O BJETIVOS PECSD

In an attempt to define a relationship in expression of PGP phenotypes isolated from different soil types, data on ACC deaminase and phytate mineralisation, and TCaP, HydroxP and FeP solubilisation, and bacterial acidification (Me-Red) from each isolate were recorded using either presence/absence data (1 and 0) or a rating system from 0 to 3 as described above (Sections 2.2.1.2 - 2.2.1.7). To visualise relationships in expression of PGP phenotypes among the samples, the data were first grouped using two-way hierarchical clustering and the organised data matrix visualised graphically as a heatmap. These analyses were conducted in the R statistics package (R Project, Version 2.12.2,

http://www.r-project.org). Each value from the raw data set listed in Appendix D of screening for PGP traits on plate assays was standardised to have a mean of zero and standard deviation of 1 using the standardisation formula: ( x – mean )/standard deviation. The clustering method used was hierarchical clustering with complete linkage. The heatmap matrix generated using this method is indicated by the colour scheme from dark red to yellow. Isolates with similar colour coding represent similar characteristics with respect to the PGP traits tested. The darker the colour (i.e. dark red) shown in the matrix, the higher the recorded value.

Statistical analysis to determine whether there was phenotypic variation in bacterial isolates based on environmental habitat (sampling location) or bacterial genera was carried out using multivariate statistics. The raw data (Appendix D) based on the high-throughput screening of 105 isolates was standardised (as before) and similarity of phenotypes among samples measured using Euclidean distance. Similarity in phenotypic attributes of the bacteria was tested using sample location as a factor. In addition, to determine if phenotypic properties were linked to bacterial genotypes, testing was also conducted using bacteria genera as a factor. In both cases, testing was conducted using analysis of multivariate similarities (ANOSIM). To further determine the relationships between environment or genera and phenotype, the data were tested using similarity percentages - species contributions (SIMPER) analysis. In this test, the variables (phenotypes) that contribute to differences between genera can be identified. Multivariate analysis was conducted in PRIMER-E (Version 6.1.150) using methods described in Clarke et al. (2006) and Clarke et al. (2008).

All numerical data from the HPLC analysis are expressed as the mean of three replicates with associated standard errors of mean (mean ± SEM). Data obtained from the glasshouse pot trial were expressed as MDW. Generalised linear modelling was used to generate pair-wise correlations between soluble P released, concentration of organic acid released by each isolate, and MDW of ryegrass from the pot trial. One-way analysis of variance (ANOVA) was used to calculate significant differences between treatments (*p < 0.05) and grouping information was achieved using the Tukey’s Method at 95% confidence. Duncans system of lettering was used where mean values that do not significantly vary (at the 5% level) between treatments share the same letter. These analyses were carried out using Minitab version 15 (Minitab, Inc., www.minitab.com).

2.3

Results

2.3.1.

Screening for plant growth-promoting traits

The raw data of the screening for PGP traits for the 105 PSB isolates are recorded in Appendix D and examples for plat screening is shown in Figure 2.3.1. The majority of isolates had some PGP traits, and this was most obvious for isolates that were able to release more than 4 mM of soluble P in the liquid culture assay. The high proportion of isolates from Haast and Eyrewell (exotic and native forest) had strong ability to solubilise DCaP, TCaP, and Hyp-G, but these isolates were not able to solubilise HydroxP using sucrose as the carbon source (Table 2.3.1). For example, isolate EE127 released 9.05 mM P from DCaP and solubilised HydroxP in glucose, but lacked the ability to utilise sucrose for HydroxP solubilisation (Hyp-S) (Table 2.3.1).

Figure 2.3.1 Sample plates of screening phosphate solubilising isolates with plant growth-promoting traits. Phosphate solubilising bacteria from Eyrewell (exotic forest soil origin) on a tricalcium phosphate plate (TCaP) (A), on Iron phosphate plate (FePA) (B) and isolates from Haast on sodium phytate plate (Na- Phy) (C). Visible halo formation indicates TCaP or iron phosphate has been solubilised by the isolate.

Isolates scored as “3” produced a large halo (diameter ≥ 8 mm) indicated by yellow box, “2” produced a

medium sized halo (5 – 7 mm) as indicated by red box, “1” a small halo (≤ 4 mm) indicated by green box,

and “0” where no halo was observed as indicated by blue box. Siderophore producing rhizobacteria detected by CAS agar plate (D) by removing Fe(III) from the Chromazurol S indicated by colour changes from blue to orange. The CAS plate indicates isolate Wi28 did not produce siderophores, whereas Ha185, Ha200 and Wh15 are siderophore producing bacteria.

Results from methyl red agar plate (Me-Red) assay showed some correlation with the amount of P released from insoluble DCaP in liquid culture assay. Isolates that produced the most acid also released higher amounts of soluble P in culture filtrate (> 6 mM). Isolates from Haast and Eyrewell that were capable of solubilising DCaP were also able to solubilise insoluble iron phosphate on FePA agar plate at low pH (pH 4). For example, isolate Ha200 released 7.56 mM soluble P from DCaP, could

solubilise TCaP, produced organic acids on Me-Red plate assay and could solubilise iron phosphate. The majority of isolates exhibited some PGP traits except for two isolates from Ballantrae pasture soil, Ba30 and Ba31 and two isolates from Winchmore pasture soil, Wi6 and Wi16, which did not exhibit any of the PGP traits tested in this study (Appendix D).

2.3.2.

Identification of bacterial genera using 16S rRNA gene sequencing

The bacterial 16S rRNA gene sequences were compared against those on the NCBI database using the BlastN search tool. The closest matches to named species on the NCBI database were inferred as being correct and were used for bacteria identification. DNA sequences were subjected to BlastN searches against the reference 16S ribosomal RNA sequence from bacteria data collection to determine bacteria genera and species (Table 2.3.1). A universal cut-off for bacterial speciation is described by Stackebrandt and Goebel (1994), where a 97% of 16S rRNA similarity level was proposed. Drancourt et al. (2000) proposed a 99% and 97% sequence similarity as the cut-off for species and genus identification, respectively. While bacterial speciation using solely 16S rRNA sequencing remains debatable (Teng et al., 2011), it remains the most practical and therefore widely used approach today. In this study, isolates were identified to genus level with the best matching 16S rRNA gene sequencing result list in Table 2.3.1 with species ID name and accession numbers from NCBI database. Isolates that are potentially new bacterial species using <97% similarity cut-off are indicated with a € sign in Table 2.3.1. In soil collected from Haast and Eyrewell, regardless of local land-use, the strongest P-solubilising bacteria (> 4 mM of P from DCaP, able to solubilise both TCaP and HydroxP in plate assays), were all in the genera Pseudomonas spp. or Burkholderia spp., with the exception of a single Serratia strain. At Winchmore, the P solubilisation taxa were Pseudomonas and

Enterobacter, and at Ballantrae Pseudomonas and Serratia were the dominant P-solubilising taxa (Table 2.3.1). Five PSB isolates belonging to the genus Arthrobacter (Ba6, Wh5, Wi30, Ha194, and Ha188, from Ballantrae, Whatawhata, Winchmore and Haast) released the lowest P concentration in the DCaP liquid culture assay (Table 2.3.1), suggesting isolates from this genus may be less effective P solubilisers. However, all isolates from this genus were able to solubilise HydroxP using both glucose and sucrose as a carbon source. This suggests Arthorobacter spp. may have multiple pathways for P solubilisation, at least in the plate assay.

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Table 2.3.1 Screening for plant growth-promoting traits and identification of phosphate solubilising bacteria.

16S rRNA gene sequencing₸ Location

& Soil type Isolate ¥ _(mM) Pcon ⱡ _ACC§ _PhyNa-₤ _TCaP₤ _RedMe-₤ _Hyp-G§ _Hyp-S§ _FePA₤ _{Species Identification#} Accession _{Number# Index} Max

Ballantrae (Pasture soil)

Ba6€ _{0.50 1 2 1 1 1 1 0 Arthrobacter globiformis strain DSM 20124 NR_026187.1 96%}

Ba40 4.51 1 2 2 2 1 1 1 Serratia grimesii strain DSM 30063 NR_025340.1 97%

Ba28 4.51 1 2 2 2 1 1 0 S. grimesii strain DSM 30063 NR_025340.1 99%

Ba21 6.24 1 2 2 3 1 1 0 Pseudomonas costantinii strain CFBP 5705 NR_025164.1 99%

Whatawhata (Pasture soil)

Wh5€ _{0.59 1 2 2 1 1 1 0 Arthrobacter methylotrophus strain TGA NR_025083.1 96%}

Wh8€ _{3.16 0 3 2 1 1 1 1 Paenibacillus amylolyticus strain NRRL NRS-290 NR_025882.1 94%}

Wh15€ _{4.86 0 3 1 1 1 0 0 Pseudomonas costantinii strain CFBP 5705 NR_025164.1 95%}

Wh22€ _{8.55 1 2 3 3 1 0 0 Pseudomonas graminis strain DSM 11363 NR_026395.1 94%}

Winchmore (Pasture soil)

Wi30 1.35 1 2 1 1 1 1 0 Arthrobacter nitroguajacolicus strain G2-1 NR_027199.1 99%

Wi11 5.95 1 1 1 2 1 0 1 Enterobacter amnigenus strain JCM1237 NR_024642.1 99%

Wi18€ _{6.36 1 2 1 3 1 1 0 Pseudomonas jessenii strain CIP 105274 NR_024918.1 95%}

Wi14 7.94 1 2 1 2 1 0 2 Pseudomonas cedrina strain CFML 96-198 NR_024912.1 98%

Wi28€ _{10.73 1 2 2 3 1 0 2 E. amnigenus strain JCM1237 NR_024642.1 94%}

Haast (Native forest)

Ha194 0.61 1 1 2 1 1 1 0 A. methylotrophus strain TGA NR_025083.1 98%

Ha188 1.02 0 1 2 1 1 1 1 A. methylotrophus strain TGA NR_025083.1 98%

Ha197 1.25 1 2 1 0 1 1 1 P. amylolyticus strain NRRL NRS-290 NR_025882.1 97%

Ha182€ _{4.16 1 2 3 0 1 0 0 Burkholderia xenovorans LB400 NR_029199.1 96%}

Ha189€ _{4.55 1 3 3 0 1 0 0 Burkholderia fungorum strain LMG 16225 NR_025058.1 95%}

Ha185€ _{5.93 1 2 3 1 1 0 0 B. xenovorans LB400 NR_029199.1 95%}

Ha183 6.05 1 2 1 1 1 0 0 B. xenovorans LB400 NR_029199.1 97%

Ha186 6.84 1 1 1 1 0 0 0 B. xenovorans LB400 NR_029199.1 97%

Ha200€ _{7.56 0 2 2 3 1 0 2 Pseudomonas frederiksbergensis strain JAJ28 NR_028906.1 96%}

Ha203 9.07 0 3 2 3 1 0 2 P. frederiksbergensis strain JAJ28 NR_028906.1 98%

41

(Exotic forest)

EE128 3.57 1 3 2 1 1 0 1 S. proteamaculans strain DSM 4543 NR_025341.1 97%

EE130 5.22 1 3 3 3 1 0 0 Pseudomonas brenneri strain CFML 97-391 NR_025103.1 98%

EE131 7.36 1 3 1 3 1 0 1 Pseudomonas umsongensis strain Ps 3-10 NR_025227.1 97%

EE132 7.66 1 3 2 3 1 0 0 P. jessenii strain CIP 105274 NR_024918.1 98%

EE133 8.06 1 3 2 3 1 0 3 P. jessenii strain CIP 105274 NR_024918.1 98%

EE127€ _{9.05 1 3 2 3 1 0 2 P. brenneri strain CFML 97-391 NR_025103.1 93%}

Eyrewell (Native forest)

EN102 4.83 1 2 2 3 1 0 1 P. brenneri strain CFML 97-391 NR_025103.1 97%

EN116€ _{5.09 0 1 2 2 1 1 0 Serratia proteamaculans strain 4364 NR_037112.1 96%}

EN101€ _{8.04 1 2 2 3 1 0 2 P. brenneri strain CFML 97-391 NR_025103.1 96%}

EN108 9.13 1 3 2 2 1 0 1 P. brenneri strain CFML 97-391 NR_025103.1 97%

Ballantrae

(internal control) PSB85 10.38 0 2 1 3 0 0 2 Pseudomonas fluorescens strain IAM 12022 NR_042199.1 99%

ⱡPhosphate released by incubating 7 days with dicalcium phosphate (CaHPO4) determined by Murphy and Riley’s colourimetric assay. § Value presented as presence/absence data, 1 and 0.

₤Rating system where scored as “3” produced a large halo (diameter ≥ 8 mm), “2” produced a medium sized halo (5 - 7 mm), “1” a small halo (≤ 4 mm) and “0” indicates no halo was observed. ¥Stains chosen for glass house pot-trial are highlighted in light green.

#Bacterial genera and species identification by 16S rRNA gene sequencing where top DNA BlastN searched accession number and ID name € Potential new species using a relaxed cut-off of <97% similarity.