ACC deaminase-producing endophytic fungal consortia promotes drought stress tolerance in M.oleifera by mitigating ethylene and H2O2

Abstract

Introduction: Drought has become more prevalent due to dramatic climate change worldwide. Consequently, the most compatible fungal communities collaborate to boost plant development and ecophysiological responses under environmental constraints. However, little is known about the specific interactions between non-host plants and endophytic fungal symbionts that produce growth-promoting and stress-alleviating hormones during water deficits.

Methods: The current research was rationalized and aimed at exploring the influence of the newly isolated, drought-resistant, ACC deaminase enzyme-producing endophytic fungi Trichoderma gamsii (TP), Fusarium proliferatum (TR), and its consortium (TP+TR) from a xerophytic plant Carthamus oxycantha L. on Moringa oleifera L. grown under water deficit induced by PEG-8000 (8% osmoticum solution).

Results: The current findings revealed that the co-inoculation promoted a significant enhancement in growth traits such as dry weight (217%), fresh weight (123%), root length (65%), shoot length (53%), carotenoids (87%), and chlorophyll content (76%) in comparison to control plants under water deficit. Total soluble sugars (0.56%), proteins (132%), lipids (43%), flavonoids (52%), phenols (34%), proline (55%), GA₃ (86%), IAA (35%), AsA (170%), SA (87%), were also induced, while H₂O₂ (-45%), ABA (-60%) and ACC level (-77%) was decreased by co-inoculation of TP and TR in M. oleifera plants, compared with the non-inoculated plants under water deficit. The co-inoculum (TP+TR) also induced the antioxidant potential and enzyme activities POX (325%), CAT activity (166%), and AsA (21%), along with a lesser decrease (-2%) in water potential in M. oleifera plants with co-inoculation under water deficit compared with non-inoculated control. The molecular analysis for gene expression unraveled the reduced expression of ethylene biosynthesis and signaling-related genes up to an optimal level, with an induction of antioxidant enzymatic genes by endophytic co-inoculation in M. oleifera plants under water deficit, suggesting their role in drought stress tolerance as an essential regulatory function.

Conclusion: The finding may alert scientists to consider the impacts of optimal reduction of ethylene and induction of antioxidant potential on drought stress tolerance in M. oleifera. Hence, the present study supports the use of compatible endophytic fungi to build a bipartite mutualistic symbiosis in M. oleifera non-host plants to mitigate the negative impacts of water scarcity in arid regions throughout the world.

Keywords: ACC deaminase; Moringa oleifera; agroforestry; antioxidants; drought stress; ethylene; hydrogen peroxide; plant-microbe interaction.

Figure 1

(A) Morphological characterization of selected fungal strains TP and TR, (B) genotyping of selected fungal strains by ITS region amplification, (C) ACC deaminase activity and ACC deaminase gene expression of TP and TR. Quantitative data represent means ± SD of three independent experiments and at least six technical replicates, (D) Phylogenetic identification of TP, and (E) TR endophytic strain.

Figure 2

Endophytic fungal (TP and TR) characterization. (A) Screening of fungal strains (TP and TR) for PEG-induced drought stress tolerance, (B) Biomass, (C) Total soluble sugars, (D) Total soluble proteins, (E) Total lipids, (F) Proline content, (G) Total flavonoids, and (H) Total phenols, measured in CF of TP and TR isolates. Quantitative data is representing the means ± SE values of at least three independent biological replications. Various letters have been presented to show the statistical differences at significance level of p¾0.05 using Duncan’s Multiple Range Test (DMRT). CF, Culture Filtrate.

Figure 3

Hormonal contents and enzymatic and non-enzymatic antioxidants in CF of TP and TR isolates. (A) IAA, (B) ABA, (C) GA₃, and (D) SA content. (E) AsA content, (F) Peroxidase enzyme activity, (G) total antioxidant capacity, and (H) Catalase enzyme activity. Quantitative data is representing the means ± SE values of at least three independent biological replications. Various letters have been presented to show the statistical differences at significance level of p¾0.05 using Duncan’s Multiple Range Test (DMRT). CF, Culture Filtrate.

Figure 4

Assessment of PEG-mediated drought stress tolerance response of M. oleifera seeds by germination test. (A) Seed germination response under PEG-induced drought conditions in the absence (above penal) and presence (lower penal) of endophytic CF inoculation, (B) cotyledon length and (C) fresh weight. Quantitative data is representing the means ± SE values of at least three independent biological replications. Various letters have been presented to show the statistical differences at significance level of p¾0.05 using Duncan’s Multiple Range Test (DMRT).

Figure 5

Effect of TP and TR inoculation on M. oleifera plant growth. (A) Schematic representation showing work plan of plant bioassay, (B) Phenotypic analysis; upper penal (intact plants) and lower penal (uproots plants), (C) Shoot and root length, (D) Total fresh and dry weight. Quantitative data is representing the means ± SE values of at least three independent biological replications. Various letters have been presented to show the statistical differences at significance level of p¾0.05 using Duncan’s Multiple Range Test (DMRT).

Figure 6

Effect of TP and TR inoculation on M. oleifera growth promoting metabolites. (A) Carotenoids, (B) Chlorophyll a/b ratio, (C) Total chlorophyll content, (D) Total soluble proteins, (E) Total phenols, (F) Total soluble sugars, (G) Total flavonoids, (H) Total lipids, and (I) Proline content. Quantitative data is representing the means ± SE values of at least three independent biological replications. Various letters have been presented to show the statistical differences at significance level of p¾0.05 using Duncan’s Multiple Range Test (DMRT).

Figure 7

Effect of TP and TR inoculation and colonization in M. oleifera phytohormones and water potential. (A) Root colonization, (B) Stem anatomy, (C) IAA, (D) GA₃, (E) SA, (F) ABA, (G) ACC content and, (H) Water potential. Quantitative data is representing the means ± SE values of at least three independent biological replications. Various letters have been presented to show the statistical differences at significance level of p¾0.05 using Duncan’s Multiple Range Test (DMRT).

Figure 8

Effect of TP and TR inoculation on M. oleifera ROS production and antioxidant potential. (A) DAB staining using leaf segment from 35-d-old plants, (B) H₂O₂, (C) AsA, (D) Peroxidase activity and (E) Catalase activity. Quantitative data is representing the means ± SE values of at least three independent biological replications. Various letters have been presented to show the statistical differences at significance level of p¾0.05 using Duncan’s Multiple Range Test (DMRT).

Figure 9

(A) Biplot of two dimensions of the principal component analysis (PCA). The scores show the contribution of TP and TR endophytic fungal isolates on reshuffling of M. oleifera plant traits, grown under normal and PEG-supplemented conditions, as indicated by the direction and magnitude of the respective vectors, and (B) Expression profiling of ethylene biosynthesis and antioxidant enzymatic genes by RT-qPCR. Quantitative data represent the means ± SD of three independent experiments and at least three technical replicates each.