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

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-sp

MINISTRY OF EDUCATION

AND TRAINING

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

Hanoi, 2019

The thesis is completed at: Vietnam Academy of Agricultural Sciences

Supervisors: Assoc.Prof.Dr Le Van Trinh and Dr Nguyen Van Liem

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

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

- 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

3.2 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 74.6- 75.3% 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

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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

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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

1.2.2.1 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)

1.2.2.2 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

1.2.2.3 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

1.2.2.5 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

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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

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CHAPTER 2 MATERIALS, CONTENTS AND METHODS OF THE STUDY

2.1 Materials, experiment instruments and equipment, location and time of study

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 P cicadae (Pae1)

- Assessing the applicability of P cicadae (Pae1) preparations to prevent

harmful coffee cicadas in the Central Highlands region

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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)

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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

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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

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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:

2.3.6 Data analysis

Statistical data analysis was obtained by using Excel program and statistical software Statistix 9.0