Poultry Litter Biochar Increases Mycorrhizal Colonisation, Soil Fertility and Cucumber Yield in a Fertigation System on Sandy Soil

: There is a continuing argument about the beneﬁts of biochar on arbuscular mycorrhizal (AM) symbiosis, crop growth, yield, and fertility of soil. There is also limited research on the e ﬀ ects of biochar on AM colonization, cucumber yield, and soil fertility improvement. Therefore, this investigation aimed to determine the impact of poultry litter biochar (PLB) on colonization of roots by indigenous AM fungi in agricultural soil and their contribution to cucumber yield, nutrition, and soil fertility improvement. A ﬁeld trial was conducted to assess the e ﬀ ect of PLB combined with compound poultry manure (CPM) and nitrophos (NP) fertilizer to investigate the response of treatments on nutrient-deﬁcient sandy soils. Plant growth responses to biochar showed better plant growth and yield of cucumber. Application of biochar with and without CPM and NP reduced the negative impact of nutrient deﬁciency stress on cucumber growth. AM fungal colonization, soil fertility, and cucumber yield were improved with the combined application of biochar, CPM, and NP fertilizer. Post-harvest, soil C, N, P, K, Ca, Mg, S, Zn, Cu, Fe, and Mn increased with application of biochar applied with CPM and NP. Biochar application with CPM and NP also increased the percent root colonization of cucumber. Use of biochar with CPM and NP has the potential to improve plant growth, yield, nutrient uptake, and soil fertility. Further studies in various agro-ecological conditions would help utilize this technology in sustainable crop production.


Introduction
Increase in atmospheric CO 2 , CH 4 , and other greenhouse gases is causing climate change, leading to global warming and related concerns. The sequestration of carbon (C) in soil is receiving more attention as a means to combat climate change. With the discovery of so-called Terra Preta, an extremely fertile soil enriched with carbonised biomass in Amazonia [1,2], biochar has appeared as a possible solution for sequestration of C in soil [3].
Biochars are products of the pyrolysis process, an irreversible thermochemical conversion of biomass from either plant or animal origins heated at very high temperature (≥250 • C) in a nil or limited oxygen environment [4,5]. Biochar properties such as pH, specific surface area, pore volume, cation exchange capacity (CEC), other nutrients, volatile matter, ash, and carbon content are mostly

Site Description
Soil (0-10 cm) used in the experiment was collected from a cucumber trial site at Geraldton, Western Australia (latitude 29 • 19 , longitude 115 • 44 ) for analysis before and after the trial. Geraldton has a Mediterranean climate with a mean annual rainfall and temperature of 400 mm and 19.7 • C, respectively. The soil was classified as Tenosol (sand over gravel) [52] and humic Dystroxerepts [53] with 92% sand, 5% silt, and 3% clay particles. Before the start of the experiment, soil was analyzed for some basic properties, including soil pH of 4.8 measured in 0.01 M CaCl 2 at 1:5 (w/v) ratio and soil organic matter of 10.2 g kg −1 measured using the dry combustion method. Soil contained 0.5 g kg −1 total N, and 6 and 4 mg kg −1 NO 3 -N and NH 4 -N, respectively. This soil was selected for its sandy texture, which was deficient in available P (7.3 mg kg −1 ) and was appropriate for a mycorrhizal response study.

Experimental Design
Cucumber (Cucumis sativus L.) was grown for 160 days under field conditions in an agricultural soil at Geraldton, Western Australia following amendment with compound poultry manure (CPM), containing N, P, K at a ratio of 5.4:1.0:2.7, and derived PLB in combination with or without nitrophos (NP) fertilizer including a no biochar added control. There were six treatments with one control applied in a randomized complete block design (RCBD) with three replicates. Details of treatments used are presented in Table 2. Biochar was applied in a field using a mini-spreader (Figure 1a,b).

Site Description
Soil (0-10 cm) used in the experiment was collected from a cucumber trial site at Geraldton, Western Australia (latitude 29°19′, longitude 115°44′) for analysis before and after the trial. Geraldton has a Mediterranean climate with a mean annual rainfall and temperature of 400 mm and 19.7 °C, respectively. The soil was classified as Tenosol (sand over gravel) [52] and humic Dystroxerepts [53] with 92% sand, 5% silt, and 3% clay particles. Before the start of the experiment, soil was analyzed for some basic properties, including soil pH of 4.8 measured in 0.01 M CaCl2 at 1:5 (w/v) ratio and soil organic matter of 10.2 g kg −1 measured using the dry combustion method. Soil contained 0.5 g kg −1 total N, and 6 and 4 mg kg −1 NO3-N and NH4-N, respectively. This soil was selected for its sandy texture, which was deficient in available P (7.3 mg kg −1 ) and was appropriate for a mycorrhizal response study.

Experimental Design
Cucumber (Cucumis sativus L.) was grown for 160 days under field conditions in an agricultural soil at Geraldton, Western Australia following amendment with compound poultry manure (CPM), containing N, P, K at a ratio of 5.4:1.0:2.7, and derived PLB in combination with or without nitrophos (NP) fertilizer including a no biochar added control. There were six treatments with one control applied in a randomized complete block design (RCBD) with three replicates. Details of treatments used are presented in Table 2. Biochar was applied in a field using a mini-spreader (Figure 1a

Sampling and Analyses of Soil and Plant
At harvest, cucumber leaves were randomly collected from each plant and dried at 60 • C for at least 72 h to determine leaves' dry weights (DW). Oven-dried leaves were ground, digested in 3:1 ratio of HNO 3 -HClO 4 and analyzed for various nutrient analysis [54]. The P concentrations in digest were measured by molybdenum-blue method [49].

Assessment of Arbuscular Mycorrhizal Fungi Colonization
After harvesting the crop, root samples (0.5 g) were washed using tap water, cut into 1 cm pieces, and cleared in 10% KOH solution. Then, root samples were acidified and stained with 0.05% trypan blue in lactoglycerol and destained in lactoglycerol [55,56]. Percent root length colonization (%) and root length colonized (m pot −1 ) were measured using a gridline intercept method under a microscope at 100× magnification [57,58].

Statistical Analyses
Analysis of variance was undertaken using Genstat (v.18) (VSN International, Hemel Hempstead, UK). One way analysis of variance was used to observe any significant impact of treatments on soil and plant parameters. A least significant difference (LSD) was applied to test any significance between the means at p ≤ 0.05.

Characteristics of Poultry Litter Biochar
The pH and EC of biochar were 9.0 and 7.7 mS cm −1 , respectively, whereas, the water-holding capacity of biochar was 70%. Total C and N concentration was 38.8% and 3.7% with a C to N ratio of 10.57. Total P, K, Ca, Mg, Na, and S levels in PLB were 2.53, 2.08, 4.5, 1.03, 1.07, and 0.46%, respectively, which indicates the presence of a significant amount of nutrients in biochar. The concentrations of micronutrients (Cu, Fe, Mn, and Zn) and some heavy metals (As, Cd, Pb, and Cr) were also found to be at appropriate and safe ranges for plants and soil (Table 1).

Biochar Effects on Plant Growth, Yield and Nutrition
Cucumber growth and yield responded to different treatments, either positively or negatively, depending on concentration of nutrients present in different applied treatments (p ≤ 0.05; Figures 2 and 3). Data regarding cucumber leaf biomass statistically increased in trend T6 > T4 > T2 and these closely resembled the effect of T1, whereas the effect of T3 and T5 was more than that of the control but not significantly different ( Figure 2). Comparison of applied treatments showed that application of T2 and T4 significantly enhanced cucumber yield, whereas the effect of T5 was positive initially and then decreased the yield. The impact of all other remaining treatments, including T3, T1, and T6, was negative on cucumber growth and yield ( Figure 3).      The nutrient concentrations in cucumber leaves were influenced by different treatments (Table 3). This indicates that application of biochar with and without poultry manure and chemical fertilizer reduced the negative impact of both macro-and micronutrient deficiency stress on cucumber growth and nutrient content of leaves, which were grown on nutrient-deficient sandy soil. Maximum N, P, and K concentration in cucumber leaves were found in plots treated with T3, which were not significantly different from other treatments; however, in comparison with T6, all treatments showed increased nutrient concentrations. The concentrations of secondary macronutrients such as S and Mg were not significantly affected by different treatments. However, Ca was substantially affected by treatments with a maximum Ca concentration of 5.2% in T2 and minimum of 3.3% in T5 ( Table 3). The concentration of micronutrients in soil showed variable effects of increases and decreases with application of PLB alone and in combined with CPM and fertilizers compared with the control.

Biochar Effects on Mycorrhizal Fungi Colonization
The mycorrhizal fungi colonization of roots (assessed as % length of root colonized) was affected by compound poultry manure, fertilizers, and biochar as shown in Figure 4. Biochar addition had variable effects on % root colonization when added with and without CPM and chemical fertilizers. The highest mycorrhizal fungi root colonization (%) was observed for T4, followed by T5 and T6. The lowest mycorrhizal colonization (%) was observed in all other treatments, including the control.

Biochar Effects on Mycorrhizal Fungi Colonization
The mycorrhizal fungi colonization of roots (assessed as % length of root colonized) was affected by compound poultry manure, fertilizers, and biochar as shown in Figure 4. Biochar addition had variable effects on % root colonization when added with and without CPM and chemical fertilizers. The highest mycorrhizal fungi root colonization (%) was observed for T4, followed by T5 and T6. The lowest mycorrhizal colonization (%) was observed in all other treatments, including the control.

Biochar Effects on Soil pH and Nutrient Availability at the End of Plant Growth Cycle
After harvesting cucumber, soil pH, total organic C, N, organic matter content, and macro-and micronutrient concentrations showed significant differences in all applied treatments compared to the control ( Table 4). The highest soil pH of 7.0 was observed in T3 and T4, which was statistically similar to pH of 6.9, 6.9, and 6.8 observed in plots that were treated with T1, T2, and T6, respectively, whereas, the lowest pH of 6.1 was observed in the control.
Soil C and N increased with application of biochar in a combination of higher doses of CPM and chemical fertilizers. The maximum concentration of N was 0.06% observed with application of T1, T2, and T4, which included biochar with higher CPM and NP, followed by 0.05% in T3 where only 13 t ha −1 PLB was added. A low concentration of N was 0.04% noted with T5 and T6, which was higher than 0.03% in the control. Similarly, the percentage total organic carbon (TOC) showed the same trend with a maximum concentration of 0.83% achieved with application of T1, which was not statistically different than the concentrations in T2, whereas T5 and T6 produced significantly low C concentrations with values of 0.45 and 0.49%, respectively. The C to N ratio (C:N) increased in T1, whereas application of all other treatments produced low C:N concentrations. Overall, the addition of biochar with CPM and NP increased the organic C concentration in soil. The presence of this organic C increased the soil organic matter (SOM) content with a maximum value of 1.43% in T1, which was statistically similar to the SOM content of T2 and T4, whereas T5 and T6 produced low SOM concentrations of 0.78 and 0.84%, respectively.
Post-harvest, soil P, K, and Ca increased with application of biochar combined with CPM only in those treatments in which a higher level of NP was applied. Maximum P and K concentrations of 58 and 218 mg kg −1 , respectively, were noted with T1 and T2, and this soil P concentration decreased with decreased doses of NP, although CPM and PLB were applied in substantial amounts. The amount of Ca, Mg, and S also increased positively with application of biochar combined with CPM and NP, while their concentration was low when only biochar was applied. All micronutrients, including Zn, Cu, Fe, and Mn, showed a general trend of increase with biochar application only when

Biochar Effects on Soil pH and Nutrient Availability at the End of Plant Growth Cycle
After harvesting cucumber, soil pH, total organic C, N, organic matter content, and macro-and micronutrient concentrations showed significant differences in all applied treatments compared to the control ( Table 4). The highest soil pH of 7.0 was observed in T3 and T4, which was statistically similar to pH of 6.9, 6.9, and 6.8 observed in plots that were treated with T1, T2, and T6, respectively, whereas, the lowest pH of 6.1 was observed in the control. Note: TOC, total organic carbon; C:N ratio, carbon nitrogen ratio; SOM, soil organic matter.
Soil C and N increased with application of biochar in a combination of higher doses of CPM and chemical fertilizers. The maximum concentration of N was 0.06% observed with application of T1, T2, and T4, which included biochar with higher CPM and NP, followed by 0.05% in T3 where only 13 t ha −1 PLB was added. A low concentration of N was 0.04% noted with T5 and T6, which was higher than 0.03% in the control. Similarly, the percentage total organic carbon (TOC) showed the same trend with a maximum concentration of 0.83% achieved with application of T1, which was not statistically different than the concentrations in T2, whereas T5 and T6 produced significantly low C concentrations with values of 0.45 and 0.49%, respectively. The C to N ratio (C:N) increased in T1, whereas application of all other treatments produced low C:N concentrations. Overall, the addition of biochar with CPM and NP increased the organic C concentration in soil. The presence of this organic C increased the soil organic matter (SOM) content with a maximum value of 1.43% in T1, which was statistically similar to the SOM content of T2 and T4, whereas T5 and T6 produced low SOM concentrations of 0.78 and 0.84%, respectively.
Post-harvest, soil P, K, and Ca increased with application of biochar combined with CPM only in those treatments in which a higher level of NP was applied. Maximum P and K concentrations of 58 and 218 mg kg −1 , respectively, were noted with T1 and T2, and this soil P concentration decreased with decreased doses of NP, although CPM and PLB were applied in substantial amounts. The amount of Ca, Mg, and S also increased positively with application of biochar combined with CPM and NP, while their concentration was low when only biochar was applied. All micronutrients, including Zn, Cu, Fe, and Mn, showed a general trend of increase with biochar application only when applied with CPM and NP, whereas their concentrations in some treatments, particularly T4 and T5, were lower than those of the other treatments and the control. These nutrients showed lower concentrations in the T6 treatment in which only 33 t ha −1 PLB was used.

Discussion
Results of this study proved that addition of biochar enhanced leaf nutrient content and minimized nutrient runoff in the sandy soil investigated. Poultry litter biochar in combination with fertilizers and CPM produced healthy fruit, and improved cucumber growth and yield, by improving soil quality, nutrient concentration in soil, and the water-holding capacity of soil, thus making the soil conducive for better plant growth and fruit development (Figures 2 and 3). Treatments T2, T4, and T6 produced a significantly larger biomass of leaves compared to T1, T3, and T5 treatments. Although several treatments produced maximum crop biomass, farmer should choose and apply those treatments that produce maximum biomass with minimum cost and environmental issues. To improve sustainable agriculture and maximize crop yield, it may be challenging for farmers to use a high dose of chemical fertilizers because this treatment is costly. Although treatment T6 produced more leaf biomass, it produced a low cucumber yield. Thus, treatment T4, which used an appropriate ratio of all ingredients, could be an excellent source of biochar with organic and inorganic fertilizers that does not require a compromise of environmental issues. Similarly, treatment T2, with 9:5:13 t ha −1 CPM, NP, and PLB, respectively, provided a maximum yield of cucumber followed by treatment T4, which included 1:2:7 t ha −1 CPM, NP and PLB. These two treatments (T2 and T4) can be the most economical response of applied treatments without compromising on environmental risks, and can thus lead to sustainable agricultural production ( Figure 3). Treatment T6, which included application of 33 t ha −1 biochar alone, did not increase growth and yield of cucumber, and showed substantial decrease in yield after one month of its application. Furthermore, an increasing trend was noted after the passage of time which might be related to the release of much of the N from the biochar in the soil.
Growth and yield of cucumber (Figures 2 and 3) signify the effect of biochar application, either alone or in combination with poultry manure and NP fertilizers [59][60][61]. The increased leaf growth may be related to the highest nutrient release from biochar in soil and the greater availability of these nutrients to plant roots [62]. This outcome is also due to the liming effect of biochar in acidic soils, changes in soil microbial activities and function, improved crop water availability, and increased cation exchange capacity of soil [63]. Biochar and poultry manure improved the physical and chemical properties of soil, including soil structure, texture, soil porosity, water-and nutrient-holding capacity, soil bulk density, and cation exchange capacity of the sandy soils used in this study [64,65]. These treatments, thus, improved soil properties and made conditions conducive for plants to obtain maximum nutrients [29]. Many previous studies have revealed that different types of biochar applied alone or in combination with inorganic and organic fertilizers has potential to considerably improve soil health [12] and increase soil aggregate stability [66]. This treatment also enhances crop productivity [67,68], nutrient availability to plants [20,69], physiochemical properties of soil, and plant growth [70].
Almost all plots showed a definite increase in nutrient concentrations in leaves compared to the control where biochar was applied with CPM and NP (Table 3). Macro-and micronutrients, including N, P, K, Ca, Mg, S, Zn, Cu, Fe, and Mn, increased with application of biochar in a combination with CPM and NP fertilizer compared to the control. Maximum nutrient contents were recorded in all treatments applied with biochar in a combination with CPM and NP. The increase in nutrient concentrations in leaves can be positively influenced directly by plant nutrient uptake because of the nutrient content in biochar, its release characteristics, availability of nutrients, and enhanced uptake of nutrients [20,65]. Mulcahy et al. [71] found remarkable development in tomato growth, yield, and nutrient concentrations in plants grown in sandy soils amended with biochar. The higher amount of biochar addition with or without organic and inorganic fertilizers improved plant uptake of P, K, Ca, Zn, and Cu, in addition to the efficient use of fertilizers, due to retention, and thus reduced nutrient leaching from soil as reported earlier [3].
In the present experiment, addition of biochar with and without CPM and NP had a variable effect on root colonization with AM fungi [39,72]. The increased % root colonization in T4 might be related to the low availability of P in this treatment in comparison to all other treatments in which biochar was either added alone in a high dose or with high amounts of CPM and NP fertilizers. Nonetheless, the mechanisms regarding the effect of biochar amendments on % root colonization by AM fungi is not known with precision [29]. Reduction of AM fungi colonization with the maximum rate of biochar is in line with findings of [73], who noted a decline in AM fungi colonization when soils were amended with a greater amount of biochar. Some scientists suggest that biochar amendments can increase % AM root colonization in plant roots [74], whereas many others note a decrease in AM fungi abundance [73]. Inhibition in AM fungal root colonization after amendment with biochar might be related to improved availability of P in soil [22]. Comparable results were reported as a decline in the mycorrhizal colonization rate with P [75].
Application of biochar with and without CPM and NP considerably increased the post-harvest soil pH, N, total organic C, soil organic matter content, and the concentrations of macro-and micronutrients in soil compared to the control (Table 4). Increase in soil pH is primarily due to the alkalinity of biochar (pH 9.0) and the presence of ashes in biochar. The ashes contain reasonable amounts of oxides and hydroxides of alkali metals, which are easily dissolvable and react quickly with soil, so they rapidly increase the soil pH [76] and release free bases (K, Ca, Mg) and other ions into soil solution [12]. Sanchez et al. [77] stated that amendments of soil with biochar raised the soil pH by 0.4-1.2 pH units, and this increase was observed mainly in sandy and loamy textured soils compared to clayey soils. AN increase in soil pH was also described by other researchers [6,78,79] who documented a positive effect of biochar on acidic soils.
The available soil N and total organic C showed a varied response to the addition of biochar with and without CPM and NP fertilizer to soil. The available soil N ranged from 0.03 to 0.06% in all treatments. Treatments T1, T2, and T4 showed available N of 0.06%. The increase in available N in the biochar system was chiefly because of the increased soil N content due to the application of inorganic N fertilizer combined with PLB and CPM. In addition to the release of N from biochar and inorganic fertilizers, biochar also has the potential to adsorb ammonia (NH 3 ) more efficiently [80] and thus perform as a binder agent for NH 3 in soil. This property of biochar lessens the volatilization of ammonia from the soil surface. Amendment of soils with biochar promotes mineralization of organic matter [10,21] and boosts the release of nutrients such as N, P, and C [81]. Chan et al. [18] stated that higher nutrient-holding capacity of biochar improves nutrient supply to soil and plants, and reduces their leaching.
The C:N ratio showed a decrease compared to the control plots and continuously decreased in plots in which biochar was applied. The decreased C:N ratio revealed a less immobilization of N in plots where biochar with organic and inorganic fertilizers was applied. The control, which recorded the highest C:N ratio, revealed more N immobilization [82,83]. Addition of biochar with poultry manure increased the soil organic C content and, ultimately, an increase in soil organic matter and fertility of soils was observed [84] due to the presence of biochar particles [12], aromatic C content, and recalcitrant C in the organic matter pool [85,86]. Release of more nutrients, particularly P, with organic amendments was also reported by Izhar Shafi et al. [87]. Increase in post-harvest soil macro-and micronutrient concentrations due to the application of biochar with poultry manures and inorganic NP fertilizers results from the nutrient reservoirs in highly weathered infertile sandy soils provided by biochar and manure [20], which improves the physical, chemical, and biological properties of soil [88]. A larger surface area with negative functional groups on organic matter facilitates nutrient capturing by biochar and stops their chelation with other nutrients. The presence of these nutrients in soil is strongly related to the mineralization of organic matter [21], and this mineralization process leads to nutrient release in soil solution [81].
Based on the biochar properties and nutrient concentrations, we found that the biochar produced from poultry litter materials is rich in C and can sequester more C in sandy soils when applied with NP fertilizer and poultry manure. However, the leaching of nutrients from organic and inorganic sources requires more experimental studies in sandy soils.

Conclusions
Results showed that application of poultry litter biochar with compound poultry manure and inorganic NP fertilizers increased yield, nutrient contents, and AM colonization, and reduced the negative impact of both macro-and micronutrient deficiency stress on cucumber growth. The results from this study indicate that the use of biochar could be an effective approach in relation to plant growth, yield, nutrient uptake, and improved soil fertility of sandy soils. However, further longer-term studies in fields of various agro-ecological conditions are needed to utilize this technology in sustainable cucumber crop production.