Leaf- and canopy-level chlorotic symptom variations are associated with differential <i>Candidatus</i> Liberibacter asiaticus infection patterns in Ponkan mandarin under field conditions

ABSTRACT Ponkan mandarin ( Citrus reticulata cv. Ponkan) is an important fruit crop planted across different countries. One of its most devastating diseases is citrus huanglongbing (HLB), which is associated with infection by the bacterium Candidatus Liberibacter asiaticus ( C Las). Although studies on HLB in Ponkan mandarin have been conducted, symptom heterogeneity and its association with C Las titer under field conditions remain underexplored. We examined the association between symptom variation and C Las infection status in a Ponkan mandarin grove by categorizing symptoms at both the leaf and canopy (tree) level. Chlorotic leaves were sampled and classified into different categories, including leaves exhibiting interveinal chlorosis (Cat. A), those showing vein yellowing (Cat. B), and blotchy mottled leaves (Cat. C). Polymerase chain reaction (PCR) and quantitative PCR (qPCR) assays targeting C Las revealed that Cat. A leaves, which resembled nutrient-deficient leaves, had higher C Las detection rates and a larger proportion of samples with high C Las titers. Canopy-level assessment of symptom diversity within each tree also revealed that plants bearing Cat. A leaves were more likely to carry C Las and have high pathogen loads. The likelihood was also greater in trees with higher leaf symptom diversity. Additional prophage typing of the detected C Las strains revealed that leaf symptom variations were independent of prophage type difference. Nutrient analyses revealed reduced magnesium, manganese, and zinc contents in Cat. A leaves, indicating that leaves prone to C Las infection also exhibited nutrient deficiencies. These findings highlight the complexity of potential cultivar-specific associations between C Las and citrus under field conditions. IMPORTANCE Although it is well-established that HLB in Ponkan mandarin is associated with C Las infection, the relationship between leaf symptom variation in this cultivar and the presence and titer of C Las under complex field conditions remains unclear. By examining symptom heterogeneity in natural field conditions at both the leaf and canopy level, this study demonstrated that HLB leaf symptom variation in naturally infected Ponkan mandarin corresponds to differences in C Las presence and titer at both levels. Notably, although blotchy mottling is considered the typical leaf symptom of HLB, we found that C Las detection rates and titers tended to be greater in leaves showing nutrient deficiency-like symptoms, which contrasts with patterns in other citrus cultivars. Recognizing inconsistencies among cultivar-specific patterns is important for interpreting HLB symptoms, devising sampling strategies, and exploring uncharacterized factors affecting symptom expression under field conditions.