Endophytic nitrogen fixation in sugarcane: present knowledge and future applications

In Brazil the long-term continuous cultivation of sugarcane with low N fertiliser inputs, without apparent depletion of soil-N reserves, led to the suggestion that N₂-fixing bacteria associated with the plants may be the source of agronomically significant N inputs to this crop. From the 1950s to 1970s, considerable numbers of N₂-fixing bacteria were found to be associated with the crop, but it was not until the late 1980s that evidence from N balance and ¹⁵N dilution experiments showed that some Brazilian varieties of sugarcane were able to obtain significant contributions from this source. The results of these studies renewed the efforts to search for N₂-fixing bacteria, but this time the emphasis was on those diazotrophs that infected the interior of the plants. Within a few years several species of such `endophytic diazotrophs' were discovered including Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae, H. rubrisubalbicansand Burkholderia sp. Work has continued on these endophytes within sugarcane plants, but to date little success has been attained in elucidating which endophyte is responsible for the observed BNF and in what site, or sites, within the cane plants the N₂ fixation mainly occurs. Until such important questions are answered further developments or extension of this novel N₂-fixing system to other economically important non-legumes (e.g. cereals) will be seriously hindered. As far as application of present knowledge to maximise BNF with sugarcane is concerned, molybdenum is an essential micronutrient. An abundant water supply favours high BNF inputs, and the best medium term strategy to increase BNF would appear to be based on cultivar selection on irrigated N deficient soils fertilised with Mo.     

Improving Sugarcane Growth and Nutrient Uptake by Inoculating Gluconacetobacter diazotrophicus

Seven Gluconacetobacter diazotrophicus strains from sugarcane roots were screened for their efficiency to promote growth and nutrient uptake in sugarcane at three levels of urea N (0, 75, and 150 kg N ha⁻¹). Inoculation by these strains improved germination, tiller number and plant height. N-uptake and apparent N-recovery increased due to inoculation and the effect was more at N₇₅ level. Gluconacetobacter diazotrophicus isolate IS100 was found to be the most efficient in promoting plant growth and nutrient uptake in sugarcane.     

Molecular assessment of diversity among endophytic diazotrophs isolated from subtropical Indian sugarcane

Twenty-two endophytic bacterial isolates from the roots of sugarcane were compared morphologically, biochemically and genetically. Gram staining, colony pigment, texture and other cultural characteristics were taken for morphological characterization. Oxidation-fermentation tests for D-glucose and D-sucrose, production of acid and hydrogen from different carbon source, oxidase activity, antibiotic and drug resistance patterns were chosen as the biochemical and physiological criteria. Twelve random decamer primers were used to analyze and compare these isolates through RAPD among themselves as well as with known standard diazotrophic strains. The isolates were compared through dendrograms constructed on the basis of similarity patterns obtained from biochemical and RAPD analysis. The estimated diversity through RAPD analysis was more evident than the diversity on the basis of morphological and biochemical characters. Within Acetobacter group, the isolates showed substantial genetic diversity for future exploitation as PGPRs and diazotrophic associative endophytes.     

Yield of micropropagated sugarcane varieties in different soil types following inoculation with diazotrophic bacteria

It is well described that the beneficial interactions between plants and bacteria are genotype and site specific. Brazilian sugarcane varieties can obtain up to 70% of their nitrogen requirement from biological nitrogen fixation (BNF), and this contribution is related to the Brazilian breeding and selection processes, by example of the variety SP70-1143. In this study the effect of two inoculation mixtures containing diazotrophic bacteria in our earlier pot experiment was evaluated with two sugarcane varieties, a known responder, SP70-1143, and a newly selected variety, SP81-3250, to investigate the sugarcane genotype effect and the role of the mixtures. The sugarcane varieties SP70-1143 and SP81-3250 were grown under commercial field conditions at three sites with contrasting soil types: an Alfisol, an Oxisol and an Ultisol that means a low, medium and high natural fertility respectively. The stem yield and BNF contribution in response to bacterial inoculation were influenced by the strain combinations in the inoculum, the plant genotype, and the soil type and nitrogen fertilization, confirming the genetic and environmental influence in PGP-bacteria interactions. Inoculation effects on the BNF contribution and stem yield increased in the variety SP70-1143 grown in the Alfisol without nitrogen fertilization for three consecutive crops, and it was equivalent to the annual nitrogen fertilization. The plants grown in the Oxisol showed small increases in the productivity of the variety SP70-1143, and in the Ultisol the sugarcane plants presented even decreases in the stem productivity due to inoculation with diazotrophic bacteria mixtures. The results demonstrate the feasibility of the inoculation technology using diazotrophic bacteria in micropropagated sugarcane varieties grown in soils with low to medium levels of fertility. In addition, the results also indicated that specific plant – bacteria – environment combinations are needed to harness the full benefits of BNF. Section Editor: C. P. Vance     

Improved methodology for isolation of Acetobacter diazotrophicus and confirmation of its endophytic habitat

Nitrogen-free, semi-solid defined medium with crystallized cane sugar (100 g/l) supplemented with cane juice (5 ml/l) was the most selective for isolating Acetobacter diazotrophicus. Surveys of A. diazotrophicus using this medium showed that >10³ cells/g fresh wt were present at all sites in all parts of the sugar cane plant and in all trash samples examined, reaching up to 10⁷/g. Additional samples, from forage grasses and cereals and from weed species collected within the sugar cane fields, were all negative. Heat treatment (50°C for 30 min) of the sugar cane setts did not affect A. diazotrophicus numbers within the plant. Nitrogenase activity of intact soil-plant systems in pots planted with heat-treated setts did not respond to inoculation with A. diazotrophicus. The endophytic habitat of this diazotroph and its propagation within the stem cuttings was confirmed.The authors are with EMBRAPA-CNPAB, Cx Postal 74.505, Seropédica, Rio de Janeiro, 23851-970, Brazil     

Ammonia-oxidizing bacteria on root biofilms and their possible contribution to N use efficiency of different rice cultivars

Ammonia-oxidizing bacteria (AOB) populations were studied on the root surface of different rice cultivars by PCR coupled with denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization (FISH). PCR-DGGE of the ammonium monooxygenase gene (amoA) showed a generally greater diversity on root samples compared to rhizosphere and unplanted soil. Sequences affiliated with Nitrosomonas spp. tended to be associated with modern rice hybrid lines. Root-associated AOB observed by FISH were found within a discrete biofilm coating the root surface. Although the total abundance of AOB on root biofilms of different rice cultivars did not differ significantly, there were marked contrasts in their population structure, indicating selection of Nitrosomonas spp. on roots of a hybrid cultivar. Observations by FISH on the total bacterial community also suggested that different rice cultivars support different bacterial populations even under identical environmental conditions. The presence of active AOB in the root environment predicts that a significant proportion of the N taken up by certain rice cultivars is in the form of NO₃ ^–-N produced by the AOB. Measurement of plant growth of hydroponically grown plants showed a stronger response of hybrid cultivars to the co-provision of NH₄ ⁺ and NO₃ ^–. In soil-grown plants, N use efficiency in the hybrid was improved during ammonium fertilization compared to nitrate fertilization. Since ammonium-fertilized plants actually receive a mixture of NH₄ ⁺ and NO₃ ^– with ratios depending on root-associated nitrification activity, these results support the advantage of co-provision of ammonium and nitrate for the hybrid cultivar.     

Influence of nitrogen fertilisation on the population of diazotrophic bacteria Herbaspirillum spp. and Acetobacter diazotrophicus in sugar cane (Saccharum spp.)

We report studies on the possible effects of fertilisation with high level of N (300 kg of N ha^-1) on the occurrence and numbers of the diazotrophic bacteria Herbaspirillum spp. and Acetobacter diazotrophicusin sugar cane plants. In the sugar cane genotype SP79-2312, the N fertilised plants generally showed higher concentrations of this element. These same plants also had lower numbers of A. diazotrophicus, while the population of Herbaspirillum spp. was not affected by N application. These differences in the concentration of N and the numbers of A. diazotrophicus due to N application were not shown in the variety SP70-1143. The numbers of A. diazotrophicus were also shown to be influenced by the harvest time, becoming reduced in the harvests that coincided with dry periods of the year.     

The effect of inoculating endophytic N2-fixing bacteria on micropropagated sugarcane plants

We investigated the effects of an autumn sowing of contrasting cover crops (oats, rye and a combination of oats and rye) on soil aggregate stability, mycorrhizal colonization, phosphorus uptake and yield of sweet corn planted the following summer. Rye is a common cover crop in the middle Atlantic region of the United States of America. It grows slowly in the autumn, survives the winter, grows rapidly in the spring and flowers in the summer. Thus, herbicide is commonly used to kill rye prior to planting spring crops. Oats, in contrast, grows rapidly in the autumn but is killed by frost during the winter. Thus, with oats, potentially less herbicide is needed to prepare the field for spring planting. When compared to fallow, oats was as effective as rye in increasing mycorrhizal colonization of sweet corn, density of mycorrhizal hyphae, and soil aggregate stability. An oats cover crop may thus be a viable alternative to rye. The combination of cover crops (rye and oats), however, was significantly better than single species of cover crops in terms of sweet corn mycorrhizal colonization, P uptake and yield of sweet corn.     

Isolation of endophytic endospore-forming bacteria from Theobroma cacao as potential biological control agents of cacao diseases

Sixty-nine endospore-forming bacterial endophytes consisting of 15 different species from five genera were isolated from leaves, pods, branches, and flower cushions of Theobroma cacao as potential biological control agents. Sixteen isolates had in vitro chitinase production. In antagonism studies against cacao pathogens, 42% inhibited Moniliophthora roreri, 33% inhibited Moniliophthora perniciosa, and 49% inhibited Phytophthora capsici. Twenty-five percent of isolates inhibited the growth of both Moniliophthora spp., while 22% of isolates inhibited the growth of all three pathogens. Isolates that were chitinolytic and tested negative on Bacillus cereus agar were tested with in planta studies. All 14 isolates colonized the phyllosphere and internal leaf tissue when introduced with Silwet L-77, regardless of the tissue of origin of the isolate. Eight isolates significantly inhibited P. capsici lesion formation (p = 0.05) in detached leaf assays when compared to untreated control leaves. ARISA with bacilli specific primers amplified 21 OTUs in field grown cacao leaves, while eubacteria specific primers amplified 58 OTUs. ARISA analysis of treated leaves demonstrated that inundative application of a single bacterial species did not cause a long-term shift of native bacterial communities. This research illustrates the presence of endospore-forming bacterial endophytes in cacao trees, their potential as antagonists of cacao pathogens, and that cacao harbors a range of bacterial endophytes.     

Biological control of take-all in wheat by endophytic Bacillus subtilis E1R-j and potential mode of action

The bacterial strain E1R-j, isolated as an endophyte from wheat roots, exhibited high antifungal activity to Gaeumannomyces graminis var. tritici (Ggt). Strain E1R-j was identified as Bacillus subtilis based on morphological, physiological and biochemical methods as well as on 16S rDNA analysis. This strain inhibited mycelium growth in vitro of numerous plant pathogenic fungi, especially of Ggt, Coniothyrium diplodiella, Phomopsis sp. and Sclerotinia sclerotiorum. In greenhouse experiments, soil drenches with cell densities of 10⁶, 10⁹ and 10¹² CFU ml⁻¹ E1R-j reduced significantly take-all disease, caused by Ggt, in wheat seedling by 62.6%, 68.6% and 70.7%, respectively, compared to the inoculated control, 4 weeks after sowing. Growth parameters such as lengths and fresh weights of roots and shoots of Ggt-inoculated control plants were significantly lower compared to Ggt-inoculated and E1R-j treated plants. Field experiments in the season 2006/2007, heights of wheat plants in the Ggt inoculated plots were significantly reduced compared to the non inoculated treatments. Yield parameters such as kernels per head and thousand kernel weight (TKW) in inoculated control plants were lower compared to the other treatments. In the experimental year 2007/2008, independent treatments with the bacterial strain E1R-j and the fungicide Triadimefon reduced take-all disease in wheat roots by 55.3% and 61.9%, compared to the inoculated control plants. In this season plant height in inoculated control was significantly lower and also the yield parameters seeds per head and especially TKW were drastically reduced compared to the other treatments. E1R-j treatment alleviated the detrimental effects of take-all on grain yield parameters to a similar extent as Triadimefon application. SEM studies revealed that in the presence of E1R-j, hyphae of Ggt showed leakage, appeared ruptured, swollen and shriveled. Following root drench, strain E1R-j was able to colonize endophytically roots and leaves of wheat seedlings. While the population of the bacterial strain in wheat roots steadily increased from the second to the fourth leaf stage, in the leaf tissue the population of the strain rapidly declined. TEM studies also showed that cells of E1R-j were present in roots of wheat seedlings and effectively retarded infection and colonization of Ggt in root tissue; suppression of Ggt by E1R-j was accompanied by disintegration of hyphal cytoplasm. In addition, in the presence of E1R-j cells in Ggt-infected root tissue morphological defense reactions were triggered such as formation of wall appositions and papillae. The results presented indicate that the endophytic strain E1R-j of B. subtilis meets demands required for biocontrol of take-all.     

Foliar Iron Fertilization of Peach (Prunus persica (L.) Batsch): Effects of Iron Compounds, Surfactants and Other Adjuvants

During a survey of nitrogen-fixing Burkholderia associated with sugarcane in Tamil Nadu, some endophytes were isolated on PCAT medium. Isolation was based on the use of the selective PCAT medium. Four isolates were studied, all belonging to the genus Burkholderia. One of them, MG43 was consistently more active in reducing acetylene and was identified as Burkholderia vietnamiensis. This isolate was used to inoculate micro-propagated sugarcane plantlets in a comparison with two other diazoptrophs, viz. Gluconacetobacter diazotrophicus^T and Herbaspirillum seropedicae^T. Inoculated plants and uninoculated controls were used in a pot experiment followed by two field experiments under different rates of nitrogen fertilisers. MG43 and G. diazotrophicus performed best in sugarcane, their natural host. Biomass increase due to MG43 inoculation reached 20% in the field. Inoculated plants were heavily colonised by the inoculated bacterium (up to 115,000 CFU g⁻¹ root fresh weight). Inoculation by a combinaison of the three strains performed less well than inoculation by a single MG43 suspension. Ecological implications are discussed, as well as the potential of these bacteria to provide a feasible alternative to higher N fertilisers rates in a low input and long term sustainable rural economy.     

Evaluation of tuber bearing Solanum species belonging to different ploidy levels for its yielding potential at low soil fertility

Virus free tubers from thirty-six clones of tuber bearing Solanum species belonging to different ploidy levels, from the world gene bank at the International Potato Center, were cultivated under field conditions at low and high fertilizer rates. Nine of the clones were chosen for their high yielding potential (advanced group) and twenty-seven for their extensiveness (native group). The tuber yields of the thirty-six clones at the two fertilizer levels were positively correlated and the advanced group yielded more than the native group, even under conditions of low soil fertility.A broad genetic variation in root size at both fertilizer levels was apparent. Although some native clones showed rather large root systems as a group, the advanced clones had a similar root dry weight to the native clones.Tuber dry weight was significantly correlated with root dry weight but the advanced clones had a higher tuber yield per unit of root dry matter produced.     

Sucrose partitioning between vascular bundles and storage parenchyma in the sugarcane stem: a potential role for the ShSUT1 sucrose transporter

A transporter with homology to the SUT/SUC family of plant sucrose transporters was isolated from a sugarcane (Saccharum hybrid) stem cDNA library. The gene, designated ShSUT1, encodes a protein of 517 amino acids, including 12 predicted membrane-spanning domains and a large central cytoplasmic loop. ShSUT1 was demonstrated to be a functional sucrose transporter by expression in yeast. The estimated K_m for sucrose of the ShSUT1 transporter was 2 mM at pH 5.5. ShSUT1 was expressed predominantly in mature leaves of sugarcane that were exporting sucrose and in stem internodes that were actively accumulating sucrose. Immunolocalization with a ShSUT1-specific antiserum identified the protein in cells at the periphery of the vascular bundles in the stem. These cells became lignified and suberized as stem development proceeded, forming a barrier to apoplasmic solute movement. However, the movement of the tracer dye, carboxyfluorescein from phloem to storage parenchyma cells suggested that symplasmic connections are present. ShSUT1 may have a role in partitioning of sucrose between the vascular tissue and sites of storage in the parenchyma cells of sugarcane stem internodes.     

Diverse endophytic nitrogen-fixing bacteria isolated from wild rice Oryza rufipogon and description of Phytobacter diazotrophicus gen. nov. sp. nov.

Twenty-three nitrogen-fixing bacteria were isolated from surface-sterilized stems and roots of wild rice Oryza rufipogon. Four clusters were defined among these bacteria by SDS-PAGE protein patterns and further confirmed by IS-PCR finger-printing analysis. Phylogenetic analysis of 16S rRNA gene sequences showed that the representative strains LS 8 and LS 18 of cluster II formed a monophyletic group sharing 94.0-97.3% similarities with defined enterobacterial species within the genera Salmonella, Citrobacter, Pantoea, Klebsiella, and Enterobacter. DNA-DNA hybridization, physiological, biochemical tests, and cell morphology also revealed that these strains could be differentiated from the related enterobacterial species. Based upon these results, we propose Phytobacter diazotrophicus gen. nov., sp. nov. to the bacterial group represented by strains LS 8 and LS 18. The type strain is LS 8(T) (=DSM 17806(T) = LMG 23328(T) = CGMCC 1.5339(T)). The DNA G+C content of strain LS 8(T) is 58.6 +/- 0.5 mol%.     

Study of sugarcane pieces as yeast supports for ethanol production from sugarcane juice and molasses

Due to the environmental concerns and the increasing price of oil, bioethanol was already produced in large amount in Brazil and China from sugarcane juice and molasses. In order to make this process competitive, we have investigated the suitability of immobilized Saccharomyces cerevisiae strain AS2.1190 on sugarcane pieces for production of ethanol. Electron microscopy clearly showed that cell immobilization resulted in firm adsorption of the yeast cells within subsurface cavities, capillary flow through the vessels of the vascular bundle structure, and attachment of the yeast to the surface of the sugarcane pieces. Repeated batch fermentations using sugarcane supported-biocatalyst were successfully carried out for at least ten times without any significant loss in ethanol production from sugarcane juice and molasses. The number of cells attached to the support increased during the fermentation process, and fewer yeast cells leaked into fermentation broth. Ethanol concentrations (about 89.73–77.13 g/l in average value), and ethanol productivities (about 59.53–62.79 g/l d in average value) were high and stable, and residual sugar concentrations were low in all fermentations (0.34–3.60 g/l) with conversions ranging from 97.67–99.80%, showing efficiency (90.11–94.28%) and operational stability of the biocatalyst for ethanol fermentation. The results of this study concerning the use of sugarcane as yeast supports could be promising for industrial fermentations. L. Liang and Y. Zhang have contributed equally to this work.     

Characterization of three endophytic, indole-3-acetic acid-producing yeasts occurring in Populus trees

Three endophytic yeast, one isolated from stems of wild cottonwood (Populus trichocarpa), two from stems of hybrid poplar (P. trichocarpa × Populus deltoides), were characterized by analyzing three ribosomal genes, the small subunit (18S), internal transcribed spacer (ITS), and D1/D2 region of the large subunit (26S). Phenotypic characteristics of the yeast isolates were also obtained using a commercial yeast identification kit and used for assisting the species identification. The isolate from wild cottonwood was identified to be closest to species Rhodotorula graminis. The two isolates from hybrid poplar were identified to be species Rhodotorula mucilaginosa. In addition, the three yeast isolates were observed to be able to produce indole-3-acetic acid (IAA), a phytohormone which can promote plant growth, when incubated with l-tryptophan. To our knowledge, the yeast strains presented in this study were the first endophytic yeast strains isolated from species of Populus.     

Genomic and transcriptomic analysis of the endophytic fungus Pestalotiopsis fici reveals its lifestyle and high potential for synthesis of natural products

Background

In recent years, the genus Pestalotiopsis is receiving increasing attention, not only because of its economic impact as a plant pathogen but also as a commonly isolated endophyte which is an important source of bioactive natural products. Pestalotiopsis fici Steyaert W106-1/CGMCC3.15140 as an endophyte of tea produces numerous novel secondary metabolites, including chloropupukeananin, a derivative of chlorinated pupukeanane that is first discovered in fungi. Some of them might be important as the drug leads for future pharmaceutics.

Results

Here, we report the genome sequence of the endophytic fungus of tea Pestalotiopsis fici W106-1/CGMCC3.15140. The abundant carbohydrate-active enzymes especially significantly expanding pectinases allow the fungus to utilize the limited intercellular nutrients within the host plants, suggesting adaptation of the fungus to endophytic lifestyle. The P. fici genome encodes a rich set of secondary metabolite synthesis genes, including 27 polyketide synthases (PKSs), 12 non-ribosomal peptide synthases (NRPSs), five dimethylallyl tryptophan synthases, four putative PKS-like enzymes, 15 putative NRPS-like enzymes, 15 terpenoid synthases, seven terpenoid cyclases, seven fatty-acid synthases, and five hybrids of PKS-NRPS. The majority of these core enzymes distributed into 74 secondary metabolite clusters. The putative Diels-Alderase genes have undergone expansion.

Conclusion

The significant expansion of pectinase encoding genes provides essential insight in the life strategy of endophytes, and richness of gene clusters for secondary metabolites reveals high potential of natural products of endophytic fungi.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-014-1190-9) contains supplementary material, which is available to authorized users.     

Isolation, molecular characterization and growth-promoting activities of endophytic sugarcane diazotroph Klebsiella sp. GR9

A group of endophytic diazotrophs were isolated from surface-sterilized roots and stems of different sugarcane varieties in the Tamilnadu region of India. From these, four isolates were selected, based on the highest acetylene reduction activity. Gene-specific PCR amplification confirmed the presence of nif-D genes in those isolates. The 16S rRNA sequence of isolates GR4 and GR7 had a 99.5% sequence similarity to the Pseudomonas sp. pDL01 (AF125317) and 16S rDNA sequence of isolate GR3 had a 100% similarity to that of Burkholderia vietnamiensis (AY973820). The 16S rDNA sequence of isolate GR9 was 99.79% similar to that of the Klebsiella pneumoniae type strain (KPY17657). Colonization by the isolates was confirmed using micropropagated sugarcane and sterile rice seedlings. Isolate GR9, identified as Klebsiella pneumoniae, was consistently more active in reducing acetylene as compared with the other isolates. The effects of GR9 and the sugarcane diazotroph Gluconacetobacter diazotrophicus were compared in inoculated micropropagated sugarcane plantlets. The effects of K. pneumoniae GR9, and four other diazotrophs, G. diazotrophicus, Herbaspirillum seropedicae, Azospirillum lipoferum 4B, and Burkholderia vietnamiensis in inoculated rice seedlings were compared. GR9 alone or in combination with the other diazotrophs performed best under pot conditions. The combined effects of nitrogen fixation and endophytic colonization of this diazotroph may be useful for the development of bio-inoculants.     

Habitat use by black bears in relation to conspecifics and competitors

Sympatric black bears (Ursus americanus) and brown bears (Ursus arctos) are common in many boreal systems; however, few predator assemblages are known to coexist on a single seasonally abundant large prey item. In lowland southwestern interior Alaska, black bears and brown bears are considered the primary cause of moose (Alces alces) calf mortality during the first 6 weeks of life. The objective of this study was to document habitat use of global-positioning system (GPS)-collared black bears during peak and non-peak seasons of black bear-induced and brown bear-induced moose calf mortality within southwestern interior Alaska, in spring 2002. We compared habitats of GPS-collared black bears to those of presumably uncollared black bears and brown bears at their moose calf mortality sites. Results from this study suggest that GPS-collared black bears use similar habitat as conspecifics more than expected during the peak period of black bear predation on moose calves, whereas they use habitat in proportion to home range availability during the peak in brown bear predation on moose calves. Sex-specific Ivlev's electivity indices describe greater than expected use of mixed-deciduous forest and needleleaf forest by male GPS-collared black bears during the peak of moose calf predation, whereas females have a tendency to use these habitats less than expected. Juvenile GPS-collared black bears largely use the same habitat as other sympatric predators during the peak of moose calf predation, whereas during the non-peak period juveniles use opposite habitats as adult GPS-collared black bears. The outcome of this study offers possible explanations (e.g., sex, age) for spatial overlap or segregation in one member of a complex predator guild in relation to a seasonal pulse of preferred prey.     

Nitrogen distribution and leaf area indices in relation to photosynthetic nitrogen use efficiency in savanna grasses

Leaf photosynthetic characteristics, distribution patterns of nitrogen content per unit leaf area (n_L) and leaf area production per unit n_Lwere measured in natural stands of a C₄ grass (Hyparrhenia rufa) from the seasonal savannas and of a C₄grass (Paspalum fasciculatum) and two C₃grasses (Leersia hexandra and Hymenachne amplexicaulis) from the flooded savannas in central Venezuela. Daily rates of canopy photosynthesis (P_cD) as well as the optimal leaf area production per unit n_Lat which P_cDfor a given total amount of nitrogen in the canopy (i.e., canopy-PNUE) is maximized were also calculated. The C₃and C₄species from the flooded savannas had similar light saturated rates of photosynthesis per unit n_L(i.e. leaf-PNUE) and similar canopy-PNUEs which was in strong contrast with previous studies. Especially H. rufa but also L. hexandra and H. amplexicaulis had leaf- and canopy-PNUEs which were considerably higher than the values calculated for most other species with the same photosynthetic pathway (i.e., C₃or C₄). In contrast to previous studies, differences in the light gradient in the canopy between stands only partially explained differences in N distribution. Measured leaf area indices were greater and the average n_L values were consequently smaller than the calculated optima. There was, however, a very strong linear correlation between the optimal and actual average n_Lindicating that even though the model overestimated average n_L, it did predict the differences in leaf area production per unit nitrogen – the inverse average n_L– very well. This result strongly indicates that leaf area production per unit of leaf nitrogen increases with leaf-PNUE and decreases with the extinction coefficient for light. Grass species from seasonal savannas have extremely high leaf-PNUEs and thus optimally produce large amounts of leaf area per unit n_L. This helps explain how stands of these species may have high leaf area indices and achieve high photosynthetic productivity despite the very low nutrient availability at which they grow.