Nghiên cứu loài nấm ký sinh côn trùng có tiềm năng trong phòng chống ve sầu hại cà phê vùng tây nguyên tt tiếng anh
MINISTRY OF EDUCATION
MINISTRY OF AGRICULTURE
AND RURAL DEVELOPMENT
VIETNAM ACADEMY OF AGRICULTURAL SCIENCES -----------------------------------------
TRAN VAN HUY
STUDY ON ENTOMOPATHOGENIC FUNGI WITH POTENTIAL FOR PREVENTION OF HARMFUL COFFEE CICADAS IN THE CENTRAL HIGHLANDS REGION Specialization Plant Protection Code: 9.62.01.12
SUMMARY OF DOCTORAL THESIS IN AGRICULTURE
The thesis is completed at: Vietnam Academy of Agricultural Sciences
Supervisors: Assoc.Prof.Dr. Le Van Trinh and Dr. Nguyen Van Liem
The thesis will be defended in front of the Academy-level Thesis Evaluation Council at:
On day..... month ..... year ....., ..... a.m./p.m
The thesis can be found at the following libraries:
1. National Library of Vietnam 2. Library of Vietnam Academy of Agricultural Science
INTRODUCTION 1. Rationale Coffee is a key export item of Vietnam with an output of over 1 million tons of green coffee, bringing the turnover of nearly 2 billion USD. Coffee is widely planted in our country with a total area of about 600,000 hectares, of which the Central Highlands region is the largest area up to 576,800 hectares. However, due to largescale concentrated planting and high intensive farming, many pests and diseases have arisen. One of the most dangerous pests of coffee is cicadas. The cicada nymphs live in the soil, bite the roots, suck the xylem sap and cause the leaves to wilt, the fruit to fall. When they occur with high density, the coffee tree may die. The increase of cicadas have caused serious damages to coffee in the provinces of Dak Lak, Dak Nong, Lam Dong, Gia Lai, with an average density of 80-100 individuls/root, some places up to 800-1000 idividulas/root. It was estimated that only in Dak Lak and Lam Dong provinces, nearly 11,000 hectares of coffee weredamaged by cicada (Pham Thi Vuong, 2010). Currently, cicadas still are causing damages in many coffee growing areas of the Central Highlands region and pose a potential risks of outbreaks. Because the cicada nymphs live in the soil, in order to preven them, coffee growers mainly use chemicals with high dosages to irrigate directly to the coffee roots,
but the effectiveess of preventing is not high and leading to polluting the environment. Currently, the direction of using parasitic fungi to control soil pests, including cicadas, is interested by many scientists and has achieved some positive results. The study on the use of beneficial fungi to prevent coffee cicadas is an urgent requirement of coffee production practices. 2. Objective and requirements of the topic 2.1. Objective To dertermine the species composition s and prevalence of parasitic fungi associated with harmful coffee cicadas and to study biological, ecological characteristics and culture conditions of high potential entomopathogenicfungi fordeveloping biological preparations oriented to prevent harmful coffee cicadas with high effectiveness in the Central Highlands region. 2.2. Requirements - Collecting, isolating and determining the species composition of natural entomopathogenic fungi on harmful coffee cicadas in the Central Highlands region - Determining potential entomopathogenic fungous species,and sudyng biological and ecological characteristics of a entomopathogenic fungus with lethal effectiveness to harmful coffee cicadas. - Determining the biomass multiplication technique for the production of biological preparations from high potential entomopathogenic fungus with high effectiveness in preventing harmful coffee cicadas. - Determining the effectiveness of preparations produced from potential entomopathogenic fungous species in preventing harmful coffee cicadas insmall and , large scale field trials and demonstration models.
3. Scientific and practicl meanings of the thesis 3.1. Scientific meanings Providing new scientific data on the species composition of entomopathogenic fungi on cicadas, and on biological and ecological characteristics and lethal ability of Paecilomyces cicadae fungus on harmful coffee cicadas in ecological conditions in Central Highlands region. Data provided is foundation for the orientation of research and application of other parasitic fungi belonging to the Paecilomyces genus in the biological control of coffee pests in particular and crop pests in general. 3.2. Pratical meaning The obtained results generated from study on appropriate conditions (environment, temperature, media pH) for the development of P. cicadae fungus will establish the basis for building a technological process to produce biological preparations for the management of harmful coffee cicadas effectively. Outcomes from this study will contributes to exploit and use of useful biological agents in preventing pests, protecting the ecological environment, increasing the coffee’s productivity and quality in our country. 4. The research’s subject and scope 4.1. The research’s subject - Paecilomyces cicadae fungus parasitizes on harmful coffee cicadas - Harmful coffee cicadasin the Central Highlands region 4.2. The research’s scope - Morphological, biological and ecological characteristics of P. cicadae fungus parasitizing on harmful coffee cicadas in the Central Highlands region. - Determining the ability to multiply and use P. cicadae fungus to develop biological preparations in preventing harmful coffee cicadas. 5. New contributions of the thesis - For the first time, 7 species of entomopathogenic fungi on cicadas in the Central Highlands region are identified and morphological, biological, ecological characteristics and parasitic potential of P. cicada fungus on harmful coffee cicadas are systematically studied. It provides new scientific data on suitable conditions and techniques for culturing of P. cicadae in culture, increasing biomass of P. cicadae (Pae1) fungus for creating preparations oriented. Initially, developing P. cicadae (Pae1) fungi‘s preparations with effectiveness in preventing harmful coffee cicada nymphsin small area of 78.2%, in large areas of 74.675.3% and in application demonstration model reaching to 67.8-68.5%. 6. Structure This thesis is 125 pages long, including a 4-page-long Introduction; Chapter 1. Scientific Foundation and Summary (38 pages); Chapter 2. Content and method of the research (26 pages); Chapter 3. Results and discussion (55 pages); Conclusion and proposals (2 pages with 34 tables and 24 illutrations); 152 reference documents including 41 Vietnamese and 111 English ones.
CHAPTER I: SCIENTIFIC BACKGROUND AND LITERATURE OVERVIEW 1.1. Scientific background of thesis Insects are often infected with a number of microorganisms such as viruses, bacteria, fungus, nematodes and protozoa. Among entomopathogenic agents, parasitic fungus on insects have proactive infect mechanism into the body of insect pests and can spread rapidly and can survive for a long time in the soil, on the body of pests, so they play an important role and are useful biological agents in an integrated pest management (IPM ) system (Gillespie, 1986; Rombach et al., 1986). According to many researches in the world and Vietnam, the number of fungi belonging to the genera Metarhizium, Beauveria and Paecilomyces are being widely applied with high effectiveness in preventing plant pests and protecting the environment and the human’s health (Noris et al., 2002). The harmful coffee cicadas have nymphal stage in the soil for 1 to 2 years, some species even live up to 17 years,therefore they are often killed by many parasitic fungi (Richard et al., 1976; Chen et al., 1991; Duke et al., 2002, Pham Thi Vuong et al., 2010; Kusavadee Sangdee, 2015). On the other hand, due to the relatively long development period of the coffee garden ecosystem, the composion of useful fungi has relatively high stability, which is convenient for the parasitic fungi to infect the cicada population. For the Paecilomyces genus, of which, Paecilomyces cicadae, a cicada parasitic fungous species, is is capable of multiplying biomass to develop biological preparations (Chen et al., 1991; Samson et al., 2004, Liang et al. ., 2005; Peter et al., 2006). Recently, there have been several studies on coffee entomopathogenic fungi (mealybugs, cicadas): M. anisopliae, B. bassiana, Paecilomyces spp. (Pham Van Nha et al., 2012, 2013; Nguyen Nhu Chuong et al., 2016, Dao Thi Lan Hoa et al., 2016, Nguyen Quang Ngoc et al., 2017). These works are important scientific and practical bases for helping to guide the implementation of this thesis. 1.2. Overview of research liturature1.2.1. Study on harmfulness of cicadas on plants in general and coffee trees in particularAccording to Westwood (1840), the cicadas are an ancient animal belonging to the Insecta class, hemiptera order, Cicadidae family. At present, cicadas are classified in Homoptera that has many different genera with thousands of species. They live in tropical and temperate regions and are the most widely distributed insects of all insects. According to Simon (1988), Marlatt (1923) and Molds (1990), cicadas lay their eggs on stems, branches grade 1 and grade 2 of coffee trees. After hatching from the eggs, the numphsfall to the ground and immediately move into the soil, dig holes along the main roots into the ground, concentrate at a depth of 10-40 cm, suck the root sap and cause the hairy root of coffee trees to break, affecting to nutrients and water absorption process of the trees. As onsequence the coffee trees are stunted growth, yellow leaves and produce low yield.
Whenmature, the nymphs move up on above of the ground, climb on the trunk of trees or lay under the dry branches, fallen leaves to molt to adults. After emerging, adultsfly to the big trees to mate, then return to the coffee gardens to lay their eggs and continue causing damage to coffee trees. 1.2.2. Study on using entomopathogenic fungi to prevent plant insect pests in general and cicadas in particular 22.214.171.124. A brief history of research on using entomopathogenic fungi to prevent plant insect pests The entomopathogenic fungi are an important biological agent in the management of harmful insect pests. The role of preventing harmful insects of entomopathogenic fungi is presented in detail in many works of authors in the world such as: Paspelop (1932-1940), Dusky (1959), Tanada (1959 - 1964). ), Hall (1964) ... (quoted by Nguyen Ngoc Tu, 1997). This is an useful factor in an integrated pest management (IPM) system (Gillespie, 1986; Rombach et al., 1986). Since the beginning of the 19th century, there have been many studies on the morphology of the parasitic fungi and their distribution in nature, focusing on research on propagation methods, nutritionmedia, culturing equipments, spore collecting method, creating preparation formulations and using them to prevent plant pests in the fields. Up to now, more than 700 species of fungi that cause insect diseases have been discovered and many useful fungal preparations have been widely commercialized, entomopathogenic fungous sources play an important role in preventing plant pests and protecting the environment (Noris et al., 2002). 126.96.36.199. Pathogenic mechanism of parasitic fungi on insects When insects infected by fungal diseases, their adipose glands and other tissues are dissolved by lipasa and protease enzymes produced by the fungi, characterized by a change in how insects move. The tissues are gradually destroyed, at first the insects move weakly, thenbecome immobile until death. When infected by a fungal infection, insects’ activities will be stopped after 2-3 days of infection, or even last up to 1 week before the fungi grow densely in the bodies of insects. Pathogenic fungi are spread through food, directly host body collision, by wind or through vectors. Fungi’s spores adhere to the bodies of insects and germinate; the fibrous system grows to the point that it covers the spiracles of pests. Spores and fungal fibrous system of entomopathogenic fungi penetrate into the bodies of insects when during its metabolic processes when developing and growing . 188.8.131.52. Study on factors affecting fungi’s pathogenic effectiveness to the hosts The most important factors that affect the pathogenic ability of Hyphomycetes on insects are solar radiation, ambient temperature, precipitation, wind and abiotic factors. Although they often focus on one of these factors, environmental factors
always interact with each other. In particular, temperature and moisture are of the two most important factors which affect the pathogenic effectiveness of fungi on the hosts. 184.108.40.206. Study on screening for an effectiveness isolate of entomopathogenic fungus in preventing insectpests Jin, Feng and Chen (2008) reported that there was a significant difference in the insecticide effectiveness of entomopathogenic fungi isolated. Fernandes (1991); Moorhouse (1993); Balavenkatasubbai et al., (1994) (quoted by Nguyen Ngoc Tu, Nguyen Cuu Huong Giang, 1997) suggested that for production of entomopathogenic fungous preparations with high yield and virulence depends a lot on the origin of isolated strain (geographic origin, isolated from insects or from the soil). 220.127.116.11. Study on entomopathogenic fungi on cicadas Cicadas are insect pests withadult stage isliving on the ground, under foliage and their nymphal stage is living in soil with a relatively high humidity, are often infected by entomopathogenic fungi. Richard et al. (1976) determined that the stages of 1, 2 and 3 nymphal starsare frequently infested by entomopathogenicfungi and spores of Paecilomyces farinosus, mainly attach cicadas at their nymphal stage in the soil, while Massospora levispora’s spores initially infect into nymphal stage and then develop to cause serious disease for adult cicadas after insects coming out of the ground. 1.2.3. Some common characteristics of Paecilomyces genus and Paecilomyces cicadae species According to Samson (1974), the Paecilomyces fungi are divided in two species groups including: Paecilomyces group and Isarioides group, of which many species in group Isarioides are parasiticfungi and cause insect disease (Samson 1974; 2004). The typical morphological characteristics of the Paecilomyces group are described in great detail by Samson (1974; 2004). Jui-Hsia Hsu et al., (2015) identified that Cordyceps ciacadae is another name of Paecilomyces cicadae under Clavicipitaceae family and Cordyceps genus, a cicada-specific parasitic genus, often distributed in tropical and subtropical regions wherethe temperature is ranged from 18-24oC and the relative humidityis > 80%. According to Samson (1974, 1988, 2004), some species groups in Paecilomyces genus have very similar morphologies, therefore, it is necessary to use the gene sequencing technique of RNA ribosoma (rDNA) to determine its scientific name. In rDNA gene segments, the ITS region is widely used to identify fungous species (Bowman et al., 1992; Hibbett, 1992; Driver et al., 2000, Fargues, 2002). 1.2.4. The method of biomass fermentation of entomopathogenic fungi to formulate biological preparations There are 3 methods of fermentation to formulate biological preparations that have been studied and applied including: submerged fermentation, non-steriled surface
fermentation and porous fermentation. One of them, the porous fermentation method with cheap materials such as white rice, brown rice, broken rice, corn flakes, rice bran, wheat, soybean, and bagasse is applied by many scientists in the world to produce entomopathgenic fungous preparations (cited by Nguyen Ngoc Tu et al., 1997). 1.2.5. Some research results on harmful coffee cicadas inViet Nam. - Thành phần loài ve sầu hại cà phê/ Species composition of harmful coffee cicadas According to Pham Thi Vuong and Nguyen Thi Thuy (2010), there are 6 species of cicadas that cause damage ro coffee treeesin the Central Highlands region, of which, 3 species that often have high occurencedensity and cause serious damages to coffee trees in the Central Highlands region. They are white cicadas (Dundubia nagarasagna), Dak Lak cicadas (Pomponia daklakensis) and small cicadas (Purana pigmentata). Research results on methods of preventing cicadas According to Phan Quoc Sung (2007), the solutions to prevent harmful coffee cicadas including: Protection of useful organisms, using light trap to catch cicada adult s, enhancing bio-organic fertilizer that help the root system recovering quickly and developing auspiciouly. According to Nguyen Manh Hung (2008), cicadas nymphs cause great damage to coffee trees but it is very difficult to prevent and eradicate them, because nymphsoften live underground at depths down about from 10 to 20cm to bite the roots and are very difficult to control by spray pesticides, and therefore the effectiveness of this method is not high easy to cause the pollution to the environment. Pham Thi Vuong et al (2010), indicated that chemical pesticides gave very low efficacy to cicada nymphs and and young cicada adults. 1.2.6. Research on the use of entomopathogenic fungous preparations to prevent insect pests The research on the production and application of useful entomopathogenic fungous preparations to preven important insect pests on plants is demonstrated through the works done by Pham Thi Thuy, 1994, 2004, 2005; Ta Kim Chinh, 1996, 2003, 2009; Tran Van Hai, 2006; Nguyen Thi Loc, 2009; Nguyen Xuan Niem, 2009; Pham Van Nha, 2012, 2013, Tran Van Huy, 2012, 2013, etc. 1.3. Issues of concern There have been many detailed studies on entomopathogenic fungi and applicability in the world.In Vietnam, the studiesfocus on two types of green fungus M. anisopliae and white fungus B. bassiana on insect pests. There have been not many studies on harmful cicadas on plants in general and on coffee trees in particular, most of these works only provided data recording on the harmfulness caused by insect pests and cicadas, and the entomopathogenic species composition found in the fields.
CHAPTER 2 MATERIALS, CONTENTS AND METHODS OF THE STUDY 2.1. Materials, study
experiment instruments and equipment, location and time of
2.1.1. Materials used for study The entomopathogenic fungi and harmful coffee cicadas; coffee trees, PDA, Czapek-Dox, Sabouraud, SDAY, N1, CMC, etc media and chemicals. 2.1.2. Experiment instruments and equipment Equipment and instruments to study microbiologies in the laboratory, and investigation and experiment instruments in the fields were used in this study. 2.1.3. / Location and time of study Location: Experiments in laboratories and in net houses were conducted at the Plant Protection Institute (PPRI), Duc Thang, Bac Tu Liem, Hanoi. The studies and surveys in the fields were carrid out in Dak Lak, Dak Nong, Gia Lai and Lam Dong provinces. + Time: From November,2013 to December, 2018. 2.2. Study ontents - Investigating to determine the species composition, prevalence and lethal ability to harmful coffee cicadas of entomopathogenic fungi in the Central Highlands region. - Determining some morphological and biological characteristics of white spawn fungus P. cicadae (Pae1) - Studying on the ecological characteristics of white spawn fungus P. cicadae - Dtermining the biomass development ability of white spawn fungus cicadae (Pae1)
- Assessing the applicability of P. cicadae (Pae1) preparations to prevent harmful coffee cicadas in the Central Highlands region. 2.3. Study methods 2.3.1. Investigation to determine the species composition prevalence and lethal ability to cicadas of entomopathogenic fungi in the Central Highlands region. Conducting surveys for determinng the spevies compositon and prevalence of entomopathgenic fungi on harmful coffee cicadas
Plant pest basic survey method of Plant Protection Institute (1997) and National Technical procedure QCVN 01-38: 2010 / BNNPTNT and survey method propsed by Samson et al., (1988) were used to conduct surveys in Dak Lak, Gia Lai, Dak Nong and Lam Dong provinces. Isolation of entomopathogenic fungi was conducted at Plant Protection Institute according to methods proposed by Barnett and Hunter (1972) and Plant Protection Institute (1997) for single-spore isolation method to entomopathogenic fungus sources on PDA environment, Sabouraud , Czapek-Dox media. Morphological classification was determine according to the documents proposed by Barnett (1955) and Samson et al., (1988,) and molecular biology techniques for sequencing genes of fungous species was used based on the method proposed by Sambrook et al., (1989); Thomas, 1999 and Peter et al., (2006). Collecting and rearing ocicadas nymphs, and identifying lethal ability of fungi were caried out according tothe method described by Pham Thi Vuong (2010) in Nam N’Jang (Dak Nong,) assessments were conducted in April, May 2014, 2015 and 2016. Determining ability of biomass development of common entomopathogenic fungous species Conducted in the Laboratory of Plant Protection Institute in August 2015 according to the method described by Plant Protection Institute (2013) on rice medium supplemented with 0.5% CaCO3 with 4 common entomopathogenic fungous species collected and isolated. Isolation and selection of white spawn fungus P. cicadae strainswith high lethal activity to cicadas nymphs Single-spore method described by Barnett and Hunter (1972) and Plant Protection Institute (1997) was used to isolate fungous strains in June 2015 to evaluate their lethal ability to cicada nymphs, from which strains with higheffectiveness were selected for further evaluation., The lethal effectiveness to cicada nymphs was evaluated according to Henderson-Tilton formula. 2.3.2. Determining some morphological and biological characteristics of white spawn fungus P. cicadae - Study some morphological characteristics of fungus P. cicadae (Pae1) Conducted at the Plant Protection Institute in May and July 2015 according to method of Liang et al., (2005). Fungi were cultured in PDA medium under room conditions. After 3, 7 and 14 days, the diameters of the colonies were measured, specimens were mad and observed under an optical microscope, the colonies’ morphology and spore branch, size and morphology of the spore were described, simultaneously were compared to description in the documents gine by Barnett, (1955) and Samson et al., (1988).
2.3.3. Biological characteristics of fungus P. cicadae (Pae1) - The ability of growth and development of white fungus P.cicadae (Pae1) Conducted at the Plant Protection Institute laboratory according to the method of Plant Protection Institute (1997) in June-July 2015, fungus was cultured on PDA mediumat a temperature of 26.1oC and relative humidity of 71, 2%, the diameters of the colonies were measure and the number of spores was counted in the red cell counting chamber. -The ability to produce extracellular enzymes of white spawn fungus P. cicadae (Pae1) Conducted at the Plant Protection Institute in July 7, 2015 based on the method stated in the microbiology collection by Nguyen Duc Luong (2004). . 2.3.4. Study on some ecological characteristics of white spawn fungus P. cicadae - Investigation to determine the lethal incidence of white fungus to harmful coffee cicada nymphs in some loctionsof the Central Highlands region Investigating the changes of the natural parasite rate of fungi in cicada nymphs Conducted according to basic survey method of the Plant Protection Institute (1997) and national Technical Procedure QCVN 01-38: 2010 / BNNPTNT in 4 locations in Dak Lak, Dak Nong, Lam Dong and Gia Lai provinces), surveys were conducte periodically in interavl of every 15 days in 2015.Investigating and evaluating the lethal incidence of white spawn fungus in cicada nymphs at different age of coffee garden.Conducted at different age coffee gardens in Dak Lak, Gia Lai and Lam Dong province by method of the Plant Protection Institute (1997) in May 2015. - Study the effect of medium factors on the growth ability of white spawn fungus P. cicadae (Pae1) Figuring out the growing ability of fungus on culturemedia: Conducted in the Laboratory of Plant Protection Institute in April 2015 by the method of Chen (1991). Study on the effect of temperature to fungus‘ growth: Conducted at Plant Protection Laboratory Laboratory from April 4, 2015, using the method of Chen (1991). Study the effect of pHmedia culture on fungus’growth Conducted at the Plant Protection Institute Laboratory in September 2015 using the method of Chen (1991). 2.3.5. Study on biomass development ability of white spawn fungus P. cicadae (Pae1) Determining the appropriate mediumcomponent to multiply fungal biomass
Conducted at the Plant Protection Institute in July 2015 according to the method of Pham Thi Thuy (2004), Nguyen Thi Loc (2009), Mascarin et al ., (2010), Sahayaraj et al., (2008). - Experiment to determine the appropriate temperature threshold to multiply fungal biomass Conducted at Plant Protection Institute from August-December 2015 according to the method of Ta Kim Chinh et al., (2003, 2009), Pham Thi Thuy (2004), Nguyen Thi Loc (2009). - Experiment to determine medium moisture conten to multiply biomass Conducted at the Plant Protection Institute in August 2015 by the method of Pham Thi Thuy (2004) and Nguyen Thi Loc (2009) - Experiment to determine appropriate mediumsubstrate mass to multiply biomass Conductedat the Plant Protection Institute in August 2015, by the method of the Plant Protection Institute (with 4 treatemntscorresponding to 4 levels of MT4 mediuml mass at ambient temperature of 25 ± 0,30C. - Experiment to determine the appropriate time to collect fungal biomass Conducted at Plant Protection Institute in September 2015 according to the method of Plant Protection Institute (2001) at ambient temperature of 25 ± 0.30C. - Study to determine multiplication technology to multiply biomass of fungus P.cicadae (Pae1) Experiment to determineappropriate technology to multiply fungal biomass Conducted at the Plant Protection Institute in October 2015, based on the reference and selection of the developing techniques of fungal biomass from the methods desribed by Ta Kim Chinh et al., (2003, 2009), Nguyen Thi Loc (2009), Sahayaraj et al. (2008), Mascarin et al. (2010) and Plant Protection Institute (2001). Study on methods for preserving the original strain (mother strain) Conducted at Plant Protection Institute from February 2015 to March 2016 according to the method of Pham Van Nha (2013). Proposing techniques for producing preparations to prevent cicadas .Developing techniques of fungal biomass based on obtained experiments’ results on determing appropriate conditions for multiplying fungal biomass of P. cicadae (Pae1) and inherited the research results reported by inhome and abroad authors to build The experiment was conducted at Plant Protection Institute in August and September 2016. + Study the effect of adhesives on the germination of whitespawn fungus
Conducted at the Laboratory of Plant Protection Institute in September 2016 according to the method of Nguyen Thi Loc (2007); Pham Thi Thuy et al., (1994) and Plant Protection Institute (2013). 2.3.6. Carrying out xperiments of using P. cicadae (Pae1) fungus to prevent harmful coffee cicadas - Determining the appropriate dosage of preparations used in the fields Conducted in the fields in Krong Pak (Dak Lak) in May 2016 according to the method of Plant Protection Institute (2001). - Determining the time to use appropriate preparations Conducted in Krong Pak district (Dak Lak) with a dosage of 30 kg / ha according to the method of Plant Protection Institute (2001) on 15-year-old coffee gardens. - Experiment to evaluate the effectiveness of white spawn fungus P. cicadae (Pae1) in preventing harmful coffee cicada nymphs in net house conditions Conducted at the Plant Protection Institute's net house in May 2016 according to the method of Plant Protection Institute (2001; 2006). - Evaluating the prevention effectiveness to harmful coffee cicadas of white spawn fungus in small scalefields Conducted in Ea Kenh commune, Krong Pak district, Dak Lak province, on a 15-year-old coffee garden.according to National Standard QCVN 01-1: 2009 / BNNPTNT - Evaluating the prevention effectiveness harmful coffee cicadas of preparations in large scale fields Conducted in Nam N ‘Jang commune (Dak Nong); Di Linh (Lam Dong) and Kong Htok commune (Gia Lai) according to National Standards (2009), QCVN 01-1: 2009 / BNNPTNT - Building an experiment demonstration model to prevent harmful coffee cicadas with white spawnfungus P. cicadae (Pae1) Conducted in Dak N'Rung commune and Nam N 'Jang commune (Dak Song, Dak Nong) with the model area of 1 ha / location from March to December / 2018 in the 15 - 20 years old coffee gardens. 2.3.6. Data analysis Statistical data analysis was obtained by using Excel program and statistical software Statistix 9.0.
CHAPTER III THE RESEARCH RESULTS AND DISCUSSION 3.1. Species composition, prevalence and lethal ability of entomopathogenic fungi to cicadas in the Central Highlands region 3.1.1. species composition, prevalence of entomopathogenic fungi on harmfull coffee cicadas From 2013 to 2017, 10 collecting sample surveys of entomopathogenic fungi on harmful coffee cicadas were carried out in 4 provinces namely Dak Lak, Dak Nong, Gia Lai and Lam Dong. The total of number samples collected was 299, divided in7 main groups: blue fungus, green fungus (Metarhizium anisopliae), white fungus (Beauveria bassiana), white spawn fungus, long spawn fungus, purple fungus (Paecilongces sp. Pae) and yellow spawn fungus. Out of 299 fungous samples collected and isolated, white spawn fungus had the highest number of samples at all locations surveyed, and was 173, accounting for 57.9%. The species composition of entomopathogenic fungi collected and isolated from 2013 to 2018 on cicada was deternimed and was 7 species. Among them, there were two species namely Nomuraea cylindrospora and Beauveria bassiana, which mainly parasitized on cicada adults s, 5 species namely Metarhizium anisopliae, Paecilomyces cicadae, Purpureocillium lilacinum, Cordyceps takaomontana and Cordyceps nutans, which mainly parasitized on cicada nymphs in coffee gardens (Table 3.2). Bảng 3.2. Species composition of entomopathogenic e fungi on harmful coffee cicadas in the Central Highlands ( Dak Lak, Dak Nong, Lam Dong, 2013-2018) Stage of frequency of No. Name of fungus cicadaparasitized encountering 1 2 3 4 5 6 7
Among the 7 species of fungi identified, the white spawn fungus Paecilomyces cicadae has many potential for prevention of harmful coffee cicadas in the Central Highlands region. It had the highest frequency of encountering (+++) in surveys in all 4 areas including Krong Pak (Dak Lak), Dak Song (Dak Nong), and Di Linh (Lam Dong) and Chu Se (Gia Lai). The results of gene sequencing were also completely consistent with the results of the fungous morphological examination under a microscope. Figure 3.6 is Phylogenetic tree based on internal transcribed spacer (ITS) sequences of Paecilomyces cicadae VS1 and other species at Genbank.
Figure 3.6. Phylogenetic tree for P. cicadae VS 01 parasite cicada harmful coffee (based on spacer (ITS) sequences and Mega 6 software) 3.1.2. Lethal ability to cicada nymph and biomass development of entomopathogenic fungi Results of evaluating the lethal ability to 2 to 4 nymphal starsof cicadas of 4 fungi with the highest frequency of encountering in the fields indicatedthat white spawn fungus (P. cicadae) gavethe highest lethal parasite effect to cicadanymphs up to 86.7%, followed by M. anisopliae green fungus with lethal parasitic effectiveness of 50.0%. When multiplied on ricemedim, biomass of P. cicadae developed quite well with the number of spore reaching to 1.8 × 109 CFU /gram of biomass, followed by M. anisopliae fungus with the number of spore reaching to 1, 4 × 106 CFU /gram, B. bassiana fungus reaching to 0.9 × 109 CFU /gram. While The N. cylindrospora fungus grew very poorly and produced very few spores in the rice medium 3.1.3. Isolation, selection of white spawn fungus P. cicadae with high lethal effectiveness to cicada nymphs From culturing on PDA medium and isolating by the single-spore purification method, 5 P. cicada strains namely Pae1, Pae2, Pae3, Pae4 and Pae5 strains were collected. Through evaluation, the Pae1 strains was found to have the highest lethal effectiveness tocicad nymphs, reaching to 87.8% after 15 days of treatment. Pae2 and Pae3 strainsreached lethal effect of 77.8% and 80.0% respectively, while Pae4 train gave a effectivnes of 73.3% and Pae5 strain gave the lowest effectiveness of 61.1%. 3.2. Some morphological and biological characteristics of P. cicadae (Pae1) fungus 3.2.1. Monophological charateristics Morphological, growth and development characteristics of white spawn fungus P. cicadae (Pae1) on PDA medium , such as: morphological characteristics of colonies, mycelium, spore branches, fungi’s spore after stably developed, were described and recordedin detail by using a microscope.
Figure 3.9. Parasitic characteristics of P. cicadae fungi on cicada larvae
Figure 3.11. The colony of P. cicadae Pae1 growing on the PDA medium plate after 14 days
Figure 3.12. Conidia of P. cicadae Pae1
Figure 3.12 Conidia of P. cicadae Pae1
3.2.2. Some biological characteristics of P.cicadae (Pae1) fungus - The ability of growth and development Table 3.10. Characteristics of growth and development of P. cicadae Pae1 after culture time on PDA medium (Plant Protection Research Institute, 2015) Time of culture
Diameters of colonies (cm)
The number of spores (CFU /cm2)
6,1 x 105
9,2 x 106
1,2 x 108
1,6 x 108
1,7 x 108
1,5 x 108
Note : T0C: air temperature, H(%): humidity
Results of evaluating the ability of mycelium’s growth and fungal spore development on PDA artificial mediumat temperature of 26.10C and relatvie humidity of 71.2% showed that the growth rate of colonies and the ability to form spores of this fungus increased rapidly from the 7th to the 9th day after culturing with the spores density reached from 9.2 x 107 to 1.2 x 108 spores / cm2. After that, the number of spores arising gradually decreased from day 12 to day 15 with the number of spores reaching from 1.6 x 109 to 1.7 x 108 spores /cm2.
- The ability to produce extracellular enzymes of some P. cicadae (Pae1) fungi Experiments on ability to produce extracellular enzymes of P. cicadae Pae1, Pae2, Pae3, Pae4, Pae5 was conducted at the Biological Control Research Center Plant Protection Research Institute. The ability to produce extracellular enzymes with chitine resolution (decomposing) ring diameter of P. cicadae (Pae1) strainreached up to 16.5mm and up to 14.6mm on Lipid substrate, 15.0 mm on cellulose substrate and also reached 12.9mm on glucose substrate.
Figure 3.15. Chitine resolution ring of P. cicadae strains
- Influence of a number of factors on the growth of P. cicadae (Pae1)
Paecilomyces cicadae (Pae1) fungus grew well on PDA in vitro culture medium with the diameters of colonies’ reaching to 5.28cm, at the optimal temperature of 20 - 25oC, the diameters of colonies reache to 5.10 and 5.52 cm, and in suitable culture medium, with pH of 6.0 and 6.5,the diamatersof colonies’ reached 5.10 and 5.57 cm, respectively After 12 days of culturing. Table. 3.14. The growth of P. cicadae (Pae1) on culture mediums (PPRI, 2015) Diameter of fungal colonies after culture days (cm)
Medium 3 days
The results of research on the conditions of fungal culture of Paecilomyces cicadae presented in tables 3.15 and 3.16 are also consistent with the previous study of Chen (1991) at Wenzhou University (China). The optimal temperature for
Paecilomyces cicadae fungal growth is between 24 - 26 ° C and its can grow at pH 4 12 but the optimum range is pH 5 to pH 6. Table. 3.15. Effect of temperature on growth of P. cicadae (Pae1) (PPRI, 2015) Diameter of fungal colonies after culture days (cm) Temperature Medium o ( C) 3 days 5 days 7 days 9 days 12 days 17 PDA 0,84b 1,62c 2,30d 3,37e 3,50e 20 PDA 0,82b 1,60c 2,47c 4,01c 5,10c 23 PDA 1,32a 2,74a 3,78a 4,48b 5,52a 25 PDA 1,28a 2,44b 3,50a 4,55a 5,36b 28 PDA 0,70b 1,64c 2,46c 3,80d 4,10d 30 PDA 0,28c 0,58d 1,24e 1,70f 2,07f CV% 8,8 0,92 0,53 0,21 0,10 Table. 3 Effect of pH medium culture on growth of P. cicadae Pae1 (PPRI, 2015) Treatment
Diameter of fungal colonies after culture days (cm) 5 day
3.3. Some ecological characteristics of white spawn fungus P. cicadae 3.3.1. Evolution of the percentage of harmful coffee cicada nymphs parasitized by P. cicadae fungus in the natureal conditionsin some areas of the Central Highlands Under natural conditions in the Central Highlands, the percentage of cicada nymphsparasitized by P. cicadae increase gradually from the end of April to 4.5% and peaked in mid - May with an average rate of 8.03%, then decrease gradually in June (6.73%). the highest percentage of cicada nymphs parasitized by fungi at 11.3% was found in Krong Pak (Dak Lak). From the beginning of September to the first half of October, the percentage of cicada nymphs parasitized by fungi increased rapidly and Figure 3.17. Fruiting bodies of P. reached the 2nd highest peak of the year in mid- cicadae growing on cicada larvae in September with the percentage was 4.88%. the soil of the Central Highland
Krông Pắk - Đắk Lắk Đắk Song - Đắk Nông Di Linh - Lâm Đồng
4 2 0
Hình 3.17. Evolution of the natural parasitization percentage of P.cicadae fungus on harmful coffee cicada nymphs in some areas (2015) 3.3.2. Lethal parasitic level to cicada nymphs in coffee gardens with differen age Among the 4 age groups of coffee gardens investigated, it was found that more than 10 year-old coffee gardens had the highest percentage of cicada nymphs parasitized by fungi, reaching from 4.3 to 5.7 % depending on each location survey 3.4. Study on biomass development ability of fungus P. cicadae (Pae1) 3.4.1. Study on the techniques of biomass multiplication - Appropriate mediumto multiply biomass Among 6 types of testing media, MT4 medium with main component of steamed rice supplemented with 0.5% CaCO3 solution gained the highest number of fungal spores, up to 1.63 x 109 spores / gram of fresh biomass and 1.81 x 109 spores / gram of dry biomass after 12 days of culture - Appropriate temperature to multiply biomass The experiment was carried out on MT4 mediumin 2015. The results showed that the fungal biomass grew well at the temperature ranged from 20- 25oC. The number of spores formed after 10 days of culture at a temperature 200C reached to 1.48 x 109 spores / gram of fresh biomass and reached to 1.69 x 109 spores / gram of dry biomass. Cultivation at a temperature of 250C obtained 1.68 x 109 spores / gram of fresh biomass and obtained1.87 x 109 spores / gram of dry biomass. On cultivation at temperatures of 17oC and 30oC, the number of spores formed was significantly lower. - Appropriate humidity of culture medium Among the moisture content levels of culture mediaincluding 31, 33, 35 and 39%, it was found that with the moisture of the mediumat 33 and 35%, the amount of fungi‘s sporesformed was equivalent (there was no statistical difference), corresponding to 1.61 x 109 and 1.75 x 109 spores /gram of fresh biomass,
corresponding to 1.82 x 109 and 1.91 x 109 spores / g of dry biomass. The results were significantly lower at 31 and 39%. -
Appropriate substrateof medium to multiply biomass
Using medium(in a ratio of 50 ml of 0.5% CaCO3 solution mixed into 100 grams of rice) in a plastic bag of size 25 x 35cm and then aseptically autoclaved it at 1210C in 30 minutes, then, cooled and mixed the mother culturesolution in bags at temperature of 25.0 ± 0.30C indicated that the highest number of spores was obtained, at the mediumweight of 150gam and 200gam /bag, corresponding to 2 doses of 1.64 x109 and 1.62 x109 spores / gram of fresh biomass, equivalent to 1.92 x109 and 1.90 x109 spores / gram of dry biomass. - Appropriate time to harvest fungi biomass The number of spores obtained when cultured at 25.0 ± 0.30C in MT4 medium (Table 3.21) show that after 5 and 7 days, fungal biomass develope with the low number of spores formed. By the time after 10 days, the highest number of spores formed with the number of spores reached to 1.92 x 109 spores/ gram, the the number of spores formed increase slowly, reaching to 1.94 x 109 spores / gram by day 12 and 1.95 x 109 spores / gram by day 15. - Determining technology to multiply fungal biomass Applying CN1 technique byporous fermentation method, propagating level 2 in a triangle flask, mixing sub-mother cultureinto CaCO3 solution and pouring submother culture solution into productionmedium resulted in the number of spores reached 2.25 x 109 spores /gram after 10 days cultured at 25.0 ± 0.3 oC. Among the 3 biomass multiplication techniques, the CN1 technique is superior because it is easy to be applied, does not require complex equipment but produced the high number of spores. - Determining method for preserving original mother culture strain Comparing between 3 methods for preserving purified spores, the method of preserving in Glycerol 10% is the best one. After 3 months of preservation, the number of spores germinated remained at 1.89 x 108 spores /ml (decreased 11.27% compared to the origina onel), after 6 months remained at1.72 x 108 spores /ml (decreased 19.24%) and after 12 months of preservation, the original P. cicadae strain(Pae1) still remained the number of spores germinated spores at 1.63 x 108 spores /ml (23.47%). - Proposing the techniques for producing preparations to preven cicadas From obtained results of the experiments on the biomass multilplication condition of P. ciacadae fungus(Pae 1), together with the inheritance of previous research results, the thesis has determined 5 technical steps for biomass development to produce biological
preparations P. ciacadae. - Effectiveness of preventing cicada nymphs of P. cicadae (Pae1) preparations after different months of preservation The experiment was conducted in September 2016. After 3 months of preservation, the validity of preparations reached to 82.5%, only decreased 5.1% compared to the original product put into storage. After 6 months, the lethal effectiveness to larvae still reached to 79.4%, decreased 8.2% and after 9 months preserved, the effectiveness remained at 76.3, dreseased 11.3%, compared to the original one. After 12 months of preservation, the effectiveness still reached at 72.2%, decreased 15.4% compared to the original. - Influence of adhesive on germination of fungi’s spores Mixing spores of P. cicadae (Pae1) fungus with adhesives at the rate of 0.01% showed that when mixing with Tween 20, the number of spores in germinated preparations reached to 3.04 x109 CFU /gr, with Enomil reached to 3.03 x109 CFU /gr and with Sunligh was to 3.02 x109 CFU /gr and there was no difference compared to those without mixing with adhesive spores not mixed (3.04 x109 CFU /gr). 3.4.2. Applicability of P. cicadae preparation to prevent harmful coffee cicadas - Appropriate dosage of preparations in the fields The obtained results of experiments using 3 different doses of preparations to control cicada in field conditions showed thats at dosage of 20 kg /ha, the effectiveness of preventing cicada nymphs only reached at 51.2;. at dosages of 30kg /ha and 40kg /ha, the effectivenes were similar, and reached 75.7% and 76.1%, respectively. Thus, it can be used at a rate of 30 kg /ha, for saving the amount of preparations used but still gain high effectiveness to prevent harmful coffee cicadas. - Appropriate time to use preparations Whenpreparations was applied in the middle of March with a dose of 30 kg / ha, the effectiveness of preventing cicadas was low, only was 41.7% at 21 days after treatment and was 60.3% after 30 days of treatment. When preparations was applied in early April (April 1, 2016), the effectiveness of preventing cicadas reached 56.3% after 21 days and reached 70.4% after 30 days of treatment. When preparations was applied in mid-April, the effectiveness reached the efficiency of 58.3% and 75.7%, respectively for 21 and 30 days after treatment. When preparations was applied in early May (May 1, 2016) , the effectiveness of preventing cicadas was very low , only reached 37.7% and 54.6%, after 21 and 30 days of treatment respectively.
- Effectiveness of preventing cicadas of preparations in net house conditions Based on the results of dertermining appropriate multiply biomass conditions and techniques, in this study P. cicadae (Pae1 preparations were produced in powder form, including spores mixed with starch auxiliary melt at ratio of 1:10. - Effectiveness of preparations in small scalefields Table 3.29.Effectivenss of P. cicadae fungi preparations in preventing harmful coffee cicadas in smaal scale fields (Ea Kenh, Krong Pak, Dak Lak - 2017) Density of Effectiveness(%) Treatment Preparations Dose cicada DBT 21 DAT 30 DAT (individua/root) I P. cicadae (Pae1) 30 kg /ha 28,1 61,7b 78,2a II M. anisopliae 30 kg /ha 26,2 25,3c 38,1c III Confidor 700WG 1g /gốc 28,7 72,8 a 61,4b IV (Control) Non-treated 27,5 CV% 11,5 12,9 Note : DBT: before the treatment, , DAT; days after treatment Results of evaluating the effectiveness of preventing harmful coffee cicadas of P. cicadae (Pae1) fungus in net house condition showed that 2 treatment in which preparations was irrigated with at concentration of 1.0 x 107 and 5.0 x 107spores /ml, equivalent to 30 kg and 40 kg /ha on used in the fields gave the lethal effectiveness to cicada of 82.0% and 86.0%, respectively. When using at the spores concentration of 0.5 × 107 spores /ml, the effectiveness only reached 67.7%, lower than those at concentrations of 1.0 x 107 and 5.0 x 107 spores /ml. Results of evaluating the effectiveness of preventing harmful coffee cicadas of preparations in narrow fields in Ea Kenh commune (Krong Pak district, Dak Lak) in 2017 showed thats P. cicadae (Pae1) preparations gave the effectiveness in preventing cicada nymphs reached to 61.7% after 21 days of treatment and reached 78.2% after 30 days of treatment, was much higher than M. green fungus treatment of using (only 25.3% after 21 days and 38.1% after 30 days of treatment). Meanwhile, using Confidor 700WG, after 21 days, the effectiveness reached 72.8 %, but after 30 days of effective of this insecticide only reached 61.4%. - Effectiveness in preventing cicadas of preparations in large scale field. Results of evaluate the effectiveness in preventing harmful coffee cicadas in large scale fields of 500m2 for each treatmentin 12-15 year old coffee gardens in Nam N 'Jang communes (Dak Song, Dak Nong) , Hoa Bac commune (Di Linh, Lam Dong) and Kong Htok commune (Chu Se, Gia Lai) in 2018 showed that
- At Nam N’Jang (Dak Song, Dak Nong) After 21 days of use, the effectiveness of preparations’ in preventing cicada nymphs reached 51.37%, and lower than those of insecticide Confidor 700WG treatment (71.3%). But after 30 days of treatment, the effectiveness of preparations reached 74.7%, meanwhile the effectiveness of Confidor 700WG decreased, and was only at 63.4%. By 45 days after treatment, the effectiveness of preparations still remained at 68.3%, whilethe effectiveness of Confidor 700WG decreased to 35.7%. Table3.30. Effectiveness of the preparations in preventing harmful coffee cicadas in large scale fields in Nam N’Jang commune, 2018 Treament
Preparations P.cicadae Control 1 Control 2
Density of cicada BT Effectiveness(%) (individuals /root) 21 DAT 30 DAT 45 DAT
30 kg /ha
Note : BT : before treatment , , DAT: day after treatment - At Di Linh (Lam Dong) The results of evaluation showed that, when using preparations with a dose of 30kg / ha after 21 days, the effectiveness in preventing cicadas reached 51.3%, but after 30 days, the effectiveness of preparations increased markedly and reached 74.7% and the validity after 45 days still reached 68.3%. Meanwhile, using chemical insecticide Oncol 20EC with a dose of 20ml / root, the effectiveness in preventing cicadas’ larvae reached 69.7% after 21 days of treatment, but markedly reduced to only 51.0% after 30 days and 35.7% left after 45 days of treatment - At Kong Htok commune( Chu Se, Gia Lai) The results also showed that after 21 days of treatment, the effectiveness of preparations was only 52.3%, after 30 days of treatment, the effectiveness or preventing cicada nymphs reached the highest level of 75.3% and on 45 days after treatment, the effectiveness was 69.0%. Meanwhile, Oncol 20 EC had the highest efficacy 73.7% at 21 days after treatment, but significantly reduced to 52.3% after 30 days and only was 37.3% after 45 days of treatment.
- Demonstration Model of preventing harmful coffee cicadas with P. cicadae (Pae1) preparations Results of experiment of using P. cicadae (Pae1) preparations to prevent harmful coffee cicadas showed that Table 3.33: Effectiveness of reducing cicada nymp density of P. cicadae (Pae1) preparations in experimental demomstraion models
(Dak Song, Dak Nong, 2018) Cicada density/ coffee (individuals/ root) 2/6 /2018 2/10/2018 1/3/2018 3 months after 7 months after (Before st 1 time of 1st time of treatment) treatment treatment
Effectiveness after 7 months
Model Control Model Control Model Control Đắk N’Rung Nâm N’Jang
- At Dak N’ Rung After 3 months of using preparations, the density of cicadanymps in the model decreased from 55.3 individulas/ root (before treatment) to 19.8 individual/ root, while in the control area, it decreased from 47.6 individuals/ root to 36.7 individuals/ root. After 7 months of the first treatment (meaning that after 3 months after the 2nd treatment), the density of cicada nymphsin the model was only 10.8 individuals/root, while in the control garden, the density was 29 , 5 individuals /root. The effectiveness in preventing cicadas in the model reached 68.5% after 7 months of handling preparations. At the same time, after 7 months of handling preparations, the rate of trees infected with yellow leave disease decreased to 5.5%> Meanwhile, in control garden, there were 35.4% of trees infected with yellow leave disease. - At Nam N’ Jang commune The density of cicada nymphs in the model from 43.7 individuals/ root at the pre-treatment time decreased to 17.8 individual/root after 3 months and only was 9.7 individuals/root after 7 months of preparation treatment. The effectiveness of preventing cicada nymphs after 7 months reached 67.8%. At the same time, the percentage of trees infected by yellow leave disease was only 4.1%. Meanwhile in the control garden, it increased to 29.6% and the efficiency of limiting trees infected by yellow leave disease only remained at 86.5% after 7 months of handling preparations.
CONCLUSIONs AND RECOMMENDATIONS 1. Conclusion 1. For the first time, 7 species of parasite fungies on cicadas in the Central Highlands region have been collected and determined. They are 2 species of Nomuraea cylindrospora Tzean and Beauveria bassiana Vuill, which parasitize mainly on adult cicadas, and 5 other species namely Metarhizium anisopliae Sorok., Paecilomyces cicadae Samson, Purpureocillium lilacinum Thom, Cordyceps takaomontana Yakush. & Kumaz and Cordyceps nutans, parasitze mainly on cicada nymphs larvae. 2.The Paecilomyces cicadae has many potential for prevention of harmful coffee cicadas in the Central Highlands region. Paecilomyces cicadae has the highest encounting frequency (+++) in surveys in all 4 locations surveyeds and the lethal parasitic effectiveness to cicada nymphs reached to 86.7%. When multiplied on ricemedium, the number of spores reached to 1.8 × 109 CFU / gram. Among 5 strainsisolated, P. cicadae (Pae1) starinshad the lethal effectiveness to cicada nymph reached to 87.8% after 15 days of treatment. 3. The morphological and biological characteristics of P. cicadae fungus (Pae1) were described in details. Spore producing ability of P. cicadae increased quickly after 7 to 9 days of culturing with the number of spore formated from 9.2 x 107 to 1.2 x 108 spores/cm2. The fungus species had an ability to produce extracellular enzymes with Chitine resolution ring‘s diameter reached up to 16.5mm, reached 14.6mm on Lipid substrate and 15.0mm on Cellulose substrate. After 12 days of cultivating, P. cicadae (Pae1) fungus grows well on PDA medium with the diameters of available colonies reached to 5.28cm, in the optimal temperature of 20 – 250C, the colonies’ diameter reached to 5.10 and 5.52 cm, and in suitable culture environment with pH of 6.0 and 6.5, the colonies’ diameters was 5.10 and 5.57cm, respectively. 4. Under natural conditions in the Central Highlands region, the percentage of cicadas infected by P. cicadae fungi reached the first peak in mid-May with an average rate of 8.03%, of which, the highest parasitic rate was to 11.3% in Krong Pak (Dak Lak). The second peak occurred in mid-September of year with 4.88% cicadas infected. Percentage of cicada nymphs infected by parasitic fungi was the highest in coffee gardens at 10 year old, from 4.3 to 5.7% depending on each location observed. 5. The nutritional requirements and other culture requirements for biomass growth of P.cicadae in solid media fermentaiton were investigated. The P. cicadae (Pae1) could produced a number of spores to 1.81 x 109 spores/gram of dry biomass after 12 days of culturing when it was culturedon MT4 medium. At 250C, the number of spores formed after 10 days reached to 1.87 x 109 spores/gram of dry biomass. With mass medium of 150gram and 200gram /bag, the number of spores produced were 1.92 x109 and 1.90 x109 spores/ gram, respectively. Ten days after cultivation was the best time for harvesting spores for making preparations with the number of spores reached 1.92 x 109 spores/gram. When the moisture of culture medium was of 33% and 35%, the number of spores produced was 1.82 x 109 and 1.91 x 109 spores /gram dry biomass, respectively. 6. Using P. cicadae preparations with a dosage of 30kg/ha was considered to be appropriate with the effectiveness of preventing of cicadas was 75.7%. Application of preparations in the middle of April gives a effectiveness up to 75.7% at 30 days after