Metagenomic analysis of soil forest microbiota on Mount Ungaran, Central Java, Indonesia

The ecological functions of forest soils are largely determined by the activities of soil microorganisms. This study aims to analyze the diversity and abundance of microbes in forest soil on Mount Ungaran, Ngesrepbalong Village, Kendal Regency, Central Java, Indonesia. This study used an exploratory observation method. Five hundred grams of forest soil was collected aseptically for chemical and metagenomic analysis. Metagenomic analysis was carried out based on 16S rRNA gene markers V3-V4 region. The result showing that the forest soil was dominated by bacteria of the order Rhizobiales (13.17%), Thermincolales (9.29%), and Vicinamibacterales (6.95%), as well as archaea of the order Nitrososphaerales (62.89%), Nitrosotaleales (4.61%), and Nitrosopumilales (1.67%). At the genera level, forest soil was dominated by Thermincola (8.42%) and Bacillus (7.39%), and one genera of archaea, namely Nitrososphaeraceae (23.17%). The bacteria that dominate forest soil are a group of Plant Growth Promoting Bacterial Agents (PGPB) in the nitrification process. Meanwhile, archaea that dominate forest soil act as ammonia oxidizing agents (AOA).


Introduction
Indonesia is one of the countries with a diversity of terrestrial ecosystems.Forest soils are vertically and horizontally structured ecosystems, consisting of many microhabitats with different physical, chemical and biological properties [1].The heterogeneity of soil ecosystems depends on the type of soil compartment [2], soil texture, plant diversity, season, and site characteristics [3].Forest soils are one of the most diverse types of ecosystems.Every gram of soil is estimated to contain 1,000 to 1,000,000 species of bacteria [4].
The ecological function of forest soil is largely determined by the activity of soil microorganisms.Microorganisms play a role in biological, biogeochemical, and soil ecological processes [5].Forest soils contain microbial communities that play a role in nutrient cycling, organic matter decomposition, and soil health [6].Acidobacteria and Actinobacteria are commonly found in forest soils and contribute to nutrient cycling, organic matter decomposition, and soil stability [7].Nitrogen-fixing bacteria, such as members of the Rhizobiaceae and Frankiaceae families, play an important role in providing nitrogen in forests [8].
Forests have an important role in carbon sequestration [9], maintaining soil quality and reducing erosion.Forests are also used as a basic ecosystem in many studies addressing changes in soil quality and ecology [10][11][12].This study aims to analyze the diversity and abundance of IOP Publishing doi:10.1088/1755-1315/1315/1/012018 2 microbes in forest soil on Mount Ungaran, Ngesrepbalong Village, Kendal Regency, Central Java, Indonesia.Metagenomics is carried out because approximately 99% of microorganisms cannot be cultured with standard culture techniques [13].This research is important to obtain markers of microbiota stability in each forest soil ecosystem.

Methods
This study used an exploratory observational design.The sample plots in this study were production forest land with an area of 180x10 m 2 (>0.18Ha), located in Gunung Sari, Ngesrepbalong Village, Limbangan District, Kendal Regency, Central Java (S o 7°09'32.3"E 110°19'48.1").The trees found within Mount Ungaran's forested area comprised of fruit-bearing trees (such as Citrus aurantiifolia, Persea americana, and Musa sp.), condiment commercial purposes plants (like Syzygium aromaticum and Coffea canephota), carbohydrate source tuber (Colocasia esculenta) and providing shade trees (such as Hibiscus tiliaceus).Soil samples were taken during the dry season.
Soil samples were taken in 3 plots of 10x10m, and the distance between plots was 50 m.Each plot was taken 5 points.At each point, 500 grams of soil samples were taken at a depth of 10 cm from the soil surface.The soil samples were then thoroughly combined (polled) until homogeneous.One thousand two hundred grams of soil sample was used for chemical analysis (C-organic, N-total, K-available, P2O5, Ca-dd, Mg-dd, Na-dd, Fe and S).Fifty grams of soil was used for metagenomic analysis.

DNA isolation and Next-Generation Sequencing (NGS) analysis
ZymoBIOMICS™ DNA Mini Kit was used for DNA extraction from forest soil samples, following the manufacturer's protocol.Microbial soil forest diversity and abundance were determined utilizing the 16S rRNA gene marker region V3-V4.The amplification process used Illumina HiSeq 2500 platform for 20 cycles using primer-forward 338F (5′-GGACTACHV GGGTWTCTAAT-3′) and primer-reverse 806R (5′-GGACTACHVGGGTWTCTAAT-3′) [14], which bound to the barcode, i.e., a sequence of eight bases specific to each sample.Furthermore, metagenomic analysis was used to generate sequencing amplification.

Sequencing data analysis
Metagenomic analysis of 16S rRNA microbial sequencing data was processed utilizing quantitative insights into microbial ecology software package (QIIME2) Ver.2019.4 [15].First, the paired-end file was demultiplexed using the demux plugin.Then quality control was performed on every sample using the Dada2 plugin [16].Furthermore, four diversity indexes were used in this study, that is Shannon [17], Simpson [18], Chao1 [19] and Observed OTUS [20].The taxonomy and species were identified based on Greengenes 13_8 99% OTU database.First, data visualization was performed in the heatmap form that was compiled using the heatmap plugin, then, barplot taxa were collected in Microsoft Excel 2010.

Results and Discussion
The forest in the Gunung Ungaran area is in a tropical forest ecosystem.The vegetation in the Ungaran Mountain forest soil samples were Citrus aurantiifolia (lime), Persea americana (avocado), Syzygium aromaticum (clove), Colocasia esculenta (taro), Coffea canephota (coffee), Hibiscus tiliaceus (sea hibiscus or coast cottonwood), Musa sp.(banana), and Poaceae.Each plant species has a characteristic root exudate, including complex organic compounds that affect the composition of bacteria around plant roots [21].The diversity of vegetation will create various microhabitats with different physical and chemical conditions [22].The density of the soil texture of the ungaran mountain forest (Table 1) also affects the diversity of bacteria.Bacteria in dense soil are dominated by bacteria that are able to adapt to dense soil [23].
The findings show that the eubacteria domain is more dominant than the Archaea domain.At the order level, 299 orders were identified (290 orders bacteria and 9 orders archaea).At the genera level, 657 genera of bacteria and 16 genera of archaea were identified.Although the number is relatively small, Archaea also has an important role in the cycle of nutrients in soil ecosystems.Some Archaea can be involved in nitrogen, methane, and sulfur cycles, and could survive in extreme environmental conditions [24].The results of the 16rRNA gene sequence analysis showed that there were 66,124 OTUs and 3476 observed species (Table 2).The Shannon and Simpson index values (Table 2) indicate a high diversity of forest soil microbes.This is related to the abundance of macro-micro chemical elements in forest soil (Table 1).According to Mercado-Blanco et al. [25], organic sources that are abundant in forest soil contain leaf litter, rotting roots, and other organic matter which provide a source of nutrition for soil microorganisms.The forest soil microbiota is dominated by various types of bacteria which are generally present in the forest soil microbiome and function to maintain the balance of the ecosystem [26].This is in accordance with the measurement data of pH, moisture and temperature of the forest soil which are not extreme (Table 1).The three orders of bacteria that dominate forest soil are Rhizobiales (13.17%),Thermincolales (9.29%), Vicinamibacterales (6.95%).Other orders found were Burkholderiales (6.29%), Clostridiales (4.81%), Gaiellales (2.60%), Paenibacillales (2.18%), Corynebacteriales (2.10%), Rokubacteriales (1, 86%), Nitrospirales (1.83%).The order Rhizobiales plays an important role in nitrogen fixation [27], increasing plant growth [28], decomposition of organic matter and recycling of nutrients in forest ecosystems [29].This causes Rhizobiales to increase the productivity of forest soil.The total N-content (2.52%; Table 1) in forest soil also supports the high abundance of Rhizobiales.Clostridiales could survive a variety of extreme environmental conditions, such as drought, high temperatures, chemical contamination, and radiation [30].The acidic conditions of the forest soil (pH 5.2; Table 1), may be the cause of the abundance of the order Gaiellales.According to Kaiser et al. [31], the order Gaiellales grows optimally at pH 6.5-7.5 and its abundance increases at pH 3-7.
The three orders of archaea that dominate forest soil are Nitrososphaerales (62.89%),Nitrosotaleales (4.61%), and Nitrosopumilales (1.67%).Other orders found were 4 Methanomassiliicoccales (0.65%), Bathyarchaeia (0.24%), Methanosarciniales (0.17%), Methanomicrobiales (0.03%), Methanocellales (0 .02%) (Figure 1).The order Nitrososphaerales with an abundance of 62.89% (Figure 1), according to Sheridan et al. [32] plays a role in soil biogeochemical processes, especially in the nitrification cycle.Archaea in forest soils that play a role in the oxidation of ammonia are referred to as ammonia-oxidizing archaea (AOA) [33].The order Nitrososphaerales, including AOA, has the enzyme ammonia monooxygenase (AMO), which allows them to oxidize ammonia under aerobic conditions [34].Likewise, the order Nitrosotaleales is included in the AOA group [35].The presence of archaea of the AOA group affects soil fertility, due to the oxidation of ammonia in forest soil [36].The two genera of bacteria that dominate forest soil are Thermincola (8.42%) and Bacillus (7.39%).The percentage of other bacterial genera is ≤1.67%.Only one archaea genus dominated forest soil, namely Nitrososphaeraceae (23.17%), while other genera were at ≤0.97% (Figure 2).The abundance of the genus Thermincola is probably due to its ability to survive in dense textured soils (Table 1).According to Hartmann et al. [37], the Thermincola genus can adapt to low oxygen conditions so that it can survive in dense soil.Members of the Bacillus genus is useful in helping plants directly or indirectly through the acquisition of nutrients, production of phytohormones, protection from pathogens and other abiotic stresses [38].Bacillus is a plant growth promoting bacteria (PGPB) because it can produce phytohormones such as indole acetic acid (IAA).Bacillus is also involved in nitrogen fixation [39][40].Nitrospira are bacteria that oxidize nitrite to nitrate, so the presence of Nitrospira in forest soil can help increase the availability of nitrogen for plants [41][42].The high N-total (Table 1) in the forest soil in this study is probably due to the role of the genus Nitrospira.
The genera Nitrososphaeraceae is a key taxon in maintaining the stability of forest soil ecosystems in driving the structure and function of the microbial community [43].Dense soil texture is the habitat of this genera.This is in accordance with the research of Wehking et al. [44] that the relative abundance of the genera Nitrososphaeraceae is higher in dense soil particles.Nitrosphaeraceae also belongs to the AOA group.The ability of the genera Nitrososphaeraceae to adapt to acidic conditions (pH 5.2; Table 1) in this study shows that this genus is resistant to stress [45].

Figure 1 .
Figure 1.Relative abundance (%) of 10 orders of bacteria and archaea from forest soil in the Mount Ungaran, Central Java, Indonesia

Figure 2 .
Figure 2. Relative abundance (%) of 10 genera of bacteria and archaea from forest soil in the Mount Ungaran, Central Java, Indonesia

Table 1 .
The physics and chemical data of forest soil in the Mount Ungaran, Central Java,

Table 2 .
Diversity and abundance of microorganism in the Mount Ungaran, Central Java,