MINSTRY OF EDUCATION AND TRAINING MINITRY OF AGRICULTURE AND RURAL DEVELOPMENT VIETNAM ACADEMY OF AGRICULTURE SCIENCES - TRAN THI MINH LOAN BIOLOGICAL AND ECOLOGICAL CHARACTERISTICS OF Meloidogyne sp PARASITISED ON EGGPLANT AND CONTROL METHODS BY INTEGRATED NEMATODES MANAGEMENT IN LAM DONG Specialization: Crop Protection Code: 9620112 SUMMARY OF PH.D AGRICULTURAL THESIS HA NOI, 2019 The work was completed at: Vietnam Academy of Agricultural Sciences Supervisor: Asscociate Professor Pham Thi Vuong Asscociate Professor Nguyen Van Ket Critic 1: Critic 2: Critic 3: The dissertation will be protected before the Institute of Dissertation Boards meets at (specify the thesis defense at the Institute level) date month year Maybe find this thesis at: Vietnam National Library Library of Vietnam Academy of Agricultural Sciences INTRODUCTION Rationale Eggplant is a fruit vegetable and is high nutrient and economical value, developed and expanded in Vietnam as well as Lam Dong province Although, eggplant is planted and took care easily but, disadvantages of eggplant production are detected by many diseases such as green wilt, leaf spot, Verticillium wilt and plant-parasitic nematodes Root-knot nematodes (Meloidogyne spp.) are common pathogens parasitised on most plants worldwide Among the nematodes discovered in Vietnam, root-knot nematodes is the most serious parasitism and thriving pest in recent years In Lam Dong, root-knot nematodes is one of the major pest parasitised on solanaceae, in generally and on eggplant, as a result, decreasing of quality and yield, increasing other diseases There have been several researches about root-knot nematodes parasitised on eggplant, however, very little has been conducted about distribution, as well as biological and ecological characteristics of root-knot nematodes on Solanaceae and eggplant in Lam Dong to control them For these reasons, we carried out the topic "Research on biological and ecological characteristics of Meloidogyne sp parasitised on eggplant and methods control by integrated nematodes management in Lam Dong" to (i) determine root-knot parasitised nematodes species composition on eggplant, (ii) study biological and ecological characteristics, and (iii) provide effective solutions for control root-knot nematodes toward integrated nematodes management in eggplant production in Lam Dong as well as in Vietnam Research aims and objectives Objectives: the objectives of the research are: (i) to identify species composition root-knot nematodes parasitised on eggplant, (ii) to determine biological and ecological characteristics of M incognita, and (iii) to provide proposing methods to prevent them toward integrated nematodes management, contributing production of eggplant and solanaceace safety, effectively and sustainably Significance Theoretical significance: The thesis has supplemented new scientific data of species composition of root-knot nematodes, biological and ecological characteristics, rules of reproduction, development and parasiticide of M incognita On that basis, effective methods and solutions to control root-knot nematodes, protecting the environment and contributing to safe production of eggplants safety in Lam Dong was proposed The thesis is a reference for agricultural students, researcher, teacher and sciences Practical significance: Proposing methods to control root-knot nematodes effectively and safely, contributing to improved process of the eggplant production toward integrated nematodes management for the purpose of stable, effective and sustainable production in Lam Dong as well as in Vietnam, particularly, chemical pesticides are abused to control nematodes today This thesis is also a document to help managers and farmers identifying symptoms of M incognita on eggplant and decide effectively management solutions of root-knot nematodes Subjects and scope of the research 4.1 Research subjects The subject of study is root-knot nematodes (Meloidogyne sp.) parasitised on eggplant 4.2 Research scope Thesis content: Identifying species composition of parasitic root-knot nematodes on eggplant; Studying biological characteristics of M incognita detected on eggplant; Accessing some of ecological conditions (soil type, organic fertilizers, rainfall, soil moisture, temperature and different eggplant varieties) affected parasitic characteristics and population of M incognita detected on eggplant; Providing solutions to control M incognita detected on eggplant according to integrated management (cultivation methods, biological methods, physical methods, chemical methods) Location: Identifying species composition, affecting ecological conditions on root-knot nematodes and open field experiments were conducted in three vegetable growing areas in Don Duong, Duc Trong and Da Lat in Lam Dong province Greenhouse experiments were conducted in the Department of Agriculture and Forestry, Dalat University In vitro experiments including idetification of root-knot nematodes, extractation vermiform root-knot nematodes from soil and root, lilfe cycle of M incognita were carried out in Plant Protection Laboratory, the Department of Agriculture and Forestry, Dalat University and laboratories of Nematology Department, Institute for Agricultural and Fisheries Research (ILVO), Flanders, Belgium Novel contributions of the thesis This thesis makes contributions: (i) To provide some new data of species composition of root-knot nematodes (Meloidogyne spp.), biological and ecological characteristics of M incognita detected on eggplant in Lam Dong province (ii) To propose methods to control root-knot nematodes detected on eggplant in Lam Dong according to integrated nematodes management to decrease damage; to reduce using chemical pesticides; to complete the protocol of eggplant production following safety and organic cultivation in Lam Dong as well as in Vietnam CHAPTER LITERATURE REVIEW 1.1 Overview Eggplant (Solanum melongena) is a fruit vegetable with high economic value In over the world, there were about 1.6 million hectares growing eggplant In Vietnam, eggplant is grown in recent years and becomes popularly in many areas throughout the country In Lam Dong, eggplant was planted reaching about 1,944 ha, with an average yield of 47.6 tons/ha in 2017 Growing eggplant can bring high income but unstable, because eggplant is a susceptible crop to many diseases, insects and nematodes, in which, Meloidogyne spp are an important group of plant parasites reduced eggplant yield and quality Root-knot nematodes are the most important and common pest worldwide, causing economic damages of agricultural crops in tropical and subtropical areas Root-knot nematodes were reduced eggplant yield up to 95% (Di Vito, 1986) There are many species of root-knot nematodes detected on eggplant, in which two species M incognita, M javanica detected in South Asia, Nepal and India, three species M incognita, M arenaria and M javanica detected in Egypt Until now, very few surveys have been conducted and evaluated the role of root-knot nematodes on crops in Vietnam, mainly focused on some industrial perennial plants Some survey reported the species composition of root-knot nematodes on vegetables in South of Vietnam and Lam Dong provine in the 1990s However, there is no report studying root-knot nematodes on eggplant On over the world, there have been a number of studies involves the different ecological conditions such as temperature, humidity, climatic factors affecting life cycle of M incognita on eggplant as well as control methods However, in Vietnam, there are no research results of biological and ecological characteristics as well as control methods root-knot nematodes on eggplant 1.2 Research situation 1.2.1 Worldwide 1.2.1.1 Research history, distribution and damage thresholds of root knot nematodes in agriculture Root-knot nematodes are obligate parasites which can be found in varieties of plants play an important role in agriculture They caused serious economic losses in agriculture The earliest report of observation of plant parasitic nematodes was in the mid-18th century (1743) when Needham observed pests on wheat by microscope However, until the middle of the 19th century, root-knot nematodes had been identified morphological characterictics clearly Typical symptoms of root-knot nematodes are many galls on the root system M incognita, M javanica and M arenaria are the major of root-knot nematodes, in which, M incognita is the most important plant parasitic nematodes Like other plant pathogens, root-knot nematodes cause estimated crop yield decline According to Taylor and Sasser (1978), for infected areas, without control methods, crop yield may be drop to 24.5% - 85.0% 1.2.1.2 Classification and identificationy root knot nematodes Root-knot nematodes (Meloidogyne) belongs to kingdom Animalia, phylum Nematoda Potts, 1932; class Chromadorea Inglis, 1983; order Rhabditida Chitwood 933; suborder Tylenchida Thorne, 1949; family Meloidogynidae Skarbilovich, 1959; genus Meloidogyne Goldi, 1987 Initially, technique identification of root-knot nematodes is mainly based on morphological characteristics, describes perineal pattern characteristics of females and the length of secondstage juveniles However, at the end of the 20th century, root-knot nematodes is identified by isozyme analysis and molecular identification methods 1.2.1.3 Biological and ecological characteristics of root-knot nematodes There are many stages growth and development of root-knot nematodes changing in different shapes which could be called sexual dimorphism Females are the pear-shaped, less moving into the root The first-stage juveniles (J1) are within the eggs The second-stage juveniles are worm shape, move in the soil After infecting roots, the second-stage juveniles (J2) develop into the third juveniles (J3) and the four juveniles (J4), then swollen to adult females or form males moving in the soil Usually, root-knot nematodes development depends on species, host and temperature In general, life cycle is 15 days to 70 days Root-knot nematode are detected on different varieties of crop with the different ways The density of root-knot nematodes in soil depends on soil type and ecological climate conditions Species composition and nematode density in soil are related to 65% of rainfall and 58% of soil temperature Soil texture and structure also affects nematodes density Sandy are more favorable than clay The density of nematodes in soil is related to organic materials added to soil In general, growth, density and survival of root-knot nematodes depend on the host, ecological conditions such as temperature, soil moisture, organic content and soil texture 1.2.1.4 Methods control root-knot nematodes Using control methods aims to limit harmful effects of nematodes on crop, stabilize yield and quality, bring high economic efficiency There are control methods of root-knot nematodes to protect crops including: crop rotation, sanitation, resistant varieties, biological methods, physical methods and nematicide Integrated nematode management is studied and implemented to efficiently control nematodes and reduce using nematicide 1.2.2 Viet Nam 1.2.2.1 Research history, classification, distribution, biological and ecological characteristics of root knot nematodes Root-knot nematodes have been called many Vietnamese names In Vietnam, the first nematode study was published by Hungarian scientist named Andrassy in 1970 with more than 30 species of plant parasitic nematodes and free living nematodes According to Nguyen Ngoc Chau and Nguyen Vu Thanh (2000), plant parasitic nematodes were divided into 30 varieties, 11 families, order In particularly, root knot nematodes parasitised in Vietnam including 10 species of M arenaria, M cynariensis, M graminicola, M incognita, M javanica, M exigua, M cofeicola, M enterolobii, M hapla and M daklakensis which belonged to genus Meloidgyne, family Heteroderidae, suborder Tylenchina and order Tylenchida Studies of investigating and classification of root-knot nematodes in Vietnam were mainly based on morphological methods In 2005, identifying nematodes by molecular methods was first applied, but there was no results of Meloidogyne identification In 2012, M graminicola and M incognita were identified by molecular methods In 2018, Trinh et al were identified new species called M daklakensis based on morphological characteristics, genes and mitochondrial ADN-based identification The study of root-knot nematodes was first published basing on biological characteristics of M arenaria parasitised on rice, M incognita, relationship between M incognita, Rotylenchulus reniformis and Tylenchorhynchus brassicae and damage thresholds parasitised on tomato and tobacco However, there are not any reports about biological and ecological characteristics of root-knot nematodes parasitised on eggplant 1.2.2.2 Methods control root-knot nematodes In Vietnam, the first report method to control of root-knot nematode was in 1981, followed by reports about control methods for root-knot nematodes on black pepper and vegetable grown in Ha Noi Control methods of root-knot nematodes in Vietnam were also carried out basing on the most popular methods on crops such as rice, tobacco, coffee, black pepper, Chinese cabbage and salad Control methods include crop rotation, resistant varieties, physical methods, biological methods, chemical methods and integrated nematodes management However, there have been publishing methods to control root-knot nematode parasitised on eggplant in Vietnam as well as Lam Dong CHAPTER METHODOLOGY 2.1 Research time Research period was from 2014 to 2017 2.2 Materials, equipments and instruments Materials were eggplant variety of TN525 Green King; sandy clay (49% sand, 10% limon and 41% clay) and clay (32% sand, 1% limon, 67% clay) and river sandy (70% sand, 20 % limon and 10% clay); nylon bags and pots; organic amendments and Yara NPK fertilizer 15-15-15; chemicals for specimens; sampling tools, sieve and laboratory equipment 2.3 Research content - Conducting to survey, collect samples, identify species composition, recognize symptoms, to determine damage threshold and to observe density change of root-knot nematodes (Meloidogyne spp.) in open field parasitised on eggplant in Lam Dong - Studying biological and ecological characteristics of M incognita detected on eggplant in Lam Dong - Studying methods to control root-knot nematodes (M incognita) following integrated nematode management - Building protocol of integrated nematodes management (M incognita) parasitised on eggplant in Lam Dong 2.4 Research methods 2.4.1 Survey and identification of root-knot nematodes parasitised on eggplant in Lam Dong 4.4.1.1 Survey Investigating farmers was carried out by interviewing directly with semi-structured questionnaires 4.1.1.2 Investigation of the main nematodes parasitised on eggplant in the field Samples were collected from 85 households On the field, an random area was chosen about 100m2 Surveying from points according to diagonal rules was to determine main pest and diseases in open field based on typical symptoms on the stem, leaf, fruit of eggplant (Daunay, (2008); Srinivasan (2009) 2.4.1.3 Sampling Soil and root samples were taken according to the W-pattern from 85 eggplant fields in Lam Dong Samples were stored in a thermostatic cabinet at a temperature of 15oC 2.4.1.4 Extraction root-knot nematodes from soil and root samples Activity second-stage juveniles were extracted from soil and root by modification Baermann funel The sample was incubated in the funnel for 48 hours at room temperature Activity second-stage juveniles were counted by stereo-microcopes with 4X magnification 2.4.1.5 Determining the ratio of root damage and root gall-index Root gall-index was assessed by the degree of infection from 1-10 according to Zeck (1971), Bridge and Page (1980) Determining the incidence of root knot nematodes 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑟𝑒𝑝𝑒𝑎𝑡 𝑓𝑖𝑒𝑙𝑑𝑠 𝑠𝑢𝑟𝑣𝑒𝑦𝑒𝑑 Incidence of root knot nematodes(%) = 100 𝑇𝑜𝑡𝑎𝑙 𝑓𝑖𝑒𝑙𝑑 𝑠𝑢𝑟𝑣𝑒𝑦𝑒𝑑 Determining the frequency of species occurrence 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑡𝑖𝑚𝑒𝑠 𝑒𝑛𝑐𝑜𝑢𝑛𝑡𝑒𝑟𝑒𝑑 𝑠𝑢𝑟𝑣𝑒𝑦 Frequency of species occurrence(%) = 100 𝑇𝑜𝑡𝑎𝑙 𝑜𝑓 𝑡𝑖𝑚𝑒𝑠 𝑠𝑢𝑟𝑣𝑒𝑦𝑒𝑑 Determining ratio of root infestation 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑟𝑜𝑜𝑡 𝑖𝑛𝑓𝑒𝑐𝑡𝑒𝑑 Ratio of root infestation (%) = 100 𝑇𝑜𝑡𝑎𝑙 𝑜𝑓 𝑟𝑜𝑜𝑡 𝑠𝑢𝑟𝑣𝑒𝑦𝑒𝑑 2.4.1.6 Extraction with perineal pattern females Females of Meloidogyne were dissected out from the root galls by using forceps under compound microscope and transferred 20 females to 0,9% NaCl 2.4.1.6 Cut perineal patterns Females were cut with a sharp knife and the body content was cleared away from the internal surface of the cuticle The cuticle of the posterior third of the body was trimmed down to a small piece containing vulva, anus and tail tip Perineal patterns were sealed with a coverslip by nail varnish 2.4.1.7 Making specimens How to make temporary and long-term specimens was been according to Bezooijen (2006) and Ravichandra (2010) 2.4.1.8 Examinations of morphological features Morphological features included the perineal pattern, stylet morphology, and distance from the base of the stylet knobs to the dorsal esophageal gland, length of second-stage juveniles, males, females by Bel-capture ruler 2.4.1.9 Identification root-knot nematodes by molecular Root-knot nematodes were identified by molecular basing on primer of tropical root-knot nematodes group 2.4.2 Biological and ecological characteristics of root-knot nematodes (M incognita) detected on eggplant 2.4.2.1 Culturing and purifying species Root-knot nematodes (M incognita) were cultured and purified on tomato roots in B5Gamborg semi-liquid media 2.4.2.2 Studying morphological and biological characteristics (life cycle, ratio of hatching of M incognita) in the laboratory Morphology characteristics and development stages of M incognita were observed at temperature about 24±1oC with different soil moisture at 30 - 40%, 40 - 50%, 50 - 60% Roots were stained to describe morphology characteristics and development stages Roots were stained by fuchsin acid Ratio of hatching was determined by counting the number of the second-stage juveniles formed on the total of hatching eggs in different environments including distilled water; extracts of 1-month eggplant roots; 6-month eggplant roots 2.4.2.3 Studying ecological conditions affected to density of root knot nematodes Preparing free-disease plants Seeds are sterilized and sown on sterilized substrates in the greenhouse Nematode inoculation in pots in the greenhouse The second-stage juveniles of M incognita were extracted from tomato roots cultured in the laboratory Initial population of M incognita was inoculated about 2000 individuals/pot Effect of soil moisture, rainfall, temperature on M incognita in the field The experiment was conducted in the open field grown eggplant TN 252 Green King variety in Suoi Thong B village, Da Ron communes, Don Duong district in Lam Dong provine Secondary data of temperature and rainfall during 2014-2017 was collected from meteorological stations of Thanh My, Don Duong and Da Lat Soil moisture is determined by weigh method The density of the second-stage juveniles was determined a 30-day period during three continuous seasons to assess correlation between the density of the second-stage juveniles of M incognita and rainfall, temperature and soil moisture Effects of soil conditions (particle composition and organic content in soil) on density of root-knot nematodes Analysis of soil texture Analysis of soil texture was deternined according to Bouyoucos (1962) and named based on soil triangle Correlation of sand content and density of second-stage juveniles in the soil was assessed Effect of different organic amendments on root- knot nematodes M incognita parasitised on eggplant Experiments were carried out in the green house Meterials included organic amendments such as chicken manure, pig manure, goat manure, cow manure, commercial organic fertilizer and eggplant “TN252 Green King” Comparison between the treatments with control was carried out, each repeated times in clay pots Root-knot nematodes (M incognita) were inoculated at weeks planting Soil and root samples were taken from planting pots and mixed to collect mixture samples Experimental measures included density second-stages juveniles in soil and roots, ratio of root galls, number of galls, flowers, fruits and eggplant yield Evaluation of eggplant varieties on root-knot nematodes The experiment was carried out in pots, in sandy clay, including six eggplant varieties including Thailand No.1, TN252 Green King, F1-033 local, black fruit NV123, Runako and Com Xanh, repeated times Inovation second-stages juveniles of M incognita after weeks planting Soil and root samples were mixed from pots to a mixture sample Experimental measures were density of the second-stages juveniles in soil and roots, root-gall index, number of flowers, fruits and ratio of fruiting 2.4.3 Control methods root-knot nematodes (Meloidogyne incognita) detected on eggplant following by integrated nematodes management in Lam Dong These experiments was conducted using eggplant ‘TN 252 Green King’ in the open field in Da Ron, Don Duong in Lam Dong The experiments were repeated times Planting density was about 17,000 plants per hectare Care and irrigation were the same in all treatments Experimental measures included density of the second-stage juveniles in soil at the time of 30 days, 60 days, 90 days planting and the end of harvesting; density of the second-stages juveniles in roots; effective control of root-knot nematode calculated by Henderson - Tilton formula; ratio of root infected, root gall index and eggplant yield 2.4.3.1 Cultivation methods to control root-knot nematodes Experimental treatments included: (i) crop rotation between eggplant with tung ho, cabbage, chili peppers, sweet corn, (ii) intercropped with the common bean Phaseolus vulgaris and eggplant 2.4.3.2 Physical methods to control root-knot nematodes Experimental treatments included: (i) soil solarization (after harvesting the previous crop, plowing and drying the soil for weeks); (ii) Keep soil dried (after harvesting the previous crop, plowing, every days upsetting soil, keep soil dry out and soil moisture lower than 35% in weeks); (iii) Covered with dark plastic (after harvesting the previous crop, deep plowing at least 25cm, irrigating and covering by plastic for weeks); (iv) Burning soil (after harvesting the previous crop for weeks, plowing the soil, making rows, covering a thin layer of 5-7cm husk into the planting groove, then a layer of 5cm thick soil, burning anaerobic, irrigating to soil moisture after 48 hours); and (v) Control (after harvesting the previous crop, did not using any method in soil, keeping for weeks, plowing soil and making row for ready to plant) 2.4.3.3 Biologicial methods to control root-knot nematodes The experimental treatments included: Jianon Chitosan super (Chitosan); Vineem 1500 EC (neem - azadirachtin); Abuna 15GR (saponin); Biosune one (Trichoderma harzianum, Trichoderma viride and other microorganism) and control 2.4.3.4 Chemiscial methods to control root-knot nematodes The experimental treatments included: Tervigo 020SC (Abamectin), Cazinon 10GR (Diazinon), Vifu-super 5GR (Carbosulfan), Map Logic 90WP (Clinoptilote) and control 2.4.4 Building a model of control root-knot nematodes (Meloidogyne incognita) according to integrated nematodes management detected on eggplant in Lam Dong The previous crop was Tung Ho After harvesting, cleaning the fields, plowing and drying for weeks, then making rows Cow manure putting down with an amount of 40m3/ha were incubated with 10 kg/ha probiotics containing T harzianum Irrigating and using plastic covered on rows, keeping them for days to allow fungi multiplied biomass rapidly, then planting after days After 10 days planting, eggplant was putted down chicken manure with amount of using about 300kg/ha The area of the model was 500 m2 compared to farmers' handling Evaluation of economic efficency of the model and control methods Calculation of production costs included costs for fertilizers, pesticides and labor Calculating total income based on yield and price which was assumed about 2,000 VND per kilogram (price assumed if profit was the lowest), the efficient economic was calculated as following: Total income (thousand VND) = selling price per kg x yield Total costs = cost of seed + cost of labor + cost of fertilizer + cost of plant protection + cost of irrigation Net income = Total income - Total costs 2.5 Data analysis Data were collected and analysed of variance (ANOVA) by Microsoft Excel 2013 software, IBM SPSS Statistics Version 22 software and MSTATC The mean comparison was done through LSD test and Duncan test with significance level of ≥95% (p≤0.05) CHAPTER RESEARCH RESULTS AND DISCUSSION 3.1 Cultivation methods and control nematode parasitited on eggplant of farmers in the studied area 3.1.1 Survey of traditional cultivation of eggplant farmer in the studied area Cultivation and control methods root-knot namtodes detected on eggplant of farmers in the studied area Table 3.1&3.3 The area and yield of eggplant varieties were grown widely in open field (Lam Dong, 4/2014-6/2017) Eggplant varieties Ratio Density Yield Infestation Yield Season of of (plants/ha) (tons/ha) level (%) losses the highest grown (%) nematode (%) populations TN252 Green King 56.47 17,000 92.72 79.16 27.13 Rainy season Runako 12.94 25,000 35,90 63.63 20.37 Rainy season Oval fruit Japanese 11.76 25,000 36,60 48.00 20.41 Rainy season Thailand No.1 8.24 19,000 74.43 71.43 25.34 Rainy season Black fruit VN123 5.88 22,000 72.00 40.00 15.11 Rainy season F1-033 local variety 3.53 19,000 78.33 33.33 Trivial losses Com Xanh 1.18 25,000 35.00 0 Rainy season There was varieties planted in Lam Dong, TN252 Green King was the most planted variety, accounting for 56.47% of the total, average yield was 92.72 tons/ha and density was 17,000 plants/ha, followed by the Runako variety accounted for 12.94% of total, average yield was 35.90 tons/ha, density was 25,000 plants/ha The lowest ratio belonged to Com xanh variety, just only 1.18% and the yield was just about 35.00 tons/ha Only 34.11% farmers checked pest and disease status of nursery stock and only checked the surface parts such as fungal disease on leaves or stems If the nursery stock was not managed 3.1.3 Density of root -knot nematodes (Meloidogyne spp.) detected on eggplant in Lam Dong Table 3.9 Second-juvenile stages density of Meloidogyne sp in soil and roots and infestation level (Lam Dong, 4/2014-6/2017) Density of second-juvenile stages Average of Average in Average in year rainy season dry season Density of J2 in soil (individual/50cm in soil) 1455 1750 827 Density of J2 in roots (individual/5 g root) 728 939 331 Damage level according to farmer estimated (%) 4.04 5.13 2.85 Yield losses (%) 12.41 14.89 7.19 During the year, density of second-juvenile stages in soil was 1455 individuals/50cm3, in roots was 728 individuals/5g of roots In the dry season, nematodes density was lower than average of year, the density of the second-juvenile stages was just only 827 individuals/50cm3 in soil lower than 628 individuals/50cm3 in soil that of average year and 331 individuals/5 g roots in roots lower than that of 397 individuals/5g in roots of average year Figure 3.12 Correlation between damage thresholds and yield crop (Lam Dong, 4/2014-6/2017) Damage level of root knot nematodes was higher, eggplant yield losses was more, they have been non-linear correlation according to the cubic equation (y = 0.02 + 3.09x-2.41x2 + 0, 49x3) In eggplant field, if root gall index of root knot nematode was 1-3, the yield would be not reduced When symptoms of root-knot were recognized visibly on roots such as stunting, fertilizer was used much higher, flowers and fruits would be reduced, yield losses would be higher Meanwhile, eggplant roots were more than 60% of root galls, approximately root-gall index, estimated yield losses up to 40 - 50% Density of the second-stage juveniles of root-knot nematodes in soil ranged from 500 individuals/50cm3 to 1500 individuals/50cm3 in soil In the 71 surveyed samples, there were soil samples with density of second-stage juveniles ranged of 2000 - 3000 individuals/50cm3, soil samples with density higher than 3000 individuals/50cm3 11 3.1.4 Surveying, collecting and identifying root-knot nematodes (Meloidogyne spp.) detected on eggplant The surveyed results showed that, root-knot nematodes were detected in the 83.52% of the soil and root samples collected There were species presented in the 71 soil and root samples Morphological characteristics were described 48 out of 71 collected samples were the same morphological characteristics of Meloidogyne incognita, 17 out of 71 collected samples were the same species of M javanica, samples were occurred mixture of the two species Figure 3.18 The frequency of Meloidogyne species (percentage) in Lam Dong (4/2014 - 6/2017) The surveyed results of root-knot nematodes species showed that occurence of M incognita was 67.71%, followed by M Javanica, accounted to 23.94% and mixture of the two species only 8.45% Table 3.12 Incidence of root knot nematodes, root gall index and density of root-knot nematodes detected on eggplant (Lam Dong, 4/2014-6/2017) Location study Incidence Root gall Density of J2 Root-knot nematodes species (%) index individials/50cm3 Don Duong 88.09 4.91 789 M incognita, M javanica Duc Trong 84.85 4.03 547 M incognita, M javanica Da Lat 60.00 3.12 356 M incognita, M javanica In of three studied location, the highest incidence of root-knot nematodes was in Don Duong (84.85%), the lowest was in Da Lat (only 60%) The density of second-stage juveniles in soil and root gall index were the highest in Don Duong, with 789 individuals/50cm3 and 4.91, respectively, otherwise, the lowest figures were in Da Lat, proved by 356 individuals/50cm3 and 3.12 respectively All of three studied areas, two species occurred were M incognita and M javanica, but infectious fields in Don Duong and Duc Trong with the mixtures of M incognita and M javanica was higher than that in Da Lat Identification results showed that DNA samples were suitable for M ethiopica, M arenaria, M enterolobii and M javanica on eggplant samples were acceptable Mi2F4/Mi1R primers, M incognita were identified Generally, 71 out of 85 collected samples were found Meloidogyne species with M incognita and M javanica Out of two, M incognita was more frequent played an important damage role with 67.61%, M javanica was lower frequent with 23.94% Mixture of them was only 8.45% 3.2 Biological and ecological characteristics of Meloidogyne incognita 3.2.1 Morphological characteristics of Meloidogyne incognita Life cycle of M incognita undergwent the first moult in the eggs to develop to the firststage juveniles (J1) to the second-stage juveniles (J2) to third-stage juveniles (J3) to fourth-stage 12 juveniles (J4) then to mature stage (adult females and males) Specific characteristics of them were showed as follows: Eggs: Egg shape was oval, contained in egg mass of the female Eggs were laid by females and usually found on the surface of galled roots sometimes inside plant tissue The first-stage juveniles: embryogenesis proceeds to the first-stage juveniles with a blunt tail tip, vermiform, curl up which mounted one in the egg Then, they developed to second-stage juveniles depended on environmental condition The second-stage juveniles: Second-stage juveniles of M incognita were movable vermiform, tapered head with stylet The tail part is transparent The third-stage juveniles: Under favorable environment, J2 hatched in roots to J3 which were tapered at the end of tails, bulged in the middle Their head were without stylet, short tail The fourth-stage juveniles: They were avocado shape, lack a functional stylet, short - small clear tail They did not feed Adult stage Females: Adult females were pear shape, sedentary, head with stylet, feed roots Males: Male were mobile, remain vermiform, leaved galls and entered soil They have spicules at the end of the tail Stylet of male is blunt, set off Table 3.13 Size of some stages of M incognita Grown stage Length (µm) Width (µm) Stylet length (µm) DEGO (µm) Eggs 77.77 32.15 Second-stage juveniles 385.13 10.64 2.62 Females 627.17 404.17 15.34 2.90 Males 1595.53 26.95 2.50 Note: DEGO (dorsal esophageal gland orifice) Morphology of M incognita has undergone different stages of development Egg stage was the smallest size The second-stage juveniles, females and males were significant differences length, width, tail, stylet and dorsal esophageal gland orifice (DEGO) The second-stage juveniles and males were vermiform, but the length of males was over times than that of the second-stage juveniles, males stylet was over 2.5 times than that of the second-stage juveniles, DEGO of males was lower than that of the second-stage juveniles Females length was shorter than that of males, longer than that of the second-stage juveniles 3.2.2 Biological characteristics of Meloidogyne incognita 3.2.2.1 Life cycle, characteristics of the sexual phases of M incognita Life cycle of M incognita started the second-stage juveniles infected into root, underwent some moults such as the third-stage juvenile, the fourth stage juveniles and adults, then hatched a new infectious second-stage juveniles when its life cycle completed Table 3.14 Development time and ratio of root inoculom of the second-stage juveniles Experiment Development time (days) Soil moiture Ratio of root (%) inoculum(%) Shortest Longest Everage ns c 8.04 36.13 85.68c b 8.00 46.77 95.46a 8.04 57.11a 93.31b Average 8.04 46.67 91.48 13 Development time of the second-stage juveniles in eggplant roots averaged 8.01 days and there was no difference between different moisture thresholds Ratio of root inoculum of the second-stage juvenile was different at three soil moisture thresholds At the average soil moisture (36.13%), ratio of root inoculum of the second-stage juveniles was the lowest, only 85.68%, the highest at the soil moisture of 46.77% reaching 95.46%, soil moisture of 57.11% achieved 93.31% Table 3.20 Life cylce of M incognita Development stage Time of development stage (days) Average (days) Experiment Experiment Experiment Second-stage juveniles 8.04 8.00 8.04 8.03 Third-stage juveniles 4.00 4.00 3.93 3.98 Fourth-stage juveniles 2.13 2.07 2.20 2.13 Females 8.00 8.00 8.07 8.02 Egg and first-stage juveniles 5.27 4.80 4.93 5.00 Life cycle 27.44 26.87 27.17 27.16 Males 19.00 20.00 19.00 19.25 o Temperature ( C) 24±1 24±1 24±1 24±1 Soil moiture (%) 36.13 46.77 57.11 46.67 In three different soil moisture at 36.13%, 46.77% and 57.11%, the average development time of the third-stage juveniles was 3.98 days, the fourth-stage juvenile was only 2.13 days, adult female was 8.02 days Time of development from fourth-stage juveniles to male was 19.25, there was not significant difference among different soil moisture Life cycle of the M incognita root knot nematode was 27.16 days at temperature at 24±1oC and soil moisture at 46.67% The development time of M incognita did not differ significantly between different soil moisture Development time of the second-stage juveniles and females were the longest with 8.03 and 8.02 days, respectively Development time of the fourth-stage juveniles was the shortest with only 2.13 days, followed by the third-stage juveniles was 3.98 days while eggs and the first-stage juveniles was for days Development time from the fourthstage juveniles to males was 19.25 days Time from the second-stage juveniles to males at conditions of 24oC ± 1oC, 46.67% was 33.39 days 3.2.2.2 Ratio of hatching egg of M incognita Table 3.21 Effect of environment condition to ratio of eggs hatch of M incognita STT Environment for eggs hatch Ratio of hatching (%) Distilled water 69.30c Root extraction of one-month root 90.72a Root extraction of over-month root 83.00b The eggs hatch ratio of M incognita was the highest in extract of one-month eggplant roots, reaching 90.72%, followed by in extract of six-month eggplant roots with 83.00% and much higher than that of distilled water with only 69.30% 3.2.3 Effect of ecological conditions on Meloidogyne incognita 3.2.3.1 Effect of soil texture on density of the second-stage juveniles in soil 14 Table 3.22 Effect of soil-particle on the second-stage juveniles detected on eggplant in Lam Dong (4/2014-6/2017) Clay content Limon content Sand content (%) Soil types Density of J2 (%) (%) (individuals/50cm3) 54.00 28.54 17.46 Sand 222 40.99 40.80 18.21 Silty clay 365 18.72 58.95 22.33 Silty loam 513 35.99 32.19 31.82 Clay loam 547 23.14 37.74 39.12 Loam 593 29.13 19.39 51.48 Silty clay loam 782 38.53 8.25 53.22 Sandy clay 891 17.34 21.05 61.61 Sandy loam 1735 Correlattion (r) between of sand and J2 0.730 Soil types affected differently on density of the second-stage juveniles While clay and limon content were not correlated with density of second-stage juveniles in soil, sand content was correlated with density of the second-stage juveniles Out of all soil types, density of the secondstage juveniles in clay was the lowest, average of 222 individuals/50cm3 in soil, followed by silty clay with average density of 365 individuals/50cm3 in soil, silty loam with 513 individuals/50cm3 in soil Density of the second-stage juveniles in sand loam was the highest, achieved 1735 individuals/50cm3 Density of the second-stage juveniles in loam was lower than in sand but higher than in clay 3.2.3.2 Effect of rainfall, air temperature and soil moisture on M incognita in soil grown eggplant in Lam Dong Table 3.23 Correlation between soil moisture, air temperature and rainfall to the density of M incognita in Lam Dong (6/2014-5/2017) Density of J2 Soil moisture Rainfall Temperature (individual/50cm ) (%) (mm) (oC) Density of r 0.672* 0.678* 0.516 J2 Sig (two ways 0.017 0.015 0.086 (individuals/ factor) 50cm3) N 12 12 12 12 * ** Soil r 0.672 0.812 0.598* moisture Sig (two ways 0.017 0.001 0.040 (%) factor) N 12 12 12 12 * ** Rainfall r 0.678 0.812 0.687* (mm) Sig (two ways 0.015 0.001 0.014 factor) N 12 12 12 12 Temperature r 0.516 0.598* 0.687* o ( C) Sig (two ways 0.086 0.040 0.014 factor) N 12 12 12 12 * Correlation at p= 0.05 (two ways factor) ** Correlation p=0.01 (two ways factor) 15 There was a close linear correlation between three factors: rainfall, soil moisture and temperature and also between these factors and the density of the second-stage juveniles in soil Rainfall and soil moisture have a very close correlation (r = 0.812) Soil moisture, rainfall are correlated with density of the second-stage juveniles in the soil (0.5