| Sumario: | Greening or Huanglongbing (HLB) is the major real biotic stress affecting citrus industry, whose causative
agent is the vascular bacterium Candidatus liberibacter, and ends with the collapse of the tree, until his
death. Microcitrus has been pointed as citrus specie tolerant to HLB due to the presence of a peptide
with a bactericidal effect that also enhances the immune system of the plant. As vascular bacterium, the
analysis and quantification of SAMP peptide in the phloem is of great interest so optimization of
extraction method is a major point in any HLB study. As Microcitrus plants show high variability in their
phenotype this work studies the relation between some biomass and physiological measurements and
the phloem sap yield in three different greenhouse plants (four‐year‐old).
Biomass fractions, number of leaves and thorns, leaf area, relative water content and phloem sap
extracted were determined from four independent replicates per plant.
In phloem sap extraction method, stems of 10–20 cm (0.5 cm diameter) were collected and cut into
pieces (1.5 length) by pruning shears. Leaves and thorns were removed, bark was stripped longitudinally
by a sterile razor blade and separated from the twig. The inner part of the bark was rinsed with
deionized water and dried with Kim wipes to exclude any contamination from the xylem sap. To collect
the phloem sap, six pieces of the bark tissue were cut into 0.5‐cm pieces using a sterile razor blade and
placed in a 0.5‐ml Eppendorf tube. A small hole was made at the bottom of the tube using a razor blade
and the tube was immersed into a 2‐ml Eppendorf tube. The sample was centrifuged at 12,000 rpm for
15 min at 4 °C and the collected phloem sap was stored at −80 °C until further use.
The highest phloem yield was obtained in the plant which presented the heaviest twigs, thorns, and
peeled bark FW, in addition to the highest leaf DW and surface, but lowest leaf number. RWC did not
correlate with any parameter measured. Low yield is correlated with high leaf number but low leaf
weight.
In this work, we develop an optimized protocol for high‐quality phloem extraction in Microcitrus stems
which can be used for a broad spectrum of HLB‐related purposes and propose larger‐leaved twigs as the
most efficient plant material to extract phloem sap.
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