Plant - Parasitic nematodes on carrots in vietnam and testing biological measures in controlling them

MINISTRY 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 Major: Nematology Code: pilot SUMMARY OF BIOLOGY DOCTORAL THESIS HA NOI – 2018 2 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 3 INTRODUCTION 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. 4 - 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 5 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 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 6 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 6 (Tamura et al., 2013); iii) statistic analysis: SPSS + 7 CHAPTER 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 TT Tên loài HN HD HY LD 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 7 Ditylenchus sp. + + + + Family Criconematidae Thorne, 1949 Genus Hemicriconemoides Chitwood & Birchfield, 1957 8 H. strictathecatus Esser, 1960 + + Genus Hemicaloosia Ray & Das, 1978 9 Hemicaloosia sp. + 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 + 8 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 + 19 Pratylenchus sp. 1 + 20 Pratylenchus sp. 2 + ORDER DORYLAIMIDA PEARSE, 1942 Family Longidoridae Thorne, 1935 Genus Longidorus Micoletzky, 1922 21 Longidorus sp. + Genus Paralongidorus Siddiqi, 1974 22 Paralongidorus sp. + Genus Xiphinema Cobb, 1913 23 X. brevicolle Lordello & Da Costa, 1961 + ORDER TRIPLONCHIDA COBB, 1920 Family Diphterophoridae (Micoletzki, 1922) Genus Diphterophora de Man, 1880 24 Diphterophora sp. + + ORDER APHELENCHIDA SIDDIQI, 1980 Family Aphelenchoididae Skarbilovich, 1947 Genus Aphelenchoides Fischer, 1894 25 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. 8 species of 7 genera were recorded in Hanoi. 7 species of 7 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. 9 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. 10 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). 11 Figure 3.1. ML phylogenetic tree (TN93+G model) based on D2-D3 of 28S rDNA of Hemicriconemoides spp. 3.2.6. Mesocriconema Andrássy, 1965 M. sphaerocephalum was recorded in Ha Noi and Hai Duong. Two populations of M. sphaerocephalum on carrots have similar morphological characteristics compared to the description of Geraert (2010) and the measurements are within the variations. Compared to the previously described 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 KM516178 H. strictathecatus 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