Characterization of the arbuscular mycorrhizal fungal community associated with rosewood (Pterocarpus tinctorius Welw.) in Miombo forests, Democratic Republic of Congo

doi:10.21203/rs.3.rs-2583352/v1

Understanding the dynamics of arbuscular mycorrhizal fungal (AMF) in response to land use change is crucial for the restoration of degraded forests. Here, we describe the diversity and structure of AMF communities in roots of Pterocarpus tinctorius sampled from aluminium- and iron-rich agricultural and forest fallow soils. AMF in root samples were identified by sequencing the large subunit region of the rRNA gene. A total of 30 operational taxonomic units (OTUs) were recovered, most of which were new to science. These OTUs belonged to five genera namely Rhizophagus, Dominikia, Glomus, Sclerocystis and Scutellospora. AMF species richness was significantly influenced by soil properties and tree density, with a low mean value (3.2) observed in acidic soils with high Al and Fe. Indicator species analyses revealed nine AMF OTUs as indicators of base saturation (4 OTUs), high aluminium (3 OTUs) and iron (2 OTUs). OTUs positively correlated with acidity (1 OTU), iron and available phosphorus (2 OTUs) were assigned to the genus Rhizophagus, and indicated Al and Fe tolerance. The results show that leguminous trees in tropical dry forests are a potential reservoir of unknown AM fungal species. The baseline data obtained in this study opens new avenues for future studies, including the use of indigenous AMF-based biostimulants to implement ecological revegetation programs and improve land use.

Miombo forest

land use

Pterocarpus tinctorius Welw.

arbuscular mycorrhizal fungal community

soil properties

Leguminous trees are important species in tropical dry forests and play a key role in agroforestry (Lebrazi and Fikri-Benbrahim 2022) and soil remediation (Bento et al. 2012) because of their dual symbiotic associations with nitrogen-fixing bacteria and arbuscular mycorrhizal fungi (AMF, Glomeromycota). Legume trees are mainly distributed between the tropics (Castro et al. 2017). In Africa, they are found in a band of tropical seasonal forest known as the Miombo forest which stretches from Angola, the southern Democratic Republic of the Congo (DR Congo), Malawi, parts of Zambia and Zimbabwe, to Mozambique and Tanzania (Timberlake et al. 2010).

In these countries, approximately 100 million people depend upon Miombo forests, mainly for agriculture and energy production (Syampungani et al. 2016; Kiruki et al. 2017). This anthropogenic pressure leads to degradation-deforestation and overexploitation of timber, particularly in the case of the rosewood Pterocarpus tinctorius (Fabaceae) (Mukenza et al. 2022). Pterocarpus tinctorius is widely distributed in Miombo woodlands and is one of the rosewoods that is illegally harvested for its multipurpose usage (Cerutti et al. 2018). This slow-growing evergreen is a source of fuelwood, poles, timber and is used in traditional medicine (Mgumia 2017).

The overexploitation of P. tinctorius threatens the tropical dry forest ecosystem and reforestation efforts need to be undertaken. The production of seedlings with the appropriate root symbionts is required to improve the field survival and sustainable growth of the seedlings. However, the AM fungal community associated with P. tinctorius has not been studied yet. AMF colonize more than 72% of plant species (Brundrett and Tedersoo 2018), facilitating access to water and nutrients, and protecting their hosts from pathogens (Gianinazzi et al. 2010). In return, AMF receive sugars and fatty acids from their host (Luginbuehl et al. 2017; Rich et al. 2021). In degraded soils, AMF increase plant biodiversity and the seedling survival and growth under water or nutrient stress (Asmelash et al. 2016). They alleviate environmental stress and improve soil properties that facilitate plant establishment (Heijden et al. 2008). The structure of AM fungal communities is influenced by the type and intensity of land use (e.g. (Sene et al. 2012b, a; Belay et al. 2013; Moora et al. 2014), edaphic properties or soil types (Oehl et al. 2010; Straker et al. 2010), plant species composition and vegetation type (Sene et al. 2012b; Martínez-García et al. 2015; Rodríguez‐Echeverría et al. 2017). These anthropogenic and natural factors act as environmental filters on the AM fungal community.

In the Zambezian ecoregion, AMF and ectomycorrhizal fungi dominate in degraded and Miombo woodlands (Hogberg and Piearce 1986). However the AM fungal diversity is higher in dry mixed forests and Miombo woodlands compared to shrub and grass savannas in Mozambique (Rodríguez-Echeverría et al. 2017). In Kenya, AMF spores are abundant in natural forests and artificial plantations compared to cropping systems (Moora et al. 2014).

A better knowledge of the AMF community associated with P. tinctorius would consolidate its domestication for restoration of degraded lands with low inherent AMF abundance and diversity (Asmelash et al. 2016). Therefore, we investigated the community structure of AMF associated with the tropical tree P. tinctorius growing in agricultural and forest fallows in relation to the host plant composition and the soil physicochemical properties. Roots were collected from three sites located in the Miombo forest, Haut-Katanga (DR Congo). Specifically, we expected different AMF communities between agricultural and forest fallows under the influence of (i) tree density and (ii) soil physicochemical variables (e.g., acidity, Al, Fe and phosphorus). The AMF community was characterized by nested PCR, cloning and Sanger sequencing of the large subunit (LSU) region of the ribosomal RNA (rRNA) gene.

Study site

The study was carried out at three sites located in the Haut-Katanga province, south-eastern DRC (Table 1, Fig. 1a and 1b). At each site, one plot categorized as agricultural fallow (AF) and one plot categorized as forest fallow (FF) were selected. The soil is dominated by haplic ferralsols with a low distribution of plinthic and rhodic ferralsols (FAO 2006; Ngongo et al. 2009). The climate of the Haut-Katanga is defined as a humid subtropical climate and corresponds to the Cwa climate (C = mild temperate w = dry winter a = hot Summer) according to the Köppen-Geiger classification (Peel et al. 2007). There is an unimodal rainfall pattern consisting of a single rainy season (from November to March), a dry season (from May to September) and transitional months (October and April) (Malaisse 1997).

Table 1. Geographical location, land-use of the sampling sites, number of root samples collected and physico-chemical characteristics of each site.

	Kasenga		Lubumbashi		Mulungwishi
	Kipeta	Musangala	Kipopo	Mikembo	Village	Center
Altitude (m)	978	984	1286	1212	1239	1206
Longitude (E)	28°32'39"	28°35'36"	27° 24' 0.2"	27°40'10"	26°33'15"	26°37'46"
Latitude (S)	10°26'34"	10°27'34"	11° 34' 22"	11° 28' 47"	10°44'31"	10°46'33"
Land-use	FF	FA	FA	FF	FA	FF
Sample no.	5	6	5	5	6	6
pH	5.43	5.27	4.79	4.67	5.50	4.89
P_tot (mg/kg)	110.14	39.81	76.48	77.94	113.44	280.96
Pbray2 (ppm)	11.10	12.90	12.37	4.49	6.05	10.11
Al (mg/kg)	2086.48	1689.05	5304.16	2961.60	3388.99	4388.48
Fe (mg/Kg)	1716.75	262.02	1272.41	1619.82	1883.50	6626.65
Textural class	Sandy clay loam	Sandy loam	Clay loam	Sandy clay loam	Silt	Sandy clay loam

Sample no.: number of samples; E: East; S: South; FA: agricultural fallow; FF: forest fallow; P_tot: total phosphorus; Pbray2: Bray 2 extractable phosphorus; Al: total aluminium; Fe: total iron.

Sampling procedure and floristic inventory

Rhizosphere soil and root samples were collected from P. tinctorius trees in July and August 2016. Five to six trees, spaced approximately 100 m apart, were sampled along a transect line. Five grams of fine roots were sampled by selecting two secondary roots on opposite sides and excavating the root from the base of the tree until the fine root branching zone was found. Roots were washed in tap water and kept fresh in glycerol-alcohol solution (50%, v/v). At the same site, 200-300 g of soil was collected for physico-chemical analyses.

A floristic inventory was carried out in a 10 m radius around each sampled tree was performed to identify the woody species associated with P. tinctorius. The number of stems was counted and recorded for each species. Species were identified in the field and unidentified specimens were collected and taken to the botanical laboratory of the Faculty of Agricultural Sciences, University of Lubumbashi, DRC, for further examination.

Soil physico-chemical analyses

The soil was dried at room temperature for one week and sieved through a 2 mm mesh. The subsequent physico-chemical analyses were carried out in the soil laboratory of the Faculty of Forestry, Geography and Geomatics of the Université Laval (Québec, Canada). Standard protocols were used for the physico-chemical analyses. Soil texture was determined from the percentage of clay (< 0.002 mm), fine silt (0.002 to 0.02 mm), coarse silt (0.02 to 0.05 mm) and sand (0.05 to 2 mm) fractions by the improved hydrometric method (Bouyoucos 1962). The pH was measured electrometrically in a suspension of 10 g of soil and 20 mL of distilled water.

Soil chemical properties, including pH, extractable cations (calcium, magnesium, potassium and sodium), cation exchange capacity (CEC) and base saturation were determined using standard procedures (Bray and Kurtz 1945). Total aluminium, iron and phosphorus were dissolved according to the manufacturer's standard protocol (CEM Corporation 2016). All elements were then measured by inductively coupled plasma spectrometry (ICP Agilent 5110 SVDV).

Available phosphorus (Pbray2) was extracted using the two-reagent system (0.03 N NH₄F: 0.1 N HCl) of Bray and Kurtz (1945) and analyzed by flow injection analysis (FIA) using Quikchem method 12-115-01-1-A (Zellweger Analytics, Lachat Instruments Division, Milwaukee, WI, USA). Analyses of total nitrogen and organic carbon were performed according to the Kjeldahl and Walkley-Black methods, respectively. Total nitrogen, carbon and sulphur were determined by high-temperature combustion and infrared detection in an elemental analyzer (TruMac CNS, LECO Instruments ULC, Mississauga, ON, Canada). The amount of soil organic matter was measured directly as the weight loss during combustion.

DNA extraction, polymerase chain reaction (PCR) amplification, cloning and sequencing of the large subunit (LSU) rRNA gene

To determine the composition of the AM fungal community in roots, total genomic DNA was isolated from 50-60 mg of root fragments (1 mm in length), ground and homogenized for 6 min using a SpeedMill PLUS (Analytik Jena AG, Jena, Germany). DNA was extracted using the QIAGEN DNeasy Plant Mini Kit protocol (QIAGEN, Mississauga, ON). The quality and quantity of the DNA was measured using a Nanodrop 1000 spectrophotometer (Thermo Scientific Inc., Wilmington, DE, USA.

The LSU region of the rRNA gene was amplified by targeting the D1 and D2 domains using a nested PCR method (Procter et al. 2014; Crossay et al. 2018). The first-round amplification was performed using the primer set LR1 (5'- GCA TAT CAA TAA GCG GAG GA - 3') / NDL22 (5'- TGG TCC GTG TTT CAA GAC G -3') (van Tuinen et al. 1998). The second-round amplification was performed with the AMF specific primer set LR1 / FLR4 (5'- TAC GTC AAC ATC CTT AAC GAA -3') was used for the second PCR (Gollotte et al. 2003).

The first-round amplification was performed in a final volume of 25 µL containing: 16.3 µL of water; 2.5 µL of 10´ buffer ; 2.5 µL of T4 gene 32 protein (25 mg/mL); 0.5 µL of deoxy-nucleoside triphosphates (dNTPs, 0.2 mM); 0.5 µL of each primer (25 mM); 0.2 µL of Taq polymerase (1 unit / reaction) and 2 µL of genomic DNA. The thermo-cycling conditions were as follows: initial denaturation at 95 °C for 4 min, followed by 35 cycles of 95 °C for 1 min, 56 °C for 1 min, 68 °C for 1 min, and final extension at 68 °C for 10 min.

The first-round PCR products were diluted 1:50 prior to the second-round amplification. The second-round amplification was performed in a final volume of 23 µL, containing 3 µL of DNA; 15.3 µL of water; 2.5 µL of 10´ buffer; 0.5 µL of BSA (bovine serum albumin, 25 mg/mL); 0.5 µL of deoxy-nucleoside triphosphates (dNTPs; 0.2 mM); 0.5 µL of each primer (25 mM); 0.2 µL of Taq platinum (1 unit / reaction). The thermo-cycling conditions were as follows: initial denaturation at 95 °C for 4 min, followed by 30 cycles of 94 °C (45 s), 60 °C (45 s), 72 °C (45 s) and a final extension at 72 °C for 5 min. Amplicons were visualized by electrophoresis on 1% agarose gel in TAE buffer after staining with ethidium bromide. PCRs were performed on an MJ Research PTC-225 Tetrad Peltier thermal cycler (USA, Canada, Mexico).

Second-round PCR products were purified using the QIAquick PCR Purification Kit (Qiagen Ltd. Crawley, UK), and then cloned into the pGEM-T or pGEM-T Easy vector according to the Promega protocol. A minimum of 16 clones per sample were amplified and visualized as previously described for the second-round amplification. Amplicons were sequenced using the Sanger method on the Genomic Analysis Platform at the Institute of Integrative and Systems Biology (IBIS, Université Laval, Québec, Canada). The LSU sequences were deposited in the NCBI GenBank database under accession numbers (available at the time of publication).

Bioinformatic and Phylogenetic analyses

The LSU sequences were edited and cleaned using the BioEdit Sequence Alignment Editor v7.2.6.1 (Hall 1999). Sequences were grouped into operational taxonomic units (OTUs), using the 95% similarity threshold in Geneious v9.0.5 (2015, Biomatters, Auckland, New Zealand). A phylogenetic analysis was performed to determine the relationships of the OTUs to the known species of AMF. For this, each OTU consensus sequence was identified with the closest sequences found in the NCBI GenBank database using BLAST (Altschul et al. 1990), in MaarjAM databases (Öpik et al. 2010) and sequences from reference cultures (Krüger et al. 2012). Sequence consensus alignments were performed using the MAFFT v7 “Auto” algorithm (Katoh and Standley 2013) as implemented in Geneious v9.0.5. A Bayesian phylogenetic tree was inferred using MrBayes v3.2 as implemented in Geneious v9.0.5. A total of 20,000 phylogenetic trees were generated and the consensus tree was calculated by excluding the first 3,000 trees.

Alpha and beta diversity analyses

AMF diversity was estimated using the number of OTUs as a proxy for species richness. Arbuscular mycorrhizal community structure was estimated Wang et al. (2019)by calculating the frequency of occurrence (FO), relative abundance (RA), importance value (IV), species richness, Shannon-Wiener index, Simpson index and Pielou evenness index as in Wang et al. (2019). The diversity indices (species richness, Shannon index, Simpson index and Pielou index) were calculated using the R package vegan v2.5-3 (Oksanen et al. 2019) in the R software v3.4.4 (R Development Core Team, 2018). Tree density data were correlated with diversity indices of AMF and edaphic properties. Rarefaction curves based on the number of samples were calculated using the specaccum function as implemented in the R package vegan.

The effects of land-use and soil parameters on the AM fungal community structure were assessed with linear mixed-effects models using the R package lme4 (Bates et al. 2015). Sites and land-use types were considered as fixed factors and P. tinctorius individuals as random factors. Significant differences between sites and their interaction were compared using Tukey's post hoc tests with Bonferroni adjustments using the R package multcomp (Hothorn et al. 2008).

Correlation analyses were performed to assess the relationships between soil properties, AM fungal diversity indices, and plant species using the function rcorr as implemented in the R package Hmisc (Harrell 2018). Correlations were plotted using the R package corrplot (Wei and Simko 2017). Normality was checked with the Shapiro-Wilk test while the homogeneity of variance, the independence of residuals and linearity were assessed with the Goldfeld-Quandt (gqtest), Durbin-Watson (dwtest) and Rainbow (raintest) tests, respectively. These tests were performed using the R package lmtest (Zeileis and Hothorn 2002).

The effects of tree density and soil properties on the presence-absence and abundance of indicator OTUs were assessed using canonical correspondence analysis (CCA) and canonical redundancy analysis (RDA), respectively. Significant AM indicator fungal species were identified using the MRT and IndVal functions as implemented in the R packages MVPARTwrap (Ouellette 2011) and labdsv (Roberts 2019).

The effect of land-use and soils on the community structure of AMF was assessed using permutational multivariate analysis of variance (PERMANOVA) with the function adonis2 as implemented in the R package vegan. The metric used was Bray-Curtis dissimilarity and 999 permutations were applied to the data. The variation in AMF composition was visualized with non-metric multidimensional scaling (NMDS), using the Bray-Curtis dissimilarity distance metric. NMDS was calculated using the function metaMDS as implemented in the R package vegan. Tree density was projected as an explanatory variable and the influence of the density of the corresponding tree species was tested with the function envifit as implemented in the R package vegan.

The overlap of the AMF communities recorded in sites categorized as agricultural fallow (FA) and forest fallow (FF) was visualized with Venn diagrams using the R package VennDiagram v1.6.20 (Chen 2018), while the bipartite networks were visualized with the R package bipartite v2.15 (Dormann et al. 2008).

Molecular diversity of AMF

Amplification, cloning and sequencing of 33 P. tinctorius field-root samples yielded 355/412 (87%) AMF sequences. These sequences were grouped into 30 OTUs, belonging to five genera, two families (Gigasporaceae and Glomeraceae) and two orders (Diversisporales and Glomerales, Fig. 2). Fourteen OTUs were assigned to the genus Rhizophagus, five to the genus Glomus-1, five to the genus Dominikia, five to the genus Sclerocystis and one to the genus Scutellospora. Only four OTUs had pairwise similarities greater than 95% with sequences from well identified herbarium cultures. Indeed, OTUs 4, 14, 15 and 19 had pairwise similarities of 95.5%, 99.2%, 97.5% and 96.4% with Rhizophagus arabicus (OTU 4), Scutellospora erythropa (OTU 14), Rhizophagus clarus (OTU 15) and Rhizophagus dalpeae (OTU 19), respectively (Table 2). The three most abundant OTUs are likely to represent new species. OTU 1, whose closest relative was Rhizophagus neocaledonicus (93.5% similarity threshold), was the most abundant (AR = 25%) and dominant (FO = 69.7% and significance value of 47.6%). OTU 2 (uncultured Sclerocystis, 95.8% similarity) and OTU 3 (uncultured Glomeromycotina, 94.7% similarity) had a relative abundance of 9.3 % and 8.45% respectively, and none of them had a pairwise similarity closely related to a known species (Table 2).

Table 2. Operational Taxonomic Units (OTUs) of AMF associated with the roots of P. tinctorius, ranked according to their total sequence abundance.

	OTU abundance										Closest GenBank BLAST
OTU	Total	KFA	KFF	LFA	LFF	MFA	MFF	FO (%)	AR (%)	IV (%)	AMF species	QC (%)	Identity (%)	Access no.
1	91	18	5	24	22	13	9	69.70	25.63	47.67	Rhizophagus neocaledonicus	98	93.61	KY362436
2	33	7	1	5	2	6	12	42.42	9.30	25.86	Uncultured Sclerocystis	99	95.81	MH504073
3	30	3	1	11	7	3	5	45.45	8.45	26.95	Uncultured Glomeromycotina	99	94.67	KT826604
4	29	1	10	1	3	8	6	39.39	8.17	23.78	Rhizophagus arabicus	98	95.45	KF154767
5	22	14	8	0	0	0	0	18.18	6.20	12.19	Uncultured Sclerocystis	100	92.88	MH504023
6	21	1	3	5	4	7	1	33.33	5.92	19.62	Rhizophagus neocaledonicus	99	93.35	KY362436
7	21	0	5	5	4	6	1	30.30	5.92	18.11	Rhizophagus neocaledonicus	97	90.35	KY362438
8	17	1	0	1	2	6	7	30.30	4.79	17.55	Uncultured Sclerocystis	97	94.34	MH504023
9	11	4	5	0	0	2	0	21.21	3.10	12.16	Uncultured Glomus	99	92.62	HE858410
10	9	0	0	0	2	1	6	18.18	2.54	10.36	Rhizophagus arabicus	99	92.68	KF154767
11	7	1	0	0	6	0	0	12.12	1.97	7.05	Glomus sp.	98	93.22	KY114652
12	7	0	0	2	0	5	0	6.06	1.97	4.02	Glomus OTU 40	99	94.18	HG996315
13	7	0	0	1	2	2	2	12.12	1.97	7.05	Uncultured Glomeromycotina	97	94.64	KJ484691
14	7	1	1	0	5	0	0	9.09	1.97	5.53	Scutellospora erythropa	95	99.20	JF816963
15	6	0	3	0	2	0	1	12.12	1.69	6.91	Rhizophagus clarus	100	97.51	FM865543
16	6	0	0	0	1	3	2	12.12	1.69	6.91	Rhizophagus arabicus	100	92.17	KF154766
17	6	1	1	0	0	4	0	12.12	1.69	6.91	Uncultured Glomus	98	95.01	FR873155
18	6	0	0	0	0	6	0	3.03	1.69	2.36	Uncultured Glomus	100	99.56	KJ701465
19	3	0	2	0	0	0	1	9.09	0.85	4.97	Rhizophagus dalpeae	96	96.40	MN130951
20	3	0	1	0	2	0	0	6.06	0.85	3.45	Glomus sp. 2 SL-2017	96	91.67	KP756522
21	3	2	1	0	0	0	0	6.06	0.85	3.45	Dominika litorea isolate Dl-8	98	90.98	MG710519
22	2	0	0	1	1	0	0	6.06	0.56	3.31	Rhizophagus neocaledonicus	96	90.64	KY362437
23	1	1	0	0	0	0	0	3.03	0.28	1.66	Uncultured Glomeromycotina	99	89.60	HQ128664
24	1	1	0	0	0	0	0	3.03	0.28	1.66	Uncultured Rhizophagus	97	93.89	AB935524
25	1	0	0	0	0	1	0	3.03	0.28	1.66	Glomus sp.	99	95.27	MK875626
26	1	0	0	0	0	1	0	3.03	0.28	1.66	Uncultured Glomus	100	98.97	AB561104
27	1	0	0	0	0	0	1	3.03	0.28	1.66	Rhizophagus neocaledonicus	100	87.80	KY362438
28	1	0	0	0	0	1	0	3.03	0.28	1.66	Uncultured Glomus	99	97.50	HQ243163
29	1	0	0	0	0	0	1	3.03	0.28	1.66	Dominikia disticha	98	91.65	KJ564145
30	1	0	0	0	0	1	0	3.03	0.28	1.66	Uncultured Glomus	99	94.81	HQ242918

K: Kasenga, L: Lubumbashi, M: Mulungwishi.

FA: Agricultural fallow, FF: forest fallow.

FO: frequency of occurrence, AR: relative abundance, IV: importance value.

Relationship between AM fungal diversity and environmental factors

The rarefaction curves showed that most of the AMF diversity associated with P. tinctorius roots was captured (Fig. 3, Fig. S1). On average, AMF species richness increased by only one OTU per newly analyzed root sample above the 8^th to 10^th root sample analyzed.

Species richness was positively correlated with the Pielou index for all woody species (r = 0.92, p < 0.001). A negative correlation was found between the species richness (r = -0.39, p = 0.02) and the Shannon index (r = -0.42, p = 0.02) of the woody species colonized with AMF (Fig. S2; Tables S2, S3). No significant correlations were observed between the physico-chemical variables and the diversity indices of AMF (Fig. S2; Tables S2 and S3).

AMF species richness was the lowest (3 to 5 OTUs, p < 0.05) in the roots of P. tinctorius growing in the AF plots from Lubumbashi and the highest (2 to 8 OTUs) in the roots of P. tinctorius growing in the AF plots from Mulungwishi (Fig. 4A). The linear mixed-effect model shows that only the interaction sites ´ fallows had a significant effect on species richness (F_2,26 = 4.09, p = 0.03). The values of the Shannon and Simpson indices recorded for the root samples from the fallows of three sites were not significantly different at p = 0.05 (Fig. 4B and 4C).

Based on the IndVal analysis, nine OTUs were identified as indicators of pH, base saturation, aluminium, iron and coarse silt (Fig. 5A). These indicator OTUs were closely related to the species R. neocaledonicus (OTUs 1 and 3), R. arabicus (OTU 4), R. irregularis MUCL43205 (OTU 9 and OTU 10), S. sinuosa MD126 (OTU 17) and to two unknown species within the genus Rhizophagus (OTU 6 and OTU 7) and Sclerocystis sp. (OTU 8) (Table S4). Canonical redundancy analysis (RDA) shows that OTUs 9 and 17 (identified as Glomus sp.) were significantly related to pH (p = 0.03); OTU 3 (unknown Glomeromycota) was significantly related to acidity (p = 0.04) (Fig. S3, F model = 1.7; p = 0.01; F first axis = 6.25; p = 0.02).

Canonical correspondence analysis (CCA) showed that the presence of four indicator OTUs (rhizo 1, 3, 4 and 10) was positively correlated with the tree density of P. tinctorius, Albizia adianthifolia, Baphia bequaertii, Combretum collinum, Strychnos inocua and Syzygium guineense (Fig. 5B). In contrast, the indicator OTUs rhizo 6, 7 and sclero 17 were only positively correlated with the tree density of Bobgunnia madagascariensis, Combretum acutifolium, Diplorhynchus condylocarpon, Erythrophleum africanum, Lannea discolor and Pseudolachnostylis maprouneifolia. In addition, the tree density of Anisophyllea boehmii was positively correlated with the presence of rhizo 9 and sclero 8.

AMF community structure

PERMANOVA shows a significant effect of sites on the taxonomic composition of AMF (Pseudo-F = 1.69, R² = 0.101, p = 0.01). Rhizophagus was the most abundant genus in the AMF community (56.6-83.9%), followed by Sclerocystis (6.2-41.1%) and Glomus (1.8-15.4%). The genera Rhizophagus, Glomus and Sclerocystis were found in all six plots of the three sites whereas Dominikia and Scutellospora were absent from the Lubumbashi and Mulungwishi sites, respectively (Fig. 6A). NMDS showed that their abundance was significantly correlated with the tree density of Albizia antunesiana (p < 0.014), Anisophyllea boehmii (p < 0.04) and Dalbergia boehmii (p < 0.04) (Fig. 6B). Tree density was the most important factor determining the composition of the AMF community.

About 50% of the OTUs (16 out of 30) were shared between sites categorized as agricultural fallow (FA) and forest fallow (FF) (Fig. S4). However, the bipartite network showed that five OTUs were present in the six plots (OTUs 1, 3, 4 and 6; genus Rhizophagus) and OTU 2 (Sclerocystis sp.), representing 57.5% of the relative abundance. Nine OTUs were detected in only one plot of these three sites, i.e., FA Kasenga (OTUs 23 and 24), FA Mulungwishi (OTUs 18, 25, 26, 28 and 30) and FF Mulungwishi (OTUs 27 and 29) (Fig. 6C).

Analysis of the AMF diversity colonizing the roots of the tropical tree P. tinctorius growing in agricultural and forest fallow showed a relatively high species richness (30 OTUs). Despite the low throughput of cloning PCR products, the rarefaction curves were close to saturation and the AM fungal richness recorded in this study is in the range of other studies targeting AMF in root samples using methods based on high-throughput sequencing. For example, 61 amplicon single variants from the Illumina sequencing of the small subunit (SSU) region of the rRNA gene were recorded in Tamarix aphylla root samples (Stefani et al. 2020a). Holste et al. (2016) recorded a total of 22 OTUs in the roots of four tree species growing in southern Costa Rica with the pyrosequencing of the SSU region of the rRNA gene. However, the number of AM fungal OTUs reported from root samples can be sometimes an order of magnitude higher. Indeed, a total of 215 OTUs were recorded in the roots of Colocasia esculenta and Pterocarpus officinalis in Guadeloupe by pyrosequencing the SSU region of the rRNA gene (Geoffroy et al. 2017). While the AMF recorded in the roots represent the taxa in direct interaction with their host, root analysis provides only a partial view of the structure of the AM fungal community present below ground. Studies investigating AM fungal diversity in root, rhizosphere and bulk soil samples have shown that each compartment is characterized by a specific set of species (e.g. Stefani et al. (2020a, b)).

About 50% of the OTUs recorded in the root samples of P. tinctorius were assigned to the genus Rhizophagus (Glomeraceae) while only one OTU was assigned to the genus Scutellospora (Gigasporaceae). The dominance of taxa assigned to the genus Rhizophagus is often observed in roots, and in this sense the genus is aptly named. Hart and Reader (2002) showed that [sic] Glomaceae isolates had high root colonization but low soil colonization, and they colonized roots before Acaulosporaceae and Gigasporaceae isolates. In contrast to our study, AMF spores belonging to the families Acaulosporaceae and Gigasporaceae dominate the AM fungal community in the Kenyan ferralsol (Mathimaran et al. 2007). AMF richness is highly dependent on the biological material analyzed (roots, spores, and extraradical mycelium) (Varela-Cervero et al. 2015) or the methodology used (spore identification, PCR-cloning, high-throughput sequencing). Therefore, to unravel the AM fungal diversity inhabiting the tropical dry forests it is necessary to analyze each belowground compartment with high-throughput sequencing methods.

The majority of the AM fungal species recorded in the roots of P. tinctorius could not be identified at the species level, suggesting the presence of many undescribed species in the Miombo region. These OTUs representing potential new species were assigned to the genera Rhizophagus, Dominikia, Glomus and Sclerocystis. Only OTUs 15 and 19 (96% similarity to Rhizophagus clarus) and OTU14 (97% similarity to Scutellospora savannicola) could be related to known species. Consequently, these results show that leguminous trees in tropical dry forests are a potential reservoir of unknown AM fungal species that need to be fully characterized in order to better assess their role in the restoration of degraded lands.

This is the first time that species of the genera Dominikia and Sclerocystis have been reported from the Miombo region. In other African regions, the genera Dominikia and Sclerocystis have been observed in alluvial plains (sand-rich) and degraded sites in secondary succession, respectively. Taxa related to the genus Sclerocystis have been observed in the roots of carob (Ceratonia siliqua) from degraded sites in Morocco (Manaut et al. 2015) and in the rhizosphere of karee tree (Searsia lancea) from mining sites in South Africa (Spruyt et al. 2014) and in three acacia species (Vachellia abyssinica, V. seyal and V. sieberiana) from a grazing pasture in Ethiopia (Belay et al. 2013). Sequences assigned to the genus Dominikia, although low in abundance, clustered into five OTUs, making the genus the second most species-rich along with the Glomus-1 clade. Dominikia disticha has been reported from marine sands in South Africa (Błaszkowski et al. 2015), which is consistent with our observations where OTUs of this genus were present in root samples from the Lufira (43-56.4% sand, FA Mulungwishi) and Luapula (54–77% sand, FA Kasenga) plains (Table S1).

Results from the CCA and NMDS biplots showed that the community structure of AMF was significantly influenced by the density of P. tinctorius trees and other coexisting host-plant species. This affected the abundance of rare genera (Dominikia and Scutellospora). For example, the AM fungal genus Dominikia and the large shrub flatbean (Dalbergia boehmii) were abundant in agriculture fallow (FA) in the Mulungwishi site (less acidic soil and high percentage of base saturation) compared to FA in the Lubumbashi site (acidic soil with high aluminium content). Plants control the AMF community during secondary succession of plant communities, and tree density determines the AMF community and diversity as has been previously reported by Martínez-García et al. (2015) and Sene et al. (2012a, 2012b, 2013). A significant positive correlation was also found between AMF species richness and the Pielou’s index of woody species. This suggests that an evenly plant community distributed promotes AMF diversity. As mycorrhizal dependence (Gaidashova et al. 2012) and AMF diversity (Dalpé et al. 2000) vary between plant species, AMF species richness is expected to be higher in sites that are co-dominated by more than two mycotrophic species (Bâ et al. 1996; Lekberg et al. 2013; Mensah et al. 2015; Rodríguez-Echeverría et al. 2017).

The community structure of AMF was significantly influenced by soil properties. Only 5 / 30 OTUs were observed in the six plots of the three sites. The presence of different AM fungal communities between sites could be due to different soil properties (see IndVal analysis). Canonical redundancy analysis showed a significant positive relationship between soil properties and the abundance of some OTUs, suggesting a functional role for acidity and aluminium (OTU 3), iron and phosphorus (OTUs 4 and 10). The preference of these OTUs for these soil conditions was confirmed by the indicator species analysis. Within Rhizophagus, OTUs 1 and 3 were indicators of acidic pH and low base saturation, while OTUs 4 and 10 were indicators of high iron concentration. OTUs 6 and 7 were also identified as indicators of high aluminium concentration. These taxa may also tolerate anthropogenic disturbance, as has been reported in other degraded or contaminated sites for Rhizophagus (Bâ et al. 1996; Mathimaran et al. 2005) and Sclerocystis (Spruyt et al. 2014; Manaut et al. 2015). However, at the genus level Rhizophagus and Sclerocystis can be considered as generalists (Oehl et al. 2010), as they were present in all plots. OTU 3 is close to R. neocaledonicus, which was first described in ultramafic soils of New Caledonia (Crossay et al. 2018). Ultramafic soils, which are rich in Ni and Fe, evolve towards ferralsols under tropical conditions (Echevarria 2017). Other similar AMF sequences have been detected in ferralsols from Kenya (Mathimaran et al. 2007), a country in the same bioclimatic zone as our sampling sites. For Fe-complexed phosphorus (strong correlation between Fe_tot and P_tot), OTU 4 (close to R. arabicus) is significantly related to Fe_tot-Pbray₂. This taxon could be involved in phosphorus uptake, as it is present in all fallows of three sites, but abundant in Mulungwishi AF and FF, and Kasenga FF, where Fe_tot and P_tot levels are high.

In conclusion, the structure of the AM fungal community did not differ between FA and FF, but it was significantly influenced by the density of P. tinctorius trees and other cohabiting woody species. Some OTUs related to the genera Rhizophagus and Sclerocystis were identified as AM indicators, probably due to their tolerance to aluminium and iron. AMF abundance was significantly correlated with certain soil properties (acidity, iron, pH and available phosphorus) and the host tree densities. These relationships would indicate co-variation of AMF diversity with host plants, apparently facilitated by soil chemical properties. Furthermore, our results suggest a functional implication for AMF diversity in terms of Al and Fe immobilization, and P uptake. The roots of tropical leguminous trees such as P. tinctorius are colonized by unknown AM fungal taxa and trap cultures are required to isolate and characterize these new taxa. These taxa could be used as biostimulants to improve the restoration of degraded soils.

Acknowledgements

This study was funded by a doctoral fellowship from the Réseau des Institutions de Formation Forestière et Environnementale d'Afrique Centrale (RIFFEAC), through its project to support the Expanded Program of Training in Natural Resource Management in the Congo Basin (PEFOGRN-BC) funded by the Congo basin forest funded, managed by the African development bank. The authors would like to thank the forest engineers Eric Lowele and Christian Kanunda for their assistance during the floristic inventory, sample collection and preparation. We also thank Stéphane Daigle, research professional in the Centre d'Étude de la Forêt (CEF) for his help with the statistical analyses. We also thank Marie-Ève Beaulieu for her help with the molecular and bioinformatics analyses. NSERC Discovery Grant (DPK) is also acknowledged. W.F.J. Parsons corrected the English.

Author contributions

JMKK and DKP conceived and designed the experiments. JMKK performed all the experiments and analyses. JMKK and GS wrote the first draft of the manuscript. FS co-supervised JMKK and rewrote the subsequent version of the manuscripts with GS. DKP was responsible for supervision and project management, as well as funding and resource acquisition. All authors provided critical input to the drafts and gave final approval for publication.

Competing interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding

This study was funded by a doctoral fellowship from the Réseau des Institutions de Formation Forestière et Environnementale d'Afrique Centrale (RIFFEAC), through its project to support the Expanded Program of Training in Natural Resource Management in the Congo Basin (PEFOGRN-BC) funded by the Congo basin forest funded, managed by the African development bank. This work also received a NSERC Discovery Grant (DPK).

Availability of data and materials

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

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No competing interests reported.

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Characterization of the arbuscular mycorrhizal fungal community associated with rosewood (Pterocarpus tinctorius Welw.) in Miombo forests, Democratic Republic of Congo

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