A 3-year field experiment was conducted in 2019 through 2022 at Agios Athanassios (northern Greece; longitude 22°23΄58΄΄ E, latitude 40°43΄32΄΄ N; 45 m above sea level) to investigate the possible interference (allelopathic effect) of silverleaf nightshade decomposed leaves/stems/fruits on durum wheat growth and yield components. The soil was a calcareous sandy loam (Typic xerorthents) with sand 55%, silt 30%, clay 15%, organic matter 1.2% and pH 7.9. During the experiment, the mean monthly temperature and the total monthly rainfall data were recorded and are presented in Fig. 1. No irrigation was applied during the experiment according to the practice of cereal farmers in the area.

A natural silverleaf nightshade infestation of 14 to 34 plants m^-2 was present as when evaluated at early summer in 2019. During this summer three treatments (A, B and C) were established in a complete randomized design with four replicates:

Plot size was 20 × 10 m. The biomass of the survived silverleaf nightshade {about 4.5-4.8 t ha^-1 (which could be compared with the concentration of 6% of bioassays) or 8.3-8.6 t ha^-1 (which could be compared with the concentration of 12% of bioassays) at 14-18 or 30-34 plants m^-2, respectively} was incorporated into the soil during the seedbed preparation in November. In particular, before the durum wheat seeding, the soil was prepared by a four-bottom moldboard plow to a depth of ~ 22 cm and a cultivator to a depth of ~ 12 cm. At the same time, nitrogen and phosphorus at 50 and 25 kg ha^-1, respectively, were incorporated.

Durum wheat var. ‘Cannavaro’ was seeded by mechanical seeder at 190 kg ha^-1 in late November of the three growing seasons. Nitrogen at 30 kg N ha^-1 was also applied post-emergence in early March. Broadleaved weeds and grasses were controlled by iodosulfuron-methyl-sodium {methyl 4-iodo-2-[3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)ureidosulfonyl] benzoate sodium salt} + mesosulfuron-methyl {methyl 2-[(4,6-dimethoxypyrimidin-2-yl)carbamoylsulfamoyl]-4-(methanesulfonamidomethyl) benzoate} at 5.6 + 5.8 g a.i. ha^-1 when durum wheat was at the middle of tillering (BBCH code 24-28; Meier, 2001) growth stage. After the destruction of silverleaf nightshade plants during the soil cultivation in November, weed regrowth was not observed during the durum wheat growing period. However, weed plants emerged at crop maturity.

On about 12 weeks after seeding (WAS) each year, durum wheat stand (plant number) was assessed at the 2-to 3 leaves growth stage (BBCH code 12-13; Meier, 2001) in a 1 m² in the centre of each plot. Also, at 16 WAS, durum wheat shoot number (tillering) and fresh weight were determined at the 2 nodes growth stage (BBCH code 32) by hand-cutting crop plants from a 1 m² area located in the centre of each plot. In late June each year, durum wheat plants from 1 m² in the centre of each plot were harvested by hand. Durum wheat yield components [total dry weight (TDW), ear number, grain yield (GY), grain number per 10 ears and 1000-grain weight (TGW)] were recorded.

The data collected from the four bioassays (one for each of the crop species) were analysed across years but separately for each crop species. In particular, a 3 × 5 (3 weed parts × 5 extract concentrations) factorial approach in a completely randomized design with three replicates was used for the four bioassay experiments.

The phytotoxic dose-response effects of silverleaf nightshade extracts on the germination and root length of the four cereals were evaluated by the use of the whole-range assessment method reported by An et al. (2005). In particular, the equation:

was used for the calculation of the inhibition indices (I). In particular, 0 to D_n was the range of the concentrations; D_c was the threshold concentration threshold which equaled the value of the control and above that the responses were inhibitory; R(0) was the response at 0 g 100 mL^-1 (control); and the f(D) represented the response function. For the calculation of the inhibition indices (I) (across the whole range of concentrations of the silverleaf nightshade extracts) the WESIA (Whole-range Evaluation of the Strength of Inhibition in Allelopathic-bioassay) software was used (Liu et al., 2007). Furthermore, for both germination and root length I value means, the differences between the cereal pairs (durum wheat and winter wheat, durum wheat and barley, durum wheat and oats, winter wheat and barley, winter wheat and oats or barley and oats) were examined using the paired student’s t-test (α = 0.05).

The field data of durum wheat were analysed across years using the one-factor randomised complete block design. Also, the linear regression equation was tested for its suitability to describe the relationship between the durum wheat parameters and silverleaf nightshade density, and also to compare wheat yield differences amongst years.

SPSS (version 17.0) was used to conduct t-tests (SPSS, 1998), Microsoft Excel was used for the linear regressions, while the ANOVAs were conducted by MSTAT (MSTAT-C, 1988). The Bartlett’s test was used to examine homogeneity of variances, while the Fisher’s protected least significant difference test procedure at p = 0.05 was used to detect and separate the mean treatment differences.

The germination and root length of the four cereals were not significantly affected by year (the two collection years of the silverleaf nightshade plants), but they were significantly (p < 0.001) affected in most cases by the silverleaf nightshade plant part (roots, leaves/stems, flowers/fruits) utilized to produce the aqueous extract, by the concentrations of the aqueous extract, and by the interaction between the plant part and the aqueous extract concentration. Thus, the plant part × aqueous extract concentration interaction means are presented. In general, the germination of the durum wheat, winter wheat and barley was not significantly affected by the extracts of the roots or the flowers/fruits (Figs. 2a, 2c and 3a). In contrast, the highest concentration of leaves/stems extracts reduced the germination of all cereals. However, the germination and the root length inhibition of all cereals increased with increasing extract concentration of the three weed parts, but this increase was not proportionally similar (Figs. 2 and 3).

The durum wheat germination inhibition caused by the leaves/stems extract concentrations of 6% and 12% was 17.0% and 25.1%, respectively (Fig. 2a). The lower concentrations did not cause any significant germination inhibition. The durum wheat root length inhibition caused by the leaves/stems extract concentrations of 6% and 12% was 63.7% and 96.6%, respectively (Fig. 2b). The corresponding reductions caused by the roots or flowers/fruits extracts were 33.1% and 51.0% or 84.4% and 92.0%, respectively. The root length reduction caused by the lower extract concentrations (0.75, 1.5 and 3%) ranged from 0% to 45.9% (Fig. 2b).

The winter wheat germination was significantly reduced (18.5%) only by the highest extract concentration (12%) of leaves/stems (Fig. 2c). However, winter wheat root length was reduced by all silverleaf nightshade extracts (Fig. 2d). In particular, the two greatest concentrations (6% and 12%) caused a 35.9% to 95.6% root length reduction, while the reduction caused by the lower concentrations ranged from 9.1% to 32.4%.

Barley germination was not significantly affected by silverleaf nightshade extracts (Fig. 3a). On the contrary, root length was reduced by 35.2% to 91.7% by the greatest extract concentrations (6 and 12%), while the root length reduction caused by the lower concentrations ranged from 8.8% to 38.3% (Fig. 3b). Similarly to the responses of durum wheat and winter wheat, the leaves/stems and flowers/fruits extracts were more phytotoxic to barley than the ones from the roots.

The extract concentrations of 0.75%, 1.5% and 3.0% did not significantly reduce the germination of oats. However, the germination reduction caused by the 6.0% and 12.0% concentrations ranged from 9.0% to 27.0% (Fig. 3c). Regarding oat root length, the highest extract concentrations (6.0% and 12.0%) reduced oat root length by 41.6% to 61.7%, while the lower concentrations caused root length reductions that ranged from 0.7% to 32.5% (Fig. 3d). The leaves/stems extracts were slightly less phytotoxic than the ones from roots or flowers/fruits.

The paired student’s t-tests conducted showed differences between I indices indicating that the extracts of silverleaf nightshade parts inhibited the germination of durum wheat and oats slightly more than that of winter wheat and barley (Table 1). No significant differences were observed amongst winter wheat, barley and oats regarding the root length inhibition, while root growth was more sensitive to the extracts than the germination. In particular, the increasing order of the silverleaf nightshade extract phytotoxicity, according to the estimated germination inhibition indices, was flowers/fruits (3.24) < roots (5.53) < leaves/stems (8.66), while the respective order, according to the estimated root length inhibition indices, was roots (29.53) < leaves/stems (45.20) < flowers/fruits (53.91).

The ANOVAs performed for durum wheat parameters indicated that crop stand (plant number) and fresh weight were significantly (p < 0.001) affected by year, silverleaf nightshade biomass (incorporated in soil) and their interaction, while the crop shoot number (tillering ability) was significantly (p < 0.001) affected by silverleaf nightshade biomass and year × silverleaf nightshade biomass interaction. Thus, the year × silverleaf nightshade biomass interaction means are presented (Fig. 4).

At 12 and 16 WAS, the silverleaf nightshade biomass that was incorporated into the soil reduced the durum wheat crop plant number, shoot number and fresh weight (Figs. 4a, 4b and 4c). The incorporated 30-34 plants m^-2 of silver nightshade caused greater growth reduction than the 14-18 plants m^-2, while the crop plant number was less affected, except for the year 3, by silverleaf nightshade biomass, compared with those of crop shoot number and fresh weight. In particular, in plots where 14-18 and 30-34 silverleaf nightshade plants m^-2 had been incorporated, durum wheat plant number was reduced by 1.4% and 11.3%, respectively (averaged across year 1 and year 2), compared with silverleaf nightshade mulch-free plots (control) (Fig. 4a). The corresponding reductions in year 3 were 36.0% and 56.5%. Durum wheat shoot number was reduced by 21.4% and 41.2% (averaged across years) by the 14-18 and 30-34 silverleaf nightshade incorporated plants m^-2, respectively (Fig. 4b). The corresponding reductions in durum wheat total fresh weight were 23.3% and 45.9%, with the lower reductions (14.5% and 19.2%) observed in year 2 (Fig. 4c).

Each year, the plant number (Fig. 4a), shoot number (Fig. 4b) and total fresh weight (Fig. 4c) of durum wheat grown under different densities of silverleaf nightshade indicated, in most cases, a proportional decrease with increasing density. This was confirmed by the high R² for the linear regression used to describe the relationship between the durum wheat characteristics and weed density.

During harvest, durum wheat TDW, ears number, GY and TGW were significantly (p < 0.001) affected by year, silverleaf nightshade biomass (incorporated in soil) and by their interaction, while the grain number per ear was significantly (p < 0.001) affected by year and by year × silverleaf nightshade biomass interaction. Thus, the year × silverleaf nightshade biomass interaction means are presented (Figs. 4 and 5).

Durum wheat TDW, GY and ear number were reduced by the silverleaf nightshade biomass that was incorporated in soil more than the TGW and grain number per 10 ears. In particular, the incorporation of 30-34 silverleaf nightshade plants m^-2 reduced durum wheat TDW, ear number and GY by 31.5%, 27.0% and 31.2% (averaged across years), respectively, when compared with the silverleaf nightshade free plots (Figs. 4d, 4e and 4f). The corresponding reductions caused by the incorporated 14-18 silverleaf nightshade plants m^-2 were 15.9%, 17.3% and 16.9%. Similarly, the durum wheat TGW and grain number per 10 ears in plots where the 30-34 silverleaf nightshade plants m^-2 were incorporated were 17.9% and 15.3% (averaged across years), respectively, lower than those in the silverleaf nightshade free plots (Fig. 5). The corresponding reductions caused by the incorporated 14-18 silverleaf nightshade plants m^-2 were 11.8% and 9.4%.

Similarly to 16 WAS in each year, the TDW (Fig. 4d), ears number (Fig. 4e) and GY (Fig. 4f) of durum wheat grown under different densities of silverleaf nightshade indicated a proportional decrease with increasing density. This result was confirmed by the high R² for the linear regression used to describe the relationship between the durum wheat characteristics and weed density.

The germination and root length inhibition of cereals by the aqueous extracts of the three silverleaf nightshade plant parts are in agreement with results reported by Alhemedy et al. (2016), who found that the powder of silverleaf nightshade had allelopathic impact on durum wheat germination and growth. Similarly, Balah et al. (2022) found that silverleaf nightshade extracts reduced germination, as well as root and shoot length of various plants such as winter wheat, barley, Egyptian clover (Trifolium alexandrinum L.), alfalfa (Medicago sativa L.), horsebean (Vicia faba L.), corn (Zea mays L.), common wild oat (Avena fatua L.), littleseed canarygrass (Phalaris minor Retz.) and common purslane (Portulaca oleracea L.).

The greatest inhibition of root length in durum wheat, winter wheat, barley and oats was caused by the leaves/stems extracts comparted with those of flowers/fruits or roots. Although allelocamicals composition or concentration were nor determined in silverleaf nightshade extracts tested in this study, the different inhibition effects on cereals caused by the leaves/stems, flowers/fruits and roots extracts could possibly be explained by the differences in amount and characteristics of physicochemicals included in these plant parts. Balah (2015) found that the allelochemicals chlorogenic acid and kaempferol β-D-(6-O-cis-cinnamoyl) glucosode, isolated from silverleaf nightshade seeds, were more phytotoxic to common purslane than coumaroyl glucoside, mangiferin, kaempferol or coumaroyl quince acid. Alhemedy et al. (2016) found that the foliage powder of silverleaf nightshade provided greater durum wheat growth inhibition than the one coming from the root. Balah et al. (2022) also found that the vegetative part extracts of silverleaf nightshade had greater allelopathic potential than the ones from the root. Anantharaju et al. (2017) found that silverleaf nightshade contains a high proportion of steroid alkaloid glycosides, including solamargin, solasonine, α-solanine and β-solanine. Eleftherohorinos et al. (1993) also found that saponins in the fruits of silverleaf nightshade exhibited allelopathic effects on cucumbers in Greece raising the possibility of allelopathic effects on other crops. In addition, Curvetto et al. (1976) found that the saponin derived from the fruits of the silverleaf nightshade reduced root growth of squirting cucumber (Ecballium elaterium A. Rich.).

In Greece, winter cereals are seeded after autumn rainfalls because the cereal fields are no-irrigated; furthermore, nitrogen fertilization is applied before seeding. Thus, the depletion of soil moisture and nitrogen because of the presence of silver nightshade during summer or the change of C/N ratio in soil because of the incorporation of silverleaf nightshade biomass could not significantly affect the durum wheat emergence and its initial growth. Consequently, the significant inhibition of durum wheat emergence and growth observed in the field could be attributed to the allelopathic effect of silverleaf nightshade incorporated biomass. This result is strengthened by the results reported by Balah et al. (2022), who found great amounts of allelochemicals in the rhizosphere of silverleaf nightshade. These allelochemicals caused significant suppression of rhizosphere bacteria. Alhemedy et al. (2016) also found that silverleaf nightshade powder had a negative effect on durum wheat emergence and growth. Similarly, Mkula (2006) found that the silverleaf nightshade extracts and soil-incorporated residues reduced the germination and early growth of cotton (Gossypium hirsutum L.). Travlos et al. (2013) found that the silverleaf nightshade competition caused 8% to 26% annual yield loss in alfalfa. Lemerle & Leys (1991) also reported that when silverleaf nightshade was chemically controlled (by repeated applications of 2,4-D or glyphosate) prior to wheat seeding, GY was increased by 14% to 69%, with the largest increases in the drought growing season.

The greater reduction in durum wheat emergence observed in year 3, caused by the incorporated biomass of silverleaf nightshade, could be attributed to the observed lower rainfall (about 50%) during November and December (the period of durum wheat emergence) (Fig. 1). This fact may lead to lower leaching of the allelochemicals of silverleaf nightshade and consequently to their greater concentration around the crop rhizosphere (Real et al., 2019; Zhang et al., 2021).

Similarly, the lower reduction of durum wheat growth and yield recorded in Year 2, compared to the reductions in Year 1 and Year 3, could be attributed to greater rainfall and temperature recorded from December to January in Year 2 (Fig. 1). These conditions could favor the durum wheat establishment, while they increased the decomposition and the leaching of the silverleaf nightshade allelochemicals (Latif et al., 2017).

The glyphosate total rate used (15840 g ai ha^-1) to suppress the continued silverleaf nightshade summer regrowth in treatment A (without weed presence) was at least twice the maximum glyphosate rate allowed in Greece. Considering the probable future limitation of herbicide use in the European Union, combining herbicides with mechanical control methods could be used for silverleaf nightshade suppression (Roberts & Florentine, 2022).

Conclusively, the results of this study indicated that the silverleaf nightshade extracts significantly reduced the root length of four winter cereals but less their germination, with the leaves/stems and flowers/fruits extracts being more phytotoxic than the ones from the roots. In field conditions, residues of the aboveground part of silver nightshade incorporated in soil by tillage before seeding significantly reduced the growth and yield components of durum wheat. This fact could be attributed to the allelopathic effect of the weed. Therefore, the effective management of silver nightshade (maybe by combined herbicide applications and mechanical control) before durum wheat seeding could increase wheat productivity.