The objectives of the thesis: To identify species composition of PPNs on carrots in major carrot growing areas in Vietnam. To determine the parasitic nematode group causing significant damage to carrots in the studied areas. To evaluate the ability to control some important PPNs on carrots by using antagonistic fungi in the laboratory conditions.
Trang 1MINISTRY OF EDUCATION
AND TRAINING
VIETNAM ACADEMY
OF SCIENCE AND TECHNOLOGY
GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY
-
NGUYEN THI DUYEN
PLANT-PARASITIC NEMATODES ON CARROTS IN VIETNAM AND TESTING BIOLOGICAL MEASURES IN CONTROLLING THEM
Trang 2This thesis can be found at:
- The library of the Graduate University of Science and Technology
- National Library of Viet Nam
Trang 3INTRODUCTION OF THESIS
1 The essence of the thesis
Carrots (Daucus carota L Him) are now widely grown and consumed worldwide due to their nutritional value and high medical value (Chen et al., 2016) In Vietnam, Lam Dong and Hai Duong have the largest area of cultivation However, in carrot-grown areas, the damage caused by plant-parasitic nematodes (PPNs) has had a negative effect on quality and productivity Currently, researches of the PPNs on carrots are relatively limited in Vietnam
In order to prevent harmful PPNs on carrots, chemical nematicides are still the prevalent choices However, the abuse of harmful chemicals has been causing negative effect to environment, human health and other organisms (Meira et al 2006) Biological measures are one of the effective measures to prevent PPNs and are of environmental safety (Perry & Moens, 2013) Therefore, it is necessary to select environmentally friendly biological products for nematode control
For the purpose of studying the PPN species composition and important nematode groups on carrots, assessing biological products for their control to provide a basis for the management of PPNs on carrots, we carry out the
research project: "Plant-parasitic nematodes on carrots in Vietnam and testing biological measures in controlling them"
2 The objectives of the thesis
- To identify species composition of PPNs on carrots in major carrot growing
areas in Vietnam
- To determine the parasitic nematode group causing significant damage to
carrots in the studied areas
- To evaluate the ability to control some important PPNs on carrots by using antagonistic fungi in the laboratory conditions
3 Scientific and practical meaning of the thesis
Scientific meaning:
- The thesis provided the composition and distribution of PPNs on carrots in some carrot cultivation areas in Vietnam, and added the species list of PPNs on carrot for the world The thesis provided morphological and molecular data of important PPNs on carrots Sequences of 19 populations of PPN on carrot in Vietnam have been deposited to Genbank
- A new species, a new genus, and 16 new records of PPNs on carrot in Vietnam were reported
- Important groups of PPNs on carrots in studied areas were determined
Trang 44
- Initially, scientific evidences of the effect of Paecilomyces sp and Lentinus
squarrosulus on two important PPNs species in the laboratory were given
Practical meaning:
- Identified of the main group of PPNs on carrots is the basis for the selection
of control measures Described specific symptoms caused by nematodes on carrots can be used as quick diagnoses Key nematode groups affecting the quality of carrots in cultivation and trade in Vietnam were determined
- Evaluated the ability to control PPNs of antagonistic fungi as a basis for the selection of effective biological control measures to reduce the amount of chemicals and to develop a sustainable agricultural ecosystem
- Data of harmful PPNs on carrots in the dissertation can be used as a source of material in teaching, as well as helping farmers to have the references in carrot production
4 The main contents of the thesis
Content 1: Determination of PPNs composition on carrots based on
morphological and molecular characteristics
Content 2: Determination of the most important PPN groups on carrots in
studied areas
Content 3: Testing Paecilomyces sp and L squarrosulus on the main
nematode groups in the laboratory
5 The main contents of the thesis
The thesis is composed of 157 pages: Introduction (6 pages); chapter 1: Literature review (19 pages); chapter 2: Methodology (9 pages); chapter 3: Resutl and discussion (121 pages); Conclusion and recommendation (2 pages) The thesis consists of: 34 tables, 49 figures and 268 references In addition, the thesis has a list of publications and appendices
CHAPTER 1 LITERATURE REVIEW
1.1 PPNs on carrots in the world
1.1.1 Important group of PPNs on carrots
In the world, PPNs on carrots have been studied since the 50s of the last century These studies were mainly concentrated in Europe, America and Australia So far, 21 genera of PPNs on carrot have been recorded (Nobbs,
2003) Particularly, Meloidogyne and Pratylenchus are the common harmful
groups in many carrot cultivation areas such as USA, Germany, Australia, Canada, etc, but the species composition was little known
1.1.2 Morphological and molecular identification of PPNs
Trang 5The combination of morphological characteristics and molecular characterization has made species identification more accurate D2D3 extension segment of 28S rDNA region was commonly used to classify some plant nematode varieties (Subbotin et al., 2008; Van Den Berg et al., 2015)
1.1.3 Controlling PPNs by using antagonistic fungi
Paecilomyces spp is saprophytic fungus in the soil, most interested and
studied, due to its parasitic potential and ability to control PPNs (Brand et al.,
2010) Mechanism of fungus Paecilomyces spp is directly parasitic by hyphae
(Jones et al., 1984), and produces substances and enzymes that are capable of decomposing the chitin layer of eggs and nematodes, creating favorable conditions for invasive fungus (Mukhtar et al., 2013)
L squarrosulus is an edible, nutritious mushroom (Omar et al., 2011)
There is no study on the ability of L squarrosulus to kill nematodes However,
in fluid culture of L squarrosulus, high levels of chitinase enzyme was found,
which can breaks down the kitin layer of nematode and their eggs (Deborah, 2018) and contains the anti-nematode ingredient 1,2 - dihydroxymintlactone (Isaka et al., 2011), phenolic (Ghatr & Sridhar, 2017)
1.2 PPNs on carrots in Vietnam
In Vietnam, the composition of PPNs on carrots has not been studied much According to N.N Chau & N.V Thanh (2000), 12 species of PPNs belonging
to 4 genera on carrots in Lam Dong were recorded In Hai Duong, V.D Phien
(2014) reported Meloidogyne and Pratylenchus on carrots in Hai Duong The
morphological characteristics is the most important diagnosis for PPN species
In addition, molecular techniques have been applied in nematode classification, making the identification to species level more accurate On the basis of DNA analysis combined with morphological characteristics, some
new species have been published for science such as R duriophilus,
R daklakensis, M daklakensis, (Trinh et al., 2004; 2012; 2018)
CHAPTER 2 MATERIALS AND METHODOLOGY
2.1 Materials and study locations
2.1.1 Study Materials
- PPNs around the rhizosphere of carrots
- Daucus carota L Him
- Paecilomyces sp and L squarrosulus
2.1.2 Locations and studying time
- 7 localities of 4 provinces were chosen for this study: Dong Anh (Ha Noi); Cam Giang and Nam Sach (Hai Duong); Van Giang (Hung Yen); Duc
Trang 66
Trong, Don Duong and Da Lat (Lam Dong) The study was conducted at the Institute of Ecology and Biological Resources The thesis is conducted from October 2014 to October 2018
Nematodes were extracted from soil and root samples following the method
of N.N Chau & N.V Thanh (1993)
2.2.3 Culturing
- Meloidogyne spp were cultured on tomatoes following López-Pérez et al (2011)
- Pratylenchus spp were cultured on carrot plates following Coyne et al (2014)
2.2.4 Fixation and mounting
Nematodes were fixed according to Courtney et al (1995), dehydrated and mounted according to the description of N.N Chau (2003)
2.2.8 Testing the efficiency of antagonistic fungi on PPNs
Experimental evaluation of the efficiency of antagonistic fungi on PPNs was conducted following the method described by Pau et al (2012)
2.2.9 Data analysis
The following softwares were used to analyze data: i) Morphometric analysis: Microsoft Excel, GENSTAT 12 (Payne et al., 2009); ii) molecular analysis: BLAST, BioEdit (Hall, 1999), MEGA 6 (Tamura et al., 2013); iii) statistic analysis: SPSS +
Trang 7CHAPTER 3 RESUTL AND DISCUSSION
3.1 List of PPNs on carrots in Vietnam
Morphological and molecular identification determined 25 species belong
to 15 genera, 9 families, and 4 orders (table 3.1)
Table 3.1 List of PPN species on carrots in studied areas
ORDER TYLENCHIDA THORNE, 1949
Family Belonolailidae Whitehead, 1960
Genus Tylenchorhynchus Cobb, 1913
1 T mashhoodi Siddiqi & Basir, 1959 + +
2 T annulatus Golden, 1971 + + + +
Family Hoplolaimidae Filipjev, 1934
Genus Helicotylenchus Steiner, 1945
3 H dihystera (Cobb 1893) Sher, 1961 +
4 H indicus Siddiqi, 1963 + + +
Genus Hoplolaimus Von Daday, 1905
5 H chambus Jairajpuri & Baqri, 1973 +
Genus Rotylenchulus Linford & Oliveira, 1940
6 R reniformis Linford & Oliveira, 1940 + + + +
Family Anguinidae Nicol, 1935 (1926)
Genus Ditylenchus Filipjev, 1936
Family Criconematidae Thorne, 1949
Genus Hemicriconemoides Chitwood & Birchfield, 1957
8 H strictathecatus Esser, 1960 + +
Genus Hemicaloosia Ray & Das, 1978
Genus Mesocriconema Andrássy, 1965
10 M sphaerocephalum (Taylor, 1936) Loof, 1989 + +
Family Meloidogynidae Filipjev, 1934
Genus Meloidogyne Goeldi, 1892
11 M incognita Chitwood, 1949 + + + +
12 M arenaria Chitwood, 1949 +
13 M graminicola Golden & Birchfield 1965 +
Family Pratylenchidae Thorne, 1949
Genus Pratylenchus Filipjev, 1936
14 P thornei Sher & Allen, 1953 +
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15 P zeae Graham, 1951 + +
16 P haiduongensis Nguyen et al., 2017 +
17 P coffeae Filipjev & Sch Stekhoven, 1941 +
18 P penestran Filipjev & Sch Stekhoven, 1941 +
ORDER DORYLAIMIDA PEARSE, 1942
Family Longidoridae Thorne, 1935
Genus Longidorus Micoletzky, 1922
Genus Paralongidorus Siddiqi, 1974
Genus Xiphinema Cobb, 1913
23 X brevicolle Lordello & Da Costa, 1961 +
ORDER TRIPLONCHIDA COBB, 1920
Family Diphterophoridae (Micoletzki, 1922)
Genus Diphterophora de Man, 1880
ORDER APHELENCHIDA SIDDIQI, 1980
Family Aphelenchoididae Skarbilovich, 1947
Genus Aphelenchoides Fischer, 1894
In this study, seven genera of PPNs on carrots were added to PPNs’ list of
Lam Dong: Ditylenchus, Tylenchorhychus, Rotylenchulus, Meloidogyne,
Hemicaloosia, Xiphinema and Diphterophora Among 15 genera of PPNs
from four studied areas, six genera have never been recorded in the world:
Diphterophora and Aphelenchoides
3.2 Morphological and molecular characteristics of PPNs on carrots
Morphological characteristics, measurements, microphotographs, molecular characteristics, host plants and distribution of PPNs on carrots from this study were described in detail and fully in the thesis
Trang 93.2.1 Tylenchorhynchus Cobb, 1913
Two species of the genus Tylenchorhynchus were recorded, namely
T annulatus and T mashoodi
T annulatus were recorded in three carrot cultivation areas in Hanoi, Hai
Duong and Lam Dong The population from Hanoi was biggest in the body length, stylet length, distance from anterior end to secretory-excretory pore and distance from anterior end to end of pharyngeal gland The measurements
of nematodes from these populations are in agreement with that of T
annulatus Geraert (2010) Few variations were observed compared to the
measurements from previous study in Vietnam (Chau & Thanh, 2000), such as larger a, c’value and stylet length
T mashhoodi were recorded in two carrot cultivation areas in Hai Duong
and Hung Yen These populations have similar morphological characteristics
compared to the original population of T mashhoodi Siddiqi & Basir, 1959
Compared to the previously described population in Viet Nam, two newly isolated populations were smaller in body length and a value (Chau & Thanh, 2000)
T annulatus can be differentiated from T mashhoodi by the following
traits: larger body length and distance from anterior end to nerve ring; labial
region is continuous to body contour vs slightly set off; secretory-excretory pore located at isthmus level vs secretory-excretory pore located at lower position; long cylindrical tail, bluntly rounded tail tip, reduced spermatheca, withou sperm, male was not found vs short conical tail, narrowed tail tip, large rounded spermatheca, full of sperm and males were present
3.2.2 Helicotylenchus Steiner, 1945
Two species of the genus Helicotylenchus were recorded on carrot, namely
H dihystera and H indicus
A population of H dihytera was isolated from carrots in Hai Duong
Compared to the populations described by Sher (1966) and Chau &Thanh
(2000), the population of H dihytera from carrots in Hai Duong has similar
morphological and morphometric characteristics, except for some small variations such as: smaller a, c value and stylet length
H indicus were recorded in three carrot cultivation areas in Vietnam:
Hanoi, Hung Yen and Lam Dong All three populations of H indicus on carrot
have similar morphological and morphometric features and the variations are not significantly different from the original population described by Siddiqi,
1963 as well as the population described by Chau & Thanh (2000)
H indicus differs from H dihystera by phasmid position Phasmid of H
Trang 1010
indicus located 1 - 4 annuli anterior to anus level Phasmid of H dihystera
located 8 - 11 annuli anterior to anus level
3.2.3 Hoplolaimus Von Daday, 1905
One species of the genus Hoplolaimus, H chambus, was recorded Only one
female of H chambus was found in Hai Duong The morphological
characteristics and measurements of this specimen are similar to the original description of Jairajpuri & Baqri, 1973 and the description of Chau & Thanh,
2000 However, the b value of this specimen is slightly smaller (Jairajpuri & Baqri, 1973; N.N Chau & N.V Thanh, 2000)
3.2.4 Rotylenchulus Linford & Oliveira, 1940
A species R reniformis of the genus Rotylenchulus was recorded This species was recorded in all 4 carrot cultivation areas Four populations of R
reniformis on carrots showed similar morphological and morphometric
characteristics compared to the description of Dasgupta et al (1968) as well as the description of Chau & Thanh (2000)
3.2.5 Hemicriconemoides Chitwood & Birchfield, 1957
H strictathecatus was recorded in Hai Duong and Hung Yen The
population from Hung Yen was smaller in body length compared to the
population from Hai Duong Two populations of H strictathecatus on carrots
in Vietnam showed similar morphological and morphometric characteristics
compared to the descriptions of Esser (1960) and Geraert (2010) H
strictathecatus has similar morphological and morphometric characteristics
compared to H litchi and H mangiferae However, Decraemer & Geraert
(1992, 1996), Crozzoli and Lamberti (2003) and most recently Van Den Berg
et al (2015) have combined these two species into the species namely H
strictathecatus due to the overlap in morphology and morphometry
Molecular charaterisation
The D2-D3 of 28S rDNA sequence of H strictathecatus on carrot is 95-99% similar to the D2-D3 of 28S rDNA sequences of H strictathecatus from GenBank The variation of D2-D3 of 28S rDNA sequence of H strictathecatus
on carrot varied 1-2% compared to other sequences of H strictathecatus (KM516173 - KM516178) and 5% compared to the sequences of H litchi
(AY780956, KF856540, MH142618 and KP192481) However, these two
species have been combined into one species, namely H strictathecatus The D2-D3 of 28S rDNA sequence of H strictathecatus on carrot was grouped
together with the sequences of H strictathecatus (96% bootstrap)
Trang 11Figure 3.1 ML phylogenetic tree (TN93+G model) based on D2-D3 of 28S
population in Vietnam, the populations of M sphaerocephalum on carrots
have some differences Measurements such as body length; a, c values; V and stylet varies widely Meanwhile, the values such as b, R, Rex are smaller (N.N Chau & N.V Thanh, 2000)
Molecular characterisation
KM516177 H strictathecatus KM516176 H strictathecatus KM516174 H strictathecatus KM516173 H strictathecatus KM516175 H strictathecatus
H strictathecatus H4359
MH142613 H strictathecatus KP192481 H strictathecatus AY780956 H strictathecatus KF856540 H strictathecatus MG029574 H parataiwanensis MG029573 H parataiwanensis MG029572 H parataiwanensis MG029571 H paracamelliae MG029570 H paracamelliae KF856534 H chitwoodi KF856533 H chitwoodi KF856539 H californianus KF856538 H californianus MG029576 H kanayaensis MG029575 H kanayaensis KF856521 H wessoni KF856520 H wessoni KF856519 H wessoni KF856527 H promissus KF856529 H promissus KF856522 H macrodorus
KT381016 Hemicaloosia guangzhouensis KT381017 H.guangzhouensis FN433861 Mesocriconema xenoplax FN433869 M xenoplax FN433856 M xenoplax DQ328717 Basiria gracilis AY780979 Aglenchus agricola
100
100
87 99
84 100 100
100
100
100
100 99
94
76 100
90 84 89 83
99 66 84 100
94
89 96 99
0.1
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The D2-D3 of 28S rDNA sequence of M sphaerocephalum (specimen ID: M4694) isolated in Hai Duong is 99% similar to M sphaerocephalum
(AB933464) on GenBank The D2-D3 of 28S rDNA sequence of
M sphaerocephalum varied 0-2% compared to M sphaerocephalum
(AB933464) on GenBank This sequence was placed together with
M sphaerocephalum from GenBank; this clade was completely separate from
other species
Figure 3.2 ML phylogenetic tree (TN93+G model) based on D2-D3 of 28S
rDNA sequences of Mesocriconema spp
3.2.7 Hemicaloosia Ray & Das, 1978
One species of the genus Hemicaloosia was recorded on carrot, namely
Hemicaloosia sp According to the identification key of Chitambar & Subbotin
(2014) Hemicaloosia sp is most similar to H luci by the following
characteristics: body length, R, Rex, stylet length and tail shape However,
Hemicaloosia sp differs from H luci by the following feartures: smaller a, b,
c, V values; larger distance from anterior end to secretory-excretory pore,
Rvan and Ran The tail of Hemicaloosia sp is also longer and more pointed compared to H luci This species is probably an unknown species
3.2.8 Xiphinema Cobb, 1913
A species of the genus Xiphinema was recorded on carrot, namely
Mesocriconema sphaerocephalum M4694
AB933464 M.sphaerocephalum AY780950 M.sphaerocephalum AB933464 M.sphaerocephalum AB933465 M.sphaerocephalum AB933465 M.sphaerocephalum AB933466 M.sphaerocephalum AB933467 M.sphaerocephalum KF430522 M.sphaerocephalum AY780969 M.solivagum AY780968 M.ornatum AY780967 Mesocriconema sp Vovlas AY780965 M.xenoplax
FN433855 M.xenoplax AY780964 M.xenoplax FN433869 M.xenoplax FN433856 M.xenoplax FN433871 M.xenoplax DQ328717 Basiria gracilis AY780979 Aglenchus agricola
Trang 13morphological and morphometric characteristics of this specimen are in
agreement with the description of Xiphinema brevicolle of Chau & Thanh (2000) and Luc & Coomans (1992)
3.2.9 Meloidogyne Goeldi, 1892
Three species of the genus Meloidogyne were recorded on carrot, namely
M incognita, M arenaria and M graminicola
M incognita was recorded in all 4 areas of carrot cultivation The
measurements of M incognita on carrots are mostly in agreement with the
original description of Chitwood (1949) Few variations were observed such as: slightly larger body length and stylet length of the juveniles; the a value of the males is larger; the a, c of the juveniles and values of the males and stylet length of the males and females are smaller These populations are similar to those in Vietnam described by Chau & Thanh, 2000
A population of M arenaria was recorded in Hai Duong This population is
mostly similar to the original description of Chitwood (1949) except for the slightly smaller body length of females, males, and juveniles, b value in males and juveniles, larger c value and stylet length in juveniles Compared to the description of Chau & Thanh (2000), the stylet length of this population is smaller
A population of M graminicola was recorded in Ha Noi Few variation were observed from the population of M graminicola on carrots compared to
the description of Golden & Birchfield (1965) such as: smaller body length of females and juveniles, and stylet length of juveniles; larger stylet length of females, body length and stylet length of the males In comparison with the
description of Chau & Thanh (2000), the stylet length of females, males and
juveniles and c value of juveniles of the population on carrots are smaller; larger a value of juveniles
Three species of Meloidogyne on carrots can be differentiated from each
other by the following traits:
- Female: M arenaria pear-shaped, short neck and narrowed at mid-body
M incognita and M.graminicola have rounded body, short neck However,
M incognita has larger body length compared to M graminicola M incognita
with high squared dorsal arch, smooth rounded ventral striae, discontinuously
M arenaria with low dorsal arch, striae formed wings M graminicola with
rounded striae, smooth, continuously, low dorsal arch M incognita has secretory-excretory pore located around basal knob level, M arenaria and
M graminicola has secretory-excretory pore located far posterior to basal knob
- Juvenile: M graminicola has more pointed tail and longer hyalin compared
to two other species Phasmid position of M incognita located around the