BIBLIOGRAPHY CAGA, RICHARD B. APRIL 2012....
BIBLIOGRAPHY
CAGA, RICHARD B. APRIL 2012. Isolation and Evaluation of
IndiginousBiological Control Agents against Coffee Leaf Rust
(Hemilieavastatrix).Benguet State University.
Adviser: Andres A. Basalong, Msc
ABSTRACT
The study was conducted at the Department of Plant Pathology at Benguet State
University, La Trinidad Benguet from November 2011 to March 2012. The study aimed
to Isolate and evaluate indigenous biological control agents against coffee rust for organic
coffee production, and to identify and characterized effective biological Control agents
against rust disease of Arabica coffee.
Result of the study showed that fungi and bacteria were isolated in different
media. Isolates from different media was used in Bioassay in order to evaluate and
characterize the effective Biological Control Agent against coffee rust on organic coffee
production.
Eight fungi and eleven bacteria were isolated from symptomatic coffee plants
that were obtained from coffee plant parts from twigs, leaves and flowers. Only 2 fungi
were identified except for Verticilliums.p. and Trichodermathat was taken from Dr.
Luciana Villanueva and Dr. Asuncion Nagpala. Furthermore, study was continuously
develop in identifying bacterial isolates except for Bacillus subtilis (81.93, 73 and158)
that was taken from Dr. Luciana Villanueva.The fungi identified were (05-F) Fusarium
sp. and (06-F) Penicillium sp.
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust
(Hemilieavastatrix)/ Richard B. Caga. 2012
TABLE OF CONTENTS
Page
Bibliography……………………………..…..……………………………………. i
Abstract……………..…………………...………………………………………….. i
Table of Contents…………………………………………………………………. ii
INTRODUCTION………………………………………………………………… 1
REVIEW OF LITERATURE…………………………………………………….. 3
MATERIALS AND METHODS………………………………………………….… 7
RESULTS AND DISCUSSION…………………………………………………... 12
Isolation and Characterization
Of Biological Control Agents ….………………............................................ 12
a. Fungal Biological Control
Agent Isolates………………………………………………………...……… 12
b. Bacterial Biological Control
Agent Isolates………………………………………………………………… 15
Bioassay on Detached Leaf ………………………………………………… 18
Leaf Disc Assay…………………………………………………………….. 20
a. Number of Lesions on Leaf
Disc (After 15 days)…………………………………………………………... 20
b. Diameter (mm) of Lesions on Leaf
Disc (After 12 days)………………………………………………………… 20
c. Number of Lesions on Leaf
Disc (After 15 days)…………………………………………………………. 21
d. Diameter (mm) of Lesions on
Leaf Disc (After 12 Days…………………………………………………… 22
SUMMARY, CONCLUSION AND RECOMMENDATION……………………. 23
Summary……………………………………………………………………. 23
Conclusion………………………………………………………………….. 23
Recommendation…………………………………………………………… 23
LITERATURE CITED……………………………………………………………. 24
APPENDICES………………………………………………………………………. 26
1
INTRODUCTION
Coffee is one of the most traded agricultural commodities in the world. It is
accepted as the most important brewed beverages due to the stimulating effect on the
human by its caffeine content. It ranks second to water (Pendergrast, 2009).
Coffee belongs to the genus Coffea L. of the family Rubiaceae. Coffee Arabica,
and coffee Canephora known as “Robusta” are now cultivated throughout the coffee
growing countries (Anonymous, 2003).Worldwide, Arabica coffee is the most important
variety. It accounts for 72% of coffee production. Arabica coffee is early bearer, that after
two years of transplanting it produce cherries. When generally managed and fully
growned, one hectare farm could yield 1,000 kilograms of green beans.
The production of coffee in the Philippines according to the National Integrated
RDE Agenda Programs (NIRDEAP) reached an all time high of 61,140 metric tons in the
year 1992 and an all time low of 37,000metric tons in 1998. In fact, such performance of
coffee in our industry was brought by the dry spells caused by El Niño phenomenon.
Coffee leaf rust caused by HemelieavastatrixBerk& Br. on Arabica coffee is one
of the important and classical diseases; it is a major disease that causes economic loss
that has been reported from over fifty coffee growing countries (Bhatet al., 2000).
Biological
control
agents
over coffee leaf rust is viewed as a progressive,
environmentallyand more eco-logically friendly way of control. Use of pesticides to
organismscausing diseases may pose many problems like toxic substances that might
harm human and may essentially providepermanent environmental damage due to
irreversible harmful effect to untargeted organisms and to ecological process. However,
before releasing a biological control agent, it is important to determine its potential for
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
2
the control of rust and conservation of non- target organisms. Hence, the need for the
evaluation of indigenous biological control agents against coffee rust will help the
farmers to maintain the quality of their crops. It will also minimize economic losses,
moreover, contributing to the restoration of biodiversity, and clean air.
The study aimed to:
1. Isolate and evaluate indigenous biological control agents against coffee rust for
organic coffee production.
2. To characterize andidentify effective biological controlagents against rust dis-
ease of Arabica coffee.
The study was conducted at the Department of Plant Pathology Laboratory at
Benguet State University from November 2011 to March 2012.
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
3
REVIEW OF LITERATURE
Climate Requirement of Coffee
Mc Mahon, et al (2002), cited that coffee Arabica requires temperature between
15°C to 24°C; trees withstand temperatures near 0°C (32°F) for only a short period.
Continuously exposure temperature below freezing, trees suffer cold damaged and with
considerable difficulty in recovering. However, temperature above 19.5°C (80°F) will
tend to reduce flowering and fruiting, and temperature below 13°C (55°F) will cause
cessation of growth and tree stunting.
Taxonomy of the Coffee Rust
Coffee leaf rust (Hemilieavastatrix) is one of the most feared pathogens to coffee
growers. It is classified under class Pucciniomycetes, order Pucciniales and family
Pucciniaceae (Conrad, 2009). According to Sangatanan (1986), Hemilieavastatrix is the
causal agent that mostly produces only uredinia stage and is prevalent. However, telial
and basidial are also observed sometimes.
Distribution and Magnitude of Damage
Of Coffee Rust
Coffee leaf rust caused by the fungus Hemelieavastatrixis an obligate parasite,
which occurs worldwide in coffee growing region (Bettencourt and Rodriguez Jr. 1988).
It is a major disease of Arabica coffee causing economic losses that was reported from
over fifty countries including India. In 1869, the fungus appeared in Ceylon now (Sri
Lanka) were it infects the foliage and the young branches, which the fungus was exists in
different physiological forms (Bhatetal., 2000). The importance is largely responsible in
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
4
increasing again when the disease invaded the Latin America countries during 1970-1985
(Bhat, 2005).
Coffee is subjected to various problems that include the important diseases which
result to reduced photosynthetic capacity of infected leaves and premature defoliation
associated with high infection levels; also it reduced the heavy carbohydrate sink created
by fruit limits which serves the amount of growth of woody tissue that gives rise to next
crop season. Thereby, the following season’s crop is reduced. In fact, it is estimated that
the losses due to coffee leaf rust can seriously reach 30 to 80% annually, although 15 %
is more typical (Kushalappa and Eskes,1989).
According to Mitchell (1988), coffee leaf rust was first to destroyed their crops in
Brazil in 1970. The disease and its symptoms were first to observed in Sri Lanka and
Ethiopia, the disease had become globally widespread by wind and rain and spores,
lesions on the underside of plant.
Mechanism of Infection and Symptoms
Brown et al (1995) reported that the urediniospores will penetrate coffee through
stomata and develop powdery orange pustules on the abaxial surface of leaves, resulting
in impaired photosynthesis, premature defoliation, and reduced floral initiation constitute
most of the damage. Arneson (2000), confirmed that Hemilieavastatrix survives primarily
as dikaryotic(having pairs of haploid nuclei that divide in tandem), nutrient absorbing
mycelium in the living tissues of the host. Hyphae are club-shaped with tips bearing
numerous pedicels on with clusters of urediniospores are produced.
The coffee leaf rust was commonly small in shape, yellowish in spots. The
disease appears on the lower surface of leaves that can produce powdery yellow to orange
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
5
spores. Although, die back was been characterize by drying of branches and twigs of the
coffee plant. In severe cases, it can cause the leaves to fall off (Stoll, 2008).
Agrios (1988), cited that the symptoms of the disease are small circular spots,
about 5mm in diameter, which are greenish yellow in the upper surface of the leaf and
yellowish to orange on the leaf’s lower surface. The spots may eventually become dry in
the center of the leaf, turn to brownish and may even leaf galls off prematurely.
Management
Effective management of this major disease is important for sustained
production and productivity of coffee. However, adopting cultural practices, planting
resistance varieties and the application of contact and systemic fungicides are
recommended for control measures (Anonymous, 1998). But in some instances,
continuous use of fungicides may pose on many problems like toxicity to non target
organisms that may involve the development of resistance in pathogen and ground water
pollution (Daivasikamani and Govindarajan, 1989). In recent years they turn to shift in
controlling the plant disease from regular use of pesticides to an alternate and more eco
friendly bio pesticides and plant based production where, many fungi and bacteria has
strains to act as a biological control agents. The indigenous strains of Bacillus subtilis and
Pseudomonas flourescens appear to function as better antagonists in disease control as
they will adapt to local conditions (Hanumanthaet al., 1989).
Rangeshwaran and Prasad (2000) reported that P. flourescens is not much
effective to work on biological agents of coffee leaf rust pathogen. Thus, present
investigation was carried out to assess the antagonistic effect of B. subtilis and P.
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
6
flourescens. Although, bacterialantagonists were tested in vitro in vivo for their
suppressing or controlling effect to the coffee leaf rust pathogen.
Since the fungicides were never used, the Hyperparasitic fungus
Verticilliumhemilieaoccurs quite frequently and able to reduce the rust inoculums under
high humid conditions. Under favorable conditions, thehyper parasite completely covers
rust uredospores and lesion with white mycelia mass. Consequently, uredosores can be
killed through necrosis of the rust lesions. In some instances, these Hyperparasites are
organisms that parasitize other parasitic and are sometimes used as biological agents
(Bhat, 2005).
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
7
MATERIALS AND METHODS
Collection and Isolation of Biological Control
Agents
Arabica plantations in Benguetwere surveyed and randomly sample healthy
anddisease Arabica coffee plants. Sampling was done in month of November to March, a
period where heavy rust infection and leaf fall occur. Every epiphyte found associated to
the coffee tissue specimens and not known as pathogen werepresumed asbeneficial or
biological control agent. These were isolated with nutrient agar (NA) or potato dextrose
agar (PDA). Pure cultures of each isolated biologicalcontrol agents were maintained
forbioassay activities and characterization.
Cultural and Morphological Characterization
The isolated biological control agents were characterized in terms of the colony
growth and development in culture media. The isolates were sampled, mounted in slides
andexamined under the microscope to characterize the appearances of their microscope
structures.
Laboratory Bioassay
Two techniques of leaf bioassay were done, the detached leaf and leaf disc
assay.Separate trials were done for fungal biological controlagents from the bacterial
biological control agent isolates.
Detached Leaf Assay. Healthy leaves of Arabica coffee were collected, washed
with sterile distilled water, blot dried and placed in transparent polyethylene bags. The
leaves were inoculated with dilutions of coffee rust uredospores, followed by spray of
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
8
biological control agents suspension, enclosed by tying the openings and incubated for 48
hours. Observations of symptom development in terms of number and size of lesions
were done.
Bioassay trials detached leaves in the laboratory were laid in the completely
randomized design (CRD) with three replications.
The treatments for the detached leaf assay were:
T1 - Kocide
T2 - Control (Plain H20)
T3
-
01-F
T4 - 01-B
T5 - Verticillium sp.
T6 - Trichoderma sp.
Leaf Disc Assay.The leaves were washed with sterile distilled water and then cut
into pieces toobtain leaf discs at size of 2.5x2.5cm. The leaf discs wereplaced in sterile
Petri dishthat wereoverlaid with wet tissue paper, inoculated with dilutions of coffee rust
uredospores, and subsequently sprayed with suspensions of biological control agents. The
treated leaf discs were incubated for48 hours. Developments of uredospores from 48
hours of incubation were monitored up to when the leaves were still green, and when
uredospores were visible.
Bioassaytrials on leaf discs werelaid in the completely randomized design (CRD)
with three replications.
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
9
Biological control agentisolates as treatment were:
Bacterial Biological ControlAgent Isolates Fungal Biological ControlAgent Isolates
T1-01-B
T1-Kocide (control)
T2- 02-B
T2-01-F
T3-03-B
T3-02-F
T4-04-B
T4-04-F
T5-05-B
T5-05-F (Fusarium sp.)
T6-06-B
T6-06-F(Penicillium sp.)
T7-07-B
T7-Verticillium sp.
T9-31 (Bacillussp.)
T8-Trichoderma sp.
T10-73(Bacillus sp.)
T9-Trichoderma sp.
T11-94 Flavobacterium
sp.
T10-Kocide
T12-158 Pseudomonas sp.
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
10
Figure 1.
Experimental set- up on Detached Leaf Assay
Figure2. Experimental set- up for Leaf Disc Assay
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
11
Data Gathered were:
1.
Isolate as biological control agents. Collection of diseased and healthy
coffee tissues was diagnosis to explore the presence of biological control agents against
coffee rust.
2.
Number of germinated and ungerminated spores of coffee rust (leaf
discand detached leaf bioassay).These were recorded starting from 48 hours after
inoculation. Lesions diameter were measured in millimeters.
3.
Number and diameter of lesions on leaf disc and detached leaf.Inhibition
of the biological control agents on the developments of the uredospores were assessed 15
days after inoculation.
4.
Morphological and Cultural Characteristics of the Isolates. The cultural
characterization was done by observing the colony growth and development on the
culture media, while the morphological characterization was done through microscopic
observations of the structures of the isolates.
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
12
RESULTS AND DISCUSSION
Isolation and Characterization
ofBiological controlAgents
Fungal Biological Control AgentIsolates. There were six fungal biological control
agents isolated and characterized. Most isolates in terms of cultural characteristics,grown
in PDA had a light orange to orange pigment while other isolates produced yellow bluish
green, light brown and white pigment in culture (Isolates 1 to 6). For morphological cha-
racteristics observed under microscope, most isolates revealed on oblate conidia (Table
1).
Table 1.Cultural and Morphological of Fungal BiologicalControl Agents.
ISOLATES
Fusarium Penicillium
CRITERIA 01-F 02-F 03-F
04-F sp. (05-F) sp. (06-F)
Fast
Slow
Slow
Slow grow- Slow
Fast grow-
Growth
growing growing growing
ing
growing
ing
From
white to
Green
Colony col-
Light
Light
or Orange
yellow
orange White Orange
Presence of
septa on
Present
mycelim Present Present Present
Present
Present
Macro-
Round
sickle
Round
Shape of
& ob-
Micro-
conidia
late oblate
oblate Rectangular oblate
Hyaline
Color of
conidia Hyaline
Hyaline
Hyaline
Hyaline Hyaline
Size of con- 1.75-5.5 1.75-3.0 2.0-5.25
1.75-3.75
idia
µm
µm
µm
µm
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
13
Figure 3. Isolate 01-F Colony (Left) Structures (Right)
Figure 4. Isolate 02-F Colony (Left) Structures (Right)
Figure 5. Isolate 03-F Colony (Left) Structures (Right)
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
14
Figure 6.Isolate 04-F Colony (Left) Structures (Right)
Figure 7.Fusarium sp. colonyIsolate
Figure 8.Penicillium sp. colon
yIsolate
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
15
Bacterial Biological Control Agent Isolates. Thebacterialbiological controlagents
were isolated in nutrient broth (NA). Pure cultures obtained were subjected to cultural
and morphological characterization. Cultural characteristics of bacterial isolates that was
grown in NA had a flat, irregular, and white in terms of their form, elevation, and color,
while other isolated produced with wavy, lobate and entire margins (Isolates 1 to 7). For
morphological characteristics, microscopic examination revealed to rod shaped, cylin-
drical and cocci shaped like (Table 2).
Table 2. Characteristics of Bacterial Biological ControlAgent Isolates
GRAM
ISOLATES FORM ELEVATION MARGIN
COLOR SHAPE (+\\-)
wavy
White (shi-
01-B Irregular
Flat
(undulate)
ny) rod -
wavy
White (shi-
02-B Irregular
Flat
(undulate)
ny) rod +
03-B Irregular
Flat lobate
transparent
rod +
wavy
Yellow
04-B Circular
Convex
(undulate)
(shiny) rod -
05-B Circular
Convex
Entire
white
cocci
-
06-B Circular
Umbonate
lobate
white rod -
07-B Circular
convex
Entire
white
cylindrical
-
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
16
Figure 9. Isolate 01-B Colonny (Left) Structures (Right)
Figure 10. Isola
te 02-B Colony (Left) Structures
Figure 11. Isolate 03-B Colony (Left) Structures
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
17
Figure 12. Isolate 04-B Colony (Left) Structures
Figure 13. Isola
ate 05-B Colony (Left) Structures
Figure 14. Isola
te 06-B Colony (Left) Structures
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
18
Figure 15. Isolate 07-B Colony (Left) Structures
Bioassay on Detached Leaf
Percentage spore germination inhibition in Table 3 shows the reaction of coffee
rust to the biological agent.The control which is plain water had the highest percentage of
spore germination inhibition with a mean of 99.6,while the lowest was from those treated
with kocidewith a spore germination inhibition of 87.1.No significant difference was
recorded in the percent inhibition of sporegermination on detached leaf.
Length of germ tube (µm) Table 3 shows that the length of germ tube of
germinated spores. The highest germ tube length of 3.7µm was obtained from those
treated with kocide and 01-F, and the lowest mean of 2.2 µm was obtained from those
treated with 01-B. Statistically there were no significant difference among the treatments
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
19
Table 3.Reaction of Coffee Rust of Biological Control Agent Against on Detached Leaf.
TREATMENTS
SPORE
LENGTTH
OF
GERM
GERMINATION
TUBE (µm)
INHIBITION
(%)
Actual
Transformed
Kocide
87.1
3.67
3.7
Control
(Plain
H20)
99.6
3.23
3.2
01-F
98.3
3.72
3.7
01-B
78.4 2.22
2.2
Verticillium sp.
98.3 2.41
2.4
Trichoderma sp.
98.2 2.52
2.5
Number of lesions on Detached Leaf (After 18 days) eighteen days after
inoculation, the number of lesions on detached leaf was counted. Most of the isolates,
plain water and kocide treatments had a lesion of which is different from those treated
with 01-F with a mean of 2. (Table 4) reveals that there were no significant differences in
the number of lesions after eighteen days of inoculation.
Diameter of lesions on detached leaf (After 18 days).The diameter of lesions on
detached leaf revealed that the highest was 2.1mm that was obtained from 01-F, and the
lowest with a mean of 1.2 mm were Verticillium sp. There were no significant differences
among the treatments (Table 4).
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
20
Table 4.Reaction of Coffee Rust of Biological Control Agent Against on Detached Leaf.
TREATMENTS
NO. OF LESIONS
DIAMETER OF LESIONS (mm)
Actual
Transformed
Actual Transformed
Kocide
0.33
1.00
1.33
1.2
Control
1.00
1.00
3.33
1.7
(Plain H20)
01-F
4.67
2.00
5.00
2.1
01-B
1.00
1.00
2.33
2.0
Verticillium sp.0.66
1.00
1.33
1.2
Trichoderma sp.1.33
1.00
2.67
1.5
Leaf Disc Bioassay
Number of lesions on Leaf disc (After 15 days)
Table 5 shows the number of lesion on leaf disc assay after 15 days of
inoculation. Treated leaf disc with 04-, 06-B, 07-B, 73, 94 and 158 had the same means
of 2.0, and mean of 1.50 was obtained from those treated with, 01-B, 02-B, 03-B, 05-B
and 31 isolates including the control. There was no significant difference.
Diameter (mm) of Lesions on Leaf disc (After 12 days)
The diameter of lesions on leaf disc is shown on table 5. The highest was obtained
from those treated with 03-B isolates with a mean of 2.79mm and the lowest was from
those treated with 01-B isolate with a mean of 1.41. Statistical analysis revealed no
significant differences.
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
21
Table 5. Effect of Bacterial BiologicalControlAgentsAgainst Coffee Rust on Leaf Disc
Assay
TREATMENTS
NO. OF LESIONS
DIAMETER OF LESIONS
(mm)
Control
(Plain
water)
1.5
2.04
01-B
(Bacteria
) 1.5
1.41
02-B
(Bacteria) 1.5
2.33
03-B
(Bacteria)
1.5
2.79
04-B
(Bacteria) 2.0
2.21
05-
B
(
Bacteria)
1.5
2.29
06-B
(Bacteria) 2.0
2.04
07-B
(Bacteria) 2.0
2.0
31(Bacillussp.) 1.5
2.58
73(Bacillus sp.) 2.0
1.71
94 Flavobacterium sp.
2.0
1.79
158 Pseudomonas sp.
2.0
2.04
Number of Lesions on Leaf disc (After 15 days)
Table 6 shows that the highest number of lesion on leaf disc was obtained from
isolate 02-F with a mean of 2.11 followed by Verticillium sp. with a mean of 1.83 and the
lowest were from Trichoderma sp. with a mean of 1.20. However, statistical analysis
revealed no significant differences on the number of lesions on leaf disc after 15 days of
inoculating.
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
22
Diameter of Lesion on Leaf disc (After12 days)
Twelve days of inoculation, the diameter of lesions on leaf disc was counted. The
04-F has the highest diameter of lesion on leaf disc with a mean of 2.67 and kocide has
the least mean diameter of lesion on leaf disc with a 1.46. In addition, there was a highly
significant difference in the mean of the diameter of the lesion leaf in terms of statistical
analysis (Table 6).
Table 6. Effect of Final Biological Control Agent Against Coffee Rust on Leaf disc
TREATMENTS
NO. OF LESIONS
DIAMETER
OF
LESIONS
(mm)
Control
1.33
1.58c
01-F
1.55
1.71b
02-F
2.11
2.33a
03-F
1.37
2.42a
04-F
1.47
2.67a
05-F (Fusarium sp.)
1.63
2.6a
06-F(Penicillium
sp.)
1.43
2.1a
Verticillium sp.
1.83
2.12a
Trichoderma sp.
1.20
2.37a
Kocide
1.37
1.46c
Means with the same are not significantly different at 5 DMRT
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
23
SUMMARY, CONCLUSION AND RECOMMENDATION
Summary
The study was conducted at the Department of Plant Pathology Laboratory at
Benguet State University, La Trinidad, Benguet from November 2011 to March 2012.
The study aimed to isolate and evaluate indigenous biological control agent against cof-
fee rust for organic Arabica coffee production, and to characterize and identify and effec-
tive biological control agent against rust disease of Arabica coffee.
There were 8 fungi and eleven bacteria isolated and presumed as Biologicalcon-
trol Agents Against the coffee rust. Two of the fungal isolates were identified as Fusa-
rium sp. and Penicilliumsp.Two of the bacterial isolates were identified as Bacillus
strains, a Flavobacteriumand Pseudomonas sp. isolate.
Conclusion
Result of the bioassays on leaf discs and detached leaves were generally not sig-
nificantly different. The data on the effectively of the fungal and bacterial isolates as bio-
logicalcontrol agents against coffee rust not conclusive.
Recommendation
Based on the findings of the study, the following are recommended:
1. Evaluation of the isolated biologicalcontrol agent against coffee rust shall
be done on seedlings.
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
24
LITERATURE CITED
AGRIOS, G . N. 1988. Plant Pathology 3rd ed. University of Florida, Gainesville. Pp.466
– 488.
ANONYMOUS. 2003. Coffee guide. Central coffee.Research institute, Chickmagalur,
Karnataka, India.P. 200.
ANONYMOUS. 1998. A compendium on pests and diseases of coffee and their
managemen in India. Coffee board research department, central coffee research
institute,Chickmagalur,Karnataka, India. P.67.
ARNESON, P. A. 2000. Coffee rust.The plant health instructor. DOI:10.1094/PHI-1-
2000-0718-02.Retrieved August 13, 2011
fromhttp://www:apsnetorg/edcenter/in/coffee rust.aspx.
BHAT, S. S. 2005. Integrated management of coffee leaf rust. Retrieved August 13,2011:
fromhttp://india coffee. Org/new letter/2005/apr/cover-story-2.Html.
BHAT, S. S., NAIDU. R.S., DAIVASIKAMANI. and NIRMALA KANNAN. 2000.
Integrated disease management in coffee. In: IPM system in agriculture. Cash
crops.Vol./
6.
BETTENCOURT, A. J. and C. J. RODRIGUEZ. 1988. Principles and practice of coffee
breeding forresistance to rust and other diseases. In: CLARKE R.J. , MACRAER,
eds, coffee: London : EL Sevier applied science. Pp. 199-234.
BROWN, J., WHAN, J., KENNY, M. and P. MERRIMAN. 1995. The effect of coffee
leaf rust onfoliation and yield of coffee in Papua New Guinea. Crop
protection.14(7);589-592.
CONRAD, J. 2009. Rust fungi. The Backyard Nature RetrievedAugust 15, 2011 from
http://www. Backyard nature.Net/f/rusts.html.
DAIVASIKAMANI, S. and T. S. GOVINDARAJAN. 1989. Field efficacy of a new
systemic fungicide PP 523 5% SC (Hexaconazole) research. 19: 65-70.
HANUMANTHA, B. T., GOVINDARAJAN, T. S. and NIRMALA KANNAN. 1989.
Comparative efficacy of four fungicides for control of coffee rust. Journal of
Coffee
Research. 19:57-64Retrieved August 15, 2011 fromhttp://en.
Wikipedia.Org/wiki/coffee
KUSHALAPPA, A. C., A. B. ESKES. 1989. Advances in coffee rust research. Ann. Rev.
Phytopathol.27:503-31.
MITCHELL, H. W. 1988. Cultivation and Harvesting of the Arabica Coffee Tree:
Agronomy. Ed. R. J CLARKE. New York:ElSeveir Applied Science.
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
25
MC MAHON, M. J., KOFRANEK, A. M., RUBATZKY, V. E. 2002. Hartmann’s plant
science.Growth, developments and utilization of cultivated plants 3rd ed. Upper,
Saddle River, New Jersey. Pp325.
PENDERGRAST, M. 2009.Tea and Coffee Trade Journal. Retrieved December 29, 2009
from http://en.wikipedia.org/wiki/Coffee
RANGESHWARAN, R. and R. D. PRASAD, 2000. Isolation and evaluation of
rhizospheric bacteria for biological control of chickpea wilt pathogens. Journal of
biological control, 14:9-15.
SANGATANAN, P. D. 1986. Agricultural arts 4.Home economics and livelihood
technology services. School graduate studies, West Visayas State College, Ilo-
ilocity. P.64.
STOLL, G. 2008. Workshop on demand workshop on module.Integrating OISAT into
agricultural training and ext. services.
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
26
APPENDICES
APPENDIX TABLE 1.Actual Percentage (%) Inhibition of Spores Germination
On Detached Leaf (After 48 hrs)
TREATMENTS REPLICATION ___
І
ІІ
ІІІ TOTAL MEAN
T˳Kocide
96.39
81.47
83.54
261.4
87.13
Tı
Control
99.46
99.68
99.55
298.69
99.56
T201-
F 98.87
98.91
97.15
294.93
98.31
T302- F
97.54
39.19 98.4 235.14
78.38
T4 Verticillium sp.
98.15
98.39
98.25
294.79
98.26
T5Trichoderma sp.
98.41
96.77
99.44
294.62
98.21
TOTAL 196.56
514.41
576.33
1679.57 559.86
ANOVA TABLE
SV DF
SS MS
FC
TABULATED
F
5%
1%
Treatment 5
1121.32
224.26 1.10ns 3.11 5.06
Error
12
2440.13
203.34
TOTAL
17
3561.45
*ns= not significant at 5% level
CV = 15.28%
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
27
APENDIX TABLE 2.Actual Length of Germ tubes of Germinated Spores on Detached
Leaf (µm) after 48 hours.
TREATMENT S
REPLICATIONS
I
II
III
T
M
T0Kocide 6
29
9
44
14.66
T1 Control
9
9
12
30
10.00
T2 01-F
3
36
10
49
16.33
T3 01-B
2
5
7
14
4.67
T4Verticillium sp.
10
4
3
17
5.67
T5Trichoderma sp.
9
5
4
18
6.00
TOTAL
39
88
45
172
57.33
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
28
APENDIX TABLE 2.1Transformed Length of Germ tubes of Germinated Spores on
Detached Leaf (µm) after 48 hours.
TREATMENT S
REPLICATION
I
II
III
T
M
T0Kocide 2.55
5.43
3.08
11.06
3.67a
T1 Control
3.08
3.08
3.54
9.7
3.23a
T2 01-F
1.87
6.04
3.24
11.15
3.72a
T3 01-B
1.58
2.35
2.74
6.67
2.22a
T4Verticillium sp.
3.24
2.12
1.87
7.23
2.41a
T5Trichoderma sp.
3.08
2.35
2.12
7.55
2.52a
TOTAL
15.4
21.37
16.39
53.36
17.79
ANOVA TABLE
SV DF
SS MS
FC
TABULATED
F
5%
1%
Treatment 5
371.78 74.36 0.91ns 3.11 5.06
Error
12
978.66
81.56
TOTAL
17
1350.44
*ns= not significant at 5% level
CV = 94.46%
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
29
APPENDIX TABLE 3.Actual Number of Rust Lesion on Detached Leaf Assay(After 18
days)
TREATMENT S
REPLICATIONS
I
II
III
T
M
0 0 1 1 0.33
T0Kocide
0 2 1 3 1.00
T1 Control
0 5 9 14
4.67
T2 01-F
0 1 2 3 1.00
T3 01-B
0 0 2 2 0.66
T4Verticillium sp.
4 0 0 4 1.33
T5Trichoderma sp.
TOTAL
4
8
15
27
9
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
30
APPENDIX TABLE 3.1TransformedNumber of Rust Lesion on Detached Leaf As-
say(After 18 days)
TREATMENT S
REPLICATIONS
I
II
III
T
M
T0Kocide 0.71
0.71
1.22
2.64
.88
T1 Control
0.71
1.58 1.22
3.51 1.17
T2 01-F
0.71
2.35 3.08
6.14 2.05
T3 01-B
0.71
1.22 1.58
3.51 1.17
T4Verticillium sp.
0.71
0.71 1.58
3.00 1.00
T5Trichoderma sp.
2.12
0.71
0.71
3.54
1.18
TOTAL
5.67
7.28
9.39
22.34
7.44
ANOVA TABLE
SV DF
SS MS
FC
TABULATED
F
5%
1%
Treatment 5
37.83 7.57 1.55ns 3.11 5.06
Error
12
58.67
4.89
TOTAL
17
96.50
*ns= not significant at 5% level
CV = 39.25%
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
31
APPENDIX TABLE 4.Actual Diameter (mm) of Rust Lesions on Detached Leaf Assay
(After 18 days)
TREATMENTS
REPLICATIONS
I
II
III
T
M
T0Kocide 0
0
4
4
1.33
T1 Control
0 8 2 10
3.33
T2 01-F
0 7 8 15
5.00
T3 01-B
0
2 5 7
2.33
T4Verticillium sp.
0 0 4 4 1.33
T5Trichoderma sp.
8 0 0 8 2.67
8
17
23
48
16
TOTAL
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
32
APPENDIX TABLE 4.1TransformedDiameter (mm) of Rust Lesions on Detached Leaf
Assay (After 18 days)
TREATMENTS
REPLICATIONS
I
II
III
T
M
T0Kocide 0.71
0.71
2.12
3.54
1.18
T1 Control
0.71 2.92 1.58 5.21 1.74
T2 01-F
0.71 2.74 2.92 6.37 2.12
T3 01-B
0.71
1.58 2.35 4.64 1.55
T4Verticillium sp.
0.71 0.71 2.12 3.54 1.18
T5Trichoderma sp.
2.92 0.71 0.71 4.34 1.45
6.47
9.37
11.8
27.64
9.21
TOTAL
ANOVA TABLE
SV DF
SS MS
FC
TABULATED
F
5%
1%
Treatment
5 28.67
5.73
0.46ns 3.11 5.06
Error
12 149.33
12.44
TOTAL
17
178.00
*ns= not significant at 5% level
CV = 132.10 %
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
33
APPENDIX TABLE 5.ActualNumber of Lesions on Leaf Disc using Bacterial Iso-
lates(After 15 days).
TREATMENTS REPLICATION
I
II
T
M
T1 Control
2 1 3
1.5
T2 01-B
1 2 3
1.5
T3 02-B
2
2
4
2
T4 03-B
1
2
3
1.5
T5 04-B 2
2
4
2
T6 05-B 1
2
3
1.5
T7 06-B 2
2
4
2
T8 07-B 3
1
4
2
T9 31
2
1
3
1.5
T10 73
2
2
4
2
T11 94 2
2
4
2
T12 158
1 3 4
2
TOTAL
21
22
43
21.5
ANOVA TABLE
SV DF
SS
MS
FC
TABULATED
F
5%
1%
Treatment
11 .91
0.08
0.17ns 2.72 4.22
Error
12 5.76
0.48
TOTAL
23
6.67
*ns= not significant at 5% level
CV = 43.57 %
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
34
APPENDIX TABLE 6.Actual Diameter (mm) of Lesions on Leaf Disc using Bacterial
Isolates(After 12 days).
TREATMENTS
REPLICATION
I
II
T
M
T1 Control
1.75 2.33 4.08 2.04
T2 01-B
1.67 1.16 2.83 1.41
T3 02-B
3.08
1.58
4.66
2.33
T4 03-B
2.58
3.0
5.58
2.79
T5 04-B 2.83
1.58
4.41
2.21
T6 05-B 2.58
2.0
4.58
2.29
T7 06-B 2.33
1.75
4.08
2.04
T8 07-B 2.58
1.41
3.99
2.0
T9 31 2.75
2.41
5.16
2.28
T10 73
1.58
1.83
3.41
1.71
T11 94 1.58
2.0
3.58
1.79
T12 158
2.08 2.0 4.08 2.04
TOTAL
27.39
23.05
50.44
25.22
ANOVA TABLE
SV DF
SS MS
FC
TABULATED
F
5%
1%
Treatment
11 3.10 0.28
Error
12 3.49 0.29
0.97ns
2.72 4.22
TOTAL 23 6.59
*ns= not significant at 5% level
CV =25.64 %
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
35
APPENDIX TABLE 7.Actual Number of Lesions on Leaf Disc using Fungal Isolates
(After 15 days)
TREATMENTS
REPLICATION
I
II
T
M
T1 Control
1 1 3
1
T2 01-F
2 1 3
2
T3 02-F
2
3
4
2
T4 03-F
1
1
3
1
T5 04-F 1
2
3
1
T6 05-F 1
2
3
2
T7 06-F 1
2
3
1
T8 Verticillium sp. 2 1 4
2
T9 Trichoderma sp. 1 1 3
1
T10Kocide 1
2
3
1
13
16
29.5
14.5
TOTAL
ANOVA TABLE
SV DF
SS MS
FC
TABULATED
F
5%
1%
Treatment
9 1.32
0.15
Error
10 2.36
0.24
0.62ns
3.02 4.95
TOTAL
19
3.68
*ns= not significant at 5% level
CV = 32.02%
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
36
APPENDIX TABLE 8.Actual Diameter (mm) of Lesions on Leaf Disc using Fungal Iso-
lates (After 12 days)
TREATMENTS
REPLICATION
I
II
T
M
T1 Control
1.41 1.75
3.16
1.58
T2 01-F
1.66 1.75
3.41
1.71
T3 02-F
2.37
2.33
4.66
2.33
T4 03-F
2.58
2.25
4.83
2.42
T5 04-F 2.42
2.91
5.33
2.67
T6 05-F 2.62
2.58
5.2
2.6
T7 06-F 1.75
2.42
4.17
2.1
T8 Verticillium sp. 2.33
1.91 4.24 2.12
T9 Trichoderma sp. 2.16
2.58 4.74
2.37
T10Kocide 1.33
1.58
2.91
1.46
TOTAL
20.63
22.06
42.69
21.35
ANOVA TABLE
SV DF
SS MS
FC
TABULATED
F
5%
1%
Treatment
9 3.25
0.36
Error
10
0.67
0.07
5.14**
3.02 4.95
TOTAL
19
3.92
**= Highly significant at 1% level
CV
=
12.42%
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
CAGA, RICHARD B. APRIL 2012. Isolation and Evaluation of
IndiginousBiological Control Agents against Coffee Leaf Rust
(Hemilieavastatrix).Benguet State University.
Adviser: Andres A. Basalong, Msc
ABSTRACT
The study was conducted at the Department of Plant Pathology at Benguet State
University, La Trinidad Benguet from November 2011 to March 2012. The study aimed
to Isolate and evaluate indigenous biological control agents against coffee rust for organic
coffee production, and to identify and characterized effective biological Control agents
against rust disease of Arabica coffee.
Result of the study showed that fungi and bacteria were isolated in different
media. Isolates from different media was used in Bioassay in order to evaluate and
characterize the effective Biological Control Agent against coffee rust on organic coffee
production.
Eight fungi and eleven bacteria were isolated from symptomatic coffee plants
that were obtained from coffee plant parts from twigs, leaves and flowers. Only 2 fungi
were identified except for Verticilliums.p. and Trichodermathat was taken from Dr.
Luciana Villanueva and Dr. Asuncion Nagpala. Furthermore, study was continuously
develop in identifying bacterial isolates except for Bacillus subtilis (81.93, 73 and158)
that was taken from Dr. Luciana Villanueva.The fungi identified were (05-F) Fusarium
sp. and (06-F) Penicillium sp.
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust
(Hemilieavastatrix)/ Richard B. Caga. 2012
TABLE OF CONTENTS
Page
Bibliography……………………………..…..……………………………………. i
Abstract……………..…………………...………………………………………….. i
Table of Contents…………………………………………………………………. ii
INTRODUCTION………………………………………………………………… 1
REVIEW OF LITERATURE…………………………………………………….. 3
MATERIALS AND METHODS………………………………………………….… 7
RESULTS AND DISCUSSION…………………………………………………... 12
Isolation and Characterization
Of Biological Control Agents ….………………............................................ 12
a. Fungal Biological Control
Agent Isolates………………………………………………………...……… 12
b. Bacterial Biological Control
Agent Isolates………………………………………………………………… 15
Bioassay on Detached Leaf ………………………………………………… 18
Leaf Disc Assay…………………………………………………………….. 20
a. Number of Lesions on Leaf
Disc (After 15 days)…………………………………………………………... 20
b. Diameter (mm) of Lesions on Leaf
Disc (After 12 days)………………………………………………………… 20
c. Number of Lesions on Leaf
Disc (After 15 days)…………………………………………………………. 21
d. Diameter (mm) of Lesions on
Leaf Disc (After 12 Days…………………………………………………… 22
SUMMARY, CONCLUSION AND RECOMMENDATION……………………. 23
Summary……………………………………………………………………. 23
Conclusion………………………………………………………………….. 23
Recommendation…………………………………………………………… 23
LITERATURE CITED……………………………………………………………. 24
APPENDICES………………………………………………………………………. 26
1
INTRODUCTION
Coffee is one of the most traded agricultural commodities in the world. It is
accepted as the most important brewed beverages due to the stimulating effect on the
human by its caffeine content. It ranks second to water (Pendergrast, 2009).
Coffee belongs to the genus Coffea L. of the family Rubiaceae. Coffee Arabica,
and coffee Canephora known as “Robusta” are now cultivated throughout the coffee
growing countries (Anonymous, 2003).Worldwide, Arabica coffee is the most important
variety. It accounts for 72% of coffee production. Arabica coffee is early bearer, that after
two years of transplanting it produce cherries. When generally managed and fully
growned, one hectare farm could yield 1,000 kilograms of green beans.
The production of coffee in the Philippines according to the National Integrated
RDE Agenda Programs (NIRDEAP) reached an all time high of 61,140 metric tons in the
year 1992 and an all time low of 37,000metric tons in 1998. In fact, such performance of
coffee in our industry was brought by the dry spells caused by El Niño phenomenon.
Coffee leaf rust caused by HemelieavastatrixBerk& Br. on Arabica coffee is one
of the important and classical diseases; it is a major disease that causes economic loss
that has been reported from over fifty coffee growing countries (Bhatet al., 2000).
Biological
control
agents
over coffee leaf rust is viewed as a progressive,
environmentallyand more eco-logically friendly way of control. Use of pesticides to
organismscausing diseases may pose many problems like toxic substances that might
harm human and may essentially providepermanent environmental damage due to
irreversible harmful effect to untargeted organisms and to ecological process. However,
before releasing a biological control agent, it is important to determine its potential for
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
2
the control of rust and conservation of non- target organisms. Hence, the need for the
evaluation of indigenous biological control agents against coffee rust will help the
farmers to maintain the quality of their crops. It will also minimize economic losses,
moreover, contributing to the restoration of biodiversity, and clean air.
The study aimed to:
1. Isolate and evaluate indigenous biological control agents against coffee rust for
organic coffee production.
2. To characterize andidentify effective biological controlagents against rust dis-
ease of Arabica coffee.
The study was conducted at the Department of Plant Pathology Laboratory at
Benguet State University from November 2011 to March 2012.
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
3
REVIEW OF LITERATURE
Climate Requirement of Coffee
Mc Mahon, et al (2002), cited that coffee Arabica requires temperature between
15°C to 24°C; trees withstand temperatures near 0°C (32°F) for only a short period.
Continuously exposure temperature below freezing, trees suffer cold damaged and with
considerable difficulty in recovering. However, temperature above 19.5°C (80°F) will
tend to reduce flowering and fruiting, and temperature below 13°C (55°F) will cause
cessation of growth and tree stunting.
Taxonomy of the Coffee Rust
Coffee leaf rust (Hemilieavastatrix) is one of the most feared pathogens to coffee
growers. It is classified under class Pucciniomycetes, order Pucciniales and family
Pucciniaceae (Conrad, 2009). According to Sangatanan (1986), Hemilieavastatrix is the
causal agent that mostly produces only uredinia stage and is prevalent. However, telial
and basidial are also observed sometimes.
Distribution and Magnitude of Damage
Of Coffee Rust
Coffee leaf rust caused by the fungus Hemelieavastatrixis an obligate parasite,
which occurs worldwide in coffee growing region (Bettencourt and Rodriguez Jr. 1988).
It is a major disease of Arabica coffee causing economic losses that was reported from
over fifty countries including India. In 1869, the fungus appeared in Ceylon now (Sri
Lanka) were it infects the foliage and the young branches, which the fungus was exists in
different physiological forms (Bhatetal., 2000). The importance is largely responsible in
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
4
increasing again when the disease invaded the Latin America countries during 1970-1985
(Bhat, 2005).
Coffee is subjected to various problems that include the important diseases which
result to reduced photosynthetic capacity of infected leaves and premature defoliation
associated with high infection levels; also it reduced the heavy carbohydrate sink created
by fruit limits which serves the amount of growth of woody tissue that gives rise to next
crop season. Thereby, the following season’s crop is reduced. In fact, it is estimated that
the losses due to coffee leaf rust can seriously reach 30 to 80% annually, although 15 %
is more typical (Kushalappa and Eskes,1989).
According to Mitchell (1988), coffee leaf rust was first to destroyed their crops in
Brazil in 1970. The disease and its symptoms were first to observed in Sri Lanka and
Ethiopia, the disease had become globally widespread by wind and rain and spores,
lesions on the underside of plant.
Mechanism of Infection and Symptoms
Brown et al (1995) reported that the urediniospores will penetrate coffee through
stomata and develop powdery orange pustules on the abaxial surface of leaves, resulting
in impaired photosynthesis, premature defoliation, and reduced floral initiation constitute
most of the damage. Arneson (2000), confirmed that Hemilieavastatrix survives primarily
as dikaryotic(having pairs of haploid nuclei that divide in tandem), nutrient absorbing
mycelium in the living tissues of the host. Hyphae are club-shaped with tips bearing
numerous pedicels on with clusters of urediniospores are produced.
The coffee leaf rust was commonly small in shape, yellowish in spots. The
disease appears on the lower surface of leaves that can produce powdery yellow to orange
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
5
spores. Although, die back was been characterize by drying of branches and twigs of the
coffee plant. In severe cases, it can cause the leaves to fall off (Stoll, 2008).
Agrios (1988), cited that the symptoms of the disease are small circular spots,
about 5mm in diameter, which are greenish yellow in the upper surface of the leaf and
yellowish to orange on the leaf’s lower surface. The spots may eventually become dry in
the center of the leaf, turn to brownish and may even leaf galls off prematurely.
Management
Effective management of this major disease is important for sustained
production and productivity of coffee. However, adopting cultural practices, planting
resistance varieties and the application of contact and systemic fungicides are
recommended for control measures (Anonymous, 1998). But in some instances,
continuous use of fungicides may pose on many problems like toxicity to non target
organisms that may involve the development of resistance in pathogen and ground water
pollution (Daivasikamani and Govindarajan, 1989). In recent years they turn to shift in
controlling the plant disease from regular use of pesticides to an alternate and more eco
friendly bio pesticides and plant based production where, many fungi and bacteria has
strains to act as a biological control agents. The indigenous strains of Bacillus subtilis and
Pseudomonas flourescens appear to function as better antagonists in disease control as
they will adapt to local conditions (Hanumanthaet al., 1989).
Rangeshwaran and Prasad (2000) reported that P. flourescens is not much
effective to work on biological agents of coffee leaf rust pathogen. Thus, present
investigation was carried out to assess the antagonistic effect of B. subtilis and P.
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
6
flourescens. Although, bacterialantagonists were tested in vitro in vivo for their
suppressing or controlling effect to the coffee leaf rust pathogen.
Since the fungicides were never used, the Hyperparasitic fungus
Verticilliumhemilieaoccurs quite frequently and able to reduce the rust inoculums under
high humid conditions. Under favorable conditions, thehyper parasite completely covers
rust uredospores and lesion with white mycelia mass. Consequently, uredosores can be
killed through necrosis of the rust lesions. In some instances, these Hyperparasites are
organisms that parasitize other parasitic and are sometimes used as biological agents
(Bhat, 2005).
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
7
MATERIALS AND METHODS
Collection and Isolation of Biological Control
Agents
Arabica plantations in Benguetwere surveyed and randomly sample healthy
anddisease Arabica coffee plants. Sampling was done in month of November to March, a
period where heavy rust infection and leaf fall occur. Every epiphyte found associated to
the coffee tissue specimens and not known as pathogen werepresumed asbeneficial or
biological control agent. These were isolated with nutrient agar (NA) or potato dextrose
agar (PDA). Pure cultures of each isolated biologicalcontrol agents were maintained
forbioassay activities and characterization.
Cultural and Morphological Characterization
The isolated biological control agents were characterized in terms of the colony
growth and development in culture media. The isolates were sampled, mounted in slides
andexamined under the microscope to characterize the appearances of their microscope
structures.
Laboratory Bioassay
Two techniques of leaf bioassay were done, the detached leaf and leaf disc
assay.Separate trials were done for fungal biological controlagents from the bacterial
biological control agent isolates.
Detached Leaf Assay. Healthy leaves of Arabica coffee were collected, washed
with sterile distilled water, blot dried and placed in transparent polyethylene bags. The
leaves were inoculated with dilutions of coffee rust uredospores, followed by spray of
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
8
biological control agents suspension, enclosed by tying the openings and incubated for 48
hours. Observations of symptom development in terms of number and size of lesions
were done.
Bioassay trials detached leaves in the laboratory were laid in the completely
randomized design (CRD) with three replications.
The treatments for the detached leaf assay were:
T1 - Kocide
T2 - Control (Plain H20)
T3
-
01-F
T4 - 01-B
T5 - Verticillium sp.
T6 - Trichoderma sp.
Leaf Disc Assay.The leaves were washed with sterile distilled water and then cut
into pieces toobtain leaf discs at size of 2.5x2.5cm. The leaf discs wereplaced in sterile
Petri dishthat wereoverlaid with wet tissue paper, inoculated with dilutions of coffee rust
uredospores, and subsequently sprayed with suspensions of biological control agents. The
treated leaf discs were incubated for48 hours. Developments of uredospores from 48
hours of incubation were monitored up to when the leaves were still green, and when
uredospores were visible.
Bioassaytrials on leaf discs werelaid in the completely randomized design (CRD)
with three replications.
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
9
Biological control agentisolates as treatment were:
Bacterial Biological ControlAgent Isolates Fungal Biological ControlAgent Isolates
T1-01-B
T1-Kocide (control)
T2- 02-B
T2-01-F
T3-03-B
T3-02-F
T4-04-B
T4-04-F
T5-05-B
T5-05-F (Fusarium sp.)
T6-06-B
T6-06-F(Penicillium sp.)
T7-07-B
T7-Verticillium sp.
T9-31 (Bacillussp.)
T8-Trichoderma sp.
T10-73(Bacillus sp.)
T9-Trichoderma sp.
T11-94 Flavobacterium
sp.
T10-Kocide
T12-158 Pseudomonas sp.
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
10
Figure 1.
Experimental set- up on Detached Leaf Assay
Figure2. Experimental set- up for Leaf Disc Assay
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
11
Data Gathered were:
1.
Isolate as biological control agents. Collection of diseased and healthy
coffee tissues was diagnosis to explore the presence of biological control agents against
coffee rust.
2.
Number of germinated and ungerminated spores of coffee rust (leaf
discand detached leaf bioassay).These were recorded starting from 48 hours after
inoculation. Lesions diameter were measured in millimeters.
3.
Number and diameter of lesions on leaf disc and detached leaf.Inhibition
of the biological control agents on the developments of the uredospores were assessed 15
days after inoculation.
4.
Morphological and Cultural Characteristics of the Isolates. The cultural
characterization was done by observing the colony growth and development on the
culture media, while the morphological characterization was done through microscopic
observations of the structures of the isolates.
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
12
RESULTS AND DISCUSSION
Isolation and Characterization
ofBiological controlAgents
Fungal Biological Control AgentIsolates. There were six fungal biological control
agents isolated and characterized. Most isolates in terms of cultural characteristics,grown
in PDA had a light orange to orange pigment while other isolates produced yellow bluish
green, light brown and white pigment in culture (Isolates 1 to 6). For morphological cha-
racteristics observed under microscope, most isolates revealed on oblate conidia (Table
1).
Table 1.Cultural and Morphological of Fungal BiologicalControl Agents.
ISOLATES
Fusarium Penicillium
CRITERIA 01-F 02-F 03-F
04-F sp. (05-F) sp. (06-F)
Fast
Slow
Slow
Slow grow- Slow
Fast grow-
Growth
growing growing growing
ing
growing
ing
From
white to
Green
Colony col-
Light
Light
or Orange
yellow
orange White Orange
Presence of
septa on
Present
mycelim Present Present Present
Present
Present
Macro-
Round
sickle
Round
Shape of
& ob-
Micro-
conidia
late oblate
oblate Rectangular oblate
Hyaline
Color of
conidia Hyaline
Hyaline
Hyaline
Hyaline Hyaline
Size of con- 1.75-5.5 1.75-3.0 2.0-5.25
1.75-3.75
idia
µm
µm
µm
µm
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
13
Figure 3. Isolate 01-F Colony (Left) Structures (Right)
Figure 4. Isolate 02-F Colony (Left) Structures (Right)
Figure 5. Isolate 03-F Colony (Left) Structures (Right)
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
14
Figure 6.Isolate 04-F Colony (Left) Structures (Right)
Figure 7.Fusarium sp. colonyIsolate
Figure 8.Penicillium sp. colon
yIsolate
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
15
Bacterial Biological Control Agent Isolates. Thebacterialbiological controlagents
were isolated in nutrient broth (NA). Pure cultures obtained were subjected to cultural
and morphological characterization. Cultural characteristics of bacterial isolates that was
grown in NA had a flat, irregular, and white in terms of their form, elevation, and color,
while other isolated produced with wavy, lobate and entire margins (Isolates 1 to 7). For
morphological characteristics, microscopic examination revealed to rod shaped, cylin-
drical and cocci shaped like (Table 2).
Table 2. Characteristics of Bacterial Biological ControlAgent Isolates
GRAM
ISOLATES FORM ELEVATION MARGIN
COLOR SHAPE (+\\-)
wavy
White (shi-
01-B Irregular
Flat
(undulate)
ny) rod -
wavy
White (shi-
02-B Irregular
Flat
(undulate)
ny) rod +
03-B Irregular
Flat lobate
transparent
rod +
wavy
Yellow
04-B Circular
Convex
(undulate)
(shiny) rod -
05-B Circular
Convex
Entire
white
cocci
-
06-B Circular
Umbonate
lobate
white rod -
07-B Circular
convex
Entire
white
cylindrical
-
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
16
Figure 9. Isolate 01-B Colonny (Left) Structures (Right)
Figure 10. Isola
te 02-B Colony (Left) Structures
Figure 11. Isolate 03-B Colony (Left) Structures
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
17
Figure 12. Isolate 04-B Colony (Left) Structures
Figure 13. Isola
ate 05-B Colony (Left) Structures
Figure 14. Isola
te 06-B Colony (Left) Structures
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
18
Figure 15. Isolate 07-B Colony (Left) Structures
Bioassay on Detached Leaf
Percentage spore germination inhibition in Table 3 shows the reaction of coffee
rust to the biological agent.The control which is plain water had the highest percentage of
spore germination inhibition with a mean of 99.6,while the lowest was from those treated
with kocidewith a spore germination inhibition of 87.1.No significant difference was
recorded in the percent inhibition of sporegermination on detached leaf.
Length of germ tube (µm) Table 3 shows that the length of germ tube of
germinated spores. The highest germ tube length of 3.7µm was obtained from those
treated with kocide and 01-F, and the lowest mean of 2.2 µm was obtained from those
treated with 01-B. Statistically there were no significant difference among the treatments
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
19
Table 3.Reaction of Coffee Rust of Biological Control Agent Against on Detached Leaf.
TREATMENTS
SPORE
LENGTTH
OF
GERM
GERMINATION
TUBE (µm)
INHIBITION
(%)
Actual
Transformed
Kocide
87.1
3.67
3.7
Control
(Plain
H20)
99.6
3.23
3.2
01-F
98.3
3.72
3.7
01-B
78.4 2.22
2.2
Verticillium sp.
98.3 2.41
2.4
Trichoderma sp.
98.2 2.52
2.5
Number of lesions on Detached Leaf (After 18 days) eighteen days after
inoculation, the number of lesions on detached leaf was counted. Most of the isolates,
plain water and kocide treatments had a lesion of which is different from those treated
with 01-F with a mean of 2. (Table 4) reveals that there were no significant differences in
the number of lesions after eighteen days of inoculation.
Diameter of lesions on detached leaf (After 18 days).The diameter of lesions on
detached leaf revealed that the highest was 2.1mm that was obtained from 01-F, and the
lowest with a mean of 1.2 mm were Verticillium sp. There were no significant differences
among the treatments (Table 4).
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
20
Table 4.Reaction of Coffee Rust of Biological Control Agent Against on Detached Leaf.
TREATMENTS
NO. OF LESIONS
DIAMETER OF LESIONS (mm)
Actual
Transformed
Actual Transformed
Kocide
0.33
1.00
1.33
1.2
Control
1.00
1.00
3.33
1.7
(Plain H20)
01-F
4.67
2.00
5.00
2.1
01-B
1.00
1.00
2.33
2.0
Verticillium sp.0.66
1.00
1.33
1.2
Trichoderma sp.1.33
1.00
2.67
1.5
Leaf Disc Bioassay
Number of lesions on Leaf disc (After 15 days)
Table 5 shows the number of lesion on leaf disc assay after 15 days of
inoculation. Treated leaf disc with 04-, 06-B, 07-B, 73, 94 and 158 had the same means
of 2.0, and mean of 1.50 was obtained from those treated with, 01-B, 02-B, 03-B, 05-B
and 31 isolates including the control. There was no significant difference.
Diameter (mm) of Lesions on Leaf disc (After 12 days)
The diameter of lesions on leaf disc is shown on table 5. The highest was obtained
from those treated with 03-B isolates with a mean of 2.79mm and the lowest was from
those treated with 01-B isolate with a mean of 1.41. Statistical analysis revealed no
significant differences.
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
21
Table 5. Effect of Bacterial BiologicalControlAgentsAgainst Coffee Rust on Leaf Disc
Assay
TREATMENTS
NO. OF LESIONS
DIAMETER OF LESIONS
(mm)
Control
(Plain
water)
1.5
2.04
01-B
(Bacteria
) 1.5
1.41
02-B
(Bacteria) 1.5
2.33
03-B
(Bacteria)
1.5
2.79
04-B
(Bacteria) 2.0
2.21
05-
B
(
Bacteria)
1.5
2.29
06-B
(Bacteria) 2.0
2.04
07-B
(Bacteria) 2.0
2.0
31(Bacillussp.) 1.5
2.58
73(Bacillus sp.) 2.0
1.71
94 Flavobacterium sp.
2.0
1.79
158 Pseudomonas sp.
2.0
2.04
Number of Lesions on Leaf disc (After 15 days)
Table 6 shows that the highest number of lesion on leaf disc was obtained from
isolate 02-F with a mean of 2.11 followed by Verticillium sp. with a mean of 1.83 and the
lowest were from Trichoderma sp. with a mean of 1.20. However, statistical analysis
revealed no significant differences on the number of lesions on leaf disc after 15 days of
inoculating.
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
22
Diameter of Lesion on Leaf disc (After12 days)
Twelve days of inoculation, the diameter of lesions on leaf disc was counted. The
04-F has the highest diameter of lesion on leaf disc with a mean of 2.67 and kocide has
the least mean diameter of lesion on leaf disc with a 1.46. In addition, there was a highly
significant difference in the mean of the diameter of the lesion leaf in terms of statistical
analysis (Table 6).
Table 6. Effect of Final Biological Control Agent Against Coffee Rust on Leaf disc
TREATMENTS
NO. OF LESIONS
DIAMETER
OF
LESIONS
(mm)
Control
1.33
1.58c
01-F
1.55
1.71b
02-F
2.11
2.33a
03-F
1.37
2.42a
04-F
1.47
2.67a
05-F (Fusarium sp.)
1.63
2.6a
06-F(Penicillium
sp.)
1.43
2.1a
Verticillium sp.
1.83
2.12a
Trichoderma sp.
1.20
2.37a
Kocide
1.37
1.46c
Means with the same are not significantly different at 5 DMRT
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
23
SUMMARY, CONCLUSION AND RECOMMENDATION
Summary
The study was conducted at the Department of Plant Pathology Laboratory at
Benguet State University, La Trinidad, Benguet from November 2011 to March 2012.
The study aimed to isolate and evaluate indigenous biological control agent against cof-
fee rust for organic Arabica coffee production, and to characterize and identify and effec-
tive biological control agent against rust disease of Arabica coffee.
There were 8 fungi and eleven bacteria isolated and presumed as Biologicalcon-
trol Agents Against the coffee rust. Two of the fungal isolates were identified as Fusa-
rium sp. and Penicilliumsp.Two of the bacterial isolates were identified as Bacillus
strains, a Flavobacteriumand Pseudomonas sp. isolate.
Conclusion
Result of the bioassays on leaf discs and detached leaves were generally not sig-
nificantly different. The data on the effectively of the fungal and bacterial isolates as bio-
logicalcontrol agents against coffee rust not conclusive.
Recommendation
Based on the findings of the study, the following are recommended:
1. Evaluation of the isolated biologicalcontrol agent against coffee rust shall
be done on seedlings.
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
24
LITERATURE CITED
AGRIOS, G . N. 1988. Plant Pathology 3rd ed. University of Florida, Gainesville. Pp.466
– 488.
ANONYMOUS. 2003. Coffee guide. Central coffee.Research institute, Chickmagalur,
Karnataka, India.P. 200.
ANONYMOUS. 1998. A compendium on pests and diseases of coffee and their
managemen in India. Coffee board research department, central coffee research
institute,Chickmagalur,Karnataka, India. P.67.
ARNESON, P. A. 2000. Coffee rust.The plant health instructor. DOI:10.1094/PHI-1-
2000-0718-02.Retrieved August 13, 2011
fromhttp://www:apsnetorg/edcenter/in/coffee rust.aspx.
BHAT, S. S. 2005. Integrated management of coffee leaf rust. Retrieved August 13,2011:
fromhttp://india coffee. Org/new letter/2005/apr/cover-story-2.Html.
BHAT, S. S., NAIDU. R.S., DAIVASIKAMANI. and NIRMALA KANNAN. 2000.
Integrated disease management in coffee. In: IPM system in agriculture. Cash
crops.Vol./
6.
BETTENCOURT, A. J. and C. J. RODRIGUEZ. 1988. Principles and practice of coffee
breeding forresistance to rust and other diseases. In: CLARKE R.J. , MACRAER,
eds, coffee: London : EL Sevier applied science. Pp. 199-234.
BROWN, J., WHAN, J., KENNY, M. and P. MERRIMAN. 1995. The effect of coffee
leaf rust onfoliation and yield of coffee in Papua New Guinea. Crop
protection.14(7);589-592.
CONRAD, J. 2009. Rust fungi. The Backyard Nature RetrievedAugust 15, 2011 from
http://www. Backyard nature.Net/f/rusts.html.
DAIVASIKAMANI, S. and T. S. GOVINDARAJAN. 1989. Field efficacy of a new
systemic fungicide PP 523 5% SC (Hexaconazole) research. 19: 65-70.
HANUMANTHA, B. T., GOVINDARAJAN, T. S. and NIRMALA KANNAN. 1989.
Comparative efficacy of four fungicides for control of coffee rust. Journal of
Coffee
Research. 19:57-64Retrieved August 15, 2011 fromhttp://en.
Wikipedia.Org/wiki/coffee
KUSHALAPPA, A. C., A. B. ESKES. 1989. Advances in coffee rust research. Ann. Rev.
Phytopathol.27:503-31.
MITCHELL, H. W. 1988. Cultivation and Harvesting of the Arabica Coffee Tree:
Agronomy. Ed. R. J CLARKE. New York:ElSeveir Applied Science.
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
25
MC MAHON, M. J., KOFRANEK, A. M., RUBATZKY, V. E. 2002. Hartmann’s plant
science.Growth, developments and utilization of cultivated plants 3rd ed. Upper,
Saddle River, New Jersey. Pp325.
PENDERGRAST, M. 2009.Tea and Coffee Trade Journal. Retrieved December 29, 2009
from http://en.wikipedia.org/wiki/Coffee
RANGESHWARAN, R. and R. D. PRASAD, 2000. Isolation and evaluation of
rhizospheric bacteria for biological control of chickpea wilt pathogens. Journal of
biological control, 14:9-15.
SANGATANAN, P. D. 1986. Agricultural arts 4.Home economics and livelihood
technology services. School graduate studies, West Visayas State College, Ilo-
ilocity. P.64.
STOLL, G. 2008. Workshop on demand workshop on module.Integrating OISAT into
agricultural training and ext. services.
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
26
APPENDICES
APPENDIX TABLE 1.Actual Percentage (%) Inhibition of Spores Germination
On Detached Leaf (After 48 hrs)
TREATMENTS REPLICATION ___
І
ІІ
ІІІ TOTAL MEAN
T˳Kocide
96.39
81.47
83.54
261.4
87.13
Tı
Control
99.46
99.68
99.55
298.69
99.56
T201-
F 98.87
98.91
97.15
294.93
98.31
T302- F
97.54
39.19 98.4 235.14
78.38
T4 Verticillium sp.
98.15
98.39
98.25
294.79
98.26
T5Trichoderma sp.
98.41
96.77
99.44
294.62
98.21
TOTAL 196.56
514.41
576.33
1679.57 559.86
ANOVA TABLE
SV DF
SS MS
FC
TABULATED
F
5%
1%
Treatment 5
1121.32
224.26 1.10ns 3.11 5.06
Error
12
2440.13
203.34
TOTAL
17
3561.45
*ns= not significant at 5% level
CV = 15.28%
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
27
APENDIX TABLE 2.Actual Length of Germ tubes of Germinated Spores on Detached
Leaf (µm) after 48 hours.
TREATMENT S
REPLICATIONS
I
II
III
T
M
T0Kocide 6
29
9
44
14.66
T1 Control
9
9
12
30
10.00
T2 01-F
3
36
10
49
16.33
T3 01-B
2
5
7
14
4.67
T4Verticillium sp.
10
4
3
17
5.67
T5Trichoderma sp.
9
5
4
18
6.00
TOTAL
39
88
45
172
57.33
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
28
APENDIX TABLE 2.1Transformed Length of Germ tubes of Germinated Spores on
Detached Leaf (µm) after 48 hours.
TREATMENT S
REPLICATION
I
II
III
T
M
T0Kocide 2.55
5.43
3.08
11.06
3.67a
T1 Control
3.08
3.08
3.54
9.7
3.23a
T2 01-F
1.87
6.04
3.24
11.15
3.72a
T3 01-B
1.58
2.35
2.74
6.67
2.22a
T4Verticillium sp.
3.24
2.12
1.87
7.23
2.41a
T5Trichoderma sp.
3.08
2.35
2.12
7.55
2.52a
TOTAL
15.4
21.37
16.39
53.36
17.79
ANOVA TABLE
SV DF
SS MS
FC
TABULATED
F
5%
1%
Treatment 5
371.78 74.36 0.91ns 3.11 5.06
Error
12
978.66
81.56
TOTAL
17
1350.44
*ns= not significant at 5% level
CV = 94.46%
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
29
APPENDIX TABLE 3.Actual Number of Rust Lesion on Detached Leaf Assay(After 18
days)
TREATMENT S
REPLICATIONS
I
II
III
T
M
0 0 1 1 0.33
T0Kocide
0 2 1 3 1.00
T1 Control
0 5 9 14
4.67
T2 01-F
0 1 2 3 1.00
T3 01-B
0 0 2 2 0.66
T4Verticillium sp.
4 0 0 4 1.33
T5Trichoderma sp.
TOTAL
4
8
15
27
9
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
30
APPENDIX TABLE 3.1TransformedNumber of Rust Lesion on Detached Leaf As-
say(After 18 days)
TREATMENT S
REPLICATIONS
I
II
III
T
M
T0Kocide 0.71
0.71
1.22
2.64
.88
T1 Control
0.71
1.58 1.22
3.51 1.17
T2 01-F
0.71
2.35 3.08
6.14 2.05
T3 01-B
0.71
1.22 1.58
3.51 1.17
T4Verticillium sp.
0.71
0.71 1.58
3.00 1.00
T5Trichoderma sp.
2.12
0.71
0.71
3.54
1.18
TOTAL
5.67
7.28
9.39
22.34
7.44
ANOVA TABLE
SV DF
SS MS
FC
TABULATED
F
5%
1%
Treatment 5
37.83 7.57 1.55ns 3.11 5.06
Error
12
58.67
4.89
TOTAL
17
96.50
*ns= not significant at 5% level
CV = 39.25%
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
31
APPENDIX TABLE 4.Actual Diameter (mm) of Rust Lesions on Detached Leaf Assay
(After 18 days)
TREATMENTS
REPLICATIONS
I
II
III
T
M
T0Kocide 0
0
4
4
1.33
T1 Control
0 8 2 10
3.33
T2 01-F
0 7 8 15
5.00
T3 01-B
0
2 5 7
2.33
T4Verticillium sp.
0 0 4 4 1.33
T5Trichoderma sp.
8 0 0 8 2.67
8
17
23
48
16
TOTAL
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
32
APPENDIX TABLE 4.1TransformedDiameter (mm) of Rust Lesions on Detached Leaf
Assay (After 18 days)
TREATMENTS
REPLICATIONS
I
II
III
T
M
T0Kocide 0.71
0.71
2.12
3.54
1.18
T1 Control
0.71 2.92 1.58 5.21 1.74
T2 01-F
0.71 2.74 2.92 6.37 2.12
T3 01-B
0.71
1.58 2.35 4.64 1.55
T4Verticillium sp.
0.71 0.71 2.12 3.54 1.18
T5Trichoderma sp.
2.92 0.71 0.71 4.34 1.45
6.47
9.37
11.8
27.64
9.21
TOTAL
ANOVA TABLE
SV DF
SS MS
FC
TABULATED
F
5%
1%
Treatment
5 28.67
5.73
0.46ns 3.11 5.06
Error
12 149.33
12.44
TOTAL
17
178.00
*ns= not significant at 5% level
CV = 132.10 %
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
33
APPENDIX TABLE 5.ActualNumber of Lesions on Leaf Disc using Bacterial Iso-
lates(After 15 days).
TREATMENTS REPLICATION
I
II
T
M
T1 Control
2 1 3
1.5
T2 01-B
1 2 3
1.5
T3 02-B
2
2
4
2
T4 03-B
1
2
3
1.5
T5 04-B 2
2
4
2
T6 05-B 1
2
3
1.5
T7 06-B 2
2
4
2
T8 07-B 3
1
4
2
T9 31
2
1
3
1.5
T10 73
2
2
4
2
T11 94 2
2
4
2
T12 158
1 3 4
2
TOTAL
21
22
43
21.5
ANOVA TABLE
SV DF
SS
MS
FC
TABULATED
F
5%
1%
Treatment
11 .91
0.08
0.17ns 2.72 4.22
Error
12 5.76
0.48
TOTAL
23
6.67
*ns= not significant at 5% level
CV = 43.57 %
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
34
APPENDIX TABLE 6.Actual Diameter (mm) of Lesions on Leaf Disc using Bacterial
Isolates(After 12 days).
TREATMENTS
REPLICATION
I
II
T
M
T1 Control
1.75 2.33 4.08 2.04
T2 01-B
1.67 1.16 2.83 1.41
T3 02-B
3.08
1.58
4.66
2.33
T4 03-B
2.58
3.0
5.58
2.79
T5 04-B 2.83
1.58
4.41
2.21
T6 05-B 2.58
2.0
4.58
2.29
T7 06-B 2.33
1.75
4.08
2.04
T8 07-B 2.58
1.41
3.99
2.0
T9 31 2.75
2.41
5.16
2.28
T10 73
1.58
1.83
3.41
1.71
T11 94 1.58
2.0
3.58
1.79
T12 158
2.08 2.0 4.08 2.04
TOTAL
27.39
23.05
50.44
25.22
ANOVA TABLE
SV DF
SS MS
FC
TABULATED
F
5%
1%
Treatment
11 3.10 0.28
Error
12 3.49 0.29
0.97ns
2.72 4.22
TOTAL 23 6.59
*ns= not significant at 5% level
CV =25.64 %
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
35
APPENDIX TABLE 7.Actual Number of Lesions on Leaf Disc using Fungal Isolates
(After 15 days)
TREATMENTS
REPLICATION
I
II
T
M
T1 Control
1 1 3
1
T2 01-F
2 1 3
2
T3 02-F
2
3
4
2
T4 03-F
1
1
3
1
T5 04-F 1
2
3
1
T6 05-F 1
2
3
2
T7 06-F 1
2
3
1
T8 Verticillium sp. 2 1 4
2
T9 Trichoderma sp. 1 1 3
1
T10Kocide 1
2
3
1
13
16
29.5
14.5
TOTAL
ANOVA TABLE
SV DF
SS MS
FC
TABULATED
F
5%
1%
Treatment
9 1.32
0.15
Error
10 2.36
0.24
0.62ns
3.02 4.95
TOTAL
19
3.68
*ns= not significant at 5% level
CV = 32.02%
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
36
APPENDIX TABLE 8.Actual Diameter (mm) of Lesions on Leaf Disc using Fungal Iso-
lates (After 12 days)
TREATMENTS
REPLICATION
I
II
T
M
T1 Control
1.41 1.75
3.16
1.58
T2 01-F
1.66 1.75
3.41
1.71
T3 02-F
2.37
2.33
4.66
2.33
T4 03-F
2.58
2.25
4.83
2.42
T5 04-F 2.42
2.91
5.33
2.67
T6 05-F 2.62
2.58
5.2
2.6
T7 06-F 1.75
2.42
4.17
2.1
T8 Verticillium sp. 2.33
1.91 4.24 2.12
T9 Trichoderma sp. 2.16
2.58 4.74
2.37
T10Kocide 1.33
1.58
2.91
1.46
TOTAL
20.63
22.06
42.69
21.35
ANOVA TABLE
SV DF
SS MS
FC
TABULATED
F
5%
1%
Treatment
9 3.25
0.36
Error
10
0.67
0.07
5.14**
3.02 4.95
TOTAL
19
3.92
**= Highly significant at 1% level
CV
=
12.42%
Isolation and Evaluation of Indiginous Biological Control Agents against Coffee Leaf Rust (Hemi‐
lieavastatrix)/ Richard B. Caga. 2012
Document Outline
- Isolation and Evaluation ofIndiginousBiological Control Agents against Coffee Leaf Rust(Hemilieavastatrix)
- BIBLIOGRAPHY
- TABLE OF CONTENTS
- INTRODUCTION
- REVIEW OF LITERATURE
- MATERIALS AND METHODS
- RESULTS AND DISCUSSION
- SUMMARY, CONCLUSION AND RECOMMENDATION
- LITERATURE CITED
- APPENDICES