Philippine Journal of Science

142 (2): 153-165, December 2013

ISSN 0031 - 7683
Date Received: ?? Feb 20??

Arbuscular Mycorrhizal Fungi and Nitrogen Fixing Bacteria

as Growth Promoters and as Biological Control Agents Against
Nematodes in Tissue-Cultured Banana var. Lakatan

Nelly S. Aggangan1, Paul Jemuel S. Tamayao2, Edna A. Aguilar3,

Julieta A. Anarna1, and Teodora O. Dizon4

1
National Institute of Molecular Biology and Biotechnology,
University of the Philippines Los Baños (UPLB), College, Laguna 4031
2
Institute of Biological Sciences, UPLB, College, Laguna
3
Office of the Vice Chancellor for Research and Extension, UPLB, College, Laguna
4
Crop Science Cluster-Institute of Plant Breeding, UPLB, College, Laguna

Banana is one of the most important food items in the Philippines, ranked fourth among food crops
after rice, wheat and maize and being first among fruits. High yield of banana plantations requires
enormous amount of chemical fertilizers and pesticides.This study was conducted to determine the
potential of arbuscular mycorrhizal fungi (AMF) and nitrogen fixing bacteria (NFB) biofertilizers
as growth promoters and biological control agents against nematodes in tissue-cultured banana var.
Lakatan under screen house conditions. Meriplants were inoculated with AMF (MykovamTM) and
NFB (Bio-NTM) during planting in individual plastic bags filled with sterile soil sand mixture. Plant
parasitic nematodes, Radopholus similis and Meloidogyne incognita suspension were poured into the
soil, two months after inoculation with biofertilizers at concentrations of 1,000 and 5,000 larvae or
eggs per seedling, respectively. Plant height, pseudostem diameter and leaf area were taken every 2
weeks. At fourth month, the plants were harvested and extent of damage due to nematodes and the
number of colony forming units of NFB were determined. Results show that AMF and AMF+NFB
inoculated seedlings grew better than the control plants. AMF treated plants were taller, had bigger
pseudostem diameter, larger leaf area, highest fine, coarse root and total plant dry weights than the
control and the other treatments. Growth of plants infectedwith R. similis alone was comparable with
M. incognita and the control. M. incognita-infected plants had numerous root galls. Root necrosis
were observed only in R. similis-infected plants. AMF reduced root galls by 33% relative to those
inoculated with M. incognita. Percent mycorrhizal colonization was not affected by either NFB or the
nematodes. The roots of AMF+NFB gave the highest bacterial colony count (8 1.02 x 104) which was
reduced by AMF+NFB+ R. similis (5 0.80 x 103) and the lowest was with AMF+NFB+ M. incognita
(5 0.50 x 103). This implies that M. incognita and R. similis reduced the NFB bacterial colonies.

Key Words: Azospirillum, biofertilizers, Bio-N, Meloidogyne incognita, Mykovam,

Radopholus similis

INTRODUCTION growth and development for efficient water and nutrient

Banana is one of the most important fruit crops, being
ranked fourth among food crops after rice, wheat and
maize and being first among fruits (Molina 2005).
Banana production mainly depends on the plants’ root
*Corresponding author: nelly_aggangan@yahoo.com
uptake. Soil constraints such as water stress, mechanical
impedance, soil acidity, and the activity of soil-borne pests
and pathogens can reduce plant uptake of such essential
substances. Arbuscular mycorrhizal fungi (AMF) form
mutualistic or symbiotic associations with almost over
90% of plant species that help increase access water and
recycle nutrients (Haystead et al.1988, Smith and Read

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Vol. 142 No. 2, December 2013
2008, Gavito and Varela 1995). Also, other benefits
can be obtained such as improved growth, drought
tolerance, pathogen resistance and fitness against polluted
environments (Smith and Read 2008). The combination
of a pathogen-free planting material and a biocontrol
agent can represent new insight on banana cultivation and
improve plant growth without increasing the input cost.
Nematodes are the most important pests of banana
throughout the world (Stanton and Pattison 2000, Ploetz
2004). Plant parasitic nematodes such as the lesion nematodes
(Pratylenchus spp. and Radopholus similis), spiral nematode
(Helicotylenchus multicenctus) and root-knot nematode
(Meloidogyne spp.) contribute to damages that may suppress
plant growth and the activity of nitrogen fixation (Lordello et
al. 1997, Ibewiro et al. 2000). Chemicals, such as nematicides,
are used to control infestations; yet such protocols can
jeopardize human health and have adverse effects on soil
fertility (Gregory et al. 2005, Jorgenson and Kuykendall
2008). Alternatives to chemical operations are devised
with the use of organic methods to avoid such constraints.
Biofertilizers such as AMF and N-fixing bacteria (NFB) with
brand names MykovamTM and Bio-NTM, respectively, are now
commercially produced at the National Institute of Molecular
Biology and Biotechnology (BIOTECH), University of the
Philippines Los Banos (UPLB), College, Laguna.
MykovamTM is a soil-based biofertilizer comprised of
eight species of AMF. This biofertilizer is very effective
in increasing yield and survival of agricultural crops,
forest species, horticultural plants, forage crops and
fruit crops. Mykovam works very well in marginal soil
conditions (Abella 2012a). Abella (2012b) reported that
this biofertilizer benefited the fruit growers in Panabo
City, Davao Del Norte. In coconut, it was reported that
previous harvest by a coconut grower from Barangay
Kipalili, San Isidro, Davao del Norte, Mindanao was
three nuts to a kilo (Abella 2013). However, few months
after the application of Mykovam, nuts weighed as much
as 1.5 to 2.5 kg a piece. Root associations with AMF
not only improve plant growth and development, but it
can also be a biocontrol agent of soil pathogens such as
infestation of Fusarium wilt organism and nematodes in
tomato (Aggangan et al. 2000a, 2000b). AMF can reduce
the plant damages caused by nematodes (Jaizme-Vega et
al.1997). In a durian farm in the eastern part of Mindanao,
durian trees infested with Phytophthora (the most dreaded
pathogen of durian), was controlled by the application of
Mykovam biofertilizer (Abella 2012c).
Bio-NTM on the other hand, is also a microbial based
biofertilizer that supplies nitrogen and enhances the growth
of rice, corn and vegetables. This biofertilizer contains two
species of Azospirillum that were isolated from the roots of
Saccharum spontaneum L., which have been screened for
their effectiveness against a variety of agricultural crops.
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Aggangan et al.: AMF and NFB as Growth Promoters and as
Biological Control Agents Against Nematodes in Lakatan
It can supply at least 50% of the nitrogen requirements of
rice and corn (Garcia and Anarna 2006).
The potential of the interaction of AMF and NFB as
biocontrol agents and growth enhancers in a single host
was determined in the study in order to improve yield
without chemical inputs which have negative effects on
soil fertility. As a pioneer experiment in the Philippines
on the interaction of AMF (Glomus and Gigaspora) and
NFB (Azospirillum spp.) in the control of nematodes M.
incognita and R. similis, the study aims to determine the
potential of these biofertilizers as growth enhancers and
as biological control agents against these organisms in
banana production under screenhouse conditions.
MATERIALS AND METHODS
Experimental design
The experiment was conducted following a Randomized
Complete Block Design (RCBD) with 12 replicates. There
were nine treatments: Control, Meloidogyne incognita
(coded as Meloi) alone, Radopholus similis (coded as
Rado) alone, AMF alone, AMF+Meloi, AMF+Rado,
AMF+NFB, AMF+NFB+Meloi and AMF+NFB+Rado.
The experiment was conducted in ascreenhouse
at the National Institute of Molecular Biology and
Biotechnology (BIOTECH), University of the Philippines
Los Baños (UPLB), College, Laguna. The experimental
pots were rearranged twice a month to minimize the error
due to light intensity differences.
Variety of banana
Banana variety Lakatan was used because of its usage
and importance to farmers and agriculturists. It is the
most favoured banana variety for desert by Filipinos
and foreigners alike. However, this variety is highly
susceptible to R. similis and M. incognita infestation
(Dizon et al. 2006).
Preparation of banana var Lakatan meriplants
Meriplants were procured from the Lapanday Foods
Corporation based in Davao City, brought to the
mycorrhiza laboratory of BIOTECH, UPLB, College,
Laguna, and planted immediately in individual plastic
bags (10.16cm x 15.24cm). Newly planted meriplants
were incubated inside a 63.5cm x 101.6cm plastic bags.
This was done to maintain relative humidity that favors
faster plant recovery. Most small scale banana growers in
Mindanao prefer banana var. Lakatan as their crop unlike
the multinational banana growers who grow Cavendish
banana for export.
Philippine Journal of Science
Vol. 142 No. 2, December 2013
Species of nematodes
Two species of nematodes used in this study were:
Meloidogyne incognita (referred onward as Meloi) and
Radopholus similis (referred onward as Rado). The former
represent the gall-forming nematode and the latter represent
the burrowing nematode. Meloidogyne spp. are the most
important pests of several crops worldwide whereas R. similis
is a lesion-forming nematode that is one of the most important
root parasites in banana (Adriano-Anaya et al. 2011).
Soil collection and preparation
The commercial garden soil mixed with sand (1:1 v/v) was
used in this experiment. The soil was air dried, passed
in wire screen and oven sterilized for 3 days at 100oC.
The physical and chemical composition of the growing
medium are as follows: color is black, pH in water is
7.21 ± 0.01, total nitrogen is 326 ± 6 mg/kg, available
phosphorus is 68.72 ± 0.15 mg/kg, organic matter is 0.69
± 0.04% and the texture is loamy sand.
Preparation and inoculation with mycorrhiza
MYKOVAMTM biofertilizer is a soil based mycorrhizal
inoculant containing spores and chopped roots colonized
by AMF belonging to the genera Glomus and Gigaspora.
This mycorrhizal inoculant is mass-produced for 5 to 6
months at BIOTECH using bahia grass as trap plant and
grown in soil and sand mixture. Inoculation was done
during transplanting of meriplants.
Five grams of mycorrhizal inoculants were placed in a
1 to 2 inches deep hole made at the center of the plastic
bags filled with 1:1 oven sterilized garden soil and sand.
One meriplant of banana var Lakatan was then directly
seated on the mycorrhizal inoculant and later filled with
the same soil medium.
Preparation and inoculation with nitrogen fixing
bacteria
Nitrogen fixing bacteria (NFB) came from the
commercially available Bio-N TM biofertilizer also
produced by BIOTECH, UPLB. Bio-N consists of
Azospirillum spp. originally isolated from the roots of
talahib (Saccharum spontaneum L.) thriving in a marginal
grassland. There are two types of Bio-N being produced
at BIOTECH. These are Bio-N for corn and Bio-N for
rice. The latter is also effective in promoting growth and
yield of vegetables. Thus, its effectiveness on banana is
explored in this study.
The roots of newly out planted meriplants of banana var
Lakatan were dipped in the slurry of Bio-N for rice until
fully covered and later put in the plastic bags with the
Mykovam. The newly planted meriplants were placed in a
tray and incubated inside a bigger plastic bags for 2 weeks
Aggangan et al.: AMF and NFB as Growth Promoters and as
Biological Control Agents Against Nematodes in Lakatan
after which the seedlings were gradually exposed to direct
sunlight inside a screenhouse. One incubation plastic
bag consisted of 20 seedlings with the same treatment.
This was done in order to minimize cross contamination
among treatments.
Transplanting from plastic bags to bigger pots
After 2 weeks of incubation inside large plastic bags, the
potted banana meriplants were transferred into a larger
pots filled with 1 kg oven sterilized soil and sand. Plastic
pots were lined inside with plastic bags to prevent the
escape of nematodes that may contaminate other on-going
experiments inside the screenhouse. Filling up of pots
with soil and watering to field capacity were done by
weight. The pots were then placed on a steel bench inside
a screenhouse. Initial height and pseudostem diameter
were measured 2 weeks after transplanting.
Preparation and inoculation with nematodes
R. similis and M. incognita were provided by Dr. Dizon.
These nematodes were collected from the roots of banana
growing in Davao City and at UPLB, College, Laguna,
respectively. M. incognita was mass-produced in situ using
okra as host plant. Extraction and maceration of nematodes
from okra galled-roots was done at the Nematology
Laboratory of the Crop Science Cluster-Institute of Plant
Breeding using the sieving method (Jenkins 1964). On the
other hand, R. silimis was mass-produced in sterilized carrot
discs in sterile Petri plates incubated at 28oC following the
procedure of Moody et al. (1973).
In each nematode treatment, the rate of inoculation for M.
incognita was 5,000 eggs or larvae/plant while for R. similis
1,000 larvae or juveniles/plant (Carlier et al. 2003, Dizon
et al 2006). Nematode suspension was pipetted to deliver
a density of 1 nematode per g dry soil and added into the
soil in four equidistant holes, 3 cm deep adjacent to the
roots (Carlier et al. 2003), applied 5 weeks after inoculation
with AMF and NFB. This timing was chosen because most
nurseries sell their nursery raised seedlings to farmers at
this age. Thus, it is postulated that the seedlings being sold
are mycorrhizal and that these are already equipped with
protection against nematodes in the field if ever.
Parameters gathered
1. Plant Growth. Plant height and pseudostem diameter
were taken 1 cm above the soil surface with the use of a
ruler and a vernier caliper, respectively. Leaf length and
leaf width of the youngest fully expanded leaf (YFEL)
were also measured for the computation of leaf area. Leaf
area was computed as the product of leaf length and width
multiplied by 0.8 (Turner 2003). Periodic measurement of
growth was done every 15 days for a period of 16 weeks.
At harvest (4 months), fresh weight of secondary roots,
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Philippine Journal of Science Aggangan et al.: AMF and NFB as Growth Promoters and as
Vol. 142 No. 2, December 2013 Biological Control Agents Against Nematodes in Lakatan

primary roots, leaves and stem were separately measured.

Dry weights were obtained after 3 days in an oven set at
70oC.
2. Assessment of root damages due to nematodes. The
extent of root damage (percent root necrosis) due to R.
similis was determined in five samples. Nematode count
inside the roots was also obtained. For M. incognita, root
galls on the whole root system were assessed using the
following root galling index: 0 – no galls, 1 – trace to very
small galls, 2 - <25% of roots galled, 3 – 25 to 50% of
roots galled, 4–51 to– 75% of roots galled, and 5->75%
of roots galled. The number of juveniles and number of
laying females were counted from fresh root samples.
3. Assessment of mycorrhizal infection and Azospirillum
population in the roots. At harvest, 0.2g fresh secondary
root samples were taken per plant to assess mycorrhizal
root colonization. Mycorrhizal infections were assessed
microscopically after the roots were cleared and stained
(Phillips and Hayman 1970). Percent mycorrhizal root
infection was quantified following the gridline intersect
method (Giovanetti and Mosse 1980). Also, the colony
forming units (cfu) of Azospirillum spp. in the Bio-N
biofertilizer as affected by the presence or absence of
mycorrhiza or nematodes was determined.

Statistical analysis
All data collected were analyzed using one-way analysis
of variance (ANOVA) in RCBD. Treatment means were
compared using Duncan’s Multiple Range Test (DMRT)
and Least Significant Difference (LSD) at p<0.05 if
ANOVA showed significant effects. Percent mycorrhizal
infection data were arcsine transformed (Gomez and
Gomez 1984) before subjecting to ANOVA analysis.
RESULTS
General appearance of Lakatan seedlings: Banana
var Lakatan seedlings inoculated with AMF alone or in
combination with NFB grew better than the uninoculated
and those inoculated with the two nematode species
(Figure 1). In terms of leaf conditions and root system,
the seedlings inoculated with mycorrhiza alone developed
very extensive secondary roots over the control and even
with the presence of nematodes (Figure 1). Secondary and
tertiary roots developed more in the AMF inoculated plants
than the control plants (Figure 2). Galls were observed in
the roots of seedlings infested with M. incognita and were
not seen in the other seedlings (Figure 2).
Plant height: Height increment was significantly affected
by the different treatments throughout the duration of the
experiment from 8 weeks after transplanting until harvest
Figure 1. General appearance of 3-month old tissue cultured banana
var. Lakatan uninoculated (a) or inoculated with AMF
(b), M. incognita (c) or AMF+Meloi (d).
(16 weeks). At 6 weeks after transplanting, the control
and those inoculated with M. incognita gave the highest
growth increments (5.49 and 4.99 cm, respectively) which
were significant as compared with those treated with
AMF+Rado (3.21 cm) (Table 1). After 2 more weeks,
the highest (8.17 cm) height increment was obtained
from AMF+NFB+Meloi inoculated plants which was
significant as compared with the control (6.51 cm),
and those inoculated with M. incognita (5.9 cm) and R.
similis (5.15 cm). Height increments in the latter three
treatments were the lowest and not significant from each
other. Height increment due to AMF+NFB+Meloi was
consistent, highest from 8 weeks after transplanting until
harvest while the control and those inoculated with Meloi
alone and Rado alone gave the lowest. On the other hand,
height increment of plants inoculated with mycorrhiza
with or without M. incognita or R. similis were comparable
with each other.
Pseudostem diameter: Pseudostem diameter was
significantly affected by the different treatments at 6, 8

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Philippine Journal of Science Aggangan et al.: AMF and NFB as Growth Promoters and as
Vol. 142 No. 2, December 2013 Biological Control Agents Against Nematodes in Lakatan

Figure 2. Close-up of roots of 4-month old tissue cultured banana var. Lakatan uninoculated (a) or inoculated
with arbuscular mycorrhizal fungi (AMF) + N-fixing bacteria (NFB) (b), AMF + NFB+ Meloi (c) or
AMF+ NFB + Rado (d). Note galls formed in the encircled portions of roots of plant treated with AMF
+ NFB + Meloi (c).

and 16 weeks after transplanting (Table 2). At 6 weeks
after transplanting, AMF with or without nematodes
gave the highest (0.62 - 0.67 cm) pseudostem
diameter which were lower (p<0.001) than the other
treatments (0.28 – 0.44 cm). Pseudostem diameter
of plants inoculated with AMF+NFB+ either Meloi
or Rado was lower (p<0.001) than those with AMF
alone or AMF+Meloi or AMF+Rado. At 8 weeks after
transplanting, inoculation with AMF+NFB+Meloi
gave the highest measurement (0.85 cm) which was
significant (p<0.05) as compared with those inoculated
with AMF+NFB (0.65 cm), AMF+NFB+Rado (0.67

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Vol. 142 No. 2, December 2013 Biological Control Agents Against Nematodes in Lakatan

Table 1. Plant height increment (cm) of banana var. Lakatan as affected by arbuscular mycorrhizal fungi (AMF), N-fixing bacteria (NFB), and
Meloidogyne incognita and Radopholus similis measured every 2 weeks. n = 12.
Week after planting
Treatment
6 8 10 12 14 16
Control 5.49 a 6.51 bcd 7.52 abc 7.93 cd 8.37 cd 9.49 cd
Meloi 4.99 a 5.9 cd 7.23 bc 8.20 cd 8.62 bcd 9.18 cd
Rado 3.97 ab 5.15d 6.54c 7.17 d 7.93 d 8.34 d
AMF 3.84 ab 6.57 a-d 7.63 abc 9.36 abc 10.33 ab 11.26 abc
AMF+Meloi 4.12 ab 6.89 abc 8.82 ab 10.28 ab 11.12 a 11.93 ab
AMF+Rado 3.21 b 5.96 bcd 7.22 bc 8.51 bcd 9.72 a-d 10.04 bcd
AMF+NFB 4.21 ab 6.98 abc 7.70 abc 8.61 bcd 9.87 abc 11.03 abc
AMF+NFB +Meloi 4.38 ab 8.17 a 9.25 a 10.80 a 11.64 a 12.48 a
AMF+NFB +Rado 4.48 ab 7.59 ab 9.08 a 10.07 ab 11.61 a 12.16 ab
LSD 1.437 1.464 1.541 1.608 1.726 1.94
P value 0.012 0.00047 0.0064 0.0002 0.000 0.0002
AMF = Mykovam, NFB = Bio-N, Meloi = Meloidogyne incognita, Rado = Radopholus similis
WAT = weeks after transplanting; 2 WAT is same as 1 month after inoculation with mycorrhizal fungi.
Treatment means with the same letter(s) are not significantly different from each other using Duncan’s Multiple Range Test at p<0.05.

Table 2. Pseudostem diameter increment (cm) of banana var. Lakatan as affected by arbuscular mycorrhizal fungi (AMF), N-fixing bacteria
(NFB), Meloidogyne incognita and Radopholus similis measured every 2 weeks. n = 12.
Week after planting
Treatment
6 8 10 12 14 16
Control 0.44b 0.61b 0.78 0.85 1.01 1.14 bc
Meloi 0.38bc 0.57b 0.74 0.80 1.02 1.01 c
Rado 0.36bc 0.56b 0.70 0.81 1.04 1.06 bc
AMF 0.62a 0.70ab 0.83 0.92 1.11 1.25 a
AMF+Meloi 0.64a 0.75ab 0.87 0.91 1.13 1.31 a
AMF+Rado 0.67a 0.74b 0.87 0.91 1.14 1.24 ab
AMF+NFB 0.28c 0.65b 0.85 0.96 1.11 1.20 ab
AMF+NFB +Meloi 0.34bc 0.85a 0.91 1.05 1.21 1.32 a
AMF+NFB +Rado 0.31bc 0.67b 0.91 0.99 1.23 1.32 a
LSD 0.138 0.1738 0.205 0.218 0.1805 0.160
P value 0.00 0.027 NS NS NS 0.0013
AMF = Mykovam, NFB = Bio-N, Meloi = Meloidogyne incognita, Rado = Radopholus similis, NS – not significant
WAT = weeks after transplanting; 2 WAT is same as 1 month after inoculation with mycorrhizal fungi.
Treatment means with the same letter(s) are not significantly different from each other using Duncan’s Multiple Range Test at p<0.05.

cm) and the control (0.61 cm), Meloi alone (0.57 cm)
and Rado alone (0.56 cm). At harvest, no significant
difference was observed on stem diameter of seedlings
treated with AMF with or without Meloi or Rado.
Pseudostem diameter of seedlings inoculated with
AMF+NFB+Meloi or AMF+NFB+Rado were the
highest and comparable with each other. The lowest
(p<0.001) pseudostem diameter was in the control (1.14
cm), M. incognita alone (1.01 cm) and the R.similis
alone (1.06 cm).
Leaf characteristics: Four to 16 weeks after transplanting,
leaf area of AMF- and/or NFB-inoculated plants with or
without nematodes was broader than the uninoculated
control or those inoculated with M. incognita alone or
R. similis alone (Figure 3). Generally, plants inoculated
with AMF alone had the largest leaf area of the youngest
fully expanded leaf at 4 weeks and the total leaf area at 16
weeks. Area of the youngest fully expanded leaf and total
leaf area of plants inoculated with AMF with or without
NFB and AMF+NFB with or without nematodes did not
differ significantly from each other (Table 3). Leaf area

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Vol. 142 No. 2, December 2013
Figure 3. Leaf area of the youngest fully expanded leaf and total
leaf area per plant of banana var. Lakatan at 1 and 4
months after inoculation with arbuscular mycorrhizal
fungi (AMF), N-fixing bacteria (NFB) with or without
nematodes Meloidogyne incognita (Meloi) or Radopholus
similis(Rado). Bars represent LSD values at p<0.05.
of the control, M. incognita and R. similis treated plants
were 2 to 3 times smaller than those inoculated with AMF
(Table 3).
Plant fresh weight: In general, inoculation with AMF and
NFB significantly increased the fresh weights of leaves,
stem, coarse roots and fine roots in relation to the control
and the nematode inoculated seedlings (Figures. 4a and
5). Combining NFB with AMF reduced (p<0.01) the total
fresh weight, but the reduction was lessened with the
presence of M.incognita or R.similis(Fig. 4a).
Plant dry weight: The total dry weight of mycorrhizal
seedlings and those inoculated with both AMF and
NFB was significantly higher as compared with the
uninoculated control and the nematode-inoculated
Aggangan et al.: AMF and NFB as Growth Promoters and as
Biological Control Agents Against Nematodes in Lakatan
Figure 4. Total fresh (a) and dry (b) weights of banana var. Lakatan
seedlings as influenced by arbuscular mycorrhiza fungi
(AMF) and N-fixing bacteria (NFB) with or without
nematodes Meloidogyne incognita (Meloi) or Radopholus
similis (Rado). Bars represent LSD values at p<0.05.
plants (Figure 4b). Stem dry weight of banana var
Lakatan was highest (2.3 g plant-1) in plants inoculated
with AMF+Rado (Figure 5a) while AMF+NFB+Rado
gave the highest leaf dry weight (2.2 g plant-1).The
control plants gave the lowest stem and leaf dry weight.
AMF-inoculation gave the highest fine root dry weight
(Figure 5b).
Fine root dry weight was reduced from 70 to 85% when
AMF was combined with either of the two nematodes.
An intermediate (40%) reduction in fine root dry weight
was observed in AMF+NFB- treated plants. Inoculation
with R. similis increase fine root dry weight by 50% over
the control plants.
Coarse root dry weight was lowest (0.15 g plant-1) in
the control plants, doubled in R. similis- infected plants
and tripled in M. incognita- infected plants (Figure 5b).
Inoculation with AMF and AMF+Rado resulted in 10
times heavier coarse dry weight but this was significantly
reduced by 50-70% with the addition of NFB.
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Vol. 142 No. 2, December 2013 Biological Control Agents Against Nematodes in Lakatan

Table 3. Leaf area of the youngest fully expanded leaf of banana var. Lakatan as affected by arbuscular mycorrhizal fungi
(AMF), N-fixing bacteria (NFB), Meloidogyne incognita and Radopholus similis measured every 2 weeks. n = 12.
Week after planting
Treatment
6 8 10 12 14
Control 18.95cd 31.62b 36.68b 37.48b 53.65b
Meloi 23.29bcd 28.03b 34.72b 40.90b 56.16b
Rado 16.10d 24.55b 36.47b 41.96b 52.24b
AMF 35.08a 71.89a 95.72a 107.57a 135.36a
AMF+Meloi 33.33ab 72.64a 87.05a 115.56a 160.81a
AMF+Rado 28.23abc 73.79a 88.30a 101.54a 134.91a
AMF+NFB 31.19ab 80.99a 86.20a 103.21a 134.70a
AMF+NFB +Meloi 26.90a-d 69.09a 97.36a 110.03a 147.59a
AMF+NFB +Rado 31.05ab 81.22a 97.99a 112.95a 138.69a
LSD 10.04 12.05 16.82 16.16 25.15
P value 0.0027 0.000 0.000 0.000 0.000
AMF = Mykovam, NFB = Bio-N, Meloi = Meloidogyne incognita, Rado = Radopholus similis
WAT = weeks after transplanting; 2 WAT is same as 1 month after inoculation with mycorrhizal fungi
Treatment means with the same letter (s) are not significantly different from each other using Duncan’s Multiple Range Test at p<0.05.

Figure 5. Partitioned dry weights of shoot (a) and root (b) of banana var. Lakatan as influenced by arbuscular
mycorrhizal fungi (AMF) and N-fixing bacteria (NFB) with or without Meloidogyne incognita (Meloi)
or Radopholus similis (Rado). Bars represent LSD values at p<0.05.

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Vol. 142 No. 2, December 2013 Biological Control Agents Against Nematodes in Lakatan

Mycorrhizal root colonization: Nematodes and NFB

greatly reduced the root colonization by AMF in the banana
var Lakatan seedlings (Figure 6). Root colonization by
AMF alone was 100% but was reduced to 91% in the
AMF+NFB+Rado and 70% in the AMF+NFB- treated
plants. Root colonization by AMF was 15-20% only in
AMF+Meloi, AMF+Rado and in AMF+NFB+Meloi.
Control, M. incognita, and R. similis- treated seedlings
(originally not inoculated with AMF) remained uncolonized
by mycorrhizal fungi (Figure 6).

Figure 7. Number of egg laying females, juveniles and total

nematode count per gram of root samples of banana var.
Lakatan, 2 months after inoculation with Meloidogyne
incognita (Meloi) as influenced by arbuscular mycorrhizal
fungi (AMF) and N-fixing bacteria (NFB).

Nematode population as influenced by mycorrhiza and

Figure 6. Mycorrhizal colonization in the roots of banana var.
Lakatan as influenced by arbuscular mycorrhizal fungi
(AMF), N-fixing bacteria (NFB), Meloidogyne incognita
(Meloi) or Radopholus similis (Rado).
bacteria: Root necrosis was observed only in seedlings
inoculated with R. similis alone. No root necrosis was
observed in the other R. similis- inoculated seedlings. As
for the M. incognita populations, root gall index of 3 in
seedlings treated with M. incognita alone was higher as
compared to 2.5 in the AMF+NFB+Meloi seedlings. The
number of egg laying female M.incognita was significantly
reduced from 79 to 27 per gram root in the presence of
AMF (Table 4 and Figure 7). Juveniles and total nematode
count were also reduced in the presence of AMF.

Table 4. Effects of arbuscular mycorrhizal fungi (AMF) and nitrogen fixing bacteria (NFB) on root necrosis, root galling, population of
juveniles and laying females of Meloidogyne incognita (Meloi) and Radopholus similis (Rado) in the roots of 4-month old tissue
cultured banana var. Lakatan. n = 6.
Root Root galling No. of juveniles No. of laying female Total Meloi
Treatment/Sample*
necrosis index per gram root per gram root count
Control 0 0 0 0 0
Meloi 0 3 423 a 79 a 502 a
Rado 1.125 0 0 0 0
AMF 0 0 0 0 0
AMF+Meloi 0 2 50 b 27b 77 b
AMF+Rado 0 0 0 0 0
AMF+NFB 0 0 0 0 0
AMF+NFB +Meloi 0 2.5 41b 62 ab 103 b
AMF+NFB +Rado 0 0 0 0 0
LSD 1.73 284 37.31 314
P value NS 0.027 0.037 0.028
*Treatments considered in statistical analyses were Meloi, AMF+Meloi, AMF+NFB+Meloi.

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Philippine Journal of Science
Vol. 142 No. 2, December 2013
Figure 8. Colonies of nitrogen fixing bacteria (NFB) Azospirillum spp. as influenced by arbuscular mycorrhizal fungi (AMF) (a), AMF +
Meloidogyne incognita (b) and AMF + Radopholus similis (c).
Table 5. Number (with standard deviation) of colony forming units
of nitrogen fixing bacteria (NFB) Azospirillum spp. in the
roots of 4-month-old tissue cultured banana var. Lakatan as
influenced by the presence of arbuscular mycorrhizal fungi
(AMF), Meloidogyne incognita (Meloi) and Radopholus
similis (Rado). n = 6.
Total number of colony
Treatment
forming units/g
AMF+NFB 8±1.02 x 104
AMF+NFB+Meloi 3±0.50 x 103
AMF+NFB+Rado 5±0.80 x 103
AMF = Mykovam, NFB = Bio-N
Azospirillum spp. population as influenced by nematodes:
Azospirillum population was greatly affected by
nematodes. The highest (81.02 x 104 cfu/g) count of
Azospirillum colonies was observed in the AMF and NFB
treatment (Table 5 and Figure 8). The presence of R. similis
reduced the colony forming units from 8 1.02 x 104 to 5
0.80 x 103 while M. incognita effected the lowest colony
forming units of 5 0.50 x 103.
DISCUSSION
The results obtained in this study clearly show that,
mycorrhiza alone was effective in promoting growth of
tissue cultured banana var. Lakatan and that the roots
became 100% infected with mycelia of mycorrhizal fungi
Glomus etunicatum, G. macrocarpum and Gigaspora
margarita. Growth promotion of micropropagated
banana due to inoculation with mycorrhizal fungi was
reported by Jaizme-Vega et al. (1997) and Pinochet et al.
(1996). Enhanced plant growth was attributed to the early
mycorrhizal inoculation. In this experiment, inoculation of
banana plantlets as early as during potting out stage proved
162
Aggangan et al.: AMF and NFB as Growth Promoters and as
Biological Control Agents Against Nematodes in Lakatan
beneficial for plant growth. Increases in plant growth was
due to improved plant nutrient uptake brought about by
the symbiotic association of plant with AMF (Jeffries et
al. 2003). AMF also benefits crop productivity through
improved access to nutrients and water and suppression
of pest and diseases (Jefwa et al. 2010).
Colonization of plant roots by AMF has been shown to
reduce damage by soil-borne plant pathogens such as
nematodes. Plant parasitic nematodes are amongst the
most common pest constraints to banana production
(Gowen et al. 2005). The benefits of AMF in nematode
management on banana have been demonstrated. Elsen
et al. (2004) reported AMF suppression of R. similis
population density by almost 50% in pots. Likewise,
Fernándes et al. (2003) have shown that colonization
by Glomus intraradices, G. manihotis and G. mosseae
reduced nematode damage caused by R. similis and M.
incognita on banana in pots.
Many reports have demonstrated the effectiveness of
AMF as biocontrol agent against nematodes due to the
suppressive effects of the fungi on their reproduction
(Elsen et al. 2003, Umesh et al. 1988, Sohrabi et al.
2012). Andrade et al. (2009) reported that the hyphae
of mycorrhiza reduced the entry points of nematodes.
Elsen et al. (2008) suggested that in Musa sp., (Grand
Nine), AMF colonization induces systemic resistance of
the plant towards plant parasitic nematodes rather than
through direct competition or inhibition. In a split root
set up, seedlings became resistant to nematodes despite
spatially separating them with AMF. This mechanism
was also proven in other experiments involving bacteria
against cysts and nematodes (Hasky-Günther et al. 1998,
Munif et al. 2001, Siddiqui and Shaukat 2002). AMF
induced systemic resistance (ISR) to root-knot nematodes
involved a transcriptional control of VCH3, a class III
chitinase (Li et al. 2006). Other studies reported ISR as a
physiological state of enhanced defensive capacity by a
Philippine Journal of Science
Vol. 142 No. 2, December 2013
range of non-pathogenic microorganisms and biological
control agents (Bakker et al. 2007). ISR was demonstrated
to be acquired by the plant when involved in mycorrhizal
association (Vierheilig 2004).
Reduced galling and nematode population in AMF-
inoculated seedlings suggest that pre-inoculation with
mycorrhiza increased plant tolerance toward these
pathogens (Pinochet et al. 1997). De la Peña et al. (2006)
reported suppression of root infection and reproduction
of a root lesion nematode, Pratylenchus penetrans by the
native AMF. Further, pre-inoculation with AMF decreased
nematode root colonization and reproduction of this root
lesion nematode.
Increased tolerance of mycorrhizal plants to nematodes can
be accounted for by the enhanced plant nutrition induced
by the mycorrhizal fungi and the excretion of a suppressive
effect by the AMF over the nematode reproduction (Cooper
and Grandison 1987, Heald et al 1989). The mechanism
on enhanced nutrition is attributed to the carbon transfers
(Harja et al. 2013) to the roots of mycorrhizal plants from
the transfer of photosynthates to the fungal partner.
It has been reported that mycorrhizal fungi produce
substantial amount of external mycelia in the soil resulting
in an efficient acquisition and transport of nutrients and
water from the soil to the plant (Smith and Read 2008). In
non-mycorrhizal plants, attacks by nematodes can destroy
and weaken the root system resulting to lack of vigor and
ultimately poor fruiting. For mycorrhizal plants, because
of the extensive external mycelia in the soil, the plant can
grow vigorously that produce better fruits.
The control plants and those inoculated with M. incognita
and R. similis grew poorly with very small leaves and
few roots. Slight increase in root biomass was due to the
presence of M. incognita and R. similis. Greatest increases
in growth and biomass were obtained from inoculation
with mycorrhiza alone. Early inoculation with mycorrhiza
efficiently controlled nematode infestation as manifested
in the number of galls produced by M. incognita and the
number of juveniles per gram root sample.
Necrosis and root galls caused by nematodes limit
nutrient and water uptake and weaken plant anchorage
(Sasser and Freckman 1987). Reduced root branching
was also manifested in seedlings inoculated with these
soil borne pathogens. In the present study, inoculation
with nematodes in the AMF+NFB treatment has slightly
increased plant biomass. This shows once again the
potential of mycorrhizal fungi as biological control
against nematodes.
In conclusion, under screenhouse conditions, AMF present
in the soil-based mycorrhizal inoculant “MykovamTM”
increased plant growth, leaf area and dry weight of
Aggangan et al.: AMF and NFB as Growth Promoters and as
Biological Control Agents Against Nematodes in Lakatan
tissue cultured banana var. Lakatan comparable with
those inoculated with combined mycorrhiza+N-fixing
bacteria. Inoculation with AMF alone gave the highest
fine and coarse root dry weight. Nematodes did not affect
the growth and biomass of mycorrhiza inoculated plants.
All roots of mycorrhiza- treated seedlings became 100%
mycorrhizal. Mycorrhiza decreased root galling index and
total nematode population counts. Nematodes reduced the
percentage of roots colonized by mycorrhizal fungi and
the population of N-fixing bacteria Azospirillum spp. but
did not affect their effectiveness as plant growth promoter.
Mycorrhiza alone is the best treatment in promoting the
growth of banana var Lakatan seedlings and effective
biocontrol agent in controlling nematode population and
infestation. Early mycorrhizal inoculation could be an
effective nursery management approach to maximize the
plant growth promoting ability of mycorrhizal fungi and
to control losses due to nematode infestation and perhaps
for other soil pathogens.
ACKNOWLEDGEMENT
This study was partly funded by the DOST-PCARRD
funded project entitled “Improving production of banana
var Saba, Lakatan and Latundan cultivars in different
cropping systems”.
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