Study on diversity of root – knot nematodes, meloidogyne spp, in tay nguyen highlands

1 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 MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY 2 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 1 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 2 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 3  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 root- knot 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 4 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 5 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, 4- Hydroxyphenylacetic (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 6 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 7 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 Multiplex- PCR 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 8 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 9.2- 17.8 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, 9.1- 16.7 µm long. 3.2.2.2. Variation of morphological characteristics and diagnosis 9 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 a