Figures (5)  Tables (4)

    • Figure 1. 

      Boxplots of the weight (kg), and number of large (diameter ≥ 60 mm) and small (diameter < 60 mm) fruits on individual trees in each year. Trees of the apple cultivar Gala, grafted onto the M9 rootstock, were planted in April 2020 with soils amended with one of nine soil treatments in addition to an unamended control.

    • Figure 2. 

      Fitted random intercept and coefficient models describing the girth increase over time of individual trees in relation to amendment with a single AMF strain or biocontrol agents (BCAs) at planting in a 2 × 2 × 2 factorial design of soil amendment (AMF [yes, no], BCA [yes, no] and plant growth-promoting rhizobacteria [yes, no]). There were significant main effects of amending soils with AMF (p < 0.01) or BCA (p < 0.05), increasing the annual girth expansion rate (the thick blue and red lines represents the main effect). Thin lines are the fitted models for individual trees. The density plots show the annual girth distribution patterns of those trees with and without (a) AMF, or (b) BCA amendment. Trees of the apple cultivar Gala, grafted onto the M9 rootstock, were planted in April 2020.

    • Figure 3. 

      Average total weight (kg) and number of large fruits per tree over the four years (2021–2024) for soil amendment with each combination of single-strain AMF and plant growth-promoting rhizobacteria (PGPR); the bar indicates one standard error. Trees of the apple cultivar Gala, grafted onto M9 rootstocks, were planted in April 2020, and soils amended at planting in a 2 × 2 × 2 factorial amendment design (a single AMF strain [yes, no], biocontrol agents [yes, no] and PGPR [yes, no]). There were significant negative interactions between AMF and PGPR amendment (p < 0.05) for the total weight and number of large fruits per tree.

    • Figure 4. 

      Scatter and density plots depicting the distribution of estimated tree height (cm) and project canopy cover area via analysis of drone-captured images in October 2023. Trees were planted in April 2020 and soils amended in a 2 × 2 × 2 factorial amendment design (a single AMF strain [yes, no], biocontrol agents [BCAs] [yes, no] and plant growth-promoting rhizobacteria [yes, no]). The main effect of AMF amendment was significant (p < 0.01) for both tree height and canopy area, whereas the main effect of BCA amendment was close to statistical significance (p < 0.1) for the tree height only.

    • Figure 5. 

      Scatter and density plots depicting distribution of (a) estimated tree height (cm), and (b) projected canopy cover area via analysis of drone-captured images in October 2023 for the untreated and Biofence soil amendment. Trees of the apple cultivars Gala, grafted onto M9 rootstocks, were planted with various soil amendment treatments in April 2020; there were 20 replicate trees per treatment. Biofence amendment significantly (p < 0.01) increased both plant height and projected canopy cover.

    • Treatment Name Components (note) Volume/amount per tree
      1 (A) East Malling (EM) AMF Diversispora sp. 25 ml (~12,500 propagules per tree)
      2 (B) Biocontrol agents Trichoderma harzianum T-22 (Trianum P, Koppert), B. subtilis (Serenade, Bayer) 100 ml water, 0.1 g Trianum, 2 ml Serenade
      3 (C) PGPR Azospirillum brassilense sp245*, Gluconacetobacter diazotrophicus Pal5 100 ml PGPR mix (~108 CFU per tree)
      4 A + B 25 ml Diversispora + 100 ml water 0.1 g Trianum, 2 ml Serenade
      5 A + C 25 ml Diversispora + 100 ml PGPR mix
      6 B + C 100 ml PGPR mix, 0.1 g Trianum, 2 ml Serenade
      7 A + B + C 25 ml Diversispora + 100 ml PGPR mix, 0.1 g Trianum, 2 ml Serenade
      8 Nothing Control treatment
      9 Biofence Brassica seed meal containing high N 300 g (2 weeks before planting)
      10 Mixed AMF Five species mix (Funeliformis geosporum, F. mosseae, Claroideoglomus claroideum, Rhizophagus Irregularis, Glomus microagregatum) 25 ml (~12,500 propagules per tree)
      * This strain was identified as Paenibacillus sp. through the 16S sequences obtained.

      Table 1. 

      Details of individual treatments applied as soil amendment at the time of apple tree planting at East Malling in April 2020. The first eight treatments form the 2 × 2 × 2 factorial design: single AMF strain (yes, no) × biocontrol agents (yes, no), and PGPR (yes, no).

    • Terms Girth (20 cm above the graft union) Height Canopy Fruit weight and number
      2021 2022 2023 2024
      2021 2022 2023 2024 Weight Number Weight Number Weight Number Weight Number
      Base model†
      + AMF& 1.94 5.26* 5.46* 6.53* 7.55** 7.38** 1.47 1.20 2.94+ 3.13+ 0.80 0.74 0.71 0.18
      + BCA 2.39 2.92+ 4.44* 1.66 3.29+ 1.07 3.14+ 2.13 0.65 1.20 0.17 0.72 0.98 1.23
      + PGPR 0.36 0.55 0.26 0.27 0.01 0.09 1.30 0.42 0.01 0.01 0.41 0.25 0.06 0.01
      + AMF : BCA 0.38 1.63 1.17 0.51 0.87 0.1 0.13 0.06 0.19 0.12 0.42 0.78 1.66 1.21
      + PGPR : BCA 8.92** 3.58+ 4.18* 1.99 0.31 0.03 0.50 0.36 0.10 < 0.01 1.84 2.49 0.72 0.69
      + AMF : PGPR 4.61* 3.56+ 3.3+ 5.64* 0.01 0.15 0.07 0.83 0.86 0.05 3.01+ 2.75+ 2.08 1.31
      + AMF : PGPR : BCA 0.81 1.11 0.39 0.33 < 0.01 0.05 0.14 0.04 0.92 0.58 0.39 0.08 0.86 0.20
      † Base model for the growth contains the fixed effects of intercept, random intercept for the two block factors (rows and columns), and girth 5 cm above the graft union measured at the planting time (April 2020) as a co-variate. & All terms have one degree of freedom. Tree girth 20 cm above the graft union and yield were assessed annually in 2021–2024. Statistical significance based on the Chi-square test in the nested linear mixed models at the level of 0.001, 0.01, 0.05, and 0.1 is indicated by ***, **, *, and +, respectively.

      Table 2. 

      Analysis of deviance of nest linear mixed models to test for statistical significance of treatment effects on annual girth and plant height/canopy area measured in October 2023 in the 2 × 2 × 2 factorial design where trees of the apple cultivar Gala, grafted onto the M9 rootstock, were planted in April 2020 and soils amended with a single strain AMF (yes, no), biocontrol agents (BCAs) (yes, no), and plant growth-promoting rhizobacteria (PGPR) (yes, no).

    • Factor level Girth (mm, 20 cm above the graft union) Height (cm) Canopy cover area Total fruit yield Large fruit (≥ 60 mm)
      2021 2022 2023 2024 Weight Number Weight Number
      Without AMF 5.39 ± 0.050 5.93 ± 0.061 6.8 ± 0.076 7.47 ± 0.093 68.6 ± 2.92 2968 ± 353.7 9.21 ± 0.352 145.4 ± 5.04 3.77 ± 0.223 35.1 ± 2.06
      With AMF 5.52 ± 0.047 6.13 ± 0.055 7.07 ± 0.073 7.83 ± 0.091 80.2 ± 3.12 4293 ± 531.8 9.66 ± 0.346 159.6 ± 5.58 3.91 ± 0.207 35.7 ± 1.80
      Without PGPR 5.45 ± 0.045 6.02 ± 0.061 6.92 ± 0.079 7.62 ± 0.100 74.3 ± 3.14 3645 ± 504.5 9.27 ± 0.331 152.1 ± 5.23 3.69 ± 0.211 34.4 ± 1.92
      With PGPR 5.45 ± 0.052 6.04 ± 0.058 6.95 ± 0.073 7.67 ± 0.088 74.5 ± 3.05 3608 ± 406.5 9.60 ± 0.366 153.1 ± 5.52 3.98 ± 0.217 36.4 ± 1.95
      Without BCA 5.40 ± 0.045 5.96 ± 0.060 6.82 ± 0.075 7.55 ± 0.097 70.6 ± 2.97 3369 ± 459.3 9.55 ± 0.364 146.7 ± 4.78 3.79 ± 0.23 35.1 ± 2.07
      With BCA 5.50 ± 0.051 6.10 ± 0.058 7.05 ± 0.075 7.74 ± 0.091 77.9 ± 3.15 3866 ± 453.4 9.33 ± 0.335 158.3 ± 5.82 3.9 ± 0.199 35.7 ± 1.80
      Trees of the apple cultivar Gala, grafted onto the M9 rootstock, were planted in April 2020 and soils amended with appropriate product(s). For each factor level, there were 80 trees.

      Table 3. 

      Individual factor level mean (± standard error) of annual tree girth, height, and canopy cover area (measured in October 2023), and total fruit weight and number over the period of 2021–2024 for three soil amendment product types in the 2 × 2 × 2 factorial design experiment: with or without single AMF strain (AMF), with or without biocontrol agents (BCAs), and with or without plant growth-promoting rhizobacteria (PGPR).

    • Model Girth Fruit production
      Large fruit (≥ 60 mm) Total
      Weight Number Weight Number
      Base model†
      + AMF& 7.37** 0.35 0.63 0.93 1.59
      + BCA 3.94* 0.11 2.18 0.8 0.93
      + PGPR 0.07 0.42 0.13 < 0.01 0.24
      + AMF : BCA 0.41 1.36 1.09 0.06 < 0.01
      + PGPR : BCA 1.49 1.36 2.16 0.59 0.22
      + AMF : PGPR 1.85 4.85* 5.1* 1.59 0.28
      + AMF : PGPR : BCA < 0.01 1.12 0.67 0.21 0.06
      † Base model for the growth contains the fixed effects of annual growth (slope) and intercept, and random intercept and slope for the two block factors (rows and columns), and individual trees. Base model for the yield contains the fixed intercept, random intercept for the two block factors (rows and columns), and individual trees, and the annual effect on the yield (i.e., years were treated as a factor instead of a continuous variable in the girth model. & For the girth, the treatment effect is on the annual expansion rate (slope); for the yield, the treatment effect was on the overall yield across years. All terms have one degree of freedom. Girth 20 cm above the graft union and yield were assessed annually in 2021–2024. Statistical significance based on the Chi-square test in the nested linear mixed models at the level of 0.001, 0.01, 0.05, and 0.1 is indicated by ***, **, *, and +, respectively.

      Table 4. 

      Analysis of deviance of nest linear mixed models to test for statistical significance of longitudinal treatment effects in the 2 × 2 × 2 factorial design where trees of the apple cultivar Gala, grafted onto the M9 rootstock, were planted in April 2020 and soils amended with a single strain AMF (yes, no), biocontrol agents (BCAs) (yes, no), and plant growth-promoting rhizobacteria (PGPR) (yes, no).