Arbuscular Mycorrhizal Fungi Associated with Wati ( Piper methysticum ) , a Medicinal Plant from Merauke Lowland , Papua , Indonesia

Indonesia is rich of natural resources, including the diversity of agricultural crops and other valuable plants. Many plant species used by local people for traditional herbal medicine and some other species used by drug industries. Among these, Wati (Piper methysticum, Piperaceae) is one of the medicinal plant found in Merauke lowland, Papua. It has been cultivated by local people because of it high value as medicinal and cultural uses. Wati plant is used to treat anti-stress, rheumatism, respiratory tract infections, tuberculosis, gonorrhea, headache etc. The habitat, including the microorganism in the soil plays an important role in the growth of this plant. Therefore, this study was conducted to identify the arbuscular mycorrhizal fungi (AMF) associated with the rhizosphere of Wati from Merauke lowland. Soil and root samples were collected from different locations and the colonization percentage on the root sample were determined. Our results showed that the number of the spores in the soil samples was 45-89 spores/50 g soil, while the colonization percentage on the root was 38.46-83.3%. Among 13 AMF morphospecies that found on the soil samples, 10 were identified to genus level such as Glomus, Clariodeoglomus, Acaulospora and Scutellospora, while the other were unidentified. Further work will be needed such as trap-plant culture method to get more information on the diversity of AMF associated with Wati.

. However, little is known about the presence of AMF on medicinal plant in Papua.It was reported that in addition to endophytic fungi, in the root of P. methysticum indicated the presence of AMF (Tanjung et al., 2014).This preliminary information becomes important to reveal the presence of AMF in medicinal plants.Therefore, this study was conducted to determine the presence AMF in the rhizosphere of wati from the lowlands of Merauke, Papua.The results of this study are important in the utilization of the types of AMF found for the purpose of endurance and enhancement of plant growth.It is only on P. methysticum plants, but also in other crops such as agriculture, plantation and forestry in general.

Study sites
The survey was conducted in the area of P. methysticum plantation in lowland area of Merauke, Papua Province.Three sites namely Wasur, Pinje and Sota were selected to sampling (Figure 1).

Soil sampling
Rhizosphere soil samples (1 kg) and roots from each individual plant were collected.Soil samples were mixed to form a composite sample for analysis of soil properties and extraction of AMF spores.

AMF colonization
The roots were separated from the stem and cleaned with running water, then were soaked in a FAA fixative solution (formalin-acetic acid-5: 5: 90) for 1 hour.Fixed roots were cleared in 10% KOH solution for 24 hours and acidified with 1% HCl for 24 hours.After washing with aqueous solution, the roots were stained with trypane blue (0.05% in lactoglycerol) for 24 hours (Vierheilig et al., 2005).Then they were examined with compound microscope for AMF structures (intraradical hyphae, extraradical hyphae, vesicles, arbuscules and intraradical spores).The estimation of root colonization percentage was done by the slide method (Kormanik & McGraw, 1984;Brundrett et al., 1996;Sun & Tang, 2012).

INTRODUCTION
Piper methysticum is one of the plant species of the Piperaceae family and known as a medicinal plant to cure rheumatism, respiratory infections, tuberculosis, gonorrhea, and headaches.The leaves and stems contain several compounds that can be used as antidepressant, analgesic, and psychoactive drug (Suharno et al., 2016), antibacteria (Zhao et al., 2010) and anticancer (Tabudravu & Jaspars, 2005).Known as "kava", this species has been widely explored in the South Pacific region, for example in Fiji, Tonga, Vanuatu, Samoa and some Micronesia regions, because it is important for community tradition events (Davis & Brown, 1999).In the lowlands of Merauke, New Guinea part of Indonesia, this species is called "Wati" and used by local communities as medicinal plants (Tanjung et al., 2014;Suharno et al., 2016).In addition, the plant is of high cultural and economic value therefore it gave impacts on the income of local communities.
Local people in Merauke have been trying to cultivate it in small scale using conventional methods (Suharno et al., 2016).Some people failed to cultivate and they believe in the existence of "superstitius powers" that only certain people can cultivate (Tanjung et al., 2014;Suharno et al., 2016).Therefore, it need a touch of technology in cultivating "wati" (Suharno et al., 2016).One is the involvement of soil microorganisms, including arbuscular mycorrhizal fungi (AMF) (Turk et al., 2006;Souza, 2015;Pagano et al., 2016).
AMF forms a symbiotic mutualism with the root system of plants.More than 80% of vascular plants form symbiotic mutualism with AMF (Turk et al., 2006;Smith & Read, 2008, Souza, 2015).Plants derive nutrients from the absorption carried out by the fungus, while the fungi derive energy sources from the photosynthesis of plants (Smith & Read, 2008;Souza, 2015).The role of AMF (Phylum Glomeromycota) in the growth of medicinal plants in various countries is significant (Sundar et al., 2011;Raei & Weisany, 2013;Chandra et al., 2014), including in China (Wang & Zhao, 2008).Several types of AMF are found to be symbiotic with the roots of medicinal plants such as ginseng (Panax ginseng, P. notoginseng, and P. quiquefolium), Datura stramonium, Atractylodes macrochepala, and several other medicinal plants (Wang & Zhao, 2008).It can even improve the composition of essential oils and antioxidant activity (da Luz et al., 2015).
In Papua, the occurence of AMF has been reported in corn, Setaria italica, weeds, and Brachiaria precumbens (Suharno et al., 2006; Su-solution containing the spores was centrifuged (2500 rpm, 10 min) by the addition of 50% sucrose.The separated spores were observed under a microscope.Morphological identification were based on spore characteristics (spore shape, spore color, glare stalk attachment, spore wall and spore reaction with Melzer's solution) according to Brundrett et al. (1996) and Schenck & Perez (1990).

Data analysis
Soil analysis was conducted at Soil Laboratory SEAMEO-Biotrop, Bogor.The observational data were analyzed qualitatively.The data is shown in the form of pictures or tables.

Soil properties
The texture of the soil in this field was categorized as loam to clay loam.The pH of the soil was acidic (5.1-5.7).The macro and micronutrient content in Sota was relatively higher than in other sites, except for available P which was the lowest compared to others (Table 1).

Spores and Colonization of AMF in P. methysticum
The results showed that AMF was found in the roots of P. methysticum and rhizosphere soil (Figure 1; Figure 2).The percentage of AMF colonization in all study sites was varied (38.46 -83.3%, average 56.65%) (Table 2).The number of spores found in the rhizosphere soil of P. methysticum varies widely, ranging from 45 to 89 spores with an average of 66,4 spores per 50 g of soil samples and 93 to 162 spores with an average of 122,2 spores per 100 g of soil samples (Table 3).

Diversity of AMF
Based on the spore morphology, 10 species of AMF that belong to 4 genera were identified but 3 species were remain unknown (Figure 2).From the known genera, the dominant genus was Glomus (4 types; Glomus sp1., Glomus sp2., Glomus sp3., Glomus sp4), followed by Acaulospora (3 types; Acaulospora foveata, Acaulospora sp1., Acaulospora sp2.), Gigaspora (1 types; Gigaspora sp.) and Scutellospora (2 types; Scutellospora sp1; Scutellospora sp2).In this study, the P. methysticum were clarified to the mycorrhizal by the presence of AMF stuctures such as hyphae, vesicles, arbuscules and spores in the root cortical cells.However, the ar-buscules were only observed in 5 of the 10 roots P. methysticum.This result was in confirmation with Maffia et al. (1993) which was found only 2 of the 8 species of Piperaceae indicated the presence of vesicles and arbuscules of AMF.The presence of colonization and development of hyphae in the root system rhizosphere affects plant growth (Torrecillas et al., 2010;Koul et al., 2012;Seema & Garampalli, 2015).This is because hyphae plays a role in helping root function in nutrient absorption (Moreira et al., 2006;Turk et al., 2006).
The number of spores in the rhizosphere soil of P. methysticum are varied.The highest number of spores was found in Pinje 4 (89 spores per 50 g soil, 162 spores per 100 g soil), while the lowest was found in Wasur 5 (45 spores per 50 g soil, 97 spores per 100 g soil).Soil condition in Pinje was sandy loam and in Wasur was sandy clay loam.The texture of soil the soil influenced the presence of AMF.The spore number and the infection of AMF was higher in loamy and sandy loam soil (Rachel et al., 1993;Sundar et al., 2011).The average number of spore was 122.2 per 100 grams of soil samples indicates the production of spores in this area was quite high.This high number was due to soil sampling is taken during the dry season.According to Cuenca & Lovera (2010) AMF in the dry season tends to produce spores than to form both intraradical and extraradical hyphae.
In this study, 4 generations have been registered namely Glomus, Acaulospora, Scutellospora and Gigaspora.However, Glomus was the dominant genus in in the rhizosphere soil of P. methysticum.This result was supported by Maffia et al. (1993) that Glomus, Acaulospora, and Gigaspora  AMF can be utilized in increasing plant growth, including medicinal plants (Thapa et al., 2015;Chandra et al., 2014).According to Gogoi & Sigh (2011), AMF has an effect on the growth of Piper longum (Gogoi & Sigh, 2011;Seema & Garampalli, 2015) and P. nigrum (da Luz et al., 2015).Significantly, AMF increases plant growth compared to uncolonized one.Several AMF species such as Glomus fasciculatum, G. versiforme, G. clarum, Glomus sp.2, G. mosseae and G. etunicatum play an important role in the growth of medicinal plants (Gogoi & Sigh, 2011).Furthermore, AMF was able to increase the composition of essential oils and antioxidant activity in Piper nigrum (da Luz et al., 2015).
The results of this study indicated that AMF were found in the lowlands of Merauke at very low pH conditions.Previously, there was not research, which has been done on AMF in rhizosphere of wati plant (P.methysticum) and in lowland area of Merauke, which having low pH.The plant species of P. methysticum are known to contain a fairly high metabolite (Tanjung et al., 2014), but AMF can be well-associated (Chandra et al., 2014).In fact, besides AMF, various plant species are also able to associate with endophytes (Cape et al, 2014;Hasanah et al., 2017).
The present study clarify the presence of AMF in the P. methysticum rhizosphere in Merauke, Papua.Further work need to be conducted such as reinoculation of the native spores to this medicinal plant in pot to test their influence in the improvement of the plant growth.In the future time, those native AMF spores may have potency to be developed as an inoculum for application as biofertilizer in cultivation of medicinal plant.These types of AMF can be utilized in the processes of land rehabilitation (Suharno et al., 2017;Setyaningsih et al., 2018), and improvement the performance of plant growth, both agricultural crops, plantations and forestry (Pagano et al., 2016).

CONCLUSION
P. methysticum which is known as traditional medicinal herb has association with AMF.The percentage of AMF colonization in all study sites was varied with average 56.65%.The number of spores found in the rhizosphere soil of P. methysticum varies widely, ranging from 93 to 162 spores with an average of 122.2 spores per 100 g of soil samples.Based on the spore morphology, there are 10 species of AMF that belong to 4 genera.The dominant genus was Glomus (4 types; Glomus sp1., Glomus sp2., Glomus sp3., Glomus sp4), followed by Acaulospora (3 types; Acaulospora foveata, Acaulospora sp1., Acaulospora sp2.), Gi-

Figure 1 .
Figure 1.Study sites in lowland area of Merauke, Papua Province.

Table 1 .
Physical and chemical properties of P. methysticum rhizosphere soil in Merauke, Papua.

Table 2 .
Percentage root colonization of AMF in P. methysticum from Merauke, Papua.

Table 3 .
AMF spores number in the rhizosphere soil of P. methysticum from Merauke, Papua.