Summary of biology doctoral thesis: Study on diversity of root –knot nematodes (Meloidogyne spp.) in Tay Nguyen Highlands

MINISTRY OF EDUCATION
VIETNAM ACADEMY
AND TRAINING
OF SCIENCE AND TECHNOLOGY
GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY
----------------------------

LE THI MAI LINH

STUDY ON DIVERSITY OF ROOT–KNOT NEMATODES,
Meloidogyne spp., IN TAY NGUYEN HIGHLANDS
Major: Nematology
Code: pilot

SUMMARY OF BIOLOGY DOCTORAL THESIS

Ha Noi – 2019

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The thesis was completed at the Graduate University of Science and
Technology, Vietnam Academy of Science and Technology

Scientific Supervisor 1: Dr. Trinh Quang Phap
Scientific Supervisor 2: Assoc. Prof. Dr. Phan Ke Long
Review 1: …
Review 2: …
Review 3: ….

The dissertation will be defended at the Council for Ph.D. thesis, meeting

at the Viet Nam Academy of Science and Technology - Graduate
University of Science and Technology. Time: Date …… month …. 2019

This thesis can be found at:
- The library of the Graduate University of Science and Technology
- National Library of Viet Nam

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INTRODUCTION
1. Essence of the thesis
Root-knot nematodes from the genus Meloidogyne are a cosmopolitan
group of obligate plant-parasites. Meloidogyne species are able to
parasitize virtually all species of higher plants in the world. Moreover,
Meloidogyne species are considered to be an important limiting factor in
vegetable protection. So, Root knot nemaotde Meloidogyne is a serious
threat to agriculture worldwide. Till 2006, 111 species had been
decribed, in which 18 species were found on coffee.
Till 2000, five species of the genus Meloidogyne were recorded on
coffee including M. incognita, M. javanica, M. exigua, M. coffeicola and
M. paranaensis. Among five species above, M. incognita causes damage
on almost all the coffee growing areas as well as all other crops. Many
studies on coffee and pepper in Vietnam indicated that M. incognita is
the most important species causing damages. Trinh et al. (2013) also
announced the presence of two species of M. exigua and M. coffeicola on
coffee, and indicated the presence of harmful Meloidogyne species in
Tay Nguyen Highlands is pretty serious and needs to be investigated in
more detail. And, there are many species of Meloidogyne have not been
discovered and published.

Morphological analysis of perineal patterns from adult Meloidogyne
females is often used as a rapid diagnostic tool. However, there are some
major limitations with this approach, including overlapping of
morphological characters, which can lead to misidentification. Some
coffee-parasitising nematodes, such as M. izalcoensis, M. paranaensis,
and M. incognita, have the overlapping of characters related to female
perineal patterns. Additional diagnostic power can be used such as
molercular characterisation of some genes including 18S, ITS, and 28S
of rDNA and COI, COII of mtDNA.
Prevention of these nematodes species in agricultural crops mostly
based on chemical pesticides that is affecting the environment as well as
causing the loss of beneficial organisms. Moreover, using chemicals is
expensive and also depends on the activity of chemicals in the soil and
depends on country-specific rules. Therefore, searching for the solutions
to effectively control nematodes will have a great significance in the
production, contributing to limit the use of chemical drugs, safeguarding
the environment. One of the effective methods of controling this

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nematode group is the method based on the biological control of
antagonistic microorganisms that can destroy and limit the development
of nematodes.
Due to the diversity of plants and topography in Tay Nguyen, as well
as the variety of root-knot nematodes and their potential damages, we
implemented the project:: "Study on diversity of root –knot nematodes
(Meloidogyne spp.) in Tay Nguyen Highlands "
2. Objectives of the thesis
 Identifying the Meloidogyne species causing damage on main

crops in Tay Nguyen Highlands
 Analyze the diversity of Meloidogyne species based on
morphology and molecular classification.
 Assessing
the
preventative
ability
of
antagonistic
microorganisms to Meloidogyne incognita in laboratory condition.
3. Contents of the thesis
 Study on distribution, frequency and density of root-knot
nematodes on some important crops: coffee, pepper, vegetables and
intercropping crops in Tay Nguyen.
 Study on the morphology and diversity of root-knot nematode
Meloidogyne in Tay Nguyen.
 Study on the genetic diversity of root-knot nematode
Meloidogyne in Tay Nguyen based on analyses of rDNA and mrDNA
genes.
 Evaluate the prevention of Meloidogyne incognita by using some
antagonistic microorganisms such as Paecilomyces javanicus,
Lysobacter antibioticus and 4-HPAA antibiotics.
4. Scientific significance and practical meaning of the thesis
Scientific significance:
 New root-knot nematodes were recorded for science not only in
Vietnam but also in global.
 New morphological and molecular characters of root-knot
nematodes in Tay Nguyen Highlands were provided. Database for
sequences of Meloidogyne spp. in Vietnam is added to the International
GeneBank. Phylogeny of root-knot nematodes in Western Highlands

with Meloidogyne species in the world was created.
 The opportunities to apply useful microorganisms for root-knot
nematodes control.
Practical meaning

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 Exactly classifying Meloidogyne species and their density
creating a basic for effective prevetion. Providing data on parasitic
ability, hosts, damages of root-knot nematodes, preventing their spread,
avoiding early infection. Creating a basic to select appropriate farming
methods for each crop especially for coffee and pepper as well as
intercropping and rotation crops.
 Providing database of morphological and molecular
characteristics of Meloidogyne spp. in Vietnam, serving as a good source
of reference.
 Evaluating the antagonistic microorganisms (Pacylomyces
javanicus and Lysobacter antibioticus HS124) in preventing
Meloidogyne spp. creating a basic for selecting prevention measure,
reducing the use of harmful chemicals, maintaining ecological balance
for sustainable development of staple crops in Tay Nguyen
5. New contributions of the thesis
 The thesis provided a complete data of the distribution, host and
morphological and molecular characteristics of root-knot nematode
species on coffee, pepper and intercropping plants in Tay Nguyen for the
first time. More new host plants infected with Meloidogyne nematode
have been recorded in Tay Nguyen as well as in Vietnam. Deposited 145
sequences of ITS, D2D3, COI, COII-16S, NAD5 gene regions of rootknot nematode species from Tay Nguyen to Genbank.
 A new species of root knot nematode has been recorded on

coffee that has been described, published and named by Meloidogyne
daklakensis n. sp. and 01 other new species has been described and
submitted.
 For the first time in Vietnam, the ability to control Meloidogyne
incognita of Pacylomyces javanicus, Lysobacter antibioticus HS124 and
4-HPAA antibiotic was assessed in laboratory conditions.
6. Composition of the thesis
The thesis is composed of 179 pages: Introduction (4 pages); chapter
1: Literature review (22 pages); chapter 2: Methodology (13 pages);
chapter 3: Resutl and discussion (119 pages); Conclusion and
recommendation (2 pages). The thesis consists of 34 tables, 39 figures,
and 183 references. In addition, the thesis has a list of publications and
appendices

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CHAPTER 1. LITERATURE REVIEW
1.1.
Root-knot nematodes in the world
1.1.1. Research history: Berkeley (1855) was the first scientist to publish
the emergence of root-knot nematodes. In 1949, Chitwood split the
genus Meloidogyne Göldi, 1887 from the genus Heterodera based on the
standard species M. exigua (Goldi, 1887). The classification of the genus
Meloidogyne is currently following the system of Karsen et al. (2013)
1.1.2. Biological characteristics, life cycle of root-knot nematode
Meloidogyne: the life cycle of root-knot nematode Meloidogyne consists
of 5 stages of development including: eggs; second stage juveniles (J2)
(mobile phase); juveniles stage 3, 4 (J3, J4) and adult stage with females
and males. Root-knot nematodes Meloidogyne spp. reproduce by 2

different ways: compulsory sexual-mating reproduction (amphimixis)
and the majority of them are bisexual reproduction (parthenogensis)
without males.
1.1.3. Damages on some crops: Root-knot nematode Meloidogyne has
the ability to parasitize almost all crops, up to 5500 different plants,
including from industrial and agricultural crops to weeds. The annual
economic losses are estimated to be hundreds of billions of dollars.
1.1.4. Identification of root-knot nematodes
1.1.4.1. Morphological analysis: Chitwood (1949) and Jebson (1983)
used the characteristics of stylet, DGO, periniel pattern, head region in
males, distance from base of stylet to dorsal gland orifice (DGO) to
describe the species. Recent studies have used detailed morphological
and morphometric characteristics.
1.1.4.2. Molecular-based methods: the regions of rDNA and mtDNA are
frequently used in identifying root-knot nematodes.
1.2. Root-knot nematodes in Vietnam
1.2.1. General root-knot nematodes in Vietnam: published studies have
mentioned many different aspects that were mainly species composition,
host range, population dynamic and control measures. According to
Nguyen and Nguyen (2000), 5 species of root-knot nematodes belonging
to the genus Meloidogyne were found in Vietnam. So far, all the studies
were mainly focused on some control methods.
1.2.2. Root-knot nematode sitution in Tay Nguyen
1.2.2.1. Overview of Tay Nguyen: Tay Nguyen or Western Highlands in
Vietnam is a plateau region consisting of 5 provinces, including Kon

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Tum, Gia Lai, Dak Lak, Dak Nong and Lam Dong from the north to the

south. Located in the savanna tropical region. The climate in Tay
Nguyen is divided into two seasons: the rainy season from May to the
end of October and the dry season from November to April of the next
year. Tay Nguyen has 2 million hectares of fertile basalt soil, which
accounts for 60% of the basalt soil area in Vietnam. Nutrient-rich basalt
soil, deep weathering layer are convenient conditions for the formation
of large-scale specialized cultivation areas of industrial crops such as
coffee, cocoa, and pepper. This is the region with the largest area of
industrial crops in Vietnam with key export crops such as coffee and
pepper.
1.2.2.2. Rooted nematode in Tay Nguyen
Meloidogyne species are considered to be a serious pest on coffee and
pepper in Tay Nguyen causing yellow leaf disease, stunting, growth
retardation on coffee, and dying slowly disease on pepper.
1.3. Biological control measures of root-knot nematode Meloidogyne
Early prevention is an important solution in controlling
nematodes. Currently, when the plant showed symptoms, the disease
caused by nematodes is already relatively serious, so the prevention will
be ineffective or slowly effective. Biological control measures can create
products that directly or indirectly affect plant-parasitic nematodes.
Paecilomyces javanicus can be used to control Meloidogyne spp.
Lysobacter antibioticus HS124 and their antibiotic product 4-HPAA
have the ability to inhibit Meloidogyne incognita.

CHAPTER 2. MATERIALS AND METHODS
2.1. Times, subject, location, content and research methods
2.1.1. Objects
Root knot nematode populations belong to the genus Meloidogyne
Paecilomyces javanicus, Lysobacter antibioticus HS124, 4Hydroxyphenylacetic (4-HPAA) (Sigma brand)
2.1.2. Location and time

The soil and root samples were collected from Gia Lai, Dak
Lak, Dak Nong, Lam Dong, Kon Tum
The study was conducted at the laboratories of Department of
Nematology and Department of Molecular Biology and Conservation
Genetics (Institute of Ecology and Biological resources).
Thesis is carried out in 4 years, from 2014 to 2018

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2.2. Methods:
2.2.1. Investigation and nematode sampling: The soil and root samples
were collected from 5 provinces in Tay Nguyen.
2.2.2. Nematodes extraction: Nematodes were extracted by using the
method of Nguyen Ngoc Chau (2003).
2.2.3 Culturing: Meloidogyne spp. were cultured on tomatoes following
López-Pérez et al. (2011).
2.2.4. Morphological analysis.
The identification of Meloidogyne species was based on the
diagnostic keys of Eisenback (1985), Hewlett & Tarjan (1981, 1983),
Jepson (1982, 1983a, b), Karssen (2002) and compared with the
descriptions of new species recently.
Statistical analysis: The measurements of Meloidogyne species were
analyzed by Canonical discriminant Analysis. The analysis was executed
in Genstad 11.
2.2.5. Molecular analysis
2.2.7.1. DNA extraction: following Holterman et al. 2009: juveniles
hatched from egg mass were used to extract DNA.
2.2.7.2. PCR
Multiplex-PCR for quick identification of Meloidogyne species: M.

arenaria, M. incognita, M. javanica can be differentiated using specific primers:
Far/Rar; Mi2F4/Mi1R1; Fjav/Rjav (Kiewnick et al. 2013).
Regions were amplified by PCR: D2D3, ITS, COI, COII-16S, NAD5
2.2.7.3. Gel electrophoresis: Multiplex PCR results were compared with
Kiewnick et al. (2013).
2.2.7.4. Purifying PCR products and sequencing DNA: GenJet PCR
Purification (Thermo Scientific- Germany) was used and sequencing was
done by Macrogen-Korea.
2.2.7.5. Alignment: sequences were aligned by ClustalW in BioEdit (Hall.
1999)
2.2.7.6. Creating phylogenetic trees: ML (Maximum Likelihood) method
was used to create phylogenetic trees with the best fit model that was
selected by Modeltest in MEGA 6.0 (Tamura et al., 2013).
2.2.8. Testing biological products on root-knot nematodes
2.3.8.1.Materials
Paecilomyces javanicus provided by Hanoi University of Sciences VNU, Lysobacter antibioticus from GCM products provided by of

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Chonnam National University (Korea)., 4-HPAA antibiotics provided by
Sigma company.
2.2.8.2. Evaluation of the ability to inhibit M. incognita: Methods were
performed according to Yoon et al., 2012 (adjusted).
2.2.8.3. Data analysis: hatching rate of eggs (%) and mortal rate of
juveniles (%) were transformed to Asin ((x / 100) ^ 1/2) and analyzed by
ANOVA in SPSS 20.

CHAPTER 3. RESUTL AND DISCUSSION
3.1. Frequency, distribution, damaging ability of Meloidogyne

3.1.1. Frequency
A total of 553 samples of 22 different host plants from Tay
Nguyen were analyzed and the result was showed in Table 3.1.
Appearance rate of Meloidogyne species on all the studied host plants
was 17/22 (55.6%). The highest appearance frequency was recorded on
pepper (84.3%), corresponding to 86/102 samples, followed by 83.3% on
tomatoes (25/30 samples), and 139/204 (68%) samples of coffee
recorded Meloidogyne spp. Root-knot nematodes were not recorded on
peanut, avocado, durian, and cassava. The symptoms on the roots of
different host plants were also different.
3.1.2. Distribution and density of root-knot nematodes
Based on morphological analysis based on the periniel pattern of
females, the tail shape of juveniles, the head of males and MultiplexPCR reaction, 7 species of the genus Meloidogyne have been recorded:
M. incognita, M. javanica, M. arenaria, M. enterolobii, M. graminicola
and 02 new species for science (Meloidogyne daklakensis and one
unpublished species)
3.2. Morphological characterisation and diversity of root-knot nematodes
(Meloidogyne spp.)
Morphological characteristics, measurements, microphotographs,
molecular characteristics, host plants and distribution of Meloidogyne
spp. from this study were described in detail and fully in the thesis.
Seven species of the genus Meloidogyne were recorded, namely M.
incognita, M. arenaria, M. javanica, M. enterolobii, M. graminicola, M.
daklakensis and Meloidogyne sp.
3.2.1. M. incognita (Kofoid & White, 1919) Chitwood, 1949
3.2.1.1. Morphological characterisation
Female: body pear-shaped, large 488 to 1151 µm long. Neck
short; stylet 10,6 to 16,4 µm long with stylet cone having anterior half

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distinctly curved dorsally. Perineal pattern oval to rounded, typically
with high dorsal arch; anus located 11 to 26 µm anterior to vulval slit;
lateral field weakly demarcated by breaks and forked striae; striae
distinct, wavy; dorsal striae condensed, smooth, wavy or zigzag;
phasmids distinct, distance between two phasmid12-24.2 µm.
Male: Labial region with large, rounded labial disc, raised above
median lips; labial disc concave to flat, high head cap nearly as wide as
labial region in lateral view; labial region usually marked by 2-5 annuli
but maybe completely smooth; labial region not distinctly separate from
rest of the body. Stylet 15 to 21 µm long.
Second-stage juveniles (J2): body 313-424 µm long. Labial
region not offset; labial disc elevated; lateral lips usually absent. Stylet
7.5-12.6 µm long. Tail 33.3-64.8 µm long with rounded tip; hyaline 9.217.8 µm long, ca 1/3 tail length.
3.2.1.2. Variation of morphological characteristics and diagnosis
Morphological and morphometric characteristics of females,
males and juveniles are highly variable between the population of M,
incognita from different hosts and locations. The characteristics of
periniel pattern in females, stylet, hyaline length in juveniles, and head
region of males are the least varied characters.
3.2.2. Meloidogyne javanica (Treub, 1885) Chitwood, 1949
3.2.2.1. Morphological characterisation
Female: body large, 522-1290 µm long, pear-shaped. Stylet
shaft cylindrical; stylet knobs not deeply indented. Dorsal striations low
and rounded to slightly squarish; perineal pattern quite unique with
distinct lateral ridges that divide the dorsal and the ventral striae, lateral
lines extended both sides of tail terminus cutting across perineal pattern.
Male: labial region with high, rounded head cap, distinctly offset
from labial region; labial disc and medial lips fused and form one

smooth, continuous structure that almost as wide as labial region in
lateral view; lip high 4.2-7.0µm. Stylet 17.8 to 19.7 µm long
Second-stage juveniles (J2): body long, slender. Head cap
anteriorly flattened; labial region posteriorly rounded; stylet knobs
transversely elongate, offset from stylet shaft. Tail 31-63 µm long,
narrow, tapering to tail end; finely rounded tail tip; hyaline distinct, 9.116.7 µm long.
3.2.2.2. Variation of morphological characteristics and diagnosis

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Morphological and morphometric characteristics of females,
males and juveniles are highly variable between the population of M.
javanica from different hosts and locations.
3.2.3. M. arenaria (Neal, 1889) Chitwood, 1949
3.2.3.1. Morphological characterisation
Female: body gourd-shaped, 630-773 µm long with short neck
protrudes anteriorly. Stylet broad and robust, 11.8-16.7µm long with
stylet cone having anterior half distinctly curved dorsally. Perineal
patterns with low dorsal arch slightly indented near lateral fields to form
rounded shoulders. No distinct lateral lines, striae short, irregular and
wavy, vulva length 16-26 21 µm.
Male: body long, vermiform; lateral lines distinct. Head cap low,
sloping posteriorly, labial region not offset from rest of body; lip high
from 4.2-5.8µm, stylet broad and straight, 14.3-20 µm long; posterior
part of stylet cone wider than stylet shaft; stylet shaft also increases in
width posteriorly; stylet knobs rounded; DGO relatively long.
Second-stage juveniles (J2): body long, slender. Head cap
anteriorly flattened but posteriorly rounded. Stylet cone and shaft wide
and robust; stylet knobs broad, rounded and posteriorly sloping. Tail

relatively long and slender, 39-60µm long; tail tip rounded. Hyaline 1215.7 µm long, hyaline indistinct; tail tip pointed.
3.2.3.2. Variation of morphological characteristics and diagnosis
In M. arenaria from our study, the characteristics such as body
length, body width, neck length, EP, periniel pattern characteristics in the
females; lip heigh, tail length, phasmid, b, b’, c, c’ in the males; sylet
length, tail length, hyaline length in the juveniles are the least varied
characteristics.
3.2.4. Meloidogyne enterolobii Yang & Eisenback., 1983
3.2.4.1. Morphological characterisation
Female: body white, pear-shaped to globubar; prominent neck
variable in length. Perineal pattern oaval-shaped with coarse and smooth
striae, dorsal arch moderately high often rounded or square; lateral lines
indistinct; perivulval region generally free of striae; tail tip visible,
phasmids large.
Male: body vermiform. Head cap high and rounded; labial
region slightly offset from body.

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Second-stage juveniles (J2): body vermiform, rather long.
Labial region slightly offset from body. Tail short, 34-48,9µm long; tail
tip rounded; hyaline 20-26.7 µm long.
3.2.4.2. Variation of morphological characteristics and diagnosis
Stylet length, DGO, and body length in juveniles are the least
varied measurements in different populations of M. enterolobii. These
measurements can be used together with the characteristics of periniel
pattern in identifying M. enterolobii.
3.2.5. Meloidogyne graminicola Golden & Birchfield 1965
3.2.5.1. Morphological characterisation

Female: Body pearly white, globular to pear-shaped with
relatively short neck. Stylet small, slender; knobs rounded, sloping
posteriotly. Secretory-excretory pore clearly distinct. Perineal pattern
egg-shaped; striae smooth, regular, continuously; dorsal arch low,
rounded, enclosing vulva region.
Male: body vermiform, 1256-2190 µm long. Labial region not
clearly offset from body; cuticular annulation distinct. Stylet stout with
rounded knobs. Stylet 13,2-17 µm long.
Second-stage juveniles ( J2): body vermiform, rather long (335400 µm). Labial region not offset from body. Tail small, 47.7-73.1µm
long; tail tip rounded; hyaline 16-24.4 µm long.
3.2.6. Meloidogyne daklakensis Trinh, Le, Nguyen, Nguyen, Liebanas &
Nguyen, 2018
3.2.6.1. Morphological characterisation
Female: body pearly white, pear-shaped and anterior body
portion commonly off–center from a median plane. Neck usually short,
not curved. Stylet short, cone base triangular and wider than shaft.
Secretory-excretory pore situated far behind from knobs of stylet within
range from level of procorpus to metacorpus. Perineal patterns of
females rounded to oval; striae smooth, regular and continuous; lateral
lines reduced, making thick prominent lateral part in SEM; dorsal arch
low, rounded and encloses distinct vulva, anus and tail tip; phasmids
indistinct; vulva slit centrally located at the un-striated area,longer than
vulva–anus distance.
Male: body vermiform, anterior end tapering and posterior region
bluntly rounded. Lip region slightly set-off from body with a high lip
region cap consists of a large labial annulus. Medial lips and labial disc
bow-tie shaped. Lateral lips large, triangular, slightly lower than labial

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disc and medial lips. Stylet robust, straight; one lateral knob projected,
two others sloping posteriorly. Testis one, occupying 58% of body
cavity. Spicules slightly curved ventrally with bluntly rounded terminus.
Gubernaculum short and crescentic shaped.

Fig 3.13. Photograph of Meloidogyne daklakensis
(A-E: female. A: body shape; B-C: labial region; D-E: perrineal pattern. F-I: J2.
F: labial region; G: lateral field; H-I: tail region; K-N: male. K, L: labial region;
M: lateral field; N: tail region; scalebar: A: 100 µm; B-N: 10 µm; B, C, E, F, G,
I, L, M, N: SEM; A, D, H, K: LM)

Second–stage juveniles (J2): body slender, tapering to an
elongated tail. Lip region narrow than body, weak and slightly offset
from body; labial disc offset; stoma slit–like; medial lips and labial disc
bow–tie shaped; labial disc rounded; amphidial openings enlarged,

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covered lateral lips. Stylet slender; cone weakly expanded at junction
with shaft; knobs rounded. Phasmids small, distinct. Tail elongateconoid.
3.2.7. Meloidogyne sp.
3.2.7.1. Morphological characterisation

Fig 3.16. Photograph of Meloidogyne sp
(A-E: female; A: shape; B-C: head; D-E: perrineal pattern. F-K: J2; F,G: head,
lip; H: line; I-K: tail; L-O:male L,M: head- lip; N: line; O: tail; scal bar:A: 100
µm; B-N: 10 µm; B, E, G, H, K, M, N, O: SEM; A, C, F, I, L: LM)

Female: body swollen with a small posterior protuberance,
pearly white, varying in shape, elongated from ovoid to saccate. Neck
prominent, bent at various angles to body. Stylet short; cone base

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triangular and wider than shaft; stylet tip normally straight, sometimes
slightly curved dorsally. Secretory-excretory pore located at level of
procorpus, behind from stylet knobs. Perineal patterns round to oval with
continuous smooth striae, and distinct whorl; lateral lines without distinct
incisures, sometimes appearing as a faint, discontinuous, sometimes
linear depression; dorsal arch low, rounded and encloses distinct vulva
and tail tip; phasmids large, distinct, far from tail terminus. The vulva slit
is centrally located at unstriated area, nearly as wide as the vulva-anus
distance; perivulval region free of striae. Tail terminus visible, wide,
surrounded by concentric circles of striae.
Male: body vermiform, anterior end tapering and posterior
region bluntly rounded. Head caps high and rounded, consists of a large
labial and two post-labial annuli, sometime with incomplete annuli. Lip
region continuous to body. Enface view of lip region show stoma slitlike, located in ovoid to hexagonal cavity surrounded by pit-like
openings of six inner labial sensilla. Subventral and subdorsal lips fuse to
form median lips, each lips with two cephalic sensilla. Lateral lips large,
triangular, lower than labial disc and medial lips. Testis one, occupying
58% of body cavity. Spicules slightly curved ventrally with bluntly
rounded terminus. The gubernaculum short and crescentic shaped. Tail
short, distinct phasmids at cloacal aperture level.
Second-stage juveniles (J2): body slender, tapering to an
elongated tail. Lip region narrower than body, weak and slightly set off.
Under SEM, prestoma opening rounded, surrounded by small, pore-like

openings of six inner labial sensilla. Medial lips and labial disc
dumbbell-shaped in face view. Tail conoid with rounded unstriated
terminus; hyaline clearly defined; rectum dilated; phasmids small,
distinct.
3.3. Morphological and morphometric variations of Meloidogyne
spp.
3.3.1. Analysis of morphometric variations in Meloidogyne spp.
Morphometrics of adult females, males and juveniles from the
same populations were used in a Canonical discriminant analysis (CDA).
For juveniles, among 16 quantitative characters, five quantitative
characters (lip height, DGO, hyaline length, b, and c’) were
taxonomically significant. For males, among 23 characters were used,
two characters (b’, c’) were taxonomically significant. There were three

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characters (Stl, EP, a) were taxonomically significant among 12 analyzed
characters in the females.
3.3.2. Morphological variations of Meloidogyne spp.

Fig 3.21. Variations in periniel patterns of Meloidogyne spp.
(Scalebar: A: 10 µm)

Fig 3.22. Variations in head regions of males of Meloidogyne spp.
(Scalebar: 10 µm)

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Fig 3.23. Variations in tail region of juveniles of Meloidogyne spp.,
(Arrows indicate the location of the phasmid, scale bar:10 µm)
3.4 . Genetic diversity of root knot nematodes (Meloidogyne spp)
3.4.1. Multiplex-PCR

Hình 3.24. MultiplexPCR results (M: Marker 100; J: M. javanica; A: M.
arenaria; I: M. incognita; 1-27: M. incognita; 28-29: M. graminicola; 30-36:
M. javanica; 37-40: M. daklakensis; 41-43: M. enterolobii; 44-48: M.
arenaria; 49-50: Meloidogyne sp; +, - )

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3.4.2. Molecular characterisation
3.4.2.1 ITS-rDNA

Fig 3.25.Phylogenetic relationships of Meloidogyne spp., based on
the ITS rDNA sequences using TN92+G model. Numbers on the
left of the each node are bootstrap values for 1000 replications.

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3.4.2.2 D2D3-rDNA

Fig 3.26. Phylogenetic relationships of Meloidogyne spp., based on
the D2-D3 of 28S rDNA sequences using K2+G model. Numbers
on the left of each node are bootstrap values for 1000 replications.

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3.4.2.3. COI mtDNA

Fig 3.27. Phylogenetic relationships of Meloidogyne spp., based on
the COI mtDNA sequences using GTR+G model. Numbers on the
left of each node are bootstrap values for 1000 replications.

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3.4.2.4. COII-16S mtDNA

Fig 3.28. Phylogenetic relationships of Meloidogyne spp., based on the
COII-16S mtDNA sequences using HKY+G+I model. Numbers on the
left of each node are bootstrap values for 1000 replications.

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3.4.2.5. NAD5 (NADH dehydrogenase subunit 5) mtDNA

Fig 3.29. Phylogenetic relationships of Meloidogyne spp., based on
the region of NAD5 sequences using GTR+G model. Numbers on
the left of each node are bootstrap values for 1000 replications.

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Discussion

In this study, the sequences from 5 gene regions of the tropical
group (M. incognita, M. javanica, M. arenaria, M. arabicida, M.
izalcoensis) was grouped together (clade Ia) with bootstrap 99% in ITS
rDNA sequences, 92% in D2-D3 of 28S rDNA sequences, 96% in COI
mtDNA sequences, 99% in COII-16S mtDNA sequences and 98% in
NAD5 mtDNA sequences, these species were recorded mostly in tropical
regions. The species in temperate group (M. hapla; M. fallax, M.
chitwoodi) were grouped together in other clade. M. incognita, M.
javanica, M. arenaria showed very closely related relationship in this
study that can be hardly differentiated (especially the similarity can be
100% in IGS region). Moreover, some studies showed the possibility to
hybridize M. incognita and M. arenaria proving their closely related
phylogenetic relationship.
Jansen et al. (2016) studied the regions of COI, COII, CO3,
NAD1, NAD3, NAD5 mtDNA and concluded that NAD5 gene can be
used to identify the species in tropical group (M. incognita, M. javanica,
and M. arenaria). Other species in clade Ia, such as M. ethiopica, M.
inornata, M. luci, can be identified by using COII-16S region.
Our study showed similar results to Jansen et al. (2016),
nucleotide variation in NAD5 gene were highest (1-2%) among analyzed
gene regions in M. incognita, M. javanica, and M. arenaria. Therefore,
this gene can be used to identify these species.
The sequences of M. enterolobii (specimen ID: 5342, 5349,
5352) showed the most closely related relationship to tropical group.
These results are in agreement with Tigano et al., 2005. The sequences of
M. enterolobii are clearly separated from the sequences of other species
in clade Ia, creating clade Ib with bootstrap 99% in ITS sequences, 92%
in D2D3 sequences, 99% in COI sequences, 100% in COII-16S and
NAD5 sequences.
Aside from species in clade I, the above gene regions can be

used to easily identify all other Meloidogyne species.Onkendi, Moleleki
(2013) proved ITS, D2D3 sequences can be used as efficient tool in
identifying M. marylandi, M. graminis, and M. naasi or M. enterolobii,
M. hapla, M. chitwoodi, and M. fallax. More closely related species can
be identified using mtDNA genes because the mtDNA genes evolve very
fast, but they are intraspecific highly conserved.

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Genetic variations within tropical group are low and the genetic
variations are higher in other species. Thus, rDNA genes (ITS, D2D3)
and mtDNA genes (COI, COII) can be used in identifying Meloidogyne
species except for M. incognita, M. javanica, M. arenaria. Only NAD5
gene can be used to separate the species in tropical group. Moreover,
specific primers can be used to identify M. incognita, M. javanica, and
M. arenaria quickly.
3.5. Testing preventative measures
3.5.1. Testing Paecylomyces javanicus on M. incognita
P. javanicus inhibited significantly the hatching rate of M.
incognita. The hatching rate reduced corresponding to the increase of the
concentration of P. javanicus. The hatching rates were 11.8%, 8.9% and
3.8% in different concentrations of P. javanicus compared to 39.1% in
distilled-water and 30% in 0.9% NaCl after 120hrs. The hatching rates in
distilled-water and 0.9% NaCl were insignificantly different.
After 24hrs, mortality rate of juveniles was highest in CT2 fomular
(16.6%). After 72hrs, the mortality rate was 6,3% in 0.9% NaCl, but can
be up to 65% in the experiments with P. javanicus and 87% after 168
hrs.

Effect of P. javanicus Table 3.29. Effect of P. javanicus to
mortality rate of juveniles of M.
to hatching rate of M. incognita
incognita
3.5.2. Testing Lysobacter antibioticus HS124 on M. incognita
After 48hrs, mortality rate of juveniles of M. incognita was 14.8% at
30% of Lysobacter antibioticus HS124, but the lower concentrations of
Lysobacter antibioticus HS124 had no effect on mortality rate of
juveniles of M. incognita. After 72hrs and 168hrs, the mortality rates of
juveniles of M. incognita were highest corresponding to 59.3 and 65.5%.
The hatching rate of M. incognita in distilled-water increased through the
Table 3.28.

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time was 59.6% after 120hrs and was 29.4% in 30% of Lysobacter
antibioticus HS124.

Table 3.30. Effect of L. antibioticus
HS124 to hatching rate of M.
incognita

Table 3.31. Effect of L. antibioticus
HS124 to mortality rate of juveniles of
M. incognita

3.5.3. Testing 4-HPAA on M. incognita
After 24hrs, the mortality rates were 31.9 and 37.7 % at 5 and 7.5
mg/ml of 4-HPAA, respectively. These mortality rates were significantly

different. At 2.5 and 5 mg/ml of 4-HPAA, mortality rate of M. incognita
were 98.1–98.2% after 72hrs. The results showed that 4-HPAA was
highly effective in controlling M. incognita.
After 24hrs, hatching rates in distilled-water and 1mg/ml of 4HPAA were insignificantly different (16.3% and 15.1%, respectively)
but in 5-7.5 mg/ml of 4-HPAA hatching rates was 0%.

Table 3.33.. Effect of 4-HPAA to
mortality rates of juveniles M.
incognita

Table 3.32. Effect of 4-HPAA to
hatching rate of M. incognita

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