Effect of a cellulose decomposing bacterium, humic acid, and wheat straw on Cucurbita pepo L. growth and soil properties

Abstract

Due to the increasing need for food security across the world, it has become urgent to develop a combination of different agricultural inputs that can enhance higher crop productivity under plant growth limiting environment. Thus, the effects of a cellulose decomposing bacterium (Geobacillus stearothermophilus SSK-2018), humic acid, and wheat straw were evaluated on the growth of Cucurbita pepo L. (squash) under an arid land condition during a two-year (2020 and 2021) field trials. Laboratory analysis revealed that G. stearothermophilus SSK-2018 was a gram-positive bacterium capable of promoting plant growth by producing indole-3-acetic acid as well as enzymatic activities including cellulase, amylase, protease, gelatinase, and phosphatase. Consequently, the inoculation of SSK-2018 significantly increased (P ≤ 0.05) squash’s fresh stem, leaf, and root weights by 14.70%, 18.63% and 11.13%, respectively, compared to the uninoculated control. Humic acid significantly (P ≤ 0.05) increased fresh stem, leaf, and root weights by 78.78%, 62.5% and 63.74%, respectively, compared to the control. Wheat straw also significantly increased (P ≤ 0.05) fresh stem, leaf, and root weights by 52.68%, 30.32% and 42.30%, respectively, compared to the control. Furthermore, the combinations of the treatments, including humic acid and straw, SSK-2018 and straw, and humic acid, SSK-2018 and straw, significantly increased the growth of squash. Soil properties including bulk density, moisture content, and basic cations were also significantly (P ≤ 0.05) improved by humic acid, SSK-2018 and wheat straw. This study suggests that like NPK, bio-organic treatments including SSK-2018, humic acid and straw, can equally improve plant’s fresh stem, leaf, and root weights as well as soil’s bulk density, moisture content, and basic cations (K⁺, Ca²⁺, Na⁺, and Mg²⁺). Therefore, the present study recommends the continuous use of bio-organic amendments to achieve a sustainable improvement in plant growth and soil properties under arid land conditions. However, the continuous use of soil bio-organic amendments should be regularly monitored for potential increase in greenhouse gas emissions that may be at par with that of N fertilizers.

Keywords:

• food security
• Geobacillus stearothermophilus
• squash growth
• organic materials
• cellulose decomposition
• arid land

1. Introduction

By 2050, projections show that the world will be a habitat for 9.8 billion people (United Nations Department of Economics and Social Affairs, UNDESA, 2017). To meet the increasing global food demand, developing a consortium of agricultural inputs that can enhance higher crop productivity, especially in plant growth-limiting environments like arid lands, is crucial (Bello et al., 2021). Attainment of optimal crop yield in arid lands is limited due to the predominantly sandy nature of the soils, harsh climatic conditions (hot, dry, and windy), salinity, and drought. Importantly, the soils are characteristically poor owing to their low cation exchange capacity, organic matter content, nutrients, water-holding capacity, and high evapotranspiration (Al-Omran et al., 2005). Particularly, in most regions in Saudi Arabia, there is a high scarcity of water for agricultural purposes due to very low rainfall amount which rarely exceeds 100 mm annually (Alkolibi, 2002). It has recently been reported that sustainable crop production in Saudi Arabia is majorly hampered by the harsh climate and water scarcity (Ma et al., 2022). Therefore, for an enhanced crop production in Saudi Arabia, it is imperative to develop effective farming practices that can promote organic matter accumulation with improved water conservation and soil structure over time. Effective farming practices involving the use of mineral fertilizers, organic amendments, and microbes can sustainably improve soil health and food production and reduce the negative impacts of agriculture on the environment (Cozzolino et al., 2021; George et al., 2016).

The utilization of organic materials like straw, humic acid, and manure can improve soil physicochemical properties, organic carbon, and microbial activities, and prevent eutrophication and groundwater pollution with inorganic fertilizers (Antonious et al., 2022). On addition to arid land soils and other soil types, humic substances (for example, humins, humic, and fulvic acids) act as soil conditioners that can control several processes including microbial activity, plant nutrition and growth, plant root initiation and emergence, soil aggregate stability, and nitrogen and carbon cycling in the agroecosystem (Dobbss et al., 2010; Piccolo et al., 2018; Sutradhar & Fatehi, 2023). The main sources of commercial humic acid are lignite, leonardite, peat and sometimes, sediments from rivers (Nieweś et al., 2022) and extraction from composted plant residues (Monda et al., 2018; Sutradhar & Fatehi, 2023). Furthermore, the inoculation of beneficial microbes alongside the application of soil organic amendments has a high potential in improving the plant-soil microbiome (Arif et al., 2020). Nutrient uptake and yield are promoted due to the integrated use of humic substances and beneficial soil microbes (Thomason et al., 2020).

Soil cellulolytic bacteria are groups of beneficial microbes that are capable of decomposing organic (cellulose-rich) materials such as wheat straw into nutrients that can be taken up by plants (Bello et al., 2022b and b). Enzymatic hydrolysis of cellulose by microorganisms is aided by the secretion of cellulase (Bhardwaj et al., 2021). Enzymes such as cellulase, amylase, phosphatase, dehydrogenase, invertase and urease are secreted by soil beneficial microbes to aid the mineralization of organic matter leading to the transformation of nutrients in organic form to inorganic form (Angelovicova et al., 2014; Veres et al., 2015). The presence of these microorganisms is a key indicator of improved soil health as they enhance higher nutrient uptake while integrating unused nutrients into the soil reservoir of nutrients—soil organic matter (Antonious et al., 2022). Cellulose decomposing microbes are also capable of plant growth-promotion and are good supplements to inorganic fertilizer application as they promote soil nutrient-use efficiency and nutrient cycling (Neumann & Römheld, 2012). Although these microorganisms are capable of decomposing plant residues, attention must be paid to carbon dioxide (CO₂) emissions that may be given off during the decomposition process. Greenhouse gas emissions such as CO₂, and methane (CH₄) can occur during straw decomposition under field conditions due to the transformation of cellulose into soil organic matter (Allen et al., 2020; Sun et al., 2022; Weil & Brady, 2017).

Cucurbita pepo L. also commonly known as “squash” is a vegetable crop that is widely grown across the globe (Saudi Arabia inclusive) due to its versatility and adaptations to varying climatic conditions in relation with the different varieties (Abd El-Mageed et al., 2016; Bello et al., 2022b; Gichana et al., 2019). However, squash has a high need for adequate nutrients which is mostly met by the application of unsustainable, non-renewable, and non-enviro-friendly chemical fertilizers. The cultivation of squash is even more challenging under arid land conditions due to the poor nature of the soils and environmental conditions. Therefore, to seek for sustainable substitutes or supplements in the cultivation of squash in Saudi Arabia, this study investigated the effects of organic amendments (humic acid and wheat straw) and a soil beneficial bacterium (Geobacillus stearothermophilus SSK-2018) on squash growth and soil properties. G. stearothermophilus SSK-2018 have been previously described as a cellulose decomposing bacterium (CDB) and further probed in this study for its cellulose decomposing traits in the laboratory (Bello et al., 2022a). Under the arid conditions of the current study, humic acid can act as a biostimulant that can have immediate effect on plant growth and soil conditioning for improved crop yield. Likewise, unlike other types of beneficial soil bacteria, CDB only needs little nitrogen to biodegrade organic materials. In addition, the use of wheat straw in the current study can help preserve soil moisture and allow for the proliferation of beneficial soil microbes. Therefore, the overall aim of this study is to contribute knowledge and practices towards the enhancement of agroecosystem functioning and increased food security in an arid environment by employing microbial inoculants in conjunction with soil organic amendments.

2. Materials and methods

2.1. Field location, layout, and experimental design

In 2019/20 and 2020/21 planting seasons, two different field trials were conducted to evaluate the impacts of a cellulose decomposing bacterium (Geobacillus stearothermophilus SSK-2018), humic acid, and wheat straw on the growth of squash and soil properties of an arid land. The field trials were conducted at the Soil Science Field, King Abdulaziz University’s Research Farm, Hada Al-Sham, Saudi Arabia (Supplementary Figure S1). The field study was conducted using a split-split plot design replicated three times. The treatments’ arrangement in the field was completely randomized and had three rates of humic acid (0, 50, and 100 L ha⁻¹) as the main plots, and two rates of bacterium inoculation (with and without) as the sub plots. At the sub-sub plots, there were three rates of wheat straw (0, 20 and 30 t ha⁻¹ straw) and one rate of NPK 20:20:20 fertilizer at 600 kg ha⁻¹. NPK 20:20:20 was added as a treatment in the sub-sub plots to allow for the evaluation of the performance of the treatments (bio-organic amendments) compared to the conventional practice of mineral fertilizer application.

2.2. Land preparation, planting, irrigation, and other cultural practices

The experimental site was prepared by first ploughing the land, then followed by harrowing, levelling, and marking out of the field into 72 plots according to the experimental layout (Bello et al., 2022b; Supplementary Figure S2). Planting was done by direct seeding using seeds of hybrid variety of Lebanese squash and the plant spacing was 1 m between rows and 0.5 m between plants, and each plot was 6 m² with 12 plant stands. The study area being an arid environment with a range of 0 to 23.6 mm rainfall per month during the growing season, a drip irrigation system was deployed in providing water to the plants (Bello et al., 2022a). The field trials were monitored throughout the growing periods to ensure that sufficient water was available for plant’s uptake and weeds as well as pests were controlled appropriately.

2.3. Treatments application

The bio-organic amendments (humic acid, G. stearothermophilus, and wheat straw) were applied differently as required. Humic acid (made from leonardite) was applied by diluting the concentrated Humacareliquid, according to the given rates (50 and 100 L ha⁻¹), in water before spraying on the respective plots. The composition of humic acid in Humacare was 12% (w/w) as supplied by the manufacturer. The humic acid application at each rate was made in four equal doses at 15 days interval each after planting. The humic acid used in the study was regarded as a biostimulant due to its overall effect on squash performance from our previous studies (Bello et al., 2022b and b). Wheat straw was incorporated into the soil at a depth of 0–30 cm about one month before planting was carried out. Wheat straw incorporation was immediately followed by bacterium inoculation to enhance faster decomposition of straw and establishment of the bacterium in the soil before planting (Supplementary Figure S3). The inoculum of G. stearothermophilus was prepared in the laboratory as described and contained 2.5 × 10⁶ colony forming units at the time of inoculation. The field inoculation of G. stearothermophilus was made at a close range to the soil by spraying. Plots containing wheat straw were regularly watered and turned over to allow for easy decomposition and aeration to support microbial activities. NPK 20:20:20 fertilizer was applied in a similar way to humic acid. The chemical constituents of humic acid and wheat straw used in this study have been previously characterized in the studies of Bello et al. (2022b) and b. In 2021, the same plots and field layout as 2020 was maintained to validate the effect of treatments over time.

2.4. Soil analysis

2.4.1. Initial soil analysis and properties

Samples were collected at 0 to 30 cm soil depth for physicochemical properties analyses according to the methods described by Pansu and Gautheyrou (2006). The initial soil analyses included soil texture, electrical conductivity (EC), pH, bulk density (BD), particle density (PD), porosity, moisture content, soil organic matter (OM) as well as nitrogen, phosphorus (P₂O₅), potassium (K⁺), calcium (Ca²⁺), sodium (Na⁺) and magnesium (Mg²⁺). Some of the soil initial properties have been previously described in the study of Bello et al. (2022b). The most probable number (MPN) of bacteria in the experimental soil was determined using the spread plate method. The initial soil physicochemical properties are as shown in (Table 1).

Table 1. Physicochemical and biological properties of the experimental soil

Soil property
pH	7.02
Electrical conductivity (dS m−1)	5.06
Bulk density (g cm−3)	1.51
Particle density (g cm−3)	3
Porosity (%)	43.44
Organic matter (%)	0.605
Total nitrogen (%)	0.11
Total phosphorus (%)	0.05
Basic cations (mg kg−1)
Calcium (Ca2+)	1250
Magnesium (Mg2+)	1100
Potassium (K+)	1000
Sodium (Na+)	2050
Particle size distribution (%)
Clay	8.1
Silt	18.2
Sand	73.7
Soil texture	Sandy loam
Bacteria population (CFU g−1 of soil)	6.4 x 10^8

CFU—colony forming units

2.4.2. Final soil analysis

Representative soil samples per treatment were taken and analyzed for pH, EC, soil moisture content, porosity, and basic cations (Ca, Mg, K and Na) according to the methods described by Pansu and Gautheyrou (2006).

2.5. Laboratory characterization of cellulose decomposing bacterium (Geobacillus stearothermophilus SSK-2018)

2.5.1. Morphological characteristics

To study the morphological characters of G. stearothermophilus, a dilute cell suspension was prepared by suspending loopful of 3–5 days old culture in sterilized distilled water and streaked on nutrient agar (NA) plates. These plates were incubated at 24°C for 3–5 days and colony characters of bacteria viz., colour and shape were recorded.

2.5.2. Physiological and biochemical tests

The physiological and biochemical tests were performed with G. stearothermophilus. Physiological tests (motility, gram stain, and spore formation) were determined according to Bergey’s Manual of Determinative Bacteriology (Hensyl, 1994). Biochemical characterization like catalase test (He et al., 1983), cellulase, amylase, protease activities (Rodarte et al., 2011; Sands, 1990), gelatin liquefaction (Stapp (1961), optimum pH test (Sands, 1990), casein hydrolysis (Cruickshank et al., 1975), esculin hydrolysis and urease test (Schaad et al., 2001), starch hydrolysis (Barrit, 1936) and phosphate solubilization, and indole acetic acid (IAA) production (Abo-Elyousr et al., 2019) were performed.

2.6. Statistical analysis

Collected data on fresh weights of leaf, stem, and root as well as some soil physicochemical properties were statistically analyzed using SAS 9.3 Software (SAS, 2011). Treatment means that were statistically different from each other were separated using LSD at the 95% significant level. The two-way interaction graphs of treatments were plotted with the aid of GraphPad Prism 9.3.1 software. The three-way interaction graphs were plotted using the R program (ggplot) 4.1.2 (R Core Team, 2021).

3. Results and discussion

3.1. Effect of treatments on squash growth

The development and growth of squash plants under field conditions requires adequate management and use of effective agricultural inputs (Supplementary Figure S4). The application of humic acid significantly (P ≤ 0.05) increased fresh stem, leaf, and root weights (Table 2). 100 L ha⁻¹ humic acid significantly increased fresh stem weight compared to the control and 50 L ha⁻¹ of humic acid by 78.78% and 38.74%, respectively, in 2019/20 season and by 73.68% and 28.89%, respectively, in 2020/21 season. Similarly, humic acid at the rate of 100 L ha⁻¹ significantly increased fresh root weight compared to the control and 50 L ha⁻¹ of humic acid by 63.74% and 22.87%, respectively, in 2019/20 season and by 61.04% and 27.22%, respectively, in 2020/21 season. The application of humic acid has been reported to cause significant improvement in plant growth (Monda et al., 2018). Humic acid can induce changes in plant roots by enhancing the emergence of lateral roots and increasing root length and hair density (Nardi et al., 2017; Olivares et al., 2017). The inoculation of SSK-2018 in both seasons significantly (P ≤ 0.05) improved fresh stem, leaf, and root weights. In 2019/20 and 2020/21 seasons, fresh leaf weights were increased due to the inoculation of SSK-2018 by 18.63% and 21.29%, respectively compared to the control. Likewise, the inoculation of SSK-2018 significantly improved fresh root weights by 11.13% and 10.52%, respectively, compared to the control (uninoculated treatment) in 2019/20 and 2020/21 planting seasons. The inoculation of beneficial microbes can enhance plant growth by increasing nutrient availability for root uptake while causing positive significant changes in the root structure (Adesemoye & Kloepper, 2009; Hansen et al., 2020).

Table 2. Effect of treatments on the stem, leaf, and root fresh weights of squash during the 2019/20 and 2020/21 planting seasons

Treatments	Fresh stem weight(g plant^−1)		Freshleaf weight (g plant^−1)		Fresh root weight (g plant^−1)
	2019/20	2020/21	2019/20	2020/21	2019/20	2020/21
Humic acid (L ha^−1)
0	97.52c	98.10c	684.79c	774.36c	24.74c	26.64c
50	128.55b	132.19b	891.88b	983.48b	32.97b	33.72b
100	174.35a	170.38a	1112.80a	1207.29a	40.51a	42.90a
LSD0.05	21.04	12.12	200.79	103.97	2.1	2.74
Cellulose decomposing bacterium (CDB)
Without	124.36b	127.16b	820.11b	893.28b	31.01b	32.70b
With	142.59a	139.96a	972.88a	1083.47a	34.46a	36.14a
LSD0.05	11.81	4.85	139.88	46.21	2.73	1.18
Wheat straw (t ha^−1)
0	95.54d	110.03d	728.27c	882.15c	24.66d	31.11d
20	119.85c	126.14c	845.65b	987.08b	30.05c	33.14c
30	145.87b	137.63b	949.08b	1016.61ab	35.09b	35.58b
600 kg ha^−1 NPK	172.63a	160.42a	1062.96a	1067.66a	41.15a	37.85a
LSD0.05	19.5	10.15	104.57	70	3.44	1.67

Means having similar letter(s) within the same column are not significantly (P ≤ 0.05) different from each other using Fisher’s LSD.

The application of wheat straw and NPK in the sub-sub plots significantly (P ≤ 0.05) improved fresh stem, leaf, and root weights compared to 0 t ha⁻¹ straw (control treatment; Table 2). Wheat is an exhaustive crop with high uptake of nutrients from the soil during growth (Akhtar et al., 2014). A significant portion of these nutrients may be locked up in the plant residues after grain harvest and made available for other plant’s use on decomposition in the soil environment. NPK application significantly increased fresh stem weight by 80.69%, 52.68% and 25.44% compared to 0 (control), 20, and 30 t ha⁻¹ wheat straw, respectively, in 2019/20 season. However, the percentage of increment in fresh stem weight was lower in 2020/21 season compared to 2019/20 season. In 2019/20 and 2020/21 seasons, the application of NPK increased fresh leaf weight by 45.96% and 21.03%, respectively, compared to the control. Likewise, in 2019/20 and 2020/21 seasons, 30 t ha⁻¹ straw significantly enhanced fresh leaf weight compared to the control by 15.73% and 15.24%, respectively. Plant residues like wheat straw are biodegradable agricultural by-products that can be used to sustainably improve fertility of soils for increased crop production (Nigussie et al., 2021).

3.2. Two-way interaction effects of treatments on fresh weights of squash

3.2.1. Humic acid and wheat straw or NPK interaction

Humic acid application at 100 L ha⁻¹ in combination with NPK and soil incorporation of straw at 30 and 20 t ha⁻¹ significantly (P ≤ 0.05) improved fresh stem weight by 52.92%, 25%, and 17.22%, respectively, compared to the combination of 0 L ha⁻¹ humic acid and 0 t ha⁻¹ straw (control treatment; Figure 1). Likewise, humic acid (50 L ha⁻¹) in combination with NPK and wheat straw incorporation at 30 t ha⁻¹ significantly improved fresh stem weight by 46.51% and 26.49%, respectively. Humic acid can promote the degradation of straw thereby enhancing the accumulation of soil organic matter for increased plant growth (Ghanney et al., 2023). However, without the application of humic acid, it was only NPK that significantly improved fresh stem weight than the control by 33.12%. This indicates that humic acid can work synergistically with both inorganic and organic sources of nutrients to improve plant growth. In addition to being a biostimulant that can directly support plant growth, humic acid application may also aid the decomposition of wheat straw by the soil native microbial population thereby resulting in a cumulative increase in growth.

Figure 1. Effects of humic acid and wheat straw levels interaction on fresh stem weight.

3.2.2. Geobacillus stearothermophilus SSK-2018 and wheat straw or NPK interaction

The inoculation of SSK-2018 combined with NPK application increased fresh root weight significantly (P ≤ 0.05) by 17.93% compared to NPK application without SSK-2018 inoculation (Figure 2). Likewise, SSK-2018 inoculation in combination with 20 t ha⁻¹ straw increased fresh root weight significantly by 9.28% compared to 20 t ha⁻¹ straw without SSK-2018 inoculation. Furthermore, with SSK-2018 inoculation, the application of NPK improved fresh root weight compared to 30 and 20 t ha⁻¹ of straw and sole SSK-2018 inoculation by 13.11%, 18.38% and 25.02%, respectively. These findings show the importance of synergy between SSK-2018 and straw or NPK in improving fresh root weight of squash over their sole application. The interaction of SSK-2018 with straw may improve the decomposition and mineralization of organic materials and biogeochemical cycling of nutrients that can lead to increased plant growth (Chaparro et al., 2012; Lou et al., 2011). NPK interaction with SSK-2018 in the rhizosphere can decrease the soil C:N ratio, to favour the activities of the bacterium in plant growth-promotion, in addition to direct uptake of N by plant’s root.

Figure 2. Effects of cellulose decomposing bacterium and wheat straw levels interaction on fresh root weight of squash.

3.3. Effect of three-way interaction of treatments on fresh leaf weight of squash

Results obtained show that interaction of humic acid, SSK-2018 and wheat straw increased the fresh leaf weight of squash significantly at P ≤ 0.05 (Figure 3). The inoculation of SSK-2018 in the presence of the combination of 600 kg ha⁻¹ NPK with 100 L ha⁻¹ of humic acid application was the most effective treatments interaction with significant impact on leaf weight. This effectiveness was followed by that of the combination of SSK-2018 with 100 L ha⁻¹ of humic acid and 30 t ha⁻¹ of wheat straw. The growth of wheat has also been shown to be significantly improved by the integrated inoculation of Pseudomonas plecoglossicida 2,4-D with humic acid application (Feoktistova et al., 2022). Similarly, the growth of maize was highly improved when humic acid was jointly applied with Rhizobium cellulosilyticum or Burkholderia gladioli which are both plant growth-promoters (Melo et al., 2018). Humic acid may contain up to 4% nitrogen, 57% organic carbon and 1% phosphorus which is sufficient to support optimal plant growth in the presence of other organic inputs (Akhtar et al., 2014). Moreover, in the presence of CDB, most of the treatment combinations outperformed those without CDB inoculation. A recent study has established that the use of bio-organic amendments composing of biochar mixed with organic materials such as sewage sludge, vermicompost, manure and organic fertilizer had significant effects on squash growth, and soil properties (Antonious et al., 2022). Another study has also indicated that the joint application of humic acid, a soil bacterium (Pseudomonas fluorescens) and fertilizers, sustainably improved leaf biomass and other growth and yield parameters of cabbage (Verma et al., 2014).

Figure 3. Effect of three-way interaction of treatments on fresh leaf weight of squash.

Columns display values (n = 3) ±SD.

3.4. Characterization of cellulose decomposing bacterium

Increased crop yield and soil fertility from traditional agricultural practices involving the application of inorganic and organic fertilizers can be sustainably attained via the introduction of beneficial soil microbes (Cozzolino et al., 2021). Table 3 presents a summary of the characterization of the CDB used in this study. G. stearothermophilus SSK-2018 is a gram-positive bacterium (Supplementary Figure S5) with positive reactions for cellulase activity, starch hydrolysis, amylase activity, protease activity, casein hydrolysis and phosphate solubilization (Supplementary Figure S6 a-f). The components of cellulases responsible for cellulose hydrolysis include endoglucanases—breakdown the strong β-glycosidic bonds to give smaller chains of cellulose with free ends, cellobiohydrolases (exoglucanases)—breakdown cellulose into cellobiose, and β-glucosidase—breakdown cellobiose into glucose monomers (Esteghlalian et al., 2002). The test for gelatin liquefaction was also positive and the bacterium (G. stearothermophilus) also showed the potential for IAA production. Thus, G. stearothermophilus SSK-2018 was regarded as a beneficial soil microbe with the capability for plant growth-promotion.

Table 3. Morphological, physiological, and enzymatic characterization of Geobacillus stearothermophilus SSK-2018

Characteristics	Result
Gram staining	Positive
Cell shape	Rod
Colour	Creamy
Motility	Positive
Sporulation	Negative
Optimum temperature	30°C
Optimum pH	7
Gelatin liquefaction	Positive
Casein hydrolysis	Positive
Cellulase activity	Positive
Starch hydrolysis	Positive
Protease activity	Positive
Asculin hydrolysis	Positive
Urease activity	Positive
Phosphate solubilization	Positive
Indole acetic acid production	Positive

3.5. Soil analysis

3.5.1. Soil pH, bulk density, electrical conductivity, gravimetric moisture content and porosity

The soil analysis results presented in this study are those of the 2020/21 planting season. The application of humic acid did not significantly (P ≤ 0.05) affect soil pH, and EC, and CDB inoculation also did not significantly (P ≤ 0.05) had effect on the soil pH (Table 4). However, soil pH was observed to be significantly higher in the 600 kg ha⁻¹ NPK and 0 t ha⁻¹ wheat straw (control) plots compared to the 30 and 20 t ha⁻¹ wheat straw applied plots. This is contrary to the study of Jia et al. (2022) where NPK was found to decrease soil pH in arid regions of China. The lower soil pH in wheat straw applied plots may be due to the release of ammonia (NH₃) during straw decomposition which reacts with soil water vapour to form ammonium hydroxide (NH₄OH) that increases soil acidity (Ghanney et al., 2023; Ogunwande et al., 2008). Although there were no significant differences in the EC obtained from the wheat straw treatments, it is expected that the decomposition of straw may result in the release of mineral salts that can increase soil EC (Ghanney et al., 2023). Likewise, CDB inoculation had significant effect on the soil EC and BD. It has been suggested that microbial inoculants are promising tools in the improvement of agroecosystem functioning for increased food security (Bello et al., 2018; Calvo et al., 2014; Hansen et al., 2020). Lower soil EC and BD are positive indicators of soil quality that can enhance plant growth under arid land conditions. The soil BD was also significantly higher in the respective control plots compared to the individual treatments including humic acid and wheat straw. Plots with 100 L ha⁻¹ humic acid treatment had an average BD of 1.40 g cm⁻³ which was statistically significantly (P ≤ 0.05) lower than the average BD of 1.90 g cm⁻³ obtained in the control plots. Soils with lower BD enhance quick emergence of plant seedlings and increased proliferation of the root system through the rhizosphere. Expectedly, wheat straw incorporation significantly reduced soil BD compared to NPK application and 0 t ha⁻¹ wheat straw. Soil incorporation of straw can improve soil fertility with an increased humus content and aggregate stability (Yang et al., 2020; Zhang et al., 2014).

Table 4. Effect of treatments on soil pH, electrical conductivity, bulk density, gravimetric moisture content, and porosity in 2020/21 planting season

Source of variation	pH	EC (dS m^−1)	Bulk density (g cm^−3)	Gravimetric moisture content (%)	Porosity (%)
Humic acid (L ha^−1)
0	7.72a	4.97a	1.90a	4.96c	28.20c
50	7.67a	5.15a	1.57b	6.84b	40.39b
100	7.65a	4.71a	1.40c	9.79a	46.73a
LSD0.05	.114	0.485	0.101	0.337	3.17
Cellulose decomposing bacteria (CDB)
Without	7.69a	4.98a	1.77a	6.71b	33.52b
With	7.67a	4.91b	1.49b	7.69a	43.36a
LSD0.05	.046	0.66	0.08	0.38	2.65
Wheat straw (t ha^−1)
0	7.74a	5.28a	1.88a	6.42d	29.31d
20	7.65b	4.93a	1.54c	7.49b	41.84b
30	7.62b	4.89a	1.36d	8.09a	48.35a
600 kg ha^−1 NPK	7.70a	4.69a	1.74b	6.80c	34.27c
LSD0.05	.046	0.722	0.118	0.275	4.40

Notes: Means having similar letter(s) within the same column are not significantly (P ≤ 0.05) different from each other using Fisher’s LSD.

Soil gravimetric moisture content and porosity were improved (P ≤ 0.05) significantly by humic acid, SSK-2018 and wheat straw (Table 4). 100 L ha⁻¹ humic acid significantly improved soil moisture content by 97.38% and 43.13% compared to 0 L ha⁻¹ humic acid (control) and 50 L ha⁻¹ humic acid, respectively. The application of humic acid may reduce soil water evaporation thereby enhancing higher soil moisture content in the rhizosphere (Goel & Dhingra, 2021). Similarly, soil incorporation of 30 t ha⁻¹ wheat straw improved soil moisture content by 26.01% and 18.97% compared to 0 t ha⁻¹ of straw (control) and NPK, respectively. Soil porosity was improved significantly (P ≤ 0.05) by the application of 50 and 100 L ha⁻¹ humic acid. Likewise, SSK-2018 inoculation significantly (P ≤ 0.05) improved soil porosity by 29.18% than the uninoculated treatment. The incorporation of 30 t ha⁻¹ wheat straw significantly improved soil porosity by 64.96% and 41.09%, respectively, compared to the control and NPK application. Similarly, soil incorporation of 20 t ha⁻¹ straw significantly (P ≤ 0.05) increased soil porosity by 42.75% and 22.09%, respectively, compared to 0 t ha⁻¹ straw (control) and NPK application.

3.5.2. Interaction effect of humic acid and Geobacillus stearothermophilus SSK-2018 on soil moisture content

The inoculation of SSK-2018 in conjunction with humic acid (100 L ha⁻¹) significantly (P ≤ 0.05) improved soil moisture content by 120.13% and 80.55%, respectively, compared to the control (0 L ha⁻¹ humic acid without SSK-2018 inoculation) and the sole application of 100 L ha⁻¹ humic acid without SSK-2018 inoculation (Figure 4). Furthermore, 50 L ha⁻¹ humic acid application in combination with SSK-2018 inoculation significantly improved soil moisture content than the control (0 L ha⁻¹ humic acid without SSK-2018 inoculation) and the sole application of 50 L ha⁻¹ humic acid without SSK-2018 inoculation. Humic substances can act as a media for microbial inoculant proliferation in the soil (Canellas & Olivares, 2014). Humic acid contains bioactive substances (for example, auxin) that can further promote the effectiveness of beneficial microbes in plant growth-promotion (Cozzolino et al., 2021).

Figure 4. Effects of humic acid and cellulose decomposing bacterium interaction on soil gravimetric moisture content.

3.5.3. Effect of three-way interaction of treatments on soil moisture content

The concept of jointly using organic and inorganic fertilizers or amendments with the inclusion of plant growth promoting microbes in crop cultivation and management of soil resources is currently a global practice (Bello & Yusuf, 2021; Vanlauwe et al., 2010). In the current study, the three-way interaction of the treatments significantly (P ≤ 0.05) impacted the gravimetric soil moisture content (Figure 5). Optimal soil moisture is essential for easy dissolution of soil nutrients, proliferation of roots in the rhizosphere and accelerated plant growth (Lee et al., 2017). The applications of 50 L ha⁻¹ humic acid and 600 kg ha⁻¹ NPK combined with SSK-2018 inoculation significantly improved soil moisture content compared to all other treatment combinations with SSK-2018 inoculation. This was followed by the applications of 100 L ha⁻¹ humic acid and 30 t ha⁻¹ of wheat straw combined with SSK-2018 inoculation. In the presence of SSK-2018 inoculation and 20 t ha⁻¹ straw, 50 L ha⁻¹ humic acid improved soil moisture content significantly (P ≤ 0.05) compared to the control and 100 L ha⁻¹ humic acid. Overall, treatments interaction involving humic acid in combination with SSK-2018 and straw outperformed those treatments without humic acid application in the combination. The obtained results indicate that there is significant synergy among the treatments (humic acid, CDB and wheat straw) in the improvement of soil moisture which is a key constraint to crop growth under arid land conditions.

Figure 5. Effect of three-way interaction of treatments on gravimetric moisture content.

Columns display values (n = 3) ±SD.

3.5.4. Effect of three-way interaction of treatments on soil pH

The three-way interaction effects of imposed treatment on soil pH were highly variable (Figure 6). With SSK-2018 inoculation and 100 L ha⁻¹ humic acid application, NPK application led to a significantly (P ≤ 0.05) higher soil pH. NPK in conjunction with manure application have been demonstrated to increase soil pH where NPK application alone decreased it (Jia et al., 2022). Likewise, in the current study, in the absence of SSK-2018 inoculation and humic acid application, NPK application resulted in a significantly reduced soil pH than all other treatments combination. This is expected as N containing fertilizers are known with soil acidification through reduction in the soil pH. In the presence of SSK-2018 inoculation and 50 L ha⁻¹ humic acid, 30 t ha⁻¹ wheat straw incorporation increased the soil pH compared to the control and 100 L ha⁻¹ humic acid. This observation was, however, reversed in the absence of SSK-2018 inoculation where the control (0 L ha⁻¹ humic acid) significantly increased the soil pH compared to 50 and 100 L ha⁻¹ humic acid. Microbial degradation of straw may result in ammonification which produces ammonium ion (NH₄⁺) that are alkaline in nature and can cause the soil pH to increase (Ghanney et al., 2023).

Figure 6. Effect of three-way interaction of treatments on soil pH.

Columns display values (n = 3) ±SD.

3.5.5. Interaction effects of humic acid and wheat straw on soil physicochemical properties

The interaction of humic acid and wheat straw had significant (P ≤ 0.05) effect on soil BD, gravimetric moisture content, and porosity (Figure 7 a-c). The interactions of 50 L ha⁻¹ humic acid with either NPK, 30 and 20 t ha⁻¹ of straw reduced the soil pH significantly compared to the interactions of 100 L ha⁻¹ humic acid with either NPK or straw (Figure 7a). However, the control treatment (0 L ha⁻¹ humic acid and 0 t ha⁻¹ straw) had significantly lowered soil BD compared to treatments that received the different rates of humic acid and wheat straw. Similarly, 100 L ha⁻¹ humic acid interaction with either NPK or 20 and 30 t ha⁻¹ of straw improved gravimetric moisture content significantly by 128.66%, 111.92% and 97.91%, respectively, compared to the control treatment (Figure 7b). The interaction of 100 L ha⁻¹ humic acid with either NPK or 20 and 30 t ha⁻¹ of wheat straw significantly improved soil porosity by 208.75%, 200.47%, and 268.27%, respectively, compared to the combination 0 L ha⁻¹ humic acid and 0 t ha⁻¹ wheat straw (control) (Figure 7c). Furthermore, 100 L ha⁻¹ humic acid application combined with either NPK or wheat straw application at 20 and 30 t ha⁻¹ significantly improved soil porosity by 143.14%, 136.61%, and 190.01%, respectively, compared to NPK application alone without humic acid. Gravimetric moisture content and porosity are key indicators of water availability for plant uptake. The ability of the treatments to improve soil moisture and porosity is an important process in the survival of plants under arid conditions. When water is unavailable for plant uptake, leave and shoot growth are severely impacted (Hsiao & Jackson, 1999).

Figure 7. Effects of humic acid and wheat straw levels interaction on soil (a) bulk density (b) gravimetric moisture content, and (c) porosity.

3.6. Soil basic cations (calcium, magnesium, potassium, and sodium)

3.6.1. Effect of treatments on soil basic cations

The effects of treatments on soil exchangeable cations (Ca²⁺, Mg²⁺, K⁺ and Na⁺) are as shown in Table 5. Most of the results obtained from the individual treatments followed the same trend where humic acid, CDB and straw all had significant (P ≤ 0.05) effect on the soil exchangeable soil cations (Ca, Mg and K) except Na. The application of humic acid at 100 L ha⁻¹ significantly increased Ca content in the soil compared to 0 (control) and 50 L ha⁻¹ humic acid by 95.31% and 52.78%, respectively. The application of humic substances has been suggested to aid the transport of nutrients from soils to the plant root for uptake (Goel & Dhingra, 2021). Furthermore, the inoculation of CDB increased soil exchangeable Ca by 13.90% compared to the uninoculated control treatment. 30 t ha⁻¹ wheat straw incorporation increased soil exchangeable Ca significantly (P ≤ 0.05) by 28.58%, 16.21%, and 7.99% compared to 0 t ha⁻¹ of straw (control), NPK, and 20 t ha⁻¹ straw, respectively. In addition, 20 t ha⁻¹ straw incorporation increased soil exchangeable Ca significantly by 19.07% compared to 0 t ha⁻¹ of straw (control).

Table 5. Effect of treatments on soil basic cations in 2020/21 planting season

Source of variation	Calcium (mg kg^−1)	Magnesium (mg kg^−1)	Potassium (mg kg^−1)	Sodium (mg kg^−1)
Humic acid (L ha^−1)
0	9947.62c	15171.62c	1262.20c	1599.39a
50	12716.58b	19153.07b	1750.22b	1616.91a
100	19428.56a	23567.75a	2909.70a	1698.46a
LSD0.05	858.57	2236.2	438.57	127.88
Cellulose decomposing bacteria (CDB)
Without	13119.26b	18534.71b	1781.02b	1623.38a
With	14942.58a	20060.24a	2167.06a	1653.13a
LSD0.05	588.73	814.3	99.88	37.70
Wheat straw (t ha^−1)
0	12246.25d	17483.75d	1717.26d	1669.86a
20	14581.11b	19903.33b	2072.05b	1669.99a
30	15746.47a	21000.73a	2217.24a	1676.16a
600 kg ha^−1 NPK	13549.86c	18802.08c	1889.54c	1673.81a
LSD0.05	795.54	805.65	13.72	75.42

Notes: Means having similar letter(s) within the same column are not significantly (P ≤ 0.05) different from each other using Fisher’s LSD.

For soil exchangeable Mg, the application of 100 L ha⁻¹ humic acid increased it by 55.34% and 23.05%, respectively, compared to the control and 50 L ha⁻¹ humic acid (Table 5). Wheat straw incorporation at 30 t ha⁻¹ increased soil exchangeable Mg significantly (P ≤ 0.05) by 20.12%, 11.69% and 5.51% compared to 0 t ha⁻¹ straw (control), NPK, and 20 t ha⁻¹ straw, respectively. In addition, 20 t ha⁻¹ straw incorporation increased soil exchangeable Mg significantly (P ≤ 0.05) by 13.83% compared to 0 t ha⁻¹ straw (control). Easily decomposable constituents of straw such as proteins and sugars stimulate soil bacterial activities which can enhance the availability of soil nutrients (Paterson et al., 2011).

3.6.2. Two-way interaction effect of treatments on soil basic cations

3.6.2.1. Cellulose decomposing bacterium and humic acid

The combination of CDB inoculation with 100 L ha⁻¹ humic acid increased soil exchangeable K significantly (P ≤ 0.05) by 170.01% and 158.77%, respectively, compared to sole inoculation of CDB and the control (Figure 8). Furthermore, CDB inoculation in conjunction with 100 L ha⁻¹ humic acid increased K significantly by 34.30% compared to sole application of 100 L ha⁻¹ humic acid. Moreover, CDB inoculation combined with 100 L ha⁻¹ humic acid application increased soil exchangeable K significantly (P ≤ 0.05) by 72.82% compared to CDB inoculation combined with 50 L ha⁻¹ humic acid. The application of humic acid combined with the inoculation of some beneficial microbes (Bacillus amyloliquefaciens and Pseudomonas spp.) have been demonstrated to improve nutrient uptake in a potted soil experiment (Cozzolino et al., 2021).

Figure 8. Effects of humic acid and cellulose decomposing bacterium interaction on soil exchangeable potassium.

3.6.2.2. Humic acid and wheat straw or NPK

The combination of humic acid (100 L ha⁻¹) with NPK, 20 t ha⁻¹, and 30 t ha⁻¹ wheat straw significantly increased soil exchangeable Ca by 157.17%, 125.85%, and 143.46%, respectively, compared to the control (Figure 9). Furthermore, the combination of humic acid (100 L ha⁻¹) with NPK, 20 t ha⁻¹, and 30 t ha⁻¹ wheat straw significantly increased Ca by 102.8%, 77.68%, and 91.53%, respectively, compared to the sole application of 30 t ha⁻¹ wheat straw. Similarly, 100 L ha⁻¹ humic acid with NPK, 20 t ha⁻¹, and 30 t ha⁻¹ wheat straw increased Ca significantly by 85.50%, 62.52%, and 75.19%, respectively, compared to the sole application of NPK. The study of Akhtar et al. (2014) has also demonstrated that combined humic acid and wheat straw application is capable of soil nutrients availability.

Figure 9. Effects of humic acid and wheat straw interaction on soil exchangeable calcium.

3.6.2.3. Geobacillus stearothermophilus SSK-2018 and wheat straw or NPK

The inoculation of SSK-2018 in conjunction with either the application of NPK or wheat straw increased soil exchangeable Ca significantly at P ≤ 0.05 (Figure 10). The inoculation of SSK-2018 in conjunction with either the application of NPK, or 20, and 30 t ha⁻¹ straw increased soil exchangeable Ca significantly by 25.39%, 24.57%, and 35.58%, respectively, compared to SSK-2018 inoculation without wheat straw application. The integrated application of straw with bacteria can improve soil quality and provide soil with resistance to diseases by suppressing the survival of pathogenic microorganisms in the rhizosphere which may hinder plant growth (Liu et al., 2021). In an arid environment in China, the application of straw alone did not improve soil properties (increased soil aggregate stability and soil organic matter accumulation). This was, however, reversed when Bacillus amyloliquefaciens was co-applied with straw (Liu et al., 2021).

Figure 10. Effects of humic acid and cellulose decomposing bacterium interaction on soil exchangeable calcium.

3.6.3. Effect of three-way interaction of treatments on soil exchangeable magnesium (Mg)

Humic acid, SSK-2018 and wheat straw combination significantly (P ≤ 0.05) improved Mg availability in the soil (Figure 11). 50 L ha⁻¹ humic acid and NPK combined with SSK-2018 inoculation increased soil Mg significantly (P ≤ 0.05) compared to all other treatment combinations. The inoculation of plant growth-promoting bacteria alongside the application of humic acid has been shown to boost nutrient uptake by plant roots (Melo et al., 2018; Pishchik et al., 2016). 30 t ha⁻¹ straw incorporation in combination with 100 L ha⁻¹ humic acid and SSK-2018 inoculation increased soil exchangeable Mg significantly compared to 0 t ha⁻¹ (control) and 20 t ha⁻¹ of wheat straw. The combination of humic acid and CDB with wheat straw soil incorporation may result in faster mineralization of wheat straw through the acidification of the rhizosphere as both humic acid and CDB can cause the plant to secrete IAA (Chaiharn & Lumyong, 2011). Humic acid in addition to organic acid released during CDB degradation of wheat straw may promote the availability of residual soil nutrients. The combined application of humic-rich organic amendments with the inoculation of plant beneficial microbes improves soil microbial diversity, crop nutrient uptake, growth, and yield (Cozzolino et al., 2021; Maji et al., 2017). Due to the positive impact of humic acid on plant roots, humic acid may influence higher microbial colonization of plants to aid an extensive root system capable of tapping available nutrients in the rhizosphere compared to inoculated plants without humic acid application. Overall, humic acid efficiency in this study may be attributed to the presence of active substances (phytohormones) which can be utilized directly by plants while simultaneously promoting soil conditioning through enhancing the proliferation of beneficial microbes for increased mineralization of wheat straw and improved squash performance.

Figure 11. Effect of three-way interaction of treatments on soil exchangeable magnesium.

Columns display values (n = 3) ±SD.

4. Conclusion

In the current study, the effects of humic acid, G. stearothermophilus SSK-2018 and wheat straw were evaluated on squash growth and soil properties. G. stearothermophilus SSK-2018 was a cellulose decomposing bacterium (CDB) that enhances plant growth through the production of indole-3-acetic acid, along with various enzymatic activities including cellulase, amylase, protease, gelatinase, and phosphatase. This study demonstrated that G. stearothermophilus SSK-2018, humic acid, and wheat straw significantly improved the fresh weights of stems, leaves, and roots compared to the control. Furthermore, the combinations of treatments involving humic acid and straw, SSK-2018 and straw, as well as humic acid, SSK-2018, and straw, significantly boost squash growth. The study also demonstrated that soil properties, including bulk density, moisture content, and basic cations, were significantly improved by humic acid, SSK-2018, and wheat straw and their different combinations. However, the improvements obtained from the treatments were at par with that of NPK fertilizer application. Therefore, this study suggests that bio-organic amendments should be used consistently for a longer period (more than two seasons) to sustainably improve soil properties that would lead to significant crop performance compared to the use of inorganic fertilizer. However, the continuous use of soil bio-organic amendments should be regularly monitored for potential increase in greenhouse gas emissions that may be like that of nitrogen-containing fertilizers. This would mean that the use of bio-organic amendments should be closely monitored for improved straw management practices that can reduce the emissions of CO₂ and CH₄ without any negative effect on crop growth and yield.

Acknowledgments

This research work was funded by Institutional Fund Projects under grant no. (IFPIP: 685-155-1443). The authors gratefully acknowledge technical and financial support provided by the Ministry of Education and King AbdulAziz University, DSR, Jeddah, Saudi Arabia. Notes on contributors Suleiman Kehinde Bello had his PhD in Field Crops and Soil Science from the Department of Arid Land Agriculture, Faculty of Environmental Sciences, King Abdulaziz University, Jeddah, Saudi Arabia. Samir Gamil Al-Solaiman is a Professor of Soil Chemistry and Fertility at the Department of Arid Land Agriculture, Faculty of Environmental Sciences, King Abdulaziz University, Jeddah, Saudi Arabia. Kamal Ahmed Mohamed Abo-Elyousr is a Professor of Plant Pathology at the Department of Arid Land Agriculture, Faculty of Environmental Sciences, King Abdulaziz University, Jeddah, Saudi Arabia.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/23311932.2023.2246182.

Additional information

Funding

This research work was funded by Institutional Fund Projects under grant no. (IFPIP: 685-155-1443) by the Ministry of Education and King AbdulAziz University, DSR, Jeddah, Saudi Arabia.