Journal of Agricultural Biotechnology and Sustainable Development Vol. 2(8) pp.

xxx-xxx, April, 2010

Available online http://www.academicjournals.org/JABSD
©2010 Academic Journals

Full Length Research Paper

Biofertilization of banana (Musa spp. L.) with free-living

N2 fixing bacteria and their effect on mycorrhization and
the nematode Radopholus similis
Adriano-Anaya ML1, Gutiérrez-Miceli FA2, Dendooven L3, Salvador-Figueroa M1*
1
Centro de Biociencias. Universidad Autónoma de Chiapas. Carretera a Puerto Madero Km 2.0 Tapachula, 30700,
Chiapas, México.
2
Departamento de Ingeniería Química y Bioquímica. Instituto Tecnológico de Tuxtla-Gutiérrez, Tuxtla-Gutiérrez,
Chiapas, México.
3
Laboratory of Soil Ecology, GIB, Dept. Biotechnology and Bioengineering, Cinvestav, México.
November 24, 2010

Banana plants (Musa spp. L.) cv 'Great dwarf' were amended with free-living N2 fixing bacteria (FLNFB)
and arbuscular mycorrhizal fungi (AMF) and the presence of the burrowing nematode Radopholus
similis was monitored in the field. Five treatments were applied by inoculating banana roots with four
strains of FLNFB, that is C1, C2, C3 and C4 isolated from the rhizoplane of the same banana cultivar, or
by keeping them uninoculated. The largest number of nematodes was found in the untreated roots and
the lowest in the roots inoculated with C4. The largest percent of mycorrhizal colonization was found
when banana roots were inoculated with C1 and the lowest in roots that were not inoculated. The
number of R. similis decreased with increased colonization with AMF. The largest percentage of
necrosis of the banana roots was found when banana roots were not inoculated and the lowest in roots
that were inoculated with C4. The necrosis of the roots increased with increased number of R. similis,
but decreased with increased colonization with AMF. It was found that inoculating the banana cv 'Great
dwarf' with FLNFB increased the root colonization with AML, and reduced the number of nematodes
and root damage.

Key words: Arbuscular mycorrhizal fungi, burrowing or banana-root nematode, free-living N2 fixing bacteria,
plant development.

INTRODUCTION

Plant development is closely related to the interaction
with microbiota in the phylosphere and rhizosphere
(Green et al., 2006). For instance, 80 and 92% of
surveyed plant species and families, respectively, were
mycorrhizal with arbuscular mycorrhizal fungi (AMF)
being dominant (Wang and Qiu, 2006). They suggested
that the origin of arbuscular mycorrhiza coincides with the
origin of land plants. In the rhizosphere, AMF and
pathogens need to be in equilibrium, so that a normal
*Corresponding author: E-mail: msalvad@hotmail.com, Tel. +52
(962) 6427972.
plant development can occur (Reyes and Valery, 2007).
The increase in pathogens populations leads to diseases
(Whipps, 2001) thereby hampering plant growth (Siddiqui
and Akhtar, 2008). The interaction in the rhizosphere
between plant and microorganisms has been studied
intensively (Mathesius, 2009) and the importance of
certain root exudates in the relationship between plants
and microorganisms, beneficial and pathogenic, has
been demonstrated (Rojas-Andrade et al., 2003).
Radopholus similis, the burrowing or banana-root
nematode, is one of the most important root parasites of
banana plants (Musa spp. L.). Although effective
chemical nematicides exist, but their high cost and
toxicity limit their use. Nematode management for
resource-poor farmers relies on the use of hot water-
treated suckers (Speijer et al., 1995), tissue culture-
derived planting material or mulching (Talwana et al.,
2003). None of these methods, however, offers complete
control of nematodes (Athman et al., 2006). The roots of
the banana, as those of most plants, are colonized by
AMF and the intensity of colonization depends on
fertilizer use and AMF species (Declerck et al., 2002).
The hyphae of AMF reduce the entry points of the
nematodes and induce physiological changes that
increase plant growth (Andrade et al., 2009). Studies
have shown that AMF might reduce the damage done to
plants by nematodes (Jaizme-Vega et al., 1997), but the
synergistic effect of soil fungi and nematodes might break
down the protection of AMF against pathogens
(Greipsson and El-Mayas, 2002). It is well known that
conventional agricultural practices might have a negative
effect on soil fertility due to the use of fertilizers, tillage
and pesticides (Gregory et al., 2005). Excessive fertilizer
use increases soil salinity, tillage disrupts soil structure
accelerating soil organic matter decomposition, while
pesticide might negatively affect beneficial organisms
(Jorgenson and Kuykendall, 2008). New agricultural
practices have been developed to reverse those negative
effects (Butler et al., 2007). The introduction of beneficial
microorganisms often through inoculation upon planning
of crops or biofertilization can improve yields without
negatively affecting soil fertility (Avis et al., 2008).
Application of organisms that are antagonistic against
pests or pathogens is another way to improve crop
development (Alabouvette and Steinberg, 2006).
Therefore, an alternative way to control R. similis, instead
of using a nematicide, is to inoculate roots with beneficial
symbiotic organisms so that through antagonisms or
simple competition the pathogen is prevented to infect
the plant (Van der Veken et al., 2008). The objective of
this work was to investigate the effect of biofertilizing
roots of the banana plant with four free-living N2 fixing
bacteria isolated from roots of the similar banana cv
'Great dwarf' and to determine how this affected the
colonization of the roots with AMF and the presence of R.
similis.
MATERIALS AND METHODS
Free-living N2 fixing bacteria
Four free-living N2 fixing bacterial strains C1, C2, C3 and C4
(Azospirillum spp.) were isolated from the rhizosphere of banana
plants cultures and selected for their high production of indole-3-
acetic acid (IAA).
Bacteria produce IAA under stress conditions promoting plant
growth and shoot water content and thus increase plant drought
tolerance (Marulanda et al., 2009). The isolated bacterial strains
were kept in nFB culture medium and cultivated in nutrient broth
(pH 7.0) at 200 rpm and at 30°C before inoculation.
Plantlets culture and inoculation
Nine hundred banana plantlets were cultivated in the greenhouse in
pots containing 5 kg soil from a banana field. The soil contained
320 g kg-1 sand, 410 g kg-1 loam and 270 g kg-1 clay. The plantlets
were fertilized with urea (10 g pot-1) 60 days after sowing. The
plantlets were divided in five groups. Hundred and eighty plants
were inoculated with 5 ml of each of the four bacteria (1 × 108 cells
ml-1) eight days after application of urea. The remaining 180
plantlets were not inoculated and served as control.
Field experiment
The plantlets were transferred to the ‘Santa Lucrecia’ farm (14° 40’
21.56” N; 92° 10’ 27.19” W), a banana plantation in Suchiate,
(Chiapas, México), 18 days after bacterial inoculation. Five 1000 m2
plots (180 plants plot-1) were outlined (one for each of the four
bacterial strains and one for control). Plots were arranged in a
complete randomized block design. Each plant was fertilized montly
with 20 g N, 72 g K, 11 g Ca, 4 g Mg and 2 g S. Eight days after the
fertilizer was applied, the roots of each plant were inoculated with
30 ml of the previously mentioned bacterial inoculums, i.e. four
different strains at 1 × 108 cells ml-1.
Sampling and assays
After 100, 128, 156, 184, 212 and 240 days, 10 banana plants were
selected at random and the rhizosphere collected (30 cm3 soil +
roots). The diazotrophic bacteria in the rhizoplane were determined
by cultivating them on nFB medium. Nematodes in the roots were
extracted with a Baermann funnel as described by Hooper et al.
(2005) and counted under a microscope. Nematodes were
identified as described by Hunt et al. (2005). The mycorrhizal
colonization was determined as described by Giovannetti and
Mosse (1980) on previous stained roots as described by Phillips
and Hayman (1970). The damage done by the nematodes to the
roots was visually determined in three categories: 1) undamaged
roots, 2) roots with < 50% damage and 3) necrotic roots with > 50%
damage.
Different phenological stages of the banana plants were defined
as follows (Soto, 1991). The infantile stage was defined as the time
between the bud outset and the first formal leaf formation and
lasted from 120 to 160 days. The juvenile stage was defined from
the first formal leaf to the floral differentiation stage and took
between 50 and 60 days, while the reproductive phase covered the
time between floral differentiation stage until the fruits were
collected.
Statistical analysis
The number of root nematodes, AMF colonization and root necrosis
was subjected to one-way analysis of variance (Tukey, p < 0.05) to
test for significant differences between the treatments, i.e. different
strains, with InfoStat Professional version 2008.
RESULTS
The number of free-living N2 fixing bacteria on the roots
8 -1
of inoculated banana plants was >10 g roots, while in
5 6 -1
the non-inoculated plants it was between 10 and 10 g
 Figure 1. The number of nematodes Radopholus similis (per 100 g roots), b) the percentage of arbuscular
mycorrhizal fungi colonization (%) and c) necrosis (%) of the roots of the banana plant (Musa spp. L.) cv
'Great dwarf' inoculated with four different free-living N2 fixing bacteria (C1 ( ), C2 ( ), C3 ( ), C4 ( )) and
left untreated ( ) grown in the field.

roots. The number of nematodes found on the roots
remained low until day 128 and started to increase
thereafter (Figure 1a). At day 240, the number of
nematodes found in the roots was significantly different
between the treatments (P<0.05). The largest amount
-
was found in the untreated roots (3618 nematodes 100 g
1
) and the lowest in the roots inoculated with C4 (969
-1
nematodes 100 g ). The colonization of the banana roots
with AMF increased with a maximum found between 128
and 156 days, and then decreased (Figure 1b). The
colonization of the banana roots with AMF was
significantly different between the treatments (P<0.05).
The largest percent colonization was found when banana
roots were inoculated with C1 (32%) and the lowest in
roots that were not inoculated (16%). The number of R.
similis decreased significantly and exponentially with
increased colonization of the roots with AMF (P<0.05)
(Figure 2a).
The necrosis of the banana roots decreased until day
184 in all treatments, except in the roots that were not
inoculated which showed an increase between day 100
and 128 (Figure 1c). After day 184, the number of roots
that showed necrosis increased in all treatments. The
necrosis of the banana roots was significantly different
between the treatments (P<0.05). The largest percentage
of necrosis of the banana roots was found when they
were not inoculated (26%) and the lowest in roots that
were inoculated with C4 (11%). The necrosis of the roots
increased significantly with an increased number of R.
similis, but decreased significantly with increased
colonization of the roots with AMF (P<0.05) (Figure 2b
and c).
DISCUSSION
Root colonization with arbuscular mycorrhizal fungi
(AMF)
The colonization of banana roots changed with time. In
 Figure 2. The correlation a) between the number of nematodes Radopholus similis (per 100 g roots) and
the percentage of arbuscular mycorrhizal fungi colonization (%), b) the number of nematodes Radopholus
similis (per 100 g roots) and necrosis (%) of the roots c) necrosis (%) of the roots and the percentage of
arbuscular mycorrhizal fungi colonization (%) of the roots of the banana plant (Musa spp. L.) cv 'Great
dwarf' inoculated with four different free-living N2 fixing bacteria.

cereals, AMF root colonization is related to the
phenological stage of the plant (Gavito and Varela, 1993;
Majic et al., 2008). Colonization increased during the
vegetative phase and decreased during the reproductive
phase. Photosynthetic products are transported to the
roots where they are used by the microorganisms in the
rhizosphere. The microorganisms proliferate and their
metabolites and/or released nutrients are taken up by the
plants. When the plant matures and the reproductive
phase starts, photosynthetic products are used for
flowering and seed development. The plant metabolites
released in the rhizosphere decreases, microbial growth
slows and the AMF loosen their association with cortical
cells.
The level of colonization with AMF found in this
experiment for banana plants not inoculated were less
than those reported. Adriano (2001) found a 58 to 88%
colonization with Glomus mosseae for the banana cv
'Great dwarf' during the reproductive phase. Jaizme-Vega
and Pinochet (1997) found a 47% colonization of roots of
the same banana cultivar with G. mosseae, 34% with G.
aggregatum and 33% with G. intraradices.
Population of Radopholus similis
Jaizme-Vega et al. (1997) reported that the banana cv
'Great dwarf' colonized by the AMF G. mosseae were
less susceptible to infections with the root-knot nematode
Meloidogyne incognita favouring plant growth. Contrarily,
Jaizme-Vega and Rodriguez-Romero (2004) reported
that the same banana plants inoculated with G. mosseae,
G. aggregatum and G. intraradices had the same number
of Pratylenchus goodeyi and M. incognita than the
untreated plants. However, Elsen et al. (2008) found that
AMF have the ability to induce systemic resistance
against plant parasitic nematodes R. similis and
Pratylenchus coffeae in the root system. AMF reduced
both nematode species by more than 50%, even when
the AMF and the plant parasitic nematodes were spatially
separated. Similar results were obtained with maize (Zea
mays L.), olive (Olea europaea L.) (Castillo et al., 2006)
and kiwi (Actinidia deliciosa) (Verdejo et al., 1990). The
mycorrhiza had neither effect on the number of
pathogenic nematodes nor was the number of AML
affected by the pathogens.
Root damage
Jaisme-Vega et al. (1997) reported a 36 to 64% reduction
in root galling caused by the root-knot nematode M.
incognita when micropropagated banana plants cv 'Great
dwarf' were inoculated with G. mosseae reaching 58 to
88% of AM root colonization. Jaizme-Vega and
Rodriguez-Romero (2004) found that banana plants cv
'Great dwarf' inoculated with G. mosseae, G. aggregatum
and G. intraradices, and infected with the nematode P.
goodeyi, had 4, 15 and 13% lesions, respectively, while
the plants that were not inoculated had 28%. The
colonization with G. mosseae was 47%, with G.
aggregatum 34% and with G. intraradices 33%. No
differences, however, in nematode populations were
found. Elsen et al. (2008) found that the number of
lesions in the roots of banana cv ‘Williams’ amended with
±1850 mycorrhizal spores and infected with 5000 eggs
and juvenile M. javanica one month later, were 29%
lower with G. mosseae, 25% with G.macrosporum and
16% with G. caledonium compared to plants not
inoculated with mycorrhizae. They suggested an
inhibitory effect of the micorrhiza on the infection with
nematodes through changes in the nutrition of the plant,
biochemical changes in the plant tissue, e.g. an increase
in aminoacids, peroxidases, quitinases, phytoalexins,
increases in lignin, stress reduction, changes in the
microbial populations in the roots and a more dense root
system. Vaast et al. (1998) found a decrease in lesions
by P. coffeae in the roots of coffee (Coffea arabica L.)
when inoculated with the mycorrhizae Acaulospora
mellea or G. clarum. Castillo et al. (2006) found similar
results when studying the effect of mycorrhiza on the
pathogenicity of M. incognita and P. vulnus on roots of
olive tree. Castillo et al. (2006) suggested that the
reduction in pathogenicity was not related to a reduction
in infection rate, but was related to a higher tolerance
towards the nematode.
Our findings conclude that inoculating roots of the
banana plant cv 'Great dwarf' with free-living N2 fixing
bacteria isolated from roots of the similar plant resulted in
a higher colonization of the roots with AMF, a lower
number of pathogenic nematodes and healthier roots.
ACKNOWLEDGEMENTS
We thank FOMIX CONACyT-Estado de Chiapas for
funding the research (Grant CHIS-2002-C01-4332).
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