FIGURE 1A; Composite PCR-DGGE Community Profiles for the Three Plant Types

FIGURE 1A; Composite PCR-DGGE community profiles for the three plant types.

Bands represent OTU (ribosomal RNA gene diversity) of rhizosphere bacterial species. Band patterns represent the rhizosphere community diversity observed in the rhizosphere of the crop plants Wheat, Pea and Sugar Beet at growth stages GS1 (seedling) through GS5 (post harvest) compared to the community observed in bulk soil sampled at each time point. N=3 as shown (lanes)

FIGURE 1B; Sub-groups within the bacterial community of the Pea rhizosphere. PCR-DGGE profiles with 16S universal eubacterial primers, alpha-proteobacterial primers and gamma-proteobacterial primers. Figure compares the profiles of seedlings (GS1) follow seed inoculation with: H = water control, WT = SBW25 wild type inocula, and GM = Phz+ 23.10 inocula. N=3 as shown (lanes)

FIGURE 1C; Fungal community diversity. Wheat rhizosphere DGGE profiling using general fungal 18S primers. Illustrates changing diversity profiles with plant growth stage (GS). N=3 as shown (lanes)

FIGURE 2.

PCR-DGGE- Molecular assessment of microbial diversity. Principle component analysis of DGGE profiles of the untreated rhizosphere of the three field grown crop species using 16S eubacterial primers (A) and 18S general fungal primers (B).

Data plotted against axis 1 and 2 (proportion of variance for each axis represented in brackets). Data points for plants species are represented in the legend by P = pea, W = wheat and S = sugar beet. Growth stages are indicated by the numbers 1(seedling), 2, 3, 4, and 5 (post harvest).

N=3, individual replicates as shown

(3.1)

(3.2) (3.3)

FIGURE 3;

CLPP (microbial community activity / metabolic profiles) of chitinase tobacco treatments. Physiological (BIOLOG) profiles demonstrate microbial community responses due to plant growth stage, applied treatment, and plant chitinase production. Principle component analysis of tobacco plant

Fig 3.1 of clustering based upon plant growth stage (GS 1 - 5), arrow showing succession path with as plant develops.

Fig 3.2 data from GS1 (seedling) showing loose clustering by treatment: white = water control, light grey = metalaxyl-M, dark grey = WT- SBW25 inocula, black = GM - 23.10 (PHZ) inocula;

Fig 3.3 GS2 comparison of microbial community profiles from all data derived from parental line and chitinase producing tobacco (white = parental line, light grey = chitinase expression).

(4.1a)(4.1b)

(4.2a) (4.2b)

(4.3a)(4.3b)

FIGURE 4;

Population densities of major groups of culturable micro organisms isolated from the rhizosphere of wheat plants from growth stages (GS) 1, 2, 3, 4, 5.

Fig 4.1 Total bacterial densities – CFU on broad range microbiological media,

Fig 4.2 Total pseudomonad bacterial densities, CFU on pseudomonas selective media

Fig 4.3 Total fungal densities, on potato dextrose agar

Populations were isolated from the rhizosphere of (a) field grown wheat (n=18) and (b) mesocosm (n=3) grown wheat.

Treatments: Dark grey bar = Water control; light grey bar = SBW25 wild type inocula; white bar = 23.10 (PHZ producing SBW25 variant).

Nb Fig 4.2 –light grey bar and white dots= CFU counts SBW25 wild type inocula; white bar grey dots = CFU counts 23.10 inocula..

(5.1)(5.2)

(5.3)(5.4)

FIGURE 5;

CLPP (microbial community activity and metabolic profiles), deliberate field release.

Response of field grown wheat rhizosphere microbial communities treated with (G) PHZ producing fluorescent pseudomonad SBW25, (W) wild type inocula or (H) water control.

Fig 5.1, data presented for each growth stage over the growing season; arrow indicates observed trend in community shift with growth stage (GS).

Fig 5.2 (GS 1), Fig 5.3 (GS 2) and Fig 5.4 (GS 3) to illustrate inocula effect within each growth stage of the wheat plant. black circles = water control; grey circles = wild type treatment and white circles = GM treatment. (n=18)

(6.1a) (6.1b)

(6.2a) (6.2b)

(6.3a) (6.3b)

(6.4a) (6.4b)

Figure 6; PCA analysis of PCR-DGGE total microbial community diversity in Wheat rhizospheres. Deliberate release experiment.

16S - Eubacteria (6.1); alpha-proteobacteria (6.2); gamma-proteobacteria (6.3); and 18S fungi (6.4).

(a) plant growth stages GS1 to GS5, arrows show the general trend; suffix (n=9)

(b) cumulative treatment effect (H = water control; W = wild type inocula; G = GM inocula) for all growth stages.

FIGURE 7. ANOSIM plots, of microbial metabolic activity (CLPP) and total microbial diversity (DGGE) under mesocosm conditions.

DGGE – 16S-total, alpha sub-group, gamma-sub-group bacterial diversity and fungal diversity (18S) for pea, wheat and sugar beet plants grown in field soil in mesocosms under controlled environmental conditions.

Each figure represents comparison of treatment (wild type inocula – SBW25 (WT) or GM – PHZ producing variant (23.10) (GM) against water control).

R values on y axis and growth stages and plant types on x axis (n=3).

7a.

7b.

7c.

7d.

7e.

FIGURE 8. ANOSIM plots, of microbial metabolic activity (CLPP) and total microbial diversity (DGGE) of field grown wheat, pea and sugar beet planted in fungicide treated soil.

Comparisons of metalaxyl treated with untreated. R values on y axis and growth stages and methodology on x axis (n=6).

FIGURE 9. ANOSIM plots, of microbial metabolic activity (CLPP) to compare the impact of fungicide (metalaxyl) and anti-fungal compound producing GM plants (chitinase) and GM bacteria (phenazine) alone or in combination.

Comparisons made between untreated, non-chitinase producing plant. R values on y axis with plant growth stages (1-5) shown for each microbial community assessment [n=18 for CLPP (activity) and n=6 for DGGE (diversity)].

It should be noted that no significant differences were recorded when wild type bacterial inocula, SBW25 (WT) performance was compared to the PHZ+ variant 23.10 (GM). This observation highlights the inocula effect rather than the effect of the additional function, the expression of phenazine. Arrows indicate the general trend.

9a Parental tobacco treated with fungicide, wt SBW25 and GM bacteria

9b Chitinase producing tobacco with fungicide, wt SBW25 and GM bacteria

FIGURE 10. Deliberate field release of genetically modified bacteria.

ANOSIM plots of microbial metabolic activity (CLPP) and total microbial diversity (DGGE) in field grown wheat rhizospheres.

Plants were treated with water (control), wild type or GM bacterial inocula at the time of seed planting. Microbial community response was monitored over the growing season. Comparisons made against untreated water control. R values on y axis with growth stages 1-5 (shown) for each assessment [n=18 for CLPP and n=6 for DGGE].

It should be noted that no significant differences were recorded when wild type bacterial inocula, SBW25 (WT), performance was compared to the PHZ+ variant 23.10 (GM). This highlights the inocula effect rather than an impact of the additional function, the expression of phenazine. Arrows indicate general trend.