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Soil Biology & Biochemistry 38 (2006) 769784 www.elsevier.


An assessment of nodulation and nitrogen xation in inoculated Inga oerstediana, a nitrogen-xing tree shading organically grown coffee in Chiapas, Mexico
J.M. Grossmana,*, C. Sheafferb, D. Wyseb, B. Bucciarellib, C. Vanceb, P.H. Grahamc
b a Department of Crop and Soil Sciences, Cornell University, 726 Bradeld Hall, Ithaca, NY 14853, USA Department of Agronomy and Plant Genetics, University of Minnesota, 411 Borlaug Hall, 1991 Upper Buford Circle, St Paul, MN 55108, USA c Department of Soil, Water and Climate, University of Minnesota, 439 Borlaug Hall, 1991 Upper Buford Circle, St Paul, MN 55108, USA

Received 27 July 2004; received in revised form 27 May 2005; accepted 14 July 2005 Available online 24 August 2005

Abstract Coffee (Coffea arabica) production provides a source of income for small-scale farmers in Chiapas, Mexico. Organic production regulations prohibit the use of synthetic nitrogen (N) fertilizers, therefore farmers are dependent upon N-sources accepted by organic certication agencies. In the state of Chiapas, little is known about effectiveness of rhizobia and nodule development, location and structure of the common coffee shade tree genus Inga. The objectives of this study were to (1) evaluate the symbiotic effectiveness of selected rhizobia isolates as inoculants for Inga oerstediana under greenhouse and eld conditions, (2) describe the morphological and histochemical characteristics of I. oerstediana root nodules, and (3) apply the 15N natural abundance technique to investigate nitrogen xation in two stands of I. oerstediana of different ages intercropped with C. arabica. To meet objectives one and two, we assessed shoot biomass, nodule number, nodule mass and total shoot N of inoculated I. oerstediana seedlings at 90 and 150 days after planting (DAP) in the greenhouse and eld. Light microscopy, and in situ hybridization of nodule sections for leghemoglobin and Nif H cDNA determination were used to describe nodule morphology and histology. Results indicated that tested isolates appear not to be xing N2 150 DAP and inoculation with isolated bacteria, and that inoculated treatment nodules lacked leghemoglobin and Nif H mRNA transcript, however contained infected bacteroids. An unidentied brown-pigmented granular substance was present in all nodules examined. I. oerstediana appears slow to nodulate, with negligible nodulation at 90 DAP, and limited nodulation at 150 DAP. Using the natural abundance method to meet objective three, recycling of xed N in older 57 year plots is thought to have caused great 15N value variation in both reference and leguminous trees and data could not be used to estimate % N derived from Biological Nitrogen Fixation (BNF). I. oerstediana found in younger 13 year old plots was found to derive 20% of its N from BNF. As xation appears to be low in young Inga, recommendations for organic C. arabica shade tree management include supplementation of N during early growth of Inga-C. arabica intercrop, and longer-term nodulation studies combined with additional N2-xation assessment using 15N natural abundance methods. q 2005 Elsevier Ltd. All rights reserved.
Keywords: Chiapas; Coffee; Mexico; Organic; Nitrogen-xation; Root nodule bacteria; Agroforestry; Leguminous trees; Inga

1. Introduction Coffee (C. arabica) cultivation is the main source of income for over two million Mexicans (Nestel, 1995), ranking Mexico within the top six most productive coffeeproducing countries globally (Waridel and St Pierre, 2002).

* Corresponding author. Tel.: C1 607 254 5389; fax: C1 607 255 2644. E-mail address: jmg225@cornell.edu (J.M. Grossman).

0038-0717/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2005.07.009

xico is an Coffee production in the state of Chiapas, Me important means of economic survival for many indigenous Mayan peasants. Adequate nitrogen (N) supply is crucial to C. arabica production (Bornemisza, 1982). Harvest of organic C. arabica beans in the Chiapas highlands has been shown to remove an average of 19 kg N haK1 yrK1, with only 10 kg N haK1 yrK1 replaced via compost additions (Perez-Grovas Garza, 1998). Two different means of N replacement that occur in C. arabica production are the unshaded system and shaded system. In modern C. arabica production found throughout Latin America, plants are often grown unshaded and with


J.M. Grossman et al. / Soil Biology & Biochemistry 38 (2006) 769784

N replacement achieved using chemical N fertilizer applied at rates often reaching 100300 kg N haK1 yrK1 (Carvajal, 1959; Vicente-Chandler et al., 1959; Parra, 1960; Reynoldsvargas et al., 1994), and leaching of excess fertilizer-derived nitrate to groundwater is prevalent (Babbar and Zak, 1995). This system is known to boost C. arabica yields, but signicantly increases production costs by necessitating purchase of synthetic inputs such as fertilizer and pesticides (Muschler, 1997), and reducing the productive life of the C. arabica plants. In Mexico, however, the more common system of coffee production is the growth of coffee under shade trees (Moguel and Toledo, 1999). In traditional shaded C. arabica plantations, native leguminous tree species are often used to supply all or a portion of the N needs of C. arabica (Roskoski, 1982; Roskoski et al., 1982; Soto-Pinto et al., 2000, 2001). The use of atmospheric nitrogen (N2) xing trees for improvement of associated crop production is integral to low-input sustainable agricultural practices in most developing countries (Sanginga et al., 1985; Kang et al., 1990; Awonaike et al., 1992; Milnitsky et al., 1997; Sprent and Parsons, 2000). Contribution of N to C. arabica systems by such trees has been well documented in Venezuela (Aranguren et al., 1982; Escalante, 1995; Escalante, 1997), Costa Rica (Babbar and xico (Roskoski, Zak, 1995; Lindblad and Russo, 1986), Me 1981 1982; Greenberg et al., 1997), Cuba (Rodriguez et al., 1991), Guatemala (Munoz, 1997), Honduras (CIDICCO, 1995), and Sri Lanka (Kathirgamathaiyah et al., 1993; Ranasinghe, 1995). In these systems, an increase in plantavailable N is the result of the symbiotic relationship between specic nodule-forming, N2-xing bacteria, known as rhizobia, with the leguminous host. The state of Chiapas is the largest producer of certied mez Cruz et al., 2000). organic C. arabica in Mexico (Go Many farmers here have chosen to organically certify their land in order to obtain the 1530% price premium that the sale of organic C. arabica provides (Janssen, 1997). Organic regulations prohibit the use of synthetic N fertilizers so farmers are dependent upon N-sources accepted by organic certication agencies. Such accepted sources of N include farmer-made compost from natural sources, and symbiotic N-xation (Naturland, 2000). The symbiotic effectiveness of the leguminous shade tree/rhizobia relationship is thus critical to high yield of C. arabica organic systems. The leguminous tree species used to shade coffee vary by region (Giller, 2001; Babbar and Zak, 1995; Escalante, 1997). In Chiapas the shade tree commonly used is Inga sp. (Nolasco, 1985) subfamily Mimosoideae. This genus has over 300 species throughout tropical America and is also valued for fuelwood, food, soil improvement, and weed control (Pennington and Fernandes, 1998). It is assumed in Chiapas that Inga species, two of which are I. oerstediana and I. pavoniana, provide N to C. arabica through symbiotic N-xation and subsequent mineralization of deposited leaf litter. Local farmers have observed trees of this genus to provide many benets to C. arabica, including

healthier coffee plants and improved soil quality (Grossman, 2003). Despite the perceived importance of Inga sp. in organic C. arabica plantations and farmer observations, little is known about its contribution to the N status of the soil and its association with N2-xing symbiotic bacteria. It has been assumed in Chiapas that Inga sp., primarily I. oerstediana, provide N to C. arabica through symbiotic nitrogen xation and subsequent mineralization of deposited leaf litter, yet the xation ability of this species has not been conrmed in situ. Nitrogen derived from the atmosphere through nitrogen xation (% Ndfa) has been estimated in Inga jinicuil at 35% using the acetylene reduction technique on N fertilized plots (Roskoski, 1982). This technique may, however, give an inaccurate picture of nitrogen xed as it disturbs root nodules before measurement, and measures only the short-term activity of the nitrogenase enzyme. Alternatively, the 15N natural abundance technique relies upon small differences in the isotopic 15N concentration that occurs between atmospheric nitrogen and the natural 15N enrichments of soils due to isotopic discrimination processes during N transformation. Atmospheric N2 is not enriched with 15N (d15NZ0) thus N2 xing species will often demonstrate lower 15N values than non-N2 xing species that use only available soil N to meet their needs. The 15N natural abundance technique has been successfully used in a number of environments to estimate N2 xation in woody legumes (Shearer et al., 1983; Domenach et al., 1989; Beaupied et al., 1990; Kurdali et al., 1990; Ladha et al., 1993; Van Kessel et al., 1994; Muofhe and Dakora, 1999; Galiana et al., 2002), and provided that tissue discrimination for 15N is considered, is regarded as the most accurate way to measure nitrogen xation in woody tree species (Boddey et al., 2000; Gathumbi et al., 2002). Accurate quantication of nitrogen xation contributions to organic C. arabica systems is needed to better understand the N economy of this system, but no data is currently available. We hypothesize that bacterial strains isolated from the root nodules of Inga oerstediana can be selected for optimized N2-xation capacity and that the characterization of I. oerstediana root nodule structure will further the understanding of nodule function and the effectiveness of the symbiotic relationship between Inga sp. and its microsymbiont. Further, we hypothesize that the contribution of xed N to the soil by Inga oerstediana can be estimated using the isotopic natural abundance technique. Such an understanding would be of value to the increased production and long-term ecological maintenance of organic C. arabica systems in Chiapas. The specic objectives of this study were to (1) evaluate the symbiotic effectiveness of selected rhizobia isolates as inoculants in Inga oerstediana, (2) describe the morphological and histochemical characteristics of I. oerstediana root nodules, and (3) apply the 15N natural abundance

J.M. Grossman et al. / Soil Biology & Biochemistry 38 (2006) 769784


technique to investigate nitrogen xation in two stands of I. oerstediana of different ages intercropped with C. arabica.

2. Materials and methods 2.1. Isolate collection and initial assessment Nodules from 4 to 10-year-old I. oerstediana (Benth, identication by Sousa, 2001) were collected from 20 organic coffee farms in two communities in the munici and Tenejapa within the state of palities of Chenalho Chiapas, Mexico (Fig. 1). Additional nodules not used for isolation were also collected from a 5-year-old Inga oerstediana tree to record general nodule morphological characteristics. Soils from the study sites were collected and analyzed for physical and chemical characteristics (Soil Testing Laboratory, El Colegio de La Frontera Sur, Chiapas, Mexico). A total of 10 nodules were collected from three trees of I. oerstediana on each farm and placed in silica gel desiccation vials. Nodules were also collected from two additional species of Inga, I. pavoniana (G. Don.) and another unidentied Inga species. Nodules were rehydrated, surface sterilized using 95% (v/v) ethanol for 30 s, 3.25% (v/v) chlorox for 3 min, rinsed 5! in sterile distilled H2O, and crushed onto YEM agar plates (Vincent, 1970). After 2 weeks, pure isolates were collected, sub-cultured until puried, and maintained on YEM agar slants (Vincent, 1970) then frozen in 15% (v/v) glycerol in AG media and stored at K70 8C. In total, 83 strains were isolated from root nodules of I. oerstediana, I. pavoniana and the unidentied

species of Inga. Aside from isolates of I. pavoniana, Inga isolates demonstrated characteristics consistent with Bradyrhizobium. Dendrograms generated using phylogenetic and phenotypic traits showed strains to exhibit signicant diversity, even among strains originating from the same farm. Using PCR genomic ngerprinting, 40 subclusters taken from four main clusters of similar strains were identied. One representative strain was chosen from each sub-cluster for the current study. Grossman (2005) provides a thorough characterization of the isolates. The 40 strains were screened for nodulation and N2 xation in August 2000. Pods of I. oerstediana were collected from two trees approximately 10 m apart, and 1000 seeds separated and wrapped in moist newspaper for transport from Mexico to the University of Minnesota. Seeds were surface sterilized with (95% v/v ethanol for 30 s and 2.0% (v/v) commercial H202 for 7 min, washed using three rinses of sterile deionized water (dH20), and left to germinate on sterile moistened course grain sand at 25 8C until the radicle reached 1.31.5 cm (Vincent, 1970). They were then planted one seed per unit in sterile Leonard jar units (Somasegaran and Hoben, 1994) containing a mixture of Sunshine Mix #4 (Sun Gro Horticulture Inc., Bellevue, WA) and course grain sand moistened with 50 mL N-free plant nutrient solution (PNS) (Broughton and Dilworth, 1971). Strains were grown in BY broth to a minimum of 108 cells mLK1, and seeds inoculated with a 105 cells mLK1 dilution of cell culture (Somasegaran and Hoben, 1994) using a sterile pipette. Plants were grown in a Model E-15 growth chamber (Conviron, Winepeg, Canada) with 14 h days and 25 8C/20 8C daynight temperature. Experimental

Fig. 1. Map of nodule collection sites.


J.M. Grossman et al. / Soil Biology & Biochemistry 38 (2006) 769784

design was a randomized complete block with four replicate units. Plant biomass and fresh nodule mass were then determined 90 days after planting (DAP). 2.2. Evaluation of best performing strains: pot and seedling preparation Five strains selected from the initial assessment above for nodule and shoot biomass were evaluated under sterile and native soil substrate conditions in Chiapas, Mexico in August 2001. Treatments included four replicates of each of the ve chosen strains in sterilized four-gallon round tree pots (Hummert International, Earth City, MO) containing either 50% sterile perlite:washed sand and 5% compost (sterile substrate), or non-sterile 50% native soil:perlite (native substrate). Soil collected from the seed source site was shown to contain 0.95% N and had a pH of 5.7. A set of positive and negative controls was used with each substrate type. The negative control (N) was not inoculated, the positive control was fertilized with commercial levels of N fertilizer (200 mg LK1 NH4NOK 3 ). One thousand seeds of Inga oerstediana were collected locally from one tree source, sterilized as described above, and left to germinate in sterile petri dishes lined with sterile moistened paper towels. Inoculants were prepared and seeds inoculated as described above. Seedlings were covered at the surface with 2 cm of sterile perlite as an anti-contamination mulch (Masutha et al., 1997). 2.3. Trials Trials took place at El Colegio de La Frontera Sur greenhouses (elevation 2100 m) using natural light conditions between October 2001 and March, 2002. Greenhouse temperature uctuation over the ve months ranged from a minimum of 214 8C to a maximum of 2346 8 C. Plants were arranged in a randomized complete block design and seedlings watered bi-weekly using a receptacle placed below the pots with a wicking system for plant uptake. The rst 60d, watering was done by placing 50 mL full-strength N-free PNS into the receptacle below. For the last 90d, a 50% concentration of PNS diluted with dH20 was used for watering. After the rst 60d we observed browning of the leaves thought to be caused by cold ambient temperatures in the greenhouse. At 90 and 150 DAP two replicates of each substrate and strain treatment combination were harvested from the greenhouse. Originally all four replicate plants were scheduled to be harvested at 90d, however, due to limited growth and treatment response after this time period, we opted to harvest two replicates at 90d and the second two at 150d. Shoots were excised and dried at 55 8C for 3 days, and weighed for biomass. Nodule fresh weight and total shoot N was also determined.

Total leaf tissue N spectra was collected with a NIRSystems (Silver Springs, MD) Model 6500 scanning monochrometer with a range of 4002500 nm.1 The Infrasoft International (ISI, Port Matilda, PA) NIRS 3 version 4.0 software program Select was used to choose 50 samples found to be representative of population means. Total N concentration of the 50 representative samples was determined using a LECO FP-528 Nitrogen Analyzer as in the Dumas method (Simone et al., 1994; Matejovic, 1995). For standard curve equation development, results of the 50 sample set were analyzed using the ISI software program Calibrate with the modied partial least squares regression option and two passes to eliminate outliers (Shenk and Westerhaus, 1991). The math treatment of 1,4,4,1 (rst derivative, gap over which derivative was calculated, number of data points used in rst smoothing, and second smoothing where one means no smooth) was used for all equations. Plants receiving no fertilizer N in the greenhouse did not perform well, necessitating combination of the small samples for adequate NIRS measurement. 2.4. Field evaluation At 90 DAP, 34 replicates of each substrate and strain treatment combination reserved for this purpose were removed from the greenhouse and transplanted to a local C. arabica eld close to where seeds were originally collected. Plants were checked for nodulation prior to replanting, and nodule number recorded. After 60 additional days of growth in the eld setting, nal nodule number and difference in nodulation from 90d were recorded in Table 2. Included in the eld trial were N-fertilized controls previously grown in the greenhouse in sterile soil, K N controls previously grown in sterile soil, and N-fertilized and KN controls previously grown in native soil. Experimental design was a randomized complete block utilizing four blocks in a total area of 4 m2. Plants were watered as needed using local untreated water sources. At 150 DAP (90 days in greenhouse C60 days in eld) eld plants were harvested and analyzed as plants grown in the greenhouse. Treatment means and standard deviations for all experiments were analyzed using a one-way ANOVA and student t-tests with SigmaPlot 2000 software. 2.5. Histochemical analysis At 150 DAP root nodules were removed from greenhouse-grown plants and xed in a paraformaldehyde gluteraldehyde solution according to the protocol described by Trepp et al. (1999). Embedded material was sectioned at 10 mm and mounted on poly-L-lysine coated glass slides.
Mention of a proprietary product does not constitute a recommendation or warranty of the product by the University of Minnesota and does not imply approval to the exclusion of other suitable products.

J.M. Grossman et al. / Soil Biology & Biochemistry 38 (2006) 769784


Tissue sections were stained with toluidine blue to evaluate overall nodule structure. Histochemical tests to detect lignin, suberin, general phenolics and tannins were conducted as follows: PhloroglucinolHCL (Faulkner and Kimmins, 1975) was used to detect lignin; Sudan IV (Jensen, 1962) was used to localize suberin; general phenolics and tannins were detected by exposing tissue for 30 min to 2 h in a solution of 0.02 M ferric chloride 0.02 M potassium ferricyanide (Sherwood and Vance, 1976). Tissue autouorescence was examined under UV illumination with a lter cube having the following wavelength parameters: Ex 330-380, DM 400, BA 420. In situ Hybridization of I. oerstediana nodule sections and probe preparation of the Leghemoglobin and Nif H cDNA was carried out according to the protocol described by Trepp et al. (1999). All tissue sections were viewed with a Labophot microscope (Nikon) with images recorded using a Nikon DXM 1200 digital camera. 2.6. Natural abundance sampling sites Sampling sites were actively farmed C. arabica plots in the state of located in the municipality of Chenalho Chiapas, Mexico (1570 m elevation), located between 168 05 0 and 168 56 0 North latitude and 918 40 0 and 928 31 0 West longitude. The area has a bimodal rainfall distribution with distinct wet and dry seasons from June to October and November to May, respectively. Annual mean precipitation a, 1973) and ranges from 2000 to 2500 mm (Garc temperature from 18 to 22 8C (Paau and Brill, 1982). Local soils are classied in the FAO/UNESCO (Food and Agriculture Organization/United Nations Educational Scientic and Cultural Organization) system as Andisols (INEGI, 1993). Soils at the study sites were acidic (average pH 5.8), with an average N content of 0.25%, and 9.49 ppm P (Grossman, 2005). The only soil amendment applied to the plots was compost that was applied to the base of the C. arabica plants annually, made from farmers household waste, C. arabica berry pulp, I. oerstediana leaves, and manure. The rst sampling was carried out in October 2000 in a 2 ha plot of 57 year old I. oerstediana and the second in October 2001 in a 1 ha plot of 13 year old I. oerstediana, both intercropped with C. arabica. The rst plot had been certied organic for 5 years, and the second was not certied organic, but had similar soil management as the rst. 2.7. Natural abundance experimental design and methods In the 57 year old plot, Liquidambar styraciua, a nonleguminous shade tree often found intercropped with C. arabica, was chosen as a reference species due to its similar branching size and rooting pattern to I. oerstediana. Plants of C. arabica were also used as additional reference species. Twelve replicate sets were chosen randomly from around the plot, each containing one tree of I. oerstediana, a C. arabica shrub planted close (!3 m) and far (O5 m)

from the trunk base of I. oerstediana (not inuenced by leaf litter deposition) and L. styraciua. The use of replicate sets increases the likelihood that both species are using soil of identical 15N enrichment. The 13 year old Inga plots were sampled by taking material from only C. arabica and I. oerstediana. Twenty leaves were collected from a radius around each tree including both older and newer leaf material. Leaves were dried at 55 8C for 3 days. A sub-sample of each set of leaf material was analyzed for total N in a LECO FP-528 Nitrogen Analyzer (Dumas method, Simone et al., 1994; Matejovic, 1995). The Stable Isotope Facility (Davis, CA) carried out the analysis for natural 15N abundance as described by Bremmer and Van Kessel (1990). 2.8. Natural abundance nitrogen isotope composition Nitrogen isotope composition was expressed as follows: d15 N Z atom%15 Nsample Katom%15 Nstandard atom%15 Nstandard ! 1000 where the standard is atmospheric N2 (0.3666 atom% 15N) (Mariotti, 1984). The percentage of N derived from N2 xation (% Ndfa) was calculated as follows: %Ndfa Z 100 X KY X KB

where X is the d15N of the non-N2-xing reference plant (C. arabica or L. styraciua), Y the d15N of the eld-grown I. oerstediana, and B is the d15N for I. oerstediana trees grown in an inorganic N-free medium. If X is sufciently large relative to B, then B can be disregarded (Shearer and Kohl, 1986). Values found to be outside the range of 1.5 standard deviations above or below the mean were considered outliers and removed. To calculate %Nfda, only values found to be greater than zero were used to calculate the mean for each treatment. Student t-test was used to estimate signicant differences between d15N values.

3. Results 3.1. Inoculant and substrate treatment effects Results from all experiments indicated that inoculation with selected strains of rhizobia does not confer any advantage to young I. oerstediana seedlings (90 DAP). However, in older seedlings (150 DAP), statistical differences in shoot biomass and nodulation between inoculated treatments and KN controls were present (Fig. 2). Nodule mass, however, was not positively correlated with shoot biomass in any treatment. N-fertilized plants had signicantly higher shoot biomass than the KN control and all inoculated plants under all treatment conditions.


J.M. Grossman et al. / Soil Biology & Biochemistry 38 (2006) 769784





Biomass (g)

C10 2

Biomass (g)




B6 3

0.4 0.2


C8P1 N B63 C102 C9Z4


C5 2 C9Z 4





Treatment (c)
0.5 0.4

Treatment (d) 1.0


Biomass (g)

Biomass (g)


0.3 0.2 0.1 0.0

N+ Native

N+ Sterile N+ Native

N+ Sterile

N Native N Sterile

C52 C9Z4


0.4 0.2 0.0




N Native N sterile








Fig. 2. (a) Effect of inoculation with selected rhizobia strains on Inga oerstediana in sterile substrate after 90d. (b) Effect of inoculation with selected rhizobia strains on Inga oerstediana in sterile substrate after 150d. (c) Shoot biomass (g) of Inga oerstediana in native substrate after 90d greenhouse growth. (d) Effect of inoculation with selected rhizobia strains on Inga oers tediana in n a tive substra te a fter 150d.

Results were essentially the same for plants grown in sterile and native substrates (Fig. 2). Exceptions in sterile substrate included C8P-1, isolated from Inga pavoniana, which demonstrated signicantly lower shoot biomass than the KN plant at 90 DAP (pZ0.015). It is noteworthy that despite the low shoot biomass of C8P-1, the plants had signicantly higher nodule biomass than any other treatment at 150 DAP (Table 1). Exceptions in native soil included 150 DAP seedlings, where four of the ve total

strains (C10-2, C8P-1, B6-3, and C9Z-4) produced plants with signicantly higher shoot biomass than KN controls and equal in biomass to the NC controls, and only one (C5-2) exhibited lower biomass than the KN controls. These results are not surprising given the high soil N of the native substrate (0.950%). Transplant of seedlings grown in sterile substrate to eld conditions at 90 DAP did not encourage production of plant biomass greater than that of the KN control plants for any treatment (pO0.05) (Fig. 3).

Table 1 Mean nodule number and mass of inoculated greenhouse Inga oerstediana (nZ2 except when noted) Treatment or strain Mean nodule number Sterile soil 90 DAP N-fertilized K N C8P-1 C5-2 B6-3 C10-2 C9Z-4 0, nZ5 0, nZ7 12 (14.1) 3.5 (4.9) 1.5 (2.1) 18.5 (26.1) 5.5 (7.7) 150 DAP 0, nZ5 0, nZ7 107 (31.1) nd 42 (4.2) 48.5 (68.6) 17.5 (3.5) Native soil 90 DAP 0 18.3 (18.0) 6.5 (0.7) 6 (0) 3.5 (4.9) 1 (1.4) 5.5 (7.8) 150 DAP 0 0a 18.8 (4.1) 7 (9.9) 13 (5.7) 21 (9.9) 15 (9.9) Mean nodule mass (mg) Sterile soil 90 DAP 0a 0a 10.8 (13.1)a 1.2 (1.7)a 2.6 (3.7)a 9 (12.7)a 6.1 (8.7)a 150 DAP 0a 0a 194.1 (19.2)b ND 72.5 (0)c 189.3a 26.8 (7.1)d Native soil 90 DAP 0a 13.2 (9.1)a 9.2 (1.1)a 4 (5.4)a 2.2 (3.2)a 0.7 (1.0)a 56 (79.2)a 150 DAP 0a 0a 35.4 (2.6)b 0.6 (0.9)a 11.8 (9.8)ab 12.5 (14.4)ab 15.2 (5.4)a

Treatment means for mass appearing in the same column and with the same letter are statistically similar at the 0.05 level of signicance. Standard deviations in parentheses. nd, not determined; DAP, days after planting. a One surviving replicate.

J.M. Grossman et al. / Soil Biology & Biochemistry 38 (2006) 769784


Inga oerstediana sterilesoil after 3 months infield

0.7 0.6



0.4 0.3 0.2 0.1 0.0


AB B C102 C9Z4

Fig. 3. Effect of transplant of Inga oerstediana to native conditions after 90d of grown under sterile conditions in greenhouse (data taken 150dap).

N-fertilized controls in both substrate treatments were signicantly taller than KN controls at both 90 (p!0.0005) and 150 DAP (p!0.012). There was no signicant difference in height between native substrate N-fertilized and KN controls at both harvests. Differences in nodule development between inoculated treatments in both substrates were observed at 150 DAP (Table 1). Most plants grown in sterile and in native substrate in the greenhouse had less than 15 discernible nodules after 90 DAP. However, 150 DAP nodules were clearly visible in both substrates, and inoculated sterile seedlings consistently demonstrated greater nodule biomass than seedlings being inoculated by the same strain under native substrate conditions, with C8P-1 and B6-3 being signicantly greater (Table 1, p!0.05). Of the K N sterile nodule-free plants transferred to the eld 90 DAP, 65% were nodulated at 150 DAP, with all having only six or fewer nodules (Table 2). In all cases nodules had white or brown interiors. No treatment transferred to the eld obtained signicantly greater nodule mass than those growing in native substrate in the greenhouse for the full 150d. Total leaf N of plants harvested from 150 DAP I. oerstediana samples in both sterile and native substrate

is found in Table 3. In only one case, strain C9Z-4, was total tissue N of surviving inoculated plants grown in sterile substrate under greenhouse conditions for 150 DAP signicantly greater than that of plants given no N. In 150 DAP sterile soil treatments, eld transplants had greater N than greenhouse plants. This increase was not surprising since eld transplants had access to higher levels of mineralized soil N in the eld site than greenhouse treatments. No signicant difference was found between the N content of sterile substrate treatments fertilized with N and any of the eld treatments originally grown in native soil. Mean %N of eld samples grown only in native soil throughout the experiment was 3.40% (sd 0.52). Sanginga et al. (1989) reported that N2-xing trees have been shown to prefer obtaining N from available soil sources to xed N2. In this study, there was no apparent plant response to inoculation, however, nodule biomass itself was increased in sterile over native soil strain treatments (Table 1). Lack of nodules present on 150 DAP native soil treatments relative to those grown in sterile soil could be caused by inhibition of nodulation due to high soil N concentration of native soils used in the greenhouse experiment (0.950%) (Dommergues, 1995; Goi et al., 1997). Further evidence supporting the idea that plants are utilizing available soil N levels over xed N2 is that plants grown in native soil throughout the 150 day experiment (Table 3, column 4), including plants never receiving inoculant nor N-fertilizer, demonstrated mean plant tissue N concentrations that were statistically indiscernible (pO 0.05) from the mean of any other N-fertilized treatment. In all inoculated treatments, at least one transplanted eld replicate had nodules visible at 90 DAP that were still present at 150 DAP. At 150 DAP, old nodules often had obtained a dark brown interior pigment, and in many cases small new nodules often had formed on lower ne roots that exhibited white exteriors and interiors. In the eld, both KN and N-fertilized sterile substrate had no visible nodules when transplanted (90 DAP) and both were found to have

Biomass (g)

Table 2 Mean nodule number and mass of inoculated eld Inga oerstediana transplanted from greenhouse, 150d (nZ4, except when noted) Treatment Original soil substrate Mean nodule number (mg) Sterile soil Nodules at 150d N-fertilized (nZ10) K N (nZ10) C8P-1 C5-2 B6-3 C10-2 C9Z-4 4.71 (7.13) 5.07 (5.17) 36 (10.40) 21.3 (26.73) 8.0 (10.46) 15.25 (18.64) 15.5 (11.33) Difference from 90d 0 C5.07 C23 C13 C26 0 C11.5 Native soil Nodules at 150d 0 8.5 (4.8) ND 10.67 (4.04) 24.5 (10.61) 15b 14.5 (10.15) Difference from 90d 0 C2.0 ND 0 C1.25 C4 C5.75 5.0 (9.0) ad 6.8 (8.1)a 90 (31)c 5.7 (0.8)ab 15.8 (23.6)ac 29.5 (46.1) bcd 44.8 (25.3)c 0a 16.25 (7.0)b ND 38.7 (15.4)b 50.0 (38.2)ba 19.0b 17.5 (12.7)b Mean nodule mass (mg) at 150 da Sterile soil Native soil

Treatment means for mass appearing in the same column and with the same letter are statistically similar at the 0.05 level of signicance. Standard deviation in parentheses. ND, not determined. a Data for mass at 90d could not be taken without destruction of nodules, therefore, difference in mass between 90 and 150 days was not recorded. b One surviving replicate.





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Table 3 Mean total tissue N of inoculated greenhouse and eld seedlings of Inga oerstediana at 150 dap Treatment or strain Mean tissue N (%) of greenhouse seedlings (nZ2 unless otherwise noted) Sterile soil N-fertilized K N C8P-1 C5-2 B6-3 C10-2 C9Z-4 Inoculated treatment mean 2.86a (nZ5) 2.19b (nZ7) 2.59ab (nZ1)a ND 1.95b 2.397 (nZ1)a 2.62a 2.39 (0.30) Native soil 2.73a 2.92 (nZ1)a 2.24bc ND 2.61ac 2.42 (nZ1)a 2.53b (nZ3) 2.57 (0.38) Mean tissue N (%) of eld seedlings (nZ3 unless otherwise noted) Sterile soil 3.19a (nZ7) 3.74b (nZ14) 3.61abc 3.80bd (nZ2) 3.70ab 3.99bd 3.44ab (nZ4) 3.70 (0.39) Native soil 3.25a 2.89a 3.69a 2.96a (nZ4) 3.56a 3.59a 3.27a 3.40 (0.52)

Treatment means appearing in the same column and with the same letter are statistically similar at the 0.05 level of signicance. ND, not determined. a Replicates with only one experimental unit were comprised of two or more very small samples within the same treatment group. Combination was required for accurate measurement using NIR instrument.

nodules when harvested at 150 DAP (Table 2.), demonstrating that nodulation in I. oerstediana does occur in situ. 3.2. morphology Nodules on 90 and 150 DAP seedlings were found on or close to lateral root junctions. This lateral root emergence suggests nodulation to occur by rhizobia infecting epidermal breaks during root growth, as occurs in other tropical N2-xing trees (Sprent et al., 1989; Ramirez and Flores, 1994). Root nodules on the mature eld-grown I. oerstediana were scarce and difcult to nd. These mature nodules were spherical or very occasionally slightly oblong, had a brown surface and interior. A red or pink interior typically indicates nitrogen-xation is occurring. However, in these mature Inga nodules taken from the eld, red/pink may have been obscured by the brown coloration thus making this simple test for nitrogen-xation inconclusive. Nodule appearance was consistent with the desmodioid type described by Corby (1988), who characterized such nodules as having lateral root juncture placement, limited determinate growth, non-branching structure, small size, and presence of lenticels. However, no lenticels were visible on I. oerstediana nodules. At 90 DAP, nodule surface and interior were white, and by 150 DAP a brown pigment was evident throughout the nodule surface and interior. On roots collected from the mature eld-grown tree of approximately 5 years of age nodules were found within 10 cm of the soil surface on both the taproot and lateral roots. This appears to be common, as nodules on L. leucocephala, a N2-xing tree, were also found within 1020 cm of the soil surface gberg and Kvarnstro m, 1982). (Ho The size of nodules of I. oerstediana in both the eld and greenhouse studies were small at !5.0 mm axial length. Corby (1988) found nodules of Inga seedlings to fall within the dimensions of 350 mm axial length and 530 mm trans for 80100 DAP nodules, and 9!7 mm for 119 DAP seedlings. Ninety days after inoculation, nodules of I. uraguensis are reported to have dimensions of 1 cm!

1.01.5 cm (Frioni et al., 1998), and nodules of up to 50 mm in length have been recorded (Norris, 1969). Nodules formed with inoculant C8P-1 were larger in size than those formed by other strains and upon microscopic evaluation were shown to have a slight lobed appearance near the meristem region. 3.3. structure and histochemistry Despite nodules round appearance, study of longitudinal nodule sections suggests these nodules to be of indeterminate type, common in N-xing tree species (Corby, 1988; Sprent, 1989, 1995; Frioni et al., 1998). Fig. 4 illustrates the following nodule characteristics. Nodules contained an apical meristematic region followed proximally by cells containing a high concentration of infection threads. Posterior to these cells are cells in the early stages of bacteroid formation followed by a central zone of mature infected and uninfected cells. In the central zone of the nodule, approximately 4080% of cells were occupied by bacteriods. One of the most important ndings was that Lb and NifH transcripts were not detected in these cells, suggesting that they are ineffective in N2 xation. Mature infected cells were always associated with large vacuoles occupying 3060% of the total cell space. However, at least one Inga species (Inga capitata) has been found to be nonvacuolated (Sprent, 1989). Consistent with other authors (Sprent, 1989; Frioni et al., 1998), our Mimosoideae nodules were found to have a thick inner and outer cortex (20200 mm and 80120 mm, respectively) containing numerous vascular bundles surrounding the perimeter of the nodule. There was extensive accumulation of a brown-pigmented material throughout the central zone and inner cortical cells of all nodules evaluated. This material appeared to be contained within a membrane and specically associated with uninfected cells within the body of the nodule. Additionally, inner cortical cells associated with the putative nodule meristem contained a dense concentration

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Fig. 4. Representative section of nodule from seedling inoculated with strain C8P-1, stained with Toluidine blue. Meristem (M), Infection Thread (IT), Early stages Bacteroid Formation (BF), Central Zone (CZ), Vacuole (V).

of this substance. This substance stained blue-green with toluidine blue, but failed to autouoresce under UV illumination. It gave a negative reaction with phloroglucinolHCl, sudan IV and fericyanide. Iodine staining was inconclusive since a positive iodine response was similar to the actual pigment color. This substance was originally thought to be a phenolic material or starch polymer. Sclereids, a lignied sclerenchyma tissue, have been linked to brittle nodule structure in other Inga nodules, rendering them prone to tearing at this region during the sectioning process (Sprent, 1989). Sprent et al. (1989) found such sclereid compounds present in the mimosoid nodules of I. capitata as well as numerous other woody mimosoid species. We observed blue-green staining with toluidine blue, a characteristic of lignin, however, the negative

staining reactions with phloroglucinolHCl, sudan IV and fericyanide indicate a lack of phenol rings indicative of a lignied or suberized substance. A common feature of ineffective nodules is a high concentration of starch in the infected zone (Vance and Johnson, 1983). However, toluidine blue is known to leave starch compounds unstained, and the granules in our nodules stained a deep blue-green. A suberized cell boundary cell layer exists between the inner and outer cortex. This cell layer was sudan IV positive and autouoresced under UV illumination. Outer cortical cell walls of I. oerstediana root nodules are phloroglucinol HCl positive, suggesting that they contain a lignin-like polymer. In contrast inner cortical cell walls are devoid of such substances. Both suberin and lignin deposits are

Table 4 Mean plant tissue N and shoot d15N () of 57 year old Inga oerstediana intercropped with coffee plants, non-leguminous Liquidumbar styraciua, and Coffea arabica on an organic farm in Chiapas, Mexico (nZ11) Plant material or control Inga oerstediana Liquidumbar styraciua (reference plant) arabica (reference plant near) Cafe arabica (reference plant far) Cafe Tissue N (%) (p!0.005) (sd) 3.49a (0.43) 2.44b (0.42) 2.84b (0.42) 2.93b (0.40) Shoot d15N () (p!0.05) (sd) 0.61a (0.61) 0.22a (0.73) 1.41b (0.80) 0.36a (0.92)

Values with different letters indicate signicant differences between I. oerstediana and reference species.


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Table 5 Mean plant tissue N, shoot d15N (), and estimated % nitrogen derived from atmosphere (Ndfa) of 13 year old Inga oerstediana intercropped with coffee plants on a farm in Chiapas, Mexico (nZ15) Plant material Inga oerstediana arabica (reference) Cafe Tissue N (mg plantK1) (p!0.01) 2.9a (0.73) 2.20b (0.43) Shoot d15N () (p!0.01) 5.72a (0.90) 7.28b (1.37) % Ndfa using coffee plants as control 20.4%

Standard deviations between means are in parenthesis following data. Student t-test was used to estimate signicant differences between coffee and I. oerstediana values. Values with different letters indicate signicant differences between I. oerstediana and coffee reference plants.

common to many woody Mimosoideae legumes (Sprent et al., 1989). 3.4.


N natural abundance

In older 57 year plots, signicant differences were found between d15N treatment means using C. arabica-near and I. oerstediana (Table 4), however, standard deviations of means were large enough to cause suspicion in the values and thus they were not used to estimate %Ndfa. There were no differences found between mean d15N of I. oerstediana and the reference plants of C. arabica-far, or L. styraciua I. oerstediana found in the younger 13 year old plot was found to derive 20.4% of its N from BNF (Table 5).

to limited soil P and N necessary for nodulation, a nding supported by others (Szott et al., 1991). This was not an issue with our seedlings as the PNS used to water contained adequate amounts of P and N. Roskoski (1982) has also reported I. vera in C. arabica plantations of central Mexico to be non-nodulating. In contrast, Fernandes (1998) and Corby (1988) describe inoculated I. edulis seedlings as profusely nodulated at 90116 DAP. As inoculated plants and nodules of treatment C8P-1 demonstrated high biomass at 150 DAP, future inoculation studies should extend beyond this period of growth. 4.2. Inga sp. root nodule structure Desmodioid nodule type as observed here is unusual for woody legumes of the Mimosoideae family (Corby, 1988). With regards to Inga, Corby described nodules of seven inoculated species as mostly mucunoid, caesalpinioid, and in only one case as desmodioid (Inga nobilis, 80100 DAP), whereas Frioni et al. (1998) found I. uraguensis nodules 90 days after inoculation to have mucunoid morphology, with large size (often exceeding 10 mm in length) and dense branching. Larger size of nodules formed with inoculant C8P-1 suggests eventual development toward elongated and branched non-desmodioid morphology, as has been observed in other N2-xing tree species (Giller, 2001). Histological examination of Inga sp. nodules formed when inoculated by indigenous root nodule bacteria showed all nodules to be well infected by rhizobia after 150 DAP. This leads us to question the lack of leghemoglobin and Nif H mRNA transcript present in the nodules. No abnormal structural characteristics linked to ineffective perennial nodules recognized by Vance and Johnson (1983) and Vance et al. (1980) were identied. It is highly unlikely the dissimilar strains tested here (Grossman, 2005) would each induce ineffectiveness as seen in tested plants. This leads us to believe that ineffectiveness is plant induced. Presence of bacteroids in infected cells indicates that bacteria were successfully released into cells via the observed infection thread. As synthesis of nitrogenase is known to occur almost immediately after bacteria release (Paau and Brill, 1982), and leghaemoglobin produced at approximately the same time (Sprent, 1989), failure in nodule development and N2xation appears to occur sometime around bacteria release into the nodule cell.

4. Discussion 4.1. Strain evaluation Native rhizobia strains appeared not to produce a growth response when inoculated onto the host I. oerstediana until 150 DAP, when only a slight, highly variable response was observed. Moreover, since plant growth and nodule biomass were not correlated, and Lb and NifH transcript not observed in nodules, none of the isolates tested appear effective in xing N2 5 months after inoculation. Nodulation was limited in seedlings planted in native soil, leading us to question the symbiotic effectiveness of the indigenous soil isolates of I. oerstediana and consequently the value of this host as a source of N to intercropped C. arabica plants. Inga oerstediana appears to be slow to nodulate, even using high concentrations of prepared inoculant as we did here. Nodulation is normally observed in herbaceous species 618 days after inoculation (Graham, 2000). Leguminous trees have been found to nodulate in as little n and as 8 days after inoculation (Acosta-Dura nez-Romero, 2002), but many often take up to Mart 100 days (Corby, 1988; Goi et al., 1997; Fernandes, 1998). Inga appears to commonly take longer to nodulate than other leguminous trees. Our observations of negligible nodulation at 90 DAP, and very limited nodulation at 150 DAP parallels those of Van Kessel and Roskoski (1981), who rst recorded nodules on I. jinicuil in a pot experiment using diluted native soil 130 DAP, and in eld experiments 200 DAP. In both cases authors attributed nodulation delay

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4.3. Natural abundance and nitrogen xation Similar d15N values of crop plants to that of intercropped legume species, as observed in older 57-year-old sampled C. arabica plots (Table 4), might suggest a transfer of Ndfa from I. oerstediana to non-leguminous crops such as C. arabica. This similarity of d15N values of both reference species to I. oerstediana supports the idea that in well established agroforestry systems; i.e. greater than 6 years of age in stands of L. leucocephalia (Van Kessel et al., 1994), or established C. arabica systems as studied here, the natural 15N abundance method may be unsuitable for evaluating % Ndfa. An important difference between the older and younger plots is effect of C. arabica harvest on d15N values. Plants in the younger plot had not yet begun to produce coffee. Thus it represented essentially a closed system as no N was being exported in the harvest. This loss of N could remove recycled xed N and available non-xed soil N from the system, of which only a fraction is replaced through C. arabica berry pulp present in annually applied compost. As nitrogen xing trees prefer to derive their N from available soil N over atmospheric sources (Sanginga et al., 1989), this export of N could possibly serve to alter system N dynamics by lowering the total available soil N and increase I. oerstedianas dependence on N-xation to meet its N needs. Future studies are necessary to evaluate overall N balances in agroforestry systems that do or do not export N in the coffee harvest, and that use inputs of N in the form of compost and N2-xation. In the younger 13 year old plots, %Ndfa (Table 5) was less than other reported for other nitrogen-xing tree species (Kadiata et al., 1998; Boddey et al., 2000), though one study reports 9-month-old G. sepium having a %Ndfa of 15.6 (Kadiata et al., 1998). Our report of low %Nfda could possibly be due to the slow onset of nodulation and nitrogen xation in Inga oerstediana when using native rhizobia found in situ, as our study indicated that nodules in inoculated tree seedlings were found to not be functional after 150d. Such low levels of nitrogen xation could suggest that at early stages of coffee production with intercropped shade trees, C. arabica may be competing with associated young legumes for soil nutrients. Age of tree was also found by Muofhe and Dakora (1999) to be a factor inuencing %N derived from xation in 1-year-old Aspalathus linearis compared to the higher %N derived from xation in 3-year-old trees. The greater variation in d15N among replicates of the same species in the 57 year old plots (Table 4) could be attributed to non-uniform placement of compost by farmers, whereas the younger plots had received fewer annual compost treatments. The roots of younger trees and shrubs are less developed and thus occupy less total soil volume. Some (Piccolo et al., 1996), but not all (Gathumbi et al., 2002) authors have reported soils at different depths having variations in d15N, meaning that larger, better developed trees might take up N from depths with different 15N

signature values. The use of younger trees with less explored soil area further ensures that reference and legume species are utilizing a homogeneous 15N-to-14N ratio soil source. Estimated d15N values for our reference species in the older plots were among the lowest reports of agroforestry woody non-legumes found in the literature (Boddey et al., 2000; Gathumbi et al., 2002), and up to 20-fold lower than reports of other woody reference species in Latin America (Yoneyama et al., 1993). It has been shown in other agroforestry systems intercropping woody legumes and non-legumes that as N accumulates in the soil following N2 xation and legume litter decomposition, N2 xation will slow (Kadiata et al., 1998), and then cease as legumes have sufcient soil N available to meet their growth needs (Dommergues et al., 1984; Van Kessel et al., 1994). C. arabica farmers in Chiapas prune I. oerstediana annually beginning when trees are 4 years old in order to manage light intensity and moisture levels in their coffee plots. Pruned material is chopped with a machete and left on the soil as a surface mulch. Low 15N values observed in 57 year reference species could possibly be due to transfer of xed N from legumes to non-legumes over time, resulting in a reduced d15N of the reference species (Van Kessel et al., 1994), however it has been shown in L. leucocephala and G. sepium that only incorporation of material into soil, not surface mulching, serves to reduce the amount of N xed per tree over time (Kadiata et al., 1998). The d15N value found in the C. arabica reference species in the younger plot was 530 fold higher than those of the older plot (using references of C. arabica-near and C. arabica-far, respectively), and falls within the range for other woody nonleguminous species in the literature (Boddey et al., 2000). This high value, when compared to the much lower value of the older plot, supports the idea that xed N had been recycled in the older system serving to lower the d15N of the reference species shoot material. In only one identied case was the d15N value of an N2xing tree found to be higher than both of our sets of data from Inga oerstediana (Gathumbi et al., 2002, d15NZ0.91), with other authors reporting leguminous trees with considerably lower legume d15N, ranging from K2.89 to K0.34 (Boddey et al., 2000). Non N2-xing C. arabica in younger plots exhibited a signicant enrichment in 15N relative to the I. oerstediana of the same age (Table 4, p! 0.01). However, in the older plot this enrichment of C. arabica was signicantly reduced d15N (p!0.001). The enrichment of younger reference species 15N relative to that of the older reference species was possibly due to the fact that younger I. oerstediana had not yet been subjected to the annual pruning and mulching cycle, and thus it is likely that little xed N was recycled to the younger reference species in the form of mineralizable N provided through legume leaf material. In the 57 year old plots, 15N was likely recycled from legume to non-legume, reducing the enrichment of 15N observed in the young plot. Values of


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I. oerstediana d15N in the older plot were found to correspond to those found by Van Kessel et al. (1994) for L. leucocephala that had been associated with non-xing reference species (and therefore taking up recycled nonxed N) for 6 years, while in younger plots our values were slightly higher than van Kessels 1 year old L. leucocephala that was more dependent upon atmospheric N2 than available soil N. It can be concluded that 13 year old trees of the younger plot had possibly not begun to x nitrogen to their fullest potential. In both young and old plots, total leaf tissue N of I. oerstediana was shown to be signicantly higher than non-leguminous control species (Tables 4 and 5), with the I. oerstediana leaf tissue in younger plots having signicantly less N than the older plots (p!0.05). This reduced total N concentration supports the idea that after 13 years, I. oerstediana is not xing N up to its fullest potential, however more research on C. arabica agroforestry systems is necessary. We found that in older more established agroforestry systems, natural abundance may not be a reliable technique for assessing nitrogen xation on farm due to recycling of xed N that occurs between leguminous and nonleguminous species. On younger plots where the xed N held in tree tissue has not been returned to the system, this technique seems to be reliable for a rough estimation of nitrogen xed by tree legumes associated with non-legume perennial crops such as coffee, which in C. arabica I. oerstediana systems appears to be low. It will be necessary to conrm this method by comparison to other methods of nitrogen xation estimation, such as the N-difference technique, as well as temporal sampling to investigate changes in plant d15N over time. 4.4. Management implications Our results may not be surprising given data collected from other low-input coffee systems in Chiapas and elsewhere. Despite organic farmer claims that Inga shade improves coffee plant health (Grossman, 2003), Romero-Alvarado et al. (2002) found no differences in C. arabica yield or soil total N content in systems dominated by I. sapindioides and those containing few to no Inga species. These ndings are in contrast to older studies showing coffee shaded with nodulated I. jinicuil to have higher yields than in an adjacent plot of non-nodulated I. vera (Roskoski, 1982). In general, the literature reports relatively low contributions of N through N2-xation of shade trees in coffee agroecosystems (Beer et al., 1998), which using the acetylene reduction technique fall within the range of 35 60 kg N haK1 yrK1 (Van Kessel and Roskoski, 1981; Escalante et al., 1984; Roskoski and Van Kessel, 1985; Lindblad and Russo, 1986). However, nodulation and nitrogen-xing activity of Inga, as in other legumes, has been shown to be heavily inuenced by the types and amounts of additional nutrients supplied, suggesting that

the quantity of N xed may be amenable to manipulation through simple management techniques (Van Kessel and Roskoski, 1983). Given the difculty of accurately measuring N2 xation in trees, the natural 15N abundance method is recommended over other methods (Boddey et al., 2000; Gathumbi et al., 2002) and should be corroborated with nodule mass and plant growth to measure actual N2xation in Inga. Farmers in the region where this study took place claim that I. oerstediana is a local species. However, farmers often participate in seed trading and propagation of Inga for specic use within their C. arabica plantations. It is therefore likely that farmer accessions of Inga have travelled in and around the southern Mexico region. If future data continues to indicate that nodules formed by symbioses with the local bacteria are non-functional, inoculation of this species with alternate bacteria sources and selection of Inga varieties is recommended. However, for an inoculant program to be accepted by local small-scale farmers, care should be taken to integrate stakeholder participation into the inoculant technology development program. Furthermore, efforts to identify centers of origin for Inga species should be increased to help match Inga species with effective symbionts. Little is understood about the symbiotic relationships between tropical legumes and their symbionts (Parker, 2003; Susilo et al., 2004). Our study indicates that further research with Ingas symbiotic relationship with indigenous Bradyrhizobia could aid in improving organic coffee production throughout Latin America. In light of results from this study, farmers claim that C. arabica plants have better growth and development under Inga sp. shade could possibly be attributed to characteristics of the deposited leaf litter itself, including its ability to control soil temperatures (Fernandes, 1998), increase soil organic matter (Nair and Rao, 1977; Araya, 1994; Beer et al., 1990, 1998), and guard against soil moisture loss and erodability on steep slopes (Beer et al., 1998), rather than to N contribution by I. oerstediana. In bean systems in Costa Rica, Rosemeyer et al. (2000) found that when added as mulch, litter from I. edulis reduced bean nodule biomass and increased bean yield in comparison to systems mulched with non-leguminous secondary vegetation, suggesting that Inga litter is contributing to improved overall soil health. I. edulis leaves have been shown to have increased concentrations of polyphenolics when compared to other agroforestry legumes (Palm and Sanchez, 1991), contributing to their slow decomposition, and likely negatively inuencing their release of N (Palm and Sanchez, 1990) when left as a surface mulch. Further, Janos (1975) reported signicant increases in height, cotyledon retention and the number and length of leaves of I. oerstediana after inoculation with mycorrhizal fungi, indicating that the tripartite relationship between mycorrhizae, rhizobia and Inga should also be investigated as a means to improve growth and development of this tree.

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Our results suggest that in order to achieve sustained organic coffee production, conservation benets, and longterm ecosystem maintenance, careful attention must be paid to shade tree species composition of agroforestry systems. Increases in coffee prices during the 1970 s stimulated transformation from diverse coffee agroecosystems into homogenous systems containing C. arabica and a limited diversity of shade trees (Fuentes-Flores, 1979; Hoffmann et al., 1987; Nestel, 1995), which in Mexico was encouraged ), the state by INMECAFE (Instituto Mexicano del Cafe agency responsible for trade and production of coffee in Mexico until 1990. INMECAFE promoted technological packages including the intensive use of agrochemicals, the reduction or complete removal of native shade trees in favor of Inga species, and the increase in density of C. arabica trees per unit of land (Toledo et al., 1985; Hoffmann et al., 1987), leaving many farms shaded by a monoculture of Inga species. Recent studies have shown that losses of diverse shade tree species in traditional coffee systems are correlated with reductions in both insect diversity (Nestel and Dickschen, 1990; Nestel et al., 1993; Perfecto and Vandeermeer, 1994; Perfecto and Snelling, 1995), and benets derived from multi-use tree species such as rewood, food, and construction materials (Soto-Pinto et al., 2001). In contrast, Greenberg et al. (1997) showed that both coffee grown under a canopy of native forest and under a planted canopy dominated by Inga spp. were found to support a high overall diversity of bird species. Together with other authors (Peeters et al., 2003), we recommend that benets derived from a diverse shade canopy be thoroughly weighed against those of using Inga spp. monoculture for provision of N via BNF. We suggest that organic coffee production should be achieved via a diverse shade cover that includes leguminous trees with high N xation potential. Historically, in Chiapas the leguminous tree genus of choice has been Inga, however, it should be noted that other quickly established leguminous species might help to improve the N economy of the system while contributing to an increased diversity in the system. Suggestions of species to be evaluated might include Cajanus cajan, Desmodium species, Calliandra species, Gliricidia sepium or Erythrina species (CIDDICO, 1995; Dommergues, 1995; Veasey et al., 1995; Budowski and Russo, 1997; Rosemeyer et al., 2000). Our conclusion is that the selected Inga isolates tested in this study lack symbiotic effectiveness with the common host tree I. oerstediana 150 DAP. Our results showing that the tested isolates appear not to be xing N2 150 DAP and inoculation with isolated bacteria is supported by histology data demonstrating that inoculated treatment nodules lacked leghemoglobin and Nif H mRNA transcript, as well as plant data showing total shoot N equal to that of the KN control plants. This serves to decrease the value of these trees for N addition in organic C. arabica farms during early growth of Inga (15 months of age), as trees will possibly be competing with the coffee crop by utilizing available soil

N (Beer et al., 1998). Further, BNF in 13 year old C. arabicaI. oerstediana systems appears to be low when measured on-farm using the 15N natural abundance technique. Our results suggesting low nodulation and nitrogen xation in Inga are consistent with other authors whose results indicate that some Inga species (I. edulis) may have delayed growth and development during the rst two years of life (Nichols et al., 2001). However Inga seems to eventually recover, as other studies have shown growth, nodulation and nitrogenase activity of I. edulis to be among the best in 38 year trials of leguminous agroforestry species (Tilki and Fisher, 1998; Carpenter et al., 2004). This is perhaps correlated to I. edulis ability to increase organic C, extractable P and total N in some soil systems after three years (Fisher, 1995). We do not know whether the symbiosis between I. oerstediana and associated isolates will over time become more effective, and no structural or histological characterization of older nodules (O150 DAP) has yet been carried out. Amount of total N added from agroforestry species mulch, including Inga, that is taken up by the crop is typically quite low, with recoveries at less than 20% (Palm, 1995). In order to achieve the goal of increased production of intercropped coffee species, it therefore becomes necessary to examine not only BNF in Inga species, but pruning nutrient release patterns as well. Recommendations for those working with organic farmers and Inga systems include increased use of composts and other sources of N accepted in organic production systems, especially during early establishment of Inga. As organic C. arabica production in low input systems is in part dependent upon the effective xation of Inga species to provide an available source of N to the coffee plants, additional research in this area would be extremely valuable.

Acknowledgements I would like to thank the MacArthur Interdisciplinary Program on Global Change, Sustainability and Justice; a Robles/US-Mexico Commission for EduFulbright-Garc cational and Cultural Exchange; the Department of Agronomy and Plant Genetics, and the Graduate School of the University of Minnesota for providing the funding that made this project possible. Sincere thanks to Michael Grossman at ESRI, who helped create the map of the study sites. El Colegio de la Frontera Sur (ECOSUR) in San Cristobal de Las Casas, Chiapas, provided me with essential xico, especially David Alvarezinstitutional support in Me a-Barrios, Miguel Angel Lopez-Anaya, and Solis, Luis Garc n Majomut, notably Walter AnzuetoLourdes Herrera. Unio Anzueto and Victor Perez-Grovas Garza, arranged invaluable communication with the study communities. Most of all, I offer my greatest sincere gratitude to the producers of n Majomut for allowing me to become acquainted with Unio


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