The cultivable endophytic bacteria from poplar and willow trees share many closely related strains, the majority of which belong to the Gammaproteobacteria
(Fig. ). The dominance of gammaproteobacteria is consistent with previous observations of the endophytic community diversity in poplar trees growing on a benzene-, toluene-, ethylbenzene-, and xylene-contaminated site (28
). However, in contrast to previous studies, we also observed a significant number of Rhodococcus
spp. (12% of the cultivable strains), including Rhodococcus equi
. As noticed previously (36
), we found that the highest number of endophytic bacteria reside in the roots, with their numbers declining in the stems, shoots, and leaves (results not shown).
Endophytic bacteria from poplar trees, representative of the dominantly observed genera Enterobacter, Serratia, Stenotrophomonas, and Pseudomonas, were tested for their capacities to improve growth of their poplar host. In addition, they were screened for the production or metabolism of plant growth-promoting compounds, phytohormones, and sugars. A better understanding of their plant growth-promoting capabilities was further obtained by initiating the sequencing of their genomes.
sp. strain 638 had the most-pronounced beneficial effect on the development and growth of poplar cuttings. This result was not only repeatable in our hands with P. deltoides
× P. nigra
DN-34 but also with the hybrid poplar clone 0P367 (Populus deltoides
× P. nigra
) (significance level, P
< 0.05) (L. Newman, unpublished results). On the other hand, while no significant plant growth-promoting effect was observed for P. putida
W619 with P. deltoides
× P. nigra
DN-34, strain W619 significantly (significance level, P
< 0.01) promoted the growth of another hybrid poplar tree [Populus deltoides
× (P. trichocarpa
× P. deltoides
) cv. Grimminge] (N. Weyens, J. Boulet, D. Adriaensen, J.-P. Timmermans, E. Prinsen, S. Van Oevelen, J. D'Haen, K. Smeets, D. van der Lelie, S. Taghavi, and J. Vangronsveld, submitted for publication). Also, the promiscuous plant growth-promoting effect of B. cepacia
Bu72 on poplar trees (this study and reference 36
) and yellow lupine (5
) is noticeable. Therefore, before the application of this concept to other poplar cultivars, preliminary studies to confirm the plant growth-promoting effect of the selected endophyte are required.
The plant growth-promoting effect of Enterobacter
sp. strain 638 might be explained by the presence of the putative alsDS
pathway for acetoin synthesis (Fig. ), a potent plant growth-promoting compound (29
). As for the rhizosphere bacterium Bacillus amyloliquefaciens
), it was unclear which function catalyzes the conversion of acetoin into 2,3-butanediol. We assumed that acetoin (produced and released by Enterobacter
sp. strain 638 and S. proteamaculans
568) can enter the poplar cells, where it can be converted into 2,3-butanediol. Enterobacter
sp. strain 638 also possesses a putative acetoin reductase for synthesis of diacetyl, whose role in plant growth promotion is unknown.
None of the other traits linked to plant growth regulation were identified in Enterobacter
sp. strain 638: the strain produces low levels of IAA, is unable to fix nitrogen, and lacks the pathways to metabolize ACC, GABA, and phosphonoacetic acid (PAA). Sequence analysis, however, revealed that Enterobacter
sp. strain 638 contains a 157.7-kb plasmid, pENT638-1, which is related to F plasmids found in other Enterobacteriaceae
. Plasmids of this family are involved in host interaction and virulence, such as the pFra plasmid of the plague microbe Yersinia pestis
). In pENT638-1, the pFra pathogenicity island has been replaced by a 23-kb putative genomic island (flanked by an integrase gene and having a GC content that is significantly different than that of the rest of the plasmid). This island contains a group of open reading frames with strong homology to hypothetical proteins of Azotobacter vinelandii
AvOP, as well as to a putative srfABC
operon, which is also present in a horizontally acquired region of Salmonella
spp. and which is believed to be involved in virulence (46
). Adjacent to this region, a putative ndvB
(8,532-bp) gene was located. ndvB
, which codes for a protein involved in the production of β-(1→2)-glucan, is required by Sinorhizobium meliloti
for bacterial invasion of the nodule (10
). Many other genes involved in plant invasion were present on pENT638-1—genes coding for proteins with an autotransporter domain (type V secretion) or virulence domains (agglutinin, pertactin, or adhesin). In addition, plasmid pENT628-1 carries many relBE
toxin/antitoxin systems, often located in the proximity of regions that presumably play a role in the successful interaction between Enterobacter
sp. strain 638 and its host.
S. proteamaculans 568 is interesting, as it is, in contrast to Enterobacter sp. strain 638, able to metabolize GABA and PAA, two compounds involved in regulating plant responses to stress. S. proteamaculans 568 is, like Enterobacter sp. strain 638, able to produce 2-acetoin but lacks the acetoin reductase for the bidirectional conversion of acetoin and diacetyl. Furthermore, it lacks the putative plant invasion functions found on plasmid pENT628-1. A further comparison between both Enterobacteriaceae strains should provide a better understanding of the observed differences in their plant growth-stimulating potentials.
In the Enterobacteriaceae
, sugar uptake dominantly occurs via PTS systems, and both S. proteamaculans
568 and Enterobacter
strain 638 contain PTS systems that are consistent with their sugar metabolism (Table ). S. proteamaculans
568 also contains a copy of PTS from the glucitol family. The high number of PTS genes found in the Enterobacteriales
compared to those found in the Pseudomonadales
and the Xanthomonadales
is well known (4
). Consistent with this observation is that both P. putida
W619 and S. maltophilia
R551-3 possessed a single gene coding for a PTS from the fructose family. This PTS family is most prevalent in proteobacteria and is believed to have evolved into other PTS systems (4
W619 seems to be well adapted to influence the phytohormone balances of its host: the strain appears to produce high levels of IAA and is able to metabolize PAA and GABA. Elevated levels of GABA and PAA, a nonindolic auxin that can account for up to one-half of the total bioassayable auxin activity in plant extracts (45
), can inhibit plant growth. The complexity of the phytohormone balance points toward the existence of a complex mechanism that fine-tunes the interactions between P. putida
W619 and other endophytes and their poplar hosts. For instance, the negative effects on poplar development observed after inoculation with M. populi
BJ001 might reflect a disturbance of this balance, e.g., caused by unnaturally high numbers of this bacterium during inoculation.
Many endophytic bacteria, such as S. maltophilia
R551-3, are closely related to pathogenic microorganisms whose genomes have been or are in the process of being sequenced. Future genome annotation and comparative genomics of endophytic bacteria and phylogenetically closely related, nonendophytic microorganisms should result in the identification of the subset of genes necessary for a successful endophytic colonization of poplar trees. Understanding the interactions between endophytic bacteria and their host plants, facilitated by the published genome sequence of Populus trichocarpa
), should ultimately result in the design of strategies for improved poplar biomass production as a feedstock for biofuels and bioremediation.