SOIL PHYSICAL FACTORS AFFECTING DISEASE SEVERITY OF ROOT-KNOT NEMATODE ON TOBACCO AND MORPHOLOGY AND MOLECULAR IDENTIFICATION OF TROPICAL NEMATODES


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.

.

SOIL PHYSICAL FACTORS AFFECTING DISEASE SEVERITY OF ROOT-KNOT NEMATODE ON TOBACCO AND MORPHOLOGY AND MOLECULAR IDENTIFICATION OF TROPICAL NEMATODES



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