Horticulture

Monitoring the health of Huanglongbing-affected citrus trees by following changes in leaf Candidatus Liberibacter asiaticus (CLas) titer has an inherent element of imprecision because CLas titer varies considerably within the tree canopy and with calendar seasons. In addition, the destructive sampling method used to determine CLas titer entails a different set of leaves per sampling period adding to the inconsistency and inexactitude of the results. To overcome these ambiguities and to reduce the numerical variability between samples, we developed an experimental method that analyzes portions of the same treated leaves for up to four sampling periods. By assaying subsamples of adjacent locations of the same leaf, random variability was significantly reduced, and comparative analysis can be carried out with greater precision.

There is accumulating evidence that root system collapse is a primary symptom associated with Huanglongbing (HLB)-induced tree decline, especially for commercial sweet orange and grapefruit trees on Swingle and Carrizo rootstocks. Maintaining root health is imperative to keep trees productive in an HLB-endemic environment. Preliminary greenhouse and field studies have shown that HLB-impacted trees had secondary and micronutrient deficiencies that were much greater in the roots than in the leaves, and that treatments containing three-times the recommended dose of manganese (Mn) improved tree health and growth and increased feeder root density in greenhouse trees. These results suggested that trees in an HLB-endemic environment have higher specific micronutrient requirements than those currently recommended. To test this hypothesis, established Vernia sweet orange grafted onto rough lemon rootstock trees were divided into eight supplemental CRF nutrition treatments (including two-times and four-times the recommended doses of Mn and boron) using a randomized complete block design in a commercial grove in St. Cloud, FL. The following supplemental nutrition treatments were used: no extra nutrition (control); Harrell’s–St. Helena mix 0.9 kg per tree; Harrell’s with 32 g of Florikan polycoated sodium borate (PSB) per tree; Harrell’s with 90 g of TigerSul® Mn sulfate (MS) per tree; Harrell’s with 32 g of PSB and 90 g of MS per tree; 180 g of MS per tree; 64 g of PSB per tree; and 180 g of MS plus 64 g of PSB per tree applied every 6 months since Fall 2015. Leaf and soil nutritional analyses were performed in Mar. 2017, Sept. 2017, and May 2018; a quantitative polymerase chain reaction was performed for Candidatus Liberibacter asiaticus (CLas) titer estimation in Nov. 2017. Significantly higher cycle threshold (Ct) values indicating reduced CLas bacterial populations were observed in trees that received the higher doses of Mn, especially those receiving four-times the recommended dosage of Mn (180 g Mn). Many trees exhibited Ct values of 32 or more, indicating a nonactive infection. Fruit yields of these trees were also increased. No significant differences in juice characteristics, canopy volume, and trunk section area were found between control plants and plants treated with 180 g Mn. Soil and leaf nutrients B, K, Mn, and Zn were significantly different among treatments at various times during the study. Our results strongly suggest that overdoses of Mn can suppress CLas bacterial titers in sweet orange trees on rough lemon rootstock, thus providing a therapeutic effect that can help restore tree health and fruit yields. This response was not observed when Mn and B were combined in the overdose, suggesting an antagonistic effect from B on Mn metabolism. When an overdose of Mn is used, biological functions and tree tolerance lost due to nutritional imbalances caused by HLB might be restored. Further studies are needed to elucidate which metabolic pathways are altered by comparing overdosed and conventionally fertilized HLB-impacted trees and to determine if the observed therapeutic effects can be achieved in trees grafted to other important commercial rootstocks.

‘ Candidatus Liberibacter asiaticus’ ( CL as) is associated with the most destructive disease of citrus, huanglongbing ( HLB ). The most widely used methods for detection of CL as are PCR ‐based and require purification of DNA from plant samples. Elution of DNA from tissue prints made on nitrocellulose membranes followed by qPCR ( TPE ‐ qPCR ) was compared to DNA extraction of plant tissue followed by PCR (X‐ PCR ) by testing the same tissue samples. The former estimated a higher CL as population in tissue prints than the latter ( t ‐test; P  =   0.009). All extracts prepared for TPE ‐ qPCR throughout the experiment were also tested by conventional PCR and 80.8% were identified as positive. A similar set of stem and petiole tissue samples was tested by TPE ‐ qPCR and immunoassay. Although the detection rate by TPE ‐ qPCR was higher than by immunoassay, about 6% of tissue prints were positive by immunoassay but not by TPE ‐ qPCR . Thus, a higher detection rate would be achieved by combining TPE ‐ qPCR with immunoassay. Significant differences were observed in the performance of nitrocellulose membranes from different manufacturers in these assays. Immunotissue prints showed that the spatial distribution pattern of CL as infection varied widely from one sample to another, but the patterns were highly correlated among serial sections from the same sample, suggesting that CL as preferentially colonizes adjacent phloem cells in a vertical rather than horizontal direction.