Plant Probiotic Bacterial Endophyte, Alcaligenes faecalis, Modulates Plant Growth and Forskolin Biosynthesis in Coleus forskohlii.

Coleus forskohlii is an herb, well-known for its medicinal compound forskolin present in its roots, with wide range of pharmaceutical applications. Here, we report, for the first time, the role of plant-probiotic bacterial endophytes of C. forskohlii, CFLB1 and CFRB1, isolated from leaf and root, which regulate plant growth and in plant forskolin content. Native bacterial endophyte, CFRB1 (Alcaligenes faecalis), significantly modulates primary plant productivity and forskolin content under pot and field conditions. Under field conditions, CFRB1 endophyte application significantly enhanced photosynthetic pigments and reduced the severity of root-knot and root rot diseases. Expression analyses of functional genes involved in the forskolin biosynthesis in C. forskohlii plants treated with CFRB1 endophyte under field conditions revealed differential upregulation of four C. forskohlii diterpene synthases (CfTPSs), CfTPS1, CfTPS2, CfTPS3 and CfTPS4, along with cytochrome P450 (CfCYP76AH15) and acyltransferase (CfACT1-8) genes. CFRB1 treatment reduced the severity of nematode infection and root rot in C. forskohlii plants by 81 and 78%, respectively. Overall, we demonstrate that cross-talk of plant-endophyte interaction in C. forskohlii is beneficial, leading to enhanced forskolin content through modulation of forskolin biosynthetic pathway genes along with increased plant yield and reduced disease incidence. Thus, endophytic isolate, A. faecalis (CFRB1), could be deployed as a novel bio-stimulant for enhancing in planta forskolin content during cultivation of C. forskohlii.

Treatments with native Coleus forskohlii endophytes improve fitness and secondary metabolite production of some medicinal and aromatic plants.

Endophytes have been shown to play a crucial role in determining the fitness of host plant during their association, yet the cross-functional effect of endophytes of one plant on another plant remains largely uncharacterized. In this study, we attempt to analyze the effect of native endophytes of Coleus forskohlii (Phialemoniopsis cornearis (SF1), Macrophomina pseudophaseolina (SF2), and Fusarium redolens (RF1), isolated from stem and root parts) on plant growth and secondary metabolite enhancement in medicinal plant Andrographis paniculata, and aromatic plants Pelargonium graveolens and Artemisia pallens. Here, we report, endophytic treatments with SF2 (21%) and RF1 (9%) in A. paniculata resulted in significant enhancement of andrographolide along with plant primary productivity. Correspondingly, application of fungal endophytes RF1, SF1, and SF2 significantly improved the plant growth (11 to 40%), shoot weight (28 to 34%), oil content (44 to 58%), and oil yield (72 to 122%) in P. graveolens. Interestingly, treatment of A. pallens with three fungal endophytes resulted in significant enhancement of plant productivity and oil content (12 to 80%) and oil yield (32 to 139%). Subsequently, the endophyte treatments RF1 and SF1 enhanced davanone (13 to 22%) and ethyl cinnamate (11 to 22%) content. However, SF2 endophyte-treated plants did not show any improvement in ethyl cinnamate content but enhanced the content of davanone (10%), a signature component of davana essential oil. Overall, results depict cross-functional role of native endophytes of C. forskohlii and repurposing of functional endophytes for sustainable cultivation of economically important medicinal and aromatic crops.

Functional Fungal Endophytes in Coleus forskohlii Regulate Labdane Diterpene Biosynthesis for Elevated Forskolin Accumulation in Roots.

Coleus forskohlii is a perennial medicinal shrub cultivated mainly for its forskolin content. The plant has been used since ancient times in ayurvedic traditional medicines for the treatment of hypertension, glaucoma, asthma, congestive heart failures, obesity, and cancer. Use of endophytic microorganisms presents a special interest for the development of value-added bioactive compounds through agriculture. Limited investigations have been undertaken on in planta enhancement of forskolin content using endophytic fungus in sustainable agriculture. Here we report specific roles of three fungal endophytes, Fusarium redolens (RF1), Phialemoniopsis cornearis (SF1), and Macrophomina pseudophaseolina (SF2), functionally acting as plant probiotic fungus, regulating secondary metabolite (forskolin) biosynthesis in C. forskohlii. The root endophyte, RF1, and shoot endophytes, SF1 and SF2, were found to enhance forskolin content by 52 to 88% in pot and 60 to 84% in field experiments as compared to uninoculated control plants. The three endophytes also enhanced total biomass owing to plant growth promoting properties. The expression of diterpene synthases (CfTPSs) like CfTPS1, CfTPS2, CfTPS3, and CfTPS4 were significantly upregulated in endophyte-treated C. forskohlii plants. Elevated expression of key diterpene synthases (CfTPS2) in the forskolin biosynthesis pathway, exclusively present in the root cork of C. forskohlii, was observed following SF2 endophyte treatment. Furthermore, endophyte treatments conferred a variety of antagonistic activity against nematode galls (80%) and plant pathogens like Fusarium oxysporum, Colletotricum gloeosporioides, and Sclerotium rolfsii. RF1 and SF1 fungal endophytes showed positive for IAA production; however, SF1 also indicated phosphate solubilization activity. Overall, the qualitative and quantitative improvement of in planta forskolin enhancement represents an area of high commercial interest, and hence, our work focused on novel insights for the application of three fungal endophytes for in planta enhancement of forskolin content for C. forskohlii cultivation by a sustainable approach.

Innate endophytic fungus, Aspergillus terreus as biotic elicitor of withanolide A in root cell suspension cultures of Withania somnifera.

In the present study, root cell suspension cultures of W. somnifera were elicited with mycelial extract (1% w/v) and culture filtrate (5% v/v) of their native endophytic fungus Aspergillus terreus 2aWF in shake flask. Culture filtrate of A. terreus 2aWF significantly elicits withanolide A at 6H (12.20 ± 0.52 µg/g FCB). However, with A. terreus 2aWF mycelial extract, withanolide A content was higher at 24H (10.29 µg/g FCB). Withanolide A content was maximum with salicylic acid (0.1 mM) treatment at 24H (8.3 ± 0.20 µg/g FCB). Further, expression analysis of withanolide pathway genes, hydrogen peroxide production, and lipid peroxidation was carried out after 48H of elicitation with 2aWF mycelial extract and culture filtrate. The expression levels of withanolides biosynthetic pathway genes, viz. HMGR, DXR, FPPS, SQS, SQE, CAS, SMT1, STE1 and CYP710A1 were quantified by real time PCR at 48H of elicitation. In all the treatments, the expression levels of key genes were significantly upregulated as compared to untreated suspension cells. Hydrogen peroxide was noticeably enhanced in SA, mycelia extract and culture filtrate, at 20% (115 ± 4.40 nM/g FCB), 42% (137.5 ± 3.62 nM/g FCB), and 27% (122.8 ± 1.25 nM/g FCB) respectively; however, lipid peroxidation was 0.288 ± 0.014, 0.305 ± 0.041 and 0.253 ± 0.007 (µM/gm FCB) respectively, higher than the control (0.201 ± 0.007 µM/gm FCB).