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Trifolium subterraneum cover cropping enhances soil fertility and weed seedbank dynamics in a Mediterranean apricot orchard

Trifolium subterraneum cover cropping enhances soil fertility and weed seedbank dynamics in a... The soils of Mediterranean semiarid environments are commonly characterized by low levels of organic matter and mineral elements, as well as severe weed infestations, which, taken together, cause an intensive use of auxiliary inputs (tillage, fertilizers, herbicides). Although cover crops are recognized to sustainably improve soil health, the impact of Trifolium subterraneum L. cover cropping needs specific attention. This research investigates for the first time the effects over 4 years of T. subterraneum and spontaneous flora cover crops, after either incorporating their dead mulches into the soil or leaving them on the soil surface, on soil organic matter (SOM), macroelements, mineral nitrogen, microelements, and weed seedbank dynamics as indicators of soil quality in an apricot orchard. Compared to a conventional management control, the T. subterraneum cover crop with the burying of dead mulch into the soil increased the amount of SOM (+ 15%), ammoniacal (+ 194%) and nitric (+ 308%) nitrogen, assimilable P O (+ 5%), exchangeable K O (+ 14%), exchangeable Na (+ 32%), exchangeable K (+ 16%), Fe (+ 15%), Mn 2 5 2 (+ 28%), Zn (+ 36%), and Cu (+ 24%), while it decreased the weed seedbank size (‒ 54%) and enhanced weed biodiversity. These findings suggest that T. subterraneum cover cropping may be an environment-friendly tool to enhance soil quality and limit auxiliary input supply in Mediterranean orchards. Keywords Cover cropping · Subterranean clover · Soil health · Soil organic matter · Soil macroelements · Soil microelements · Weed soil seedbank 1 Introduction * Umberto Anastasi The weaknesses of agricultural activity (i.e., release of agro- umberto.anastasi@unict.it chemicals, loss of water resources, generation of ammonia Aurelio Scavo from grazing (GAG) emission, soil degradation, and loss aurelio.scavo@unict.it of biodiversity) are attributed to the irrational or ineffi- Alessia Restuccia cient use of auxiliary inputs and to the excessive biological a.restuccia@unict.it homogeneity typical of intensive farming systems. There- Cristina Abbate fore, in agreement with the United Nations (UN) Sustain- cristina.abbate@unict.it able Development Goals (SDGs) and with the strategies of Sara Lombardo the European Commission (EC) Green Deal, the transition saralomb@unict.it of agricultural systems towards sustainability requires an Stefania Fontanazza agroecological and multifunctional approach in the manage- stefania.fontanazza@yahoo.it ment of agroecosystems (EC 2019; UN 2015). It is widely Gaetano Pandino recognized that polyculture supports the stability and lesser g.pandino@unict.it dependence on external inputs of agroecosystems by using Giovanni Mauromicale environmental resources (solar radiation, rainfall, N , CO , 2 2 g.mauromicale@unict.it etc.) more effectively than monoculture (Finney and Kaye Department of Agriculture, Food and Environment (Di3A), 2017). A number of researchers have demonstrated that the University of Catania, Via Valdisavoia, 5, 95123 Catania, inclusion of cover crops in no-till farming systems ensures Italy Vol.:(0123456789) 1 3 70 Page 2 of 16 A. Scavo et al. various ecosystem services (soil erosion control, carbon studied managements, while it increased the amount sequestration, reduction of nutrient leaching, degradation of ammonia-oxidizing (Nitrosomonas europaea) and of agrochemicals, increase of biodiversity, pollinator attrac- N-fixing (Azotobacter vinelandii) bacteria involved in the tion, etc.) (Blanco-Canqui et al. 2015; Daryanto et al. 2019; soil N-cycle, which resulted in a significant enhancement Kaye and Quemada 2017; Mauromicale et al. 2010; Sharma of ammoniacal and nitric N-availability in the soil. et al. 2018a, b; Wulanningtyas et al. 2021). Given that soil quality is a function of a complex Areas under semiarid Mediterranean-type climates network of interrelationships among physical, chemi- are commonly characterized by irregular rainfall cal, and biological properties, in which the organic regimes with intense autumnal events and drought in matter (SOM) content plays a key role (Jiménez et al. late spring and summer. In these climatic conditions, 2002), and keeping in mind the benefits deriving from soils are often clayey, poor in organic matter, and cover crops on soil health, especially under an inte- subjected to erosion, which contribute to lowering the grated management strategy (Scavo and Mauromicale macro- and microelements’ content (Lombardo et  al. 2020), in this research, we have hypothesized that 2014; Madejón et al. 2007). The loss of soil minerals, the optimization of T. subterraneum cover cropping particularly relevant for macronutrients such as nitrogen management can allow lowering the environmental (N) and phosphorous (P), combined with the severe impact and enhancing the productivity of Mediterra- weed infestations, represents two critical aspects of nean orchard agroecosystems through an equilibrium Mediterranean agroecosystems, where an intensive and between the main soil quality indicators and weed pres- indiscriminate use of auxiliary inputs (tillage, fertilizers, sure. Hence, the goals were to compare subterranean and herbicides) is consequently adopted. In this clover and spontaneous f lora cover crops, burying or framework, subterranean clover (Trifolium subterraneum leaving their dead mulches on the soil surface, to a L.), a self-pollinated annual legume with remarkable conventional management on a Mediterranean apricot geocarpism, native to the Mediterranean Basin and orchard by monitoring the changes of SOM, macro- widely diffused throughout the world in regions with and microelements, and weed seedbank over a 4-year Mediterranean-type climates (Australia, New Zealand, period (Fig. 1). The Americas and South Africa), is considered an eligible cover crop, owing to its self-reseed and N-fixation ability, rapid growth, and weed suppressive ability (Enache and Ilnicki 1990; Restuccia et al. 2020; Scavo et al. 2020). Subterranean clover has also proved to adapt well under organic farming and high tree density orchards thanks to its sciaphilic habitus (Mauro et al. 2011; Mauromicale et  al. 2010). However, the magnitude of cover crop effects on soil quality closely depends on soil properties, management practices, climatic conditions, etc., thus making it important to locally evaluate these potential impacts over a medium-long-term period (Sharma et al. 2018a, b). Cover cropping effects on soil properties under Mediterranean semiarid conditions have already been investigated (Moreno et  al. 2009; Ramos et  al. 2020), but the influence of T. subterraneum cover cropping in Mediterranean apricot (Prunus armeniaca L.) orchards requires specific attention. In recent researches conducted in Sicily (southern Italy), important improvements have been observed in the weed control and nutritional status of soil in an apricot orchard managed with subterranean clover compared to spontaneous flora cover cropping and conventional soil management (disc ploughing and shallow chopping for weed control) (Restuccia et al. 2020; Scavo et al. 2020). Overall, the incorporation of T. subterraneum dead mulches into Fig. 1 a View of Trifolium subterraneum L. cover cropping in cen- the soil significantly decreased the weed seedbank size tral Sicily (Italy). Detail of subterranean clover (b). Photographs by and the weed aboveground biomass compared to other S. Fontanazza. 1 3 Trifolium subterraneum cover cropping enhances soil fertility and weed seedbank dynamics… Page 3 of 16 70 (119 mm), February 2018 (109 mm), and October 2018 2 Materials and methods (189 mm). Air temperatures were always optimal for the establishment and growth of T. subterraneum. Indeed, 2.1 Site and weather description minima temperatures fell below 3 °C only in January of season IV, while the mean maxima were 26 °C at emer- The experiment was carried out over four growing sea- gence stage (October) and 21.5  °C at flowering stage sons (I, II, III, and IV) from 2015/2016 to 2018/2019 in (April) of subterranean clover. central Sicily [37°13′ N., 14°05′ E., 290 m a.s.l.], south- ern Italy. The area has a semiarid Mediterranean climate 2.2 Experimental design and cover crop with mild winters, mean annual rainfall of ~ 500  mm, management mostly concentrated in the fall-winter period, and sum- mer droughts. According to the USDA soil taxonomy (Soil A completely randomized block design was adopted, involv- Survey Staff 1999), the soil is classified as Regosoil, and, ing 5 treatments and 4 replicates. Cover cropping treatments at the beginning of the experiment, in the 0–40-cm layer, included T. subterraneum with the leaving of dead mulch on it showed a clayey texture (25.7% sand, 30.6% silt, 43.7% the soil surface (TCC-S), T. subterraneum with the burying clay), a 25% of gravel, and had the following mean char- of dead mulch into the soil (TCC-B), spontaneous flora with acteristics: 1.9% organic matter; 6.1% total CaC O ; 5.6% the leaving of dead mulch on the soil surface (SCC-S), and active limestone; pH 8.0; electrical conductivity 1.3 dS ‒1 −1 spontaneous flora with the burying of dead mulch into the m ; total N 1.1‰; available P O 13 mg  kg ; exchange- 2 5 −1 soil (SCC-B). The control was a conventional management able KO 422 mg  kg ; cation exchange capacity (CEC) ‒1 (CM) ordinarily adopted for apricot orchards in the area 49.6 meq 100  g ; exchangeable bases (Ca, Mg, Na, and ‒1 where the experiment took place, which consists of ‒ 15 cm K) 42.2, 5.4, 1.1, and 0.9 mg 100  g , respectively; Fe −1 −1 −1 winter disc ploughing (late September) and three shallow 12.47 mg  kg ; Mn 3.75 mg  kg ; Cu 2.36 mg  kg ; and −1 choppings (on November, February, and May, respectively) Zn 1.34 mg  kg . The typical crops of the zone are cereals, for weed control. The experiment was carried out in a large legumes, olive, grape vineyard, carob, apricot, and peach area of about 1 ha within a Mediterranean orchard. In par- orchards. In the last 3  years  before the apricot orchard ticular, the area of each plot was 10 × 8.7 m (87 m ), for a planting, the experimental field has been cultivated with net plot size of 348 m , which intercepted 6 trees per treat- wheat, chickpea, and a fallow year. ment, and 1740 m in total (20 plots). Furthermore, in order Climatic data including maxima and minima monthly to avoid interference during the soil sampling, a buffer area temperatures and mean monthly rainfall were recorded of 2 m in width was considered around each experimental from November 2015 to July 2019 by a meteorological plot (Fig. 3). station (Mod. Multirecorder 2.40; ETG, Firenze, Italy) Apricot (cv. Wonder, together with ‘Pinkcot®’ and ‘Big located ~ 15 m from the experimental area (Fig. 2). About Red®’ as pollinators) planting was conducted in January 23% of the total 4-year rainfall fell in January 2016 2012 in a 3.5 × 4.5 m layout. At the start of season I and Fig. 2 Maxima and minima monthly temperatures and monthly total rainfall during the growing seasons. 1 3 70 Page 4 of 16 A. Scavo et al. Fig. 3 (a) Schematic representation of the field experimental layout folium subterraneum cover cropping leaving dead mulch on the soil according to a randomized complete block design, and (b) detail of surface; TCC-B, T. subterraneum cover cropping burying dead mulch the experimental unit with scheme of soil sampling. Each soil sample in the soil; SCC-S, spontaneous flora cover cropping leaving dead is the sum of 5 subsamples per plot collected along the diagonals of mulch on the soil surface; SCC-B, spontaneous flora cover cropping the central part. CM, conventional apricot management; TCC-S, Tri- burying dead mulch in the soil. during the experiment, apricot trees were 3.5–4.0-m high. Self-compensating drip irrigation replacing 100% of the Subterranean clover seeding was done in November 2015 by daily evapotranspiration and twice chopping per year for −2 hand at 2–3-cm depth using 2000 germinable seeds m of weed control (in early spring and mid-summer) were carried the cultivar ‛Seaton Park’, a common Australian early-mid out. Incorporation of dead mulches in TCC-B and SCC-B season genotype presenting an autumn–winter-spring cycle was carried out in September of each season at − 15 cm soil and vernalization requirement for flowering. The length of depth by disc ploughing. Insect and fungal control were done the biological cycle (i.e., from the beginning of plant emer- by low-dose applications of captano, tebuconazole, propi- gence until all the plants within a plot had completely dried conazole, and lambda-cyhalothrin only when required. up) was 200 days in season I, 220 days in season II, 250 in season III, and 240 in season IV (Restuccia et al. 2020). Its 2.3 Soil sampling and analyses −2 −2 total biomass was 44 g DW m in season I, 283 g DW m −2 −2 in season II, 239 g DW m in season III, and 159 g DW m Before each soil sampling, a field scouting was conducted to in season IV, thus confirming a good establishment of the locate the sampling units by excluding the non-representa- cover crop after the self-reseeding (Restuccia et al. 2020). tive areas and the outer 3 m for each plot. Soil sampling was In all plots, in October, a shallow hoeing at 10-cm deep carried out using a core sampler by collecting 5 randomly followed by rigid tine harrowing was performed, together distributed subsamples (each of 0.75 dm ) along the diago- −1 with a fertilization program consisting of 40 kg  ha P O , nals of the central part per plot, which were pooled to form 2 5 −1 −1 14 kg  ha  N, and 10 kg  ha K O. Then, in each season, a composite sample. The following soil depths and times the following fertilizers were provided through fertirriga- were adopted: tion from the developing apricot fruit phase until harvesting: −1 300 kg  ha Ponimag® (11% N, 41% K O and 3% MgO), • Two depths (0‒20 and 21‒40 cm) in March of season II −1 −1 50 kg  ha simple perphosphate (P O), 100 kg  ha liq- and season IV to determine the contents of soil organic 2 5 −1 uid Ca, and 50 kg  ha Hergoton Plus® (8% total organic matter and macro- and microelements N, 26% biologic C, and 44.45% total organic matter). 1 3 Trifolium subterraneum cover cropping enhances soil fertility and weed seedbank dynamics… Page 5 of 16 70 −2 From top soil (0‒15 cm) twice per season [March (T ) m ), relative density (RD), relative frequency (RF), and the and June (T )] to monitor the dynamics of the soil N relative abundance index (RAI): + − mineral fractions (NH and NO ), in accordance with �∑ � 4 3 Scavo et al. (2020) RD(%) = × 100 (1) From top soil (0‒15 cm) twice per season [April (T ) and September (T )] for the weed seedbank � � RF(%) = ∑ × 100 (2) A total of 400 soil cores (20 plots × 2 seasons × 2 sam- pling depths or 2 sampling times × 5 subsamples) was taken. Samples were collected in season II and IV to RD + RF obtain a mid-term and a final evaluation. RAI(%) = (3) Fresh soil samples were initially air-dried at room tem- perature for about 2 weeks, crushed, and sieved through where ∑Y = sum of the number of seeds for a weed a 2-mm mesh sieve before laboratory analyses. The species; S = total number of weed seeds within the plot; exchangeable cations (K, Ca, Mg) were determined fol- F = absolute frequency of a species (i.e., number of sam- lowing the ISO 13,536 (1995) procedure while the avail- pling units in which the species i occurred) i; and ∑F = sum able P O according to the ISO 11,263 (1994) method. The of the absolute frequencies of all species. 2 5 soil organic carbon and total N analyses were conducted Richness and evenness were used to describe the within- according to the ISO 14,235 (1998) and ISO 11,261 (1995) community species diversity (α-diversity) (Travlos et al. methods, respectively. The N mineral fractions were 2018). Species richness was calculated as the total number determined in the extractable 10 g soil samples treated of weed seeds counted in the five soil cores for each plot with 50  ml of 0.5  M K SO into 250-ml bottles. After (Moonen and Bàrberi 2004), while evenness was estimated 2 4 1 h shake, filtration with glass fiber Whatman GF/A and by computing the Shannon–Wiener (H′) and the Pielous (J) 0.45-μm millipore filter, and centrifugation for 15  min diversity indices, as suggested by Adeux et al. (2019): + − at 3000 rpm, the ammoniacal (NH ) and nitric (N O ) 4 3 H = −p lnp (4) N-levels of the extracts were colorimetrically measured i i by diffusion (Crooke and Simpson 1971). The bioavail- able fraction of the main micronutrients (Cu, Fe, Mn, Zn) � J = was measured after extraction with buffered diethylen- (5) max etriaminepentaacetic acid (DTPA) solution following the th Lindsay and Norvell (1978) method. where p = proportional abundance of the i species; and H' = logarithm of species richness. max 2.4 Analysis of the soil weed seedbank 2.5 Statistical analysis The analysis of the soil weed seedbank followed Scavo et al. For each soil variable, analysis of variance (ANOVA) (2019a). Firstly, 20 soil samples from each treatment (5 subsam- was used to evaluate the effect of studied factors and their ples × 4 replicates) were mixed giving a final volume of 15 dm , interaction. In particular, a generalized linear mixed model and then they were freed from inert components such as stones, (GLMM) was applied considering “cover cropping” and pebbles, and dead debris. Secondly, seeds were separated from “sampling depth” as fixed factors and “season” as random soil by using a metal tube (Karcher, K 3500 model, Winnenden, factor (Gomez and Gomez 1984). At each season, one- Germany) working at 20–120 bar adjustable washer pressure way ANOVAs were performed (one with “cover cropping” and equipped with a removable cap consisting of a steel mesh of and another with “sampling depth”). Moreover, in order to 250 μm. Thirdly, weed counts and identification were performed observe the overall effect of cover cropping on each soil in Petri dishes after 24 h air-drying with a MS5 Leica stereomicro- variable under study, one-way ANOVAs was carried out scope (Leica Microsystems, Wetzlar, Germany). Seed identifica- by pooling data over seasons and sampling depths. The tion and grouping followed Conti et al. (2005), while the seedbank ANOVA basic assumptions of homoscedasticity and nor- −2 size was calculated as the number of seeds m for each plot. mality were checked by graphically inspecting the residuals, In accordance with Nkoa et al. (2015) and Scavo et al. which showed no significant deviations, together with the (2020), the soil weed seedbank was analyzed taking account Bartlett’s test. Post hoc comparison of means was performed both species abundance and diversity. Abundance was meas- through the Fisher’s protected least significant difference ured considering the seedbank size (number of weed seeds 1 3 70 Page 6 of 16 A. Scavo et al. (LSD) test at α = 0.05 by using the statistical package CoS- seasons, was not influenced by factors under study (data not tat® 6.003 (CoHort Software, Monterey, CA, USA). shown). Ca was the prevailing exchangeable cation (~ 88% Soil seedbank data, species richness, RAI data, and of CEC), followed in decreasing order by Mg (~ 9%), Na diversity indices were subjected to two-way ANOVAs (~ 2%), and K (~ 1%). No significant differences among treat- (“cover cropping” × “sampling time”) for each season. ments were observed for exchangeable Ca and Mg concen- Prior to ANOVA, data about seedbank size needed log trations, while exchangeable Na and K were influenced by transformation, whereas an arcsine-square root transfor- cover cropping type. mation was used for RAI of major weeds. Moreover, H′ In season II, the GLMM showed how cover cropping had and J data were square root and logit-transformed, respec- high significance on all the soil variables, except for total N tively. Data on species richness did not need any transfor- and exchangeable Mg (Table 1). In particular, regardless of mation before ANOVA. Multivariate statistics was con- sampling depth, the highest levels of SOM were detected ducted to analyze the weed species composition. In each in SCC-S and TCC-B (+ 13% and + 10% than CM, respec- season, multivariate analysis of variance (MANOVA) on tively), the highest concentrations of assimilable P O in 2 5 log (x + 1)-transformed data was carried out to evaluate the SCC-S (+ 15%, even if not statistically significant) and the effect on variance of cover cropping, sampling time, and highest ones of exchangeable K O in TCC-B (+ 15%), of their interaction. Significance levels in MANOVA were exchangeable Ca in SCC-B and SCC-S (+ 7% and + 5%, determined according to Wilks’ criterion. The effects of respectively), of exchangeable Na in SCC-B and SCC-S MANOVA were visualized by applying a principal com- (+ 51% and + 48%, respectively), and of exchangeable K ponent analysis (PCA) on 8 major weeds, considering the in TCC-B (+ 17%) (Table  1). Concerning soil microele- means for each “cover cropping × sampling time” com- ments (Table 2), SCC-S promoted the highest concentra- −1 −1 bination. PCA was performed on the covariance matrix tion of Mn (7.97  mg  kg ), Zn (0.22  mg  kg ), and Cu −1 of log (x + 1)-transformed density data, and the results (3.18 mg  kg ), while Fe-level was the highest in SCC-B −1 were displayed on “distance” biplots (Legendre and Leg-(9.55 mg  kg ). Higher amounts of soil microelements than endre 2012). The computer package Minitab® version 16 CM were found also in TCC-B, whereas TCC-S caused a (Minitab Inc., State College, PA, USA) was used to per- decrease. No significant differences were observed regard- form both MANOVA and PCA. ing soil layers, except for exchangeable K O and exchangea- ble Na. The two-way interaction “cover cropping × sampling depth” was significant only for SOM, exchangeable K O, exchangeable Na, exchangeable K, and Fe (Tables 1 and 2). 3 Results In this view, except for exchangeable Na and Fe, TCC-B and SCC-S performed better in the 0‒20-cm soil layer than in 3.1 Cover‑cropping effects on soil organic matter, the 21‒40 cm one. macro‑ and microelements The cumulated cover cropping effects of season IV on the above-mentioned soil variables were more marked than ANOVA demonstrated a significant effect of cover cropping those of season II. Indeed, GLMM indicated that TCC-B on SOM and soil nutrient content, even if with seasonal- ensured a significant increase, compared to CM, in the dependent results. From the one-way ANOVA applied on amount of SOM (+ 19%), total N (+ 17%), exchangeable ʻcover croppingʼ and pooling over seasons and sampling K O (+ 13%), exchangeable Na (+ 30%), exchangeable K depths (Fig. 4), emerged that TCC-B significantly increased (+ 27%), Fe (+ 12%), Mn (+ 46%), Zn (+ 52%), and Cu the amount of SOM by 15%, total N by 7%, exchangeable (+ 36%) (Tables 1 and 2). Spontaneous flora cover crop- K O by 14%, exchangeable Na by 32%, exchangeable K by ping (SCC-B and SCC-S) provided the highest content of −1 16%, Fe by 15%, Mn by 28%, Zn by 36% and Cu by 24%, assimilable P O (58.2 and 42.0 mg  kg , respectively) 2 5 −1 as compared to those of CM. The highest levels of assimila- and Mn (8.1 mg  kg ), whereas exchangeable Ca and Mg −1 ble P O were found in SCC-B (39.8 mg  kg ) and SCC-S did not differ significantly, as observed in season II. More- 2 5 −1 (32.7 mg  kg ). Also, SCC-B and SCC-S showed signifi- over, in this season, the effect of sampling depth was gen- cant increments in SOM and soil macro- and microelement erally significant, except for exchangeable Ca, exchange- levels with respect to CM, while TCC-S was interestingly able Mg, exchangeable Na, Fe, and Mn. The 0 − 20-cm soil associated with lower amounts of total N (‒ 4%), assimilable layer showed higher amounts of SOM (+ 19.5%), total N P O (‒ 26%), exchangeable K O (‒ 5%), exchangeable K (+ 11.4%), assimilable P O (+ 139%), exchangeable K O 2 5 2 2 5 2 (‒ 3%), and Cu (‒ 17%), as compared to CM (Fig. 4). CEC, (+ 34%), exchangeable K (+ 35%), Zn (+ 46%), and Cu ‒1 on average equal to ~ 50 meq 100  g throughout the four (+ 53%) than the 21 − 40 cm one. 1 3 Trifolium subterraneum cover cropping enhances soil fertility and weed seedbank dynamics… Page 7 of 16 70 Fig. 4 Effect of cover-cropping, pooling over seasons, and soil sam- TCC-S, Trifolium subterraneum cover cropping leaving dead mulch pling depths, on organic matter and macro- and micro-elements. Bars on the soil surface; TCC-B, T. subterraneum cover cropping bury- are standard error of the mean (SEM, n = 8). Different letters indicate ing dead mulch in the soil; SCC-S, spontaneous flora cover cropping statistical significance by applying a one-way analysis of variance leaving dead mulch on the soil surface; SCC-B, spontaneous flora with LSD test at P ≤ 0.05. CM, conventional apricot management; cover cropping burying dead mulch in the soil. TCC-S better performing in season II and TCC-B in season 3.2 Cover‑cropping effects on soil mineral nitrogen IV. Averaged over sampling times (March and June), in season dynamics II, TCC-B determined an increase of 148% in soil NH and 244% in soil NO , compared to CM. Such positive effect The GLMM indicated that the effect of cover cropping was became stronger in season IV, especially for NH , with highly significant (P ≤ 0.001) for both mineral N forms and a + 194% than CM and + 133% than the same treatment in sea- in both seasons, while the sampling time was significant only son II. Also, the soil from SCC-S and SCC-B plots contained in season IV (Table 2). Subterranean clover cover cropping more mineral N than CM (+ 49 and + 76%, respectively). The always showed the highest levels of total soil mineral N, with 1 3 70 Page 8 of 16 A. Scavo et al. Table 1 Effect of cover cropping (CC) and soil sampling depth (SD) subterraneum cover cropping leaving dead mulch on the soil surface, on organic matter (SOM), macroelements, and exchangeable bases TCC-B T. subterraneum cover cropping burying dead mulch in the with analysis of variance (ANOVA, P values). Values are means soil, SCC-S spontaneous flora cover cropping leaving dead mulch on (n = 2) with standard deviation (in brackets). Values within a column the soil surface, SCC-B spontaneous flora cover cropping burying followed by the same letters are not significantly different at P ≤ 0.05 dead mulch in the soil, season II 2016/2017, season IV 2018/2019. (LSD test). CM conventional apricot management, TCC-S Trifolium ‒1 ‒1 ‒1 ‒1 Treatment SOM (g kg ) Total N (g kg )Assimilable P O (mg kg )Exchangeable K O (mg kg ) 2 5 2 Season II Season IV Season II Season IV Season II Season IV Season II Season IV CC CM 18.38 (0.48) 18.63 (1.49) 1.32 (0.21)a 1.30 (0.08) 20.4 (5.6)a 18.0 (9.9)c 368.0 (46.1) 403.9 (28.1)bc bc bc bc b TCC-S 17.75 (0.96)c 19.50 (2.08) 1.27 (0.10)a 1.25 (0.13)c 14.5 (1.7)b 14.0 (5.1)c 329.5 (11.4) 401.5 (37.0)c b c TCC-B 20.25 (1.50) 22.25 (5.62)a 1.27 (0.05)a 1.52 (0.28)a 21.7 (1.7)a 18.5 (9.8)b 421.7 (53.0) 456.5 (95.5)a ab a SCC-S 20.75 (2.75)a 17.0 (1.41)c 1.17 (0.28)a 1.35 (0.06) 23.5 (2.9)a 42.0 (15.8)a 382.7 (40.5) 441.5 (72.1)ab bc b SCC-B 19.75 (2.50) 19.25 (0.96) 1.30 (0.08)a 1.45 (0.10) 21.5 (2.5)a 58.2 (23.8)a 370.5 (31.4) 420.2 (68.4) abc b ab b abc SD 0‒20 cm 19.66 (2.08)a 21.05 (3.42)a 1.32 (0.15)a 1.45 (0.18)a 20.5 (4.9)a 42.5 (25.4)a 392.3 (53.1) 486.7 (57.8)a 21‒40 cm 19.10 (2.02)a 17.60 (1.35) 1.22 (0.15)b 1.30 (0.12)b 20.2 (3.8)a 17.8 (8.5)b 356.7 (31.5) 362.8 (28.2)b b b ANOVA CC 0.0345 0.0024 0.6188 0.0207 0.0474 0.0004 0.0028 0.0343 SD 0.3556 < 0.001 0.1358 0.0084 0.8815 0.0003 0.0055 < 0.001 CC × SD 0.0151 0.0049 0.1376 0.1812 0.7902 0.0291 0.0118 0.0017 Treatment Exchangeable Ca Exchangeable Mg Exchangeable Na Exchangeable K ‒1 ‒1 ‒1 ‒1 (meq 100  g )(meq 100  g )(meq 100  g )(meq 100  g ) Season II Season IV Season II Season IV Season II Season IV Season II Season IV CC CM 41.7 (0.7)ab 46.3 (4.5)a 4.55 (0.56)a 6.25 (0.37) 0.83 (0.04)d 1.80 (0.49) 0.75 (0.03) 0.85 (0.02)b ab bc bc TCC-S 40.2 (1.0)b 43.4 (2.8)a 4.32 (0.10)a 6.15 (0.48) 0.93 (0.05)c 2.90 (0.64)a 0.70 (0.06)c 0.85 (0.02)b ab TCC-B 40.5 (4.0)b 42.6 (1.8)a 4.32 (0.22)a 6.40 (0.08)a 1.13 (0.09)b 2.35 (0.50) 0.88 (0.04)a 0.98 (0.11)a ab SCC-S 44.0 (1.7)a 43.6 (2.9)a 4.20 (0.18)a 6.40 (0.71)a 1.23 (0.04)a 1.80 (0.28) 0.78 (0.04)b 0.93 (0.04)ab bc SCC-B 44.5 (0.6)a 46.9 (2.8)a 4.25 (0.17)a 5.60 (0.18)b 1.25 (0.11)a 1.45 (0.49)c 0.80 (0.07)b 0.88 (0.04)b SD 0‒20 cm 42.3 (1.6)a 44.8 (3.1)a 4.39 (0.38)a 6.06 (0.46)a 1.03 (0.05)b 1.88 (0.42)a 0.80 (0.03)a 1.03 (0.6)a 21‒40 cm 42.0 (3.4)a 43.3 (3.0)a 4.28 (0.17)a 6.26 (0.51)a 1.11 (0.12)a 2.24 (0.54)a 0.76 (0.05)a 0.76 (0.03)b ANOVA CC 0.0363 0.6250 0.5231 0.1698 < 0.001 0.0230 0.0031 0.0548 SD 0.7466 0.3262 0.4535 0.3607 0.0085 0.1605 0.0734 < 0.001 CC × SD 0.3387 0.6692 0.4669 0.8113 0.0034 0.4499 0.0226 0.0015 two-way interaction and the sampling time were significant at members (32%), annual (77%), and therophytes (77%) (Table 3). + ‒ P ≤ 0.001 in season IV, with higher values of NH , NO and Sonchus sp., Anagallis arvensis L., and Phalaris paradoxa L. 4 3 their sum in the June sampling than the March one. had the highest relative abundance values. Centaurea napifolia L. had the higher relative abundance values in season IV than 3.3 Cover‑cropping effects on the weed soil in season II, while Portulaca oleracea L. showed an opposite seedbank trend. No consistent trends were observed for the other weeds. Nevertheless, the effect of cover cropping on species rich- Over the two seasons under study, the 0 − 15-cm soil seed- ness was inconsistent, and, for this reason, these data were not bank was composed of 22 taxa in total, mainly Asteraceae shown. Among the 22 species and genera recorded, only a few 1 3 Trifolium subterraneum cover cropping enhances soil fertility and weed seedbank dynamics… Page 9 of 16 70 Table 2 Effect of cover cropping (CC) and soil sampling depth (SD) (LSD test). CM conventional apricot management, TCC-S Trifolium + ‒ on major microelements and ammoniacal (NH ) and nitric (NO ) subterraneum cover cropping leaving dead mulch on the soil surface, 4 3 nitrogen with analysis of variance (ANOVA, P values). Values are TCC-B T. subterraneum cover cropping burying dead mulch in the means (n = 2 for soil microelements and n = 3 for mineral nitrogen) soil, SCC-S spontaneous flora cover cropping leaving dead mulch on with standard deviation (in brackets). Values within a column fol- the soil surface, SCC-B spontaneous flora cover cropping burying lowed by the same letters are not significantly different at P ≤ 0.05 dead mulch in the soil, season II 2016/2017, season IV 2018/2019. ‒1 ‒1 ‒1 ‒1 Treatment Fe (mg kg ) Mn (mg kg ) Zn (mg kg ) Cu (mg kg ) Season II Season IV Season II Season IV Season II Season IV Season II Season IV CC CM 7.75 (0.56)b 14.42 (3.44)b 5.57 (1.52)bc 5.46 (2.24)b 0.19 (0.02)ab 0.25 (0.07)b 2.79 (0.49)ab 2.68 (0.62)b TCC-S 8.40 (0.05)b 15.18 (1.39)b 4.41 (0.88)c 7.85 (2.35)a 0.13 (0.03)b 0.35 (0.11)ab 1.76 (0.05)c 2.77 (0.33)b TCC-B 9.33 (0.31)a 16.16 (3.10)a 6.14 (0.75)bc 7.97 (3.09)a 0.21 (0.03)a 0.38 (0.18)a 3.12 (0.22)ab 3.65 (1.59)a SCC-S 9.29 (1.02)a 14.68 (1.89)b 7.97 (0.95)a 6.17 (2.55)ab 0.22 (0.03)a 0.29 (0.09)ab 3.18 (0.29)a 3.66 (1.20)a SCC-B 9.55 (1.27)a 14.35 (0.92)b 6.16 (0.28)b 8.10 (0.91)a 0.19 (0.02)ab 0.34 (0.10)ab 2.60 (0.28)b 3.58 (0.78)a SD 0‒20 cm 8.76 (1.06)a 14.92 (2.15)a 5.83 (2.12)a 7.90 (2.24)a 0.19 (0.05)ab 0.38 (0.13)a 2.66 (0.59)a 3.95 (0.98)a 21‒40 cm 8.97 (0.96)a 15.00 (2.37)a 6.28 (0.81)a 6.30 (2.31)a 0.18 (0.03)ab 0.26 (0.05)b 2.72 (0.62)a 2.58 (0.35)b ANOVA CC 0.0024 0.0010 0.0142 0.0127 0.0006 0.0427 0.0012 0.0316 SD 0.3930 0.7060 0.3866 0.1129 0.2551 0.0046 0.7092 < 0.001 CC × SD 0.0090 0.9254 0.2651 0.2432 0.0369 0.0223 0.9091 0.0275 + ‒ + ‒ Treatment NH (ppm) NO (ppm) NH + NO (ppm) 4 3 4 3 Season II Season IV Season II Season IV Season II Season IV CC CM 2.64 (0.86)d 5.17 (0.33)d 5.22 (1.23)d 5.03 (0.43)e 7.86 (1.66)d 10.20 (0.67)e TCC-S 7.61 (0.94)a 15.66 (0.46)a 18.51 (1.12)a 18.73 (0.45)b 26.12 (1.21)a 34.39 (0.56)b TCC-B 6.54 (1.09)b 15.22 (0.53)a 17.98 (1.14)a 20.54 (0.54)a 24.52 (1.25)a 35.76 (0.90)a SCC-S 3.91 (0.31)c 5.87 (0.55)c 11.38 (1.60)b 9.31 (0.52)d 15.29 (1.84)b 15.17 (0.54)d SCC-B 3.71 (0.34)cd 7.24 (0.31)b 9.38 (1.54)c 10.68 (0.59)c 13.10 (1.67)c 17.91 (0.89)c SD March 4.48 (0.68)b 7.81 (0.41)b 12.89 (1.26)a 12.04 (0.53)b 17.37 (1.64)a 19.85 (0.79)b June 5.33 (0.81)a 11.85 (0.46)a 12.10 (1.39)a 13.67 (0.48)a 17.43 (1.42)a 25.52 (0.63)a ANOVA CC < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 ST 0.0174 < 0.001 0.1485 < 0.001 0.9100 < 0.001 CC × ST 0.0854 < 0.001 0.1246 < 0.001 0.0703 < 0.001 evidenced high enough density to be included in the multivariate SCC-B > TCC-S > TCC-B (Table 4). Regarding sampling analysis for species composition. In particular, only for 8 taxa time, in both seasons, the seedbank size was significantly −2 was an RD > 4% (i.e., major weeds) found: Amaranthus retro- higher in April than in September (3380 vs. 1020 seeds m −2 flexus L., A. arvensis, C. napifolia, P. paradoxa, P. oleracea, in season II and 2920 vs. 1655 seeds m in season IV). Sinapis arvensis L., Sonchus sp., and Veronica cymbalaria Bod- Significant effects were also observed in terms of seed- ard, altogether accounting for 91.2% of the whole soil seedbank bank diversity and evenness (Table  4). The two studied throughout the two seasons. However, the ANOVA computed biodiversity indices were differently affected depending on on RAI data of major weeds highlighted no significant effects the season. In season II, TCC-B evidenced the highest H either for cover cropping or for sampling time (data not shown). (1.43) and J (0.89), indicating a high weed diversity and no Abundance was also analyzed by calculating the seed- dominant species. In this season, both H and J were greater bank size (Table  4). The interactive effect “cover crop- in September than in April (1.53 vs. 0.93 for H and 0.91 vs. ping × sampling time” two-way ANOVA was significant in 0.64 for J). In season IV, the two-way interaction was sig- season II (P ≤ 0.001) and season IV (P ≤ 0.05), with both nificant only for H (P ≤ 0.001); CM highlighted the highest factors showing significance at P ≤ 0.001. Concerning the H (1.8) and did not differ significantly from TCC-B for J effect of cover cropping, the lowest seedbank size was (0.93 vs. 0.87, respectively). The lowest H and J were found −2 detected in TCC-B plots (1469 seeds m on the average of in SCC-S (1.04 and 0.56) and SCC-B (1.33 and 0.7) plots. seasons), with a reduction of 55.4% in season II and 57.4% Contrariwise to season II, H was higher in April than in in season IV, as compared to CM. Overall, in both seasons, September (1.54 vs. 1.42), whereas the sampling time was the general trend in decreasing order was CM > SCC-S > not consistent for J. 1 3 70 Page 10 of 16 A. Scavo et al. 1 3 Table 3 Binomial name, botanical family, life cycle, biological group (BG), mean relative abundance values, and mean relative densities (RD) of the weed species under 5 cropping systems and 2 seasons. CM conventional apricot management, TCC-S Trifolium subterraneum cover cropping leaving dead mulch on the soil surface, TCC-B T. subterraneum cover cropping burying dead mulch in the soil, SCC-S spontaneous flora cover cropping leaving dead mulch on the soil surface, SCC-B spontaneous flora cover cropping burying dead mulch in the soil, season II 2016/2017, a b season IV 2018/2019. Biological groups: T therophytes, H hemicryptophytes. Averaged over all treatments. RAI data were averaged over sampling times. a b Binomial name Botanical family Life cycle BG CM TCC-S TCC-B SCC-S SCC-B RD (%) II IV II IV II IV II IV II IV Amaranthus retroflexus L. Amaranthaceae Annual T − 0.13 − < 0.1 − < 0.1 0.1 < 0.1 0.12 < 0.1 4.1 Anagallis arvensis L. Primulaceae Annual T 0.1 0.13 0.15 < 0.1 0.32 < 0.1 0.21 < 0.1 < 0.1 0.23 12.7 Artemisia vulgaris L. Asteraceae Perennial H < 0.1 − − − − − < 0.1 − − − 0.9 Avena sp. Poaceae Annual T − < 0.1 − − − − − − − − 0.2 Centaurea napifolia L. Asteraceae Annual T − < 0.1 − 0.15 < 0.1 0.22 < 0.1 < 0.1 < 0.1 < 0.1 5.2 Euphorbia helioscopia L. Euphorbiaceae Annual T − − − < 0.1 − − − − − − 0.2 Fallopia convolvulus (L.) Á. Löve Polygonaceae Annual T − − − − − − − − 0.1 < 0.1 0.7 Fumaria sp. Fumariaceae Annual T < 0.1 < 0.1 − − − − < 0.1 < 0.1 − − 0.7 Galium aparine L. Rubiaceae Annual T − − − − < 0.1 − − − 0.13 − 2.2 Helminthotheca echioides (L.) Holub Asteraceae Annual T − − − < 0.1 − − − − − < 0.1 0.3 Lamium amplexicaule L. Lamiaceae Annual T − − − − − − < 0.1 − − − 0.1 Medicago polymorpha L. Fabaceae Annual T − − − − − < 0.1 − − − − 0.9 Phalaris paradoxa L. Poaceae Annual T < 0.1 0.1 0.22 < 0.1 0.28 − < 0.1 0.21 0.14 0.13 9.1 Polygonum aviculare L. Polygonaceae Annual T − − − − − − − < 0.1 − − 0.1 Portulaca oleracea L. Portulacaceae Annual T 0.18 < 0.1 0.18 < 0.1 0.11 < 0.1 0.12 − 0.14 < 0.1 7.3 Reichardia picroides (L.) Roth Asteraceae Perennial H − − − − − − − < 0.1 − < 0.1 0.3 Silene sp. Caryophyllaceae Perennial H − < 0.1 − < 0.1 − < 0.1 − − − − 0.8 Sinapis arvensis L. Brassicaceae Annual T − < 0.1 0.13 0.11 − < 0.1 0.15 < 0.1 0.3 < 0.1 8.7 Sonchus sp. Asteraceae Biennial H 0.27 0.22 0.41 0.35 0.25 0.24 0.43 0.49 0.34 0.38 40.0 Taraxacum sp. Asteraceae Perennial H − < 0.1 − < 0.1 − − − − − − 1.1 Veronica cymbalaria Bodard Plantaginaceae Annual T − < 0.1 < 0.1 < 0.1 < 0.1 0.19 − 0.1 − < 0.1 4.2 Xanthium sp. Asteraceae Annual T − < 0.1 − − − − − − − − 0.4 Trifolium subterraneum cover cropping enhances soil fertility and weed seedbank dynamics… Page 11 of 16 70 Table 4 Effect of cover cropping (CC) and soil sampling time (ST) TCC-S Trifolium subterraneum cover cropping leaving dead mulch on the weed seedbank size, Shannon diversity index (H), and Pielou’s on the soil surface, TCC-B T. subterraneum cover cropping burying evenness index (J) with analysis of variance (ANOVA, P values). Val- dead mulch in the soil, SCC-S spontaneous flora cover cropping leav - ues are means (n = 4) with standard deviation (in brackets). Values ing dead mulch on the soil surface, SCC-B spontaneous flora cover within a column followed by the same letters are not significantly dif- cropping burying dead mulch in the soil, season II 2016/2017, season ferent at P ≤ 0.05 (LSD test). CM conventional apricot management, IV 2018/2019. −2 Treatment Seedbank size (seeds m ) H J Season II Season IV Season II Season IV Season II Season IV CC CM 3250 (256)a 3488 (307)a 1.10 (0.37)b 1.80 (0.06)a 0.80 (0.06)ab 0.87 (0.03)ab TCC-S 1750 (202)c 1788 (380)cd 1.12 (0.46)b 1.56 (0.42)b 0.69 (0.28)ab 0.77 (0.10)bc TCC-B 1450 (281)d 1488 (409)d 1.43 (0.05)a 1.66 (0.21)b 0.89 (0.03)a 0.93 (0.01)a SCC-S 2325 (203)b 2425 (329)b 1.13 (0.71)b 1.04 (0.32)d 0.63 (0.40)b 0.56 (0.07)c SCC-B 2225 (164)b 2250 (349)bc 1.37 (0.54)a 1.33 (0.58)c 0.84 (0.19)ab 0.70 (0.27)bc ST April 3380 (358)a 2920 (364)a 0.93 (0.29)b 1.54 (0.43)a 0.64 (0.21)b 0.75 (0.18)a September 1020 (85)b 1655 (346)b 1.53 (0.16)a 1.42 (0.39)b 0.91 (0.05)a 0.78 (0.18)a ANOVA CC < 0.001 < 0.001 < 0.001 < 0.001 0.1156 0.0054 ST < 0.001 < 0.001 < 0.001 0.0062 < 0.001 0.7875 CC × ST < 0.001 0.0131 < 0.001 < 0.001 0.0949 0.0759 MANOVA indicated a strong significance of the two-way reducing the number of weed seeds in the soil, as compared interaction (P ≤ 0.001) in seasons II and IV on the compo- to the conventional management. Such results were strongly sition of the weed seedbank, with sampling time contribut- influenced by cover crop type and season, together with the ing more to variance than cover cropping in both seasons sampling depth for the soil variables and sampling time for (F = 3360 vs. 439.5 in season II and 6.6 vs. 3.7 in season IV). the weed seedbank. Such differences in the weed communities of the soil seed - bank were represented by two ordination biplots, one per sea- 4.1 Soil organic matter and soil macro‑ son, derived from PCA on major weeds (Fig. 5). In season II, and micro‑elements most weeds and treatments were discriminated along the PC1 (42.4% of variance). In particular, the weeds A. retroflexus and The role of cover cropping on soil physical and chemical S. arvensis, together with the treatments SCC-S, SCC-B, and properties has been investigated (Adeli et al. 2020; Mauro CM at the first sampling time (T1), were negatively correlated et al. 2015; Nascente and Stone 2018; Ramos et al. 2020; with PC1 (left side of the biplot), while all the other weeds Wulanningtyas et al. 2021). In the present study, the best and treatments were discriminated on the right side. CM-T1 results were obtained by adopting T. subterraneum with dead was associated with S. arvensis, SCC-B-T1 and SCC-S-T1 mulches incorporated into the soil (TCC-B). The intensity of showed no association with any weeds, and in T2, all treat- these increases was much higher in season IV than II, likely ments showed a lower seedbank size, except for TCC-B-T2 due to a cumulative effect, although subterranean clover bio- −2 and CM-T2. In season IV, the discriminations were clearer. As mass showed an opposite trend (283 g DW m in season II −2 observed in season II, the treatments at the second sampling and 159 g DW m in season IV). Therefore, in contrast with time (T2) showed a lower seedbank, as evidenced by the fact the work of Roldán et al. (2003), reporting higher values in that TCC-B-T2, TCC-S-T2, and CM-T2 were not infested by the soil physio-chemical properties on increasing the amount any weeds. SCC-S-T1 was highly infested with C. napifolia; of residue cover, here no relationship was detected between TCC-B-T1 with Sonchus sp.; TCC-S-T1 with P. oleracea; and soil variables and cover crop biomass. Moreover, higher the spontaneous flora cover cropping (SCC-B-T2, SCC-B-T1 amounts of SOM and soil chemical variables were generally and SCC-S-T2) with S. arvensis, P. paradoxa and A. arvensis. found in the 0 − 20-cm layer than in the deeper one, as com- All the weeds, except for C. napifolia and V. cymbalaria, were monly indicated in literature (Wang et al. 2008). Our results discriminated by the PC2 along the negative side of the axe. were corroborated by Wulanningtyas et  al. (2021), who reported that the combination of no tillage and rye (Secale cereale L.) cover crop was very effective in increasing the 4 Discussion levels of soil organic carbon, total N, available P, exchange- able bases (K, Ca, Mg, Na), cation exchange capacity, and The results obtained in this 4-year field research indicate that some soil physical and biological properties, mainly in the cover cropping had remarkable effects in enhancing the lev - 25 − 75-cm layer. Similarly, Adeli et al. (2020) indicated that els of SOM and soil macro- and microelements as well as in the integration of winter cover crops and soil amendments 1 3 70 Page 12 of 16 A. Scavo et al. atmosphere via photosynthesis of the cover crop (Moebius- Clune et al. 2016), and direct root exudation of C-containing compounds into the rhizosphere (Scavo et al. 2019b). In this regard, Marschner (1995) estimated that about 5 − 21% of all photosynthetically fixed carbon is released into the rhizosphere through root exudation. The increase in SOM 2+ on one side and the release of rhizodeposits (e.g., Ca , 2+ 3+ 3+ + Fe and Fe, Al, K , as well as mucilages and several organic compounds) from the cover crop on the other con- tribute to promote the formation of aggregates and, thus, to improve the soil structure, the cation exchange capacity, and the microbial activity (Scavo et al. 2019b). The stimula- tion of the soil enzymatic-complex activity and promotion of the soil microbial biomass are often reported in cover cropping field experiments (Adeli et al. 2020; Nunes et al. 2018). The incorporation of plant residues into the soil, in particular, increases soil N richness, soil temperature, and aeration, thus creating favorable conditions for microorgan- isms (Turmel et al. 2015), including plant-promoting bac- teria and arbuscular mycorrhizal fungi, which are of key importance in enhancing soil P availability and biological fixation. Overall, these effects may explain the increase in soil macro- and microelement levels. Furthermore, buried plant residues show a faster decomposition and mineraliza- tion rate than surface ones. In our previous research, TCC-B ensured a major increase in the amount of Nitrosomonas europaea and Azotobacter vinelandii, two of the most important bacteria involved in the soil N-cycle, in addition to an enhancement of the soil mineral N (Scavo et al. 2020). After another year of cover cropping, the NH and the total mineral N content were fur- ther increased respectively by 56% and 14% in March and by Fig. 5 Principal components analysis ordination biplot with the 8 34% and 12% in June, compared to season III. The increases most abundant weed species and genera. Red names and arrows highlight the discrimination of weeds along the principal compo- in mineral N were more marked than those detected for the nents. Season II, 2016/2017; season IV, 2018/2019; T , April; T , 1 2 total N, since the latter is largely composed of organic N, September; TCC-S, Trifolium subterraneum cover cropping leav- which is unavailable for plants and therefore of less impor- ing dead mulch on the soil surface; TCC-B, T. subterraneum cover tance for plant nutrition. The higher amounts of soil mineral cropping burying dead mulch in the soil; SCC-S, spontaneous flora cover cropping leaving dead mulch on the soil surface; SCC-B, N in the June sampling can be attributed to weather condi- spontaneous flora cover cropping burying dead mulch in the soil; tions, since the higher temperature and the lower soil mois- CM, conventional apricot management. AMAR, Amaranthus retro- ture of late spring are favorable for N mineralization. Given flexus; ANAAR, Anagallis arvensis; CENTNA, Centaurea napifolia; the low number of Fabaceae members detected in the soil PHAPA, Phalaris paradoxa; PORTOL, Portulaca oleracea; SINAR, Sinapis arvensis; SONCH, Sonchus sp.; VEROCY, Veronica cymba- weed seedbank, it is reasonable to assume that the higher laria. amount of N released into the soil in TCC-B and TCC-S than the other treatments was caused by the massively inclu- into no-till cropping systems improved the soil total car- sion of a N-fixing species like T. subterraneum. The higher bon and several soil physical, hydrological, and microbial soil N-content caused by T. subterraneum than spontaneous properties. Ramos et al. (2020) also found that oat-vetch flora cover cropping is in accordance with Kuo and Jellum cover crops enhanced soil quality (SOM and total N content, (2002), who indicated that as plant tissue N increased and phosphatase, and β-glucosidase activities) in semiarid envi- tissue C/N ratio decreased (typical characteristics of legumi- ronments compared to frequently tilled management. Here, nous species), the N mineralization rate increased. Similarly the significant improvement of SOM in TCC-B plots was to SOM and macro- and microelements, TCC-B performed probably caused by a multiple cascading effect of carbon better than TCC-S in enhancing the mineral N content, likely input from residue decomposition, carbon capture from the due to the faster decomposition rate of subterranean clover 1 3 Trifolium subterraneum cover cropping enhances soil fertility and weed seedbank dynamics… Page 13 of 16 70 residues into the soil. The work of Kuo and Jellum (2002) of available food resources from grain crops, which are nor- also states that the incorporation of cover crop residues mally harvested in the same period, may have promoted the shows higher soil organic C and NO than removing them. predation of weed seeds by birds and edaphic fauna. Under In a recent study, Lombardo et al. (2021) found that TCC-B rainfed conditions and low-input agricultural regime such as improved the nutritional status of the apricot by significantly that of our experiment, several studies have found signic fi ant increasing the content of macro- and micronutrients in both weed seed loss from predation when seeds remained on the leaves and fruits than spontaneous flora and CM, thus dem- soil surface, up to 60 − 70% (Buhler et al. 1997). In addi- onstrating how the beneficial effects on soil properties were tion, in the late spring and summer, the level of subterranean reflected in the plants. clover allelochemicals accumulated into the rhizosphere (~ ‒ 15 cm) reach the climax due to a cumulative effect and to 4.2 Weed soil seedbank the low rainfall which limits their leaching, thus increasing the fatal seed germination. Moreover, the soil seedbank here The effects of 4 years cover cropping on weed management detected was primarily composed by annual spring–summer were evaluated on the soil seedbank at a depth of 10 − 15 cm, therophytes needing a cold period over winter to broke seed where most weed seeds are commonly concentrated (Swan- dormancy. For such weeds, the dormancy is broken in late ton et al. 2000). In accordance with Scavo et al. (2020), a spring so that their seeds are less detected in autumn (Buh- total of 22 taxa was detected, mainly belonging to the Aster- ler et al. 1997). Interestingly, the long-term effect of cover aceae family, the most diffused weed botanical family in cropping was not limited only to weeds with the same ger- Mediterranean agroecosystems (Restuccia et al. 2019). Weed mination seasonality (i.e., autumn/winter germinating spe- communities were dominated by therophytes and annual cies), but subterranean clover had an effect independently spring–summer weeds such as Sonchus sp., A. retroflexus , from its phase. A similar result was found by Bàrberi and P. oleracea, P. paradoxa, C. napifolia, S. arvensis, and V. Mazzoncini (2001). Furthermore, in their work, the authors cymbalaria, which showed an RD > 4%. However, in agree- reported a not-significant reduction of the seedbank density ment with our previous study (Scavo et al. 2020) and other by using subterranean clover in a conventional tillage system researches (Swanton et al. 1999), cover cropping and sam- while becoming relevant (‒ 22%) if combined with no till- pling time did not have significant effects on RAI data, as no age. It is known that cover crops can be differently managed consistent effects were either observed on species richness. within the orchards by moving or incorporating the plant The lack of substantial changes in the number of weed spe- biomass in order to mitigate their competition with trees. cies among cover cropping treatments is a wide phenomenon A study conducted in an apricot orchard in Turkey using (Ngouajio et al. 2003; Swanton et al. 1999) that some authors Vicia villosa Roth, V. pannonica, V. pannonica + Triticale attribute to the “buffering” effect of the weed seedbank, basi- (70 + 30%), Phacelia tanacetifolia Benth. and Fagopyrum cally caused by weed seed longevity (Bàrberi and Lo Cascio esculentum Moench cover cropping evidenced that these spe- 2001). On the contrary, such effects are often evident on the cies effectively reduced weed richness and density (Tursum emerged weed flora, as found in our previous research (Res- et al. 2018). In addition, the management of these cover crops tuccia et al. 2020) where TCC-S reduced by 21% species through the residue incorporation into the soil enhanced the richness compared to CM. Concerning the seedbank size, weed suppression compared to herbicidal (glyphosate) or TCC-B proved to be the most efficient treatment in reduc- mechanical control (Tursun et al. 2018). The effects obtained ing the number of weed seeds in the soil, even after another here on the soil seedbank were reflected also on the real weed growing season with respect to our previous research, with flora, given that T. subterraneum significantly decreased the a mean reduction of ‒ 57.4% compared to CM, followed by aboveground weed biomass up to 86%, with a positive cor- spontaneous flora cover cropping. The phytotoxic effect of relation between weed growth suppression and cover crop subterranean clover was stronger in April than September, biomass (Restuccia et al. 2020). The higher phytotoxicity of also due to the higher seedbank size in spring. It is likely that subterranean clover cover cropping than spontaneous flora is the differences in the seedbank size between April and Sep- explained by physio-chemical processes. The physical inter- tember are due to (i) the high presence of annual spring–sum- ference is caused by its strong competitive capacity for water, mer weeds with high dormancy rate during autumn, (ii) seed nutrients, light, and space, while the secondary metabolites germination of annual weeds promoted by late summer-early (essentially phenolic compounds and polyphenols) released autumn rainfall, and (iii) species whose seeds massively into the soil via root exudation and decomposition from plant germinate with autumn rainfall are hard to detect in spring residues—especially when incorporated into the soil (TCC- (Buisson et al. 2018). Anyway, a higher seedbank size in B)—are involved in the chemical interference. spring than autumn is a common situation in Mediterranean The eventual presence of associations between treatments environments. Probably, the severe drought conditions typi- under study and weeds was analyzed by PCA on those spe- cal of the late spring and summer associated with the lack cies showing a high enough density (i.e., major weeds). 1 3 70 Page 14 of 16 A. Scavo et al. Data availability The datasets analyzed during the current study are Most variation in the species composition was explained by available from the corresponding author on reasonable request. sampling time, considering that several treatments showed no weed infestation in the autumn sampling, whereas during Declarations spring, the treatments were highly infested. As observed for the emerged weed flora (Restuccia et al. 2020), TCC-B-T2 Ethics approval Not applicable. did not show any particular association with weeds, and its soil seedbank evenness was very high (0.93), indicating the Consent to participate Not applicable. absence of dominant species. The lowest biodiversity was Consent for publication Not applicable. found in the spontaneous flora cover cropping treatments, which at the same time also showed the lowest evenness, Conflict of interest The authors declare no competing interests. thus confirming the association emerging from the multi- variate analysis. Overall, cover cropping did not determine Open Access This article is licensed under a Creative Commons Attri- relevant shifts in weed populations. Other studies have bution 4.0 International License, which permits use, sharing, adapta- shown no clear patterns in weed communities during cover tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, cropping experiments (Moonen and Bàrberi 2004; Shrestha provide a link to the Creative Commons licence, and indicate if changes et al. 2002; Swanton et al. 1999). Indeed, cover crop type, were made. The images or other third party material in this article are management practices, and several biotic and abiotic fac- included in the article's Creative Commons licence, unless indicated tors closely influence both species composition and weed otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not patterns in the soil seedbank. Moreover, it is necessary to permitted by statutory regulation or exceeds the permitted use, you will keep in mind also that the measured soil seedbank does not need to obtain permission directly from the copyright holder. To view a necessarily represent the weed species able to germinate and copy of this licence, visit http://cr eativ ecommons. or g/licen ses/ b y/4.0/ . growth. References 5 Conclusions Adeli A, Brooks JP, Read JJ, Feng G, Miles D, Shankle MW, Barks- dale N, Jenkins JN (2020) Management strategies on an upland This study highlights the benefits deriving from the use of soil for improving soil properties. Commun Soil Sci Plan subterranean clover cover cropping, especially when incor- 51:413–429. https:// doi. org/ 10. 1080/ 00103 624. 2019. 17094 90 porating its dead mulches into the soil, in Mediterranean Adeux G, Vieren E, Carlesi S, Bàrberi P, Munier-Jolain N, Cord- orchards. The cumulative T. subterraneum cover cropping eau S (2019) Mitigating crop yield losses through weed diversity. Nat Sustain 2:1018–1026. h t t p s : / / d o i . o r g / 1 0 . 1 0 3 8 / for four consecutive years led to a significant improvement s41893- 019- 0415-y in SOM, macro- and microelement levels, a decrease in Bàrberi P, Mazzoncini M (2001) Changes in weed community com- weed pressure, and an enhancement in weed biodiversity, position as influenced by cover crop and management system in which have also resulted in positive effects on the productive continuous corn. Weed Science 49(4):491–499. https:// doi. org/ 10. 1614/ 0043- 1745 performance of the apricot orchard. These principles can Bárberi P, Lo Cascio B (2001) Long-term tillage and crop rotation be applied to similar agroecosystems with a Mediterranean effects on weed seedbank size and composition. 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Agr Syst 134:6–16. https://doi. or g/10. 1016/j. agsy .2014. 05. 009 Tursun N, Işık D, Demir Z, Jabran K (2018) Use of living, mowed, and soil incorporated cover crops for weed control in apricot 1 3 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Agronomy for Sustainable Development Springer Journals

Trifolium subterraneum cover cropping enhances soil fertility and weed seedbank dynamics in a Mediterranean apricot orchard

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Abstract

The soils of Mediterranean semiarid environments are commonly characterized by low levels of organic matter and mineral elements, as well as severe weed infestations, which, taken together, cause an intensive use of auxiliary inputs (tillage, fertilizers, herbicides). Although cover crops are recognized to sustainably improve soil health, the impact of Trifolium subterraneum L. cover cropping needs specific attention. This research investigates for the first time the effects over 4 years of T. subterraneum and spontaneous flora cover crops, after either incorporating their dead mulches into the soil or leaving them on the soil surface, on soil organic matter (SOM), macroelements, mineral nitrogen, microelements, and weed seedbank dynamics as indicators of soil quality in an apricot orchard. Compared to a conventional management control, the T. subterraneum cover crop with the burying of dead mulch into the soil increased the amount of SOM (+ 15%), ammoniacal (+ 194%) and nitric (+ 308%) nitrogen, assimilable P O (+ 5%), exchangeable K O (+ 14%), exchangeable Na (+ 32%), exchangeable K (+ 16%), Fe (+ 15%), Mn 2 5 2 (+ 28%), Zn (+ 36%), and Cu (+ 24%), while it decreased the weed seedbank size (‒ 54%) and enhanced weed biodiversity. These findings suggest that T. subterraneum cover cropping may be an environment-friendly tool to enhance soil quality and limit auxiliary input supply in Mediterranean orchards. Keywords Cover cropping · Subterranean clover · Soil health · Soil organic matter · Soil macroelements · Soil microelements · Weed soil seedbank 1 Introduction * Umberto Anastasi The weaknesses of agricultural activity (i.e., release of agro- umberto.anastasi@unict.it chemicals, loss of water resources, generation of ammonia Aurelio Scavo from grazing (GAG) emission, soil degradation, and loss aurelio.scavo@unict.it of biodiversity) are attributed to the irrational or ineffi- Alessia Restuccia cient use of auxiliary inputs and to the excessive biological a.restuccia@unict.it homogeneity typical of intensive farming systems. There- Cristina Abbate fore, in agreement with the United Nations (UN) Sustain- cristina.abbate@unict.it able Development Goals (SDGs) and with the strategies of Sara Lombardo the European Commission (EC) Green Deal, the transition saralomb@unict.it of agricultural systems towards sustainability requires an Stefania Fontanazza agroecological and multifunctional approach in the manage- stefania.fontanazza@yahoo.it ment of agroecosystems (EC 2019; UN 2015). It is widely Gaetano Pandino recognized that polyculture supports the stability and lesser g.pandino@unict.it dependence on external inputs of agroecosystems by using Giovanni Mauromicale environmental resources (solar radiation, rainfall, N , CO , 2 2 g.mauromicale@unict.it etc.) more effectively than monoculture (Finney and Kaye Department of Agriculture, Food and Environment (Di3A), 2017). A number of researchers have demonstrated that the University of Catania, Via Valdisavoia, 5, 95123 Catania, inclusion of cover crops in no-till farming systems ensures Italy Vol.:(0123456789) 1 3 70 Page 2 of 16 A. Scavo et al. various ecosystem services (soil erosion control, carbon studied managements, while it increased the amount sequestration, reduction of nutrient leaching, degradation of ammonia-oxidizing (Nitrosomonas europaea) and of agrochemicals, increase of biodiversity, pollinator attrac- N-fixing (Azotobacter vinelandii) bacteria involved in the tion, etc.) (Blanco-Canqui et al. 2015; Daryanto et al. 2019; soil N-cycle, which resulted in a significant enhancement Kaye and Quemada 2017; Mauromicale et al. 2010; Sharma of ammoniacal and nitric N-availability in the soil. et al. 2018a, b; Wulanningtyas et al. 2021). Given that soil quality is a function of a complex Areas under semiarid Mediterranean-type climates network of interrelationships among physical, chemi- are commonly characterized by irregular rainfall cal, and biological properties, in which the organic regimes with intense autumnal events and drought in matter (SOM) content plays a key role (Jiménez et al. late spring and summer. In these climatic conditions, 2002), and keeping in mind the benefits deriving from soils are often clayey, poor in organic matter, and cover crops on soil health, especially under an inte- subjected to erosion, which contribute to lowering the grated management strategy (Scavo and Mauromicale macro- and microelements’ content (Lombardo et  al. 2020), in this research, we have hypothesized that 2014; Madejón et al. 2007). The loss of soil minerals, the optimization of T. subterraneum cover cropping particularly relevant for macronutrients such as nitrogen management can allow lowering the environmental (N) and phosphorous (P), combined with the severe impact and enhancing the productivity of Mediterra- weed infestations, represents two critical aspects of nean orchard agroecosystems through an equilibrium Mediterranean agroecosystems, where an intensive and between the main soil quality indicators and weed pres- indiscriminate use of auxiliary inputs (tillage, fertilizers, sure. Hence, the goals were to compare subterranean and herbicides) is consequently adopted. In this clover and spontaneous f lora cover crops, burying or framework, subterranean clover (Trifolium subterraneum leaving their dead mulches on the soil surface, to a L.), a self-pollinated annual legume with remarkable conventional management on a Mediterranean apricot geocarpism, native to the Mediterranean Basin and orchard by monitoring the changes of SOM, macro- widely diffused throughout the world in regions with and microelements, and weed seedbank over a 4-year Mediterranean-type climates (Australia, New Zealand, period (Fig. 1). The Americas and South Africa), is considered an eligible cover crop, owing to its self-reseed and N-fixation ability, rapid growth, and weed suppressive ability (Enache and Ilnicki 1990; Restuccia et al. 2020; Scavo et al. 2020). Subterranean clover has also proved to adapt well under organic farming and high tree density orchards thanks to its sciaphilic habitus (Mauro et al. 2011; Mauromicale et  al. 2010). However, the magnitude of cover crop effects on soil quality closely depends on soil properties, management practices, climatic conditions, etc., thus making it important to locally evaluate these potential impacts over a medium-long-term period (Sharma et al. 2018a, b). Cover cropping effects on soil properties under Mediterranean semiarid conditions have already been investigated (Moreno et  al. 2009; Ramos et  al. 2020), but the influence of T. subterraneum cover cropping in Mediterranean apricot (Prunus armeniaca L.) orchards requires specific attention. In recent researches conducted in Sicily (southern Italy), important improvements have been observed in the weed control and nutritional status of soil in an apricot orchard managed with subterranean clover compared to spontaneous flora cover cropping and conventional soil management (disc ploughing and shallow chopping for weed control) (Restuccia et al. 2020; Scavo et al. 2020). Overall, the incorporation of T. subterraneum dead mulches into Fig. 1 a View of Trifolium subterraneum L. cover cropping in cen- the soil significantly decreased the weed seedbank size tral Sicily (Italy). Detail of subterranean clover (b). Photographs by and the weed aboveground biomass compared to other S. Fontanazza. 1 3 Trifolium subterraneum cover cropping enhances soil fertility and weed seedbank dynamics… Page 3 of 16 70 (119 mm), February 2018 (109 mm), and October 2018 2 Materials and methods (189 mm). Air temperatures were always optimal for the establishment and growth of T. subterraneum. Indeed, 2.1 Site and weather description minima temperatures fell below 3 °C only in January of season IV, while the mean maxima were 26 °C at emer- The experiment was carried out over four growing sea- gence stage (October) and 21.5  °C at flowering stage sons (I, II, III, and IV) from 2015/2016 to 2018/2019 in (April) of subterranean clover. central Sicily [37°13′ N., 14°05′ E., 290 m a.s.l.], south- ern Italy. The area has a semiarid Mediterranean climate 2.2 Experimental design and cover crop with mild winters, mean annual rainfall of ~ 500  mm, management mostly concentrated in the fall-winter period, and sum- mer droughts. According to the USDA soil taxonomy (Soil A completely randomized block design was adopted, involv- Survey Staff 1999), the soil is classified as Regosoil, and, ing 5 treatments and 4 replicates. Cover cropping treatments at the beginning of the experiment, in the 0–40-cm layer, included T. subterraneum with the leaving of dead mulch on it showed a clayey texture (25.7% sand, 30.6% silt, 43.7% the soil surface (TCC-S), T. subterraneum with the burying clay), a 25% of gravel, and had the following mean char- of dead mulch into the soil (TCC-B), spontaneous flora with acteristics: 1.9% organic matter; 6.1% total CaC O ; 5.6% the leaving of dead mulch on the soil surface (SCC-S), and active limestone; pH 8.0; electrical conductivity 1.3 dS ‒1 −1 spontaneous flora with the burying of dead mulch into the m ; total N 1.1‰; available P O 13 mg  kg ; exchange- 2 5 −1 soil (SCC-B). The control was a conventional management able KO 422 mg  kg ; cation exchange capacity (CEC) ‒1 (CM) ordinarily adopted for apricot orchards in the area 49.6 meq 100  g ; exchangeable bases (Ca, Mg, Na, and ‒1 where the experiment took place, which consists of ‒ 15 cm K) 42.2, 5.4, 1.1, and 0.9 mg 100  g , respectively; Fe −1 −1 −1 winter disc ploughing (late September) and three shallow 12.47 mg  kg ; Mn 3.75 mg  kg ; Cu 2.36 mg  kg ; and −1 choppings (on November, February, and May, respectively) Zn 1.34 mg  kg . The typical crops of the zone are cereals, for weed control. The experiment was carried out in a large legumes, olive, grape vineyard, carob, apricot, and peach area of about 1 ha within a Mediterranean orchard. In par- orchards. In the last 3  years  before the apricot orchard ticular, the area of each plot was 10 × 8.7 m (87 m ), for a planting, the experimental field has been cultivated with net plot size of 348 m , which intercepted 6 trees per treat- wheat, chickpea, and a fallow year. ment, and 1740 m in total (20 plots). Furthermore, in order Climatic data including maxima and minima monthly to avoid interference during the soil sampling, a buffer area temperatures and mean monthly rainfall were recorded of 2 m in width was considered around each experimental from November 2015 to July 2019 by a meteorological plot (Fig. 3). station (Mod. Multirecorder 2.40; ETG, Firenze, Italy) Apricot (cv. Wonder, together with ‘Pinkcot®’ and ‘Big located ~ 15 m from the experimental area (Fig. 2). About Red®’ as pollinators) planting was conducted in January 23% of the total 4-year rainfall fell in January 2016 2012 in a 3.5 × 4.5 m layout. At the start of season I and Fig. 2 Maxima and minima monthly temperatures and monthly total rainfall during the growing seasons. 1 3 70 Page 4 of 16 A. Scavo et al. Fig. 3 (a) Schematic representation of the field experimental layout folium subterraneum cover cropping leaving dead mulch on the soil according to a randomized complete block design, and (b) detail of surface; TCC-B, T. subterraneum cover cropping burying dead mulch the experimental unit with scheme of soil sampling. Each soil sample in the soil; SCC-S, spontaneous flora cover cropping leaving dead is the sum of 5 subsamples per plot collected along the diagonals of mulch on the soil surface; SCC-B, spontaneous flora cover cropping the central part. CM, conventional apricot management; TCC-S, Tri- burying dead mulch in the soil. during the experiment, apricot trees were 3.5–4.0-m high. Self-compensating drip irrigation replacing 100% of the Subterranean clover seeding was done in November 2015 by daily evapotranspiration and twice chopping per year for −2 hand at 2–3-cm depth using 2000 germinable seeds m of weed control (in early spring and mid-summer) were carried the cultivar ‛Seaton Park’, a common Australian early-mid out. Incorporation of dead mulches in TCC-B and SCC-B season genotype presenting an autumn–winter-spring cycle was carried out in September of each season at − 15 cm soil and vernalization requirement for flowering. The length of depth by disc ploughing. Insect and fungal control were done the biological cycle (i.e., from the beginning of plant emer- by low-dose applications of captano, tebuconazole, propi- gence until all the plants within a plot had completely dried conazole, and lambda-cyhalothrin only when required. up) was 200 days in season I, 220 days in season II, 250 in season III, and 240 in season IV (Restuccia et al. 2020). Its 2.3 Soil sampling and analyses −2 −2 total biomass was 44 g DW m in season I, 283 g DW m −2 −2 in season II, 239 g DW m in season III, and 159 g DW m Before each soil sampling, a field scouting was conducted to in season IV, thus confirming a good establishment of the locate the sampling units by excluding the non-representa- cover crop after the self-reseeding (Restuccia et al. 2020). tive areas and the outer 3 m for each plot. Soil sampling was In all plots, in October, a shallow hoeing at 10-cm deep carried out using a core sampler by collecting 5 randomly followed by rigid tine harrowing was performed, together distributed subsamples (each of 0.75 dm ) along the diago- −1 with a fertilization program consisting of 40 kg  ha P O , nals of the central part per plot, which were pooled to form 2 5 −1 −1 14 kg  ha  N, and 10 kg  ha K O. Then, in each season, a composite sample. The following soil depths and times the following fertilizers were provided through fertirriga- were adopted: tion from the developing apricot fruit phase until harvesting: −1 300 kg  ha Ponimag® (11% N, 41% K O and 3% MgO), • Two depths (0‒20 and 21‒40 cm) in March of season II −1 −1 50 kg  ha simple perphosphate (P O), 100 kg  ha liq- and season IV to determine the contents of soil organic 2 5 −1 uid Ca, and 50 kg  ha Hergoton Plus® (8% total organic matter and macro- and microelements N, 26% biologic C, and 44.45% total organic matter). 1 3 Trifolium subterraneum cover cropping enhances soil fertility and weed seedbank dynamics… Page 5 of 16 70 −2 From top soil (0‒15 cm) twice per season [March (T ) m ), relative density (RD), relative frequency (RF), and the and June (T )] to monitor the dynamics of the soil N relative abundance index (RAI): + − mineral fractions (NH and NO ), in accordance with �∑ � 4 3 Scavo et al. (2020) RD(%) = × 100 (1) From top soil (0‒15 cm) twice per season [April (T ) and September (T )] for the weed seedbank � � RF(%) = ∑ × 100 (2) A total of 400 soil cores (20 plots × 2 seasons × 2 sam- pling depths or 2 sampling times × 5 subsamples) was taken. Samples were collected in season II and IV to RD + RF obtain a mid-term and a final evaluation. RAI(%) = (3) Fresh soil samples were initially air-dried at room tem- perature for about 2 weeks, crushed, and sieved through where ∑Y = sum of the number of seeds for a weed a 2-mm mesh sieve before laboratory analyses. The species; S = total number of weed seeds within the plot; exchangeable cations (K, Ca, Mg) were determined fol- F = absolute frequency of a species (i.e., number of sam- lowing the ISO 13,536 (1995) procedure while the avail- pling units in which the species i occurred) i; and ∑F = sum able P O according to the ISO 11,263 (1994) method. The of the absolute frequencies of all species. 2 5 soil organic carbon and total N analyses were conducted Richness and evenness were used to describe the within- according to the ISO 14,235 (1998) and ISO 11,261 (1995) community species diversity (α-diversity) (Travlos et al. methods, respectively. The N mineral fractions were 2018). Species richness was calculated as the total number determined in the extractable 10 g soil samples treated of weed seeds counted in the five soil cores for each plot with 50  ml of 0.5  M K SO into 250-ml bottles. After (Moonen and Bàrberi 2004), while evenness was estimated 2 4 1 h shake, filtration with glass fiber Whatman GF/A and by computing the Shannon–Wiener (H′) and the Pielous (J) 0.45-μm millipore filter, and centrifugation for 15  min diversity indices, as suggested by Adeux et al. (2019): + − at 3000 rpm, the ammoniacal (NH ) and nitric (N O ) 4 3 H = −p lnp (4) N-levels of the extracts were colorimetrically measured i i by diffusion (Crooke and Simpson 1971). The bioavail- able fraction of the main micronutrients (Cu, Fe, Mn, Zn) � J = was measured after extraction with buffered diethylen- (5) max etriaminepentaacetic acid (DTPA) solution following the th Lindsay and Norvell (1978) method. where p = proportional abundance of the i species; and H' = logarithm of species richness. max 2.4 Analysis of the soil weed seedbank 2.5 Statistical analysis The analysis of the soil weed seedbank followed Scavo et al. For each soil variable, analysis of variance (ANOVA) (2019a). Firstly, 20 soil samples from each treatment (5 subsam- was used to evaluate the effect of studied factors and their ples × 4 replicates) were mixed giving a final volume of 15 dm , interaction. In particular, a generalized linear mixed model and then they were freed from inert components such as stones, (GLMM) was applied considering “cover cropping” and pebbles, and dead debris. Secondly, seeds were separated from “sampling depth” as fixed factors and “season” as random soil by using a metal tube (Karcher, K 3500 model, Winnenden, factor (Gomez and Gomez 1984). At each season, one- Germany) working at 20–120 bar adjustable washer pressure way ANOVAs were performed (one with “cover cropping” and equipped with a removable cap consisting of a steel mesh of and another with “sampling depth”). Moreover, in order to 250 μm. Thirdly, weed counts and identification were performed observe the overall effect of cover cropping on each soil in Petri dishes after 24 h air-drying with a MS5 Leica stereomicro- variable under study, one-way ANOVAs was carried out scope (Leica Microsystems, Wetzlar, Germany). Seed identifica- by pooling data over seasons and sampling depths. The tion and grouping followed Conti et al. (2005), while the seedbank ANOVA basic assumptions of homoscedasticity and nor- −2 size was calculated as the number of seeds m for each plot. mality were checked by graphically inspecting the residuals, In accordance with Nkoa et al. (2015) and Scavo et al. which showed no significant deviations, together with the (2020), the soil weed seedbank was analyzed taking account Bartlett’s test. Post hoc comparison of means was performed both species abundance and diversity. Abundance was meas- through the Fisher’s protected least significant difference ured considering the seedbank size (number of weed seeds 1 3 70 Page 6 of 16 A. Scavo et al. (LSD) test at α = 0.05 by using the statistical package CoS- seasons, was not influenced by factors under study (data not tat® 6.003 (CoHort Software, Monterey, CA, USA). shown). Ca was the prevailing exchangeable cation (~ 88% Soil seedbank data, species richness, RAI data, and of CEC), followed in decreasing order by Mg (~ 9%), Na diversity indices were subjected to two-way ANOVAs (~ 2%), and K (~ 1%). No significant differences among treat- (“cover cropping” × “sampling time”) for each season. ments were observed for exchangeable Ca and Mg concen- Prior to ANOVA, data about seedbank size needed log trations, while exchangeable Na and K were influenced by transformation, whereas an arcsine-square root transfor- cover cropping type. mation was used for RAI of major weeds. Moreover, H′ In season II, the GLMM showed how cover cropping had and J data were square root and logit-transformed, respec- high significance on all the soil variables, except for total N tively. Data on species richness did not need any transfor- and exchangeable Mg (Table 1). In particular, regardless of mation before ANOVA. Multivariate statistics was con- sampling depth, the highest levels of SOM were detected ducted to analyze the weed species composition. In each in SCC-S and TCC-B (+ 13% and + 10% than CM, respec- season, multivariate analysis of variance (MANOVA) on tively), the highest concentrations of assimilable P O in 2 5 log (x + 1)-transformed data was carried out to evaluate the SCC-S (+ 15%, even if not statistically significant) and the effect on variance of cover cropping, sampling time, and highest ones of exchangeable K O in TCC-B (+ 15%), of their interaction. Significance levels in MANOVA were exchangeable Ca in SCC-B and SCC-S (+ 7% and + 5%, determined according to Wilks’ criterion. The effects of respectively), of exchangeable Na in SCC-B and SCC-S MANOVA were visualized by applying a principal com- (+ 51% and + 48%, respectively), and of exchangeable K ponent analysis (PCA) on 8 major weeds, considering the in TCC-B (+ 17%) (Table  1). Concerning soil microele- means for each “cover cropping × sampling time” com- ments (Table 2), SCC-S promoted the highest concentra- −1 −1 bination. PCA was performed on the covariance matrix tion of Mn (7.97  mg  kg ), Zn (0.22  mg  kg ), and Cu −1 of log (x + 1)-transformed density data, and the results (3.18 mg  kg ), while Fe-level was the highest in SCC-B −1 were displayed on “distance” biplots (Legendre and Leg-(9.55 mg  kg ). Higher amounts of soil microelements than endre 2012). The computer package Minitab® version 16 CM were found also in TCC-B, whereas TCC-S caused a (Minitab Inc., State College, PA, USA) was used to per- decrease. No significant differences were observed regard- form both MANOVA and PCA. ing soil layers, except for exchangeable K O and exchangea- ble Na. The two-way interaction “cover cropping × sampling depth” was significant only for SOM, exchangeable K O, exchangeable Na, exchangeable K, and Fe (Tables 1 and 2). 3 Results In this view, except for exchangeable Na and Fe, TCC-B and SCC-S performed better in the 0‒20-cm soil layer than in 3.1 Cover‑cropping effects on soil organic matter, the 21‒40 cm one. macro‑ and microelements The cumulated cover cropping effects of season IV on the above-mentioned soil variables were more marked than ANOVA demonstrated a significant effect of cover cropping those of season II. Indeed, GLMM indicated that TCC-B on SOM and soil nutrient content, even if with seasonal- ensured a significant increase, compared to CM, in the dependent results. From the one-way ANOVA applied on amount of SOM (+ 19%), total N (+ 17%), exchangeable ʻcover croppingʼ and pooling over seasons and sampling K O (+ 13%), exchangeable Na (+ 30%), exchangeable K depths (Fig. 4), emerged that TCC-B significantly increased (+ 27%), Fe (+ 12%), Mn (+ 46%), Zn (+ 52%), and Cu the amount of SOM by 15%, total N by 7%, exchangeable (+ 36%) (Tables 1 and 2). Spontaneous flora cover crop- K O by 14%, exchangeable Na by 32%, exchangeable K by ping (SCC-B and SCC-S) provided the highest content of −1 16%, Fe by 15%, Mn by 28%, Zn by 36% and Cu by 24%, assimilable P O (58.2 and 42.0 mg  kg , respectively) 2 5 −1 as compared to those of CM. The highest levels of assimila- and Mn (8.1 mg  kg ), whereas exchangeable Ca and Mg −1 ble P O were found in SCC-B (39.8 mg  kg ) and SCC-S did not differ significantly, as observed in season II. More- 2 5 −1 (32.7 mg  kg ). Also, SCC-B and SCC-S showed signifi- over, in this season, the effect of sampling depth was gen- cant increments in SOM and soil macro- and microelement erally significant, except for exchangeable Ca, exchange- levels with respect to CM, while TCC-S was interestingly able Mg, exchangeable Na, Fe, and Mn. The 0 − 20-cm soil associated with lower amounts of total N (‒ 4%), assimilable layer showed higher amounts of SOM (+ 19.5%), total N P O (‒ 26%), exchangeable K O (‒ 5%), exchangeable K (+ 11.4%), assimilable P O (+ 139%), exchangeable K O 2 5 2 2 5 2 (‒ 3%), and Cu (‒ 17%), as compared to CM (Fig. 4). CEC, (+ 34%), exchangeable K (+ 35%), Zn (+ 46%), and Cu ‒1 on average equal to ~ 50 meq 100  g throughout the four (+ 53%) than the 21 − 40 cm one. 1 3 Trifolium subterraneum cover cropping enhances soil fertility and weed seedbank dynamics… Page 7 of 16 70 Fig. 4 Effect of cover-cropping, pooling over seasons, and soil sam- TCC-S, Trifolium subterraneum cover cropping leaving dead mulch pling depths, on organic matter and macro- and micro-elements. Bars on the soil surface; TCC-B, T. subterraneum cover cropping bury- are standard error of the mean (SEM, n = 8). Different letters indicate ing dead mulch in the soil; SCC-S, spontaneous flora cover cropping statistical significance by applying a one-way analysis of variance leaving dead mulch on the soil surface; SCC-B, spontaneous flora with LSD test at P ≤ 0.05. CM, conventional apricot management; cover cropping burying dead mulch in the soil. TCC-S better performing in season II and TCC-B in season 3.2 Cover‑cropping effects on soil mineral nitrogen IV. Averaged over sampling times (March and June), in season dynamics II, TCC-B determined an increase of 148% in soil NH and 244% in soil NO , compared to CM. Such positive effect The GLMM indicated that the effect of cover cropping was became stronger in season IV, especially for NH , with highly significant (P ≤ 0.001) for both mineral N forms and a + 194% than CM and + 133% than the same treatment in sea- in both seasons, while the sampling time was significant only son II. Also, the soil from SCC-S and SCC-B plots contained in season IV (Table 2). Subterranean clover cover cropping more mineral N than CM (+ 49 and + 76%, respectively). The always showed the highest levels of total soil mineral N, with 1 3 70 Page 8 of 16 A. Scavo et al. Table 1 Effect of cover cropping (CC) and soil sampling depth (SD) subterraneum cover cropping leaving dead mulch on the soil surface, on organic matter (SOM), macroelements, and exchangeable bases TCC-B T. subterraneum cover cropping burying dead mulch in the with analysis of variance (ANOVA, P values). Values are means soil, SCC-S spontaneous flora cover cropping leaving dead mulch on (n = 2) with standard deviation (in brackets). Values within a column the soil surface, SCC-B spontaneous flora cover cropping burying followed by the same letters are not significantly different at P ≤ 0.05 dead mulch in the soil, season II 2016/2017, season IV 2018/2019. (LSD test). CM conventional apricot management, TCC-S Trifolium ‒1 ‒1 ‒1 ‒1 Treatment SOM (g kg ) Total N (g kg )Assimilable P O (mg kg )Exchangeable K O (mg kg ) 2 5 2 Season II Season IV Season II Season IV Season II Season IV Season II Season IV CC CM 18.38 (0.48) 18.63 (1.49) 1.32 (0.21)a 1.30 (0.08) 20.4 (5.6)a 18.0 (9.9)c 368.0 (46.1) 403.9 (28.1)bc bc bc bc b TCC-S 17.75 (0.96)c 19.50 (2.08) 1.27 (0.10)a 1.25 (0.13)c 14.5 (1.7)b 14.0 (5.1)c 329.5 (11.4) 401.5 (37.0)c b c TCC-B 20.25 (1.50) 22.25 (5.62)a 1.27 (0.05)a 1.52 (0.28)a 21.7 (1.7)a 18.5 (9.8)b 421.7 (53.0) 456.5 (95.5)a ab a SCC-S 20.75 (2.75)a 17.0 (1.41)c 1.17 (0.28)a 1.35 (0.06) 23.5 (2.9)a 42.0 (15.8)a 382.7 (40.5) 441.5 (72.1)ab bc b SCC-B 19.75 (2.50) 19.25 (0.96) 1.30 (0.08)a 1.45 (0.10) 21.5 (2.5)a 58.2 (23.8)a 370.5 (31.4) 420.2 (68.4) abc b ab b abc SD 0‒20 cm 19.66 (2.08)a 21.05 (3.42)a 1.32 (0.15)a 1.45 (0.18)a 20.5 (4.9)a 42.5 (25.4)a 392.3 (53.1) 486.7 (57.8)a 21‒40 cm 19.10 (2.02)a 17.60 (1.35) 1.22 (0.15)b 1.30 (0.12)b 20.2 (3.8)a 17.8 (8.5)b 356.7 (31.5) 362.8 (28.2)b b b ANOVA CC 0.0345 0.0024 0.6188 0.0207 0.0474 0.0004 0.0028 0.0343 SD 0.3556 < 0.001 0.1358 0.0084 0.8815 0.0003 0.0055 < 0.001 CC × SD 0.0151 0.0049 0.1376 0.1812 0.7902 0.0291 0.0118 0.0017 Treatment Exchangeable Ca Exchangeable Mg Exchangeable Na Exchangeable K ‒1 ‒1 ‒1 ‒1 (meq 100  g )(meq 100  g )(meq 100  g )(meq 100  g ) Season II Season IV Season II Season IV Season II Season IV Season II Season IV CC CM 41.7 (0.7)ab 46.3 (4.5)a 4.55 (0.56)a 6.25 (0.37) 0.83 (0.04)d 1.80 (0.49) 0.75 (0.03) 0.85 (0.02)b ab bc bc TCC-S 40.2 (1.0)b 43.4 (2.8)a 4.32 (0.10)a 6.15 (0.48) 0.93 (0.05)c 2.90 (0.64)a 0.70 (0.06)c 0.85 (0.02)b ab TCC-B 40.5 (4.0)b 42.6 (1.8)a 4.32 (0.22)a 6.40 (0.08)a 1.13 (0.09)b 2.35 (0.50) 0.88 (0.04)a 0.98 (0.11)a ab SCC-S 44.0 (1.7)a 43.6 (2.9)a 4.20 (0.18)a 6.40 (0.71)a 1.23 (0.04)a 1.80 (0.28) 0.78 (0.04)b 0.93 (0.04)ab bc SCC-B 44.5 (0.6)a 46.9 (2.8)a 4.25 (0.17)a 5.60 (0.18)b 1.25 (0.11)a 1.45 (0.49)c 0.80 (0.07)b 0.88 (0.04)b SD 0‒20 cm 42.3 (1.6)a 44.8 (3.1)a 4.39 (0.38)a 6.06 (0.46)a 1.03 (0.05)b 1.88 (0.42)a 0.80 (0.03)a 1.03 (0.6)a 21‒40 cm 42.0 (3.4)a 43.3 (3.0)a 4.28 (0.17)a 6.26 (0.51)a 1.11 (0.12)a 2.24 (0.54)a 0.76 (0.05)a 0.76 (0.03)b ANOVA CC 0.0363 0.6250 0.5231 0.1698 < 0.001 0.0230 0.0031 0.0548 SD 0.7466 0.3262 0.4535 0.3607 0.0085 0.1605 0.0734 < 0.001 CC × SD 0.3387 0.6692 0.4669 0.8113 0.0034 0.4499 0.0226 0.0015 two-way interaction and the sampling time were significant at members (32%), annual (77%), and therophytes (77%) (Table 3). + ‒ P ≤ 0.001 in season IV, with higher values of NH , NO and Sonchus sp., Anagallis arvensis L., and Phalaris paradoxa L. 4 3 their sum in the June sampling than the March one. had the highest relative abundance values. Centaurea napifolia L. had the higher relative abundance values in season IV than 3.3 Cover‑cropping effects on the weed soil in season II, while Portulaca oleracea L. showed an opposite seedbank trend. No consistent trends were observed for the other weeds. Nevertheless, the effect of cover cropping on species rich- Over the two seasons under study, the 0 − 15-cm soil seed- ness was inconsistent, and, for this reason, these data were not bank was composed of 22 taxa in total, mainly Asteraceae shown. Among the 22 species and genera recorded, only a few 1 3 Trifolium subterraneum cover cropping enhances soil fertility and weed seedbank dynamics… Page 9 of 16 70 Table 2 Effect of cover cropping (CC) and soil sampling depth (SD) (LSD test). CM conventional apricot management, TCC-S Trifolium + ‒ on major microelements and ammoniacal (NH ) and nitric (NO ) subterraneum cover cropping leaving dead mulch on the soil surface, 4 3 nitrogen with analysis of variance (ANOVA, P values). Values are TCC-B T. subterraneum cover cropping burying dead mulch in the means (n = 2 for soil microelements and n = 3 for mineral nitrogen) soil, SCC-S spontaneous flora cover cropping leaving dead mulch on with standard deviation (in brackets). Values within a column fol- the soil surface, SCC-B spontaneous flora cover cropping burying lowed by the same letters are not significantly different at P ≤ 0.05 dead mulch in the soil, season II 2016/2017, season IV 2018/2019. ‒1 ‒1 ‒1 ‒1 Treatment Fe (mg kg ) Mn (mg kg ) Zn (mg kg ) Cu (mg kg ) Season II Season IV Season II Season IV Season II Season IV Season II Season IV CC CM 7.75 (0.56)b 14.42 (3.44)b 5.57 (1.52)bc 5.46 (2.24)b 0.19 (0.02)ab 0.25 (0.07)b 2.79 (0.49)ab 2.68 (0.62)b TCC-S 8.40 (0.05)b 15.18 (1.39)b 4.41 (0.88)c 7.85 (2.35)a 0.13 (0.03)b 0.35 (0.11)ab 1.76 (0.05)c 2.77 (0.33)b TCC-B 9.33 (0.31)a 16.16 (3.10)a 6.14 (0.75)bc 7.97 (3.09)a 0.21 (0.03)a 0.38 (0.18)a 3.12 (0.22)ab 3.65 (1.59)a SCC-S 9.29 (1.02)a 14.68 (1.89)b 7.97 (0.95)a 6.17 (2.55)ab 0.22 (0.03)a 0.29 (0.09)ab 3.18 (0.29)a 3.66 (1.20)a SCC-B 9.55 (1.27)a 14.35 (0.92)b 6.16 (0.28)b 8.10 (0.91)a 0.19 (0.02)ab 0.34 (0.10)ab 2.60 (0.28)b 3.58 (0.78)a SD 0‒20 cm 8.76 (1.06)a 14.92 (2.15)a 5.83 (2.12)a 7.90 (2.24)a 0.19 (0.05)ab 0.38 (0.13)a 2.66 (0.59)a 3.95 (0.98)a 21‒40 cm 8.97 (0.96)a 15.00 (2.37)a 6.28 (0.81)a 6.30 (2.31)a 0.18 (0.03)ab 0.26 (0.05)b 2.72 (0.62)a 2.58 (0.35)b ANOVA CC 0.0024 0.0010 0.0142 0.0127 0.0006 0.0427 0.0012 0.0316 SD 0.3930 0.7060 0.3866 0.1129 0.2551 0.0046 0.7092 < 0.001 CC × SD 0.0090 0.9254 0.2651 0.2432 0.0369 0.0223 0.9091 0.0275 + ‒ + ‒ Treatment NH (ppm) NO (ppm) NH + NO (ppm) 4 3 4 3 Season II Season IV Season II Season IV Season II Season IV CC CM 2.64 (0.86)d 5.17 (0.33)d 5.22 (1.23)d 5.03 (0.43)e 7.86 (1.66)d 10.20 (0.67)e TCC-S 7.61 (0.94)a 15.66 (0.46)a 18.51 (1.12)a 18.73 (0.45)b 26.12 (1.21)a 34.39 (0.56)b TCC-B 6.54 (1.09)b 15.22 (0.53)a 17.98 (1.14)a 20.54 (0.54)a 24.52 (1.25)a 35.76 (0.90)a SCC-S 3.91 (0.31)c 5.87 (0.55)c 11.38 (1.60)b 9.31 (0.52)d 15.29 (1.84)b 15.17 (0.54)d SCC-B 3.71 (0.34)cd 7.24 (0.31)b 9.38 (1.54)c 10.68 (0.59)c 13.10 (1.67)c 17.91 (0.89)c SD March 4.48 (0.68)b 7.81 (0.41)b 12.89 (1.26)a 12.04 (0.53)b 17.37 (1.64)a 19.85 (0.79)b June 5.33 (0.81)a 11.85 (0.46)a 12.10 (1.39)a 13.67 (0.48)a 17.43 (1.42)a 25.52 (0.63)a ANOVA CC < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 ST 0.0174 < 0.001 0.1485 < 0.001 0.9100 < 0.001 CC × ST 0.0854 < 0.001 0.1246 < 0.001 0.0703 < 0.001 evidenced high enough density to be included in the multivariate SCC-B > TCC-S > TCC-B (Table 4). Regarding sampling analysis for species composition. In particular, only for 8 taxa time, in both seasons, the seedbank size was significantly −2 was an RD > 4% (i.e., major weeds) found: Amaranthus retro- higher in April than in September (3380 vs. 1020 seeds m −2 flexus L., A. arvensis, C. napifolia, P. paradoxa, P. oleracea, in season II and 2920 vs. 1655 seeds m in season IV). Sinapis arvensis L., Sonchus sp., and Veronica cymbalaria Bod- Significant effects were also observed in terms of seed- ard, altogether accounting for 91.2% of the whole soil seedbank bank diversity and evenness (Table  4). The two studied throughout the two seasons. However, the ANOVA computed biodiversity indices were differently affected depending on on RAI data of major weeds highlighted no significant effects the season. In season II, TCC-B evidenced the highest H either for cover cropping or for sampling time (data not shown). (1.43) and J (0.89), indicating a high weed diversity and no Abundance was also analyzed by calculating the seed- dominant species. In this season, both H and J were greater bank size (Table  4). The interactive effect “cover crop- in September than in April (1.53 vs. 0.93 for H and 0.91 vs. ping × sampling time” two-way ANOVA was significant in 0.64 for J). In season IV, the two-way interaction was sig- season II (P ≤ 0.001) and season IV (P ≤ 0.05), with both nificant only for H (P ≤ 0.001); CM highlighted the highest factors showing significance at P ≤ 0.001. Concerning the H (1.8) and did not differ significantly from TCC-B for J effect of cover cropping, the lowest seedbank size was (0.93 vs. 0.87, respectively). The lowest H and J were found −2 detected in TCC-B plots (1469 seeds m on the average of in SCC-S (1.04 and 0.56) and SCC-B (1.33 and 0.7) plots. seasons), with a reduction of 55.4% in season II and 57.4% Contrariwise to season II, H was higher in April than in in season IV, as compared to CM. Overall, in both seasons, September (1.54 vs. 1.42), whereas the sampling time was the general trend in decreasing order was CM > SCC-S > not consistent for J. 1 3 70 Page 10 of 16 A. Scavo et al. 1 3 Table 3 Binomial name, botanical family, life cycle, biological group (BG), mean relative abundance values, and mean relative densities (RD) of the weed species under 5 cropping systems and 2 seasons. CM conventional apricot management, TCC-S Trifolium subterraneum cover cropping leaving dead mulch on the soil surface, TCC-B T. subterraneum cover cropping burying dead mulch in the soil, SCC-S spontaneous flora cover cropping leaving dead mulch on the soil surface, SCC-B spontaneous flora cover cropping burying dead mulch in the soil, season II 2016/2017, a b season IV 2018/2019. Biological groups: T therophytes, H hemicryptophytes. Averaged over all treatments. RAI data were averaged over sampling times. a b Binomial name Botanical family Life cycle BG CM TCC-S TCC-B SCC-S SCC-B RD (%) II IV II IV II IV II IV II IV Amaranthus retroflexus L. Amaranthaceae Annual T − 0.13 − < 0.1 − < 0.1 0.1 < 0.1 0.12 < 0.1 4.1 Anagallis arvensis L. Primulaceae Annual T 0.1 0.13 0.15 < 0.1 0.32 < 0.1 0.21 < 0.1 < 0.1 0.23 12.7 Artemisia vulgaris L. Asteraceae Perennial H < 0.1 − − − − − < 0.1 − − − 0.9 Avena sp. Poaceae Annual T − < 0.1 − − − − − − − − 0.2 Centaurea napifolia L. Asteraceae Annual T − < 0.1 − 0.15 < 0.1 0.22 < 0.1 < 0.1 < 0.1 < 0.1 5.2 Euphorbia helioscopia L. Euphorbiaceae Annual T − − − < 0.1 − − − − − − 0.2 Fallopia convolvulus (L.) Á. Löve Polygonaceae Annual T − − − − − − − − 0.1 < 0.1 0.7 Fumaria sp. Fumariaceae Annual T < 0.1 < 0.1 − − − − < 0.1 < 0.1 − − 0.7 Galium aparine L. Rubiaceae Annual T − − − − < 0.1 − − − 0.13 − 2.2 Helminthotheca echioides (L.) Holub Asteraceae Annual T − − − < 0.1 − − − − − < 0.1 0.3 Lamium amplexicaule L. Lamiaceae Annual T − − − − − − < 0.1 − − − 0.1 Medicago polymorpha L. Fabaceae Annual T − − − − − < 0.1 − − − − 0.9 Phalaris paradoxa L. Poaceae Annual T < 0.1 0.1 0.22 < 0.1 0.28 − < 0.1 0.21 0.14 0.13 9.1 Polygonum aviculare L. Polygonaceae Annual T − − − − − − − < 0.1 − − 0.1 Portulaca oleracea L. Portulacaceae Annual T 0.18 < 0.1 0.18 < 0.1 0.11 < 0.1 0.12 − 0.14 < 0.1 7.3 Reichardia picroides (L.) Roth Asteraceae Perennial H − − − − − − − < 0.1 − < 0.1 0.3 Silene sp. Caryophyllaceae Perennial H − < 0.1 − < 0.1 − < 0.1 − − − − 0.8 Sinapis arvensis L. Brassicaceae Annual T − < 0.1 0.13 0.11 − < 0.1 0.15 < 0.1 0.3 < 0.1 8.7 Sonchus sp. Asteraceae Biennial H 0.27 0.22 0.41 0.35 0.25 0.24 0.43 0.49 0.34 0.38 40.0 Taraxacum sp. Asteraceae Perennial H − < 0.1 − < 0.1 − − − − − − 1.1 Veronica cymbalaria Bodard Plantaginaceae Annual T − < 0.1 < 0.1 < 0.1 < 0.1 0.19 − 0.1 − < 0.1 4.2 Xanthium sp. Asteraceae Annual T − < 0.1 − − − − − − − − 0.4 Trifolium subterraneum cover cropping enhances soil fertility and weed seedbank dynamics… Page 11 of 16 70 Table 4 Effect of cover cropping (CC) and soil sampling time (ST) TCC-S Trifolium subterraneum cover cropping leaving dead mulch on the weed seedbank size, Shannon diversity index (H), and Pielou’s on the soil surface, TCC-B T. subterraneum cover cropping burying evenness index (J) with analysis of variance (ANOVA, P values). Val- dead mulch in the soil, SCC-S spontaneous flora cover cropping leav - ues are means (n = 4) with standard deviation (in brackets). Values ing dead mulch on the soil surface, SCC-B spontaneous flora cover within a column followed by the same letters are not significantly dif- cropping burying dead mulch in the soil, season II 2016/2017, season ferent at P ≤ 0.05 (LSD test). CM conventional apricot management, IV 2018/2019. −2 Treatment Seedbank size (seeds m ) H J Season II Season IV Season II Season IV Season II Season IV CC CM 3250 (256)a 3488 (307)a 1.10 (0.37)b 1.80 (0.06)a 0.80 (0.06)ab 0.87 (0.03)ab TCC-S 1750 (202)c 1788 (380)cd 1.12 (0.46)b 1.56 (0.42)b 0.69 (0.28)ab 0.77 (0.10)bc TCC-B 1450 (281)d 1488 (409)d 1.43 (0.05)a 1.66 (0.21)b 0.89 (0.03)a 0.93 (0.01)a SCC-S 2325 (203)b 2425 (329)b 1.13 (0.71)b 1.04 (0.32)d 0.63 (0.40)b 0.56 (0.07)c SCC-B 2225 (164)b 2250 (349)bc 1.37 (0.54)a 1.33 (0.58)c 0.84 (0.19)ab 0.70 (0.27)bc ST April 3380 (358)a 2920 (364)a 0.93 (0.29)b 1.54 (0.43)a 0.64 (0.21)b 0.75 (0.18)a September 1020 (85)b 1655 (346)b 1.53 (0.16)a 1.42 (0.39)b 0.91 (0.05)a 0.78 (0.18)a ANOVA CC < 0.001 < 0.001 < 0.001 < 0.001 0.1156 0.0054 ST < 0.001 < 0.001 < 0.001 0.0062 < 0.001 0.7875 CC × ST < 0.001 0.0131 < 0.001 < 0.001 0.0949 0.0759 MANOVA indicated a strong significance of the two-way reducing the number of weed seeds in the soil, as compared interaction (P ≤ 0.001) in seasons II and IV on the compo- to the conventional management. Such results were strongly sition of the weed seedbank, with sampling time contribut- influenced by cover crop type and season, together with the ing more to variance than cover cropping in both seasons sampling depth for the soil variables and sampling time for (F = 3360 vs. 439.5 in season II and 6.6 vs. 3.7 in season IV). the weed seedbank. Such differences in the weed communities of the soil seed - bank were represented by two ordination biplots, one per sea- 4.1 Soil organic matter and soil macro‑ son, derived from PCA on major weeds (Fig. 5). In season II, and micro‑elements most weeds and treatments were discriminated along the PC1 (42.4% of variance). In particular, the weeds A. retroflexus and The role of cover cropping on soil physical and chemical S. arvensis, together with the treatments SCC-S, SCC-B, and properties has been investigated (Adeli et al. 2020; Mauro CM at the first sampling time (T1), were negatively correlated et al. 2015; Nascente and Stone 2018; Ramos et al. 2020; with PC1 (left side of the biplot), while all the other weeds Wulanningtyas et al. 2021). In the present study, the best and treatments were discriminated on the right side. CM-T1 results were obtained by adopting T. subterraneum with dead was associated with S. arvensis, SCC-B-T1 and SCC-S-T1 mulches incorporated into the soil (TCC-B). The intensity of showed no association with any weeds, and in T2, all treat- these increases was much higher in season IV than II, likely ments showed a lower seedbank size, except for TCC-B-T2 due to a cumulative effect, although subterranean clover bio- −2 and CM-T2. In season IV, the discriminations were clearer. As mass showed an opposite trend (283 g DW m in season II −2 observed in season II, the treatments at the second sampling and 159 g DW m in season IV). Therefore, in contrast with time (T2) showed a lower seedbank, as evidenced by the fact the work of Roldán et al. (2003), reporting higher values in that TCC-B-T2, TCC-S-T2, and CM-T2 were not infested by the soil physio-chemical properties on increasing the amount any weeds. SCC-S-T1 was highly infested with C. napifolia; of residue cover, here no relationship was detected between TCC-B-T1 with Sonchus sp.; TCC-S-T1 with P. oleracea; and soil variables and cover crop biomass. Moreover, higher the spontaneous flora cover cropping (SCC-B-T2, SCC-B-T1 amounts of SOM and soil chemical variables were generally and SCC-S-T2) with S. arvensis, P. paradoxa and A. arvensis. found in the 0 − 20-cm layer than in the deeper one, as com- All the weeds, except for C. napifolia and V. cymbalaria, were monly indicated in literature (Wang et al. 2008). Our results discriminated by the PC2 along the negative side of the axe. were corroborated by Wulanningtyas et  al. (2021), who reported that the combination of no tillage and rye (Secale cereale L.) cover crop was very effective in increasing the 4 Discussion levels of soil organic carbon, total N, available P, exchange- able bases (K, Ca, Mg, Na), cation exchange capacity, and The results obtained in this 4-year field research indicate that some soil physical and biological properties, mainly in the cover cropping had remarkable effects in enhancing the lev - 25 − 75-cm layer. Similarly, Adeli et al. (2020) indicated that els of SOM and soil macro- and microelements as well as in the integration of winter cover crops and soil amendments 1 3 70 Page 12 of 16 A. Scavo et al. atmosphere via photosynthesis of the cover crop (Moebius- Clune et al. 2016), and direct root exudation of C-containing compounds into the rhizosphere (Scavo et al. 2019b). In this regard, Marschner (1995) estimated that about 5 − 21% of all photosynthetically fixed carbon is released into the rhizosphere through root exudation. The increase in SOM 2+ on one side and the release of rhizodeposits (e.g., Ca , 2+ 3+ 3+ + Fe and Fe, Al, K , as well as mucilages and several organic compounds) from the cover crop on the other con- tribute to promote the formation of aggregates and, thus, to improve the soil structure, the cation exchange capacity, and the microbial activity (Scavo et al. 2019b). The stimula- tion of the soil enzymatic-complex activity and promotion of the soil microbial biomass are often reported in cover cropping field experiments (Adeli et al. 2020; Nunes et al. 2018). The incorporation of plant residues into the soil, in particular, increases soil N richness, soil temperature, and aeration, thus creating favorable conditions for microorgan- isms (Turmel et al. 2015), including plant-promoting bac- teria and arbuscular mycorrhizal fungi, which are of key importance in enhancing soil P availability and biological fixation. Overall, these effects may explain the increase in soil macro- and microelement levels. Furthermore, buried plant residues show a faster decomposition and mineraliza- tion rate than surface ones. In our previous research, TCC-B ensured a major increase in the amount of Nitrosomonas europaea and Azotobacter vinelandii, two of the most important bacteria involved in the soil N-cycle, in addition to an enhancement of the soil mineral N (Scavo et al. 2020). After another year of cover cropping, the NH and the total mineral N content were fur- ther increased respectively by 56% and 14% in March and by Fig. 5 Principal components analysis ordination biplot with the 8 34% and 12% in June, compared to season III. The increases most abundant weed species and genera. Red names and arrows highlight the discrimination of weeds along the principal compo- in mineral N were more marked than those detected for the nents. Season II, 2016/2017; season IV, 2018/2019; T , April; T , 1 2 total N, since the latter is largely composed of organic N, September; TCC-S, Trifolium subterraneum cover cropping leav- which is unavailable for plants and therefore of less impor- ing dead mulch on the soil surface; TCC-B, T. subterraneum cover tance for plant nutrition. The higher amounts of soil mineral cropping burying dead mulch in the soil; SCC-S, spontaneous flora cover cropping leaving dead mulch on the soil surface; SCC-B, N in the June sampling can be attributed to weather condi- spontaneous flora cover cropping burying dead mulch in the soil; tions, since the higher temperature and the lower soil mois- CM, conventional apricot management. AMAR, Amaranthus retro- ture of late spring are favorable for N mineralization. Given flexus; ANAAR, Anagallis arvensis; CENTNA, Centaurea napifolia; the low number of Fabaceae members detected in the soil PHAPA, Phalaris paradoxa; PORTOL, Portulaca oleracea; SINAR, Sinapis arvensis; SONCH, Sonchus sp.; VEROCY, Veronica cymba- weed seedbank, it is reasonable to assume that the higher laria. amount of N released into the soil in TCC-B and TCC-S than the other treatments was caused by the massively inclu- into no-till cropping systems improved the soil total car- sion of a N-fixing species like T. subterraneum. The higher bon and several soil physical, hydrological, and microbial soil N-content caused by T. subterraneum than spontaneous properties. Ramos et al. (2020) also found that oat-vetch flora cover cropping is in accordance with Kuo and Jellum cover crops enhanced soil quality (SOM and total N content, (2002), who indicated that as plant tissue N increased and phosphatase, and β-glucosidase activities) in semiarid envi- tissue C/N ratio decreased (typical characteristics of legumi- ronments compared to frequently tilled management. Here, nous species), the N mineralization rate increased. Similarly the significant improvement of SOM in TCC-B plots was to SOM and macro- and microelements, TCC-B performed probably caused by a multiple cascading effect of carbon better than TCC-S in enhancing the mineral N content, likely input from residue decomposition, carbon capture from the due to the faster decomposition rate of subterranean clover 1 3 Trifolium subterraneum cover cropping enhances soil fertility and weed seedbank dynamics… Page 13 of 16 70 residues into the soil. The work of Kuo and Jellum (2002) of available food resources from grain crops, which are nor- also states that the incorporation of cover crop residues mally harvested in the same period, may have promoted the shows higher soil organic C and NO than removing them. predation of weed seeds by birds and edaphic fauna. Under In a recent study, Lombardo et al. (2021) found that TCC-B rainfed conditions and low-input agricultural regime such as improved the nutritional status of the apricot by significantly that of our experiment, several studies have found signic fi ant increasing the content of macro- and micronutrients in both weed seed loss from predation when seeds remained on the leaves and fruits than spontaneous flora and CM, thus dem- soil surface, up to 60 − 70% (Buhler et al. 1997). In addi- onstrating how the beneficial effects on soil properties were tion, in the late spring and summer, the level of subterranean reflected in the plants. clover allelochemicals accumulated into the rhizosphere (~ ‒ 15 cm) reach the climax due to a cumulative effect and to 4.2 Weed soil seedbank the low rainfall which limits their leaching, thus increasing the fatal seed germination. Moreover, the soil seedbank here The effects of 4 years cover cropping on weed management detected was primarily composed by annual spring–summer were evaluated on the soil seedbank at a depth of 10 − 15 cm, therophytes needing a cold period over winter to broke seed where most weed seeds are commonly concentrated (Swan- dormancy. For such weeds, the dormancy is broken in late ton et al. 2000). In accordance with Scavo et al. (2020), a spring so that their seeds are less detected in autumn (Buh- total of 22 taxa was detected, mainly belonging to the Aster- ler et al. 1997). Interestingly, the long-term effect of cover aceae family, the most diffused weed botanical family in cropping was not limited only to weeds with the same ger- Mediterranean agroecosystems (Restuccia et al. 2019). Weed mination seasonality (i.e., autumn/winter germinating spe- communities were dominated by therophytes and annual cies), but subterranean clover had an effect independently spring–summer weeds such as Sonchus sp., A. retroflexus , from its phase. A similar result was found by Bàrberi and P. oleracea, P. paradoxa, C. napifolia, S. arvensis, and V. Mazzoncini (2001). Furthermore, in their work, the authors cymbalaria, which showed an RD > 4%. However, in agree- reported a not-significant reduction of the seedbank density ment with our previous study (Scavo et al. 2020) and other by using subterranean clover in a conventional tillage system researches (Swanton et al. 1999), cover cropping and sam- while becoming relevant (‒ 22%) if combined with no till- pling time did not have significant effects on RAI data, as no age. It is known that cover crops can be differently managed consistent effects were either observed on species richness. within the orchards by moving or incorporating the plant The lack of substantial changes in the number of weed spe- biomass in order to mitigate their competition with trees. cies among cover cropping treatments is a wide phenomenon A study conducted in an apricot orchard in Turkey using (Ngouajio et al. 2003; Swanton et al. 1999) that some authors Vicia villosa Roth, V. pannonica, V. pannonica + Triticale attribute to the “buffering” effect of the weed seedbank, basi- (70 + 30%), Phacelia tanacetifolia Benth. and Fagopyrum cally caused by weed seed longevity (Bàrberi and Lo Cascio esculentum Moench cover cropping evidenced that these spe- 2001). On the contrary, such effects are often evident on the cies effectively reduced weed richness and density (Tursum emerged weed flora, as found in our previous research (Res- et al. 2018). In addition, the management of these cover crops tuccia et al. 2020) where TCC-S reduced by 21% species through the residue incorporation into the soil enhanced the richness compared to CM. Concerning the seedbank size, weed suppression compared to herbicidal (glyphosate) or TCC-B proved to be the most efficient treatment in reduc- mechanical control (Tursun et al. 2018). The effects obtained ing the number of weed seeds in the soil, even after another here on the soil seedbank were reflected also on the real weed growing season with respect to our previous research, with flora, given that T. subterraneum significantly decreased the a mean reduction of ‒ 57.4% compared to CM, followed by aboveground weed biomass up to 86%, with a positive cor- spontaneous flora cover cropping. The phytotoxic effect of relation between weed growth suppression and cover crop subterranean clover was stronger in April than September, biomass (Restuccia et al. 2020). The higher phytotoxicity of also due to the higher seedbank size in spring. It is likely that subterranean clover cover cropping than spontaneous flora is the differences in the seedbank size between April and Sep- explained by physio-chemical processes. The physical inter- tember are due to (i) the high presence of annual spring–sum- ference is caused by its strong competitive capacity for water, mer weeds with high dormancy rate during autumn, (ii) seed nutrients, light, and space, while the secondary metabolites germination of annual weeds promoted by late summer-early (essentially phenolic compounds and polyphenols) released autumn rainfall, and (iii) species whose seeds massively into the soil via root exudation and decomposition from plant germinate with autumn rainfall are hard to detect in spring residues—especially when incorporated into the soil (TCC- (Buisson et al. 2018). Anyway, a higher seedbank size in B)—are involved in the chemical interference. spring than autumn is a common situation in Mediterranean The eventual presence of associations between treatments environments. Probably, the severe drought conditions typi- under study and weeds was analyzed by PCA on those spe- cal of the late spring and summer associated with the lack cies showing a high enough density (i.e., major weeds). 1 3 70 Page 14 of 16 A. Scavo et al. Data availability The datasets analyzed during the current study are Most variation in the species composition was explained by available from the corresponding author on reasonable request. sampling time, considering that several treatments showed no weed infestation in the autumn sampling, whereas during Declarations spring, the treatments were highly infested. As observed for the emerged weed flora (Restuccia et al. 2020), TCC-B-T2 Ethics approval Not applicable. did not show any particular association with weeds, and its soil seedbank evenness was very high (0.93), indicating the Consent to participate Not applicable. absence of dominant species. The lowest biodiversity was Consent for publication Not applicable. found in the spontaneous flora cover cropping treatments, which at the same time also showed the lowest evenness, Conflict of interest The authors declare no competing interests. thus confirming the association emerging from the multi- variate analysis. Overall, cover cropping did not determine Open Access This article is licensed under a Creative Commons Attri- relevant shifts in weed populations. Other studies have bution 4.0 International License, which permits use, sharing, adapta- shown no clear patterns in weed communities during cover tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, cropping experiments (Moonen and Bàrberi 2004; Shrestha provide a link to the Creative Commons licence, and indicate if changes et al. 2002; Swanton et al. 1999). Indeed, cover crop type, were made. The images or other third party material in this article are management practices, and several biotic and abiotic fac- included in the article's Creative Commons licence, unless indicated tors closely influence both species composition and weed otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not patterns in the soil seedbank. Moreover, it is necessary to permitted by statutory regulation or exceeds the permitted use, you will keep in mind also that the measured soil seedbank does not need to obtain permission directly from the copyright holder. To view a necessarily represent the weed species able to germinate and copy of this licence, visit http://cr eativ ecommons. or g/licen ses/ b y/4.0/ . growth. References 5 Conclusions Adeli A, Brooks JP, Read JJ, Feng G, Miles D, Shankle MW, Barks- dale N, Jenkins JN (2020) Management strategies on an upland This study highlights the benefits deriving from the use of soil for improving soil properties. Commun Soil Sci Plan subterranean clover cover cropping, especially when incor- 51:413–429. https:// doi. org/ 10. 1080/ 00103 624. 2019. 17094 90 porating its dead mulches into the soil, in Mediterranean Adeux G, Vieren E, Carlesi S, Bàrberi P, Munier-Jolain N, Cord- orchards. 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Journal

Agronomy for Sustainable DevelopmentSpringer Journals

Published: Dec 1, 2021

Keywords: Cover cropping; Subterranean clover; Soil health; Soil organic matter; Soil macroelements; Soil microelements; Weed soil seedbank

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