Biomolecular mycorrhizal diversity in Azadirechta excelsa (Jack) Jacobs roots in habitats with different altitude

Azadirechta excelsa is a member of the Meliaceae family with high economic value. This tree is widely planted in South Sumatra, especially in Bengkulu Province. The mutualism symbiosis between several Meliaceae plants and mycorrhiza plants has been studied. However, the diversity of mycorrhiza in the roots of A. excelsa is not well known. We conducted a biomolecular analysis of the roots of this tree species to find out in more detail the types of mycorrhiza associated with the roots and investigated habitat differences in altitude (highland and lowland) that affect mycorrhizal diversity. We used the DNA sequencing method from the roots of A. excelsa and amplified it through PCR using ITS primers forward ITS5.8S and reverse ITS4. We took 14 root samples of E. excelsa; 6 trees from the highland (959 m asl) and eight trees from the lowland (63m asl). The average age of the tree is eight years. The results of the sequencing identification from NCBI using the BLAST method found ten types of mycorrhiza from 4 families associated with A. excelsa roots. They are seven species from the Mycenaceae family (Mycena sp, Filoboletus manipularis, M. amicta, M. pura, M. rosea, M. citrinomarginata, and Favolaschia manipularis), one species from the Psathyrellaceae family (Psathyrella sp), one species from the Tricholomataceae family (Tricholomataceae sp), and one species from the Agaricaceae family (Agaricales sp). All of those types of mycorrhiza belong to the Agaricales order, the Agaricomycetes class from the Basidiomycota phylum. There was no specific type of mycorrhiza that occupied the roots of the tree. Thus, the difference in altitude did not affect the type of mycorrhiza on A. excelsa roots.

Most trees develop symbiotic relationships with ectomycorrhizal fungi (EMF), affecting soil resource availability that limits growth [14).The effects of different EMF species on seedling development differ by order of magnitude [16][17][18][19], and these effects may impact how specific soil communities are managed and preserved to promote tree growth in actual forests [20][21].
Ectomycorrhizal fungi composition is a potential biomarker of the underlying forces that drive tree growth and suggests that variations in forest EMF communities are responsible for variations in tree growth [14].
Azadirachta excelsa (Jack) Jacobs) is a member of the Meliaceae family that grows naturally widespread from Sumatra to Papua New Guinea [22].This species is one of the most widely promoted trees for planting in Bengkulu Province.It is well-known in this region and has a variety of uses, 1255 (2023) 012059 IOP Publishing doi:10.1088/1755-1315/1255/1/012059 2 including insecticidal ones.The seeds can produce the chemical compound azadirachtin, which can be used as a natural pesticide that is relatively more environmentally friendly.
A. excelsa is a fast-growing species being promoted as a plantation species in community forests in Bengkulu.The community generally develops this tree through agroforestry patterns with crops and plantation crops, such as rubber or oil palm.The community's high interest in planting A. excelsa, especially outside their natural distribution, requires data and information support related to the characteristics of the species and the conditions where it grows.This information helps determine the right location for development or having biophysical conditions relatively the same as their natural distribution area.It will reduce the possibility of failure in developing this species.The low fertility of the majority of native soils, however, poses a significant obstacle to the successful establishment of this species in this region.
A. excelsa is known to have a mutualistic symbiosis with mycorrhizal fungi.The results of a study by [23] found mycorrhizal associations in A. excelsa of 81.25%.[24] also reported an association of mycorrhizal fungi in A. excelsa seedlings.However, little is known about the relationship between mycorrhizal fungi and A. excelsa.Based on the literature search results, we found no molecular studies of mycorrhizal fungi associated with A. excelsa.
Investigating ectomycorrhizal fungi in a plant community demands identification methods to accurately determine the identity and provide information certainty of ectomycorrhizal fungi as symbionts in mycorrhizal associations.Characterization of fungi ectomycorrhizal fungi morphologically is not enough to be used to determine the identity [25].The results of the fruiting body description also make it difficult to completely know the whole community of mycorrhizal fungi ecto-fungal community [26].Meanwhile, not all fruiting bodies are associated with ecto mycorrhizae, and a fungal mycorrhizal fungus may not always form fruiting bodies [27], as well as inconsistent occurrence [28].
Molecular approaches are now widely used to identify types of mycorrhizal fungi aiming to increase knowledge about the diversity and ecology of fungi [29].This approach can also complement the morphological analysis of fruiting bodies and allow the identification of genera or species of ectomycorrhizal fungi through studies performed on their DNA [30].The identity of ectomycorrhizal fungi is obtained through comparison with a DNA sequence database of transverse transcribed spacer sequences (ITS) or mitochondrial ribosomes [31].It is further explained that the molecular approach can also be applied to samples of ectomycorrhizal fungi in the form of mycelium because the colonization of some fungi is more often found in the form of mycelium rather than spores.In addition, some of the ectomycorrhizal fungi abundant in the soil rarely produce fruiting bodies, and vice versa.Conversely, species commonly forming fruiting bodies may not be found in underground soil [32].
There has not been much research using the molecular approach to the presence of ectomycorrhizal fungi in A.excelsa trees in tropical rainforest ecosystems, especially in Bengkulu.Therefore, this research used a molecular system to investigate ectomycorrhizal fungi associated with A. excelsa species at different altitudes.It is an important issue related to enhancing the growth quality of A. excelsa trees in community forests.

Sampling sites
The root samples of A. excelsa roots were collected in two places with different altitude characteristics.The first location was at an altitude of ± 959 masl in Pal Delapan Village, Bermani Ulu Raya District, Rejang Lebong Regency, with coordinates 219137 E and 9627231 N (highlands).The second one was at an altitude of ± 63 masl in Kertapati Village, Air Besi District, North Bengkulu Regency, with coordinates 179575 E and 9612028 N (lowlands).The average rainfall from January 2021 to January 2022 at the two sampling locations ranged from 253. 38

Data collection
The sample trees were selected using the census method on six healthy adult trees at the first location.The growth range of tree height was between 18 to 26 m, with a range of tree diameters between 24 to 35 cm. A. excelsa trees were around eight years old.In the second location, there were eight trees.The growth range of tree height was from 13 to 28 m, with a range of tree diameters between 17 to 35 cm.The age of A. excelsa trees ranged from 7 to 9 years.
A. excelsa's roots were extracted using their root tips by tracing the roots from the stem base as Anwar's procedure [33].The roots taken were fine fresh roots marked with bright colours with a length of 10cm.About 20 root samples were taken randomly in 4 (four) compass point directions in each tree at a soil depth of 0-30 cm.The roots were washed with water, air-dried, and placed in labeled paper bags.Then, the samples were brought to the laboratory.
The root samples were re-cleaned with tap water and dried, cut into 1 cm lengths.The sample was weighed at 0.03 g and then pulverized using a mortar, mixing it with liquid nitrogen, and then put into a microtube.The samples were used to prepare their DNA.Mycorrhizal fungal DNA was isolated using the Geneaid Fungal Kit following the manufacturer's protocol.The isolated DNA samples were then amplified through polymerase chain reaction (PCR) using ITS 5 forward primers.8S and reverse ITS4 with a reaction composition of 25µL Go Taq Green Master Mix (Promega, USA). 5 µL of DNA template extracted DNA, 2 µL each of forward and reverse primers, and H2O to reach a final volume of 50 µL.PCR started with denaturation at 95℃ for 5 minutes, followed by 30 cycles consisting of 30 seconds at 95℃, 45 seconds at 58℃, and 1 minute at 72℃, followed by final elongation at 72℃ for 10 minutes and terminated at 4℃.The DNA was visualized by electrophoresis on 1.5% agarose gel with 1X TAE buffer (Tris-Acetate-EDTA) at 100 V for 32 minutes.The DNA samples were sent to PT Genetika Science Indonesia in Jakarta for sequencing.

Data analysis
Nucleotide sequences (forward and reverse) sequencing results were edited by alignment using the Clustal W menu in the MEGA 11 software program.The resulting DNA sequences were then compared with the GenBank database at the National Center for Biotechnology Information (NCBI) through the Barcode of Life Data System (BoLD System) to see the similarity of the samples tested.The phylogeny diagram was reconstructed with MEGA 11 software using the neighbour-joining method with 1000 replications.

Results and discussions
Based on the electrophoresis results (figure 2), the CO1 gene DNA sequence length ranged from 300 to 700 bp.PCR products with bright bands were continued through sequencing to obtain the CO1 gene DNA sequence.Based on the sequencing results obtained, the length of the nucleotide sequence entirely ranged from 321 to 409 bp for forward and reverse, ranging from 316 to 398 bp.Then proceed with the editing stage using Mega 11 software.Many nucleotide sequence bands obtained in this study were faded or thin, indicating poor sequence quality.Bright or thick sequence bands indicate good sequence quality.Sequences that have good quality are then used in the data analysis stage.
M= marker; PKB= samples from the highland site (Pal Delapan Village KKB= samples from the lowland site (Kertapati Village));

Figure 2. Mycorrhizal gene bands detected by electrophoresis results
Based on the description above, it is known that the detected mycorrhizal fungi sequences generally appear thicker and brighter.However, there were many damaged samples, so the band length was short.The nucleotide sequences obtained were then compared with the nucleotides contained in the GenBank data to determine the level of similarity of the samples tested.From the 14 samples tested, only five produced PCR products with good conditions, and their sequences could be identified.Four samples were from the lowland site (Kertapati Village), namely KKB1, KKB5, KKB6, and KKB8.Then only 1 sample from the highland site (Pal Delapan Village) was detected, namely PKB1.The sequences of mycorrhizal fungi species detected are presented in table 1.All mycorrhizal fungi detected are part of the Basidiomycota phylum, a fungus with diploid stages in their life cycle that includes all species that produce spores in a box-shaped body called a basidium [34].Basidiomycota fungi are one of the fungi that can be seen with the naked eye because of their large body size.Generally, these fungi live as saprophytes on the remains of living things, but some live symbiotically with plant roots to form mycorrhizal fungi, which help the growth of fungi and their host plants [35].Based on the results of the sequences, there were four families of fungi: Mycenaceae, Psathyrellaceae, Tricholomataceae, and Agaricaceae.All these families are in the class Agaricomycetes and belong to the order Agaricales.Marasmiaaceae, Psathyrellaceae, Agaricaceae, Mycenaceae, and Crepidotaceae are classified as dominant Agaricales in pine and mixed forests [36].Agaricales is one of the orders with the highest level of diversity found in Indonesia, consisting of 6 families, such as Agaricaceae, Hygrophoraceae, Marasmiaaceae, Mycenaceae, Pleurotaceae, and Psathyrellaceae [37].[38] mentioned that Mycena sp is a mycorrhizal fungus associated with the roots of wild Dendrobium officinale.[36] stated that the Mycenaceae family is associated with Engelhardia serrata and Schima wallichii trees.S. walichii is also associated with the Psathyrellaceae family.Based on the detected species, 1 type of mycorrhizal fungi belongs to this family, namely Psathyrella sp.The Tricholomataceae family detected only one species, namely Tricholomata sp.[39] mentioned that 35 sequences of mycorrhizal fungi species from BLAST are associated with Vaccinium oldhami (Japanese blueberry), including Tricholomata sp.Meanwhile, the Agaricaceae family has one detected species, namely Agarica sp.[37] mentioned in his research that the Agaricaceae family is associated with several types of dipterocarps.
[40] stated that the types of ectomycorrhizal fungi in the tropics still face obstacles in the availability of data in Genbank.According to [41], Genbank data on ectomycorrhizal fungi in the forest ecosystems in tropical Asia and especially Southeast Asia is still limited.In addition, some identification results are still based on matches with the type of ectomycorrhizal fungi associated with plants from the Pinaceae [42].
There was no significant difference between the types of ectomycorrhizal fungi present in the roots of A. excelsa in the highlands and lowlands.A. excelsa trees growing in these two locations have the same type of ectomycorrhiza from the Mycenaceae family, although the types of species are different.It is probably because the ecological conditions in both locations are almost the same.Soil acidity in both locations is around 4, with low to medium nutrient content.It indicates that A. excelsa can grow in a wide range of altitudes, and the types of ectomycorrhizae associated with it are similar.[43] stated that the branch relationship on the phylogeny diagram illustrates the level at which different sequences are interconnected.Sequences with a high similarity level will be located on one branch.Meanwhile, sequences with a low level of similarity will be found on different branches.Based on the results of the phylogeny tree reconstruction shown in figure 3, the bootstrap value of species that have been detected ranges from 19-100%.If the bootstrap value is above 95%, it is categorized as stable, and unstable if it is below 70%.The long genetic distance between the detected mycorrhizal fungi species and the host plant samples indicates a gap that means species differences.Mycorrhizal fungal sequences on adjacent branches indicate that the sequences have closely clustered, including Tricholomata sp. and Agarica sp., which have a bootstrap value of 98%.The mycorrhizal sequences of Psathyrella sp. and Mycena sp. are also on adjacent branches, with a bootstrap value of 98%.Filoboletus manipularis and Favolaschia manipularis were adjacent mycorrhizal fungi sequences, with a bootstrap value 92.The phylogeny tree reconstruction results show that each mycorrhizal fungus sequence detected is closely clustered.It is in line with the results of the BOLD system identification, which shows that all mycorrhizal fungal sequences come from 1 order Agaricales, phylum Basidiomycota.
In light of the constraints identified in this research, it is recommended that further biomolecular studies be conducted to explore the correlation between mycorrhizal fungi and A. excelsa, taking into account diverse environmental variables that impact the growth of both entities.A. excelsa exhibits variation in mycorrhizal associations across different geographical locations and ecological conditions.

Conclusion
Among the 14 root samples tested, there were seven ectomycorrhizal species from the Mycenaceae family, including Mycena sp, Filoboletus manipularis, Mycena amicta, Mycena pura, Mycena rosea, Mycena citrinomarginata, Favolaschia manipularis, one species from the Psathyrellaceae family, namely Psathyrella sp, one species from the Tricholomataceae family namely Tricholomataceae sp., and one species from the Agaricaceae family, namely Agaricales sp.All ten species belong to the order Agaricales, class Agaricomycetes of the Basidiomycota phylum.
The site where A. excelsa grows in the highlands and lowlands, only affected by the differences in the species of ectomycorrhizal fungi associated with its roots, but they came from the same family.

Figure 1 .
Figure 1.The sampling site

Figure 3 .
Figure 3. Phylogenetic tree reconstruction of mycorrhizal sequences detected in NCBI GenBank

Table 1 .
Types of ectomycorrhizal fungi found associated with A. excelsa trees