Improvement of plant growth and seed yield in Jatropha curcas by a novel nitrogen-fixing root associated Enterobacter species

Abstract

Background: Jatropha curcas L. is an oil seed producing non-leguminous tropical shrub that has good potential to be a fuel plant that can be cultivated on marginal land. Due to the low nutrient content of the targeted plantation area, the requirement for fertilizer is expected to be higher than other plants. This factor severely affects the commercial viability of J. curcas.

Results: We explored the feasibility to use endophytic nitrogen-fixing bacteria that are native to J. curcas to improve plant growth, biomass and seed productivity. We demonstrated that a novel N-fixing endophyte, Enterobacter sp. R4-368, was able to colonize in root and stem tissues and significantly promoted early plant growth and seed productivity of J. curcas in sterilized and non-sterilized soil. Inoculation of young seedling led to an approximately 57.2% increase in seedling vigour over a six week period. At 90 days after planting, inoculated plants showed an average increase of 25.3%, 77.7%, 27.5%, 45.8% in plant height, leaf number, chlorophyll content and stem volume, respectively. Notably, inoculation of the strain led to a 49.0% increase in the average seed number per plant and 20% increase in the average single seed weight when plants were maintained for 1.5 years in non-sterilized soil in pots in the open air. Enterobacter sp. R4-368 cells were able to colonize root tissues and moved systemically to stem tissues. However, no bacteria were found in leaves. Promotion of plant growth and leaf nitrogen content by the strain was partially lost in nifH, nifD, nifK knockout mutants, suggesting the presence of other growth promoting factors that are associated with this bacterium strain.

Conclusion: Our results showed that Enterobacter sp. R4-368 significantly promoted growth and seed yield of J. curcas. The application of the strains is likely to significantly improve the commercial viability of J. curcas due to the reduced fertilizer cost and improved oil yield.

Figure 1

Taxonomical classification of isolated Enterobacter strains. Phylogenetic tree was based on 16S rRNA gene sequences, of which the Genebank accession numbers are shown in the brackets. Bootstrap values (expressed as percentages of 1000 replications) greater than 30% are shown at the branch points. Bar: 0.005 substitutions per nucleotide position. Potential nitrogen-fixing strains, which were positive for the nifH gene as judged by PCR amplifications and sequence confirmation, are shown in indicated in blue. The number of isolates highly related to E. ludwigii and E. asburiae are indicated by the number in the bracket, respectively.

Figure 2

Nitrogenase activities of selected isolates from Jatropha cultivars. Acetylene reduction (AR) activity was measured in vitro (pure cultures) and in planta conditions. Error bars represent SD (n = 4).

Figure 3

Growth promotion ofJatropha curcasby R4-368. Seedlings were inoculated with R4-368 and grown germination in trays and then transferred to pot with one seedling/pot with unsterilized garden soil and grown in open air for 1.5 years. Plant height (A), number of leaves (B), relative chlorophyll content (C) and stem volume (D) were recorded once in 30 days. * means significant difference at 5% threshold between treated and control using DMRT. NS-not significant.

Figure 4

Effect of Enterobacte rR4-368 inoculation on seed production. Plants were planted in pots in sterilized soil (compost/sand mix at 1:1 ratio and in ɸ23 cm, 18 cm height pots; named as Trial I) or none-sterilized soil (nutrient poor clay soil in ɸ30 cm, 28 cm height pots; named as Trial II). Trial I and Trial II were maintained in different locations and started in different seasons. R4-368 cell suspension (1.2 OD_600nm, 50 ml/pot) was applied to the plant root system at 21 days after seed germination. Fertiliser was applied regularly at about half of the recommended dose of approximately 50:30:30 g/plant/year. (A) Plant height (B) leaf canopy. Seed set numbers per plant (C) (n = 8 in Trial I and n = 12 in Trial II) were measured at 480 and 520 DAI in Trail I and Trail II respectively and single seed weight (D) was calculated based the average of 180 randomly selected seeds per treatment except the non-treated for Trial I where only 117 seeds were measured. Error bars indicate standard error. * and ** indicate statistically significant between the treated and non-treated populations (*P < 0.05 for seed set per plant and **P < 0.01 for single seed weight) by Student’s t-test.

Figure 5

nif gene operon in Enterobactersp. R4-368.(A) Organization of nif gene cluster. The coding DNA sequence (CDS) was predicted with RAST [39] and the CDS diagram was generated with Vector NTI v10. (B) Comparison of nif genes between Enterabacter sp. R4-368 and K. pneumonia (GenBank: ×13303.1).

Figure 6

Plant Colonization and in vitro endoglucanase activity of Wt and ∆ nifK mutants. (A) and (B): An optical section of root tissue infected of Wt R4-368 at 7 and 20 DAI respectively. (C) and (D): An optical section of root tissue infected of Wt R4-368 ΔnifH at 7 and 20 DAI respectively. (E) and (F) are the mock-inoculated control showing no GFP signal. Scale bars (bar, 20 μm) are shown in each image. Mc: microcolony; Rh: root hair; Ag: cell aggregates; IC: intercellular colonization. (G) and (H): Congo-red stained KM agar plate [42] without and with glucose respectively.