1. Introduction
Perennial crops, grown on agricultural land and harvested in short rotations, can provide substantial amounts of biomass [
1,
2,
3,
4]. Until now, biomass was usually used as energy feedstock and its use for industrial purposes and in integrated biorefineries has only recently been contemplated [
5,
6,
7,
8].
There are three major categories of perennial crops grown for biomass. These are short rotation woody crops (SRWCs) such as willow, poplar, eucalyptus, black locust; herbaceous crops such as Virginia mallow, willow-leaf sunflower, cup plant; grasses such as giant miscanthus, prairie cordgrass, switchgrass and giant reed [
9,
10,
11]. Although such a large diversity of perennial crops offers the advantage of providing farmers with many choices, one has to possess sufficient knowledge in this regard. Obtaining a high biomass yield throughout the period of the plantation use (which has a significant impact on the production process profitability) requires making a proper selection of species and cultivars suitable for specific weather and site conditions [
12,
13,
14].
Willow has several advantages as an SRWC, including a wide range of genetic diversity, easy reproduction, tolerance to a wide range of site conditions, ability of new shoots to rapidly regrow after multiple harvests, possibility of harvests in different rotations, resistance to disadvantageous environmental conditions, e.g., morning frost, wet snow and strong winds [
4,
15,
16]. Therefore, willow biomass production in short harvest rotations has been researched in many countries, including Europe [
17,
18,
19], Canada and the USA [
20,
21,
22,
23]. Production of willow as an SRWC can be profitable [
24,
25], its energy balance is positive [
26,
27] and it brings measurable environmental benefits [
15,
28,
29,
30].
The advantages of willow and benefits from its cultivation as an SRWC are very important and can encourage potential producers to decide to start willow biomass production. However, for willow production to be economically, energy-efficiently and environmentally viable, it has to provide stable and high biomass yield per unit area [
31]. The main factors that determine the willow biomass yield have been analysed for years and they include: (1) the choice of a suitable species and cultivar [
18,
23,
31,
32]; (2) soil conditions [
13,
18]; (3) weather conditions [
31,
33]; (4) agrotechnical procedures, including the fertilisation type and rate [
17,
34]; (5) planting density and harvesting frequency [
9,
31]. It should be noted that all of these factors are important, and they have a combined impact on the final effect, i.e., the willow biomass yield. However, when attempting to rank these factors, the choice of the species and variety should be identified as the most important. In consequence, if a biomass producer chooses a wrong willow species or variety, he will not achieve a high biomass yield even if the plantation is set up at a very good site, with the optimum weather and agrotechnical conditions. Moreover, the biomass producer, who has a very limited influence (or sometimes none) on the site or weather conditions, enjoys the full (100%) responsibility for the choice of plant species and variety.
Therefore, because of the large diversity of willow species, varieties and genotypes fit for cultivation as SRWCs and since willow production produces higher yield (per year of plantation use) in three-year harvest rotations [
4,
9,
31], research was conducted to determine (1) survival rate and biometric features; (2) yield of fresh and dry biomass of 15 willow genotypes (including seven varieties and eight clones), cultivated at two different sites in two consecutive three-year harvest rotations. These findings were subsequently used to: (3) quantitatively determine the relative contribution of genetic and site-related factors and their interactions in explaining the variance of willow survival rate, biometric features and biomass yield.
2. Materials and Methods
2.1. Field Experiments
Two identical single-factorial field experiments were conducted in 2013–2018 at two experimental stations of the University of Warmia and Mazury (UWM), located near the villages of Bałdy and Obory in Poland. The experiment at Bałdy (Warmińsko-Mazurskie Province, 53°35′48″ N, 20°36′12″ E) was set up on mud-muck soil developed on calcareous gyttja in loamy subsoil. Willow cuttings were planted in April 2008 at a high density of 48 thousand per ha. The cuttings were planted in two-row strips with rows spaced every 0.75 m. The strips were spaced every 0.90 m. The cuttings in each row were planted every 0.25 m. Willows were coppiced in 2008–2012 in one-year rotations because it was believed at the time that it was a potentially interesting method for willow cultivation in small farms. However, as the market situation has changed since 2013, it was decided to extend the willow harvest cycle, which is why the plants were harvested twice in two subsequent three-year rotations: the first one (I) covering the years 2013–2015, and the second (II) the years 2016–2018. An identical field experiment was set up in April 2009 at Obory (Pomorskie Province, (53°43′34″ N, 18°53′55″ E) on humic heavy alluvial soil, formed from silty clay. The planting method was the same as at the Bałdy site. The willow at Obory was also coppiced in one-year rotations in 2009–2012. Subsequently (like at Bałdy), starting with 2013, willow was harvested twice in consecutive three-year rotations (I—2013–2015, II—2016–2018). Therefore, it must be explained that although the Bałdy experiment was set up a year earlier (2008) than the Obory experiment (2009), it had no significant direct impact on the results analysis in later three-year harvest rotations (2013–2018). This was a consequence of the fact that both at Bałdy and Obory, willows had been harvested earlier every year: five and four times, respectively. Plants harvested in the first three-year rotation grew on eight- and seven-year old stools, so the root systems can be regarded as very well-developed and the plants were giving the full yield. The stools in the second three-year rotation were 10 and 11 years old.
Fifteen identical willow genotypes of nine different species and interspecies hybrids were tested at each site (Bałdy and Obory) (
Table 1). The 15 willow genotypes included seven varieties registered at the Centre for Cultivar Testing in Słupia Wielka, and eight clones—all of them bred and kept at the collection of UWM. At each site (Bałdy and Obory), each genotype was planted in a 150-m
2 strip. At both sites, within each strip, three 40-m
2 plots were randomly designated to conduct biometric measurements and determine the plant density and biomass yield.
The same mineral fertilisation was applied in each experiment before the next three-year rotation was begun. Therefore, the following fertilisers were applied in April 2013 and 2016: nitrogen as ammonium nitrate (90 kg ha−1 N), phosphorus as triple superphosphate (13 kg ha−1 P) and potassium—as potassium salt (50 kg ha−1 K).
2.2. Willow Survival Rate, Biometric Features and Biomass Yield
The plant density on three plots at each site for each harvest rotation was determined for each genotype after the growing seasons ended and it was subsequently calculated per 1 ha. Moreover, the plant survival rate (%) was determined. The height (m) and diameter (mm) of shoots were measured in ten randomly selected plants on each plot. The height was measured from the ground level to the top of the tallest plant shoot. The shoot diameter was measured 0.50 m above the ground level. The number of shoots per stool was also determined in ten replicates on each plot. Only live shoots taller than 1.50 m were taken into account, whereas dry shoots were excluded. Thus, each analysed biometric feature (height, diameter, number of shoots) was measured 30 times for each genotype at each site and for each harvest rotation. This makes up a total of 1800 measurements of each feature, which constituted input data for further analyses.
The plants were harvested in winter (February 2016 and 2017) manually with a chainsaw after each three-year rotation. Immediately after harvest, the whole mass of shoots from each plot was weighed (within an accuracy of 0.01 kg) and the fresh biomass yield was calculated (Mg ha−1 f.m.). Representative biomass samples (approximately 3 kg) were collected during harvest from entire plants of each genotype from each plot to determine the moisture content in it. The biomass moisture content was determined at 105 °C using the oven dry method (EN ISO 18134-1:2015). Subsequently, the moisture content and the fresh biomass yield were used to calculate the dry biomass yield (Mg ha−1 d.m. (dry matter yield)). The biomass yields from the three-year rotations were divided by three to present the biomass yield per one year of plantation use. Moreover, a ranking of the mean dry biomass yield (Mg ha−1 year−1 d.m.) for 15 willow genotypes from two sites in two consecutive three-year rotations was developed.
2.3. Statistical Analysis
All statistical analyses were conducted with STATISTICA 13.3 software (TIBCO Software Inc., Palo Alto, CA, USA 2017). Such variables as survival rate, number of shoots per stool, shoot diameter, plant height, fresh matter yield (f.m.) and dry matter yield (d.m.) were analysed statistically by a repeated-measures ANOVA, with site and genotype as the grouping factors and the replicates were nested in the site effect. The rotation effect was the only factor used for repeated measures. The significance of the factors and their interactions were tested at the significance level of α = 0.05. The statistics F from these analyses are shown in the table of results. The percentage share of all the analysis effects under study in the total sum square (total SS) was calculated. This measure explained the share of individual factors in the trait variance. Moreover, Tukey′s honest significant difference (HSD) test with p < 0.05 was used to evaluate the significance of differences between the subsequently determined means and homogeneous groups. Multiple regression analysis was applied in an assessment of the relationship between fresh and dry matter yield and the morphological features of plants. The variability of the analysed features in relation to the experimental conditions was evaluated using coefficients of variation (CV%).
2.4. Soil and Weather Conditions during the Experiments
Soil examinations at Bałdy showed that the mud-muck soil was neutral, with pH 7.2 in KCl. The humus content was 16.3%. The content of P2O5, K2O and Mg was: 1435 mg, 560 mg and 637 mg kg−1 of soil, respectively. The complete humic heavy alluvial soil at Obory was also neutral with pH 7.0 in KCl. The humus content was lower: 7.09%. The content of P2O5, K2O and Mg was also lower than at Bałdy: 488 mg, 300 mg and 157 mg kg−1 of soil, respectively. However, despite the differences in the contents of these elements, the soil at both sites (Bałdy and Obory) was good and fertile.
The sites were about 150 km apart, which is why the weather conditions were different. The average air temperature during six growing seasons (April–October) at Obory ranged from 13.3 °C to 16.0 °C, in 2017 and 2018, respectively (
Table S1). The average temperature at Obory was always higher compared to Bałdy by 0.1 °C to 1.3 °C. Therefore, the annual average air temperature during the six years of the experiment at Obory (9.0 °C) was higher by approximately 0.7 °C compared to Bałdy. July and August were always the warmest months, with average monthly temperatures reaching 21 °C. The highest temperatures at both sites during the six growing seasons of the experiment were recorded in 2018.
It was the opposite case with precipitation because the average precipitation over six years at Bałdy (598 mm) was higher by approximately 20% than at Obory (
Figure S1). The average precipitation during the growing season (April–October) at Bałdy (414 mm) was approximately 18% higher than Obory. Moreover, particularly large differences in the precipitation between the study sites were recorded during the second three-year harvest rotation (2016–2018) because precipitation during all the three growing seasons was higher at Bałdy than at Obory by 80–159 mm. The largest difference was recorded during the hottest growing season of 2018, when 419 mm of precipitation was recorded at Bałdy and only 260 mm at Obory. On the other hand, the highest precipitation during the whole six-year study period was recorded during the growing season of 2017 (533 and 612 mm, at Obory and Bałdy, respectively). The annual precipitation was also the highest in 2017: 640 and 803 mm, respectively.
4. Discussion
In the present study, the willow plant survival rate decreased naturally with increasing plantation age. It was also found in other research by the authors that the willow survival rate over 12 consecutive years was significantly differentiated by the genotype, harvest rotation and the initial planting density [
31]. The willow survival rate in that research decreased rapidly in the first and second harvest rotation (88% and 58%). It was more stable in the third and fourth rotation, with a slightly decreasing trend (51% and 47%). The highest survival rate in the four three-year harvest rotations was determined for the UWM 095 genotype in the first and fourth rotation (91% and 53%, respectively).
The mean survival rate of 30 willow genotypes in research conducted in Canada was also highly varied [
35]. The mean willow survival rate after the second three-year rotation in that research was 60% (ranging from 3% to 94%) and it was lower by 25% compared to the first rotation. Furthermore, the survival rate of 12 willow genotypes in the first three-year harvest rotation in a study conducted in the USA ranged from 63% to 100%, and from 60% to 97% in the subsequent rotation [
36]. In another study, the mean survival rate of seven willow genotypes after the first three-year rotation was 75%, but the inter-genotype differences were large, ranging from 44% to 85% [
37].
In the present study, the number of shoots per rootstock ranged widely from 1.8 to 6.6, whereas in research on 25 willow plantations in Denmark, the number of shoots also ranged widely—from 1.4 to 9.9 shoots per stool depending on the cultivar, plantation age and harvest rotation [
38]. The number of three-year-old willow shoots per stool ranged from 2.1 to 3.1. A similar number of shoots per stool (2.7) for seven willow genotypes in a three-year harvest rotation were found in another study [
37]. An even smaller mean number of shoots per stool (1.4–2.3) were found in the willow harvested in the first five-year harvest rotation in Spain [
39].
In the present study, the Żubr variety was particularly distinctive since it produced the tallest plants with the largest shoot diameters. Moreover, this plant′s survivability was slightly higher, although the number of shoots was lower than the average for all the 15 genotypes. In other studies, three-year-old plants of the Żubr variety (formerly UWM 006) were also the tallest (7.28 m) and the thickest (48.6 mm) [
37]. Willows were found to be shorter in the research conducted in Canada, where the mean height for 30 genotypes was found to be 2.55 m, with the best three genotypes being 3.76 m tall [
35]. The shoot diameter for 30 genotypes in Canada measured only 11.9 mm and 15 mm for the best three genotypes, which was markedly lower than in the present study.
Willow harvest in three-year rotations can give a higher dry biomass yield compared to the yield in shorter one- or two-year rotations [
4,
31]. Moreover, the willow biomass yield in the first three-year rotation is generally lower compared to subsequent three-year harvest rotations [
13,
21,
36,
40,
41]. The yield increase in the second and subsequent harvest rotations was particularly manifest in plantations that were not coppiced in the first growing year to increase the number of shoots per stool. However, in some cases, the biomass yield during the first three-year harvest rotation was much higher than in the subsequent harvest rotations [
9,
31,
42,
43].
It must be stressed that the current study dealt with two consecutive three-year harvest rotations and one cannot (and must not) regard them as the first and the second three-year harvest rotation in a direct meaning of the word. This is a consequence of the fact that plants in the experiment were obtained earlier in one-year harvest rotations, and a change of the harvest rotation to three years took place in the fifth and the sixth year of the plantation use, when the plants′ root systems were strong (which is also explained in the Materials and Methods section). Plants harvested in the first three-year rotation grew on eight-and seven-year-old stools, so the root systems can be regarded as very well-developed and the plants were giving the full yield. Nevertheless, the study showed that the duration of a willow harvest rotation can be changed. This knowledge is also important to potential investors, as it gives them, in a sense, some flexibility in choosing the willow harvest rotation.
The mean biomass yield from two three-year harvest rotations in this study for most of the 15 genotypes under study (except one) ranged from 8 to 18 Mg ha
−1 year
−1 d.m., with the mean yield being 11.4 Mg ha
−1 year
−1 d.m. However, because of the plantation (and the plants′ root systems) age, the biomass yield obtained in the current experiment should be compared with the findings of other experiments, starting with the second three-year harvest rotation. For this reason, literature data were used and
Figure 8 shows a comparison of the willow biomass yield mainly in the second and the third harvest rotation [
9,
13,
21,
31,
35,
36,
38,
40,
41,
44,
45,
46,
47,
48,
49,
50,
51,
52], although the harvest rotation was shorter or longer by one year in some studies. Since the studies were conducted in different countries, the site conditions (soil and weather) were clearly different. Moreover, the plantations were set up with various clones and various agrotechnical and plantation management procedures were applied. Nevertheless, the findings of this study concerning the mean dry biomass yield in the three-year harvest rotation were on a medium or high level compared to the other studies cited in
Figure 8. Moreover, the current results were similar to those of a study conducted at five different sites in the USA (mean range 10.0–11.6 Mg ha
−1 year
−1 d.m.) [
9].
In the present study, the best-yielding variety—Żubr—gave a higher biomass yield at both sites and harvest rotations (range: 16–20 Mg ha
−1 year
−1 d.m.) compared to the levels achieved for the best genotypes in the USA (range 11–14 Mg ha
−1 year
−1 d.m.) [
9]. A large increase in the biomass yield in the second three-year harvest rotation was also achieved in Canada, mean 13.3 Mg ha
−1 year
−1 d.m. [
44]. Similar mean yield as in Canada was obtained in the authors’ earlier research of five willow genotypes grown at four planting densities and harvested in four three-year rotations [
31]. The highest mean yield (14.5 Mg ha
−1 year
−1 d.m.) was achieved in the cited experiment from the UWM 095 genotype, i.e., it was higher only by one Mg ha
−1 year
−1 d.m. compared to the mean value for this genotype achieved in the authors’ research. The other genotypes gave lower yields of 2.3%, 5.1%, 8.2% and 24.7%, respectively, compared to the UWM 095 genotype [
31]. However, one must add that the willow yield in four consecutive three-year harvest rotations, depending on the experimental factors as determined in the cited research, varied strongly and ranged from 4.8 to 23.2 Mg ha
−1 year
−1 d.m.
Considerable diversity (from 3.5 to 13.6 Mg ha
−1 year
−1 d.m.) was also shown in other research conducted in the USA using 18 willow genotypes at two sites in a three-year harvest rotation [
23]. Furthermore, in a study conducted in Sweden, the mean yield for two willow cultivars grown at five sites with no fertilisation amounted to 5.9 Mg ha
−1 year
−1 d.m. [
17]. The highest yield (13.2 Mg ha
−1 year
−1) was achieved with intensive fertilisation. The willow yield obtained in Denmark also ranged widely from 2.4 to 15.1 Mg ha
−1 year
−1 d.m. [
38]. Moreover, as in other studies, the yield in the second rotation was higher than in the first one.