Effectiveness of Mycorrhiza to the Growth and Essential Oil Content of Eucalyptus citriodora seedling

. Eucalyptus citriodora is one of the forest plant produced essential oil which contains various beneficial chemical compounds, including 1,8-cineol. Arbuscular Mycorrhizal Fungi (AMF) is a biofertilizer widely used to increase biomass and bioactive content in cultivated plants. The objective of this study was to determine the impact of an AMF consortium on the growth, biomass yield, and 1,8-cineol content of E. citriodora seedlings. Research was conducted six months, in the greenhouse of Indonesian Spice and Medicinal Crops Research Institut (ISMCRI), Bogor, by using randomized complete block design, with six types of inoculum treatments and four replications. The AMF consortium inoculum were explored from various acidity land under several stand of plants, namely consortium inoculum of M1, M2, M3, M4 and M5. The result showed that AMF inoculum treatments had positive effect to the growth (plant height, number of leaves and branch) and weight of leaf, stem and root of E. citriodora. The oil, citronellal and 1.8 cineole content of Eucalyptus sp leaves were increased by the application of AMF. The highest oil content, citronellal and 1.8 cineole content of E. citriodora leaf were resulted from M4 consortium application, were 2.38%, 76.07%, 0.52% respectivelly. M4 consortium application showed the highest effectivenesss to the E. citriodora.


Introduction
Eucalyptus citriodora is one of the ten most significant Eucalypts for the annual increase in world wood production, and it has a significant role in the production of wood in Asian plantations, where it ranks ninth overall.[1]; [2].Eucalyptus produces essential oils from its leaves in addition to wood.About 1.36% of the essential oil found in E. citriodora's leaves is citronellal (57%), followed by citronellol (15.89%), citronellyl acetate (15.33%), and other substances.[3]; [4].The oil contains several volatile monoterpenes, including cineole, citronellol, citronellal, limonene, linalool, and -terpinene [5].The perfumery and pharmaceutical industries use Eucalyptus essential oil, which has significant commercial value and ranks highly in both quality and quantity.In addition, it exhibited a broad spectrum of antimicrobial [6]; [7], antifungal [8], anticandidal [9], antibacterial [10]; [11], expectorant, and cough stimulant activity [12] properties.E. citriodora is one of the cultivated Eucalyptus species which developed by using plant material derived from vegetative propagation such as cuttings.The plant is widely cultivated in areas, which have low soil fertility statusTo successfully grow and develop in the field, it needs suitable seed conditions.AMF make up a significant portion of the soil microbial biomass; they are widespread throughout various ecosystems and have the potential to infect the roots of more than 80% of terrestrial plants, resulting in a mutualistic symbiosis.. AMF can broaden plant roots to increase the uptake of various macro and micronutrients, which affects plant development, growth, productivity, and quality.[13].The AMF acquires up to 20% of photosynthates from plants in exchange for mineral nutrients and water [14]; [15];.Some evidence suggests that AMF is a biofertilizer that is widely used on cultivated plants to enhance nutrient availability, plant yield, and bioactive content [16]; [17].AMF may significantly contribute to accelerating seedling growth in nurseries and helping plants survive in hostile environments.
The research aimed to identify the effectivity of several AMF consortiums toward the growth, biomass yield, and the content of oil, 1.8-cineole and citronellal of E. citriodora at 6 months after planting.

Materials and methods
Research was conducted for seven months, in the greenhouse and laboratory of Indonesian Spice and Medicinal Crops Research Institute, Bogor, West Java, since February to October 2021.
One month of E. citriodora seedlings (Manoko accession) were planted in polybags (3 kg volume) containing media soil (sterilized by using Basamid D soil fumigant) : cow dung (3:1) for six months.The AMF consortium inoculums were explored from various acidity land under several stand of plants, namely consortium inoculum of M1, M2, M3, M4 and M5.
The research was conducted using a random block design that was repeated four times.The factor was mycorrhiza application, which consisted of five consortium types.: M0 = no AMF applied M1 = Mycorrhiza isolated from vanilla rhizosphere in West Java M2 = Mycorrhiza isolated from clove rhizosphere in North Sumatera M3 = Mycorrhiza isolated from Ultisol soil M4 = Mycorrhiza isolated from Cinnamomum rhizosphere in West Java M5 = Mycorrhiza isolated from pine rhizosphere in forest soil The AMF inoculum was prepared using colonized Zea mays root fragments in quantities of 50 g per polybag of rhizosphere soil containing spores in order to obtain high levels of root colonization..There were 120 plants in the population, and five plants were used and observed for each treatment and repetition.
Six months after planting (MAP), a number of quantitative traits of the seedlings were evaluated, including plant height, the number of leaves and branches, the weight of the leaves, stems, and roots, the oil content of the leaves, the concentration of 1.8-cineol and citronella, the content of macronutrients nitrogen, phosphorus, potassium, calcium, and magnesium (N, P, K, Ca, Mg), and the infectivity of the mycorrhizal roots (AMF colonization).Oil and citral content were measured by distillation, and Gas Chromatography method [18]; [19].Nutrient uptake of leave was calculated from nutrient content and leaves weight.The infectivity of mycorrhizal roots was assessed using the [20] approach in [21].The presence of hyphae, vesicles, arbuscules, or any combination of the three, is a sign of root colonization.Using the Kjeldahl, spectrophotometer, and AAS techniques, the N, P, K, Ca, and Mg content of leaves was evaluated [22].To ascertain whether there were significant differences, the statistical studies used analysis of variance (Anova) and Duncan's Multiple Range Test (DMRT) at the 5% level.

Results and discussion
The plant height, number of leaves, and number of branches of Eucalyptus species at 6 MAP were not substantially impacted by the inoculation of AMF consortium treatments, and the control treatment had the slowest growth among the five AMF consortium treatments.(Table1).This can be caused by the young age of the plant, however there were still positive effect of AMF application on the growth of Eucalyptus sp. by increasing plant height (3.2% to 21.8%), number of leaves (27.3% to 41.6%) and branches (50% to 107.7%).[23] also found that the mycorrhizae application did not significantly affect the growth of Eucalyptus sp.(height, diameter and root shoot ratio) at 4 months after planting.AMF application resulted in the higher fresh weight of leaves, stems and roots of E. citriodora compared to non mycorrhiza treatments (Figure 1).The increasing fresh weight, stems and roots of E. citriodora leaves were 6.2% to 25.2%, 74% to 151% and 78% to 160% respectively, compared to non mycorrhiza treatments.[24] found an increase of 45% of E. globulus shoot dry weight, inoculated with Glomus deserticola in greenhouse condition.The oil, cineole and citronella content of six months plant leaves treated with mychorrhiza consortium were varied (Table 2).The oil content ranged from 0.85 to 2.38%, the cineole content range from 0.01 to 0.52%, and citronella content ranged from 48.28 to 76.07%.M4 consortium application resulted in the highest content of oil, cineole and citronella, were 2.38%, 0.52% and 76.07%.Control treatment showed the lowest content of oil, cineole and citronella, were 0.85%, 0.01% and 48.28%.The oil yield of three different Eucalyptus species (E.citriodora, E. urophylla, and E. viminalis) from diverse areas in Indonesia varies from 1.6 percent to 3.3 percent (w/w, dry weight) depending upon the type, plant location, and leaf age.1,8-cineole (18.2%-45.5%)and citronellal (69.27-82.81%)have been named as the two main ingredients of eucalyptus oil [25].Mycorrhizal fungus may be used as a bioinoculant to increase the essential oil concentration of various herbal medicine since it likely triggered or encouraged the isopentenyl diphosphate production pathway.[26]; [27].The macronutrients N, P, K, Ca and Mg content and uptake of E. citriodora leaves showed that N and K were the highest nutrient content and uptake, followed by P, Ca and Mg nutrients (Table 3 and 4).The M4 consortium treatment showed higher N, P, K, Ca and Mg uptake of E citriodora leaves.Most NO3-N that is transported to leaves is quickly transformed to amino N, which can either be used in the leaf to create protein or transferred directly to other plant parts for protein synthesis.[32].One of the key macronutrients for plant growth and development is potassium, which also activates over 60 plant enzymes and participates in the osmotic regulation of plant cells, particularly the opening and shutting of stomata [28,33].In the symbiotic connection with plants, AM hyphae are critical because they extend from the root and make it easier for water and vital inorganic nutrients like phosphate, nitrogen, and micronutrients to be transported from the soil to the plant.AM hyphae play a crucial role in the symbiotic relationship with plants because they extend from the root and facilitate the transportation of water and essential inorganic nutrients including phosphate, nitrogen, and micronutrients from the soil to the plant.They actively take up carbon from the host plants to maintain their own existence, as detailed in reference [29].AM hyphae are important in the symbiotic relationship with plants because they extend from the root and make it easier for water and vital inorganic nutrients like phosphate, nitrogen, and micronutrients to be transported from the soil to the plant.For their own survival, they actively take up carbon from the host plants, as mentioned in reference [29].Better seedling growth of Eucalyptus pellita f.Muell was obtained with double inoculation of Glomus sp. and P. arrhizus, which might also improve nutrient uptake of N and P by 64% and 155%, respectively, in comparison to non-inoculated control seedlings [30].The average mycorrhiza infectivity on root of E. citriodora inoculated with M4, M5 and M2 consortiums were 72.5%, 67.5%, and 46.8% whereas the control was 15%.M4 consortium showed significantly the highest AMF root colonization compared to others AMF consortium.A positive response to inoculation was expressed by an increase in AM root colonization levels and in yields as compared to non-inoculated controls [31].

Conclusion
Treatments with AMF inoculum led to greater growth in terms of plant height, the number of leaves and branches, and the weight of the leaves, stem, and root of E citriodora.AMF application boosted the E. citriodora leaf's oil, 1.8 cineole, and citronellal contents.The M4 consortium application produced E citriodora leaf oil with the greatest concentrations of citronellal and 1.8 cineole, which were 2.38 percent, 76.07 percent, and 0.52 percent, respectively.The M4 consortium application demonstrated the greatest efficacy with E citriodora.
We grateful to thank Mr. Redy Aditya Permadi and Mr. Hikmat Mulyana from Indonesian Spice and Medicinal Crops Research Institute.

Fig. 1 .
Fig. 1.The weight of leaves, stem and root of E. citriodora on mycorrhiza treatments at 6 MAP

Table 1 .
E. citriodora plant height, leaves, and branches on mycorrhiza treatments at 6 MAP

Table 2 .
The oil, cineole and citronella content of E. citriodora on mycorrhiza treatments

Table 3 .
The macro nutrients content of E. citriodora leaves

Table 4 .
Macro nutrients uptake of E citriodora leaves