Optimizing In vitro Culture Conditions for the Biotrophic Fungi Exobasidium vexans Through Response Surface Methodology

Abstract

The blister blight disease caused by the fungus, Exobasidium vexans has serious implications on the quality of tea production. The disease however, has been poorly studied and hence there is very limited information on the pathogen and as such the pathogenesis of blister blight infection. One of the major roadblocks in understanding E. vexans is the obligate and biotrophic nature of the fungus which limits the establishment and maintenance of in vitro cultures. To address this issue, a Central Composite Design based Response Surface Methodology (RSM) was adopted to study the modification of three fungal culture media viz. czapek dox, potato dextrose, and v8 juice, and the effect of altered media composition on growth conditions and media compositions were assessed. The response parameter for the RSM experiments was the mycelial biomass produced under different culture conditions. The uni and bi-parametric interactions among the experimental variables provided the basis for the statistically optimized conditions for maximal fungal growth. The study thus presents the recommended modifications of existing media that can lead to the successful establishment and maintenance of E. vexans in vitro cultures.

Keywords: Blister blight; Exobasidium vexans; Growth media; Optimization; RSM.

Fig. 1

Dorsal (a) and ventral (b) surfaces of blister blight infected tea leaves (Ananda Tea Estate, Lakhimpur, India). Light micrographs of E. vexans basidiospores at different phases of germination on agar. Basidiospores (c); basidiospores with germ tube growth on single end (d); basidiospores with germ tube growth from both ends (e); and hyphal growth of E. vexans showing branching (f). Extensive hyphal growth observed on infected leaf part (g–h) and appressorium formation (i) (Scale: 10 µm, gt germ tube, br branching of hyphae, hy hyphae, app appresorium)

Fig. 2

Effect of univariate interactions described by the model for czapek dox broth a–c, potato dextrose broth d–f and v8 juice broth g–i

Fig. 3

Effect of multivariate interactions: Response surface 3D plots and response surface contour plot described by the model for czapek dox broth a–d and a′–d′ and v8 juice broth e–g and e′–g′ respectively

Fig. 4

Pareto graphic analysis for mycelial biomass described by model for czapek dox (a), potato dextrose (b) and v8 juice (c)