Increased Biomass of Nursery-grown Douglas-fir Seedlings upon Inoculation with Diazotrophic Endophytic Consortia

Douglas-fir (Pseudotsuga menziesii) seedlings are periodically challenged by biotic and abiotic stresses. The ability of endophytes to colonize the interior of plants could confer benefits to host plants that may play an important role in plant adaptation to environmental changes. In this greenhouse study, nursery-grown Douglas-fir seedlings were inoculated with diazotrophic endophytes previously isolated from poplar and willow trees and grown for fifteen months in nutrient-poor conditions. Inoculated seedlings had significant increases in biomass (48%), root length (13%) and shoot height (16%) compared to the control seedlings. Characterization of these endophytes for symbiotic traits in addition to nitrogen fixation revealed that they can also solubilize phosphate and produce siderophores. Colonization was observed through fluorescent microscopy in seedlings inoculated with gfp-and mkate-tagged strains. Inoculation with beneficial endophytes could prove to be valuable for increasing the production of planting stocks in forest nurseries.


Introduction
Millions of Douglas-fir (Pseudotsuga menziesii) seedlings are grown in nurseries for reforestation purposes in North America, Europe and elsewhere [1].The majority of seedlingsare grown for more than a year as bare root seedlings, although some are also grown in containers.Environmental disturbances and stresses contribute to seedling failure when transplanted in the field, and in order to maximize production, nurseries apply intensive cultural practices, fertilization being one of the most common [1,2].This can result in additional costs to nursery operators and could also have negative environmental repercussions if nitrogen leaches from nurseries situated in areas near lakes and rivers and/or where ground water reservoirs are found.An inexpensive and environmentally-benign alternative for enhancing the productivity of newly-established forest plantations involves nursery inoculation of seedlings with plant growth-promoting microorganisms.In forestry, this has traditionally been restricted to inoculation with mycorrhizal fungi [3,4]; however, it is reported that in many cases, mycorrhizal inoculation resulted in low to no measurable benefit in outplanting success.Recently, root-associated bacteria have been shown to stimulate tree seedling growth in addition to improving mycorrhizal colonization and may be valuable in current reforestation efforts [5,6].
Some species of conifers are able to inhabit harsh and nutrient-poor subalpine sites where few other plants grow.A key component of their ecological success may be attributed to the presence of endophytes.Endophytes are microbes (bacteria and fungi) that reside inside plants and do not cause disease [7][8][9][10], and there is increasing evidence of their profound impact on plant development, physiology, evolution and adaptation [11].An important trait of some endophytes is the ability to supply nitrogen to their host plant through biological nitrogen fixation.Nitrogen-fixing endophytes have been isolated from a variety of species, such as sugarcane [12], wild rice [13], corn [14,15], African sweet potato [16], kallar grass [17], coffee [18], cactus [19] and woody plants, including poplar, willow and coniferous trees [20][21][22][23].It has been demonstrated that N fixed by diazotrophic bacteria can be utilized by plants [24].Endophytic bacteria have been found in different parts of tree tissues, such as roots [25], stems [26], shoots [27], leaves [28] and buds [29].More recently, diazotrophic endophytes were isolated from conifers growing in nitrogen-poor soils.Inoculation of lodgepole pine and western red cedar seedlings with the isolate Paenibacillus polymyxa P2b2R resulted in significant foliar 15 N dilution, indicative of biological nitrogen fixation [30,31].The importance of endophytes to the establishment and persistence of long-lived western conifers is unknown; however, endophytes may allow establishment and growth on nutrient-limited and environmentally-stressed sites; for example, similar endophytes were found in subalpine conifers from distant sites in California and Colorado [32].
Phosphorous is another essential macronutrient promoting plant growth and development.Many woody plants are dependent on ectomycorrhizal fungi for their growth and survival.Mycorrhizae inoculation of Douglas-fir seedlings is a common practice in commercial nurseries [33,34].However, commercial nursery treatments, including frequent addition of fertilizer and water, may not be favorable for mycorrhizae colonization of container-grown seedlings [35].The importance of phosphate-solubilizing endophytes in increasing the phosphorous availability by solubilization of inorganic phosphate and mineralization of organic phosphate has recently been demonstrated as one of the important mechanisms of increased plant growth [36].Siderophores are low molecular weight ferric iron-specific chelating agents produced by bacteria and fungi [37].Microbial siderophores have been reported to have a positive correlation with plant growth promotion, and the production of siderophores is being considered as one of the key traits for primary screening of beneficial endophytes [38].
In this preliminary study, we chose a consortia of diazotrophic endophytes, originally isolated from poplar and willow trees [20][21][22], because they had shown robust growth-promoting activity with agricultural crops, grasses and poplar plants grown under abiotic stress [22,[39][40][41].Our objective was to determine if these endophytes enhance the growth of Douglas-fir seedlings under nutrient-poor conditions.

Plant Material and Endophytes
Douglas-fir container (1 + 0) (4 cu. in ; Ray Leach "cone-tainers") seedlings from a local western WA provenance obtained from Silvaseed Company Foresters (Roy, WA, USA) were individually planted into plastic pots containing Sunshine Mix #2 (Steubers, WA, USA).The propagation protocol for seedling production is described elsewhere [42].Seven bacterial strains and one yeast strain (Table 1) were chosen based on strong growth in nitrogen-free media, the presence of the nitrogen fixation gene (nifH) [20,21], production of phytohormones [43] and/or their robust plant growth-promoting abilities.
Table 1.Poplar and willow endophytes used in this study and their phosphate solubilization index (PSI) in the plate assay [20,21].

Methods
Inoculation suspensions were prepared by first growing the individual endophyte strains on nutrient-rich media from −80 °C glycerol stocks.Bacteria were grown on mannitol glutamate/Luria-Bertani (MG/L) medium [44].The yeast strain was grown on yeast extract peptone dextrose (YPD) medium.Single colonies were inoculated into liquid broth and grown overnight on a rotary shaker (150 rpm) and agitated for 24 h at 30 °C.Bacteria were harvested by centrifugation at 8000 rpm for 10 min, resuspended in nitrogen-free Murashige and Skoog (NFMS) (Caisson Labs, USA) broth and washed three times in NFMS.The endophyte consortia treatment was prepared by mixing equal concentrations of each inoculation suspension as determined by measuring the optical density at 600 nm (OD600) of the individual strains and adjusting to a final OD600 of 0.1.Using a sterile conical tube, a 50-mL sample of the endophyte consortia was then delivered 5-10 cm below the surface and in close proximity to the roots.Mock-inoculated control seedlings received the same volume of sterile liquid broth.A total of 24 pots, 12 replications of inoculated or mock-inoculated control plants, were arranged in a randomized fashion.Seedlings were kept in an environment with an 18h photoperiod, a 20/14 °C day/night temperature cycle and a relative humidity of 70%-85%.Drip trays were placed under each individual pot to prevent cross-contamination through run-off.Each pot was individually irrigated with tap water.Irrigation frequency and duration were adjusted, such that the pots were not allowed to flood nor dry out between watering.Additionally, each pot was fertigated with received 100 mL of 1/2 strength, 1/8 nitrogen modified Hoagland's medium containing (g L −1 ): CaCl2•2H2O, 0.22; K2SO4, 0.17; MgSO4•7H2O, 0.26; KH2PO4, 0.136; NaFeEDTA(10% Fe), 0.015; with 1 mL•L −1 micronutrient solution containing (g•L −1 ): H3BO3, 0.773; MnSO4, 0.169; ZnSO4•7H2O, 0.288; CuSO4•5H2O, 0.062; H2MoO4 (83% MoO3), 0.04; weekly for the first two months post-inoculation, then 250 mL twice monthly until the end of the experiment.
Height measurements were recorded after inoculation and every month thereafter.After 15 months, the plants were removed from the soil, and roots and shoots were gently washed to remove dirt and debris.Roots and stems were then blotted dry, cut into root and shoot sections, and the lengths and wet weights were determined.Foliar samples were taken from each plant and oven dried at 65 °C, ground to a fine powder and analyzed for nitrogen content using a PE 2400 series II CHN elemental analyzer (Perkin-Elmer, MA, USA) at the University of Washington's School of Environmental and Forest Sciences Analytical Soils laboratory.
Potential plant growth-promoting traits, such as phosphate solubilization and siderophore production, were determined for the selected endophytes.The ability of the isolates to solubilize tricalcium orthophosphate (TCP) was tested on the National Botanical Research Institute's growth medium [45] containing g•L −1 : glucose, 10; Ca3(PO4)2, 5; MgCl2•6H2O, 5; MgSO4•7H2O, 0.25; KCl, 0.2; (NH4)2SO4, 0.1.The inoculum was added in quadruplicate on the medium in Petri dishes.The halo and colony diameters were measured after 14 days of incubation of the plates at 25 °C.The ability of the microbes to solubilize the insoluble phosphate is described by the solubilization index (the ratio of the total diameter (halo + colony diameter (mm) to the colony diameter (mm)).Analysis of siderophore production by the endophyte isolates was performed following the chrome azurol S (CAS) method of Alexander and Zuberer [46].For each isolate, 10 µL of inoculum were placed in quadruplicate on the medium.After incubation at 25 °C for 3 days, the discoloration of the medium (blue to orange) indicated siderophore-producing endophytes.
To evaluate endophytic colonization by some of the isolates, we used strains that were labeled with fluorescent markers with a broad host range plasmid.The mkate and gfp plasmids were introduced into Acinetobacter calcoaceticus strain WP19 and Rahnella sp.strain WP5, respectively, via triparental mating using E. coli DH5α as the donor and E. coli HB101 (pRK 2073) as the helper strain [47].The inoculations were done as described above, and the colonization of root tissue and needles was verified through fluorescent microscopy using a Zeiss Imager M2 equipped with an AxioCam MRM and recorded with Zeiss AxioVision software (Karl Zeiss, LLC, Thornwood, NY, USA).

Statistical Analysis
One-way ANOVA (analysis of variance) and Tukey's honestly significant difference (HSD) test were used to identify significant differences between inoculated and mock-inoculated control seedlings.

Results and Discussion
Differences in plant heights and the health of inoculated and mock-inoculated control plants were apparent after fifteen months of growth in the greenhouse under N-limited conditions.As shown in Figure 1, the mock-inoculated control plants were stunted, with some being chlorotic and showing signs of nutrient deficiency, whereas the inoculated plants appeared taller, more robust and greener than the controls.The inoculated plants were 16% (p = 0.008) taller, had 13% longer roots (p = 0.024) (Figure 2) and a significant 48% (p = 0.019) increase in biomass (Figures 2 and 3) when compared to their mock-inoculated control plants.The concentration of N in plant tissues was higher in the inoculated plants compared to the controls, although it was not statistically significant (p = 0.156) (Figure 4).Our results demonstrate that inoculation with the poplar and willow endophyte consortia stimulated growth of Douglas-fir seedlings under low nutrient conditions.It was previously concluded that these endophytes are not host-specific colonizers [39].Poplar and willow plants are angiosperms, which diverged from gymnosperms more than 100 million years ago [48].Remarkably, the endophytes from poplar and willow plants colonized Douglas-fir, a gymnosperm.It is possible that the required plant-microbe communication may be ancient, pre-dating the divergence of angiosperms and gymnosperms.Interestingly, the response of Douglas-fir to inoculation with the endophytes took more than a year to develop, which suggests that this plant species may require a period of growth under controlled conditions to facilitate the establishment of the symbiotic relationship with these endophytes.Parker and Dangerfield [49] also reported a delayed response to microbial inoculation in Douglas-fir.In studies done by Chanway et al. [50], rhizospheric bacteria isolated from perennial rye grass and white clover stimulated the growth of container Douglas-fir seedlings after 12 weeks of inoculation.More recently, Anand et al. [31] showed that seedling growth enhancement in other conifers, lodgepole pine and western red cedar occurred only after 13 months.
To assess other potential mechanisms in addition to N fixation for the improved growth of the inoculated plants, we investigated the potential of phosphate solubilization by the endophytes.The majority of the isolates were able to produce a clear zone in minimal medium containing insoluble phosphorous.As shown in Table 1, the PSI of isolate WP19 was the highest, indicating the high solubilization capacity of this endophyte compared to the rest of the endophytes.There have been a number of reports on plant growth promotion by bacteria that have the ability to convert the insoluble inorganic forms of P into soluble forms through acidification, secretion of organic acids or protons and exchange reactions [36].Many symbiotic phosphate-solubilizing microorganisms belonging to species of the genera Bacillus, Pseudomonas, Rhizobium, Enterobacter and Burkholderia and certain fungi produce phytase to mineralize organic phosphate [51].In our study, phosphate solubilization may be one of the important mechanism through which these endophytes promoted growth of Douglas-fir seedlings; however, additional studies including phosphorous quantification and mutant analysis will be necessary to determine what symbiotic factors contribute the most to growth enhancement.The role of mycorrhizal symbiosis in increasing phosphorous uptake has been studied in a number of conifer species, with recent studies showing how mycorrhizal effects on seedling establishment change with soil conditions [52].In dry Douglas-fir forests, reduced mycorrhizal diversity and abundance have been associated with reduced survival and growth of newly-planted conifer seedlings [53,54].Inoculating nursery seedlings with beneficial endophytes may help offset some of these disadvantages.Furthermore, endophytes may increase mycorrhizal development by affecting root colonization, as well as by enhancing N and P uptake.
Rungin et al. [55] recently demonstrated plant growth-enhancing effects by a siderophore-producing endophytic streptomycete isolated from Thai jasmine rice plants.Significant increases in total biomass of rice and mung bean plants were observed in the inoculated controls compared to the untreated controls and siderophore-deficient mutant treatment.Most of the endophytes in our study produced siderophores, which may have been beneficial in plant growth promotion.However, this is largely speculative and warrants further research.Siderophore-producing microbes may also have significant activity against plant pathogens [56,57].Interestingly, we found that Douglas-fir seeds inoculated with these endophytes were significantly less affected by Fusarium, a common Douglas-fir pathogen [58].Therefore, these endophytes merit further investigation, as reducing mortality by inoculations could prove valuable to tree nurseries.
To evaluate endophytic colonization, Douglas-fir seedlings were inoculated with fluorescent-labelled strains of WP5 and WP19.As seen from the fluorescent microscope images, mkate-tagged cells of Acinetobacter strain WP19 were localized in the intercellular spaces of root tissue of the inoculated seedlings (Figure 5A).In addition, GFP-tagged cells of Rahnella sp.strain WP5 colonized the needles (Figure 5B), suggesting that this endophyte is able to move up the plant into aerial parts after inoculation.Similar colonization zones have been reported for other endophytes [30].
The enhanced growth in inoculated seedlings seen in this preliminary study merits further research in larger greenhouse trials and field trials to assess the long-term benefits of endophyte inoculations.

Conclusions
The results of the present study indicate that inoculation with a diazotrophic endophytic consortia significantly increased Douglas-fir seedling growth under nutrient-limited conditions.Most commercial nursery treatments add high rates of fertilizers to maximize seedling shoot and total growth to increase establishment and success in the field [59] which is not only expensive, but also suppresses mycorrhiza development of container seedlings [60].Therefore, endophyte inoculation of seedlings may be considered as a cost-effective technique to increase growth in the early stage for the nursery stock, which can lead to later stage successes in transplanting and establishment after transplanting.

Figure 1 .
Figure 1.Representative photograph of mock-inoculated (left) and inoculated (right) Douglas-fir seedling after 15 months of growth in nutrient-poor soil.

Figure 2 .
Figure 2. Root and shoot lengths of inoculated and control Douglas-fir seedlings at harvest.Data are the mean of 12 replicates.Error bars show the standard deviation.Means designated with different letters are significantly different.

Figure 3 .
Figure 3. Root and shoot weights (g) of inoculated and control Douglas-fir seedlings at harvest.Data are the mean of 12 replicates.Error bars show the standard deviation.Means designated with different letters are significantly different.

Figure 4 .
Figure 4. Total N concentration in the inoculated and control Douglas-fir seedlings needles at harvest.Data are the mean of 12 replicates.Error bars show the standard deviation.(p = 0.156).

Figure 5 .
Figure 5. Fluorescence micrographs showing intercellular colonization of mkate-tagged strain WP19 in the root tissue of the inoculated seedling of Douglas-fir (A) and GFP-tagged strain WP5 in the needle of the inoculated Douglas-fir (B).Samples were surface-sterilized and visualized (1000×) three weeks after inoculation.