High Incidence of Diplodia Infection in HLB Symptomatic Orange Fruit and Its Implication in the Pre-harvest Fruit Drop

Huanglongbing (HLB, also known as greening), presumably caused by the α-Proteobacteria Candidatus Liberibacter, is one of the most devastating diseases of citrus and is currently having significant impact on the Florida citrus industry.  In an investigation conducted to identify fungal species in juice from HLB-symptomatic fruit, we found that Diplodia natalensis (Diplodia) was consistently present in HLB-symptomatic fruit (harvested from HLB symptomatic trees), but not in non-symptomatic fruit (harvested from trees showing no symptoms of HLB).  Diplodia is the causal organism of citrus stem end rot; the pathogen infects citrus fruit under the calyx, and is associated with cell wall hydrolytic enzymes involved in abscission.  Stem end rot caused by Diplodia is rarely seen in fruit on the tree, but typically occurs following harvest and is exacerbated by exposure to ethylene.  One symptom of HLB is excessive fruit drop, and as the severity of HLB has increased throughout Florida, preharvest fruit drop has become the most severe ever recorded.  Based  the consistent incidence of Diplodia in HLB-affected fruit and  the relationship between Diplodia and cell wall hydrolytic enzymes in the calyx abscission zone, we conducted experiments to determine the potential role of Diplodia infection in fruit drop.  Based on qPCR analysis using primers specific for the ß-tubulin gene of Diplodia, we found a significantly greater incidence of the pathogen in the columella of HLB-symptomatic fruit than in non-symptomatic fruit (70% and 5%, respectively). Diplodia infection was confirmed by morphology of conidia in isolates from the calyx abscission zone of HLB-symptomatic fruit. Two weeks following exposure to ethylene (10 ppm, 4 days) the incidence of stem end rot in HLB-symptomatic fruit (66.7%) was significantly greater than in non-symptomatic fruit (6.7%). Fruit detachment force for 88% of the HLB-symptomatic fruit was between 0-20N, whereas for non-symptomatic fruit, detachment force was 30-45N. Ethylene production from non-HLB-symptomatic was below the limit of detection, however, ethylene production was detected from 60% of symptomatic fruit. Fruit detachment force was negatively correlated with Diplodia titer (R² = 0.79), and positively correlated with ethylene production (R² = 0.70), suggesting that Diplodia infection may contribute to the HLB-associated pre-harvest fruit drop.  In addition to the potential impact on fruit drop, an increase in Diplodia infection could lead to increased incidence of stem end rot in fruit destined for the fresh market because such fruit are typically treated with ethylene to enhance chlorophyll degradation (“degreening”).