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A report on the project which aimed to develop a biological control- and induced resistance-based disease management in onion, garlic and shallot in order to reduce the farmers' dependence on fungicides.
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Management of Purple Blotch of Onion,
Garlic and Shallot Using Antagonists
and Systemic Acquired Resistance
Project Leader & Study Leader:
Rizaldo G. Bayot
IMPLEMENTING AGENCY:
National Crop Protection Center,
University of the Philippines Los Baños
PROJECT FUNDING:
Department of Agriculture – BAR #88-521-21
BACKGROUND OF RESEARCH PROJECT
Project was envisioned because of the following
circumstances:
Vegetable RDE Network identified purple blotch
caused by Alternaria porri as a priority research area
in 2000.
Considered as a major constraint in bulb Allium crops
production
Farmers rely heavily on fungicides
“environmental protection should be non-negotiable-
disease management should be done with as little impact
on the environment as possible”
Microbial antagonists and yield increasing bacteria (YIB)
are already used extensively in China
Several biological control agents have been registered in
the USA against plant diseases
These are plant-associated microorganisms that work on
the principle of the “exclusionary” system of plant defense.
Findings of researchers abroad that certain plants can be
induced to become resistant against a broad spectrum of
leaf and root pathogens using biological and chemical
inducers.
If plants can be “immunized” or induced to produce natural
products to combat infection, the use of fungicides can be
reduced; ensuring public safety and environmental
integrity
During project implementation:
Purple blotch was no longer a problem.
Leaf anthracnose caused by Colletotrichum sp.
became very serious in onion and shallot
Project evaluators were informed.
Emphasis of the project shifted to anthracnose.
Objectives:
General:
To develop a biological control- and induced resistance-based
disease management in onion, garlic and shallot in order to
reduce the farmers' dependence on fungicides
Specific:
1) To isolate, characterize and evaluate the efficacy of
indigenous microbial antagonists against Alternaria porri
and Colletotrichum sp. of garlic, onion and shallot.
2) To screen commercially available varieties of garlic, onion
and shallot for systemic acquired resistance (SAR) traits
using SAR-inducing compounds against A. porri and
Colletotrichum sp.
Methodology and Results
1. Biological Control of Purple Blotch Using Microbial
Antagonists
a. Survey of Allium farms and specimen
collection
Onion and shallot farms in Bongabon, Gabaldon, Muñoz and
San Jose City, Nueva Ecija
Garlic farms in Batac, Ilocos Norte
Purple blotch did not develop in the areas surveyed due to
dry weather condition that prevailed
Few plants in Bongabon and Gabaldon developed purple
blotch symptoms late in the season.
b. Isolation of pathogens and pathogenicity
Isolation methods: tissue planting, streaking of spore
suspension, cutting hyphal tip of germinating conidia
Culture media: water agar, potato dextrose agar (PDA),
acidified PDA, PDA amended with onion leaf extract, PDA
amended with 100 ppm streptomycin sulfate, onion leaf extract
amended with 1% glucose.
Spore suspension was prepared and sprayed to onion
seedlings
Inoculated seedlings were incubated in a moist chamber for 48
hours and observed for symptom development in greenhouse.
Alternaria sp. was isolated but did not develop typical
symptoms of purple blotch.
Delayed the other activities of the project
Anthracnose caused by Colletotrichum sp. was severe in the
areas surveyed
The isolated pathogen was highly virulent
Used in subsequent tests
c. Isolation of microbial antagonists
Roots and leaves of garlic, onion and shallot collected
from various locations were used for isolation.
Cut into small pieces 5 gms placed in test tubes or
flasks with sterile water incubated for 30 minutes
with intermittent shaking in vortex mixer suspension
streaked on King’s B medium (KB), nutrient yeast agar
(NYA) and potato dextrose peptone agar (PDPA).
Representative colonies were transferred to PDPA
slants, purified and stored in mineral oil at room
temperature.
80 probable antagonists were isolated from various
sources and maintained in the laboratory using mineral
oil
d. In vitro screening of isolates for antagonism
Dual culture test was used
Garlic Isolates1 Degree of Inhibition2
Against Alternaria sp. Against Colletotrichum sp.
GLC1 - NT
GLC2 ++ NT
GL1W2 + +
GLM1 - -
GLM3 - NT
GLM3a - -
GLM3b + -
GLM4b - -
GLNE1 + +
GLNE2 + -
GRC1a - -
GRC1b - -
GRC2a + +
GRC2b ++ NT
Table 1. In vitro test of probable antagonists for mycelial growth inhibition
of Alternaria sp. and Colletotrichum sp. of Allium using the dual
culture method.
Garlic Isolates1 Degree of Inhibition2
Against Alternaria sp. Against Colletotrichum sp.
GRC3 - NT
GRC4 + +
GR1W1a + +
GR1W2 - -
GR1W2a ++ NT
GR1W3 - -
GR1W3b + NT
GR1W5 + -
GRM3a + -
GRM3b + -
GRM4a + -
GRM4b - -
GRM5 + -
GRM6a - -
GRM6b - -
GRNE1a - -
GRNE1b - -
GRNE2b ++ +
GRNE3 - -
GRNE3b - NT
GRNE4a + +
GRNE4b + -
GRNE5a - -
Table 1. Cont…
Garlic Isolates1 Degree of Inhibition2
Against Alternaria sp. Against Colletotrichum sp.
GRNE5b - -
OLNE1 - -
OLNE2 + -
OLNE3 - -
OLNE4 - NT
OLNE4a + +
OLNE4b + -
ORNE1a + NT
ORNE1b + NT
ORNE2a ++ +
ORNE2b + +
ORUBS1 - +
ORUBS2 - -
Leek Isolates1
LL3 + NT
LR1 + NT
LR2 + NT
LR3 + NT
LR4 ++ NT
Shallot Isolates1
SL1 - NT
SL2 - NT
Table 1. Cont…
Garlic Isolates1 Degree of Inhibition2
Against Alternaria sp. Against Colletotrichum sp.
SL3 - NT
SL4 - NT
SL5 - NT
SL6 - NT
SL7 + NT
SL8 - NT
BC1 NT +++
BC2 NT ++
B2HT3 ++ ++
B3HT NT +++
B2HTRiFR1 NT +++
BRIHT +++ +++
Bp +++ NT
B. subtilis (Biotech) NT +
B. polymixa (Biotech) NT -
Table 1. Cont…
Garlic Isolates1 Degree of Inhibition2
Against Alternaria sp. Against Colletotrichum sp.
CHRHT +++ NT
CS2HT ++ ++
ESHT1 ++ NT
ISO2HT ++ ++
M3HT ++ ++
M3MTRifR3 NT +++
ANT1 +++ ++
ANT 2 NT ++
1/ Mycelial disks of the pathogen were placed at the center of PDA plates. Cells of 24-48 hr-old
cultures of antagonists were spotted on the medium about 40 mm from the fungal disk in a X
pattern. Growth inhibition was determined after 3-5 days.
Table . Cont…
d. In vitro screening of isolates for antagonism
Out of 80 isolates, 72 were tested against Alternaria sp. in dual culture
test
4 had strong inhibition, 11 had moderate inhibition, 26 had slight
inhibition, the rest had no effect
53 isolates were tested against Colletotrichum sp.
5 had strong inhibition, 7 had moderate inhibition, 12 had slight
inhibition.
Figure 3. Verification of antagonism of biocontrol agents stored in mineral oil for one year using the dual culture method against the anthracnose pathogen.
Inhibitory effects were retained after 1 year of storage in mineral oil
Figure 4. Paper disk assay of supernatants of biocontrol agents grown in nutrient broth for fifteen days against the anthracnose pathogen.
Filter-sterilized culture filtrate of some antagonists inhibited mycelial growth of
anthracnose pathogen
Spore Germination Test• Prepared spore suspension of Alternaria sp.
100 ul placed on clean glass slides
Antagonist growth (24-48 hr old) was mixed with spore
suspension
Observed microscopically
Germinating conidia of Alternaria sp. after six hours of incubation showing normal germ tube formation in untreated slide (control)(1000x).
Germinating conidia of Alternaria sp. showing swollen germ tube six hours after treatment with B2HT3 isolate (1000x).
Germinating conidia of Alternaria sp. showing large swelling germ tube 18 hours after treatment with B2HT3
isolate (1000x).
Table 2. Influence of selected bacterial antagonists on conidial germination
of Alternaria sp.
1About 100 µL spore suspension of A. porri was placed on a clean glass slide. Small
amount of bacterial growth from 24-48 hours old culture was mixed with the spore
suspension of the pathogen. Glass slides were incubated in petri plates with moist
tissue paper and examined microscopically at different time intervals for any abnormality in spore germination.
Isolate Tested1 Reaction
B2HT3 Swollen germ tubes after 5 hours
Bp Normal spore germination after 5 hours
BR1HT Few spores germinated after 5 hours, swollen germ tubes
ChrHT Few spores germinated after 5 hours, swollen germ tubes
CS2HT Some spores have normal germination, some have swollen germ tubes after 5.5 hours
ESHT1 Most spores have swollen germ tubes after 5.5 hours
ISO2HT Few spores germinated after 5 hours, swollen germ tubes
M3HT Normal spore germination after 5 hours
8 isolates were tested for effects on conidial germination of Alternaria
sp.
5 isolates had strong inhibitory effects on conidial germination .
Germinated conidia have swollen germ tubes
Treatment No. of Lesions 4 Days After Inoculation1
I II Mean
M3HTRifR3 0 0 0
ISO2HT 1.6 0.2 0.9
BRIHT 0.6 2.2 1.4
B2HT3RifR1 2.8 0.8 1.8
CHRHT 4.0 0.6 2.3
B3HT 1.6 3.2 2.4
ESHT1 3.8 3.2 3.5
Control 3.4 4.0 3.7
Table 3. Greenhouse evaluation of antagonists against
Colletotrichum sp. using the “Tanduyong” variety of
shallot (Sept. 2002).
1Each figure is the mean of five leaves.
Greenhouse evaluation of promising antagonists
against anthracnose
First trial used 7 potential antagonists
4 isolates reduced anthracnose lesions in leaves of Tanduyong
variety.
Treatment No. of Lesions 8 Days After Inoculation1
I II III MeanCHRHT 0 0 0 0.01CS2HT 0 0.2 0.2 0.131BC1 0.6 1.0 0.4 0.67 kESHT1 0.2 0.8 1.4 0.8 kBC II 1.0 0.8 1.2 1.0 jB3HT 1.2 1.8 0.5 1.17ijM3HT 1.4 1.2 1.2 1.27 hiB2HT3 1.2 0.4 2.6 1.4 hGR4 1.8 0.6 2.4 1.6 gANT1 1.8 1.2 2.2 1.73 gOLNE3 2.0 2.2 1.8 2.0 fB. poly (Biotech) 2.0 2.8 1.4 2.07 fISO2HT 2.0 4.2 2.0 2.73 eBRIHT 4.0 2.0 2.6 2.87 deANT2 2.2 1.2 5.4 2.93 deSL7 3.0 3.0 2.8 2.93 dB. substilis (Biotech) 3.4 3.4 2.6 3.13 eBp 4.2 3.8 2.2 3.4 bControl (water) 2.0 5.6 1.4 3.0 cdControl (N.B.) 5.4 4.8 7.2 5.8 a
Table 4. Greenhouse evaluation of antagonists against Colletotrichum sp.
using the “Tanduyong” variety of shallot (January, 2003).
1Each figure is the mean of five leaves.
Greenhouse evaluation of promising antagonists
against anthracnose
Second trial used 18 antagonists
8 isolates reduced anthracnose lesions on leaves of shallot
(Tanduyong) ranging from 0 to 1.4 lesions per leaf compared to 3
lesions in untreated plants.
Treatment
Disease Severity (%)
Mean1 2 3 4 5
1. Control 50 60 60 60 65 59 ab
2. QST (liquid) 30 40 20 20 20 26 f
3. QST (solid) 50 55 40 45 50 48 cde
4. BC1 (liquid) 50 55 50 40 30 45 de
5. BCI (solid) 50 60 60 60 50 56 abc
6. EPSP (liquid) 30 50 50 45 50 45 de
7. EPSP (solid) 50 60 60 55 40 53 bcd
8. Ant 1 (liquid) 50 60 60 65 65 60 ab
9. Ant 1 (solid) 45 50 45 50 50 48 cde
10. CS2HT (liquid) 50 30 45 45 45 43 e
11. CS2HT (solid) 40 50 55 60 60 53 bcd
12. ESHT l (liquid) 60 65 65 70 65 65 a
13. ESHT l (solid) 60 60 60 70 65 63 a
14. Avirulent
Alternaria
50 60 60 50 40 52 bcde
15. 150 ppm
difenoconazole
5 10 5 10 5 7 g
Table 5. Greenhouse evaluation of bacterial antagonists grown in liquid
and solid media against shallot anthracnose.
Comparison of bacterial antagonists grown
in solid and liquid media
Antagonists grown in liquid medium (PDP broth) were more
effective against anthracnose than those grown in solid
medium (PDPA) except for 1 isolate)
Treatment
Disease severity (%)
Mean1 2 3 4 5
Control 70 75 75 75 80 75 a
QST (liquid) 20 30 30 40 30 30 d
BC1 (liquid) 50 50 50 55 40 49 c
BCl (solid) 55 55 55 70 65 60 b
B2HT3 (liquid) 60 50 55 60 60 57 bc
B2HT3 (solid) 60 70 70 65 60 65 b
150 ppm
(score)
20 40 40 45 45 38 d
Table 6. Greenhouse evaluation of selected promising bacterial
antagonists grown in solid and liquid media against
shallot anthracnose.
M3HT B2HT3 B3HT ChrHT C2H
T
Bp Br1HT B.
poly
B.
sub
ISO2
HT
ESHT1
Motility (-) (-) (-) Slight (-) (-) (-) (-) (-) (-) (-)
Growth in 7%
NaCl
(+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+)
Citrate Utilization (+) (+) (-) (-) (-) (-) (+) (-) (+) (+) (+)
Acid From:
Arabinose
Mannitol
Xylose
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
(+)
Table 7. Partial characterization of selected potential antagonists.
Study 2. Systemic Acquired Resistance in Disease
Management of Onion and Shallot
Preliminary evaluation of compounds for induction
of resistance in shallot. Two varieties of shallot
were evaluated (Tanduyong and Batanes)
Various concentrations of acetylacetate and
K2HPO4 were evaluated
Results are not conclusive although the
Tanduyong and Batanes varieties showed some
degree of induced resistance
Mean No. of Lesions 6 Days After Inoculation1
Treatment 1 Day After Induction 3 Days After
Induction
Tanduyong Batanes Tanduyong Batanes
0.1% Acetylsalicylate 1.0 1.0 1.0 2.2
0.075% Acetylsalicylate 0 0.4 2.4 3.4
0.05% Acetylsalicylate 0.8 2 2.6 2.0
100 mM K2HPO4 1.6 1.6 4.4 2.8
75 mM K2HPO4 3.6 1.0 0.8 1.4
50 mM K2HPO4 1.4 1.8 4.0 3.2
Control (untreated) 1.0 1.0 6.4 4.4
Table 8 Efficacy evaluation of various concentrations of selected
chemicals for induction of resistance of shallot varieties
against Colletotrichum sp (September 2002).
1Each figure is the mean of five leaves.
Treatment Disease Severity (%) 8 days after inoculation
I II III Mean
Control 30 42 48 40.0 abc
100 ppm (BTH) 25 26 19 23.3 d
10 mM INA 38 28 36 34.0 abcd
20 mM INA 29 23 35 29.0 bcd
10 mM SA 46 44 33 41.0 ab
20 mM SA 44 40 35 39.7 abc
10 mM Oxal 43 35 44 40.7 abc
20 mM Oxal 33 24 26 27.7 cd
50 mM K2HPO4 44 39 54 46.7 a
75 mM K2HPO4 24 46 36 35.3 abcd
Table 9. Efficacy evaluation of various compounds for induced resistance
of shallot (cv. Tanduyong) against Allium anthracnose.
1Plants were inoculated four days after induction.
Second trial, isonicotinic acid (INA), salicylic acid (SA),
oxalic acid and K2HPO4 were compared with
benzothiodiazole (BTH) or acibenzolar-s-methyl
Only BTH reduced anthracnose severity (23.3%) compared to
40% in untreated plants
Plants sprayed with 20 mM oxalic acid had 27.7% disease
severity
Treatment % Disease Severity 5 Days After Inoculation Mean
1 2 3 4 5
Control (untreated) 50 40 30 40 30 38 de
150 ppm BTH 40 30 30 30 45 35 e
Acacia (fresh) 50 55 60 60 60 57 ab
Acacia (compost) 50 60 60 50 50 54 abc
(fresh) 30 40 40 30 30 34 e
(compost) 60 60 60 70 60 62 a
Chromolaena (fresh) 50 50 40 40 30 42 cde
Chromolaena
(compost)
60 40 55 60 60 55 ab
Narra (fresh) 60 60 30 60 30 48 bcd
Narra (compost) 60 70 30 60 55 55 bcd
Colletotrichum
(mango)
30 30 40 50 40 38 de
Table 10. Greenhouse evaluation of various plant extracts as possible
inducers of shallot resistance against anthracnose.
None of the plant extracts (fresh or compost extract) increased the resistance
of test plants against onion anthracnose
Field evaluation of promising antagonists and chemical inducers
against shallot anthracnose
Set-up of field experiment for evaluation of selected antagonists and
chemical inducers against leaf anthracnose of shallot.
Treatment
Disease Severity (%)1
MeanI II III
Control 50 56 62 56.0 a
150 ppm difenoconazole 36 44 34 38.0 c
BTH (150 ppm) 39 38 36 37.7 c
BC1 54 48 50 50.7 ab
B2HT3 46 40 44 43.3 bc
QST 38 42 44 41.3 c
Oxalic acid (20 mM) 38 32 46 38.7 c
Table 11. Field evaluation of selected bacterial antagonists and
chemical inducers against shallot anthracnose.
1Each figure is the mean of five plant samples.
Field evaluation of promising antagonists and chemical inducers
against shallot anthracnose
Antagonists evaluated were: BC1, B2HT3 and QST (commercial
product)
Chemical inducers evaluated: oxalic acid and BTH
Antagonist B2HT3 was comparable to QST (commercial product)
Chemical inducer oxalic acid (20mM) was comparable to BTH
(commercial product)
The treatments (except BC1) were comparable to the effect of
fungicide difenoconazole (150 ppm)
Conclusion and Recommendation
Purple blotch of bulb Allium crops caused by Alternaria porri was no longer a
serious threat to bulb Allium crops production from 2002 to 2005.
Foliar anthracnose caused by Colletotrichum sp., and later on, twister disease
caused by Colletotrichum sp. and Fusarium sp. became serious fungal diseases of
these crops.
Some indigenous bacterial antagonists may be effective against foliar
anthracnose of shallot
“Tanduyong” variety of shallot may be induced to become resistant to
anthracnose by certain chemical activators
Further studies are necessary to fine-tune the biocontrol-based and SAR-based
disease management of foliar anthracnose in bulb Allium crops..