Evidence To Illustrate The Beneficial Yield Response Of Spring Wheat And Rice To Inoculation With Nitrogenfixing Bacterial Endophytes

P.J. Riggs, R. Mortiz, Y. Dong, M. Chelius, E.W. Triple«

Dept of Agronomy, University of Wisconsin, Madison, WI 53706, USA

Two experiments were performed to examine the potential of various endophytic nitrogen-fixing bacteria to increase yield on spring wheat and dry land rice. The long-term goal of these experiments is to locate bacteria and grass genotypes that can form biological nitrogen-fixing symbioses, and thereby decrease the level of industrial fertilizer required by modern agriculture. The strains P101 (.Panteoa sp.), P102 (Panteoa sp.), K342 (Klebsiella sp.), K342 nif-hll (Klebsiella sp. nif h-11 mutant), and PAL5 (Acetobacter diazotrophicas) were chosen as inoculants. Previous experiments have illustrated these strains ability to positively affect yield of maize, sugarcane and, to a lesser extent, spring wheat in the field and greenhouse. These bacterial strains were isolated from a variety of plant sources including maize, switch grass, and sugarcane.

Strain K342 wild type produced a significant increase in dry weight (25%) in spring wheat (cv. Trenton) compared to both the uninoculated control and the K342 nif-hl 1 mutant. Nitrogen fixation may be the mechanism underlying this stimulation in yield. Nitrogen yield data illustrate that plants inoculated with the K342 wild type had 20% more N in their shoot tissues than the plants inoculated with K342 nif-hl 1 mutant and the uninoculated controls. These data suggest that there is an interaction between the bacteria and the wheat that is providing nitrogen to the wheat plants inoculated with the K342 wild type, relative to the control and nif-hl 1 mutant. The next steps will be to perform 15N2 reduction assays to directly measure the amount of biological nitrogen fixation that is occurring in this system and to place a fluorescent tag on the nitrogen-fixing genetic apparatus within the bacterium to illustrate the expression of the enzymes of nitrogen fixation in vivo. This will allow us to state confidently that there is biological nitrogen fixation occurring in this system.

Results from the rice (cv. Nipponbare) experiment illustrate several points. First, that strain P101 produces a large increase in the dry weight of rice shoot tissue (50%) compared to the uninoculated control. The mechanism for this yield increase may be due to a biological nitrogen fixation symbiosis between PI01 and the host rice plants. This hypothesis is supported by the total nitrogen yield data, which indicated that compared to the uninoculated control the rice plants inoculated with PI01 contained 70% more nitrogen on a dry weight basis. Strain PAL5 also produced a significant increase in total nitrogen per unit dry weight of shoot tissue (55%) when compared to the uninoculated control. This supports the idea that these two bacteria are potentially fixing nitrogen for the rice plant. The next steps will be to produce and test PI01 and PAL5 mutant strains against an uninoculated control and the wild type. This will help to rule out other plausible reasons for the observed yield increase such as the production of plant growth promoting compounds. Finally, 15N2 reduction assays will be completed to directly measure the amount of biological nitrogen fixation that is occurring in this system, and the use of GFP-labeled bacteria will allow use to verify that the inoculants are pursuing an endophytic lifestyle.

ASSOCIATION BETWEEN NITROGEN-FIXING BACTERIA AND CONZATTIA MULTIFLORA

E.T. Wang1, S. Sarabia1, E. Martinez-Romero2, O. Dorado3, G. Boll1, A. Lemos1

'Dept Microbiol. ENCB, LPN, Mexico

2Program Ecologia Molecular y Microbiana, CIEN, UNAM, Mexico 3Dept Biol., UAEM, Mexico

1. Introduction

Among different nitrogen fixation systems, the symbiosis of legumes-rhizobia is the most important one and has been studied thoroughly. Compared with the nodulating species, nitrogen nutrient of the non-nodulating leguminous plants is almost ignored, although they are also important in the natural ecosystems. Conzattia multiflora is a leguminous tree belonging to the subfamily of Caesalpinioideae. It grows only in Mexico. There is no record about its symbiotic or pathogenic microbes and no information about its economic value. In this study, we isolated and characterized some endophytic bacteria from this tree.

2. Isolation and Inoculation of Bacteria

Twelve isolates with yellow or colorless colonies were isolated from 27 pieces of surface sterilized cortex using semisolid nitrogen-free medium (Tapia-Hernandez et al. 2000). All were gram-negative, facultative anaerobic rods. They grow in LB medium at 37°C. In McConkey medium, the isolates with colorless colonies in PY could absorb the dye and had rose to purple colors (similar to those of Klebsiella). The four isolates with yellow colonies did not absorb the dye in McConkey medium. Four rDNA types were identified among the 12 isolates from the analysis of PCR-RFLP of 16S rDNAs. Representative isolates for the 4 rDNA types were inoculated onto the seedlings and were reisolated. The number of bacteria varied from 105 to 106 cells per gram of plant tissue in the inoculated plants while no bacteria were counted from the control plants when they were grown in cotton-sealed tubes. Two isolates, NF5 and NF9, could significantly improve the growth of seedlings as shown by the increased height of the plants. No nodules were observed on seedlings in the laboratory or on trees in fields and in pots. We also found these bacteria inside the C. multiflora seeds and these bacteria could be eliminated by germinating the seeds in a mixture of streptomycin (100 g mL"1) and tetracycline (5 g ml"1).

3. Nitrogen Fixation by the Bacteria

Low, but stable, acetylene reduction activity was detected in the four representative isolates when the bacteria were grown in semisolid nitrogen-free media. Modification of the carbon source or the addition of low concentration of yeast-extract did not enhance the acetylene reduction activity. In PCR-amplification of nifH using the primers nifH-1 and nifH-2, a band with molecular size similar to the nifH fragment of if. etli CFN42 was obtained from all the 4 isolates. The sequence of this 500 bp PCR-fragment from NF1 and NF9 was 67.7 and 73.8% similar to the nifH gene of R. leguminosarum and was 91.1% similar to each other. However, acetylene reduction in the cortex was not detected.

We concluded that: (i) this tree had no nodules, but contained a large number of endophytic bacteria in the cortex; (ii) these bacteria are facultative anaerobic nitrogen fixers; and (iii) these bacteria contained a nifH gene most similar to that of R. leguminosarum.

4. Reference

Tapia-Hernandez A et al. (2000) Micro. Ecol. 39, 49-55

ACTIN FILAMENTS ARE REQUIRED FOR SUCCESSFUL INFECTION THREAD DEVELOPMENT DURING NODULATION OF ALFALFA BY SINORHIZOBIUM MELILOTI

'Dept Biology, Lehman College, CUNY, Bronx, NY 10468, USA 2Dept Biology, MIT, Cambridge, MA 02139, USA

1. Introduction

The Sinorhizobium meliloti and Medicago sativa (alfalfa) establish symbiosis after a series of chemical signal exchanges. The S. meliloti cells enter alfalfa root nodules through plant produced infection threads in the middle root hairs and convert atmospheric nitrogen into ammonia inside the nodules. The process of root hair invasion that results in the formation of infection thread is a key step in the establishment of this symbiosis. While the development of infection threads can now be visualized using green fluorescence protein (GFP) expressing S. meliloti cells, the mechanism of infection thread development remains unclear. We explored the possibility that the development of infection threads is an inward growth of root hair and thus shares some of the same mechanism as root hair growth.

Root hairs grow at their tips and require cytoplasmic streaming to transport cell wall and membrane materials from the base of cells. The cytoplasmic streaming is driven by myosin traveling along actin filaments, which are part of cell cytoskeleton. Actin filaments are long, contractile proteins that are supported by a framework of microtubules and they are depolymerized in the presence of cytochalasin B (CB), a mold metabolite. Depolymerization of actin filaments using CB stops cytoplasmic stream in plant root hair cells.

2. Results and Discussion

The effect of CB on alfalfa root hair cytoplasmic streaming was examined by directly exposing root hairs to CB under phase contrast microscope. Alfalfa seedlings were put on microscope slides in clear liquid growth media. Root hair cytoplasmic streaming is clearly visible under a phase contrast microscope and it was stopped immediately after the addition of CB.

To test the effect of CB on the development of infection threads, alfalfa seedlings were grown on microscope slides covered with a layer of solid growth media to facilitate live viewing. Slides were inoculated with GFP expressing S. meliloti cells that enables the real time monitoring of the development of infection threads using fluorescence microscopy. Root hairs with infection threads were exposed to CB through the application of CB containing agarose gel disks. The development of infection threads was blocked completely by the presence of CB at 0.1 pg/mL concentration. In the presence of lower concentration of CB, some infection threads formed spherical balls at the end of the infection thread and then formed new branches of infection threads.

Our findings that CB stops the cytoplasmic streaming and infection thread development suggest that the development of infection thread requires the presence of actin filaments. These findings and our early findings that the formation of infection thread depends on the presence of bacterial polysaccharide raise the possibility the formation of infection threads is the result of the communication between the bacterial cells and alfalfa root hair cells.

3. Acknowledgements

We thank corresponding author, Dr Hai-Ping Cheng.

This work is supported by anNIH Grant 41353-09-16 to HC and an NIH grant GM 31030 to GCW.

CHEMICAL CHARACTERIZATION OF ROOT EXUDATES FROM RICE (iORIZA SATIVA) AND THEIR EFFECTS ON THE CHEMOTACTIC CAPACITY OF ENDOPHYTIC BACTERIA AND PLANT GROWTH-PROMOTING BACTERIA (AZOSPIRILLUM AND BACILLUS SPP.)

M. Bacilio-Jiménez1'2, S. Aguilar-Flores3, E. Zenteno4

'Dept of Mierob. Center for Biol., Res. of the Northwest (CIB), La Paz, BCS, Mexico

2Dept of Biochem., Nat. hast, of Resp., Disease. Tlalpan, 14080 D.F. Mexico

3Dept of Botany, Nat. School of Biol., Sciences. IPN, 45873 Mexico

4Lab. Immunol. Dept of Biochem., Faculty of Medicine, UNAM, 04510 Mexico

Root exudates are an important source of nutrients for the microorganisms present in the rhizosphere and participate in the colonization process through chemotaxis caused on soil microorganisms. The colonizing capacity of plant growth-promoting bacteria (PGPB) could be favored by the chemotaxis exerted by root exudates; however, competition for nutrients and colonization sites between native strains present in the rhizosphere and endophytic bacteria seems to be an important factor, which could determine their colonizing capacity. In this work, we performed the chemical characterization of amino acids and sugars released by rice plantlets. We also evaluated the effect of these exudates on the chemotactic response of PGPB such as Azospirillum brasilense and Bacillus spp., and rice endophytes such as Bacillus pumilus and Corynebacterium flavescens.

Rice seeds (Oryza sativa L.) were surface-disinfected, soaked in a solution of 150 mg nalidixic acid/L and rinsed with sterile distilled water. After germination seeds were aseptically transferred to sterile hydroponic systems. Roots were kept in half-strength complete Hoagland's solution containing 50 mg/L nalidixic acid. Plants were kept for 7, 14, 21 and 28 days. The nutrient solution was lyophilized and used for identification of amino acids in an automatic amino acid analyzer and sugars by gas-chromatography. We evaluated the chemotactic capacity towards root exudates of rice plant using an acrylic chamber provided with two holes and a well. In the holes, two capillary tubes were introduced, one containing the sample of root exudates and the other, as a control. In the well, 0.5 mL of adjusted bacterial suspension (104 cells/mL) was placed.

The root exudates contained higher concentrations and a larger variety of carbohydrates and amino acids during the first two weeks of culture. The main amino acids identified were histidine, proline, valine, alanine, and glycine. The main carbohydrates identified were glucose, arabinose, mannose, galactose and glucuronic acid. Glucose was the main sugar derivative identified.

The four bacterial strains investigated showed positive chemoattractant capacity toward the root exudates from rice, however endophytes are better fit to respond to these attractants and can be explained based on their origin: A. brasilense was isolated from maize rhizosphere and Bacillus spp. from soil in which rice had been grown. This effect of root exudates also gives them a clear ecological advantage during the first colonization stages over Azospirillum and Bacillus spp., allowing them to compete better with applied PGPB, such as the rice soil bacterium Bacillus sp. These results strongly suggest that the attractant characteristics favor endophytes and, therefore, could induce exclusion of other colonizing microorganisms from the ecological niche, explaining in part why only endophytes could colonize the rhizosphere (Bacilio-Jimenez et al. 2001).

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