Nuclear acridine orange fluorescence in Rhizoctonia isolates from rice

The genus Rhizoctonia DC (1805) has long been studied as an important soilborne pathogen that causes a wide variety of symptoms because it is a non-specialized pathogen³. Rhizoctonia sensu lato is characterized by the lack of conidiogenous cells and this taxon is composed of two groups based on the number of nuclei per cell: the multinucleate group that belongs to Rhizoctonia s. str. and the binucleate group that belongs to Ceratorhiza⁵. Currently, other authors consider the group a Ceratobasidium–Rhizoctonia complex⁷ and divide it into two groups: BNR (binucleate Rhizoctonia-like) and MNR (multinucleate Rhizoctonia-like)⁹. Many methods are used to observe the number of nuclei in fungal cells, e.g. safranine O, aniline blue, HCl-Giemsa. Some of these methods apply a staining solution involving laborious, time-consuming procedures that require no equipment (Fig. 1). Other methods use fluorophores, which are rapid and precise^1,2,4,6,8,10.

Figure 1.

Multinucleate cells stained with lactophenol cotton blue. The arrows show the position of the septa.

Since a reliable and rapid method was needed to explore the MNR and BNR groups associated with rice crops in Argentina, we applied an accurate technique to observe the number of nuclei in the strain cells belonging to the Ceratobasidium–Rhizoctonia complex.

Small portions of mycelia grown on PDA for 24 h were submerged in 0.01 % acridine orange (Sigma–Aldrich, USA) aqueous solution during 10 s. The method applied was a modification of the Yamamoto and Uchida's staining method¹¹, the latter consisting of veronal buffer. The treated mycelium was observed under epifluorescence light using an OLYMPUS BX 51 microscope (Olympus, Japan). Digital photographs were taken using the Cool Snap-Pro System (Media Cybernetics, USA) (Fig. 2). The nuclei showed green fluorescence and the other cell components orange fluorescence. Thirty isolates from a collection of 36 were multinucleate and 6 were binucleate.

Figure 2.

Cells stained with acridine orange: (a) binucleate isolate; (b) multinucleate isolate. The arrows show the nuclei.

In comparison with the other methods mentioned above, the present method is characterized by easy handling and lower cost.

To our knowledge, this is the first time this methodology has been applied to observe nuclei in cells of Rhizoctonia isolates obtained from rice cultivars from Corrientes province, Argentina.

Ethical disclosures

Protection of human and animal subjects

The authors declare that no experiments were performed on humans or animals for this study.

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The authors declare that no patient data appear in this article.

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The authors declare that no patient data appear in this article.

Funding, Financial support and acknowledgements

The present research was supported by the Governments of Argentina (Instituto Nacional de Tecnología Agropecuaria) and Japan (Japan International Cooperation Agency) in a bilateral collaboration program.

Conflict of interest

The authors declare that they have no conflicts of interest.