Customized Plant Growth Promotion with Soil- and Cultivar-Compatible Microbial Biofertilizers

Abstract

Organic fertilizers and microbial biofertilizers are now widely recognized to effectively complement traditional mineral fertilizers for plant growth. The present study shows that bio-organic fertilizers can be enhanced by the addition of functional plant-growth-promoting rhizobacteria (PGPR) that provide additional benefits to plants. We hypothesized that not all beneficial soil bacteria are functional in different farm soils and plant varieties; hence, the most effective PGPR that are suitable to each farm’s individual cropping conditions were selected. Five different field soils and their respective crops were tested for compatibility with six microbial biofertilizers (including three new bacterial strains) to supplement a commercially available bio-organic fertilizer. In pot trials with lucerne plants, four out of the six microbial treatments led to significant (p < 0.05) growth promotion benefits (up to 79.8% more leaves and dry weight) compared to mock-treated or bio-organic fertilizer-only-treated control plants. A trial with industrial hemp demonstrated that compatibility with PGPR occurs in a cultivar-specific manner, leading to growth promotion ranging from −3.4% to 68.9%, with each cultivar displaying a preference for a different PGPR. Finally, pot trials with Rhodes grass and two different soils demonstrated high yield increases compared to control plants, with Bacillus amyloliquefaciens 33YE being most effective for one soil and Bacillus velezensis UQ9000N/Pseudomonas lini SMX2 for the other soil. Yield advantages reduced after several cuts of grass, but a repeat biofertilizer treatment at 69 days after the initial treatment restored high yield advantages, with the same PGPR again being most effective. These results demonstrate the importance of customization of microbial inoculants to identify the most compatible PGPR–cultivar–soil interaction. The customization of microbial biofertilizers to soils and plant cultivars, combined with complementary fertilizer applications, can potentially lead to more reliable and more sustainable agricultural practices.

1. Introduction

Soil rhizobacteria can provide significant benefits to plants by promoting growth and defending against pathogens. This is achieved by fixing nitrogen from the air, solubilizing phosphorous that plants cannot normally access, producing siderophores for iron supply, producing plant growth hormones (e.g., auxin), increasing abiotic stress tolerance, priming plants for systemic induced resistance, and producing a range of antimicrobial compounds that assist plants to defend against pathogens [1].

However, not all soil bacteria are compatible with the farm soil and plant variety used, and it is important to select the most effective plant-growth-promoting rhizobacteria (PGPR) or fungi that can act synergistically with the microbiomes supplied by the soil or externally applied biofertilizers that are compatible to each farmer’s individual farming conditions [2]. “Microbe-friendly soils” generally harbor a high content of organic carbon leading to high microbial diversity and functionality [3], and “microbe-friendly plants” have adapted to provide root exudates and suppress defense responses when interacting with PGPR or beneficial mycorrhizal fungi [4].

Combinations of different fertilizers, such as organic fertilizers, microbial biofertilizers, and mineral fertilizers can achieve highly efficient plant growth promotion that is based on the complementary mode-of-actions of these fertilizers [5,6,7,8]. The application of mineral fertilizers provides direct plant nutrition typically over a short period, while fertilizers that include organic matter, such as compost, are typically slow release and require microbial action for decomposition before most nutrients can be taken up by the plant, although direct uptake of organic matter has also been reported [8,9,10,11].

Organic fertilizers provide slow-release nutrients to plants when decomposed. The advantage of bio-organic (pre-digested) fertilizers is that the process of decomposition of organic matter has already begun through the activity of microbes, providing partially freed-up nutrients to the plants, while the presence of compatible PGPR can provide additional sources of nutrients (e.g., via nitrogen fixation, phosphate solubilization, siderophore production), stimulate plant growth via plant hormones, defend against pathogens, and provide resilience against abiotic stress [5,6,12,13]. Based on these complementary modes of action to plant nutrition, the present study combined predigested organic fertilizer (SoiLife^®, SoiLife Group, South Murwillumbah, NSW, Australia) with the strategic use of microbial PGPR inoculants to provide superior plant growth promotion benefits. Using this formulation, we hypothesized that a customization of the use of PGPR that takes compatibility to soil and plant genotype into consideration can be a streamlined approach to identify effective PGPR for farming conditions with specific soil–cultivar combinations [2].

Hence, in the present study, the combined use of predigested organic fertilizer with various microbial biofertilizers was tested in a customized approach to determine the most effective PGPR that are effective for both the soil and plant cultivar used. Pot trials with the farm soils and their respective plant varieties revealed significant yield increases with specific microbial biofertilizers that differed for each variety and soil type used.

2. Materials and Methods

2.1. Strain Material and Isolation and Identification of New PGPR

Bacterial strains Bacillus velezensis UQ9000N, Achromobacter spanius UQ283, Bacillus amyloliquefaciens UQ154, and Bacillus amyloliquefaciens 33YE were used in this study as they have previously shown good plant promotion or pathogen control results with several horticultural crops [14,15,16].

A new bacterial strain, SMX2, was isolated from the rhizosphere soil of wheat fields at the University of Queensland Gatton Campus, Queensland, Australia. Bacterial strains d3 and d4 were isolated from the rhizosphere of wheat (cv Suntop) plants cultivated for 2 weeks in pots with silty clay loam soil from Brisbane, Australia (27°31′37.0″ S 152°59′51.8″ E) in the presence of 50 µM 1-aminocyclopropane-1-carboxylate [15]. The identity of the bacterial strains was determined via 16S rRNA gene amplicon sequencing following a BLAST search in NCBI, and the most likely identity of isolates SMX2, d3, and d4, were Pseudomonas lini, Pseudomonas chlororaphis, and Pectobacterium sp., respectively. These potential microbial biofertilizers were chosen for this study based on preliminary data with wheat and banana plants, where they achieved significant growth promotion under abiotic stress conditions.

All bacteria were isolated and cultivated in YEP medium (10 g/L yeast extract, 10 g/L peptone, 5 g/L NaCl) prior to plant treatments which are summarized in

Table 1. Overview of microbial biofertilizer/bio-organic fertilizer treatments.

Crop/Farm	Replicates per Treatment	Pooled Plants per Replicate	Measurements	PGPR Used
Sunflower (field trial)	60	n/a	Plant height [cm]	SL, SL autoclaved, SL-9000N, SL-33YE
Lucerne (alfalfa)	3 1	25 75	Leaf number Dry weight [g]	SL, SL-d4, SL-SMX2, SL-9000N, SL-283, SL-154, SL-33YE
Industrial hemp (3 varieties)	13–30 13–30	n/a n/a	Leaf growth [cm/d] Fresh weight [g]	SL-d4, SL-SMX2, SL-9000N, SL-283, SL-154, SL-33YE
Rhodes grass (Ryan Farm)	2 1	45 45	Shoot growth [g/d] Shoot weight [g]	SL, SL-d3, SL-d4, SL-SMX2, SL-9000N, SL-154, SL-33YE
Rhodes grass (Bennett Farm)	30 6 4	3 45–75 270–450	Plant height [cm] Fresh weight [g] Shoot growth [g/d]	SL-d4, SL-SMX2, SL-9000N, SL-283, SL-154, SL-33YE

SL refers to a sole or combined use with the bio-organic fertilizer SoiLife^®.

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2.2. Sunflower Trial

A field trial was carried out on sunflower plants at the Sonnschein Farm, Wamuran, QLD, Australia, to compare different combinations of the bio-organic fertilizer SoiLife^® with microbial biofertilizers. Treatments included unmodified SoiLife^® product, autoclaved SoiLife^®, and SoiLife^® in combination with life bacterial cultures UQ9000N and 33YE (referred to as SL-9000N and SL-33YE, respectively). Sunflower plants (approx. 60 plants per treatment) were treated 3 weeks after sowing by adding 4 mL of microbial inoculant (or water) at the base of each plant followed by watering with 5 L/m² diluted SoiLife^® product (SoiLife^® Inoculum plus SoiLife^® Feeder, each 1:40 diluted, according to the manufacturer’s instructions (SoiLife, Murwillumbah, NSW, Australia)).

To prepare microbial inoculants, pure bacterial cultures were first streaked on YEP agar medium on Petri dishes before using single colonies to inoculate 5 mL YEP liquid cultures that were grown on an orbital shaker at room temperature for 48 h, followed by scaling up to 300 mL cultures that were incubated for 24 h at room temperature and diluted with deionized water to an optical density at 600 nm of 0.1. Controls included SoiLife^® or autoclaved SoiLife^® product without microbial inoculants. Plant heights were measured at 5 weeks after treatments.

2.3. Lucerne Trial

A customized approach was used to determine the most effective microbe combinations that are active in the soil of the Jindalee Farm (Kings Plain, New England Tablelands, Australia), by using a pot trial with soil and the plant variety L70-Goldstrike supplied by the Jindalee Farm. Lucerne plants were tested in pot trials using this farm’s soil with six different soil rhizosphere bacteria in combination with SoiLife^® bio-organic fertilizer. These included bacterial strains d4, SMX2, UQ9000N, UQ283, UQ154, and 33YE. Water-only treatments and SoiLife^®-only treatments were used as controls for comparison. A total of 30 biological replicates were used by using 30 small (100 mL) pots for each product combination. Each pot contained two to three plants, and 600 plants were used in total. Lucerne (L70-Goldstrike) seeds were germinated in 30-cell trays (75 seeds per treatment) using the farm soil from the Jindalee Farm. At 5 days after sowing, plants were treated with various SoiLife^®-microbe combinations and controls using approx. 17 mL of diluted biofertilizer per pot.

To generate SoiLife^®-microbe biofertilizer combinations, pure bacterial cultures were first streaked on YEP agar medium on Petri dishes before using single colonies to inoculate 5 mL YEP liquid cultures that were grown on an orbital shaker at room temperature for 48 h. Fresh cultures (5 mL each) were added to 500 mL freshly prepared diluted SoiLife^® product (SoiLife^® Inoculum plus SoiLife^® Feeder, each 1:100 diluted) to generate each SoiLife^®-microbe combination.

Plant growth was first measured at 19 days after treatments by counting the number of leaves for each plant. Plant shoots were then harvested and left to dry, and combined dry biomass was weighed for each treatment. Average values and standard errors were calculated for the number of leaves per plant. Yield increases were determined either in relation to the water-only (business-as-usual) control plants or SoiLife^®-only treated plants. Average yield increases were determined from the number of leaves and shoot dry weight measurements.

2.4. Industrial Hemp Trial

The most effective crop-variety-microbe combinations for industrial hemp were determined using the soil of the Thirty Three Fruits Farm (33 Fruits, Stokers Siding, NSW, Australia). A total of approx. 540 plants, including Mountain Mango, ACDC, and Maverick varieties, were tested in pot trials using this farm’s soil with five different soil rhizosphere bacteria in combination with the SoiLife^® product. These included strains UQ154 and 33YE, which have previously shown good plant promotion results with several vegetable crops, and the new isolates d3, d4, and SMX2. Approx. 30 biological replicates were used for each product-variety combination to gain sufficient statistical significance.

Hemp seeds (cv. Mountain Mango, ACDC, Maverick) were germinated in 30-cell trays (approx. 180 seeds per variety for each trial; ~100 seeds for ACDC) using the specific farm soil. At 10 days after sowing, plants were treated with various SoiLife^®-microbe combinations using approx. 9 mL of biofertilizer per plant. A repeat treatment was performed 10 days later (20 days after germination) with a reduced dosage (33% less rhizosphere bacteria) using only the SoiLife^® feeder product as the carrier for the microbes.

To generate SoiLife^®-microbe biofertilizer combinations, pure bacterial cultures were first streaked on YEP agar medium on Petri dishes before using single colonies to inoculate 4 mL YEP liquid precultures that were grown on an orbital shaker at room temperature for 48 h. These were then used as inoculum for 50 mL cultures that were grown for another 48 h. Fresh cultures (15 mL each) were added to 250 mL freshly prepared SoiLife^® product (SoiLife^® Inoculum plus SoiLife^® Feeder, each 1:40 diluted, as per the manufacturer’s instructions) to generate each SoiLife^®-microbe combination. For the repeat inoculation, SoiLife^® Feeder product (1:40 diluted, 250 mL per treatment) was supplemented with 10 mL of freshly grown cultures.

Plant growth was visually monitored and to account for differences in germination, increases in leaf growth (rather than leaf sizes) were measured. For this purpose, a baseline measurement of leaf diameters (first leaves after cotyledons) was performed on the day of treatments just prior to the treatments. Leaf size increases were then measured for the primary and secondary leaves (excluding the cotyledons) 11 days after treatments, and the increase in leaf size was calculated by subtracting the base values. Following growth for another 12 days, leaf fresh weights were then measured for each plant (34 days after germination).

2.5. Rhodes Grass Trials

The most effective microbe combinations were determined for soils of the Ryan Farm (Doubtful Creek, New South Wales, Australia) and the Bennett Farm (Helidon, Queensland, Australia) by using pot trials with Rhodes grass variety FineCut used by these farms. Rhodes grass plants were tested in pot trials using the specific farm soils with five different soil rhizosphere bacteria in combination with the SoiLife^® product. These included strains UQ9000N, UQ154, and 33YE, which had previously shown good plant promotion results with wheat and several vegetable crops, and the new isolates d3, d4, and SMX2. Water-only treatments and SoiLife^®-only treatments were used as controls for comparison.

For the first trial using soil from the Ryan Farm, 15 biological replicates were used by using 15 small (100 mL) pots for each product combination. Each pot contained three plants on average, and 359 plants were used in total. Rhodes grass seeds were germinated on the soil surface in 30-pot trays (0.1 g seeds for 15 pots) using the specific farm soil and then watered in with the various SoiLife^®-microbe combinations and controls using approx. 3.5 mL of biofertilizer per pot. A repeat inoculation was carried out 2 weeks later. To generate SoiLife^®-microbe biofertilizer combinations, pure bacterial cultures were first streaked on YEP agar medium on Petri dishes before using single colonies to inoculate 5 mL YEP liquid cultures that were grown on an orbital shaker at room temperature for 48 h. Fresh cultures (5 mL each) were added to 50 mL freshly prepared diluted SoiLife^® product (SoiLife^® Inoculum plus SoiLife^® Feeder, each 1:40 diluted) to generate each SoiLife^®-microbe combination. Plant growth was first measured at 20 days after germination by cutting the grass followed by fresh weight measurements. The regrowth of grass was measured 9 days later to determine biomass fresh weight gain for each treatment.

For the second trial, soil and FineCut Rhodes grass seeds were used from the Bennett Farm. Thirty biological replicates were used by using 30 small (100 mL) pots for each product combination to gain sufficient statistical significance. Strain d3 and SoiLife^®-only treatments were not included in this trial. Each pot contained 13 plants on average, and 2357 plants were used in total. Rhodes grass seeds were germinated in 30-cell trays (~13 seeds per cell) using the specific farm soil. Seeds (75 mg/pot) were added to the soil surface and were then gently pushed manually into the wet soil. At 10 days after germination, plants were treated with various SoiLife^®-microbe combinations using approx. 9 mL of biofertilizer per pot. A repeat treatment was performed 12 days later (24 days after germination) with a reduced dosage (33% less rhizosphere bacteria) using the 1:40 diluted SoiLife^® feeder product as the carrier. Another repeat inoculation was carried out 79 days after germination.

To generate SoiLife^®-microbe biofertilizer combinations, pure bacterial cultures were first streaked on YEP agar medium on Petri dishes before using single colonies to inoculate 4 mL YEP liquid precultures that were grown on an orbital shaker at room temperature for 48 h. These were then used as inoculum for 50 mL cultures that were grown for another 48 h. Fresh cultures (15 mL each) were added to 250 mL freshly prepared diluted SoiLife^® product (SoiLife^® Inoculum plus SoiLife^® Feeder, each 1:40 diluted) to generate each SoiLife^®-microbe combination.

To account for uneven germination across different pots, a baseline measurement was performed on 10-day-old seedlings just before biofertilizer applications. For each pot, the number of plants, the number of plants with two leaves, and the number of plants that reached the pot edge were determined. Plant growth measurements were first taken 14 days after the initial treatments (24-day-old plants) by measuring the lengths of the three tallest plants for each pot (height of plants over soil), and values were normalized to the number of plants that reached the pot edge at the time of treatments. Nine days later (23 days after initial treatments; 34 days after germination), the grass was cut at the pot edge and fresh biomass was weighed. This was followed by regrowth measurements of plants by repeat harvesting by cutting on days 44, 52, 79, and 90 days after germination using the same technique.

2.6. Statistical Analyses

Average values and SEs were calculated for each dataset for each variety and treatment. Student’s t-test was used to determine significant (p < 0.05) differences of treatments to the control treatments without PGPR supplementation.

3. Results

3.1. Microbial Inoculants Show Different Effectiveness to Promote Growth of Sunflower and Lucerne Plants

A small-scale sunflower field trial was set up to compare the use of bio-organic and microbial bio-fertilizers in a sunflower field where mineral fertilizer was applied in the past. To determine the role of microbes in bio-organic fertilizer for plant growth promotion, SoiLife^® bio-organic fertilizer treatments were compared with autoclaved SoiLife^® applications. When comparing plant growth from bio-organic fertilizer to autoclaved fertilizer treatments, there was an insignificant (p > 0.05) reduction of plant growth by 4.3% (Figure 1). When microbial PGPR inoculants UQ9000N or 33YE were also added to plants, growth was significantly (p < 0.05) increased for SL-9000N treatments by 24% compared to SoiLife^®-only treatments, but no significant (p > 0.05) difference was found for SL-33YE treatments. These results showed that it is important to choose microbial inoculants that are functional for plant growth promotion and compatible with the soil and plants grown in the field.

To test the specificity of microbial inoculants in more detail, a pot trial was carried out with lucerne plants using field soil from a lucerne farm, which allowed rapid screening for plant growth promotion using different SoiLife^®-microbe combinations. At 19 days after microbial treatments, a trend for an increased number of leaves per plant was measured for SoiLife^® fertilizer treatment compared to the mock-treated (water-only) control plants (6.2% increase; p > 0.05; Figure 2). Increases in leaf numbers with microbial treatments compared to the water-only control plants were significant (p < 0.05) for SoiLife^® supplemented with UQ9000N, and 33YE, (31.9% and 51.7% higher, respectively). Total dry weights of combined lucerne shoots for each treatment showed a similar pattern (Figure 2). The water-only control plants also showed visibly less vigor compared to plants treated with microbial products, displaying lower plant heights and thinner stems resulting in plants often not standing upright.

To determine which biofertilizer is most effective and consistent in promoting plant growth of L70-Goldstrike in this pot trial, yield increases were determined for the average number of leaves per plant and shoot dry weights. When both measurements were taken together, the average yield showed a trend to increase by 19.6% for SoiLife^®-only treatments (p > 0.05) and significant (p < 0.05) increases were observed of 31.7%, 43.9%, and 65.8% for SoiLife^® supplemented with SMX2, 33YE, and UQ9000N, respectively. When comparing SoiLife^®-only treatment to supplementation of this organic fertilizer with customized microbes, the additional growth promotion was significantly (p < 0.05) higher by 39% for UQ9000N.

3.2. Preference for Microbial Inoculants Is Cultivar-Specific for Industrial Hemp

In this trial, three different plant cultivars were compared to identify the most effective organic fertilizer-PGPR combinations for each cultivar that are also compatible with the specific farm soil used.

The strongest leaf growth for cultivar Mountain Mango was for plants treated with SoiLife^® supplemented with d4. These plants displayed, on average, 16.7 mm wider leaves, leading to a significant (p < 0.05) growth increase of 16.2% compared to mock-treated control plants (Figure 3). This result was reflected when fresh weights of harvested leaves were measured, where leaves of plants treated with SoiLife^®-d4 also showed a trend (p > 0.05) to weigh 28.9% more than those of control plants.

For the ACDC variety, there were fewer seeds, and the germination rate was poor. Nevertheless, there were sufficient numbers (78 plants) to perform trials (Figure 3). Significant (p < 0.05) improvements in leaf growth were observed for SoiLife^®-33YE and SoiLife^®-UQ154 treatments with 28.5% and 26.3% yield increases, respectively (Figure 3). These plants displayed, on average, 26.6 mm and 24.9 mm wider leaves, respectively, compared to mock-treated control plants. SoiLife^®-UQ154 treatments also led to the highest weight gain for harvested leaves that significantly (p < 0.05) yielded 68.9% higher fresh weight than water-only control plants.

For Maverick plants, the strongest leaf growth was observed for plants treated with SoiLife^® supplemented with SMX2, d4, or UQ9000. These plants displayed, on average, 28 mm, 21 mm, and 11 mm wider leaves (22.2%, 17.0%, and 8.9%, respectively, p < 0.05) compared to mock-treated control plants (Figure 3). These results were reflected when fresh weights of harvested leaves were measured (Figure 3). Leaves of Maverick plants treated with SoiLife^®-SMX2 weighed, on average, 23.0% more (p < 0.05) than the control plants. This was followed by d4 treatments that resulted in 17.2% more leaf biomass (p < 0.05; Figure 3).

Figure 4 provides an overview of the growth promotion achieved when measuring areal growth and fresh weight increases of leaves for the three different cultivars and six treatments used in comparison to water-only control treatments. Notably, the highest growth promotion could be observed with SoiLife^®-UQ154 treatment for cultivar ACDC, which resulted in 26.3% and 68.9% increases for areal and gravimetric leaf growth, respectively. Figure 4 also shows that SoiLife^®-d4 treatments resulted in trends for yield increases for all cultivars used, while UQ154, UQ9000N, and 33YE supplementation was most effective in cultivar ACDC, but not Mountain Mango or Maverick, suggesting that compatibility with the plant genotype may be important for these microbial biofertilizers.

3.3. Customization of Microbial Biofertilizers for Rhodes Grass

The customization of microbial biofertilizers was tested for plant growth promotion of Rhodes grass using soils from two different farms. Using plant seeds and soil from the Ryan Farm, total fresh weights of cut grass were measured at 20 days after PGPR-supplemented bio-organic fertilizer treatments. Regrowth was then measured at 29 days after treatments, and growth rates were calculated to investigate if yield increases could still be observed for a second harvest following regrowth of the Rhodes grass plants. Daily growth rates per plant were significantly (p < 0.05) higher for SoiLife^®-SMX2 treatment compared to controls (Figure 5). Average fresh weight increases ranged from 10.9% for SoiLife^® product-only treatments to 101% for SoiLife^®-9000N treatments when compared to water-only control plants. When comparing growth promotion of the various combinations of SoiLife^® products enhanced with customized microbes to SoiLife^®-only treatments over both harvesting events, average increases ranged from 13.1% for SoiLife^®-d4 to 86.9% for SoiLife^®-9000N.

Using plant seeds and soil from the Bennett Farm, Rhodes grass plant height values were significantly (p < 0.05) higher for treatments with SoiLife^® supplemented with 33YE, SMX2, or UQ9000N when compared to mock-treated control plants at 14 days after microbial treatments (Figure 6 and Figure S1). To account for differences in germination, the baseline measurements (number of plants that reached the pot edge at the time of treatment) were used for normalization. Plant growth increases (determined by the height of the three tallest plants per pot; 90 plants per treatment) with microbial treatments for 33YE, SMX2, and UQ9000N, compared to the control plants were 75.2%, 38.5%, and 31.8%, respectively. A similar result was obtained when just comparing the top 50 plants per treatment. Yield increases compared to control plants without accounting for differences in germination were similar for all microbial treatments, ranging from 31.8% for SL-9000N to 36.9% for SL-d4.

Fresh weights of cut grass on a per-plant basis were then measured at 23 days after microbial treatments. As shown in Figure 7 (top left panel), all microbial treatments led to significantly (p < 0.05) higher fresh weights compared to the control plants, with SL-d4 being the most effective. The control plants also displayed visibly less growth with thinner and sometimes curly leaves that had less vigor than the plants that received the treatments.

Regrowth was then measured at 34, 44 and 52 days after treatments to investigate how long the effectiveness of the treatments can last (Figure 7). All microbial treatments (except SL-154 at 44 days) still showed significant (p < 0.05) growth promotion effects compared to the control plants, even at 52 days after the initial treatment. However, the yield advantage compared to the control gradually decreased over time, suggesting that repeat treatments may benefit plants over time (see section below).

To determine which biofertilizer is most effective and consistent to promote plant growth of Rhodes grass, average daily growth rates were determined over a longer time period (Figure 8).

When comparing the average daily regrowth of cut grass measured over a time period of 52 days on a per-plant basis, SoiLife^®-d4 treatment stood out as it led to the highest yield increases (129%) compared to the control plants (Figure 8). This was followed by SoiLife^®-9000N and SoiLife^®-33YE treatments with 63.5% and 59.27% increases, respectively. As the plants, when densely grown, compete over nutrients, growth rates were also compared on an areal basis (per pot), which could be more relevant to a field situation. When comparing the average daily regrowth of cut grass measured over the same time period on an areal basis (per pot), similar results were obtained for all microbial treatments, with SoiLife^®-33YE leading to the highest yield increases (63.6%) compared to the control plants. This was followed by SoiLife^®-SMX2 and SoiLife^®-9000N treatments with 59.1% and 55.7% increases, respectively.

Yield increases in biofertilizer-treated plants compared to control plants were observed at all time points tested (Figure 7). However, repeated harvesting by cutting the grass gradually reduced the yield advantage over time (Figure 7). Hence, a repeat biofertilizer treatment was applied 69 days after the initial treatment (79-day-old plants). This restored the higher yields for four out of the five PGPR treatments when measured 11 days later, with SoiLife^®-d4 and SoiLife^®-33YE again being the most effective biofertilizers on a per-plant and areal basis, respectively. The dynamics of yield advantages over time are shown in Figure 9.

4. Discussion

Mineral fertilizers, organic fertilizers, and microbial biofertilizers (PGPR and mycorrhizal inoculants) are now widely used for crop production [5,6,9]. Their mode of action to supply nutrients to the plants is complementary as their nutrients are derived from different sources [17,18], so their combined use can potentially lead to higher crop yields and a reduced environmental footprint. Hence, there is a current effort to develop customized fertilizer mixes that achieve an effective supply of nutrients to the plant, resulting in enhanced growth performance and yields [5,6,11,12]. The benefits of a combined use of mineral and organic fertilizers have been widely demonstrated to provide increased yields and less reliance on mineral fertilizers [17,18]. However, the supplementation with specific microbial biofertilizers requires further analyses to reveal the interplay between these different types of fertilizers and the role of plant–microbe interactions in providing effective plant nutrition. The optimization of these processes has enormous potential for more sustainable agricultural practices that result in less harm to the environment and higher yields.

Compared to mineral fertilizer applications, the use of organic fertilizers mainly relies on microbial action to degrade biomass before nutrients become available to plants [7]. Microbial biofertilizers (e.g., PGPR) can access nutrients from other sources, such as atmospheric nitrogen and insoluble phosphate, provide plant growth hormones, and assist plants in coping with biotic and abiotic stresses [4,11]. However, several studies report on the lack of consistency of microbial biofertilizers to provide these services to plants, which is likely related to compatibility issues with the soil and/or the plant host [4,19,20,21]. Hence, there is uncertainty for farmers when adopting the application of microbial biofertilizers to their farming practices.

We have previously proposed that “customized soil microbiome engineering” can potentially lead to higher yields and more resilient crop production [2]. This includes the steps of (1) preparing fertile “microbe-friendly soils”, (2) identifying “microbe-friendly plants”, and (3) choosing “plant-friendly microbes”. The current study focused on the third aspect and shows that the customization of beneficial soil rhizosphere microbes to the specific soil and plant cultivar used at a farm can significantly assist plants in growth promotion. The results suggest that identifying those PGPRs that are functional in the soil and compatible with the cultivar used, can potentially make the use of microbial biofertilizers more effective and reliable. By using a range of PGPRs from different taxa, we were able to identify those microbes that were most effective in promoting plant growth for a range of soils and plant genotypes. While results from pot trials do not always translate into results achieved in the field, the method used here can provide rapid screening of microbial biofertilizers before field trials are performed for validation.

As expected, the use of bio-organic fertilizer alone resulted in plant growth promotion trends (Figure 2 and Figure 5), most likely because it contains predigested/fermented organic matter whose freed-up nutrients are bioavailable to plants. Although the bio-organic fertilizer contains diverse microbiota, this form of plant nutrition could be significantly enhanced when it was supplemented with PGPR that can provide additional nutrients from other sources, produce plant growth hormones, and potentially provide biotic and abiotic stress mitigation. Several of the PGPRs used in the current study are able to fix nitrogen, solubilize phosphorous, produce siderophores, produce the plant growth hormone indole acetic acid, and control soil pathogens [14,15,16]. 33YE, UQ283, UQ9000N, and UQ154 have also previously been shown to promote plant growth in the absence of bio-organic fertilizer as a carrier, while plant growth promotion from the newly isolated strains Pseudomonas chlororaphis d3, Pectobacterium sp. d4, and Pseudomonas lini SMX2 had not previously been reported.

The trial on lucerne plants (L70-Goldstrike) has determined the most effective soil-microbe combinations that are both compatible with the farm soil and the variety used. Four out of the six microbial treatments tested (d4, SMX2, UQ9000N, and 33YE) led to significant (p < 0.05) growth promotion benefits compared to the control plants. It appears that the soil at Jindalee Farm is highly suitable for microbial treatments and that L70-Goldstrike is compatible to interact with a number of free-living PGPR outside nodules. Compared to water-only control treatments, yield increases were highest for the combined SoiLife^®-PGPR treatment with UQ9000N (51.7% higher number of leaves and 79.8% more shoot dry weight). Yields from this treatment were also 42.9% and 35.2% higher than SoiLife^®-only treatments for the number of leaves and dry weight, respectively. These pot trial results demonstrate that supplementation of SoiLife^® bio-organic fertilizer with PGPR is a feasible approach to achieve higher yields. However, not all PGPR were equally effective or compatible with the soil/cultivar combination used, and hence, the use of SoiLife^® -9000N has been recommended for use at the Jindalee Farm.

The trial on industrial hemp varieties has determined the most effective soil-microbe combinations that are both compatible to the farm soil and the varieties used. The three varieties tested, Mountain Mango, ACDC, and Maverick, displayed very different preferences for plant-growth-promoting rhizosphere bacteria-SoiLife^® combinations (Figure 4). SoiLife^®-d4 was the most effective application for Mountain Mango leaf growth and fresh weight yields, while SoiLife^®-154 was most effective for ACDC, and SoiLife^®-SMX2 worked best for Maverick in the farm soil tested. This information is valuable for farmers, as it enables the streamlined selection of the most effective PGPR in a pot trial simulating farming conditions with the farm soil, the varieties, and other fertilizers (e.g., SoiLife^®) applied. Indeed, the application of SoiLife^®-154 at the 33 Fruits Farm for commercial hemp production has already shown considerable differences, including larger leaves, more leaves, thicker stems, and increased root development based on observations [22].

The trials on Rhodes grass have determined the most effective soil-microbe combinations that are both compatible with the variety and the two different farm soils used. Notably, in both farm soils, SoiLife^®-SMX2 and SoiLife^®-9000N led to growth promotion benefits compared to the control plants (Figure 5, Figure 7, Figure 8 and Figure 9), suggesting that both farm soils are highly suitable for these microbial treatments. SoiLife^® supplemented with UQ9000N provided the highest plant growth promotion for soil from the Ryan Farm (Figure 5), while d4-enhanced SoiLife^® product was the preferred biofertilizer for plants grown in soil from the Bennett Farm (Figure 6, Figure 7, Figure 8 and Figure 9). This suggests that these soils have different capacities to host different microbial biofertilizers. As our current knowledge of the highly complex microbial soil ecology is insufficient to predict the stability of certain microbial strains, the performed pot trial can potentially use this rapid screening as a decision-making tool.

For the pot trial with Ryan Farm soil (Figure 5), the strain Pseudomonas chlororaphis d3 is of particular interest as it has previously been isolated from wheat rhizosphere soil using the ethylene precursor 1-aminocyclopropane-1-carboxylate as a carbon substrate, suggesting that this strain may also assist plants under abiotic stress conditions [15].

Rhodes grass plant heights and fresh weights of cut grass were consistently higher for treated plants across different measurements and time points using the Bennett Farm soil. Repeated harvesting by cutting the grass allowed for a rapid assessment of biomass yields over time and the calculation of growth rates. Growth promotion dynamics varied for different PGPR inoculants but generally followed the same pattern of high initial growth promotion benefits (Figure 9). The yield advantage of treated plants compared to the control plants then diminished gradually over time which suggested that repeat treatments may benefit plants over time. Indeed, a repeat biofertilizer treatment at 69 days after the initial treatment restored higher yields for four out of the five treatments, with the same microbes again being most effective (Figure 9). Interestingly, repeat treatment with SoiLife^®-154 did not result in restored higher yields but still displayed 37% and 22% yield advantages at the end of the trial compared to water-only control plants. This further emphasizes the usefulness of these pot trials as decision-making tools for actual farm applications. However, the way yield data are collected and analyzed is important as it is influenced by plant architecture and the development of each plant species [23,24]. A comparison of yield data per pot (area) rather than per plant could be more relevant to a field situation; for example, for grasses where planting density can influence the formation of new shoots. The comparison of both analyses for Rhodes grass showed that SL-d4 treatment was superior when analyzed on a per-plant basis but not on a per-pot basis (Figure 8 and Figure 9). This could be because this treatment had fewer plants, and the lower planting density may have led to more new shoot developments, emphasizing the importance of using consistent planting density for trials.

The results from this study suggest that customization of microbial inoculants to farm soil and plant genotype is feasible and useful to identify suitable plant-growth-promoting biofertilizer applications. Further improvements can be made to microbial soil inoculants by improving the shelf-life, competitiveness, and compatibility to soil and plant host by developing suitable formulations [25,26]. A promising area is the development of formulations that include combinations of slow-release mineral fertilizers, organic fertilizers/biochar, and microbial biofertilizers that can be optimized for different cropping systems [27,28]. Apart from enhanced crop yields, the improved nitrogen-use efficiency would result in significant environmental benefits through reduced nitrogen pollution and reduced greenhouse gas emissions by lowering the reliance on energy-intensive mineral fertilizers and lower N₂O emissions [17,18].

“Microbe-friendly” soils that benefit plant growth and suppress soil-borne plant pathogens are typically characterized by high organic carbon content and microbial biodiversity [29,30,31], but the compatibility of different soils to host specific PGPR is currently not well understood. A recent study has attempted to predict the compatibility of different soils to promote plant growth with arbuscular mycorrhizal fungi (AMFs) by measuring different soil parameters [32]. Interestingly, in some soils where AMPs were ineffective in promoting yields, plants may become more susceptible to soil-borne diseases as AMF inoculation potentially displaced other naturally-occurring endophytic fungi with biocontrol functions. The data from the present study has confirmed the notion that PGPR inoculants have different effectiveness in promoting plant growth in different soils that differ from farm to farm. As it will be difficult to provide customized microbial biofertilizer recommendations to each farm, it would be useful in the future to identify and categorize features of soil types and plant genotypes that make these more amenable to beneficial interactions with PGPR. This is a big task, but the knowledge gained could potentially lead us toward the development of novel biostimulants and farm practices that promote the compatibility of PGPR to soil and plant genotypes.

5. Conclusions

The use of bespoke soil applications using fertilizers with complementary modes of action (mineral, organic, microbial) to provide nutrients to plants is a promising emerging area, and our study has demonstrated that the combined use of bio-organic fertilizer with microbial biofertilizers (PGPR) can lead to higher yields and potentially more resilient and sustainable cropping systems. Our study also supports the notion that customization of microbial inoculants to the specific farm soil and plant genotype is necessary and feasible to identify those microbial biofertilizers that provide the highest growth benefits to plants. The approach used in this study has the potential to improve the reliability of microbial biofertilizers for yield increases and contributes towards the emerging area of customized microbiome engineering. Further research will be required to manifest the feasibility of microbial biofertilizer customization in field trials and to identify the common factors underlying host and soil compatibility of PGPR in different cropping systems. This may enable the designation of microbe-friendly soils and plant genotypes to specific microbial biofertilizers.