There is a keen interest in nut tree crops worldwide, because their production shows a massive increasing trend in many countries. During the last two decades the global Persian or English dried walnut (Juglans regia L.) production increased by 20% annually. Currently, the harvested quantity of dried walnuts with shells is 3.5 million tons globally (FAO, 2023). In Hungary, the ecological demands of walnut are similar to wine grape (Vitis vinifera L.) (Nyitrainé Sárdy et al., 2022) and apricot (Prunus armeniaca L.) (Mendelné Pászti et al., 2023). The growing area doubled to 6,000 ha during the past three decades, and the production reached the 6,000 tons dried shelled nuts annually (FAO, 2023).

The cultivars with large, at least 32 mm in diameter nut size are the most valuable product on the market (Akca, Y et al., 2016; Vahdati et al., 2019; Iordănescu et al., 2021; Özcan et al., 2022; Sokolova, 2022; Sutyemez et al., 2022; Paunović and Rade, 2023). There are some possibilities for growers to increase the nut size and to enrich the phenolic compounds of kernels. Among the phenolics, ellagic acid, ferulic acid, gallic acid, catechin, vanillic acid, caffeic acid, sinapic acid, salicylic acid, rutin, and epicatechin were abundant in the kernel and seed coat (Colaric et al., 2005; Bujdosó et al., 2014; Gharibzahedi et al., 2014; Kónya et al., 2015; Trandafir et al., 2016/a; Rahmani et al., 2018; Kafkas et al., 2020; Trandafir and Cosmulescu, 2020; Shen et al., 2021; Medic et al., 2021/a; Medic et al., 2021/b; Li et al., 2023; Wu et al., 2023). Different papers reported quite similar quantities of phenolics in three different forms, however their sequence is always the same. The largest quantity of the phenolics is in free form, which rate is 51.1%-75.8%, followed by bound form (17.7%-38.0%) and esterified form (1.3%-18.7%) (Persic et al., 2018/a; Wu et al., 2021; Wang et al., 2022; Wu et al., 2023). The main source of the phenolic compounds (Colaric et al., 2005; Persic et al., 2018/a; Sheng et al., 2021; Medic et al., 2021/a; Jin et al., 2022) is pellicle, which contains almost 95% of the phenolic compounds, which can be detected in kernel (Slatnar et al., 2015). Positive and very strong correlations were found between the total phenolic content in free, esterified, and bound forms in pellicle and the pellicle color (0.920, 0.990, 0.940) (Wang et al., 2022). Kernels with a yellow pellicle contain more total phenolic content and flavonoids than a kernel with red pellicle (Trandafir et al., 2016/b; Peršić et al., 2018/b).

Beside the genetic backgrounds of the cultivars, just usage of the biostimulants is the only way to reach both aims described above, as they do not apply mechanical and chemical thinning in the nut tree production nowadays. The biostimulants have some positive effects on the plant characteristics, which can be seen from outside and can be measured inside. Usage of them is typical for herbaceous species mostly. In the nursery production usage of biostimulants (benzyladenine in 0.02%) increased feathering of the woody fruit bearing species such as apple, European plum, and cherries (Magyar and Hrotko, 2002). Furthermore, biostimulants had a positive influence on the growth of container grown shrubs, this application (applied biostimulants: Kelpak^® in 0.3%, Yeald Plus^® in 1.5%, Bistep in 0.5% dosages) improved their market value (Kovács et al., 2017). They increased the yield of different crops such as on faba bean (Lammas et al., 2022.), barley (Lakić et al., 2022), spring wheat (Lozowicka et al., 2022), wheat (Iwaniuk et al., 2022), sweet pepper (Ombódi and Toók, 2022), and potato (Mystkowska et al., 2022). Yield of peanuts was increased 25% to 296% after usage of the biostimulant treatments (Khudaykulov et al., 2021) compared to the control. Length of spike on spring barley plants increased significantly, and weight of grains of spring barley increased also up to 30% to 31% (Lammas et al., 2022) with 7% up to 14% on corn (Ajaj et al., 2020). Number of seeds per pods of faba bean become more with 22% (Lakić et al., 2022), oil content of peanut increased 2% to 7% after the application (Khudaykulov et al., 2021) compared to the control. Protein content of spring barley was increased by 11.9% to 12.8% by usage of Restart Zh (Lammas et al., 2022.). On ‘Oita 4’ mandarin easily extractable glomalin-related soil protein application combined with some other agricultural treatments (fruit bagging, reflective film mulching, and grass-proof cloth mulching) had positive effects on fruit quality (coloration value, hardness, fruit size, weight of pulp, peel and single fruit) (Lei et al., 2023). On strawberry, biostimulant applications had an effect on the yield and total anthocyanin content, but there were no effects on the total soluble sugar and acid content (Popovic et al., 2022). The biostimulant treatments decreased the mycotoxins contamination in the seeds of spring wheat (Lozowicka et al., 2022), and spring barley (Lammas et al., 2022). Among Eastern Bulgarian conditions ‘Lozen 1’ coriander contained from 2.9% to 9.6% more essential oil content after applying the biostimulant Fertigrain – 1.26% and the foliar fertilizer Masterblend – 1.25% (Georgieva et al., 2022). On oilseed plants usage of HL100, HLN55, and TH1-20% biostimulants decreased the saturated (by 3.5%, 1.74%, and 4.7%) and polyunsaturated fatty acids (by 2.74%, 0.59%, and 3.1%) content, however the monounsaturated fatty acid content increased (by 0.99%, 0.58%, 1,47%) (Petrova et al., 2023). Application of potassium (K) foliar fertilizers (such as Alga K Plus – 1%) had a moderate increase of cluster weight of wine grape cultivar ‘Zéta’. It also increased the sugar content in its juice and acceleration of ripening processes compared to the control vines. These results pointed out the positive effects of K foliar fertilization during the ripening process due to K deficiency of grapevines (Zsigrai and Juhász, 2000).

Currently, there is no data about effects of biostimulants’ applications on walnut, therefore the possible responses of this nut crop should be checked. Aim of this study was to examine the responses of nut size characters and some phenolic compounds of walnut kernel on usage of different biostimulants applied in a bearing orchard.

The applied biostimulants had different effects on the physical parameters of the nuts. In the case of nut length, all treatments had positive effects compared to the control during both years ( Figure 1 ).

All treatments resulted to a larger nut diameter than the control. In both years, the diameter of the examined nuts reached the lowest border of first grade category ( Figure 2 ).

In both observed years, all treatments resulted to heavier dried nut weight compared to control. Among the treatments, the heaviest dried nut weight was produced by Wuxal Ascofol in 2020. In 2021, nuts after all treatments had the same value of dried nut weight. The Alga K Plus treatments reached the same values in both observed years; there was no effect of year in this case ( Figure 3 ).

All treated kernels were heavier than the control in 2020 and in 2021. Among the Wuxal Ascofol treatments, a heavier kernel weight was produced in 2020, than in 2021. Among the Kondisol, Alga K Plus, and control treatments, heavier dried kernel weights were measured in 2021 compared to 2020 ( Figure 4 ).

The applied biostimulant treatments had positive effects on the kernel recovey in both observed years, except the Kondisol treatment had a significantly negative effect in 2020 compared to the other treatments ( Figure 5 ).

There were big differences in the quantity of phenolic compounds detected in the kernel between the two years during our research. In 2021, all observed compounds had higher concentrations compared to their quantities measured in 2020.

Figure 6 shows a typical HPLC chromatogram of ‘Alsószentiváni 117’ (retention times of observed phenolic compounds; pirocathecin 8.6 min, catechin 9,5 min, chologenic acid 12,2 min, rutin 16.3 min, quercetin 18.5 min, juglon 19.4 min, cinnamic acid 21.2 min, gallic acid 32.9 min).

In 2021, the pirocatechin concentration was 5 to 14-fold higher than in 2020. In 2020, the highest concentration of pirocatechin was measured in control, followed kernels treated with Alga K Plus, Wuxal Ascofol, and Kondisol. In 2021, the quantity of this compound decreased in all kernels with applied biostimulants treatments compared to the control ( Figure 7 ).

For catechin, kernels from trees treated with Wuxal Ascofol and Alga K Plus reached the highest concentration in 2020. In the case of 2021, the Kondisol treatment had the highest concentration followed by control, Alga K Plus, and Wuxal Ascofol treatments. The concentration of catechin was 7-fold higher in the control and Kondisol treatment, followed by a 4-fold after Wuxal Ascofol and Alga K Plus treatments in 2021 compared to 2020 ( Figure 8 ).

The sequence of chlorogenic acid content was similar in both observed years, but the chlorogenic acid concentration was 4-fold higher after all treatment applications in 2021, than in 2020. Kernels treated with Kondisol reached the highest concentration followed by Alga K Plus, as well as Wuxal Ascofol treatments and control ( Figure 9 ).

There were large differences in the rutin concentration between 2020 and 2021. Kernels from Alga K Plus treatment and the control produced 17-fold, Wuxal Ascofol-treatment 15-fold, and Kondisol treatment had 10-fold higher rutin quantity in 2021 than in 2020. Among the biostimulants the sequence was different in both observed years. In 2020, kernels with Kondisol had the highest rutin concentration, followed by the two other biostimulants and control, which reached the same amount. In 2021, kernels with Alga K Plus application produced the highest concentration of rutin, followed by Kondisol, control, and Wuxal Ascofol treatments ( Figure 10 ).

The quercetin concentration increased 4-fold from 2020 to 2021 after all treatments. Within the observed years, there were quite similar values. In 2020, the Kondisol treatment reached the highest value, followed by control, Wuxal Ascofol, and Alga K Plus applications. In 2021, Alga K Plus, Kondisol treatments, control, and Wuxal Ascofol applications were in descending order ( Figure 11 ).

Juglon is one of the most important compounds of walnut, therefore we put more attention on it. The control, kernels with Wuxal Ascofol, and Alga K Plus treatments produced 3-fold, the kernels with Kondisol reached a 2-fold higher juglon concentration in 2021 than in 2020. Among the treatments the sequence of juglon concentration was different between 2020 and 2021. In 2020, Kondisol, Wuxal Ascofol, control, and Alga K Plus treatments were the decreasing order. However, the control reached the highest concentration of juglon in 2021, followed by Wuxal Ascofol, Alga K Plus, and Kondisol applications ( Figure 12 ).

The cinnamic acid concentration showed a 1.2 to 1.4-fold increase from 2020 to 2021. During 2020, the control produced the highest cinnamic acid quantity followed by samples with Kondisol, Wuxal Ascofol, and Alga K Plus treatments. In 2021, again the control reached the highest cinnamic acid concentration followed by Kondisol, Alga K Plus, and Wuxal Ascofol treatments ( Figure 13 ).

In 2021, we detected 3 to 4-fold higher gallic acid concentration after all treatments than in 2020. In 2020, the control had the highest gallic acid quantity, followed by samples treated with Wuxal Ascofol, Alga K Plus, and Kondisol. In the next year, we measured the highest concentration in the control and in a sample with Wuxal Ascofol, Alga K Plus and Kondisol treatments ( Figure 14 ).

The applied biostimulants had different effects on nut and kernel characteristics as well as the quantity of phenolic compounds in the kernels during the two trial years. All nut size and kernel traits increased after the biostimulants treatments in both observed years, except in the case of kernel recovery after Kondisol treatment in 2020. There was a significant difference between the treated nut length and the control in 2020, this difference was not observed in 2021. There were no significant differences in the nut diameter during the observed two years. There were significant difference in dried nut weight between the control and nuts treated with Wuxal Ascofol in 2020. There were significant difference in dried kernel weight between the control and nuts treated with Wuxal Ascofol in 2020. In 2021, the Alga K Plus produced a significantly positive effect on kernel recovery than the other treatments and control. It is clear, that there were huge differences between years of 2020 and 2021. The early spring weather during the pistillate flowering receptivity was warmer in 2020 than in 2021, which might have caused this difference.

According to Wielgolaski (2001) climatic factors correlated well to the phenological stages, and the air temperature is reported to be the major force for the onset of the early spring phenological stages (Rodriguez-Rajo et al., 2003; Wielgolaski, 2003; Crepinsek et al., 2009; Cosmulescu et al., 2010; Crepinsek et al., 2012; Bîrsanu Ionescu and Cosmulescu et al., 2017; Cosmulescu et al., 2015; Bujdosó et al., 2022) such as pistillate flower receptivity. Negative relationships between the heat demand of early spring phenological stages and daily air temperatures indicate that warm weather is required for early flowering (Emberlin et al., 2007). ‘Alsószentiváni 117’ has one of the earliest pistillate flowering receptivity listed on the Hungarian walnut assortment.

Respect to phenolic content, this study analyzed eight compounds. In 2020, pirocatechin content was significantly lower than the control. In 2021, again the control had significantly higher pirocatechin content, than those treated with Wuxal Ascofol and Kondisol. Catechin content of kernels treated with Wuxal Ascofol and Alga K Plus were significantly higher than in control and Kondisol in 2020. In the next year, there was no significant difference in the catechin content in all treated kernels. The chlorogenic acid content was higher in kernels treated with Kondisol in both years. In 2020, the Kondisol treatment had significant higher value in chologenic acid content. In 2021, the difference in chologenic acid content was only significant between kernels treated with Wuxal Ascofol and Kondisol. In rutin content, significant differences were only observed in kernels treated with Kondisol in 2020. For the case of quercetin content, kernels treated with Kondisol and Alga K Plus had the highest quantity. They were significantly different from those treated with Wuxal Ascofol and the control in 2021. Significant difference in juglon content was observed between kernels treated with Alga K Plus and Wuxal Ascofol in 2020. In the next year, the untreated kernels had the highest juglon content, followed by kernels treated by Wuxal Ascofol, both were significant different from kernels treated with Kondisol. There was no significant difference in the cinnamic acid content in both observed years. In the case of gallic acid, untreated kernels had the highest content, which were significantly different from all treated kernels in 2020. There was no significant difference in gallic acid in 2021.

Our results confirm statements of Popovic et al., 2022, and Petrova et al., 2023 that different effects of the biostimulants on the compounds can be detected.

The biostimulants affect the plants at different levels, and we have to underline that responses of woody fruit species on biostimulants depend not only on biostimulant – host plant interactions, but also on weather conditions during and after their application. When warm spring weather conditions occurred, not only significant differences in the nut size characteristics were observed, but all examined characters increased.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

GS: Conceptualization, Data curation, Formal Analysis, Methodology, Supervision, Writing – original draft, Writing – review & editing. GB: Supervision, Validation, Writing – original draft, Writing – review & editing. MC: Formal Analysis, Writing – original draft. OT: Data curation, Formal Analysis, Writing – original draft. LK: Data curation, Supervision, Writing – original draft. RO: Data curation, Formal Analysis, Writing – original draft. GF: Data curation, Formal Analysis, Methodology, Supervision, Validation, Writing – original draft, Writing – review & editing. GV: Conceptualization, Data curation, Formal Analysis, Methodology, Supervision, Validation, Writing – original draft, Writing – review & editing.