Arbuscular Mycorrhizal Fungi and Nitrogen Fixing Bacteria ...philjournalsci.dost.gov.ph/pdf/pjs_pdf/vol142no2/pdf/Arbuscular... · Nematodes in Tissue-Cultured Banana var. Lakatan

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Philippine Journal of Science142 (2): 153-165, December 2013ISSN 0031 - 7683Date Received: ?? Feb 20??

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

*Corresponding author: [email protected]

Nelly S. Aggangan1, Paul Jemuel S. Tamayao2, Edna A. Aguilar3, Julieta A. Anarna1, and Teodora O. Dizon4

Arbuscular Mycorrhizal Fungi and Nitrogen Fixing Bacteria as Growth Promoters and as Biological Control Agents Against

Nematodes in Tissue-Cultured Banana var. Lakatan

1National Institute of Molecular Biology and Biotechnology,University of the Philippines Los Baños (UPLB), College, Laguna 4031

2Institute of Biological Sciences, UPLB, College, Laguna3Office of the Vice Chancellor for Research and Extension, UPLB, College, Laguna

4Crop 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.

INTRODUCTIONBanana 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

growth and development for efficient water and nutrient 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

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.

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 designThe 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 bananaBanana 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 meriplantsMeriplants 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.

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Philippine Journal of ScienceVol. 142 No. 2, December 2013

Aggangan et al.: AMF and NFB as Growth Promoters and as Biological Control Agents Against Nematodes in Lakatan

Species of nematodesTwo 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 preparationThe 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 mycorrhizaMYKOVAMTM 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 bacteriaNitrogen fixing bacteria (NFB) came from the commercially available Bio-NTM 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

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 potsAfter 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 nematodesR. 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 gathered1. 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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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 analysisAll 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.

RESULTSGeneral 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

(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

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).

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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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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

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.

TreatmentWeek after planting

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 similisWAT = 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.


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 significantWAT = 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.

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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.

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

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.

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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.


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 similisWAT = weeks after transplanting; 2 WAT is same as 1 month after inoculation with mycorrhizal fungiTreatment 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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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 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).

Nematode population as influenced by mycorrhiza and 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.

Treatment/Sample* Root necrosis

Root galling index

No. of juvenilesper gram root

No. of laying female per gram root

Total Meloi count

Control 0 0 0 0 0Meloi 0 3 423 a 79 a 502 aRado 1.125 0 0 0 0AMF 0 0 0 0 0AMF+Meloi 0 2 50 b 27b 77 bAMF+Rado 0 0 0 0 0AMF+NFB 0 0 0 0 0AMF+NFB +Meloi 0 2.5 41b 62 ab 103 bAMF+NFB +Rado 0 0 0 0 0LSD 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.

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).

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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.

Treatment Total number of colony 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

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).

Azospirillum spp. population as influenced by nematodes: Azospirillum population was greatly affected by nematodes. The highest (81.02 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.

DISCUSSIONThe 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

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

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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

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.

ACKNOWLEDGEMENTThis 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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