Juglone is believed to be the only allelochemical in walnut and the reason for its allelopathy. Nevertheless, to this day, experts disagree on whether juglone is truly an allelochemical or the only allelochemical in walnut precisely because of conflicting research findings. Therefore, our first goal was to determine whether naphthoquinones are present in all parts of the walnut plant or only in certain ones. To verify this, we developed a new LC-MS/MS analytical method to identify naphthoquinones. We also determined when in the growing season and in which parts of walnut the content of naphthoquinones and other phenolics is the highest. Thus, naphthoquinones were identified and quantified in the leaves, petiole, husk, dormant buds, bark (annual and biennial), catkins (male flowers), female flower, and roots (main and lateral roots), whereas we did not detect them in the pellicle or in the peeled kernel. The content of naphthoquinones was the highest in the first months after budbreak (May), but later it decreased. The aim of the study was also to determine whether hydrojuglone-β-D-glucopyranoside and α-hydrojuglone are indeed precursors of juglone. We found that both hydrojuglone-β-D-glucopyranoside and α-hydrojuglone are indeed precursors of juglone. We also observed that the conversion of hydrojuglone-β-D-glucopyranoside to α-hydrojuglone and then to juglone in walnut husk at room temperature occurs in a time interval of less than 40 minutes. We also found that juglone is not the only allelochemical in walnut leaf extract and that there are others that can stimulate the germination of certain plants while inhibiting the germination of others. The data obtained will be an important basis for determining the plant species that do not grow well in the soils containing the aforementioned allelochemicals, especially in the case of a change of purpose of the land where walnuts were grown, and thus also a basis for selecting appropriate solutions and crops in the first years of planting.
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