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MINISTRY OF EDUCATION

MINISTRY OF AGRICULTURE

AND TRAINING

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

Reviewer 1:

Reviewer 2:


Reviewer 3:

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.

1


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.

2


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.

3


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

4


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.
1.2.2.4. 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).
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

5


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.

6



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)


P.

- 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

7


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).

8


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

9



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

10


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.

11


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

Metarhizium anisopliae Sorok.
Nomuraea cylindrospora Tzean.
Beauveria bassiana Vuill.
Paecilomyces cicadae Samson
Cordyceps takaomontana Yakush
Purpureocillium lilacinum Thom.

Cordyceps nutansPat.

Nymph
Adult
Adult
Nymp
Nymph
Nymph
Nymp

+
++
+
+++
+
-

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.

12


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.

13



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)

3 days

1,31


very few

5 days

2,18

6,1 x 105

7 days

3,18

9,2 x 106

9 days

4,15

1,2 x 108

12 days

5,26

1,6 x 108

15 days

5,29


1,7 x 108

21 days

5,27

1,5 x 108

Note : T0C: air temperature, H(%): humidity

14

T0 C

H (%)

26,1

71,2


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)

ToC

H (%)

25,0


61,0

Medium
3 days

5 days

7 days

9 days

12 days

Saboraud

0,72b

1,84b

2,76b

3,33b

3,98 b

PDA

1,24a


2,24a

3,10a

4,20c

5,28a

SDAY

0,44c

1,18c

2,05c

2,13c

2,33c

Czapek-Dox

0,70b

1,81b

2,70b

3,28b


3,68b

CV%

7,68

0,28

0.19

0.25

0.79

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

15


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

pH
medium

Diameter of fungal colonies after
culture days (cm)
5 day


7 day

9 day

H
(%)

25,0

61,0

12 day

I

5,5

1,21d

2,02c

3,01d

3,41d

II

6,0


2,09b

3,21a

4,15b

5,10b

III

6,5

2,51a

3,24a

4,31a

5,57a

IV

7,0

1,75c

2,81b

3,85c


4,15c

V

7,5

0,70e

1,35d

2,12e

2,54e

1,57

0,71

0,77

0,72

CV%

T0 C

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

16


12

10

Krông Pắk
- Đắk Lắk
Đắk Song
- Đắk Nông

Di Linh
- Lâm Đồng

8
6

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,

17


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

18


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.

19



- 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


20


- 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

Active
gradients

Preparations P.cicadae
Control 1
Control 2

Confidor
700WG

Dosage

Density of cicada BT
Effectiveness(%)
(individuals /root) 21 DAT 30 DAT 45 DAT


30 kg /ha

25,7

51,7

74,6

70,7

1g /root

24,3

71,3

63,3

25,3

25,0

-

-

-

Non-treated


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.

21


- 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

Location


(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

55,3

47,6

19,8


36,7

10,8

29,5

68,5

43,7

42,2

17,8

32,4

9,7

29,1

67,8

- 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.

22


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

23


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