In Vitro Morphogenesis of Arabidopsis to Search for Novel Endophytic Fungi Modulating Plant Growth

Abstract

Fungal endophytes have shown to affect plant growth and to confer stress tolerance to the host; however, effects of endophytes isolated from water plants have been poorly investigated. In this study, fungi isolated from stems (stem-E) and roots (root-E) of Mentha aquatica L. (water mint) were identified, and their morphogenetic properties analysed on in vitro cultured Arabidopsis (L.) Heynh., 14 and 21 days after inoculation (DAI). Nineteen fungi were analysed and, based on ITS analysis, 17 isolates showed to be genetically distinct. The overall effect of water mint endophytes on Arabidopsis fresh (FW) and dry weight (DW) was neutral and positive, respectively, and the increased DW, mainly occurring 14 DAI, was possibly related to plant defence mechanism. Only three fungi increased both FW and DW of Arabidopsis at 14 and 21 DAI, thus behaving as plant growth promoting (PGP) fungi. E-treatment caused a reduction of root depth and primary root length in most cases and inhibition-to-promotion of root area and lateral root length, from 14 DAI. Only Phoma macrostoma, among the water mint PGP fungi, increased both root area and depth, 21 DAI. Root depth and area 14 DAI were shown to influence DWs, indicating that the extension of the root system, and thus nutrient uptake, was an important determinant of plant dry biomass. Reduction of Arabidopsis root depth occurred to a great extent when plants where treated with stem-E while root area decreased or increased under the effects of stem-E and root-E, respectively, pointing to an influence of the endophyte origin on root extension. M. aquatica and many other perennial hydrophytes have growing worldwide application in water pollution remediation. The present study provided a model for directed screening of endophytes able to modulate plant growth in the perspective of future field applications of these fungi.

Fig 1. Endophyte effects on fresh weights.

(a, b) Boxplots illustrating variability of fresh weight in E-treated and control (C) Arabidopsis plants 14 (a) and 21 (b) DAI. The reference hatched line represents the median of controls. Differences were considered significant at a probability level of *: p<0.05; **: <0.01; and ***: <0.001. (c, d) Pooled data for controls (C) and plants treated with all (E), stem (stem-E) and root (root-E) endophytes and 14 (a) and 21 (b) DAI.

Fig 2. Endophyte effects on dry weights.

(a, b) Boxplots illustrating variability of dry weight in E-treated and control (C) Arabidopsis plants 14 (a) and 21 (b) DAI. The reference hatched line represents the median of controls. Differences were considered significant at a probability level of *: p<0.05; **: <0.01; and ***: <0.001. (c, d) Pooled data for controls (C) and plants treated with all (E), stem (stem-E) and root (root-E) endophytes and 14 (a) and 21 (b) DAI.

Fig 3. Endophyte effects on percentage dry weights.

(a, b) Boxplots illustrating variability of percentage dry weight in E-treated and control (C) Arabidopsis plants 14 (a) and 21 (b) DAI. The reference hatched line represents the median of controls. Differences were considered significant at a probability level of *: P<0.05; **: <0.01; and ***: <0.001. (c, d) Pooled data for controls (C) and plants treated with all (E), stem (stem-E) and root (root-E) endophytes and 14 (a) and 21 (b) DAI.

Fig 4. Endophyte-related root phenotypes in Arabidopsis.

Plants and fungus co-cultures 14 DAI (top row, a-f) and 21 DAI (bottom row, a’-f’).

Fig 5. Endophyte effects on root areas.

(a, b) Boxplots illustrating root area variability in E-treated and control (C) Arabidopsis plants 14 (a) and 21 (b) DAI. The reference hatched line represents the median of controls. Differences were considered significant at a probability level of *: p<0.05; **: <0.01; and ***: <0.001. (c, d) Pooled data for controls (C) and plants treated with all (E), stem (stem-E) and root (root-E) endophytes and 14 (a) and 21 (b) DAI.

Fig 6. Endophyte effects on root depth.

(a, b) Boxplots illustrating variability of root depth in E-treated and control (C) Arabidopsis plants 14 (a) and 21 (b) DAI. The reference hatched line represents the median of controls. Differences were considered significant at a probability level of *: p<0.05; **: <0.01; and ***: <0.001. (c, d) Pooled data for controls (C) and plants treated with all (E), stem (stem-E) and root (root-E) endophytes and 14 (a) and 21 (b) DAI.

Fig 7. Cadophora luteo-olivacea related root phenotypes in Arabidopsis.

Plants and fungus co-cultures 21 DAI with SA (a) and SL (b).

Fig 8. Endophyte effects on root system architecture.

Boxplots illustrating root system architecture variability in E-treated and control (C) Arabidopsis plants, 14 DAI. (a) number of first order lateral root; (b) total length of lateral root; (c) primary root length and (d) primary root branching. The reference hatched line represents the median of controls. Differences were considered significant at a probability level of *: p<0.05; **: <0.01; and ***: <0.001.