Revisiting Johnson 1993

In a paper published in Ecological Applications in 1993, Nancy Collins Johnson showed, experimentally, that fertilization of soil leads to the selection of Arbuscular Mycorrhizal (AM) fungi that are inferior mutualists. Johnson found that fertilization, both, alters the species composition of AM fungal communities and that big bluestem plants colonized with AM fungi and other associated soil organisms from fertilized soil were smaller and produced fewer inflorescences. Twenty-three years after the paper was published, I spoke to Nancy Johnson about her motivation to do this study, memories of field work and what we have learnt since about the effect of fertilization on mutualistic mycorrhizae.

Citation: Johnson, N. C. (1993). Can fertilization of soil select less mutualistic mycorrhizae?. Ecological applications, 3(4), 749-757.

Date of interview: 20th December 2016 (via Skype), with some updates via email in August 2020.

Hari Sridhar: How did you get interested in Mycorrhizae? I notice that it is something you studied even for your Master’s thesis.

Nancy Collins Johnson: I started studying mycorrhizae in 1983 for my master’s degree in Botany at the University of Wisconsin, Madison. My advisor, Mike Adams, was open minded about my thesis topic and encouraged me to explore ideas during my first semester in graduate school.  I wanted to study some sort of mutualistic symbiosis and I knew that I had found my research topic when I read about mycorrhizae in my Plant Physiology textbook (Salisbury and Ross, second edition).  It was just a short description, but it really caught my interest, and within two weeks I had read everything that I could find about vesicular arbuscular mycorrhizae (back then we called them VAM, now we just call them AM because some species of AM fungi don’t make vesicles). I was convinced that I wanted to study the role of VAM in mine reclamation, and was fortunate to meet Tom Hunt, a master’s student in the Landscape Architecture program who, a year earlier, had established a field experiment at the Jackson County Iron mine, 125 miles northwest of Madison. The mine processed iron ore into taconite and generated huge quantities of fine, grey tailings. Tom’s thesis research focused on developing strategies to revegetate taconite tailings, and he had established experimental plots to compare various combinations of plant species along with different types of amendments. Tom was happy to have me study the VAM in his experiment, so I brought samples from his plots back to the lab to measure VAM fungal structures in roots and examine their spores in the soil (aka taconite tailings). Then everything fell apart. I carefully followed the published instructions, but when I tried to stain VAM fungi in roots, I ended up with a bunch of disconnected xylem elements and cortical cells. When I tried to extract spores from the tailings – and looked at my spore extracts through the microscope – all I could see was a bunch of sand and random organic matter. It was a seriously low period of my master’s studies.  In the early 80’s, nobody at UW-Madison studied mycorrhizae, and I had no idea what VAM fungi REALLY looked like! I suddenly realized how naïve I had been to think that I could simply read about mycorrhizal techniques in a book and then successfully measure them. Mike Adams recommended that I call Tony Liberta at Illinois State University, and Tony recommended that I call Mike Miller at Argonne National Laboratory. So, I cold-called Mike Miller and explained my situation. I’ll always remember that phone call. Mike said “oh, you think it’s hard to study VAM? We think it’s hard too, and we’re experts.” Mike invited me to visit his lab to work with his postdoc Anne-Cressey (A-C) McGraw. I immediately accepted the offer, and visited their lab. Within just a few days A-C had taught me the methods I needed to complete my MS research. Also, A-C agreed to be on my committee, and she was a co-author on two (Johnson & McGraw 1988a; Johnson & McGraw 1988b) of the three papers that were published from my MS thesis. Mike Miller and I became good friends, and we continue to collaborate on research to this day.

HS: How did you come up with the idea of testing the effect of fertilizers on these fungi?

NJ: I became interested in the effects of fertilizer on mycorrhizae during my PhD studies with Dave Tilman at the University of Minnesota. Dave had recently established the long-term ecological research (LTER) site at Cedar Creek and he wanted to do more below-ground research. I often think that the reason Dave Tilman accepted me as a graduate student was because I had experience studying soil organisms. I recall him saying something like “I don’t care what you study for your dissertation as long as you keep working in the soil.” So, I decided to keep studying mycorrhizae at Cedar Creek. Dave started the famous Experiment 001 (Long-Term Nitrogen Addition to Undisturbed Vegetation) in 1982, and by the time I began my PhD research in 1987, the above-ground responses of vegetation to fertilization was quite dramatic. Which begged the question, what is happening below-ground?  Also, at this time I was working with some agronomists to study the influence of crop rotation on mycorrhizal fungal communities and that work suggested that mycorrhizae in highly fertilized agricultural systems might not always be beneficial to crops. These observations set the stage for the experiment that led to the 1993 publication. 

HS: What was the level of involvement of Dave Tilman in your work?

NJ: Dave Tilman was a great advisor for me because he appreciated that I liked to work independently. Although he was very busy, when I had a question or a problem, Dave would always help me solve it. The 1993 paper was the last chapter of my dissertation, and when I gave Dave my proposed experimental design and research plan, he carefully read it, gave me some good suggestions, and said, “Nancy, you’re going to need help to do all of this.” So, Dave hired three undergraduates to work with me. I had no idea what a huge effort it would be to complete the experiment, but Dave knew, and looking back, the help by undergraduates was key to the experiment’s success.

I got a dissertation fellowship from the University of Minnesota that paid me a small stipend during the last year of my PhD, but Dave Tilman paid for many of the supplies that I used in the greenhouse. I asked Dave to be a co-author on the paper, but he said “no, Nancy, this is all your idea and your work.” He was a co-author on two other papers from Cedar Creek (Johnson et al. 1991; Johnson et al. 1992) but even on those papers I had to twist his arm to be a co-author. During his own PhD, Dave published a single-authored Science paper, and I think he recognized the importance of single-author work. Also, by that time, Dave was so successful that he didn’t need more papers on his CV.

HS: Did you do all the writing when you were in New Mexico?

NJ: Yes. My husband got a job with the New Mexico chapter of the Nature Conservancy and we moved to Santa Fe in January 1991, so I did most of the microscopy and all of the writing in our spare room. I finished my dissertation and defended my PhD in August, and our son was born in December 1991. I was an unemployed full-time mother until 1993, that’s why my home address, 212 Spruce Street, Santa Fe, New Mexico is listed as my current address on the publication.  Eventually, I started to teach classes in Microbial Ecology and Ecosystem Ecology as an adjunct professor at the University of New Mexico. In 1994, I got an NSF postdoctoral fellowship to continue to explore ideas from my dissertation, and that was my lifeline back into science! Our daughter was born in 1995 and salary from the postdoctoral fellowship covered the babysitter and transportation to the University in Albuquerque. During that time I wrote my most cited paper with Jim Graham and Andrew Smith titled Functioning of mycorrhizal associations along the mutualism to parasitism continuum.

HS: What do you remember of the peer-review of this paper at Ecological Applications?

NJ: I can’t remember it being overly difficult but I do remember that they were really slow.

HS:  Did you continue to work at Cedar Creek after this study?

NJ: Oh yes, I continue to work at Cedar Creek to this day. Gail Wilson, Mike Miller and I got an NSF grant to test a “unified hypothesis of mycorrhizal function” based on stoichiometry (Johnson 2010). That work led to some really important findings that help predict mycorrhizal functioning from both a resource economics perspective (Johnson et al. 2015) and also an evolutionary perspective (Johnson et al. 2010). In addition to studying mycorrhizal responses to fertilization, my students and I have collaborated with Peter Reich to study mycorrhizae in his Free Air CO2 Enrichment (FACE) experiment at Cedar Creek (Wolf et al. 2003; Antoninka et al. 2011). I am currently on sabbatical, and I received a Bullard Fellowship from Harvard Forest. If it weren’t for COVID, I would be starting a comparative study of the microbiomes of AM symbioses in long-term fertilization experiments at several LTER sites, including Cedar Creek. Right now, that study is on pause until travel gets easier.

HS: Has Cedar Creek changed a lot from the time you did this study?

NJ: The change is just tremendous. In the 80’s, on a busy summer day, there might have been a maximum of 10 or 12 researchers working at Cedar Creek. In the 90’s, Dave Tilman expanded the research enterprise to include a huge biodiversity experiment and about that time many other researchers came to work at the LTER site, including Peter Reich with his FACE experiment called “BioCON,” which is still in operation today. Many new buildings have been added, and now on a busy day at Cedar Creek there can be hundreds of people – primarily summer interns – working on many different research projects.

HS: Are the experimental sites protected from external disturbance?

NJ: Yes thankfully, Cedar Creek Ecosystem Science Reserve is well protected because it belongs to the University of Minnesota. Back in the 80’s the area around Cedar Creek was rural with lots of farmland, but now suburban residential developments are creeping ever closer to the Reserve.

HS: Could we go over the names in the Acknowledgements to get a sense of how you knew these people and how they helped?

NJ: Certainly. The first person listed is Barb Pecenka; she was one of the three undergraduates that Dave hired to help me. Barb was key to the success of the experiment because she had a green thumb. Barb watched the plants like a hawk, and it was phenomenal how well they grew. And of course, I acknowledge Dave Tilman for all of his help that I described earlier. Rick Johnson is my husband, and he helped in many ways – especially when I needed moral support. Abderrahman El Haddi, who we always just called El Haddi, was the data Manager for Dave Tilman. Back then, there wasn’t R, and El Haddi helped do some of the statistical analyses. Frank Pfleger was a Plant Pathology professor who was on my committee. Frank was very interested in mycorrhizae and I worked many hours working with him analyzing spores. Frank would get official type specimens of mycorrhizal fungal species sent to his lab and this resource was incredibly helpful for identifying the spores that we isolated from Cedar Creek. Anna Shoeman and Lisa Vogel were the other two undergraduates, and they helped set-up and maintain the greenhouse experiment and were a huge help with harvesting, cleaning and weighing the plants. Ron Bowen donated the seeds. He has a prairie restoration company very close to Cedar Creek, so the seeds were of a local variety of big bluestem. A Graduate School fellowship from the University of Minnesota provided me a small monthly stipend while I was working in my spare room in Santa Fe, New Mexico.

HS: If you were to redo these experiments today, what would you do differently?

NJ: If I redid the experiment today, I would use molecular genetics to identify which mycorrhizal fungi are inside the roots.  These days my lab group looks at plant responses to the whole microbial community and not just mycorrhizal fungi so I would also look at the microorganisms interacting with the fungi. One additional variable that I would measure if I redid the experiment would be the extraradical hyphae present in the pots because oftentimes the amount of hyphae in the soil can be more insightful than the colonization inside roots. I thank Gail Wilson for showing me the value of that measurement. She is a co-author on the PNAS paper that showed that coadapted plant-fungus-soil combinations produce more extraradical hyphae than novel combinations (Johnson et al. 2010). Other than that, I would do the experiment the same if I did it today. Actually, I don’t think that I could do the experiment as well today because now there are so many other distractions in my life and I just don’t have the focus and energy that I had 30 years ago.

HS: At the time when you were doing the work, did you anticipate at all that it would become such an important paper in the field? Do you have a sense of what it mostly gets cited for?

NJ: We didn’t have H-values and Impact Factors back then, so I didn’t think about the paper’s impact in those terms. But I do remember thinking that it was an important finding to share. I expect that the paper is probably cited most by people who are interested in context dependency of mutualism. Scientific knowledge advances incrementally, and looking back now, I can see how this paper contributes to our understanding of how mycorrhizae function in ecosystems. This empirical study was the beginning of my efforts to articulate economic and evolutionary mechanisms for the range of outcomes of mycorrhizal interactions from beneficial mutualism to plant growth depression (aka parasitism). I don’t want to give the impression that I was the first person to see that mycorrhizal symbioses function along a mutualism to parasitism continuum. Others before me used the word ‘parasitism’ to describe plant growth depression caused by mycorrhizal fungi. The pioneers in this field of research were Bethlenfalvay, Peng, Eissenstat &  Graham, and Modjo & Hendrix. My work builds upon the shoulders of those important researchers.

HS: You mention a taxonomic dilemma in the paper. You say that the spores of Glomus intraradix formed a continuum with G. aggregatum spores. Do we know more about this today, and has this taxonomic dilemma been sorted out?

NJ: The taxonomy of AM fungi has evolved a great deal in the past 30 years – and it continues to evolve. Nowadays, molecular genetics and sequence data can help distinguish among the many taxa that have small laminated spores with simple hyphal attachments.  Glomus intraradices changed to Glomus intraradix for about a year – unfortunately for me it was the year I published this paper – and then it got changed back to Glomus intraradices. So now it looks like I don’t know how to spell. Anyway, according to a frequently updated species list, it is now called Rhizophagus intraradices, and Glomus aggregatum is now Rhizophagus aggregatum. One thing to keep in mind is that genotypes of the same AM fungal species vary widely in their symbiotic performance. Different spore isolates from the same clone – much less the same species – may vary so much that some may be beneficial while other spore isolates provide little benefit to plant growth.

HS: You used Sorghum sudanense as a host plant. Is that plant still used as a host for lab cultures?

NJ: Yes, Sorghum sudanense and Sorghum bicolor are common host plants for AM fungal cultures. Like most C-4 grasses, they are highly dependent on mycorrhizae. Leeks and onions are also commonly used hosts for trap cultures – especially in northern areas with less intense sunlight. 

HS: Is big bluestem still a fairly common plant in the Cedar Creek plots?

NJ: It depends on the field and the experimental treatment. Little bluestem is more common in many of the fields, and both big and little bluestem disappear from highly fertilized plots and quack grass (Elymus repens) takes their place. But big bluestem is a very common in true prairie and savannah, especially in areas that get burned regularly.

HS: If you redid this experiment today  would you still use big bluestem?

NJ: Yes, I would because it is highly dependent on mycorrhizae. That makes it an excellent indicator of mycorrhizal function and local adaptation of plants through their mycorrhizae. We compared the mycorrhizal responsiveness of a plant species that increased in abundance (quackgrass) with big bluestem that decreased in abundance in response to nitrogen enrichment, and you can see very different root morphologies and allocation patterns. Check out the photo of quackgrass and big bluestem roots in Figure 1 of Johnson et al. 2008. You can see that quackgrass has very fine roots while big bluestem has coarse roots. It essentially uses mycorrhizal fungal hyphae in place of root hairs.

HS: Do you still have the slides of the spores you made for this study?

NJ: Yes. In fact, they’re sitting in a cupboard not five feet away from me. You’ve really sent me down memory lane. I pulled out my old dissertation, my old notebook, my old plant physiology book. I could pull out those slides and take a look at them. It would be fun to see them again. I used to take such good notes and I have notes on which spores are on every slide.

HS: I think all the material related to important studies in the field should be archived somewhere.

NJ: I agree, that’s a really good idea. I know that the Cedar Creek LTER has a very effective Data Archive system. I remember sending them the data from my dissertation field experiments ( Johnson et al. 1991; Johnson et al. 1992), but this was a greenhouse experiment, so I doubt that they have these data in their archives.  It would be nice if there could be someplace to archive this stuff. People might want them when I am gone.

HS: You say, “Although it is clear that soil microorganisms are able to suppress mycorrhizal responses, the mechanisms responsible for this phenomenon remain a mystery.” Do we know more about the mechanisms today?

NJ: It’s still largely a mystery, but we now have some tools to start to solve it. Recent advances in metagenomics, transcriptomics, proteomics and advanced imaging technologies are helping us to begin to see the microbial players. There is no question that plants and their associated mycorrhizal fungi host complex microbiomes, and that the performance of mycorrhizae is really an emergent property of these complex adaptive systems. We know that resource availability and environmental thresholds create the ecological and evolutionary theater for these complex adaptive systems to assemble and disassemble. Other than that, we understand very little. It is interesting that you asked me this question because it is the topic of the Bullard Fellowship that I am just now starting for my sabbatical. Next year I hope to be able to provide a more satisfying answer.

HS: You say, “If heavily fertilized agricultural systems have the potential to develop inferior mycorrhizal associations, then effective management requires manipulation of VAM fungal communities through inoculation, or cultural practices that favor proliferation of the most beneficial VAM fungi. Do you see the findings from such studies being incorporated into agriculture practice?

NJ: Oh, I am happy to say that the recent Restorative Agriculture movement is paying close attention to mycorrhizae. Over the years I have become convinced that adding commercial inoculants composed of exotic AM fungi is a big mistake. Companies that sell inoculum give the impression that all AM fungi are the same and that they disappear because of soil disturbance, but that just isn’t true. These fungi are amazingly abundant and resilient. Agriculture and other anthropogenic disturbances will generally not eradicate AM fungi, but it will cause the composition of AM fungal communities to change. Commercial inoculum is expensive, the fungi are often maladapted to the environment, and even worse, it introduces exotic fungi that could potentially become undesirable invasive species.

The goal of Restorative Agriculture should be to manage the system so that indigenous communities of AM fungi thrive and generate mutualistic symbioses. I think that the most important thing to recognize is the balance of trade between plants and AM fungi. We have discovered that if you want to select for mutualistic mycorrhizae it’s best if the system has plenty of light, is phosphorus-limited, and is not nitrogen-limited. This combination of resource availabilities maximizes the plant benefits of the symbiosis (access to phosphorus/water) and minimizes the costs. The reason you don’t want to have light or nitrogen-limitation is because the currency that the plant is delivering to the fungus is photosynthate. And for a plant to be effective at photosynthesizing it has to have lots of light, chlorophyll and ribulose bisphosphate carboxylase and those compounds require a lot of nitrogen. So, the advice I always give when asked how to encourage AM mutualisms is to strive for a phosphorus-limited system that’s not light or nitrogen-limited.

HS: In the 27 years since this paper was published, have you ever read the paper again?

NJ: Actually, until you asked me for this interview, I don’t remember reading it again. But this experiment was highlighted in the chapter on mutualisms in the early editions of Manuel Molles’ Ecology: Concepts and Applications textbook that I used to use when I taught Ecology. It was actually kind of fun because a textbook artist remade all of my figures, so during the lectures on mutualism I could talk about my own work, and the students thought it was pretty cool that their teacher’s experiment was featured in the textbook. I’m glad you gave me the opportunity to read the original paper. It was fun to see that – to a large extent – I am still picking away at solving the same mysteries.

HS: When you read it now, what strikes you the most about it?

NJ: Well, we no longer call them VAM. That sort of stands out, you know, this archaic terminology. Also, the spelling of a lot of the fungal species has changed and that is irritating. But, you know, I was actually pleasantly surprised. I realized that I don’t write that much differently today. I think that the paper was fairly well-crafted. It was much more difficult for me to write back then compared to today. But when I reread the paper, I realized that there’s entire sentences in here that I’ve definitely written again. The sentence: “A mycorrhiza includes both plant and a fungus, so a “co-adapted mycorrhiza-soil complex” can be defined as a dynamic system in which both plant and fungal communities have adjusted and continue to adjust to the soil conditions and to one another so that mycorrhizal associations within the complex become increasingly more mutualistic over time” was the inspiration for our PNAS paper published in 2010. So, a lot of the things that I did later, started in this paper. I’m glad that you reminded me about it.

HS: Would you count this as one of your favorite pieces of work?

NJ: Combined with the 2010 PNAS paper that I wrote with Gail and Jackie Wilson, Mike Miller and Matt Bowker, it would be one of my favorites. I really worked hard on the paper, and the experimental design has been the basis of a lot of the experiments that we’ve done since then. This design recognizes that mycorrhizae and their microbiomes are complex invisible systems, and a really good approach to study them is to observe their patterns in nature first, and then try to manipulate the functioning of the “black-box” microbial system in a greenhouse using natural materials as much as possible. The results of mycorrhizal studies that use a mail-order fungus and soil bought at a hardware store are not as useful for ecological studies, because I believe the sentence that I mentioned in the previous question is really true.

HS: How was the paper received when it was published? Did it attract a lot of attention?

NJ: That’s an interesting question. It was published in 1993 when my son was two and I was an unemployed stay-home mom living in Santa Fe. Back then we didn’t have the internet, and I was really kind of out of touch. I’m sure that I got some reprint request postcards, but I don’t remember getting a flood of reprint requests in the mail like I got for the Agronomy Journal paper  (Johnson et al. 1992) which was also part of my dissertation. So, it was just kind of low key.

HS: What would you say to a student who’s about to read this paper today? Would you guide their reading in any way? Would you point them to other things they should read along with this paper? Would you add any caveats to their reading?

NJ: I would recommend that they read the 1993 paper in combination with my 2010 Tansley review, and I would suggest that they also read a beautiful paper describing a field experiment complemented by a greenhouse study by Shengjing Jiang and Yongjun Liu (2018). That study takes two important steps forward through manipulating mycorrhizae in the field using Benomyl, and using molecular genetics to determine the impacts of nitrogen fertilization on the composition of the AM fungal communities in their experimental field plots. Little-by-little, we are linking microbial community composition to their function in ecosystems. With all of the new technologies that are now available, we can expect some major breakthroughs in the next decade.

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