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Bulletin 1. Mandaamin Institute, Elkhorn, Wisconsin;

Published on the Internet, January, 2016.

Partnerships between Maize and Bacteria for Nitrogen

Efficiency and Nitrogen Fixation.

By Walter Goldstein, Ph.D., Executive Director12

.

Abstract: Partnerships between maize plants and diazotrophic (N2 fixing) bacteria can result in

greater plant N efficiency through enhanced root growth and N2 fixation. The literature on these

relationships was reviewed, from early Brazilian studies to the present interest in genetically

engineering nodules from legumes into maize.

Despite inconsistencies, the overall literature shows that N2 fixation can occur naturally in maize

in variable and sometimes substantial amounts. Errors in methodology may explain some of the

divergent results. However, many studies showed that the choice of cultivar is critical for a

response to inoculation. Modern hybrids and inbreds vary greatly in their response to

diazotrophic bacteria.

The successful, natural relationship between plant and diazotrophic bacteria is a dialogue leading

to a meshing of metabolic activities. The plant modulates its root exudates and down regulates

its active defense systems while the bacteria provides various services (N2 fixation, hormone

production, etc.) either in the rhizosphere, the rhizoplane or inside the root or stem. The maize

plants’ response to N deficiency results in production of aerenchyma in roots and low levels of

ethylene, both of which may be conducive to endophytic colonization. Lack of establishment of

relationship may partly have to do with pre-existing endophytic communities dominated by seed-

borne Fusarium spp.

Some accessions from various landraces of maize appear to be especially N-efficient/N2 fixing.

Mexican scientists discovered that under certain conditions they may develop exaggerated brace

roots covered with copious quantities of mucigel, which harbors diazotrophic bacteria. Visual

evidence suggests that they also possess an oxidative system for scavenging microbial N from

brace roots. These varietal adaptations may enhance N2 fixing activity.

The history of the Mandaamin Institute’s breeding program to develop N-efficient/N2 fixing

maize varieties from these landraces is described. N-efficient plants developed in the program

are associated with more chlorophyll and less barrenness under N-stress conditions.

1 Mandaamin Institute, Inc., W2331 Kniep Road, Elkhorn, WI 53121 USA. wgoldstein@mandaamin.org (email). 262-

248-1533 (phone). 2 The author acknowledges and is thankful for funding from USDA-NIFA-OREI competitive grants program and

from the Ceres Trust for supporting the research leading to this paper.

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Finally, in summary, a hypothetical framework of maize-diazotroph relationships, drawn from

the literature and our findings, is presented to stimulate further research on developing

responsive varieties of maize.

Introduction: Microbes called plant beneficial rhizobacteria live in the root zone (rhizosphere)

and on the root surface (rhizoplane) of plants. Similar bacteria called endophytes live inside

plant roots and shoots. These bacteria live in dynamic relationships with their plant hosts. The

growth of the bacteria in the rhizosphere is regulated by the quantity and composition of root

exudates (Mendes et al. 2013; Santi et al. 2013). The growth of bacteria inside the plant is

limited by the plant’s own defense systems (Rosenblueth and Martinez-Romero 2006). Services

the bacteria provide include hormone production, stimulation of root growth, better mineral

availability (mediated by iron chelating substances called siderophores or by microbially induced

solubilization of phosphorus), better uptake of nutrients, N2 fixation, protection from pathogens,

induced systemic resistance to diseases, reduction in stress by reduction in ethylene levels, and

through other mechanisms (Fuentes-Ramirez and Caballero-Mellado 2005; Barea et al. 2005,

Gaiero et al. 2013, Mendes et al. 2013, Santi et al. 2013). Amongst different varieties of maize,

a great diversity and potential of these bacteria to provide services has been established

(Johnston-Monje and Raizada 2011; Ogbow and Okonkwo 2012).

Various ‘biofertilizers’ are used to inoculate maize and other cereals with diazotrophic bacteria

in order to improve nitrogen efficiency and the availability of nutrients in the face of reduced

levels of fertilizer (Dobbelaere et al. 2001; Kennedy et al. 2004; [ICRISAT], 2004; Fuentes-

Ramirez and Caballero-Mellado 2005; Gaiero et al. 2013, Santi et al. 2013). In several

documented cases (which will be reviewed below) there has been no apparent effect of

inoculation on the N balance or productivity of the maize plant; however, in many other cases

there are profound effects including growth stimulation and substantial N2 fixation. It has not

been apparent why there have been erratic results. However, it has been argued that many

external and internal factors can influence the effects of inoculation with bacteria and that

application of fertilizers does not always produce significant yield increases either (Dobbelaere

et al. 2001).

Hidden in the literature, however, are numerous cases where it has been established that the

relationship between these microbes and maize differs according to the variety of maize.

Modern cultivars of maize differ widely in their ability to establish beneficial relationships with

inoculated diazotrophs that leads to great N efficiency. In part, this may be due to pre-existing

relationships such as a seed-borne endophyte community that includes antagonistic Fusarium

species. On the other hand, ancient races of maize or teosinte have been shown to harbor diverse

rhizosphere and endophytic communities rich in native diazotrophs and antagonistic to Fusarium

species. Furthermore, methods for detecting N2 fixation in the real world are less than perfect,

and some of the differences in findings in the scientific literature are associated with inadequate

sampling technique. There is evidence that farming systems may affect the potential for N2

fixation in maize, with conventional systems less conducive than organic systems.

The following is a review of the research on maize-microbial partnerships leading to greater N-

efficiency and N2 fixation. It describes the history of research, leading from early Brazilian work

to the present interest in developing transgenic N2 fixing maize. It then explores natural N2

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fixation in maize, including early and modern research on effects of inoculation with different

diazotrophs, and problems with methods to detect N2 fixation. In the second part of the paper

physiology and ecology will be addressed, including maize adaptation to N stress, exudate and

defense systems for fostering and diazotrophs, the nature of plant-microbial interactions, the

origin of diazotrophs, and their recruitment, the possibility that endophytic Fusarium species

may inhibit diazotrophs, and the effects of farming practices on the partnerships. The third part

describes N2 efficient landraces with brace roots that culture and harvest diazotrophs in their

mucigel and some results from breeding with these landraces. An optimal breeding program for

N-efficiency/N2 fixation is described. Finally, the results of the review are synthesized into an

overall summary of the relationships between maize plants and diazotrophs.

Evidence is presented that the capacity to partner with microbes for greater N efficiency and N2

fixation has existed in maize landraces and in wild maize (teosinte), probably for millennia. This

knowledge should be utilized to help farmers reduce fertilizer use as soon as possible. Therefore,

it is hoped that this review will attract attention and funding to the task of breeding modern

maize cultivars that optimize partnerships with bacteria for accomplishing this task. Areas where

more research is needed are identified at the end of this review.

Historical context: During the preceding century, the interest in N2 fixation by maize/bacterial

associations was stimulated by three developments.

The first was the pioneering work of J. Doebereiner and her colleagues in Brazil on the N2

fixation that occurred in the roots of grasses/cereals with the help of bacteria. The major

accomplishments of that work, which began around 1953 (Baldani and Baldani 2005) were:

1) The discovery of new diazotrophic bacteria. These were a) free-living, diazatrophic

bacteria in the rhizosphere (Beijerinckia fluminensis and Azotobacter paspali); b)

associative diazotrophs colonizing the rhizoplane (Azospirillum lipoferum, A. brasilense,

A. amazonense); and c) endophytic diazotrophs having the ability to live inside the plant

(Herbaspirillum seropedicae, H. rubrisubalbicans, Gluconacetobacter diazotrophicus,

Burkholderia brasilensis and B. tropica).

2) A better understanding of the ecology, physiology, biochemistry, and genetics of these

diazotrophs, including their mechanisms of colonization and infection of the plant tissues.

3) Quantification of N2 fixation.

4) The discovery that endophytic bacteria have a much greater potential for N2 fixation than

associative diazotrophs.

5) The discovery that the plant genotype strongly affects the plant/bacteria association.

In Brazil, N2 fixation associated with an endophytic bacteria (G. diazatrophicus) was found to be

a major factor contributing to the N economy of sugarcane, fixing 60 to 80% of the plant’s total

N and obviating the need for N fertilizer (Boddey et al.1991).

A second, social and environmental, development contributing to interest in N2 fixation in

cereals in the preceding century in the USA was the growing realization that the widespread use

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of mineral N fertilizers is environmentally unsustainable and energetically expensive. This

debacle was probably argued most clearly by Commoner and his colleagues at Washington

University (Commoner 1971, 1976, and 1977; Kohl et al.1971). There was a growing realization

that N fertilizer not only ensured high yields, it also led to substantial pollution of ground and

surface waters and to hypoxia in the Gulf of Mexico (Goldstein et al.1996; Liu et al.1997; Nolan,

et al. 1998; Burkart and James 1999).

A third development was new technology that made research on microbial N2 fixation easier.

These included the acetylene reduction assay (ARA) which allowed microbial nitrogenase (the

enzyme that fixes N2) to be measured relatively easily (Hardy et al.1968), and growth media

which allowed the culturing of N2 fixing microorganisms that lived under low oxygen conditions

in the root zone (Baldani and Baldani 2005). Around the turn of the millennium new ‘-omics’

methods also became available which allowed identification and quantification of microbial

species living with plants and their physiology without needing to resort to culturing them. The

application of these methods continues to broaden understanding and reveal details of

plant/microbial ecology and dynamics.

The consequences of these developments set the stage for scientists to consider whether

microbial N2 fixation could also be effective in replacing N fertilizer for maize. In fact, in 1996

Triplett stated that “The development of nitrogen fixation in maize can be considered the holy

grail of nitrogen fixation research.” The term ‘Holy Grail’ has been reused since then ([EUFIC],

1999; Arnason 2013; Vezina 2013), and most recently by Monsanto (Stephens 2015), but the

term has changed to mean pursuing transgenic modification of cereals to fix N2 or otherwise

increase N efficiency.

Present efforts to develop N efficiency: In the preceding four decades total cereal production

tripled but use of N fertilizers increased from 12 to 104 Tg per year (Mulvaney et al.2009). Most

of those fertilizers are in the form of ammonia and they are often used formulaically to ensure

high crop yields irrespective of actual need.

Since the turn of the millennium awareness of the negative impacts of synthetic N fertilizers has

grown to include negative impacts of nitrogen fertilizer on reducing soil organic matter and

nitrogen content (Mulvaney et al. 2009) and on increasing nitrous oxide, a potent greenhouse gas

increasing global warming.

In April 2011, an international conference on Nitrogen and Global Change was held in

Edinburgh, Scotland, and was attended by 350 scientists, policymakers, and industry and NGO

representatives (NitroEurope 2011). The massive impacts of nitrogen fertilizer on health and

ecology were discussed. The resulting Edinburgh Declaration on Reactive Nitrogen stated that:

“An overall strategy to reduce the losses and adverse impacts of reactive nitrogen on society

should be focused on improving nitrogen use efficiency, particularly in agriculture, which can

provide significant financial benefits to farmers and society as a whole.”

There are also clear financial grounds for improving nitrogen efficiency. It has been estimated

that nitrogen fixation saves the Brazilian sugarcane industry $1 billion per year in costs of N

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fertilizer (Arnason 2014). According to Birger (2011) in the U.S.A. the major seed breeding

conglomerates Pioneer Hi-Bred, Monsanto, and Syngenta are engaged in a costly race to develop

‘super corn’ that is highly N-efficient. Dow-Agroscience is pursuing a similar track together

with Arcadia Biotechnologies (Arcadia 2015). Obviously, a patentable trait that would help

farmers reduce their fertilizer bill would be compelling and would quickly lead to market

dominance. Recent advances in genomic and metabolomic methods enable greater dissection of

traits (Boddu 2011). A prime target for these companies are novel genes affecting nitrogen use

efficiency (Moose and Below 2008; McAllister et al. 2012).

Are transgenics the solution? According to McAllister et al. (2012), the response of crops to N

has plateaued. Bioengineering crops for nitrogen use efficiency has become rolled into the

concept of a second green revolution (McAllister et al. 2012). The Improved Maize for African

Soils (IMAS) project involves a partnership between the international maize center (CIMMYT),

the Kenya Agricultural Research Institute, the South African Agricultural Research Council, and

the DuPont Pioneer Company ([CIMMYT] 2012). This 10 year project, which started in 2010

and costs $19.5 million, is taking conventional and transgenic approaches to breeding N-efficient

maize (Gilbert, 2014). It is funded by the Bill and Melinda Gates Foundation and USAID.

The Gates Foundation met with a group of researchers in 2011 to discuss ways of reducing the

need for N fertilizer application to cereals by increasing biological N2 fixation (Beatty and Good

2013; Arnason 2014). Three strategies were discussed: 1) Bioengineering nodules and plant-

microbe signaling pathways into cereal crops from legumes; 2) Understanding and improving

relationships between endophytic nitrogen fixing bacteria and cereals and developing

biofertilizers with these bacteria; 3) Bioengineering nitrogenase into cereal crop organelles such

as chloroplasts or mitochondria. The Gates Foundation chose to fund the first option through

joint funding projects with the US National Science Foundation and by funding the Engineering

Nitrogen Symbiosis for Africa (ENSA) project.

Monsanto is also engaged in a major parallel project to bioengineer N2 fixation into maize

(Stephens 2015). Furthermore, the Danforth Center, a non-profit organization funded by

Monsanto, is part of the ENSA team.

In the past there was a brief interest in producing nodules in cereals based on observation that

application of 2,4-D or other auxin-like substances could induce tumor formation in roots and

that N fixing bacteria could actively colonize such sites and fix N2 (Kennedy and Tchan1992,

Kennedy 1994, Kennedy et al. 1997). But at the recent 19th

International Congress on N2

fixation, many contributions from significant microbial labs around the world were focused on

legume/microbe signaling and nodule dynamics. The ultimate objective is to transfer legume

genetic information into cereals so they can fix N2. This objective was conceived as a long-term

project equivalent to travelling to the moon. In fact, the thematic logo shown repeatedly to the

conference attendees was of astronauts climbing up a rope to the moon. That parallels a

generally held agreement that this approach is a long-term (decades-long) endeavor that may or

may not succeed (Arnason 2014).

Practical problems with transgenics: In the context of transferring genetic information from

legumes to cereals it is presupposed that the host cereal plant will eventually perform in an

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economically competitive way despite massive multi-genic transformations. However,

preceding empirical results suggest that transgenic modification may result in imbalances leading

to lower fitness and non-competitive yields. With the exception of Bt maize, which protects

plants from insects, there is little evidence for yield gains associated with transgenic crops

despite massive efforts by industry to develop them (Gurian-Sherman 2009) and public relations

efforts to promote that idea. Extensive yield trials in Wisconsin showed that in many cases

(again with the exception of Bt corn), transgenic maize hybrids appear to be associated with

yield reduction (“yield drag”) (Shi et al. 2013) and gene stacking resulted in unpredictable

intergenic interactions that are not additive. Substantial evidence exists that glyphosate tolerant

soybeans have some yield drag (Gurian-Sherman 2009; Ching and Mathews 2015) and the

Roundup Ready transgene is associated with reduced N2 fixation (Hungria et al. 2013).

Between 1987 and 2008 there were 11,275 US approved field trials with transgenics, of which

only 3,022 were not concerned with herbicide or insect resistant traits (Gurian-Sherman 2009).

The success rate for these novel traits, which were intended to cause bacterial, fungal, nematode,

virus, and abiotic stress resistance as well as yield increase has been apparently very low in terms

of commercial releases.

Alan Good, from the University of Alberta, stimulated transgenic research on nitrogen use

efficiency by apparently increasing N efficiency in canola with genes from barley (McAllister et

al. 2012; Vezina 2013; Arnason 2014). He has promoted transgenic technology together with

Eric Rey, founder of Arcadia Biotechnology, a company that markets transgenic products

(Vezina 2013).

“Yet Good, who is still trying to engineer NUE plants in his own lab, has become much more

reserved than Rey in his outlook for nitrogen efficiency in plants. “There have not been any big

developments in NUE technology in the real world,” despite the “huge surge of interest in this

area and a surge of investment,” he told me.

NUE crops are notoriously difficult to work with, he noted. “You only see the [nitrogen-efficient]

phenotype some of the time; it works very well in some environments but not well in others.

There’s a very strong genotype-environment interaction.” And with this sort of biotechnology,

which requires major upfront investment, plants need to perform well in a wide variety of

environments if they’re to be successful commercially.

At the moment, Good contends, most of the companies working on NUE plants are “slamming a

gene in under a set of promoters,” essentially “throwing genes in left, right, and center” to see

what sticks. There are a lot of wasted experiments, a few promising results, and these tend to

fizzle or prove much more difficult to get working consistently in real-world settings than

anticipated. “People are finding out how hard it is.”

As far as all the talk about a second green revolution, Good admitted to being “partners in

crime” with Rey in hyping the new technology. Now, with the benefit of hindsight, Good believes

the fundamental complexity of attempting to fiddle with a plant’s nitrogen metabolism will push

the timeline for NUE plants back considerably past the already-gone predictions of 2010 by

Ridley and others.” Vezina (2013).

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What kind of trait does the world need? The world needs cultivars that need less N fertilizer

inputs and have greater protein production. That can be achieved by projects that breed the

naturally occurring capacity for partnering with microbes for N-efficiency/N2 fixation into

modern, high yielding cultivars. The results of breeding projects do not have to be transgenic to

help humanity and our planet. In fact, a large sector of the public does not want to eat transgenic

food. CIMMYT’s efforts to breed drought resistance and N efficiency in maize appear to be

quicker and more productive using classical breeding methods than efforts using transgenic

technology (Gilbert 2014). Furthermore, promoting microbial partnership and N2 fixation as a

naturally occurring trait freely available in the public domain, rather than promoting the

development of novel, patented transgenes, might prove to be a greater boon for all of humanity

Table 1. A summary of the reviewed research testing N2 fixation in maize.

-38, 1996.

Researcher Year Place

Bacteria

genera Method result Ndfa %

Raju et al. 1972 USA Enterobacter ARA

N'ase activity; difference

in foliage color NABuelow &

Dobereiner 1975 Brazil Azospirillum? ARA

22 x difference in N'ase;

max at flowering NA

Barber et al. 1976 USA Azospirillum ARA

Little N'ase or effect on

plants NA

Rennie 1980 Canada Azospirillum 15N dilution

sugary substrate

increased fixation 13 to 38%

Ela et al. 1982 USA Azotobacter ARA cultivar effect NAAlexander &

Zuberer 1989 USA Azospirillum ARA

Little N'ase or effect of

bacteria on plants NAG. de Salamone

et al. 1996 Argentina Azospirillum 15N dilution cultivar effect up to 58%

El Komy 1998 Egypt Azospirillum 15N dilution

cultivar effect. A.

lipoferum > A. brasilense 0 to 35%Chelius and

Triplett 2000 USA Klebsiella nifH detection

sugary substrate fostered

expression of nifH NA

Riggs et al 2000 USA

multiple

species

growth

parameters

yield increases but no

relief of N defic. NA

Montanez et al 2009 Uruguay

indigenous

bacteria 15N dilution cultivar effect 0 to 33%

Schwartz 2009 Mexico

indigenous

bacteria

15N natural

abundance management effects 0 to 98%

Goldstein 2009 USA Azospirillum

15N natural

abundance cultivar effect 0 to 48%

Yamprai et al. 2014 Thailand

Azotobacter &

Azospirillum

difference and

ARA substantial N fixation 29 to 52%

Araujo et al 2014 Brazil Herbaspirillum NUE cultivar effect 34, 64%

Guimares et al 2014 Brazil Herbaspirillum

growth

parameters little effect NA

Puri 2014 Canada Paenibacillus15

N dilution fixation. 17%

Cavalcanti et al 2015 Brazil Herbaspirillum nat abundance fixation. up to 37%

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and the planet in the long and short term because the results are more freely available to all,

including small, medium, and large sized seed companies.

Where and how does N2 fixation occur in corn? Studies to test N2 fixation in maize are

summarized in Table 1.

Raju et al. (1972) observed that where hybrid Pioneer 3773 grew in Oregon, USA under N

deficient conditions, some plants had unusually dark-green foliage. They took whole plant

samples, measured nitrogenase activity with ARA and found that the darker green plants reduced

10 times more acetylene than lighter green, deficient appearing plants. They consistently

isolated a N2 fixing, gel producing Enterobacter cloacae strain from roots and rhizospheres of

the green, N2 fixing plants. The gel produced by the bacteria was hypothesized to serve as

protection against oxygen and thereby facilitate N2 fixation in the rhizosphere.

Von Bülow and Doebereiner (1975) examined fixation amongst 138 S1 lines of a breeding

population called UR1. Roots were sequentially removed for analysis through a portion of the

growing season without digging up whole plants. Following a pre-incubation period, acetylene

reduction was measured on the roots. They found very large and consistent differences between

the lines for nitrogenase activity over the sampling period. One breeding line had 23 times

higher nitrogenase activity than the control population; those high rates were comparable to

soybeans. A second study by the same authors with a brachytic cultivar (Piranao) showed that

nitrogenase levels were highest at the 75% silk stage and then again at grain fill. Roots that were

3-5 mm in diameter with many laterals were usually the most active. Azospirillum spp. were

isolated from roots that showed nitrogenase activity.

Barber et al. (1976) did greenhouse and field trials in Oregon to test the effects of inoculation

with Azospirillum strains from Brazil on numerous Maize Belt inbreds and hybrids of maize. N2

fixation activity was very low and inoculation did not substantially affect yield or N

accumulation. Application of ammonium nitrate fertilizer eliminated fixation. Fixation rates

obtained with excised roots were higher than those obtained from intact plants measurements.

Incubating excised roots overnight before measuring N2 fixation resulted in a 30 fold increase in

the number of N2 fixing bacteria. The authors concluded that the consequences of pre-incubation

(similar to that used by von Buelow and Doebereiner (1976)) were artificially high estimates of

N2 fixation using the acetylene reduction method. Tjepkema and van Berkum (1977) had similar

results in Brazil with ARA measurements of roots and core samples.

De-Polli et al. (1982) grew maize in New Jersey, including some of the same inbreds that Barber

had grown as well as other hybrids, and obtained strikingly different results. They found

significant nitrogenase activity on intact plants, root-soil cores, lower stem segments, and

excised roots. Excised roots showed the lowest activity. Neither reduced oxygen levels nor pre-

incubation were necessary to obtain acetylene reduction. There were no significant differences

in activity between inbreds or hybrids. Whole plants had a diurnal rhythm of fixation with

activity almost ceasing at night. This rhythm was not apparent with detached roots and appeared

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to not be caused by differences in daytime and night-time temperatures. Bacteria were isolated

from roots and lower stems with morphology similar to Azospirillum spp. These results provided

strong evidence for the occurrence of nitrogenase activity under temperate conditions with

indigenous N2-fixing bacteria.

Rennie (1980) studied N2 fixation in an in vitro system using the classical difference method

(based on N determination) and the 15

N isotope dilution method. Both methods could accurately

measure N2 fixation at 13%. If sucrose, malate or succinate were added to the growing media

the N2 fixed increased to 19%, 28% and 38% of the total N in the plant, respectively.

Alexander and Zuberer (1989) measured N2 fixation in the Maize Belt hybrid B73 x Mo17

grown under greenhouse conditions. In one set of experiments the plants were grown in artificial

soil then transferred to hydroponic solutions. In the solutions the microbes in roots fixed N2 at

low levels. Fixation was 200 times higher at a partial oxygen pressure pO2 of 2 versus 10. At

the lower oxygen level, fixation increased 10 fold if malate was added to the solution. However

the high rate found with malate was equivalent to the normal rate found if the plants were

allowed to grow undisturbed in artificial soil. 15

N2 trials showed uptake of N by fixation in roots

and later mobilization to shoots. Inoculation with Azospirillum lipoferum did not increase dry

matter content nor N content of the roots.

Ela et al. (1982) in Wisconsin, grew 1450 maize plants from 180 seed lots that had been

inoculated with Azotobacter vinelandii OP, a free-living N fixer, and screened them for

nitrogenase activity. Plants were grown in pots with vermiculite or sand media and fertilized

with N-deficient nutrient solution. Though conventional Maize Belt inbreds did not show

nitrogenase activity, a few maize varieties from South or Central America harbored nitrogenase

activity. 15

N was incorporated into the plants, proving that N2 fixation was occurring. The South

American maize was crossed with the Wisconsin inbred W22 and over several cycles of

selection a line was bred with 50x enhanced nitrogenase activity, but still with much lower

nitrogenase activity than for soybeans.

It did not matter whether inoculation was with Azotobacter vinelandii, Raju et al.’s (1972)

Enterobacter cloacae strain, a Klebsiella pneumoniae strain, or later with Azospirillum spp.

(Brill, 1983). All were effective at promoting nitrogenase expression with this original maize or

breeding lines developed from it. This indicates some level of promiscuity on behalf of the

active plants as these bacteria are primarily thought to inhabit different niches: Azotobacter

vinelandii (rhizosphere), Klebsiella pneumoniae (endophyte), Azospirillum spp. (rhizoplane).

Further observations from the Wisconsin researchers (Brill, 1982, 1984) on their research, given

in subsequent summary reports to the US Department of Energy without numerical data, were: 1)

Fixation levels plateaued after 5 generations of selection. 2) Genetic control of the trait by the

plant was complex rather than single gene. 3) Fixation occurred mostly in areas of young,

rapidly growing roots, and root diameter in active plants was smaller than for inactive plants; 4)

The root mucigel was the site of active growth of many unidentified bacteria species possessing

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a halo-zone around them in electron microscope investigations. 5) Microscopic and monoclonal

antibody studies revealed Azospirillum-like organisms in the mucigel on roots but most-

probable-number determinations of Azospirillum-like bacteria showed no significant difference

in numbers on active or inactive roots. 6) Two groups of unidentified microorganisms were

isolated on a semi-solid selective media from active roots; none of them were the inoculated

species mentioned above. 7) Combining strains from those two groups were necessary for in

vitro expression of nitrogenase. 8) Furthermore, it was not possible to grow sterile plants;

despite doing surface sterilization of seed, bacteria of the genera Bacillus, Pseudomonas, and

Arthrobacter were isolated from roots; these were apparently seed-borne endophytes. 9)

Attempts to suppress these bacteria with antibiotics had negative effects on plant development,

even causing chlorosis. 10) Anaerobic conditions in pots did not increase nitrogenase activity.

11) Fixation also occurred under hydroponic conditions. 12) Breeding lines that were

nitrogenase-active or inactive in a pot system had the same performance under hydroponic

conditions. 13) Under the hydroponic conditions, nitrogenase activity was higher at 2% oxygen

rather than 20% oxygen. 14) When active plants were grown together with non-active plants,

nitrogenase was not active, suggesting that rhizosphere effects or exudates from neighboring

plants affected the process (Brill, 1982).

Chelius and Triplett (2000) studied the endophyte Klebsiella pneumoniae found in maize inbred

Mo17 in the context of a greenhouse in vitro system. This endophyte was localized in the inter-

cortical regions of the stem, in the zone of maturation of root hairs, residing on the epidermis and

growing into the cortex of the roots. Manipulation of plant tissues was found to decrease

colonization in plant stems. Dinitrogenase reductase (nifH), an enzyme that is part of the

nitrogenase system, was expressed if the bacteria were supplied with 0.1% sucrose in the agar

medium.

Inoculation studies with diazotrophs and their effects on N2 fixation: Okon and Kapulnick

(1986) followed the effects of inoculation of maize with Azospirillum brasilense on the

development of root systems. Initially, the bacteria were found around root hairs, later in the

cortical tissues, including the inner cortex and xylem vessels and zones of secondary root

formation. Inoculation increased root area and root length, the number of lateral roots, and

increased nutrient uptake. Okon (1994) reviewed an extensive literature on Azospirillum spp.

and concluded that many of the benefits of the species are due to stimulation of root growth, in

part associated with the production of auxin. Trials on 13 different sites (mostly in the USA)

with different levels of N fertilization showed that yield responses to inoculation with A.

brasilense mostly occurred on lighter-textured soils with lower rates of fertilization (Fallik and

Okon 1996). Large increases in yield due to inoculation with A. brasilense (average

approximately 30%) were found in large-scale trials in different states in Mexico over a two year

period where no N fertilizer or low rates were applied. There were much lower responses where

high rates of N were applied (Dobbelaere et al. 2001, Fuentes-Ramirez and Caballero-Mellado

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2005). Effects of inoculation on the yields of maize were larger where maize landraces were

used instead of hybrids and where maize was grown on poor, light-textured soils.

In Argentina, field trials were carried out with Azospirillum inoculants on different cultivars of

maize from Argentina and Brazil (Garcia de Salamone and Doebereiner 1996). Inoculation

significantly affected yield, total nitrogen content, and nitrate reductase activity, but there were

large differences in response between different cultivars. Parallel experiments tested the effects

of these Azospirillum species and their nitrate reductase negative mutants (NR-) on four cultivars

of maize in pot experiments (Garcia de Salamone et al.1996). Two of the hybrids responded

neither to inoculation nor fertilizer. The two other hybrids had similar responses to inoculation

as they had to fertilization with 100 kg N ha -1

. Inoculation strongly increased their N

accumulation and there was no difference whether the bacteria was NR- or normal. Furthermore,

even though there were difficulties with stabilizing 15

N enrichment in the soil, the results with 15

N dilution showed very significant fixation (up to 58% Ndfa) for the responsive cultivars.

In Egypt, El Komy et al. (1998) studied the effects of different rates of farmyard manure and

inoculation with two different strain of Azospirillum lipoferum and one strain of A. brasilense on

the growth of two cultivars of maize. They utilized 15

N dilution to estimate %Ndfa. N2 fixation

caused by inoculation varied from 6% to 31% where no manure was applied, 5% to 35% where 5

t/ha was applied, and -15% to -2% where 10 t/ha were applied. Inoculation with A. lipoferum

resulted in consistently greater fixation than did A. brasilense, and one cultivar fixed more than

the other.

In Thailand, Yamprai et al. (2014) carried out pot studies on the effects of inoculating a sweet

maize cultivar (Suwan 5) with Azotobacter croococcum and Azospirillum lipoferum versus an

un-inoculated control. Differences between initial and final total soil N and plant N indicated

that substantial N2 fixation had occurred due to inoculation with both microbes relative to

uninoculated pots. There was 47% more N in the soil and 29% more N in plants with A.

croococcum and 24% more N in the soil and 52% more N in plants with A. lipoferum than in the

control pots where no inoculum was used. Fixation estimates based on the difference method

correlated with ARA measurements of nitrogenase. There was no apparent difference in rates of

fixation associated with time of sampling in the day.

Roncato-Maccari et al. (2003) modified the nifH gene of a strain of the endophytic, diazotrophic

bacteria Herbaspirillum seropedicae to express the gus A-kanamycin reporter system. Using

histochemical methods they could visualize the expression of the nifH gene when

Herbaspirillum grew in or on the tissues of plants. In the first days following inoculation, the

bacteria grew in sites of active root exudation such as on root surfaces and in zones where

secondary roots were formed. Subsequently, it colonized the root cortex, parenchyma and xylem

of the stem. The bacteria expressed the nifH gene.

In a greenhouse trial, the effects of inoculating three hybrids with the diazotrophic bacteria H.

seropedicae were assessed under different rates of fertilization with N (Araujo et al. 2014a).

Inoculation coupled with fertilization increased shoot N contents 32% and 62% for the hybrids

P3646H and BRS3035, respectively. Without any fertilization, N use efficiency was increased by

12

34% and 64%, for the two respective hybrids. The volume of roots, root length, dry weight of

the shoot, and chlorophyll content were also increased. Treatment with the strain Z-94 of H.

seropedicae plus 80 kg N ha -1

raised the N content of the tops by up to 25%.

In another set of greenhouse inoculation trials with A. brasilense (AbV5) and H. seropedicae

(SmR1), no effects of inoculation were noted on the morphology, productivity, or chlorophyll

content of DeKalb (Monsanto) hybrid ‘DKB 390', grown until 30 days after emergence

(Guimares et al. 2014).

Fernando de Araujo et al. (2014) screened 35 different Brazilian cultivars for their response to H.

seropedicae inoculation. Only nine of the commercial hybrids responded with increases in dry

matter production or N content.

Different strains of the H. seropedicae were tested for their effects on maize (Cavalcanti et al.

2015). N2 fixation was assessed using natural abundance. Strain ZAE94 produced the best

results under controlled conditions. Field inoculation with this strain increased maize yield up to

34%, depending on the plant genotype, and fixed 37% of the N in the hybrid SHS5050.

It is possible to establish the endophyte Gluconacetobacter diazotrophicus, which is responsible

for N2 fixation in sugarcane, in maize cultivars (Tian 2009). Maize cultivars differ in their

susceptibility. Inter-cellular colonization occurs at low rates and is positively affected by the

sugar content of tissues. Furthermore, colonization was not associated with measurable N2

fixation (Eskin 2012). However, Cocking et al. (2005) have inoculated with G. diazotrophicus

and found them present inside meristematic root cells of maize and other cereals. They found

that the bacteria are localized in intracellular vesicles and express the nifH gene.

Riggs et al. (2001) studied the effects of inoculation with different strains of diazotrophic

bacteria (Klebsiella pneumoniae, Pantoea agglomerans, Gluconacetobacter diazotrophicus, and

Herbaspirillum seropedicae) on the productivity of maize grown with different levels of N

fertilizer in five states in the Midwestern USA. Though significant increases in yield were

obtained none of the inoculations could relieve the N deficiency status associated with non-

fertilized conditions.

These inoculation studies have been predominantly carried out with single species diazotrophs.

However, inoculation of other grass species with consortia of organisms conveying different

services and occupying different niches has at times been provided more stable and larger

responses to inoculation by promoting endophytic colonization, plant productivity, and N2

fixation (Zlotnikov et al. 1998; Minamisawa et al. 2004; Minamisawa et al. 2005; Raja et al.

2006; Oliveira et al. 2009). Research on endophytes occurring in wild rice, wild sorghum, and

other grasses in many Asian countries have shown that Clostridia spp. present as obligate

anaerobes can fix N even under aerobic conditions if they are grown together with certain

aerobic endophytes that are non-fixers (Enterobacter cloacae, Bacillus, Burkholderia, and

Microbacterium sp. often in multiple combinations). Similar partnerships were reported earlier

(Brill, 1984; Zlotnikov et al.1998).

13

N2 fixation by indigeneous diazotrophs: The 15

N dilution method was employed to estimate N2

fixation in ten maize cultivars commonly grown in Uruguay (Montañez et al.2009). Plants were

grown under controlled conditions in poor sandy soils with additions of two different levels of

the 15

N fertilizer. The cultivar with the highest uptake of tracers was used as a reference for

calculating the % Ndfa. Fixation ranged from 12 to 33%. Eleven diazotrophic endophytes were

also extracted from seed, root, stem, and leaf tissues of 19 cultivars using selective media. These

were closely related to the genera Pantoea, Pseudomonas, Rahnella, Herbaspirillum,

Azospirillum, Rhizobium and Brevundimonas.

In a second study, endophytic species were described that produce IAA, nifH, and siderophores,

and had phosphate solubilizing abilities (Montañez et al. 2012). Diazotrophs were in the genera

Rahnella, Pantoea, Rhizobium, Pseudomona, Herbaspirillum, Enterobacter, Brevundimonas and

Burkholderia. Inoculation trials showed positive effects of ten of the strains on shoot growth but

little positive effect on root growth. There was a positive interaction between strains and maize

cultivars for both shoot and root dry weight.

Problems measuring N2 fixation: Experiments have utilized several different methods, and in

many of the cases reviewed here there is an indication of substantial N2 fixation and in some

cases there is not. A confounding factor is that the different methods employed by different

authors produce artefactual conditions that distort measurements of N2 fixation.

It is not unreasonable to expect that excavation and washing of maize roots would have greater

effects on reducing nitrogenase activity in the rhizosphere, rhizoplane of maize roots than they

would for legume roots and nodules as the relevant diazotrophs in the cereal system are not

buffered inside nodules. Von Bülow and Doebereiner (1975) compensated for disruption

associated with excavation and washing of the root systems by adding an incubation period

before analyzing excised roots with ARA. However, incubation was criticized for producing

unrealistically high ARA values (Barber et al. (1976); Tjepkema and van Berkum (1977) or low

(De-Polli, et al. 1982) values.

In fact, physical disruption of roots has been demonstrated to drastically reduce N2 fixation (Von

Bülow and Doebereiner 1975; Wani 1983; Chelius and Triplett 2000). This may explain

divergent ARA values obtained by Alexander and Zuberer (1989), where plants were switched

from soil systems to hydroponic systems before measurement of ARA. Wani et al. (1983)

studied the effects of mechanical disturbance on N fixation in sorghum and millet. Mechanical

disturbance associated with putting in place or transporting cores reduced activity significantly.

Where the core with millet was put in place 15 days after planting and removed 56 days after

planting, ARA values were 10 times higher than where cores were both put in place and removed

after 56 days. This calls into question results obtained by Barber et al. (1976) and Tjepkema and

van Berkum (1977), where core samples were taken shortly before measurement of ARA. ARA

sampling is best done on intact, undisturbed plants, or in core samples that are established early

and handled very carefully (Wani et al 1983; Wani 1986; Boddey et al.1998).

Additional complications of using the ARA, which is based on the conversion of acetylene to

ethylene by nitrogenase, are that:1) plants and soils produce ethylene; and 2) soils oxidize

14

ethylene but ethylene oxidation is inhibited by acetylene, causing it to become available during

the ARA. Methods for obviating these complications through check procedures are discussed by

Boddey et al. (1998).

A more recent objection to the method was proposed in a review on N2 fixation by endophytes,

Doty (2011). Many endophytic strains test negative for ARA but can be grown on N-free-media,

and this might be because they have the ability to degrade the ethylene that they produce. This

issue may be resolved in the future by isotopic experiments.

In the studies reviewed here, natural isotope abundance based on the ratios of naturally occurring 15

N and 14

N ratios (δ15

N ratio) was also applied to quantify fixation. Problems with the method

were outlined by Boddey et al. (2001), with special reference to homogeneity of soil exploration

by roots and N isotope uptake. Additional problems are that diazotrophs stimulate root growth

of maize and thereby stimulate N-uptake from soils. Thus a plant-bacterial association that is

fixing N2 may at the same time be taking more N from soil organic matter because of the larger

root system and extra demands associated with greater growth. The net effect would be that

actual increases in 14

N due to fixation of N2 would be balanced out by greater uptake of 15

N from

soil organic matter and there would be no change in the δ15

N ratio. Hence, fixation could be

occurring but would not be detected by the method. A remedy to this problem is to grow the

plants in a very low organic matter soil where N uptake from soil organic matter is minimized.

A further problem with the method is choosing which reference plant to use. The reference

plants chosen may also foster N2 fixation by bacteria. The ideal reference plant is probably a

cultivar of the same species with similar rooting patterns that has demonstrated no or little N2

fixing ability.

The availability of N2 that is fixed by diazotrophs to plant growth is unclear. However

Pankievicz et al. (2015), recently utilized 13

N isotope in a system with Setaria viridis as the host

plant and Azospirillum brasilense and H. seropedicae as associated N2 fixers. They were able to

show highly efficient fixation by the microbes and turnover of N into plant tissues. Efficient

turnover of labelled endophyte protein was also demonstrated in Agave tequilana (Beltran-

Garcia et al. 2014).

Plant physiology: ethylene, N deprivation, aerenchyma: Ethylene buildup signals and

activates several different plant defense systems against microbial pathogens (Ecker and Davis

1987). The establishment of commensal relationships between plants and diazotrophic bacteria

probably depends on low levels of tissue ethylene. The effects of ethylene are mediated through

the APATALA2/Ethylene response factor (Nunez-Pastrana et al. 2013). These are a family of

transcription factors that play a role in the response to pathogens, and also play roles in

mediating relationships with diazotrophs, including Herbaspirillum and Rhizobium, and

including nodule formation in legumes. These response factors integrate cross-talk between

signaling pathways mediated by ethylene, jasmonic acid, salicylic acid, ABA, and cytokinins.

15

Plant microRNAs regulate gene expression post-transcriptionally, and they seem to sense N

deficiency by shifting patterns of metabolic regulation (Xu et al. 2011; Liang et al. 2012).

Another major maize plant response to N deficiency is to make aerenchyma and thereby provide

low-ethylene sites for diazotrophic bacteria. Aerenchyma are normally formed in response to

waterlogged (hypoxic) conditions which enhance the synthesis and accumulation of tissue

ethylene (Yamauchi et al. 2013 ). The ethylene triggers a programmed death response behind

the growing tip of young crown roots. Ethylene signals production of reactive oxygen species

(ROS), including hydrogen peroxide, and the production of enzymes (cellulose, pectinase,

xylanase). Amazingly, specific sets of cortex cells are digested leaving contiguous hollowed out

cell walls in regular spacing around the cortex. The resulting gas-filled, connected, tubular

lacunae connect the atmosphere with the growing roots and enable such roots to live and grown

under anoxic conditions.

Similar lysigenous aerenchyma are also formed in response to deprivations in nitrogen,

phosphorous, and sulfur (Siviannis et al. 2012). Such rooting systems have lower energy

requirements and greater exploratory activity (Postma and Lynch 2011). The creation of

aerenchyma whether under hypoxic or nutrient deficiency conditions appears to be metabolically

similar (Siviannis et al. 2012) except that in the N response: 1) lysigenous aerenchyma are

formed under conditions where there is a strong reduction in ethylene production (Drew, et

al.1989), and 2) the formation of lysigenous aerenchyma stops at the base of the root so there is

no direct connection to the atmosphere (Siyiannis et al. 2012). Ethylene accumulation under N

deficient conditions is one-half to one-third that of roots grown under normal, non-hypoxic

conditions, and there are parallel reductions in the activity of ethylene forming enzymes (Drew et

al.1989, 2000). Under N deficient conditions the root tissues become sensitized to ethylene; and

the formation of aerenchyma proceeds as a response to lower doses of endogenous ethylene (He

et al.1992). Responses to P deficiency also involve some decrease in ethylene production and

sensitization of tissues to ethylene, but these responses are considerably less than for N

deficiency (Drew et al.1989, 2000; He et al.1992).

Furthermore, localization studies have shown that aerenchyma provide sites for colonization by

diazotrophs including Azoarcus sp. in Kallar grass (Reinhold and Hurek 1987) and in rice

(Egener et al.1999), and Enterobacter gergoviae in maize (An et al. 2007).

The nature of the host-bacteria relationship: The composition of root exudates and the

lipopolysaccharide composition of cell walls of bacteria are part of the dialogue between the

plant and the bacteria that leads to and fosters colonization (Rosenblueth and Martinez-Romera,

2006). Inoculation with Azospirillum brasilense was shown to reduce the superoxide

concentration of the roots by 30%, and this reduction in oxidative stress defenses was caused by

the cell wall composition of the bacteria (Mendez-Gomez et al. 2015). Transcriptome studies of

the infection of rice with Herbaspirillum seropedicae (Brusamarello et al. 2011) have shown that

inoculation with the bacteria represses production of plant defense proteins (thionin and PBZ1)

and controls expression of auxin and ethylene-responsive genes.

16

Flavonoids in the exudates appear to increase colonization by free-living diazotrophs and

inoculation with Azospirillum sp. has been shown to induce plant production of flavonoids (Santi

et al. 2013). The composition of maize root exudates changed when plants were inoculated with

Herbaspirillum seropedicae, with and without humic acids (da Silva Lima et al. 2014). Plants

with the Herbaspirillum produced lower quantities of overall exudate, but higher contents of

carbohydrates, heterocyclic N compounds, steroids, terpene precursors of gibberellic acid and

lower quantities of fatty acids. The auxin, gibberellic acid, and nitric oxide produced by the

bacteria cause greater plant growth (Dobbelaere et al. 2001; Di Palma et al. 2011).

Mandimba et al. (1986) isolated Enterobacter cloacae and Azospirillum lipoferum from maize

and rice. Strains isolated from maize were more strongly attracted to maize mucigel than were

strains of the same species isolated from rice or the E.coli control. These results suggest that

host-strain specific partnerships had developed between maize and these bacteria.

Genome sequencing of A. brasilense and A. lipoferum (Wisniewski-Dyé et al. 2012) showed that

genes involved in adaptation to specific niches (colonizing roots, promoting plant growth with

phytohormones, taking up iron) are specific to different strains and species. Chamam et al.

(2012) tested the effects of two Azospirillum strains isolated from two different rice cultivars.

The strains had their strongest growth promoting effects on the rice cultivar they were selected

from. One of the strains was a rhizoplane inhabitant and the other was more of an endophyte.

The rhizoplane inhabiting strain had stronger effects on plant secondary metabolism of the

cultivar it was isolated from. Further studies of the transcriptome of inoculated and non-

inoculated rice (Drogue et al. 2014) showed that 16% of the genes in the rice genome were

affected by inoculation, but of those genes affected 83% of them were affected in a combination

specific manner. This included genes affecting ethylene signaling and auxin production. These

results suggest significant co-evolution and fine-tuning of regulation and metabolism has

occurred between host-and bacteria.

On the other hand, diazotrophs do not have to be isolated from maize to have a beneficial effect

on maize. Sharon Doty’s research team at the University of Washington in Seattle isolated

diazotrophs from willow (Salix sitchensis) and Poplar (Populus trichocarpa) (Khan, et al.2012;

Knoth et al. 2012). These included Rahnella sp ., Rhizobium tropici, Sphingomonas yanoikuye,

and the yeast species Rhodotorula graminis and Rhodotorula mucilaginosa . These species were

tested for their effects on crops individually and as consortia. Following inoculation, the poplar

endophytes were found present in root stem and leaf tissue and especially in vascular tissues

(Knoth et al. 2012) of maize plants. Inoculation had variable effects but appeared to increase

early growth of roots and shoots and to increase total dry matter production. It also increased N

content of shoots and the rate of CO2 assimilation.

Furthermore, Puri (2014) inoculated maize with a Paenibacillus polymyxa strain isolated from

lodgepole pine from British Columbia, Canada. P. polymyxa colonized the rhizosphere (5.52 x

106 cfu/g dry root) and internal tissues (4.54 x 105 cfu/g fresh weight) and increased root length

by 35% and biomass by 31% by 30 days after inoculation. A15

N dilution study showed that the

inoculated seedlings derived 17% of foliar nitrogen from the atmosphere, 30 days after

inoculation.

17

Maize has been traditionally grown together with beans (Phaseolus vulgaris) by native cultures.

Rhizobium etli is the main symbiont in beans. It colonizes the rhizosphere and lives as an

endophyte in maize roots. Several Mexican landraces were tested for their ability to form

endophytic relationships with this bacterium (Gutierrez-Zamora and Martinez-Romero 2001).

N2 fixation was not detected using the ARA. The growth of Puebla 162 (maize landrace

Cacahuacintle) was strongly affected by inoculation with some strains of the bacteria. The

presence of this bacterium as an endophyte in maize roots is less if the maize is grown in

monoculture than if co-cropped with beans. Furthermore, the strains isolated from maize grown

in monoculture had lost the ability to fix N2.

Where do the diazotrophic bacteria come from? Diazotrophic bacteria in the plant and

rhizosphere come from the seed or are recruited from the soil. The rhizosphere is a competitive

and selective site with strong recruitment for certain bacteria. Microorganisms inhabiting the

rhizosphere of maize (hybrid Pioneer 34B43 grown with normal fertilization in Illinois, USA)

have been studied using genetic microarrays (Li et al. 2014). The rhizosphere was naturally

enrichened with organisms that express 47% more nifH than the bulk soil. Most of the nifH is

produced by uncultured microorganisms, suggesting that many N2-fixing microorganisms are yet

unknown. Microbial N2 fixation, ammonification, denitrification, and N reduction were all

higher in the rhizosphere than in the bulk soil. The genes responsible for cycling C, N, P, and S

were attributed to the phyla Proteobacteria and Actinobacteria, and especially to the genera

Bradyrhizobium, Pseudomonas, Rhodopseudomonas, and Mycobacterium. The authors

surmised that microbial competition is intense in the rhizosphere because there is high

abundance of antibiotic resistance genes there and resistance may convey colonization

advantages. The genera Burkholderia, Mycobacterium, Bacillus, and Yersinia were abundant

antibiotic producers in the rhizosphere, and they may thereby provide the plant with protection

from pathogens.

Peiffer et al. (2013) examined the rhizosphere composition of 27 different inbred lines grown on

five sites in three states in the USA by pyrosequencing of bacterial 16S rRNA genes from both

bulk soils and rhizospheres. There was a great deal of variation in microbial composition

between the bulk soil and the rhizosphere and between sites. There was a small, significant

proportion of heritable variation in total diversity across fields and between replicates.

Seed inherited endophytes: Johnston-Monje and Raizada (2011) investigated the endophyte

populations found in the seed, roots, and stems of four teosinte species, 10 landraces and a

modern cultivar by culturing and non-culturing approaches using terminal-restriction-length-

polymorphisms (TRFLP) found in 16S rDNA. TRFLP signals showed that a core set of

Clostridium and Paenibacillus species were conserved in all groups. Culturing studies showed

that the γ-proteaceae were prevalent with widespread representation from Methylobacteria,

Pantoea, Enterobacter and Pseudomonas species. Two thirds of the endophytes that would

grow on N-free media belonged to the latter two genera. Enterobacter and Pantoea were the

most common genera. Endophytes demonstrated variable potential for multiple physiological

functions such as the abilities to stimulate plant growth, grow on N deficient media, solubilize

phosphate, sequester iron, secrete RNase, antagonize pathogens, breakdown the precursor of

ethylene, and produce acetoin/butanediol and auxin. Enterobacter asburiae was found to be able

18

to have the ability to leave roots and colonize the rhizosphere. A second study (Johnston-Monje

et al. 2014) found that some of the endophytes found in plants of wild corn (teosinte), a Mexican

Landrace, and a Pioneer hybrid were vertically transmitted from one generation to another

through seed tissues.

Endophytes are present even during seed development. A study of the endophytic populations in

reciprocal hybrids using molecular techniques showed that there was an apparent dynamic in

microbial populations from the pro-embryo phase to the hard dough phase with Undibacterium

sp. predominating early and Burkholderia and Pantoae spp. predominating at later phases (Liu et

al. 2013).

Liu et al. (2012) investigated the endophytes in seed of four hybrids and their parents by building

non-culture 16S rDNA libraries. The endophytes found differed between inbreds. They

included species in the genera Pantoea, Sphingomonas, Stenotrophomonas, Acinetobacter,

Pseudomonas, Leclercia, and Enterobacter. In some cases, hybrids had lower diversity than

their inbred parents. However, the dominant endophyte genera in the parental species were

conserved in the seed of their offspring hybrids. Results did not disprove that there might be an

endophytic contribution from the male parent.

However, a closer look at maize endophytes in Parana, Brazil (Ikeda et al.2013), suggests that

their origin is complex, and strongly affected by hybridization. Endophytes were studied in four

inbreds and three hybrids made with crossing those inbreds. Endophytes were isolated in four

different N-free culture media from roots of inbreds and hybrids. The 217 strains that were

isolated were reduced to 98 based on morphological commonalities. Biochemical characteristics

and BOX-PCR showed great variation between the remaining isolates. The 16S rRNA gene was

sequenced for a representative set of 35 isolates. The bacteria belonged to the genera Pantoea,

Bacillus, Burkholderia, and Klebsiella. None of the strains found in the inbreds were present in

any of the hybrids. Moreover, there were qualitative differences in the generic composition of

different breeding lineages and hybrids. For example, inbred LA possessed Bacillus,

Burkholderia, and Pantoea species; inbred LC possessed Bacillus and Klebsiella spp. The cross

LA x LC possessed only Pantoea spp.

Adding to the complexity of interpreting the origin of the endophytes from generation to

generation is the fact that bacteria routinely generate new, heritable, epi-genotypes especially

under stress conditions (Casadesús and Low 2013).

It could be that heterosis (hybrid vigor) in maize strongly affects endophyte populations. Picard

et al. (2004) and Picard and Bosco (2005) also found that heterosis in maize strongly influences

the composition of rhizosphere-inhabiting Pseudomonads. Relative to their parent inbreds,

hybrids had greater Pseudomonad populations that were more diverse. These strains could

provide greater benefits to the host plant including antibiotic production against root pathogens

and greater production of auxin.

Fusarium infestation as a gatekeeper for maize/diazotroph relationships: Some fungal

endophyte Fusarium species are both mutualistic and latently pathogenic (Kuldau and Yates

19

2000). Races of F. verticillioides and other Fusarium live as symptomless endophytes in corn;

these races can pass from generation to generation by seed (Wilke et al. 2007) or infection can

occur through environmental inoculation. The F. verticillioides found in maize has a high level

of genomic polymorphism; it appears to co-evolve together with different maize breeding

families and inbred lines (Pamphile and Azevedo 2002). Races of Fusarium spp. range in their

pathogenicity, but they can produce mycotoxins in grain and silage. Such Fusarium include the

Gibberella fujikuroi teliomorph species complex (especially F. verticillioides (=F. moniliforme),

F. proliferatum, and F. subglutinans), which can cause root, stalk, and Fusarium ear rot and the

F. graminearum species complex (teleomorph, G. zeae), which can cause Gibberella stalk rot

and Gibberella ear rot (Moretti 2009; Mesterhazy et al. 2012). Under alternating drought/wet

conditions or borer pressure F. verticillioides produces toxic fumonisins and under warm, wet

conditions, F.graminearum (Gibberella zea) produces toxic deoxynivalenol (Koenning and

Payne 2012) in both silage and grain.

It is hypothesized that systemic infestation by Fusarium endophytes may be a partial reason why

Corn Belt maize does not fix N2 nor respond to inoculation with diazotrophs. The establishment

of dominating Fusarium races may be an indirect effect of selection by breeders under N-

fertilized conditions for the following positive attributes:

1) Selection for stiff stalks and strong roots. Non-symptomatic F. verticillioides increases the

stiffness of stalks through lignification of vascular tissue and stimulates root growth (Yates et al.

1997).

2) Selection for resistance to other diseases. F. verticillioides endophytes probably protect

maize plants from common smut fungi (Ustilago maydis) (Lee et al. 2009). Fusarium produced

fusaric acid also reduces the diversity of other fungal endophytes (Saunders et al. 2010).

3) Selection for greater grain yield: Yates et al. (2005) tested yields under fertilized conditions

in south Georgia during 1997, 1998, and 1999 and concluded that: “Yield and vegetative growth

of mature maize plants grown from F. verticillioides-inoculated seed were equal to or greater

than plants grown from non-inoculated seed.” There was a yield advantage for maize inoculated

with one of the fungal strains in 1998 under growing conditions with severe heat stress. It is

conceivable that a breeder would select for maize that fosters this strain because it is more stress

resistant.

4) Selection for resistance to maize borers and root worms. Maize has been selected to produce

high levels of toxic compounds called benzoxazinoids in order to chemically control first

generation European Maize Borers (Reid 1988) and Western Maize Rootworm (Xie et al.1992).

Fusarium (especially F. verticillioides) thrive as endophytes in the presence of benzoxazinoids

as they can tolerate and metabolize them (Glenn et al. 2001; Saunders and Kohn 2009). The

presence of these compounds results in up to 35x higher levels of Fusarium in tops and roots

(Saunders and Kohn 2009). The secretion of benzoxazinoids into the rhizosphere also has the

benefit of attracting beneficial pseudomonads which may control other plant pathogens (Neal et

al. 2012).

5) Benefits of extra gibberellins. Gibberellin hormones can be produced by G. fujikuroi and F.

verticillioides (Yabuta et al.1934, Rangaswamy and Balu 2010) and may account for some

effects of asymptomatic infections (Yates et al.1997). Breeders might inadvertently select for

plants that exhibit a phenotype caused by extra gibberellin production irrespective of whether the

gibberellin was produced by the plant or the Fusarium. Extra gibberellins can increase vigor, the

20

numbers of crown roots, suppress tillering, and reduce both tassel branching (Nickerson and

Embler 1960) and the expression of male inflorescences (Rood and Pharis 1980). All these traits

are sought after by breeders as improved agronomic characteristics.

Negative interactions and effects for Fusarium: Aside from these positive services, F.

verticillioides also has negative effects on the health and ecology of the maize plant:

1) It strongly reduces the protein and chlorophyll content of the shoot, reducing photosynthetic

potential and producing overall symptoms of N deficiency (Pinto et al.2000; Rodriguez-Brljevich

et al. 2010; Ketabchi and Shahrtash 2011). This may be due to a disruption of plant sphingolipid

production by toxic fumonisins (Williams et al. 2007) or to rot and disease of the mesocotyl or

rooting system (Oren et al. 2002; Rodriguez-Brljevich et al. 2010).

2) Production of fusaric acid by Fusarium antagonizes gram-negative bacterial endophytes

(Bacon et al. 2001, 2004), many of which may be beneficial endophytes providing mutualistic

functions. Because endophytic diazotrophs in maize are predominantly gram-negative bacteria,

Fusarium may thereby inhibit N2 fixation or other beneficial activities of these organisms. This

might contribute to the low protein and chlorophyll contents that are a result of infection.

3) In stress years Fusarium cause Fusarium and Gibberella ear rot (FER and GER).

Resistance and antagonists to Fusarium: According to a recent comprehensive review

(Mesterhazy et al. 2012) most commercial hybrids are susceptible or highly susceptible to FER

and GER; full resistance has not been found. Though dominant, di-genic, and polygenic modes

of inheritance of resistance have been hypothesized, and heritability estimates for GER and FER

are high, marker assisted selection for breeding resistance is not feasible at this time because

effects of individual QTL’s are too small (Mesterhazy, et al. 2012). It is difficult to predict

resistance of hybrids based on resistance of inbreds (Eller et al. 2009; Mesterhazy et al. 2012).

Some level of maternal inheritance of FER resistance exists (Headrick and Pataky, 1991) but it is

not known whether this is associated with endophytic bio-control. Strains of the endophytic

fungus Acremonium zea living in maize landraces from heat and drought-prone areas in the USA

are potent antagonists of F. verticillioides (Wicklow et al. 2008; Wicklow and Poling, 2009). A

bacterial endophyte (Bacillus subtilis = B. mojavensis) found in one maize landrace can displace

F. verticillioides (Bacon et al. 2001). Bacterial isolates with antagonistic potential have been

found in many maize landraces (Johnston-Monje and Raizada 2011) and in teosinte (Mousa et al.

2015). Some Mexican landraces have been found (like teosinte) to produce low levels of

benzoxazinoids and to be more resistant to Fusarium/Gibberella sp than Corn Belt dent cultivars

(Reid 1988; Reid et al.1990a, 1990b).

Farming systems, Fusarium, and diazotrophic bacteria: N2 fixation in maize may not work

as well under currently practiced, conventional, maize intensive farming conditions as under

more extensive organic conditions. Studies with a native Mexican landrace showed that no-till

coupled with previous glyphosate use reduced N2 fixation substantially (Schwartz 2009).

Haahtela et al.1988 found that glyphosate and Roundup repressed nitrogenous activity by

21

Klebsiella pneumoniae and Pantoea agglomerans. They did not affect the growth of A.

lipoferum and Pseudomonas sp., but stimulated their nitrogenase activity.

Systems effects may be caused by differences in plant health associated with soil structure and

chemical residues. The rhizosphere has been described as a battleground between pathogenic and

beneficial microbes. The dynamic is determined by crop rotations and decomposing organic

materials (Raaijmakers et al. 2008) which affect a range of soil biological and physical

characteristics. Root disease can be suppressed by using residue and organic matter management

(Bailey and Lazarovits 2003).

Problems with Fusarium induced root rot are associated with a complex of Fusarium species

found in conventional, corn-intensive systems with the dominant species being F. oxysporum

(Warren and Kommendahl 1973). In studies of the long-term Wisconsin Integrated Cropping

Systems Trials, root-rot associated with Fusarium spp. was suppressed by forage-based crop

rotations and addition of manure (Goldstein 2000). Conventional maize had more diseased roots

in early stages of growth than organic maize (Goldstein 2000), causing the former plants to

produce more sets of shoot-borne roots during grain production. This fits the general Fusarium

pattern of inciting root disease and stimulating root growth.

Establishing successful plant/ diazotrophic relationships involves complex ‘cross-talk’-type

regulation and depends mainly on regulated suppression of plant defense biochemicals (Lambais

2001). It is possible that aggressive infection by Fusarium may disrupt diazotrophic activity

because it triggers the plant to massively produce superoxide and hydrogen peroxide in oxidative

bursts that also trigger systemic resistance (Alvarez et al.1998; de Gara et al. 2003; Kumar et al.

2009; Pechanova and Pechan, 2015). Infection with Fusarium spp. also induces a wide range of

other plant defense secondary chemistry (Pechanova and Pechan, 2015) which might also

negatively affect diazotrophic bacteria.

Conventional farming uses soluble N fertilizers while organic farmers rely more on slowly

available organic fertilizers for providing N to crops. High levels of N (especially ammonium)

in the soil and plant are associated with lower N2 fixation and fewer diazotrophic endophytes in

sugarcane (Carvallo et al. 2014) and maize (Roesch et al. 2006). On the other hand, organic

manuring may be conducive to diazotrophs. Nested PCR-denaturing gradient gel electrophoresis

(DGGE) analyses were used to fingerprint the endophytic community in maize that was grown in

long-term trials of mineral fertilization and organic fertilization. Organic manuring with

compost especially increased the diversity of methanotrophic, γ-proteobacteria species in both

the bulk soil and in the maize root endophyte community (Seghers et al. 2003, 2004). The γ-

proteobacteria species made up the bulk of bacterial endophytes in the maize root (Seghers et al.

2004). Mineral and organic fertilized plants had roots with different patterns of γ-proteobacteria

species which could be differentiated 100%. They include endophytes of the genera

Enterobacter, Rahnella, and Pseudomonas, genera known to have strains that have the potential

for N2 fixation. Though root endophyte populations were strongly affected, fertilization did not

affect the composition of endophytes in seed. Herbicide application did not appear to affect the

composition of endophytes in roots or seed.

An issue hitherto not considered is the impact N fertilizer is having on the evolution of

diazotrophs. A long term experiment in Michigan revealed that Rhizobia isolated from soils that

22

had been treated with N fertilizer were less effective symbionts for clover (Weese et al. 2015). It

remains to be seen whether this is also valid for associative bacteria.

Shifts in the other direction have been noted for maize inbreds developed under conventional, N

fertilized conditions then switched to organic, N-limited conditions. For a number of seasons the

author of this paper and a cooperating USDA-ARS breeder had noticed an “Inbred Culture-

Shock Syndrome.” (Goldstein 2012). Some inbred cultivars perform poorly (e.g., they display

yellow-green leaves and are relatively stunted) the first year they are grown under organic

conditions, but when they are multiplied a second or third year under organic conditions their

color and vigor improves. Explanations for this phenomenon, if it truly exists, could include

undetected outcrosses, epigenetic changes, shifts in the relationship of plants to microbes

including the balance of endophytes in plants, or subtle effects of selection practices or complex

interactions between these factors. Growing maize on organic sites may affect genetic and

epigenetic variation and/or the ability to distinguish such variation. In 2010, eight inbred or

partly inbred cultivars that had been grown and selected under organic or conventional

conditions were compared. Four were conventional commercial inbreds which either had been

produced at the organic farm or were the original conventionally produced inbreds. The other

four were derived from conventionally grown S3 lines and further bred either under organic

conditions or under conventional conditions by USDA-ARS. The resulting lines, most of which

had been under numerous years of selection, were grown side by side in 2010 with replications

on an organic field without fertilizer. Plants from seed that had been grown or bred under organic

conditions had consistently higher chlorophyll content of leaves in early September during grain

fill with an average positive increase of 16% (range 9 to 28%) relative to the conventionally

grown/bred lines (difference significant at P<0.001). These results suggest that the organically

grown/bred maize is more efficient at obtaining N because chlorophyll content is linearly

equated to the N content of plants (Schepers et al., 1992).

Mexican landraces, mucilage on brace roots, and diazotrophs: A landrace grown in the

village of Totontepec in Oaxaca, Mexico, is locally famous for its ability to be grown for years

on the same site without mineral N fertilizers (personal communication, Boone Hallberg,

Oaxaca, 2011). This landrace is Mixeño, according to classification by Bruce F. Benz (Texas

Wesleyan University, Fort Worth, Texas) who collected from the village; and by Flavio Aragon

Cuevas, (Central Valley CIRPAS INIFAP, Oaxaca, Mexico) who has studied collections from

the village. However, in the literature it is also referred to as Oloton (Vega-Segovia and Ferrera-

Cerrato, 1993; Gonzalez-Ramirez, 1994). The crop can grow up to 6 meters tall with whorls of

brace roots being formed up to 1.5 meters above the soil surface. The brace roots are covered

with mucigel (Gonzalez-Ramirez, 1994).

Thomas Boone Hallberg, from Ixtlan, Oaxaca (photo 1), a re-known naturalist, has been active

for decades for his work studying the biology of Oaxacan ecosystems, supporting local farming

communities, and supporting higher educational institutions of Oaxaca. He observed plants in

Totontepec and hypothesized that the mucigel might harbor N2 fixing activity that would support

vigorous growth under unfertilized conditions (Hallberg, 1995). He communicated this

hypothesis to microbiologist G.J. Bethenfalvay (USDA-ARS, Albany, California), who gathered

samples. On October 15th

, 1990, based on laboratory analysis, Bethenfalvay confirmed

Hallberg’s suspicions about the existence of N2 fixing bacteria in the aerial roots of the

Totontepec corn (Hallberg, 2016). Hallberg also shared this information with Ronald Ferrera-

23

Cerrato (Graduate School in Agricultural Science, Montecillo, State of Mexico), Jesus Caballero-

Mellado (Center for Nitrogen fixation at the Universidad Nacional Autonoma de Mexico,

Photo 1. Thomas Boone Hallberg and roots of a cross with Mixeño.

Cuernavaca, Morelos, Mexico), Howard Shapiro (Mars Corporation), the author of this article,

and many others (Cummings, 2002). Hallberg encouraged and helped them to test his hypothesis

(see acknowledgements in Gonzalez-Ramirez (1994), Gonzalez-Ramirez and Ferrera-Cerrato

(1995), Martinez-Romero et al. (1997)). The author has also had personal communications with:

Margaret Smith (Plant Breeding and Genetics, Cornell University) in Jan. 2012; Thomas Boone

Hallberg in 2012, 2014, and 2015; Flavio Aragon Cuevas , (INIFAP, Oaxaca, Mexico) in Dec.

2015, and Paulina Estrada de los Santos (Instituto Politecnico Nacional, Escuela Nacional de

Ciencias Biologicas, Mexico, DF) in Dec. 2015 that confirm Hallberg’s role as described above.

Vega-Segovia and Ferrera-Cerrato (1993) made a preliminary study of the microbial

composition of the root exudate from the brace roots, from the rhizoplane and from the

rhizosphere of these plants. The brace root exudate had high levels of bacteria of the genera

Azotobacter, Azospirillum, Derxia, Pseudomonas, and Beijerinckia, in order of progressively

descending population densities. These genera are known to possess diazotrophic species. The

highest population levels in exudate were of Azotobacter (6.3 x 107). Population counts of those

genera in the rhizosphere and rhizoplane were in the 106

range. Though fungal species could not

be isolated either from the exudate or from the rhizoplane, they were present at a low level in the

rhizosphere (5.5 x 103), suggesting some level of antibiosis by the bacteria in the mucigel and

rhizoplane.

In a second, more detailed study (Gonzalez-Ramirez 1994, Gonzalez-Ramirez and Ferrera-

Cerrato 1995), 93 strains of bacteria were isolated, 46 from the mucigel on the brace roots and 47

from the rhizosphere. Of these, many could fix N2 (36 in the mucigel and 38 from the

rhizosphere), and some showed high rates of nitrogenase in vitro. In the mucigel the species

identified were Pseudomonas fluorescens, Klebsiella pneumoniae, Azospirillum sp., Azospirillum

lipoferum, Enterobacter cloacae, Derxia gummosa, Xanthobacter and Flavobacterium. All of

these except the Flavobacterium had strains that demonstrated nitrogenase activity with the

24

ARA. Strains of the first three species produced antibiotics active against Rhizoctonia solani,

Sclerotium rolfsii, and Fusarium spp.

In the rhizosphere they identified E. cloacae, Citrobacter freundii, E. agglomerans,

Azospirillum sp., K. pneumoniae, D. gummosa, P. fluorescens, A. lipoferum, Beijerinckia indica,

and Xanthobacter. The first 6 species had strains that fixed N2, and the first two had strains that

produced antibiotics active against R. solani, S. rolfsii, and Fusarium sp. Three strains had the

capacity to fix N2, produce antibiotics, and solubilize phosphorus (Azospirillum spp., C. freundii,

E. cloacae).

Estrada et al. (2002) also isolated N2 fixing strains of E. cloacae, Azospirillum sp., K.

pneumoniae, as well as B. cepacia from Mixeño rhizospheres. These bacteria could live as

endophytes in the roots of maize. Estrada et al. (2001 and 2002) and Reis et al. (2004), also

discovered Burkholderia tropica that lived as a rhizosphere inhabitant and endophyte in the

Totontepec landrace Mixeño, in other maize cultivars, and in teosinte (Estrada et al. 2002; Reis

et al. 2004). B. tropica was found at similar levels in inoculated laboratory grown plants, as was

found in uninoculated, field grown plants of the same landrace and in teosinte tissues (up to 103

or 105

CFU/g root or shoot fresh weight, respectively) (Estrada et al. 2002). This species was

identical to B. tropicalis isolated from the sugarcane rhizosphere in Brazil, some strains of which

had been demonstrated to fix N2, inhibit nematodes, and stimulate the growth of maize plants

(Guyon et al. 2003). B. tropica was found in sugarcane in Brazil, and in maize from Mexico, but

it was not found in maize in Brazil (Reis et al. 2004; Perin et al. 2006), though a related species

(B. silvatlantica) was found (Perin et al. 2006). Furthermore, maize strains of this species from

Mexico were found to produce volatile compounds that inhibit the fungal pathogens Fusarium

culmorum, F. oxysporum, S. rolffsi, and Colletotricum gloeosporoioides (Tenorio et al. 2013)

while strains of the species from sugarcane in Brazil did not inhibit smut (Ustilago scitaminea)

or Fusarium spp. (Guyon et al. 2003).

There exist other beneficial, non-pathogenic Burkholderia species (including B. unamae, B.

phytofirmans) that live in rhizospheres and as endophytes in Mexican maize landraces (Estrada

et al. 2002, Reis et al. 2004; Caballero et al. 2004; Johnston-Monje and Raizada 2011). These

species and B. tropica (as well as Azospirillum spp) can potentially reduce ethylene levels in

their hosts (Blaha et al. 2006; Onofre et al. 2009). They have been shown to have a quorum

sensing system which facilitates interaction with their host and regulates bacterial biological

responses according to bacterial population size (Suarez 2010). Recruitment of the beneficial

Burkholderia species into the rhizosphere may occur through the secretion of oxalate by the

maize plant as these species are oxalotrophic (Kost et al. 2014). Apparently, B. tropica is not

transferred through seed to its progeny (Estrada et al. 2002), though B. phytofirmans can be

(Johnston-Monje and Raizada 2011).

Schwarz (2009) studied the N dynamics of the Mixeño landrace in Totontepec and in greenhouse

trials at the University of California-Davis using the 15

N natural abundance method. In the first

set of field trials in Totontepec in 2004, estimates of percent of N in the plant derived from the

air (% Ndfa) ranged from -255± 155.3 to 8±33. A greenhouse experiment showed biological

nitrogen fixation (BNF) estimates that ranged from -21±14 to 15±10% Ndfa. In 2006 trials it

25

was discovered that management significantly affected results. The system of tillage coupled

with no-herbicide application resulted in 40 ±6% Ndfa, while there was less appreciable fixation

associated with no tillage or no-tillage coupled with herbicide use. However, plants randomly

sampled from farmer fields had 85±28% Ndfa or 96±24% Ndfa in accordance with which

reference plant was chosen to make the comparison.

Brace root dynamics in N-efficient maize. N-efficient maize breeding lines derived from a

wide set of Latin American landraces were grown near Elkhorn, Wisconsin, in 2014 at the

Mandaamin Institute (Goldstein, unpublished data). Breeding lines derived from crosses with

multiple Mexican and South American landraces produced 3 to 4 whorls of brace roots with

exaggerated growth and often covered with mucigel excretions. Inspection in September of

some of these plants suggested a dynamic harvest of N might be taking place. While new sets of

brace roots on recently emerged whorls appeared healthy and were exuding mucigel, the roots on

lower, earlier-formed whorls had dark tips that appeared burned, like the ends of cigarettes or

burned matchsticks, suggesting intense oxidative activity. In some cases these roots with

‘burned’ tips produced new sets of short, above-ground lateral roots, often in the proximity of the

tip, and these new secondary roots were sometimes also be covered with mucigel (see photos 2-

6).

The generation of massive quantities of superoxide (O2·−) and hydrogen peroxide in a kind of

oxidative burst is the common primary mechanism by which plants destroy pathogens and

localized tissues (de Gara et al. 2003). It is hypothesized that massive oxidative bursts are

happening here. Note in photo 6 that the ‘burned’ root tip area corresponds to areas of greatest

accumulation of mucilage, where the greatest bacterial growth may be expected to trigger rapid

and localized death of plant and bacterial tissues.

Photos 2 and 3. A single F4 plant derived from crossing Corn Belt Inbred x landrace

Cacahuazintle. Picture on the left shows the third above-ground whorl which is exuding

mucigel. Picture on the right shows the second above-ground whorl with burned appearing tips.

26

Photos 4 and 5. Left Corn Belt inbred x Gordo-Cristalino de Chihuahua landrace, F4 plant on

left, Corn Belt inbred x Azul landrace, F6 plant on right. Plants show ‘burned’ tips on brace

roots.

Photo 6 shows 4th

whorl brace roots in a F2 plant that was derived from crossing a Maize Belt

inbred with Mixeño. Note the overlay of mucigel over the darkened root tips.

27

White et al. (2012) found that grasses excrete hydrogen peroxide into their rhizospheres,

apparently to harvest N from diazotrophic bacteria. Maize, however, was not shown to excrete

hydrogen peroxide into its rhizosphere. However, as has been described above, maize roots are

fully capable of generating reactive oxygen species to produce significant tissue death in the

cortex when constructing aerenchyma.

Inspection of brace roots developed on N-efficient breeding lines derived from crosses of Maize

Belt maize with Brazilian and Peruvian landraces in 2014 suggested that for the South American

derivatives there was less formation of mucigel and a much lower degree of development of the

burned tip syndrome. This may reflect differences in the way that plants interact with microbes

in brace roots. As some kind of oxidative, N-scavenging trait is accentuated in derivatives of

Mexican maize, we intend to do further research to determine whether it occurs in the crown

roots that are growing in the soil. Recent transcriptome research on brace roots of maize

suggests that brace root physiology is similar to crown roots but dissimilar from the primary root

system (Li et al. 2011).

This trait may be more deeply seated in the grass family. Brace roots associated with copious

production of mucigel have also been found in sorghum cultivars under selection for N2 fixation

(see cover photo in Wani and Patancheru 1986). It would be useful to compare these cultivars

with maize for their N dynamics.

Breeding for nitrogen efficiency/fixation: Maximizing plant-bacterial partnerships means to

engage in a co-evolutionary approach to breeding. Diazotrophic species and strains have been

isolated from cultivars of maize with demonstrated N efficiency. These strains have co-evolved

with their hosts. Such strains of bacteria differ in the services they can provide and they may be

plant variety-specific in the expression of those services. It would be fruitful to screen strains

found in maize landraces that appear to be N-efficient and to test the best of these bacteria on

multiple responsive cultivars across multiple N-limited sites.

The selection work done by von Bülow and Doebereiner (1975) and by Ela et al.1982, were the

first attempts to select maize for the ability to foster N2 fixing bacteria, but these selection

programs apparently did not continue. Successful efforts to select millet and sorghum for N2

fixation in India were documented in an ICRISAT report (Wani and Patancheru 1986). Wani

(1986) acknowledged that preceding N2 fixation research had been microbially-focused and that

little was known about the plant’s involvement in the process. His preference for methods of

selection were to couple ARA measurement on intact, pot-grown plant samples with 15

N isotope

dilution in greenhouse trials in addition to field testing for yield potential under low fertility

conditions.

Selection for N efficiency/N2 fixation in maize is done at the Mandaamin Institute, a non-profit

organization located in Wisconsin (www.mandaamin.org). Breeding lines are inoculated with

appropriate consortia of diazotrophs then selected for fitness under stress conditions. Steps in

the breeding process have been to: 1) Create an artificial consortium of diazotrophs (including

28

variable rhizosphere, rhizoplane, and endophyte inhabiting species) based on testing of selected

strains and combinations of strains on responsive maize cultivars; 2) Evaluate different cultivars

or breeding lines for responses to inoculant under N-deficient conditions. 3) Select responsive

cultivars and make appropriate crosses between them. 4) Inoculate and evaluate breeding lines

from those crosses, evaluate their performance under N-limited conditions on multiple sites, and

select and self-pollinate plants from individual lines that perform well across all sites. This

process of inoculation, grow-out, selection, and self-pollination is carried out sequentially for at

least eight generations, all the time selecting for maximum performance as inbreeding depression

accumulates. A further set of criteria for selection are provided by performance of the breeding

lines in hybrid test-crosses in replicated breeding trials on high and low-fertility soils. Yield data

from those trials is used to select which breeding lines to carry forward.

In 2009 exotic maize landraces were identified that were N sufficient on an N limiting site

relative to deficient maize belt maize varieties. Analysis of the stable δ-15

N isotopes from grain

samples suggested that some of the exotic cultivars fixed up to half of their N from the air

(Goldstein, 2012; Young and Szeliga, 2012) and that this trait could be transferred through

hybridization. The landraces that showed N efficiency/N2 fixation from highland Mexico include

Azul, Gordo, Cristalino de Chihuahua, Cacahuacintle, Celaya, Conico, and Mixeño; as well as

races from upland and lowland South America; and from the Southwestern USA. This broad

phylogenetic diversity (Vigouroux, et al. 2008) suggests that the trait is being expressed in

multiple cultivars placed throughout the native evolution of the crop.

Hybrid crosses with selected exotics produced approximately 40% more protein, lysine,

methionine, and cysteine per hectare than did conventional hybrids (p < 0.001) on an unfertilized

site in 2010 (Goldstein, 2012). Inoculation of seed with a mix of N2 fixing bacteria increased

yields of grain and protein for the exotic crosses by 18 and 19%, respectively (p<5%) but did not

affect conventional hybrids (Goldstein, 2012). Families and lines bred from these hybrids were

inoculated with a mix of diazotrophic bacteria and grown in a replicated trial on N-deficient, P

and K and moisture sufficient, sandy soils in 2013. The two conventionally bred check

populations averaged 77% barren plants. In contrast the top 20% of N-efficient lines had 10 to

40% barren plants (Goldstein, 2015a, 2015b).

Some of the breeding lines that have been derived from crosses with these races and are putative

N-efficient/N2 fixing show very dark green foliage indicating a high N content when grown on

multiple N deficient sites. Expression of the trait and nitrogenase activity in roots in breeding

progeny is variable. Photo 7 shows two N-efficient breeding lines growing on the left and two,

related, less-efficient breeding lines on the right, grown under N limited conditions). The

effective lines showed earlier flowering, and superior ear set on multiple sites under low N and

drought-stress conditions than sister lines that do not possess the trait (Goldstein, 2015a, 2015b).

Ongoing field trials are establishing which of the lines have yield potential in order to produce

commercial grade hybrids for seed companies, and the trait is being backcrossed into adapted

cultivars. The first set of yield trials in 2015 showed competitive yields in some hybrid

29

combinations. Aside from grain yield, the quantity of protein harvested is an important criterion

for the end user. The breeding effort for N efficiency is evaluated in terms of increasing the

stability of production of high protein grain with high levels of the essential amino acids

methionine and lysine.

The trait for producing multiple sets of brace roots is being tested as a potential help for N

efficiency/fixation. Preliminary observations suggest that year and environment affect

expression of the brace roots and lines that appear to be N-efficient under N-limiting conditions

in successive years do not always express exaggerated brace roots.

Discussion, hypotheses, and conclusions: Nitrogen efficiency may be improved by inoculating

plants with diazotrophs like Azospirillum spp, and this may or may not be due to N2 fixation.

Nevertheless, though results from the scientific literature were variable, there is enough data to

conclude that N2 fixation in maize-microbial associations occurs, and that in some cases it can be

a significant contribution to the plant’s N balance.

Table 1 shows a summary of the research surveyed here to test N2 fixation in maize. The

research has been carried out in numerous countries with most of it in the USA or in South

America. Many of the publications indicate that varietal differences play a major role in

determining the efficacy of the plant-microbial relationship. In several cases nitrogenase activity

appears to be substrate limited, however, the nature of in vitro trials makes it difficult to

extrapolate this conclusion to normal field conditions.

Plants somehow perceive whether a microbe is pathogenic or beneficial and either turn up or turn

down their defense systems. The signaling cascades that are invoked result in chromatin

remodeling which, in turn, regulates the expression of various defense systems (Ma et al. 2011).

30

Studies showed that the inoculation of rice and Azotobacter spp. produces profound changes in

the metabolism of plants effecting large parts of the rice genome including and going beyond

defense chemistry (Brusamarello et al. 2011; Chamam et al. 2012; Drogue et al. 2014). It is

significant that that the metabolic consequences are so diverse and so specific to plant variety

and bacterial strain. This indicates that there existed an evolving ‘dialogue’ between the partners

leading to greater fitness of the team.

Breeding is directed evolution and fitness is a function of partnership between plant and

microbial flora. At question is under which conditions do we select for fitness? The argument

has been made that selection for fitness under conventionally managed, N-fertilized conditions

has inadvertently resulted in stronger partnerships between maize and Fusarium species. The

Fusarium endophyte induces N-deficiency but that deficiency may be covered up by N-fertilizer

use.

An ideal breeding program: So what form should a breeding program take that breeds for

optimal partnerships between maize and bacteria leading to better N efficiency/N2 fixation under

N-limited growing conditions?

It is best that we begin combining field and lab research to find out. To begin with, we lack a

basic understanding of the impacts of our breeding methods on maize-microbial partnerships.

Questions are: Does substantial inbreeding lead to less fit partnerships? What is the impact of

hybridization and heterosis on such partnerships? How do such partnerships develop? What are

the epigenetic as well as genetic changes made in developing partnership processes?

The Mandaamin Institute has instigated a co-evolutionary breeding program that is strong in

directed evolution but weak on understanding the dynamics of the plant-bacterial evolution.

Having a greater understanding of those dynamics might make its breeding process more

successful and efficient. It might be helpful to:

1) Isolate, identify, and test diazotrophs from promising landraces and cultivars for

widespread use in inoculant consortia. It is important to go beyond understanding of the

potential services a bacterial strain can offer, to knowing whether that microbe exercises

such services in the context of different varietal backgrounds and actual field conditions.

2) Specifically develop molecular screening tools to identify nifH or nitrogenase-

expressing plant/microbial partnerships and to track individual microbial strains in order

to learn which bacteria actively fix N2.

3) To track the protein production benefits of microbial-plant associations. Stable

production of high quantities of grain or silage protein is a potential outcome of these

associations.

4) To better screen breeding lines and confirm that N2 fixation occurs with ARA, 15

N

dilution methods, and field trials.

5) Define and track plant genetic and epigenetic contributions to the partnership.

31

6) Expand the testing protocol to more sites to gain information on environmental effects,

stability of traits, and yield.

7) Further investigate the impact and utilization of N fixation in brace roots and their

mucilage and determine whether the plants are scavenging N by use of an oxidative

system.

It is important to maintain the main emphasis of such a program on overall fitness. Fitness can

best be evaluated through field trials. Cultivars with N efficiency traits will have to produce

similar yields to conventional hybrids under all conditions if they are to be accepted by seed

companies and farmers. There are some indications that the trait being worked with may

contribute to less barrenness and hence greater yield under N-limited stress conditions. Maize-

bacterial partnerships should improve overall fitness and hence greater yield and yield stability

under N-limited conditions, and they should be evaluated from that perspective.

Summarizing relationships:

Based on integrating relevant literature, and observations made in research at the Mandaamin

Institute, several facts and hypotheses presented in this paper are synthesized here below into a

picture of how maize-bacterial relationships might work. Hopefully this will stimulate more

testing and research:

1) In some cases N2 fixation can contribute a significant portion of the N budget of the

plant. In some cases it does not. Internal and external conditions are critical

determinants for a successful partnership and N2 fixation to occur.

2) Maize can interact with and foster soil diazotrophic bacteria by altering its production of

exudates and by lowering defense systems; simultaneously, the bacteria can signal the

plant to reduce its active defense systems while providing it with services such as

nutrients and hormones.

3) The basic physiological response of maize to N deficiency is creating conditions

(aerenchyma and low ethylene conditions) that are favorable to endophyte development.

4) Maize has a core set of endophytes that are vertically transferred from generation to

generation with seed, but rhizosphere inhabitants and endophytes are also actively

recruited from the bulk soil.

5) The composition of root and rhizosphere inhabitants is effected by different soils and by

organic manuring.

6) Hybridization strongly affects endophyte composition.

7) Farming systems can affect N2 fixation, and organic systems may encourage it.

8) Modern maize cultivars differ in their ability to have relationships with bacteria leading

to N2 fixation.

32

9) Breeders may have inadvertently selected modern cultivars for strong relationships with

Fusarium endophytes. These endophytes may serve as gatekeepers, antagonizing

diazotrophs and preventing N2 fixation through antibiosis and by increasing plant

generated oxidative stress.

10) Specific landraces (including several Mexican and South American landraces) may be

better at cultivating diazotrophic bacteria and their N2 fixing activity than others, but the

trait appears to exist in multiple cultivars across the maize phylogenetic spectrum.

11) Those responsive landraces may form promiscuous relationships with diazotrophs that

inhabit different niches (endophyte, rhizoplane, rhizosphere inhabitants).

12) N efficiency/N fixation is heritable and can be bred into modern cultivars using a co-

evolutionary approach. The trait probably has complex inheritance as it is the product of

a meshing of plant and bacteria metabolisms. It involves epigenetic and genetic

components on both plant and microbial components and is an active process strongly

affected by environment.

13) Scientifically established methods to assist breeding for N2 fixation include measurement

of nitrogenase in undisturbed plants, 15

N dilution trials, and field trials under low fertility

conditions, but could well be expanded to include molecular methods for identifying and

tracking diazotrophic species, the expression of beneficial metabolic traits such as N2

fixation, or plant genetic and epigenetic elements important in establishing relationships.

14) Mucigel secretion on crown roots and on a well-developed brace root system harbor

diazotrophs and contribute to fixation. Some Mexican landraces and cultivars of maize

are capable of producing exaggerated growth of brace roots and production of mucigel.

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