BIBLIOGRAPHY ANAS, JOSEPH C. APRIL...
BIBLIOGRAPHY
ANAS, JOSEPH C. APRIL 2013. Follow-up Study on the Efficiency of Honeybees
(Apis mellifera Linnaeus) in Disseminating Trichoderma KA to Manage Gray Molds
(Botrytis cinerea Pers) and Pollination in Strawberries (Fragaria x ananassa Duch).
Benguet State University, La Trinidad, Benguet.
Adviser: Lita Molitas – Colting, PhD.
ABSTRACT
The study aimed to: a) determine how far the Trichoderma spores will be
disseminated by Apis mellifera; b) determine if the Trichoderma spores “dropped” by
Trichoderma-laden honeybees are enough to prevent the rotting of the berries; and c)
determine the pollination efficiency of the Trichoderma-laden honeybees. A modified
Trichoderma Dissemination Box was used to contain the Trichoderma.
The honeybees successfully dessiminated the Trichoderma spores to the strawberry
flowers from 20m to 70m away from the hive.
The fruit rot incidence in strawberry fruits was reduced by 58% from fruit setting
to maturity and increased by 45% marketable yield.
There was no significant increase in the number of fruit set between the pollinated
and unpollinated flowers. The Trichoderma spores that were disseminated were effective
in significantly reducing fruit rot incidence by about 60%. However, an average of 20%
disease reduction on marketable fruits was recorded. As a result, a significant increased in
marketable yields by 45% was realized at harvest.
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
RESULTS AND DISCUSSION
Distances by which Honeybees can Disseminate
Trichoderma KA Spores
The treatment was found positive with Trichoderma KA growth. Mycelium and
spore germination was observed from the different isolation done in each treatment.
Honeybees are efficient in disseminating Trichoderma KA at a given distance. In T1 which
has a distance of 20m, colonies of Trichoderma KA were observed. The spores germinated
at surface were surrounded by its mycelium (Fig. 18) while in T2 which has a 40m distance
away from the hive several colonies of Trichoderma KA growth were observed at the sides
of the petriplates. The mycelium bears its spores as it continous to spread on the plated
PDAs (Fig. 19). Moreover, in farther treatments the growth of the Trichoderma KA was
noticed to have a small number of spores than the previous treatments which was closer to
the hive. Spore germination was moderately compact at T3 which has a 60m distance from
the hive (Fig. 20) while in T4 – 70m the mycelium growth of Trichoderma KA was
dominant after incubation (Fig. 21). Indeed, the concentration of Tichoderma KA spores
was observed from 20m to 40m distances away from the hive (Fig. 18 & 19). Based on
these results, honeybees as they flew out to forage in farther distances it is possible that
their was a loss of inocula from the bees resulting to lesser inocula to be deposited to the
strawberry flowers. However, it is still evident that honeybees are efficient in disseminating
the Trichoderma KA at a given distances.
According to Bilu et al. (2004) the amount that a bee deposits on a flower per visit
is not known, but must be considerably less than that found on the bee’s body upon leaving
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
the hive. Inoculum may be lost as the bee flies towards the flowers, and the amount
delivered per flower is diluted by the many flowers that a bee visits per trip.
On the other hand, Saclangan claimed that honeybees can disseminate Trichoderma
up to 30m away from the hive but, the result of this study showed that bees can disseminate
Trichoderma spores from 20m distance and even as much farther as 40m, 60m and 70m
away from the hive (Fig. 18-21).
The findings of Kovach (1999) provides further support to the result of this study
where he proved that bees exiting from the hive on their way to the field, can pick up as
many as 100,000 Trichoderma spores each. As they forage for nectar and pollen, the bees
leave spores of Tichoderma 22 behind and on the flowers. Trichoderma 22 fights the
familiar gray mold known as Botrytis fruit rot by outcompeting the rot, spore for spore.
In addition, the designed Biocontrol Agent Box which was based on Biocontrol
Introduction box (BCAB) enabled the honeybees to disseminate Trichoderma KA spores
five hours after their release at different distances from the hive reaching up to 70m.
The effectiveness of using bees as a biocontrol agent depends on several factors.
Honeybees can disseminate Trichoderma inoculum to strawberry and effectively control
gray mold (Maccagnani et al., 1999; Kovach et al., 2000). The success of the technique,
however, depends on the type of inoculum dispenser (Bilu et al., 2004; Maccagnani et al.,
2005), the Trichoderma strains, the carrier, and the attractiveness of the strawberry cultivar
to the bees (Kovach et al., 2000).
Meanwhile, the occurrence of the contaminants such as fungus and bacteria in the
isolated suspensions were relatively high since the flowers and honeybees cannot be
sterilized and were highly exposed to an open environment.
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
Figure 18. Mycelium and spore germination of Trichoderma KA
isolated from strawberry flowers at 20m distance from the
hive five hours after foraging
Figure 19. Mycelium and spore germination of Trichoderma KA isolated from strawberry
flowers at 40m distance from the hive five hours after foraging
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
Figure 20. Colony growth of Trichoderma KA isolated from
strawberry flowers at 60m distance from the hive five
hours after foraging
Figure 21. Mycelia of Trichoderma KA isolated from strawberry
flowers at 70m distance from the hive five hours of
foraging
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
Evaluation of Trichoderma KA for Botrytis
Management
The result in Table 1 shows the Botrytis cinerea rate of infection in both treatments.
There was no Botrytis infection observed on the tagged sample flowers. However, after the
fruit set, formation of Botrytis cinerea was observed. Symptoms of rotting started on the
developing fruits on both treatments. This corroborates with the findings of Legard et al.
(2005) as cited by Saclangan (2008) showed that Botrytis cinerea Pers causes severe
preharvest losses primarily due to infections of fruits, especially under humid conditions
when daytime temperatures are moderate to warm (60oF to 75oF).
The use of honeybees as an applicator of the Trichoderma KA for the Botrytis
management was evaluated on fruits. Out of the samples, the treatment using honeybees
had a 42% disease incidence from fruit set to maturity while 68% of infected fruit incidence
was recorded on the treatment without using bees. Significantly, the study found a 58%
reduction of disease infection when using bees as disseminators of Trichoderma KA.
It corroborates with the early study of Saclangan (2008) wherein he conducted a
bioassay test on fruits to evaluate the efficacy of Trichoderma Koniingi in suppressing the
disease. A suspension of Botrytis cinerea was sprayed in the sterilized cotton contained in
plastic containers in which the sample fruits, which were dipped on Trichoderma
suspension, were then placed and got incubated for three days. About 56% of the untreated
fruits were infected with the fruit rot disease as compared to the treated fruits with
Trichoderma Koniingi, a total of 33% of the fruits were infected while an average of 67%
disease management reduction on fruit samples.
The disease assessment using honeybees in disseminating Trichoderma KA was
nearly similar to the result of the bioassay test. The established BAB which was modified
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
from the former BCA box was one of the factors that made the study successful in attaining
the percentage reduction of Botrytis cinerea in strawberries.
Previous studies showed that the effectiveness of using bees as a biocontrol agent
depends on several factors. Honeybees can disseminate Trichoderma inoculum to
strawberry and effectively control grey mold (Maccagnani et al., 1999; Kovach et al.,
2000). The success of the technique, however, depends on the type of inoculum dispenser
(Bilu et al., 2004; Maccagnani et al., 2005), the Trichoderma strains, the carrier, and the
attractiveness of the strawberry cultivar to the bees (Kovach et al., 2000).
Table 1. Percentage Botrytis infection on tagged samples from fruit setting to maturity
TREATMENTS FLOWERS FRUITS
Without honeybees
0%
68%
With Honeybees loaded with
0%
42%
Trichoderma KA
Pollination Efficiency of Honeybees
The flowers that were exposed to Apis mellifera produce a true-to-type shape of
strawberries. Sweet Charlie Strawberry Variety has two types of shape – wedge and conical
shape (Fig. 23a & 23b). Pollination and fertilization of the ovules resulted to normal
development of fruits.
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
Number of Fruit Set
The number of fruits that set after the flowers were secluded or exposed to the bees
is shown in Table 2. The number of fruit set in plants secluded from the bees was 50 out
of 50 which has a computed percentage fruit set of 100%. On the other hand, the fruit set
in sample flowers exposed to the bees was 50 which is also equivalent to 100%. The data
showed that there was no difference between the fruit set percentages of both exposed and
covered strawberry flowers. However, in order to achieve complete pollination, self fertile
strawberries require pollinators such as bees to reduce deformities and to attain bountiful
quality of fruits for strawberry production.
The flowers of all the current commercial strawberry cultivars (Fragaria x
ananassa Duch) are hermaphrodite and self-fertile. However, these flowers may not be
completely self-fertilized; thus, the role of bees as pollinators is important in order to
reduce deformities and increase yield for commercial strawberry production (McGregor,
1976).
Table 2. Number of fruit sets and its total percentage
TREATMENTS FRUIT SET PERCENTAGE
Without honeybees
50
100%
With Honeybees loaded with
50
100%
Trichoderma KA
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
Number of Marketable and Deformed Harvest
Table 3 shows the percentage of harvests noted on marketable (Fig. 23a & 23b) and
not marketable fruits (Fig. 23c) on both treatments. Based on the tagged samples, 26%
were deformed (not marketable) and only 6% were normal fruits among all the fruits which
were treated without using the bees. Whereas, a total of 8% were deformed and 50% were
normal fruits among all the fruits treated using honeybees. Basing from these data
percentages on both treatments, there was a 69% calculated reduction of deformities using
honeybees and an average of 88% increase in true-to-type fruits was calculated on the
exposed strawberry samples for pollination.
Honeybees pollinated 11% of achenes on their first visit, so to produce a normal
berry (fertilization rate of 87%) about 11 visits by honeybees/flower are required to achieve
a normal-sized berry (Anonymous, 2013).
This result determines the pollination rate of honeybees in reducing deformities and
promoting a good quality for strawberry production. However this was attained by using
bees as pollinator and with the aid of other pollinators since the field was open for insect
visitation.
Strawberry flowers are hermaphroditic (having both male and female reproductive
organs) and self-fertile and 80% of fruit production is due to abiotic factors such as gravity
and wind; however, pollinating insects play as essential role in obtaining maximum fruit
as well as reducing deformities (Chagnon et al., 1989).
The use of pollinator such as bees is important to improve pollination by increasing
mobility of pollen grains and consequently, ensuring ovule fertilization and achene
development (Leech et al., 2000). Pollination by insects often enhances yield and fruit
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
quality compared with non-pollinated crops. In addition, Chagnon et al. (1989) stated that
honey bees (Apis mellifera L.) are recognized as the main pollinator of the strawberry crop.
Table 3. Percentage of marketable fruits and non-marketable fruits at harvest
TREATMENTS NOT MARKETABLE MARKETABLE
(Deformed Fruits)
Without honeybees
26%
6%
With Honeybees loaded with
8%
50%
Trichoderma KA
Pollination and Dissemination Evaluation
on overall Harvests
The result in Table 4 shows the total harvest that includes the normal, deformed
and the infected fruits in both treatments. Of all the berries exposed to bees in the treatment
process, 58% of the harvests were normal while 24% were infected. On the side of the
berries secluded from the bees, a total of 32% were normal while a total of 30% was
infected with Botrytis cinerea.
Out of these results, the idea of combined pollination and dissemination of
beneficial fungi using bees was evaluated in this study. The calculated total average
increase in marketable fruits out of the treatments was 45% while the use of Trichoderma
KA was proven effective in managing the Botrytis cinerea by a 20% disease reduction in
the harvest. This means that out of 45% marketable harvests, the combined pollination and
dissemination comprises 20% reduction of the disease and increase in percentage harvests.
In relation to the previous study by Lab-oyan in 2001, he noted that pollination alone by
honeybees increased yield by 25%; therefore, the efficiency of honeybees as pollinator and
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
32
disseminator of the Trichoderma KA for Botrytis control increased marketable fruits up to
45%.
Table 4. Percentage of infected and uninfected fruits at harvest
TREATMENT UNINFECTED INFECTED
Without honeybees
32%
30%
With Honeybees loaded with
58%
24%
Trichoderma KA
.
100
100%
80
100 %
80%
60
100%
60%
40
40%
50%
38%
20
30%
20%
6%
26%
18%
24%
0
8%
0%
Category 1
Category 2
Category 3
Category 4
Fruit
Infected Harvest
Deform
Set
with
(Normal)
Infected
Botrytis
with Botrytis
Exposed to hone
ybees (Blue)
Covered with mesh cloth (Red)
Figure 22. Total percentage of normal, deformed and infected strawberries from fruit set
to harvest
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
a
b
c
d
Figure 23. Sample of true-to-type shape of Sweet Charlie Strawberry Fruit a) wedge shape
b) cone shape c) deformed fruit d) infected fruit with Botrytis rot
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
SUMMARY, CONCLUSIONS AND RECOMMENDATIONS
Summary
The study aimed to a) determine how far the Trichoderma spores will be
disseminated by honeybees; b) determine if the Trichoderma spores “dropped” by
Trichoderma-laden honeybees are enough to prevent the rotting of the berries; and c)
determine the pollination efficiency of the Trichoderma-laden honeybees.
The efficiency of the honeybees as a disseminator of the Trichoderma spp. that was
reported by Saclangan (2008) confirms the success of this study. As recommended, the
researcher modified the box prior to the release and assessment of Trichoderma KA in
managing Botrytis cinerea in strawberries.
The isolated dilutions from different distances were positive of Trichoderma KA
growth. Honeybees can disseminate Trichoderma on different distances ranging from 20m,
40m, and 60m up to 70m distances away from the hive.
In relation to the dissemination of the Trichoderma KA for Botrytis management
on the tagged samples, a total of 68% of the berries secluded from bees got infected while
a total of 42% of the berries subjected to bee dissemination were found infected. However,
the calculated percentage of disease reduction from fruit set to maturity was 58%.
Therefore using bees as disseminators of Trichoderma KA reduced the incidence of
Botrytis from fruit set to maturity; and this was attained by using the modified Biocontrol
Agent Box (BAB). Indeed, the previous studies were correct on stating that the design of
the dispenser affects the results of the treatment.
Additionally, on the number of fruit sets after the flowering of the strawberries,
both the exposed and covered flowers attained 50 out of 50 or 100% fruit sets as recorded.
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
The combined pollination and dissemination of honeybees promoted true-to-type
shape of fruits by 88% and reduced deformities by 69% on strawberry fruits. The data
recorded on the overall harvest was 58% for the exposed samples while 32% was recorded
on the samples secluded from the bees. Based on the disease assessment using Trichoderma
KA to suppress Botrytis cinerea in strawberries, there was an average of 20% increase in
marketable fruits at harvest. However, out of this percentage, the calculated average on the
efficiency of honeybees as disseminator of Trichoderma KA for Botrytis management and
as pollinators of strawberries was 45% increased in the number of true-to-type marketable
fruits at harvest.
Conclusions
The Biocontrol Agent Box (BAB) successfully enables the honeybees to deliver
Trichoderma KA resulting to the significant reduction of fruit rot incidence on the
developing strawberries until harvests that were located as far as 70m away from the hive.
Marketable fruits also significantly increased.
Recommendations
The Biocontrol Agent Box (BAB) can be used in similar studies. The amount of
Trichoderma KA deposited by honeybees to the flower per visit is not known. Further
36
studies are recommended to take a sample of a honeybee loaded with Trichoderma KA at
a certain distance, likewise, to take a sample of the flower which received pollination first
and dissemination of the biocontrol agent.
It is strongly recommended that bee colony density would be relatively high, in
order to achieve saturation pollination and a higher percentage of large, well-formed fruits.
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
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Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
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Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
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WrcYbAnOMN&sa=X&oi=bookresult&ct=result&resnum=5&ved=0CDUQ6AE
wBA#v=onepage&q&f=false.
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
ANAS, JOSEPH C. APRIL 2013. Follow-up Study on the Efficiency of Honeybees
(Apis mellifera Linnaeus) in Disseminating Trichoderma KA to Manage Gray Molds
(Botrytis cinerea Pers) and Pollination in Strawberries (Fragaria x ananassa Duch).
Benguet State University, La Trinidad, Benguet.
Adviser: Lita Molitas – Colting, PhD.
ABSTRACT
The study aimed to: a) determine how far the Trichoderma spores will be
disseminated by Apis mellifera; b) determine if the Trichoderma spores “dropped” by
Trichoderma-laden honeybees are enough to prevent the rotting of the berries; and c)
determine the pollination efficiency of the Trichoderma-laden honeybees. A modified
Trichoderma Dissemination Box was used to contain the Trichoderma.
The honeybees successfully dessiminated the Trichoderma spores to the strawberry
flowers from 20m to 70m away from the hive.
The fruit rot incidence in strawberry fruits was reduced by 58% from fruit setting
to maturity and increased by 45% marketable yield.
There was no significant increase in the number of fruit set between the pollinated
and unpollinated flowers. The Trichoderma spores that were disseminated were effective
in significantly reducing fruit rot incidence by about 60%. However, an average of 20%
disease reduction on marketable fruits was recorded. As a result, a significant increased in
marketable yields by 45% was realized at harvest.
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
RESULTS AND DISCUSSION
Distances by which Honeybees can Disseminate
Trichoderma KA Spores
The treatment was found positive with Trichoderma KA growth. Mycelium and
spore germination was observed from the different isolation done in each treatment.
Honeybees are efficient in disseminating Trichoderma KA at a given distance. In T1 which
has a distance of 20m, colonies of Trichoderma KA were observed. The spores germinated
at surface were surrounded by its mycelium (Fig. 18) while in T2 which has a 40m distance
away from the hive several colonies of Trichoderma KA growth were observed at the sides
of the petriplates. The mycelium bears its spores as it continous to spread on the plated
PDAs (Fig. 19). Moreover, in farther treatments the growth of the Trichoderma KA was
noticed to have a small number of spores than the previous treatments which was closer to
the hive. Spore germination was moderately compact at T3 which has a 60m distance from
the hive (Fig. 20) while in T4 – 70m the mycelium growth of Trichoderma KA was
dominant after incubation (Fig. 21). Indeed, the concentration of Tichoderma KA spores
was observed from 20m to 40m distances away from the hive (Fig. 18 & 19). Based on
these results, honeybees as they flew out to forage in farther distances it is possible that
their was a loss of inocula from the bees resulting to lesser inocula to be deposited to the
strawberry flowers. However, it is still evident that honeybees are efficient in disseminating
the Trichoderma KA at a given distances.
According to Bilu et al. (2004) the amount that a bee deposits on a flower per visit
is not known, but must be considerably less than that found on the bee’s body upon leaving
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
the hive. Inoculum may be lost as the bee flies towards the flowers, and the amount
delivered per flower is diluted by the many flowers that a bee visits per trip.
On the other hand, Saclangan claimed that honeybees can disseminate Trichoderma
up to 30m away from the hive but, the result of this study showed that bees can disseminate
Trichoderma spores from 20m distance and even as much farther as 40m, 60m and 70m
away from the hive (Fig. 18-21).
The findings of Kovach (1999) provides further support to the result of this study
where he proved that bees exiting from the hive on their way to the field, can pick up as
many as 100,000 Trichoderma spores each. As they forage for nectar and pollen, the bees
leave spores of Tichoderma 22 behind and on the flowers. Trichoderma 22 fights the
familiar gray mold known as Botrytis fruit rot by outcompeting the rot, spore for spore.
In addition, the designed Biocontrol Agent Box which was based on Biocontrol
Introduction box (BCAB) enabled the honeybees to disseminate Trichoderma KA spores
five hours after their release at different distances from the hive reaching up to 70m.
The effectiveness of using bees as a biocontrol agent depends on several factors.
Honeybees can disseminate Trichoderma inoculum to strawberry and effectively control
gray mold (Maccagnani et al., 1999; Kovach et al., 2000). The success of the technique,
however, depends on the type of inoculum dispenser (Bilu et al., 2004; Maccagnani et al.,
2005), the Trichoderma strains, the carrier, and the attractiveness of the strawberry cultivar
to the bees (Kovach et al., 2000).
Meanwhile, the occurrence of the contaminants such as fungus and bacteria in the
isolated suspensions were relatively high since the flowers and honeybees cannot be
sterilized and were highly exposed to an open environment.
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
Figure 18. Mycelium and spore germination of Trichoderma KA
isolated from strawberry flowers at 20m distance from the
hive five hours after foraging
Figure 19. Mycelium and spore germination of Trichoderma KA isolated from strawberry
flowers at 40m distance from the hive five hours after foraging
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
Figure 20. Colony growth of Trichoderma KA isolated from
strawberry flowers at 60m distance from the hive five
hours after foraging
Figure 21. Mycelia of Trichoderma KA isolated from strawberry
flowers at 70m distance from the hive five hours of
foraging
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
Evaluation of Trichoderma KA for Botrytis
Management
The result in Table 1 shows the Botrytis cinerea rate of infection in both treatments.
There was no Botrytis infection observed on the tagged sample flowers. However, after the
fruit set, formation of Botrytis cinerea was observed. Symptoms of rotting started on the
developing fruits on both treatments. This corroborates with the findings of Legard et al.
(2005) as cited by Saclangan (2008) showed that Botrytis cinerea Pers causes severe
preharvest losses primarily due to infections of fruits, especially under humid conditions
when daytime temperatures are moderate to warm (60oF to 75oF).
The use of honeybees as an applicator of the Trichoderma KA for the Botrytis
management was evaluated on fruits. Out of the samples, the treatment using honeybees
had a 42% disease incidence from fruit set to maturity while 68% of infected fruit incidence
was recorded on the treatment without using bees. Significantly, the study found a 58%
reduction of disease infection when using bees as disseminators of Trichoderma KA.
It corroborates with the early study of Saclangan (2008) wherein he conducted a
bioassay test on fruits to evaluate the efficacy of Trichoderma Koniingi in suppressing the
disease. A suspension of Botrytis cinerea was sprayed in the sterilized cotton contained in
plastic containers in which the sample fruits, which were dipped on Trichoderma
suspension, were then placed and got incubated for three days. About 56% of the untreated
fruits were infected with the fruit rot disease as compared to the treated fruits with
Trichoderma Koniingi, a total of 33% of the fruits were infected while an average of 67%
disease management reduction on fruit samples.
The disease assessment using honeybees in disseminating Trichoderma KA was
nearly similar to the result of the bioassay test. The established BAB which was modified
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
from the former BCA box was one of the factors that made the study successful in attaining
the percentage reduction of Botrytis cinerea in strawberries.
Previous studies showed that the effectiveness of using bees as a biocontrol agent
depends on several factors. Honeybees can disseminate Trichoderma inoculum to
strawberry and effectively control grey mold (Maccagnani et al., 1999; Kovach et al.,
2000). The success of the technique, however, depends on the type of inoculum dispenser
(Bilu et al., 2004; Maccagnani et al., 2005), the Trichoderma strains, the carrier, and the
attractiveness of the strawberry cultivar to the bees (Kovach et al., 2000).
Table 1. Percentage Botrytis infection on tagged samples from fruit setting to maturity
TREATMENTS FLOWERS FRUITS
Without honeybees
0%
68%
With Honeybees loaded with
0%
42%
Trichoderma KA
Pollination Efficiency of Honeybees
The flowers that were exposed to Apis mellifera produce a true-to-type shape of
strawberries. Sweet Charlie Strawberry Variety has two types of shape – wedge and conical
shape (Fig. 23a & 23b). Pollination and fertilization of the ovules resulted to normal
development of fruits.
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
Number of Fruit Set
The number of fruits that set after the flowers were secluded or exposed to the bees
is shown in Table 2. The number of fruit set in plants secluded from the bees was 50 out
of 50 which has a computed percentage fruit set of 100%. On the other hand, the fruit set
in sample flowers exposed to the bees was 50 which is also equivalent to 100%. The data
showed that there was no difference between the fruit set percentages of both exposed and
covered strawberry flowers. However, in order to achieve complete pollination, self fertile
strawberries require pollinators such as bees to reduce deformities and to attain bountiful
quality of fruits for strawberry production.
The flowers of all the current commercial strawberry cultivars (Fragaria x
ananassa Duch) are hermaphrodite and self-fertile. However, these flowers may not be
completely self-fertilized; thus, the role of bees as pollinators is important in order to
reduce deformities and increase yield for commercial strawberry production (McGregor,
1976).
Table 2. Number of fruit sets and its total percentage
TREATMENTS FRUIT SET PERCENTAGE
Without honeybees
50
100%
With Honeybees loaded with
50
100%
Trichoderma KA
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
Number of Marketable and Deformed Harvest
Table 3 shows the percentage of harvests noted on marketable (Fig. 23a & 23b) and
not marketable fruits (Fig. 23c) on both treatments. Based on the tagged samples, 26%
were deformed (not marketable) and only 6% were normal fruits among all the fruits which
were treated without using the bees. Whereas, a total of 8% were deformed and 50% were
normal fruits among all the fruits treated using honeybees. Basing from these data
percentages on both treatments, there was a 69% calculated reduction of deformities using
honeybees and an average of 88% increase in true-to-type fruits was calculated on the
exposed strawberry samples for pollination.
Honeybees pollinated 11% of achenes on their first visit, so to produce a normal
berry (fertilization rate of 87%) about 11 visits by honeybees/flower are required to achieve
a normal-sized berry (Anonymous, 2013).
This result determines the pollination rate of honeybees in reducing deformities and
promoting a good quality for strawberry production. However this was attained by using
bees as pollinator and with the aid of other pollinators since the field was open for insect
visitation.
Strawberry flowers are hermaphroditic (having both male and female reproductive
organs) and self-fertile and 80% of fruit production is due to abiotic factors such as gravity
and wind; however, pollinating insects play as essential role in obtaining maximum fruit
as well as reducing deformities (Chagnon et al., 1989).
The use of pollinator such as bees is important to improve pollination by increasing
mobility of pollen grains and consequently, ensuring ovule fertilization and achene
development (Leech et al., 2000). Pollination by insects often enhances yield and fruit
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
quality compared with non-pollinated crops. In addition, Chagnon et al. (1989) stated that
honey bees (Apis mellifera L.) are recognized as the main pollinator of the strawberry crop.
Table 3. Percentage of marketable fruits and non-marketable fruits at harvest
TREATMENTS NOT MARKETABLE MARKETABLE
(Deformed Fruits)
Without honeybees
26%
6%
With Honeybees loaded with
8%
50%
Trichoderma KA
Pollination and Dissemination Evaluation
on overall Harvests
The result in Table 4 shows the total harvest that includes the normal, deformed
and the infected fruits in both treatments. Of all the berries exposed to bees in the treatment
process, 58% of the harvests were normal while 24% were infected. On the side of the
berries secluded from the bees, a total of 32% were normal while a total of 30% was
infected with Botrytis cinerea.
Out of these results, the idea of combined pollination and dissemination of
beneficial fungi using bees was evaluated in this study. The calculated total average
increase in marketable fruits out of the treatments was 45% while the use of Trichoderma
KA was proven effective in managing the Botrytis cinerea by a 20% disease reduction in
the harvest. This means that out of 45% marketable harvests, the combined pollination and
dissemination comprises 20% reduction of the disease and increase in percentage harvests.
In relation to the previous study by Lab-oyan in 2001, he noted that pollination alone by
honeybees increased yield by 25%; therefore, the efficiency of honeybees as pollinator and
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
32
disseminator of the Trichoderma KA for Botrytis control increased marketable fruits up to
45%.
Table 4. Percentage of infected and uninfected fruits at harvest
TREATMENT UNINFECTED INFECTED
Without honeybees
32%
30%
With Honeybees loaded with
58%
24%
Trichoderma KA
.
100
100%
80
100 %
80%
60
100%
60%
40
40%
50%
38%
20
30%
20%
6%
26%
18%
24%
0
8%
0%
Category 1
Category 2
Category 3
Category 4
Fruit
Infected Harvest
Deform
Set
with
(Normal)
Infected
Botrytis
with Botrytis
Exposed to hone
ybees (Blue)
Covered with mesh cloth (Red)
Figure 22. Total percentage of normal, deformed and infected strawberries from fruit set
to harvest
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
a
b
c
d
Figure 23. Sample of true-to-type shape of Sweet Charlie Strawberry Fruit a) wedge shape
b) cone shape c) deformed fruit d) infected fruit with Botrytis rot
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
SUMMARY, CONCLUSIONS AND RECOMMENDATIONS
Summary
The study aimed to a) determine how far the Trichoderma spores will be
disseminated by honeybees; b) determine if the Trichoderma spores “dropped” by
Trichoderma-laden honeybees are enough to prevent the rotting of the berries; and c)
determine the pollination efficiency of the Trichoderma-laden honeybees.
The efficiency of the honeybees as a disseminator of the Trichoderma spp. that was
reported by Saclangan (2008) confirms the success of this study. As recommended, the
researcher modified the box prior to the release and assessment of Trichoderma KA in
managing Botrytis cinerea in strawberries.
The isolated dilutions from different distances were positive of Trichoderma KA
growth. Honeybees can disseminate Trichoderma on different distances ranging from 20m,
40m, and 60m up to 70m distances away from the hive.
In relation to the dissemination of the Trichoderma KA for Botrytis management
on the tagged samples, a total of 68% of the berries secluded from bees got infected while
a total of 42% of the berries subjected to bee dissemination were found infected. However,
the calculated percentage of disease reduction from fruit set to maturity was 58%.
Therefore using bees as disseminators of Trichoderma KA reduced the incidence of
Botrytis from fruit set to maturity; and this was attained by using the modified Biocontrol
Agent Box (BAB). Indeed, the previous studies were correct on stating that the design of
the dispenser affects the results of the treatment.
Additionally, on the number of fruit sets after the flowering of the strawberries,
both the exposed and covered flowers attained 50 out of 50 or 100% fruit sets as recorded.
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
The combined pollination and dissemination of honeybees promoted true-to-type
shape of fruits by 88% and reduced deformities by 69% on strawberry fruits. The data
recorded on the overall harvest was 58% for the exposed samples while 32% was recorded
on the samples secluded from the bees. Based on the disease assessment using Trichoderma
KA to suppress Botrytis cinerea in strawberries, there was an average of 20% increase in
marketable fruits at harvest. However, out of this percentage, the calculated average on the
efficiency of honeybees as disseminator of Trichoderma KA for Botrytis management and
as pollinators of strawberries was 45% increased in the number of true-to-type marketable
fruits at harvest.
Conclusions
The Biocontrol Agent Box (BAB) successfully enables the honeybees to deliver
Trichoderma KA resulting to the significant reduction of fruit rot incidence on the
developing strawberries until harvests that were located as far as 70m away from the hive.
Marketable fruits also significantly increased.
Recommendations
The Biocontrol Agent Box (BAB) can be used in similar studies. The amount of
Trichoderma KA deposited by honeybees to the flower per visit is not known. Further
36
studies are recommended to take a sample of a honeybee loaded with Trichoderma KA at
a certain distance, likewise, to take a sample of the flower which received pollination first
and dissemination of the biocontrol agent.
It is strongly recommended that bee colony density would be relatively high, in
order to achieve saturation pollination and a higher percentage of large, well-formed fruits.
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
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Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
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Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013
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http://books.google.com.ph/books?id=vebPZKAJODAC&pg=PA326&lpg=PA32
6&dq=honeybee+disseminating+trichoderma+in+strawberry&source=bl&ots=Ph
s6W3oP7&sig=VdWbsM5hYqGOIsOi31HNggrzxJQ&hl=tl&ei=OZKtTLejBM
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wBA#v=onepage&q&f=false.
Follow-up Study on the Efficiency of Honeybees (Apis mellifera Linnaeus) in Disseminating
Trichoderma KA to Manage Gray Molds (Botrytis cinerea Pers) and Pollination in
Strawberries (Fragaria x ananassa Duch) | ANAS, JOSEPH C. APRIL 2013