# An Untargeted Metabolomic Approach to Characterize the Emerging Kernel Disorder “Orange Spot” in Walnut (Juglans regia L.) cv. Chandler

## TL;DR

This study investigates the cause of 'orange spot' in walnut kernels and finds it is a non-infectious disorder linked to oxidative stress and lipid changes.

## Contribution

The study provides the first metabolomic characterization of orange spot in walnuts, linking it to oxidative stress and lipid disorganization.

## Key findings

- Orange spot is not caused by bacterial infection.
- Affected kernels show metabolic changes in glyoxylate, glutamate, and glycerophospholipid pathways.
- Increased fatty acid content in affected kernels suggests oxidative stress.

## Abstract

Background/Objectives: The physiological disorder known as “orange spot” in Juglans regia L. cv. Chandler is an emerging kernel quality issue of increasing concern for the Chilean walnut industry. Characterized by a localized orange discoloration of the embryo, its etiology remains unknown. This study aimed to determine whether orange spot is associated with bacterial infection and to elucidate the metabolic alterations underlying its development, testing the hypothesis that it results from oxidative imbalance and lipid disorganization. Methods: Untargeted metabolomic profiling and fatty acid methyl ester (FAME) analysis were applied to compare affected (orange) and unaffected (white) walnut kernels collected from a commercial orchard in Melipilla, Chile, during the 2018 and 2022 seasons. Bacterial DNA was screened by PCR amplification of the 16S rRNA gene (799F/1193R). Results: Orange spot incidence reached 31–34% across seasons. No bacterial-sized amplicons were detected in either kernel type, indicating the absence of detectable bacterial DNA under the analyzed conditions. Metabolomic analyses revealed significant alterations in glyoxylate and dicarboxylate metabolism, glutamate-related pathways, and glycerophospholipid metabolism. Affected kernels exhibited reduced levels of L-glutamic acid and antioxidant-associated metabolites, alongside increased gluconic, citric and quinic acid, consistent with the redox imbalance and membrane oxidative degradation. FAME analysis showed higher total fatty acid content in affected kernels, suggesting intensified lipid turnover associated under oxidative stress. Conclusions: Orange spot is a physiological, non-infectious disorder linked to premature oxidative stress and lipid disorganization, negatively affecting walnut kernel quality.