BIBLIOGRAPHY SACLANGAN, DAN A. APRIL...
BIBLIOGRAPHY
SACLANGAN, DAN A. APRIL 2008. Preliminary Study on the Field
Application of Trichoderma spp. on Strawberry Flowers Using Honeybees (Apis
mellifera Linnaeus). Benguet State University, La Trinidad, Benguet.
Adviser: Luciana Villanueva, PhD
ABSTRACT
The study was conducted to establish the efficiency of delivering Trichoderma
spp. to strawberry flowers using honey bees, Apis millefera, and to observe any
detrimental effect of the biological control agent (BCA) on the bee colony.
A BCA introduction box was designed and attached to a bee colony containing
eight frames. The colony was situated on a strawberry farm of about 450 m2 area. Two
plots were covered with a white net to prevent the bees to forage on the flowers. Trial
and observation were done for the first few days. The commercial Trichoderma spp.
“Biocon” in powder form was placed in the BCA introduction box. After three days,
strawberry flowers from different distances away from the bee colony and in the netted
area were collected and isolated.
The isolated samples collected from the farm were positive of Trichoderma
growth while samples from the netted area had other fungal and bacterial growths. The
bees from the colony were able to disseminate Trichoderma to the strawberry farm as far
as 30 meters away. Although the amount of food on the frames was reduced, bee
population increase was noted during hive inspections on subsequent weeks after the
experiment. There were no adverse effects of the BCA on the bee colony.
TABLE OF CONTENTS
Page
Bibliography ………………………………………………………..………………….. i
Abstract ..………………………………………………………………………………. i
Table of Contents ……………………………………………………………………… ii
INTRODUCTION ……………………………………………………………………... 1
REVIEW OF LITERATURE
Biological Control ……………………………………………………………… 5
Trichoderma ……………………………………………………………………. 5
Bees for Delivery ………………………………………………………………. 6
Honeybee Pollination ………………………………………………………….. 6
The Disease ……………………………………………………………………. 7
Survival ………………………………………………………………………... 7
Cultural Control ……………………………………………………………….. 8
MATERIALS AND METHODS
Bioassay Test ………………………………………………………………….. 9
BCA Introduction Box ………………………………………………………… 11
Experimental Set Up…………………………………………………………… 12
Location of Strawberry Field …………………………………………………. 12
Field Setup ……………………………………………………………………. 14
Collection of Strawberry Flowers ……………………………………………. 18
Isolation
of
Trichoderma spp ……….………………………………………… 19
Bee Colony Examination ……………………………………………………... 19
RESULTS AND DISCUSSION
Bioassay Test …………………………………………………………………. 21
Isolation
of
Trichoderma spp. on Strawberry Flowers ………………………. 25
Meteorological Data …………………………………………………………. 26
Colony Examination …………………………………………………………. 27
SUMMARY, CONCLUSION AND RECOMMENDATION
Summary …………………………………………………………………….. 30
Conclusion ………………………………………………………………….... 31
Recommendation …………………………………………………………….. 31
LITERATURE CITED ………………………………………………………………. 32
ii
1
INTRODUCTION
Biological control is a promising approach for the management of plant diseases
but its use in commercial agriculture remains limited. Among the factors that limit the
adoption of biological control into mainstream agriculture is the effective means of
delivery to the crop. According to Dr. Gary E. Harman of the Cornell University (1992),
delivery systems must ensure that biocontrol agents will grow well and achieve their
purpose. Delivery and application processes must be developed on a crop by crop and
application by application basis. No general solutions exist such that biocontrol systems
must be developed for each crop.
Thorough knowledge and complete analysis of all the factors involved is
necessary for developing the most effective biocontrol system. According to Dr. Miriam
Zilberstain, an expert of Integrated Pest Management in Israel, the most effective way to
manage a disease is to obtain all the needed information about the crop, the pathogen, the
control measures implemented by the farmer and the conditions in the area. A
comprehensive assessment of all the factors that affect the disease development should be
done by the researcher.
The fungus that causes Botrytis fruit rot or gray mold of strawberry is widespread
locally. It can infect strawberry flowers when spores landing on them are exposed to free
water and cool temperatures. The disease is damaging because it extensively sporulates in
strawberry flowers and can spread quickly, particularly under warm, wet conditions
common at bloom time (McCandless, 1999). Spores that lay dormant in the flower
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
Honeybees (Apis mellifera Linnaeus) / Dan A Saclangan.. 2008
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resume activity on the berry later in the season anytime before or after harvest when
sugars increase and conditions become favorable to disease development.
Trichoderma spp. are free-living fungi that are common in soil and root
ecosystems. Recent discoveries show that these are opportunistic, avirulent plant
symbionts, as well as being parasites of other fungi (Harman, 2004). Most Trichoderma
strains produce volatile and non-volatile toxic metabolites that impede colonization by
antagonized microorganisms. Botrytis cinerea spore germination is impeded (fungistasis)
by hydrolytic enzymes produced by Trichoderma. The mechanism of action involved
in the suppression of B. cinerea by Trichoderma are prevention of spore germination and
deactivation of harmful enzymes (Elad, 2000). Therefore, Trichoderma should be
applied during bloom time so it would colonize the flower and prevent any activity of the
pathogen on its surface. As the flower develops into a fruit, the volatile compounds
produced by Trichoderma would inhibit Botrytis infection.
Honeybees are the most effective pollinators of the majority of agricultural crops.
One reason for this is due to their physical characteristics. Their bodies are covered with
fine hairs where pollen grains are attached to as they visit flowers (Fig. 9). The pollen
grains that are attached to their bodies are easily dislodged as they visit other flowers.
Another reason why bees are effective pollinators is their so called flower constancy or
the flower fidelity behavior. Once the field bees decide to forage on one type of plant,
they would visit that same kind of plant for the rest of the day. Therefore, pollen grains
from one species are transferred to the same species. There would be no exchange of
pollen grains with different plants because the foraging bee would only visit one type of
plant per day.
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
Honeybees (Apis mellifera Linnaeus) / Dan A Saclangan.. 2008
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It is in this context why the A. mellifera was thought of to be used in applying
Trichoderma in the field. The Trichoderma combined in a powdery material was placed
in a specially designed box connected to the entrance of the hive. As the forager bees
exit the hive, they pass through the BCA. The bees, being naturally hairy, will pick up
the dust material containing the Trichoderma and as they forage on strawberry flowers,
the material would be deposited on each of the flowers visited (Fig. 13). Secondly, A.
mellifera colonies for pollination contain thousands of foragers which would facilitate in
the application of Trichoderma. Third, the honeybees are in movable hive boxes. These
can be easily transferred to different farms and situated on the desired place. Lastly,
honeybees improve strawberry pollination. Berries pollinated by bees were noted to be
larger, perfect in shape and true to color. Thus, the use of A. mellifera to apply
Trichoderma as BCA against Botrytis will not only minimize, if not prevent fruit rot but
it will increase yield.
The information acquired from this research favors reduced reliance on chemicals
for the control of fungal pathogens by farmers, reduced labor, enhanced environmental
conservation and prolonged duration of disease management. Furthermore, most of our
farmers do not put much attention on the significance of the pollination of crops. This
study advocates the integration of using honeybee pollination on local agriculture and
promotes heightened awareness of the benefits that beekeeping brings.
The objectives of this study are to establish the efficiency of delivering
Trichoderma spp. to strawberry flowers using honey bees, Apis millefera, and to observe
any detrimental effects of the biological control agent on the bee colony.
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
Honeybees (Apis mellifera Linnaeus) / Dan A Saclangan.. 2008
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The study was conducted from September 2007 to March 2008. Field
experiments were done on the strawberry field of Mr. Locloc Pa-at located behind the
College of Arts and Sciences building at Benguet State University, La Trinidad Benguet,
with an estimated area of more than 450m2. Laboratory experiments were done at the
Biocon Laboratory of the Horticulture Research and Training Institute (HORTI) and at
Plant Pathology Laboratory of the College of Agriculture, Benguet State University, La
Trinidad, Benguet.
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
Honeybees (Apis mellifera Linnaeus) / Dan A Saclangan.. 2008
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REVIEW OF LITERATURE
Biological Control
Biocontrol is often viewed as a progressive and environmentally friendly way to
control pest organisms because it leaves behind no chemical residues that might have
harmful impacts on humans or other organisms, and when successful, it can provide
essentially permanent, widespread control with a very favorable cost-benefit ratio
(Newman et al., 1998).
One of the requirements for successful biocontrol is delivery and application
methods should permit the full expression of the biocontrol agent. Delivery systems must
ensure that biocontrol agents will grow well and achieve their purpose. In our experience,
delivery and application processes must be developed on a crop by crop and application
by application basis. No general solutions exist, and so biocontrol systems must be
developed for each crop (Harman, 1992).
Trichoderma
Trichoderma strains exert biocontrol against fungal phytopathogens either
indirectly, by competing for nutrients and space, modifying the environmental conditions,
or promoting plant growth and plant defensive mechanisms and antibiosis, or directly, by
mechanisms such as mycoparasitism. These indirect and direct mechanisms may act
coordinately and their importance in the biocontrol process depends on the Trichoderma
strain, the antagonized fungus, the crop plant, and the environmental conditions,
including nutrient availability, pH, temperature, and iron concentration (Benítez, 2004).
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
Honeybees (Apis mellifera Linnaeus) / Dan A Saclangan.. 2008
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Bees for Delivery
Over the past few years, several studies have shown that honeybees can
successfully disseminate beneficial fungi, bacteria, and viruses to strawberries, pome
fruits, and clover respectively. One of which is the study by Kovach and his colleagues
at Cornell University from 1994-1997. They installed a 'footbath' in the box that fits
across the bee hive entrance. As bees exit the hive on their way to the field, they walk
across the 'footbath', picking up spores of the biocontrol agent.
Honey bee delivery of Trichoderma harzianum can be considered a useful
technique for the biological control of Botrytis fruit rot. Bee delivered Trichoderma
provides equivalent control as the present chemical fungicides available to growers and
also maximizes strawberry yield through better pollination (Kovach).
There were no significant differences in the bee hive health parameters measured
(bee longevity, brood size, and hive weight) between untreated hives and hives exposed
to over 150 g of Trichoderma harzianum strain T.39 (Trichodex) over a 30 day period.
Given the several years of observational data and the results of the experiment, the
researchers believe that T. harzianum does not significantly impact honey bee health and
that the risk associated with bees visiting crops sprayed with T. harzianum formulations
or exposed to the concentrated product of this biocontrol agent are minimal (Kovach).
Honeybee Pollination
Self pollination of strawberry fruits amounts to 53%. Wind furthermore adds
14% of pollination and an additional 24% with the help of the honeybees (Delaplane and
Mayer, 2000). Imagine that additional 24% to all the fruits that would be produced. The
yield of the crop would increase significantly.
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
Honeybees (Apis mellifera Linnaeus) / Dan A Saclangan.. 2008
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Strawberries plucked from plants that had been visited by bees weighed 26 to 40
percent more than those from plants that had not been treated. Strawberry growers do not
generally keep hives because strawberry pollination occurs primarily via wind and
gravity. Study from earlier research indicates that sending in the bees results in better
pollination (Wong, 2000).
The Disease
Botrytis fruit rot (gray mold) is caused by the fungus Botrytis cinerea and is the
most important disease of strawberry worldwide. This disease causes severe pre-harvest
losses primarily due to infections of fruit and flowers, especially under humid conditions
when daytime temperatures are moderate to warm (60º to 75ºF). Botrytis fruit rot is also
an important post harvest disease, since the fungus grows at refrigeration temperatures.
This pathogen infects a wide range of plants including many fruit, vegetable, and weed
species (Legard et al., 2005).
In wet seasons, 80-90% losses of flowers and fruit can occur on unsprayed plants.
The disease thrives during prolonged rainy and cloudy periods just before or during
harvest, and on dense, lush, foliar growth (Ries, 1995).
Survival
B. cinerea is both very common and well adapted for survival. It occurs on a wide
range of hosts and overwinters in dead leaves and decaying plant tissue. As the
temperatures warm in the spring, infective spores are produced and disseminated to
susceptible strawberry tissue by air currents, splashing rain or insects. When these spores
contact water, they germinate and infect plants within hours. B. cinerea has an advantage
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
Honeybees (Apis mellifera Linnaeus) / Dan A Saclangan.. 2008
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over many pathogens in that it has the ability to colonize either living or dead tissue.
Many times, the fungus first becomes established in dead or dying tissue and then moves
into healthy tissue (Hartman and Hershman, 1996).
Cultural Control
Removal and destruction of dead or infected plant material is necessary to help
reduce the amount of inoculum capable of producing new infections. It is also
recommended to remove all ripe fruits during harvest as well as any fruit with signs of
decay or rain damage. Use of plastic mulches prevents berry-soil contact, thus reducing
disease. In addition, some cultivars have flowers and fruits that develop with an upright
stature. This allows fruit to be exposed to better air movement and sunlight and reduces
the risk of infection (Browne et al., 2005).
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
Honeybees (Apis mellifera Linnaeus) / Dan A Saclangan.. 2008
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METHODOLOGY
Each of the factors that are involved in the study (the plant, the farm, the BCA,
the bee colony and the BCA introduction box) was thoroughly considered and planned.
Internationally, it is established that Trichoderma harzianum T65 strain is the
most effective Trichoderma species for Botrytis cinerea. But locally there has been no
established Trichoderma species recommended for the management of B. cinerea. In
search for different Trichoderma species, the researcher came across two locally
available species: one from the Plant Pathology Laboratory of BSU, Trichoderma
koniingi from Dr. Asuncion Nagpala and Trichoderma isolated from the commercial
BCA named “Biocon”, which was given by Dr. Virginia Cuevas of the University of the
Philippines, Los Banos.
Prior to the field set up, bioassay tests were conducted and dusting of the BCA on
strawberry flowers was done.
Bioassay Test
On Petri Plates
Bioassay tests were conducted to compare the efficacy of the two Trichoderma
species which shall be used in the field application.
T0
no
Trichoderma
T1
Trichoderma koniingi
T2
Trichoderma isolated from “Biocon”
A suspension of Botrytis spores (1.7 x 107 spore/ml) were placed on previously
plated PDA. Then four paper discs dipped in two Trichoderma suspensions (T. koniingi
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
Honeybees (Apis mellifera Linnaeus) / Dan A Saclangan.. 2008
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and “Biocon” isolate) were placed on the PDA. The plates were properly labelled and
incubated at 33°. After two days, the plates were observed for fungal growths.
Botrytis is a slow growing fungus on artificial media. It took a week for the
fungus to cover about 70% of the surface of PDA on petri plates. Another bioassay test
was performed using 6 day old B. cinerea. Two suspensions were prepared, one
containing T. koniingi and another containing Trichoderma isolated from commercial
product Biocon. Three paper discs were dipped in each of the suspension then placed
above the Botrytis growths. Observations were done after 2 and 4 days of incubation.
On Fruits
To evaluate further the efficacy of Trichoderma species in suppressing the
disease, additional bioassay test was done using strawberry fruits. There were three
treatments, with three replications, each containing three half-ripe strawberry fruits:
For each of the treatments sterile cotton was placed on 3 sets of plastic containers.
A suspension of Botrytis spores were sprayed on the cotton of all the treatments. For the
control, three half ripe fruits were placed above the damp cotton in each of the three
plastic containers. For T1, the fruits were first dipped in a suspension containing T.
koniingi (1.7 x 107 spore/ml) and placed on properly labeled containers. While in T2, the
fruits were dipped in a suspension of Trichoderma isolated from Biocon then placed on
separate containers. The plastic containers were then covered and incubated at room
temperature. Observation was done after 3 and 5 days of incubation (Fig. 1).
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
Honeybees (Apis mellifera Linnaeus) / Dan A Saclangan.. 2008
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Fig. 1. Dipping of fruits on Trichoderma suspension
BCA Introduction Box
The researcher believes that the success of the study revolves around the design of
the box. He proposed that the box should contain a pollen trap to reduce the entry of the
biocon agent in dust form into the colony. The researcher conferred the purpose of the
study with local beekeepers on how the box should be designed. Mr. Ricky Tayaotao
who conducts beekeeping trainings in La Trinidad, Benguet volunteered to design and
build a box for the experiment.
This box is called the BCA introduction box since it is where the biological
control agent was introduced (Fig. 2). The box is a modified pollen trap. It contains a
movable plastic screen where the bees’ body fits through. Before the bees would leave
the box, they would pass through an area where the BCA is introduced. When the bees
enter the hive coming from the field, the pollen packed on their hind legs are scraped off
by this screen. This would prevent the entry of the pollens exposed to the BCA in dust
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
Honeybees (Apis mellifera Linnaeus) / Dan A Saclangan.. 2008
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form. The pollens that were scraped off drop through another screen and into a sliding
box or a drawer (Fig. 2).
Fig. 2. The unmodified BCA Introduction Box at different angles
Experimental Set Up
Location of Strawberry Field
The strawberry field needed was one with a wide area having numerous
strawberry flowers. No chemicals should have been applied on the farm prior to and
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
Honeybees (Apis mellifera Linnaeus) / Dan A Saclangan.. 2008
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during the field setup. Fortunately, the researcher was able to find a strawberry field
satisfying these requirements. The farm is owned by Mr. Locloc Pa-at who agreed to
cancel some of the needed farm activities in favor of the study.
The study was conducted during the peak months of strawberry bloom (December
2007 and January and February 2008) which reconcile with the best time to observe the
behavior of bees foraging on strawberry flowers. It was noted that bees visited the crop
starting at 8:45am until 3:00pm depending on the weather conditions.
Fig. 3. Mr. Locloc Pa-at’s strawberry farm with an area of about 450 m2.
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
Honeybees (Apis mellifera Linnaeus) / Dan A Saclangan.. 2008
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Field Set Up
Before the sun was up at 5:30am in the morning, the ten framer box containing
eight frames of bees was positioned on the western part of the strawberry field (Fig. 10).
The purpose for situating the hive there is to make the flowers of the crop the first
flowers that the bees would be exposed to. When the bees would commence their
orientational flights, the strawberry crop would be in between their hive and the sun
which is their main basis for direction. The hive is two meters away from the first row of
strawberries. One of the factors affecting the visit of bees to flowers is the proximity to
their hive (Fig. 4).
Two plots in the center of the strawberry field were covered with a white net to
prevent the bees to forage on the flowers. This netted area served as control (Fig. 5).
The BCA introduction box was connected into the entrance of the hive box before
the bees came out and oriented on the new location (Fig. 6). The setup was maintained in
that condition for two days. On the third day, trial was done to test the box. A small
amount of the Biocon was placed onto the box and observation was made. From the
result of the trial, the researcher made modifications on the box to improve its
effectiveness (Fig. 7). On the fourth day at 8:40 am, the BCA was placed on the box. At
10:00am, Biocon that dropped on the drawer was again used. At 11:00am, Biocon was
refilled. On the fifth day at 8:40 am, the BCA introduction box was filled with Biocon
and refilled throughout the day when necessary (Fig. 8). During this two days of BCA
introduction, the amount placed in the BCA introduction box was 45 grams. There were
still Biocon left on the drawer of the box after the field application.
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
Honeybees (Apis mellifera Linnaeus) / Dan A Saclangan.. 2008
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Fig. 4. The bee colony placed two meters near the strawberry plants
Fig. 5. Netted area that served as the control
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
Honeybees (Apis mellifera Linnaeus) / Dan A Saclangan.. 2008
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Fig. 6. The bee colony with the attached BCA introduction box
Fig. 7. Modification of the BCA introduction box
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
Honeybees (Apis mellifera Linnaeus) / Dan A Saclangan.. 2008
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Fig. 8. The BCA introduction box containing the commercial Trichoderma (Biocon)
Fig. 9. Bees that had contact with the BCA in powder form; note the fine hair of
the bees’ body
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
Honeybees (Apis mellifera Linnaeus) / Dan A Saclangan.. 2008
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Collection of Strawberry Flowers
On the sixth day, strawberry flowers were collected in different distances away
from the hive. To accomplish this, a tagged string was used (Fig. 10). Two and one-
day old flowers were collected. Each of the samples was placed on sterile plastic
cellophane to prevent contact among the flowers. The different distances where
strawberry samples were collected from are: 2-6m, 7-12m, 13-18m, 19-24m, 24-30m
and the netted area.
Legend
bee colony
strawberry plots
netted strawberry plots
tagged string
Fig. 10. Field set up and collection from different distances away from the hive
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
Honeybees (Apis mellifera Linnaeus) / Dan A Saclangan.. 2008
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Isolation of Trichoderma spp.
The flower samples were handled aseptically. These were placed on sterilized
paper and sliced into four pieces using sterilized scalpel and forceps. The four pieces
were then planted on previously plated potato dextrose agar (PDA). After each of the
tissue planting method was done, the scalpel and the forceps were dipped in denatured
alcohol and heated on alcohol lamps to prevent the transfer of spores from one flower to
another. The sterile paper used was replaced after each flower isolated (Fig. 11). The
plates were then incubated in an incubation cabinet with a temperature of 33° to promote
fungal growth.
Fig. 11. Isolation of Trichoderma spp. from strawberry flowers
Bee Colony Examination
The hive used was examined to determine any detrimental effect of exposing it to
the BCA. Inspections were performed a week after the BCA introduction and the
following weeks thereafter. The colony components inspected were: egg laying of the
queen, brood cycle, increase or decrease of adult bees and stored food.
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
Honeybees (Apis mellifera Linnaeus) / Dan A Saclangan.. 2008
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Fig. 12. The researcher on the field set up of the study
Fig. 13. Bees foraging on strawberry flowers
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
Honeybees (Apis mellifera Linnaeus) / Dan A Saclangan.. 2008
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RESULTS AND DISCUSSION
Bioassay Test
Using the Same Day Old B. cinerea
and Trichoderma Isolates
After
three
days
of incubation, the Trichoderma isolates grew rapidly covering
about 80% of the media. There was no Botrytis growth seen on both treatments. (Fig.
14a & 14b)
Fig. 14a. Treatment using T. koniingi
Fig. 14b. Treatment using the commercial preparation of Trichoderma spp. (“Biocon”)
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
Honeybees (Apis mellifera Linnaeus) / Dan A Saclangan.. 2008
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Using Six Day Old B. cinerea
After two days of incubation, mycelial growth was observed on the paper discs
containing T. koniingi. The advance of the T. koniingi was hindered when it was placed
above Botrytis growth (Fig. 15a). While in T2 fruiting structures were already evident on
the fungal growth from the Trichoderma isolated from Biocon (Fig. 15b). Therefore the
Biocon isolate grows more rapid compared to the T. knoniingi when placed above
Botrytis growth.
Fig. 15a. Bioassay using T. koniingi against B. cinerea after two days of incubation
Fig. 15b. Bioassay using Biocon isolate against B. cinerea after two days of incubation
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
Honeybees (Apis mellifera Linnaeus) / Dan A Saclangan.. 2008
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After four days of incubation, it is evident that the growth of the Trichoderma on
the T2 treatment is more rapid compared to the Trichoderma on the T1. In T2, the
Trichoderma isolate has already covered the total surface of the media. (Fig. 16b)
Fig. 16a. Bioassay using T. koniingi after four days of incubation
Fig. 16b. Bioassay using Biocon isolate after four days of incubation
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
Honeybees (Apis mellifera Linnaeus) / Dan A Saclangan.. 2008
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Bioassay Test Using Half Ripe Fruits
After three days of incubation, there were severe infections of Botrytis on the
treatment without Trichoderma. T1 had three fruits infected with the disease while T2
had two fruits infected. (Fig 17a, 17b and 17c)
T0
Fig. 17a. Control
T1
Fig. 17b. T. koniingi
T2
Fig. 17c. Trichoderma isolated from “Biocon”
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
Honeybees (Apis mellifera Linnaeus) / Dan A Saclangan.. 2008
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Isolation of Trichoderma spp. on Strawberry Flowers
There
were
no
Trichoderma growths on the flowers that were collected from the
netted area: instead other fungal and bacterial growths were noted (Fig. 18a). All the
flower sample’s collected from 7-12m had Trichoderma growths (Fig. 18c). There was
only 1 flower that had Trichoderma from 13-18m, while the other flowers that were
collected from 2-6m, 19-24m and 25-30m had 2 to 3 flower samples with Trichoderma
growth. The results show that the bees were able to disseminate the Trichoderma at
different distances from the hive reaching as far as 30 meters away while no Trichoderma
was observed from the flowers planted in the area secluded from the bees.
Fig. 18a. Netted
Fig. 18b. 2-6m
Fig. 18c. 7-12m
Fig. 18d. 13-18m
Fig. 18e. 19-24m
Fig. 18f. 25-30m
Fig. 18. Isolated Trichoderma from different areas in the strawberry field
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
Honeybees (Apis mellifera Linnaeus) / Dan A Saclangan.. 2008
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Meteorological Data
Foraging behavior of the honey bees is greatly influenced by the weather. Thus,
meteorological data were acquired from Philippine Atmospheric, Geophysical and
Astronomical Services Administration (PAG-ASA) Balili, La Trinidad, Benguet (Table
1). The prevailing temperature favors bee foraging. However, there were frequent
occurrences of rain shower in the afternoon. Hence, minimal bees were seen in the field
during this time. The light rain may have limited the foraging time for the bees. Wind
speed was also high. This may have affected the detachment of the powder material on
the bees as they forage. Though it was cloudy, there was no fog occurrence.
Table 1. Meteorological data gathered during the introduction of the BCA
02- 21- 2008
02- 22- 2008
Relative Humidity
76%am
68%am
Average Temperature
20.65°C
21.5°C
Rainfall
traize 2:00pm
rainshower 2:00pm
Wind Speed
04 knots/sec
06 knots/sec
Wind Direction
180°S
180°S
Daylength
753 min
752 min
Fog Occurrence
None
None
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
Honeybees (Apis mellifera Linnaeus) / Dan A Saclangan.. 2008
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Colony Examination
Inspection of the hive shows that the egg laying of the queen was not hindered
(Fig. 22). The presence of different brood stages proves that the brood cycle was
continuous. The increase in the number of adult bees was also evident on the hive
entrance. Prior to the field setup, there was high quantity of stored food. After the study,
it was observed that there was a slight decrease. Nevertheless, the food left was still high
(Fig. 21). The maintained food collected proves that there was no sudden loss of foragers
that were in direct contact to the BCA. Two weeks after the study, a new frame was
added to the colony (Fig. 23). The colony increase was not interrupted. Overall, this
would mean that there was no adverse effect on the hive after its exposure to the BCA.
Fig. 19. The colony one week after the experiment
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
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Fig. 20. The noted increase in the number of adult bees a week after the experiment
Fig. 21. A frame showing that the stored food was still abundant
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
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Fig. 22. Hive inspection show uniform laying of the queen after the experiment
Fig. 23. The bee colony two weeks from the experiment; note one frame was added
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
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30
SUMMARY, CONCLUSION AND RECOMMENDATION
Summary
The study was conducted to establish the efficiency of delivering Trichoderma
spp. to strawberry flowers using honey bees, Apis mellifera, and to observe any
detrimental effect of the BCA on the bee colony. The study was conducted from
September 2007 to March 2008. Field experiments were done at the strawberry field of
Mr. Locloc Pa-at located within the Benguet University grounds. On the other hand,
laboratory experiments were conducted at the Biocon Laboratory of HORTI and at Plant
Pathology Laboratory of the College of Agriculture, Benguet State University, La
Trinidad, Benguet.
The preparation of the colony to be used, BCA selection, designing and
modification of the BCA introduction box and locating an experimental field were all
accomplished prior to the main phase of the study.
Using a bee hive colony containing eight frames, the BCA introduction box was
attached and placed in the strawberry farm. After three days of orientation for the bees,
the commercial BCA in powder form was introduced for two days. For the untreated
control, an area was covered with net to prevent bees from the hive to have contact with
these flowers. On the sixth day of the field setup, samples were collected on different
distances away from the bee hive and on the netted area. The samples were placed on
previously plated PDA to observe the presence of the BCA on the flowers.
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
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31
The isolated samples collected from the farm at different distances from the bee
colony were positive of Trichoderma growth while samples from the netted area showed
growth of other fungi and bacteria.
Hive inspections show that the food stored was slightly reduced but an increase of
bee population was noted on subsequent weeks after the experiment.
Conclusion
Using the BCA introduction box, the commercial Trichoderma “Biocon” was
successfully delivered to strawberry flowers using honeybees. Thus, it is feasible to use
A. mellifera in the field application of Trichoderma to strawberry flowers. The BCA that
the bees were exposed for four days had no adverse effect on the colony.
Recommendation
Since the study is only preliminary, further experiments should be conducted prior
to the promotion of the use of this method of delivery. There is no established local BCA
that has proven effective against B. cinerea. It is strongly recommended to first evaluate
other potential antagonists against this disease of strawberry. There is also a need to
modify and improve the BCA introduction box. The big challenge is to design it in such
a way that the bees would contact the BCA as they exit the colony but will prevent the
BCA’s entry into the colony when bees return to the hive. Further studies should also be
conducted to compare the effectiveness of this method and spraying as a technique of
delivering the biocon as control against B. cinerea gray mold of strawberry with chemical
fungicides.
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
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32
LITERATURE CITED
BENITEZ, T. 2004. Biocontrol Mechanisms for Trichoderma Strains. Retrieved
February 20, 2008 from http://www.im.microbios.org/0704/0704249.pdf
BROWNE, G., S. KOIKE and W. GUBLER. 2005. UC IPM Pest Management
Guidelines:
Strawberry. Strawberry Botrytis Fruit
Rot.
UC
ANR
Publication
3468.
Retrieved July 9, 2007 from http://www.ipm.
ucdavis
.edu/PMG/r734100111.html#SYMPTOMS
DELAPLANE, K. and MAYER, D. 2000. Crop Pollination by Bees. CAB Int.
Wallingford Oxon OX108DE, UK
ELAD, Y. 2000. Biological control of foliar pathogens by means of Trichoderma
harzianum and potential modes of action. Crop Prot. 19:709-714.
HARMAN, G. E., C. R. HOWELL, C. R.VITERBO, I. CHET, and M. LORITO. 2004.
Trichoderma species—Opportunistic, |Avirulent Plant Symbionts. Retrieved
February 20, 2008 from http://www.weizmann.ac.il/Biological_Chemistry/
scientist
/Chet/NR.pdf
HARMAN, G.E. Trichoderma
for Biocontrol of Plant Pathogens:
From Basic Research to Commercialized Products. Retrieved July 20, 2007 from
http://www.nysaes.cornell.edu/ent/bcconf/talks/harman.html
HARTMAN, J.R and D.E. HERSHMAN. Gray Mold of Strawberry. Retrieved July 20,
2007 from http://www.ca.uky.edu/agc/pubs/ppa/ppa31/ppa31.htm
KOVACH, J. Using Bees to Disseminate Trichoderma to Strawberries for the Control of
Botrytis Fruit Rot. Retrieved July 16, 2007 from
http://www.nysipm.cornell.edu/nysipm/publications/beedissem/default.asp
KOVACH, J. The Effect of Trichoderma harzianum on Honey Bee Survival. Retrieved
July 14, 2007 from http://www.nysipm.cornell.edu/nysipm/publications
/trich/default.asp
LEGARD. Integrated Management of Strawberry Diseases in Winter Fruit
Production Areas. Retrieved July 25, 2007, from http://strawberry
.ifas.ufl.edu/prodguidedis.htm
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Honeybees (Apis mellifera Linnaeus) / Dan A Saclangan.. 2008
33
MCCANDLESS, L.1999. Entomologist's honeybee 'footbath' delivers beneficial fungi to
strawberries
Retrieved July 14, 2007 from http://66.218.69.11/search/
cache?ei=UTF-8&p=effects+of+bee+pollination+on+strawberry&fr=yfp-t-01&fp
_ip=PH&u= www.nysaes.cornell.edu / pubs / press/ 1999/ bees.html&w =effects+
effect
+bee+bees+pollination+strawberry&d= S6NLkurnO482&icp= 1&.intl=us
MERTELY, J.C. and N.A. PERES. Botrytis Fruit Rot or Gray Mold of Strawberry.
Retrieved August 1, 2007 from http://edis.ifas.ufl.edu/PP152
NEWMAN, R.M., D.C. THOMPSON, and D.B. RICHMAN. 1998. Conservation
Strategies for the biological control of weeds. Pp. 371-396. In P. Barbosa (ed.)
Conservation
biological
control. Academic Press, San Diego.
RIES, S.M. 1995. Gray Mold of Strawberry. July 20, 2007 from
http://www.ipm.uiuc.edu/fruits/diseases/gray_mold/index.html
WONG, K. 2000. Delivering Fungicides on Bee Feet. July 14, 2007 from
http://www.sciam.com/article.cfm?articleId=000572F-76C8-1C61- B882809EC
588
ED9F&ec=ypi
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
Honeybees (Apis mellifera Linnaeus) / Dan A Saclangan.. 2008
SACLANGAN, DAN A. APRIL 2008. Preliminary Study on the Field
Application of Trichoderma spp. on Strawberry Flowers Using Honeybees (Apis
mellifera Linnaeus). Benguet State University, La Trinidad, Benguet.
Adviser: Luciana Villanueva, PhD
ABSTRACT
The study was conducted to establish the efficiency of delivering Trichoderma
spp. to strawberry flowers using honey bees, Apis millefera, and to observe any
detrimental effect of the biological control agent (BCA) on the bee colony.
A BCA introduction box was designed and attached to a bee colony containing
eight frames. The colony was situated on a strawberry farm of about 450 m2 area. Two
plots were covered with a white net to prevent the bees to forage on the flowers. Trial
and observation were done for the first few days. The commercial Trichoderma spp.
“Biocon” in powder form was placed in the BCA introduction box. After three days,
strawberry flowers from different distances away from the bee colony and in the netted
area were collected and isolated.
The isolated samples collected from the farm were positive of Trichoderma
growth while samples from the netted area had other fungal and bacterial growths. The
bees from the colony were able to disseminate Trichoderma to the strawberry farm as far
as 30 meters away. Although the amount of food on the frames was reduced, bee
population increase was noted during hive inspections on subsequent weeks after the
experiment. There were no adverse effects of the BCA on the bee colony.
TABLE OF CONTENTS
Page
Bibliography ………………………………………………………..………………….. i
Abstract ..………………………………………………………………………………. i
Table of Contents ……………………………………………………………………… ii
INTRODUCTION ……………………………………………………………………... 1
REVIEW OF LITERATURE
Biological Control ……………………………………………………………… 5
Trichoderma ……………………………………………………………………. 5
Bees for Delivery ………………………………………………………………. 6
Honeybee Pollination ………………………………………………………….. 6
The Disease ……………………………………………………………………. 7
Survival ………………………………………………………………………... 7
Cultural Control ……………………………………………………………….. 8
MATERIALS AND METHODS
Bioassay Test ………………………………………………………………….. 9
BCA Introduction Box ………………………………………………………… 11
Experimental Set Up…………………………………………………………… 12
Location of Strawberry Field …………………………………………………. 12
Field Setup ……………………………………………………………………. 14
Collection of Strawberry Flowers ……………………………………………. 18
Isolation
of
Trichoderma spp ……….………………………………………… 19
Bee Colony Examination ……………………………………………………... 19
RESULTS AND DISCUSSION
Bioassay Test …………………………………………………………………. 21
Isolation
of
Trichoderma spp. on Strawberry Flowers ………………………. 25
Meteorological Data …………………………………………………………. 26
Colony Examination …………………………………………………………. 27
SUMMARY, CONCLUSION AND RECOMMENDATION
Summary …………………………………………………………………….. 30
Conclusion ………………………………………………………………….... 31
Recommendation …………………………………………………………….. 31
LITERATURE CITED ………………………………………………………………. 32
ii
1
INTRODUCTION
Biological control is a promising approach for the management of plant diseases
but its use in commercial agriculture remains limited. Among the factors that limit the
adoption of biological control into mainstream agriculture is the effective means of
delivery to the crop. According to Dr. Gary E. Harman of the Cornell University (1992),
delivery systems must ensure that biocontrol agents will grow well and achieve their
purpose. Delivery and application processes must be developed on a crop by crop and
application by application basis. No general solutions exist such that biocontrol systems
must be developed for each crop.
Thorough knowledge and complete analysis of all the factors involved is
necessary for developing the most effective biocontrol system. According to Dr. Miriam
Zilberstain, an expert of Integrated Pest Management in Israel, the most effective way to
manage a disease is to obtain all the needed information about the crop, the pathogen, the
control measures implemented by the farmer and the conditions in the area. A
comprehensive assessment of all the factors that affect the disease development should be
done by the researcher.
The fungus that causes Botrytis fruit rot or gray mold of strawberry is widespread
locally. It can infect strawberry flowers when spores landing on them are exposed to free
water and cool temperatures. The disease is damaging because it extensively sporulates in
strawberry flowers and can spread quickly, particularly under warm, wet conditions
common at bloom time (McCandless, 1999). Spores that lay dormant in the flower
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resume activity on the berry later in the season anytime before or after harvest when
sugars increase and conditions become favorable to disease development.
Trichoderma spp. are free-living fungi that are common in soil and root
ecosystems. Recent discoveries show that these are opportunistic, avirulent plant
symbionts, as well as being parasites of other fungi (Harman, 2004). Most Trichoderma
strains produce volatile and non-volatile toxic metabolites that impede colonization by
antagonized microorganisms. Botrytis cinerea spore germination is impeded (fungistasis)
by hydrolytic enzymes produced by Trichoderma. The mechanism of action involved
in the suppression of B. cinerea by Trichoderma are prevention of spore germination and
deactivation of harmful enzymes (Elad, 2000). Therefore, Trichoderma should be
applied during bloom time so it would colonize the flower and prevent any activity of the
pathogen on its surface. As the flower develops into a fruit, the volatile compounds
produced by Trichoderma would inhibit Botrytis infection.
Honeybees are the most effective pollinators of the majority of agricultural crops.
One reason for this is due to their physical characteristics. Their bodies are covered with
fine hairs where pollen grains are attached to as they visit flowers (Fig. 9). The pollen
grains that are attached to their bodies are easily dislodged as they visit other flowers.
Another reason why bees are effective pollinators is their so called flower constancy or
the flower fidelity behavior. Once the field bees decide to forage on one type of plant,
they would visit that same kind of plant for the rest of the day. Therefore, pollen grains
from one species are transferred to the same species. There would be no exchange of
pollen grains with different plants because the foraging bee would only visit one type of
plant per day.
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It is in this context why the A. mellifera was thought of to be used in applying
Trichoderma in the field. The Trichoderma combined in a powdery material was placed
in a specially designed box connected to the entrance of the hive. As the forager bees
exit the hive, they pass through the BCA. The bees, being naturally hairy, will pick up
the dust material containing the Trichoderma and as they forage on strawberry flowers,
the material would be deposited on each of the flowers visited (Fig. 13). Secondly, A.
mellifera colonies for pollination contain thousands of foragers which would facilitate in
the application of Trichoderma. Third, the honeybees are in movable hive boxes. These
can be easily transferred to different farms and situated on the desired place. Lastly,
honeybees improve strawberry pollination. Berries pollinated by bees were noted to be
larger, perfect in shape and true to color. Thus, the use of A. mellifera to apply
Trichoderma as BCA against Botrytis will not only minimize, if not prevent fruit rot but
it will increase yield.
The information acquired from this research favors reduced reliance on chemicals
for the control of fungal pathogens by farmers, reduced labor, enhanced environmental
conservation and prolonged duration of disease management. Furthermore, most of our
farmers do not put much attention on the significance of the pollination of crops. This
study advocates the integration of using honeybee pollination on local agriculture and
promotes heightened awareness of the benefits that beekeeping brings.
The objectives of this study are to establish the efficiency of delivering
Trichoderma spp. to strawberry flowers using honey bees, Apis millefera, and to observe
any detrimental effects of the biological control agent on the bee colony.
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The study was conducted from September 2007 to March 2008. Field
experiments were done on the strawberry field of Mr. Locloc Pa-at located behind the
College of Arts and Sciences building at Benguet State University, La Trinidad Benguet,
with an estimated area of more than 450m2. Laboratory experiments were done at the
Biocon Laboratory of the Horticulture Research and Training Institute (HORTI) and at
Plant Pathology Laboratory of the College of Agriculture, Benguet State University, La
Trinidad, Benguet.
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REVIEW OF LITERATURE
Biological Control
Biocontrol is often viewed as a progressive and environmentally friendly way to
control pest organisms because it leaves behind no chemical residues that might have
harmful impacts on humans or other organisms, and when successful, it can provide
essentially permanent, widespread control with a very favorable cost-benefit ratio
(Newman et al., 1998).
One of the requirements for successful biocontrol is delivery and application
methods should permit the full expression of the biocontrol agent. Delivery systems must
ensure that biocontrol agents will grow well and achieve their purpose. In our experience,
delivery and application processes must be developed on a crop by crop and application
by application basis. No general solutions exist, and so biocontrol systems must be
developed for each crop (Harman, 1992).
Trichoderma
Trichoderma strains exert biocontrol against fungal phytopathogens either
indirectly, by competing for nutrients and space, modifying the environmental conditions,
or promoting plant growth and plant defensive mechanisms and antibiosis, or directly, by
mechanisms such as mycoparasitism. These indirect and direct mechanisms may act
coordinately and their importance in the biocontrol process depends on the Trichoderma
strain, the antagonized fungus, the crop plant, and the environmental conditions,
including nutrient availability, pH, temperature, and iron concentration (Benítez, 2004).
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Bees for Delivery
Over the past few years, several studies have shown that honeybees can
successfully disseminate beneficial fungi, bacteria, and viruses to strawberries, pome
fruits, and clover respectively. One of which is the study by Kovach and his colleagues
at Cornell University from 1994-1997. They installed a 'footbath' in the box that fits
across the bee hive entrance. As bees exit the hive on their way to the field, they walk
across the 'footbath', picking up spores of the biocontrol agent.
Honey bee delivery of Trichoderma harzianum can be considered a useful
technique for the biological control of Botrytis fruit rot. Bee delivered Trichoderma
provides equivalent control as the present chemical fungicides available to growers and
also maximizes strawberry yield through better pollination (Kovach).
There were no significant differences in the bee hive health parameters measured
(bee longevity, brood size, and hive weight) between untreated hives and hives exposed
to over 150 g of Trichoderma harzianum strain T.39 (Trichodex) over a 30 day period.
Given the several years of observational data and the results of the experiment, the
researchers believe that T. harzianum does not significantly impact honey bee health and
that the risk associated with bees visiting crops sprayed with T. harzianum formulations
or exposed to the concentrated product of this biocontrol agent are minimal (Kovach).
Honeybee Pollination
Self pollination of strawberry fruits amounts to 53%. Wind furthermore adds
14% of pollination and an additional 24% with the help of the honeybees (Delaplane and
Mayer, 2000). Imagine that additional 24% to all the fruits that would be produced. The
yield of the crop would increase significantly.
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Strawberries plucked from plants that had been visited by bees weighed 26 to 40
percent more than those from plants that had not been treated. Strawberry growers do not
generally keep hives because strawberry pollination occurs primarily via wind and
gravity. Study from earlier research indicates that sending in the bees results in better
pollination (Wong, 2000).
The Disease
Botrytis fruit rot (gray mold) is caused by the fungus Botrytis cinerea and is the
most important disease of strawberry worldwide. This disease causes severe pre-harvest
losses primarily due to infections of fruit and flowers, especially under humid conditions
when daytime temperatures are moderate to warm (60º to 75ºF). Botrytis fruit rot is also
an important post harvest disease, since the fungus grows at refrigeration temperatures.
This pathogen infects a wide range of plants including many fruit, vegetable, and weed
species (Legard et al., 2005).
In wet seasons, 80-90% losses of flowers and fruit can occur on unsprayed plants.
The disease thrives during prolonged rainy and cloudy periods just before or during
harvest, and on dense, lush, foliar growth (Ries, 1995).
Survival
B. cinerea is both very common and well adapted for survival. It occurs on a wide
range of hosts and overwinters in dead leaves and decaying plant tissue. As the
temperatures warm in the spring, infective spores are produced and disseminated to
susceptible strawberry tissue by air currents, splashing rain or insects. When these spores
contact water, they germinate and infect plants within hours. B. cinerea has an advantage
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over many pathogens in that it has the ability to colonize either living or dead tissue.
Many times, the fungus first becomes established in dead or dying tissue and then moves
into healthy tissue (Hartman and Hershman, 1996).
Cultural Control
Removal and destruction of dead or infected plant material is necessary to help
reduce the amount of inoculum capable of producing new infections. It is also
recommended to remove all ripe fruits during harvest as well as any fruit with signs of
decay or rain damage. Use of plastic mulches prevents berry-soil contact, thus reducing
disease. In addition, some cultivars have flowers and fruits that develop with an upright
stature. This allows fruit to be exposed to better air movement and sunlight and reduces
the risk of infection (Browne et al., 2005).
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METHODOLOGY
Each of the factors that are involved in the study (the plant, the farm, the BCA,
the bee colony and the BCA introduction box) was thoroughly considered and planned.
Internationally, it is established that Trichoderma harzianum T65 strain is the
most effective Trichoderma species for Botrytis cinerea. But locally there has been no
established Trichoderma species recommended for the management of B. cinerea. In
search for different Trichoderma species, the researcher came across two locally
available species: one from the Plant Pathology Laboratory of BSU, Trichoderma
koniingi from Dr. Asuncion Nagpala and Trichoderma isolated from the commercial
BCA named “Biocon”, which was given by Dr. Virginia Cuevas of the University of the
Philippines, Los Banos.
Prior to the field set up, bioassay tests were conducted and dusting of the BCA on
strawberry flowers was done.
Bioassay Test
On Petri Plates
Bioassay tests were conducted to compare the efficacy of the two Trichoderma
species which shall be used in the field application.
T0
no
Trichoderma
T1
Trichoderma koniingi
T2
Trichoderma isolated from “Biocon”
A suspension of Botrytis spores (1.7 x 107 spore/ml) were placed on previously
plated PDA. Then four paper discs dipped in two Trichoderma suspensions (T. koniingi
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and “Biocon” isolate) were placed on the PDA. The plates were properly labelled and
incubated at 33°. After two days, the plates were observed for fungal growths.
Botrytis is a slow growing fungus on artificial media. It took a week for the
fungus to cover about 70% of the surface of PDA on petri plates. Another bioassay test
was performed using 6 day old B. cinerea. Two suspensions were prepared, one
containing T. koniingi and another containing Trichoderma isolated from commercial
product Biocon. Three paper discs were dipped in each of the suspension then placed
above the Botrytis growths. Observations were done after 2 and 4 days of incubation.
On Fruits
To evaluate further the efficacy of Trichoderma species in suppressing the
disease, additional bioassay test was done using strawberry fruits. There were three
treatments, with three replications, each containing three half-ripe strawberry fruits:
For each of the treatments sterile cotton was placed on 3 sets of plastic containers.
A suspension of Botrytis spores were sprayed on the cotton of all the treatments. For the
control, three half ripe fruits were placed above the damp cotton in each of the three
plastic containers. For T1, the fruits were first dipped in a suspension containing T.
koniingi (1.7 x 107 spore/ml) and placed on properly labeled containers. While in T2, the
fruits were dipped in a suspension of Trichoderma isolated from Biocon then placed on
separate containers. The plastic containers were then covered and incubated at room
temperature. Observation was done after 3 and 5 days of incubation (Fig. 1).
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Fig. 1. Dipping of fruits on Trichoderma suspension
BCA Introduction Box
The researcher believes that the success of the study revolves around the design of
the box. He proposed that the box should contain a pollen trap to reduce the entry of the
biocon agent in dust form into the colony. The researcher conferred the purpose of the
study with local beekeepers on how the box should be designed. Mr. Ricky Tayaotao
who conducts beekeeping trainings in La Trinidad, Benguet volunteered to design and
build a box for the experiment.
This box is called the BCA introduction box since it is where the biological
control agent was introduced (Fig. 2). The box is a modified pollen trap. It contains a
movable plastic screen where the bees’ body fits through. Before the bees would leave
the box, they would pass through an area where the BCA is introduced. When the bees
enter the hive coming from the field, the pollen packed on their hind legs are scraped off
by this screen. This would prevent the entry of the pollens exposed to the BCA in dust
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
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form. The pollens that were scraped off drop through another screen and into a sliding
box or a drawer (Fig. 2).
Fig. 2. The unmodified BCA Introduction Box at different angles
Experimental Set Up
Location of Strawberry Field
The strawberry field needed was one with a wide area having numerous
strawberry flowers. No chemicals should have been applied on the farm prior to and
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
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during the field setup. Fortunately, the researcher was able to find a strawberry field
satisfying these requirements. The farm is owned by Mr. Locloc Pa-at who agreed to
cancel some of the needed farm activities in favor of the study.
The study was conducted during the peak months of strawberry bloom (December
2007 and January and February 2008) which reconcile with the best time to observe the
behavior of bees foraging on strawberry flowers. It was noted that bees visited the crop
starting at 8:45am until 3:00pm depending on the weather conditions.
Fig. 3. Mr. Locloc Pa-at’s strawberry farm with an area of about 450 m2.
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
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Field Set Up
Before the sun was up at 5:30am in the morning, the ten framer box containing
eight frames of bees was positioned on the western part of the strawberry field (Fig. 10).
The purpose for situating the hive there is to make the flowers of the crop the first
flowers that the bees would be exposed to. When the bees would commence their
orientational flights, the strawberry crop would be in between their hive and the sun
which is their main basis for direction. The hive is two meters away from the first row of
strawberries. One of the factors affecting the visit of bees to flowers is the proximity to
their hive (Fig. 4).
Two plots in the center of the strawberry field were covered with a white net to
prevent the bees to forage on the flowers. This netted area served as control (Fig. 5).
The BCA introduction box was connected into the entrance of the hive box before
the bees came out and oriented on the new location (Fig. 6). The setup was maintained in
that condition for two days. On the third day, trial was done to test the box. A small
amount of the Biocon was placed onto the box and observation was made. From the
result of the trial, the researcher made modifications on the box to improve its
effectiveness (Fig. 7). On the fourth day at 8:40 am, the BCA was placed on the box. At
10:00am, Biocon that dropped on the drawer was again used. At 11:00am, Biocon was
refilled. On the fifth day at 8:40 am, the BCA introduction box was filled with Biocon
and refilled throughout the day when necessary (Fig. 8). During this two days of BCA
introduction, the amount placed in the BCA introduction box was 45 grams. There were
still Biocon left on the drawer of the box after the field application.
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
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Fig. 4. The bee colony placed two meters near the strawberry plants
Fig. 5. Netted area that served as the control
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
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Fig. 6. The bee colony with the attached BCA introduction box
Fig. 7. Modification of the BCA introduction box
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
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Fig. 8. The BCA introduction box containing the commercial Trichoderma (Biocon)
Fig. 9. Bees that had contact with the BCA in powder form; note the fine hair of
the bees’ body
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Collection of Strawberry Flowers
On the sixth day, strawberry flowers were collected in different distances away
from the hive. To accomplish this, a tagged string was used (Fig. 10). Two and one-
day old flowers were collected. Each of the samples was placed on sterile plastic
cellophane to prevent contact among the flowers. The different distances where
strawberry samples were collected from are: 2-6m, 7-12m, 13-18m, 19-24m, 24-30m
and the netted area.
Legend
bee colony
strawberry plots
netted strawberry plots
tagged string
Fig. 10. Field set up and collection from different distances away from the hive
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
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Isolation of Trichoderma spp.
The flower samples were handled aseptically. These were placed on sterilized
paper and sliced into four pieces using sterilized scalpel and forceps. The four pieces
were then planted on previously plated potato dextrose agar (PDA). After each of the
tissue planting method was done, the scalpel and the forceps were dipped in denatured
alcohol and heated on alcohol lamps to prevent the transfer of spores from one flower to
another. The sterile paper used was replaced after each flower isolated (Fig. 11). The
plates were then incubated in an incubation cabinet with a temperature of 33° to promote
fungal growth.
Fig. 11. Isolation of Trichoderma spp. from strawberry flowers
Bee Colony Examination
The hive used was examined to determine any detrimental effect of exposing it to
the BCA. Inspections were performed a week after the BCA introduction and the
following weeks thereafter. The colony components inspected were: egg laying of the
queen, brood cycle, increase or decrease of adult bees and stored food.
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
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Fig. 12. The researcher on the field set up of the study
Fig. 13. Bees foraging on strawberry flowers
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
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RESULTS AND DISCUSSION
Bioassay Test
Using the Same Day Old B. cinerea
and Trichoderma Isolates
After
three
days
of incubation, the Trichoderma isolates grew rapidly covering
about 80% of the media. There was no Botrytis growth seen on both treatments. (Fig.
14a & 14b)
Fig. 14a. Treatment using T. koniingi
Fig. 14b. Treatment using the commercial preparation of Trichoderma spp. (“Biocon”)
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
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Using Six Day Old B. cinerea
After two days of incubation, mycelial growth was observed on the paper discs
containing T. koniingi. The advance of the T. koniingi was hindered when it was placed
above Botrytis growth (Fig. 15a). While in T2 fruiting structures were already evident on
the fungal growth from the Trichoderma isolated from Biocon (Fig. 15b). Therefore the
Biocon isolate grows more rapid compared to the T. knoniingi when placed above
Botrytis growth.
Fig. 15a. Bioassay using T. koniingi against B. cinerea after two days of incubation
Fig. 15b. Bioassay using Biocon isolate against B. cinerea after two days of incubation
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
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After four days of incubation, it is evident that the growth of the Trichoderma on
the T2 treatment is more rapid compared to the Trichoderma on the T1. In T2, the
Trichoderma isolate has already covered the total surface of the media. (Fig. 16b)
Fig. 16a. Bioassay using T. koniingi after four days of incubation
Fig. 16b. Bioassay using Biocon isolate after four days of incubation
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Bioassay Test Using Half Ripe Fruits
After three days of incubation, there were severe infections of Botrytis on the
treatment without Trichoderma. T1 had three fruits infected with the disease while T2
had two fruits infected. (Fig 17a, 17b and 17c)
T0
Fig. 17a. Control
T1
Fig. 17b. T. koniingi
T2
Fig. 17c. Trichoderma isolated from “Biocon”
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
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Isolation of Trichoderma spp. on Strawberry Flowers
There
were
no
Trichoderma growths on the flowers that were collected from the
netted area: instead other fungal and bacterial growths were noted (Fig. 18a). All the
flower sample’s collected from 7-12m had Trichoderma growths (Fig. 18c). There was
only 1 flower that had Trichoderma from 13-18m, while the other flowers that were
collected from 2-6m, 19-24m and 25-30m had 2 to 3 flower samples with Trichoderma
growth. The results show that the bees were able to disseminate the Trichoderma at
different distances from the hive reaching as far as 30 meters away while no Trichoderma
was observed from the flowers planted in the area secluded from the bees.
Fig. 18a. Netted
Fig. 18b. 2-6m
Fig. 18c. 7-12m
Fig. 18d. 13-18m
Fig. 18e. 19-24m
Fig. 18f. 25-30m
Fig. 18. Isolated Trichoderma from different areas in the strawberry field
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
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Meteorological Data
Foraging behavior of the honey bees is greatly influenced by the weather. Thus,
meteorological data were acquired from Philippine Atmospheric, Geophysical and
Astronomical Services Administration (PAG-ASA) Balili, La Trinidad, Benguet (Table
1). The prevailing temperature favors bee foraging. However, there were frequent
occurrences of rain shower in the afternoon. Hence, minimal bees were seen in the field
during this time. The light rain may have limited the foraging time for the bees. Wind
speed was also high. This may have affected the detachment of the powder material on
the bees as they forage. Though it was cloudy, there was no fog occurrence.
Table 1. Meteorological data gathered during the introduction of the BCA
02- 21- 2008
02- 22- 2008
Relative Humidity
76%am
68%am
Average Temperature
20.65°C
21.5°C
Rainfall
traize 2:00pm
rainshower 2:00pm
Wind Speed
04 knots/sec
06 knots/sec
Wind Direction
180°S
180°S
Daylength
753 min
752 min
Fog Occurrence
None
None
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Colony Examination
Inspection of the hive shows that the egg laying of the queen was not hindered
(Fig. 22). The presence of different brood stages proves that the brood cycle was
continuous. The increase in the number of adult bees was also evident on the hive
entrance. Prior to the field setup, there was high quantity of stored food. After the study,
it was observed that there was a slight decrease. Nevertheless, the food left was still high
(Fig. 21). The maintained food collected proves that there was no sudden loss of foragers
that were in direct contact to the BCA. Two weeks after the study, a new frame was
added to the colony (Fig. 23). The colony increase was not interrupted. Overall, this
would mean that there was no adverse effect on the hive after its exposure to the BCA.
Fig. 19. The colony one week after the experiment
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
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Fig. 20. The noted increase in the number of adult bees a week after the experiment
Fig. 21. A frame showing that the stored food was still abundant
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
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Fig. 22. Hive inspection show uniform laying of the queen after the experiment
Fig. 23. The bee colony two weeks from the experiment; note one frame was added
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
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SUMMARY, CONCLUSION AND RECOMMENDATION
Summary
The study was conducted to establish the efficiency of delivering Trichoderma
spp. to strawberry flowers using honey bees, Apis mellifera, and to observe any
detrimental effect of the BCA on the bee colony. The study was conducted from
September 2007 to March 2008. Field experiments were done at the strawberry field of
Mr. Locloc Pa-at located within the Benguet University grounds. On the other hand,
laboratory experiments were conducted at the Biocon Laboratory of HORTI and at Plant
Pathology Laboratory of the College of Agriculture, Benguet State University, La
Trinidad, Benguet.
The preparation of the colony to be used, BCA selection, designing and
modification of the BCA introduction box and locating an experimental field were all
accomplished prior to the main phase of the study.
Using a bee hive colony containing eight frames, the BCA introduction box was
attached and placed in the strawberry farm. After three days of orientation for the bees,
the commercial BCA in powder form was introduced for two days. For the untreated
control, an area was covered with net to prevent bees from the hive to have contact with
these flowers. On the sixth day of the field setup, samples were collected on different
distances away from the bee hive and on the netted area. The samples were placed on
previously plated PDA to observe the presence of the BCA on the flowers.
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
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The isolated samples collected from the farm at different distances from the bee
colony were positive of Trichoderma growth while samples from the netted area showed
growth of other fungi and bacteria.
Hive inspections show that the food stored was slightly reduced but an increase of
bee population was noted on subsequent weeks after the experiment.
Conclusion
Using the BCA introduction box, the commercial Trichoderma “Biocon” was
successfully delivered to strawberry flowers using honeybees. Thus, it is feasible to use
A. mellifera in the field application of Trichoderma to strawberry flowers. The BCA that
the bees were exposed for four days had no adverse effect on the colony.
Recommendation
Since the study is only preliminary, further experiments should be conducted prior
to the promotion of the use of this method of delivery. There is no established local BCA
that has proven effective against B. cinerea. It is strongly recommended to first evaluate
other potential antagonists against this disease of strawberry. There is also a need to
modify and improve the BCA introduction box. The big challenge is to design it in such
a way that the bees would contact the BCA as they exit the colony but will prevent the
BCA’s entry into the colony when bees return to the hive. Further studies should also be
conducted to compare the effectiveness of this method and spraying as a technique of
delivering the biocon as control against B. cinerea gray mold of strawberry with chemical
fungicides.
Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
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LITERATURE CITED
BENITEZ, T. 2004. Biocontrol Mechanisms for Trichoderma Strains. Retrieved
February 20, 2008 from http://www.im.microbios.org/0704/0704249.pdf
BROWNE, G., S. KOIKE and W. GUBLER. 2005. UC IPM Pest Management
Guidelines:
Strawberry. Strawberry Botrytis Fruit
Rot.
UC
ANR
Publication
3468.
Retrieved July 9, 2007 from http://www.ipm.
ucdavis
.edu/PMG/r734100111.html#SYMPTOMS
DELAPLANE, K. and MAYER, D. 2000. Crop Pollination by Bees. CAB Int.
Wallingford Oxon OX108DE, UK
ELAD, Y. 2000. Biological control of foliar pathogens by means of Trichoderma
harzianum and potential modes of action. Crop Prot. 19:709-714.
HARMAN, G. E., C. R. HOWELL, C. R.VITERBO, I. CHET, and M. LORITO. 2004.
Trichoderma species—Opportunistic, |Avirulent Plant Symbionts. Retrieved
February 20, 2008 from http://www.weizmann.ac.il/Biological_Chemistry/
scientist
/Chet/NR.pdf
HARMAN, G.E. Trichoderma
for Biocontrol of Plant Pathogens:
From Basic Research to Commercialized Products. Retrieved July 20, 2007 from
http://www.nysaes.cornell.edu/ent/bcconf/talks/harman.html
HARTMAN, J.R and D.E. HERSHMAN. Gray Mold of Strawberry. Retrieved July 20,
2007 from http://www.ca.uky.edu/agc/pubs/ppa/ppa31/ppa31.htm
KOVACH, J. Using Bees to Disseminate Trichoderma to Strawberries for the Control of
Botrytis Fruit Rot. Retrieved July 16, 2007 from
http://www.nysipm.cornell.edu/nysipm/publications/beedissem/default.asp
KOVACH, J. The Effect of Trichoderma harzianum on Honey Bee Survival. Retrieved
July 14, 2007 from http://www.nysipm.cornell.edu/nysipm/publications
/trich/default.asp
LEGARD. Integrated Management of Strawberry Diseases in Winter Fruit
Production Areas. Retrieved July 25, 2007, from http://strawberry
.ifas.ufl.edu/prodguidedis.htm
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MCCANDLESS, L.1999. Entomologist's honeybee 'footbath' delivers beneficial fungi to
strawberries
Retrieved July 14, 2007 from http://66.218.69.11/search/
cache?ei=UTF-8&p=effects+of+bee+pollination+on+strawberry&fr=yfp-t-01&fp
_ip=PH&u= www.nysaes.cornell.edu / pubs / press/ 1999/ bees.html&w =effects+
effect
+bee+bees+pollination+strawberry&d= S6NLkurnO482&icp= 1&.intl=us
MERTELY, J.C. and N.A. PERES. Botrytis Fruit Rot or Gray Mold of Strawberry.
Retrieved August 1, 2007 from http://edis.ifas.ufl.edu/PP152
NEWMAN, R.M., D.C. THOMPSON, and D.B. RICHMAN. 1998. Conservation
Strategies for the biological control of weeds. Pp. 371-396. In P. Barbosa (ed.)
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Preliminary Study on the Field Application of Trichoderma spp. on Strawberry Flowers Using
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Document Outline
- Preliminary Study on the FieldApplication of Trichoderma spp. on Strawberry Flowers Using Honeybees (Apismellifera Linnaeus)
- BIBLIOGRAPHY
- ABSTRACT
- TABLE OF CONTENTS
- INTRODUCTION
- REVIEW OF LITERATURE
- Biological Control
- Trichoderma
- Bees for Delivery
- Honeybee Pollination
- The Disease
- Survival
- Cultural Control
- RESULTS AND DISCUSSION
- SUMMARY, CONCLUSION AND RECOMMENDATION
- LITERATURE CITED