References

Allaway D et al. (2000) Mol. Microbiol. 36, 508-515

Atkins CA, Smith PMC (2000) In Triplett E (ed), Prokaryotic Nitrogen Fixation, pp. 559-587,

Horizon Scientific Press, Wymondham, UK Day DA et al (2001) Cell. Mol. Life Sci. 58, 61-71 Li Y et al (2001) Microbiol. 147, 663-670 Waters JK et al (1998) Proc. Nat. Acad. Sci. USA 95, 12038-12042

Section 11: Endophytic / Associative Plant-Microbe Interactions

CHAIR'S COMMENTS: ENDOPHYTIC AND ASSOCIATIVE PLANT-MICROBE INTERACTIONS

C. Kennedy

Dept Plant Pathology, University of Arizona, Tucson, AZ 85721, USA

Associative nitrogen-fixing bacteria colonize the rhizosphere and sometimes the interior surfaces of outer root cortical cells of host plants. Those identified include species of several genera, mainly members of Proteobacteria groups. The best studied of these is Azospirillum brasilense and other Azospirillum species. Plant hosts for these root-colonizing bacteria include wheat, maize and other monocots and a few dicots.

Diazotrophs colonizing the interior of plants, mainly in intercellular spaces, are endophytes, the best studied being Gluconacetobacter diazotrophicus (formerly Acetobacter diazotrophicus), and Herbaspirillum species. These have been mainly isolated from sugarcane grown agriculturally throughout the world, and also from a few other plants, both monocots and dicots, including coffee, pineapple, sorghum and others. Another endophytic relationship occurs between a nitrogen-fixing species of Azoarcus, originally isolated from Kallar grass in Pakistan and now studied as a colonizer of rice.

Results from several laboratories have shown that many associative and endophytic diazotrophs do enhance the growth of their plant partners in plant inoculation experiments. A challenge to this field of research is to establish, definitively, whether the transfer of fixed nitrogen from the identified associative or endophytic colonizers is significant. N balance and isotope dilution experiments would suggest this is true. It is also true that other factors such as indole acetic acid (IAA) production can be beneficial to plant growth, possibly by increasing efficiency of fixed N source uptake by plant roots.

This field of research is growing rapidly - about 20% of the posters at this meeting concern associative and endophytic diazotrophs, studied not only for their benefit to plant growth but also to address basic questions of bacterial physiology, genetics, and gene regulation in these organisms. The next few years will be important in establishing the exact nature of the contribution of individual endophytes to plant health and nutrition. The papers presented in this session address some of these and other issues. An overview follows.

Eric Triplett described diazotrophic endophytes isolated from maize and switchgrass. A strain of K. pneumoniae isolated from maize, named 342, and its nifH mutant derivative, along with switchgrass isolate Pantoea sp. P102 were used to inoculate rice and wheat varieties. These plants were tested because in previous experiments, K. pneumoniae isolates failed to relieve N-deficiency in maize although inoculated plants showed growth responses to inoculation in N-sufficient conditions. The smaller size of rice and wheat might allow the benefits of bacterial nitrogen fixation to be more easily evident. Gfp-tagged bacteria were observed to colonize intercellular spaces of the root cortex in both host plants. Growth benefit to both plants grown under N deficiency was observed with wild-type strains but not with the nifH mutant of strain 342, consistent with but not proving that bacterially fixed nitrogen was supplied to host plants. This work adds additional members to the growing family of diazotrophic endophytes and widens the potential for beneficial plant-microbe associations.

Paula Bonfante and colleagues have discovered diazotrophic bacteria to be endosymbionts of arbuscular mychorrhizal fungi: non-culturable species of Burkholderia appear in the cytoplasm of Gigaspora margarita BEG34 and other species of Gigasporaceae. These bacteria were found to contain DNA encoding the three nitrogenase subunits, the nifHDK, genes. The fungi apparently transmit the bacteria vertically from one generation to the next. This novel association could represent a mechanism for extending the known benefit of AM fungi on plant growth to conditions where nitrogen is otherwise limiting.

Species of Azospirillum were the first associative diazotrophs to be isolated from the rhizosphere of monocot host plants and remain the best characterized of this category of nitrogen-fixing bacterial types. It is now widely considered that while little bacterially fixed nitrogen is provided for host plant growth, Azospirillum is a beneficial organism by its ability to produce the auxin, indole acetic acid (IAA). Jos Vanderleyden and co-workers extended these studies and report that wheat growth promotion by A. brasilense occurs under sub-optimal levels of fixed N supply but not in the absence of provided fixed N. IAA production by Azospirillum brasilense is growth phase dependent, highest at pH 5.5 and is stimulated by external supply of IAA. The ipdC gene encodes a key enzyme in the IAA biosynthetic pathway, indole pyruvate decarboxylase. Two promoter-swapping strategies were described for constitutitive IPD expression and for plant-induced IPD expression. Plants inoculated with A. brasilense carrying these elements had shorter, more highly developed roots with greater root hair proliferation than plants inoculated with wild-type. Also, the total mass of plants was greater in those inoculated with the ipdC expression construct strains than in those inoculated with wild-type. These studies confirm and extend the hypothesis that IAA is a plant-growth promoting substance produced by A. brasilense and further indicate that genetic manipulation of levels/patterns of genes involved in IAA biosynthesis can be used to improve the degree of benefit that associative rhizosphere bacteria have on plant growth.

Barbara Reinhold-Hurek and colleagues originally isolated Azoarcus sp. strain BH72 from Kallar grass in Pakistan. Here she reported that further attempts to isolate the strain again from Kallar grass were not successful. However, nifH mRNA was abundant in plant tissues; preparation of cDNA from RNA and its sequencing showed that the nifH transcript was from Azoarcus sp. strain BH72. Thus this organism apparently has different phases during which it can be culturable or unculturable, depending on factors not known at this stage. One speculation is that the cells in plants are in a state of hyperderepression, characterized by nitrogenase being bound to a membrane array in a diazosome structure; such cells could possibly be unable to form colonies. Turning to the use of rice as a host for Azoarcus sp. strain BH72, results were shown suggesting that the degree of interaction between the bacteria and rice plants is cultivar-specific, with the suggestion that the degree of expression of nifHDK correlates with the degree to which the host plant carries out a defense response, typified by lignification and browning. Finally, the three PH-like proteins in Azoarcus sp. strain BH72, GlnB, GlnK and GlnY, were further characterized, along with the ammonium transporter encoded by amtB adjacent to glnK. Mutations in these genes variably influence the capacity for switch-off of nitrogenase activity that occurs after ammonium addition to nitrogen-fixing cultures. In addition both GlnK and GlnY have an ability to associate with cell membranes according to N status, apparently by binding to the membrane spanning AmtB protein.

Future work on these and other associative and endophytic/endosymbiotic diazotrophs will focus on identifying specific factors in both the bacteria and plant partners that lead to beneficial effects and effective colonization.

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