SOIL PHYSICAL FACTORS AFFECTING DISEASE SEVERITY OF ROOT-KNOT NEMATODE ON TOBACCO AND MORPHOLOGY AND MOLECULAR IDENTIFICATION OF TROPICAL NEMATODES
ABSTRACT
In Malaysia, tobacco industry is very crucial in uplifting the socio-economic status of farmer in Kelantan and Terengganu. Root-knot nematodes (Meloidogyne spp.) lower the production and quality of tobacco. In this study, 24 tobaccos cultivation areas were surveyed for root gall disease and species diversity of root- knot nematode. Soil physical and pH analysis were carried out to investigate their correlation to disease severity and number of root-knot nematode number in soil. Isolated Meloidogyne spp. was observed for morphology for genus identification and further analyses via Touchdown Polymerase chain reaction (TD-PCR) for species identification. From 24 cultivation area, 22 showed infestation of root-knot nematode. Tobacco plant infected with root-knot nematode showed root gall, yellowing leaves and stunted growth. One- Way ANOVA analysis showed that soil physical properties, and soil pH affect disease severity and Meloidogyne spp. number in soil. Meloidogyne spp. numbers in soil also affect disease severity. From Pearson correlation analysis, there was significant correlation at 0.01 between Meloidogyne spp. number in soil (r = 0.753), soil pH (r = 0.238), soil moisture (r = 0.203) and soil bulk density (r = 0.227) to disease severity. There was also significant correlation at
0.01 between soil pH (r = 0.373), soil moisture (r = 0.359), soil particle density (r = - 0.404) and soil pore spaces (r = -0.332) to Meloidogyne spp. number in soil. Root- knot nematode species identification was identified using Touchdown PCR with primer 194/195 which amplified 5s-18s ribosomal region and species specific SCAR
primers. All 22 samples showed amplification using primer 194/195 yielding 720 bp which is categorized as Tropical Meloidogyne spp. SCAR primers showed high specificity to the related species reliably determined species composition, detecting mixed population of Meloidogyne javanica and Meloidogyne incognita. A population amplified with the SCAR primer Fjav/Rjav yielding 720 bp products was categorized in the M. javanica group while population amplified with the SCAR primer MiF/MiR yielding 999 bp was categorized in the M. incognita group. Out of 22 samples, 15 samples showed occurrence of M. javanica while 7 samples showed mixture of M. javanica and M. incognita.
TABLE OF CONTENTS
ACKNOWLEDGEMENTS ii
TABLE OF CONTENTS iii
LIST OF TABLES vii
LIST OF FIGURES ix
LIST OF SYMBOLS AND ABBREVIATIONS xiii
ABSTRAK xiv
ABSTRACT xvi
CHAPTER 1: INTRODUCTION 1
CHAPTER 2: LITERATURE REVIEW 3
Tobacco in Malaysia3
Tobacco cultivation in Malaysia3
Importance of tobacco in Malaysia4
Plant parasitic nematodes related to Tobacco (Nicotiana tabacum)5
Root knot nematode, Meloidogyne spp.7
General morphology7
Male7
Female9
Second stage juveniles (J2)11
Life cycle13
Distribution and importance of Meloidogyne spp.17
Effect of Meloidogyne spp. on tobacco19
Identification of Meloidogyne spp.20
CHAPTER 3: MATERIALS AND METHODS 26
Study site and soil sampling26
Disease incidence evaluation27
Soil physical analysis and pH28
Soil pH28
Soil moisture28
Soil bulk density29
Soil particle density and pore percentage30
Soil texture31
Observation of tobacco samples34
Tobacco seedling preparation34
Evaluation of Root knot (gall) index on the planted tobacco35
Statistical Analysis35
Inoculation and Pathogenicity test of Meloidogyne spp.36
Isolation of Second stage juvenile (J2)36
Isolation of Meloidogyne spp. female from root sample37
Morphological observation Meloidogyne spp.38
Female38
Second stage juveniles (J2)38
Eggs38
Molecular identification Meloidogyne spp.39
DNA extraction39
DNA purity and concentration40
PCR component and primer41
Touchdown PCR42
Gel electrophoresis44
Gel DNA extraction45
Sequencing and Identification using Basic Local Alignment46
Search Tool (BLAST))
CHAPTER 4: RESULTS 47
The root disease and disease incidence of tobacco47
Soil physical analysis and pH52
Analysis of soil samples, number of Meloidogyne spp and the disease55
of tobacco plants
Evaluation of Root knot (gall) index on the planted tobacco59
Statistical Analysis61
Relationship of soil physical, pH and Meloidogyne spp. to disease61
severity
Relationship of soil physical properties and pH to the number of64
Meloidogyne spp.
Correlation of disease severity and number of Meloidogyne spp.67
Pathogenicity test of Meloidogyne spp.69
Morphological observation of Meloidogyne spp.72
Female72
Second stage juveniles (J2)75
Eggs77
Morphological analysis of different Meloidogyne sp.80
Molecular identification Meloidogyne spp.82
DNApurity and concentration82
Touchdown PCR84
Touchdown PCR using primer 194/19584
Touchdown PCR using SCAR primer88
Sequencing and Identification using Basic Local Alignment95
Search Tool (BLAST)
BLAST of sequenced DNA amplified from primer 194/19595
BLAST of sequenced DNA amplified from SCAR primer96
CHAPTER 5: DISCUSSION 101
The effect of Meloidogyne spp. on tobacco growth101
Effect of soil physical properties and pH to disease severity and102
Meloidogyne spp. number in soil
Effect of soil pH102
Effect of soil moisture103
Effect of soil bulk density105
Effect of soil particle density and pore percentages106
Effect of soil texture107
Morphological observation110
Female110
Second stage juveniles (J2)111
Eggs112
Molecular identification113
Touchdown PCR using primer 194/195113
Touchdown PCR using SCAR primer114
Sequencing and Identification using Basic Local Alignment115
Search Tool (BLAST)
CHAPTER 6: CONCLUSION 117
REFERENCES 119
APPENDICES 131
LIST OF TABLES
Table 3.1 The list of location and the collection date of the soil samples
for the study 27
Table 3.2 PCR component (PROMEGA) 41
Table 3.3 Primer used for the TD-PCR 42
Table 3.4 Touchdown PCR (TD-PCR) programs 42
Table 3.5 Annealing Temperature for each primer 43
Table 4.1 Root disease and disease incidence on sampling site 48
Table 4.2 Soil physical and pH analysis 54
Table 4.3 Disease severity of tobacco and the number of Meloidogyne 56
spp. in root gall and soil.
Table 4.4 Morphological analysis of planted tobacco and disease severity 58
Table 4.5 Disease severity and the number of Meloidogyne spp. of 59
planted tobacco
Table 4.6 Pearson correlation value (r) of soil physical properties and pH 61
to disease severity
Table 4.7 Pearson correlation value of soil physical properties and pH to 64
Meloidogyne spp. number in soil
Table 4.8 Duncan analysis of Meloidogyne spp. to disease severity 67
Table 4.9 Morphology observation of Meloidogyne spp. female 72
Table 4.10 Morphology observation of Meloidogyne spp. J2 75
Table 4.11 Morphological observation of eggs 77
Table 4.12 Morphological comparison between M. javanica, M. incognita, 81
M. arenaria, and M. thailandica
Table 4.13 DNA purity and concentration extracted from female and J2 of 83
Meloidogyne spp. using Qiagen DNA extraction kit
Table 4.14 BLAST result from National Center for Biotechnology 97
Information (NCBI) for female of Meloidogyne spp. using
primer Fjav/Rjav
Table 4.15 BLAST result from National Center for Biotechnology 98
Information (NCBI) for J2 of Meloidogyne spp. using primer
Fjav/Rjav
Table 4.16 BLAST result from National Center for Biotechnology 99
Information (NCBI) for female of Meloidogyne spp. using
primer MiF/MiR
Table 4.17 BLAST result from National Center for Biotechnology 99
Information (NCBI) for J2 of Meloidogyne spp. using primer MiF/MiR
LIST OF FIGURES
Figure 2.1 Active tobacco cultivation area in Malaysia 5
Figure 2.2 Gross morphology and anatomy of a male Meloidogyne spp. 8
Figure 2.3 Gross morphology and anatomy of a female Meloidogyne spp. 10
Figure 2.4 Gross morphology and anatomy of a Meloidogyne spp. J2 12
Figure 2.5 Life cycle of root knot nematode (Meloidogyne species). J2: 16
Second stage juvenile; J3: Third stage juvenile; J4: Fourth stage
juvenile
Figure 3.1 Moisture can with soil samples used for soil moisture analysis 29
Figure 3.2 USDA Textural Triangle 33
Figure 3.3 Modified Baermann isolation used for Meloidogyne spp. J2 37
isolation from infected soil and root.
Figure 3.4 Nanodrop machine used to check concentration and purity of 40
extracted DNA.
Figure 3.5 BIO RAD MyCycler machine used to run TD-PCR reaction. 43
Figure 4.1 Disease incidence of tobacco plant from various places in 48
Kelantan and Terengganu
Figure 4.2 Percentage of root disease observed from various places in 49
Kelantan and Terengganu.
Figure 4.3 Tobacco fields infected with root gall disease showing stunted 49
growth and yellowing leaf.
Figure 4.4 Tobacco root infected with root gall disease. Arrow shows root 50
gall caused by Meloidogyne spp.
Figure 4.5 Tobacco root gall and root rot disease complex 50
Figure 4.6 (A) Tobacco root gall. Red arrow shows large root gall while 51
green arrow shows small root gall. (B) Large root gall (C) Small root gall observe under compound microscope.
Figure 4.7 Stage or level of disease severity of root gall on tobacco root. 60
(A) Healthy tobacco or stage 0, (B) Disease severity stage 1,
(C) Disease severity stage 2, (D) Disease severity stage 3, (E) Disease severity stage 4
Figure 4.8 Graph to show correlation between disease severity and soil 62
pH.
Figure 4.9 Graph to showed correlation between disease severity and soil 62 moisture percentage
Figure 4.10 Line graph to show correlation between disease severity and 63
soil bulk density
Figure 4.11 Disease severity of tobacco plant of sandy soil and loamy sand 63
Figure 4.12 Line graph to show correlation between soil pH and number of 65
Meloidogyne spp. in soil.
Figure 4.13 Line graph to show correlation between soil moisture and 65
Meloidogyne spp. number in soil
Figure 4.14 Line graph to show correlation between soil particle density 66
and Meloidogyne spp. number in soil
Figure 4.15 Line graph to show correlation between soil pore spaces and 66
Meloidogyne spp. number in soil.
Figure 4.16 Number of Meloidogyne spp. in sandy soil and loamy sand 67
Figure 4.17 Graph to show correlation between disease severity and number 68 of Meloidogyne spp. in soil.
Figure 4.18 Pathogenicity test of Meloidogyne spp.(A) Healthy tobacco leaf 70
(B) Healthy tobacco root (C) Infected tobacco leaf (D) Infected tobacco root
Figure 4.19 (A) Root gall on tobacco root (B) Egg mass of Meloidogyne 71 spp. on the infected tobacco root (C) Female of Meloidogyne
spp. teased out from the root gall (D) Female of Meloidogyne
spp.
Figure 4.20 Female of Meloidogyne sp. in the dissected tobacco root. (A) 73 Female embedded in root gall (B) Red arrow shows J2 became saccate after getting nutrient from root while green arrow shows mature pear shaped like female of Meloidogyne sp.
Figure 4.21 Female of Meloidogyne spp. observed under compound 74 microscope. (A) Female under 10x magnification compound microscope (B) Female stylet in the circle observed under 40x magnification.
Figure 4.22 (A) J2 of Meloidogyne spp. under 20x magnification compound 76 microscope (B) Stylet under 100x magnification compound microscope (C) Hyaline tail under 100x magnification compound microscope
Figure 4.23 (A) Egg mass of Meloidogyne spp. on infected tobacco root (B) 78 Teased out of Meloidogyne spp. egg mass under 10x
magnification compound microscope
Figure 4.24 (A-D) Morphology of Meloidogyne spp. eggs at different stage 79
(D) First stage juveniles of Meloidogyne spp.(J1)
Figure 4.25 Gel photograph showing bands obtain from isolated 84
Meloidogyne spp. female DNA from Kelantan using primer
194/195.
Figure 4.26 Gel photograph showing bands obtain from isolated 85
Meloidogyne spp. J2 DNA from Kelantan using primer
194/195.
Figure 4.27 Gel photograph showing bands obtain from isolated 86 Meloidogyne spp. female DNA from Terengganu using primer 194/195
Figure 4.28 Gel photograph showing bands obtain from isolated 87
Meloidogyne spp. J2 DNA from Terengganu using primer
194/195
Figure 4.29 Gel photograph showing bands obtain from isolated female 89
DNA of Meloidogyne spp. from Kelantan using specific primer
Fjav/Rjav
Figure 4.30 Gel photograph showing bands obtain from isolated J2 DNA of 90
Meloidogynespp.fromKelantanusingspecificprimer
Fjav/Rjav
Figure 4.31 Gel photograph showing bands obtain from isolated female 91
DNA of Meloidogyne spp. from Terengganu using specific
primer Fjav/Rjav
Figure 4.32 Gel photograph showing bands obtain from isolated J2 DNA of 92
Meloidogyne spp. from Terengganu using specific primer
Fjav/Rjav
Figure 4.33 Gel photograph showing bands obtain from isolated female 93
DNA of Meloidogyne spp. using specific primer MiF/MiR
Figure 4.34 Gel photograph showing bands obtain from isolated J2 DNA of 94
Meloidogynespp.fromKelantanusingspecificprimer
MiF/MiR
Figure 4.35 Meloidogyne species composition on sampled tobacco cultivation around Kelantan and Terengganu.
100
LIST OF SYMBOLS AND ABBREVIATIONS
ng : Nanogram
µm : Micrometre
µl : Microlitre
nm : Nanometre
mM : Milimollar
ml : Millilitre
pmol : Picomolar
kb : Kilobase
bp : Base pair
se : Standard error
J2 : Second stage juveniles
DEGO : Dorsal esophageal gland orifices DNA : Deoxyribonucleic acid
IGS : Intergenic spacer
PCR : Polymerase Chain Reaction
AFLP : Amplified fragment length polymorphism RAPD : Random amplified polymorphic DNA RFLP : Restriction fragment length polymorphism AFLP : Amplified fragment length polymorphism SCAR : Sequence characterised amplified region BLAST : Basic local alignment search tool
DMRT : Duncan multiple range test
CHAPTER 1: INTRODUCTION
Tobacco (Nicotiana tabacum) is considered to be one of the most important industrial crops and highly demanded throughout the world (Luc et al., 2005). In Malaysia, tobacco industry is very crucial in uplifting the socio-economic status of farmer in Kelantan and Terengganu. Domestic tobacco demand and economic value of tobacco in Malaysia has enabled farmers to benefit from the lucrative crop. National Kenaf & tobacco board described the economical value in the Q & A,’ Why not grow tobacco and other food crops?’ It is said that the farmers who grow tobacco earn up to RM10,000 per hectare and for the farmer-curers able to earn up to RM25,000 per hectare (http://www.lktn.gov.my/page.php?140).
Meloidogyne spp. (root- knot nematode) that cause root gall has been known to pose a serious threat of tobacco production in the world often lower the quality and yield (Luc et al., 2005).. Meloidogyne spp. has been reported to cause infection on more than 5500 plant species including crop and weeds (Trudgill and Blok, 2001; Van Biljon, 2003; Adam et al., 2007). Annual crop losses cause by Meloidogyne spp. estimated to exceed $US 50 billion (Bent et al., 2008). In Malaysia, Meloidogyne spp. not only showed infection on tobacco but also on other crop such as guava (Psidium guajava L.) in Perak (Razak and Lim, 1987; Tahery et al., 2011), chilli (Capsicum frutuscen) (Tahery et al., 2011), black pepper (Piper nigram L.), turfgrass on golf courses (Tahery et al., 2011), kenaf (Hibiscus cannabinus) (Tahery et al., 2011) and banana (Razak, 1994; Tahery et al., 2011).
Although chemical control is the most reliable method to control Meloidogyne spp., chemicals are toxic to human and environment (Sirias, 2011). Therefore, new strategies to control Meloidogyne spp. such as integrated management practice, tolerant tobacco varieties and biological control are needed (Bertrand et al., 2000). Correct species identification is basic to efficient nematode control and successful plant quarantine operations. Previous studies have been carried out to identify Meloidogyne spp. using various morphological character but they are unpractical and insufficient (Adam et al., 2007; Sirias, 2011). Besides, species identification using morphology might overlap between species (Adam et al., 2007; Sirias, 2011). Therefore, nowadays, molecular method base on utilization of DNA and PCR have many advantages and have been used for reliable Meloidogyne spp. identification. Meloidogyne spp. identification could be useful to select the best strategy for their management (Adam et al., 2007). Considering the importance of Meloidogyne spp., this study was done with the following objective:
1. To find relationship of soil physical properties and pH to disease severity and population density of Meloidogyne spp..
2. To observe morphology of Meloidogyne spp. for genus identification
3. To identify species of Meloidogyne spp. (root-knot nematode) via molecular method.
.