et al

THESIS
EFFECTIVENESS OF LOCAL ENTOMOPATHOGENIC FUNGI AS
BIOINSECTICIDE FOR TOMATO INSECT PESTS
CHEERAPHA PANYASIRI
A Thesis Submitted in Partial Fulfillment of
the Requirements for the Degree of
Master of Science (Agriculture)
Graduate School, Kasetsart University
2005
ISBN 974-9846-84-2

ACKNOWLEDGMENTS
I wish to express my deep gratitude to my advisor, Associate Professor Dr. Tipvadee
Attathom who extends valuable encouragement, suggestions and criticism throughout the course
of my study. Her constant assistance and kindness in editing this thesis manuscript is highly
appreciated.
I would like to deeply thank my major committee, Assistant Professor Dr. Wiboon
Chongratanameteekul and my minor committee, Dr. Wanwilai Intanoo for their advices and
valuable comments for the completion of this study.
Many thanks are expressed to Miss Kannika Srinaunmak and members of insect
pathology laboratory for their helpfulness and friendship during my graduate study, to Mrs.
Apinun Sonong and Mrs. Yupin Srihirun for their technical assistant on electron microscopic
study and their friendliness.
Most of all, I am gratefully and sincerely thanks to my parents and my sister who
mercifully provided me everything, especially encouragement and educational supports
throughout my study.
This study was funded by the National Research Council of Thailand (NRCT) and the
Deutsche Forschungsgemeinschift (DFG, German Research Council) within frame work of the
joint research project on “Integrated Management of Tomato Pests under Protected Cultivation
Using Biological Products”. Special thanks are extended to Professor Dr. Michael Poehling,
Institute of Plant Diseases and Plant Protection, Faculty of Horticulture, Hannover University,
Germany for his valuable suggestions and effort in providing MS. scholarship through the DFG
project. This study was financially supported, in part by the Graduate School, Kasetsart
University.
Cheerapha Panyasiri
October 2005

TABLE OF CONTENTS
Page
TABLE OF CONTENTS…………………………………………………………………… i
LIST OF TABLES…………………………………………………………………………. ii
LIST OF FIGURES………………………………………………………………………… iv
INTRODUCTION…………………………………………………………………………. 1
LITERATURE REVIEW………………………………………………………………….. 4
MATERIALS AND METHODS………………………………………………………….. 22
RESULTS AND DISCUSSION…………………………………………………………... 32
Insect survey and sample collection……………………………………………… 32
Fungal isolation and identification ………………………………………………. 33
Efficacy of entomopathogenic fungi against tomato insect pests……………..….. 49
Pathological observation………………………………………………………….. 55
Mass production of entomopathogenic fungi………….…………………………. 60
Spray application in screen cage for insect control………………………………. 79
CONCLUSION……………………………………………………………………………. 84
LITERATURE CITED…………………………………………………………………….. 87
APPENDIX………………………………………………………………………………… 104
i

LIST OF TABLES
Table Page
1 Newly recovered of entomopathogenic fungi isolated from insect collected from
field and greenhouse grown tomato. …………………………………………….. 34
2 Isolates of entomopathogenic fungi obtained from governmental institutes ……. 53
3 Efficacy of entomopathogenic fungi against tomato insect pests.……… ………. 54
4 Number of conidia of the fungus, Paecilomyces fumosoroseus (Acc. no. FWA3)
produced on different substrates and harvested at different period of time………… 62
5 Number of conidia of the fungus, Paecilomyces fumosoroseus (Acc. no. BCC
7058) produced on different substrates and harvested at different period of time…. 64
6 Number of conidia of the fungus, Beauveria bassiana (Acc. no. BCC 1658)
produced on different substrates and harvested at different period of time……… 66
7 Number of conidia of the fungus, Metarhizium anisopliae (Acc. no. KKU 2)
produced on different substrates and harvested at different period of time……… 68
8 Number of conidia of the fungus, Hypocrella hypocreoidea (Acc. no. BCC
11370) produced on different substrates and harvested at different period of time… 70
9 Number of conidia produced by the five effective isolates of entomopathogenic
fungi cultured on different substrates used as growth medium……………………. 71
10 Analysis of LC 50 value of entomopathogenic fungi against tomato insect pests at 4
days after fungal spraying to tomato infested plants in insect screen cage………... 83
ii

LIST OF TABLES (cont’d)
Appendix Table Page
1 Cost of each substrate used for conidia production………………………….......... 105
2 Spray application of Paecilomyces fumosoroseus (Acc. no. FWA3) against thrips,
Ceratothripoides claratris in insect screen cages………………………………… 106
3 Spray application of Metarhizium anisopliae (Acc. no. KKU 2) against mealybug,
Pseudococcus cryptus in insect screen cages……………………………………… 106
4 Spray application of Paecilomyces fumosoroseus (Acc. no. FWA3) against
whitefly, Bemisia tabaci in insect screen cages…………………………………… 107
iii

LIST OF FIGURES
Figure Page
1 Colony characteristics of the entomopathogenic fungi newly recovered from
naturally infected insect pests of tomato. ……………………………………. 40
2 Morphological diagram of the isolates of entomopathogenic fungi newly
recovered from insect pests of tomato..………………………………………… 44
3 Scanning electron micrographs demonstrated infection process of
entomopathogenic fungi………………………………………………………… 57
4 Scanning electron micrographs of insects infected with entomopathogenic fungi 58
5 Scanning electron micrographs of conidiophores and conidia of highly effective
fungal isolates produced on the surface of insect cadavers. …………………….. 59
6 Mycelium growth and sporulation of Paecilomyces fumosoroseus (Acc. no.
FWA3) on different solid substrates used as culture medium…………………… 72
7 Mycelium growth and sporulation of Paecilomyces fumosoroseus (Acc. no.
BCC 7058) on different solid substrates used as culture medium………………. 73
8 Mycelium growth and sporulation of Beauveria bassiana (Acc. no. BCC 1658)
on different solid substrates used as culture medium……………………………. 74
9 Mycelium growth and sporulation of Hypocrella hypocreoidea (Acc. no. BCC
11370) on different solid substrates used as culture medium……………………. 75
10 Mycelium growth and sporulation of Metarhizium anisopiae (Acc. no. KKU 2)
on different solid substrates used as culture medium……………………………. 76
iv

EFFECTIVENESS OF LOCAL ENTOMOPATHOGENIC FUNGI AS
BIOINSECTICIDE FOR TOMATO INSECT PESTS
INTRODUCTION
Tomato (Lycopersicon esculentum) is one of the economic important crops of Thailand. It is
consumed as fresh table tomato and as an essential raw material for a variety of food processing
industries. Tomato production faces many problems from several causes such as seasonal weather,
temperature, humidity, diseases and insect pests. There are several insect species feed on tomato for
examples: thrips, whitefly, tomato fruitworm, leaf miner, leafhopper, aphid, mites and mealybug.
Thrips, mealybug and whitefly are particularly important pests and are the major causes for the
reduction of tomato production.
Thrips attack leads to the wilting of young plants. Leaves are silvered and flecked. It is also a
vector of spotted wilt virus (Morgan, 1979). In recent year, it has become one of the most
economically damaging pest to greenhouse crops (Osborne et al., 1990). In Thailand the crops most
seriously attacked by thrips are chili, watermelon, cucumber, onion, shallot, garlic, eggplant,
mushroom, potato, asparagus, yard-long bean, garden pea, okra and tomato (Bansiddhi and
Poonchaisri, 1991). Mealybug is an important pest in greenhouse. Outbreaks often occur in
greenhouses even when densities are low in field. Several natural enemies of mealybug have been
reported (Itioka and Inoue, 1996) and are thought to be important for suppressing the pest in the field.
Whitefly is an important pest of horticultural crops in greenhouse. The problems are increasing on
outdoor crops such as strawberry, raspberry, pepper, cucumber, tomato, lettuce, citrus and lima bean.
Economic damage to crops are caused by ingestion of plant sap and contamination of crop products
with honeydew that forms a substrate for the development of sooty mold (Johnson et al., 1992; Liu et
al., 1993)
In many countries, control of tomato pests is based mainly on chemical insecticides.
Chemical residues in fresh tomato raise awareness of adverse effects of chemical insecticides to
1

2
human health and environment. In addition, extensive and indiscriminate uses of chemical
insecticides had created resistant problem in many insect species. Growing tomatoes under protected
cultivation is increasing due to the significant market size for pesticide-free tomatoes. However,
some tiny, plant sucking insect such as thrips, Ceratothripoides claratris; mealybug, Pseudococcus
cryptus and whitefly, Bemisia tabaci can increase rapidly in greenhouse and cause costly damage to
tomato. Concerns about the negative impacts of chemical insecticides have led to alternative
strategies for pest control, the biological control using entomopathogens. There are many naturally
occuring entomopathogens that are effective against insects, such as virus, bacteria, nematode and
fungi. Entomopathogenic fungi are well-recognized as effective biocontrol agents for tiny plant
sucking insects in particular, for example the fungi, Beauveria bassiana and Paecilomyces
fumosoroseus were reported to be effective against whiteflies (Bemisia tabaci), Beauveria bassiana,
Verticillium lecanii, Paecilomyces fumosoroseus and Hirsutella sp. were effective against thrips
(Thrips tabaci) and Verticillium lecanii, Aschersonia sp. and Hypocrella sp. were effective against
mealybug (Pseudococcus cryptus) (Butt, 1997). This study aims to search for local strains of
entomopathogenic fungi and evaluate their effectiveness against some tomato insect pests. Attempts
are made to develop those potential fungi as bioinsecticide for tomato pests control under protected
cultivation.

OBJECTIVES
1. To search for local strains of fungi that are pathogenic to tomato insect pests.
2. To evaluate and compare efficacy of the fungus isolates against some important tomato insect
pests in greenhouse.
3. To develop the effective fungus strains as bio-insecticide for the control of insect pests of tomato
cultivated in greenhouse.
3

Insect pests of tomato
LITERATURE REVIEW
The vast majority of tomato pests are aphids (Aulacorthum solani), leaf miner (Liromyza
bryoniae), leafhopper (Zygina pallidfrous), red spider mite (Tetramychus urticae), springtails, thrips
(Thrips tabaci), tomato moth (Lacanobia oleracea), whitefly (Trialeuriodes vaporiorum), wire worm
(Agriotes obscurus), and woodlice (Morgan, 1979) but the important pests are thrip (Thrips tabaci),
whitefly (Bemisia tabaci (Genn.)), mealybug (Ferrisia virgata (Ckall)) and aphids (Aulacorthum
solani) (Jones, 1999).
1. Thrips
Thrips is a world wide distributed insect species. It is classified in the order Thysanoptera.
Larvae are pale-yellow, adult thrips are small, dark brown insect with elongated bodies, and fringed
wings (Poonchaisri, 1990). The leaves of attacked plants become silvered and flecked. Heavy attacks
lead to the wilting of young plants. They are vectors of spotted wilt virus (Morgan, 1979). In
Thailand, plant crops that are seriously attacked by thrips are chili, watermelon, cucumber, onion,
shallot, garlic, eggplant, tomato, potato, asparagus, yard-long bean, garden pea, okra and mushroom
(Bansiddhi and Poonchaisri, 1991). Similar records of plants species attacked by thrips were also
reported in Taiwan (Chang, 1991).
Life cycle: Thrips undergoes complete metamorphosis in which the life cycle composed of
egg, larva, pupa and adult stage. Hill (1983) described life cycle of thrips as follow: white eggs are
laid in notch in the epidermis of the leaves and stems of young plants. They take 4-10 days to hatch.
The larva feed on the leave by rasping the epidermis of the leaves and sucking the exudate. White or
yellow nymphs moult twice in about five days. Pupation occurs in the soil, and takes 4-7 days. The
adult is a small, yellow-brown thrips, with darker transverse bands of 1 mm long across the thorax
4

5
and abdomen. It takes about 3 weeks for one generation and there are generally several generations
per year.
Distribution: Thrips is almost completely cosmopolitan insect species. Hill (1983) reported
the distribution of thrips in West Africa, Canada and south Scandinavia to South Africa and New
Zealand. In Asia, there were records in Philippines which revealed that thrips feed only on tomato,
pepper, eggplant, watermelon, mushmelon, cucumber, garlic and potato in low elevated areas.
Species attacking several corps are predominantly Thrips palmi Karny and Megalurothrips usitatus
Bagnall (Bernardo, 1991). In Indonesia, T. tabaci Lind, T. palmi Karny and T. parvispinus Karny are
the major thrips species on vegetables which include chili, potato, tomato, melons and amaranth
(Sastrosiswojo, 1991). In Malaysia, T. palmi Karny was found on cucumber (Cucumis salivus), chili
(Capsicum annuum), brinjal (Salanum melongena) and tomato (Lycopersicon esculentum). It is the
only vector reported to transmit tomato spotted wilt virus (TSWV) (Fauziah and Saharan, 1991). In
Thailand, the important thrips species are Scirtothrips dorsalis, T. parvispinus, T. tabaci, Haplothrips
floricola and T. flavus (Poonchaisri, 1990).
2. Mealybug
Mealybugs are in the order Homoptera, family Pseudococcidae. Lindquist (1997)
described that mealybugs are the least scale-like of the group, mainly because they are soft-bodied,
without the outer shell associated with insects as in the other scale insect families. Instead, mealybugs
are usually covered with a white waxy powder, and have filamentous projections around the
perimeter. Plant crops that are seriously attacked by mealybugs are coffee, cocoa, citrus, cotton, jute,
groundnut, beans, cassava, sugarcane, sweet potato, cashew, guava and tomato. Moderate damage
also occurs in many other plants. They feed on young shoots, berries and leaves. Heavy attacks of
mealybug usually follow periods of prolonged drought.
Life cycle: Hill (1983) described that female mealybug lays 300-400 eggs, the egg hatches in
a few hours and the young nymph can move away quite rapidly. They are full grown in about 6

6
weeks. The female is a distinctive mealybug with a pair of conspicuous longitudinal sub-median dark
strips, long glassy wax threads, a pronounced tail, and a powdery waxy secretion. The entire life cycle
takes about 40 days.
Distribution: Borror et al. (1989) described that mealybug is a large group, with more than
300 species in America. There are three important pest species in this group. The citrus mealybug,
Planococcus citri (Risso) and the citrophilus mealybug, Pseudococcus fragilis Brain, are serious pests
of citrus and also attack greenhouse plants. The longtailed mealybug, Pseudococcus longispinus
(Targioni- Tozzetti), is often found in greenhouses, where it attacks a variety of plants. They are
destructive insect pests in some area such as Australia and South America (Hill, 1983). Species of
mealybug that are distributed throughout southern Europe, the Middle East, parts of Africa, South
America and North America were first identified in California in the Coachella Valley in the early
1990s (Gill, 1994).
3. Whitefly
Whitefly belongs to the order Homoptera, family Aleyrodidae and is close relatives of
aphids, scale insects, mealybugs, hoppers and cicadas. Shetlar (2003) described whitefly as a white
insect with pale yellow body of approximately 2 mm long. It feeds by extracting plant fluids with
sucking mouth parts. Feeding damage appears as yellow, stunted growth and, in severe cases,
honeydew and sooty mold can develop on plant parts. Sooty mold is a black fungus that grows on
honeydew. Honeydew and sooty mold can reduce photosynthesis and crop value. Plant death can
occur if large populations of whitefly are left untreated. Plants that are seriously attacked by whitefly
are cotton, tomato, tobacco, sweet potato and cassava. Many wild and other cultivated plants are also
attacked by whitefly.
Life cycle: Life cycle of whitefly had been described by Borror (1989) and Shetlar (2003).
In brief, the white egg is about 0.2 mm long and pear-shaped. It is laid on upper part of the leaf and
egg hatches after about 7 days. The newly hatch larva can move a very short distance before setting

7
down and starting to feed. The last instar larva is about 0.7 mm long and red eye of the adult can be
seen. The nymphal stage lasts 2-4 weeks according to temperature and hatch to adult in a minute.
The adults of both sexes are winged, and the wings are covered with the white dust or waxy powder.
The adult is usually active whitish insect that feed on leaves. The female may lay 100 or more eggs.
Distribution: The whiteflies are most abundant in the tropics and subtropics. It is a
cosmopolitan insect species widespread occurring from Europe to Asia. Their population dynamics
strongly suggested that biological control might provide the greatest capacity to reduce damage
caused by Bemisia argentifolii (B. tabaci, strain B) in both greenhouse and field crops (van Lenteren
and Woets, 1988; Baumgartner and Yano, 1990; Gerling, 1990).
Recently its importance as a pest to field crops has increased. Damage estimates in 1991 by
B. argentifoli to crops in the United State exceeded $500 million. Moreover its tranmission ability of
a virus to the Florida tomato crops was also reported (Perring et al., 1993). Entomogenous fungi,
Paecilomyces fumosoroseus, Verticillium lecanii and Beauveria bassiana were used successfully for
control of whitefly nymphs (Landa et al., 1994).
4. Aphid
Aphids are in the order Homoptera, family Aphididae. They infest a wide range of host
plants. Palmer and Smith (1967) listed some important cultivated hosts which include potato, tomato,
eggplant, sunflower, pepper, pea, bean, apple, turnip, corn, sweet potato, asparagus, clover and rose.
Weeds such as ragweed, lambsquarters, jimsonweed, pigweed, shepherdspurse and wild lettuce are
also common food plants. Sporadic in occurrence, aphid infestations are rarely severe enough to kill
plants. Aphids pierce veins, stems, growing tips, and blossoms with their needle-like mouthparts. As
a result, blossoms are shed and yield is reduced. New growth becomes stunted and curled. Heavily
infested plants turn brown and die from the top down. Aphids tend to spread rapidly from field and
transmitting a number of viral diseases. These include various mosaics, leaf roll, spindle tuber, and
unmottled curly dwarf diseases (Griffin, 2004).

8
Aphids are soft-bodied, pear-shaped insect usually wingless. The body color may be solid
pink, green, yellow and pink mottled, or light green with a dark stripe. It is about 2.5 to 3.5 mm long
and has a pair of long, slender tailpipe-like appendages known as cornicles at the dorsal side of the
fifth or sixth abdominal segment. These cornicles secrete a defensive fluid. Aphid also excrete
honeydew, which is emitted from the anus. Adult females give birth to live young, called nymphs.
Although slightly smaller than adults, nymphs are similar in color and shape (Griffin, 2004).
Life cycle: In Thailand, female aphids feed and reproduce year round. No eggs or males are
produced. Without mating, wingless females give birth to about 50 live nymphs. During warm
weather, each of these nymphs matures in 2 or 3 weeks. The life cycle continues in this manner until
overcrowding occurs or food becomes scarce. At this time nymphs develop into winged adults and
migrate to new host plants. Once settled down, these aphids begin reproducing and the life cycle
continues as before. During winter, however, feeding and reproduction occur at a much slower rate.
Many generations are produced each year (Lowe, 1973 and Borror, 1989).
Distribution: Aphids are found almost widespread from Europe to Asia and occur throughout
North American (Lowe, 1973). The aphids are tended by the ants, which transfer them from one food
plant to another. The ants feed on the honeydew produced by the aphids. In many species the winged
form migrates to a different host plant, and the reproductive process continues.
Entomopathogenic Fungi
Many insect pests are susceptible to infection by naturally occurring insect pathogenic fungi.
Several fungi have been studied as potential mycoinsecticides. These fungi are very specific to
insects, often to particular species and do not infect animals or plants. Fungi provide the only
satisfactory microbial means of biocontrol of plant sucking insects such as aphids and whiteflies that
are not susceptible to bacteria and viruses. The well-studied insect pathogenic fungi include
Beauveria bassiana for locusts and beetles, Metarhizium anisopliae and M. flavoviride for locusts and
Verticillium lecanii for control of aphid. Other possible fungal candidates of biocontrol agents

9
include B. brongniartii, Hirsutella thompsonii, Paecilomyces fumosoroseus, P. farinosus, Nomuraea
rileyi and Aschersonia aleyrodis.
1. History
Boucias and Pendland (1998) summarized history of the entomogenous fungi as they
originated more than 2000 years ago. The first record, Cordyceps(Ascomycota) was found to infect
Lepidopteran larvae in ancient China. The colored fruiting body of this fungus usually protrudes
from the mouth or anus of the insect cadaver and is therefore very conspicuous. Throughout history,
it has been used for religious, medicinal purposes and as food. Cordyceps was the subject of the first
published account of an entomopathogenic fungus by Reaumus in 1726. Another fungus, Beauveria
bassiana (Deuteromycetes) was observed in about 900 AD in silkworms in Japan. It was used for
medical purposes as an antiseptic for wounds and sore throats. Moreover, it was through a study of
this fungus that the germ theory of disease was postulated by A. Bassi in 1834. In the 1870’s, E.
Metchnikoff pioneered the field of cellular immunity through his earlier research on other fungal
entomopathogen, the green muscardine fungus Metarhizium anisopliae (Deuteromycetes).
The use of B. bassiana and M. anisopliae as biological control agent has been studies with
many insect pests. Both fungi also have potential use against soil insects such as curculionids,
scarabs and lepidopterous (Ferron, 1981)
In Brazil, M. anisopliae has been used effectively against spittle bugs on sugarcane and the
spittle bug population were reduced following spraying conidia products, usually at 10 12 conidia/ha.
Incidence of mortality from M. anisopliae followed treatment was expected to be at least 40% which
increase sugarcane content sufficiently to justify fungal use (Roberts et al., 1991). Zimmermann
(1993) reported that the host list of M. anisopliae was more than 200 insect species from seven
orders.

10
B. bassiana is available commercially as a microbial insecticide since B. bassiana can now
be mass produced by a fermentation process and formulated to enable the fungus to withstand
ultraviolet light, temperature and humidity extreme commonly encountered in the field. There are
several products that contain B. bassiana and registered with EPA. B. bassiana takes 3-7 days to kill
an insect and will take some time to suppress the pest population when using these product. Through
spray coverage is essential because fungal spore must contact the insect for infection to occur
(Hofmann and Frodsham, 1993).
2. Natural incidence
The fungi isolated from or known to infect insects belong either to the class Hyphomyces
or Zygomyces in the subdivisions Deuteromycotina and Zygomycotina, respectively.
2.1 Zygomycetes
Observations of natural epizootics in thrips populations had largely been restricted to
Entomophthorales. Neozygites parvispora, formerly known as Entomophthora parvispora, had been
found frequenly in Thrips tabaci and T. fuscipennis populations on onion crops in central and
southern Europe. Attempts to utilize this agent for management of thrips under field conditions were
not successful, although better control of T. tabaci was obtained in greenhouses where environmental
conditions favoured infection and spread of the disease (Carl, 1975; MacLeod et al., 1976). In the
Netherlands, epizootics caused by Entomophthora thripidum had been observed in T. tabaci
infestations on a variety of greenhouse crops, but in field trails the fungus failed to suppress thrips
populations below an economically acceptable level (Ramakers, 1976; Samson et al., 1979).
Saito et al. (1989) reported a low incidence of Neozygites parvispora in field-collected
specimens of T. palmi. This pathogen was also recovered from Frankliniella occidentalis infesting
glasshouse-growth in Italy (Magano di san Lio et al., 1992). In glasshouse studies, Vacante et al.
(1994) noted that N. parvispora caused up to 60 per cent mortality in motile developmental stages of

11
F. occidentalis and reduced both the insect population density and the proportion of leaves and
flowers infested. Development of an epizootic appeared to be less dependent upon high ambient
relative humidity than for other isolates of this species.
2.2 Hyphomycetes (Imperfect fungi)
Vertillium lecanii, Metarhizium anisopliae, Paecilomyces farinosus, P. lilacinus and
Hirsutella sp. had been isolated from Taeniothrips inconsequens, a recurrent pest of sugar maple in
the north-eastern USA (Skinner et al., 1991; Brownbrigde, 1995). Beauveria bassiana had been
recovered from Thrips calcaratus, Frankliniella occidentalis and Haplothrips tritici (Lyubenov,
1961; Humber, 1992). Hall (1992) and Hall et. al. (1994) had reported Hirsutella sp. nov. causing an
epizootic in T. palmi in Trinidad and the occasional incidence of P. fumosoroseus in collected
specimens. Hirsutella sp. was also isolated from Liothrips mikaniae (Greenwood and Mill, 1989).
Although not generally regarded as a virulent insect pathogen, an Aspergillus sp. had also been
reported to infect thrips (Dyadechko, 1977).
A number of species of hyphomycetous imperfect fungi (Deuteromycetes) had been reported
to attack aphids. The fungi, Cephalosporium aphidicola Petch, C. muscarium Petch, Cladosporium
aphidis Thuem., Hirsutella aphidis Petch and Paecilomyces (Spicaria) farinosus (Dicks ex. Fr) Brown
and Smith were recorded from aphid hosts by Petch (1932, 1948), Since they are seldom reported by
researchers, it can be assumed that they occur rarely in nature and play a minimal role in the
suppression of aphid populations which they are reported to attack.
Numerous field trails were conducted with the fungus, Acrostalagmus aphidum Oud in
Puerto Rico against the aphid, Myzus persicae (Sulzer) and Aphis gossypii Glover, on egg plant by
Nolla (1929), who reported good control following spraying of infested plants with spore
suspensions. Results of the studies conducted in the 1970’s by Wolcott using A. aphidum against
heavy infestations of Sipha flava (Forbes) on young sucarcane in Puerto Rico were much more
impressive. He found that under equally optimum weather conditions of extended periods of rainfall

12
or high humidity at tropical temperatures, application of infective stages of the fungus could bring
mass infection and total destruction of the pest population on the treated foliage.
The only other report on trails with A. aphidum is that of Shands et al. (1958), who claimed
successful infection of small numbers of Macrosiphum euphorbiae (Thomas) following application of
the fungus. Moreover, the collection of a few infected aphids in check plots adjacent to the treated
areas led them to believe that some spread of the fungus had taken place. This claim of the
establishment and possible spread of the fungus were in doubt. Since during the following 10 years
of study, thousands of dead, diseased aphids of several species infesting potatoes in northeastern
Maine were collected and examined, none was found to be infected by A. aphidum (Shand et al.,
1962; Shands et al., 1972). It is possible that the fungus requires tropical weather conditions for
development.
Not all stages in an insect’s life cycle are equally susceptible to infection by
entomopathogenic Hyphomycetes. In some situations, immature insects are more susceptible to
infection than mature insects. For example, young larvae of the European corn-borer (Ostrinia
nubilalis) were more susceptible to B. bassiana than older larvae (Feng et al., 1985). In contrast,
adults western flower thrips (Frankliniella occidentalis) were more susceptible to V. lecanii than
larvae (Vestergaard et al., 1995). Most host factors, such as insect developmental rates, cannot be
considered independent of environment (e.g. temperature). High temperatures accelerate insect
development and will reduce the time between molts, which can subsequently reduce the prevalence
of infection due to loss of inoculum on exuviae. Insect density is of particular importance in the
epizootiology of disease. As the density of insects increases, there is a higher probability of an insect
coming into contact with a pathogen (i.e. with infected individuals or with the pathogen directly).
The behavior of insects can influence epizootic development, and can affect the dispersal of an
entomopathogen.

3. Infection path way of entomopathogenic fungi
Poinar and Thomas (1984) described infection process of entomopathogenic fungi which
is typical for most of the fungi found to attack insect species. Most entomogenous fungi initiate their
infection by a germinating spore or conidia which penetrate the cuticle of an insect. The invasive
hypha enters the host’s tissues (often the fat body is first attacked) and ramifies through the hemocoel.
In some fungi such as Metarrhizium anisopliae, Beauveria bassiana, Conidiobolus coronata, and C.
apiculatus, “hyphal bodies” or segments of hyphae break off and circulate in the host’s hemocoel
during the early stages of infection. Upon contact with insect, the spore responds to biochemical cues
present in the waxy epicuticle and germinates within 8-16 hrs. Soon the fungus stops growing
horizontally on the surface of the cuticle and initiate penetration, using a combination of mechanical
pressure and a mixture of cuticle degrading enzymes, which attack and dissolve the cuticle. Once the
fungus breaks through the cuticle and underlying epidermals, it tends to invade in haemocoel of the
insect and proliferate in the haemolymph. The insect’s defense system in the haemocoel employs
phagocytosis and the secretion. Usually, within 24 hrs. of germination, the fungus rapidly proliferates
through the insect. The infected insects stop feeding and become lethargic. They may die relatively
rapidly within 2-7 days (Jaronski, 1997). After filling the dying or dead insect with mycelium,
hyphae emerge out through the insect’s integument, grow rapidly and produce spores on the external
surface of the host. These spores are dispersed by wind or rain and even by the parasitized insect
during feeding or mating. In some instances the fungi are localized in special organs of the host; e.g.,
the fungi Massospora cicadina and Strongwellsea castrans occur only in the abdomen of adult hosts.
Some fungi are host or stage specific, while others (e.g., Beauveria bassiana and Metarrhizium
anisopliae) exhibit a wide host range. Fungal infection and especially epizootic are mainly dependent
on a large host population and ideal climatic conditions. The life cycle of the fungus is completed
when it sporulates on the cadaver of the host. Under the right conditions, particularly higher relative
humidity, the fungus will break out through the body wall of the insect producing aerial spores. High
humidity is critical to spore germination, fungal survivorship and transmission from host to host. The
dead insect’s body may be form an empty shell, often but not always with green, red or brown fungal
growth, either enveloping the body or emerging from joints and body segments. These external
13

14
hyphae produce conidia that ripen and are released into the environment completing the cycle.
Adequate moisture and temperature are usually required for successful sporulation and spore
germination.
Fungus as biological control agent
1. Mass propagation of the fungus
In laboratory, entomopathogenic fungi were usually maintained as pure culture on Saboraud
Dextrose Agar (SDA) and Potato Dextrose Agar (PDA). But field application for insect control, a
large amount of spores or conidia is required. Therefore, mass production of entomopathogenic fungi
that is cost-effective and provides numbers of viable spores is important for the successful
development of fungi as mycoinsecticides.
Daoust et al. (1983) described the procedure in which insect fungi produced as spore
suspension containing approximately 5 x 10 7 – 1 x 10 8 conidia/ml. They were used to inoculate PDA
agar (0.5 ml/plate) or rice medium (5 ml/bag). The rice medium, which contained 150 g of long-grain
rice and 150 ml of water was sterilized high-density polyethylene autoclave bag for 20 minutes at
121°c. The bags were loosely sealed with metal ties during autoclaving, cooled down, and then,
tightly sealed with only a small amount of air using the same ties. Each bag was injected with 5ml of
the conidial suspension using a hypodermic syringe and the puncture was sealed with tape. Bags
were shaked vigorously to distribute the inoculum, and incubated for 14 days at 25°c - 26°c in the
dark. Conidia were harvested and seived through 125 m particle size by the method of Daoust and
Roberts (1983). Conidia used in all dry formulations were grown on rice medium while all other
formulations contained PDA-grown conidia. The other fungus growth substrates are almond, coconut,
corn, cottonseed, linseed, olive, peanut, soybean, sunflower, wheat germ and sorghum.
Agudelo et al. (1983) maintained P. farinosus in solid media. The solid media employed
were autoclaved moist bran (McCoy et al., 1941). The bran was thoroughly mixed with tap water

15
(1.5:1 w/w) and autoclaved in a capped Erlenmeyer flask for 30 min. Potato egg yolk (Getzin, 1961)
containing 5% potato, 1% fresh egg yolk, 1% active dry yeast, 1% Bacto peptone (Difco), and 1.5%
agar. These ingredients were mixed in a Waring Blender, dispensed into Petri dishes, then sterilized
for 20 min. Beall et al. (1939) reported the use of soil bean mass as fungal medium. One hundred
grams of dry soybeans were soaked in 250 ml of water for 24 hr. The surplus water was poured off
and the swollen beans were ground in Waring blender. The bean mash was put into Petri dishes, and
autoclaved for 30 min. The mycelia produced in solid media were sparse and form a tight mat-like
growth with the substrate, which was difficult to suspend in water.
Bourassa (1998) had identified B. bassiana as one of a series of isolated pathogenic to P.
truncates. Conidia were produced by use of standard two-stage system (Jenkins et al., 1998) with
rice as the solid substrate. Extracted and dried conidia were sieved with a 106 µm sieve to remove
large hyphal fragments. Dried conidia powder contained 4.59 x 10 10 conidia/g and had a germination
rate above 98%.
2. Formulation of entomopathogenic fungus
The development of a suitable formulation is essential to the successful utilization of
mycoinsecticides. Of primary importance is the retention of viability and virulence of the infective
units during storage and application. Formulation is mandatory in order to enhance spore application
and efficacy. The type of formulation ultimately selected depends upon the biology and physical
properties of the pathogen and the location and habits of the target pest (Daoust et al., 1983).
Roberts and Yendol (1971) described the formulation that spore can be applied in dust,
sprays or granules. The best formulation depends primarily upon the fungal species. Dust offer a
slight advantage, in that they can be stores in a formulated condition, and their swirling in air jets
tends to adhere them to lower as well as upper surfaces of foliage. Selection of appropriate diluents is
important since some materials inhibit spore germination. Talc, flour and milk powder have served as
suitable diluents for dusts. Aqueous sprays of B. bassiana spores are generally unsuitable unless a

16
wetting agent is included, although it is advisible to test its effect on spore viability first. Some
compounds which have proved satisfactory to be used as wetting agent include Triton X-100, DuPont
Spreader-sticker. Triton X-155 and the household detergent “Trend” at 1% in water. They had no
detectable adverse effect on the spores of B. bassiana. Granules (corn meal or attapulgite) are more
effective than sprays or dusts when treating corn with B. bassiana for corn borer control. Compounds
which shield spores from ultra-violet rays of the sun, increase moisture in the microclimate of the
spores, and reduce the allergenicity hazard from spores should receive special consideration by
formulators of entomogenous fungi. Encapsulation, which conceivably could satisfy some of these
requirements, has not been reported for fungi.
Inglis et al. (1993) found that Beauveria bassiana conidia dispersed better in oil than in
0.05% aqueous Tween80, while substantial clumping occurred in a 5% emulsion of the oil in water,
even after homogenization.
Smith (1994) reported that in dry conditions (45-100% RH day-night), Paecilomymes
fumosoroseus conidia sprayed in oil (Shellsol T: rape seed oil, 7:3) and in two emulsions (1 and 10%
Cadacide emulsifiable oil in water) killed 80-100% whitefly (Bemisia tabaci), but killed none when
sprayed in 0.1% aqueous Tween80 (all sprays applied at 2 ml per cucumber leaf on potted plants)
M. anisopliae var. acridum is being developed as a Biological Control Agent of acridids in
variuos regions of the world, including Africa (e.g. Lomer et al., 1997a), Australia (e.g. Milner, 1997)
and Brazil (e.g. Magalhaes et al., 2000). In a large research program conducted in Africa, conidia
were produced on rice (Jenkins et al., 1998) and they were dried and stored for various periods of
time at low (>18 months) and/or ambient (~ 12 months) temperatures (Lomer et al., 1997a). Conidia
were formulated in oils (primarily paraffinic oils used alone or blends with botanical oils) and applied
at ultra-low volumes, using hand-held Micro-Ultra applicators. The efficacy of M. anisopliae var.
acridum was probably enhanced by secondary spore pick-up. There were observations that acridids
coming in contact with vegetation infested with conidia pick up inoculum and often became infected
and died by mycosis (Bateman et al., 1998; Thomas et al., 1998). Furthermore, the fungus can persist

17
in fragments of infected grasshopper cadavers and survive adverse as a source of secondary inoculum
(i.e. horizontal transmission), enhancing the efficacy of M. anisopliae var. acridum against acridids
(Thomas et al., 1996). However, the biotic and abiotic factors regulating horizontal transmission in
field setting are still poorly understood.
3. Efficacy test against insect species
James (2003) described application technique for entomopathogenic fungi in laboratory as
follow: leaves with insects were sprayed using a Potter Precision Laboratory Spray Tower (Burkhard
Manufaturing, Rickmansworth, England) with 0.7 kg/cm 2 of pressure and the fine-mist nozzle (as
described by James and Jaronski, 2000). Each leaf was laid out flat on an acrylic plate with the
abaxial surface facing up, and then sprayed with 1 ml of each treatment preparation. After being
sprayed, all the leaves were set upright by placing the water picks in test tube racks such that the
leaves did not touch or overlap each other. The leaves were allowed to air dry in the laboratory, and
each rack of leaves was covered with a plastic bag to raise the humidity to ≥ 95%. The leaves were
not arranged in any particular order within each rack. All the racks were incubated at 25 ๐ c with a
photoperiod of 14:10 (L:D) h, and a small temperature and relative humidity recorder was placed in
one of the bags. The leaves were removed from the bag after 24 h and incubated at 70% RH, 25 ๐ c
with photoperiod of 14:10 (L:D) h. Insect mortality was recorded 7 days after each spray application.
Burges (1998) described laboratory work on the infection of Metarhizium and Beauveria
conidia formulated in oil which has provided much evidence of infection at low humidities and the
importance of penetration at intersegmental membranes. Previous reports indicated that conidial
germination on cinch bugs (Blissus leucopterus) tended to be localized at the articulation between
coxa and trochanter (Ramoska, 1984). Topical appication of conidia in oil was more effective than in
water plus wetting agent on the mouth parts of the cocoa weevil by 36 times (Prior et al., 1988) and
under the pronotal shield of locusts by 146 times at ca 35% RH (Bateman et al., 1993). The LT 50
(lethal time to kill half the insects) was lower if beetles were inoculated under the elytra than on the
elytra where conditions were shown to be more fungistatic (Butt et al., 1995). Efficient killing in the

18
presence of oil was indicated by steep dose-mortality curves more akin to those for chemicals than for
microbial agents (Bateman et al., 1993)
In field experiment, de la Rosa, et al. (2000) revealed that conidia were applied between
06.00 and 08.00 h with a previously calibrated manual pressure knapsack sprayer. A dose of 1x10 9
conidia per plant was applied for both B. bassiana and M. anisopliae; the number of conidia in
suspension was determined with a Neubauer chamber.
A domestic strain of the fungal pathogen V. lecanii in Korea was tested for the control of
aphid and whitefly (Kim et al., 2001). Of the six isolates collected in Korea, V. lecanii caused the
highest mortality of cotton aphid (Aphid gossypii). V. lecanii was selected as a biological control
agent for whitefly, based on bioassay and field tests. The pesticides dimethomorph and procymidone
did not affect spore germination and mycelial growth of V. lecanii, provided they were used at the
recommended concentration.
Fargues et al. (1991) demonstrated that the reproductive potential of Colorado potato beetle
females surviving infection by B. bassiana was reduced at 22 ๐ C but not at 25 ๐ C. Adults of the
hymenopteran parasitoid of Russian wheat aphid (Aphelinus asychis) treated with P. fumosoroseus
and incubated at high humidity were significantly less active (e.g. percentage of time walking,
walking speed and distance covered) than untreated insects (Lacey et al., 1997). Developmental
times were prolonged and the predation efficacy of the coccinellid predator, Serangium
parcesetosum, was similarly reduced in individuals sprayed with B. bassiana (Poprawski et al.,
1998). Authurs and Thomas (1999) observed that down locusts (Locusta pardalina) infected by M.
anisopliae var. acridum were more susceptible to predation. Similar effects had been shown for
Sahelian grasshoppers (Thomas et al., 1998). These recent reports demonstrated that sublethal effects
occured in insect pests infected with entomopathogenic fungi.
Foliar application of B. bassiana was used effectively in conjunction with the bacterium B.
thuringiensis. Densities of beetles in the fungus-bacterium-treated plots declined yearly relative to

19
other treatments (Drummond and Groden, 1996). Poprawski et al.(1997) observed that applications
of B. bassiana conidia at 3-4-day intervals early in the season effectively reduced densities of older
larvae and provided substantial foliar protection; larval densities were 10, 21 and 41 larvae per plant
for the B. bassiana, insecticide (i.e. esfenvalerate, piperonyl butoxide, oxamyl and carbofuran) and
control treatment, respectively. Lacey et al. (1999a) also observed significant effects of B. bassiana
against Colorado potato beetle.
P. fumosoroseus causes rapid infection and death of all whitefly stages. Under optimal
condition, hyphae are present in the haemocoel within 24 h of inoculation, death occurs between 24
and 48 h, hyphae emerges and conidiogenesis occurs on the surface of the cadaver within 72 h
(Osborne et al., 1990a). Optimal growth rates are between 20 and 30 ๐ C, with optima related to the
microclimate of the fungal isolate’s biotype (Vidal et al., 1997a). Highly virulent isolates of P.
fumosoroseus with considerable control potential against whiteflies are widespread and numerous
(Lacey et al., 1996; Vidal et al., 1997b; Wraight et al., 1998) Wraight et al. (2000) demonstrated that
infection can take place at ambient relative humidities as low as 25%. Hyphal bodies are more
virulent than conidia (Lacey et al., 1999b) and can be rapidly produced in liquid culture, remaining
viable and virulent following drying (Jackson et al., 1997). P. fumosoroseus demonstrated limited
lethal and sub-lethal effects on Serangium parcesstosum, an important coccinelid predator of
whiteflies, suggesting that the integration of these two control agents in IPM may be possible
(Poprawski et al., 1998).
Inundative application of P. fumosoroseus conidia or hyphal bodies had been used
successfully to control whiteflies in field crops. In small-scale field trails using portable air-assist
sprayer, multiple applications of P. fumosoroseus at 4-7-day intervals provided > 90% mortality of
late-instar whiteflies on cucumber, cantaloupe melons and zucchini squash (Wraight et al., 2000).
Although effects on nymphs were highly significant, the effects on adult whiteflies were minimal.
Commercial products based on this fungus are now available for whitefly control (Shah and Goette,
1999; Wraight et al., 2000). Mycotech Corporation, Burtte, Montana has developed an emulsifiable
oil formulation, which can be readily targeted and applied with low-volume air-assist sprayers or

20
moderate- to high-volume hydraulic sprayer (Wraight and Carruthers, 1999). Because whiteflies
primarily inhabit the undersides of leaves, a special effort has to be made to adequately target this
area. This can be accomplished by spraying upward from below the canopy level, using nozzles
mounted on swivels on vertical tubes. For crops with low canopies (e.g. cucurbits), high-pressure
hydraulic sprayers, fitted with drop nozzles carried at or slightly above canopy levels are effective.
Application of a wettable powder formulation of B. bassiana (Mycotrol) at 560 g/ha at 2-4-weekly
applications in cucumbers and five to seven applications in cantaloupe melons consistenly provided
65-75% control of first-generation whitefly larvae (Wraight et al., 2000).
Several taxa of fungus had demonstrated excellent suppression of insect pests in
greenhouses, including Aschersonia spp., B. bassiana, M. anisopliae, P. fumosoroseus and V. lecanii.
Considerable research has focused on the development of V. lecanii against a variety of insect pests of
glasshouse crops, including chrysanthemum (Hall and Burges, 1979; Hall, 1981)
P. fumosoroseus (PEF-97®) and B. bassiana (Botaniguard®) are two other fungi that have
recently been registered against an array of greenhouse pests, including aphids, thrips, whiteflies and
spider mites (Shah and Goettel, 1999).
4. Factors affecting the efficacy of entomopathogenic fungi
4.1 Dose
Vestergaard et al. (1995) and Brownbridge et al. (1994) suggested that mortality of
thrips exposed to B. bassiana, M. anisopliae or V. lecanii was dose dependent. High mortality was
obtained using fungal concentrations of 10 7 and 10 8 conidia/ml. In reality, few thrips would be
expected to receive this dose in greenhouse because of their cryptic habit (i.e. hiding in flower), but
some control was obtained with M. anisopliae even at 10 5 conidia/ml (Vestergaard et al., 1995).
Cadavers containing fungus could then act as a source of inoculum for infecting healthy thrips.

4.2 Temperature
Thrips were susceptible to M. anisopliae at all temperatures tested, the optimum being
23°C (Vestergaard et al., 1995). A fall in temperatures of 3 to 5° C increases the time to death by 1
day, which could be critical in a heavily infested glasshouse, particularly if high humidities could not
be sustained long enough for infection to occur (Vestergaard et al., 1995). Dramatic fluctuations in
temperature will stunt fungal growth.
4.3 Humidity
Helyer et al. (1992) observed that four consecutive nights of high humidity per week
or a cycle of two nights of high humidity and two nights of ambient humidity resulted in excellent
control of aphids, thrips and whiteflies by V. lecanii with no adverse impacts on the crop. The
efficacy of V. lecanii for controlling pests other than aphids, thrips and whiteflies (e.g. mites,
nematode and rusts) has also been demonstrated in a number of glasshouse crops (Verhaar et al.,
1996). The ability of V. lecanii to infect other fungi (i.e. mycoparasites) appeard to be unique among
the entomopathogenic Hyphomycetes (Askara et al., 1998).
4.4 The environment
A variety of environmental factors have been shown to have dramatic effects on the
efficacy of entomopathogens against insect pests. Salient parameters influencing the success of
entomopathogenic Hyphomycetes against insects are solar radiation, temperature, water availability,
precipitation and wind. Although, attentions often focus on a particular variable, environmental
parameters interact with each other in their impact on entomopathogens and, where possible, these
factors should be addressed interactively (Butt et al., 2001).
21

1. Insect survey and collection
MATERIALS AND METHODS
Insect surveys were undertaken in important tomato plantations in Thailand. They were in
Pathum Thani, Nakhon Pathom, Ratchaburi, Petchaburi, Suphan Buri, Nakhon Ratchasima and Ubon
Ratchatani provinces. In addition, the surveys were made in tomato greenhouses in Chiang Mai and
Phrae provinces. Alive and dead insects were collected from tomato plants using sweep net and
aspirator. Collected insects which included insect pests and beneficial insects were identified and
observed for disease infection. Cadavers of the dead insect were kept in the vial with tight cover for
identification of the causative agents.
2. Fungal isolation and identification
The objective of this study is to search for biological agents that are effective against insect
species that attack tomato cultivated in protected condition such as in greenhouse. It is well known
that entomopathogenic fungi are far more effective in controlling insect species under greenhouse
condition than other pathogens of insect. This study was therefore, focused on searching and
screening for effective entomopathogenic fungi against tomato insect pests in greenhouse or under
protected cultivation. All collected alive and dead insects were examined for strains of
entomopathogenic fungi. Fungus recovery was made by placing the collected insects in moisture
chamber, within 1-2 days the fungal mycelia appeared on the insect cadavers and grew rapidly to
cover the whole bodies. The mycelia were picked up from the insect cadavers and cultured on potato
dextrose agar (PDA). Subculturing was performed several times until pure cultures were obtained.
Strains of fungi were maintained on PDA and incubated at 25 ๐ c for further study. The recovered
fungus strains were identified to species level based on their morphology and growth appearance on
medium according to Domsch et al. (1993) and de Hoog et al. (2000) with the kind assistance of
Associate Professor Poonpilai Suwannarit at the Department of Microbiology, Faculty of Science,
Kasetsart University, Bangkhen Campus, Bangkok. Morphological characteristics of the fungi were
22

23
confirmed by observation under scanning electron microscope provided at Central Laboratory and
Greenhouse Complex, Kasetsart University Research and Development Institute, Kasetsart
University, Kamphaengsaen Campus, Nakhon Pathom.
3. Insect mass rearing
Tomato (Lycopersicon esculentum) was used as host plant for mass rearing all experimental
insects. Tomato plants were cultivated in clay pot, three plants per pot and maintained in
screenhouse. To initiate insect stock culture, field collected populations of whitefly (Bemisia tabaci),
tomato thrips (Ceratothripoides claratris) and mealybug (Pseudococcus cryptus) were separately
released on one month old potted tomato plants. Individual pot of tomato plant which had been
already infested with each insect species was placed in insect screen cages to protect against entry of
its natural enemies. All of these experimental units were then maintained in the air-conditioned
glasshouse with the controlled temperature of approximately 25 ºC. Pots of fresh tomato plant were
replaced when the old plants were no longer served as food. Thrips and whitefly would automatically
move to the newly supplied food plants and the life cycle took about three weeks to one month. For
mealybug, they were moved to new tomato plants with the aid of fine paint brush and the entire life
cycle took about 40 days. General caring of tomato plant in glasshouse was practiced regularly.
Colony of each insect species was maintained in the glasshouse as described to obtain adequate
number of insects for further experiments.
4. Efficacy test of fungi against tomato insect pests
Preliminary evaluation was performed to determine efficacy of all fungus isolates against
each target insect species. Bioassays were used for this preliminary screening.

4.1 Preparation of fungus inoculum
The fungus isolates used in this study were strains that recovered from insect species
collected from tomato field and greenhouse, as above-described and strains that were kindly provided
by
1.) National Center for Genetic Engineering and Biotechnology (NCGEB), Ministry of
Science, Technology and Energy
2.) Associate Professor Dr. Sivilai Saksirirat, Department of Entomology, Faculty of
Agriculture, Khon Kaen University
3.) Department of Agriculture (DOA), Ministry of Agriculture and Cooperative.
The fungal suspensions used as inoculum were prepared as follow : each fungus isolates was
cultured in PDA medium till the diameter of the colony reach 5 cm. Five millimeters of 0.1% tween
20 was dropped onto the fungus colony in culture plate. The colony was scraped out from the medium
by fine paint brush. The suspension was then collected and vortexed to make a homogeneous fungal
suspension. Spore count was made using an Improved Neubauer Bright Line haemacytometer and
fungal concentration of approximately 10 5 to 10 7 conidia/ml was prepared. This suspension was used
as inoculum for the efficacy tests of each fungus isolate.
4.2 Bioassay procedure
a. Bioassay against thrips, Ceratothripoides claratris
Circular plastic containers, measuring 5 cm in diameter and 2.5 cm in height with tight
cover were prepared and sterilized before use with 70% alcohol. Piece of moisten cotton was
placed into each container to provide humidity and tomato leaf was provided to serve as food for
tested insect. Thrips at immature stage were dipped into each fungus inoculum prepared as
24

25
described in 4.1 for 30 seconds and placed on fresh tomato leaves in the containers. Ten
individual thrips were used in each treatment. The experimental containers were tightly closed
and placed at room temperature. Three replications were performed for each fungus isolate. The
0.1%Tween 20 suspension was used in control units. Mortality was recorded in 4 days post
inoculation. Evaluation of the causative agent was accessed by placing the dead insect on PDA
plate and the recovered fungus grew on the medium was observed, identified and compared with
that in the original inoculum. Percent mortality was calculated and corrected using Abbott’s
formula (Abbott, 1925).
b. Bioassay against mealybug, Pseudococcus cryptus
Twelve wells cell culture plates with cover were prepared and sterilized before use with
70% alcohol. To absorb humidity in the well, the plaster cement was poured into the wells about
0.3 mm and covered with filter paper. Two or three drops of water were applied onto the filter
paper to provide moisture for tomato leaves and pieces of tomato stalk which were served as food
for tested insects. Mealybugs at immature stage were dipped into each fungus inoculum for 30
second and placed on fresh tomato leaves or piece of tomato stalk in each well of the culture
plate. Ten individual mealybugs were used in each treatment. Three replications were made for
each fungus isolate. In control unit, fungus inoculum was replaced by 0.1% Tween 20. All
experimental units were sealed by paraffin tape and were maintained at room temperature.
Mortality was recorded in the fourth day post inoculation. The bioassay against each fungus
isolate was repeated three times. Percent mortality was calculated and corrected using Abbott’s
formula (Abbott, 1925).
c. Bioassay against whitefly, Bemisia tabaci
Circular plastic containers, measuring 5 cm in diameter and 2.5 cm in height with tight
cover were prepared and sterilized before use with 70% alcohol. Piece of moisten cotton was
placed into each container to provide humidity and tomato leaf was provided to serve as food for

26
tested insect. A tomato leaf with ten whiteflies at immature stage (lower surface of the leaf) was
dipped into each fungus inoculum prepared as described in 4.1 for 30 second, air-dried for the
seconds and placed on fresh tomato leaves in the containers. The experimental containers were
tightly closed and placed at room temperature. Three replications were performed for each
fungal isolate. The 0.1%Tween 20 suspension was used in control units. Mortality was recorded
in 4 days post inoculation. Evaluation of the causative agent was accessed by placing the dead
insect on PDA plate and the recovered fungus grew on the medium was observed, identified and
compared with that in the original inoculum. Percent mortality was calculated and corrected
using Abbott’s formula (Abbott, 1925).
5. Specimen preparation for scanning electron microscope (SEM) observation
Healthy insects, fungal infected insects and all fungal isolates were observed using scanning
electron microscope (SEM). The fungi were grown on PDA medium until sporulation. The media
with arising conidiophores and conidia were then cut into cube of approximately 5 × 5 mm. The
samples were prepared according to the method described by Hoppert et al. (1998) which included
the following processes:
5.1 Fixation
Osmium staining for SEM
1. Place the sample in a Petri dish at the periphery and incline the dish so that the sample will
be located at the highest point.
2. Place a few drops of OsO 4 in the dish, cover with a lid, seal tightly and incubate 2 hrs or
overnight at 4 °C.
3. Using a pipette, remove the OsO 4 and carefully rinse the lower area with water.
4. Level the dish out on a bench and surround the sample with water, always making sure that
no water ever contacts the upper surface of the sample. Incubate for 15 min and repeat twice.

5. Excess of liquid is removed by transferring the sample on to filter paper.
6. Dehydrate with ethanol series. Incubate for 15 min in each series (30%, 50%, 70%, 80%,
90% and 100%).
7. Place a few drops of amylacetate in the dish. Incubate for 7 min.
5.2 Dehydration and drying
Dehydration was accomplished in critical point dryer, Model HITACHI HCP-2. The
consecutive steps were as the followings:
1. Precool the apparatus to 5-10°c below the ambient temperature by flushing with liquid CO 2.
2. Load samples immersed in ethanol (or acetone) and seal the chamber.
3. Open the CO 2 inlet valve and allow the chamber to fill with CO 2.
4. Slightly open the exhaust valve to expel any ethanol.
5. Close the exhaust valve and fill the chamber with CO 2 again.
6. Close the inlet valve and allow the specimen to remain in liquid CO 2 for ~30 min, to allow
time for the CO 2 to replace any dehydration fluid.
7. Flush again with CO 2, until there is no ethanol left.
8. Close both valves
9. Turn on the heater and allow the temperature to rise above 31°c and the pressure to build up
to 1100-1200 p.s.i.
10. Once the critical temperature and pressure are exceeded, slowly vent off the CO 2 while
keeping the temperature elevated.
11. The specimens are now ready to be mounted on stubs and sputter coated.
27

5.3 Sputtering
Conductivity of biological specimens has to be increased prior to visualization in the
electron microscope. The fixation procedure may provide sufficient conductivity, but normally,
sputtering (Gold, Wolfram or Platinum, the latter two have a smaller particle size) or vacuum
evaporation of Gold-Palladium is necessary.
Sputtering of the metal is done in a vacuum chamber equipped with a gold-plated cathode
and a glow-discharge unit. Glow discharging results in the release of gold atoms which subsequently
cover the sample (anode). All of the specimens used in this study were gold coated in FINE COAT
ion sputter Model JEOL JFC-1100.
The specimens were then observed and studied using scanning electron microscope
(SEM) Model JEOL JSM-35CF operated at 10-15 kv.
6. Mass production of fungi for greenhouse application
6.1 Media preparation
White-rice, brown rice (unmilled rice), broken-milled rice, corn and sorghum grains were
evaluated for their potential as the best substrate for fungus propagation. White-rice, brown rice and
broken-milled rice were cooked in automatic rice cooker (rice : water = 1:1). Corn and sorghum
grains were soaked in water for 48 hrs, then boiled until the grains were soft and swollen. Two
hundred grams of each substrate were put into heat resistant plastic bags. One tiny hole was made on
the bag for fungus inoculation afterward. The bag was perforated about 8-10 pores for ventilation,
then tightly closed with a rubber band and the hole was immediately covered with tape. The substrate
bags were then autoclaved for 30 min at 121°c and cooled down to normal at room temperature
before inoculation.
28

6.2 Fungal inoculum
Five fungus isolates were selected for mass production experiments on various substrates.
They were two isolates of Paecilomyces fumosoroseus, Acc. no. BCC 7058 and FWA3 and one
isolate of Beauveria bassiana, Acc. no. BCC 1658. These three isolates were proved to be effective
against tomato thrips, Ceratothripoides claratris (more than 80% mortality). The other two isolates
were Metarhizium anisopliae, Acc no. KKU 2 and Hypocrella hypocreoidea, Acc. no. BCC 11370
which were proved to be effective against mealybug, Pseudococcus cryptus (more than 70%
mortality). The fungal suspension used as inoculum was prepared as follow: each fungus isolate was
cultured on PDA medium till the diameter of the colony reach 5 cm. One percent Tween 20 solution
was dropped onto the fungal colony in culture plates. Suspension was prepared by gently scraping
conidia from surface of the medium. The suspension was collected and vortexed in 0.1% Tween 20
for 1 min and filtered through sterilized funnel equipped with screen to eliminate hyphae and pieces
of agar. Fungal concentrations were measured using an Improved Neubauer Bright Line
haemocytometer and concentration of approximately 10 7 conidia/ml was prepared in double distilled
water. This suspension was used as inoculum for further mass production trials.
6.3 Inoculation procedure
In laminar flow carbinet, one milliliter of each fungal suspension was quickly pipetted
and inoculated into the bags containing fungus medium through the hole previously made. The bags
were then shook several times to make thorough distribution of fungus conidia in the substrate mass.
Three bags of each substrate were made for each fungus isolate. All medium bags were incubated at
25 ° C and >80% RH. After 48 hrs, the medium bags were shook again to increase space between
grains which accelerated spore germination and production. Conidia were harvested at 10, 12, 15 and
20 days post inoculation by washing with 1% Tween 20.
29

6.4 Evaluation
Potential substrates for fungus mass production were evaluated on the basis of their
ability to produce viable spores or conidia. The conidia were harvested at 10, 12, 15 and 20 days
after inoculation by randomly collecting the contaminated medium from the substrate bags, three pick
up points per bag and one gram for each point. The collected medium was then washed with 200 ml
of 1% household detergent to release the conidia. Conidia or spore count was made under light
microscope using an Improved Neubauer Bright Line haemacytometer. Results were statistically
analyzed using one way analysis of variance (ANOVA) and compared the efficiency of each substrate
for conidia production using Duncan New Multiple Range Test.
7. Utilization of fungi for tomato insect pests control
Based on the result from the bioassays, the most effective fungal isolates against each insect
species were selected and mass produced on the most suitable growth substrate. Utilization of those
selected fungal isolates against important tomato insect pests were performed in insect screen cages.
7.1 Preparation of the tomato plants infested with insect species
a. Thrips, Ceratothripoides claratris
To obtain thrips-infested tomato plant for the trials, one month old healthy tomato
plants, 1 plant per pot, were moved into thrips-rearing greenhouse. They were maintained in this
greenhouse to allow infestation of thrips on the plants. The immature stages of thrips were observed
on those healthy plants within two weeks. Number of thrips in each plant were counted and recorded.
b. Mealybug, Pseudococcus cryptus
Mealybug’s infestations on one month old healthy tomato plants were accomplished
30

31
by transferring five adult mealybugs onto each plant. The infested plants were maintained in insect
cage for 2 weeks for mealybug colonization. Number of mealybug in each plant were counted and
recorded.
c. Whitefly, Bemisia tabaci
To obtain whitefly-infested tomato plants, ten adult whiteflies were transferred to each
of one month old healthy tomato plants using aspirator. The infested plants were maintained in insect
cage. The immature stages of whitefly colonized on those healthy plants within two or three weeks.
Number of whitefly on each plant were counted and recorded.
7.2 Spray application and evaluation
The fungal conidia harvested from the most suitable growth substrate (Result of Section
6) were suspended in d.H 2O and the fungal concentrations were calculated. Serial dilutions were
made and fungal concentrations of 10 1 , 10 3 , 10 5 , 10 7 and 10 9 conidia/ml were used. These prepared
fungus inoculums were sprayed on the insect-infested tomato plants, 15 ml per plant. The application
were thoroughly made on both, the upper surface and lower surface of neath. The plants were then
brought into the insect screen cages. A cup of water was placed in each cage to provide humidity for
conidia germination and fungal growth acceleration. Clean water was sprayed to plants in control
unit. Mortality was recorded in 4 days post inoculation. Evaluation was accessed by direct counting
of the alive and dead insects left on the plants. Results of the spray application and fungal
pathogenicity test in insect screen cage were subjected to statistical analysis with the aid of computer
program and the data were analyzed using probit analysis (Finney, 1962). The median lethal
concentration (LC 50) was finally estimated.

1. Insect survey and sample collection
RESULTS AND DISCUSSION
Insect species collected from the surveys which included insect pests and beneficial insects
were identified. The insect species found on tomato plants were whitefly, thrips, aphids, mealybug,
planthopper, tomato fruitworm, beet armyworm, diamond-back moth, leaf miners, grasshopper and
flea beetle. The beneficial insects collected in tomato fields were dragonfly, lady-bird beetle, antlion,
ant, long legged fly and some parasitic hymenopteran insects.
The pest complex of tomato consists mainly of lepidopteran fruit and stemborers and a
variety of plant sucking herbivores. Polyphagous species like the fruit borer, Helicoverpa (Heliothis)
armigera (Lepidoptera: Noctuidae) and Phthorimea operculella (Lepidoptera: Gelechiidae), as well
as Spodoptera spp. (Lepidoptera: Noctuidae) are the main lepidopteran pests of tomatoes in Southeast
Asia (Deang, 1969; Gomaa et al., 1978; Krishnaiah et al., 1981; Kakar et al., 1990). Berlinger et al.
(1993) reported that field-grown tomatoes, as well as those under protected cultivation, are attacked
by aphids (e.g. Myzus persicae, Aphid gossypii), whiteflies (e.g. Trialeurodes vaporariorum, Bemisia
tabaci) and thrips (e.g. Thrips tabaci, T. flavus, T. palmi, Frankliniella occidentalis). These pests not
only cause direct damage, but are also important vectors of different virus diseases of tomatoes. In
addition to these pests, spider mites and nematodes, in particular Meloidogyne spp., can be major
constraints of tomato production in tropics and subtropics.
Jones (1999) reported that insect species commonly affected tomato plants were aphids,
Colorado potato beetles, corn earworms (tomato fruitworm), flea beetles, fruit flies, hornworms, leaf
miners, pinworms, spider mites, stink bugs and whiteflies. Another insect that is becoming
increasingly difficult to control is the thrips, a very small insect that can damage the tomato plant as
well as carry virus diseases.
32

33
Chaimongkol (n.d.) reported that in Thailand the key pests of tomato are whitefly (Bemisia
tabaci), horn worm (Protoparce quingue, Maculate and P. sexta) and tomato fruitworm (Helicoverpa
(Heliothis) zea).
Most of the insect species previously reported to attack tomato plant were also found in this
study to cause damage to tomato, particularly insects that were able to attack both field tomato and
tomato greenhouse. They were those tiny, plant sucking insects for example: whitefly, thrips,
mealybug and aphid. They were target insects for this study since they caused serious direct damage
(consuming plant juice) and indirect damage (vector of virus diseases) to tomato in protected
cultivation. It is worth to note that there were several insects found in tomato field and greenhouse
that were identified as beneficial insects. Their roles either as parasite or predator in nature may be
important in controlling some destructive insect species of tomato. It is interesting to further
investigate their potential as biological control agents for controlling insect pests of tomato especially
in an integrated approach with entomopathogens.
2. Fungal isolation and identification
Diseased insect collected from tomato field and greenhouse were investigated for the
causative agents. Twelve isolates of fungi were recovered from insect cadavers which include 6
isolates from whiteflies and 6 isolates from thrips (Table 1). The accession number (Acc. no.) FWA
and FWN were designated to the isolates recovered from adult and nymph of whitefly respectively.
The accession number (Acc. no.) FTA was designated to the isolates recovered from adult thrips.
These naturally occurring entomopathogenic fungi were identified on the basis of their morphology
and growth appearance on medium according to Domsch et al. (1993) and de Hoog et al. (2000).
Their colony characteristics and growth appearances as shown on both sides of the culture plates
(upper surface and lower surface) and their morphology observed microscopically were described as
follow:

Table 1 Newly recovered of entomopathogenic fungi from insect collected from field and
greenhouse grown tomato.
Accession no. 1/
FWA1
FWA2
FWA3
FWA4
FWA5
FWN1
FTA1
FTA2
FTA3
FTA4
FTA5
FTA6
Scientific name Host
Fusarium solani
Acremonium potronii
Paecilomyces fumosoroseus
Paecilomyces fumosoroseus
Paecilomyces fumosoroseus
Paecilomyces lilacinus
Fusarium incarnatum
Penicillium citrinum
Penicillium verruculosum
Fusarium solani
Acremonium hyalinulum
Mucor hiemalis
1/ FWA = fungus isolates recovered from adult whitefly.
FWN = fungus isolates recovered from nymph whitefly.
FTA = fungus isolates recovered from adult thrips.
Whitefly
Whitefly
Whitefly
Whitefly
Whitefly
Whitefly
Thrips
Thrips
Thrips
Thrips
Thrips
Thrips
34

1. Fusarium solani (Acc. no. FWA1)
Colony characteristics: Colonies growing rapidly, with white to cream-colored aerial
mycelium, usually green to bluish-brown when sporodochia are present; the lower surface usually
colorless (Figure 1a, b).
Microscopy: Conidiophores arising laterally from aerial hyphae, monophialides mostly
with a rather distinct collarette. Macroconidia produced on elongated, branched conidiophores which
soon formed sporodochia, usually moderately curved, with short, blunt apical and indistinctly
pedicellate basal cells, mostly 3-septate, occasionally 5-septate. Microconidia usually abundant,
produced on shorter, sometimes verticillate conidiophores. Chlamydospores frequent, singly or in
pairs, terminal or intercalary, smooth or rough-walled (Figure 2.1a, b).
2. Acremonium potronii (Acc. no. FWA2)
Colony characteristics: Colonies restricted, pink slimy, becoming powdery to
granulose; the lower surface colorless (Figure 1c, d).
Microscopy: Phialides simple, erect, arising from creeping hyphae. Conidia aggregating
in slimy head, obovoidal or tear shaped, smooth-walled (Figure 2.2).
3. Paecilomyces fumosoroseus (Acc. no. FWA3, FWA4 and FWA5)
Colony characteristics: Colonies grow considerably slower than the very fast growing
fungi, for example Fusarium solani, mycelium floccose and pink in color (Figure 1e), at first colonies
restricted and later fluffy with undefined boundery; the lower surface colorless (Figure 1e, f, g, h, i, j).
Microscopy: Conidiophores erect, smooth-walled, hyaline, bearing several compact
35

36
whorls of phialides which have a strongly inflated base tapering into a long and narrow neck.
Conidia cylindrical to fusiform, smooth-walled, hyaline to pale pink (Figure 2.3).
4. Paecilomyces lilacinus (Acc. no. FWN1)
Colony characteristics: Colonies growing rapidly, floccose, vinaceous to violet; the
lower surface yellow-brown (Figure 1k, l).
Microscopy: Conidiophores erect, mostly arising from submerged hyphae, occasionally
forming tufts up to 2 mm high, bearing branches with densely clustered phialides; conidiophore stipes
3-4 µm wide, yellow to purple, rough and smooth-walled. Phialide consisting of a swollen basal part,
tapering into a thin neck. Conidia ellipsoidal to fusiform, smooth-walled to slightly roughened,
hyaline, purple in mass, in divergent chains (Figure 2.4a, b).
5. Fusarium incarnatum (Acc. no. FTA1)
Colony characteristics: Colonies growing rapidly, aerial mycelium floccose, at first
whitish, later becoming avellaneous to buff-brown; the lower surface pale, becoming peach-colored
(Figure 1m, n).
Microscopy: Conidiophores scattered in the aerial mycelium, loosely branched;
polyblastic conidiogenous cells abundant. Sporodochail macroconidia slightly curved, with foot cell,
3-7-septate. Conidia on aerial conidiophores (blastoconidia) usually born singly on scattered
denticles, fusiform to falcate, mostly 3-5-septate. Microconidia sparse or absent. Chlamydospores
sparse, spherical, becoming brown, intercalary, single or in chain (Figure 2.5a, b,c).

6. Penicillium citrinum (Acc. no. FTA2)
Colony characteristics: Colonies with slow to moderate growth, velutinous to floccose;
mycelium white to grayish-orange. Conidial masses grayish-turquoise, frequently a pale yellow to
reddish-brown soluble pigment is produced; the lower surface colorless, pale (Figure 1o, p).
Microscopy: Conidiophore stipes smooth-walled, penicilli biverticillate. Metulae 12-15
µm long, divergent, in whorls of 3-5. Phialides flask-shaped. Conidia spherical to subsperical,
smooth-walled or finely roughened (Figure 2.6a, b).
7. Penicillium verruculosum (Acc. no. FTA3)
Colony characteristics: Colonies velutinous or somewhat floccose to funiculose,
mycelium white to bright yellow, conidial mass green; the lower surface pale to yellow (Figure 1q, r).
Microscopy: Conidiophore stipes, smooth-walled; penicilli usually biverticillate.
Metulae in whorls of 7-10, 8-15 µm long. Phialides in whorls of 7-10, ampulliform to acerose.
Conidia spherical to subspherical, with roughened walls (Figure 2.7a, b).
8. Fusarium solani (Acc. no. FTA4)
Colony characteristics: Colonies growing rapidly, with white to cream-colored aerial
mycelium, usually green to bluish-brown when sporodochia are present; the lower surface usually
colorless, becoming peach-colored (Figure 1s, t).
Microscopy: Conidiophores arising laterally from aerial hyphae. Monophialides mostly
with a rather distinct collarette. Macroconidia produced on elongated, branched conidiophores which
soon form sporodochia, usually moderately curved, with short, blunt apical and indistinctly
pedicellate basal cells, mostly 3-septate, occasionally 5-septate. Microconidia usually abundant,
37

38
produced on shorter, sometimes verticillate conidiophores. Chlamydospores frequent, singly or in
pairs, terminal or intercalary, smooth or rough-walled (Figure 2.8a, b).
9. Acremonium hyalinulum (Acc. no. FTA5)
Colony characteristics: Colonies growing rapidly, white to ochraceous, powdery to
slightly floccose; the lower surface ochraceous to grayish-brown (Figure 1u, v).
Microscopy: Conidiophores simple or repeatedly branched. Phialides in whorls on very
short side branches, developing short, apical, polyphialidic branches with age, tip with localized wall
thickenings. Conidia in chains, spindle-shaped with rounded upper ends, hyalide, relatively thickwalled
(Figure 2.9).
10. Mucor hiemalis (Acc. no. FTA6)
Colony characteristics: Colonies expanding, the mycelium tiered, grayish-ochraceous;
the lower surface grayish-ochraceous (Figure 1w, x).
Microscopy: Sporangiophores hyaline, up to 15 mm high and 15 µm wide, erect,
unbranched at first, later sparingly branched. Sporangia yellowish at first, becoming dark brown,
with diffluent membranes, columellae ellipsoidal, spherical when young. Sporangiospores
ellipsoidal, sometimes flattened at one side, smooth-walled. Oidia present in substrates hyphae
(Figure 2.10a, b).
The entomopathogenic fungi used in this study were also obtained from other laboratories in
Thailand. Nineteen isolates were from BIOTEC Culture Collection, National Center for Genetic
Engineering and Biotechnology (NCGEB), Thailand. They were designated with the Acession
number BCC (Acc. no. BCC). Five isolates designated the accession number KKU were from
Department of Entomology, Khonkaen University and two isolates of Hirsutella thompsonii

39
designated the accession number DOA were from Department of Agriculture (DOA), Ministry of
Agriculture and Cooperatives (Table 2). The total of 33 fungal isolates were subjected to efficacy
tests against major insect pests of tomato cultivated in greenhouse.

Fusarium solani (Acc. no. FWA1)
Acremonium protronii (Acc. no. FWA2)
Paecilomyces fumosoroseus (Acc. no. FWA3)
a
c
e
Fusarium solani (Acc. no. FWA1)
Acremonium protronii (Acc. no. FWA2)
Paecilomyces fumosoroseus (Acc. no. FWA3)
Figure 1 Colony characteristics of the entomopathogenic fungi newly recovered from naturally
infected insect pests of tomato.
Left = top view of PDA plates; right = bottom view of PDA plates
b
d
f
40

Paecilomyces fumosoroseus (Acc. no. FWA4)
Paecilomyces fumosoroseus (Acc. no. FWA5)
Paecilomyces lilacinus (Acc. no. FWN1)
Figure 1 (continued)
g
i
k
Paecilomyces fumosoroseus (Acc. no. FWA4)
Paecilomyces fumosoroseus (Acc. no. FWA5)
Paecilomyces lilacinus (Acc. no. FWN1)
h
l
j
41

Fusarium incarnatum (Acc. no. FTA1)
Penicillium citrinum (Acc. no. FTA2)
Penicillium verruculosum (Acc. no. FTA3)
Figure 1 (continued)
m
o
q
Fusarium incarnatum (Acc. no. FTA1)
Penicillium citrinum (Acc. no. FTA2)
Penicillium verruculosum (Acc. no. FTA3)
n
p
r
42

Fusarium solani (Acc. no. FTA4) s Fusarium solani (Acc. no. FTA4)
Acremonium hyalinulum (Acc. no. FTA5)
Mucor hiemalis (Acc. no. FTA6)
Figure 1 (continued)
u
Acremonium hyalinulum (Acc. no. FTA5)
w Mucor hiemalis (Acc. no. FTA6)
x
t
v
43

2.1a
macroconidia
conidiophores
microconidia
septate hypha
Figure 2 Morphological diagram of the isolates of entomopathogenic fungi newly recovered from
insect pests of tomato.
2.1 Fusarium solani (Acc. no. FWA1) showing:
2.1 a macroconidia on conidiophores
2.1 b microconidia on conidiophores
conidia
2.1b
44
septate hypha (creeping hypha)
phialide
Figure 2 (continued) 2.2
conidia.
2.2 Acremonium protronii (Acc. no. FWA2) showing creeping hyphae, phailaides and

Figure 2 (continued)
phialides
metula
conidia
conidiophore
2.3 Paecilomyces fumosoroseus (Acc. no. FWA3, FWA4, FWA5) showing: conidiophores,
metula, conidia.
conidia
phialides
metulae
conidiophores
2.4a 2.4b
Figure 2 (continued)
2.4 Paecilomyces lilacinus (Acc. no. FWN1) showing:
2.4a. rough-walled conidiophore, phailides and conidia
2.4b. smooth-walled conidiophore, phailides and conidia
2.3
45

Figure 2 (continued)
Figure 2 (continued)
septate hypha
2.5a
microconidia
macroconidia
phialides
conidiophores
aerial conidiophore
( )
2.5b
2.5 Fusarium incarnatum (Acc. no. FTA1) showing:
2.5a. conidiophores, aerial conidiophore, macroconidia
2.5b. scattered denticles of blastoconidia
2.5c. microconidia
conidia
phialides
metulae
phialophores
septate hypha
2.6a 2.6b
2.6 Penicillium citrinum (Acc. no. FTA2) showing:
2.6a. conidiophores, conidia, metulae, penicilli biverticillate
2.6b. conidiophores, conidia, metulae, penicilli divergent
2.5c
46

2.7a
Figure 2 (continued)
conidia
phialides
metulae
conidiophores
septate hypha
2.7 Penicillium verruculosum (Acc. no. FTA3) showing:
2.7a. conidiophores, conidia, metulae
2.7b. conidiophores, conidia, bimetulae
Figure 2 (continued)
macroconidia
conidiophores
Branched conidiophore
microconidia
septate hypha
2.7b
2.8a 2.8b
2.8 Fusarium solani (Acc. no. FTA4) showing:
2.8a. conidiophore, macroconidia and microconidia
2.8b. brached conidiophore and macroconidia
47

conidia
Figure 2 (continued)
Septate hypha
phialides
conidiophores
2.9 Acremonium hyalinulum (Acc. no. FTA5) showing conidiophores, phailide and conidia.
sporangia
sporangiospores
Figure 2 (continued)
2.10a
columellae
sporangiophores
2.10 Mucor hiemalis (Acc. no. FTA6) showing:
2.10a. sporagiophores and sporangia
2.10b. sporagiophores, sporangiospores and columellae
2.10b
2.9
48

3. Efficacy of entomopathogenic fungi against tomato insect pests.
Thirty three isolates of the entomopathogenic fungi which included those from BIOTEC
Culture Collection, NCGEB, from Khonkaen University, from DOA of Thailand and the newly
recovered isolates from insect collected from tomato plants (table 2) were bioassayed against each
species of tomato insect pests. In this study, efficacies of the fungi were classified into 4 categories;
insects.
1. Highly effective strains are those that caused more than 90% mortality of the tested
2. Effective strains are those that caused less than 90% but more than 70% mortality of the
tested insects.
3. Low effective strains are those that caused less than 60% mortality of the tested insects.
4. Ineffective strains are those that caused no mortality of the tested insects.
3.1 Tomato thrips, Ceratothripoides claratris
According to the purposed criteria for efficacy evaluation, the bioassays against tomato thrips
provided the following results (table3):
A. Highly effective isolates are Paecilomyces fumosoroseus Acc. no. FWA3, FWA5 and
BCC 7058
B. Effective isolates (less than 90% but more than 70% mortality) are Beauveria bassiana
(Acc. no. BCC 1658) and Paecilomyces fumosoroseus (Acc. no. BCC 1659 and FWA4) which caused
80% mortality and B. bassiana (Acc. no. KKU 1) which caused 70% mortality of C. claratris.
49

50
C. Low effective isolates (less than than 60% mortality) are Hypocrella hypocreoidea (Acc.
no. BCC 11370) and Hirsutella thompsonii (Acc. no. DOA 2064) which caused 40% mortality;
Paecilomyces lilacinus (Acc. no. FWN1) which caused 30% mortality; Metarhizium anisopliae (Acc.
no. KKU 3 and KKU 5) and Fusarium solani (Acc. no. FTA4) which caused 20% mortality;
Verticillium lecanii (Acc. no. BCC 1535), M. anisopliae (Acc. no. KKU 2 and KKU 4), F. solani
(Acc. no. FWA1) and Penicillium verruculosum (Acc. no. FTA3) which caused 10% mortality.
D. Ineffective fungus isolates against tomato thrips are:
Beauveria bassiana (Acc. no. BCC 1442), M. anisopliae (Acc. no. BCC 1555), V. lecanii
(Acc. no.BCC 1560), Aschersonia hypocreoidea (Acc. no. BCC 1650), Nomuraea rileyi (Acc.
no.BCC 2054 and BCC 2062), A. badia (Acc. no. BCC 2170), A. samoensis (Acc. no. BCC 2905),
Metarhizium sp. (Acc. no. BCC 9735), Hirsutella thompsonii (Acc. no. DOA 1813), Acremonium
protronii (Acc. no. FWA2), F. incarnatum (Acc. no. FTA1), P. citrinum (Acc. no. FTA2), A.
hyalinulum (Acc. no. FTA5) and Mucor hiemalis (Acc. no. FTA6).
3.2 Mealybug, Pseudococcus cryptus
According to the purposed criteria for efficacy evaluation, the bioassays against mealybug
provided the following results (table3):
A. Effective isolates (less than 90% but more than 70% mortality) are Hypocrella
hypocreoidea (Acc. no. BCC 11370) and M. anisopliae (Acc. no. KKU 2).
B. Low effective isolates (less than 60% mortality) are Fusarium solani (Acc. no. FTA4)
which caused 60% mortality; P. fumosoroseus (Acc. no. BCC 7058), M. anisopliae (Acc. no. KKU
3) and P. lilacinus (Acc. no. FWN1) which caused 40% mortality; Paecilomyces fumosoroseus
(Acc. no. FWA3) which caused 30% mortality; Beauveria bassiana (Acc. no. BCC 1658),
Aschersonia badia (Acc. no. BCC 2170), Hirsutella thompsonii (Acc. no. DOA 2064), B. bassiana

51
(Acc. no. B1), P. fumosoroseus (Acc. no. FWA5), Penicillium verruculosum (Acc. no. FTA3) and
Mucor hiemalis (Acc. no. FTA6) which caused 20% mortality; Verticillium lecanii (Acc. no. BCC
1535), A. hypocreoidea (Acc. no. BCC 1650), P. fumosoroseus (Acc. no. BCC 1659), A. samoensis
(Acc. no. BCC 2905), Metarhizium sp. (Acc. no. BCC 9735), H. thompsonii (Acc. no. 1813), M.
anisopliae (Acc. no. KKU 4 and KKU 5), Acremonium protronii (Acc. no. FWA2), P. fumosoroseus
(Acc. no. FWA4), Fusarium incarnatum (Acc. no. FTA1), Penicillium citrinum (Acc. no. FTA2) and
A. hyalinulum (Acc. no. FTA5) which caused 10% mortality.
C. Ineffective isolates against mealybugs are:
B. bassiana (Acc. no. BCC 1442), M. anisopliae (Acc. no. BCC 1555), V. lecanii (Acc.
no.BCC 1560), Nomuraea rileyi (Acc. no.BCC 2054 and BCC 2062), F. solani (Acc. no. FWA1).
3.3 Whitefly, Bemisia tabaci
According to the purposed criteria for efficacy evaluation, the bioassays against whitefly
provided the following results (table 3):
A. Effective isolates isolates against whiteflies (less than 90% but more than 70% mortality)
are Fusarium solani (Acc. no. FWA1) and P. fumosoroseus (Acc. no. FWA3) which caused 80%
mortality; P. fumosoroseus (Acc. no. FWA4 and FWA5) which caused 70% mortality.
B. Low effective isolates (less than 60% mortality) are P. fumosoroseus (Acc. no. BCC
7058) which caused 60% mortality; P. fumosoroseus (Acc. no. BCC 1659) which caused 40%
mortality; V. lecanii (Acc. no. BCC 1560), B. bassiana (Acc. no. BCC 1658), Metarhizium sp. (Acc.
no. BCC 9735), Penicillium verruculosum (Acc. no. FTA3) and Fusarium solani (Acc. no. FTA4)
which caused 20% mortality; Verticillium lecanii (Acc. no. BCC 1535), Aschersonia hypocreoidea
(Acc. no. BCC 1650), A. badia (Acc. no. BCC 2170), Beauveria bassiana (Acc. no. KKU 1),
Acremonium protronii (Acc. no. FWA2), Paecilomyces lilacinus (Acc. no. FWN1), Fusarium

52
incarnatum (Acc. no. FTA1) and Acremonium hyalinulum (Acc. no. FTA5) which caused 10%
mortality.
C. Ineffective isolates against whitefly are:
B. bassiana (Acc. no. BCC 1442), M. anisopliae(Acc. no. BCC 1555), Nomuraea rileyi
(Acc. no.BCC 2054 and BCC 2062), A. samoensis (Acc. no. BCC 2905), Hypocrella hypocreoidea
(Acc. no. BCC 11370), Hirsutella thompsonii (Acc. no. DOA 1813), Hirsutella thompsonii (Acc. no.
DOA 2064), M. anisopliae (Acc. no. KKU 2, KKU 3, KKU 4 and KKU 5), Penicillium citrinum
(Acc. no. FTA2) and Mucor hiemalis (Acc. no. FTA6).

Table2 Isolates of entomopathogenic fungi obtained from governmental institutes
No. Accession No. Scientific name Insect host *
1 BCC 1442 Beauveria bassiana Hemiptera-Podopidae
2 BCC 1535 Verticillium lecanii Homoptera - scale insect
3 BCC 1555 Metarhizium anisopliae Homoptera
4 BCC 1560 Verticillium lecanii Homoptera - scale insect
5 BCC 1650 Aschersonia hypocreoidea Homoptera - scale insect
6 BCC 1658 Beauveria bassiana -
7 BCC 1659 Paecilomyces fumosoroseus Buprestidae/leaf litter
8 BCC 2054 Nomuraea rileyi -
9 BCC 2062 Nomuraea rileyi -
10 BCC 2170 Aschersonia badia Homoptera - scale insect
11 BCC 2905 Aschersonia samoensis Homoptera - nymph
12 BCC 7058 Paecilomyces fumosoroseus -
13 BCC 9735 Metarhizium sp. -
14 BCC 11370 Hypocrella hypocreoidea Homoptera - nymph
15 DOA 1813 Hirsutella thompsonii Mite
16 DOA 2064 Hirsutella thompsonii Mite
17 KKU 1 Beauveria bassiana -
18 KKU 2 Metarhizium anisopliae -
19 KKU 3 Metarhizium anisopliae -
20 KKU 4 Metarhizium anisopliae -
21 KKU 5 Metarhizium anisopliae -
BCC = BIOTEC Culture Collection, National Center for Genetic Engineering and National
Science Technology and Development Agency
DOA = Department of Agriculture, Ministry of Agriculture and Cooperative
KKU = KhonKaen University
* - = no information
53

Table 3 Efficacy of entomopathogenic fungi against tomato insect pests.
Average % corrected mortality 2/
Accession
1/
no.
Control
BCC 1442
BCC 1535
BCC 1555
BCC1560
BCC1650
BCC 1658
BCC 1659
BCC 2054
BCC 2062
BCC 2170
BCC 2905
BCC 7058
BCC 9735
BCC 11370
DOA 1813
DOA 2064
KKU 1
KKU 2
KKU 3
KKU 4
KKU 5
FWA1
FWA2
FWA3
FWA4
FWA5
FWN1
FTA1
FTA2
FTA3
FTA4
FTA5
FTA6
Scientific name
-
Beauveria bassiana
Verticillium lecanii
Metarhizium anisopliae
Verticillium lecanii
Aschersonia hypocreoidea
Beauveria bassiana
Paecilomyces fumosoroseus
Nomuraea rileyi
Nomuraea rileyi
Aschersonia badia
Aschersonia samoensis
Paecilomyces fumosoroseus
Metarhizium sp.
Hypocrella hypocreoidea
Hirsutella thompsonii
Hirsutella thompsonii
Beauveria bassiana
Metarhizium anisopliae
Metarhizium anisopliae
Metarhizium anisopliae
Metarhizium anisopliae
Fusarium solani
Acremonium protronii
Paecilomyces fumosoroseus
Paecilomyces fumosoroseus
Paecilomyces fumosoroseus
Paecilomyces lilacinus
Fusarium incarnatum
Penicillium citrinum
Penicillium verruculosum
Fusarium solani
Acremonium hyalinulum
Mucor hiemalis
Thrips Mealybug Whitefly
0.00 0.00 0.00
0.00 0.00 0.00
13.33 13.33 13.33
3.33 0.00 3.33
0.00 0.00 23.33
0.00 6.67 10.00
76.67 20.00 23.33
80.00 10.00 36.67
0.00 0.00 0.00
0.00 0.00 0.00
0.00 23.33 6.67
0.00 10.00 0.00
93.33 43.33 63.33
0.00 13.33 23.33
40.00 70.00 0.00
3.33 6.67 0.00
26.67 23.33 0.00
73.33 20.00 10.00
13.33 73.33 3.33
23.33 36.67 0.00
6.67 13.33 0.00
20.00 10.00 0.00
6.67 0.00 76.67
0.00 10.00 10.00
90.00 33.33 76.67
80.00 13.33 73.33
93.33 26.67 73.33
26.67 43.33 13.33
0.00 13.33 6.67
0.00 6.67 0.00
13.33 23.33 23.33
16.67 56.67 23.33
3.33 10.00 13.33
3.33 23.33 0.00
1/ Accession no. BCC : from Biotech Culture Collection
Accession no. DOA : from Department of Agricultur
Accession no. KKU : from Department of Entomology, Khonkaen University
Accession no. FW and FT : newly recovered isolates
2/ Corrected mortality using Abbott’s formula
The most effective isolates against thrips.
The most effective isolates against mealybug.
The most effective isolates against whitefly.
54

4. Pathological observation
The fungus isolates that have been proved to be effective against tomato insect pests in
protected cultivation or greenhouse were observed for their pathogenicity using scanning electron
microscope. Routes of infection for all isolates were similar and followed the typical process
occurred in the majority of entomopathogenic fungi. The infection process could be described as
follow:
The fungal conidia are able to adhere to the insect cuticle, especially on rough, less-waxy
cuticle or cuticle with numbers of setae and hairs. At the favourable environment, the conidia
germinated by forming a germ tube which afterward penetrated through the cuticle and gained entry
into the insect body cavity or hemocoel (Figure 3a). In some occasion, the conidia may never
germinate for a number of reasons, including environmental factors such as low humidity or the
presence of inhibitory factors on the cuticle. If germination does take place, germ tube may grow on
the surface of the insect for short distances but never get through the insect cuticle because the fungus
lacks the enzymes required to soften the cuticle. On the cuticle of the tested insects it was found that,
after attachment of the conidia, germinated germ tube pass through either spiracle (Figure 3b) or the
cuticle and the epidermal regions of the insect integument into the hemocoel (Figure 3c). Extensive
vegetative growth of the fungus, usually by replication of yeast-like hyphal bodies or blastospores,
then occured in the hemocoel. Nutrients in the hemolymph and surrounding fat body were depleted
by such extensive fungal growth, and the host insects died of starvation, fungal invasion or
toxification depended on species of the fungi. At or just prior to the time of death, hyphal bodies
converted to mycelia, which grew and ramified throughout host tissues and exited the cadaver
through the cuticle. The mycelium branched and grew on the surface of the cuticle which finally
covered the entire insect body (Figure 3d). Infective propagules or conidia were produced by the
fungus on the exterior of the cadaver (Figure 3e). In nature these spores or conidia dispersed by
environmental agents (wind, water) or by animals. The healthy and mummified cadaver of thrips,
mealybug and whitefly were shown in Figure 4. Conidiophores and conidia of Paecilomyces
fumosoroseus (Acc. no. FWA3), Beauveria bassiana (Acc. no. BCC 1658), Hypocrella hypocreoidea
55

56
(Acc. no. BCC 11370) and Metarhizium anisopliae (Acc. no. KKU 2) on insect cadavers were shown
in Figure 5.
Spore germination and penetration can be clearly illustrated with the aid of scanning electron
microscope. St Legar et al. (1988) using scanning electron microscope to study germination of
Metarhizium anisopliae conidia on the surface of the cuticle of the tobacco budworm. Once on the
cuticle, the conidia responded to biochemical cues present in the waxy epicuticle and germinates
within 8-16 hrs. Soon the fungus stopped growing horizontally on the surface of the cuticle and
initiated penetration, using a combination of mechanical pressure and a mixture of cuticle degrading
enzymes (lipases, proteases and chitinases), which attack and dissolve the cuticle. It tended to invade
in haemocoel of the insect and proliferated in the hemolymph. Usually, within 24 hrs of germination,
the fungus rapidly proliferated through the insect. Growth could be in the form of mycelium or yeastlike
blastospores. The life cycle of the fungus was completed when it sporulated on the cadaver of
host. McCoy et al., (1988) illustrated germination process of conidia of the white-muscardine fungus,
Beauveria bassiana on the epicuticular surface of the corn earworm, Heliothis zea using scanning
electron microscope. The germinating conidia, attached to the host, formed a germ tube and
appressorium. The germination structures also strengthened the adhesion of the fungus. Successful
germination and penetration depend, not necessarily, on the total percentage of germination but also
on the duration of the germination time, mode of germination, aggressiveness of the fungus, the type
of fungal spore and host susceptibility.

1 µm
10 µm
1 µm
a
1 µm
c 100 µm
d
Figure 3 Scanning electron micrographs demonstrated infection process of entomopathogenic fungi
(a) Conidia of Beauveria bassiana germinated as germ tube on cuticle of thrips.
(b) Penetration of germ tube of Paecilomyces fumosoroseus into hemocoel of thrips
through the spiracle (arrow).
(c) Penetration of germ tube of B. bassiana into hemocoel of thrips through the cuticle (arrow).
(d) Mummified cadaver of thrips infected with B. bassiana at the second day post inoculation.
(e) Mycelium of B. bassiana ramified on the cuticle and produced conidia after penetration out
from insect cadaver.
e
57
b

100 µm
100 µm
100 µm c
100 µm
Figure 4 Scanning electron micrographs of insects infected with entomopathogenic fungi
a
100 µm e 100 µm
f
(a) Healthy thrips, Ceratothripoides claratris
(b) Cadaver of thrips covered with Paecilomyces fumosoroseus
(c) Healthy mealybug, Pseudococcus cryptus
(d) Cadaver of mealybug covered with Metarhizium anisopliae
(e) Healthy whitefly, Bemisia tabaci
(f) Cadaver of whitefly covered with Paecilomyces fumosoroseus
d
58
b

10 µm a 10 µm
b
100 µm
c d
100 µm
Figure 5 Scanning electron micrographs of conidiophores and conidia of highly effective fungal
isolates produced on the surface of insect cadavers.
(a) Paecilomyces fumosoroseus (Acc. no. FWA3)
(b) Beauveria bassiana ( Acc. no. BCC 1658)
(c) Hypocrella hypocreoidea (Acc. no. BCC 11370)
(d) Metarhizium anisopliae (Acc. no. KKU 2)
59

5. Mass production of entomopathogenic fungi
Three effective isolates of entomopathogenic fungi against thrips namely, Paecilomyces
fumosoroseus (Acc. no. FWA3 and BCC 7058) and Beauveria bassiana (Acc. no. BCC 1658) and
two isolates against mealybugs namely, Hypocrella hypocreoidea (Acc. no. BCC 11370) and
Metarhizium anisopiae (Acc. no. KKU 2) were mass produced using different substrates to support
fungus growth and sporulation. The substrate selected for this study were, white-rice, broken-milled
rice, brown rice, corn grains and sorghum grains. All fungal isolates were cultured on these
substrates and incubated at room temperature (25 – 30 ºC). Fungal growth on each substrate was
observed and the conidia were harvested in 10, 12, 15 and 20 days of incubation period. Conidia
counts were made using Improved Neubauer Bright Line haemacytometer. The results could be
summarized as the followings:
5.1 Mass production of entomopathogenic fungi, Paecilomyces fumosoroseus (Acc. no.
FWA3) on different substrates used as growth medium.
On different substrates, P. fumosoroseus (Acc. no. FWA3) grew in similar manner. Two
days after inoculation, the fungal mycelium formed on the substrates and covered parts of the
substrates. They were discrete unit which are easy to disperse the conidia. But in five days post
inoculation, the mycelium growth over-crowded on the substrate. While the mycelia were produced
well on all substrates, number of the conidia obtained from each substrate varied considerably. The
average number of conidia produced on white-rice, broken-milled rice, brown rice, corn grains and
sorghum grains were 0.97 x 10 7 , 0.44 x 10 7 , 0.32 x 10 7 , 0.32 x 10 7 and 0.48 x 10 7 , conidia/ml
respectively (Table 4). Figure 6 illustrated mycelium growth and sporulation of P. fumosoroseus
(Acc. no. FWA3) on different substrate used in this study. It was observed that when grew on whiterice,
the fungus produced abundant mycelia and conidia. The sporulation process was very fast, only
after the day 10 th , all of the substrates turned into pink or violet color and floccose. The mycelia
produced on corn grain and brown rice were sparse and formed tightly with the substrate (Figure 6),
60

61
which was difficult to wash off 1% household detergent. Results indicated that white-rice was the
best substrate for P. fumosoroseus (Acc. no. FWA3) mass propagation.
Statistical analysis indicated that there were significant differences on the number of conidia
produced by P. fumosoroseus (Acc. no. FWA3) when cultured on different substrates. However,
when using brown rice and corn grains as medium, there were no significant differences. Results also
indicated that there were no significant differences on amounts of the conidia harvested at 10, 12, 15
and 20 days after inoculation (Table 9).

62
Table 4 Number of conidia of the fungus, Paecilomyces fumosoroseus (Acc. no. FWA3) produced
on different substrates and harvested at different period of time.
Harvesting time
Number of conidia (x10 7 conidia/ml)*
(day) White-rice Broken -milled rice Brown rice Corn grains Sorghum grains
10 1.12 0.50 0.28 0.41 0.57
12 1.09 0.35 0.26 0.24 0.49
15 1.06 0.42 0.33 0.35 0.35
20 0.61 0.48 0.42 0.30 0.51
Total 3.88 1.74 1.28 1.30 1.92
Average 0.97 0.44 0.32 0.32 0.48
* average from three sets of one gram of cultured medium per 200 ml of clean water supplemented
with 1% household detergent

63
5.2 Mass production of entomopathogenic fungi, Paecilomyces fumosoroseus (Acc. no. BCC
7058) on different substrates used as growth medium.
P. fumosoroseus (Acc. no. BCC 7058) grew on different substrates used in this study,
demonstrated similar vegetative growth and sporulation. The sporulation process was very similar to
P. fumosoroseus (Acc. no. FWA3). After 10 days of incubation, the whole substrates turned into
white color and cheese-liked. The mycelia were crowded on every grain especially white rice,
broken-milled rice and sorghum grains. Number of conidia produced on white-rice, broken-milled
rice, brown rice, corn grains and sorghum grains were 0.44 x 10 7 , 0.25 x 10 7 , 0.24 x 10 7 , 0.04 x 10 7
and 0.17 x 10 7 conidia/ml respectively (Table 5). Similar to P. fumosoroseus (Acc. no. FWA3) when
grew on white rice, the fungus produced abundant mycelia and conidia. The mycelia formed on
brown rice and corn grain adhered less tightly with the substrate than on the other cereals but the
conidia were produced on brown rice more than those produced on corn grains (Figure 7).
Statistical analysis indicated that there were significant differences on the number of conidia
produced by P. fumosoroseus (Acc. no. BCC 7058) when cultured on different substrates which were
similar to P. fumosoroseus (Acc. no. FWA3). However, when using broken-milled rice and sorghum
grains as medium, there were no significant differences. Results also indicated that there were no
significant differences on amounts of the conidia harvested at 10, 12, 15 and 20 days after inoculation
(Table 9).

Table 5 Number of conidia of the fungus, Paecilomyces fumosoroseus (Acc. no. BCC 7058)
produced on different substrates and harvested at different period of time.
Number of conidia (x10 7 Harvesting time
conidia/ml)*
(day) White-rice Broken -milled rice Brown rice Corn grains Sorghum grains
10 0.49 0.24 0.25 0.04 0.14
12 0.53 0.18 0.26 0.05 0.13
15 0.39 0.33 0.25 0.03 0.20
20 0.35 0.27 0.22 0.03 0.19
Total 1.76 1.02 0.98 0.15 0.66
Average 0.44 0.25 0.24 0.04 0.17
* average from three sets of one gram of cultured medium per 200 ml of clean water supplemented
with 1% household detergent
64

65
5.3 Mass production of entomopathogenic fungi, Beauveria bassiana (Acc. no. BCC 1658)
on different substrates used as growth medium.
All substrates used in this study could support growth and sporulation of Beauveria bassiana
(Acc. no. BCC 1658). The fungus grew in similar manner on all substrates. The vegetative growth
and sporulation process was similar to that of P. fumosoroseus (Acc. no. FWA3). Two days after
inoculation, the fungus mycelia occurred on the substrate. They grew rapidly covered parts of the
substrates. After 10 days, the whole substrates turned into white color and cheese-liked. The mycelia
were not floccose. They looked like some dust covered the periphery. Then the whole substrates
turned into pale color. Number of conidia produced on white-rice, broken-milled rice, brown rice,
corn grains and sorghum grains were 0.90 x 10 7 , 0.98 x 10 7 , 0.88 x 10 7 , 0.37 x 10 7 and 0.29 x
10 7 conidia/ml respectively (Table 6). This fungus differed from P. fumosoroseus (Acc. no. FWA3), it
produced abundant mycelia and conidia when grew on broken-milled rice. The mycelia produced in
sorghum grains were over-crowded but beared a few conidia when compared to other substrates.
Results indicated that broken-milled rice was the best substrate for growing B. bassiana (Acc. no.
BCC 1658) and sorghum grains was the least efficient substrate for fungus sporulation (Figure 8).
Statistical analysis indicated that there were no significant differences on the number of
conidia produced by B. bassiana (Acc. no. BCC 1658) when cultured on white rice, broken-milled
rice and brown rice but significantly different from those produced on corn grains and sorghum
grains. The two latter cereals could produced no significant different amounts of conidia. The
amount of conidia produced within 10, 12, 15 and 20 days after inoculation were significantly
differences. The best time for harvesting the conidia was 10 days after inoculation (Table 9).

Table 6 Number of conidia of the fungus, Beauveria bassiana (Acc. no. BCC 1658) produced
on different substrates and harvested at different period of time.
Number of conidia (x10 7 Harvesting time
conidia/ml)*
(day) White-rice Broken -milled rice Brown rice Corn grains Sorghum grains
10 1.47 1.57 1.60 0.50 0.41
12 1.14 0.82 0.94 0.23 0.31
15 0.29 1.00 0.33 0.36 0.29
20 0.69 0.55 0.66 0.38 0.15
Total 3.58 3.94 3.53 1.47 1.16
Average 0.90 0.99 0.88 0.37 0.29
* average from three sets of one gram of cultured medium per 200 ml of clean water supplemented
with 1% household detergent
66

67
5.4 Mass production of entomopathogenic fungi, Metarhizium anisopiae (Acc. no. KKU 2)
on different substrates used as growth medium.
On most of the tested substrates, the mycelia of the fungus, M. anisopiae (Acc. no. KKU 2)
occurred, grew and covered parts of the substrates within the second day after inoculation. However,
on brown rice, the mycelia grew very slow. Sporulation process was fast on white-rice in which after
10 days, the whole substrates turned into white color and then into green color when they produced
many conidia in the next day. The mycelia grew on broken-milled rice, corn grains and sorghum
grains were floccose. Number of conidia produced on white-rice, broken-milled rice, brown rice,
corn grains and sorghum grains were 0.29 x 10 7 , 0.15 x 10 7 , 0.05 x 10 7 , 0.03 x 10 7 and 0.03 x
10 7 conidia/ml respectively (Table 7). The mycelium produced on brown rice was sparse and formed
tightly with the substrate. On white-rice, broken-milled rice and brown rice, the conidia were
difficult to wash off from the substrate with 1% household detergent since the mycelium bearing
conidia was deep-rooted into the substrate. Results indicated that white-rice was the best substrate for
propagating M. anisopiae (Acc. no. KKU 2), corn grains and sorghum grains were the least efficient
substrate for fungus sporulation (Figure 9).
Statistical analysis indicated that there were no significant differences on the number of
conidia produced by M. anisopiae (Acc. no. KKU 2) when cultured on brown rice, corn grains and
sorghum grains but were significant different from those cultured on white-rice and broken-milled
rice. M. anisopiae (Acc. no. KKU 2) produced conidia on all of the tested substrates less than the
other isolates of fungi used in this study. There were significant differences on amounts of conidia
harvested at 10, 12, 15 and 20 days after inoculation in which the best time for harvesting is 15 days
after inoculation (Table 9).

68
Table 7 Number of conidia of the fungus, Metarhizium anisopliae (Acc. no. KKU 2) produced on
different substrates and harvested at different period of time.
Number of conidia (x10 7 Harvesting time
conidia/ml)*
(day) White-rice Broken -milled rice Brown rice Corn grains Sorghum grains
10 0.38 0.09 0.05 0.05 0.05
12 0.25 0.13 0.08 0.01 0.04
15 0.22 0.31 0.04 0.06 0.03
20 0.33 0.06 0.05 0.02 0.03
Total 1.18 0.58 0.22 0.14 0.14
Average 0.29 0.15 0.05 0.03 0.03
* average from three sets of one gram of cultured medium per 200 ml of clean water supplemented
with 1% household detergent

69
5.5 Mass production of entomopathogenic fungi, Hypocrella hypocreoidea (Acc. no. BCC
11370) on different substrates used as growth medium.
Hypocrella hypocreoidea (Acc. no. BCC 11370) grew slowly on all experimented substrates.
It was considered a slow growing fungus. The mycelia were produced on all substrate but not
floccose. After 10 days, the whole substrates turned into white color. Number of conidia produced
on white-rice, broken-milled rice, brown rice, corn grains and sorghum grains were 0.18 x 10 7 , 0.24 x
10 7 , 0.17 x 10 7 , 0.04 x 10 7 and 0.11 x 10 7 conidia/ml respectively (Table 8). Similar to B. bassiana
(Acc. BCC 1658) this fungus produced abundant mycelia and conidia when grew on broken-milled
rice. The mycelia produced in brown rice, corn grains and sorghum grains were sparse and formed
tightly with the substrate (Figure 10), hence difficult to wash off from the substrates with 1%
household detergent. Results indicated that broken-milled rice was the best substrate for producing
H. hypocreoidea (Acc. no. BCC 11370) and corn grains was the least efficient substrate for fungus
sporulation (Figure 9).
Statistical analysis indicated that there were significant differences on the number of conidia
produced by H. hypocreoidea (Acc. no. BCC 11370) when cultured on different substrates especially
when using corn grains and sorghum grains. However, when using white-rice and brown rice, there
were no significant differences. Corn grains provided the least numbers of conidia of H.
hypocreoidea (Acc. no. BCC 11370). The amount of conidia produced within 10, 12, 15 and 20 days
after inoculation were significantly differences. The highest number of conidia was obtained when
cultured the fungus for 20 days after inoculation (Table 9) which confirmed that fungus, H.
hypocreoidea (Acc. no. BCC 11370) was a very slow growing fungus.

Table 8 Number of conidia of the fungus, Hypocrella hypocreoidea (Acc. no. BCC 11370)
produced on different substrates and harvested at different period of time.
Harvesting time
Number of conidia (x10 7 conidia/ml)*
(day) White-rice Broken -milled rice Brown rice Corn grains Sorghum grains
10 0.12 0.21 0.16 0.04 0.08
12 0.10 0.18 0.07 0.02 0.07
15 0.08 0.35 0.07 0.06 0.12
20 0.42 0.23 0.38 0.05 0.16
Total 0.72 0.98 0.68 0.16 0.42
Average 0.18 0.24 0.17 0.04 0.11
* average from three sets of one gram of cultured medium per 200 ml of clean water supplemented
with 1% household detergent
70

Table 9 Number of conidia produced by the five effective isolates of entomopathogenic fungi cultured on different substrates used as growth
medium.
Factor Paecilomyces fumosoroseus
(Acc. no. FWA3)
media (A)
White-rice
Broken -milled rice
Brown rice
Corn grains
Sorghum grains
F-test
97.05 a 1/
43.58 cb
32.05 c
32.38 c
48.03 b
Paecilomyces fumosoroseus
(Acc. no.BCC 7058)
44.10 a 1/
25.47 b
24.43 c
3.83 d
16.52 b
No. of conidia (x10 5 conidia/ml)
Beauveria bassiana
(Acc. no. BCC 1658)
89.57 a 1/
98.52 a
88.22 a
36.65 b
28.97 b
Metarhizium anisopliae
(Acc. no. KKU 2)
29.45 a 1/
14.52 b
5.42 c
3.47 c
3.47 c
Hypocrella hypocreoidea
(Acc. no.BCC 11370)
18.12 b 1/
21.73 a
16.98 b
3.99 d
10.60 c
** ** ** ** **
C.V. (%) 31.38 33.81 43.03 26.96 28.81
time of harvesting (B)
10 days
57.65 a
12 days
15 days
20 days
1/
23.00 a
48.65 a
49.89 a
46.28 a
1/
11.09 a
23.92 a
24.16 a
21.41 a
1/
1/
12.17 ba 12.24 b
68.64 b
10.09 bc
45.43 c
13.17 a
48.56 cb
9.61 c
1/
8.69 c
13.55 b
24.79 a
F-test
ns ns ** * **
C.V. (%) 31.38 33.81 43.03 26.96 28.81
1/ Means follow by the same letter in a column are not significantly different by Tukey’s LSD test (P < 0.05).
* 0.01 > P < 0.05
** P < 0.01
ns P > 0.01
Paecilomyces fumosoroseus (Acc. no. FWA3 and BCC 7058) and Beauveria bassiana (Acc. no. BCC1658) were effective against thrips,
Ceratothripoides claratris and whitefly, Bemisia tabaci.
Metarhizium anisopliae (Acc. no. KKU 2) and Hypocrella hypocreoidea (Acc. no.BCC 11370) were effective against mealybug, Pseudococcus
cryptus.
71

white rice a broken-milled rice
brown rice
sorghum grains
c
corn grains
Figure 6 Mycelium growth and sporulation of Paecilomyces fumosoroseus (Acc. no. FWA3) on
different solid substrates used as culture medium.
e
b
d
72

white rice
broken-milled rice
brown rice c corn grains
d
sorghum grains
a
Figure 7 Mycelium growth and sporulation of Paecilomyces fumosoroseus (Acc. no. BCC 7058) on
different solid substrates used as culture medium.
e
b
73

white rice a broken-milled rice
brown rice c corn grains
sorghum grains
Figure 8 Mycelium growth and sporulation of Beauveria bassiana (Acc. no. BCC 1658) on
different solid substrates used as culture medium.
e
b
d
74

white rice
broken-milled rice
brown rice c corn grains
d
sorghum grains
a
Figure 9 Mycelium growth and sporulation of Metarhizium anisopiae (Acc. no. KKU 2) on
different solid substrates used as culture medium.
e
b
75

white rice
broken-milled rice
brown rice c corn grains
d
sorghum grains
a
Figure 10 Mycelium growth and sporulation of Hypocrella hypocreoidea (Acc. no. BCC 11370) on
different solid substrates used as culture medium.
e
b
76

77
Results from this study indicated that among all cereals tested, white-rice was the best
substrate for fungus production. However, broken-milled rice and brown rice were also good
substrates for some fungus isolates. Mass production of any entomopathogen as bioinsecticide,
cost/benefit ratio is a significant factor in determining the acceptance of both the producers and
consumers. Since broken-milled rice is cheaper than white-rice, it is suggested that broken-milled
rice can be used to mass produce fungus as bioinsecticide alternative to white-rice (Appendix table 1).
The optimum time for spore or conidia production is 10 days of incubation period for most of the
tested fungus isolates.
Conidia productions of several fungi were depended on surface area of substrate provided as
growing medium. Therefore, uniform distribution of fungus inoculum into the substrate masses was
necessary. To accomplish this objective, frequently shaking the medium bags was recommended.
Shaking the bags every 2-3 days during incubation period would ensure a homogeneous development
of the fungus on all grains. Moreover, the mycelium overgrown clumps of grains were crushed which
facilitating mycelium growth and exposing more surface area of grains. It was observed that the
enormous surface and the granular structure of the medium could be controlled more easily and the
clumps better crushed in the bags than in other containers. Heat-resistant bag was, considered, a
suitable container for fungus production. Increasing surface area of the substrate and controlling
substrate clumping were two parameters that had significant effects on fungus productivity.
Results from this study indicated that large quantities of conidia were produced faster on rice
than other kinds of grain. This may be due to the smaller size of rice which provide higher ratio of
surface per volume. There are enormous spaces for mycelium growth and at the same time small size
of rice grains provide less nutrition for fungus growth. Nutrients were depleted quickly, the
mycelium was unable to develop and sporulation occurred.
Among grains used as substrate in this study, the white rice had more surface area and space
for fungus growth than broken-milled rice. Corn grains and sorghum grains had caryopsis cover the
seed which might protect it from fungus penetration through seed coat for food supply, as a

78
consequence, the fungus produced less mycelium and conidia. Brown rice had the pericarp cover
which might have effect on mycelium growth and conidia productivity.
Various kinds of cereal have been used extensively to mass produce the entomopathogenic
fungi as bioinsecticide because they are locally available and cheap. Fungal mass productions had
been successfully developed in many laboratories around the world and were scaled up to commercial
level using several methods (Feng et al, 1994).
This study agreed with McCoy et al. (1988) who reported that conidia of M. anisopliae were
successfully produced in sealed plastic bag of rice for control of rhinoceros beetle, Oryctes
rhinoceros, a serious pest of coconut palms. The fungus M. anisopliae could be grown on
commercially available media (e.g., Sabouraud dextrose agar) as solid substrate or in liquid or
submerged culture. For inexpensive mass production, M. anisopliae conidia have been grown on
steriled rice and membranes saturated with suspension of skim milk powder, dextrose or sucrose and
potassium nitrate (Bailey and Rath, 1994). Using rice as fungal medium was also reported by Meikle
et al. (2001) who used the entomopathogenic fungus, B. bassiana to treat maize ears placed in
traditional grain stores against Prostephanus truncates. The experiments were conducted from
September 1997 to March 1998 in the Benin Republic, West Africa. The conidia produced on whiterice
were extracted and dried conidia were sieved through a 106-µm sieve to remove large hyphal
fragments. Dried conidia powder contained 4.59 x 10 10 conidia/g and had a germination rate above
98%. Conidia were formulated on the day of application.
In addition to the nutrition of each substrate used as medium for fungal propagation, it might
be necessary to supplement in the medium with some additives to accelerate fungal growth and
sporulation. Feng et al. (2004) had used B. bassiana and P. fumosoroseus for the control of
Trialeurodes vaporariorum on greenhouse grown lettuce. Streamed rice with hard rubber-like
firmness was used as a solid substrate for conidiation. To initiate the solid cultures, the streamed rice
was mixed with 15% (v/w) of a 2-day liquid culture (shaken in Sabouraud dextrose broth at 23 – 25
°C) supplemented with 0.6% KNO 3 (w/w) as a conidiation-stimulating agent, and then gently poured

79
into steriled plastic trays, approximately 1.5 cm in depth. The inoculated tray plates were placed on
shelves in an incubation room and were maintained at 25 ± 1 °C for 10 days. The conidia were
harvested and formulated into dried conidia powder and reported in conidia per gram unit. The
resulting product contained 1.48 × 10 11 conidia/g for B. bassiana and 1.35 × 10 11 conidia/g for P.
fumosoroseus. In comparison, number of conidia they obtained when using rice as substrate was
higher than that reported in this study. This was definitely due to the liquid culture (Sabouraud
dextrose broth) supplemented with 0.6% KNO 3 (w/w) added into the rice medium. In this study, only
cooked rice was used without any supplemented substances. Although some additives might
accelerate fungal growth and sporulation, cost effectiveness of the production process should be
awared. Moreover, other factors, for example, incubation period and incubation conditions could be
adjusted in order to obtain the optimal conidial production with high cost-effectiveness.
6. Spray application in screen cage for insect pest control
From bioassay studies the most effective fungus isolates against thrips, Ceratothripoides
claratris were Paecilomyces fumosoroseus (Acc. no. FWA3, FWA4 and FWA5), against mealybug,
Pseudococcus cryptus were Metarhizium anisopliae (Acc. no. KKU 2) and Hypocrella hypocreoidea
(Acc. no. BCC 11370) and against whitefly, Bemisia tabaci were P. fumosoroseus (Acc. no. FWA3)
and Fusarium solani (Acc. no. FWA1). Two of the most virulent fungi against the target insect pests
of tomato were selected as representative to explore their potential for spray application as
bioinsecticide. The experiments were made in insect screen cage. P. fumosoroseus (Acc. no. FWA3)
was used for spray application against thrips and whiteflies and M. anisopliae (Acc. no. KKU 2) was
used against mealybug. The results were as follow:

6.1 The fungus, Paecilomyces fumosoroseus (Acc. no. FWA3) against thrips,
Ceratothripoides claratris.
P. fumosoroseus (Acc. no. FWA3) was mass produced on white-rice and conidia were
harvested after 10 days incubation time. The concentration of the initial fungal suspension harvested
from the substrate was 5 × 10 9 conidial/ml and serial dilutions for five different concentrations were
made for spray trials. These fungal suspensions were sprayed to thrips-infested tomato plants in
insect screen cages. Percent mortality of tomato thrips, C. claratris was observed in 4 days after
spray application. Fungal suspensions at the concentration of 5 × 10 1 , 5 × 10 3 , 5 × 10 5 , 5 × 10 7 and 5 ×
10 9 conidial/ml caused 77.8, 54.5, 61.9, 62.1 and 80.7% mortality of tomato thrips respectively (table
10). The lethal concentration (LC 50) of P. fumosoroseus was calculated as 9.51 × 10 2 conidia/ml
(Table 13). Result from this study suggested that P. fumosoroseus (Acc. no. FWA3) was very
effective against thrips. The fungus concentration as low as 10 3 conidia/ml could cause 50%
mortality of thrips larvae. When higher percent mortality is needed, as in actual field condition,
higher concentration of the fungus is required for spray application.
6.2 The fungus, Metarhizium anisopliae (Acc. no. KKU 2) against mealybug,
Pseudococcus cryptus
After mass propagation on white-rice for 10 days, the conidia of M. anisopliae (Acc. no.
KKU 2) were harvested. The concentration of the initial fungal suspension was 4.5 × 10 9 conidial/ml
and the serial dilutions for five different concentrations were made for spray trials. These fungal
suspensions were sprayed to mealybug-infested tomato plants in insect screen cages. Percent
mortality of mealybug, P. cryptus was observed in 4 days after spray application. Fungal suspensions
at the concentration of 4.5 × 10 1 , 4.5 × 10 3 , 4.5 × 10 5 , 4.5 × 10 7 and 4.5 × 10 9 conidial/ml caused 48.8,
24.8, 57.3, 23.5 and 49.2% mortality of mealybug respectively (table 11). Since percent mortalities
of mealybug highly varied and did not correspond with the concentrations of the fungus inoculum.
Statistical analysis indicated that the lethal concentration (LC 50) of M. anisopliae against mealybug
could not be calculated (Table 13).
80

6.3 The fungus, Paecilomyces fumosoroseus (Acc. no. FWA3) against whitefly,
Bemisia tabaci.
The concentration of the initial suspension of P. fumosoroseus (Acc. no. FWA3)
harvested from the substrate was 6.85 × 10 9 conidial/ml and the serial dilutions were made for spray
trials. These fungal suspensions were sprayed to whitefly-infested tomato plants in insect screen
cages. Percent mortality of whitefly, Bemisia tabaci was observed in 4 days after spray application.
Fungal suspensions at the concentration of 6.85 × 10 1 , 6.85 × 10 3 , 6.85 × 10 5 , 6.85 × 10 7 and 6.85 ×
10 9 conidial/ml caused 29.6, 37.0, 48.1, 74.1 and 92.6% mortality of whiteflies respectively (table
12). The lethal concentration (LC 50) of P. fumosoroseus was calculated as 9.41 × 10 4 conidia/ml
(table 13). Result from this study could suggest that fungus at the concentration of at least 10 4
conidia/ml or higher is needed for the control of whitefly in tomato cultivated in greenhouse.
Variation in the susceptibility of different insect stages may contribute to the differences in
LC 50 values. For example, Vestergaard et al. (1995) found that immatures of western flower thrips,
Frankliniella occidentalis (Pergande), were less susceptible to V. lecanii and M. anisopliae compared
with adults. Brownbrigde et al. (2001) note that 2-d old Bemisia argentifolii nymphs were more
sensitive to B. bassiana compared with neonates. Liu et al. (2002) using the second instar nymphs of
Lygus lineolaris (Hemiptera: Miridae) to demonstrate pathogenicity of several isolates of B. bassiana.
The fungal suspensions at the concentration ranging from 2 × 10 4 to 2 × 10 7 conidia/ml caused
mortality ranging from 35 to 98%. Among the isolates, three of them produced mortality not
significantly different from the water control. The LC 50 values of the five most pathogenic isolates
range from 0.8 to 5.0 × 10 5 conidia/ml. Hernandez and Garza (1994) reported the LC 50 of 4.3 × 10 8
conidia/ml for P. fumosoroseus isolate PF2 sprayed against B. tabaci nymphs feed on Hibiscus rosasinensis.
However, the amount of suspension sprayed was not indicated. In the assays, doses of 100
conidia/mm 2 are produced by spraying suspensions containing approximately 1 × 10 7 conidia/ml. In
previous reports, larval stage was often used for pathogenicity assays and spray trials. This confirmed
that larval stage is more susceptible to fungus infection than adult, which is hold true for plants
sucking insects, the target insects for this study.
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82
Actual field conditions are needed to confirm whether laboratory results reflect isolate
performance in the field. Moreover, other fungal characteristics such as spore production,
germination and hyphal growth rates and effects of environmental conditions ranging that influence
persistence must also be evaluated so that the most appropriate strains can be further developed.
Additionally, it should take into account that most bioassay are carried out at different climate
settings, with different host-plant species. Meekes et al. (2000) found that bioassays carried out on
poinsettia seemed to have a more hostile environment for entomopathogenic fungi than on cucumber
and gerbera. It was, therefore, difficult to compare the result obtained from this study to the others
since there were many pathogenicity-affecting factors involved which were differ in each situation.
Simply apply the materials using conventional spray methods may not deliver sufficient
inoculum to the insect to provide adequate control. None of the reported study gave evidence to
convince that every insect test touched or received the fungi solution. In addition, the migratory
behavior of the insect pest may also be a hindrance to management because multiple applications may
be required to target the newly invading individuals, which occurs throughout much of the growing
period. Therefore, future research and development focused on spray application techniques to
overcome the concern factors is essential in order to achieve successful control of insect pests using
entomopathogenic fungi.

83
Table 10 Analysis of LC 50 value of entomopathogenic fungi against tomato insect pests at 4 days
after fungal spraying to tomato infested plants in insect screen cage.
Tomato
Thrips,
Mealybug,
Whitefly,
insect Ceratothripoides claratris Pseudococcus cryptus Bemisia tabaci
Fungi Paecilomyces fumosoroseus Metarhizium anisopliae Paecilomyces fumosoroseus
(Acc. no. FWA3) (Acc. no. KKU 2) (Acc. no. FWA3)
LC50 9.51 × 10 2
N/A* 9.41 × 10 4
Slope 0.105 ± 0.026 N/A 0.235 ± 0.044
A 5.347 ± 5.483 N/A 5.143 ± 0.117
M 16.281 N/A 15.582
Regressive line : Y = A + Slope × (X+M)
* Not available

CONCLUSION
Thirty three isolates of entomopathogenic fungi were screened for their efficacy as biological
control agent for important tomato insect pests under protected condition or in greenhouse. These
insect pest species were tomato thrips, Ceratothripoides claratris, mealybug, Pseudococcus cryptus
and whitefly, Bemisia tabaci. Twelve isolates of these fungi were newly recovered from naturally
diseased insect collected from tomato field and greenhouse. Twenty one isolates were obtained from
different governmental institutes in Thailand. Bioassays of each fungal isolate against each target
insect species indicated that the highly effective fungi against thrips which caused 93.33% mortality
were Paecilomyces fumosoroseus (Acc. no. FWA5 and BCC 7058), against mealybug which caused
73.33% mortality was Metarhizium anisopliae (Acc. no. KKU 2) and against whitefly which caused
76.67% mortality were Fusarium solani (Acc. no. FWA1) and P. fumosoroseus (Acc. no. FWA3).
The three effective isolates of entomopathogenic fungi against thrips and two isolates against
mealybugs were selected for mass propagation study using different cereals as solid substrate to
support fungus growth and sporulation. Five different cereals, white rice, broken-milled rice, brown
rice, corn grains and sorghum grains were prepared for mass production of Paecilomyces
fumosoroseus (Acc. no. FWA3, BCC 7058), Beauveria bassiana (Acc. no. BCC 1658), H.
hypocreoidea (Acc. no. BCC 11370) and M. anisopliae (Acc. no. KKU 2). Number of conidia
produced on each substrate were recorded at 10, 12, 15 and 20 days of incubation period at 25 - 30°C.
It was found that conidia production tended to increase as the time progressed and reached the
maximum number on day 10. P. fumosoroseus (Acc. no. FWA3 and BCC 7058) cultured on white
rice gave the maximum number of 0.97 × 10 7 and 0.44 × 10 7 conidia/ml, respectively. B. bassiana
(Acc. no. BCC 1658) and M. anisopliae (Acc. no. KKU 2) gave the maximum number of 0.98 × 10 7
and 0.24 × 10 7 conidia/ml when cultured on broken-milled rice. While H. hypocreoidea (Acc. no.
BCC 11370) gave the maximum number of 0.29 × 10 7 conidia/ml when cultured on white-rice.
Based on conidia productivity, it was suggested that white-rice was the most suitable medium for
fungus mass production. The described fungal mass production procedure was considered the
efficient one. The substrates used as fungal medium were cheap and locally available. Particular
84

85
important, the method was precise, simple and provided fast production of the conidia. This method
can be further developed to plant scale production to make available entomopathogenic fungi-based
bioinsecticide commercially.
The highly effective fungus isolates against thrips, mealybug and whitefly were selected for
spray trials in insect screen cages. The fungus, Paecilomyces fumosoroseus (Acc. no. FWA3) was
used against thrips and whitefly and Metarhizium anisopliae (Acc. no. KKU 2) against mealybugs.
For thrips control, P. fumosoroseus (Acc. no. FWA3) at the concentration of 5 × 10 1 , 5 × 10 3 , 5 × 10 5 ,
5 × 10 7 and 5 × 10 9 conidial/ml, were applied to thrips-infested tomato plants in insect screen cages.
The fungus caused 77.8, 54.5, 61.9, 62.1 and 80.7% corrected mortality respectively and the lethal
concentration (LC 50) was estimated as 9.51 × 10 2 conidia/ml. The same fungus was used for whitefly
control and it was found that P. fumosoroseus (Acc. no. FWA3) at the concentration of 6.85 × 10 1 ,
6.85 × 10 3 , 6.85 × 10 5 , 6.85 × 10 7 and 6.85 × 10 9 conidial/ml, caused 29.6, 37.0, 48.1, 74.1 and 92.6%
mortality respectively. The lethal concentration (LC 50) of P. fumosoroseus for whitefly control was
estimated as 9.41 × 10 4 conidia/ml. For mealybug control, M. anisopliae (Acc. no. KKU 2) at the
concentration of 4.5 × 10 1 , 4.5 × 10 3 , 4.5 × 10 5 , 4.5 × 10 7 and 4.5 × 10 9 conidial/ml, caused 48.8, 24.8,
57.3, 23.5 and 49.2% mortality respectively Since percent mortalities of mealybug highly varied and
did not correspond with the concentrations of the fungus inoculum, therefore, the lethal concentration
(LC 50) of M. anisopliae for mealybug control was not available.
The study of entomopathogenic fungi for controlling insect pest is one branch of the
biological control science. The role of entomopathogenic fungi in insect control has encouraged the
interest of scientists to explore for their potential as bioinsecticide. This interest increased
significantly in recent days in which chemical control of insect pests had led to the problems of toxic
residues and pollution threaten to human and animal’s lifes and environment. However, the use of
entomopathogenic fungi for controlling the insect has some limitations. These limitations are biotic
factors such as strains of fungus, fungal virulence, host specific and biotic factors such as the
environmental conditions, including light, humidity, temperature and application method. Macro and
microclimate differences between field and protected condition (greenhouse or screenhouse) should

86
have received particular attention in order to form an appropriate and most efficient insect control
method for each condition. For tomato insect pest control using entomopathogenic fungi, the
investigation on the potential strain, economical production method, formulation and application
strategies are very important for effective use of this biological agent.

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APPENDIX
104

Potato Dextrose Agar
Composition per liter:
Potato Infusion:
Composition per 10 ml:
APPENDIX A
Fungal media
Agar 20.0 g
Glucose 20.0 g
Potato infusion 200.0 g
Potato, unpeeled and sliced 200.0 g
Preparation of Potato Infusion: Add potato slices to 1.0 l of distilled/deionized water. Gentlely heat
and bring to boil. Continue boiling for 30 min. Filter through cheesecloth.
105
Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0 l. Mix
thoroughly. Gentlely heat and bring to boil. Distribute into tubes or flasks. Autoclave for 15 min at
15 psi, pressure at 121 °C. Pour into steriled Petri dishes or leave in tubes.
Appendix table 1 Cost of each substrate used for conidia production.
Substrates Cost (Baht/kg)
White rice
12
Broken-milled rice
9
Brown rice
12
Corn grains
9
Sorghum grains
15

APPENDIX A
Appendix table 2 Spray application of Paecilomyces fumosoroseus (Acc. no. FWA3) against thrips,
Ceratothripoides claratris in insect screen cages.
concentration Replication 1 Replication 2 Replication 3
(spores/ml) Total treated Killed Total treated Killed Total treated Killed
Control
5 x 10 1
5 x 10 3
5 x 10 5
5 x 10 7
5 x 10 9
56 0 56 0 56 0
22 14 20 17 66 53
36 30 78 30 29 18
71 67 60 33 50 12
54 40 50 27 20 10
10 10 49 35 50 43
Appendix table 3 Spray application of Metarhizium anisopliae (Acc. no. KKU 2) against mealybug,
Pseudococcus cryptus in insect screen cages.
concentration Replication 1 Replication 2 Replication 3
(spores/ml) Total treated Killed Total treated Killed Total treated Killed
Control
4.5 x 10 1
4.5 x 10 3
4.5 x 10 5
4.5 x 10 7
4.5 x 10 9
53 0 53 0 53 0
31 22 20 20 31 24
66 57 27 6 26 22
27 4 47 24 49 22
31 19 47 34 9
9
41 10 48 34 43 20
106

Appendix table 4 Spray application of Paecilomyces fumosoroseus (Acc. no. FWA3) against
whitefly, Bemisia tabaci in insect screen cages.
concentration Replication 1 Replication 2 Replication 3
(spores/ml) Total treated Killed Total treated Killed Total treated Killed
Control
6.85 x 10 1
6.85 x 10 3
6.85 x 10 5
6.85 x 10 7
6.85 x 10 9
9
0
9
0
9
0
9
2
9
4
9
2
9
3
9
5
9
2
9
4
9
3
9
6
9
7
9
7
9
6
9
8
9
8
9
9
107

CURRICULUM VITAE
NAME : Miss Cheerapha Panyasiri
BIRTH DATE : May 21, 1979
BIRTH PLACE : Lampang, Thailand
EDUCATION : B.S. (Agriculture), Kasetsart University (2002)
SCHOLARSHIP/AWORD : Scholarship from the Deutsche Forschungsgemeinschift (DFG,
German Research Council), under Protected Cultivation Using
Biological Products”. Special thanks are extended to Professor
Dr. Michael Poehling, Institute of Plant Diseases and Plant
Protection, Faculty of Horticulture, Hannover University,
Germany (2003-2005)
: Scholarship for Master degree research from the Graduate School,
Kasetsart University 2004
108