Table 1.
Results of the PCRs with the specific primers for the genus Burkholderia.
Of each plant structure, three biological replicates were tested. If the sequences were identified as Candidatus Burkholderia kirkii, the presence of the host specific endophyte is indicated with a +, otherwise the absence is indicated with a–in the last column.
Fig 1.
In situ detection of Burkholderia endophytes in transverse sections of Psychotria punctata leaves using FISH.
(A) Detail of a leaf showing nodules scattered throughout the abaxial leaf surface. (B) TBO-stained section through a nodule, which contains endophytes, as well as parenchymatous cells. (C) Merged epifluorescence image of the nodule showing FISH-labelled bacteria (red, arrows) and autofluorescence of parenchymatous cells (green). (D) Same section as (C), stained with TBO after FISH labelling, which stains the mucus surrounding the bacteria (arrows correspond with the same bacteria indicated on the fluorescent image (C)). (E) Merged epifluorescence image of the spongy parenchyma showing the intercellular space, in which no endophytes were detected. (F) Same section as (E), stained with TBO after FISH labelling. n, nodule; pa, elongated parenchymatous cells; sp, spongy mesophyll.
Fig 2.
In situ detection of Burkholderia endophytes in longitudinal sections of P. punctata vegetative buds using FISH.
(A) Overview of an excised vegetative bud revealing the shoot apical meristem and two leaf primordia, and the leaf enclosed chamber (LEC) filled with mucus. The LEC is enclosed by decussate arranged leaf pairs and their interpetiolar stipules. The stipules of the second distalmost node are not visible due to their parallel placement with the sectioning plane. (B) TBO-stained section through the LEC, enclosed by stipules and the leaves of the first distalmost node and the regions visualised in (C and E). (C) Merged epifluorescence image of the LEC (B), showing FISH-labelled bacteria (red, arrows) between the colleters in the mucus. (D) Same section as (C) stained with TBO after FISH labelling. (E) Merged epifluorescence image of the abaxial leaf surface (B), showing Burkholderia (red labelling, arrows) in the mucus between the trichomes. (F) Same detail as (E) stained with TBO after FISH labelling. The detected endophytes occur in mucus residue. c, colleters; l1, leaves of first distalmost node; l2, leaves of second distalmost node; lec, leaf enclosed chamber; lp, leaf primordia; m, mucus; sam, shoot apical meristem; st, stipules of the leaf primordia; st1, stipules of the first distalmost node; st3 stipules of third distalmost node; t, trichomes.
Fig 3.
In situ detection of Burkholderia endophytes in longitudinal sections of P. punctata flower buds using FISH.
(A) TBO-stained section through the flower bud. (B) Detail of (A) showing a locule with ovule. (C) Detail of (A) showing the absence of mucus between the anthers and the location of detail (D) between the sepals and petals. (D) Merged epifluorescence image of the space between the sepals and petals (C), showing FISH-labelled endophytes (red, arrows) between colleters. (E) Same detail as (D) stained with TBO after FISH labelling, visualising mucus between the bacteria and colleters. a, anthers; c, colleters; f, filament; lo, locule; o, ovule; p, petals; se, sepals.
Fig 4.
In situ detection of Burkholderia endophytes in longitudinal sections of P. punctata embryos using FISH.
(A) Overview of a longitudinal section through the pyrene showing the embryo, located in the embryonic cavity, above the ventral intrusion. (B) Overview of an embryo showing the site of the section (red line). (C) TBO-stained section through the embryo of which the sectioning plane is indicated in (B), showing the intercotyledonary space (*). At the shoot meristem, between the cotyledons, mucus is stained between endosperm cell residue. (D) Merged epifluorescence images of the bacterial isolation method, showing a FISH-labelled endophyte (red, arrow). (E) Same detail as (D) stained with TBO after FISH labelling. (F) Merged epifluorescence images of the intercotyledonary space (C; *), showing FISH-labelled endophyte (red, arrow). (G) Same detail as (F) stained with TBO after FISH labelling. (H) Merged epifluorescence images of the intercotyledonary space of a different embryo, showing FISH-labelled endophytes (red, arrows) located between the cotyledons at the outer cell wall surface of degrading endosperm cells. (I) Same detail as (H) stained with TBO after FISH labelling. co, cotyledon; d, degrading endosperm cell; e, embryo; ec, embryonic cavity; en, endocarp; es, endosperm; vt, ventral intrusion.