Biostimulants, eugenol, seaweed extract, caryophyllene.

The 'sacred basil' or 'holy basil' Ocimum sanctum Linn (2n = 32) is an aromatic shrub belonging to the family Lamiaceae. Tulsi, the Queen of Herbs, the legendary 'Incomparable One' of India, is one of the most sacred and revered of the many healing and health-giving herbs of the Orient. Tulsi, the sacred basil, is revered for its religious and spiritual sanctity, as well as its importance in the East's traditional Ayurvedic and Unani systems of holistic health and herbal medicine. In Charaka Samhita, an Ayurvedic text, Charaka mentions it as a life-saving herb-"the elixir of life." Tulasi is native to the Indian subcontinent and widely cultivated in Southeast Asian tropics (Staples and Kristiansen 1999). The essential oils from the genus Ocimum have wide applications in the perfumery and cosmetic industries, as well as indigenous medical systems (Ved and Goraya 2008).

The experiment was carried out with the two varieties viz., CIM- Ayu and CIM- Angana which was procured from the Central Institute of Medicinal and Aromatic Plants (CIMAP), Boduppal, Hyderabad. The experiment was laid out in a Factorial randomized block design (FRBD) with 14 treatments and 3 replications. The treatments include two varieties CIM- Ayu (V1) and CIM- Angana (V2) and seven biostimulants B1: Chitosan @ 0.1%, B2: Chitosan @ 0.5%, B3 : Humic acid @ 0.2%, B4: Humic acid @ 0.4%, B5 : Seaweed extract @ 5%, B6 : Seaweed extract @ 10%, B7 : Water spray (control). The treatment combinations includes T1(V1B1) : CIM- Ayu  + Chitosan @ 0.1 % , T2 (V1B2): CIM- Ayu + Chitosan @ 0.5%, T3 (V1B3) : CIM- Ayu + Humic acid @ 0.2%, T4 (V1B4): CIM- Ayu  +  Humic acid @ 0.4 %, T5 (V1B5) : CIM- Ayu  +  Seaweed extract @ 5%, T6 (V1B6): CIM- Ayu +  Seaweed extract @ 10%, T7 (V1B7): CIM- Ayu  +  Water spray (control) , T8 (V2B1): CIM- Angana + Chitosan @ 0.1 %, T9 (V2B2): CIM- Angana + Chitosan @ 0.5%, T10 (V2B3): CIM- Angana   + Humic acid @ 0.2%, T11 (V2B4): CIM- Angana  +  Humic acid @ 0.4 %, T12 (V2B5): CIM- Angana  +  Seaweed extract @ 5%, T13 (V2B6): CIM- Angana   +  Seaweed extract @ 10%, T14 (V2B7): CIM- Angana  +  Water spray (control).

A. Oil yield per plant (g/plant)

It is evident from the data that varieties, biostimulants and their combination had significant impact on oil yield during the years 2021, 2022 and pooled is presented in the Table 1. Among the varieties maximum oil yield per plant was recorded in CIM – Ayu (V1) (1.07, 1.08 and 1.08) than CIM - Angana (V2) (1.02, 1.00 and 1.01) during the years 2021, 2022 and pooled respectively. The application of biostimulants also showed a significant effect on oil yield per plant during both the years and pooled, in which the maximum (1.58, 1.59 and 1.59) oil yield per plant was recorded in B6, followed by B4 (1.39, 1.38 and 1.39), B2 (1.19, 1.21 and 1.20) and B3 (1.07, 1.05 and 1.06) during 2021, 2022, and pooled respectively. Likewise, the minimum oil yield per plant was recorded in B7(control) (0.53, 0.52 and 0.53) during 2021, 2022 and pooled respectively.

The interaction effect between varieties and biostimulants was observed to be significant during the year 2021. The maximum oil yield per plant(1.59) was registered in T6 (V1B6) and was on par with T13 (V2B6) (1.57) Whereas, the lowest oil yield per plant(0.51) was registered in T14 (V2B7) and it was on par with T7 (V1B7) (0.55). During the year 2022 and pooled significantly maximum oil yield per plant (1.65 and 1.62) was recorded in T6 (V1B6) followed by T13 (V2B6) (1.52 and 1.55), T4 (V1B4) (1.38 and 1.40) and was at par with T11 (V2B4) (1.38 and 1.37) during 2022 and pooled respectively. Whereas, the minimum oil yield per plant (0.49 and 0.50) was recorded in T14 (V2B7) during the year 2022 and pooled, respectively.  

B. Oil yield per plot (g/plot)

Among the varieties, maximum oil yield per plot (40.23, 40.40 and 40.32) was recorded in V1 than V2 (38.20, 37.38 and 37.79) during the year 2021, 2022 and in pooled, respectively. It has been found from the results that the application of biostimulants had a considerable influence on oil yield per plot. Among the biostimulants, B6- recorded maximum (59.41, 59.51 and 59.46) oil yield per plot followed by B4 (52.08, 51.80 and 51.94), B2 (44.70, 45.36 and 45.03), B3 (39.98, 39.41 and 39.69) and the lowest oil yield per plot was recorded in B7 - Water spray (control) (19.89, 19.54 and 19.71) during 2021, 2022 and pooled respectively.

The interaction effect between varieties and biostimulants was observed to be significant during the year 2021. Maximum oil yield per plot(59.78) was recorded in T6 (V1B6) and was on par with T13 (V2B6) (59.05) Whereas, the lowest oil yield per plot (19.10) was recorded in T14 (V2B7) and it was on par with T7 (V1B7) (20.68).During the year 2022 and pooled significantly maximum oil yield per plot (62.05 and 60.91) was recorded in T6 (V1B6) followed by T13 (V2B6) (56.98 and 58.01) during 2022 and pooled respectively. Whereas, the minimum oil yield per plot (18.48 and 18.79) was recorded in T14 (V2B7) during the year 2022 and pooled, respectively.  

C. Oil yield per hectare (Kg/ha)

Among the varieties maximum oil yield per hectare was recorded in V1 (67.05, 67.33 and 67.19) than V2 (63.67, 62.29 and 62.98) during the years 2021, 2022 and pooled respectively. The application of biostimulants also showed a significant effect on oil yield per hectare during both the years and pooled, in which the maximum (99.02, 99.19 and 99.10) oil yield per hectare was recorded in B6, followed by B4 (86.79, 86.33 and 86.56), B2 (74.50, 75.60 and 75.05) and B3 (66.63, 65.69 and 66.16) Likewise, the minimum oil yield per hectare was recorded in B7 (33.15, 32.56 and 32.85) during 2021, 2022 and pooled respectively.

The interaction effect between varieties and biostimulants was observed to be significant during the year 2021. The maximum oil yield per hectare(99.63) was registered in T6 (V1B6) and was on par with T13 (V2B6) (98.42) Whereas, the lowest oil yield per hectare(31.83) was registered in T14 (V2B7) and it was on par with T7 (V1B7) (34.46). During the year 2022 and pooled significantly maximum oil yield per hectare(103.42 and 101.52) was recorded in T6(V1B6) followed by T13 (V2B6) (94.96 and 96.69) during 2022 and pooled respectively. Whereas, the minimum oil yield per hectare (30.79 and 31.31) was recorded in T14 (V2B7) during the year 2022 and pooled, respectively.  

D. Essential oil profiling

The results pertaining to the essential oil profiling as influenced by varieties, biostimulants and their interaction during 2021 is presented in Table 2. There was a significant effect of varieties on the essential oil profiling during 2021. Among the varieties V1 recorded maximum (31.42, 20.27 and 9.96 respectively) content of eugenol, methyl eugenol and caryophyllene respectively, than V2 (23.94, 3.87 and 9.58 respectively). In case of limonene, linalool, methyl chavicol and β-elemene, the maximum content was recorded in V2 (1.64, 1.37, 1.26 and 13.38 respectively) compared to V1 (1.28, 0.24, 0.51 and 6.74).

The application of biostimulants was found to be significant on the essential oil profiling. The maximum content of limonene, linalool, methyl chavicol, eugenol, methyl eugenol, β-elemene  and caryophyllene  respectively was observed by the application of  B6 (1.66, 0.89, 1.00, 33.07, 14.17, 10.64 and 10.95 respectively), while, the minimum content of limonene, linalool, methyl chavicol, eugenol, methyl eugenol, β-elemene  and caryophyllene was observed in B7 (1.28, 0.71, 0.73, 23.18, 10.02, 9.16 and 8.83 respectively).

In the present study among the interactions, Treatment T6(V1B6) recorded significantly maximum content of (38.65, 24.17 and 11.63 respectively) eugenol, methyl eugenol and caryophyllene respectively, followed by T4 (V1B4) (35.47, 23.07 and 11.18), while the minimum (20.19, 3.67 and 8.73 respectively) was recorded in T14 (V2B7). In case of limonene, linalool, methyl chavicol and β-elemene maximum content (1.77, 1.49, 1.40 and 14.19 respectively) was noticed in T13 (V2B6), while, the minimum (1.11, 0.18, 0.41 and 6.16 respectively) was recorded in T7 (V1B7).

The results pertaining to the essential oil profiling as influenced by varieties, biostimulants and their interaction during 2022 is presented in Table 3. There was a significant effect of varieties on the essential oil profiling during 2022. Among the varieties, V1 recorded maximum (33.59, 21.23 and 10.43 respectively) content of eugenol, methyl eugenol and caryophyllene respectively, than V2 (26.16, 4.78 and 10.06 respectively). In case of limonene, linalool, methyl chavicol and β-elemene, the maximum content was recorded in V2 (1.69, 1.42, 1.30 and 13.86 respectively) compared to V1 (1.31, 0.29, 0.56 and 7.20).

The application of biostimulants was found to be significant on the essential oil profiling. The maximum content of limonene, linalool, methyl chavicol, eugenol, methyl eugenol, β-elemene  and caryophyllene  respectively was observed by the application of  B6 (1.63, 0.93, 1.04, 35.23, 15.13, 11.10 and 11.42 respectively), while, the minimum content of limonene, linalool, methyl chavicol, eugenol, methyl eugenol, β-elemene  and caryophyllene was observed in B7 (1.35, 0.75, 0.77, 25.53, 10.79, 9.70 and 9.33 respectively).

In the present study among the interactions, Treatment T6(V1B6) recorded significant maximum content of (40.82, 25.13 and 12.10 respectively) eugenol, methyl eugenol and caryophyllene respectively, followed by T4 (V1B4) (37.64, 24.03 and 11.65), while the minimum (22.72, 4.25 and 9.26 respectively) was recorded in T14 (V2B7). In case of limonene, linalool, methyl chavicol and β-elemene maximum content (1.82, 1.54, 1.45 and 14.65 respectively) was noticed in T13(V2B6), while, the minimum (1.16, 0.23, 0.46 and 6.62 respectively) was recorded in T7 (V1B7).

The results concerned to the essential oil profiling as influenced by varieties, biostimulants and their interaction during Pooled analysis over two years is presented in Table 4. Among the varieties, V1 recorded maximum (32.50, 20.75 and 10.20 respectively) content of eugenol, methyl eugenol and caryophyllene respectively, than V2 (25.05, 4.32 and 9.82 respectively). In case of limonene, linalool, methyl chavicol and β-elemene, the maximum content was recorded in V2 (1.67, 1.40, 1.28 and 13.62 respectively) compared to V1(1.30, 0.26, 0.54 and 6.97).

The application of biostimulants was found to be significant on the essential oil profiling. The maximum content of limonene, linalool, methyl chavicol, eugenol, methyl eugenol, β-elemene  and caryophyllene  respectively was observed by the application of  B6 (1.64, 0.91, 1.02, 34.15, 14.65, 10.87 and 11.18 respectively), while, the minimum content of limonene, linalool, methyl chavicol, eugenol, methyl eugenol, β-elemene  and caryophyllene was observed in B7(1.32, 0.73, 0.75, 24.36, 10.41, 9.43 and 9.08 respectively).

In the present study among the interactions, Treatment T6 (V1B6) recorded significantly maximum content of (39.74, 24.65 and 11.87 respectively) eugenol, methyl eugenol and caryophyllene respectively, followed by T4 (V1B4) (36.56, 23.55 and 11.42), while the minimum (21.45, 3.96 and 9.00 respectively) was recorded in T14 (V2B7). In case of limonene, linalool, methyl chavicol and β-elemene maximum content (1.79, 1.51, 1.42 and 14.42 respectively) was noticed in T13(V2B6), while, the minimum (1.13, 0.20, 0.43 and 6.39 respectively) was recorded in T7 (V1B7).

E. Chlorophyll content (SPAD value)

It is evident from the data that varieties, biostimulants and their interaction had a significant impact on chlorophyll content during the years 2021, 2022 and pooled is presented in the Table 5. Among the varieties, maximum chlorophyll content (34.90, 37.32 and 36.11) was recorded in V1 than V2 (33.60, 35.70 and 34.65) during the year 2021, 2022 and in pooled, respectively

It has been found from the results that application of biostimulants had a considerable influence on chlorophyll content. Among the biostimulants, B6 recorded maximum (45.80, 48.76 and 47.28) chlorophyll content followed by B4 (40.78, 42.59 and 41.68), B2 (35.86, 38.10 and 36.98) and B3 (33.50, 35.65 and 34.57) during 2021, 2022, and pooled respectively. Likewise, the minimum chlorophyll content was recorded in B7 (26.21, 28.38 and 27.29) during 2021, 2022 and pooled respectively. Among the interactions, Treatment T6 (V1B6) recorded maximum chlorophyll content (46.64, 50.80 and 48.72), followed by T13 (V2B6) (44.96, 46.71 and 45.84), T4 (V1B4) (42.10, 43.58 and 42.84) and T11 (V2B4) (39.45, 41.60 and 40.52), Whereas the minimum chlorophyll content (26.06, 28.25 and 27.16) was recorded in T14 (V2B7) and it was on par with T7 (V1B7) (26.35, 28.50 and 27.43) during the year 2021, 2022 and pooled, respectively.  

DISCUSSION

Mafakheri (2017) reported that using Seaweed extract increased the percentage and yield of essential oil (EO) in an experiment on the fenugreek plant. In parsley (Petroselinum crispum (Mill.) Fuss), chlorophyll content, seed yield, EO percentage and yield were significantly affected by SWE foliar spray (Aly et al., 2021). Mostafa (2015) found that foliar application of SWE increased yield, yield components and EO content (EOC) of fennel (F. vulgare) when compared to control plants. The reason for improving the performance of plants with biological stimuli can be related to the improvements in the biochemical processes in the plants and soil, the activation of some growth-stimulating enzymes, the transfer of ions and as a result, the promotion of photosynthesis potential (Haeusler et al., 2018). 

Seaweed extracts can be applied directly to the soil or as a foliar spray. The increase in plant yield caused by seaweed fertilizer application is linked to hormonal components, specifically cytokinins found in seaweeds (Featon by-Smith and Van Staden 1984). On the other hand, it was reported that when seaweed extract was applied as a foliar spray, the application positively affected root growth, allowing plants to get more water and nutrients from the soil, resulting in increased yield (Mancuso et al., 2006). In this study, plant height, chlorophyll content, fresh herbage yield and essential oil yield increased because of seaweed application. The increase in Ocimum plant growth could be attributed to the presence of micro and macronutrients, cytokinins, auxins and betaines in seaweed extracts, which increase photosynthetic rate and aid in vegetative growth (Devi and Mani 2015). The application of seaweed increased essential oil ratios and similar results were also claimed by Jhariya and Jain (2017) in coriander, Garg (2007), for fennel and Gharib et al. (2008) for marjoram. Moreover, in this study, it was found that by seaweed application, an increase in the oil content of Ocimum plants was achieved.

The presence of major and trace elements, as well as secondary metabolite elicitors, in A. nodosum extracts may have contributed to the increase in essential oil content and modified chemical composition in the study. Major elements such as nitrogen and phosphorus, as well as trace elements such as zinc and boron, were found in the composition analysis of A. nodosum SWE, which may stimulate growth and enrich the composition of treated plants. The presence of nitrogen and phosphorus has been linked to increased oil production and improved essential oil quality in a variety of medicinal plants (Singh et al., 2002; Anwar et al., 2005; Sotiropoulou and Karamanos 2010; Chrysargyris et al., 2016). Trace elements such as boron have been associated with carbohydrate metabolism and hormone functions in plants (Dordas and Brown 2005). Misra and Sharma (1991) reported that Zinc concentration in nutrient application was important for oil yield and menthol concentration in Mentha arvensis L.

A. nodosum is a brown macroalgae composed of polysaccharides such as alginates, fucans, laminarin, and carrageenan, which play an important role in activating salicylic acid, jasmonic acid, and ethylene signaling pathways in plants (Vera et al., 2012). Early studies revealed that metabolic elicitors bind to receptor proteins on plant cell membranes, resulting in increased production of secondary metabolites such as essential oils (Vera et al., 2012; Sharma et al., 2014). Elicitors include polysaccharides like alginic acid, laminarans, and carrageenans, which are the main components of polysaccharides in several commercial seaweed liquid fertilizers like A. nodosum. These carbohydrates have been linked to higher levels of secondary metabolites in plants, such as saponins, essential oils, and phytoalexins (Gururaj et al., 2012; Hashmi et al., 2012).

At the vegetative stage, A. nodosum application resulted in increased chlorophyll content of leaves, which was most likely due to inhibition of chlorophyll degradation caused by betaines present in the extract (Blunden et al., 1996 ; Whapham et al., 1993). These betaine compounds in seaweed extracts prevent the loss of photosynthetic activity by inhibiting chlorophyll degradation (Genard et al., 1991). Similarly, asparagus plants treated with A. nodosum showed a significant increase in chlorophyll content, stomatal conductance, photosynthetic rate, and transpiration rates.

Table 1: Impact of biostimulants on oil yield of sacred basil (Ocimum sanctum L.).

Factor I :Varieties Factor II : Biostimulants

V1: CIM - Ayu                  B1: Chitosan @ 0.1%  B5: Seaweed extract  @ 5%

V2: CIM - Angana            B2: Chitosan @ 0.5%    B6: Seaweed extract @ 10%

B3: Humic acid @ 0.2%    B7: Water Spray (Control)      

B4: Humic acid@ 0.4%

Table 2: Impact of biostimulants on essential oil profiling of sacred basil (Ocimum sanctum L.) during the year 2021.

Factor I :Varieties Factor II : Biostimulants

V1: CIM - Ayu                     B1:Chitosan @ 0.1%      B5: Seaweed extract  @ 5%

V2: CIM - Angana                B2:Chitosan @ 0.5%       B6: Seaweed extract @ 10%

B3: Humic acid @ 0.2%      B7: Water Spray (Control)      

B4: Humic acid@ 0.4%

Table 3: Impact of biostimulants on essential oil profiling of sacred basil (Ocimum sanctum L.) during the year 2022.

Factor I :Varieties Factor II : Biostimulants

V1: CIM - Ayu                     B1:Chitosan @ 0.1%   B5: Seaweed extract  @ 5%

V2: CIM - Angana                B2:Chitosan @ 0.5%   B6: Seaweed extract @ 10%

B3: Humic acid @ 0.2%        B7: Water Spray (Control)      

B4: Humic acid@ 0.4%

Table 4: Impact of biostimulants on essential oil profiling (pooled) of sacred basil (Ocimum sanctum L.).

Factor I :Varieties Factor II : Biostimulants

V1: CIM - Ayu                     B1:Chitosan @ 0.1%    B5: Seaweed extract  @ 5%

V2: CIM - Angana                B2:Chitosan @ 0.5%    B6: Seaweed extract @ 10%

B3: Humic acid @ 0.2%        B7: Water Spray (Control)      

B4: Humic acid@ 0.4%

Table 5:  Impact of biostimulants on chlorophyll content (SPAD value) of sacred basil (Ocimum sanctum L.)      

Factor I :Varieties Factor II : Biostimulants

V1: CIM - Ayu                     B1:Chitosan @ 0.1%    B5: Seaweed extract  @ 5%

V2: CIM - Angana               B2:Chitosan @ 0.5%    B6: Seaweed extract @ 10%

B3: Humic acid @ 0.2%      B7: Water Spray (Control)      

 B4: Humic acid@ 0.4%

Plate 1.  Extraction of oil from Ocimum sanctum. 

 T. Srikanth, N. Seenivasan,  Veena Joshi, K. Venkatalaxmi, D. Anitha Kumari and V. Suresh  (2024). Impact of Biostimulants on Oil Yield and Composition of Sacred Basil (Ocimum sanctum L.). Biological Forum – An International Journal, 16(4): 20-27.