Isolation of three genera of microorganisms in lahar-laden soils of Sta. Rita, Pampanga, Philippines through the 16s rRNA gene sequence analysis

ABSTRACT

A lahar-laden soil is described as marginal soil due to its poor physicochemical and biological properties and is hypothesised to have low biodiversity of soil microbes that would help support plant growth. Thus, the study aimed to assess the presence of soil microorganisms in lahar-laden soils in Sta. Rita, Pampanga, with their ability as beneficial microbes for crop production. Vigna radiata was used as the trap crop and grown for a maximum of 35 days in a microcosm experiment where all variables were similar except for the location of the sampling site. Through the sequence analysis of the 16S rRNA gene, five representative isolates revealed close resemblance to the genus Bradyrhizobium and Rhizobium, while the others were classified as non-rhizobial endophytes, namely, Pseudomonas, Agrobacterium, and Leifsonia. These results indicate that the lahar-laden soils in the sampling site harbour some agronomically-important genera of microorganisms. Surprisingly, this is the first time that the species Leifsonia xyli was identified and isolated in acidic lahar-laden soil. In contrast, it is hypothesised that the presence of L.xyli in this study might be attributed to the acidic soil pH, leading to the suppression of rhizobia in root nodules.

KEYWORDS:

• Acid soil
• Bradyrhizobium
• Leifsonia xyli
• life on land
• Vigna radiata

Introduction

In the Philippines, one of the most remarkable events that changed the physical attributes of the soil, such as relief, and the accessibility of erodible materials (Westen van and Daag 2005), is the eruption of Mt. Pinatubo in June 1991, which resulted in the massive deposits of lahar in different regions of affected areas. Lahar usually refers to volcanic debris and mainly contains sand, rocks, mud, and water or simply mudflow, which originates on the steep flanks of a volcanic edifice (Procter et al. 2020). Due to poor physicochemical qualities, low yield, higher input requirements, and substantial nutrient losses, lahar deposits are deemed marginal. As a result, the environment formed by the lahar impacts the lives of those who reside in the region, particularly in production methods such as land preparation and crop planting (Garcia et al. 2017).

In this context, the natural restoration of marginal soil to increase soil productivity is a lengthy process; consequently, a rehabilitation programme would be more appropriate to influence food production significantly (Yoshinaga et al. 1994), and the utilisation of microorganisms might be the key to restoring marginal soil in a natural process. Thus, soil microbes, a novel contributor to soil fertility, agricultural output, and environmental quality, demonstrate an essential role in nature. However, their importance in agriculture appears to be critical due to current intensive farming practices, such as the unwise use of agrochemicals (synthetic fertiliser and pesticides), which assisted in achieving a specific arduous production target but hampered the agroecosystem in the long run. Thus, sustainable management is highly recommended to utilise beneficial microbes as inoculants or biofertilizers as a holistic approach that improves crop yield and soil fertility.

One of the most prominent organisms used in crop production is rhizobia, wherein symbiosis involving leguminous crops and bacteria (Rhizobiaceae) can fix almost 80% of biological Nitrogen (Prasuna 2014). The nitrogenase enzyme's exclusive ability to fix atmospheric Nitrogen and convert it into plant usable ammonia can reduce synthetic mineral nitrogen in food production. Rhizobia inoculation in specific legumes with compatible, effective, modified, and well-matched rhizobia could be the most ecological, productive, and economical to advance plant yield (Chianu et al. 2011; Collino et al. 2015; Saturno et al. 2017). Agricultural management such as the utilisation of fertilisers, pesticides, tillage, crop, and other determinants like salinity (Song et al. 2017), the origin of cultivar (Muhammad et al. 2012), the availability of Phosphorus (Zhang et al. 2011), is the significant constraints that affect the diversity of rhizobia (Yan et al. 2014). Specifically, environmental stress limits rhizobia's existence and population diversity in soil ecosystems, reducing their nitrogen fixation capacity (Sindhu et al. 2019) and eventually limiting crop productivity (Barrios et al. 2008). Thus, identifying the locally adapted rhizobia with a higher population, which shows a promising result, could be used to upsurge yield in legumes (Mason et al. 2020).

Another noble soil microbe in crop production is Pseudomonas spp which helps to increase crop biomass, nodulation, nitrogen, phosphorus uptake, chlorophyll content, and increase in shoot ratio of several crops, and enhances the capability to survive in contaminated soil (Mishra et al. 2011; Zahir et al. 2011; Egambrdieva et al. 2013). Hence, if this technology is used correctly, it will serve as a holistic method to restore degraded soil due to exhaustive agri- practices in food production or possible remedy to improve marginal soils such as lahar laden and ultimately enhance the fertility and soil biodiversity, which helps to mitigate climate change in the country. Thus, the objective of the study was to detect, after a few decades, if the lahar-laden soil may have a sufficient population of beneficial microorganisms to be utilised for crop production. Specifically, the study aims to isolate and characterise some local and beneficial bacteria with an abundant population in the lahar-laden soils of Sta. Rita.

Materials and methods

Soil collection and analysis

The data were collected in three identified sites in Sta Rita (San Juan, San Isidro, and San Jose) and were cleared before obtaining a soil bar with a thickness of 2–3 cm and a dimension of 10–20 depth. After the soil was collected, it was homogenised to produce a single composite sample weighing roughly 1 kg of soil and samples were brought to the Regional Soil laboratory in Pampanga. The soil parameters were subjected to different methods, soil pH was measured using potentiometric, electrical conductivity (E.C.) was analyzed via conductometric, and Cation Exchange Capacity (CEC) was measured thru the cation displacement method. On the other hand, soil nutrients such as Nitrogen (N) were calculated using Kjeldahl. In contrast, the ammonium acetate extraction method was used for Potassium (K), while Phosphorus (P) was measured via the Olsen method. Lastly, soil textures (sand, silt, clay) were analyzed using the Bouyoucos Hydrometer method.

Experimental setup

Mung bean was used as a host plant to trap the bacteria in the lahar-laden soil. The mung bean seed was sterilised with a 5% bleach solution for 3 min and washed with sterile distilled water four times. Afterward, the seeds were sown in a pot weighing 5 kg of lahar-laden soil. The pot was irrigated with sterile distilled water and placed in the designated experimental area, and the plant was supplied with a mineral solution which is Nitrogen (N) free for 30 days based on the standard procedure of Doran and Parkin 1994. After 30 days, destructive sampling was conducted to collect root nodules.

Isolation, purification, and morphological characterization

The morphological test followed the identification criteria based on Mason and Saeki (2019) and Arora's (2003) reports. Random nodules with a diameter of 2 mm and above were collected (preferably between 20–40 nodules). Some nodules were crushed to check the pink discolouration that will indicate the presence of leghemoglobin, and yeast extract mannitol agar (YMA) was used as culture media. Root nodules of selected samples were washed thoroughly to remove soils, and the nodules were surface-sterilised with 5% diluted bleach solution for 10 min and rinsed several times in sterile distilled water. Nodules were individually crushed into a test tube with distilled water, streaked onto a YMA, and incubated within 7 days based on procedures set by Martyniuk and Oroń 2011. After incubation, individual isolates were maintained on YMA plates, and universal plating procedures described by Reghuvaran et al. 2011 and Tak et al. 2020 were utilised to isolate nodulating bacteria. On YMA plates, well-separated, single colonies and dominant isolates were restreaked to create a pure culture of isolates. These nitrogen-fixing isolates were produced by repeated subculturing on a YMA plate and validated by Gram staining method. However, this study only stored the refined culture and did not do the inoculation test.

DNA extraction and amplification of 16S rRNA gene

A bacterial cell grown on the YMA plate was cultured on YMA with Congo Red, and repeated re-streaking was performed until a single pure colony was obtained. The genomic DNA of isolates was extracted from 28° C at four day- old bacterial strains from a YMA broth medium followed by the protocol of Artigas et al. (2020). Then, using a wire loop, the isolated bacterial colony was resuspended in 1 ml of autoclaved distilled water, and centrifuged for 1 min at 10,000–12,000 rpm, followed by the removal of the supernatant and addition of 200 µl of InstaGene matrix to the pellet then incubation at 56 ° C for 15–30 min, it was lysed by vortex at high speed (15,000 x g) for 10 s. The tube was then placed in a 100 °C heat block or boiling water bath for 8 min, and the vortex was repeated at high speed for 10 s. Afterward, it was centrifuged at 10,000–12,000 rpm for 2–3 min, and then 20 µl of the resulting supernatant was used per 50 µl PCR reaction.

PCR amplification of the full-length 16S rRNA gene was executed using MACROGEN Species identification in which 16S rRNA genes were first amplified by using 27F 5′ (AGA GTT TGA TCM TGG CTC AG 3;’ Wilson et al. 1990) and 1492R 5′ primer (TAC GGY TAC CTT GTT ACG ACT T) 3′. Then it was followed by sequencing primers 785F 5′ (GGA TTA GAT ACC CTG GTA) 3′ and 907R 5′ (CCG TCA ATT CMT TTR AGT TT) 3′. The target gene for 16S rRNA gene amplification was approximate 1500 bp PCR product. At the same time, the reaction mixture was based on the protocol of Sankhla et al. (2017). The amplified PRC product was subjected to the Gel electrophoresis with 1.5% agarose gel for 20 min running at 300 V, 200A, 2 μL of DNA loaded

Selection of representative isolates for sequence analysis

Representative isolate/s were collected for PCR amplification and sequence analysis. However, before sequencing, the amplified PCR products were cleansed using a PRC Multiscreen filter plate (Millipore Corp.) based on the manufacturer's protocol, while the amplified PCR product was sequenced at MACROGEN (Sout Korea). At the same time, the identification of bacteria was carried out molecularly using the 16S rRNA gene analysis method. The representative isolate/s were selected per location based on dominance on the morphology test. Only one representative isolate was chosen if the dominance was greater than 70%. If the dominance is less than 60%, the two most dominant isolates were selected for the PCR test.

Basic Local Alignment Search Tool (BLAST) was utilised to determine the closely related species using the BLASTn of the U.S National Centre for Biotechnology Information (NCBI) (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Further, the entire sequence analysis was submitted to the NCBI GeneBank database to obtain accession numbers, and then the building of a phylogenetic tree was followed.

Result and discussion

Soil chemical and physical properties

This study collected soil samples in three lahar-affected localities in Sta Rita, Pampanga. The pH of the soil samples ranged from very acidic to moderately acidic (4.95–5.40), indicating that some critical nutrients are limited in the soil, as seen by low N, P, K, and CEC values. The electrical conductivity (E.C.) (dS/m) of samples from San Jose (1.4 dS/m) was recognised as non-saline soil, while San Isidro and San Juan ranged between 4.5 and 4.7 dS/m and were classified as strong saline to very strong saline according to SRDI (2010). In addition, macroelements such as Nitrogen, Phosphorus, and Potassium were deficient, and these values correspond to low fertility, which may cause a restriction in microbial activities toward nodule formation. These data indicate that the Sta Rita lahar soil used in this study has low fertility, highlighting the importance of soil reclamation methods.

More so, the distribution of soil texture (%) varied between locations; samples from San Jose contained more proportion of sand (54.79%) than those from San Isidro (44.75%) and San Juan (33.01); San Jose showed a lower % of silt (24.54), San Isidro registered 36.53%, and San Juan recorded the highest rate of silt with 48.35%. This suggests that they vary in soil texture; San Isidro and San Juan had a similar textural class of loam soil, whereas San Jose revealed sandy loam soil (see Table 1). In a study by Mason et al. (2017), acidic to neutral soil conditions provided a controlling factor for the distribution of bradyrhizobia. In addition, it was revealed that soil texture could also play an essential role in the abundance and dominance of certain species and even strains of rhizobia in the soil.

Table 1. Soil physical-chemical properties of the three (3) locations in Sta Rita, Pampanga, after being laden with lahar.

LOCATION	PARAMETERS						SOIL TEXTURE (%)			TEXTURAL CLASS
Sta Rita, Pampanga	pH	N (%)	P (ppm)	K (cmol/kg)	CEC (cmol/kg)	E.C. (mS/cm)	Sand	Silt	Clay
1. San Jose	5.32	0.08	115.95	0.35	5.76	0.14	54.79	24.54	20.67	Sandy loam
2. San Isidro	5.4	0.07	23.61	0.27	4.97	0.45	44.75	36.54	18.7	Loam
3. San Juan	4.95	0.15	8.83	0.24	7.77	0.47	33.01	48.35	18.64	Loam

Morphological characterization of isolates

In Figure 1, individual colonies were characterised morphologically based on their colour, form, consistency, elevation, opacity, and margin. The growth morphology of each strain's pure colony was listed and described using the criteria reported by Mason and Saeki (2019) and Arora (2003). After streaking on YMA- BTB plate, isolates were incubated in a dark room for 2–7 days to form pure colonies. Based on their reactivity to Yeast Extract Mannitol Agar (YMA) supplemented with bromothymol blue (BTB), isolates were tentatively classified as slow (medium turned blue) due to the production of alkaline substances. In contrast, the medium that turned yellow due to the production of acid compound was classified as fast grower isolates (Somasegaran and Hoben 1994), as seen in Figure 2. Isolates that responded to medium with YMA-BTB after 48 h were classed as fast growers, whereas medium that turned blue after 96 h owing to alkaline production was categorised as slow growers. The reports of Hamza and Alebejo (2017) served as the foundation for identifying the isolates as either rapid or slow growers.

Figure 1. (A) The pink colour in nodules indicates leghemoglobin, (B) sterilisation of nodules in microtubes, (C) Growth of isolates in Yeast Extract Mannitol Agar (YMA) plate.

Figure 2. (A) Root nodules of mung bean under lahar-laden soil, (B) Rhizobia grew on YMA+ Congo red, (C) Rhizobia grew on YMA + Bromothymol Blue (BTB).

Isolates were classified into four groups based on their growth morphology in YMA. Group I, was isolated in San Jose (S.Jo), where isolates had a transparent, white, round form with a slightly convex elevation and an entire edge. When handled with a needle, the colony was liquid and generated a sticky mucus-like substance (mucoid). On the other hand, Group II isolates were obtained from San Isidro (S.I). They were transparent, cream irregular in form, smooth with a little convex elevation along the whole margin, and manipulated in a needle; the colony was liquid. Simultaneously, the Group III (S.Is) isolates from San Isidro exhibited a similar morphology, being transparent and cream in colour.

By comparison, the form was uneven, smooth, and slightly convex across the edge. Finally, Group IV isolates were recovered from San Juan (S.Ju). They were reported opaque, smooth, and cream in colour, round in shape, with a convex elevation and complete edge. It was viscid when handled with a needle (sticks to loop) as presented in Table 2.

Table 2. Characterisation of isolates based on their growth morphology on Yeast Extract Mannitol Agar (YMA) plate medium, including a number of pure isolates with an average number of nodules indicated in parenthesis, isolated from lahar-laden soil of Sta Rita, Pampanga.

REPRESENTATIVE ISOLATES		NO OF PURE ISOLATES	COLONY MORPHOLOGY	FORMATION OF ACID /ALKALINE SUBSTANCES
San Jose	S.Jo	4 (34)	Translucent, offwhite, circular, liquid, mucoid, slightly convex, elevation with entire margin	Alkaline
San Isidro	S.Is	2 (8)	Translucent, pink, irregular, liquid, smooth, slightly convex, elevation with entire margin	Acid
	S.I	1 (8)	Translucent, cream, rod-shaped, liquid, smooth, convex, elevation with entire margin	Acid
San Juan	S.Ju	3 (32)	Opaque, white cream smooth, viscid circular, convex elevation with entire margin	Alkaline
Total No. of pure Isolates and nodules		10 (82)

The isolates were then examined on a YMA plate with bromothymol blue (BTB) to detect fast and slow-growing rhizobia and their ability to produce acid and alkaline compounds. After streaking in YMA-BTB and incubating for 48 h in the darkroom, San Isidro isolates (S.I & S.Is) developed an acid substance in YMA-BTB. The medium changed to yellow colour, suggesting the presence of fast-growing isolates. The isolates from San Jose (S.Jo) and San Juan (S.Ju) generated alkaline compounds and appeared blue on a YMA plate containing BTB, suggesting they were slow-growing isolates. YEMA-BTB has been used as an indicator of rhizobia to characterise them as slow or fast-growing rhizobia based on alkali and acidic compound production Shahzad et al. (2012); Saeki et al. (2005).

Sequence analysis based on 16S rRNA genes

Morphologically distinct bacterial colonies were purified from the YMA plate, and the relative distribution of nitrogen-fixing bacteria within root nodules of mung bean were elucidated in different areas of Sta Rita which were affected by lahar through 16S rRNA gene sequencing, wherein it showed that the study isolates belong to other genera, namely, Bradyrhizobium, Pseudomonas, Rhizobium, Agrobacterium and Leifsonia as chief micro symbionts of mung bean. The alignment and sequence comparison of the 16S rRNA gene in a study with the obtained known bacterial sequences in the National Centre for Biotechnology Information (NCBI) database showed that the study isolates were closely related to known bacterial lineages. It is evident in Figure 3 that mung bean nodule isolates from lahar soil collected in San Jose (S.Jo) were confirmed as nitrogen-fixing bacteria dominated by the genus Bradyrhizobium as the predominant symbiont of mung bean.

Figure 3. The phylogenetic analysis of mung bean isolates using 16S rRNA gene sequence from San Jose under lahar sediments.

Also, the Bradyrhizobium genus has separated into several species/strains with accession numbers, such as B. elkanii (strain NBRC 14791, USDA 76), B. embrapense (SEMIA 6208), B. viridifuturi,(SEMIA 690) B. valentinum,(LmjM3) B.pachyrhizi (PAC 48) and B.jicamae, (PAC 68) which was based on a phylogenetic tree from sequence analysis of the 16S rRNA gene.

As demonstrated in Figure 3, the most common rhizobial population was grouped around Bradyrhizobium elkanii, the dominant mungbean isolates from San Jose's lahar soil (S.Jo). These findings corroborated prior observations by Favero et al. (2022) that Bradyrhizobium was a prevalent and frequent genus in mungbean nodules. Additionally, isolation of nitrogen-fixing bacteria from mungbean nodules grown in various soils revealed a majority of Bradyrhizobium strains (Risal et al. 2012; Yang et al. 2019). Furthermore, the study indicates that San Jose's lahar soil contains a greater proportion of sand (59.79%); hence, the quantity and dispersion of isolates belonging to the B. elkanii cluster are mostly correlated to a greater proportion of sand and high temperature (Mason et al. 2018).

The diversity and population of isolates belonging to the B. elkanii were influenced by soil pH and the change of some dominating bradyrhizobia species in San Jose's lahar soil. Therefore, it is recognised that soil characteristics and climate, as a factor, have a considerable effect on the diversity of legume symbionts in natural environments. In line with this, substantiated research indicates that soils with a modest to moderate acidity might have a significant impact on the diversity of B. elkanii in the tropics and subtropics (Saeki et al. 2005; Adhikari et al. 2012; Shiro et al. 2013 Yan et al. 2014; Lemaire et al. 2015; Mason et al. 2018; Pires et al. 2018.). In India, the Bradyrhizobium yuanmingense was frequently isolated from strong acid to alkaline soils under the species of Vigna and soybean. Similarly, in the Eastern Himalayan region, the diverse strain of bradyrhizobia was found in highly acidic soil with high precipitation and altitude using phylogenetic analysis (Vinuesa and Rojas-Jimenez 2008; Appunu et al. 2009b; Rathi et al. 2018). This is also consistent with Beukes et al. (2016) and Dos Santos et al. (2017), who reported that Bradyrhizobium strains discovered on native species of Chamaecrista in South Africa and Brazil, respectively, were genetically diverse. Therefore, compatibility with host plants depends on a symbiotic relationship which controlled by edaphic properties such as soil pH (Yang et al. 2001), accessible soil nutrients, and precipitation (Pires et al. 2018). However, in Brazil, Mimosa species were typically found nodulated with Paraburkholderia in low-pH soils, whereas Rhizobium with neutral-alkaline soil did not nodulate in native species of M. claussenii; thus, they concluded that soil pH affects plant compatibility, leading to particular symbionts (Pires et al. 2018)

Previous investigations by Mason et al. (2017, 2018) indicated that B. elkanii is the most dominating and widely distributed species of bradyrhizobia in Philippines soil; hence they hypothesised that in the Philippines, B. elkanii is the most valuable species for symbiosis and an efficient N-fixer. Meanwhile, recent research by Favero et al. (2022) revealed that Bradyrhizobium strains derived from the tropical soils of Brazil had the capacity to inoculate mung beans; however, the biological nitrogen fixation contribution was insufficient to fulfil the plant's nitrogen need. In addition, Delamuta et al. (2015) reported that some novel Bradyrhizobium species discovered in Brazil exhibited better symbiotic performance than commercial inoculants, whereas, Gyogluu et al. (2017) stated that some native bradyrhizobia isolates from Mozambican soil were symbiotically efficient and had the potential to be used as an inoculant.

In Figure 4, it is illustrated that isolates from San Isidro (S.Is) were dominated by one genus and clustered into Pseudomonas with different species, namely, P.monteilii (strain NBRC 103158 and CIP 104883), P.taiwanensis (DSM 21245), P. plecoglossicida (FPC951 and NBRC 103162), P.knackmussii (B13), P. mosselii (CFML 90-83) P. entomophilia (L48) while the other isolates classified in the genus Rhizobium with specific species such as R.tropici (strain, NBRC 15247 and CIAT 899), R.anhuiense, (CCBAU 23252) R.laguerreae (FB206), and R.lusitanum (P1-7) and lastly, few of the isolates were categorised in a genus of Agrobacterium rhizogenes (strain IFO 13257, ATCC 11325 and NBRC 13257).

Figure 4. Dendrogram of genetic diversity of mung bean isolates from San Isidro based on 16S rRNA gene with accession numbers.

The 16S rRNA gene sequence analysis shows the genetic diversity of isolates. Despite the high degree of specificity between legumes and Rhizobium, non-rhizobial endophytes such as Pseudomonas and Agrobacterium are frequently observed and present in root nodules of mungbean. In line with the result above, non-rhizobial strains may enter root nodules of legumes by infecting rhizobial bacteria and establishing an ecological niche for non-rhizobial bacteria to survive and grow (Muresu et al. 2008; Deng et al. 2011; Pandya et al. 2013). Additionally, Chidebe et al. (2018) and Leite et al. (2017) previously reported the presence of a diverse array of non-rhizobial endophytes (NRE) linked with cowpea root nodules.

Also, non-rhizobial bacterial strains have previously been isolated from string bean nodules in Brazil (Chidebe et al. 2018) and common bean nodules in Western Kenya (Kawaka et al. 2018); thus, as Martínez-Hidalgo and Hirsch (2017) hypothesised, non-rhizobial bacteria are highly diverse and abundant in legume crop nodules.

In this study, Pseudomonas was the most common and frequent genus in mungbean root nodules as NRE in San Isidro's lahar soil. Recent reports validated by Hakim et al. (2018, p. 2020) that Pseudomonas strains have previously been identified in mungbean produced in Pakistan and other legumes (Cardoso et al. 2018).

Even though this species may produce nodules, the mechanisms of colonisation by NRE are unknown; however, it appears that the plant plays a significant role (Wang et al. 2018; Gage 2020). Pseudomonas and Agrobacterium as NRE in mungbean nodules revealed that the plant regulates bacterial diversity, implying that genotype-dependent specialisation toward microsymbionts may occur (Leite et al. 2017; Liu et al. 2019). Nonetheless, the high frequency of Agrobacterium and Rhizobium in the sequencing study might be explained by horizontal symbiotic gene transfer, which is facilitated by the adaptability and co-evolution of legume-rhizobia compatibility (Velázquez et al. 2010; Chidebe et al. 2018). Meanwhile, analyses revealed that Rhizobium was the dominant rhizobial genus associated with the root nodules of mungbean; however, their relative distribution is site-specific.

In Figure 5, a dendrogram based on 16S rRNA sequence analysis showed isolates collected in mungbean nodules from lahar soils of San Isidro were characterised and classified under the genus of Rhizobium, forming 60% of rhizobial sequences with the leading species R. tropici.

Figure 5. The dendrogram shows the genetic diversity of isolates with accession numbers based on 16S rRNA sequence analysis.

Unlike the genus Pseudomonas, Rhizobium contributes to nitrogen fixation with mutual relation on legumes. According to studies, Rhizobium strains isolated from root nodules of mungbean using culture media could form nodules under manipulated environments (Yang et al. 2008; Zhang et al. 2008; Lu et al. 2009).

The findings of Hakim et al. (2018) in Pakistan showed that a small percentage of sequence belonging to Rhizobium (2.06%) was found in mung bean nodules, which is similar to the findings of Cuadrado et al. (2009) that Rhizobium, Bradyrhizobium, and Mesorhizobium in Colombia mainly nodulated string bean. Previously, it was demonstrated that the soil's characteristics and microbial community affected the variety of bacteria found within root nodules (Bulgarelli et al. 2012; Leite et al. 2017). Additionally, the distribution and genetic diversity of certain rhizobia in legumes were primarily controlled by soil parameters such as nutrient content, soil pH, and soil type (Mason et al. 2017; Saeki et al. 2017), as well as salinity and temperature (Berrada et al. 2012). Akatsu et al. (2014) discovered a high incidence of Ensifer and Rhizobium strains in root nodules of legume crops grown in sandy soil, such as cowpea and mung bean.

Among agro-environmental parameters, soil pH was the most frequently mentioned since it significantly affects mineral nutrient absorption, resulting in the specificity of legume rhizobial variety (Zhang et al. 2011; Mason et al. 2018; Wang et al. 2018). The genus Rhizobium, dominated by R.tropicii species, was found as a microsymbiont of mung bean in lahar soil using 16S rRNA sequence analysis. According to soil analysis, the lahar taken from San Isidro was acidic (5.4). Hence, soil acidity has been recognised as the primary determinant in rhizobia populations such as Rhizobium. As previously stated, soil pH affects the microbial community, including certain bacteria.

According to the study, abiotic stressors such as high salt and acidity concentrations influenced the diversity of certain rhizobia; for example, Cerro et al. (2017) reported that the prevalence of Rhizobium tropici in common beans was frequently observed under tropical acidic soils.

As seen in Figure 6, the genus Leifsonia was the predominant bacterium found in root nodules of mung beans extracted from San Juan (S.Ju) lahar soil. The genetic diversity of Leifsonia was determined using 16S rRNA gene sequence analysis. The dendrogram below shows that Leifsonia has been classified into many species. Simultaneously, strains of Leifsonia were retrieved from the National Centre for Biotechnology Information (NCBI) with accession numbers, Leifsonia poae accounting for 20% (strain VKM Ac-1401) of total Leifsonia, L.lichenia accounting for 10% (2Sb), L.xyli accounting for 10% (JCM 9733), L. shinshuensis accounting for 20% (DB 102), L.aquatica accounting 20% and lastly L.naganoensis accounting 20% (DB103).

Figure 6. The dendrogram shows the phylogenetic diversity of bacteria on 16S rRNA gene sequencing with accession numbers. Isolates were collected in root nodules of mung beans under the lahar-laden soil of San Juan (S.Ju).

Unlike other micro symbionts classified in several genera, such as Bradyrhizobium, Rhizobium, Pseudomonas, and Agrobacterium, which were previously highlighted in the study, it is uncommon that the occurrence of Leifsonia occupies root nodules of mung bean.

Few studies reported that Leifsonia had been implicated in causing sugarcane ratoon stunting disease (Gagliardi and Camargo 2009).

As a result, Leifsonia was taxonomically categorised as a member of phylum Actinobacteria, which has been characterised as a common resident of the root endosphere of plants and as containing a high diversity of diazotrophs. Notably, the genus Leifsonia has not been isolated commonly in legume crops such as mung bean. Liaqat and Eltem (2016) reported that Leifsonia shinshuensis was discovered in the rootstock of peach using molecular 16S rRNA sequence analysis. In contrast, Leifsonia xyli was unexpectedly discovered in Phaseolus vulgaris L. and described as an effective symbiotic strain (Cardoso et al. 2012).

Recent findings of Rodríguez-Rodríguez et al. (2021) showed that endophytic bacteria such as Paenibacillus, Pantoea, and Leifsonia isolated from the soil of Eastern Brazil show a positive result in both nodulations, rhizobial and endophytic bacterial diversity, which is probably due to more suitable edaphic properties of the area. However, due to the scarcity of research on Leifsonia residents in mungbean root nodules, particularly in the Philippines, this study cannot correlate the influence of soil parameters such as pH and texture with the diversity of the genus Leifsonia. This provides a prospect to use this isolate for other related studies.

In summary, the study provides the following observations: the dominance of Bradyrhizobium elkanii strain inhabiting mung bean nodules on lahar soil of San Jose is detected to be the primary micro symbionts which are correlated with a high proportion of sand and slightly acidic soil pH. Meanwhile, the genus Pseudomonas is the most abundant non-rhizobial bacteria observed in root nodules from lahar of San Isidro, with Agrobacterium and Rhizobium as inhabitants of mung bean nodules. Interestingly, uncommon microbes are found in lahar of San Juan; Leifosonia dominated the whole root nodules of mung bean. These observations suggest that a soil pH of 4.95 can significantly influence the diversity leading to specific bacteria. Acidity may be why rhizobia did not colonise the root nodules showing occupancy of Leifsonia sp. Notably, these non-rhizobial endophytes are also reported as pathogenic to some crops, while others state that Leifsonia has biotechnological potential to be used as an inoculant due to its high capability to fix Nitrogen and symbiosis.

Acknowledgment

The Department of Science and Technology- Science Education Institute funded the study under the Accelerated Science and Technology Human Resource Development Programme (ASTHRDP). The author would like to thank also the Plant Disease Diagnostic and Biocontrol Laboratory, Pampanga State Agricultural University, for providing the facilities for completing the research work.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability

The data supporting this study's findings are available upon reasonable request.

Additional information

Funding

This work was supported by DEPARTMENT OF SCIENCE AND TECHNOLOGY - SCIENCE EDUCATION INSTITUTE (DOST-SEI), Republic of the Philippines.

Notes on contributors

Israel M. Guanzon

Israel Guanzon is currently a faculty in Dept. of Crop Science, College of Agriculture Systems and Technology at Pampanga State Agricultural University, Pampanga, Philippines, He holds an MS in Soil Science (Soil biology) with a cognate of Crop Seed physiology and is a scholar.

Maria Luisa T. Mason

Dr. Maria Luisa T. Mason is currently holding a position as an Associate professor in the Department of Soil Science, College of Agriculture at Central Luzon State University, Nueva Ecija, Philippines. She obtained her graduate degree (MS/Ph.D) from the University of Miyazaki, Japan, under the Japanese Government (MEXT) Scholarship program in 2019. She has published several articles in international journals since 2017 and served as a reviewer in some National & International journals.

Purisima P. Juico

Dr. Purisima P. Juico is also a Associate professor in Department of Soil Science in College of Agriculture at Central Luzon State University. She took her graduate degree in Soil Science with specialization on Soil Chemistry and Mineralogy at the University of the Philippines, Los Banos in Laguna Philippines she also published several articles both national and abroad.

Fernan T. Fiegalan

Dr. Fernan T. Fiegalan- have an in depth knowledge on soil geomorphology, soil conservation and environmental soil science. He is a MS /Ph. D holder in Environmental Science at the University of the Philippines, he also published numerous journal both national and international. Dr. FIEGALAN is currently associate professor at the Department of Soil Science, College of Agriculture at Central Luzon State university, Nueva Ecija, Philippines.