Summary of biology doctoral thesis: Plant-parasitic nematodes on carrots in Vietnam and testing biological measures in controlling them

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.

MINISTRY OF EDUCATION VIETNAM ACADEMY AND TRAINING 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 Major: Nematology Code: pilot SUMMARY OF BIOLOGY DOCTORAL THESIS HA NOI – 2018 The thesis was completed at the Graduate University of Science and Technology, Vietnam Academy of Science and Technology Scientific Supervisor 1: Assoc Prof Dr Nguyen Ngoc Chau Scientific Supervisor 2: Dr Trinh Quang Phap Reviewer 1: … Reviewer 2: … Reviewer 3: … The dissertation will be defended protected 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 … 2018 This thesis can be found at: - The library of the Graduate University of Science and Technology - National Library of Viet Nam INTRODUCTION OF THESIS 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 plantparasitic 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" 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 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 - 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 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 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 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 The 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 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 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 - localities of provinces were chosen for this study: Dong Anh (Ha Noi); Cam Giang and Nam Sach (Hai Duong); Van Giang (Hung Yen); Duc 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 2.2 Methods 2.2.1 Sampling The samples were collected randomly following a cross-diagonal pattern (N.N.Chau, 2003; Perry & Moens, 2013) 2.2.2 Extraction 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) 2.2.5 SEM Nematode samples were prepaired for taking SEM pictures according to Abolafia (2015) 2.2.6 Morphological identification Morphological identification was based on Siddiqi (2000) Classification key of N.N Chau & N.V Thanh (2000), Castillo & Vovlas (2007), Kazachenko & Mukhina (2013), Geraert (2010; 2011), Chitambar & Subbotin (2014) were used to identify to species level 2.2.7 Molecular analysis DNA were extracted according to Holterman et al (2009) Target genes were amplified according to De Ley et al (1999) PCR products were purified by GeneJet PCR Purification kit and sent to the Macrogen Company – Korea for sequencing 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 (Tamura et al., 2013); iii) statistic analysis: SPSS + CHAPTER RESUTL AND DISCUSSION 3.1 List of PPNs on carrots in Vietnam Morphological and molecular identification determined 25 species belong to 15 genera, families, and orders (table 3.1) Table 3.1 List of PPN species on carrots in studied areas TT 10 11 12 13 14 Tên loài ORDER TYLENCHIDA THORNE, 1949 Family Belonolailidae Whitehead, 1960 Genus Tylenchorhynchus Cobb, 1913 T mashhoodi Siddiqi & Basir, 1959 T annulatus Golden, 1971 Family Hoplolaimidae Filipjev, 1934 Genus Helicotylenchus Steiner, 1945 H dihystera (Cobb 1893) Sher, 1961 H indicus Siddiqi, 1963 Genus Hoplolaimus Von Daday, 1905 H chambus Jairajpuri & Baqri, 1973 Genus Rotylenchulus Linford & Oliveira, 1940 R reniformis Linford & Oliveira, 1940 Family Anguinidae Nicol, 1935 (1926) Genus Ditylenchus Filipjev, 1936 Ditylenchus sp Family Criconematidae Thorne, 1949 Genus Hemicriconemoides Chitwood & Birchfield, 1957 H strictathecatus Esser, 1960 Genus Hemicaloosia Ray & Das, 1978 Hemicaloosia sp Genus Mesocriconema Andrássy, 1965 M sphaerocephalum (Taylor, 1936) Loof, 1989 Family Meloidogynidae Filipjev, 1934 Genus Meloidogyne Goeldi, 1892 M incognita Chitwood, 1949 M arenaria Chitwood, 1949 M graminicola Golden & Birchfield 1965 Family Pratylenchidae Thorne, 1949 Genus Pratylenchus Filipjev, 1936 P thornei Sher & Allen, 1953 HN HD HY LD + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 15 16 17 18 19 20 21 22 23 24 25 P zeae Graham, 1951 P haiduongensis Nguyen et al., 2017 P coffeae Filipjev & Sch Stekhoven, 1941 P penestran Filipjev & Sch Stekhoven, 1941 Pratylenchus sp Pratylenchus sp ORDER DORYLAIMIDA PEARSE, 1942 Family Longidoridae Thorne, 1935 Genus Longidorus Micoletzky, 1922 Longidorus sp Genus Paralongidorus Siddiqi, 1974 Paralongidorus sp Genus Xiphinema Cobb, 1913 X brevicolle Lordello & Da Costa, 1961 ORDER TRIPLONCHIDA COBB, 1920 Family Diphterophoridae (Micoletzki, 1922) Genus Diphterophora de Man, 1880 Diphterophora sp ORDER APHELENCHIDA SIDDIQI, 1980 Family Aphelenchoididae Skarbilovich, 1947 Genus Aphelenchoides Fischer, 1894 Aphelenchoides sp + + + + + + + + + + + + + Note: “+” symbol presenting availability; HN: Ha Noi, HD: Hai Duong, HY: Hung Yen and LD: Lam Dong The highest number of PPNs on carrots was recorded in Hai Duong with 16 species that belong to 12 genera, followed by Lam Dong with 12 species of 10 genera species of genera were recorded in Hanoi species of genera were recorded in Hung Yen These results are also the first records of PPNs on carrots in Hanoi, Hung Yen and Hai Duong 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: Hemicriconemoides, Hemicaloosia, Hoplolaimus, Paralongidorus, 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 3.2.1 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 indicus located - annuli anterior to anus level Phasmid of H dihystera located - 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 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) 10 morphological 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 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 13 center of hyaline region, while the phasmid of M arenaria located above hyaline and the phasmid of M graminicola located near tail tip Molecular characterisation: The D2-D3 sequence of populations (ID: M4859, M3656, M4907 and M5269) are 100% similar compared to the populations of M incognita on GenBank The population M4806 is 100% similar to M arenaria and the population M4856 is 99% similar to M graminicola on GenBank KY882485 M morocciensis KP306531 M incognita KY882486 M morocciensis KX752373 M javanica KX752374 M javanica KX752375 M javanica KX752330 M incognita 96 KX752332 M incognita M incognita M3656 M incognita M5269 M incognita M4859 KX752382 M incognita 99 M incognita M4907 KX752383 M incognita KX151138 M arenaria EU364889 M arenaria 93 AF435803 M arenaria M arienara M4806 KP901082 M arenaria KY882484 M ethiopica 91 KY882481 M ethiopica 99 KF482372 M ethiopica KP411230 M enterolobii KX823404 M enterolobii 99 KX823403 M enterolobii 91 KX823401 M enterolobii KX823400 M enterolobii 95 KC241978 M minor KC241976 M minor KC241979 M naasi 96 JN019266 M naasi 84 KP901068 M naasi M graminicola M4856 94 KY660544 M graminicola 97 KY250089 M graminicola KY660545 M graminicola KF751066 M graminicola 99 JX465616 M trifoliophila JX465617 M trifoliophila 66 JX465618 M trifoliophila EU555406 Radopholus similis 0.05 Figure 3.3 ML phylogenetic tree (HKY+G model) based on the D2-D3 of 28S rDNA sequences of Meloidogyne spp There is no nucleotide variation between the populations M4859, M3656, M4907 and M5269, the variation are only to varied nucleotides compared to the population M4806, and varied from 72 to 73 nucleotides compared to the population M4856 14 The sequences of the populations M4859, M3656, M4907 and M5269 were placed in the same clade with M incognita from GenBank The sequence of the population M4806 was placed together with M arenaria Notably, the sequences of M incognita and M arenaria were placed in the same clade and separate from other species The sequence of M4856 was placed together with M graminicola 3.2.10 Pratylenchus Filipjev, 1936 species of Pratylenchus were recorded on carrot, namely P coffeae, P penetrans, P thornei, P zeae, P haiduongensis, Pratylenchus sp and Pratylenchus sp Pratylenchus coffeae A population of P coffeae was recorded in Hung Yen The population of P coffeae on carrots showed few variation compared to the previously described population of P coffeae in Vietnam such as: smaller body length, stylet length and V, b, c values in the females The tail tip shapes of this population vary from smooth to truncate The population of P coffeae also showed some variations compared to the populations of Sher & Allen, (1953) and Ryss, (1988) such as: smaller body length, stylet length and V value in females Pratylenchus penetrans P penetrans was recorded in a carrot cultivation area in Lam Dong The population of P penetrans on carrots is mostly similar to the population of Loof (1960) and Vovlas & Troccoli (1990) Few variations were observed such as: smaller body length, stylet length and a, c, V values in the females In comparison with the population of Chau & Thanh (2000), a and b values are larger Pratylenchus thornei One female of P thornei was isolated from carrot in Hai Duong with specimen ID P4358 The morphology and morphometry of the specimen P4358 isolated from carrot are totally in agreement with the description of P thornei of Sher & Allen, 1953 In comparison with the population of P thornei of Chau & Thanh (2000), the specimen on carrot has smaller body length, stylet length and c value Pratylenchus zeae populations of P zeae were isolated from carrots: population code P4854 isolated in Hanoi and population P3656 isolated in Hai Duong These populations have smaller body length, stylet length, a and b values compared 15 to the original description (Graham, 1951); Body length, b and V value were larger than previously isolated population in Vietnam (Chau & Thanh, 2000) Pratylenchus haiduongensis Nguyen et al., 2017 population codes: P3655, P3658 and P4728 were isolated in Hai Duong These three populations belong to a new species (described as P haiduongensis) and have distinct morphological characteristics from all other species according to Castillo & Vovlas (2007) This species can be described by the following morphological characteristics: Female: Head region bearing three annuli On the en face view showed by the SEM, lateral margins of oral disc were distinct and prominent, subdorsal and subventral segments slightly separated from the labial disc by two grooves Stylet is robust and strongly sclerotized Lateral field with four lateral lines at midbody, central band sometimes illustrated with oblique striae Lateral field bearing areolation at the pharynx region and tail region and sometimes appears in the vulval region Spermatheca ovalin shape with round central cavity, without sperm or reduced in some specimens Postuterine branch is long, ca - 3,5 times body width at vulva Vulva position is high (66-75%) The tail shape can be subhemispherical with Figure 3.4 Pratylenchus haiduongensis a smooth terminus, slightly ndented, (LM ♀) A: Pharyngeal region, B-D: broadly smooth, or a cleft terminus Stylet, F-G: Lateral field, E: Post-uterine observed sac, H-I: Genital tract, J-L: Tail, L: Male: Not found phasmids (Scalebar = 10 µm) P haiduongensis is most similar to P parazeae (Wang et al., 2015) However, P haiduongensis can be differentiated from P parazeae (Wang et al., 2015) by having smaller body length and lip width; slightly separated lip region with slightly separated labial disc vs continuous lip region with plain 16 and smooth labial disc; labial region with distinct and prominent lateral margins of oral disc, subdorsal and subventral segments are slightly separated from the labial disc by two grooves, and four short lines marked in ventral and dorsal segment vs smooth and undivided labial region; longer stylet; stylet knobs are rounded or slightly anteriorly flattened, never indented anteriorly vs rounded to indented anteriorly knobs; and the lateral field having prominent ridges vs lateral field having narrow incisures Pratylenchus sp Pratylenchus sp was isolated on carrots in Lam Dong with population codes: P4861, P4981, P5189 and P5208 Morphological and morphometric characteristics of these four populations can be described as below: Figure 3.5 Pratylenchus sp (Scalebar: 10 µm) (LM ♀) A: Pharyngeal region, B-C: Stylet, D: Lateral field, E: Spermatheca, G: Post-uterine sac, H-I: Tail region, K: phasmids (LM ♂) J: Tail region, L: phasmids Female: Head region bearing three annuli Stylet 14 - 16 µm long, strong, robust; basal knobs rounded or sloping anteriorly Lateral field with lateral lines forming ridges, ca one-third body width Outer part of lateral field with areolation, central ridge without broken lines Spermatheca oval to rounded, full of sperms Post-uterine sac short, ca 0.8 - 1.5 times body width at vulva Vulva position low, located 77 - 82% lower part of body Phasmids located at one-second of tail Tail long, cylindrical, subcylindrical, or clavated; 19 - 28 annuli at ventral side Tail tip varied in shape: subcylindrical with conical tail tip; subcylindrical with rounded, smooth tail tip; concave tail with smooth tail tip; clavated tail with smooth tail tip; truncate tail tip; subcylindrical with crenated tail tip 17 Male: similar to females except for sexual characteristics According to the identification key of Castillo & Vovlas (2007), Pratylenchus sp is most closely resembled to P penetrans However, Pratylenchus sp differs from P penetrans by the following feartures: (1) Lateral field without broken lines; (2) Tail shape varies from smooth to truncate; and (3) The appearance of a groove in submedian lip (Figure 3.6) Figure 3.6 Enface structure of Pratylenchus sp and P penetrans (Hernándezet al., 2000) Pratylenchus sp Pratylenchus sp was isolated on carrots in Da Lat - Lam Dong with specimen ID: P3130 This population can be described by the following morphological characteristics: Female: Labial region bearing three annuli Labial disc separated from labial region Stylet strong, 15.2 - 16.6 µm long, basal knob rounded or sloping anteriorly Lateral field with lines (sometimes divided to lines) forming ridges, center ridge large without broken lines; Lateral field with areolation Spermatheca well developed, rounded or oval, full of sperm Post-uterine sac short, ca - 1.5 times body width Figure 3.7 Pratylenchus sp Vulval position low (77 - 83%), (Scalebar:10 µm) vulva lips short Tail (LM ♀) A: vùng Pharyngeal region, B-C: subcylindrical with rounded or Stylet, D: Lateral field, E: Spermatheca, conical tail tip, smooth, 18 - 30 F: Post-uterine sac, G-H: Tail region annuli ventral side (LM ♂) J: Tail region 18 Male: similar to females except for sexual characteristics According to the identification key of Castillo & Vovlas (2007), Pratylenchus sp is most closely resembled to P penetrans However, Pratylenchus sp can be distinguished from P penetrans by the following traits: (1) Lateral field without broken lines, sometimes divided to lines vs Lateral field with lines, with broken lines; (2) Submedian lip separated from lateral lips by grooves in a obtuse angle vs perpendicular grooves (Figure 3.8) Figure 3.8 Enface structure of Pratylenchus sp and P penetrans (Hernándezet al., 2000) Pratylenchus sp differs from Pratylenchus sp by the following features: (1) Tail tip varies in shape; (2) Lateral field sometimes with lines vs Lateral field with lines; (3) The absence of a groove in submedian lip (Figure 3.9) Figure 3.9 Enface structure of Pratylenchus sp and Pratylenchus sp Host: carrot (Daucus carota) (in this study) Canonical discriminant analysis - CDA Research results showed that for the females: populations were separated into different groups, of which Pratylenchus sp 2, P zeae and P haiduongensis were separated into separate groups; Pratylenchus sp and P penetrans separated from other species but have overlapping parts Four populations (ID: P4861, P4981, P5189 and P5208) were not separated from the population of Pratylenchus sp Seven morphological characteristics of females including stylet length, V, EP, b, b ', c' and G2 / VBD are the most 19 significant indicators (> and