BIBLIOGRAPHY AGLUYA, CRESCINTI P. APRIL...
BIBLIOGRAPHY
AGLUYA, CRESCINTI P. APRIL 2012. Isolation and Characterization of
Entomopathogenic Microorganism Affecting Insect Pests in Organic Farms in La Trinidad
Benguet.Benguet State University, La Trinidad, Benguet.
Adviser: Jocelyn C. Perez, MSc.
ABSTRACT
The study was conducted to identify pathogens infecting insect pest in organic vegetable
farms and to characterized the pathogens and symptoms exhibited by the host insets. The study
was conducted at the Plant Pathology Laboratory Benguet State University from November to
March 2012. The growth of microorganisms isolated from white grubs (Phyllophagaspp) (B.
bassiana and N. rileyi) and semi looper(Trichoplusiani) (Trichoderma, Aspergillus and
Penicilliumand the bacteria coded as S4, S6, S8 and S10)in three different media ( Sabraud
Dextrose Agar, Malt Extract Agar and Potato Carrot Agar) were evaluated. Their efficacy was
tested in their host insect.
Two fungal species were isolated from infected white grubs (Phyllophagaspp) while
three fungal species from semi looper(Trichoplusiani). These fungi were Beuvariabassiana,
Nomuraearileyi in white grubs and Trichoderma sp., Aspergillus sp. and Penicillium sp. from
semi looper. Four bacterial pathogens were likewise isolated from semi looper.
The four bacterial isolates have similar colony form, edge and catalase test in the three
different media but differ in color of the colony, B. bassiana has cottony growth in SDA and no
growth observed in MEA and PCA, Trichoderma has a whitish to graycolor on the bottom and
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in Organic
Farms in La Trinidad Benguet /Crescinti P. Agluya. 2012
green spores on the top view in MEA and PCA while in SDA it is color orange on the bottom
and yellow green on top view, Penicilliumhas green spores in MEA and PCA while in SDA it is
creamy white to yellow green while Aspergillus has a black color on the top and bottom view in
MEA and PCA while in SDA it is yellow to dark green on the top view.
Diamond back moth exposed to Trichoderma, Aspergillus and Penicilliumbecome
sluggish unequal in size, rapidly when disturbed and transfer from one place to another, while
diamond back moth exposed in bacterial isolates (S4, S6, S8 and S10) were observed to have
unequal in size, discoloration and decayed if the infection is severe.
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in Organic
Farms in La Trinidad Benguet /Crescinti P. Agluya. 2012
TABLE OF CONTENTS
Page
Bibliography………………………………………………………………….…… i
Abstract…………………………………………………………………………... i
Table of Contents ………………………………………………………………...
iii
INTRODUCTION
1
REVIEW OF LITERATURE 4
Organic Agriculture ………………………………………………………. 4
Entomopathogens/ Biopesticide…………………………………………..
4
Diseased Insects ………………………………………………………….. 5
Entomopathogenic Microorganism…….....................................................
6
Metarhiziumanisopleae……………..........................................................
7
Nomureaerileyi…………………………………………………………..
7
Beavariabassiana…………………..........................................................
8
Nuclear Polyhedrosis Virus …………………………................................ 9
MATERIALS AND METHODS
Culture of Potential Microorganisms ……………………………………
11
Characterization of microorganisms ……………………………………..
11
Growth in different medium …………………………………………….
12
Preparation of fungal Suspension
For Bio-assay Test ……………………………………………………….
12
Preparation of Bacterial Suspension
For Bio-assay Test…………………………………………………… ….
12
Mass Production of Entomopathogenic
Microorganism in Target insect .…………………………………………
13
Data Gathered ……..……………………………………………………
14
RESULT AND DISCUSSION …………………………………………………
16
Microorganisms Isolated from
White Grub and Semi looper ………………………………………. ……
16
Bacteria isolated from Semi looper ……………………………………...
16
Fungi isolated from white grub
and semi lopper…………………………………………………………...
16
Mortality of white grubs exposed to
Beauvariabassianaand Nomureaerileyi………………………………..
24
Mortality of diamond back moth larvae exposed to
25
Penicilium, Aspergillus and Trichoderma………………………………..
Mortality of diamond back moth Treated
with Bacterial Isolates……………………………………………………
29
SUMMARY, CONCLUSIONS AND RECOMMENDATION……………….
31
LITERATURE CITED ……………………………………………………......... 33
APPENDICES……………………………………………………………………
35
1
INTRODUCTION
Vegetables
grown
without
pesticides are natural foods that are grown with
consciousness for the earth and the environment. Studies have proven that organic foods
have a much higher nutritional quality than other fruits and vegetables, and they
minimize the risk of exposing your family or even other people to chemical that could
harm our health (Bolling, 2010).
Organic farming is a system of agriculture that uses environmentally sound
materials and techniques in raising crops or even livestock that are free from growth
hormones and antibiotics. Organic farmers rely on pesticides and fertilizers derived from
plants, animal waste and minerals. They incorporate use of biological organism for pest
management. These methods used in organic farming seek to increase soil fertility,
balance insect population and reduce air, soil and water pollution (Hynes, 2007).
Organic agriculture as defined by International Federation of Organic Agriculture
Movement (IFOAM, 2008) is a sustainable farming system that embraces
environmentally, socially and economically sound production methods for food , fibber
and livestock, recycling nutrient and strengthening natural processes that help to control
pests and diseases, and maintain long term soil fertility to ensure successful production.
Organically grown vegetables are in demand due to the increasing awareness of
hazards of pesticides to the environment, animals and human health (Tad-awanet al.,
2007).Surveys show that organically grown vegetable demand higher price than that
conventionally produced.
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
Organic Farms in La Trinidad Benguet / Crescinti P. Agluya. 2012
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Cotthem (2009) stated that organic vegetables are important because crops grown
organically have fifty percent more nutrient and vitamins compared to crops produced by
conventional farming methods.
Organically produced food are fairly easy to find. But it can be more expensive
compared to conventionally produced food because growing organic food means using
organic garden fertilizers, organic pesticide and organic soil that may cause more produce
(Douglas, and Anderson, 2010).
Entomopathogens (fungi, bacteria, nematode and viruses) are widespread in the
natural environment and cause infection in many pest species. Entomopathogens are safe
and selective insecticides, asit only kills its host insect (Steinnhaus, 1946).
Bacillus thuringensis (Bt) is one of the most widely used pathogen for biological
control of insects. It is a naturally occurring soil bacterium that causes disease in insect
pests (pan Germany, OISAT, 2005) and is the so called insecticide that is acceptable for
managementof insects in organic farming systems (Steinhaus, 1963). It is accepted as an
alternative in organic farming and is considered ideal for pest management because it is
host specific and is non-toxic on natural enemies and on humans (Pan Germany, OISAT,
2005).
The result of this experiment would provide alternatives to the farmers that can
lower overall unit cost of production and allow the farmer to be more competitive.
The study was conducted to:
1. Identify pathogens infecting insect pest in organic vegetable farms, and
2. Characterize the pathogens and symptoms exhibited by the host insects.
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
Organic Farms in La Trinidad Benguet / Crescinti P. Agluya. 2012
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The experiment was conducted at the College of Agriculture, Plant Pathology
Laboratory, Benguet State University, La Trinidad, Benguetfrom November 2011 to
April 2012.
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
Organic Farms in La Trinidad Benguet / Crescinti P. Agluya. 2012
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REVIEW OF LITERATURE
Organic Agriculture
Organic agriculture is the integration of our responsibilities to the present and
future generations in the way we produce the food and fiber we all require and our duties
to enhance and maintain the natural environment which is both our resource based and
our personal setting. It extends beyond the farm gate to the community, local and global
(Bolling, 2010).
Entomopathogens/ Biopesticides
Much have been said and done about chemical approach to disease and insect
control and the problems associated with the use of hazardous chemicals for pest control.
There is considerable pressure on growers to reduce or eliminate the use of pesticide
residues on human health and environment an alternative to chemical control is biological
control with entomopathogens (Koul and Cuperus, 2007).
Biopesticides are pest management tools that are based on beneficial
microorganism (fungi, bacteria, viruses and protozoan), beneficial nematodes or other
safe, biologically based active ingredients. They can be specific to disease, weeds and
arthropods pest. Biopesticides are usually inherently less toxic than conventional
pesticides and it degrades rapidly in the environment. They are effective in very small
quantities and often decomposed quickly, thereby resulting in lower exposure and largely
avoiding the problem caused by environment pesticides (Steinhaus, 1946).
Entomopathogens are microorganisms that caused disease on arthropods,
particularly insects and mites. Entomopathogens are used for microbial control have
range of desirable characteristics including safety to people, compatibility with other
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
Organic Farms in La Trinidad Benguet / Crescinti P. Agluya. 2012
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natural enemies and a lack of toxic residues. They also offer the possibility of providing
persistent control by multiplying in the pest population (Chandler, 2005).
Diseases of Insect Pest
Insects suffering from bacterial diseases exhibit lack of motility, a diminished
appetite, and rectal and oral discharges. In most cases, the infecting bacterium eventually
invades the body cavity of the insects, and infection ends in septicemia. After death, the
body (of the larvae specially) darkens rapidly to a brown to black color. It is usually very
soft and becomes more or less shapeless. The internal tissues may breakdown to a viscid
consistency, sometimes accompanied by odor, but ordinarily they do not “melt” or
liquefy to the extent characteristics of certain virus infections. Usually the insect dries
and become shriveled, with the integument remaining intact. One of the factors that
affects the effectiveness of biopesticides is microbes enters the body of the insects.
Disease producing microorganisms frequently have some special part of the body that
afford them ready entrance into the insects body. This vulnerable point may be the
integument, broken or intact, the intestinal tract, and the spiracle of their body openings.
The portal of the entry may, in general, vary also according to the group of
microorganism concerned. Whereas, most fungi enter the body cavity by penetrating the
integument of body wall of the insect, the spore of the fungus developing in a diseased
insect are disseminated in such a way that they come into direct contact with the
integument of a healthy specimen which is the invaded (Steinhaus, 1946).
Norris
et al. (2003) stated that pathogenic microorganisms, primarily the fungi,
bacteria and viruses, disrupt the normal function of their host, resulting in reduced growth
or even death. Pathogens that use insect pest as hosts, therefore they have the potential to
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
Organic Farms in La Trinidad Benguet / Crescinti P. Agluya. 2012
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provide biological control of their hosts. Several different types of parasitic pathogen
infect arthropods and can provide substantial level of biological control. An epidemic of
pathogen against arthropods is referred to as an epizootic. Under favorable conditions, an
epizootic can kill all susceptible individuals in a population, resulting in a population
crash of the target insect pest.
Entomopathogenic Microorganisms
Most of the bacteria pathogenic to insects are confined to the family: Bacillaceae,
Enterobacteriaceae, Bacteriaceae, Lactobacteriaceae, Micrococcaceae, and
Pseudomona-ceae. This group consist of simple undifferentiated cells, where many of the
species are pleomorphic, one-celled plant-like organisms that multiply by fission
(Steinhaus, 1963).
Entomopathogenic viruses are either DNA or RNA infective unit surrounded by
occlusion body (OB). Viruses have been used for classical and augmentative biological
control for insect pest they are specific and environmentally safe. The transfer of a virus
to a host usually requires the virus to survive in soil litter and on the plant surfaces,
before they are moved passively by biotic or biotic agents. Additionally, the efficacy of
many viruses as biological control is adversely affected by direct sunlight (Rechcigl and
Rechigl, 2000).
Fungi known to cause infection and disease in insects are Phycomycetes,
Ascomycetes, Basidiomycetes and Deoteromycetes. Some fungi infecting insects are
obligate parasites, others were semi-parasitic and others are common saprophytic species
which under certain conditions, are able to cause frank infection in susceptible insects.
Rechcigl and Rechigl (2000) stated that fungi differ from most of the other types of insect
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
Organic Farms in La Trinidad Benguet / Crescinti P. Agluya. 2012
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pathogens in that they do not have to be ingested in order to invade their host. Fungi can
enter their host through natural openings in the insect cuticle and spread to the homocoel.
Because fungi infect insects by penetrating the cuticle, direct contact between the fungi
and the insect host is necessary. The ability of fungi to infect the insect external
integument makes them good candidates for controlling piercing/ sucking herbivores,
which are usually immune to other pathogens due to their feeding behavior (Koul and
Cuperus, 2007).
Metarhiziumanisopliae
Metarhiziumanisopliaes also known asEntomophoraanosplia in the early1990s
named after the insect species it was originally isolated from the
beetle,Anisopliaaustriaca (Steinhaus, 1963).
The disease caused by this fungus is called green muscardine disease because of
the green color of its spores. When the sexual spore of the fungus come in contact with
the body of an insect host, they germinate and the hyphae that emerge penetrate the
cuticle. The fungus develops inside the body eventually killing the insect. The cuticles of
the cadaver often become red. If the ambient humidity is high enough, a white mold then
grows on the cadaver which soon turns green as spores are produced.
Metarhiziumaniopliae is known to have worldwide distribution and is capable of
infecting more than one hundred different specie belonging to a variety of insect orders
(Chandler, 2005).
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
Organic Farms in La Trinidad Benguet / Crescinti P. Agluya. 2012
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Nomureaerileyi (Farlow) Samson
Nomureaerileyi hasa pale green conidiophores ona white basal felt of mycelium.
The conidiaare broadly ellipsoidand in fry chain. They are 3.5-4.5 x 2-3micrometer
long. The conidiophores have branches contains 2-5 phialidesor conidial chains.
The germ tube passes directly into the spicuticle. There is also evidence of direct
penetration of the cuticle. Points of entry darken indicating lysis, presumably due to
enzymatic action. Hyphalbodies formby budding from pre-existinghyphae and are
distinctly nucleated. At the death of the host, hyphae began to grow outward. Mohamed
and Mohamed (1978) also reported that in vitro enzymatic tests the fungus secrets
chitinase, protease and lipase.
Nomuraearileyi attacks larvae of rice insect such as leaf folder, stem borer, green
caterpillar army worm and case worm (Rechigl and Rechigl, 2000).
Beauvariabassiana
Beauvariabassiana (Balsamo) Vuillemin, white muscardinefungus, was the first
microorganism to be recognized as a disease agent.
Beauvariabassiana is a naturally occurring fungi, it generally infects
throughtheintegument.Mahr(1997)explainedthat under favorable environment and
moistureconditions, a conidium adhere into the host cuticle will germinate. The hypha
growing from the spore secrets enzymes which attack and dissolve the cuticle allowing it
to penetrate the skin and grow into the insect body. Once inside the host, it produces
toxin called beauvarian that weakens the host immune system. After the insect dies,
anantibiotic (oosperin) is produced that enables the fungus mass. When conditions are
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
Organic Farms in La Trinidad Benguet / Crescinti P. Agluya. 2012
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favorablethey will growth through the softer parts ofthe insect bodyproducing
thecharacteristics “white bloom”. It was further found out that it infects the highest
number of potato weevil (Mayapit, 2007).
Beauvariabassiana occurs worldwide. It has an extensive host list that include
such important pest as white flies, aphids, grass hopper, termites, Colorado beetle,
Mexican bean beetle, Japanese beetle, ball weevil, cereal leaf beetle, bark beetles, lygus
bugs, cinch bug, fire ant, European corn borers, codling moth and douglas fir cassock
moth (Mahr, 1997).
Other fungi that attacks insects belong to species in the genus Entomophthora and
are the bestknown that provide very high levels of control of many insects, such as
aphids, lepidopterous caterpillars, and grasshoppers (Steinhaus, 1963). Other species
belong to Penicilliumand Trichoderma(Sullivan, 2004).
Nuclear Polyhedrosis Virus
Nuclear Polyhedrosis Virus (NPV) belongs to family Baculviridea. These are
double strand-DNA virus (ds-DNA) with rod-shaped nucleocapsids. This enables the
virus to infect cells more easily, and aids in reproduction of the virus.
When the insect eats the virus cell (occlusion body), its protein is degraded by
gut proteases, freeing virions, which penetrate the midgut cells of theinsect. The virions
invade the cell nucleus, and multiplying causing cell rapture. Virions are then passed into
the haemocoel, invades and form OBs in additional susceptible tissues including the
tracheal matrix, fat body and hypodermis causes rapture of the intersegmental membrane
and oozing of the larval contents to the exterior of the insect, once this fracture begins,
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
Organic Farms in La Trinidad Benguet / Crescinti P. Agluya. 2012
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the insect melts and contents containing OBs that will be dispersed by abiotic and biotic
agents (Rechigl and Rechigl, 2000).
Nuclear polyhedrosis virus is naturally occurring virus that infects many
caterpillar pests, alfalfa looper, corn earworm, imported cabbageworm, cabbage lopper,
cotton bollworm, cotton leafworm, tobacco budworm, armyworms, European corn borer,
almond moth, spruce budworm, Douglas fir tussock moth, pine sawfly andgypsy moth.
Preparations of granulosis virus has been isolatedfrom several caterpillar species,
including imported cabbageworm, cabbage looper, armyworm, fall webworm, and
mosquitoes, among many others (Rechigl and Rechilg, 2000).
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
Organic Farms in La Trinidad Benguet / Crescinti P. Agluya. 2012
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MATERIALS AND METHODS
Collection of Diseased Insects in Organic Farm
Organic farms (BSU Organic Farm, BSU Experimental Area and Swamp
Experimental Area) in La Trinidad were visited. Diseased insects belonging to the order
Lepidoptera and Coleoptera were collected. Infected larvae that were usually sluggish,
hanging on the leaves, or present on the upper surface of the leaves were collected. In
addition dead insects that wereattachedon the leavesand larvaewhich were smelly,
discolored and flaccidwere likewise collected randomly and were brought to the
laboratory for microscopy of microbial growth in the gut.
Culture of Potential Microorganisms
Diseased insects collected from different organic farms were incubated at room
temperature and were initially isolated in Nutrient Agar (NA) for bacteria and water Agar
(WA) for fungi. The pure culture for bacteria was obtained using NA for bacteria and
PDA for fungi.
Characterization of Microorganisms
Microorganisms isolated from the different diseased insects were grown in
different media (SDA, MEA and PCA) characterization was based on their structures, for
fungi color and shape of the spores and hyphae and for bacteria shape of the cell catalase
test and the gram stain reaction.
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
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Growth in Different Medium
Pure culture of the microorganisms were cut using a cork borer or blade and were
grown in three different culture media (Sabraud Dextrose Agar, Malt Extract Agar and
Potato Carrot Agar). Characterization of growth was done.
Preparation of Fungal Suspension
For Bioassay Tests
The seven-day old pure cultures of fungal isolates were used as treatments for the
bioassay test. In preparing the suspensions, the plates containing pure culture of the
fungus were scraped with sterilized wire loop and dispensed in Sterilized Distilled Water
(SDW) on test tubes. The spore count (Table 1) was standardized by counting the number
of the spores per ml using the haemocytometer.
Preparation of Bacterial Suspension
For Bioassay Test
Twenty ml SDW were poured on the plates containing pure culture of bacteria. A
wire loop was used to scrape the bacterial growth on the NA and poured into the new
sterilized Petri plates ready for the bioassay test.
Table 1. Spore count of the fungal isolates used in bioassay tests
FUNGAL
ISOLATES SPORE
COUNT
PER
ML
Beauvariabassiana 3.56 x 106
Nomuraearileyi 4.45 x 106
Trichoderma sp.
5.40 x106
Aspergillus sp.
5.32 x 106
Penicillium sp.
5.08 x 106
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
Organic Farms in La Trinidad Benguet / Crescinti P. Agluya. 2012
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Mass Production of Entomopathogenic Microorganism
In Target Insects
White Grub (Phyllophaga spp)
Healthy white grubs were collected from infested sweetpotato roots at the
Mountain Province Provincial Nursery Area located at OtucanBauko Mountain Province.
These were placed in sterile plastic container with sterile soil and roots of sweet potato,
ten (10) larvae were placed in each container.Fifteen ml of the fungal organism
(Beauvariabassiana and Nomuraearileyi) weresprayed in each container. A separate set-
up without inoculum was designated as control. Appearance of symptoms was observed
at different duration of time (48, 72, 120,168 and 240 hours).
Diamond back moth (Plutellaxylostella)
Healthy diamond back moth were collected in the field. After gathering sufficient
semi lopper, ten (10) larvae were introduced in sterilized plastic container containing
three leaves of Chinese cabbage that was dipped on the fungal suspension (Penicillium
sp. Aspergillussp. and Trichoderma sp.) and bacterial suspension (S4, S6, S8 and S10).
Addition of three leaves daily was done to ensure enough supply of food likewise, the
tissue paper was replaced regularly during the gathering of larval mortality at 24, 48,72,
120, 168 and 240 hours (method1). Likewise one Chinese cabbage leaf was dipped on
the residual fungal suspension used in method 1 and one larva was introduced (method
2). The number of dead larvae was recordedafter 24, 48, 72, 120, 168 and 240 hours.
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
Organic Farms in La Trinidad Benguet / Crescinti P. Agluya. 2012
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A.
B.
Figure1. Bioassay testsset-up. A. Bioassay ofmicroorganism on semi diamond back
moth larvae B. Bioassay of microorganism on white grub
Data Gathered
1.Identification and Characterization of Pathogen
a. Identification of pathogen. Thepure culture of the microorganism grown in
PDA or NA will be identified using morphological, culture characteristics and gram stain
(for bacteria only)
b. Characteristics of the Pathogen
b.1. Pure culture of the isolates will be transferred in three different media
(Sabraud Dextrose Agar, Malt Extract Agar and Potato Carrot Agar) to characterize their
growth on what medium they prefer.
b.2. Morphological characteristics of the pathogen in term of fruiting body,
mycelia branching (for fungus), reaction to stain and shape of the cells (for bacteria).
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
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c. Cultural characteristics of the pathogen in term of production of pigments.
2.Description of Diseased Insects. This was done by observing and listing the
appearance of the collected insects from organic farm that are showing positive infection
(this will be based on the review of literature).
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
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RESULTS AND DISCUSSION
Microorganisms Isolated from White Grub and
Semi looper
Fungi and Bacteria were isolated from white grub and semi lopper. The fungal
isolates include Beauvariabassiana, Numoraearileyi, Penicillium sp., Trichoderma sp.,
and bacteria coded as S4, S6, S8 and S10 were likewise isolated.
Bacteria isolated from Semi looper.The cultural and morphological characteristics
of the different isolated bacteria are summarized in Table 1 and Table 2 respectively.
The four bacterial isolates have similar colony form, edge and surface in the three
different media used (Sabraud Dextrose Agar, Malt Extract Agar and Potato Carrot
Agar).
Bacterial cells of the four isolates have the same form, catalase test and staining
reaction (Plate 2, 3, 4, and 5). In terms of cell arrangement isolates S4, S6, and S8 are
single or in pair while isolates S10 are in chain. However, regarding the cell size the
diameter of the four isolatesis ranging from 1-2 u in diameter.
Fungi isolated from white grub and semi looper. The fungi that were isolated in
white grubs were Beauvariabasianaand Nomuraearileyi while Trichoderma, Penicillium
and Aspergilluswere isolated in the semi looper. The different genera of fungi are shown
in Figures 6, 7, 8 and 9 respectively.
Beauvariabassiana is a slow-growing fungus and is difficult to grow in agar.
Mycelium appears white in culture and bears masses of powdery spores. It exhibits
cottony growth in Sabraud Dextrose Agar (Figure 6) while in Malt Extract Agar and
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
Organic Farms in La Trinidad Benguet / Crescinti P. Agluya. 2012
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Potato Carrot Agar no growth was recorded. The one-celled colorless conidia are born
along a thin filament in a zigzag fashion as the conidia are produced. Conidiogenous cells
are flask-shaped, rachiform, proliferating sympodialy aggregated into sporodochia.
Conidia are hyaline and globose in shape and are easily detached, a condition leading to
scattered colonies (Figure 6).
Table 2. Cultural characteristics of bacterial isolates in different media
CULURAL
CHARACTERISTICS
TREATMENTS/ Form Surface Edge Elevation Color
ORGANISMS
S4
SDA Circular Slimy, glistening Entire Raised Light yellow
MEA Circular Slimy, glistening Entire Raised Light yellow
PCA Circular Slimy, glistening Entire Raised Light yellow
S6
SDA Circular Slimy, glistening Entire Raised Yellow
MEA Circular Slimy, glistening Entire Convex Yellow
PCA Circular Powdery, dry Entire Flat Yellow
S8
SDA Circular Slimy, glistening Entire Raised Creamy white
MEA Circular Slimy, glistening Entire Raised Creamy white
PCA Circular Slimy, glistening Entire Convex Creamy white
S10
SDA Circular Slimy, glistening Entire Raised White
MEA Circular Slimy, glistening Entire Flat White
PCA Circular Slimy, glistening Wavy Flat White
Nomuraearileyi is a fast growing fungus and is easily to grown in Sabraud
Dextrose Agar the species produces green spores while in Potato Carrot Agar and Malt
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
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Extract Agar no growth was observed. A young culture has white mycelia which turns
green as the culture ages, (Figure 6). Nomuraearileyi is composed of pale green to gray
conidiophores on a white basal felt mycelium. The conidia are broadly ellipsoid and in
dry chains. Size ranges from 3.5-4.5 x 2.3 um long. The conidiophores have branches
were each branch contains phialide spores, (Figure 6).
Table 3. Morphological characteristics of bacterial isolates in different media
MORPHOLOGICAL
CHARACTERISTICS
TREATMENTS/ Form Arrangement Staining Reaction Catalase test
ORGANISMS
S4
SDA Coccus Single/ pair Negative Positive
MEA Coccus Single/ pair Negative Positive
PCA Coccus Single/ pair Negative Positive
S6
SDA Coccus Single/ pair Negative Positive
MEA Coccus Single/ pair Negative Positive
PCA Coccus Single/ pair Negative Positive
S8
SDA CoccusSingle/ pair Negative Positive
MEA CoccusSingle/ pair Negative Positive
PCA CoccusSingle/ pair Negative Positive
S10
SDA CoccusChainNegative Positive
MEA CoccusChain Negative Positive
PCA CoccusChain Negative Positive
A
B
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
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C
D
Figure 2. Bacterial isolate S4. Isolated from semi lopper. (a) Colony growth in PCA (b)
colony growth in MEA (c) colony growth in SDA and (d) gram staining
A
B
C
D
Figure 3. Bacterial isolate S6. Isolated from semi lopper. (a) Colony growth in PCA (b)
colony growth in MEA (c) colony growth in SDA and (d) gram staining
A
B
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
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C
D
Figure 4. Bacterial isolate S8. Isolated from semi lopper. (a) Colony growth in PCA (b)
colony growth in MEA (c) colony growth in SDA and (d) gram staining
A
B
C
Figure 5. Bacterial isolate S10. Isolated from semi lopper. (a) Colony growth in PCA (b)
colony growth in MEA (c) gram staining
Trichoderma is a fast growing fungus and easily grown in agar media. It has a
whitish to gray color on the bottom and green spores on the top view in MEA and PCA
while in SDA it is color orange on the bottom and yellow green on top view (Plate3).
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
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Penicillium is a fast growing fungus and easily grown in agar media. It has green
spores in MEA and PCA while in SDA it is creamy white to yellow green.Penicillium
species are fast growing fungi and have aconidial structures that resembles brushes
(Plate8.d).
2
A 1
3
B
1
2
Figure 6. Bottom view(1), and top view (2), conidiopore and chain of spore (3) of the
different fungal genera. A. Beauvaria bassiana, B. Nomuraea rileyi
A
B
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
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C
D
Figure 7.Trichoderma sp. Isolated from semi lopper. (a) Colony growth in PCA (b)
Colony growth in MEA (c) colony growth in SDA and (d) morphological
structure
A
B
C
D
Figure 8.Penicillum sp. Isolated from semi lopper. (a) Colony growth in PCA (b) colony
growth in MEA (c) colony growth in SDA and (d) morphological structure
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
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A
B
C
D
Figure 9.Aspergillus sp. Isolated from semi lopper. (a) Colony growth in PCA (b) colony
growth in MEA (c) colony growth in SDA and (d) morphological structure
Mortality of white grubs larvae exposed to
Beauvariabassiana and Nomureaerileyi
Figure 10 shows that mortality of white grubs exposed to B. bassiana and N.
rileyi.Beauvariabassiana infects the highest number of white grubs in 168 hrs and 240
hrs as compared to Nomuraearileyi.
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60
50
40
B. bassiana
30
N. rileyi
20
Control
10
0
24 hours 48 hours 72 hours 120 hours 168 hours 240 hours
Figure10. Percent mortality of White grubs larvae treated with fungal isolates at different
duration of time (hours)
Mortality of diamond back moth larvae exposed to
Penicilium,Aspergillusand Trichoderma
Figure 11 shows the percent mortality of larvae that fed on Chinese cabbage
leaves dipped in fungal suspension. The result showed that Penicillium, Aspergillus and
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
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Trichoderma caused infection to the larvae. The result further showed that
microorganisms are more host specific in causing fatalities to other organisms.
The result shows that semi looper mortality increases as the duration of exposure
to the treated leaves up to 168 hrs of exposure except for Trichoderma were mortality
decreased on 168 hrs of exposure. This showed that the virulence or of the
microorganisms decreases as exposure of time increases.
The mode of entry of the fungi could also affect the mortality rate of the larvae.
The fungi needs to penetrate the insect cuticle before it produces toxins that cause the
mortality of larvae. The low population of dead larvae could be attributed to the virulence
of the microorganisms.
35
30
Trichoderma
25
Aspergillus
20
15
Penicillium
10
Control
5
0
12 hours 24 hours 72 hours
120
168
240
hours
hours
hours
Figure 11. Percent mortality of Diamond back moth larvae treated with fungal isolates at
different duration of time
The larvae become sluggish after 24 hours and respond less rapidly when
disturbed. The larvae ceases to eat. Likewise, the infected larvae show migration from
one position to another while other infected larvae climbed to the top of the container or
highest tip of leavesand they hang their pro-legs and die (Stainhuas, 1946) then molds
were observed growing on the insect cuticle after a day. The dead larva then was covered
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
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with mycelium and later turns green due to accumulation of green spores (Shepard et. al,
1999)
Figure 12 shows the feeding and dead larvae after 24 hour of exposure to the left
over fungal suspension (Method 2) bioassay. The larvae consume a small portion of the
leaves that were inoculated with fungal and bacterial isolates. It was also observed that
larvae excrete feces and regurgitate yellow substances just after feeding on the leaves.
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
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A
B
Exposed to Aspergillus isolates
Exposed to Aspergillus isolates
A
B
Exposed to Penicillium isolates
Exposed to Penicillium isolates
A
Exposed to Trichoderma isolates
B
Exposed to Trichoderma isolates
Plate12. Dead larvae after 24 hours of exposure to fungal suspensionA method 1 B.
Method
2
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S4
S4
A
B
S6
S6
A
B
S8
S8
A
B
S10
S10
A
B
Figure 13. Dead larvae after 24 hours exposure to bacterial isolate A. method 1 B.
Method
2.
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Mortality of diamond back moth Treated
with Bacterial Isolates
Figure 14 shows the mortality of larvae exposed to different bacterial isolates.The
resultshowsthat all the bacterial isolates (S4, S6, S8 and S10) caused mortality to the
larvae.
Larvae were observed to have discoloration and unequal sizes despite their
uniformity during the start of the bioassay. Yellow discharges from the mouth and also
from the anus were observed as symptoms of larvae infected with bacteria (Steinhaus,
1946).
70
60
50
S4
40
S6
30
S8
20
S10
10
Control
0
12
24
48
72
120
168
240
hours hours hours hours hours hours hours
Figure 14.Percent mortality of Diamond back moth larvae treated with bacterial isolates
at different duration of time(hours)
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
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The result shows that S6, S8 and S10 caused mortality in semi looper when it is
exposed up to 168 hours. However, more dead larvae were observed on the treatment S10
after 72 hours of exposure. Larvae exposed to S6 and S10 had the highest mortality at
240 hours. The mortality of larvae increased up to 168 hours in all treatments and
decreased at 240 hrs.
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
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SUMMARY, CONCLUSION AND RECOMMENDATION
Summary
The study was conducted to identify the pathogens infecting insect pests in
organic vegetable farms, to characterize the pathogens isolated and symptoms exhibits by
the infected white grubs and semi looper and to determine the efficacy of these
microorganisms in the control of white grub and semi looper.
Based on the results, all the fungal and bacterial isolates grew on the three
different media (SDA, PCA and MEA) but grew faster in SDA.
The microorganisms isolated from white grub wereBeauvariabassiana and
Numoreaerileyi and in semi looper include Trichoderma, Aspergillus and Penicillium and
the bacterial isolates coded as S4, S6, S8 and S10. The fungal and bacterial isolates
causepathologies to its host white grub (B. bassiana and N. rileyi) and semi lopper
(Trichoderma, Aspergillus, Penicillium and the bacterial isolates S4, S6, S8, and S10).
Conclusion
The identified entomogenous fungi that can caused pathologies to white grub
were Beauvariabassiana and Nomuraearileyi while in semi lopper were
Trichoderma,Aspergillus and Penicillium while the entomogenous bacteria were S4, S6,
S8 and S10.
The isolated entomogenous microorganisms can thrive in the three different
media the Sabraud Dextrose Agar, Potato Carrot Agar and Malt Extract Agar.
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
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Recomendation
Further studies on the identification of bacteria and fungi up to species level
isrecommended. Field evaluation trial of the microorganisms must be conducted to
furtherevaluate their efficacies on insects and reaction of host plants towards
microorganisms.
In addition, other media including the utilization of indigenous materials for the
multiplication of these microorganisms should be explored.
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
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LITERATURE CITED
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COTTHEM, W. V. 2009.The Importanceof OrganicVegetable gardening.Retrieved
December
12,2010from
http//www.gadeningtips.afrugalgardener.com/.
DOUGLAS, G. and G. ANDERSON 2010.Organic gardening. Retrieved December15,
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HYNES, E.2007.Microsoft Encarta (DVD), Microsoft Corporation2006.Microsoft
Publisher New York.
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(IFOAM).2008Retrieved December 13,2010 from
http//www.ifoam.org/growing_organic/definitions/sdhw/pdf/Definition of organi
c_agricultureReport.pdf//
KOUL,OandG.W. CUPERUS.2007.EcologicallyBasedIntegrated Pest
Management Biddles Ltd, Kings Lynn United Kingdom. Pp. 230-234
MAHR, S.1997.KnowYourFriends:Beauveriabassiana. MidwestBiological
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13,2010fromwww.entomology.
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MAYAPIT, E. B. 2007. Isolation, Characterazation and Mass Culture of the
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University, La TrinidaBenguet.P. 25.
MOHAMED J. C.AND K.G.MOHAMED.1978.EntomopathogenicFungiRetrieved
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NORRIS, R. F., E. P.CASWELL_CHEN and M.KOGAN.2003.Concepts in
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Education,
Inc.
Pp.234-236
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http//www.oisat.org/control_methods/other_methods/compost_tea.html.
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RECHCIGL,J.E.andN. A.RECHCIGL.2000.InsectPest Management
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SULLIVAN, P. 2004.Sustainable management of soil-borne Plant Diseases: Soil Systems
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STEINHAUS, E.A.(2nd Ed.)1963. Insect Pathology:An Advance Treatise.
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STEINHAUS, E. A.1946.Principlesof Insect Pathology.New York. McGraw-hill book
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J. P. PABLO., J. P. SIMONGO., C. G. KISWAAnd C. S.SHAGOL 2007
Organic PotatoVarieties andPractices inBenguet Philippines P.2.
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35
APPENDICES
Appendix Table 1. Mortality of white grubs exposed to fungal isolates at different
duration of time
DURATION OF EXPOSURE
FUNGAL
24
48 72
120 168 240
ISOLATES hours hourshourshourshourshours
Control 0 0 0 0 0 0
B. bassiana 0 0 0 0 30 50
N. rileyi 0 0 0 0 20 30
Appendix Table 1. Mortality of diamond back moth exposed to fungal isolates at
different duration of time
DURATION OF EXPOSURE
FUNGAL
12 24
72
120
168 240
ISOLATES hours hourshourshourshourshours
Control 0 0 0 0 0 0
Trichoderma 0
10 20 25
20 15
Aspergillus
0
5 15 15 25 15
Penicillium
0 10 15 25 30 20
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
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Appendix Table 3. Mortality of diamond back moth exposed to bacterial isolates at
different duration of time
DURATION
OF
EXPOSURE
BACTERIAL 24
48 72
120 168 240
ISOLATES hours hourshourshourshourshours
Control 0 0 0 0 0 0
S4
20 30
30 30 40 30
S6
30
40 40 40 60 40
S8
10
30
40 30
50
40
S10
20
40
50 40
50
30
Isolation and Characterization of Entomopathogenic Microorganism Affecting Insect Pests in
Organic Farms in La Trinidad Benguet / Crescinti P. Agluya. 2012
Document Outline
- Isolation and Characterization ofEntomopathogenic Microorganism Affecting Insect Pests in Organic Farms in La TrinidadBenguet
- BIBLIOGRAPHY
- TABLE OF CONTENTS
- INTRODUCTION
- REVIEW OF LITERATURE
- MATERIALS AND METHODS
- RESULTS AND DISCUSSION
- SUMMARY, CONCLUSION AND RECOMMENDATION
- LITERATURE CITED
- APPENDICES