Study of influence of genus and mycorrhizae dosage on the growth of patchouli (Pogostemon cablin Benth.) in the seedling and post-seedling phases in inceptisols soil

Pogostemon cablin Benth. (Lamiaceae) is the source of patchouli essential oil, which is an important and unique commodity in the fragrance industry. Patchouli plants are also cultivated on inceptisol with low nutrient content and CEC exchange. The use of mycorrhiza is expected to increase nutrient absorption, and plant performance of patchouli. This research is divided into 2 phases, namely the seedling and post-seedling phases. This study applies a Randomized Block Design (RBD) with 3 x 3 factorial and 3 replications. The observed factors were genus mycorrhizae; namely M1 (Glomus mossae), M2 (Gigaspora cf gigantea), and M3 (Glomus mossae + Gigaspora cf gigantea); and mycorrhizal dose; namely D1 (5 g), D2 (10 g), and D3 (15 g). Data were subjected to analysis of ANOVA using SPSS ver. 21.0 with Tukey’s test. In the seedling and post-seedling phases, the initiation of Glomus mossae had a significant impact on the growth of patchouli and mycorrhiza colonization. The best mycorrhizae dose is 5g which has a significant effect on the number of leaves at 135 and 150 DAP. The interaction of Glomus mossae at a dose of 10 g mycorrhizae has a significant effect on the stem diameter 135 and 150 DAP.


Introduction
Patchouli (Pogostemon cablin Benth.) is one of the potential plants to produce essential oils which are used as a blending agent in various industrial sectors [1][2].Aceh patchouli is widely cultivated because it has a higher of oil quality [3].Patchouli cultivation in Aceh is cultivated from the lowlands to the highlands on various soil types, such as entisols, andisols and inceptisols.Conditions of different soil types can contribute different nutrients to patchouli plant growth.
In this study, observations were made on the growth of patchouli plants grown on Inceptisol soil.Inceptisol soil that is spread in Indonesia is around 37.5% with the characteristics of low nutrient content, low C/N content, soil acidity (low pH), CEC (Cation Exchange Capacity) for the elements Ca, Mg, K and Low-moderate Na, the presence of the nutrient phosphate (P2O5) is fixed by free Al and Fe and the oxyhydroxides of Al and Fe form Al and Fe-P compounds which cause the nutrient P to be insoluble and unavailable to plants, and the content of the element potassium which can be exchange rate is low due to the leaching [4][5][6].This condition is a critical point for improving the quality of inceptisol soil to support the growth of patchouli plants.
Improvements that can be made to improve the condition of the inceptisol soil are by providing Ca and Mg nutrients to increase soil pH, and adding nutrients through inorganic or organic fertilizers such as biofertilizers [7][8].The use of biofertilizer is one of the efforts to help the growth and increase of agricultural crop yields [9].Biofertilizers not only increase the assimilation of nutrients from plants (total N, P, and K), but also improve soil properties, and total N in the soil [10].The content of biological fertilizers are microorganisms that have a positive role for plants.Microbial groups that are often used are microbes that fix N from the air, microbes that dissolve nutrients (especially P and K), microbes that stimulate plant growth, such as rhizobacteria and mycorrhizae.Mycorrhiza is a symbiotic association between fungi and plant roots which colonize the plant root cortex tissue to form a hartig net, that function for exchange of plantderived carbohydrates and fungus-derived nutrients [11][12].There are two types of mycorrhizae, namely ectomycorrhizae and endomycorrhizae.One of the subtypes of endomycorrhizae is arbuscular mycorrhizae which can be associated with 80% of higher plant species [13][14].
The use of mycorrhizae has been utilized by several farmers and researchers in Indonesia to increase nutrient uptake from the surface of the rhizosphere and contribute to the supply of water and nutrients to plants, reducing mineral fertilizer use [15][16][17][18].Intake of nutrients as phosphorus, nitrogen, and other macronutrients and micronutrients [19], by involving hyphae of fungi which able to improve plant growth performance and crop yield [20][21][22].Mycorrhizae colonization that is formed will increase the possibility of more effective absorption of nutrients, especially when the solubility and compound of mineral concentrations in the soil are low, such as in inceptisol soils [23][24][25][26].So, this study aims to quantify the effect of mycorrhizae genus and doses on the growth of patchouli plants on inceptisol soil.

Research Implementation
This research was divided into 2 phases, namely the seedling and post-seedling phases.In the seedling phase, cuttings of patchouli plants of the same length are put into seedling polybags which contained planting media in the form of inceptisol top soil and have been given a mycorrhizal starter that was adjusted to the mycorrhizal genus and the dose given of 5 gr for each poplybag.Patchouli plant seedlings were planted in a shady place to maintain soil moisture.Observations during the seedling phase for 60 days, with parameters; the number of leaves, number of shoots, and number of branches of patchouli plants which were measured and observed at 15, 30, and 45 DAP.
In the post-seedling phase, the patchouli plants used were aged 60 DAP which had the same homogenity performance and growth criteria.The patchouli plants had been grown on Inceptisol soil and had been initiated by mycorrhizal inoculation during the transplantation period with the appropriate dosage of treatment.Patchouli plants were observed for their growth from 120-150 DAP on the parameters of plant height, number of leaves, number of branches, stem diameter and percentage of root colonization of patchouli plants.The plant cultivation carried out, involved giving NPK fertilizer with 25% of the recommended dose (150 kgs ha -1 ) for each plant, pruning unproductive branches, and management of weeds and pathogens.
Mycorrhizae colonization analysis was observed at the Plant Physiology Laboratory, Syiah Kuala University, Banda Aceh.The sample used was 10-20 cm from the length of the plant roots.The root samples were cleaned with 10% KOH solution (10g KOH in 100 ml aquades) for 5-10 minutes [27].The cleaned root samples were stained with trypan blue solution overnight, and then 1 cm root samples were cut, to be observed under a microscope with an objective magnification of 10x and 40x.Percentage of root colonization by mycorrhizae was calculated by the method of Giovannethi and Mosse (1980) [28]. (1) The percentage of infected roots was determined based on the criteria as follows.Very High 5

Effect of the mycorrhizae genus on the growth of patchouli plant in the seedling phases
The results of the F test showed that the mycorrhizae genus on patchouli plant seedlings (Pogostemon cablin Benth) had a significant influence on the number of leaves at 30 and 45 DAP, the number of shoots at 30 and 45 DAP, and the number of branches at 30 and 45 DAP.Table 2 shows the growth of patchouli plants in the seedling phase due to inoculation from the mycorrhizal genus.
The initiation of the mycorrhizal genus of the Glomus mossae had a significant effect on the number of leaves, number of shoots and number of branches of patchouli plant seedlings at 30 and 45 DAP which were significantly different from other treatments.Initiation of mycorrhiza in the seedling phase can increase plant resistance, seed adaptation to the environment by reducing the mortality ratio [16].Mycorrhizae in plants generally grow and develop rapidly and show healthier plant performance than plants without mycorrhiza initiation, especially in conditions of low soil fertility, such as Inceptisol soil [13].

Effect of the mycorrhizae genus on the growth of patchouli plant in the post-seedling phases
The result showed that there is a significant effect of inoculation of genus mycorrhizae on the plant height 135 and 150 DAP, number of branches 150 DAP, stem diameter on 135 and 150 DAP, and root colonization, but not significant on the other parameters.Tabel 3 shows patchouli growth as affected by the inoculation of mycorrhizae genus.Initiation of the mycorrhizal genus of the Glomus mossae had a significant effect on plant height of 135 and 150 DAP, number of branches 150 DAP and stem diameter of 135 and 150 DAP which were significantly different from the inoculation treatment of mycorrhizal genus Gigaspora cf gigantea and mixed mycorrhiza Glomus mossae + Gigaspora cf gigantea, however the percentage of mycorrhizae colonization was highest in the mixture of Glomus mossae + Gigaspora cf gigantea, which was not significantly different from Glomus mossae.This is due to the low acidity of the inceptisol, and the high adaptability of Glomus mossae to these conditions causing the best patchouli plant growth to be found in treatments initiated by Glomus mossae.This is in line with Kaya et al. (2009) that mycorrhiza can help to improve the acidity of the rhizosphere and help dissolve soil macronutrients and micronutrients that can be absorbed by the plant root system [29].Mycorrhizae Glomus mossae also increase the availability of Mg, Cu, Fe and Zn elements in the soil to be uptake by plants [30].Glomus mossae arbuscular inoculation on patchouli plants affected the growth of chlorophyll content, gas exchange in photosynthesis and increased the availability of phosphate nutrients in inceptisol soil conditions which had P-limited soil conditions.Nutrient absorption by mycorrhizae is determined by the mycorrhizal colonization that is formed.In a study by Long et al, (2010) stated the percentage of colonization formed by Glomus mossae was higher than that of Gigaspora cf gigantea [31][32][33].

Effect of mycorrhizae dosages on the growth of patchouli plants in inceptisol soil
The result showed that there is a significant effect of dosages mycorrhizae on number of leaves 120 and 135 DAP, stem diameter 135 and 150 DAP but not significant on the plant height 120, 135, and 150 DAP, number of leaves 150 DAP, number of branches 120, 135, and 150 DAP, and stem diameter 120 DAP.Tabel 4 shows patchouli average growth as affected by the dosages of mycorrhizae.The best dose of mycorrhizae that had a significant effect on the growth of patchouli plants was at a dose of 5 g for the number of leaves at 120 and 135 DAP which was significantly different from the mycorrhizal dose of 10 g.The best stem diameter of patchouli plants aged 135 and 150 DAP, and root colonization was found in 10 g mycorrhizal dose which was significantly different from 5 g mycorrhizal dose.Doses of 5 g of mycorrhizae appear as the minimum effective dose of this organic fertilizer that ensures optimal performance of the castor bean in the field [34].The number of mycorrhizal doses given will affect the number of spores contained in the given dose.Spores from potential fungi with plant roots are influenced by inceptisol soil physical, chemical, and biological factors.So that the dose of mycorrhizae used affects the effectiveness of mycorrhizae in absorbing nutrients and water for the growth of patchouli plants.

Interactions between genus and dosages mycorrhizal of patchouli plants on inceptisol soil
The result showed that there is a significant effect in the interaction of mycorrhizae genus Glomus mossae and dosages mycorrhizae of 10 g on the stem diameter 135 and 150 DAP, but not significant on the others parameter.Tabel 5 shows patchouli average growth as affected by the interaction of mycorrhizae genus and doses of mycorrhizae.Based on Table 5 showed that there was an interaction between Glomus mossae and a mycorrhizae dose of 10 g on the average stem diameter of patchouli plants 135 and 150 DAP, which was significantly different from the other combinations.Selection of the right genus according to the characteristics of the inceptisol soil will determine the growth of patchouli plants, especially organic patchouli.Mycorrhizae colonization of the genus Glomus mossae + Gigaspora cf gigantea can cause the host plant to interact with the fungus to exchange nutrients for better plant growth.In present study, the higher root colonization in Glomus mossae can improve all growth parameters of sapota seeds (Manilkaraachras mill Forsberg) [35].Arbuscular mycorrhizae fungi (e.g Glomus intraradices and Glomus mossae) are important components of plant root ecosystems that have a significant effect on increasing ecosystem efficiency [36].Latef and Chaoxing (2011) [37] found that tomato plants infected by Glomus mosseae under low temperature stress improved plant growth.The presence of Glomus mosseae peanuts significantly improved plant growth (28%), pod production (22%), and seed weight (12%) [38].This research in line with Zhang, et al. (2018) [39] showed that the interaction of the mycorrhizal genus has an effected on increasing the growth of several types of plants, both food, horticultural and perennial crops which produce production value.

Conclusions
Inoculation of Glomus mossae increases the vegetative growth of patchouli plans in the seedling and post-seedlings phases.The best dose of mycorrhizae that had a significant effect on the growth of patchouli plants was at a dose of 5 g for the number of leaves at 135 and 150 DAP which was significantly different from the mycorrhizal dose of 10 g.There was a significant effect between the interaction of the Glomus mossae arbuscular mycorrhizae with a dose of 10 g on the growth of patchouli stem diameter.The percentage of mycorrhizae colonization was highest in the mixture of Glomus mossae and Gigaspora cf gigantea.

Table 1 .
Classification of mycorrhizae colonization percentage.

Table 2 .
Mycorrhizae genus impact on the growth of patchouli (Pogostemon cablin Benth.) in the seedling phase.Note, the values are average ± standard deviation for 3 replications in each group; the same letter is not significantly different at the 0.05 level tested by HSD.

Table 3 .
Mycorrhizae genus impact on the average growth of patchouli (Pogostemon cablin Benth.) in the post-seedling phases.

Table 4 .
Dosages of mycorrhizae impact on the average growth of patchouli (Pogostemon cablin Benth.) in inceptisol soil.Note.The values are average ± standard deviation for 3 replications in each group; the same letter is not significantly different at the 0.05 level tested by HSD.

Table 5 .
Mean stem diameter of patchouli plants aged 135 and 150 DAP due to the treatment of mycorrhizae genus and mycorrhizal doses on inceptisol soil.