INTEGRATED EFFECTS OF VESICULAR-ARBUSCULAR MYCORRHIZA AND Nostoc sp. IRRI 59 COMBINED WITH INORGANIC FERTILIZER
ON SOIL MICROBIAL POPULATION AND GROWTH OF LOWLAND RICE (Oryza sativa L.)
NIKO CALIENTA CEDICOL
SUBMITTED TO THE FACULTY OF THE INSTITUTE OF BIOLOGICAL SCIENCES, COLLEGE OF ARTS AND SCIENCES, UNIVERSITY OF
THE PHILIPPINES, LOS BAÑOS IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
BACHELOR OF SCIENCE IN BIOLOGY Major in Microbiology
APRIL 2010
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The thesis attached hereto, entitled Integrated Effects of Vesicular-Arbuscular Mycorrhiza and Nostoc sp. IRRI 59 Combined with Inorganic Fertilizer on Soil Microbial Population and Growth of Lowland Rice (Oryza sativa L.) prepared and submitted by Mr. Niko C. Cedicol in partial fulfillment of the requirements for the degree of Bachelor of Science in Biology, major in Microbiology, is hereby accepted.
___________________________ ___________________________
LUCILLE C. VILLEGAS JOCELYN T. ZARATE
Adviser Co-Adviser
____________________ _____________________
Date Signed Date Signed
___________________________
LUCILLE C. VILLEGAS Head, Microbiology Division ________________________
Date Signed
____________________________
DAMASA M. MACANDOG Director, Institute of Biological Sciences
________________________
Date Signed
Accepted in partial fulfillment of the requirements for the degree of Bachelor of Science in Biology, major in Microbiology.
______________________________
ASUNCION K. RAYMUNDO Dean, College of Arts and Sciences University of the Philippines Los Banos
_______________________
Date Signed
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TABLE OF CONTENTS
PAGE
TITLE PAGE i
BIOGRAPHICAL SKETCH iii
ACKNOWLEDGMENTS iv
TABLE OF CONTENTS vi
LIST OF TABLES ix LIST OF FIGURES xi
LIST OF APPENDICES xiv
ABSTRACT xv
INTRODUCTION 1
REVIEW OF RELATED LITERATURE 5
I. Rice (Oryza sativa L.) 5
II. Mycorrhiza 6
A. Endomycorrhiza 7
B. Morphology and Infection Patterns of VAM Fungi 10
C. Morphology of the Plant Symbiont 13
III. Cyanobacteria (Blue-Green Algae) 14
A. Biological Nitrogen Fixation 16
B. Effect of Cyanobacteria on Rice 16
C. Nostoc sp. 18
IV. Inorganic Fertilizer 20
A. Effect of Inorganic Fertilizer on Algal Population 21 B. Effect of Inorganic Fertilizer on Bacterial, Actinomycete 22 and Fungal Population
MATERIALS AND METHODS 24
I. Purification and Mass Production of Nostoc sp. IRRI 59 24
II. Pot and Soil Preparation 26
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III. Inorganic Fertilizer Application 27
IV. Rice Seed Preparation and Germination 27
V. Biofertilizer Application 28
VI. Treatment Composition and Experimental Design 28 VII. Assessment of the Combined Effect of Nostoc sp. IRRI 59 29
and VAM on Growth of Lowland Rice Under Different Inorganic Fertilizer Levels
A. Effect of Preliminary Inoculation of Mykovam Biofertilizer 29 on Rice Seed Germination
B. Qualitative Examination of Rice Leaf Using Leaf Color Chart 29 C. Effects of Microbial Inoculation Combined with 29
Different Levels of Inorganic Fertilizer on Rice Growth
D. Mycorrhization 30
VIII. Descriptive Assessment of the Fluctuations in Microbial 30 Population at Different Growth Stages of Lowland Rice
A. Soil Sampling 30
B. Soil Dilution 31
C. Plating Technique and Most Probable 31
Number Method (MPN)
IX. Assay for Plant Growth-Promoting Activity of Different Treatments 32
X. Statistical Analyses 33
RESULTS AND DISCUSSION 37
I. Assessment of the Combined Effect of Nostoc sp. IRRI 59 37 and VAM on Growth of Lowland Rice Under Different
Inorganic Fertilizer Levels
A. Effect of Preliminary Inoculation of Mykovam Biofertilizer 37 on Rice Seed Germination
B. Qualitative Examination of Rice Leaf Using Leaf 39 Color Chart
C. Effects of Microbial Inoculation Combined with 40 Different Levels of Inorganic Fertilizer on Rice Growth
1.Effect of Soil Sterilization (Factor A) 41 2.Effect of Biofertilization (Factor B) 43 3.Effect of the use of Chemical/Inorganic 45 Fertilizer (Factor C)
ix
4.Effect of Interactions among the Three 46 Factors (Soil Sterilization, Biofertilization
and Chemical Fertilization)
D. Mycorrhization 51
II. Descriptive Assessment of Fluctuations in Microbial 55 Population at Different Growth Stages of Lowland Rice
1. Descriptive Assessment of Bacterial Population 55 2. Descriptive Assessment of Actinomycete and Fungal Population 66 3. Descriptive Assessment of Algal Population 66 III. Assay for Plant Growth-Promoting Activity of Different 78
Treatments on Rice Seed Germination
SUMMARY AND CONCLUSION 80
LITERATURE CITED 84
APPENDICES 89
x
LIST OF TABLES
Table Title Page
1 Schematic representation of morphological diversity among blue-green algae (Roger, 1983).
15
2 Major groups of N2-fixing microorganisms in lowland rice fields (Roger and Watanabe, 1986).
19
3 Some physicochemical properties of BIOTECH soil used for pot experiment (Analysis performed by Analytical Services Laboratory, Soils and Agro-Systems Division, College of Agriculture, UPLB).
26
4 Qualitative N macronutrient deficiency examination of rice in different treatments using the Leaf Color Chart.
40
5a Effect of soil sterilization (Factor A) on growth of lowland rice. 42 5b Effect of different biofertilizer treatments (Factor B) on growth of
lowland rice.
43
5c Effect of the use of chemical or inorganic fertilizer (Factor C) on growth of lowland rice.
45
5d Effect of sterilization and biofertilization (A x B) on growth of lowland rice
46
5e Effect of sterilization and the use of chemical fertilizer (A x C) on growth of lowland rice.
47
5f Effect of biofertilization and the use of inorganic/chemical fertilizer (B x C) on growth of lowland rice.
47
5g Effect of sterilization, biofertilization and the use of inorganic/chemical fertilizer (A x B x C) on growth of lowland rice. Each value is mean of 12 replicates.
48
6 Influence of different inorganic fertilizer levels on bacterial flora of both unsterilized and sterilized soils in Control (Uninoculated) Treatment (expressed as cfu g-1 dry soil x 105)**.
57
7 Influence of different inorganic fertilizer levels together with inoculated Nostoc sp. IRRI 59 on bacterial flora of both unsterilized and sterilized soils (expressed as cfu g-1 dry soil x 105)**.
58
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8 Influence of different inorganic fertilizer levels together with inoculated Mykovam (BIOTECH) biofertilizer, on bacterial flora of both unsterilized and sterilized soils (expressed as cfu g-1 dry soil x 105)**.
60
9 Influence of different inorganic fertilizer levels together with inoculated Mykovam (BIOTECH) biofertilizer and Nostoc sp. IRRI 59 on bacterial flora of both unsterilized and sterilized soils (expressed as cfu g-1 dry soil x 105)**.
64
10 Influence of different inorganic fertilizer levels on algal flora of both unsterilized and sterilized soils in Control Treatment (MPN g-1 soil x 103).*
67
11 Influence of different inorganic fertilizer levels together with inoculated Nostoc sp. IRRI 59 on algal flora of both unsterilized and sterilized soils (expressed as MPN g-1 soil x 103)*.
69
12 Influence of different inorganic fertilizer levels together with inoculated Mykovam (BIOTECH) biofertilizer, on algal flora of both unsterilized and sterilized soils (expressed as MPN g-1 soil x 103)*.
72
13 Influence of different inorganic fertilizer levels together with inoculated Mykovam (BIOTECH) biofertilizer and Nostoc sp. IRRI 59 on algal flora of both unsterilized and sterilized soils (expressed as MPN g-1 soil x 103)*.
73
14 Mean height of rice seedlings treated with Nostoc sp. IRRI 59 extracts together with Mykovam biofertilizer, for plant growth-promoting activities.
78
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LIST OF FIGURES
Figure Title Page
1 Root of host plant with arbuscules (a) and vesicles (b) from VAM.
Linear association in root of Allium porrum with arbuscules (A) and vesicles (V) on longitudinal hyphae near the entry point shown in arrow (c) (Brundrett 2008).
12
2 Photomicrograph of Nostoc sp. IRRI 59 filaments in BG-11 medium showing presence of heterocysts shown in arrows (400X).
25
3 General appearance of rice seedlings. Without Mykovam biofertilizer (a and b), and with Mykovam biofertilizer (c and d) inoculated prior to transplanting in individual pots.
38
4 Effect of soil sterilization, biofertilization and chemical fertilizer application on different growth parameters in rice. (a) Tiller Count, (b) Shoot Dry Weight, (c) Root Dry Weight, and (d) Height.
50
5a Rice root hair infection by fungal hyphae, 120 days after planting.
Arrows point to fungal hyphae present on both inside and outside of root hairs.
52
5b Presence of putative vesicles and arbuscules inside the rice root, characteristic of VAM fungi.
52
6 Aerenchyma in rice shoots (arrows) are responsible for oxygen transport from environment to the root system in waterlogged soil thereby allowing establishment of mycorrhiza.
54
7a Fluctuations in bacterial population in unsterilized soil for the Control (Uninoculated) Treatment at different growth stages of rice under the influence of different levels of inorganic fertilizers.
56
7b Fluctuations in bacterial population in sterilized soil for the Control (Uninoculated) Treatment at different growth stages of rice under the influence of different levels of inorganic fertilizers.
56
8 Presence of other algae and cyanobacteria in Nostoc sp. only treatment which might have come from the tap water used in the pot experiment.
59
9a Fluctuations in bacterial population in unsterilized soil for the Nostoc sp. Treatment at different growth stages of rice under the influence of different levels of inorganic fertilizers.
61
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9b Fluctuations in bacterial population in sterilized soil for the Nostoc sp.
Treatment at different growth stages of rice under the influence of different levels of inorganic fertilizers.
61
10a Fluctuations in bacterial population in unsterilized soil for the Mykovam Treatment at different growth stages of rice under the influence of different levels of inorganic fertilizers.
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10b Fluctuations in bacterial population in sterilized soil for the Mykovam Treatment at different growth stages of rice under the influence of different levels of inorganic fertilizers.
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11a Fluctuations in bacterial population in unsterilized soil for the Combined Nostoc sp. + Mykovam Treatment at different growth stages of rice under the influence of different levels of inorganic fertilizers.
65
11b Fluctuations in bacterial population in sterilized soil for the Combined Nostoc sp. + Mykovam Treatment at different growth stages of rice under the influence of different levels of inorganic fertilizers.
65
12 Sample Martin’s Rose Bengal Medium showing countable fungal growth using Miles and Misra plating method.
66
13a Fluctuations in algal population in unsterilized soil for the Control (Uninoculated) Treatment at different growth stages of rice under the influence of different levels of inorganic fertilizers (expressed as MPN g-1 soil x 103).
68
13b Fluctuations in algal population in sterilized soil for the Control (Uninoculated) Treatment at different growth stages of rice under the influence of different levels of inorganic fertilizers (expressed as MPN g-1 soil x 103).
68
14a Fluctuations in algal population in unsterilized soil for the Nostoc sp.
Treatment at different growth stages of rice under the influence of different levels of inorganic fertilizers (expressed as MPN g-1 soil x 103).
70
14b Fluctuations in algal population in sterilized soil for the Nostoc sp.
Treatment at different growth stages of rice under the influence of different levels of inorganic fertilizers (expressed as MPN g-1 soil x 103).
70
15a Fluctuations in algal population in unsterilized soil for the Mykovam Treatment at different growth stages of rice under the influence of different levels of inorganic fertilizers (expressed as MPN g-1 soil x 103).
71
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15b Fluctuations in algal population in sterilized soil for the Mykovam Treatment at different growth stages of rice under the influence of different levels of inorganic fertilizers (expressed as MPN g-1 soil x 103).
71
16a Fluctuations in bacterial population in unsterilized soil for the Integrated Nostoc sp. + Mykovam Treatment at different growth stages of rice under the influence of different levels of inorganic fertilizers (expressed as MPN g-1 soil x 103).
74
16b Fluctuations in bacterial population in sterilized soil for the Integrated Nostoc sp. + Mykovam Treatment at different growth stages of rice under the influence of different levels of inorganic fertilizers (expressed as MPN g-1 soil x 103).
74
17a Sample growth on MPN tube after tillering stage (45 do) showing positive for growth.
75
17b Green algae predominate in most of the MPN tubes. 75
18a Sample green algal growth in pot experiment set up after tillering stage. 76 18b Nostoc sp. can still be observed clinging on rice stems. 76
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LIST OF APPENDICES
Appendix Title Page
1 Culture media used in the experiment. 89
2 Clearing and staining of roots outlined by Philips and Hayman (1970).
91
3 Fertilizer calculations from “Growing Lowland Rice: A Production Handbook”-Africa Rice Center (Nwilene, et al, n.d.).
92
4 Leaf Color Chart ( LCC) from PhilRice. 93
5 MPN Table taken from Ogram and Al-Agely (2004). 94
6 Typhoon “Santi’ Specifications (Data taken from Agromet, UPLB).
95
7 Rice Height Analysis of Variance and Duncan’s Multiple Range Test.
96
8 Rice Shoot Dry Weight Analysis of Variance and Duncan’s Multiple Range Test.
100
9 Rice Root Dry Weight Analysis of Variance and Duncan’s Multiple Range Test.
105
10 Rice Tiller number Analysis of Variance and Duncan’s Multiple Range Test
110
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CEDICOL, NIKO C. March 2010. Integrated Effects of Vesicular-Arbuscular Mycorrhiza and Nostoc sp. IRRI 59 Combined with Inorganic Fertilizer on Soil Microbial Population and Growth of Lowland Rice (Oryza sativa L.) College of Arts and Sciences. University of the Philippines Los Banos, College, Laguna. Bachelor of Science in Biology major in Microbiology.
Major Adviser: Dr. Lucille C. Villegas Co-Adviser: Dr. Jocelyn T. Zarate
ABSTRACT
Improvement in growth of lowland rice (var. NSIC-122/Angelica) and fluctuations in soil microbial population at different growth stages were studied under the influence of varying inorganic fertilizer levels, and/or using VAM fungi (Mykovam BIOTECH) and Nostoc sp. IRRI 59 treatment.
Inoculation of rice seeds with Mykovam resulted to increased seedling height, greener leaf blades and longer and finer root hairs as compared to uninoculated at 10 days after seed germination. Leaf Color Chart (LCC) reading 7 days after tillering stage (45 days old), revealed 100% N deficiency for uninoculated Control and Mykovam treatments which did not receive either chemical fertilizer or Nostoc sp. IRRI 59.
Chemical fertilizer addition at both 90:60:60 and 45:30:30 kg NPK ha-1 significantly promoted growth of almost all growth parameters measured. Soil sterilization, biofertilization (especially with combined Nostoc sp.+Mykovam), and chemical fertilizer at the rate of 90:60:60 kg NPK ha-1 significantly improved rice growth as indicated by increase of 83% in tiller count, 30% in height, 102% in root dry weight and 204% for shoot dry weight. VAM infection was observed through the presence of inter and intracellular fungal hyphae; however, presence of vesicles and arbuscules inside rice roots were putatively identified through microscopy. Assay for plant growth-promoting activity of different treatments revealed highly significant result in mean seedling height difference from negative control for oven-dried Nostoc sp. extracts, Mykovam only, and integrated air-dried Nostoc sp. + Mykovam extracts, with maximum value (in cm) obtained from former.
Soil bacterial population was increased by inoculation with combined Mykovam and Nostoc sp. IRRI 59 under 90:60:60 kg NPK ha-1 in unsterilized soil compared to other treatments after grain-yielding stage of rice (120 days old) while inoculation with Nostoc sp. IRRI 59 alone under 0:0:0 kg NPK ha-1 increased bacterial population for sterilized soil. Actinomycete and fungal population were not determined since counts were below the detection limit of the plating technique used. Algal population in sterilized soil was higher than unsterilized soil based on mean effects of plant age.
Increase in algal population was observed at tillering stage then gradually declined at grain-yielding stage. Inverse relationship was observed between algal population and levels of inorganic fertilizer.
