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agriculture
Article
Arbuscular Mycorrhization in Colombian and Introduced
Rubber (Hevea brasiliensis) Genotypes Cultivated on Degraded
Soils of the Amazon Region
Clara P. Peña-Venegas *, Armando Sterling and Tatiana K. Andrade-Ramírez
Citation: Peña-Venegas, C.P.;
Sterling, A.; Andrade-Ramírez, T.K.
Arbuscular Mycorrhization in
Colombian and Introduced Rubber
(Hevea brasiliensis) Genotypes
Cultivated on Degraded Soils of the
Amazon Region. Agriculture 2021,11,
361. https://doi.org/10.3390/
agriculture11040361
Academic Editors:
Isabelle Trinsoutrot-Gattin
and Babacar Thioye
Received: 3 March 2021
Accepted: 26 March 2021
Published: 16 April 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
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iations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Sinchi Amazonic Institute of Scientific Research, Leticia 910001, Colombia; asterling@sinchi.org.co (A.S.);
tatiannita17@gmail.com (T.K.A.-R.)
*Correspondence: cpena@sinchi.org.co; Tel.: +57-3108149907
Abstract:
Rubber (Hevea brasiliensis, (Willd. Ex Adr. de Juss) Muell. Arg, Euphorbiaceae) is an
important commercial latex-producing plant. Commercially, rubber is reproduced from a limited
number of grifting genotypes. New promising genotypes have been selected to replace traditional
genotypes. In addition, rubber has been promoted to recuperate Amazon soils degraded by extensive
cattle ranching. Arbuscular mycorrhizal (AM) symbiosis is an important alternative for improving
plant nutrition in rubber trees and recuperating degraded soils, but AM fungal communities on
different plantations and in rubber genotypes are unknown. Spore abundance, root colonization and
AM fungal community composition were evaluated in rubber roots of Colombian and introduced
genotypes cultivated in degraded soils with different plantation types. Traditional (spore isolation
and description; clearing and staining roots) and molecular techniques (Illumina sequencing) were
used to assess AM fungi. Rubber roots hosted a diverse AM fungal community of 135 virtual taxa
(VT) in 13 genera. The genus Glomus represented 66% of the total AM fungal community. Rubber
genotype did not affect the arbuscular mycorrhization, hosting similar AM fungal communities.
The composition of the AM fungal community on old and young rubber plantations was different.
Diversity in AM fungi in rubber roots is an important characteristic for restoring degraded soils.
Keywords: Amazon; grifting plants; soil restoration
1. Introduction
Rubber (Hevea brasiliensis, (Willd. Ex Adr. de Juss) Muell. Arg, Euphorbiaceae) is a
native species from the Amazon region and an important commercial plant species. It is
cultivated in more than 40 countries for latex production [
1
]. Global consumption of natural
rubber is increasing. Between 2000 and 2018, rubber consumption increased 96.23% [2].
Global rubber production is based on the cultivation of a few rubber clones that are
reproduced from grifting stumps. Although it secures homogeneity in latex production,
the lack of genetic diversity increases susceptibility to phytosanitary problems and reduces
rubber latex yielding. Producers are creating new plantations in areas that were covered
by natural tropical forests [
3
], avoiding common diseases such as the South American leaf
blight (SALB), caused by Pseudocercospora ulei (Henn.),with negative consequences for
natural ecosystems. Others are developing new rubber genotypes with more resistance
to diseases and good latex production [
4
] for cultivation in traditional areas. The genetic
improvement of commercial rubber is not enough to guarantee good latex production
because cultivated plants require good nutrition. Commercial plantations require regular
fertilization during the 25-year production cycle, which increases latex production cost.
Although Asiatic countries are the main latex rubber producers, rubber is still an
important crop in Amazonian countries. In the Amazon region of Colombia, 4534 Ha
are cultivated with rubber, producing 183 tons of latex per year [
5
]. In Colombia, rub-
ber production is based mainly on three introduced genotypes—IAN 873, IAN 710, and
Agriculture 2021,11, 361. https://doi.org/10.3390/agriculture11040361 https://www.mdpi.com/journal/agriculture
Agriculture 2021,11, 361 2 of 17
FX 3864 [6]
. These rubber genotypes have been planted in areas with a high incidence of
SALB, resulting in a high presence of the disease and low local latex production [4].
In the Colombian Amazon, rubber has been promoted as a key crop recuperating
degraded areas after years of extensive cattle ranching. Therefore, around 70 Ha of rubber
trees were planted on degraded pastures with the purpose of transforming these unpro-
ductive areas into sustainable productive ones. Naturally, most of the Amazon basin has
low-fertility soils [
7
], in which phosphorus (P) is usually the limiting soil nutrient. De-
graded Amazon soils have very low P levels that are sometimes undetectable by traditional
P analyses (e.g., less than 0.09 mg/Kg soil by Bray II). Although rubber is a native species
from the Amazon region, it can experience P deficiencies, which manifest as lower leaf
production in young rubber trees, brown color on leaf edges, less growth, and lower latex
production in mature rubber trees [
8
]. Low-cost alternatives that improve soil conditions
for rubber plantations are becoming an important issue for producers.
Arbuscular mycorrhization seems to be a feasible low-cost alternative that improves
latex production locally because it occurs naturally in rubber [
9
,
10
]. Arbuscular mycorrhiza
is a symbiosis between plant roots and Glomeromycota obligate endosymbiont fungi [
11
],
occurring in about 80% of all plants. Arbuscular mycorrhiza is identified as the main plant–
root association in crop species that mobilizes nutrients to plants, especially P, improving
nutrition even in low fertile soils [
12
]. This association also reduces stress in host plants
produced by water or nutrient limitations or the presence of contaminants or pathogens [
13
].
Although it seems to be the best alternative for a more sustainable and chipper rubber tree
agriculture, there are still some unsolved issues that limit our knowledge on how grifting
plants react to arbuscular mycorrhizal associations.
Grifting plants as commercial rubber are still physiologically poorly understood.
Some authors consider them as individual plants that interact, while others consider them
as hybrid plants with unique and individual autonomy [
14
]. The latter is based on the
observed root-top relationship of grafting plants, in which upward water and mineral
nutrient supply, downward flow of photosynthates, and root-top interchange of hormonal
signals change [
15
]. Symbiotic associations such as arbuscular mycorrhiza might offer
insights into how gifting plants behave. If grifting plants act as different individuals
downward and upward, arbuscular mycorrhizal (AM) associations will not be affected by
upward genotypic variations. However, if grifting plants act as hybrids, upward genotypic
variations will affect root affinity by AM fungi and therefore AM associations among
different clones.
Ecologically, plants with a high affinity for arbuscular mycorrhizal (AM) fungi ben-
efit themselves and the plant community around them [
16
]. Therefore, AM symbiosis
might play an important role in recovering degraded soils [
17
]. Different results support
or deny the importance of rubber as an AM host plant in the restoration of environ-
ments. Based on soil spore-borne AM fungi, Feldmann et al. [
18
] found higher AM fungal
diversity in natural rubber stands than in commercial monoclonal plantations. These
differences were attributed to crop management practices on rubber plantations. How-
ever,
Herrmann et al. [9]
found highly diverse communities of AM fungi on commercial
plantations of rubber cultivated in Thailand despite the high levels of applied fertilizers.
Since arbuscular mycorrhiza occurs at the root level, it is not well understood whether
genomic variation or environmental and abiotic factors affect more AM symbiosis in rubber.
Rubber production in the Amazon region offers a good environment for addressing these
issues. Since 2009, the Sinchi Institute has selected promising introduced and Colombian
rubber genotypes that are less susceptible to common pathogens and have good potential
for latex production [
19
]. Sterling and Rodriguez [
19
] compared plant growth, resistance to
phytopathogens, and potential latex production between promising rubber genotypes and
the introduced genotype IAN 873 (control) indicating that new genotypes performed better
than IAN 873. For a better understanding of arbuscular mycorrhizal association in rubber,
this study (i) established differences in the AM fungal community associated with rubber
roots cultivated on different plantation types, (ii) determined the influence of soil variables
Agriculture 2021,11, 361 3 of 17
on the AM fungal community associated with these rubber plantations, and (iii) evaluated
the root colonization and AM fungal community composition of Colombian and introduced
rubber genotypes cultivated in degraded soils of the Colombian Amazon region.
2. Materials and Methods
2.1. Sites and Samples
Samples were collected in five municipalities of the Caquetástate, Colombian Amazon
region: Albania, Belen de los Andaquíes, Florencia, El Paujil, and San Vicente del Caguán.
The study area was located at 1
◦
17
0
15” N to 02
◦
02
0
41” N, and 74
◦
54
0
39” W to 75
◦
54
0
2” W
(Figure 1). The elevation in this area ranges between 245 and 559 m above sea level. The
average annual rainfall is around 3000 mm, with a dryer period between December and
February. The average annual temperature is up to 24
◦
C [
20
]. The area corresponded to
the upper Amazon basin and is influenced by the Andes mountain chain. Soils in the study
area are classified as Inceptisols and Oxisols (USDA soil classification). The area is covered
by introduced pastures for extensive cattle ranching with different levels of degradation,
commercial rubber plantations, and some relicts of natural secondary forests.
Figure 1. Map with the location of the study area. Scale: 1:2,000,000.
Four types of rubber plantations were visited to collect soil and root samples—three
monoclonal plantations (MPs) between 30 and 50 years old with introduced rubber geno-
types; two six-year-old agroforestry systems (ASs) in which introduced genotypes of rubber
were intercropped with copoazu fruit trees (Theobroma grandiflomum (Willd.
Ex Spreng.
)
K.Schum.); three nine-year-old clonal trials with introduced genotypes (CTIGs); and three
one-year-old clonal trials with Colombian rubber genotypes (CTCGs), plus the intro-
duced genotype IAN 873 (traditional cultivar) as a control for comparisons (Table S1,
Supplementary Materials).
Rubber plantations differed in their agronomic management. MPs were cleaned pe-
riodically of weeds and fertilized once per year with an inorganic NPK fertilizer. ASs
received weed control every three months and fertilization twice per year with organic fer-
tilizers. Additionally, two commercial insecticides were applied every 15 days to copoazu
trees when insects were causing phytosanitary problems [
21
]. CTCGs and CTIGs received
weed control every three months and fertilization with a mix of organic and inorganic
fertilizers every six months. Phytosanitary controls were not performed on the latter two
plantation types [22].
Agriculture 2021,11, 361 4 of 17
On each plantation, two types of samples were collected—soil samples and rubber
tree root samples. A composed topsoil sample of about 500 g from 5 sub-samples of 100 g
was collected to evaluate (i) the physicochemical composition of the soils where rubber
tree was growing, (ii) the relative abundance of soil-borne AM fungal spores, and (iii) the
AM fungal community composition at the family level. Soil samples were dried at room
temperature to reduce the percentage of humidity and transported to the laboratory in
plastic bags.
Root samples were used to estimate (i) the percentage of AM root colonization and
(ii) the
AM fungal community composition of rubber roots. Between 5 to 15 fine root
samples of individual rubber trees were collected in each site from the bulky roots present
in the first 20 cm of soil depth. The length of fine roots collected from each plant ranged
between 3 and 10 cm approximately. A total of 255 root samples were collected. Root
samples were transported in paper bags to the laboratory where root samples were divided
in two: one-half of each sample was fixed in a Formalin-alcohol solution (Formaldehyde-
ethanol-water 1:1:2) to estimate the percentage of AM root colonization. From the second
half, a subset of 160 samples was stored at
−
70
◦
C and used to estimate the AM fungal
community composition with molecular approaches. The subset included root samples of
at least one replicate of each plantation type, with root samples of all rubber tree genotypes.
The number of soil and root samples that were collected and analyzed is summarized
in Table S1, Supplementary Materials.
2.2. Soil Physicochemical Analysis
Soil samples were processed to estimate the following soil physicochemical properties:
soil texture (Bouyoucos); pH (1:1 in water); percentage of organic carbon (with potassium
dichromate solution); cation exchange capacity (CEC), Ca, Mg, K, and Na expressed in
mg/kg of dry soil (with 1N ammonium acetate at pH = 7); and available phosphorus (P)
expressed as mg/kg of dry soil (Bray II).
2.3. Extraction of AM Spores from Field Soils
About 25 g of each soil sample was used to extract AM fungal spores present in
each soil. Five replicates of each soil sample were processed. Spores were obtained by
wet sieving, followed by centrifugation in a saturated sucrose solution [
23
]. Isolated
soil-borne AM fungal spores were placed in Petri dishes and counted directly with a stere-
oscope (
SZ40 Olympus
, Allentown, PA, USA). Distinctive AM fungal spore morphotypes
were mounted on slides with lactoglycerol (LG) (Lactic acid: Glycerol: water 1:2:1) and
LG + Melzer’s
reagent (Micro-science, London, UK) (1:1 v/v) and observed under a com-
pound microscope (BX53 Olympus, Allentown, PA, USA) in order to classify the spores
into particular AM fungal families.
2.4. AM Fungal Colonization of Rubber Roots
Root samples were processed by clearing and staining roots with a solution of 0.05%
trypan blue in lactoglycerol [
24
]. The percentage of AM root colonization was estimated by
the gridline intersect method [
25
], obtaining the total percentage of AM root colonization
in 100 root intersections. The procedure was repeated thrice per sample.
2.5. Molecular Analysis of Rubber Roots
DNA was obtained from 0.2 g of dried roots using the PowerMax
®
Soil DNA Isola-
tion Kit (Qiagen, Germantown, MD, USA). This DNA was quantified with Nanodrop
®
(
Thermo Fisher Scientific
, Madison, WI, USA) before sequencing. The AM fungal DNA
was amplified from samples using AM fungal specific primers for the small-subunit (SSU)
ribosomal RNA gene—WANDA [
26
] and AML2 [
27
]. The PCR mixture contained 5
µ
L of
5XHOT FirePol Blend Master Mix (Solis Biodyne, Tartu, Estonia); 0.5
µ
L of each 20
µ
M
primer; 1
µ
L of template DNA; and nuclease-free water to reach a total reaction volume
of 25
µ
L. The PCR was performed following the thermocycling conditions reported by
Agriculture 2021,11, 361 5 of 17
Davison et al. [
28
]: 95
◦
C for 15 min, 30 cycles of 95
◦
C for 30 s, 55
◦
C for 30 s, and 72
◦
C
for 30 s, followed by 72
◦
C for 5 min. PCR products and the amplification success were
checked on 1% agarose gel. Both negative (distilled water) and positive (synthetic double-
stranded DNA with relevant priming sites) controls were included in the PCR to secure
the quality of the PCR products. A DNA concentration of 5 ng/
µ
L was used for the library
preparation. A library was prepared for each sample. The library preparation was carried
outusing a modified Illumina 16S rRNA protocol, standardized by Asper Biogene. Each
library was ligated with Illumina adaptors using the TruSeq DNA PCR-free library prep kit
(
Illumina Inc.
, San Diego, CA, USA). The second PCR used a reaction mix composed of
5µL
of PCR product of fist PCR, 5
µ
L of Nextera XT index 1 primer (N7xxx), 5
µ
L of Nextera XT
index 2 primer (E5xxx), and 15
µ
L of 2
×
KAPA HiFi Hotstart PCR mix (without KAPA
primer). The PCR program was run as follows: 3 min at 95
◦
C, followed by 7 cycles of 30 s
at 95
◦
C, 30 s at 55
◦
C, and 30 s at 72
◦
C, with a final extension of 5 min at
72 ◦C
and held at
4
◦
C. Libraries were sequenced on the Illumina MiSeq platform (
Illumina Inc.,
San Diego,
CA, USA) using a 2
×
300 bp paired-read sequencing approach at Asper Biogene laboratory
(Tartu, Estonia). A total of 22,810,852 raw sequences and 17,632,788 clean sequences were
obtained, which corresponded to 77.3% of the total sequences. The raw sequences are
available under request.
2.6. Bioinformatics
Demultiplexed paired-end reads were analyzed according to the bioinformatics steps
described by Vasar et al. [
29
]. Primer sequences were matched allowing one mismatch
in forward or reverse chains. Removal of barcode and primer sequences was conducted
using the PEAR v. 0.9.8. program. Singletons were removed, and amplicons between
170 and 540 pb
in length were included in the analysis. Amplicons longer than 540 bp
were cut at that length and conserved for further analysis. Chimeric sequences were de-
tected and removed using UCHIME v7.0.1090 [
30
] in the reference database mode using
the default parameters and MaarjAM database (https://maarjam.botany.ut.ee, accessed
on
8 January 2021
, status June 2019, 384 virtual taxa (VT)). Sequence alignment was per-
formed using the MAFFT v. 7 multiple sequence alignment web service in JALVIEW
version 2.8 [31]
, subjected to a neighbor-joining phylogenetic analysis in TOPALi v2.5 [
32
]
using the default parameters with the MaarjAM and the International Nucleotide Sequence
Database Collaboration (INSDC). Retained reads were subjected to a BLAST+ search
v 2.8.1 [33]
using 97% identity and 95% alignment length thresholds. Representative se-
quences of Glomeromycota virtual taxa (VT) were deposited in the NCBI GenBank under
the accession numbers (MW900266 to MW900422).
2.7. Statistical Analysis
Differences in the abundance of soil-borne spores and in the percentage of AM root
colonization among the plantation types were assessed using linear mixed-effects mod-
els (LMEs) (plantation identity as a random effect) (function lme from the R package
v. 3.5.1. nlme
). When significant differences were obtained, a Tukey honest significant
difference (HSD) test was performed with a p< 0.05 value to discriminate significant
differences. Additionally, comparisons in the percentage of root colonization and richness
between the Colombian and introduced rubber tree genotypes within the same plantation
type were assessed using a Kruskal-Wallis test from the R program v. 3.5.1.
Sampling efficacy was assessed with species accumulation curves (method “exact”)
using the functions accumcomp and ggplot from the R packages BiodiversityR and ggplot2,
respectively [
34
,
35
]. The AM fungal richness between the plantation types and between
the genotype types was visualized with Venn diagrams using the function ggvenn from R
package ggven [
36
]. The AM fungal richness (S) and diversity indices (Shannon-Wiener,
InvSimpson, and Pielou’s evenness) were estimated using the functions Specnumber and
diversity from the R package vegan [
37
]. The Shannon index (H’) was exponentially
transformed to obtain a variable (expH’) that satisfied the replication principle, following
Agriculture 2021,11, 361 6 of 17
Herrmann et al. [
9
] recommendations. An LME (function lme from the R package nlme) [
38
]
was adjusted to analyze the effect of the plantation type or genotype type on ecological
indices. The effect of the plot (plantation identity or genotype identity) was included as a
random effect in each model for the nested study design. The differences in mean variables
among the plantation types or between the genotype types were analyzed with the Fisher’s
least significant difference LSD post hoc test (α= 0.05).
A nested permitational multivariate analysis of variance PERMANOVA using the
function nested.npmanova from the R package BiodiversityR [
34
] was performed to compare
the AM fungal community composition among the plantations and between the genotypes.
A non-metric multidimensional scaling ((NMDS), 50 iterations) was used (Bray-Curtis
distance) and plotted with two axes and standard deviations ellipses (
α
= 0.05) to visualize
changes in the AM fungal community composition among the plantation types. The
functions metaMDS and ordiellipse (R package vegan), and ggplot and theme (R package
ggplot2) were used [
34
,
37
]. The indicator species value analysis was used to study the
strength of the relationship between the AM fungal VT and the rubber plantations or the
AM fungal VT and the rubber genotypes (function indval from the R package labdsv [
39
]).
An indicator value species of at least 0.25 was considered as a good indicator taxon [9].
Soil physicochemical variables were analyzed with LME and the means pairs were
compared with Fisher’s LSD post hoc test (
α
= 0.05). Spearman’s correlation was used
to relate the AM fungal diversity with the soil variables. The AM fungal community
composition data were Hellinger transformed and included with all soil variables in a
redundancy analysis (RDA) in order to assess the effect of soil variables on the AM fungal
community composition (function rda from the R package vegan) [
37
]. Sequential ANOVA
tests (1000 iterations) were performed for the contribution of each soil variable. The
importance of individual AM fungal VT was determined using the parameter r
≥
0.5
(
α= 0.05
) to better explain the variation among the plantation types. RDA was plotted with
two axes in a triplot graphic using functions ordiellipse and envfit (R package vegan) [
37
],
and ggplot and theme (R package ggplot2) [35].
The analyzes were performed in R language, v. 4.0.3 [
40
] using the interface in
InfoStat v. 2020 [
41
] for the LME, Fisher’s LSD, and Spearman’s tests; the interface in Qeco
v. 2018 [42]
for the formal ecological analyses; and the interface in RStudio v. 1.3.1093 [
43
]
for the graphical analyses.
3. Results
3.1. Soil Conditions of Rubber Plantations
Soil physicochemical analysis indicated that the soils where the rubber was cultivated
had nutritional limitations. The available P was one of the most limited soil nutrients for
plant growth, with values lower than 2 mg/kg of soil (Table 1). There was a significant
difference in the content of sodium among the plantation types. AS and MP had more
sodium than CTCG and CTIG.
Table 1.
Soil physicochemical composition of the rubber tree plantations: clonal trial with Colombian genotypes (CTCG);
clonal trial with introduced genotypes (CTIG); agroforestry system (AS); and monoclonal plantation (MP).
Plantation Type SOIL Physicochemical Variables
pH OC (%) CEC
(meq/100g) Loam (%) Clay (%) Sand (%)
CTCG 4.62 ±0.04a 1.37 ±0.24a 10.46 ±1.62a 14.14 ±0.46a 47.77 ±3.55a 38.71 ±4.03a
CTIG 4.62 ±0.05a 0.85 ±0.29a 7.05 ±1.67a 8.14 ±0.66b 43.33 ±4.38a 48.33 ±4.86a
AS 4.55 ±0.08a 1.17 ±0.29a 5.94 ±1.65a 13.98 ±0.35a 49.00 ±5.36a 40.71 ±4.99a
MP 4.55 ±0.05a 1.15 ±0.24a 7.13 ±1.54a 8.00 ±0.00b 49.33 ±4.37a 42.67 ±3.94a
K (mg/kg) Ca (mg/kg) Mg (mg/kg) Na (mg/kg) P (mg/kg)
CTCG 67.48 ±8.37a 330.45 ±30.47a 58.90 ±6.58a 32.79 ±3.26b 1.31 ±0.46a
CTIG 40.13 ±10.41a 212.29 ±34.99a 35.99 ±7.72a 29.64 ±4.03b 0.59 ±0.58a
AS 38.60 ±9.78a 255.18 ±40.88a 46.00 ±7.55a 46.03 ±4.00a 1.09 ±0.55a
MP 55.51 ±9.13a 238.00 ±27.78a 32.67 ±6.16a 49.32 ±3.96a 0.73 ±0.45a
OC: organic carbon, CEC: cation exchange capacity, K: potassium, Ca: calcium, Mg: magnesium, Na: sodium, and P: available phosphorus.
Values in columns corresponded to mean and standard error. Values followed by the same letter do not differ statistically (Fisher’s least
significant difference LSD test, p< 0.05).
Agriculture 2021,11, 361 7 of 17
3.2. Abundance of Arbuscular Mycorrhizal Spores in Soils
Significant differences in the abundance of AM fungal spores were found among the
rubber plantations (p= 0.001). Spore abundance in MP was significantly lower than in AS
and CTIG (Figure 2).
Figure 2.
Spore abundance of arbuscular mycorrhizal fungi in soils on different rubber tree planta-
tions: AS: agroforestry; MP: monoclonal plantation; CTIG: clonal trial with introduced genotypes;
and CTCG: clonal trial with Colombian genotypes. Spore abundance expressed as the number of
spores per 100 g of dry soil. Letters express significant differences.
3.3. Arbuscular Mycorrhizal Fungal Colonization of Rubber Roots
All rubber root samples were colonized by AM fungi, with percentages of colonization
between 1 and 96%. There were significant differences in the percentage of AM root colo-
nization among the different rubber plantations (p < 0.0001). MP and AS had significantly
higher percentages of AM root colonization (66
±
17% and 57
±
23%, respectively,
Figure 3
)
than the clonal trials. Root colonization on the other rubber plantations was between 36
and 47% on average.
Figure 3.
Percentage of arbuscular mycorrhizal colonization of rubber roots cultivated on dif-
ferent plantation types: clonal trial with Colombian genotypes (CTCG); clonal trial with intro-
duced genotypes (CTIG); monoclonal plantation (MP); and agroforestry system (AS). Letters express
significant differences.
Agriculture 2021,11, 361 8 of 17
There were no significant differences in the percentage of AM root colonization be-
tween the Colombian (46%) and introduced (44%) rubber genotypes (p= 0.665), even when
Colombian and IAN 873 were growing on the same plantation (p= 0.368;
Kruskal-Wallis).
3.4. Arbuscular Mycorrhizal Fungal Community in Rubber Roots
Accumulation curves of VT in rubber roots cultivated on different plantations indi-
cated that AM fungal community composition on CTCG was properly estimated but not
on the other rubber tree plantations (Figure 4). According to the Chao index, 95% of the
total AM fungal community colonizing rubber tree roots in CTCG was estimated, 85% in
CTIG, 84% in MP, and 81% in AS.
Figure 4.
Accumulation curves of the virtual taxa colonizing rubber root samples collected on
different types of plantations: clonal trial with Colombian genotypes (CTCG); clonal trial with
introduced genotypes (CTIG); agroforestry system (AS); and monoclonal plantation (MP).
Glomeraceae represented more than 77% of the total AM fungal community associated
with rubber tree roots independent of the approach used to estimate the AM fungal
community composition. However, the molecular approach increased the number of
VT of the families Archaeosporacee and Gigasporaceae, compared to the soil-born spore
approach (Figure 5).
The sequencing approach estimated a total of 419,089 Glomeromycotina sequences.
Sequences corresponded to 135 VT of 13 genera. Rubber plantations were highly diverse,
with more than seven different AM fungal genera per plantation. Glomus with 89 VT was
the most diverse and the most frequent genus, representing 66% of the total AM fungal
community in rubber roots. Three additional important genera colonizing rubber roots
were Acaulospora with 10 VT, Paraglomus with nine VT, and Archaeospora with eight VT,
representing 20% of the total AM fungal community. Other AM fungal genera in rubber
roots, with less frequency, were Claroideoglomus and Scutellospora with five VT, Rhizophagus
with three VT, and Ambispora,Diversispora, Gigaspora, Kuklospora, Redeckera, and Viscospora,
Agriculture 2021,11, 361 9 of 17
with one VT each. On average, each rubber plantation and each rubber root sample hosted
22 and 23 VT, respectively. The complete list of VT obtained in this study is summarized in
Table S2, Supplementary Materials.
Figure 5.
Community composition of arbuscular mycorrhizal fungi associated with rubber roots
cultivated on different types of plantations: clonal trial with Colombian genotypes (CTCG); clonal
trial with introduced genotypes (CTIG); monoclonal plantation (MP); and agroforestry system (AS).
Community composition was estimated by two different methodologies—soil spore-borne approach
and sequencing approach.
The PERMANOVA analysis showed significant differences between the AM fungal
community composition associated with different rubber plantations (p= 0.027) (
Table 2
)
but did not in those colonizing different rubber genotypes (p= 0.068). The NMDS plot
separated the AM fungal communities of MP from AS, CTIC, and CTCG (Figure 6). In
addition, the AM fungal communities of AS were separated according to the location of
the AS.
Table 2. Characteristics of rubber plantations and their arbuscular mycorrhizal (AM) fungal composition.
Plantation
Type Rubber Tree Genotypes Plantation Identity Age (Years) Geographic Location Number
Samples aTotalNumber of AM
Fungal Reads
Number of
Vitual Taxa
CTCG
Promising Colombian genotypes: ECC 25, ECC 29, ECC 35,
ECC 60, ECC 64, ECC 66, ECC 73, ECC 83, ECC 90 and IAN 873
(traditional cultivar)
CTCG 1 1 El Paujil 10 37,631 53
CTCG 2 1 San Vicente del Caguán 10 32,814 80
Promising Colombian genotypes (in a continuous sequence):
ECC 101 to ECC 199, and IAN 873 (traditional cultivar). The
genotype ECC 169 had not samples for molecular analysis
CTCG 3 1 El Paujil 99 140,362 99
CTIG
Promising introduced genotypes: CDC 312, CDC 56, FDR 4575,
FDR 5597, FDR 5788, FX 3899 P1, FX 4098, GU 198 and MDF
180, and IAN 873 (traditional cultivar)
CTIG 1 9 Florencia 10 40,249 78
CTIG 2 9 San Vicente del Caguán 10 26,622 78
AS Promising introduced genotypes: FX 4098 and FDR 5788, and
IAN 873 (traditional cultivar)
AS 1 6 Albania 4 30,670 44
AS 2 6 San Vicente del Caguán 4 4784 35
MP Introduced genotypes planted as traditional cultivars: FX 3864,
FX 25 or IAN 873
MP 1 30 San Vicente del Caguán 4 36,188 32
MP 2 50 Belén de los Andaquíes 4 25,226 44
MP 3 50 Florencia 4 44,543 50
CTCG: clonal trial with Colombian genotypes; CTIG: clonal trial with introduced genotypes; AS: agroforestry system; MP: monoclonal
plantation. aOne representative sample for each genotype was obtained.
Agriculture 2021,11, 361 10 of 17
Figure 6.
Non-metric multidimensional scaling (NMDS) plots displaying AM fungal communities colonizing rubber roots.
Ellipses indicate one standard deviation around the centroid position of each rubber plantation.
The most abundant VT colonizing rubber roots was Paraglomus VT444, with
50,279 reads
.
Some VT were identified as indicator species of plantation types or rubber tree genotypes
(Table S3, Supplementary Materials). Most of the indicator species identified were
Glomus
(82% of all indicator species), Acaulospora and Archaeospora (each representing 5% of all in-
dicator species), Gigaspora, and Scutellospora (each representing 2% of all indicator species).
3.5. Richness and Diversity of Arbuscular Mycorrhizal Fungal in Rubber Roots
Around 27% of the total VT was shared among all rubber tree plantations. Only CTCG
showed a high number of exclusive VT (around 19%), while the other plantation types
had less than 5% of exclusive VT (Figure 7a). The AM fungal community compositions in
roots of Colombian and introduced rubber genotypes were very similar. The Colombian
and introduced rubber genotypes shared around 70% of the total AM fungal community
(Figure 7b).
VT richness colonizing rubber roots was significantly different among the plantation
types (p < 0.001) but not between the rubber tree genotypes (p= 0.665), even if they
(Colombian vs. IAN 873) were growing on the same plantation (p= 0.429). CTIG and MP
hosted significantly more AM fungal VT than the other plantation types (Table 3). Only the
exponential Shannon index showed significant differences in the diversity of the different
rubber plantations (Table 3).
Agriculture 2021,11, 361 11 of 17
Figure 7.
Arbuscular mycorrhizal fungal community colonizing rubber roots in (
a
) different plantation types (CTCG: clonal
trial with Colombian genotypes; MP: monoclonal plantation; AS: agroforestry system; CTIG: clonal trial with introduced
genotypes) and (b) between Colombian and Introduced rubber genotypes.
Table 3.
Richness and diversity indexes of AM fungal colonizing roots of Colombian and introduced rubber genotypes and
different plantation types: CTCG (clonal trial with Colombian genotypes); CTIG (clonal trial with introduced genotypes);
AS (agroforestry system); and MP (monoclonal plantation).
Factor Level Richness (S) Exponential
Shannon (expH0)
Inverse Simpson
1/D Evenness (Piélou)
Genotype type Colombian 20.75 ±2.78a 5.26 ±0.73a 3.68 ±0.40a 0.53 ±0.03a
Introduced 25.08 ±1.88a 6.85 ±0.55a 4.67 ±0.35a 0.58 ±0.02a
Plantation type
CTCG 20.80 ±2.04b 5.42 ±0.44b 3.82 ±0.22a 0.53 ±0.02a
CTIG 30.55 ±2.63a 8.13 ±0.66a 5.33 ±0.43a 0.61 ±0.04a
AS 19.63 ±3.13b 4.90 ±0.94b 3.33 ±0.66a 0.51 ±0.05a
MP 25.08 ±2.55ab 7.31 ±0.77ab 5.00 ±0.54a 0.60 ±0.04a
Values in the columns corresponded to mean and standard error. Values followed by the same letter do not differ statistically (Fisher’s LSD
test, p< 0.05).
3.6. Arbuscular Mycorrhizal Fungi and Soil Properties
Three diversity indexes (inverse Simpson (1/D), exponential Shannon (ExpH
0
), and even-
ness) were negatively correlated with pH, Ca and Mg (r<
−
0.64; p< 0.05),
two diversity
indexes
(1/D and ExpH
0
) were negatively correlated with P (r<
−
0.65; p< 0.05), and the VT richness
was negatively correlated with Ca (r=−0.72; p< 0.05) (Table S4, Supplementary Materials).
Redundancy analysis (RDA) of AM fungal communities associated with soil variables
(inertia proportion constrained = 23.77%) showed a clear separation of MP from the
other rubber plantations (Figure 8a). The AM fungal communities on MP were strongly
associated with a higher soil Na content (Figure 8b), where Glomus VT269 was favored by
this condition. RDA plot also showed that the AM fungal communities on CTCG were
associated with sandier soils (Figure 8a), where Glomus VT280 was favored by soils with
coarse texture. There was a weak relationship between the AM fungal community on
CTCG and P (Figure S1, Supplementary Materials).
Agriculture 2021,11, 361 12 of 17
Figure 8.
(
a
) Redundancy analysis (RDA) of AM fungal communities associating with rubber tree
plantations, constrained by soil variables. Blue arrows indicate significant soil variables for the
constrained ordination (p< 0.05). Red arrows indicate significant AMF VT species with maximum
correlation with ordination scores (r
≥
0.5; p< 0.05). Ellipses indicate one standard deviation around
the centroid position of each rubber tree plantation. (
b
) Redundancy analysis (RDA) of AM fungal
communities associated with rubber tree plantations according to Na soil gradient.
4. Discussion
Rubber planted on degraded soils of the Colombian Amazon harbored highly diverse
AM fungal communities. The AM fungal communities in rubber roots differed among the
plantation types but not between the rubber genotypes.
The molecular approach to assess AM fungal community composition was more
sensitive than the soil-borne spore approach. The molecular approach recorded a higher
number of AM fungi from the Archaeosporaceae and Gigasporaceae families. The Ar-
chaeosporaceae and Gigasporaceae families produced low numbers of large-spore size [
44
]
than the Glomeraceae family, which was the best represented and most diverse in the study
area (with 77% of the total AM fungal community). Additionally, the molecular approaches
provided information for the presence of AM fungi that do not sporulate frequently or do
not sporulate at all [
45
], offering a more accurate picture of the AM fungal community. The
AM fungal community in rubber roots was composed of 135 VT in 13 genera. There was a
higher number of AM fungi associated with rubber roots than in previous studies (
111 VT
of 10 genera [
9
]), even on the rubber plantations planted on areas previously covered by
pastures (on average, 22 VT of 7 genera per plot; 16 VT of 5 genera per plot [
10
]). It is
important to indicate that this is the first study evaluating the AM fungal composition of
rubber roots using WANDA and AML2 primers, which limits comparisons with results
from previous publications. Differences in the number of VT between this study and others
Agriculture 2021,11, 361 13 of 17
might have resulted from the molecular technique used to assess the AM fungi and should
not be interpreted as real differences.
Glomus was the dominant genus, representing 66% of the total AM fungal community,
followed by Acaulospora, Paraglomus, and Archaeospora.Glomus and Acaulospora were
reported previously as frequent genera associated with rubber [
9
,
10
], and the main genera
composing the AM fungal community of Amazon soils [46–48].
In the study area, rubber plantations were planted on degraded pastures after years of
cattle ranching. Soils where rubber tree grows presented low fertility, with very low levels
of available P and significant differences in the amount of Na among plantations. Pfeiffer
and Bloss [
49
] found that phosphate fertilizers included low concentrations of Cu, Zn, Na,
K, and SO
4
and were a source of those elements. A significantly higher amount of Na on the
older plantations (AS and MP) might have resulted from periodical fertilization in earlier
years and later soil accumulation. MP and AS also had significantly higher percentages of
root colonization (66% and 57%, respectively). Higher percentages of root colonization in
MP and AS might have been the consequence of an accumulative effect from fertilization,
through which Na, in combination with P, increased the AM root colonization [
49
]. On the
other plantations, rubber roots presented percentages of AM root colonization around 47%,
similar to previous reports [50].
The AM fungal richness and diversity (ExpH
0
) were significantly higher in CTIG and
MP than in the other plantation types. Feldmann et al. [
18
] found lower spore diversity
on commercial rubber plantations than in natural tree stands. They attributed differences
to agronomic practices on commercial plantations and mainly to the use of pesticides
and extensive weeding. Although there were differences in the agronomic practices used
on the different rubber plantations (e.g., MP received less fertilization than the other
plantations), our results are best explained by differences in age between rubber plantations.
Plantations over nine years old (MP and CTIG) had higher AM fungal richness and diversity
than younger plantations. Even with the limitations to compare results obtained from
different molecular techniques, our result was similar to the one reported by Herrmann and
collaborators. Herrmann et al. [
9
] also found differences in AM fungal richness related to
plant age in a rubber chronosequence of 3, 6, and 16 years old. The NMDS analysis clearly
showed how the AM fungal community composition of very old rubber plantations was
sufficiently different from the others, forming a separate cluster (Figure 6). Additionally,
the low numbers of AM fungal spores in MP resembled the abundance of AM fungal
spores in soils of mature Amazon forests [46].
Diversity indexes were negatively correlated with pH, Ca, Mg, and P. Similar results
had been reported previously for rubber plantations [
9
]. The edaphic condition is one of the
main factors affecting AM fungal community composition [
51
,
52
]. Soil pH has been clearly
identified as one of the most important abiotic drivers of AM fungal communities [
28
,
53
].
pH directly influences the availability of Ca, Mg, and P in soil. In soils with a low pH, P
became particularly less available as it is sorbed in insoluble compounds [
54
]. Some specific
AM fungi are commonly associated with acid soils, such as Acaulosporaceae, and some
Glomeraceae, such as Rhizophagus manihotis [
28
,
55
]. Particularly, Rhizophagus manihotis is
commonly associated with acidic poor-nutrient soils [56,57].
In clonal trials, selected Colombian rubber genotypes performed better (growth, early
latex production, and resistance to pathogens) than the IAN 873 (control) genotype [
19
].
Our results indicated that AM root colonization, richness, and composition of AM fun-
gal communities in roots of different rubber genotypes were similar, even if different
rubber genotypes (Colombian vs. IAN 873) were under the same conditions (location,
plantation type, and age). The AM fungal community composition in roots of Colom-
bian and introduced genotypes shared around 70% of the total community (Figure 7b).
Herrmann et al. [9]
identified four VT as indicator species for rubber, namely, Glomus
VT399, Acaulospora VT227, Rhizophagus manihotis VT90, and Glomus VT124. From those VT,
three were recorded in this study. Rhizophagus manihotis was one of the 20 more abundant
VT colonizing rubber tree roots. Glomus VT399 was an indicator species of introduced
Agriculture 2021,11, 361 14 of 17
rubber tree genotypes, and Acaulospora VT227 was an indicator species of MP, but both had
a low number of reads. The most abundant VT colonizing rubber roots was Paraglomus
VT444, which was not reported before in rubber tree roots. Paraglomus VT444 was an
indicator taxon of Colombian rubber genotypes and AS. This VT was isolated before in the
study area from pastures, and its abundance increased in soils of secondary forests that
originated from the natural regrowth of those pastures [
58
]. Since AS was similar to natural
secondary forests, Paraglomus VT444 persisted in AS soils. The particular association of
this VT with Colombian rubber genotypes might be related to the fact that the Colombian
genotypes were the youngest rubber trees and were cultivated on recently used pastures.
Rubber preferences for a particular AM fungal taxon were weak. Strong relationships
between particular AM fungi and rubber seem to be context dependent, varying from
one site to another. Apparently, plant host-AM fungal preferences occur at the plant
level (functional) groups [
59
] rather than at the level of individual plant species. Even
if grifting plants such as rubber tree clones behave as particular individuals [
15
], the
arbuscular mycorrhizal association would not be affected by genotypic differences between
genotypes. Rubber tree genotypes apparently behave as natural varieties, even if genotypic
variability was induced artificially. The similarity in arbuscular mycorrhization among
manioc varieties (Manihot esculenta Crantz), another important commercial Euphorbiaceae
species, has been reported [60], supporting the interpretation of our results.
According to Chen et al. [
3
], agroforestry systems for rubber trees are the best al-
ternative for improving soil properties. Our results indicated that none of the rubber
plantations improved the degraded condition of the soils. Significant differences in the soil
composition occurred only on rubber plantations older than 30 years. Herrmann et al. [
9
]
also found that the soil fertility of rubber plantations did not change over time despite
fertilization. Frequent weed control and other agronomic practices could be responsible for
this result. Although soil condition is not improved during the productive cycle of rubber,
its roots host significantly more AM fungi (in average 22 VT per plot) than other commercial
crops cultivated in Amazon, i.e., mahogany (Swietenia macrophylla; 19 VT [
10
]), eucalyptus
(
16 VT [10]
), and soybean (17 VT [
61
]). A higher AM fungal diversity will enhance the
external mycelial network, improving the nutrition of the plant community [
16
]. Addi-
tional traits (e.g., soil aggregation, soil organic C, biomass C sequestrated) and ecological
services (plant, enthomofauna, and bird diversity) have been evaluated on different rubber
plantations and genotypes [
3
,
22
,
62
], corroborating the importance of this plant species for
soil restoration.
Supplementary Materials:
The following are available online at https://www.mdpi.com/article/
10.3390/agriculture11040361/s1, Table S1: Soil and rubber tree samples collected and analyzed,
Table S2
: Arbuscular mycorrhizal fungal virtual taxa recovered from different rubber plantations,
Table S3: Arbuscular mycorrhizal fungal virtual taxa with significant indicator species value for
rubber genotypes and plantation types, Table S4: Spearman’s correlation coefficients between soil pa-
rameters, and richness and diversity indexes of arbuscular mycorrhizal fungi, Figure S1: Redundancy
analysis (RDA) of arbuscular mycorrhizal fungal communities associated with rubber plantations
according to phosphorus soil gradient.
Author Contributions:
Conceptualization, C.P.P.-V. and A.S.; methodology, A.S., C.P.P.-V. and
T.K.A.-R
.; software, A.S.; validation, C.P.P.-V. and A.S.; formal analysis, A.S. and C.P.P.-V.; investiga-
tion, C.P.P.-V.; resources, A.S. and C.P.P.-V.; data curation, T.K.A.-R. and A.S.; writing—original draft
preparation, C.P.P.-V.; writing—review and editing, C.P.P.-V. and A.S.; visualization, T.K.A.-R. and
A.S.; supervision, C.P.P.-V.; project administration, A.S.; funding acquisition, A.S. All authors have
read and agreed to the published version of the manuscript.
Funding:
This study was funded by FCTeI-SGR, Contract 59/2013 Instituto Amazónico de Investiga-
ciones Científicas SINCHI–Gobernación del Caquetá–Universidad de la Amazonía–Asociación de
Reforestadores y Cultivadores de Caucho del Caquetá; and by the Government of Colombia through
the project BPIN 2017011000137 “Investigación en conservación y aprovechamiento sostenible de la
diversidad biológica, socioeconómica y cultural de la Amazonia colombiana.”
Agriculture 2021,11, 361 15 of 17
Acknowledgments:
We thank Daniela León and Martti Vasar for their help with bioinformatic ana-
lyzes. We also thank all the farmers of the study area for their help and support during
the fieldwork.
Conflicts of Interest: The authors declare no conflict of interest.
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