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MINISTRY OF EDUCATION AND TRAINING (MOET) MINISTRY OF AGRICULTURE AND RURAL DEVELOPMENT (MARD) VIETNAM ACADEMY OF AGRICULTURAL SCIENCE (VAAS) --------- NGUYEN THE HINH RESEARCH ON PESTS, DISEASES AND WEEDS IN THE INTERCROPPING SYSTEMS OF MONDO GRASS (Ophiopogon japonicus Wall.) AND OTHER CROPS IN PHU THO PROVINCE Branch of Study: Plant Protection Code No.: 62.62.01.12 SUMMARY OF AGRICULTURAL DOCTORAL THESIS HÀ NỘI - 2014 The thesis has been completed in: VIETNAM ACADEMY OF AGRICULTURAL SCIENCE (VAAS) Scientific supervisors: 1. Prof. Dr. Nguyen Van Tuat 2. Dr. Nguyễn Đình Vinh Independent Evaluator 1: Independent Evaluator 2: Independent Evaluator 3: This thesis shall be presented before Institutional Thesis Evaluation Committee at: Vietnam Academy of Agricultural Science (VAAS) Date: ... day... month... year This thesis can be referred at the library of: Vietnam National Library Library of Vietnam Academy of Agricultural Science 1 INTRODUCTION 1. The demand of the research subject: Mondo grass is a perennial grass with the scientific name Ophiopogon Japonicus Wall, which belong to the Ophiopogon genus, Ruscaceae family (Liliaceae family before). The species of this genus mainly originated from the regions of East, Southeast and South Asia. Nowadays, the mondo grass is polulated in the world, frequently seen in tropical and semitropical areas. Mondo grass is used as soil coverage plants and ornamental plants in the parks, office gardens of the countries of China, United States, Gemany, Thailand and Japan. In some other countries such as Vietnam and China, mondo grass’s tubes and roots are used in making medicines. Mondo grass is a crop with many advantages and be able to grow well when intercropping with other crops. Initial survey on agri-ecological conditions and the market demand showed that the mondo grass has a huge potential to intercrop in the gardens of fruit crops and other industrial crops in Northern Mountainous areas. Dispite of the huge potential, the mondo grass was grown spontaneously by the mountainous farmers without any research on pests, diseases and other intercropping tactics to maximize the benefits of this crop. Therefore, the implementation of the research subject “Research pests, diseases and weeds in the interopping systems of Mondo grass (Ophiopogon japonicus Wall.) and other crops in Phu Tho province” is highly needed for sustainable intercropping systems in this province. 2. The purposes of the research To study the composition and damage level of pests, diseases and weeds on mondo grass, tea and pomelo in 02 intercropping systems of mondo grass in tea plantation and mondo grass in pomelo garden. To research the mondo grass disease (stem rot disease) and control measures. To study the supression impact of mondo grass to weeds and some integrated crop management tactics to control weeds in the intercropping systems with mondo grass. To evaluate the economic and environment benfits of the mondo grass intercropping systems in Phu Tho province. 3. 3.1. Scientific and practical values of the research Scientic value (i) The research results contributed to the database of pests and diseases on mondo grass in Phu Tho province. (ii) The research identified the pathogen causing stem rot disease of mondo grass is the fungus Pythium helicoides Drechsler, through the molecular analysis and DNA classification, the research found a fungus which has never been found on the mondo grass in Vietnam. The research showed the description of stem rot disease symptoms and some 2 biological characteristics of the fungus P. helicoides Drechsler, testing of some biological and chemical control measures on the fungus to enrich the scientific basis for development of the stem rot disease control procedures on the mondo grass. (iii) The research studied the impacts of mondo grass intercropping to some major pests on the main crops (tea and pomelo) in the intercropping systems, initially determined the suppression impacts of mondo grass on weeds through the physical influence (shading) and chemical influence (mondo grass root’s powder). 3.2. Practical value (i) The results on the pest composition and damage level as well as the experiments on stem rot disease control measures helped development of some crop protection procedures to enhance the yields of mondo grass. (ii) The results on the influence of mondo grass intercropping on some major pests and weeds in tea and pomelo plantations helped in development of technical cultural procedures for the intercropping of mondo grass in the tea and pomelo gardens. (iii) Analysis on economic and environment values helped in advising farmers on the benefits of mondo grass intercropping in tea and pomelo plantations in Phu Tho province. 3.3. New findings of the research  Determined the pathogen causing the mondo grass’s stem rot disease is the fungus Pythium helicoides Drechsler. This fungus has been announced as pathogens in some crops in the world but this is the first time this fungus was found as the pathogen causing stem rot disease in mondo grass in Vietnam  Determined some biological characteristics of the fungus Pythium helicoides Drechsler and experimented some biological and chemical measures to control the fungus in the laboratory which found the basis for development of the mondo grass’s stem rot disease in the field.  Mondo grass intercropping did not influence the major pests in the tea plantations such as Empoasca flavescens Fabr., Physothrips stiventris Bagn, Oligonychus coffeae Niet, Helopentis theivora Waterhouse, Colletotrichum Camelliae Masse, Colletotrichum theae Petch as well as pests on pomelo plantations such as Phyllocnistis citriella Saintion and Xanthomonas campestris.  Mondo grass can suppress the weed growth through the physical mechanism (shading) and chemical mechnism (root powder). This result is in line with the research result of Lin D., Tsuzuki et al (2003) that the root powder of mondo grass did suppressed the germination and growth of the weeds in the rice fields. Therefore, mondo grass intercropping can efficiently control weeds in tea and pomelo plantations.  Initially proposed some mondo grass’s intercropping tactics (optimal planting time, density and distance between rows) to reach the highest weed control towards the integrated crop management (ICM) strategies. 3 4. The thesis structure The main content consists of 146 pages (excluding the reference and appendices) – Introduction: 5 pages; Chapter 1: Scientific basis and Document overview – 29 pages; Chapter 2: Materials, Main research contents and Methodologies – 19 pages; Chapter 3: Research results and Discussion – 90 pages; Conclusions and Suggestions – 3 pages; Reference – 23 pages. The thesis consists of 35 tables, 28 pictures and referred to 71 Vietnamese documents and 110 English documents. CHAPTER 1 SCIENTIFIC BASIS AND DOCUMENT OVERVIEW 1.1. Scientific basis of the research The mondo grass originated from China and Japan but was introduced into Vietnam hundred years. The plant adapted well with the climate and soil conditions of many various eco-agricultural areas from the Central to the North of Vietnam. The mondo grass has many advantages of a good intercropping plant and high potential to become an important intercropping crop in the large areas of perennial fruits and industrial crop gardens in Northern mountainous areas. However, there is no record of research in mondo grass pests as well as its intercropping systems in Vietnam. 1.2. National and International document overview 1.2.1. International document overview 1.2.1.1. Research on the mondo grass’s uses Many research on using mondo grass as the ornamental plants for soil coverage, fens in family gardens, parks or offices. Many research and findings on using tubes and roots of mondo grass for making medicines have been carried out by pharmaceutical researchers in China and other Eastern countries. 1.2.1.2. Research on mondo grass pests and control measures Some research on mondo grass pests have been implemented and the results showed that the mondo grass was pested by a few species such as scale Pinnaspis carisis Ferris and snail. Some control measures have been suggested such as spraying soap pesticides to control scale, using granular bait to control snail. 1.2.1.3. Research on mondo grass’s disease and control measures Some fungi have been found as the mondo grass’s pathogen such as the Collectotrichum sp. causing yellow leaves, the fungus Fusarium sp. found in roots and stems of the disease mondo grass plants, the fungi Phytophthora palmivova and Phytophthora 4 nicotiana found in the mondo grass’s nurseries and glass houses, the fungus Pythium splendens causing mondo grass’s root rot disease. Spraying chemical pesticide Aliette can control the fungus Pythium splendens. The fungus Pythium helicoides Drechsler has never found as a pathogen in mondo grass in the world. 1.2.1.4. Research in the pests on tea, pomelo and the intercropping systems There are many research in the pests on tea and pomelo crops have been carried out internationally. There are specific pests for each ecological areas even though some major pests can be found in most tea growing regions. There are records of over a thousand of insect and 400 fungi species identified in the world tea growing areas. Many pests and diseases on pomelo crop have been studied in the countries of Malaysia, Indonesia, China, Japan, India, Fiji, Philippines and Thailand. Many international research confirmed the benfits of the various intercropping systems. However, there is no research results recorded on the influence of the intercropping to pests and diseases on tea and pomelo crops. The study of Jey Deputy (1998) showed that the mondo grass can frequently maintain the completed soil coverage, therefore, well competed with weeds. Lin D., Tsuzuki et al (2003) recorded that the solution from mondo grass’s root powder could suppress the germination and growth of weeds in the rice fields without the negative impact to the growth of rice plant. 1.2.2. National document overview 1.2.2.1. Research on the mondo grass’s uses Many national pharmaceutical documents mentioned the mondo grass as a major ingredient in traditional medicines. Mondo grass’s tube in fact has been used in many traditional medicines for hundred years in Vietnam. 1.2.2.2. Research in mondo grass’s pests and diseases and the control measures There are a few studies recorded on the mondo grass’s pests and diseases. The survey results of Nguyen Dinh Vinh and Nguyen Thi Thanh Hai (2011) showed that the majority (72%) of farmers in Phu Tho and Yen Bai provinces mentioned the mondo grass as a crop with a few pests, only the stem rot disease is the most dangerous which could damage the crop yield at the dangerous level of 8/10. No research result on the fungus Pythium helicoides Drechsler as a pathogen causing mondo grass disease in Vietnam has been recorded. 1.2.2.3. Research in the pests on tea, pomelo and the intercropping systems Tea and pomelo are the traditional crops in Vietnam, therefore, many studies on their cultivation tactics, pests and diseases have been found and refered during the document overview. 5 There are many research works on various intercropping systems in Vietnam. However, studying on the intercropping systems between mondo grass and other crops is very limited (Nguyen Dinh Vinh et al, 2007, 2009). 1.2.2.4. Climate and soil conditions of the research site Phu Tho is a province with large mondo grass plantations, where the farmers have longterm experience in growing this crop. There are also many mondo grass processing units in the districts of Ha Hoa and Thanh Ba. Therefore, Phu Tho has large potential in development of mondo grass, particularly in the areas of unfertile soil, the mondo grass is considered as the most suitable crop which can maximize the use of land and bring good income to poor farmers. CHAPTER 2 MATERIALS, RESEARCH CONTENTS AND METHODOLOGY 2.1. Materials 2.1.1. Laboratory materials Mondo grass plants (healthy and disease infected) of variety M2 which are polpularly grown in Ha Hoa district, Phu Tho province. Media for tissue culture are WA, CA, PCA, PDA, CMA. Chemical pesticides of Ridomil 72 MZ, Viben-C 50 BTN, Aliette 80WP 0,1%; Copper - Zinc 85 WP and biological pesticides of Trichoderma asperellum, Bacillus amyloliquefaciens and Streptomyces misionensis. Other common laboratory equipment and tools. 2.1.2. Field research materials Tea plantations of Phuc Van Tien variety of 2-4 years with the mondo grass intercropped between two tea rows; Pomelo gardens of Dien variety of 1 – 3 years intercropped with the mondo grass. 2.1.3. Research site and duration Surveys and field research are carried out at Bang Gia commune, Ha Hoa district, Phu Tho province. The experiments and analysis are implemented at Plant Protection Research Institute (PPRI). The research duration is from August 2009 to March 2013. 2.2. Research contents - To study the composition and damage level of pests on mondo grass, tea and pomelo plants in the two (02) intercropping systems of mondo grass in tea plantations and pomelo gardens. Comparison of the pests on tea and pomelo plants between intercropped plantations and non-intercropped plantations in Phu Tho province. 6 - To study specifically the Mondo grass’s stem rot disease: pathogen identification, some biological characteristics and laboratory testing as well as field trials of chemical and biological fungicides. - To survey the composition and damage level of weeds in the two (02) intercropping systems of mondo grass in the tea and pomelo plantations. The weed suppression mechanism of mondo grass is also studied. - To experiment some integrated crop management (ICM) measures for managing weeds in the intercropping system between mondo grass and pomelo in Phu Tho province. - To evaluate the economic and environment benefits of the intercropping systems between mondo grass with tea and pomelo. 2.3. Methodology 2.3.1. Survey and sample collection Surveys of pests and diseases in the intercropping systems are implemented by the basic survey methodology of Plant Protection Research Institute (1999-2000). The samples were sent to PPRI’s specialists for analysis and classification. The damage level is determined by the pest density or level of leaf damage. 2.3.1.1 Identification of the survey sites The surveys selected are some communes in Ha Hoa district, Phu Tho province where mondo grass is popularly grown. To comprehensively survey the mondo grass’s stem rot disease, the supplementary surveys have been carried out in some other places in Ha Noi and Bac Giang. The selection of 3 – 5 gardens per location and 5 cross-cutting points per each garden was inplemented during the survey. 2.3.1.2 Survey methodology for pests and diseases in tea and pomelo plants For tea pests: Survey frequency is one time per 7 days. The oil tray is used for sampling green leafhopper and natural enemies. The thrip is collected in the tea buds, the red mite is collected in the tea leaves; the tea buds eaten by mosquito bugs were counted; the tea leaves infected with brown blight were collected and the tea buds infected with bud rot disease were counted for analysis. For pomelo pests: Survey frequency is one time per two months in the selected young pomelo gardens. The selection of 20 pomelo plants per garden were made and the collected pests through catching by net and sampling were sent to laboratory for analysis. For the mondo grass pests: Carefully watching the mondo grass in the intercropped plantations to collect pests and diseases for analysis. The apprearance frequency of the pests below 20% of the samples is rated less popular (+), 20-50% of the samples is rated popular (++), more than 50% is rated most popular (+++) 7 2.3.2. The assessment methodology of the impacts of mondo grass intercropping to some major pests in the main crops. To survey the 06 major pests in the tea plantations (green leafhopper, mosquito bug, thrip, red mite, brown blight, bud rot) and 02 major pests in the pomelo plantations (leafminer, black rot). The comparision of the pest apprearance and damages between the plantations with and without mondo grass intercropping was made. To carefully observe the growth (height, shading, leaf number) of tea and pomelo plants with and without the mondo grass intercropping. 2.3.3. Methodology for researching the mondo grass’s stem rot disease Methodology for identification of pathogen: The Koch’s principles have been followed [Drenth và Sendall, 2004] including 4 steps: Symptom description and initial clasification; (ii) Isolation of the disease’s pathogen and identification; (iii) Artificial infection of the isolated pathogen to the healthy plants and observe the similar disease symptoms; (iv) Reisolation of the pathogen from artificial infected plants and comparison with the intitial isolated pathogen above. The isolation of pathogen from the disease plants was done through direct isolation and using trapping methods [Drenth và Sendall, 2004]. The artificial infection through soil is followed the methodology of Drenth và Sendall (2004). Methodology of identification of the mondo grass’s stem rot disease: using the molecular technology which consists the following steps: (i) Prepartion of the pathogen sources [Drenth và Sendall, 2004]; (ii) Isolation of DNA [Doyle và Doyle methodology (1987)]; (iii) PCR and DNA sequencing analysis [Sambrook methodology (1983)]; (iv) Seeking the similar sequences in Genbank database to identify the pathogen [Using the on-line sofware BLAST in the NCBI]. Methodology for studying some biological characteristics of the pathogen: To study the influence of temperature to the fungus growth: the temperature levels popular in the province were set for the experiment: 14oC, 18oC, 22oC, 26oC, 30oC, 34oC. To study the influence of the light condition to the fungus growth: continuously 12 hours in light and 12 hours in dark. To study the influence of pH level to the fungus growth: the pH levels popular in the provincial soil were set for the experiment: 5,0 - 5,5 - 6,0 - 6,5 - 7,0 - 7,5. Methodology for the control measures of the mondo grass’s stem rot diease: - To study the suppression efficacy of the anti-biotic fungus Trichoderma asperellum, vi khuẩn Bacillus amyloliquefaciens and bacterial Streptomyces misionensis, Streptomyces aureofaciens, on the growth of the pathogen fungus Pythium helicoides Drechsler in the invitro condition: the diameter of the pathogen fungus was measured in the experiments. - To study the control efficacy of chemical fungicides on the pathogen Pythium helicoides Drechsler: the experiment consists of 5 formula: (i) Ridomil 72 MZ 0,2%; (ii) Viben - C 50 BTN 0,2%; (iii) Aliette 80WP 0,1%; (iv) Copper - Zinc 85 WP 0,2%; (v) 8 Control: No chemical; Culture the fungus P. helicoides Drechsler in the PDA media and observe the growth of the fungus. The fungicide efficacy is calculated by Abbott methodology. - To study the efficacy of the fungicides in the fields: The trial consists of 6 formula: (i) Spray Aliette 80 WP 0,2%; (ii) Spray Ridomil gold 68 WG 0,3%; (iii) Spray biological fungicide Trichoderma asperellum; (iv) Spray biological fungicide Streptomyces aureofaciens; (v) Spray biological fungicide Bacillus amyloliquefaciens; (vi) Control: spray with water. The narrow trials were arranged by completed random blocks, each plot is 50 m2, 3 replications, the disease levels (%) were monitored before and after treatment (Plant Protection Research Institute, 1999). The fungicide efficacy is calculated by Henderson-Tilton. 2.3.4. Study the weeds and some control measures of weeds in the intercropping systems Assessment of the apprearance frequency and the damage level of weeds: To survey the weeds one per two months. To select 5 cross-cutting points per each plantation, put the wooden spare 100 x 100 cm and collect all the weeds within the square, bring to laboratory for cleaning, classification and weigh. Classification of weeds by comparison with the pictures or consultantion with the specialists. The weed with the apprearance frequency below 20% is rated (+); from 20-50% is rated (++) and over 50% is rated (+++). The damage level of weeds is considered by the weight of weeds (more weight resulted to more nutrient competition with crops). The more weight of weeds, the higher damage levels to crops. To study the suppression impact of the mondo grass root powder: The experiment consists of 2 formula by the completed randomization block with 3 replications. The area of each plot is 50 m2. The dry mondo grass’s root powder is diluted into water at the dose of 50g/m2 and sprayed evenly on all plots [Lin D., Tsuzuki và cộng sự (2003)], the control plot was sprayed with water. The composition and weight of weeds are monitored at 60 days after spraying (Plant Protection Research Institute, 1999). To study the intercropping cultural practices of mondo grass in the pomelo plantations for weed controlling and enhancing farmer income: The experiments for studying the influence of mondo grass growing time, density and row distance in the intercropping systems have been designed. The soil coverage level of mondo grass is measured by square 1m x 1m, which consists of 100 small dots 1cm x 1cm. The dot partly covered is calculated at 50% coverage (the land coverage measurement methodology in the forestry area). 2.3.8. Assessment of the economic, environment and plant protection aspects of the intercropping systems of the mondo grass in pomelo and tea plantations To study the economic efficiency of the intercropping systems through the profits achieved by the mondo grass harvest. To assess the environment impact and plant protection meaning through the reduction of herbicides and observing of the overall environment and soil in the plantations. 2.4. Data analysis: The research data are analysed by the software of Excel, IRRISTAT 5.0. 9 CHAPTER 3 RESEARCH RESULTS AND DISCUSSION 3.1. Research pests and diseases in the system of mondo grass intercropped in the tea plantations 3.1.1. Composition and damage level of mondo grass’s pests and diseases The survey results of the mondo grass intercropped tea plantations in Ha Hoa district, Phu Tho province showed that there are 4 mondo grass’s of stem rot (Pythium sp.), fired leaf (Macrophoma sp.), stunt (not yet identified the cause) and dry leaf tip (not yet identified the cause) and 3 pests of scale (Pseudaulacaspis cockerelli Cooley), small snail (Achatina fulica) and leaf hopper (Caelifera sp.). Overall, the mondo grass has a few pests with low damage level except the stem rot disease which can cause heavy yield losses if no prevention is taken from the growing time. 3.1.2.Composition and damage level of tea pests and diseases in the intercropping systems Through the surveys of the tea intercropped with mondo grass in Ha Hoa district, Phu Tho province, there are 04 insect pests identified including green leafhopper (Empoasca flavescens Fabr.), mosquito bug (Helopentis theivora Waterhouse), thrip (Physothrips stiventris Bagn), red mite (Oligonychus coffeae Niet) and 04 diseases including blister blight (Exobasidium vexans Masse), brown blight (Colletotrichum Camelliae Masse), grey blight (Pestalossia theae Sawada), bud rot (Colletotrichum theae Petch). Initial assessment showed that except the blister blight and grey blight have low damage level, other pests and diseases have medium damage levels and could become epidemics. 3.1.3. Comparison of the mondo grass’s and tea pests and diseases in Phu Tho province The comparison results showed that the tea’s pests and diseases are different from the mondo grass’s ones. The observation in the plantations showed that the tea’s pests did not cause damage to the mondo grass and vice versa, the mondo grass’s pests did not cause damage to the tea plants in the intercropping system. 3.1.4. Comparison of the damage level of the tea’s major pests in the tea plantations with and without intercropped mondo grass The research results showed that the intercropping of mondo grass in the tea plantations did not cause significant changes of the density of green leafhopper, thrip, red mite as well as the damage level of mosquito bug. On the other hand, intercropping of mondo grass caused lightly increase of brown blight and bud rot. The observation results showed that the tea’s growth (height, shading size, leaf number) was similar between the mondo grass intercropped tea plantations and the control (without intercropping). Generally, the mondo grass is not eaten by green leafhopper, thrip, red mite and mosquito bug in the intercropping system. 10 3.2. Research pests and diseases in the system of mondo grass intercropped in the pomelo plantations 3.2.1. Composition and damage level of the mondo grass’s pests and diseases The research result showed that the composition of mondo grass’s pests and diseases in the pomelo plantation was similar to that in the tea plantation. Among the pests and diseases detected in the mondo grass, the stem rot disease has the medium damage level and could become epidemic in the favorable weather. Supplementary surveys in the concentrated mondo grass plantation areas showed that there was the difference in terms of disease levels, of which, the highest disease level is 9,1%. The disease infection usually caused death of newly planted mondo grass, which resulted to supplementary seedlings. The disease also caused partly death of the grown up mondo grass which resulted to the losses of yields. 3.2.2. Composition and damage level of pomelo’s pests and diseases The result showed that there are 7 pest species including citrus leafminer (Phyllocnistis citriella Saintion), citrus long-horned beetle (Anoplophora chinensis), red mite (Panonychus citri), black citrus aphid (Toxoptera aurantii), citrus mealybug (Planococcus citri), citrus tree borer (Chelidonium argentatum Dalmann), weevils (Hypomeces squamosus Fabr) and 4 diseases of black rot (Xanthomonas campestris), brown rot (Phytophthora citriphora), citrus scab (Elsinoe fawcettii), greasy spot (Mycosphaerella citri) identified in the pomelo gardens intercropped with mondo grass. Some pests of citrus tree borer, red mite, black citrus aphid had low apprearance frequency and caused non-significant damage. Citrus leafminer appreared more frequently and caused significant damage to young pomelo leaves. Among the dectected diseases, black rot was frequently found and caused significant damage to the young pomelo leaves. The remaining citrus scab, brown rot, greasy spot were also found in the young pomelo plantations in Phu Tho province but caused insignificant damage during the survey time. 3.2.3. Comparison of the mondo grass’s and pomelo’s pests and diseases in Phu Tho province The comparison showed that the composition of pests and diseases of pomelo and mondo grass is not similar. The observation revealed that the pomelo’s pests did not damage mondo grass and vice versa, the mondo grass’s pests did not cause damages for pomelo plants in the intercropping system. 3.2.4. Comparison of the damage level of the pomelo’s major pests in the pomelo garden with and without intercropped mondo grass The research result showed that the damage level caused by citrus leafminer and black rot is not significantly different between pomelo gardens intercropped with and without mondo grass. Therefore, it is concluded that mondo grass intercropping does not influence the damage level of the citrus leafminer and black rot to the pomelo plants. 11 3.3. Research in mondo grass’s stem rot disease and control measures 3.3.1 The mondo grass’s stem rot disease’s symptoms The initial symptom is the stains in young leaf, the junction between leaf and base. Then, the leaf changes to yellow to dark color and the leaf base is rotten, gently pulling can separate the upper leaves from the base. During the rainy season, the upper leaves are still green but the leaf base was already rotten due to high atmosphere moisture. The disease spreaded from young stem base down to the roots and caused the rotten roots and tubes. When pulling the diseased plant, roots become black color and tubes totally rotten. The disease is quickly developed and spreaded in the season between Winter and Spring as well as in Autumn with the wet weather and rainny. The disease can kill young mondo grass and caused yellow, rotten leaf and serious reduction of mondo grass yields. 3.3.2. The research in the pathogen isolation of mondo grass’s stem rot disease Through analysis of the disease samples, the fungi of Fusarium sp. and Pythium sp. were found in the disease stains. Isolation of pathogens and re-infection into the healthy plants have been implemented in the laboratory of Plant Protection Research Institute. The methodogy of using various trapping materials (apple, rose flower, pear, papaya) has been tested to purify fungus source of Pythium sp. for re-infection and identification. The result showed that the rose flower is the best trapping materials for purify the pathogen source Pythium sp. from the disease samples with the isolation ratio at 76,2%. 3.3.3. The artificial infection of isolated pathogens from the mondo grass’s stem rot disease samples 3.3.3.1. Direct infection to the mondo grass’s leaf base in the laboratory condition With total 20 fungal sources of isolated Pythium sp., 08 fungal souces of various morphology have been used for infection. The isolated fungal sources were cultured in the PDA media and independently infected into the healthy mondo grass’s leaves. The results have been shown in the table 3.14. There are 4 pathogen sources of Pythium sp. caused the symptoms similar to the disease symptoms in the field: after the incubation time from 24 – 32 hours, the infected leaf changed to the brown color. The remaining pathogen sources did not cause the disease symptoms, of which, one is Pythium sp. and 3 are Fusarium sp. Therefore, it is concluded that the pathogen causing the mondo grass’s stem rot disease is the fungus Pythium sp. Although many pathogen sources of the fungus Fusarium sp. were found in the collected samples, the experiment result showed that this fungus is not the pathogen of the mondo grass’s stem rot disease. 12 Table 3.14. Results of artificial infection into the mondo grass leaf (2011-2012) TT 1 2 Souces of Pythium sp. MM 1 MM 2.1 Percentage (%) of infection 100 100 Time of disease appreared (hours) Percentage (%) of reisolation Symptoms 24 The leaf tissue changed brown and the leaf base rotten 100 28 The leaf tissue changed brown and the leaf base rotten 100 100 3 MM 2.2 100 32 The leaf tissue changed brown and the leaf base rotten 4 MM 3.2 0 0 0 0 5 MM 5.1 0 0 0 0 6 MM 6 0 0 0 0 7 MM 8 0 0 0 0 100 0 8 MM 26 100 20 The leaf tissue changed brown and the leaf base rotten 9 Control 0 0 0 3.3.3.2. Artificial infection of the mondo grass’s stem rot disease in the net house The 8 pathogen sources have been used to artificially infect the mondo grass plants in the net houses to confirm the determined pathogen. The result is shown in the table 3.15. The research result showed that among the pathogens, the fungus Pythium sp. provided the highest disease infection at 46,6%. The disease symptoms appreared 63 - 86 days after artificial infection with the incubation time averaged at 77 days. The disease infected more in the young plants. The symptoms are similar to that of the stem rot disease observed in the fields of Ha Hoa district – the stem base changed brown then rotten, the young plants died after disease become serious. The fungus sources of Fusarium sp. did not cause the disease 13 symptoms as that of the mondo grass’s stem rot disease in the fields. It is concluded that the fungal Fusarium sp. is not the pathogen of the mondo grass’s stem rot disease. Table 3.15. The artificial infection results of the isolated pathogen sources (Hanoi, 2011) No 1 2 Pathogens Disease rate (%) Symptoms 46,6% 63 - 86 The stem base is rotten, gentl pulling can separate the upper stem from the base, the whole stem and roots rotten when the disease becomes serious. 0 0 Pythium sp. Fusarium sp. % of reisolated samples Incubation time (days) 100 No disease symptom 0 To precisely identify which species of Pythium sp. caused the stem rot disease, the molecular technology has been used to classify the pathogen. 3.3.4. Application of molecular technology in identification of the mondo grass’s stem rot disease 3.3.4.1. PCR analysis: All the pathogen sources were extracted DNN through PCR and sequencing analysis. After fine-tunning, the 4 samples owned the DNA length from s. These sequences contained the ITS 1 and ITS 2 needed for analysis. The result is shown in the table 3.16. Table 3.16. The results of DNA analysis for the 4 pathogen sources of Pythium sp. isolated from the mondo grass’s stem rot disease samples (Ha Noi, 2011) No Sample Sequence codes 1 MM1 2 MM2.1 3 MM2.2 4 MM26 1406ZAC000 1406ZAC001 1406ZAC002 1406ZAC003 1406ZAC004 1406ZAC005 1406ZAC006 1406ZAC007 Primer Sequence quality ITS4 ITS5 ITS4 ITS5 ITS4 ITS5 ITS4 ITS5 Good Average Good Average Good Average Good Average Readable sequences (bp) 623 689 689 641 14 3.2.4.2. Searching of the matched sequences from Gen Bank The online software (Blast search) was used to search the matched sequences, using the found sequences as the query. The result showed that the 4 pathogen sources are the fungus Pythium helicoides Drechsler with the preciseness of 99 % overall the compared sequences. The result shown in the table 3.17. Table 3.17. The result of searching the matched sequences from the Genbank through the online software Blast (2012) No. Sample Results of searching from Genbank Determined species Query code Comparison length (%) Matching ratio (%) Pythium helicoides HQ643383 100 99 02 MM2.1 Pythium helicoides HQ643383 100 99 03 MM2.2 Pythium helicoides HQ643383 100 99 04 MM26 HQ643383 100 99 01 MM1 Pythium helicoides 15 3.3.4.3. Classification structural analysis Pythium helicoides-HQ643383 MachMon1 HQ643383 MachMon1 Pythium helicoides-AY598665 MachMon2.1 AY598665 Pythium helicoides -AB217659 MachMon2.1 1 MachMon2.2 AB217659 helicoides-AB108057 Pythium MachMon2.2 100 Pythium 2 helicoid MachMon26 AB108057 es Pythium helicoides-AB108057 MachMon26 Pythium helicoides-FJ348741 AB108057 Pythium helicoides-AB108032 FJ348741 56 Pythium helicoides-AB108051 AB108032 Pythium helicoides-AB1080345 AB108051 Pythium helicoides-HQ643383 AB1080345 Pythium cinnamomi-GU191211 HQ643383 Pythium vexans-GU931701 GU191211 GU931701Pythium arrhenomanes-HQ643471 Pythium aphanidermatum-HQ643439 HQ643471 100 57 20 Pythium HQ643439myriotylum-HQ643704 HQ643704 Diagram 3.12. Classification structural analysis based on the ITS of 4 fungal sources Oomyces isolated from the disease mondo grass 3.3.5 Morphology of the fungus Pythium helicoides Drechsler Observation of the fungus P. helicoides Drechsler through the microscopes showed that the fungal spown has white color, spongy, no wall along the spown and the round sporages developed on the top or middle of the fungal spowns. 3.3.6. Influence of some environment conditions to the growth and development of the fungus Pythium helicoides Drechsler The result showed that the fungus P. helicoides Drechsler can adapt with a large range of pH from 5 to 8 and the various light conditions as well as the most suitable temperature from 25 - 350C, which matched the weather conditions of Spring in Phu Tho province. Therefore, the possibility of stem rot disease epidemic outbreak in the Spring is rather high when widely growth of mondo grass in Phu Tho province. 16 3.3.7. Research the measures to control mondo grass’s stem rot disease 3.3.7.1-2. Result of experiment on the suppression efficacy of the biological pesticide Trichoderma asperellum and anti-biotic bacteria to the fungus Pythium helicoides Drechsler in the laboratory conditions: The experiment resulted that the biological pesticide products Trichoderma asperellum and antibiotic bacteria Streptomyces misionensis, Streptomyces aureofacien, Bacillus amyloliquefaciens brought the high efficacy in the suppression of the fungus P. helicoides Drechsler at the high rate of 75%. 3.3.7.3. Field trials of some chemical fungicides to the fungus Pythium helicoides Drechsler in the laboratory conditions: The chemical fungicides such as Aliette 80WP 0,1%, Ridomil 72 MZ 0,2%, and Viben - C 50 BTN 0,1% provided the controlling efficacy to the fungus P. helicoides Drechsler more than 78%. The chemical Copper - Zinc 85 WP 2% brought the low controlling efficacy of 31,9%. The result is presented in the table 3.23. Table 3.23. The controlling efficacy of some chemical fungicides to the fungus Pythium helicoides Drechsler (PPRI, Hanoi, 2012) Diameter of colony No 1 day 2 days Controlling efficacy (%) Chemical fungicides 1 Ridomil 72 MZ 0,2% 0,0 0,0 100 2 Viben - C 50 BTN 0,2% 0,0 0,6 93,3 3 Aliette 80WP 0,1% 0,8 2,0 78,1 4 Copper - Zinc 85 WP 0,2% 3,6 6,1 31,9 5 Control (Water) 5,6 9,0 0 CV (%) 3,2 1,6 3.3.7.4. The trial results of controlling efficacy of some biological and chemical fungicides to the mondo grass’s stem rot disease in the fields: The trial result showed that the biological pesticide products of Streptomyces aureofaciens, Bacillus amyloliquefaciens, Trichoderma asperellum delivered the medium suppression efficacy as high as 63% and 69% after 3 months and 6 months relatively. However, the biological fungicides do not deliver the satisfactory impacts in the cases of epidemic outbreaks due to the slow action. The result is presented in the table 3.24. 17 Table 3.24. The controlling efficacy of some biological pesticides against the mondo grass’s stem rot disease (Phu Tho, 2013) No Trial formula 1 Trichoderma asperellum Streptomyces aureofaciens Bacillus amyloliquefaciens Controlc(water) CV (%) 2 3 4 Before treatment Disease ratio (%) 0,7 After treatment 3 months 6 months Disease Efficacy Disease Efficacy ratio (%) (%) ratio (%) (%) 2,7 63,0 3,3 69,2 1,3 3,3 75,7 4,0 79,9 1,3 4,7 65,3 5,3 73,3 0,7 7,3 25,7 0 10,7 24,2 0 The chemical fungicides such as Ridomil gold 68 WG 0,3% and Aliette 80 WP 0,2% brought the disease control efficacy more than 70% after 1 month. The controlling impact of Aliette 80WP still remained in the fields after 2 months while the efficacy of Ridomil Gold 68WG reduced after 2 months since spraying. Overall, these 2 chemical fungicides delivered the fair suppression impacts and quick action, which can be efficient in the cases of epidemic outbreak. The trial results are presented in the table 3.25. Table 3.25. The controlling efficacy of some chemical fungicides to the mondo grass’s stem rot disease in the fields (Phu Tho, 2013) Before treatment No Trial formula After treatment 1 month Disease ratio (%) Disease ratio (%) Efficacy (%) 2 months Disease Efficacy ratio (%) (%) 1 Spray Aliette 80 WP 0,2% 4,8 5,7 70,3 5,7 73,6 2 Spray Ridomil gold 68 WG 0,3% 3,8 2,9 75,0 4,8 63,2 3 Control (water) 3,8 15,2 17,1 11,7 20,6 CV (%) 3.4. Some measures for controlling the weeds in the intercropping systems 3.4.1. The composition and popularity of the weeds in the intercropping system between mondo grass and tea 18 The weed composition is very diversified in the tea plantations including many annual and perenial weeds. After 3 years of studying the tea plantations grown in the feralit soil in Ha Hoa district, the 15 weed species have been identified including Digitaria marginata Link, Imperata cylindrica PB, Commedia nudiflora L., Zigodium scandans, Cyperus rotundus L., Cyclosorus parasiticus, Ageratum conyzoides L., Paspalum conjugatum L., Cynodon dactylon Pers, Celtella asiatica Urs, Panicum repens L., Bellis perennis, Eupatorium odoratum L., Oxalis cornicullata L., Lygodium sp., Centranthera cochinchinensis L.. The most popular weeds are Commedia nudiflora L, Ageratum conyzoides L, Bellis perennis, Centranthera cochinchinensis L; the popular weeds are Cyperus rotundus L., Paspalum conjugatum L, Celtella asiatica Urs, Panicum repens L and the less popular weeds are Imperata cylindrica PB, Digitaria marginata Link, Lygodium sp.. The weeds are well grown in the spring including Commedia nudiflora L, Cyperus rotundus L., Bellis perennis, Panicum repens L. The perennial weeds well gorwn the whole year are Imperata cylindrica PB, Lygodium sp., Paspalum conjugatum L.. The Ageratum conyzoides L. grown strongly in the season of autumn-winter and the Centranthera cochinchinensis L., Bellis perennis died in the winter but germinated in the next spring. Therefore, in the tea plantation always consists of many weed species with the various levels of growth and damage. 3.4.2. The damage level of weeds in the tea plantations The monitoring of the weed weight in the tea plantations showed that the weeds grown faster than the young tea plants. After 2 months, the weed weight is 0,73 kg/ m2, the peak was reached at 1,41 kg/m2 after 7months. The weeds strongly competed with the young tea plants and become the environment for tea’s pests and dieseases. The comparison showed that after 7 months of intercropping, the tea plantations intercropped with the mondo grass always had the weed weight lower than the tea plantations without mondo grass intercropping. When the mondo grass grown up, the leaf shading had larger soil coverage, which compressed the weed growth significantly, particularly the annual weeds such as cỏ cứt lợn, cỏ vừng và hoa cúc dại. Therefore, the mondo grass intercropping reduced the weeds significantly in the comparison with the non-intercropped tea plantations. 3.4.3. The composition and popularity of the weeds in the pomelo platations intercropped with the mondo grass The survey results showed that there are 24 weed species in the pomelo plantations intercropped with the mondo grass including Digitaria marginata Link, Imperata cylindrica PB), Commedia nudiflora L., Cyperus rotundus L., Ageratum conyzoides L., Paspalum conjugatum L., Cynodon dactylon Pers, Celtella asiatica Urs, Panicum repens L., Bellis perennis, Oxalis cornicullata L., Centranthera cochinchinensis L., Eclipta alba Hassk, Zigodium scandans, Jussiaea repens, Tadehagi triquetrum L. Ohashi, Eleusine indica, Lophatherum gracile Brongn, Eupatorium odoratum L., Achyranthes aspera L., Portulaca oleracea L., Marsilea quaurifolia L., Scoparia dulcis Linn. Of which, 05 species of
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