, nitrogen is the major limiting factor for plant growth and

productivity. Further, insect herbivory exacerbates nitrogen deprivation since it removes

nitrogen from plants. Here we show a novel mechanism by which plants form a symbiotic

relationship with the EIPF Metarhizium and Beauveria and reacquire this lost nitrogen. These

73  
fungi are globally ubiquitous in soil ecosystems and are able to infect a wide range of insects.

The results suggest that EIPF play an important role in the ecological cycling of insect

nitrogen back to plant communities.

 
 
 
 

 
 

 
 
 
 

 
 
 

 
 
 
 

 
 

74  
 Chapter 4: Trading Insect Nitrogen for Photosynthate- Carbon
Translocation from Plant to Endophytic, Insect Pathogenic,
Fungus

Prepped as: Behie SW, Moreira CC, Sementchoukoca I, Barelli L, Zelisko PM, & Bidochka
MJ. Trading insect nitrogen for photosynthate: Carbon translocation from plant to insect
pathogenic, endophytic fungus.
Behie developed experimentation under supervision of Bidochka and Zelisko. Behie
performed/supervised all experimentation and analyses. Behie wrote the manuscript with additions,
edits, and guidance from Bidochka and Zelisko.

75  
4.1 Abstract
 
Many vascular plants are able to form close symbiotic associations with endophytic fungi.

Metarhizium is a common soil fungus that has a bifunctional role as a plant endophyte and an

insect pathogen. Previously we had shown that the endophytic capability and insect

pathogenicity of Metarhizium are coupled to provide an active method of nitrogen transfer to

a host plant via fungal mycelia. We speculated that in a reciprocal exchange for insect-

derived nitrogen, the plant provided photosynthate to the fungus. We used 13CO2, in airtight

plant growth chambers, to track the incorporation of 13C into plant (haricot bean; Phaseolus

vulgaris) carbohydrates and the subsequent translocation of 13C into fungal-specific

carbohydrates (trehalose and chitin) in the root/endophytic fungal complex. The amount of
13
C present in root-associated fungal biomass was determined over a 21 day period by

sequentially extracting trehalose and N-acetylglucosamine (GlcNAc) from roots and

analyzing for 13C-trehalose and 13C-GlcNAc using nuclear magnetic resonance (NMR)

spectroscopy. The translocation of photosynthate to endophytic Metarhizium occurred in the

absence of a host insect in the soil; however, in the presence of an insect host (as a nitrogen

source), we observed a significant increase in plant derived photosynthate in fungal-specific

carbohydrates. These findings are evidence that the host plant is trading photosynthate for

insect-derived nitrogen in this tripartite symbiotic interaction that involves a plant, an

endophytic insect pathogenic fungus and soil insects.

76  
4.2 Introduction
 
Soil fungi form intimate symbioses with over 90% of all vascular plant species

(Malloch et al. 2008). These interactions have evolved over 400 million years, and are of

tremendous ecological importance as they enhance plant nutrient acquisition (Parniske 2008).

Nitrogen is considered one of the more critical limiting nutrients for plant growth, and

generally must be fixed by nitrogen fixing bacteria for plant consumption (Cocking 2003).

On the other hand there is a scarcity of easily utilizable carbon in the soil and subsequently,

there is tremendous competition amongst soil microbes for carbon (Pearson et al. 1993).

Plants are typically photosynthetically efficient when provided with adequate nitrogen

(Hikosaka 2005). This sets up a potential symbiotic exchange of nutrients between plants and

fungi; plants require nitrogen and fungi require carbon.

Although intuitive in nature, there are few documented examples of nitrogen-carbon

exchange in fungal-plant symbioses. The majority of fungal plant symbionts, including the

mycorhizal fungi, are obligate biotrophs and unable to survive outside of the plant. In these

associations, the fungus requires carbon from the plant for survival since it is unable to

efficiently scavenge carbon from the soil (Smith et al. 2009). In exchange for plant-derived

sugars, the fungus provides its plant host with soil nutrients such as phosphate and nitrogen,

however, this reciprocal exchange has only been inferred from gene expression or transporter

staining experiments (Fellbaum et al. 2012, Jones et al. 2009, Balestrini et al. 2006) and has

never been specifically shown in terms of molecular transport (Karandashov & Bucher 2005,

Guether et al. 2009).

77  
 Metarhizium spp. form a unique group of fungi that are plant endophytes as well as

insect pathogens. Previously we found that Metarhizium transferred insect derived nitrogen

to their plant hosts (Behie et al. 2012). That is, Metarhizium was able to infect a soil-borne

insect and subsequently transferred the insect-derived nitrogen to plants via fungal hyphae

through an endophytic association (Behie et al. 2012, Behie & Bidochka 2014). This

transaction is most probably widespread in nature since Metarhizium is a globally distributed

soil fungus with a broad insect host range, infecting over 200 species of insects (Hajek & St.

Leger 1994, Roberts & Hajek 1992) and is able to form endophytic associations, under

laboratory and field conditions, with monocots (wheat, switchgrass) and dicots (haricot bean,

soy bean) (Behie & Bidochka 2014). Here, I hypothesized that, in exchange for the insect-

derived nitrogen transferred to host plants, Metarhizium receives plant-derived photosynthate.

4.3 Materials and Methods

 
4.3.1 Fungal Cultures and Plant Material
 
Metarhizium robertsii strain Ma2575 was obtained from the U. S. Department of Agricultural

Research Service Collection of Entomopathogenic Fungal Cultures, Ithaca, New York. Stock

cultures were grown at 27°C in potato dextrose agar (PDA; Difco laboratories, Mississauga,

Onatario, Canada. Phaseolus vulgaris (Haricot Bean) seeds were obtained from OSC seeds,

Waterloo, Ontario, Canada.

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4.3.2 Seed sterilization and plating
 
Seeds were surface sterilized before use in order to prevent any unwanted fungal or bacterial

growth. Seeds were immersed in sterile distilled water for 30 min in a 50 mL capped plastic

tube. They were then washed in a 4% sodium hypochlorite solution three times, ten minutes

each wash. The seeds were rinsed with sterile distilled water in between each wash. The fluid

was drained and seeds were washed with sterile distilled water. The seeds were then placed

in 15% hydrogen peroxide for thirty minutes and then washed three times with sterile

distilled water. The seeds were then kept overnight at -5°C to allow for synchronization

of growth, then plated on water agar and kept at 25°C for a photoperiod of 16 h a day for a

minimum of 7 days in order to obtain seedlings which were further used in the study.

4.3.3 Microcosm set up and plant growth

 
Microcosms were created as previously described (Chapter 2). All microcosms were

sterilized by UV light for 2.5 hours prior to use. Once sterilized the Petri dishes were filled

with sterile soil and five fungal infected, 15N injected larvae and sealed with parafilm.

Control treatments contained petri dishes with 5 1 cm x 1 cm agar plugs, or only sterile soil.

Petri dishes were then placed in the pot and covered with soil completing the microcosm. The

soil was filled to 3 cm from the top and a 7 day old seedling was planted into the soil. Plants

were watered daily with sterile, distilled water and once a week with 50 mL of 50% MMN

solution [(0.05 g CaCl2, 0.025 g NaCl, 0.05 g KH2PO4, 0.5 g (NH4)2PO4, 0.15 g

MgSO4•7H2O, 1 mg FeCl3•6H2O, 5 g glucose monohydrate, 10 mL trace elements solution

(3.728 g KCl, 1.546 g H3BO3, 0.845 g MnSO4•H2O, 0.05 g ZnSO4•7H20, 0.0125 G CuSO4,

0.05 g (NH4)6Mo7024•4H2O per 1 L) per 1 L].

79  
4.3.4 Assimilation chamber set up and CO2 addition
 
Plant assimilation chambers were created in order to provide plants with a 1500 ppm 13CO2

environment. All chambers were air tight with a volume of 60L (Supp. Fig. 4). Plants were

placed in the assimilation chambers and sealed, once inside the chambers, 13CO2 was added

until 1500 ppm was reached. Plants were allowed to grow in 13CO2 rich environment for 8

hours a day, during maximum photosynthetic period. Plants were given 13CO2 every 48 h.

4.3.5 Sequential Extraction of soluble sugars and N-acetylglucosamine

 
Soluble sugars and N-acetylglucosamine extractions were carried out following protocols

previously described (Govindarajulu et al. 2011). Plants were harvested at appropriate time

points (1, 7, 14, 21 days) and lyophilized to remove all moisture. Plants were then ground,

frozen, with a mortar and pestle in 4-6 mL of MeOH/H2O (7/3). The mixture was then

filtered through Fischer brand P4 filter paper, and methanol was removed by evaporation

under reduced pressure. The aqueous solution was freeze-dried and stored at 4oC for NMR

analysis. The solid plant residue was then used for N-acetylglucosamine extraction. Plant

tissue was treated for 20 minutes in boiling 1M KOH. KOH was then decanted and the solids

were washed in deionized water and soaked in potassium phosphate buffer (pH 6). One unit

of chitinase, 1 unit of chitosinase, and 0.1 unit of glucanase were added to the samples (each

enzyme was prepared as a stock solution in potassium phosphate buffer). Sodium azide

(0.01% ) was added to the to each sample. Samples were then incubated at 37°C for 7 days

under continuous agitation. After enzymatic digestion, samples were centrifuged and

supernatants were lyophilized and stored at 4°C for NMR analysis.

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4.3.6 Nuclear magnetic resonance spectroscopy
 
All NMR spectra were recorded in D2O (900 μL) on a Bruker Ultra Shield Plus AV-600

spectrometer (1H at 600 MHz, 13C at 151 MHz) using methanol as an internal reference for

all spectra at 49.5 ppm. Acquisition parameters were the same for all samples run. Samples

were run for 27, 000 scans to obtain maximum resolution of 13C resonance. NMR spectra

were analyzed using the Bruker Topspin v2.0 software interface.

4.3.7 Soil analysis and plating

 
Soil was analyzed throughout the experiment to ensure Metarhizium was the only microbial

species present in microcosms. Soil was taken from pots containing microcosms with M.

robertsii and plant seedlings. Soil was sampled 2 cm from the plant roots at a depth of 2 cm.

One gram of soil was sampled from 5 separate plants every two days. Metarhizium soil

samples were plated on PDA media to ensure growth of all fungal species potentially present.

Only Metarhizium was found to be present in microcosms.

4.3.8 Fungus colonized root staining

 
Harvested roots were washed in sterile distilled water until all soil removed and then fixed in

a 1:1:1 formalin:acetic acid:alcohol solution for 24 h. The roots were then dehydrated in

increasing concentrations of ethanol (30%, 50%, 70%) for 30 min, then in 90% ethanol for

60 min. After dehydration, the roots were cut into 2 cm sections and cleared in methyl

salicylate for 12 h. The roots were then embedded in paraffin wax and sectioned using a

81  
microtome. The wax was removed by washing the samples with xylene for 15 min. The roots

were then placed in 10% (wt/vol) KOH solution and heated at 65°C for 60 min. After one

hour, the KOH was decanted and replaced with Chorazol E Black staining solution (80%

lactic acid, glycerol, and distilled water with 0.1% Chlorazol black E, 1:1:1:1) and heated at

65oC for 60 min.

4.3.9 Root colonization assay

 
Roots were harvested form soil microcosms at 1, 7, 14, and 21 days and washed in sterile

distilled water to remove all soil, air dried and weighed. Roots were then weighed, and

crushed using liquid nitrogen. The root powder was suspended in 0.1% triton x solution and

plated on PDA. PDA plates were allowed to incubate at 27°C for 6 days. Colony forming

units were then counted.

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4.4 Results and Discussion

I used 13CO2 to track the incorporation of carbon into plant carbohydrates (Phaseolus

vulgaris), and the subsequent translocation of 13C into fungal-specific carbohydrates

(trehalose and chitin) in the root/endophytic fungal complex. The 13CO2 was added to airtight

plant growth chambers that contained plants with root-associated Metarhizium in soil

microcosms (Supp. Fig. 4). Soil microcosms contained seedlings and Metarhizium added to

the soil as agar plugs (1cm x 1cm, 5 per pot). 13CO2 was provided to plants, in assimilation

chambers, during the optimal photosynthetic period (8 hours, during maximum sunlight) at

1500 ppm. The amount of 13C present in root-associated fungal biomass was determined over

a 21 day period (1, 7, 14, and 21 days) by sequentially extracting trehalose and N-

acetylglucosamine (GlcNAc) from roots (Bago et al. 2003) and analyzing for 13C-trehalose

and 13C-GlcNAc using 13C nuclear magnetic resonance (NMR) spectroscopy. After 7, 14,

and 21 days in the presence of 13CO2, we observed 13C incorporation into Metarhizium

produced carbohydrates (Fig. 11). All experimental spectra showed 13C peaks present at

intensities over what was observed in natural abundance samples, indicating high levels of

carbon movement from the plant to its fungal partner.

Neither 13C labelled-trehalose nor -GlcNAc were observed in plants grown in the

absence of Metarhizium (Fig 11). However, 13C resonances are present in the spectra

representative of soluble plant sugars including fructose, glucose, and sucrose. Control plants

without 13CO2 did not demonstrate 13C NMR resonances for carbohydrate species that were

above natural abundance levels (Sup Figure 2). This indicated that the enhanced levels of 13C

present in the plant samples were derived from the 13CO2 in the assimilation chambers.

In order to ensure there was no CO2 fixation by Metarhizium present in the soil,
83  
microcosms were set up with Metarhizium without the addition of a haricot bean seedling,

and placed in assimilation chambers with 13CO2. The soil was then sampled at 1, 7, 14, and

21 days, and sequentially extracted for trehalose and GlcNAc. No 13C was present above

natural abundance in soil samples (Fig. 11) indicating that all 13C present in fungal biomass

was obtained through the transfer of photosynthetically fixed carbon into root-colonized

Metarhizium.

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Figure 11. Relative percent 13C in Metarhizium and plant derived sugars. Resonances present
in NMR spectra were relatively integrated against an internal standard (MeOH, run in all
samples at equal concentrations) and values were then calculated as a percentage of internal
standard amplitude. A) Closed circles represent 13C in trehalose extracted from root
colonized Metarhizium with no insect present. Squares represent 13C in trehalose extracted
from root colonized Metarhizium with an insect present. Open circles represent 13C in
trehalose extracted from roots without Metarhizium present. B) Closed circles represent 13C
in GlcNAc extracted from root colonized Metarhizium with no insect present. Squares
represent 13C in GlcNAc extracted from root colonized Metarhizium with an insect present.
Open circles represent 13C in GlcNAc extracted from roots without Metarhizium present. C)
Closed circles represent 13C GlcNAc extracted from soil with Metarhizium without a plant.
Squares represent 13C trehalose extracted from soil with Metarhizium without a plant. Open
circles represents 13C in all sugars extracted from soil without Metarhizium or plant present
(N=20, error bars represent S.E.).

85  
 At 21 days, in the presence of a host insect there was a significant increase in the

amount of 13C translocated into Metarhizium when compared to microcosms without an

insect (ANOVA, p<0.05) (Fig. 1). Furthermore, in the presence of an insect, we observed

larger root systems than in microcosms without insects, and thus more fungal root

colonization (ANOVA, p<0.05) (Fig. 12). This suggested that Metarhizium was able to

efficiently barter insect-derived nitrogen for plant-produced carbon, and thus the overall

plant/fungal complex is healthier. An analogous situation has been observed in arbuscular

mycorrhizal fungi (AM). AM colonized roots typically lead to healthier plant systems with

respect to nutrient acquisition,  and an overall increase in primary production (Javot et al.

2006).

86  
Figure 12. Relative percent 13C in fungal produces sugars as a function of plant root weight.
Closed circles represent root weights when an insect is present in the microcosm, open
circles represent root weights without an insect in the microcosm.

87  
 Figure 13 shows that there is an increase in overall 13C accumulation in trehalose

when compared to plant synthesized sugars (sucrose), and that when an insect is present in

the system, there is an overall increase in 13C accumulation in the fungus, 13C transported

into the fungal biomass from the plant. This provides further evidence to suggest that soil

insects increase overall soil nutrient economy; increasing the amount of nitrogen the fungus

is able to provide the plant in exchange for carbon.

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Figure 13. Ratio of trehalose to sucrose in 13C labeled plants colonized by Metarhizium.
Closed circles represent 13C trehalose/sucrose ratios with an insect present. Open circles
represent 13C trehalose/sucrose ratios without an insect present. Signifigant differences exist
at 7 and 21 days (p<0.05) (N=20).

89  
 In Metarhizium, a disaccharide transporter, Metarhizium raffinose transporter (mrt),

has been shown to play a role in the uptake of raffinose from the surrounding environment

(Fang & St. Leger 2010). However, in well-defined plant/fungal relationships, it is thought

that plant synthesized monosaccharides are shuttled into the fungal biomass from the root

(Helber et al. 2011). In mycorrhizal fungi, for example, there is a specific increase in the

monosaccharide transporter MST2 during plant root colonization (Helber et al. 2011).

Metarhizium has three defined monosaccharide transporters, and while these transporters

have not been characterized in terms of specific function or location, they share sequence

identity with transporters in other root colonizing fungi such as Beauveria bassiana, and

Glomerella graminicola, suggesting a potential role in root colonization, and potentially in

nutrient cycling.

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Figure 14. Internal cross section of M. robertsii colonized haricot bean plant roots stained
with Chlorazol black. A) Metarhizium colonized plant roots, sectioned with microtome and
subsequently stained with Chlorazol black. B) Plant roots grown without Metarhizium
stained with Chlorazol black. Images were taken at 400 x magnification.

91  
 Previously we have shown that plants can derive a significant portion of nitrogen

from soil insects through their endophytic associations with the insect pathogen Metarhizium

spp. (Behie et al. 2012, Behie & Bidochka 2014). Here we have shown that plants

reciprocate with an exchange of carbon to their endophytic associates. Plants

photosynthetically fix carbon while Metarhizium can obtain scarce nitrogen via specialization

as insect pathogens. Metarhizium is phylogenetically related to other plant endophytes (Behie

et al. 2013) and we suggest that their evolution as insect pathogens and subsequent

translocation of nitrogen (Hu et al. 2014) allowed them to effectively barter with host plants

for photosynthate.

92  
 Chapter 5: Nutrient Transfer to Plants by Phylogenetically
Diverse Fungi Suggests Convergent Evolutionary Strategies in
Rhizospheric Symbionts

Published as: Behie SW, Padilla-Guerrero IE, & Bidochka MJ. Nutrient transfer to plants by
phylogenetically diverse fungi suggests convergent evolutionary strategies in rhizospheric
symbionts. Communicative Integrative Biology 2013 6, e22321

Behie and Padilla-Guerrero performed all phylogenetic analyses and tree construction. Behie wrote the
manuscript with additions, edits, and guidance from Bidochka.

93  
5.1 Introduction

More than 90% of all terrestrial plant species engage in symbiotic relationships with

fungi and are dependent upon these interactions for survival. These relationships with plants

are formed with mycorrhizal (endo- and/or ectomycorrhizal fungi) or endophytic fungi.

Endophytic fungi live within plant tissues including root and leaves. Many fungi are host and

tissue specific (Carroll 1988). Generally, endophytic fungi may be defined as asymptomatic

plant colonizers, and an endophyte may not remain within the plant tissue throughout its life.

Functional endomycorrhizal fungi, however, are explicitly excluded as “endophytes”, as they

are specialized for nutrient transfer from sources outside of the root (Brundrett 2002) and,

more specifically, are phylogenetically distinct from most groups of endophytes (Arnold &

Lutzoni 2007, Parros-Alfaro et al. 2008). The endophytic arbuscular mycorrhizal fungi (AM)

are placed in a separate monophyletic phylum, the Glomeromycota (Shubler et al. 2001).

The term endophyte is thus defined by location and does not address the nature of the

relationship with the plant or its phylogenetic status. Therefore, AM fungi are a specialized

group of endophytic fungi with a unique phylogenetic status. The ectomycorrhizal fungal

species, on the other hand, are among the Basidiomycota as well as the Ascomycota. Here,

the definition includes that ectomycorrhizal fungi produce a hyphal sheath, or mantle,

covering the root tip and a hartig net of hyphae surrounding the plant cells within the root

cortex (Carlile et al. 2001). Endophytic associations differ from mycorrhizas primarily by

the absence of a localized interface of specialized hyphae and the absence of synchronized

plant-fungus development (Brundett 2004). Brundett (2004) also suggests that in endophytic

fungal associations there is little nutrient transfer to plants. However, several non-

mycorrhizal endophytic fungi can provide nutrients to plants (Behie et al. 2012).

94  
 Here we explore recent findings that endophytic fungi can also specialize in nutrient

transfer from outside of the root, specifically, from soil-borne insects that they can infect

(Behie et al. 2012). These endophytic, insect pathogenic fungi (EIPF) are phylogenetically

distinct from AM fungi. In this minireview, we suggest convergent evolutionary strategies

for nutrient transfer to plants with some species of EIPF, AM fungi as well as

ectomycorrhizal fungi.

5.2 Nutrient transfer in AM and endophytic fungi

 
AM fungi are well known as mediators of nutrient transfer to plants. The majority of

vascular plants, (80-90%) (Malloch et al. 1980) are able to form associations with AM fungi,

and these associations have been described, in part, by nutrient exchange. Initial studies on

nutrient transfer focused on the ability of AM fungi to provide roots with phosphate and it

was found that the arbuscule of these fungi, specifically the peri-arbuscular membrane,

contained phosphate transporters (Gaude et al. 2012). Providing the fungus with increased

levels of plant-derived carbon resulted in increased transference of phosphate (Bucking &

Shachar-Hill 2005). This suggests a coordinated barter of nutrients between plant and fungus.

Some fungi can also provide nitrogen to plants (Govindarajulu et al. 2005). Glomus species

have the ability to transfer nutrients, including nitrogen to the roots of leguminous plants

such as Medicago (Gaude et al. 2012, Cox & Tinker 1976). Ceratobasidium cornigerum has

also been shown to provide orchids (Goodyera repens) with nitrogen in exchange for carbon

(Cameron et al. 2006).

Some fungal endophytes can affect plant growth and plant responses to pathogens,

herbivores, and environmental change; others produce useful or novel secondary metabolites.

95  
Root endophytes colonize healthy plant roots, however, one of the defining characteristics

has been that endophytes do not have nutrient transfer interfaces commonly observed in AM

fungi (Brundrett 2006, Schultz & Boyle 2006). This definition is not without exception. An

increase in nutrient content and growth was observed for Carex spp., Pinus contorta, and

Vulpia ciliata when inoculated with dark septate endophytic fungi (Haselwandter & Read

1982, Jumponen et al. 1998, Newsham 1999).

For example, the dark septate endophytic fungus Heteroconium chaetospira forms a

functional symbiosis with Brassica campestris (Chinese cabbage) where the fungus transfers

nitrogen to, and receives carbon from, the plant (Usuki & Narisawa 2007). The Brassicaceae

do not usually form mycorrhizal associations so this association with H. chaetospira, as well

as others (Behie et al. 2012), suggests that endophytes can also transfer nutrients to plants.

Endophytic associations can also result in more efficient nutrient acquisition since root

associated fungal hyphae are able to obtain soil nutrients from areas too small for plant roots

to penetrate (Majdi et al. 2001).

Recently, some insect pathogenic fungi have been shown to form endophytic

associations with plant roots (Sasan & Bidochka 2012, Akello et al. 2007, Posada & Vega

2005). In particular, one EIPF, Metarhizium robertsii, can transfer insect-derived nitrogen to

plants (Behie et al. 2012). M. robertsii infected a soil insect after which the fungal mycelia

colonized the host plant (switchgrass and haricot bean) where nitrogen transfer was detected

(Behie et al. 2012). The ectomycorrhizal fungus, Laccaria bicolor was also able to transfer

insect derived nitrogen from springtails to the roots of eastern white pine trees (Klironomos

& Hart 2001). However, it was not shown that L. bicolor is an insect pathogen and it is

possible that L. bicolor transferred nitrogen to plants from insect cadavers. The ability of
96  
EIPF to transfer insect derived nitrogen to plant roots indicates a fundamental shift in the

way these fungi are viewed within the ecosystem as they represent the ability of the plant to

regain nitrogen previously lost by insect herbivory.

5.3 Convergent evolution in nutrient transfer in AM and endophytic fungi

 
The earliest fossil evidence of mycorrhizal associations with plant roots is ca. 460

million years ago (Wilkinson 2001). The early evolutionary origin of these mutually

beneficial partnerships suggests that fungal/root associations, specifically mycorrhizae, may

have benefited plants as well as fungi and has significantly affected the global distribution of

flora (Cairney 2000). These associations are not trivial since these symbioses enhance the

overall fitness of the plant (Cairney 2007). However, not all root-associated fungi are

mycorrhizae, and despite the fact that mycorrhizal fungi have been ubiquitously noted, other

root associated fungi (i.e. endophytes) have been discovered. These non-Glomeromycota

fungi are most likely not the product of continued co-evolution with plants followed by

fungal divergences, but have separately undergone convergent evolution as plant root

associates. That is, beneficial plant/fungal associations have evolved separately several times

over the past 400 my.

A fungal phylogeny constructed in Figure 15 shows the phylogenetic position of the

Glomeromycota in relation to EIPF and ectomycorrhizal fungi. The EIPF are

phylogenetically distant from the Glomeromycota but are also involved in nutrient transfer to

plants. Endophytic fungi may have multiple ecological niches since they are not restricted to

the host plant. Metarhizium is taxonomically related to other endophytic fungi, notably

Claviceps (Fig. 15). We suggest that these fungi have undergone convergent evolution as

97  
plant symbionts. However, Metarhizium is also a generalist insect pathogen that can infect

over 200 insect species (St. Leger 1993). Metarhizium is potentially a plant symbiont due to

phylogenetic heritage (i.e. that they are closely related to other endophytes), and it has also

evolved mechanisms as a generalist insect pathogen. This bifunctional lifestyle as a plant

endophyte and an insect pathogen may have allowed Metarhizium to provide plants with

insect-derived nitrogen.

98  
Figure 15. Phylogenetic positioning of 4 major fungal phyla with examples of plant
symbionts, ability of these symbionts to transfer nutrients, insect pathogens and plant
pathogens. The tree shown was created using ITS1 5.8s rRNA and ITS2 sequences. Green
bars indicate plant symbionts. Blue bars indicate nutrient transfer between plant and fungal
symbiont. Red bars denote insect pathogens. Yellow bars indicate plant pathogens. Asterisks
above bars indicate potential, untested, capacity of the fungus.

99  
 A number of saprotrophs obtain carbon from decaying plant matter, but this method

of acquisition is inefficient for uncompetitive fungi (Tiunev & Scheu 2004). Several root

associated fungal species are able to utilize plant-derived carbon (Cameron et al. 2006,

Gavito & Olsson 2003, Hawkes et al. 2008, Bago et al. 2003), which could lead to a

nitrogen-carbon barter.

However, not all endophytes behave in the same manner with respect to nutrient

acquisition. Generally, endophytic fungi can be placed into two groups, clavicipitaceous, and

non-clavicipitaceous (Rodriguez et al. 2009). EIPF are clavicipitaceous fungi and are able to

acquire nutrients from the insects they infect and can then transfer those nutrients to host

plants (Behie et al. 2012). Non-insect pathogens that are plant symbionts, however, are

generally decomposers of decaying plant materials found in soil; through this decomposition

they are able to acquire nutrients necessary for potential exchange with plant roots (Dighton

1991). The fungal phylogeny shown in Figure 15 indicates that the divergence between

Ascomycete EIPF and Glomeromycota occurred early in fungal evolutionary history. The

recent discovery that several ascomycetous insect pathogens are also able to form plant root

associations suggests a possible convergence in plant symbiosis and nutrient exchange

strategies between these fungi and the Glomeromycota as well as some of the

ectomycorrhizal fungi. More specialized insect pathogens are found in the Zygomycetes,

specifically in the Entomophthorales. Most of these fungi are fastidious and obligate insect

pathogens (Bidochka et al. 2000) and have not yet been reported as plant associates (Fig. 15).

The ability of AM fungi, EIPF, and some species of ectomycorrhizal fungi, three

genetically disparate fungal groups, to act as mediators of plant nutrient transfer suggests

convergent evolution as root colonizers and conduits of nutrient transfer. The challenge is to
100  
identify convergent evolutionary strategies at the molecular level in order to identify fungal

genes that mediate root colonization as well as nutrient transfer. However, by using well-

known mycorrhizal species as genetic models for EIPF, gene homologues involved in

plant/fungus communication and/or nutrient transfer may be identified. It has been shown

that in Glomus and Laccaria species there is an up-regulation of ammonium transporters

during plant root association (Gaude et al. 2012, Lucic et al. 2008). These fungi are from

distinct fungal lineages and this finding indicates they have developed similar methods for

nitrogen transfer to plant roots. It has also been shown that in phylogenetically disparate

fungal plant symbionts there is an increase in simple sugar transporters during root

colonization that coincides with an increase in monosaccharide transporters in plant roots

(Harrison 1996).

With respect to phosphate translocation, in some Glomus species there was an

increase in the expression of phosphate transporters in the extraradical and intraradical

mycelia during root association (Benedetto 2005). This increase in expression has also been

observed in Hebeloma cylindrosporum during root symbiosis (Tatry et al. 2009). This up-

regulation of phosphate transporters in fungal symbionts during root colonization

corresponds to an increase in plant root phosphate transporters (Karandashov & Bucher

2005). Similar expression patterns have also been seen in root-associated fungus with respect

to sulphate transporters (Allen & Shachar-Hill 2009). It is likely that numerous fungal

species have developed transporters and evolved similar nutrient exchange programs in order

to acquire carbon from plants. The next step is to elucidate the molecular mechanisms by

which plants sense the acquisition of fungal-derived nutrients and allow a symbiotic

association that draws carbon from the plant.

101  
 Most arbuscular mycorrhizal fungi are obligate biotrophs (Azcon-Aguilar et al. 1999)

and are unable to grow in pure culture and transformation systems are not available for many

of these fungi. This provides an opportunity for Metarhizium to act as a potential model to

test the role of nutrient transporters and plant permissiveness in these endophytes since gene

knockout systems (Staats et al. 2007) as well as the fungal genome (Gao et al. 2011) are

readily available (Fang et al. 2006).

5.4 Materials and Methods

Phylogentic analysis of fungal species
 
Molecular phylogenentic analysis of the ITS1, 5.8s rRNA, and ITS2 sequences was

conducted to identify the relatedness of Metarhizium to other plant colonizing fungi.

Sequence alignments were made using clustal_x 2.1 using default settings (Wyrebek et al.

2011). A maximum parsimony tree was constructed using Molecular Evolutionary Genetics

Analysis (MEGA) version 5.

5.5 Conclusion
 
Traditional research on insect pathogenic fungi has generally been focused on fungal

virulence toward insect hosts and therefore largely ignored other aspects of their soil ecology.

The discovery that some insect pathogenic fungi are able to form endophytic associations

with the capacity of nutrient transfer indicates a convergence among fungal lineages, most

likely as a method to obtain carbon from plant roots. Subsequent findings that EIPF are

capable of nutrient transfer to plant roots fundamentally changes the way these fungi must be

102  
viewed in soil habitats. These fungi must now be seen as an integral part of plant biological

environments and an important part of the soil nutrient cycle.

103  
Chapter 6: Conclusions and Perspectives

104  
 The overall hypothesis of this thesis was that Metarhizium root colonization requires

reciprocal nutrient exchange with plant hosts. I wanted to determine the efficacy of the EIPF

Metarhizium in providing its plant hosts with insect-derived nitrogen, and to determine

whether the fungus received carbon from the plant in return. As a result of the nutrient

exchange described in well-studied plant/fungal symbioses, as well the fact that nitrogen is

critical to plant health, yet often limited in soils, it was hypothesized that Metarhizium could

act as a nitrogen vector in soil, whereby Metarhizium provides plant roots with insect-derived

nitrogen, in exchange for carbon. While it was previously known that Metarhizium was a

potent insect pathogen and a plant endophyte (St. Leger 1993, Sasan & Bidochka 2012), no

previous studies had shown a link between these two life styles.

In this thesis, I determined that five Metarhizium species, as well as Beauveria

bassiana, were able to infect insects, harvest organically bound insect nitrogen, and transfer

it to plant. I showed that Metarhizium spp. were able to cycle nitrogen to a number of

different plant species, including both monocots and dicots, and were able to furnish plant

roots with insect-derived nitrogen in field studies. In addition, I was also able to show that

Metarhizium is provided with photosynthetically derived carbon from its plant hosts, thus

describing a reciprocal exchange between the two symbionts.

6.1 Nitrogen transfer by EIPF

Nitrogen is limiting to plants in most ecosystems globally (Mary et al. 1996). Some

insects that feed on plant tissue may exacerbate the nitrogen deficiency as they remove plant-

incorporated nitrogen. However, as EIPF, such as Metarhizium and Beauveria, are capable of

infecting insects, collecting insect-derived nitrogen, and subsequently furnishing plants with

105  
this nitrogen, this symbiosis provides plants with a system by which they are able to

reacquire nitrogen lost to insect herbivory. As these fungal species are globally ubiquitous,

these results suggest that EIPF play an important role in the cycling of nitrogen to plants.

Not all species of Metarhizium or Beauveria bassiana, however, transfer nitrogen

with the same efficacy. Variation among species indicated that there were potentially a

number of ecophysiological conditions that might influence EIPF nitrogen transfer efficiency.

Previous work has suggested that a strong Metarhizium robertsii association with soybean

requires increased soil salinity (Khan et al. 2012), a finding that reflects the nitrogen transfer

results between M. robertsii and soybean in the present study (Fig. 5). It has also been shown

that other environmental conditions such as the seasonality, soil moisture, and nutrient

availability, can affect both plants and fungi with respect to fungal-mediated nutrient cycling

(Rodrigues 1994, Elamo et al. 1999, Marks & Clay 1996).

It was important to determine the ability of Metarhizium to transfer insect-derived

nitrogen to plants in field natural settings, where the microbial competition for root space is

potentially high. Plant roots are capable of hosting a wide variety of microbial symbionts at

any one time (Parniske 2008), however, the competitive ability of Metarhizium in the

rhizosphere has not yet been specifically tested, and therefore nitrogen transfer experiments

in a field setting were important. It was found that, in a field environment Metarhizium was

able to transfer insect nitrogen to its plant host, suggesting its ability to effectively compete,

in the microenvironment of the rhizosphere, for space on plant roots.

106  
6.2 Reciprocal exchange: Carbon transfer from plant to Metarhizium
 
In numerous other fungal symbionts, it has been shown that root colonized fungi

receive carbon, in the form of carbohydrates, from their plant hosts in exchange for soil

nutrients (Cameron et al. 2006). However, the majority of studies have inferred carbon

transfer through gene expression or staining experiments (Fellbaum et al. 2012, Jones et al.

2009, Balestrini et al. 2006). Here, labeled carbon was specifically tracked from the

atmosphere, through the plant, and into fungal produced sugars. It was found that root

colonized Metarhizium receives photosynthetically fixed carbon from their plant hosts, and

that Metarhizium is capable of metabolizing this carbon for use in fungal processes. This

suggests that the nutrient transfer between symbionts is reciprocal. It has yet to be shown,

however, if nitrogen transfer is specifically critical to overall root colonization, or if other

nutrients, such as phosphorus or sulfites, are cycled through Metarhizium as well. Regardless,

these results indicate that Metarhizium is capable of acquiring plant-derived carbon in

exchange for insect-derived nitrogen (Fig. 16).

107  
Figure 16. Representation of the flow of nutrients between Metarhizium and its plant host.
Metarhizium provides insect-derived nitrogen to its plant host in exchange for plant sugars.
In this way plants have been granted access to large nitrogen reserves in soil, in the form of
insects, while Metarhizium is provided with a carbon source (I thank M. K. Bidochka for Fig.
16).

108  
6.3 Evolution of EIPF
 
A number of fungal species are at a specific competitive disadvantage with respect to

acquiring organically bound carbon in the soil therefore the ability of EIPF to colonize plant

roots may have been evolutionarily driven (Tiunov & Scheu 2004). Plant root exudate

contains a large number of plant-synthesized sugars (Rovira 1969), and could have

potentially drawn Metarhizium into the rhizosphere. Once in this unique microenvironment,

microbial competition may have provided further selection pressure, pushing rhizosphere

competent microbes towards plant root colonization (Hibbing et al. 2010).

Phylogenetic analysis of Metarhizium (Fig. 15) shows that it is more closely related to

other plant colonizing fungal genera, such as Gibberella and Epichloe, than pure insect

pathogens such as Zoophthora, but that the divergence between the Glomeromycota and the

Ascomycete EIPF occurred early in overall fungal evolutionary history. This suggests a

possible convergence of plant root colonization ability, and nitrogen transfer, between these

phyla, a convergence driven by the need for carbon acquisition.

109  
 Potential Agricultural Benefits Through Biotechnological
Manipulation of Plant-Fungal Associations

Published as: Behie SW, & Bidochka MJ. Potential agricultural benefits through
biotechnological manipulation of plant fungal associations. Bioessays 2013 35, 328-331.
Behie wrote the manuscript with additions, edits, and guidance from Bidochka.

110  
6.4 Introduction
 
Over 90% of vascular plants form symbioses with soil fungi (Gaude et al. 2012).

These associations confer a number of benefits to the plant; benefits that include increased

overall plant health, protection from phytopathogens, and improved nutrient acquisition

(Sasan & Bidochka 2012, Vance 2001). In natural ecosystems, many plants rely on soil fungi

for the acquisition of important nutrients, such as nitrogen and phosphorus (Vance 2001).

Mycorrhizal fungi are able to transfer nutrients to plants through root colonization; these

fungi reside almost exclusively in the phylum Glomeromycota (Shubler et al. 2001). More

recently, research has demonstrated that some ascomycetous insect pathogenic fungi have

endophytic capability (Sasan & Bidochka 2012) and are able to transfer insect-derived

nitrogen to plants (Behie et al. 2012). A number of these insect pathogenic fungi are

currently used as biological control agents in agricultural settings. The finding that

Metarhizium sp., an ascomycetous insect pathogen, is also able to transfer insect derived

nitrogen to plants through an endophytic association (Behie et al. 2012) leads to the

possibility that this fungus can be utilized in agricultural settings not only to protect plants

from harmful insect herbivory, but also to contribute to overall plant health. By elucidating

the underlying genetic mechanisms by which symbioses are formed between plants and fungi,

resultant biotechnological manipulations could confer beneficial traits to agricultural crops.

Examples of these traits include the reduction of environmentally harmful chemical

pesticides, limiting the use of fertilizers and increasing plant resistance to saline conditions.

In this article we discuss ways to potentially manipulate plant-fungal interactions, at the

molecular level, in order to benefit agricultural crops.

111  
6.5 Could Biotechnology Be Used To Facilitate Beneficial Fungal-Crop Symbioses?

Most plant species are able to form beneficial symbiotic relationships with soil fungi;

the challenge would be to genetically modify agricultural crops, or their symbiotic fungi, in

order to facilitate this association. First, fungal or plant genes responsible for the symbiotic

relationship need to be elucidated in order to develop a biotechnological strategy to facilitate

the interaction. For example, in barrel clover (Medicago truncatula), 29 genes were up

regulated during mycorrhizal association, 11 of which were not up regulated in plants during

bacterial colonization, suggesting that only certain genes play a role in plant-fungal

interactions (Wiedmann et al. 2004). Furthermore, some plant genes were up regulated prior

to physical contact with the fungus, indicating that secreted fungal molecules prime the plant

for association (Wiedmann et al. 2004).

During the initial stages of fungal association with plants, there is an increase in plant

defense systems, particularly the jasmonic acid (JA) and salicylic acid (SA) biosynthetic

pathways (Pozo & Azcon-Aguilar 2007). The up regulation of the SA pathway is generally

considered to be a response to biotrophic pathogens (Pozo & Azcon-Aguilar 2007). During

sustained symbiosis however, equilibrium is achieved and the activity of this pathway falls to

a lower steady state. If this were not the case, the plant defense mechanism would continue to

be activated and could potentially eliminate the fungal symbiont (Medina et al. 2003). This

suggests that beneficial fungi suppress the SA pathway, which is essential for sustained

association. Harmful phytopathogens are unable to suppress the SA pathway, indicating

specific communication between the plant and beneficial fungal symbionts (Pozo & Azcon-

Aguilar 2007). Identifying and controlling genes responsible for fungus-plant communication

could enhance the relationship.

112  
 Several plant and fungal genes related to symbiosis have been identified. MtScp1 is a

plant gene that encodes a carboxypeptidase involved in the transmission of fungal specific

signals (Liu et al. 2003). Fungal genes that play a role in plant adherence, such as mad2

(Wang & St. Leger 2007) and myc are integral to fungal/plant association since they control

the regulation of molecules secreted from the fungus prior to association that stimulate root

development and expression of plant genes required for intercellular fungal interaction

(Bucher et al. 2009) (Fig. 17). These genes could be manipulated on a molecular level to

facilitate fungal-root associations.

In order to elucidate the complete set of genes involved in this complex, commutative,

and beneficial partnership between plant and fungus, plants with, and without, mycorrhizal

fungi could be genetically assessed using proteomic analysis and next generation sequencing.

An increased understanding of the genes involved in plant-fungal communication could then

be studied in, for example, the insect pathogenic, endophytic fungus Metarhizium; a good

candidate fungus for testing genetic manipulations. Most arbuscular mycorrhizal fungi,

recognized as beneficial plant fungi, are obligate biotrophs (Bucking & Shachar-Hill 2005),

difficult to grow in pure culture and not amenable to traditional transformation systems.

Metarhizium, on the other hand, is not a fastidious fungus and is easily genetically

manipulated (Fang et al. 2006).

113  
Figure 17. Potential plant benefits from symbiotic fungal gene manipulations or plant gene
manipulations. Manipulation of fungal or plant genes could lead to improved protection from
drought/salinity (MtScp1, myc, mad2), increased nutrient acquisition (MtPt4, GmosPT, GvPT,
GmAMT4, MtGim7) or protection against insects (pr1, ste1, chy1, try1). Red denotes fungal
genes, green denotes plant genes. Genes shown are representative of numerous potentially
important genes in the different functional groups (Wang & St. Leger 2007, Hohnjec et al.
2005, Wulf et al. 2003, St. Leger & Wang 2010)

114  
6.6 Agricultural Sustainability in Drought or Saline Conditions through Fungal
Symbiosis

Agricultural productivity is susceptible to drought and soil salinity (Ruiz-Lozano &

Aroca 2010). Mycorrhizal fungi can improve water uptake and retention in crops under

drought conditions (Kothari et al. 1990). For example, corn plants with mycorrhizal fungi

had a 30% higher rate of transpiration, and water uptake in relation to root size was double

that of control plants (Kothari et al. 1990). Fungal inoculation was also shown to improve

water uptake in red clover, apple, rose, and rangeland grass (Kothari et al. 1990). It is

thought that colonization of plant roots by soil fungi increases stomatal efficiency of plants.

Furthermore, extraradical mycelia allow mycorrhizal associated roots to become more

efficient at water uptake in soil, extending the water depletion zone and providing plant roots

with access to water previously unavailable (Ruiz-Lozano & Aroca 2010).

Soil salinity can damage plants through increased accumulation of reactive oxygen

species created during prolonged exposure to stress (Waqas et al. 2012). Endophytic

associations can induce the production of antioxidants such as glutathione (Waqas et al.

2012). For example, soybean with fungal symbionts have higher proline content in their

roots, which is required for maintaining cellular osmotic balance under saline conditions

(Porcel & Ruiz-Lozano 2004). Metarhizium was shown to form an endophytic association

with soybean roots under saline conditions (Khan et al. 2012). The challenge is to use a

biotechnological approach in order to establish or strengthen this fungal partnership with

other crop plants.

115  
6.7 Nutrient Exchange in Plant-Fungal Symbiosis
 
Plants have been shown to provide carbon to root colonizing fungi in exchange for

soil nutrients (Hawkes et al. 2008). This exchange has been studied primarily in mycorrhizal

fungi, where the fungus provides nutrients obtained from the soil to plant roots in exchange

for plant derived monosaccharides (Parniske 2008). Research on this symbiotic exchange

initially focused on the provision of phosphorus to the plant by Glomus spp. that received

plant monosaccharides from a number of plants including carrots, wheat, and onion (Bucking

& Shachar-Hill 2005, Koide & Kabir 2000). During the symbiotic exchange there was

coordinated up regulation of plant (Medicago truncatula) and fungal (Glomus mosseae,

Glomus intraradices) nutrient transporters (Hohnjec et al. 2005). MtPt4, a phosphate

transporter, was found only in plant roots during mycorrhizal association (Hohnjec et al.

2005), this coincided with the up regulation of the fungal phosphate transporters GmosPT

and GvPT during colonization of inner cortical root cells (Benedetto et al. 2005). Plant cells

that have been colonized by fungal symbionts show an increased expression in the putative

hexose transporter Mtst1 (Harrison 1996) as well as two putative sucrose transporters (Gaude

et al. 2012). This synchronized response was also observed with plant nitrogen transporters

and the intraradical mycelia of Glomus (Gaude et al. 2012, Vance 2001). In soybean, it was

found that five ammonium transporters were induced during fungal association, the most

abundant of which was GmAMT4 (Kobae 2010). Putative ammonium transporters were also

expressed in the fungus during root colonization, along with the nitrate transporter MtGim7

(Wulf et al. 2003). This nutrient exchange is significant as limited phosphorous or nitrogen

acquisition of plants limits overall plant health and productivity (Vance 2001). Recently it

was shown that the endophytic insect pathogenic fungus, Metarhizium was able to transfer

116  
nitrogen directly from infected insects to bean and switchgrass (Behie et al. 2012). It is

speculated that the plant provides carbon in exchange for insect-derived nitrogen.

A major question is the fidelity of plant-fungal symbiosis. Is the level of fungal

provision of nutrient to the plant recognized and reciprocated by the plant and vice versa?

And does nutrient exchange physically strengthen the symbiosis? Restricting nutrient flow

via knockout of nutrient transporters genes and measuring reciprocal nutrient exchange can

test this.

6.8 Potential of Endophytic Fungi to Protect Crops against Insect Pests

 
There are numerous examples of insect pathogenic fungi used as biological control

agents (BCA) under agricultural conditions. A major benefit of BCA is that they reduce

chemical pesticides in the environment, many of which are harmful to human health, or are

increasingly ineffective through the development of insect resistance (Butt & Copping 2000).

Metarhizium spp. have a bifunctional lifestyle as a plant endophyte and an insect pathogen.

The potential for genetic engineering of such filamentous fungi provides the opportunity for

studying overexpression of genes responsible for pathogenicity. Pr1 and chy1, genes that

encode insect cuticle degrading enzymes (St. Leger & Wang 2010), st1, a gene involved in

lipid metabolism (Shah et al. 2005), and mad1, a gene responsible for fungal adherence to

insect cuticle (Wang & St. Leger 2007) (Fig. 17), lend themselves to biotechnological

manipulation and could lead to strain improvement with respect to virulence and insect host

range.

117  
6.9 Conclusion
 
In this minireview we have suggested that biotechnological manipulation of plant and

symbiotic fungal genes could result in improved crop sustainability. The majority of current

knowledge on plant fungal symbioses focuses on mycorrhizal fungi and their plant partners.

However, these fungi are not amenable to biotechnological manipulations. Recent work

suggests that several ascomycetous insect pathogenic fungi are able to form endophytic

associations and are responsive to novel biotechnological manipulations, which could then be

applied to improve crop sustainability.

118  
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Appendix A
 

 
Supplementary Figure 1. Microcosm design and experimental setup. A) Empty, open
microcosm. B) Closed microcosm and pot. C) Closed microcosm inside pot. D) Closed
microcosm containing soil and 15N injected, M. robertsii infected larvae.

 
 
 

 
 

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Supplementary Figure 2. Galleria melonella larvae infected with Metarhizium robertsii and
Aspergillus flavus. Images were taken six days after initial infection. Insects were infected by
placing them on fungal growth plates and agitating for two minutes. A), B), and C) Images of
M. robertsii infection of wax moth larvae. D), E), and F) Images of A. flavus infection of
wax moth larvae. G), H), and I) Images of wax moth larvae with no fungal infection.
 
 

138  
  

Supplemenatry Figure 3. Time course, after insect infection of M. robertsii (close circles)
and A. flavus (open circles) propagules found within 0.5 cm from plant roots at a depth of 2
cm.

139  
Supplementary Figure 4. Microcosm design and experimental setup. A) Empty petri dish,
with 30 micron mesh adhered to the surface with silicon adhesive. B) Closed petri dish plant
container. C) Microcosm showing petri dish I plant container. This would be filled with soil.
D) 13C CO2 compressed gas bottle and gas injection syringe, used to addgas volumetrically
into gas assimilation chambers. E) 7 day plants in assimilation chamber at 1500 ppm 13C CO2.

140  
Supplementary Figure 5. 13C NMR spectra of both trehalose and N-acetylglucosamine (1, 7,
14, and 21 days) obtained from Metarhizium colonized plant roots or Metarhizium colonized
soil grown in 13C or natural CO2 environment, as well as control treatments. A) Trehalose
spectra without insect present in soil microcosms. B) Trehalose spectra with insect present in
soil microcosms. C) NAcGlc spectra without insects present in soil microcosms. D) NAcGlc
spectra with insects present in soil microcosms. All spectra were run at 27, 000 scans, with
100uL MeOH control (49.5 ppm).

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Supplementary Figure 6. 13C NMR spectra of soluble sugars extracted from plants grown
without 13C CO2. A) Plant grown without fungus or insect present in soil micocosms. B)
Plant grown with fungus present in soil microcosms, with no insect. C) Plants grown with
both fungus and insect present in soil microcosms.

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Appendix B

Supplementary Calculation 1. Calculation of insect nitrogen in soil

The soils of grasslands and woodlands contain, on average, just over 3.5 million insects per
acre (Schulze et al. 2001). This translates to ca. 900 insects per square meter. If we take an
average weight of insects of between 5- 50 mg and each insect is approx. 10% nitrogen
(Klironomos & Hart 2001), then there is approx. 0.4-4 g (by weight) of available insect
nitrogen per square meter of habitat

Supplementary Calculation 2. Calculation of insect nitrogen available to plants through

EIPF
The estimated amount of atmospheric nitrogen that is fixed in an ecosystem varies between
10-160 kg/ha/yr (Boring et al. 1988). Approximately 31% of this fixed, plant incorporated,
nitrogen is then lost to insect herbivores (Lovett et al. 2002). Therefore the amount of plant
nitrogen lost through insect herbivory varies between 3.1kg/ha/yr (10kg/ha/yr x 0.31) to
49.6kg/ha/yr (160kg/ha/yr x 0.31). It is estimated that insects are able to incorporate 50% of
ingested nitrogen (Fox & Macauley 1977), therefore the amount of plant nitrogen
incorporated into insect biomass varies between 1.55kg/ha/yr (10kg/ha/yr x 0.31) to
24.8kg/ha/yr (160kg/ha/yr x 0.31).
We then estimated that approximately 30% of soil insects may become infected by EIPF
(Bidochka et al. 1998) however this number could be as high as 66%. Therefore, the amount
of insect derived nitrogen potentially provided to plants by EIPF varies between
0.465kg/ha/yr (1.55kg/ha/yr x 0.30) and 7.44kg/ha/yr (24.8kg/ha/yr x 0.30).
We then calculated that the percentage of the total fixed nitrogen provided to plants
through EIPF as 4.65%. That is 0.93kg/ha/yr/insect derived nitrogen provided to plants
through EIPF divided by 10kg/ha/yr average atmospheric fixed nitrogen. Similarly,
7.44kg/ha/yr insect derived nitrogen provided to plants through EIPF divided by 160kg/ha/yr
average atmospheric fixed nitrogen in an ecosystem is also 4.65%.
 

 
 
 

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