Improving nutrient uptake in wheat through cultivar specific interaction with Azospirillum
Hoagland, L., Murphy, K.2, Carpenter-Boggs, L.3, Jones, S.4
Key words: nitrogen, plant breeding, organic farming, diazotrophs
Abstract
Obtaining sufficient plant available nitrogen in organic dryland wheat cropping systems is difficult.  This study was conducted to determine whether inoculation with Azospirillum could improve nitrogen uptake and increase crop yield, and whether there are differences among wheat cultivars in the ability to benefit from inoculation of these diazotrophic bacteria.  Seed from twenty historic and modern wheat cultivars were either left untreated, or treated with a commercial inoculant of Azospirillum, and planted at two locations under certified organic management.  In one location with lower fertility, inoculation significantly increased yield and protein, and clear differences existed among individual cultivars in response to the inoculant.  In another location with higher fertility, none of the cultivars responded as favorably to the inoculant, and yield in some cultivars was reduced.  Plant breeders should be able to select for beneficial cultivar interactions with Azospirillum to increase wheat yield and protein levels.  Additional research is needed to determine the impact of site-specific soil conditions on the effectiveness of Azospirillum in organic systems.
Introduction
In low-input organic dryland grain cropping systems, obtaining sufficient plant available nitrogen (N) can be problematic.  Organic producers often utilize manure or compost to meet N needs, but in many areas the cost of transportation and application make bulky organic fertilizers uneconomical.  Low rainfall also limits the ability of some growers to utilize leguminous green manure crops.  One alternative to help supplement N is through exploitation of diverse diazotrophic bacteria capable of biological nitrogen fixation (BNF).  Associative BNF can contribute 10-50% of the total N requirement of wheat (Solimon et al., 1995; Kennedy and Islam, 2001).  In addition to BNF, these bacteria may increase plant growth through production of phytohormones, phosphorous release, increased nutrient uptake, enhanced stress resistance, biocontrol of both major and minor plant pathogens (Dobbelaere et al., 2003), and improved water status (Creus et al, 2004).  However, these plant-microbial interactions are dependent on plant genotype (Iniguez et al., 2004) and site-specific soil conditions (de Oliviera et al., 2006).  
Historically, plant breeders have not selected directly for interaction with beneficial soil bacteria, yet in Brazil, sugarcane breeders selecting for high yield under low-input conditions inadvertently selected for interaction with native diazotrophic bacteria (Baldani et al., 2002).  Prior to the advent of chemical fertilizers that have high plant available nitrogen, breeding programs of other graminaceous crops like wheat, may also have indirectly selected for this association.  However, after decades of cultivar selection under conditions that utilize chemical fertilizers, modern cultivars may not interact efficiently with these bacteria, and relevant levels of native diazotrophic bacteria may no longer be present in the soil.
To improve nutrient uptake and yield in low-input organic wheat systems, we tested the efficacy of a commercial inoculant of Azospirillum, a diazotrophic bacteria that has been shown to positively interact with wheat.  Twenty historic and modern winter wheat cultivars were evaluated for the ability to interact with, and benefit from inoculation, and to identify cultivars for use in organic production systems.  
Materials and methods
In autumn 2006, field trials were established in two certified organic fields with moderate rainfall near Pullman, WA.  Each trial contained five historic and five modern hard red cultivars, and five historic and five modern soft white cultivars, each replicated four times.   Each trial was arranged in a split block design, with seed from all cultivars in one block receiving a commercial inoculant of Azospirillum brasiliense (EMD Crop Bioscience) according to the manufacturer’s recommendation (9.8 ml kg-1), and cultivars in the other block left untreated.  A previous winter pea plowdown provided approximately 40 kg ha-1 of N.  Weed control was accomplished by hand weeding throughout the growing season.  
Plant emergence, leaf greenness using a SPAD chlorophyll meter, plant height, yield and protein were recorded.  In each of the four blocks within each split block, ten soil samples were randomly collected to a depth of 10-cm and pooled for analysis.  Soil samples were analyzed for total carbon (C) and nitrogen (N), inorganic N, and potentially mineralizable N, and organic matter (OM) was estimated.  Field plot locations were recorded permanently using GPS coordinates to track survival of the introduced bacteria in the soil over time.  
Results
Seed treatment with a commercial inoculant of Azospirillum had little overall impact on seedling emergence, leaf greenness, or plant height in either location, but did have an impact on crop yield and protein.  In location A, average crop yield was increased among all cultivars by 538 kg ha-1; however, yield increase ranged from 134 to 1,209 kg ha-1 among individual cultivars, and was significantly increased in only five cultivars (Figure 1). In location B, yield data was compromised due to harvest difficulties resulting from the slope of the land, but in general, the cultivars did not respond as favorably to the inoculant, and yield was decreased in some cultivars (data not shown).  In location B, average crop yields among all cultivars were 638 kg ha-1 greater than average yields in nursery A. Only two historic hard red cultivars responded favorably to the inoculant in both locations, but yield increases were not significant.
In both locations, protein levels were increased in response to the inoculant.  Overall protein levels were greater in location B, but increases in response to the inoculant were greater in location A. In location A, six cultivars had significantly increased protein levels (data not shown).  Modern cultivars, Masami and Bauermeister had both significantly increased yield and protein in location A.  In location B, only two cultivars had significantly increased protein levels, and correspondingly, these two cultivars had significantly reduced yield in response to inoculation. Only one cultivar, Buchanan, had significantly increased protein levels in both locations.
Total C and N, inorganic N, and OM were lower in nursery A than in nursery B (Table 1).

Figure 1: Wheat cultivars with significantly increased yield as a result of Azospirillum inoculation in location A (P<0.05).
Tab. 1:  Soil nutrient levels in Azospirillum inoculation trials
 	Total C	Total N	Inorganic N	Organic Matter	
Location	(mg kg-1)	(%)	
A	13,700	980	9.31	2.37	
B	20,200	1460	14.35	3.49	

Discussion
Treatment of wheat seeds with a commercial inoculant of Azospirillum increased crop yield and protein in organically managed systems, but the interaction was dependent on plant genotype.  Five of the twenty wheat cultivars inoculated with Azospirillum had significantly increased crop yield, suggesting that wheat breeders can select for this interaction.  The ability to benefit from Azospirillum inoculation was present in some modern cultivars, indicating that this trait can co-exist with other beneficial plant traits like high yield and disease tolerance that have been traditionally selected for in modern breeding programs.  Inoculation also resulted in increased protein levels in some cultivars, suggesting that this interaction can be used to select wheat cultivars for use in the hard red market class, where high protein levels are required. Site-specific soil conditions also had an impact on wheat yield and protein in response to inoculation.  In our study, greater soil fertility levels were correlated with an insignificant, or deleterious response in some cultivars to inoculation with Azospirillum.  This is consistent with the findings of Martinez de Olivera et al. (2006) who found sugarcane productivity to be reduced as a result of inoculation with diazotrophic bacteria in a high fertility soil.  However, these findings contradict company reports on the activity of the Azospirillum inoculant under conventional wheat management.  More research is needed to determine how site-specific soil conditions affect interaction of graminaceous crops with diazotrophic bacteria. To better meet our objectives, we expanded our trials in autumn 2007.  Ten advanced lines from our organic breeding program were included and the trials were planted in five different locations that include regions of low and intermediate rainfall, and organic and conventional management.  Soil samples from all 2006 and 2007 planted sites will be evaluated for additional chemical and biological factors to help determine how site-specific soil conditions influence the interaction of wheat and Azospirillum.  A subset of the cultivars used in our field studies were planted with and without inoculation in greenhouse trials using zero fertilizer, or either organic or chemical fertilizer at both a low and high rate.  Root abundance of Azospirillum and mycorrhizae, and nitrogen fixation by Azospirillum among individual cultivars will be determined.  Abundance and diversity of native diazotroph communities will be evaluated in soils from across Washington State.   
Conclusions
Initial results from the first year of this study suggest that inoculation of wheat with Azospirillum can improve nutrient uptake and increase crop yield and protein under organic management, but this is dependent upon genotype and site-specific soil conditions.  Wheat breeders may be able to select for cultivars that benefit from association with these bacteria, but additional research is needed to validate the findings of cultivars differences, and determine the impact of site-specific soil conditions before recommending this to organic farmers as a management tool.  
Acknowledgments
We thank the USDA-CSREES Organic and Integrated Grant Program for support of this research; EMD Crop Bioscience for donation of Azospirillum inoculant; and, Steve Lyon, Kerry Balow, and Meg Gollnick for assistance in field and lab experiments.
References
Baldani, J.I., Reis, V.M., Baldani, V.L.D., Dobereiner, J. (2002): A brief story of nitrogen fixation in sugarcane – reasons for success in Brazil. Funct. Plant Biol. 29:417-423.
Creus, D.M., Sueldo, R.J., Rolando, J., Barassi, C.A. (2004): Water relations and yield in Azospirillum inoculated wheat exposed to drought in the field. Can. J. of Bot. 82:273-281.
de Oliveira, A.L.M, Canuto, E.D., Urquiga, S., Reis, V.M., Baldani, J.I. (2006): Yield of micropropogated sugarcane varieties in different soil types following inoculation with diazotrophic bacteria.  Plant Soil 284:23-32. 
Dobbelaere, S., Vanderleyden, J., Okon Y. (2003): Plant growth-promoting effects of diazotrophs in the rhizosphere. Crit. Rev. of Plant Sci. 22:107-149.
Iniguez, A. L., Dong, Y., Triplett, E.W. (2004): Nitrogen fixation in wheat provided by Klebsiella pneumoniae 342.  Mol. Plant Mic. Int. 17:1078-1085.
Kennedy, I.R., Islam, N. (2001): The current and potential contribution of asymbiotic nitrogen fixation to nitrogen requirements on farms: a review.  Aust. J. of Exp. Ag. 41:447-457.
Solimon, S., Seeda, M.A., Aly, S.S.M., Gadalla, A.M. (1995): Nitrogen fixation by wheat plants as affected by nitrogen fertilizer levels and non-symbiotic bacteria. Egyption J. of Soil Sci. 35:401-413.