Although the effects of fertilization and microbiota on plant growth have been widely studied, our understanding of the chemical fertilizers to alter soil chemical and microbiological properties in woody plants is still limited. The aim of the present study is to investigate the impact of long-term application of chemical fertilizers on chemical and microbiological properties of root-associated soils of walnut trees. The results show that soil organic matter (OM), pH_kcl, total nitrogen (TN), nitrate-nitrogen (NO₃⁻), and total phosphorus (TP) contents were significantly higher in non-fertilized soil than after chemical fertilization. The long-term fertilization led to excessive ammonium-nitrogen (NH₄⁺) and available phosphorus (AP) residues in the cultivated soil, among which NH₄⁺ resulted in soil acidification and changes in bacterial community structure, while AP reduced fungal diversity. The naturally grown walnut trees led to an enrichment in beneficial bacteria such as Burkholderia, Nitrospira, Pseudomonas, and Candidatus_Solibacter, as well as fungi, including Trichoderma, Lophiostoma, Phomopsis, Ilyonectria, Purpureocillium, Cylindrocladiella, Hyalorbilia, Chaetomium, and Trichoglossum. The presence of these bacterial and fungal genera that have been associated with nutrient mobilization and plant growth was likely related to the higher soil OM, TN, NO₃⁻, and TP contents in the non-fertilized plots. These findings highlight that reduced chemical fertilizers and organic cultivation with beneficial microbiota could be used to improve economic efficiency and benefit the environment in sustainable agriculture.

Rejuvenation is an efficient approach used in the cuttings of trees and horticultural crops, to improve their rooting ability, especially in difficult-to-root trees. WOX gene family members are involved in cell-fate transformation through balancing the maintenance and proliferation of the stem cells. However, there are no reports about the WOX gene family in Walnut (Juglans regia L.) and its relationship between rejuvenation and adventitious roots formation (ARF). Here, a genome-wide identification of JrWOX genes and their physical and chemical properties, phylogeny, and expression profiles in different organs and during rejuvenation-induced ARF is reported. The phenotype and histology characteristics of mature and rejuvenated cuttings (Mc and Rc) are also observed. In this study, 12 genes were identified and clustered into three groups based on phylogenetics, special domains, and conserved motifs. The gene structures and conserved motifs were relatively conserved, while the 12 sequences of the JrWOXs domain were diversified. Gene expression in root, stem, leaf, female flower, immature fruit, and zygotic embryo revealed that the expression levels of JrWOX4a, JrWOX4b, JrWOX5, JrWOX11, and JrWOX13 in the root were significantly higher than those of other JrWOXs, while only the expression of JrWOX11 was exclusive to the root organ. Additionally, rejuvenation treatment significantly induced almost all JrWOX genes, except JrWOX4a, JrWOX4b, and JrWOX13 (Rc 0 vs. Mc 0). During the ARF process, the transcripts of JrWOX11 and JrWOX5 were consecutively increased on a significance level; in contrast, the transcription levels of the other JrWOXs decreased or changed insignificantly. The phenotype and histology observation indicate that rejuvenation treatment made the base of the stem expand and reduced the thickness and density of sclerenchyma between the cortex and phloem. This might provide the conditions for the formation of new meristem niches. The results provided insight into the JrWOX genes’ general characteristics and their roles in rejuvenation-induced ARF.

Plant growth characteristics after grafting are mainly dependent on photosynthesis performance, which may be influenced by grafting combinations with different rootstocks and scions. In this study, we used one-year-old walnut grafts to investigate the grafting compatibility between precocious (‘Liaoning 1’, L) and hybrid (‘Zhong Ning Sheng’, Z) walnut, as well as rootstock and scion impact on the growth and photosynthetic properties of walnut trees. The results showed that grafting compatibility between the two varieties is high, with survival rates upward of 86%. Overwintering survival of grafted seedlings was as high as 100%, which indicated that the allopolyploid had good resistance to low-temperature stress. The homograft of the hybrid walnut had the highest net photosynthesis rate (18.77 μmol·m⁻²s⁻¹, Z/Z) and growth characteristics, which could be due to its higher transpiration rate and stomatal conductance, whereas the homograft of precocious walnut presented the lowest net photosynthesis rate (15.08 μmol·m⁻²s⁻¹, L/L) and growth characteristics. Significant improvements in the net photosynthesis rate (15.97 and 15.24 μmol·m⁻²s⁻¹ for L/Z and Z/L, respectively) and growth characteristics of precocious walnut were noticed during grafting of the hybrid walnut, which could have been contributed by their transpiration rate. The results of this study serve as a guide for the selection and breeding of good rootstock to improve plant growth characteristics and photosynthetic efficiency. We conclude that good rootstock selection improves plant growth potential and could play an important role in sustainable production.

Dioscorea opposita cultivar Tiegun is an economically important crop with high nutritional and medicinal value. Plants can activate complex and diverse defense mechanisms after infection by pathogenic fungi. Moreover, endophytic fungi can also trigger the plant immune system to resist pathogen invasion. However, the study of the effects of endophytic fungi on plant infection lags far behind that of pathogenic fungi, and the underlying mechanism is not fully understood. Here, the black spot pathogen Alternaria alternata and the endophytic fungus Penicillium halotolerans of Tiegun were identified and used to infect calli. The results showed that A. alternata could cause more severe membrane lipid peroxidation, whereas P. halotolerans could rapidly increase the activity of the plant antioxidant enzymes superoxide dismutase, peroxidase, and catalase; thus, the degree of damage to the callus caused by P. halotolerans was weaker than that caused by A. alternata. RNA sequencing analysis revealed that various plant defense pathways, such as phenylpropanoid biosynthesis, flavonoid biosynthesis, plant hormone signal transduction, and the mitogen-activated protein kinase signaling pathway, play important roles in triggering the plant immune response during fungal infection. Furthermore, the tryptophan metabolism, betalain biosynthesis, fatty acid degradation, flavonoid biosynthesis, tyrosine metabolism, and isoquinoline alkaloid biosynthesis pathways may accelerate the infection of pathogenic fungi, and the ribosome biogenesis pathway in eukaryotes may retard the damage caused by endophytic fungi. This study lays a foundation for exploring the infection mechanism of yam pathogens and endophytic fungi and provides insight for effective fungal disease control in agriculture.