Cunde Pan

Walnut (Juglans regia Linn.) leaf scorch (WLS) was first reported in 2012 in Hotan, Xinjiang, China (Zhang et al., 2012). Initially, brown spots appear at the apex of the leaflets with slight shrinking. These spots spread inward along the leaf margins in a flame-like pattern. The scorched areas curl inward with a yellow halo. No powdery or bacterial signs were observed on the leaf surface. In severe cases, the leaves dried up and shrank, affecting the entire tree. However, new leaves did not show any signs of scorching. We collected 300 symptomatic leaf samples from 10-12 year-old trees of the susceptible WLS species Wen185, located in Daryaboyi (40°72'N, 80°49'E), Xakal (40°69'N, 80°52'E), and Karatal (40°73'N, 80°39'E) for X. fastidiosa PCR detection analysis. X. fastidiosa was detected in asymptomatic leaves of trees with severe WLS, as well as in asymptomatic leaves of trees exhibiting mild WLS symptoms, and it was even found in asymptomatic leaves of trees without any WLS symptoms.To isolate X. fastidiosa, walnut leaves with petioles were disinfected with 3% bleach for 10 minutes, followed by four washes in autoclaved deionized water. The midrib and petiole of the leaf were cut off with a sterile blade, and a 2 mm to 3 mm section was excised. Then, the cut ends were squeezed with a pair of pliers, and the sap was blotted onto Periwinkle Wilt (PW) plates (Davis et al. 1983). The plates were sealed with parafilm and incubated at 28°C in the dark, with daily observations for the development of individual colonies. The sap collected from 5 out of 20 leaf samples with scorch symptoms and from 20 leaves without symptoms never showed bacterial growth in PW.. Six colonies were tested and confirmed positive for X. fastidiosa using the RST31 and RST33 primer pair (Minsavage et al., 1994). The uploaded sequence accession numbers are PP871340-PP871342. Blast analysis of Multilocus Sequence Typing (MLST) sequences from the isolated strain using the X. fastidiosa MLST Database (http://pubmlst.org/xfastidiosa) revealed a perfect match with sequences of alleles leuA_3, petC_3, malF_, cysG_3, holC_4, nuoL_3, and gltT_3 (PP871343-PP871349). Using MEGA software to concatenate the MLST single gene fragments and construct a ML multi-gene fragment phylogenetic tree through Maximum Likelihood (ML) analysis, the bacterium was identified as X. fastidiosa subsp. multiplex To fulfill Koch's postulates, a purified colony from the PW plate was suspended in 500 μL of deionized water at 1×10^8 CFU·mL-1. A sterilized 5 ml medical needle was used to prick a small hole on the main branch, 5 cm from the base, of three-year-old walnut seedlings, and the bacterial solution was injected into the small hole. Three biological replicates were performed for each treatment (5 μL and 10 μL of bacterial solution, deionized water), and the experiment was repeated three times. Scorching symptoms were observed three months after inoculation in 11 out of the 15 seedlings inoculated with a 10 μL bacterial solution, and isolation of X. fastidiosa occurred in leaf samples from 5 of these seedlings, completing Koch's postulates. Walnut seedlings with water stayed asymptomatic, PCR negative. To our knowledge, X. fastidiosa has been found infecting grapes (Chu, 2001) in Shaanxi, China and pears (Su et al., 2016) in Taiwan, China. This is the first report of X. fastidiosa involvement in WLS in China. Further research on the occurrence of the disease will help prevent the spread of the disease.

Fruit quality is known to be regulated by the balance between leaf number/area and fruit number, but less is known about the effects of fruit–bearing branch agronomic traits, particularly for walnuts (Juglans regia L.). We assessed nut quality, physiological and biochemical indexes of leaves, and microstructure of leaves and fruit stalks at various leaf–to–fruit ratios to gain insight into the relationships between branch agronomic traits and nut quality, to identify those traits that made a better contribution to nut quality and to find out the range of the leaf–to–fruit ratio and the object of fruit to be removed for thinning. We found that the top fruit on the fruit stalk had a higher longitudinal diameter and kernel weight than the bottom at the low leaf–to–fruit level, and branches with more pinnate compound leaves had a better capacity for carbohydrate assimilation and transportation to produce better quality fruits. Specifically, with the increasing leaf number, the branch diameter, total leaf area, net photosynthetic rate, chlorophyll content, fruit weight, fruit diameters, and kernel protein content also increased. Moreover, at the microscopic level, the fruit stalk vascular bundle, leaf thickness, palisade mesophyll thickness, and ratios of palisade mesophyll to spongy mesophyll thickness in the leaf also showed the same trend. Therefore, when the ratio of leaf area to the fruit number was less than 181.5 square centimeters per fruit on the branches, reducing the fruit number could improve the size and the crude fat content of fruits. While the ratio was more than 247.8 square centimeters per fruit, the fruit number had no significant effect on fruit quality, but increasing leaf area and branch diameter could improve the fruit size and yield.

The Gly-Asp-Ser-Leu (GDSL)-lipase family is a large subfamily of lipolytic enzymes that plays an important role in plant growth and defense against environmental stress. However, little is known about their function in pecans (Carya illinoensis K. Koch). In this study, 87 CilGDSLs were identified and divided into 2 groups and 12 subgroups using phylogenetic analysis; members of the same sub-branch had conserved gene structure and motif composition. The majority of the genes had four introns and were composed of an α-helix and a β-strand. Subcellular localization analysis revealed that these genes were localized in the extracellular matrix, chloroplasts, cytoplasm, nucleus, vacuole, and endoplasmic reticulum, and were validated by transient expression in tobacco mesophyll cells. Furthermore, the analysis of the promoter cis-elements for the CilGDSLs revealed the presence of plant anaerobic induction regulatory, abscisic acid response, light response elements, jasmonic acid (JA) response elements, etc. The qRT-PCR analysis results in “Pawnee” with salt treatment showed that the CilGDSL42.93 (leaf) and CilGDSL39.88 (root) were highly expressed in different tissues. After salt stress treatment, isobaric tags for relative and absolute quantitation (iTRAQ) analysis revealed the presence of a total of ten GDSL proteins. Moreover, the weighted gene co-expression network analysis (WGCNA) showed that one set of co-expressed genes (module), primarily CilGDSL41.11, CilGDSL39.49, CilGDSL34.85, and CilGDSL41.01, was significantly associated with salt stress in leaf. In short, some of them were shown to be involved in plant defense against salt stress in this study.

Carbohydrate content and metabolism‐related enzyme activities were analysed in walnut fruits during fruit growth by artificial manipulation of the leaf‐to‐fruit ratio (LFR) to investigate the relationship between carbohydrate utilisation and LFR. LFR significantly affected glucose and sucrose content, and sucrose synthetase (SS) activity in the pericarp or kernel. When the leaf number was the same, sucrose content and SS activity in the pericarp and kernel decreased with increasing fruit load, while glucose and fructose content and acid invertase (AI) activity in the pericarp increased gradually. When the fruit number was the same, glucose and sucrose content and SS activity in the pericarp and kernel increased gradually as the number of leaves increased, while fructose content and AI and sucrose phosphate synthetase (SPS) activity in the pericarp increased, thereby indicating that enhanced AI activity in the pericarp improved the competitive ability of the sink when LFR was low during the fast fruit‐growing period, while, high AI activity rapidly decomposed sucrose into hexose to supply the energy required for cell multiplication and growth during the fruit fast‐growing period, when LFR was high. During the oil transformation period, the increase in sucrose content in the pericarp resulted from SS and SPS activities.

Based on 13 C isotope tracer technology, we investigated the 13 13 C, 13 Camount, and 13 CPCT were recorded in sucrose. These results suggested that sucrose was the main form of carbohydrate assimilation and transportation in walnut fruit, and large amounts of sucrose are accumulated in the peel and in the kernel, where it is also the base for the transformation of oil.