Growth inhibition of an Araucaria angustifolia (Coniferopsida) fungal seed pathogen, Neofusicoccum parvum, by soil streptomycetes

Abstract

Background: Araucariaceae are important forest trees of the southern hemisphere. Life expectancy of their seedlings can largely be reduced by fungal infections. In this study we have isolated and characterized such a fungus and investigated the potential of Streptomyces Actinobacteria from the respective rhizosphere to act as antagonists.

Results: The pathogenic fungus from Araucaria angustifolia seeds was identified by morphological markers (pore-associated Woronin-bodies) as belonging to the Pezizomycotina. Molecular data identified the fungus as Neofusicoccum parvum (Botryosphaeriaceae). Co-cultures on agar of this fungus with certain streptomycete isolates from the rhizosphere, and from the surface of Araucaria roots significantly reduced the growth of the fungus. HPLC analysis of the agar yielded streptomycete-specific exudate compounds which were partly identified. There were differences in compounds between single (bacteria, fungus) and dual cultures (bacteria + fungus).

Conclusion: Streptomycetes from the rhizosphere of Araucariaceae produce exudates which can suppress the development of pathogenic fungi in their seeds.

Figure 1

Neofusicoccum parvum infection of A. angustifolia seedlings (Bar = 1 cm in A, B, F). A, Seedling; B, Megagametophyte and cotyledons infected with the fungus; C, Scanning electron microscopy of infected megagametophyte tissue that surrounds the cotyledon; D, Starch grains covered by hyphae; E-F, Non infected tissues. All images were taken from plants/tissues after 50 days of germination. ct - haustorial cotyledon, se - seed, mg – megagametophyte, st - starch grain.

Figure 2

Section through a septum of Neofusicoccum parvum showing a simple pore associated with Woronin-bodies (one is indicated by an arrow). Scale bar = 0.5 μm.

Figure 3

Co-cultures of streptomycete isolates with the plant pathogenic fungus Neofusicoccum parvum. The fungal isolate is located in the center of the Petri dish. Mxy identifies the different Streptomycete isolates. M, isolates from rhizosphere soil; MW, isolates from the surface of Araucaria roots.

Figure 4

Association statistics of the 24 exudate compounds, listed in Table 1. This approach illustrates that the inhibition of the fungus in co-culture was dependent on the presence of compounds of group 1 (component 1–4; □) and group 2 (component 16–18; ◊). For numbers of the relevant compounds see Table 1: □ 1,2,3,4; ◊ 16–18; ○ 22; Δ 13; ӿ 5–12, 14–15, 19–21, 23–24.

Figure 5

Association statistics of the streptomycete isolates or their co-cultures with N. parvum and the respective exudates. Fungus-inhibiting bacteria together with their exudates (singly or in combination with the fungus; □, ○, Δ) separate well from those causing little or no inhibition (◊). □ M2, 4, 5; MW 2, 4, 9; ○ BM2, 4, 5; Δ BMW2, 4, 9; ◊ M7, 8; MW1, 6; BM7, 8; BMW1, 6. M, isolates from rhizosphere soil; MW, isolates from the surface of Araucaria roots. B, co-cultures with the Brazilian fungus (N. parvum).

Figure 6

HPLC analysis of agar extracts obtained from single and dual cultures in Petri dishes. The eluate was monitored at 210 and 310 nm. A)Neofusicoccum parvum, B) bacterial isolate M5, C) co-culture of bacterium and fungus. Peaks labelled with retention times of 7.7 and 8.4 min represent tetraene-polyene macrolides of the nystatin-type, those with an asterix indicate agar constituents.