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.
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 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 INTRODUCTION 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 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 " 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 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 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 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 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 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 CHAPTER 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 stages of development including: eggs; second stage juveniles (J2) (mobile phase); juveniles stage 3, (J3, J4) and adult stage with females and males Root-knot nematodes Meloidogyne spp reproduce by 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), 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 provinces, including Kon 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 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 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 years, from 2014 to 2018 2.2 Methods: 2.2.1 Investigation and nematode sampling: The soil and root samples were collected from 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 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 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, 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 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 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, 11 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 12 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 13 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) 14 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; +, - ) 15 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 16 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 17 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 18 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 19 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 20 Discussion In this study, the sequences from 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 21 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 22 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 and 7.5 mg/ml of 4-HPAA, respectively These mortality rates were significantly different At 2.5 and 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 23 CHAPTER CONCLUSION 4.1 CONCLUSION The thesis recorded 50 populations of Meloidogyne spp on 17 different host plants that belong to 07 species of Meloidogyne namely: M incognita, M javanica, M arenaria, M graminicola, M enterolobii 01 new species (M daklakensis) and 01 probably new species (Meloidogyne sp.) were recorded M cynariensis, a species was previously found in Lam Dong, was not found in this study Density and frequency of Meloidogyne spp were high on almost all the regions Thus, preventative measures should be applied before planting Morphologically intraspecific variations of Meloidogyne spp are relatively high EP, periniel pattern in females, lip height, stylet length, lip region in males, tail length, hyaline length, tail shape are useful features in identifying Meloidogyne spp Genetic variations between M incognita, M javanica, and M arenaria are low and can be hardly identified by using ITS, D2D3, COI, COII sequences NAD5 gene is a good marker in identifying these species Genetic variations of ITS, D2D3, COI, COII, NAD5 regions are high, and these regions can be used efficiently in analyzing the evolution of all other Meloidogyne species Multiplex-PCR can be effeciently used to identify three species M incognita, M arenaria, M javanica quickly with high sensitivity and accuracy Their sequence lengths are 300, 420, and 670 bp, respectively P javanicus at 2×106 spores/ml had the highest effect in controlling M incognita, inhibiting 96% of egg hatching and causing 87% mortality of M incognita after 168hrs L antibioticus at 3×105 spores/ml killed 65.5 % juveniles of M incognita and reduced the hatching rate to 30.4% after 168hrs 4-HPAA at 2.5mg/ml can kill 90% juveniles and reduced the hatching rate to 11.8% after 96hrs 4.2 Recommendation A more detailed description of the species Meloidogyne sp in this study needs to be prepared and published All the experiments of testing biological products were executed in laboratory condition and need to be conducted on the field condition to have more precise conclusion 24 LIST OF WORKS HAS BEEN PUBLISHED International journal (SCI) Trinh, Q P., Le, T M L., Nguyen, T D., Nguyen, H T., Liebanas, G., & Nguyen, T A D Meloidogyne daklakensis n sp.(Nematoda: Meloidogynidae), a new root-knot nematode associated with Robusta coffee (Coffea canephora Pierre ex A Froehner) in the Western Highlands, Vietnam Journal of helminthology, 2018, 1-13 National journal Le Thi Mai Linh, Nguyen Thi Duyen, Trinh Quang Phap, Nguyen Thi Phuong Anh, Pham Van Ty Biologycal control of Meloidogyne incognita on coffee by Paecilomyces javanicus Journal Of Biotechnology 13(4): 1025-1029, 2015 Le Thi Mai Linh, Nguyen Thi Duyen, Nguyen Huu Tien, Trinh Quang Phap First record of Meloidogyne incognita on maize (Zea mays L.) in western highland, Vietnam Journal Of Biology 2017, Volume 39, Number 1: 15-23 Le Thi Mai Linh, Nguyen Thi Duyen, Phan Ke Long, Trinh Quang Phap Genetic diversity of COII-16S-rRNA sequence of some Meloidogyne spp species on coffee in Dak Lak Proceedings of the 7th national scientific conference on ecology and biological resources Hanoi 2017, 768-773 Le Thi Mai Linh, Trinh Quang Phap Genetic diversity 28s-rDNA sequence of some Meloidogyne spp species in the Western Highlands Proceedings of the 16th Vietnamese plant diseases-2016; 298-303 Le Thi Mai Linh, Nguyen Thi Duyen, Trinh Quang Phap, Nguyen Van Toan Affect of Lysobacter antibioticus to Meloidogyne incognita in Laboratory condition Journal of Plant Protection, 2017 Number (271) 18-24 25 ... 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... preventative ability of antagonistic microorganisms to Meloidogyne incognita in laboratory condition Contents of the thesis Study on distribution, frequency and density of root- knot nematodes on some important... 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 " Objectives of the thesis