Biochar-fertilizer mixture: does plant life history trait determine fertilizer application rate?

ABSTRACT

The annual cumin and perennial fennel are economically important medicinal crops of cold dry regions of Pakistan. We hypothesized that the cumin, which produces 2–3 times less biomass, will respond to lower rates of mixture of biochar with synthetic NPK fertilizer or manure, compared to fennel. The NPK, poultry manure and their mixture with wood-derived or cow manure-derived biochars were applied for three consecutive years. No positive relation between application rate of biochar-mixed fertilizers and yield of both crops was observed over three years of study, except that manure-derived biochar-NPK mixture had a positive relation (R² = 0.99, P = 0.01) with the yield of fennel only during the third year. Significant positive influences of biochar-based fertilizers compared to control were observed for cumin and fennel of third year cropping. The co-amendment of NPK (0.14 kg ha⁻¹) with manure-derived biochar (6.6 t ha⁻¹) consistently increased the yield of cumin during the first two years of cropping, as opposed to NPK fertilizer. Cumin had a greater seed:stover biomass ratio when it received the co-amendment of wood-derived biochar with NPK or poultry manure. Our findings indicate that there is some potential for biochar-fertilizer amendments to improve the growth of these high-value medicinal crops.

KEYWORDS:

• Cuminum cyminum
• Foeniculum vulgare
• medicinal crops
• soil mineral nitrogen
• Olsen phosphorus

Introduction

Biochar improves soil quality and crop yield in arid regions when it is co-amended with synthetic fertilizer or organic wastes such as manure, crop residue or composts. For instance, a field-based study was conducted in semi-arid region, at Agricultural Research Farm, Arid Agriculture University, Rawalpindi, Pakistan, on sandy loam soil. The amendment of sugarcane bagasse-derived slow-pyrolized biochar (production temperature 400°C), at 1% C ha⁻¹ rate with urea at 46 kg N ha⁻¹ for two years, significantly (P ≤ 0.05) reduced soil bulk density by 4%, increased total nitrogen by 20% and microbial biomass carbon by 9% [1]. Similarly, 39,reported that amendment of biochar, which was derived from the twigs from vineyard at 12 t ha⁻¹ rate in sandy loam soil, increased soil nitrogen, phosphorus and potassium by 18%, 13% and 31% respectively (P ≤ 0.05). Interestingly, when biochar was co-amended at 12 t ha⁻¹ rate with urea as 0.087 t ha⁻¹ rate, it increased concentrations of phosphorus and potassium in soil by 8% and 7% respectively than when biochar was amended in soil alone [2]. The total biomass of maize was increased in biochar-amended soil by 45%; whereas, when biochar was co-amended with urea, it increased yield by 72% than control [2]. In another study that was conducted in cold arid agricultural research farm, University of Balochistan Quetta, Pakistan, application of small particle-sized wood-derived slow-pyrolyzed biochar (particle size < 0.65 mm) at 5 and 10 t ha⁻¹ rates, as mixtures with goat manure in 1:1 and 1:2 manure:biochar ratio, in sandy loam soil, increased second year yield of garden pea (Pisum sativum) by 59% and 63% respectively (P ≤ 0.05) than when only goat manure was applied to soil [3].

Crops from the Apiaceae family include economically important medicinal crops, such as the annual Cuminum cyminum (cumin seeds) and perennial Foeniculum vulgare (fennel seeds). The cold dry climatic conditions of Balochistan, Pakistan, is suitable for the growth of these two crops. Although there is lack of information regarding influence of biochar on yield of these two crops, such amendments are reported to have a positive influence on crop growth under dry climatic conditions [3–5]. For this reason, we expect that co-amendment of biochar with fertilizer could be a good option to improve the growth of these crops. Furthermore, properties of biochars vary mainly due to feedstock and production temperature. In general, porosity and concentration of nutrients in biochars are in the order of manure > stover > wood [6,7]. Empirical evidences exist, which demonstrate that different biochars have variable influences on crops [8, Adekiya et al., 2020a]. According to the meta data analysis by 22, in nutrient-poor soils of dry regions, nutrient-rich biochars (e.g. manure-derived biochars) tend to have more positive influence on crops than the biochars that have lower nutrient concentrations [e.g. wood-derived biochars). However, exceptions exist; for example, two years of field study in arid environment using farmyard manure-derived biochar and wood-derived biochar as soil amendments, 30,found no difference in grain yield of garden pea between treatments of using these biochars. Furthermore, there is a knowledge gap whether biochars from different sources and their application rates have different or similar influences on economically important crops that belong to same family but have different life history traits (i.e. phenology such as vegetative growth period and the time reproductive growth initiation occurs and life form such as annual or perennial] e.g. C. cyminum and F. vulgare. This type of knowledge can improve yield and the cultivation of these crops on a large scale can generate substantial income to the local farmers.

The objective of the study is to evaluate the influence of amendment rates of wood-derived and cow manure-derived biochars as mixtures with synthetic NPK fertilizer or poultry manure, on yield and stover biomass production of C. cyminum and F. vulgare under field conditions for three years. The hypotheses of this study are: (1) crop with high biomass requires more fertilizer amendment (i.e. F. vulgare) than the crop that has low biomass (i.e. C. cyminum), (2) consistent low application rates of fertilizers for three years have a gradual positive influence on crop growth while high application rates of fertilizers have positive influence during first cropping year and (3) co-amendment of biochar with fertilizers increases concentration of N and P in soil; whereas, concentrations of mineral N and soluble inorganic P are higher in the soil cultivated with C. cyminum than F. officinalis because higher biomass acquires nutrients in higher concentration and, therefore, leaving less nutrients in soil.

Materials and methods

Study area

The experiment was conducted at the Balochistan Agricultural Research and Development Centre (BARDC), Quetta, Pakistan (66° 57’ 20” E, 30° 11’ 39” N). The climate in Quetta is Mediterranean type with dry warm summers (maximum 31.7 ± 0.66°C in August during study period) and cold winters (minimum 2.02 ± 0.67°C in December during study period) with rain and snow, giving a total annual precipitation of 214.9 mm on average of three years of study. Soil at the experimental site was Brown Chernozem silt loam with 50 g clay kg⁻¹, 500 g silt kg⁻¹ and 450 g sand kg⁻¹ containing 9.9 g organic matter kg⁻¹ soil. It had pH 7.9, electrical conductivity of 0.25 dS m⁻¹, 0.68 mg NO₃-N kg⁻¹ and 4.7 mg Olsen-P kg⁻¹.

Biochars and manure

Cow and buffalo manure from Quetta was purchased and burned to black mass (biochar) in a domestic kiln at 350–550°C, based on the temperature range recorded for this type of kiln [9]. Slow-pyrolysis wood-derived biochar was obtained from timber market of Quetta city and is produced by burning Acacia nilotica L. wood in underground kilns, locally referred to ‘Bhatti’. Poultry manure (not bedded) of boiler chicken was obtained from poultry farm of Quetta city. In poultry farm, manure was piled up in a room with open windows and was air-dried when collected. Physical and chemical properties of biochars and poultry manure are presented in Table 1.

Table 1. Properties of biochar and manure used as soil amendments for Cuminum cyminum and Foeniculum vulgare in Quetta, Pakistan.

Properties	Wood biochar	Cow manure biochar	Poultry manure
pH	8.15	9.15	8.21
EC (dS m^−1)	14.6	8.55	11.2
Ash (g kg^−1)	45.0	293	449
Nitrogen (mg g^−1)	4.3	2.9	20.8
Phosphorus (mg g^−1)	0.3	5.6	13.6
Copper (mg g^−1)	0.041	0.054	0.12
Zink (mg g^−1)	0.046	0.047	0.039
Chromium (mg g^−1)	0.023	0.098	0.087

Experimental design

The experiment was conducted in October 2017 and ended in August 2019. The crops (C. cyminum and F. vulgare) were cultivated separately and treatments were assigned randomly to the 1.5 × 1.5 m plots. There was approximately 0.25 m buffer between plots and no opening between plots to prevent horizontal flow of water (Supplementary Figure S1). The treatments were control (no inorganic and organic amendment), inorganic fertilizer (Zarkhez 17% N, 17% P (as P₂O₅) and 17% K (as K₂O), Engro fertilizers Limited Pakistan) applied at 666.6 kg ha⁻¹ (100 g fertilizer per 1.5 × 1.5 m plot), which contained 113 kg ha⁻¹ N (source of N was not specified and was kept secret by manufacturer), 113 kg ha⁻¹ P₂O₅ and 113 kg ha⁻¹ K₂O, NPK fertilizer mixed with wood-derived and manure-derived biochars (amount of NPK fertilizer and biochars in NPK fertilizer-biochar mixture treatments are provided in Table 2), and following organic-based fertilizers; mixture of wood-derived biochar and inorganic fertilizer, mixture of cow manure-derived biochar and inorganic fertilizer, poultry manure, mixture of poultry manure and wood-derived biochar, mixture of cow manure-derived biochar and poultry manure. Each organic-based fertilizer was applied at 0.25 kg plot⁻¹ (1.6 t ha⁻¹), 0.5 kg plot⁻¹ (3.3 t ha⁻¹) and 1 kg plot⁻¹ (6.6 t ha⁻¹) rates. The poultry manure-biochar mixtures were made as 1:1 fertilizer:biochar ratio on dry weight bases. To ensure consistency in nutrient concentrations of fertilizers for replications, before amendment, these fertilizers were well-mixed and carefully weighed each time. In a factorial experimental design, with two factors (fertilizer type and application rates), each treatment was replicated three times and the total number of plots were 42 for each crop. The biochars, manures and biochar-manure mixtures were amended in soil followed by their mixing in soil to 2–3 cm. The fertilizers of a given treatment were amended in the same plots for three consecutive years in autumn season (mid-October to early November). Treatments and their abbreviations are given in Table 2.

Table 2. Application rates of biochar co-amended with NPK fertilizer and poultry manure on field plots cultivated with Cuminum cyminum and Foeniculum vulgare. Treatments were applied to the same plot once a year for three years in the field experiment. Amendment rates are on dry weight bases.

Treatment	Application rate of NPK fertilizer	Application rate of manure	Application rate of biochar
Control	0	0	0
Synthetic fertilizer (NPK)	70 kg ha^−1 N, 70 kg ha^−1 P2O5 and 70 kg ha^−1 K2O	0	0
NPK + wood-derived biochar	8.75 kg ha^−1 N, 8.75 kg ha^−1 P2O5 and 8.75 kg ha^−1 K2O	0	0.83 t ha^−1 (3.57 kg ha^−1 N, 0.25 t ha^−1 P2O5)
	17.5 kg ha^−1 N, 17.5 kg ha^−1 P2O5 and 17.5 kg ha^−1 K2O	0	1.66 t ha^−1 (7.13 kg ha^−1 N, 0.49 kg ha^−1 P2O5)
	35 kg ha^−1 N, 35 kg ha^−1 P2O5 and 35 kg ha^−1 K2O	0	3.32 t ha^−1 (14.2 kg ha^−1 N, 0.99 kg ha^−1 P2O5)
NPK + manure-derived biochar	8.75 kg ha^−1 N, 8.75 kg ha^−1 P2O5 and 8.75 kg ha^−1 K2O	0	0.83 t ha^−1 (2.40 kg ha^−1 N, 4.64 kg ha^−1 P2O5)
	17.5 kg ha^−1 N, 17.5 kg ha^−1 P2O5 and 17.5 kg ha^−1 K2O	0	1.66 t ha^−1 (4.81 kg ha^−1 N, 9.29 kg ha^−1 P2O5)
	35 kg ha^−1 N, 35 kg ha^−1 P2O5 and 35 kg ha^−1 K2O	0	3.32 t ha^−1 (9.62 kg ha^−1 N, 18.59 kg ha^−1 P2O5)
Poultry manure	0	1.66 t ha^−1 (34.5 kg ha^−1 N, 22.5 kg ha^−1 P2O5)	0
	0	3.32 t ha^−1 (69 kg ha^−1 N, 45 kg ha^−1 P2O5)	0
	0	6.64 t ha^−1 (138 kg ha^−1 N, 90 kg ha^−1 P2O5)	0
Poultry manure + wood-derived biochar	0	0.83 t ha^−1 (17.2 kg ha^−1 N, 11.2 kg ha^−1 P2O5)	0.83 t ha^−1 (3.57 kg ha^−1 N, 0.25 t ha^−1 P2O5)
	0	1.66 t ha^−1 (34.5 kg ha^−1 N, 22.5 kg ha^−1 P2O5)	1.66 t ha^−1 (7.13 kg ha^−1 N, 0.49 kg ha^−1 P2O5)
	0	3.32 t ha^−1 (69 kg ha^−1 N, 45 kg ha^−1 P2O5)	3.32 t ha^−1 (14.2 kg ha^−1 N, 0.99 kg ha^−1 P2O5)
Poultry manure + manure-derived biochar	0	0.83 t ha^−1 (17.2 kg ha^−1 N, 11.2 kg ha^−1 P2O5)	0.83 t ha^−1 (2.40 kg ha^−1 N, 4.64 kg ha^−1 P2O5)
	0	1.66 t ha^−1 (34.5 kg ha^−1 N, 22.5 kg ha^−1 P2O5)	1.66 t ha^−1 (4.81 kg ha^−1 N, 9.29 kg ha^−1 P2O5)
	0	3.32 t ha^−1 (69 kg ha^−1 N, 45 kg ha^−1 P2O5)	3.32 t ha^−1 (9.62 kg ha^−1 N, 18.59 kg ha^−1 P2O5)

Sowing of seeds, harvest of plants and biomass estimation

The seeds of both crops were sown during the optimum time for their germination. The seeds of C. cyminum were sown on 12, 15 and 14 February 2017, 2018 and 2019 respectively; while the seeds of F. officinalis were sown on 06, 06 and 5 March 2017, 2018 and 2019 respectively. For both crops, 0.006 t ha⁻¹ seeds were broadcasted to plots. Sowing was carried out for three consecutive years for both crops. The F. vulgare is perennial but was sown for three years also as plants were harvested with roots. The plots were irrigated once or twice (depending on need based on apparent dryness of soil and signs of leaf wilting) every week with groundwater. The plots were watered to saturation point (Supplementary Figure 1). Plants were harvested at seed maturation stage. The harvest of C. cyminum was carried out in the first week of June (04, 06 and 07 of June in 2017, 2018 and 2019 respectively); whereas, F. vulgare was harvested after mid of August (17, 23 and 21 of August 2017, 2018 and 2019 respectively). Stover and seeds were oven-dried at 40°C for 48 hours followed by measurement of their biomass.

Soil sampling, processing and analysis for physicochemical properties

Soil samples were taken from 0 to 10 cm depth from the center of each plot after harvest of third year crop (July 2019 for C. cyminum and August 2019 for F. officinalis) (Oladele et al., 2019). Soil samples were collected in zip-lock bags, air-dried in laboratory and passed through 2 mm mesh sieve. Soil samples were extracted with 2 M KCl solution as 1:5 soil:KCl solution ratio [10]. The soil mineral N (cumulative of nitrate (NO₃⁻) and ammonium (NH₄⁺)) were analyzed by microtiter plate technique [11]. Soluble inorganic soil phosphorus was analyzed with the method of 12. Instead of microplate reader, samples were analyzed with spectrophotometer (UV-1700 pharma spec, UV–visible, Shimadzu) [12,13].

Statistical analysis

The D’Agostino-Pearson K² test was applied to assess normal distribution of data sets. Due to large differences between values of treatments, data sets could not be transformed for normal distribution. Therefore, non-parametric Kruskal-Wallis test was applied to assess if significant difference in a given data set exists. The data sets with significant difference were subsequently subjected to the pairwise comparison between treatment means of individual data sets, using Mann–Whitney test with Bonferroni correction. The raw data of all studied parameters, range values, mean separation letters and skewness of each treatment of a given study parameter are provided in supplementary files (Table S1-S3). Regression analysis to evaluate the effect of biochar application on the crop growth (i.e. yield, stover biomass production and seed:stover ratio) was done separately for plots that received NPK fertilizer and poultry manure. All analyses were carried out using CoStat software and Microsoft Excel.

Results

Hypotheses 1 and 2: Rate of fertilizer has a positive relation with plant biomass and consistent low application rate shows positive influence over time

With few exceptions, regression analysis showed no significant relation between yield, stover biomass and seed:stover biomass ratio of both crops over three years of cropping. For C. cyminum, of third year crop, significant negative relation was observed between application rate of mixture of NPK with manure-derived biochar and yield (R² = 0.99, P ≤ 0.03; Figure 1). For F. vulgare of third year crop, a significant positive relation was observed between yield and application rate of mixture of NPK with manure-derived biochar (R² = 0.99, P ≤ 0.01; Figure 2). The seed:stover biomass ratio of first year crop of C. cyminum had a significant positive relation with application rate of poultry manure (R² = 0.99, P ≤ 0.01; Figure 3). The seed:stover biomass ratio of second year crop of F. vulgare had a significant positive relation with application rate of mixture of poultry manure with wood-derived biochar (R² = 0.99, P ≤ 0.01; Figure 4). Significant positive influence of fertilizers applied at lower application rate was observed for yield of both crops (Table 3 and Table S1)’. Moreover in Table 3, results of C. cyminum could be found.

Figure 1. Regression analysis between application rates of NPK-mixed biochar fertilizers with yield, stover biomass and seed:stover ratio of C. cyminum for three years of cropping. NPK, synthetic NPK fertilizer; WB, wood-derived biochar; MB, cow manure-derived biochar; PM, poultry manure; +, mixture of NPK or PM with biochar; 1.6, 3.3 and 6.6, fertilizer amendment rates in t ha−1.

Figure 2. Regression analysis between application rates of NPK-mixed biochar fertilizers with yield, stover biomass and seed:stover ratio of F. vulgare for three years of cropping. NPK, synthetic NPK fertilizer; WB, wood-derived biochar; MB, cow manure-derived biochar; PM, poultry manure; +, mixture of NPK or PM with biochar; 1.6, 3.3 and 6.6, fertilizer amendment rates in t ha⁻¹.

Figure 3. Regression analysis between application rates of poultry manure-mixed biochar fertilizers with yield, stover biomass and seed:stover ratio of C. cyminum for three years of cropping. NPK, synthetic NPK fertilizer; WB, wood-derived biochar; MB, cow manure-derived biochar; PM, poultry manure; +, mixture of NPK or PM with biochar; 1.6, 3.3 and 6.6, fertilizer amendment rates in t ha⁻¹.

Figure 4. Regression analysis between application rates of poultry manure-mixed biochar fertilizers with yield, stover biomass and seed:stover ratio of F. vulgare for three years of cropping. NPK, synthetic NPK fertilizer; WB, wood-derived biochar; MB, cow manure-derived biochar; PM, poultry manure; +, mixture of NPK or PM with biochar; 1.6, 3.3 and 6.6, fertilizer amendment rates in t ha⁻¹.

Hypothesis 3: co-amendment of biochar with fertilizers increases concentration of N and P in soil and concentration depends on crop biomass

Table 3. Mean ± SD of yield (t ha⁻¹), stover biomass (t ha⁻¹) and seed:stover biomass ratio of first, second and third year cropping of C. cyminum.

Treatments	Yield			Stover biomass			.Seed:stover biomass ratio
	Year 1	Year 2	Year 3	Year 1	Year 2	Year 3	Year 1	Year 2	Year 3
Control	414 ± 81.5^abcd	371 ± 87^abcd	57 ± 17^c	407 ± 131^abcd	492 ± 123^abc	69 ± 31^e	1.06 ± 0.20^abc	0.79 ± 0.25^abc	0.90 ± 0.34^a
NPK	239 ± 116^e	310 ± 49^d	84 ± 53^bc	362 ± 224^abcd	475 ± 180^abc	109 ± 75^de	0.71 ± 0.14^b	0.70 ± 0.21^bc	1.04 ± 0.68^abc
NPK+WB1.6	247 ± 133d^e	297 ± 103 ^cd	162 ± 48^b	244 ± 82^d	413 ± 116^bc	404 ± 100^b	0.98 ± 0.22^abc	0.72 ± 0.14^bc	0.40 ± 0.06^c
NPK+WB3.3	479 ± 3.07^b	450 ± 12^bc	168 ± 51^ab	445 ± 16^c	399 ± 53^c	306 ± 106^b	1.07 ± 0.03^a	1.14 ± 0.19^a	0.55 ± 0.10^abc
NPK+WB6.6	334 ± 54.8^de	396 ± 55^c	204 ± 101^ab	356 ± 74^d	588 ± 165^abc	296 ± 92^bc	0.95 ± 0.16^abc	0.70 ± 0.15^c	0.72 ± 0.35^abc
NPK+MB1.6	552 ± 185^bc	466 ± 39^abc	122 ± 21^b	595 ± 208^ab	606 ± 117^ab	287 ± 118^bc	0.93 ± 0.03^bc	0.80 ± 0.24^abc	0.51 ± 0.33^abc
NPK+MB3.3	377 ± 202^abcde	446 ± 149^abcd	112 ± 28^b	482 ± 197^abcd	615 ± 126^ab	172 ± 31 ^cd	0.74 ± 0.13^c	0.75 ± 0.27^bc	0.65 ± 0.14^abc
NPK+MB6.6	508 ± 0.38^a	481 ± 31^ab	96 ± 67^bc	541 ± 16^a	620 ± 83^a	176 ± 92 ^cde	0.94 ± 0.02^b	0.78 ± 0.12^c	0.49 ± 0.13^abc
PM1.6	577 ± 150^abcde	525 ± 125^bcd	97 ± 110^ab	757 ± 140^abcd	583 ± 112^abc	187 ± 216^abcd	0.76 ± 0.09^c	0.90 ± 0.09^bc	0.52 ± 0.06^abc
PM3.3	526 ± 124^abcde	423 ± 38^bc	487 ± 194^ab	578 ± 159^abcd	687 ± 84^a	1114 ± 487^abc	0.91 ± 0.16^bc	0.62 ± 0.06^c	0.44 ± 0.15^abc
PM6.6	469 ± 84 ^cd	575 ± 140^abcd	103 ± 70^bc	333 ± 63^d	621 ± 198^abc	313 ± 137^bcde	1.41 ± 0.26^ab	0.93 ± 0.15^abc	0.33 ± 0.10^c
PM+WB1.6	277 ± 197^bcde	401 ± 152^abcd	287 ± 106^ab	291 ± 220^abcd	571 ± 158^abc	895 ± 353^abc	0.95 ± 0.08^ab	0.70 ± 0.18^abc	0.32 ± 0.16^abc
PM+WB3.3	541 ± 54^abc	523 ± 74^ab	110 ± 128^abc	492 ± 106^abcd	565 ± 112^abc	389 ± 171^bcde	1.10 ± 0.14^ab	0.92 ± 0.08^ab	0.28 ± 0.22^abc
PM+WB6.6	307 ± 26^de	495 ± 67^abc	288 ± 127^abc	261 ± 170^abcd	491 ± 128^abc	920 ± 462^abcde	1.18 ± 0.35^abc	1.01 ± 0.18^abc	0.31 ± 0.52^abc
PM+MB1.6	435 ± 33^c	391 ± 33^c	45 ± 143^abc	367 ± 73^bcd	500 ± 135^ab	81 ± 234^abcde	1.18 ± 0.18^abc	0.78 ± 0.18^bc	0.56 ± 0.08^b
PM+MB3.3	443 ± 94 ^cd	323 ± 74^c	509 ± 182^a	459 ± 24^b	457 ± 156^ab	988 ± 289^a	0.97 ± 0.00^b	0.71 ± 0.26^bc	0.51 ± 0.09^c
PM+MB6.6	333 ± 211^abcd	520 ± 11^a	288 ± 109^ab	369 ± 933^abcd	524 ± 101^a	525 ± 199^ab	0.90 ± 0.61^abc	0.99 ± 0.16^abc	0.55 ± 0.09^c

Within column, values with different uppercase letters are significantly different at P < 0.05. Control, no fertilizer amendment; NPK, synthetic NPK fertilizer; WB, wood-derived biochar; MB, cow manure-derived biochar; PM, poultry manure; +, mixture of NPK or PM with biochar; 1.6, 3.3 and 6.6, fertilizer amendment rates in t ha⁻¹.

As compared to NPK, its co-amendment with wood-derived or manure-derived biochars decreased concentration of mineral N significantly in soil cultivated with C. cyminum (P ≤ 0.05, Figure 5) while for F. vulgare, no significant difference was observed. As compared to poultry manure at lower application rate, its mixture with biochars at most application rates, significantly increased mineral N of soil cultivated with both crops (P ≤ 0.05; Figure 5). Concentration of soluble mineral P of soil cultivated with C. cyminum was significantly higher in response to the amendment of poultry manure with manure-derived biochar at higher application rate (P ≤ 0.05, Figure 5). For soil cultivated with F. vulgare, significant higher concentration of P was observed in response to co-amendment of poultry manure with manure-derived biochar at higher application rate (i.e. 6.6 t ha⁻¹) as compared to when pultry manure was applied at the same rate (P ≤ 0.05, Figure 5).

Figure 5. Mean ± SD of concentration of mineral N and soluble mineral P of soil grown for three years with C. cyminum and F. officinalis. Bars with different letters show significant differences at P ≤ 0.05. Control, no fertilizer amendment; NPK, synthetic NPK fertilizer; WB, wood-derived biochar; MB, cow manure-derived biochar; PM, poultry manure; +, mixture of NPK or PM with biochar; 1.6, 3.3 and 6.6, fertilizer amendment rates in t ha⁻¹. The mean separation letters are based on Kruskal–Wallis test, followed by pairwise comparison between treatment means of individual data sets, using Mann–Whitney test with Bonferroni correction.

With one exception, there was no difference in concentration of mineral N between soils of both crops. Concentration of mineral N was significantly lower under cultivation of C. cyminum in response to poultry manure applied at 1.56 t ha⁻¹ as compared to the soil cultivated with F. vulgare (P ≤ 0.05; Figure 6). Similarly, no difference in soluble mineral P was observed between soils of both crops except for NPK fertilizer and mixture of poultry manure with manure-derived biochar at 6.6 t ha⁻¹ amendment rate, which increased concentration in C. cyminum cultivated soil as compared to F. vulgare (P ≤ 0.05; Figure 6).

Figure 6. Mean ± SD of comparison between soils grown with C. cyminum and F. vulgare for three years for concentration of mineral N and soluble mineral P. Bars with * show significant differences between C. cyminum and F. officinalis-grown soil (P ≤ 0.05). Control, no fertilizer amendment; NPK, synthetic NPK fertilizer; WB, wood-derived biochar; MB, cow manure-derived biochar; PM, poultry manure; +, mixture of NPK or PM with biochar; 1.6, 3.3 and 6.6, fertilizer amendment rates in t ha⁻¹. The Kruskal–Wallis test, followed by pairwise comparison between two treatment means was performed using Mann–Whitney test with Bonferroni correction.

Discussion

Hypotheses 1 and 2: Rate of fertilizer has a positive relation with plant biomass and consistent low application rate shows positive influence over time

The C. cyminum and F. vulgare are grown in arid to semi-arid regions; dry warm summer is required for their growth and seed maturation. Mediterranean climate and well-drained fertile soil are suitable for their cultivation [14,15]. In our study, yield and stover biomass of control treatments of both crops in third year of cropping was lower than first and second year of cropping (P ≤ 0.05; Table 3,Table S2). Likewise, for C. cyminum, except for three treatments i.e. poultry manure, mixture of poultry manure with wood-derived and manure-derived biochars, amended at 3.3 t ha⁻¹, 1.6 t ha⁻¹ and 3.3 t ha⁻¹ respectively, which did not show year-wise difference in yield, under other fertilizer treatments yield of third year crop was significantly lower than first and/or second year cropping (P ≤ 0.05; Table 3,Table S1 and S2). However, contrary to the results for C. cyminum; for F. vulgare, except NPK and its mixture with wood-derived biochar, which showed significantly lower yield for third year than first and/or second year crop, for other fertilizer treatments, no year-wise difference was found (Table S1 and S2). In third year, study area received unexpected high rainfall (520.6 mm in 2019 versus 90.2 mm and 34 mm in 2017 and 2018 respectively) and unexpected rainfall in May (16.4 mm), July (15.9 mm) and August (17.4 mm). The temperatures were also lower than usual during growing season of crops. Wet soil conditions under Mediterranean climatic conditions may reduce crop yield by reducing aeration to roots and causing diseases to crops [16–18]. Organic amendments reduce bulk density and increase porosity of soil [16]. Furthermore, biochar, due to its dark color, keeps soil temperature warmer. For instance, soil amended with straw-derived slow-pyrolyzed biochar had 2°C higher temperature and difference between day and night temperatures was lower [19]. These factors allow drainage of excess water and promote soil aeration, which in return helps crops cope with unexpected high rainfall-induced soil water saturation [16]. In our study, contrary to C. cyminum, year-wise difference under most fertilizer treatments was not observed for F. vulgare. Since F. vulgare has greater biomass, possible higher rate of evapotranspiration as compared to C. cyminum might also helped this crop cope with unexpected high rainfall [18] under biochar-based fertilizer amendments.

In Balochistan, Pakistan, commonly urea and P₂O₅ are applied together in 1:1 ratio with an application rate of 60 kg ha⁻¹ each. In this province, organic fertilizer as air-dried farmyard or poultry manure is applied at 1–2 t ha⁻¹ rate. The application rates of inorganic fertilizer (as N, P₂O₅ and K₂O) used in this study are within range or higher than the rates previous studies have reported for sandy loam soils (0.03 t ha⁻¹ to 0.3 t ha⁻¹ where N as urea or N, P as P₂O₅ or super phosphate and K₂O or K₂SO₄ reported in previous studies) [20, 21]. Likewise, amendment rate of poultry manure and poultry manure-biochar mixture of this study are within the range of application rates reported in previous studies for sandy to sandy loam soils [22–24]. The yield of C. cyminum of our study is similar with the other published reports [25,26]. However, yield of F. vulgare was many times lower than the yield reported in other studies [27–30]. Similar to our study, 8,sowed F. vulgare for three years in field; however, they observed approximately 4 times more yield each year than the yield obtained in our study. The lower yield in our study may be due to wild variety of this crop because significant differences in yield between varieties of F. vulgare are reported [31].

Due to space limitation and because of mostly non-significant and inconsistent results, data of comparison between treatment means are presented in supplementary material. Contrary to our hypothesis, with few exceptions, no relation was observed between application rate of mixture of NPK fertilizer and biochars, with plant growth performance parameters (i.e. yield stover biomass and seed:stover biomass ratio) over three years of study for both crops (Figure 1–4). The exception was observed for C. cyminum for third year crop only, in which application rate of mixture of NPK with manure-derived biochar had a negative relation with yield (R² = 0.99, P = 0.01; Figure 1). However, as compared to NPK, for C. cyminum, this fertilizer at 1.6 t ha⁻¹ and 6.6 t ha⁻¹ amendment rates, improved yield by 57% and 53% respectively of first year and by 33.5% and 35.6% respectively of second year crop (Figure S 1, Table S1). For C. cyminum, as compared to NPK fertilizer, its mixture with wood-derived biochar at 3.75 t ha⁻¹ application rate also improved yield by 50% for first year of crop (P < 0.05; Table 3,Table S1) and at 3.75 t ha⁻¹ and 7.5 t ha⁻¹ amendment rates improved yield by 31%, 22% respectively for second year crop (P < 0.05; Table 3,Figure S1). Although not consistent over three years of study, our findings for C. cyminum are in agreement with empirical evidences that co-amendment of biochars with inorganic NPK fertilizers improves yield significantly [8, Adekiya et al., 2020 a,b; 23].

For F. vulgare, as compared to NPK, its mixture with biochars did not improve yield (Table S2). For this crop, mixture of NPK with manure-derived biochar had a significant positive relation with yield for third year crop only (R² = 0.99, P = 0.00; Figure 2). Furthermore, application rate of this fertilizer treatment had a positive relation with stover biomass of first year crop only (R² = 0.99, P = 0.01; Figure 2). Stover biomass was improved significantly only of third year crop in response to mixture of NPK with wood-derived biochar at 1.6 t ha⁻¹ and manure-derived biochar at 6.6 t ha⁻¹ rate as compared to NPK (P < 0.05; Figure S1, Table S2).

For C. cyminum, non-significant relationship was found for yield and stover biomass with application rate of poultry manure or its co-amendment with biochars. A positive relation of seed:stover biomass ratio was observed with application rate of only poultry manure for only first year crop of C. cyminum (R² = 0.99, P = 0.01; Figure 3). For F. vulgare, application rate of co-amendment of poultry manure with wood-derived biochar had a positive relation with stover biomass of third year crop only (R² = 0.99, P = 0.02; Figure 4). A positive relation was observed for Seed:stover biomass ratio of F. vulgare with application rate of co-amendment of poultry manure with wood-derived biochar for second year crop only (R² = 0.99, P = 0.01; Figure 4).

For F. vulgare, yield and stover biomass of first year and second crop was significantly lower in response to amendment of poultry manure at higher application rate (i.e. 3.75 t ha⁻¹) as compared to NPK fertilizer and most of biochar-based NPK fertilizer treatments. However, when poultry manure was co-amended with wood-derived biochar at 7.5 t ha⁻¹ rate for first year crop and 3.75 t ha⁻¹ rate for second year crop, it improved yield by 19.4% and 40% respectively (P < 0.05; Figure 2). This indicates that amendment of wood-derived biochar improved the positive influence of poultry manure on yield of F. vulgare. A 14% significant improvement in the yield of radish in response to co-amendment of poultry manure with biochar (produced from the woods of Khaya senegalensis, Parkis biglosa, Terminalia glaucescens and Prosopis Africana) (50 t ha⁻¹ biochar mixed with 5 t ha⁻¹ poultry manure) as compared to poultry manure (amended at 5 t ha⁻¹ rate) was reported [32,33]. The authors attributed this positive influence on radish yield to the improvement of crop for utilization of nutrients from poultry manure. The amendment of biochar manure mixture improves soil quality. It reduces bulk density, increases water and nutrient holding capacity and improves air infiltration of soil [13, Adekiya et al., 2020 a, b; 34]. All these factors improve plant root-microbiome interaction, nutrient cycling in rhizosphere, plant defense mechanism and ultimately plant growth performance [6,8,35]. Due to fund limitation, we did not examine the influence of biochar-poultry manure mixture on the interrelated factors such as soil health, biological and biochemical functions in rhizosphere and plant growth performance. This phenomenon however, merits future investigation.

Co-amendment of wood-derived biochar with NPK at 3.3 t ha⁻¹ rate increased yield and seed:stover ratio of C. cyminum of first two years of cropping by ~34% and ~36% respectively than NPK fertilizer (P < 0.05; Figure 4). Similarly, for first year crop, as compared to poultry manure applied at 1.6 t ha⁻¹ rate, co-amendment of poultry manure with wood-derived biochar at 1.6 t ha⁻¹ and 3.3 t ha⁻¹ rates increased seed:stover ratio by ~28% and ~33% respectively (P < 0.05; Figure 4). Furthermore, ~31% increase in seed:stover biomass ratio was also observed for the second year crop under treatment of wood-derived biochar mixed with poultry manure applied at 3.32 t ha⁻¹ rate than the poultry manure applied at same amendment rate (P < 0.05; Figure 4). Interestingly, there was no difference in stover biomass between these treatments. This indicates that amendment of wood-derived biochar with NPK or poultry manure improved plant growth performance. To our knowledge, it is the first report that shows positive influence of wood-derived biochar as mixture with NPK or poultry manure on seed:stover biomass ratio without reducing stover biomass than when NPK or poultry manure were applied alone. However; for F. vulgare, although mixture of wood-derived biochar with NPK at 3.32 t ha⁻¹ amendment rate increased seed:stover ratio than NPK fertilizer by 42%, stover biomass under this treatment was significantly lower than NPK fertilizer treatment. This indicates that the increase in seed:stover biomass ratio was may be stress-induced, as plants invest more resources for seed production under stress conditions [21,36].

For first two years of cropping of both crops, there was no difference between control treatment and most of fertilizer treatments for yield and stover biomass production. As compared to control, yield of first year crop of C. cyminum was significantly lower under NPK fertilizer treatment. However, cumulative aboveground plant biomass (seed + stover) was not different (Table S1, S3,). Inorganic fertilizer was applied at very high rates in field, which probably had negative influence on seed production. Significant differences in yield and stover biomass between control and most of biochar-based fertilizer treatments were obvious for third year cropping of both crops (Table 4,Table S1 and S2). We applied fertilizers in autumn season. Being highly porous, when biochar is applied in soil as a mixture with synthetic or organic fertilizers in autumn season, it allows microbes to dwell and grow in pores and on surfaces of these fertilizers. Their activities such as decomposition of organic matter and nutrient cycling help improve soil quality for crop cultivation in spring [8]. Therefore, fertilizers were applied in autumn season (mid-October to early November) of three consecutive years. However, due to high concentrations of silt and sand and low concentration of organic matter (9.9 g kg⁻¹ soil), soil might not retained nutrients from these fertilizers for the following spring of two cropping years. This factor might had resulted in no difference in aboveground plant biomass between control and fertilizer treatments. However, continuous organic amendments for three years might increased the nutrient-holding capacity of soil. When biochar is mixed with organic or inorganic fertilizers, it captures nutrients from fertilizers and acts as slow-release fertilizer in soil [37–40]. High absorption capacity of biochar possibly caused accumulation of nutrients in biochar-NPK or biochar-poultry manure mixture over time in soil. On the other side, continuous cropping in soil with no fertilizer input (i.e. control treatment) may have resulted in depletion of nutrients. These both factors possibly had contributed in higher yield and stover biomass production in organic-based fertilizer-amended soils as compared to control for the third year crops. Unfortunately we did not examine this hypothesis; however, after three years of cropping, no difference in concentration of mineral nitrogen and soluble mineral phosphorus in soil of all treatments, despite significantly higher yield and stover biomass production under organic-based fertilizers than control treatment, may explain our phenomenon in this regard (Figure 5 and Figure 6).

Hypothesis 3: co-amendment of biochar with fertilizers increases concentration of N and P in soil and concentration depends on crop biomass

Table 4. Mean ± SD of yield (t ha⁻¹), stover biomass (t ha⁻¹) and seed:stover biomass ratio of first, second and third year cropping of F. vulgare.

Treatments	Yield			Stover biomass			Seed:stover biomass ratio
	Year 1	Year 2	Year 3	Year 1	Year 2	Year 3	Year 1	Year 2	Year 3
Control	79.06 ± 8.9^b	75.3 ± 18.6^bcde	53.73 ± 7.9^e	1630 ± 183^cde	1050 ± 523^bc	722 ± 180^c	0.049 ± 0.01^ab	0.079 ± 0.020^a	0.077 ± 0.017^ab
NPK	103.77 ± 59.15^abc	116 ± 27.0^ab	90 ± 4.1^a	3399 ± 2106^ab	2584 ± 2104^ab	1386 ± 230^b	0.031 ± 0.003^c	0.060 ± 0.02^ab	0.066 ± 0.010^ab
NPK+WB1.6	83.33 ± 24.1^abc	112 ± 7.3^a	86.6 ± 9.3^ab	1812 ± 488^bcd	2566 ± 492^a	1829 ± 134^a	0.045 ± 0.001^ab	0.044 ± 0.005^b	0.047 ± 0.002^c
NPK+WB3.3	90.66 ± 17.77^ab	112.2 ± 17.89^ab	81.7 ± 8.7^ab	1687 ± 221 ^cd	2936 ± 785^bc	1545 ± 351^ab	0.053 ± 0.005^a	0.039 ± 0.004^b	0.054 ± 0.010^bc
NPK+WB6.6	76 ± 6.9^b	69.3 ± 38.8^bcde	58 ± 8.3^d	1834 ± 139^c	1342 ± 975^bc	1173 ± 390^abc	0.041 ± 0.004^bc	0.067 ± 0.032^ab	0.051 ± 0.009^bc
NPK+MB1.6	72.44 ± 26.0^abc	75.3 ± 6.7^c	68 ± 9.8^bcde	1980 ± 204^bc	1134 ± 260^c	1120 ± 384^bc	0.035 ± 0.009^bc	0.068 ± 0.012^a	0.066 ± 0.026^abc
NPK+MB3.3	85.33 ± 27.35^abc	89.77 ± 36.43^abcde	75.5 ± 11.4^bcd	2236 ± 897^abcd	1787 ± 1406^abc	1599 ± 452^b	0.039 ± 0.004^bc	0.060 ± 0.019^ab	0.048 ± 0.006^c
NPK+MB6.6	98 ± 7.21^a	75.5 ± 19.14^bcde	90.4 ± 15.0^abc	2722 ± 48^a	1448 ± 697^bc	2027 ± 173^a	0.036 ± 0.003^c	0.060 ± 0.028^ab	0.044 ± 0.006^bc
PM1.6	83.3 ± 35.5^abc	86.8 ± 48.5^abcde	70.4 ± 29.3^abcde	2482 ± 1177^abcde	1672 ± 1205^abc	1448 ± 837^abc	0.034 ± 0.002^c	0.066 ± 0.030^ab	0.056 ± 0.020^abc
PM3.3	58.22 ± 7.4^c	45.7 ± 9.89^e	66.6 ± 5.8 ^cd	1739 ± 491^de	715 ± 246^c	1108 ± 199^bc	0.034 ± 0.004^c	0.065 ± 0.008^ab	0.061 ± 0.007^ab
PM6.6	76.6 ± 20.6^abc	60.6 ± 26.7^cde	69.5 ± 10.5^cde	1752 ± 429^bcde	1087 ± 815^bc	1267 ± 226^b	0.043 ± 0.007^c	0.064 ± 0.017^ab	0.055 ± 0.005^b
PM+WB1.6	64.4 ± 6.6^bc	79.1 ± 42.48^abcde	77.3 ± 15.6^bcd	1164 ± 233^e	1473 ± 1064^bc	909 ± 417^bc	0.057 ± 0.014^ab	0.074 ± 0.043^ab	0.091 ± 0.021^a
PM+WB3.3	65.77 ± 22.95^bc	76.4 ± 15.4 ^cd	81.5 ± 16.2^abc	1678 ± 456^bcde	1130 ± 436^bc	1026 ± 529^bc	0.038 ± 0.003^bc	0.070 ± 0.012^ab	0.087 ± 0.022^ab
PM+WB6.6	72 ± 4.6^b	124.2 ± 87.9^abcde	69.3 ± 12.1^bcd	1666 ± 830^bcde	1299 ± 915^bc	1298 ± 804^abc	0.049 ± 0.018^abc	0.147 ± 0.168^ab	0.063 ± 0.027^abc
PM+MB1.6	68.22 ± 10.1^bc	44.6 ± 12.34^e	66.2 ± 3.2^cd	1756 ± 529^bcde	811 ± 344^c	1050 ± 235^bc	0.041 ± 0.014^abc	0.058 ± 0.012^ab	0.064 ± 0.012^ab
PM+MB3.3	73.33 ± 56.72^abc	53.5 ± 14.0^de	57.3 ± 34.5^abcde	2667 ± 2422^abcde	1063 ± 429^c	1287 ± 695^abc	0.030 ± 0.010^c	0.052 ± 0.008^ab	0.043 ± 0.002^c
PM+MB6.6	76.44 ± 40.75^abc	54.22 ± 14.36^de	73.5 ± 12.1^bcde	2239 ± 1607^abcde	1152 ± 547^c	1358 ± 633^abc	0.036 ± 0.010^bc	0.051 ± 0.015^ab	0.059 ± 0.016^abc

Within column, values with different uppercase letters are significantly different at P < 0.05. Control, no fertilizer amendment; NPK, synthetic NPK fertilizer; WB, wood-derived biochar; MB, cow manure-derived biochar; PM, poultry manure; +, mixture of NPK or PM with biochar; 1.6, 3.3 and 6.6, fertilizer amendment rates in t ha⁻¹.

Our fifth hypothesis was that concentrations of mineral N and soluble inorganic P are higher in response to co-amendment of biochar with fertilizers in the soil cultivated with C. cyminum than F. officinalis as plant with higher biomass acquire nutrients in higher concentration, leaving less nutrients in soil. Our results are not in agreement to our hypothesis except for only one treatment, i.e. co-amendment of manure-derived biochar with poultry manure at higher application rate (P < 0.05; Figure 6) (7.5 t ha⁻¹). Plant species vary in their response to fertilizer treatments. Published reports suggest that plant species vary in their interaction to their microbiome under a given environmental condition [41]. Therefore, type and application rate of fertilizer is not a stronger predictor of their influence on plant-soil microbe interaction [41]. It merits further investigation to evaluate microbial abundance and community structure in the rhizosphere of these crops under various fertilizer types and their application rates. Such a study will help get an insight into crop-specific responses to fertilizers and their application rates in the context of N and P cycling in rhizosphere. Such a study will help understand how to modulate plant-microbe interaction to increase N and P cycling with different fertilizers and their amendment rates.

Recommendations

Our results that are related to the influence of co-amendment of biochars, with synthetic and organic fertilizers and their application rates on plant growth performance, with regard to plant life history traits such as phenology (vegetative growth period and the time reproductive growth initiation occurs) and life form (annual versus perennial), did not confirm our hypotheses. Interrelations of plant biomass and its life history traits with the type and application rate of biochar-based fertilizers need to be evaluated in the context of influence of fertilizers on N and P cycling in soil and other physicochemical and biological properties of soil, including rhizobiome. Such a study will help get an insight into best fertilizer management practice for better agronomic output of a given crop.

The influence of amendment of poultry manure and its co-amendment with biochars, on the yield of both crops, was comparable to NPK fertilizer as well as with the co-amendment of NPK with biochars. Furthermore, co-amendment of fertilizers with biochars tended to increase concentration of soluble inorganic P in soil. Poultry and farmyard manures in the province of Balochistan are highly economic compared to synthetic fertilizers. For example, one kilogram of synthetic NP (nitrogen and phosphorus) fertilizer costs approximately 0.65 US$; whereas one ton of air-dried poultry and cow manure costs US$ 35 (price varies slightly in various places of this province). One kilogram of wood-derived biochar as leftover broken small pieces, available in local timber markets, is approximately 0.2 US$. Owing to the high prices of synthetic fertilizers, use of poultry manure and biochars produced from farmyard manure and wood will be a cost-efficient approach and as an alternative to synthetic fertilizers for agronomic purpose.

Conclusions

Contrary to our hypotheses, amendment rates of fertilizers did not have relation with yield of both crops except for over-time non-consistent few exceptions. The exceptions were significant negative relation of C. cyminum and significant positive relation of F. vulgare for yield of third year crops with amendment rate of mixture of manure-derived biochar with NPK. As compared to control, significant positive influences of biochar-based fertilizers on yield and stover biomass of both crops was obvious only for third year cropping. As compared to NPK fertilizer, an obvious positive influence of its co-amendment with manure-derived biochar on yield of C. cyminum at higher application rate (6.6 t ha⁻¹) was observed consistently for the first two years. This indicates that this fertilizer has potential for higher agronomic output of C. cyminum. Another interesting finding was, as compared to NPK or poultry manure, their co-amendments with wood-derived biochar at higher application rates (3.3 t ha⁻¹ and 6.6 t ha⁻¹), consistently for first and second years, significantly increased seed:stover biomass ratio of C. cyminum without reducing stover biomass. Our results show that application rates of biochar-poultry manure- or biochar-NPK-mix fertilizers have no relation with plant growth performance or with plant life history trait.

Author Contributions

Abdul Ghani Achakzai:- PhD Candidate and conduct the experiment

Abdul Hanan Buriro:- field supervisor who supervises the experiments in the field

Shamim Gul:- Principal Supervisor of the project who designs the experiments

Hidayatullah Khan:- Lab fellow helped in Sample analysis

Sadiq Agha:- Lab fellow helped in Sample analysis

Sadaf Aslam Ghori:- Field data collection

Zsolt Ponya:- statistical analysis and result interpretation

Tariq Ismail:- Data Analysis and discussion writing and correspondence

Acknowledgments

This research is funded by National Research Program for Universities (NRPU) Grant # 9664.

Disclosure statement

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

Additional information

Funding

This research is funded by National Research Program for Universities (NRPU) Grant # 9664.