Pastos y forrajes Vol 40 No. 4 del 2017

Vol. 40, No. 4, October-December 2017 / NRS 0099
ISSN 0864-0394 (printed version) / ISSN 2078-8452 (online version)
Quarterly journal. Official organ of the Ministry of Higher Education for pastures and forages | 1978
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Vol. 40, No. 4, October-December / 2017
Revista Trimestral. Órgano oficial del Ministerio de Educación Superior para el área de los pastos y forrajes
Quarterly journal. Official organ of the Ministry of Higher Education for pastures and forages
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CONTENT
| review paper |
Identification and degradation of mimosine, a toxic compound
in Leucaena leucocephala (Lam.) de Wit
Luis Alejandro Ospina-Daza, María Eugenia Buitrago-Guillen and Julio Ernesto
Vargas-Sánchez.................................................................................................241
| scientific paper |
Soil macrofauna as indicator of agroecological conversion of a productive
system of Moringa oleifera Lam. in Nicaragua
Álvaro Noguera-Talavera, Nadir Reyes-Sánchez, Bryan Mendieta-Araica and
Martha Miriam Salgado-Duarte....................................................................249
| TECHNICAL NOTE |
Seed characteristics of five mulberry (Morus alba L.) varieties harvested
in Matanzas, Cuba
Jorge Jesús Reino-Molina, Laura A. Montejo-Valdés, Jorge Alberto Sánchez-
Rendón and Giraldo Jesús Martín Martín....
..................................................259
Effect of controlled conditions on the germination of five Morus alba L.
varieties
Jorge Alberto Sánchez-Rendón, Jorge Jesús Reino-Molina, Mayté Pernús-
Alvarez, Dariel Morales-Queroland Giraldo Jesús Martín-Martín........264
| TECHNICAL NOTE |
Agronomic evaluation and selection of Brachiaria spp. accessions
on moderate fertility soils
Lisset Castañeda-Pimienta, Yuseika Olivera-Castro and Hilda Beatriz Wencomo-
Cárdenas............................................................................................................272
| SHORT Communication |
Control of Sitophilus zeamais Motschulsky with marble powder in stored
corn grains
Yandy Rodríguez-Ledesma, Raúl Mirabal-García, Claribel Suárez-Pérez,
Marcos Tulio García-González, Anayansi Albert-Rodríguez and Idelfonso
Orrantia-Cárdenas............................................................................................277
Evaluation of secondary metabolites in the meal of Stizolobium
aterrimum forage, for its use in animal feeding
Idania Scull-Rodríguez, Lourdes Lucila Savón-Váldes and Josefa Victoria
Hormaza-Montenegro...........................................................................................282
Effect of non-conventional diets with native microorganisms on pig rearing
Iván Lenin Montejo-Sierra, Luis Lamela-López, Javier Arece-García, María
Teresa Lay-Ramos and Diosnel García-Fernández.....................................287
Effect of zootechnical additives on productive and health indicators in broilers
Grethel Milián-Florido, Ana Julia Rondón-Castillo, Manuel Pérez-Quintana,
Fátima Graciela Arteaga-Chávez, Ramón Boucourt-Salabarría, Yadileiny
Portilla-Tundidor, Marlen Rodríguez-Oliva, Yoenier Pérez-Fernándezand
Marta Elena Laurencio-Silva..........................................................................293
Technology management in the university-Cuban state animal husbandry
enterprise relation. Part II. Implementation and validation of the model
Mileisys Benitez-Odio, José Andrés Díaz-Untoria, Raúl Ricardo Fernández-
Concepción, Alexei Yoan Martínez-Robaina and Álvaro Celestino Alonso-
Vázquez..............................................................................................................301

Pastos y Forrajes, Vol. 40, No. 4, October-December, 241-248, 2017 / Indentification and degradation of mimosine 241
Review paper
Identification and degradation of mimosine, a toxic compound
in Leucaena leucocephala (Lam.) de Wit
Luis Alejandro Ospina-Daza, María Eugenia Buitrago-Guillen and Julio Ernesto Vargas-Sánchez
Universidad de Caldas, Calle 65 No. 26-10. apdo. aéreo 275, Manizales, Colombia
E-mail: alejo.mvz7@gmail.com
Abstract
Leucaena leucocephala is known for being a shrub that generates nutritional benefits for animal husbandry in
the world. Nevertheless, its consumption has been limited due to antinutritional factors such as mimosine, a toxic that
exists in the plant. This review compiles studies by different authors who identified and characterized bacteria found in
the rumen and in the rhizosphere of the plants, capable of degrading mimosine and its degradation products, 3,4- and
2,3-dihydroxypyridone (3,4 and 2,3 DHP). Mimosine (non-protein aminoacid which is found in L. leucocephala leaves
and seeds) degradation by bacteria is a fundamental factor in the protection of animals when consuming leucaena. Mi-
mosine and its degradation products have been used in the rhizosphere by the strain TAL1145 of Rhizobium, as carbon
and nitrogen source; while at rumen level bacteria such as Synergistes jonesii can degrade it. Recent studies have proven
that other rumen bacteria, like Streptococcus lutetiensis have the capacity to degrade this compound. Such advances will
take the species L. leucocephala to another knowledge level, in order to search for higher efficiency and safety in its use
for animal feeding.
Keywords: animal feeding, Streptococcus lutetiensis, Synergistes jonesii.
Introduction
In the tropics ruminant nutrition is based mainly
on the consumption of pastures, harvest byproducts
and, to a lower extent, preserved feedstuffs, such as
silages and meals. Nevertheless, the grasses used in
feeding have high fiber contents and low protein per-
centages, bringing about a considerable decrease in
the general development of the animal, particularly
in the dry season (Villanueva et al., 2013). A large
number of forage trees, mainly legumes, are used
as supplement in the diets of low nutritional quality;
they show high protein content and good digestibili-
ty, compared with grasses (Pereyra et al., 2015).
Besides their favorable chemical composition,
forage trees and shrubs have other positive
properties, because they help to recover degraded
soils (Rangel et al., 2016), increase their nutrient
content due to the symbiosis they establish with
bacteria of the Rhizobium genus (Higashide, 2014),
protect against erosion, and can be also used as fuel
and construction materials.
All the legume species have secondary
metabolites, some of which are called antinutritional
factors (ANF), that can cause a negative effect on
the nutritional value of the feedstuff, as well as
on animal health. ANFs can be defined as those
substances generated by the natural metabolism of
plant species, and which, by different mechanisms,
exert adverse results on the optimum nutrition of the
animals, because they decrease the digestive and/
or metabolic effects (Rodríguez and Ledesma,
2014).
Legumes generate these substances through
their secondary metabolism, as a defense mechanism
against the attack of molds, bacteria, insects and
birds; or, in some cases, as product of metabolism
when they are subject to stress conditions. These
plants, when contained in the diet ingredients, reduce
intake and prevent nutrient digestion, absorption and
utilization by the animal (Casso and Montero, 1995).
Among the most studied ANFs, present in most
legume species, the following can be cited: lecithins,
tannins, cyanogen glycosides, vicine and convicine,
galactosyl sucrose oligosaccharide, galactomannan
gums, saponins, non-protein aminoacids, neurolatheri-
gens, arginine analogues, alkaloids, phytic acid, anti-
genic proteins and aromatic aminoacids; and within
the last ones mimosine stands out [3-N-(3-hydroxy-
4-pyridone) aminopropionic-ao-acid], present in
Leucaena leucocephala (Lam.) de Wit. (Casso and
Montero, 1995).
The objective of this review was to compile
studies conducted by different authors who identi-
fied and characterized bacteria capable of degrad-
ing mimosine and its degradation products, which
were found in the rumen and in the rhizosphere of
the plants.

242 Pastos y Forrajes, Vol. 40, No. 4, October-December, 241-248, 2017 / Luis Alejandro Ospina-Daza
Characteristics of mimosine
Mimosine is a non-protein aminoacid, present in
the tropical forage legume L. leucocephala, which
has been responsible for toxicity symptoms in some
species of domestic animals; among these symptoms
the following stand out: alopecia, anorexia, weight
loss, deep salivation, lesions through the esophagus,
necrotic papillae in the rumen and reticulum, hyper-
plasia of the thyroid gland and low levels of circulat-
ing thyroxine hormone (Xuan et al., 2013).
Mimosine has an aromatic ring of 3-hydroxy-
4-(1H)-pyridone (3,4 DHP) (Nguyen and Tawata,
2016). This compound plays an important role in the
plant resistance to a large variety of phytopathogen
agents; besides, as it is structurally analogue to thy-
roxine, it allows an inhibitor or antagonist behavior
in many processes in which it is intermediary. In
animals intoxicated by mimosine consumption an
effect is shown on production, which includes low
reproductive values due to precocious embryo mor-
tality and perinatal death (Hammond, 1995).
The aromatic ring of 3,4 DHP is free in the
rumen and in the circulation of intoxicated ani-
mals, which indicates that mimosine is easily hy-
drolyzed in the rumen and excreted at renal level
(Barros-Rodríguez et al., 2012). Small quantities
of mimosine have also been found in the nitrifying
nodules of L. leucocephala and in the exudate adja-
cent to its roots, which is degraded through Rhizo-
bium strains; on the other hand, when non-adapted
animals are fed with leucaena over 30 % of dry
matter in their diet, this can induce toxicity cases
and cause death (Soedarjo and Borthakur, 1998).
Recently published studies show the efforts
to decrease the mimosine contents of the L. leu-
cocephala leaves using ethyl methanesulfonate
(EMS), and thus improve the nutritional value of
the plant. With this advance foresters intend to re-
duce significantly the price of animal feeding in the
future (Zayed et al., 2014).
Molecular identification of mimosine
AlthoughL.leucocephalaisconsideredapromising
alternative source of protein, and that it can also help to
mitigate the emission of rumen methane (CH4) in the
tropics (Soltan et al., 2013), the presence of mimosine
in 2-10 % DM in the leaf and 2-5 % DM in the seed
limits the quantity of foliage that can be supplied to
cattle, because mimosine (depilatory agent) and its
degradation product (3-hydroxy-4 (DHP)3
(strong
goitrogenic agent) are considered toxic for many
species. The partial resistance to mimosine-caused
toxicity in ruminants from certain geographical
areas has been ascribed to the capacity of their
rumen microorganisms, which, restrictively,
metabolize mimosine and DHP. An example is the
report in Hawaii, in which resistant goats, with
efficient microorganisms that counteracted the
effects of mimosine-caused toxicity, transferred the
microorganisms to the rumen of Australian cattle,
which was susceptible to such toxicity (Lalitha et
al., 1993).
For the identification of mimosine and DHP,
the most commonly used reaction method is the
colorimetric one with FeCl3
(Ilham et al., 2015). It
has little sensitivity and specificity, because its poor
solubility in aqueous and organic solvents limits
its application, as it requires a high concentration
to have accurate measurements. Liquid gas chro-
matography, liquid chromatography and ion-pair
reversed-phase high performance liquid chroma-
tography require sophisticated equipment and do
not offer an improvement in sensitivity (Lalitha et al.,
1993).
The spectrophotometric method for mimosine
and/or DHP estimation is more sensitive than other
reported ones, and can be most adequately coupled
to an ion-exchange chromatography and paper
chromatography for the specific essay of these
compounds.Itisessentiallybasedontheformationof
an intense azoic yellow coloring between mimosine
and/or DHP and p-nitroaniline diazonium salt
(Ilham et al., 2015).
Applying spectrophotometry it was estimated
that the quantity of mimosine in the L. leucocephala
leavesvariesbetween3,75and5,5%DM,depending
on the type of leaves, season, soil quality, etc. Due
to the high sensitivity of the procedure, mimosine
was detectable in the discolored extracts with
activated carbon even after high dilutions, and
the concentrations of other interfering compounds
became negligible for these dilutions. A useful
application of this technique was extended to the
evaluation of mimosine toxicity in the experimental
feeding material based on L. leucocephala leaves.
The early observations indicated that, during
the anaerobic degradation of these leaves using
mixed specific inoculant, mimosine was actively
metabolized, as shown by the method with FeCl3
after the separation from paper chromatography.
When applying the above-described method it
was confirmed that only traces of mimosine and
DHP remained, which indicated almost 99 % in 48
h, and the solid biomass made an adequate choice

of feeding material with high protein content. In
the comparison of the method reported with other
techniques of mimosine estimation it was proven
that the colorimetric method with FeCl3
requires a
solution of 20-500 µl for an accurate measurement
(Lalitha et al., 1993).
Mimosine degradation from Rhizobium sp.
strains
Mimosine is a toxin found in large quantities in
the seeds and leaves of legume trees and shrubs of
the Leucaena genus. In its structure it is analogous
to dihydroxyphenylalanine (L-Dopa) with a 3-hy-
droxy-4-pyridone ring instead of a 3,4 dihydroxy-
phenil ring (Nguyen and Tawata, 2016). This toxin
is distributed throughout the plant, and is found from
4 to 5 % in the seeds (dry basis); in different parts of
the plant, such as stems and leaves, it can vary from
1 to 12 %, and in the root from 1 to 1,5 % (Soedarjo
et al., 1994). It has been proven that some Rhizobium
strains can degrade the substance mimosine.
According to the work conducted in Hawaii
by Soedarjo et al. (1994), a total of 32 strains have
been collected and cultivated in optimum media,
such as TY (Beringer, cited by Martínez et al.,
2015); complete medium for rhizobia growth, YEM
(Abrahamovich et al., 2014); yeast-mannitol extract
(also used for rhizobia culture) and a Rhizobium-mi-
mosine medium (RM), in order to know which of
the collected strains had the capacity to grow in a
medium with the toxin. It was determined that the
strains TAL1145 and TAL1566 had capacity to use
mimosine as only source of carbon and nitrogen.
The growth rates of those strains in RM medium
with different mimosine concentrations were de-
termined through the inoculation of 50 mL of this
medium in 250-mL bottles with screw top, which
contained 0,5 mL of Rhizobium culture. The cul-
tures were subject to 28 ºC, with agitation, and the
growth was determined every 6 h by measuring the
cell density as the optical density in a colorimeter.
It was determined that only a limited number of
Rhizobium strains could use mimosine as selective
growth substance; in contrast, the strains which
did not have this capacity formed nitrogen-fixing
nodules, as in the case of MS13. The utilization of
mimosine can be a specialized mechanism which
has been developed by some rhizobia that live in
the rhizosphere of leucaena to survive, which pro-
vides a competitive advantage to certain Rhizobium
strains. A direct relation could not be established
between the capacity to catabolize mimosine and
the capacity to fix nitrogen, because two strains,
TAL1145 and TAL1566, degrade mimosine and
use it as nitrogen and carbon source, but only
TAL1145 is good forming nitrifying nodules, un-
like TAL1566 which has deficiency to form them
(Soedarjo et al., 1994).
In a study conducted later than this one, in the
island of Guam, the strain with the best results re-
garding nodulation and utilization of mimosine for
itself, in terms of nitrogen and carbon (TAL1145),
was used. For such purpose native Rhizobium were
isolated from the L. leucocephala nodules, in order
to verify whether they had the capacity to degrade
mimosine and examine them for the midA gene. As
it was mentioned above leucaena nodulates with
several types of Rhizobium, such as Rhizobium
strain TAL1145, Rhizobium strain NGR234, and
with strains of Rhizobium tropici, such as CIAT899.
Among these rhizobia, only the strain TAL1145 and
some related strains can degrade mimosine and use
it as carbon and nitrogen source, while strains like
CIAT899 and NGR234 cannot degrade it (Soedarjo
et al., 1994).
After isolating the rhizobia, culturing the
found strains and subjecting them in a RM liquid
medium, and isolating and amplifying the genomic
DNA of the Rhizobium strains that utilized the mi-
mosine, it was concluded that eight of the 11 isolat-
ed strains used it, because the RM medium changed
from brownish yellow to colorless. This was cor-
roborated by Soedarjo et al. (1994), who proved that
mimosine was not detected in the medium by high
performance liquid chromatography (HPLC), when
the medium became colorless, with the presence of
rhizobia that degrade it. As shown in table 1, from
the eight mimosine-degrading strains only three
generated the PCR fragment 1055-pb, which sug-
gests that these strains contained the gen midA of
the strain TAL1145 (Marutani et al., 1999).
According to the above described facts, the im-
portance of knowing further the strains belonging
to the Rhizobium genus, which have the capacity
to utilize mimosine and its degradation product 3,4
and 2,3 hydroxypyridone, was proven.
Soedarjo et al. (1994) isolated in different
parts of the world some Rhizobium strains from
the leucaena nodules which fulfilled this condition
and which, additionally, could use it as carbon and
nitrogen source. Although the capacity to catabolize
mimosine for nodulation and nitrogen fixation is
not required, this provides a competitive advantage
to mimosine-degrading Rhizobium (mid+) in the

rhizosphere of leucaena, by supplying a selective
source of nutrients and, at the same time, inhibiting
the growth of other microorganisms and rhizobia
(Soedarjo and Borthakur, 1998). The Rhizobium
strain TAL1145 is one of the mid+ strains, known
for being competitive for the occupation of the
nodule in leucaena (Siddiqi and Athar, 2013), and
has been described as a very efficacious nitrogen
fixing nodules in Leucaena spp. The coincidence
in its efficient N2
fixation capacity and its competitive
capacity make it an ideal choice to be used in inoculant
preparations.
To identify and characterize the mid genes
present in the fragment from 12 to 6 kb of TAL1145,
which are required for mimosine degradation, the
cosmid pUHR181 was isolated from a clone library
of TAL1145 (Borthakur et al., 2003). When this
cosmid was transferred to non-degrading (mid-)
strains, such as TAL182 and CIAT899, the deg-
radation product 3-hydroxy-4-pyridone (HP) was
accumulated in the culture medium. This suggested
that pUHR181 contained genes for the degrada-
tion of mimosine to HP. The plasmid pUHR191 is
a derivative from pUHR181 which contains an in-
sert of 12 to 6 kb, constructed by the elimination
of a fragment of approximately 10 kb of pUHR181.
The transconjugants of TAL182 and CIAT899
contain pUHR191 which turn mimosine into HP
(Borthakur et al., 2003).
Initially 12 defective TAL1145 in mimosine
degradation (mid-) were established; they were
made through Tn3Hogus (transposon), TnphoA
(bacteriophage) or insertion of kanamycin
resistance cassette. A PstI fragment (a restriction
enzyme) of 5-0 kb of TAL1145, subcloned from a
cosmid clone that contains mid genes for mimosine
degradation, complements most mid- mutants. The
sequencing of this fragment and the PstI fragment
of 0-9 kb which is adjacent identified five genes:
midA, midB, midC, midD and midR, from which
the first three codify ABC transporter proteins
implied in the mimosine absorption; while midD
Table 1. Ability to utilize mimosine and detection of the midA gene of TAL1145 in different strains.
Sampling site/ soil type N° of strain Ability to use mimosine
as C and N source
Detection of the
fragment 1055 pb
Barrigada/ B1 + -
Pulantat B2 + -
B4 + -
B5 + -
B7 ND (contaminated) + (weak)
B9 + +
B10 + + (weak)
B12 + +
B19 - -
B24 - +/- (very weak)
B26 + -
Yigo/ Y1 - ND
Guam cobbly clay Y2 - ND
Y3 - ND
Y4 + ND
Y5 - +
Y7 - ND
Y9 - ND
Control/ Rhizobium TAL1145 + +
Bradyrhizobium sp. - ND
Source: Marutani et al. (1999).
+: strain with capacity to utilize mimosine as nutrient, and indicates the positive detection of the fragment 1055 pb.
-: strain without capacity to catabolize mimosine, and does not indicate the detection of the fragment 1055 pb.
ND: strain that was not included in the bioessay or in the PCR analysis.

codifies an aminotransferase required to degrade
mimosine into HP, and midR is a regulating gene
whichcodifiesaLysR-typetranscriptionalactivator.
Thus, mid genes are specific for the Rhizobium of
leucaena and are absent in the Rhizobium strains
and in Bradyrhizobium spp. (Borthakur et al., 2003).
Just as in the strain TAL1145 the presence of
genes responsible for mimosine degradation and
utilization could be determined, nodulation-effi-
cient strains, which have potential to degrade other
antinutritional factors, should be further studied
(Xu et al., 2013).
Degradation of mimosine from rumen bacteria
The tree legume L. leucocephala is a high-
quality feedstuff used in ruminants, which is
extremely important for livestock production
in the tropics, in spite of the presence of mimosine
in its leaves. This non-protein toxic aminoacid
limits productivity and adversely affects animal
health (Halliday et al., 2013). Given the fact that L.
leucocephala has a high potential in production,
studies have been conducted in order to know more
specifically which rumen microorganisms could be
involved in the degradation of the products derived
from mimosine and thus reduce toxicity in ruminants.
In Venezuela, Domínguez-Bello and Stewart
(1991) isolated a bacteria belonging to the
Clostridium genus in sheep fed leucaena that
did not develop toxic symptoms, which had the
capacity to degrade 3-4 DHP. For the isolation
of the bacteria clarified rumen content was used,
which was subject to the growth of bacteria in a
culture medium; it had micro- and macrominerals,
resazurin, cysteine, phytone (soybean peptone)
and sugars, which as a whole promoted bacterial
growth. Additionally, the medium was gassed
using CO2
and became oxygen-free. For the
identification of the anaerobic bacteria two systems
of commercial tests were used (API-20A and 32A
ATB), consisting in strips of essay domes that
contained dehydrated substrates and propitiated
the growth of bacteria from the Clostridium genus;
mimosine, 3,4 DHP and 2,3 DHP were added to the
bacteria that grew in the API systems. As a result
the strain that degraded the above-mentioned toxics
corresponded to a Clostridium (called strain 162),
given its low proportion of guanine and cytosine.
The strain 162 degraded mimosine, 3,4 DHP, 2,3
DHP and dihydroxyphenylalanine (DOPA), but
no 3-hydroxypyridine or catechol when these
compounds were added to the RPF medium (Baird-
Parker rabbit plasma and fibrinogen base agar). The
highest degradation was to mimosine (50-60 %)
with regards to 2,3 DHP or 3,4 DHP (35-45 %).
Later, Allison et al. (1992) identified and
characterized a bacterium isolated from the rumen
of a goat (in Hawaii), capable of degrading 3-4
DHP and its isomer 2-3 DHP, which was named
Synergistes jonesii. This research was based on the
toxicity generated by the consumption of the legume
L. leucocephala in ruminants; nevertheless, in some
parts of the world resistant animals to the toxicity
of the plant components, due to their capacity to
degrade3-4 DHP and its isomer 2-3 DHP, were found.
S. jonesii was originated from four strains
(78-1, 100-6, 113-4, 147-1) isolated from a goat, in
Hawaii (Jones and Megarrity, cited by Halliday et
al., 2014); these strains were cultivated in anaero-
bic media similar to the above-described ones for
Clostridium, which were subject to essay kits (API-
ZIM, AN-IDENT, API-20A) for the identification
of anaerobic Gram-negative bacteria and of the
enzymes they produced. Afterwards, the identified
bacteria were subject to an antibiotic-sensitivity
test, based on the inhibition of strain growth exert-
ed by 3,4 DHP and visualized by colorimetry. This
study allowed to conclude that S. jonesii differed
from any organism, after having compared it with
600 different bacteria, because the 16S sequence of
its ribosomal RNA is very singular; besides using
as main energy source such aminoacids as arginine
and histidine, factor that also separates it from the
other bacteria with which it was compared.
When comparing among S. jonesii strains, the
microscopic exams indicated that all of them had
similar morphology, and no differences were ob-
served between the strains 78-1, 100-6 and 113-4;
while only 78-1 was subject to the complete range
of physiological tests. The most definite and unique
tests were considered for these bacteria with the
four strains and no physiological differences were
detected among the latter. All degraded 2,3 and 3,4
DHP, and used arginine, histidine and DHP, besides
other energy substrates for growth.
From this work, it was described that S. jonesii
is an anaerobic, non-spore forming, non-motive,
Gram-negative bacteria; likewise, it has the capacity
to ferment pyridinediols, its only known habitat
is the rumen and it was originally isolated for its
function of helping the Hawaiian goats that grazed
L. leucocephala (Holland-Moritz et al., 2014).
The organism is not omnipresent in the rumen
populations, but defined geographical limits for its
distribution have been proven (Allison et al., 1992).

When this bacterium was inoculated in the ru-
men of animals which were not adapted to the con-
sumption of leucaena, the toxic effects were reduced
(Palmer et al., 2010). Nevertheless, in spite of the
inoculation, the animals from different latitudes (for
example, Australia, Indonesia and Ethiopia) can only
tolerate up to30 % of leucaena in the diet (Dalzell
et al., 2012). In such cases, the hypothesis that the
populations of rumen bacteria capable of degrading
mimosine to DHP are saturated by the high levels of
leucaena (mimosine) ingestion, has been stated.
It is known that the bacteria S. jonesii has the
capacity to degrade 3-4 and 2-3 DHP; however, its
presence or absence has not been confirmed in the
animals fed leucaena in other places where this
plant is native. In addition, it is not known whether
some bacteria have the capacity to degrade mimo-
sine, because the above-mentioned ones only have
the capacity to degrade 3-4 and 2-3 DHP, but not
mimosine (Jetana et al., 2012). Also, Dalzell et al.
(2012) suggested that other bacteria with degrading
capacity for mimosine and its metabolites can exist.
Mimosine degradation is ascribed to a type
of mimosinase, enzyme which is found in the leu-
caena leaf; although the activation mechanism is
not known, there is the belief that at the moment
of chewing the leaf this enzyme is activated, when
mimosine is hydrolyzed and 3-4 DHP is produced
(Pereira et al., 2013). Reports have also been made
about the hydrolytic activity in the rumen of the
animals fed with leucaena; thus, it is assumed that
there are some bacteria which could degrade mi-
mosine (Kudo et al., 1984). Along with the above
explained facts, the intoxication by mimosine is
believed to be scarce, because the animals are ca-
pable of regulating the intake of L. leucocephala,
with which they avoid the possibility of intoxication
(Bacab et al., 2013).
The statements by Kudo et al. (1984) and Dalzell
et al. (2012) about the idea of finding more bacteria
responsible for the degradation of the most wide-
ly known toxics of L. leucocephala, were recently
concreted. In this sense, in 2015 a study revealed
four bacterial isolates (Streptococcus lutetiensis,
Clostridium butyricum, Lactobacillus vitulinus and
Butyrivibrio fibrisolvens) which could completely
degrade mimosine within the seven days of incu-
bation. It was also observed that C. butyricum and
L. vitulinus were capable of partially degrading 2,3
DHP within the 12 days of incubation, while S. lu-
tetiensis was capable of completely degrading 3,4
as well as 2,3 DHP (Derakhshani et al., 2016).
Conclusions
Mimosine which is found in the rhizosphere
of L. leucocephala is beneficial as it is captured by
certain Rhizobium strains, which favors mimosine
degradation and nitrifying nodulation. Thus, a se-
lective isolation of these strains could contribute to
the elaboration of specific inoculants for intensive
L. leucocephala crops.
The DHP degradation activity by the bacteria
mentioned in this review confirms the importance
of the catabolic processes that occur in the rumen,
for the tolerance of ruminants to the secondary me-
tabolites of the plants.
An exhaustive tracing of the rumen content
of livestock grazing L. leucocephala is needed, in
order to identify other mimosine-transforming bac-
teria and to contribute to the development of most
effective inoculants which could be used by farmers
against mimosine toxicity.
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Received: April 4, 2017
Accepted: October 29, 2017

Pastos y Forrajes, Vol. 40, No. 4, October-December, 249-258, 2017 / Álvaro Noguera-Talavera 249
Scientific Paper
Soil macrofauna as indicator of agroecological conversion of a productive
system of Moringa oleifera Lam. in Nicaragua
ÁlvaroNoguera-Talavera,NadirReyes-Sánchez,BryanMendieta-AraicaandMarthaMiriamSalgado-Duarte
Universidad Nacional Agraria km 12 ½ Carretera Norte, Apdo. 453, Managua, Nicaragua
E-mail: nogueratalavera@yahoo.es
Abstract
In order to determine the diversity and functionality of soil macrofauna as biological indicator of soil health and
the effect of management practices in productive systems of Moringa oleifera Lam., a study was conducted in areas of
the National Agricultural University, Nicaragua. The essays lasted nine months, in which agroecological management
and conventional management practices were implemented. The soil macrofauna was sampled through the methodology
developed by the International Tropical Soil Fertility and Biology Program. Taxonomic identification at phylum, class,
order and family level was performed, as well as of functional groups: detritivores, soil engineers, herbivores and
predators. The non-parametric Kruskal-Wallis test was applied to determine statistical differences in the variation of
density per taxon and functional group per management system. The density of individuals was statistically different
(p < 0,05) between management systems at class, order and family level. Diversity was higher in agroecological
conversion, and a higher equitability of families stood out. The functional groups were different between systems,
with dominance of soil engineers (64,22 %) in agroecological conversion, and of detritivores (74,19 %) in conventional
management. Association was found of the management practices with the density and diversity of the soil macrofauna;
and the organisms from the families Formicidae and Termitidae and order Coleoptera, which interacted with M. oleifera
in different development stages, were identified. The number of taxonomic units constituted an indicator that allowed
to distinguish between the management systems, soil health and transformation dynamics of the chemical, physical and
biological properties of each system.
Keywords: biodiversity, soil management, indicator organisms
Introduction
Conventional agriculture is contextualized in dif-
ferentstudies(Nietoetal.,2013;Altierietal.,2015;Hatt
et al., 2016) as the causative factor of the current envi-
ronmental crisis, which leads to the low sustainability
of agriculture (Gliessman, 2013), soil degradation, loss
of biodiversity due to simplification of the ecosystems
(Hatt et al., 2016; Altieri et al., 2017) and the distur-
bance of the hydrological cycle; in this sense, agro-
ecology, as science with multidisciplinary approach
(Nieto et al., 2013), represents the resurfacing of so-
cially, environmentally and culturally sustainable pro-
ductive systems, through the promotion of ecological
processes beneficial for the soil, water conservation
and biodiversity management (Altieri et al., 2015; Al-
tieri et al., 2017).
In the transition towards ecological agriculture,
a predominant principle, according to the report by
Blanco et al. (2013) and Nicholls et al. (2016), is the
improvement in the transformation of the physical
and chemical properties and in the biological
functionality of the soil (Matienzo-Brito et al.,
2015), because the capacity of a crop to withstand
or tolerate the attack of pest insects and diseases is
linked to the biological properties of this resource
(Nicholls and Altieri, 2008).
According to Navarrete et al. (2011), the ability
of the soil to support biological productivity should
be evaluated based on its specific functionality,
because it integrates the biological, chemical and
physical components in certain management situations,
which suggests a relation between biodiversity and
productivity. These authors refer that the evaluation of
soil quality allows to understand the degree to which
management practices contribute to sustainability.
From this approach, the soil macrofauna is an
indicatorfortheappraisaloftheefficiencyofsustainable
agriculture.
The soil macrofauna groups invertebrates larger
than 2 mm of diameter (Cabrera-Dávila, 2012) and
shows the following characteristics: sedentary
habit, short-term variability in their diversity and
population size (Cabrera-Dávila, 2014), a short period
between generations, high density and reproduction
capacity which allow intensive sampling. Such
characteristics permit it to be used as monitoring
and diagnosis indicator of the soil use intensity

250 Pastos y Forrajes, Vol. 40, No. 4, October-December, 249-258, 2017 / Soil macrofauna as indicator of agroecological conversion
(Díaz et al., 2014), its conservation or disturbance
status and health (Cabrera, 2012; Vieira da Cunha et
al., 2012); as well as of the effect, in time, of productive
practices (Socarrás and Izquierdo, 2014; Gómez et
al., 2016), which is related to the report by Cabrera-
Dávila (2014) about a higher variety and quantity of
organisms in the soils with adequate management.
The objective of this study was to determine
the diversity and functionality of soil macrofauna
as biological indicator of soil health, in productive
systems of Moringa oleifera Lam.
Materials and Methods
Geographical location and edaphoclimatic
characteristics of the study area
The study was conducted between June, 2013,
and March, 2014, in the experimental unit San-
ta Rosa of the National Agricultural University,
geographically located in Managua, in the coordi-
nates 12o
09’ 30.65” N and 86o
10’ 06.32” W, at an
altitude of 50 m.a.s.l. (INETER, 2015)
The recorded historical annual mean rainfall
and temperature are 1 099 mm and 27 ºC, respec-
tively, and the relative humidity is 74 % (INETER,
2015); with predominance of two seasons defined
by a dry season from November to April and a rainy
season from May to October.
In 2013 the annual rainfall in the area was
1 070,4 mm (fig. 1), lower compared with the
historical mean; June (285,3 mm) and September
(330,2) were the months with higher rainfall
(INETER, 2015).
The soil belongs to the taxonomic order Ando-
sol; due to its textural class it is sandy loam-clayey,
and it has good drainage.
Description of the experiments
The essays lasted nine months, with establish-
ment date in June, 2013, and harvest date in March,
2014, time during which agroecological manage-
ment and conventional agriculture practices were
applied.
The area with agroecological management
corresponded to a one-hectare lot, and the
agroecosystem with conventional management
consisted in a plantation area, with an extension of
5 ha; in both cases an effective sampling area of
0,18 ha was used. In each system four rectangular
sampling units were delimited (15 x 30 m). The defined
method for sampling the soil macrofauna was
systematicwithmonolithsseparatedat15mbetweenthem,
distributedindiagonaltranseptwithinthesamplingunits.
Adjacent vegetation with regards to the
management systems
• Agroecological conversion. The agrosystem
complementary flora was composed by living
fences with such tree species as Eucalyptus ca-
maldulensis (Dehnh.), Azadirachta indica (A.
Juss.), Cordia dentata (Poir.), Pithecellobium
dulce (Roxb.) Benth., Albizia saman (Jacq.)
Merr. and Stemmadenia obovata (Hook. & Arn.)
K. Shum.; while the surrounding lots correspon-
ded to agricultural land use with Moringa oleife-
ra (Lam.) and Sorghum bicolor (L.).

• Conventional management. The complementary
flora was constituted by living fences with tree
species such as A. indica (Neem), Spondias sp.
and A. saman; while the surrounding lots corres-
ponded to pasture production, like CT-15 and
Cynodon nlemfuensis (Vanderyst.), in addition
to such crops as Saccharum officinarum (L.), M.
oleifera and S. bicolor (L.).
Experimentaldesignandtreatments.Thedesign
consisted in experimental units of rectangular shape,
randomly selected. Each unit had plants established at
a distance of 3 x 3 m, in lineal arrangement.
• Treatment 1. Conventional management, which
consisted in an area of M. oleifera monocrop, with
activities of mechanized soil preparation, mecha-
nized and chemical weed control, inorganic ferti-
lization and irrigation.
• Treatment 2. Agroecological conversion, whose
approach was the establishment of a polycrop sys-
tem, minimum tillage in soil preparation, legume
rotation, organic fertilization with compost, in-
corporation of green manures, without irrigation,
and weed control with legume cover.
Evaluations. Collections were made of the soil
macrofauna before the harvest or the system ex-
ploitations. The collection period was between De-
cember, 2013, and January, 2014, at the end of the
rainy season, defined from the criteria proposed by
Cabrera et al. (2011).
For the collection the methodology proposed by
the International Tropical Soil Biology and Fertility
Program (Lavelle et al., 2003) was used. From each
management system 12 soil monoliths of 25 x 25 cm
were extracted, up to a depth of 30 cm.
The collected specimens were deposited in
vials with alcohol at 70 %, and were later identified
at phylum, class, order and family level, through the
use of keys developed by Roldan (1988) and Castner
(2000), in the biology laboratory of the School of
Natural Resources and Environment.
The macrofauna was classified into detritivores,
herbivores, soil engineers and predators, according to
the functional groups proposed by Cabrera et al. (2011).
The results corresponded to the indicators:
density (individuals per m2
) of each identified taxon
and functional group, and diversity per management
system; diversity and composition at family level
were analyzed as indicators related to the soil
health, according to Rendón et al. (2011), and their
interaction to the M. oleifera crop.
For the evaluation of management systems,
from the set of monoliths two samples were collect-
ed composed by 2 kg of soil and the chemical and
physical properties were determined in the soil and
water laboratory (LABSA) of the National Agricul-
tural University (table 1).
Experimental procedure. The composition of the
area with agroecological management was constituted
by a M. oleifera plantation with density of 1 111 plants,
managed in polycrop with rotations of Canavalia
ensiformis (L.), Canavalia brasiliensis (Mart. ex Benth.),
Cajanus cajan (L.) Millsp., and Vigna unguiculata (L.)
Walp.
The establishment and management consisted
in minimum tillage, manual weed control, organic
fertilization with compost (N: 35 %; P: 0,22 ppm; K:
0,86 meq/100 g of soil; Cu: 96 ppm, and humidity:
32,07 %); 0,03 kg were applied to each plant at the
moment of sowing and six months later, for a total
application of 33,3 kg/ha, incorporation of legumes
and harvest and weed residues, and weed and insect
management through legume cover.
The conventional system corresponded to a
M. oleifera plantation in monocrop with density of
1 111 plants/ha. The establishment and management
were defined by a set of practices, such as mechanized
Table 1. Chemical and physical properties of the soil in two management systems of M. oleifera Lam.
Soil property Conventional management Agroecological conversion
pH 6,54 6,58
OM (%) 3,11 4,40
N (%) 0,14-0,16 0,16-0,22
P (ppm) 35,70 ND
Humidity (%) 48,60 43,87
Textural class Sandy loam Sandy loam clay
ND: not detected, pH: soil-water ratio 1:25, Hester (1930) en AOAC (1980). OM: humid combustion of Walkey and
Black (1934), in AOAC (1980). N: Kjendalh (1883) in AOAC (1980). P: OLSEN (1954) in AOAC (1980). Texture:
Bouyoucos hydrometer (AOAC, 1980). Humidity: drying in oven and weight difference AOAC (1980).

tillage in the soil preparation (weed control with
weeder); soil turning, which consisted in three harrow
activities; and soil breakup between 20 and 30 cm of
depth for furrow elaboration, with mechanical sub-
soil plow.
Inorganic fertilization was applied (N:15-P: 30-
K:10 at a rate of 50 kg/ha) at the moment of sowing
and after one year of establishment of the agrosys-
tem; besides, sprinkler irrigation, pruning prac-
tices, pest control through inorganic chemicals
(Cypermethrin 100) were used and weed control
with weeder, with a frequency from two to three
times during the essay, in addition to chemical con-
trol (herbicide 2-4-D and glyphosate).
Statistical processing. Non-parametric statis-
tics (Kruskal-Wallis) was used to determine dif-
ferences in the variation of density per taxon and
functional group per management system. As part
of the macrofauna diversity, at management system
level for the taxa, classes and orders, the domi-
nance index of the community (D) was determined
through the method proposed by Turner and Gar-
ner (1991), with t-Student comparisons in the PAST
program version 1.29.
A diversity analysis was made by the cluster
method, to determine the probability of similarity
of families per management system and Jaccard in-
dex. This index expresses the degree in which two
or more samples are similar due to the species present
in them, and it was used for the family level.
Results and Discussion
The system agroecological conversion recorded
thehighesttaxonomicdiversityofthesoilmacrofauna,
with three phyla, five classes, nine orders and 19
families; compared with a phylum, three classes,
two orders and four families identified in the
conventional system (table 2).
Thedensityofindividualswasstatisticallydifferent
(p < 0,05) between management systems; 7 424 ind/m2
were recorded in agroecological conversion, compared
with 1 984 ind/m2
in conventional management (table
2). This result coincides with the ones reported by Díaz-
Porres et al. (2014) and Matienzo-Brito et al. (2015),
who reported differences in the density and diversity
of the soil macrofauna between diverse systems and
simplified animal husbandry systems, with regards
to intensive cropping systems, as consequence of the
complexity and management of the systems. These
authors found that with higher diversification there
was higher biological activity, and this applies in this
study to the agroecological conversion system.
At class level, the diversity components showed
higher total density in agroecological conversion,
associated to the dominance of specimens of the
taxonomic group Insecta, which was expressed in
significant differences (p < 0,05) in the communi-
ty dominance index (D = 0,68); the dominance of
the class Insecta was also observed in conventional
management (D = 0,59, table 3).
Rendónetal.(2011),whenanalyzingthedominance
of the phylum Arthropoda, and within it of the
class Insecta, explained that its reproductive,
feeding habits and its distribution and ecological
intervention at soil level make it useful as biological
indicator of the status of such resource.
The richness of classes was higher in agroeco-
logical conversion (five classes), compared with
conventional management and, thus, low similarity
probability was determined (J = 0,16) between the
management systems.
Sheibani and Gholamalizadeh (2013) reported
that the soil turning during tillage has effects on the
physical-chemical indicators that promote the func-
tional diversity of the macrofauna; while Ayuke et
al. (2009) and Díaz-Porres et al. (2014) associated the
low diversity of the soil macrofauna to such manage-
ment practices as the use of agrochemicals and the
modification (simplification) of the habitat when es-
tablishing M. oleifera in monocrop, elements which
allow to explain the low equity values recorded.
The lower diversity in agroecological conver-
sion, compared with the results reported by Ayuke
et al. (2009), was ascribed to the age of the system.
In this regard, Nicholls et al. (2016) stated that pro-
ductivity based on the functional diversity tends
to be low during the first three to five years in di-
versification schemes, compared with conventional
management, to be later increased due to efficient
designs regarding facilitation relations among
crops, which contributes not only to the increase of
diversity, but also to its functionality in favor of the
system.
The above-explained fact is an indicator of the
progressive increase in the diversity values as part
of the evolution of the system, according to Nicholls
et al. (2016), who recorded decrease of diversity
after a few years of starting the practices based on
organic inputs, crop rotation and incorporation of
legumes.
In this sense, it is stated that biodiversity in agri-
culture differs among agroecosystems, which in turn
differ in indicators relative to establishment time,
constitution of the species and sustainable practices.

Table2.Taxonomicclassification,trophicgroupsanddiversityofsoilmacrofaunaorganismsintwomanagementsystems.
ManagementsystemPhylumClassOrderFamilyDensity(ind/m2
)Relativedensity(%)Trophicgroup
Agroecologicalconversion
AnellidaOligochaetaHaploxidaLumbricidae3524,74Engineer
Arthropoda
Arthropoda
MalacostracaIsopodaArmadillidiidae4325,81Detritivore
DiplopodaJulidaJulidae961,29Detritivore
Insecta
ColeopteraElmidae
Chrysomelidae
Curculionidae
Dermestidae
Scarabaeidae
Hydrophilidae
Ptilodactylidae
176
16
16
16
576
16
448
2,37
0,22
0,22
0,22
7,76
0,22
6,03
Detritivore
Engineer
Engineer
Detritivore
Herbivore
Detritivore
Detritivore
DipteraDrosophilidae
Noctuidae
16
16
0,22
0,22
Detritivore
Herbivore
Hymenoptera
Isoptera
Formicidae
Vespidae
Termitidae
784
32
3600
10.55
0,43
48,49
Engineer
Herbivore
Engineer
Mollusca
GastropodaMesogastropodaPhysidae
Planorbidae
Thiaridae
16
16
352
0,22
0,22
4,74
Detritivore
Detritivore
Detritivore
GastropodaLittorinimorphaHydrobiidae4486,03Detritívoro
D=0,21a
H’=1,86a
J=0
∑7424a∑10064,22%SE
27,37%Det
8,41%Her
ConventionalArthropoda
Arachnida-Salticidae46423,39Predator
ChilopodaScolopendromorphaScolopendridae482,42Predator
InsectaTrichopteraHydropsychidae
Leptoceridae
272
1200
13,71
60,48
Detritivore
Detritivore
D=0,43b
J=0
∑1984b∑10074,19%Det
25,81
%Pre
D:dominanceindexatcommunitylevel,J:Jaccardindex,H’:Shannon-Wienerindex
SE:soilengineer,Det:detritivore,Her:herbivore,Pre:predator

Table 3. Density and diversity of classes and orders of the soil macrofauna in two management systems.
Taxonomic group
Management system
Agroecological conversion Conventional management
Class Density (ind/m2
) Diversity Density (ind/m2
) Diversity
Malacostraca 432 Dominance index
D = 0,68a
NR Dominance index
D = 0,59b
Insecta 5 712 1 472
Diplopoda 96 NR
Chilopoda NR Similarity index
J = 0,16
48 Similarity index
J = 0,16Gastropoda 832 NR
Arachnida NR 464
Oligochaeta 352 NR
Order Dominance index Dominance index
Haploxida 352 D = 0,33a
NR D = 0,93b
Isopoda 432 NR
Julida
Isoptera
96
3 600
NR
Coleoptera 1 264 NR
Diptera 32 NR
Hymenoptera
****
816
NR
Similarity index NR
464
Similarity index
Mesogastropoda 384 J = 0 NR J = 0
Littorinimorpha 448 NR
Scolopendromorpha NR 48
Trichoptera NR 1 472
Equal letters in the rows indicate not significant differences in the index value.
NR: not recorded, **** unidentified Arachnida order.
The class Insecta recorded the orders with
higher representativeness, in which Isoptera (3 600
ind/m2
), Coleoptera (1 264 ind/m2
) and Hymenop-
tera (816 ind/m2
) stood out for their high density in
the agroecological conversion management. These
orders, according to Ayuke et al. (2009), occupy
an important proportion at soil macrofauna level,
which confers a good transformation dynamics
of soil properties in this management system; in
contrast, in conventional management Trichoptera
(1 472 ind/m2
) showed the highest density.
Due to the higher density of Trichoptera, the
dominance index of the community recorded a
higher value (D = 0,93) in conventional manage-
ment, compared with agroecological conversion
(D = 0,33), with significant differences (p < 0,05)
between the systems; there was no probability of
similarity between management practices, because
they did not show common orders (table 3). The
low value of the dominance index in agroecological
conversion proved that the structure with different
strata, low-impact management and, thus, habitat
diversity and quality promote equity in the distri-
bution of the soil macrofauna and high potential of
interactions within the system.
The diversity of families was different between
systems. In that sense, the community dominance
(D = 0,43) was approximately double in conventional
management,comparedwithagroecologicalconversion
(D = 0,21); for which, in the latter management system,
equity in the density of individuals per family was
higher (H’ = 1,86 vs. 1,0; p < 0,05).
From the 19 identified families in agroecological
conversion, eight represented 94,15 % of the popu-
lation of the soil macrofauna (table 2); among which
Termitidae (48,49 %), Formicidae (10,55 %) and
Scarabaeidae (7,76 %) stood out. The dominance of
these families was reported by Ayuke et al. (2009)
and Cabrera et al. (2011), with representatives that
perform important functions within the agrosys-
tems, from the point of view of participation in the
regulation of the physical-biological dynamics of the

soil, as well as in their interaction with the crops,
mainly as pest organisms.
In conventional management, from the four
identified families, Leptoceridae represented 60,48 %
of the macrofauna population, followed by Saltici-
dae (23,39 %) and Hydropsychidae (13,71 %); while
Scolopendridae was the family with lower propor-
tion of individuals.
Different authors, among them Díaz-Porres et al.
(2014),provedthatthe incorporation of harvest waste,
especially when they have high nitrogen content
(legumes), promotes an increase in the density of
individuals. On the contrary, when in the system
there is high cover of pastures, which frequently
have high C/N ratio (Díaz-Porres et al., 2014), the
density is lower. The results of this study coincide
with the above-presented report, because in agro-
ecological conversion periodical incorporations
to the soil of legumes and pruning waste from M.
oleifera, whose leaves have high nitrogen content
(2 g in 100 g of fresh matter) were made; while in
conventional management, the soil cover was main-
ly star grass (C. nlemfuensis).
The distribution of families per functional group
was different between management systems with
predominance of soil engineers (64,22 %) and detri-
tivores (27,37 %) in agroecological conversion; and
of detritivores (74,19 %) and predators (25,81 %) in
conventional management (fig. 2).
Matienzo-Brito et al. (2015) stated that the num-
ber of functional groups differs due to the complexity
in the composition of the ecosystems, with advan-
tage for the diversified systems with management
of axillary biota; this allows to explain the presence
of groups with functions of biomass accumulation
and transformation, such as soil engineers and de-
tritivores. In that sense, Díaz-Porres et al. (2014)
concluded that the conditions with higher influence
on the diversity and functionality of the soil macro-
fauna groups are the organic matter content and the
carbon/nitrogen ration contained in the soil.
Association has been reported between the pre-
dominance of detritivores and the little intensive soil
use and, thus, with good organic matter content (fig. 2),
contributes complementarily to the function of soil
engineers, and confirms that the difference in the
composition of functional groups was associated to
the habitat conditions.
The proportion of detritivores confirmed the
organic matter content recorded in the soil analy-
sis (table 1) for both systems, as well as its quality
regarding the N content and decomposition status,
because representative species of the families Hy-
drobiidae, Hydrophilidae and Physidae are indica-
tors of sites with decomposing sediments (manure,
snail excreta and decomposing plant material).
Soil humidity (table 1) was a condition which,
according to Cabrera et al. (2011) and Matien-
zo-Brito et al. (2015), influenced the presence of cer-
tain organisms. This factor was assumed due to the
presence of snail families, such as Hydrophilidae,
Planorbidae and Leptoceridae, in conventional man-
agement (table 2); and of water coleopterans, like El-
midae, in agroecological conversion (table 2). Some

species show a distribution associated to flooded
sites or in water without current and to humid en-
vironments; for which they are useful as indicators
of soil humidity, of the decomposition degree of or-
ganic matter and, thus, of the available nutrients in
the system.
The effect of a high presence of detritivores is
related to their feeding activities, because, as part
of the trophic network in agrosystems, this func-
tional group increases the efficiency of the mobility
and acquisition of nutrients by the plants; this influ-
ences indirectly the presence of leaf-eating insects
and crop pests (Altieri and Nicholls, 2003), condi-
tion that is not desirable from the point of view of
crop health and productivity.
In practical sense, detritivores make efficient
the concentration of resources expressed in biomass,
organic matter and, thus, available nutrients for the
plants,forwhichtheconcentrationofresourcesattracts
more potential pest organisms; however, the activities
of diversification, crop rotation and promotion of
natural enemies contribute to the decrease of pests
and, thus, to the productive sustainability of the
systems.
Silva et al. (2012) concluded that the diversity of
predators, which in this study was composed mainly by
the classes Arachnida (Fam. Salticidae, 23,39 %), Ar-
thropoda (family Formicidae, 10, 55 %) and Chilo-
poda (family Scolopendridae, 2,42 %), indicates
availability of prey. Due to their epigeal habit, they
functionally contribute to the regulation of popula-
tions of potential pest species, condition which was also
reported by Díaz-Porres et al. (2014) in agricultural sys-
tems, compared with naturalized systems.
An important population of tailless whip scor-
pions was recorded in the agroecological conver-
sion system, as well as predators of the family
Formicidae, which influenced a high proportion of
herbivores, in spite of the existence of a high con-
centration of resources (diversity of legumes and
weeds, among others). This allows to prove that
the reduction of pest populations in agroecological
systems is a consequence of the nutritional changes
induced in the crop by organic fertilization, as well
as of the increase of natural pest controls (Altieri
and Nicholls, 2003).
The remarkable incidence of individuals of the
family Formicidae in agroecological conversion
was due to the presence of M. oleifera, legumes and
flowering weeds; this group, called soil engineers,
indicates particular conditions, such as the degree
of disturbance of the ecosystem and, in turn, the
potential interaction between crops and organisms.
Chávez et al. (2016) stated that Formicidae in-
dividuals are organisms of remarkable specific di-
versity, because they are commonly found in high
density and constitute useful indicators, because
they experience fast responses to different agricul-
tural practices.
On the other hand, Castro et al. (2008) reported
that high densities of Formicidae (leaf-cutter ants)
can move the same quantity of soil as earthworms,
this explains the fact that, in the management sys-
tems Solenopsis sp. and Camponotus sp. transport-
ed detritus originated by the decomposition of M.
oleifera and the legumes into the soil.
Regarding the interaction of M. oleifera with
Formicidae representative organisms, the plant
phenology, manifested in two profuse annual
flowerings, guaranteed the presence and function-
ality of this family; which was shown in activities
such as defoliation, which had higher incidence on
young plants, and the foraging of flowers in adult
plants, mainly in the dry season. The predation
of individuals of the Formicidae family by small
spiders of the family Salticidae was also observed,
with which one of the population regulation
mechanisms was identified.
As part of the functional group soil engineers, the
density of Termitidae in agroecological conversion
shows the conditions under which this system
was originated, regarding the predominance of
shrubby vegetation of forestry fallow, leading to
the concentration of branches and other organic
material, favorable for the proliferation of these
organisms.
The interaction of organisms from the Termitidae
family with M. oleifera was negative, because
Termitidae constituted a pest that quickly weakened
the plants of different ages and led to the dry rot of
branches and stems. Associated to the parasitism
of Termitidae on M. oleifera, Lepidoptera larvae
appeared which exerted herbivory, in addition to
Hydrobiidae that acted as detritivore.
The incorporation and continuous existence
of litter in the soil in the form of harvest wastes
increased the density of some Formicidae orders,
which transported them into the soil; and in the pro-
cess predation of other macrofauna organisms, such
as Termitidae and Coleoptera larvae, occurred. Other
predators, like Julidae and Scolopendridae, used the
galleries to exert their function, the former in the
comminution of plant remains (Chávez et al., 2016).

Contrary to the report by Leyva-Rodríguez et al.
(2012) and Cabrera (2012), humidity did not influence
the presence of Oligochaeta, although low abun-
dance of earthworm was observed in agroecological
conversion; this coincides with the values reported
by Matienzo-Brito et al. (2015) in diversified plots,
in systems of soil use conversion from conventional
to agroforestry, which showed low density at first
and an evident increase 10 years after conversion.
Individuals from this functional group were not re-
corded in the conventional management system.
Chávez et al. (2016) stated that the presence of
Coleoptera is important, because it participates in the
comminution of plant wastes, for which it is an indicator
of biomass and organic matter accumulation. The
presenceofherbivores,mainlyColeopteralarvae(family
Scarabaeidae, Phyllophaga ssp.), was associated to
different agricultural soil uses and, according to Leyva-
Rodríguez et al. (2012), to the incorporation of wastes
and to litter quality, as a product of the presence of trees
and shrubs with high protein levels in the systems.
The interaction between Scarabaeidae larvae and
adults with M. oleifera in conventional management
and with M. oleifera plus legumes in agroecological
conversion was different between the growth
stages, because in larval stage they fed from the
roots of young plants; while in adult stage the
species Cotinis mutabilis fed from the nectar of
M. oleifera and of the legumes, which contributed
to the pollination process.
Noctuidae, for being represented by larvae or
caterpillars that live on the soil and feed from leaves,
flowers, fruits and sometimes from seeds, represented
affectation risks for the M. oleifera crop in the initial
growth stage, but, on the other hand, some species in
adult stage participate in the pollination processes of
companion tree species and, thus, attract pollinators to
the system.
Conclusions
The number of taxonomic units, soil macrofauna
density and diversity constituted indicators that allowed
to distinguish between the agroecological management
and conventional agriculture of M. oleifera.
Likewise, the high proportion of individuals
from the functional groups soil engineers and de-
tritivores in agroecological conversion was an indi-
cator of good soil health, as well as of a remarkable
dynamics of physical-biological transformation.
In systems with conventional management, the
lack of individuals from the functional group soil
engineers originated a slow physical-biological trans-
formation, in spite of the high organic matter decom-
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Received: October 4, 2016
Accepted: September 28, 2017

Pastos y Forrajes, Vol. 40, No. 4, October-December, 259-263, 2017 / Seed characteristics of mulberry varieties 259
Technical note
Seed characteristics of five mulberry (Morus alba L.) varieties
harvested in Matanzas, Cuba♦
Jorge Jesús Reino-Molina1
, Laura A. Montejo-Valdés2
, Jorge Alberto Sánchez-Rendón2
and
Giraldo Jesús Martín Martín1
1
Estación Experimental de Pastos y Forrajes Indio Hatuey, Universidad de Matanzas, Ministerio de Educación Superior
Central España Republicana, CP 44280, Matanzas, Cuba
2
Instituto de Ecología y Sistemática, La Habana, Cuba
E-mail: jreino@ihatuey.cu
Abstract
The objective of the study was to characterize the morphological and physiological traits of the seeds of five Morus
alba L. varieties (cubana, tigreada, universidad, universidad mejorada and yu-62), which were harvested at the Pastures
and Forages Research Station Indio Hatuey –Matanzas, Cuba–. A completely randomized design was used, and the type
of embryo, seed size and mass, moisture content, allocation of biomass to the seed reserves (embryo-endosperm) and
desiccation tolerance index, were determined. Simple classification variance analysis was performed on the data. The
results indicated that the seeds had ovate to round shape; while the embryo was developed, of folded type. The fresh mass
varied between 1,30 and 1,46 mg, and most of the seed resources were aimed at reserve formation (between 62,3 and
68,1 %). The moisture content varied between 11,5 and 13,2 %, and the desiccation tolerance index was lower than
0,5; value which is in correspondence with that of orthodox seeds. It is concluded that the information obtained on the
biology of the M. alba seed is highly useful for the conservation of the germplasm bank of this species and for its sexual
propagation.
Keywords: plant embryos, moisture, seeds.
♦
This result corresponds to the project «Studies with Morus sp. for the development of sustainable human and animal feeding and
health technologies in Cuba», National Science and Technology Program «Feed production», of the Ministry of Science, Technol-
ogy and Environment, Cuba.
Introduction
Since the 90’s of last century, mulberry (Morus
alba L. Moraceae) is studied in Cuba for sustaina-
ble forage production, due to its high acceptability
by animals, and at present it is researched for its
outstanding medicinal and silk industry potential
(Martín et al., 2014). For such purpose, the coun-
try has a germplasm of 20 varieties preserved at the
Pastures and Forages Research Station Indio Hatuey
(EEPFIH), which were introduced from Costa Rica,
Ethiopia, Brazil, South Korea, China and Spain
(Martín et al., 2014).
This germplasm has been characterized, with
the aim of evaluating its growth and development
under the soil and climate conditions of different
zones of Cuba and for its inclusion in the sustain-
able technologies which contribute to biomass
production and the obtainment of bioproducts of
interest for human, animal and plant health (Martín
et al., 2014). However, if the mulberry crop is to be
intensively and effectively exploited in the country,
its seeds, as well as the germination mechanisms,
should be accurately characterized.
The sexual reproduction of this species through
seeds is essential to maintain genetic diversity. The
knowledge of their biology is an important tool in
order to be successful in the establishment of the
seedlings in agricultural and silvopastoral systems,
preserve a viable seed bank, make breeding programs
and face climate change (Jiménez-Alfaro et al., 2016).
Among the seed traits the morphophysiological
ones stand out, because they can be rapidly deter-
mined and have an important predictive value con-
cerning plant adaptation (Sánchez et al., 2015). In this
sense, Baskin and Baskin (2007; 2014) acknowledge
that the internal structure of seeds, particularly em-
bryo morphology, is a valuable piece of information
for the classification of seed dormancy. Meanwhile,
Jiménez-Alfaro et al. (2016) also state that seed size
and mass are vitally important traits in the life cy-
cle of a plant, because they have implications in the
dispersal, establishment and survival mechanisms of
the species. In addition, the hydration degree of dis-
seminules plays a fundamental role in their longevity
and germination performance.

260 Pastos y Forrajes, Vol. 40, No. 4, October-December, 259-263, 2017 / Seed characteristics of mulberry varieties
Nevertheless, other seed characteristics could
also show the responses of the species to the envi-
ronment; for example, the physical defense struc-
tures (testa/endocarp) and the nutrient content in
the seed reserves –embryo/endosperm– (Daws et
al., 2006; Montejo et al., 2015). For such reasons,
the objective of this study was to characterize the
different morphophysiological traits of the fresh
seeds of five mulberry varieties which were har-
vested at the EEPFIH.
Materials and Methods
Plant material. The evaluated fresh M. alba
seeds were from the genetic resource bank of the
EEPFIH. As treatments seeds of five varieties were
used: cubana, tigreada, universidad, universidad
mejorada and yu-62, which were harvested in March,
2014. A completely randomized design was used for
the trials, and they were performed immediately
after collection, in the seed laboratory of the Institute
of Ecology and Systematics –Havana, Cuba.
Seed characterization. The seed shape was
described according to Niembro (1988), and the
characteristics of the testa surface were detailed in
agreementwiththereportbyStearn(1992).Thedescrip-
tion of the embryo type was carried out based on
morphology (shape) and its degree of development
(size), through the embryo length-seed length (S)
relation, according to the classification criterion
proposed by Baskin and Baskin (2007). For such
purpose a sample of 30 seeds per variety was used,
from which the embryos were extracted with a sur-
gical scalpel. Afterwards, they were examined in a
stereoscopic microscope equipped with microme-
ter, to measure the embryo length (mm). It was con-
sidered that the seed showed non-developed embryo
(in terms of size) when it was small, but with differ-
entiated organs, and the relation between the embryo
size with regards to the seed (E-S) was lower than 0,5
mm. On the other hand, a seed was considered to
have fully developed embryo when it occupied more
than 50 % of the seed cavity (E-S > 0,5 mm) or the
whole seed cavity (Baskin and Baskin, 2007).
Afterwards, from each variety a sample of
100 seeds was randomly taken, in which the seed
dimensions were determined (length, width and
diameter) with a caliper (Mitutoyo, of 0,02 mm
accuracy). With these values the variance index of
the seed dimensions was calculated, according to
the method proposed by Thompson et al. (1993).
Before the calculation of variance, each seed size
value was divided by the length value, for the latter
to be equal to the unit. Thus, in a spherical seed the
variance is 0; while in an elongated or flattened one,
the variance can be up to 0,33.
The other seed variables studied were: to-
tal fresh mass (mg), total dry mass (mg), initial
moisture content (%) and dry mass of the reserves
(embryo-endosperm, mg). The fresh mass was de-
termined by individually weighing the seeds on
a scale (Sartorius, with 10-4
g accuracy). The dry
mass and moisture content were obtained from dry-
ing the seeds during 17 h, in a stove at 103 ± 2 ºC,
according to the regulations of the International
Seed Testing Association (ISTA, 2007). To calcu-
late the fraction (or allocation) of the seed dry mass
aimed at the seed reserves the value of this seed
component was divided by the total seed dry mass
(Sánchez et al., 2009), and the resulting values were
multiplied by 100 to facilitate data interpretation.
The desiccation sensitivity probability index, P
(D-S) was also determined, based on biometric data
of the seeds, according to the formula proposed by
Daws et al. (2006):
P (D-S)=
e 3,269-9,974a+2,156b
1+e 3,269-9,974a+2,156b
where a represented the seed mass fraction aimed
at the seed coats (MSC) and b is the log10
of the total
seed dry mass. Therefore: if P (D-S) > 0,5 it is proba-
ble that the seeds are desiccation sensitive; if P (D-S)
< 0,5 the seeds are likely to be desiccation tolerant;
while if P (D-S) = 0,5 the seeds have the same proba-
bility to be desiccation sensitive or tolerant.
Statistical analysis. All the quantitative data
were processed through a simple classification variance
analysis, and in the case of the data expressed in
percentage (moisture content and allocation to re-
serves) they were transformed with the arcsine of
the square root of the proportion. For such purpose
the program InfoStat v. 2015 (Di Rienzo et al.,
2015) was used, taking into consideration that the
fixed significance level was p ≤ 0,05.
Results and Discussion
The M. alba seeds which were subject to
analysis showed ovate to round shape; their color
was light brown, and the testa surface, granulated to
colliculate. Inside they showed a small layer of en-
dosperm which completely surrounded the embryo,
located in the central axis of the disseminule and
whose organs were differentiated; which allows to
state that it corresponds to a folded embryo (fig. 1),

Pastos y Forrajes, Vol. 40, No. 4, October-December, 259-263, 2017 / Seed characteristics of mulberry varieties 261
according to the characterization made by Baskin
and Baskin (2007). This, in turn coincides with
the characterization made by Baskin and Baskin
(2014), for the Moraceae family. The embryonic
axis was continuous, and the cotyledons, incum-
bent. The embryo size with regards to the inside
of the seed cavity (E-S) was 50 % higher; thus, it
is considered a developed embryo. This indicates
that the dormancy classes the species can present,
once the fruit is dispersed by the mother plant, are
the physical, the physiological one, or combinations
of these classes (Baskin and Baskin, 2014). In fact,
for the fresh and aged M. alba seeds it has been
reported that physical and physiological dormancy
can exist (Barbour et al., 2008), although they can
be non-dormant (Permán et al., 2013).
The length, width and diameter values of the
M. alba seeds did not show significant differences
among the varieties (table 1). The seed length varied
in a range from 2,15 to 1,77 mm, and the seeds were
thicker than wide. As the seed length was lower than
5 mm, this places the species in the smallest seed
size category (class A), proposed by Hladik and
Miquel (1990) for tree species. On the other hand,
the variance of the dimensions showed that the
seeds of all the varieties tended to be spherical, or
ovate to round, as commented above.
The total (fresh and dry) seed mass, moisture
content, biomass allocation to the seed reserves and
desiccation sensitivity index did not show signifi-
cant differences among the varieties (table 2). The
average value of the fresh mass of the varieties was
1,41 mg, which corresponds to the second category
of seed size (1,0-9,9 mg) proposed by Montejo et al.
(2015) for tree species.
The initial moisture content varied between 11,5
and 13,2 %, with an average of 12,3 %; these moisture
percentages were adjusted to the ones established for
species with orthodox or desiccation tolerant seeds
during storage (Dickie and Pritchard, 2002). In fact,
the orthodox behavior of M. alba seeds is known
(Permán et al., 2013; Royal Botanic Garden, 2015).
The values for the desiccation sensitivity in-
dex (< 0,5) also indicated that the seeds of the five
varieties could be tolerant to desiccation. Accord-
ing to Permán et al. (2013), mulberry seeds can be
maintained viable during two to three years under
common environmental conditions. Likewise, it
has been reported that in the Moraceae family more
than 50 % of the species show orthodox or desic-
cation tolerant seeds (Dickie and Pritchard, 2002).
The seeds of the five varieties allocated more
than 60 % of the total dry mass to the formation of
Table 1. Average values of the seed dimensions in the M. alba varieties.
Variety Length (mm) Width (mm) Diameter (mm) Variance of the dimensions
Cubana 2,06 (0,02) 0,96 (0,03) 1,63 (0,02) 0,07 (0,01)
Tigreada 2,07 (0,02) 0,92 (0,01) 1,58 (0,02) 0,07 (0,009)
Universidad 1,77 (0,02) 1,10 (0,02) 1,48 (0,02) 0,03 (0,01)
Universidad mejorada 1,83 (0,05) 1,03 (0,05) 1,46 (0,04) 0,04 (0,008)
Yu-62 2,15 (0,02) 1,00 (0,03) 1,65 (0,03) 0,07 (0,01)
( ): SE

262 Pastos y Forrajes, Vol. 40, No. 4, October-December, 259-263, 2017 / Seed characteristics of mulberry varieties
nutritional reserves (embryo/endosperm), as occurs
in other species of very small seeds (Sánchez et
al. (2009). This could guarantee that the seedlings
have a certain amount of resources to grow during
the first stages of their growth in nursery, phenomenon
recorded in seeds from Talipariti elatum (Sw.) and
Ceiba pentandra (L.) (Sánchez et al., 2009). In ad-
dition, a considerable quantity of resources in the
seed reserves could be an advantage for the beginning
of growth on nutrient-poor soils (Sánchez et al., 2015).
Some of these traits have been identified in
seeds from neotropical pioneer species, which grow
in tropical evergreen and semideciduous forests,
and also in M. alba seeds which grow in cultivated sys-
tems and wildly in semiarid Mediterranean zones
(Permán et al., 2013).
Conclusions
The M. alba seeds showed a developed folded
embryo, which occupied more than 50 % of the in-
side of the seed cavity, characteristic which showed
that they did not show morphological or morpho-
physiological dormancy; but they could exhibit
physiological dormancy, as occurs in other species
of the Moraceae family. On the other hand, the
seed mass values, moisture content and desiccation
sensitivity index were in the range reported for the
species with orthodox or desiccation tolerant seeds
during the storage. For such reason, the information
that was obtained about the biology of the M. alba
seed is highly useful for the conservation of the
germplasm bank of this species and for its sexual
propagation.
Acknowledgements
The authors thank Alejandro Gamboa for the
technical assistance in the laboratory.
Barbour, J. R.; Read, R. A. & Barnes, R. L. Morus
L. In: F. T. Bonner and R. P. Karrfalt, eds. The
Table 2. Average values of the morphophysiological traits of the seeds in the M. alba varieties
Variety
Fresh mass
(mg)
Dry mass
(mg)
Moisture
content (%)
Allocation to
reserves (%)
Sensitivity to desiccation
Cubana 1,46 (0,05) 1,28 (0,05) 12,3 (2,1) 68,1 (3,3) 0,0011 (1,6E-07)
Tigreada 1,29 (0,03) 1,15 (0,03) 11,5 (2,5) 64,8 (2,2) 0.0012 (1,6E-07)
Universidad 1,43 (0,07) 1,24 (0,06) 13,2 (1,9) 63,9 (2,8) 0.0010 (1,6E-07)
Universidad mejorada 1,40 (0,08) 1,22 (0,07) 12,8 (1,6) 67,8 (2,3) 0,0012 (1,6E-07)
Yu-62 1,48 (0,04) 1,21 (0,04) 11,9 (1,9) 62,3 (2,3) 0.0012 (1,6E-07)
( ): SE The percentage data are in correspondence with the original ones.
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