Enhancing the Quality of Monoterpenes in (Cymbopogon nardus (L.) Rendle) Through Rhizobacteria Application Toward Sustained Soil Health

Cymbopogon nardus is an aromatic plant which produces essential oil. Citronellal, citronellol, and geraniol as well-known as monoterpenes. The component of monoterpenes in this research were triggered by organic matter, such as organic manure and rhizobacteria. Organic fertilizers are used for environmentally friendly by maintaining soil and plant health and it’s necessary to achieve agricultural sustainability. Rhizobacteria are beneficial microorganisms for plants and soil for environmental management of both soil, plants, and soil micro biodiversity. Rhizobacteria contributes to soil fertilization through bio-fixation and bio-solubilization of essential nutrients along with the production of growth regulators. Microorganisms regulate the mechanism of absorption and mobility of nutrients in soil. The objective of this research is to investigate the effects of rhizobacteria and organic manure on the component of monoterpenes as well as the content of citronellal, citronellol, and geraniol. The results research show that C. nardus treated with rhizobacteria accumulating of nitrogen 1% and geraniol 5% in leaves which is more than those without rhizobacteria. Interestingly, content of citronellal and citronellol in leaves increased reach at 10% treated by without rhizobacteria and it higher than those supplied with rhizobacteria. In that case, citronellal, citronellol, geraniol formed from the carbon which was seems supplied by organic manure. Furthermore, rhizobacteria treatment shows a stable content of oil components in various rates of organic manure, but able to increase the oil yield at about 30% of C. nardus. Meanwhile, without supply of rhizobacteria, the result of oil yield relatively similar in various rates of organic manure. Regarding to the results, found that application of rhizobacteria able to increase the fresh weight, dry weight, and oil content. As consequently, contribution of rhizobacteria and organic manure to the mechanism of mineralization and improve soil health indirectly. Hence, plant able to uptake and accumulation nutrients in particular nitrogen, phosphorous and potassium.


Introduction
Cymbopogon nardus (L.) Rendle is a herbal plant that produce essential oil.C.nardus contains of main oil components to determine quality and purity, such as citronellal, citronellol, and geraniol.These components are secondary metabolites categorized to terpenoid group, which are synthesized through 1299 (2024) 012007 IOP Publishing doi:10.1088/1755-1315/1299/1/012007 2 monoterpene pathway.The essential oil contains monoterpene such as citral, geraniol, citronellol, citronellal, linalool, elemol, 1,8 cineole, limonene, β caryophyllen, methyl heptonone, geranyl acetate, and geranyl format from several species of Cymbopogon such kind C.nardus, C. citratus, and C. martinii [1].Monoterpenes accumulation in leaves can be induced by an organic material such as organic manure and rhizobacteria.Organic manure is mineral sources contain essential nutrients for plant growth and affects soil quality as well-known physical, chemical and biological properties.Nutrient availability in soil can be improved through rhizobacteria supply.Earlier study by Leountidou [2], rhizobacteria contribute to several mechanisms in the rhizosphere such as nutrient cycling, degradation of organic matter, increasing soil fertility hence it encourages plant growth.The combination of organic manure and rhizobacteria able to induce growth-promoting hormones in roots and the availability of nutrients to stimulate plants growth and content of essential oil particularly in citronellal, citronellol, and geraniol.Previous study by Liao [3] stated that rhizobacteria have positively correlates to plant biomass.Organic manure in various rates on Cymbopogon martini shows that the total of biomass and essential oil significantly increased [4].The metabolism of medicinal plants can be increased by application of organic manure which affect plant morphology and biomass [5].
Nutrient management through application organic material is one of the agricultural practices to enhance essential oil components.The induction of monoterpene in C.nardus strongly depends on growth phase and the availability of soil nutrients.Biosynthesis of essential oil from Cymbopogon species is accumulating in young leaves, which are deeply influenced by the plant's development stage, and environment factors like the nutrient availability.Converting the nutrients availability and partitioning carbon as result of photosynthesis product into biomass and synthesis into secondary metabolism [6].The availability of main components of secondary metabolism gained from photosynthesis directly affects essential oil synthesis.This research objectives is to investigate the effects of rhizobacteria and organic manure on monoterpenes and obtain the optimal rate of organic manure for C. nardus growth hence produce high quality of essential oil.Based on these considerations, the management of environmental between C. nardus, rhizobacteria, and organic manure are expected to improve the quality particularly in component of citronellal, citronellol, and geraniol.

Materials and Methods
The research was conducted from August 2021 to March 2022 in Experimental Garden of Agricultural Faculty, Brawijaya University, Jatimulyo, Malang, with the altitude at about 460 meters above sea level.The soil type at experimental locations is Andisols that the correct type of soil to C. nardus optimal growth.The research method used Randomized Factorial Design with three-time repetitions.The first factor is PGPR which contain some of microbes there are Rhizobium sp., Lactobacillus sp., Trichoderma sp., Aspergillus sp., and Penicillium sp.Rhizobacteria treatment arranged with two different levels: P0: Without Rhizobacteria dan P20: Supply of 20 mL plant -1 Rhizobacteria.The second factor is organic manure with five levels there are K10: 10 t ha -1 , K15: 15 t ha -1 , K20: 20 t ha -1 , K25: 25 t ha -1 , K30: 30 t h -1 .The data was analysed using the variance analysis (F Test) with 5% level.If there is a significant effect, then continue to use the DMRT test with 5% level.
The data observed in sixth month after planting, there is a dry weight parameter (g plant -1 ) because of the separation process between leaves and roots, was placed in an oven at 80°C for 2 x 24 hours until dry-stable weight.Fresh weight (g plant -1 ) was measured using a digital scale.Then, the oil yields was observed by weighing 500 g of dry C.nardus at 6 months after planting and putting it into a distillatory for 6 hours for each treatment.Afterward, the distilled oil was put into a bottle, recorded the results, and calculated using the following formula below : Oil Yield (%) = Oil Weight (kg) Distilled Leaves Weight (kg) x 100% Leaves area (cm 2 ) was measured at first until sixth month after planting using LAM (Leaf Area Meter) tools by putting each leaf in the device and recording the value of the leaves area displayed on the device.Meanwhile, the measurement of nitrogen was carried out destructively at harvest in 6 MAP by weighing 1 g of leaves, putting them into a Kjeldahl flask with a mixture of 1 g selenite and 5 mL H2SO4, and destructed them at 300ºC.After the mixture was done, diluted it with 50 mL of H2O, added 20 mL of 40% NaOH, distilled it, and contained it with 20 mL of boric acid.The distillation was stopped when the storage volume reached the limit of 50 mL, and the mixture turned green.The next step was titration of the mixture with H2SO4 until burgundy color marked the titration endpoint and calculated the total of nitrogen using the following formula: Total N (%) =  −   ℎ x 0.14 x N. H2SO4 x 100 x Water Content The observation of carbon was measureated in 6 MAP by weighing 1 g of C. nardus for every treatment and putting it into Erlenmeyer tube to be mixed with 5 mL of K2Cr2O7 1 N solution before shaking it.Afterward, 5 mL H2SO4 was added to the solution before being shaken again and set aside for 30 minutes.Then 15 mL of aquadest and 5 mL of H3PO4 were added to the sample.Next, 1 drop of diphenylamine was added.The sample underwent titration using FeSO4 1 N until the solution became light blue.The following formula measures the C-organic content. C-Organic= ℎ  0,77 x 0,33 = K2Cr2O7 volume The essential oil observation at harvest i.e. 6 MAP through distillation of leaves then extracted oil use the oil dilution added N-Hexane as the diluent for oil composition analysis by GC-MS instrument (QP-2010 Ultra/Shimadzu).C. nardus's components test was done 6 months after the planting at Institut Atsiri, Brawijaya University, Malang, using the steam distillation method.The C. nardus's components to test were citronellal, citronellol, and geraniol, and other component.

The Biomass of Root and Shoot Ratio C. nardus
Ratio of C. nardus shoot and roots at 6 MAP presents in Figure 1.In general, without-rhizobacteria (P0) and with-rhizobacteria (P20) present the ratio of dry weight of shoot (65%) and roots (35%) have relatively same trend in both treatments.On treatment without rhizobacteria (P0), the shoot roots ratio at K15, K25, and K30 have the same average range about at 45% to 55%.On the other hand, rhizobacteria (P20) treatment on K25 and K30 show a higher percentage between shoot (45%) than roots (55%) than other dosage of organic manure.In comparison, the ratio of shoot at K10, K15, and K20 show a lower percentage reach at about 60% and roots by 40% in average.

The Fresh Weight, Dry Weight, and Oil Yield
The average of fresh weight and dry weight (Figure 2) and oil yield of C. nardus presented in Figure 3 were measured at 6 MAP.Fresh weight (1970 g) and dry weight (508 g) without rhizobacteria (P0) treatment shows relatively similar pattern at all various levels of manure.Supply of organic manure at P0 treatment shows remind stable of dry weight as the biomass of plant, however the treatment of rhizobacteria (P20) is able to increase dry weight and fresh weight by 30%, respectively.The maximum dry weight 862 g and fresh weight 3185 g was reached at both K25 and K30 by 2 fold at 6 MAP.
The oil yield was produced at 6 MAP by C. nardus on rhizobacteria (P20) at K10 (1.94%) hold down to K15 (1.62%), and the pattern was increased at K20, K25, and K30 at about 1.9%.While, without rhizobacteria (P0) at K10 shows that minimum point of oil yield at 0.5% while 3-fold of oil yield reached at K15, K20, and K30.Furthermore, the oil yield relatively reminds stable at P20 (K10, K20, and K25) were reached 2.1%.The enhancement of fresh weight and dry weight were followed by increasing oil yield trend on maximum rate of K25 and declined with adding 5 t ha -1 organic manure.

Leaves Area and Nitrogen Content of C. nardus
The observation of leaves area and nitrogen content in C. nardus at 6 MAP are present on Figure 4 and 5.In general, rhizobacteria treatment (P20) shows that increasing leaves area is followed by nitrogen accumulation.Enhancing of leaves area at about 30.000 cm 2 and nitrogen at 1.7% reach at K20 to K30 in respectively.Meanwhile, nitrogen content of K10 and K15 show a relatively remind stable at around 1.3%.Moreover, without rhizobacteria (P0), the leaves area reached at K25 and gradually enhance by K10, K15, K20, and K30.Nitrogen content in leaves is presented in Figure 5. Interestingly, rhizobacteria (P20) able to enhance 10% of nitrogen content reached by K30.Meanwhile, the rate of organic manure K10, K15, K20, and K25 had the similar trend of nitrogen content with P0 in average 1.3%.

Carbon
Figure 6 presents of carbon content on C. nardus's leaves.The carbon content without rhizobacteria (P0) shows that the application of various levels of organic manure incline at about 30% carbon in leaves compared to P20.While the treatment of rhizobacteria (P20) shows the same pattern of carbon content particularly 1.50% in average.Interestingly, the treatment of K10, K25, and K30 are two folds increase compared to the treatment rate of K15 and K20 at about 30%.

Oil Components
The C. nardus oil components, such as citronellal, citronellol, and geraniol are presented in Figure 7.In general, both of treatments without-rhizobacteria (P0) and with-rhizobacteria (P20) have a higher citronellal 20% compared to the other components.Furthermore, the citronellol component have the same range of 15.95-16.83%with rhizobacteria (P20) or without rhizobacteria (P0).By contrast, rhizobacteria (P20) produces the oil components relative stable at all various rates of organic manure.Interestingly, citronellal level gradual increases with adding 5 t ha -1 of organic manure reach at 40,38% in K30.

Discussion
The measurement of nitrogen content has the same trend under rhizobacteria (P20) and increased while supplying of organic manure.It was showing an increasing nitrogen content followed by expand of vegetative plant, such as leaves area, fresh weight, and dry weight.By contrast, without rhizobacteria (P0), present in fluctuated trends of leaves area obtains under all dosages of organic manure.The nitrogen in leaves is required and converted to form of organs immediately during 3 and 4 MAP, hence, the leaves formed completely to formulate secondary metabolites.The organic manure indirectly affects to observation variables in this research which have a role in nutrients availability.However, organic manure provides nutrients is relatively slow availability, hence rhizobacteria can take role nutrients uptake by plant through produce some hormones.Address to Hasan [7], stated that rhizobacteria colonization trigger plant development, such as hormones indole-3-acetate, cytokinin, and gibberellin by the roots.Furthermore, higher rhizobacteria population tend to enrich mineralization and nutrition availability much earlier in rhizosphere by dissolved phosphates, oxidation of sulphur, and availability of ammonium.Refers to Bhardwaj [8] stated that inoculation of rhizobacteria directly on rhizosphere affect to nutrients absorption mechanism through several mechanisms, such as nitrogen fixation, mineralization of organic compounds, and dissolution of mineral nutrients.Meanwhile, it supported and triggered by the ion absorption system, accessibility, and concentration of nutrients in the roots [9] as well as plant growth and productivity [10].The result of research present that the biomass significantly increased under treatment of organic manure at 6 MAP.It might be occurred due to the primary metabolism closely related to carbohydrate formed in leaves and it converted to organ of plant.Based on El-Sayed [11], mentioned that the influence of organic manure enhances plant height due to the nitrogen level may be a precursor of hormones to formed protein synthesis for cell elongation.Regard to Nandapure [12], C. nardus is a kind of plant which able to produce and accumulate biomass rapidly five months after planting, even though nutrients uptake reach maximum around at ten months.It was clearly that increasing biomass following by the carbon accumulation which continue to synthesis monoterpenes as secondary metabolites.The application of organic manure encourages the proliferation of soil microbes and inducing root lateral hence increases the biomass of the root system which following by carbon content accumulation [13].Rhizobacteria simultaneously involves in various stages of plant growth, such as germination until harvest phase [14] The cultivation of aromatic plants with organic fertilizers can improve soil health and determine factor for quality of essential oils [15].Rhizobacteria was added to roots contained bacteria and fungi, there are Rhizobium sp., Lactobacillus sp., Trichoderma sp., Aspergillus sp., and Penicillium sp.Microorganism in rhizobacteria play a role the mechanism of absorption and mobility of nutrients.In a previous study conducted by Kutlu [16], PGPR inoculation with various bacterial strains increased such kind fresh weight, dry weight, and essential oil yields in (Origanum onites L.).Based on our research, we also found the same results, fresh weight, dry weight, and oil content increased after PGPR added compared without PGPR.Nurzynska-Wierdak [15], states the availability of essential nutrients N, P, K, Ca, and Mg effect on composition of essential oils particular like citronellal, citronellol, and geraniol.The role of rhizobacteria support the dissolution of nutrients, especially Nitrogen and Phosphorus in the soil thus it impacts on growth and induction of oil components in aromatic plants.Nutrient absorption in aromatic plants will trigger oil biosynthesis and affect to plant growth characteristics [17,18].The accumulation of macronutrients N, P, K are limiting factors which had a role particular on plant metabolism, as well known forming of tissue, organs, and converting primary products to secondary metabolism.Indeed, nutrient status such as nitrogen content in leaves and accumulation of monoterpenes, such as citronellal, citronellol, and geraniol, were influenced by the availability of soil nutrients and nutrient accumulation in leaves [19].In this case, the nutrients include an essential macro nutrient, as nitrogen.The average accumulation of nitrogen reached at about 10% in leaves by rhizobacteria (P20) at all dosage levels.Carbon and nitrogen regulate on modulating plant growth and development, as organic manure compound which is required rhizobacteria form to be energy.Organic matter had manipulated and provide condition in term storage of water, which induce vegetative plant growth as well as secondary metabolites such kind monoterpenes [20].Internal factors of plant morphology such leaves area, clearly contributes in biomass accumulation.It assumed that there is interrelationship between carbohydrates and monoterpenes as secondary metabolism [21].Therefore, much higher accumulation of biomass result substrate availability for monoterpenes biosynthesis [22].The secondary metabolite pathway involves precursors, co-factors, energy (ATP), (NADPH) from primary metabolism [23].The glycolysis of carbohydrates leads to the production of ATP and reduce nucleotides, and substrates for monoterpene biosynthesis as well as the abundant carbon, thus will be synthesized into secondary metabolism such kind citronellal, citronellol, and geraniol.Therefore, regards to Santoro [24], the availability of nutrients obtained from rhizobacteria inoculation enhance indirectly photosynthetic efficiency and primary metabolism accumulation, which results to enrich secondary metabolite concentrations such kind of essential oils.
The low percentage of oil yield closely related to density of C. nardus oil.By contrast, the high oil component of C. nardus was not correlated by the percentage of oil yield.Deeply stated by Rochim [25], the higher production of oil yield causes lacks quality of C. nardus oil.Metabolism and synthesis of essential oils are rapid during early growth and development of young leaves in C. nardus.Based on Mukkaram [26], research clearly that young plant tissues had more essential oils compared to old leaves.Moreover, plant phase development affects to biosynthesis and accumulation of essential oils.In previous research conveyed by Gershenzon [27], the trend of monoterpenes accumulation in Peppermint increases rapidly in young leaves for 21 days at the stage of leaf development.In addition Cymbopogon sp.monoterpenes patterns accumulation enhanced in young leaves as well as in the Lamiaceae family [28].Address to another investigation by Gupta and Ganjewala [29], a large amount of essential oil in lemongrass plays a role by actively biogenetic young leaves and leaves position include anabolic and catabolic processes.Therefore, the role of monoterpenes metabolism regards to Moser and Pichler [30], induced by the formation of terpenoid isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) as precursor through carbon sources.It was triggered by the esterase enzyme which can convert geranyl acetate.Thus, two biosynthetic pathways by Abdallah and Quax [31], involve the 2Cmethyl-D-erythritol-4-phosphate (MEP) pathway which is known well as the 1-deoxy pathway-Dxylulose-5-phosphate (DXP) pathway and mevalonate pathway (MVA).In MVA pathway, the formation of IPP precursors is obtained and delivered to the formation of monoterpenes higher in young leaves.Meanwhile, the acetate pathway enzyme MVA mevalonate-5pyrophosphate (MVAPP and PMD) decarboxylase occurs strongly influenced by environmental conditions such as soil microbes' population.Supply of organic manure and rhizobacteria in C. nardus indirectly trigger mineralization in soil, hence the large uptake of nutrient by plants.Furthermore, plants are able to absorb nutrients rapidly during growth and development while rhizobacteria supply.Evidently, in this result research shows that monoterpenes remain stable in leaves under rhizobacteria application.Carbon and macronutrients are used in two kinds of metabolism as well-known primary and secondary metabolism.During the plant growth phase, nitrogen and carbohydrates initiate monoterpenes biosynthesis, and those depend on resources and utilization by partitioning of primary secondary metabolism.Address to Anggraini [32], that the production of primary and secondary metabolites used carbon and macronutrients such as nitrogen, phosphorus, and potassium in plants are closely interlinked.This result is supported by Thirumurugan [33] and Cui [34], that sufficient nutrients, particularly carbon and nitrogen, clearly induce the synthesis of primary and secondary metabolites.
Obviously, monoterpene components namely citronellal, citronellol, and geraniol, were found to be like this research results, which were observed under the same conditions.Supported by Ganjewala and Gupta [35], stated that the MEP (Methyl erythritol) pathway, IPP in plastids will be synthesized into components of citronella, namely citronellall, citronellol, and geraniol.Indeed, besides being formed into oil components, IPP is formed into other products such as chlorophyll, carotenoids, and growth regulators.Therefore, plant secondary metabolites tend to be the same and deeply influenced by carbon content in leaves and nutrient availability in rhizosphere

Figure 1 .
Figure 1.The Percentage of Biomass on C. nardus's Shoot and Root Ratio at 6 MAP