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Bulletin 1. Mandaamin Institute, Elkhorn, Wisconsin; Published on the Internet, January, 2016. Partnerships between Maize and for Nitrogen Efficiency and . By Walter Goldstein, Ph.D., Executive Director12.

Abstract: Partnerships between maize and diazotrophic (N2 fixing) bacteria can result in greater N efficiency through enhanced 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 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. [email protected] (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. 2

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 3 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 4

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 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 5 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, th Kennedy 1994, Kennedy et al. 1997). But at the recent 19 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 6 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). 7

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.

Bacteria Researcher Year Place genera Method result Ndfa % N'ase activity; difference Raju et al. 1972 USA Enterobacter ARA in foliage color NA Buelow & 22 x difference in N'ase; Dobereiner 1975 Brazil Azospirillum? ARA max at flowering NA Little N'ase or effect on Barber et al. 1976 USA Azospirillum ARA plants NA sugary substrate Rennie 1980 Canada Azospirillum 15N dilution increased fixation 13 to 38% Ela et al. 1982 USA Azotobacter ARA cultivar effect NA Alexander & Little N'ase or effect of Zuberer 1989 USA Azospirillum ARA bacteria on plants NA G. de Salamone et al. 1996 Argentina Azospirillum 15N dilution cultivar effect up to 58% cultivar effect. A. El Komy 1998 Egypt Azospirillum 15N dilution lipoferum > A. brasilense 0 to 35% Chelius and sugary substrate fostered Triplett 2000 USA Klebsiella nifH detection expression of nifH NA multiple growth yield increases but no Riggs et al 2000 USA species parameters relief of N defic. NA indigenous Montanez et al 2009 Uruguay bacteria 15N dilution cultivar effect 0 to 33% indigenous 15N natural Schwartz 2009 Mexico bacteria 15Nabundance natural management effects 0 to 98% Goldstein 2009 USA Azospirillum abundance cultivar effect 0 to 48% Azotobacter & difference and Yamprai et al. 2014 Thailand Azospirillum ARA substantial N fixation 29 to 52% Araujo et al 2014 Brazil Herbaspirillum NUE cultivar effect 34, 64% growth Guimares et al 2014 Brazil Herbaspirillum parameters little effect NA Puri 2014 Canada Paenibacillus 15N dilution fixation. 17% Cavalcanti et al 2015 Brazil Herbaspirillum nat abundance fixation. up to 37% -38, 1996. 8 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 9 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 15N 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 15 allowed to grow undisturbed in artificial soil. 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 15 activity. 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 10 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 11

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 15N enrichment in the soil, the results with 15N 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 15 the growth of two cultivars of maize. They utilized 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

15 N2 fixation by indigeneous diazotrophs: The 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 15N 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 15N and 14N ratios (δ15N 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 14 15 actual increases in N due to fixation of N2 would be balanced out by greater uptake of N from soil organic matter and there would be no change in the δ15N 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 13N 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 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. A15N 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 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 15N 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 15N 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-15N 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.

15 4) To better screen breeding lines and confirm that N2 fixation occurs with ARA, 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, 15N 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. Such cultivars may scavenge N from colonies of diazotrophs on and in those brace roots by use of oxidative bursts similar to a plant defense response.

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