Colonization and growth promotion of outplanted spruce seedlings pre ...

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Abstract: Seeds of two hybrid spruce (Picea glauca (Moench) Voss × Picea engelmannii Parry ex Engelm.) ecotypes were inoculated with one of six plant ...
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Colonization and growth promotion of outplanted spruce seedlings pre-inoculated with plant growth-promoting rhizobacteria in the greenhouse Masahiro Shishido and Christopher P. Chanway

Abstract: Seeds of two hybrid spruce (Picea glauca (Moench) Voss × Picea engelmannii Parry ex Engelm.) ecotypes were inoculated with one of six plant growth-promoting rhizobacteria (PGPR) strains previously shown to be able to stimulate spruce growth in controlled environments. The resulting seedlings were grown in the greenhouse for 17 weeks before outplanting at four reforestation sites. Inoculation with five of the six strains caused significant seedling growth promotion in the greenhouse, which necessitated analysis of relative growth rates (RGR) to evaluate seedling performance in the field. Four months after outplanting, most strains enhanced spruce shoot or root RGRs in the field, but seedling growth responses were strain specific. For example, Pseudomonas strain Ss2-RN significantly increased both shoot and root RGRs by 10–234% at all sites, but increases of 28–70% were most common. In contrast, Bacillus strain S20-R was ineffective at all outplanting sites. In addition, seedlings inoculated with four of the six strains had significantly less shoot injury than control seedlings at all sites. Evaluation of root colonization by PGPR indicated that bacterial population declines were not related to spruce growth response variability in the field. Our results indicate that once plant growth promotion is induced in the greenhouse, seedling RGR can increase by more than 100% during the first growing season in the field. However RGR increases of 21–47% were more common and may be more representative of the magnitude of biomass increases that can result from PGPR inoculation. Résumé : Les graines de deux écotypes d’épinette hybride (Picea glauca (Moench) Voss × Picea engelmannii Parry ex Engelm.) ont été inoculées avec chacune des six races de rhizobactéries capables de promouvoir la croissance des plantes et dont la capacité de stimuler la croissance de l’épinette dans un environnement contrôlé a déjà été démontrée. Les semis ainsi produits ont été cultivés en serre pendant 17 semaines avant d’être transplantés sur quatre sites de reboisement. L’inoculation avec cinq des six races a provoqué une augmentation importante de la croissance des semis en serre; ce qui a nécessité une analyse des taux relatifs de croissance pour évaluer la performance des semis au champ. Quatre mois après la transplantation, la plupart des races ont augmenté le taux relatif de croissance de la tige et des racines de l’épinette mais la réaction de croissance des semis était spécifique selon la race. Par exemple, la race Ss2-RN de Pseudomonas a provoqué une augmentation du taux relatif de croissance de la tige et des racines de 10 à 234% dans tous les sites mais une augmentation de 28 à 70% était plus fréquente. Au contraire, la race S20-R de Bacillus n’a eu aucun effet dans aucun des sites. De plus, les semis inoculés avec quatre des six races ont subi significativement moins de dommages à la tige que les semis témoins dans tous les sites. L’évaluation de la colonisation des racines par des rhizobactéries capables de promouvoir la croissance des plantes a montré que les chutes de population bactérienne n’étaient pas reliées à la variabilité de la réaction de croissance de l’épinette au champ. Nos résultats indiquent qu’une fois que la stimulation de la croissance a été induite en serre, le taux relatif de croissance des semis peut augmenter de plus de 100% pendant la première saison de croissance au champ. Cependant, une augmentation du taux relatif de croissance de 21–47% était plus fréquente et serait plus représentative de l’ampleur de l’augmentation de biomasse provoquée par l’inoculation de rhizobactéries capables de promouvoir la croissance des plantes. [Traduit par la Rédaction]

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Introduction Plant growth-promoting rhizobacteria (PGPR) (Kloepper and Schroth 1978) have been evaluated primarily on agricultural crops (Kloepper 1993; Ogoshi et al. 1997), but recent

investigations with tree species have also yielded promising results (Chanway 1997; Enebak et al. 1998). For agricultural or forestry applications, the ultimate evaluation of PGPR

Received February 19, 1999. Accepted January 15, 2000. M. Shishido.1 Department of Forest Sciences, University of British Columbia, Suite 270-2357 Main Mall, Vancouver, BC V6T 1Z4, Canada. C.P. Chanway.2 Department of Forest Sciences and Department of Soil Science, University of British Columbia, Vancouver, BC V6T 1Z4, Canada. 1 2

Present address: Faculty of Horticulture, Chiba University, 648 Matsudo, Matsudo-city, Japan 271-8510. Corresponding author. e-mail: [email protected]

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must be made in the field under environmental conditions similar to those that target plants are typically exposed. In forestry, where rotation times may be measured in decades, PGPR inoculation would not necessarily be expected to have a direct positive influence on timber production. Instead, the goal of forest seedling inoculation with PGPR is to improve survival and establishment of outplanted seedlings, often through enhancing root system development, although reduction of disease incidence in the early years after outplanting is also desirable. However, seedling growth response variability is an undesirable characteristic of PGPR studies that has impeded commercial development of bacterial inoculants for trees (Chanway and Holl 1993, 1994). A basic tenet often cited in studies of PGPR with annual crops is that introduced bacteria must colonize plant root systems and persist in the rhizosphere during the growing season for plant growth promotion to be manifested (Suslow and Schroth 1982). Population sizes of certain PGPR may decline rapidly in the rhizosphere (e.g., Kloepper et al. 1980) and could contribute to seedling growth response variabilty. However, in some cases, no concomitant reduction in plant growth promotion efficacy is accompanied by reductions in PGPR population sizes (Reddy and Rahe 1989). For tree species, Holl and Chanway (1992) demonstrated the ability of Bacillus strain L6-16R to colonize the rhizosphere of lodgepole pine 1 month after inoculation but observed large monthly population declines thereafter. Interestingly, seedling growth promotion coincided with the period of PGPR population decline. On the other hand, in a growth chamber study, Bacillus strains L6-16R and Pw-2R established large populations (i.e., >5 × 107 colony-forming units (cfu)/g fresh root tissue) in the lodgepole pine (Pinus contorta Dougl. ex Loud.) rhizosphere 2 weeks after inoculation, with only slight declines in population size 4 weeks after inoculation (Shishido et al. 1995). Fluctuations in the size of the root-associated bacterial populations did not appear to be related to seedling growth responses, but in general, very little is known about the population dynamics of PGPR on tree roots at reforestation sites and the possible effects of such fluctuations on seedling growth. In addition to colonizing the root surface and associated rhizosphere soil, certain plant growth-promoting bacteria are also capable of entering plant tissues and colonizing microsites inside “host” plants (Hallmann et al. 1997; Chanway 1998). Some bacterial endophytes are diazotrophic and are thought to satisfy most of the host plant’s nitrogen requirements through nitrogen fixation within plant tissues, e.g., Acetobacter (Baldani et al. 1997; James et al. 1997). Other endophytic bacteria of the genera Bacillus and Pseudomonas are known to act as biological control agents either directly, by inhibiting plant pathogenic microorganisms such as Pythium and Rhizoctonia (Hagedorn et al. 1989, 1993) or, indirectly, by inducing plant systemic resistance to fungal and bacterial pathogens such as Fusarium and Pseudomonas syringae (Wei et al. 1991, 1996). Because competition from indigenous soil microorganisms can reduce or eliminate the population of introduced beneficial microorganisms (Kloepper et al. 1989), growth promotion may occur more consistently if PGPR colonize the comparatively buffered environment within plant tissues. In addition, it is possible

Can. J. For. Res. Vol. 30, 2000

that a post-inoculation, pre-outplanting growth period in the nursery or greenhouse may facilitate improved PGPR population establishment and result in enhanced bacterial persistence in the field. However, work is only beginning on the possible practical applications of endophtyic PGPR, particularly for tree species. The primary objectives of this experiment were to determine if “pre-inoculating” spruce seedlings in the greenhouse 17 weeks in advance of outplanting enhanced PGPR efficacy after outplanting and if root colonization was related to growth and survival of spruce seedlings in the field.

Materials and methods Microorganisms We used six bacterial strains previously shown to be able to stimulate seedling growth: Bacillus polymyxa strains L6-16R, S20-R, and Pw-2R, which are rifamycin-resistant derivatives of strains L6, S20, and Pw-2, respectively, and Pseudomonas fluorescens strains Sm3-RN, Ss2-RN, and Sw5-RN, which are nalidixic acid and rifamycin resistant strains derived from Sm3, Ss2, and Sw5, respectively (Shishido et al. 1996a, 1996b). These antibiotic-resistant strains were similar to their respective wild-type strains in colony morphology, 23S-rDNA sequences, Biolog™ (Biolog Inc., Hayward, Calif.) carbon substrate utilization, profiles of gas chromatography of fatty acid methyl esters, and intrinsic antibiotic resistance to streptomycin, kanamycin, tetracycline, and nalidixic acid. Strains L6-16R, S20-R, Pw-2R, and Ss2-RN are known external root colonists and are unable to enter internal plant tissues, while strains Pw-2R and Sm3-RN have been detected inside gymnosperm seedling tissues (Shishido et al. 1995; Shishido 1997).

Seedling preparation Seeds of two hybrid spruce (white spruce (Picea glauca (Moench) Voss) × Engelmann spruce (Picea engelmannii Parry ex Engelm.)) ecotypes (Mackenzie, B.C.: 55°11′ N, 122°58′ W, 780 m elevation; Williams Lake, B.C.: 52°33′ N, 122°06′ W, 884 m elevation) were used to grow seedlings for this experiment. Seeds were stratified at 4°C for 30 days before sowing in plastic containers (RLC-3 Fir Cells, 2.5 cm diameter × 12 cm deep, 45 cm3; Stuewe & Sons, Inc., Covallis, Oreg.) filled with autoclaved Sunshine-Mix (Fisons Horticulture Inc., Vancouver, B.C., Canada) soil medium, a mixture of vermiculite, bark, peat moss, quartz, gypsum, perlite, and CaCO3 (organic matter, 44%; total N, 0.72% (pH 5.7); available nutrients (µg·g–1): P, 540; K, 1260; Ca, 9000; Mg, 2820; Cu, 2; Zn, 24; Fe, 120; Mn, 144; B, 4.8; and SO4-S, 2254). Bulk density of this medium was 0.10 g·cm–3. Four seeds were planted in each container. PGPR inoculum was prepared using 50% strength tryptic soy (TS) broth for Bacillus strains and full-strength King’s B (KB) broth for Pseudomonas strains. Cultures were grown for 3 days on a rotary shaker (120 rpm) at room temperature. Cells were harvested by centrifugation (approximately 3000 × g for 15 min) and washed by resuspending the resultant bacterial pellet in sterile 10 mM phosphate buffer (SPB) and centrifuging as described above. After a second washing cycle, cells were resuspended in 10 mM SPB at the following inoculum densities (cfu/mL): L616R, 2.0 × 105; Pw-2R, 3.3 × 105; S20-R, 1.1 × 105; Sm3-RN, 1.8 × 107; Ss2-RN, 2.2 × 107; and Sw5-RN, 4.2 × 106. Containers were inoculated by pipetting 1.0 mL of one of the strains directly over the soil covering seeds. Control containers received 1.0 mL of sterile 10 mM SPB. One week later, after most germinants had emerged and cotyledons were visible, a second inoculation was performed following the same protocol to ensure seedlings © 2000 NRC Canada

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Table 1. Site description of the four field sites and properties of topsoils. Property

Smithers Blunt Creek

Smithers Shoe-house

Williams Lake regular cut-block

Williams Lake landing

Latitude (N) Longitude (W) Elevation (m) Slope (%) Landform Soil texture Coarse fragments (>2 mm) (%) Disturbance type pH* EC (dS·m–1) Total carbon (%) Total nitrogen (%) Phosphorus (µg·g–1) Potassium (µg·g–1) Calcium (µg·g–1) Magnesium (µg·g–1) Iron (µg·g–1) Sulfate sulfur (µg·g–1)

55°10′ 127°02′ 1200 5–10 Glacial till — 59 Scalp, wheel ruts 5.1 0.44 32.2 0.79 57 380 6100 980 35 15.1

55°55′ 127°34′ 730 0–5 Fluvial veneer over till Loam 57 Landing 6.3 0.48 8.9 0.12 52 190 2900 390 205 3.2

52°37′ 121°45′ 950 0–5 Colluvial veneer over till Clay loam 61 Scalp, wheel ruts 5.3 0.42 12.0 0.24 71 290 2300 480 155 4.0

52°37′ 121°45′ 950 0–5 Colluvial veneer over till Loam 48 Landing 6.3 0.48 2.0 0.06 52 190 1500 290 220 6.5

*Ratio of ovendried soil to deionized H2O of 1:2.

received an adequate bacterial population to stimulate seedling growth (Shishido et al. 1996a, 1996b). Cell densities for the second inoculation were (cfu/mL): L6-16R, 2.9 × 106; Pw-2R, 4.9 × 107; S20-R, 9.8 × 106; Sm3-RN, 6.6 × 108; Ss2-RN, 9.9 × 108; and Sw5-RN, 8.5 × 108. Each inoculation treatment had 180 containers (90 seedlings per spruce ecotype), which were contained in 30 × 60 cm racks (RL200 Tray, Stuewe & Sons, Inc., Oreg.). Racks were placed on the greenhouse bench ca. 30 cm apart and were rotated every week to reduce potential positional effects in the greenhouse. Seedlings were thinned to the largest single germinant per cone after the second inoculation treatment. Seedlings were watered every 3 days and fertilized every 2 weeks with a solution containing the following nutrients (µg/seedling): N, 1000; P, 250; K, 500; Ca, 500; Mg, 25; S, 48; Fe, 2; Mn, 0.25; Cu, 0.1; Zn, 0.1; B, 0.1; Mo, 0.025 (van den Driessche 1989). Temperature in the greenhouse ranged from 14 to 28°C, and an extended photoperiod of 19 h light : 5 h dark was achieved through artificial light with a minimum of 130 µmol·m–2·s–1 photosynthetically active radiation at bench level. Fifteen weeks after sowing (and 2 weeks before outplanting), external and internal root colonization was assessed on eight randomly selected seedlings per bacterial treatment (four seedlings per spruce ecotype). To evaluate external root colonization, i.e., in the rhizosphere and on the root surface, the primary root including the apical meristem was sampled from each seedling. Each root was placed in a culture tube containing 10 mL of 10 mM SPB with glass beads and agitated for 1 min. Root washings were then serially diluted and plated onto 50% strength TS agar amended with 100 mg·L–1 rifamycin and cycloheximide and 50 mg·L–1 nystatin for Bacillus and KB agar amended with 100 mg·L–1 nalidixic acid, rifamycin, and cycloheximide and 50 mg·L–1 nystatin for Pseudomonas. To evaluate internal root colonization by bacteria, roots were washed in 10 mM SPB and surface sterilized by immersion in 1.8% NaClO for 1 min followed by three rinses in sterile water. Tissues were then aseptically triturated in 10 mL SPB with a mortar and pestle, and serial dilutions of the resulting homogenates were plated (0.1-mL aliquots) on 50% strength TS agar (for Bacillus) or KB agar (for Pseudomonas) amended with antibiotics as de-

scribed above. Dilution plates were incubated for up to 4 days at 28°C before colony numbers were assessed.

Evaluation of root colonization and seedling performance of PGPR-inoculated spruce grown in the field PGPR-inoculated seedlings were 4 months old at the time of outplanting. Two field sites located near Smithers, B.C. (Blunt Creek site and Shoe-house site), and two near Williams Lake, B.C. (regular cut-block site and landing site), were used for this experiment. Site descriptions including soil properties are presented in Table 1. Seedlings were matched to the region of their origin, i.e., the Williams Lake spruce ecotype was planted at the Williams Lake sites and the Mackenzie ecotype was planted at the Smithers sites. Seedlings were not subjected to dormancy-inducing treatments before outplanting, because they were scheduled for harvest before fall frosts would normally occur in these regions. Before outplanting at each site during the first week of June, 74 seedlings of uniform height were selected from each PGPR treatment. Twenty of these were used for determining preplanting seedling biomass and the remainder (54) were used for planting (27 at each of two sites near Smithers and Williams Lake). At each site, existing vegetation was cleared and inoculated seedlings were planted according to a randomized complete block design (n = 27) with seven inoculation treatments (six bacterial treatments plus the control). After gently removing root plugs from plastic containers, seedlings were planted at a 1-m spacing with care to reduce the possibility of cross contamination between PGPR treatments, e.g., entire treatments were established one at a time at each site, and planting tools were disinfected before starting a new treatment. Four months after outplanting (late September or early October), seedlings were excavated, taking care to include the whole root system. Seedlings were placed in individual plastic bags and stored at 4°C before being processed in the laboratory within 2 weeks of harvesting. External and internal root colonization by each strain was assessed on four seedlings in each treatment using the dilution plate assays described above. All seedlings in each treatment were also visually assessed for shoot injury using a four-rank scale based on the percentage of dead needles. Category 0 seedlings had © 2000 NRC Canada

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Fig. 1. PGPR recovery from (a) external and (b) internal root tissues of 8-month-old spruce seedlings grown at the Smithers Blunt Creek site for 4 months. Error bars are SEs.

Fig. 2. PGPR recovery from (a) external and (b) internal root tissues of 8-month-old spruce seedlings grown at the Smithers Shoehouse site for 4 months. Error bars are SEs.

91–100% needle mortality; category 1 had 51–90% needle mortality; category 2 had 10–50% needle mortality, and category 3 had less than 10% needle mortality. Root systems were then washed free of soil and separated from shoots. Root and shoot dry weights were assessed after drying at 70°C for 4 days.

Statistical analyses of seedling performance and root colonization by PGPR strains Shoot injury ranks were analyzed using the non-parametric Kruskal–Wallis test. Shoot and root growth responses to PGPR treatment in the greenhouse were assessed using ANOVA and Fisher’s protected least significant difference (LSD) for each spruce ecotype. Shoot and root relative growth rates (RGR) of seedlings during their greenhouse growth period were calculated according to Hunt’s (1978) relationship:

dW  d(ln W ) RGR = W −1   =  dT  dT

where W is the seedling shoot or root biomass (n = 20) at T, the time interval between sowing and harvesting (112 days). The RGR of seedlings grown in the field was also calculated according to Hunt (1978), using the formula:

RGR =

ln W2 − ln W1 dT

where W2 is dry seedling weight (n = 27) at the final harvest (i.e., the end of the field growth period), W1 is the mean seedling shoot or root biomass at the end of the greenhouse growth period (standard error was 5% of the mean for both shoots and roots), and dT is the number of days seedlings grew in the field (approximately 112, depending on the site). Biomass of dead seedlings was treated as unchanged. Because planting sites were selected on the basis of differences in environmental conditions (Table 1) and spruce ecotypes were nested within sites, data from each site were analyzed separately. © 2000 NRC Canada

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Fig. 3. PGPR recovery from (a) external and (b) internal root tissues of 8-month-old spruce seedlings grown at the Williams Lake regular cutblock site for 4 months. Error bar are SEs.

Fig. 4. PGPR recovery from (a) external and (b) internal root tissues of 8-month-old spruce seedlings grown at the Williams Lake landing site for 4 months. Error bar are SEs.

Results Root colonization by PGPR and plant growth responses to inoculation under greenhouse conditions Four months after inoculation (i.e., at the end of the greenhouse growth period), Bacillus and Pseudomonas inoculant strains were detected with >2 × 104 and >6 × 105 cfu/g rhizosphere soil, respectively (Figs. 1a, 2a, 3a, and 4a). Strains Pw-2R and Sm3-RN also colonized internal root tissues with 1 × 102 – 6 × 104 cfu/g root tissue (Figs. 1b, 2b, 3b, and 4b). Inoculation with all strains except Sm3-RN significantly enhanced seedling growth (either shoot or root biomass) of gymnosperm ecotypes by the end of the greenhouse phase of the experiment (Table 2). Strain L6-16R was the most effective PGPR in the greenhouse, causing root and shoot biomass increases of 29 and 35%, respectively, on

both spruce ecotypes. Root and shoot biomass increases caused by the other strains in the greenhouse ranged from 12 to 22%. Root colonization by PGPR and plant growth responses to inoculation under field conditions In general, external root colonization levels declined during growth in the field. Population levels of Bacillus strains occasionally dropped below the assay detection limit (104 cfu/g rhizosphere soil), which resulted in low mean values and large SEs (Figs. 1a, 2a, 3a, and 4a). Of the six strains evaluated, Pseudomonas strain Sw5-RN was recovered with the highest external root populations at every site, while Bacillus strain S20-R had the lowest population sizes at all but the Williams Lake regular cutblock site. Internal root colonization by Pseudomonas strain Sm3RN was detected in seedlings sampled at each of the four © 2000 NRC Canada

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1.60±0.29 –0.60±0.56 1.33±0.15 3.30±0.47 1.03±0.59 1.63±0.20 2.73±0.47 0.12±0.56 1.44±0.20

107.8±5.4 123.4±6.6

2.37±0.37 0.96±0.54 1.82±0.16 3.91±0.42† 0.96±0.53 1.93±0.17 3.10±0.34† 0.76±0.39 1.70±0.18 4.36±0.55** 1.68±0.46 1.85±0.22

143.4±5.81** 153.7±5.06**

2.97±0.34 0.55±0.62 2.04±0.17 4.34±0.31* 0.71±0.51 1.85±0.16 3.00±0.37 1.06±0.49 1.89±0.20 3.11±0.40 2.10±0.41* 1.93±0.21

2.95±0.54 0.54±0.51 1.26±0.18

107.5±4.7 135.3±6.27*

115.9±6.4 136.6±6.92*

125.1±6.52* 129.4±6.1

S20-R

139.4±5.13** 150.7±6.53**

Pw-2R

4.72±0.39** 1.71±0.65 1.78±0.12

2.78±0.42 0.88±0.47 1.52±0.15

3.94±0.36† 0.86±0.45 1.52±0.14

2.48±0.44 0.19±0.69 1.74±0.19

103.8±5.7 125.6±5.2

101.7±4.8 119.3±5.1

Sm3-RN

4.26±0.47** 2.44±0.67* 1.85±0.20

3.79±0.31† 1.12±0.47 1.96±0.15

4.74±0.52* 1.52±0.55 2.00±0.14

1.94±0.50 1.67±0.43† 1.85±0.17

118.9±5.2 137.8±6.08*

121.1±6.17* 132.3±5.92†

Ss2-RN

3.44±0.56 1.31±0.74 1.56±0.23

3.28±0.32† 0.55±0.40 1.82±0.16

4.42±0.44* 0.88±0.44 1.96±0.16

3.09±0.40 0.98±0.54 1.81±0.18

118.8±5.9 138.6±6.68**

125.6±6.36** 136.1±6.51*

Sw5-RN

2.40±0.51 0.73±0.41 1.30±0.18

2.23±0.26 0.84±0.42 1.48±0.17

2.92±0.41 0.70±0.33 1.41±0.19

2.18±0.42 0.01±0.60 1.44±0.19

107.9±4.7 116.0±5.4

103.1±6.2 116.8±6.3

Control

850

Note: Shoot injury ranks were as follows: 0, dead; 1, more than 50% dead needles; 2, up to 50% dead needles; 3, no detectable injury. † p < 0.1. *p < 0.05. **p < 0.01.

In greenhouse Mackenzie ecotype Shoot biomass (mg) Root biomass (mg) Williams Lake ecotype Shoot biomass (mg) Root biomass (mg) In fields Smithers Blunt Creek site RGR for shoot (mg·g–1 per day) RGR for root (mg·g–1 per day) Shoot injury rank Smithers Shoe-house site RGR for shoot (mg·g–1 per day) RGR for root (mg·g–1 per day) Shoot injury rank Williams Lake regular cutblock site RGR for shoot (mg·g–1 per day) RGR for root (mg·g–1 per day) Shoot injury rank Williams Lake landing site RGR for shoot (mg·g–1 per day) RGR for root (mg·g–1 per day) Shoot injury rank

L6-16R

Table 2. Spruce seedling growth (mean and standard error) under greenhouse and field conditions after inoculations with PGPR strains.

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sites. Bacillus strain Pw-2R was detected inside seedlings from three of the four sites. Pseudomonas strains Ss2-RN and Sw5-RN were occasionally detected in homogenates from surface sterilized roots (Figs. 1b, 3b, and 4b). Because seedling biomass increased in response to PGPR inoculation in the greenhouse, seedling growth in the field was evaluated using RGRs rather than absolute growth rates. In general, shoot RGRs were more responsive to bacterial treatment than root RGRs at the end of the outplanting period (Table 2). Except for a reduction in shoot RGR at the Blunt Creek site in response to strains S20-R and Ss2-RN, mean shoot RGR increased in response to bacterial inoculation by 9–97% at all sites, with most increases in the 25– 75% range (Table 2). Root RGRs were more variable than shoot RGRs, but many of these strains caused increases of root RGR in excess of 100%, particularly at the Williams Lake landing site. Individual strains were found to perform differently at these sites. For example, strain Ss2-RN significantly increased either shoot or root RGR at all sites, while seedling RGR was not enhanced by S20-R at any of the field sites, notwithstanding significant biomass increases caused by this strain in the greenhouse. The effect of other strains varied by site so that strains Pw-2R and Sm3-RN, for example, were ineffective at the Smithers Blunt Creek site but were effective RGR promoters at the other three sites. Seedlings planted at landing sites (Smithers Shoe-house and Williams Lake landing) were more likely to benefit from PGPR inoculation than those planted at less compacted, mechanically scarified Smithers Blunt Creek and Williams Lake regular cutblock sites (Table 2). Shoot injury ranks showed large variations among inoculation treatments (Table 2). However, seedlings inoculated with strains L6–16R, Pw-2R, Ss2-RN, and Sw5-RN had less shoot injury than non-inoculated controls at every site. Shoot injury of strain S20-R-treated seedlings did not differ from controls, and strain Sm3-RN had variable effects among the four sites.

Discussion The primary objectives of this experiment were to determine if “pre-inoculating” spruce seedlings in the greenhouse 17 weeks in advance of outplanting would enhance PGPR efficacy and if root colonization was related to growth and survival of spruce seedlings in the field. Spruce seedling RGR was significantly enhanced in the field by all PGPR, except strain S20-R. Although the growth rates observed in the field were lower than those in the greenhouse, these results indicate that inoculating seedlings some time in advance of outplanting results in significant growth enhancement in the field. The latter point gains particular relevance in view of a concurrent experiment we performed at the same outplanting sites with 1-year-old spruce seedlings inoculated 1 or 2 days before outplanting with five of the six strains used in the current work (strains L6-16R, S20-R, Pw2R, Ss2-RN, and Sm3-RN; Chanway et al. 1997). Three months after outplanting, spruce shoot growth was unaffected by bacterial inoculation at the Smithers Shoe-house and Williams Lake regular sites and significantly inhibited by strain Sm3-RN at the Williams Lake landing site

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(C. Chanway, unpublished data). Only two significant increases in shoot weight occurred in that trial, both at the Smithers Blunt Creek site. Spruce root growth responded in similar fashion to shoot growth. It is possible that carry-over effects due to nutrient loading of seedlings before outplanting (van den Driessche 1985; Malik and Timmer 1996) or supplementary nutrients and moisture in the root plug (e.g., Burdett et al. 1984; Nilsson and Örlander 1995) influenced seedling growth in the shortterm trials described in this paper. Such effects could obscure nutrient differences between sites. However, based on the paucity of positive responses to PGPR inoculation in the study by Chanway et al. (1997), which would also have been affected by such nursery carry-over effects, “preinoculation” of seedlings appears to be a much superior strategy for enhancing PGPR efficacy after outplanting. One possible reason why “pre-inoculated” seedlings showed superior performance in the field compared with non-inoculated controls or seedlings inoculated immediately before outplanting may relate to their larger size after the greenhouse growth period. Many studies have demonstrated the importance of seedling size in relation to soil moisture stress at planting sites. For example, Hines and Long (1986) reported that smaller Engelmann spruce seedlings, based on diameter of the root collar, exhibited significantly higher drought stress 4 weeks after outplanting as well as lower survival rates during the first and second growing seasons. Van den Driessche (1992) also observed significant correlations between shoot dry weight of conifer seedlings in the nursery and outplanted seedling growth at xeric sites. Our results with strains L6-16R, Pw-2R, Ss2-RN, and Sw5-RN agree with these previous studies in terms of the importance of seedling size and subsequent performance in the field. However, the unique aspect of our study is that preplanting seedling biomass was increased by PGPR inoculation. Our results with Pseudomonas strain Sm3-RN are particularly noteworthy, because this strain colonized external and internal seedling tissues without affecting seedling growth in the greenhouse but caused large, statistically significant increases in spruce RGR after outplanting. These observations are consistent with our finding that seedlings inoculated well in advance of outplanting perform better than seedlings inoculated immediately before planting but do not support the hypothesis that the superior performance is simply related to larger seedling size at planting. Perhaps this strain is capable of alleviating certain environmental stresses once seedlings are outplanted, and in the absence of such stresses, i.e., in the greenhouse, no growth promotion is observed. Alternatively, because it is endophytic, strain Sm3-RN may require the post-inoculation, pre-planting period to establish an effective internal bacterial population size for plant growth promotion to occur. External root population sizes of Bacillus and Pseudomonas were ca. 104 and 106 cfu/g soil, respectively, by the end of the greenhouse growth period. Since the inoculum densities of the Bacillus and Pseudomonas strains were ca. 105 and 107 cfu/plant cone, respectively, only approximately 10% of the initial PGPR population remained after the greenhouse phase of this experiment. Similar root-zone declines in PGPR population have been observed in studies on agricultural plants, particularly under conditions of water © 2000 NRC Canada

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stress (Kloepper et al. 1980; Juhnke et al. 1987, 1989; Reddy and Rahe 1989; Bashan et al. 1991; Kumar and Dube 1992), as well as for strain L6-16R in the rhizosphere of lodgepole pine (Holl and Chanway 1992). PGPR population sizes associated with spruce roots decreased a further 10- to 100-fold during the 4-month period of field growth, suggesting that field conditions were severe, even in the rhizosphere of host seedlings. However, not all strains persisted in the rhizosphere with equal tenacity. For example, Pseudomonas strains Sw5-RN and Sm3-RN generally maintained larger population sizes than the other strains, while populations of Pseudomonas strain Ss2-RN declined drastically at most of the sites. The reason for the rapid decline of the Ss2-RN population is not clear. It is possible that this strain was more susceptible to harsh environmental factors such as low water potentials and large soil temperature fluctuations characteristic of cutover sites than the other two pseudomonads. Strain Ss2-RN was originally isolated from the rhizoplane of a spruce root (Shishido et al. 1996b), and may be adapted to a narrower range of environmental conditions than the other two Pseudomonas strains, which were isolated from the rhizosphere. The population sizes of Bacillus strains recovered from the rhizosphere also generally declined after outplanting. Bacillus are well known to sporulate under adverse environmental conditions (Roszak and Colwell 1987), and the rapid decline in vegetative cell population soon after soil inoculation has been documented (Siala et al. 1974; Van Elsas et al. 1986; Roszak and Colwell 1987). In general, these colonization data clearly support the view by Kloepper (1993) that “one cannot assume from a report on a single strain of any genus or species that all strains of the taxon are capable of colonizing roots.” Nevertheless, in spite of declining rhizosphere populations in the field, bacterial inoculation generally had the desired affect of stimulating seedling growth, which suggests that there is no positive relationship between rhizosphere population size and growth promotion efficacy at these sites. Growth promotion by our strains in the field was reflected primarily in the relative shoot growth rates, and less so in relative root growth rates. This may have been, in part, due to the high light intensity at the cutover sites or the supplementary nutrients and moisture in the root plug (e.g., Burdett et al. 1984; Nilsson and Örlander 1995). However, the technical difficulties associated with harvesting root samples after a period of field growth, i.e., possible incomplete root system recovery, may have obscured trends in root RGR at these sites, which may have been higher than we were able to detect. It is interesting that PGPR mean biomass increases were greater at both landing sites (Smithers Shoe-house and Williams Lake landing) compared with the nearby, site-prepared (scarified) cutblock sites, although no formal site comparisons were made (see Statistical analysis of seedling performance section). Surface organic matter may impede the growth of planted seedlings by reducing the available soil– root contact area in duff layers for nutrients and water (McMinn and Hedin 1990) or by increasing the incidence of low soil temperature. Dobbs and McMinn (1977) indicated that, because of its high thermal diffusivity, mineral soil facilitates larger heat re-radiation during the night and reduces

Can. J. For. Res. Vol. 30, 2000

the incidence of frost damage. Although it is difficult to pinpoint exact environmental factors that differentiated the apparent seedling growth response to PGPR inoculation between these “landing” and “regular” sites, PGPR inoculation may be more useful in soils that have been degraded physically, such as landing sites compared with relatively undisturbed environments. In the only other similar studies performed to date, Chanway and Holl (1993, 1994) also observed greater PGPR efficacy at hybrid spruce and lodgepole pine at sites of lower productivity and hypothesized that such inocula may be most useful at sites with harsh seedling growth conditions. Interestingly, seedlings used in those studies were also inoculated in a controlled environment 4–5 weeks in advance of outplanting. However, our most recent results with spruce inoculated on site and outplanted on landing sites do not support this hypothesis (Chanway et al. 1997), possibly due to the lack of a postinoculation, pre-outplanting period (i.e., seedlings were inoculated 1 or 2 days before outplanting). We were also interested in comparing growth promotion efficacy in the field of the two endophytic strains, Pw-2R and Sm3-RN, to that of the other non-endophytic PGPR strains. Endophytic strains appeared to show greater seedling growth promotion efficacy compared with non-endophytic PGPR at the Williams Lake landing site but not at the other outplanting sites. Therefore, the hypothesis that endophytic PGPR strains are protected from adverse environmental conditions in the host tissues and improve the persistence of plant growth promotion efficacy was not supported, at least for the short term after outplanting. In addition, the population sizes of Pw-2R and Sm3-RN recovered from internal root tissues were almost always smaller than the corresponding rhizosphere population, which suggests that, notwithstanding their endophytic capabilities, these two strains appear to be more efficacious external root colonists than endophytes. However, there is no known threshold population size for endophytic PGPR to affect plant growth promotion, and it could be hypothesized that endophytic PGPR may be efficacious even with relatively small population sizes because of their strategic location within the host plant. Therefore, in the longer term, the ability of endophytic strains to diversify their habitat by colonizing internal and external host tissues could provide a significant survival and persistence advantage over their external root colonizing counterparts.

Conclusions PGPR-inoculated spruce seedlings frequently outperformed non-inoculated controls under field conditions based on RGR. The survival of endophytic PGPR was confirmed after the first season in the field, but efficacy of endophytic PGPR was not significantly greater that external root colonizing PGPR, at least in the short term. In addition, the population sizes of most of PGPR in the rhizosphere declined significantly in the greenhouse and field and were not correlated to seedling growth responses after outplanting. We conclude that a post-inoculation, pre-outplanting period of seedling growth in the greenhouse or nursery has a significant, positive effect on PGPR efficacy in the field. However, long-term field studies are required to demonstrate this with a greater degree of certainty. © 2000 NRC Canada

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Acknowledgements Funding for this project was provided by a Natural Sciences and Engineering Research Council of Canada Research Grant to C.P.C. and a postgraduate scholarship from the Foundation for Advanced Studies on International Development of Japan to M.S.

References Baldani, J.I., Caruso, L., Baldani, V.L.D., Goi, S.R., and Döbereiner, J. 1997. Recent advances in BNF with non-legume plants. Soil Biol. Biochem. 29: 911–922. Bashan, Y., Levanony, H., and Whitmoyer, R.E. 1991. Root surface colonization of non-cereal crop plants by pleomorphic Azospirillum brasilense Cd. J. Gen. Microbiol. 137: 187–196. Burdett, A.N., Herring, L.J., and Thompson, C.F. 1984. Early growth of planted spruce. Can. J. For. Res. 14: 644–651. Chanway, C.P. 1997. Inoculation of tree roots with plant growth promoting soil bacteria: an emerging technology for reforestation. For. Sci. 43: 99–112. Chanway, C.P. 1998. Bacterial endophytes: ecological and practical implications. Sydowia, 50: 149–170. Chanway, C.P., and Holl, F.B. 1993. First year field perfomance of spruce seedlings inoculated with plant growth promoting rhizobacteria. Can. J. Microbiol. 39: 1084–1088. Chanway, C.P., and Holl, F.B. 1994. Growth of outplanted lodgepole pine seedlings one year after inoculation with growth promoting rhizobacteria. For. Sci. 40: 238–246. Chanway, C.P., Shishido, M., Jungwirth, S., Nairn, J., Markham, J., Xiao, G., and Holl, F.B. 1997. Second year growth responses of outplanted conifer seedlings inoculated with PGPR. In Proceedings of the 4th International Workshop on Plant GrowthPromoting Rhizobacteria: Plant Growth-Promoting Rhizobacteria—Present Status and Future Prospects, 5–10 Oct. 1997, Sapporo, Japan. Edited by A. Ogoshi, K. Kobayashi, Y. Homma, F. Kodama, N. Kondo, and S. Akino. Japan-OECD Joint Workshop, Paris. pp. 172–176. Dobbs, R.C., and McMinn, R.G. 1977. Effects of scalping on soil temperature and growth of white spruce seedlings. B.C. Ministry of Agriculture, Victoria. Rep. B.C. Soil Sci. Workshop No. 6. pp. 66–73. Enebak, S.A., Wei, G., and Kloepper, J.W. 1998. Effects of plant growth-promoting rhizobacteria on loblolly and slash pine seedlings. For. Sci. 44: 139–144. Hagedorn, C., Gould, W.D., and Bardinelli, T.R. 1989. Rhizobacteria of cotton and their repression of seedling disease pathogens. Appl. Environ. Microbiol. 55: 2793–2797. Hagedorn, C., Gould, W.D., and Bardinelli, T.R. 1993. Field evaluations of bacterial inoculants to control seedling disease pathogens on cotton. Plant Dis. 77: 278–282. Hallmann, J., Quadt-Hallmann, A., Mahafee, W.F., and Kloepper, J.W. 1997. Bacterial endophytes in agricultural crops. Can. J. Microbiol. 43: 895–914. Hines, F.D., and Long, J.N. 1986. First- and second-year survival of containerized Engelmann spruce in relation to initial seedling size. Can. J. For. Res. 16: 668–670. Holl, F.B., and Chanway, C.P. 1992. Rhizosphere colonization and seedling growth promotion of lodgepole pine by Bacillus polymyxa. Can. J. Microbiol. 38: 303–308. Hunt, R. 1978. Plant growth analysis. Edward Arnold Ltd., London. James, E.K., Olivares, F.L., Baldani, J.I., and Döbereiner, J. 1997. Herbaspirillum, an endophytic diazotroph colonizing vascular

853 tissue in leaves of Sorghum bicolor L. Moench. J. Exp. Bot. 48: 785–797. Juhnke, M.E., Mathre, D.E., and Sands, D.C. 1987. Identification and characterization of rhizosphere-competent bacteria of wheat. Appl. Environ. Microbiol. 53: 2793–2799. Juhnke, M.E., Mathre, D.E., and Sands, D.C. 1989. Relationship between bacterial seed inoculum density and rhizosphere colonization of spring wheat. Soil Biol. Biochem. 21: 591–595. Kloepper, J.W. 1993. Plant growth-promoting rhizobacteria as biological control agents. In Soil microbial ecology—applications in agricultural and environmental management. Edited by F.B. Metting, Jr. Marcel Dekker, New York. pp. 255–274. Kloepper, J.W., and Schroth, M.N. 1978. Plant growth promoting rhizobacteria on radishes. In Proceedings of the 4th International Conference on Plant Pathogenic Bacteria Vol. 2, 27 Aug. – 2 Sept. 1978, Angers, France. Edited by Station de Pathologie Vegetale et Phytobacteriologie. Gilbert-Clarey, Tours, France. pp. 879–882. Kloepper, J.W., Schroth, M.N., and Miller, T.D. 1980. Effects of rhizosphere colonization by plant growth-promoting rhizobacteria on potato plant development and yield. Phytopathology, 70: 1078–1082. Kloepper, J.W., Lifshitz, R., and Zablotowicz, R.M. 1989. Freeliving bacterial inocula for enhancing crop productivity. Trends Biotechnol. 7: 39–44. Kumar, B.S.D., and Dube, H.C. 1992. Seed bacterization with a fluorescent Pseudomonas for enhanced plant growth, yield and disease control. Soil Biol. Biochem. 24: 539–542. Malik, V., and Timmer, V.R. 1996. Growth, nutrient dynamics, and interspecific competition of nutrient-loaded black spruce seedlings on a boreal mixedwood site. Can. J. For. Res. 26: 1651– 1659. McMinn, R.G., and Hedin, I.B. 1990. Site preparation: mechanical and manual. In Regenerating British Columbia’s forests. Edited by D.P. Lavender, R. Parish, C.M. Johnson, G. Montgomery, A.Vyse, R.A. Willis, and D. Winston, Univeristy of British Columbia Press, Vancouver. pp. 150–163. Nilsson, U., and Örlander, G. 1995. Effects of regeneration methods on drought damage to newly planted Norway spruce seedlings. Can. J. For. Res. 25: 790–802. Ogoshi, A., Kobayashi, K., Homma, Y., Kodama, F., Kondo, N., and Akino, S. (Editors). 1997. Proceedings of the 4th International Workshop on Plant Growth-Promoting Rhizobacteria: Plant Growth-Promoting Rhizobacteria—Present Status and Future Prospects, 5–10 Oct. 1997, Sapporo, Japan. Japan-OECD Joint Workshop, Paris. Reddy, M.S., and Rahe, J.E. 1989. Growth effects associated with seed bacterization not correlated with populations of Bacillus subtilis inoculant in onion seedling rhizospheres. Soil Biol. Biochem. 21: 373–378. Roszak, D.B., and Colwell, R.R. 1987. Survival sterategies of bacteria in the natural environment. Microbiol. Rev. 51: 365–379. Shishido, M. 1997. Plant growth promoting rhizobacteria (PGPR) for interior spruce (Picea engelmannii × P. glauca) seedlings. Ph.D. thesis, University of British Columbia, Vancouver. Shishido, M., Loeb, B.M., and Chanway, C.P. 1995. Rhizosphere and internal root colonization of lodgepole pine by two seedling growth-promoting Bacillus strains originating from different root microsites. Can J. Microbiol. 41: 701–713. Shishido, M., Massicotte, H.B., and Chanway, C.P. 1996a. Effect of plant growth promoting Bacillus strains on pine and spruce seedling growth and mycorrhizal infection. Ann. Bot. (London), 77: 433–441. © 2000 NRC Canada

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854 Shishido, M., Petersen, D.J., Massicotte, H.B., and Chanway, C.P. 1996b. Pine and spruce seedling growth and mycorrhizal infection after inoculation with plant growth promoting Pseudomonas strains. FEMS Microbiol. Ecol. 21: 109–119. Siala, A., Hill, L.R., and Gray, T.R.G. 1974. Populations of sporeforming bacteria in an acid forest soil, with special reference to Bacillus subtilis. J. Gen. Microbiol. 81: 183–190. Suslow, T.V., and Schroth, M.N. 1982. Rhizobacteria of sugar beets: effect of seed application and root colonization on yield. Phytopathology, 72: 199–206. van den Driessche, R. 1985. Late-season fertilization, mineral nutrient reserves, and retranslocation in planted Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings. For. Sci. 31: 485–496. van den Driessche, R. 1989. Nutrient deficiency symptoms in container-grown Douglas-fir and white spruce seedlings. B.C. Ministry of Forests, Victoria. Canada–B.C. Forest Resource Development Agreement (FRDA) Rep. 100.

Can. J. For. Res. Vol. 30, 2000 van den Driessche, R. 1992. Changes in drought resistance and root growth capacity of container seedlings in response to nursery drought, nitrogen, and potassium treatments. Can. J. For. Res. 22: 740–749. Van Elsas, J.D., Dijkstra, A.F., Govaert, J.M., and Van Veen, J.A. 1986. Survival of Pseudomonas fluorescens and Bacillus subtilis introduced into two soils of different texture in field microplots. FEMS Microbiol. Ecol. 38: 151–160. Wei, G., Kloepper, J.W., and Tuzun, S. 1991. Induction of systemic resistance of cucumber to Colletotrichum orbiculare by selected strains of plant growth-promoting rhizobacteria. Phytopathology, 81: 1508–1512. Wei, G., Kloepper, J.W., and Tuzun, S. 1996. Induced systemic resistance to cucumber diseases and increased plant growthpromoting rhizobacteria under field conditions. Phytopathology, 86: 221–224.

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